\\text{Prob}(E_v \\mid \\neg C_v)$. But that’s similar to saying $E_v$ and $C_v$ are correlated—they tend to occur together; and it is a well-known mantra that correlation is not causation. High candy consumption in one month is correlated with low divorce rate the next. But eating candy does not prevent divorce. The correlation is due to the fact that candy eating is correlated with being young and young people have generally not been married long enough to get divorced. So, something more needs to be said.\n\nSuppose you were able to control for all the other factors causing or preventing $E_v$.\\(^1\\) Imagine you could look in a population where all of these took some fixed value. So everyone is the same age, the same religion, is undergoing the same stress at work, has the same number of children, etc. In this population it seems reasonable to expect that if candy consumption does cause low divorce rate, it will increase the probability; and the probability will increase only if it does so since in this population there’s no other way to account for an increase in probability. Somewhat more formally, let $K_i$ represent a population where all the causes of $E_v$ other than $C_i$ take some fixed value, where $i$ ranges over all the sets of values these causes can take. Then $C_i$ causes $E_v$ in $K_i$ if and only if $\\text{Prob}(E_v \\mid C_i + K_i) > \\text{Prob}(E_v \\mid \\neg C_i + K_i)$.\n\nThis is the gist of the probabilistic theory of causation, though more details need to be ironed out. For instance, note that this characterization is relative to the population satisfying $K_i$. What then about a larger population that contains $K_i$? That is one of the points of dispute. Some philosophers and social scientists insist on keeping the relativization to the $K_i$. Others allow that $C_i$ causes $E_v$ in any population that contains $K_i$, though this can lead to it being true both that $C_i$ causes $E_v$ in a population and also prevents $E_v$ there in cases where in one subpopulation $C_i$ increases the probability of $E_v$ and in another it decreases it. Under any circumstances this kind of characterization will always be relativized to a population since different populations will have different probabilities over the same variables and also they may well have other general causal relations holding, hence different factors for determining the $K_i$s. Chapter 3 worries about taking studies that show that a policy worked—i.e. caused the targeted outcomes—in one situation as evidence that it will have the same effects elsewhere. It is just this kind of relativity of causal claims to other causal factors (those that pick out the $K_i$s) that creates this worry: whether $C_i$ causes $E_v$ or prevents it or does nothing to $E_v$ at all in a given situation can depend very much on what other causal factors are there in the situation along with $C_i$.\n\n\\(^1\\) Except those on the causal pathway between $C_i$ and $E_v$ if it exists.\n\nNote also that seen as a characterization of general level causality—as a way of providing clear unambiguous sense for the concept—the probabilistic theory of causation is not reductive since we need to refer to general causal relations in specifying what $K_i$ is and thus we refer to other general causal relations in explaining what is required for any given general causal relation to hold. It is, however, very constraining. Each of the factors in $K_i$ must satisfy a similar formula with respect to $C_i$ and the remaining factors in $K_i$ since each of these is itself meant to be a cause of $E_v$. Although this may not narrow the choice of causes for $E_v$ to a single choice, it will rule out a huge number of alternatives. And adding some information about a few factors, that they are indeed a cause of $E_v$ or that they are not, can sometimes fix the set entirely given the probabilities.\n\nThis way of characterizing causality has the advantage that it connects immediately with standard statistical methods used in the social sciences to test for causal relations. These methods are used in what are called ‘observational studies’, which means that the data come from populations in their natural settings and not from specially selected populations enrolled in experiments. In the populations under study, social scientists measure correlations or regressions between factors to begin to test whether there is a causal connection between them in that population. These are weaker notions than conditional probabilities, like $\\text{Prob}(E_v \\mid C_i)$ and $\\text{Prob}(E_v \\mid \\neg C_i)$, but are closely related. In a better test, they measure partial correlations or partial regressions, holding fixed other variables that they hope represent the other causes. This is akin to the partial conditional probabilities $\\text{Prob}(E_v \\mid C_i + K_j)$ and $\\text{Prob}(E_v \\mid \\neg C_i + K_j)$. Similar kinds of ideas are used in econometrics, in estimating the coefficients that appear in economic equations that will hopefully represent causal relations.\n\nThe two assumptions of the probabilistic theory—that an effect is always probabilistically dependent on its cause and that this kind of dependency disappears when other causes are held fixed—are also at the core of what are called ‘Bayes nets methods’ for causal inference, for which computer programmes can generate all sets of causal relations among a given set of variables that are possible, given information about probabilistic dependencies among the variables, supposing the fundamental assumptions linking causes and probabilities are satisfied.\n\nThese two assumptions are also at the core of the reasoning behind randomized controlled trials (RCTs), which are highly touted as the gold standard for causal testing in medicine and, as we see in Chapter 3, in evidence-based policy, and are being pushed throughout the social sciences now, especially in development economics. The neat thing about RCTs is that they help with one of the central problems that the statistical methods discussed so far face: that we generally don’t know what the other causal factors are and so don’t know what to hold fixed.\n\nIn an RCT, the individuals in a population enrolled in the experiment—which could be individuals, schools, countries, etc.—are randomly assigned either to the treatment group, which will be subject to the cause ($C_i$), or to the control group, which will not have the cause ($\\neg C_i$), but may perhaps receive a placebo. There should be as much masking as possible: the individuals in the experiment should not know which group they are in, nor should anyone involved in further monitoring or treatment, or in reading out the results to see if $E_v$ obtains or not, or in carrying out the statistical analysis. This is to guard against conscious or unconscious bias that may influence the results that are finally recorded. The aim is to ensure that both groups have the same distribution for all the other causal factors influencing the outcome, so that every arrangement of them—every $K_i$—has the same frequency in both groups. This aim can very rarely be achieved and moreover, we won’t know when it has been: statistics can tell us how often in the long run random assignment will produce any particular imbalance but we do not have even that kind of assessment when it comes to how well the masking has succeeded. So, as always in scientific work, we must not place too much confidence in the results of a single study, even one that has been very well conducted.\n\nIn order to see the logic of the causal inference, let us suppose, though, that the other causal factors have the same frequency in the treatment and control groups. The $\\text{Prob}(E_v)$ in the treatment group will then be an average across the probability of $E_v$ given $C_i$ in each of the subpopulations in it—i.e. each of the $K_i$. So it will be an average over $\\text{Prob}(E_v \\mid C_i + K_j)$. Similarly, the $\\text{Prob}(E_v)$ in the control is the average of the probability of $E_v$ given $\\neg C_i$, in each of the subpopulations represented there—each of the $K_i$. So it is an average over $\\text{Prob}(E_v \\mid \\neg C_i + K_j)$. By our hypothesis that the other causal factors have the same distribution in the two groups, the frequency of each $K_i$ subpopulation is the same in both. So if the probability of $E_v$ is greater in the treatment group than in the control group, that implies that for one of the $K_i$ subpopulations, $\\text{Prob}(E_v \\mid C_i + K_j) > \\text{Prob}(E_v \\mid \\neg C_i + K_j)$. It follows that there is at least one subpopulation—one $K_i$—of the experimental population in which ‘$C_i$ causes $E_v$ in $K_i$’ is true, or at least is true under the probabilistic theory’s way of characterizing causality.\n\nCan we conclude from the experiment ‘$C_i$ causes $E_v$ in the experimental population’? That depends on the decision referred to earlier about what to say about ‘$C_i$ causes $E_v$’ in a population given that $C_i$ causes $E_v$ in one of its subpopulations. What matters is that, however this decision is made, those using the claim should understand it and not read more out of it than the study supports. In particular, the higher probability in the treatment over the control group shows only that $C_i$ causes $E_v$ in some subpopulation of the population enrolled in the experiment. It may hold in specific other populations\n\nor even across most. But $C_t$ may have exactly the opposite effect on $E_v$ in some subpopulations than it does in others. Finding out whether that is true requires a great deal more social science work.\n\n2.2 Intervention and Manipulation\n\nThe manipulation view of causation revolves around the idea that causes give us effective strategies for producing effects we want, or preventing those we do not; by manipulating the cause we can manipulate the effect in a predictable way. So manipulation theories characterize general-level causation roughly this way, where again there is a dispute about the exact details of the formulation: for any two times $t$ and $t'$, ‘$C_t$ causes $E_{v'}$ in situations of type $S'$ just in case manipulating $C$ at $t$—making it bigger or smaller, or bringing it in or taking it away—is regularly followed by appropriate changes in $E$ at $t'$. For this to be true, we must be careful how these manipulations are carried out. ‘Reducing class sizes causes improved reading scores’ is true in many populations. But not if you lower teacher quality at the same time as reducing class size. Alternatively the manipulation of one factor may be followed by improvement in another without any causal connection. For instance new programmes for teaching reading may be followed by better reading scores not because the programmes cause better scores but because the teachers adopting them are regularly the better teachers or they become more enthusiastic when trying out new programmes.\n\nThis is where the concept of intervention comes in. ‘Intervention’ has been given a variety of formal definitions, but the basic idea is that an intervention is a manipulation that is done in the ‘right way’ to make the causal relation, or lack of it, apparent. The right way will be one in which neither other causes nor other preventative of the effect change, an idea we are familiar with from the probabilistic theory. Nor can any causal relations involving the effect change during the intervention. That’s because it is not a fair test of a causal relation if suddenly there are a lot of new causal relations produced between the effect and other factors that were not causes before. This includes changes in the very relation under test. One familiar way in which the requirement that causal relations involving the effect not change during intervention is violated is when the manipulation of the cause is so ham-fisted that it busts up the causal relation we are trying to test. One familiar example is when we wind up the toy soldier to see if that will make it march, but we wind too tightly and break the mechanism that makes it work.\n\nNobel-prize-winning Chicago School economist Robert Lucas claims this is frequently true with attempts to manipulate economic variables to bring about desired change. For instance, inflation, when it occurs naturally, can in the short run cause reductions in unemployment. That’s because, so the story goes, entrepreneurs mistakenly see the universal rise in prices as a real rise in prices in their sector and so hire more workers to produce more goods to meet increased demand. If, though, the government manipulates inflation to improve unemployment, entrepreneurs will recognize the rise in prices for what it is—just inflation—and will not open new jobs. The government’s very attempt to use the causal relation between inflation and unemployment will break it. Or at least so says Lucas’s model.\n\nAs with probabilistic theory, the characterization of general causation using the concept of manipulation is not reductive since the definition of an intervention, however the details are worked out, will have to refer to other general causal relations.\n\nAlso, as with the probabilistic theory, the characterization of causality in terms of manipulation is closely linked with familiar methods for testing, in this case with real non-statistical experiments where other causes are held fixed and only the cause under test is varied. Here too we are often stymied by our lack of knowledge of what these other causes are. This is the problem that plagues standard before-and-after studies. In before-and-after studies the cause is administered and we look to see if the effect changes. But, are we sure that nothing else changed at the same time as the cause? One of the tricks of social science is to find situations where we can have reasonable confidence that nothing else that could influence the effect has changed even if we do not have a catalogue of what all the other influences might be to check on them. This is the strategy that is used in what are called ‘instrumental variables’ models in economics. For instance Joshua Angrist in a classic paper uses the Vietnam era draft lottery to measure the effect of veteran status on earnings (Angrist 1990). Since whether an individual was drafted was determined on the basis of a randomly assigned number, we can be fairly confident that factors that affect earnings, like education, were equally distributed between the group of individuals who were drafted and the group of individuals who were not, as in an RCT. And so we can be fairly confident that the comparison between the average earnings in one group and the average earnings in the other tells us something about the effect of veteran status on earnings.\n\nThe manipulation theory of general-level causation is closely akin to the counterfactual theory of singular causation, which is based on the idea that one specific event, $c$, is a cause of a later event, $e$, just in case $e$ would not have occurred unless $c$ had. You can see immediately that this kind of claim will be hard to test since $c$ and $e$ are specific events that either occur or do not; so we can never observe what would have happened had things been different. There are also huge problems of formulation. There is a vast literature available in philosophy on counterfactuals and causes in which you can read more about these (see e.g. Paul 2009 and references therein).\n\nSimilar counterfactuals have come to the fore in social science lately because of the rise of the evaluation industry for social policy. As Chapter 3 explains, there is a great demand to know whether the policies we have implemented have ‘worked’: Has the policy genuinely produced the change intended? To answer that, it helps to know what would have happened otherwise. RCTs are often employed to this end. But of course they cannot tell us of a specific case whether the policy brought about the effect there, since they only look at groups. If an RCT shows more positive results in the treatment group, the group in which the policy was implemented, than in the group where it was not, we can conclude that in some of the cases in the treatment group the policy produced the effect. But we do not know which; we have no way to sort cases where the policy brought about the effect from those where something else was responsible. So for reliable evaluation of the single case, some different way of reasoning is required. This often involves process tracing which we shall discuss in the next section.\n\nNot only do we not know from the RCT for which individuals the policy worked. We do not know what sets of characteristics matter. We know that there is at least one arrangement of characteristics that individuals might have—one $K_i$—for which the policy promotes the desired outcome. We call these ‘support factors’. But we do not know which it is. This matters when it comes to putting the claims established in RCTs to use in predicting what will happen if the policy were to be implemented elsewhere. If the population elsewhere has no individuals with just the right arrangement of support factors (no individuals described by the successful $K_i$s), or not enough to make it pay, the policy will not produce the desired outcomes in the new setting. Given good reason to think that the new setting does have enough individuals with the right arrangements, we have good reason to suppose the policy will work for some individuals there. But without good reason to suppose this, the RCT results will not be much use in predicting results in the new setting.\n\nConsider for instance Oportunidades, a Mexican programme for poverty reduction. Because Oportunidades was itself designed as an RCT, there is very good evidence that it did contribute to reducing poverty where it was implemented. Does the success of Oportunidades provide evidence supporting predictions regarding what would happen were a similar programme implemented in some other population? Only if enough individuals in this target population have the right arrangement of support factors. And one cannot assume without evidence that this will be so. This point is illustrated by the case of Opportunity NYC, a poverty reduction programme modelled—and named—after Oportunidades which was implemented in New York City in 2007 but discontinued in 2010 because it failed to produce the expected effects. The success of Oportunidades in Mexico did not, by itself, provide sufficient evidence to support the prediction that Opportunity NYC would produce similar results, and the mistaken assumption that it did led to a poor policy decision.\n\n### 2.3 Mechanisms\n\nThere are three senses of the term ‘mechanism’ in play currently in work on causation in philosophy and in the social and in the biomedical sciences. These include notions of *causal process*, *invariance*, and *underlying structure*.\n\n**CAUSAL PROCESSES**\n\nFor singular causation it is widely held that a cause must be connected with its effect by some spatio-temporally continuous process between them. So tracing the causal process between them can be a good way to tell whether two events are related as cause and effect, a good way IF we know how to tell a causal process when we see one. So what distinguishes a continuous sequence of events that constitutes a causal process from one that does not? One popular philosophical account answers that in causal processes energy is transferred at each step in between. But this is not of much help in social science (see e.g. Dowe 2009).\n\nA standard procedure in social science and in policy evaluation is to break the sequence into small steps where it is easier to determine for each step whether it is a genuine cause of the next. For instance, the Global Environment Facility evaluated the effectiveness of one of its programmes, the end-goal of which was to ‘establish a long-term conservation finance mechanism to support biodiversity conservation’ in the Bwindi national park (Uganda), by determining whether this programme had an effect on four intermediate outcomes (e.g. the establishment of a Bwindi Trust) which in turn were supposed to contribute to achieving the end-goal of the program (GEF 2007: 6).\n\nProcess tracing necessarily focuses on singular causation. But it can be a tool for general causation as well. If in situations of kind $S$, an event of kind $C$ at $t$ is regularly followed by an event of kind $E$ at $t'$ and it regularly happens that the same kind of causal process connects the individual events, then we can conclude at the general level that ‘In $S$-type situations, $C_t$ causes $E_{t'}$.’ This kind of reasoning has played a significant role in the biomedical sciences. For instance, tracing the process by which the chemicals in cigarette smoke lead to lung cancer provided a central piece of the evidence that showed that smoking causes lung cancer.\n\n**INVARIANCE**\n\nSuppose we observe that in a particular kind of situation $S$ one kind of feature $F_2$ regularly changes after another, $F_1$, changes. That is mere correlation and we know that correlation does not equal causation. But suppose that $F_1$\n\nchanges by intervention. If F1 causes F2 we expect that F2 will change following the intervention in the way that it always has; but not, if F1 does not. Changing levels of candy consumption may be regularly followed by changing probabilities of divorce. But this relationship is not invariant under intervention. If we intervene to change candy consumption—‘intervene’, so the only change is in the amount of candy consumed—the divorce rate will not change in tandem. This observation has given rise to the invariance account of causality now in fashion in philosophy of science. The basic idea is that, supposing C\\textsubscript{i} and E\\textsubscript{t} are regularly associated in S, then ‘C\\textsubscript{i} causes E\\textsubscript{t} in S’ just in case the association between C\\textsubscript{i} and E\\textsubscript{t} is invariant under interventions on C\\textsubscript{i}.\n\nThis account is clearly closely connected with the manipulation account, and it shares one major drawback with that account that we have not yet discussed. The manipulation account is grounded in the idea that causes provide strategies for changing the world. Suppose we do characterize causation as invariance under intervention. Then changing C\\textsubscript{i} will be a good way to change E\\textsubscript{t}—but that follows only if C\\textsubscript{i} is changed by intervention. But interventions are hard to come by. We are in a far more powerful position to predict what will happen and to engineer what we want if we know about a regular association that is invariant under the method of implementation that we will in fact employ. This gives rise to another, different characterization of general-level causation, one that we can find in contemporary economics (see e.g. Hoover 2001: ch. 2). Roughly, if C\\textsubscript{i} and E\\textsubscript{t} are regularly associated in S, then C\\textsubscript{i} causes E\\textsubscript{t} in S relative to φ just in case the association between C\\textsubscript{i} and E\\textsubscript{t} is invariant when C\\textsubscript{i} is changed by φ, where φ can then represent the method by which we will in fact change C\\textsubscript{i}. Knowing that C\\textsubscript{i} causes E\\textsubscript{t} under this characterization of causation immediately provides an effective strategy for changing E at t’.\n\nNotice that these different versions of the invariance account can give different verdicts about whether C\\textsubscript{i} causes E\\textsubscript{t} in any given situation. This underlines the importance of being clear exactly what we mean when we make causal claims in social science.\n\n**UNDERLYING STRUCTURES**\n\nDifferent causal relations hold in different social, cultural, and economic settings. What is a required politeness in one culture can cause insult in another. Why? What causal relations hold in a social setting depends on the underlying system, the underlying social, economic, and cultural structure. It is now standard in philosophy of biology to call the structures that underlie causal relations there ‘mechanisms’, and sometimes this usage is adopted in the social sciences as well. Though there is no widely used mechanistic characterization of causation, there is widespread sense that, if we are to follow the injunction of the LSE motto, to know the causes of things, we should come to understand the underlying mechanism that make the particular causal principles that hold for a given population possible.\n\nThis is clearly of central importance in social planning and policy. Consider recent problems in child welfare in the UK, where attention tended to focus on causal processes. When a tragic child death occurs, ‘The standard response is to hold an inquiry…trying to get a picture of the causal sequence of events that ended in the child’s death…We are tracing a chain of events back in time to understand how it happened.’ So says Eileen Munro (2005: 377), author of Chapter 3 and of the 2012 UK government report on child protection. Munro recommends instead a ‘systems’ approach. That means focusing on the underlying mechanism or social structure that makes these kinds of processes possible, or even probable. As a US National Academy of Science report on building safer health systems explains, ‘The focus must shift from blaming individuals for past errors to a focus on preventing future errors by designing safety into the system’ (Kohn et al. 2000: 5).\n\nSo it is clear that it is important to study social mechanisms in social science. What is not clear is what kinds of concepts should be used to describe these mechanisms and their operation, nor what tool kit of methods will help us to understand them. This is one of the central tasks right now on which philosophy and social science intersect.\n\n### 2.4 Powers\n\nJohn Stuart Mill argued that women have the same powers of leadership and imagination as men, and we have these powers even should they be seldom displayed. Give women the same education, upbringing, situation, and opportunities as men and they will display these qualities equally with men. And he used this claim to argue for dramatic changes in the social policies and practices in play at the time regarding the role of women in society.\n\nAs with all concepts, if the concept *power* is to play an important role like this in social science or in social policy deliberation, we should be clear what is meant by it. This is a big topic in metaphysics nowadays and, to a lesser extent, in philosophy of science (see e.g. Mumford 2009). It is usual to make at least four assumptions about powers. First, a system—an object, a person, an institution—can have a power without displaying it. Second, the power will be displayed if the conditions are right. Third, what actually happens when a power is displayed depends on the setting. So what happens when a woman displays her imagination will depend on whether she is at a laboratory work bench or addressing Parliament or tending her children. Fourth, when a power operates in a situation, what happens there will depend in some intelligible way on the display of the power (as when a heavy metal object does not fall to the ground because the power of a magnet is displayed).\n\nThe most typical characterizations of powers ‘back’ define them from their displays. Roughly, A has the power to $\\alpha$ just in case when properly enabled and failing something stopping it doing so (which we sometimes express as ‘nothing interferes with the display’), A does $\\alpha$. This characterization is dramatically non-reductive because of the concepts of *enablement* and *interference*. Contrast this with an alternative: ‘A has the power to $\\alpha$ just in case there are some circumstances $C\\alpha$ such that it is a law of nature that in $C\\alpha$, A does $\\alpha'$, where $C\\alpha$ is to be filled in by some possibly very long list of features that can be characterized without any power-related notions, like ‘is schooled at Eton’, ‘learns Latin’, ‘does not play with dolls’, etc. Many powers advocates do not adopt this alternative because, they argue, there is never a list that does the job. In each actual situation there is a fact of the matter about whether the circumstances that obtain there constitute an interference and whether they are enabling, but there is nothing these facts share in common that we could fill in for $C\\alpha$ other than that they are enabling or they interfere. This is taken by many to be particularly problematic since philosophers have not succeeded in having much enlightening to say about interferences and enablers at the general level.\n\nWhy then take powers seriously? Philosophers offer a panoply of metaphysical arguments. But for social science the answer seems to lie in the usefulness of the concept, as with Mill, who argued for the centrality of the related notion of *tendency* in political economy. Another word used for much the same idea is ‘capacity’. In order to link it with our contemporary discourse, I shall use the word ‘power’ throughout for all these related notions. Mill’s model for powers comes from Newtonian mechanics. The sun has the power to attract the planets. Nothing ever interferes with it so the sun constantly displays this power (called ‘gravity’). What happens when it displays it? The planets move around it in elliptical orbits. By contrast, when the earth displays its power to attract a cannonball shot at an enemy ship, the cannonball moves along a parabola. What actually happens when the power of gravitational attraction is displayed (i.e. when the sun or earth attract other objects) depends on the setting. To make a link with our immediately previous discussion, we may think of the earth–cannonball pair as a mechanism, in the sense of an underlying structure, giving rise to the causal principle, ‘The earth displaying its power of gravitational attraction causes cannonballs to move along parabolas’; and the planetary system as a mechanism giving rise to the causal principle ‘The sun displaying its power of gravitational attraction causes the planets to move along ellipses around it’.\n\nHow do we know what happens when a power is displayed in a given setting? That’s easy, at least in principle, in Newtonian mechanics. All that’s relevant to how a power affects motions is what other powers are displayed in the situation. The display of the power is represented by what we call a force, represented by a vector. When several forces are displayed at once, we add these vectors, call the result ‘the total force, $F_{\\text{tot}}$’, and calculate the acceleration of any object in the situation by the familiar formula $F_{\\text{tot}} = ma$, where $m$ is the mass of the object. That is what we do, for instance, when we want to know how a pin will behave when a magnet attracts it upward and the earth attracts it downward, or how an electron in an atom behaves when it is attracted by both the mass of the nucleus and the charge on the protons there.\n\nThat’s all physics. Life is not so easy in social science, which may have some knowledge of powers but little knowledge of methods for calculating just what happens in different situations when the powers are displayed. Consider an economics example already mentioned, the one involving skill loss during unemployment. Earlier I suggested that economics entertains a causal principle that skill loss perpetuates high levels of unemployment. That is probably a mistaken way to look at it. It is seldom the case that skill loss is the only thing going on that affects unemployment. There are, for instance, sometimes vigorous successful government efforts to combat unemployment. In these cases skill loss may be followed by improving levels of employment, contrary to what is suggested by this causal principle. A better way to put it might be ‘Skill loss has the power to cause continuing high unemployment’. This will not lead us to expect that skill loss actually produces unemployment whenever it occurs, but only that, to understand the levels of employment that actually occur, we shall have to take the downward push of skill loss into account.\n\nSo, we return to the question, ‘How do we take it into account?’ How do we predict what happens when a socio-economic power is displayed? There is no systematic answer. This is one of the problems social scientists constantly confront in trying to put their isolated parcels of hard-won knowledge—like the knowledge of specific powers such as the power of skill loss to perpetuate unemployment—to use.\n\n### 3. Putting Causal Knowledge to Use\n\nChapter 14 in this book teaches us that the meaning we assign a scientific concept must be closely paired with our methods for finding out whether it obtains. We should have good arguments to show that the methods we employ provide a good way to find out about just what we claim we are finding out about. That message has been echoed throughout this chapter with respect to the concept of causation when it is used in the social sciences. It is not just meaning and method that must match, however. So too must use. The use to which we put our social scientific claims—the inferences we draw from them and the practices we adopt on the basis of them—should be\n\nsupported by what those claims actually say; and what they say that supports our inference should in turn be supported by our methods for deciding that what they say is true. It is no good making a very broad claim whose first half is well supported by the evidence but then draw our inferences from the second half, which is unsupported.\n\nWe have looked at a number of accounts of what causal claims say. The claims say different things because they employ different concepts of causation. These concepts may all go under the same title, ‘general causal relation’, but they require different conditions to be met in order to obtain. So different methods must be used to find out about them and different uses made of them. So, of what use is knowledge of the causes of things? Knowing causes helps us to understand how things work. But it is also supposed to be of practical value. Knowing the causes of things should help us to change the world.\n\nHere we must be cautious though. There is a tendency to too much haste, to try to read off directly from our causal claims just what we must do to bring about the change we desire. That will work with some kinds of causal knowledge but not most. Consider the manipulation concept of causation found in economics that I mentioned in discussing invariance: $C_t$ causes $E_t$ relative to $\\phi$ just in case manipulating $C_t$ by $\\phi$ is followed by $E'_t$, where $\\phi$ is one of the methods available to us to change $C$ at $t$. Suppose we know that. Then we are in a powerful position to change $E$ at $t'$.\n\nBut suppose we have established instead a different causal claim, say a manipulationist claim of the kind favoured in philosophy—$C_t$ causes $E_t$ just in case if $C_t$ changes by intervention $E_t$ changes—or in invariance-type claim—$C_t$ causes $E_t$ just in case the association between them is invariant when $C_t$ changes by intervention. That too can be of immediate use—if we have an intervention to hand, which we usually do not. We have seen the same kind of problem with knowledge of powers. Knowing about what powers a system has will not by itself tell us what to expect to happen in particular circumstances nor how to build circumstances to get what we want. Nor does knowing the causal relations, on the probabilistic theory, that hold in one population, perhaps the population enrolled in an RCT, give us immediate guidance about how to bring about change in a different population.\n\nThis does not, however, in any way make causal knowledge in social science useless. It just puts it more on a par with similar knowledge in the natural sciences, where strategies are built by splinting together a very great many different pieces of knowledge and often undertaking designated new researches and developing new theory to fill in gaps. The great nineteenth-century physicist Lord Kelvin laid the first Atlantic cable in 1866. His knowledge of causes in physics was essential to his success but it definitely did not tell him just what to do. Similarly, the large team of physicists, engineers, mathematicians, and technicians who developed the US Second World War radar took many months to do so, despite the vast repository of causal knowledge the team members brought to the project. The conclusion I propose is that the causal knowledge we work so hard to gain in the social sciences can be of immense use. But there is no reason to think it should be easier to put our social science knowledge to use than it is to use our knowledge in the natural sciences.\n\nReferences\n\nAngrist, J. (1990). ‘Lifetime Earnings and the Vietnam Era Draft Lottery: Evidence from Social Security Administrative Records’, *American Economic Review*, 80(3): 313–36.\n\nDowe, P. (2009). ‘Causal Process Theories’ in H. Beebee et al. (eds), *Oxford Handbook of Causation*. Oxford: Oxford University Press, 213–33.\n\nGarrett, D. (2009). ‘Hume’, in H. Beebee et al. (eds), *Oxford Handbook of Causation*. Oxford: Oxford University Press, 73–91.\n\nGEF (2007). *Case Study: Bwindi Impenetrable National Park and Mgahinga Gorilla National Park Conservation Project*. Impact Evaluation Information Document, 7. Washington, DC: Global Environment Facility, Evaluation Office.\n\nHoover, K. (2001). *Causality in Macroeconomics*. Cambridge: Cambridge University Press.\n\nKohn, L., Corrigan, J., and Donaldson, M., eds (2000). *To Err is Human: Building a Safer Health System*. Washington, DC: Committee on Quality of Health Care in America, Institute of Medicine.\n\nMumford, S. (2009). ‘Causal Powers and Capacities’, in H. Beebee et al. (eds), *Oxford Handbook of Causation*. Oxford: Oxford University Press, 265–78.\n\nMurro, E. (2005). ‘Improving Practice: Child Protection as a Systems Problem’, *Child and Youth Services Review*, 27: 375–91.\n\nPaul, L. A. (2009). ‘Counterfactual Theories’, in H. Beebee et al. (eds), *Oxford Handbook of Causation*. Oxford: Oxford University Press, 158–84.\n\nReiss, J. (2013). *Philosophy of Economics: A Contemporary Introduction*. London: Routledge.\n\nRoberts, C. (1995). *The Logic of Historical Explanation*. University Park, PA: Pennsylvania State University Press.\n\nRussell, B. (1912–13). ‘On the Notion of Cause’, *Proceedings of the Aristotelian Society*, 13: 1–26.\n\nFurther Readings\n\nBeebee, H., Hitchcock, C., and Menzies, P., eds. (2009). *Oxford Handbook of Causation*. Oxford: Oxford University Press.\n\nCampaner, R., and Galavotti, M. C. (2007). ‘Plurality in Causality’, in P. Machamer and G. Wolters (eds), *Thinking about Causes: From Greek Philosophy to Modern Physics*. Pittsburgh: University of Pittsburgh Press. 178–99.\n\nCartwright, N. (2007). *Hunting Causes and Using Them*. Cambridge: Cambridge University Press.\n\nMcKim, V., and Turner, S., eds (1997). *Causality in Crisis: Statistical Methods and the Search for Causal Knowledge in the Social Sciences*. South Bend, IN: University of Notre Dame Press.\n\nRusso, F. (2009). *Causality and Causal Modelling in the Social Sciences*. New York: Springer.\n\n**Index**\n\naggression 104–5, 107, 114–16, 119 \nagmatology 259 \nAnany, Roger 71–2 \narchaeology 68–77, 79–82\n\nbehavioural traits 115, 118 \nenvironmental factors 102–3, 106–7, 118 \nheritable factors 107–8 \nindividual differences 104, 116–17, 119 \npluralism in the study of 113, 115–17 \npopulation differences 115–17, 119 \npopulation-level factors 109–10, 116 \nsex differences in heritability 115\n\nbehaviour genetics: \nmolecular 105, 108, 112–13 \nquantitative 104–5, 107–8, 112\n\nBoghossian, Paul 71–3 \nboundary objects 238–9 \nBrannan, Michael 93 \nBurt, Cyril 172–3\n\nCampbell, Donald 294–5 \nCartwright, Nancy 61–2 \ncase studies 288–307 \ncombinath laboratory experiments 291–2, 297, 301, 304 \ncritiques of 292–3\n\ncategories: \ncharacterization of 267–70, 272–3, 276, 278–81, 284–5 \nprocedures for measurement 276–8, 280–1, 284–5 \nrepresentation of 271–6, 279–80, 284–5 \ncausal principles 62, 310–11, 322–3 \ncausal processes 63, 305, 319 \ncausal spaces 111–13, 115 \ncausation 102–6, 115, 273, 308–10, 312–13, 319–20, 323–5 \nmanipulation view 316–18 \nprobabilistic theory of 312–14, 324 \nsingular 317, 319 \nchoice models 185–7, 189, 191, 193, 195, 197–9, 201, 203, 205–6 \nEU models 199, 201 \ngame models 191–201, 203–5\n\nindividual decision 186–91 \ncivil war 265–73, 277–8, 284 \nethnicity 270, 282–4, 311–12 \ncivil war studies 265, 275–6 \nClark, Geoff 71, 73, 81 \nclass sizes 55–7, 61–2 \nclimate change 2, 31–46, 133–4, 253 \nadaptation 39–41, 43 \nadaptive capacity 39–41 \ncost of future generations 42, 45, 130–3 \nintegrated assessment models 38, 41 \nmitigation 41–3 \nscenarios 37–41 \nvulnerability to 39–41 \nclimate change impacts 38–9, 43 \ncollectivism 7–9, 20, 95 \ncommunity-based participatory research (CBPR) 68, 79–80 \nconcepts 1, 97, 238, 268–72, 274, 277–8, 283–4, 308–9 \nBallung 268–72, 273–8, 284 \nconventional networks 96–7, 100 \nconstraint satisfaction network 97 \nconsensus conferences 251–3, 261 \nconventions 215–18, 221–6, 229 \ncrime 103–4, 108–10, 119 \nCrombie, Alistair 288–9\n\nDasgupta, Partha 24 \nDaston, Lorraine 127, 155 \nDavies, Huw 52, 64 \ndecision theory 185, 201, 203 \ndegrees of freedom 295 \nDeleuze, Gilles 139–40 \nDeVault, Marjorie 192–3 \ndevelopmental systems theory (DST) 106, 112–14 \ndisability-adjusted life year (DALY) 276, 280–1 \ndiscipline 234–5, 237–40, 244 \ndiscount rate 42, 130–1, 133 \ndiscovery 299–300, 302, 306 \ndoctrine 234–5, 242, 244 \nDouglas, Heather 70 \nDouglas, Jack 137–8 \nDupré, John 179 \nDurkheim, Émile 136–8"}}},{"rowIdx":204695,"cells":{"text":{"kind":"string","value":"NEUROENDOCRINE REGULATION OF GASTRIC ENDOCRINE CELL FUNCTION\n\nThesis submitted in accordance with the requirements of The University of Liverpool for the degree of Doctor of Philosophy by Andrew Damian Higham B.Sc., M.B. Ch.B., M.R.C.P.\n\nMarch 1998\n\nLIVERPOOL UNIVERSITY LIBRARY\n\nLATIVX\n\nThe last decade has seen major advances in the understanding of diseases of the proximal gastrointestinal tract. Not least of these has been the recognition of the significance of *Helicobacter pylori* (*H. pylori*) gastritis in the pathogenesis of peptic ulceration, chronic atrophic gastritis and gastric carcinoma. In addition the development of proton pump inhibitors (PPI) has been heralded as a major therapeutic advance. However both *H. pylori* gastritis and PPI therapy elevate plasma gastrin concentrations and this has led to increasing interest in the physiological role of the antral hormone gastrin.\n\nIn this thesis, the role of intrinsic antral neurons in the regulation of gastrin release in response to a variety of stimuli has been examined. In addition, the effects of antral denervation on gastric emptying were clearly apparent and prompted investigation of the relationship between antral neurons and the cholecystokinin-independent mechanisms that have been well described as regulators of gastric emptying in the intact rat. It was found that antral neurons are essential for normal gastric emptying but are not required for the CCK-mediated inhibition of gastric emptying.\n\nIn addition, following antral denervation there was elevated fasting and meal stimulated plasma gastrin, the post-prandial rise in gastrin release occurring by a non-gastrin releasing peptide mediated mechanism. Moreover, non-nutrient\n\ndistension of the stomach was an adequate stimulus for gastrin release following antral denervation, suggesting that in the intact rat, antral neurons normally inhibit gastrin release to non-nutrient distension.\n\nThe effects of antral denervation on the endocrine cells of the stomach were also studied as were the effects of hypergastrinaemia on the Enterochromaffin-like (ECL) cells of the gastric corpus. Of particular relevance was the demonstration by Ashahara et al., (1996) of the *reg* gene in the ECL cells of the rat. Expression of this gene has been associated with pancreatic endocrine cell growth and differentiation. Data is presented that indicates that expression of this gene is regulated by gastrin and that it may play a role in promoting the differentiation of endocrine cells of the proximal gastrointestinal tract. Mutations of *reg* were associated with the development of gastric carcinoid (ECL cell) tumours in patients with hypergastrinaemia.\n\nOverall it is clear that the antral innervation plays a central role in the control of gastrin release and that loss of the normal innervation of the G-cell leads to fasting and meal-stimulated hypergastrinaemia. The effects of persistently elevated gastrin on other cell populations of the stomach are likely to be of increasing importance with respect to understanding the mechanisms of tumour development.\n\nThe work presented in this thesis has appeared in part in the following publications:\n\n**Papers**\n\nHigham A.D., Vaillant C., Yegen B., Thompson D.G., Dockray G.J.: Relationship between CCK and antral innervation in the control of gastric emptying in the rat. *Gut* 41:24-32 1997\n\nHigham A.D., Noble P., Thompson D.G., Dockray G.J.: Increased sensitivity of gastrin cells to gastric distension following antral denervation in the rat. *J Physiol* 503(1):169-175 1997\n\nHigham A.D., Dimaline R., Varro A., Attwood S, Armstrong G, Dockray G.J., Thompson D.G.: Atrophic gastritis and large carcinoid nodules: Octreotide suppression test predicts beneficial outcome from antrectomy. *Gastroenterology (In press)*.\n\n**Abstracts**\n\nHigham, A.D. Thompson, D.G., Dockray, G.J.: Antral denervation by benzalkonium chloride leads to gastric retention of solids in the rat. *Gut* 36(1):A52 1995.\n\nHigham, A.D. Thompson, D.G., Dockray, G.J.: Peptidergic afferent and intrinsic innervation of the rat stomach following antral denervation. *Neurogastroenterol Mot* 7:9 1995.\n\nHigham, A.D. Valliant, C. Thompson, D.G., Dockray, G.J.: Complete loss of peptidergic afferent fibre innervation of the rat stomach following antral denervation. *Reg Peptides* 57:199 1995.\n\nHigham, A.D., Thompson, D.G., Dockray, G.J.: Post-prandial gastrin release after antral denervation: no role for gastrin-releasing peptide. *J Physiol* 487:34P 1996.\n\nHigham A.D., Noble P.J., Thompson D.G., Dockray G.J. Increased gastrin release following distension after antral denervation. *J Physiol* 499: 104P 1997.\n\nHigham A., Thompson D.G., Dimaline R. & Dockray G.J.: Increased *Reg* gene expression in the rat gastric corpus following omeprazole treatment. *Reg Peptides*. 71:55 1997.\n\n**Chapters and Invited Reviews:**\n\nHigham A.D.: Clinical endocrinology of gastric function. *Surgery* (in press) 1997.\n\nStatement of Originality\n\nAll of the data in this thesis that is presented as results has arisen wholly from my own work apart from the following contributions from colleagues within the Department of Physiology, University of Liverpool. The tissue culture of AR42J and HIT cells was performed by Dr Lisa Bishop as was the permanent transfection of the HIT cells. The plasma CCK assay was performed by Mrs C McLean. On occasion, the preparative surgery of the gastric fistula rats was performed by myself in conjunction with P J Noble.\n\n| Abbreviation | Description |\n|--------------|-------------|\n| BAC | Benzalkonium chloride |\n| CAG | Chronic atrophic gastritis |\n| CCK | Cholecystokinin |\n| CGRP | Cacitonin gene-related peptide |\n| CRF | Chronic renal failure |\n| EC | Enterochromaffin |\n| ECL | Enterochromaffin-like |\n| EGF | Epidermal growth factor |\n| GRP | Gastrin-releasing peptide |\n| HGF | Hepatocyte growth factor |\n| LOH | Loss of heterozygosity |\n| MEN1 | Type I multiple endocrine neoplasia syndrome |\n| NO | Nitric oxide |\n| PA | Pernicious anemia |\n| PAP | Pancreatitis associated protein |\n| PPI | Proton pump inhibitor |\n| PSP | Pancreatic stone protein |\n| PTP | Pancreatic thread protein |\n| SP | Substance P |\n| TGF-α | Transforming growth factor alpha |\n| VIP | Vasoactive intestinal polypeptide |\n| ZE | Zollinger Ellison syndrome |\n\n# Contents\n\n## Chapter 1\n\n### Introduction\n\n- Historical review of gastrointestinal peptide physiology: 2\n- Innervation of the stomach: 5\n- Neuroendocrine regulation of gastric emptying: 15\n- Neuroendocrine regulation of gastric acid secretion: 16\n- Clinical significance of hypergastrinaemia: 22\n- Gastrin, endocrine cell growth and differentiation in the stomach and pancreas: 26\n- Aims and objectives: 34\n\n## Chapter 2\n\n### Materials and methods\n\n- Materials: 37\n- Methods: 39\n\n## Chapter 3\n\n### Chemical denervation of antral neurons\n\n- Introduction: 57\n- Methods: 59\n- Results: 60\n- Discussion: 69\n- Summary: 72\n\nChapter 4\n\nAntral denervation and gastric emptying\n\nIntroduction ................................................................. 75\nMethods .................................................................. 77\nResults .................................................................... 78\nDiscussion ................................................................. 85\nSummary .................................................................. 88\n\nChapter 5\n\nAntral denervation and G-cell function\n\nIntroduction ................................................................. 91\nMethods .................................................................. 93\nResults .................................................................... 94\nDiscussion ................................................................. 105\nSummary .................................................................. 110\n\nChapter 6\n\nControl of reg expression in rat corpus mucosa and AR42J cells\n\nIntroduction ................................................................. 112\nMethods .................................................................. 113\nResults .................................................................... 114\nDiscussion ................................................................. 124\n\nChapter 7\n\nReg gene expression in human gastric mucosa\n\nIntroduction 131\nMethods 133\nResults 136\nDiscussion 145\nSummary 152\n\nChapter 8\n\nConclusion and Implications\n\nConclusions 155\nImplications and future studies 156\n\nChapter 9\n\nBibliography 162\n\nAcknowledgements 188\n\nChapter 1\n\nIntroduction\n\nAt the beginning of this century two schools of thought were prominent with regard to the mechanisms that controlled visceral function. Langley provided evidence that responses to feeding were mediated by nervous reflexes (Langley, 1898) and his conclusions were supported by the work of Sherrington (1899) and Pavlov (1910). Set against this were the experiments of Bayliss and Starling (1902) and Edkins (1905) who proposed that blood borne agents (named secretin and gastrin respectively) were responsible for stimulating digestive secretions from the pancreas and stomach. Although Bayliss and Starling (1899) previously had reported the reciprocal effects of the vagal and splanchnic innervation on gastric motility, it took almost forty years before it was considered that the nervous and endocrine systems might interact closely to regulate visceral functions such as gastric acid secretion (Uvnas 1942).\n\nWhilst the work of Bayliss and Starling was readily accepted, Edkins’ proposal that an antral hormone stimulated gastric acid secretion remained unconfirmed until the work of Komarov in the late 1930’s and of Grossman et al., a decade later (1948). Meanwhile several groups attempted to purify to homogeneity secretin, the hormone proposed by Bayliss and Starling to stimulate pancreatic secretion in response to a meal. This was achieved in 1961 by Jorpes and Mutt who isolated secretin and at the same time Gregory and Tracy isolated and characterised the peptide hormone gastrin (Gregory and Tracy, 1964). Within a few years it was shown conclusively that gastrin was produced by and localised within a defined\n\npopulation of endocrine cells in the gastric antral mucosa (McGuigan, 1968). Not surprisingly at the time, gastrin was considered to be strictly a gastrointestinal hormone. Furthermore, the similarities between gastrin and secretin seemed to confirm the prevailing view that peptides acted as classical hormones.\n\nAlmost coincidentally however, Burnstock et al., (1963) described neurotransmission in the gut that was mediated by a nonadrenergic and noncholinergic agent. Morphological studies of the myenteric plexus suggested the existence of peptide-containing nerve fibres (Baumgarten et al., 1970; Cook and Burnstock 1976). Subsequently, considerable evidence has been provided that has generated widespread support for the idea that peptides act as neurotransmitters in neurons of the enteric, sympathetic, parasympathetic and central nervous systems.\n\nAlthough von Euler and Gaddum as early as 1931, had suggested that agents with identical biological activity were present in extracts of both gut and brain, the work of Vanderhaeghen et al., (1975) raised again the idea that a peptide previously considered to be a classic gut hormone (gastrin) might be present within neurons of the central nervous system. The technical and analytical advances in the last two decades have now established fundamental new concepts in the distribution of regulatory peptides. It is now clear that any one peptide may have a specific function at a specific site, determined by the cell type responsible for its synthesis, mode of release and subsequent tissue distribution, but the same peptide may occur at another independent site within the body and have a separate function and mode of action determined again by the local environment. Hence peptides such as\n\ncholecystokinin (CCK) and gastrin, previously considered as hormones may act elsewhere as neurotransmitters or paracrine mediators, the converse also being true.\n\nWith the general acceptance of similarity between the spectrum of peptides that function as hormones and as neurotransmitters, it is perhaps less surprising that there is such close interaction between neurons and endocrine cells in the regulation of visceral functions such as gastric acid secretion. The main objective of the studies described in this thesis was to determine the role of antral neurons in the control of antral G-cell responses to physiological stimuli. The experimental model of antral denervation that was developed to investigate the role of intrinsic neurons in the regulation of the G-cell produced chronic hypergastrinaemia and delayed gastric emptying. This prompted further studies on the role of antral neurons in the regulation of gastric emptying and the effects of sustained elevations in plasma gastrin on endocrine cell gene expression with particular relevance to enterochromaffin-like (ECL) cell growth and carcinoid tumour development.\n\nThe following account will describe the normal gastric innervation and its role in regulating G cell function and gastric emptying, review the physiology of gastrin and the pathophysiology of hypergastrinaemia in particular the development of ECL cell tumours, review the evidence for the role of gastrin in endocrine cell growth and differentiation and finally introduce putative regulators of cell growth and differentiation in gastroentero-pancreatic endocrine cells.\n\nLangley (1921) first recognised the importance of the intrinsic nervous system of the gut, considering it a separate division of the autonomic nervous system in addition to the sympathetic and parasympathetic divisions. Considerable evidence in support of this has been presented (Cooke, 1987; Furness and Costa, 1987; Wood, 1987) and it is clear that processes such as absorption, secretion and coordinated motor activity may be regulated wholly by the enteric nervous system. The extrinsic innervation of the gut interacts with enteric neurons exerting an overriding influence when necessary so that gut function may be integrated with the prevailing needs of the body as a whole. Extrinsic neuronal inputs are supplied by vagal and spinal nerve trunks each providing both afferent and efferent nerve fibres.\n\ni) Vagal innervation of the stomach\n\nThe majority of nerve fibres in the vagi are afferent fibres having their nerve cell bodies in the nodose ganglia. These afferent fibres have their peripheral endings within the muscle and mucosal layers of the gut wall and act as mechano-, thermo- and chemo-receptors conveying signals to the central nervous system (Andrews, 1986; Blackshaw and Grundy, 1990; El Ouazziani and Mei, 1979; 1891). Almost all vagal afferent fibres are unmyelinated (Andrews, 1986) but a small population of nerve fibres within the vagus are small diameter myelinated fibres believed to be of sympathetic origin (Lundberg et al., 1976).\n\nUsing retrograde tracing and immunohistochemistry it has been shown that vagal afferent fibres projecting from the anterior wall of the stomach have their cell bodies in the left nodose ganglion and those that project from the posterior gastric wall have their cell bodies in the right nodose ganglion (Dockray et al., 1988; Green 1988). A number of peptides have been identified in vagal afferent fibres including substance P (SP) (Katz and Karten, 1980; Lundberg et al., 1978; 1983; 1985) CCK (Mantyh and Hurt, 1984), vasoactive intestinal polypeptide (VIP) (Lundberg, 1978; Said and Rosenberg, 1976) and gastrin (Dockray et al., 1981). Despite this variety, the majority of peptide-containing vagal nerve fibres project to the thoracic viscera (Lundberg, 1983; Mulderry 1985; Cadieux 1986) such that less than 10% of gastric vagal afferent fibres are peptidergic (Green, 1988).\n\nThe vagus also carries efferent fibres to the stomach but these account for only 10% of the total number of vagal fibres. The majority of vagal efferent fibres are excitatory cholinergic fibres that are involved with the normal regulation of gastric tone as well as secretion of gastrin and gastric acid. In addition however, there are also inhibitory pathways such as those causing release of nitric oxide (NO) (Desai et al., 1991) and VIP (Grider et al., 1985).\n\nii) Spinal afferent innervation of the stomach\n\nSimilar to the vagal innervation of the stomach, spinal afferent neurons for the most part are unmyelinated (Kuo et al., 1982). Gastric spinal afferents have their nerve cell bodies in the thoracolumbar dorsal root ganglia (Green and Dockray, 1988). Conversely however, there is no lateralisation of innervation such that\n\nretrograde tracing from one surface of the stomach labels equal numbers of nerve cell bodies in the right and left dorsal root ganglia. The spinal efferent innervation of the stomach is approximately 95% adrenergic and projects from cell bodies contained within the coeliac ganglion (Macrae et al., 1986).\n\nSeveral different peptides have been identified within spinal afferent neurons but the most important are calcitonin gene-related peptide (CGRP) (Gibson et al., 1984; Lee et al., 1987; Rosenfeld et al., 1983) and substance P (Hokfelt et al., 1976; Price 1985; Seybould and Elde, 1980). CGRP is of primary interest because of its specific distribution within the stomach and is relevant to the work contained within this thesis. A brief review of the physiology of CGRP is given below before describing the intrinsic innervation of the stomach.\n\n**Discovery and distribution of CGRP:**\n\nThe existence of CGRP was predicted on the basis of the discovery of a novel cDNA sequence corresponding to an mRNA transcript that was shown to arise from alternative splicing of the gene encoding calcitonin (Amara et al., 1982; Rosenfeld et al., 1983). Subsequently a related peptide β-CGRP was isolated (Amara et al., 1985; Steenbergh et al., 1985) whose primary gene does not encode for calcitonin. The distribution of CGRP within the stomach has important implications. CGRP-immunoreactive fibres may be found in all layers of the stomach (Clague et al., 1985; Green and Dockray 1988; Sternini et al., 1987) but there are no intrinsic neurons or endocrine cells that contain CGRP in the rat stomach. In fact in rodent stomach almost all CGRP is contained within spinal\n\nafferent extrinsic nerve fibres (Green and Dockray, 1988; Sternini et al., 1987; Varro et al., 1988). This makes CGRP a useful marker for an intact spinal afferent gastric innervation.\n\n**Biological actions of CGRP in the stomach:**\n\nIt is well established that CGRP acts as a peripheral neurotransmitter from spinal afferent nerve terminals (Holzer, 1992). Much of the available experimental evidence points to a role for CGRP in gastric mucosal protection. Hence, installation of noxious chemicals into the gastric lumen results in increased mucosal damage if the spinal afferent innervation has been lesioned (Holzer and Sametz, 1986) or the action of CGRP is blocked by immunoneutralisation (Forster and Dockray, 1991). Conversely, stimulation of CGRP release protects against mucosal damage (Holzer and Lippe, 1988; Holzer et al., 1989; 1990) induced by installation of noxious chemicals.\n\nThe gastroprotective effect of CGRP acts partly by increasing gastric mucosal blood flow possibly by the release of NO (Lambrecht et al., 1993). In addition however, CGRP has been shown to be a potent inhibitor of gastric acid secretion (Tache et al., 1984; Pappas et al., 1986; Helton et al., 1989), a factor that also contributes to the gastroprotective effects of this peptide. The inhibition of acid secretion is mediated in part by release of somatostatin from D cells of the gastric corpus (Dunning and Taborsky, 1987; Inui et al., 1991). In addition it has been shown that CGRP regulates somatostatin synthesis by corpus D cells at the level of gene transcription (Sandvik et al., 1993).\n\niii) Intrinsic innervation of the stomach\n\nThe intrinsic nerve supply of the gastrointestinal tract, collectively termed the enteric nervous system, comprises two main plexi of nerves described first by Meissner (1857) and Auerbach (1864). Meissner described a submucous plexus and Auerbach a plexus of nerves lying between the circular and longitudinal muscle layers generally referred to as the myenteric plexus. The structure and distribution of the enteric nervous system were described in detail by Gaskell (1886) and the functional significance of the enteric nervous system as a separate branch of the autonomic nervous system was proposed later by Langley (1921).\n\nStudies of the anatomy of intrinsic gut neurons were facilitated greatly by the techniques of myotomy and myomectomy coupled with immunohistochemistry first described by Furness and Costa (1979). The variety and colocalisation of neuropeptides present in enteric neurons, their projections and putative actions have received much interest over the past two decades (for review see Dockray 1994; Furness and Costa 1987). Generally speaking, the tachykinin/substance P family of peptides are present in intrinsic excitatory neurons (Katayama and North, 1978; Bartho et al., 1982; Bartho and Holzer, 1985; Holzer, 1989) and the VIP/peptide histidine isoleucine family is present in intrinsic inhibitory neurons (Brookes et al., 1991; Ekblad et al., 1987; Shultzberg et al., 1989; Grider, 1989). Gastrin-releasing peptide (GRP) is present in secretor motor neurons and this is of particular relevance as GRP-containing neurons stimulate the release of gastrin from antral G\n\ncells (Bunnett et al., 1985; Kovacs et al., 1995; Varner et al., 1991). In the lesioning experiments described later in this thesis, substance P, VIP, GRP and CGRP were assayed as markers of different classes of intrinsic or extrinsic neurons to enable confirmation of denervation. There follows a brief review of substance P, VIP and GRP in relation to the enteric nervous system.\n\n**Substance P**\n\nSubstance P (SP) was the first gut neuropeptide to be discovered. Although extracts of horse intestine had been shown to stimulate atropine resistant contraction of rabbit ileum as early as 1931 (von Euler and Gaddum, 1931), SP was isolated and sequenced first from the bovine hypothalamus (Chang and Leeman, 1970; Chang and Leeman, 1971). The identical peptide was shown later to be present in small intestine (Carraway and Leeman, 1979). Evidence for its neuronal localisation was suggested by Ehrenpreis and Pernow (1953) who reported reduced levels of SP bioactivity in the aganglionic segment of colon affected by Hirschsprung’s disease. Lembeck (1953) suggested that SP was located within the primary afferent neurons on the basis of findings of higher concentrations of peptide in dorsal compared with ventral spinal roots. Although there is compelling evidence that a subset of primary afferent neurons contain both SP and CGRP (Gibbins et al., 1985; Green and Dockray, 1988), lesioning experiments using capsaicin, have shown that in the rat stomach and small intestine the majority (>90%) of SP is present in intrinsic neurons (Holzer et al., 1980).\n\nThe presence of SP within primary afferent neurons has prompted studies that suggest that SP released from these neurons may act as a central neurotransmitter of pain and may play a role as a peripheral transmitter during inflammatory reactions in the gut (Dockray, 1994; Holzer, 1988; Sharkey, 1992). The predominant localisation of SP within intrinsic gut neurons has directed several groups towards identifying its possible role within the gut. There is evidence that SP directly stimulates intestinal smooth muscle contraction (Taylor and Bywater, 1986) and that SP release is involved in the contraction that occurs on the oral side of a distending bolus as part of the peristaltic reflex (Bartho et al., 1982; Holzer, 1989; Yokayama and North, 1983). In the rat colon, SP release appears to mediate only the noncholinergic contraction that occurs with high pressure distension as this could be inhibited by immunoneutralisation (Grider, 1989b).\n\nIn addition to the direct effects of SP on smooth muscle, there is also evidence that release of SP may stimulate other myenteric plexus neurons (Yan and Youther, 1982; Fosberg et al., 1984). Regardless of the precise physiological role of SP in the control of motility, intrinsic SP neurons generally can be considered to be excitatory and SP is the most useful marker of this class of neuron.\n\n**Vasoactive intestinal polypeptide**\n\nVasoactive intestinal polypeptide (VIP) was discovered by Said and Mutt by screening of extracts of gut and lung for vasodilator activity (Said and Mutt, 1970; 1972). Vasodilatation induced by VIP is only one manifestation of its ability to relax smooth muscle. In the gut VIP is localised to a population of myenteric neurons that\n\nproject distally and innervate the circular muscle (Brookes et al., 1991; Ekblad et al., 1987), and to a population of submucosal neurons that project to the mucosa (Schultzberg et al., 1980; Costa and Furness, 1983). There is evidence from immunoneutralisation studies that VIP may mediate the descending relaxation of the peristaltic reflex (Grider and Makhlouf, 1986; Grider et al., 1985; Grider, 1989a). In addition, VIP may be one of the final mediators of the vagovagal, non-adrenergic, non-cholinergic relaxation of the stomach (Faherenkug et al., 1978; Ito et al., 1988), and play a role in the control of gastric emptying (Forster et al., 1991; D’Amato et al., 1992).\n\nSubmucosal VIP-containing neurons that project to the mucosa stimulate secretion from enterocytes (Cooke, 1992). In terms of gastric endocrine cell function, VIP has been shown to inhibit gastric acid secretion by stimulating the release of somatostatin from mucosal D cells (Saffouri et al., 1984). From the above description therefore, in contrast to SP, VIP can be considered as a marker of inhibitory myenteric plexus neurons.\n\n**Gastrin-releasing peptide**\n\nIn 1971, Espramer and Anastasi isolated bombesin from amphibian skin (Anastasi et al., 1971). A major effect of this peptide was stimulation of acid secretion that was shown in the dog to be mediated by gastrin (Bertaccini et al., 1974). The mammalian counterpart of bombesin was identified using C-terminally directed antibodies to the amphibian peptide (Walsh et al., 1979). Coincidentally, McDonald and co-workers discovered that extracts of the non-antral porcine\n\nstomach were a potent stimulus to gastrin release (McDonald et al., 1978; McDonald et al., 1979). They proposed that the peptide responsible was gastrin-releasing peptide (GRP) and subsequently the C-terminal sequence of GRP and bombesin have been shown to be almost identical. However, GRP is not strictly the mammalian equivalent of amphibian bombesin as amphibia have separate genes encoding GRP and bombesin (Nagalla et al., 1992).\n\nIn the mammalian gut, GRP-immunoreactivity is found exclusively within neurons (Dockray et al., 1979). The majority of GRP immunoreactive fibres are present in the mucosa with some fibres present in the circular muscle and myenteric plexus. (Dockray et al., 1979). Most GRP-containing neurons have their cell bodies in the myenteric plexus and project their axons distally (Costa et al., 1984; Ekblad et al., 1984). There is good evidence that the major physiological role of this neuropeptide in the stomach is to stimulate gastric acid secretion via release of gastrin from antral G cells (Bunnett et al., 1985; Hildebrand et al., 1991; Hirschowitz and Molina, 1983; Kovacs et al., 1995).\n\nA schematic representation of the major classes of neuron present within the gastric antrum is shown in Figure 1.1.\n\nFigure 1.1: Schematic representation of the major classes of intrinsic and extrinsic neurons that innervate the gastric antrum in the rat and are susceptible to lesioning by serosal application of the neurotoxin BAC. The shaded area depicts the likely depth of penetration of BAC. LM - longitudinal muscle; CM - circular muscle; SM - submucosa. Excitatory (SP) and inhibitory (VIP) myenteric neurons are shown and receive inputs from spinal afferent (CGRP) and vagal nerve fibres. GRP is present predominantly in secretomotor nerves that supply the mucosa (see text for full details).\n\nIn recent years a substantial body of evidence has emerged detailing the interactions between the intestinal hormone cholecystokinin (CCK) and vago-vagal reflexes that together mediate the delayed gastric emptying in response to fat- and protein-rich meals (Dockray, 1988; Mayer, 1994). Exogenous CCK inhibits gastric emptying in both rat (Conorver et al., 1988; Green et al., 1988) and man (Chey et al., 1969; Liddle et al., 1986). Protein-rich meals are known to stimulate secretion of endogenous CCK (Liddle et al., 1984; 1986) and delay gastric emptying (Green et al., 1988; Moran and McHugh, 1981), an effect that is reversed by specific CCK-A receptor antagonists (Green et al., 1988; Liddle et al., 1989). In addition there is good evidence that vagal afferent fibres express CCK-A receptors (Corp et al., 1993; Moriarty et al., 1997) and that the CCK-mediated delay in gastric emptying is initiated by stimulation of vagal afferent fibres (Raybould and Tache, 1988; Forster et al., 1990; Forster et al., 1991; Holzer et al., 1994). Furthermore, the efferent limb of the reflex that results in relaxation of the gastric body also requires an intact vagus nerve (Raybould and Tache, 1988; Raybould et al., 1987). Vagal efferent relaxation of the gastric corpus is mediated by non-adrenergic and non-cholinergic neurotransmission that probably involves both NO (Desai et al., 1991; Forster et al., 1991; D’Amato et al., 1992) and VIP (Forster et al., 1991; D’Amato et al., 1992).\n\nIn addition to the CCK-vagal pathway that regulates gastric relaxation in response to feeding, gastric emptying may also be affected by spinal afferent innervation, since lesioning of spinal afferent neurons by capsaicin pre-treatment\n\nreduces the emptying rate of liquid test meals (Forster et al, 1990; Forster and Dockray, 1992).\n\nOverall the rate of gastric emptying depends on the pressure gradient between the duodenum and stomach and the resistance to flow across the pylorus and antrum (Mayer, 1994). The content of the ingested meal determines the rate of emptying and CCK-mediated vagovagal reflexes play an important role in delaying gastric emptying by relaxation of the gastric corpus. The importance of antropyloric motility in regulating gastric emptying has received less attention although the pulsatile manner of delivery of gastric content to the duodenum can be attributed to the phasic motor pattern of the antrum (Houghton et al., 1988; Malbert et al., 1991; Prather et al., 1993). There is some evidence that CCK may act on the distal stomach both by a direct action on smooth muscle and indirectly via release of neurotransmitters (Scheurer et al., 1983; Yamagishi and Debas, 1978). However the relationships between CCK and antral neurons in the regulation of gastric emptying has not been examined previously in any detail and represents one of the objectives of the studies presented in this thesis.\n\n**Neuroendocrine Regulation of Gastric Acid Secretion.**\n\nThe G cells, D cells and ECL cells of the stomach together with the extrinsic and intrinsic innervation of the stomach, provide an intricate regulatory mechanism for the control of gastric pH (Fig 1.2).\n\nFigure 1.2: Following meal ingestion, intraluminal pH rises and somatostatin (SOM) release from the D cell is inhibited. The G cell secretes gastrin that leads to histamine release from the ECL cell and H⁺ release from the parietal cell. Vagal neurons also stimulate the parietal cell (1.2a). As luminal pH then falls, antral D cells release SOM that inhibits further gastrin release. Spinal afferent neurons regulate SOM release from corpus D cells. SOM inhibits the ECL cell and parietal cell (1.2b). See text for full description.\n\nG cells\n\nThese cells are present in the antrum of the stomach and are also found in lesser numbers in the proximal duodenum (McGuigan, 1968; Solcia et al., 1975). Their primary function is to synthesise, store and release the amidated 17 amino acid peptide gastrin. G cells process the prohormone precursor peptide progastrin by sequential steps involving sulfation, phosphorylation, cleavage, C-terminal amidation or glycine extension and further cleavage (for review see Dockray et al., 1996). The acid secretory activity of gastrin is dependent on the presence of the amidated carboxyl terminus (Soll et al., 1984; Takeuchi et al., 1980). Alternative forms of gastrin, extended at their C-terminus by glycine also have been reported to possess biological activity other than stimulation of acid secretion, namely stimulation of expression of the gene encoding the H⁺-K⁺-ATPase present in parietal cells (Kaise et al., 1995). Furthermore, recent studies suggest that amidated gastrin, glycine extended gastrin and progastrin itself are all putative growth factors (Seva et al., 1994; Singh et al., 1996; Wang et al., 1996).\n\nG cells have a luminal brush border and secrete gastrin in response to the presence of protein and amino-acids within the gastric lumen (Dockray and Gregory, 1989; Walsh, 1994). In keeping with these observations are the reduction in gastrin mRNA reported with fasting and increases in gastrin mRNA abundance seen with feeding (Wu et al., 1991). In addition G cells respond to changes in luminal pH although this may be indirect, being mediated by changes in somatostatin release from adjacent antral D cells (Karnik et al., 1989). G cells\n\nexpress several different receptors on their basolateral membrane including receptors for gastrin releasing peptide (Vigna et al., 1990; Weigart et al., 1992).\n\n**ECL cells**\n\nThese cells are present only in the gastric corpus and their main function is to synthesise histamine by the decarboxylation of the amino acid histidine (Rubin and Schwartz, 1979). This reaction is catalysed within the cell cytosol by the enzyme histidine decarboxylase (Kubota et al., 1984). Histamine is then transported into secretory vesicles probably by the vesicular monoamine transporter VMAT$_2$ (Merickel and Edwards., 1995). Unequivocal evidence for the stimulation of acid secretion by histamine was provided by the discovery of H$_2$ receptor antagonists and their ability to block acid secretion (Black et al., 1972). Gastrin stimulates the ECL cell to release histamine (Chuang et al., 1992) and if the gastrin stimulus is sustained, ECL cells increase their production and storage capacity of histamine, in part by upregulating HDC and VMAT$_2$ gene expression (Dimaline and Struthers, 1996; Swarovsky et al., 1994). The secretion of histamine by the ECL cell can be inhibited by release of somatostatin from corpus D cells (Chuang et al., 1993). Finally ECL cells increase in number in response to continued gastrin stimulation (Hava, 1986; Poynter and Selway, 1991; Wangberg et al., 1995).\n\n**D cells**\n\nD cells are present in both the antrum and corpus of the stomach. In both sites, their function is to synthesise, store and release somatostatin (Dockray et al., 1996). Antral D cells have a luminal brush border and respond to changes in pH in\n\nthe stomach (Gustavsson and Lundquist, 1978; Holst et al., 1987; Schubert et al., 1988). In contrast, corpus D cells do not have a luminal brush border and somatostatin mRNA abundance in corpus D cells is much less affected by changes in luminal pH that occur with feeding or treatment with omeprazole (Sandvik et al., 1993). It appears that corpus D cell somatostatin gene transcription is regulated primarily by neurohumoral influences of which the most significant may be those of the CGRP-containing spinal afferent neurons (Sandvik et al., 1993).\n\n**Regulation of acid secretion**\n\nThe parietal cells are responsible for the release of acid ($\\text{H}^+$) into the gastric lumen. They can be stimulated to release $\\text{H}^+$ by cholinergic neurons and by histamine (Black et al., 1972; Black and Shankley, 1987; Kabekar et al., 1969; Main and Pearce, 1982; Soll, 1978). Gastrin receptors have been demonstrated on parietal cells (Soll et al., 1984) suggesting that gastrin also may act directly on these cells to induce acid secretion. As intragastric pH rises (such as with ingestion of a meal) antral G cells release gastrin in response to a variety of subsequent stimuli. These include rising luminal pH (Dockray et al., 1991), increased cholinergic stimulation (Kabekar et al., 1969; Main and Pearce, 1982), release of GRP from non-cholinergic neurons (Saffouri et al., 1984; Schubert et al., 1992), the presence of intraluminal protein and amino-acids (Chiba et al., 1980; Saffouri et al., 1980; Walsh, 1994), and a reduction in somatostatin release from adjacent antral D cells that are themselves inhibited by the rising luminal pH (Gustavsson and Lundquist, 1978; Holst et al., 1987; Schubert et al., 1988).\n\nGastrin passes via the circulation to the ECL cell and stimulates these cells to release histamine (Chuang et al., 1992). ECL cells may also respond to the increased cholinergic drive that accompanies meal ingestion (Maine and Pearce, 1982; Soll, 1982). Histamine released from ECL cells acts via parietal cell H\\textsubscript{2} receptors causing H\\textsuperscript{+} release (Black et al., 1972, Black and Shankley, 1987). This co-ordinated response provides the acid pH required for gastric digestion by pH dependent enzymes and as digestion and gastric emptying proceeds, returns gastric pH to its normal basal level (Fig 1.2a).\n\nAs the gastric pH falls, antral D cells respond by releasing somatostatin that in turn inhibits gastrin release from G cells (Gustavsson and Lundquist, 1978; Holst et al., 1987; Schubert et al., 1988). As a result of reduced intraluminal pH, gastrin levels fall, permitting somatostatin release from corpus D cells which in turn prevents further secretion of histamine from the ECL cell and H\\textsuperscript{+} from the parietal cell (Fig. 2b). The spinal afferent innervation of the corpus may also stimulate corpus D cell somatostatin release as it has been shown to functionally regulate corpus rather than antral D cell somatostatin gene expression (Sandvik et al., 1993).\n\nIt may be readily appreciated from the above description that disorders of gastric acid secretion are likely to be associated with elevated plasma gastrin levels. Hence, primary disorders of the G cell or ectopic gastrin secretion that result in hypergastrinaemia will result in increased acid output. Conversely, decreased acid output from whatever cause will result in elevated plasma gastrin as antral G cells\n\nrespond to the high intragastric pH. There follows a discussion of conditions associated with hypergastrinaemia and the related pathophysiology of the ECL cell.\n\n**Clinical significance of chronic hypergastrinaemia**\n\nFrom a clinical viewpoint the major conditions of the stomach that are associated with altered endocrine cell function are encompassed by the conditions associated with hypergastrinaemia (Figure 1.3). The commonest of these are the conditions associated with *H pylori* infection with or without concurrent treatment with a proton pump inhibitor (PPI). However, Zollinger Ellison syndrome (ZE) and pernicious anaemia are the conditions associated with the highest and most sustained elevations in plasma gastrin. In these conditions ECL cell (carcinoid) tumours have been described (see below) and it appears that it is the trophic action of gastrin on the ECL cell that is of pathophysiological importance. The elevations of plasma gastrin in *H pylori* or with PPI therapy are of lesser magnitude and rarely sustained and ECL cell tumours have not been reported. However, *H pylori* infected patients treated with a PPI may develop significant elevations in plasma gastrin and ECL cell hyperplasia has been documented in up to a third of such patients taking a PPI for five years (Eiselle et al., 1997). Although hyperplasia precedes dysplasia and neoplasia, the time course of this progression is not known and it may take several years before carcinoid tumours develop. Hence carcinoid tumours developing in patients rendered hypergastrinaemic by *H pylori* and PPI therapy remains a remote possibility.\n\n| Associated Condition | Representative Range of Plasma Gastrin* (pM) | Causal mechanism for hypergastrinaemia |\n|----------------------|---------------------------------------------|----------------------------------------|\n| Pernicious Anaemia (PA) | 200 - >1000 | Atrophic gastritis leads to achlorhydria and secondary rise in plasma gastrin |\n| Zollinger Ellison (ZE) Syndrome | Normal - >1000 | Functioning gastrin secreting tumour usually of the duodenum or pancreas causes increased acid output. |\n| Chronic Renal Failure. (CRF) | Normal - 600 | Kidneys are the major site of breakdown of circulating gastrins. |\n| Proton Pump Inhibitor Treatment (PPI) # | Normal - 350 | Reduces acid output by direct action on the parietal cells’ H⁺/K⁺ ATPase. |\n| H. pylori infection | Normal - 200 | a) antral gastritis reduces antral somatostatin with loss of inhibition of gastrin release by gastric acid. \nb) Corpus gastritis leads to atrophy and hypochlorhydria |\n\nFigure 1.3: Conditions associated with hypergastrinaemia and the primary causal mechanism for the elevated plasma gastrin.\n\n* Amidated gastrin; normal fasting <30pM; normal post-prandial <90pM.\n\n# depends on duration of treatment and presence of *H pylori*. (6-8 week treatment with PPI raises plasma gastrin up to twofold).\n\nECL cell (Carcinoid) tumours and Hypergastrinaemia.\n\nChronic atrophic gastritis (CAG) with or without pernicious anemia (PA) is the commonest cause of ECL cell hyperplasia and in 5-10% of patients there is a progression to gastric carcinoid tumors (Borch et al., 1985; Bordi et al., 1991; 1995). The achlorhydria of CAG leads to hypergastrinemia which is generally accepted to be a stimulus to ECL cell growth (Bordi et al., 1995; Creutzfeld, 1988; Hakanson and Sundler, 1990; Solcia et al., 1986). The natural history of these tumors in man has been extensively reviewed (Modlin et al., 1995; Rindi, 1995; Solcia et al., 1991; Thomas et al., 1994), prompted at least in part by the demonstration that in rodents hypergastrinemia secondary to long-term treatment with H$_2$-receptor antagonists or proton pump inhibitors, may lead to gastric carcinoid tumors (Hava, 1986; Poynter and Selway, 1991; Wangberg et al., 1995).\n\nEnterochromaffin-like cell tumors associated with CAG are usually small (<1cm), may be single or multiple, are most often present incidentally in patients over the age of 60, and rarely progress to metastatic disease (Bordi et al., 1995; Solcia et al., 1991; Modlin et al., 1995). This has led to conservative management for the majority of patients with or without endoscopic screening. Some authors have advocated twice daily subcutaneous injections of the long acting somatostatin analogue octreotide (Ferraro et al., 1996; Bordi et al., 1993). This therapy has been shown to reverse ECL cell hyperplasia in patients with CAG without ECL cell tumors, and to inhibit gastrin release from the antral gastrin (G-) cell (Ferraro et al., 1996; Bordi et al., 1993). Surgical antrectomy to remove G-cells is also effective in\n\nreducing plasma gastrin levels and is reported to result in complete resolution of small ECL cell tumors (Hirschowitz et al., 1992; Modlin et al., 1995; Scully et al., 1997). In contrast, the management of large ECL cell tumours (>2cm) associated with CAG is less well defined. Antrectomy has been advocated since it may induce tumor regression, or at least halt metastatic spread (Eckhauser et al., 1988; Scully et al., 1997) However, reports of a metastasis rate of >20% for primary tumours that are >2cm in diameter, together with the demonstration that in a few cases the tumors fail to regress following antrectomy, has persuaded others to recommend total gastrectomy (Borch et al., 1985; Thomas et al., 1995).\n\nAlthough all PA patients are hypergastrinaemic and the majority show ECL cell hyperplasia some other factor(s) must also be involved in the progression of hyperplasia to neoplasia as the incidence of carcinoid tumours in PA patients is 5-10% (Borch et al., 1985; Bordi et al., 1991; 1995). Azzoni has suggested that the expression of BCL-2 by hyperplastic ECL cells may be of importance as it may prolong exposure to other oncogenic factors (Azzoni et al., 1996). However relatively little is known of the underlying genetic risk factors for ECL cell tumour development in CAG.\n\nIn the sporadic form of ZE the incidence of carcinoid tumours is similar to that in PA. In contrast patients with ZE as part of the type I multiple endocrine neoplasia syndrome (MEN1) have a much higher incidence of carcinoid tumour development (up to 35%) presumably due to an inherited susceptibility to endocrine cell tumour development (Lehy et al., 1992). The recent positional cloning of the\n\nMEN1 gene (Chandrasekharappa et al., 1997) has facilitated the study of abnormalities of this gene in the development of carcinoid tumours. Loss of heterozygosity (LOH) at the MEN1 locus could be demonstrated in the majority of carcinoid tumours arising in patients with ZE as part of the MEN1 syndrome (Debelenko et al., 1997). However very few gastric carcinoids arising from CAG had associated LOH at the MEN1 gene locus suggesting other genes may be important in the pathogenesis of these tumours. Data is presented in this thesis that is in favour of a potential candidate gene, reg, being of importance to the development of carcinoid tumours. Before reviewing the available information on the reg family of genes, the role of gastrin in the differentiation and growth of endocrine cells of the stomach and pancreas is discussed.\n\nGastrin, endocrine cell growth and differentiation in the stomach and pancreas.\n\nIn addition to the well established role of amidated gastrin in regulating gastric acid output, there is an increasing body of evidence to suggest that gastrin also regulates the differentiation of some gut endocrine cells and the growth and overall numbers of certain endocrine and epithelial cell populations within the stomach.\n\nThe first direct evidence of the trophic effect of amidated gastrin came from the observation that pentagastrin administration to rats increased the functioning\n\nparietal cell mass thereby contributing to the long term increase in acid output. (Crean et al., 1969). Prior to this, gland atrophy following antrectomy had been reported (Lees and Grandjean, 1958), indirectly suggesting a trophic role for gastrin in maintaining the acid secreting mucosa. Gastrin also stimulates the growth of ECL cells (Bordi et al., 1995; Tielemans et al., 1990), a subject that has received much interest due to the theoretical risk of ECL cell tumour development in patients receiving long term pharmacological acid suppression.\n\nMore recent studies using transgenic mice clearly demonstrate the importance of amidated gastrin acting via the CCK-B receptor in the maintenance of normal cell populations within the gastric mucosa. Hence, overexpression of amidated gastrin leads to increased proliferation of precursor cells in the proliferative zone of the gastric mucosa and increased parietal and ECL cell numbers (Wang et al., 1993). Conversely, targeted disruption of the CCK-B receptor produced marked gastric mucosal atrophy with reductions in the numbers of parietal and ECL cells (Langhans et al., 1997; Nagata et al., 1996). Similarly, targeted disruption of the gastrin gene resulted in a marked reduction in ECL cell and parietal cell number but did not affect the proliferative index in the stomach (Koh et al., 1997). The latter study is of particular interest as it highlights the importance of gastrin in regulating the differentiation of gastric parietal and ECL cells independent of the degree of stem cell proliferation.\n\nThere is also good evidence that gastrin is responsible for the development in the embryo of endocrine cells of the pancreas (Wang et al., 1993). In the case of\n\npancreatic endocrine cell development candidate genes have been proposed to be involved in the growth and differentiation of islet β-cells. (Terazono et al., 1988; Otonkoski et al., 1994; Watanabe et al., 1994). The *reg* gene was first isolated by Terazono and colleagues in 1988 by differential screening of a rat regenerating pancreatic islet cDNA library. This gene has been renamed *reg Iα*, following the discovery of several other homologous genes that appear to comprise a multigene family.\n\n**The *reg* gene family**\n\nThe rat *reg Iα* cDNA contained a single open reading encoding for a 165 aminoacid protein, of which the first 21 aminoacids appeared to constitute a signal peptide. The human homologue of rat *reg Iα* was identified by screening of a human pancreatic cDNA library with a 60 base pair complement of the rat cDNA (Terazono et al., 1988). Human *reg Iα* encodes a 166 aminoacid protein with a 22 aminoacid signal peptide. Human *reg Iα* normally is expressed in the pancreas, gastric mucosa and kidney; ectopic expression of *reg* has been described in tumours of the colon and rectum (Watanabe et al., 1990). The homology between rat and human *reg* cDNA was 75% (68% in AA sequence) including similar homology in the 5’ untranslated region. Perhaps of significance was the observation that the position of all seven cysteine residues within the predicted protein were conserved. Subsequent cloning of the respective genes indicated another common feature, each consisting of 6 exons and 5 introns. These two features are common to all members of the *reg* gene family.\n\nFollowing the cloning and sequencing of *reg* it became clear that the previously described pancreatic stone protein (PSP) was a truncated product of the same gene (Watanabe *et al.*, 1990). In fact PSP consists of 144 amino acids (23-166 of the parent *reg*) and has been renamed PSP$_I$. A shorter variant also has been identified and is 11 amino acids shorter (34-166 of the parent *reg*). This protein is identical to the pancreatic thread protein (PTP) also referred to as PSP$_{II}$ (Watanabe *et al.*, 1990).\n\nOther members of the human *reg* family that have been identified so far, include *reg Iβ* (Moriizumi *et al.*, 1994), *reg* related sequence (Watanabe *et al.*, 1990) and pancreatitis associated protein (Iovanna *et al.*, 1991; Morizumi *et al.*, 1993). Confusingly, pancreatitis associated protein (PAP) has an identical aminoacid sequence to peptide 23 (Katsumata *et al.*, 1995) and HIP, a protein that was first identified by its expression ectopically in primary hepatocellular carcinoma (Lasserre *et al.*, 1992). These members of the gene family are tandemly ordered in a 95kbp DNA region of human chromosome 2p12 as determined by fluorescence in situ hybridisation (Miyashita *et al.*, 1995).\n\nThe murine *reg* gene family has also been studied but reveals unexpected differences. The murine homologue of *reg Iα* has been identified and localised to chromosome 12 (Unno *et al.*, 1993). A second member of the family *reg II* has also been cloned. Although the gene consists of 5 introns and 6 exons with conservation of all cysteine residues, the whole sequence is <60% homologous to *reg I*, has no human homologue and maps to mouse chromosome 3 (Unno *et al.*, 1993). Further\n\nmembers of the mouse *reg* gene family have recently been described. Hence murine *reg IIIα*, *reg IIIβ* and *reg IIIγ* bear reasonable homology to each other but represent a separate gene group and are localised on Chromosome 6 (Narushima et al., 1997). Unlike other members of the *reg* family, the *reg III* genes are preferentially expressed in the intestine and are expressed only at low levels in regenerating pancreatic islets.\n\nThe available evidence suggests that members of the *reg* gene family are expressed within endocrine cells of the stomach and pancreas. In particular, *reg Iα* (*reg*) expression appears to be markedly upregulated during regeneration following surgical resection (Terazono et al., 1988; or mucosal ulceraton induced by waster immersion stress (Ashahara et al., 1997) or treatment with non-steroidal anti-inflammatory drugs (Kawanami et al., 1997). However, the potential relationship between gastrin and expression of the *reg* gene has not been explored.\n\n**Reg gene expression and endocrine cell growth and differentiation**\n\nThe *reg* gene was first proposed as a regulator of pancreatic islet cell growth following its isolation from regenerating islets (Terazono et al., 1988). In mature adult pancreas, *reg* expression is limited to acinar cells. The Reg protein is secreted into pancreatic juice where it has been suggested to act as an inhibitor of pancreatic stone formation (Patard et al., 1996; Bertrand et al., 1996). During islet cell regeneration the Reg protein also appears to be produced by endocrine cells as it has been shown to colocalise almost exclusively with insulin in β-cell secretory granules (Terazono et al., 1990).\n\nOther models of islet cell growth such as hyperplasia following surgical wrapping of the pancreas (Zenilman et al., 1996) and islet cell regeneration following growth suppression by insulinoma (Miyaura et al., 1991), have shown a consistent relationship between expression of reg and increasing islet cell number. None of these studies attempted to determine whether reg may act as a growth factor or as a promoter of differentiation.\n\nOtonkoski and colleagues (1994b) proposed that reg expression was a marker of proliferation rather than differentiation based on observations using nicotinamide to stimulate differentiation and hepatocyte growth factor (HGF) to stimulate proliferation in 7 day cultures of human foetal pancreatic cells. Reg expression was significantly increased by HGF. However, the effects of HGF are not purely trophic. In fact, the in vitro mitogenic effect of HGF is limited to the first three to four days of culture (Beattie et al., 1996). Thereafter, HGF has been shown to increase the formation of islet cell clusters in 6 day cultures of human foetal pancreatic cells and increase their insulin content (Otonkoski 1994a). Thus HGF appears to stimulate both growth and differentiation in this model. HGF also stimulates the differentiation of AR42J cells into insulin secreting cells (Mashima et al., 1996). The pancreatic cell line AR42J has recently been shown to express reg (Zenilman et al., 1996) and can be manipulated by appropriate culture conditions to differentiate towards either acinar or endocrine cell phenotype (Christophe, 1994; Mashima et al., 1996). Interestingly, differentiation towards an acinar cell phenotype is associated with reduced reg expression (Zenilman et al., 1997).\n\nIn 1994, Watanabe et al., described the amelioration of diabetes (induced surgically in rats by 90% pancreatectomy) by daily administration of recombinant rat Reg protein. Administration of Reg protein led to a significant increase in islet cell size, insulin positive β-cell number and amelioration of diabetes. They also demonstrated a threefold increase in the incorporation of $^{3}$H-thymidine into cultured rat islet cells exposed to recombinant rat Reg protein. Autoradiography demonstrated a limited increase in the proliferative index of insulin positive cells from 0.1% in controls to 0.2% in Reg treated cultures. Based on these experiments, Watanabe et al., claimed that the trophic effects of Reg protein explain its ability to stimulate β-cell regeneration and ameliorate diabetes.\n\nIn contrast, Smith et al., (1994) argue against reg playing a role in islet cell growth from experimental evidence employing a model of selective β-cell growth induced by glucose infusion for 96hrs. Such a procedure produces a 50% increase in the rat islet β-cell mass with a fivefold increase in the proliferative index of islet β-cells (Bonner-Weir et al., 1989). Using this model Smith et al., (1994) could find no evidence of increased reg expression during or after the glucose infusion. Isolated rat islets in culture however, do respond to glucose exposure, both by increased reg expression and increased $^{3}$H-thymidine incorporation (Francis et al., 1992).\n\n**Reg expression in endocrine cells of the stomach**\n\nIn 1996, Ashahara and colleagues reported the presence of reg in the endocrine cells of the rat stomach. In fact, by using the techniques of in situ\n\nhybridisation and immuno-histochemistry they clearly demonstrated that the large majority of *reg*-positive cells were the ECL cells. Furthermore, *reg* mRNA abundance was upregulated during the period of mucosal repair following stress ulceration induced by water immersion. Plasma gastrin concentrations and acid output were not measured. Nevertheless the presence of *reg* in ECL cells and its upregulation during ECL cell regeneration suggest that *reg* may play a role in the growth and/or differentiation of the gastric ECL cell.\n\nIn summary, *reg Iα* is a major member of the *reg* family and is expressed during pancreatic islet cell regeneration and in the rat gastric ECL cell. The protein product of the gene stimulates the formation of mature endocrine cells in the regenerating pancreas. Expression of *reg* is associated with growth and differentiation of endocrine cells but its exact function as trophic factor or promoter of differentiation remains to be determined. The ectopic expression of *reg* and related family members in tumours of the gastrointestinal tract and liver justifies the further studies required to elucidate the exact role of the *reg* gene and its protein product. The possibility that the *reg* gene is involved in the development of gastric carcinoid tumours in patients with hypergastrinaemia is examined in this thesis.\n\nAims and Objectives\n\n1. To develop a model of localised denervation of the gastric antrum in the rat in order to study the role of intrinsic antral neurons in the regulation of gastric function.\n\n2. To examine the effects of lesioning of antral denervation on gastric emptying in the rat to test the hypothesis that intrinsic neurons of the gastric antrum regulate gastric emptying and mediate the actions of extrinsic reflexes already known to influence gastric emptying.\n\n3. To examine the effects of antral denervation on the control of gastrin release in the rat in order to test the hypothesis that antral neurons regulate the responses of the G-cell to feeding and gastric distension.\n\n4. To investigate *reg* gene expression in the pancreatic cell line AR42J in order to test the hypothesis that gastrin and EGF may regulate *reg* expression.\n\n5. To determine the relationship between *reg* expression and hypergastrinaemia in both rat and man to test the hypothesis that gastrin stimulation of ECL cell function and growth are associated with upregulation of *reg* expression.\n\n6. To investigate the possibility that mutations of the *reg* gene (a putative regulator of endocrine cell differentiation) are associated with carcinoid tumour development as a consequence of chronic hypergastrineamia.\n\nChapter Two\n\nMaterials and Methods\n\nMaterials\n\nAll laboratory chemicals that can be considered as in routine use were obtained from either BDH chemicals, Poole U.K., or from Sigma Chemicals limited, Poole, U.K.. The materials used for routine molecular biology experiments that were not available from the above companies were obtained from either Gibco GBRL, Renfrewshire, Scotland, or from Severn Biotech, Worcester, U.K.. All radioactive materials were obtained from Amersham Life Sciences, Buckinghamshire, U.K.. Other specific items are listed below.\n\nDrugs/Chemicals:\n\n- Benzalkonium chloride: Sigma Chemicals Ltd.\n- Bovumin: First link, West Midlands, U.K.\n- BW2258U89: Dr J Leban, Burroughs Wellcome, U.S.A.\n- Capsaicin: Sigma Chemicals Ltd.\n- Diazepam (Valium): Roche products Ltd., Derbyshire U.K.\n- Enrofloxacin: Bayer Health Business Group, Suffolk, U.K.\n- Fentanyl/Fluanisone (Hypnorm): Janssen Animal Health, Buckinghamshire, U.K.\n- L-740093: Merk Sharp and Dohme, Harlow, U.K.\n\nPeptides:\n\nGastrin Peninsula Laboratories, St Helens, UK.\n\nporcine GRP\\textsubscript{14-28} Bachem (uk) Ltd., Saffron Walden, U.K.\n\nrat Tyr\\textsuperscript{0}\\alpha-CGRP\\textsubscript{28-37} Bachem (uk) Ltd., Saffron Walden, U.K.\n\nSubstance P Peninsula Laboratories, St Helens, UK.\n\nporcine VIP Peninsula Laboratories, St Helens, UK\n\nRestriction endonucleases:\n\nApa I Promega, Southampton, U.K.\n\nNot I Gibco GBRL, Renfrewshire, Scotland\n\nNru Gibco GBRL, Renfrewshire, Scotland\n\nSpe I Gibco GBRL, Renfrewshire, Scotland\n\nXba Promega, Southampton, U.K.\n\nCloning Vectors:\n\npGEMT-Easy Vector Promega, Southampton, U.K.\n\npcDNA3.1 ZEO Invitrogen, De Schelp 12, Netherlands\n\nMethods\n\nAnimals\n\nAdult Wistar rats (approximately 250g) were kept on a 12hr light/dark cycle and fed standard laboratory rat chow. Rats were fasted overnight before operations and refeeding experiments but allowed free access to water. Rats were anaesthetized by i.m. injection of a mixture of fentanyl citrate (0.32 mg.kg$^{-1}$), fluanisone (4 mg.kg$^{-1}$) and diazepam (2mg.kg$^{-1}$). Post-operatively, rats were given buprenorphine (0.1mg.kg$^{-1}$) for analgesia, antibiotic prophylaxis (enrofloxacin 5mg.kg$^{-1}$) and 10ml of saline by subcutaneous injection.\n\nBenzalkonium chloride treatment\n\nThe stomach was exteriorised through a midline upper abdominal incision. The serosal surface of the region to be denervated was covered with a layer of gauze cut to size. Benzalkonium chloride (0.02%, 0.1%, or 0.5% w/v) dissolved in 0.9% NaCl was painted onto the gauze, which was kept moist with the BAC solution for 3 minutes, and then removed. The serosal surface of the stomach was then rinsed thoroughly with normal saline and returned to the peritoneal cavity and the incision closed in layers. The two faces of the stomach were treated separately; in some experiments only the anterior face of the antrum was treated. A similar method was used for denervation of the corpus employing a strip of gauze (approximately 5mm wide) placed over the corpus alongside the border of the glandular and non-glandular regions. In order to selectively lesion the afferent innervation of the stomach, the same experimental approach was employed using\n\nthe sensory neurotoxin capsaicin (1% w/v in olive oil) in place of BAC. Control animals underwent a sham operation with serosal application of normal saline (or olive oil). Animals were allowed to recover for at least 11 days before experiments.\n\n**Refeeding experiments**\n\nControl and BAC-treated rats were fasted for 24hrs on wire-bottomed cages, and were then either maintained as fasted controls or allowed to refeed for 30 minutes. In some experiments rats were injected subcutaneously 30 minutes before refeeding with either a GRP antagonist BW2258U89 at a dose of 2mg.kg$^{-1}$ (Moody et al., 1995) or with vehicle (0.14M saline). Rats were killed by cervical dislocation, decapitated and trunk blood was taken for assay of plasma gastrin. Samples of antral and corpus mucosa were taken for mRNA extraction (see below) and the stomach content was removed. Gastric retention was determined by the dry weight of solid material in the stomach.\n\n**Gastric fistula rats**\n\nRats were treated by application of either BAC or saline to the antral serosa as described above and a small stainless steel Gregory cannula was installed in the corpus as previously described (Dimaline et al., 1986). The cannula was exteriorised through a midline stab incision and the incision closed in layers. Rats began training in Bollman cages on the tenth post operative day.\n\nGastric emptying studies\n\nGastric fistula rats were allowed at least 11 days to recover from surgery before experiments. Prior to experiments the stomach was washed with warm 0.14M NaCl to remove solids. On the experimental day, the emptying of test solutions (saline, 50mM HCl, 4.5% peptone, containing phenol red, 60mg.l\\(^{-1}\\)) instilled into the stomach via the gastric fistula, was determined as previously described (Forster et al., 1990).\n\nGastric distension studies\n\nPrior to experiments the stomach was washed extensively via the gastric cannula with warm 0.14M NaCl to remove solids. Rats were left for at least 90 minutes before experiments began. Plasma gastrin responses were determined after gastric distension with the following test solutions: 2% (w/v) methyl cellulose (2%MC); 2%MC at pH6 (50mM ammonium acetate); 2% MC with 50mM HCl; 4.5% w/v peptone. In each case the stomach was distended by connecting the cannula to a reservoir of test solution and elevating the reservoir to 5cm H\\(_2\\)O pressure for 30mins. Immediately following distension, rats were killed by cervical dislocation and decapitation and 1.5ml of trunk blood was then taken for gastrin radioimmunoassay. Samples of antrum from control and BAC-treated rats were taken to confirm denervation.\n\nMeasurement of acid output\n\nRats fitted with a gastric cannula were trained in Bollman cages to permit the required handling of the cannula before acid output could be measured. Prior to experiments, rats were fasted overnight. On the day of experiment the stomach was\n\nwashed extensively with warm 0.14M NaCl to remove solids. Rats were left for at least 90 minutes before experiments began. Gastric acid output was determined by collecting gastric secretion over four sequential 15 min periods and titrating to pH 7.0 with 20mM NaOH using a pH autotitrator as previously described (Dimaline, et al., 1986).\n\n**Omeprazole treatment**\n\nRats were given omeprazole (400μmol.kg\\(^{-1}\\)) by gavage under light Halothane anaesthesia. Rats were fed *ad libitum* and received omeprazole daily for five days. On the fifth day rats were killed by cervical dislocation and decapitation and 1.5ml of trunk blood was taken for gastrin radioimmunoassay. Samples of gastric corpus were taken for total RNA extraction (see below).\n\n**Radioimmunoassay**\n\nThe neuropeptide content of tissues was determined after extraction with boiling water (0.1 g.ml\\(^{-1}\\)), homogenizing and centrifuging and re-extraction of the pellet with 0.5M acetic acid, as previously described (Hutchison et al., 1981). Water or acid extracts were assayed at the appropriate dilution for the following peptides: Gastrin releasing peptide (GRP) was assayed using antibody 1078, with porcine GRP 14-28 as standard and iodinated Tyr\\(^1\\) COOH-terminal decapeptide of bombesin as label, as previously described (Dockray et al., 1979). Substance P was assayed with antibody L83, using undecapeptide substance P as standard and \\(^{125}\\)I-Bolton Hunter labelled substance P as label. Vasoactive intestinal polypeptide (VIP) was assayed using antibody L25, with porcine VIP as standard and \\(^{125}\\)I labelled VIP as label as previously described (Hutchison\n\nCalcitonin gene related peptide (CGRP) was assayed with antibody L273, using rat Tyr\\textsuperscript{0}a-CGRP 28-37 as standard and the same peptide labelled with \\textsuperscript{125}I as label as previously described (Varro et al., 1988). The concentration of amidated gastrins in plasma gastrin was assayed using antibody L-2, using synthetic human non-sulphated G-17 as standard and for labelling with \\textsuperscript{125}I as previously described (Dockray et al., 1991). The standard conditions for each assay are shown in Figure 2.1\n\n**Extraction of RNA**\n\n**Extraction of Tissue RNA:**\n\nTissue mRNA was extracted and quantified as described previously (Dimaline et al., 1991). Samples of antral or corpus mucosa were homogenised in 3mls of 4M guanadinium isothiocyanate buffer containing 25mM sodium acetate pH 6.0 and 0.84% (vol/vol) β-mercaptoethanol and frozen at -80°C until required. After thawing at room temperature, samples were pipetted onto a 5.7M caesium chloride cushion (1.3ml) in an ultracentrifuge tube. Samples were centrifuged overnight at 41,100 rpm to pellet the RNA. The mRNA pellet was dissolved in 300µl of Tris-EDTA (TE) buffer with 0.2% SDS and precipitated with 0.3M sodium acetate under 100% ethanol overnight.\n\n**Extraction of Cell RNA**\n\nRNA extraction from cultured cells was performed using the Quickprep™ total RNA extraction kit (Pharmacia Biotech, Herts, U.K.) according to the manufacturers instructions. The media (2ml) was removed from cultured cells and replaced by 900µl of a mixture of lithium chloride buffer, β-mercaptoethanol and extractoin buffer. Cells were\n\n| Peptide Assay | Antibody/Dilution | BUFFER | INCUBATION | SEPARATION |\n|---------------|-------------------|--------|------------|------------|\n| | | | | Ch:Dx:MPr* |\n| GASTRIN | L2 - 1:100,000 | Na⁺Bicarbonate pH 8.4, 0.5% Bovumin, 10mM Na⁺ Azide | 72hrs : 4 °C | 10 : 1 : 0.5 |\n| CGRP | L271 - 1:80,00 | 50mM PO₄, pH 7.4, 0.5% Bovumin, 0.25% EDTA, 0.14M NaCl | 72hrs : 4 °C | 10 : 1 : 1 |\n| GRP | 1078 - 1:80,000 | Na⁺Bicarbonate pH 8.4, 0.5% Bovumin, 10mM Na⁺ Azide | 48hrs : 4 °C | 10 : 1 : 0.5 |\n| SUBSTANCE P | L-83 - 1:400,000 | 50mM PO₄, pH 7.4, 0.5% Bovumin, 0.14M NaCl, 10mM Na⁺ Azide | 48hrs : 4 °C | 10 : 1 : 0.5 |\n| VIP | L-25 - 1:40,000 | 20mM PO₄, pH 6.0, 0.4% BSA, 0.1% EDTA, 10mM Na⁺ Azide, 0.025% w/v polybrene | 48hrs : 4 °C | 10 : 1 : 0.5 |\n\nFig 2.1 Conditions for each radioimmunoassay are provided in the table. *For separation of the assay a mixture of charcoal dextran and milk protein (Marvel) in the above ratio (g/100ml) was added to each tube.\n\nresuspended in the solution by scraping and then pipetted into a universal container. The cells were then homogenised and the lysate transferred to a 1.5ml eppendorf containing 600µl of caesium trifluoroacetate solution and left to stand on ice for ten minutes. RNA was pelleted by centrifugation at 15000rpm for 15 minutes in a microcentrifuge. The RNA pellet was washed thoroughly, allowed to dry and resuspended in 10-20µl of diethyl pyrocarbonate-treated (DEPC) water before quantification. Any unused RNA was precipitated with 0.3M sodium acetate under 100% ethanol and stored at -80°C.\n\n**RNA electrophoresis**\n\nStored precipitated RNA was pelleted by centrifugation at 13000rpm for 15 minutes, washed twice in 70% ethanol/DEPC H₂O and once in 100% ultrapure ethanol. Pellets were air dried at room temperature and dissolved in 10-50µl of DEPC H₂O. RNA samples were quantified by spectrophotometer and 10µg aliquots added to denaturing buffer (formamide: 37% formaldehyde: 10 x MOPS (3-(4-Morpholino) propane sulfonic acid) in ratio 4:1.75:1 and heated to 55 °C for 15 minutes. Samples were then placed on ice and 2µl of RNA loading buffer added to each sample. The denatured RNA was then electrophoresed in a 1x MOPS/2% formaldehyde 1% agarose gel.\n\n**Northern Blot and Hybridisation**\n\nAfter electrophoresis, RNA was electroblotted to nylon membranes (Nytran 0.5) overnight in 0.5% TAE buffer (containing 0.2M Tris base, 1M acetate and 0.1MEDTA) at 4 °C. RNA was crosslinked to the nylon membrane by ultraviolet light (UV Cross Linker, Hoeffer scientific instruments, San Francisco, USA). Membranes were placed in\n\nhybridisation buffer (50% formamide containing 5 x Denhardts solution, 5 x sodium chloride sodium phosphate EDTA buffer (SSPE), 0.5% SDS and sonicated salmon sperm DNA (200μg.ml\\(^{-1}\\)) and warmed to 65 °C for 4 hours prior to the addition of \\(^{32}\\text{P}\\)-labelled cRNA probe (2x10\\(^6\\) cpml\\(^{-1}\\)). Membranes were hybridised overnight and then washed twice with 2xSSPE containing 0.1% SDS at room temperature for 20min, and once with 0.1xSSPE/0.1% SDS at 65°C for 20min. Signals were removed from membranes using boiling 0.1% SDS and membranes rehybridized with alternative specific probes as required. Signals were quantified using a Phosphor Imager.\n\n**Reverse transcription/PCR**\n\n*Reverse transcription* of RNA was performed by first annealing 1μl oligo dTs (500μg.ml\\(^{-1}\\)) to ≈50μg of total RNA by heating to 55°C for 5 minutes and cooling slowly. cDNA was generated by incubating the following reaction at 42°C for 1 hour.: oligo dT and RNA (10μl), 2μl DTT(100mM), 1μl dNTPs (25 mM), 1μl RNAsin, 4μl 5x AMV RT Buffer and 2μl AMV reverse transcriptase. The cDNA generated was purified by phenol/chloroform extraction, precipitated with 0.5M ammonium acetate and washed with 100% ethanol. The cDNA was then resuspended in 100μl of TE. 5μl of this solution was used as template for PCR.\n\nHuman *reg* cDNA was generated by reverse transcription of total RNA extracted from gastric mucosal biopsies taken from either normal corpus or from carcinoid nodules. Two different size products were generated by separate pairs of primers chosen to include either a 297bp central portion of the coding region.\n\ncontaining an *EcoR* I restrictiton site, or the full length coding sequence. The smaller fragment was used as template for riboprobe synthesis (see below) and the larger fragment (557bp) used for cloning and sequencing (see below). Primers were chosen based on the full coding sequence of the published human *reg* mRNA (Terazono *et al.*, 1988) as follows: The 297 bp fragment: sense - TCTCCTGCCTGATGTTTCTGTCTC antisense - GGCGGTTCTTTTTGGGGTCAT, The 557bp fragment: sense GATTGTTGATTTGCCCTCTTA, antisense - TCCAGCTGCCTCTAGTTTTTGAA.\n\nRat *reg* cDNA was generated by reverse transcription of total RNA extracted from rat pancreas. PCR primers were chosen to include the full coding sequence of the published rat *reg* mRNA (Terazono *et al.*, 1988) as follows: sense - AGCCTGCAGAGATTGTTGACTTTG; antisense - AGGGGGTTGACTTTGCTTTTGATA and gave a predicted product size of 651bp (Figure 2.2). Restriction digestion of the 651bp PCR product with *EcoR*1 cleaved the product into 253 & 398bp fragments.\n\n*Polymerase chain reaction* was performed under standard conditions in a final volume of 100µl containing 10x *taq* polymerase buffer (10µl), MgCl (1.5mM), dATP (0.2mM), dCTP (0.2mM), dGTP (0.2mM), dTTP (0.2mM), template cDNA (5µl), sense and antisense primers (5ng. µl$^{-1}$) and *taq* polymerase (0.5µl). 60µl of mineral oil was layered over the reaction mixture which was then placed into a thermal cycler (Hybaid, Middlesex UK). After a 5 minute denaturation at 95°C, thirty cycles consisting of annealing (1 minute at 58 °C), extension (2 minutes at 72°C) and denaturation (1 minute at 95°C) were performed. A final extension step (72 °C) was performed for ten minutes before determining the outcome by agarose gel electrophoresis.\n\nFigure 2.2 PCR products of human and rat *reg* cDNA using human carcinoid tumour RNA and rat pancreatic RNA as template for reverse transcription. The predicted sizes of the human PCR products were 557bp (lane 1) and 297bp (lane 2) depending on the primers used. The predicted size of the rat PCR product was 651bp (lane 3). See text for full details.\n\nDNA Ligation\n\nThe PCR fragments of human and rat *reg* cDNA were ligated into the pGEM-T Easy vector by incubating approximately equimolar concentrations of PCR product and vector in the presence of DNA ligase at 4°C overnight according to the manufacturer's instructions.\n\nCloning and Sequencing\n\n*Bacterial Transformation:* Chemically competent JM109 bacterial cells were stored at -80°C. Prior to transformation, cells were thawed on ice for 20 minutes before addition of 2 µl of 8.7M β-mercaptoethanol and 1 µl of DNA ligation mixture. Incubation on ice was continued for a further 30 minutes. The cells were then heated to 42°C for 45 seconds and immediately placed on ice for a further 2 minutes. Thereafter, 450 µl of SOC medium (containing 2% Tryptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM Kcl, 10 mM MgCl₂·6H₂O and 20 mM glucose) was added and the reaction incubated at 37°C for one hour with constant gentle agitation.\n\nAfter this incubation a 100 µl aliquot was spread onto petri dishes containing 20 mls of LB/agar (1% Tryptone, 0.5% Yeast extract, 1.0% NaCl, 1.5% agar) that had been treated previously with 100 µl isopropyl-β-D-thiogalactosidase (IPTG 0.1 M), 30 µl 5-bromo-4-chloro-3-indolyl-β-D-galactosidase (XGAL 0.2 M) and 200 µl of ampicillin (10 mg.ml⁻¹). The remaining cell suspension was pelleted by centrifugation (5000 rpm for 3 minutes) and the cells resuspended in 200 µl of SOC medium. A further 100 µl aliquot\n\nof cells, now at a higher cell density, was plated onto a LB/agar plate. Plates were then incubated overnight at 37°C and selection of successfully transformed colonies was by colour selection and antibiotic resistance. Successfully transformed colonies (white) were sub-cloned onto a second LB/agar plate and into 3ml of LB medium containing ampicillin (0.05mg.ml\\(^{-1}\\)). Following a further overnight incubation at 37 °C, the amplified plasmid DNA was extracted and purified for restriction digestion (to confirm presence of the expected cDNA sequence insert) and sequencing.\n\n**Plasmid DNA extraction** Small, medium and large scale plasmid DNA extraction was performed using commercially available kits according to manufacturers instructions (Wizard Minipreps, Midipreps and Maxipreps). The protocol is similar for each with adjustments for scale depending on the yield of plasmid DNA required. In general, small scale preparations were sufficient for sequencing purposes. Medium scale preparations were used to generate templates for cRNA probe synthesis and large scale preparations were used for generating linearised plasmid DNA for permanent cell transfection (see below).\n\nFollowing overnight culture at 37 °C, bacterial cells were pelleted by centrifugation, the supernatant discarded and the cells resuspended in resuspension buffer. The cells were then lysed by addition of lysis solution (NaOH/SDS) and protein and bacterial DNA were precipitated by addition of a neutralising solution (potassium acetate). The precipitate was removed by centrifugation and the plasmid DNA extracted from the supernatant by addition of a high affinity resin. This resin with bound DNA was\n\nimmobilized to a column by vacuum suction. The column was then washed twice (80% ethanol) and the DNA eluted by addition of TE buffer (68 °C) followed by centrifugation with collection of the eluate. The DNA content and purity was measured by spectrophotometry.\n\n**Riboprobe synthesis**\n\nTemplates for generating cRNA probes for gastrin, HDC, somatostatin and GAPDH were provided by Dr R. Dimaline and contained the appropriate template as described previously (Dimaline *et al.*, 1991; 1993a; 1994). The template for generating the cRNA probes for rat and human *reg* were prepared by restriction fragment digestion of midiprep purified pGEM t-Easy vector containing the full length rat *reg* sequence or the 253bp human *reg* sequence as described above. After sequencing the vector to determine orientation of the insert, 10µg of each sample was digested with restriction enzyme *ApaI* in appropriate enzyme buffer in a volume of 20µl for 3 hours at 37°C. Complete linearisation of plasmid DNA was confirmed by gel electrophoresis. 0.5µl of template was used for riboprobe synthesis. The reaction conditions were as follows: 2µl 5x SP6 buffer, 1µl DTT(10mM), 2.5µl DNTPs (100µM each ATP, UTP, GTP), 1µl CTP (10µM), 0.5µl RNase, 0.5µl template DNA, 0.5 µl RNA polymerase (1-5 units) and 2.5µl $^{32}$P-CTP ($\\approx$ 5µM). The reaction mixture was incubated at 37°C for 1 hour and then RNase free DNase added for a further 15 minutes. The synthesised probe was purified by size exclusion and 1µl of purified probe placed in 5ml of scintillation fluid and\n\nincorporated radioactivity counted. Labelled probe was used at a concentration of $2 \\times 10^6$ cpm per ml of hybridisation buffer.\n\n**Preparation of DNA for permanent transfection**\n\nTwo sequences of the human *reg* gene were chosen for permanent transfection into the hamster insulinoma tumour cell (HIT) line. These were the full length coding sequence of the normal (wild type) *reg* and the sequence in which the first methionine residue (initiator methionine) had been mutated to a valine (ATG→GTG). Representative clones containing each sequence were prepared by small scale DNA extraction (Miniprep). The purified plasmid DNA (>600ng in 15μl) was then restriction digested with the combined endonucleases *Spe*I and *Not*I at 37 °C for 3 hours. The restriction fragment containing the *reg* sequence was then purified by gel electrophoresis (1.5% agarose). The DNA band of correct size (∼600bp) was cut from the gel and DNA removed from the gel onto a column by centrifugation (13,000rpm for 60 seconds), and eluted from the column by the addition of 20μl sterile water followed by centrifugation at 13,000rpm for 20 seconds. A small aliquot of the DNA was again gel electrophoresed to assess purity and concentration (20ng.μl$^{-1}$).\n\nEach restriction fragments was then ligated into the mammalian expression vector pcDNA3.1 Zeo. The vector circular DNA was linearised by concomitant digestion with the restriction endonucleases *Xba*I and *Not*I. (The *Xba*I restriction leaves an identical sequence overhang to that of *Spe*I used to create the insert DNA). Complete digestion by both enzymes was presumed by serial examination of control vector digests.\n\nby each restricton endonuclease separately. After 4 hours, the linearised vector was gel purified and eluted. The concentration of vector and its purity was estimated by gel electrophoresis.\n\nLigation of the wild type and mutant *reg* inserts was achieved by addition of equimolar concentrations of insert and vector DNA in the presence of DNA ligase and appropriate buffer. The ligation reaction was incubated overnight at 14°C. In addition, a control ligation reaction was also performed to ensure that complete digestion by both endonucleases occurred whilst preparing the vector DNA. This consisted of linearised vector without any insert. As the *Xba* and *Not I* restriction site overhangs are incompatible, re-ligation should not occur without the presence of insert DNA. Hence no colonies should form with cloning of the control ligation. Conversely, any colonies present from the *reg* insert ligations should all be positive for insert DNA. This selection process was required as the pcDNA3.Zeo vector does not incorporate the lac operon gene that allows for colour selection.\n\nFollowing cloning into JM109 cells, colonies from each of the wild type and mutant *reg* transformations were selected for small scale DNA preparation. The presence of a *reg* insert was shown by restriction digestion with *EcoRI* and then confirmed by sequencing. Positive clones for each sequence were then processed for large scale DNA preparation to yield $\\approx 300 \\mu l$ of concentrated DNA (3-5 $\\mu$cg.$\\mu l^{-1}$). For each permanent transfection, 10 $\\mu g$ of linearised DNA (in <20 $\\mu l$) was required. Therefore, the appropriate volume of each vector containing wild type and mutant *reg* inserts were linearised by\n\ndigeston using the restriction endonuclease *Nru*. Linearised DNA was incorporated into cultured HIT cells by electroporation (see below).\n\n**Tissue culture and permanent transfection**\n\nThe hamster insulinoma cell line HIT T15 was grown in RPMI 1640 medium supplemented with 11mM glucose, 10% v/v foetal calf serum, 2.5ug.ml\\(^{-1}\\) fungizone, 100U.ml\\(^{-1}\\) penicillin and 100µg.ml\\(^{-1}\\) streptomycin (all from GIBCO BRL, Life Technologies, Paisley, Scotland). Stably transfected cell lines expressing wild type or mutant human *reg* were produced by electroporation of \\(5 \\times 10^6\\) cells in 0.8ml at 200V and 1070µF in incomplete medium containing 10µg of linearised pcDNA *reg* construct. Cells were grown in complete RPMI in 10cm dishes for 48 hours prior to selection with zeocin (750µg/ml\\(^{-1}\\)) (Invitrogen). Six colonies expressing each construct were cloned and screened for *reg* expression by northern blot.\n\nThe rat pancreatic cell line AR42J was grown in RPMI 1640 medium supplemented with 10% v/v foetal calf serum. For experiments, cells were plated out at \\(1.5 \\times 10^6\\) cells in 5ml culture medium / 60ml dish and grown at 37°C/ 5% CO2. Following a 24 hour growth period, the cells were washed and their media replaced with incomplete RPMI with the following stimulii or appropriate vehicle (control). Human gastrin-17 (HG17), rat gly-extended gastrin-9 (G-Gly) in 50mM ammonium bicarbonate, wortmannin, mouse salivary gland extract (EGF) in RPMI, L740093 in 1x10\\(^{-6}\\)% DMSO. In all cases except for wortmannin, the cells were incubated for 24\n\nhours at 37°C / 5%CO₂ prior to RNA extraction. In the case of wortmannin, the cells were incubated for 4 hours prior to RNA extraction.\n\n**Statistical analysis**\n\nThe statistical test used for each experiment is indicated in the appropriate figure legend. In general, for the experiments involving denervation, the data was analysed using Mann Whitney U test and for other analyses Student’s t test was used.\n\nChapter Three\n\nChemical denervation of antral neurons\n\nIntroduction\n\nThe gastric emptying of liquids is dependent on the pressure gradient between the lumen of the stomach and proximal duodenum, and the resistance to flow between the two (Mayer et al., 1994). It has long been known that the rate of emptying depends on the nature of the luminal contents; thus, acid, hyperosmolal solutions, high viscosity, protein- and fat-rich meals all delay gastric emptying (Mayer et al., 1994). The emptying of liquids is pulsatile in nature and this can be attributed to phasic contraction of the pyloric antral region of the stomach (Houghton et al., 1988; Malbert et al., 1991; Prather et al., 1993). Alterations in the flow of gastric content across the antrum can be effected by changing either resistance to flow generated by antral contractions or the overall tone of the gastric body and the duodenum. There is a significant body of evidence to indicate the importance of vagal afferent innervation of the proximal gut (Raybould et al., 1988; Forster et al., 1990; Holzer et al., 1994) and vagal efferent innervation of the gastric corpus (Forster et al., 1991; D’Amato et al., 1992) in regulating delayed gastric emptying by inducing gastric corpus relaxation in response to hyperosmolal, protein-rich or fat-rich solutions in the duodenum. However, much less is known of the role of intrinsic antral neurons in the control of pyloric flow.\n\nAntral neurons however are known to innervate both the muscle layers of the antrum and the mucosal epithelial cells including endocrine cells (Gabella, 1987; Furness and Costa, 1987; Ekblad et al., 1987). The clearest example of this and the most relevant in terms of digestive physiology of the stomach is the innervation of Gcells of the gastric antrum by GRP-immunoreactive neurons that have their cell bodies in the myenteric plexus (Dockray et al., 1979; Miller et al., 1989). Systemically administered GRP has been shown to stimulate gastric acid output by the release of gastrin from G-cells in both animals and man (Varner et al., 1981; Bunnett et al., 1985). Further evidence that GRP exerts physiologically regulates the G-cell comes from the demonstration that G-cells express GRP receptors (Vigna et al., 1990) and GRP is capable of stimulating the release of gastrin from G cells in primary culture (Giraud et al., 1987).\n\nThe experiments described in this chapter were undertaken to define the anatomical lesion induced by chemical denervation of the rat antrum. This would then allow potential study of the role of the antral innervation in gastric emptying and the regulation of mucosal endocrine cell function. To this end I used the detergent benzalkonium chloride (BAC) which has already been used to lesion small intestinal myenteric neurons (Fox et al., 1983; Dahl et al., 1987). In keeping with the observations of others (Fox et al., 1983; Dahl et al., 1987) I was able to demonstrate that BAC application to the rat stomach produces a highly specific, reproducible and permanent loss of the all neurons in the treated region. Following antral denervation, the intrinsic innervation of the corpus is spared, but there was loss of the spinal afferent innervation of the gastric corpus. Conversely, when a band of proximal gastric corpus was denervated, intrinsic and extrinsic antral innervation was preserved.\n\nMethods\n\nAll operations were performed on Wistar rats. For antral denervation rats were starved overnight, but for corpus denervation rats were fed *ad libitum*. Following exposure of the stomach, BAC (0.02%, 0.1%, or 0.5% w/v) dissolved in 0.9% NaCl was painted onto gauze, which was kept in contact with the region to be treated and kept moist with the BAC solution for 3 minutes, and then removed. The two faces of the stomach were treated separately; in some experiments only the anterior face of the antrum was treated. In order to selectively lesion the afferent innervation of the stomach, the same experimental approach was employed using the sensory neurotoxin capsaicin (1% w/v in olive oil) in place of BAC. Control animals underwent a sham operation with serosal application of vehicle (normal saline or olive oil). Animals were allowed to recover for at least 11 days before experiments. Denervation was assessed by radioimmunoassay of neuropeptides in extracts of BAC-treated and adjacent tissues.\n\nResults\n\nDose-Response Relationship.\n\nInitial experiments were directed at determining the optimal dose of BAC for antral denervation. The tissue concentrations of GRP, CGRP, substance P and VIP measured by radioimmunoassay of antral extracts were reduced in a dose dependent manner 11 days after prior administration of BAC (Fig 3.1). At a concentration of 0.02% BAC had no effect; 0.1% BAC produced a 25% loss of CGRP, but following 0.5% w/v BAC all neuropeptides were depleted. The concentrations of substance P, VIP and GRP in extracts of the gastric corpus were not affected by prior application of BAC to the antrum (Fig 3.2). However, the concentration of CGRP in the corpus was significantly reduced by application of 0.5% BAC to the antrum. (Fig 3.2)\n\nTime Dependency of Responses\n\nIn order to determine the duration of responses to 0.5% BAC, neuropeptide concentrations were examined in extracts of tissues sampled 11 to 25 days after treatment. All neuropeptides assayed were depleted in antral extracts 11 days after BAC, and remained substantially reduced at all times up to 25 days (Fig 3.3). Similarly there was loss of CGRP in both antrum and corpus in rats treated with BAC on the antrum at all times examined (Fig 3.4).\n\nFig 3.1 Neuropeptide concentrations (CGRP, GRP, substance P and VIP) in extracts of antrum following serosal application to the antrum of BAC or vehicle (control). Only at 0.5% w/v BAC was there a significant loss of neuropeptides derived from both intrinsic and extrinsic afferent neurons of the antrum. (n = 6 all groups; * p<0.05, compared with control, Mann Whitney U test; in this and subsequent figures, values are means ± SEM).\n\nFig 3.2 Neuropeptide concentrations (CGRP, GRP, substance P and VIP) in extracts of the antrum 11 (n=5), 18 (n=5) and 25 (n=3) days after application of 0.5% w/v BAC or vehicle (control, n=6) to the serosa of the antrum. The loss of all neuropeptides was complete at day 11 and unchanged thereafter. (* p<0.05, compared with control, Mann Whitney U test)\n\nFig 3.3 Neuropeptide concentrations (CGRP, GRP, substance P and VIP) in extracts of corpus following serosal application to the antrum of BAC or vehicle (control). Even at 0.5% w/v BAC there was no significant loss of neuropeptides derived from intrinsic neurons of the Corpus. However at 0.5% w/v BAC there is a significant loss of corpus afferent innervation as shown by the significant reduction in corpus CGRP levels. (n = 6 all groups; * p<0.05, compared with control, Mann Whitney U test)\n\nFig 3.4 Neuropeptide concentrations (CGRP, GRP, substance P and VIP) in extracts of corpus 11 (n=5), 18 (n=5) and 25 (n=3) days after application of 0.5% w/v BAC or vehicle (control, n=6) to the serosa of the antrum. As with the results for the antrum, the selective loss of corpus CGRP after antral BAC was complete at day 11 and unchanged thereafter (* p<0.05, compared with control, Mann Whitney U test)\n\nSpecificity of Lesion\n\nTo examine the specificity of the effects described above, we studied the effect of BAC applied to a circumferential band (approximately 5mm) of the gastric corpus immediately distal to the non-glandular part of the stomach. In the BAC-treated region there was substantial depletion of all the neuropeptides studied (Fig 3.5). In the untreated corpus, and in the antrum, the tissue concentrations of VIP, substance P, CGRP and GRP were similar to control (Fig 3.5). It is worth emphasising that following BAC treatment of the corpus, antral CGRP was unaffected. Given that CGRP is present only in spinal afferent neurons, this result seemed to indicate a specific lesion in the corpus projections of these neurons induced by antral BAC treatment. To confirm this, the effects of the sensory neurotoxin capsaicin applied in a similar fashion to BAC, were examined as this neurotoxin is known to lesion only small diameter afferents leaving intrinsic innervation unaffected. Serosal application of 1% w/v capsaicin to the antrum, produced complete loss of CGRP in extracts of antrum and corpus (Fig 3.6). In contrast, neuropeptides found in intrinsic neurons (VIP and GRP) were not changed by treatment of the antrum with capsaicin (Fig 3.6).\n\nFinally, in some experiments BAC was applied to a single side of the antrum. In this case, there was depletion of neuropeptides on the treated side as already noted, but the concentrations in the untreated side of the antrum were similar to controls (Fig 3.7).\n\nFig 3.5 Neuropeptide concentrations (CGRP, GRP, substance P and VIP) in extracts of antrum, control corpus and a band of proximal corpus treated with either 0.5% BAC (hatched bars) or vehicle (open bars). There was complete loss of the neuropeptides in the band of corpus exposed to BAC but not in the remainder of the corpus, or the antrum. Note in particular the preservation of CGRP in the antrum. (* p<0.05, compared with vehicle, Mann Whitney U test; n = 6 all groups).\n\nFig 3.6 Neuropeptide concentrations (CGRP, GRP and VIP) in extracts of antrum and corpus following topical 0.1% w/v capsaicin (hatched bars), or vehicle (open bars), to the serosal surface of the antrum. The spinal afferent innervation of both the antrum and corpus was lost as shown by CGRP concentrations, while neuropeptides of the intrinsic innervation (GRP, VIP) were unaffected in both regions. (n = 4, both groups; * p<0.05, compared with vehicle, Mann Whitney U test).\n\nFig 3.7 Neuropeptide concentrations (CGRP and GRP) in extracts of either the anterior (a) or posterior (p) surfaces of the antrum (A) and ipsilateral corpus (C) following topical 0.5% w/v BAC (hatched bars) or vehicle (open bars) to the anterior serosal surface of the antrum. Neuropeptides of the intrinsic innervation (GRP, VIP) were reduced only in the BAC treated region. The spinal afferent innervation of both the antrum and corpus was lost as shown by CGRP concentrations predominantly on the side of the stomach that received antral BAC (n = 4, both groups; * p<0.05, compared with vehicle, Mann Whitney U test).\n\nThe experimental data obtained in these experiments provided substantial evidence that application of 0.5% w/v BAC to the serosal surface of the antrum for 5 minutes produced a chronic, profound and sustained loss of both the intrinsic and extrinsic afferent innervation. Although this is in keeping with the studies that have reported the effects of BAC on the rat small intestine (Fox et al., 1983; Dahl et al., 1987; Holle et al., 1990; Jodal et al., 1993), rat colon (Sato et al., 1978, Sakata et al., 1979) and opossum oesophagus (Gaumnitz et al., 1993) the duration of exposure and concentration of BAC used was different. In the previous reports in the rat, the preferred method has been that described by Fox et al. (1983) requiring 0.062% w/v BAC and an exposure time of 30 minutes for each surface of the intestine. Others have used higher concentrations of BAC (0.2%) with the same exposure time (Zuculoto et al., 1991). Fox et al. (1983) demonstrated clearly that the degree of denervation produced by BAC was concentration dependent. Extending these observations, See et al. (1988) confirmed that BAC induces myenteric denervation by disrupting the outer layers of the intestine following application to the serosa. The depth of damage to the intestine was dependent on the concentration used and the length of exposure time.\n\nIn order to reduce the time under anaesthetic, it seemed reasonable to use a higher concentration of BAC and to determine the appropriate dose by a dose response study. The effective dose to induce comprehensive denervation with a 5\n\nminute exposure time was found to be 0.5% w/v BAC as this dose significantly reduced neuropeptide levels to less than 10% of control values.\n\nThe neuropeptides assayed were GRP, CGRP, VIP and substance P. These neuropeptides are representative markers of separate populations of intrinsic and spinal afferent neurons although there is a degree of overlap. Hence GRP is a good and specific marker in the rat antrum for intrinsic myenteric neurons that are secretomotor and stimulate release of gastrin from G cells (Dockray et al., 1979). A proportion of these neurons also contain VIP which, in addition is also found separately in inhibitory motor neurons of the rat myenteric plexus (Ekblad et al., 1987). CGRP is present almost exclusively in spinal afferent neurons (Varro et al., 1988; Sternini et al., 1986; Sternini et al., 1987) in the rat although a proportion of these neurons also contain substance P (Green and Dockray, 1988). However the majority of substance P is found in intrinsic neurons of the myenteric and submucosal plexi (Holzer et al., 1980).\n\nThe distribution of neuropeptides within the wall of the stomach explains to some extent the pattern of loss of as determined by radioimmunoassay. Thus, GRP present predominantly in myenteric neurons was reduced to undetectable levels. Similarly CGRP present in spinal afferent neurons that innervate the serosa and muscle layers of the gut also was reduced to undetectable levels. However VIP and substance P are known to be present also in submucosal neurons and the latter also\n\nmay be found in inflammatory cells. Although there was a significant reduction in VIP and substance P, these peptides were still detectable in tissue extracts.\n\nThe earliest time point that denervation was assessed was 11 days and the latest time point was 27 days. Antral denervation was complete at 11 days and there was no change at the later times studied. This observation is in agreement with previously published experiments on the rat small intestine that have shown complete denervation as early as 24 hours after exposure to BAC (See et al., 1988) and persistence of denervation for up to 160 days (Zuculoto et al., 1991; See et al., 1988). In all the published studies on denervation induced by BAC it has been assumed that the effect was localised to the region of exposure. The data presented here indicate clearly that the spinal afferent innervation of the whole stomach is lesioned by antral BAC treatment. This was shown by loss of CGRP in antral and corpus tissue extracts following antral BAC.\n\nThree lines of evidence suggest that this observation was not due to inadvertent exposure of the corpus to BAC. Firstly, other neuropeptide levels in corpus tissue extracts remained unaffected by antral BAC treatment. Secondly, in the reverse experiment lesioning a circumferential band of proximal corpus did not produce a loss of antral CGRP. Thirdly, exposure to BAC of only the anterior surface of the antrum caused a significant reduction of intrinsic neuropeptides only in tissue extracts of the anterior and not posterior antrum.\n\nThe validity of the observation that lesioning antral spinal afferents removes corpus spinal afferent innervation was confirmed by using the specific small diameter afferent fibre neurotoxin capsaicin. Again exposure of antral spinal afferents to capsaicin reduced CGRP levels in antrum and corpus leaving other neurotransmitters levels unaffected. Taken together these observations suggest that CGRP-containing nerve fibres that supply the gastric corpus also supply the antrum, either by passage over the antrum or by projection of branches to both regions.\n\nFinally, confirmation of the effect of BAC on the gastric innervation described above were supported by immunohistochemical studies performed by Dr. C Vaillant. Samples of antrum and corpus were taken from rats that had either received antral BAC or BAC to a circumferential band of proximal corpus. The results of Dr. Vaillant’s studies essentially confirm the data obtained by radioimmunoassay. Hence there was a complete loss of immunoreactive nerve cell bodies and fibres only in the treated region following exposure to 0.5% BAC. However, specific staining for CGRP demonstrated that following antral application of BAC there was loss of CGRP-immunoreactive fibres from both the antrum and corpus whereas denervation of a band of corpus with BAC resulted in complete loss of immunoreactive fibres in the treated region, but not in the untreated antrum (Higham et al., 1997).\n\nSummary\n\nAntral BAC treatment effectively lesions all myenteric neurons and spinal afferent fibres of the antrum, as well as the spinal afferent innervation of the\n\nremainder of the stomach. The intrinsic innervation of the proximal stomach is unaffected. Consequently there is a loss of innervation of the antral musculature and antral mucosal endocrine cells. The primary aim of developing such a model of complete antral denervation was to study the effects that this would have on gastric motility and antral endocrine cell function. The results of these studies are presented in the next two chapters.\n\nChapter Four\n\nAntral denervation and Gastric Emptying\n\nFor many years, antro-pyloric motility has been recognised to be an important factor controlling the rate of gastric emptying (Canon 1911). More recently, the pulsatile nature of antral contractions and the resting pressure of the pyloric sphincter have been shown to be significant factors governing the delivery of gastric content to the duodenum (Houghton et al., 1988; Malbert and Ruckebusch, 1991; Prather et al., 1993; Dent et al., 1994; Mayer et al., 1994). The likely role of the intrinsic antral innervation in determining the pattern of antral motor contractions has received little attention. This has been primarily because of the lack of available methodology to selectively lesion only antral neurons.\n\nIn contrast the extrinsic innervation of the stomach has been more amenable to study but have been shown to have little long term effects on the pattern of antro-pyloric motility (Quigley and Louckes, 1951; Stoddard et al., 1973). Indeed the majority of studies on the extrinsic reflex control of gastric emptying have concentrated on duodeno-gastric reflexes that regulate gastric emptying by effecting changes in gastric corpus tone either through vagally mediated (Raybould and Tache, 1988; Raybould et al., 1987; Holzer et al., 1994) or spinal afferent pathways (Forster et al., 1990; Forster et al., 1991).\n\nHowever, BAC has been shown to lesion myenteric neurons in a defined segment of rat jejunum and disrupt motor patterns that are dependent only upon an intact intrinsic innervation (Holle and Forth, 1990). In this chapter the data presented are from experiments that have used BAC to lesion the antral innervation to determine the role of antral innervation on the control of gastric emptying. Initially the retention of solid food was assessed and compared to the effects of the lesion produced by antral application of capsaicin. Finally the gastric emptying of a variety of liquid test meals was determined in control and BAC treated rats. The results demonstrate the requirement for an intact antral innervation for normal gastric emptying.\n\nMethods\n\nAntrally denervated and capsaicin treated rats were prepared and after each experiment denervation confirmed by radioimmunoassay of appropriate neuropeptide markers (see methods and chapter 3). Gastric emptying was determined either by studies of the retention of solid material in the stomach of fasted rats or by the determination of the emptying of liquids instilled into the stomach of rats with a gastric cannula. Gastric cannulas were placed at the time of antral denervation and all rats were allowed at least 11 days to recover after surgery.\n\nFor the determination of gastric emptying of solids, rats were fasted for 48 hrs on wire bottomed cages, killed by cervical dislocation and the stomach content weighed wet and again after prolonged drying at $50^0C$. For the determination of gastric emptying of liquids in gastric fistula rats, the stomach was washed extensively with warm 0.14M NaCl to remove solids prior to experiments and then the emptying of test solutions (saline, HCl, peptone: containing phenol red, 60mg.l$^{-1}$) instilled into the stomach via the gastric fistula was determined.\n\nResults\n\nIn fasted rats previously treated with 0.5% BAC on the antrum, there was a striking retention of solids in the stomach compared to controls (Fig 4.1). The wet and dry weight of gastric content was variable in BAC treated rats but all 0.5% BAC-treated rats exhibited overtly distended stomachs (by solid, gas or liquid) after a 48 hr fast. The retention of solid was not apparent after treatment with lower concentrations of BAC (Fig 4.1). However with 0.5% BAC the retention of solids was seen at all time points examined (Fig 4.2). In 48 hr fasted BAC treated rats that were allowed to refeed for 30 minutes, the dry weight of gastric content increased further (Fig 4.3). The dry weight of gastric content was similar to refed control rats.\n\nGiven that BAC causes loss of intrinsic and extrinsic afferent innervation whereas capsaicin lesions only extrinsic afferents (see chapter 3), the latter was used to determine whether the effects of BAC might simply reflect the loss of afferent innervation. Rats that had been pre-treated with capsaicin on the antrum did not exhibit gastric retention after fasting (Fig 4.4). To confirm the importance of the whole antrum in regulating gastric emptying the effects of denervation of a circumferential band of corpus and of one antral surface was also examined. Interestingly, these lesions had little effect on gastric retention of solid (Fig 4.4)\n\nThe emptying of several different types of test meal was significantly delayed in rats previously treated with 0.5% BAC on the antrum (Fig 4.5). It is worth noting\n\nthat in control rats, the emptying of HCl and peptone was inhibited compared with saline. However, in BAC-treated rats there was no significant difference in the emptying of saline, HCl and peptone, even though all of these emptied less rapidly than the corresponding rates in control animals.\n\nIt is known that in intact rats, prior administration of peptone just before an emptying trial with peptone (ie peptone plus preload) produces an exaggerated inhibition of emptying due to enhanced CCK release (Forster and Dockray, 1992). The same effect was observed in the present control rats; in addition, however, peptone plus preload produced a significant inhibition of emptying compared with saline in BAC-treated rats (Fig 4.5). This suggests that CCK might be mediating this effect in BAC-treated rats implying an intact vagally mediated duodeno-gastric reflex. To ensure that the gastric retention in BAC-treated rats was not primarily due to enhanced CCK release, plasma CCK was measured in fasted BAC and control rats. The concentration in control rats ($1.0 \\pm 0.1$ pM) was not significantly different from that in BAC-treated rats ($1.2 \\pm 0.1$ pM).\n\nFig 4.1 Gastric retention after a 48 hr fast in control rats (open bars, n=6) and in rats treated with increasing concentrations of BAC to the antrum (hatched bars, n=6). Only at the highest dose of 0.5% w/v BAC was there significant retention of solid in the stomach (*p < 0.05, Mann Whitney U test).\n\nFig 4.2. Gastric retention at various time points following antral BAC (hatched bars, n=6) or sham operation (open bars, n=6) and after a 48 hr fast. At all time points there was a significant retention of solid within the stomach only in rats treated with BAC to the antrum (*p < 0.05, Mann Whitney U test).\n\nFig 4.3 Gastric retention in control rats (open bars, n=6) and in rats treated with 0.5% w/v BAC to the antrum (hatched bars, n=6) after a 48 hr fast and following a 30 minute refeed. There was a significant retention of solid in the stomach of fasted BAC treated rats compared to controls but after refeeding the dry weight of gastric content was similar in both groups (*p < 0.05, Mann Whitney U test).\n\nFig 4.4 Gastric retention after a 48 hr fast in control rats and rats treated with BAC either to the whole of the antrum, or to the anterior surface of the antrum only, or to a band of corpus adjacent to the non-glandular stomach, or with capsaicin to the antrum.\n\nData shown are for wet weight (hatched bars) and dry weight (open bars). Only BAC treatment of the whole antrum leads to gastric retention (*p < 0.05, Mann Whitney U test).\n\nFig 4.5 Gastric emptying rates for different liquids in gastric fistula rats pre-treated with either 0.5% w/v BAC (n=8, hatched bars) or vehicle (n=6, open bars). For all liquids shown emptying rates are slower in BAC treated rats. In control rats, peptone and HCl empty more slowly than saline, and peptone after a peptone preload empties more slowly than peptone alone. In BAC treated rats, saline, HCl and peptone empty at the same rate, but peptone after a peptone preload induces further delay in gastric emptying.\n\nThe present data provides evidence that the integrity of the intrinsic innervation of the rat antrum is obligatory for the normal emptying of solids and liquids from the rat stomach. Specifically, it was found that the loss of intrinsic antral neurons caused by the detergent BAC resulted in retention of solids, and delayed emptying of both nutritive (peptone) and non-nutritive (saline, HCl) solutions. The retention of solids and liquids could not be attributed to changes in plasma CCK, which is the hormone best known for its ability to inhibit gastric emptying (Dockray 1988) since plasma CCK was normal. In a further series of experiments performed on antrally denervated gastric fistula rats, Dr B Yegen studied the effects of the specific CCK-antagonist (Green et al., 1988) on the emptying of liquid test meals. In these experiments there was no significant difference in the emptying rates of saline, HCl and peptone after L-364,718 in BAC-treated rats compared to BAC rats that did not receive the antagonist. Interestingly, however, the delayed emptying of peptone after a preload was reversed and was comparable to the emptying of saline. It would appear therefore that the action of CCK in controlling gastric emptying can be exerted independently of the antral innervation.\n\nThe application of BAC to the antrum lesioned both the intrinsic and extrinsic afferent innervation; however loss of the latter cannot explain the retention of solids, since there was no retention in rats treated with capsaicin which lesions only extrinsic afferent fibres. Taken as a whole, therefore, the present data lead to the conclusion that\n\nthe antral innervation is essential for the gastric emptying of solid and of both nutritive and non-nutritive solutions, but not for the delaying actions of CCK on gastric emptying.\n\nThe neuroendocrine mechanisms that determine gastric emptying have attracted increasing attention in recent years. In the case of CCK such studies have suggested a primary site of action at receptors on vagal afferent nerve fibres, the activation of which evokes a vago-vagal reflex leading to relaxation of the gastric corpus (Raybould and Tache, 1988; Tache et al., 1988; Forster et al., 1990). The possible involvement of the distal stomach in mediating the effect of CCK on gastric emptying has been suggested by previous studies (Yamagishi and Debas, 1978) but little has been done to determine whether - in this case - CCK acts directly on smooth muscle or indirectly via the innervation. The findings here suggest that any effects of CCK on the innervation of the antro-pyloric region are not of central importance in its role as an inhibitor of gastric emptying.\n\nThe effect of antral denervation on emptying can be considered specific since denervation of a fully circumferential band of proximal corpus (which would also lesion vagal efferent fibres) did not lead to gastric retention. In part the retention of solids after antral denervation may be attributable to a decreased capacity of the antrum for trituration and therefore the reduction of solids to a form suitable for emptying. In addition, however, there is also plainly a marked delay in emptying of liquids. This effect is distinguishable from the reduced emptying of liquids seen after\n\nlesioning of afferent neurons by capsaicin (Forster et al., 1990; Forster et al., 1991) since it is only in antrally denervated rats that there is a substantial delay in the emptying of isosmotic saline.\n\nThe pumping action of the antrum is well known, and contributes to the episodic delivery of gastric contents to the duodenum (Malbert and Ruckebusch, 1991; Dent et al., 1994). We suggest that the co-ordination of this activity requires intrinsic antral neurons and possibly intact efferent vagal neurons and that in the absence of these neurons the antrum generates an unremitting high-resistance to the passage of both liquids and solids. In view of the non-specific effect of BAC in lesioning all neuron populations within the area of exposure (excluding the submucosal neurons) it is not possible to speculate which if any neuron population is essential to maintain normal gastric emptying. It is interesting to note however that targeted disruption of the neuronal nitric oxide synthase gene leads to a phenotype essentially indistinguishable from the BAC treated antrally denervated rat (Huang et al., 1993). NO has been shown to mediate the nonadrenergic, noncholinergic smooth muscle relaxation throughout the gastrointestinal tract (Boeckxstaens et al., 1991) including relaxation of the gastric corpus (Desai et al., 1991). In the NOS knockout mouse, the presumed loss of smooth muscle relaxation in the region of the antrum and pylorus had similar effects to those documented here.\n\nA further possibility that may explain the high pressure resistance in the BAC treated antrum is that the BAC treatment has a direct effect on the muscle itself. It has\n\nbeen apparent from early studies with BAC that following denervation of the small intestine there is significant thickening of both longitudinal and circular muscle layers of the intestine (Fox et al., 1983; See et al., 1988). In addition the muscle became spontaneously contractile with altered mechanical properties including the ability to generate three times the active stress of control smooth muscle in response to muscarinic agonists (Osinski and Bass, 1994). This was later shown to be a consequence of an inability to resequester Ca$^{2+}$ in the sarcoplasmic reticulum leading to increased intracellular calcium (Osinski and Bass, 1995). Although it has been impossible to distinguish between the direct effect of BAC and the secondary effects of loss of normal innervation on smooth muscle function, Hadzijahic et al. (1993) have compared different methods of inducing myenteric denervation (Surgical vs BAC) and inflammation (intraluminal stasis and 5% acetic acid) to determine the underlying stimulus to the increased muscle thickness. Their results together with the observation of muscle thickening in the NOS knockout mice indicate that the changes in muscle thickness at least are a consequence of denervation and not a direct effect of BAC on smooth muscle.\n\n**Summary**\n\nAntral BAC produces selective lesions of defined populations of neurons in the stomach and leads to dramatic changes in gastric emptying. After antral BAC treatment there was retention of solids in rats fasted for 48 hr. Moreover, in antrally denervated rats with a gastric fistula, the emptying of saline, acid and peptone was substantially delayed. The cholecystokinin-dependent inhibition of gastric emptying\n\nof peptone was preserved after antral BAC. Hence it appears that the innervation of the antrum is essential for normal emptying of both liquids and solids, but the inhibition of gastric emptying produced by cholecystokinin is not dependent on antral neurons.\n\nChapter Five\n\nAntral denervation and G-cell Function\n\nIntroduction\n\nThe release of the antral hormone gastrin is controlled by the luminal contents of the stomach and by neurohumoral agents acting at the basolateral membrane of the G-cell. The main luminal stimulus is the presence of protein and protein-digestion products while the main luminal inhibitory factor is gastric acid (Dockray & Gregory, 1989; Walsh, 1994). The available evidence suggests that gastric acid releases somatostatin from antral D-cells which in turn inhibits gastrin release by a paracrine mechanism (Saffouri et al., 1980; Chiba et al., 1980; Walsh, 1994). The mechanisms by which luminal nutrients stimulate the G-cell are less certain. The luminal surface of the G-cell consists of tufts of microvilli that project into the lumen and this has led to the idea that luminal nutrients might act directly on the cell (Lichtenberger et al., 1982; Delvalle & Yamada, 1990; Lichtenberger, 1982). However, it is also clear that luminal stimulation of the G-cell can be indirectly mediated by nervous pathways (Saffouri et al., 1984; Schubert et al., 1992).\n\nIt is well recognised that vagal efferent neurons stimulate gastrin secretion in the cephalic phase of digestion and in addition, there are local intrinsic nervous reflexes controlling the G-cell. There is evidence for cholinergic-muscarinic stimulation of the G-cell, particularly in the rat (Schubert & Makhlouf, 1993; Schubert et al., 1992; Saffouri et al., 1984), but in dog and man atropine typically has no effect on nerve-mediated gastrin release, or stimulates it - indicating the importance of non-cholinergic inputs to the G-cell (Dockray & Tracy, 1980; Feldman et al., 1979;\n\nSchiller *et al.*, 1980; Farooq & Walsh, 1975). The neuropeptide gastrin releasing peptide (GRP) which is well represented in antral mucosal nerve fibres (Dockray *et al.*, 1979), is thought to be the main non-cholinergic neurotransmitter stimulating the G-cell. In keeping with this, both antibodies and more recently selective antagonists to GRP have been shown to inhibit gastrin secretion in response to either luminal peptone, or electrical field stimulation of the antrum or vagal nerve stimulation (Schubert, *et al.*, 1985; Holst, *et al.*, 1990; Schubert, *et al.*, 1992). The experiments described in this chapter were performed in an attempt to define the relative importance of neural mechanisms compared to the direct stimulation by luminal factors on gastrin release. To this end I have studied the luminal factors that influence gastrin release in control and antrally denervated rats. The results indicate increased sensitivity to non-nutrient distension after antral denervation and suggest the existence of an antral inhibitory innervation in the rat that normally suppresses G-cell responses to non-nutrient distension.\n\nMethods\n\nAntrally denervated rats were prepared and after each experiment denervation confirmed by radioimmunoassay of appropriate neuropeptide markers in tissue extracts of antral muscle (see Methods). Gastric cannulas were placed at the time of antral denervation and all rats were allowed at least 11 days to recover after surgery. For the refeeding experiments, rats were fasted for 48 hrs on wire-bottomed cages and were then allowed to refeed for 30 minutes. The GRP antagonist (BW2258U89) {Moody et al Life Sci 56: 521-529} (2mg.kg$^{-1}$ in 0.5ml of 0.14M saline) or saline, were injected subcutaneously 30mins before refeeding and then trunk blood was taken for assay of plasma gastrin. Samples of antral and corpus mucosa were taken for RNA extraction for analysis of gastrin, somatostatin, HDC and GAPDH mRNA abundance by Northern blot.\n\nGastric fistula rats:\n\nAs before, (chapter 4) the stomach was washed with warm 0.14M NaCl to remove solids at least one hour prior to experiments. Gastric acid output was determined by collecting gastric secretion over four sequential 15 min periods and titrating to pH 7.0 with 20mM NaOH using a pH autotitrator. Plasma gastrin responses were determined after gastric distension with the following test solutions: 2% (w/v) methyl cellulose (2%MC); 2%MC at pH6 (50mM ammonium acetate); 2% MC with 50mM HCl; 4.5% w/v peptone. In each case the stomach was distended by connecting the cannula to a reservoir of test solution and elevating the reservoir to 5cm H$_2$O pressure for 30mins.\n\nResults\n\nBasal plasma gastrin\n\nBasal plasma gastrin concentrations were similar in control, fasted rats (7.6 ± 1 pM; Fig5.1) and control rats with a gastric fistula (11.8 ± 2 pM; Fig5.4). In BAC treated rats fasted for 48hrs plasma gastrin was significantly elevated (28.4 ± 7pM, p<0.05 vs control; Fig 5.1). In these animals there was also marked gastric retention compared to controls (see Chapter 4). It seemed likely that gastric retention in BAC-treated rats accounted for the increased plasma gastrin, since in fasted BAC-treated rats with a gastric fistula, plasma gastrin was similar to control (13.5 ± 3.2pM; Fig 5.4) when the gastric contents were removed by lavage via the cannula.\n\nRefeeding experiments\n\nWhen control rats were fed for 30min after fasting for 48hr, plasma gastrin increased approximately 3-fold. There was a comparable increase in plasma gastrin in BAC-treated rats but the absolute increase after refeeding was higher than that in control rats, due to the elevation of basal plasma gastrin concentration (Fig 5.1). In keeping with the idea that GRP mediates food-stimulated release of gastrin in the rat, I found that the GRP antagonist BW2258U89 (0.5 mg.kg⁻¹) inhibited the rise in plasma gastrin in control rats seen after refeeding for 30min following a 48 hr fast. In contrast, in BAC-treated rats, the post-prandial rise in gastrin was not dependent on GRP since there was no significant difference compared with BAC-treated rats that received vehicle in place of the GRP antagonist (Fig 5.2).\n\nFigure 5.1 Plasma gastrin in fasted and refed rats 11 days after either sham operation (open bars) or antral denervation by 0.5% BAC (hatched bars). In fasted BAC-treated rats there was elevated fasting plasma gastrin. After refeeding, plasma gastrin increased approximately three-fold in both control and BAC-treated rats. (n=>6 for each group, * p<0.05 vs control response, Mann Whitney U test).\n\nFigure 5.2 Plasma gastrin concentrations in control and antrally-denervated rats after administration of the specific GRP antagonist BW225U86 (hatched bars) or saline (open bars) 30mins prior to refeeding. In control rats, but not antrally denervated rats, the GRP antagonist significantly attenuated the post-prandial rise in plasma gastrin. (n=6 for all groups, * p<0.05 Mann Whitney U test).\n\nBasal acid output:\n\nUnder normal circumstances gastrin release is inhibited by luminal acid so that one potential explanation for the elevated plasma gastrin in BAC-treated rats was that BAC treatment of the antrum influenced acid secretion. In fact the range of basal acid output in BAC-treated rats overlapped with that in controls but the mean was approximately 3-fold lower (Fig 5.3). Complete antral denervation was required to reduce mean basal acid output as rats that received BAC only to the anterior surface of the stomach had a mean basal acid output comparable with controls.\n\nDistension experiments\n\nIn control rats distension with the non-nutritive solution 2% MC had no effect on plasma gastrin. In contrast, distension of BAC-treated rats with 2% MC produced a significant rise in plasma gastrin (Fig 5.4). Although this appeared to substantiate the view that distension stimulates gastrin release in antrally denervated rats, there was also the possibility that alterations in pH may play a part in the response given the reduced acid output in BAC-treated rats. Therefore distension experiments were performed with buffered 2% MC (acidified and pH6) and with peptone as a positive control. In control rats distension with 4.5% w/v peptone increased plasma gastrin 3-fold but distension with the non-nutritive solutions 2 % methylcellulose (2%MC), or 2%MC pH 6 or 2%MC with 50 mM HCl did not increase plasma gastrin (Fig 5.5).\n\nIn BAC-treated rats, the effects of distension with peptone and 2%MC with 50mM HCl in methylcellulose were similar to control. However distension by either 2%MC or 2%MC pH 6.0 did stimulate gastrin release, the increase in plasma gastrin being comparable to that produced by peptone (Fig 5.5).\n\nFigure 5.3 Mean basal acid output (mmol.15min$^{-1}$) in control and antrally denervated rats. Two groups of denervated rats were studied: either total antral denervation, or denervation of the anterior surface only (hemi-BAC). Each data point represents the mean acid output of four sequential 15min collections from one rat. Only in the rats with complete antral denervation was there a significant reduction in basal acid output. (* p<0.05 ANOVA).\n\nFigure 5.4 Plasma gastrin responses in control and BAC-treated rats after gastric lavage to remove residual gastric content. Rats were either left undistended (open bars) or were distended with the non-nutritive solution 2%MC for 30 minutes at a pressure of 5cm of H$_2$O (hatched bars). In BAC-treated rats but not controls, plasma gastrin was increased by distension with 2%MC. (n=6 or more for each group, * p<0.05 vs control response).\n\nFigure 5.5 Plasma gastrin responses in control (open bars) and BAC-treated rats (hatched bars). Rats were either left undistended or distended for 30 mins with either 2%MC (buffered with 50mM HCL or to pH6) or 4.5% w/v peptone. Plasma gastrin after distension with 4.5% peptone was similar in control and antrally-denervated gastric fistula rats. In BAC-treated rats, but not controls, plasma gastrin was also increased by distension with 2%MC buffered to pH6.0. Neither group showed an increase in plasma gastrin after distension with 2%MC with 50mmol HCl. (n=6 or more for each group, * p<0.05 vs control response, Mann Whitney U test).\n\nGastrin, somatostatin HDC and GAPDH mRNA abundance\n\nIt is known that gastric endocrine cell responses frequently involve altered changes in the synthesis of active products, as seen by changes in mRNA abundance (Dockray et al. 1996). To determine whether antral denervation might alter the synthesis of regulatory molecules in gastric endocrine cells Northern blots of mRNA’s encoding gastrin, somatostatin, HDC and GAPDH were analysed (Figures 5.6 & 5.7). There was no difference in the expression of GAPDH between control and BAC treated rats. In the antrum there was a significant reduction in antral mucosal somatostatin mRNA abundance in BAC-treated rats compared with controls and a similar decrease in gastrin mRNA abundance. In the corpus however, somatostatin mRNA abundance was unchanged in BAC-treated rats compared with controls, while HDC mRNA was variably increased. The latter is compatible with the sustained increase in plasma gastrin in these animals.\n\nFigure 5.6 Representative Northern blots of total antral mRNA hybridised with specific cRNA probes for gastrin (upper panel) and somatostatin (lower panel). Lanes 1-6 are control and lanes 7-12 BAC-treated rats. BAC treatment significantly lowered gastrin and somatostatin mRNA abundance. These changes were considered specific as BAC treatment had no effect on GAPDH mRNA abundance (results not shown).\n\nFig 5.7 Relative mRNA abundance in the antrum of GAPDH, gastrin and somatostatin, and in the corpus of GAPDH, somatostatin and HDC in control (open bars) and BAC-treated rats (hatched bars). The relative abundance of each mRNA species has been normalised by taking as 100% the abundance in controls. Note the abundance of the non-regulated marker GAPDH was similar in control and BAC treated rats. In the antrum BAC decreased gastrin and somatostatin mRNA (n=6 in each group; * p<0.05, Students t test).\n\nDiscussion\n\nIn previous studies that have examined the neuronal regulation of G-cell function the extrinsic innervation has been manipulated by stimulation, lesioning or pharmacological blockade (Dockray & Gregory, 1989; Walsh, 1994); the role of intrinsic neurons has been largely determined by pharmacological approaches using neurotransmitter antagonists or antibodies (Schubert et al., 1985; Schubert et al., 1992). In the present experiments antral denervation by BAC was used to lesion both the intrinsic and extrinsic innervation of the rat antrum. The subsequent experimental data support the conclusions that the post-prandial gastrin response in control rats is largely mediated by the neuropeptide transmitter GRP; in antrally denervated rats, a post-prandial gastrin response is maintained but this is no longer inhibited by a GRP antagonist. Instead, it appears that non-nutritive distension becomes a strong stimulus for the denervated G-cell and this explains for the most part the fasting and meal stimulated hypergastrinaemia seen in the antrally denervated rat. The results are unexpected given that it is generally thought that G-cell responses to distension are mediated by nervous reflexes (Dockray & Gregory, 1989; Walsh, 1994; Schubert & Makhlouf, 1993).\n\nSince gastrin release is inhibited by luminal acid, one potential explanation for the elevated plasma gastrin in antrally denervated rats might be reduced acid output. In fact there was a decrease in basal acid secretion after denervation. However, the capacity of the denervated G-cell to respond to distension was increased even when intragastric pH was buffered at pH 6.0 suggesting that G-cell responses secondary to\n\nchanges in acid output are unlikely to be important in the response to denervation. Furthermore, in undistended BAC-treated rats with a gastric fistula plasma gastrin was comparable to control rats suggesting that basal acid output was sufficient to maintain inhibition of gastrin release. In man, gastric distension is a good stimulus to acid secretion (Soares et al., 1977), but in the conscious rat, it has been reported that non-nutritive distension of the stomach inhibits acid secretion by activating a sympathetic reflex (Dimaline et al., 1986). Hence the chronic gastric retention after antral denervation may lead to inhibition of acid output by this mechanism.\n\nIn support of this argument, it is clear that after lesioning the antral innervation there was a pronounced gastric retention of solids, even after 48hr fasting. After complete emptying of the stomach in antrally denervated rats fitted with a gastric fistula, basal plasma gastrin was similar to control levels, suggesting that the hypergastrinaemia in denervated rats without a gastric cannula was attributable to the gastric retention.\n\nStudies in the isolated perfused rat stomach have provided evidence that GRP mediates the release of gastrin in response to luminal peptone (Schubert et al., 1992). The observation that the post-prandial increase in plasma gastrin concentration was blocked by a GRP antagonist suggests that a similar mechanism operates in conscious rats *in vivo*. However, the post-prandial increase in gastrin in antrally denervated rats was not blocked by a GRP antagonist suggesting that an additional mechanism comes into play after denervation. Interestingly, the gastrin response to gastric distension\n\nwith peptone in antrally denervated rats was no greater than that to non-nutritive distension. Two inferences may be drawn from this: First, that the stimulatory effect of nutrient is normally mediated by antral neurons, and second that antral neurons normally inhibit G-cell responses to non-nutrient distension. Studies in the isolated perfused rat stomach have shown that antral neurons mediate stimulation of gastrin release after distension at low pressures, and inhibition at high pressures (Schubert & Makhlouf, 1993). The latter may explain why methylcellulose at pH6 did not stimulate gastrin release in the intact stomach. The novel finding in the present experimental series is that there exists a mechanism for distension-evoked release of gastrin which is independent of the antral innervation.\n\nIt is generally thought that gastrointestinal responses to mechanical distension are mediated by nervous reflexes. Two possible mechanisms might account for the unexpected observation that the denervated G-cell responds to non-nutritive distension. First, the G-cell or a nearby cell in the antral mucosa might be mechanosensitive (which is suppressed by the antral innervation), or develops such sensitivity after denervation. In this context it has been recognised for many years that enterochromaffin cells (EC) cells are mechano-sensitive (Bulbring and Lin, 1958; Kirchgessner et al., 1992); denervated G-cells might therefore take on EC-cell like properties, or alternatively antral EC cells might regulate G-cell function for example through a paracrine mechanism. Second, non-nutritive distension of the stomach could release an endocrine factor from the gastric corpus, which increases gastrin release.\n\nfrom the denervated, but not innervated, antrum. In the dog, Debas *et al.* (1975) showed that distension of the corpus increased gastrin release from an extrinsically denervated antral pouch suggesting the existence of an oxynto-pyloric endocrine reflex. The existence of a similar reflex in the rat might explain the present findings. If so, however, it would appear that such a reflex is not normally activated in the innervated antrum.\n\nHypergastrinaemia stimulates the enterochromaffin-like (ECL) cells of the gastric corpus which are an important source of histamine. The increased HDC mRNA abundance observed in BAC-treated rats is compatible with the hypergastrinaemia of antral denervation. Increased histamine synthesis and secretion might be expected to stimulate increased acid output, and the reduced acid secretion observed would therefore appear not to be secondary to inhibition at the level of the ECL cell. In the antrum we found marginally reduced gastrin and somatostatin mRNA abundance. Gastrin synthesis is regulated both at the level of gene transcription and at the level of mRNA translation (Bate *et al.*, 1996). The present data raise the possibility that after denervation there is independent control of release of synthesis at a post-transcriptional level. Furthermore, given that somatostatin and gastrin mRNA abundance appeared to be reduced following denervation without an overall depression in gene transcription (indicated by similar abundance of GAPDH in control and BAC treated rats) it seems possible that the innervation of the antral mucosa normally may regulate transcription of specific genes in target endocrine cells. Taken together with the possibility that following denervation G-cells may themselves\n\ntake on the EC-cell property of mechanosensitivity, it would appear that antral neurons play some role in the maintenance of the normal differentiated state of the G-cell.\n\nUnfortunately the factors that regulate the differentiation of G-cells have proved difficult to study not least because of the problems of generating G-cell lines. However the mechanisms of differentiation in endocrine cells per se have been studied in pancreatic islet cells. Interestingly, gastrin is expressed in the foetal pancreas at the time of endocrine cell development (Brand and Fuller, 1988) and there is good evidence to suggest that gastrin acts in conjunction with TGF-α to promote normal pancreatic endocrine cell differentiation (Wang et al., 1993). In adult rats, following partial pancreatectomy, pancreatic islet cell regeneration occurs and is associated with expression of the Reg gene (reg) (Terazono et al., 1988) implying that reg expression may be involved in differentiation. The latter is of interest because the reg gene has been shown recently to be present in the gastric ECL cell (Ashahara et al., 1996), a cell whose gene expression is well known to be regulated by gastrin. In addition reg expression is increased during the regeneration phase following acute gastric mucosal injury (Ashahra et al., 1996).\n\nIt is plausible to suppose therefore that the remodelling of gastric mucosal function after denervation involves gastrin itself, acting in conjunction with other factors to regulate differentiation of endocrine cells, including the ECL cell. The\n\nstudies detailed in the next chapter begin to investigate the possibility that gastrin regulates *reg* expression.\n\n**Summary**\n\nIn the intact rat, luminal nutrient releases gastrin via activation of neurons secreting GRP whilst the antral innervation normally inhibits gastrin release in response to non-nutrient distension. After antral denervation, post-prandial gastrin release is maintained but by a mechanism independent of neurons secreting GRP. Moreover, gastric distension with a non-nutrient solution becomes an adequate stimulus for gastrin release. Despite reductions in gastrin and somatostatin mRNA abundance following BAC treatment, hypergastrinaemia occurred and produced changes in ECL-cell gene expression although basal acid output was reduced. The altered G-cell function that occurred after denervation and the possibility that this effected changes in gastric endocrine cell differentiation remains to be determined.\n\nChapter Six\n\nControl of reg expression in rat corpus mucosa and AR42J cells\n\nThe work of several laboratories indicates that the *reg* gene is expressed during pancreatic or gastric mucosal regeneration (Terazono et al., 1988; Miyaura et al., 1991; Ashahara et al., 1996). Since *reg* is expressed in pancreatic β cells and gastric ECL cells it is reasonable to suppose that it may be implicated in endocrine cell growth or differentiation. Gastrin is known to stimulate growth of ECL cells both in the human (Borch et al., 1985; Bordi et al., 1986; Roucayrol and Cattan, 1989; Solcia et al., 1991) and the rat (Havu 1986; Hakanson and Sundler, 1990) and is essential for normal gastric (Koh et al., 1997) and pancreatic (Wang et al., 1993) endocrine cell development. In the latter case, gastrin must act in conjunction with growth factors for normal differentiation to occur. However, the possible interactions between gastrin, growth factors and *reg* expression have not been directly studied.\n\nAR42J cells are an amphicrine cell line with the capability to differentiate into either acinar or endocrine cells depending on the tissue culture conditions (Christophe, 1994). These cells express both the gastrin/CCK-B receptor and the *reg* gene (Lambert et al., 1991; Zenilman et al., 1996), suggesting that they might be a useful system in which to investigate the relationships outlined above. The experiments performed in this chapter were undertaken first to determine whether gastrin might regulate *reg* gene expression in rat gastric corpus and second to study in the AR42J cell line some of the features of gastrin-induced *reg* expression. The results indicate that *reg* expression is regulated by gastrin acting via the CCK-B receptor. In addition,\n\nalthough EGF potentiated gastrin-stimulated *reg* expression the reported data suggest that *reg* expression is associated with endocrine cell differentiation and not growth.\n\n**Methods**\n\n**Animal Studies**\n\nRats were either fasted for 48 hrs on wire-bottomed cages or fed ad libitum and given omeprazole (400μmol.kg$^{-1}$) by gavage for 5 days. Rats fed *ad libitum* were used as controls. At the end of treatment, trunk blood was taken for assay of plasma gastrin and samples of corpus mucosa were taken for RNA extraction and analysis of HDC and *reg* mRNA abundance by Northern blot.\n\n**Tissue Culture**\n\nCulture of AR42J cells and treatment with amidated and gly-extended gastrin, L740093 (1nM), EGF (10pM-1μM) and wortmannin (5 x 10$^{-8}$M) was performed by Dr. LA Bishop. Cells were cultured at a density of 1x10$^6$ for 24 hrs in depleted media before appropriate treatment. All treatments were given for 24 hrs except for wortmannin (4 hrs). At the end of the culture period total cell mRNA was extracted by the lithium chloride precipitation method and *reg* mRNA abundance analysed by Northern blot (see Chapter 2).\n\nResults\n\nReg expression in rat gastric corpus\n\nIn keeping with previously published observations (Dimaline et al., 1993; Swarovsky et al., 1994), treatment of Wistar rats with omeprazole (400μmol.kg\\(^{-1}\\)) for five days lead to a five to ten-fold increase in plasma gastrin and an associated three-fold increase in HDC mRNA in gastric corpus mucosa, compared to rats fed ad libitum. Conversely, fasting significantly reduced plasma gastrin and HDC mRNA abundance (Fig 6.1). In addition however, omeprazole-induced hypergastrinaemia also resulted in altered reg gene expression in the gastric mucosa, the changes in magnitude of reg mRNA abundance being almost identical to those of HDC (Fig 6.1).\n\nReg expression in AR42J cells\n\nTo examine in more detail how gastrin might regulate reg gene expression, AR42J cells were cultured in the presence of increasing concentrations of both amidated (G17-NH\\(_2\\)) and glycine-extended gastrin (G-gly). Amidated and glycine-extended gastrin both produced dose-dependent increases in reg expression with G-gly being approximately 1000-fold less potent than its amidated counterpart (Fig 6.2 & 6.3). To determine the relevant gastrin receptor mediating the reg response, AR42J cells were treated with either G17-NH\\(_2\\) (10\\(^{-8}\\) M) or G-gly (10\\(^{-6}\\) M) in the presence of the specific CCK-B receptor antagonist L-740093. The increase in reg mRNA abundance stimulated by G17-NH\\(_2\\) was completely inhibited by the CCK-B receptor antagonist (Fig 6.4). The response to G-gly, although significantly attenuated by L-740093, remained increased by approximately two-fold (Fig 6.5).\n\nFigure 6.1 **upper panel**: Effect of fasting and omeprazole treatment on plasma gastrin (open bars), *reg* mRNA (hatched bars) and HDC mRNA abundance (stippled bars). Note *reg* mRNA abundance was decreased by fasting and increased by omeprazole treatment, the magnitude of the change paralleling that of HDC mRNA. (*n=6 for each group, * p<0.05 vs control response, Mann Whitney U test). **lower panel**: Representative Northern blot of *reg* mRNA abundance in 48 hr fasted (lanes 1-4), fed ad lib. (lanes 5-8) and omeprazole treated rats (lanes 9-12).\n\nFigure 6.2 Representative Northern blots for *reg* mRNA abundance in total RNA extracted from duplicate cultures of AR42J cells treated with increasing molar concentrations of either G17-NH$_2$ (upper panel) or G-gly (lower panel). Note the dose range of the response is approximately 3 log units higher for G-gly.\n\nFigure 6.3 Dose response relationship between G17-NH$_2$ (closed circles) and G-gly (open circles) and $reg$ mRNA abundance in AR42J cells. The $A_{50}$ for G17-NH$_2$ was approximately $10^{-8}$M and that for G-gly approximately $5 \\times 10^{-5}$M.\n\nFigure 6.4 Effect of $10^{-9}$M L-740093 (hatched bars) on the *reg* mRNA response to $10^{-8}$M G17-NH$_2$ (upper panel) and a representative Northern blot (lower panel) of *reg* mRNA abundance in total RNA extracted from duplicate cultures of AR42J cells treated with vehicle (ammonium bicarbonate; lanes 1&2), L-740093 (lanes 3&4), G17-NH$_2$ (lanes 5&6) and G17-NH$_2$ and L-740093 (lanes 7&8). The twenty five-fold increase in mRNA abundance produced by G17-NH$_2$ was completely reversed by L-740093.\n\nFigure 6.5 Effect of $10^{-9}$M L-740093 (hatched bars) on the *reg* mRNA response to $10^{-6}$M G-gly (upper panel) and a representative Northern blot (lower panel) of *reg* mRNA abundance in total RNA extracted from cultures of AR42J cells treated with ambic (lanes 1&2) G-gly (lanes 3&4) and G-gly and L-740093 (lanes 5-7). Although the response to G-gly was significantly attenuated by L-740093, *reg* mRNA abundance remained increased by approximately two-fold.\n\nEGF and *Reg* expression in AR42J cells\n\nThe effects of EGF on *reg* expression is shown in Fig 6.6. At the highest dose of EGF ($10^{-6}$M) there is no significant upregulation of *reg* expression even though this concentration of EGF has been shown to induce proliferation of AR42J cells. However, EGF, at a concentration of $10^{-9}$M potentiated the effects of amidated and glycine-extended gastrin on *reg* expression (Fig 6.7). Hence the ten to twenty-fold increase in *reg* expression induced by $10^{-8}$M G17-NH$_2$ was further enhanced ten-fold by EGF ($10^{-9}$M).\n\nWortmannin and *Reg* expression in AR42J cells\n\nInhibition of the PI3 kinase in cultured human foetal pancreatic cells has been shown to induce differentiation towards an endocrine cell phenotype (Ptasznik et al., 1997). Treatment of AR42J cells with the PI3 kinase inhibitor wortmannin ($5 \\times 10^{-8}$ M) for four hours, increased *reg* expression in both unstimulated and G17-NH$_2$-stimulated AR42J cells (Fig 6.8).\n\nFigure 6.6 **upper panel**: Relationship between increasing doses of EGF and *reg* mRNA abundance in AR42J cells. Results are expressed relative to mRNA expression in control cultures arbitrarily defined as 5% of a predicted maximum response. Even at $10^{-6}$M EGF there is no increase in *reg* mRNA abundance.\n\n**lower panel**: Representative Northern blot of *reg* mRNA in duplicate cultures of AR42J cells exposed to increasing concentrations of EGF.\n\nFig 6.7 **upper panel**: relationship between G17-NH$_2$ (10$^{-8}$M) or G-gly (10$^{-7}$M), EGF (10$^{-9}$M) and *reg* mRNA abundance in AR42J cells. Results have been normalised to the response to 10$^{-8}$M G17-NH$_2$ taken as 100%. The response of AR42J cells to either G-gly or EGF alone was approximately 10% of the G17-NH$_2$ response. However, EGF increased the response of AR42J cells to G17-NH$_2$ by fifteen-fold and to G-gly by seven-fold.\n\n**lower panel**: Representative Northern blots for *reg* mRNA in duplicate cultures of AR42J cells treated with G17-NH$_2$ (lanes 1&2), G-gly (lanes 3&4), EGF (lanes 5&6), G17-NH$_2$ and EGF (lanes 7&8) and G-gly and EGF (lanes 9&10).\n\nFig 6.8 **upper panel**: relationship between G17-NH$_2$ (10$^{-8}$M), wortmannin (5 x 10$^{-8}$M) and *reg* mRNA abundance in AR42J cells. The gastrin-stimulated response in *reg* mRNA is further increased two-fold by wortmannin. Note that wortmannin also stimulates *reg* mRNA expression in control cultures. **lower panel**: Representative Northern blots for *reg* mRNA in duplicate cultures of AR42J cells treated with Ambic (lanes 1&2), wortmannin (lanes 3&4), G17-NH$_2$ (lanes 5&6) and G17-NH$_2$ and wortmannin (lanes 7&8).\n\nThe data presented in this chapter provide evidence that *reg* expression in rat corpus mucosa responds to changes in plasma gastrin and in AR42J cells is regulated by amidated gastrin acting through the gastrin/CCK-B receptor. In addition, whilst EGF alone had no effect on *reg* expression it markedly potentiated gastrin stimulated *reg* mRNA abundance. Furthermore, in culture conditions that have been shown to favour differentiation of AR42J cells into insulin-secreting endocrine cells, *reg* gene expression was increased.\n\nRegulation of ECL cell gene expression by plasma gastrin has been demonstrated for a variety of genes including HDC (Dimaline *et al.*, 1991; Swarovsky *et al.*, 1994;). The observed threefold increase in HDC mRNA abundance following 5 day omeprazole treatment was in keeping with previously reported data. The presence of *reg* in rat ECL cells has been reported only recently (Ashahara *et al.*, 1996) and the results presented here demonstrate for the first time that *reg* gene expression in vivo can be regulated by plasma gastrin.\n\nThe present results contrast with those of Zenilman *et al.*, (1997) who showed that gastrin, CCK and glucagon (all 10nM) had no effect on reg mRNA abundance in AR42J cells. However, the tissue culture protocol in the experiments reported by Zenilman did not incorporate 24 hrs of culture in serum depleted media prior to the period of treatment. AR42J cells have been reported to synthesise and secrete gastrin\n\nthat may then act in an autocrine manner (Blackmore and Hirst, 1992) and this may explain the discrepancy between the above findings and the lack of effect of gastrin seen by Zenilman and colleagues. An additional explanation of the results of Zenilman et al., is the influence of the cell density of AR42J cells on *reg* gene expression. In preliminary experiments it was found that *reg* gene expression was upregulated (approximately tenfold) as cell density reached confluence. Zenilman et al. conducted their experiments on confluent cells unlike the experiments reported here.\n\nThe gastrin stimulated *reg* expression in AR42J cells appears to act via the gastrin/CCK-B receptor known to be present on these cells (Lambert et al., 1991). The reported dissociation constant for gastrin at this receptor in AR42J cells is 4nM (Zhou et al., 1992). The $A_{50}$ of the *reg* response to G17-NH$_2$ was $\\approx 10$ nM and the complete inhibition of the response by the specific gastrin/CCK-B receptor antagonist L-740093 indicates that this response is mediated via the gastrin/CCK-B receptor. The *reg* response to G-gly also was inhibited by L-740093 but only by $\\approx 80\\%$. The dose of G-gly required to increase *reg* expression was much higher than that for G17-NH$_2$ and given that L-740093 is a competitive antagonist at this receptor it seems likely that the effects of G-gly on *reg* expression also are mediated via the gastrin/CCK-B receptor. The parallel rightward displacement of the G-gly dose-response curve compared to that of G17-NH$_2$ also suggested that G-gly was acting at the same receptor as G17-NH$_2$ but with lower affinity.\n\nPerhaps by coincidence, the magnitude of change in *reg* mRNA abundance in the rat gastric mucosa was identical almost to that of HDC. Given that gastrin regulates HDC gene expression via the gastrin/CCK-B receptor (Hollande et al., 1994), this might suggest a common intracellular pathway regulating HDC and *reg* gene expression. The rat and human HDC promoter has been shown to be regulated by a protein kinase C dependent pathway (Hocker et al., 1996; Zhang et al., 1996). For the human HDC gene a palindromic sequence (5'-CCCTTTAAATAAAGGG-3') that confers transcriptional responsiveness to gastrin has been identified and defined as a gastrin response element. A similar sequence is present within the rat HDC promoter region (5'-CCCTTAAATAAGAGGG-3'). In each case the putative response element lies within 25 base pairs of the transcriptional start site. The 5'-regulatory region of the rat *reg* gene has also been described in AR42J cells (Miyashita et al., 1994). The regulatory sequence of most significance indicates a region -256 to -237 upstream of the transcriptional start site that has significant sequence homology with the consensus sequence of the pancreatic exocrine enhancer (Boulet et al., 1986). Although this would suggest a separate regulatory mechanism for *reg* gene transcription in AR42J cells, the response to gastrin was not studied. However, there does not appear to be a sequence similar to the putative gastrin response element of the rat HDC gene within the reported promoter region of the rat *reg* gene. Nevertheless the data clearly indicate that gastrin regulates *reg* expression in AR42J cells via a gastrin/CCK-B receptor mediated pathway.\n\nOther effects of gastrin on AR42J cells that have been shown to depend on a gastrin/CCK-B receptor mediated pathway include stimulation of amylase secretion (Lambert et al., 1991) and stimulation of growth (Pradel et al., 1993; Seva et al., 1994; Zenilman et al., 1997). This raises the possibility that *reg* gene expression may be part of the growth response of AR42J cells to gastrin. This seems unlikely for two reasons: Firstly, the growth promoting effects of G17-NH$_2$ on AR42J cells is relatively weak compared to that of EGF (Watson et al., 1993). Despite this, the data above demonstrate that $10^{-6}$M EGF had no effect on *reg* gene expression although this dose would be expected to produce a large trophic response. This indicates that *reg* expression, although present in AR42J cells, is not required for cell growth. Secondly, treatment of AR42J cells with the PI3 kinase inhibitor wortmannin which has been shown to inhibit cell growth and promote endocrine cell differentiation (Ptasznick et al., 1997) was found to increase *reg* gene expression. The effect of wortmannin on AR42J cell *reg* expression was not confined to unstimulated cells as it also potentiated the effects of gastrin on *reg* expression.\n\nSeveral other lines of evidence indicate that *reg* gene expression is associated with differentiation towards an endocrine cell phenotype. Treatment of AR42J cells with dexamethasone has been shown to promote differentiation of an acinar cell phenotype and concurrently reduced *reg* expression (Zenilman et al., 1997). Conversely, treatment with activin A, betacellulin or HGF stimulates differentiation of AR42J cells towards an endocrine cell phenotype (Mashima et al., 1996a; Ptasznik et al., 1997; Mashima et al., 1996b) and HGF has been shown to increase *reg* expression.\n\nin human foetal pancreatic cells (Otonkoski et al., 1994). Taken together, the experimental evidence suggests that *reg* expression in AR42J cells is associated with differentiation towards an endocrine cell phenotype. If so, then this process would be expected to be favoured by treatment with gastrin and potentiated by either EGF or wortmannin. The data are of interest as AR42J cells may provide a useful model to elucidate the intracellular pathways that mediate these effects.\n\nThe interaction between growth factors, gastrin and the differentiation of endocrine cells has been described in the mouse pancreas (Wang et al., 1993). Here at least, both are required in conjunction for normal development of the endocrine pancreas. In the stomach, gastrin does not seem to be essential for normal ECL cell development as these cells can still be found in the gastric mucosa of gastrin knockout mice (Koh et al., 1997). However, in this model ECL cell numbers were significantly reduced implying that gastrin regulates the number of mature ECL cells in the stomach perhaps by influencing the growth and differentiation of precursor cells. There are no data as yet on *reg* expression in such experimental situations but the provisional data presented thus far indicate that *reg* expression may be a marker for ECL cell function and more specifically may play a role in the maintenance of a normal ECL population within the gastric mucosa. The clinical implications of this are the subject of the next chapter.\n\nSummary\n\nReg expression in rat gastric corpus was increased by 5 day omeprazole treatment. In AR42J cells reg expression was shown to be regulated by amidated gastrin acting through the CCK-B receptor. EGF alone had no effect on reg expression but markedly potentiated the gastrin stimulated response. In culture conditions that favour differentiation of AR42J cells into insulin-secreting endocrine cells, reg gene expression was increased. The observations of others clearly indicate that growth factors acting together with gastrin may regulate endocrine cell growth and differentiation in the gastrointestinal tract. AR42J cells may provide a useful model to elucidate the intracellular pathways that mediate these effects.\n\nChapter Seven\n\nReg gene expression in human gastric mucosa.\n\nIn rodents hypergastrinemia secondary to long-term treatment with H$_2$-receptor antagonists or proton pump inhibitors (PPI) may lead to gastric carcinoid tumours (Hava, 1986; Poynter and Selway, 1991; Wangberg et al., 1995). This has prompted extensive reviews of the natural history of these tumours in man (Solcia et al., 1991; Thomas et al., 1994; Rindi, 1995; Modlin et al., 1995). It is now clear that hypergastrinaemia from whatever cause may result in hyperplasia of the gastric ECL cell. Significant ECL-cell hyperplasia has been demonstrated in one third of patients rendered hypergastrinaemic by *H pylori* infection and coincident treatment with a PPI (Eiselle et al., 1997). The majority of patients with CAG display some degree of ECL cell hyperplasia, the achlorhydria of CAG leading to hypergastrinemia that is generally accepted to be a stimulus to ECL cell growth (Creutzfeld, 1988; Hakanson and Sundler, 1990; Bordi et al., 1995). In fact, chronic atrophic gastritis (CAG) with or without pernicious anaemia is the commonest cause of enterochromaffin-like (ECL-) cell hyperplasia and in 5-10% of patients there is a progression to gastric carcinoid tumours (Borch et al., 1985; Bordi et al., 1991; Bordi et al., 1995).\n\nThe recent demonstration of *reg* gene expression in rat ECL cells together with the evidence suggesting that the reg protein may be important for pancreatic endocrine cell growth and differentiation imply a possible role for *reg* in the control of\n\nECL cell growth and differentiation. Gastrin has been shown to be important for entero-pancreatic endocrine cell development (Wang et al., 1993; Koh et al., 1997), acting in concert with growth factors such as TGF-α. In keeping with these observations, the results presented in the previous chapter showed gastrin stimulation of *reg* expression, potentiation of this response by EGF and upregulation of *reg* mRNA in AR42J cells exposed to culture conditions that favour differentiation towards an endocrine cell phenotype. The experiments performed in this chapter attempt to determine whether *reg* expression in the human gastric corpus is affected by plasma gastrin concentration and whether *reg* may have a pathophysiological role in ECL cell tumour development in patients with chronic hypergastrinaemia.\n\nHuman studies\n\nThe details of the patients that provided samples for the experimental studies described in this chapter are shown in Table 7.1 Plasma gastrin was assayed by RIA using antibody L2. Endoscopic biopsies were taken from the gastric corpus and from any corpus nodules if present from hypergastrinaemic patients that required a gastroscopy. Duplicate biopsies were sent to Dr G Armstrong (Consultant Histopathologist, Hope Hospital) for Histology. Total mRNA was extracted from biopsies and reg mRNA analysed by Northern blot and RT/PCR, cloning and sequencing (see Chapter 2). For the Northern blots, reg mRNA is expressed relative to the non-regulated mRNA for GAPDH.\n\nOctreotide Suppression Test\n\nTwo of the patients with carcinoid tumours (JH & KD) consented to an octreotide suppression test. The intended study was explained and informed consent obtained from both patients. Endoscopic biopsies were taken from ECL cell nodules and apparently normal corpus prior to and at the end of a 72hr infusion of octreotide (25mg.hr\\(^{-1}\\)). Plasma samples were taken before, during and after the octreotide infusion for assay of gastrin.\n\n| PATIENT | AGE | SEX | DIAGNOSIS | PLASMA GASTRIN (pM) | ECL Cell Polyps | Polyp size |\n|---------|-----|-----|------------------------------------------------|---------------------|-----------------|------------|\n| J.H. | 44 | F | P.A. Nodules | 850 | >50 | >1cm |\n| K.D. | 42 | M | P.A. Nodules | 600 | 2 | 1cm + <5mm |\n| K.W. | 72 | F | P.A. Nodules | 1200 | >20 | <1cm |\n| P.S. | 47 | M | Chronic Renal Failure Diabetes Mellitus | 650 | <10 | <5mm |\n| M.B. | 56 | F | Z.E./MEN1 | 280 | <10 | <5mm |\n| P.G. | 65 | F | P.A. | 450 | <10 | <5mm |\n| P.M. | 61 | F | P.A. | 400 | <10 | <5mm |\n| W.S. | 72 | M | P.A. | 480 | <10 | <5mm |\n| J.C. | 47 | M | Z.E. | 250 | Nil | |\n| T.O. | 72 | M | P.A. | 570 | Nil | |\n| D.K. | 46 | M | P.A. | 440 | Nil | |\n| H.W. | 75 | F | P.A. | 180 | Nil | |\n| B.C. | 62 | M | P.A. | 250 | Nil | |\n| S.B. | 47 | F | P.A. | 1300 | Nil | |\n| E.H. | 84 | F | P.A. | 850 | Nil | |\n| P.T. | 56 | F | Cystic fundic gland polyps | 7 | Nil | <5mm |\n| M.L. | 70 | F | Cystic fundic gland polyps | 28 | Nil | upto 1cm |\n| M.L. | 66 | F | Cystic fundic gland polyps | 20 | Nil | <5mm |\n| H.B. | 86 | F | Cystic fundic gland polyps | 14 | Nil | <5mm |\n| J.T. | 43 | F | Cystic fundic gland polyps | 20 | Nil | <5mm |\n| A.M. | 63 | F | Dyspepsia, Normal OGD | 15 | Nil | |\n| E.H. | 72 | F | Iron Deficiency, Normal OGD | 26 | Nil | |\n| E.Q. | 50 | F | Dyspepsia, Normal OGD | 13 | Nil | |\n| T.B. | 43 | M | Dyspepsia, Normal OGD | 25 | Nil | |\n| A.L. | 63 | M | Dyspepsia, Normal OGD | 12 | Nil | |\n\nTable 7.1 Details for all patients that provided tissue biopsies for either Northern blotting of *reg* mRNA abundance or RT/PCR to generate cDNA transcripts of *reg* mRNA for sequencing purposes. Hypergastrinaemic patients are shown above the control subjects that were selected on the basis of a normal oesophago-gastric-duodenoscopy (OGD) and normal plasma gastrin.\n\nPermanent cell transfection\n\nTwo constructs of human *reg* were selected for permanent transfection. These were the normal full length construct and the *reg* sequence that included a missense mutation at the initiator methionine residue (ATG→GTG). Stably transfected hamster insulinoma tumour (HIT) cell lines expressing wild type or mutant human *reg* were produced by electroporation of $5 \\times 10^6$ cells in 0.8ml at 200V and 1070μF in incomplete medium containing 10μg of linearised pcDNA *reg* construct. Cells were grown in complete RPMI in 10cm dishes for 48 hours prior to selection with zeocin ($750\\mu g/ml^{-1}$) (Invitrogen). Six colonies expressing each construct were cloned and screened for *reg* expression by northern blot.\n\nResults\n\nReg expression in human gastric corpus.\n\nReg mRNA abundance was determined in hypergastrinaemic patients and compared to that of control subjects (figure 7.1). In patients with sustained hypergastrinaemia (plasma gastrin <400pM) reg mRNA abundance was increased approximately threefold. In addition, suppression of plasma gastrin by a 72hr infusion of the somatostatin analogue octreotide, was associated with a significant downregulation of HDC (n=2) and reg mRNA (n=1) in tumour biopsies. The effect of octreotide on plasma gastrin and HDC mRNA abundance are shown for patient JH in Figure 7.2. The effect of octreotide on reg expression was determined in patient KD and is shown in Figure 7.3.\n\nReg expression in endocrine and non-endocrine cell polyps\n\nTotal RNA extracted from biopsies that were confirmed histologically to be non-endocrine in type (fundic cystic gland hyperplasia) showed a reduction in reg mRNA abundance compared to controls. Conversely, reg mRNA abundance was greatly increased in some ECL cell nodules compared to the patient’s own corpus but not in all cases. In fact the abundance of reg mRNA in ECL cell tumour biopsies was very variable (Figure 7.4) and did not relate directly to plasma gastrin concentrations.\n\nFig 7.1 *Upper panel* Reg mRNA abundance in patients with hypergastrinaemia (plasma gastrin >400pM) compared to control subjects (plasma gastrin <100pM). There is a threefold increase in reg mRNA in patients with hypergastrinaemia (n = 10 for each group; * p<0.05, compared with control, Mann Whitney U test). *Lower panel* Representative Northern blot of reg mRNA in controls (lane 1-4) and hypergastrinaemic patients (lanes 5-8) demonstrating the increased reg mRNA in hypergastrinaemic corpus.\n\nFig 7.2 *Upper panel* Effect of octreotide infusion on plasma concentrations of amidated gastrin in patient JH. Plasma gastrin concentrations decreased during the infusion but remained above the upper limit of normal (indicated by dashed horizontal lines). The dose of octreotide is indicated by the black bar graph. *Lower panel* Northern blot for HDC mRNA in total RNA extracted from biopsies of carcinoid nodule (lane 1) and corpus (lane 2) pre-octreotide and from carcinoid nodule (lane 3) and corpus (lane 4) post-octreotide. HDC mRNA was reduced in both nodule and corpus following the octreotide infusion.\n\nFig 7.3 Upper panel Effect of octreotide infusion on plasma concentrations of amidated gastrin in patient KD. Plasma gastrin concentrations decreased during the infusion falling to within the normal range (indicated by dashed horizontal lines). The dose of octreotide is indicated by the black bar graph. Lower panel a) Northern blot for reg mRNA in total RNA extracted from biopsies of carcinoid nodule (lane 1) and corpus (lane 2) pre-octreotide and from carcinoid nodule (lane 3) and corpus (lane 4) post-octreotide. Reg mRNA was reduced in carcinoid nodule following the octreotide infusion. b) Northern blot of the same membrane for GAPDH mRNA.\n\nFig 7.4 Upper panel Reg mRNA abundance in fundic cystic gland polyps (FCGP n=5) compared to controls (n=6). Reg mRNA was reduced by $\\approx$50% in the non-endocrine polyps (* p<0.05 Mann Whitney U test). To the right of the axis break is shown the reg mRNA abundance in ECL cell tumours in individual patients (initials on X axis) relative to their own corpus reg mRNA abundance normalised to 100 arbitrary units. Note the wide variation in tumour reg mRNA abundance Lower panel Representative Northern blot for reg mRNA in total RNA extracted from biopsies of normal corpus (lanes 1,3 and 5) non-endocrine cell polyps (lanes 2,4 and 6) hypergastrinaemic corpus (lane 7) and ECL cell nodule (lanes 8).\n\nReg Sequence and ECL cell tumours\n\nThe cDNA sequence of clones derived from RT/PCR of reg mRNA was determined in six hypergastrinaemic patients without ECL cell tumours and five patients with tumours. At least two clones were sequenced and data on the full sequence length obtained. All patients without ECL cell tumours had a normal reg sequence. Three of the five patients with nodules had at least one mutation of their reg sequence in either corpus or nodule. The sequence information from patients with ECL cell nodules is presented in Table 7.2. JH had multiple presumably acquired mutations identified from the biopsies of her nodules. PS had a single mutation identified in both corpus and nodule suggesting a germ line mutation. Interestingly, this mutation also occurred in JH and immediately appeared to suggest a pathophysiological mechanism. The mutation was a point mutation in the codon for the initiator methionine (ATG → GTG) and this would result in either the failure of initiation of protein translation or initiation of protein translation at a suitable upstream methionine residue. If protein translation commenced at the next upstream methionine, this would result in loss of the first eight amino acids of the signal peptide with a failure of the protein translated to enter the secretory pathway (Fig 7.5).\n\nTable 7.2 Sequence data from cDNA transcripts of *reg* mRNA extracted from hypergastrinaemic patients with ECL cell tumours. Three of the five patients with nodules had at least one mutation in their *reg* gene identified from either nodule or corpus. All patients without nodules had normal *reg* sequence. PS had a single mutation identified from both nodule and corpus suggesting a germ line mutation. Patient JH had multiple, presumably acquired mutations identified from her tumour RNA. Note the same mutation in the initiator methionine residue occurred in both JH and PS.\n\nFig 7.5 Upper panel Hydrophobicity plot of normal Reg protein showing the hydrophobic tail of the signal peptide (bold line) and below this the first ten amino acids of the protein sequence. Lower panel Initiator methionine mutant reg showing the point mutation resulting in the initiator methionine residue being substituted by valine. Above this is shown the predicted hydrophobicity plot assuming protein translation occurs at the next upstream methionine. Note loss of the membrane spanning hydrophobic signal peptide.\n\nFig 7.6 Northern blot of *reg* mRNA abundance in 5 clones of HIT cells permanently transfected with mutant *reg* (lanes 1-5) and 6 clones of HIT cells permanently transfected with wild type *reg* (lanes 6-11). Although there is variability, all clonal cell lines express *reg* mRNA.\n\nThe data presented in this chapter demonstrate for the first time that *reg* expression in the human gastric corpus is increased in patients with hypergastrinaemia and may be suppressed in carcinoid tumour nodules by a 72hr infusion of octreotide. Perhaps of more significance was the demonstration of an association between mutations of *reg* and the development of carcinoid tumours in patients with hypergastrinaemia.\n\nIt is now generally accepted that gastrin has a trophic action on the ECL cell and high circulating levels of gastrin promotes ECL cell hyperplasia. Hypergastrinaemia induced by H$_2$-receptor antagonists leads to the progressive development of ECL cell hyperplasia and gastric carcinoid tumours in rodents (Hava 1986; Poynter and Selway, 1991; Wangberg et al., 1995). Anti-secretory therapy in man may lead to significant ECL cell hyperplasia (Eiselle et al., 1997) but no cases of carcinoid tumour have been reported. This may be because the hypergastrinaemia induced by such therapy is rarely high enough or sustained. Conversely, conditions associated with persistent and large elevations in plasma gastrin are associated with carcinoid tumour development.\n\nSeveral genes expressed by ECL cells, including those for HDC, VMAT2 and chromogranin A, are upregulated by an elevated plasma gastrin (Dimaline et al., 1993a; Dimaline and Struthers, 1996; Dimaline et al., 1993b). The ability of gastrin to\n\nstimulate new protein synthesis and growth of ECL cells can be blocked by specific gastrin/CCK-B receptor antagonists or by somatostatin and its analogues (Dimaline and Sandvik, 1991; Bordi et al., 1993; Ferraro et al., 1996). The data presented here confirm that HDC gene expression within the human gastric corpus mucosa was increased in patients with hypergastrinaemia and may be downregulated by a 72hr infusion of octreotide. Reg mRNA was downregulated in carcinoid nodule but not in normal corpus by octreotide (see below). In both cases, plasma gastrin fell during octreotide treatment although in the case of JH, plasma gastrin remained elevated above the upper limit of normal. Despite this there was profound depression in ECL cell function in the face of elevated plasma gastrin. This observation suggests that in addition to reducing the stimulus to ECL cell function mediated by gastrin, octreotide may itself exert direct inhibitory effects on ECL cell function. The ability of octreotide to influence gene expression within carcinoid tumour tissue suggests that this tissue is still under normal regulatory control.\n\nIn patient KD, reg expression changed in parallel with HDC. There was a marked increase in reg mRNA abundance in the patient’s carcinoid nodule compared to his corpus mucosa and in his nodule at least, this was reduced by the octreotide infusion. Perhaps surprisingly, octreotide appeared to increase reg mRNA in gastric corpus. One possible explanation for this may be the variation in tissue sampling (see below). The elevated reg mRNA in gastric carcinoid tissue seen in KD was clearly not the situation for all patients with nodules as the abundance of reg mRNA in nodule\n\ntissue was highly variable between patients. There may be several reasons for this. In rodents it has been suggested that as carcinoid tumours develop and progress, they develop autonomy and become less dependent on gastrin as a trophic stimulus. The variability in *reg* expression in patients perhaps may indicate patients at a different stage of their disease. Alternatively, the degree of inflammation in the surrounding corpus was not taken into account. The ability of growth factors to potentiate the gastrin stimulated *reg* mRNA response also may explain the observed variability in nodule *reg* mRNA abundance. Hence, perhaps as CAG progresses and the parietal cell mass falls, the degree of inflammation also subsides. Tissue sampling introduces another unknown variable although this maybe more relevant to corpus mRNA as macroscopic nodules represent reproducible sites to biopsy. Although biopsies were taken from corpus mucosa that appeared macroscopically normal through an endoscope, the presence of microscopic carcinoids within biopsies that were processed for corpus mRNA cannot be excluded. This probably explains not only the variability in *reg* expression between patients but also the somewhat surprising increase in corpus *reg* mRNA in KD after his octreotide infusion. Furthermore, part or all of the increase in corpus *reg* mRNA in patients with hypergastrinaemia may be explained by an increase in ECL cell number as a consequence of hyperplasia and also due to concurrent atrophy of other mucosal cell types in patients with CAG.\n\nCarcinoid tumours occur most commonly in patients with CAG, but are also associated with ZE and CRF. The incidence of carcinoid nodules in CAG is\n\napproximately 5% and is similar to the incidence of carcinoid tumour occurring in the sporadic form of ZE syndrome. The incidence of carcinoid nodules in ZE associated with MEN1 syndrome rises to $\\approx 30\\%$ (Lehy et al., 1992). This suggests a genetic predisposition to carcinoid tumour development in this patient subgroup and is in keeping with the recently published observations of (Debelenko et al., 1997). They demonstrated that 75% of ZE patients with MEN1 syndrome have loss of heterozygosity at the MEN1 gene locus with deletion of the wild type allele. The MEN1 gene has recently been sequenced (Chandrasekharappa et al., 1997) and is the tumour suppressor gene that carries the inherited risk for developing endocrine tumours that are the feature of MEN1 syndrome. Of interest also however, was the observation of a low incidence of loss of heterozygosity at the MEN1 gene locus (1 of 6 patients) in patients with CAG and carcinoid nodules (Debelenko et al., 1997). This implies that another gene may be responsible for the risk of tumour development in these patients.\n\nThere is a growing body of evidence to suggest that the *reg* gene may play a role in the growth and differentiation of entero-pancreatic endocrine cells. It seemed reasonable to examine therefore whether mutations of *reg* may be associated with the development of carcinoid tumours in patients with hypergastrinaemia. The sequence data presented show that in 3 of 5 patients with hypergastrinaemia and carcinoid tumours there was at least one identifiable mutation in *reg* whereas in all hypergastrinaemic patients without carcinoid nodules the wild type *reg* coding\n\nsequence was found. This identifies an association between mutations of *reg* and a risk of developing carcinoid tumours in the presence of hypergastrinaemia. However, a significant number of normal *reg* sequences were also obtained from both the corpus and the nodules of patients with tumours. Again this may reflect sampling error as each biopsy of tumour tissue will also have normal mucosal cells within it. To overcome this, microscopic dissection of fresh biopsy material to obtain clonal tumour cells will be required. It is also likely that the *reg* gene is not the only genetic mutation required or necessary to develop carcinoid tumours just as mutations of menin do not appear to be solely responsible for the development of carcinoid tumours in ZE associated with MEN1 (Debelenko *et al.*, 1997).\n\nIt seems likely that both acquired and germ line mutations of *reg* may occur in association with carcinoid tumours. Patient JH had multiple mutations of *reg* in several clones sequenced indicating multiple acquired mutations. Patient PS had only one mutation of his *reg* but it occurred in three of four sequences in both his corpus and nodule. This is suggestive of a germ line mutation. Interestingly the mutation identified in PS also occurred in patient JH and occurred at the methionine residue that initiates protein synthesis. The point mutation of A → G resulted in a loss of the initiator methionine, it being replaced by a valine residue. The possible results of this mutation include; i) a complete failure of protein translation, ii) protein translation occurs at the next upstream methionine that is “in frame”, iii) protein translation occurs at the most appropriate methionine residue that may be “out of frame”. In the case of the first and last instance either no protein or a\n\ndifferent protein will be produced with an expected loss of the normal function of *reg*. In the case of the second instance the protein produced will be missing eight amino acids of the N-terminal hydrophobic signal peptide. This should result in failure of the protein product to enter the secretory pathway as the signal peptide will be too short to span the hydrophobic membrane domain. The subsequent protein product may then either be degraded within the cytosol or act intracellularly. This could result theoretically in either a loss or gain in function.\n\nAccording to the scanning model of mRNA translation proposed by Kozac (1978 and 1989), the sequence immediately adjacent to the initiator methionine codon determine the likelihood of protein translation occurring. The most important requirement is for a purine at position -3. In the absence of a purine at position -3, a G at +4 is essential for efficient translation (Kozak, 1989 & 1991; Iida and Masuda, 1996). Examination of the transfected mutant *reg* sequence fails to identify any alternative ATG codon with a surrounding sequence that meets these criteria. This would suggest that translation is unlikely to occur at an alternative start site and if it does occur at the next upstream methionine residue then translation will be inefficient. To confirm that the initiator methionine mutant *reg* resulted in failure of the translated protein product to enter the secretory pathway, constructs of human *reg* were inserted into a mammalian expression vector (pcDNA3-ZEO$^+$). The constructs were then permanently transfected into the hamster insulinoma tumour cell line (HIT) by Dr L Bishop. Cells were cultured\n\nFig 7.7 Photomicographs of HIT cells permanently transfected with either wild type (a) or mutant (b) *reg* constructs and stained sequentially with monoclonal anti-human *reg* antibody (1:2000; gift from Dr J Wand, Boston USA) and FITC conjugated horse anti mouse antibody (1:50). Note, only in the cells expressing wild type *reg* is there clearly discernible staining within the golgi apparatus and secretory granules. (x 400 approx., Courtesy of Colin Blackmore)\n\nunder zeocin selection and surviving cell lines were confirmed as expressing the *reg* gene by Northern blot of total mRNA extracted from $10^6$ cells (Fig 7.6). Clonal cell lines were then stained immuno-histochemically with a monoclonal anti-human reg antibody followed by FITC rabbit-anti-human secondary antibody. Intracellular distribution of reg protein was determined by immunofluorescence microscopy. (This work was performed by C. Blackmore, Dept of Physiology, Liverpool). The results of this work demonstrate that in the cell lines expressing wild type *reg* the protein enters the secretory pathway with clear staining of the Golgi apparatus and secretory granules (see photomicrograph 7.7a). In contrast, in cells expressing mutant *reg* there is no discernible pattern of staining (see photomicrograph 7.7b). Although this suggests that the protein either is not produced or is rapidly degraded, it remains possible that the shortened signal peptide remains attached to the N-terminal of the protein product and prevents the anti-human reg antibody from recognising the normal N-terminus of the reg protein. The specificity of the human reg antibody is being determined.\n\n**Summary**\n\nThe *reg* gene is known to be present in the human stomach and there is an increasing body of evidence to suggest that it may play a role in the growth and differentiation of endocrine cells in the gut including the ECL cell. Gastrin is also known to influence enteropancreatic endocrine cell differentiation and has a trophic effect on the ECL cell. The data presented demonstrate that plasma gastrin influences *reg* gene expression in human gastric corpus mucosa. In addition, the development of\n\ngastric carcinoid tumours in patients with hypergastrinaemia is associated with mutations of the *reg* gene. This raises the possibility of identifying patients at risk of carcinoid tumour development in the presence of sustained hypergastrinaemia.\n\nChapter Eight\n\nConclusions and Implications\n\nThe main objectives of the work contained within this thesis were to examine the role of antral neurons in the control of gastric emptying and the mechanisms that regulate gastrin release from G cells of the antrum, using a model of antral denervation in the rat. In addition, the significance of *reg* gene expression and carcinoid tumour development was examined by studying the relationships between gastrin and *reg* expression in the ECL-cells of the rat, in human gastric corpus and in a rat pancreatic cell line (AR42J).\n\n**Conclusions**\n\n1. Serosal application of BAC to the gastric antrum effectively lesions all myenteric and spinal afferent fibres of the antrum and the spinal afferent innervation of the gastric corpus. The intrinsic innervation of the proximal stomach is unaffected. Such chemically induced denervation provides a model for experimental studies of the intrinsic innervation of antral muscle and the antral mucosal endocrine cells of which the major cell of interest is the G cell.\n\n2. In the antrally denervated rat there is retention of solid within the stomach that is apparent after a 48hr fast. In antrally denervated rats fitted with a gastric fistula, the emptying of liquid test meals such as saline, acid and peptone was delayed. However, the CCK-dependent inhibition of gastric emptying of peptone was preserved after antral denervation by BAC. It appears that innervation of the antral musculature is\n\nessential for normal gastric emptying of liquids and solids but not CCK-mediated inhibition of gastric emptying.\n\n3. In the intact rat, antral neurons normally inhibit gastrin release in response to non-nutrient distension of the gastric corpus and luminal nutrient produces gastrin release via activation of neurons that release GRP. Following antral denervation, post-prandial gastrin release was maintained but was no longer dependent on either antral neurons or release of GRP. Moreover, non-nutrient distension of the stomach becomes an adequate stimulus for gastrin release.\n\n4. In AR42J cells *reg* mRNA expression was stimulated, in a dose-dependent manner, by amidated gastrin acting through the CCK-B receptor. *Reg* expression in rat and human gastric corpus was also dependent on plasma gastrin concentrations. Mutations of *reg* (a putative regulator of differentiation of gut endocrine cells) were associated with gastric carcinoid tumour development in patients with hypergastrinaemia. A major limitation of the studies thus far are that mRNA only has been measured and quantification of the protein product has not been performed.\n\n**Implications and future studies**\n\nMyenteric denervation by BAC was described first in the rat colon (Sato *et al.*, 1988), where its serosal application produced a phenotype similar to that of Hirschsprung’s disease. The effects of small intestinal denervation by BAC have also\n\nbeen described both by serosal application (myenteric plexus ablation) and luminal application (submucosal plexus ablation). The particular advantages that have been described for BAC are that it can be applied to a strictly defined area of the gut and its application lesions only the myenteric intrinsic neurons and the extrinsic innervation of the region when applied to the serosal surface. Clearly, the effects of BAC on extrinsic afferent fibres may be more widespread depending on the anatomy of innervation. Nevertheless the lesion produced relies upon the ability of intestinal smooth muscle to recover whilst neuronal loss persists. There is a significant inflammatory element to the lesion during the first 7 days (See et al., 1988). Thereafter, histologically inflammation subsides but muscle hypertrophy begins, although active stress generation by hypertrophied muscle does not exceed normal values until after the second week postoperatively (Herman and Bass, 1990).\n\nMost of the studies performed in this thesis used animals towards the end of their second post-operative week. However, the possible confounding factors of continued inflammation and antro-pyloric muscle hypertrophy could conceivably contribute to some of the observations described. In addition, because of a lack of a neuropeptide marker for extrinsic vagal fibres, no attempt was made to determine the extent of vagal damage induced by BAC. Although antral BAC treatment almost certainly disrupts the antral vagal innervation, the preservation of CCK-mediated delay in gastric emptying suggests that the duodenal vagal afferent and gastric vagal efferent fibres were intact.\n\nInflammation of the gastric mucosa is known to result in increased levels of growth factors such as TGF-α (Polk et al., 1992). Growth factors such as TGF-α and EGF have been shown to both stimulate gastrin release and inhibit acid secretion (Konturek et al., 1984; Rhodes et al., 1986). Furthermore, chronic gastric retention and delayed gastric emptying are well recognised complications of hypertrophic pyloric stenosis. In fact, chronic gastric retention is also associated with fasting and meal-stimulated hypergastrinaemia in the myenteric neuropathy that occurs as a result of *Trypanosomal* infection (Oliveira et al., 1980; Troncon et al., 1984).\n\nPerhaps of more immediate relevance may be the mucosal inflammation produced by *H.pylori*. There have been reports that antral gastritis due to *H.pylori* may also induce loss of mucosal nerve fibres and hence lead to denervation (Stead et al., 1996). Interestingly, and in keeping with the observations here, *H. pylori* infection produces basal and meal stimulated increases in plasma gastrin (Levi et al., 1989; McKoll et al., 1989; Tarnasky et al., 1993) and reduces antral somatostatin mRNA levels (Kaneko et al., 1990; Moss et al., 1992) although gastrin mRNA apparently is unaffected. The exaggerated response to GRP in *H. pylori* infection (Graham et al., 1991; McKoll and El-Omar, 1995) is in keeping with increased sensitivity seen following chronic denervation. The potential role of inflammation and growth factors in the BAC-treated rat was not examined.\n\nOf more concern however, may be the potential relationship between trophic factors, elevated plasma gastrin and the growth of gastric mucosal cells given the association between *H. pylori* and the development of gastric tumours. Certainly, the\n\nmarked potentiation by EGF of the gastrin-stimulated expression of *reg* in AR42J cells is preliminary evidence for a similar potentiation occurring in gastric mucosal endocrine cells or their precursors. The wide variability of *reg* expression in hypergastrinaemic patients may indicate different degrees of mucosal inflammation, independent of the level of their plasma gastrin and was a variable that was not controlled for in these studies.\n\nFinally, the role of gastrin and growth factors in the control of differentiation of gastric epithelial (endocrine and non-endocrine) cells is likely to become of increasing importance given the relationship between these factors, PPI therapy, *H. pylori* gastritis and gastric malignancy. The possible role of *reg Iα* as a tumour suppressor gene for endocrine cells of the stomach remains to be clarified but further studies of the *reg* gene family are likely to provide new insights into the cellular and molecular mechanisms that regulate the fundamental processes that lead to the differentiation and maintenance of cell phenotype. The role of *reg Iα* in the stomach and in AR42J cells provides a model system for testing the following hypotheses:\n\ni. expression of the *reg* gene is regulated by gastrin,\n\nii. expression of the *reg* gene influences the progressive differentiation of precursor endocrine cells towards in the gastric mucosa,\n\niii. mutations of *reg* result in failure of differentiation and may cause endocrine cell tumour development in the gastric mucosa.\n\nThe following provides an experimental outline of the future studies that will test these hypotheses:\n\ni) Does gastrin directly control *reg* expression?\n\na) Using the same techniques of tissue culture, mRNA extraction and Northern blot it would be possible to study gastrin receptor mediated events in AR42J cells using recognised gastrin receptor agonists, antagonists and inhibitors. The effects of somatostatin on *reg* expression could also be examined.\n\nb) Gastrin response elements in the promoter region of human *reg* could be identified by transfection of AR42-J cells with different constructs of the promoter region coupled to a reporter luciferase gene. Exact localisation of the relevant sequences would then require scanning mutagenesis and DNA footprinting.\n\nii) Is an intact *reg* essential for normal differentiation of an endocrine cell?\n\na) The ability of *reg* to induce differentiation will be assessed. The ability of recombinant reg protein to induce differentiation in responsive precursor cell lines will be studied and in addition, transfection of undifferentiated but responsive cells with wild type *reg* will also be performed. Expression of markers of differentiation will be assessed following stimulation with gastrin.\n\nb) Immunohistochemical markers of differentiation (CGA, HDC) and proliferation (PCNA, MIB) will be correlated with *reg* expression in precursor stem cells and mature ECL cells in patients with and without hypergastrinaemia.\n\niii Does a *reg* mutation produce abnormal physiology?\n\na) The normal biosynthesis of the Reg protein will be assessed *in vitro* in gastric biopsies by pulse chase experiments incorporating $^{35}$S methionine. Comparisons will be made between patients with and without mutations of *reg*.\n\nb). The processing of wild type and mutant *reg* by Hamster Insulinoma Tumour (HIT) cells will be assessed by morphological and biosynthetic analysis. We have already permanently transfected wild type and mutant *reg* into HIT cells that are capable of processing and secreting transfected proteins.\n\nc). 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(1996) Pancreatic thread protein is mitogenic to pancreatic-derived cells in culture. Gastroenterology 110: 1208-1214.\n\nZENILMAN ME, PERFETTI R, SWINSON K, MAGNUSON T & SHULDINER MD. (1996) Pancreatic regeneration (reg) gene expression in a rat model of islet hyperplasia. Surgery 119: 576-584.\n\nZHANG Z, HOCKER M, KOH T & WANG TC. (1996) The human histidine decarboxylase promoter is regulated by gastrin and phorbol 12-myristate 13-acetate through a downstream cis-acting element. J Biol Chem 271: 14188-14197.\n\nZHOU W, POVOSKI SP, LONGNECKER DS & BELL RH. (1992) Novel expression of gastrin (cholecystokinin-B) receptors in azaserine-induced rat pancreatic carcinoma: receptor determination and characterisation. Cancer Res 52: 6905-6911.\n\nZUCOLOTO S, SILVA JC, OLIVIERA SM & MUCCILLO G. (1991) The chronological relationship between the thickening of smooth muscle, epithelial cell proliferation and myenteric neural denervation in the rat jejunum. Cell Prolif 24: 15-20.\n\nAcknowledgements\n\nI would like to acknowledge Professor Graham Dockray for his supervision of my project and for his advice, support and encouragement over the last three years. I also wish to thank Professor David Thompson and Drs. Rod Dimaline and Andrea Varro for their help and collaboration. I am grateful for the technical assistance of Mrs Cath MacLean, Mark Houghton and particularly David Trafford whose patience and willingness to help seemed infinite. I also would like to recognise the MRC for their financial support of this research.\n\nOn a personal note, I wish to offer special thanks to my colleagues in the Department of Physiology (University of Liverpool) for their helpful advice and friendship and in particular to Lisa Bishop, PJ Noble, Atsushi Murai, Peter Djali and Colin Blackmore each of whom in their own way have made the last three years unforgettable."}}},{"rowIdx":204696,"cells":{"text":{"kind":"string","value":"AN ORDINANCE APPROVING, ADOPTING AND ENACTING AMERICAN LEGAL PUBLISHING'S OHIO BASIC CODE, 2017 EDITION, AS THE CODE OF ORDINANCES FOR THE MUNICIPALITY OF Amanda Village, OHIO, AND DECLARING AN EMERGENCY.\n\nWHEREAS, the present general and permanent ordinances of the municipality are inadequately arranged and classified and are insufficient in form and substance for the complete preservation of the public peace, health, safety and general welfare of the municipality and for the proper conduct of its affairs.\n\nWHEREAS, American Legal Publishing Corporation publishes a Code of Ordinances suitable for adoption by municipalities in Ohio.\n\nWHEREAS, it is necessary to provide for the usual daily operation of the municipality and for the immediate preservation of the public peace, health, safety and general welfare of the municipality that this ordinance take effect at an early date.\n\nNOW, THEREFORE, BE IT ORDAINED BY THE LEGISLATIVE AUTHORITY OF THE MUNICIPALITY OF Amanda Village, OHIO:\n\nSection 1. American Legal Publishing's Ohio Basic Code, 2017 Edition, as reviewed and approved by the Legislative Authority, is hereby adopted and enacted. Any prior version of the Ohio Basic Code which may have been previously adopted by the municipality is hereby repealed as obsolete and is hereby replaced in its entirety by this Ohio Basic Code, 2017 Edition.\n\nSection 2. One copy of American Legal Publishing's Ohio Basic Code, 2017 Edition, certified as correct by the Mayor and Clerk of the Legislative Authority, as required by Ohio Revised Code § 731.23, shall be kept in its initial form on file in the office of the Clerk of the municipality and retained as a permanent ordinance record of the municipality. The Clerk of the municipality is authorized and directed to publish a summary of all new matters contained in the Code of Ordinances as required by Ohio Revised Code § 731.23. Such summary is attached hereto and marked as \"Exhibit A\".\n\nSection 3. All ordinances and resolutions or parts thereof which are in conflict or inconsistent with any provision of the Ohio Basic Code, 2017 Edition, as adopted in Section 1 hereof, are hereby repealed as of the effective date of this ordinance, except as follows:\n(A) The enactment of the Ohio Basic Code, 2017 Edition, shall not be construed to affect a right or liability accrued or incurred under any legislative provision prior to the effective date of such enactment, or an action or proceeding for the enforcement of such right or liability. Such enactment shall not be construed to relieve any person from punishment for an act committed in violation of any such legislative provision, nor to affect an indictment or prosecution therefor. For such purposes, any such legislative provision shall continue in full force notwithstanding its repeal for the purpose of revision and codification.\n\n(B) The repeal provided above shall not affect:\n(1) The grant or creation of a franchise, license, right, easement or privilege;\n(2) The purchase, sale, lease or transfer of property;\n(3) The appropriation or expenditure of money or promise or guarantee of payment;\n(4) The assumption of any contract or obligation;\n(5) The issuance and delivery of any bonds, obligations or other instruments of indebtedness;\n(6) The levy or imposition of taxes, assessments or charges;\n(7) The establishment, naming, vacating or grade level of any street or public way;\n(8) The dedication of property or plat approval;\n(9) The annexation or detachment of territory;\n(10) Any legislation enacted subsequent to the adoption of this ordinance.\n(11) Any legislation specifically superseding the provision of the Ohio Basic Code.\n\nSection 4. Whenever reference is made in any documents, publications, or signs of the municipality, including but not limited to traffic tickets and traffic-control signs, to a section as it existed in a former edition of the Ohio Basic Code, the reference shall extend and apply to the section referred to as subsequently amended, revised, recodified, or renumbered.\n\nSection 5. This ordinance is declared to be an emergency measure necessary for the immediate preservation of the peace, health, safety and general welfare of the people of this municipality, and shall take effect at the earliest date provided by law.\n\nDate Passed: 2-6-17\n\nAttest:\n\nMayor\n\nClerk of the Legislative Authority\n\nNotice is hereby given that on the 6th day of February, 2017, there was enacted by the Legislative Authority of the Municipality of Amanda Village, Ohio, an ordinance entitled “An Ordinance Approving, Adopting and Enacting American Legal Publishing’s Ohio Basic Code, 2017 Edition, as the Code of Ordinances for the Municipality of Amanda Village, Ohio.”\n\nA summary of the subjects, including all new matters contained in the Code of Ordinances, as adopted, are as follows. The majority of Basic Code provisions are based directly on state law.\n\n**TITLE I: GENERAL PROVISIONS**\n\n**Chapter 10: General Provisions**\n\n| Section | Title |\n|---------|--------------------------------------------|\n| 10.01 | Short titles |\n| 10.02 | Definitions |\n| 10.03 | Rules of construction |\n| 10.04 | Revivor; effect of amendment or repeal |\n| 10.05 | Construction of section references |\n| 10.06 | Conflicting provisions |\n| 10.07 | Severability |\n| 10.08 | Reference to offices |\n| 10.09 | Errors and omissions |\n| 10.10 | Ordinances repealed |\n| 10.11 | Ordinances unaffected |\n| 10.12 | Ordinances saved |\n| 10.13 | Application to future ordinances |\n| 10.14 | Interpretation |\n| 10.15 | Amendments to code; amendatory language |\n| 10.16 | Statutory references |\n| 10.17 | Preservation of penalties, offenses, rights and liabilities |\n| 10.18 | Determination of legislative intent |\n| 10.99 | General penalty |\n\n**TITLE III: ADMINISTRATION**\n\n**Chapter 30: General Provisions**\n\n| Section | Title |\n|---------|--------------------------------------------|\n| 30.01 | Application of Title III |\n| 30.02 | Qualifications; oaths |\n| 30.03 | Bonds of officers and employees; amount |\n| 30.04 | Additional bond; where bonds recorded and kept |\n| 30.05 | Approval of bonds |\n| 30.06 | Sufficiency of form of bond |\n| 30.07 | Filling vacancies in offices |\n| 30.08 | Public records available |\n| 30.09 | Records Commission |\n| 30.10 | Meetings of public bodies to be open; exceptions; notice |\n| 30.11 | Municipal officers may attend conference or convention; expenses |\n| 30.12 | Residency requirements prohibited; exceptions |\n\n**Chapter 31: Executive Authority**\n\n**General Provisions**\n\n| Section | Title |\n|---------|--------------------------------------------|\n| 31.001 | Executive power; where vested |\n\n**Mayor**\n\n31.015 Term of Mayor; power and duties \n31.016 General duties of the Mayor \n31.017 Communications to the Legislative Authority \n31.018 Protest against excess of expenditures \n31.019 Supervision of conduct of officers \n31.020 Annual report to the Legislative Authority \n31.021 Mayor to file charges against delinquent officers \n31.022 Vacancies in office of Mayor \n31.023 Disposition of fines and other moneys\n\n**Clerk**\n\n31.040 Election, term, qualifications of the Clerk \n31.041 Powers and duties of Clerk \n31.042 Books and accounts; merger of offices \n31.043 Seal of Clerk \n31.044 Combined offices of Clerk and Treasurer; Fiscal Officer\n\n**Treasurer**\n\n31.060 Election, term, qualifications of the Treasurer \n31.061 Accounts of Treasurer \n31.062 Powers and duties \n31.063 Quarterly account; annual report \n31.064 Receipt and disbursement of funds \n31.065 Duty of delivering money and property\n\n**Street Commissioner**\n\n31.080 Qualifications \n31.081 General duties \n31.082 Assistants\n\n**Other Officials**\n\n31.100 Legal counsel \n31.101 Administrator \n31.102 Board of Trustees of Public Affairs \n31.103 Fire Engineer, Engineer and Superintendent of Markets\n\n---\n\n**Chapter 32: Legislative Authority**\n\n**General Provisions**\n\n32.001 Members of the Legislative Authority; election; terms of office \n32.002 President Pro Tempore; employees \n32.003 Vacancy when President Pro Tempore becomes Mayor \n32.004 Qualifications of members of the Legislative Authority \n32.005 Compensation and bonds of municipal officers and employees \n32.006 Vacancy \n32.007 Judge of election and qualification of members; quorum and special meetings \n32.008 Rules; journal; expulsion of members \n32.009 Meetings \n32.010 General powers \n32.011 Failure to take oath or give bond \n32.012 Notice when new bond required \n32.013 Care, supervision and management of public institutions\n\nContracts, Bids and Proceedings\n\n32.025 Contracts by the Legislative Authority or Administrator\n32.026 Bids and proceedings\n32.027 Alterations or modifications of contract\n32.028 Contract restrictions\n32.029 Award to lowest responsive and responsible bidder\n\nOrdinances and Resolutions\n\n32.040 Ordinances and resolutions as evidence\n32.041 Passage procedure\n32.042 Style of ordinances\n32.043 Subject and amendment of ordinances and resolutions\n32.044 Authentication and recording of ordinances and resolutions\n32.045 Publication of ordinances and resolutions; proof of publication and circulation\n32.046 Notice for proposed amendments to the municipal Charter\n32.047 Times of publication required\n32.048 Publication and certification of ordinances in book form\n32.049 Adoption of technical ordinances and codes\n32.050 Certificate of Clerk as to publication\n32.051 Publication when no newspaper published in municipality\n32.052 Effect of not making publication\n32.053 Ordinances providing for appropriations or street improvements; emergency ordinances\n\nInitiative and Referendum\n\n32.070 Initiative petitions\n32.071 Referendum petitions\n32.072 More than one ordinance required; application of subchapter\n32.073 Presentation of petitions\n32.074 Copy of proposed ordinance or measure to be filed with Clerk\n32.075 Words to be printed in red\n32.076 Designation of committee filing petition; public inspection of petitions; ordinances passed or repealed prior to election\n32.077 Itemized statement by petition circulator\n32.078 Prohibited practices relative to petitions\n32.079 Accepting premiums for signing\n32.080 Threats in securing signatures\n32.081 Application of subchapter if Charter adopted\n\nChapter 33: Judicial Authority\n\nGeneral Provisions\n\n33.01 Jurisdiction in ordinance cases and traffic violations\n33.02 Powers of Mayor and Mayor's Court Magistrate in criminal matters\n33.03 Duties of Mayor and Mayor's Court Magistrate; fees; office; seal\n33.04 Mayor's Court Magistrate\n33.05 Powers to suspend driver's license in OVI cases\n\nContempt of Court\n\n33.20 Summary punishment for contempt\n33.21 Acts in contempt of court\n33.22 Hearing\n33.23 Contempt action for failure to pay support, failure to comply or interference with a visitation order; summons\n33.24 Right of accused to bail\n33.25 Hearing on contempt; penalties; support orders; failure to withhold or deduct money pursuant to support order\n33.26 Imprisonment until order obeyed\n33.27 Proceedings when party released on bail fails to appear\n\n33.28 Release of prisoner committed for contempt\n33.29 Judgment final\n33.30 Alternative remedy\n\nChapter 34: Police Department\n\nSection\n\n34.01 Marshal and Police Chief synonymous\n34.02 Appointment of Marshal\n34.03 Deputy marshals and police officers\n34.04 Auxiliary police units\n34.05 Offenses affecting employment of law enforcement officers; probationary period; final appointment\n34.06 Removal proceedings; suspension; appeals\n34.07 General powers\n34.08 Powers and duties of Marshal\n34.09 Disposition of fines and penalties\n34.10 Property recovered by police\n34.11 Disposition to claimant\n34.12 Sale of unclaimed property; disposition of proceeds\n34.13 Expenses of storage and sale; notice\n34.14 Contracts for police protection; nonresident service without contract\n34.15 Peace officer administering oaths; acknowledging complaints, summonses, affidavits and returns of court orders\n\nChapter 35: Fire Department\n\nSection\n\nGeneral Provisions\n\n35.01 Municipal fire regulations; fire department\n35.02 Fire Chief; Fire Prevention Officer; employment of firefighters; criminal records check for firefighters\n35.03 Schooling of officers and firefighters of fire department\n35.04 Legislative Authority may purchase engines and equipment\n35.05 Buildings for department\n35.06 Records\n35.07 Maximum consecutive hours for firefighters on duty\n35.08 Investigation of cause of fire\n35.09 Right to examine buildings, premises, and vehicles\n35.10 Burning buildings for firefighting instruction or research\n35.11 Impersonating fire safety inspector\n35.12 Standards for equipment\n35.13 Persons entitled to be known as firefighters\n35.14 Firefighting and emergency services agreements\n35.15 Regulation of construction in fire limits\n\nVolunteer Firefighters’ Dependents Fund Board\n\n35.30 Definitions\n35.31 Establishment\n35.32 Membership; vacancies\n35.33 Election and term of members\n35.34 Organization; rules and regulations; roster\n35.35 Compensation and expenses of Board; legal advisor\n\nChapter 36: Civil Actions Against the Municipality\n\nSection\n\n36.01 Definitions\n36.02 Nonliability of municipality; exceptions\n36.03 Defenses and immunities\n36.04 Limitation of actions\n36.05 Damages\n36.06 Satisfaction of judgments\n36.07 Provision of employees’ defense; consent judgments\n36.08 Liability insurance\n36.09 Certain actions unaffected\n36.10 Certain charges against municipal officers filed with Probate Judge; proceedings\n\nTITLE VII: TRAFFIC CODE\n\nChapter 70: General Provisions\n\nGeneral Provisions\n\n70.01 Definitions\n70.02 Compliance with order of police officer\n70.03 Emergency vehicles to proceed cautiously past red or stop signal\n70.04 Exceptions generally; emergency, public safety and coroner vehicles exempt\n70.05 Persons riding or driving animals upon roadways\n70.06 Prohibitions against pedestrians and slow-moving vehicles on freeways\n70.07 Use of private property for vehicular travel\n70.08 Names of persons damaging real property by operation of vehicle to be provided to owner\n70.09 Limited access highways; barriers along; vehicles to enter and leave at designated intersections\n70.10 Through highways\n70.11 Officer may remove ignition key\n70.12 Removal of vehicles after accidents\n\nTraffic-Control Devices\n\n70.30 Obeying traffic-control devices\n70.31 Signal lights\n70.32 Signals over reversible lanes\n70.33 Ambiguous or non-working traffic signals\n70.34 Pedestrian-control signals\n70.35 Unauthorized signs and signals prohibited\n70.36 Alteration, defacement, or removal prohibited\n70.37 Unauthorized possession or sale of devices\n70.38 Signal preemption devices; prohibitions\n70.99 Penalty\n\nChapter 71: Licensing Provisions\n\nMotor Vehicle Licensing\n\n71.01 Display of license plates or validation stickers; registration\n71.02 Improper use of noncommercial motor vehicle\n71.03 Operating motor vehicle ordered immobilized; forfeiture\n71.04 Operation or sale without certificate of title\n71.05 Display of certificate of registration\n71.06 Use of unauthorized plates\n71.07 Operating without dealer or manufacturer license plates\n\nDriver’s Licenses\n\n71.20 Prohibited acts\n71.21 Permitting minor to operate vehicle prohibited; temporary instruction permit; probationary license\n71.22 License required as driver or commercial driver on public or private property; nonresident exemption\n71.23 Employment of a minor to operate a taxicab prohibited\n71.24 Restriction against owner lending vehicle for use of another\n71.25 Suspension of driver’s licenses; license suspended by court of record\n71.26 Display of license\n71.27 Prohibition against false statements\n71.28 Driving under suspension or in violation of license restriction\n\n71.29 Operating motor vehicle or motorcycle without valid license\n71.30 Driving under OVI suspension\n71.31 Driving under financial responsibility law suspension or cancellation; driving under a nonpayment of judgment suspension\n71.32 Failure to reinstate license\n\n**Commercial Driver's Licenses**\n\n71.45 Definitions\n71.46 Use of actual gross weight in lieu of rating\n71.47 Prohibited acts\n71.48 Prerequisites to operation of commercial motor vehicle\n71.49 Physical qualification to operate commercial motor vehicles\n71.50 Criminal offenses\n71.51 Application of federal regulations\n71.52 Employment of drivers of commercial vehicles\n\n71.99 Penalty\n\n**Chapter 72: Traffic Rules**\n\n**General Provisions**\n\n72.001 Lanes of travel upon roadways\n72.002 Driving through safety zone\n72.003 Vehicles traveling in opposite directions\n72.004 Rules governing overtaking and passing of vehicles\n72.005 Permission to overtake and pass on the right\n72.006 Driving to left of center line\n72.007 Prohibition against driving upon left side of roadway\n72.008 Hazardous zones\n72.009 One-way highways and rotary traffic islands\n72.010 Rules for driving in marked lanes\n72.011 Space between moving vehicles\n72.012 Divided roadways\n72.013 Rules for turns at intersections\n72.014 U-turns and turning in roadway prohibited\n72.015 Starting and backing vehicles\n72.016 Turn and stop signals\n72.017 Hand and arm signals\n\n**Right-of-Way**\n\n72.030 Right-of-way at intersections\n72.031 Right-of-way when turning left\n72.032 Right-of-way at through highways; stop signs; yield signs\n72.033 Stop at sidewalk area; stop signs on private roads and driveways\n72.034 Right-of-way on public highway\n72.035 Pedestrian on sidewalk has right-of-way\n72.036 Right-of-way of public safety vehicles\n72.037 Funeral procession has right-of-way\n72.038 Pedestrians yield right-of-way to public safety vehicle\n72.039 Pedestrian on crosswalk has right-of-way\n72.040 Right-of-way yielded to blind person\n72.041 Right-of-way yielded by pedestrian\n\n**Pedestrians**\n\n72.055 Pedestrian movement in crosswalks\n72.056 Pedestrian walking along highway\n72.057 Prohibition against soliciting rides; riding on outside of vehicle\n72.058 Pedestrian on bridge or railroad crossing\n72.059 Persons operating motorized wheelchairs\n\n72.060 Intoxicated or drugged pedestrian hazard on highway\n72.061 Operation of electric personal assistive mobility devices\n\n**Grade Crossings**\n\n72.075 Stop signs at grade crossings\n72.076 Driving vehicle across railroad grade crossing\n72.077 Vehicles required to stop at grade crossings\n72.078 Slow-moving vehicles or equipment crossing railroad tracks\n\n**School Buses**\n\n72.090 Regulations concerning school buses\n72.091 Violation of regulations; report; investigation; citation; warning\n72.092 Restrictions on the operation of school buses\n72.093 School bus inspection\n72.094 School bus not used for school purposes\n72.095 Licensing by Department of Public Safety\n72.096 Registration and identification of school buses\n72.097 School bus marking\n72.098 Flashing light signal lamps\n72.099 Occupant restraining device for operator\n\n**Prohibitions**\n\n72.115 Obstruction and interference affecting view and control of driver\n72.116 Occupying travel trailer while in motion\n72.117 Driving upon closed highway prohibited\n72.118 Driving upon sidewalk area or paths exclusively for bicycles\n72.119 Obstructing passage of other vehicles\n72.120 Following an emergency or public vehicle prohibited; approaching stationary public safety vehicle with caution\n72.121 Driving over unprotected fire hose\n72.122 Placing injurious material on highway or depositing litter from motor vehicle\n72.123 Transporting child not in child-restraint system prohibited\n72.124 Occupant restraining devices\n72.125 Use of engine brakes prohibited\n72.126 Operating motor vehicle while wearing earphones or earplugs\n72.127 Chauffeured limousines and livery services\n72.128 Operating traction engine upon improved highway\n72.129 Cracking exhaust noises; peeling out\n72.130 Shortcutting across private property\n72.131 Texting while driving prohibited\n72.132 Use of electronic wireless communication devices by minors or probationary drivers while driving prohibited\n\n**Chapter 73: Motor Vehicle Crimes**\n\n**General Provisions**\n\n73.01 Driving under the influence of alcohol or drugs\n73.02 Implied consent\n73.03 Physical control of vehicle while under the influence\n73.04 Driving commercial vehicle with impaired alertness or ability; use of drugs\n73.05 Reckless operation of vehicles\n73.06 Reckless operation off streets and highways; competitive operation\n73.07 Operator to be in reasonable control\n73.08 Immobilizing or disabling device violation\n73.09 Street racing defined; prohibited on public highways\n73.10 Speed limits\n73.11 Slow speed or stopping\n73.12 Emergency vehicles excepted from speed limitation\n73.13 Speed regulations on bridges\n73.14 Presenting false name or information to officer\n\n73.15 Prohibition against resisting officer\n73.16 Operation restricted for mini-trucks and low-speed, under-speed, or utility vehicles\n\n**Stopping After Accident**\n\n73.30 Failure to stop after accident\n73.31 Stopping after accident on other than public roads or highways\n73.32 Accident involving damage to realty\n73.33 Failure to report accident\n\nChapter 74: Equipment and Loads\n\n**Equipment**\n\n74.01 Unsafe vehicles, prohibition against operation\n74.02 Bumpers on motor vehicles\n74.03 Lighted lights required\n74.04 Headlights\n74.05 Tail lights and illumination of rear license plate\n74.06 Red reflectors required\n74.07 Safety lighting of commercial vehicles\n74.08 Stoplight regulations\n74.09 Obscured lights on vehicles\n74.10 Red light or flag required\n74.11 Lights on parked vehicles\n74.12 Lights and emblem on slow-moving vehicles; lights and reflectors on multi-wheel agricultural tractors or farm machinery\n74.13 Spotlight and auxiliary driving lights\n74.14 Cowl, fender, and back-up lights\n74.15 Two lights displayed\n74.16 Headlights required\n74.17 Lights of less intensity\n74.18 Number of lights permitted; red and flashing lights\n74.19 Standards for lights on snow removal equipment and oversize vehicles\n74.20 Flashing lights permitted for certain types of vehicles\n74.21 Lights and sign on transportation for preschool children\n74.22 Focus and aim of headlights\n74.23 Brake equipment; specifications\n74.24 Brake fluid\n74.25 Minimum standards for brakes and components\n74.26 Horns, sirens, and warning devices\n74.27 Mufflers; excessive smoke or gas\n74.28 Rearview mirrors\n74.29 Windshields and wipers\n74.30 Solid tire requirements\n74.31 Requirements for safety glass in motor vehicles; use of tinted glass or reflectorized material\n74.32 Directional signals\n74.33 Installation and sale of seat safety belts required; definition\n74.34 Requirements for extra signal equipment\n74.35 Display of warning devices on disabled vehicles\n74.36 Requirements for vehicles transporting explosives\n74.37 Studded tires; seasonal use permitted\n74.38 Safety inspection decals for buses\n74.39 Air bags\n\n**Loads**\n\n74.50 Permit required to exceed load limits\n74.51 Limitation of load extension on left side of vehicle\n74.52 All loads shall be properly secured\n74.53 Towing requirements; exception to size and weight restrictions\n74.54 Weighing of vehicle; removal of excess load\n74.55 Operation of vehicle exceeding weight limits prohibited\n\n74.56 Load limits\n74.57 Maximum width, height, and length\n74.58 Statement of gross vehicle weight\n74.59 Wheel protectors required on heavy commercial vehicles\n74.60 Liability for damages; prosecution; application of monies\n74.61 Weight exceptions for certain vehicles\n\nChapter 75: Bicycles, Motorcycles and Off-Road Vehicles\n\nGeneral Provisions\n\n75.01 Bicycles; application of Title VII\n75.02 Operation of motorized bicycle\n75.03 Rules for bicycles, motorcycles, and snowmobiles\n75.04 Prohibition against attaching bicycles and sleds to vehicles\n75.05 Riding bicycles; motorcycles abreast\n75.06 Equipment of bicycles\n\nSnowmobiles, Off-Highway Motorcycles, and All-Purpose Vehicles\n\n75.25 Definitions\n75.26 Equipment\n75.27 Code application; prohibited operation\n75.28 Permitted operation\n75.29 Licensing requirements of operator\n75.30 Maintenance of vehicles for hire\n75.31 Accident reports\n75.32 Impounding of vehicle\n75.33 Local control within police power\n75.34 Registration of vehicles\n75.35 Certificate of title; prohibitions\n\nChapter 76: Parking Regulations\n\nSection\n\n76.01 Prohibition against parking on highways\n76.02 Condition when motor vehicle left unattended\n76.03 Police may remove illegally parked vehicle\n76.04 Parking prohibitions\n76.05 Parking near curb; privileges for persons with disabilities\n76.06 Parking on private property in violation of posted prohibition\n76.07 Selling, washing or repairing vehicle upon roadway\n76.08 Truck loading zones\n76.09 Bus stops and taxicab stands\n76.10 Parking in alleys and narrow streets; exceptions\n76.11 Registered owner prima facie liable for unlawful parking\n76.12 Waiver\n\nTITLE IX: GENERAL REGULATIONS\n\nChapter 90: Animals\n\nAnimals Running at Large\n\n90.01 Dogs or other animals running at large; nuisance, dangerous or vicious dogs; hearings\n90.02 Confining animal found at large; publication of notice; lien\n90.03 Unavoidable escapes\n90.04 Fees\n90.05 Rabies quarantine orders of Mayor\n90.06 Interfering with enforcement of quarantine orders\n90.07 Dogs may be killed for certain acts\n\nOffenses Relating to Animals\n\n90.20 Abandoning animals\n90.21 Injuring animals\n90.22 Poisoning animals\n90.23 Cruelty to animals; cruelty to companion animals\n90.24 Animal fights\n90.25 Trapshooting\n90.26 Loud dog\n90.27 Dog tags\n90.28 Nuisance, dangerous and vicious dog defined; transfer of ownership certificate; form stating dog's prior behavior\n90.29 Failure to register dog or dog kennel\n90.30 Hindering the capture of unregistered dog\n90.31 Unlawful tag\n90.32 Rights of blind, deaf or hearing impaired, or mobility impaired person, or trainer with assistance dog\n90.33 Retail sale and transportation of dogs\n90.34 Coloring rabbits or baby poultry; regulation of sale and display\n90.35 Jacklighting prohibited\n90.36 Restrictions on dog ownership for certain convicted felons\n\n90.99 Penalty\n\nChapter 91: Fireworks, Explosives, Fire Prevention\n\nFireworks and Explosives\n\n91.01 Definitions\n91.02 Possession, sale, and use of fireworks\n91.03 Permit to use fireworks\n91.04 Manufacturing or wholesale sale without a license; prohibitions\n91.05 Purchasers to comply with law; unauthorized purchases\n91.06 Exhibition without a license; prohibitions\n91.07 Unauthorized transportation or shipping\n91.08 Application of subchapter\n91.09 Arrest of offender; seizure and forfeiture of fireworks; distribution of fines\n91.10 Safety requirements for fireworks showroom structures\n91.11 Storage of explosives\n91.12 Blasting permit\n\nFire Prevention\n\n91.30 Removal of flammable materials or obstructions\n91.31 Protective appliances\n91.32 Compliance with order\n91.33 Waste receptacles\n91.34 Hotel to have fire warning device producing visible signal\n91.35 Fire suppression systems\n91.36 Violations of State Fire Code prohibited\n91.37 Posting arson notices in hotels, motels and other places\n91.38 Negligent burning\n91.39 Spreading alarm of unfriendly fire\n91.40 Unvented heaters\n\nOpen Burning\n\n91.55 Definitions\n91.56 Relations to other prohibitions\n91.57 Open burning in restricted areas\n91.58 Permission and notice to open burn\n\n91.99 Penalty\n\nChapter 92: Intoxicating Liquors\n\nSection\n\n92.01 Definitions\n92.02 Exemptions from chapter\n92.03 Restrictions applicable to sale of beer and intoxicating liquor for consumption on the premises\n92.04 Restrictions on sale of beer and liquor\n92.05 Permit required; activities prohibited without permit\n92.06 Illegal transportation prohibited\n92.07 Open container prohibited; exception\n92.08 Underage person shall not purchase intoxicating liquor or beer\n92.09 Prohibitions; minors under 18 years; low-alcohol beverages\n92.10 Alcohol vaporizing devices prohibited\n92.11 Misrepresentation to obtain alcoholic beverage for a minor prohibited\n92.12 Misrepresentation by a minor under 21 years\n92.13 Sale to underage persons prohibited\n92.14 Posting of card\n92.15 Good faith acceptances of spurious identification\n92.16 Consumption in motor vehicle prohibited\n92.17 Hours of sale or consumption\n92.18 Obstructing search of premises prohibited\n92.19 Illegal possession of intoxicating liquor prohibited\n92.20 Sale or possession of diluted liquor and refilled containers prohibited\n92.21 Keeping place where beer or intoxicating liquors are sold in violation of law\n92.22 Intoxicating liquors shall not be sold in brothels\n92.23 Use of intoxicating liquor in a public dance hall prohibited; exceptions\n92.24 Poisonously adulterated liquors\n92.25 Tavern keeper permitting rioting or drunkenness\n92.26 Notice of action to prohibit liquor business\n92.27 Procedure when injunction violated\n92.28 Liquor transaction scans\n92.29 Affirmative defenses\n\n92.99 Penalty\n\nChapter 93: Nuisances\n\nSection\n\nGeneral Provisions\n\n93.01 Application of the chapter\n93.02 Definitions\n93.03 Nuisances generally; injunctions; violation; contempt\n93.04 Maintaining certain nuisances\n93.05 Collection of cost of abating dangerous property condition; injunction; rehabilitation\n93.06 Trimming of trees and shrubbery to prevent obstruction\n\nSeptic Tanks, Cesspools, and Refuse\n\n93.20 Location of privy vaults, cesspools, and septic tanks\n93.21 Unsanitary vaults\n93.22 Removal of contents of vault\n93.23 Deposit of dead animals, offal upon land or water\n93.24 Defiling spring or well prohibited\n93.25 Dumping of refuse in municipality prohibited\n93.26 Abandoned refrigerators\n93.27 Discarding litter prohibited\n93.28 Power of municipality to fill or drain land\n\nWeeds and Litter on Private Property\n\n93.40 Keeping down weeds\n93.41 Notice to owner to cut noxious weeds, remove litter; service\n93.42 Fees for service and return\n93.43 Procedure when owner fails to comply with notice\n93.44 Written return to County Auditor; amount as a lien upon property\n\nUnclean Habitations\n\n93.60 Permitting unclean habitations\n93.61 When habitations are deemed unsanitary\n93.62 Order for abatement or vacation of premises\n93.63 Enforcement of vacation order by Fire Chief or Police Chief\n93.64 Enforcement through court proceedings\n\n93.99 Penalty\n\nChapter 94: Streets and Sidewalks\n\nGeneral Provisions\n\n94.01 Conditions precedent to improving streets\n94.02 Opening permit required\n94.03 Application and cash deposit\n94.04 Restoration of pavement\n94.05 Barriers around excavations\n94.06 Warning lights\n94.07 Sidewalk construction by the municipality\n94.08 Unloading on street or sidewalk\n94.09 Street or sidewalk obstruction\n94.10 Materials on street or sidewalk\n94.11 Duty to keep sidewalks in repair and clean of ice and snow\n94.12 Ramped curbing for persons with disabilities\n94.13 Flagpole along right-of-way\n94.14 Altering or injuring marker or monument\n\nConstruction and Repair\n\n94.25 Construction and repair may be required\n94.26 Resolution of necessity\n94.27 Notice to construct or repair\n94.28 Assessments of costs against owner\n94.29 Proceedings may include different owners\n94.30 Making and levying assessments\n\nChanges in Streets\n\n94.40 Change of name, vacating or narrowing streets by petition\n94.41 Change of name, vacating or narrowing streets without petition\n94.42 Notice; exception\n94.43 Publication of notice\n94.44 Effect of order of vacation\n94.45 Effect on public utility easements\n\n94.99 Penalty\n\nChapter 95: Unclaimed and Abandoned Vehicles\n\nSection\n\n95.01 Impounding motor vehicle on private property; requirements\n95.02 Impounding abandoned motor vehicle on public property; notice; disposition\n95.03 Disposition of vehicle ordered into storage\n95.04 Disposition of abandoned junk motor vehicles\n95.05 Abandonment of junk motor vehicle prohibited\n95.06 Junk motor vehicle; order to cover or remove; notice; exceptions\n\nChapter 96: Watercraft\n\nSection\n\n96.01 Definitions; applicability\n96.02 Failure to comply with law enforcement order; fleeing\n96.03 Duty upon approach of law enforcement vessel\n96.04 Flashing lights prohibited; exceptions\n\n96.05 Siren prohibited; exceptions\n96.06 Regulations for operation of powercraft of more than ten horsepower\n96.07 Restrictions on child operators; duty of supervisory adult\n96.08 Reckless operation; maintaining sufficient control; wakes restricted\n96.09 Unsafe conditions\n96.10 Marking of bathing and vessel areas\n96.11 Mooring prohibited in certain areas\n96.12 Operating under influence of alcohol or drugs prohibited\n96.13 Implied consent\n96.14 Incapacitated operators prohibited\n96.15 Water skiing confined to ski zones\n96.16 Observer required when towing skier\n96.17 Water skiing after dark prohibited\n96.18 Personal flotation device required for towed person\n96.19 Ski jumps prohibited\n96.20 Permit for special water events\n96.21 Sale of single celled inflatable vessels prohibited\n96.22 Sitting, standing, walking on moving vessels restricted\n96.23 Engine warm-up required\n96.24 Personal flotation devices for children under ten\n96.25 Operation without personal flotation devices prohibited\n96.26 Distress signal or flag required\n96.27 Anchor requirements\n96.28 Specification for fire extinguishers\n96.29 Backfire flame control device required\n96.30 Ventilation requirement on powercraft\n96.31 Abandonment of junk vessels or outboard motors\n96.32 Exhaust muffler required; noise levels; exceptions\n96.33 Safety equipment on rental vessels\n96.34 Capacity plate\n96.35 Littering prohibited\n96.36 Dwellings; sanitary systems\n96.37 Prima facie evidence of negligence\n96.38 Requirements for operating personal watercraft\n96.39 Numbering\n96.40 Registration\n96.41 Tags indicating expiration date; attachment of identification number\n96.42 Altering of serial numbers; false information prohibited\n96.43 Accident reports\n96.44 Enforcement\n96.45 Firearms offenses; signaling devices\n96.46 Tampering with navigation aid or vessel prohibited\n96.47 Certificate of title; exceptions\n96.48 Manufacturer's or importer's certificate\n96.49 Prohibitions relating to certificates of title\n96.50 Permanently displayed hull identification number\n\n96.99 Penalty\n\nTITLE XI: BUSINESS REGULATIONS\n\nChapter 110: General Provisions\n\nSection\n\n110.01 Licenses required to engage in certain businesses; exceptions\n110.02 Application for license\n110.03 Issuance of license\n110.04 Date and duration of license\n110.05 License not transferable\n110.06 License certificate to be displayed\n110.07 Revocation or suspension\n110.08 Appeal and review\n\n110.99 General penalty for Title XI\n\nChapter 111: Taxicabs\n\nSection\n\n111.01 Definitions\n111.02 Certificate of public convenience and necessity required\n111.03 Application for certificate\n111.04 Issuance of certificate\n111.05 Liability insurance required\n111.06 License fees\n111.07 Transfer of certificates and licenses\n111.08 Suspension and revocation of certificates\n111.09 Taxicab driver's license\n111.10 Application for driver's license\n111.11 Examination of applicant; motor vehicle operator's permit required\n111.12 Police investigation of applicant; traffic and police record\n111.13 Consideration of application\n111.14 Issuance of license; duration; annual fee\n111.15 Display of license\n111.16 Suspension and revocation of license\n111.17 Failure to comply with federal, state and municipal laws\n111.18 Vehicle equipment and maintenance\n111.19 Designation of taxicabs\n111.20 Taximeter and display of rates required\n111.21 Number of passengers allowed\n111.22 Articles left in vehicles\n111.23 Vehicles from other municipalities\n111.24 Receipts\n111.25 Refusal of passenger to pay legal fare\n111.26 Solicitation, acceptance and discharge of passengers\n111.27 Open stands; use\n111.28 Taxicab service\n111.29 Manifests\n111.30 Records and reports of holders\n111.31 Police department; duty to enforce chapter\n111.32 Disposition of vehicle license fees\n\nChapter 112: Peddlers, Itinerant Merchants, and Solicitors\n\nSection\n\n112.01 Definitions\n112.02 License requirement\n112.03 Application procedure\n112.04 Standards for issuance\n112.05 Revocation procedure\n112.06 Standards for revocation\n112.07 Appeal procedure\n112.08 Exhibition of identification\n112.09 Municipal policy on soliciting\n112.10 Notice regulating soliciting\n112.11 Duty of solicitors\n112.12 Uninvited soliciting prohibited\n112.13 Time limit on soliciting\n\nChapter 113: Commercial Amusements\n\nSection\n\n113.01 Bowling; billiards and pool\n113.02 Circuses, carnivals, shows and other such entertainment\n113.03 Deposit required\n113.04 License fee for public entertainment or exhibition\n113.05 License fee may be waived for civic interest\n\nChapter 114: Tattooing and Body Piercing Services\n\nSection\n\n114.01 Definitions\n114.02 Prohibitions\n114.03 Application for license; fees; issuance\n\n114.04 Inspection of facilities\n114.05 Suspension or revocation of license\n114.06 Consent for performing procedures on persons under 18\n114.07 Prohibitions relating to persons under 18\n114.08 Defenses to violations\n114.09 Training standards; records; safety and sanitation; equipment\n114.10 Application of local regulation on chapter\n\n114.99 Penalty\n\nTITLE XIII: GENERAL OFFENSES\n\nChapter 130: General Provisions\n\nSection\n\n130.01 Application of Title XIII\n130.02 Definitions\n130.03 Classification of offenses\n130.04 Common law offenses abrogated\n130.05 Rules of construction\n130.06 Limitation of criminal prosecutions\n130.07 Requirements for criminal liability; voluntary intoxication\n130.08 Culpable mental states\n130.09 Organizational criminal liability\n130.10 Personal accountability for organizational conduct\n130.11 Attempt\n130.12 Complicity\n130.13 Presumption of innocence; proof of offense; affirmative defense\n130.14 Battered woman syndrome\n130.15 Delinquency adjudications deemed convictions\n130.16 Criminal law jurisdiction\n130.17 Disposition of unclaimed or forfeited property held by Police Department\n130.18 Imposing sentence for misdemeanor\n130.19 Multiple sentences\n130.20 Apprehension, detention, or arrest of persons on bond\n130.21 Self defense: limitations on duty to retreat prior to using force\n\n130.99 Penalty for Title XIII\n\nChapter 131: Offenses Against Property\n\nSection\n\n131.01 Definitions\n131.02 Arson; determining property value or amount of physical harm\n131.03 Criminal damaging or endangering; vehicular vandalism\n131.04 Criminal mischief\n131.05 Damaging or endangering aircraft or airport operations\n131.06 Criminal trespass; aggravated trespass\n131.07 Tampering with coin machines\n131.08 Theft\n131.09 Unauthorized use of a vehicle\n131.10 Unauthorized use of property, including telecommunication property and computers; possession of municipal property\n131.11 Passing bad checks\n131.12 Misuse of credit cards\n131.13 Making or using slugs\n131.14 Prima facie evidence of purpose to defraud\n131.15 Tampering with records\n131.16 Securing writings by deception\n131.17 Defrauding creditors\n131.18 Receiving stolen property\n131.19 Value of stolen property\n131.20 Degree of offense when certain property involved\n131.21 Injuring vines, bushes, trees, or crops\n131.22 Detention and arrest of shoplifters and those committing motion picture piracy; protection of institutional property\n\n131.23 Insurance fraud; workers' compensation fraud; Medicaid fraud\n131.24 Injury to property by hunters\n131.25 Evidence of intent to commit theft of rented property; evidence of lack of capacity to consent\n131.26 Forgery of identification cards\n131.27 Criminal simulation\n131.28 Personating an officer\n131.29 Trademark counterfeiting\n131.30 Diminishing or interfering with forfeitable property\n131.31 Recording credit card, telephone or Social Security numbers\n131.32 Prosecutions for theft of utilities\n131.33 Motion picture piracy\n\nChapter 132: Offenses Against Public Peace\n\nSection\n\n132.01 Riot\n132.02 Failure to disperse\n132.03 Justifiable use of force to suppress riot\n132.04 Disorderly conduct\n132.05 Disturbing a lawful meeting\n132.06 Misconduct at an emergency\n132.07 Telecommunications harassment\n132.08 Inducing panic\n132.09 Making false alarms\n132.10 Inciting to violence\n132.11 Unlawful display of law enforcement emblem\n132.12 Impersonating a peace officer\n132.13 Safety of crowds attending live entertainment performances\n132.14 Misconduct involving a public transportation system\n\nChapter 133: Sex Offenses\n\nSection\n\n133.01 Definitions\n133.02 Unlawful sexual conduct with a minor\n133.03 Sexual imposition\n133.04 Public indecency\n133.05 Voyeurism\n133.06 Polygraph examinations for victims: restrictions on use\n133.07 Procuring\n133.08 Soliciting; loitering to engage in\n133.09 Prostitution\n133.10 Disseminating matter harmful to juveniles\n133.11 Displaying matter harmful to juveniles\n133.12 Deception to obtain matter harmful to juveniles\n133.13 Rules of evidence\n133.14 Declaratory judgment\n133.15 Injunction; abatement of nuisance\n133.16 Unlawful operation of viewing booths depicting sexual conduct\n133.17 Juveniles on the premises of adult entertainment establishments prohibited\n133.18 Sexually oriented businesses; illegal operation and activity\n133.19 Unlawful advertising of massage\n\n133.99 Sentencing for sexually oriented offenses; sexual predators; registration\n\nChapter 134: Gambling Offenses\n\nSection\n\n134.01 Definitions\n134.02 Prohibitions against gambling; exception\n134.03 Operating a gambling house\n134.04 Public gaming\n134.05 Cheating\n134.06 Regulations concerning operation of licensed bingo game\n134.07 Records to be kept\n134.08 Requirements for bingo game operators\n134.09 Bingo games for amusement only\n\n134.10 Prohibitions where instant bingo game is conducted\n134.11 Raffle drawings\n134.12 Instant bingo other than at bingo sessions\n134.13 Restrictions on owner or lessor of location at instant bingo\n134.14 Skill-based amusement machines; prohibited conduct\n\nChapter 135: Offenses Against Persons\n\nSection\n\n135.01 Definitions\n135.02 Negligent homicide\n135.03 Vehicular homicide; vehicular manslaughter\n135.04 Assault; negligent assault\n135.05 Injury to persons by hunters\n135.06 Menacing; aggravated menacing; menacing by stalking\n135.07 Unlawful restraint\n135.08 Criminal child enticement\n135.09 Coercion\n135.10 Bigamy\n135.11 Unlawful abortion; failure to perform viability testing\n135.12 Abortion trafficking\n135.13 Nonsupport of dependents\n135.14 Endangering children\n135.15 Interference with custody; interference with support orders\n135.16 Domestic violence\n135.17 Hazing prohibited\n135.18 Contributing to unruliness or delinquency of a child\n135.19 Failure to provide for functionally impaired person\n135.20 Patient abuse or neglect; patient endangerment; exceptions; false statements; retaliation\n135.21 Interference with right of person to engage in housing transactions because of race, religion, or the like\n135.22 Ethnic intimidation\n135.23 Violating a protection order, consent agreement, anti-stalking protection order or order issued by a court of another state\n135.24 Adulteration of food\n135.25 Illegal distribution of cigarettes, other tobacco products, or alternative nicotine products; transaction scans\n135.26 Nonsmoking areas in places of public assembly\n135.27 Spreading contagion\n135.28 Abuse of a corpse\n135.29 Unlawful collection of bodily substances\n\nChapter 136: Offenses Against Justice and Administration\n\nSection\n\n136.01 Definitions\n136.02 Falsification\n136.03 Compounding a crime\n136.04 Failure to report a crime\n136.05 Failure to aid a law enforcement officer\n136.06 Obstructing official business\n136.07 Obstructing justice\n136.08 Resisting arrest\n136.09 Having an unlawful interest in a public contract\n136.10 Soliciting or receiving improper compensation\n136.11 Dereliction of duty\n136.12 Interfering with civil rights\n136.13 Illegal conveyance of prohibited items onto grounds of a detention facility or other specified governmental facilities\n136.14 False report of child abuse or neglect\n136.15 Assaulting police dog or horse, or assistance dog\n136.16 Disclosure of confidential peace officer information\n136.17 Intimidation of crime victim or witness\n136.18 Using sham legal process\n136.19 Making false allegation of peace officer misconduct\n136.20 Misuse of 9-1-1 system\n136.21 Failure to disclose personal information\n\nChapter 137: Weapons Control\n\nSection\n\n| Section | Description |\n|---------|-------------|\n| 137.01 | Definitions |\n| 137.02 | Carrying concealed weapons |\n| 137.03 | Using weapons while intoxicated |\n| 137.04 | Improperly handling firearms in a motor vehicle |\n| 137.05 | Possessing criminal tools |\n| 137.06 | Failure to secure dangerous ordnance |\n| 137.07 | Unlawful transactions in weapons |\n| 137.08 | Underage purchase of firearm or handgun |\n| 137.09 | Pointing and discharging firearms and other weapons |\n| 137.10 | License or permit to possess dangerous ordnance |\n| 137.11 | Possession of an object indistinguishable from a firearm in a school safety zone |\n| 137.12 | Possession of deadly weapon while under detention |\n| 137.13 | Concealed handgun licenses: possession of a revoked or suspended license; additional restrictions; posting of signs prohibiting possession |\n| 137.14 | Defaced firearms |\n\nChapter 138: Drug Offenses\n\nSection\n\n| Section | Description |\n|---------|-------------|\n| 138.01 | Definitions |\n| 138.02 | Trafficking in controlled substances; gift of marihuana |\n| 138.03 | Drug possession offenses |\n| 138.04 | Possessing drug abuse instruments |\n| 138.05 | Permitting drug abuse |\n| 138.06 | Illegal cultivation of marihuana |\n| 138.07 | Abusing harmful intoxicants |\n| 138.08 | Illegal dispensing of drug samples |\n| 138.09 | Federal prosecution bar to municipal prosecution |\n| 138.10 | Nitrous oxide: improper dispensing or distribution; possession in a motor vehicle |\n| 138.11 | Laboratory report required |\n| 138.12 | Counterfeit controlled substances |\n| 138.13 | Use, possession, or sale of drug paraphernalia |\n| 138.14 | Controlled substance or prescription labels |\n| 138.15 | Possession, sale and disposal of hypodermics |\n| 138.16 | Controlled substance schedules |\n| 138.17 | Unlawful furnishing of prescription to enable persons to be issued handicapped parking placards or license plates |\n| 138.18 | Pseudoephedrine sales |\n\nTITLE XV: LAND USAGE\n\nChapter 150: General Provisions\n\nSection\n\nParks and Recreation\n\n| Section | Description |\n|---------|-------------|\n| 150.01 | Recreation Board |\n| 150.02 | Board of Park Trustees |\n\nPlanning and Zoning\n\n| Section | Description |\n|---------|-------------|\n| 150.15 | Planning Commission |\n| 150.16 | Board of Zoning Appeals |\n\nPARALLEL REFERENCES\n\nOhio Legislative History References – Master Table\n\nINDEX\n\nThis summary of contents has been verified and authorized for publication by the Legislative Authority of the Municipality of Amanda Village, Ohio.\n\nSigned: \nMayor \nClerk of the Legislative Authority\n\nCERTIFICATION OF CODIFIED ORDINANCES\n\nWe, Mark A Moore, Mayor, and Carrie A Ayers, Clerk of the Legislative Authority, of the Municipality of Amanda Village, Ohio, pursuant to Ohio Revised Code §§ 731.23 and 731.42, hereby certify that the general and permanent ordinances of the Municipality, as revised, rearranged, compiled, renumbered as to sections, codified and printed herewith in component codes and titles are correct as and constitute the Code of Ordinances for the Municipality of Amanda Village, Ohio.\n\nMark A Moore\nMayor\n\nClerk of the Legislative Authority\n\nOrdinance to set rates for outside of village limits\n\nAmend Ordinance #1-2014\n\nWhereas the legislative authority deems it necessary to amend water rates.\n\nWhereas the village would like to set rates for outside the village limits.\n\nWhereas the legislative authority would like to set a 20% monthly surcharge for water on any tap outside the village limits.\n\nWhereas the legislative authority would like to set a 20% monthly surcharge for sewer on any tap outside the village limits.\n\nNow therefore let it be ordained by VILLAGE OF AMANDA, OHIO:\n\nThat the village Legislative Authority does hereby amend the water rates listed above. To add the surcharge fees for water and sewer for any tap outside of the village limits. This surcharge will be a 20% surcharge on water and sewer. That this ordinance shall take effect upon the earliest date allowed by law.\n\nMayor Mark A. Moore\n\nVote: 4 yes 0 no\n\nAttest: Carrie Ayers Fiscal Officer\n\nThe three reading requirement was waived/not waived:\nYeas: 4 nays: 0\n\nThe undersigned, Fiscal Officer of the Legislative Authority, does hereby certify that the foregoing legislation was posted in not less than five (5) public places, as determined by the legislative authority, for a period of not less than fifteen (15) days prior to the effective date thereof.\n\nFiscal Officer\n\n1st 4-3-17\n2nd Read 5-1-17\n3rd Wnd 5-1-17\n\nORDINANCE TO AMEND PROPERTY MAINTENANCE CODE\n\nWHEREAS THE VILLAGE COUNCIL DESIRES TO AMEND ORDINANCE 02-2013 THE INTERNATIONAL PROPERTY MAINTENANCE CODE\n\nWHEREAS THE LEGISLATIVE AUTHORITY DEEMS IT NECESSARY TO ADD ADDITIONAL PROVISIONS TO THE EXISTING CODE\n\nWHEREAS THE ATTACHED EXHIBITS B, C, AND D ARE TO BE AMENDED TO THE EXISTING CODE 02-2013\n\nNOW THEREFORE, LET IT BE ORDAINED BY THE VILLAGE OF AMANDA, OHIO:\n\nTHAT THE VILLAGE COUNCIL AUTHORIZES AMENDMENTS TO THE INTERNATIONAL PROPERTY MAINTENANCE CODE 02-2013, WITH THE ADDITIONS OF EXHIBIT B, C, AND D\n\nTHE ORDINANCE SHALL BE IN EFFECT AND BE IN FULL FORCE FROM AND AFTER THE EARLIEST PERIOD ALLOWED BY LAW.\n\nMark A. Moore\nMAYOR\n\nVOTE: YES 5 NO 0\n\nCarrie Ayers\nFISCAL OFFICER\n\nTHE THREE READING REQUIREMENT WAS WAIVED / NOT WAIVED:\n\nYEAS: 5 NAYS: 0\n\nFISCAL OFFICER\n\nTHE UNDERSIGNED, FISCAL OFFICER OF THE LEGISLATIVE AUTHORITY, DOES HEREBY CERTIFY THAT THE FOREGOING LEGISLATION WAS POSTED IN NOT LESS THAN FIVE (5) PUBLIC PLACES, AS DETERMINED BY THE LEGISLATIVE AUTHORITY FOR THE PERIOD OF NOT LESS THAN FIFTEEN (15) DAYS PRIOR TO THE EFFECTIVE DATE THEREOF.\n\nCarrie Ayers\nFISCAL OFFICER\n\n6-5-17 1st Reading\n\nSECTION 302\nEXTERIOR\nPROPERTY AREAS\n\n302.13 STORAGE OF JUNK VEHICLES AND OTHER MOTOR VEHICLES:\n\nDEFINITIONS\n\n(A) \"Junk motor vehicles\" means any motor vehicle which meets any three of the following requirements:\n 1. Is five years old or older;\n 2. Is extensively damaged, such as damage including but not limited to any of the following: missing wheels, tires, motor, or transmission;\n 3. Is not able to be legally operated upon a public street, road, or highway for any reason, whether mechanical, operational, or otherwise;\n 4. Does not have validly issued license plates and a valid registration which allows it to be operated legally upon public streets, roads, or highways;\n 5. Has a fair market value of fifteen hundred dollars ($1,500.00) or less;\n 6. Is not running and not operable (\"operable\" meaning able to be started and driven under its own power).\n\n(B) A junk motor vehicle left in the open for 72 hours is declared a nuisance and shall be abated in accordance with provisions of this chapter.\n\n(C) \"Motor vehicle\" means every vehicle propelled or drawn by power other than muscular power, except motorized bicycles, road rollers, traction engines, power shovels, power cranes, and other equipment used in construction work and not designed for or employed in general highway transportation, hole-digging machinery, well-drilling machinery, ditch-digging machinery, farm machinery, and trailers designed and used exclusively to transport a boat between a place of storage and a marina, or in and around a marina, when drawn to towed on a street or highway for a distance of no more than ten miles.\n\nand at a speed of twenty-five miles per hour or less\n(See ORC. Section 4511.OI(B).\n\n(D) \"In the open\" shall be defined as not garaged.\n\nPROHIBITONS.\n\n(A) No person shall willfully permit a junk motor vehicle to remain in the open on private property which the person owns, occupies, or controls after receipt of an order to remove the junk motor vehicle. The order shall state that a hearing appealing the order to remove the junk motor vehicle maybe had, shall describe the vehicle to be removed, and shall be served by a Code Enforcement Officer or Police Officer.\n\n(B) If any recipient of an order to remove a junk motor vehicle shall fails to appeal the same to the Mayor in writing within ten (10) days after the receipt of the order, it shall be conclusively presumed to establish the junk motor vehicle as a nuisance and the junk motor vehicle shall be removed immediately upon order by a code official designated by the Mayor, the Fire Department, Police Department, or their designee. The fact that a junk motor vehicle is left on private property without the filing of an appeal by the recipient of an order to remove a junk motor vehicle is prima facie evidence of willful failure to comply with the order.\n\n(C) No person shall leave a junk motor vehicle for any period of time on private property to which such person does not have the right of possession without the authorization of the person having the right of possession of such property.\n\nCOLLECTOR'S VEHICLE\n\nA collector's vehicle as defined by O.R.C. Section 4501.01 is a junk motor vehicle for purposes of this section, regardless if it is licensed or unlicensed, if the collector's vehicle meets the definition of a junk motor vehicle as set forth herein.\n\nSTORAGE AND DISPOSAL OF JUNK MOTOR VEHICLES.\n\n(A) After a motor vehicle has been determined to be a junk motor vehicle, as provided herein, the Health Department, Fire Department: Code Enforcement Officer, Police Officer, or their designee shall immediately cause the junk motor vehicle to be removed from the property where the junk motor vehicle is located. The place of storage for any junk motor vehicle which has been removed by this section shall be designated by the Mayor.\n\n(B) Any junk motor vehicle which has been stored by the Police Department pursuant to this chapter, shall be disposed of in accordance with the procedures set forth in O.R.C. Section 4513.63.\n\n**APPEALS**\n\n(A) The Board of Zoning Appeals (BZA) Shall hear any appeals within thirty (30) days of the filing of an appeal by the recipient of an order to remove a junk motor vehicle left in the open. The BZA shall rule whether or not the motor vehicle is a junk motor vehicle left in the open. If the vehicle is declared by the BZA to be a junk motor vehicle left in the open, the BZ shall order the removal of the particular vehicle by the Police Department.\n\n(B) Any appeal from a decision of the BZA shall be made pursuant to O.R.C. Section 2506.\n\n**EXCEPTIONS**\n\nThis chapter shall not apply to vehicles stored inside a garage, in a licensed towing service, in a licensed motor vehicle salvage facility, in a licensed scrap processing yard, in a licensed auto repair garage yard, in a licensed paint spray shop yard, in a licensed gasoline station yard, or in a licensed vehicle dealership yard, or any other lawful storage area not within the public view as prescribed by O.R.C. Section 4737.09.\n\n**PENALTY**\n\nWhoever fails to remove a junk motor vehicle left in the open, after receipt of a proper order to remove the same, shall be subject to the penalties provided here in. Each junk motor vehicle left in the open in violation here of shall constitute a separate offense: Every twenty (20) days that this section is violated shall constitute a separate offense.\n\nSECTION 302\nEXTERIOR PROPERTY AREAS\n\n302.10 MISCELLANEOUS PROVISIONS:\n\n(A) All exterior parts of every dwelling or structure, including exterior walls, parapet walls, decorative additions, chimneys and all other exterior structures, either above or below the roof line, shall be maintained in a safe condition, weather-tight and so as to resist decay or deterioration.\n\n(B) Any dwelling, structure or accessory building whose exterior surface is bare, deteriorated, ramshackle, tumble-down, decaying, disintegrating or in poor condition shall be repaired or razed.\n\n(C) All buckled, rotted or decayed walls, doors, windows, porches, floors, steps, railing trim and their missing members shall be replaced and put in good condition. All replacements shall match and conform to current design or be replaced completely.\n\n(D) All exterior wood or unfinished surface shall be sealed and painted or the surface covered with other protective coating or treated to prevent rot and decay and to conform and match the existing paint or surface covering and original design or replacement thereof. All exterior walls and surfaces shall be properly protected against the weather where such are defective or lack weather protection, including lack of paint or surface covering or have weathered due to lack of proper protective coating.\n\n(E) Any dwelling structure or secondary or appurtenant structure whose exterior surface is deteriorated, decaying, disintegrating, or whose exterior surface has weathered with dirt and grime or has been impaired through the peeling or flaking of paint or other protective coating, and which exterior surfaces consist of an area of twenty-five percent (25%) or more of the external surface of the structure, shall be repaired, repainted or recovered with an approved protective coating or surface. All exterior surfaces shall be replaced or repaired in good condition preparatory to painting or coating. All bare exterior surfaces, which are flaking or crumbling shall be replaced or sealed in a good and workmanlike manner. All new or repaired bare surfaces shall be painted or coated. All exterior surfaces weathered\n\nwith dirt or grime, which are peeling or flaking, shall be painted or covered with approved protective coating or surface.\n\n(F) All repairs shall be completed in a timely manner consistent with accepted construction practices.\n\n(G) All dwelling structures and the premises thereof shall be maintained free from sources of breeding, harborage and infestation by insects, vermin or rodents.\n\n(H) No owner, operator or resident agent of any premises shall maintain or permit to be maintained at or on the exterior areas of any such premises any condition which deteriorates or debases the appearance of the neighborhood, reduces property values in the neighborhood, adversely alters the appearance and general character of the neighborhood, creates a fire, safety or health hazard or which is a public nuisance. Such conditions include but are not limited to the following:\n\n1. Broken or dilapidated fences, walls or other structures;\n2. Out-of-use or non-usable appliances and machinery;\n3. Rugs, rags or other materials hung on lines or in other places on the premises, which materials are not being used for general household or housekeeping purposes;\n4. Broken, dilapidated or unusable furniture, mattresses or other household furniture, broken glass, plastic materials, paints, miscellaneous coverings and/or any other materials, including those described in this section, placed at or on the premises in such a manner as to be patently unsightly, grotesque or offensive to the senses;\n5. No unregistered motor vehicles and/or junk vehicles shall be parked on any property for more than thirty (30) days;\n6. Lawns and landscaping shall be so maintained so as not to constitute a blighting or deteriorating effect on the neighborhood. Grass brush, briers, burrs shall be maintained at a height not to exceed twelve (12) inches. This provision shall not apply to vegetable gardens, bushes, vines, shrubs or trees that produce food for human consumption;\n7. No building materials or materials, earth, sand or dirt intended for use in landscaping, gardening or construction shall be left standing open or covered upon any premises for a period of time exceeding two (2) months without specific written authorization from the Village Zoning Department.\n\n(I) New construction initiated on the exterior of any structure or surrounding yard, after the effective date of the adoption of this provision, shall be completed within one (1) year. In the event of unforeseeable delays in construction, the Code Enforcement Officer is authorized to extend the one (1) year time period for an additional time period of six (6) months or less.\n\nSection 302 Exterior Property Areas\n\n302.11 STORAGE OF CERTAIN MATERIALS DECLARED A NUISANCE:\n\n(A) Definitions\n\n1. Automobile parts\" means and includes any portion or parts of any motor driven vehicle as detached from the vehicle as a whole.\n\n2. Motor vehicle in an inoperative condition\" means and includes any style or type of motor-driven vehicle used or useful for the conveyance of persons or property which is unable to move under its own power due to defective or missing parts, and which have remained in such condition for a period of not less than 30 consecutive days.\n\n3. Motor vehicle unfit for further use\" means and includes any style type of motor-driven vehicle used for the conveyance of persons or property, which is in a dangerous condition, has defective or missing parts, or is in such a condition generally as to be unfit for further (1se as a conveyance.\n\n4. Refuse\" embraces only such matter as was either in fact noxious or has been refused and abandoned by its owner as worthless.\n\n5. Rubbish\" means and includes wire, chips, shavings, bottles, broken glass, crockery, tin, cast or wooden ware, boxes, rags, dead weeds, paper circulars, handbills, boots, shoes, ashes or any waste material other than garbage or offal.\n\n6. Scrap metal\" means and includes pieces of or parts of steel, iron, tm, zinc, copper, aluminum, or any alloy thereof, whether covered with porcelain\n\nor any other material, whether intact or in parts, which has served its usefulness in its original form and can no longer be used or useful for its originally intended purpose.\n\n7. \"Used building materials\" means and includes any materials, such as wood, stone, brick, cement block, asphalt, blacktop, concrete, construction debris, or any composition thereof, used or useful in the erection of any building or structure, which have been used previously for such erection or construction, by the same persons or by any other person.\n\n(B) Storage of Certain Materials Declared a Nuisance.\n\nIt shall be unlawful and is hereby declared a nuisance for any reason to store, place or allow to remain, automobile parts, motor vehicles in an inoperative condition, motor vehicles unfit for further use, refuse, rubbish, scrap metal, or used building materials, as defined herein, on any lot, lots, parts of lots, or parcel of land, within the corporate limits if the Village.\n\n(C) Order for Removal.\n\n(1) In the event of a violation of division (b) of this section, the Mayor or Property Maintenance Code Officer shall give notice to the owner, occupant or person having charge of the premises upon which the violation occurs to cease such violation. Such notice shall be in writing and shall be served upon the owner, occupant or person having charge of the premises either personally or at the usual place of residence of such owner, occupant or person having charge of such premises or by registered or certified mail addressed to such person's last known place of residence.\n\n(2) If the person served with the notice as provided in division (1) fails to cause such violation to cease within ten days of the date upon which the notice w\n\nissued, he or she shall be subject to the penalties provided herein, and a separate offense shall be deemed committed upon each day during or on which the violation occurs or continues beyond such ten-day period; provided that no additional notice of violation is required to be given.\n\n(D) Exception\n\nNotwithstanding the provisions of division (b) of this section, it shall not be unlawful for any person to purchase used building materials and place or store them on any lot, lots parts of lots, or parcel of land, when such materials are stored within the confines of a closed garage, or building or are to a used by the purchaser or owner in later construction on the same lot or any lot owned or controlled by such person; provided that such materials shall not remain on the lot, lots, parts of lots, or parcel of land, for a period of more than 30 days, unless the construction or erection planned for the use of the materials has commenced; and provided further that such materials are used or consumed in the construction or removed from the premises within a period of four months from the time the materials are first placed on the lot, lots, parts of lots or parcel off and. It shall be unlawful for any person or persons to move any materials so stored or placed to another location within the village for the purpose of avoiding the intent of this section, except that any such materials may be moved to another lot, lots, parts of lots or parcel of land, when the same have been sold to a bona fide purchaser for value for such purchaser's own use.\n\nSECTION 302\nEXTERIOR\nPROPERTY AREAS\n\n302.13 STORAGE OF JUNK VEHICLES AND OTHER MOTOR VEHICLES:\n\nDEFINITIONS:\n(A) \"Junk motor vehicles\" means any motor vehicle which meets any three of the following requirements:\n 1. Is five years old or older;\n 2. Is extensively damaged, such as damage including but not limited to any of the following: missing wheels, tires, motor, or transmission;\n 3. Is not able to be legally operated upon a public street, road, or highway for any reason, whether mechanical, operational, or otherwise;\n 4. Does not have validly issued license plates and a valid registration which allows it to be operated legally upon public streets, roads, or highways;\n 5. Has a fair market value of fifteen hundred dollars ($1,500.00) or less;\n 6. Is not running and not operable (\"operable\" meaning able to be started and driven under its own power);\n\n(B) A junk motor vehicle left in the open for 72 hours is declared a nuisance and shall be abated in accordance with provisions of this chapter.\n\n(C) \"Motor vehicle\" means every vehicle propelled or drawn by power other than muscular power, except motorized bicycles, road rollers, traction engines, power shovels, power cranes, and other equipment used in construction work and not designed for or employed in general highway transportation, hole-digging machinery, well-drilling machinery, ditch-digging machinery, farm machinery, and trailers designed and used exclusively to transport a boat between a place of storage and a marina, or in and around a marina, when drawn to towed on a street or highway for a distance of no more than ten miles.\n\nand at a speed of twenty-five miles per hour or less\n(See ORC. Section 451I.Ol(B).\n\n(D) \"In the open\" shall be defined as not garaged.\n\nPROHIBITIONS.\n\n(A) No person shall willfully permit a junk motor vehicle to remain in the open on private property which the person owns, occupies, or controls after receipt of an order to remove the junk motor vehicle. The order shall state that a hearing appealing the order to remove the junk motor vehicle maybe had, shall describe the vehicle to be removed, and shall be served by a Code Enforcement Officer or Police Officer.\n\n(B) If any recipient of an order to remove a junk motor vehicle shall fails to appeal the same to the Mayor in writing within ten (10) days after the receipt of the order, it shall be conclusively presumed to establish the junk motor vehicle as a nuisance and the junk motor vehicle shall be removed immediately upon order by a code official designated by the Mayor, the Fire Department, Police Department, or their designee. The fact that a junk motor vehicle is left on private property without the filing of an appeal by the recipient of an order to remove a junk motor vehicle is prima facie evidence of willful failure to comply with the order.\n\n(C) No person shall leave a junk motor vehicle for any period of time on private property to which such person does not have the right of possession without the authorization of the person having the right of possession of such property.\n\nCOLLECTOR'S VEHICLE\n\nA collector's vehicle as defined by O.R.C. Section 4501.01 is a junk motor vehicle for purposes of this section, regardless if it is licensed or unlicensed, if the collector's vehicle meets the definition of a junk motor vehicle as set forth herein.\n\nSTORAGE AND DISPOSAL OF JUNK MOTOR VEHICLES.\n\n(A) After a motor vehicle has been determined to be a junk motor vehicle, as provided herein, the Health Department, Fire Department: Code Enforcement Officer, Police Officer, or their designee shall immediately cause the junk motor vehicle to be removed from the property where the junk motor vehicle is located. The place of storage for any junk motor vehicle which has been removed by this section shall be designated by the Mayor.\n\n(B) Any junk motor vehicle which has been stored by the Police Department pursuant to this chapter, shall be disposed of in accordance with the procedures set forth in O.R.C. Section 4513.63.\n\nAPPEALS\n\n(A) The Board of Zoning Appeals (BZA) Shall hear any appeals within thirty (30) days of filing an appeal by the recipient of an order to remove a junk motor vehicle left in the open. The BZA shall rule whether or not the motor vehicle is a junk motor vehicle left in the open.\n\nIf the vehicle is declared by the BZA to be a junk motor vehicle left in the open, the BZ shall order the removal of the particular vehicle by the Police Department.\n\n(B) Any appeal from a decision of the BZA shall be made pursuant to O.R.C. Section 2506.\n\nEXCEPTIONS\n\nThis chapter shall not apply to vehicles stored inside a garage, in a licensed towing service, in a licensed motor vehicle salvage facility, in a licensed scrap processing yard, in a licensed auto repair garage yard, in a licensed paint spray shop yard, in a licensed gasoline station yard, or in a licensed vehicle dealership yard, or any other lawful storage area not within the public view as prescribed by O.R.C. Section 4737.09.\n\nPENALTY\n\nWhoever fails to remove a junk motor vehicle left in the open, after receipt of a proper order to remove the same, shall be subject to the penalties provided here in. Each junk motor vehicle left in the open in violation here of shall constitute a separate offense. Every twenty (20) days that this section is violated shall constitute a separate offense.\n\nORDINANCE TO ADOPTING AN UPDATED VERSION OF THE (2018) INTERNATIONAL PROPERTY MAINTENANCE CODE, AS AMENDED, TOGETHER WITH ALL UPDATED REVISIONS OF SAID CODE.\n\nWHEREAS THE VILLAGE COUNCIL DESIRES TO AMEND ORDINANCE 02-2013 THE INTERNATIONAL PROPERTY MAINTENANCE CODE TO ADD ALL REVISIONS AND UPDATED VERSIONS;\n\nWHEREAS THE VILLAGE COUNCIL DESIRES TO ADOPT THE UPDATED 2018 INTERNATIONAL PROPERTY MAINTENANCE CODE;\n\nWHEREAS THE LEGISLATIVE AUTHORITY DEEMS IT NECESSARY TO ADD ADDITIONAL PROVISIONS TO THE EXISTING CODE IN ORDER TO MAINTAIN THE SAFETY AND WELLBEING OF THE CITIZENS;\n\nNOW THEREFORE, LET IT BE ORDAINED BY THE VILLAGE OF AMANDA, OHIO:\n\nTHAT THE VILLAGE COUNCIL AUTHORIZES AMENDMENTS AND MOVES TO ADOPT THE 2018 INTERNATIONAL PROPERTY MAINTENANCE CODE,\n\nTHE ORDINANCE SHALL BE IN EFFECT AND BE IN FULL FORCE FROM AND AFTER THE EARLIEST PERIOD ALLOWED BY LAW.\n\nMAYOR\n\nVOTE: YES 5 NO 0\n\nCARRIE AYERS\nFISCAL OFFICER\n\nTHE THREE READING REQUIREMENT WAS WAIVED / NOT WAIVED:\n\nYEAS: 5 NAYS: 0\n\nFISCAL OFFICER\n\nTHE UNDERSIGNED, FISCAL OFFICER OF THE LEGISLATIVE AUTHORITY, DOES HEREBY CERTIFY THAT THE FOREGOING LEGISLATION WAS POSTED IN NOT LESS THAN FIVE (5) PUBLIC PLACES, AS DETERMINED BY THE LEGISLATIVE AUTHORITY FOR THE PERIOD OF NOT LESS THAN FIFTEEN (15) DAYS PRIOR TO THE EFFECTIVE DATE THEREOF.\n\nFISCAL OFFICER\n\nTo: Village Officials\n\nRe: Subscription to the Ohio Basic Code, 2018 Edition\n\nThank you for your subscription to the *Ohio Basic Code, 2018 Edition*. This model Code of Ordinances has been adopted by the Village as their own Code, and the 2018 Edition replaces any previous editions of the Village Code. Please note, however, that if you have received a looseleaf “Title 17: Municipal Regulations,” this **Title 17 is not affected by this 2018 Edition and should be retained**. Please mail any Village ordinances to update your Title 17 to our office at your earliest convenience.\n\nEnclosed you will also find a sample adopting ordinance, a summary of new matter (for publication notification), and a certification of codified ordinances, all provided for your convenience in adopting the *Ohio Basic Code, 2018 Edition*.\n\nWe are pleased to provide the *Ohio Basic Code, 2018 Edition*, and we look forward to continuing to serve Ohio villages.\n\nVery truly yours,\n\nTodd Paul Myers, J.D.\nVice President, Major Client Services\nemail@example.com\n\nCERTIFICATION OF CODIFIED ORDINANCES\n\nWe, ________________, Mayor, and ________________, Clerk of the Legislative Authority, of the Municipality of ________________, Ohio, pursuant to Ohio Revised Code §§ 731.23 and 731.42, hereby certify that the general and permanent ordinances of the Municipality, as revised, rearranged, compiled, renumbered as to sections, codified and printed herewith in component codes and titles are correct as and constitute the Code of Ordinances for the Municipality of ________________, Ohio.\n\n__________________________\nMayor\n\n__________________________\nClerk of the Legislative Authority\n\nAN ORDINANCE APPROVING, ADOPTING AND ENACTING AMERICAN LEGAL PUBLISHING’S OHIO BASIC CODE, 2018 EDITION, AS THE CODE OF ORDINANCES FOR THE MUNICIPALITY OF ________, OHIO, AND DECLARING AN EMERGENCY.\n\nWHEREAS, the present general and permanent ordinances of the municipality are inadequately arranged and classified and are insufficient in form and substance for the complete preservation of the public peace, health, safety and general welfare of the municipality and for the proper conduct of its affairs.\n\nWHEREAS, American Legal Publishing Corporation publishes a Code of Ordinances suitable for adoption by municipalities in Ohio.\n\nWHEREAS, it is necessary to provide for the usual daily operation of the municipality and for the immediate preservation of the public peace, health, safety and general welfare of the municipality that this ordinance take effect at an early date.\n\nNOW, THEREFORE, BE IT ORDAINED BY THE LEGISLATIVE AUTHORITY OF THE MUNICIPALITY OF ________, OHIO:\n\nSection 1. American Legal Publishing’s Ohio Basic Code, 2018 Edition, as reviewed and approved by the Legislative Authority, is hereby adopted and enacted. Any prior version of the Ohio Basic Code which may have been previously adopted by the municipality is hereby repealed as obsolete and is hereby replaced in its entirety by this Ohio Basic Code, 2018 Edition.\n\nSection 2. One copy of American Legal Publishing’s Ohio Basic Code, 2018 Edition, certified as correct by the Mayor and Clerk of the Legislative Authority, as required by Ohio Revised Code § 731.23, shall be kept in its initial form on file in the office of the Clerk of the municipality and retained as a permanent ordinance record of the municipality. The Clerk of the municipality is authorized and directed to publish a summary of all new matters contained in the Code of Ordinances as required by Ohio Revised Code § 731.23. Such summary is attached hereto and marked as “Exhibit A”.\n\nSection 3. All ordinances and resolutions or parts thereof which are in conflict or inconsistent with any provision of the Ohio Basic Code, 2018 Edition, as adopted in Section 1 hereof, are hereby repealed as of the effective date of this ordinance, except as follows:\n(A) The enactment of the Ohio Basic Code, 2018 Edition, shall not be construed to affect a right or liability accrued or incurred under any legislative provision prior to the effective date of such enactment, or an action or proceeding for the enforcement of such right or liability. Such enactment shall not be construed to relieve any person from punishment for an act committed in violation of any such legislative provision, nor to affect an indictment or prosecution therefor. For such purposes, any such legislative provision shall continue in full force notwithstanding its repeal for the purpose of revision and codification.\n\n(B) The repeal provided above shall not affect:\n (1) The grant or creation of a franchise, license, right, easement or privilege;\n (2) The purchase, sale, lease or transfer of property;\n (3) The appropriation or expenditure of money or promise or guarantee of payment;\n (4) The assumption of any contract or obligation;\n (5) The issuance and delivery of any bonds, obligations or other instruments of indebtedness;\n (6) The levy or imposition of taxes, assessments or charges;\n (7) The establishment, naming, vacating or grade level of any street or public way;\n (8) The dedication of property or plat approval;\n (9) The annexation or detachment of territory;\n (10) Any legislation enacted subsequent to the adoption of this ordinance.\n (11) Any legislation specifically superseding the provision of the Ohio Basic Code.\n\nSection 4. Whenever reference is made in any documents, publications, or signs of the municipality, including but not limited to traffic tickets and traffic-control signs, to a section as it existed in a former edition of the Ohio Basic Code, the reference shall extend and apply to the section referred to as subsequently amended, revised, recodified, or renumbered.\n\nSection 5. This ordinance is declared to be an emergency measure necessary for the immediate preservation of the peace, health, safety and general welfare of the people of this municipality, and shall take effect at the earliest date provided by law.\n\nDate Passed: 2-5-17\n\nAttest:\n\nMayor\n\nClerk of the Legislative Authority\n\nNotice is hereby given that on the 5th day of Feb., 2018, there was enacted by the Legislative Authority of the Municipality of Village of Avon, Ohio, an ordinance entitled “An Ordinance Approving, Adopting and Enacting American Legal Publishing’s Ohio Basic Code, 2018 Edition, as the Code of Ordinances for the Municipality of Village of Avon, Ohio.”\n\nA summary of the subjects, including all new matters contained in the Code of Ordinances, as adopted, are as follows. The majority of Basic Code provisions are based directly on state law.\n\nTITLE I: GENERAL PROVISIONS\n\nChapter 10: General Provisions\n\nSection\n\n10.01 Short titles\n10.02 Definitions\n10.03 Rules of construction\n10.04 Revivor; effect of amendment or repeal\n10.05 Construction of section references\n10.06 Conflicting provisions\n10.07 Severability\n10.08 Reference to offices\n10.09 Errors and omissions\n10.10 Ordinances repealed\n10.11 Ordinances unaffected\n10.12 Ordinances saved\n10.13 Application to future ordinances\n10.14 Interpretation\n10.15 Amendments to code; amendatory language\n10.16 Statutory references\n10.17 Preservation of penalties, offenses, rights and liabilities\n10.18 Determination of legislative intent\n10.99 General penalty\n\nTITLE III: ADMINISTRATION\n\nChapter 30: General Provisions\n\nSection\n\n30.01 Application of Title III\n30.02 Qualifications; oaths\n30.03 Bonds of officers and employees; amount\n30.04 Additional bond; where bonds recorded and kept\n30.05 Approval of bonds\n30.06 Sufficiency of form of bond\n30.07 Filling vacancies in offices\n30.08 Public records available\n30.09 Records Commission\n30.10 Meetings of public bodies to be open; exceptions; notice\n30.11 Municipal officers may attend conference or convention; expenses\n30.12 Residency requirements prohibited; exceptions\n\nChapter 31: Executive Authority\n\nSection\n\nGeneral Provisions\n\n31.001 Executive power; where vested\n\n**Mayor**\n\n31.015 Term of Mayor; power and duties \n31.016 General duties of the Mayor \n31.017 Communications to the Legislative Authority \n31.018 Protest against excess of expenditures \n31.019 Supervision of conduct of officers \n31.020 Annual report to the Legislative Authority \n31.021 Mayor to file charges against delinquent officers \n31.022 Vacancies in office of Mayor \n31.023 Disposition of fines and other moneys\n\n**Clerk**\n\n31.040 Election, term, qualifications of the Clerk \n31.041 Powers and duties of Clerk \n31.042 Books and accounts; merger of offices \n31.043 Seal of Clerk \n31.044 Combined offices of Clerk and Treasurer; Fiscal Officer\n\n**Treasurer**\n\n31.060 Election, term, qualifications of the Treasurer \n31.061 Accounts of Treasurer \n31.062 Powers and duties \n31.063 Quarterly account; annual report \n31.064 Receipt and disbursement of funds \n31.065 Duty of delivering money and property\n\n**Street Commissioner**\n\n31.080 Qualifications \n31.081 General duties \n31.082 Assistants\n\n**Other Officials**\n\n31.100 Legal counsel \n31.101 Administrator \n31.102 Board of Trustees of Public Affairs \n31.103 Fire Engineer, Engineer and Superintendent of Markets\n\n---\n\n**Chapter 32: Legislative Authority**\n\n**General Provisions**\n\n32.001 Members of the Legislative Authority; election; terms of office \n32.002 President Pro Tempore; employees \n32.003 Vacancy when President Pro Tempore becomes Mayor \n32.004 Qualifications of members of the Legislative Authority \n32.005 Compensation and bonds of municipal officers and employees \n32.006 Vacancy \n32.007 Judge of election and qualification of members; quorum and special meetings \n32.008 Rules; journal; expulsion of members \n32.009 Meetings \n32.010 General powers \n32.011 Failure to take oath or give bond \n32.012 Notice when new bond required \n32.013 Care, supervision and management of public institutions\n\n### Contracts, Bids and Proceedings\n\n32.025 Contracts by the Legislative Authority or Administrator \n32.026 Bids and proceedings \n32.027 Alterations or modifications of contract \n32.028 Contract restrictions \n32.029 Award to lowest responsive and responsible bidder \n\n### Ordinances and Resolutions\n\n32.040 Ordinances and resolutions as evidence \n32.041 Passage procedure \n32.042 Style of ordinances \n32.043 Subject and amendment of ordinances and resolutions \n32.044 Authentication and recording of ordinances and resolutions \n32.045 Publication of ordinances and resolutions; proof of publication and circulation \n32.046 Notice for proposed amendments to the municipal Charter \n32.047 Times of publication required \n32.048 Publication and certification of ordinances in book form \n32.049 Adoption of technical ordinances and codes \n32.050 Certificate of Clerk as to publication \n32.051 Publication when no newspaper published in municipality \n32.052 Effect of not making publication \n32.053 Ordinances providing for appropriations or street improvements; emergency ordinances \n\n### Initiative and Referendum\n\n32.070 Initiative petitions \n32.071 Referendum petitions \n32.072 More than one ordinance required; application of subchapter \n32.073 Presentation of petitions \n32.074 Copy of proposed ordinance or measure to be filed with Clerk \n32.075 Words to be printed in red \n32.076 Designation of committee filing petition; public inspection of petitions; ordinances passed or repealed prior to election \n32.077 Itemized statement by petition circulator \n32.078 Prohibited practices relative to petitions \n32.079 Accepting premiums for signing \n32.080 Threats in securing signatures \n32.081 Application of subchapter if Charter adopted \n\n### Chapter 33: Judicial Authority\n\n#### General Provisions\n\n33.01 Jurisdiction in ordinance cases and traffic violations \n33.02 Powers of Mayor and Mayor’s Court Magistrate in criminal matters \n33.03 Duties of Mayor and Mayor’s Court Magistrate; fees; office; seal \n33.04 Mayor’s Court Magistrate \n33.05 Powers to suspend driver’s license in OVI cases \n\n#### Contempt of Court\n\n33.20 Summary punishment for contempt \n33.21 Acts in contempt of court \n33.22 Hearing \n33.23 Contempt action for failure to pay support, failure to comply or interference with a visitation order; summons \n33.24 Right of accused to bail \n33.25 Hearing on contempt; penalties; support orders; failure to withhold or deduct money pursuant to support order \n33.26 Imprisonment until order obeyed \n33.27 Proceedings when party released on bail fails to appear\n\n33.28 Release of prisoner committed for contempt\n33.29 Judgment final\n33.30 Alternative remedy\n\nChapter 34: Police Department\n\nSection\n\n34.01 Marshal and Police Chief synonymous\n34.02 Appointment of Marshal\n34.03 Deputy marshals and police officers\n34.04 Auxiliary police units\n34.05 Offenses affecting employment of law enforcement officers; probationary period; final appointment\n34.06 Removal proceedings; suspension; appeals\n34.07 General powers\n34.08 Powers and duties of Marshal\n34.09 Disposition of fines and penalties\n34.10 Property recovered by police\n34.11 Disposition to claimant\n34.12 Sale of unclaimed property; disposition of proceeds\n34.13 Expenses of storage and sale; notice\n34.14 Contracts for police protection; nonresident service without contract\n34.15 Peace officer administering oaths; acknowledging complaints, summonses, affidavits and returns of court orders\n\nChapter 35: Fire Department\n\nGeneral Provisions\n\nSection\n\n35.01 Municipal fire regulations; fire department\n35.02 Fire Chief; Fire Prevention Officer; employment of firefighters; criminal records check for firefighters\n35.03 Schooling of officers and firefighters of fire department\n35.04 Legislative Authority may purchase engines and equipment\n35.05 Buildings for department\n35.06 Records\n35.07 Maximum consecutive hours for firefighters on duty\n35.08 Investigation of cause of fire\n35.09 Right to examine buildings, premises, and vehicles\n35.10 Burning buildings for firefighting instruction or research\n35.11 Impersonating fire safety inspector\n35.12 Standards for equipment\n35.13 Persons entitled to be known as firefighters\n35.14 Firefighting and emergency services agreements\n35.15 Regulation of construction in fire limits\n\nVolunteer Firefighters’ Dependents Fund Board\n\nSection\n\n35.30 Definitions\n35.31 Establishment\n35.32 Membership; vacancies\n35.33 Election and term of members\n35.34 Organization; rules and regulations; roster\n35.35 Compensation and expenses of Board; legal advisor\n\nChapter 36: Civil Actions Against the Municipality\n\nSection\n\n36.01 Definitions\n36.02 Nonliability of municipality; exceptions\n36.03 Defenses and immunities\n36.04 Limitation of actions\n36.05 Damages\n36.06 Satisfaction of judgments\n36.07 Provision of employees’ defense; consent judgments\n36.08 Liability insurance\n36.09 Certain actions unaffected\n36.10 Certain charges against municipal officers filed with Probate Judge; proceedings\n\nTITLE VII: TRAFFIC CODE\n\nChapter 70: General Provisions\n\nGeneral Provisions\n\n| Section | Description |\n|---------|-------------|\n| 70.01 | Definitions |\n| 70.02 | Compliance with order of police officer |\n| 70.03 | Emergency vehicles to proceed cautiously past red or stop signal |\n| 70.04 | Exceptions generally; emergency, public safety and coroner vehicles exempt |\n| 70.05 | Persons riding or driving animals upon roadways |\n| 70.06 | Prohibitions against pedestrians and slow-moving vehicles on freeways |\n| 70.07 | Use of private property for vehicular travel |\n| 70.08 | Names of persons damaging real property by operation of vehicle to be provided to owner |\n| 70.09 | Limited access highways; barriers along; vehicles to enter and leave at designated intersections |\n| 70.10 | Through highways |\n| 70.11 | Officer may remove ignition key |\n| 70.12 | Removal of vehicles after accidents |\n\nTraffic-Control Devices\n\n| Section | Description |\n|---------|-------------|\n| 70.30 | Obeying traffic-control devices |\n| 70.31 | Signal lights |\n| 70.32 | Signals over reversible lanes |\n| 70.33 | Ambiguous or non-working traffic signals |\n| 70.34 | Pedestrian-control signals |\n| 70.35 | Unauthorized signs and signals prohibited |\n| 70.36 | Alteration, defacement, or removal prohibited |\n| 70.37 | Unauthorized possession or sale of devices |\n| 70.38 | Signal preemption devices; prohibitions |\n| 70.99 | Penalty |\n\nChapter 71: Licensing Provisions\n\nMotor Vehicle Licensing\n\n| Section | Description |\n|---------|-------------|\n| 71.01 | Display of license plates or validation stickers; registration |\n| 71.02 | Improper use of noncommercial motor vehicle |\n| 71.03 | Operating motor vehicle ordered immobilized; forfeiture |\n| 71.04 | Operation or sale without certificate of title |\n| 71.05 | Display of certificate of registration |\n| 71.06 | Use of unauthorized plates |\n| 71.07 | Operating without dealer or manufacturer license plates |\n\nDriver’s Licenses\n\n| Section | Description |\n|---------|-------------|\n| 71.20 | Prohibited acts |\n| 71.21 | Permitting minor to operate vehicle prohibited; temporary instruction permit; probationary license |\n| 71.22 | License required as driver or commercial driver on public or private property; nonresident exemption |\n| 71.23 | Employment of a minor to operate a taxicab prohibited |\n| 71.24 | Restriction against owner lending vehicle for use of another |\n| 71.25 | Suspension of driver’s licenses; license suspended by court of record |\n| 71.26 | Display of license |\n| 71.27 | Prohibition against false statements |\n| 71.28 | Driving under suspension or in violation of license restriction |\n\n71.29 Operating motor vehicle or motorcycle without valid license\n71.30 Driving under OVI suspension\n71.31 Driving under financial responsibility law suspension or cancellation; driving under a nonpayment of judgment suspension\n71.32 Failure to reinstate license\n\n**Commercial Driver's Licenses**\n\n71.45 Definitions\n71.46 Use of actual gross weight in lieu of rating\n71.47 Prohibited acts\n71.48 Prerequisites to operation of commercial motor vehicle\n71.49 Physical qualification to operate commercial motor vehicles\n71.50 Criminal offenses\n71.51 Application of federal regulations\n71.52 Employment of drivers of commercial vehicles\n71.99 Penalty\n\n**Chapter 72: Traffic Rules**\n\n**General Provisions**\n\n72.001 Lanes of travel upon roadways\n72.002 Driving through safety zone\n72.003 Vehicles traveling in opposite directions\n72.004 Rules governing overtaking and passing of vehicles\n72.005 Permission to overtake and pass on the right\n72.006 Driving to left of center line\n72.007 Prohibition against driving upon left side of roadway\n72.008 Hazardous zones\n72.009 One-way highways and rotary traffic islands\n72.010 Rules for driving in marked lanes\n72.011 Space between moving vehicles\n72.012 Divided roadways\n72.013 Rules for turns at intersections\n72.014 U-turns and turning in roadway prohibited\n72.015 Starting and backing vehicles\n72.016 Turn and stop signals\n72.017 Hand and arm signals\n\n**Right-of-Way**\n\n72.030 Right-of-way at intersections\n72.031 Right-of-way when turning left\n72.032 Right-of-way at through highways; stop signs; yield signs\n72.033 Stop at sidewalk area; stop signs on private roads and driveways\n72.034 Right-of-way on public highway\n72.035 Pedestrian on sidewalk has right-of-way\n72.036 Right-of-way of public safety vehicles\n72.037 Funeral procession has right-of-way\n72.038 Pedestrians yield right-of-way to public safety vehicle\n72.039 Pedestrian on crosswalk has right-of-way\n72.040 Right-of-way yielded to blind person\n72.041 Right-of-way yielded by pedestrian\n\n**Pedestrians**\n\n72.055 Pedestrian movement in crosswalks\n72.056 Pedestrian walking along highway\n72.057 Prohibition against soliciting rides; riding on outside of vehicle\n72.058 Pedestrian on bridge or railroad crossing\n72.059 Persons operating motorized wheelchairs\n\n72.060 Intoxicated or drugged pedestrian hazard on highway\n72.061 Operation of electric personal assistive mobility devices\n72.062 Operation of personal delivery device on sidewalks and crosswalks\n\n**Grade Crossings**\n\n72.075 Stop signs at grade crossings\n72.076 Driving vehicle across railroad grade crossing\n72.077 Vehicles required to stop at grade crossings\n72.078 Slow-moving vehicles or equipment crossing railroad tracks\n\n**School Buses**\n\n72.090 Regulations concerning school buses\n72.091 Violation of regulations; report; investigation; citation; warning\n72.092 Restrictions on the operation of school buses\n72.093 School bus inspection\n72.094 School bus not used for school purposes\n72.095 Licensing by Department of Public Safety\n72.096 Registration and identification of school buses\n72.097 School bus marking\n72.098 Flashing light signal lamps\n72.099 Occupant restraining device for operator\n\n**Prohibitions**\n\n72.115 Obstruction and interference affecting view and control of driver\n72.116 Occupying travel trailer while in motion\n72.117 Driving upon closed highway prohibited\n72.118 Driving upon sidewalk area or paths exclusively for bicycles\n72.119 Obstructing passage of other vehicles\n72.120 Following an emergency or public vehicle prohibited; approaching stationary public safety vehicle with caution\n72.121 Driving over unprotected fire hose\n72.122 Placing injurious material on highway or depositing litter from motor vehicle\n72.123 Transporting child not in child-restraint system prohibited\n72.124 Occupant restraining devices\n72.125 Use of engine brakes prohibited\n72.126 Operating motor vehicle while wearing earphones or earplugs\n72.127 Chauffeured limousines and livery services\n72.128 Operating traction engine upon improved highway\n72.129 Cracking exhaust noises; peeling out\n72.130 Shortcutting across private property\n72.131 Texting while driving prohibited\n72.132 Use of electronic wireless communication devices by minors or probationary drivers while driving prohibited\n\n**Chapter 73: Motor Vehicle Crimes**\n\n**General Provisions**\n\n73.01 Driving under the influence of alcohol or drugs\n73.02 Implied consent\n73.03 Physical control of vehicle while under the influence\n73.04 Driving commercial vehicle with impaired alertness or ability; use of drugs\n73.05 Reckless operation of vehicles\n73.06 Reckless operation off streets and highways; competitive operation\n73.07 Operator to be in reasonable control\n73.08 Immobilizing or disabling device violation\n73.09 Street racing defined; prohibited on public highways\n73.10 Speed limits\n73.11 Slow speed or stopping\n73.12 Emergency vehicles excepted from speed limitation\n73.13 Speed regulations on bridges\n\n73.14 Presenting false name or information to officer\n73.15 Prohibition against resisting officer\n73.16 Operation restricted for mini-trucks and low-speed, under-speed, or utility vehicles\n\n**Stopping After Accident**\n\n73.30 Failure to stop after accident\n73.31 Stopping after accident on other than public roads or highways\n73.32 Accident involving damage to realty\n73.33 Failure to report accident\n\n**Chapter 74: Equipment and Loads**\n\n**Equipment**\n\n74.01 Unsafe vehicles, prohibition against operation\n74.02 Bumpers on motor vehicles\n74.03 Lighted lights required\n74.04 Headlights\n74.05 Tail lights and illumination of rear license plate\n74.06 Red reflectors required\n74.07 Safety lighting of commercial vehicles\n74.08 Stoplight regulations\n74.09 Obscured lights on vehicles\n74.10 Red light or flag required\n74.11 Lights on parked vehicles\n74.12 Lights and emblem on slow-moving vehicles; lights and reflectors on multi-wheel agricultural tractors or farm machinery\n74.13 Spotlight and auxiliary driving lights\n74.14 Cowl, fender, and back-up lights\n74.15 Two lights displayed\n74.16 Headlights required\n74.17 Lights of less intensity\n74.18 Number of lights permitted; red and flashing lights\n74.19 Standards for lights on snow removal equipment and oversize vehicles\n74.20 Flashing lights permitted for certain types of vehicles\n74.21 Lights and sign on transportation for preschool children\n74.22 Focus and aim of headlights\n74.23 Brake equipment; specifications\n74.24 Brake fluid\n74.25 Minimum standards for brakes and components\n74.26 Horns, sirens, and warning devices\n74.27 Mufflers; excessive smoke or gas\n74.28 Rearview mirrors\n74.29 Windshields and wipers\n74.30 Solid tire requirements\n74.31 Requirements for safety glass in motor vehicles; use of tinted glass or reflectorized material\n74.32 Directional signals\n74.33 Installation and sale of seat safety belts required; definition\n74.34 Requirements for extra signal equipment\n74.35 Display of warning devices on disabled vehicles\n74.36 Requirements for vehicles transporting explosives\n74.37 Studded tires; seasonal use permitted\n74.38 Safety inspection decals for buses\n74.39 Air bags\n\n**Loads**\n\n74.50 Permit required to exceed load limits\n74.51 Limitation of load extension on left side of vehicle\n74.52 All loads shall be properly secured\n74.53 Towing requirements; exception to size and weight restrictions\n74.54 Weighing of vehicle; removal of excess load\n\n74.55 Operation of vehicle exceeding weight limits prohibited\n74.56 Load limits\n74.57 Maximum width, height, and length\n74.58 Statement of gross vehicle weight\n74.59 Wheel protectors required on heavy commercial vehicles\n74.60 Liability for damages; prosecution; application of monies\n74.61 Weight exceptions for certain vehicles\n\nChapter 75: Bicycles, Motorcycles and Off-Road Vehicles\n\nSection\n\nGeneral Provisions\n\n75.01 Bicycles; application of Title VII\n75.02 Operation of motorized bicycle\n75.03 Rules for bicycles, motorcycles, and snowmobiles\n75.04 Prohibition against attaching bicycles and sleds to vehicles\n75.05 Riding bicycles; motorcycles abreast\n75.06 Equipment of bicycles\n\nSnowmobiles, Off-Highway Motorcycles, and All-Purpose Vehicles\n\n75.25 Definitions\n75.26 Equipment\n75.27 Code application; prohibited operation\n75.28 Permitted operation\n75.29 Licensing requirements of operator\n75.30 Maintenance of vehicles for hire\n75.31 Accident reports\n75.32 Impounding of vehicle\n75.33 Local control within police power\n75.34 Registration of vehicles\n75.35 Certificate of title; prohibitions\n\nChapter 76: Parking Regulations\n\nSection\n\n76.01 Prohibition against parking on highways\n76.02 Condition when motor vehicle left unattended\n76.03 Police may remove illegally parked vehicle\n76.04 Parking prohibitions\n76.05 Parking near curb; privileges for persons with disabilities\n76.06 Parking on private property in violation of posted prohibition\n76.07 Selling, washing or repairing vehicle upon roadway\n76.08 Truck loading zones\n76.09 Bus stops and taxicab stands\n76.10 Parking in alleys and narrow streets; exceptions\n76.11 Registered owner prima facie liable for unlawful parking\n76.12 Waiver\n\nTITLE IX: GENERAL REGULATIONS\n\nChapter 90: Animals\n\nSection\n\nAnimals Running at Large\n\n90.01 Dogs or other animals running at large; nuisance, dangerous or vicious dogs; hearings\n90.02 Confining animal found at large; publication of notice; lien\n90.03 Unavoidable escapes\n90.04 Fees\n90.05 Rabies quarantine orders of Mayor\n90.06 Interfering with enforcement of quarantine orders\n90.07 Dogs may be killed for certain acts\n\nOffenses Relating to Animals\n\n90.20 Abandoning animals\n90.21 Injuring animals\n90.22 Poisoning animals\n90.23 Cruelty to animals; cruelty to companion animals\n90.24 Animal fights\n90.25 Trapshooting\n90.26 Loud dog\n90.27 Dog tags\n90.28 Nuisance, dangerous and vicious dog defined; transfer of ownership certificate; form stating dog's prior behavior\n90.29 Failure to register dog or dog kennel\n90.30 Hindering the capture of unregistered dog\n90.31 Unlawful tag\n90.32 Rights of blind, deaf or hearing impaired, or mobility impaired person, or trainer with assistance dog\n90.33 Retail sale and transportation of dogs\n90.34 Coloring rabbits or baby poultry; regulation of sale and display\n90.35 Jacklighting prohibited\n90.36 Restrictions on dog ownership for certain convicted felons\n90.37 Sexual conduct with an animal\n90.99 Penalty\n\nChapter 91: Fireworks, Explosives, Fire Prevention\n\nFireworks and Explosives\n\n91.01 Definitions\n91.02 Possession, sale, and use of fireworks\n91.03 Permit to use fireworks\n91.04 Manufacturing or wholesale sale without a license; prohibitions\n91.05 Purchasers to comply with law; unauthorized purchases\n91.06 Exhibition without a license; prohibitions\n91.07 Unauthorized transportation or shipping\n91.08 Application of subchapter\n91.09 Arrest of offender; seizure and forfeiture of fireworks; distribution of fines\n91.10 Safety requirements for fireworks showroom structures\n91.11 Storage of explosives\n91.12 Blasting permit\n\nFire Prevention\n\n91.30 Removal of flammable materials or obstructions\n91.31 Protective appliances\n91.32 Compliance with order\n91.33 Waste receptacles\n91.34 Hotel to have fire warning device producing visible signal\n91.35 Fire suppression systems\n91.36 Violations of State Fire Code prohibited\n91.37 Posting arson notices in hotels, motels and other places\n91.38 Negligent burning\n91.39 Spreading alarm of unfriendly fire\n91.40 Unvented heaters\n\nOpen Burning\n\n91.55 Definitions\n91.56 Relations to other prohibitions\n91.57 Open burning in restricted areas\n91.58 Permission and notice to open burn\n91.99 Penalty\n\nChapter 92: Intoxicating Liquors\n\nSection\n\n| Section | Description |\n|---------|-------------|\n| 92.01 | Definitions |\n| 92.02 | Exemptions from chapter |\n| 92.03 | Restrictions applicable to sale of beer and intoxicating liquor for consumption on the premises |\n| 92.04 | Restrictions on sale of beer and liquor |\n| 92.05 | Permit required; activities prohibited without permit |\n| 92.06 | Illegal transportation prohibited |\n| 92.07 | Open container prohibited; exception |\n| 92.08 | Underage person shall not purchase intoxicating liquor or beer |\n| 92.09 | Prohibitions; minors under 18 years; low-alcohol beverages |\n| 92.10 | Alcohol vaporizing devices prohibited |\n| 92.11 | Misrepresentation to obtain alcoholic beverage for a minor prohibited |\n| 92.12 | Misrepresentation by a minor under 21 years |\n| 92.13 | Sale to underage persons prohibited |\n| 92.14 | Posting of card |\n| 92.15 | Good faith acceptances of spurious identification |\n| 92.16 | Consumption in motor vehicle prohibited |\n| 92.17 | Hours of sale or consumption |\n| 92.18 | Obstructing search of premises prohibited |\n| 92.19 | Illegal possession of intoxicating liquor prohibited |\n| 92.20 | Sale or possession of diluted liquor and refilled containers prohibited |\n| 92.21 | Keeping place where beer or intoxicating liquors are sold in violation of law |\n| 92.22 | Intoxicating liquors shall not be sold in brothels |\n| 92.23 | Use of intoxicating liquor in a public dance hall prohibited; exceptions |\n| 92.24 | Poisonously adulterated liquors |\n| 92.25 | Tavern keeper permitting rioting or drunkenness |\n| 92.26 | Notice of action to prohibit liquor business |\n| 92.27 | Procedure when injunction violated |\n| 92.28 | Liquor transaction scans |\n| 92.29 | Affirmative defenses |\n| 92.99 | Penalty |\n\nChapter 93: Nuisances\n\nSection\n\nGeneral Provisions\n\n| Section | Description |\n|---------|-------------|\n| 93.01 | Application of the chapter |\n| 93.02 | Definitions |\n| 93.03 | Nuisances generally; injunctions; violation; contempt |\n| 93.04 | Maintaining certain nuisances |\n| 93.05 | Collection of cost of abating dangerous property condition; injunction; rehabilitation |\n| 93.06 | Trimming of trees and shrubbery to prevent obstruction |\n\nSeptic Tanks, Cesspools, and Refuse\n\n| Section | Description |\n|---------|-------------|\n| 93.20 | Location of privy vaults, cesspools, and septic tanks |\n| 93.21 | Unsanitary vaults |\n| 93.22 | Removal of contents of vault |\n| 93.23 | Deposit of dead animals, offal upon land or water |\n| 93.24 | Defiling spring or well prohibited |\n| 93.25 | Dumping of refuse in municipality prohibited |\n| 93.26 | Abandoned refrigerators |\n| 93.27 | Discarding litter prohibited |\n| 93.28 | Power of municipality to fill or drain land |\n\nWeeds and Litter on Private Property\n\n| Section | Description |\n|---------|-------------|\n| 93.40 | Keeping down weeds |\n| 93.41 | Notice to owner to cut noxious weeds, remove litter; service |\n| 93.42 | Fees for service and return |\n| 93.43 | Procedure when owner fails to comply with notice |\n| 93.44 | Written return to County Auditor; amount as a lien upon property |\n\nUnclean Habitations\n\n93.60 Permitting unclean habitations\n93.61 When habitations are deemed unsanitary\n93.62 Order for abatement or vacation of premises\n93.63 Enforcement of vacation order by Fire Chief or Police Chief\n93.64 Enforcement through court proceedings\n\n93.99 Penalty\n\nChapter 94: Streets and Sidewalks\n\nGeneral Provisions\n\n94.01 Conditions precedent to improving streets\n94.02 Opening permit required\n94.03 Application and cash deposit\n94.04 Restoration of pavement\n94.05 Barriers around excavations\n94.06 Warning lights\n94.07 Sidewalk construction by the municipality\n94.08 Unloading on street or sidewalk\n94.09 Street or sidewalk obstruction\n94.10 Materials on street or sidewalk\n94.11 Duty to keep sidewalks in repair and clean of ice and snow\n94.12 Ramped curbing for persons with disabilities\n94.13 Flagpole along right-of-way\n94.14 Altering or injuring marker or monument\n\nConstruction and Repair\n\n94.25 Construction and repair may be required\n94.26 Resolution of necessity\n94.27 Notice to construct or repair\n94.28 Assessments of costs against owner\n94.29 Proceedings may include different owners\n94.30 Making and levying assessments\n\nChanges in Streets\n\n94.40 Change of name, vacating or narrowing streets by petition\n94.41 Change of name, vacating or narrowing streets without petition\n94.42 Notice; exception\n94.43 Publication of notice\n94.44 Effect of order of vacation\n94.45 Effect on public utility easements\n\n94.99 Penalty\n\nChapter 95: Unclaimed and Abandoned Vehicles\n\nSection\n\n95.01 Impounding motor vehicle on private property; requirements\n95.02 Impounding abandoned motor vehicle on public property; notice; disposition\n95.03 Disposition of vehicle ordered into storage\n95.04 Disposition of abandoned junk motor vehicles\n95.05 Abandonment of junk motor vehicle prohibited\n95.06 Junk motor vehicle; order to cover or remove; notice; exceptions\n\nChapter 96: Watercraft\n\nSection\n\n96.01 Definitions; applicability\n96.02 Failure to comply with law enforcement order; fleeing\n96.03 Duty upon approach of law enforcement vessel\n96.04 Flashing lights prohibited; exceptions\n\n| Section | Description |\n|---------|-------------|\n| 96.05 | Siren prohibited; exceptions |\n| 96.06 | Regulations for operation of powercraft of more than ten horsepower |\n| 96.07 | Restrictions on child operators; duty of supervisory adult |\n| 96.08 | Reckless operation; maintaining sufficient control; wakes restricted |\n| 96.09 | Unsafe conditions |\n| 96.10 | Marking of bathing and vessel areas |\n| 96.11 | Mooring prohibited in certain areas |\n| 96.12 | Operating under influence of alcohol or drugs prohibited |\n| 96.13 | Implied consent |\n| 96.14 | Incapacitated operators prohibited |\n| 96.15 | Water skiing confined to ski zones |\n| 96.16 | Observer required when towing skier |\n| 96.17 | Water skiing after dark prohibited |\n| 96.18 | Personal flotation device required for towed person |\n| 96.19 | Ski jumps prohibited |\n| 96.20 | Permit for special water events |\n| 96.21 | Sale of single celled inflatable vessels prohibited |\n| 96.22 | Sitting, standing, walking on moving vessels restricted |\n| 96.23 | Engine warm-up required |\n| 96.24 | Personal flotation devices for children under ten |\n| 96.25 | Operation without personal flotation devices prohibited |\n| 96.26 | Distress signal or flag required |\n| 96.27 | Anchor requirements |\n| 96.28 | Specification for fire extinguishers |\n| 96.29 | Backfire flame control device required |\n| 96.30 | Ventilation requirement on powercraft |\n| 96.31 | Abandonment of junk vessels or outboard motors |\n| 96.32 | Exhaust muffler required; noise levels; exceptions |\n| 96.33 | Safety equipment on rental vessels |\n| 96.34 | Capacity plate |\n| 96.35 | Littering prohibited |\n| 96.36 | Dwellings; sanitary systems |\n| 96.37 | Prima facie evidence of negligence |\n| 96.38 | Requirements for operating personal watercraft |\n| 96.39 | Numbering |\n| 96.40 | Registration |\n| 96.41 | Tags indicating expiration date; attachment of identification number |\n| 96.42 | Altering of serial numbers; false information prohibited |\n| 96.43 | Accident reports |\n| 96.44 | Enforcement |\n| 96.45 | Firearms offenses; signaling devices |\n| 96.46 | Tampering with navigation aid or vessel prohibited |\n| 96.47 | Certificate of title; exceptions |\n| 96.48 | Manufacturer’s or importer’s certificate |\n| 96.49 | Prohibitions relating to certificates of title |\n| 96.50 | Permanently displayed hull identification number |\n| 96.99 | Penalty |\n\n---\n\n**TITLE XI: BUSINESS REGULATIONS**\n\n**Chapter 110: General Provisions**\n\n| Section | Description |\n|---------|-------------|\n| 110.01 | Licenses required to engage in certain businesses; exceptions |\n| 110.02 | Application for license |\n| 110.03 | Issuance of license |\n| 110.04 | Date and duration of license |\n| 110.05 | License not transferable |\n| 110.06 | License certificate to be displayed |\n| 110.07 | Revocation or suspension |\n| 110.08 | Appeal and review |\n| 110.99 | General penalty for Title XI |\n\nChapter 111: Taxicabs\n\nSection\n\n111.01 Definitions\n111.02 Certificate of public convenience and necessity required\n111.03 Application for certificate\n111.04 Issuance of certificate\n111.05 Liability insurance required\n111.06 License fees\n111.07 Transfer of certificates and licenses\n111.08 Suspension and revocation of certificates\n111.09 Taxicab driver's license\n111.10 Application for driver's license\n111.11 Examination of applicant; motor vehicle operator's permit required\n111.12 Police investigation of applicant; traffic and police record\n111.13 Consideration of application\n111.14 Issuance of license; duration; annual fee\n111.15 Display of license\n111.16 Suspension and revocation of license\n111.17 Failure to comply with federal, state and municipal laws\n111.18 Vehicle equipment and maintenance\n111.19 Designation of taxicabs\n111.20 Taximeter and display of rates required\n111.21 Number of passengers allowed\n111.22 Articles left in vehicles\n111.23 Vehicles from other municipalities\n111.24 Receipts\n111.25 Refusal of passenger to pay legal fare\n111.26 Solicitation, acceptance and discharge of passengers\n111.27 Open stands; use\n111.28 Taxicab service\n111.29 Manifests\n111.30 Records and reports of holders\n111.31 Police department; duty to enforce chapter\n111.32 Disposition of vehicle license fees\n\nChapter 112: Peddlers, Itinerant Merchants, and Solicitors\n\nSection\n\n112.01 Definitions\n112.02 License requirement\n112.03 Application procedure\n112.04 Standards for issuance\n112.05 Revocation procedure\n112.06 Standards for revocation\n112.07 Appeal procedure\n112.08 Exhibition of identification\n112.09 Municipal policy on soliciting\n112.10 Notice regulating soliciting\n112.11 Duty of solicitors\n112.12 Uninvited soliciting prohibited\n112.13 Time limit on soliciting\n\nChapter 113: Commercial Amusements\n\nSection\n\n113.01 Bowling; billiards and pool\n113.02 Circuses, carnivals, shows and other such entertainment\n113.03 Deposit required\n113.04 License fee for public entertainment or exhibition\n113.05 License fee may be waived for civic interest\n\nChapter 114: Tattooing and Body Piercing Services\n\nSection\n\n114.01 Definitions\n114.02 Prohibitions\n114.03 Application for license; fees; issuance\n\n114.04 Inspection of facilities\n114.05 Suspension or revocation of license\n114.06 Consent for performing procedures on persons under 18\n114.07 Prohibitions relating to persons under 18\n114.08 Defenses to violations\n114.09 Training standards; records; safety and sanitation; equipment\n114.10 Application of local regulation on chapter\n\n114.99 Penalty\n\nTITLE XIII: GENERAL OFFENSES\n\nChapter 130: General Provisions\n\nSection\n\n130.01 Application of Title XIII\n130.02 Definitions\n130.03 Classification of offenses\n130.04 Common law offenses abrogated\n130.05 Rules of construction\n130.06 Limitation of criminal prosecutions\n130.07 Requirements for criminal liability; voluntary intoxication\n130.08 Culpable mental states\n130.09 Organizational criminal liability\n130.10 Personal accountability for organizational conduct\n130.11 Attempt\n130.12 Complicity\n130.13 Presumption of innocence; proof of offense; affirmative defense\n130.14 Battered woman syndrome\n130.15 Delinquency adjudications deemed convictions\n130.16 Criminal law jurisdiction\n130.17 Disposition of unclaimed or forfeited property held by Police Department\n130.18 Imposing sentence for misdemeanor\n130.19 Multiple sentences\n130.20 Apprehension, detention, or arrest of persons on bond\n130.21 Self defense: limitations on duty to retreat prior to using force\n\n130.99 Penalty for Title XIII\n\nChapter 131: Offenses Against Property\n\nSection\n\n131.01 Definitions\n131.02 Arson; determining property value or amount of physical harm\n131.03 Criminal damaging or endangering; vehicular vandalism\n131.04 Criminal mischief\n131.05 Damaging or endangering aircraft or airport operations\n131.06 Criminal trespass; aggravated trespass\n131.07 Tampering with coin machines\n131.08 Theft\n131.09 Unauthorized use of a vehicle\n131.10 Unauthorized use of property, including telecommunication property and computers; possession of municipal property\n131.11 Passing bad checks\n131.12 Misuse of credit cards\n131.13 Making or using slugs\n131.14 Prima facie evidence of purpose to defraud\n131.15 Tampering with records\n131.16 Securing writings by deception\n131.17 Defrauding creditors\n131.18 Receiving stolen property\n131.19 Value of stolen property\n131.20 Degree of offense when certain property involved\n131.21 Injuring vines, bushes, trees, or crops\n131.22 Detention and arrest of shoplifters and those committing motion picture piracy; protection of institutional property\n\n131.23 Insurance fraud; workers' compensation fraud; Medicaid fraud\n131.24 Injury to property by hunters\n131.25 Evidence of intent to commit theft of rented property; evidence of lack of capacity to consent\n131.26 Forgery of identification cards\n131.27 Criminal simulation\n131.28 Personating an officer\n131.29 Trademark counterfeiting\n131.30 Diminishing or interfering with forfeitable property\n131.31 Recording credit card, telephone or Social Security numbers\n131.32 Prosecutions for theft of utilities\n131.33 Motion picture piracy\n\nChapter 132: Offenses Against Public Peace\n\nSection\n\n132.01 Riot\n132.02 Failure to disperse\n132.03 Justifiable use of force to suppress riot\n132.04 Disorderly conduct\n132.05 Disturbing a lawful meeting\n132.06 Misconduct at an emergency\n132.07 Telecommunications harassment\n132.08 Inducing panic\n132.09 Making false alarms\n132.10 Inciting to violence\n132.11 Unlawful display of law enforcement emblem\n132.12 Impersonating a peace officer\n132.13 Safety of crowds attending live entertainment performances\n132.14 Misconduct involving a public transportation system\n\nChapter 133: Sex Offenses\n\nSection\n\n133.01 Definitions\n133.02 Unlawful sexual conduct with a minor\n133.03 Sexual imposition\n133.04 Public indecency\n133.05 Voyeurism\n133.06 Polygraph examinations for victims: restrictions on use\n133.07 Procuring\n133.08 Soliciting; loitering to engage in\n133.09 Prostitution\n133.10 Disseminating matter harmful to juveniles\n133.11 Displaying matter harmful to juveniles\n133.12 Deception to obtain matter harmful to juveniles\n133.13 Rules of evidence\n133.14 Declaratory judgment\n133.15 Injunction; abatement of nuisance\n133.16 Unlawful operation of viewing booths depicting sexual conduct\n133.17 Juveniles on the premises of adult entertainment establishments prohibited\n133.18 Sexually oriented businesses; illegal operation and activity\n133.19 Unlawful advertising of massage\n\n133.99 Sentencing for sexually oriented offenses; sexual predators; registration\n\nChapter 134: Gambling Offenses\n\nSection\n\n134.01 Definitions\n134.02 Prohibitions against gambling; exception\n134.03 Operating a gambling house\n134.04 Public gaming\n134.05 Cheating\n134.06 Regulations concerning operation of licensed bingo game\n134.07 Records to be kept\n134.08 Requirements for bingo game operators\n134.09 Bingo games for amusement only\n\n134.10 Prohibitions where instant bingo game is conducted\n134.11 Raffle drawings\n134.12 Instant bingo other than at bingo sessions\n134.13 Restrictions on owner or lessor of location at instant bingo\n134.14 Skill-based amusement machines; prohibited conduct\n\nChapter 135: Offenses Against Persons\n\nSection\n\n135.01 Definitions\n135.02 Negligent homicide\n135.03 Vehicular homicide; vehicular manslaughter\n135.04 Assault; negligent assault\n135.05 Injury to persons by hunters\n135.06 Menacing; aggravated menacing; menacing by stalking\n135.07 Unlawful restraint\n135.08 Criminal child enticement\n135.09 Coercion\n135.10 Bigamy\n135.11 Unlawful abortion; failure to perform viability testing\n135.12 Abortion trafficking\n135.13 Nonsupport of dependents\n135.14 Endangering children\n135.15 Interference with custody; interference with support orders\n135.16 Domestic violence\n135.17 Hazing prohibited\n135.18 Contributing to unruliness or delinquency of a child\n135.19 Failure to provide for functionally impaired person\n135.20 Patient abuse or neglect; patient endangerment; exceptions; false statements; retaliation\n135.21 Interference with right of person to engage in housing transactions because of race, religion, or the like\n135.22 Ethnic intimidation\n135.23 Violating a protection order, consent agreement, anti-stalking protection order or order issued by a court of another state\n135.24 Adulteration of food\n135.25 Illegal distribution of cigarettes, other tobacco products, or alternative nicotine products; transaction scans\n135.26 Nonsmoking areas in places of public assembly\n135.27 Spreading contagion\n135.28 Abuse of a corpse\n135.29 Unlawful collection of bodily substances\n\nChapter 136: Offenses Against Justice and Administration\n\nSection\n\n136.01 Definitions\n136.02 Falsification\n136.03 Compounding a crime\n136.04 Failure to report a crime\n136.05 Failure to aid a law enforcement officer\n136.06 Obstructing official business\n136.07 Obstructing justice\n136.08 Resisting arrest\n136.09 Having an unlawful interest in a public contract\n136.10 Soliciting or receiving improper compensation\n136.11 Dereliction of duty\n136.12 Interfering with civil rights\n136.13 Illegal conveyance of prohibited items onto grounds of a detention facility or other specified governmental facilities\n136.14 False report of child abuse or neglect\n136.15 Assaulting police dog or horse, or assistance dog\n136.16 Disclosure of confidential peace officer information\n136.17 Intimidation of crime victim or witness\n136.18 Using sham legal process\n136.19 Making false allegation of peace officer misconduct\n136.20 Misuse of 9-1-1 system\n136.21 Failure to disclose personal information\n\nChapter 137: Weapons Control\n\nSection\n\n| Section | Description |\n|---------|-------------|\n| 137.01 | Definitions |\n| 137.02 | Carrying concealed weapons |\n| 137.03 | Using weapons while intoxicated |\n| 137.04 | Improperly handling firearms in a motor vehicle |\n| 137.05 | Possessing criminal tools |\n| 137.06 | Failure to secure dangerous ordnance |\n| 137.07 | Unlawful transactions in weapons |\n| 137.08 | Underage purchase of firearm or handgun |\n| 137.09 | Pointing and discharging firearms and other weapons |\n| 137.10 | License or permit to possess dangerous ordnance |\n| 137.11 | Possession of an object indistinguishable from a firearm in a school safety zone |\n| 137.12 | Possession of deadly weapon while under detention |\n| 137.13 | Concealed handgun licenses: possession of a revoked or suspended license; additional restrictions; posting of signs prohibiting possession |\n| 137.14 | Defaced firearms |\n\nChapter 138: Drug Offenses\n\nSection\n\n| Section | Description |\n|---------|-------------|\n| 138.01 | Definitions |\n| 138.02 | Trafficking in controlled substances; gift of marihuana |\n| 138.03 | Drug possession offenses |\n| 138.04 | Possessing drug abuse instruments |\n| 138.05 | Permitting drug abuse |\n| 138.06 | Illegal cultivation of marihuana |\n| 138.07 | Abusing harmful intoxicants |\n| 138.08 | Illegal dispensing of drug samples |\n| 138.09 | Federal prosecution bar to municipal prosecution |\n| 138.10 | Nitrous oxide: improper dispensing or distribution; possession in a motor vehicle |\n| 138.11 | Laboratory report required |\n| 138.12 | Counterfeit controlled substances |\n| 138.13 | Use, possession, or sale of drug paraphernalia |\n| 138.14 | Controlled substance or prescription labels |\n| 138.15 | Possession, sale and disposal of hypodermics |\n| 138.16 | Controlled substance schedules |\n| 138.17 | Unlawful furnishing of prescription to enable persons to be issued handicapped parking placards or license plates |\n| 138.18 | Pseudoephedrine sales |\n\nTITLE XV: LAND USAGE\n\nChapter 150: General Provisions\n\nSection\n\nParks and Recreation\n\n| Section | Description |\n|---------|-------------|\n| 150.01 | Recreation Board |\n| 150.02 | Board of Park Trustees |\n\nPlanning and Zoning\n\n| Section | Description |\n|---------|-------------|\n| 150.15 | Planning Commission |\n| 150.16 | Board of Zoning Appeals |\n\nPARALLEL REFERENCES\n\nOhio Legislative History References – Master Table\n\nINDEX\n\nThis summary of contents has been verified and authorized for publication by the Legislative Authority of the Municipality of __________, Ohio.\n\nSigned: _________________________ _________________________\nMayor Clerk of the Legislative Authority\n\nOrdinance #4- 2018\n\nOrdinance to Increase Trash Rates\n\nWhereas the legislative authority deems it necessary to increase trash rates.\n\nWhereas the new contract price has increased deeming it necessary to increase rates.\n\nWhereas the legislative authority would like increase residential rates from 13.47 to 15.37 and senior rates from 12.47 to 14.37\n\nWhereas the legislative authority would like increase dumpster rates 2 yd to $42.00 and 4 yd to $65.00\n\nWhereas all other trash rates will remain the same.\n\nNow therefore let it be ordained by VILLAGE OF AMANDA, OHIO:\n\nThat the village Legislative Authority does hereby pass the trash rates listed above. That this ordinance shall take effect upon the earliest date allowed by law.\n\nMayor Mark A. Moore\n\nVote 5 yes 0 no\n\nAttest: Carrie Ayers Fiscal Officer\n\nThe three reading requirement was waived/not waived:\n\nYeas 5 nays 0\n\nThe undersigned, Fiscal Officer of the Legislative Authority, does hereby certify that the foregoing legislation was posted in not less than five (5) public places, as determined by the legislative authority, for a period of not less than fifteen (15) days prior to the effective date thereof.\n\nFiscal Officer\n\nORDINANCE TO RESTRICT PARKING ON PARTS OF LUTZ ST.\n\nWhereas the village legislative authority wishes to restrict parking on the north side of Lutz St from Halderman St to the side doors of Midwest Fabricating to state no parking zone from 7am to 2pm.\n\nWhereas the legislative authority deems it in the best interest of the residents.\n\nNOW THEREFORE BE IT ORDAINED BY THE VILLAGE OF AMANDA, OHIO:\n\nThat the area along side of Midwest Fabricating on the north side of Lutz St. be restricted parking-- no parking zone during 7am-2pm from Halderman St to side doors of Midwest Fabricating.\n\nThat this ordinance shall take effect upon the earliest date allowed by law.\n\nMayor Mark A. Moore\n\nVote 5 yes 0 no\n\nAttest:\nCarrie Ayers Fiscal Officer\n\nThe three reading requirement was waived/not waived:\nYea 5 nays 0\n\nThe undersigned, Fiscal Officer of the Legislative Authority, does hereby certify that the foregoing legislation was posted in not less than five (5) public places, as determined by the legislative authority, for a period of not less than fifteen (15) days prior to the effective date thereof.\n\nFiscal Officer\n\nORDINANCE TO RAISE EMPLOYEES SALARY\n\nWHEREAS the village Legislative Authority give employees a raise.\n\nWHEREAS a 3% increase in wages for salary and hourly employees.\n\nNOW THEREFORE, BE IT ORDAINED BY THE VILLAGE OF AMANDA, OHIO:\n\nThat the village council feel a 3% increase in salary is warranted and shall be implemented on the current pay cycle. For hourly and salary employees.\n\nThat this ordinance shall take effect and be in force from and after the earliest period allowed by law.\n\nMayor Mark A. Moore\n\nVote: Yes 5 No 0\n\nAttest:\nCarrie Ayers\nFiscal Officer\n\nThe undersigned, Fiscal Officer of the Legislative Authority, does hereby certify that the foregoing legislation was posted in not less than (5) five public places as determined by the Legislative Authority, for a period of not less than (15) fifteen days prior to the effective date thereof.\n\nFiscal Officer\n\nOrdinance to regulate all complaints\n\nWhereas the legislative authority deems it necessary to regulate incoming complaints.\n\nWhereas the legislative authority following legal counsel and Ohio Municipal League advise, would like to require any complaints coming into the village office whether for zoning, property maintenance, or any other village business identify themselves by name and address.\n\nWhereas identifying the complainant can serve to establish that complaints are made in good faith.\n\nNow therefore let it be ordained by VILLAGE OF AMANDA, OHIO:\n\nThat the village Legislative Authority does hereby regulate incoming complaints. That all incoming complaints are required to identify themselves by name and address. That this ordinance shall take effect upon the earliest date allowed by law.\n\nMayor Mark A. Moore\n\nVote 5 yes 0 no\n\nAttest:\n\nCarrie Ayers Fiscal Officer\n\nThe three reading requirement was waived/not waived:\n\nYeas 5 nays 0\n\nThe undersigned, Fiscal Officer of the Legislative Authority, does hereby certify that the foregoing legislation was posted in not less than five (5) public places, as determined by the legislative authority, for a period of not less than fifteen (15) days prior to the effective date thereof.\n\nFiscal Officer\n\nOrdinance to regulate all record requests\n\nWhereas the legislative authority deems it necessary to regulate incoming requests as well as public record request.\n\nWhereas the legislative authority following legal counsel and Ohio Municipal League advise, would like to require any requests coming into the village office whether for zoning, property maintenance, or any other village business identify themselves by name and address.\n\nWhereas identifying the requests can serve to establish that request are made in good faith.\n\nNow therefore let it be ordained by VILLAGE OF AMANDA, OHIO:\n\nThat the village Legislative Authority does hereby regulate all incoming requests. That all incoming requests are required to identify themselves by name and address. That this ordinance shall take effect upon the earliest date allowed by law.\n\nMayor Mark A. Moore\n\nVote 4 yes 0 no\n\nAttest:\n\nCarrie Ayers Fiscal Officer\n\nThe three reading requirement was waived/not waived:\n\nYea 4 nays 0\n\nThe undersigned, Fiscal Officer of the Legislative Authority, does hereby certify that the foregoing legislation was posted in not less than five (5) public places, as determined by the legislative authority, for a period of not less than fifteen (15) days prior to the effective date thereof.\n\nFiscal Officer\n\nOrdinance to Set Garbage Guidelines\n\nWhereas the Village Legislative Authority would like to set guidelines to regulate the garbage collection.\n\nWhereas the guidelines will regulate the garbage pick-up dates and times, as well as set limits how much refuse is allowed.\n\nWhereas the guidelines further address required containers, in addition to what items are suitable/not suitable for collection.\n\nNow Therefore let it be ordained by VILLAGE OF AMANDA, OHIO:\n\nThat the village Legislative Authority does hereby set Garbage Guidelines set forth in the attached appendix A, to regulate garbage collection.\n\nThat this ordinance shall take effect upon the earliest date allowed by law.\n\nMayor Mark A. Moore\n\nVote 5 yes 0 no\n\nAttest:\n\nCarrie Ayers Fiscal Officer\n\nThe three reading requirement was waived/not waived:\n\nYea 5 nays 0\n\nThe undersigned, Fiscal Officer of the Legislative Authority, does hereby certify that the foregoing legislation was posted in not less than five (5) public places, as determined by the legislative authority, for a period of not less than fifteen (15) days prior to the effective date thereof.\n\nFiscal Officer\n\n1st Reading 11/05/18\n2nd Reading 12-3-18\n\nGarbage Guidelines\n\n- Garbage is collected 1 day per week on your scheduled pick up day (Tuesday). Selective dumpsters are set up on a 2 day a week pick up the alternate day is (Friday). Pick up times runs between 6 A.M. and 5 P.M.\n\n- Garbage must be set out no earlier than the night before pick-up. Containers are to be removed the same day.\n\n- All garbage must be placed within 5 feet of curb or alley. (Do not block road or sidewalk).\n\n- Major holiday of Thanksgiving and Christmas collection will run one day behind.\n\n- City residents are limited to 8-45 gallon cans or bags or 2-96-gallon rollout containers a week. Weighing no more than 50 pounds per container. Excluding yard waste. Cardboard boxes and burning barrels are not acceptable containers. Ashe containers shall be made of metal; ash and rubbish containers shall be of a kind suitable for collection purposes and shall be of such weight that can be handled by one person.\n\n- Home cleanouts are in excess of regular trash service and will be subject to additional charges.\n\n- Brush and tree limbs, carpets, etc. must be bundled and tied in lengths no longer than 3 to 4 feet.\n\n- One large item a month at no cost. Any additional large items will be charged. 24-hour advance notice to notify drivers of large item pick-up.\n\n- Special and Hazardous waste including but not limited to construction material, rocks, dirt, metal pipe, concrete, liquids, paint, batteries, tires, hazardous chemicals, anything with Freon (refrigerators and air conditioners), or any Epa prohibited items will NOT be collected.\n\n- If animals or weather scatter trash from your trash cans it is NOT the trash haulers responsibility to clean up, Residents are responsible for cleaning their area. To avoid this make sure all refuse is in a lidded container.\n\nJanuary 2019\n\nRe: The Ohio Basic Code, 2019 Edition\n\nEnclosed please find your subscription order for the *Ohio Basic Code, 2019 Edition*. Please note that the *Ohio Basic Code, 2019 Edition*, has been designed to replace, in its entirety, any previous editions of the Ohio Basic Code.\n\nWe are pleased to provide the *Ohio Basic Code, 2019 Edition*. Should you have any questions, please do not hesitate to contact our office.\n\nVery truly yours,\n\nTodd Paul Myers, J.D.\nVice President, Major Client Services\nfirstname.lastname@example.org\n\nAN ORDINANCE APPROVING, ADOPTING AND ENACTING AMERICAN LEGAL PUBLISHING’S OHIO BASIC CODE, 2019 EDITION, AS THE CODE OF ORDINANCES FOR THE MUNICIPALITY OF Village of Amanda, OHIO, AND DECLARING AN EMERGENCY.\n\nWHEREAS, the present general and permanent ordinances of the municipality are inadequately arranged and classified and are insufficient in form and substance for the complete preservation of the public peace, health, safety and general welfare of the municipality and for the proper conduct of its affairs.\n\nWHEREAS, American Legal Publishing Corporation publishes a Code of Ordinances suitable for adoption by municipalities in Ohio.\n\nWHEREAS, it is necessary to provide for the usual daily operation of the municipality and for the immediate preservation of the public peace, health, safety and general welfare of the municipality that this ordinance take effect at an early date.\n\nNOW, THEREFORE, BE IT ORDAINED BY THE LEGISLATIVE AUTHORITY OF THE MUNICIPALITY OF Village of Amanda, OHIO:\n\nSection 1. American Legal Publishing’s Ohio Basic Code, 2019 Edition, as reviewed and approved by the Legislative Authority, is hereby adopted and enacted. Any prior version of the Ohio Basic Code which may have been previously adopted by the municipality is hereby repealed as obsolete and is hereby replaced in its entirety by this Ohio Basic Code, 2019 Edition.\n\nSection 2. One copy of American Legal Publishing’s Ohio Basic Code, 2019 Edition, certified as correct by the Mayor and Clerk of the Legislative Authority, as required by Ohio Revised Code § 731.23, shall be kept in its initial form on file in the office of the Clerk of the municipality and retained as a permanent ordinance record of the municipality. The Clerk of the municipality is authorized and directed to publish a summary of all new matters contained in the Code of Ordinances as required by Ohio Revised Code § 731.23. Such summary is attached hereto and marked as “Exhibit A”.\n\nSection 3. All ordinances and resolutions or parts thereof which are in conflict or inconsistent with any provision of the Ohio Basic Code, 2019 Edition, as adopted in Section 1 hereof, are hereby repealed as of the effective date of this ordinance, except as follows:\n(A) The enactment of the Ohio Basic Code, 2019 Edition, shall not be construed to affect a right or liability accrued or incurred under any legislative provision prior to the effective date of such enactment, or an action or proceeding for the enforcement of such right or liability. Such enactment shall not be construed to relieve any person from punishment for an act committed in violation of any such legislative provision, nor to affect an indictment or prosecution therefor. For such purposes, any such legislative provision shall continue in full force notwithstanding its repeal for the purpose of revision and codification.\n\n(B) The repeal provided above shall not affect:\n (1) The grant or creation of a franchise, license, right, easement or privilege;\n (2) The purchase, sale, lease or transfer of property;\n (3) The appropriation or expenditure of money or promise or guarantee of payment;\n (4) The assumption of any contract or obligation;\n (5) The issuance and delivery of any bonds, obligations or other instruments of indebtedness;\n (6) The levy or imposition of taxes, assessments or charges;\n (7) The establishment, naming, vacating or grade level of any street or public way;\n (8) The dedication of property or plat approval;\n (9) The annexation or detachment of territory;\n (10) Any legislation enacted subsequent to the adoption of this ordinance.\n (11) Any legislation specifically superseding the provision of the Ohio Basic Code.\n\nSection 4. Whenever reference is made in any documents, publications, or signs of the municipality, including but not limited to traffic tickets and traffic-control signs, to a section as it existed in a former edition of the Ohio Basic Code, the reference shall extend and apply to the section referred to as subsequently amended, revised, recodified, or renumbered.\n\nSection 5. This ordinance is declared to be an emergency measure necessary for the immediate preservation of the peace, health, safety and general welfare of the people of this municipality, and shall take effect at the earliest date provided by law.\n\nDate Passed: 2-1-19\n\nAttest:\n\nMayor\n\nClerk of the Legislative Authority\n\nNotice is hereby given that on the 4th day of Feb., 2019, there was enacted by the Legislative Authority of the Municipality of Village of Avon, Ohio, an ordinance entitled “An Ordinance Approving, Adopting and Enacting American Legal Publishing’s Ohio Basic Code, 2019 Edition, as the Code of Ordinances for the Municipality of Village of Avon, Ohio.”\n\nA summary of the subjects, including all new matters contained in the Code of Ordinances, as adopted, are as follows. The majority of Basic Code provisions are based directly on state law.\n\n---\n\n**TITLE I: GENERAL PROVISIONS**\n\nChapter 10: General Provisions\n\n| Section | Title |\n|---------|--------------------------------------------|\n| 10.01 | Short titles |\n| 10.02 | Definitions |\n| 10.03 | Rules of construction |\n| 10.04 | Revivor; effect of amendment or repeal |\n| 10.05 | Construction of section references |\n| 10.06 | Conflicting provisions |\n| 10.07 | Severability |\n| 10.08 | Reference to offices |\n| 10.09 | Errors and omissions |\n| 10.10 | Ordinances repealed |\n| 10.11 | Ordinances unaffected |\n| 10.12 | Ordinances saved |\n| 10.13 | Application to future ordinances |\n| 10.14 | Interpretation |\n| 10.15 | Amendments to code; amendatory language |\n| 10.16 | Statutory references |\n| 10.17 | Preservation of penalties, offenses, rights and liabilities |\n| 10.18 | Determination of legislative intent |\n| 10.99 | General penalty |\n\n---\n\n**TITLE III: ADMINISTRATION**\n\nChapter 30: General Provisions\n\n| Section | Title |\n|---------|--------------------------------------------|\n| 30.01 | Application of Title III |\n| 30.02 | Qualifications; oaths |\n| 30.03 | Bonds of officers and employees; amount |\n| 30.04 | Additional bond; where bonds recorded and kept |\n| 30.05 | Approval of bonds |\n| 30.06 | Sufficiency of form of bond |\n| 30.07 | Filling vacancies in offices |\n| 30.08 | Public records available |\n| 30.09 | Records Commission |\n| 30.10 | Meetings of public bodies to be open; exceptions; notice |\n| 30.11 | Municipal officers may attend conference or convention; expenses |\n| 30.12 | Residency requirements prohibited; exceptions |\n\nChapter 31: Executive Authority\n\nSection\n\n*General Provisions*\n\n31.001 Executive power; where vested\n\n**Mayor**\n\n31.015 Term of Mayor; power and duties \n31.016 General duties of the Mayor \n31.017 Communications to the Legislative Authority \n31.018 Protest against excess of expenditures \n31.019 Supervision of conduct of officers \n31.020 Annual report to the Legislative Authority \n31.021 Mayor to file charges against delinquent officers \n31.022 Vacancies in office of Mayor \n31.023 Disposition of fines and other moneys\n\n**Clerk**\n\n31.040 Election, term, qualifications of the Clerk \n31.041 Powers and duties of Clerk \n31.042 Books and accounts; merger of offices \n31.043 Seal of Clerk \n31.044 Combined offices of Clerk and Treasurer; Fiscal Officer\n\n**Treasurer**\n\n31.060 Election, term, qualifications of the Treasurer \n31.061 Accounts of Treasurer \n31.062 Powers and duties \n31.063 Quarterly account; annual report \n31.064 Receipt and disbursement of funds \n31.065 Duty of delivering money and property\n\n**Street Commissioner**\n\n31.080 Qualifications \n31.081 General duties \n31.082 Assistants\n\n**Other Officials**\n\n31.100 Legal counsel \n31.101 Administrator \n31.102 Board of Trustees of Public Affairs \n31.103 Fire Engineer, Engineer and Superintendent of Markets\n\n**Chapter 32: Legislative Authority**\n\n**General Provisions**\n\n32.001 Members of the Legislative Authority; election; terms of office \n32.002 President Pro Tempore; employees \n32.003 Vacancy when President Pro Tempore becomes Mayor \n32.004 Qualifications of members of the Legislative Authority \n32.005 Compensation and bonds of municipal officers and employees \n32.006 Vacancy \n32.007 Judge of election and qualification of members; quorum and special meetings \n32.008 Rules; journal; expulsion of members \n32.009 Meetings \n32.010 General powers \n32.011 Failure to take oath or give bond \n32.012 Notice when new bond required \n32.013 Care, supervision and management of public institutions\n\n### Contracts, Bids and Proceedings\n\n| Section | Description |\n|---------|-------------|\n| 32.025 | Contracts by the Legislative Authority or Administrator |\n| 32.026 | Bids and proceedings |\n| 32.027 | Alterations or modifications of contract |\n| 32.028 | Contract restrictions |\n| 32.029 | Award to lowest responsive and responsible bidder |\n\n### Ordinances and Resolutions\n\n| Section | Description |\n|---------|-------------|\n| 32.040 | Ordinances and resolutions as evidence |\n| 32.041 | Passage procedure |\n| 32.042 | Style of ordinances |\n| 32.043 | Subject and amendment of ordinances and resolutions |\n| 32.044 | Authentication and recording of ordinances and resolutions |\n| 32.045 | Publication of ordinances and resolutions; proof of publication and circulation |\n| 32.046 | Notice for proposed amendments to the municipal Charter |\n| 32.047 | Times of publication required |\n| 32.048 | Publication and certification of ordinances in book form |\n| 32.049 | Adoption of technical ordinances and codes |\n| 32.050 | Certificate of Clerk as to publication |\n| 32.051 | Publication when no newspaper published in municipality |\n| 32.052 | Effect of not making publication |\n| 32.053 | Ordinances providing for appropriations or street improvements; emergency ordinances |\n\n### Initiative and Referendum\n\n| Section | Description |\n|---------|-------------|\n| 32.070 | Initiative petitions |\n| 32.071 | Referendum petitions |\n| 32.072 | More than one ordinance required; application of subchapter |\n| 32.073 | Presentation of petitions |\n| 32.074 | Copy of proposed ordinance or measure to be filed with Clerk |\n| 32.075 | Words to be printed in red |\n| 32.076 | Designation of committee filing petition; public inspection of petitions; ordinances passed or repealed prior to election |\n| 32.077 | Itemized statement by petition circulator |\n| 32.078 | Prohibited practices relative to petitions |\n| 32.079 | Accepting premiums for signing |\n| 32.080 | Threats in securing signatures |\n| 32.081 | Application of subchapter if Charter adopted |\n\n### Chapter 33: Judicial Authority\n\n#### General Provisions\n\n| Section | Description |\n|---------|-------------|\n| 33.01 | Jurisdiction in ordinance cases and traffic violations |\n| 33.02 | Powers of Mayor and Mayor's Court Magistrate in criminal matters |\n| 33.03 | Duties of Mayor and Mayor's Court Magistrate; fees; office; seal |\n| 33.04 | Mayor's Court Magistrate |\n| 33.05 | Powers to suspend driver's license in OVI cases |\n\n#### Contempt of Court\n\n| Section | Description |\n|---------|-------------|\n| 33.20 | Summary punishment for contempt |\n| 33.21 | Acts in contempt of court |\n| 33.22 | Hearing |\n| 33.23 | Contempt action for failure to pay support, failure to comply or interference with a visitation order; summons |\n| 33.24 | Right of accused to bail |\n| 33.25 | Hearing on contempt; penalties; support orders; failure to withhold or deduct money pursuant to support order |\n| 33.26 | Imprisonment until order obeyed |\n| 33.27 | Proceedings when party released on bail fails to appear |\n\n33.28 Release of prisoner committed for contempt\n33.29 Judgment final\n33.30 Alternative remedy\n\nChapter 34: Police Department\n\nSection\n\n34.01 Marshal and Police Chief synonymous\n34.02 Appointment of Marshal\n34.03 Deputy marshals and police officers\n34.04 Auxiliary police units\n34.05 Offenses affecting employment of law enforcement officers; probationary period; final appointment\n34.06 Removal proceedings; suspension; appeals\n34.07 General powers\n34.08 Powers and duties of Marshal\n34.09 Disposition of fines and penalties\n34.10 Property recovered by police\n34.11 Disposition to claimant\n34.12 Sale of unclaimed property; disposition of proceeds\n34.13 Expenses of storage and sale; notice\n34.14 Contracts for police protection; nonresident service without contract\n34.15 Peace officer administering oaths; acknowledging complaints, summonses, affidavits and returns of court orders\n\nChapter 35: Fire Department\n\nSection\n\nGeneral Provisions\n\n35.01 Municipal fire regulations; fire department\n35.02 Fire Chief; Fire Prevention Officer; employment of firefighters; criminal records check for firefighters\n35.03 Schooling of officers and firefighters of fire department\n35.04 Legislative Authority may purchase engines and equipment\n35.05 Buildings for department\n35.06 Records\n35.07 Maximum consecutive hours for firefighters on duty\n35.08 Investigation of cause of fire\n35.09 Right to examine buildings, premises, and vehicles\n35.10 Burning buildings for firefighting instruction or research\n35.11 Impersonating fire safety inspector\n35.12 Standards for equipment\n35.13 Persons entitled to be known as firefighters\n35.14 Firefighting and emergency services agreements\n35.15 Regulation of construction in fire limits\n\nVolunteer Firefighters’ Dependents Fund Board\n\n35.30 Definitions\n35.31 Establishment\n35.32 Membership; vacancies\n35.33 Election and term of members\n35.34 Organization; rules and regulations; roster\n35.35 Compensation and expenses of Board; legal advisor\n\nChapter 36: Civil Actions Against the Municipality\n\nSection\n\n36.01 Definitions\n36.02 Nonliability of municipality; exceptions\n36.03 Defenses and immunities\n36.04 Limitation of actions\n36.05 Damages\n36.06 Satisfaction of judgments\n36.07 Defending and indemnifying employees\n36.08 Liability insurance\n36.09 Certain actions unaffected\n36.10 Certain charges against municipal officers filed with Probate Judge; proceedings\n\nTITLE VII: TRAFFIC CODE\n\nChapter 70: General Provisions\n\nGeneral Provisions\n\n| Section | Description |\n|---------|-------------|\n| 70.01 | Definitions |\n| 70.02 | Compliance with order of police officer |\n| 70.03 | Emergency vehicles to proceed cautiously past red or stop signal |\n| 70.04 | Exceptions generally; emergency, public safety and coroner vehicles exempt |\n| 70.05 | Persons riding or driving animals upon roadways |\n| 70.06 | Prohibitions against pedestrians and slow-moving vehicles on freeways |\n| 70.07 | Use of private property for vehicular travel |\n| 70.08 | Names of persons damaging real property by operation of vehicle to be provided to owner |\n| 70.09 | Limited access highways; barriers along; vehicles to enter and leave at designated intersections |\n| 70.10 | Through highways |\n| 70.11 | Officer may remove ignition key |\n| 70.12 | Removal of vehicles after accidents |\n\nTraffic-Control Devices\n\n| Section | Description |\n|---------|-------------|\n| 70.30 | Obeying traffic-control devices |\n| 70.31 | Signal lights |\n| 70.32 | Signals over reversible lanes |\n| 70.33 | Ambiguous or non-working traffic signals |\n| 70.34 | Pedestrian-control signals |\n| 70.35 | Unauthorized signs and signals prohibited |\n| 70.36 | Alteration, defacement, or removal prohibited |\n| 70.37 | Unauthorized possession or sale of devices |\n| 70.38 | Signal preemption devices; prohibitions |\n| 70.99 | Penalty |\n\nChapter 71: Licensing Provisions\n\nMotor Vehicle Licensing\n\n| Section | Description |\n|---------|-------------|\n| 71.01 | Display of license plates or validation stickers; registration |\n| 71.02 | Improper use of noncommercial motor vehicle |\n| 71.03 | Operating motor vehicle ordered immobilized; forfeiture |\n| 71.04 | Operation or sale without certificate of title |\n| 71.05 | Display of certificate of registration |\n| 71.06 | Use of unauthorized plates |\n| 71.07 | Operating without dealer or manufacturer license plates |\n\nDriver's Licenses\n\n| Section | Description |\n|---------|-------------|\n| 71.20 | Prohibited acts |\n| 71.21 | Permitting minor to operate vehicle prohibited; temporary instruction permit; probationary license |\n| 71.22 | License required as driver or commercial driver on public or private property; nonresident exemption |\n| 71.23 | Employment of a minor to operate a taxicab prohibited |\n| 71.24 | Restriction against owner lending vehicle for use of another |\n| 71.25 | Suspension of driver's licenses; license suspended by court of record |\n| 71.26 | Display of license |\n| 71.27 | Prohibition against false statements |\n| 71.28 | Driving under suspension or in violation of license restriction |\n\n71.29 Operating motor vehicle or motorcycle without valid license\n71.30 Driving under OVI suspension\n71.31 Driving under financial responsibility law suspension or cancellation; driving under a nonpayment of judgment suspension\n71.32 Failure to reinstate license\n\n**Commercial Driver's Licenses**\n\n71.45 Definitions\n71.46 Use of actual gross weight in lieu of rating\n71.47 Prohibited acts\n71.48 Prerequisites to operation of commercial motor vehicle\n71.49 Physical qualification to operate commercial motor vehicles\n71.50 Criminal offenses\n71.51 Application of federal regulations\n71.52 Employment of drivers of commercial vehicles\n\n71.99 Penalty\n\n---\n\n**Chapter 72: Traffic Rules**\n\n**General Provisions**\n\n72.001 Lanes of travel upon roadways\n72.002 Driving through safety zone\n72.003 Vehicles traveling in opposite directions\n72.004 Rules governing overtaking and passing of vehicles\n72.005 Permission to overtake and pass on the right\n72.006 Driving to left of center line\n72.007 Prohibition against driving upon left side of roadway\n72.008 Hazardous zones\n72.009 One-way highways and rotary traffic islands\n72.010 Rules for driving in marked lanes\n72.011 Space between moving vehicles\n72.012 Divided roadways\n72.013 Rules for turns at intersections\n72.014 U-turns and turning in roadway prohibited\n72.015 Starting and backing vehicles\n72.016 Turn and stop signals\n72.017 Hand and arm signals\n\n**Right-of-Way**\n\n72.030 Right-of-way at intersections\n72.031 Right-of-way when turning left\n72.032 Right-of-way at through highways; stop signs; yield signs\n72.033 Stop at sidewalk area; stop signs on private roads and driveways\n72.034 Right-of-way on public highway\n72.035 Pedestrian on sidewalk has right-of-way\n72.036 Right-of-way of public safety vehicles\n72.037 Funeral procession has right-of-way\n72.038 Pedestrians yield right-of-way to public safety vehicle\n72.039 Pedestrian on crosswalk has right-of-way\n72.040 Right-of-way yielded to blind person\n72.041 Right-of-way yielded by pedestrian\n\n**Pedestrians**\n\n72.055 Pedestrian movement in crosswalks\n72.056 Pedestrian walking along highway\n72.057 Prohibition against soliciting rides; riding on outside of vehicle\n72.058 Pedestrian on bridge or railroad crossing\n72.059 Persons operating motorized wheelchairs\n\n72.060 Intoxicated or drugged pedestrian hazard on highway\n72.061 Operation of electric personal assistive mobility devices\n72.062 Operation of personal delivery device on sidewalks and crosswalks\n\n**Grade Crossings**\n\n72.075 Stop signs at grade crossings\n72.076 Driving vehicle across railroad grade crossing\n72.077 Vehicles required to stop at grade crossings\n72.078 Slow-moving vehicles or equipment crossing railroad tracks\n\n**School Buses**\n\n72.090 Regulations concerning school buses\n72.091 Violation of regulations; report; investigation; citation; warning\n72.092 Restrictions on the operation of school buses\n72.093 School bus inspection\n72.094 School bus not used for school purposes\n72.095 Licensing by Department of Public Safety\n72.096 Registration and identification of school buses\n72.097 School bus marking\n72.098 Flashing light signal lamps\n72.099 Occupant restraining device for operator\n\n**Prohibitions**\n\n72.115 Obstruction and interference affecting view and control of driver\n72.116 Occupying travel trailer while in motion\n72.117 Driving upon closed highway prohibited\n72.118 Driving upon sidewalk area or paths exclusively for bicycles\n72.119 Obstructing passage of other vehicles\n72.120 Following an emergency or public vehicle prohibited; approaching stationary public safety vehicle and certain other vehicles with caution\n72.121 Driving over unprotected fire hose\n72.122 Placing injurious material on highway or depositing litter from motor vehicle\n72.123 Transporting child not in child-restraint system prohibited\n72.124 Occupant restraining devices\n72.125 Use of engine brakes prohibited\n72.126 Operating motor vehicle while wearing earphones or earplugs\n72.127 Chauffeured limousines and livery services\n72.128 Operating traction engine upon improved highway\n72.129 Cracking exhaust noises; peeling out\n72.130 Shortcutting across private property\n72.131 Texting while driving prohibited\n72.132 Use of electronic wireless communication devices by minors or probationary drivers while driving prohibited\n\n**Chapter 73: Motor Vehicle Crimes**\n\n**General Provisions**\n\n73.01 Driving under the influence of alcohol or drugs\n73.02 Implied consent\n73.03 Physical control of vehicle while under the influence\n73.04 Driving commercial vehicle with impaired alertness or ability; use of drugs\n73.05 Reckless operation of vehicles\n73.06 Reckless operation off streets and highways; competitive operation\n73.07 Operator to be in reasonable control\n73.08 Immobilizing or disabling device violation\n73.09 Street racing defined; prohibited on public highways\n73.10 Speed limits\n73.11 Slow speed or stopping\n73.12 Emergency vehicles excepted from speed limitation\n73.13 Speed regulations on bridges\n\n73.14 Presenting false name or information to officer\n73.15 Prohibition against resisting officer\n73.16 Operation restricted for mini-trucks and low-speed, under-speed, or utility vehicles\n\n**Stopping After Accident**\n\n73.30 Failure to stop after accident\n73.31 Stopping after accident on other than public roads or highways\n73.32 Accident involving damage to realty\n73.33 Failure to report accident\n\n**Chapter 74: Equipment and Loads**\n\n**Equipment**\n\n74.01 Unsafe vehicles, prohibition against operation\n74.02 Bumpers on motor vehicles\n74.03 Lighted lights required\n74.04 Headlights\n74.05 Tail lights and illumination of rear license plate\n74.06 Red reflectors required\n74.07 Safety lighting of commercial vehicles\n74.08 Stoplight regulations\n74.09 Obscured lights on vehicles\n74.10 Red light or flag required\n74.11 Lights on parked vehicles\n74.12 Lights and emblem on slow-moving vehicles; lights and reflectors on multi-wheel agricultural tractors or farm machinery\n74.13 Spotlight and auxiliary driving lights\n74.14 Cowl, fender, and back-up lights\n74.15 Two lights displayed\n74.16 Headlights required\n74.17 Lights of less intensity\n74.18 Number of lights permitted; red and flashing lights\n74.19 Standards for lights on snow removal equipment and oversize vehicles\n74.20 Flashing lights permitted for certain types of vehicles\n74.21 Lights and sign on transportation for preschool children\n74.22 Focus and aim of headlights\n74.23 Brake equipment; specifications\n74.24 Brake fluid\n74.25 Minimum standards for brakes and components\n74.26 Horns, sirens, and warning devices\n74.27 Mufflers; excessive smoke or gas\n74.28 Rearview mirrors\n74.29 Windshields and wipers\n74.30 Solid tire requirements\n74.31 Requirements for safety glass in motor vehicles; use of tinted glass or reflectorized material\n74.32 Directional signals\n74.33 Installation and sale of seat safety belts required; definition\n74.34 Requirements for extra signal equipment\n74.35 Display of warning devices on disabled vehicles\n74.36 Requirements for vehicles transporting explosives\n74.37 Studded tires; seasonal use permitted\n74.38 Safety inspection decals for buses\n74.39 Air bags\n\n**Loads**\n\n74.50 Permit required to exceed load limits\n74.51 Limitation of load extension on left side of vehicle\n74.52 All loads shall be properly secured\n74.53 Towing requirements; exception to size and weight restrictions\n74.54 Weighing of vehicle; removal of excess load\n\n74.55 Operation of vehicle exceeding weight limits prohibited\n74.56 Load limits\n74.57 Maximum width, height, and length\n74.58 Statement of gross vehicle weight\n74.59 Wheel protectors required on heavy commercial vehicles\n74.60 Liability for damages; prosecution; application of monies\n74.61 Weight exceptions for certain vehicles\n\nChapter 75: Bicycles, Motorcycles and Off-Road Vehicles\n\nSection\n\nGeneral Provisions\n\n75.01 Bicycles; application of Title VII\n75.02 Operation of motorized bicycle\n75.03 Rules for bicycles, motorcycles, and snowmobiles\n75.04 Prohibition against attaching bicycles and sleds to vehicles\n75.05 Riding bicycles; motorcycles abreast\n75.06 Equipment of bicycles\n\nSnowmobiles, Off-Highway Motorcycles, and All-Purpose Vehicles\n\n75.25 Definitions\n75.26 Equipment\n75.27 Code application; prohibited operation\n75.28 Permitted operation\n75.29 Licensing requirements of operator\n75.30 Maintenance of vehicles for hire\n75.31 Accident reports\n75.32 Impounding of vehicle\n75.33 Local control within police power\n75.34 Registration of vehicles\n75.35 Certificate of title; prohibitions\n\nChapter 76: Parking Regulations\n\nSection\n\n76.01 Prohibition against parking on highways\n76.02 Condition when motor vehicle left unattended\n76.03 Police may remove illegally parked vehicle\n76.04 Parking prohibitions\n76.05 Parking near curb; privileges for persons with disabilities\n76.06 Parking on private property in violation of posted prohibition\n76.07 Selling, washing or repairing vehicle upon roadway\n76.08 Truck loading zones\n76.09 Bus stops and taxicab stands\n76.10 Parking in alleys and narrow streets; exceptions\n76.11 Registered owner prima facie liable for unlawful parking\n76.12 Waiver\n\nTITLE IX: GENERAL REGULATIONS\n\nChapter 90: Animals\n\nSection\n\nAnimals Running at Large\n\n90.01 Dogs or other animals running at large; nuisance, dangerous or vicious dogs; hearings\n90.02 Confining animal found at large; publication of notice; lien\n90.03 Unavoidable escapes\n90.04 Fees\n90.05 Rabies quarantine orders of Mayor\n90.06 Interfering with enforcement of quarantine orders\n90.07 Dogs may be killed for certain acts\n\nOffenses Relating to Animals\n\n90.20 Abandoning animals\n90.21 Injuring animals\n90.22 Poisoning animals\n90.23 Cruelty to animals; cruelty to companion animals\n90.24 Animal fights\n90.25 Trapshooting\n90.26 Loud dog\n90.27 Dog tags\n90.28 Nuisance, dangerous and vicious dog defined; transfer of ownership certificate; form stating dog's prior behavior\n90.29 Failure to register dog or dog kennel\n90.30 Hindering the capture of unregistered dog\n90.31 Unlawful tag\n90.32 Rights of blind, deaf or hearing impaired, or mobility impaired person, or trainer with assistance dog\n90.33 Retail sale and transportation of dogs\n90.34 Coloring rabbits or baby poultry; regulation of sale and display\n90.35 Jacklighting prohibited\n90.36 Restrictions on dog ownership for certain convicted felons\n90.37 Sexual conduct with an animal\n\n90.99 Penalty\n\nChapter 91: Fireworks, Explosives, Fire Prevention\n\nSection\n\nFireworks and Explosives\n\n91.01 Definitions\n91.02 Possession, sale, and use of fireworks\n91.03 Permit to use fireworks\n91.04 Manufacturing or wholesale sale without a license; prohibitions\n91.05 Purchasers to comply with law; unauthorized purchases\n91.06 Exhibition without a license; prohibitions\n91.07 Unauthorized transportation or shipping\n91.08 Application of subchapter\n91.09 Arrest of offender; seizure and forfeiture of fireworks; distribution of fines\n91.10 Safety requirements for fireworks showroom structures\n91.11 Storage of explosives\n91.12 Blasting permit\n\nFire Prevention\n\n91.30 Removal of flammable materials or obstructions\n91.31 Protective appliances\n91.32 Compliance with order\n91.33 Waste receptacles\n91.34 Hotel to have fire warning device producing visible signal\n91.35 Fire suppression systems\n91.36 Violations of State Fire Code prohibited\n91.37 Posting arson notices in hotels, motels and other places\n91.38 Negligent burning\n91.39 Spreading alarm of unfriendly fire\n91.40 Unvented heaters\n\nOpen Burning\n\n91.55 Definitions\n91.56 Relations to other prohibitions\n91.57 Open burning in restricted areas\n91.58 Permission and notice to open burn\n\n91.99 Penalty\n\nChapter 92: Intoxicating Liquors\n\nSection\n\n| Section | Description |\n|---------|-------------|\n| 92.01 | Definitions |\n| 92.02 | Exemptions from chapter |\n| 92.03 | Restrictions applicable to sale of beer and intoxicating liquor for consumption on the premises |\n| 92.04 | Restrictions on sale of beer and liquor |\n| 92.05 | Permit required; activities prohibited without permit |\n| 92.06 | Illegal transportation prohibited |\n| 92.07 | Open container prohibited; exception |\n| 92.08 | Underage person shall not purchase intoxicating liquor or beer |\n| 92.09 | Prohibitions; minors under 18 years; low-alcohol beverages |\n| 92.10 | Alcohol vaporizing devices prohibited |\n| 92.11 | Misrepresentation to obtain alcoholic beverage for a minor prohibited |\n| 92.12 | Misrepresentation by a minor under 21 years |\n| 92.13 | Sale to underage persons prohibited |\n| 92.14 | Posting of card |\n| 92.15 | Good faith acceptances of spurious identification |\n| 92.16 | Consumption in motor vehicle prohibited |\n| 92.17 | Hours of sale or consumption |\n| 92.18 | Obstructing search of premises prohibited |\n| 92.19 | Illegal possession of intoxicating liquor prohibited |\n| 92.20 | Sale or possession of diluted liquor and refilled containers prohibited |\n| 92.21 | Keeping place where beer or intoxicating liquors are sold in violation of law |\n| 92.22 | Intoxicating liquors shall not be sold in brothels |\n| 92.23 | Use of intoxicating liquor in a public dance hall prohibited; exceptions |\n| 92.24 | Poisonously adulterated liquors |\n| 92.25 | Tavern keeper permitting rioting or drunkenness |\n| 92.26 | Notice of action to prohibit liquor business |\n| 92.27 | Procedure when injunction violated |\n| 92.28 | Liquor transaction scans |\n| 92.29 | Affirmative defenses |\n| 92.99 | Penalty |\n\nChapter 93: Nuisances\n\nSection\n\nGeneral Provisions\n\n| Section | Description |\n|---------|-------------|\n| 93.01 | Application of the chapter |\n| 93.02 | Definitions |\n| 93.03 | Nuisances generally; injunctions; violation; contempt |\n| 93.04 | Maintaining certain nuisances |\n| 93.05 | Collection of cost of abating dangerous property condition; injunction; rehabilitation |\n| 93.06 | Trimming of trees and shrubbery to prevent obstruction |\n\nSeptic Tanks, Cesspools, and Refuse\n\n| Section | Description |\n|---------|-------------|\n| 93.20 | Location of privy vaults, cesspools, and septic tanks |\n| 93.21 | Unsanitary vaults |\n| 93.22 | Removal of contents of vault |\n| 93.23 | Deposit of dead animals, offal upon land or water |\n| 93.24 | Defiling spring or well prohibited |\n| 93.25 | Dumping of refuse in municipality prohibited |\n| 93.26 | Abandoned refrigerators |\n| 93.27 | Discarding litter prohibited |\n| 93.28 | Power of municipality to fill or drain land |\n\nWeeds and Litter on Private Property\n\n| Section | Description |\n|---------|-------------|\n| 93.40 | Keeping down weeds |\n| 93.41 | Notice to owner to cut noxious weeds, remove litter; service |\n| 93.42 | Fees for service and return |\n| 93.43 | Procedure when owner fails to comply with notice |\n| 93.44 | Written return to County Auditor; amount as a lien upon property |\n\nUnclean Habitations\n\n93.60 Permitting unclean habitations\n93.61 When habitations are deemed unsanitary\n93.62 Order for abatement or vacation of premises\n93.63 Enforcement of vacation order by Fire Chief or Police Chief\n93.64 Enforcement through court proceedings\n\n93.99 Penalty\n\nChapter 94: Streets and Sidewalks\n\nGeneral Provisions\n\n94.01 Conditions precedent to improving streets\n94.02 Opening permit required\n94.03 Application and cash deposit\n94.04 Restoration of pavement\n94.05 Barriers around excavations\n94.06 Warning lights\n94.07 Sidewalk construction by the municipality\n94.08 Unloading on street or sidewalk\n94.09 Street or sidewalk obstruction\n94.10 Materials on street or sidewalk\n94.11 Duty to keep sidewalks in repair and clean of ice and snow\n94.12 Ramped curbing for persons with disabilities\n94.13 Flagpole along right-of-way\n94.14 Altering or injuring marker or monument\n\nConstruction and Repair\n\n94.25 Construction and repair may be required\n94.26 Resolution of necessity\n94.27 Notice to construct or repair\n94.28 Assessments of costs against owner\n94.29 Proceedings may include different owners\n94.30 Making and levying assessments\n\nChanges in Streets\n\n94.40 Change of name, vacating or narrowing streets by petition\n94.41 Change of name, vacating or narrowing streets without petition\n94.42 Notice; exception\n94.43 Publication of notice\n94.44 Effect of order of vacation\n94.45 Effect on public utility easements\n\n94.99 Penalty\n\nChapter 95: Unclaimed and Abandoned Vehicles\n\nSection\n\n95.01 Impounding motor vehicle on private property; requirements\n95.02 Impounding abandoned motor vehicle on public property; notice; disposition\n95.03 Disposition of vehicle ordered into storage\n95.04 Disposition of abandoned junk motor vehicles\n95.05 Abandonment of junk motor vehicle prohibited\n95.06 Junk motor vehicle; order to cover or remove; notice; exceptions\n\nChapter 96: Watercraft\n\nSection\n\n96.01 Definitions; applicability\n96.02 Failure to comply with law enforcement order; fleeing\n96.03 Duty upon approach of law enforcement vessel\n96.04 Flashing lights prohibited; exceptions\n\n96.05 Siren prohibited; exceptions\n96.06 Regulations for operation of powercraft of more than ten horsepower\n96.07 Restrictions on child operators; duty of supervisory adult\n96.08 Reckless operation; maintaining sufficient control; wakes restricted\n96.09 Unsafe conditions\n96.10 Marking of bathing and vessel areas\n96.11 Mooring prohibited in certain areas\n96.12 Operating under influence of alcohol or drugs prohibited\n96.13 Implied consent\n96.14 Incapacitated operators prohibited\n96.15 Water skiing confined to ski zones\n96.16 Observer required when towing skier\n96.17 Water skiing after dark prohibited\n96.18 Personal flotation device required for towed person\n96.19 Ski jumps prohibited\n96.20 Permit for special water events\n96.21 Sale of single celled inflatable vessels prohibited\n96.22 Sitting, standing, walking on moving vessels restricted\n96.23 Engine warm-up required\n96.24 Personal flotation devices for children under ten\n96.25 Operation without personal flotation devices prohibited\n96.26 Distress signal or flag required\n96.27 Anchor requirements\n96.28 Specification for fire extinguishers\n96.29 Backfire flame control device required\n96.30 Ventilation requirement on powercraft\n96.31 Abandonment of junk vessels or outboard motors\n96.32 Exhaust muffler required; noise levels; exceptions\n96.33 Safety equipment on rental vessels\n96.34 Capacity plate\n96.35 Littering prohibited\n96.36 Dwellings; sanitary systems\n96.37 Prima facie evidence of negligence\n96.38 Requirements for operating personal watercraft\n96.39 Numbering\n96.40 Registration\n96.41 Tags indicating expiration date; attachment of identification number\n96.42 Altering of serial numbers; false information prohibited\n96.43 Accident reports\n96.44 Enforcement\n96.45 Firearms offenses; signaling devices\n96.46 Tampering with navigation aid or vessel prohibited\n96.47 Certificate of title; exceptions\n96.48 Manufacturer’s or importer’s certificate\n96.49 Prohibitions relating to certificates of title\n96.50 Permanently displayed hull identification number\n\n96.99 Penalty\n\nTITLE XI: BUSINESS REGULATIONS\n\nChapter 110: General Provisions\n\nSection\n\n110.01 Licenses required to engage in certain businesses; exceptions\n110.02 Application for license\n110.03 Issuance of license\n110.04 Date and duration of license\n110.05 License not transferable\n110.06 License certificate to be displayed\n110.07 Revocation or suspension\n110.08 Appeal and review\n\n110.99 General penalty for Title XI\n\nChapter 111: Taxicabs\n\nSection\n\n111.01 Definitions\n111.02 Certificate of public convenience and necessity required\n111.03 Application for certificate\n111.04 Issuance of certificate\n111.05 Liability insurance required\n111.06 License fees\n111.07 Transfer of certificates and licenses\n111.08 Suspension and revocation of certificates\n111.09 Taxicab driver's license\n111.10 Application for driver's license\n111.11 Examination of applicant; motor vehicle operator's permit required\n111.12 Police investigation of applicant; traffic and police record\n111.13 Consideration of application\n111.14 Issuance of license; duration; annual fee\n111.15 Display of license\n111.16 Suspension and revocation of license\n111.17 Failure to comply with federal, state and municipal laws\n111.18 Vehicle equipment and maintenance\n111.19 Designation of taxicabs\n111.20 Taximeter and display of rates required\n111.21 Number of passengers allowed\n111.22 Articles left in vehicles\n111.23 Vehicles from other municipalities\n111.24 Receipts\n111.25 Refusal of passenger to pay legal fare\n111.26 Solicitation, acceptance and discharge of passengers\n111.27 Open stands; use\n111.28 Taxicab service\n111.29 Manifests\n111.30 Records and reports of holders\n111.31 Police department; duty to enforce chapter\n111.32 Disposition of vehicle license fees\n\nChapter 112: Peddlers, Itinerant Merchants, and Solicitors\n\nSection\n\n112.01 Definitions\n112.02 License requirement\n112.03 Application procedure\n112.04 Standards for issuance\n112.05 Revocation procedure\n112.06 Standards for revocation\n112.07 Appeal procedure\n112.08 Exhibition of identification\n112.09 Municipal policy on soliciting\n112.10 Notice regulating soliciting\n112.11 Duty of solicitors\n112.12 Uninvited soliciting prohibited\n112.13 Time limit on soliciting\n\nChapter 113: Commercial Amusements\n\nSection\n\n113.01 Bowling; billiards and pool\n113.02 Circuses, carnivals, shows and other such entertainment\n113.03 Deposit required\n113.04 License fee for public entertainment or exhibition\n113.05 License fee may be waived for civic interest\n\nChapter 114: Tattooing and Body Piercing Services\n\nSection\n\n114.01 Definitions\n114.02 Prohibitions\n114.03 Application for license; fees; issuance\n\n114.04 Inspection of facilities\n114.05 Suspension or revocation of license\n114.06 Consent for performing procedures on persons under 18\n114.07 Prohibitions relating to persons under 18\n114.08 Defenses to violations\n114.09 Training standards; records; safety and sanitation; equipment\n114.10 Application of local regulation on chapter\n\n114.99 Penalty\n\nTITLE XIII: GENERAL OFFENSES\n\nChapter 130: General Provisions\n\nSection\n\n130.01 Application of Title XIII\n130.02 Definitions\n130.03 Classification of offenses\n130.04 Common law offenses abrogated\n130.05 Rules of construction\n130.06 Limitation of criminal prosecutions\n130.07 Requirements for criminal liability; voluntary intoxication\n130.08 Culpable mental states\n130.09 Organizational criminal liability\n130.10 Personal accountability for organizational conduct\n130.11 Attempt\n130.12 Complicity\n130.13 Presumption of innocence; proof of offense; affirmative defense\n130.14 Battered woman syndrome\n130.15 Delinquency adjudications deemed convictions\n130.16 Criminal law jurisdiction\n130.17 Disposition of unclaimed or forfeited property held by Police Department\n130.18 Imposing sentence for misdemeanor\n130.19 Multiple sentences\n130.20 Apprehension, detention, or arrest of persons on bond\n130.21 Self defense: limitations on duty to retreat prior to using force\n\n130.99 Penalty for Title XIII\n\nChapter 131: Offenses Against Property\n\nSection\n\n131.01 Definitions\n131.02 Arson; determining property value or amount of physical harm\n131.03 Criminal damaging or endangering; vehicular vandalism\n131.04 Criminal mischief\n131.05 Damaging or endangering aircraft or airport operations\n131.06 Criminal trespass; aggravated trespass\n131.07 Tampering with coin machines\n131.08 Theft\n131.09 Unauthorized use of a vehicle\n131.10 Unauthorized use of property, including telecommunication property and computers; possession of municipal property\n131.11 Passing bad checks\n131.12 Misuse of credit cards\n131.13 Making or using slugs\n131.14 Prima facie evidence of purpose to defraud\n131.15 Tampering with records\n131.16 Securing writings by deception\n131.17 Defrauding creditors\n131.18 Receiving stolen property\n131.19 Value of stolen property\n131.20 Degree of offense when certain property involved\n131.21 Injuring vines, bushes, trees, or crops\n131.22 Detention and arrest of shoplifters and those committing motion picture piracy; protection of institutional property\n\n131.23 Insurance fraud; workers' compensation fraud; Medicaid fraud\n131.24 Injury to property by hunters\n131.25 Evidence of intent to commit theft of rented property; evidence of lack of capacity to consent\n131.26 Forgery of identification cards\n131.27 Criminal simulation\n131.28 Personating an officer\n131.29 Trademark counterfeiting\n131.30 Diminishing or interfering with forfeitable property\n131.31 Recording credit card, telephone or Social Security numbers\n131.32 Prosecutions for theft of utilities\n131.33 Motion picture piracy\n\nChapter 132: Offenses Against Public Peace\n\nSection\n\n132.01 Riot\n132.02 Failure to disperse\n132.03 Justifiable use of force to suppress riot\n132.04 Disorderly conduct\n132.05 Disturbing a lawful meeting\n132.06 Misconduct at an emergency\n132.07 Telecommunications harassment\n132.08 Inducing panic\n132.09 Making false alarms\n132.10 Inciting to violence\n132.11 Unlawful display of law enforcement emblem\n132.12 Impersonating a peace officer\n132.13 Safety of crowds attending live entertainment performances\n132.14 Misconduct involving a public transportation system\n\nChapter 133: Sex Offenses\n\nSection\n\n133.01 Definitions\n133.02 Unlawful sexual conduct with a minor\n133.03 Sexual imposition\n133.04 Public indecency\n133.05 Voyeurism\n133.06 Polygraph examinations for victims: restrictions on use\n133.07 Procuring\n133.08 Soliciting; loitering to engage in\n133.09 Prostitution\n133.10 Disseminating matter harmful to juveniles\n133.11 Displaying matter harmful to juveniles\n133.12 Deception to obtain matter harmful to juveniles\n133.13 Rules of evidence\n133.14 Declaratory judgment\n133.15 Injunction; abatement of nuisance\n133.16 Unlawful operation of viewing booths depicting sexual conduct\n133.17 Juveniles on the premises of adult entertainment establishments prohibited\n133.18 Sexually oriented businesses; illegal operation and activity\n133.19 Unlawful advertising of massage\n\n133.99 Sentencing for sexually oriented offenses; sexual predators; registration\n\nChapter 134: Gambling Offenses\n\nSection\n\n134.01 Definitions\n134.02 Prohibitions against gambling; exception\n134.03 Operating a gambling house\n134.04 Public gaming\n134.05 Cheating\n134.06 Regulations concerning operation of licensed bingo game\n134.07 Records to be kept\n134.08 Requirements for bingo game operators\n134.09 Bingo games for amusement only\n\n134.10 Prohibitions where instant bingo game is conducted\n134.11 Raffle drawings\n134.12 Instant bingo other than at bingo sessions\n134.13 Restrictions on owner or lessor of location at instant bingo\n134.14 Skill-based amusement machines; prohibited conduct\n\nChapter 135: Offenses Against Persons\n\nSection\n\n135.01 Definitions\n135.02 Negligent homicide\n135.03 Vehicular homicide; vehicular manslaughter\n135.04 Assault; negligent assault\n135.05 Injury to persons by hunters\n135.06 Menacing; aggravated menacing; menacing by stalking\n135.07 Unlawful restraint\n135.08 Criminal child enticement\n135.09 Coercion\n135.10 Bigamy\n135.11 Unlawful abortion; failure to perform viability testing\n135.12 Abortion trafficking\n135.13 Nonsupport of dependents\n135.14 Endangering children\n135.15 Interference with custody; interference with support orders\n135.16 Domestic violence\n135.17 Hazing prohibited\n135.18 Contributing to unruliness or delinquency of a child\n135.19 Failure to provide for functionally impaired person\n135.20 Patient abuse or neglect; patient endangerment; exceptions; false statements; retaliation\n135.21 Interference with right of person to engage in housing transactions because of race, religion, or the like\n135.22 Ethnic intimidation\n135.23 Violating a protection order, consent agreement, anti-stalking protection order or order issued by a court of another state\n135.24 Adulteration of food\n135.25 Illegal distribution of cigarettes, other tobacco products, or alternative nicotine products; transaction scans\n135.26 Nonsmoking areas in places of public assembly\n135.27 Spreading contagion\n135.28 Abuse of a corpse\n135.29 Unlawful collection of bodily substances\n\nChapter 136: Offenses Against Justice and Administration\n\nSection\n\n136.01 Definitions\n136.02 Falsification\n136.03 Compounding a crime\n136.04 Failure to report a crime\n136.05 Failure to aid a law enforcement officer\n136.06 Obstructing official business\n136.07 Obstructing justice\n136.08 Resisting arrest\n136.09 Having an unlawful interest in a public contract\n136.10 Soliciting or receiving improper compensation\n136.11 Dereliction of duty\n136.12 Interfering with civil rights\n136.13 Illegal conveyance of prohibited items onto grounds of a detention facility or other specified governmental facilities\n136.14 False report of child abuse or neglect\n136.15 Assaulting police dog or horse, or assistance dog\n136.16 Disclosure of confidential peace officer information\n136.17 Intimidation of crime victim or witness\n136.18 Using sham legal process\n136.19 Making false allegation of peace officer misconduct\n136.20 Misuse of 9-1-1 system\n136.21 Failure to disclose personal information\n\nChapter 137: Weapons Control\n\nSection\n\n137.01 Definitions\n137.02 Carrying concealed weapons\n137.03 Using weapons while intoxicated\n137.04 Improperly handling firearms in a motor vehicle\n137.05 Possessing criminal tools\n137.06 Failure to secure dangerous ordnance\n137.07 Unlawful transactions in weapons\n137.08 Underage purchase of firearm or handgun\n137.09 Pointing and discharging firearms and other weapons\n137.10 License or permit to possess dangerous ordnance\n137.11 Possession of an object indistinguishable from a firearm in a school safety zone\n137.12 Possession of deadly weapon while under detention\n137.13 Concealed handgun licenses: possession of a revoked or suspended license; additional restrictions; posting of signs prohibiting possession\n137.14 Defaced firearms\n\nChapter 138: Drug Offenses\n\nSection\n\n138.01 Definitions\n138.02 Trafficking in controlled substances; gift of marihuana\n138.03 Drug possession offenses\n138.04 Possessing drug abuse instruments\n138.05 Permitting drug abuse\n138.06 Illegal cultivation of marihuana\n138.07 Abusing harmful intoxicants\n138.08 Illegal dispensing of drug samples\n138.09 Federal prosecution bar to municipal prosecution\n138.10 Nitrous oxide: improper dispensing or distribution; possession in a motor vehicle\n138.11 Laboratory report required\n138.12 Counterfeit controlled substances\n138.13 Use, possession, or sale of drug paraphernalia\n138.14 Controlled substance or prescription labels\n138.15 Possession, sale and disposal of hypodermics\n138.16 Controlled substance schedules\n138.17 Unlawful furnishing of prescription to enable persons to be issued handicapped parking placards or license plates\n138.18 Pseudoephedrine sales\n\nTITLE XV: LAND USAGE\n\nChapter 150: General Provisions\n\nSection\n\nParks and Recreation\n\n150.01 Recreation Board\n150.02 Board of Park Trustees\n\nPlanning and Zoning\n\n150.15 Planning Commission\n150.16 Board of Zoning Appeals\n\nPARALLEL REFERENCES\n\nOhio Legislative History References – Master Table\n\nINDEX\n\nThis summary of contents has been verified and authorized for publication by the Legislative Authority of the Municipality of Village of Avon, Ohio.\n\nSigned: [Signature] Mayor\n\nClerk of the Legislative Authority\n\nCERTIFICATION OF CODIFIED ORDINANCES\n\nWe, Mark A. Mear, Mayor, and Carrie Ayers, Clerk of the Legislative Authority, of the Municipality of Village of Anarole, Ohio, pursuant to Ohio Revised Code §§ 731.23 and 731.42, hereby certify that the general and permanent ordinances of the Municipality, as revised, rearranged, compiled, renumbered as to sections, codified and printed herewith in component codes and titles are correct as and constitute the Code of Ordinances for the Municipality of Village of Anarole, Ohio.\n\n[Signature]\nMayor\n\n[Signature]\nClerk of the Legislative Authority"}}},{"rowIdx":204697,"cells":{"text":{"kind":"string","value":"Project Overview\n\nWhether traveling to school, running errands, or enjoying a stroll through the park, many people in Pekin choose to walk and bike, and many more would choose to do so if conditions were better.\n\nA safe and connected system of sidewalks, trails, and bikeways not only supports people to traveling by bike and foot, but also contributes to the safety of all road users and to the quality of life for Pekin residents.\n\nThe Pekin Bicycle and Pedestrian Master Plan will serve as the City’s vision and blueprint for creating a safer, more comfortable transportation environment for people of all ages, abilities, and backgrounds, especially for people who walk, bike, and use mobility assistance devices like wheelchairs or walkers.\n\nThis long-range planning document will fuse inspiration and ideas from Pekin residents and community stakeholders with technical analysis of current conditions and best practices in bicycle and pedestrian planning and facility design.\n\nThe final plan will include recommendations for new trails, bikeways, sidewalks, and intersection improvements, as well as supporting policies, programs, and implementation strategies. These recommendations will guide future investments in infrastructure projects and help the City secure funding from state, federal, and other sources.\n\nPlanning Inputs\n\n- Field Analysis of Current Conditions\n- Public Workshops and Survey Forms\n- Existing & Previously Planned Facilities\n- Network Quality (LTS) Analysis\n- Existing Bike/Ped Activity & Demand Analysis\n- Direction from Municipalities and MPO\n- Steering Committee & Stakeholder Input\n\nProject Timeline\n\n**PHASE 1: Existing Conditions, Needs Assessment, Visioning**\n- FEB\n- MAR\n- APR\n- MAY\n- JUN\n\nThe first phase of the plan focuses on creating a shared understanding of current conditions for walking and biking and learning from Pekin residents about community needs, issues, and aspirations relating to active transportation. The project team will establish a vision for the Bicycle and Pedestrian Master Plan and supporting goals to help bring the vision to life. These early stages in the planning process will form the foundation for identifying and prioritizing recommendations for new trails, sidewalks, bikeways, and supporting programs.\n\n**PHASE 2: Plan Recommendations & Implementation Strategies**\n- JUL\n- AUG\n- SEP\n\nThe second phase of the project focuses on developing recommendations for projects that improve walking and bicycling safety, connectivity, and access, like new sidewalks, bike lanes, and trails. The project team will also identify policy, programming, and project development strategies to support the plan’s vision and integrate walking and biking into transportation decision-making and projects.\n\n**PHASE 3: Draft Plan Development & Review**\n- OCT\n- NOV\n- DEC\n\nThe third and final phase of the project focuses on refining the plan recommendations, prioritization, and implementation strategies based on feedback from the open house public meeting and creating a draft plan document for review. After final revisions, the plan will be considered for official adoption.\n\nPekin Bicycle & Pedestrian Master Plan\n\nSeptember 28, 2023\nOpen House #2\n\nTHE VISION: The City of Pekin strives to make walking and bicycling safe, accessible, convenient, and equitable transportation options that connect people to places, foster recreational and economic development opportunities, support healthy and active living, and elevate quality of life.\n\nGOAL #1 - SAFETY: Create a safe travel environment for everyone, especially vulnerable road users like people walking, rolling, and bicycling.\n\nObjective 1.1. Improve pedestrian and bicycle safety through the installation of sidewalks, trails, dedicated bikeways, and intersection enhancements.\n\nObjective 1.2. Create a safety action plan to evaluate crash history, identify trends, and develop a high-injury network for future safety investments.\n\nObjective 1.3. Reduce fatal and serious injury crashes through proven safety countermeasures.\n\nObjective 1.4. Support safe and responsible travel through education programs and campaigns.\n\nGOAL #2 - NETWORK: Develop a complete, convenient, and interconnected active transportation network.\n\nObjective 2.1. Expand the Pekin Bike Trail to increase community access to one of the community’s most valued recreation and active transportation amenities.\n\nObjective 2.2. Target sidewalk infill, sidewalk system expansion, and crossing improvements on pedestrian priority corridors.\n\nObjective 2.3. Develop a low-stress on-street bikeway system to support access to everyday destinations throughout Pekin.\n\nObjective 2.4. Coordinate with community partners and local, regional, and state agencies to increase regional connectivity and access to destinations in neighboring communities.\n\nObjective 2.5. Develop a community-wide active transportation wayfinding system to guide people to schools, parks, Downtown Pekin, the Pekin Bike Trail, and other major destinations.\n\nObjective 2.6. Prioritize and pursue projects in a manner that balances community impact, project feasibility, and available resources and staff capacity.\n\nGOAL #3 - DESIGN: Design active transportation facilities that are accessible and comfortable for people of all ages and abilities.\n\nObjective 3.1. Apply current standards and best practices for low-stress bicycle and pedestrian facility design.\n\nObjective 3.2. Prioritize projects that address known ADA deficiencies, align with the recommendations in the ADA Transition Plan, and promote safe and accessible pathways for people with disabilities, children, seniors, and people with limited mobility.\n\nObjective 3.2. Evaluate Pekin Bike Trail crossings to improve user safety and to increase motorist awareness.\n\nGOAL #4 - POLICY: Develop policies that integrate active transportation into city decision-making processes.\n\nObjective 4.1. Develop internal procedures to consider and integrate active transportation facilities in capital improvement planning, programming, and project development.\n\nObjective 4.2. Support active transportation through revisions to the zoning ordinance, subdivision regulations, and street standards.\n\nObjective 4.3. Establish baseline community outreach and engagement protocols for transportation projects.\n\nGOAL #5 - PROGRAMS: Foster an environment that supports and embraces walking and biking through community partnerships and education, encouragement, and awareness programs.\n\nObjective 5.1. Foster collaboration and partnerships with community organizations and agencies throughout the region to identify and implement education, encouragement, and awareness programs and campaigns to increase walking and bicycling activity and safety.\n\nObjective 5.2. Develop Safe Routes to School program in partnership with Pekin Public Schools to increase walking and bicycling to school.\n\nObjective 5.3. Create an awareness campaign to raise the profile of active transportation, promote safe travel behaviors, and highlight the benefits of walking and bicycling.\n\nOnline Survey Results\n\nWalking-Related Questions\n\nHow easy is it to walk in your neighborhood and to nearby destinations?\n\n- Very Easy: 11%\n- Moderately Easy: 31%\n- Neutral: 15%\n- Moderately Difficult: 24%\n- Very Difficult: 10%\n- Other (please specify): 6%\n\nHow far do you typically travel when walking?\n\n- Less than 0.5 miles: 10%\n- 0.5 to 1 mile: 17%\n- 1 to 2 miles: 36%\n- 2 to 5 miles: 31%\n- 5 to 10 miles: 3%\n- Over 10 miles: 0%\n- N/A: 3%\n\nFor which of the following reasons do you choose to walk for work or recreation trips? (Check all that apply)\n\n- Safety/security: 23%\n- Convenience: 24%\n- Health benefits/fitness: 95%\n- Environmental benefits: 39%\n- For fun/enjoyment: 72%\n- Don't have access to a vehicle: 1%\n- Don't have access to public transit: 0%\n- Other (please specify): 4%\n\nWhich of the following barriers prevent you from walking more? (Please select all that apply)\n\n- I do not feel safe and worry about: 55%\n- I am physically unable to walk: 1%\n- I have no place to park: 68%\n- Lack of sidewalks: 18%\n- Obstruction to vision: 15%\n- Lack of lighting: 43%\n- I don't like walking: 27%\n- I don't like walking because of children: 8%\n- I don't like walking because of crime: 4%\n- I don't like walking because of weather: 16%\n- Noise: 6%\n- I walk too much as it is: 4%\n- Other (please specify): 7%\n\nPlease evaluate the following sentence based on how it represents you: \"I would like to walk more than I do now for my daily commute, errands, and other activities.\"\n\n- Agree: 42%\n- Somewhat Agree: 24%\n- Neutral: 21%\n- Somewhat Disagree: 7%\n- Disagree: 6%\n\nPlease evaluate the following sentence based on how it represents you: \"I would like to walk more than I do now for fun, exercise, and other recreational activities.\"\n\n- Agree: 78%\n- Somewhat Agree: 16%\n- Neutral: 5%\n- Somewhat Disagree: 1%\n- Disagree: 1%\n\nWhat destinations would you like to be able to walk to? (Choose all that apply)\n\n- Work: 12%\n- Bus Stop: 4%\n- School: 11%\n- Trails: 75%\n- Parks, playgrounds, and recreation centers: 75%\n- Grocery stores, local shops, and restaurants: 59%\n- Government services (city hall, police station, social services, library, office, etc.): 23%\n- Place of worship (church, synagogue, mosque, etc.): 21%\n- Other (please specify): 5%\n\nPekin Bicycle & Pedestrian Master Plan\n\nSeptember 28, 2023\nOpen House #2\n\nOnline Survey Results\n\nBicycling-Related Questions\n\nHow easy is it for you to bike in your neighborhood and to nearby destinations?\n\n- Very easy: 8%\n- Moderately Easy: 21%\n- Neutral: 24%\n- Moderately Difficult: 31%\n- Very difficult: 16%\n\nHow far do you typically travel when biking?\n\n- Less than 0.5 miles: 5%\n- 0.5 to 1 mile: 2%\n- 1 to 2 miles: 11%\n- 2 to 5 miles: 25%\n- 5 to 10 miles: 23%\n- Over 10 miles: 11%\n- N/A: 22%\n\nFor which of the following reasons do you choose to bike for work, school, or recreation trips? (Check all that apply)\n\n- Safety concerns: 28%\n- Convenience: 20%\n- Health benefits/exercise: 92%\n- Environmental benefits: 49%\n- For fun/entertainment: 72%\n- Don’t have access to a vehicle: 1%\n- Don’t have access to public transit: 1%\n- Other (please specify): 2%\n\nWhat type of cyclist best describes you? (One selection allowed)\n\n- Strong and Fearless (Comfortable biking on major streets without bike lanes): 16%\n- Enthusiastic and Confident (Comfortable biking on major streets only with bike lanes): 16%\n- Interested but Concerned (Only comfortable biking if separated from cars and/or major streets): 54%\n- No Way, No How (Not interested in biking at all): 15%\n\nPlease evaluate the following sentence based on how it represents you: “I would like to bike more than I do now for my daily commute, errands, and other activities.”\n\n- Agree: 38%\n- Somewhat Agree: 24%\n- Neutral: 21%\n- Somewhat Disagree: 6%\n- Disagree: 11%\n\nPlease evaluate the following sentence based on how it represents you: “I would like to bike more than I do now for fun, exercise, and other recreational activities.”\n\n- Agree: 63%\n- Somewhat Agree: 21%\n- Neutral: 8%\n- Somewhat Disagree: 5%\n- Disagree: 4%\n\nWhich of the following bikeway types would you like to see in Pekin? (Check all that apply)\n\n- Shared Lane: 6%\n- Painted Shoulder/Shoulder Bike Lane: 50%\n- Marked and Signed Bike Route: 49%\n- Bike Lane: 59%\n- Buffered Bike Lane: 47%\n- Separated Bike Lane (Cycle Track): 65%\n- Shared Use Path / Speed Hump: 84%\n\nWhich of the following barriers prevent you from biking more? (Please select all that apply)\n\n- I don't have time/space to ride: 11%\n- Distractions from traffic: 10%\n- I live too far from the places I need to go: 4%\n- Lack of dedicated bike lanes: 52%\n- Lack of safe places to park: 69%\n- There isn't an entire place to ride: 58%\n- Not enough parking: 32%\n- Too much traffic: 22%\n- I don't feel safe biking around pedestrians: 22%\n- Disability or other health issues: 19%\n- Weather prevents me: 17%\n- None. I like as much as I want to bike (like I currently do): 4%\n- Other (please specify): 15%\n\nWhat destinations would you like to be able to bike to? (Choose all that apply)\n\n- Work: 27%\n- Bus Stop: 3%\n- School: 12%\n- Trails: 94%\n- Parks, playgrounds, and recreation centers: 77%\n- Grocery stores, local shops, and restaurants: 55%\n- Government services (city hall, library, social services, post office, etc.): 28%\n- Place of worship (church, temple, mosque, etc.): 17%\n- Other (please specify): 6%\n\nBicycle Improvements Toolkit\n\nThere is no one-size-fits-all approach to bicycle improvements. Each bicycle facility or intersection improvement must be compatible with roadway characteristics, traffic patterns, and adjacent land uses. The following bicycle facilities and intersection improvements highlight the diversity of options available to better support bicycling in Pekin.\n\n**BICYCLE FACILITY TYPES**\n\n- **Shared Travel Lanes**\n- **Paved Shoulders**\n- **Separated Bicycle Lanes**\n- **Bicycle Wayfinding & Direction Signs**\n- **Bicycle Boulevards**\n- **Bicycle Lanes / Buffered Bicycle Lanes**\n- **Shared Use Paths & Trails**\n- **Bicycle Parking**\n\n**INTERSECTION IMPROVEMENTS**\n\n- **Bike Boxes**\n- **Bike Lane Markings/Conflict Markings/Green Paint**\n- **Bicycle Signals**\n- **Protected Intersections**\n\nPekin Bicycle & Pedestrian Master Plan\n\nSeptember 28, 2023\nOpen House #2\n\nProposed Bicycle Network\n\nPekin Bicycle & Pedestrian Master Plan\n\nCommunity Features\n- City Hall\n- Hospital\n- Library\n- Post Office\n- School\n- Place of Worship\n- Full Service Grocery Store\n- Park\n- CityLink Bus Stop\n\nBicycle Recommendations\n- Existing Trail\n- Shared Use Path\n- Side Path\n- Separated Bike Lane\n- Bike Lane\n- Bike Blvd\n- Shared Travel Lane\n- Crossing Improvements\n\nPedestrian Improvements Toolkit\n\nCreating a safe, comfortable, accessible, and attractive environment for walking does not happen overnight. It takes years to develop a high-quality pedestrian system. With the right plans and policies in place, the City of Pekin can direct public infrastructure investments and private development to support people walking, regardless of their age, ability, or use of mobility assistance devices.\n\n**NETWORK ENHANCEMENTS**\n\n- **Sidewalks**\n - New Construction\n - Sidewalk Infill & Repairs\n\n- **Street Furniture**\n - Benches\n - Planters\n\n- **Pedestrian-Scale Lighting**\n - Streetlights\n - Pathway Lights\n\n- **Shade Trees**\n - Trees along sidewalks\n - Trees in parks\n\n**INTERSECTION IMPROVEMENTS**\n\n- **Curb Extensions**\n - Curbs extended into intersections\n\n- **ADA-Accessible Curb Ramps**\n - Ramps for wheelchair access\n\n- **Mid-Block Crosswalks**\n - Crosswalks at intersections\n\n- **Pedestrian Signals & Countdown Timers**\n - Traffic signals with pedestrian signals\n\n- **Marked Crosswalks**\n - Painted crosswalks\n\n- **Raised Crosswalks**\n - Elevated crosswalks\n\n- **Leading Pedestrian Interval**\n - Traffic lights timed for pedestrians\n\n- **Median Refuge Islands**\n - Islands in the middle of intersections\n\n---\n\nPekin Bicycle & Pedestrian Master Plan\n\nSeptember 28, 2023\nOpen House #2\n\nThe ADA Transition Plan will be the primary driver of investments in sidewalks and intersection improvements.\n\nWith that in mind, the pedestrian network in the Pekin Bicycle and Pedestrian Master Plan focuses on pedestrian priority corridors.\n\nPedestrian priority corridors consist primarily of arterial and collector roadways that connect neighborhoods to retail, shopping, schools, parks, and other vital destinations.\n\nThis approach acknowledges that not all roads are created equal. Pedestrian priority corridors generally carry higher volumes of motor vehicle traffic at higher speeds, and safe and accessible pedestrian facilities are necessary to support pedestrian activity.\n\nAlso included in the pedestrian network are shared use paths, sidepaths, and the existing Pekin Bike Path.\n\nCommunity Features\n\n- City Hall\n- Hospital\n- Library\n- Post Office\n- School\n- Full Service Grocery Store\n- Place of Worship\n- Park\n- CityLink Bus Stop\n\nPedestrian Recommendations\n\n- Pedestrian Priority Corridor\n- Existing Trail\n- Shared Use Path\n- Side Path\n- Crossing Improvements\n\nProject Prioritization\n\nActive transportation infrastructure projects must compete with other capital improvements and municipal services, as well as with one another, for limited internal and external resources. In order to maximize investment and provide the greatest benefit, the City of Pekin should take a prioritized approach to invest in active transportation infrastructure and plan implementation.\n\nImpact Factors:\n- Access to schools, parks, commercial districts/hubs, transit\n- Low stress/all ages and abilities\n- Connections to existing facilities\n- Safety (addressing high-crash corridors & intersections)\n- Public support\n\nEffort Factors:\n- Project Cost\n- Project Complexity\n- Ownership\n- Coordination with Programmed Improvements\n\nImplementation Categories:\n\n| Priority | Feasibility | Description |\n|----------|-------------|-------------|\n| High | High | Long Term Improvement |\n| | | Projects for further study and evaluation. Seek grant funding to advance projects. |\n| Low | Low | Short Term Improvement |\n| | | High priority and easy to implement projects for short term development. |\n| Low | High | Opportunity Improvement |\n| | | Lower priority projects that may become an opportunity with the necessary funding or partnerships. |\n\nWhat are your Priorities?\n\nSupporting Programs\n\nEARN-A-BIKE PROGRAM\nEarn-a-Bike programs give students the opportunity to learn basic bike maintenance and bicycling skills, as well as route selection and mapping. Students who participate in these programs typically learn to build a bicycle from the ground up, and upon completion of the program receive a refurbished bike (usually the one they build), along with a helmet, bike lock, and bike lights.\n\nTRAIL USER ETIQUETTE CAMPAIGN\nStatewide, local parks and trails are available for use by bicyclists, walkers, runners, skateboarders, rollerbladers, parents with strollers, dogs, children, and, in some cases, equestrians, as well as other modes. A campaign for trail user etiquette works to ensure that all users safely and responsibly share the trail. The campaign may include media advertisements, trail signage, brochures, an “ambassador” program, etc.\n\nHAPPY TRAILS TO HEALTHY FOODS\nMany communities have developed programs that promote physical activity and healthy eating goals in improving overall public health and wellness. This important link can be highlighted in a fun and interactive manner through promoting healthy food outlets along the bicycle, pedestrian, trail, and transit network and partnering with health food providers to identify safe routes for active transportation to their locations.\n\nCAR-FREE STREET EVENTS\nCar-free street events are temporary “openings” that create a temporary park that is open to the public for walking, bicycling, dancing, etc. The purpose of the event is to encourage biking and other forms of physical activity to the general public by providing a fun, welcoming environment for activity. Car-free street events have been very successful internationally and are rapidly becoming popular in the U.S.\n\nFAMILY BIKING PROGRAMS\nFamily Biking programs help parents figure out how to safely transport children by bicycle and help children learn bicycling skills. Activities may include bicycle safety checks, a group ride or parade, “freedom from training wheels” clinics, and opportunities to try out different ways to transport children (e.g., trailers, cargo bicycles, kid seats, etc.).\n\nSAFE ROUTES TO SCHOOL PROGRAM\nThe City of Pekin can benefit from a comprehensive Safe Routes to School program that combines local facility, safety, and route planning with the development of school and travel events, like walking school buses or bike trains. Both of these strategies require a commitment and support from school leadership and staff, the City of Pekin, and families with school-aged children.\n\nSILVER SNEAKERS SENIORS PROGRAM\nInterested agencies, nonprofits, health departments and senior centers can partner to develop an active lifestyles program for senior citizens utilizing the bicycle, pedestrian, and greenways network. Activities could include adult tricycle or bicycle rides, nature walks, walks to lunch, and safety education.\n\nOUTDOOR PUBLIC ART\nOutdoor art along a bicycle, pedestrian, and trail network can bring attention to the network and attract newcomers to bicycling and walking, while also enhancing outdoor space. By adding art to trail facilities, the community is creating a unique interactive amenity for both residents and visitors. Such programs can also attract new partners, promoters, and sponsors of the active transportation network.\n\nINTERPRETIVE SIGNAGE\nInterpretive signage along a trail and greenway network serves as an education tool. Information related to the history of an area, its cultural significance, or natural features is provided on a graphically appealing sign. Topics could range from native species of plants to river currents to famous historical figures.\n\nBICYCLE FRIENDLY BUSINESS PROGRAM\nBusinesses in Pekin have an important role to play in fostering a bicycle friendly community. From supporting employees with secure bicycle storage facilities, showers, and lockers to incentivizing customers that arrive on bike with discounts or other promotions, there are many ways businesses can promote bicycling. The League of American Bicyclists Bicycle Friendly Business program will be a valuable resource, and businesses should strive to achieve recognition as a Bicycle Friendly Business.\n\nBIKE MONTH EVENTS\nCities and towns across the country participate in National Bike Month annually, during May. The League of American Bicyclists (LAB) hosts a website for event organizers. The website contains information on nationwide and local events, an organizing handbook, and promotional materials. Events can vary widely, including events to promote cycling like morning-commute energizer stations and an end-of-day rally or celebration, a group ride with the mayor, discounts at local businesses for bicycle commuters, short, themed community bicycle rides, mountain bike skills clinics, and commuter courses for adults.\n\nPERSONAL TRAVEL ENCOURAGEMENT\nPTE programs are designed to educate the public on how to approximately 10% and increase bicycling, walking and transit use within a target area. The program:\n\n- Delivers customized travel information packets;\n- Hosts fun events such as guided rides, walks, and classes; and\n- Sends trained outreach staff to farmers’ markets and other community events\n\nBICYCLE COUNT PROGRAM\nBy utilizing both a manual and automated counting practices, Pekin can better measure and understand how people travel in the community, which can provide insight into heavily utilized, and where to invest in future transportation improvements. The Pedestrian and Bicycle Information Center (PBIC) has a wealth of information and resources to help the City develop a comprehensive counting program.\n\nTARGETED TRAFFIC ENFORCEMENT\nBy focusing traffic enforcement efforts on areas of high travel around schools, senior centers, business districts, and high-crash locations, the Pekin Police Department can support bicycle and pedestrian safety, especially for the most vulnerable road users in our community.\n\nTHEMED WALKING/BIKING MAPS & GUIDES\nWalking and biking maps and guides can be used to explore the city’s rich history, unique character, and beautiful parks. The guides would be targeted to a variety of ages and abilities, offering varying routes for families, touring cyclists, and others. Themes can include history, architecture, parks, and culture and suited for both Pekin residents and visitors.\n\nTRAFFIC TICKET DIVERSION CLASS\nA diversion class is offered to those offenders of certain bicycle-related traffic violations, such as running a stop sign on a bike. It can be aimed just at bicyclists or at bicyclists and motorists, or pedestrians. In lieu of a citation and/or fine, individuals can take a one-time, free or inexpensive class.\n\nCREATE-A-COMMUTER PROGRAM\nCreate-A-Commuter programs equip people with the tools to overcome common barriers to bicycling to work, school, and other everyday destinations. A successful program often includes bicycle skills and maintenance courses, mentorship pairing with experienced cyclists, and Guaranteed Ride Home (GRH) element to provide participants with taxi, rideshare, or transit service if an unexpected need arises.\n\nBICYCLE + PEDESTRIAN RESOURCE WEBSITE\nThe City could create a website that serves as a one-stop resource for bicyclists, pedestrians, and trail users of all types. Information could include tips for commuters, route planning services, community events, message boards, and more.\n\nBICYCLE PARKING PROGRAM\nPeople traveling by bicycle rely on safe and secure bicycle parking facilities to support their trips. To incentivize bicycle parking installation, the City of Pekin should consider the development of a bicycle parking program that provides reduced-cost bicycle racks for local businesses and community destinations.\n\nKEY QUESTION: ARE THERE OTHER PROGRAM IDEAS YOU WOULD LIKE TO SEE IN PEKIN?\n(write your thoughts or place a dot beside an existing idea to show your support)\n\nLAUNCH PARTIES FOR NEW BIKEWAYS\nThe area’s cities and counties should partner with local advocacy groups to generate media attention and engage local citizens in each incremental expansion of the bikeway network. Popular launch parties in other jurisdictions have included bicycle-handling workshops, free bike mechanic services, live music, bicycle giveaways, and other activities.\n\nWe Need Your Input!\n\nWhat will make this plan a success?\n\nWhat’s your top priority or project?\n\nWhat else do we need to know?"}}},{"rowIdx":204698,"cells":{"text":{"kind":"string","value":"Cardiac Myocyte Diversity and a Fibroblast Network in the Junctional Region of the Zebrafish Heart Revealed by Transmission and Serial Block-Face Scanning Electron Microscopy\n\nPascal J. Lafontant \n*DePauw University*, email@example.com\n\nFollow this and additional works at: [https://scholarship.depauw.edu/bio_facpubs](https://scholarship.depauw.edu/bio_facpubs)\n\nPart of the Biology Commons\n\n**Recommended Citation**\nLafontant PJ, Behzad AR, Brown E, Landry P, Hu N, et al. (2013) Cardiac Myocyte Diversity and a Fibroblast Network in the Junctional Region of the Zebrafish Heart Revealed by Transmission and Serial Block-Face Scanning Electron Microscopy. *PLoS ONE* 8(8): e72388. doi:10.1371/journal.pone.0072388\n\nThis Article is brought to you for free and open access by the Biology at Scholarly and Creative Work from DePauw University. It has been accepted for inclusion in Biology Faculty publications by an authorized administrator of Scholarly and Creative Work from DePauw University.\n\nCardiac Myocyte Diversity and a Fibroblast Network in the Junctional Region of the Zebrafish Heart Revealed by Transmission and Serial Block-Face Scanning Electron Microscopy\n\nPascal J. Lafontant¹*, Ali R. Behzad², Evelyn Brown³, Paul Landry³, Norman Hu⁴, Alan R. Burns³,⁵\n\n¹ Department of Biology, DePauw University, Greencastle, Indiana, United States of America, ² Imaging and Characterization Core Lab, King Abdullah University of Science and Technology, Thuwal, Kingdom of Saudi Arabia, ³ College of Optometry, University of Houston, Houston, Texas, United States of America, ⁴ Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, Utah, United States of America, ⁵ Department of Pediatrics, Baylor College of Medicine, Houston, Texas, United States of America\n\nAbstract\n\nThe zebrafish has emerged as an important model of heart development and regeneration. While the structural characteristics of the developing and adult zebrafish ventricle have been previously studied, little attention has been paid to the nature of the interface between the compact and spongy myocardium. Here we describe how these two distinct layers are structurally and functionally integrated. We demonstrate by transmission electron microscopy that this interface is complex and composed primarily of a junctional region occupied by collagen, as well as a population of fibroblasts that form a highly complex network. We also describe a continuum of uniquely flattened transitional cardiac myocytes that form a circumferential plate upon which the radially-oriented luminal trabeculae are anchored. In addition, we have uncovered within the transitional ring a subpopulation of markedly electron dense cardiac myocytes. At discrete intervals the transitional cardiac myocytes form contact bridges across the junctional space that are stabilized through localized desmosomes and fascia adherent junctions with adjacent compact cardiac myocytes. Finally using serial block-face scanning electron microscopy, segmentation and volume reconstruction, we confirm the three-dimensional nature of the junctional region as well as the presence of the sheet-like fibroblast network. These ultrastructural studies demonstrate the previously unrecognized complexity with which the compact and spongy layers are structurally integrated, and provide a new basis for understanding development and regeneration in the zebrafish heart.\n\nCitation: Lafontant PJ, Behzad AR, Brown E, Landry P, Hu N, et al. (2013) Cardiac Myocyte Diversity and a Fibroblast Network in the Junctional Region of the Zebrafish Heart Revealed by Transmission and Serial Block-Face Scanning Electron Microscopy. PLoS ONE 8(8): e72388. doi:10.1371/journal.pone.0072388\n\nEditor: Leonard Eisenberg, New York Medical College, United States of America\n\nReceived March 1, 2013; Accepted July 9, 2013; Published August 23, 2013\n\nCopyright: © 2013 Lafontant et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.\n\nFunding: PJL is supported by the Fisher and Faculty Development Funds at DePauw University. A. Burns is supported National Institutes of Health/National Eye Institute grants EY017120 and P30EY007551. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\nCompeting Interests: The authors have declared that no competing interests exist.\n\n* E-mail: firstname.lastname@example.org\n\nIntroduction\n\nThe zebrafish heart consists of a spongy ventricular myocardium made of trabecular bundles projecting radially into the ventricular lumen, and of an outer compact heart layer that encases the spongy trabeculae. The proportion of spongy to compact heart varies significantly between fish and appears to strongly correlate to each species particular ecological physiology [1,2,3,4]. The hearts of fish with relatively low activity consist primarily or exclusively of a spongy myocardium with a rudimentary compact myocardium fed by deoxygenated luminal blood flow (type-1 heart). More active fish display a thicker compact myocardium invested with vessels carrying oxygenated blood (type-2 heart). The two myocardial segments have been historically and empirically considered distinct anatomical structures that form a functional unit supporting the physiological demands of fish. Because of its hypothesized importance in fish ventricular function, the characteristics of the spongy-compact interface (SCI) between the two ventricular layers have been an area of considerable interest and controversy.\n\nThe zebrafish has emerged as an important model of heart development and regeneration. The adult zebrafish heart is a type-2 heart with a compact layer perfused by capillaries. An extensive number of studies have provided insights into the molecular mechanisms underlying the development of the zebrafish ventricle [5,6,7] and its spongy trabeculae [8,9]. Other studies have explored the molecular mechanisms that orchestrate the zebrafish’s remarkable ability to regenerate [10,11]. More recently, the emergence of the mature zebrafish ventricle has been demonstrated by genetic approach to be driven by clonally-dependent cardiac myocytes, illustrating diversity within the ventricular myocyte population [12]. A number of studies have also provided insight into the ultrastructural characteristics of the developing and adult zebrafish heart [13,14,15]. However to date the nature of SCI in the zebrafish heart has received little attention.\n\nStudies in a variety of fish using different approaches have resulted in markedly diverse understandings and models of this\n\ninterface region. Early studies in the albacore tuna suggested that the two myocardial layers were attached by a connective tissue layer [16,17]. Transmission electron microscopy studies in four different fish species, not including the tuna, provided the first detailed evidence not only for connective tissue at the interface of the two myocardial layers [18,19], but of the presence of fibroblasts and a distinct set of flattened transitional cardiac myocytes occupying this complex junctional region (JR). On the other hand, in a more recent study of the sockeye salmon and rainbow trout, similar structures in the interface region were not observed [20]. Whether these studies underscore the structural diversity of the JR in fish, or reflect differences in study methodologies is not clear. The different findings in the salmon and trout heart may be indicative of the intra- and inter-species diversity in the architectural arrangements of the two layers.\n\nThe purpose of our study was to ascertain the cellular and ultrastructural nature of the interface region between compact and spongy heart of adult zebrafish using light and transmission electron microcopy (TEM). Here, we provide evidence of a complex and previously unrecognized JR containing a network of fibroblasts, a subpopulation of phenotypically transitional cardiac myocytes at the base of the spongy myocardium, and describe the spatial distribution of adherens junctions linking the spongy and compact layers. The three-dimensional structure of the JR was confirmed by serial block-face scanning electron microscopy (SBF-SEM) and computerized segmentation. To our knowledge, this is the first detailed description of the JR of the zebrafish ventricle, as well as the first ultrastructural 3D reconstruction of this region using SBF-SEM.\n\n**Materials and Methods**\n\n**Animals**\n\nZebrafish were obtained from Aquatic Research Organisms (Hampton, NH), and maintained in 10-gallon tanks, with 15 to 20 fish per tank, at 28 degrees Celsius on a 14/10 hour day/night cycle. Experimental procedures were approved by the Committee for the Care and Use of Laboratory Animals at DePauw University.\n\n**Transmission Electron Microscopy**\n\nFor studies of the myocardium in plastic sections and by transmission electron microscopy tissue samples were processed as described previously [21]. Zebrafish were euthanized using 0.2% MS Tricaine. Once all body and opercula movements had ceased, the heart was removed by grasping and pulling the aorta proximal to the bulbus arteriosus. The hearts were fixed in 100 mM cacodylate buffer containing 2.5% glutaraldehyde overnight at 4 degrees C. The hearts were washed the next day in cacodylate buffer and stored at 4 degrees C for later processing. Hearts were post-fixed in 1% tannic acid and transferred to 1% osmium tetroxide, dehydrated in an acetone series, and then embedded in Embed-812 resin (Electron Microscopy Sciences, Hatsfield, PA). Two-micron thick sagittal sections were cut and toluidine-blue stained for light microscopy analysis. Stained sections were visualized and imaged using a Nikon Optiphot (Nikon Instruments Inc., Melville, NY). For electron microscopy analysis, ultra-thin sections 100 nm thick were cut, set on single slot or 200-mesh copper or nickel grids, and imaged on a Tecnai G2 Spirit BioTWIN electron microscope (FEI Company, Hillsboro, OR). Additional fixed and embedded hearts (n = 9) were obtained from the University of Utah and processed as previously described [14]. Briefly, these hearts were perfusion-fixed with 2.5% glutaraldehyde-2% paraformaldehyde in 0.1 M cacodylate buffer, and 4×10⁻⁴ g/kg dose of diltiazem for 4 hours. The hearts were then post-fixed in 2% osmium tetroxide, dehydrated, and embedded in Spurr’s epoxy resin. Ultra-thin sections 100 nm thick were cut and mounted on single slot or 200-mesh copper or nickel grids and imaged on a Tecnai G2 Spirit BioTWIN electron microscope (FEI Company, Hillsboro, OR).\n\n**Immunofluorescence Imaging**\n\nFollowing isolation, zebrafish hearts were cryoprotected in 30% sucrose in PBS overnight. The following day, the hearts were embedded in freezing medium (Tissue-tek OCT, Torrance, CA), kept at −80 degrees C and later sectioned using a Leica CM 1900 cryostat (Leica Microsystems, Bannockburn, IL). Ten-micron sagittal sections were fixed for 3 minutes in 4% paraformaldehyde, permeabilized with 0.5% triton X in PBS, blocked with 3% BSA for 2 hours, and immunoreacted overnight with anti-MYH1 antibody or MEF antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA), followed by Texas-Red conjugated anti-mouse or anti-rabbit secondary antibody. Next, the sections were incubated with FITC-labeled wheat germ agglutinin (WGA, Triticum vulgaris, Sigma-Aldrich, Saint Louis, MO) for 2 hours. Other sections were stained with TRITC-labeled WGA. Following Hoechst staining, sections were imaged on a Zeiss Axio Imager.M (Carl Zeiss Microscopy, Thornwood, NY) or a Leica TCS SP5 confocal microscope (Leica Microsystems, Bannockburn, IL).\n\n**Serial Block-face Scanning Electron Microscopy Imaging**\n\nTissue fixation and embedding were performed using the previously published protocols with some modifications [22]. Briefly, excised zebrafish hearts were fixed in 2.5% glutaraldehyde in 100 mM sodium cacodylate buffer at 4 degrees C overnight. The hearts were rinsed the next day in 100 mM sodium cacodylate. The hearts were post-fixed and heavy metal-contrasted with potassium ferrocyanide, osmium tetroxide, thiocarbohydrazide, uranyl acetate and lead aspartate. Next, the hearts were dehydrated in ethanol followed by acetone and embedded in Durcupan resin. Approximately 2×2 mm, 100 nm thick sections were obtained, mounted on slot grids and surveyed by TEM. Blocks with optimal tissue orientation and easily identifiable landmarks in the region of interest were selected for further trimming and imaging. Serial block-face sectioning (at 50 or 100 nm imaging intervals) and imaging were performed using a Gatan 3View system (Gatan, Pleasanton, CA) mounted in an FEI Quanta FEG 200 SEM (FEI Company, Hillsboro, OR). The SEM was operated at an accelerating voltage of 2.0 kV and 30 pascals pressure in low vacuum mode. A solid state backscatter detector was used to acquire serial section images (stacks of 800, 632, and 500 sections) from three regions of interest. Three-dimensional segmentation and reconstruction of fibroblasts and cardiac myocytes was performed using Amira 5.2 software.\n\n**Results**\n\nI. **Electron Translucent and Electron Dense Cardiac Myocytes Are Organized into a Transitional Cell Ring**\n\nTo determine the nature of the interface region between the compact and spongy heart of the adult zebrafish, we first studied the ventricle under light microscopy. Using two-micron thick toluidine blue-stained plastic embedded sections, we observed the expected organization: a relatively thin layer of the compact heart (approximately 10–20 um thick) framing the trabeculae which projected radially into the ventricular lumen (Fig. 1. A). However closer inspection revealed an unexpected number of darkly-stained elongated structures, at irregular intervals at the interface between\n\nFigure 1. Low magnification of the adult zebrafish heart by light and TEM. (A), light microscopy view of the apical region of the zebrafish ventricle showing a thin layer compact heart (CH) with trabeculae (Tr) projecting within the lumen (Lu). (B), higher magnification of the apex showing darkly stained linear structures (arrows) close to the spongy-compact interface of the ventricular myocardium. (C), low magnification TEM of compact ventricular myocardium constituted of four to five overlapping cardiac myocytes (CM) layers from epicardium (Epi) to lumen (Lu), with electron dense cells (arrow) within a complex junctional region (JR, brackets) at the interface of compact and spongy myocardium. (D), higher magnification show mitochondria (Mt) filled section of electron dense transitional cardiac myocyte (EDTCM). EDTCM are apposed to trabecular cardiac myocytes and endocardial cells (Endo) on the luminal side. EDTCMs are separated from the compact cardiac myocytes (CCM) by a junctional space, an interstitial space within the JR. (E), low magnification TEM of the JR (brackets) showing flattened electron translucent transitional cardiac myocytes (ETTCM) in contact with trabeculae and in proximity to endocardium (Endo) on the luminal side. (F), higher magnification of a flattened cells show presence of actin and myosin filaments, apposition to trabeculae and endocardial cells as well as separation from the compact myocardium by the junctional space.\n\ndoi:10.1371/journal.pone.0072388.g001\n\nFigure 2. Electron dense and electron translucent transitional cardiac myocytes interactions. (A), example of end-to-end interaction between EDTCM and electron translucent cardiac myocytes (ETTCM) forming a continuum at the spongy-compact interface. (B), higher magnification of contact area with complex adhesion junctions including desmosomes between the two cells. (C), example of interaction between mitochondria filled and actin-myosin filament (AMF) containing EDTCM cells and ETTCM via adhesion junctions (arrows). (D), higher magnification of EDTCM and ETTCM showing gap junctions (arrows), and (E), desmosomes. (F), AMF in a transitional cell. (G), higher magnification (inset in F, rotated) showing the hexagonal array of myosin in cardiac myocytes. (F) sarcomere and z-band in the same cell. AMF, actin-myosin filament; CCM, compact cardiac myocytes; CW, cardiac myocytes; EDTCM, electron dense transitional cardiac myocytes; ETTCM, electron translucent transitional cardiac myocytes; Endo, endocardium; Epi, IF, intermediate filaments; Lu, lumen; Mt, mitochondrion; Tr, trabeculae.\n\ndoi:10.1371/journal.pone.0072388.g002\n\nthe compact and spongy segments of the ventricle (Fig. 1, B). To elucidate the nature of these darkly-stained structures, we studied their characteristics and precise location within the zebrafish junctional region (JR) using transmission electron microscopy (TEM). Low magnification TEM reveals a compact myocardium of 4–5 layers of cardiac myocytes (CM) (Fig. 1, C). The compact and spongy interface is revealed as a complex JR containing a set of flattened CM as compared to the compact or trabecular CM. Ultrastructurally, the darkly stained structures appear as electron dense CM running along the adjacent base of the trabeculae (Fig. 1, D). We have called these cells: “dark cells” or electron dense transitional cardiac myocytes (EDTCM). Except for their high electron density, these cells display similar flattened and rectangular phenotypes as the transitional CM described previously [18]. In adjacent regions, the ventricle displays a similar arrangement but with electron translucent transitional cardiac myocytes (ETTCM) (Fig. 1, E). Actin-myosin filament bundles were easily identified within the ETTCM confirming their CM phenotype (Fig. 1 F), whereas the cytoskeletal contents of the EDTCM were obscured by the electron dense material contained in these cells.\n\nII. Transitional Cell Ring and Trabeculae Contacts\n\nThe EDTCM were in direct contact with the ETTCM (Fig. 2, A). Within the plane of observation, shorter segments of EDTCM alternated with longer segments of ETTCMs, and together these two cell populations appear to form a continuous ring of transitional CM within the JR. The transitional CM could be seen linked at their tapered ends through complex adherent junctions resembling fascia adherentes (Fig. 2, B). Additionally, the transitional CM were in direct contact or in close apposition to the base of CM sheets of projecting spongy trabeculae (Fig. 2, A, C), and in the intertrabecular lacunar spaces they were bounded in their luminal aspect by the endocardium (Fig. 2, C). However in contrast to their connections to trabeculae, no contacts were observed between transitional CM of the transitional ring and the adjacent endocardial cells. In some instances, EDTCM and ETTCM minimally overlapped at their tapered ends (Fig. 2, C), with connections occurring through discrete gap junctions (Fig. 2, D) and desmosomes (Fig. 2, E). Detailed observations of the cytoplasm of these cells show the presence of actin-myosin filaments oriented in different planes (Fig. 2, F). The presence of caveolae and the hexagonal array of equidistant thick myosin filaments characteristic of cardiac muscle cells can be appreciated (Fig. 2, G). Z-bands bordering sarcomeres oriented perpendicularly or obliquely in the plane of observation (Fig. 2, H) can also be seen in these cells, further confirming their cardiac myocyte phenotype. Finally the cardiac nature of these narrow width transitional cells was also confirmed using immunostaining with anti-myosin heavy chain-1 (MYH1) and anti-myocyte enhancer factor-2 (MEF) antibodies to specifically identify cardiac myocytes (Fig. 3).\n\nWe further investigated the nature of the arrangement between the transitional CM ring and the CM forming the trabeculae. Transitional CM can be found linked to trabecular myocytes through a variety of adherens junctions. These junctions included fascia adherentes (Fig. 4, A, B) that link actin-myosin filaments of adjacent cells. Various amounts of amorphous material were observed along transitional CM and trabecular CM areas of apposition (Fig. 4 C, D). However discrete electron dense areas...\n\nwith a sub-domain containing desmosomes were also observed (Fig. 4 E, F).\n\nIII. Fibroblast Network and Collagen in the Junctional Region\n\nThe transitional ring of myocytes on its abluminal side is bounded by a narrow (0.5–1 um) connective tissue space. This space separates the EDTCM and ETTCM from the adjacent CM of the compact layer, creating a junctional space within the JR (Fig. 5. A). The junctional space appears to create an extensive structural discontinuity between the spongy and the compact myocardium. It contains numerous cells arranged longitudinally and running parallel to the transitional CM ring. The absence of basement membranes, their elongated shape, and their location within this interstitial space suggest they are fibroblasts (Fig. 5. B, C). These fibroblasts contained ovoid nuclei with scant perinuclear cytoplasm that appeared mostly devoid of organelles, except for occasional vesicles and polyribosomes. In the perinuclear region of the fibroblasts, the junctional space widens to 2–3 microns. The cytoplasm of these fibroblasts is stretched bi-directionally into long thin filopodia-like processes. These fibroblasts mostly form a single layer in the junctional space with little to minimal overlap of their cytoplasm. The fibroblasts and their cytoplasmic processes fit tightly within the junctional space, however a measurable space was always observed between the fibroblast and the adjacent transitional and the compact ventricular CM.\n\nThe fibroblasts were also found in close proximity or in direct contact with parallel or perpendicularly-oriented collagen fibers (Fig. 5. B, C). At irregular intervals, terminal profiles displaying long cytoplasmic extensions were in close proximity with the membranes of long cytoplasmic processes originating from another fibroblast (Fig. 5. D). In some instances, cytoplasmic terminal ends of fibroblasts overlapped, or could be seen in contact with the same bundle of collagen. Occasionally fibroblasts terminal ends were found in close contact; however we could not unequivocally confirm specialized adhesion junctions between interacting terminal ends. Occasionally close approximation between fibroblast filopodia and CM could be seen; however no direct contact between fibroblasts and CM and no specialized myocytes-fibroblasts junctions were observed. Close observation of many of the transitional CM however revealed the presence of caveolae within the membranes orientated toward the fibroblasts within the junctional space (Fig. 5. C). Across the junctional space, the compact CM membranes were observed to have numerous caveolae facing toward the fibroblasts (Fig. 5. D, E). More examples of caveolae could be seen on the membranes of CM facing the junctional space at a distance from the fibroblast processes (Figure S1). While most fibroblast nuclei and cytoplasmic processes resided within the JR, some cytoplasmic projections could be seen invested in the compact myocardium adjacent to small vessels, suggesting that fibroblasts may not be locally restricted to the junctional space.\n\nFigure 5. Fibroblasts network in the junctional region of the zebrafish ventricle. (A), TEM of a fibroblast (Fb) cytoplasm and its thin extended process/filopodium (FP) 100 to 200 nm thick spanning the junctional space (JS) in between the transitional CM ring and the adjacent compact cardiac myocyte (CCM). Note vesicle (*) in an enlarged region (~300 nm) of the fibroblasts filopodium (FP). (B), fibroblast profile with nucleus (Nu) and its cytoplasm extending into a long thin process, and collagen fiber (CF) bundles running perpendicular to the section’s plane. (C), another fibroblast and its nucleus (Nu) and with cytoplasm extending into a long process, and collagen fiber (CF) bundles running parallel to the section’s plane. (D), fibroblast filopodial termini in close approximation. Electron dense region in the close approximation area suggestive of specialized adhesion junction (arrow). Note the presence of caveolae (*) on the membrane of the CCM facing the fibroblast in the junctional space.\n\ndoi:10.1371/journal.pone.0072388.g005\n\nFigure 6. Transitional CM ring and compact CM contacts. (A), ETTCM and Compact CM direct contacts mediated by adhesion junctions (arrows). Note the JS with fibroblast (Fb) on the right and narrow JS of the left of the contact region. (B), higher magnification of inset in (A) showing adhesion junctions associated with fascia adherentes (thick arrow), and also with desmosomes (thin arrow). Note the abundance of caveolae (*) in the transitional CM membrane in the contact region. (C), another narrow region of contact (arrow) between a transitional and a compact cardiac myocyte.\n\nforming a bridge between the two cells and adjacent Z-band in the CCM. A fibroblast process is seen on each side of the junction. Note a vesicle (V) in the fibroblast process. (D), higher magnification of the contact area (arrow) showing a desmosome associated with actin, myosin, and intermediate filaments on the ETTCM and abundant ribosomes (rb) in the CCM. (E), a tri-cellular junction between two transitional cardiac CM and one compact CM. (F), higher magnification of (E, inset) showing desmosomes between the ETTCM and each ETTCM with the compact CM (arrows). (G), discrete adhesion junction between a transitional and compact CM (arrows). (H), higher magnification of inset 1 of (F). (I), higher magnification of inset 2 of F, showing compact CM contact within an invagination of the transitional CM.\n\ndoi:10.1371/journal.pone.0072388.g006\n\nIV. Transitional Cardiac Myocytes Contacts across the Junctional Space\n\nFrom our observations, we estimated that the junctional space containing fibroblasts and collagen occupies 80 to 90% of the junctional region between compact and spongy heart, so that the majority of the CM membranes in the transitional ring are not directly linked to the compact heart. The remaining portion of the spongy-compact interface consisted of cell membrane appositions between transitional CM and the most adjacent CM of the compact heart (Fig. 6. A). These regions of apposition contained specialized adherens junctions including closely spaced desmosomes and fascia adherents (Fig. 6. B). These areas of contact between the transitional ring and compact myocardium circumscribed the junctional space and interrupted the course of the fibroblasts and their filopodia in the plane of the sections (Fig. 6. C, D). Occasionally a region of tri-cellular contact was observed, where a compact myocyte was joined at the end-to-end contact of two transitional CM through desmosomes (Fig. 6. E, F). However most transitional ring myocytes and compact CM contacts occurred through apposition of their lateral borders via desmosomes (Fig. 6. G, H), membrane protrusions, and interdigitations (Fig. 6. I).\n\nV. Serial Block-face SEM, Segmentation, and Volume Reconstruction of the Junctional Region\n\nTo better assess the three-dimensional architecture of the junctional region and confirm the three-dimensional nature of the fibroblast network, we performed serial block-face scanning electron microscopy (SBF-SEM) of the adult zebrafish heart. A preparatory survey of the fibroblast network in the JR of the zebrafish ventricle was performed by TEM in 100 nm sections from heavy metal-contrasted Durcupan-embedded hearts. The junctional region and the fibroblasts were easily identifiable. Image stacks generated with the FEI Quanta 200 FEG SEM equipped with the Gatan 3View (Fig. 7. A) were used to generate three-dimensional reconstructions of the JR architecture (Fig. 7. B, Movie S1), and confirm the 3D projection of fibroblasts in the junctional volume space. Observation of a subset of serial sections demonstrated the predicted discontinuous nature of cellular contacts between the compact heart and transitional myocytes that anchor the luminal trabeculae, and confirmed fibroblast-fibroblast contacts in the junctional space (Fig. 7. C-I, Movie S2). Segmentation of fibroblasts from serial sections followed by three-dimensional reconstruction demonstrated the sheet-like nature of fibroblasts arranged in a network occupying the junctional space of the zebrafish heart (Fig. 7. J, K). Additional segmentation of transitional and compact cardiac myocytes demonstrated contact surfaces between compact and spongy heart through the fibroblast network layer (Fig. 7. L).\n\nDiscussion\n\nThis paper documents a previously unrecognized connective tissue space located at the interface of the spongy and compact myocardial of the adult zebrafish heart. It extends our understanding of the cellular and ultrastructural nature of the ventricular JR of an important research model. The existence of a connective tissue layer in the junctional region of type-2 fish hearts has remained controversial. A continuous fibrous membrane interposed between the compact and spongy layers has been reported in the hearts of tuna [16,17], atlantic salmon and rainbow trout [23]. These observations led to the hypothesis of the connective tissue as an important adhesive substrate between the two myocardial layers. In these early studies, detailed composition of the connective tissue layer was not described. Midttun using TEM demonstrated in four type-2 fish hearts species the presence of fibroblasts and collagen fibers within a 6 to 7 um wide space existing in the junctional region [18]. By contrast in a study by Pieperhoff et al., using light and scanning electron microscopy, a connective tissue layer at the interface of the two myocardial layers in the sockeye salmon and trout was not observed [20], beyond small focal regions of extracellular matrix enrichment. Unlike the previous studies, these authors challenged the hypothesis of a connective tissue layer separating the two myocardial layers. In both Midtun’s and our studies, the junctional space and its content were not readily detected by light microscopy; however, they could be observed by transmission electron microscopy, suggesting that ultrastructural thin sections imaging may be necessary to describe the structure of the JR of fish ventricles.\n\nOur ultrastructural observations reveal a junctional space measuring for the most part less than one micrometer wide, populated by a single layer of fibroblasts organized into a network, and framed by CM belonging the compact myocardium distally and the spongy myocardium proximally. Fibroblasts are an important component of the mammalian heart and play an essential role in heart structure and physiology. They are a source of extracellular matrix molecules and growth factors, and they serve as sensors for mechanical signals [24,25,26,27,28]. While it has been suggested in early studies that collagen accumulation modulates regeneration in the zebrafish ventricle [10], fibroblasts in the fish heart have received little attention. More recently studies of cardiac injury in the zebrafish [29,30,31] and giant danio [21] demonstrated accumulation and resorption of collagen during fish ventricular regeneration, suggesting an important role for fibroblasts. Indeed the presence of activated fibroblasts was observed during ventricular remodeling in the zebrafish [31] and giant danio heart [21]. The presence of fibroblasts has previously been noted in the sub-epicardial space of fish species, including the zebrafish [14]. In the present study, the fibroblasts’ arrangement suggests a highly organized three-dimensional network exists at the interface of the compact and spongy ventricle. The function of this fibroblast network is presently unclear.\n\nFibroblast networks have been documented in a variety of organs and species, with direct fibroblast-fibroblast contacts being observed in mammalian corneas [32,33]. Fibroblast interactions suggestive of a network have also been reported in the mammalian heart [34]. Fibroblasts have also been implicated in physiological mammalian cardiac muscle growth [27] as well as in injury responses [35,36,37,38]. Our study by TEM and the reconstruction following segmentation of the SBF-SEM micrographs demonstrate that fibroblasts form a three-dimensional network in the JR of the adult zebrafish heart. In addition to fibroblastfibroblast interactions, direct CM and fibroblast contacts have also been reported in normal and diseased hearts and in culture [39,40,41]. In this study however, we did not uncover direct CM-fibroblasts contacts in the uninjured zebrafish heart. When fibroblast processes were seen in close proximity and in apparent apposition to myocytes, the intervening myocyte basement membrane was always present. Yet we cannot rule out that these contacts exist and may occur at a frequency too low to be easily...\n\ndetected. Nevertheless, we found a remarkable number of caveolae in the sarcolemma of CM bordering the junctional space and facing adjacent fibroblasts. This suggests possible mechanisms by which paracrine signaling may take place between adjacent CM and fibroblasts occupying the junctional space.\n\nThe present study also establishes that the junctional space occupies the majority of the overall SCI, and creates an extensive volume of non-contact between the two myocardial layers. Given the sparsity of the collagen fibers observed in the junctional space, we question whether in the zebrafish, the connective tissue would provide sufficient support to maintain the structural integrity of the two layers. We speculate that the connective tissue compartment may not be the primary mechanism by which adhesion between the two myocardia is maintained. Indeed, the junctional space is interrupted by contact bridges between the two layers at discrete intervals through membrane apposition of adjacent CM, and that desmosomes and fascia adherentes are frequently found within the contact regions between transitional zebrafish CM and compact CM. These findings are consistent with the demonstration of highly enriched adhesion junction molecules between the two myocardial layers observed in salmon and trout [20].\n\nAn important finding is the transitional ring of cardiac myocytes similar to that previously described by Midrunn, that underscores the phenotypic diversity of cardiac myocytes present in the zebrafish heart, suggesting specific structure-function relationship. The notion of cardiac myocyte diversity is apparent in recent work demonstrating the clonal contribution of cardiac myocytes to the emergence of the zebrafish heart [12]. In that study two populations of peripheral cardiac myocytes were described by genetic labeling and spatio-temporal characteristics: a primordial and a cortical set. The localization of the primordial layer suggests it occupies a space similar to the transitional ring population described in the present study. Consequently, it would be interesting to determine whether these two populations spatially interact or are one and the same. Another intriguing finding is the discovery of a subset of “dark” electron dense myocytes within the transitional CM ring that supports the trabeculae. To our knowledge, these electron dense cardiac myocytes have not been reported in fish or mammalian hearts. A set of dark cells was observed in the ventricle of icefish; however, they were believed to be non-contractile in nature [42]. Observations of the zebrafish electron dense cells at high magnification show clear evidence of myofibrils. In addition these cells form a continuum with adjacent electron translucent CM as well as other electron dense CM in the transitional ring and myocytes of adjacent trabeculae via adhesive junctions. These observations raise the intriguing possibility of further phenotypic specialization within the transitional CM population bordering the JS. We speculate this particular phenotypic change may be regulated by regional stress distribution within the myocardium or may reflect their metabolic state. The understanding of the functional and the molecular differences between the dark and light cells within the transitional ring warrants further study.\n\nConclusions\n\nIn conclusion, this study sheds light on the phenotypic diversity of CM at the JR of the adult zebrafish heart. It documents the existence of a fibroblast network that contributes to the architectural complexity of the region (Fig. 8). We suggest the transitional CM form the main anchoring substrate for the trabecular and compact heart. It is possible that in type-2 fish hearts, both the connective tissue layer and the adherens junctions account for the integrity of the spongy-compact interface. It is also possible that in fish, the ratio of connective tissue and direct CM adhesive contact, as well as the distribution of adherens junctions might be dependent on the species, their age and ecological\n\nphysiology. In the end it is intriguing to consider how the articulation of the compact and spongy interface progresses during development and how the re-articulation occurs during regeneration of the zebrafish heart.\n\nSupporting Information\n\nFigure S1 Transitional myocyte abluminal caveolae. (A), Junctional space (JS) with a fibroblast process (FP). (B), Higher magnification of inset in (A) showing caveolae (*) on transitional CM facing the JS. (TIF)\n\nMovie S1 Architecture of the junctional region of zebrafish heart. The sheet-like nature of fibroblasts can be appreciated. (MP4)\n\nMovie S2 Segmented fibroblasts and transitional CM. (MP4)\n\nReferences\n\n1. Sauter RM (1980) Morphological studies on the ventricle of teleost and elasmobranch hearts. J Zool, Lond 190: 259–272.\n2. Sanchez-Quintana D, Garcia-Martinez V, Climent V, Hurle JM (1995) Morphological analysis of the fish heart ventricle: myocardial and connective tissue architecture in three species. Ann Anat 177: 267–274.\n3. Sanchez-Quintana D, Hurle JM (1997) Ventricular myocardial architecture in marine fishes. Anat Rec 217: 263–273.\n4. Sauter RM (1983) Morphology and innervation of the fish heart. Adv Anat Embryol Cell Biol 89: 1–102.\n5. 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PLoS Biol 2: e329.\n\nAcknowledgments\n\nWe thank Margaret Gondo for TEM help, Lynn Bedard and Tamara Beauboeuf for editorial suggestions, Tanmoy Das and Henning Schneider for valuable discussions, the Prindle Institute for Ethics at DePauw University for dedicated space for scholarly work, and also John Eskew, Wendy Tomamichel, and Clay Higginbotham for technical help and with animal care. We thank Jayne Williams for logistical help. We thank Barry Stein of The Electron Microscopy Center and Jim Powers of the Light Microscopy Imaging Center at Indiana University-Bloomington for their help.\n\nAuthor Contributions\n\nConceived and designed the experiments: PJL A. Behzad EB PL NH A. Burns. Performed the experiments: PJL A. Behzad EB PL NH A. Burns. Analyzed the data: PJL PL A. Burns. Contributed reagents/materials/analysis tools: PJL A. Behzad EB PL NH A. Burns. Wrote the paper: PJL. Designed and performed TEM experiments: PJL A. Burns EB NH. 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Dev Cell 16: 233–244.\n28. MacKenna DA, Dolli F, Vuori K, Ruoslahri E (1998) Extracellular signal-regulated kinase and c-Jun NH2-terminal kinase activation by mechanical stretch is integrin-dependent and matrix-specific in rat cardiac fibroblasts. J Clin Invest 101: 301–310.\n29. Schnabel K, Wu CC, Kurth T, Weidinger G (2011) Regeneration of cryoinjury induced necrotic heart lesions in zebrafish is associated with epicardial activation and cardiomyocyte proliferation. PLoS One 6: e18503.\n30. Gonzalez-Rosa JM, Martin V, Peralta M, Torres M, Mercader N (2011) Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish. Development 138: 1663–1674.\n31. Chabials F, Veit J, Rainer G, Jazwinska A (2011) The zebrafish heart regenerates after cryoinjury-induced myocardial infarction. BMC Dev Biol 11: 21.\n32. Haasenstiel J (1982) On the ultrastructure of the developing and adult mouse corneal stroma. Anat Embryol (Berl) 168: 291–305.\n33. Nishiida T, Yasumoto K, Otori T, Desaki J (1983) The network structure of corneal fibroblasts in the rat as revealed by scanning electron microscopy. Invest Ophthalmol Vis Sci 29: 1837–1890.\n34. Goldsmith EC, Hoffman A, Morales MO, Potts JD, Price RL, et al. (2004) Organization of fibroblasts in the heart. Dev Dyn 230: 787–794.\n35. Dobaczewski M, Frangogiannis NG (2009) Chemokines and cardiac fibrosis. Front Biosci (Schol Ed) 1: 391–405.\n36. Kohr S (2009) Myofibroblasts in diseased hearts: new players in cardiac arrhythmias? Heart Rhythm 6: 845–856.\n37. Porter KE, Turner NA (2012) Cardiac fibroblasts: at the heart of myocardial remodeling. Circ Res Ther 12: 255–278.\n38. van den Borne SW, Dirx J, Blanksteijn WM, Verjans J, Hoštůra L, et al. (2010) Myocardial remodeling after infarction: the role of myofibroblasts. Nat Rev Cardiol 7: 30–37.\n39. Chilton L, Giles WR, Smith GL (2007) Evidence of intercellular coupling between co-cultured adult rabbit ventricular myocytes and myofibroblasts. J Physiol 583: 225–236.\n40. Kohl P, Cannellini P (2007) Cardiac myocyte-nonmyocyte electrotonic coupling: implications for ventricular arrhythmogenesis. Heart Rhythm 4: 233–235.\n41. Miraglia M, Salvatore N, Rohr S (2007) Myofibroblasts induce ectopic activity in cardiac tissue. Circ Res 101: 755–758.\n42. Harrison P, Zummo G, Farina F, Tota B, Johnston IA (1991) Gross-Anatomy, Myoarchitecture, and Ultrastructure of the Heart Ventricle in the Haemoglobinless Icefish Chaenocephalus-Acraatus. Canadian Journal of Zoology-Revue Canadienne De Zoologie 69: 1339–1347."}}},{"rowIdx":204699,"cells":{"text":{"kind":"string","value":"Cellular characterization of the outgrowth and connectivity of the brain serotonin system\n\nJeroen Dudok\n\nThe research described in this thesis was conducted at the Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, The Netherlands, and was supported by a grant from the Dutch Organization of Scientific Research (NWO-MW-PIO900-01-001).\n\nPublication of this thesis was financially supported by:\n\nCNCR\nDepartment of Functional Genomics\nVrije Universiteit Amsterdam\n\nCover: Serotonergic neurons in the dorsal raphe nucleus\nPrint: Wöhrmann Print Service\nISBN: 978-90-8570-585-7\n\n© Jeroen Dudok 2010. All rights reserved. No part of this thesis may be reproduced, stored in a retrieval system, or transmitted in any form or by any means without prior permission of the author.\n\nCellular characterization of the outgrowth and connectivity of the brain serotonin system\n\nACADEMISCH PROEFSCHRIFT\n\nter verkrijging van de graad Doctor aan\nde Vrije Universiteit Amsterdam,\nop gezag van de rector magnificus\nprof.dr. L.M. Bouter,\nin het openbaar te verdedigen\nten overstaan van de promotiecommissie\nvan de faculteit der Aard- en Levenswetenschappen\nop woensdag 29 september 2010 om 15.45 uur\nin de aula van de universiteit,\nDe Boelelaan 1105\n\ndoor\n\nJacobus Johannis Dudok\ngeboren te Zierikzee\n\npromotor: prof.dr. M. Verhage\ncopromotor: dr. A.J.A. Groffen\n\nAan mijn ouders\n\n# Contents\n\n| Chapter | Title | Page |\n|---------|----------------------------------------------------------------------|------|\n| 1 | General introduction | 11 |\n| 2 | Chronic activation of the 5-HT$_2$ receptor reduces 5-HT neurite density as studied in organotypic slice cultures | 37 |\n| 3 | Dynamic vesicular trafficking of the serotonin reuptake transporter in hippocampal neurons | 57 |\n| 4 | Presynaptic localization of tryptophan hydroxylase 2-EGFP in mature hippocampal neurons | 73 |\n| 5 | The effect of a polymorphism in Piccolo on localization and trafficking dynamics of the serotonin reuptake transporter | 85 |\n| 6 | Deletion of Munc18-1 in 5-HT neurons results in rapid degeneration of the 5-HT system and early postnatal lethality | 105 |\n| 7 | Towards a genetic approach to study serotonergic outgrowth and connectivity in vivo | 127 |\n| 8 | General discussion | 140 |\n| | Nederlandse samenvatting | 154 |\n| | List of abbreviations | 159 |\n| | Dankwoord | 161 |\n| | Curriculum Vitae | 164 |\n\nChapter 1\n\nGeneral introduction\n\nGeneral introduction\n\nThe mammalian brain is by far the most complex system in the body. The human brain consists of approximately 100 billion nerve cells (neurons), which form a highly complex wiring scheme. How this complex wiring is achieved is still poorly understood. The brain contains several clusters of cells which contain a certain neurotransmitter, such as dopaminergic cells in the substantia nigra or acetylcholinergic cells in ventral and dorsal striatum.\n\nAnother example of such clusters are the raphe nuclei in the midbrain that secrete serotonin (5-hydroxytryptamine, 5-HT). 5-HT has diverse modes of action in the central nervous system and is involved in brain development as will be described in this introduction. There are a few remarkable characteristics of the 5-HT system. First of all, the 5-HT system consists of only a few neurons (~1000 in rodents, ~0.5 million in humans) compared to the total number of neurons. Nevertheless, these neurons send projections to virtually every other brain area. It is estimated that each 5-HT neuron makes more than $1 \\times 10^6$ connections. Secondly, 5-HT is predominantly released extrasynaptically (so called volume or paracrine transmission), which implies that 5-HT acts on a slower and longer-lasting time scale compared to classical neurotransmitters like glutamate. Thirdly, emerging evidence shows that 5-HT has a role during brain development. Finally, the 5-HT system is involved in certain behavioural and psychopathological processes including anxiety, depression, aggression and obsessive-compulsive disorder (OCD).\n\nIn this chapter the main aspects of the 5-HT system will be introduced and the diverse modes of action which 5-HT has are described. First, there will be a description about the anatomical organization and development of the 5-HT system. Subsequently, the role of 5-HT in brain development will be introduced and the relation between the 5-HT system and behavioural and psychopathological processes will be discussed. Finally, the aim and scope of this thesis will be outlined.\n\nAnatomical organization of 5-HT cell clusters\n\nAfter the initial discovery of 5-HT (see box 1), it took about 20 years before the cellular distribution of 5-HT was described by Dahlstrom and Fuxe using the Falck-Hillarp technique. They showed that 5-HT cell bodies are clustered in nine cell groups close to the midline in the rhombencephalon, called the raphe nuclei. The nine nuclei are divided in two groups, the rostral and caudal group. The caudal group consists of clusters B1 to B5 and send projections predominantly in the midbrain itself and towards the spinal cord. On the other hand, the rostral\n\ncell group consists of clusters B6 to B9 and sends projections to the forebrain. The largest clusters of cells are the dorsal raphe nucleus (DRN) and the median raphe nucleus (MRN) (B7 and B8 respectively). These raphe nuclei also contain several types of non-5-HT neurons, such as GABA-ergic and noradrenergic neurons. The 5-HT neurons densely innervate virtually every brain area, with the most dense 5-HT innervations in the cortex and limbic structures including the hippocampus and basal ganglia.\n\n**Box I**\n\n**The discovery of 5-HT**\n\nIn the 1930’s Dr. Vittorio Erspamer tried to extract chemical compounds from enterochromaffin cells capable of causing smooth muscle contraction in the gut. In an extract of rabbit gastric mucosa, he identified a substance that could cause smooth muscle contraction. He called this substance enteramine, since it was an amino acid derivative (amine) found in enterochromaffin cells.\n\nIn the 1940’s another group identified a substance which had vasoconstrictive activity in serum. This serum influenced the tone, hence the name serotonin. When analyzing both substances, it was found that enteramine and serotonin were the same substance.\n\nThe first indication for a role of 5-HT in the brain came from Woolley, who showed that 5-HT and the hallucinogenic compound lysergic acid diethylamide (LSD) had comparable actions in the cortex of the cat. This eventually led to the discovery of 5-HT in the vertebrate brain like man and dog.\n\nDahlstrom and Fuxe were the first to describe the distribution of 5-HT cell bodies in the brain by histochemical detection of the 5-HT derivative indoleamine. The description of 5-HT projections from the 5-HT cell bodies towards the telencephalon and the diencephalon followed one year later. It was found that in the brain stem a relatively small population of 5-HT neurons was present, with the largest clusters in the dorsal and median raphe nuclei.\n\n**The development of the 5-HT system**\n\nIn the mouse brain, 5-HT neurons are generated around embryonic day (E) 10, and these cells are among the first neurons expressing a specific neurotransmitter. In the neural tube, 5-HT precursor cells are formed by the combined action of sonic Hedgehog, Fgf4 and Fgf8. The proper position of these 5-HT precursor cells is defined by the midbrain-hindbrain organizing centre (MHO), Shh signaling and the transcription factor Nkx2.2 (see figure 1).\n\nIn a knockout (KO) mouse for Nkx2.2, no 5-HT neurons are present in the midbrain raphe nuclei, except in the DRN, showing that Nkx2.2 is essential for 5-HT neuron differentiation (Briscoe et al., 1999). Another transcription factor that is involved in the initial development of 5-HT neurons is GATA3 (van Doorninck et al., 1999). After 5-HT precursor cells are formed, other\n\nfactors are required to establish mature 5-HT neurons. Among these factors are two transcription factors, LIM homeobox transcription factor 1 (Lmx1) and PC12 Ets factor (Pet1).\n\nLmx1b expression starts at E10.75 in the ventral part of the hindbrain including the floor plate and from E13.5 Lmx1b is present in the raphe nuclei (Ding et al., 2003). Lmx1b KO mice die within 24 hours after birth (Chen et al., 1998). Lmx1b-deficient mice lack all 5-HT neurons, even when Lmx1b was only deleted in 5-HT neurons, (Ding et al., 2003; Zhao et al., 2006). These studies show that Lmx1b is not required for the initial generation of 5-HT neurons, but is necessary for the differentiation and survival of 5-HT neurons during postnatal development.\n\nPet1 was found in a screen in PC12 cells to identify novel ETS domain transcription factors (Fyodorov et al., 1998). Pet1 expression is restricted to the raphe nuclei and precedes the expression of 5-HT by approximately half a day (Hendricks et al., 1999). Furthermore, several genes specific for 5-HT neurons, such as the rate limiting enzyme tryptophan hydroxylase (Tph) and the 5-HT reuptake transporter (SERT) have a conserved Pet1 binding site in their promoter (Hendricks et al., 1999). This suggests that Pet1 is involved in the final\n\ndifferentiation of 5-HT neurons. In a Pet1 KO mouse, there is an approximately 70% reduction in number of 5-HT neurons. The fact that there are still 30% of 5-HT neurons remaining suggests that these are either independent of Pet1 or there is another unknown transcription factor.\n\nAfter this genetic cascade, at approximately E11.5, 5-HT neurons start expressing the enzymes necessary for 5-HT synthesis (see box II) and proteins necessary for reuptake (SERT) and degradation of 5-HT (Monoamine-A (MAO-A)). Shortly after initiation of expression of these genes, 5-HT neurons start to\n\n**Box II**\n\n**Synthesis of 5-HT**\n\n5-HT is a neurotransmitter which belongs to the group of the biogenic amines, to which also the catecholamines (dopamine, adrenaline and noradrenaline) belong. All biogenic amines are derived from amino acids: the catecholamines from tyrosine, and 5-HT from tryptophan. 5-HT and the precursor tryptophan belong to a group of chemical compounds called the indoles, with a benzene ring joined to a five-member ring containing nitrogen. Tryptophan is an essential amino acid, which means that this amino acid cannot be made in the body from other amino acids and must therefore be ingested through the diet.\n\nFor synthesis of 5-HT in the brain, tryptophan is actively transported through the blood brain barrier via the large neutral amino acid transporter (LAT1). Once in the brain, tryptophan is metabolized to 5-HT using two different enzymes. First, tryptophan is hydroxylated in the 5-position to generate 5-hydroxy-L-tryptophan. This reaction is catalyzed by Tph, which is the rate limiting enzyme in 5-HT synthesis. Subsequently 5-hydroxy-L-tryptophan is converted to 5-hydroxytryptamine (5-HT) by the enzyme 5-hydroxytryptophan decarboxylase. 5-HT is stored in synaptic vesicles and large dense cored vesicles and transported into these vesicles by the enzyme Vesicular Monoamine Transporter 2 (VMAT2). After being released, 5-HT is transported back into the terminal using SERT, and either recycled or converted to 5-hydroxyindolacetic acid (5-HIAA) by MAO-A.\n\nThe rate limiting enzyme Tph has a (6R)-L-erythro-5,6,7,8-Tetrahydrobiopterin (Tetrahydrobiopterin, BH4) binding site. BH4 is an essential cofactor for the proper functioning of Tph. Without BH4, Tph cannot hydroxylate its substrate and thus 5-HT cannot be synthesized. BH4 is also required for dopamine synthesis by acting as a cofactor for tyrosine hydroxylase. Mice deficient for the BH4 gene die within 48 hours after birth and have extremely low levels of 5-HT and dopamine.\n\nIt has long been thought that only one Tph gene was present in the mammalian brain, although in zebrafish, two Tph genes were identified. However, in a mouse deficient for the Tph gene, no difference in brain 5-HT levels was found compared to control mice. This led to the discovery of the second Tph gene, called neuronal Tph or Tph2 (Walther et al., 2003). By using immunohistochemical analysis and in situ hybridization, it has been shown that Tph1 is expressed in the periferic system in the enterochromaffin cells and in the pineal gland in the brain, whereas Tph2 is exclusively expressed in the raphe nuclei in brain and in the myenteric plexus in the gut (Patel et al., 2004; Malek et al., 2005; Gutknecht et al., 2009).\n\nrelease 5-HT and start to send axon branches towards the spinal cord and via the medial forebrain bundle towards the cortex. Only after birth the 5-HT network is fully matured.\n\nIn the human brain, 5-HT neurons are for the first time detected when the embryo is five weeks old (Sundstrom et al., 1993). At ten weeks of age, 5-HT neurons are detected in all nuclei of the reticular formation and after 15 weeks, the 5-HT neurons are clustered in the raphe nuclei (Shen et al., 1989). After birth, 5-HT levels increase during the first two years and then decline to adult levels at the age of five years (Hedner et al., 1986).\n\n**5-HT release is both synaptic and extrasynaptic**\n\nSeveral classical neurotransmitters such as GABA, acetylcholine and glutamate are released from small synaptic vesicles (SSVs) at synaptic sites with a postsynapse in close proximity. However, neurotransmitters such as dopamine and 5-HT are released not only synaptically, but there is also so-called extrasynaptic release (also called paracrine transmission or volume transmission; here there will be referred to this type of release as volume transmission). A major difference between synaptic release and volume transmission is that there is no postsynapse present, so upon release the neurotransmitters diffuse away and can mediate effects distant from the release site. In this manner volume transmission can produce paracrine effects. In contrast to synaptic release, where SSVs are only released from axonal terminals, volume transmission of 5-HT occurs also in the soma and in dendrites, the so-called somatodendritic release. In 5-HT neurons, vesicles storing 5-HT are not only present in axonal terminals, but also in somata, dendrites and axonal varicosities (Hery and Ternaux, 1981). It was shown in Retzius neurons of the leech that 5-HT is released from both SSVs and large dense core vesicles (LDCVs), where there is an average discharge of 4700 and 80,000 molecules of 5-HT for SSVs and LDCVs respectively (Bruns and Jahn, 1995). In another study of the same authors, several differences between SSV and LDCV release of 5-HT were shown. First of all there was a difference in distribution: while SSVs were exclusively found in axons, LDCVs were also found at the soma. Furthermore LDCV release was slower compared to SSV release (Bruns et al., 2000).\n\nThere are several lines of evidence for volume transmission of 5-HT, some of which are indirect. First of all it has been shown that 5-HT varicosities contain 5-HT vesicles, but lack a postsynaptic specialization in close proximity (Moukhles et al., 1997). 5-HT receptors and the SERT are localized at sites away from release sites, indicating that 5-HT diffuses over larger distances to activate these receptors (Ridet et al., 1993). The presence of SERT distant from release sites\n\nsuggests that these are required to transport 5-HT back into the cells to terminate the action of 5-HT in the extracellular space. Secondly, it has been shown by carbon fiber amperometry that upon stimulation an increase of 5-HT levels in the extracellular medium occurs (Bunin and Wightman, 1998). This suggests that upon stimulation, not all 5-HT is buffered by synaptic receptors and transporters, but diffuses away into the extracellular medium (Bunin and Wightman, 1998).\n\nIn contrast to extrasynaptic release, synaptic release occurs predominantly from SSVs. Apart from vesicular release of 5-HT, there is also evidence for non-vesicular release of 5-HT using a carrier-mediated mechanism (Levi and Raiteri, 1993).\n\nThe ratio between volume transmission and synaptic transmission of 5-HT depends on the location in the brain. In the DRN itself, 5-HT release is predominantly paracrine, and here somatodendritic 5-HT release activates 5-HT\\textsubscript{A} autoreceptors which are present at the somata and dendrites. These activated receptors activate a G protein-activated inwardly rectifying K\\textsuperscript{+} (GIRK) current. In target areas of the 5-HT system, the ratio between volume transmission and synaptic transmission is still under debate. However, there are electron microscopy studies that quantified the percentage of 5-HT varicosities which formed a complex with a postsynaptic specialization. This revealed that ~25%, 48% and 38% of 5-HT varicosities formed a synaptic complex in rat hippocampus, suprachiasmatic nuclei and supraoptic nuclei of the hypothalamus respectively (Oleskevich and Descarries, 1990; Boulaiach et al., 1994). It is noteworthy that 5-HT is not released exclusively from 5-HT neurons. During development, thalamocortical fibers transiently express SERT, capture 5-HT and use it as a borrowed transmitter (Lebrand et al., 1996). In the striatum, dopamine transporters can take up 5-HT and subsequently co-release dopamine and 5-HT from dopaminergic terminals (Zhou et al., 2005a).\n\n**5-HT has several effects on synaptic transmission**\n\n5-HT not only functions to activate postsynaptic receptors, but also has diverse functions in the presynapse. Several studies were performed on invertebrates, such as crayfish, lamprey and aplysia. It was observed at the crayfish neuromuscular junction that a brief exposure to 5-HT resulted in increased release of glutamate (Dixon and Atwood, 1989a). Injection of a protein kinase A inhibitor or an adenylate cyclase inhibitor abolished this effect, suggesting that the facilitatory effect of 5-HT required the action of both the phosphatidylinositol and adenylate cyclase second messenger pathway (Dixon and Atwood, 1989b). In a study by Wang and colleagues, they made use of the styryl dye FM1-43, a fluorescent dye which is taken up by endocytosis following vesicle release, thus\n\nspecifically labeling recycled vesicles (Betz and Bewick, 1992). Using this approach, application of 5-HT at the crayfish neuromuscular junction did not affect the rate of recycling. Instead, 5-HT increased the number of vesicles which are available for release, the ready-releasable pool (RRP) of vesicles (Wang and Zucker, 1998).\n\nIn contrast to this stimulatory effect in the crayfish neuromuscular junction, 5-HT has an inhibitory action on synaptic transmission in the lamprey giant synapse (Buchanan and Grillner, 1991). This effect of 5-HT is likely mediated via G protein βγ subunits, since injection of these subunits mimic the effect of 5-HT (Blackmer et al., 2001). Furthermore the presynaptic protein SNAP25 is involved in this effect, since injection of Botulimum toxin A, which cleaves SNAP25, abolished the effect of 5-HT (Gerachshenko et al., 2005). Again using FM-dyes it was demonstrated that the inhibitory effect of 5-HT on the lamprey giant synapse is mediated via a change in vesicle fusion properties since 5-HT seemed to prevent full fusion of vesicles (Photowala et al., 2006).\n\nIn aplysia the effect of 5-HT on the gill-withdrawal reflex is well studied. In the 70’s it was shown for the first time that local application of 5-HT sensitized the gill-withdrawal reflex. This effect was also shown after application of cyclic adenosine monophosphate (cAMP), suggesting that this effect of 5-HT is mediated via an increase in cAMP (Brunelli et al., 1976). Both 5-HT and cAMP can close single potassium channels in aplysia sensory neurons, resulting in an increased transmitter release (Siegelbaum et al., 1982). This K⁺ channel, called the S (serotonin)-type K⁺ channel is modulated by 5-HT, cAMP and protein kinase A (PKA) (Shuster et al., 1985). Using calcium imaging on isolated synapses in culture, it was shown that 5-HT application results in a calcium influx in the terminal (Eliot et al., 1993). A brief single pulse of 5-HT resulted in short-term facilitation lasting minutes, whereas five subsequent pulses of 5-HT over 1.5 hours resulted in long-term facilitation lasting more than one day (Montarolo et al., 1986). 5-HT does not only affect release characteristics at single synapses, but upon application also activates silent presynaptic terminals. This results in the generation of new functional release sites within hours after application (Kim et al., 2003). This 5-HT-related growth of new synapses requires Cdc42, N-WASP and PAK (Udo et al., 2005).\n\nIn summary, 5-HT affects synaptic transmission in several ways. In crayfish neuromuscular junctions, 5-HT increases synaptic transmission by increasing the size of the RRP of vesicles. In the lamprey giant synapse 5-HT inhibits transmission by affecting single vesicle release kinetics. Finally, in aplysia sensory neurons, application of 5-HT can induce both short term facilitation and long term facilitation via an increase in transmitter release and the formation of new functional release sites.\n\n5-HT has a role during development in maturation of the brain.\n\nAn increasing amount of evidence points towards a role of 5-HT in brain development and maturation. Additionally, several studies show that 5-HT has an important role during early embryogenesis. 5-HT is present at early stages in sea urchins, chick embryos and rodents (Emanuelsson et al., 1988; Buznikov, 1991). Application of a 5-HT antagonist to sea urchin embryos significantly delayed cell division, implicating a role in the regulation of cell division (Renaud et al., 1983). In sea urchin blastulae, 5-HT, but also dopamine and noradrenaline stimulate ciliary activity. Application of these monoamines was associated with changes in intracellular calcium and adenyl cyclase activity (Soliman, 1983). In the chick embryo, 5-HT is synthesized by the notochord (Wallace, 1982). Monoamine inhibitors cause malformation in the chick embryo such as neural tube defects. Exposure of mouse embryos to 5-HT resulted in craniofacial malformations and increased cell death in craniofacial and cardiac mesenchyme (Yavarone et al., 1993); (Shuey et al., 1993). Several studies in rodents used injections of either p-chlorophenylalanine (PCPA) to inhibit Tph or the 5-HT-specific neurotoxin 5,7-dihydroxytryptamine. Injection of PCPA in mouse embryos from E12-E17 resulted in subtle alterations in pyramidal and non-pyramidal neuronal populations (Vitalis et al., 2007). 5,7-dihydroxytryptamine was injected at the day of birth in mice embryos in the medial forebrain bundle. These injected mice had a decreased 5-HT innervation towards the cortex and hippocampus and male mice showed a decreased novelty-induced exploration, suggesting an anxiety-like phenotype (Hohmann et al., 2007).\n\nKO mice have shown to be a valuable tool in studying the role of the 5-HT system in development. KO mice for tryptophan hydroxylase 1 (Tph1), the rate limiting enzyme involved in periferic 5-HT synthesis, lack periferic 5-HT (Walther et al., 2003). Although these KO mice do not have an overt phenotype, approximately 80% of pups born from homozygous mothers are smaller and show developmental abnormalities compared to embryos born from heterozygous or wildtype mothers (Cote et al., 2007).\n\nThis phenotype was only observed in Tph1 KO embryos born from Tph1 KO mothers, showing that maternal 5-HT is crucial for normal embryonic development. Tryptophan hydroxylase 2 (Tph2) is the Tph isoform that is exclusively expressed in the raphe nuclei (Gutknecht et al., 2009). Deletion of Tph2 does not result in an overt phenotype, nor does deletion of both Tph1 and Tph2, although in another study a reduced viability in these mice was observed (Savelieva et al., 2008; Alenina et al., 2009). Thus it appears that maternal 5-HT is indispensable for proper embryonic (brain) development, but 5-HT synthesis in the embryo is not.\n\n5-HT as a neurotrophic factor\n\nIn addition to the role of 5-HT in (early) neurodevelopment, 5-HT also functions as a neurotrophic factor. Application of 5-HT to buccal ganglion neuron 19 in helisoma caused a cessation in filopodial motility and an inhibition of neurite outgrowth (Haydon et al., 1984). Application of 5-HT results in a rise in intracellular calcium levels in the growth cone which probably links directly to the inhibition in outgrowth (McCobb et al., 1988). Application of 5-HT to 5-HT neurons also resulted in decreased outgrowth (Whitaker-Azmitia and Azmitia, 1986). In contrast to this inhibitory effect of 5-HT on growth of 5-HT neurons, 5-HT acts as a stimulatory signal on thalamic neurons which display more processes per cell and increased length of processes upon 5-HT administration (Lieske et al., 1999).\n\nMouse models with an impaired 5-HT system\n\nSince the introduction of the knockout technique in mice, several genes of the 5-HT system have been assessed for their effect on development and behaviour. The most important genes with striking effects on brain development or behaviour will be mentioned here. Mice lacking the MAO-A gene involved in the degradation of 5-HT to 5-hydroxyindolacetic acid (5-HIAA) display increased levels of 5-HT (Cases et al., 1996). These mice have a lack of barrels in the somatosensory cortex that is restored by application of PCPA, suggesting that this effect is due to excess levels of 5-HT (Cases et al., 1996). Additionally, mice lacking MAO-A display increased aggressive behaviour (Cases et al., 1995).\n\nOn the contrary, deleting the Vesicular Monoamine Transporter 2 (VMAT2) which is necessary for 5-HT packaging in vesicles, results in mice with brain 5-HT levels of ~1% compared to control (Wang et al., 1997; Fon et al., 1997). These KO mice are significantly smaller compared to wildtype littermates and the majority dies within a few days after birth. 5-HT fibers are almost completely absent in the cortex at that age. VMAT2 heterozygotes display prolonged immobility times in the forced swim test, suggesting depressive-like behaviour (Fukui et al., 2007). This behaviour was normalized by the antidepressant imipramine.\n\nDeletion of the SERT which is essential for 5-HT reuptake results in mice with increased extracellular 5-HT levels (Mathews et al., 2004). These mice have a reduced number of 5-HT neurons in the DRN, a reduced firing rate of these neurons and these mice display behavioural alterations (Lira et al., 2003). Notably, these behavioural effects were mimicked by applying the SERT blocker fluoxetine to young control mice between postnatal day 4 and 21 (Ansorge et al., 2004).\n\nIn addition to these genes involved in 5-HT synthesis, packaging, breakdown or reuptake, several 5-HT receptor genes have also been ablated in mice (for an overview of 5-HT receptors see Box III). The 5-HT\\textsubscript{1A} receptor acts as a heteroreceptor on non-5-HT neurons, but also as a 5-HT autoreceptor in the DRN. Mice with a deletion of the 5-HT\\textsubscript{1A} receptor spent less time in the center of the open field in the open field test and avoid the open arms in the elevated plus maze, suggesting that these mice display an increased anxiety-related behaviour (Parks et al., 1998; Ramboz et al., 1998). A very elegant study demonstrated that this effect on behaviour in the 5-HT\\textsubscript{1A} receptor KO occurs during development. In this study a second mouse line was made in which the expression of the 5-HT\\textsubscript{1A} receptor could be restored specifically in the forebrain. Rescue of 5-HT\\textsubscript{1A} receptor expression in the forebrain was sufficient to restore normal anxiety-related behaviour (Gross et al., 2002). In contrast, deletion of the 5-HT\\textsubscript{1A} receptor during development and rescue of the receptor after postnatal day 21 did not restore normal anxiety-related behaviour (Gross et al., 2002). This clearly shows that the 5-HT\\textsubscript{1A} receptor is required during development to establish normal anxiety-related behaviour.\n\n**Box III**\n\n**5-HT receptors**\n\nAt least 14 different 5-HT receptor subtypes are known which are divided into 6 different groups. The first and largest group of 5-HT receptors is the 5-HT\\textsubscript{1} group consisting of the 1A, 1B, 1D, 1E, 1F and 1P receptors. All receptors in this group are coupled to G proteins, and activation results in a decrease in cAMP levels. The most important receptor of this group is the 5-HT\\textsubscript{1A} receptor, which is expressed in the raphe nuclei on 5-HT cell bodies and functions as a 5-HT autoreceptor. Binding of 5-HT to the 5-HT\\textsubscript{1A} receptor results in activation of a GIRK channel which results in hyperpolarization of the 5-HT neuron; in this way, the 5-HT\\textsubscript{1A} receptor is involved in fine-tuning 5-HT release.\n\nTo the 5-HT\\textsubscript{2} receptor group belong the 2A, 2B and 2C receptors. These receptors are also coupled to G proteins, and activation of these receptors results in an increase of inositol 3-phosphate (IP3). The 5-HT\\textsubscript{3} receptor is the only 5-HT receptor which is a (Na\\textsuperscript{+}/K\\textsuperscript{+}) ion channel. The 5-HT\\textsubscript{4} and 5-HT\\textsubscript{6,7} receptors are coupled to Gs proteins and activation results in an increase of cAMP. Finally, activation of the 5-HT\\textsubscript{5A,B} receptor results in cAMP decrease. For the majority of these 5-HT receptors there are agonists and antagonists known. For example, the 5-HT\\textsubscript{1A} receptor is agonized by 8-OH-DPAT whereas it is antagonized by WAY-100635.\n\nMice lacking the 5-HT\\textsubscript{1B} receptor display an increased aggressive-related behaviour: they have a shorter latency to attack in the resident intruder paradigm (Saudou et al., 1994). The 5-HT innervation is increased in the hippocampus and the amygdala of these mice, possibly contributing to the aggressive phenotype of these mice (Ase et al., 2001). Another receptor known to\n\nbe localized in the DRN is the 5-HT\\textsubscript{2C} receptor (Clemett et al., 2000). This receptor is localized on GABA-ergic neurons and activation of this receptor results in a decreased firing of 5-HT neurons (Boothman et al., 2006). Mice that lack the 5-HT\\textsubscript{2C} receptor are overweight and suffer from seizures (Tecott et al., 1995). Moreover, studies have been performed to show that these mice display compulsive-like behaviour (Chou-Green et al., 2003).\n\nAn additional study showed that 5-HT\\textsubscript{2C} KO mice display decreased inhibition in the conflict anxiety paradigm (Weisstaub et al., 2006). This decreased inhibition was rescued by selective restoration of the 5-HT\\textsubscript{2C} receptor in the cortex, suggesting that 5-HT\\textsubscript{2C} receptors in the DRN are not involved in this effect (Weisstaub et al., 2006).\n\nThus, 5-HT receptor KO mice do not exhibit overt developmental defects, but anxiety- or aggressive-related behaviour of these animals is affected as assessed in various behavioural paradigms.\n\n**The 5-HT system in relation to psychopathological processes**\n\n**Anxiety & depression**\n\nThe notion that the 5-HT system could be involved in psychopathological conditions such as major depression and anxiety-related disorders came only after the discovery that drugs used to treat these conditions exerted their function (partially) via the 5-HT system. In the late 50’s, compounds such as iproniazide and G22355 (later named imipramine) were prescribed to patients suffering from tuberculosis. However, serendipitously it was found that these compounds were also effective in treating depression (Bloch et al., 1954; Kuhn, 1958). Only several years after it was found that these drugs had an antidepressant response, the mode of action of these drugs was elucidated; iproniazide inhibited the degradation of mono-aminergic compounds (5-HT, noradrenaline and dopamine), whereas imipramine blocked the reuptake of 5-HT and noradrenaline into the terminal. These findings led to the postulation of the monoamine hypothesis of depression in the late 60’s, which stated that a decreased concentration of 5-HT and noradrenaline in the extracellular space might be responsible for depression. It was poorly understood, however, why there was such a discrepancy between the immediate modes of action of these compounds in contrast to the delayed therapeutic effects. Also, it was not clear why only $\\sim$2/3 of all patients responded to these drugs.\n\nThese considerations abandoned the monoamine hypothesis and the so-called network hypothesis of depression was postulated. This hypothesis states that depression results from an alteration in certain neuronal networks, and antidepressants restore these networks, which explains the time lag between\n\nstarting antidepressant administration and the onset of therapeutic actions. Evidence for this hypothesis, however, is still limited and mostly indirect. Human studies using functional magnetic resonance imaging (fMRI) or positron emission tomography (PET) imaging on patients suffering from major depression have shown volume reductions in cortex and hippocampus. Although farfetched, one could argue that this reflects a decreased network density.\n\nThe last few years a new hypothesis was proposed, largely based on the work by Santarelli and colleagues: the neurogenesis hypothesis. It had already been shown that treatment with antidepressants increased adult neurogenesis in the dentate gyrus (Malberg et al., 2000; Perera et al., 2007). Santarelli and colleagues showed that blocking neurogenesis in the dentate gyrus by X-irradiation blocked the behavioural effects of the antidepressant fluoxetine (Prozac) (Santarelli et al., 2003). In other words neurogenesis is required for the behavioural effects of antidepressants. However, several studies have shown that neurogenesis is not involved in the etiology of depression.\n\nThus, although it is clear that there is a link between the 5-HT system and anxiety and depression, it is still far from clear what this exact link is.\n\n**Aggression**\n\nAggressive behaviour is defined as violent and impulsive behaviour often resulting in breaking the law. 5-HT is one of the neurotransmitters which is implicated in the etiology of aggression. A mutation in the MAO-A gene caused aggression-related abnormal behaviour (impulsive aggression, arson, attempted rape, and exhibitionism) in five males of a large kindred (Brunner et al., 1993). Several studies have shown a reduced concentration of the 5-HT metabolite 5-HIAA in cerebrospinal fluid (CSF) of individuals suffering from aggressive behaviour (Asberg et al., 1976; Coccaro et al., 1997). In rhesus monkeys, there was a negative relation found between high aggression and CSF 5-HIAA levels (Higley et al., 1992). A number of studies implicate the 5-HT$_2$ receptor subtypes in aggressive behaviour. A study by Winstanley and colleagues showed that 5-HT depletion increased the number of premature responses in the 5-choice serial reaction time task in rats (Winstanley et al., 2004). Also, in this study it was found that application of a 5-HT$_{2A}$ antagonist decreased impulsive behaviour, whereas application of a 5-HT$_{2C}$ antagonist had the opposite effect (Winstanley et al., 2004). A clinical study showed that antipsychotic drugs which had anti-aggressive properties were 5-HT$_{2A}$ antagonists (Krakowski et al., 2006). PET imaging studies show an increased 5-HT$_{2A}$ receptor binding in aggressive patients or in borderline personality disorder patients (Siever et al., 2002; Soloff et al., 2007). These abovementioned studies indicate increased 5-HT$_{2A}$ receptor sensitivity in aggressive behaviour patients.\n\nAutism\n\nAutism is a behavioural disorder with unknown etiology, although impaired brain development may underlie this disorder. The link between autism and 5-HT was made after the discovery that autistic patients had elevated platelet 5-HT levels (Boullin et al., 1970; Ritvo et al., 1970). Additionally, depletion of the 5-HT precursor tryptophan resulted in a deterioration in autistic patients (McDougle et al., 1993). Chugani and colleagues used alpha-[11C]methyl-L-tryptophan as a tracer for PET to measure 5-HT synthesis. They showed that in autistic boys there is a decreased 5-HT synthesis in frontal cortex, thalamus, and dentate nucleus of the cerebellum, although there is an increased 5-HT synthesis in the contralateral dentate nucleus (Chugani et al., 1997). Possibly increased levels of 5-HT during early brain development result in the loss of 5-HT terminals which could explain why several drugs which show great efficacy in treating autism enhance 5-HT neurotransmission (Brodkin et al., 1997; Fatemi et al., 1998). More evidence for this hypothesis is the fact that prenatal exposure to cocaine, which is a releaser of 5-HT, is a risk factor for developing autism (Davis et al., 1992). Furthermore, several studies using the 5-HT agonist 5-methoxytryptamine during development in rodents show that this results in autistic-like behaviour (Winslow and Insel, 1990; Kahne et al., 2002).\n\nPolymorphisms in genes involved in the 5-HT system\n\nSERT\n\nIn several genes involved in the 5-HT system, polymorphisms and short nucleotide polymorphisms (SNPs) have been found. An example of a gene in which polymorphisms have been found which correlates with psychopathological processes is SERT. Soon after the cloning of the human SERT, several polymorphisms have been found (Furlong et al., 1998; Battersby et al., 1999). These include two SNPs in the promoter region, a variable number of tandem repeats polymorphism in intron 2, and rare Ile-425-Val and Pro-339-Leu SNPs which affects function and surface expression (Kilic et al., 2003; Prasad et al., 2005). One polymorphism has been found in the promoter region. This polymorphism, a 44 bp insertion has been called the 5-HTT gene-linked polymorphic region, 5-HTTLPR. The two polymorphisms, called the short (s) and long (l) variant have been shown to modulate SERT transcriptional activity (Lesch et al., 1996). In a luciferase assay in human placental choriocarcinoma cells it was shown that the l variant has approximately a threefold higher transcriptional activity compared to the s variant (Heils et al., 1996). Additionally, cells expressing the l/l variant of the SERT displayed a twofold\n\nhigher uptake rate of 5-HT compared to cells expressing the s/s variant. In human postmortem brain material, reduced mRNA concentrations of SERT were found in the raphe nuclei from subjects having the s/s variant of the SERT promoter polymorphism. This shows that both in vitro and in vivo, the s/s variant is associated with a decreased SERT expression and reduced 5-HT reuptake (Little et al., 1998).\n\nIn 1996, the first study was published which demonstrated a link between the SERT promoter polymorphism and anxiety, aggression and depression traits (Lesch et al., 1996). Since then, several studies have demonstrated a link between the s/s promoter variant and aggression, anxiety and depression. Moreover, several studies show that individuals carrying the s/s variant have a poorer response to antidepressant treatment than individuals carrying the l/l or l/s variant (Smeraldi et al., 1998; Zanardi et al., 2000).\n\n**MAO-A**\n\nMAO is involved in the degradation of 5-HT to 5-HIAA, and there are 2 isoforms encoded by different genes, MAO-A and MAO-B. MAO-A has a higher affinity for 5-HT than MAO-B. These genes are both localized on the X chromosome (Lan et al., 1989). In humans a point mutation in the MAO-A gene has been found in the 8th exon, resulting in a truncated MAO-A protein. The family in which the point mutation was identified suffered from abnormal behaviour including disturbed regulation of impulsive aggressiveness (Brunner et al., 1993). In the promoter region of MAO-A a 30 bp repeat polymorphism was found. The low-activity 3-repeat allele was associated with antisocial behaviour in a German population (Samochowiec et al., 1999). In the rhesus macaque, a repeat polymorphism in the promoter affected transcription of the MAO-A gene and was associated with aggressive behaviour (Newman et al., 2005).\n\n**Tph2**\n\nTph1 and Tph2 are the rate limiting enzymes for 5-HT synthesis. Tph1 is expressed in the enterochromaffin cells in the gut and in the pineal gland, whereas Tph2 is the neuronal form and exclusively expressed in the raphe nuclei (Patel et al., 2004; Zill et al., 2004a). In Tph2 a C1473G mutation, resulting in a Pro-447-Arg substitution results in a reduction in 5-HT synthesis. BALB/cJ mice, which are homozygous for the 1473G mutation, have a 50 to 70% reduction in 5-HT synthesis in the frontal cortex resulting in ~40% reduction in 5-HT content in the frontal cortex (Zhang et al., 2004). In the human Tph2 gene, a G1463A SNP resulting in a Arg441His replacement was identified which, when transfected in PC12 cells, resulted in a ~80% loss of function in 5-HT synthesis.\n\n(Zhang et al., 2005). In a cohort of 87 patients suffering from major depression, 9 were found to carry this SNP, while among 219 controls, only 3 subjects had this SNP (Zhang et al., 2005). Knockin mice carrying this human mutation displayed behavioural abnormalities (Beaulieu et al., 2008). In contrast to SNPs found in exons, also SNPs in introns of the Tph2 gene have been found. In intron 5, a SNP has been found which was associated with major depression and suicide (Zill et al., 2004b; Harvey et al., 2004).\n\nAs in the SERT promoter, also in the Tph2 promoter polymorphisms have been found. One of these polymorphisms, rs4570625, has been associated with increased amygdala reactivity to emotional stimuli (Canli et al., 2005). Two polymorphisms, rs4570625 and rs11178997, were linked to anxiety disorder and major depression (Zhou et al., 2005b). As shown by a luciferase assay in 5-HT neurons, the polymorphism rs11178997 significantly reduced Tph2 transcriptional activity by 22% (Scheuch et al., 2007).\n\n**Conclusions**\n\n5-HT is a transmitter that has several modes of action in the central nervous system. First of all, 5-HT has a role during brain development and maturation. Secondly, due to the volume transmission, 5-HT can influence several aspects of behaviour, depending on the receptor(s) to which 5-HT binds. 5-HT can also regulate synaptic transmission. KO mice for the 5-HT\\textsubscript{1A}, 5-HT\\textsubscript{1B} or SERT gene display altered anxiety-related, depression-related and aggression-related behaviour. Finally, polymorphisms in several genes functioning in the 5-HT system are implicated in depression, anxiety, aggression and autism.\n\n**Aim and outline of this thesis**\n\nIn this thesis the outgrowth and connectivity of the brain 5-HT system is studied. To study these aspects we used a number of different approaches. *First* of all, we used organotypic slice cultures to study the outgrowth of the 5-HT system in vitro. *Secondly*, we used neuronal cultures to study trafficking of two components of the 5-HT system, SERT and Tph2. *Thirdly*, we investigated whether a SNP in a presynaptic gene which associates with major depressive disorder, affects trafficking of the SERT. *Fourthly*, we used a genetic approach in mice to study the effect of a silenced 5-HT system on mouse development. *Finally*, we describe our approach to generate a mouse model in which the 5-HT system is labeled with fluorescent reporter genes to study the outgrowth and connectivity in vivo.\n\nIn chapter 2, we used organotypic slice cocultures of the dorsal raphe nucleus and the hippocampus as a target area. We have used this model system to study the outgrowth of the 5-HT system in vitro. Moreover, we investigated whether this system could be used to assess the effect of pharmacological manipulations on the outgrowth of the 5-HT system.\n\nIn chapter 3, we tagged SERT with the red fluorescent protein mCherry, and expressed this protein in hippocampal neurons to investigate the trafficking dynamics of SERT.\n\nIn chapter 4, we tagged Tph2 with EGFP and took the same approach as in chapter 3 to investigate the subcellular distribution and trafficking of Tph2 in neurons.\n\nIn chapter 5, we studied whether knockdown of the presynaptic gene Piccolo affects SERT trafficking and incorporation into the membrane. A SNP in the C2A domain of this protein was found which associated with major depressive disorder. We expressed these two variants and investigated whether there is a difference in SERT localization. Also, we investigated whether knockdown of Piccolo affects SERT trafficking.\n\nIn chapter 6, we used a genetic approach to silence the 5-HT system. 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Verhage$^1$\n\n$^1$Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, the Netherlands\n$^a$Department of Anatomy and Neurosciences, Vrije Universiteit Medical Center, Amsterdam, the Netherlands\n$^b$Present address: Kavli Institute for Systems Neuroscience and Centre for the Biology of Memory, Norwegian University of Science and Technology NTNU, Trondheim, Norway\n\nPublished in Brain Research (2009) Dec 11;1302:1-9\n\nAbstract\n\nThe serotonin system densely innervates the brain and is implicated in psychopathological processes. Here we studied the effect of serotonin and serotonin pharmacological compounds on the outgrowth of serotonergic projections using organotypic slice co-cultures of hippocampus and dorsal raphe nuclei. Immunohistochemical analysis showed that several serotonergic neurites had grown into the target slice within 7 days in culture, after which the neurite density stabilized. These projections expressed the serotonin-synthesizing enzyme tryptophan hydroxylase and the serotonin transporter and contained several serotonin positive varicosities that also accumulated presynaptic markers. Chronic application of a 5-HT$_2$ agonist reduced the serotonergic neurite density, without effects on survival of serotonergic neurons. In contrast, application of a 5-HT$_{1A}$ agonist or the serotonin transporter inhibitor fluoxetine did not affect serotonergic neurite density. We conclude that serotonergic connectivity was reproduced in vitro and that the serotonin neurite density is inhibited by chronic activation of the 5-HT$_2$ receptor.\n\nIntroduction\n\nThe serotonin (5-hydroxytryptamine (5-HT)) system has its cell bodies clustered in the midbrain raphe nuclei and sends abundant projections to virtually every brain area (Hornung, 2003; Rubenstein, 1998). The 5-HT system influences a wide variety of physiological processes, such as sleep-wake rhythm, feeding, sexual behaviour and nociception (Jacobs and Azmitia, 1992). Moreover, the 5-HT system is implicated in several psychopathological processes, such as anxiety, aggression, obsessive compulsive disorder and depression (Castren, 2005).\n\nDuring brain development, the 5-HT neurons are one of the first groups of cells expressing a specific neurotransmitter (Lauder, 1990; Gaspar et al., 2003). Accumulating evidence shows that alterations in brain 5-HT levels during development could result in alterations in brain development and behavioural alterations. 5-HT reuptake transporter (SERT) knockout (KO) mice have an increase in extra neuronal 5-HT but a reduction in overall brain 5-HT levels (Bengel et al., 1998; Mathews et al., 2004). They exhibit behavioural alterations, reduced number of 5-HT neurons in the dorsal raphe nuclei (DRN) and reduced firing of 5-HT neurons in the DRN (Lira et al., 2003). These behavioural alterations observed in SERT KO mice can be mimicked by application of the\n\nSERT inhibitor fluoxetine during development between postnatal day 4 and 21 (Ansorge et al., 2004).\n\nIn contrast to reduction in brain 5-HT content after disruption of the SERT gene, there is a significant increase in brain 5-HT levels after inactivation of the Monoamine Oxidase-A (MAO-A) gene (Cases et al., 1996). The chronic elevation of 5-HT levels during brain development results in lack of barrels in the barrel cortex (Cases et al., 1996). These data suggest that either reductions in brain 5-HT levels or excess 5-HT levels during a critical period in the development of the brain can lead to abnormalities in brain development and behavioural alterations. In vitro experiments have shown that 5-HT can inhibit the outgrowth of neurites or even cause growth cone collapse on 5-HT neurons (Haydon et al., 1984; Koert et al., 2001). Whether alterations in brain 5-HT levels during development of the brain result in alterations in 5-HT outgrowth, branching or connectivity, or whether the behavioural alterations observed are a result of alterations in the 5-HT outgrowth and connectivity is currently unknown. Organotypic slice cultures of the DRN and a target slice could be a valuable reduced model system to study 5-HT outgrowth in vitro.\n\nTherefore the aim of this study was to investigate whether organotypic slice co-cultures of DRN and hippocampus, a method that has previously been characterized, can be used to study 5-HT outgrowth and the effect of pharmacological manipulations in vitro (Papp et al., 1995; Guthrie et al., 2005). We show that within seven days of culturing several 5-HT neurites have grown into the hippocampal slice. Using these slice co-cultures, we studied whether chronic application of 5-HT pharmacological compounds affects the outgrowth of 5-HT neurites from the DRN. To this end we used a 5-HT\\textsubscript{1A} receptor agonist, a 5-HT\\textsubscript{2} receptor agonist and the SERT inhibitor fluoxetine since it has been shown before, also on non-5-HT cells, that (chronic) activation of a 5-HT\\textsubscript{1A} or 5-HT\\textsubscript{2} receptor or blockade of the SERT can affect (5-HT) outgrowth or synaptogenesis (Wilson et al., 1998; Kondoh et al., 2004; Fricker et al., 2005; Zhou et al., 2006). We show here that chronic application of a 5-HT\\textsubscript{2} agonist results in a reduction in 5-HT neurite density. We conclude that these organotypic slice co-cultures can be used to study 5-HT outgrowth and the effect of 5-HT pharmacological compounds on this outgrowth in vitro.\n\n**Results**\n\n*Development and outgrowth of 5-HT neurons in organotypic slice co-cultures*\n\nTo study the outgrowth of 5-HT neurites ex vivo, we used organotypic slice co-cultures of the DRN and a hippocampal slice. We positioned the slices close to each other to allow 5-HT neurites to grow into the hippocampal slice. Within\n\nseven days of culturing the slices flattened out and several non-neuronal cells migrated away from the slices (Fig. 1).\n\n**Figure 1. Overview of the culturing method**\nThe DRN and hippocampal slice were positioned close to each other to allow 5-HT neurites to grow into the hippocampal slice. After culturing for 7 days, the slices flatten and several cells (e.g. macrophages) migrate away from the slices. Scale bar: 500 µm.\n\nWe performed immunohistochemistry for 5-HT to investigate whether 5-HT neurons survived in the slices. This showed that in several DRN slices 5-HT neurons were present, which resembled the topology of 5-HT neurons in the DRN in vivo (Fig. 2A). However, there were also DRN slices which contained only few 5-HT neurons, possibly resulting from a variation in the dissection of the DRN slice. A Z-stack of 5-HT cell somata showed that these cells have several primary neurites which contain numerous varicosities (Fig. 2B). In the growth cones of 5-HT neurites, 5-HT was also detected (Fig. 2C). Next, we focussed on the expression of tryptophan hydroxylase (Tph) and SERT, two markers for the 5-HT system. In 5-HT immunopositive cell bodies and in the 5-HT neurites and varicosities Tph was expressed (Fig. 2D,E). We found that all the 5-HT neurites were positive for SERT labelling, with the highest level of SERT staining on the soma and in the 5-HT varicosities (Fig. 2F,G). Moreover, SERT was also expressed in a punctate pattern in the 5-HT growth cones. (Fig. 2H). In vivo, 5-HT is released from varicosities in 5-HT axons and dendrites and 5-HT release is predominantly paracrine (Bunin and Wightman, 1999). In the slices, the 5-HT neurites contain several round and fusiform shaped varicosities (Fig. 3A,B).\n\nFigure 2. 5-HT neurons survive in the slice and grow out several 5-HT neurites\n\n(A) A DRN slice cultured for 7 days which is stained for 5-HT shows that several 5-HT cells are present in the slice. Note the presence of the lateral wings of the DRN (arrows) and the group of 5-HT neurons in the ventral part of the DRN (arrowhead). (B) A Z stack of two cells shows that the cells project several neurites that are 5-HT positive. (C) The growth cones also contain 5-HT, both in the core (arrowhead) and in the filopodia (arrows). (D,E) Staining for the rate limiting enzyme Tph shows that Tph is present both in the cell bodies and in the varicosities in the neurites. (F,G) The other marker for the 5-HT system, SERT, is present in 5-HT cell bodies and 5-HT neurites. (H) Moreover, there is also a punctate SERT staining in 5-HT growth cones, both in the core (arrowhead) and in the filopodia (arrows). Scale bars: 200 µm in A, 50 µm in B and D, 20 µm in F and G, 5 µm in C and E, 2 µm in H.\n\nImmunohistochemistry revealed that in these varicosities the calcium sensor Synaptotagmin and the active zone marker Bassoon are present, suggesting that the varicosities are presynaptic sites where calcium dependent 5-HT release occurs (Fig. 3C,D) (Perin et al., 1991; tom Dieck et al., 1998). This showed that in organotypic slice co-cultures of DRN and hippocampus 5-HT neurons survive and grow out neurites that contain 5-HT varicosities with immunoreactivity for all tested components of the 5-HT release machinery.\n\nDense ingrowth of 5-HT neurites into hippocampal slices\n\nTo study the outgrowth, we fixed slice cultures at different time points and quantified the 5-HT outgrowth. To this end the images were converted to binary images and the 5-HT neurite density in the slices was quantified (Fig. 4A-C).\n\nFigure 3. 5-HT neurites contain several varicosities which are positive for presynaptic markers\n\n(A) A Z-stack in the hippocampal slice shows that several thin, highly branching 5-HT neurites have grown into the slice. (B) A blow-up of the red box in A shows that these neurites contain several varicosities. (C,D) These varicosities are positive for the protein involved in calcium dependent secretion, Synaptotagmin (Syt), and for the active zone marker Bassoon. Scale bars: 50 µm in A, 10 µm in B and D and 2 µm in C.\n\nWe quantified the 5-HT neurite density as the area in the slice occupied by 5-HT neurites compared to the total area of the slice. Immunohistochemistry for 5-HT on hippocampal slices cultured without the DRN for seven days revealed that there were no 5-HT neurites present (Fig. 4E). Thus, 5-HT neurites observed in the hippocampal slice in co-cultures are the result of re-growth of 5-HT neurites from the DRN slice. In co-cultures fixed at four days in vitro (DIV) we observed that the DRN slice already contained several 5-HT neurites, and the first 5-HT neurites started to grow into the hippocampal slice (Fig. 4F,I). At DIV7, also the\n\nhippocampal slice contained several 5-HT neurites (Fig. 4G,J). Quantification of the 5-HT neurite density in the slices at DIV14 and DIV21 revealed that this was not significantly different from DIV7 (Fig. 4D,H; DIV7 DRN 8.42 ± 0.8%, HIP 6.31 ± 0.84% n=16; DIV14 DRN 9.49 ± 1.42%, HIP 5.12 ± 0.9% n=12; DIV21 DRN 9.26 ± 1.16%, HIP 6.61 ± 1.43% n=4). We investigated whether there is regional variation in the 5-HT neurite distribution in the hippocampus. At DIV4 the first 5-HT neurites which start growing into the hippocampal slice do not appear to display a preference for a certain region. (Fig. 4K). At DIV7, 5-HT neurites have grown in a uniform distribution into the hippocampus, and there are no regions which contain a higher density of 5-HT neurites than other regions (Fig. 4L). Thus, the initial outgrowth of the 5-HT neurites occurs in the first 7 days, after which the network stabilizes and the homogenous distribution suggests that 5-HT neurites do not have any preferential target region within the hippocampal slice in vitro.\n\n**The effect of 5-HT pharmacological compounds on the outgrowth of 5-HT projections**\n\nFinally, we studied whether this culture model system can be used to study the effect of chronic application of 5-HT pharmacological compounds on the outgrowth of 5-HT neurites. There are several 5-HT receptors which are expressed in both the DRN and the hippocampus.\n\n**Figure 4 next page. Dense ingrowth of 5-HT neurites into hippocampal slices within seven days of culturing**\n\nAn original image of a hippocampal slice co-cultured with a DRN slice shows that there is abundant ingrowth of 5-HT neurites, but there is also a lot of red background staining. (B) When this image is converted to a binary image using the steps described in experimental procedures, the 5-HT neurites are positively labelled (black), whereas the background is removed (white). (C) A blow up of part of the binary image shows that all 5-HT neurites are detected, and the red background is removed. (D) Quantification of the 5-HT neurite density (Y-axis) in the DRN and hippocampal slices at DIV7, DIV14 and DIV21. (E) A hippocampal slice cultured without the DRN contains only background staining, and no 5-HT neurites. (F,I) Maximal 5-HT ingrowth of the slices is reached in the first seven days of culturing. At DIV4, the DRN slice already contains several 5-HT neurites, and the first 5-HT neurites start to grow into the hippocampal slice. (G,J) At DIV7, both the DRN and hippocampal slice contain abundant 5-HT neurites. (H) At DIV21 the 5-HT neurite density in the slices has not changed. (K,L) No regional variation in 5-HT neurite density in the hippocampal slice at DIV4 (K) and DIV7 (L). Figures I and J are a blow up of the black box in F and G respectively. Figures K and L are blow ups of the hippocampal slices in figure F and G respectively. Scale bars: 500 µm in A, B, E, F, G, H, I, J, K and L, 20 µm in C.\n\nThese include the 5-HT\\textsubscript{1A} receptor, the 5-HT\\textsubscript{2} receptor and the 5-HT\\textsubscript{7} receptor (Gustafson et al., 1996; Clemett et al., 2000; Xu and Pandey, 2000; Bickmeyer et al., 2002; Garcia-Alcocer et al., 2006). Since it has been shown before, also on non-5-HT cells, that (chronic) activation of a 5-HT\\textsubscript{1A} or 5-HT\\textsubscript{2} receptor or blockade of the SERT can affect (5-HT-ergic) outgrowth or synaptogenesis, we decided to use a 5-HT\\textsubscript{1A} receptor agonist 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT), a 5-HT\\textsubscript{2} receptor agonist (+)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI hydrochloride) and the SERT inhibitor fluoxetine (Wilson et al., 1998; Kondoh et al., 2004; Fricker et al.,\n\nWe chronically applied the pharmacological compounds in 10 µM concentrations during the first seven days of outgrowth. First of all, we studied the effect of chronic blockade of the SERT by fluoxetine. Fluoxetine did not affect 5-HT neurite density (Fluoxetine: DRN: 8.44 ± 0.72%, HIP: 6.12 ± 1.12% n=10) compared to control condition (Fig. 5A,B,E,F). Subsequently, we tested the effect of chronically activating the 5-HT\\textsubscript{1A} and 5-HT\\textsubscript{2} receptor, two receptors which are present on 5-HT neurons. Moreover, it has been shown previously that activation of these receptors can affect outgrowth of Purkinje cells (Kondoh et al., 2004). First we investigated the effect of the 5-HT\\textsubscript{1A} receptor agonist 8-OH-DPAT. Activation of the autoreceptor by 5-HT or an agonist results in a reduction in 5-HT release (Quick, 2003). However, 8-OH-DPAT did not affect 5-HT neurite density (DRN 10.67 ± 0.79%, HIP 7.21 ± 0.9% n=9) (Fig. 5 C,E,F).\n\n**Figure 5. Chronic application of a 5-HT\\textsubscript{2} agonist reduces 5-HT neurite density in the slices**\n\n(A) In control condition, after seven days of culturing several 5-HT neurites have grown into the slices. (B) Chronic blockade of the SERT with the SERT inhibitor fluoxetine did not affect 5-HT neurite density in the slices. (C) Chronic activation of the 5-HT\\textsubscript{1A} receptor with 8-OH-DPAT also did not affect 5-HT neurite density in the slices. (D) However, chronic activation of the 5-HT\\textsubscript{2} receptor with DOI significantly reduced 5-HT neurite density both in the DRN and hippocampal slice. (E,F) Quantification of 5-HT neurite density in the DRN slice and hippocampal slice, respectively. Scale bar: 500 µm. * p< 0.05, ** p<0.01, *** p<0.001.\n\nNext, we investigated the effect of chronic 5-HT\\textsubscript{2} receptor activation. Activation of the 5-HT\\textsubscript{2} receptor results in an increased protein kinase C activity and release of calcium from internal stores. We chronically activated the 5-HT\\textsubscript{2} receptor by applying the 5-HT\\textsubscript{2} receptor agonist DOI hydrochloride. Chronic application of the 5-HT\\textsubscript{2} receptor agonist DOI hydrochloride resulted in a significantly decreased 5-HT neurite density (Fig. 5 D,E,F) (DRN 2.94 ± 1.01%, HIP 2.29 ± 1.05% n=5). To exclude the possibility that pharmacological treatment affected the survival of 5-HT neurons, we counted the number of 5-HT cells per group. This showed that treatment with fluoxetine, 8-OH-DPAT or DOI hydrochloride did not affect the number of 5-HT neurons in the slices (data not shown). Hence, changes in viability of 5-HT neurons cannot explain the effect of DOI hydrochloride. We conclude that these organotypic slice co-cultures can be used to study the 5-HT outgrowth and ingrowth into a target area in vitro and that chronic activation of the 5-HT\\textsubscript{2} receptor results in a decreased 5-HT neurite density.\n\n**Discussion**\n\n*An organotypic co-culture model system to study 5-HT outgrowth ex vivo*\n\nWe used organotypic slice co-cultures to study the outgrowth of the 5-HT system ex vivo. Neurite projections of 5-HT neurons grew into the slices and at 7 days a dense network had formed with many serotonergic boutons that had accumulated presynaptic markers. The regional differences in 5-HT innervation observed in the hippocampus in vivo were not reproduced ex vivo and it has been observed that fewer synapses form in slices than in vivo (see discussion in Papp et al., 1995). However, also in vivo the majority of the 5-HT release sites (70-80%, (Oleskevich and Descarries, 1990), are not classical synapses, i.e. with a juxtaposed postsynaptic specialization. Hence, the in vivo connectivity of the 5-HT system appears to depend largely on non-synaptic release. Therefore, despite some discrepancies compared to the in vivo situation, the slice co-culture system is an excellent, reduced model system to unravel the basic principles that orchestrate 5-HT neurite outgrowth and connectivity. Such a reduced system is a valuable supplementation of in vivo studies, which are often complicated by the exceptionally complex and dynamic connectivity of the 5-HT system, with for instance large fluctuations in innervation over the course of a few days (transient hyper-innervation, see (Fujimiya et al., 1986; D’Amato et al., 1987).\n\n*Chronic activation of the 5-HT\\textsubscript{2} receptor decreased 5-HT neurite density*\n\nWe found that chronic application of a 5-HT\\textsubscript{2} agonist decreased the neurite density of 5-HT neurites. Since DOI hydrochloride treatment did not affect 5-HT neuron survival, 5-HT mediated cytotoxicity cannot explain the decrease in 5-HT\n\nneurite density. Hence, the decreased neurite density appears to be the result of a specific effect of the activation of the 5-HT$_2$ receptor on the neurites (decreased outgrowth/increased pruning). In contrast, chronic activation of the 5-HT$_{1A}$ receptor did not mimic the effects of 5-HT, although the 5-HT$_{1A}$ receptor has often been implicated in shaping 5-HT connectivity (Gross et al., 2000; Gross et al., 2002). Activation of somatodendritic 5-HT$_{1A}$ receptors results in hyperpolarisation and a decrease in 5-HT release (Quick, 2003). However, chronic activation results in rapid desensitization (Assie et al., 2006) which may explain why we do not observe an effect of chronic 5-HT$_{1A}$ receptor activation. On the other hand, 5-HT$_2$ receptor activation increases intracellular calcium and activates protein kinase C (Tamir et al., 1992). This suggests that (chronic) activation of the 5-HT$_2$ receptor affects outgrowth, since elevation of calcium levels in growth cones reduces outgrowth (Gomez and Spitzer, 1999). Since DOI hydrochloride has a comparable affinity for the 5-HT$_{2A}$ and 5-HT$_{2C}$ receptor subtype, based on these data we cannot conclude which of the two subtypes is involved in the effect of DOI hydrochloride.\n\nHowever, although the 5-HT$_{2A}$ receptor is widely expressed throughout the brain, the receptor seems not to be expressed in the DRN (Xu and Pandey, 2000). In contrast, the 5-HT$_{2C}$ receptor is present in the DRN (Clemett et al., 2000; Serrats et al., 2005). Moreover, it has been shown previously, that activation of the 5-HT$_{2C}$ receptor in the DRN results in a reduction in 5-HT neuron firing (Boothman et al., 2006). Therefore, based on the anatomical distribution we speculate that the effect of DOI hydrochloride is predominantly mediated via the 5-HT$_{2C}$ receptor. Fluoxetine did not affect 5-HT neurite density. It could be expected that fluoxetine application mimics a 5-HT$_{1A}$ receptor agonist, i.e. SERT blockade results in an increase in extracellular 5-HT which could activate 5-HT$_{1A}$ autoreceptors. However, possibly upon application of fluoxetine extracellular 5-HT is rapidly diluted in the culture medium in vitro, and therefore extracellular 5-HT will not reach high enough concentration levels to mimic the effect of a 5-HT$_{1A}$ receptor agonist. Also chronic fluoxetine treatment in vivo failed to produce effects similar to chronic 5-HT application (Zhou et al., 2006). In fact, chronic fluoxetine enhanced neurite density in this case (Zhou et al., 2006).\n\n**Conclusions**\n\nWe have shown here that in DRN – hippocampus organotypic slice co-cultures 5-HT projections grow into the target slice. After four days of culturing in the DRN slice already several 5-HT projections were present, and the first 5-HT projections started to grow into the target slice. Within seven days of culturing also the target slice contained several 5-HT neurites. These 5-HT neurites,\n\ncontained several varicosities, which are presumably sites of 5-HT release, since presynaptic markers were present in these varicosities. Finally, we have shown that this culture model system can be used to study the effect of pharmacological manipulations on the outgrowth of 5-HT neurites, and that application of a 5-HT$_2$ agonist results in a reduction in 5-HT neurite density in the slices.\n\n**EXPERIMENTAL PROCEDURES**\n\n*Laboratory animals*\n\nWildtype C57BL/6 mouse fetuses were obtained from caesarean section at embryonic day 18 or postnatal day 1. Animals were housed and bred according to institutional, Dutch and U.S. governmental guidelines.\n\n*Pharmacological compounds and antibodies*\n\nMouse monoclonal anti-Tph antibody, which detects both Tph1 and Tph2, was obtained from Sigma-Aldrich and used in a 1:1000 dilution. Rabbit anti 5-HT polyclonal and mouse anti-SERT monoclonal were obtained from Immunostar/Diasorin and used in a 1:1000 dilution. Bassoon and Synaptotagmin monoclonal antibodies were used in a 1:500 and 1:1000 dilutions respectively and obtained from Stressgen. As secondary antibodies Alexa593-conjugated goat anti rabbit and Alexa488-conjugated goat anti mouse were used in a 1:1000 dilution (Molecular probes). 8-OH-DPAT and DOI hydrochloride were obtained from Sigma-Aldrich. Fluoxetine was obtained from Tocris.\n\n*Organotypic slice co-cultures*\n\nOrganotypic slice cultures were made as follows. After decapitation of the fetuses, the heads were immediately transferred to ice-cold dissection Gey’s balanced salt solution (dGBSS, Invitrogen, supplemented with 0.65 g/l glucose and 200 µM kynurenic acid). For isolation of a DRN slice, the brains were dissected in dGBSS and the midbrain cut into 400 µm thick slices using a McIlwain tissue chopper (Mickle Engineering, Gomshall, UK). Individual slices were separated in dGBSS and the hindbrain slice containing the rostral DRN was identified by visual inspection. The DRN was dissected out using a dissection knife. The entire hippocampus was dissected out of the brain and individual slices (400 µm) were sagitally cut perpendicular to the hippocampus’ longitudinal axis. The hippocampal and DRN slices were allowed to recuperate in dGBSS at 4°C for 60 minutes. A hippocampal and DRN slice were cultured in close proximity on a poly-D-lysine coated 12x24 glass coverslip (O. Kindler GmbH & Co. Mikroskopische Gläser, Freiburg, Germany) in a plasma clot (chicken plasma, Cocalico Biologicals Inc. Reamstown, U.S.) which was coagulated with thrombin (Merck, Darmstadt, Germany). Culturing medium\n\nconsisted of 50% BME Hanks, 25% Hanks balanced salt solution and 25% horse serum supplemented with 1.3 g/L glucose and 200 µM glutamine. For culturing 700 µL of culturing medium was added to the slices in a Nunc flat-bottomed tube. The slices were incubated in a roller drum at 36°C. After the first week, medium was changed twice a week.\n\n**Pharmacological treatments**\nAll pharmacological compounds were dissolved in ddH$_2$O, filter sterilized and aliquots stored at -80°C. After DIV1 10 µM of the pharmacological compound was added to the slices. Every day a new aliquot of pharmacological compound was added to the slices. In control slices only vehicle was added (sterile ddH$_2$O). At DIV7 the slices were fixed, processed and analyzed as described below.\n\n**Immunohistochemistry**\nSlices were fixed for 20 minutes in 4% paraformaldehyde dissolved in phosphate buffered saline. After fixation the slices were washed three times five minutes in phosphate buffered saline (PBS) and nonspecific binding was blocked by incubating the slices in PBS containing 0.1% Triton X-100 and 2% normal goat serum for two hours. The slices were incubated overnight with the primary antibodies diluted in PBS containing 0.1% Triton X-100 supplemented with 2% normal goat serum at 4°C. After incubation in the primary antibodies, the slices were washed three times two hours in PBS on a shaking platform. The secondary antibodies were diluted in PBS supplemented with 2% normal goat serum and incubated for one hour. After incubation in the secondary antibodies, the slices were washed again three times two hours in PBS and mounted in Dabco-Mowiol (Sigma) on glass coverslips. All reactions were carried out at room temperature unless otherwise stated. No labeling was observed when omitting the primary antibodies.\n\n**Confocal analysis**\nConfocal analysis of the slices was performed on a LSM 510 microscope (Carl Zeiss b.v. Weesp, the Netherlands) and a 63x Plan-Neofluar lens (Numerical aperture 1.4, Carl Zeiss). To excite the Alexa 488 antibody a HeNe1 laser was used and for excitation of the Alexa 593 antibody a HeNe2 laser was used. For analysis of the 5-HT neuron morphology and the 5-HT neurites, Z-stacks of 1 µm were made. Images were analyzed and further processed in Zeiss CLSM software.\n\n**Histological quantification of 5-HT neurite density**\nQuantification of 5-HT immunopositive neurites and cells was performed using an MCID Elite imaging system (Imaging Research Inc., Ont., Canada). Images of the immunostained co-cultures were digitized using an objective magnification\n\nof 20x on a Leica DM/RBE photo-microscope with a Sony (DXC-950P, 640 x 512 pixels) camera using epifluorescence microscopy. The 5-HT-immunopositive neurites were segregated from background using several point operators and spatial filters combined in an algorithm designed to detect local changes in relative optical density. Briefly, images underwent histogram equalization and smoothing (low-pass filter, kernel size 7x7). The unfiltered image was subtracted from the smoothed image, followed by a series of steps to optimize the processed image and make it a suitable measuring template for detecting objects the size and shape of 5-HT neurites or cell bodies. This algorithm was preferred over relative optical density thresholding since it does not involve an observer-dependent operation. Finally, a line was manually drawn around the DRN or hippocampal slice and the number of positive pixels (i.e. pixels representing 5-HT neurites) compared to the total number of pixels was calculated. The number of 5-HT neurons was counted manually in the slices.\n\nData analysis\nIn order to analyze differences in neurite density between different pharmacological treatments, ANOVA was used with the Bonferonni test for post hoc analysis. Data shown are mean values ± standard error of the mean (SEM). 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Synapse 12:155-168.\n\ntom Dieck S, Sammarti-Vila L, Langnaese K, Richter K, Kindler S, Soyke A, Wex H, Smalla KH, Kampf U, Franzer JT, Stumm M, Garner CC, Gundelfinger ED (1998) Bassoon, a novel zinc-finger CAG/glutamine-repeat protein selectively localized at the active zone of presynaptic nerve terminals. J Cell Biol 142:499-509.\n\nWilson CC, Faber KM, Haring JH (1998) Serotonin regulates synaptic connections in the dentate molecular layer of adult rats via 5-HT1a receptors: evidence for a glial mechanism. Brain Res 782:235-239.\n\nXu T, Pandey SC (2000) Cellular localization of serotonin(2A) (5HT(2A)) receptors in the rat brain. Brain Res Bull 51:499-505.\n\nZhou L, Huang KX, Kecojevic A, Welsh AM, Koliatsos VE (2006) Evidence that serotonin reuptake modulators increase the density of serotonin innervation in the forebrain. *J Neurochem* 96:396-406.\n\nChapter 3\n\nDynamic vesicular trafficking of the serotonin reuptake transporter in hippocampal neurons\n\nJ.J. Dudok\\textsuperscript{1}, A.J.A. Groffen\\textsuperscript{1} and M. Verhage\\textsuperscript{1}\n\n\\textsuperscript{1}Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, the Netherlands\n\n\\textit{In preparation}\n\nAbstract\n\nThe serotonin (5-HT) reuptake transporter (SERT) has an important role in 5-HT transmission by terminating the action of 5-HT in the synaptic cleft. Here we address the question how SERT is transported in neuronal cells. We studied the trafficking of SERT in hippocampal neurons by tagging SERT with a fluorescent protein. We show that SERT displays a punctate distribution in axons and a more uniform distribution in dendrites. The majority of SERT puncta do not co-localize with the synaptic marker Synapsin, showing that SERT is predominantly localized extra-synaptically. SERT is transported in secretory vesicles as SERT co-localizes with the secretory vesicle marker tissue plasminogen activator (tPA). Live cell imaging revealed a highly dynamic trafficking of SERT along the neurites. Approximately 35% of the vesicles moved, with an average velocity of $\\sim$1.2 µm/s. SERT vesicles do not display a preference for direction. We conclude that SERT, tagged with a fluorescent protein, displays a highly dynamic character in hippocampal neurons. These data are important to achieve a better understanding of the dynamics of treatment with 5-HT reuptake inhibitors.\n\nIntroduction\n\nThe projections of the 5-HT system abundantly innervate several brain areas. 5-HT is released both synaptically and extra-synaptically. After being released, 5-HT is cleared from the extracellular space by the SERT. Since this protein regulates the termination of the action of 5-HT, it is very important in regulating efficacy of 5-HT transmission. Moreover, SERT is the primary target for the selective serotonin reuptake inhibitors (SSRIs) class of antidepressants, several tricyclic antidepressants, and may mediate the effects of amphetamines such as 3,4-methylenedioxy-N-methylamphetamine (MDMA).\n\nSERT is a sodium and chloride neurotransmitter symporter and belongs to the solute carrier family 6, to which also transporters for dopamine, noradrenaline/adrenaline, glycine and GABA belong (Chen et al., 2004). In the adult brain, SERT is expressed in 5-HT neurons, but during development also transiently in the cortex, hippocampus, thalamocortex and the lateral geniculate nucleus (Lebrand et al., 1996; Zhuang et al., 2005). In 5-HT neurons, SERT is predominantly localized in axons but also in the cytoplasm of the soma and dendrites (Zhou et al., 1998; Tao-Cheng and Zhou, 1999). In axons SERT is not only associated with varicosities, sites where 5-HT is released, but also with the plasma membrane of the axons (Zhou et al., 1998).\n\nSeveral lines of evidence indicate that 5-HT has a role during brain development. In mice, deletion of SERT results in a 60-80% reduction in total\n\nbrain tissue 5-HT level, although there is a 7-8 fold increase in extracellular 5-HT levels in the striatum and substantia nigra (Fabre et al., 2000; Mathews et al., 2004). Analysis of emotional behaviour of these mice revealed that deletion of SERT results in abnormalities in anxiety-related behaviour (Holmes et al., 2003a; Holmes et al., 2003b). Additionally, blockade of SERT during development with the SSRIs fluoxetine, clomipramine or citalopram results in mice which display altered emotional behaviour in adulthood (Ansorge et al., 2004; Ansorge et al., 2008). These studies show that SERT is important in establishing normal emotional behaviour during development, presumably by regulating the extracellular concentration of 5-HT in the brain.\n\nThe level of SERT present at the cell membrane at synaptic boutons determines the rate of 5-HT reuptake and hence the efficacy of 5-HT transmission. Therefore, there is extensive regulation of the level of cell-surface expressed SERT. The trafficking and regulation of SERT cell-surface expression has been studied in many different cell types, including HEK293 cells, iC11 cells, basophilic leukemia 2H3 cells and 5-HT neurons (Zhu et al., 2004; Lau et al., 2008a; Lau et al., 2008b). A study using biotinylated SERT in 5-HT neurons showed that citalopram reduced the amount of cell surface-expressed SERT (Lau et al., 2008b) However, live cell imaging to investigate the trafficking dynamics of SERT has not been studied in neurons before.\n\nHere we investigated the trafficking of SERT in hippocampal neurons. To this end we expressed SERT tagged with the fluorescent protein mCherry in hippocampal neurons. SERT displays a polarized distribution, as in axons SERT is distributed in a punctate manner, whereas in dendrites SERT is present in a uniform distribution. SERT is transported in secretory vesicles through the neurites, as it co-localized with the secretory vesicle marker tPA. Live cell imaging revealed that SERT displays a highly dynamic vesicular trafficking. Analysis of direction of movement showed that SERT does not display a preference for a direction of movement. In future experiments, live cell imaging of mCherry-SERT can be used to address whether 5-HT reuptake inhibitors affect the highly dynamic SERT trafficking.\n\n**Results**\n\n*Localization of mCherry-SERT in hippocampal neurons differs between axons and dendrites*\n\nTo study the trafficking we tagged the N-terminus of SERT with the red fluorescent protein mCherry, which is an improved variant of monomeric red fluorescent protein in terms of maturation and photostability (Shaner et al., 2004). Tagging the N-terminus of SERT with a fluorescent protein does not interfere with the function of SERT (Schmid et al., 2001; Just et al., 2004;\n\nWe used hippocampal neurons that do not express SERT, although it is expressed transiently in a subpopulation of hippocampal neurons during development (Narboux-Neme et al., 2008).\n\nThe expression of mCherry-SERT in dissociated hippocampal neurons was analyzed between 9-12 days in vitro (DIV) using live cell imaging and immunocytochemistry on fixed neurons. This revealed that the whole neuron was extensively labelled with mCherry-SERT (Fig. 1A). Immunocytochemistry for the dendritic marker Microtubule Associated Protein 2 (MAP2) was used to discern between the dendrite and axon (Fig. 1B,C). SERT localization differed between axons and dendrites. In dendrites SERT is predominantly uniformly distributed where only occasionally a puncta of SERT is observed. However in axons, SERT displays a more punctate distribution suggesting that SERT is transported in vesicles through the axon (Fig. 1D,E,F). Alternatively, the punctate pattern may reflect local accumulation of SERT in varicosities. To further explore whether these puncta are varicosities or vesicles, we used immunocytochemistry and live cell imaging.\n\n**Figure 1. Distribution of mCherry-SERT differs between axons and dendrites**\n\n(A-C) Expression of mCherry-SERT in a hippocampal neuron (DIV11). (A) mCherry-SERT is present in all the neurites and the cell body. (B) MAP2 labelling to discern between axons and dendrites. (C) Merged image. (D-F) Blow up of the white box in C. (D) mCherry-SERT distribution differs between axons and dendrites, as in the axon mCherry-SERT displays a punctate distribution, whereas in dendrites mCherry-SERT displays a more uniform distribution where only occasionally a mCherry-SERT puncta is observed. (E) MAP2 labelling. (F) Merged image. Scale bars in C and F are 10 µm.\n\nSERT is transported in secretory vesicles\n\nSince SERT displayed a punctate distribution in neurites, we investigated whether these puncta are secretory vesicles. To this end mCherry-SERT was co-expressed with tPA-GFP, which is transported in secretory vesicles in hippocampal neurons (Lochner et al., 1998; Silverman et al., 2005). In neurons expressing both mCherry-SERT and tPA-GFP, the majority of SERT puncta co-localized with tPA-GFP (Fig. 2A-F). Therefore, these data strongly suggest that SERT is transported in secretory vesicles along the axons in hippocampal neurons.\n\nFigure 2. mCherry-SERT is transported in secretory vesicles\n\n(A-C) Co-expression of mCherry-SERT and tPA-GFP in a hippocampal neuron (DIV11). (A) mCherry-SERT displays a punctate distribution. (B) tPA-GFP is transported in secretory vesicles. (C) The majority of mCherry-SERT puncta co-localize with tPA-GFP, suggesting that mCherry-SERT is transported in secretory vesicles. (D-F) Blow-up of the white box in C. (D) mCherry-SERT expression. (E) tPA-GFP expression. (F) Merged image. Arrows show the co-localization between mCherry-SERT and tPA-GFP. Scale bars in C and F are 10 μm.\n\nTo investigate whether vesicles of SERT co-localized with presynaptic terminals, we performed immunocytochemistry for the synaptic marker Synapsin on neurons transfected with mCherry-SERT. Although several mCherry-SERT puncta are in close vicinity of Synapsin puncta (Fig. 3 A-F), only few mCherry-SERT puncta co-localized with Synapsin. Thus, mCherry-SERT is transported in secretory vesicles and several of those vesicles localize to the vicinity of presynaptic terminals.\n\nFigure 3. Several mCherry-SERT vesicles are localized in the vicinity of presynaptic terminals\n\n(A-C) Expression of mCherry-SERT in a hippocampal neuron (DIV11). (A) mCherry-SERT expression, (B) Synapsin labelling, (C) Merged image. Several mCherry-SERT vesicles are localized in the vicinity of presynaptic terminals, and some co-localized with the presynaptic marker Synapsin. (D-F) Blow up of the white box in C. (D) mCherry-SERT expression, (E) Synapsin labelling, (F) Merged image. Arrows in F show mCherry-SERT vesicles which are localized in the vicinity of presynaptic terminals. Arrowhead shows a mCherry-SERT vesicle which co-localizes with a Synapsin puncta. Scale bars in C and F are 10 µm.\n\nFigure 4 next page. Trafficking dynamics of mCherry-SERT secretory vesicles\n\nLive cell imaging of mCherry-SERT trafficking dynamics in hippocampal neurons (DIV 9-11). (A) Two frames 5 seconds apart (green is 1 s, red is 5 s) from a 1 minute movie reveal the different modes of trafficking of mCherry-SERT. There are mCherry-SERT vesicles which move in the anterograde direction (arrow), in the retrograde direction (arrowhead) or are stationary (yellow puncta, asterisks). (B) Corresponding kymograph of part of the neurite displays the different trafficking modes of mCherry-SERT. Vesicle 1 is a stationary vesicle, vesicle 2 moves in the anterograde direction, whereas vesicle 4 moves in the retrograde direction. Vesicle 3 is a vesicle which pauses for a while and then continues moving. Vesicle numbers are shown in the individual images taken 10 seconds apart and vesicle trajectories are highlighted in the kymograph. (C) No difference between average anterograde and retrograde velocity as can be observed in the histogram of the average velocity. (D) No difference between average velocity of mCherry-SERT vesicles which move in the anterograde or retrograde direction or which display bidirectional movements. (E) Percentage moving vesicles does not differ between vesicles which move anterogradely, retrogradely or bidirectionally. (F) Also, percentage pausing time for moving vesicles is not different between vesicles which move anterogradely, retrogradely or bidirectionally. Scale bars in A and B are 10 µm.\n\nSERT trafficking is highly dynamic\n\nWe analyzed the trafficking dynamics of SERT using live-cell imaging of hippocampal neurons expressing mCherry-SERT. We observed rapid movements of mCherry-SERT vesicles in both the anterograde and retrograde direction. Additionally, also stationary mCherry-SERT puncta were observed (Fig. 4A). To illustrate different types of movement we show part of a neurite with 10 second time intervals. In this neurite a stationary vesicle can be observed (vesicle 1), but also a vesicle which moves in an anterograde direction (vesicle 2) and a vesicle which moves in a retrograde direction (vesicle 4). Additionally, vesicles are...\n\nobserved which are temporarily stationary but then continue moving (vesicle 3 in Fig. 4B). In the corresponding kymograph of this part of the neurite the trajectories of the numbered vesicles are shown (Fig. 4B). We quantified the direction of vesicle movement, percentage of moving vesicles, average velocity of moving vesicles and percentage pausing time of moving vesicles using the automated vesicle detection program Fluotrack (Broeke et al., 2008). First we measured the average velocity of anterogradely and retrogradely moving vesicles. The frequency histogram plotting the velocities of mCherry-SERT vesicles moving in the anterograde or retrograde direction showed that the velocity distributions for anterogradely and retrogradely moving vesicles did not differ (Fig. 4C). The average velocity of all moving vesicles was $1.22 \\pm 0.03$ µm/s. The average velocity of anterograde vesicles was $1.31 \\pm 0.05$ µm/s compared to $1.34 \\pm 0.06$ µm/s in retrograde direction. Vesicles moving bidirectional (vesicles that changed direction during imaging) moved with an average velocity of $1.12 \\pm 0.055$ µm/s (Fig. 4D). Next, we determined the percentage of puncta which moved in the anterograde or retrograde direction, moved bidirectional or were stationary. This revealed that on average $34.52 \\pm 5.31\\%$ of mCherry-SERT puncta were moving during one minute of imaging. Further analysis of fraction of moving mCherry-SERT vesicles which moved in the anterograde or retrograde direction or moved bidirectional, revealed that there was no difference (Fig. 4E, see also table 1). Finally, we calculated the pausing time for the moving vesicles, which is the fraction of time moving vesicles pause during the 1 minute movie. There was no difference in percentage pausing time between mCherry-SERT vesicles which moved anterogradely, retrogradely or bidirectionally (Fig. 4F, see also table 1).\n\nWe conclude that mCherry-SERT displays highly dynamic vesicular trafficking in hippocampal neurons and that these mCherry-SERT vesicles do not have a preference for a direction of movement.\n\n**Table 1: Overview of mCherry-SERT vesicle dynamics**\n\n| | Velocity (µm/s) | Percentage of total | Percentage pausing time |\n|------------------|-----------------|---------------------|-------------------------|\n| Anterograde | $1.31 \\pm 0.05$ | 13.83 | 42.13 |\n| Retrograde | $1.34 \\pm 0.06$ | 12.06 | 39.21 |\n| Bidirectional | $1.12 \\pm 0.06$ | 8.63 | 41.04 |\n| Stationary | - | 65.48 | - |\n\nVelocity data are shown as mean ± standard error of the mean (SEM). Data of 927 mCherry-SERT puncta in 6 neurons.\n\nDiscussion\n\nHere we studied the trafficking of SERT in hippocampal neurons. We used human SERT cDNA tagged at its N-terminus with the red fluorescent protein mCherry. Overexpression of mCherry-SERT in hippocampal neurons resulted in a distinct pattern of expression between axons and dendrites; in dendrites SERT displayed a uniform distribution, whereas in axons SERT displayed a punctate distribution. Using electron microscopy, it has been shown that in 5-HT neurons endogenous SERT predominantly localized to varicosities and the membrane in axons, and in soma and dendrites SERT localized predominantly in the cytoplasm (Zhou et al., 1998; Tao-Cheng and Zhou, 1999). Thus, although we are using hippocampal neurons, mCherry-SERT distribution seems to mimic the distribution of endogenous SERT in 5-HT neurons.\n\nUltrastructural studies revealed that SERT labeling was associated with vesicular organelles, which possibly are large dense cored vesicles (LDCVs) (Pickel and Chan, 1999). Here, we co-transfected mCherry-SERT with tPA-GFP for which it has been shown that these are transported in LDCVs (also called secretory vesicles) (Silverman et al., 2005). The majority of mCherry-SERT puncta co-localized with tPA-GFP, strongly suggesting that SERT is transported in secretory vesicles. Several mCherry-SERT vesicles are localized in close proximity (~1 µm), and some co-localized, with presynaptic terminals. This suggests that the majority of SERT is not localized in the presynaptic terminal itself, but rather is localized close to a synapse. Indeed, previous research found that presynaptic membranes lacked SERT, but SERT was present on extrasynaptic domains, either in close proximity or at some distance away from synapses (Zhou et al., 1998).\n\nLive cell imaging revealed that mCherry-SERT vesicles display highly dynamic transport, i.e. there are vesicles which move anterogradely or retrogradely without interruptions, stationary vesicles or vesicles which move bidirectional. Analysis of average velocity revealed that both anterograde and retrograde vesicles moved with an average velocity of ~1.3 µm/s. The average velocity is in analogy with other transport or transport associated proteins which are transported in secretory vesicles, like the p75 neurotrophin receptor and Gephyrin (Maas et al., 2006; Formaggio et al., 2008). Surprisingly, we found that mCherry-SERT vesicles did not display a preference for direction of movement. For other releasable fluorescent cargos that are transported in secretory vesicles, such as Sema3A, BDNF and CCL21 it has been shown that transport is predominantly anterogradely (Adachi et al., 2005; de Wit et al., 2006; de Jong et al., 2008). In contrast, the p75 neurotrophin receptor, which is transported in vesicular structures, does not display a preference for direction of movement (Formaggio et al., 2008). Moreover, the scaffold protein Gephyrin,\n\nwhich is associated and co-transported with the glycine receptor also does not display a preference for anterograde or retrograde movement (Maas et al., 2006). Therefore, it appears that proteins which can be secreted and are transported in secretory vesicles predominantly move in the anterograde direction, whereas receptor/transporter proteins move both in the anterograde and retrograde direction.\n\nAn explanation for this could be that cargos which can be secreted are transported towards synaptic release sites distant from the cell soma. After a vesicle has been released, the secreted compounds either diffuse in the extracellular space (as is the case for Sema3A) or are taken up again and degraded in the presynaptic terminal. Hence, releasable cargos are hardly transported back towards the cell soma. On the other hand, the level of membrane expression for receptor and transporter protein is tightly regulated, and these proteins can be rapidly internalized (Loder and Melikian, 2003; Melikian, 2004; Zhu et al., 2004). Upon internalization, transporters are packaged in vesicles again and are recycled. Finally, receptors and transporters can maintain functioning without a need for intracellular transport, whereas secreted components are dependent on a new supply of vesicles. Therefore, vesicles containing transporters or receptors can be transported both in anterograde and retrograde directions.\n\nIn conclusion, expression of mCherry-SERT in hippocampal neurons differs between axons and dendrites. In axons mCherry-SERT is distributed in puncta, which co-localize with tPA-GFP and therefore presumably are secretory vesicles. Several of these mCherry-SERT vesicles are localized in close proximity of presynaptic terminals, but only few mCherry-SERT vesicles co-localized with presynaptic terminals. Live cell imaging revealed that mCherry-SERT displays highly dynamic trafficking behaviour with no preference for direction of movement.\n\n**EXPERIMENTAL PROCEDURES**\n\n*Plasmids*\n\nConstruction of the human SERT cDNA with ECFP at the 5’ end (ECFP-SERT in the pECFP-C1 backbone (Clontech)) has been described before ((Schmid et al., 2001); a kind gift of Dr. H. Sitte). To construct mCherry-SERT, ECFP was excised with AseI and BsrGI and replaced for mCherry.\n\n*Neuronal culturing and transfection*\n\nHippocampi from embryonic day 18 C57/Bl6 mouse embryos were dissected free of meninges and collected in Hanks Balanced Salt Solution (HBSS; Invitrogen) supplemented with 7 mM HEPES (Invitrogen). Hippocampi were digested with\n\n0.25% trypsin (Invitrogen) in HBSS supplemented with 7 mM HEPES for 15 minutes at 37°C. After digestion tissue was washed three times with HBSS supplemented with 7 mM Hepes and subsequently triturated with a fire-polished glass pipette. Dissociated neurons were plated at a density of 25,000 cell/well on rat glial cells on 18 mm glass coverslips (Menzel Glaser, Braunschweig, Germany) in 12 well plates. Cells were cultured in neurobasal medium supplemented with B27 supplement, 18 mM HEPES, 0.5 mM Glutamax and penicillin/streptomycin (Invitrogen). Once a week half of the medium was replaced. The hippocampal cultures were transfected using Lipofectamine 2000 (Invitrogen). Before transfection, half of the neurobasal medium was collected from each well and replaced with new neurobasal medium. For one well, 1 µl of Lipofectamine 2000 was added to 25 µl of serum free glutamax medium (Invitrogen) and incubated for 5 minutes. Subsequently, from each construct 1 µg of DNA was added to 25 µl of serum free glutamax and immediately mixed with the 25 µl of serum free glutamax containing Lipofectamine 2000. This mixture was incubated for 30 minutes at room temperature before adding to the cells. After 4-6 hours of incubation on the cells at 37°C, half of the neurobasal medium was replaced again with the earlier collected neurobasal medium. Cells were routinely transfected at DIV2-7 and imaged at DIV9-12.\n\n**Live cell imaging**\n\nCells were imaged in a chamber perfused with Tyrode’s solution (119 mM NaCl, 2.5 mM KCl, 2 mM MgCl$_2$, 2 mM CaCl$_2$, 30 mM glucose, 25 mM HEPES, pH 7.4) on an Axiovert II Microscope (Zeiss, Oberkochen, Germany) equipped with a Coolsnap HQ camera (Photometrics, Tucson, Arizona, USA) and a Polychrome IV illumination unit (TILL photonics, Grafelfing, Germany). Images were acquired in MetaMorph 6.2 software (Universal Imaging, Downingtown, Philadelphia, USA) using a 100x oil objective.\n\n**Immunocytochemistry and confocal microscopy**\n\nFor immunocytochemistry, neurons were fixed by incubation in 4% paraformaldehyde in phosphate buffered saline (PBS) for 20 minutes. Subsequently, to block aspecific binding and for permeabilization, the neurons were incubated in PBS containing 0.2% Triton X-100 and 4% fetal calf serum for 20 minutes. Neurons were incubated in primary antibody diluted in PBS containing 0.2% Triton X-100 for one hour. After washing three times in PBS, neurons were incubated in secondary antibodies in PBS for one hour. Neurons were washed again three times in PBS and mounted in Dabco-Mowiol. All reactions were carried out at room temperature. As primary antibodies, monoclonal MAP2 (Chemicon) and polyclonal Synapsin (E028) were used. As secondary antibodies Alexa fluor-conjugated antibodies were used (Invitrogen).\n\nFor confocal analysis, neurons were examined on a confocal LSM510 microscope (Zeiss, B.V. The Netherlands)\n\n**Trafficking analysis**\n\nIn order to analyze the trafficking dynamics of SERT, one minute movie image stacks with 1 s interval between images, were analyzed in the custom written software program Fluotrack (Broeke et al., 2008). To this end, metamorph files (.stk) were converted to .avi files using ImageJ, and analyzed using Fluotrack. Data analysis was performed using the SPSS statistical package. Data shown are mean values ± SEM.\n\n**References**\n\nAdachi N, Kohara K, Tsumoto T (2005) Difference in trafficking of brain-derived neurotrophic factor between axons and dendrites of cortical neurons, revealed by live-cell imaging. BMC Neurosci 6:42.\n\nAnsorge MS, Morelli E, Gingrich JA (2008) Inhibition of serotonin but not norepinephrine transport during development produces delayed, persistent perturbations of emotional behaviours in mice. J Neurosci 28:199-207.\n\nAnsorge MS, Zhou M, Lira A, Hen R, Gingrich JA (2004) Early-life blockade of the 5-HT transporter alters emotional behaviour in adult mice. Science 306:879-881.\n\nBroeke JH, Ge H, Dijkstra IM, Cemgil AT, Riedl JA, Niels Cornelisse L, Toonen RF, Verhage M, Fitzgerald WJ (2008) Automated quantification of cellular traffic in living cells. J Neurosci Methods.\n\nChen NH, Reith ME, Quick MW (2004) Synaptic uptake and beyond: the sodium- and chloride-dependent neurotransmitter transporter family SLC6. Pflugers Arch 447:519-531.\n\nde Jong EK, Vinet J, Stanulovici VS, Meijer M, Wesseling E, Sjollema K, Boddeke HW, Biber K (2008) Expression, transport, and axonal sorting of neuronal CCL21 in large dense-core vesicles. Faseb J 22:4136-4145.\n\nde Wit J, Toonen RF, Verhaagen J, Verhage M (2006) Vesicular trafficking of semaphorin 3A is activity-dependent and differs between axons and dendrites. Traffic 7:1060-1077.\n\nFabre V, Beaufour C, Evrard A, Rioux A, Hanoun N, Lesch KP, Murphy DL, Lanfumey L, Hamon M, Martres MP (2000) Altered expression and functions of serotonin 5-HT1A and 5-HT1B receptors in knock-out mice lacking the 5-HT transporter. Eur J Neurosci 12:2299-2310.\n\nFjorback AW, Pla P, Muller HK, Wiborg O, Saudou F, Nyengaard JR (2009) Serotonin transporter oligomerization documented in RN46A cells and neurons by sensitized acceptor emission FRET and fluorescence lifetime imaging microscopy. Biochem Biophys Res Commun 380:724-728.\n\nFormaggio E, Cantu C, Chiamulera C, Fumagalli GF (2008) p75 neurotrophin receptor distribution and transport in cultured neurons. Neurosci Res 62:32-42.\n\nHolmes A, Murphy DL, Crawley JN (2003a) Abnormal behavioural phenotypes of serotonin transporter knockout mice: parallels with human anxiety and depression. Biol Psychiatry 54:953-959.\n\nHolmes A, Yang RJ, Lesch KP, Crawley JN, Murphy DL (2003b) Mice lacking the serotonin transporter exhibit 5-HT(1A) receptor-mediated abnormalities in tests for anxiety-like behaviour. Neuropsychopharmacology 28:2077-2088.\n\nJust H, Sitte HH, Schmid JA, Freissmuth M, Kudlacek O (2004) Identification of an additional interaction domain in transmembrane domains 11 and 12 that supports oligomer formation in the human serotonin transporter. J Biol Chem 279:6650-6657.\n\nLau T, Horschitz S, Bartsch D, Schloss P (2008a) Monitoring mouse serotonin transporter internalization in stem cell-derived serotonergic neurons by confocal laser scanning microscopy. Neurochem Int.\n\nLau T, Horschitz S, Berger S, Bartsch D, Schloss P (2008b) Antidepressant-induced internalization of the serotonin transporter in serotonergic neurons. Faseb J 22:1702-1714.\n\nLebrand C, Cases O, Adelbrecht C, Doye A, Alvarez C, El Mestikawy S, Seif I, Gaspar P (1996) Transient uptake and storage of serotonin in developing thalamic neurons. Neuron 17:823-835.\n\nLochner JE, Kingma M, Kuhn S, Meliza CD, Cutler B, Sealettar BA (1998) Real-time imaging of the axonal transport of granules containing a tissue plasminogen activator/green fluorescent protein hybrid. Mol Biol Cell 9:2463-2476.\n\nLoder MK, Melikian HE (2003) The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines. J Biol Chem 278:22168-22174.\n\nMaas C, Tagnaouti N, Loebrich S, Behrend B, Lappe-Sieke C, Kneussel M (2006) Neuronal cotransport of glycine receptor and the scaffold protein gephyrin. J Cell Biol 172:441-451.\n\nMathews TA, Fedele DE, Coppelli FM, Avila AM, Murphy DL, Andrews AM (2004) Gene dose-dependent alterations in extraneuronal serotonin but not dopamine in mice with reduced serotonin transporter expression. J Neurosci Methods 140:169-181.\n\nMelikian HE (2004) Neurotransmitter transporter trafficking: endocytosis, recycling, and regulation. Pharmacol Ther 104:17-27.\n\nNarboux-Neme N, Pavone LM, Avallone L, Zhuang X, Gaspar P (2008) Serotonin transporter transgenic (SERTcre) mouse line reveals developmental targets of serotonin specific reuptake inhibitors (SSRIs). Neuropharmacology 55:994-1005.\n\nPickel VM, Chan J (1999) Ultrastructural localization of the serotonin transporter in limbic and motor compartments of the nucleus accumbens. J Neurosci 19:7356-7366.\n\nSchmid JA, Schölze P, Kudlacek O, Freissmuth M, Singer EA, Sitte HH (2001) Oligomerization of the human serotonin transporter and of the rat GABA transporter 1 visualized by fluorescence resonance energy transfer microscopy in living cells. J Biol Chem 276:3805-3810.\n\nShaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY (2004) Improved monomeric red, orange and yellow fluorescent proteins\n\nderived from *Discosoma* sp. red fluorescent protein. Nat Biotechnol 22:1567-1572.\n\nSilverman MA, Johnson S, Gurkins D, Farmer M, Lochner JE, Rosa P, Scalettar BA (2005) Mechanisms of transport and exocytosis of dense-core granules containing tissue plasminogen activator in developing hippocampal neurons. J Neurosci 25:3095-3106.\n\nTao-Cheng JH, Zhou FC (1999) Differential polarization of serotonin transporters in axons versus soma-dendrites: an immunogold electron microscopy study. Neuroscience 94:821-830.\n\nZhou FC, Tao-Cheng JH, Segu L, Patel T, Wang Y (1998) Serotonin transporters are located on the axons beyond the synaptic junctions: anatomical and functional evidence. Brain Res 805:241-254.\n\nZhu CB, Hewlett WA, Feoktistov I, Biaggioni I, Blakely RD (2004) Adenosine receptor, protein kinase G, and p38 mitogen-activated protein kinase-dependent up-regulation of serotonin transporters involves both transporter trafficking and activation. Mol Pharmacol 65:1462-1474.\n\nZhuang X, Masson J, Gingrich JA, Rayport S, Hen R (2005) Targeted gene expression in dopamine and serotonin neurons of the mouse brain. J Neurosci Methods 143:27-32.\n\nChapter 4\n\nPresynaptic localization of tryptophan hydroxylase 2 in mature hippocampal neurons\n\nJ.J. Dudok\\textsuperscript{1}, A.J.A. Groffen\\textsuperscript{1} and M. Verhage\\textsuperscript{1}\n\n\\textsuperscript{1}Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, the Netherlands\n\n\\textit{In preparation}\n\nAbstract\n\nTryptophan hydroxylase (Tph) is the rate limiting enzyme in serotonin (5-HT) synthesis. Two genes named Tph1 and Tph2 synthesize 5-HT in peripheral tissues and the central nervous system, respectively. When expressed in neurons, Tph1 displays a cytoplasmic distribution in the cell body and is associated with microtubules in the axon and dendrites. Axonal Tph1 is associated with 5-HT varicosities. Here, we studied the subcellular distribution of Tph2. To this end we expressed Tph2-EGFP in hippocampal neurons. In young neurons, before synaptogenesis has occurred, Tph2-EGFP displayed a cytoplasmic distribution throughout the neurites. Live cell imaging revealed that Tph2-EGFP is not transported in vesicles. In mature neurons, Tph2-EGFP accumulated in presynaptic terminals of axons, whereas in dendrites it was distributed throughout the cytoplasm, also in spines. In live cell imaging, Tph2-EGFP hardly displayed any movement. These results suggest that Tph2 is freely diffusible in the dendrites and soma, but accumulates at the synaptic terminal by a mechanism that deserves further study.\n\nIntroduction\n\nThe neurons of the 5-HT system are among the first neurons in the brain synthesizing a specific neurotransmitter. Two enzymes are necessary to synthesize 5-HT from its precursor L-tryptophan. Of these, the rate limiting enzyme is Tph which converts L-tryptophan to 5-hydroxy-L-tryptophan. For decades only one Tph isoform was known in the mammalian brain. However, deletion of this gene did not result in a significant decrease in brain 5-HT levels, which led to the discovery of a second Tph gene called Tph2 (Walther et al., 2003). Tph2 is exclusively expressed in the raphe nuclei, whereas Tph1 is expressed in the pineal gland and in the peripheral 5-HT system (Patel et al., 2004; Gutknecht et al., 2009).\n\nSince 5-HT projections are distributed throughout the brain, it is unlikely that Tph2 is only present in the cell body to synthesize 5-HT. Indeed, previous research showed that Tph was present in the cytoplasm of the cell body, and in axons and dendrites where it was associated with microtubules (Joh et al., 1975). Additionally, Tph was associated with 5-HT varicosities, suggesting local synthesis of 5-HT (Pickel et al., 1976; Pickel et al., 1977).\n\nAlthough the subcellular distribution of Tph1 is known, it is currently unknown how Tph is transported to synaptic terminals and whether Tph2 also\n\nlocalizes to presynaptic terminals. Therefore we tagged Tph2 with EGFP, and expressed this chimaeric protein in hippocampal neurons. We show here that in young neurons (before synaptogenesis), Tph2-EGFP is homogenously distributed throughout the neurites. However, in mature neurons, Tph2-EGFP accumulated at presynaptic terminals in the axon. In the dendrites of mature neurons, Tph2-EGFP displayed a homogenous distribution, and also localized to spines. Live cell imaging suggested that Tph2 is a soluble protein and is not transported in vesicles to presynaptic terminals. We conclude that Tph2-EGFP accumulates at presynaptic terminals in mature hippocampal neurons.\n\n**Results**\n\n*Cytoplasmic distribution of Tph2-EGFP in young neurons*\n\nTo study the distribution of Tph2 in neurons, we inserted EGFP at the N-terminus of Tph2 and expressed Tph2-EGFP in hippocampal neurons. First, we started with imaging Tph2-EGFP in neurons at 9 days in vitro (DIV), which are still immature neurons in terms of synapse development (Basarsky et al., 1994) Transfected neurons showed a uniform distribution of Tph2-EGFP through the whole neuron, indicating that Tph2-EGFP is cytoplasmic (Fig. 1A). We did not observe Tph2-EGFP puncta, suggesting that Tph2-EGFP is not transported in\n\n\n\nTph2-EGFP was expressed in young hippocampal neurons before synaptogenesis and imaged at DIV9. (A) Tph2-EGFP displays a cytoplasmic distribution throughout all the neurites. (B) Live cell imaging revealed that Tph2-EGFP is not transported in vesicles, and only little movement is detected. Images are taken 10 seconds apart, first image is green and image 10 seconds later is in red. (C) Zooming in on the axon (arrow in A), as identified by its smaller diameter compared to dendrites shows that already puncta are observed which might be immature synapses or varicosities (arrows). (D) In the dendrites, Tph2-EGFP distribution is cytoplasmic, and Tph2-EGFP also localized to dendritic filopodia. Scale bars: 10 µm in B, 5 µm in C and D.\n\nA\n\nTph2-EGFP\n\nB\n\nSynaptophysin-mCherry\n\nC\n\nMerge\n\nD\n\n*\n\n***\n\nE\n\nF\n\nG\n\nH\n\nI\n\nJ\n\n*\n\n*\n\nK\n\nL\n\nM\n\n1s 30s\n\nN\n\n1s 30s\n\nScale bars: A, B, C = 5 μm; D, E, F = 10 μm; G, H, I = 20 μm; J, K, L = 20 μm; M, N = 10 μm.\n\nNeurons expressing Tph2-EGFP and the presynaptic marker Synaptophysin-mCherry were imaged at DIV23. (A) Tph2-EGFP was expressed in the cytoplasm of dendrites. (B) Expression of Synaptophysin-mCherry in the same neuron. (C) Merged image. (D) Blow up of the white box in C shows that some Tph2-EGFP appears to accumulate in the tip of dendritic spines (asterisks). (E) Synaptophysin-mCherry expression. (F) Merged image. (G) Axon of a neuron expressing Tph2-EGFP. Tph2-EGFP displays a punctate expression, indicative of presynaptic localization. (H) Indeed, Tph2-EGFP co-localizes with Synaptophysin-mCherry. (I) Merged image. (J) A EGFP expressing neuron as a control shows that EGFP is homogenously distributed in the dendrites (asterisks) and in the axon as identified by the absence of MAP2 staining (arrows). (K) MAP2 staining. (L) Merged image. (M) Live cell imaging revealed that Tph2-EGFP is static. Images are taken 30 seconds apart, with the green image after 1 second, and the red image after 30 seconds. (N) Blow up of the white box in J shows that some dendrites which contain Tph2-EGFP displayed some movement (arrows). Scale bars: 10 µm in C, 5 µm in F, 10 µm in I, 10 µm in L, 10 µm in M and 5 µm in N.\n\nvesicles, but instead is a soluble protein. Live cell imaging revealed that Tph2-EGFP is rather static, and no Tph2-EGFP puncta or other movements were observed (Fig. 1B). The axon, as identified by its smaller diameter and larger length as compared to dendrites, also had a uniform distribution of Tph2-EGFP, although some Tph2-EGFP appeared to accumulate in puncta along the axon (arrows in Fig. 1C). As in axons, in dendrites Tph2-EGFP displayed a uniform distribution and also filopodia were labelled (Fig. 1D).\n\n**Tph2-EGFP localizes to presynaptic terminals in mature neurons**\n\nNext, we investigated the distribution of Tph2-EGFP in mature neurons after synaptogenesis had occurred. To this end we transfected hippocampal neurons with Tph2-EGFP at DIV18 and imaged Tph2-EGFP at DIV23. Tph2-EGFP was uniformly distributed in the cell body, dendrites and dendritic spines (Fig. 2A-C). We expressed EGFP in neurons as a control to compare the distribution of EGFP with Tph2-EGFP (Fig. 2J-L). In contrast to EGFP, which labels the whole dendritic spine, it appeared that in neurons transfected with Tph2-EGFP in some spines Tph2-EGFP accumulated in the tip of the spine, suggesting that Tph2-EGFP was enriched in postsynaptic structures (Fig. 2D-F). We co-transfected Tph2-EGFP with Synaptophysin-mCherry, which is a marker for synaptic vesicles and presynaptic terminals (Jahn et al., 1985; Granseth et al., 2006). In the axon, identified by the punctate labelling of Synaptophysin-mCherry, Tph2-EGFP displayed a punctate distribution (Fig. 2G). The Tph2-EGFP puncta co-localized with Synaptophysin-mCherry puncta, strongly suggesting that Tph2-EGFP accumulated at presynaptic terminals (Fig. 2H,I). In neurons transfected\n\nwith EGFP as a control, EGFP displayed a uniform distribution throughout the axon (Fig. 2J-L). Next, we imaged the neurons expressing Tph2-EGFP during 1 minute. Hardly any Tph2-EGFP movement could be observed (Fig. 2M). The only movement which could be observed was some motility in postsynaptic spines (Fig. 2N).\n\nTo further test if Tph2-EGFP is localized to presynaptic terminals in mature neurons, we fixed neurons expressing Tph2-EGFP at DIV23 and performed immunocytochemistry for the synaptic marker Synapsin and the dendritic marker MAP2 to discriminate dendrites and axons (Fig. 3A-C). In the axon, as identified by the absence of MAP2 labelling, Tph2-EGFP displayed a punctate distribution (Fig. 3D-F). Immunocytochemistry for Synapsin showed that in dendrites of neurons expressing Tph2-EGFP, spines were labelled, and some spine heads were localized in close proximity with Synapsin puncta, suggesting that this is a synapse (Fig. 3G-I). Finally, we focussed on the axon of a neuron expressing Tph2-EGFP which we stained for Synapsin. The boutons with Tph2-EGFP co-localized with Synapsin, strongly suggesting that Tph2-EGFP localized to presynaptic terminals (Fig. 3J-L).\n\n**Discussion**\n\nWe have shown here that Tph2, the rate limiting enzyme in 5-HT synthesis localized to presynaptic terminals in mature hippocampal neurons. Moreover, in dendrites and in the cell body Tph2-EGFP had a cytoplasmic distribution and appeared to accumulate in the tip of some dendritic spines. Thus, it appears that Tph2-EGFP displayed a polarized distribution in mature hippocampal neurons. Live cell imaging revealed that hardly any Tph2-EGFP movement could be observed, and no Tph2-EGFP vesicles were observed, indicating that Tph2 is a soluble protein. Accordingly, Tyrosine hydroxylase, an enzyme related to Tph2, also was shown to be a soluble protein (Mockus et al., 1997). Electron microscope studies on Tph and TH localization revealed that these proteins were enriched on presynaptic terminals, and were associated with microtubules in somatodendritic compartments (Pickel et al., 1976). Therefore, Tph2-EGFP seems to mimic the endogenous distribution of Tph in 5-HT neurons.\n\nTph2-EGFP displayed a polarized distribution in mature hippocampal neurons, i.e. in dendrites the protein is distributed throughout the cytoplasm, whereas in the axon the protein accumulated in presynaptic terminals. This suggests that in presynaptic terminals and in 5-HT varicosities, there is local synthesis of 5-HT. In dendrites, Tph2-EGFP localized to spines, which prompts the question whether 5-HT could be synthesized in dendrites.\n\nFigure 3. Immunocytochemistry on a mature neuron expressing Tph2-EGFP\n\nNeurons (DIV23) expressing Tph2-EGFP were fixed and immunocytochemistry was performed for MAP2 and Synapsin. (A) Tph2-EGFP expression. (B) MAP2 immunostaining to distinguish between dendrites and the axon. (C) Merged image. (D) Blow up of the white box in C shows a dendrite (asterisk) which has Tph2-EGFP through the whole cytoplasm, and the axon (arrows) in which Tph2-EGFP displays a punctate distribution. (E) MAP2 staining. (F) Merged image. (G) Tph2-EGFP expressing neurons were stained for the presynaptic marker Synapsin. (H) Synapsin staining. (I) Merged image, which shows that in the close proximity of some dendritic spines there is a Synapsin puncta, suggesting that these are true synapses (asterisks). (J) Distribution of Tph2-EGFP in an axon. (K) Synapsin staining. (L) Tph2-EGFP puncta were positive for Synapsin (arrows), showing that these are presynaptic terminals. Scale bars: 20 µm in C, 10 µm in F, 5 µm in I and L.\n\nTph is localized in dendrites, where it associated with microtubules (Joh et al., 1975). Correspondingly, 5-HT vesicles are detected in dendrites, either associated with a synaptic specialization or with the membrane (Chazal and Ralston, 1987). After stimulation of Retzius neurons in the leech, which contain large dense core vesicles (LDCVs) filled with 5-HT, several vesicles are localized close to the cell membrane as studied by electron microscopy (Trueta et al.,\n\nTherefore, this strongly suggests that 5-HT release can occur from the somatodendritic compartment. Our results indicate that 5-HT can also be synthesized in the somatodendritic compartment.\n\nTph2-EGFP was not associated with vesicles and the protein interactions for its presynaptic accumulation remain unclear. Tph is associated with microtubules, suggesting it could interact with molecular motors of the dynein and kinesin family which move along microtubules (Hirokawa and Takemura, 2005). For Tph2, several polymorphisms are known, some of which associate with neuropsychiatric conditions (Zhang et al., 2006). Both in humans and in mice a polymorphism in the Tph2 gene is found which results in a marked reduction in Tph2 activity (Zhang et al., 2004; Zhang et al., 2005). Finally, it would be interesting to investigate whether these polymorphisms not only affect Tph2 activity, but could also affect Tph2 transport and localization.\n\n**EXPERIMENTAL PROCEDURES**\n\n*Plasmids*\n\nTph2 cDNA in pCR4-TOPO vector was obtained from Geneservice. To clone Tph2 in an expression vector, EGFP was digested using PstI and cloned in the PstI site of pBiotag-EGFP-C1. Synaptophysin-mCherry was a kind gift from Dr. A. Jeromin (Allen Brain Institute).\n\n*Neuronal culturing and transfection*\n\nHippocampi from embryonic day 18 C57/Bl6 mouse embryos were dissected free of meninges and collected in Hanks’ Balanced Salt Solution (HBSS; Invitrogen) supplemented with 7 mM HEPES (Invitrogen). Hippocampi were digested with 0.25% trypsin (Invitrogen) in HBSS supplemented with 7 mM HEPES for 15 minutes at 37°C. After digestion tissue was washed three times with HBSS supplemented with 7 mM Hepes and subsequently triturated with a fire-polished glass pipette. Dissociated neurons were plated at a density of 25,000 cell/well on rat glial cells on 18 mm glass coverslips (Menzel Glaser, Braunschweig, Germany) in 12 well plates. Cells were cultured in neurobasal medium supplemented with B27 supplement, 18 mM HEPES, 0.5 mM Glutamax and penicillin/streptomycin (Invitrogen). Once a week half of the medium was replaced. The hippocampal cultures were transfected using Lipofectamine 2000 (Invitrogen). Before transfection, half of the neurobasal medium was collected from each well and replaced with new neurobasal medium. For one well, 1 µl of Lipofectamine was added to 25 µl of serum free glutamax medium and incubated for five minutes. Subsequently, from each construct 1 µg of DNA was added to 25 µl of serum free glutamax and immediately mixed with the 25 µl of serum free\n\nglutamax containing Lipofectamine. This mixture was incubated for 30 minutes before adding to the cells. After 4-6 hours of incubation on the cells, half of the neurobasal medium was replaced again with the earlier collected neurobasal medium. Cells were routinely transfected at DIV2-7 and DIV18 and imaged at DIV10-14 and DIV23.\n\n**Live cell imaging**\n\nCells were imaged in a chamber perfused with Tyrode’s solution (119 mM NaCl, 2.5 mM KCl, 2 mM MgCl$_2$, 2 mM CaCl$_2$, 30 mM glucose, 25 mM HEPES, pH 7.4) on an Axiovert II Microscope (Zeiss, Oberkochen, Germany) equipped with a Coolsnap HQ camera (Photometrics, Tucson, Arizona, USA) and a Polychrome IV illumination unit (TILL photonics, Grafelfing, Germany). Images were acquired in MetaMorph 6.2 software (Universal Imaging, Downingtown, Philadelphia, USA) using a 100x oil objective.\n\n**Immunocytochemistry**\n\nFor immunocytochemistry, primary antibodies against MAP2 (monoclonal, Chemicon) and synapsin (polyclonal Eo28) were used in a 1:1000 dilution. As secondary antibodies, goat-anti-mouse Alexa488 and goat-anti-rabbit Alexa 543 were used (Invitrogen). To perform immunocytochemistry, cells were incubated in 4% paraformaldehyde dissolved in phosphate buffered saline (PBS, pH 7.4), for 20 minutes. Subsequently, cells were washed two times with PBS and incubated in PBS containing 0.2% Triton X-100 and 4% fetal calf serum for 20 minutes. Cells were incubated in primary antibody in PBS containing 0.2% Triton X-100 for one hour, washed three times with PBS and incubated in the secondary antibody in PBS for one hour. After washing the cells three times with PBS, coverslips were mounted using Dabco-Mowiol. All reactions were performed at room temperature. Cells were analyzed on a confocal LSM510 microscope (Zeiss, B.V. The Netherlands) using a 63x oil immersion lens and appropriate lasers and filters to visualize EGFP fluorescence and secondary antibodies.\n\n**References**\n\nBasarsky TA, Parpura V, Haydon PG (1994) Hippocampal synaptogenesis in cell culture: developmental time course of synapse formation, calcium influx, and synaptic protein distribution. J Neurosci 14:6402–6411.\n\nChazal G, Ralston HJ, 3rd (1987) Serotonin-containing structures in the nucleus raphe dorsalis of the cat: an ultrastructural analysis of dendrites, presynaptic dendrites, and axon terminals. J Comp Neurol 259:317–329.\n\nGranseth B, Odermatt B, Royle SJ, Lagnado L (2006) Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron 51:773–786.\n\nGutknecht L, Kriegebaum C, Waider J, Schmitt A, Lesch KP (2009) Spatio-temporal expression of tryptophan hydroxylase isoforms in murine and human brain: convergent data from Tph2 knockout mice. Eur Neuropsychopharmacol 19:266–282.\n\nHirokawa N, Takemura R (2005) Molecular motors and mechanisms of directional transport in neurons. Nat Rev Neurosci 6:201-214.\n\nJahn R, Schiebler W, Ouimet C, Greengard P (1985) A 38,000-dalton membrane protein (p38) present in synaptic vesicles. Proc Natl Acad Sci U S A 82:4137-4141.\n\nJoh TH, Shikimi T, Pickel VM, Reis DJ (1975) Brain tryptophan hydroxylase: purification of, production of antibodies to, and cellular and ultrastructural localization in serotonergic neurons of rat midbrain. Proc Natl Acad Sci U S A 72:3575-3579.\n\nMockus SM, Kumer SC, Vrana KE (1997) A chimeric tyrosine/tryptophan hydroxylase. The tyrosine hydroxylase regulatory domain serves to stabilize enzyme activity. J Mol Neurosci 9:35-48.\n\nPatel PD, Pontrello C, Burke S (2004) Robust and tissue-specific expression of TPH2 versus TPH1 in rat raphe and pineal gland. Biol Psychiatry 55:428-433.\n\nPickel VM, Joh TH, Reis DJ (1976) Monoamine-synthesizing enzymes in central dopaminergic, noradrenergic and serotonergic neurons. Immunocytochemical localization by light and electron microscopy. J Histochem Cytochem 24:792-306.\n\nPickel VM, Joh TH, Reis DJ (1977) A serotonergic innervation of noradrenergic neurons in nucleus locus coeruleus: demonstration by immunocytochemical localization of the transmitter specific enzymes tyrosine and tryptophan hydroxylase. Brain Res 131:197-214.\n\nTrueta C, Sanchez-Armass S, Morales MA, De-Miguel FF (2004) Calcium-induced calcium release contributes to somatic secretion of serotonin in leech Retzius neurons. J Neurobiol 61:309-316.\n\nWalther DJ, Peter JU, Bashammakh S, Hortnagl H, Voits M, Fink H, Bader M (2003) Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science 299:76.\n\nZhang X, Beaulieu JM, Gainetdinov RR, Caron MG (2006) Functional polymorphisms of the brain serotonin synthesizing enzyme tryptophan hydroxylase-2. Cell Mol Life Sci 63:6-11.\n\nZhang X, Beaulieu JM, Sotnikova TD, Gainetdinov RR, Caron MG (2004) Tryptophan hydroxylase-2 controls brain serotonin synthesis. Science 305:217.\n\nZhang X, Gainetdinov RR, Beaulieu JM, Sotnikova TD, Burch LH, Williams RB, Schwartz DA, Krishnan KR, Caron MG (2005) Loss-of-function mutation in tryptophan hydroxylase-2 identified in unipolar major depression. Neuron 45:11-16.\n\nChapter 5\n\nThe effect of a polymorphism in Piccolo on localization and trafficking dynamics of the serotonin reuptake transporter\n\nJ.J. Dudok\\textsuperscript{1}, A.J.A. Groffen\\textsuperscript{1} and M. Verhage\\textsuperscript{1}\n\n\\textsuperscript{1}Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, the Netherlands\n\nAbstract\n\nThe active zone protein Piccolo is involved in formation of synapses and stabilization of existing synapses. Recently, a short nucleotide polymorphism (SNP) in the C2A domain of Piccolo was found in association with depression. In this study we addressed whether knockdown of Piccolo affected the localization and trafficking of the serotonin reuptake transporter (SERT) in hippocampal neurons. We co-expressed a short nucleotide RNA which resulted in an efficient knockdown of Piccolo with mCherry-SERT to evaluate the effect of knockdown of Piccolo on SERT localization and trafficking in hippocampal neurons. We found that knockdown of Piccolo did not alter SERT trafficking dynamics. Subsequently, we expressed the two variants of the Piccolo C2A domain together with SERT in heterologous cells. SERT displayed a uniform distribution at the membrane upon co-expression of the wildtype C2A variant. However, upon co-expression of the mutant C2A variant, SERT displayed a more patchy distribution at the membrane, indicating that membrane levels of SERT might be decreased. These data suggest that knockdown of Piccolo does not alter SERT trafficking dynamics, but the mutant variant of the Piccolo C2A domain might alter SERT membrane localization.\n\nIntroduction\n\nMajor depression is a neuropsychiatric disorder affecting millions of people world wide each year. The serotonin (5-HT) system is implicated in depression. An important protein in the 5-HT system is the SERT which regulates the transport of 5-HT back into the synapse after its release, thus regulating the extracellular 5-HT concentration. Therefore, SERT is an important factor in determining efficacy of 5-HT neurotransmission. Several polymorphisms have been described in SERT which associate with psychopathological processes such as depression, anxiety, aggression and autism (Murphy et al., 2004; Serretti et al., 2006).\n\nSeveral decades ago it was hypothesized that psychopathological processes such as depression and anxiety were the result of an alteration in extracellular 5-HT content. This hypothesis, the so-called monoamine hypothesis, was based on the fact that blockade of the SERT by selective 5-HT reuptake inhibitors (SSRIs) resulted in an alleviation in depressive symptoms, possibly due to the increase in extracellular 5-HT levels. However, between starting SSRI treatment and alleviation of depressive symptoms was a time period of 3-4 weeks, whereas 5-\n\nHT levels increased immediately after SSRI intake. Therefore the monoamine hypothesis was abandoned and shifted towards the so-called network hypothesis, which stated that psychopathological processes such as anxiety and depression could be the result of an altered 5-HT network connectivity (Castren, 2005). The evidence for this hypothesis was based on human imaging studies which showed that humans with depression had a reduced grey matter volume in cortex and hippocampus. However, evidence for this hypothesis on a cellular level is still lacking. Recently, a SNP in the presynaptic gene Piccolo was found which might be associated with major depressive disorder (Sullivan et al., 2008). Therefore, this revitalizes the network hypothesis for depression and opens up new opportunities to study the relation between the presynaptic terminal, proper formation of networks, and the relation between 5-HT network (formation) and psychopathological processes such as depression.\n\nPiccolo is a 420 kDa protein which is enriched in presynaptic terminals where it is a structural component of the presynaptic cytoskeletal cytomatrix (PCM) (Cases-Langhoff et al., 1996). Piccolo is a multidomain zinc finger protein which is structurally related to Bassoon, another component of the PCM (tom Dieck et al., 1998; Fenster et al., 2000). Piccolo contains several coiled coil domains, a PDZ domain and two C2 domains (Fenster and Garner, 2002). The C2A domain functions as a low-affinity calcium sensor in presynaptic terminals (Gerber et al., 2001). Piccolo and Bassoon, along with other presynaptic constituents, are transported in 80 nm large dense core vesicles (LDCVs) to nascent synapses, and 2 or 3 of these vesicles are sufficient to form an active synapse (Zhai et al., 2001; Shapira et al., 2003; Tao-Cheng, 2007). The recent described nonsynonymous SNP rs2522833 in Piccolo encodes a common variation in the C2A domain, where a serine is substituted for an alanine (Sullivan et al., 2008).\n\nPiccolo is involved in the regulation of the internalization of the dopamine transporter (DAT) in heterologous cells. Specifically, expression of the C2A domain of Piccolo reduces methamphetamine-induced DAT internalization. (Cen et al., 2008). DAT and SERT are structurally related proteins, which belong to the solute carrier superfamily. It could be likely that membrane incorporation and internalization of these proteins is regulated in the same manner. Additionally, Piccolo influences synaptic function by negatively regulating synaptic vesicle exocytosis (Leal-Ortiz et al., 2008). These data led us to hypothesize that possibly, Piccolo might contribute to the overall risk of depression by affecting the membrane incorporation of SERT.\n\nTo test this hypothesis we expressed mCherry-SERT in hippocampal neurons and investigated whether knockdown of Piccolo or the variation rs2522833 affected the localization and trafficking of SERT. We show here that\n\nknockdown of Piccolo in neurons did not affect SERT trafficking. However, expression of the Piccolo C2A domain variation rs2522833 in heterologous cells might affect membrane localization of SERT.\n\nResults\n\n*Piccolo localized to neurotransmitter release sites in hippocampal and 5-HT neurons*\n\nFirst, we analyzed the cellular localization of Piccolo in hippocampal neurons and 5-HT neurons. Hippocampal neurons (14 days in vitro (DIV)) were fixed and immunocytochemistry was performed for Piccolo and for the presynaptic terminal marker Synaptobrevin-2 (VAMP2). Piccolo immunoreactivity was distributed in a punctate pattern, indicative for labeling of presynaptic terminals (Fig. 1A). Indeed, the majority of Piccolo puncta co-localized with VAMP2 puncta, showing that these represented presynaptic terminals (Fig. 1B,C). Zooming in on part of a neurite showed that >90% of Piccolo puncta co-localized with VAMP2 (Fig. 1D-F). Thus, Piccolo was present in presynaptic terminals.\n\nNext, we investigated the localization of Piccolo in 5-HT neurons. We used a coronal brain slice from an adult mouse through the dorsal raphe nucleus (DRN), the region in the brain where the majority of 5-HT neurons is localized. We performed immunocytochemistry for tryptophan hydroxylase (Tph), the rate limiting enzyme for 5-HT synthesis and a marker for the 5-HT system. In a DRN slice, several 5-HT neurons were present (Fig. 1G). Immunocytochemistry for Piccolo resulted in a punctate labeling in the DRN slice (Fig. 1H). Piccolo staining was barely detectable in the 5-HT cell bodies (Fig. 1I). However, Piccolo puncta co-localized with Tph puncta. Tph is present in varicosities and presynaptic terminals (chapter 4 of this thesis) (Pickel et al., 1976).\n\n**Figure 1 next page. Piccolo localizes to presynaptic terminals in neurons**\n\n(A-C) Hippocampal neurons were fixed at DIV14, and immunocytochemistry was performed for Piccolo (A) and the presynaptic marker VAMP2 (B). (C) Merged image shows that several Piccolo puncta co-localized with VAMP2 puncta. (D-F) Blow up of the white box in C shows that almost every Piccolo puncta co-localizes with a VAMP2 puncta. (D) Piccolo staining. (E) VAMP2 staining. (F) Merged image. (G) A coronal brain slice through the DRN from an adult mouse was stained against the 5-HT marker Tph. This shows that 5-HT cell bodies and fibers are present. (H) Piccolo staining on this slice shows that Piccolo expression is low in 5-HT cell bodies. (I) Merged image. (J) Blow up of the white box in I shows a 5-HT cell body and several Tph puncta, which might be 5-HT varicosities. (K) Piccolo staining shows that in several of the Tph puncta Piccolo is present. (L) Merged image. Scale bars: 20 µm in C and I, 10 µm in F and L.\n\nThese Tph puncta might therefore represent 5-HT varicosities, axonal sites where 5-HT release occurs, often without a post-synaptic specialization. Therefore, it seems that also in 5-HT neurons Piccolo is localized in release sites (Fig 1J-L).\n\n**Efficient knockdown of Piccolo in hippocampal neurons**\n\nNext, we investigated whether knockdown of Piccolo affected trafficking of mCherry-SERT. In hippocampal neurons, mCherry-SERT is transported in secretory vesicles which move through the axon with no preference for direction of movement (Chapter 3, this thesis). To achieve efficient knockdown of Piccolo,\n\nwe designed six short hairpin RNA (shRNA) nucleotides, targeted towards the N-terminus of Piccolo. These short hairpins were inserted in a vector containing also an internal ribosomal entry site (IRES) and EGFP, as a control for transfected neurons. We tested the shRNAs for their Piccolo knockdown efficiency. To this end we transfected the shRNA constructs in hippocampal neurons, and four days later fixed and processed the neurons for immunocytochemistry. Several constructs tested did not show any knockdown of Piccolo. We decided to use shRNA#5 as a negative control. Analysis of Piccolo expression in MAP2 negative axons of neurons transfected with shRNA#5 was not reduced compared to untransfected cells (Fig. 2A-D). The axon had a punctate labeling of Piccolo, which was indistinguishable from untransfected neurons. On the other hand, transfection of shRNA#6 caused a reduction in levels of Piccolo in axons (Fig. 2E-H, 2I-L).\n\nQuantification of the number of Piccolo puncta per μm neurite revealed that in control condition there was on average one Piccolo puncta per two μm neurite, whereas in shRNA#6 mediated knockdown there was on average one Piccolo puncta per ten μm neurite (control $0.49 \\pm 0.11$ Piccolo puncta per μm axon; knockdown $0.11 \\pm 0.02$ Piccolo puncta per μm axon, $p=0.002$) (Fig. 2M). Thus, shRNA#6 resulted in an efficient, approximately 80%, reduction in number of Piccolo puncta. Therefore, we used this construct to test the effect of Piccolo knockdown on mCherry-SERT localization and trafficking.\n\n**Knockdown of Piccolo does not affect SERT trafficking**\n\nWe used shRNA#6 to investigate the effect of Piccolo knockdown on mCherry-SERT localization and trafficking. SERT tagged with mCherry is transported in secretory vesicles, which move in anterograde and retrograde direction, with no preference for direction of movement (chapter 3 of this thesis). We transfected mCherry-SERT and shRNA#6 in hippocampal neurons, and used EGFP as a control. One minute movies of mCherry-SERT trafficking were made, and the movies were analyzed using the custom written Fluotrack software program (Broeke et al., 2008). The velocity of moving puncta was quantified, as well as the direction of movement. For direction we discerned anterograde direction, retrograde direction and bidirectional, vesicles which changed direction during imaging. Both in neurons expressing shRNA#6 and EGFP, we observed several puncta of mCherry-SERT, which displayed rapid movements in both the anterograde and retrograde direction. Additionally, also stationary vesicles were observed (Fig. 3A-D). We quantified the average velocity of mCherry-SERT moving puncta in control neurons expressing EGFP, and neurons expressing shRNA#6. A velocity histogram shows the distribution of the average velocity and direction of movement between control and knockdown (Fig. 3E).\n\nFigure 2. Efficient shRNA-mediated knockdown of Piccolo\n\nSeveral shRNA constructs against Piccolo were tested for their efficacy to reduce Piccolo expression in cultured hippocampal neurons. ShRNA#5 did not result in Piccolo knockdown. (A) EGFP expression, showing that the shRNA construct is expressed. (B) Piccolo staining. (C) Neurite is MAP2 negative, showing that this is an axon. (D) Merged image. (E) EGFP expression in a neuron transfected with shRNA#6-EGFP. (F) Piccolo staining. (G) MAP2 staining. (H) Merged image, showing that shRNA#6 results in efficient knockdown of Piccolo in the axon. (I-L) Blow up of the white box in H, with EGFP, Piccolo staining, MAP2 staining and merged image respectively. (M) Quantification of the level of Piccolo knockdown shows that shRNA#6 resulted in a ~80% reduction in Piccolo puncta (control $0.49 \\pm 0.11$ Piccolo puncta per $\\mu m$ axon ($n=7$); knockdown $0.11 \\pm 0.02$ Piccolo puncta per $\\mu m$ axon ($n=8$)). Scale bars: 5 $\\mu m$ in D and L, 10 $\\mu m$ in H.\n\nTrafficking of mCherry-SERT did not differ between control and knockdown (average velocity control 1.30 ± 0.042 µm/s (n=8 neurons); knockdown 1.36 ± 0.039 µm/s, (n=18 neurons) (Fig. 3F). Also, percentage of moving vesicles was not different between control and knockdown (control 46.69 ± 5.03%; knockdown 39.88 ± 3.39%) (Fig. 3G). Additionally, percentage of vesicles moving anterogradely, retrogradely or bidirectionally did not differ between control and knockdown (data not shown).\n\nFigure 3 previous page. Piccolo knockdown does not affect SERT trafficking dynamics\n\n(A) Neurons transfected with EGFP and mCherry-SERT. Images are taken 5 seconds apart. Several mCherry-SERT puncta are observed which are either stationary (yellow), or are moving anterogradely or retrogradely. (B) A neuron transfected with shRNA#6EGFP and mCherry-SERT. Images taken 5 seconds apart show that after Piccolo knockdown also several mCherry-SERT puncta are observed, some of which are stationary, and other which display anterograde or retrograde movements. (C) and (D) are blow ups of the white boxes in A and B, respectively. (E) Velocity histogram of mCherry-SERT puncta in control neurons and Piccolo knockdown neurons. Negative values are retrograde movements, positive values are anterograde movements. (F) Average velocity of mCherry-SERT puncta in control neurons and Piccolo knockdown neurons. (G) Percentage moving mCherry-SERT puncta in control neurons and Piccolo knockdown neurons. Scale bars: 10 µm in A and B, 5 µm in C and D.\n\nExpression of C2A variant rs2522833 alters SERT distribution at the membrane in heterologous cells\n\nFinally, we were interested whether the SNP in the C2A domain of Piccolo which was found to associate with major depression, affected the membrane localization of SERT. Therefore, we constructed the C2A\\textsubscript{wt} domain of Piccolo in an IRES2-EGFP vector and a C2A\\textsubscript{mut} domain with the rs2522833 SNP which results in a serine to alanine substitution at amino acid 4684. We transfected these constructs, together with mCherry-SERT, in heterologous Neuro2A cells, which do not express endogenous Piccolo. In Neuro2A cells transfected with mCherry-SERT together with Piccolo C2A\\textsubscript{wt}, SERT was localized predominantly at the cell membrane. The majority of cells displayed this SERT distribution, although also in some cells mCherry-SERT was homogenously distributed. This may be due to the level of expression, since in several cells which showed this mCherry-SERT distribution, also EGFP fluorescence intensity was increased compared to other cells (Fig. 4A-C). Zooming in on a few cells expressing EGFP and mCherry-SERT showed that SERT is localized at the membrane, as mCherry fluorescence appeared like a ring at the membrane. Moreover, mCherry-SERT also seemed to associate with intracellular organelles, as some mCherry-SERT appeared as puncta in the cytoplasm. (Fig. 4D-F). Expressing mCherry-SERT together with Piccolo C2A\\textsubscript{mut} resulted in cells in which mCherry-SERT was localized at the membrane, but also some cells in which mCherry-SERT displayed a more uniform distribution in the cytoplasm which might again be due to the level of expression (Fig. 4G-I). A typical example of three cells expressing both constructs showed that in one cell mCherry-SERT was localized at the membrane, and in the other cells mCherry-SERT displayed a more uniform distribution in the cytoplasm (Fig. 4J-L). Strikingly, membrane bound mCherry-SERT in Piccolo C2A\\textsubscript{mut} expressing cells displayed a more patchy\n\ndistribution than in Piccolo C2Awt expressing cells (Fig. 4M-O). A line scan profile of a typical cell co-expressing mCherry-SERT with Piccolo C2Awt showed that the highest level of fluorescence was at the membrane (Fig. 4P). In contrast, a line scan profile of a cell co-expressing mCherry-SERT with Piccolo C2Amut showed that fluorescence levels in the cytoplasm were comparable with fluorescence levels at the membrane (Fig. 4Q).\n\nThus, it appeared that co-expression of the Piccolo C2Amut with the rs2522833 SNP resulted in an altered distribution of mCherry-SERT at the membrane. These data suggest that Piccolo is not involved in the trafficking of SERT, but might be involved in regulating SERT membrane levels.\n\n**Discussion**\n\nPiccolo is a large protein, which is part of the active zone in the presynaptic terminal. There, Piccolo probably has a scaffolding role, but Piccolo also contains a PDZ domain and two C2 domains. Piccolo can be alternatively spliced, resulting in absence or presence of the C2B domain (Wang et al., 1999). Additionally, in the C2A domain a nine-residue sequence has been found which is subject of alternative splicing, resulting in variants of the C2A domain with different calcium affinities (Garcia et al., 2004). In this study we focused exclusively on the long C2A variant. The gene region in which Piccolo is localized has been linked to autism (Nabi et al., 2003). The SNP rs2522833 is localized in the C2A domain (Sullivan et al., 2008). The C2A domain has a low calcium affinity, suggesting an involvement in presynaptic plasticity processes (Gerber et al., 2001). It is currently unknown whether the SNP affects PIP$_2$ or Ca$^{2+}$ binding to the C2A domain.\n\n*Figure 4 next page. Expression of Piccolo C2Awt and C2Amut with mCherry-SERT in heterologous cells*\n\n(A-C) Expression of C2Awt–IRES-EGFP and mCherry-SERT in Neuro2A cells. (D-F) Blow up of white box in C shows that in the majority of cells mCherry-SERT is localized at the membrane. In the cytoplasm, mCherry-SERT displays a punctate distribution, suggesting association with intracellular organelles. (G-I) Expression of C2Amut–IRES-EGFP and mCherry-SERT in heterologous cells. (J-L) Blow up of white box #1 in I shows that in several Neuro2A cells expressing C2Amut–IRES-EGFP, mCherry-SERT seems to have a more uniform cytoplasmic distribution. (M-O) Blow up of white box #2 in I shows that in cells in which mCherry-SERT is localized at the membrane, mCherry-SERT is not uniformly distributed at the membrane, but instead is distributed in patches along the membrane. (P,Q) Line scan profiles of a typical cell expressing mCherry-SERT with C2Awt–IRES-EGFP or C2Amut–IRES-EGFP (lines in figures E and K respectively). Arrows indicate cell membrane. Scale bars: 20 µm in C and I, 10 µm in F and L and 5 µm in O.\n\n**EGFP**\n\n- **A**: EGFP expression in cells.\n- **B**: mCherry-SERT expression in cells.\n- **C**: Merge of EGFP and mCherry-SERT expressions.\n\n**C2Awt-IRESGFP**\n\n- **D**: EGFP expression in cells.\n- **E**: mCherry-SERT expression in cells.\n- **F**: Merge of EGFP and mCherry-SERT expressions.\n\n**C2Amut-IRESGFP**\n\n- **G**: EGFP expression in cells.\n- **H**: mCherry-SERT expression in cells.\n- **I**: Merge of EGFP and mCherry-SERT expressions.\n\n**P**: Fluorescence intensity profile across a cell with a nucleus.\n\n**Q**: Fluorescence intensity profile across a cell without a nucleus.\n\nThe regulation of SERT membrane localization and internalization is important in determining efficacy of 5-HT transmission. Increases in membrane localization of SERT increase 5-HT reuptake and thereby reduce extracellular levels of 5-HT and binding of 5-HT to postsynaptic 5-HT receptors. In contrast, internalization of SERT results in increased extracellular levels of 5-HT and increased postsynaptic activation of 5-HT receptors.\n\nProtein kinase C (PKC) reduces the expression level of SERT, presumably by altering the membrane localization of SERT (Qian et al., 1997). Indeed, application of phorbol esters results in increased internalization of SERT. In contrast, substrates of SERT, such as 5-HT, reduce PKC dependent SERT internalization, whereas SERT antagonists, such as antidepressants, block this 5-HT mediated reduction in SERT internalization (Ramamoorthy and Blakely, 1999). On the other hand, protein kinase G (PKG) activity increases the membrane localization of SERT (Zhu et al., 2004). Therefore, there is an extensive level of regulation of membrane expression of SERT. There is an indirect link between Piccolo and SERT; Syntaxin1A is known to interact with SERT, and to regulate SERT activity (Quick, 2003). The C2A domain of Synaptotagmin, which shares similarity with the C2A domain of Piccolo, interacts with Syntaxin1A (Shao et al., 1997). Therefore this makes it likely that also the C2A domain of Piccolo is able to interact with Syntaxin1A, and thus possibly with SERT.\n\nWe used heterologous Neuro2A cells, which do not express Piccolo endogenously, to study the effect of expression of both C2A variants on SERT localization. We showed that distribution of SERT at the membrane might be altered after co-expression with the mutant C2A variant. Previously it has been shown that DAT trafficking and internalization is not altered in heterologous cells upon Piccolo knockdown. However, upon treatment of cells with 3,4-methylenedioxy-N-methylamphetamine (MDMA), there is a significant increase in DAT internalization. Thus, Piccolo seems to regulate the MDMA dependent internalization of DAT. For SERT, it has been shown that application of antidepressants and the PKC activator β-phorbol 12-myristate 13-acetate (β-PMA) results in an increased internalization of SERT (Lau et al., 2008a; Lau et al., 2008b). In view of this, it would be interesting to challenge SERT with an antidepressant after expression of both C2A variants, to investigate whether this results in an altered distribution of SERT between the two C2A variants.\n\nSeveral studies show that the expression level, membrane level, or transport activity of SERT might be affected in psychopathological processes. Levels of SERT expression in DRN are reduced in brains of depressed suicide victims (Arango et al., 2001). In contrast, long-term administration of antidepressants seemed to also reduce SERT surface expression (Hirano et al., 2005). Recent\n\nstudies showed that a SNP in SERT which associated with obsessive compulsive disorder resulted in increased transport activity of SERT (Kilic et al., 2003). Thus, further studies are required to investigate the relation between the SNP in Piccolo, SERT expression and activity, and major depression disorder.\n\nEXPERIMENTAL PROCEDURES\n\nPlasmids\n\nHuman SERT (hSERT) cDNA tagged with ECFP at the 5' end in the Clontech ECFP-C1 vector was a kind gift from Dr. H. Sitte. To clone a red fluorescent version of hSERT we replaced ECFP for the red fluorescent protein mCherry. To this end mCherry was digested out of the vector using AseI and BsrGI and cloned sticky in the ECFP-hSERT vector from which ECFP had been excised. For construction of pSuper-CMV-EGFP-shRNA-Piccolo, pSuper was digested using EcoRI and PstI and CMV-EGFP was inserted after isolation of this fragment from pEGFP-C3 (Clontech) using BsrGI and NsiI. The shRNA against Piccolo was constructed using the following primers: forward 5'-GATCCCCACCCCAAAATATAAAGCTTTCAAGAGAAGCCTTATATTITGTGGGTTTTTTTGGAAGA-3' and reverse 5'-AGCTTTTCTCAAAGCTTTATATTGTGGGGTGGG-3'. These primers were annealed by heating to 100°C, cooling down to room temperature and inserted in pSuper-CMV-EGFP by digesting this vector with BglII and HindIII. For construction of C2Awt, this fragment was isolated from a Yeast-2-Hybrid mouse cDNA bank using the following primers: forward 5'-GGCGTGGATCCCAAGCAGCTG-3' and reverse 5'-GTTTTGACTGGAGTGGAGACTG-3' and subsequently a PCR was performed using the following primers: forward 5'-AAGGATCCGCCTCTCACCCAATTACAGGA-3' and reverse 5'-TTTCGGGCCGATGCTTTCACTGCTGTITTTTCAG-3' to add BamHI and NotI sites. For C2Amut (S468A), the C2A domain was isolated using two PCR reactions using the following primers: forward 5'-AAGGATCCGCCTCTCACCCAATTACAGGA-3' and reverse 5'-CGAGGAGTGTTGTCCAGATGAGCAGTACTAGATAAAATCAATCAATAC-3' and forward 5'-GTATGATGTGATTATCTAGTACTGCTCATCTGGACAACACTCCTCG-3' and reverse 5'-TTTCGGGCCGATGCTTTCACTGCTGTITTTTCAG-3'. These two PCR products were annealed and a PCR was performed on this product using primers: forward 5'-AAGGATCCGCCTCTCACCCAATTACAGGA-3' and reverse 5'-TTTCGGGCCGATGCTTTCACTGCTGTITTTTCAG-3' to add BamHI and NotI sites and C2Awt and C2Amut fragments were ligated in pGEX. To construct pCMV-C2A-IRES2EGFP, both fragments were isolated from pGEX by\n\nPCR using the following primers: forward 5'-AAAGGATCCACATGGCCTCTCACCCCAATTACAGG-3' and reverse 5'-TTTGAATTCTCAGCTTTTCAGTCTGTCTTTTCAG-3' to add BamHI and EcoRI sites and a Kozak and stop sequence. Fragments were inserted in pIRES2EGFP by ligating this vector with BglII and EcoRI.\n\n**Cell culture and transfections**\n\nHippocampi from embryonic day 18 C57/Bl6 mouse embryos were dissected free of meninges and collected in Hanks’ Balanced Salt Solution (HBSS; Invitrogen) supplemented with 7 mM HEPES (Invitrogen). Hippocampi were digested with 0.25% trypsin (Invitrogen) in HBSS supplemented with 7 mM HEPES for 15 minutes at 37°C. After digestion tissue was washed three times with HBSS supplemented with 7 mM Hepes and subsequently triturated with a fire-polished glass pipette. Dissociated neurons were plated at a density of 25,000 cell/well on rat glial cells on 18 mm glass coverslips (Menzel Glaser, Braunschweig, Germany) in 12 well plates. Cells were cultured in neurobasal medium (Invitrogen) supplemented with B27 supplement (Invitrogen), 18 mM HEPES, 0.5 mM Glutamax (Invitrogen) and penicillin/streptomycin (Invitrogen). Once a week half of the medium was replaced. The hippocampal cultures were transfected using Lipofectamine 2000 (Invitrogen). Before transfection, half of the neurobasal medium was collected from each well and replaced with new neurobasal medium. For one well, 1 µl of Lipofectamine was added to 25 µl of serum free glutamax (Invitrogen) medium and incubated for 5 minutes. Subsequently, from each construct 1 µg of DNA was added to 25 µl of serum free glutamax and immediately mixed with the 25 µl of serum free glutamax containing Lipofectamine. This mixture was incubated for 30 minutes before adding to the cells. After 4–6 hours of incubation on the cells, half of the neurobasal medium was replaced again with the earlier collected neurobasal medium. Cells were routinely transfected at DIV2–7 and imaged at DIV10–14.\n\n**Immunocytochemistry**\n\nFor immunocytochemistry, neuronal cells were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) (pH 7.4) for 20 minutes at room temperature. Cells were washed in PBS and blocked in PBS containing 4% fetal calf serum and 0.2% Triton X-100 for 20 minutes at room temperature. Cells were incubated overnight with primary antibodies at 4°C in PBS containing 0.2% Triton X-100. As primary antibodies monoclonal MAP2 (Chemicon), and polyclonal Piccolo (Synaptic Systems) and Synapsin (Eo28) were used. After incubation in primary antibodies, cells were washed three times using PBS. Subsequently, cells were incubated in Alexa Fluor-conjugated secondary antibodies (Invitrogen) in PBS\n\nfor one hour at room temperature. All antibodies were used in a 1:1000 dilution. Finally, cells were washed three times in PBS and mounted using Dabco-Mowiol.\n\nFor immunohistochemistry on mouse brain slices, mice were anaesthetized and transcardially perfused with 4% paraformaldehyde (PFA) in PBS (pH 7.4). Brains were post fixed in PFA for one day, and subsequently cryoprotected in increasing concentrations of sucrose. Thirty μm coronal adult brain slices were made using a Cryostat (Leica). Midbrain slices containing the dorsal and medial raphe nucleus were blocked for two hours in PBS containing 10% normal goat serum and 0.5% Triton X-100. Subsequently, the slices were incubated overnight in primary antibodies in PBS containing 0.5% Triton X-100 at 4°C. Brain slices were washed three times two hours in PBS and incubated in secondary antibodies in PBS overnight at 4°C. After washing three times two hours, slices were mounted using Dabco-Mowiol. As primary antibodies monoclonal Tph (Sigma, recognizing both Tph2 and Tph2) and polyclonal Piccolo (Synaptic systems) were used in a 1:1000 dilution. Alexa Fluor-conjugated secondary antibodies (Invitrogen) were used in a 1:1000 dilution. Cells and slices were examined on a Zeiss LSM 510 confocal laser scanning microscope (Zeiss, BV The Netherlands).\n\n**Live cell imaging**\n\nCells were imaged in a chamber perfused with Tyrode’s solution (119 mM NaCl, 2.5 mM KCl, 2 mM MgCl$_2$, 2 mM CaCl$_2$, 30 mM glucose, 25 mM HEPES, pH 7.4) on an Axiovert II Microscope (Zeiss, Oberkochen, Germany) equipped with a Coolsnap HQ camera (Photometrics, Tucson, Arizona, USA) and a Polychrome IV illumination unit (TILL photonics, Graefling, Germany). Images were acquired in MetaMorph 6.2 software (Universal Imaging, Downingtown, Philadelphia, USA) using a 100x oil objective.\n\n**Image analysis**\n\nTo analyze the trafficking of mCherry-hSERT, image stacks were converted to .avi movie files using ImageJ software. Movies were analyzed using the Fluotrack software program (Broeke et al., 2008) to quantify the average number of moving puncta, the average velocity of moving puncta, the average pausing time of moving puncta and the direction of moving puncta.\n\n**References**\n\nArango V, Underwood MD, Boldrini M, Tamir H, Kassir SA, Hsiung S, Chen JJ, Mann JJ (2001) Serotonin 1A receptors, serotonin transporter binding and\n\nserotonin transporter mRNA expression in the brainstem of depressed suicide victims. *Neuropsychopharmacology* 25:892-903.\n\nBroeke JH, Ge H, Dijkstra IM, Cemgil AT, Riedl JA, Niels Cornelisse L, Toonen RF, Verhage M, Fitzgerald WJ (2008) Automated quantification of cellular traffic in living cells. *J Neurosci Methods*.\n\nCases-Langhoff C, Voss B, Garner AM, Appeltaner U, Takei K, Kindler S, Veh RW, De Camilli P, Gundelfinger ED, Garner CC (1996) Piccolo, a novel 420 kDa protein associated with the presynaptic cytomatrix. *Eur J Cell Biol* 69:214-223.\n\nCastrén E (2005) Is mood chemistry? *Nat Rev Neurosci* 6:241-246.\n\nCen X, Nitta A, Ibi D, Zhao Y, Niwa M, Taguchi K, Hamada M, Ito Y, Ito Y, Wang L, Nabeshima T (2008) Identification of Piccolo as a regulator of behavioural plasticity and dopamine transporter internalization. *Mol Psychiatry* 13:349, 451-363.\n\nFenster SD, Garner CC (2002) Gene structure and genetic localization of the PCLO gene encoding the presynaptic active zone protein Piccolo. *Int J Dev Neurosci* 20:161-171.\n\nFenster SD, Chung WJ, Zhai R, Cases-Langhoff C, Voss B, Garner AM, Kaempf U, Kindler S, Gundelfinger ED, Garner CC (2000) Piccolo, a presynaptic zinc finger protein structurally related to bassoon. *Neuron* 25:203-214.\n\nGarcia J, Gerber SH, Sugita S, Sudhof TC, Rizo J (2004) A conformational switch in the Piccolo C2A domain regulated by alternative splicing. *Nat Struct Mol Biol* 11:45-53.\n\nGerber SH, Garcia J, Rizo J, Sudhof TC (2001) An unusual C(2)-domain in the active-zone protein piccolo: implications for Ca(2+) regulation of neurotransmitter release. *Embo J* 20:1605-1619.\n\nHirano K, Seki T, Sakai N, Kato Y, Hashimoto H, Uchida S, Yamada S (2005) Effects of continuous administration of paroxetine on ligand binding site and expression of serotonin transporter protein in mouse brain. *Brain Res* 1053:154-161.\n\nKilic F, Murphy DL, Rudnick G (2003) A human serotonin transporter mutation causes constitutive activation of transport activity. *Mol Pharmacol* 64:440-446.\n\nLau T, Horschitz S, Bartsch D, Schloss P (2008a) Monitoring mouse serotonin transporter internalization in stem cell-derived serotonergic neurons by confocal laser scanning microscopy. *Neurochem Int*.\n\nLau T, Horschitz S, Berger S, Bartsch D, Schloss P (2008b) Antidepressant-induced internalization of the serotonin transporter in serotonergic neurons. *Faseb J* 22:1702-1714.\n\nLeal-Ortiz S, Waites CL, Terry-Lorenzo R, Zamorano P, Gundelfinger ED, Garner CC (2008) Piccolo modulation of SynapsinIa dynamics regulates synaptic vesicle exocytosis. *J Cell Biol* 181:831-846.\n\nMurphy DL, Lerner A, Rudnick G, Lesch KP (2004) Serotonin transporter: gene, genetic disorders, and pharmacogenetics. *Mol Interv* 4:109-123.\n\nNabi R, Zhong H, Serajee RJ, Huq AH (2003) No association between single nucleotide polymorphisms in DLX6 and Piccolo genes at 7q21-q22 and autism. *Am J Med Genet B Neuropsychiatr Genet* 119B:98-101.\n\nPickel VM, Joh TH, Reis DJ (1976) Monoamine-synthesizing enzymes in central dopaminergic, noradrenergic and serotonergic neurons.\n\nImmunocytochemical localization by light and electron microscopy. J Histochem Cytochem 24:792-306.\n\nQian Y, Galli A, Ramamoorthy S, Risso S, DeFelice LJ, Blakely RD (1997) Protein kinase C activation regulates human serotonin transporters in HEK-293 cells via altered cell surface expression. J Neurosci 17:45-57.\n\nQuick MW (2003) Regulating the conducting states of a mammalian serotonin transporter. Neuron 40:537-549.\n\nRamamoorthy S, Blakely RD (1999) Phosphorylation and sequestration of serotonin transporters differentially modulated by psychostimulants. Science 285:763-766.\n\nSerretti A, Calati R, Mandelli L, De Ronchi D (2006) Serotonin transporter gene variants and behaviour: a comprehensive review. Curr Drug Targets 7:1659-1669.\n\nShao X, Li C, Fernandez I, Zhang X, Sudhof TC, Rizo J (1997) Synaptotagmin-synaptaxin interaction: the C2 domain as a Ca2+-dependent electrostatic switch. Neuron 18:133-142.\n\nShapira M, Zhai RG, Dresbach T, Bresler T, Torres VI, Gundelfinger ED, Ziv NE, Garner CC (2003) Unitary assembly of presynaptic active zones from Piccolo-Bassoon transport vesicles. Neuron 38:237-252.\n\nSullivan PF, de Geus EJ, Willemsen G, James MR, Smit JH, Zandbelt T, Arolt V, Baune BT, Blackwood D, Cichon S, Coventry WL, Domschke K, Farmer A, Fava M, Gordon SD, He Q, Heath AC, Heutink P, Holstboer F, Hoogendijk WJ, Hottenga JJ, Hu Y, Kohli M, Lin D, Lucae S, Macintyre DJ, Maier W, McGhee KA, McGuffin P, Montgomery GW, Muir WJ, Nolen WA, Nothen MM, Perlis RH, Pirlo K, Posthuma D, Rietschel M, Rizzu P, Schosser A, Smit AB, Smoller JW, Tzeng JY, van Dyck R, Verhage M, Zitman FG, Martin NG, Wray NR, Boomsma DI, Penninx BW (2008) Genome-wide association for major depressive disorder: a possible role for the presynaptic protein piccolo. Mol Psychiatry.\n\nTao-Cheng JH (2007) Ultrastructural localization of active zone and synaptic vesicle proteins in a preassembled multi-vesicle transport aggregate. Neuroscience 150:575-584.\n\ntom Dieck S, Sammarti-Vila L, Langnaese K, Richter K, Kindler S, Soyke A, Wex H, Smalla KH, Kampf U, Franzer JT, Stumm M, Garner CC, Gundelfinger ED (1998) Bassoon, a novel zinc-finger CAG/glutamine-repeat protein selectively localized at the active zone of presynaptic nerve terminals. J Cell Biol 142:499-509.\n\nWang X, Kibschull M, Laue MM, Lichte B, Petrasch-Parwez E, Kilimann MW (1999) Acczonin, a 550-kD putative scaffolding protein of presynaptic active zones, shares homology regions with Rim and Bassoon and binds profilin. J Cell Biol 147:151-162.\n\nZhai RG, Vardinon-Friedman H, Cases-Langhoff C, Becker B, Gundelfinger ED, Ziv NE, Garner CC (2001) Assembling the presynaptic active zone: a characterization of an active one precursor vesicle. Neuron 29:131-143.\n\nZhu CB, Hewlett WA, Feoktistov I, Biaggioni I, Blakely RD (2004) Adenosine receptor, protein kinase G, and p38 mitogen-activated protein kinase-dependent up-regulation of serotonin transporters involves both transporter trafficking and activation. Mol Pharmacol 65:1462-1474.\n\nChapter 6\n\nDeletion of Munc18-1 in 5-HT neurons results in rapid degeneration of the 5-HT system and early postnatal lethality\n\nJ.J. Dudok\\textsuperscript{1}, A.J.A. Groffen\\textsuperscript{1}, R.F.T. Toonen\\textsuperscript{1}, Neuro-BSIK Mouse Phenomics consortium and M. Verhage\\textsuperscript{1}\n\n\\textsuperscript{1}Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, the Netherlands\n\nSubmitted\n\nAbstract\n\nThe serotonin (5-HT) system densely innervates many brain areas and is important for proper brain development. To specifically ablate the 5-HT system we generated mutant mice carrying a floxed Munc18-1 gene and Cre recombinase driven by the 5-HT-specific serotonin reuptake transporter (SERT) promoter. Mutant mice were smaller than their control littermates and the majority died within a few days after birth. Immunohistochemical analysis of brains of these mice showed that initially 5-HT neurons are formed and the cortex is innervated with 5-HT projections. From embryonic day (E) 16 onwards, however, 5-HT neurons started to degenerate and at postnatal day (P) 2 hardly any 5-HT projections were present in the cortex. The 5-HT system of mice heterozygous for the floxed Munc18-1 allele was indistinguishable from control mice. Behavioural analysis of these heterozygous mice showed that there is no difference in basal behaviour, conditioning and anxiety-related behaviour compared to control mice. These data show that deletion of Munc18-1 in 5-HT neurons results in rapid degeneration of the 5-HT system and suggests that the 5-HT system is important for postnatal survival.\n\nIntroduction\n\nThe 5-HT system consists of clusters of cell bodies in the midbrain raphe nuclei, with the largest clusters in the median raphe nucleus (MRN) and the dorsal raphe nucleus (DRN). Several brain areas receive dense 5-HT innervation and 5-HT is released both synaptically and as volume transmission (Ridet et al., 1993; Bunin and Wightman, 1998). Due to this and to the several 5-HT receptor subtypes which are present in the brain, 5-HT has many roles and influences many processes in the brain (Jacobs and Azmitia, 1992).\n\nNeurogenesis of 5-HT neurons in the mouse brain occurs in the ventral rhombencephalon around E10 (Wallace and Lauder, 1983). One day later, 5-HT neurons begin to synthesize and secrete 5-HT and start growing out axons. Around birth, target areas such as the forebrain and the hippocampus are densely innervated with 5-HT projections. Only after birth, the maturation of the 5-HT network is completed.\n\nSeveral studies have addressed the role of 5-HT on the development of the 5-HT system and brain development. In a conditional Lmx1b knockout (KO) mouse, almost all 5-HT neurons fail to survive, resulting in a significant decrease in brain tissue 5-HT levels (Zhao et al., 2006). However, these mice do not show\n\nan overt phenotype and survive to adulthood (Zhao et al., 2006). In contrast, it was shown that maternal 5-HT is required for embryonic development (Cote et al., 2007). Furthermore, in tryptophan hydroxylase 2 (Tph2) KO mice 5-HT neurons are completely devoid of 5-HT, but the morphology and neurite distribution of the 5-HT system is not affected, and these mice do not show any behavioural phenotype (Gutknecht et al., 2008). Neonatal depletion of 5-HT by the neurotoxin 5,7-Dihydroxytryptamine results in rather subtle changes in behavioural response and brain development (Hohmann et al., 2007; Bennett-Clarke et al., 1995).\n\nIn this study we silenced the 5-HT system by conditional deletion of Munc18-1 in 5-HT neurons. Neurons that lack the presynaptic protein Munc18-1 have a complete absence of neurotransmitter secretion and these mice are born paralyzed and die immediately after birth (Verhage et al., 2000). In these mice, initially synapses are formed and the assembly of the brain is normal. However, in later stages of brain development there is massive neuronal cell death and brain degeneration (Verhage et al., 2000).\n\nIn SERT-Cre\\textsuperscript{wt} Munc18-1\\textsuperscript{lox/lox} mice 5-HT neurons were initially generated and 5-HT projections innervated the midbrain and cortex, later followed by degeneration and loss of 5-HT projections in the cortex. The majority of these mice died within a few days after birth. These data suggest that the 5-HT system contributes importantly to postnatal brain development.\n\n**Results**\n\n*Deletion of Munc18-1 in SERT expressing neurons results in postnatal lethality*\n\nWe have shown previously that Munc18-1 mutant mice lack regulated secretion and have a lethal phenotype (Verhage et al., 2000). Therefore, we have generated Munc18-1\\textsuperscript{lox/lox} mutant mice in order to conditionally delete Munc18-1. Crossing these mice with a L7-Cre line, with Cre expressed in Purkinje neurons in the cerebellum, resulted in mice which developed severe ataxia, suggesting a cerebellar phenotype (Heeroma et al., 2004). Here we crossed Munc18-1\\textsuperscript{lox/lox} mice with SERT-Cre mice, which express Cre in SERT expressing neurons. These are the 5-HT neurons in the raphe nuclei, but also some hippocampal neurons and thalamocortical neurons which express SERT transiently during development (Zhuang et al., 2005; Narboux-Neme et al., 2008). Crossing SERT-Cre mice with Munc18-1\\textsuperscript{lox/lox} mice results in mice in which Munc18-1 is specifically removed in SERT expressing neurons (Fig. 1A). We crossed SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/wt} mice with SERT-Cre\\textsuperscript{wt/wt} Munc18-1\\textsuperscript{lox/wt} mice which should result in 12.5% of offspring which have SERT-Cre\\textsuperscript{cre/wt} and Munc18-1\\textsuperscript{lox/lox}\n\ngenotypes. Genotyping mice sacrificed at E16 or E18 revealed that there were more SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} and SERT-Cre\\textsuperscript{wt/wt} Munc18-1\\textsuperscript{lox/lox} mice and less SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/wt} mice than expected (p = 0.007) (Fig. 1B). Genotyping 101 mice three weeks after birth revealed that genotype frequencies were not distributed at Mendelian ratio (p = 0.027). Only three SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice were found, whereas based on Mendelian ratio the expected number of SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice should be 13, giving a 77% mortality rate in SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice (Fig. 1C). Also, SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice were smaller compared to littermates (data not shown). These three remaining SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice all died in the fourth postnatal week. This shows that Munc18-1 deletion in SERT expressing neurons results in postnatal lethality.\n\nFigure 1. Postnatal lethality in SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice\n\n(A) Overview of breeding approach. Crossing SERT-Cre with Munc18-1\\textsuperscript{lox/lox} mice results in deletion of Munc18-1 only and specifically in SERT expressing neurons, whereas all other neurons still express Munc18-1.\n\n(B) Genotypes of mice sacrificed at E16 or E18 differed from the expected Mendelian ratio. (C) Genotyping mice after weaning at 3 weeks of age revealed that only few SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice survived up to three weeks.\n\n5-HT neurons are initially generated but then quickly degenerate\n\nTo assess the effect of Munc18-1 deletion in 5-HT neurons on the development of the 5-HT system, we made coronal sections of paraformaldehyde (PFA) fixed brains at different developmental stadia and performed immunohistochemistry for 5-HT. As developmental time points we chose E16, E18 and P2. Expression of SERT, and thus of Cre recombinase, starts at E11 and the earliest recombination observed in the DRN in SERT-Cre mice was at E12.5 (Narboux-Neme et al., 2008). Since it will take some days before all remaining Munc18-1 mRNA and protein is degraded, we chose E16 as the first time point. We first focused on the 5-HT cell bodies in the DRN. Immunohistochemistry for 5-HT on brain slices from E16 SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} and control mice showed that in the DRN 5-HT neurons are present and these showed the characteristic DRN topology (Fig. 2A,B). The number of 5-HT neurons did not differ from control (control 312 ± 17.67, SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} 305 ± 25.43; Fig. 2G,H,O). However, morphological analysis of the neurons showed that these were already degenerating, as assessed by the round morphology and reduced number of primary neurites. At E18, there was a ~80% decrease in the number of 5-HT neurons in SERT-Cre\\textsuperscript{cre/wt} M18\\textsuperscript{lox/lox} mice compared to control (control 358 ± 10.82, SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} 59 ± 4.84, Fig. 2C,D,O). At P2 there were only few 5-HT neurons left in the DRN (control 364 ± 19.68, SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} 25 ± 2.89, Fig. 2E,F,O). At E18 and P2 the remaining 5-HT neurons in SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} brain sections displayed an altered morphology compared to control 5-HT neurons (Fig. 2I,J,K,L). 5-HT neurons in control brains had a characteristic fusiform or ovoid shape and grew out several primary neurites (Fig. 2M). In contrast, the mutant 5-HT neurons had a rounded morphology and the majority did not contain primary neurites, indicative of degenerating neurons (Fig. 2N). This showed that between E16 and P2 there is a massive degeneration of 5-HT neurons in the DRN.\n\n5-HT projections innervate the DRN and cortex but are degenerated at P2\n\nNext, we focused on the innervation of the midbrain and cortex with 5-HT projections. Midbrain and cortex are the first regions which are densely innervated with 5-HT projections during development. We quantified 5-HT neurite density first in DRN midbrain sections at E16, E18 and P2. At E16 the sections from both control and SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice contained several 5-HT neurites and there was no significant difference in 5-HT innervation density (1.31 ± 0.51% and 0.63 ± 0.24% respectively, Fig. 3A,B). At E18, in control sections the 5-HT innervation density was slightly increased compared to E16 (Fig. 3C). However, in SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} sections the 5-HT innervation density was decreased compared to control (control 2.32 ± 0.86%, SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} 0.31 ± 0.27%, Fig. 3D).\n\nFigure 2. Rapid degeneration of 5-HT neurons in SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice\n\nWe compared the number of 5-HT cell bodies in the DRN in control and SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice at E16, E18 and P2. (A-F) At E16, in both control mice (A) and SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} (lox/lox) mice (B) the 5-HT cell bodies are distributed in the DRN topology. At E18 and P2, however, in midbrain sections from SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} brains (D, F respectively) only few 5-HT neurons are present compared to control sections (C, E respectively). (G-L) Zooming in on 5-HT cell bodies shows that in control sections at E16 (G), E18 (I) and P2 (K) the cell bodies grow out several neurites and have a fusiform or ovoid morphology. However, remaining 5-HT cell bodies in SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} sections at E16 (H), E18 (J) and P2 (L) have a round morphology with hardly any neurites. (M,N) Blow up of some 5-HT cell bodies in control and SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice sections shows the differences in 5-HT cell body morphology. (O) Analysis of the number of 5-HT cell bodies in the sections showed that there is no difference at E16, but at E18 and P2 there is a decrease of 70% to 80% in SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} sections. Scale bars: 200 µm in F, 50 µm in L and 20 µm in N. Data shown are mean ± standard error of the mean (SEM). * p < 0.05.\n\nAt P2 only few 5-HT neurites are left in SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} sections (control 1.6 ± 0.41%, SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} 0.33 ± 0.14%, Fig. 3E,F). Next, we focused on the 5-HT innervation density in the cortex. This revealed that already at E16 there is a reduction in 5-HT innervation density in SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} sections (control 3.27 ± 1.14%, SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} 0.65 ± 0.19%, Fig. 3G,H). At E18 and P2 the reduction in 5-HT innervation density is augmented, with only very few 5-HT neurites left at P2 in the cortex (E18 control 1.73 ± 0.45%, SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} 0.36 ± 0.18%, P2 control\n\n1.78 ± 0.52%, SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsubscript{lox/lox} 0.19 ± 0.04%, Fig. 3I,J,K,L). Quantification showed that in the midbrain sections the 5-HT innervation density was reduced to ~20% and ~10% at E18 and P2 respectively (Fig. 3M). In the cortex, the 5-HT innervation density was reduced to ~20%, ~10% and ~5% at E16, E18 and P2 respectively (Fig. 3N).\n\nIn summary, deletion of Munc18-1 in 5-HT neurons resulted in rapid degeneration of 5-HT cell bodies and the absence of 5-HT projections in the postnatal brain.\n\n**Figure 3. Degeneration of 5-HT neurites in DRN and cortex**\n\n(A-F) At E16 in the midbrain containing the DRN, there was no difference in 5-HT neurite density between control (A) and SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsubscript{lox/lox} (B) mice. In control sections at E18 (C) and P2 (E) several 5-HT neurites were present. However, in SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsubscript{lox/lox} sections at E18 (D) and P2 (F) only very few 5-HT neurites are left. (G-L) In the cortex already at E16 there is a reduction in 5-HT neurite density in sections from SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsubscript{lox/lox} (H) mice compared to control (G). At E18 (J) and P2 (L), in cortical sections from SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsubscript{lox/lox} mice only very few remaining 5-HT neurites are present, in contrast to E18 (I) and P2 (K) control cortical sections. (M) Quantification of the 5-HT neurite density in the DRN revealed that at E16 there is no difference, but at E18 and P2 5-HT neurite density is reduced to ~20% and ~10% respectively. (N) In cortical sections, at E16 the 5-HT neurite density is reduced to ~20% and at E18 and P2 the 5-HT neurite density is reduced to ~10% and ~5% respectively. Scale bar 50 µm in L. Data shown are mean ± SEM * p < 0.05.\n\nBasal behaviour is not affected in SERT-Cre\\textit{er/cut} Munc18-1\\textit{fl/ut} mice\n\nFinally, we were interested whether deletion of one allele of Munc18-1 in SERT expressing cells affected mouse behaviour. Previously, we have shown that Munc18-1 expression levels regulate the readily releasable pool (RRP) size. In Munc18-1 heterozygous mice there is a faster synaptic rundown during episodes of high activity, a decreased RRP size and a decrease in docked vesicles at the synapse (Toonen et al., 2006). Therefore it is likely that deletion of one functional Munc18-1 allele in SERT expressing neurons affects the efficacy of 5-HT neurotransmission.\n\nFigure 4. Deletion of one functional allele of Munc18-1 in SERT expressing neurons does not affect number of 5-HT cell bodies and 5-HT innervation density\n\n(A-D) Midbrain sections containing the DRN from control (A) and hz (B) mice shows that 5-HT neurons are distributed in the characteristic DRN topology. Zooming in on 5-HT cell bodies show that both in control (C) and hz sections (D) these neurons have a fusiform or ovoid morphology and grow out several neurites. (E-H) 5-HT neurite density is not different between control and hz mice in the midbrain containing the DRN (E and F respectively) or in cortical sections from control and hz mice (G and H respectively). (I) Number of 5-HT cell bodies per section does not differ between control and hz. (J) 5-HT neurite density in midbrain containing DRN and cortical sections is not different between control and hz. Scale bars: 200 µm in B, 50 µm in D and H.\n\nFirst, we investigated whether deletion of one allele of Munc18-1 in SERT expressing neurons affected the number of 5-HT cell bodies in the DRN, or the 5-HT fiber density in the DRN and cortex. Analysis of midbrain sections from control and SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/wt} (hz) mice showed that 5-HT neurons are distributed in the characteristic DRN topology, and control and hz are indistinguishable (Fig. 4A,B). 5-HT cell bodies in both control and hz had a fusiform or ovoid shape (Fig. 4C,D). Quantification of the number of 5-HT cell bodies in the DRN revealed no difference between control and hz (control 364 ± 14.78, hz 361 ± 15.01, Fig. 4I). Likewise, the 5-HT neurite density in the midbrain and cortex were similar (midbrain control 4.38 ± 0.93%, hz 4.67 ± 0.55%; cortex control 2.18 ± 0.35%, hz 2.87 ± 0.99%, Fig. 4E-H,I,J). Thus, deletion of one allele of Munc18-1 does not affect 5-HT neuronal survival, 5-HT neurite outgrowth or 5-HT projection density.\n\nWe tested several behavioural characteristics of hz mice by monitoring them for six days in home cages equipped with a video camera on top to visualize and analyze mouse behaviour. We analyzed the behaviour of eleven hz mice and compared several behavioural parameters with eleven control mice. First, we analyzed whether there was a difference in total distance moved in the open space and time spent under the shelter. Control and hz mice moved a similar total distance in the open space (control dark phase 2231.78 ± 139.39 cm/hour, light phase 483.91 ± 32.30 cm/hour; hz dark phase 2150.61 ± 112.70 cm/hour, light phase 433.69 ± 26.15 cm/hour; Fig. 5A,B), spent a similar proportion of time in a sheltered compartment (control dark phase 58.99 ± 2.03%, light phase 86.37 ± 2.36%; hz dark phase 60.04 ± 1.11%, light phase 90.38 ± 0.45%; Fig. 5C) and spent a similar time drinking (control dark phase 33.92 ± 1.99 s/hour, light phase 7.50 ± 0.52 s/hour; hz dark phase 41.06 ± 4.72 s/hour, light phase 8.06 ± 0.79 s/hour; Fig. 5D). Also, time spent in the feeder was indistinguishable between control and hz (control dark phase 476.77 ± 15.17 s/hour, light phase 160.55 ± 6.58 s/hour; hz dark phase 497.73 ± 18.41 s/ hour, light phase 165.33 ± 6.99 s/hour; Fig. 5E). Thus, basal behavioural parameters were indistinguishable between control and hz.\n\n\\textit{Conditioning and anxiety related behaviour is not affected in SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/wt} mice}\n\nTo investigate whether hz mice display altered conditioning behaviour, a free operant conditioning task was performed, where a jump on the shelter resulted in the drop of a sucrose pellet. The number of directed short movement bouts (SMBs) increased during day four and five compared to day 1-3, but this conditioning behaviour did not differ between control and hz.\n\nFigure 5. Hz mice do not display altered basal behaviour\n\n(A) Six day plot of average distance moved (cm / 12 hours) during dark and light phase. Note the increase in distance moved during the dark phase at day 4 due to the free operant conditioning test. (B) No difference in average distance moved (cm/hour) between control and Hz mice during the dark and light phase. (C) Proportion of time spent under the shelter does not differ between control and Hz mice. (D) There is no difference in time spent drinking (s/hour) between control and Hz mice. (E) Also, there is no difference in time spent in the feeder (s/hour). Data shown are mean ± SEM.\n\n(day 1-3 control $17.07 \\pm 2.12$, hz $16.33 \\pm 2.17$; day 4-5 control $45.3 \\pm 5.27$, hz $46.32 \\pm 5.78$; Fig. 6A). Number of pellets dropped during pre-training phase (session 1-10, day four) did not differ between control and hz mice (control $4.88 \\pm 0.43$, hz $5.99 \\pm 0.45$; Fig. 6B). Also during the sessions at day five (session 11-12), there was no difference in the number of pellets dropped (control $20.36 \\pm 2.81$, hz $19.95 \\pm 2.34$; Fig. 6B).\n\nTo assess whether hz mice displayed altered anxiety related behaviour, a one trial anxiety task was performed at day six. To this end a lightspot was created at the feeder place, and the duration of time the animal spent in the lightspot and the feeder was recorded and compared with the time the animal spent at the feeder without a lightspot on. At day five (baseline) and day six (test) there was no difference between control and hz, but both groups spent less time in the lightspot at day six (day five control $1798.72 \\pm 208.1$ s, hz $2108.01 \\pm 284.63$ s; day six control $668.76 \\pm 61.28$ s, hz $682.19 \\pm 83.98$ s; Fig. 6C). Next, we investigated whether the frequency of entering the feeder zone at day five (baseline) and day six (test) was different. Frequency of entering the feeder zone did not differ between control and hz mice, but both groups displayed a decreased frequency during the lightspot test during day six (day five control $16.64 \\pm 2.51$, hz $23.09 \\pm 3.51$; day six control $7.15 \\pm 1.40$, hz $8.12 \\pm 1.56$; Fig. 6D). Also, time spent in the feeder zone during day five and day six did not differ between control and hz (day five control $528.15 \\pm 50.21$ s, hz $621.91 \\pm 65.99$ s; day six control $191.01 \\pm 30.36$ s, hz $198.46 \\pm 33.53$ s; Fig. 6E). Finally, analysis of latency to enter the lightspot zone after lightspot off revealed that this did not differ between control and hz (control $1567.71 \\pm 377.86$ s; hz $1293.87 \\pm 329.17$ s; Fig. 6F). Thus, mice in which one allele of Munc18-1 is deleted in SERT expressing neurons do not display altered basal, conditioning or anxiety-related behaviour.\n\n**Discussion**\n\nIn this study, we showed that SERT-Cre$^{cre/cre}$wt Munc18-1$^{lox/lox}$ mice display a postnatal lethality phenotype, accompanied by a rapid degeneration of the 5-HT system. Inactivation of one allele of Munc18-1 in SERT expressing neurons does not affect 5-HT cell number or 5-HT innervation density, and does not affect anxiety related behaviour or conditioning related behaviour.\n\nCrossing Munc18-1$^{lox/lox}$ mice with SERT-Cre mice results in deletion of Munc18-1 from SERT expressing cells. It was shown previously that crossing SERT-Cre mice with loxSTOPlox-EYFP mice results in >99% of 5-HT neurons in the DRN which are EYFP positively labeled (Zhuang et al., 2005).\n\nFigure 6. Hz behaviour on free operant conditioning test and one trial anxiety test does not differ from control\n\n(A) The number of directional SMB’s is increased at day 4 and 5 compared to day 1-3, but does not differ between control and hz. (B) Number of sucrose pellets acquired is increased at session 11 and 12 (day 5), compared to session 1-10 (day 4, pre-training day), but does not differ between control and hz. (C) Time spent in the lightspot (s) is decreased at day 6 (lightspot on) compared to day 5 (baseline, without lightspot on), but does not differ between control and hz. (D) Frequency of entering the feeder zone is decreased at day 6 compared to day 5, but there is no difference between control and hz mice. (E) During lightspot on on day 6 mice spent less time in the feeder (s) which does not differ between control and hz. (F) No difference in latency (s) to enter the lightspot zone after lightspot off. Data shown are mean ± SEM.\n\nHowever, SERT is also transiently expressed in some other brain regions during development, such as the thalamocortical neurons (Lebrand et al., 1996). In SERT-Cre ROSA-loxstoplox-LacZ double positive mice, strong LacZ expression was not only observed in the DRN, but also in the thalamus, cingulate cortex and\n\nin the CA3 region of the hippocampus (Zhuang et al., 2005; Narboux-Neme et al., 2008). Therefore, in SERT-Cre Munc18-1\\textsuperscript{lox/lox} mice also in some non-5-HT neurons Munc18-1 will be deleted. Genotyping sacrificed mice at E16 or E18 revealed that there were more SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice than expected, showing that these mice do not die during embryonic development. However, genotyping mice three weeks after birth showed that the majority of SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice had died, and the remaining SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice died within the fourth postnatal week. SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice were smaller compared to control mice which is in analogy with the VMAT2 KO mice which are hypoactive, smaller compared to control littermates, do not feed and die within a few days after birth (Fon et al., 1997; Wang et al., 1997).\n\nPreviously we showed that in Munc18-1 KO mice, the neuromuscular junction initially develops, but at later stages most of the motor neuronal cell bodies in the spinal cord degenerate (Heeroma et al., 2003). In analogy with this, in the absence of regulated secretion the hypothalamo-neurohypophysial system is initially normally formed, but in later stages of development degenerates (Korteweg et al., 2004). This is consistent with our observation that Munc18-1 is dispensable for initial generation and outgrowth of 5-HT neurons, but required for their survival.\n\nSince also in some other neurons Munc18-1 is deleted, it is unclear whether the observed phenotypes can be attributed to the degeneration of the 5-HT system. In Tphz2 KO mice, 5-HT neurons are completely devoid of 5-HT, yet there are no alterations in 5-HT neuron morphology and 5-HT neurite distribution (Gutknecht et al., 2008). Although these mice can survive into adulthood, approximately 50% does not survive the first 4 weeks and surviving mice have growth retardation (Alenina et al., 2009). Additionally, in a conditional Lmx1b KO mouse, 5-HT neurons are initially formed but almost all central 5-HT neurons fail to survive and brain 5-HT levels are reduced to ~10% (Zhao et al., 2006). Remarkably, these mice display normal locomotor behaviour and show no abnormalities in gross brain morphology. Recently, it was shown that these mice have severe apnea and have a ~20% mortality rate during the neonatal period, although the majority of these mice survive beyond P28 (Hodges et al., 2008; Hodges et al., 2009).\n\nApparently, even the absence of central 5-HT synthesis or an almost complete removal of 5-HT neurons does not result in a severe (postnatal) lethality phenotype. Thus, although several of these mice display high (postnatal) mortality, the phenotypes observed are not as severe as in the SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice.\n\nOn the other hand, mice which lack GAP43, display disrupted barrel cortex formation and thalamocortical connections fail to form. These mice display a\n\npostnatal lethality phenotype, with 50% of homozygotes which die between P0 and P2, and >95% of homozygotes which die before P21 (Maier et al., 1999). Although these mutant mice display a reduced 5-HT innervation in the cortex and hippocampus, they have no significant difference in the number of 5-HT neurons, and other areas such as the piriform cortex and amygdale receive normal 5-HT innervation (Donovan et al., 2002). Thus, based on these data it is unclear whether the postnatal lethality phenotype observed in this study is attributable to a degeneration of thalamocortical or 5-HT neurons. It would be very interesting to delete the Munc18-1 gene specifically in 5-HT neurons using the Pet-Cre transgenic line, in which expression of Cre is restricted to 5-HT neurons (Scott et al., 2005). This approach could be used to investigate whether the phenotype we observed could indeed be attributed to degeneration of the 5-HT system rather than degeneration of thalamocortical neurons.\n\nWe have shown that mice in which one allele of Munc18-1 is deleted in SERT expressing neurons did not display altered basal, conditioning or anxiety-related behaviour. In these mice number of 5-HT neurons and 5-HT neurite density was indistinguishable from control mice. However, we have shown previously in neurons cultured from Munc18-1 heterozygous mice that there is a subtle secretion phenotype (Toonen et al., 2006). Thus, one could argue that possibly deletion of one functional allele of Munc18-1 affects 5-HT release and thus efficacy of 5-HT neurotransmission.\n\nThere could be a few explanations why reduced 5-HT neurotransmission in the heterozygous mice used in this study does not affect behaviour. First of all, the reduction in 5-HT neurotransmission might just be too subtle to affect mouse behaviour. In Munc18-1\\(^{-/-}\\) neuronal cultures, there is a ~25% reduction in synaptic vesicle release (Toonen et al., 2006). In contrast, expression of tetanus toxin light chain in 5-HT neurons in vivo results in a robust (>90%) inhibition in 5-HT neurotransmission and these mice display decreased anxiety-related behaviour and enhanced associative learning (Kim et al., 2009). This suggests that a substantial reduction in 5-HT release is required to affect behaviour and that deletion of only one allele of Munc18-1 is not sufficient to achieve this reduction in 5-HT release.\n\nSecondly, in Munc18-1 null mice synaptic vesicle release is completely abolished. However, at least in chromaffin cells lacking Munc18-1, there is still some large dense core vesicle (LDCV) release, although this is reduced to ~10% (Voets et al., 2001). In Munc18-1 heterozygous mice LDCV release is presumably largely unaffected. In the 5-HT system, the bulk amount of 5-HT is released from LDCVs, which contain up to ~17 times as much 5-HT molecules as a single synaptic vesicle (Bruns and Jahn, 1995). Thus, if 5-HT released from synaptic vesicles is more severely affected than 5-HT released from LDCVs, there is only a\n\nminor reduction in release of 5-HT and thus in efficacy of 5-HT neurotransmission. Finally, (minor) reductions in extracellular 5-HT levels might be compensated for, such as by reduced SERT levels at the synaptic membrane and/or increased expression of postsynaptic 5-HT receptors, to guarantee proper levels of 5-HT neurotransmission.\n\nIn conclusion, deletion of one allele of Munc18-1 in 5-HT neurons does not affect mouse behaviour presumably due to the fact that there is a too subtle reduction in 5-HT neurotransmission in these mice. However, deletion of both alleles of Munc18-1 in SERT expressing neurons results in a rapid degeneration of the 5-HT system and postnatal lethality.\n\n**EXPERIMENTAL PROCEDURES**\n\n*Laboratory animals*\n\nGeneration and characterization of SERT-Cre mice has been described (Zhuang et al., 2005). Briefly, immediately upstream of the SERT translational start codon a cassette containing Cre and a FRT-flanked neomycin cassette was inserted. To generate conditional Munc18-1 mice (Heeroma et al., 2004), floxed Munc18-1 mice were generated by insertion of LoxP sites flanking the second exon using homologous recombination. To obtain SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice, SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/wt} mice were crossed with SERT-Cre\\textsuperscript{wt/wt} Munc18-1\\textsuperscript{lox/wt} mice. Pregnant females were sacrificed by cervical dislocation. Mouse embryos were obtained by caesarean section of pregnant females from timed matings. To investigate the effect of deletion of Munc18-1 in the 5-HT system on the development of the 5-HT system, SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice were used. SERT-Cre\\textsuperscript{wt/wt} Munc18-1\\textsuperscript{lox/lox}, SERT-Cre\\textsuperscript{wt/wt} Munc18-1\\textsuperscript{lox/wt}, or SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{wt/wt} littermates were not different from SERT-Cre\\textsuperscript{wt/wt} Munc18-1\\textsuperscript{wt/wt} mice and were used as controls. SERT-Cre mice were genotyped as has been previously described (Zhuang et al., 2005). For genotyping of Munc18\\textsuperscript{lox/lox} mice the following primers were used: 5’-ttgtggtcgaatggccagtag-3’, 5’-cctgtatgggtactgttcgttcaactaaataa-3’ and 5’-tctgaaccttgaggccagtcgagacacag-3’. Animals were housed and bred according to institutional and Dutch guidelines.\n\n*Immunohistochemistry*\n\nFor immunohistochemical analysis of 5-HT, embryonic mouse brains were dissected and immediately fixed by immersion in 4% PFA in phosphate buffered saline (PBS, pH 7.4). For analysis of 5-HT neurons and projections in adult brains, mice were anaesthetized and transcardially perfused with 4% PFA in PBS. For post-fixation, brains were incubated in 4% PFA in PBS overnight. For cryoprotection, brains were incubated in increasing concentrations of sucrose.\n\nSubsequently, coronal slices of 40 µm were made. For immunohistochemistry, brain slices were first incubated in blocking buffer containing PBS supplemented with 0.5% Triton X-100 and 10% normal goat serum for two hours. Subsequently, slices were incubated overnight with primary antibodies in PBS containing 0.5% Triton X-100 at 4°C in under gentle agitation. The next day, the slices were washed three times two hours in PBS and incubated with secondary antibodies in PBS for one hour under gentle agitation. Finally, slices were washed again three times two hours in PBS and mounted in Dabco-Mowiol for analysis. All procedures were performed at room temperature unless otherwise stated. Polyclonal anti-5-HT (1:1000) (Immunostar/Diasorin) was used as the primary antibody, and as a secondary antibody GAR-546 (1:1000) was used (Invitrogen). To analyze number of 5-HT cell bodies in DRN sections, at least three sections per experimental group were imaged using a CLSM 510 microscope. Number of 5-HT cell bodies was manually counted in the slices. For analysis of 5-HT fiber density in DRN and cortex a Z-stack of at least three positions per group was made. To analyze the 5-HT fiber density, the Z-stacks were projected in a single image and binarized. The 5-HT fiber density was quantified as the area in the image occupied by 5-HT neurites compared to the total area.\n\nMouse behaviour\n\nBehavioural analysis of mice was tested in the Phenotyper® (Noldus Information Technology, Wageningen, The Netherlands). For the home cage test, one animal per cage was tested for six days. Eleven SERT-Cre\\textsuperscript{wt/wt} Munc18-1\\textsuperscript{lox/wt} mice were used in the experimental group and eleven SERT-Cre\\textsuperscript{wt/wt} Munc18-1\\textsuperscript{lox/wt} mice were used as a control group. Each cage contains a top unit equipped with built-in hardware for videotracking, containing a digital infrared sensitive video camera and infrared lights. Using this method the animal behaviour can be continuously assessed, both in the light and in the dark phase. Ethovision 3.0 software was used as videotracking software. The phenotyper cages (30x30x35 cm) were made of transparent Perspex walls with an aluminium floor, and a feeding station and a water bottle attached to the outside. To the waterspout a lickometer was attached to determine the total duration the animal is touching the waterspout. A shelter unit (diameter: 9 cm, height: 10 cm, non-transparent material) was placed in the cage, which was defined as a hidden zone in Ethovision, and the software program could distinguish between in the shelter and on the shelter. Videotracking was performed at a rate of 12.5 samples per second and the following parameters were determined: average velocity of movement, total distance moved in the open space, proportion of time spent in the feeder, proportion of time spent in the shelter, and time spent drinking.\n\nThese parameters were calculated in two hour bins for both the dark and light phase. For the analysis of movement and velocity, only movement was scored when the mouse moved at a velocity of 3.5 cm/s and higher, averaged over twelve samples. Furthermore, total proportion of time spent in a zone of the home cage (on shelter, in shelter, feeder, pellet or waterspout), frequency of zone visits and frequency on the shelter during the dark period was quantified.\n\nOn day four and five a free operant conditioning task was performed as follows. The number of SMBs was quantified, which is a directed movement of the animal from one point in the cage to another. A distinction is made between raw and directional SMBs; When the animal moves with a higher velocity than the threshold value, which is determined by dividing the total distance moved by the total duration of movement, the SMB is considered to be a directional SMB. When the animal is moving at a lower velocity, it is considered as a raw SMB. Thus, in a directional SMB the animal is either moving with a greater velocity, or is taking a shorter route. At day four and five, when the mice gets on its shelter, this will result in the drop of a sucrose pellet. Thus, the mouse ‘learns’ that it has to jump onto its shelter in order to receive a new sucrose pellet.\n\nOne day six a one trial anxiety test was performed as follows. Fifteen minutes after the change from light to dark phase, a lightspot is on the feeder, thus creating a lightspot zone. The lightspot lasts for three hours, and the total amount of time the animal is in the lightspot zone in this period is recorded and compared with a baseline reference. The baseline reference is acquired by recording the amount of time the animal is in the feeder zone at day five at the corresponding hours, without a lightspot on.\n\n**Statistical analysis**\n\nData were analyzed using SPSS 17.0. For analysis of number of 5-HT cell bodies in DRN and 5-HT neurite density in DRN and cortex, data was analyzed using the independent samples t-test. To analyze whether observed genotype frequencies differed significantly from a Mendelian ratio, a one-way chi-square test was performed. Mouse behaviour parameters were analyzed using the non-parametric Wilcoxon test after checking for normal distribution. Data shown are mean ± SEM. Significance level was set at p <0.05.\n\n**References**\n\nAlenina N, Kikic D, Todiras M, Mosienko V, Qadri F, Plehm R, Boye P, Vilianovitch L, Sohr R, Tenner K, Hortnagl H, Bader M (2009) Growth retardation and altered autonomic control in mice lacking brain serotonin. Proc Natl Acad Sci U S A 106:10332–10337.\n\nBennett-Clarke CA, Lane RD, Rhoades RW (1995) Fenfluramine depletes serotonin from the developing cortex and alters thalamocortical organization. Brain Res 702:255-260.\n\nBruns D, Jahn R (1995) Real-time measurement of transmitter release from single synaptic vesicles. Nature 377:62-65.\n\nBunin MA, Wightman RM (1998) Quantitative evaluation of 5-hydroxytryptamine (serotonin) neuronal release and uptake: an investigation of extrasynaptic transmission. J Neurosci 18:4854-4860.\n\nCote F, Fligny C, Bayard E, Launay JM, Gershon MD, Mallet J, Vodjdani G (2007) Maternal serotonin is crucial for murine embryonic development. Proc Natl Acad Sci U S A 104:329-334.\n\nDonovan SL, Mamounas LA, Andrews AM, Blue ME, McCasland JS (2002) GAP-43 is critical for normal development of the serotonergic innervation in forebrain. J Neurosci 22:3543-3552.\n\nFon EA, Pothos EN, Sun BC, Killeen N, Sulzer D, Edwards RH (1997) Vesicular transport regulates monoamine storage and release but is not essential for amphetamine action. Neuron 19:1271-1283.\n\nGutknecht L, Waider J, Kraft S, Kriegebaum C, Holtmann B, Reif A, Schmitt A, Lesch KP (2008) Deficiency of brain 5-HT synthesis but serotonergic neuron formation in Tph2 knockout mice. J Neural Transm 115:1127-1132.\n\nHeeroma JH, Plomp JJ, Roubos EW, Verhage M (2003) Development of the mouse neuromuscular junction in the absence of regulated secretion. Neuroscience 120:733-744.\n\nHeeroma JH, Roelandse M, Wierda K, van Aerde KI, Toonen RF, Hensbroek RA, Brussaard A, Matus A, Verhage M (2004) Trophic support delays but does not prevent cell-intrinsic degeneration of neurons deficient for munc18-1. Eur J Neurosci 20:623-634.\n\nHodges MR, Wehner M, Aungst J, Smith JC, Richerson GB (2009) Transgenic mice lacking serotonin neurons have severe apnea and high mortality during development. J Neurosci 29:10341-10349.\n\nHodges MR, Tattersall GJ, Harris MB, McEvoy SD, Richerson DN, Denneris ES, Johnson RL, Chen ZF, Richerson GB (2008) Defects in breathing and thermoregulation in mice with near-complete absence of central serotonin neurons. J Neurosci 28:2495-2505.\n\nHohmann CF, Walker EM, Boylan CB, Blue ME (2007) Neonatal serotonin depletion alters behavioural responses to spatial change and novelty. Brain Res 1139:163-177.\n\nJacobs BL, Azmitia EC (1992) Structure and function of the brain serotonin system. Physiol Rev 72:165-229.\n\nKim JC, Cook MN, Carey MR, Shen C, Regehr WG, Dyamecki SM (2009) Linking genetically defined neurons to behaviour through a broadly applicable silencing allele. Neuron 63:305-315.\n\nKorteweg N, Maia AS, Verhage M, Burbach JP (2004) Development of the mouse hypothalamo-neurohypophysial system in the munc18-1 null mutant that lacks regulated secretion. Eur J Neurosci 19:2944-2952.\n\nLebrand C, Cases O, Adelbrecht C, Doye A, Alvarez C, El Mestikawy S, Seif I, Gaspar P (1996) Transient uptake and storage of serotonin in developing thalamic neurons. Neuron 17:823-835.\n\nMaier DL, Mani S, Donovan SL, Soppet D, Tessarollo L, McCasland JS, Meiri KF (1999) Disrupted cortical map and absence of cortical barrels in growth-associated protein (GAP)-43 knockout mice. Proc Natl Acad Sci U S A 96:9397-9402.\n\nNarboux-Neme N, Pavone LM, Avallone L, Zhuang X, Gaspar P (2008) Serotonin transporter transgenic (SERTcre) mouse line reveals developmental targets of serotonin specific reuptake inhibitors (SSRIs). Neuropharmacology 55:994-1005.\n\nRidet JL, Rajaofetra N, Teilhac JR, Geffard M, Privat A (1993) Evidence for nonsynaptic serotonergic and noradrenergic innervation of the rat dorsal horn and possible involvement of neuron-glia interactions. Neuroscience 52:143-157.\n\nScott MM, Wylie CJ, Lerch JK, Murphy R, Lobur K, Herlitze S, Jiang W, Conlon RA, Strowbridge BW, Deneris ES (2005) A genetic approach to access serotonin neurons for in vivo and in vitro studies. Proc Natl Acad Sci U S A 102:16472-16477.\n\nToonen RF, Wierda K, Sons MS, de Wit H, Cornelisse LN, Brussaard A, Plomp JJ, Verhage M (2006) Munc18-1 expression levels control synapse recovery by regulating readily releasable pool size. Proc Natl Acad Sci U S A 103:18332-18337.\n\nVerhage M, Maia AS, Plomp JJ, Brussaard AB, Heeroma JH, Vermeer H, Toonen RF, Hammer RE, van den Berg TK, Missler M, Geuze HJ, Sudhof TC (2000) Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science 287:864-869.\n\nVoets T, Toonen RF, Brian EC, de Wit H, Moser T, Rettig J, Sudhof TC, Neher E, Verhage M (2001) Munc18-1 promotes large dense-core vesicle docking. Neuron 31:581-591.\n\nWallace JA, Lauder JM (1989) Development of the serotonergic system in the rat embryo: an immunocytochemical study. Brain Res Bull 10:459-479.\n\nWang YM, Gainetdinov RR, Fumagalli F, Xu F, Jones SR, Bock CB, Miller GW, Wightman RM, Caron MG (1997) Knockout of the vesicular monoamine transporter 2 gene results in neonatal death and supersensitivity to cocaine and amphetamine. Neuron 19:1285-1296.\n\nZhao ZQ, Scott M, Chiechio S, Wang JS, Renner KJ, Gereau RWt, Johnson RL, Deneris ES, Chen ZF (2006) Lmx1b is required for maintenance of central serotonergic neurons and mice lacking central serotonergic system exhibit normal locomotor activity. J Neurosci 26:12781-12788.\n\nZhuang X, Masson J, Gingrich JA, Rayport S, Hen R (2005) Targeted gene expression in dopamine and serotonin neurons of the mouse brain. J Neurosci Methods 143:27-32.\n\nChapter 7\n\nTowards a genetic approach to study serotonergic outgrowth and connectivity in vivo\n\nJ.J. Dudok\\textsuperscript{1}, A.J.A. Groffen\\textsuperscript{1}, J. Wortel\\textsuperscript{1} and M. Verhage\\textsuperscript{1}\n\n\\textsuperscript{1}Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, the Netherlands\n\nAbstract\n\nAltered connectivity of the serotonin (5-HT) system might underlie psychopathological processes such as anxiety and depression. Therefore, it would be very interesting to be able to visualize all 5-HT fibers, axons or presynaptic terminals/varicosities in living mouse brain. To specifically label 5-HT neurons and fibers, our aim is to express Cre recombinase in the 5-HT system. We describe here the generation of a tryptophan hydroxylase 2 (Tph2)-iCre targeting construct, the targeting of mouse embryonic stem (ES) cells and the blastocyst injections of positively targeted clones. Moreover, to study 5-HT connectivity in vivo, we generated several transgenic lines using the loxSTOPlox (LSL) methodology.\n\nEventually, we hope to use these techniques to create mice in which specifically in the 5-HT system reporter genes are expressed, so that these can be used to study the 5-HT outgrowth and connectivity in vivo.\n\nIntroduction\n\nThe 5-HT system has its cell bodies in the midbrain raphe nuclei. Although only few 5-HT neurons are present, virtually every brain area receives dense 5-HT innervation which triggers signaling pathways through several different 5-HT receptors. For these reasons the 5-HT system influences a wide variety of processes. Moreover, the 5-HT system is implicated in major depressive disorder and anxiety. One of the current hypotheses for psychopathological processes such as anxiety and depression is that 5-HT connectivity might be affected. Therefore, it would be very interesting to study the connectivity of the 5-HT system in vivo.\n\nTo study 5-HT outgrowth and connectivity in vivo, our aim is to generate a mouse line with expression of improved Cre recombinase (iCre) specifically in 5-HT neurons. As locus for the iCre knockin, we selected the Tph2 gene, which is selectively expressed in the 5-HT neurons in the raphe nuclei (Patel et al., 2004; Zhang et al., 2005; Clark et al., 2006). To be able to specifically express transgenes in the 5-HT system, we relied on the Cre-loxP technique (Tsien et al., 1996; Nagy, 2000). We selected to use iCre instead of Cre, since iCre gives improved Cre expression due to the application of mammalian codon usage (Shimshek et al., 2002).\n\nIn addition we generated several transgenic lines that carry a ubiquitous promoter, a LSL sequence and synaptically or axonally targeted fluorescent fusion proteins. Our ultimate goal is to combine the Tph2-iCre knockin allele with these transgenes, which would constitute an ideal model to study 5-HT\n\nconnectivity and dynamics in the living mouse brain. Here we describe the generation of the Tph2-iCre targeting construct, and the generation of Tph2-iCre chimaeric mice. Additionally, we describe the generation of several LSL transgenic lines.\n\nResults\n\nConstruction and design of the Tph2-iCre targeting construct\nIn order to specifically express iCre in 5-HT neurons, we selected the locus for Tph2 which is localized at chromosome 10 D2. Tph2 is the rate limiting enzyme in 5-HT synthesis and is specifically expressed in 5-HT neurons in the brain. To generate mice with expression of iCre specifically in 5-HT neurons, our approach was to generate a targeting construct in which iCre cDNA is inserted at the endogenous Tph2 start codon. We designed the targeting construct in a way that the 1.2 kb short arm at the 5’ side of the construct stops just upstream of the endogenous Tph2 start codon. The 5.5 kb long arm was designed to start 53 bp downstream of the short arm, so that only 53 bp of endogenous DNA, including the endogenous Tph2 start codon, was deleted in case of homologous recombination (Fig. 1A). In between the short and long arm the iCreVenus cDNA was inserted and a neomycin phosphotransferase cassette (neo) for positive selection, flanked by FRT sites. These can be used to later on remove the neo cassette using the flippase/FRT system (O’Gorman et al., 1991).\n\nIn case of homologous recombination of the targeting construct in ES cells and subsequent generation of Tph2-iCre mice our approach was to use heterozygous Tph2-iCre mice. These contain one functional allele with Tph2 expression, whereas in the other allele, part of the first exon of Tph2 including the start codon is replaced for iCreVenus. In this case these mice will still have a functional Tph2 allele and expression of iCreVenus from the Tph2 promoter (Fig. 1B).\n\nGeneration of Tph2-iCre chimaeric mice\nAfter designing and constructing the Tph2-iCre targeting construct, the construct was sequence verified. This showed that the construct had been correctly made and that the iCreVenus cDNA contained no mutations. The linearized targeting construct was used for ES cell transfection. The antibiotic G418 was used for positive selection of the neomycin resistance marker (Gossler et al., 1986). Eventually five clones were found in which homologous recombination had occurred. Blastocyst injection of clone 1C11 resulted in several chimaeric mice. Although we performed several rounds of chimaeric breeding, we have unfortunately not achieved germline transmission of the Tph2-iCre allele until now.\n\n**Figure 1. Tph2-Cre knockin approach**\n\n(A) The targeting construct was created to replace the start codon of the first exon of Tph2 with a iCre cassette and a neomycin resistance cassette flanked with FRT sites. The targeting construct consisted of a short arm of 1.2 kb, a long arm of 5.5 kb and a iCreVenus and neo cassette. In case of homologous recombination only 53 bp of endogenous DNA is deleted and replaced for the iCreVenus cassette and the neomycin resistance cassette. To identify ES cell clones in which homologous recombination occurred, a PCR screening approach was used. A forward primer (arrow) outside the short arm of the targeting construct and a reverse primer (arrow) in the iCre cassette were used to amplify a 1.5 kb fragment of DNA in case of homologous recombination (dotted line). (B) Approach for Southern blot screening of ES cell clones. An inside and an outside probe were used (grey boxes). For the inside probe, genomic DNA is digested with BsrGI. This will result in a 5.3 kb fragment in WT DNA, but in a 3.3 kb fragment in clones in which homologous recombination occurred due to the presence of an additional BsrGI site in the targeting construct. For the outside probe, genomic DNA is digested with SwaI, which will result in a 14.2 kb fragment in WT DNA, but in a 9.8 kb fragment in clones in which homologous recombination occurred, due to the presence of an additional SwaI site in the targeting construct.\n\n**Generation of LSL transgene lines**\n\nFor construction of LSL transgene lines we used several marker genes tagged with either EGFP or mCherry, which is an improved variant of mRFP that matures more completely and is more photostable (Shaner et al., 2004). As marker genes we used SynaptophysinpHluorin (SypHy), TMSyxEGFP and TMSyxmCherry, which all specifically label axons and presynaptic terminals, and VenusKras4b which is a general membrane marker.\n\n**Figure 2. Validation of transgene constructs**\n\nTo investigate whether the LSL approach worked, we expressed LSL transgene construct with or without iCre cDNA in HEK293 cells. (A,B) No Expression of SypHy or TMSyxEGFP in HEK293 cells after transfection of LSL-SypHy or LSL-TMSyxEGFP without iCre. (C,D) After transfection of LSL-SypHy or LSL-TMSyxEGFP with iCre, SypHy and TMSyxEGFP were expressed in HEK293 cells respectively. (E,F) In neurons, VenusKras4b or TMSyxEGFP are not expressed when transfected without iCre. (G) Upon transfection of LSL-VenusKras4b with iCre, VenusKras4b is expressed in neurons, and the membrane is labeled, also in growth cones (inset). (H) Upon transfection of LSL-TMSyxEGFP with iCre, TMSyxEGFP is expressed in axons, as shown by the absence of MAP2 staining. Immunocytochemistry for the synaptic marker Synapsin shows that there is colocalization between TMSyxEGFP and Synapsin, showing that TMSyxEGFP is targeted to synapses. Scale bars: A-G 10 µm, inset in G 2 µm, H 5 µm.\n\nSypHy is the improved variant of Synaptophysin and detects secretion events due to the increase in pH in synaptic vesicles after exposure to the extracellular medium (Miesenbock et al., 1998; Granseth et al., 2006). TMSyxEGFP contains part of the transmembrane domain of SyntaxinA coupled to EGFP; TMSyxmCherry is the same but with a red fluorophore. VenusKras4b is the EYFP derived Venus fused with the 20 amino acid C-terminal plasma membrane targeting domain of Kras4b (Welman et al., 2000). As ubiquitous promoter either the chicken β-actin promoter or the Thy1 promoter was used (Aigner et al., 1995; Ludin et al., 1996). In between the promoter and the transgene, we inserted a floxed stop sequence in order to acquire iCre mediated transgene expression (Lakso et al., 1992). Some of the constructs were expressed in heterologous cells or neurons to test whether the floxed stop sequence prevented expression and whether co-transfection of iCre resulted in expression of the transgene. Transfection of β-actin-LSL-SypHy or Thy1-LSL-TMSyxEGFP alone in heterologous cells did not result in expression of the transgene (Fig. 2A,B). In contrast, after co-transfection with iCre, there was strong expression of\n\nboth transgenes (Fig. 2C,D). Also in neurons, expression of β-actin-LSL-VenusKras4b or Thy1-LSL-TMSyxEGFP alone did not result in detectable transgene expression (Fig. 2E,F). However, after co-expression with iCre, VenusKras4b expression was detected (Fig. 2G). After co-expression of iCre and Thy1-LSL-TMSyxEGFP neurons were found which expressed TMSyxEGFP and this localized exclusively to axons and synapses as shown by the absence of staining for the dendritic marker MAP2 and co-localization with the synaptic marker Synapsin (Fig. 2H).\n\nThese transgenic constructs were linearized and injected in oocytes to generate seven founder lines of β-actin-LSL-SypHy, five founder lines of β-actin-LSL-VenusKras4b, five founder lines of Thy1-LSL-TMSyxEGFP and eleven founder lines of Thy1-LSL-TMSyxmCherry.\n\n**Screening of expression of transgenes in founder lines**\n\nIn order to screen the expression of the transgenes per founder line, and to find out whether there are lines with expression of the transgene in the raphe nuclei, we first removed the LSL cassette by crossing the founder lines with the CMV-Cre mouse line, a line in which Cre is ubiquitously expressed (Schwenk et al., 1995).\n\nThis breeding strategy resulted in mice in which the LSL cassette was removed in all cells (Fig. 3A). This in contrast to crossing with Tph2-iCre mice, which is expected to result in removal of the LSL cassette only and specifically in 5-HT neurons. Thus, in 5-HT neurons the transgene will be expressed, whereas in all other cells the LSL is still present and the transgene is not expressed (Fig. 3B).\n\nAfter screening all transgenic founder lines, we found several lines in which the transgene was very low and mosaically expressed. For β-actin-LSL-SypHy we found one founder line with low expression in the Purkinje cell layer of the cerebellum, and two founder lines with expression in the hippocampal CA1-CA3 region and dentate gyrus (data not shown). For β-actin-LSL-VenusKras4b we only found very low and mosaic expression of the transgene (data not shown). For Thy1-LSL-TMSyxEGFP and TMSyxmCherry, we found one line with TMSyxEGFP ubiquitously expressed in neocortex, and one line with TMSyxmCherry mosaically expressed in cortex and hippocampus (data not shown). Unfortunately, not one founder line did show expression of the transgene in midbrain or raphe nuclei areas.\n\n**Discussion**\n\nHere we have described the generation of Tph2-iCre chimaeric mice and several LSL transgenic mouse lines. We would like to eventually create mice with expression of a marker gene specifically in 5-HT neurons. This would allow us to\n\nFigure 3. Approach for screening expression pattern of transgenic mice\n\n(A) To screen the expression pattern of LSL transgenic mice, these mice were crossed with a CMV-Cre mouse, which ubiquitously expressed Cre recombinase. This results in mice in which the LSL is removed in all cells, thus allowing an analysis of transgene expression pattern. (B) Once LSL mice are identified with expression in the 5-HT system, they can be used to cross with Tph2-Cre mice to drive transgene expression specifically in the 5-HT system.\n\nWe choose to target iCreVenus to the first exon of Tph2, so that the endogenous start codon and small part of the first exon of Tph2 is replaced for iCreVenus. In this way we anticipated that Tph2 expression relies on endogenous Tph2 promoter activity and is specifically expressed in 5-HT neurons in which Tph2 is expressed. In contrast to this approach we could also have chosen to target iCreVenus to the 3’ side of the Tph2 open reading frame using an internal ribosomal entry site (IRES). We did, however, not use this approach because the expression of a reporter gene behind an IRES is often rather low. Although using our approach we sacrifice one functional Tph2 allele, we expect that this will not significantly affect brain development and behaviour in Tph2 heterozygotes. Deletion of both Tyrosine hydroxylase alleles results in mid-gestational lethality.\n\nHowever, TH heterozygotes showed no obvious anatomical or behavioural defects (Zhou et al., 1995). Therefore, we reason that our approach will not significantly affect brain development or behaviour.\n\nUsing a transgenic approach with a LSL cassette to allow transgene expression only in cells in which iCre is also present, we aimed to express these marker genes specifically in 5-HT neurons. However, none of the founder lines we screened showed expression of the transgene construct in the midbrain or raphe nuclei. It has been shown for both the chicken β-actin and mouse Thy1 promoter that these are able to induce expression of transgenes in the cortex and hippocampus (Feng et al., 2000; Roelandse et al., 2003). However, it appears more difficult to also induce expression in midbrain and raphe nuclei for these promoters, since none of our transgenic lines expressed the transgene in these regions. Recently, it was shown that a 1.8 kb region immediately upstream the Pet-1 gene is sufficient to acquire 5-HT neuron specific expression of a transgene (Scott et al., 2005a; Scott et al., 2005b). Therefore, in new experiments we could try to rely on the Pet-1 promoter to express transgenes in the 5-HT neurons. Possibly, future experiments with novel blastocyst injections of Tph2-iCre targeted ES clones and transgene injections using the Pet-1 promoter will result in a Tph2-iCre mouse line and transgenic mouse lines with expression of reporter genes in 5-HT neurons. These mouse lines can then be used to create mouse lines with expression of reporter genes specifically in 5-HT neurons to possibly resolve the longstanding question whether psychopathological processes are a result of altered 5-HT network connectivity.\n\n**EXPERIMENTAL PROCEDURES**\n\n*Generation of Tph2-iCre targeting construct*\n\nThe Tph2-iCre targeting construct was designed in such a way that in the wildtype Tph2 locus, 53 bp in exon 1 are deleted, including the start codon, and replaced for a iCreVenus cassette. The Tph2-iCre targeting construct was generated as follows. The 1.2 kb short arm upstream the endogenous start codon of Tph2 was generated with PCR primers forward 5’CATGCATAGAAAGTGTCCAG3’ reverse 5’TTCTITCTCAGCAGCAGGGG3’. The 5.5 kb long arm starting 34 bp behind the Tph2 start codon was generated with PCR primers forward 5’GGGCCAGGAGAGGGTTGTCTCTTGGAITICTG3’ and reverse 5’CCGTACATGAGGACTCGGTGAGAGCATCTG3’. Both arms were subcloned in the pGEM-T easy vector (Promega). In the piCreVenus vector the NotI site behind Venus was removed and a linker containing SwaI, BsiWI, MluI and NotI sites was placed in the AflII site behind the polyA tail. The short arm was cloned sticky using EcoRI in front of iCreVenus in the modified iCreVenus vector. A neomycin resistance cassette flanked by FRT sites was removed from\n\nthe ploxPFRTNEOFRTloxP vector (a kind gift from Jeroen Pasterkamp, Utrecht) using partial digestion with BamHI and cloned blunt in the unique SpeI site behind the long arm in the pGEM-T easy vector. Finally, the FRTNEOFRT long arm Tph2 was cloned sticky behind the short arm iCreVenus using NotI. The construct was verified by sequence analysis. The construct was linearized with KspI for targeting.\n\n**Targeting ES cells**\n\nIn order to target ES cells with the Tph2-iCre targeting construct, approximately $1 \\times 10^6$ ES cells (line E14.1) were electroporated with 100 µg of linearized Tph2-Cre targeting construct and plated on murine embryonic feeders. The subsequent day, neomycin selection was started by adding 400 µg/ml G418. After 10 days, non targeted clones had died and 700 surviving clones were picked and individually grown. DNA of all these clones was screened to distinguish between clones in which the targeting construct was randomly inserted and clones in which homologous recombination had occurred.\n\nTo select for clones in which homologous recombination occurred, the clones were screened with a nested PCR approach using the following primers: 1st PCR forward 5’ CACTTTTCTTCACACCCITCTC3’ and reverse 5’ CCTGACTTTCATCAGAGGTGGCATC3’ nested PCR forward 5’ TTATTTGTGTGAGACACGTGA3’ and reverse 5’ TTTTGGTGCAACAGTCAGCAG3’ which results in a 1.6 kb PCR fragment in positively targeted clones. From the 700 targeted clones, eventually 5 clones were selected in which homologous recombination occurred designated 2F11, 1A1, 1C11, 4E1 and 2E11. Homologous recombination was verified using a 5’ primer outside the forbidden construct and sequence analysis of this region. ES cell clones 1C11 and 4E1 were injected in blastocysts to generate chimaeric mice.\n\n**Generation of transgenic mice**\n\nIn order to generate transgenic mice for conditional expression of reporter genes, the LSL cassette derived from the Pbs302 vector was used (Sauer, 1993). As promoter, the chicken β-actin and regulatory elements of the mouse Thy1 gene were used. As reporter genes, SypHy (a kind gift from Andreas Jeromin), VenusKras4b, TMSyx (transmembrane Syntaxin1A) EGFP and TMSyxmCherry were used. The constructs were cloned as follows. For β-actin-LSL-SypHy, the LSL cassette was excised from the Pbs302 vector using EcoRI and SpeI and cloned blunt in the unique XbaI site in the pβ-actin-SypHy vector. For β-actin-LSL-VenusKras4b, the LSL cassette was excised from the Pbs302 vector using EcoRI and SpeI and cloned blunt in the BglII site in the pVenusKras4b vector. Subsequently, LSL-VenusKras4b was excised using NheI and XbaI and cloned blunt in BlnI and XbaI in pβ-actin-EGFP to replace EGFP. Both β-actin-LSLSypHy and β-actin–LSL-VenusKras4b were linearized with AatII for transgene injection. For Thy1-LSL-TMSyxEGFP, TMSyxEGFP was excised with NheI and SspI and cloned blunt in the unique NotI site in pThy1-LSL (a kind gift from Christiaan Levelt, Amsterdam). To clone TMSyxmCherry, TMSyx was excised from pTMSyxEGFP using NheI and EcoRI and cloned blunt in pmCherry-N3 in the EcoXI site. Then TMSyxmCherry was excised using NheI and SspI and cloned blunt in the NotI site the in pThy1-LSL vector. Both Thy1-LSL-TMSyxEGFP and Thy1-LSL-TMSyxmCherry were linearized with AatII for transgene injection. To create transgenic mice, linearized DNA constructs were injected in the pronucleus of fertilized C57BL/6 oocytes.\n\n**Transfection of transgene plasmids in heterologous cells and neurons**\n\nTransgene plasmids were transfected in HEK293 cells or neurons using Lipofectamine 2000 reagent according to the manufacturer’s protocol. HEK293 cells and neurons were cultured as has been published before (Groffen et al., 2004; de Wit et al., 2006). In HEK293 cells, one day post-transfection expression of transgene constructs was visualized using an inverted microscope fitted with fluorescence filters. In neurons and HEK293 cells, expression of transgene constructs was analyzed using a LSM510 confocal laser scanning microscope (Zeiss, B.V. The Netherlands). Immunocytochemical analysis of neurons was performed as has been described before using monoclonal MAP2 (Chemicon) and polyclonal Synapsin I (E028) as primary antibodies and goat-anti-mouse Alexa 543/644 and goat-anti-rabbit Alexa 543 (Molecular Probes) (de Wit et al., 2006).\n\n**Screening of expression pattern**\n\nIn order to investigate the expression pattern of the transgene construct per transgenic line, the founder lines were crossed with the 129-Cre mouse line, which has ubiquitous expression of Cre (Schwenk et al., 1995). Pups positive for Cre and the transgene were used for in vivo paraformaldehyde (PFA) perfusion between two and four months age. For in vivo PFA fixation, mice were anaesthetized and transcardially perfused with 4% PFA dissolved in phosphate buffered saline (PBS) (pH 7.4). After fixation, brains were removed and postfixed in 4% PFA for 24 hours at 4°C. Subsequently, brains were cryoprotected in increasing concentrations of sucrose dissolved in PBS at 4°C. For screening of expression pattern of the transgenes, the fixed and cryoprotected brains were sagitally cut in 30 µm slices using a microtome. In case the EGFP fluorescence was too low, Immunohistochemistry was performed using the free floating method. To this end, slices were first blocked using blocking solution which contained 0.1% Triton X-100 and 10% normal goat serum for two hours under gentle agitation. Slices were incubated overnight at 4°C using primary antibody,\n\nwashed three times two hours the next day with PBS 0.1% Triton X-100 and incubated with the secondary antibody for two hours. After washing for three times two hours in PBS, slices were positioned on a glass slide, allowed to dry and overlaid with a glass coverslip using Dabco Mowiol. All reactions were carried out at room temperature unless otherwise stated.\n\n**Analysis of transgene expression**\n\nTo analyze the expression pattern of the transgene constructs, brain slices were analyzed using an inverted microscope equipped with fluorescence filters and a Sony camera. A LSM510 confocal laser scanning microscope was used to investigate the expression of the transgene constructs in individual neurons.\n\n**References**\n\nAigner L, Arber S, Kapfhammer JP, Laux T, Schneider C, Botteri F, Brenner HR, Caroni P (1995) Overexpression of the neural growth-associated protein GAP-43 induces nerve sprouting in the adult nervous system of transgenic mice. Cell 83:269–278.\n\nClark MS, McDevitt RA, Neumaier JF (2006) Quantitative mapping of tryptophan hydroxylase-2, 5-HT1A, 5-HT1B, and serotonin transporter expression across the anteroposterior axis of the rat dorsal and median raphe nuclei. J Comp Neurol 498:611–623.\n\nde Wit J, Toonen RF, Verhaagen J, Verhage M (2006) Vesicular trafficking of semaphorin 3A is activity-dependent and differs between axons and dendrites. Traffic 7:1060–1077.\n\nFeng G, Mellor RH, Bernstein M, Keller-Peck C, Nguyen QT, Wallace M, Nerbonne JM, Lichtman JW, Sanes JR (2000) Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28:41–51.\n\nGossler A, Doetschman T, Korn R, Serfling E, Kemler R (1986) Transgenesis by means of blastocyst-derived embryonic stem cell lines. Proc Natl Acad Sci U S A 83:9065–9069.\n\nGranseth B, Odermatt B, Royle SJ, Lagnado L (2006) Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron 51:773–786.\n\nGroffen AJ, Brian EC, Dudok JJ, Kampmeijer J, Toonen RF, Verhage M (2004) Ca(2+)-induced recruitment of the secretory vesicle protein DOC2B to the target membrane. J Biol Chem 279:23740–23747.\n\nKerr JN, Denk W (2008) Imaging in vivo: watching the brain in action. Nat Rev Neurosci 9:195–205.\n\nLakso M, Sauer B, Mosinger B, Jr., Lee EJ, Manning RW, Yu SH, Mulder KL, Westphal H (1992) Targeted oncogene activation by site-specific recombination in transgenic mice. Proc Natl Acad Sci U S A 89:6232–6236.\n\nLudin B, Doll T, Meili R, Kaech S, Matus A (1996) Application of novel vectors for GFP-tagging of proteins to study microtubule-associated proteins. Gene 173:107–111.\n\nMiesenbock G, De Angelis DA, Rothman JE (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394:192–195.\n\nNagy A (2000) Cre recombinase: the universal reagent for genome tailoring. Genesis 26:99–109.\n\nO’Gorman S, Fox DT, Wahl GM (1991) Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science 251:1351-1355.\n\nPatel PD, Pontrullo C, Burke S (2004) Robust and tissue-specific expression of TPH2 versus TPH1 in rat raphe and pineal gland. Biol Psychiatry 55:428-433.\n\nRoelandse M, Welman A, Wagner U, Hagmann J, Matus A (2003) Focal motility determines the geometry of dendritic spines. Neuroscience 121:39-49.\n\nSauer B (1993) Manipulation of transgenes by site-specific recombination: use of Cre recombinase. Methods Enzymol 225:890-900.\n\nSchwenk F, Baron U, Rajewsky K (1995) A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells. Nucleic Acids Res 23:5080–5081.\n\nScott MM, Krueger KC, Deneris ES (2005a) A differentially autoregulated Pet-1 enhancer region is a critical target of the transcriptional cascade that governs serotonin neuron development. J Neurosci 25:2628–2636.\n\nScott MM, Wylie CJ, Lerch JK, Murphy R, Lobur K, Herlitze S, Jiang W, Conlon RA, Strowbridge BW, Deneris ES (2005b) A genetic approach to access serotonin neurons for in vivo and in vitro studies. Proc Natl Acad Sci U S A 102:16472–16477.\n\nShaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22:1567–1572.\n\nShimshick DR, Kim J, Hubner MR, Spergel DJ, Buchholz F, Casanova E, Stewart AF, Seeburg PH, Sprengel R (2002) Codon-improved Cre recombinase (iCre) expression in the mouse. Genesis 32:19–26.\n\nTsien JZ, Chen DF, Gerber D, Tom C, Mercer EH, Anderson DJ, Mayford M, Kandel ER, Tonegawa S (1996) Subregion- and cell type-restricted gene knockout in mouse brain. Cell 87:1317–1326.\n\nWelman A, Burger MM, Hagmann J (2000) Structure and function of the C-terminal hypervariable region of K-Ras4B in plasma membrane targeting and transformation. Oncogene 19:4582–4591.\n\nZhang X, Gainetdinov RR, Beaulieu JM, Sotnikova TD, Burch LH, Williams RB, Schwartz DA, Krishnan KR, Caron MG (2005) Loss-of-function mutation in tryptophan hydroxylase-2 identified in unipolar major depression. Neuron 45:11-16.\n\nZhou QY, Quaife CJ, Palmiter RD (1995) Targeted disruption of the tyrosine hydroxylase gene reveals that catecholamines are required for mouse fetal development. Nature 374:640-643.\n\nChapter 8\n\nGeneral discussion\n\nGeneral discussion\n\nThe aim of the studies described in this thesis was to investigate different cellular aspects of the outgrowth and connectivity of the brain 5-HT system. The 5-HT system has the remarkable feature that although there are only few 5-HT neurons, localized in the midbrain raphe nuclei, virtually every brain area receives dense 5-HT innervations. These innervations contain several 5-HT varicosities, where 5-HT is released both synaptically and as volume transmission. Due to several 5-HT receptors which are present in the brain, 5-HT has many functions and influences a wide variety of processes. Moreover, the 5-HT system is implicated in several psychopathological processes such as anxiety and depression. Despite the high social and economical importance of these conditions, there is a lot of uncertainty about the involved mechanisms. Therefore, a further cellular characterization of the outgrowth and connectivity of the 5-HT system is of high interest. We used several experimental approaches to study different cellular aspects of the outgrowth and connectivity of the 5-HT system.\n\nIn chapter 2 we used organotypic slice cultures to study 5-HT outgrowth and connectivity and the effect of pharmacological manipulations in vitro. This revealed that the 5-HT neurite density was not affected by blocking the SERT or application of a 5-HT$_{1A}$ agonist, but the density was reduced by application of a 5-HT$_2$ agonist. This was presumably due to inhibited outgrowth of the 5-HT system.\n\nIn chapter 3, we investigated the trafficking of SERT. We expressed SERT tagged with the fluorescent protein mCherry in hippocampal neurons. We showed that mCherry-SERT is transported in vesicles which displayed a dynamic trafficking.\n\nIn chapter 4 we used the same approach as in chapter 3, but now we studied the localization and trafficking of Tph2, the rate limiting enzyme in 5-HT synthesis. We tagged Tph2 with EGFP and studied the localization of Tph2-EGFP in hippocampal neurons. In axons Tph2-EGFP displayed a punctate distribution and co-localized with Synaptophysin-mCherry, a marker for synaptic terminals, suggesting that Tph2-EGFP localized to synaptic terminals.\n\nIn chapter 5 we studied the effect of a SNP in the presynaptic gene Piccolo on the trafficking and localization of SERT. Knockdown of Piccolo in neurons did not affect mCherry-SERT trafficking. The SNP in Piccolo possibly affects the localization of SERT, although future research is needed to further resolve this issue.\n\nIn chapter 6 we crossed SERT-Cre mice with floxed Munc18-1 mice to specifically abolish Munc18-1 in SERT expressing neurons. This resulted in early postnatal lethality and a rapid degeneration of the 5-HT system. We used heterozygous mice to study the effect of removal of one Munc18-1 allele in SERT expressing neurons on behaviour using a phenotyping experiment. Basal behaviour, conditioning and anxiety related behaviour were not affected.\n\nIn chapter 7 we describe our approach to create mice which have Cre expression specifically in 5-HT neurons. Moreover, we show that we have generated transgenic lines with a floxed stop cassette which can be used for spatial regulation of transgene expression upon Cre mediated recombination. In the future, these mice can be used to specifically label 5-HT neurons, or to specifically remove a gene in 5-HT neurons.\n\nRoles of 5-HT in outgrowth, connectivity and behaviour\n\nOne of the current hypotheses for psychopathological processes such as depression is that altered 5-HT network connectivity might be involved in these processes. Furthermore, it is hypothesized that antidepressants such as fluoxetine might restore aberrant 5-HT network connectivity (Castren, 2005). We used organotypic slices to study these processes in an in vitro model system. Our data showed that fluoxetine did not affect 5-HT innervation density.\n\nIn adult rats, chronic fluoxetine application did result in an increased density of 5-HT projections in the forebrain (Zhou et al., 2006). Interestingly, an in vivo study using chronic fluoxetine treatment either during development or during adulthood, showed that when applied during adulthood, fluoxetine did not affect behaviour. In contrast, fluoxetine treatment during development resulted in increased anxiety-related behaviour during adulthood (Ansorge et al., 2004; Ansorge et al., 2008). Our data suggests, although it is in vitro, that this effect of fluoxetine on behaviour does not result from an altered innervation of 5-HT projections. However, in our model system we were unable to investigate whether the number of 5-HT varicosities was affected by fluoxetine application. Thus, although 5-HT projection density is unaffected, connectivity of the 5-HT system might be affected due to an altered number of 5-HT varicosities or an altered rate of 5-HT release.\n\nAt least in the snail Helisoma, 5-HT acts as an autotrophic factor on its own outgrowth; 5-HT applied at µM concentrations to the growth cones of 5-HT neurons resulted in an abrupt cessation of growth cone motility and outgrowth (Haydon et al., 1984; Haydon et al., 1987).\n\nThe 5-HT\\textsubscript{IA} and 5-HT\\textsubscript{3C} receptor might mediate this effect of 5-HT, as these receptors are expressed in the DRN (Wright et al., 1995; Clemett et al., 2000). Our data showed that 5-HT\\textsubscript{IA} receptor activation slightly increased 5-HT neurite\n\ndensity whereas chronic activation of the 5-HT\\textsubscript{2} receptor decreased 5-HT neurite density. It is tempting to speculate that these receptors act in concert during outgrowth to regulate a proper development of 5-HT projections towards target areas mediated by 5-HT. Interestingly, 5-HT\\textsubscript{1A} and 5-HT\\textsubscript{2} receptors have different affinities for 5-HT; 5-HT\\textsubscript{1A} receptors are high affinity 5-HT receptors which have an EC\\textsubscript{50} value in the nM range, whereas 5-HT\\textsubscript{2} receptors are low affinity 5-HT receptors, which have an EC\\textsubscript{50} value in the \\(\\mu\\)M range (Fargin et al., 1989; Marek and Aghajanian, 1994; Azmitia, 2001). The 5-HT\\textsubscript{3C} receptor is present on GABA-ergic cells in the DRN, and it is unknown whether this receptor is present on 5-HT neurons (Serrats et al., 2005). This suggests that the inhibitory effect of 5-HT\\textsubscript{3C} receptor activation is not a direct effect on 5-HT neurons, but might be an indirect effect via GABA-ergic neurons. Indeed, in vivo data supports that 5-HT\\textsubscript{3C} receptor activation inhibits neuronal firing of 5-HT neurons via GABA-ergic neurons (Boothman et al., 2006). Thus, in the DRN \\(\\mu\\)M levels of 5-HT might inhibit outgrowth, whereas nM levels might stimulate outgrowth of 5-HT innervations.\n\nMice in which the 5-HT\\textsubscript{1A} receptor is deleted have increased anxiety-related behaviour, as they spend less time in the center in the open field test and avoid the open arms in the elevated plus maze (Parks et al., 1998; Ramboz et al., 1998). Overexpression of the 5-HT\\textsubscript{1A} receptor, on the other hand, results in reduced anxiety-related behaviour (Deng et al., 2007). A very elegant study was performed by Gross and colleagues, who showed that restoring 5-HT\\textsubscript{1A} receptor expression in the forebrain during development, but not during adulthood rescued the behavioural phenotype of 5-HT\\textsubscript{1A} KO mice. (Gross et al., 2002). Clearly, the 5-HT\\textsubscript{1A} receptor is important during development to establish normal anxiety-like behaviour. Unfortunately, no studies were performed to resolve whether 5-HT connectivity is affected in these mice.\n\nDeletion of the 5-HT\\textsubscript{3C} receptor, on the other hand, results in compulsive-like behaviour, and these mice are obese and suffer from seizures (Tecott et al., 1995; Chou-Green et al., 2003). Unfortunately, also for these mice it is not known whether 5-HT connectivity is affected.\n\n**Trafficking of SERT and Tph2 in neurons**\n\nAs the 5-HT system has long projections to several brain areas, proteins involved in 5-HT synthesis and reuptake are transported over large distances towards axonal growth cones and varicosities. In chapter 3 and 4 we focused on SERT and Tph2 transport in hippocampal neurons and observed that SERT and Tph2 displayed a distinct distribution. SERT was predominantly localized extrasynaptically and some SERT puncta localized in the vicinity of presynaptic terminals. In contrast, Tph2 accumulated in puncta which colocalized with\n\nsynaptic markers. Obviously, Tph2 is required at presynaptic terminals and varicosities for local synthesis of 5-HT. Indeed in 5-HT neurons Tph is enriched in presynaptic terminals (Pickel et al., 1976; Pickel et al., 1977). On the other hand, SERT is localized extra-synaptically to transport extracellular 5-HT which has diffused away from its release sites back into the neuron. An electron microscopy study showed that the majority of SERT in 5-HT neurons was located outside synapses (Zhou et al., 1998). Thus, the distribution of Tph2 and SERT which we found in hippocampal neurons seems to mimic the distribution of endogenous SERT and Tph2 in 5-HT neurons.\n\nWe also showed that SERT and Tph2 are transported in distinct ways through the neuron. In contrast to Tph2, SERT displayed a vesicular trafficking. Apparently, SERT contains a signal peptides which mediates the transport in vesicles. Alternatively, SERT contains transmembrane domains, which requires that SERT is transported in vesicles as these lipophilic domains are insoluble.\n\nAs SERT is transported in secretory vesicles, there should be one or more motor proteins responsible for this transport. There are several families of motor proteins which are involved in the transport of several different proteins through neurites (for a review see (Schlager and Hoogenraad, 2009). The average velocity of SERT transport vesicles and the fact that these do not display a preference for direction of movement is in accordance with the average velocity of transport vesicles containing glycine (Maas et al., 2006). Also in this study, it was shown that dynein motors are responsible for the retrograde transport of glycine (Maas et al., 2006). Dynein motors are involved in transporting other cargo molecules through neurons, such as the Trkb receptor and Bassoon (Yano et al., 2001; Fejtova et al., 2009). The trafficking dynamics of Trkb and Bassoon are comparable with the trafficking dynamics of SERT, strongly suggesting that the dynein motor protein is responsible for SERT transport (Ha et al., 2008; Fejtova et al., 2009).\n\n**A SNP in Piccolo associates with depression**\n\nRecently, a SNP in Piccolo was found which was associated with depression (Sullivan et al., 2008; Bochdanovits et al., 2009). Piccolo is a structural component of the presynaptic cytoskeletal cytomatrix (PCM) and structurally related to Bassoon, another component of the PCM (Cases-Langhoff et al., 1996; tom Dieck et al., 1998; Fenster et al., 2000). Piccolo is a multidomain zinc finger protein which contains several coiled coil domains and a PDZ domain (Fenster and Garner, 2002). Piccolo also contains two C2 domains, C2A and C2B, of which C2A functions as a low-affinity calcium sensor in presynaptic terminals (Gerber et al., 2001). The SNP in Piccolo was found to be closely localized to the C2A domain (Sullivan et al., 2008).\n\nIn the presynaptic terminal, Piccolo is involved in different processes. First of all, Piccolo, together with Bassoon, is involved in presynaptic terminal formation. Piccolo and Bassoon are transported in Piccolo-Bassoon transport vesicles together with other constituents of presynaptic terminals, and two or three of these vesicles are sufficient to form an active synapse (Zhai et al., 2001; Shapira et al., 2003; Tao-Cheng, 2007). In chapter 5 we have shown that 5-HT neurons contain Piccolo. Thus, in 5-HT varicosities Piccolo might be involved in formation of varicosities, regulating 5-HT secretion and regulation of SERT internalization. We have shown that knockdown of Piccolo did not affect SERT trafficking dynamics, suggesting that Piccolo is not involved in SERT transport. Secondly, since Piccolo is present in the presynaptic terminal, Piccolo might be involved in the modulation of synaptic vesicle exocytosis. Indeed, Piccolo influences synaptic function by negatively regulating synaptic vesicle exocytosis, possibly via a calmodulin kinase II-dependent mechanism, mediated through the modulation of Synapsin1A dynamics (Leal-Ortiz et al., 2008). Thus, Piccolo might be involved in modulating 5-HT release from synaptic vesicles. However, the majority of 5-HT is secreted as bulk release from large dense core vesicles (LDCVs) (Bruns and Jahn, 1995). Therefore, although it might be that a SNP in Piccolo could modulate 5-HT release from synaptic vesicles, this would probably only have minor effects on extracellular 5-HT levels.\n\nThirdly, it was shown in heterologous cells that Piccolo is involved in the regulation of DAT internalization. Specifically, the C2A domain was involved in this process (Cen et al., 2008). As DAT and SERT belong to the same gene family and are structurally related it is conceivable that Piccolo could be involved in SERT internalization. In chapter 5, we have investigated in heterologous cells whether the SNP in the C2A domain of Piccolo alters the localization of mCherry-SERT. This showed that expression of the C2A domain of Piccolo containing the SNP seemed to alter the membrane distribution of SERT compared to the control C2A variant. It would be very interesting to further investigate whether the SNP in the C2A domain affects varicosity density of 5-HT projections or regulation of SERT membrane localization. If so, this would be the first cellular evidence that altered 5-HT network connectivity might be involved in the etiology of depression.\n\n**Deletion of Munc18-1 in SERT expressing neurons results in early postnatal lethality**\n\nOur data in chapter 6 showed that deletion of Munc18-1 in SERT expressing neurons resulted in early postnatal lethality. Although neurons are initially formed and there is normal brain development, deletion of Munc18-1 eventually results in degeneration (Verhage et al., 2000; Heeroma et al., 2003; Korteweg et\n\nOur data showed that also 5-HT neurons in which Munc18-1 is deleted initially develop and grow out projections which innervated the cortex. However, after the initial generation, the 5-HT neurons undergo rapid degeneration, and at P2 only very few 5-HT neurons were left. This is the first mouse model which has a complete removal of the 5-HT system. This in contrast to the Tph2 KO mouse, which has no 5-HT synthesis in the central nervous system, but still 5-HT neurons and projections are unaffected.\n\nDeletion of Tph2 resulted in ~50% lethality in the first four weeks (Alenina et al., 2009). Surprisingly, however, another research group did not find any increased lethality in Tph2−/− mice ((Savelieva et al., 2008) and personal communication). Deletion of Pet1 and Lmx1b results in mice in which ~70% and >99% of 5-HT neurons fail to survive respectively. Concerning the marked reduction in 5-HT neurons and projections in these mice, it is surprising that these mice only display subtle phenotypes (Ding et al., 2003; Hendricks et al., 2003; Zhao et al., 2006)\n\nUnfortunately, as also thalamocortical neurons and some hippocampal neurons briefly express SERT, and thus Cre recombinase, during development, it is difficult to assess whether the observed phenotype is attributable to a degenerating 5-HT system. Therefore, crossing floxed Munc18-1 mice with Pet-Cre or Tph2-Cre mice which express Cre exclusively in 5-HT neurons should finally resolve whether complete removal of the 5-HT system affects brain development and survival.\n\n**Altered connectivity of the 5-HT system in human psychopathological processes?**\n\nDescriptive studies on the effect of psychopathological processes on human 5-HT connectivity rely on postmortem brain tissue. The last decade, however, more and more human imaging studies are performed using functional magnetic resonance imaging (fMRI) and positron emission tomographic (PET) imaging to resolve whether brain circuitry is affected in psychopathological processes associated with the 5-HT system.\n\nResearch on postmortem brain material from depressive people who committed suicide predominantly focused on mRNA expression levels or binding of radiolabeled 5-HT to 5-HT receptors. Several studies showed that there is an elevated 5-HT2A and 5-HT2C receptor abundance in the frontal cortex of depressed suicide victims (Arango et al., 1990; Xu and Pandey, 2000; Prasad et al., 2005). Other studies reported either reduced or increased binding or availability of 5-HT1A receptors, depending on the brain region studied (Drevets et al., 2007). From these data, however, it cannot be deduced whether the altered\n\nlevels of mRNA expression or altered levels of receptor binding are the result of an alteration in expression levels or of an altered 5-HT projection density.\n\nPET imaging is used to measure cerebral blood flow and rate of glucose metabolism as a measure for brain activity. Functional MRI is used to measure blood oxygen level dependent (BOLD) fluctuations which are a measure for activity and connectivity of brain regions. PET and fMRI studies have reported a decreased activation of cortical areas such as the dorsolateral prefrontal cortex and the anterior cingulate cortex in depressed subjects (Drevets et al., 1997; Mayberg et al., 1999). On the other hand, other studies reported an increased activation of limbic areas such as the amygdala in depression (Sheline et al., 2001; Siegle et al., 2002). Several studies have explored whether antidepressants restore aberrant activation patterns observed in depression. Antidepressant treatment increases glucose metabolism in prefrontal cortex and reduces activity in limbic areas (Sheline et al., 2001; Harvey et al., 2004b).\n\nAlthough it is difficult to conclude from these imaging studies if altered brain network connectivity is a cause or consequence of the depression, one could at least speculate that connectivity changes occur at some stage. Clearly, more research is needed to further elucidate whether network connectivity alterations underlie psychopathological processes associated with the 5-HT system.\n\n**Future perspectives**\n\nThere are a number of approaches which could be taken to further elucidate the connectivity of the 5-HT system, the relation between the 5-HT system and behaviour, and the involvement of the 5-HT system in psychopathology. First of all, it would be of great interest to label all 5-HT neurons and neurites with fluorescent markers. This could be achieved by creating knockin mice using the Tph2 promoter to drive expression of Cre and to cross these mice with floxed stop fluorescent reporter mice. Additionally, it has already been shown that Pet-Cre mice express Cre specifically in 5-HT neurons, and crossing with reporter mice showed that >99% of 5-HT neurons were labeled (Scott et al., 2005). These mice could then be used to reconstruct the connectivity of the 5-HT system. Secondly, there are several subpopulations of 5-HT neurons that can be distinguished based upon their precursor genes (Jensen et al., 2008). If Cre mouse lines could be made for these specific subpopulations, these could be crossed with reporter mice or with the so-called brainbow mice (Livet et al., 2007). It would be very interesting to find out whether different subpopulations of 5-HT neurons innervate different brain regions, and are involved in different aspects of behaviour.\n\nAdditionally, these mouse lines could be used to specifically inhibit neurotransmission from these populations of 5-HT neurons, or to inhibit neurotransmission from all 5-HT neurons using Tph2-Cre mice. Expressing tetanus toxin in neurons blocks synaptic activity since tetanus toxin cleaves VAMP2 which is a protein necessary for vesicle fusion. It has been shown that this approach works in vivo, e.g. in the retina, but also for 5-HT neurons (Kerschensteiner et al., 2009; Kim et al., 2009). The work of Kim et al showed that silencing a subset of 5-HT neurons resulted in mice with behavioural alterations (Kim et al., 2009). These mice could be used to further explore the role of the 5-HT system and subsets of the 5-HT system on behaviour and to what extend these contribute to pathophysiological processes such as anxiety and depression. The other way around, these mice could be used to study whether silencing of 5-HT neurons affects 5-HT connectivity, i.e. 5-HT projection and varicosity density.\n\nA third interesting future direction is to use mice which express Cre specifically in 5-HT neurons to inactivate 5-HT autoreceptors only in 5-HT neurons. A lot of research has been conducted in elucidating the role of 5-HT autoreceptors in determining 5-HT transmission and behaviour. The 5-HT\\textsubscript{IA} and 5-HT\\textsubscript{IB} knockout mice display altered anxiety-related and aggression-related behaviours. However, in these mice it is difficult to assess whether these phenotypes observed are due to deletion of 5-HT autoreceptors, or deletion of these receptors from non-5-HT neurons. A far more elegant approach, however, would be to generate floxed 5-HT\\textsubscript{IA} and 5-HT\\textsubscript{IB} mice and cross these with Pet-Cre or Tph2-Cre mice to selectively inactivate these receptors only in 5-HT neurons. This will allow to distinguish between the role of 5-HT autoreceptors and heteroreceptors, and to investigate to what extend the deletion of 5-HT autoreceptors affects mouse behaviour.\n\nRecently, also a tamoxifen inducible Tph2-Cre mouse line has been generated, which allows to temporally regulate Cre expression in 5-HT neurons (Weber et al., 2009). This mouse line allows to investigate whether deleting a gene in the 5-HT system either during development or during adulthood affects 5-HT outgrowth, connectivity or mouse behaviour. Finally, it would be interesting to further investigate whether connectivity is affected in anxiety and depression in human subjects. 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J Neurochem 96:396-406.\n\nNederlandse samenvatting\n\nCellulaire karakterisatie van de uitgroei en connectiviteit van het serotonine systeem in het brein\n\nNederlandse samenvatting\n\nDe menselijke hersenen bestaan uit ongeveer 100 miljard zenuwcellen (neuronen). Neuronen zijn de basis elementen die bijvoorbeeld gedrag en geheugen mogelijk maken. Neuronen hebben een aantal uitlopers waarmee ze met elkaar kunnen communiceren. Een neuron heeft een axon, waarmee het signalen naar een ander neuron kan versturen, en meerdere dendrieten, waarmee een neuron signalen van andere neuronen kan ontvangen. Neuronen communiceren met elkaar door het uitscheiden van signaalstoffen, neurotransmitters genaamd. Deze neurotransmitters kunnen binden aan bepaalde receptoren in een ontvangende cel, waardoor een signaal doorgegeven kan worden. De punten waar twee cellen met elkaar communiceren worden synapsen genoemd. Hier wordt door de zendende cel een presynaptische terminal gevormd op de ontvangende cel. Vanuit deze presynaptische terminal worden de neurotransmitters uitgescheiden, en deze worden opgevangen door receptoren in de postsynaptische terminal in de ontvangende cel.\n\nEr zijn veel verschillende soorten neuronen, deze worden onder andere in groepen ingedeeld door de neurotransmitter die ze uitscheiden. Een van de groepen neuronen zijn de zogenaamde serotonerge neuronen, neuronen die de neurotransmitter serotonine uitscheiden. Deze neuronen zijn in een aantal clusters gelokaliseerd in de hersenstam. In tegenstelling tot vele andere groepen neuronen, hebben de serotonerge neuronen een aantal opvallende kenmerken. Ten eerste is het een erg kleine groep neuronen, in de menselijke hersenen gaat het ongeveer om een half miljoen cellen. Ten tweede zijn ondanks dit kleine aantal in bijna alle andere hersengedeelten uitlopers van deze serotonerge neuronen aanwezig. Ten derde wordt deze neurotransmitter in deze hersengebieden in grote hoeveelheden uitgescheiden, waardoor grote groepen andere neuronen door deze serotonerge neuronen beïnvloed kunnen worden. Ten vierde zijn deze serotonerge neuronen, het zogenaamde serotonerge systeem, betrokken bij het beïnvloeden van verschillende soorten gedrag. Als laatste is het serotonerge systeem betrokken bij aandoeningen zoals depressiviteit, angststoornissen, autisme en obsessief compulsief gedrag. Een welbekend antidepressivum zoals Prozac grijpt bijvoorbeeld aan op de serotonerge neuronen, door de heropname van uitgescheiden serotonine terug het neuron in te blokkeren.\n\nHet idee achter aandoeningen zoals depressie is dat er een verandering ontstaat in de uitlopers van deze serotonerge neuronen naar andere hersengebieden en dat antidepressiva de uitlopers weer in de oude, gezonde staat terugbrengen. In dit proefschrift hebben we op een aantal verschillende\n\nmanieren geprobeerd te kijken naar de dynamiek van de uitgroei van deze serotonerge neuronen.\n\nIn hoofdstuk 2 hebben we uit muizenhersenen, die we als modelsysteem hebben gebruikt, de serotonerge neuronen gesneden in een hersenplakje, een zogenaamde organotypische slice. Dit plakje hebben we naast een plakje van een ander hersengebied, de hippocampus, gelegd om te kijken of in dit kweeksysteem de serotonerge neuronen uitlopers vormden het hippocampale plakje in. Inderdaad bleken de uitlopers van de serotonerge neuronen binnen vier dagen het andere plakje in te groeien. Vervolgens hebben we onderzocht of door toediening van stoffen zoals het hierboven genoemde Prozac de uitgroei van de serotonerge neuronen geremd of juist gestimuleerd werd. Voor de stof Prozac bleek dat het toedienen geen effect had op de uitgroei van de serotonerge neuronen. Echter, toediening van een andere stof die aan een bepaalde serotonerge receptor bindt, bleek een effect te hebben op de serotonerge uitlopers. Na zeven dagen waren er namelijk minder serotonerge uitlopers in het hippocampale plakje dan zonder de toediening van deze stof. Hieruit blijkt dat chronische activatie van deze serotonerge receptor leidt tot een remming in de uitgroei van de serotonerge neuronen.\n\nIn hoofdstuk 3 hebben we een belangrijk eiwit in serotonerge neuronen, het eiwit dat zorgt voor heropname van serotoninine in het neuron na afgifte (de serotoninine heropname transporter, SERT) voorzien van een rood fluorescerend eiwit (mCherry). Dit zogenaamde fusie-eiwit, mCherry-SERT genaamd, hebben we tot expressie gebracht in neuronen om te onderzoeken hoe de verdeling van dit eiwit er in neuronen uitziet. Het bleek dat dit eiwit in het neuron aanwezig was in vesicles. Dit zijn pakketjes waarmee eiwitten in een neuron getransporteerd worden. Vervolgens hebben we door middel van microscopie ‘live’ naar de beweging van pakketjes mCherry-SERT gekeken. Uit de analyse van de snelheid en de richting van de beweging bleek dat de pakketjes zich voortbewogen met een gemiddelde snelheid van 1.2 µm/s (=4.32 mm/uur) en dat de pakketjes geen voorkeur hadden voor de richting waarin ze zich voortbewogen in de uitlopers.\n\nIn hoofdstuk 4 hebben we een soortgelijke aanpak gekozen als in hoofdstuk 3. Echter, nu hebben we gekeken naar het eiwit dat belangrijk is voor de aanmaak van serotoninine, tryptophan hydroxylase 2 (Tph2). Dit eiwit hebben we voorzien van een groen fluorescerend eiwit, (Enhanced Green Fluorescent Protein, EGFP). Dit fusie-eiwit hebben we tot expressie gebracht in neuronen om de verdeling van dit eiwit, ook in vergelijking met mCherry-SERT, te onderzoeken. Hieruit bleek dat, in tegenstelling tot mCherry-SERT, Tph2-EGFP zich door de hele cel bevond, en niet in vesicles zat. In jonge neuronen had Tph2-EGFP geen voorkeur voor een bepaald deel van de cel. In oudere neuronen echter, bleek dat Tph2-EGFP voornamelijk aanwezig was in presynaptische\n\nterminals, maar tegelijkertijd ook in de dendrieten. Ook in deze cellen hebben we door middel van microscopie live naar de beweging van Tph2-EGFP gekeken. Hieruit bleek dat Tph2-EGFP, in tegenstelling tot mCherry-SERT, zich niet in vesicles bevond en daardoor ook geen beweging liet zien.\n\nIn hoofdstuk 5 hebben we onderzocht of een variatie in een gen dat codeert voor het eiwit Piccolo, effect heeft op de verdeling en dynamiek van het eiwit SERT. Deze variatie in het gen wordt geassocieerd met depressie, waardoor het erg interessant is om te onderzoeken of de variatie in dit gen verantwoordelijk is voor een verandering in de dynamiek van het serotonine systeem.\n\nHiertoe hebben we eerst onderzocht of het eiwit Piccolo voorkwam in de serotonerge neuronen en uitlopers, en dit bleek inderdaad het geval. Vervolgens hebben we een zogenaamde knockdown construct gemaakt waarmee de hoeveelheid Piccolo in een cel grotendeels gereduceerd kan worden. Na het tot expressie brengen van dit construct in neuronen bleek inderdaad dat de hoeveelheid Piccolo in een cel met meer dan 80% gereduceerd was. Vervolgens hebben we onderzocht of een vermindering in de hoeveelheid Piccolo van invloed is op de beweging en dynamiek van het eiwit SERT. Om dit te onderzoeken hebben we mCherry-SERT tot expressie gebracht in neuronen waarin de hoeveelheid Piccolo gereduceerd was en hebben we door middel van live microscopie naar de dynamiek van het eiwit mCherry-SERT gekeken. Dit hebben we vergeleken met neuronen waarin de hoeveelheid Piccolo niet gereduceerd was. We concludeerden uit deze vergelijking dat een vermindering in de hoeveelheid Piccolo geen effect heeft op de beweging en snelheid van bewegen van mCherry-SERT. Als laatste hebben we gekeken, in een ander cel type, of de mutante variant van het eiwit Piccolo, in vergelijking met de normale variant de verdeling van het eiwit SERT beïnvloedt. Om dit te onderzoeken hebben we de mutante variant van Piccolo en mCherry-SERT in cellen tot expressie gebracht en gekeken of de verdeling van mCherry-SERT veranderd was. In cellen waarin mCherry-SERT tot expressie gebracht was met de normale variant van Piccolo was mCherry-SERT, zoals verwacht, gelocaliseerd aan het membraan. Na expressie van mCherry-SERT met de mutante variant van Piccolo bleek echter dat de hoeveelheid mCherry-SERT aan het membraan verminderd was. Hieruit concludeerden wij dat mogelijk in serotonerge neuronen de mutante variant van het eiwit Piccolo ervoor zorgt dat er een verandering is in de hoeveelheid SERT aan het membraan. Hierdoor zou de signaaldoorgifte van serotonerge neuronen naar andere neuronen verstoord kunnen zijn.\n\nIn hoofdstuk 6 hebben we onderzocht wat het effect is van het uitschakelen van het serotonine systeem in de muis. Om dit te onderzoeken hebben we door muizen te kruisen een muis verkregen waarbij een gen in het serotonine systeem uitgeschakeld is. Uit onderzoek naar de embryonale ontwikkeling van het serotonerge systeem in deze muizen bleek dat het serotonerge systeem in eerste\n\ninstantie tot ontwikkeling kwam. De serotonerge neuronen waren aanwezig, en serotonerge vezels groeiden uit naar andere hersengebieden. We hebben het serotonerge systeem ook bestudeerd iets later in de hersenontwikkeling van deze muizen. Dit toonde aan dat de serotonerge neuronen een veranderde morfologie vertoonden, waaruit afgeleid kan worden dat deze neuronen degenereren. Uiteindelijk zijn bijna al de serotonerge neuronen verdwenen. Ook de projecties van deze neuronen verdwenen later tijdens de hersenontwikkeling. Hieruit concludeerden wij dat het uitschakelen van dit gen uiteindelijk leidt tot een verdwijning van de serotonerge neuronen. Vervolgens hebben we gekeken wat het effect hiervan is op de ontwikkeling en het overleven van deze muizen. Dit toonde aan dat de meeste muizen vlak na de geboorte doodgingen. Enkele muizen die wel overleefden waren kleiner dan controle muizen die dit gen nog wel hadden, en gingen uiteindelijk ook binnen drie weken dood. We waren ook geïnteresseerd of het gedrag van de muizen veranderd was. Echter, aangezien de meeste muizen al snel na de geboorte dood gingen, was dit niet mogelijk. Daarom hebben we het gedrag geanalyseerd van muizen die nog een kopie van dit gen hadden, en dit vergeleken met controle muizen. Om het gedrag van deze muizen te analyseren hebben we deze muizen zeven dagen in een speciale kooi gehouden. Deze kooien bevatten een videocamera zodat later een groot aantal parameters geanalyseerd kan worden, zoals de hoeveelheid beweging, hoe vaak deze muizen drinken of hoeveel deze muizen eten. Verder konden we ook nog het angst gerelateerde gedrag en het leergedrag van deze muizen analyseren. Uit analyses van muizen met een kopie van het gen bleek dat deze geen veranderd gedrag vertoonden ten opzichte van controle muizen.\n\nHet doel in hoofdstuk 7 was om een muis te maken waarbij alle serotonerge neuronen en uitlopers naar andere hersengebieden gelabeld waren. Om dit doel te bereiken hebben we een gen gebruikt dat specifiek tot expressie komt in serotonerge neuronen, Tph2. Een stuk DNA voor een gen, een promoter genoemd, is verantwoordelijk voor waar en wanneer het gen afgeschreven wordt. Achter de promoter van Tph2 hebben een eiwit genaamd Cre recombinase gezet, zodat de expressie van Cre recombinase wordt gereguleerd door de Tph2 promoter en zodoende alleen tot expressie komt in serotonerge neuronen. In dit hoofdstuk hebben we beschreven hoe we embryonale stamcellen gemanipuleerd hebben en hoe we deze hebben geïnjecteerd in muizen blastocysten. Helaas is het nog niet gelukt om deze muis te genereren. Om specifiek serotonerge neuronen te labelen hebben we nog een aantal andere muizen gemaakt, waarbij het eiwit alleen aangemaakt wordt als in de cell Cre recombinase tot expressie komt. Door deze muizen met elkaar te kruisen zou Cre recombinase alleen in serotonerge neuronen ‘aan’ staan, en zou alleen in deze neuronen een zogenaamd reporter eiwit aangezet worden. Op deze manier hopen we uiteindelijk muizen te hebben waarbij specifiek in serotonerge neuronen reporter genen tot expressie komen,\n\nwaardoor we deze uiteindelijk kunnen gebruiken om in levende muizen naar de dynamische uitgroei en connectiviteit van het serotonerge systeem te kijken. Verder kunnen we dan onderzoeken of manipulatie aan dit systeem leidt tot een verandering in uitgroei van serotonerge neuronen en tot muizen die bepaalde aspecten van humane depressiviteit nabootsen. Aan de andere kant kunnen we symptomen van humane depressiviteit in muizen induceren, en onderzoeken of dit leidt tot veranderingen in het serotonerge systeem. Uiteindelijk zouden we op deze manier ook kunnen onderzoeken of medicatie tegen depressiviteit leidt tot een verandering in de connectiviteit van het serotonerge systeem en deze weer terugbrengt in de oude staat.\n\n| Abbreviation | Description |\n|--------------|-------------|\n| 5-HIAA | 5-hydroxyindolacetic acid |\n| 5-HT | serotonin |\n| 5-HT | 5-hydroxytryptamine |\n| 8-OH-DPAT | 8-hydroxy-2-(di-n-propylamino)tetraline |\n| BOLD | blood oxygen level dependent |\n| cAMP | cyclic adenosine monophosphate |\n| CSF | cerebrospinal fluid |\n| DAT | dopamine transporter |\n| dGBSS | dissection Gey’s balanced salt solution |\n| DIV | days in vitro |\n| DOI hydrochloride | (+)-2,5-dimethoxy-4-iodoamphetamine hydrochloride |\n| DRN | dorsal raphe nucleus |\n| E | embryonic day |\n| EGFP | enhanced green fluorescent protein |\n| ES | embryonic stem |\n| fMRI | functional magnetic resonance imaging |\n| GIRK | G protein-activated inwardly rectifying K⁺ |\n| HBSS | Hanks Balanced Salt Solution |\n| iCre | improved Cre recombinase |\n| IP₃ | inositol 3-phosphate |\n| IRES | internal ribosomal entry site |\n| KO | knockout |\n| l | long |\n| LAT₁ | large neutral amino acid transporter |\n| LDCV | large dense core vesicle |\n| Lmx1 | LIM homeobox transcription factor 1 |\n| LSD | lysergic acid diethylamide |\n| LSL | loxSTOPlox |\n| MAO-A | monoamine Oxidase-A |\n| MAP2 | microtubule associated protein 2 |\n| MDMA | 3,4-methylenedioxy-N-methylamphetamine |\n| MHO | midbrain-hindbrain organizing centre |\n| MRN | median raphe nucleus |\n| neo | neomycin phosphotransferase cassette |\n| OCD | obsessive-compulsive disorder |\n| p | postnatal day |\n| PBS | phosphate buffered saline |\n| PCM | presynaptic cytoskeletal cytomatrix |\n\n| Abbreviation | Description |\n|--------------|-------------|\n| PCPA | p-chlorophenylalanine |\n| PET | positron emission tomography |\n| Pet1 | PC12 Ets factor |\n| PFA | paraformaldehyde |\n| PKA | protein kinase A |\n| PKC | Protein kinase C |\n| PKG | protein kinase G |\n| β-PMA | β-phorbol 12-myristate 13-acetate |\n| RRP | readily releasable pool |\n| s | short |\n| SEM | standard error of mean |\n| SERT | 5-HT reuptake transporter |\n| SMB | short movement bouts |\n| SNP | short nucleotide polymorphism |\n| SSRI | selective serotonin reuptake inhibitor |\n| SSV | small synaptic vesicle |\n| Tetrahydrobiopterin BH4 (6R)-L-erythro-5,6,7,8-Tetrahydrobiopterin | |\n| TH | tyrosine hydroxylase |\n| tPA-GFP | tissue plasminogen activator-GFP |\n| Tph | tryptophan hydroxylase |\n| Tph1 | tryptophan hydroxylase 1 |\n| Tph2 | tryptophan hydroxylase 2 |\n| VAMP2 | synaptobrevin-2 |\n| VMAT2 | Vesicular Monoamine Transporter 2 |\n\nDankwoord\n\nNu ik aan het einde van mijn promotietraject ben gekomen en het boekje af is, besef ik dat ik een ontzettend leuke en leerzame tijd heb gehad, en daar wil ik graag een aantal mensen voor bedanken.\n\nIn de eerste plaats mijn promotor Matthijs. Beste Matthijs, ik wil je bedanken voor het feit dat ik dit project op je lab heb mogen uitvoeren, de begeleiding en de dingen die ik van je geleerd heb. Verder ben ik je dankbaar dat ik ook de ruimte kreeg om mijn eigen invulling aan dit project te geven.\n\nIn de tweede plaats mijn copromotor Sander: Beste Sander, door een stage die ik bij jou gelopen heb ben ik op dit lab terecht gekomen, ik ben je daar nog steeds dankbaar voor. Tijdens mijn promotieproject dank ik je voor de begeleiding en het nakijken van mijn manuscripten.\n\nDe leescommissie dank ik voor de aandacht die ze aan mijn proefschrift hebben willen besteden.\n\nVerder heb ik veel gehad aan de suggesties en input van Ruud en Joris tijdens onze meetings, mijn dank daarvoor. Oliver, ik zou je graag willen bedanken voor de suggesties met betrekking tot gedragsproeven in muizen en de hulp met de perfusies. Ik dank Pieter Voorn en Menno Witter voor het meedenken met het slice hoofdstuk en Pieter en Allert tevens voor alle hulp op de MCID microscoop.\n\nMijn dank gaat uit naar mijn beide paranimfen. Beste Joost, bedankt voor al die keren dat je me op het lab een hoetje(s) liet schrikken 😂 Ik heb altijd met heel veel plezier met jou samengewerkt op het lab, maar we hebben ook heel veel lol gehad buiten het werk om.\n\nBeste Arthur, ondanks dat je pas in mijn laatste jaar mijn kamergenoot werd, bleek al snel dat we het qua humor goed met elkaar konden vinden. Behalve onze liefde voor flauwekul bleek ook onze liefde voor (speciale) bier(en) overeen te komen (zou daar een relatie tussen zitten?). Ik wens je alle succes met jouw verdere afronding van je promotietraject en met de voortzetting van het door ons opgezette ‘bureau voor flauwekul’.\n\nBeste Ineke, ik wil je danken voor alle gezelligheid, maar ook voor alle goede gesprekken over de wetenschap en aanverwante zaken. Ik wens je heel veel succes met de verdere afronding van je opleiding tot klinisch chemicus.\n\nBuiten het feit dat ik veel geleerd heb, heb ik ook de sfeer op de afdeling en het lab altijd als zeer positief ervaren. Op het DNA lab, het kloppend hart van de afdeling, heb ik altijd met heel veel plezier gewerkt. Niet in de laatste plaats door\n\nalle gezelligheid die daar altijd te vinden was. Voor al deze gezelligheid, maar zeker ook voor alle hulp ben ik de analisten grote dank verschuldigd.\n\nBeste Robbie, Mr. Salmon, ik ben je dankbaar voor de stage die ik bij jou gelopen heb waarin je me hebt ingewijd in de geheimen van het kloneren. Hoewel ik nog niet eens jouw pipetten mag vasthouden denk ik dat ik inmiddels aardig kan kloneren.\n\nBeste Ingrid, bedankt voor je humor op het lab en voor de hulp met de kloneringen. N.B. Ik heb in de wandelgangen vernomen dat zelfs *jij* nu getrouwd bent :-)\n\nIk ben ook veel dank verschuldigd aan Desiree en later Boukje voor alle hulp met de neuronen kweek. Bastijn, jou dank ik voor het uitwerken van de Phenotyper data.\n\nBeste Joke, bedankt voor alle hulp met de muizen, maar zeker ook voor je altijd vriendelijke houding.\n\nDit proefschrift was ook zeker niet mogelijk geweest zonder alle hulp vanuit de muizenstallen. Dus Chris, Bert en Lieselotte, ook jullie hartelijk dank voor alle hulp met de muizen.\n\nMijn mede aio’s Jurjen, Juliane, Sabine, Marieke, Asiya, Rhea, Jiun, Tony en Gregoire dank ik voor alle discussies en ik wens jullie allen veel succes met de verdere voortzetting van jullie project en de afronding van jullie proefschrift.\n\nTina en Els jullie dank ik voor alle administratieve ondersteuning. Els, als ik een vraag had wist je die altijd binnen no time te beantwoorden.\n\nAlle andere nog niet genoemde collega’s van het lab dank ik voor alle discussies tijdens labmeetings, gezelligheid tijdens de dagelijkse lablunch en het jaarlijkse labuitje.\n\nRoel, ik dank je voor alle gezelligheid tijdens de treinreizen vanuit Hoofddorp, en ik vind het erg leuk dat we elkaar nog weleens tegenkomen.\n\nMijn nieuwe lab Neuromedische Genetica op het NIN dank ik voor de gastvrijheid waarmee ik in hun lab ben opgenomen en de plezierige werksfeer.\n\nGijs, Ray en Robert, we hebben altijd ontzettend veel lol gehad tijdens onze studieperiode. Ik ben blij dat we nog weleens afspreken. Wanneer gaan we weer poolen (of was het nou snooker...?).\n\nAls laatste zou ik graag een aantal mensen willen bedanken uit mijn persoonlijke omgeving.\n\nDe mannen uit Renesse: Alex, Bart, Kenneth, Michael; bedankt voor alle nachtelijk stapuurtjes en (wintersport) vakanties.\n\nBeste familie Visser, ik dank jullie voor alle gezelligheid en alle borreltjes die we al met elkaar gedronken hebben. Wat is het bij jullie toch altijd lekker rustig... :-)\n\nIk dank mijn schoonouders, Jan en Christien, mijn schoonzussen en zwagers voor de interesse in mijn proefschrift en alle feesten, familiedagen en andere gezellige activiteiten. Is die Eiffeltoren nou al gevonden?\n\nMijn broer(tje) en zus(je), ik dank jullie voor de interesse in mijn proefschrift, de gezelligheid tijdens het meerijden naar Renesse en ik wens jullie alle succes met de afronding van jullie studie.\n\nMijn ouders, ‘promoveren zit dat erin dan?’, Frans en Lia, ik ben heel trots op jullie en ik ben blij jullie zoon te zijn. Ik realiseer me maar al te vaak wat een rijkdom het was om zo te mogen opgroeien. Ik dank jullie voor alle steun en vertrouwen, maar ook voor alle gezelligheid. Als we elkaar zien is het altijd erg gezellig en vaak ineens midden in de nacht als we zitten te kletsen onder het genot van een wijntje.\n\nAls laatste dank ik natuurlijk mijn lieve vrouw Judith. Sinds die kapotte printer zijn we alweer bijna 7 jaar bij elkaar, wonen we al ruim 3 jaar in Hoofddorp en zijn we al ruim 1 jaar getrouwd. Time flies when having fun, wil ik daar alleen maar even mee zeggen. Ik wens jou ook alle succes met de verdere afronding van jouw promotieproject en boekje, en heb zin om samen de toekomst tegemoet te gaan!\n\nJeroen\n\nHoofddorp, Juli 2010\n\nCurriculum vitae\n\nJacobus Johannis Dudok werd geboren op 13 juni 1981 te Zierikzee. In 1993 begon hij aan zijn VWO opleiding aan het Buys Ballot College te Goes, waar hij in 1999 zijn Gymnasium Beta diploma haalde. In augustus 1999 begon hij aan de Bachelor Biomedische Wetenschappen aan de Vrije Universiteit te Amsterdam. In deze opleiding liep hij zijn Bachelor stage van februari 2002-juni 2002 aan de vakgroep pathologie in het VU medisch centrum. Hier bestudeerde hij, begeleidt door Dr. R. Veerhuis en Ing. E. van Elk, de verschillende aggregatie vormen van Aβ1-42, het eiwit betrokken bij de ziekte van Alzheimer. In september 2002 begon hij aan de Master of Neurosciences aan de Vrije Universiteit te Amsterdam. Tijdens deze master deed hij twee stages bij de afdeling Functionele Genoomanalyse. Tijdens de eerste masterstage deed hij onderzoek naar de calcium afhankelijke translocatie van het presynaptische eiwit Doc2B, begeleidt door Dr. S. Groffen. Tijdens zijn tweede masterstage, begeleidt door Ing. R. Zalm, cloneerde hij een targeting construct en gebruikte deze om muis embryonale stamcellen mee te targetten. Zijn masterscriptie schreef hij onder leiding van Dr. J. Pronk en ging over de in silico clonering en karakterisatie van het eiwit Tph2. In augustus 2004 studeerde hij af aan de Vrije Universiteit te Amsterdam. Van oktober 2004 tot april 2009 deed hij zijn promotieonderzoek naar de uitgroei en connectiviteit van het serotonerge systeem. Dit onderzoek deed hij onder begeleiding van professor Verhage aan de vakgroep Functionele Genoomanalyse aan de Vrije Universiteit te Amsterdam. De resultaten van dit promotieonderzoek zijn in dit proefschrift beschreven. Vanaf april 2009 werkt hij als postdoctoraal onderzoeker op het Nederlands Instituut voor Neurowetenschappen (NIN) bij de vakgroep Neuromedische Genetica onder leiding van Dr. J. Wijnholds. Hier doet hij onderzoek naar de functionele rol van het eiwit MPP3 in het Crumbs complex en in neuronale cellen.\n\nJacobus Johannis Dudok was born on the 13th of June 1981 in Zierikzee, the Netherlands. In 1993 he started his secondary education at the Buys Ballot College in Goes, where he graduated in 1999. In August 1999 he started the Bachelor Medical Biology at the Vrije Universiteit Amsterdam. During this Bachelor he carried out a research project from February 2002 to June 2002 at the research department of pathology at the VU medical center. Here he studied the different aggregation states of Aβ1-42, the peptide involved in Alzheimer’s disease, supervised by Dr. R. Veerhuis and Ing. E. van Elk. In September 2002 he started the Master of Neuroscience at the Vrije Universiteit Amsterdam. During this master he carried out two research projects at the department of Functional Genomics. During his first research project he studied the calcium dependent translocation of the presynaptic protein Doc2B, supervised by Dr. S. Groffen. During his second research project he constructed a targeting construct and used this to target mouse embryonic stem cells, supervised by Ing. R. Zalm. He wrote his Master Thesis under supervision of Dr. J. Pronk about the in silico cloning and characterization of the protein Tph2. In August 2004 he graduated at the Vrije Universiteit Amsterdam. From October 2004 until April 2009 he did his PhD project on the outgrowth and connectivity of the serotonin system. This PhD project was supervised by professor Verhage and carried out at the department of Functional Genomics at the Vrije Universiteit Amsterdam. The results of that study are described in this thesis. He is currently working as a postdoctoral fellow at the Netherlands Institute for Neuroscience (NIN) in the department of Neuromedical Genetics headed by Dr. J. Wijnholds. 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Reckless thrust by Quality
Quality recording act Reckless are on release with Heart Of Steel, an LP produced by Ross Munro at Metalworks in Mississauga, Ont. At press time, the label had not chosen a single, although a video is imminent for the Hot 'n Ready track. Following a couple of lineup changes, Reckless are currently Steve Madden (principal writer, guitars, vocals), Douglas Lang Adams (lead vocals, guitar), Steven Wayne Lederman (drums), Brian Beggs (bass), and Nick Brewerton (guitar). The LP, recorded and mixed by Hugh Cooper, qualifies as 4 parts Cancon.
Reckless have given their label a powerful and surprisingly melodic album — for which considerable credit is due producer Ross Munro. Munro, a former professional musician, has applied his production savvy to create an energetic and cleanly produced product that will undoubtedly find favour with fans of "thoughtful" hard rock — loud and proud, but occasionally attentive to dynamics.
Reckless recently performed at Toronto's Diamond Club, an opportunity to present the new material with the added profile of a video shoot at the end of the evening — the forthcoming Hot 'n Ready.
Currently managed by Steve Thompson of Backstage Productions, Reckless released an LP in 1981 that garnered interest in the U.K. and Europe, and toured with acts like Iron Maiden and Saga. It was Madden's hassles with Saga's management firm that kept him from recording the music of Reckless as "... heavy pop with a European feel ... but incorporating an American sound as well."
Indeed, judging from their Diamond date, the band does render homage to a variety of people — notably Germany's Scorpions, Triumph, and any number of American hard-rock acts.
Madden cites The Yardbirds, Jeff Beck, and early Led Zeppelin as influences, while in concert he emulates the frenetic upper-fret riffing and buzzsaw chording epitomized by Queen's Brian May.
The Diamond show — performed at relentlessly high volume — did give the band's adherents and the media an opportunity to see what all the fuss is about.
Vocalist Adams, when he could be heard, displayed a powerful voice that, to this set of ears, generally held to the recorded version. Guitarists Brewerton and Madden, although cliche in dress and stage manner, did deliver some interesting arrangements — despite the atrocious sound.
Live chat aside, Heart Of Steel stands well, considering how long it took to record. Three completed songs were taken by manager Thompson to MIDEM in early '84, and the band resumed work on a first LP when a British deal had been inked. The record has a surprisingly spontaneous feel and should certainly land Munro kudos for his participation.
Munro, a former business partner of Thompson's, came in on the project after Thompson asked him to audition the band's demo. "I saw their rehearsals and I liked them," Munro said in a recent telephone interview. "I worked primarily on pre-production with Steve (Madden) and changed a few arrangements around so we had it down basically after pre-production. He (Madden) gave me a lot of flexibility; and I asked his opinion from time to time. It worked out well."
The result, Heart Of Steel, is impressive. So buy your neighbour a plane ticket, close the windows, and crank up the turntable — it's time to get Reckless.
At retail, Quality has virtually sold through on their initial shipment. At radio, AOR playlisting has been attained in Toronto, Ottawa, Halifax, and Newfoundland, with favourable response garnered in Edmonton, Calgary, and Regina. Having released a 12" AOR sampler of the Hot 'n Ready track, backed with an interview side, Quality is currently preparing a promo only 7" edit of Hot 'n Ready.
A national tour is presently in the works.
Also included on the Reckless LP are Drivin' You Mad, Feel The Fire, Need You Next To Me, and Shadows Of You - KCW
Darkroom
THE STUNNING NEW LP
“A TEST OF TIME”
PRODUCED BY
TOM TREUMUTH
INCLUDES “TEST OF TIME”, “IN A DREAM”, “BROKEN ENGLISH”, AND “DON’T PLAY WITH BREAKING HEARTS”
available on cassette and disc from
wea
Music of Canada, Ltd.
The following is a list of the most important and frequently used terms in the field of computer science.
WALT SAYS!
Just a matter of time!!!
All the I's are dotted and T's crossed for the biggest takeover in the history of Canadian broadcasting. All they need now is the nod from the Ottawa "nodders" (EC: I've noticed they've been nodding off quite frequently, lately) and we'll have coast to coast balanced programming.
Watch for it!!!
There will shortly be an announcement of a major, major television awards presentation that will involve a star-studded cast. One of the major, major players in this costly little project is a label that prides itself in being first - and they are in very, very deep. Even CBC-TV has been caught up in the excitement.
No need to panic . . . !!
Contrary to what many of us believed, John Spragge has control of the situation at CFRB. He may have shaken up listeners with a move to the right of the expected in music policy and the dropping of Charles Doering's 10 a.m. "news and comments", (he's still got the early risers with his 7 a.m. news slot), but the adjustment hasn't been that bad. Wally Crouter is fitting in well with the Commodores' Nightshift, Manhattan's Tuxedo Junction and Culture Club's Mistake No. 3 - a very nice mix with the Lee Greenwood/Tammy Wynette duet of It Should Have Been Love By Now and Wake Up Little Suzie by the Everly Brothers.
The move should be to A/C
While Samantha Taylor captures a whopping big national audience for her CBC-TV Video Hits series and the list of copycats grows and grows - all trying for the same age group - it just doesn't make sense. It's no secret that a couple of TV music channels in the U.S. have successfully launched music video shows aimed at the 18 to 49 age group. Obviously there's a market for this type of show - all it needs is a little creative from the people who claim to know what's going on. The potential is awesome.
What's NOT hot!!!
That "thumb" which always looked to me like a middle finger aimed at the record industry, may disappear this Friday when the Toronto Star launches their new What's On section. They'll be asking the industry if this new section is HOT??? or NOT??? I personally will not be attending this party . . . feeling that it is NOT HOT and that the BamBoo Club is a little too disco-like for a non-trendy like myself. The Bam-Boo Club is NOT HOT!!! The Diamond is HOT! The Copa is NOT! The Globe continues to be HOT! Wilder Penfield is HOT! Greg Quill is NOT! Stay tuned for my finger pointing and my evaluation of what's HOT and . . . etc!!!
The ultimate critique and review!!
Finally, an album came into our offices which, when reviewed, netted four words. Since these are words not usually found in children's books, the album will not appear on our album page. The ultimate review . . . it was not worthy of a mention.
True value for charity monies!!
The Variety Club and the Canadian Recording Industry Association held their annual gourmet brunch and auction at Toronto's Inn On The Park (Mar. 10). The food was the finest, the guests were charming, and the cause is most worthy. But . . . I would think it be very appropriate for the auction to be more specific of any limitations in the items they are offering. In the true spirit of the Variety Club, the items should be sold so that the bidders know exactly what the limitations are - on the trips, memberships, auto leasing etc. There were some pretty pricey items up for bids, and after paying thousands, you don't want to find out that the item can only be used on a Friday in August, if it rains. My job is to criticize, I'm doing my job!!!
CORRECTION
Right players - wrong movie. It was reported last week in RPM that B.J. Cook and David Tyson would have one of their compositions included in the Porky's Revenge (Porky's III) soundtrack. This information is not correct. The Cook/Tyson composition, Has Heaven Had A Change Of Heart, will be used in the Meatballs III movie, with Liberty Silver doing the vocals.
RPM
published weekly since February 24th, 1964 by RPM MUSIC PUBLICATIONS LTD.
6 Brentcliffe Road
Toronto, Ontario, M4G 3Y2
416-425 0257 - Telex: 06 22756
Walt Gercals - Editor & Publisher
Rita Drago - Subscriptions
Peter Martin - Chart Compilation
Kevin O'Wardine - Video & General News
Stan Klees - Special Projects
The following codes are used throughout RPM's charts as a key to record distributors:
A&M - W POLYGRAM - Q
CBS - H QUALITY - M
CAPITOL - F RCA - N
MCA - WEA - P
MAPL logos are used throughout RPM's charts to define Canadian content on record releases.
M - Music was composed by a Canadian
A - Artist who is featured is a Canadian citizen
P - Production was wholly recorded in Canada
L - Lyrics were written by a Canadian citizen
Advertising rates on request. Second Class Postage paid in Toronto. Registration No. 1351 PRINTED IN CANADA
An L.P. from one of Canada's foremost singer/songwriters and mainstay of Canadian radio ...
RICH DODSON
(Former original member of The Stampeders & Juno Award-winning composer of "SWEET CITY WOMAN")
FEATURING HIS CURRENT HIT SINGLE
"NO TIME TO SAY GOODBYE"
Currently playlisted at over 200 radio stations including:
CHFI (FM) CKEY CKFM (FM)
CJOB CJOY CKBB CKJS CKKW CKOY
CKSL CHSC CKTB CKXM (FM) CKWW VOCM
PROMOTED NATIONALLY BY MPL PROMOTIONS
Contact: MARY LYNN
Telephone: (416) 484-8789
DISTRIBUTED BY: THE MOSS MUSIC GROUP (CDA) INC. 510 CORONATION DRIVE, UNIT 17, WEST HILL, ONTARIO, CANADA M1E 4X6 / TELEPHONE: (416) 284-0126
THANK YOU PROGRAMMERS FOR THE OVERWHELMING SUPPORT!
The following is a list of the most important and frequently used terms in the field of computer science:
- Algorithm: A step-by-step procedure for solving a problem or performing a task.
- Data structure: A way of organizing data in a computer program to make it easier to access, manipulate, and store.
- Database: A collection of data organized in a structured manner that can be accessed and managed by a computer program.
- Database management system (DBMS): A software application that provides services for creating, maintaining, and managing databases.
- Database query: A request made to a database to retrieve specific information.
- Database schema: The logical structure of a database, including the tables, columns, and relationships between them.
- Database normalization: A process of organizing data in a database to minimize redundancy and improve data integrity.
- Database transaction: A sequence of database operations that must either all succeed or all fail, ensuring data consistency.
- Database indexing: A technique used to speed up database queries by creating a separate index on one or more columns of a table.
- Database replication: The process of copying data from one database to another to ensure data availability and fault tolerance.
- Database security: The measures taken to protect databases from unauthorized access, modification, or destruction.
- Database backup: The process of creating a copy of a database to restore it in case of data loss or corruption.
- Database recovery: The process of restoring a database to a previous state after a failure or disaster.
- Database concurrency control: Techniques used to manage simultaneous access to a database by multiple users or processes.
- Database optimization: The process of improving the performance of a database by optimizing its design, indexing, and query execution.
- Database administration: The management and maintenance of a database system, including planning, implementation, and monitoring.
- Database modeling: The process of creating a conceptual model of a database to represent real-world entities and their relationships.
- Database querying: The process of retrieving data from a database using a query language such as SQL.
- Database schema design: The process of designing the logical structure of a database, including the tables, columns, and relationships between them.
- Database schema evolution: The process of modifying the schema of a database over time to adapt to changing requirements and data needs.
- Database schema validation: The process of checking the validity and consistency of a database schema before implementing it.
- Database schema versioning: The process of tracking changes to a database schema over time, allowing for easy comparison and rollback.
- Database schema migration: The process of transferring a database schema from one environment to another, such as from development to production.
- Database schema refactoring: The process of restructuring a database schema to improve its design and maintainability.
- Database schema reverse engineering: The process of generating a database schema from an existing database, often used to understand legacy systems.
- Database schema transformation: The process of converting a database schema from one format to another, such as from relational to NoSQL.
- Database schema validation: The process of checking the validity and consistency of a database schema before implementing it.
- Database schema versioning: The process of tracking changes to a database schema over time, allowing for easy comparison and rollback.
- Database schema migration: The process of transferring a database schema from one environment to another, such as from development to production.
- Database schema refactoring: The process of restructuring a database schema to improve its design and maintainability.
- Database schema reverse engineering: The process of generating a database schema from an existing database, often used to understand legacy systems.
- Database schema transformation: The process of converting a database schema from one format to another, such as from relational to NoSQL.
The following is a list of the most important and frequently used terms in the field of computer science.
Virgin confirms Minds' 7"
Virgin Records Canada have released a 7" of Don't You (Forget Me) by Simple Minds, the track that is included on the Breakfast Club Soundtrack, distributed by A&M. Originally, the company had intended only releasing the 12", which is already on the market and which contains the extended album version and the edited version plus A Brass Band In Africa. The latter was produced by Steve Lillywhite, while the soundtrack was produced by Keith Forsey.
MCA awards promo bucks
Ellen McGrath of the Mr. Sound store at Limeridge Mall in Hamilton was the winner of $1,000 in the MCA Merry Music Program, a major retail campaign that ran over the Christmas season. The national promotion involved even high level executives at MCA who were each assigned a retail outlet. With MCA supplying the in-store point-of-purchase material, each store had to rack 10 titles from MCA at the front of the outlet including both albums and cassettes. The campaign was not only designed to boost MCA's current product, but to make company reps more familiar with the various store managers. Bonus prizes went to other outlets.
Young resigns General Publishing
Gerry Young, former Publicity and Advertising Manager at General Publishing, has resigned this position. He will now devote his full time to his 2 groups, The Parachute Club and M+M. As President of Current Records, a division of Current Entertainment, Young has announced the signing of Double Dare to the Current roster.
Reggae show for Massey Hall
Reggae acts Third World, Dennis Brown, Gregory Isaacs, and Lloyd Parks with We, The People will perform at Massey Hall Apr. 3. This aggregation is the touring version of the renowned Reggae Sunsplash show. The same roster participated in a similar event held last year at the Crystal Palace in London, England.
MCA push on Go West
New from MCA is the first single from the U.K. duo, Go West, titled We Close Our Eyes. The single was taken from their self-titled LP. A video of the single, directed by former 10cc members Kevin Godley and Lol Creme, is also available. Go West comprises Peter Cox and Richard Drumming, both songwriters for Chrysalis, their release label. The album was produced in London by Gary Stevenson.
Radio
New appointments at Rawlco
Harry Dekker has been appointed PD at CJME Regina. Harry has been with Rawlco Communications since 1977, and is well known for his popular morning show, which he will continue with. Brenda Selzer joins the programming staff as Assistant PD. She will continue as CJME Promo Director. Mike MacNaughton is moved up to Creative Director for CJME/Z99. He joined Rawlco in 1980, working first in production for CJME, and then as PD for Z99. Mike will oversee all creative aspects of both stations. Ed Walker takes on the duties of PD at Z99. He has been with Rawlco for 12 years, of which 9 of them were on-air with CJME. He was appointed Music Director at CJME in 1982, and held the afternoon drive shift. He will continue with on-air contributions along with his new duties.
CHQB Radio Bruins aids Variety Club
The Variety Club Night, recently held in Powell River, B.C. raised over $2,000 for the local tent. The CHQB Radio Bruins took on the Leisure Services staff in a hockey game, drawing one of the largest crowds of the year - over 1200 to see a 4-4 tie game. Darcy Roat of the Vancouver Canucks was in attendance to present prizes which included two pairs of tickets to the Canuck/Boston game in Vancouver. Air travel was courtesy of Air B.C. The evening also featured a breakdance demonstration as well as air band competitions.
Unique clearance sale at CJBK
Steve Garrison, Promotions Manager at CJBK London asks the question: "What does a promotion manager do with left over albums, t-shirts, movie posters, radios, watches, and more?" He answers his own question with: "Why, he holds a January Clearance Sale." The station conducted just such a sale (Jan. 28) and asked listeners to call in to win some of the station's left over promotional giveaways. The prizes ranged from 10 Kim Mitchell concert t-shirts to left over dinner give certificates. Concludes Garrison, "The day proved to be a winner on the air, and our listeners just loved it."
CFCY's Joseph - around the world
CFCY Charlottetown's afternoon personality, Hilary Joseph, sets off on his around the world trip on Feb. 14. He will visit Toronto, London, Bombay, Singapore, Bangkok, Hong Kong, and Vancouver. He will be accompanying Al Rach, Air Canada Manager for P.E.I., and Cathay Pacific's Peter Quinn. The CFCY listener who is closest to predicting the duration of the trip will win a trip for two around the world, plus spending money from the two airlines. Hilary will phone in reports from each city. Close to 1,000 qualifiers will be following Hillary's progress to see if they are in the running. Writes Garry Barker, Program Director for CFCY, "This promotion has been one of the most talked about in CFCY's 60 year history."
Q94 listeners off to the sun
Q-94, in cooperation with P.S. Holidays of Winnipeg, recently sent 12 Q-94 listeners to Club Maeva in Manzanillo, Mexico for a Suntan Tournament. The tournament began when the 12 listeners stepped off the plane in Mexico. Prizes were awarded for various suntan categories, e.g. best left arm tan, darkest tan, palest ring finger tan etc.
don’t you forget about...
don’t you
(forget about me)
the new 7” & 12” single from
SIMPLE MINDS
“This is the hit we’ve all been waiting for!”
Terry Trojek, Saturn Distributing
Minds over Radio
| FM | AM |
|------|------|
| CFOX | CICH |
| CILA | CIHI |
| CFMC | CKGM |
| CITI | CKLC |
| Q107 | CKSL |
| CFNY | CFRA |
| CKCU | CFGO |
| CHEZ | CKPT |
| CKOI | CKOM |
| CJMF | AM106|
| C100 | CKRA |
Minds over Retail
“No.1 selling piece of product in the store, period - in its first week out.”
Peter Roffman - Records On Wheels
621 Yonge Street, Toronto
“Simple Minds’ first North American smash hit single.”
Ian Marchant - Sunrise Warehouse, Toronto
“Retail response already on this hit,
so DON’T YOU forget about it.”
Margo Fraser - DJ’s Music Services, Calgary
“It’s fandabbydozy, a good bird’s-eye tatty fritter -
all in all a smash hit single selling like hotcakes.”
Stewart Hay - Roblans Distributors
Minds over America
R&R CHR = BREAKER! (166/30)
R&R AOR/Hot Tracks 11*
The Album Network - Powercuts 11*
Minds over Medium rotation
it’s SIMPLE MINDS over matter!
PolyGram
Virgin Records Canada
The following is a list of the most important and commonly used terms in the field of computer science:
1. Algorithm: A step-by-step procedure for solving a problem or performing a task.
2. Data Structure: A way of organizing data that allows efficient access, modification, and manipulation.
3. Database: An organized collection of data stored in a computer system.
4. Database Management System (DBMS): Software that manages databases and provides an interface for users to interact with them.
5. Encryption: The process of converting information into a code so that only authorized parties can understand it.
6. Hashing: A process of converting data into a fixed-size string of characters.
7. Interface: A way for two systems to communicate with each other.
8. Network: A collection of computers and devices connected together.
9. Operating System (OS): A software program that controls the hardware and software resources of a computer.
10. Programming Language: A set of instructions that a computer can understand and execute.
11. Query: A request for information from a database.
12. Security: The protection of data and systems from unauthorized access, use, disclosure, disruption, modification, or destruction.
13. Software: A set of instructions that tell a computer what to do.
14. System: A collection of related components that work together to achieve a common goal.
15. User: An individual who interacts with a computer system.
16. Virtualization: The creation of virtual versions of physical resources such as servers, storage, and networks.
17. Web Application: A software application that runs on a web server and is accessed through a web browser.
18. Wireless Network: A network that uses radio waves to transmit data between devices.
19. XML: eXtensible Markup Language, a markup language that defines a set of rules for encoding documents in a format that is both human-readable and machine-readable.
20. YAML: Yet Another Markup Language, a markup language that is used to represent structured data.
The following is a list of the most important and frequently used terms in the field of computer science:
- Algorithm: A step-by-step procedure for solving a problem or performing a task.
- Data structure: A way of organizing data in a computer program to make it easier to access, manipulate, and store.
- Database: A collection of data organized in a structured manner that can be accessed and managed by a computer program.
- Database management system (DBMS): A software application that provides services for creating, maintaining, and managing databases.
- Database query: A request made to a database to retrieve specific information.
- Database schema: The logical structure of a database, including the tables, columns, and relationships between them.
- Database normalization: A process of organizing data in a database to minimize redundancy and improve data integrity.
- Database transaction: A sequence of database operations that must either all succeed or all fail, ensuring data consistency.
- Database indexing: The process of creating indexes on database tables to speed up query execution.
- Database replication: The process of copying data from one database to another to ensure data availability and fault tolerance.
- Database security: The measures taken to protect databases from unauthorized access, modification, or destruction.
- Database backup: The process of creating a copy of a database to restore it in case of data loss or corruption.
- Database recovery: The process of restoring a database to a previous state after a failure or disaster.
- Database concurrency control: The mechanisms used to manage simultaneous access to a database by multiple users or processes.
- Database performance tuning: The process of optimizing database queries and configurations to improve response time and throughput.
- Database administration: The management of database systems, including planning, implementation, maintenance, and monitoring.
- Database design: The process of creating a database schema that meets the requirements of an organization or application.
- Database modeling: The creation of diagrams and models to represent the structure and relationships of a database.
- Database optimization: The process of improving the efficiency of database operations and queries.
- Database partitioning: The division of a large database into smaller, more manageable parts.
- Database clustering: The grouping of database servers together to improve performance and availability.
- Database sharding: The process of dividing a database into multiple smaller databases to distribute the load and improve scalability.
- Database federation: The combination of multiple databases into a single logical entity to provide a unified view of data.
- Database virtualization: The use of virtualization technologies to create and manage multiple databases on a single physical server.
- Database migration: The process of transferring data and schema from one database to another.
- Database portability: The ability to move a database between different platforms or environments without losing functionality.
- Database interoperability: The ability of different databases to exchange data and share information with each other.
- Database interoperability standards: The set of rules and protocols that allow different databases to communicate and exchange data.
- Database interoperability tools: The software applications and libraries that facilitate the exchange of data between different databases.
- Database interoperability challenges: The difficulties and limitations encountered when trying to achieve interoperability between different databases.
- Database interoperability solutions: The techniques and approaches used to overcome the challenges and improve interoperability between databases.
These terms are fundamental to understanding the concepts and practices related to database management and are widely used in the field of computer science.
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|
IN THE DISTRICT COURT OF CLEVELAND COUNTY
STATE OF OKLAHOMA
STATE OF OKLAHOMA, ex rel.,
MIKE HUNTER,
ATTORNEY GENERAL OF OKLAHOMA,
Plaintiff,
vs.
(1) PURDUE PHARMA L.P.;
(2) PURDUE PHARMA, INC.;
(3) THE PURDUE FREDERICK COMPANY;
(4) TEVA PHARMACEUTICALS USA, INC.;
(5) CEPHALON, INC.;
(6) JOHNSON & JOHNSON;
(7) JANSSEN PHARMACEUTICALS, INC.;
(8) ORTHO-McNEIL-JANSSEN
PHARMACEUTICALS, INC., n/k/a
JANSSEN PHARMACEUTICALS, INC.;
(9) JANSSEN PHARMACEUTICA, INC.,
n/k/a JANSSEN PHARMACEUTICALS, INC.;
(10) ALLERGAN, PLC, f/k/a ACTAVIS PLC,
f/k/a ACTAVIS, INC., f/k/a WATSON
PHARMACEUTICALS, INC.;
(11) WATSON LABORATORIES, INC.;
(12) ACTAVIS LLC; and
(13) ACTAVIS PHARMA, INC.,
f/k/a WATSON PHARMA, INC.,
Defendants.
Case No. CJ-2017-816
Judge Thad Balkman
STATE OF OKLAHOMA}
CLEVELAND COUNTY } S.S.
FILED
AUG 26 2019
In the office of the
Court Clerk MARILYN WILLIAMS
JUDGMENT AFTER NON-JURY TRIAL
Beginning May 28, 2019 and ending July 15, 2019, the Court conducted a non-jury trial in the above-captioned matter. From the time this action was commenced on June 30, 2017, and through and including today, this Court has been the beneficiary of exemplary professionalism and legal work on the part of counsel for each of the parties – certainly on par with what one would hope for and expect in a case of this magnitude. For that, I wish to express my sincere
appreciation to each of you. The Court, having heard testimony of the witnesses sworn and examined in open court, having observed their demeanor and credibility, having reviewed the exhibits admitted into evidence, and being fully advised in the premises, finds as follows:
**INTRODUCTION**
1. The State’s sole claim for relief against the Defendants was for causing a public nuisance pursuant to Okla. Stat. tit. 50, §1 et seq., and the State sought relief in the form of abatement of the nuisance. The Defendants sought no counterclaims at trial and contended the State failed to meet its burden of proof. In addition the Defendants asserted multiple theories of defense under both statutory and common law.
2. Over the course of 33 days of trial, the parties called 42 witnesses, submitted 874 exhibits into evidence and presented an additional 225 court exhibits.
3. The parties agree Oklahoma is suffering a crisis related to opioid drug abuse. The parties agree on certain statistics and data substantiating the crisis. The parties agree that from 1994 to 2006, prescription opioid sales increased fourfold and that from 2011-2015, more than 2,100 Oklahomans died of an unintentional prescription opioid overdose. It was undisputed that in 2015, over 326 million opioid pills were dispensed to Oklahoma residents, enough for every adult to have 110 pills. Oklahoma dispenses the most prescription fentanyl per capita. In 2017, 4.2% of babies born covered by SoonerCare were born with Neonatal Abstinence Syndrome (also called NAS), a group of conditions caused when a baby withdraws from certain drugs it’s exposed to in the womb before birth.
4. The State, primarily under the leadership of the Oklahoma Department Mental Health and Substance Abuse Services (“ODMHSAS”), as well as through other agencies,
undertook substantial efforts to implement programs, plans and measures to combat and mitigate the consequences of the opioid overdose crisis.
5. Pursuant to a grant awarded in 2008, ODMHSAS conducted a statewide assessment that identified the prevention of underage drinking and prescription drugs as the two most pressing issues facing the State of Oklahoma. In 2012, the State formed a Prescription Drug Working Group that identified a series of recommendations and issues to address and implement and proposed a “State plan to address prescription drug abuse.” In 2016, the State released a second prescription drug plan for “reducing prescription drug abuse in Oklahoma” and “a review of progress and updated State plans.” Some of the actions taken by the State included establishing opioid prescribing guidelines and a naloxone opioid reversal and overdose response program. And, in 2017, the Oklahoma Attorney General and Oklahoma Legislature assembled and convened the Oklahoma Opioid Commission, which issued a report that outlined and detailed recommendations for policies, legislation, regulations and other programs aimed at “combating the opioid crisis in the State of Oklahoma. Those actions achieved some success in reducing the rate of unintentional opioid overdose deaths in the State.
6. On March 29, 2017, President Donald J. Trump established the President’s Commission on Combating Drug Addiction and the Opioid Crisis. The Commission studied ways to combat and treat drug abuse, addiction, and the opioid crisis. It assessed the availability and accessibility of treatment services and overdose reversal throughout the country and reported on best practices for addiction prevention, including healthcare provider education and evaluation of prescription practices, and the use and effectiveness of State prescription drug monitoring programs.
FINDINGS OF FACT
To the extent any evidence in the record conflicts with one of the facts found below, the Court has weighed the competing evidence and found that the greater weight of the evidence weighs in favor of the facts set forth below.
1. The State of Oklahoma and the public in general are currently experiencing an opioid crisis and epidemic (hereinafter referred to as the “Opioid Crisis”). See, e.g., Trial Tr. (6/25/19 a.m., Commissioner White) at 62:10-73:25, 105:14-108:3; Trial Tr. 6/26/19 p.m., Commissioner White) at 45:13-46:4, 47:17-48:19, 53:20-56:22, 65:17-22; Trial Tr. (5/29/19 p.m., J&J: Deem-Eshleman) at 15:18-16:04; Trial Tr. (5/30/19 p.m., J&J: Deem-Eshleman) at 36:21-22; Trial Tr. (6/5/19 a.m., J&J: Deem-Eshleman) at 80:08-13, 81:19-23; Trial Tr. (7/2/19 p.m., Diesselhorst) at 30:23-33:15; Trial Tr. (5/29/19 a.m., Courtwright) at 22:15-18.
2. This current stage of the Opioid Crisis was started by and still primarily involves prescription opioids. See, e.g., Trial Tr. (6/11/19 a.m., Kolodny) at 74:11-15; Trial Tr. (5/29/19 a.m., Courtwright) at 22:19-21; Trial Tr. (6/6/19 a.m., Mazloomdoost) at 22:15-23; Trial Tr. (6/3/19, a.m. J&J: Deem-Eshleman) at 126:16-18.
3. Through the mid-1990s, there was no opioid epidemic. Trial Tr. (6/26/19 p.m., Commissioner White) at 29:15-18, 66:25-67:6; see also, e.g., Trial Tr. (6/17/19 p.m., Beaman) at 73:8-19.
4. Since at least the mid-1990s, Defendants have marketed, promoted and sold opioid drugs in Oklahoma. See, e.g., Ct. Ex. 0092 (Mashett) at 401:4-16. During this time period, Defendants specifically manufactured and sold certain of their own branded opioid drugs as a part of its pain franchise, including: (i) Duragesic—a transdermal patch made out of the active
pharmaceutical ingredient ("API"), fentanyl; (ii) Ultram and Ultram Extended Release ("ER")—tablets made out of the API, tramadol; (iii) Ultracet—tablets made out of the APIs, tramadol and acetaminophen; (iv) Nucynta and Nucynta ER—tablets made out of the API, tapentadol; (v) Tylenol with Codeine—tablets made out of the APIs, acetaminophen and codeine; (vi) Tylox—capsules made out of the APIs, acetaminophen and oxycodone. See, e.g., S-1073 at 10; J-2769 at 1; Trial Tr. (5/30/19 a.m., J&J: Deem-Eshleman) at 43:4-9; Trial Tr. (6/27/19 a.m., Moskovitz) at 21:7-22:13; Trial Tr. (6/11/19 a.m., Kolodny) at 108:5-7.
5. Dr. Paul Janssen originally invented fentanyl in the 1950s. Trial Tr. (6/3/19 p.m., J&J: Deem-Eshleman) at 67:17-23; Trial Tr. (6/5/19 a.m., J&J: Deem-Eshleman) at 80:17-20. Fentanyl is a highly addictive opioid. Trial Tr. (6/3/19 a.m., J&J: Deem-Eshleman) at 47:02-05. Fentanyl can always be abused. Trial Tr. (6/3/19 a.m., J&J: Deem-Eshleman) at 47:06-12. As a Schedule II opioid comprised of fentanyl, Defendants’ Duragesic “has the highest potential for abuse.” See Trial Tr. (6/28/19 p.m., Moskovitz) at 93:03-07.
6. As part of its “pain management franchise,” from the 1990s through at least 2016, Defendant Johnson & Johnson wholly owned two subsidiaries that, together, supplied other opioid manufacturers with opioid APIs to be used in opioid drugs. See, e.g., S-0340; S-1048; S-0006. First, Johnson & Johnson owned a subsidiary based in Tasmania, Tasmanian Alkaloids Limited (“Tasmanian Alkaloids”), which cultivated and processed opium poppy plants to manufacture narcotic raw materials that were imported into the U.S. to be processed and made into APIs necessary to manufacture opioid drugs. See, e.g., S-0340; S-1048; S-0006. Second, Johnson & Johnson owned a subsidiary based in the U.S., Noramco, Inc. (“Noramco”), which imported the narcotic raw materials produced by Tasmanian Alkaloids, processed these materials
into APIs, then sold these APIs to other opioid manufacturers in the U.S. See, e.g., S-0340; S-1048; S-0006.
7. Up until 2016, when Johnson & Johnson sold its Noramco/Tasmanian Alkaloids businesses, Tasmanian Alkaloids and Noramco were “sister companies,” as “both of them were” members of Defendants’ “family of companies.” Ct. Ex. 220 (Martin) at 9:17-23, 12:17-13:8, 104:5-107:2. Testimony from Noramco at trial demonstrated that Noramco employees did not believe Noramco maintained its own bank accounts, separate from Defendants’ treasury. Ct. Ex. 220 (Martin) at 101:19-24. Defendants, Noramco and Tasmanian Alkaloids shared employees and resources that were “required to operate the business.” S-1048 at 9. Noramco employees, including Mr. Martin, physically worked at Defendants’ facilities in New Jersey at various times. Ct. Ex. 220 (Martin) at 8:20-9:1. Noramco employees “were with Johnson & Johnson.” Ct. Ex. 220 (Martin) at 9:17-18, 9:21-23. Further, employees simultaneously held positions at multiple companies within the Johnson & Johnson Family of Companies at times. See, e.g., Trial Tr. (6/11/19 p.m., Kolodny) at 15:11-20. During this time, Noramco and Tasmanian Alkaloids were key parts of Defendants’ “pain management franchise” or “pain franchise.” Ct. Ex. 0092 (Mashett) at 75:3-11; S-0340. This “pain franchise” included all of Defendants’ pain products and “was an important part of [Defendants’] business from the mid-1990s to after 2010.” Ct. Ex. 0092 (Mashett) at 75:1-11.
8. Defendants, through these subsidiaries, supplied the following opioid APIs to other drug manufacturers in the U.S., including Purdue and Teva: oxycodone, hydrocodone, morphine, codeine, fentanyl, sufentanil, buprenorphine, hydromorphone, and naloxone. See, e.g., S-0340 at 4; S-1048 at 7, 10, 22; S-0006 at 6-7; Ct. Ex. 220 (Martin) at 155:2-162:15, 184:24-185:16; Ct. Ex. 0092 (Mashett) at 219:18-220:8, 230:8-24. By 2015, Defendants’ “Noramco
World Wide Narcotics Franchise,” comprised of Noramco and Tasmanian Alkaloids, had become “the #1 supplier of Narcotic APIs in the United States, the world’s largest market.” S-1048 at 6.
9. In the 1980s, Johnson & Johnson acquired and formed Tasmanian Alkaloids and Noramco, in order to ensure a “reliable source of [narcotic] raw materials” and “security of supply” for its Tylenol with Codeine range of pain medications. See S-0006 at 3; S-1048 at 13; Trial Tr. (6/11/19 a.m., Kolodny) at 108:13-17.
10. Noramco, located in the U.S., imports the narcotic raw materials produced by Tasmanian Alkaloids, like morphine or thebaine, into the U.S., processes them into API, then sells them to drug manufacturers in the U.S. See, e.g., S-340, S-1048. Noramco was “an important part of J&J’s business” from the mid-1990s until at least after 2010. Ct. Ex. 0092 (Mashett) at 75:3-11. Johnson & Johnson’s ownership of these subsidiaries uniquely positioned its pain management franchise to provide U.S. drug manufacturers, including Johnson & Johnson itself, with “Security of Supply”—“Direct Access to Narcotic Raw Material – From Our Fields to Your Formulations.” S-1048 at 11-13. Through its subsidiary, Noramco, Johnson & Johnson supplied oxycodone API to other drug manufacturers. See Trial Tr. (5/29/19 p.m., J&J: Deem-Eshleman) at 36:22-37:01, 44:02-04; see also, e.g., Trial Tr. (6/11/19 a.m., Kolodny) at 63:10-65:06, 100:1-134:11.
11. In 1994, Defendants, in concert with subsidiary, Tasmanian Alkaloids, “anticipated demand” for oxycodone. See S-0006 at 6; Trial Tr. (5/29/19 p.m., J&J: Deem-Eshleman) at 59:19-24; Trial Tr. (6/11/19 a.m., Kolodny) at 113:2-13. Specifically, Defendants’ scientists at Tasmanian Alkaloids began a project “in 1994 in order to develop a high thebaine poppy variety to meet the anticipated demand.” S-0006 at 6. The result of Defendants’ research
project was the creation of a “high thebaine” poppy, called the “Norman Poppy,” which Defendants internally described as “a transformational technology that enabled the growth of oxycodone.” See S-0006 at 6-7; S-340 at 7; Trial Tr. (5/29/19 p.m., J&J: Deem-Eshleman) at 42:14-62:02; Trial Tr. (6/11/19 a.m., Kolodny) at 106:4-111:18. In 1994, Purdue filed its new drug application (“NDA”) for OxyContin. See Trial Tr. (6/11/19 a.m., Kolodny) at 113:2-13.
12. Through Noramco, Defendants met the anticipated opioid demand by selling API, including oxycodone, to Purdue. Ct. Ex. 0092 (Mashett) at 222:3-16; see also, e.g., S-1788; Trial Tr. (5/29/19 p.m., J&J: Deem-Eshleman) at 42:14-62:02; Trial Tr. (6/11/19 a.m., Kolodny) at 109:9-115:8.
13. Noramco and Tasmanian Alkaloids were an important part of Defendants’ pain management enterprise that included all of Defendants’ pain products and “was an important part of [Defendants’] business from the mid-1990s to after 2010.” See S-0340 Ct. Ex. 0092 (Mashett) at 75:1-11.
14. Through Noramco, Defendants supplied API to other opioid manufacturers, including Teva. Ct. Ex. 0092 (Mashett) at 219:18-220:8, 230:8-24. Noramco sold the majority of its “controlled substance” via “long-term agreements” and had such agreements “with all 7 of the top U.S. generic companies.” S-1048 at 18. Through Noramco, Defendants supplied other U.S. opioid manufacturers with opioid APIs, including: oxycodone, hydrocodone, morphine, codeine, buprenorphine, hydromorphone and naloxone. See, e.g., S-1048; Trial Tr. (6/11/19 a.m., Kolodny) at 127:4-134:11.
15. Defendants’ subsidiary, Noramco, grew to become the No. 1 narcotic API supplier of oxycodone, hydrocodone, codeine and morphine in the United States. S-1048; Trial Tr. (5/29/19 p.m., J&J: Deem-Eshleman) at 70:09-75:16.
16. Through the mid-1990s, there was no opioid epidemic. Trial Tr. (6/26/19 p.m., Commissioner White) at 29:15-18, 66:25-67:6; see also, e.g., Trial Tr. (6/17/19 p.m., Beaman) at 73:8-19.
17. In 1997, after seeing the success that Purdue had in marketing OxyContin for chronic non-cancer pain, Defendants re-launched their fentanyl-based Duragesic patch for the chronic, non-cancer market as well. S-2355; see also Trial Tr. (5/30/19 a.m., J&J: Deem-Eshleman) at 78:07-81:05; Trial Tr. (6/3/19 p.m., J&J: Deem-Eshleman) at 25:01-03; Trial Tr. (6/13/19 p.m., Kolodny) at 16:15-25.
18. Defendants, acting in concert with others, embarked on a major campaign in which they used branded and unbranded marketing to disseminate the messages that pain was being undertreated and “there was a low risk of abuse and a low danger” of prescribing opioids to treat chronic, non-malignant pain and overstating the efficacy of opioids as a class of drug. Trial Tr. (6/26/19 p.m., Commissioner White) at 29:15-31:9, 67:13-68:9, 82:7-21; see also, e.g., Trial Tr. (6/10/19 p.m., Stone) at 27:15-40:8; see also, e.g., Trial Tr. (6/13/19 p.m., Kolodny) at 11:25-12:05, 17:2-23:13; Trial Tr. (6/17/19 a.m., Kolodny) at 109:4-25.
19. Defendants’ marketing and promotional efforts were designed to reach Oklahoma doctors through multiple means and at multiple times over the course of the doctor’s professional education and career in Oklahoma. See Trial Tr. (5/30/19 a.m., J&J: Deem-Eshleman) at 63:01-66:20. Examples of such marketing and promotion include, among other things, “education”
from Defendants’ sales representatives, literature funded by Defendants in medical journals and publications, materials from professional societies/patient advocacy groups, continuing medical education funded by Defendants, unbranded marketing materials, and Defendants’ paid speakers. See Trial Tr. (5/30/19 a.m., J&J: Deem-Eshleman) at 63:01-66:20. Other avenues included dinners and presentations where doctors spoke to other doctors, partnering with third-party advocacy groups or academic groups to hold seminars, symposiums and conferences. Ct. Ex. 2 (Portenoy) at 40:24-41:11. All of these many different efforts were intended to influence the prescribing behavior of physicians and, thus, increase Defendants’ profits from opioids. See, e.g., Ct. Ex. 2 (Portenoy) at 43:8-19; Trial Tr. (6/13/19 p.m., Kolodny) at 11:25-12:5, 17:2-23:13; Trial Tr. (6/13/19 a.m., Kolodny) at 63:2-64:04; S-2364; S-1246; S-1372; S-1844; S-3961; S-3960; S-881; S-903; S-510; S-1163; S-1780.
20. A key element in Defendants’ opioid marketing strategy to overcome barriers to liberal opioid prescribing was its promotion of the concept that chronic pain was undertreated (creating a problem) and increased opioid prescribing was the solution. See, e.g., Trial Tr. (6/10/19 p.m., Stone) at 83:17-22; S-1239 at 4-6; S-0982; Trial Tr. (6/12/19 p.m., Kolodny) at 46:23-47:5.118. For example, Defendants’ unbranded marketing campaigns frequently focused on [h]eightening awareness of the under treatment of pain and its consequences.” See, e.g., S-0223 at 1; S-1239 at 5-6; S-2358. Defendants trained their Oklahoma sales representatives on how to use these campaigns, including through the use of “emotional selling” for opioids by convincing physicians that undertreated pain was harming patients. See, e.g., S-0223 at 3.
21. Another unbranded marketing message Defendants used to accomplish the “[b]ehavior [c]hange” of “increase[d] opioid use” was that undertreated acute pain inevitably would turn into chronic pain. See 1163 at 17; S-1780; Trial Tr. (6/11/19 p.m., Kolodny) at
Defendants emphasized this message in their marketing materials that promoted opioids generally as a class of drug. See, e.g., S-0760; Trial Tr. (6/3/19 a.m., J&J: Deem-Eshleman) at 54:20-63:25.
22. Defendants used the phrase, “pseudoaddiction,” to convince doctors that patients who exhibited signs of addiction—e.g., asking for “higher and higher doses” of opioids or returning to the doctor “early” before a prescription should have run out—were not actually suffering from addiction, but from the undertreatment of pain; and the solution, according to Defendants’ marketing, was to prescribe the patient more opioids. See, e.g., Trial Tr. (6/11/19 a.m., Kolodny) at 87:3-88:6; Trial Tr. (6/13/19 a.m., Kolodny) at 74:25-89:11; Trial Tr. (6/6/19 a.m., Mazloomdoost) at 35:21-36:5, 44:7-45:4; Ct. Ex. 2 (Portenoy) at 215:24-219:8. Defendants repeatedly promoted the concept of “pseudoaddiction” in various publications over time. See, e.g., S-954 at 2; S-0740 at 6; S-0760 at 3.
23. Defendants ran a website called Prescribe Responsibly as a form of unbranded marketing. S-0974; Trial Tr. (6/3/19 a.m., J&J: Deem-Eshleman) at 90:17-91:07; see also S-0954; Trial Tr. (6/11/19 p.m., Kolodny) at 139:1-147:25. Information on the Prescribe Responsibly website promoted Defendants’ messaging that the solution to “pseudoaddiction” was “to prescribe more opioids.” See S-0954; Trial Tr. (6/11/19 p.m., Kolodny) at 139:1-147:25.
24. Another unbranded marketing initiative that Defendants employed was the dissemination of a brochure, titled “Finding Relief.” See S-1247; Trial Tr. (6/11/19 p.m., Kolodny) at 40:6-14. The Finding Relief brochure, which was widely disseminated, did not differentiate between different kinds of opioids and discussed them as a class of drugs without reference to any of the differences between them. See S-1247; Trial Tr. (6/28/19 a.m., Moskovitz) at 108:02-110:09, 112:12-113:02. The Finding Relief brochure actively promoted the
concept that pain was undertreated. See S-1247; Trial Tr. (6/11/19 p.m., Kolodny) at 40:6-14. The brochure downplayed any risks associated with opioids. See, e.g., Trial Tr. (6/11/19 p.m., Kolodny) at 98:17-99:22.
25. As part of Defendants’ marketing and advocacy programs aimed at increasing opioid prescriptions, in addition to influencing doctors, Defendants employed strategies to influence a wide range of governmental agencies, through messages aimed at “optimizing the benefits of prescription opioids for pain management [and] minimizing their risks,” including the risk of addiction, abuse and diversion. See, e.g., Trial Tr. (6/26/19 p.m., Commissioner White) at 57:21-61:4; S-1161 at 10; Trial Tr. (6/26/19 p.m., Commissioner White) at 110:2-111:8; Trial Tr. (6/10/19 p.m., Stone) at 36:3-37:22; Trial Tr. (6/3/19 a.m., J&J: Deem-Eshleman) at 83:24-90:13.
26. Defendants used a sales force in Oklahoma to promote, market and sell various types of opioids, including the branded opioid drugs that Defendants, themselves, manufactured: Duragesic, Ultram, and Nucynta. See Trial Tr. (5/30/19 a.m., J&J: Deem-Eshleman) at 43:10-16.
27. Defendants’ training of their sales representatives in Oklahoma included teaching sales representatives to avoid the so-called “addiction ditch”—i.e., to avoid the negatives (addiction) and emphasize the positives (supposed efficacy) in sales calls—and to use a study from Dr. Portenoy “to create dialogue about Opiophobia as a barrier.” S-1364 at 16; Trial Tr. (5/29/19 p.m., J&J: Deem-Eshleman) at 30:14-33:11; see also Trial Tr. (7/2/19 p.m., Diesselhorst) at 46:10-16; S-1162.
28. As part of this training, Defendants trained their sales representatives that there was a 2.6% or lower risk of addiction when using opioids prescribed by a doctor. See S-1364; Trial Tr. (5/29/19 p.m., J&J: Deem-Eshleman) at 30:14-33:11. As part of this same training, Defendants trained sales representatives to “establish that moderate to severe acute pain continues to be undertreated.” S-1364 at 10; Trial Tr. (6/3/19 p.m., J&J: Deem-Eshleman) at 7:02-14.
29. Defendants’ corporate representative was not aware of any training provided to Defendants’ sales force in Oklahoma on the disease of addiction. See Trial Tr. (5/30/19 a.m., J&J: Deem-Eshleman) at 46:19-51:06. Nor was Defendants’ corporate representative aware of any training provided to the sales force related to the history of opioid use and epidemics in the U.S. or human history. See Trial Tr. (5/30/19 a.m., J&J: Deem-Eshleman) at 46:19-51:06.
30. Defendants trained their sales reps to target high-opioid-prescribing physicians, including pain specialists and primary care physicians. See S-2514; S-2515; S-2538; Trial Tr. (5/30/19 p.m., J&J: Deem-Eshleman) at 116:04-152:25. Defendants particularly targeted primary care physicians with their opioid marketing, identifying them as “Key Customer[s]” for Defendants’ pain franchise. S-2358 at 15 (defining “Prescribers” as a “Key Customer Segment”); Trial Tr. (5/30/19 a.m., J&J: Deem-Eshleman) at 129:20-130:23; Trial Tr. (6/12/19 p.m., Kolodny) at 115:10-14; see also, e.g., Ct. Ex. 2 (Portenoy) at 213:25-214:10 (testifying that after 1996, the pharmaceutical industry targeted primary care physicians with their marketing efforts in order to convince these doctors to prescribe opioids for chronic non-cancer pain).
31. Defendants’ Oklahoma call notes document that sales representatives distributed visual aids citing the Allan, Simpson and Milligan studies thousands of times, including, at a minimum: (i) 726 times between June 2002 and December 2002; (ii) 1,683 times in 2003; and
(iii) 754 times in 2004. See S-2481 – S-2492; see also Ct. Ex. 223. Defendants’ Oklahoma call notes further document their sales representatives using the Allan, Simpson and Milligan studies over 1,000 times in sales visits to Oklahoma doctors between 1998 and 2004. See S-2481 – S-2492; see also Ct. Ex. 223.
32. The representations in these marketing materials related to functionality and low abuse rates, DAWN data, and the Milligan, Allan, and Simpson Studies, were later described as false and misleading by the FDA. See, e.g., S-0038; see also Section F.3, infra. Defendants funded each of these studies. S-2517; S-2521; S-2523; Trial Tr. (5/30/19 p.m., J&J: Deem-Eshleman) at 152:23-152:25.
33. Defendants did not train their sales representatives regarding red flags that could indicate a “pill mill,” including, for example, pain clinics with patients lined up out the door or patients passed out in the waiting room. See Trial Tr. (6/3/19 a.m., J&J: Deem-Eshleman) at 29:07-09; Trial Tr. (7/2/19 p.m., Diesselhorst) at 86:22-87:7, 170:6-172:6.
34. Marketing strategies developed for Defendants’ sales force included utilizing a coupon program as a marketing tool for Duragesic and sample voucher programs, in which a sales representative delivered to a physician a “sample voucher for a box of 25mcg or 50mcg patches redeemed at pharmacy for a free 15-day trial of DURAGESIC.” S-2366; Trial Tr. (5/30/19 p.m., J&J: Deem-Eshleman) at 66:18-79:08; see also S-0582; and S-1358 at 14.
35. Defendants’ sales representatives called on Oklahoma medical professionals hundreds of thousands of times while selling opioids as evidenced by 35 boxes of call notes from Defendants’ Oklahoma sales representatives over the last two decades. Defendants’ Oklahoma sales representatives brought breakfast, lunch, coffee and snacks to Oklahoma doctors’ offices
and used speaker programs as part of their sales strategies. See S-2481 – S-2492; see also Trial Tr. (7/2/19 p.m., Diesselhorst) at 184:1-185:19; see also S-4497
36. Defendants made substantial payments of money to a variety of different pain advocacy groups and organizations that influenced prescribing physicians and other health care professionals. The organizations included the American Academy of Pain Medicine (“AAPM”), American Pain Society (“APS”), American Pain Foundation (“APF”), American Geriatrics Society, American Chronic Pain Association, National Pain Foundation, Pain and Policies Study Group (“PPSG”), Pain Care Forum, American Society of Pain Management Nursing, American Academy of Pain Management/Academy of Integrative Pain Management (“AIPM”), Center for Practical Bioethics, and Joint Commission on Accreditation of Healthcare Organizations (“JCAHO”). See, e.g., S-1349
37. Two organizations Defendants funded, the AAPM and APS, issued a “Consensus Statement” in 1996 that was drafted by a committee that included Robert Angarola, an attorney who at one time represented Defendants on opioid-related issues. See S-0900; Trial Tr. (6/11/19 p.m., Kolodny) at 20:10-39:8. Specifically, the Consensus Statement was written by a committee including David Haddox (former Purdue Pharma medical director), David Joranson (founder of PPSG), Richard Payne (KOL, co-leader of Defendants’ NPEC program), Matthew Midcap (who had a financial relationship with Defendants), Daniel Carr (who had a financial relationship with Defendants), and Robert Angarola (outside counsel to Defendants in 1990 related to thebaine imports from Tasmania). Dr. Portenoy consulted on the Consensus Statement as well. Trial Tr. (6/11/19 p.m., Kolodny) at 41:03-44:24.
38. The Consensus Statement suggests pain is undertreated and doctors should prescribe more opioids and described a fear of addiction, regulatory action and diversion as “impediments” to the use of opioids. S-0900; Trial Tr. (6/11/19 p.m., Kolodny) at 20:10-39:08.
39. Defendants actively promoted the Consensus Statement, ratifying and repeating its statements in Defendants’ own marketing. See, e.g., S-0760
40. Part of Defendants’ marketing strategy included medical education activities. See, e.g., S-1358; S-2364; Trial Tr. (6/13/19 a.m., Kolodny) at 52:20-68:18. This included the creation and funding of a group known as “NPEC” (National Pain Education Council) whose purposes was to provide Continuing Medical Education (“CME”) related to pain and opioids. See S-0975, S-0582; Trial Tr. (5/29/19 p.m., J&J: Deem-Eshleman) at 23:06-28:12; see also Ct. Ex. 2 (Portenoy) at 87:25-89:9.
41. The target audience for Defendants’ NPEC initiative included primary care physicians, pain specialists, oncologists, residents, nurses and pharmacists. S-0881 at 3. In Defendants’ 2003 Business Plan Summary for Duragesic, Defendants described NPEC as serving “to benefit not only DURAGESIC but also all future Janssen pain products.” S-1358 at 10.
42. CME materials for Defendants’ NPEC program in 2002 disseminated false and misleading statements regarding opioids and pain management. See, e.g., Trial Tr. (6/6/19 a.m., Mazloomdoost) at 48:12-62:24.
43. Defendants viewed the efforts to schedule tramadol by agencies within the State of Oklahoma as a “threat.” S-0463; see Trial Tr. (6/3/19 a.m., J&J: Deem-Eshleman) at 49:07-54:15. In 2008, in response to Defendants learning that “the Oklahoma Board of Pharmacy is
threatening to schedule tramadol again,” Defendants’ Therapeutic Area Head and expert witness, Dr. Bruce Moskovitz, recommended that Defendants “mobilize” and send a “‘swat’ team” to Oklahoma to deal with the threat. S-0463; see Trial Tr. (6/3/19 a.m., J&J: Deem-Eshleman) at 49:07-54:15.
44. Defendants’ opioid marketing, in its multitude of forms, was false, deceptive and misleading. See, e.g., Trial Tr. (6/11/19 a.m., Kolodny) at 69:6-72:23, 85:10-21, 90:21-91:25; Trial Tr. (6/13/19 p.m., Kolodny) at 17:2-23:13; Trial Tr. (6/17/19 a.m., Kolodny) at 109:4-25; Trial Tr. (6/25/19 a.m., Commissioner White) at 66:10-19; Trial Tr. (6/26/19 p.m., Commissioner White) at 112:21-113:15, 117:14-120:12, 129:2-13, 130:22-132:7; Trial Tr. (6/17/19 p.m., Beaman) at 64:20-71:12, 80:18-85:7-20; S-0760; S-0037; S-0038; S-2481 – S-2492; S-2524; S-2538; S-2515; S-0974; S-0954; S-1247; S-0712; S-4128; S-1249; S-1706; S-2354; S-2372.
45. In 1998, the FDA found three different convention posters Defendants used to promote Duragesic to contain marketing messages that were “false and misleading” for numerous reasons including using misleading comparative efficacy claims without substantial evidence, taking data out of context to deliver misleadingly incomplete impressions, promoting unapproved uses, emphasizing the “chronic pain” indications without the limitations and restrictions, and deceptively minimizing risks and safety issues. See S-4128.
46. In 2001, Defendants were advised by Defendants’ own hired scientific advisory board that many of the primary marketing messages Defendants used to promote opioids in general, and Duragesic specifically, were misleading and should not be disseminated. See S-0035. Specifically, Defendants were advised not to market opioids, including fentanyl-based Duragesic, using messages related to abuse or with claims about supposedly low abuse potential.
See S-0035; Trial Tr. (5/30/19 a.m., J&J: Deem-Eshleman) at 94:16-124:21. Defendants were advised that no data existed that could support these claims, that the data Defendants pointed to (DAWN data) was incapable of supporting these claims, that aggressively marketing OxyContin on this same basis was what had gotten Purdue “in trouble,” that minimizing the risk of abuse of Duragesic was “dangerous” due to its lethal nature, and that an increase of Duragesic sales would surely cause an increase in abuse of and addiction to the drug. S-0035. The “Conclusion: Do not include the abuse message. Do not sell opioids on the abuse issue.” S-0035.
47. In 2004, the FDA sent Defendants a letter stating that a professional file card that Defendants used to promote Duragesic (“Duragesic file card”) contained “false or misleading claims about the abuse potential and other risks of [Duragesic], and include[d] unsubstantiated effectiveness claims for Duragesic.” S-0038 at 1. The FDA found that the Duragesic file card misbranded the drug by “suggesting that Duragesic has a lower potential for abuse compared to other opioid products,” and “the file card could encourage the unsafe use of the drug, potentially resulting in serious or life-threatening hypoventilation.” S-0038 at 1.
48. Substantiating the advice of Defendants’ advisors in 2001, in 2004, the FDA found Defendants’ suggestion that Duragesic was “less abused than other opioid drugs” was “false or misleading” because: (i) the FDA was “not aware of substantial evidence or substantial clinical experience to support this comparative claim”; (ii) “DAWN data cannot provide the basis for a valid comparison” among opioid products; and (iii) “DAWN is not a clinical database” but, rather, a “national public health surveillance system that monitors drug-related emergency department visits and deaths.” S-0038 at 2; see also S-0035; S-1703.
49. The FDA concluded that Defendants’ Duragesic file card made “false or misleading safety claims and unsubstantiated effectiveness claims for Duragesic” and “thus
misbrand[ed] Duragesic in violation of the Act (21 U.S.C. § 352(a)).” S-0038 at 3. The FDA requested that Defendants “immediately cease the dissemination of promotional materials for Duragesic the same as or similar to those described” in this 2004 letter. S-0038 at 3. The FDA further mentioned that the “violations discussed” in the letter did not “necessarily constitute an exhaustive list” and it was Defendants’ responsibility to “ensure that [its] promotional materials for Duragesic comply with each applicable requirement of the Act and FDA implementing regulations.” S-0038 at 4.
50. Many other promotional materials that Defendants used in Oklahoma contained the same false and misleading messaging as the file card. The file card was not the only piece of marketing that contained these materials. Evidence was presented of a variety of visual aids distributed in Oklahoma and utilized by sales representatives containing identical false and misleading messages. See, e.g., S-2524; S-2538.
51. Defendants’ marketing materials repeatedly used the Porter and Jick letter and the Milligan, Allan and Simpson Studies in deceptive ways to support misleading claims that downplay the risk of addiction and overstate the efficacy of opioids. See, e.g., S-1706; S-1710; S-1364; Trial Tr. (6/13/19 a.m., Kolodny) at 69:16-72:20.
52. Defendants additionally executed their strategy of targeting high-opioid-prescribing physicians in Oklahoma, including doctors who ultimately faced disciplinary proceedings or criminal prosecution. See, e.g., S-1358; S-2357; S-1844; S-0510; S-903; Trial Tr. (6/10/19 p.m., Stone) at 53:20-54:17.
53. Both Drs. Beaman and Mazloomdoost testified that the multifaceted marketing misinformation campaign by the opioid industry, including Defendants, influenced their
practices and caused them to liberally and aggressively write opioid prescriptions they would never write today. See, e.g., Trial Tr. (6/17/19 p.m., Beaman) at 40:22-41:13, 68:7-69:6, 79:1-81:23; Trial Tr. (6/6/19 a.m., Mazloomdoost) at 72:17-73:2.
54. The increase in opioid addiction and overdose deaths following the parallel increase in opioid sales in Oklahoma was not a coincidence; these variables were “causally linked.” Trial Tr. (6/25/19 a.m., Commissioner White) at 73:19-23. Dr. Beaman also testified that, in his opinion, the increase in opioid overdose deaths and opioid addiction treatment admissions in Oklahoma was caused by the oversupply of opioids through increased opioid sales and overprescribing since the late 1990s. See Trial Tr. (6/17/19 p.m., Beaman) at 69:2-73:19.
55. Commissioner White testified that the oversupply and “significant widespread rapid increase in the sale of opioid prescription medications” beginning in the mid-1990s caused the “significant rise in opioid overdose deaths” and “negative consequences” associated with opioid use, including addiction, opioid use disorder, the rise in NAS, and children entering the child welfare system. Trial Tr. (6/25/19 a.m., Commissioner White) at 62:10-63:5.
56. With respect to the prescription opioid epidemic in the U.S., on November 1, 2017, the President’s Commission on Combating Drug Addiction and the Opioid Crisis issued its final report and recommendations. See S-1574; Trial Tr. (6/3/19 p.m., J&J: Deem-Eshleman) at 61:17-91:22.
57. The President’s Commission on Combating Drug Addiction and the Opioid Crisis found “Contributors to the Current Crisis” in the U.S. to include, among other things:
• the use of the Porter & Jick letter to make “unsubstantiated claims” by pharmaceutical companies;
• the lack of “[h]igh quality evidence demonstrating that opioids can be used safely for chronic non-terminal pain”;
• the use of the phrase, “pain as the ‘fifth vital sign,’” by the APS, JCAHO and others; and
• the fact that, “[t]o this day, the opioid pharmaceutical industry influences the nation’s response to the crisis. For example, during the comment phase of the guideline developed by the [CDC] for pain management, opposition to the guideline was more common among organizations with funding from opioid manufacturers than those without funding from the life sciences industry.”
See S-1574; Trial Tr. (6/3/19 p.m., J&J: Deem-Eshleman) at 68:21-69:21, 70:5-23, 81:13-23, 84:22-87:20; see also, e.g., S-1349; S-1350. Defendants did all of these things in Oklahoma. For example, Defendants used the Porter & Jick letter to make “unsubstantiated claims” about the risk of addiction when using opioids. Defendants made claims, unsupported by any high quality evidence, that opioids could be used safely for chronic non-terminal pain. Defendants used the phrase, “pain as the ‘fifth vital sign,’” to influence doctors to liberally prescribe opioids.
58. By no later than 2001, “a significant number of Oklahoma physicians, the healthcare community, law enforcement, medical advisory boards, the DUR Board” and others in Oklahoma were “being pushed and pushed and marketed [to] and misled” about opioids by Defendants. Trial Tr. (6/26/19 p.m., Commissioner White) at 47:17-48:19. For example, Dr. Terrell Phillips, gave a CME presentation to the Oklahoma State Medical Association (“OSMA”) in October 2016 about how to avoid addiction in pain management, in which Dr. Phillips stated:
“Everyone here knows how we got in this situation. They told us we were underprescribing. We need to prescribe more. It’s the patient’s rights to have pain medicine, so we all got on board. And when someone said they were hurting, we said,
Okay, we are going to give you something. Now it’s just the opposite. Not everyone deserves pain medicine.”
Trial Tr. (7/12/19 a.m., Phillips) at 71:2-23; see also S-4743 at 7:20-7:48.
CONCLUSIONS OF LAW
1. In Oklahoma, nuisance law is defined by statute. 50 O.S. 1981 §1, defines a nuisance as follows:
A nuisance consists in unlawfully doing an act, or omitting to perform a duty, which act or omission either:
First. Annoys, injures or endangers the comfort, repose, health, or safety of others; or
Second. Offends decency; or
Third. Unlawfully interferes with, obstructs or tends to obstruct, or renders dangerous for passage, any lake or navigable river, stream, canal or basin, or any public park, square, street or highway; or
Fourth. In any way renders other persons insecure in life, or in the use of property, provided, this section shall not apply to preexisting agricultural activities.
50 O.S. §2, states that a public nuisance “is one which affects at the same time an entire community or neighborhood, or any considerable number of persons, although the extent of the annoyance or damage inflicted upon the individuals may be unequal.”
2. The plain text of the statute does not limit public nuisances to those that affect property. Unlike other states’ statutes that limit nuisances to the “habitual use or the threatened or contemplated habitual use of any place,” Oklahoma’s statute simply says “unlawfully doing an act, or omitting to perform a duty.” There is nothing in this text that suggests an actionable nuisance requires the use of or a connection to real or personal property. See Epps v. Ellison,
1921 OK 279, ¶ 3, 200 P. 160, 161 (“Section 4250, Rev. Laws 1910 [(former numbering for 50 O.S. § 1)] defines a nuisance to be any act which annoys, injures, or endangers the comfort, repose, health, or safety of others, or in any way renders other persons insecure in life or in the use of property.” (emphasis added)); see also *Hall v. Galmor*, 2018 OK 59, ¶ 45, 427 P.3d at 1070 (“Our task is to determine the ordinary meaning of the words that the Legislature chose in the provision s of law at issue.”); *Cox v. State ex rel. Oklahoma Dep’t of Human Servs.*, 2004 OK 17, ¶ 26, 87 P.3d 607, 617 (“This Court does not read exceptions into a statute nor may we impose requirements not mandated by the Legislature.”).
3. Supreme Court precedent also supports the conclusion that Oklahoma’s nuisance law extends beyond the regulation of real property and encompasses the corporate activity complained of here. “Section 4250, Rev. Laws 1910 [(former numbering for 50 O.S. § 1)] defines a nuisance to be any act which annoys, injures, or endangers the comfort, repose, health, or safety of others, or in any way renders other persons insecure in life or in the use of property.” *Epps v. Ellison*, 1921 OK 279, ¶ 3, 200 P. at 161.
“Nuisance, as defined at 50 O.S. 1981 §1, consists in unlawfully doing an act, or omitting to perform a duty, which act or omission annoys, injures or endangers the comfort, repose, health or safety of others; or, in any way renders other persons insecure in life, or in the use of property. Thus, the term “nuisance” signifies in law such a use of property or such a course of conduct irrespective of actual trespass against others, or of malicious or actual criminal intent, which transgresses the just restrictions upon use or conduct which the proximity of other persons or property imposes. *Briscoe*, 1985 OK 43, ¶ 9, 702 P.2d 33 at 36. See also *Reaves v. Territory*, 1903 OK 92, 74 P. 951 “no claim of damages to property rights” [existed].
4. However, and in the alternative, in the event Oklahoma’s nuisance law does require the use of property, the State has sufficiently shown that Defendants pervasively, systemically and substantially used real and personal property, private and public, as well as the public roads, buildings and land of the State of Oklahoma, to create this nuisance.
5. The State presented substantial evidence—which Defendants did not attempt to dispute—that Defendants’ sales representatives were trained in their Oklahoma homes how to spread Defendants’ marketing messages (see, e.g., Trial Tr. (5/30/19 a.m., J&J: Deem-Eshleman) at 44:13-46:17, 51:7-9); they conducted their deceptive marketing and sales efforts in doctors’ offices, hospitals, restaurants, and other venues; S-2481 – S-2492; Trial Tr. (6/13/19 a.m., Kolodny) at 92:13-25); they used company cars traveling on State and county roads to disseminate those misleading messages (see, e.g., Trial Tr. (7/2/19 p.m., Diesselhorst) at 168:10-170:4); Defendants paid speakers to deliver Defendants’ messages to doctors in their Oklahoma offices (Trial Tr. (5/30/19 a.m., J&J: Deem-Eshleman) at 52:20-53:3, 55:17-20, 62:12-22; Trial Tr. (7/2/19 p.m., Diesselhorst) at 168:10-170:4); S-3080); and Defendants sent their messages into the homes of thousands of Oklahomans via computers, smart phones or other devices (see, e.g., S-2358; S-1073; S-0974; S-0954; S-1239; Trial Tr. (5/30/19 a.m., J&J: Deem-Eshleman) at 137:14-139:04; Trial Tr. (6/3/19 a.m., J&J: Deem-Eshleman) at 90:17-91:17, 102:23-103:8) Ct. Ex. 0092 (Mashett) at 302:5-304:11), all of which involve the use of property, real and personal, to create and exacerbate the public nuisance.
6. The challenged conduct here is Defendants’ misleading marketing and promotion of opioids. The State claims that Defendants engaged in a false, misleading, and deceptive marketing campaign designed to convince Oklahoma doctors, patients, and the public at large that opioids were safe and effective for the long-term treatment of chronic, non-malignant pain.
The greater weight of the evidence shows that Defendants did, in fact, engage in such false and misleading marketing and the law is clear that such conduct qualifies as the kind of act or omission capable of sustaining liability under Oklahoma’s nuisance law. See *Epps v. Ellison*, 1921 OK 279, ¶¶ 3, 200 P. 160, 161.
7. Defendants promoted their specific opioids using misleading marketing. Among other things, they sent sales representatives into Oklahoma doctors’ offices to deliver misleading messages, they disseminated misleading pamphlets, coupons, and other printed materials for patients and doctors, and they misleadingly advertised their drugs over the internet—all of which occurred here in Oklahoma. But Defendants also pervasively promoted the use of opioids generally. This “unbranded” marketing included things like print materials that misleadingly touted the safety and efficacy of opioids as a class of pain medication, as well as online materials that promoted opioids generally. Defendants used and viewed medical education events (including Speakers Bureau sessions and CME opportunities) as promotional endeavors that Defendants leveraged to increase the market for opioids through misleading messaging.
8. According to Defendants’ own internal training documents, Defendants concede that “False and Misleading” promotion includes at least the following types of conduct: Broadening of product indication; Data taken out of context; Minimization of safety issues; Omission of material information; Comparative efficacy or safety claims without substantial evidence; and Overstatements of efficacy or safety. See S-2376 at 20. The greater weight of the evidence demonstrated that Defendants engaged in promotional activities that violated each one of these rules.
9. Based upon my finding that the Defendants’ false, misleading, and dangerous marketing campaigns have caused exponentially increasing rates of addiction, overdose deaths,
and Neonatal Abstinence Syndrome, I conclude these are unlawful acts which “annoys, injures, or endangers the comfort, repose, health, or safety of others.” 50 O.S. §1.
10. The facts show Defendants engaged in false and misleading marketing of both their drugs and opioids generally, and the law makes clear that such conduct is more than enough to serve as the act or omission necessary to establish the first element of Oklahoma’s public nuisance law.
11. Accordingly, based on the foregoing, I conclude (a) that Defendants engaged in false and misleading marketing of both their drugs and opioids generally; and (b) this conduct constitutes a public nuisance under extant Oklahoma law.
12. The First Amendment to the United States Constitution does not prohibit imposing liability for the acts complained of here. “First Amendment rights may not be used as the means or the pretext for achieving substantive evils which the legislature has the power to control.” *Cal. Motor Transport Co. v. Trucking Unltd.*, 404 U.S. 508, 515(1971) (internal citation omitted). “The fact that dissemination of information and opinion on questions of public concern is ordinarily a legitimate, protected and indeed cherished activity does not mean, however, that one may in all respects carry out that activity exempt from sanctions designed to safeguard the legitimate interests of others. . . . Federal securities regulation, mail fraud statutes, and common-law actions for deceit and misrepresentation are only some examples of our understanding that the right to communicate information of public interest is not unconditional.” *Curtis Pub. Co. v. Butts*, 388 U.S. 130, 150 (1967) (plurality op.).
13. Moreover, Supreme Court precedent recognizes a “commonsense distinction between speech proposing a commercial transaction”—i.e., commercial speech—"which occurs in an area traditionally subject to government regulation, and other varieties of speech.” *Cent.
Hudson Gas & Elec. Corp. v. Pub. Serv. Comm'n, 447 U.S. 557, 562 (1980). "The Constitution, therefore, accords a lesser protection to commercial speech than to other constitutionally guaranteed expression." Id. at 562-63. As a threshold test, "for commercial speech to come within [the First Amendment], it at least must concern lawful activity and not be misleading." Id. at 563-564. "Consequently, there can be no constitutional objection to the suppression of commercial messages that do not accurately inform the public about lawful activity." Id. at 563.
14. This understanding of the First Amendment has led the Supreme Court to consistently "emphasize that some forms of commercial speech regulation are surely permissible," including "restrictions on false, deceptive, and misleading commercial speech." Friedman v. Rogers, 440 U.S. 1, 9 (1979). See Virginia State Bd. of Pharmacy v. Virginia Citizens Consumer Council, Inc., 425 U.S. 748, 771-72 (1976)("Untruthful speech, commercial or otherwise has never been protected for its own sake. Obviously, much commercial speech is not provably false, or even wholly false, but only deceptive or misleading. We foresee no obstacle to a State’s dealing effectively with this problem. The First Amendment, as we construe it today, does not prohibit the State from insuring that the stream of commercial information flow cleanly as well as freely."); Bates v. State Bar of Ariz., 433 U.S. 350, 383 (1977) ("Advertising that is false, deceptive, or misleading of course is subject to restraint. Since the advertiser knows his product and has a commercial interest in its dissemination, we have little worry that regulation to assure truthfulness will discourage protected speech. And any concern that strict requirements for truthfulness will undesirably inhibit spontaneity seems inapplicable because commercial speech generally is calculated. Indeed, the public and private benefits from commercial speech derive from confidence in its accuracy and reliability. This, the leeway for untruthful or misleading expression that has been allowed in other contexts has little force in the
commercial arena.”); *Bolger v. Yongs Drug Prods. Corp.*, 463 U.S. 60, 69 (1983) (“The State may deal effectively with false, deceptive, or misleading sales techniques.”); *44 Liquormart v. R.I.*, 517 U.S. 484, 501 (1996) (plurality op.) (“When a State regulates commercial messages to protect consumers from misleading, deceptive, or aggressive sales practices, or requires the disclosure of beneficial consumer information, the purpose of its regulation is consistent with the reasons for according constitutional protection to commercial speech and therefore justifies less than strict review.”); *Cent. Hudson Gas & Elec. Corp. v. Pub. Serv. Comm’n*, 447 U.S. 557, 563-64 (1980) (“[T]here can be no constitutional objection to the suppression of commercial messages that do not accurately inform the public about lawful activity. The government may ban forms of communication more likely to deceive the public than to inform it, or commercial speech related to illegal activity.”); *Thomas v. W. States Med. Ctr.*, 535 U.S. 357, 367 (2002) (“Although commercial speech is protected by the First Amendment, not all regulation of such speech is unconstitutional. In Central Hudson, supra, we articulated a test for determining whether a particular commercial speech regulation is constitutionally permissible. Under that test we ask as a threshold matter whether the commercial speech concerns unlawful activity or is misleading. If so, then the speech is not protected by the First Amendment.”).
15. The record proves the speech at issue here was clearly commercial in nature as defined by the Supreme Court as speech that “propos[es] a commercial transaction.” See *Cent. Hudson Gas & Elec. Corp. v. Pub. Serv. Comm’n*, 447 U.S. 557, 562 (1980).
16. The First Amendment does not protect Defendants’ messages which were misleading in that they were told by their own experts that marketing opioids on their abuse potential was dangerous and that Purdue had already shown that such a message was prone to mislead. S-0035. They were told that the data they cited did not support their claims before they
made them, and then again by the FDA after they had already started spreading that misleading message. Id. They knew the studies they were citing were incomplete, unsound, or fraught with misrepresentations. S-2511. The Defendants’ sales reps delivered those messages, and as the call notes and the sales trends demonstrate, Oklahoma physicians were influenced by the misleading messages Defendants were delivering. S-2357 at 11-12. Accordingly, I conclude that the speech at issue here is commercial in nature and that it is therefore not protected speech under the First Amendment.
17. As a matter of law I find that Defendants actions caused harm and those harms are the kinds recognized by 50 O.S. §1 because those actions annoyed, injured or endangered the comfort, repose, health or safety of Oklahomans. This statute requires the State to prove that Defendants’ actions caused harm and that those harms are of the kind recognized under the statute. I further find that the State has satisfied its burden of proof and that the Defendants’ actions were the cause-in-fact of its injuries.
18. There are no intervening causes that supervened or superseded Defendants’ acts and omissions as a direct cause of the State’s injuries, or otherwise defeat a finding of direct and proximate cause. See, e.g., Graham v. Keuchel, 1993 OK 6, ¶ 9, 8447 P. 2d 342, 348 (“To rise to the magnitude of a supervening cause, which will insulate the original actor from liability, the new cause must be (1) independent of the original act, (2) adequate of itself to bring about the result and (3) one whose occurrence was not reasonably foreseeable to the original actor.”).
19. I further find that the facts of this action show by the greater weight of the evidence a public nuisance that “affects at the same time an entire community or neighborhood, or any considerable number of persons, although the extent of the annoyance or damage inflicted upon the individuals may be unequal.” 50 O.S. §2. There can be no question that this nuisance
affects entire communities, neighborhoods, or a considerable number of persons. This nuisance has negatively impacted the entire State.
20. I further find that the public nuisance created by the Defendants has affected and continues to affect at the same time entire Oklahoma communities and neighborhoods, as well as a considerable number of Oklahomans, although the extent of the harm inflicted upon individual Oklahomans may be unequal.
21. The Court further finds no act or omission by the State was a direct or proximate cause of public nuisance created by the Defendants.
22. The public nuisance can be abated.
23. The proper remedy for the public nuisance is equitable abatement.
24. The Court finds that the appropriate remedy to address the Opioid Crisis is the abatement of the nuisance.
25. Wherefore it is the order of this Court that the contours of the State’s proposed Abatement Plan are reasonable and necessary to abate the public nuisance and to execute the aforesaid Abatement Plan the Court orders as follows:
**ABATEMENT OF NUISANCE**
1. Commissioner Terri White, the “primary architect of the State’s Abatement Plan,” testified that the public nuisance in Oklahoma can be and “must be abated.” Trial Tr. (6/25/19 a.m., Commissioner White) at 89:15-16, 101:13-102:4; Trial Tr. (6/26/19 p.m., Commissioner White) at 129:19-130:1.
2. As the President’s Commission on Combating Drug Addiction and the Opioid Crisis found: “Historical precedent demonstrated that this crisis can be fought with effective medical education, voluntary or involuntary changes in prescribing practices, and a strong
regulatory and enforcement environment.” S-1574; see also Trial Tr. (6/3/19 p.m., J&J; Deem-Eshleman) at 68:5-9.
3. The State’s experts used a public health approach to develop the State’s Abatement Plan. Trial Tr. (6/20/19 p.m., Hawkins) at 106:10-13. The experts drew upon best practice documents from Johns Hopkins, the White House, the Oklahoma Commission, the Surgeon General and the CDC, among others. Trial Tr. (6/21/19 p.m., Hawkins) at 72:8-15; see also Ct. Ex. 116. The experts reviewed other State plans, academic literature, research, and produced an outline of recommendations. Id. The experts then engaged other stakeholders, such as other State agencies and professionals, in reviewing and contributing to the recommendations. Id.
4. Commissioner White testified that, in her opinion, the State’s Abatement Plan will abate the nuisance, save countless lives of Oklahomans in the future, save countless people from becoming addicted to opioids in the future, and eliminate the negative impact this nuisance has had on the State of Oklahoma. Trial Tr. (6/25/19 a.m., Commissioner White) at 112:8-17; see also, e.g., Trial Tr. (6/26/19 p.m., Commissioner White) at 129:21-130:1. Oklahomans’ lives can be saved if the State obtains the resources needed “to use evidence-based programs to abate this crisis.” Trial Tr. (6/25/19 a.m., Commissioner White) at 117:13-118:14.
5. Opioid Use Disorder Prevention, Treatment & Recovery Services are the central feature of the State’s Abatement Plan. (6/20/19 p.m., Hawkins) at 114:21-22.
6. Establishment of a comprehensive Opioid Use Disorder (“OUD”) treatment program serving all Oklahoma residents who need OUD treatment services is necessary to abate the nuisance. Trial Tr. (6/20/19 p.m., Hawkins) at 116:22-117:2 & 117:15-20; Trial Tr. (6/25/19
a.m., Commissioner White) at 91:17-19 & 110:21-112:7; Trial Tr. (6/17/19 p.m., Beaman) at 83:17-20; Trial Tr. (6/13/19 p.m., Kolodny) at 125:4-19; S-4734 at 19.
7. As part of the Addiction Treatment Services, all Oklahoma residents in need of treatment services will be eligible to receive a biopsychosocial assessment based on the American Society of Addiction Medicine ("ASAM") level of care placement criteria, and comprehensive treatment and recovery services based on the ASAM level of care needed, including early intervention, outpatient services, ambulatory detoxification, intensive outpatient, partial hospitalization, residential care, medically managed detoxification and medication. Trial Tr. (6/280/19 p.m., Hawkins) at 118:7-23; S-4734 at 19; S-3924.
8. The total yearly cost for these services in 2019 dollars is $232,947,710.
9. Addiction Treatment - Supplementary Services are necessary to abate the nuisance. Trial Tr. (6/21/19 a.m., Hawkins) at 9:8-12; S-4734 at 20; Trial Tr. (6/25/19 a.m., Commissioner White) at 100:3-7; Trial Tr. (6/26/19 p.m., Commissioner White) at 129:21-130:1. These supplementary addiction treatment services include housing services, employment services, as well as additional personnel for the juvenile justice system to support with assessing and guiding youthful offenders and their family members in receiving mental health and addiction services and care navigators to coordinate with opioid affected youth in the juvenile justice and other State systems and their family to improve recovery outcomes. Trial Tr. (6/21/19 a.m., Hawkins) at 6:21-7:7; S-4734 at 20.
10. The total yearly cost for these services in 2019 dollars is $31,796,011.
11. Public medication and disposal programs are necessary to abate the nuisance. Trial Tr. (6/21/19 a.m., Hawkins) at 16:13-18; Trial Tr. (6/25/19 a.m., Commissioner White) at 100:3-7; Trial Tr. (6/26/19 p.m., Commissioner White) at 129:21-130:1; S-4734 at 22. This
includes maintaining existing programs like Safe Trips for Scripts and developing home based medication take-back and disposal programs.
12. The total yearly cost for these services in 2019 dollars is $139,883.
13. Enabling all primary care practices and emergency departments to enroll in the Screening, Brief Intervention and Referral to Treatment (“SBIRT”) practice dissemination program for academic detailing, continuing education, electronic medical record integration consultation and embedded practice facilitation services, and implementing universal substance use patient screening and intervention for SoonerCare patients is necessary to abate the nuisance. Trial Tr. (6/21/19 a.m., Hawkins) at 25:11-20; Trial Tr. (6/25/19 a.m., Commissioner White) at 100:3-7; Trial Tr. (6/26/19 p.m., Commissioner White) at 129:21-130:1; S-4734 at 26-27.
14. Universal Screening has two components. Trial Tr. (6/21/19 a.m., Hawkins) at 22:3-9. The first component of universal screening supports the costs of providing SBIRT services to SoonerCare members in the State of Oklahoma. Trial Tr. (6/21/19 a.m., Hawkins) at 22:10-23:22; S-4734 at 26. The second component of universal screening is to widely disseminate the practice of SBIRT within primary care practices and emergency departments throughout the State. Trial Tr. (6/21/19 a.m., Hawkins) at 23:23-25:7; S-4734 at 27.
15. The total yearly cost for these Universal Screening services in 2019 dollars is $56,857,054.
16. Pain prevention and non-opioid pain management therapies, including cognitive behavioral therapy for pain, physical therapy, and exercise programs are necessary to abate the nuisance. Trial Tr. (6/21/19 a.m., Hawkins) at 32:22-33:1; Trial Tr. (6/25/19 a.m., Commissioner White) at 100:3-7; Trial Tr. (6/26/19 p.m., Commissioner White) at 129:21-130:1; S-4734 at 29-30.
17. The first component is a pain management benefit program for SoonerCare members, this component includes administrative and personnel costs to oversee and administer the pain management benefits program. Trial Tr. (6/21/19 a.m., Hawkins) at 28:24-31:51; S-4734 at 29. The second component of Pain Services is the cost of physical therapists or occupational therapists or similar providers to operate within the Oklahoma county health department system. Trial Tr. (6/21/19 a.m., Hawkins) at 31:6-21; S-4734 at 30. The third component of Pain Services is cognitive behavioral therapy for the treatment of chronic pain. Trial Tr. (6/21/19 a.m., Hawkins) at 32:4-21; S-4734 at 30.
18. The total yearly cost for these services in 2019 dollars is $103,277,835.
19. Expanded and targeted naloxone distribution and overdose prevention education to those at high risk of experiencing or witnessing overdose is necessary to abate the nuisance. (Trial Tr. (6/21/19 a.m., Hawkins) at 54:19-22; Trial Tr. (6/25/19 a.m., Commissioner White) at 100:3-7; Trial Tr. (6/26/19 p.m., Commissioner White) at 129:21-130:1; Trial Tr. (6/13/19 a.m., Kolodny) at 125:20-126:02; S-4734 at 39-40.
20. This component of the Abatement Plan is twofold: First, it includes continued naloxone programming at ODMHSAS (the cost of the medication, overdose education services, and administrative costs) Trial Tr. (6/21/19 a.m., Hawkins) at 52:10-53; S-4734 at 39.
21. Second, the naloxone distribution/education component of the Abatement Plan includes expanding the naloxone program through OSDH involving emergency medical services, rural fire, and rural EMS services in Oklahoma to include all volunteer and fire departments in the State and continue the emergency medical rural response program. Trial Tr. (6/21/19 a.m., Hawkins) at 53:7-19; S-4734 at 39. This requires personnel including a coordinator,
epidemiologist, a naloxone training coordinator for these emergency responders, and support staff. Trial Tr. (6/21/19 a.m., Hawkins) at 53:16-23; S-4734 at 39.
22. The total yearly cost for these services in 2019 dollars is $1,585,797.
23. Medical Case Management/Consulting (Project Echo) is necessary to abate the nuisance. Trial Tr. (6/20/19 a.m., Croff) at 77:7-12; Trial Tr. (6/20/19 p.m., Hawkins) at 116:15-21; Trial Tr. (6/25/19 a.m., Commissioner White) at 100:3-7; Trial Tr. (6/26/19 p.m., Commissioner White) at 129:21-130:1; see also S-4734 at 48.
24. The total yearly cost for these services in 2019 dollars is $3,953,832.
25. Developing and disseminating NAS treatment evaluation standards, including continuing education courses is necessary to abate the nuisance. Trial Tr. (6/21/19 p.m., Hawkins) at 9:2-5; Trial Tr. (6/24/19 a.m., Hawkins) at 33:4-7 & 36:1-5; S-4734 at 53; Trial Tr. (6/25/19 a.m., Commissioner White) at 100:3-7; Trial Tr. (6/26/19 p.m., Commissioner White) at 129:21-130:1.
26. This component of the Abatement Plan is based on a high-quality national program of quality improvement, called the Vermont Oxford Network Quality Improvement package. Trial Tr. (6/21/19 p.m., Hawkins) at 8:10-16; 9:9-10; S-4734 at 53, n.119. This program will provide intensive training in and support to Oklahoma birthing hospitals to help certify them or accredit them as centers of excellence in NAS evaluation and assessment and shall be overseen by the University of Oklahoma Health Sciences Center as part of their perinatal quality improvement project. Trial Tr. (6/21/19 p.m., Hawkins) at 8:10-20.
27. The total yearly cost for these services in 2019 dollars is $107,683,000.
28. Funding the development of NAS as a required reportable condition, including OSDH and hospital-level management and infrastructure costs, is necessary to abate the
nuisance. Trial Tr. (6/21/19 p.m., Hawkins) at 37:11-14; Trial Tr. (6/24/19 a.m., Hawkins) at 37:19-22, 38:6-9 & 38:19-24; S-4734 at 64; Trial Tr. (6/25/19 a.m., Commissioner White) at 100:3-7; Trial Tr. (6/26/19 p.m., Commissioner White) at 129:21-130:1. The purpose of this component is to fund the costs for the oversight of NAS birth documentation and reporting. Trial Tr. (6/24/19 a.m., Hawkins) at 37:9-10.
29. The total yearly cost for these services in 2019 dollars is $181,983.
30. Implementing universal substance use screening for pregnant women and enabling all OB/GYN practices and hospitals to enroll in the SBIRT practice dissemination program for academic detailing, continuing education, electronic medical record consultation, and embedded practice facilitation services is necessary to abate the nuisance. Trial Tr. (6/21/19 p.m., Hawkins) at 12:16-21; Trial Tr. (6/25/19 a.m., Commissioner White) at 100:3-7; Trial Tr. (6/26/19 p.m., Commissioner White) at 129:21-130:1; S-4734 at 54-55.
31. The first component of Prenatal Screening is practice dissemination and the second component is to support universal screening for pregnant SoonerCare members. Trial Tr. (6/21/19 p.m., Hawkins) at 10:8-11; 11:20-21; S-4734 at 54.
32. The total yearly cost for these services in 2019 dollars is $1,969,000.
33. Medical treatment for infants born with NAS or suffering from opioid withdrawal is necessary to abate the nuisance. Trial Tr. (6/21/19 p.m., Hawkins) at 14:22-15:2; Trial Tr. (6/24/19 a.m., Hawkins) at 17:18-20; Trial Tr. (6/25/19 a.m., Commissioner White) at 100:3-7; Trial Tr. (6/26/19 p.m., Commissioner White) at 129:21-130:1; S-4734 at 56.
34. This component of the Abatement Plan funds additional costs above and beyond costs for an ordinary birth for infants born with NAS due to the nuisance. Trial Tr. (6/21/19 p.m., Hawkins) at 13:22-25; (6/24/19 a.m., Hawkins) at 16:21-22.
35. The total yearly cost for these services in 2019 dollars is $20,608,847.
36. Funding for investigatory and regulatory actions related to the nuisance are necessary to abate it. Trial Tr. (6/21/19, p.m., Hawkins) at 51:8-15; Trial Tr. (6/24/19 p.m., Hawkins) at 22:3-23; Trial Tr. (6/25/19 a.m., Commissioner White) at 100:3-7; Trial Tr. (6/26/19 p.m., Commissioner White) at 129:21-130:1; S-4734 at 65-70.
37. Oklahoma law enforcement agencies, licensure boards and the Oklahoma Office of the Chief Medical Examiners ("OCME") have been overwhelmed by cases related to opioids and the need to conduct investigations and do not have the necessary staffing and resources in place to respond to this nuisance. Trial Tr. (6/21/19, p.m., Hawkins) at 38:2-14. This component of the plan allows these agencies, offices and boards to meet the massive demands on their time from heavy caseloads due to the nuisance. Trial Tr. (6/21/19, p.m., Hawkins) at 38:11-14.
38. The first component of Enforcement/Regulatory relates to the ODMHSAS. Trial Tr. (6/21/19 p.m., Hawkins) at 38:15-20; S-4734 at 65. Crisis Intervention Team Training (CIT) is an activity that is performed with law enforcement agencies. Trial Tr. (6/21/19 p.m., Hawkins) at 38:20-22. The cost of $500,000 per year allows ODMHSAS to bring this training statewide for all law enforcement officers in the State. Trial Tr. (6/21/19 p.m., Hawkins) at 38:24-39:2; S-4734 at 65. CIT is training in recognizing addiction and addiction crises, intervening, deescalating situations, assisting those in the community with finding referrals and resources and improving overall law enforcement and community relations with regard to what these officers are seeing in the community with addictive behaviors. Trial Tr. (6/21/19, p.m., Hawkins) at 39:2-8.
39. The second component of Enforcement/Regulatory relates to OBN. Trial Tr. (6/21/19 p.m., Hawkins) at 39:9-14; S-4734 at 65. OBN needs additional staff to deal with the burdensome caseload resulting from the nuisance. Trial Tr. (6/21/19, p.m., Hawkins) at 38:2-10.
40. There are personnel and non-personnel costs. The personnel costs include a criminal/civil analyst and compliance inspectors/agents. The non-personnel costs include the cost to cover OBN’s registration system technology and its collaborative work with local law enforcement for a heroin and opioid task force at an annual cost. Trial Tr. (6/21/19, p.m. Hawkins) at 40:15-19; S-4734 at 65.
41. The Oklahoma licensure boards also are overwhelmed by the nuisance in terms of their capacity to investigate each case and the number of complaints they are receiving. Trial Tr. (6/21/19 p.m., Hawkins) at 43:16-44:7. They require additional staff to deal with their high caseloads as a result of this nuisance. Id.
42. In addition, many of the boards participate in peer-assistance programs, where they are working with their licensees who are affected with addiction. Id. Opioids are a significant driver of their licensees needing these peer-assistance programs. Id. The nuisance has placed an undue burden on these boards and their ability to process cases and serve their licensees effectively. Id. To adequately address the nuisance, the boards need additional personnel and training. Trial Tr. (6/21/19 p.m., Hawkins) at 41:1-3.
43. The Oklahoma Veterinary Board requires an additional investigator. Trial Tr. (6/21/19 p.m., Hawkins) at 41:3-5; S-4734 at 66. The Board also requires non-personnel costs including equipment and training for this additional investigator. Trial Tr. (6/21/19 p.m., Hawkins) at 41:5-10; S-4734 at 66. The Veterinary Board also requires annual training costs for its investigators, the executive director, board counsel and one board member. Id.
44. The Oklahoma State Osteopathic Board requires a full-time prosecutor, support staff to assist the prosecutor, two full-time investigators, investigator support staff and additional
office space for these additional staff members. Trial Tr. (6/21/19 p.m., Hawkins) at 41:14-21; S-4734 at 66.
45. The Oklahoma Board of Nursing requires a full-time prosecutor, nurse investigators, a legal secretary, and a nurse case manager for the Peer Assistance Program. Trial Tr. (6/21/19 p.m., Hawkins) at 41:25-42:6; S-4734 at 67. The Peer Assistance Program is to assist their licensees who are struggling with substance use disorder. Trial Tr. (6/21/19 p.m., Hawkins) at 41:25-42:9; S-4734 at 67. The Board also requires an educator for the Board that will work with licensees through the State. Id.
46. In addition, the Nursing Board requires IT development costs for education materials and administrative costs, including office equipment for these new personnel. Trial Tr. (6/21/19 p.m., Hawkins) at 42:9-13; S-4734 at 67.
47. The Oklahoma Board of Medical Licensure and Supervision requires additional personnel to address the nuisance including, additional investigators, an assistant for these investigators, and a support services administrator. Trial Tr. (6/21/19 p.m., Hawkins) at 42:17-20; S-4734 at 67. The Board also requires specialized training and professional development for investigators. Trial Tr. (6/21/19 p.m., Hawkins) at 42:21-23; S-4734 at 67. It also requires additional expert medical reviews to help with the Board’s heavy caseload of prescription opioid complaints against licensees. Trial Tr. (6/21/19 p.m., Hawkins) at 42:23-43:1; S-4734 at 67. Finally, it requires one-time costs in surveillance equipment. Trial Tr. (6/21/19 p.m., Hawkins) at 43:1-2; S-4734 at 67.
48. The Oklahoma Board of Dentistry requires additional full-time experienced investigators and training for Board members, Board staff, investigators and attorneys and its licensees. Trial Tr. (6/21/19 p.m., Hawkins) at 43:3-12; S-4734 at 67.
49. The OCME also has been overburdened with heavy caseloads due to the nuisance. Trial Tr. (6/21/19, p.m., Hawkins) at 44:9-45:3. In order to keep up with this heavy caseload, the OCME requires additional equipment for autopsies and toxicology tests and personnel. Id.
50. First, OCME requires additional salary for their medical examiner physicians to encourage retention. Trial Tr. (6/21/19, p.m., Hawkins) at 44:14-45:6; S-4734 at 68. OCME also requires forensic pathologists, a full-time forensic chemist and full-time medicolegal death scene investigators. Trial Tr. (6/21/19, p.m., Hawkins) at 45:7-11; S-4734 at 68.
51. In addition, OCME requires costs for maintenance on instruments it purchased in order to address the heavy caseload in the toxicology lab and a new CT scanner due to the nuisance. Trial Tr. (6/21/19, p.m., Hawkins) at 45:12-25; S-4734 at 68.
52. The Office of the Attorney General also requires services, programs and personnel to abate the nuisance. S-4734 at 69. The criminal justice division requires salary and benefits for additional assistant attorneys general, investigators and support staff as well as non-personnel costs for these employees in the form of equipment and training. Trial Tr. (6/21/19, p.m., Hawkins) at 46:2-16; S-4734 at 69.
53. The Medicaid Fraud Control division requires salary and benefits for additional personnel including one assistant attorney general and investigators as well as non-personnel costs for these employees in the form of equipment and training. Trial Tr. (6/21/19, p.m., Hawkins) at 46:17-23; S-4734 at 69.
54. The Legal/Agency Counsel division requires salary and benefits and non-personnel costs of equipment and training for assistant attorneys general. Trial Tr. (6/21/19, p.m., Hawkins) at 46:24-47:6; S-4734 at 69.
55. Victim Services requires addiction and substance abuse training and travel for 7 individuals. Trial Tr. (6/21/19, p.m., Hawkins) at 47:1-3; S-4734 at 69.
56. The Policy & Legislative Development and Tracking division requires salary and benefits and non-personnel costs in the form of equipment and training for one assistant attorney general. Trial Tr. (6/21/19, p.m., Hawkins) at 47:4-6; S-4734 at 69.
57. The total yearly costs for these services in 2019 dollars is $11,101,076.
58. The costs of Enforcement/Regulatory are reasonable and necessary expenses to implement this component of the Abatement Plan. Trial Tr. (6/21/19, p.m., Hawkins) at 51:16-22; Trial Tr. (6/25/19, a.m., Commissioner White) at 100:3-7; Trial Tr. (6/26/19, p.m., Commissioner White) at 129:21-130:1.
59. The Court finds that the sum necessary to carry out the Abatement Plan in year one is the sum of $572,102,028.
60. Though several of the State’s witnesses testified that the plan “will take at least 20 years” to work, the State did not present sufficient evidence of the amount of time and costs necessary, beyond year one, to abate the Opioid Crisis. Trial Tr. (6/24/19, a.m., Hawkins) at 87:20-88:1.
61. To the extent any argument or objection set forth by Defendants in their Renewed Motion for Judgment, or any other motion, filing or pleading, is not specifically addressed by the Court’s Findings of Fact and Conclusions of Law, such argument and/or objection made by Defendants is hereby denied and overruled.
62. The Court concludes that, pursuant to agreement between the State and its Outside Counsel governing the Oklahoma Action, Outside Counsel is expressly entitled to collect attorneys’ fees from the abatement proceeds as a result of the Judgment entered in this
action. Outside Counsel is also entitled to compensation for all reasonable costs incurred in prosecuting this action as hereinafter determined by the Court. The attorneys’ fees owed to Outside Counsel are fair, reasonable and appropriate under Oklahoma law. Outside Counsel are entitled to payment of the contractually agreed amounts from the abatement proceeds without further order from this Court and may obtain such payment by presenting this judgment.
Consistent with the terms of that agreement, Outside counsel is also permitted to seek reimbursement of any future costs they may incur in prosecuting this action by subsequent application to this Court.
63. The Court retains jurisdiction over the parties and the abatement proceeds, to the extent allowed by law, as a result of the Court’s Final Judgment.
64. The Court will enter such further Orders pertaining to the implementation of the Abatement Plan as necessary and in due course.
It is so ORDERED.
DATED this 26th day of August, 2019.
Honorable Thad Balkman, District Judge
|
PHOTODYNAMICS STUDIES OF LIGAND-PROTECTED GOLD NANOCENTERS BY USING ULTRAFAST TRANSIENT INFRARED SPECTROSCOPY
BY
SATU MUSTALAHTI
Academic Dissertation for the Degree of Doctor of Philosophy
To be presented, by permission of the Faculty of Mathematics and Science of the University of Jyväskylä, for public examination in KEM4, on December 11th, 2015 at 12 noon.
UNIVERSITY OF JYVÄSKYLÄ
Copyright ©, 2015
University of Jyväskylä
Jyväskylä, Finland
ISBN 978-951-39-6418-4
ISSN 0357-346X
ABSTRACT
Mustalahti, Satu
Photodynamics studies of ligand-protected gold nanoclusters by using ultrafast transient infrared spectroscopy
Jyväskylä: University of Jyväskylä, 2015, 58 p.
(Department of Chemistry, University of Jyväskylä Research Report Series
ISSN 0357-346X)
ISBN 978-951-39-6418-4
Highly monodisperse samples of three ligand-protected gold nanoclusters Au_{102}(pMBA)_{44}, Au_{144}(SC_2H_4Ph)_{60}, and a cluster tentatively identified as Au_{130}(pMBA)_{50} were characterized by UV/vis and infrared spectroscopy, and their photodynamics was studied by transient absorption spectroscopy. The dynamics study for each cluster was performed by electronically exciting the cluster with a pump pulse in the visible or near infrared region and by monitoring the transient absorption of vibrational modes of the ligands with a mid-IR probe pulse. The photodynamics studies were used to determine the molecular or metallic behavior of the cluster, and also to gain important size dependent information about the relaxation processes and energy states involved in them.
A drastic difference between the relaxation dynamics is observed between Au_{102}(pMBA)_{44} and Au_{130}(pMBA)_{50} clusters when compared to Au_{144}(SC_2H_4Ph)_{60}, which clearly shows that the two smaller clusters show molecular behavior, while the Au_{144} species shows relaxation typical for metallic species. Based on this, the transition region between molecular and metallic behavior for gold can be narrowed to occur between 130 and 144 gold atoms.
By combining the experimental results with energies of the electronic states of the clusters obtained from DFT calculations, the observed photodynamics for the molecular clusters could be explained. For both clusters, the relaxation was shown to involve relaxation via triplet states, and also revealed a vital role of the relative energies of different energy states in singlet and triplet manifolds. Vibrational cooling was also found to have a significant role in the relaxation, since the excess vibrational energy in the hot system was found to facilitate the observed relaxation processes.
Keywords: gold nanocluster, femtosecond, ultrafast spectroscopy, electronic relaxation, transient absorption, Au_{102}(pMBA)_{44}, Au_{144}(SR)_{60}, Au_{130}, vibrational spectroscopy
Author’s address Satu Mustalahti
Department of Chemistry
Nanoscience Center
University of Jyväskylä
P.O. Box 35
FI-40014 University of Jyväskylä
email@example.com
Supervisors Professor Mika Pettersson
Department of Chemistry
Nanoscience Center
University of Jyväskylä
P.O. Box 35
FI-40014 University of Jyväskylä
Reviewers Professor Nikolai Tkatchenko
Department of Chemistry and Bioengineering
Tampere University of Technology
Finland
Dr. Jan Helbing
Department of Chemistry
University of Zürich
Switzerland
Opponent Associate Professor Kenneth L. Knappenberger, Jr.
Department of Chemistry and Biochemistry
Florida State University
USA
PREFACE
The work presented in this thesis has been carried out at the Department of Chemistry, Nanoscience Center, University of Jyväskylä in 2012–2015, and was funded by The National Doctoral Programme in Nanoscience (NGS-NANO), which is gratefully acknowledged.
I am deeply grateful for my supervisor Prof. Miika Pettersson for introducing me to this very interesting thesis project. I would also like to thank him for all his guidance and for believing in me throughout the project. In addition, I am extremely grateful for Pasi Myllyperkiö for his guidance and priceless help throughout the project, and for teaching me everything I know about practical laser spectroscopy. I am also grateful for the reviewers of this thesis for their valuable comments that really helped to improve this thesis.
None of the work presented here could have been done without a very inspiring collaboration that I have been privileged to be a small part of. I would like to thank Prof. Hannu Häkkinen for helping me to understand the fascinating world of nanoclusters. In particular, I would also like to thank Tanja Lahtinen for providing me with the samples, even if I asked for ridiculously large amounts of them, and Sami Malola for all nice figures and also for the computational results that proved out to be essential for all of my work. Kirsi Salorinne, Tiia-Riikka Tero and Jaakko Koivisto are also gratefully acknowledged for analyzing the samples and for their support. I am also grateful for Gerrit Groenhof for broadening my scientific knowledge by introducing me to my almost two-day side project.
In addition, I am very grateful for several past and present colleagues. I would especially like to thank Heli Lehtivuori and Liisa Antila for their guidance, support, and friendship during my thesis work. I would also like to thank Heikki Häkkänen for many interesting discussions and useful tips regarding the experiments and Jukka Aumanen for his company on various conference trips. A special thank you is also owed to my office roommates Anna Kausamo and Mikko Vääärämäki for their support, several interesting discussions, and for tolerating my company on a daily basis. Finally, I would also like to thank everyone else at nanoscience center and chemistry department for all your contributions in creating an interesting and supporting working environment.
For everything else needed to complete this thesis I am indebted beyond words to my family and relatives. I would like to express my heartfelt gratitude especially to my parents Tarja and Hannu, and my beloved siblings Kati, Pauli and Anni for their endless support, encouragement, and understanding.
Jyväskylä, November 2015
Satu Mustalahti
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on original publications listed below. In the following text, they are referred to by their Roman numerals.
I Satu Mustalahti, Pasi Myllyperkiö, Tanja Lahtinen, Kirsi Salorinne, Sami Malola, Jaakko Koivisto, Hannu Häkkinen and Mika Pettersson, Ultrafast Electronic Relaxation and Vibrational Cooling Dynamics of Au$_{144}$(SC$_3$H$_4$Ph)$_{60}$ Nanocluster Probed by Transient Mid-IR Spectroscopy, *J. Phys. Chem. C*, **2014**, 118, 18233–18239.
II Satu Mustalahti, Pasi Myllyperkiö, Sami Malola, Tanja Lahtinen, Kirsi Salorinne, Jaakko Koivisto, Hannu Häkkinen and Mika Pettersson, Molecule-like Photodynamics of Au$_{102}$(pMBA)$_{44}$ Nanocluster, *ACS Nano*, **2015**, 9, 2328–2335.
III Satu Mustalahti, Pasi Myllyperkiö, Tanja Lahtinen, Sami Malola, Kirsi Salorinne, Tiia-Riikka Tero, Jaakko Koivisto, Hannu Häkkinen and Mika Pettersson, Photodynamics of a Molecular Water-Soluble Nanocluster Identified as Au$_{130}$(pMBA)$_{50}$, *J. Phys. Chem. C*, **2015**, 119, 20224–20229.
The author is the primary author of papers I–III. She has performed most of the spectroscopic experiments and data-analysis in all papers and participated in writing papers I and II. She wrote the first draft of paper III.
| Abbreviation | Description |
|--------------|-------------|
| pMBA | para-mercaptobenzoic acid |
| UV | ultraviolet |
| vis | visible |
| IR | infrared |
| DFT | density functional theory |
| CD | circular dichroism |
| HOMO | highest occupied molecular orbital |
| LUMO | lowest unoccupied molecular orbital |
| LDM | liquid drop model |
| LSPR | localized surface plasmon resonance |
| NMR | nuclear magnetic resonance |
| FTIR | Fourier transform infrared |
| MCT detector | mercury cadmium tellurium (HgCdTe) detector |
| BS | beam splitter |
| GS | ground state |
| VR | vibrational relaxation |
| IC | internal conversion |
| ISC | intersystem crossing |
| FL | fluorescence |
| PH | phosphorescence |
| SDS- | sodium dodecyl sulfate |
| PAGE | polyacrylamide gel electrophoresis |
| DCM | dichloromethane |
| OPA | optical parametric amplifier |
| NIR | near infrared |
| PET | phenylethanethiol (HSC₂H₄Ph) |
CONTENTS
ABSTRACT
PREFACE
LIST OF ORIGINAL PUBLICATIONS
ABBREVIATIONS
CONTENTS
1 INTRODUCTION ........................................................................................................... 11
2 BACKGROUND ............................................................................................................. 13
2.1 General chemical and physical properties of gold .............................................. 13
2.2 Gold nanoclusters and motivation for their studies ............................................ 14
2.3 Thiol protected gold nanoclusters ....................................................................... 15
2.3.1 Synthesis .................................................................................................. 15
2.3.2 General structure of thiol-protected gold nanoclusters ......................... 16
2.3.3 Electronic properties ............................................................................... 18
2.3.4 Stability and the superatom model .......................................................... 20
2.3.5 Optical properties ..................................................................................... 21
2.3.6 Molecular and metallic behavior ............................................................. 21
2.3.7 The ligand layer ....................................................................................... 22
2.4 Studied clusters .................................................................................................... 23
2.4.1 Au_{102}(pMBA)_{44} .................................................................................... 23
2.4.2 Au_{144}(SC_2H_4Ph)_{60} ............................................................................. 23
2.4.3 Clusters with Au_{130} core ....................................................................... 24
2.5 Optical spectroscopy and its utilization in cluster studies .................................. 25
2.5.1 The spectrum of electromagnetic radiation .............................................. 25
2.5.2 Spectroscopy and hierarchy of energy levels in molecules ..................... 25
2.5.3 UV/vis spectroscopy ................................................................................ 26
2.5.4 Infrared spectroscopy ............................................................................... 27
2.5.5 Electronic and vibrational spectroscopy in cluster studies ..................... 28
2.5.6 Time-resolved spectroscopy ..................................................................... 28
18.104.22.168 Electronic excitation and relaxation .................................................. 29
22.214.171.124 Molecular systems ............................................................................. 29
126.96.36.199 Metallic systems ................................................................................ 30
188.8.131.52 The two-temperature model ............................................................... 31
2.5.7 Transient absorption spectroscopy ............................................................ 31
2.5.8 Transient absorption in nanocluster studies ............................................. 33
3 EXPERIMENTAL METHODS ......................................................................................... 34
3.1 Samples and sample characterization ................................................................. 34
3.2 Sample preparation and handling ....................................................................... 34
3.3 Spectroscopy studies ........................................................................................... 35
4 RESULTS AND DISCUSSION ................................................................. 38
4.1 Au$_{144}$(SC$_2$H$_4$Ph)$_{60}$ ................................................................. 38
4.2 Au$_{102}$(pMBA)$_{44}$ ................................................................. 42
4.3 Au$_{130}$(pMBA)$_{50}$ ................................................................. 47
4.4 Changing from molecular to metallic behavior ....................... 51
4.5 Implications of the results for future work ............................. 51
5 SUMMARY AND CONCLUSION ......................................................... 53
REFERENCES .................................................................................. 55
1 INTRODUCTION
The utilization of gold in human history spans from ancient civilizations to present day society.\textsuperscript{1} Due to its many unique properties and high value, gold has always been one of the most sought after elements. It has been widely used, for example, as currency and jewelry and has also been given significant symbolic value. Although already studied and utilized for millennia, gold has found a central role and renewed research interest in modern nanoscience research in the form of nanoclusters and particles.
Nanoscience is a relatively new research field that focuses on a variety of sample systems that fall in the size range of about 1–100 billionths of a meter, namely 1–100 nanometers. Nanoscience is by nature a highly interdisciplinary field combining knowledge from chemistry, physics, and biology in an effort to study various nanoscale systems from supramolecular assemblies, carbon nanotubes, and nanoclusters to biological entities, such as proteins and viruses. One of the popular research topics in this field is nano-sized clusters that lie on the borderline between chemistry, physics and material science.\textsuperscript{2} The clusters represent a form of matter different from both the bulk form and the atomic systems. This type of clusters can be formed from atoms or molecules and from a variety of elements, including gold.
Nano-sized objects can be studied by several methods to obtain versatile information about their structure and properties. One of the methods commonly used is optical spectroscopy, in which the interaction between light and matter is utilized to determine properties of the sample that are related to energy states.\textsuperscript{3–5} Spectroscopy can also be extended to obtain time dependent information by utilizing short excitation pulses. In many cases, time-resolved information in the time scale relevant to nuclear motions is required. For these studies, ultrafast spectroscopy on the femtosecond time scale is needed. This type of research falls into the field of femtochemistry\textsuperscript{6}, which is a modern research field dedicated to processes occurring in billionths of a microsecond.
In the work presented in this thesis,\textsuperscript{7–11} ultrafast femtosecond spectroscopy in mid-IR region is used to study in detail the photophysics of ligand-protected gold nanoclusters in three different sizes. The studied clusters are of particular
interest since they fall into a size range close to the transition between molecular and metallic cluster behavior. The study presented here involves experiments, in which the relaxation dynamics of the electronic excitation of the clusters is monitored by studying the changes in the vibrational modes of the ligand molecules. These results can be used to obtain fundamental size-dependent information on the clusters and to narrow the transition region between molecular and metallic behavior.
2 BACKGROUND
2.1 General chemical and physical properties of gold
The atomic number of gold (Au) is 79, and it is a member of group 11 in the periodic table of elements together with copper and silver. In the electronic ground state, a gold atom has a filled 5d electronic shell and the electronic configuration [Xe]4f¹⁵d¹⁰6s¹. In chemical compounds, the oxidation state of gold is usually either Au(III) or Au(I), of which Au(III) is dominant. Rarer oxidation states observed for gold include Au(V), which is found only in AuF₅ and AuF₆, and Au(II). In some compounds, gold can also be found in the anionic form as the auric anion Au(-I). Metallic gold has an fcc (face-centered cubic) crystal lattice. In the metallic form, gold has a characteristic yellow color and is a highly malleable non-magnetic and non-toxic metal that can be alloyed with other metals. Chemically, gold is very inert, allowing it to be observed in pure metallic form in nature.
Gold has many unique properties among metals and even among the group 11 metals. Many of these properties can be explained by electronic structure and relativistic effects that play a significant role for gold. The electronic structure of metallic gold is inherited from the electronic structure of individual gold atoms as the increasing number of atoms causes the electronic levels to form a band structure. Electrons of the filled gold d orbitals form a d-band filled with electrons. The conduction band of gold is then formed from 6s and 6p orbitals. The interband transition threshold between the d-band and the conduction band is 1.84 eV, which corresponds to red part of the visible spectrum. The interband transition is also responsible for the characteristic color of metallic gold. The chemical bonding in metallic systems is generally described by free valence electrons moving in the potential provided by the nuclei.
2.2 Gold nanoclusters and motivation for their studies
Clusters are a unique form of material on the borderline between the atomic level and bulk material.\textsuperscript{2} In principle, clusters can be obtained by cutting metallic gold into smaller pieces or by growing them by combining individual atoms. The exact definition of a cluster varies slightly depending on the source, but in general, cluster research concerns the study of particles with size varying from few atoms to up to thousands of atoms.\textsuperscript{2,9} Particles of this size fall into the nanoscale region for which they can be referred to as nanoclusters. Clusters can be divided into different size ranges depending on the number of atoms and hence the size of the cluster. The division is often made so that the small clusters with properties closer to those of molecules and individual atoms are separated from the larger nanoparticles and nanocrystals.\textsuperscript{10} Although this thesis concerns only gold species, it should be noted that clusters can also be made from other metals, molecules, or even noble gases.\textsuperscript{2}
At the moment, clusters have gathered an enormous amount of interest in the scientific community, and the field is developing and expanding rapidly. Both fundamental and applied research is currently performed. Gold clusters can be obtained by using several methods, and they can be studied in gas phase, in solution, and by supporting them on surfaces.\textsuperscript{1,2,10} By using modern research methods, many different cluster properties can now be addressed, which has significantly contributed to the development of the field.
Much of the interest directed towards gold clusters is related to their properties and their utilization in various applications.\textsuperscript{1,11,12} Due to their size and structure, gold clusters have unique properties that differ greatly from both the properties of small chemical gold compounds and the metallic gold presented in the previous chapter. These properties include, for example, the size dependent optical, electronic, and photothermal properties, and catalytic activity, which is significantly increased when compared to that of metallic gold.\textsuperscript{1,2} These features are highly attractive towards many applications, for example, in photonics, sensors, medicine, and biochemistry.\textsuperscript{1,11,12}
From the point of view of fundamental research, clusters provide an ideal class of compounds to study the evolvement of the bulk properties from the properties of individual atoms.\textsuperscript{2} Especially for metals, this provides a possibility to study the evolvement of the atomic geometry to the fcc lattice and the emergence of common metallic properties, such as the electric conductivity.\textsuperscript{2} One of the important fundamental aspects that can also be addressed is determining the number of metal atoms that is needed to make the system metallic. This type of possibility to correlate the size of the cluster with its properties also serves as motivation for the research presented in this thesis.
2.3 Thiol protected gold nanoclusters
One of the problems related to gold nanoclusters is that some sort of protection is generally needed in order to prevent aggregation into larger particles at high concentrations.\textsuperscript{10} Therefore, in recent years, particular interest has been directed towards monolayer protected particles and especially thiolate protected nanoclusters.\textsuperscript{10,13} In these systems, a layer of covalently bonded ligands is attached to the cluster surface to protect the metallic core. The protecting ligand layer prevents cluster aggregation making them stable in ambient conditions. Compared to unprotected gas phase clusters, the protected ones are attractive for many reasons. The additional stability allows the clusters to be handled like chemicals, which facilitates studies and applications inaccessible to gas phase clusters. In addition, the ligand layer has a significant effect on the cluster properties and can also be modified.
The interaction between gold and sulfur plays a key role in the thiol protected clusters.\textsuperscript{14} The strength of covalent Au–S interaction is close to the Au–Au bond. Therefore, the bonding in the gold surface is modified by the interaction with sulfur. In addition to protected clusters, the bonding between gold and sulfur has been studied in self-assembled monolayers on gold surfaces and in molecular junctions in gold electrodes in molecular electronics applications.\textsuperscript{14,15} In addition to sulfur, phosphorus also has high affinity towards gold. Hence, phosphines can also be used as ligands for gold clusters.\textsuperscript{10}
2.3.1 Synthesis
Colloidal solutions of gold have been known for hundreds of years.\textsuperscript{1} However, the synthesis of nanometer-sized thiol protected clusters was first introduced in 1994 by Brust et al.\textsuperscript{16} The introduction of a synthesis method has facilitated a large experimental effort focusing on these systems, and different modifications of the synthesis are used to obtain clusters with different sizes and different ligands.\textsuperscript{17–19} For example, thiols with different alkyl chains and thiols containing carboxylic acid groups are generally used as ligands.\textsuperscript{17–19} In addition, more complex molecules, such as glutathione, and protein units with thiol group containing cysteine residues have been used.\textsuperscript{20–22}
In general, the cluster synthesis includes two major steps.\textsuperscript{16} Gold is added to the reaction mixture as Au(III) ions by using AuCl\textsubscript{4}\textsuperscript{−} salt. The gold ions are first reduced by thiols to form a Au–S–Au–S polymer. The polymer is then further reduced to form the clusters by adding sodium borohydride (NaBH\textsubscript{4}) to the mixture. The exact reaction mechanism of the cluster formation and the ligand induced effects on this process are not fully known. Variations to the synthesis can be introduced, for example, by changing the molar ratio between gold and thiols.\textsuperscript{19} Other methods to produce thiol protected clusters include a synthesis of phosphine protected clusters followed by a ligand exchange with thiols, and
a method in which polymer stabilized clusters are first prepared and then thiolated by adding the thiol ligand.\textsuperscript{10}
The synthesis procedures generally produce a polydisperse cluster size distribution. To separate different cluster sizes, partial precipitation procedures or liquid chromatography can be used.\textsuperscript{17,19,23} Much of the recent research effort in synthesis has been dedicated to optimizing the synthesis to produce monodisperse cluster samples with different cluster sizes and different ligands.\textsuperscript{17,18,24} Some highly stable cluster sizes can also be made by first making polydisperse samples of larger clusters and then using excess amount of thiol to etch the cluster to form the most stable species. This kind of procedure is called the size-focusing method.\textsuperscript{23}
### 2.3.2 General structure of thiol-protected gold nanoclusters
Due to their structure, thiol-protected gold clusters provide a quite unique sample system, because they combine metallic gold to small organic ligand molecules. As stated, the thiol-protected clusters consist of a metallic gold core protected by covalently bonded thiolate ligands. The exact structure of all thiol protected clusters was not known until the crystal structure of Au\textsubscript{102}(pMBA)\textsubscript{44} (pMBA=\textit{para}-mercaptobenzoic acid) was solved in 2007 by x-ray crystallography.\textsuperscript{25} Following the first structure determination, the structures of several clusters having different size and different ligands have been determined. These species include, for example, Au\textsubscript{25}, Au\textsubscript{38}, Au\textsubscript{68}, Au\textsubscript{130}, and Au\textsubscript{133}.\textsuperscript{26–30} The exact structure determination can be done by x-ray crystallography or by high resolution electron microscopy.\textsuperscript{25,30} To deduce an exact cluster composition in the form of core size and the number of ligand molecules, mass spectrometry with help of elemental analysis can also be used.\textsuperscript{19,31} Because the exact structure of many of the small thiol-protected clusters is known, special interest has been directed towards studying them.
The general structure of the clusters can be understood to consist of three parts: an inner gold core, which is covered by a surface layer that includes the gold atoms bonded to the ligands, and finally the outer ligand layer.\textsuperscript{25,32} The inner core is generally metallic, and the gold atoms have a formal charge of 0. The surface layer consists of gold-sulfur motifs, where the gold has a formal +1 charge, while the sulfur atoms have a formal -1 charge. The surface layer consists of protecting gold-sulfur motifs that can have different structures. These structures commonly include shorter S–Au–S units and longer S–Au–S–Au–S units.\textsuperscript{25–29} This type of structure where the gold core of the cluster is divided into metallic part and the protecting motifs, is referred to as the divide and protect model.\textsuperscript{32} The outer ligand layer consists of the organic parts of the ligands. As an example, the structure of Au\textsubscript{102}(pMBA)\textsubscript{44} is shown in Fig. 1.
The structure of the gold core varies with the size of the particle.\textsuperscript{19,33} In the larger particles (> 144 Au atoms), the core typically adopts an fcc structure of the metallic gold.\textsuperscript{19} For smaller ones, the core structures have been found to be, for example, dodecahedral or icosahedral.\textsuperscript{19,33} For example, the Au\textsubscript{102} species shown in Fig. 1 has a Marks decahedron based core of 79 gold atoms. In addition to nearly spherical clusters, a prolate shaped core has been observed, for example, for Au\textsubscript{138} and Au\textsubscript{130} species.\textsuperscript{26,29} It has also been shown that in some cases, the choice of ligand molecule affects the size and geometry of the cluster.\textsuperscript{24}
Clusters larger than Au\textsubscript{25} species are generally intrinsically chiral even if the ligand itself is achiral.\textsuperscript{25,34} This is due to the arrangement of the protecting gold–sulfur motifs around the metallic core. The chiral isomers have been separated by chiral liquid chromatography and studied by CD spectroscopy for Au\textsubscript{40}(SC\textsubscript{2}H\textsubscript{4}Ph)\textsubscript{24,34} This property has been of interest for potential use in enantioselective catalysis.
2.3.3 Electronic properties
Many of the interesting features of clusters are intimately related to their electronic properties. The electronic energy level structure affects the stability of the cluster and determines, for example, the electron affinity and ionization energy of the cluster.\textsuperscript{2,35} In addition, the optical and photothermal cluster properties depend on the electronic structure of the cluster.\textsuperscript{36}
Atoms and small molecules have discrete energy states, while the metals have a continuum of electronic states with no energy gap between the valence and conduction bands.\textsuperscript{1,2} Clusters represent a size-dependent intermediate form between these extremes. The evolution of metallic bands from the discrete energy states can be qualitatively understood by starting from discrete states of an atom and observing a gradually increasing density of electronic states as the number of atoms contributing to the electronic structure increases. As the density of states increases, the discrete states group to form bands, and the gap between occupied and unoccupied states vanishes to form a continuum of states.
In material science, the distinction between metallic, semiconductor, or insulating materials is generally done based on the size of the energy or band gap of the material.\textsuperscript{2} The existence and non-existence of the energy gap between occupied and unoccupied states or a HOMO-LUMO gap can also be used to divide clusters into metallic gapless species and insulating or molecular species with an energy gap. The evolution of the density of electronic states and the distinction between different cluster sizes is shown in Fig. 2. On the borderline between the molecular and metallic behavior, the energy gap is small. In this case, electrons can be excited to the unoccupied states by the thermal energy. Therefore, for the cluster to show molecular behavior, the energy gap has to be large enough.
The difference between electronic state structure of clusters and bulk metals is induced by quantum confinement, which is the effect of limiting the electric field to small systems.\textsuperscript{1,2,9} Several models can be utilized to study the electronic structure of clusters. The simplest model is the liquid drop model (LDM), which is a classic electrostatic model that considers the cluster as a conducting sphere.\textsuperscript{2} LDM works well for the larger clusters and can be used to qualitatively describe trends for size-dependence of many cluster properties, such as ionization energies and melting temperatures.\textsuperscript{2} Due to its classic nature, LDM fails to describe small clusters with diameters less than $\sim 3$ nm in which quantum size effects play a significant role.\textsuperscript{2} The quantum effects can be taken into account by using the spherical jellium model in which the cluster is described as a positively charged sphere filled with electron gas.\textsuperscript{2} For this system, the electronic energy states can be determined by utilizing an attractive potential induced by the core and using it to solve the Schrödinger equation. Other versions of the jellium model that are not limited to spherical geometry have also been developed.\textsuperscript{37}
Another important feature of the electronic energy state structure of clusters is the formation of a shell structure.\textsuperscript{2,35} Based on experimental\textsuperscript{38} and computational results, it has been observed that the electronic states group into bands and form shells analogous to atomic structures. These types of shells are referred to as supershells. These results have led to the use of superatom theory, which will be described in more detail in the next section.\textsuperscript{35}
For the small thiol-protected clusters with known structure, the electronic energy levels can be studied in more detail, since they can be calculated based on the specific cluster structure.\textsuperscript{1,25,39} This type of study can be performed by utilizing DFT calculations, and can be used to explain experimental results accurately.\textsuperscript{1,28,35,39,40} In the case of the ligand protected clusters, the ligand induced effects on the electronic levels have to be taken into account. For example, electronic states that are localized in the ligands or the interface are introduced to the system.
### 2.3.4 Stability and the superatom model
Stability of nanoclusters depends on the combined effect of cluster geometry and electronic properties.\textsuperscript{2,35} For small clusters, the electronic effects generally dominate due to increased importance of quantum mechanical effects, while the geometric effects are more important for larger particles. The increased stabilities of certain cluster sizes has led to a concept of magic numbers, a sequence of numbers indicating particularly stable cluster sizes.
As stated in the previous section, the electronic levels of the clusters form bands, which in turn form supershells.\textsuperscript{35} This type of concept is analogous to electronic shells of atoms and is used in the superatom theory. In this theory, the bands of states of clusters are treated as superatom orbitals, and in analogy to atoms, the most stable species correspond to the shell closings. According to the model, the lowest energy superatom orbitals, which are mainly derived from gold 6s orbitals are $1S^2 | 1P_0 | 1D_{10} | 2S^2 1F_{14} | 2P_0 1G_{18} | 2D_{10} 3S^2 1H_{22} | \ldots$, where the letters indicate the angular-momentum characters. Based on this, the magic numbers are 2, 8, 18, 34, 58, 92, 138, \ldots.
For the thiol protected clusters, the stability consideration and the number of electrons is affected by the ligand layer and the electron withdrawing nature of the thiol ligands. The magic numbers for these clusters can be described by utilizing the divide and protect model for the structure of the clusters.\textsuperscript{35} The electron counting rule for the protected clusters is shown in equation (1).
$$n^* = Nv_A - M - z,$$
where $n^*$ is the shell-closing electron count of the metallic core, $N$ is the number of core atoms, $v_A$ is the atomic valence, $M$ is the number of electron withdrawing monodentate ligands, and $z$ is the overall charge of the cluster.
Stable thiol-protected clusters that do not follow the above mentioned magic numbers have also been observed.\textsuperscript{34} This indicates that for certain cluster sizes, the geometric effects are important even though the cluster is relatively small. Recently, it has also been shown that the ligand can also affect the size and geometry of the gold core.\textsuperscript{34}
2.3.5 Optical properties
The first application of colloidal gold particles, which have been used for centuries, has been their use as red pigment in stained glass.\textsuperscript{1} This example demonstrates the unique optical properties of the particles compared to bulk properties. Since the optical properties of clusters are closely related to their electronic properties, quantum confinement and the density of electronic states play a significant role.\textsuperscript{1-2,9,41} When studying the optical features of the clusters, a division between small clusters and larger species is important, since the optical response of the clusters in different size regions is also different.
The most interesting and most prominent optical feature supported by the larger gold nanoclusters is the localized surface plasmon resonance (LSPR).\textsuperscript{9,12,42} The LSPR is a collective charge density oscillation of the conduction electrons that are localized on the surface of metals.\textsuperscript{9,12,42} LSPR is responsible for strong scattering of light and also dominates the absorption spectrum of gold clusters with a single intense peak at $\sim 530$ nm. This absorption is responsible for the red color observed for the clusters. In addition, LSPR causes field-enhancement of the local electromagnetic fields. The plasmonic species have attracted tremendous interest in recent years, and the study of plasmons has broadened into its own research field. LSPR is also very attractive for various applications including sensors that are based on the field-enhancement effects.\textsuperscript{12}
For small clusters with a significant energy gap, no plasmon band is observed and the optical properties reflect the electronic energy structure of the system.\textsuperscript{1,28,40} The clusters generally show strong absorption. Due to the fact that electrons can be optically excited from the occupied states to the unoccupied states and due to the relatively large density of electronic states, the cluster spectrum is generally continuous with features characteristic to the specific band structure of the cluster. Because of the continuous spectrum in the visible range, the clusters appear black or brown.
2.3.6 Molecular and metallic behavior
As mentioned in Section 2.2, clusters are in many contexts divided to molecule-like and metallic particles based on their size, and one of the general research questions in cluster studies is to determine the exact number of atoms required to induce the transition from molecular to metallic behavior.\textsuperscript{2} This question has been addressed for gold by using different methods and also for different type of samples.\textsuperscript{41-45} In addition, different types of criteria for molecular and metallic behavior based on the electronic and optical cluster properties have been used.\textsuperscript{41-45} The results of some of these studies are briefly discussed here together with the criteria and methods used in the work presented in this thesis.
The evolution of the metallic electronic energy bands has been studied in the gas phase for a large number of mass selected clusters.\textsuperscript{44} The results show discrete absorptions for the smallest clusters with sizes up to few tens of atoms. For larger sizes, formation of the d-band and the sp-band typical for bulk metals is clearly seen. On the other hand, the energy gap closing has been studied by STM for two-dimensional clusters on MgO on Ag surface.\textsuperscript{45} For these clusters, the gap closing was found to occur between 70 and 100 gold atoms. Another possibility to characterize the metallic nature is to study the geometry of the gold core and the evolution of the structure towards the fcc lattice.\textsuperscript{19} For thiol-protected clusters, the Au\textsubscript{144} species has shown to be the largest cluster with molecular arrangement of core atoms with an icosahedral core. In the same study, the Au\textsubscript{187} species was shown to have a core with the fcc lattice indicating that the transition region is somewhere between these cluster sizes. The emergence of the plasmon band can also be used as a criterion for metallic behavior.\textsuperscript{41,42} Based on computational results, the metallic LSPR band is observed for clusters larger than the Au\textsubscript{144} species.\textsuperscript{42}
For the purpose of the work presented in this thesis, the crucial property used to define the molecular or metallic behavior is the existence of electronic energy gap or a HOMO–LUMO gap. One of the techniques that can be used to determine the existence of the gap is transient absorption spectroscopy.\textsuperscript{27,36,43,46–54} Recently, the transition from molecular to metallic behavior for monodisperse samples of the ligand-protected clusters has been of great interest, and the work presented in this thesis is part of this effort.\textsuperscript{1,111} Based on transient absorption studies, Varnavski et al.\textsuperscript{54} narrowed the molecular behavior to cluster size of about 2.2 nm, which corresponds to $\sim$300 atoms. The small gold clusters with 11, 25, and 28 atoms have been identified as molecular.\textsuperscript{48–53} In other studies, Au\textsubscript{102}, Au\textsubscript{115}, Au\textsubscript{117}, and Au\textsubscript{133} species have been shown to present non-metallic behavior,\textsuperscript{27,47} while the Au\textsubscript{144} cluster has been identified as a metallic species.\textsuperscript{46,47} For phosphine protected clusters, the transition between different behaviors has been indicated to occur between 13 and 55 gold atoms.\textsuperscript{43} The use of transient absorption in clusters studies is described in more detail in Section 2.5.8.
### 2.3.7 The ligand layer
The protecting ligand layer has an effect on the cluster structure, and its electronic and optical properties as presented in previous sections. In addition, the ligand molecules are responsible, in particular, for many of the chemical properties of protected clusters, since the ligand layer is directly in contact with the surrounding environment. Hence, the ligand layer has to be taken into account when handling the cluster samples. For example, the solubility of the clusters to different solvents depends on the ligand molecule and its protonation state.\textsuperscript{55} The ligand layer and the protecting units have not been studied as widely as the gold core, but for example, vibrational and NMR spectroscopy have been used in their studies.\textsuperscript{40,56–58}
The ligand layer also offers versatile possibilities for utilizing the clusters in applications, since the ligand layer can be modified chemically to achieve desired functionality. It has been shown, for example, that by utilizing a ligand
exchange reaction, the ligands of existing clusters can be changed into other molecules.\textsuperscript{59} One example of chemical modification has been presented by Marjomäki et al., who modified the ligand layer of $p$MBA protected clusters to include a linker molecule that can selectively be attached to cysteine residues on a surface of a virus.\textsuperscript{60}
\subsection*{2.4 Studied clusters}
In the work presented in this thesis, three clusters with different sizes were studied.\textsuperscript{1–3} These clusters were Au$_{102}(p$MBA)$_{44}$, Au$_{144}$(SC$_2$H$_4$Ph)$_{60}$, and a previously unreported cluster tentatively identified as Au$_{130}(p$MBA)$_{30}$. The structure and some previously reported electronic properties of the clusters are briefly discussed in this chapter.
These clusters were primarily chosen based on their size, which falls into the size region close to the expected transition region between molecular and metallic behavior. For the first two studies presented in papers I and II, well known stable cluster compositions were chosen to accurately link the photodynamics of the system to a particular structure and also to previously obtained results. In addition, the synthetic procedure of the cluster also had to be well established to produce large enough quantities of the highly monodisperse sample for the performed experiments. Following the work presented in papers I and II, an intermediate cluster size was chosen to bridge the region between the previously studied cluster sizes.
\subsubsection*{2.4.1 Au$_{102}(p$MBA)$_{44}$}
The Au$_{102}(p$MBA)$_{44}$ cluster has been a subject of many studies since the determination of its structure, which is shown in Fig. 1.\textsuperscript{25} The cluster is composed of a Au$_{79}$ core protected by 19 S–Au–S units and two S–Au–S–Au–S units. The cluster is stable, and it can be understood as a superatom complex for electronic shell closing of 58 electrons.\textsuperscript{35} Based on electronic structure calculations and experimental electronic spectrum, the Au$_{102}$ species has a HOMO–LUMO gap of 0.45 eV, indicating a molecular behavior.\textsuperscript{40} It has also been shown that the cluster is water-soluble in partly deprotonated form.\textsuperscript{55}
\subsubsection*{2.4.2 Au$_{144}$(SC$_2$H$_4$Ph)$_{60}$}
The exact cluster composition for the Au$_{144}$ species has been determined by mass spectrometry studies,\textsuperscript{17} but the exact structure of the cluster has not been determined despite active effort.\textsuperscript{61} However, a widely accepted model for the structure has been proposed based on DFT calculations.\textsuperscript{61} The proposed structure includes an icosahedral Au$_{114}$ core protected by 30 S–Au–S units.\textsuperscript{61} A
presentation of the structure based on this model is shown in Fig. 3. Based on calculations\textsuperscript{61} and electrochemical experiments,\textsuperscript{62} the cluster has no HOMO-LUMO gap, thus indicating metallic behavior. However, the cluster has been shown to have an optical gap of $\sim$0.27 eV due to selection rules.\textsuperscript{56,63}
### 2.4.3 Clusters with Au$_{130}$ core
Au$_{130}$(SR)$_{50}$ was first introduced when the species was isolated by chromatography and identified by mass spectrometry.\textsuperscript{31} After that, clusters with Au$_{130}$ core have been reported with different organosoluble ligands.\textsuperscript{23–24,26,64} Tsukuda and Negishi also proposed a structure of the cluster, which was later confirmed by calculations to be reasonable.\textsuperscript{31,65} Recently, the proposed structure was also confirmed by x-ray crystallography.\textsuperscript{26} The structure of the cluster core is slightly prolate shaped and closely resembles the structure of the core of the Au$_{102}$ cluster.\textsuperscript{26,31} The optical spectrum of these organosoluble clusters has been reported and shows no significant plasmon band, but no detailed study of the energy gap magnitude has been reported. Electrochemical studies for this cluster size indicate non-molecular behavior.\textsuperscript{66}
2.5 Optical spectroscopy and its utilization in cluster studies
2.5.1 The spectrum of electromagnetic radiation
Light is a form of electromagnetic radiation. Classically it can be understood as perpendicular oscillating electronic and magnetic fields. Due to wave-particle duality light can also be described by photons. The energy of the electromagnetic radiation and the photon depends on the wavelength of the radiation according to equation (2).\textsuperscript{3,4}
\[
E = \frac{hc}{\lambda},
\]
(2)
where \(E\) is the photon energy, \(h\) is the Planck constant, \(\lambda\) is the wavelength of the radiation, and \(c\) is the speed of light.
The full spectrum of electromagnetic radiation spans over a wide wavelength range, which is generally divided into different parts based on the use and effects of the radiation.\textsuperscript{3,4} The full spectrum includes different regions from low energy radio frequencies and microwaves to higher energy visible light and high energy x-rays and cosmic rays. The spectrum of electromagnetic radiation as a function of wavelength is shown in Fig. 4.
2.5.2 Spectroscopy and hierarchy of energy levels in molecules
Spectroscopy considers the interaction between light and matter including absorption, scattering, and emission processes.\textsuperscript{3,4} The light and matter interaction is mainly mediated by the electric field component of the light. A spectroscopy measurement generally includes the study of the interaction of light as a function of wavelength. This type of experiment can be used to access information about the energies required to transfer the system between different energy states. Different regions of the electromagnetic spectrum have a different type
of effects on the samples and utilization of various spectral regions yields information on different properties of the sample. These interactions in different wavelength regions with molecules are shown in Fig. 4.
The interaction between different wavelengths regions of light and molecules is induced by a hierarchy of energy levels in molecules.\textsuperscript{3,4} The electronic energy levels of molecules are determined by the structure of the molecule and the electronic states of the individual atoms. Each electronic state is associated with it several possible vibrational states, which in turn have several rotational states that are relevant in the gas phase. The energy required for vibrational or rotational transition is, therefore, significantly smaller than the energy required for electronic excitation. The work presented in this thesis concentrates on electronic excitation in the visible and near infrared regions and on vibrational modes of the clusters in the mid-IR region.
### 2.5.3 UV/vis spectroscopy
The radiation in the UV/vis region generally matches the differences between electronic energy levels in the molecules.\textsuperscript{3,4} Therefore, electronic energy levels and molecular properties related to the electronic structure can be studied by this method. The absorption spectrum in this region can be obtained by measuring the intensity of light at different wavelengths with and without the sample. The amount of light absorbed by the sample is usually determined by absorbance, which can be calculated according to equation (3). The second part of equation (3) shows how the absorbance value depends on the sample concentration and optical path of the sample. This part is known as the Beer–Lambert law.\textsuperscript{3,4}
\[
A = \log \frac{I}{I_0} = \varepsilon cl,
\]
(3)
where \( A \) is the absorbance, \( I \) is the transmitted intensity, \( I_0 \) is the intensity entering the sample, \( \varepsilon \) is the molar absorption coefficient of the sample, \( c \) is the sample concentration, and \( l \) is the optical path length of the sample.
The spectrum can be measured by using a UV/vis spectrometer, a device designed for absorption experiments in this region. A schematic presentation of the measurement is shown in Fig. 5.
2.5.4 Infrared spectroscopy
Molecules are in constant vibrational motion and have several vibrational modes depending on the number of atoms.\textsuperscript{3,4} Since the vibrational modes of many functional groups are highly localized, IR spectrum can be used to identify these groups in molecules. Infrared spectrum can also be used to identify the sample, since the spectrum is characteristic for each molecule. Due to selection rules, only the vibrational modes that induce a change in the dipole moment of the sample can be studied by this method.
In addition, the IR spectroscopy can be used to obtain versatile information, for example, about the vibrational energy level structures and anharmonicity of the vibrational modes.\textsuperscript{3,4} Since populations of different vibrational energy states depend on temperature according to the Boltzmann distribution, changes in the peak positions and widths can be used to analyze the effects of temperature in the system.
Measurement of an IR spectrum is generally done by using a FTIR spectrometer, a device based on a Michelson interferometer. In this technique, the spectrum is first measured in a time domain as an interferogram and then calculated in frequency domain by using a Fourier transformation. A schematic presentation of an FTIR spectrometer is shown in Fig. 6.
2.5.5 Electronic and vibrational spectroscopy in cluster studies
UV/vis spectroscopy can be used to study the electronic properties of the clusters. For large plasmonic clusters, the plasmon absorption dominates the UV/vis spectrum with a single strong and broad absorption at $\sim 530$ nm.\textsuperscript{1,2,41} Due to the strong plasmon peak, UV/vis spectrum can also be used to study the existence or non-existence of a plasmon band and, therefore, used to estimate the molecular or metallic behavior of the cluster. Since the optical properties of small non-plasmonic clusters depend on the electronic properties of the cluster, the spectrum can also be used in sample characterization for these cluster sizes.\textsuperscript{1,17,40} Due to the high density of states in the clusters, the optical spectra of the clusters generally show broad continuous absorption in the entire UV/vis region. By comparing the experimental spectra with computational results, the different spectral features can be identified in more detail.\textsuperscript{19,28,40} As an example, the UV/vis spectrum of Au$_{102}(p$MBA)$_{44}$ is shown in Fig. 7.
Figure 7. UV/vis spectrum of Au$_{102}(p$MBA)$_{44}$.
The vibrational modes of the clusters in the mid-IR region are mainly localized in the ligand molecules.\textsuperscript{40,56,63} Therefore, the studies in this region are generally concentrated on the ligands. To study the vibrational modes of the Au–S interface, far-IR spectroscopy can be used.\textsuperscript{67} In addition, the onset for electronic absorbance of the clusters can be studied in this region for many cluster sizes.\textsuperscript{40,56,63}
2.5.6 Time-resolved spectroscopy
To understand energy relaxation processes occurring after the electronic excitation, it is useful to study the time dependence of sample properties. Time can be
added as another dimension in spectroscopy experiments by using pulsed light. The time scale in which nuclear motion and molecular relaxation of the atoms occur is in the femtosecond to picosecond range ($10^{-15}$–$10^{-12}$ s).\textsuperscript{5} Therefore, femtosecond pulses can be utilized to study molecular relaxation and photochemical reactions in time scale relevant to molecules. This type of pulses in different spectral regions can be obtained by using pulsed lasers.\textsuperscript{58,69} To understand the time resolved experiments and their results, different electronic relaxation processes and the role of different energy states in the relaxation must be understood. These are discussed below.
### 184.108.40.206 Electronic excitation and relaxation
When a photon with energy matching the energy difference between two electronic states of the sample interacts with a sample system, the photon can be absorbed and electronically excite the sample.\textsuperscript{3,5} Depending on the electronic energy state structure of the molecule, the excess energy provided by the photon can induce different relaxation processes and photochemical reactions. Following the excitation, the molecule relaxes back to its electronic ground state or to photoproducts. Different relaxation processes depend on the electronic energy structure of the molecule and can be non-radiative or radiative. The initial excitation energy can be lost as heat, emitted photons, or be used to form photoproducts. The existence or non-existence of the HOMO–LUMO gap also has a significant role in the relaxation processes, and the time scales of relaxation can vary several orders of magnitude between molecular and metallic systems.
### 220.127.116.11 Molecular systems
For molecules, the electronic relaxation can be generally understood by single electron excitation and subsequent electronic and vibrational relaxation processes.\textsuperscript{5} Following an absorption of a photon, the molecule is generally excited to higher energy electronic state with the same spin state as the ground state. Most commonly, this is a singlet state. The molecule also generally has excess vibrational energy in the electronic state. The relaxation back to the electronic ground state (GS) can occur via different pathways. In each of the electronic states, the energy can relax via vibrational relaxation (VR) towards the minimum vibrational energy of the state. In addition, the molecule can change its electronic state to lower energy states with different spin states. Non-radiative transition between electronic states having the same spin is called internal conversion (IC), while the formally forbidden transition between different spin states is referred to as intersystem crossing (ISC). For molecules, the ISC generally occurs between singlet and triplet manifolds. Once the molecule is relaxed to the lowest excited state, it can return to the ground state by emitting a photon. The emission from the states with the same spin state is called fluorescence (FL), while the transition from a state with different spin is called phosphorescence.
(PH). The relaxation via the forbidden transitions is generally long-lived when compared to the allowed transitions. Due to a HOMO-LUMO gap in molecules, the radiative relaxation either as fluorescence or as phosphorescence is a common phenomenon. Typical time scales for the different relaxation processes vary from sub-picosecond IC for high energy electronic states and picosecond scale for vibrational cooling to nanoseconds for fluorescence from lowest excited states. The spin forbidden radiative relaxation from a different manifold of spin states is generally slower than fluorescence and may occur even in seconds or minutes.
The relaxation processes in molecules are generally described by Jablonski diagrams, which show the electronic energy states as lines and different processes as arrows. This kind of presentation is shown in Fig. 8. Alternatively, the electronic states of the system can be presented as potential energy surfaces.
**Figure 8.** The relaxation processes involving singlet and triplet manifolds of molecular systems presented as a Jablonski diagram. The thinner lines represent the vibrational energy levels of the molecule. See text for description of various processes.
### 18.104.22.168 Metallic systems
For metallic systems, there is no significant energy gap. This facilitates a different type of relaxation compared to molecular systems.\textsuperscript{36} In metals the excitation energy is first rapidly distributed via electron-electron scattering. In this process, the energy is non-thermally distributed between electrons to create a hot electron distribution corresponding to high temperatures. This process generally occurs in the femtosecond time scale. Due to electron-electron scattering, the information about the original excitation is rapidly lost. Subsequently, the energy is transferred to vibrational motions of the atoms via electron-phonon coupling. The efficiency of this coupling is determined by the electron–phonon
coupling constant, and it affects the relaxation time. Finally, the energy is dissipated into the surrounding medium via collisions. With this type of metallic relaxation, the energy of the photon is efficiently converted to heat, since there is no significant relaxation via emission.
22.214.171.124 The two-temperature model
The photothermal behavior of metals after electronic excitation by light has been studied for a long time and can be successfully explained by the so-called two temperature model.\textsuperscript{36,46,47,70} The model describes heat transfer from the optically excited electrons to the unexcited metallic lattice. In this model, two coupled differential equations (4a–b) can be used to describe the energy flow from the excited electrons to the lattice.
\begin{align}
C_e(T_e) \frac{dT_e}{dt} &= -g(T_e - T_l) \\
C_l \frac{dT_l}{dt} &= g(T_e - T_l) \\
C_e(T_e) &= \gamma T_e
\end{align}
In these equations, $C_e$ and $C_l$ are the heat capacities of the electrons and the lattice, respectively, $g$ is the electron-phonon coupling constant, and $\gamma$ is a constant, which for gold is $\gamma = 66 \text{ J m}^{-3} \text{ K}^{-2}$.\textsuperscript{46} The electronic heat capacity depends linearly on the temperature as shown in equation (4c). Because the heat capacity of the excited electrons is temperature-dependent, the rate of heat transfer depends on the excitation energy. This kind of behavior can be used as an indication of metallic behavior in the clusters and can also be used to determine the electron–phonon coupling constant for the cluster. Generally, the electron-phonon coupling constant is determined by using low excitation energies\textsuperscript{71} or by measuring the relaxation time of the system with different excitation energies and then extrapolating the results to zero energy.\textsuperscript{36,46,47}
2.5.7 Transient absorption spectroscopy
Transient absorption spectroscopy can be used to obtain a time-dependent absorption spectrum of the sample by utilizing pulsed light.\textsuperscript{4,68,69} This type of experiment can be used to study especially the non-radiative photochemical and photophysical properties of the systems.
Transient absorption experiment is a pump–probe measurement in which the sample is first excited by a pump pulse.\textsuperscript{4,68,69} The changes induced by the pump pulse are then monitored by a probe pulse, which is detected. Time dependence can be introduced to the experiment by delaying the probe pulse with respect to the pump pulse by modifying the distance travelled by the beams.
and repeating the measurement with several time delays. This is done by using a movable delay line. A schematic presentation of a transient absorption measurement is shown in Fig. 9.
Figure 9. A schematic presentation of a transient absorption experiment.
Transient absorption spectrum is generally presented as a difference spectrum between sample absorption with and without excitation. The transient absorption signal can then be calculated as shown in equation (5).
\[
\Delta A[OD](\lambda, \Delta t) = \log \frac{I_0(\lambda)}{I(\lambda, \Delta t)_{\text{excitation}}} - \log \frac{I_0(\lambda)}{I(\lambda, \Delta t)_{\text{no excitation}}}
\]
(5)
where \( I_0 \) is the intensity entering the sample and \( I_{\text{excitation}} \) and \( I_{\text{no excitation}} \) are the intensities transmitted with and without excitation. To improve the shot-to-shot signal to noise ratio, the probe beam can be split into two separate beams that enter the sample at different spots and are detected separately. This type of method allows simultaneous measurement of intensities with and without excitation.
The transient signal includes positive and negative features when the absorbance of the sample is increased or decreased after the excitation. The changes in absorbance can be due to the ground state bleach, stimulated emission, excited state absorption, or by photoproduct absorption. In general, no signal is observed when the probe pulse enters the sample before the pump pulse. However, in the mid-IR region a non-zero transient signal is sometimes observed. This signal is induced by perturbed free induction decay, which is a coherent effect that is observed when the time resolution of the experiment is shorter than the phase relaxation of the vibrational transition.\(^{72}\)
To obtain information about the photodynamics of the sample, the obtained time dependent absorption signal needs to be analyzed. The relaxation
time constants for different relaxation processes can be obtained from the data by fitting. In many cases, the signal decays exponentially and a sum of exponential components is fitted to the data to extract the time constants for different processes. The fitting is done according to equation (6).
\[
\Delta A[OD](\lambda, \Delta t) = \sum_{i=1}^{n} A_i(\lambda) \exp(-\Delta t/\tau_i),
\]
(6)
where \(A_i\) is the amplitude of component \(i\), \(\Delta t\) is the time delay, and \(\tau_i\) is the relaxation time constant of component \(i\).
### 2.5.8 Transient absorption in nanocluster studies
Larger plasmonic clusters with different cluster sizes have been studied extensively by UV/vis region transient absorption experiments.\(^{36}\) Relaxation dynamics typical for metallic systems have been observed for these clusters. In addition, the two temperature model has been used to extract the electron–phonon coupling constants for the clusters based on the excitation energy dependence of the recovery of plasmon bleach band absorption.
The use of transient absorption experiments in determining the molecular and metallic behavior was already mentioned in Section 2.3.6. The use of this method for this purpose is based on the differences in the relaxation time scales in molecular and metallic systems described in Sections 126.96.36.199 and 188.8.131.52. Generally, fast relaxation in time scale of a few tens of picoseconds has been observed for metallic species, while the relaxation of molecular clusters extends to hundreds of picoseconds or even nanoseconds.\(^{27,36,43,46-54}\) In addition, the excitation fluence dependence or independence based on the two temperature model has been used as an indicator of metallic or molecular behavior.\(^{27,46,47}\)
The UV/vis region transient absorption of Au\(_{35}\) species has been studied rather extensively by several groups in anionic and neutral form and also in rod shaped form.\(^{48-51}\) The relaxation dynamics of this species was observed to depend on the charge state of the cluster. Some relaxation processes were also interpreted. These processes involve the initial relaxation from the excited state to lower exited states referred to as semi-ring states, which are localized in the protecting gold-sulfur units, and radiative relaxation to the ground state.
In addition to UV/vis region transient absorption, mid-IR region probing has been used for studying energy transfer from gold clusters to protein capping layer and villin capped clusters.\(^{21,22}\) Based on these studies, it has been shown that the energy introduced to the clusters as electronic excitation is transferred to heat and proceeds in the protein chain in the form of vibrational energy,\(^{22}\) and that the protein folding of villin is not affected by the laser induced heating of the system.\(^{21}\)
3 EXPERIMENTAL METHODS
3.1 Samples and sample characterization
The studied cluster samples were Au$_{102}(p$MBA)$_{44}$, Au$_{144}$(SC$_2$H$_4$Ph)$_{60}$, and a previously unreported cluster identified as Au$_{130}(p$MBA)$_{50}$. Au$_{144}$(SC$_2$H$_4$Ph)$_{60}$ and Au$_{102}(p$MBA)$_{44}$ samples were synthesized according to previously published procedures.\textsuperscript{17,55} The Au$_{130}$ species was obtained as a by-product of Au$_{102}(p$MBA)$_{44}$ synthesis\textsuperscript{57} and was isolated by a separate precipitation procedure explained in detail in paper III. All cluster samples were obtained via an in-house collaboration.
For the performed studies, it is important to know the sample composition, and to confirm the high monodispersity of the samples. Therefore, composition and the monodisperse nature of all samples were first studied with several methods. In each case, the sample was characterized by UV/vis and infrared spectroscopy together with NMR spectroscopy.\textsuperscript{73} In addition, composition of the organosoluble Au$_{144}$ sample was confirmed with mass spectrometry.\textsuperscript{73} For the water-soluble $p$MBA protected clusters, size and monodispersity of the sample were confirmed by SDS-PAGE analysis.\textsuperscript{11-13} In each analysis, the results of used samples were compared to a reference for monodisperse samples and also compared to computational results when possible.
3.2 Sample preparation and handling
All clusters were studied in solutions. The solvents were mainly chosen based on the solubility of the sample and on the solvent IR spectrum. High enough solubility of the sample was needed to allow high cluster concentrations, and the solvent IR spectrum was required to provide a suitable transparent window to allow the sample absorption to be monitored. In the experiments the organosoluble Au$_{144}$(SC$_2$H$_4$Ph)$_{60}$ cluster was dissolved in deuterated dichloromethane (DCM-D2), while the $p$MBA-protected clusters were dissolved in D$_2$O.\textsuperscript{11-13}
It has been shown that in a D$_2$O solution, the carboxylic acid groups of the $p$MBA ligands are partly in deprotonated form and that the immediate surrounding solvent layer depends on the counter cation.\textsuperscript{55} In addition, the cluster is insoluble in methanol.\textsuperscript{18,55} In the present work, Na$^+$ was used as the counter cation in all solutions. The cations can be incorporated to the cluster sample by precipitating the sample in deprotonated form from a water-methanol mixture. Na$^+$ counter cation can be obtained by using NaOH to precipitate the sample. Before the spectroscopic measurements the sample was dried and then dissolved in D$_2$O. Since it was shown that the dichloromethane solution of cluster samples can be contaminated if it is in frequent contact with polymeric labware\textsuperscript{63}, special care was taken to avoid sample contamination of the Au$_{144}$(SC$_2$H$_4$Ph)$_{80}$ solution by only using materials suitable for chlorinated solvents when handling the solution.
### 3.3 Spectroscopy studies
Transient absorption was chosen as the research method to study the photodynamics of the clusters. Visible and NIR light was chosen for pumping to electronically excite the cluster core, and the mid-IR region was chosen for probing to obtain information about the vibrational relaxation and to monitor the energy flow from the initially excited gold core to the ligands. Due to the drastically different relaxation time scales for molecular systems and gapless metallic systems, transient absorption method is also well suited for the distinction between molecular and metallic cluster behavior as stated in Section 2.5.8. Since the time scale of the relaxation dynamics is highly dependent on the existence of the energy gap, transient absorption is a sensitive and relatively straightforward method for this type of studies. In previous studies, the existence and magnitude of the energy gap has been determined by linear spectroscopy.\textsuperscript{40,56,63} However, the determination of the onset for electronic absorbance is hindered by the broad absorption bands of the clusters and the weak electronic absorbance close to the gap. This type of difficulty in using the linear spectra in the determination of the existence of the gap was also observed for the studied Au$_{130}$ species.\textsuperscript{11}
Prior to the photodynamics studies each cluster sample was characterized by UV/vis and FTIR spectroscopy. For the electronic spectrum measurements a 1 mm optical path quartz cuvette was used. The measurements in the mid-IR region were performed in a flow cell\textsuperscript{74} with an 80 µm optical path length. The solvent absorption was removed from all measured spectra by subtraction.
The transient absorption experiments were performed by using a home-built UV/vis pump–mid-IR probe spectrometer. The used laser setup is based on a Ti:Sapphire amplifier from which ~100 fs laser pulses at ~800 nm are obtained with a 1 kHz repetition rate. The mid-IR beam used for probing is generated from the fundamental beam by using a double-pass optical parametric amplifier (OPA) combined with difference frequency mixing of the signal and
idler beams from the OPA.\textsuperscript{75} Before the sample, the beam is divided into a probe and a reference beam, which enter the sample in different spots and are detected separately. The detection is done by using a double array MCT (HgCdTe) detector. The pump pulse is produced from the fundamental beam by using a TOPAS device (travelling-wave optical parametric amplification of superfluorescence) and is aligned to hit the sample at the same spot as the probe beam. Every second pump pulse is blocked by a synchronized chopper. The time resolution of the experiment was determined by the rise time of electron signal in thin silicon wafer and was found to be $\sim 250$ fs. A schematic representation of the used setup is shown in Fig. 10, and a more detailed description of the used laser setup is described in Ref. 76.
\begin{figure}[h]
\centering
\includegraphics[width=\textwidth]{transient_absorption_setup.png}
\caption{A schematic presentation of the transient absorption setup used in the experiments. The pump is shown in gray and the probe in black. The thinner black line represents the reference beam.}
\end{figure}
The sample solution was moved back and forth in the flow cell\textsuperscript{74} by utilizing a microfluidic flow controller system during each measurement to prevent laser induced sample damage. This technique also facilitated the measurements to be performed with small sample volumes, since only 50–100 µl of sample solution was used. The transient spectrum for each sample was measured by averaging at least five spectra to improve the signal-to-noise ratio. In addition, different excitation energies were used to determine the pump fluence dependence of the transient absorption spectrum of the samples.
4 RESULTS AND DISCUSSION
The main results of the work reported in papers I-III are presented and discussed in this chapter. For each of the studied clusters, the results of spectroscopic characterization obtained by electronic and vibrational spectroscopy are shown in addition to the results of the photodynamics studies. Finally, the results for different particle sizes are combined to address the transition from molecular to metallic behavior of gold clusters, and to discuss the implications of the results.
4.1 Au\textsubscript{144}(SC\textsubscript{2}H\textsubscript{4}Ph)\textsubscript{60}\textsuperscript{I}
The UV/vis spectrum and the molar absorption coefficients of the Au\textsubscript{144} species are shown in Fig. 11A. The spectrum nicely matches with the previously reported spectrum, thus partly confirming the quality of the sample.\textsuperscript{17} The spectrum clearly shows strong continuous absorption from the NIR to UV region with no significant plasmon band. FTIR spectra of both the sample and the ligand molecule measured in DCM-D2 solution are shown in Fig. 11B. It can be seen that the vibrational spectrum of the cluster closely resembles that of the PET (PET= phenylethanthiol) ligand. This result is reasonable since the vibrational modes of the cluster in this region are expected to be highly localized on the ligands. The comparison between the spectrum of PET and Au\textsubscript{144} species also shows that the absorption band induced by the S–H stretching vibration in PET, which is observed at $\sim$2600 cm\textsuperscript{-1}, disappears for the cluster, thus indicating the formation of the Au–S bond. In addition to the absorption of the vibrational modes, electronic absorption can also be seen from the FTIR spectrum as continuously rising absorption in the higher energy region of the spectrum. The onset of the electronic absorption is observed at $\sim$2200 cm\textsuperscript{-1} and is marked with a red arrow in Fig. 11B. This result also matches nicely with previously reported results.\textsuperscript{63}
Figure 11. A) UV/Vis spectrum of Au$_{14}$(SC$_2$H$_4$Ph)$_{60}$. B) FTIR spectrum Au$_{14}$(SC$_2$H$_4$Ph)$_{60}$ and PET ligand in DCM-D2. C) Enlarged view of the vibrational mode used for probing.
For the transient absorption experiment a suitable visible pump and mid-IR probe wavelengths can be determined from the steady-state spectra. The pump wavelength was chosen to be 652 nm, which is marked with a red arrow in Fig. 11A. DFT calculations were used to confirm that the excitation is highly localized in the gold core. The probe wavelength was chosen based on the vibrational spectrum. In order to be able to study the electronic relaxation, the probed vibrational mode was selected from the region of the spectrum where there is no electronic absorption. Based on its strong absorbance, the vibrational mode at $\sim$1495 cm$^{-1}$ was chosen. An enlarged view of the FTIR spectrum of the mode is shown in Fig. 11C. The vibrational mode was identified based on calculations and comparison to identified vibrational modes for monosubstituted benzenes as one of the vibrational modes of the benzene ring of the ligand molecules.\textsuperscript{77}
The mid-IR transient absorption spectrum of Au$_{144}$(SC$_3$H$_4$Ph)$_{60}$ is shown in Fig. 12A. The spectrum shows a strong broad positive feature in the short time delays together with a negative bleach signal and a positive hot band signal of the probed mode. The kinetics for different features is shown in Fig. 12B. By using global fitting, two time constants were obtained for the relaxation processes. The faster time constant describing the initial positive feature was found to be 1.5 ps, while the longer time constant describing the behavior of the vibrational band was determined to be 29 ps.
**Figure 12.** A) A contour plot of the transient absorption spectrum of Au$_{144}$(SC$_3$H$_4$Ph)$_{60}$. The color scale is in mOD. B) Kinetics at the hot band (red) and bleach band (blue). The fits to kinetics are shown in black. The inset shows kinetics at 1510 cm$^{-1}$.
The broad and short-lived low energy absorption might be indicative of generation of hot electron gas after excitation, which has been previously observed for gold clusters and also for conduction band electrons in semiconductors.\textsuperscript{22,78} The strong broad positive signal is, therefore, attributed to the excited state absorption of the electrons of the gold core. Behavior of this type of signal
can be explained by relaxation in metallic systems as described in Section 184.108.40.206. The relaxation in metallic systems first proceeds via electron–electron scattering. This process is too fast to be observed separately in the performed experiment. The following electron–phonon scattering during which the energy is transferred to vibrational energy is observed as a decreasing excited state absorption signal. The faster time constant can, therefore, be assigned to relaxation of the initially excited electrons.
According to the two-temperature model presented in Section 220.127.116.11, for metallic systems the electronic relaxation time depends on the initial electronic temperature and hence from the total excitation energy deposited to the cluster by the pump pulse. Based on the model and on the interpretation of the short lived electron signal, a linear excitation energy dependence of the relaxation time constant should be observed. To test this, a set of experiments in which the excitation pulse energy was changed was carried out. The results shown in Fig. 13 can be used to confirm the expected linear dependence.
**Figure 13.** The excitation energy dependence of the electronic relaxation time constant for Au$_{144}$(SC$_2$H$_4$Ph)$_{80}$. The red line shows a linear fit to the presented data points.
The electronic relaxation is followed by the vibrational cooling of the system as the energy is transferred from the vibrations of the core to the ligands and is subsequently dissipated to the surrounding solvent system. The longer time constant (29 ps) can be attributed to the vibrational cooling of the cluster. Based on the transient absorption results, it can be shown that the energy is rapidly transferred from the excited electrons to phonons and that the absorption of the ligand vibrational mode is shifted from its ground state position. This implies that the vibrational modes of the ligands are strongly coupled to the vibrational modes of the Au–S interface and the modes of the gold core. It can
also be seen that the electronic excitation energy is rapidly and efficiently transferred into heat.
Based on the observed photodynamics and their interpretation, it can be stated that the Au\textsubscript{144}(SC\textsubscript{2}H\textsubscript{4}Ph)\textsubscript{80} cluster behaves like a metallic system with no significant energy gap. The obtained photodynamics results confirm the reported results from transient absorption in the UV/vis region for Au\textsubscript{144} species with organosoluble and water soluble ligands.\textsuperscript{46,47} Based on these results, the Au\textsubscript{144} species has been shown to be the smallest known cluster to exhibit metallic behavior. In addition to the metallic behavior, information about the vibrational relaxation is obtained.
### 4.2 Au\textsubscript{102}(pMBA)\textsubscript{44}\textsuperscript{II}
The UV/vis spectrum of Au\textsubscript{102}(pMBA)\textsubscript{44} is shown in Fig. 7. Since the electronic spectrum of this species has been previously studied in detail\textsuperscript{40}, the measured spectrum could be used to confirm the monodispersity of the sample by comparing the results. FTIR spectrum of the sample was also measured and is shown in Fig. 14. The spectrum is consistent with previously published spectra and also the spectrum of the pMBA ligand.\textsuperscript{40} Due to strong solvent absorption, the spectral region from $\sim$2000 cm\textsuperscript{-1} to $\sim$3000 cm\textsuperscript{-1} could not be measured. Again, the electronic absorption of the cluster is clearly seen at the higher energy region of the spectrum.
The appropriate pump and probe wavelengths could be chosen based on the measured spectra for the purpose of the photodynamics studies as done for Au$_{144}$(SC$_2$H$_3$Ph)$_{60}$. The primary excitation wavelength was chosen to be 652 nm. The experiment was also repeated with two NIR excitation wavelengths, 1304 nm and 1518 nm, to assist the assignment of the relaxation dynamics. Again computational results were used to confirm that the excitation is mainly localized in the gold core. The absorption band at $\sim$1400 cm$^{-1}$ was chosen as the probed vibrational mode.
A contour plot of the transient absorption spectrum is shown in Fig. 15. The transient absorption spectrum shows a negative bleach band that seems to disappear in about 20 ps, and a hot band that extends beyond the experimental window of the experiment and shows relaxation processes in different time scales.
**Figure 15.** A contour plot of mid-IR transient absorption spectrum for Au$_{102}$(pMBA)$_{44}$. The color scale is in mOD.
Relaxation kinetics was analyzed by separately fitting the hot band maximum, and the bleach signal. The resulting time constants were 6.8 ps for the bleach and 1.5 ps, 84 ps, and $\sim$3.5 ns for the hot band. The fitted kinetics is shown in Fig. 16. The kinetics results of the NIR excitation experiments were analyzed similarly, and the shortest relaxation time constant was found to decrease as the excitation wavelength increases. The results are shown in Fig. 16, and the obtained time constants and the relative amplitudes of different components for the hot band kinetics are shown in Table 1. The relaxation time constants were found to be independent of the pump fluence.
Figure 16. The kinetics obtained with different excitation wavelengths for hot band maximum (upper panel) and for bleach (lower panel). Exponential fits to data are shown in black. Reprinted with permission from paper II. Copyright 2015 American Chemical Society.
Table 1. The relaxation time constants obtained with different excitation wavelengths. The percentage of the relaxation component is also shown.
| Excitation wavelength | $\tau_1$ | $\tau_2$ | $\tau_3$ | $\tau_4$ |
|-----------------------|----------|----------|----------|----------|
| **652 nm** | 1.5 ps | 84 ps | ~3.5 ns | 6.8 ps |
| | 55.3 % | 16.1 % | 28.6 % | |
| **1304 nm** | 600 fs | 84 ps | ~3.5 ns | 6.8 ps |
| | 35.3 % | 28.7 % | 36.0 % | |
| **1518 nm** | 500 fs | 84 ps | ~3.5 ns | 6.8 ps |
| | 40.2 % | 33.2 % | 26.5 % | |
To more closely analyze the bleach kinetics, which appears to significantly differ from the hot band kinetics, the transient spectrum at different time delays was fitted. The fitted spectra for few a time delays are shown in Fig. 17A. Amplitude of the fitted bleach component shows long-lived kinetics hidden by the overlapping bands, which explains the seemingly asymmetric bands. The kinetics obtained for the bleach band by fitting is shown in Fig. 17B.
Figure 17. A) The fitted transient spectra with different time delays. B) Bleach recovery kinetics obtained by spectral fitting and extracting the time dependent amplitude of the bleach component. The red dotted line shows the bleach recovery kinetics fitted with a sum of obtained time constants 6.8 ps, 84 ps, and 3.5 ns. Reprinted with permission from paper II. Copyright 2015 American Chemical Society.
Since the observed relaxation shows long-lived excited species and the relaxation time constants are observed to be independent of the pump fluence,
the relaxation of the cluster is consistent with molecular behavior and the existence of a HOMO-LUMO gap. The observed kinetics also matches nicely with reported UV/vis transient absorption results.\textsuperscript{47} Since the relaxation dynamics confirms the molecular behavior of the cluster, the observed kinetics was explained by using processes commonly used for molecules. For this purpose, the electronic energy levels of the cluster in singlet and triplet states were calculated. Based on this, it was observed that the geometric arrangement of the core atoms slightly changes as the spin state of the cluster changes. This was taken into account by also relaxing the triplet state structure and by calculating the energy states in different spin states in both geometries. The calculated energy levels and a summary of the interpretation of the relaxation dynamics is presented in Fig. 18.
**Figure 18.** The computational energy levels and a summary of the interpretation of the relaxation dynamics for Au\textsubscript{102}(pMBA)\textsubscript{14} cluster. The T* and S* indicate the first excited triplet state in singlet state geometry and the ground state in triplet state geometry, respectively.
The fastest relaxation time constant (0.5–1.5 ps) was interpreted to represent the electronic relaxation from the originally excited states to the lower singlet and triplet states. The time constant cannot be assigned to a specific process but is attributed to various relaxation processes facilitated by the heating of the cluster due to simultaneous vibrational relaxation. The fast relaxation time constant is consistent with the high density of electronic states in the cluster and corresponds well with the electronic relaxation observed for Au\textsubscript{144} species.\textsuperscript{14,47} The high density of states can also facilitate relaxation via one-phonon processes, and the initial relaxation is interpreted to involve several processes including multiple internal conversions and intersystem crossings between singlet and triplet manifolds. The intersystem crossing is presumed to occur rapidly and effectively due to the high spin–orbit coupling for the heavy gold atoms.
The excitation wavelength dependence of the shortest time constant can also be explained by this type of interpretation. It is assumed that the loss of
amplitude is associated with relaxation to the ground state, which is facilitated by excess vibrational energy that promotes sampling of curve crossings. As the amount of excess energy provided by the excitation is decreased, the potential energy surfaces of different electronic states are not sampled as efficiently; hence, the amount of time that the system spends possessing enough excess energy for curve crossings is reduced, which seemingly leads to a shorter relaxation time constant.
The longer time constants obtained from the hot band kinetics (84 ps and ~3.5 ns) can then be assigned to relaxation from the excited singlet and triplet states to the ground state. The shape of the potential energy surface of the ground state and the $S_1$ state are expected to be similar; hence, they are not expected to cross. On the other hand, based on the calculated energy levels, the energy difference between the ground state and the $T_1$ state geometry is small. Therefore, a crossing between the ground state and the $T_1$ state exists close to the $T_1$ state minimum as shown in Fig. 18. Based on this, the 84 ps time constant is assigned to relaxation from the $T_1$ state to the ground state. The remaining ~3.5 ns time constant is then assigned to the relaxation from the $S_1$ to the ground state. The $S_1$ to ground state relaxation can potentially proceed via emission, which would result in fluorescence in the mid-IR region. The situation in which the relaxation from the triplet state is faster than from the singlet state is uncommon, but based on the presented interpretation, it can be explained by the computed energy states. The remaining 6.8 ps time constant obtained from the bleach kinetics could be attributed to the vibrational cooling of the cluster.
### 4.3 Au$_{130}$(pMBA)$_{50}$
The UV/vis and FTIR spectra of the Au$_{130}$ cluster are shown in Fig. 19, in which a comparison to the computational electronic spectrum of the Au$_{130}$ species is also shown. The computational results were obtained by utilizing DFT calculations and a model for the cluster structure proposed in Ref. 31.
Based on computational results, the HOMO–LUMO gap of the Au$_{130}$(SH)$_{50}$ cluster is 0.22 eV, but the stronger absorptions appear with energies higher than 0.55 eV. This result is consistent with the FTIR spectrum of the studied cluster shown in Fig. 19B. An accurate value for the onset of the electronic absorption for this cluster could not be obtained due to the solvent absorption and the absorption of OH stretching mode of the residual water in the region of interest. However, optical gap of the studied cluster can be narrowed to 2000–4300 cm$^{-1}$ region.
Figure 19. A) The UV/vis spectrum of the studied cluster (black), the computational spectrum for Au$_{130}$(SH)$_{50}$ (blue). B) FTIR spectrum of the studied cluster (black) and the computational electronic spectrum for Au$_{130}$(SH)$_{50}$ (blue). The absorption band marked with an asterisk (*) is induced by the OH stretching vibration of residual water. The inset shows an enlarged image of the optical absorption region of the spectrum. Reprinted with permission from paper III. Copyright 2015 American Chemical Society.
The transient absorption experiment was, again, performed by using 652 nm as excitation wavelength. The vibrational mode used for probing for Au$_{102}$ cluster was also used for Au$_{130}$ species. A contour plot of the transient spectrum of the sample is shown in Fig. 20A. The spectrum qualitatively resembles that obtained for Au$_{102}$(pMBA)$_{44}$, since a strong, positive hot band and a bleach signal that seemingly disappears after ~20 ps are observed. The kinetics obtained for the hot band maximum and the bleach are shown in Fig. 20B.
No pump fluence dependence of the kinetics was observed when the excitation energy was changed between ~100–350 nJ.
Figure 20. A) A contour plot of transient absorption spectrum for Au$_{130}$ species. The color scale is in mOD. B) The kinetics obtained for the hot band maximum and the bleach signals for Au$_{130}$ cluster. Reprinted with permission from paper III. Copyright 2015 American Chemical Society.
Five different relaxation processes could be resolved for the cluster by fitting the kinetics for the bleach and the hot band. The obtained time constants were 5.6 ps for the bleach kinetics and 1.2 ps, 20 ps, and 1.2 ns for the hot band kinetics. In addition, a long-lived constant component was observed in the hot band kinetics. Relaxation time constant for this process was estimated to be >10 ns.
The long-lived excited species and the pump energy independence of the kinetics clearly indicate molecular behavior and the existence of a significant energy gap. To explain the observed relaxation dynamics, a procedure similar to that used for Au$_{102}$ species was utilized. The interpretation of the occurring relaxation processes was again strongly based on electronic energy states calculated for the singlet and triplet states of the Au$_{130}$(SH)$_{50}$ cluster, which was chosen as a model based on the cluster assignment. A small change in the geometry of the core atoms is associated with the change in the spin state also for the Au$_{130}$ cluster. Therefore, the triplet state geometry is relaxed separately and the energy states are calculated also in this geometry. The computational energy levels and a summary of the interpretation of the relaxation dynamics is presented in Fig. 21.
The shortest relaxation time constant (1.2 ps) can be explained by using interpretation similar to the one used for the corresponding time constant observed for Au$_{130}(p$MBA)$_{44}$ cluster. Based on this, the time constant can be attributed to electronic relaxation from the initially excited state to the lowest excited singlet and triplet states. Again, the relaxation time constant cannot be assigned to a single transition but to multiple internal conversion and intersystem crossing processes leading to population in both singlet and triplet manifolds. The time constant compares well with the 1.5 ps time constant observed for the Au$_{102}(p$MBA)$_{44}$ cluster.\textsuperscript{11} The second shortest time constant (5.6 ps) obtained for the bleach band was assigned to vibrational relaxation.
Based on the calculated energy levels in singlet and triplet manifolds, the longer time constants could be assigned to relaxation processes occurring from the S$_1$ and T$_1$ states. For the relaxation from the S$_1$ state to the ground state, the process was estimated to occur faster than for Au$_{102}$ because of the reduced size of the gap. Therefore, the 1.2 ns time constant was assigned to this process. The remaining time constants were then related to the relaxation from the triplet state to the ground state. Based on the calculations, the ground state energy level in the triplet state geometry is 0.24 eV higher than in the triplet state geometry. Therefore, an energy barrier is presumed to exist between the triplet state and the ground state, which significantly slows down the relaxation due to the required activation energy. The long-lived excited state with a relaxation time constant >10 ns is, therefore, assigned to the relaxation from the triplet state. The 20 ps time constant is assigned to the relaxation from the triplet state during the vibrational relaxation. During the vibrational cooling of the system, there is excess vibrational energy, which facilitates the crossing of the barrier and hence a rapid intersystem crossing from the triplet state to the ground state. After the vibrational cooling, the population is trapped in the triplet state and relaxes more slowly through a thermally activated process.
The presented interpretation of the results implies that there is a long-lived excited triplet state involved in the relaxation processes. This would imply that the cluster can become transiently magnetic. In addition, it is possible that there is a small population in the triplet state, since the energy difference between the ground state and the first excited triplet state is small. This idea was
tested by measuring the EPR spectrum of the sample. However, no signal that could be assigned to the cluster was observed.
4.4 Changing from molecular to metallic behavior
By combining the size-dependent information obtained for the studied clusters,\textsuperscript{1-3} the fundamental question of the molecular or metallic nature of the systems can be addressed. As stated in Section 2.3.6, this question for the thiol-protected clusters has been of high interest during the past few years\textsuperscript{19,27,46,47} and the work presented in this thesis makes a significant contribution to this discussion.
The Au\textsubscript{144} species has been identified as the smallest known metallic cluster.\textsuperscript{1,46,47} On the other hand, Au\textsubscript{102} has clearly been identified as a molecular species.\textsuperscript{47,11} The results of two different research groups agree well on these cluster sizes. The drastic difference between the behaviors of the two cluster sizes differing only by 42 gold atoms in size is fundamentally important and can be used to significantly narrow the transition region between molecular and metallic behavior.
The size region between 102 and 144 gold atoms has been addressed in simultaneous work by different research groups.\textsuperscript{11,27,47} Based on the work presented in this thesis, the region can be further narrowed to be between 130 and 144 gold atoms.\textsuperscript{11} Based on the results of other research groups, it has been concluded that Au\textsubscript{133} cluster and clusters with approximate size of 115 and 117 gold atoms are non-metallic.\textsuperscript{27,47} However, the relaxation dynamics of these clusters show no long-lived relaxation components. To narrow the transition region even further more studies are needed.
4.5 Implications of the results for future work
The results presented in this thesis show that the mid-IR transient absorption is a good method for the determination of the photodynamics of the thiol protected clusters and can be used to provide evidence about the existence or non-existence of the energy gap. Compared to the UV/vis transient absorption the method provides additional information about the vibrational relaxation and its role in the relaxation process. In addition, some of the spectral features of the clusters overlap severely in the UV/Vis region, making it difficult to analyze the results. In these cases, a study in the mid-IR region might provide additional information about the relaxation processes.
The presented interpretation of the relaxation dynamics for the Au\textsubscript{102}(pMBA)\textsubscript{44} cluster implies that the relaxation of the long-lived population in the excited singlet state can relax via emitting a photon. Since the HOMO-LUMO gap corresponds to emission in the mid-IR region, it would be interesting to study this further since fluorescence is rarely observed in this energy region. In addition, the implied magnetic properties of the Au\textsubscript{130} species could be potentially utilized in applications. In addition, further evidence about the magnetic properties would be important for verifying the interpretation shown in paper III.
5 SUMMARY AND CONCLUSION
Three thiol-protected gold nanoclusters, Au$_{144}$(SC$_2$H$_4$Ph)$_{60}$, Au$_{102}$(pMBA)$_{44}$ and Au$_{130}$(pMBA)$_{50}$, were characterized by optical and electronic spectroscopy and their photodynamics was studied by transient absorption in the mid-IR region with visible excitation. The observed relaxation dynamics for each cluster was analyzed, and the relaxation processes involved were identified. The results were also used to determine the molecular or metallic behavior of the cluster.
Based on the drastic difference between relaxation dynamics of Au$_{102}$(pMBA)$_{44}$ and Au$_{130}$(pMBA)$_{50}$ when compared to the Au$_{144}$(SC$_2$H$_4$Ph)$_{60}$ cluster, it could be clearly seen that the Au$_{102}$ and Au$_{130}$ species show molecular behavior while the Au$_{144}$ cluster is metallic. The results presented in this thesis can, therefore, be used to narrow the size region in which the transition from molecular to metallic occurs to be between 130 and 144 gold atoms. This information is of fundamental importance when considering not only the clusters, but also gold as a metal.
The relaxation dynamics of the Au$_{144}$ species could be explained by processes typically occurring in metallic systems. The photodynamics observed for the molecular clusters, on the other hand, could be explained by the specific electronic energy level structure of the clusters. For both clusters, the relaxation was shown to include multiple relaxation processes involving both the singlet and triplet states. Based on the relative energies of the singlet and triplet states in different spin state cluster geometries, the observed differences in relaxation kinetics could also be explained. For the Au$_{102}$ species, a fast (84 ps) relaxation via a triplet state was observed due to energy surface crossing, while a slow relaxation (>10 ns) due to an energy barrier and a thermally activated process were observed for Au$_{130}$. For both clusters, the role of vibrational cooling was also found to be significant, since the excess energy controlled by the vibrational cooling facilitates many of the observed relaxation processes.
The interpretations of the observed relaxation also have interesting implications for potential future work. The potential fluorescence in the mid-IR region from the Au$_{102}$ cluster, and the transient magnetic properties implied for
the Au$_{130}$ species may provide interesting possibilities for fundamental research and also for applications.
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|
Ambient Occlusion and Shadows for Molecular Graphics
Robert Easdon
A thesis submitted for the degree of
Master of Science
at the University of East Anglia
September 2013
Ambient Occlusion and Shadows for Molecular Graphics
Robert Easdon
© This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that no quotation from the thesis, nor any information derived therefrom, may be published without the author’s prior, written consent.
Computer based visualisations of molecules have been produced as early as the 1950s to aid researchers in their understanding of biomolecular structures. An important consideration for Molecular Graphics software is the ability to visualise the 3D structure of the molecule in a clear manner.
Recent advancements in computer graphics have led to improved rendering capabilities of the visualisation tools. The capabilities of current shading languages allow the inclusion of advanced graphic effects such as ambient occlusion and shadows that greatly improve the comprehension of the 3D shapes of the molecules.
This thesis focuses on finding improved solutions to the real time rendering of Molecular Graphics on modern day computers. The methods of calculating ambient occlusion and both hard and soft shadows are examined and implemented to give the user a more complete experience when navigating large molecular structures.
Acknowledgements
I would like to thank both my primary supervisor Dr. Stephen Laycock and my secondary supervisor Dr. Steven Hayward for their support and advice throughout my degree, helping advance both my computer graphics knowledge, as well as my understanding of the biological aspects of this research. Further thanks in particular to Dr. Stephen Laycock for his consistent encouragement and guidance, which has helped me greatly over the past two years.
# Table of Contents
Abstract i
Acknowledgements ii
1 Introduction 1
1.1 Motivations and Research Objectives 3
1.2 Contributions 4
1.3 Thesis Outline 5
1.4 Terminology 5
1.4.1 HaptiMOL iSAS 5
1.4.2 Molecular Graphics (MG) 5
1.4.3 OpenGL Shading Language (GLSL) 5
1.4.4 Screen Space Ambient Occlusion (SSAO) 6
2 Literature Review 7
2.1 Introduction 7
2.2 Early Molecular Graphics on Computers 8
2.3 Modern Molecular Graphics Systems and Techniques 14
2.4 Ambient Occlusion 23
2.4.1 Geometry Based Methods 27
2.4.2 Image-Space Techniques 30
2.5 Shadows 34
2.5.1 Shadow Volumes 36
2.5.2 Shadow Mapping 38
2.6 Conclusion 41
3 Techniques for the Rendering of Molecules in Space-Filling Representation 43
3.1 Introduction 43
3.2 HaptiMOL iSAS 44
3.3 Ray Casting using 2D Billboards 45
3.3.1 Point Sprites 50
3.4 Culling Molecules with a Clipping Plane at an Arbitrary Angle 50
3.4.1 Multiple Clipping Planes 53
3.5 Clipping Molecules to a Navigation Cube 56
3.6 Conclusion 59
4 Ambient Occlusion for Molecules in Space-Filling Representation 62
4.1 Introduction 62
4.2 Ray Traced Ambient Occlusion 63
4.2.1 Single Sample Ray Traced Ambient Occlusion 63
4.3 Screen Space Ambient Occlusion 66
4.4 Analytically Calculating the Ambient Occlusion of Spheres 67
4.4.1 Implementation Decisions 69
4.5 Hybrid Ambient Occlusion 71
4.6 Comparison of Ambient Occlusion Techniques 72
4.7 Deferred Rendering 76
4.8 Conclusion 79
5 Real Time Shadows for Molecules in Space-Filling Representation 81
5.1 Introduction 81
5.2 GPU Ray Tracing with GLSL 82
5.2.1 Grid Creation 83
5.2.2 Grid Traversal 84
5.2.3 Grid Storage 84
5.2.4 Performance 86
5.3 Aiding User Interaction with a Shadow Casting Probe Light 90
5.4 Dynamic Shadows for Navigation Cube Exploration 91
5.5 Soft Shadows for Space-Filling Molecules using Ray Tracing 96
5.6 Future Advances in the Rendering of Space-Filling Molecules using Path Tracing 102
5.7 Conclusion 106
6 Conclusions 107
6.1 Discussion and Conclusions 107
6.2 Future Work 111
Bibliography 113
| Table | Description | Page |
|-------|-----------------------------------------------------------------------------|------|
| 3.1 | Here we compare the rendering time of a small molecule, 1CRN, with a number of larger molecules clipped to the same amount of atoms using this method. | 59 |
| 4.1 | Ray traced ambient occlusion at full and half resolution. | 65 |
| 4.2 | AO data layout. | 69 |
| 4.3 | Analytical ambient occlusion with varying number of occluding atoms. | 70 |
| 4.4 | A comparison of ambient occlusion techniques we have presented. | 72 |
| 4.5 | G-Buffer data layout. | 79 |
| 5.1 | Here we compare the shadow calculation time of a small molecule, 1CRN, with a number of larger molecules clipped to the same amount of atoms using this method. | 95 |
| 5.2 | A comparison of the performance between hard and soft shadows. | 102 |
| 5.3 | Path tracing performance with 100 samples per pixel, 2 light sources, and the light path allowed to bounce up to 3 times in the scene. Render times are in seconds. | 106 |
2.1 Overview of the molecular modelling system, with the KLUGE in the lower left corner, and a space-filling model by the display. Photo courtesy of Martin Zwick. ........................................ 10
2.2 A close up view of the CRT screen, KLUGE, with the globe device that controls the direction and speed of a structure’s rotation. Photo courtesy of Martin Zwick. ........................................ 10
2.3 A stereoscopic pair of images depicting the amylose polymer generated by ORTEP [Joh65]. ........................................ 11
2.4 A diagram of the ray tracing algorithm. View rays are sent from the camera to find the closest intersection point. Rays to test for shadows or other illumination effects are then sent out from this point. ........................................ 16
2.5 Due to the random paths of the eye rays in the second pass, they are unlikely land on the exact spots stored in the photon map. The solution is to average several photons hits within a circumference about the eye ray’s intersection point. ........................................ 20
2.6 The radiosity algorithm applied to a scene. Each pass adds additional illumination to the scene. Image by Hugo Elias. ........................................ 20
5.5 Here we compare four molecules to look for similar patterns in performance in relation to the number of grid cells. Being able to choose an optimal cell size (indicated above selected points) when initialising the grid can lead to significant memory savings with a relatively small increase in shadow calculation time. ........................................... 90
5.6 This figure shows how the grid is traversed for a point light. Once the light’s grid cell has been reached, we can stop the traversal. ............... 92
5.7 The red area highlighted on the molecule shows the pixels that the point light illuminates. Limiting shadow calculations to only these pixels ensures no unnecessary processing, therefore improving performance. ................................................................. 92
5.8 This image shows the probe sphere (dark blue) with a point light at its center. The attenuation of the light is set to illuminate atoms in the molecule that are in close proximity, giving the user a better sense of depth when navigating the molecular structure. However, without shadows, there are areas that receive light even though they are occluded by other atoms (highlighted by the arrows). ............... 93
5.9 Here is the same image, but with shadows enabled. The position of the probe sphere relative to other atoms is made clearer, allowing the user to navigate the structure with more confidence. .................. 93
5.10 The light blue cells indicate the bounding cells of the navigation cube. A shadow ray (the green line), traverses the grid until it is outside of the workspace, instead of traversing the entire grid. ...................... 94
5.11 The backbone structure of the molecule is rendered to give the user a better understanding of the molecular structure. Space-filling atoms, and therefore complex lighting and shadow calculations, are limited to those atoms inside the navigation cube. ..................................... 96
5.12 The inner sphere casts the umbra of the shadow. An outer sphere creates a larger shadow, with the area between the two spheres creating the penumbra. ................................................................. 97
5.13 To produce the soft shadow effect, we surround our sphere with center $c$ and radius $R$, with a larger sphere with radius $Rb$. We then scale the shadow value based on the distance $d$ from the shadow ray to the center of the sphere. ........................................... 99
5.14 Here is an example of traditional hard shadows, calculated by casting a single shadow ray towards a point light source. If the shadow ray collides with any spheres in the molecule, then both the diffuse and specular terms are set to zero, leaving just the ambient term to light the pixel. ................................................................. 100
5.15 Here the hard shadows have been replaced with the soft shadows using the technique described above. The smooth change between shadowed and unshadowed areas can be seen, aiding the spatial perception of the scene. ................................................................. 100
5.16 Here we see our new rendering techniques integrated into the HaptiMOL iSAS software. The user interacts with the molecule by controlling a probe sphere using a PHANTOM Omni haptic device. ............ 101
5.17 Here we see a molecule rendered using path tracing. The light path was allowed to bounce up to three times in the scene. Atoms were given different material properties, helping to quickly distinguish between element types. Reflections can be seen on the red oxygen atoms and blue nitrogen atoms. .................................................. 103
5.18 This diagram illustrated how the colour of a pixel is determined using path tracing. The light source is directly sampled at each intersection point. Indirect illumination is gathered along the light path, which is based on the material BRDF. ............................................. 104
Chapter 1
Introduction
Computer generated images of molecules first appeared in the 1950s [AW59]. Up until this point, molecules were hand drawn or modelled by physical objects, for study in structural biology and biochemistry. These models were limited by practicalities such as complexity of construction as well as the large space they consumed. Molecules can consist of thousands of individual atoms, making it hard to study areas of the model when they are occluded by other atoms. Although not known at the time, computers would hold the answer to the problem of displaying the atomic structure of molecules. Early computing hardware, devices to display the images, as well as devices for interactive input were all limitations for computer graphics engineers at the time. However, as computing power increased and new graphics hardware was developed, real time rates could be achieved to allow interaction with a molecular structure on personal computers. Exploration of molecules no longer required physical models or expensive and large mainframe systems. Researchers were able to load newly developed graphics programs onto their own devices, allowing for more efficient work.
Appreciating the 3D form of a molecule from a 2D image on a display is often difficult. Following advancements in graphics algorithms and hardware, improved lighting and stereoscopic techniques are commonly employed in molecular visualisation software. These techniques are useful to aid biologists in understanding the 3D
structure of proteins by enhancing the simulation of depth in the image. One such technique, popularised by the games industry and now seen in molecular visualisations, is ambient occlusion [ZIK98].
Ambient occlusion is a shading method used in computer graphics which helps to improve upon local illumination models by taking into account attenuation of light due to occlusion. It attempts to approximate the way light radiates in real life, producing effects such as contact shadows, unattainable using standard lighting methods. Although a very basic approximation to full global illumination, it can be calculated much faster whilst still enhancing the visual representation of the scene.
Ambient occlusion is commonly calculated in offline rendering software by casting rays in every direction from a point on the surface. Rays that do not intersect geometry in the scene and reach the background or “sky” increase the brightness of the surface, whereas rays that do intersect with other objects in the scene contribute no illumination. This results in points surrounded by large amounts of geometry rendered darker than points with little geometry on the visible hemisphere. Attempts to incorporate ambient occlusion into real time applications have seen a number of techniques developed, such as image space approximations of the algorithm introduced by Mittring [Mit07], to reduce calculation time of the effect. Although, as with many real time approximations, these techniques tend to suffer from inaccuracies and are of a lower quality than a ray traced approach.
Whilst ambient occlusion deals with light with no specific direction or source, it may also be desirable to handle light that has a clear origin, such as a torch or the sun. In real time graphical applications today, Shadow Mapping is a popular choice to generate shadows in a scene.
Shadow Mapping has been used both in pre-rendered scenes and real time scenes in many graphics applications, including molecular graphics programs. Shadows are
created by testing whether a pixel is visible from the light source, by comparing it to a z-buffer from the light source’s view, stored in the form of a texture. The ease of implementation combined with the lack of knowledge needed about the scene’s geometry has made this technique a popular choice for real time graphics applications, including molecular visualisation programs. However, additional techniques are required to correct visible aliasing at shadow edges where projected shadow map texels cover a noticeably large area in screen space.
To give users the best experience when visualising molecular structures, ambient occlusion and shadowing methods are both included into molecular graphics packages to enhance user interaction with the structure. However, there are challenges in creating real time graphics applications of large biomolecules on a variety of computer hardware. The rendering algorithms need to maintain a frame rate of 30 Hertz to ensure the simulation remains interactive. This is not a simple task considering molecules can contain many thousands of atoms.
1.1 Motivations and Research Objectives
Many software programs exist which attempt to convey the 3D form of biomolecules. However, few manage to provide the sense of depth that is imperative to help navigate large molecular structures and understand how their geometry relates to their function. Using complex lighting techniques, such as ambient occlusion, to achieve a high quality visual representation is vital in aiding a user’s perception of a molecule, although producing these results is commonly limited to images generated offline. However, speed of rendering is of the highest importance in real time applications, with modern day computers giving the expectation of a fast and smooth interactive frame rate.
With these motivations in mind, the aim of this thesis is to improve upon clarity
of the existing HaptiMOL iSAS software and bring an easier understanding of the 3D shape and structure of large molecules or complex proteins, whilst still producing a real time visualisation. This will be achieved through implementing advanced, yet efficient, lighting techniques including ambient occlusion, and hard and soft shadows, that will be key to improving the realism of the molecular visualisation. The techniques developed will be incorporated into HaptiMOL iSAS, improving upon its traditional rendering methods. HaptiMOL iSAS is a software package that provides haptic rendering with molecular graphics to give a deeper appreciation of the shape of biomolecules by augmenting the sense of sight with touch [SHL09]. By using a spherical probe that is the same size as a water molecule, rolling it over the biomolecule is equivalent to exploration of the solvent accessible surface.
1.2 Contributions
The main contributions of this thesis include; the investigation of the visualisation problems associated with rendering large structures encountered in biomolecular study. The technical comparison of popular ambient occlusion and shadow techniques and how they perform when used in molecular graphics. Ray tracing on the GPU with the use of the OpenGL graphics library and the GLSL shading language is investigated as a method to create these effects, focussing on the space-filling molecular representation. Knowing we are rendering spheres to represent atoms allows us to make several optimisations to allow real time performance using this technique. Finally, the integration of the most promising results into existing software that has been developed for the study of arbitrary size biomolecules.
1.3 Thesis Outline
Chapter 2 gives a comprehensive review of first the history behind the graphical visualisation of molecules, and then moves on to current methods being employed to solve the problems associated with it. Chapter 3 looks at efficient ways of rendering space-filling representations of molecules, and how clipping planes can be implemented to remove atoms from view whilst still maintaining a realistic visual representation of the molecule. In Chapter 4, a variety of ambient occlusion methods are compared based on performance and visual quality. Shadows play a key role in 3D perception in graphics, therefore Chapter 5 covers shadow techniques, including a ray traced approach on the GPU. The thesis concludes in Chapter 6, including a section on further work that can be done to extend on the research described in previous chapters.
1.4 Terminology
1.4.1 HaptiMOL iSAS
Software developed on throughout the thesis. Further explained in Section 3.2. Haptic MOLecular interactive Solvent Accessible Surface.
1.4.2 Molecular Graphics (MG)
Molecular Graphics (MG) is the term used to describe the study of molecules and their properties through graphical representation.
1.4.3 OpenGL Shading Language (GLSL)
OpenGL Shading Language (GLSL) is a high-level shading language to give developers more direct control of the graphics pipeline. It allows for increased flexibility in the rendering pipeline at the vertex and fragment level.
1.4.4 Screen Space Ambient Occlusion (SSAO)
Screen space ambient occlusion (SSAO) is a rendering technique for approximating the computer graphics ambient occlusion effect in image space.
Chapter 2
Literature Review
2.1 Introduction
Before attempting to find solutions to the real time rendering of Molecular Graphics on modern day computers, it is important to firstly understand where the problems came from and what methods have already been employed to solve them. Therefore this chapter focuses on reviewing the literature relevant to Molecular Graphics, and in particular two graphical techniques, ambient occlusion and shadows, that are the focus of this thesis. In Section 2.2, early examples of Molecular Graphics on computers are looked at to help give an overview of the importance of Molecular Graphics. Current and popular molecular visualisation systems are then analysed in Section 2.3, as well as the modern rendering techniques that have been developed for these applications. The final two sections look at graphics techniques that link directly to this thesis. Section 2.4 discusses Ambient Occlusion, methods to apply this lighting technique to real time applications, and how it can be used with good effect to improve the comprehension of the 3D shapes of the molecules. Finally, Section 2.5 investigates the current methods of implementing shadows into a 3D scene, and looks at why they are so important to a person’s perception of the environment.
2.2 Early Molecular Graphics on Computers
Molecular Graphics is the field of visualising molecules and their properties on graphical display devices [IUP]. There are many uses for visualising molecules, with these models playing an important role in the study of proteins and nucleic acids, and allowing hypotheses to be tested. Molecular Graphics assisted drug design is another extremely important area of research aided by computers to speed up the development process.
Before the time of computers, molecules were modelled using physical objects, perhaps the best known is the DNA model built from rods and sheets by Watson and Crick [WC+53]. Physical models allow easy interactivity, are tactile, and their visual aspects can not be easily reproduced on computers. However, computer rendering of molecules is now the standard method to present graphical molecular data. This is due to the practicalities of building physical models, with large molecules possibly taking weeks to build. Computer visualisations are also very good at representing computed properties such as atomic charge and electrostatic potential, needed for applications such as drug design. Haptic devices are sometimes employed to reproduce the missing tactile element found in physical models, and has been applied to molecular graphics in three main areas. The first is the teaching of structural biology, where a user can explore all areas of geometry with an extra sense of touch [SWS+03]. A second application is particle steering with the haptic device used to manipulate a molecular structure [BKB+07]. Finally haptics are used to explore the docking of proteins and ligands [SB08]. Combined with 3D stereoscopic displays and realistic lighting techniques giving the illusion of depth, haptics can improve visualisations for molecular graphics researchers.
It was in 1966 at Massachusetts Institute of Technology (MIT) that computers were first used to graphically render molecular data to a CRT display. Using Project
MAC (Multi-Access Computer), an early mainframe computer [FC66], a model building program was designed to work with protein structures [Lev66]. The program allowed the animation and interaction of a molecular structure, with a 3D effect achieved by rotating the wireframe model constantly on the screen. Figure 2.1 shows an overview of the molecular modelling system, with the visual display known as the ‘KLUGE’ in the bottom left of the image.
Hardware constraints limited the system, offering only a small amount of interaction to the user with a trackball-like device to control the rotation speed of the molecule on the screen. Storage and processing power at the time limited the viewing of structures to simplified versions of more complex molecules. The lack of colour also limited the ability to distinguish between atom types. Figure 2.2 provides a close up view of the display, as well as the input device that controls the molecule direction and speed of rotation.
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Also in the 1960s, Carroll K. Johnson of Oak Ridge National Laboratory released ORTEP (Oak Ridge Thermal Ellipsoid Plot Program), a program to produce stereoscopic drawings of molecular and crystal structures with a pen-plotter (a vector graphics printing device) [Joh65]. It was able to produce ball-and-stick illustrations of crystal structures at a quality high enough for publication uses. An appealing feature of this application was the ability to produce stereoscopic pairs of images. This technique generates a 3D view of an image by creating a single image for each eye. This stereo rendering aids greatly in the visualisation of complex arrangements.
Figure 2.1: Overview of the molecular modelling system, with the KLUGE in the lower left corner, and a space-filling model by the display. Photo courtesy of Martin Zwick.
Figure 2.2: A close up view of the CRT screen, KLUGE, with the globe device that controls the direction and speed of a structure’s rotation. Photo courtesy of Martin Zwick.
of atoms, such as proteins. Figure 2.3 shows a pair of images generated by ORTEP. Later revisions to ORTEP added features such as colouring and additional output formats, however, it remained a static modelling package. Interaction with a molecule is achieved through the command line, updating the image based on certain inputs, meaning it could not provide a true interactive experience.
![Figure 2.3: A stereoscopic pair of images depicting the amylose polymer generated by ORTEP [Joh65].](image)
By the 1990s, technological developments in hardware allowed for high quality visualisations of molecules on personal computers. The availability of the personal computer (PC) removed the need for expensive and large mainframe systems, requiring full-time staff to maintain. Researchers could load molecular graphics programs onto their general purpose device to explore molecules of interest whenever desired, allowing more effective and efficient work.
RasMol, developed by Roger Sayle [SB92] in 1992, was an important development in the molecular graphics field at the time. Before RasMol, visualisation programs
required expensive dedicated workstations to run, meaning they were not easily accessible to researchers due to the large costs involved. RasMol quickly became a widely used tool for creating images for research publications, and for education purposes. The source code was made open source, and was continued to be revised years after its release by many contributors to support different types of computers.
Sayle’s original interest during his undergraduate degree was in the problem of depth perception of solid objects in computer graphics. This interest was then applied to molecular graphics in particular, leading to the creation of a shadowing program that would generate shadows for hard sphere molecular representations. RasMol’s rendering algorithm worked by first calculating the visible surfaces using a scanline z-buffer algorithm similar to the one described by Porter [Por78], then calculating the shadowed pixels using ray tracing [Whi80]. These shadows provided an enhanced 3D effect that could help in the discovery of pockets in the molecular structure, which are of interest to researchers when looking at how the geometry of the molecule relates to its function.
RasMol’s efficient rendering code has been used in a number of molecular visualisations programs, including MDL’s Chime [CHI]. Chime, made available in 1996, takes around 16,000 lines of lines of code from Sayle’s original source, with many thousands more added by MDL to add new features whilst improving the user experience. Chime differs from RasMol in that Chime is a web browser plug-in that runs inside the browser, with visualisations happening directly on a page of a website. Websites providing the ability to run this plug-in would display molecules provided by the author of the site, however dedicated websites and interfaces provided support for showing any molecule desired, such as Protein Explorer.
Protein Explorer is ideal for beginners, with the goal of the program to make the power of Chime available to scientists, educators, and students alike [Pro]. It is a
RasMol-like interface to help load and navigate structures, providing the full power of Chime, without requiring advanced technical knowledge and a large amount of time to use. It can make the visual exploration of the protein much easier for novice users as well as more convenient for specialist users. Chime proves more effective than RasMol for presenting structural information, mainly because the plug-in can be accompanied by text and keys within the same web page. Buttons in the text can control Chime’s functions through the JavaScript web language, allowing tasks to be automated, and providing an excellent educational resource. One negative feature of Chime is the code base has not been released publicly, which would have allowed external code contributions for new functionality to keep the program relevant.
A more modern open-source molecular viewer came in the form of Jmol in the early 2000s [Jmo]. It is a cross-platform program, that can be run as a standalone Java application on the desktop, or as an applet that can be integrated into web pages of all major browsers. This offered an alternative to the Chime plug-in which was no longer in development. With a basic understanding of web technologies such as HTML, Jmol can be easily incorporated into a website to display molecular graphics visualisations to a wide audience.
Similar to RasMol, Jmol uses a custom rendering engine optimised specifically for drawing core geometric primitives that are used in molecular representations. Instead of using universal graphics APIs such as DirectX or OpenGL, building a custom rendering engine allows more efficient rendering as the engine does not need to be generic for all graphics. This also means Jmol can perform high-performance 3D rendering with no hardware requirements and no need for 3D acceleration plug-ins, making it a highly adopted tool even today.
While Jmol was an improvement to existing rendering systems at the time such as RasMol, there was a growing desire for the use of advanced rendering techniques
to improve the aesthetics and perception of a molecule. Rapid advancements in dedicated graphics hardware would allow these effects to be achieved at real time rates. The next section will look at modern systems that employ these techniques that allow for enhanced high quality publication images to be generated, as well as interactive study of molecular structures.
2.3 Modern Molecular Graphics Systems and Techniques
Modern molecular graphics applications commonly split the rendering of molecular data into two areas: real time and offline rendering. Offline rendering is generally used to produce high quality images of structures used for publication, or combined to create scripted animations. Performance is a second priority to image quality with pre-rendering, allowing more complex algorithms to be used to improve the image. Of course, user interaction will be impaired, however it is typical for molecular graphics programs to provide the option for a simplified interactive display, as well as an offline solution allowing the user to create high quality images and movies.
Visual Molecular Dynamics (VMD) [HDS96] is a molecular modelling and visualisation system, primarily designed for viewing and analysing the results of molecular dynamics simulations. A common theme in molecular graphics applications, the program uses OpenGL [Grob] to render real time visualisations, and incorporates external rendering tools such as POV-Ray [Pov] and RenderMan [Stu], to create high quality ray traced images for publication use. Another system able to create presentation quality images, as well as analyse molecules in real time is PyMol [Del02]. It is seen as the current industry standard for molecular graphics software. Again OpenGL is used to generate interactive visualisations, with PyMol implementing Shadow Mapping for shadow generation. Offline renders can be created using PyMol’s own ray
tracing engine, which has made it widely used for molecular images in much of the scientific literature.
Ray tracing is a graphics technique that takes a different approach to rendering a 3D scene than the traditional rasterisation technique. The rasterisation algorithm takes a stream of vertices, transforms them into corresponding 2D points on the viewer’s monitor, and finally the transformed 2D triangles are filled in as appropriate. Ray tracing, however, is a technique for generating an image by tracing a ray through pixels in an image plane and simulating the effects of its encounters with the objects in the scene. The technique is capable of producing a very high degree of realism, far higher than the popular rasterisation method, but this comes at a greater computational cost. Therefore it is not common to see the technique employed for real time graphics applications. However, due to ray tracing’s ability to simulate a wide variety of optical effects, such as reflection, refraction and scattering, it has become a widely used tool for use in film special effects as well as molecular graphics renders, where the image can be rendered offline.
The first ray tracing algorithm used for rendering was presented by Arthur Appel in 1968 [App68], although what was presented is now generally considered ray casting, as no secondary rays such as shadow rays were used. The idea behind the algorithm is for each pixel, rays are cast from the eye to find the closest object in the scene intersecting the ray. Using the material properties and the effect of the lights in the scene, this algorithm can determine the shading of this object. The simplifying assumption is made that if a surface faces a light, the light will reach that surface and not be blocked or in shadow. The important advantage Appel showed was what ray casting offered over older scan line algorithms in its ability to easily deal with non-planar surfaces and solids, such as cones and spheres. If a mathematical surface can be intersected by a ray, it can be rendered using ray casting. More complex
objects can be created by using solid modelling techniques and easily rendered.
**Figure 2.4:** A diagram of the ray tracing algorithm. View rays are sent from the camera to find the closest intersection point. Rays to test for shadows or other illumination effects are then sent out from this point.
The next important development came from Turner Whitted in 1980 [Whi80]. Whilst Appel cast rays from the eye into the scene until they hit an object, the rays were traced no further. Whitted continued the process. When a ray hit a surface, it could generate up to three new types of rays: reflection, refraction, and shadow. A reflected ray continues on in the mirror-reflection direction from a shiny surface. It is then intersected with objects in the scene and the closest object it intersects is what will be seen in the reflection. Refraction rays travelling through transparent material work similarly, with the addition that a refractive ray could be entering or exiting a material. The shadow ray is used to test if a surface is visible to a light. A ray is traced between this intersection point and the light. If any opaque object is found in between the surface and the light, the surface is in shadow and so the light does not contribute to its shade. This new layer of ray calculations added more realism to ray traced images, however the time to calculate all ray paths in the scene meant it could take many minutes or hours to produce just a single frame.
With the advancement in graphics hardware, with fixed function pipelines replaced with programmable vertex and fragment processors, the graphics pipeline evolved into a general programmable stream processor capable of more than simple triangle rendering. Purcell et al. showed how ray tracing could be mapped to graphics hardware in an efficient way using textures to store the scene structure, and a uniform grid to reduce intersection tests [PBMH02]. They note that the uniform grid enables constant-time access to the grid cells and takes advantage of coherence using the blocked memory system of the GPU. However, it is not an optimal acceleration structure for scenes with non-uniform distributions of geometry.
Relative performances of acceleration structures in ray tracing have been studied widely. Havran [Hav00] compares a number of acceleration structures across a variety of scenes and finds that the k-d tree is the best general purpose acceleration structure for CPU ray tracers. Foley et al. [FS05] applied a k-d tree acceleration structure to GPU ray tracing and showed how a k-d tree yields far better performance than a uniform grid for complex scenes.
The first implementation of a real time ray tracer was credited at the 2005 SIGGRAPH computer graphics conference as the REMRT/RT tools developed for the BRL-CAD solid modelling system [Muu87]. The BRL-CAD ray tracer is the first known implementation of a parallel network distributed ray tracing system that achieved several frames per second in rendering performance. Since then, there have been considerable efforts and research towards implementing ray tracing in real time speeds for a variety of purposes. This research has been extended into the field of molecular graphics by Marsalek et al. [MDG+10], who worked on the ray tracing component of BALLView.
BALLView is the Biochemical Algorithms Library’s standalone molecular modelling and visualisation application. It offers standard visualisation models for atoms,
bonds, and surfaces as well as grid based visualisation. At CEBIT 2009, BALLView was prominently presented as the first complete integration of real time ray tracing technology into a molecular viewer and modelling tool, using RTfact: a real time ray tracing library. For best results however, a very high specification computer is required to produce an interactive frame rate.
A drawback of conventional ray tracing is the unrealistically sharp nature of reflections and shadows, due to the use of single rays to sample these domains. Distributed ray tracing, also called distribution ray tracing and stochastic ray tracing, proposed by Cook et al. [CPC84] takes a different approach. It uses multiple rays to sample area lights, glossy and diffuse reflection together with other effects, and allows for the rendering of “soft” phenomena such as soft shadows.
Shadows in traditional ray tracing are discrete, limited by the use of a single shadow ray per pixel. It produces shadows with sharp edges, known as “hard” shadows. Shadows in the real world transition from fully shadowed to partially shadowed in a gradual fashion. This is due to the finite area of real light sources, and scattering of light from other surfaces. By modelling light sources with a finite area, and using a set of rays that are cast in a random fashion about the projected area of the light source, the amount of light transmitted from the source to the surface can be approximated by the ratio of rays that hit the light source to the total number of rays cast. Of course, many rays are needed to produce smooth results, and can therefore lead to lengthy render times.
An alternative approach, path tracing, proposed by Kajiya [Kaj86], allows for interactive previewing of a scene. Instead of expensive but accurate estimates of the radiance from surface interactions seen in distributed ray tracing, path tracing combines a large number of cheap but individually inaccurate samples to produce a good estimate. It can produce very fast but noisy initial results. It then refines them
over time as the number of samples increases.
As the power of CPUs and GPUs increased, so did the interest in ray tracing and path tracing algorithms for real time applications. This was aided by the maturing of GPGPU programming toolkits such as CUDA [Cor] and OpenCL [Groa] and GPU ray tracing SDKs such as OptiX [PBD+10].
Ray tracing is just one of many methods to calculate illumination in a scene. Photon mapping, developed by Jensen [Jen96], is a two-pass global illumination algorithm that approximately solves the rendering equation. Rays from the light source and rays from the camera are traced independently until some termination criterion is met, then they are connected in a second step to produce a radiance value. In the first rendering pass, packets of light, or photons, are cast into the scene from a light source. When a photon hits a surface, the intersection point and incoming direction are stored in a cache called a photon map. In the second rendering pass, the scene is rendered by a Monte Carlo ray tracer, with the photon map used to estimate the radiance of each pixel to produce the final image. Figure 2.5 illustrates the algorithm. The advantage of photon mapping is that it is easier to solve global illumination problems that are traditionally hard to solve from the observer’s standpoint, for example: intersecting a small light source with random bounces, or simulating the refraction of light through a transparent substance such as glass or water.
Radiosity is another popular algorithm to calculate global illumination in computer graphics. Introduced by Goral et al. [GTGB84], it divides the scene into smaller surfaces, or patches. All the patches are considered to be diffuse reflectors, emitters or a combination of both. A view factor is computed for each pair of patches, which defines how well each patch can see each other. This indicates the amount of radiant light energy that can be transferred between the patches. The smaller the patches are the more accurate the result will be, at the cost of longer computation time. Figure
Figure 2.5: Due to the random paths of the eye rays in the second pass, they are unlikely land on the exact spots stored in the photon map. The solution is to average several photons hits within a circumference about the eye ray’s intersection point.
Figure 2.6 illustrates the radiosity method. The downside with radiosity is that unlike rendering methods such as path tracing, which handle all types of light paths, radiosity only accounts for paths which leave a light source and are reflected diffusely before hitting the eye. Therefore radiosity based methods are generally not used to solve the complete rendering equation.
Figure 2.6: The radiosity algorithm applied to a scene. Each pass adds additional illumination to the scene. Image by Hugo Elias.
Real time rendering means ensuring each rendered frame is computed fast enough to allow the viewer to interact with the virtual environment. A variety of tools and libraries are available to help produce graphics in real time, helping to take advantage of modern graphics hardware. New graphics techniques have allowed higher quality
visual results to be achieved, while still maintaining a 30 Hertz frame rate, generally considered the minimum for interactive applications.
QuteMol is an open-source molecular visualisation program that focuses on applying modern rendering techniques to achieve a comprehensive array of visual effects [TCM06]. It makes heavy use of modern graphics card features including programmable shaders and frame and vertex buffers, to help achieve these effects at real time rates.
The program uses two popular molecular representations for its visualisations: space-fill, where atoms are represented as spheres, and ball-and-stick, where bonds between atoms are also displayed. An efficient method to render these models is vital considering molecules can contain many thousands of atoms. QuteMol uses a technique called “impostors” to greatly reduce the amount of geometry needed to represent atoms. An impostor is a term used to describe the use of 2D geometry to subtly replace a real 3D object in a scene. The impostor will always face the camera, and therefore appears to look natural from any angle. QuteMol’s impostors are procedurally calculated in the fragment shader, meaning they look perfect regardless of how enlarged they are on the screen. The reduction in triangles needed to represent an object translates into significant performance gains, especially for large molecular structures, meaning more rendering time can be spent on complex lighting effects.
Efficient methods used by Botsch and Kobbelt [BK03], and Guennebaud et al. [GP+03] need just one vertex call to generate an impostor. Further improvements by Sigg et al. [SWBG06] introduce methods to compute a tight screen-space bounding box of the quadric in the vertex shader to allow for perspective warping seen in tessellated geometry. For each fragment generated during the rasterisation of this box, the ray-quadric intersection is computed in the fragment shader to classify pixels belonging to the shape. To avoid lighting calculations for fragments which are subsequently
overdrawn (a drawback of this technique), deferred shading [DWS+88] is employed to reduce lighting calculations needed to one per pixel.
QuteMol offers a range of non-photorealistic effects with the goal of producing clearer, more informative images of molecules. There are many of these effects, inspired by artistic styles such as painting or drawing, that can be found in feature films as well as video games. QuteMol shows how some of these effects, particularly edge highlighting, can also be used for scientific visualisation to enhance an image. For example, a thin border around the edge of atoms can be helpful in detecting atom intersections (top left of Figure 2.7). Simple border detection is achieved by identifying polygons that are facing away from the viewer, and colouring them a darker shade. Depth-aware borders attempt to bring more information about the structure in an initiative way, with line thickness increasing at atom borders as the distance between the atoms increases. This effect is shown in the top right image of Figure 2.7. QuteMol also features a depth-aware halo effect, where halos irradiate from each atom, again the strength of the effect indicating depth distance. They are similar to the depth-aware border effect, but it maps z-distance over transparency rather than line thickness. This is illustrated in the bottom left image of Figure 2.7. Unlike photorealistic techniques, these effects are generally fast to compute, taking advantage of fragment shaders to perform fast per-pixel effects.
As well as offering non-photorealistic effects, QuteMol uses many of the common shading techniques, including Phong shading and self-shadowing using shadow maps. However, it also includes more advanced methods, namely ambient occlusion, that is considered a challenge for real time rendering. Ambient occlusion, explored further in Section 2.4, proves to be particularly useful in improving image clarity. In QuteMol, the occlusion factors are computed on the GPU, stored in a specially formatted texture to allow use with the impostors technique, then used at run-time to
cover atoms’ surfaces. Figure 2.8 shows a comparison of the lighting methods available on a space-filling model. The left image is rendered using the basic lighting equation, with the lack of depth perception clearly being an issue. The center image shows how self-shadowing, similar to RasMol’s rendering algorithm, can improve depth cueing. However the right image shows how ambient occlusion can provide significant improvements to depth perception. The lower image of Figure 2.8 shows a mix of non-photorealistic techniques as well as simple global illumination models to greatly improve the perception of the model, and provide helpful images for research purposes.
This thesis focusses on two graphical techniques that have already been shown to improve realism and depth perception in molecular graphics applications: ambient occlusion and cast shadows. There has been a vast amount of research into both by the graphics community, and we will now look at the development of both in greater detail.
### 2.4 Ambient Occlusion
Ambient occlusion is a technique that approximates the amount of indirect light reaching a point on the surface of an object [ZIK98]. The amount of light reaching the surface is based on how much light is being occluded by other objects in the world. Unlike local lighting effects where only light coming directly from a light source is taken into account, ambient occlusion is a global method, taking into account other geometry in a scene. It can therefore produce effects otherwise unattainable to standard lighting methods, such as contact shadows. Although it is a very basic approximation to full global illumination, it can be calculated far faster, and is still able to enhance the visual quality of the scene. This has made it a popular and well researched technique, used in graphics applications such as video games and molecular
Figure 2.7: Four images showing QuteMol’s array of post-processing techniques to enhance depth perception. The top left image shows thin borders around each atom to help in detecting atom intersections. The top right image shows depth aware borders with line thickness increasing based on distance. The bottom left image show a similar effect, but uses halos with varying transparency to achieve the same result. Finally the bottom right image shows these effects combined with ambient occlusion to greatly aid the users perception of the molecule.
Figure 2.8: Three illustrations comparing the same molecule rendered using QuteMols standard rendering (left), self shadowing (middle) and ambient occlusion rendering modes (right). The right image uses ambient occlusion to darken areas of the molecule that light cannot access, therefore giving a better perception of depth and creating a better 3D appearance to the molecule. The bottom image combines all of what QuteMol offers to provide the user many cues to the structure of the molecule.
Ambient occlusion helps to give perceptual clues to the spatial proximity of geometry in a scene. Langer and Bültthoff [LB00] found that the depth discrimination under diffuse lighting is superior to that predicted by standard direct illumination. They found combining static ambient and dynamic lighting using a simple multiplication gives perceptually plausible results at high frame rates.
The occlusion $A_p$ at a point $\bar{p}$ on a surface with normal $\hat{n}$ can be computed by integrating the visibility function over the hemisphere $\Omega$ with respect to projected solid angle:
$$A_p = \frac{1}{\pi} \int_{\Omega} V_{\bar{p}, \hat{\omega}} (\hat{n} \cdot \hat{\omega}) \, d\omega$$
(2.4.1)
where $V_{\bar{p}, \hat{\omega}}$ is the visibility function at $\bar{p}$, defined to be zero if $\bar{p}$ is occluded in the direction $\hat{\omega}$ and one otherwise, and $d\omega$ is the infinitesimal solid angle step of the integration variable $\hat{\omega}$. The classical ambient term [Pho75] is commonly combined with $1 - A_p$, improving the visual quality greatly over its traditional constant value.
A vast number of techniques have been developed that can be used to approximate this integral. The most simple and accurate method is the use of the Monte Carlo method by casting rays from the point $\bar{p}$ in a hemisphere around the surface normal, and testing for intersections with other geometry in the scene. After a large number of rays have been cast, we get an accurate approximation of the occlusion value at this point.
One of the first applications of ambient occlusion in production rendering was for spatial effects in motion pictures [Lan02], using commercialised software [Chr02]. This software uses pre-rendered occlusion maps accessed when the scene needs to be rendered. Ray traced ambient occlusion is also available for offline renders in molecular graphics software, such as PyMol, for high quality publication images. While it
is simple to implement these techniques for offline rendering applications, doing so for real time dynamic scenes has proven to be difficult. We will look at methods that attempt to speed up occlusion calculations to enable real time performance, noting if they have been used in molecular graphics applications.
2.4.1 Geometry Based Methods
Techniques to calculate ambient occlusion can generally be categorised into two distinct areas: geometry based methods and image based methods. Geometry based techniques, the focus of this section, are methods where occlusion calculations use the full information of the 3D geometry in the scene. Computing the occlusion at this stage avoids view dependant errors seen in image-space methods, although performance can be impacted for scenes with large complexity.
Pharr and Green [PG04] use techniques developed for offline rendering by Landis [Lan02], pre-processing the scene to compute the accessibility at each vertex. The accessibility value is then interpolated for each pixel in the fragment shader, giving a smooth occlusion effect with minimal run-time overhead. As these values are pre-calculated, the method will not produce the correct lighting results for dynamic meshes, limiting this technique to static scenes. The pre-processing of the accessibility values is performed using ray tracing, which can lead to lengthy pre-processing time for larger scenes. For a scene containing 150,000 triangles, the pre-process took approximately four minutes, using 512 rays per triangle to compute the accessibility values.
Sarletu and Klein [SK04] present a similar technique for calculating self-occlusion of static meshes. Their work differs by computing the accessibility by rendering the object from a number of directions positioned uniformly over the bounding sphere of the object. The vertex accessibility from these positions is found using occlusion
queries to take advantage of graphics hardware. The downside to this technique is the need to render the object many times to generate occlusion uniformly, however this method is faster than the software ray tracer in the previous method. This method has been implemented into QuteMol’s molecular visualiser as a fast way to compute occlusion for static molecules, with successful results.
To enable occlusion for dynamic scenes, Bunnel [Bun05] proposes a disc-based approach to approximate faces in a mesh. This method again requires a large pre-computation step to calculate the disc elements from the polygon data. For each vertex, shadow values from all emitters are gathered to determine the ambient occlusion. Figure 2.9 illustrates this method. Gathering the visibility from all emitters can cause areas that are too dark, because elements that are in shadow can themselves cast shadows. A second pass is used to correct this overshadowing by multiplying each form factor by the emitter element’s accessibility from the last pass, meaning elements that are in shadow will cast fewer shadows on other elements. As the visibility values are estimated per-vertex, highly tessellated meshes are needed to produce higher quality detailed occlusion. Although this method is able to produce dynamic occlusion in real time, performance is again limited by the geometric complexity of the scene.
Similar themed approaches from Ren et al. [RWS+06] and Shanmugam and Arikan [SA07] use spheres instead of discs as proxy occluders. Both these approaches approximate complex geometry with spheres to compute a directional visibility function. As proxy geometry is far simpler than the original, this method will miss high frequency occlusion effects that can help define small details. This method also tends to suffer in performance when there are a large number of objects. Shanmugam and Arikan split up the ambient occlusion calculations into two different parts: a high frequency approximation for nearby surface detail in image space, and a low frequency
approximation using spherical occluders for a soft occlusion effect caused by distant objects. Defining occlusion as two distinct areas has been employed successfully in work by Reinbothe et al. [RBA09], where rays are cast against a voxelized scene for low frequency effects, then high-frequency features are highlighted in a screen space occlusion pass.
A novel method by Kontkanen and Laine [KL05], Ambient Occlusion Fields, computes inter-object occlusion in real time. For each occluding object, they pre-compute a 3D grid in the surrounding space that encodes an approximation of the occlusion caused by the object. This volumetric information is then used in the fragment shader at run-time to quickly determine an occlusion value on the receiving object. While this method achieves real time performance, it has similar issues to other methods we have seen, specifically long pre-computation time and large amounts of memory from the usage of a large number of textures.
We have seen a number of the popular geometry based ambient occlusion methods. These methods each have their advantages in fulfilling specific roles, although a common theme is the limitations in performance from geometric complexity, as well as pre-processing being required for many of these methods. We will now assess the
second major category of ambient occlusion techniques that calculate occlusion in screen space, which have been a popular topic of research due to their simplicity and support for arbitrary scene complexity.
2.4.2 Image-Space Techniques
Screen Space Ambient Occlusion (SSAO) is a rendering technique to approximate the ambient occlusion factor by analysing the scene’s depth buffer. It was developed by Mittring [Mit07] and was used for the first time in a video game in the 2007 PC game Crysis [Fra]. The algorithm runs in a fragment shader for each pixel on the screen, sampling the depth values around the current pixel and computing the amount of occlusion from each of the sampled points based on differences in depth. The resulting approximation is relatively successful in adding an ambient occlusion effect to a scene in real time, and has made it a very popular choice in a number of interactive applications such as video games.
Figure 2.10: Resulting image from Mittring’s implementation of SSAO [Mit07]. Incorrect shading of geometry is clearly visible due to over occlusion, resulting in an overly dark image. This is corrected in later implementations.
Mittring found that by comparing depths of surrounding pixels, it was possible to compute a darkening factor to create silhouettes around objects. By limiting this
effect to nearby receivers, a local ambient occlusion effect could be produced. To create an image with high visual quality, a large number of samples would be needed per pixel, which would not be acceptable for a real time application. To reduce the number of samples, they suggested randomly distributing sample positions around the origin in a sphere. The high-frequency noise that is produced is then blurred, using information from the depth buffer to preserve edge details. This method allows a reduction in the number of depth samples per pixel, eight being used in this algorithm, while maintaining performance and visual quality.
There are a number of key advantages SSAO has over the ambient occlusion methods we have previously discussed. Firstly, as the occlusion factor is calculated in screen space, the process can be done entirely on the GPU taking advantage of its large processing power. The process is also completely dynamic, requiring no pre-computation, meaning no loading times and no requirements for system memory. This allows the technique to be integrated very easily into a modern pipeline. Finally, the method is completely independent of scene complexity, ideal for scenes with high numbers of polygons where other methods would struggle to achieve real time performance.
Of course, there are notable disadvantages to this method that stop it becoming the preferred choice in certain applications. The technique is still a relatively expensive process after considering the need for depth aware blurring stages to smooth the output before lighting calculations happen. The effect is view dependant, not able to take into account geometry that is not rendered on the screen, such as geometry clipped by the frustum or discarded due to back-face culling. The technique is also only effective for local occlusion, with a large sampling radius proving too costly due to missed cache reads in the GPU hardware.
Whilst Mitrting was the first to introduce SSAO, there have since been a vast
number adaptations of the original algorithm to tackle some of the disadvantages listed above. Filion and McNaughton [FM08] introduce a method inspired by the work from Crytek, with key changes made to improve both image quality and performance.
A problem with Crytek’s original technique is self-occlusion. Using random offset vectors generated in a sphere around the test point leads to some samples penetrating through the surface of the object itself. Filion and McNaughton solve this issue by generating vectors around a hemisphere centred around the normal at that point on the screen. This approach does not produce incorrect shadowing that occurs in Crytek’s technique, seen in Figure 2.10, but does require an extra buffer to store the image space normals. However, as normals are commonly stored as well as depth in a deferred rendering pipeline, this is generally of little concern. They also found the main bottleneck of their algorithm to be texture sampling, noting how increasing the sampling area size reduces the GPU’s texture cache effectiveness yielding poorer performance. To combat this, they down sample the depth buffer to a quarter of the original size, improving texture cache performance significantly at the cost of a loss in detail.
Horizon Based Ambient Occlusion (HBAO), introduced by Bavoil et al. [BSD08], takes another approach to estimating occlusion values. This technique uses the depth buffer as a height map, calculating the amount of occluding geometry for each point by searching for the horizon. The horizon is defined by the average slope in the height field around this point, with heavily occluded points having a steep horizon angle. Sample directions around the point of interest are chosen in image space and sample points are created along those directions, producing a circular sampling area around the pixel. This method produces higher quality occlusion effects than standard SSAO, but is more expensive to compute.
Another novel technique described in their research includes the use of an angle
bias, which is used to remove artifacts that sometimes appear when geometry is rendered at low tessellation. The bias allows occlusion near the tangent plane of the point’s hemisphere to be ignored. This geometry could provide false occlusion values, which would previously have been difficult to exclude using older methods.
Like Filion and McNaughton, they source a half resolution depth buffer for the occlusion calculations. They then render the occlusion pass at half resolution as well, whilst blurring at full resolution to avoid bleeding across edges. They apply the cross bilateral filter, developed by Kopf et al. [KCLU07], separately in the X and Y directions. Although it is a non-separable technique, using this method greatly reduces the performance cost for a small drop in blurring quality.
Bavoil and Sainz [BS09] build on the horizon-based occlusion algorithm, attempting to solve view dependant issues seen in other SSAO algorithms. They use multiple depth layers, with a technique called depth peeling, in an attempt to remove view dependant artifacts caused by the use of a single depth layer. The technique takes samples from all the depth layers for each test point. By taking the largest occlusion value from all the layers, they greatly reduce the issue of missing information behind the Z-Buffer in the vast majority of cases, providing there are enough depth layers.
Bavoil and Sainz also introduce a method to solve missing occlusion caused at the edges of the screen, due to the lack of depth information outside the view frustum. They suggest enlarging the depth images and the field of view, using a simple constant value, although this will increase time to render the depth image by a small amount. To help increase the performance of the technique, they also suggest a max footprint parameter, discarding samples outside this area, giving a performance boost due to better caching.
Volumetric Obscurance published by Loos and Sloan [LS10], improves upon the SSAO technique by making better use of each depth buffer sample. Instead of treating
samples as points, with a binary comparison between the depth buffer and the sampled depth, each sample is treated as a line sample. This is used to estimate occlusion as proportional to the volumetric occlusion of a point. However, the performance of this technique is not competitive with other methods we have seen.
As we can see, there has been a vast amount of research into screen space methods to calculate ambient occlusion. This is because of its desirable traits such as predictable rendering time, ease of integration, and being completely dynamic. The separation of rendering time from geometric complexity makes it desirable for real time applications with dynamic scenes, and therefore has become a very common technique employed in video games, but has yet to be integrated into any popular molecular graphics programs.
### 2.5 Shadows
Shadows occur when an object partially or fully blocks light falling on another surface. Unlike ambient occlusion, where light is considered to have no specific direction or source, we commonly tend to think of shadows that are cast from a clear origin, with a consistent direction, such as the sun or a torch light. Shadows are important in computer graphics to help create a realistic image of a scene. They provide the user with important visual cues about the 3D structure of objects, and their relative positions to each other, to enhance a user’s depth perception of the world.
Depth perception is defined as the visual ability to perceive the relative distance of objects in one’s visual field. It arises from a variety of depth cues, commonly split into two categories: binocular cues, providing depth information when viewing a scene with both eyes, and monocular cues, providing depth information when viewing a scene with one eye. Binocular cues include binocular disparity and convergence.
Monocular cues include occlusion, perspective, size, reference frames, as well as lighting effects including the shadows that are cast by objects, providing an effective cue for the brain to determine the shape of objects and their position in space. Figure 2.11 illustrates how powerful shadows can be in providing depth cues in a scene.
There have been numerous studies into the role shadows play in helping us understand the 3D world. Wanger et al. [WFG92] assessed the influence of pictorial cues on the perceived spatial relations in computer generated images. They found that hard shadows provided the dominant cue for spatial and scaling tasks compared with other common visual cues such as including projection, motion and frames of reference. This work was further developed by Wanger [Wan92], to assess the effect of shadow quality on the perception of space. Their results indicated that less physically accurate hard edged shadows may be preferable in tasks requiring accurate perception of an object’s shape.
When an object moves, the apparent relative motion against a stationary object gives hints about their relative distance. Kersten et al. [KMK94] analysed how cast
shadow motion provides information for the inference in object motion, and measures the human observers’ use of this information. They demonstrate that simply adjusting the motion of a shadow is enough to dramatically change the apparent trajectories of the shadow casting object. Mamassian et al. [MKK98] also found a shadow’s motion to have a profound effect on the spatial understanding. They also show how shadows can be informative about the shape of the object, the shape of the object receiving the shadow, and the spatial arrangement between the two.
Clearly these psychophysical experiments strongly show that shadows play a vital role in computer graphics to produce an enhanced visual representation of the scene. Since the survey of shadow algorithms by Woo et al. [WPF90], there has been dramatic development in both graphics hardware and technology allowing shadows to become part of real time applications, including molecular graphics. However, recreating realistic shadows is still a difficult task, and still generates much research.
Ray tracing is perhaps the easiest method to comprehend when adding shadows to a scene. A shadow ray is traced from each surface point toward each light. If any opaque object lies between these two points, then the surface is in shadow. Shadows created using this technique were first seen in work by Whitted [Whi80], but whilst graphics hardware has advanced rapidly, creating ray traced shadows at real time speeds is still a challenge today. We will now turn to investigate popular rendering techniques employed in modern applications to recreate real time shadows in a graphical environment.
2.5.1 Shadow Volumes
Shadow volumes, proposed by Frank Crow [Cro77], is a method that calculates the geometry of the area occluded from a light source to determine shadowed areas. The basic process to calculate the shadow volume is to first find all silhouette edges of the
mesh. These are the edges that separate front facing and back facing faces. These edges are then extended in the direction away from the light source to a point at infinity. The volume is then capped at the front or back depending on the implementation. If a point lies within this volume, then it is in shadow of the light. Figure 2.12 illustrates the shadow volumes of a basic scene.
Shadow volumes were a popular method for real time shadowing, and while shadow maps have become the more popular choice, shadow volumes still have their advantages. The main being they are accurate to the nearest pixel unlike shadow maps, that suffer aliasing due to limitations in texture resolution.
The original algorithm was adapted by Heidmann [Hei91] to use the stencil buffer to compute the per-pixel count for the point in volume test. Front facing areas of the shadow volume increment the stencil buffer, and back facing areas decrement it. After rendering all the volume, the stencil buffer holds a mask that indicates areas of shadow. However, their method does produce incorrect results when the viewport intersects with the shadow volume. Everitt and Kilgard [EK03] resolved this by moving the far clipping plane to infinity, producing a more robust and hardware
accelerated technique.
There are, however, issues with the shadow volume technique that are still present today. The additional geometry needed for the volumes is costly not only for the CPU but also in fill rate time, as these polygons tend to cover large portions of the visible scene. These issues are compounded for more complex shadow casters. There are also cases where shadow casters do not have a mesh that accurately represents their shape, such as billboards. As shadow volumes are based on the object’s mesh, the shadow of the object will not be represented correctly. Finally, shadow volumes can not produce realistic soft shadow effects seen in real-world environments, which are important for creating a realistic visualisation. A technique called Shadow Mapping takes a different approach to creating shadows in computer graphics, which we will now look at in detail.
2.5.2 Shadow Mapping
Shadow mapping is one of the popular techniques to add shadows to a 3D scene. Introduced by Lance Williams [Wil78], it has been used extensively in real time and offline graphics, including many popular molecular graphics applications such as QuteMol and RasMol. The technique requires at least two stages. The scene is firstly rendered from the light’s point of view, with the depth buffer saved to a texture. The scene is then rendered again from the camera’s viewpoint, with each pixel tested against the depth map of the light, after being projected into the right coordinates. If the z-value is greater than the stored value in the depth map, then the object can be considered behind the occluding object, and therefore can be drawn in shadow. Figure 2.13 gives a visual breakdown of this process.
There are many reasons as to why shadow mapping has become a very popular technique for shadow generation in graphics, especially for real time applications.
They are very easy to implement, and need only a single texture to hold depth information for each light. There is no processing or knowledge of the scene’s geometry, as shadow calculations happen in image space. This means the technique will work automatically for geometry created or altered on the GPU. Their cost is also less sensitive to geometric complexity than other techniques, which is vital in today’s applications with ever increasing polygon counts.
There are disadvantages to the shadow mapping technique however. They are generally less accurate than shadow volumes, with the accuracy of the shadow map limited by its resolution. This drop in quality, especially for small shadow maps, is usually visible as aliasing at shadow edges where the projected shadow-map texels cover a noticeably large area in screen space. A desirable way to overcome this would be to increase the shadow map size, but given memory and computational constraints, is not always viable. The scene geometry must also be rendered once per light, and for point lights many more times to capture its omnidirectional depth. For scenes with many lights, shadows normally have to be restricted to the main light source in the scene, normally the sun. There has been much research to improve many of the problems listed here, and we will look at the more notable techniques in more detail.
A particular area of shadow map research looks at warping algorithms to tackle the problem of insufficient shadow map resolution in regions near to the eye. A technique
called Perspective Shadow Maps by Stamminger and Drettakis [SD02] attempts to wrap the light frustum to exactly coincide with the view frustum by applying standard shadow mapping in post-perspective space of the camera. This approach improves shadow quality in some cases, however encounters difficulties in dynamic environments. Light Space Perspective Shadow Maps by Wimmer et al. [WSP04] wrap the camera frustum in a way that leaves the directions of light sources unchanged. A new light frustum is built that is parallel to the shadow map, sized to include the camera frustum and potential shadow casters. This method is more stable than the Perspective method, but does not use the shadow map fully. Trapezoidal Shadow Maps by Martin and Tan [MT04] build a bounding trapezoid of the camera frustum instead of a frustum in the Light Space method to improve shadow map usage.
Another area of shadow mapping research looks at splitting the view frustum into multiple depth layers. A separate shadow map is then generated for each of these layers. Introduced by Zhang et al. [ZSXL06] and further developed by Dimitrov [Dim07], the motive behind this idea is the observation that points at different distances from the view need different shadow map sampling densities. By splitting up the frustum, each shadow map can focus in a smaller area and therefore provide a better match between sampling frequencies in view space and texture space.
The work described above looks at how best to choose shadow map samples to reduce artefacts, however, even aliasing free these methods still produce hard shadow effects. In order to better recreate real world soft shadow effects, several solutions have been developed. Percentage Closer Filtering by Reeves et al. [RSC87] is a method where multiple shadow map comparisons are made per pixel and are then averaged together, calculating the percentage of the surface that is not in shadow. The original algorithm used random samples over the region to be shaded. Bunnell and Pellacini [BP04] optimised this technique for modern GPU hardware, using a 4x4
sample region to reduce aliasing.
Variance shadow maps by Donnelly and Lauritzen [DL06] try to address the problem of efficiently filtering shadow maps. They note that standard shadow maps can only store the depth of a single point, and present a method to improve on this by representing a distribution of depths for each pixel. By doing this, the shadow map can be represented in a manner that can be filtered linearly, giving the ability to take advantage of graphics hardware’s built-in capabilities such as mipmapping and anisotropic filtering. However, this technique has the potential issue of light bleeding, meaning areas of light occur that should be fully in shadow. Attempts have been made to fix this issue by Lauritzen [Lau07], and VSM certainly remains a promising direction for future research.
In this section we have looked at a variety of popular shadowing techniques and their developments over time. While ray tracing is used to create realistic and accurate shadows in offline renders, shadow mapping has become the dominant method for recreating shadow effects in real time applications. A combination of Cascaded Shadow Maps and Percentage Closer Filtering seems to be the current method of choice to produce shadows for current generation real time graphics applications, as they have been highly optimised for the current generation of GPUs.
2.6 Conclusion
The overarching goal of Molecular Graphics is to provide a scientist an enhanced way to study molecules and their properties through graphical representation. Graphical systems have been developed to provide the ability to manipulate a molecule by rotating its geometry and hiding atoms that obscure others. Advanced lighting methods have been adopted to provide a better sense of depth, with ambient occlusion and shadows being two of the more popular methods due to their simplicity in
comparison to a full global illumination model. In the next chapter, we will look at efficient methods to render molecules in the space-filling representation, to improve both performance and appearance in our HaptiMOL iSAS software.
Chapter 3
Techniques for the Rendering of Molecules in Space-Filling Representation
3.1 Introduction
In chemistry a space-filling model, also known as calotte model, is a type of 3D molecular model where the atoms are represented by spheres. The radius of the sphere is proportional to the radius of the atom, commonly defined by the van der Waals Radii [Bon64]. The center-to-center distances of the atoms are proportional to the distances between the atomic nuclei.
The colouring of atom types is important to help distinguish individual atoms in the molecule. Most molecular graphics programs use the CPK colour system, named after the designers of the original space-filling models created by Corey and Pauling in the 1950s [RBC53] and later revised by Koltun [Kol65]. This system provides a consistent standard for atomic colour for atoms of different chemical elements.
Our HaptiMOL iSAS software uses the space-filling representation, and CPK colour system, for its molecular visualisation. Knowing we are only working with spheres allows us to make several optimisations to the rendering process, whilst also enhancing the visual appearance of the molecules.
3.2 HaptiMOL iSAS
HaptiMOL iSAS is software that provides haptic rendering with molecular graphics to give a deeper appreciation of the shape of biomolecules by augmenting a user’s sense of sight with touch. By using a spherical probe that is the same size as a water molecule, the process of exploration has the benefit of being able to determine regions on the molecular surface that are accessible to the solvent. This gives insight into how awkward it is for a water molecule to gain access to or escape from channels and cavities, indicating possible entropic bottlenecks.
The software uses the OpenGL 2.0 API to render the molecular structure, using display lists to render triangle meshes representing the spherical atoms. Phong shading is used for lighting, with two light sources illuminating the molecule. One light source is located at the centre of the probe, giving the user a better understanding of its position in relation to other atoms in the molecule. In this thesis, we aim to improve upon these rendering techniques currently used to display molecules to the user. This section focuses on how to render spheres that represent atoms efficiently.
Later sections look at adding advanced lighting effects to the software, including ambient occlusion and shadows, in the hope of further aiding the depth perception of the molecule to improve navigation of a biomolecule.
3.3 Ray Casting using 2D Billboards
A common way for molecular visualisation programs to render a sphere in a 3D scene is to generate a triangle mesh. This mesh would likely need hundreds of triangles to produce a smooth enough shape to appear spherical to the human eye. In some cases it may be viable to generate a sphere with an arbitrary number of triangles, and thus improve the approximation, but this will always be an approximation. Per-pixel lighting can also be used to help disguise a low polygon mesh with the smooth lighting effects it provides, however the individual triangles in the mesh may still be visible where the spheres intersect. In the general case, memory usage and rendering time have to be taken into account, therefore we would need to limit the number of triangles rendered to maintain an interactive frame rate, consequently hurting the visual representation.
A sphere can be defined as a set of points in space that are an equal distance from a common center point. A technique that is employed by some molecular visualisation programs such as QuteMol to approximate this definition is Impostors. This is where the geometric shape is simply a place-holder providing a way to invoke the fragment shader over a certain region of the screen.
Given a 2D square, we can invoke a fragment shader over this region, culling away fragments that are further away from the center of the square than the radius of the sphere. This square will be in the same position and have the same dimensions as the actual circle, and it will always face the camera. In the fragment shader, we then compute the position and normal of each point along the sphere’s surface. By doing
this, we can map each point on the square to a point on the sphere we are trying to render. This technique produces spheres that appear perfectly round regardless of how close the sphere is to the camera. This also helps to drastically reduce the number of triangles per sphere from hundreds to just two. However, the problem with this technique is that the sphere will always appear perfectly circular to the viewer, no matter the viewing angle. The problem is illustrated in 3.2.
The technique described above, whilst producing seemingly perfect spheres, is simply creating a circle in window space. This will not reproduce the elongation effects seen on the real geometry in Figure 3.2. It is a good approximation if the spheres appear small on the screen. However, if the spheres are large, or close to the camera, then inaccuracies can be clearly seen with this method. We will instead use the ray casting technique to get the position and normal of a sphere for each fragment. This will also prove important in later chapters, where ray tracing is used for ambient occlusion and shadows. It will ensure the shadows cast by the spheres will match exactly the geometry being rendered.
Ray casting, first coined by Scott Roth in 1982 [Rot82], is a technique for generating an image by casting a ray through each pixel in an image plane, then intersecting these rays with objects in the scene. Once the nearest intersection point has been found, the final pixel colour is determined based on the incoming light and material properties of the object. Instead of performing the ray casting algorithm for each pixel of the screen, we perform it on each billboard. This removes the need for a acceleration structure to ensure the first object hit by the ray can be found quickly.
We can compute the intersections between rays and spheres using their parametric equations.
\[
\mathbf{P} = \mathbf{P}_0 + t\hat{\mathbf{v}} \tag{3.3.1}
\]
\[
|\mathbf{P} - \mathbf{C}|^2 - r^2 = 0 \tag{3.3.2}
\]
We can substitute the ray equation into the sphere equation:
\[
|(\mathbf{P}_0 + t\hat{\mathbf{v}}) - \mathbf{C}|^2 - r^2 = 0 \tag{3.3.3}
\]
This can then be written in the form of a quadratic equation:
\[ \mathbf{v}^2 t^2 + 2\mathbf{\hat{v}} \cdot (\mathbf{P}_0 - \mathbf{C})t + |\mathbf{P}_0 - \mathbf{C}|^2 - r^2 = 0 \]
(3.3.4)
The equation can be solved using the quadratic formula. Equation 3.3.5 is the general form of the quadratic equation. We can then substitute in the values in order to solve:
\[ at^2 + bt - c = 0 \]
(3.3.5)
\[ a = |\mathbf{\hat{v}}|^2 \]
(3.3.6)
\[ b = 2\mathbf{\hat{v}} \cdot (\mathbf{P}_0 - \mathbf{C}) \]
(3.3.7)
\[ c = |\mathbf{P}_0 - \mathbf{C}|^2 - r^2 \]
(3.3.8)
Solving this quadratic equation for each fragment of the billboard will produce a sphere that is identical to one made up from traditional geometry, but will be pixel perfect regardless of its position to the camera. We can compare this technique with the more traditional triangle mesh that was previously being used to represent atoms in the HaptiMOL iSAS software. The time to render a single frame was measured, with no lighting effects used, and camera positioned to fit the whole structure on screen. The results are shown in Figure 3.4.
It is clear from Figure 3.4 that even the most poorly tessellated triangle mesh cannot reproduce the performance of the billboard technique. This is especially apparent with larger molecules with many tens of thousands of atoms. Frame rates drop below what is considered interactive when using triangle meshes, but with billboards can remain interactive to the user.
Figure 3.4: This graph compares the performance of Point Sprites, Billboards aligned on the GPU, and triangle meshes when rendering molecules with varying amount of atoms, from hundreds to tens of thousands.
3.3.1 Point Sprites
In order to create the camera aligned billboards described in section 3.3, we can calculate how to position the four points to align them to face the camera on the CPU. We then send these points to the GPU to render them. However, OpenGL provides an alternative way produce view aligned geometry on the GPU, using Point Sprites. This saves doing billboard calculations in the main application and reduces the number of vertices that have to be sent to the vertex program.
The comparison of the two rendering techniques, also shown in Figure 3.4, shows the advantage Point Sprites have over billboarded quads. This is due to the billboarding calculations taking place on the GPU for the Point Sprites, as well as less data needing to be transferred through the pipeline. However, a large drawback to Point Sprites arises when they are clipped from the screen. OpenGL discards the entire Point Sprite when the vertex is outside of the view frustum, which leads to a noticeable disappearance of atoms around the edges of the screen. This leads to the conclusion that both Billboard and Point Sprite rendering methods could be included into the software, with Point Sprites being used on lower end graphics cards where an interactive frame rate takes precedence over visual appearance.
3.4 Culling Molecules with a Clipping Plane at an Arbitrary Angle
The iSAS software allows the user to interact with a molecule with a probe sphere. As the user moves the probe into the pockets and channels of the molecule, the user can lose sight of it. If we are able to clip the structure away as the probe moves inside, we could always see the probe in relation to the protein structure. Another motivation for clipping planes includes being able to see channels that may have been impossible to detect without culling parts of the structure away. Other software packages either
do not provide clipping functionality, or do not employ a nice clipping strategy, and so the visual results can appear unnatural.
For our software, it would be desirable for a clipping plane to be fixed to the probe sphere, meaning as the user moves the probe into channels and pockets, spheres between the probe and the camera are culled away. This would ensure the user’s view of the probe sphere is never restricted. It would be beneficial if the user has the option to cull the spheres entirely if they intersect the plane, or alternatively display the partially culled sphere as if it had been cut. This may be advantageous in cases where the user wants to know exactly how far the probe is in relation to other atoms in the molecule. This approach will also produce a more visually correct culling effect, where instead of whole atoms unnaturally popping out of view when they intersect the plane, the atoms gradually get smaller as the plane passes through them.
The improvements in the rendering of atoms in Section 3.3 show how 2D ray traced billboards can be used in place of traditional triangle meshes. This makes the implementation of rendering partially cut spheres easier, as we can use the intersection points of the ray-sphere intersection to our advantage in order to change the shape of the atom.
When a ray intersects a sphere, it has two points of intersection, where it enters and exits the sphere (unless the ray is tangent to the sphere, meaning there is just one intersection point). As a billboard will always face the camera, it is common when rendering spheres using this technique to only calculate the intersection point nearest to the camera, as we know the second intersection point will always be occluded. This approach will work fine when the spheres do not need to be culled, however the 3D effect breaks if fragments are discarded that are behind the plane, seen in the left image of Figure 3.5.
A solution to rendering spheres that have been cut by a plane at an angle can
be seen in Figure 3.6. As described above, a ray-sphere intersection produces two intersection points, $P_{min}$ and $P_{max}$, where $P_{min}$ defines the first intersection, closest to the camera, and $P_{max}$ defines the second intersection. If both $P_{min}$ and $P_{max}$ are behind the clipping plane, the fragment can be discarded, seen as the red section of Figure 3.6. If $P_{min}$ is behind the plane, but $P_{max}$ is in front, then we can render the fragment at position $P_{max}$, depicted as the green section of Figure 3.6. If both $P_{min}$ and $P_{max}$ are in front of the plane, we simply render $P_{min}$ shown as the blue section of Figure 3.6.
The result of calculating both $P_{min}$ and $P_{max}$ can be seen in the center image of Figure 3.5. However the appearance is still not what would be expected from a sphere cut in two. Most people intuitively imagine the atom modelled as “full” solid sphere rather than as a thin empty shell. To make the sphere appear solid, as in the right image of Figure 3.5, we simply calculate the distance from $P_{max}$ to the plane, in the direction of the camera, and move $P_{max}$ onto the plane accordingly. We also set the normal of the fragment to that opposite of the plane, to produce the correct lighting results. The result of this technique on a full molecule can be seen in Figure 3.8.
It will be important to ensure fully dynamic ambient occlusion and shadows when
the atoms are culled, as these atoms no longer produce occlusion or cast shadows on atoms still visible. This will aid in producing a more visually believable scene, where lighting effects change dynamically in response to the user moving the clipping plane. Work on both dynamic ambient occlusion and shadows will be covered in later chapters. These lighting effects would not be possible in many other software packages such as QuteMol, where ambient occlusion effects are baked into textures in a pre-processing stage.
The results of this technique provide real time user interaction between a clipping plane and a molecule. A realistic visualisation is maintained by modelling the atoms to appear solid when culled by the clipping plane. This helps to produce a more immersive and useful molecular visualisation for the user.
### 3.4.1 Multiple Clipping Planes
As well as a clipping plane fixed to the probe sphere, it may also be desirable for the user to hide other sections of a molecule that they may not need to view. The algorithm above can support multiple clipping planes by moving $P_{max}$ onto the closest
Figure 3.7: This is the full structure of a molecule that has not been clipped by the clipping plane.
Figure 3.8: Here the protein has been culled by a clipping plane. It is possible to see pockets and channels that were previously hidden when viewing the full structure.
plane. The result of two clipping planes on an atom is shown in Figure 3.9.
The performance impact of adding a dynamic clipping plane to the scene is small. For each atom, we perform a simple sphere-plane test on the CPU to determine if the atom is fully behind the plane. These atoms do not need to be rendered, therefore the number of draw calls can be reduced and this means we keep the more complex fragment shader computations to a minimum. If the atom partially intersects the clipping plane, a vertex attribute is set and passed to the fragment shader to indicate further processing. For fragments that intersect the clipping plane, we perform two point-plane tests, for both $P_{min}$ and $P_{max}$, to determine which pixels we need to discard or alter. Atoms ahead of the clipping plane are rendered normally without these tests, again reducing the amount of fragment operations. However, the real bottleneck in performance tends to be the number of OpenGL draw calls. In practise, this means that adding more clipping planes to the scene will likely reduce the draw call count, and therefore boost performance.
Figure 3.10: This graph shows the performance impact on the GPU of adding clipping planes to the scene. The clipping planes were positioned around the 6 faces of the navigation cube (introduced in the next section) on a molecule containing 58870 atoms. 327 atoms were left unclipped in the navigation cube after all clipping planes were added. This performance test was done without culling atoms on the CPU to highlight the GPU performance impact of the algorithm described in Section 3.4.
3.5 Clipping Molecules to a Navigation Cube
HaptiMOL uses a navigation cube to visualise the boundaries of the safe working area of the haptic device being used. Moving the device out from the edges of the cube will translate the molecule so that the desired area of the protein can be safely studied. The navigation cube is illustrated in Figure 3.11.
In the previous section, we looked at how clipping planes can be used to remove areas of the molecule to ensure the user is always able to see the probe sphere. Through experimentation, we noted how the inclusion of a simple sphere-plane test per atom
can help to improve performance by reducing draw calls. The performance benefits are especially noticeable when rendering large molecules with tens of thousands of atoms, where vertex processing tends to become the bottleneck of the graphics pipeline. Figure 3.4 shows how, even using the efficient billboarding techniques to render the atoms, we still reach a point when the time to render a frame drops out of what is considered the minimum for real time performance.
For systems unable to render the entirety of a molecule in real time, we have developed a method to only render atoms inside a scalable navigation cube. This provides the user an easy way to reduce the number of atoms rendered to suit their needs, without having to position clipping planes manually, and without having to perform sphere-plane tests per atom. Once the user has navigated to the area of the molecule that they are interested in exploring, this method can be employed to remove atoms outside the area of interest, and therefore boost performance to real time levels for interaction with the molecule.
To render only atoms inside the selected area, we use a uniform grid to allow us to quickly determine the specified area of the molecule the user has navigated to. We talk more about the uniform grid in Chapter 5, including its generation and choice on grid cell density. Figure 3.12 gives a 2D illustration of how the grid cells are chosen. Each of the navigation cube corners are transformed into the grid’s local space. For each corner, we find their grid index, indicated by the green cells. The minimum and maximum index of the bounding cells are then updated accordingly, shown as the blue cells. Once these cells have been determined, we then traverse through each cell, testing spheres contained in each cell with the navigation cube. Figure 3.13 shows this technique implemented in our software.
It is important to determine the performance overhead of this technique. Table 3.1 shows the clipping method applied to a number of molecules. We first measured the rendering time of a small molecule of 327 atoms, without the clipping method employed. We then used the clipping method on a number of much larger molecules, moving the navigation cube to select a 327 atom section of each. We noted only a very small increase in rendering time, showing this method can be applied effectively on all molecule sizes in our target range.
We also show how we now have an effective method to render sections of very large molecules in real time. 3IYN, an molecule containing almost 100,000 atoms, takes 60 milliseconds render time per frame using the billboard technique. The user can now enable this technique to achieve real time rates that they could get when rendering smaller size molecules.
Table 3.1: Here we compare the rendering time of a small molecule, 1CRN, with a number of larger molecules clipped to the same amount of atoms using this method.
| Name | Amount | Frame Time (ms) | Overhead (ms) |
|------|--------|-----------------|---------------|
| 1CRN | 327 | 0.32 | 0.0 |
| 1BKS | 5231 | 0.36 | 0.04 |
| 3ETS | 9152 | 0.36 | 0.04 |
| 1JB0 | 24198 | 0.36 | 0.04 |
| 1AON | 58870 | 0.37 | 0.05 |
| 3IYN | 99595 | 0.38 | 0.06 |
3.6 Conclusion
In this chapter, we cover a variety of techniques to render atoms being represented by spheres. The use of billboards as a platform to perform ray casting provides an enhanced visual benefit over polygonal meshes by ensuring the spheres are accurate to the nearest pixel, whilst sustaining a far greater frame rate than even the most poorly tessellated triangle meshes. We also show how billboards can be produced cheaply using point sprites, to further reduce processing time in the creation of camera facing geometry.
Whilst this billboarding technique provides many advantages over traditional ray casting methods, it can only be used for the primary ray. Further rays to create shadows, reflections or refraction effects will still need to rely on the traditional methods described in the following chapters.
The clipping plane is a vital tool for exploring large data sets, ensuring the user
Figure 3.13: These four images show how clipping to the navigation cube is achieved. The top left image shows the full molecule, 1ADG, and the top right image includes the uniform grid of the molecule. The bottom left image highlights the bounding cells of the navigation cube, whilst the bottom right image shows the final result of clipping to this cube.
can always see the probe sphere, whilst also finding pockets and channels in the structure that may be hidden from view. The use of ray casting has allowed for spheres to appear partially culled, giving the user a precise visual cue to the location of the clipping plane in relation to the atoms. Additional clipping planes can easily be implemented with minimal performance impact.
Finally, we have shown a simple technique to render small portions of molecules using a uniform grid to quickly determine areas to cull. This is especially useful for interacting with larger molecules, that are hard to render in real time, even with the billboarding method. This technique will also prove useful for limiting complex lighting calculations to a manageable area, discussed further in Chapter 5.
The next chapters will look at lighting techniques, including realistic ambient occlusion effects to help improve the perception of depth in the scene, and ray traced soft shadows.
Chapter 4
Ambient Occlusion for Molecules in Space-Filling Representation
4.1 Introduction
In graphics, it is often difficult to appreciate the 3D form of a scene from a 2D image. In our HaptiMOL iSAS software especially, it becomes increasingly necessary when visualising molecules with many thousands of atoms to ensure the user has a good visual understanding of the structure. This will benefit researchers looking at how the geometry of the molecule relates to its function, and also aid users of our software to spot pockets and channels the probe can access. To aid in the depth perception of a molecule, advanced lighting and stereoscopic techniques are sometimes employed in molecular visualisation software. These techniques, now available due to the advancements by modern graphics algorithms and hardware, are used to help achieve the simulation of depth that aid in helping biologists understand the 3D structure of proteins.
The ambient occlusion shading model is perhaps the most popular lighting technique to help the perception of the 3D scene. Experiments have shown that depth perception under uniform diffuse lighting conditions are superior to a direct lighting
model [LB00]. In this chapter, we will discuss a number of ambient occlusion implementations with the goal of improving the user’s perception of depth in our software.
4.2 Ray Traced Ambient Occlusion
The most simple, yet most expensive technique to calculate ambient occlusion is to cast rays from a point, and to test for an intersection with other geometry in the scene. After many hundreds of samples generated randomly over the point’s hemisphere, an accurate approximation of the ambient occlusion is produced. Figure 4.1 shows molecule 1CRN rendered using this method, using the ray tracer developed in the next chapter. With 100 rays cast per pixel, the noise pattern from the randomly cast rays is reduced to an acceptable level, however the image takes over 3 seconds to generate. We use cosine-weighted sampling, where samples are weighted by the cosine of the angle between the ray and the normal. This generates samples weighted more heavily towards the normal, and reduces noise for the same amount of computation. Of course, for a real-time application, this is still not a viable solution. We will investigate the possibility of reducing the number of samples, as well as other methods to try to reduce the rendering time to real-time rates.
4.2.1 Single Sample Ray Traced Ambient Occlusion
It is common for ambient occlusion methods such as SSAO to use a much lower number of samples per pixel, with an additional blurring pass taking into account depth discontinuities to remove the high-frequency noise produced as a consequence. This solution allows a reduction in the number of depth samples per pixel to around 16 while maintaining an acceptable quality.
However, 16 samples per pixel will still be expensive if ray traced. Figure 4.2 shows how a single ray traced sample can produce an ambient occlusion approximation,
Figure 4.1: Ray traced ambient occlusion using 100 random samples per pixel. The image took around 3 seconds to generate. This image will be used as a reference throughout this chapter.
albeit very noisy. Figure 4.2b is calculated at full screen resolution, whilst Figure 4.2c is calculated at half resolution. The performance of each of these can be seen in Table 4.1.
(a) 100 samples at full resolution. (b) 1 sample at full resolution. (c) 1 sample at half resolution.
Figure 4.2: Ray traced ambient occlusion of the 1CRN molecule, with varying samples and resolutions.
A smart blur filter can then be used to remove the noise, whilst retaining sharp edges. The blur pass gives the impression of using multiple samples for a fraction of the cost. A common smart blurring technique used today is a cross bilateral
Table 4.1: Ray traced ambient occlusion at full and half resolution.
| Name | Amount | Full Resolution (ms) | Half Resolution (ms) | Change (%) |
|------|--------|----------------------|----------------------|------------|
| 1CRN | 327 | 21.67 | 7.04 | -67.51 |
| 1BKS | 5231 | 47.28 | 15.51 | -67.19 |
| 3ETS | 9152 | 56.89 | 17.30 | -69.59 |
| 1JB0 | 24198 | 58.33 | 16.68 | -71.40 |
| 1AON | 58870 | 64.07 | 20.50 | -68.00 |
| 3IYN | 99595 | 71.90 | 23.64 | -67.12 |
filter. This is a depth dependant Gaussian blur that diffuses the ambient occlusion whilst avoiding blurring across edges. This is achieved by using lower weights for samples with a large difference in depth from the current pixel. Figure 4.3 shows the effectiveness of the blurring stage to remove noise from the image.
(a) 100 samples at full resolution. (b) 1 sample at full resolution with bilateral blur. (c) 1 sample at half resolution with bilateral blur.
Figure 4.3: Ray traced ambient occlusion of the 1CRN molecule, after applying a bilateral filter to remove the noise caused by the reduced number of samples.
The results of using a single ray traced ambient occlusion sample per pixel are promising. The more global ambient occlusion effect that ray tracing provides is desirable, especially to help gain an understanding of a large molecular structure. With a smart blurring stage, it is possible to produce smooth results from an original noisy source image.
There are notable issues with this method. Flickering can be seen caused by the randomly generated ray direction for each pixel. This is exaggerated when calculating the ambient occlusion at a lower resolution to the screen size. A working solution
is to ensure the blur radius is large, and to apply a secondary blur pass to further smooth the flickering effects. The consequence of the heavy blurring is local ambient occlusion detail is lost, and in Section 4.5 we look at ways to tackle this.
### 4.3 Screen Space Ambient Occlusion
Screen space ambient occlusion (SSAO) is a rendering method to approximate ambient occlusion in real time. It has become the standard approach for computing ambient occlusion in real time applications such as video games. First introduced by Mitrtring [Mit07], the technique uses the depth buffer as an approximation of the geometry in the scene. The occlusion factor is then calculated in screen space, meaning the process can be done entirely on the GPU. The process is dynamic, requires no pre-computation, and is independent of scene complexity. These features are desirable for our HaptiMOL iSAS software, where molecules can contain many thousands of individual atoms.
Whilst calculated completely on the GPU, screen space ambient occlusion is still a relatively expensive process. It is common to see SSAO calculated at half screen resolution, and with perhaps only 16 samples per pixel. A depth aware blur such as a cross bilateral filter will then be used to smooth the image before using it in lighting calculations. Figure 4.4 shows SSAO calculated at different resolutions after a blur pass has been performed.
Compared with other ambient occlusion solutions, SSAO has a set of features that make it desirable for molecular graphics software. The independence from scene complexity makes it ideal for molecular structures of all sizes. The fully dynamic nature makes it excellent for scenes with moving objects such as the probe sphere in our software. It is also easily integrated into a modern graphics pipeline.
The limitation of SSAO, however, is the distances at which the method produces
results that can be interpreted as occlusion. An extreme case can be seen in the differences between Figures 4.10 and 4.12, where atoms are too far away for the SSAO technique to capture, giving a false impression of the structure of the molecule. Increasing the sampling radius to try and capture a larger area of objects decreases performance significantly, and leads to fine ambient occlusion detail being lost. SSAO is a useful technique for solving occlusion at relatively small distances, meaning another solution needs to be found if occlusion at higher distances is desired.
4.4 Analytically Calculating the Ambient Occlusion of Spheres
The ambient occlusion equation is most easily approximated by sending out test rays from a point on a surface of an object, and finding if the rays intersect with other objects in the scene. The ambient occlusion value will then be dependant on how far away the objects are, and how many intersections are found. To produce sufficiently accurate results using this method, many hundreds of rays need to be tested per pixel, which rules out this method for real time applications.
Work by Iñigo Quílez [Qui06] shows how it is possible to analytically calculate the occlusion of a sphere to a point on an arbitrary surface. This is especially useful for our iSAS software, which uses spheres to represent atoms of the molecule. We will
therefore investigate the possibility of using this method for a space-filling molecular representation, with the goal to create high quality and dynamic occlusion effects.
The equation below describes the blocking factor for a sphere with radius $r$ that is at a distance $d$ from this point. The blocking factor, $bl(r,d)$, is the area that the sphere projects into the hemisphere divided by the area of the hemisphere.
\begin{figure}[h]
\centering
\includegraphics[width=0.5\textwidth]{sphere_ambient_occlusion.png}
\caption{The ambient occlusion of a sphere to a point on a surface can be calculated knowing the radius of the sphere and the distance from the sphere’s center to the surface.}
\end{figure}
\begin{equation}
bl(r,d) = 1 - \sqrt{1 - (r/d)^2}
\end{equation} \hspace{1cm} (4.4.1)
The expression is simply a function of $r/d$. This is logical, as making a sphere bigger means it will have to move further to have the same projected shadow or occlusion factor. A sphere $x$ times bigger will project the same shadow if it is $x$ times further away. The effect can be seen in Figure 4.6.
4.4.1 Implementation Decisions
To integrate this technique into our software, for each atom, we store the atoms nearest to it. As the atoms in the molecule do not need to move, we can calculate an atom’s nearest neighbours in an initialisation stage as the molecule is loaded. The sphere IDs of these neighbouring atoms are then stored in a texture to be accessed in the lighting stage of our graphics pipeline. These IDs point to a second pre-calculated texture that stores the position and radius of each atom. A final texture is used to store a flag for each atom indicating if the atom has been culled by a clipping plane. This will ensure the lighting is dynamic as atoms are removed from view. The occlusion of the movable probe sphere is calculated on all nearby pixels, in a similar fashion to a point light detailed in the next chapter.
Table 4.2: AO data layout.
| Red | Green | Blue | Alpha | Texture Format |
|-----|-------|------|-------|----------------|
| Culled Flag | | | | GL_R8 |
| Position | Radius | | | GL_RGBA32F |
| Atom Occluders | | | | GL_R32F |
The amount of occluding atoms to store for each atom is based both on performance and storage limitations. OpenGL 2.1 API implementations require as a minimum a 2048x2048 texture size to be supported. Based on this minimum size, we can store up to 32 occluding atoms per atom whilst still supporting molecules well over 100,000 atoms in size. Table 4.3 shows the performance of this ambient occlusion method with a varying number of occluding atoms and over a range of molecules.
Table 4.3: Analytical ambient occlusion with varying number of occluding atoms.
| Name | Amount | 8 atoms (ms) | 16 atoms (ms) | 32 atoms (ms) |
|------|--------|--------------|---------------|---------------|
| 1CRN | 327 | 2.41 | 4.19 | 6.66 |
| 1BKS | 5231 | 2.44 | 4.31 | 7.02 |
| 3ETS | 9152 | 2.72 | 4.83 | 7.61 |
| 1JB0 | 24198 | 2.74 | 4.90 | 7.97 |
| 1AON | 58870 | 2.48 | 4.41 | 7.68 |
| 3IYN | 99595 | 2.39 | 4.36 | 6.58 |
(a) 100 samples at full resolution. (b) Analytical AO using 8 nearest spheres. (c) Analytical AO using 32 nearest spheres.
Figure 4.7: Analytical ambient occlusion of the 1CRN molecule.
Analytically calculating the ambient occlusion of spheres has many appeals. The formula to calculate the occlusion factor is very simple, and performs well at a per pixel basis. The technique produces occlusion with no artifacts, and therefore fits well with our pixel perfect spheres from the previous chapter. This also means no additional blur stage is required. However, this method does require additional pre-processing and memory overhead, although a lower occluder count could be used to
tackle this if needed. Due to the occluder limit, this technique is only useful for occlusion in localised areas as it is unable to capture a more global occlusion value.
4.5 Hybrid Ambient Occlusion
There has been interesting work investigating hybrid ambient occlusion techniques, such as Shanmugam and Arikan [SA07]. They look at separating the ambient occlusion problem into detailed high-frequency occlusion, and distant low-frequency occlusion domains, demonstrating good results.
In the previous sections we have presented a number of techniques to calculate the ambient occlusion of molecular structures. They each have their own unique characteristics, and each have advantages and disadvantages over each other. Taking two of these methods and using them together in a hybrid solution could provide an ambient occlusion solution that no technique on their own could provide.
The single sample ray traced ambient occlusion seen in Section 4.2.1 calculates occlusion that is too far for other techniques to produce, but lacks fine detail as a result of heavy blurring. The analytical solution from Section 4.4 is ideal for calculating occlusion at smaller distances, but lacks a more global reach. Combining these two methods with a simple multiplication can produce results much closer to the reference ray traced image that either could on their own. Figure 4.8 illustrates this method.
(a) Analytical AO with 8 (b) 1 ray traced sample at (c) Multiplication of (a) occluding spheres. half resolution, with blur. and (b).
Figure 4.8: Combination of two ambient occlusion techniques.
4.6 Comparison of Ambient Occlusion Techniques
The previous sections have detailed a number of possible solutions to calculate ambient occlusion for space-filling molecules. Choosing the best solution for our HaptiMOL iSAS software will need to be based on a number of areas. Performance is the key factor for the software. The application needs to run in real time to allow the user to navigate the structure with a probe sphere. Aiding the depth perception is an important secondary factor to take into account. A good visual understanding on the molecule will be of great benefit to the user, helping them move the probe with more confidence and efficiency. Visual quality also needs to be factored in, with a smooth natural appearance important as to not distract the user.
Figure 4.9 shows the relative performances of each ambient occlusion method that has been implemented, and Table 4.4 shows their advantages and disadvantages. It is clear that both the SSAO and analytical methods have a significant performance advantage over the ray traced and hybrid solutions. The performance of the ray traced solutions is also affected by the number of atoms in the molecule. The SSAO and analytical methods are not affected by the molecule size. This predictable constant performance is very appealing for software that visualises molecules that vary by a large amount in size.
Table 4.4: A comparison of ambient occlusion techniques we have presented.
| Technique | Advantages | Disadvantages |
|--------------------|-------------------------------------------------|--------------------------------------|
| Ray Traced 100 samples | High accuracy, no noise | Not real time |
| Ray Traced 1 sample | Real time, captures global occlusion | Noisy, needs strong blurring |
| SSAO | Real time, constant rendering time | View dependant, local occlusion only |
| Analytical | Real time, high quality | Local occlusion only |
| Hybrid | Captures both near and far occlusion | Not realistic for real time use yet |
The choice between SSAO and the analytical technique comes down to visual quality, as performance wise they are similar, and both provide localised occlusion. SSAO requires calculation at half resolution, with a blurring pass to achieve an acceptable level of performance. The analytical solution produces a pixel perfect effect, with no artifacts or flickering due to randomised sampling methods. For our software the analytical method is the best solution, with constant fast performance, and high quality occlusion.
For users with access to higher end PCs, the hybrid solution may be a viable option, and could be considered for future updates in our software.
Figure 4.9: This graph shows the performance of calculating ambient occlusion using a number of techniques.
Figure 4.10: 100 samples at full resolution.
Figure 4.11: 1 sample at half resolution with blur.
Figure 4.12: SSAO at half resolution with blur.
Figure 4.13: Analytical technique with 32 occluding atoms.
Figure 4.14: Hybrid solution with ray tracing and analytical methods.
Figure 4.15: Hybrid solution with diffuse colours.
4.7 Deferred Rendering
In the previous chapter ray tracing was used to produce pixel perfect spheres onto billboards giving a large visual improvement to the scene. However, when this technique is combined with relatively complex lighting calculations, performance can be an issue. This is largely due to the need to set the fragment depth manually in the fragment shader. If the driver knows that the output fragment depth is the same as the one generated, it can perform a simple depth test before executing the fragment shader, known as an early depth test. This means that it can discard fragments before computing potentially complex fragment shaders. Indeed, most modern hardware has complicated early z culling hardware that can discard multiple fragments with a single test. However, as soon as we set the fragment depth manually, further along in the pipeline, these optimisations cannot be performed.
The lighting calculations performed per pixel on the molecules, whilst not inherently expensive on their own, can become very expensive to compute when combined with the large overdraw of pixels as a consequence of no early depth testing. A technique called deferred rendering can be employed in this case, to replace the traditional forward rendering approach.
Forward rendering is a straightforward approach where we apply a set of transformations on the vertices of every object in the vertex shader followed by a lighting calculation per pixel in the fragment shader. Since each pixel of every object gets only a single fragment shader invocation we have to provide the fragment shader with information on all light sources and take all of them into account when calculating the light effect per pixel. This is a simple approach but it has its downsides. If the scene is highly complex with many objects and a large depth complexity (same screen pixel covered by several objects) we get a lot of wasted GPU cycles.
Deferred rendering is a popular technique which targets the specific problem above.
The key point behind deferred rendering is the decoupling of the geometry calculations and the lighting calculations. Instead of taking each object from the vertex buffer into its final resting place in the framebuffer, we separate the processing into two major passes. In the first pass we run the usual vertex shader, but instead of sending the processed attributes (such as positions, normals, colours) into the fragment shader for lighting calculations, we forward them into what is known as the Geometry Buffer or G-Buffer. This is a logical grouping of several 2D textures that are written to all at once using a capability of OpenGL called Multiple Render Targets (MRT). Since we are writing the attributes in the fragment shader, the values that end up in the G-Buffer are the result of the interpolation performed by the rasterizer on the vertex attributes. This stage is called the Geometry Pass. Every object is processed in this pass, and because of the depth test, when the geometry pass is complete the textures in the G-Buffer are populated by the interpolated attributes of the closest pixels to the camera. This means that all the irrelevant pixels that have failed the depth test have been dropped and what is left in the G-Buffer are only the pixels for which lighting must be calculated. This will solve the issue of overdraw that is caused when rendering our geometry.
Using deferred rendering over the traditional forward rendering provides other benefits. Lighting as a 2D post process means we can apply potentially thousands of lights efficiently, combining different types such as point and spot, and mixing shadowed and unshadowed lights, without many different combinations in of lighting shaders as previously required. It also allows us to spatially optimise lighting and complex shading, meaning we only apply light to the pixels which are actually in the light’s area of effect. The information stored in the G-Buffer can also be used for other post processing effects, that may also be useful to improve the viewers’ perception of the 3D structure of the molecule.
Anti-aliasing is an important consideration for graphics applications. Aliasing occurs as monitors display an image in the form of uniformly coloured pixels, with geometry in the scene not aligned to these pixels appearing jagged. FXAA has become one of the most popular techniques to combat aliasing. Created by Timothy Lottes [Lot11], FXAA runs as a single full screen pixel shader post process, that can be easily be integrated into the deferred rendering pipeline. On our development system, this shader takes 1.5 milliseconds to run on a 1280x720 image at default settings. This is a very acceptable cost for the excellent edge smoothness it produces. The only downside to this technique in the form of a slight loss of detail in textures will not effect our software.
There are notable downsides to deferred rendering that need to be discussed. Deferred rendering has an inherent overhead in terms of memory use, due to the storage requirements of the G-Buffer. It also has potentially reduced material flexibility. Unlike in forward rendering, it is not possible to change the shader which computes lighting to make changes to one material, as we have to do everything en-masse in a 2D post process. Another problem is that any transparent objects in the scene cannot be handled in the deferred renderer. These objects need to be dealt with in a second, forward rendered pass meaning we still need a working forward renderer.
In our case however for the HaptiMOL software, the disadvantages were not enough of a concern to outweigh the vast benefits deferred rendering provides. There is little to no use of the alpha channel in the application. The material properties are very simple for the molecules, meaning we are not disadvantaged from a loss of material flexibility. Finally, the concern about memory use is still valid, but with careful decisions on what data we store in the G-Buffer, as well as texture formats for the render targets, memory use can be kept to a minimum. Table 4.5 breaks down the G-Buffer layout of our pipeline, with the total GPU memory usage of the G-Buffer
at around 25MB at 1280x720 resolution.
Table 4.5: G-Buffer data layout.
| Red | Green | Blue | Alpha | Texture Format |
|-------|-------|-------|-------|----------------|
| Diffuse Colour | | | | GL_RGBA8 |
| Position | Atom ID | | | GL_RGBA32F |
| Normal | Material ID | | | GL_RGBA16F |
4.8 Conclusion
In this chapter we hope to have shown how ambient occlusion can greatly improve visual depth perception of molecules, aiding the user to navigate the structure with more efficiency. We have explored a variety of methods all able to run in a real time environment.
Analytically calculating the ambient occlusion of spheres provides the best overall solution for our software. We show how this technique can perform similar to other modern ambient occlusion techniques such as Screen Space Ambient Occlusion, and compares favourably when comparing visual quality, benefiting from the lack of blurring required to produce a smooth outcome. Ray traced results are shown to be promising, and will become a viable solution as users of our software adopt powerful modern graphics cards.
Ambient occlusion works well as a basic approximation of indirect illumination in this application. However the effect is lost when direct lights are added to the scene without shadows. Producing shadows for our molecular visualisation software will be the focus of the next chapter.
Figure 4.16: The scene is rendered into a G-Buffer, storing material properties (left top), positions (left middle) and normals (left bottom) of the scene. The information in these textures is then used to produce the final image, seen on the right.
Chapter 5
Real Time Shadows for Molecules in Space-Filling Representation
5.1 Introduction
Shadows are vital for the human perception of a 3D environment. Research such as work from Mamassian et al. [MKK98] and Wanger et al. [WFG92] explore, through psychophysical experiments, the role shadows play in helping understand an object’s relative position and size in a graphical scene. Work by Kersten et al. [KMK94] shows how the movement of the shadows play an important role in producing apparent motion in depth. Shadows can also reveal geometry hidden from view, or geometry that is outside the field of view. The ability to achieve accurate, dynamic and real time shadows in our HaptiMOL iSAS software would greatly enhance the users’ ability to navigate a molecular structure.
However, reproducing realistic shadows in a real time graphics application has been and remains a difficult task. In Section 3.3 we described a technique to produce ray cast spheres using camera aligned billboards. In this chapter we will look to continue exploring the ray tracing theme to produce an elegant solution for dynamic shadows, whilst still maintaining an interactive frame rate.
5.2 GPU Ray Tracing with GLSL
Ray tracing offers a much more elegant and accurate solution for our problem domain compared to other popular shadow techniques such as shadow mapping. A scene needs to be rendered once per directional light source, and six times for each omnidirectional light source using shadow mapping. Using ray tracing, shadows are a simple extension to the scene, especially for simple geometry such as spheres. A shadow ray is used to test if a surface is visible to a light. When a primary ray hits a surface, a ray is traced between this intersection point and the light. If any opaque object is found in between the surface and the light, the surface is in shadow and so the light does not contribute to its shade.
Ray tracing is generally an expensive process due to the high number of intersection tests per frame. A brute force approach to ray tracing is to test each ray with all objects in the scene. This becomes infeasible to compute in real time as the number of objects in the scene increases. The primary approach to accelerate ray tracing is to reduce the total number of intersection tests. This usually involves traversing some form of spatial data structure to find objects nearby the ray.
Currently the HaptiMOL iSAS software uses OpenGL and OpenGL Shading Language (GLSL) for rendering a molecular structure with Phong shading and no shadows. GLSL is a high-level shading language created to give developers more direct control of the graphics pipeline at the vertex and fragment level. It is desirable to carry on using these technologies for ease of integration, as well as compatibility with older hardware. Shader Model 3.0 introduced the ability to perform GPGPU in the shaders, and work by Purcell et al. [PBMH02] and Christen [Chr05] have shown how it is possible to perform ray tracing techniques on the GPU with shaders, using a uniform grid to reduce intersection tests.
The uniform grid is one of the easier acceleration structures to implement on a
GPU. First introduced by Fujimoto and Iwata [FI85], the grid evenly divides the scene into same size cells, with each cell containing references to all objects that intersect it. The structure works best when the grid cells closely match all the objects in size. This can be achieved in our case as the dimensions of the atoms in a molecule vary only by a relatively small amount. Atoms sizes defined by Bondi [Bon64] range from Hydrogen with an atomic radius of 1.2 angstroms, to potassium with a radius of 2.75 angstroms. The structure also maps well to GPU memory by using textures as storage, and the algorithm to traverse the structure is simple enough to implement in a pixel shader, meaning it is still a popular choice of acceleration structure on the GPU. For these reasons it makes a good choice of acceleration structure for our software.
### 5.2.1 Grid Creation
The creation of the uniform grid will need to be performed each time a user loads a molecule into the program. The creation needs to be fast enough to ensure there is minimal delay between loading and the molecule being rendered. The method of the uniform grid’s creation is shown below. For each Sphere $S_i$:
1. Find the grid cell containing the center of Sphere, $S_i$
2. Perform collision tests between Sphere, $S_i$, and all the cells directly next to the initial cell from step 1, with cells represented by Axis Aligned Bounding Boxes (AABB)
3. Add a reference to Sphere, $S_i$, in the grid texture for the initial cell, as well as cells where Sphere-AABB test returns true
One of the most important choices for the uniform grid is the cell size. It is generally the case that the cell size will be large enough to contain the largest object.
This ensures the number of cells an object overlaps is no more than eight and limits the cell intersection tests, important for the initialisation speed. However, performance during the grid traversal stage is of most importance, as this needs to happen in real time. The decision of cell size is further discussed in Section 5.2.4.
### 5.2.2 Grid Traversal
A simple way to traverse a uniform grid was first introduced by Amanatides and Woo [AW87], using a 3D-DDA algorithm. This algorithm only requires a small amount of operations to guide the ray between neighbouring cells with the cost of traversal being linear to the number of cells visited. Another feature of the algorithm is a valid hit will occlude hits in the following cells as voxels are visited in order of the ray passing through the grid, seen in Figure 5.1.
### 5.2.3 Grid Storage
To access the uniform grid structure as well as the geometry data in the pixel shader, the data is mapped to texture memory. Previous work on GPU ray tracing from Purcell et al. [PBMH02], Christen [Chr05] and Nguyen [Ngu07], use a method where each cell in the grid index structure contains a pointer to a grid cell list. The grid cell list contains atom IDs that point to the actual geometry data. This method can potentially handle molecules with hundreds of thousands of atoms in size, which will ensure support for a large proportion of molecules currently recorded.
As these data textures are generated on CPU, the current implementation is limited to rendering static scenes only. This is acceptable in our software as the atoms in a molecule do not move. However, clipping planes can be placed in the scene stopping atoms behind the plane from being rendered. Solutions to ensure dynamic lighting effects for the molecule are explored in Section 5.4.
Figure 5.1: A representation of the 3D-DDA algorithm. The green line represents the ray, and the highlighted blue cells are cells the ray intersects.
Figure 5.2: This is the structure of the data that is mapped to texture memory. Based on a texture size of 2048x2048 pixels, this structure ensures we can perform ray tracing calculations on molecules hundreds of thousands of atoms in size.
Algorithm 1 Shadow calculation algorithm in light pass
1: for all pixels containing geometry do
2: calculate $N \cdot L$
3: if $N \cdot L > 0$ then
4: if Sphere ID > 0 then
5: calculate grid index of ray origin
6: if grid index is outside grid then
7: project ray origin onto grid
8: end if
9: while inside grid do
10: for all spheres in grid cell do
11: if ray intersects sphere then
12: pixel is in shadow
13: end if
14: end for
15: end while
16: end if
17: end if
18: end for
5.2.4 Performance
Choosing a suitable cell size for the uniform grid is vital to achieve real time performance for shadow calculations. A smaller cell size should help reduce the number of intersection tests needed per cell. A larger cell size will help reduce memory required to store the grid data, as the number of empty grid cells, as well as the likelihood of spheres being in multiple cells is reduced. If memory usage is a concern, the grid cell size can be increased at the cost of a likely decrease in performance.
Figure 5.3 compares the performance of a variety of grid cell sizes for a test molecule, from a single cell brute force approach, to cells that are the smallest possible size, meaning they are able to fully contain the largest atom in the molecule.
The most common molecules explored using HaptiMOL generally contain thousands or tens of thousands of atoms. GroEL, containing nearly sixty thousand atoms, is considered a large molecule. By default we use the minimum cell size available, as
for the vast majority molecules we have the memory available to achieve maximum performance. This size is a good starting point to reduce memory requirements, as atoms can be in no more than eight cells in the worst case. However, as we can see, there is room for more memory savings if required.
In Figure 5.3, we can see for low levels of subdivision, the shadow calculations are very expensive. However, with only a small increase in subdivision the performance can increase dramatically. These results closely resemble those of Amanatides and Woo [AW87], although, with the minimum cell size set to contain the largest atom in the molecule, we do not see the reduced performance of having too many grid cells they encounter.
The most notable feature of the performance results is that we see from roughly halfway between the minimum and maximum amount of cells, the performance increase becomes negligible. For 1CRN, the time to calculate shadows with 567 grid cells takes 12.60 milliseconds, whilst for 294 grid cells, this time increases only slightly to 14.32 milliseconds. Almost halving the number of cells with less than a 2 millisecond drop in performance is significant, especially if this trend is repeated for molecules with tens of thousands of grid cells. For very large molecules or molecules with unique structures that make the uniform grid inefficient, with lots of empty cells, finding this optimal zone between memory usage and performance is appealing, allowing smaller textures to store the grid to be used.
To help investigate why the performance gains drop off so dramatically, Figure 5.4 shows the relationship between the number of grid cells and the average number of atoms per cell for 1CRN.
We can see a nice correlation between Figures 5.3 and 5.4. This shows a direct link in performance and the number of atoms per cell. Traversing the grid is relatively cheap, whilst the intersection tests needed per cell is where we incur the most cost. What we also learn is that increasing the number of cells beyond a certain point does little to reduce the average number of atoms per cell, which explains the reduced performance gains for higher grid densities. For this particular molecule, a cell size of 50 seems to produce the best performance to memory ratio. It would be desirable to know if these findings can be used for other molecules of varying sizes.
Looking at the performance results of this new selection of molecules, we see a similar pattern as before. A cell size in the region of 50 seems to produce the best memory to performance ratio. It is important to note, however, for larger molecules especially, the performance difference between the minimum cell size and our optimal suggestion is still significant when considering real time performance. With 1AON,
Figure 5.4: Here the relationship between the average number of atoms per cell and the number of grid cells is shown. There is clearly a link between the number of atoms per cell, and shadow calculation time. Cell sizes are shown above selected points.
for example, this difference in around 10 milliseconds, which is a large amount of time considering real time targets 33 milliseconds rendering time.
Our ultimate goal is to achieve real time shadows to improve the users’ perception of the scene. We have shown we can achieve real time rates for small and medium sizes molecules. However, for large molecules, ray traced shadows drop below these rates on our development system. In Chapter 3 we looked at rendering only a small portion of a molecule, clipped to a navigation cube to reduce draw calls and therefore rendering time. In Section 5.4, we will look at building on this technique to achieve real time and dynamic shadows on larger molecules such as GroEL.
Figure 5.5: Here we compare four molecules to look for similar patterns in performance in relation to the number of grid cells. Being able to choose an optimal cell size (indicated above selected points) when initialising the grid can lead to significant memory savings with a relatively small increase in shadow calculation time.
5.3 Aiding User Interaction with a Shadow Casting Probe Light
The HaptiMOL iSAS software includes a point light positioned at the center of the probe sphere. The attenuation of the light is set to illuminate atoms near to the probe, giving the user a better understanding of its position relative to other atoms in the molecule. Currently however, the light does not cast shadows, meaning areas of the molecule that are occluded from the light are still being illuminated. A shadow
casting probe light would give the user a better understanding of where the probe sphere is positioned relative to the molecular structure.
Creating shadows for a point light using the Shadow Mapping technique requires the scene to be rendered six times to cover areas the light can potentially illuminate. Ray tracing greatly simplifies the calculation of point light shadows, needing a single shadow ray per pixel. Algorithm 1 describes ray tracing for directional lights, where the ray needs to traverse the grid until it exits, as the light source is effectively outside the grid. For point lights, the algorithm can be modified slightly to reduce the number of voxels traversed. This is achieved by adding an extra condition to stop the traversal once the ray reaches the light’s grid cell, as we know everything after the light’s grid cell cannot occlude the pixel we are testing. Figure 5.6 illustrates this method.
Another small change that can be made is limiting the area of shadow calculation to pixels in the illuminated area of the point light. A pre-set cut-off is defined manually in the light pass shader to ensure expensive shadow calculations are performed only when necessary. An example of the reduced area of pixels to perform processing on is shown in Figure 5.7.
### 5.4 Dynamic Shadows for Navigation Cube Exploration
One of our aims for our software is to achieve fully dynamic shadows when atoms are culled, either by a clipping plane or by the navigation cube introduced in Chapter 3. This will aid in producing a better interactive experience, where lighting effects change in response to users’ actions.
In the previous chapter, we described a simple way to achieve a dynamic ambient occlusion effect by using an additional texture to store a culled flag for each atom. We can use the same technique here to ensure atoms that have been culled from view
Figure 5.6: This figure shows how the grid is traversed for a point light. Once the light’s grid cell has been reached, we can stop the traversal.
Figure 5.7: The red area highlighted on the molecule shows the pixels that the point light illuminates. Limiting shadow calculations to only these pixels ensures no unnecessary processing, therefore improving performance.
Figure 5.8: This image shows the probe sphere (dark blue) with a point light at its center. The attenuation of the light is set to illuminate atoms in the molecule that are in close proximity, giving the user a better sense of depth when navigating the molecular structure. However, without shadows, there are areas that receive light even though they are occluded by other atoms (highlighted by the arrows).
Figure 5.9: Here is the same image, but with shadows enabled. The position of the probe sphere relative to other atoms is made clearer, allowing the user to navigate the structure with more confidence.
can no longer cast shadows on atoms still visible in the scene. We can make a small but important change to Algorithm 1 to reduce unnecessary traversal of the shadow ray through the uniform grid. Figure 3.12 shows how we calculate the bounding cells for the navigation cube. We can send the minimum and maximum indexes of these cells to the light shader, and are then able to traverse the grid until we are outside of these bounding cells, instead of outside of the entire grid as was previously needed. Figure 5.10 illustrates this change.
The bounding cells of the navigation cube are an efficient way of culling large portions of a molecule, as traversal of a shadow ray can be stopped when it is outside of these cells. However, we still need to test for atoms that may be in the bounding cells but are not in the navigation cube. Table 5.1 shows the performance overhead of the dynamic shadows, with 6 milliseconds being the average on a variety of molecules. This decrease in performance comes from the need to check if an atom has been culled, meaning an additional texture read and branch in the light pass shader.
What can be seen is that we have an effective way to produce dynamic ray traced shadows on molecules with tens of thousands of atoms by reducing atoms rendered
Table 5.1: Here we compare the shadow calculation time of a small molecule, 1CRN, with a number of larger molecules clipped to the same amount of atoms using this method.
| Name | Amount | Shadow Calculation Time (ms) | Overhead (ms) |
|------|--------|------------------------------|---------------|
| 1CRN | 327 | 12.68 | 0.0 |
| 1BKS | 5231 | 18.77 | 6.09 |
| 3ETS | 9152 | 18.53 | 5.85 |
| 1JB0 | 24198 | 19.21 | 6.53 |
| 1AON | 58870 | 18.84 | 6.16 |
| 3IYN | 99595 | 19.23 | 6.55 |
to those in the navigation cube. Molecules that would be impossible to render in real time on our system, can now be explored at interactive rates. Reducing shadow calculation time also allows for the possibility of adding both a directional light source, as well as a point light source located at the center of the probe sphere, to the scene.
However, by limiting the atoms rendered to those inside the navigation cube, the user can not appreciate the full structure of the molecule. Our solution is to render the backbone structure of the molecule, using only simple lighting effects. The backbone structure illustrates the main chain of the structure which many biologists use when visualising a molecule. The main chain often adopts substructures called secondary structures which are mainly helices and sheets, and their arrangement in proteins makes their structure easy to identify. These can not be seen in space-filling models, as they are occluded by the dense packing of the atoms. Figure 5.11 shows how the backbone structure is rendered alongside the space-filling representation, with the complex lighting calculations only being performed on a much smaller scale. This helps boost performance whilst still allowing ray traced shadows and ambient occlusion effects for areas of the molecule the user is interacting with.
Figure 5.11: The backbone structure of the molecule is rendered to give the user a better understanding of the molecular structure. Space-filling atoms, and therefore complex lighting and shadow calculations, are limited to those atoms inside the navigation cube.
5.5 Soft Shadows for Space-Filling Molecules using Ray Tracing
One of the limitations of ray tracing is the hard edges it computes for shadows. This is because the light source is usually defined by a single point, meaning the surface is either fully lit or fully shadowed. In reality, a surface can have a partial view of a light, where only a fraction of the light source is visible from that point. Traditionally we need to cast multiple rays from these area light sources in order to model how they interact with the scene. This technique will produce an accurate soft shadow effect if enough rays are used, however this dramatically increases computation time.
Knowing we are only ray tracing with spheres provides the opportunity to calculate plausible soft shadows with little extra computation. Parker et al. [PSS98] introduced an algorithm that creates a shadow of a soft-edged object from a point light source, rather than creating a correct soft-edged shadow from an area light source. We can integrate this technique into our previous ray tracing work with minimal extra code, providing a simple way to create visually plausible soft shadows with little extra computation. Although these soft shadows are approximate, they are robust and have penumbra widths that behave in a believable way. The main benefit of this technique is that only one shadow ray is needed per pixel.
**Figure 5.12:** The inner sphere casts the umbra of the shadow. An outer sphere creates a larger shadow, with the area between the two spheres creating the penumbra.
As seen in Figure 5.12, the penumbra is the shadow of the semi-opaque (outer) object that is not also shadowed by the opaque (inner) object. The transparency
of the outer object increases from no transparency next to the inner object, to full transparency at the boundary of the outer object.
The intensity gradient of the penumbra needs to look natural. This can be achieved by computing a variable $s$ that begins on the surface of the inner object, and increases with distance to the surface of the outer object. Parker et al. suggest a linear gradient between the two boundaries, however a Hermite curve provided a smoother transition when tested.
There is also a need for the algorithm to remain simple and robust for multiple objects. For example, if a point is in the penumbra region for two objects, we can make a composite attenuation factor based on the individual factors $s1$ and $s2$. We can take the minimum value between $s1$ and $s2$, which will yield continuous intensity transitions and visually pleasing results for the shadow of multiple objects.
For a shadow ray $\mathbf{p} = \mathbf{o} + t\hat{\mathbf{v}}$ toward a light with position $\mathbf{l}$, and a sphere with center $\mathbf{c}$ and radius $R$, we need to decide whether we are in the penumbra region, and if so, what is the value of $s$, the fractional distance between umbra and penumbra boundaries. We first compute the distance $t_0$ to the point on the shadow ray closest to $\mathbf{c}$:
$$t_0 = (\mathbf{c} - \mathbf{o}) \cdot \hat{\mathbf{v}} \quad (5.5.1)$$
If $t_0$ is negative, then $s$ is set to 1, as there is no collision with the sphere. We then compute the value of the minimum distance $d$ from the center of the sphere $\mathbf{c}$ to the ray:
$$d = \|t_0\hat{\mathbf{v}} - \mathbf{c} + \mathbf{o}\| \quad (5.5.2)$$
If this distance is between $R$ and $Rb$ then we calculate $s$ as:
Figure 5.13: To produce the soft shadow effect, we surround our sphere with center $c$ and radius $R$, with a larger sphere with radius $Rb$. We then scale the shadow value based on the distance $d$ from the shadow ray to the center of the sphere.
\[ s = \frac{(d - R)}{b} \]
(5.5.3)
If $d$ is less than $R$, $s$ is set to 0 as the ray is colliding with the inner sphere. If $d$ is greater than $Rb$, $s$ is set to 1, as the ray is not colliding with either the inner or outer sphere. The value of $s$ is multiplied by the specular and diffuse terms to produce the soft shadow effect in the scene.
Table 5.2 compares the performance of this soft shadow technique with hard shadows. For small molecules with hundreds of atoms, we can see around a 20 percent increase in rendering time. This increases to around 30 percent for molecules with tens of thousands of atoms. This relatively small hit in performance is offset by the realistic and more natural shadows that help the spatial perception of the molecule.
Figure 5.14: Here is an example of traditional hard shadows, calculated by casting a single shadow ray towards a point light source. If the shadow ray collides with any spheres in the molecule, then both the diffuse and specular terms are set to zero, leaving just the ambient term to light the pixel.
Figure 5.15: Here the hard shadows have been replaced with the soft shadows using the technique described above. The smooth change between shadowed and unshadowed areas can be seen, aiding the spatial perception of the scene.
Figure 5.16: Here we see our new rendering techniques integrated into the HaptiMOL iSAS software. The user interacts with the molecule by controlling a probe sphere using a PHANTOM Omni haptic device.
Table 5.2: A comparison of the performance between hard and soft shadows.
| Name | Amount | Hard Shadows (ms) | Soft Shadows (ms) | Difference (%) |
|------|--------|-------------------|-------------------|----------------|
| 1CRN | 327 | 12.72 | 15.54 | 19.95 |
| 1BKS | 5231 | 26.68 | 33.77 | 23.45 |
| 3ETS | 9152 | 33.53 | 43.69 | 24.52 |
| 1JB0 | 24198 | 38.29 | 49.33 | 25.19 |
| 1AON | 58870 | 38.04 | 50.14 | 27.44 |
| 3IYN | 99595 | 35.93 | 47.14 | 26.98 |
5.6 Future Advances in the Rendering of Space-Filling Molecules using Path Tracing
Path tracing, proposed by Kajiya [Kaj86], is a computer graphics technique similar to ray tracing, in that rays are cast from a camera into the scene. Path tracing differs by generating light transport paths using a chain of rays. Rays are cast recursively along these paths to estimate the transported radiance. These rays are traced recursively until hitting a light source, with the rendering equation being evaluated along the path. There is no distinction made between illumination emitted from a light source and illumination reflected from a surface. The ray directions are governed by a bidirectional reflectance distribution function (BRDF) that defines how light is reflected at the surface of an object. Path tracing naturally simulates global illumination effects such as indirect lighting, soft shadows and caustics, that have to be specifically added to other rendering methods. Some of these effects are visible in Figure 5.17, including colour bleeding and reflections. When combined with physically accurate models of both objects and materials, path tracing can produce photorealistic images, and is commonly used to generate reference images for other rendering algorithms due to its unbiased nature.
To avoid artifacts caused by the random sampling of light paths, a large amount of samples are needed per pixel to produce a noise free image. Biased path tracers accept the loss of a small amount of accuracy in return for far fewer samples needed.
Figure 5.17: Here we see a molecule rendered using path tracing. The light path was allowed to bounce up to three times in the scene. Atoms were given different material properties, helping to quickly distinguish between element types. Reflections can be seen on the red oxygen atoms and blue nitrogen atoms.
per pixel to generate a noise free image. There are many techniques employed to help reduce the samples needed per pixel, and therefore rendering time of the image.
In unbiased path tracing, a path’s radiance is considered zero if it does not hit a light source. However, it is unlikely to hit a light source using randomly generated ray directions, especially if the light source is small. As lights in scene are likely to effect surface radiance the most, lights can be sampled directly at each intersection point along the path.
Rays in an unbiased path tracer are generated uniformly over a surface. However, it is far more efficient to generate rays in the directions the luminance would have been greater. Importance sampling is a technique commonly used to cast less rays while still converging to a correct luminance value. If the density of rays cast in certain directions matches the strength of contributions in those directions, the result
will be the same, but far fewer rays were cast.
In an unbiased path tracer, light paths bounce infinitely in the environment until a light source is hit, or they leave the scene. However, as every bounce has less energy, a light path that has bounced many times will have little impact of the surface radiance. Limiting the number of light path bounces, commonly to three or four per pixel, helps reduce rendering time dramatically with little impact on the final image. Figure 5.18 illustrates the basic principles of path tracing.
**Figure 5.18:** This diagram illustrated how the colour of a pixel is determined using path tracing. The light source is directly sampled at each intersection point. Indirect illumination is gathered along the light path, which is based on the material BRDF.
Recent advances in graphics hardware have started to allow straightforward and intuitive algorithms such as path tracing to be considered for real-time applications. Our ray tracer, detailed in Section 5.2, can be easily extended to allow for multiple bounces of light rays per pixel. The two main light sources in our scene, a directional light and a probe light, can be sampled directly when calculating the direct lighting.
Algorithm 2 describes the simple path tracing method used in our GLSL lighting shader.
**Algorithm 2 Path tracing algorithm**
1: **for all** pixels containing geometry **do**
2: **for** number of bounces **do**
3: get minimum intersection point
4: **if** no hit **then**
5: return total accumulated colour
6: **end if**
7: calculate direct lighting
8: colour accumulation *= current surface colour
9: total accumulated colour += colour accumulation * direct lighting
10: set ray origin to intersection point
11: set ray direction based on material BRDF
12: **end for**
13: pixel colour = mix(accumulated colour, previous frame’s colour, number of samples)
14: **end for**
We measured the rendering speeds of path tracing on a number of molecules, using our development machine from previous performance tests, as well as a desktop PC with a Nvidia GeForce GTX 580 graphics card. For our implementation, around 100 samples per pixel are needed to reduce noise to an acceptable level, however real time rates with this number of samples are clearly not yet achievable even when using a high end GPU. Progressive rendering, a method for incremental computation of the image, can allow for the scene to remain interactive, gathering additional samples as the camera stops moving. Even this can not be achieved on our development machine, taking 83 milliseconds to calculate one sample on a small molecule. However, using the GTX 580 we can achieve a minimum of one sample per pixel on all the molecules tested, while achieving at least 30 frames per second. This highlights the speed of progression seen in modern graphics cards.
Table 5.3: Path tracing performance with 100 samples per pixel, 2 light sources, and the light path allowed to bounce up to 3 times in the scene. Render times are in seconds.
| Name | Amount | ATI Mobility Radeon HD 5650 | GeForce GTX 580 |
|------|--------|-----------------------------|-----------------|
| ICRN | 327 | 8.33 | 1.53 |
| 1BKS | 5231 | 19.62 | 1.75 |
| 3ETS | 9152 | 24.77 | 2.13 |
| 1JB0 | 24198 | 26.85 | 2.34 |
| 1AON | 58870 | 31.89 | 2.86 |
5.7 Conclusion
In this chapter we hope to have shown how shadows play an important role in improving the visual depth perception of molecules, aiding the user in navigating the structure with more efficiency. Shadows were achieved using ray tracing techniques implemented on the GPU with GLSL, including a uniform grid acceleration structure to help achieve real time frame rates. Giving the probe sphere the ability to cast shadows onto the molecular structure allows the user to gain a better understanding to the position of the probe relative to other atoms. Clipping a molecule to the navigation cube plays an important role in exploring large molecules at an interactive frame rate. We have looked at methods to ensure shadow effects change dynamically in response to the user moving the cube, creating a more visually believable scene. Additional improvements to the shadows to give them a more natural soft feel were also incorporated with only a small increase in processing cost. We finally looked at path tracing as a future rendering method in molecular graphics, noting how graphics hardware is progressing towards real time rates for simple scenes using this method.
Chapter 6
Conclusions
6.1 Discussion and Conclusions
The results of this thesis have been to improve the rendering methods for the HaptiMOL iSAS software. The software enables users to interact with the solvent accessible surface of biomolecules, by probing the surface with a sphere. These rendering improvements provide a number of benefits to users when exploring and navigating a molecular structure.
Using a billboarded approach to render spheres that represent atoms in the molecule gave the most significant performance benefit to the software. With this technique, we can reduce the number of vertices that represent the atom down from hundreds in the original software, to just four using this method. Even with this technique, however, frame rates drop below real time rates on our development machine when rendering molecules above 50,000 atoms. The use of point sprites has been shown as a possible way to render a sphere to the screen with just a single vertex sent through the graphics pipeline, with significant performance gains.
There are other notable advantages to using this billboard technique over a traditional triangle mesh. A ray-sphere intersection test is performed per pixel onto the billboard, giving a noticeable visual benefit by ensuring a sphere’s representation is
accurate to the nearest pixel. This is desirable as the haptic feedback provided by the device will exactly match the visual representation on the screen.
The software features a clipping plane to aid the user in exploring large data sets. In its old form, the software supported a single clipping plane attached to the probe sphere, helping the user maintain a clear view of the probe, whilst aiding in finding pockets and channels in the structure that may have been hidden from view. Experimentation has shown how these planes can also be used to help reduce rendering time by reducing draw calls, especially for large molecules, where the system is limited by the number of vertices in the scene. Our improvements to the clipping plane implementation include allowing atoms to appear partially culled, giving the user a precise visual cue to the location of the plane in relation to the atoms. The clipping plane can also be placed at any angle chosen by the user, with additional clipping planes supported if needed.
While the performance and appearance of the molecule’s geometry is a very important aspect of the software, we were also interested in modernising the lighting techniques currently employed in the system. Ambient occlusion, although only an approximation of full global illumination, has been shown with great effect by Tarini et al. [TCM06] to enhance a user’s perception of a molecular structure.
Their chosen method was to pre-compute the ambient occlusion, storing the values in a texture map for each atom. However, our software allows a user to interact with a molecule, through moving a probe sphere, as well as removing areas of the molecule with clipping planes. We needed a ambient occlusion solution that could handle these dynamic scenes, to enable the user to receive real-time visual feedback of the lighting effects based on their input with the device.
We have spent time investigating a number of real-time solutions for calculating
ambient occlusion. Each of these methods has their own advantages and disadvantages, and our goal was to find a solution that would best fit our requirements. One choice was to use a popular technique called screen space ambient occlusion. This method is ideal for large scenes due to its independence from scene complexity, although calculating occlusion in screen space leads to local and view dependant results. We also investigated ray tracing as a tool to capture the occlusion. This method provides a better way to capture a more global occlusion effect, however it comes at a large performance cost. We looked at reducing the number of samples, whilst incorporating a blurring stage to reduce this cost to a more acceptable level. Although we do see a notable loss of localised detail due to the lower sample count and blurring filter. A more unique and specialised method we considered was to analytically calculate the ambient occlusion of spheres, demonstrated by Inigo Quilez [Qui06]. This proved to be a very effective way at producing high quality local ambient occlusion effects with relatively small performance costs, although unable to capture global occlusion effectively. We finally looked at using a combination of these methods, allowing us to take advantages from different techniques to produce an outcome that a single technique on their own could not achieve. A full comparison can be seen in Section 4.6, with Figure 4.9 giving a visual comparison of all the techniques.
Shadows are perhaps the most important of visual cues to perceive both depth and location of objects in a 3D scene. We have looked at using shadows to aid the user navigate the structure with more efficiency.
In Chapter 3, we looked at efficient methods to produce ray traced spheres using billboards. In Chapter 5, we looked at the possibility at continuing the ray tracing theme, to produce real time ray traced shadows on the GPU. We had to consider memory requirements, and an existing structure proposed by Purcell et al. [PBMH02] was chosen, meaning we could support molecules well over 100,000 atoms in size. We
also looked at acceleration structures that would map well to GLSL, with a uniform grid proving the easiest to implement. The results are promising, able to maintain real time rates on our development machine. There are limitations to using a uniform grid as an acceleration structure, namely a large amount of empty grid cells are produced as the cell sizes decrease. We therefore suggest a cell size that balances performance with memory requirements.
We also looked at adding shadows to another existing light source in the software, located at the center of the probe sphere, illuminating areas of the atoms in close proximity to the probe. Allowing this light source to produce shadows would give the user a better understanding to the position of the probe relative to other atoms. Techniques to optimise the shadow calculations of a point light were developed, such as performing calculations only on certain pixels, and reducing grid traversals.
A technique for calculating soft shadows using a single shadow ray was implemented, with a small impact on rendering performance. To help reduce shadow calculation times on larger molecules, we added an option to render only a small section of a molecule. This allows a user to still use these advanced lighting techniques on molecules that would otherwise not be rendered at interactive rates. We also added the option to render the backbone trace in areas outside the navigation cube, helping the user visualise the full structure.
Given current rates of GPU development, it will not be long before accurate global illumination techniques, such as path tracing, become possible to achieve in real time. We extended our ray tracing system to one that can bounce multiple rays per pixel. With this technique, we could achieve effects such as reflection and refraction, as well as colour bleeding. Atoms were given different material properties to help distinguish between them, as well as aid in depth perception. The rendering speed using a modern high end GPU proved to be vastly superior to the medium range laptop we
were developing on previously, taking a large step towards real time performance. However, a new generation of graphics cards will be required for interactive usage of this method.
Overall, we have investigated and implemented rendering methods that will benefit users of our software. The system built allows graphics effects to be tuned to the user's taste, as well as turned off completely. This will allow the software to run on a large variety of computers, both low and high end. The GLSL shaders are built dynamically, ensuring the minimal and optimal code is run each frame, reducing costly operations such as branching. The effects are high quality and real time, both goals set from the outset.
6.2 Future Work
Ambient occlusion and shadows are just two of many graphical effects that enhance depth perception. Whilst our approach was to reproduce a realistic visualisation of the molecule, we could have also included techniques such as depth of field. This is the distance between the nearest and farthest objects in a scene that appear acceptably sharp in an image. This could be used based on the location of the probe sphere, blurring objects a certain distance from the sphere, allowing the user to focus on just a small area of the molecule. Other post processing techniques that may prove useful could be depth cueing techniques such as depth aware contour lines or haloing effects proposed by Tarini et al. [TCM06].
Even using point sprites, large molecules become hard to render in real time on lower specification PCs. Occlusion culling could be looked at as a way to reduce the number of draw calls needed to render the structure.
HaptiMOL ENM is software which allows forces to be applied to atoms of an elastic
network model, using a haptic feedback device. It used a ball and stick representation to visualise the molecule. Our ray tracing techniques may be able to be implemented into with software, adding the ability to produce ray traced cylinders that represent the stick.
We have shown how path tracing can be applied to molecular graphics to provide global illumination in the scene. While we could not achieve a real time rate of 30 Hertz using this method, a combination of future hardware, as well as filtering and importance sampling techniques to reduce the number of samples required per pixel, will soon allow this method to be used in interactive molecular graphics programs.
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|
THE FAMILY OF
Hans and Margaret Zum (Bach) Biettschi
THE FAMILY OF
Hans and
Margaret Zum (Bach)
Biettschi
Compiled by Raymond and Darla Beachy
1626 - 2004
© 2004 Darla Kizer
All rights reserved. No portion of this book may be reproduced by any means, electronic or mechanical, including photocopying, recording, or by any information storage retrieval system, without written permission of the copyright's owner, except for the inclusion of brief quotations for a review.
For additional copies of this book contact:
Darla Kizer
4911 Old Oak Leaf Drive
Sarasota, FL 34233
941-925-4548
DEDICATION
To Raymond Noah Beachy,
his parents, and thirteen siblings,
who form the nucleus of a remarkable family.
Raymond Noah Beachy
December 16, 1928–March 7, 1996
PREFACE
For those who are acquainted with other family record books, this one will need a little explaining. The computer software program used to compile this record is the *World Family Tree Maker*, Version #11. It arranges the families much the same way that other record keeping systems do.
The families are numbered from 1 to 1697 at the beginning of each paragraph. The number assigned to the family member is followed by the person’s name. In (parenthesis) following the person’s name is the name of the immediate ancestor(s), or parent and his or her generation number.
Where there is no number assigned it is because the individual has no immediate descendants. This can be due to being a child in their parent’s home yet; single and not married yet, or married but having no children born to them yet.
There are fourteen generations listed beginning with Hans Biettschi in 1626 and ending with the youngest child Brian Zachary Beachy born on Sept. 12, 2002. The very last child born and listed is in the thirteenth generation.
Included with some of the notes is information regarding burial place, occupation, education, medical, marriage, residence, church, ordination for deacon, preacher, minister and bishop. Where there was background information that was included, also.
An index is provided giving the page or pages where the person is listed. It also includes a listing of the many multiple births recorded in this family. There is one set of triplet girls and one set of triplet boys born along with multiple sets of twins recorded. There are five families that have given birth to two sets of twins each. The first double set of twins born to a family is the Moses Beachy family, son of Peter Beachy II. Abraham and Sarah were born November 26, 1815 and the second set, David and Peter Beachy were born on May 5, 1823.
INTRODUCTION
This work is submitted to the public in good faith. What began as recording of genealogy of his immediate family has now become a major work that has taken ten years and much research to complete. Initially it was the vision and work of my husband, Raymond Noah Beachy (1928-1996). His vision began to take shape after a very successful second family reunion of his siblings.
He began by using the family information contained in two books on the Peter Beachy family—the *Memorial History of Peter Bitsche* by Samuel M. Peachy and *Descendants of Peter Beachy 1823-1971* by Mr. and Mrs. Henry J. Schrock and Freda Schrock. Some family background was documented from the book *The Beachy Family—Our American Roots* by William V. and Betty K. Beachy. All other family genealogy information was obtained directly from each family through correspondence or personal contact. The computer software used is *Family Tree Maker Version 9 and 11*.
Intensely interested in family Ray enjoyed looking up and visiting with family members, no matter where they lived. In our travels during the eight plus years of our marriage, he always seemed to know where some of his family members or former friends lived. Many times we would drive miles off of our chosen path to go, visit and spend time with them before continuing our journey. Always made richer because of those times it was never a burden, but always a joy, to share this part of life with him.
Those visits afforded the author the opportunity of learning to know cousins who otherwise would have been strangers and only names on a paper. It has allowed me to be able to piece the family members together more intelligently. This has been such a fun project and has fostered interest in the genealogy of my own parents and their families.
Ray was a great “story teller” and it was always interesting to hear his stories even after the third or fourth telling. Possessing a sharp and quick mind he could hold you spellbound as he related the details and events of the story. His sense of humor was always present at those times too. Sensitive to family and friends, I never heard him utter a negative statement about another person. If he could not say something positive about the person, he said nothing. He simply remained quiet. This seems to be a Beachy family trait and is a tribute to the loving parents who raised this remarkable family. Music is, and always has been, an important part of this family. Most possess natural musical talent and love to sing as a group. Many play instruments of various kinds.
After a few years of talking about having a family reunion the first one was organized by, and held at the church of the oldest living child, Eli Walter Beachy. Eli and his children, along with some of his church members, hosted the first event. It was held in the summer of 1988 at the Beachy Amish/Mennonite Church in Franklin, Kentucky, where Eli and his family live, and where he served as bishop.
Present were 165 immediate descendants of Noah Eli and Lydian (Miller) Beachy. They gathered at the church on a warm summer Friday afternoon. They came from Ohio, Florida, Pennsylvania, California, Kentucky, Virginia, Arizona, Kansas, Washington State, Alaska, and Canada. As family members began to leave on Sunday afternoon each looked forward in anticipation of the next reunion. The decision had been made to meet again in three years. A committee composed of three couples was selected to plan and host the next reunion. Since that time the reunions have continued to be held every three years at various places with different family members hosting the event. One has been held in Ohio, one in Florida, and two in Pennsylvania and this year it will be in Tennessee. It continues to be the highlight for family members as they each look forward to renewing family relationships each time. It has helped to strengthen family ties for the younger generations as well as the older family members.
The family was privileged to have the patriarch of the family, Noah Eli Beachy, present at the first reunion. He was father, grandfather, and great-grandfather and was eighty-nine years of age on that memorable occasion. The mother, Lydian, had died on July 7, 1982, at the age of eighty-one years, nine months, and two days, succumbing to congestive heart failure. Both are buried side by side in the King Church Cemetery located in Hartville, Stark County, Ohio.
Nine months prior to the second reunion Noah Eli Beachy died. His life ended on October 18, 1990, in Pennsylvania following abdominal surgery. He was ninety-one years, one month, and eight days of age and had been in good health just prior to this, while living with his daughter and son-in-law, Nancy and Willis Troyer. Present at the second reunion was the only surviving member of the E. N. Beachy family. He is Daniel E. Beachy who was born on September 1, 1915, in Bay Minette, Baldwin County, Alabama. At this printing, he still lives in Hartville, with his wife, Ella S. (Slabaugh) Beachy. On July 9, 2002, they celebrated their sixty-third wedding anniversary. As this book is printed he and Ella remain alert, active, and involved with the family.
In July 1991, the second reunion was held and attended by many of those present at the first one, plus some new additions to the family through births and marriages. It too was a joyous occasion as stories were shared of the past and new persons introduced to the family. Photo albums were shared so those living in distant places could feel the love, warmth, and heartbeat of this wonderful family.
Following this reunion Ray began to compile by hand, on genealogy sheets, the members of his family. He was interested in ancestors and descendants and was recording each generation as he remembered them and researched others. A visit to Leroy Beachy in Walnut Creek, Holmes County, Ohio, provided much background information on family members. We are deeply indebted to Leroy for his valuable assistance in providing information and background on the family members who had come from Europe.
In 1993, a computer was purchased and we began transferring the information onto it, using the *Family Tree Maker* software. Little did we know then how large the project would become. Today it includes more than 13,700 names.
Many feel a deep interest in knowing names, places of residence and occupations of their ancestors and descendants. That search for knowledge is the reason for this work to be completed and made available to anyone who desires to have it in his or her home. In the Bible we are commended to honor our parents, and it does not seem to be out of place to go a little further and honor and cherish the memory of our forefathers and friends still living today.
Many letters were written to collect the material for this family record and many hours were consumed in arranging the material correctly. Even with the greatest of care mistakes can hardly be avoided on account of the great many dates and numbers to be arranged. Often letters of information were poorly written with names misspelled or dates incorrect, etc. We ask for your indulgence and patience as you use the information. It is the desire of the author to extend heartfelt thanks to all that sent in the records and assisted in many ways.
The author trusts that this book, although not perfect, may be useful, helpful and interesting, to those who read it. We have attempted to include some interesting stories and events about some of the family members. Also included are bits of information on church membership, church ordinations, occupations and who performed the ceremony for couples united in marriage. Some burial, educational and medical information is included. Some medical notes are made available, where known, in order to assist future families in knowing what genetic disease exist in the families. Twins abound in this family tree, and we have listed one hundred twenty-five sets. Five families had two sets of twins born to each of them while one family had three sets of twins born to them. One family had triplet daughters born to them prematurely, all failed to survive. Another family gave birth to triplet boys, all of whom survived.
We are hopeful that this book will help to answer questions regarding kinship. We also hope that the younger generations will benefit from learning of their ancestors. An assignment in some schools is given for the students to research their family trees. We prayerfully present this to them, to assist them with their research and documentation.
—Darla Beachy Kizer, December 1, 2004
# TABLE OF CONTENTS
| Section | Page |
|--------------------------|------|
| Dedication | III |
| Preface | V |
| Introduction | VII |
| Family History | XI |
| Genealogy | 1 |
| Index | 615 |
|
COMPTOMETER
medley
A COPY of each edition is sent free to every operator on Felt & Tarrant's mailing list. The intention is to interest and encourage Comptometer Operators for the benefit of themselves and their employers.
May We Remind You—
the name "Comptometer" belongs to Felt & Tarrant and cannot rightly be used for any other make of adding and calculating machine. For many years, in fact, Felt & Tarrant's slogan has been
"If It's Not Made By Felt & Tarrant It's Not A Comptometer"
Reunion Dates
Since the end of the war, Felt & Tarrant have entertained over fifty thousand guests and now have pleasure to invite all Comptometer Operators to a further series of reunions. If you have not received details ten days before your date, as shown below please get in touch with your school.
| 1950 | 1951 |
|---------------|-----------------|
| Cardiff | Swansea |
| Liverpool | Nottingham |
| Birmingham | Hull |
| Bradford | London |
| Leeds | Coventry |
| Manchester | Middlesbrough |
| Edinburgh | Newcastle |
| Bristol | Sheffield |
| Glasgow | Belfast & Dublin |
Blazer Badges
So many operators have asked for blazer badges like we used to supply before the war that we have managed to obtain new stocks in a most attractive shield design. There are two qualities, one in gold and silver wire at 15/- and the other in a silk thread at 5/6. These are the exact cost prices. Badges can be inspected and purchased at your local school.
Lest We Forget
1. If you change your name or address, let us know so that you will receive future Medleys and reunion invitations.
2. If you can introduce girls to our schools you will receive a bonus of 10/6d. for every girl accepted.
3. If you want occasional work your local school will be glad to arrange it for you. Please get in touch with them.
Reunions
Right
“HERE’S HOWARD.”
That bright star of many radio shows, Michael Howard, makes a point at Birmingham.
(Photograph by Yeldham Unwin, Coventry.)
Left
PETER BROUGH has won well-deserved popularity on stage and radio. At Leeds and Bradford he introduced the not-so-dummy Archie Andrews to Yorkshire operators.
Below
NICK NISSEN, the “Fiddling Fool,” caused great hilarity among our Birmingham guests.
(Photograph by Yeldham Unwin, Coventry.)
Below
—from L to R—Michael Moore (“Ignorance is Bliss”) and Cyril Fletcher entertained us right merrily at Manchester. Alfred Swain, eminent B.B.C. baritone, sang at Birmingham, Manchester, Leeds and Bradford. Cyril Addison, on the right, was a splendid accompanist.
(Photograph by Manchester Weekly Newspapers.)
Your Old Friend
Bill Cargill
is invited to sample the ice-cream before it is distributed to Leeds and Bradford audiences.
World Archery Champion
MISS BARBARA WATERHOUSE, a twenty-one years old Operator employed by Accles & Pollock Ltd., Oldbury, Staffs., brought honour and fame to this country, and to all Comptometer Operators, by winning the Women's World Archery Championship in Paris last year. It was only after twenty-eight strenuous hours of archery, spread over four days, that Barbara won the title, and she achieved the further distinction of breaking eight previous world's records at the same time. Packed in her luggage when she arrived home were no less than twelve gold medals, three silver medals, one bronze medal, two silver cups, one silver casket and the inevitable nylons—two pairs of them.
Barbara’s fiancé is Jack Thompson, a twenty-one years old engineer also employed by Accles & Pollock Ltd. He is a keen archer himself and has spent a lot of spare time coaching Barbara to competition standard. No doubt Barbara’s training and experience as a Comptometer Operator also helped her agile fingers and quick eye to spot and correct any errors of trajectory or flight.
Accles & Pollock Ltd., famous for metal tubes of all descriptions, made the bows and arrows which Barbara used with such telling accuracy and encouraged her efforts at every stage.
Congratulations, Barbara, and more power to your elbow!
British Legion Queen
HIGHLIGHT of the special British Legion week at Aintree was the crowning of their Queen for 1949/50. The honour fell upon Miss Joyce Allen, a Comptometer Operator at Dunlop Rubber Co. Ltd., Walton, Liverpool. Joyce, who is eighteen and auburn-haired, wore a lovely white gown with a ballerina skirt of picot net decorated with silver sequins. The satin bodice had a square neckline. White elbow-length mitts, an attractive head-dress and a bouquet of pink roses completed as charming a picture as one could wish to see. The crowning ceremony took place before a big audience and was accompanied by the rousing music of bagpipes.
Queen Joyce and her escort of pipers
(Photograph by Louis Samson, Liverpool.)
ATTRACTIVE NEW BROOCH
THE Comptometer shield, showing the four arithmetical symbols, has been newly-designed as a neat brooch with strong clasp. You can see it at your local school and buy it for 1/6d. if you wish.
Reunions North
GLASGOW reunion committee courageously introduced some new features at St. Andrews Hall. There is little doubt that the fashion parade was popular and much credit is due to Lewis's Royal Polytechnic Co. by whose courtesy it was staged.
A display by the Scottish Country Dance Society, and an attempt to get all operators to join in the Eightsome Reel, were innovations that aroused interest and excitement but which were somewhat handicapped by lack of space. However, the buffet was available for those who did not feel like Highland Flinging.
Fashion Show at Glasgow.
Photo by Scottish Studios, Glasgow.
Edinburgh operators apparently were not thinking of country dancing. They had a nice cosy reunion with the Rae Mackintosh Orchestra to play for them and the B.B.C. singers, Ruth Morrison and John Jeffrey, to help the evening to pass pleasantly. O, and the usual interval for meeting old friends and making a lot of refreshments disappear quickly.
Buffet-time at Edinburgh.
Photo by Edinburgh Evening News
and Reunions West
THE introduction of novelty was also a feature of CARDIFF reunion. The programme included presentations by Cardiff Comptometer Dramatic Society which received well-earned approval and will no doubt lead to further efforts by these talented young people. Some good singing and an exhibition of ballroom dancing helped to make a memorable evening for Cardiff district operators.
A section of Cardiff Comptometer Dramatic Society. The lady with the pipe is Miss B. Dittmer, Principal of Cardiff Comptometer School.
Photo by Western Mail & Echo
Bristol
Even though the photographer did not turn up, Bristol operators certainly did, and there were several parties from outlying districts. Ken Lewis brought his orchestra and the programme also included Tommy Ware with his wiscracks, Harold H. Poole with his deciets and Leonard Nott with tenor songs. From what we saw of these West Country operators we think the camera missed a treat. No doubt they will give us pictorial evidence of this next time.
Swansea
OPERATORS here were delighted to have their own reunion again instead of journeying to Cardiff. They made themselves thoroughly at home at the Mackworth Hotel and renewed many old friendships in the intervals between music and refreshments. Phil Bacon and his Dance Orchestra played for them, Mat Evans ventrilquised, Mel Daniell dispensed light comedy and the art of "personality voice" was expounded by Wynn Thomas.
BOANAMARY has no shops, cinema, cafes, dance halls or anything like that. The nearest town is Majunga about thirty miles away. We make the journey in a camionnet—a large van made into a small bus—on an unsurfaced road."
So writes Mrs. Crawford, formerly Miss Joan Bellis, after two years in Madagascar where her husband works at a large meat-packing plant. Joan was recently on leave in her home town of Liverpool where, before her marriage, she was employed for some years by J. Russell & Co., as a Comptometer Operator. She left at Liverpool school in 1942. Life and climate in Madagascar are very different from England, for it is hot and wet from October to April and cool and dry from May to September. About twenty British and fifty French live in the little village of Boanamary, their houses being one-storey stone and wood buildings surrounded by verandahs.
"We never have fires," says Joan, "for it is always warm. Water in the tap is cold and only for washing. Drinking water is brought daily by the water-cart and has to be boiled, then bottled and put in the icebox or refrigerator. Cooking is done on a log fire. Native 'boys' do the shopping and cooking while 'ramatoa' (native women) do the washing and mind the babies."
Although the shops are thirty miles away, Joan says there is a village canteen for little odds and ends, and there is a golf-course and a club for dancing and other social gatherings. A quiet remote life for anyone who likes it.
If you are good at geography you will know that Madagascar is a large island about a thousand miles long. It lies in the Indian Ocean to the East of Africa.
ABOUT a thousand miles to the west of Madagascar is the town of Salisbury in Southern Rhodesia. Here also are "boys" to do the chores while the lady of the house goes to town.
One lady, at least, goes to town as a Comptometer Operator and when on leave recently she called at Glasgow School for some decimal cards to take back with her. Scottish operators knew her as Stella Burns. She was at the Scottish Milk Marketing Board until she joined the Army in the last war. During her service she met a Rhodesian and changed her name to Mrs. H. G. West and her residence to Rhodesia. Much to the delight of her parents, Stella was accompanied on her Glasgow visit by her three-year old daughter.
Stella likes the Rhodesian way of life. Salisbury has fine spacious streets recipe which she is taking back with her. She got it from friends in Yorkshire—and where else can you get real parkin?
The lady is Miss Verna Rutledge who is a Comptometer Operator with the Melbourne Herald, Victoria's only evening newspaper. She obtained twelve months' leave to visit England but likes it so much that she applied for an extension to next October.
Having visited France and Switzerland, Miss Rutledge spent a fortnight at an agricultural camp picking tomatoes and potatoes in Wiltshire. This was a memorable experience because of its opportunities to make friends and share opinions with people of this country. Miss Rutledge was amazed, and other Australian visitors have said the same, at the mistaken ideas about Australia. In most places their lighting, heating and water services are as good as ours. Women's fashions are always up-to-date and materials, imported from England, usually better than ours. The Melbourne shops are "marvellous" and prices about the same as here.
Miss Rutledge, who is keeping in touch with operating by pounding a Comptometer at Australia House in London, tells us her own favourite recreations in Australia are golf, surfing and all-the-year-round tennis.
We hope the memories she will take back will be as delectable as the parkin.
continued from previous page
THE GIRL FROM RHODESIA
and suburbs reaching about seven miles from the centre. The climate is delightful and the high altitude keeps the heat down in summer. What it is that keeps the income tax down we are not sure. We can only make you squirm a little by mentioning that a married couple with one child pay nothing on any salary up to £1,000. After that, the rate is only two shillings in the pound.
This seems to be as near paradise as any place you will find in the world today.
London Reunions
The two pictures on this page show the true spirit of reunion. Joy shared is joy doubled. There is so much to talk about when old friends meet. Betty has a new hairdo; Margaret is engaged to the boy she met at Eastbourne; Pam and her hubby have found a house at Wembley; Mary has a lovely baby boy. So it goes on all through the intervals—not only at Kingsway Hall, but at gatherings all over the British Isles. No wonder that guests look forward to their next "do." This great family spirit is one of the happy things in a troubled world.
Yes, London had ices for the first time—four thousand ices all alike—yet this girl insisted on sampling her friend's ration.
The programme this time was mainly comedy. How could it be otherwise with Dick Bentley and Cyril Fletcher right at the top of their form? There was also Peter Brough extracting a quieter kind of humour from that remarkably lifelike dummy, Archie Andrews. Our old friends Jack Leon and his orchestra again delighted us and introduced some clever solo turns, the trumpeter being unusually skilled. Rita Williams found time between her B.B.C. dates to sing for us and then encouraged us all to "put another penny in the nickelodeon" and to raise the roof with other popular items. Redvers Llewellyn, operatic baritone, was warmly applauded for songs on a rather higher plane.
You must see my snaps—souvenirs of happy holidays—wedding groups—all part of the pleasure of reunions.
Photos by London News Agency Photos Ltd.
Twelve Thousand Watched
You will agree that seventeen-year-old Miss Marjorie Giles, Comptometer Operator at Spillers Ltd., Liverpool, was well worth watching as she won the sixth heat of the "Miss New Brighton" competition. Her photograph on this page confirms the judges' decision.
There were eight weekly heats in this popular Merseyside contest and each of them brought some twelve thousand people to the New Brighton Pool.
Marjorie thinks August is an eventful month in her life. It was August, 1946, when she entered Liverpool Comptometer School. It was August of last year when she was acclaimed winner of the weekly contest and tumultuously applauded by the large crowds. It was still August when she appeared as one of the eight finalists in the Beauty Contest. And that really was a great day. As guests of the Wallasey Publicity and Entertainments Committee they were given a royal reception and dined later at the Grand Hotel, New Brighton, with Harry Lester and His Haysseeds, the Mayor of Wallasey and other notable people. They were filmed by Gaumont British and had numerous offers of contracts for personal appearances, advertising and modelling.
Marjorie, who is as modest as she is charming, told us that none of these offers had tempted her to forsake her career as a Comptometer Operator and her pastimes of skating and swimming.
Things they Do
PUBLIC SPEAKING
The above picture shows the Southern Region team being presented with a trophy in the finals of a Public Speaking contest at Butlin's Holiday Camp, Filey. The lady is Miss Anita Dribble, a former pupil in London Comptometer School, and now an operator at The Pyrene Ltd., Brentford. There was also a medal and a money prize for each member of the team.
PUBLIC SINGING
Miss Edna Andrews (above right) is an operator at the South Eastern Gas Board, London. Trained at the Blackheath Conservatoire of Music, Miss Andrews holds the Associated Board Certificates for singing and has performed at several concerts and social functions. Entering an amateur talent contest, she was chosen for a week's appearance at New Cross Empire and received second prize.
SISTERS TWO
Miss S. G. Chant of Ebby Vale sent this charming snap of herself and her sister Ruth who had just become Mrs. Williams. Miss Chant is an operator with Shell-Mex, Leeds, and Mrs. Williams with Richard Thomas & Baldwins, Ebby Vale.
MOTHER AND DAUGHTER
From Paisley, Scotland, came the picture below of a somewhat different operator relationship—Mrs. M. King and her daughter Miss Moira King.
SISTERS THREE
And there are no prizes for deciding which are the three sisters in the above merry group. The camera caught Mary, Rose and Greta Clements enjoying a joke with friends at the Glasgow reunion.
Photo by Scottish Studios.
TWINS—OF COURSE!
We never go to press without some twins to grace our pages. Muriel (left) and Barbara Dexter are operators at Halex Ltd., London.
Table Tennis
Mrs. Doreen Rae (left) last season won the North of England Open Championship and several Manchester and County trophies both for singles and team play. The latter included the rose bowl for the champion ladies' team of England. Mrs. Rae was trained at Manchester Comptometer School.
Photo by Manchester Weekly Newspapers.
Miss Doris Reavey (right), trained in Liverpool School, is an operator at Warrington Corporation. Last season she won the North Western District N.A.L.G.O tennis trophy which only once before, in 1934, had been won by a local competitor.
Photo by Warrington Guardian.
More Reunions
NOTTINGHAM (Top Right)
Sparti, the lightning cartoonist, enjoys a joke with some of the guests and no doubt finds more material for his clever drawings. It was a happy evening at the Elite Cinema Ballroom.
Photo by Yeldham Unwin
COVENTRY (Below)
All these girls made the journey from Rugby to join in the fun at the Civic Restaurant. They are operators at British Thomson Houston Co., Ltd.
SHEFFIELD (Below)
And Stan Hatton has a smile that is "all lit up" judging by the electric light bulb over his head. Lit-wit rather than nit-wit! The large gathering much appreciated the resumption of refreshments and we expect the Committee will go all out for a similar 'do' next time.
Photo by Sheffield Telegraph & Star
HULL (Below)
Some serious business gets "sandwiched" in between the music and entertainment. Judging by the smiles, there were no complaints.
Photo by Hull Daily Mail
It's On The Cards — We hope.
CAN you spot yourself in the above picture? It is a section of our London audience waving their bonus postcards. We are grateful to those operators who filled them up and returned them but we still want hundreds more. In case you have lost your card, we are sending you another. This applies not only to London but to all areas where the operator shortage is acute.
SO PLEASE—if there is a reply-paid postcard in this magazine, will you be good enough to help us. Lay your cards on the table, fill in your name and the name of some girl leaving school or in a job she does not like, and then pop the card in the post. No stamp is needed.
If the girl is enrolled as a pupil you will receive an award of 10/6. This applies however many girls you help us to enrol. We have now paid thousands of half-guineas but the demand for operators grows and grows as more Comptometers are sold and as girls leave business to take up domestic duties.
SO PLEASE FILL UP AND POST THOSE CARDS — AND DO IT NOW.
TRIBUTE TO OUR MUSIC MAKERS
BELOW is an excellent picture of the Blue Stars Orchestra who gave much pleasure to the reunion guests at LEICESTER. We cannot help thinking of the scores of similar orchestras up and down the country who have added so much enjoyment to operators' gatherings. There is not room for all their photos, but will they please accept this "honourable mention" as a grateful tribute to their performances.
More Good Companies
We have previously shown many reunion parties grouped by the firms they work for. We have covered several Counties, north, south, east and west. This time we have pleasure to show operators from Lancashire firms and to rejoice in the cheerfulness that has shone out from every picture in this series. There are some more groups on Pages 28 and 29 taken at Birmingham. Have YOU appeared yet?
Newcastle and Middlesbrough
The above operators deserve a space in our pages not only for their charm but for their cross-country coach journey all the way from Carlisle to Newcastle to join the northeastern reunion. This was held on two nights in April and enjoyed a similar programme to Middlesbrough operators who celebrated on the previous evening. There were large audiences everywhere.
Broadcast with Kenneth Horne
Known as "The Allegros" these three charming brunettes broadcast with Kenneth Horne in the "Spot the Winner" programme in January last. They must have been heard by many of their sister-operators, for they were "on the air" again later in a "Spot the Winner" request programme. Their songs were "Dry Bones," "Carolina in the Morning" and "Music, Music, Music." They have appeared in the Caroll Levis road show and are naturally in great demand at entertainments and dinners. You will see that Sylvia is also a prize-winner in our essay competition.
They are Comptometer Operators with D. Napier & Son, London, and have all studied music seriously. Sylvia does their song arrangements and plays the piano. They tell us that this "swing harmony" calls for more difficult and concentrated work than most people would imagine.
We are grateful to Alice for sending the photograph and story and are glad to know that this brilliant team particularly like singing to old folk and hospital patients.
Three Wallasey Sisters
On your right, reading downwards and also in order of age, are Eileen, Audrey and Brenda Gresham. These comely sisters were trained in Liverpool Comptometer School and live across the Mersey in Wallasey.
Eileen was trained ten years ago, and was followed about a year later by Audrey. That leaves seventeen-year-old Brenda as a comparative baby, but there was no doubt whatever about her business career. She took the course last year and is now happily launched on the seas of commerce in pursuit of her sisters.
This is another fine family record, and we are sure Eileen speaks for her sisters, and many thousands of sister-operators, when she writes:-
"As 1950 marks the end of a decade in my career as a Comptometer Operator, I should like to say in these pages a sincere 'Thank you' to the staff of the School who have been so kind and helpful, particularly during the blitzes when my family were bombed out and I was compelled to find a position in Chester to where I was evacuated. The School lost no time in providing me with a suitable position."
Operators' Baseball Team
Many good things come from America and the Felt & Tarrant Comptometer School at Cardiff think Baseball is one of them. Their team, shown below, was organised last year by Miss J. C. Sadler, a teacher at Cardiff School. They played several matches and have now been accepted into the South Wales Ladies' Baseball League. Their first captain was Miss Eva Smith and they are coached by Mr. E. G. Cottrell. Miss B. Dittmer, School Principal, is secretary. We hope their games will be as rousing as some we have seen in American films.
Bournemouth Regatta Queen
The slim and lovely lady opposite is a Comptometer Operator in charge of analysis work at Bournemouth Corporation Transport. Much of her spare time is spent on the beach at Honeycombe Chine, Boscombe, where she acquires that delightful tan that appeals to judges of feminine charm and beauty competitions.
Wearing a royal blue two-piece swimsuit, this twenty-two year old Miss Margaret Welch won the title of Bournemouth Regatta Queen of 1949. She thus achieved a childhood ambition, for just ten years earlier she was junior queen of Boscombe Carnival and thereupon decided that one day she would attain the higher honours that she now holds. Another distinction in 1949 was second place in the Carnival beauty competition.
The crowning was part of a big Regatta programme and Margaret's selection was cheered by thousands who had waited in the Pleasure Gardens for two hours while the judges made their choice. Among the crowd were many French students on holiday, and they yelled their approval when Monsieur Charles Antoni, Mayor of the Riviera town of Cannes, placed the crown on Margaret's head. In the excitement of the usual kiss of congratulation the crown fell to the ground. Monsieur Charles swiftly retrieved it and, with typical French gallantry, gave Margaret a second kiss—all for luck. The crowd roared their appreciation.
Margaret, whose hobbies are dancing, skating, fencing, riding and swimming, said later "This is the happiest moment of my life so far. I have been sun-bathing for weeks in preparation for the competition but never thought I stood a chance." To a further enquiry she replied "No, I'm not engaged or anything."
Her reward for winning the competition, apart from the honour of the title, was a Max Factor beauty case valued at ten guineas. Fellow-operators may like to know that Margaret has brown eyes, dark hair with natural curls and is 5ft. 6½ins. tall. Bust and hips are 34 inches and waist 23 inches.
Best of luck to you, Margaret, wherever you go!
Irish Loveliness
Throughout the green length and breadth of Ireland there are Comptometer Operators as fair as their own fair counties.
The lady on your left is Miss Catherine Mary Blackmore, winner of the Dawn Beauty Competition of Ireland in 1947. Catherine has been an operator with Clover Meats Ltd., Waterford, for six years. This is in Southern Ireland.
We are happy to receive evidence of Irish interest in this magazine and hope it will encourage many more entries from the Emerald Isle.
Margaret Welch after the Crowning Ceremony
Essay Competition
1. MY MOST EXCITING EXPERIENCE
Holidays abroad, however exciting to those taking them, often sound rather like travel brochures when recounted to friends later. Several essays came within this category and none was quite good enough. It was finally decided that the following two episodes are well written and strike the right note of excitement that should appeal to everyone. Three guineas has been awarded to each competitor.
A NOCTURNAL VISIT
By Margaret Bannister, Cardiff
FIVE interesting years in the W.A.A.F.s as a Radio Telephony Operator on Direction Finding Stations has left me with many incidents to look back on, but one particular episode comes to my mind whenever I remember those long night duties.
It was during the time that the enemy was at his most troublesome and rumours were rife of impending invasion. We D/F operators had been instructed on our line of action should German Para-troopers land near the radio tower. We were rather isolated from everyone else, the nearest help being at the camp three miles away across four or five fields. We were only two girls in charge of valuable secret radio equipment which could be destroyed rather than fall into German hands. Two hatchets were provided for this purpose.
Here, then, you have the scene. The rolling Yorkshire moors, the dark fields with sheep and cows dotted about, and in one field a small wooden tower with a blast wall around it, looking like a windmill without sails, and inside that tower, two little W.A.A.F.s busily making a cup of tea during a lull.
It had been a hectic couple of hours, and now that the crackle of the headphones had died down, the silence was something that could almost be felt.
And then—from outside came the unmistakable sound of footsteps on the concrete path between the blast wall and the tower. We stopped, looked questioningly at each other, and listened. Yes, there it was again—stealthy footsteps slowly making their way around the tower, and an occasional gusty sigh as if the intruder were carefully releasing his breath in an endeavour to make as little noise as possible.
The word "Paratroopers" flashed across my mind as I reached for the shiny hatchet, and my heart sank to my heavy W.A.A.F. shoes. One glance at my companion told me that she too had come to the same conclusion for she was grasping the other hatchet.
So with bated breath we waited. The intruder seemed to be waiting too, only a gentle rubbing on the wooden walls under the shuttered window indicating that he was still there.
At last I could bear the inactivity no longer. Summoning my courage I yelled "Who's there?" That brought results quickly enough.
All at once there was a violent commotion, a pair of scurrying feet, and the intruder found his voice in a loud "Baa-aa." Going to the exit, we heard our nocturnal visitor tearing away down the field as fast as his four spindly legs could carry him, uttering terrified bleats all the way.
We laughed shamefacedly, replaced our weapons, and soothed our shattered nerves with the much-needed cup of tea.
MEETING DANNY KAYE
By Sylvia Kerr, Hayes (London)
IT is always a thrill to meet one's favourite film or stage star, and it was certainly an achievement for my three Comptometer friends and myself when, through our hobby of modelling, we had the pleasure of presenting personally to Danny Kaye, in his dressing room at the London Palladium, a doll model of him that we had made completely ourselves, even to the clothes he was wearing.
We have all been great admirers of this comic genius from the time we saw him in his film "Up in Arms" to the time when, a few years later, he came to these shores and captured the hearts of all from our Royal Family to the working class with his charm and personality. After having the wonderful chance of seeing him perform in person, we all wished that we could find some way of expressing our appreciation of him. It was then that the idea of making a model was born. Plans were made and we all started work, my friends stitching the body, and myself on the actual clay modelling. I sat with a photograph as a guide and almost gave up in despair before the face really began to resemble him. The hands were also an important feature and I had to be careful with the moulding of them.
Gradually the model, twenty inches high, began to take shape. He was dressed in a perfectly tailored suit, white shirt, brown knitted tie, knitted socks and brown shoes. No detail was overlooked, even to the handkerchief in his breast pocket and the gold signet ring on his little finger.
Finally, I added the hair, which was a golden curly wig made from doll's hair. After weeks of work and concentration, and a terrific amount of fun, our model was complete. Now we faced the biggest problem. We were very anxious to deliver our gift personally, and it proved no easy task. The Palladium stage door was always closely guarded and we received no replies to numerous letters asking for an interview.
Towards the end of Danny's performances at the Palladium we made our last effort and, taking the doll with us, went to see the Manager. He was impressed enough with our story to send a message backstage asking if Danny Kaye would see us. It seemed like years waiting for a reply, and when we found ourselves being escorted to the great star's dressing-room we still couldn't quite believe it.
We soon felt at ease, and spent half-an-hour chatting with this friendly and surprisingly quiet American who has become one of the most admired artists of our generation. When we said goodbye, he shook hands and said he was very moved by our gesture and would take the doll back to Hollywood for his little daughter's bedroom. We felt that all our work had been worth while. It was a great thrill and an evening we shall never forget.
2. REUNIONS
Some essayists suggested competitions, dances and games, but these things are mostly impracticable owing to the large numbers involved and lack of space. For the same reason a buffet is often impossible. Almost every entry, however, emphasised the pleasure of meeting old friends. Some pointed out that newcomers may feel lonely at first, so we appeal here and now to all operators to chat with their neighbours, especially the youngsters, and forget formality. A prize of three guineas is awarded for the following essay because it expresses the true spirit of "Reunion" more clearly and concisely than other entries.
WHAT I THINK OF REUNIONS
By K. M. Bragg, Birmingham
I ENJOY the Reunions, not mainly because we have a good variety show or a free night out, but because we renew old acquaintances and recall many happy days. I especially enjoyed the 1949 one, and I have been to them all, as I met friends I worked with six or seven years ago that I had not seen since we worked together. It is grand to recall experiences with folks we worked with.
The main questions among the girls were "Where are you working now?" "Are you married yet?" and "Did you marry that fellow you were courting when I
worked with you?" Typical questions for a girls' natter session, but all part of the "happy family" spirit. It is something to realise that we are all working the same machines and are given the chance to catch up on past history.
In all my twelve years as an operator, I have never regretted learning the machine in spite of regular cries of "The Compts. will do it" and "Give it to the Compts." We are some of the most hard-worked people in offices, but the majority of us enjoy it.
I think in the stage show which is given us there is always some artiste who appeals to every one of us, thus making it all a thoroughly enjoyable evening.
3. THE MEDLEY
The Editor selected this subject in the hope of getting suggestions for the improvement of your magazine. Surprisingly few ideas have been sent. One or two competitors suggested beauty hints and knitting patterns. These would be printed if they seemed good enough, and were operators' own work. The main distinction of the Medley is that it is for, and by, Comptometer Operators. We do not go to experts for articles on special subjects; these are amply provided in regular publications. Nor do we favour fashion notes. Fashion changes so quickly that its rightful chroniclers are the daily and weekly press.
To the essayist who thought her locality was not sufficiently in the news, all we can say is "if you don't send it we can't print it."
For the above suggestions we thank the writers and hope they will regard these answers as satisfactory. The remaining essays were, we modestly think, too fulsome in their praise. We have selected one of the less effusive examples because it neatly associates the Medley with the average operator's chores and thoughts. It expresses what your Editor always has in mind, which is, to create added interest and zest for all Comptometer Operators by letting them know what goes on in their great world-wide family. A prize of three guineas has been sent to Mrs. Evans.
MY CANDID OPINION OF THE MEDLEY
By Olive Evans, Selsdon (London)
When I slipped my Comptometer Medley out of its envelope at breakfast the other morning, my husband enquired if it was another flower catalogue. He was disappointed, because we enjoy browsing through them together in the winter evenings, but to me it brought the same feeling as if I had been given a bouquet on a special occasion. I could not settle to the housework properly until I had read it through, and now I write to express my deep appreciation of it.
It is said "The best things in life are free." In these days of high cost of living, it warms the heart to receive such a beautifully produced magazine "With the compliments of Felt and Tarrant."
My first reaction on receiving the Medley is to scan the pages for photographs of operators I have personally known. You can imagine that my joy is heightened when, as with the last two copies, there was a photograph of twins who were at Aldwych House with me in 1940, and mention was made of one of their bridesmaids who sat next to me in School.
The standard of contributions, like the standard of operators, is high, and the sketches and photographs are excellent.
I shall have to wait years before I can visit Montana or Australia, but I got a real thrill out of reading the experiences of fellow-operators in these distant lands. I have not even been to a Reunion yet, but that does not lessen my pleasure of seeing the photos of the lucky ones who have. And oh! how I laughed over that delightfully odd ode. I shall hate parting with my copy of the Medley, but those verses are too good to be missed by my ballet-mane friends. And how versatile my fellow-operators are!—not only on their machines but in their hobbies. I am merely a good housewife—or so my husband says—but I take my hat off to those who have achieved success in so many spheres.
Now to end as I started, with flowers. A bumper bouquet to the Editor for a splendid Medley—a grand magazine that stimulates interest in fellow-operators everywhere.
Speed the next issue!
Is it cold down there?
Our only male competitor humorously recalls that he was "fortunate enough to spend fourteen blissful days surrounded by pretty faces and fascinating curls" at our London School. He thinks the Medley is "jolly good" but finds most of the photos too formal. For his special benefit, therefore, we have chosen the above and hope he will like it. Testing the ice are June Bushell (right) sitting pretty with her friend Joan Cheslim (left). Behind them is another friend, Betty Cockayne. June was trained last year at Birmingham Comptometer School.
Good News for Belfast and Dublin
Felt & Tarrant are happy to announce that they hope to arrange reunions in Dublin and Belfast in February, 1951.
We hope that all operators who possibly can will attend, even if they live some distance away. If you receive the Medley it means you are on the Mailing List and will get an invitation. If you know any operators who may not be on the Mailing List, would you be good enough to tell them to get in touch with Felt & Tarrant at once.
We send to all operators "across the water" our cordial greetings and hope to hear that there have been bumper gatherings and high spirits at their first post-war reunions. In addition to invitations, there will be announcements in local papers.
It Had to Come
FROM time to time we have put the spotlight on Comptometer relationships of several kinds—mothers and daughters, wives and husbands, aunts and nieces, cousins, sisters (including lots of twins) and no doubt many more will appear in these columns from time to time.
This page, however, is in honour of grandmothers who are still working as operators. We have tracked down some of these gallant ladies at two or three reunions and the audiences have paid worthy tribute to them as they stepped up for their vouchers. Now, for the first time, we are able to show a picture of a grandmother and two of her grandchildren. Mrs. L. Watkins, pictured below, lives at Hounslow and works for a well-known firm on the Great West Road. She has a third grandchild in Australia but has not seen her yet.
Mrs. Watkins learned the Comptometer in 1914 when her name was Miss Notman. In the first world war she was employed in a Command Paymaster’s Office. The Comptometer was then a comparative novelty and senior officers would crowd round to see how it worked. In the second world war Mrs. Watkins was in charge of all female staff in the cost office of Heston Aircraft.
Since her marriage in 1920, Mrs. Watkins has brought up four children, taught ballet dancing and sung at numerous concerts. We congratulate this evergreen operator on her full and interesting life so far, and hope she will realise her next ambition, which is to visit her eldest daughter and the grand-daughter in Australia.
A worthy Gallery of Honour portrait —
Gallery of Honour
THE distinction of being trained in Cardiff as the first Comptometer Operator for the Newport district seems to belong to Miss Dorothea Algar. That was thirty-four years ago, and Miss Algar recalls it in a letter which also tells us how much she enjoys reunions and these magazines, and of her happiness in being associated so long with the Felt & Tarrant “family.”
Miss Algar is now chief operator with Messrs. F. James (Newport) Ltd., and was recently presented with a clock on the completion of twenty-five years service with this Firm. We heartily wish her continued happiness and good health.
No “time” like this “present” for Miss D. Algar when tribute is paid to her long and excellent record
Photo by courtesy of South Wales Argus
She Flies Solo
CANADIAN born Mrs. Stanton was a Comptometer Operator in Montreal but has been working for some time in the Newcastle area where she lives with her husband and daughter, Patricia Anne.
In 1942, while still Miss Kelly, this lady made the headlines on both sides of the Atlantic as the first woman wireless operator appointed to R.A.F. Ferry Command. She also realised her ambition to fly solo and gained her pilot’s certificate. While stationed in Newfoundland she met her future husband and they were married in Montreal.
Mrs. Stanton misses her sailing, skiing and ice hockey, but speaks highly of our social services, concerts and theatres. She was hoping to fit in some weekend flying at Longbenton.
Mrs. Harriet Stanton, Newcastle.
HERE are four more pictures in our good "Company" series, but with the added interest that all these operators came to Birmingham reunion by coach. It is highly satisfactory to your reunion committee that many girls think it worth while to make a lengthy journey to spend a happy evening with their sister-operators.
Stanton Ironworks Co., Ltd., Stanton, Derbyshire.
Return Journey 100 miles
English Electric Co., Ltda., Stafford.
Return Journey 60 miles
Marston Excelsior (I.C.I.) Ltd., Wolverhampton and I.C.I. Ltd., Bilston, Staffs.
Return Journey 50 miles
These four photos by Yeldham Unwin, Coventry
In one coach from Wolverhampton, Staffs., came these operators of Wolverhampton Steel Co., Weldless Steel Tubes, Ltd., and H. M. Hobson, Ltd..
Return Journey 50 miles
From the Postbag
Liverpool.
"I would like to express my thanks for the copy of the "Comptometer Medley." This magazine is, in my opinion, one of the best I have read. Its homeliness and layout are most attractive.
This letter enables me to express appreciation of the excellent service and kindly cooperation extended to us by you personally and your mechanics, and I would also like to take this opportunity to place on record our pleasure in being so satisfied with the young ladies recommended from your organization. I am sure you will feel gratified to note that I cannot recollect a single case where we have been disappointed.
J. Bibby & Sons, Ltd."
Clifton, Manchester.
"My wife and I tender you and your staff our very sincere thanks for all your care and painstaking attention towards our daughter Muriel during her initial training at your school. This has enabled her to be now happily and comfortably situated in a post as a Junior Operator. We hope she will avail herself of the facilities to gain further knowledge of future advancement in the career now open to her by keeping in close touch with the School, and being always a credit to her teachers.
We shall never hesitate to recommend the course to any parents who may be interested.
I. & E. L. Anderton."
Operator: "Sometimes I wish Felt & Tarrant had found something better to do with their time"
"Basic" Sea Shanty
In wintertime the cold air blows
With fogs and winds and icy snows;
Then spring breaks through, like gentle laughter,
And bids the summer follow after.
Then shoals of merry motorists start
(No more can coupons break their heart)
To hatch new plans for far-off pranks,
Their "spirit" high as in their tanks.
There's Pa and half a dozen nippers,
Aunts and uncles packed like kippers,
Buckets and spades and bathing slippers,
Sun-tanned beauties and lovely dippers:
Shrieks of joy from a hundred lips
When Ma goes down to the sea and strips.
And here's a coach in blue and grey,
"Its cheaper by coach," the posters say:
This is a trip for the Male Voice Choir
No longer dressed in their Sunday attire,
They sing of the sands and salty dips,
These folk who go down to the sea in slips.
Now look at what the road reveals—
An endless line of automobiles
Stopped outside the local pub,
The smart hotel or country club:
Progress here is rather slow
But that is the way of the world you know.
Some folk plaster their trips with nips
And many go down to the sea in sips.
Anne Walters.
Operators’ Prize List
The Editor takes pleasure in congratulating operators once more on many excellent entries. The best of these are printed in this issue and prizes have been awarded as follows.
**GENERAL**
Miss B. Waterhouse, Birmingham—
“World Archery Champion” £3.3.0
Miss M. Giles, Liverpool—
“Twelve Thousand Watched” £3.3.0
Miss M. Welch, Bournemouth (London)—
“Bournemouth Regatta Queen” £3.3.0
Miss J. Allen, Aintree, Liverpool—
“British Legion Queen” £2.2.0
Mrs. J. Crawford, formerly Liverpool—
“Girl from Madagascar” £2.2.0
Miss A. Walters, Manchester—
“Basic Sea Shanty” £2.2.0
Miss C. M. Blackmore, Dublin—
“Irish Loveliness” £2.2.0
Miss A. Robins, London—
“Broadcast with Kenneth Horne” £2.2.0
Miss E. Piercey, London—
“Broadcast with Kenneth Horne” £1.1.0
Miss S. Kerr, London—
“Broadcast with Kenneth Horne” £1.1.0
Mrs. H. G. West, formerly Glasgow—
“Girl from Rhodesia” £1.1.0
Miss A. Dribble, London—
“Public Speaking” £1.1.0
Miss D. Reavy, Warrington (Liverpool)—
“Tennis Trophy” £1.1.0
Mrs. D. Rae, Droylsden (Manchester)—
“Table Tennis” £1.1.0
Miss E. Andrews, London—
“Public Singing” £1.1.0
Mrs. H. Stanton, Fenham (Newcastle)—
“She Flies Solo” £1.1.0
Mrs. H. Smith, formerly Sheffield—
“Girl in Egypt” £1.1.0
Miss V. Rutledge, Leeds—
“Parkin for Australia” £1.1.0
**GALLERY OF HONOUR**
Mrs. L. Watkins, London—
“It Had to Come” £3.3.0
Miss D. Algar, Newport (Cardiff)
See Page 27
£2.2.0
**RELATIVES’ PHOTOGRAPHS**
£1.1.0 each to
Mrs. M. King, Paisley (Glasgow)
Miss Moira King, Paisley (Glasgow)
Miss S. G. Chant, Ebbw Vale (Cardiff)
Mrs. R. Williams, Ebbw Vale (Cardiff)
Miss E. Gresham, Wallasey (Liverpool)
Miss A. Gresham, Wallasey (Liverpool)
Miss B. Gresham, Wallasey (Liverpool)
Miss M. Dexter, (London)
Miss B. Dexter, (London)
**ESSAY COMPETITION**
£3.3.0 each to
Mrs. K. M. Bragg, (Birmingham)
Miss M. Bannister, (Cardiff)
Mrs. O. Evans, Selsdon (London)
Miss S. Kerr, Hayes (London)
**LET’S HEAR FROM YOU**
The above prize money is more than we ever paid before. Next time we hope it will be bigger still—but have we heard from YOU yet? You can see from this issue what is wanted—photos, exciting experiences, championships, competition winners, in fact, anything of outstanding interest. Prizes up to £3.3.0 are given for operators’ own original entries, and all will be acknowledged if sent to:
The Editor, Comptometer Medley,
Felt & Tarrant, Ltd.,
Aldwych House, London, W.C.2.
**AND MAY WE SAY?**
We do hope you have enjoyed this magazine. Rather than throw it away perhaps you could pass it to a girl or her parents and so help to introduce her close to the progressive career of Comptometer Operator. Or we will gladly post copies to your friends if you will send us their names and addresses. Your co-operation will help to meet industry’s demand for operators. What is more, you may also share in the bonus scheme, 10/6 being awarded for every new pupil you introduce provided she is accepted.
Treyarnon Bay, Treyarnon Bay,
Where slanting crag meets flashing spray,
Your lustrous waters wash the shore
And flood the creeks with joyous roar;
But when the lusty tide withdraws,
When all the swirling echoes pause,
Then softly falls, on sea-carved bowers,
A symphony of halcyon hours,
And like a turquoise, rock-set, cool,
There shines a pool—a sky-blest pool—
Where swimmers meet; the buoyant air
Vibrates with fluting laughter there.
No cares obtrude, nor any fears,
No phantom night, no grisly years,
But only sweet abiding dawn,
Eternal nymph and ageless faun.
Treyarnon Bay, Treyarnon Bay,
Though summers fade and go their way,
Still those auroral visions seem
To thrill with rose each fleeting dream.
K.F.J.
Printed by Edwin Jones & Sons (London) Ltd.
THE CONTROLLED KEY
Comptometer
HIGH SPEED ADDING AND CALCULATING MACHINE
FELT & TARRANT LTD.
THESE ARE YOUR OFFICES AND SCHOOLS
BELFAST : 69, Howard Street
Telephone : Belfast 27032
BIRMINGHAM : 11, Albert Street
Telephone : Colmore 4448 (Birmingham)
BRADFORD : Britannia House, Leeds Road
Telephone : Bradford 57888
BRISTOL : Peasants' Insurance Buildings, Marsh Street
Telephone : Bristol 1083
CARDIFF : 15, Working Street
Telephone : Cardiff 2287
COVENTRY : Halifax Chambers, High Street
Telephone : Coventry 4806
DUBLIN : College Park Chambers, 11, Nassau Street
Telephone : Dublin 82443
EDINBURGH : 42, George Street
Telephone : Central 5467
GLASGOW : 82, Robertson Street
Telephone : Central 8094 (Glasgow)
HULL : Bank Chambers, Lowgate
Telephone : Central 36246 (Hull)
LEEDS : Merrion Chambers, 31-37, Merrion Street
Telephone : Leeds 33463
LEICESTER : Eagle House, Friar Lane
Telephone : Leicester 2277
LIVERPOOL : 3, The Albany, Old Hall St.
Telephone : Central 1893 (Liverpool)
LONDON : Aldwych House, Aldwych, W.C.2
Telephone : Holborn 4374
MANCHESTER : 198, Deansgate
Telephone : Deansgate 3887 (Manchester)
MIDDLESBROUGH : Bank Chambers, Wilson Street
Telephone : Middlesbrough 2813
NEWCASTLE-ON-TYNE : 12, Ellison Place
Telephone : Newcastle 21831
NOTTINGHAM : Gordon House, Carrington Street
Telephone : Nottingham 43684
SHEFFIELD : Parade Chambers, East Parade
Telephone : Sheffield 26052
WE ARE ALWAYS GLAD TO SEE YOU
|
(54) USE OF MONOCYTES DERIVED CELLS, ANTIGENS AND ANTIBODIES FOR OPTIMAL INDUCTION OF IMMUNOTHERAPEUTIC EFFICIENCY
ANWENDUNG VON MONOCYTE-ABSTAMMENDE ZELEN, ANTIGENE UND ANTIKÖRPER ZUR OPTIMALEN INDUZIERUNG EINER IMMUNTHERAPEUTISCHEN LEISTUNGSFÄHIGKEIT
UTILISATION DE CELLULES DERIVEES DE MONOCYTES, D'ANTIGENES ET D'ANTICORPS AFIN D'INDUIRE UNE EFFICACITE IMMUNOTHERAPEUTIQUE OPTIMISEE
(84) Designated Contracting States:
AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE
(30) Priority: 09.04.1998 EP 98400883
(43) Date of publication of application:
17.01.2001 Bulletin 2001/03
(73) Proprietor: I.D.M. IMMUNO-DESIGNED MOLECULES
75011 Paris (FR)
(72) Inventors:
• BARTHOOLEYNS, Jacques
F-91440 Bures-sur-Yvette (FR)
• FOURON, Yves
Marlborough, MA 01752 (US)
• ROMET-LEMONNE, Jean-Loup
F-75003 Paris (FR)
(74) Representative: Grosset-Fournier, Chantal
Catherine et al
Grosset-Fournier & Demachy,
54, rue Saint-Lazare
75009 Paris (FR)
(56) References cited:
WO-A-87/00054 WO-A-91/05871
WO-A-92/05793 WO-A-94/26875
WO-A-96/22781 WO-A-97/10002
WO-A-97/44441
• MAYORGA L.SL ET AL: "Reconstitution of endosomal proteolysis in a cell-free system"
JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 264, no. 10, 1989, pages 5392-5399, XP002077582
• "Biologie Moléculaire de la cellule (third edition)"
FLAMMARION EDITION * page 622 *
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).
Description
[0001] The invention relates to the use of monocytes derived cells, antigens and antibodies for optimal induction of immunotherapeutic efficiency.
[0002] Macrophages play a major role in the antitumoral response, and they are able to be activated by immunological activators against cancer cells (Adams D. and Hamilton T.: "Activation of macrophages for tumor cell kill: effector mechanism and regulation"; in Heppner & Fulton (eds), Macrophages and cancer. CRC Press, 1988, p. 27; Fidler M. Macrophages and metastases. A biological approach to cancer therapy. Cancer Res. 45: 4714, 1985).
[0003] Furthermore, macrophages, or other cells derived from monocytes or from their precursors, with their strong capacity for endocytosis, digestion, and surface antigen presentation, are capable of inducing a specific immune response.
[0004] Macrophages represent the first natural line of defense against infectious agents (bacteria, virus) which are normally killed. However, resistant pathogens can develop ways to escape recognition and killing by macrophages. Protection cannot be very effectively achieved with the attenuated infectious agents or with their recombinant peptidic components.
[0005] Monocytes derived cells (MDCs) are immune cells such as obtained by culture of blood mononuclear cells in non adherent gas permeable plastic or Teflon bags for 5 to 10 days at 37°C in O₂/CO₂ atmosphere. Their culture medium (RPMI, IMDM, AIM5 (Gibco) or X-VIVO (Biowhittaker)) contains eventually cytokines or ligands as defined in patents n° PCT/EP93/01232, n° WO94/26875, EP 97/02703 or WO 97/44441 or in the articles mentioned below:
- "Autologous lymphocytes prevent the death of monocytes in culture and promote, as do GM-CSF, IL-3 and M-CSF, their differentiation into macrophages". (Lopez M., Martinache Ch., Canepa S., Chokri M., Scotto F., Bartholeyns J.; J. of Immunological Methods, 159: 29-38, 1993);
- "Immune therapy with macrophages: Present status and critical requirements for implementation" (Bartholeyns J., Romet-Lemonne J-L., Chokri M., Lopez M.; Immunobiol., 195: 550-562, 1996);
- "In vitro generation of CD83⁺ human blood dendritic cells for active tumor immunotherapy" (Thurnher M., Papesh C., Ramoner R., Gastlt G. and al.; Experimental Hematology, 25: 232-237, 1997);
- "Dendritic cells as adjuvants for immune-mediated resistance to tumors" (Schuler G. and Steinman R. M.; J. Exp. Med., 186: 1183-1187, 1997).
All these patents applications and articles are included herein for references.
[0006] They can be activated by INF-γ at the end of culture to obtain in particular cytotoxic macrophages. They can be centrifuged to be concentrated and purified before resuspension in isotonic solution.
[0007] Monocytes derived cells (MDCs) can either be killer macrophages, phagocytizing cells, growth factors and cytokines releasing cells, or dendritic cells according to their conditions of differentiation. Dendritic cells can for example be obtained as described in "In vitro generation of CD83⁺ human blood dendritic cells for active tumor immunotherapy" (Thurnher M., Papesh C., Ramoner R., Gastlt G. and al.; Experimental Hematology, 25: 232-237, 1997) and "Dendritic cells as adjuvants for immune-mediated resistance to tumors" (Schuler G. and Steinman R. M.; J. Exp. Med., 186: 1183-1187, 1997), and EP n° 97/02703.
[0008] Mature dendritic cells are very potent antigen presenting cells to initiate an immune response. The dendritic cells can be characterized by the induction of T cell proliferation and by their phenotype (presence of CD80, CD86, CD83, MHC-I, MHCII on their membranes).
[0009] A bispecific agent could be employed to target antigens (WO 92/05793) and human IgM or IgG antitumoral antibodies are developed to target tumors or metastases. Clinical anti-cancer responses achieved with these monoclonal human antibodies are not impressive due to unsolved problems of tissue distribution and disseminated sites, limited access to the tumor or to low local concentration, inadequate pharmacokinetics or ineffective dosage, and mainly to the lack of effector cells impairing cellular and humoral immune responses against the relevant tumor.
[0010] IgG, IgA and IgM antibodies directed against infectious agents can be isolated in human plasma and are prepared as monoclonal antibodies. These antibodies do very seldom neutralize the infection as such; a cellular immune response is required. Therapeutic vaccines are therefore difficult to develop with attenuated infectious agents or with their recombinant peptidic components.
[0011] Pan carcinomic tumor antigens (present in breast, prostate, lung, melanoma, glioma, neuroblastoma and colorectal tumors for example) are described and can be targeted, at least in vitro, by specific human monoclonal antibodies. These pan antitumor antibodies do not react with normal cells and tissues such as epithelium, fibroblasts, neuroectodermal or muscle cells. However, these antibodies bind through their Fc part to reticuloendothelial cells such as macrophages bearing Fc receptors. Therefore most antibodies do not effectively reach the tumor target antigen.
[0012] The inefficiency of anti-tumor or anti-infection antibodies is overcome by the present invention which enables to enhance cellular and humoral anti-tumor and anti-infection immune response by initiating ex vivo and controlling the interaction between target antigens, specific antibodies and effector monocyte derived cells.
[0013] One aim of the invention is to provide a process for preparing monocytes derived cells presenting antigenic epitopes on their membrane.
The invention relates in a general embodiment to a process for the preparation of monocytes derived cells, in a purified form and substantially free of contaminants, presenting antigenic epitopes on their membranes after interiorization and processing of at least an antigen-antibody complex formed between an antigen and a non-bispecific antibody, under appropriate conditions, said epitopes corresponding to proteolytic degradation products of said antigen,
- with said non-bispecific antibody being directed against a tumor or against an infectious agent, and
- with said antigen being fragments of tumor or of infectious agent, including membranes or tumor apoptotic bodies, or infectious antigens or being recombinant tumor or infectious antigens.
The present invention describes an ex-vivo method to favor and control the interaction between autologous macrophages or monocytes derived antigen presenting cells, antibodies such as pan anti-tumor antibodies and relevant tumor antigen(s). This controlled interaction (e.a. tumor Ag-pan anti-tumor Ab-effector macrophage) allows a synergistic enhancement of the relevant biological activities and as a result induces in vivo cellular and humoral anti-tumor or anti-infection immune response. Tumor apoptotic bodies are particularly adequate as source of tumor antigen.
The invention takes advantage of an antigen, particularly the availability of the pan tumor antigen, or the tumor apoptotic body as source of tumor antigen, and of the corresponding specific human antibody, to create in vitro a molecular complex which is readily internalized by monocytes derived macrophages or antigen presenting cells. The endocytosis of the immune complex mainly via Fc receptors of the macrophages allows effective processing in vitro. The invention enables also the favored ex vivo interaction between fragments of a patient's tumor membrane, IgM monoclonal antibodies recognizing tumor epitopes on these membranes, and autologous macrophages, resulting in adequate processing of the tumor.
In the case of cancer, by way of example, the immune response is forced in the present invention by initial in vitro interaction between the complex pan tumoral Ag-antitumoral Ab-autologous monocytes derived antigen presenting cells or macrophages. This interaction allows endocytosis of the carcinoma antigen or of the apoptotic body via Fc receptors, adequate processing, interaction with MHC I and MHC II molecules and presentation of antigenic tumor peptides on the cell membrane. The tumor epitopes presented on the membrane of the effector cells are recognized by autologous T lymphocytes of the patient. The pan tumoral nature of the antigen avoids the MHC restriction which limits the use of purified tumor antigens/epitopes to a few selected patients. The injection of this preparation to patients with most types of carcinomas induces potent specific humor-
al and cellular immune responses against the relevant tumor.
In the case of infection, an alternative approach to induce effective immunotherapy is to inject a ternary complex formed between the antibody, the infectious agent and macrophages processing the complex, presenting adequately epitopes of the pathogen and forcing the induction of a long lasting immunity against the pathogen and against subsequent infections.
The expression "purified form" means that the ternary complex formed by tumor or infectious antigens, antitumor or anti-infectious IgM, IgA, IgG antibodies, and effector macrophages or monocyte derived cells is segregated from other cell types or complexes.
The expression "tumor apoptotic bodies" refers to microbodies isolated from tumor cells or cell lines induced to apoptosis, containing mitochondria and DNA within a membrane exposing tumor antigens (Bellone M., Iezzi G., Roverel Galati G. et al. - "Processing of engulfed apoptotic bodies yields T cell epitopes", Journal of Immunology, 159: 5391-5399, 1997).
The expression "substantially free of contaminants" means that macrophages or monocyte derived cells are 90% pure and that no contaminants other than the antibody and the tumor Ag or apoptotic bodies or infectious antigens are present.
The expression "antigen-antibody complex formed between an antigen and an antibody under appropriated conditions" means that antigen and corresponding antibody associate by high affinity (10^4 to 10^11 L/M) non covalent binding, the antigen can be particular as in the case of apoptotic bodies, increasing the avidity of the complex formed.
The expression "epitopes corresponding to proteolytic degradation products of said antigen" means that the antigen phagocytosed in vacuoles of macrophages or monocyte derived cells is digested by proteases into smaller polypeptide fragments.
The monocyte derived cells of the invention can be identified by the following method: presence of Fcγ receptors (CD16, CD32, CD64), of Fcμ and Fcγ receptors (CD89) and of CR3, of CR5 and of mannose receptor on their membranes at the same time as high levels of MHC I and of MHC II molecules.
The invention also relates to a process for the preparation of monocyte derived cells presenting antigenic epitopes on their membranes after interiorization and processing of at least an antigen-antibody complex, formed between an antigen and an antibody, said antibody being exclusively a non-bispecific antibody and said process comprising the following steps:
a) preparation of the monocytes derived cells according to the following method:
1) recovery of blood derived mononuclear cells directly from blood apheresis or from blood bag collection, followed if necessary by centrifugation, to eliminate a substantial part of red blood cells granulocytes and platelets, and collection of peripheral blood leukocytes;
2) washing peripheral blood leukocytes obtained at the preceeding steps for instance by centrifugation (to remove 90% of platelets, red blood cells and debris) to obtain mononuclear cells;
3) resuspension of the total mononuclear cells obtained at the preceeding step in culture medium (AIMV, RPMI or IMDM type) at $10^6$ to $2 \times 10^7$ cells/ml, possibly completed by cytokines and/or autologous serum, and culture for 5 to 10 days at 37°C under O$_2$/CO$_2$ atmosphere in hydrophobic gas permeable bags, to obtain monocyte derived cells and contaminating lymphocytes;
b) addition of antigens and antibodies to the monocyte derived cells obtained at the preceeding step to form a ternary complex between monocyte derived cells, an antigen and an antibody;
c) incubation of said ternary complex for a time and at a temperature sufficient to allow endocytosis into intracellular vacuoles of the monocyte derived cells and processing of the antigen-antibody complex, with said processing consisting of digestion of the antigen-antibody complex and association of the epitopes of the antigen resulting from the digestion with MHC molecules, to obtain monocyte derived cells presenting antigenic epitopes on their membranes.
[0026] The preparation of the monocyte derived cells can be carried out as described in patents n° PCT/EP93/01232, n° WO94/26875, EP 97/02703 or WO 97/44441 or in the articles mentioned below:
- "Autologous lymphocytes prevent the death of monocytes in culture and promote, as do GM-CSF, IL-3 and M-CSF, their differentiation into macrophages" (Lopez M., Martinache Ch., Canepa S., Chokri M., Scotto F., Bartholeyns J.; J. of Immunological Methods, 159: 29-38, 1993);
- "Immune therapy with macrophages: Present status and critical requirements for implementation" (Bartholeyns J., Romet-Lemonne J-L., Chokri M., Lopez M.; Immunobiol., 195: 550-562, 1996);
- "In vitro generation of CD83+ human blood dendritic cells for active tumor immunotherapy" (Thurnher M., Papesch C., Ramoner R., Gastl G. and al.; Experimental Hematology, 25: 232-237, 1997);
- "Dendritic cells as adjuvants for immune-mediated resistance to tumors" (Schuler G. and Steinman R. M.; J. Exp. Med., 186: 1183-1187, 1997).
[0027] According to an advantageous embodiment of the invention, the process is such that
- said antibody is directed against a tumor or against an infectious agent,
- with said antigen is fragments of tumor or of infectious agent, including membranes or tumor apoptotic bodies, or purified tumor or infectious antigens or is a recombinant tumor or infectious antigen.
[0028] According to an advantageous embodiment of the invention, in the step of addition, said antigens and antibodies are in the form of a complex.
[0029] According to an another embodiment of the invention, in the step of addition, said antigens and antibodies are not in the form of a complex.
[0030] According to an advantageous embodiment of the invention, the process comprises the additional following step:
d) centrifugation of the monocyte derived cells presenting antigenic epitopes on their membranes, washing and resuspension, for instance in isotonic medium, to obtain a suspension of the above defined monocyte derived cells.
[0031] According to an another advantageous embodiment of the invention, in the process above defined, the step of centrifugation is followed by
e) freezing at temperature below or equal to -80°C aliquots of the above said suspension, with the addition of a cryopreservative.
[0032] According to an another advantageous embodiment of the process, the step of freezing is followed by
f) melting said above frozen aliquots at a temperature enabling to obtain a suspension of monocyte derived cells presenting antigenic epitopes on their membranes, for instance at 4°C, washing said suspension and resuspending it, for instance in an isotonic medium, to obtain a suspension of monocyte derived cells presenting antigenic epitopes on their membranes.
[0033] Advantageously, in the process of the invention, the antibodies are human or humanised IgG, IgA or preferably IgM (for induction of primary immune response) directed against tumor antigens or against infectious antigens (viral or bacterial).
[0034] Advantageously, in the process of the invention, the antigens are purified tumor, viral or bacterial antigens (polypeptides, glycopeptides, oligosaccharide) or membrane fragments serving as complex antigens.
[0035] Advantageously, in the process of the invention, the antigens are pan tumor antigens present on different tumor types.
[0036] The invention also relates to a ternary complex in a purified form, and substantially free of contaminants, between monocytes derived cells, an antigen and an anwith said antibody being exclusively a non-bispecific antibody and being directed against a tumor or against an infectious agent,
with said antigen being fragments of tumor or of infectious agent including membranes or tumor apoptotic bodies, or purified tumor or infectious antigens or being recombinant tumor or infectious antigens, and
with said antigen and said antibody being liable to form an antigen-antibody complex under appropriate conditions.
[0037] The expression "ternary complex" designates a complex formed *ex vivo* between an antigen (tumor or infectious origin), an antibody recognizing that antigen and a macrophage or monocyte derived cell binding the previous binary complex on its membrane before internalization.
[0038] A ternary complex can be identified by electron microscopy or by FACS analysis (fluorescence cell analysis) of the monocyte derived cell and of the molecules expressed on its membrane. High affinity binding between the antigen and the antibody occurs before binding of the complex to the monocyte derived cell membrane. Binding occurs at a temperature below 10°C while internalization occurs only at a temperature above 20°C.
[0039] The expression "purified form" means that the ternary complex formed by tumor or infectious antigens, anti-tumor or anti-infectious IgM, IgA, IgG antibodies, and effector macrophages or monocyte derived cells is segregated from other cell type or complexes.
[0040] The expression "substantially free of contaminants" means that macrophages or monocyte derived cells are 90% pure and that no contaminants other than the antibody and the tumor or infectious antigens are present.
[0041] A process for the preparation of a ternary complex between monocyte derived cells, an antigen and an antibody as above defined, comprising the following steps:
1) recovery of blood derived mononuclear cells directly from blood apheresis or from blood bag collection, followed if necessary by centrifugation, to eliminate a substantial part of red blood cells granulocytes and platelets, and collection of peripheral blood leukocytes;
2) washing peripheral blood leukocytes obtained at the preceeding steps for instance by centrifugation (to remove 90% of platelets, red blood cells and debris) to obtain mononuclear cells;
3) resuspension of the total mononuclear cells obtained at the preceeding step in culture medium (AIMV, RPMI or IMDM type) at $10^6$ to $2 \cdot 10^7$ cells/ml, possibly completed by cytokines and/or autologous serum, and culture for 5 to 10 days at 37°C under $\text{O}_2/\text{CO}_2$ atmosphere in hydrophobic gas permeable bags, to obtain monocyte derived cells and contaminating lymphocytes;
[0042] b) addition of antigens and antibodies to the monocyte derived cells obtained at the preceeding step to form of a ternary complex between monocyte derived cells, an antigen and an antibody.
[0043] In the process above defined, said antigens and antibodies are advantageously in the form of a complex.
[0044] In another embodiment of the process above defined, said antigens and antibodies are not in the form of a complex.
[0045] The invention also relates to a ternary complex between monocyte derived cells, an antigen and an antibody such as obtained according to the process above defined.
[0046] The invention also relates to a pharmaceutical composition containing as active substance a ternary complex according to the invention, in association with a pharmaceutically acceptable vehicle.
[0047] The pharmaceutical compositions of the invention are advantageously in the form of sterile injectable preparations.
[0048] The monocyte derived cells are administered at a dose of about $10^6$ to about $10^9$ cells/kg of body weight, particularly from about $10^7$ to about $10^8$ cells/kg of body weight.
[0049] The invention also relates to a vaccine containing as active substance a ternary complex according to the invention, in association with a pharmaceutically acceptable vehicle.
[0050] The invention also relates to a ternary complex according to the invention, for the preparation of a medicament for treating cancer or infectious diseases.
**EXAMPLES**
[0051] Macrophages or monocyte derived cells of the following examples are prepared according to the methods described in the above-mentioned references.
1) SCID mice are inoculated subcutaneously with human tumor cells. They are also "humanized" by i.v. injection of about 10 million human total mononuclear cells. No immune response is seen and the tumor keeps growing to reach 1 square cm size. Mice are then injected intravenously either with
a) 10 µg of pan tumor carcinoma antigen,
b) with 0.1 mg of IgG anti-pan carcinoma Ag,
c) with both,
d) with 1 million activated autologous macrophages,
e) with both Ag plus Ab and macrophages.
The absence of objective response is seen in groups a), b), and c); partial transient response in groups
d) and a tumor regression in group e) are documented by measurement of tumor size with calipers in two perpendicular directions.
2) Anti-HBV antibodies are mixed with HBs particles in a ratio 10/1 with about $10^7$ HBs/ml and incubated for 4h at 37°C in IMDM medium with about $10^6$/ml monocyte derived cells. The resulting monocyte derived cells of the invention induce the proliferation of cytotoxic T lymphocytes (autologous to the monocyte derived cells) specific for HBV ($10^6$ monocyte derived cells resuspended per ml or RPMI medium added to $5.10^5$ T lymphocytes during 5 days, followed by measurement of tritiated thymidine incorporation).
3) The emergence of resistance to antibiotics in hospitals, even after triple therapies, requires the development of new vaccinal immunotherapies of infections. Monocyte derived cells obtained ex vivo are resuspended in culture medium in the presence a: of monoclonal or polyclonal antibodies specific for the resistant infectious agent (e.a. virus, staphylococci) or of anti-LPS antibodies for Gram negative bacteria, and b: in the presence of killed pathogens (pasteurisation) or membrane extracts.
A ternary complex is formed between the infectious antigen, the antibodies and the monocyte derived cells. These monocyte derived cells cultured at about $5.10^6$/ml in IMDM medium process the antibody-infectious antigen complex during 4h incubation time at 37°C. The cells are centrifuged, resuspended in isotonic saline and injected locally or systemically to the patient to induce protective therapeutic immunity against the infectious agents that can be illustrated by the decrease in the infection titer and the presence of anti-infectious antibodies in peripheral blood. It is shown that in mice with lethal Gram(-) infections, the injection of macrophages having processed anti-LPS antibodies and heat killed bacteria results in immune protection of the animals.
The above-mentioned experiments have been performed with different subsets of monocyte derived cells expressing immunoglobulin Fc receptors, and in particular with macrophages.
4) Monocyte-derived macrophages of HLA-A2 haplotype, are incubated with the S protein of Hepatitis B virus in the presence or not of an anti-HBS immunoglobulin which displays a high affinity for the FcγRI, CD64. After 4 hours of incubation, the macrophages are mixed with increasing numbers of an HLA-A2 restricted T cell clone specific for HBS. The stimulation of the T cell clone is assessed by measuring the secretion of cytokines, e.g. IFNγ. It is shown that in the presence of the anti-HBS antibody, the stimulation of the T cell clone is 100 fold higher than in absence of the immunoglobulin.
**Claims**
1. Process for the preparation of monocyte derived cells presenting antigenic epitopes on their membranes after interiorization and processing of at least an antigen-antibody complex, formed between an antigen and an antibody, said antibody being exclusively a non-bispecific antibody, and said process comprising the following steps:
a) preparation of the monocytes derived cells according to the following method:
1) recovery of blood derived mononuclear cells directly from blood apheresis or from blood bag collection, followed if necessary by centrifugation, to eliminate a substantial part of red blood cells granulocytes and platelets, and collection of peripheral blood leukocytes;
2) washing peripheral blood leukocytes obtained at the preceding steps for instance by centrifugation to remove 90% of platelets, red blood cells and debris to obtain mononuclear cells;
3) resuspension of the total mononuclear cells obtained at the preceding step in culture medium such as AIMV, RPMI or IMDM type at $10^6$ to $2.10^7$ cells/ml,
b) addition of antigens and antibodies to the monocyte derived cells obtained at the preceeding step to form a ternary complex between monocyte derived cells, an antigen and an antibody;
c) incubation of said ternary complex for a time sufficient and at a temperature above 20°C to allow endocytosis into intracellular vacuoles of the monocyte derived cells and processing of the antigen-antibody complex, with said processing consisting of digestion of the antigen-antibody complex and association of the epitopes of the antigen resulting from the digestion with MHC molecules, to obtain monocyte derived cells presenting antigenic epitopes on their membranes.
2. Process for preparation of monocyte derived cells according to claim 1, wherein in the step of resuspension of the total mononuclear cell, the culture medium is completed by cytokines and/or autologous serum.
3. Process according to claims 1 or 2, wherein said antibody is directed against a tumor or against an infectious agent, with said antigen being fragments of tumor or of infectious agent, including membranes or tumor apoptotic bodies, or purified tumor or infectious antigens or is a recombinant tumor or infectious antigen.
4. Process according to claim 1 or 2, wherein in the step of addition, said antigens and antibodies are in the form of a complex.
5. Process according to anyone of claims 1 to 4, comprising the additional following step:
d) centrifugation of the monocyte derived cells presenting antigenic epitopes on their membranes, washing and resuspension, for instance in isotonic medium, to obtain a suspension of the above defined monocyte derived cells.
6. Process according to claim 5, wherein the step of centrifugation is followed by
e) freezing at temperature below or equal to -80°C aliquots of the above said suspension, with the addition of a cryopreservative.
7. Process according to claim 6, wherein the step of freezing is followed by
f) melting said above frozen aliquots at a temperature enabling to obtain a suspension of monocyte derived cells presenting antigenic epitopes on their membranes, for instance at 4°C, washing said suspension and resuspending it, for instance in an isotonic medium, to obtain a suspension of monocyte derived cells presenting antigenic epitopes on their membranes.
8. Process according to anyone of claims 1 to 7, wherein the antibodies are human or humanised IgG, IgA or preferably IgM for induction of primary immune response directed against tumor antigens or against infectious antigens, such as viral or bacterial antigens.
9. Process according to anyone of claims 1 to 7, wherein the antigens are purified tumor, viral or bacterial antigens such as polypeptides, glycopeptides, oligosacharides, or membrane fragments serving as complex antigens.
10. Process according to claims 8 or 9, wherein the antigens are pan tumor antigens present on different tumor types.
11. Ternary complex in a purified form, being 90% pure and no contaminants other than antibody and the tumor antigen or apoptotic bodies or infectious antigens being present, between monocytes derived cells, an antigen and an antibody
with said antibody being exclusively a non-bispecific antibody and being directed against a tumor or against an infectious agent,
with said antigen being fragments of tumor or of infectious agent including membranes or tumor apoptotic bodies, or purified tumor or infectious antigens or being recombinant tumor or infectious antigens, and
with said antigen and said antibody being liable to form an antigen-antibody complex under appropriate conditions.
12. Process for the preparation of a ternary complex between monocyte derived cells, an antigen and an antibody according to claim 11, comprising the following steps:
a) preparation of monocyte derived cells according to the following method:
1) recovery of blood derived mononuclear cells directly from blood apheresis or from blood bag collection, followed if necessary by centrifugation, to eliminate a substantial part of red blood cells granulocytes and platelets, and collection of peripheral blood leukocytes;
2) washing peripheral blood leukocytes obtained at the preceeding steps for instance by centrifugation to remove 90% of platelets, red blood cells and debris to obtain mononuclear cells;
3) resuspension of the total mononuclear cells obtained at the preceeding step in culture medium such as AIMV, RPMI or IMDM type at $10^6$ to $2.10^7$ cells/ml, possibly completed by cytokines and/or autologous serum, and culture for 5 to 10 days at 37°C under O$_2$/CO$_2$ atmosphere in hydrophobic gas permeable bags, to obtain monocyte derived cells and contaminating lymphocytes;
b) addition of antigens and antibodies to the monocyte derived cells obtained at the preceding step to form of a ternary complex between monocyte derived cells, an antigen and an antibody.
13. Process according to claim 12, wherein said antigens and antibodies are either in the form of a complex or not.
14. Ternary complex between monocyte derived cells, an antigen and an antibody such as obtained according to claims 12 or 13.
15. Pharmaceutical composition containing as active
substance a ternary complex according to claim 14, in association with a pharmaceutically acceptable vehicle.
16. Pharmaceutical composition according to claim 15, in the form of sterile injectable preparations.
17. Vaccine containing as active substance a ternary complex according to claim 14, in association with a pharmaceutically acceptable vehicle.
18. Use of a ternary complex according to claim 14, for the preparation of a medicament for treating cancer or infectious diseases.
Patentansprüche
1. Verfahren zur Herstellung von monozytenabgeleiteten Zellen, die Antigenepitope auf ihren Membranen, nach Aufnahme und Verarbeitung von zumindest einem Antigen-Antikörper-Komplex, gebildet zwischen einem Antigen und einem Antikörper, präsentieren, wobei besagter Antikörper ausschließlich ein nicht bispezifischer Antikörper ist und besagtes Verfahren die folgenden Schritte umfasst:
a) Präparation der monozytenabgeleiteten Zellen gemäß dem folgenden Verfahren:
1) Erhalten von blutabgeleiteten mononuklearen Zellen direkt durch Blutapherese oder durch Blutbeutelentnahme, wenn notwendig gefolgt von Zentrifugation, um einen wesentlichen Teil der roten Blutzellen, Granulozyten und Blutplättchen zu entfernen, und Sammeln von peripheren Blutleukozyten;
2) Waschen der peripheren Blutleukozyten, die durch den vorhergehenden Schritt erhalten wurden, z. B. durch Zentrifugation, um 90 % der Blutplättchen, roten Blutzellen und Debris zu entfernen, um mononukleare Zellen zu erhalten;
3) Resuspension der gesamten durch den vorhergehenden Schritt erhaltenen mononuklearen Zellen, in Kultivierungsmedium wie z. B. AIMV, RPMI oder IMDM Typ bei $10^6$ bis $2.10^7$ Zellen/ml,
b) Zugabe von Antigenen und Antikörpern zu den monozytenabgeleiteten Zellen, die bei dem vorhergehenden Schritt erhalten wurden, um einen ternären Komplex zwischen monozytenabgeleiteten Zellen, einem Antigen und einem Antikörper zu bilden;
c) Inkubation von besagtem ternären Komplex für eine ausreichende Zeit und bei einer Temperatur über 20 °C, um Endozytose in intrazelluläre Vakuolen der monozytenabgeleiteten Zellen und die Prozessierung des Antigen-Antikörper-Komplexes zu erlauben, wobei besagte Prozessierung aus der Verdauung des Antigen-Antikörper-Komplexes und der Assoziation der Epitope des Antigens, resultierend aus der Verarbeitung mit MHC Molekülen besteht, um monozytenabgeleitete Zellen, die Antigenepitope auf ihren Membranen präsentieren zu erhalten.
2. Verfahren für die Herstellung von monozytenabgeleiteten Zellen nach Anspruch 1, wobei bei dem Schritt der Resuspension der gesamten mononuklearen Zellen, das Kultivierungsmedium durch Zytokine und/oder autologes Serum vervollständigt ist.
3. Verfahren nach Anspruch 1 oder 2, wobei besagter Antikörper gegen einen Tumor oder gegen ein infektiöses Agenz gerichtet ist, und wobei besagte Antigene Fragmente von Tumor oder von infektiösem Agenz sind, beinhaltend Membranen oder tumorapoptotische Körper, oder gereinigte Tumor- oder infektiöse Antigene oder ein rekombinantes Tumor- oder infektiöses Antigen sind.
4. Verfahren nach Anspruch 1 oder 2, wobei bei dem Schritt der Zugabe, besagte Antigene und Antikörper in der Form eines Komplexes vorliegen.
5. Verfahren nach einem der Ansprüche 1 bis 4, umfassend den zusätzlichen folgenden Schritt:
d) Zentrifugation der monozytenabgeleiteten Zellen, die Antigenepitope auf ihren Membran präsentieren, Waschen und Resuspension, z. B. in isotoni schem Medium, um eine Suspension der oben beschriebenen monovzytenabgeleiteten Zellen zu erhalten.
6. Verfahren nach Anspruch 5, wobei der Schritt der Zentrifugation gefolgt wird von
c) Einfrieren von Aliquoten der oben genannten Suspension bei Temperaturen unter oder gleich -80 °C, mit der Zugabe von einem Kühl schutzmittel.
7. Verfahren nach Anspruch 6, wobei der Schritt des Einfrierens gefolgt wird von
f) Auftauen besagter oben gefrorener Aliquote bei einer Temperatur, die es erlaubt eine Suspension von monozytenabgeleiteten Zellen, die Antigenepitope auf ihren Membranen präsentieren, zu erhalten, z. B. bei 4 °C, Waschen besagter Suspension und Resuspension davon, z. B.
in einem isotonischen Medium, um eine Suspension von monozytenabgeleiteten Zellen, die Antigenepitope auf ihren Membranen präsentieren zu erhalten.
8. Verfahren nach einem der Ansprüche 1 bis 7, wobei die Antikörper menschliches oder humanisiertes IgG, IgA oder bevorzugt IgM, für die Induktion einer primären Immunantwort, gerichtet gegen Tumoranatigene oder gegen infektiöse Antigene, wie z. B. virale oder bakterielle Antigene sind.
9. Verfahren nach einem der Ansprüche 1 bis 7, wobei die Antigene gereinigte Tumor-, virale oder bakterielle Antigene, wie z. B. Polypeptide, Glykoproteide, Oligosaccharide oder Membranfragmente sind, die als Komplexantigene dienen.
10. Verfahren nach Anspruch 8 oder 9, wobei die Antigene Pantumor-Antigene, vorhanden bei verschiedenen Tumortypen sind.
11. Ternärer Komplex in einer gereinigten Form, der 90 % rein ist und wo keine Kontaminanten, außer den Antikörpern und den Tumoranatigenen oder apoptotischen Körperchen oder infektiösen Antigenen vorhanden sind, zwischen monozytenabgeleitete Zellen, einem Antigen und einem Antikörper,
- wobei besagter Antikörper ausschließlich ein nicht bispezifischer Antikörper ist und gegen einen Tumor oder gegen ein infektiöses Agenz gerichtet ist,
- und wobei besagtes Antigen Fragmente eines Tumors oder eines infektiösen Agenz, beinhaltend Membranen oder tumorapoptotische Körper, oder gereinigtes Tumor- oder infektiöses Antigen ist, oder rekombinantes Tumor- oder infektiöses Antigen ist, und
- wobei besagtes Antigen und besagter Antikörper geeignet sind einen Antigen-Antikörper-Komplex unter geeigneten Bedingungen zu bilden.
12. Verfahren für die Herstellung eines ternären Komplexes zwischen monozytenabgeleiteten Zellen, einem Antigen und einem Antikörper nach Anspruch 11, umfassend die folgenden Schritte:
a) Präparation der monozytenabgeleiteten Zellen gemäß dem folgenden Verfahren:
1) Erhalten von blutabgeleiteten mononuklearen Zellen direkt durch Blutapherese oder durch Blutbeutelentnahme, wenn notwendig gefolgt von Zentrifugation, um einen wesentlichen Teil der roten Blutzellen, Granulozyten und Blutplättchen zu entfernen, und Sammeln von peripheren Blutleukozyten;
2) Waschen der peripheren Blutleukozyten, die durch den vorhergehenden Schritt erhalten wurden, z. B. durch Zentrifugation, um 90 % der Blutplättchen, roten Blutzellen und Debris zu entfernen, um mononukleare Zellen zu erhalten;
3) Resuspension der gesamten durch den vorhergehenden Schritt erhaltenen mononuklearen Zellen, in Kultivierungsmedium wie z. B. AIMV, RPMT oder IMDM Typ bei $10^6$ bis $2 \cdot 10^7$ Zellen/ml, eventuell vervollständigt durch Zytokine und/oder autologes Serum und Kultivierung für 5 bis 10 Tage bei 37 °C unter einer O$_2$/CO$_2$ Atmosphäre in hydrophoben, gasdurchlässigen Beuteln, um monozytenabgeleitete Zellen und kontaminierende Lymphozyten zu erhalten;
b) Zugabe von Antigenen und Antikörpern zu den monozytenabgeleiteten Zellen, erhalten bei dem vorhergehenden Schritt, um einen ternären Komplex zwischen monozytenabgeleiteten Zellen, einem Antigen und einem Antikörper zu bilden.
13. Verfahren nach Anspruch 12, wobei besagte Antigene und Antikörper entweder in der Form eines Komplexes sind oder nicht.
14. Ternärer Komplex zwischen monozytenabgeleiteten Zellen, einem Antigen und einem Antikörper, wie erhalten nach Ansprüchen 12 oder 13.
15. Pharmazeutische Zusammensetzung beinhaltend als aktive Substanz einen ternären Komplex nach Anspruch 14 in Verbindung mit einem pharmazeutisch akzeptablen Träger.
16. Pharmazeutische Zusammensetzung nach Anspruch 15, in der Form eines sterilen, injzierbaren Präparats.
17. Impfstoff beinhaltend als aktive Substanz einen ternären Komplex nach Anspruch 14, in Verbindung mit einem pharmazeutisch akzeptablen Träger.
18. Verwendung eines ternären Komplexes nach Anspruch 14, für die Herstellung eines Medikaments zur Behandlung von Krebs oder infektiösen Krankheiten.
Revendications
1. Procédé de préparation de cellules dérivées de monocytes présentant des épitopes antigéniques sur
leurs membranes après intériorisation et protéolyse d'au moins un complexe antigène-anticorps, formé entre un antigène et un anticorps, ledit anticorps étant exclusivement un anticorps non bispécifique, et ledit procédé comprenant les étapes suivantes :
a) la préparation de cellules dérivées de monocytes selon la méthode suivante :
1) la récupération de cellules mononucléées dérivées de sang directement à partir d'une aphérèse sanguine ou à partir d'une collecte de poches de sang, suivie si nécessaire par une centrifugation, pour éliminer une partie substantielle des hématies, des granulocytes et des plaquettes, et collecte des leucocytes du sang périphérique.
2) le lavage des leucocytes du sang périphérique obtenus aux étapes précédentes, par exemple par centrifugation, pour éliminer 90% des plaquettes, des hématies et des débris, pour obtenir des cellules mononucléées ;
3) la resuspension de la totalité des cellules mononucléées obtenues à l'étape précédente dans un milieu de culture tel que AIMV, RPMI ou le type IMDM à $10^6$ à $2.10^7$ cellules/ml,
b) l'addition d'antigènes et d'anticorps aux cellules dérivées de monocytes obtenues à l'étape précédente pour former un complexe ternaire entre les cellules dérivées de monocytes, un antigène et un anticorps ;
c) l'incubation dudit complexe ternaire pendant un temps suffisant et à une température supérieure à 20°C pour permettre l'endocytose des cellules dérivées de monocytes dans des vacuoles intracellulaires et la protéolyse du complexe antigène-anticorps, ladite protéolyse consistant en la digestion du complexe antigène-anticorps et en l'association des épitopes de l'antigène résultant de la digestion avec des molécules du CMH, pour obtenir des cellules dérivées de monocytes présentant des épitopes antigéniques sur leurs membranes.
2. Procédé de préparation de cellules dérivées de monocytes selon la revendication 1, dans lequel dans l'étape de resuspension de la totalité des cellules mononucléées, le milieu de culture est complété par des cytokines et / ou un sérum autologue.
3. Procédé selon la revendication 1 ou 2, dans lequel ledit anticorps est dirigé contre une tumeur ou contre un agent infectieux, ledit antigène étant des fragments de tumeur ou d'agent infectieux, incluant des corps apoptotiques de membrane ou de tumeur, ou des antigènes infectieux ou tumoraux purifiés ou étant un antigène infectieux ou tumoral recombinant.
4. Procédé selon la revendication 1 ou 2, dans lequel dans l'étape d'addition, lesdits antigènes et anticorps sont sous la forme d'un complexe.
5. Procédé selon l'une quelconque des revendications 1 à 4, comprenant l'étape supplémentaire suivante :
d) la centrifugation des cellules dérivées de monocytes présentant des épitopes antigéniques sur leurs membranes, le lavage et la resuspension, par exemple dans un milieu isotonique, pour obtenir une suspension de cellules dérivées de monocytes définies ci-dessus.
6. Procédé selon la revendication 5, dans lequel l'étape de centrifugation est suivie de
c) la congélation d'aliquotes de ladite suspension ci-dessus à une température inférieure ou égale à -80°C, avec l'addition d'un cryoconservateur.
7. Procédé selon la revendication 6, dans lequel l'étape de congélation est suivie de
f) la décongélation desdits aliquotes congelés ci-dessus à une température permettant d'obtenir une suspension de cellules dérivées de monocytes présentant des épitopes antigéniques sur leur membranes, par exemple à 4°C, le lavage de ladite suspension et la resuspension de celle-ci, par exemple dans un milieu isotonique, pour obtenir une suspension de cellules dérivées de monocytes présentant des épitopes antigéniques sur leur membranes.
8. Procédé selon l'une quelconque des revendications 1 à 7, dans lequel les anticorps sont des IgG, IgA ou, de préférence, IgM humaines ou humanisées, pour induire une réponse immunitaire primaire, dirigés contre des antigènes tumoraux ou contre des antigènes infectieux, tels que les antigènes viraux ou bactériens.
9. Procédé selon l'une quelconque des revendications 1 à 7, dans lequel les antigènes sont des antigènes tumoraux, viraux ou bactériens purifiés tels que des polypeptides, des glycopeptides, des oligosaccharides ou des fragments membranaires servant d'antigènes complexes.
10. Procédé selon la revendication 8 ou 9, dans lequel les antigènes sont des antigènes tumoraux pan présents sur différents types de tumeurs.
11. Complexe ternaire sous une forme purifiée, pur à 90% et sans aucun contaminant autre qu’un anticorps et l’antigène tumoral ou des corps apoptotiques ou des antigènes infectieux, entre les cellules dérivées de monocytes, un antigène et un anticorps
- le dit anticorps étant exclusivement un anticorps non bispécifique et étant dirigé contre une tumeur ou contre un agent infectieux,
- le dit antigène étant des fragments de tumeur ou d’agent infectieux incluant des corps apoptotiques membranaires ou tumoraux, ou des antigènes tumoraux ou infectieux purifiés ou étant des antigènes tumoraux ou infectieux recombinants, et
- le dit antigène et le dit anticorps étant capables de former un complexe antigène-anticorps dans des conditions appropriées.
12. Procédé de préparation d’un complexe ternaire entre des cellules dérivées de monocytes, un antigène et un anticorps selon la revendication 11, comprenant les étapes suivantes :
a) la préparation de cellules dérivées de monocytes selon la méthode suivante :
1) la récupération de cellules mononucléées dérivées du sang directement à partir d’une aphasèse sanguine ou d’une collecte de poches de sang, suivie si nécessaire d’une centrifugation, pour éliminer une partie substantielle des hématies, des granulocytes et des plaquettes, et collecte de leucocytes du sang périphérique ;
2) le lavage des leucocytes du sang périphérique obtenus aux étapes précédentes par exemple par centrifugation, pour éliminer 90% des plaquettes, des hématies et des débris, pour obtenir des cellules mononucléées;
3) la resuspension de la totalité des cellules mononucléées obtenues à l’étape précédente dans un milieu de culture tel que AIMV, RPMI ou le type IMDM à $10^6$ à $2.10^7$ cellules/ml, éventuellement complétée par des cytokines et/ou un sérum autologue, et la culture pendant 5 à 10 jours à 37°C sous atmosphère O$_2$/Co$_2$ dans des sacs hydrophobes perméables au gaz, pour obtenir des cellules dérivées de monocytes et des lymphocytes contaminants;
b) l’addition d’antigènes et d’anticorps aux cellules dérivées de monocytes obtenues à l’étape précédente pour former un complexe ternaire entre des cellules dérivées de monocytes, un antigène et un anticorps.
13. Procédé selon la revendication 12, dans lequel lesdits antigènes et anticorps sont sous la forme d’un complexe ou ne sont pas sous la forme d’un complexe.
14. Complexe ternaire entre des cellules dérivées de monocytes, un antigène et un anticorps tel qu’obtenu selon la revendication 12 ou 13.
15. Composition pharmaceutique contenant comme substance active un complexe ternaire selon la revendication 14, en association avec un excipient pharmaceutiquement acceptable.
16. Composition pharmaceutique selon la revendication 15, sous forme de préparations injectables stériles.
17. Vaccin contenant comme substance active un complexe ternaire selon la revendication 14, en association avec un excipient pharmaceutiquement acceptable.
18. Utilisation d’un complexe ternaire selon la revendication 14, pour la préparation d’un médicament pour traiter le cancer ou des maladies infectieuses.
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AN ORDINANCE ADOPTING AND ENACTING A NEW CODE OF ORDINANCES OF THE CITY OF ANDERSON, COUNTY OF MCDONALD, STATE OF MISSOURI; ESTABLISHING THE SAME; PROVIDING FOR THE REPEAL OF CERTAIN ORDINANCES NOT INCLUDED THEREIN, EXCEPT AS HEREIN EXPRESSLY PROVIDED; PROVIDING FOR THE MANNER OF AMENDING SUCH CODE OF ORDINANCES; PROVIDING PENALTY FOR THE VIOLATION THEREOF; AND PROVIDING WHEN THIS ORDINANCE SHALL BECOME EFFECTIVE
Be it ordained by the Board of Aldermen of the City of Anderson, County of McDonald, State of Missouri, as follows:
Section 1. Approval, Adoption and Enactment of Code.
Pursuant to Section 71.943 of the Revised Statutes of Missouri, the codification of ordinances, as set out in Titles I through VII, each inclusive, of the "Code of Ordinances of the City of Anderson, County of McDonald, State of Missouri," is hereby adopted and enacted as the "Code of Ordinances of the City of Anderson"; which shall supersede all other general and permanent ordinances of the City passed on or before May 17, 2016, to the extent provided in Section 3 hereof.
Section 2. When Code Provisions Effective.
All provisions of such Code shall be in full force and effect from and after the effective date of this ordinance as set forth herein.
Section 3. Repeal of Legislation Not Contained in Code; Legislation Saved From Repeal; Matters Not Affected By Repeal.
A. All ordinances of a general and permanent nature of the City adopted on final passage on or before May 17, 2016, and not included in such Code or recognized and continued in force by reference therein, are hereby repealed from and after the effective date of this ordinance, except those which may be specifically excepted by separate ordinance, and except the following which are hereby continued in full force and effect, unless specifically repealed by separate ordinance:
1. Ordinances promising or guaranteeing the payment of money for the City, or authorizing the issuance of any bonds or notes of the City or any other evidence of the City's indebtedness, or authorizing any contract or obligation assumed by the City.
2. Ordinances levying taxes or making special assessments.
3. Ordinances appropriating funds or establishing salaries and compensation, and providing for expenses.
4. Ordinances granting franchises or rights to any person, firm or corporation.
5. Ordinances relating to the dedication, opening, closing, naming, establishment of grades, improvement, altering, paving, widening or vacating of streets, alleys, sidewalks or public places.
6. Ordinances authorizing or relating to particular public improvements.
7. Ordinances respecting the conveyances or acceptance of real property or easements in real property.
8. Ordinances dedicating, accepting or vacating any plat or subdivision in the City or any part thereof, or providing regulations for the same.
9. Ordinances annexing property to the City.
10. All zoning and subdivision ordinances not specifically repealed and not included herein.
11. Ordinances establishing TIF districts or redevelopment districts.
12. Ordinances relating to traffic schedules (e.g., stop signs, parking limits, etc.).
13. All ordinances relating to personnel regulations (e.g., pensions, retirement, job descriptions and insurance, etc.).
14. Ordinances authorizing the establishment of industrial development corporations.
15. Ordinances establishing tax rates for the City.
B. The repeal provided for in this Section shall not be construed to revive any ordinance or part thereof that has been repealed by a subsequent ordinance which is repealed by this ordinance.
C. The repeal provided for in this Section shall not affect any offense or act committed or done or any penalty or forfeiture incurred or any contract or right established or accruing before the effective date of this ordinance, nor shall it affect any prosecution, suit or proceeding pending or any judgment rendered prior to such date.
Section 4. Amendments To Code.
Any and all additions and amendments to such Code when passed in such form as to indicate the intention of the Board of Aldermen to make the same a part thereof shall be deemed to be incorporated in such Code so that reference to the "Code of Ordinances of the City of Anderson" shall be understood and intended to include such additions and amendments.
Section 5. Violations and Penalties.
A. Except as hereinafter provided, whenever in any rule, regulation or order promulgated pursuant to such ordinances of the City, any act is prohibited or is made or declared to be unlawful or an offense or a misdemeanor, or whenever in such City ordinance, rule, regulation or order doing of any act is required or the failure to do any act is declared to
be unlawful, where no specific penalty is provided therefor, the violation of any such ordinance of the City, or of any rule, regulation or order promulgated pursuant to such City ordinance, shall be punished by a fine of not less than five dollars ($5.00) and not more than five hundred dollars ($500.00) or by imprisonment for a period not to exceed ninety (90) days, or by both such fine and imprisonment.
B. Whenever any provision of the Revised Statutes of Missouri or other Statute of the State limits the authority of the City to punish the violation of any particular provision of these ordinances or rules, regulations or orders promulgated pursuant thereto to a fine of less amount than that provided in this Section or imprisonment for a shorter term than that provided in this Section, the violation of such particular provision of these ordinances or rules, regulations or orders shall be punished by the imposition of not more than the maximum fine or imprisonment so authorized, or by both such fine and imprisonment.
C. Whenever any provision of the Revised Statutes of Missouri or other Statute of the State establishes a penalty differing from that provided by this Section for an offense similar to any offense established by these ordinances, rules, regulations or other orders of the City, the violation of such City law, ordinance, rule, regulation or order shall be punished by the fine or imprisonment established for such similar offense by such State law.
D. Each day any violation of these ordinances, rules, regulations or orders promulgated pursuant thereto shall continue shall constitute a separate offense, unless otherwise provided.
E. Whenever any act is prohibited by this Code, by an amendment thereof, or by any rule or regulation adopted thereunder, such prohibition shall extend to and include the causing, securing, aiding or abetting of another person to do said act. Whenever any act is prohibited by this Code, an attempt to do the act is likewise prohibited.
Section 6. Applicability of General Penalty.
In case of the amendment by the Board of Aldermen of any Section of such Code for which a penalty is not provided, the general penalty as provided in Section 5 of this ordinance shall apply to the Section as amended; or in case such amendment contains provisions for which a penalty other than the aforementioned general penalty is provided in another Section in the same Chapter, the penalty so provided in such other Section shall be held to relate to the Section so amended, unless such penalty is specifically repealed therein.
Section 7. Filing of Copy of Code; Codes To Be Kept Up-To-Date.
A copy of such Code shall be kept on file in the office of the City Clerk, preserved in loose-leaf form or in such other form as the City Clerk may consider most expedient. It shall be the express duty of the City Clerk, or someone authorized by said officer, to insert in their designated places all amendments and all ordinances or resolutions which indicate the intention of the Board of Aldermen to make the same part of such Code when the same have been printed or reprinted in page form and to extract from such Code all provisions which from time to time may be repealed by the Board of Aldermen. This copy of such Code shall be available for all persons desiring to examine the same.
Section 8. Altering or Tampering With Code; Violations and Penalties.
It shall be unlawful for any person to change or alter by additions or deletions any part or portion of such Code, or to insert or delete pages or portions thereof, or to alter or tamper with such Code in any manner whatsoever which will cause the law of the City of Anderson to be misrepresented thereby. Any person violating this Section shall be punished as provided in Section 5 of this ordinance.
Section 9. Severability.
It is hereby declared to be the intention of the Board of Aldermen that the Sections, paragraphs, sentences, clauses and phrases of this ordinance and the Code hereby adopted are severable, and if any phrase, clause, sentence, paragraph or Section of this ordinance or the Code hereby adopted shall be declared unconstitutional or otherwise invalid by the valid judgment or decree of a court of competent jurisdiction, such unconstitutionality or invalidity shall not affect any of the remaining phrases, clauses, sentences, paragraphs and Sections of this ordinance or the Code hereby adopted.
Section 10. Effective Date.
This ordinance and the Code adopted hereby shall become effective July 19, 2016.
PASSED by the Board of Aldermen of the City of Anderson this 19th day of July 2016.
APPROVED by the Mayor of the City of Anderson this 19th day of July 2016.
______________________________
John Sellers
Mayor of the City of Anderson
ATTEST:
Andrea Browning
City Clerk
Section DL.010. Disposition of Legislation.
The following is a chronological listing of submitted ordinances of the City of Anderson adopted since the 1974 City Code, indicating for each either its inclusion in the 2016 Code or the reason for its exclusion. [Enabling legislation which is not general and permanent in nature is considered to be non-Code material (NCM).] The Board of Aldermen Minutes of May 17, 2016, which included approval of substantive Code amendments, was the last legislation reviewed for the 2016 Code publication.
| Ord. No. | Adoption Date | Subject | Disposition |
|----------|---------------|----------------------------------------------|----------------------|
| 45.042 | 2-8-1982 | City officials: City Collector amendment | Ch. 115, Art. III |
| 45.048 | 6-30-1982 | City officials: Chief of Police amendment | Ch. 115, Art. IV |
| 45.050 | 3-14-1983 | Zoning amendment | Ch. 405 |
| 45.053 | 11-13-1984 | Sewer regulations | Ch. 700 |
| 45.054 | 8-16-1986 | Traffic Code: speed limits amendment | Traffic Sch. I |
| 45.057 | 1-26-1987 | Zoning amendment | Ch. 405 |
| 411-94 | 5-9-1994 | Zoning amendment | Ch. 405 |
| 711-94 | 8-8-1994 | General building provisions: miscellaneous provisions amendment | Ch. 500, Art. V |
| 031715 | 3-17-2015 | Taxation and finance: sales tax and applicability to utilities | Ch. 140, Art. IV |
| 62915 | 6-29-2015 | Annexation | NCM |
| 21616 | 2-17-2016 | General building provisions: miscellaneous provisions amendment | Ch. 500, Art. V |
| 21616A | 2-17-2016 | Violations and penalties amendment | Ch. 100, Art. III; Ch. 130, Art. I; Ch. 130, Art. III; Ch. 300 |
| 41916 | 4-19-2016 | Reestablishing City limits | NCM |
| Resolution | 5-17-2016 | Licenses and occupational taxes amendment | Ch. 605 |
| Ord. No. | Adoption Date | Subject | Disposition |
|---------|---------------|----------------------------------------------|----------------------|
| Motion | 5-17-2016 | Mayor and Board of Aldermen: meetings amendment | Ch. 110, Art. II |
| 71916 | 7-19-2016 | Adopting Ordinance | Front of Code |
Chapter 100
GENERAL PROVISIONS
ARTICLE I
City Incorporation and Seal
Section 100.010. Municipal Incorporation.
Section 100.020. City Seal.
ARTICLE II
General Code Provisions
Section 100.030. Contents of Code.
Section 100.040. Citation of Code.
Section 100.050. Official Copies of Code.
Section 100.060. Altering or Amending Code.
Section 100.070. Numbering of Code.
Section 100.080. Definitions and Rules of Construction.
Section 100.090. Words and Phrases — How Construed.
Section 100.100. Headings.
Section 100.110. Continuation of Prior Ordinances.
Section 100.120. Effect of Repeal of Ordinance.
Section 100.130. Repealing Ordinance Repealed — Former Ordinance Not Revived — When.
Section 100.140. Severability.
Section 100.150. Tense.
Section 100.160. Notice.
Section 100.170. Notice — Exceptions.
Section 100.180. Computation of Time.
Section 100.190. Gender.
Section 100.200. Joint Authority.
Section 100.210. Number.
ARTICLE III
Penalty
Section 100.220. General Penalty.
ARTICLE I
City Incorporation and Seal
Section 100.010. Municipal Incorporation.
The inhabitants of the City of Anderson, as its limits now are or may hereafter be defined by law, shall be and continue a body corporate by the name of "The City of Anderson" and as such shall have perpetual succession, may sue and be sued, implead and be impleaded, defend and be defended in all courts of law and equity and in all actions whatever; may receive and hold property, both real and personal, within such City and may purchase, receive and hold real estate within or without such City for the burial of the dead; and may purchase, hold, lease, sell or otherwise dispose of any property, real or personal, it now owns or may hereafter acquire; may receive bequests, gifts and donations of all kinds of property; and may have and hold one (1) common Seal and may break, change or alter the same at pleasure; and may do any act, exercise any power and render any service which contributes to the general welfare, and all courts of this State shall take judicial notice thereof.
Section 100.020. City Seal.
[CC 1995 §100.100; CC 1974 §20.010]
A. The Seal of the City shall, as heretofore, be the words "City Seal" in Roman capitals, inside of and surrounded by a scroll or circular impression having inscribed therein the words "City of Anderson, Missouri." The Seal shall be circular and about two (2) inches in diameter. The City Seal shall be safely kept in the office of the City Clerk.
B. The City Clerk shall be the keeper of the common Seal of the City of Anderson, and any impression of said Seal to any contract or other writing shall have no validity or binding obligation upon the City unless such impression be accompanied by the attestation and signature of the City Clerk, and then only in cases authorized by law or the ordinances of this City.
ARTICLE II
General Code Provisions
Section 100.030. Contents of Code.
This Code contains all ordinances of a general and permanent nature of the City of Anderson, Missouri, and includes ordinances dealing with municipal administration, municipal elections, building and property regulation, business and occupations, health and sanitation, public order and similar subjects.
Section 100.040. Citation of Code.
This Code may be known and cited as the "Municipal Code of the City of Anderson, Missouri."
Section 100.050. Official Copies of Code.
At least three (3) copies of the published book shall be kept on file in the office of the City Clerk and kept available for inspection by the public at all reasonable business hours.
Section 100.060. Altering or Amending Code.
A. It shall be unlawful for any person to change or amend by additions or deletions any part or portion of this Code, or to insert or delete pages or portions thereof, or to alter or tamper with such Official Copy of the Code in any manner whatsoever which will cause the law of the City to be misrepresented thereby. Any person, firm or corporation violating this Section shall be punished as provided in Section 100.220 of this Code.
B. This provision shall not apply to amendments, additions or deletions to this Code, duly passed by the Board of Aldermen, which may be prepared by the City Clerk for insertion in this Code.
Section 100.070. Numbering of Code.
Each Section number of this Code shall consist of two (2) parts separated by a period; the figure before the period referring to the Chapter number, and the figure after the period referring to the position of the Section in the Chapter.
Section 100.080. Definitions and Rules of Construction.
A. In the construction of this Code and of all other ordinances of the City, the following definitions shall be observed, unless it shall be otherwise expressly provided in any Section or ordinance, or unless inconsistent with the manifest intent of the Board of Aldermen, or unless the context clearly requires otherwise:
BOARD OF ALDERMEN — The Board of Aldermen of the City of Anderson, Missouri.
CERTIFIED MAIL or CERTIFIED MAIL WITH RETURN RECEIPT REQUESTED — Includes certified mail carried by the United States Postal Service or any parcel or letter carried by an overnight, express or ground delivery service that allows a sender or recipient to electronically track its location and provides a record of the signature of the recipient.
CITY — The words "the City" or "this City" or "City" shall mean the City of Anderson, Missouri.
COUNTY — The words "the County" or "this County" or "County" shall mean the County of McDonald, Missouri.
DAY — A day of twenty-four (24) hours beginning at 12:00 Midnight.
MAY — Is permissive.
MAYOR — An officer of the City known as the Mayor of the Board of Aldermen of the City of Anderson, Missouri.
MONTH — A calendar month.
OATH — Includes an affirmation in all cases in which an affirmation may be substituted for an oath, and in such cases the words "swear" and "sworn" shall be equivalent to the words "affirm" and "affirmed."
OFFENSE — Shall mean and be the same as ordinance violation and is punishable as provided in Section 100.220 of this Code.
OWNER — The word "owner," as applied to a building or land, shall include any part owner, joint owner, tenant in common, joint tenant or tenant by the entirety of the whole or a part of such building or land.
PERSON — May extend and be applied to bodies politic and corporate, and to partnerships and other unincorporated associations.
PERSONAL PROPERTY — Includes money, goods, chattels, things in action and evidences of debt.
PRECEDING, FOLLOWING — When used by way of reference to any Section of this Code, shall mean the Section next preceding or next following that in which the reference is made, unless some other Section is expressly designated in the reference.
PROPERTY — Includes real and personal property.
PUBLIC WAY — Includes any street, alley, boulevard, parkway, highway, sidewalk or other public thoroughfare.
REAL PROPERTY — The terms "real property," "premises," "real estate" or "lands" shall be deemed to be co-extensive with lands, tenements and hereditaments.
SHALL — Is mandatory.
SIDEWALK — That portion of the street between the curb line and the adjacent property line which is intended for the use of pedestrians.
STATE — The words "the State" or "this State" or "State" shall mean the State of Missouri.
STREET — Includes any public way, highway, street, avenue, boulevard, parkway, alley or other public thoroughfare, and each of such words shall include all of them.
TENANT, OCCUPANT — The words "tenant" or "occupant," applied to a building or land, shall include any person who occupies the whole or a part of such building or land, whether alone or with others.
WRITTEN, IN WRITING and WRITING WORD FOR WORD — Includes printing, lithographing, or other mode of representing words and letters, but in all cases where the signature of any person is required, the proper handwriting of the person, or his/her mark, is intended.
YEAR — A calendar year, unless otherwise expressed, and the word "year" shall be equivalent to the words "year of our Lord."
B. Newspaper. Whenever in this Code or other ordinance of the City it is required that notice be published in the "official newspaper" or a "newspaper of general circulation published in the City," and if there is no newspaper published within the City, the said notice shall be published in a newspaper of general circulation within the City, regardless of its place of publication. Such newspaper shall not include an advertising circular or other medium for which no subscription list is maintained.
C. Delegation Of Authority. Whenever a provision appears in this Code requiring the head of a department or an officer of the City to do some act or make certain inspections, it may be construed to authorize the head of the department or officer to designate, delegate and authorize subordinates to perform the required act or make the required inspections, unless the terms of the provision or Section designate otherwise.
Section 100.090. Words and Phrases — How Construed.
Words and phrases shall be taken in their plain or ordinary and usual sense, but technical words and phrases having a peculiar and appropriate meaning in law shall be understood according to their technical import.
Section 100.100. Headings.
The headings of the Chapters and Sections of this Code are intended as guides and not as part of this Code for purposes of interpretation or construction.
Section 100.110. Continuation of Prior Ordinances.
The provisions appearing in this Code, so far as they are in substance the same as those of ordinances existing at the time of the adoption of this Code, shall be considered as a continuation thereof and not as new enactments.
Section 100.120. Effect of Repeal of Ordinance.
No offense committed and no fine, penalty or forfeiture incurred, or prosecution commenced or pending previous to or at the time when any ordinance provision is repealed or amended, shall be affected by the repeal or amendment, but the trial and punishment of all such offenses and the recovery of the fines, penalties or forfeitures shall be had, in all respects, as if the provision had not been repealed or amended, except that all such proceedings shall be conducted according to existing procedural laws.
Section 100.130. Repealing Ordinance Repealed — Former Ordinance Not Revived — When.
When an ordinance repealing a former ordinance, clause or provision is itself repealed, it does not revive the former ordinance, clause or provision, unless it is otherwise expressly provided; nor shall any ordinance repealing any former ordinance, clause or provision abate, annul or in anywise affect any proceedings had or commenced under or by virtue of the ordinance so repealed, but the same is as effectual and shall be proceeded on to final judgment and termination as if the repealing ordinance had not passed, unless it is otherwise expressly provided.
Section 100.140. Severability.
It is hereby declared to be the intention of the Board of Aldermen that the Chapters, Sections, paragraphs, sentences, clauses and phrases of this Code are severable, and if any phrase, clause, sentence, paragraph, Section or Chapter of this Code shall be declared unconstitutional or otherwise invalid by the valid judgment or decree of any court of competent jurisdiction, such unconstitutionality or invalidity shall not affect any of the remaining phrases, clauses, sentences, paragraphs, Sections and Chapters of this Code since the same would have been enacted by the Board of Aldermen without the incorporation in this Code of any such unconstitutional or invalid phrase, clause, sentence, paragraph or Section.
Section 100.150. Tense.
Except as otherwise specifically provided or indicated by the context, all words used in this Code indicating the present tense shall not be limited to the time of adoption of this Code but shall extend to and include the time of the happening of any act, event or requirement for which provision is made herein, either as a power, immunity, requirement or prohibition.
Section 100.160. Notice.
A. Whenever notice may be required under the provisions of this Code or other City ordinance, the same shall be served in the following manner:
1. By delivering the notice to the person to be served personally or by leaving the same at his/her residence, office or place of business with some person of his/her family over the age of fifteen (15) years;
2. By mailing said notice by certified or registered mail to such person to be served at his/her last known address; or
3. If the person to be served is unknown or may not be notified under the requirements of this Section, then by posting said notice in some conspicuous place at least five (5) days before the act or action concerning which the notice is given is to take place. No person shall interfere with, obstruct, mutilate, conceal or tear down any official notice or placard posted by any City Officer, unless permission is given by said officer.
Section 100.170. Notice — Exceptions.
The provisions of the preceding Section shall not apply to those Chapters of this Code wherein there is a separate definition of notice.
Section 100.180. Computation of Time.
In computing any period of time prescribed or allowed by this Code or by a notice or order issued pursuant thereto, the day of the act, event or default after which the designated period of time begins to run is not to be included. The last day of the period so computed is to be included unless it is a Saturday, Sunday or a legal holiday, in which event the period runs until the end of the next day which is neither a Saturday, Sunday nor a legal holiday. When the period of time prescribed or allowed is less than seven (7) days, intermediate Saturdays, Sundays and legal holidays shall be excluded in the computation.
Section 100.190. Gender.
When any subject matter, party or person is described or referred to by words importing the masculine, females as well as males, and associations and bodies corporate as well as individuals, shall be deemed to be included.
Section 100.200. Joint Authority.
Words importing joint authority to three (3) or more persons shall be construed as authority to a majority of such persons unless otherwise declared in the law giving the authority.
Section 100.210. Number.
When any subject matter, party or person is described or referred to by words importing the singular number, the plural and separate matters and persons and bodies corporate shall be deemed to be included; and when words importing the plural number are used, the singular shall be included.
ARTICLE III
Penalty
Section 100.220. General Penalty.
A. Whenever in this Code or any other ordinance of the City, or in any rule, regulation, notice or order promulgated by any officer or agency of the City under authority duly vested in him/her or it, any act is prohibited or is declared to be unlawful or an offense, misdemeanor or ordinance violation or the doing of any act is required or the failure to do any act is declared to be unlawful or an offense, misdemeanor or ordinance violation, and no specific penalty is provided for the violation thereof, upon conviction of a violation of any such provision of this Code or of any such ordinance, rule, regulation, notice or order, the violator shall be punished by a fine not exceeding five hundred dollars ($500.00) or by imprisonment in the City or County Jail not exceeding ninety (90) days, or by both such fine and imprisonment; provided, that in any case wherein the penalty for an offense is fixed by a Statute of the State, the statutory penalty, and no other, shall be imposed for such offense, except that imprisonments may be in the City prison or workhouse instead of the County Jail.
B. Every day any violation of this Code or any other ordinance or any such rule, regulation, notice or order shall continue shall constitute a separate offense.
C. Whenever any act is prohibited by this Code, by an amendment thereof, or by any rule or regulation adopted thereunder, such prohibition shall extend to and include the causing, securing, aiding or abetting of another person to do said act. Whenever any act is prohibited by this Code, an attempt to do the act is likewise prohibited.
D. Minor Traffic Violations. The punishment of a "minor traffic violation," as defined by Section 300.010 of the Anderson Municipal Code, shall be subject to the following:
[Ord. No. 21616A, §2, 2-17-2016]
1. The maximum fine and Court costs that can be imposed for the violation of any minor traffic violation shall be three hundred dollars ($300.00). This does not include additional fees that may be added, i.e., warrant fees and deferred payment fees.
2. Minor traffic violations shall not be punishable by imprisonment, unless the violation:
a. Involved alcohol or controlled substances,
b. Endangered the health or welfare of others, or
c. Involved eluding or giving false information to a law enforcement officer.
3. A person convicted of a minor traffic violation shall not be placed in confinement for failure to pay a fine unless such nonpayment violates the terms of the person's probation.
4. Court costs shall be assessed against such person unless the Court finds that the defendant is indigent.
Chapter 105
ELECTIONS
ARTICLE I
In General
Section 105.010. Conformance of City Elections With State Law.
All City elections shall be conducted and held in conformance with the provisions of Chapter 115, RSMo.
Section 105.020. Date of Municipal Election.
A. A municipal election for the qualified voters of this City shall be held on the first (1st) Tuesday after the first (1st) Monday in April of each year.
B. On the first (1st) Tuesday after the first (1st) Monday in April of even-numbered years, a municipal election of the qualified voters of the City of Anderson shall be held for the purpose of electing a Mayor who shall hold his/her office for a term of two (2) years and until his/her successor is elected and qualified.
C. On the first (1st) Tuesday after the first (1st) Monday in April of odd-numbered years, a municipal election of the qualified voters of the City of Anderson shall be held for the purpose of electing one (1) Alderman from each ward who shall hold his/her office for a term of two (2) years and until his/her successor is elected and qualified.
D. On the first (1st) Tuesday after the first (1st) Monday in April of even-numbered years, a municipal election of the qualified voters of the City of Anderson shall be held for the
purpose of electing one (1) Alderman from each ward who shall hold his/her office for a term of two (2) years and until his/her successor is elected and qualified.
Section 105.030. Declaration of Candidacy — Dates for Filing.
Any person who desires to become a candidate for an elective City office at the general City election shall file with the City Clerk, not prior to the hour of 8:00 A.M., on the sixteenth (16th) Tuesday prior to, nor later than 5:00 P.M., on the eleventh (11th) Tuesday prior to the next City municipal election, a written declaration of his/her intent to become a candidate at said election. The City Clerk shall keep a permanent record of the names of the candidates, the offices for which they seek election, and the date of their filing, and their names shall appear on the ballots in that order.
Section 105.035. Disqualification as Candidate for Elective Public Office, When — Disqualification From Participation in Election, When — Affidavit To Be Filed, Requirements — Investigation of Alleged Delinquency.
A. No person shall qualify as a candidate for elective public office in the State of Missouri who has been found guilty of or pled guilty to a felony or misdemeanor under the Federal laws of the United States of America or to a felony under the laws of this State or an offense committed in another state that would be considered a felony in this State.
B. Any person who files as a candidate for election to a public office shall be disqualified from participation in the election for which the candidate has filed if such person is delinquent in the payment of any State income taxes, personal property taxes, municipal taxes, real property taxes on the place of residence, as stated on the declaration of candidacy, or if the person is a past or present corporate officer of any fee office that owes any taxes to the State.
C. Each potential candidate for election to a public office shall file an affidavit with the Department of Revenue and include a copy of the affidavit with the declaration of candidacy required under Section 115.349, RSMo. Such affidavit shall be in substantially the form as set out in Section 115.306, RSMo.
D. Upon receipt of a complaint alleging a delinquency of the candidate in the filing or payment of any State income taxes, personal property taxes, municipal taxes, real property taxes on the place of residence, as stated on the declaration of candidacy, or if the person is a past or present corporate officer of any fee office that owes any taxes to the State, the Department of Revenue shall investigate such potential candidate to verify the claim contained in the complaint. If the Department of Revenue finds a positive affirmation to be false, the Department shall contact the Secretary of State, or the election official who accepted such candidate's declaration of candidacy, and the potential candidate. The Department shall notify the candidate of the outstanding tax owed and give the candidate thirty (30) days to remit any such outstanding taxes owed which are not the subject of dispute between the Department and the candidate. If the candidate fails to remit such amounts in full within thirty (30) days, the candidate shall be disqualified from participating in the current election and barred from refiling for an
entire election cycle even if the individual pays all of the outstanding taxes that were the subject of the complaint.
Section 105.040. Declaration of Candidacy — Notice to Public.
The City Clerk shall, on or before the sixteenth (16th) Tuesday prior to any election at which City offices are to be filled by said election, notify the general public of the opening filing date, the office or offices to be filled, the proper place for filing, and the closing filing date of the election. Such notification may be accomplished by legal notice published in at least one (1) newspaper of general circulation in the City.
Section 105.050. Notice of Elections.
In City elections, the City Clerk shall notify the County Clerk prior to 5:00 P.M. on the tenth (10th) Tuesday prior to any City election except as noted in Section 115.125.1, RSMo. The notice shall be in writing, shall specify that the Board of Aldermen is calling the election, the purpose of the election, the date of the election, and shall include a certified copy of the legal notice to be published including the sample ballot. The notice and any other information required by this Section may, with the prior notification to the election authority receiving the notice, be accepted by facsimile transmission prior to 5:00 P.M. on the tenth (10th) Tuesday prior to the election, provided that the original copy of the notice and a certified copy of the legal notice to be published shall be received in the office of the election authority within three (3) business days from the date of the facsimile transmission.
ARTICLE II
Wards
Section 105.060. Wards.
[CC 1995 §120.010; CC 1974 §§30.010 — 30.030]
A. The City is hereby divided into two (2) Wards, as hereinafter delineated.
1. Ward Number One. Ward Number One shall be comprised of that portion of the City east of the railroad tracks.
2. Ward Number Two. Ward Number Two shall be comprised of that portion of the City west of the railroad tracks.
Chapter 110
MAYOR AND BOARD OF ALDERMEN
ARTICLE I
Mayor and Board of Aldermen — Generally
Section 110.010. Aldermen — Qualifications.
Section 110.020. Mayor — Qualifications.
Section 110.025. Duties of Mayor as President of Board.
Section 110.030. Board to Select an Acting President — Term.
Section 110.040. Acting President to Perform Duties of Mayor — When.
Section 110.050. Mayor and Board — Duties.
Section 110.060. Mayor May Sit in Board.
Section 110.070. Ordinances — Procedure to Enact.
Section 110.080. Bills Must Be Signed — Mayor's Veto.
Section 110.090. Board to Keep Journal of Proceedings.
Section 110.100. Board Shall Publish Semi-Annual Statements.
Section 110.110. No Money of City to Be Disbursed Until Statement Is Published — Penalty.
Section 110.120. Board May Compel Attendance of Witnesses — Mayor to Administer Oaths.
Section 110.130. Mayor to Sign Commissions.
Section 110.140. Mayor Shall Have the Power to Enforce Laws.
Section 110.150. Mayor — Communications to Board.
Section 110.160. Mayor May Remit Fine — Grant Pardon.
Section 110.170. Proclamations, Meetings, Elections.
Section 110.180. Appoint Certain Officers — Control Police.
Section 110.190. (Reserved)
ARTICLE II
Board of Aldermen Meetings
Section 110.200. Regular Meetings.
Section 110.210. Special Meetings.
Section 110.220. Quorum Must Be Present.
Section 110.230. Compelling Attendance.
Section 110.240. Effect of Adjourned Meetings.
Section 110.250. Rules of Order.
Section 110.260. Decorum.
Section 110.270. Voting.
Section 110.280. Permission Required for Members to Leave Chamber.
Section 110.290. Order of Business.
Section 110.300. Expression of Dissent or Protest by Member.
Section 110.310. Procedure as to Objections Upon Second Reading of Bill.
Section 110.320. When Bills May Be Amended.
Section 110.330. Rules of Procedure.
ARTICLE I
Mayor and Board of Aldermen — Generally
Section 110.010. Aldermen — Qualifications.
No person shall be an Alderman unless he/she be at least eighteen (18) years of age, a citizen of the United States, and an inhabitant and resident of the City for one (1) year next preceding his/her election, and a resident, at the time he/she files and during the time he/she serves, of the ward from which he/she is elected.
Section 110.020. Mayor — Qualifications.
No person shall be Mayor unless he/she be at least twenty-five (25) years of age, a citizen of the United States, and a resident of the City at the time of and for at least one (1) year next preceding his/her election.
Section 110.025. Duties of Mayor as President of Board.
[CC 1995 §115.060; CC 1974 §24.050]
A. At the hour designated for Board meetings, the Mayor shall call the Board of Aldermen to order, and he/she shall act as President of the Board.
B. The Mayor shall appoint all committees, subject to the concurrence of the Board of Aldermen, the appointment or election of which is not otherwise provided for by this Code or other ordinance.
Section 110.030. Board to Select an Acting President — Term.
The Board shall elect one (1) of their own number who shall be styled "Acting President of the Board of Aldermen" and who shall serve for a term of one (1) year.
Section 110.040. Acting President to Perform Duties of Mayor — When.
When any vacancy shall happen in the office of Mayor by death, resignation, removal from the City, removal from office, refusal to qualify, or from any other cause whatever, the Acting President of the Board of Aldermen shall, for the time being, perform the duties of Mayor, with all the rights, privileges, powers and jurisdiction of the Mayor, until such vacancy be filled or such disability be removed; or, in case of temporary absence, until the Mayor's return.
Section 110.050. Mayor and Board — Duties.
The Mayor and Board of Aldermen of each City governed by this Chapter shall have the care, management and control of the City and its finances and shall have power to enact and ordain any and all ordinances not repugnant to the Constitution and laws of this State, and such as they shall deem expedient for the good government of the City, the preservation of peace and good order, the benefit of trade and commerce, and the health of the inhabitants thereof, and such other ordinances, rules and regulations as may be deemed necessary to carry such powers into effect and to alter, modify or repeal the same.
Section 110.060. Mayor May Sit in Board.
The Mayor shall have a seat in and preside over the Board of Aldermen but shall not vote on any question except in case of a tie, nor shall he/she preside or vote in cases when he/she is an interested party. He/she shall exercise a general supervision over all the officers and affairs of the City and shall take care that the ordinances of the City, and the State laws relating to such City, are complied with.
Section 110.070. Ordinances — Procedure to Enact.
A. The style of the ordinances of the City shall be: "Be it ordained by the Board of Aldermen of the City of Anderson, as follows:" No ordinance shall be passed except by bill, and no bill shall become an ordinance unless on its final passage a majority of the members elected to the Board of Aldermen shall vote for it, and the "ayes" and "nays" be entered on the journal. Every proposed ordinance shall be introduced to the Board of Aldermen in writing and shall be read by title or in full two (2) times prior to passage, both readings may occur at a single meeting of the Board of Aldermen. If the proposed ordinance is read by title only, copies of the proposed ordinance shall be made available for public inspection prior to the time the bill is under consideration by the Board of Aldermen. No bill shall become an ordinance until it shall have been signed by the Mayor, or person exercising the duties of the Mayor's office, or shall have been passed over the Mayor's veto as herein provided.
B. The provisions of this Section shall not apply to ordinances proposed or passed under Section 79.135, RSMo.
Section 110.080. Bills Must Be Signed — Mayor's Veto.
Every bill duly passed by the Board of Aldermen and presented to the Mayor and by him/her approved shall become an ordinance, and every bill presented as aforesaid, but returned with the Mayor's objections thereto, shall stand reconsidered. The Board of Aldermen shall cause the objections of the Mayor to be entered at large upon the journal and proceed at its convenience to consider the question pending, which shall be in this form: "Shall the bill pass, the objections of the Mayor thereto notwithstanding?" The vote on this question shall be taken by "ayes" and "nays" and the names entered upon the journal, and if two-thirds (2/3) of all the members-elect shall vote in the affirmative, the City Clerk shall certify the fact on the roll, and the bill thus certified shall be deposited with the proper officer and shall become an
ordinance in the same manner and with like effect as if it had received the approval of the Mayor. The Mayor shall have power to sign or veto any ordinance passed by the Board of Aldermen; provided, that should he/she neglect or refuse to sign any ordinance and return the same with his/her objections, in writing, at the next regular meeting of the Board of Aldermen, the same shall become a law without his/her signature.
Section 110.090. Board to Keep Journal of Proceedings.
The Board of Aldermen shall cause to be kept a journal of its proceedings, and the "ayes" and "nays" shall be entered on any question at the request of any two (2) members. The Board of Aldermen may prescribe and enforce such rules as it may find necessary for the expeditious transaction of its business.
Section 110.100. Board Shall Publish Semi-Annual Statements.
The Board of Aldermen shall semi-annually each year, at times to be set by the Board of Aldermen, make out and spread upon their records a full and detailed account and statement of the receipts and expenditures and indebtedness of the City for the half year ending with the last day of the month immediately preceding the date of such report, which account and statement shall be published in some newspaper in the City.
Section 110.110. No Money of City to Be Disbursed Until Statement Is Published — Penalty.
In the event the financial statement of the City is not published as required by Section 110.100, the Treasurer of the City shall not pay out any money of the City on any warrant or order of the Board of Aldermen after the end of the month in which such financial statement should have been published until such time as such financial statement is published. Any Treasurer violating the provisions of this Section shall be deemed guilty of a ordinance violation.
Section 110.120. Board May Compel Attendance of Witnesses — Mayor to Administer Oaths.
The Board of Aldermen shall have power to compel the attendance of witnesses and the production of papers and records relating to any subject under consideration in which the interest of the City is involved and shall have power to call on the proper officers of the City, or of the County in which such City is located, to execute such process. The officer making such service shall be allowed to receive therefor such fees as are allowed by law in the Circuit Court for similar services, to be paid by the City. The Mayor or Acting President of the Board of Aldermen shall have power to administer oaths to witnesses.
Section 110.130. Mayor to Sign Commissions.
The Mayor shall sign the commissions and appointments of all City Officers elected or appointed in the City and shall approve all official bonds unless otherwise prescribed by ordinance.
Section 110.140. Mayor Shall Have the Power to Enforce Laws.
The Mayor shall be active and vigilant in enforcing all laws and ordinances for the government of the City, and he/she shall cause all subordinate officers to be dealt with promptly for any neglect or violation of duty; and he/she is hereby authorized to call on every male inhabitant of the City over eighteen (18) years of age and under fifty (50) to aid in enforcing the laws.
Section 110.150. Mayor — Communications to Board.
The Mayor shall, from time to time, communicate to the Board of Aldermen such measures as may, in his/her opinion, tend to the improvement of the finances, the Police, health, security, ornament, comfort and general prosperity of the City.
Section 110.160. Mayor May Remit Fine — Grant Pardon.
The Mayor shall have power to remit fines and forfeitures and to grant reprieves and pardons for offenses arising under the ordinances of the City; but this Section shall not be so construed as to authorize the Mayor to remit any costs which may have accrued to any officer of said City by reason of any prosecution under the laws or ordinances of such City.
Section 110.170. Proclamations, Meetings, Elections.
[CC 1995 §110.140; CC 1974 §21.220]
The Mayor shall have the power to issue proclamations, call mass meetings and regular and special elections in such a manner as this Code or other ordinances or State law may provide.
Section 110.180. Appoint Certain Officers — Control Police.
[CC 1995 §110.160; CC 1974 §21.250]
The Mayor, with the advice and consent of the Board of Aldermen, shall have the power to appoint all appointive officers of the City except the City Clerk. He/she shall have authority to give such orders to the Chief of Police and Policemen of the City as in his/her judgment the public good may require, and it shall be the duty of the Chief of Police and Police Officers to obey such orders.
Section 110.190. (Reserved)
ARTICLE II
Board of Aldermen Meetings
Section 110.200. Regular Meetings.
[CC 1995 §115.010; CC 1974 §24.010; 5-17-2016]
A. The Board of Aldermen of this City shall meet in regular session in the Board Room of the City Hall at the hour of 6:00 P.M. on the third Tuesday of each month.
1. When any such meeting day is a holiday, the regular meeting shall be held at such time as may be provided by the Board on motion at the previous meeting.
2. The Board may, by motion, dispense with any regular meeting, but at least one (1) meeting, regular or special, must be held in each calendar month.
Section 110.210. Special Meetings.
Special meetings may be called by the Mayor or by any two (2) members of the Board by written request filed with the City Clerk who shall thereupon prepare a notice of such special meeting in conformance with Chapter 120, Open Meetings and Records Policy, of this Code.
Section 110.220. Quorum Must Be Present.
At the hour appointed, the Mayor, or in his/her absence the Acting President of the Board of Aldermen, shall call the Board to order, the Clerk shall call the roll of members and announce whether or not a quorum is present. A majority of the members elected to the Board shall constitute a quorum. If a quorum not be present, a smaller number may lawfully adjourn the meeting from day to day until a quorum is present.
Section 110.230. Compelling Attendance.
[CC 1995 §115.040; CC 1974 §24.040]
In case that a lesser number than a quorum shall convene at a regular or special meeting of the Board of Aldermen, the majority of the members present are authorized to direct the Chief of Police or other City Officer to send for and compel the attendance of any or all absent members upon such terms and conditions and at such time as such majority of the members present shall agree. It shall be prima facia evidence that any member who has not attended three (3) consecutive meetings shall have vacated his/her office.
Section 110.240. Effect of Adjourned Meetings.
[CC 1995 §115.050; CC 1974 §24.045]
All adjourned meetings of the Board shall, to all intents and purposes, be continuations of the meetings of which they are adjournments, and the same proceedings may be had at such adjourned meetings as at the meeting of which they are adjournments.
Section 110.250. Rules of Order.
[CC 1995 §115.090; CC 1974 §24.065]
Except as otherwise provided by law or ordinance, the proceedings of the Board of Aldermen shall be controlled by Robert's Rules of Order, as revised.
Section 110.260. Decorum.
[CC 1995 §115.100; CC 1974 §24.070]
The Presiding Officer of the Board of Aldermen shall preserve decorum and shall decide all questions of order subject to appeal to the Board of Aldermen. Any member may appeal to the Board from a ruling of the Presiding Officer upon a question of order. If the motion for an appeal is seconded, the member making the appeal may briefly state his/her reason for the same and the Presiding Officer may briefly express his/her ruling, but there shall be no debate on the appeal and no other member shall participate in the discussion. The Presiding Officer shall then put the question to vote as to whether the decision of the Chair shall be sustained. If a majority of the members present vote "aye", the ruling of the Chair is sustained; otherwise, it is overruled.
Section 110.270. Voting.
[CC 1995 §115.110; CC 1974 §24.075]
Every member of the Board shall vote upon every question and when requested by any two (2) members the vote upon any question shall be taken by "ayes" and "nays" and be recorded.
Section 110.280. Permission Required for Members to Leave Chamber.
[CC 1995 §115.120; CC 1974 §24.080]
No member of the Board of Aldermen may leave the Board Chamber while in regular or special session without permission from the Presiding Officer.
Section 110.290. Order of Business.
[CC 1995 §115.130; CC 1974 §24.090]
A. At the meetings of the Board of Aldermen, the order of business shall be as follows:
1. Call the meeting to order.
2. Roll call.
3. Petitions, remonstrances, complaints and requests and the hearing of any person or group desiring to address the Board. All petitions, remonstrances, complaints and requests shall be presented to the Board in writing. However, the Board may decide to act on oral discussions of any petitions, remonstrances, complaints or requests presented by the interested parties appearing in open meeting. In this case such discussion by the interested parties shall be limited to five (5) minutes per
speaker, except where an extension of time for oral discussion is granted by the Board.
4. Opening of bids.
5. Public hearing as required by law or ordinance.
6. Reading and acting upon unapproved minutes of previous meetings.
7. Acting on unfinished business. The unfinished business from the last preceding meeting shall take precedence over any new business.
8. Reading of communications. All communications which in any manner whatever pertain to the business or functions of the City or any of its elected or appointed employees shall be read.
9. Reports of special boards, committees, and City Officers.
10. Resolutions which require action by the Board.
11. Reading of bills requiring a second (2nd) reading for passage.
12. Introduction and first (1st) reading of bills.
13. Miscellaneous business.
14. Adjournment.
Section 110.300. Expression of Dissent or Protest by Member.
[CC 1995 §115.140; CC 1974 §24.100]
Any member of the Board of Aldermen shall have the right to express dissent from or protest against any ordinance or resolution of the Board and to have the reason therefor entered upon the journal. Such dissent or protest must be filed in writing and presented to the Board not later than the next regular meeting following the date of the passage of the ordinance or resolution to which objection is taken.
Section 110.310. Procedure as to Objections Upon Second Reading of Bill.
[CC 1995 §115.170; CC 1974 §24.130]
Upon the announcement of a second (2nd) reading of any bill, if there are objections to it, the question shall be put by the Chairman, "Shall the proposal be rejected?" If a majority of the Board vote in favor of rejection, the bill shall be defeated.
Section 110.320. When Bills May Be Amended.
[CC 1995 §115.180; CC 1974 §24.140]
Any bill shall be subject to amendment until the vote upon final passage.
Section 110.330. Rules of Procedure.
[CC 1995 §115.210; CC 1974 §24.180]
The Board of Aldermen may by resolution prescribe and enforce such rules as it may find necessary for the expeditious transaction of its business, but such rules shall not contravene the requirements of this Code or other ordinance.
Section 110.340. Amendment or Suspension of Rules.
[CC 1995 §115.230; CC 1974 §24.200]
Any rule of the Board of Aldermen may be repealed, altered or amended by a majority vote of the members. Every amendment offered shall lie on the table until the next meeting of the Board of Aldermen before being voted upon except by the unanimous consent of all elected members of the Board of Aldermen (including the Mayor). Any rule may be suspended by a majority vote of the members of the Board, or quorum being present by unanimous consent.
Section 110.350. Alderman — To Attend Meetings Unless Excused.
[CC 1995 §115.240]
The members of the Board of Aldermen shall attend all meetings of the Board unless leave of absence be granted or unless excused for illness or other special reasons.
Chapter 115
CITY OFFICIALS
ARTICLE I
General Provisions
Section 115.010. Elective Officers — Terms.
Section 115.020. Appointive Officers.
Section 115.030. Removal of Officers.
Section 115.040. Officers to Be Voters and Residents — Exceptions.
Section 115.050. Officers' Oath — Bond.
Section 115.060. Salaries Fixed by Ordinance.
Section 115.070. Vacancies in Certain Offices — How Filled.
Section 115.080. Powers and Duties of Officers to Be Prescribed by Ordinance.
ARTICLE II
City Clerk
Section 115.090. City Clerk — Election — Duties — Term.
Section 115.100. Duties Generally.
Section 115.110. Compensation.
Section 115.120. Temporary City Clerk.
Section 115.130. (Reserved)
ARTICLE III
City Collector
Section 115.140. Appointment.
Section 115.150. Duties Generally.
Section 115.160. Compensation.
Section 115.170. Deputy Collector.
Section 115.180. through Section 115.230. (Reserved)
ARTICLE IV
Chief of Police
Section 115.240. Duties.
Section 115.250. Appointment of Reserve Officers by Chief of Police.
Section 115.260. Chief of Police to Make Report.
ARTICLE V
City Attorney
Section 115.270. Appointment — Term.
Section 115.280. Qualifications.
Section 115.290. Duties Generally.
Section 115.300. Report to Board of Aldermen.
Section 115.310. Temporary Absence — Acting City Attorney.
Section 115.320. Compensation.
ARTICLE VI
Miscellaneous Provisions
Section 115.330. Officers To Report Receipts and Expenditures.
Section 115.340. Mayor or Board May Inspect Books and Records of Officers.
ARTICLE I
General Provisions
Section 115.010. Elective Officers — Terms.
The elective officers of the City and their terms shall be those set out in Section 105.020 of this Code.
Section 115.020. Appointive Officers.
The Mayor, with the consent and approval of the majority of the members of the Board of Aldermen, shall have power to appoint a City Treasurer, City Attorney, Municipal Judge, Chief of Police, City Engineer, Fire Chief, City Collector and such other officers as he/she may be authorized by ordinance to appoint, and if deemed for the best interests of the City, the Mayor and Board of Aldermen may, by ordinance, employ special counsel to represent the City, either in a case of a vacancy in the office of City Attorney or to assist the City Attorney, and pay reasonable compensation therefor.
Section 115.030. Removal of Officers.
A. The Mayor may, with the consent of a majority of all the members elected to the Board of Aldermen, remove from office, for cause shown, any elective officer of the City, such officer being first given opportunity, together with his/her witnesses, to be heard before the Board of Aldermen sitting as a Board of Impeachment. Any elective officer, including the Mayor, may in like manner, for cause shown, be removed from office by a two-thirds (2/3) vote of all members elected to the Board of Aldermen, independently of the Mayor's approval or recommendation. The Mayor may, with the consent of a majority of all the members elected to the Board of Aldermen, remove from office any appointive officer of the City at will, and any such appointive officer may be so removed by a two-thirds (2/3) vote of all the members elected to the Board of Aldermen, independently of the Mayor's approval or recommendation. The Board of Aldermen may pass ordinances regulating the manner of impeachments and removals.
B. Nothing in this Section shall be construed to authorize the Mayor, with the consent of the majority of all the members elected to the Board of Aldermen, or the Board of Aldermen by a two-thirds (2/3) vote of all its members, to remove or discharge any chief, as that term is defined in Section 106.273, RSMo.
Section 115.040. Officers to Be Voters and Residents — Exceptions.
All officers elected to offices or appointed to fill a vacancy in any elective office under the City Government shall be voters under the laws and Constitution of this State and the ordinances of the City except that appointed officers need not be voters of the City. No person shall be elected or appointed to any office who shall at the time be in arrears for any unpaid
City taxes or forfeiture or defalcation in office. All officers, except appointed officers, shall be residents of the City.
Section 115.050. Officers' Oath — Bond.
Every officer of the City and his/her assistants and every Alderman, before entering upon the duties of his/her office, shall take and subscribe to an oath or affirmation before some court of record in the County, or the City Clerk, that he/she possesses all the qualifications prescribed for his/her office by law; that he/she will support the Constitution of the United States and of the State of Missouri, the provisions of all laws of this State affecting Cities of this class, and the ordinances of the City, and faithfully demean himself/herself while in office; which official oath or affirmation shall be filed with the City Clerk. Every officer of the City, when required by law or ordinance, shall, within fifteen (15) days after his/her appointment or election, and before entering upon the discharge of the duties of his/her office, give bond to the City in such sum and with such sureties as may be designated by ordinance, conditioned upon the faithful performance of his/her duty, and that he/she will pay over all monies belonging to the City, as provided by law, that may come into his/her hands. If any person elected or appointed to any office shall fail to take and subscribe such oath or affirmation or to give bond as herein required, his/her office shall be deemed vacant. For any breach of condition of any such bond, suit may be instituted thereon by the City, or by any person in the name of the City, to the use of such person. The bond provisions of this Section may be satisfied by the securing of a blanket bond or blanket bonds, approved by the Board of Aldermen, covering such officers by name or position.
Section 115.060. Salaries Fixed by Ordinance.
[CC 1995 §110.080; CC 1974 §21.150]
The Board of Aldermen shall fix the compensation of all the officers and employees of the City by ordinance. The salary of an officer shall not be changed during the time for which he/she was elected or appointed. In addition to the fees allowed by this Code or other law or ordinance, the City Officers shall receive such compensation for their services as the Board of Aldermen shall from time to time provide.
Section 115.070. Vacancies in Certain Offices — How Filled.
If a vacancy occurs in any elective office, the Mayor or the person exercising the duties of the Mayor shall cause a special meeting of the Board of Aldermen to convene where a successor to the vacant office shall be selected by appointment by the Mayor with the advice and consent of a majority of the remaining members of the Board of Aldermen. If the vacancy is in the office of Mayor, nominations of a successor may be made by any member of the Board of Aldermen and selected with the consent of a majority of the members of the Board of Aldermen. The Board of Aldermen may adopt procedures to fill vacancies consistent with this Section. The successor shall serve until the next regular municipal election. If a vacancy occurs in any office not elective, the Mayor shall appoint a suitable person to discharge the duties of such office until the first (1st) regular meeting of the Board of Aldermen thereafter, at which time such vacancy shall be permanently filled.
Section 115.080. Powers and Duties of Officers to Be Prescribed by Ordinance.
The duties, powers and privileges of officers of every character in any way connected with the City Government, not herein defined, shall be prescribed by ordinance. Bonds may be required of any such officers for faithfulness in office in all respects.
ARTICLE II
City Clerk
Section 115.090. City Clerk — Election — Duties — Term.
The Board of Aldermen shall elect a Clerk for such Board, to be known as "the City Clerk", whose duties and term of office shall be fixed by ordinance. Among other things, the City Clerk shall keep a journal of the proceedings of the Board of Aldermen. He/she shall safely and properly keep all the records and papers belonging to the City which may be entrusted to his/her care; he/she shall be the general accountant of the City; he/she is hereby empowered to administer official oaths and oaths to persons certifying to demands or claims against the City.
Section 115.100. Duties Generally.
[CC 1995 §110.300; CC 1974 §21.420]
A. The City Clerk shall, in addition to other duties which are or may be required of him/her by this Code or other ordinance, attend all meetings of the Board of Aldermen.
1. He/she shall have the custody of the books, records, papers and documents belonging to the City.
2. He/she shall prepare all certificates of election or appointment of the City Officers, and deliver the same to the persons elected or appointed.
3. He/she shall countersign all City bonds, warrants, drafts and orders upon the Treasury for money, and shall see that all ordinances appropriating money out of the Treasury are endorsed by the Treasurer before passage, and shall affix thereto the Seal of the City and keep a record thereof showing the number, date and amount thereof, the name of the person to whom, and on what account issued, and when redeemed.
4. He/she shall record the certificates, oaths and bonds of all the City Officers.
5. He/she shall keep an index of the records of the proceedings of the Board of Aldermen.
6. He/she shall prepare semi-annually a statement of the receipts and expenditures of the City, and cause the same to be published in a newspaper published in the City.
7. He/she shall prepare blank licenses for all purposes for which licenses are required to be issued, and when required, shall cause the same to be issued, signing his/her name and affixing the Seal of the City thereto, and shall keep an account with the Collector for such licenses and the amount of the license tax thereon.
8. He/she shall furnish without delay to any person, when called upon during business hours to do so, certified copies of any records, books, or papers which are in his/her custody, for which services a reasonable fee to be set by City ordinance may be charged, and which shall be paid by the person demanding such certified copy into the Treasury of the City.
Section 115.110. Compensation.
[CC 1995 §110.310; CC 1974 §21.430]
The salary of the City Clerk shall be such as from time to time the Board of Aldermen shall fix by ordinance as a certain amount per month.
Section 115.120. Temporary City Clerk.
[CC 1995 §110.320; CC 1974 §21.440]
A. Upon temporary disability or inability of the City Clerk to perform his/her duties as set forth in this Code or other ordinances of the City due to illness, absence from the City or other cause, the Board of Aldermen shall, in the same manner as the City Clerk is elected as set forth in Section 115.090, proceed to elect a temporary City Clerk who shall have the same powers and duties as the regular City Clerk and shall hold office until the disability of the City Clerk is removed.
B. Such temporary City Clerk shall receive as compensation such salary as the Board of Aldermen shall provide for at the time of the election of such officer.
Section 115.130. (Reserved)
ARTICLE III
City Collector
Section 115.140. Appointment.
The Mayor with the approval of a majority of the members of the Board of Aldermen shall appoint a City Collector.
Section 115.150. Duties Generally.
[CC 1995 §110.330; CC 1974 §21.520; Ord. No. 45-042 §21.550, 2-8-1982]
A. The Collector shall perform all the duties specified in this Code and shall perform such other duties as may be directed by the City Clerk and/or Mayor.
B. The hours of the City Collector's position are from 9:00 A.M. to 4:30 P.M., Monday through Friday.
Section 115.160. Compensation.
[CC 1995 §110.340; CC 1974 §21.530]
The City Collector shall receive as compensation for his/her services a fee or percentage, as may be fixed by ordinance.
Section 115.170. Deputy Collector.
[CC 1995 §110.350; CC 1974 §21.540]
The Mayor may appoint a Deputy Collector to be approved by the Board of Aldermen, and when such Deputy Collector shall have taken and subscribed to the oath provided by this Code, he/she shall possess all the qualifications and powers and be charged with the same duties as the Collector.
Section 115.180. through Section 115.230. (Reserved)
ARTICLE IV
Chief of Police
Section 115.240. Duties.
[CC 1995 §110.420; CC 1974 §21.800]
The Chief of Police shall perform all duties required of the City Marshal by law.
Section 115.250. Appointment of Reserve Officers by Chief of Police.
[CC 1995 §110.430; Ord. No. 45-048 §21.850, 6-30-1982]
A. The Chief of Police may appoint, subject to the approval of a majority of the Board of Aldermen, one (1) or more assistants, not to exceed four (4) in number, who shall have the same police powers as the Marshal, and shall perform any and all duties proposed upon them, and whose term of office shall continue at the pleasure of the Board of Aldermen, and shall be entitled to such compensation as a majority of the Board of Aldermen shall fix.
B. Upon appointment, a police commission card shall be issued to the officers, signed jointly by the Chief of Police and the Mayor.
1. The Chief of Police may further appoint, subject to the approval of a majority of the Board of Aldermen, one (1) or more assistants, not to exceed twelve (12) in number, who shall serve as Reserve Police Officers, and who shall have the same police powers as the Marshal, and shall perform any and all duties imposed upon them, and whose term of office shall continue at the pleasure of the Board of Aldermen.
2. Upon appointment, a police commission card shall be issued to the officers, signed jointly by the Chief of Police and the Mayor.
Section 115.260. Chief of Police to Make Report.
[CC 1995 §110.440]
The Chief of Police shall attend and make a report to the Board of Aldermen of his/her department's activities during the preceding month at the formal meeting of the Board of Aldermen. In the event the Chief of Police is unable to attend, he/she shall cause a member of his/her department to attend and deliver said report.
ARTICLE V
City Attorney
Section 115.270. Appointment — Term.
[CC 1995 §110.220; CC 1974 §21.300]
The Mayor, with the advice and consent of the Board of Aldermen, at the first (1st) meeting after each annual City election shall appoint a suitable person as City Attorney who shall hold office for one (1) year, unless sooner removed from office, and until his/her successor is appointed and qualified.
Section 115.280. Qualifications.
[CC 1995 §110.230; CC 1974 §21.310]
No person shall be appointed to the office of City Attorney unless he/she be a licensed and practicing attorney at law in this State.
Section 115.290. Duties Generally.
[CC 1995 §110.240; CC 1974 §21.320]
A. The City Attorney shall, in addition to his/her other duties which are or may be required by this Code or other ordinance, prepare all charges or complaints against any party, or parties, charged with violation of this Code or other ordinance of the City, and, when ordered by the Mayor or Board of Aldermen to do so, to prosecute or defend all suits and actions originating or pending in any court of this State, to which the City is a party, or in which the City is interested.
1. It shall be the duty of the City Attorney to prosecute all persons charged with a violation of this Code or other ordinance of the City.
2. The City Attorney shall make, and he/she is hereby authorized and empowered to make, affidavits on behalf of the City in all cases where the same may be necessary in taking an appeal or change of venue or any other matter necessary to proper legal proceedings.
3. The City Attorney shall give his/her opinion to all City Officials.
Section 115.300. Report to Board of Aldermen.
[CC 1995 §110.250; CC 1974 §21.330]
A. The City Attorney shall attend meetings of the Board of Aldermen when called upon to do so by the Mayor or majority of the Board of Aldermen. Any member of the Board of Aldermen may at any time call upon the City Attorney for an oral or written opinion to decide any question of law, but not to decide upon any parliamentary rules or to resolve any dispute over the propriety of proposed legislative action.
B. The City Attorney may be requested to report to the Board of Aldermen the condition of any matters pending or unsettled in the City Municipal Court, or any other proceeding pending in any other court of which he/she may have charge under orders of the Mayor or Board of Aldermen.
Section 115.310. Temporary Absence — Acting City Attorney.
[CC 1995 §110.260; CC 1974 §21.340]
In case of absence, sickness or other inability of the City Attorney to attend court, or when, before assuming his/her official duties, he/she shall have been counsel adverse to the City, he/she shall inform the Mayor thereof, in writing, and the Mayor shall appoint some other attorney to represent the City in such cases, or during temporary absence, sickness or inability. Should the City Attorney fail, neglect, or refuse to give such notice, as above provided, and the interests of the City in case of such failure, neglect or refusal, need the immediate services of an attorney, then the Mayor may appoint some other attorney to attend to such cases, who shall receive the compensation allowed to the City Attorney for like services.
Section 115.320. Compensation.
[CC 1995 §110.270; CC 1974 §21.350]
A. The City Attorney shall be allowed compensation such as from time to time shall be fixed by the Board of Aldermen. The City Attorney shall not receive compensation contingent upon the outcome of any case in the Municipal Court.
B. In the event of a case in which the City is interested being tried in any Circuit Court, Supreme Court or Court of Appeal, then the Board of Aldermen shall allow the City Attorney the usual and customary fees and necessary expenses allowed in like or similar cases.
ARTICLE VI
Miscellaneous Provisions
Section 115.330. Officers To Report Receipts and Expenditures.
It shall be the duty of all the officers of the City to report annually to the Board of Aldermen, such reports to embrace a full statement of the receipts and expenditures of their respective
offices and such other matters as may be required by the Board of Aldermen by ordinance, resolution or otherwise.
Section 115.340. Mayor or Board May Inspect Books and Records of Officers.
The Mayor or Board of Aldermen shall have power, as often as he/she or they may deem it necessary, to require any officer of the City to exhibit his/her accounts or other papers or records and to make report to the Board of Aldermen, in writing, touching any matter relating to his/her office.
Chapter 120
OPEN MEETINGS AND RECORDS POLICY
ARTICLE I
In General
Section 120.010. Definitions.
Section 120.020. Meetings, Records and Votes To Be Public — Exceptions.
Section 120.030. Electronic Transmissions — Public Record — When.
Section 120.040. Notices of Meetings.
Section 120.045. Notice Required for Public Meeting on Tax Increases, Eminent Domain, Creation of Certain Districts, and Certain Redevelopment Plans.
Section 120.050. Closed Meetings — How Held.
Section 120.060. Journals of Meetings and Records of Voting.
Section 120.070. Accessibility of Meetings.
Section 120.080. Segregation of Exempt Material.
Section 120.090. Custodian Designated — Response to Request for Access to Records.
Section 120.100. Fees for Copying Public Records — Limitations.
ARTICLE II
Law Enforcement Arrest Reports and Records, Incident Reports, Etc.
Section 120.110. Definitions.
Section 120.120. Police Department Records.
Section 120.130. Effect of Nolle Pros, Dismissal and Suspended Imposition of Sentence on Records.
Section 120.140. Public Access of Closed Arrest Records.
Section 120.150. "911" Telephone Reports.
Section 120.160. Daily Log or Record Maintained by Police Department of Crimes, Accidents or Complaints — Public Access to Certain Information.
ARTICLE I
In General
Section 120.010. Definitions.
As used in this Chapter, unless the context otherwise indicates, the following terms mean:
CLOSED MEETING, CLOSED RECORD or CLOSED VOTE — Any meeting, record or vote closed to the public.
COPYING — If requested by a member of the public, copies provided as detailed in Section 120.100 of this Chapter, if duplication equipment is available.
PUBLIC BUSINESS — All matters which relate in any way to performance of the City's functions or the conduct of its business.
PUBLIC GOVERNMENTAL BODY — Any legislative, administrative or governmental entity created by the Constitution or Statutes of this State, orders or ordinances of the City, judicial entities when operating in an administrative capacity or by executive order, including:
1. Any advisory committee or commission appointed by the Mayor or Board of Aldermen.
2. Any department or division of the City.
3. Any other legislative or administrative governmental deliberative body under the direction of three (3) or more elected or appointed members having rulemaking or quasi-judicial power.
4. Any committee appointed by or at the direction of any of the entities and which is authorized to report to any of the above-named entities, any advisory committee appointed by or at the direction of any of the named entities for the specific purpose of recommending, directly to the public governmental body's governing board or its Chief Administrative Officer, policy or policy revisions or expenditures of public funds.
5. Any quasi-public governmental body. The term "quasi-public governmental body" means any person, corporation or partnership organized or authorized to do business in this State pursuant to the provisions of Chapters 352, 353 or 355, RSMo., or unincorporated association which either:
a. Has as its primary purpose to enter into contracts with public governmental bodies or to engage primarily in activities carried out pursuant to an agreement or agreements with public governmental bodies; or
b. Performs a public function, as evidenced by a statutorily or ordinance-based capacity, to confer or otherwise advance, through approval, recommendation or other means, the allocation or issuance of tax credits, tax abatement, public debt, tax exempt debt, rights of eminent domain, or the contracting of lease-back agreements on structures whose annualized payments commit public tax revenues; or any association that directly accepts the appropriation of money from the City, but only to the extent that a meeting, record or vote relates to such appropriation.
PUBLIC MEETING — Any meeting of a public governmental body subject to this Chapter at which any public business is discussed, decided or public policy formulated, whether such meeting is conducted in person or by means of communication equipment including, but not limited to, conference call, video conference, Internet chat or Internet message board. The term "public meeting" shall not include an informal gathering of members of a public governmental body for ministerial or social purposes when there is no intent to avoid the purposes of this Chapter, but the term shall include a vote of all or a majority of the members of a public governmental body, by electronic communication or any other means, conducted in lieu of holding a public meeting with the members of the public governmental body gathered at one (1) location in order to conduct public business.
PUBLIC RECORD — Any record, whether written or electronically stored, retained by or of any public governmental body including any report, survey, memorandum, or other document
or study prepared for the public governmental body by a consultant or other professional service paid for in whole or in part by public funds, including records created or maintained by private contractors under an agreement with a public governmental body or on behalf of a public governmental body. The term "public record" shall not include any internal memorandum or letter received or prepared by or on behalf of a member of a public governmental body consisting of advice, opinions and recommendations in connection with the deliberative decision-making process of said body, unless such records are retained by the public governmental body or presented at a public meeting. Any documents or study prepared for a public governmental body by a consultant or other professional service as described in this Subdivision shall be retained by the public governmental body in the same manner as any other public record.
PUBLIC VOTE — Any vote, whether conducted in person, by telephone, or by any other electronic means, cast at any public meeting of any public governmental body.
Section 120.020. Meetings, Records and Votes To Be Public — Exceptions.
A. All meetings, records and votes are open to the public, except that any meeting, record or vote relating to one (1) or more of the following matters, as well as other materials designated elsewhere in this Chapter, shall be closed unless the public governmental body votes to make them public:
1. Legal actions, causes of action or litigation involving a public governmental body and any confidential or privileged communications between a public governmental body or its representatives and its attorneys. However, any minutes, vote or settlement agreement relating to legal actions, causes of action or litigation involving a public governmental body or any agent or entity representing its interests or acting on its behalf or with its authority, including any insurance company acting on behalf of a public governmental body as its insured, shall be made public upon final disposition of the matter voted upon or upon the signing by the parties of the settlement agreement, unless, prior to final disposition, the settlement agreement is ordered closed by a court after a written finding that the adverse impact to a plaintiff or plaintiffs to the action clearly outweighs the public policy considerations of Section 610.011, RSMo., however, the amount of any monies paid by, or on behalf of, the public governmental body shall be disclosed; provided however, in matters involving the exercise of the power of eminent domain, the vote shall be announced or become public immediately following the action on the motion to authorize institution of such a legal action. Legal work product shall be considered a closed record.
2. Leasing, purchase or sale of real estate by a public governmental body where public knowledge of the transaction might adversely affect the legal consideration therefor. However, any minutes or vote or public record approving a contract relating to the leasing, purchase or sale of real estate by a public governmental body shall be made public upon execution of the lease, purchase or sale of the real estate.
3. Hiring, firing, disciplining or promoting of particular employees by a public governmental body when personal information about the employee is discussed or
recorded. However, any vote on a final decision, when taken by a public governmental body, to hire, fire, promote or discipline an employee of a public governmental body shall be made available with a record of how each member voted to the public within seventy-two (72) hours of the close of the meeting where such action occurs; provided however, that any employee so affected shall be entitled to prompt notice of such decision during the seventy-two (72) hour period before such decision is made available to the public. As used in this Subsection, the term "personal information" means information relating to the performance or merit of individual employees.
4. Non-judicial mental or physical health proceedings involving an identifiable person, including medical, psychiatric, psychological, or alcoholism or drug dependency diagnosis or treatment.
5. Testing and examination materials, before the test or examination is given or, if it is to be given again, before so given again.
6. Welfare cases of identifiable individuals.
7. Preparation, including any discussions or work product, on behalf of a public governmental body or its representatives for negotiations with employee groups.
8. Software codes for electronic data processing and documentation thereof.
9. Specifications for competitive bidding, until either the specifications are officially approved by the public governmental body or the specifications are published for bid.
10. Sealed bids and related documents, until the bids are opened; and sealed proposals and related documents or any documents related to a negotiated contract until a contract is executed, or all proposals are rejected.
11. Individually identifiable personnel records, performance ratings or records pertaining to employees or applicants for employment, except that this exemption shall not apply to the names, positions, salaries and lengths of service of officers and employees of public agencies once they are employed as such.
12. Records which are protected from disclosure by law.
13. Meetings and public records relating to scientific and technological innovations in which the owner has a proprietary interest.
14. Records relating to municipal hotlines established for the reporting of abuse and wrongdoing.
15. Confidential or privileged communications between a public governmental body and its auditor, including all auditor work product; however, all final audit reports issued by the auditor are to be considered open records pursuant to this Chapter.
16. Operational guidelines, policies and specific response plans developed, adopted, or maintained by any public agency responsible for law enforcement, public safety, first response, or public health for use in responding to or preventing any critical
incident which is or appears to be terrorist in nature and which has the potential to endanger individual or public safety or health. Financial records related to the procurement of or expenditures relating to operational guidelines, policies or plans purchased with public funds shall be open. When seeking to close information pursuant to this exception, the public governmental body shall affirmatively state in writing that disclosure would impair the public governmental body's ability to protect the security or safety of persons or real property, and shall in the same writing state that the public interest in non-disclosure outweighs the public interest in disclosure of the records.
17. Existing or proposed security systems and structural plans of real property owned or leased by a public governmental body, and information that is voluntarily submitted by a non-public entity owning or operating an infrastructure to any public governmental body for use by that body to devise plans for protection of that infrastructure, the public disclosure of which would threaten public safety.
a. Records related to the procurement of or expenditures relating to security systems purchased with public funds shall be open.
b. When seeking to close information pursuant to this exception, the public governmental body shall affirmatively state in writing that disclosure would impair the public governmental body's ability to protect the security or safety of persons or real property and shall in the same writing state that the public interest in non-disclosure outweighs the public interest in disclosure of the records.
c. Records that are voluntarily submitted by a non-public entity shall be reviewed by the receiving agency within ninety (90) days of submission to determine if retention of the document is necessary in furtherance of a State security interest. If retention is not necessary, the documents shall be returned to the non-public governmental body or destroyed.
18. The portion of a record that identifies security systems or access codes or authorization codes for security systems of real property.
19. Records that identify the configuration of components or the operation of a computer, computer system, computer network or telecommunications network and would allow unauthorized access to or unlawful disruption of a computer, computer system, computer network or telecommunications network of a public governmental body. This exception shall not be used to limit or deny access to otherwise public records in a file, document, data file or database containing public records. Records related to the procurement of or expenditures relating to such computer, computer system, computer network or telecommunications network, including the amount of monies paid by, or on behalf of, a public governmental body for such computer, computer system, computer network or telecommunications network, shall be open.
20. Credit card numbers, personal identification numbers, digital certificates, physical and virtual keys, access codes or authorization codes that are used to protect the security of electronic transactions between a public governmental body and a
person or entity doing business with a public governmental body. Nothing in this Section shall be deemed to close the record of a person or entity using a credit card held in the name of a public governmental body or any record of a transaction made by a person using a credit card or other method of payment for which reimbursement is made by a public governmental body.
Section 120.030. Electronic Transmissions — Public Record — When.
Any member of a public governmental body who transmits any message relating to public business by electronic means shall also concurrently transmit that message to either the member's public office computer or the custodian of records in the same format. The provisions of this Section shall only apply to messages sent to two (2) or more members of that body so that, when counting the sender, a majority of the body's members are copied. Any such message received by the custodian or at the member's office computer shall be a public record subject to the exception of Section 610.021, RSMo.
Section 120.040. Notices of Meetings.
A. All public governmental bodies shall give notice of the time, date and place of each meeting and its tentative agenda in a manner reasonably calculated to advise the public of the matters to be considered, and if the meeting will be conducted by telephone or other electronic means, the notice of the meeting shall identify the mode by which the meeting will be conducted and the designated location where the public may observe and attend the meeting. If a public body plans to meet by Internet chat, Internet message board or other computer link, it shall post a notice of the meeting on its website in addition to its principal office and shall notify the public how to access that meeting. Reasonable notice shall include making available copies of the notice to any representative of the news media who requests notice of meetings of a particular public governmental body concurrent with the notice being made available to the members of the particular governmental body and posting the notice on a bulletin board or other prominent place which is easily accessible to the public and clearly designated for that purpose at the principal office of the body holding the meeting, or if no such office exists, at the building in which the meeting is to be held.
B. Notice conforming with all of the requirements of Subsection (A) of this Section shall be given at least twenty-four (24) hours, exclusive of weekends and holidays when City Hall is closed, prior to the commencement of any meeting of a governmental body unless for good cause such notice is impossible or impractical, in which case as much notice as is reasonably possible shall be given.
C. The City shall allow for the recording by audiotape, videotape or other electronic means of any open meeting. The City may establish guidelines regarding the manner in which such recording is conducted so as to minimize disruption to the meeting. No audio recording of any meeting, record or vote closed pursuant to the provisions of Section 120.020 shall be permitted without permission of the City; any person who violates this provision shall be guilty of an ordinance violation.
D. Each governmental body proposing to hold a closed meeting or vote shall give notice of the time, date and place of such closed meeting or vote and the reason for holding it by reference to a specific exception allowed pursuant to Section 120.020 hereof. The notice shall be the same as described in Subsection (A) herein.
E. A formally constituted subunit of a parent governmental body may conduct a meeting without notice during a lawful meeting of the parent governmental body, a recess in that meeting, or immediately following that meeting if the meeting of the subunit is publicly announced at the parent meeting and the subject of the meeting reasonably coincides with the subjects discussed or acted upon by the parent governmental body.
Section 120.045. Notice Required for Public Meeting on Tax Increases, Eminent Domain, Creation of Certain Districts, and Certain Redevelopment Plans.
For any public meeting where a vote of the Board of Aldermen is required to implement a tax increase, or with respect to a retail development project when the Board of Aldermen votes to utilize the power of eminent domain, create a transportation development district or a community improvement district, or approve a redevelopment plan that pledges public funds as financing for the project or plan, the Board of Aldermen, or any entity created by the City, shall give notice conforming with all the requirements of Subsection (1) of Section 610.020, RSMo., at least four (4) days before such entity may vote on such issues, exclusive of weekends and holidays when the facility is closed; provided that this Section shall not apply to any votes or discussion related to proposed ordinances which require a minimum of two (2) separate readings on different days for their passage. The provisions of Subsection (4) of Section 610.020, RSMo., shall not apply to any matters that are subject to the provisions of this Section. No vote shall occur until after a public meeting on the matter at which parties in interest and citizens shall have an opportunity to be heard. If the notice required under this Section is not properly given, no vote on such issues shall be held until proper notice has been provided under this Section. Any legal action challenging the notice requirements provided herein shall be filed within thirty (30) days of the subject meeting, or such meeting shall be deemed to have been properly noticed and held. For the purpose of this Section, a tax increase shall not include the setting of the annual tax rates provided for under Sections 67.110 and 137.055, RSMo.
Section 120.050. Closed Meetings — How Held.
A. Except as set forth in Subsection (D) of Section 120.040, no meeting or vote may be closed without an affirmative public vote of the majority of a quorum of the public governmental body. The vote of each member of the governmental body on the question of closing a public meeting or vote and the specific reason for closing that public meeting or vote by reference to a specific Section of this Chapter shall be announced publicly at an open meeting of the governmental body and entered into the minutes.
B. Any meeting or vote closed pursuant to Section 120.020 shall be closed only to the extent necessary for the specific reason announced to justify the closed meeting or vote. Public governmental bodies shall not discuss any business in a closed meeting, record or vote which does not directly relate to the specific reason announced to justify the closed meeting or vote. Public governmental bodies holding a closed meeting shall close only an
existing portion of the meeting facility necessary to house the members of the public governmental body in the closed session, allowing members of the public to remain to attend any subsequent open session held by the public governmental body following the closed session.
Section 120.060. Journals of Meetings and Records of Voting.
A. Except as provided in Section 120.020, rules authorized pursuant to Article III of the Missouri Constitution and as otherwise provided by law, all votes shall be recorded, and if a roll call is taken, as to attribute each "yea" and "nay" vote, or abstinence if not voting, to the name of the individual member of the public governmental body. Any votes taken during a closed meeting shall be taken by roll call. All public meetings shall be open to the public and public votes and public records shall be open to the public for inspection and duplication. All votes taken by roll call in meetings of a public governmental body, consisting of members who are all elected, shall be cast by members of the public governmental body who are physically present and in attendance at the meeting or who are participating via video-conferencing. When it is necessary to take votes by roll call in a meeting of the public governmental body, due to an emergency of the public body, with a quorum of the members of the public body physically present and in attendance and less than a quorum of the members of the public governmental body participating via telephone, facsimile, Internet, or any other voice or electronic means, the nature of the emergency of the public body justifying that departure from the normal requirements shall be stated in the minutes. Where such emergency exists, the votes taken shall be regarded as if all members were physically present and in attendance at the meeting.
B. A journal or minutes of open and closed meetings shall be taken and retained by the public governmental body including, but not limited to, a record of any vote taken at such meeting. The minutes shall include the date, time, place, members present, members absent, and a record of votes taken. When a roll call vote is taken, the minutes shall attribute each "yea" and "nay" vote, or abstinence if not voting, to the name of the individual member of the public governmental body.
Section 120.070. Accessibility of Meetings.
A. Each meeting shall be held at a place reasonably accessible to the public and of sufficient size to accommodate the anticipated attendance by members of the public and at a time reasonably convenient to the public unless for good cause such a place or time is impossible or impractical. Every reasonable effort shall be made to grant special access to the meeting to handicapped or disabled individuals.
B. When it is necessary to hold a meeting on less than twenty-four (24) hours' notice, or at a place that is not reasonably accessible to the public, or at a time that is not reasonably convenient to the public, the nature of the good cause justifying that departure from the normal requirements shall be stated in the minutes.
Section 120.080. Segregation of Exempt Material.
If a public record contains material which is not exempt from disclosure, as well as material which is exempt from disclosure, the custodian shall separate the exempt and non-exempt material and make the non-exempt material available for examination and copying in accord with the policies provided herein. When designing a public record the custodian shall, to the extent practicable, facilitate a separation of exempt from non-exempt information. If the separation is readily apparent to a person requesting to inspect or receive copies of the form, the custodian shall generally describe the material exempted unless that description would reveal the contents of the exempt information and thus defeat the purpose of the exemption.
Section 120.090. Custodian Designated — Response to Request for Access to Records.
A. The City Clerk shall be the custodian of records and will be responsible for maintenance and control of all records. The custodian may designate deputy custodians in operating departments of the City and such other departments or offices as the custodian may determine. Deputy custodians shall conduct matters relating to public records and meetings in accord with the policies enumerated herein.
B. Each public governmental body shall make available for inspection and copying by the public of that body's public records. No person shall remove original public records from the office of a public governmental body or its custodian without written permission of the designated custodian. No public governmental body shall, after August 28, 1998, grant to any person or entity, whether by contract, license or otherwise, the exclusive right to access and disseminate any public record unless the granting of such right is necessary to facilitate coordination with, or uniformity among, industry regulators having similar authority.
C. Each request for access to a public record shall be acted upon as soon as possible, but in no event later than the end of the third (3rd) business day following the date the request is received by the custodian of records of a public governmental body. If records are requested in a certain format, the public body shall provide the records in the requested format, if such format is available. If access to the public record is not granted immediately, the custodian shall give a detailed explanation of the cause for further delay and the place and earliest time and date that the record will be available for inspection. This period for document production may exceed three (3) days for reasonable cause.
D. If a request for access is denied, the custodian shall provide, upon request, a written statement of the grounds for such denial. Such statement shall cite the specific provision of law under which access is denied and shall be furnished to the requester no later than the end of the third (3rd) business day following the date that the request for the statement is received.
Section 120.100. Fees for Copying Public Records — Limitations.
A. Except as otherwise provided by law, each public governmental body shall provide access to and, upon request, furnish copies of public records subject to the following:
1. Fees for copying public records, except those records restricted under Section 32.091, RSMo., shall not exceed ten cents ($.10) per page for a paper copy not larger than nine (9) by fourteen (14) inches, with the hourly fee for duplicating time not to exceed the average hourly rate of pay for clerical staff of the public governmental body. Research time required for fulfilling records requests may be charged at the actual cost of research time. Based on the scope of the request, the public governmental body shall produce the copies using employees of the body that result in the lowest amount of charges for search, research, and duplication time. Prior to producing copies of the requested records, the person requesting the records may request the public governmental body to provide an estimate of the cost to the person requesting the records. Documents may be furnished without charge or at a reduced charge when the public governmental body determines that waiver or reduction of the fee is in the public interest because it is likely to contribute significantly to public understanding of the operations or activities of the public governmental body and is not primarily in the commercial interest of the requester.
2. Fees for providing access to public records maintained on computer facilities, recording tapes or disks, videotapes or films, pictures, maps, slides, graphics, illustrations or similar audio or visual items or devices, and for paper copies larger than nine (9) by fourteen (14) inches shall include only the cost of copies, staff time, which shall not exceed the average hourly rate of pay for staff of the public governmental body required for making copies and programming, if necessary, and the cost of the disk, tape or other medium used for the duplication. Fees for maps, blueprints or plats that require special expertise to duplicate may include the actual rate of compensation for the trained personnel required to duplicate such maps, blueprints or plats. If programming is required beyond the customary and usual level to comply with a request for records or information, the fees for compliance may include the actual cost of such programming.
B. Payment of such copying fees may be requested prior to the making of copies.
ARTICLE II
Law Enforcement Arrest Reports and Records, Incident Reports, Etc.
Section 120.110. Definitions.
As used in this Article, the following terms shall have the following definitions:
ARREST — An actual restraint of the person of the defendant, or by his/her submission to the custody of the officer, under authority of a warrant or otherwise for a criminal violation which results in the issuance of a summons or the person being booked.
ARREST REPORT — A record of a law enforcement agency of an arrest and of any detention or confinement incident thereto together with the charge therefor.
INACTIVE — An investigation in which no further action will be taken by a law enforcement agency or officer for any of the following reasons:
1. A decision by the law enforcement agency not to pursue the case.
2. Expiration of the time to file criminal charges pursuant to the applicable statute of limitations or ten (10) years after the commission of the offense, whichever date earliest occurs.
3. Finality of the convictions of all persons convicted on the basis of the information contained in the investigative report, by exhaustion of or expiration of all rights of appeal of such persons.
INCIDENT REPORT — A record of a law enforcement agency consisting of the date, time, specific location, name of the victim, and immediate facts and circumstances surrounding the initial report of a crime or incident, including any logs of reported crimes, accidents and complaints maintained by that agency.
INVESTIGATIVE REPORT — A record, other than an arrest or incident report, prepared by personnel of a law enforcement agency inquiring into a crime or suspected crime either in response to an incident report or in response to evidence developed by Law Enforcement Officers in the course of their duties.
Section 120.120. Police Department Records.
A. The Police Department of the City shall maintain records of all incidents reported to the Police Department and investigations and arrests made by the Police Department. All incident reports and arrest reports shall be open records. Notwithstanding any other provision of law other than the provisions of Subsection (C) of this Section or Section 320.083, RSMo., investigative reports of the Police Department are closed records until the investigation becomes inactive. If any person is arrested and not charged with an offense against the law within thirty (30) days of the person's arrest, the arrest report shall thereafter be a closed record except that the disposition portion of the record may be accessed and except as provided in Section 120.140.
B. Except as provided in Subsections (C) and (D) of this Section, if any portion of a record or document of a Police Department Officer or the Police Department, other than an arrest report which would otherwise be open, contains information that is reasonably likely to pose a clear and present danger to the safety of any victim, witness, undercover officer or other person; or jeopardize a criminal investigation, including records which would disclose the identity of a source wishing to remain confidential or a suspect not in custody; or which would disclose techniques, procedures or guidelines for Police Department investigations or prosecutions, that portion of the record shall be closed and shall be redacted from any record made available pursuant to this Chapter.
C. Any person, a family member of such person within the first degree of consanguinity of such person if deceased or incompetent, attorney for a person, or insurer of a person involved in any incident or whose property is involved in an incident may obtain any records closed pursuant to this Section or Section 120.140 for purposes of investigation.
of any civil claim or defense as provided by this Subsection. Any individual, his/her attorney, or insurer involved in an incident or whose property is involved in an incident, upon written request, may obtain a complete unaltered and unedited incident report concerning the incident and may obtain access to other records closed by the Police Department pursuant to this Section. Within thirty (30) days of such request, the Police Department shall provide the requested material or file a motion pursuant to this Subsection with the Circuit Court having jurisdiction over the Police Department stating that the safety of the victim, witness or other individual cannot be reasonably ensured, or that a criminal investigation is likely to be jeopardized. Pursuant to Section 610.100(4), RSMo., if, based on such motion, the court finds for the Police Department, the court shall either order the record closed or order such portion of the record that should be closed to be redacted from any record made available pursuant to this Subsection.
D. Any person may apply pursuant to this Subsection to the Circuit Court having jurisdiction for an order requiring a Law Enforcement Agency to open incident reports and arrest reports being unlawfully closed pursuant to the Section. If the court finds by a preponderance of the evidence that the Law Enforcement Officer or Agency has knowingly violated this Section, the officer or agency shall be subject to a civil penalty in an amount up to one thousand dollars ($1,000.00). If the court finds that there is a knowing violation of this Section, the court may order payment by such officer or agency of all costs and attorneys' fees, as provided by Section 610.027, RSMo. If the court finds by a preponderance of the evidence that the Law Enforcement Officer or Agency has purposely violated this Section, the officer or agency shall be subject to a civil penalty in an amount up to five thousand dollars ($5,000.00) and the court shall order payment by such officer or agency of all costs and attorney fees, as provided in Section 610.027, RSMo. The court shall determine the amount of the penalty by taking into account the size of the jurisdiction, the seriousness of the offense, and whether the Law Enforcement Officer or Agency has violated this Section previously.
E. The victim of an offense as provided in Chapter 566, RSMo., may request that his/her identity be kept confidential until a charge relating to such incident is filed.
Section 120.130. Effect of Nolle Pros, Dismissal and Suspended Imposition of Sentence on Records.
A. If the person arrested is charged but the case is subsequently nolle prossed, dismissed, or the accused is found not guilty, or imposition of sentence is suspended in the court in which the action is prosecuted, official records pertaining to the case shall thereafter be closed records when such case is finally terminated, except as provided in Subsection (B) of this Section and Section 120.140 and except that the court's judgment or order or the final action taken by the prosecutor in such matters may be accessed. If the accused is found not guilty due to mental disease or defect pursuant to Section 552.030, RSMo., official records pertaining to the case shall thereafter be closed records upon such findings, except that the disposition may be accessed only by law enforcement agencies, child care agencies, facilities as defined in Section 198.006, RSMo., and in-home services provider agencies as defined in Section 660.250, RSMo., in the manner established by Section 120.140.
B. If the person arrested is charged with an offense found in Chapter 566, RSMo., Sections 568.045, 568.050, 568.060, 568.065, 568.080, 568.090 or 568.175, RSMo., and an imposition of sentence is suspended in the court in which the action is prosecuted, the official records pertaining to the case shall be made available to the victim for the purpose of using the records in his/her own judicial proceeding or if the victim is a minor to the victim's parents or guardian, upon request.
Section 120.140. Public Access of Closed Arrest Records.
A. Except as otherwise provided under Section 610.124, RSMo., records required to be closed shall not be destroyed; they shall be inaccessible to the general public and to all persons other than the defendant except as provided in this Section and Section 43.507, RSMo. The closed records shall be available to: criminal justice agencies for the administration of criminal justice pursuant to Section 43.500, RSMo., criminal justice employment, screening persons with access to criminal justice facilities, procedures and sensitive information; to law enforcement agencies for issuance or renewal of a license, permit, certification, or registration of authority from such agency including, but not limited to, watchmen, security personnel, private investigators, and persons seeking permits to purchase or possess a firearm; those agencies authorized by Section 43.543, RSMo., to submit and when submitting fingerprints to the central repository; the Sentencing Advisory Commission created in Section 558.019, RSMo., for the purpose of studying sentencing practices in accordance with Section 43.507, RSMo.; to qualified entities for the purpose of screening providers defined in Section 43.540, RSMo.; the Department of Revenue for driver license administration; the Department of Public Safety for the purposes of determining eligibility for crime victims' compensation pursuant to Sections 595.010 to 595.075, RSMo.; Department of Health and Senior Services for the purpose of licensing and regulating facilities and regulating in-home services provider agencies and Federal agencies for purposes of criminal justice administration, criminal justice employment, child, elderly, or disabled care, and for such investigative purposes as authorized by law or presidential executive order.
B. These records shall be made available only for the purposes and to the entities listed in this Section. A criminal justice agency receiving a request for criminal history information under its control may require positive identification, to include fingerprints of the subject of the record search, prior to releasing closed record information. Dissemination of closed and open records from the Missouri criminal records repository shall be in accordance with Section 43.509, RSMo. All records which are closed records shall be removed from the records of the Police Department and Municipal Court which are available to the public and shall be kept in separate records which are to be held confidential and, where possible, pages of the public record shall be retyped or rewritten omitting those portions of the record which deal with the defendant's case. If retyping or rewriting is not feasible because of the permanent nature of the record books, such record entries shall be blacked out and recopied in a confidential book.
Section 120.150. "911" Telephone Reports.
Except as provided by this Section, any information acquired by the Police Department or a first responder agency by way of a complaint or report of a crime made by telephone contact using the emergency number "911" shall be inaccessible to the general public. However, information consisting of the date, time, specific location, and immediate facts and circumstances surrounding the initial report of the crime or incident shall be considered to be an incident report and subject to Section 120.120. Any closed records pursuant to this Section shall be available upon request by law enforcement agencies or the Division of Workers' Compensation or pursuant to a valid court order authorizing disclosure upon motion and good cause shown.
Section 120.160. Daily Log or Record Maintained by Police Department of Crimes, Accidents or Complaints — Public Access to Certain Information.
A. The City of Anderson Police Department, if it maintains a daily log or record that lists suspected crimes, accidents or complaints, shall make available the following information for inspection and copying by the public:
1. The time, substance and location of all complaints or requests for assistance received by the Police Department;
2. The time and nature of the Police Department's response to all complaints or requests for assistance; and
3. If the incident involves an alleged offense or infraction:
a. The time, date and location of occurrence;
b. The name and age of any victim, unless the victim is a victim of a crime under Chapter 566, RSMo.;
c. The factual circumstances surrounding the incident; and
d. A general description of any injuries, property or weapons involved.
Section 125.010. Federal Old-Age and Survivors Insurance.
[CC 1995 §125.010; CC 1974 §§23.010 — 23.050]
A. **Policy.** It is hereby declared to be the policy and purpose of the City of Anderson, Missouri, to extend to all eligible employees and officials of said City who are not excluded by law or by this Section and whether employed in connection with a governmental or proprietary function of said City the benefits of the system of Federal Old-Age and Survivors Insurance as authorized by the Social Security Act Amendments of 1950, and by Senate Committee Substitute for Senate Bill No. 3 of the 66th General Assembly of the State of Missouri and amendments thereof, as the same may be now and hereafter in effect.
B. **Plan Authorized.** The Mayor and City Clerk of the City of Anderson, Missouri, are hereby authorized and directed, on behalf of this City, to prepare, execute and submit to the Office of Administration, Division of Accounting, as State Agency of the State of Missouri, a plan and agreement for extending said benefits to said eligible employees and officials of the City of Anderson, Missouri, in the form prepared by the State Agency and hereby approved and adopted by the Board of Aldermen of this City, which plan and agreement are to become effective upon approval thereof by the State Agency, and are further authorized and directed to execute agreements and modifications and amendments thereof with said State Agency, providing for the extension of said benefits to said employees and officials as set forth in said plan and agreement, as provided for in Subsection (A) hereof, said plan and agreement to provide that said extension of benefits is to be effective on January 1, 1957.
C. Commencing on the first (1st) day of the month following the date of the approval of the plan and agreement of this City by the State Agency, there shall be deducted from the wages of all employees and officials of the City of Anderson, Missouri, to whom the benefits of said system of Federal Old-Age and Survivors Insurance are extended, by virtue of the plan and agreement hereinbefore provided for, the amount of each of said employees' and officials' contributions, as determined by the applicable State and Federal laws and by said plan and agreement, the aggregate amount of said deductions to be paid into the Contributions Fund created by Senate Committee Substitute for Senate Bill No. 3 of the 66th General Assembly of the State of Missouri; provided however, that from the first (1st) payment of wages made to each of said employees and officials after the benefits of said system have been extended to such employees and officials, there shall be deducted a sum equal to the amount which would have been due and payable from each of said employees and officials had said extension of benefits been provided and effective on January 1, 1957.
D. Commencing on the first (1st) day of the month following the date of the approval of the plan and agreement of this City by the State Agency, there is hereby authorized to be appropriated from the General Fund of the City of Anderson, Missouri, and there is, and shall be, appropriated the sum or sums of money necessary to pay the contributions of the City of Anderson, Missouri, which shall be due and payable by virtue of the extension of the benefits of the Federal Old-Age and Survivors Insurance System to the eligible employees and officials of said City, said sum or sums of money to be paid into the Contributions Fund created by Senate Committee Substitute for Senate Bill No. 3 of the 66th General Assembly of the State of Missouri; provided however, that in making the first (1st) payment to said Contributions Fund, after the benefits of said system have been extended to such employees and officials, said first (1st) payment shall include a sum equal to the amount which would have been due and payable had said extension of benefits been provided and effective on January 1, 1957. The fund from which said appropriation is made will, at all times, be sufficient to pay the contributions of the City by this Section directed to be paid to said Contributions Fund.
E. The City of Anderson, Missouri, from and after the approval of the plan and agreement of this City by the State Agency, shall fully comply with, and shall keep such records, make such reports and provide such methods of administration of said plan and agreement as may be required by all applicable State and Federal laws, rules and regulations, now and hereafter in effect with respect to the extension of the benefits of the Federal Old-Age and Survivors Insurance System to the employees and officials of this City. For the purpose of administering said plan and agreement, the City Clerk of this City shall be the official who shall make all required reports, keep all records, and be responsible for the administration of said plan and agreement on behalf of this City, and any and all notices and communications from the State Agency to this City with respect to said plan and agreement shall be addressed to "City Clerk of Anderson, Missouri".
Section 125.020. Workers' Compensation.
[CC 1995 §125.020; CC 1974 §23.060]
The City of Anderson, Missouri, hereby elects to accept the Workers' Compensation Law of the State of Missouri.
Chapter 130
MUNICIPAL COURT
ARTICLE I
General Provisions
Section 130.010. Court Established.
Section 130.020. Jurisdiction.
Section 130.030. Selection of Municipal Judge.
Section 130.040. Municipal Judge — Vacation of Office.
Section 130.050. Municipal Judge — Qualifications for Office.
Section 130.060. Superintending Authority.
Section 130.070. Report to Board of Aldermen.
Section 130.080. Docket and Court Records.
Section 130.090. Municipal Judge — Powers and Duties Generally.
Section 130.100. Prosecutions Based on Information Only, Proceedings.
Section 130.110. Violations Bureau.
Section 130.120. Issuance and Execution of Warrants.
Section 130.130. Arrests Without Warrants.
Section 130.140. Jury Trials.
Section 130.150. Duties of the City's Prosecuting Attorney.
Section 130.160. Summoning of Witnesses.
Section 130.170. Municipal Judge Without Jurisdiction, When.
Section 130.180. Jailing of Defendants.
Section 130.190. Parole and Probation.
Section 130.200. Right of Appeal.
Section 130.210. Appeal From Jury Verdicts.
Section 130.220. Breach of Recognizance.
Section 130.230. Disqualification of Municipal Judge From Hearing a Particular Case.
Section 130.240. Absence of Judge — Procedure.
Section 130.250. Failure To Appear in Municipal Court.
Section 130.260. (Reserved)
ARTICLE II
Court Clerk
Section 130.270. Office Established.
Section 130.280. Selection and Term of Court Clerk.
Section 130.290. Hours and Authorization of Compensation.
Section 130.300. Bond.
Section 130.310. Clerk of Court — Duties.
Section 130.320. Clerk of Court — Powers.
ARTICLE III
Fines and Court Costs
Section 130.330. Installment Payment of Fine.
Section 130.340. Court Costs.
ARTICLE I
General Provisions
Section 130.010. Court Established.
There is hereby established in the City of Anderson a Municipal Court to be known as the "Anderson Municipal Court, a Division of the 40th Judicial Circuit Court of the State of Missouri." In the event a Police Court existed prior to the establishment of a Municipal Court, this Court is a continuation of the Police Court of the City as previously established and is termed herein "The Municipal Court."
Section 130.020. Jurisdiction.
Violations of municipal ordinances shall be heard and determined only before Divisions of the Circuit Court as hereinafter provided in this Chapter. "Heard and determined," for purposes of this Chapter, shall mean any process under which the Court in question retains the final authority to make factual determinations pertaining to allegations of a municipal ordinance violation.
Section 130.030. Selection of Municipal Judge.
[CC 1995 §130.040]
A. The Judge of the City's Municipal Court shall be known as a Municipal Judge of the 39th Judicial Circuit Court and shall be selected by appointment to the position by the Mayor with approval of a majority of the members of the Board of Aldermen for a term as specified herein.
B. The Municipal Judge shall hold his/her office for a period of two (2) years and shall take office biannually from April, 1994. If for any reason a Municipal Judge vacates his/her office, his/her successor shall complete that term of office, even if the same be for less than two (2) years.
Section 130.040. Municipal Judge — Vacation of Office.
A. The Municipal Judge shall vacate his/her office under the following conditions:
1. Upon removal from office by the State Commission on the Retirement, Removal and Discipline of Judges as provided in Missouri Supreme Court Rule 12;
2. Upon attaining his/her seventy-fifth (75th) birthday; or
1. State Law Reference — As to certain violations concerning an accused with special needs, §479.040, RSMo.
3. If he/she should lose his/her license to practice law within the State of Missouri or if he/she should fail to complete the course of instruction as required by Section 130.060, Subsection (A)(1) hereof.
Section 130.050. Municipal Judge — Qualifications for Office.
A. The Municipal Judge shall possess the following qualifications before he/she shall take office:
1. He/she must be a licensed attorney, qualified to practice law within the State of Missouri or within six (6) months after selection for the position, each Municipal Judge who is not licensed to practice law in this State shall satisfactorily complete the course of instruction for Municipal Judges prescribed by the Supreme Court.
2. He/she need not reside within the City.
3. He/she must be a resident of the State of Missouri.
4. He/she must be between the ages of twenty-one (21) and seventy-five (75) years.
5. He/she may serve as a Municipal Judge for any other municipality.
6. He/she may not hold any other office within the City Government.
7. The Municipal Judge shall be considered holding a part-time position and as such may accept other employment.
Section 130.060. Superintending Authority.
The Municipal Court of the City shall be subject to the rules of the Circuit Court of which it is a part and to the rules of the State Supreme Court. The Municipal Court shall be subject to the general administrative authority of the Presiding Judge of the Circuit Court, and the Judge and Court personnel of said Court shall obey his/her directives.
Section 130.070. Report to Board of Aldermen.
The Municipal Judge shall cause the Court Clerk to prepare, within the first ten (10) days of every month, a report indicating the following:
A list of all cases heard or tried before the Judge during the preceding month, giving in each case the name of the defendant, the fine imposed if any, the amount of costs, the names of defendants committed, and the cases in which there was an application for trial de novo, respectively. The Court Clerk or the Judge shall verify such lists and statements by affidavit and shall file the same with the City Clerk who shall lay the same before the Board of Aldermen of the City for examination at its first (1st) session thereafter. The Municipal Court shall, within the ten (10) days after the first (1st) of the month, pay to the Municipal Treasurer the full amount of all fines collected during the preceding month, if not previously paid to the Municipal Treasurer.
Section 130.080. Docket and Court Records.
The Municipal Judge shall be a conservator of the peace. He/she shall keep a docket in which he/she shall enter every case commenced before him/her and the proceedings therein and he/she shall keep such other records as may be required. Such docket and records shall be records of the Circuit Court of McDonald County. The Municipal Judge shall deliver said docket, records and all books and papers pertaining to his/her office to his/her successor in office or to the Presiding Judge of the Circuit.
Section 130.090. Municipal Judge — Powers and Duties Generally.
A. The Municipal Judge shall be and is hereby authorized to:
1. Establish a Violations Bureau as provided for in the Missouri Rules of Practice and Procedure in Municipal and Traffic Courts and Section 479.050, RSMo.
2. Administer oaths and enforce due obedience to all orders, rules and judgments made by him/her and may fine and imprison for contempt committed before him/her while holding Court in the same manner and to the same extent as a Circuit Judge.
3. Stay execution of any fine or sentence, suspend any fine or sentence, and make such other orders as the Municipal Judge deems necessary relative to any matter that may be pending in the Municipal Court.
4. Make and adopt such rules of practice and procedure as are necessary to implement and carry out the provisions of this Chapter, and to make and adopt such rules of practice and procedure as are necessary to hear and decide matters pending before the Municipal Court, and to implement and carry out the provisions of the Missouri Rules of Practice and Procedure in Municipal and Traffic Courts.
5. The Municipal Judge shall have such other powers, duties and privileges as are or may be prescribed by the laws of this State, this Code or other ordinances of this City.
B. In addition to such other authority as exists to order conditions of probation, the Court may order such conditions as the Court believes will serve to compensate the victim, any dependent of the victim, or society.² [Ord. No. 21616A, §7, 2-17-2016]
1. Such conditions may include, but shall not be limited to:
a. Restitution to the victim or any dependent of the victim, in an amount to be determined by the Court;
b. The performance of a designated amount of free work for a public or charitable purpose, or purposes, as determined by the Court;
c. The assessment of shock jail time, so long as such jail time is within the range of punishment authorized by ordinance or law for the offense charged;
---
2. Editor's Note: See also §130.190 of this Chapter.
d. The assessment of a shock fine so long as such amount is within the range of punishment authorized by ordinance or law for the offense charged;
e. Completion of alcohol or substance abuse education, evaluation or treatment programs at the expense of the defendant; and
f. Completion of anger management, evaluation or treatment programs at the expense of the defendant.
2. The defendant may refuse probation conditioned on the performance of free work. If he/she does so, the Court shall decide the extent or duration of sentence or other disposition to be imposed and render judgment accordingly. Any County, City, person, organization, agency, or employee of a County, City, organization or agency charged with the supervision of such free work or who benefits from its performance shall be immune from any suit by the defendant or any person deriving a cause of action from him/her if such cause of action arises from such supervision of performance, except for an intentional tort or gross negligence. The services performed by the defendant shall not be deemed "employment" within the meaning of the provisions of Chapter 288, RSMo.
Section 130.100. Prosecutions Based on Information Only, Proceedings.
All prosecutions for the violation of municipal ordinances shall be instituted by information and may be based upon a complaint. Proceedings shall be in accordance with the Supreme Court Rules governing practice and procedure in proceedings before Municipal Judges.
Section 130.110. Violations Bureau.
Should the Municipal Judge determine that there shall be a Violations Bureau, the City shall provide all expenses incident to the operation of the same.
Section 130.120. Issuance and Execution of Warrants.
A. All Courts must issue a summons prior to the issuance of a warrant. A uniform citation is not a warrant. [Ord. No. 21616A, §4, 2-17-2016]
B. In the event an arrest is made due to a traffic stop, a warrant must be issued within twenty-four (24) hours or the offender is to be released. Only the Judge may order and sign a warrant. Time runs from the time the offender is taken into custody. Once a warrant is issued, defendants must appear before a Judge within forty-eight (48) hours if the offense is a "minor traffic violation" and within seventy-two (72) hours for all other offenses or be released. [Ord. No. 21616A, §4, 2-17-2016]
C. In the event a warrant is issued, the Court may assess a warrant fee in the amount of fifty dollars ($50.00) for each warrant issued to be added to the balance of any fines and costs due and collected in the same manner as provided by law. [Ord. No. 21616A, §3, 2-17-2016]
D. All warrants issued by a Municipal Judge shall be directed to the City Marshal, Chief of Police or any other Police Officer of the municipality or to the Sheriff of the County. The warrants shall be executed by the Marshal, Chief of Police, Police Officer or Sheriff at any place within the limits of the County and not elsewhere, unless the warrants are endorsed in the manner provided for warrants in criminal cases and, when so endorsed, shall be served in other Counties as provided for in warrants in criminal cases.
Section 130.130. Arrests Without Warrants.
The City Marshal, Chief of Police or other Police Officer of the City may, without a warrant, make arrest of any person who commits an offense in his/her presence, but such officer shall, before the trial, file a written complaint with the Judge hearing violations of municipal ordinances.
Section 130.140. Jury Trials.
Any person charged with a violation of a municipal ordinance of this City shall be entitled to a trial by jury as in prosecutions for misdemeanors before an Associate Circuit Court Judge. Whenever a defendant accused of a violation of a municipal ordinance has a right to and demands such trial by jury, the Municipal Court shall certify the case to the Presiding Judge of the Circuit Court for reassignment.
Section 130.150. Duties of the City's Prosecuting Attorney.
It shall be the duty of an attorney designated by the City to prosecute the violations of the City's ordinances before the Municipal Judge or before any Circuit Judge hearing violations of the City's ordinances. The salary or fees of the attorney and his/her necessary expenses incurred in such prosecutions shall be paid by the City. The compensation of such attorney shall not be contingent upon the number of cases tried, the number of guilty verdicts reached, or the amount of fines imposed or collected.
Section 130.160. Summoning of Witnesses.
It shall be the duty of the Municipal Judge to summon all persons whose testimony may be deemed essential as witnesses at the trial and to enforce their attendance by attachment, if necessary. The fees of witnesses shall be the same as those fixed for witnesses in trials before Associate Circuit Court Judges and shall be taxed as other costs in the case. When a trial shall be continued by a Municipal Judge, it shall not be necessary to summon any witnesses who may be present at the continuance, but the Municipal Judge shall orally notify such witnesses as either party may require to attend before him/her on the day set for trial to testify in the case and enter the names of such witnesses on his/her docket, which oral notice shall be valid as a summons.
Section 130.170. Municipal Judge Without Jurisdiction, When.
A. If, in the progress of any trial before the Municipal Judge, it shall appear to the Judge that the accused ought to be put upon trial for an offense against the criminal laws of the State and not cognizable before him/her as Municipal Judge, he/she shall immediately stop all further proceedings before him/her as Municipal Judge and cause the complaint to be made before some Associate Circuit Court Judge of the County.
B. For purposes of this Section, any offense involving the operation of a motor vehicle in an intoxicated condition as defined in Section 577.001, RSMo., shall not be cognizable in Municipal Court, if the defendant has been convicted, found guilty, or pled guilty to two (2) or more previous intoxication-related traffic offenses as defined in Section 577.023, RSMo., or has had two (2) or more previous alcohol-related enforcement contacts as defined in Section 302.525, RSMo.
Section 130.180. Jailing of Defendants.
If, in the opinion of the Municipal Judge, the City has no suitable and safe place of confinement, the Municipal Judge may commit the defendant to the County Jail, and it shall be the duty of the Sheriff, if space for the prisoner is available in the County Jail, upon receipt of a warrant of commitment from the Judge to receive and safely keep such prisoner until discharged by due process of law. The municipality shall pay the board of such prisoner at the same rate as may now or hereafter be allowed by law to such Sheriff for the keeping of other prisoners in his/her custody. The same shall be taxed as cost.
Section 130.190. Parole and Probation. 3
A. Any Judge hearing violations of municipal ordinances may, when in his/her judgment it may seem advisable, grant a parole or probation to any person who shall plead guilty or who shall be convicted after a trial before such Judge. When a person is placed on probation, he/she shall be given a certificate explicitly stating the conditions on which he/she is being released.
B. In addition to such other authority as exists to order conditions of probation, the Court may order conditions which the Court believes will serve to compensate the victim of the crime, any dependent of the victim, or society in general. Such conditions may include, but need not be limited to:
1. Restitution to the victim or any dependent of the victim in an amount to be determined by the Judge; and
2. The performance of a designated amount of free work for a public or charitable purpose or purposes as determined by the Judge.
C. A person may refuse probation conditioned on the performance of free work. If he/she does so, the Court shall decide the extent or duration of sentence or other disposition to be imposed and render judgment accordingly. Any County, City, person, organization or
3. Editor's Note: See also §130.090(B) of this Chapter.
agency or employee of a County, City, organization or agency charged with the supervision of such free work or who benefits from its performance shall be immune from any suit by the person placed on parole or probation or any person deriving a cause of action from him/her if such cause of action arises from such supervision of performance, except for intentional torts or gross negligence. The services performed by the probationer or parolee shall not be deemed employment within the meaning of the provisions of Chapter 288, RSMo.
D. The Court may modify or enlarge the conditions of probation at any time prior to the expiration or termination of the probation term.
Section 130.200. Right of Appeal.
In any case tried before the Municipal Judge, except where there has been a plea of guilty or where the case has been tried with a jury, the defendant shall have a right of trial de novo before a Circuit Court Judge or upon assignment before an Associate Circuit Court Judge. An application for a trial de novo shall be filed within ten (10) days after judgment and shall be filed in such form and perfected in such manner as provided by Supreme Court rule.
Section 130.210. Appeal From Jury Verdicts.
In any case tried with a jury before an Associate Circuit Judge, a record of the proceedings shall be made, and appeals may be had upon that record to the appropriate Appellate Court.
Section 130.220. Breach of Recognizance.
In the case of a breach of any recognizance entered into before a Municipal Judge or an Associate Circuit Court Judge hearing a municipal ordinance violation case, the same shall be deemed forfeited and the Judge shall cause the same to be prosecuted against the principal and surety, or either of them, in the name of the municipality as plaintiff. Such action shall be prosecuted before a Circuit Court Judge or Associate Circuit Court Judge, and in the event of cases caused to be prosecuted by a Municipal Judge, such shall be on the transcript of the proceedings before the Municipal Judge. All monies recovered in such actions shall be paid over to Municipal Treasury to the General Revenue Fund of the municipality.
Section 130.230. Disqualification of Municipal Judge From Hearing a Particular Case.
A Municipal Judge shall be disqualified to hear any case in which he/she is in any way interested or, if before the trial is commenced, the defendant or the prosecutor files an affidavit that the defendant or the municipality, as the case may be, cannot have a fair and impartial trial by reason of the interest or prejudice of the Judge. Neither the defendant nor the municipality shall be entitled to file more than one (1) affidavit or disqualification in the same case.
Section 130.240. Absence of Judge — Procedure.
If the Municipal Judge or Provisional Judge be absent, sick or disqualified from acting pursuant to the general administrative authority of the Presiding Judge of the Circuit Court over the Municipal Divisions within the Circuit contained in Section 478.240, RSMo., a special Municipal Judge may be designated in accordance with the provisions of Section 479.230, RSMo., until such absence or disqualification shall cease.
Section 130.250. Failure To Appear in Municipal Court.
A. A person commits the offense of failure to appear in Municipal Court if:
1. He/she has been issued a summons for a violation of any ordinance of the City of Anderson and fails to appear before the Judge of the Municipal Court at the time and on the date on which he/she was summoned, or at the time or on the date to which the case was continued;
2. He/she has been released upon recognition of bond and fails to appear before the Judge of the Municipal Court at the time and on the date on which he/she was summoned, or at the time or on the date to which the case was continued;
3. He/she has been placed on Court supervised probation and fails to appear before the Judge of the Municipal Court at the time specified by said Judge as a condition of the probation.
B. The Court cannot apply failure to appear charges for "minor traffic violations." This does not include fees and costs associated with the initial offense. [Ord. No. 21616A, §3, 2-17-2016]
C. Nothing in this Section shall prevent the exercise of the Municipal Court of its power to punish for contempt.
Section 130.260. (Reserved)
ARTICLE II
Court Clerk
Section 130.270. Office Established.
There is hereby established the office of Court Clerk for the City of Anderson Municipal Division of the McDonald County Circuit Court.
Section 130.280. Selection and Term of Court Clerk.
The Court Clerk shall be appointed by the Mayor with the consent of a majority of the members of the Board of Aldermen to serve for an unspecified term at the will of the Mayor and Board of Aldermen.
Section 130.290. Hours and Authorization of Compensation.
The Court Clerk shall attend all sessions of the Anderson Municipal Division of the 39th Judicial Circuit Court and may be required to be present at the Anderson City Hall to perform the duties of the office at such additional times as the Mayor or Board of Aldermen may specify. Compensation for the Court Clerk shall be established by ordinance from time to time.
Section 130.300. Bond.
Within fifteen (15) days after appointment and before entering upon the discharge of the above-described duties of office, the Court Clerk shall give bond to the City in the sum of fifty thousand dollars ($50,000.00) conditioned upon the faithful performance of said duties and the said Court Clerk will pay over all monies belonging to the City, as provided by law, that may come into the Court Clerk's hands.
Section 130.310. Clerk of Court — Duties.
[CC 1995 §130.180; CC 1974 §72.160]
A. The duties of the Clerk of the Municipal Court shall be as follows:
1. To prepare, under the supervision of the City Attorney, the following:
a. Complaints under this Code or other City ordinances;
b. Trial dockets;
c. All necessary appeal and recognizance forms;
d. Transcripts of records; and
e. Any and all other forms necessary in any action pending in the Municipal Court.
2. To maintain permanent files and records of the Municipal Court proceedings and to have the care and custody of same.
3. To prepare, under the supervision of the Municipal Judge, all reports required of the Municipal Judge or of the Municipal Court by State Statutes, this Code or other City ordinances.
4. To keep complete records regarding fines, penalties, costs and any other charges arising out of any action in the Municipal Court.
5. To assist the Chief of Police in the preparation of any reports or records required of him/her in connection with the operation of the Municipal Court.
6. To tax all costs, make out all bills and executions and make out and issue all processes of the Municipal Court.
7. To keep an appeal docket in which shall be recorded the proceedings in any Municipal Court case appealed to the Circuit Court.
8. To fulfill such other duties as may be assigned to him/her by the Mayor or Board of Aldermen.
Section 130.320. Clerk of Court — Powers.
[CC 1995 §130.190; CC 1974 §72.170]
A. The Clerk of the Municipal Court, and any Deputy Clerk that may serve under him/her, shall be and is hereby authorized to:
1. Take oaths and affirmations.
2. Accept and sign complaints signed and sworn to or affirmed before him/her.
3. Sign and issue warrants.
4. Sign and issue commitments to jail.
5. Sign and issue subpoenas requiring the attendance of a witness and sign and issue subpoenas duces tecum.
6. Fix the amount of bail and admit to bail.
7. Accept the appearance, waiver of trial and plea of guilty and payment of fine and costs in Traffic Violations Bureau cases or as directed by the Municipal Judge; generally act as Violations Clerk of the Traffic Violations Bureau.
8. Perform such other duties as provided for by ordinance, by rules of practice and procedure adopted by the Municipal Judge; and as provided for by the Missouri Rules of Practice and Procedure in Municipal and Traffic Courts.
ARTICLE III
Fines and Court Costs
Section 130.330. Installment Payment of Fine.
A. When a fine is assessed for violation of an ordinance, it shall be within the discretion of the Judge assessing the fine to provide for the payment of the fine on an installment basis under such terms and conditions as he/she may deem appropriate.
B. If installments or deferred payment is allowed, the defendant shall be placed on probation until the total fines and Court costs are paid in full. [Ord. No. 21616A, §6, 2-17-2016]
C. If the total amount due is not paid within one (1) month of the agreement to pay, there shall be an additional administrative fee of fifty dollars ($50.00). [Ord. No. 21616A, §6, 2-17-2016]
Section 130.340. Court Costs.
A. In addition to any fine that may be imposed by the Municipal Judge in any case filed in the Anderson Municipal Division of the 39th Judicial Circuit Court, and in addition to all other fees authorized or required by law, there shall be assessed as costs the following:
1. Costs of Court in the amount of twelve dollars ($12.00).
2. *Police Officer training fee.* A fee of three dollars ($3.00) is hereby established and assessed as additional Court costs in each Court proceeding, except that no such fee shall be collected when the proceedings against the defendant have been dismissed.
a. Two dollars ($2.00) of each such Court cost shall be transmitted monthly to the Treasurer of the City and used to pay for Police Officer training as provided by Sections 590.100 to 590.180, RSMo. The City shall not retain for training purposes more than one thousand five hundred dollars ($1,500.00) of such funds for each certified Law Enforcement Officer or candidate for certification employed by the City. Any excess funds shall be transmitted quarterly to the City's General Fund.
b. One dollar ($1.00) of each such Court cost shall be sent to the State Treasury to the credit of the Peace Officers Standards and Training Commission Fund created by Section 590.178, RSMo.
3. *Crime Victims' Compensation Fund.* An additional sum of seven dollars fifty cents ($7.50) shall be assessed and added to the basic costs in Subsection (1) of this Section, provided that no such cost shall be collected in any proceeding when the proceeding or the defendant has been dismissed by the Court. All sums collected pursuant to this Subsection shall be paid at least monthly as follows:
a. Ninety-five percent (95%) of such fees shall be paid to the Director of Revenue of the State of Missouri for deposit as provided in Section 595.045.5, RSMo.
b. Five percent (5%) shall be paid to the City Treasury.
4. There may also be assessed a cost of up to four dollars ($4.00) per case for each criminal case, including violations of any County or municipal ordinance for the purpose of providing operating expenses for shelters for battered persons as set out in Section 488.607, RSMo.
5. Other costs, such as for the issuance of a warrant, a commitment or a summons, as provided before the Associate Circuit Judge in criminal prosecutions.
6. Actual costs assessed against the City by the County Sheriff for apprehension or confinement in the County Jail or costs assessed against the City by any other detention facility.
7. Mileage, in the same amount as provided to the Sheriff in criminal violations, for each mile and fraction thereof the officer must travel (both directions) in order to serve any warrant or commitment or order of this Court.
8. Any other reasonable cost as may be otherwise provided by ordinance including, but not limited to, costs of confinement, including any necessary transportation related thereto, medical costs incurred by the City while a defendant is in City custody, and costs related to the arrest and testing of any person for any intoxication-related traffic offense as set out in Section 130.340(9) hereof.
9. *Reimbursement of certain costs of arrest.*
a. Upon a plea or a finding of guilty of violating the provisions of Sections 342.020 or 342.030 of this Code or any ordinance of the City of Anderson involving alcohol- or drug-related traffic offenses, the Court may, in addition to imposition of any penalties provided by law, order the convicted person to reimburse the Police Department for the costs associated with such arrest.
b. Such costs hereby authorized shall include the reasonable cost of making the arrest, including the cost of any chemical test made as authorized or required by law or ordinance to determine the alcohol or drug content of the person's blood, and the costs of processing, charging, booking and holding such person in custody.
c. The Chief of Police may establish a schedule of such costs hereby authorized and shall submit the same to the Municipal Judge. However, the Court may order the costs reduced if it determines that the costs are excessive.
10. *Judicial Education Fund.* Cities by ordinance may provide for fees in an amount per case to be set pursuant to Sections 488.010 to 488.020, RSMo., for each municipal ordinance violation case filed before a Municipal Judge, and in the event a defendant pleads guilty or is found guilty, the Judge may assess costs against the defendant except in those cases where the defendant is found by the Judge to be indigent and unable to pay the costs. The fees authorized in this Subsection are in addition to service charges, witness fees and jail costs that may otherwise be authorized to be assessed, but are in lieu of other Court costs. The fees provided by this Subsection shall be collected by the Municipal Division Clerk in municipalities electing or required to have violations of municipal ordinances tried before a Municipal Judge pursuant to Section 479.020, RSMo., or to employ judicial personnel pursuant to Section 479.060, RSMo., and disbursed as provided in Subsection (1) of Section 479.080, RSMo. Any other Court costs required in connection with such cases shall be collected and disbursed as provided in Sections 488.010 to 488.020, RSMo.; provided that each Municipal Court may establish a Judicial Education Fund and an Appointed Counsel Fund, each in separate accounts under the control of the Municipal Court to retain one dollar ($1.00) of the fees collected on each case. The fees collected shall be allocated between the two (2) funds as determined by the Court. The Judicial Education Fund shall be used only to pay for the continuing education and certification required of the Municipal Judges by law or Supreme Court Rule; and judicial education and training for the Court Administrator and Clerks of the Municipal Court.
The Appointed Counsel Fund shall be used only to pay the reasonable fees approved by the Court for the appointment of an attorney to represent any
defendant found by the Judge to be indigent and unable to pay for legal representation, and where the Supreme Court rules or the law prescribes such appointment. Provided further, that no Municipal Court shall retain more than one thousand five hundred dollars ($1,500.00) in the Judicial Education Fund for each Judge, Administrator or Clerk of the Municipal Court and no more than five thousand dollars ($5,000.00) in the Appointed Counsel Fund. Any excess funds shall be transmitted quarterly to the General Revenue Fund of the County or Municipal Treasury.
11. **Inmate Security Fund.**
a. A surcharge of two dollars ($2.00) shall be assessed as costs in each Court proceeding filed in any Court in any City adopting such a surcharge, in all violations of any municipal ordinance; except that no such fee shall be collected in any proceeding in any Court when the proceeding or the defendant has been dismissed by the Court or when costs are to be paid by the City. A surcharge of two dollars ($2.00) shall be assessed as costs in a Juvenile Court proceeding in which a child is found by the Court to come within the applicable provisions of Subdivision (3) of Subsection (1) of Section 211.031, RSMo.
b. The Treasurer shall deposit funds generated by the surcharge into the "Inmate Prisoner Detainee Security Fund." Funds deposited shall be utilized to acquire and develop biometric verification systems and information sharing to ensure that inmates, prisoners, or detainees in a holding cell facility or other detention facility or area which holds persons detained only for a shorter period of time after arrest or after being formally charged can be properly identified upon booking and tracked within the local law enforcement administration system, criminal justice administration system, or the local jail system. Upon the installation of the information sharing or biometric verification system, funds in the Inmate Prisoner Detainee Security Fund may also be used for the maintenance, repair, and replacement of the information sharing or biometric verification system, and also to pay for any expenses related to detention, custody, and housing and other expenses for inmates, prisoners, and detainees.
B. The Municipal Court may request the Director of the Department of Revenue to intercept a person's tax refund for delinquent Court costs, fines and fees or other sums ordered by Municipal Court in excess of twenty-five dollars ($25.00) in accordance with any rules developed by the Department of Revenue. [Ord. No. 21616A, §5, 2-17-2016]
Chapter 135
PROCUREMENT, CONFLICT OF INTEREST
Section 135.010. Purchasing Agent Designated.
[CC 1995 §135.010; CC 1974 §25.010]
A. The City Clerk and Public Works Director are hereby designated as Purchasing Agents for the City. They, when authorized, shall procure for the City bids for supplies and services needed by the City, in accordance with the procedures prescribed by this Chapter or required by law.
B. Except as provided in this Chapter, it shall be unlawful for any City Officer or employee to order the purchase of any supplies or make any contract within the purview of this Chapter other than through the City Clerk, and any purchase ordered or contract made contrary to the provisions hereof shall not be approved by City Officials, and the City shall not be bound thereby.
Section 135.020. Duties Generally.
[CC 1995 §135.020; CC 1974 §25.020]
A. In addition to the purchasing authority conferred in Section 135.010, and in addition to any other powers and duties conferred by this Code or other ordinance, the Purchasing Agents shall:
1. Act to procure for the City the highest quality in supplies and contractual services at the least expense to the City.
2. Discourage uniform bidding and endeavor to obtain as full and open competition as possible on all purchases and sales.
3. Keep informed of current developments in the field of purchasing, prices, market conditions and new products, and secure for the City the benefits of research done in the field of purchasing by other governmental jurisdictions, national technical societies, trade associations, and by private businesses and organizations.
4. Prepare, adopt and maintain a vendors' catalog file. Said catalog shall be filed according to materials and shall contain descriptions of vendors' commodities, prices, and discounts.
5. Exploit the possibilities of buying "in bulk" so as to take full advantage of discounts.
6. Act so as to procure for the City all Federal and State exemptions to which it is entitled.
7. Have the authority to declare vendors who default on their quotations irresponsible bidders and to disqualify them from receiving any business from the municipality for a stated period of time.
Section 135.030. Requisitions and Estimates.
[CC 1995 §135.030; CC 1974 §25.030]
A. Each City department or agency shall file with a Purchasing Agent detailed requisitions or estimates of their requirements in supplies and contractual services in such manner, at such times, and for such future periods as the City Clerk shall prescribe.
1. A City department or agency shall not be prevented from filing, in the same manner, with the City Clerk at any time a requisition or estimate for any supplies and contractual services, the need for which was not foreseen when the detailed estimates were filed.
2. The Purchasing Agent shall examine each requisition or estimate and shall have the authority to revise it as to quantity, quality, or estimated cost.
Section 135.040. Conflict of Interest.
[CC 1995 §135.040; CC 1974 §25.040]
No officer or employee of the City shall transact any business in his/her official capacity with any business entity of which he/she is an officer, agent or member or in which he/she owns a substantial interest; nor shall be/she make any personal investments in any enterprise which will create a substantial conflict between his/her private interest and the public interest; nor shall be/she or any firm or business entity of which he/she is an officer, agent or member, or the owner of substantial interest sell any goods or services to any business entity which is licensed by or regulated in any manner by the City.
Section 135.050. Conflict of Interest — Officers and Employees Not To Deal With Certain Entities.
[CC 1995 §135.050; CC 1974 §25.050]
No officer or employee of this City shall enter into any private business transaction with any person or entity that has a matter pending or to be pending upon which the officer or employee is or will be called upon to render a decision or pass judgment. If any officer or employee is already engaged in the business transaction at the time that a matter arises, he/she shall be disqualified from rendering any decision or passing any judgment upon the same.
Section 135.060. Conflict of Interest — Penalties.
[CC 1995 §135.060; CC 1974 §25.060]
Any person who violates the provisions of Sections 135.040 — 135.050 shall, upon conviction thereof, be punished as provided in Section 100.220 of this Code.
Section 135.070. Gifts and Rebates.
[CC 1995 §135.070; CC 1974 §25.070]
The Purchasing Agents and every other officer and employee of the City are expressly prohibited from accepting, directly or indirectly, from any person, company, firm or corporation to which any purchase order or contract is, or might be awarded, any rebate, gift, money, or anything of value whatsoever, except where given for the use and benefit of the City. Violation of the provisions of this Section shall upon conviction thereof be punished as provided in Section 100.220 of this Code.
Section 135.080. Competitive Bidding Required.
[CC 1995 §135.080; CC 1974 §25.080]
All purchases of, and contracts for supplies and contractual services, and all sales of personal property which has become obsolete and unusable shall, except as specifically provided herein, be based wherever possible on competitive bids.
Section 135.090. Formal Contract Procedure.
[CC 1995 §135.090; CC 1974 §25.090]
All supplies and contractual services, except as otherwise provided in this Chapter, when the estimated cost thereof shall exceed five thousand dollars ($5,000.00), shall be purchased by formal, written contract from the lowest responsible bidder, after due notice inviting proposals. All sales of personal property which has become obsolete and unusable, when the estimated value shall exceed five thousand dollars ($5,000.00), shall be sold by formal written contract to the highest responsible bidder, after due notice inviting proposals.
Section 135.100. Notice Defined.
[CC 1995 §135.100; CC 1974 §25.100]
A. The notice required by Section 135.090 shall consist of the following:
1. Notice inviting bids shall be published once in at least one (1) official newspaper of the City and at least five (5) days preceding the last day set for the receipt of proposals. The newspaper notice required herein shall include a general description of the articles to be purchased or sold, shall state where bid blanks and specifications may be secured, and the time and place for opening bids.
2. The Purchasing Agents shall also solicit sealed bids from all responsible prospective suppliers who have requested their names to be added to a "Bidders' List", which the Purchasing Agents shall maintain, by sending them a copy of such newspaper notice or such other notice as will acquaint them with the proposed purchase or sale. In any case, invitations sent to the vendors on the bidders' list shall be limited to commodities that are similar in character and ordinarily handled by the trade group to which the invitations are sent.
3. The Purchasing Agents shall also advertise all pending purchases or sales by a notice posted on the public bulletin board in the City Hall.
4. The Purchasing Agents shall also solicit sealed bids by:
a. Direct mail request to prospective vendors, or
b. Internet, or
c. By telephone,
as may seem to them to be in the best interest of the City.
Section 135.110. Bid Opening Procedure.
[CC 1995 §135.110; CC 1974 §25.110]
Bids shall be submitted sealed to the City Clerk and shall be identified as bids on the envelope. They shall be opened in public at the time and place stated in the public notices. A tabulation of all bids received shall be posted for public inspection.
Section 135.120. Lowest Responsible Bidder.
[CC 1995 §135.120; CC 1974 §§25.120 — 25.125]
A. Contracts shall be awarded to the lowest responsible bidder. Bids shall not be accepted from, nor contracts awarded to, a contractor who is in default on the payment of taxes, licenses or other monies due the City. In determining "lowest responsible bidder," in addition to price, the following shall be considered:
1. The ability, capacity and skill of the bidder to perform the contract or provide the service required;
2. Whether the bidder can perform the contract or provide the service promptly, or within the time specified, without delay or interference;
3. The character, integrity, reputation, judgment, experience and efficiency of the bidder;
4. The quality of performance of previous contracts or services;
5. The previous and existing compliance by the bidder with laws and ordinances relating to the contract or service;
6. The sufficiency of the financial resources and ability of the bidder to perform the contract or provide the service;
7. The quality, availability and adaptability of the supplies, or contractual services to the particular use required;
8. The ability of the bidder to provide future maintenance and service for the use of the subject of the contract;
9. The number and scope of conditions attached to the bid.
B. **Bids Accepted.** All bids shall be accepted or rejected by the Board of Aldermen. When the award is not given to the lowest bidder, a full and complete statement of the reasons for placing the order elsewhere shall be entered upon the journal of the Board.
**Section 135.130. Tie Bids.**
[CC 1995 §135.130; CC 1974 §25.130]
A. If all bids received or the lowest bids received are for the same total amount or unit price, quality and service being equal, the contract shall be awarded to a local bidder.
B. Where there is no local low bidder, the award shall be made on the basis of a drawing of lots, to be held in public.
**Section 135.140. Open Market Procedure.**
[CC 1995 §135.140; CC 1974 §25.140]
A. All purchases of supplies and contractual services, and all sales of personal property which has become obsolete and unusable for which competitive bidding is not required by Section 135.090 of this Code shall be made in the open market, without newspaper advertisement and without observing the procedure prescribed by Section 135.110 for the award of formal contracts.
1. All open market purchases shall, whenever possible, be based on at least three (3) competitive bids, and shall be awarded to the lowest responsible bidder in accordance with the standards set forth in Section 135.120.
2. The City shall solicit bids by:
a. Direct mail request to prospective vendors;
b. By telephone;
c. By public notice posted on the bulletin board of the City Hall; and
d. By internet.
3. The City Clerk shall keep a record of all open market orders and the bids submitted in competition thereon, and such records shall be open to public inspection.
Section 135.150. Emergency Purchases.
[CC 1995 §135.150; CC 1974 §25.150]
In case of an apparent emergency which requires immediate purchase of supplies or contractual services, the Board of Aldermen may authorize the purchase at the lowest obtainable price, any supplies or contractual services regardless of the amount of the expenditure. A full explanation of the circumstances of an emergency purchase shall be recorded in the journal of the Board of Aldermen.
Section 135.160. Cooperative Procurement.
[CC 1995 §135.160; CC 1974 §25.160]
To the maximum extent practicable, the purchases of this City shall be made under the provisions of the Missouri State-Local Technical Services Act. The provisions of this Chapter requiring competitive bidding at the local level shall not apply to such purchases.
Chapter 140
TAXATION AND FINANCE
ARTICLE I
Fiscal Year
Section 140.010. Fiscal Year.
[CC 1995 §140.010; CC 1974 §22.010]
The fiscal year of this City shall begin on October first (1st) and end on September thirtieth (30th) of the next succeeding year.
ARTICLE II
Budget
Section 140.020. Budget Required — Contents — Expenditures Not to Exceed Revenues.
A. Prior to the commencement of each fiscal year, a budget for the City shall be prepared and the same will be presented to and approved by the Board of Aldermen.
B. The annual budget shall present a complete financial plan for the ensuing fiscal year and shall include at least the following information:
1. A budget message describing the important features of the budget and major changes from the preceding year;
2. Estimated revenues to be received from all sources for the budget year, with a comparative statement of actual or estimated revenues for the two (2) years next preceding, itemized by year, fund and source;
3. Proposed expenditures for each department, office, commission, and other classification for the budget year, together with a comparative statement of actual or estimated expenditures for the two (2) years next preceding, itemized by year, fund, activity and object;
4. The amount required for the payment of interest, amortization and redemption charges on the debt of the City; and
5. A general budget summary.
C. In no event shall the total proposed expenditures from any fund exceed the estimated revenues to be received plus any unencumbered balance or less any deficit estimated for the beginning of the budget year; provided that nothing herein shall be construed as requiring the City to use any cash balance as current revenue or to change from a cash basis of financing its expenditures.
Section 140.030. Budget Officer.
A. The budget shall be prepared under the direction of a Budget Officer. Except as otherwise provided by law or ordinance, the Budget Officer shall be designated by the Board of Aldermen of the City. All officers and employees shall cooperate with and provide to the Budget Officer such information and such records as he/she shall require in developing the budget. The Budget Officer shall review all the expenditure requests and revenue estimates, after which he/she shall prepare the proposed budget as defined herein.
B. After the Budget Officer has prepared the proposed budget, he/she shall submit it, along with such supporting schedules, exhibits, and other explanatory material as may be necessary for the proper understanding of the financial needs and position of the City, to the Board of Aldermen. He/she shall submit at the same time complete drafts of such orders, motions, resolutions or ordinances as may be required to authorize the proposed expenditures and produce the revenues necessary to balance the proposed budget.
Section 140.040. Board of Aldermen May Revise Budget, Limits — Approval.
The Board of Aldermen may revise, alter, increase or decrease the items contained in the proposed budget, subject to such limitations as may be provided by law; provided that in no event shall the total authorized expenditures from any fund exceed the estimated revenues to be received plus any unencumbered balance or less any deficit estimated for the beginning of the budget year. Except as otherwise provided by law, the Board of Aldermen shall, before the beginning of the fiscal year, approve the budget and approve or adopt such orders, motions, resolutions or ordinances as may be required to authorize the budgeted expenditures and produce the revenues estimated in the budget.
Section 140.050. Increase of Expenditure Over Budgeted Amount to Be Made Only on Formal Resolution.
After the City has approved the budget for any year and has approved or adopted the orders, motions, resolutions or ordinances required to authorize the expenditures proposed in the budget, the City shall not increase the total amount authorized for expenditure from any fund, unless the Board of Aldermen adopts a resolution setting forth the facts and reasons making the increase necessary and approves or adopts an order, motion, resolution or ordinance to authorize the expenditures.
ARTICLE III
Levy of Taxes
Section 140.060. Board to Provide for Levy and Collection of Taxes — Fix Penalties.
The Board of Aldermen shall, from time to time, provide by ordinance for the levy and collection of all taxes, licenses, wharfage and other duties not herein enumerated and, for neglect or refusal to pay the same, shall fix such penalties as are now or may hereafter be authorized by law or ordinance.
Section 140.070. Fixing Ad Valorem Property Tax Rates, Procedure.
The Board of Aldermen shall hold at least one (1) public hearing on the proposed rates of taxes at which citizens shall be heard prior to their approval. The Board of Aldermen shall determine the time and place for such hearing. A notice stating the hour, date and place of the hearing shall be published in at least one (1) newspaper qualified under the laws of the State of Missouri of general circulation in the County within which all or the largest portion of the City is situated, or such notice shall be posted in at least three (3) public places within the City; except that in any County of the First Class having a Charter form of government, such notice may be published in a newspaper of general circulation within the City even though such newspaper is not qualified under the laws of Missouri for other legal notices. Such notice shall be published or posted at least seven (7) days prior to the date of the hearing. The notice shall include the assessed valuation by category of real, personal and other tangible property.
1. Cross Reference — As to notice required for public meeting on tax increases, eminent domain, creation of certain districts, and certain redevelopment plans, §120.045.
in the City for the fiscal year for which the tax is to be levied as provided by Subsection (3) of Section 137.245, RSMo., the assessed valuation by category of real, personal and other tangible property in the City for the preceding taxable year, for each rate to be levied the amount of revenue required to be provided from the property tax as set forth in the annual budget adopted as provided by Chapter 67, RSMo., and the tax rates proposed to be set for the various purposes of taxation. The tax rates shall be calculated to produce substantially the same revenues as required in the annual budget adopted as provided in this Chapter. Following the hearing the Board of Aldermen shall fix the rates of taxes, the same to be entered in the tax book. Failure of any taxpayer to appear at such hearing shall not prevent the taxpayer from pursuit of any other legal remedy otherwise available to the taxpayer. Nothing in this Section absolves the City of responsibilities under Section 137.073, RSMo., nor to adjust tax rates in event changes in assessed valuation occur that would alter the tax rate calculations.
Section 140.080. Assessment — Method of.
In the absence of a City Assessor, and until such City Assessor is duly appointed and qualified, it shall be the duty of the Mayor of the City to procure from the County Clerk of McDonald County, Missouri, on or before the first (1st) day of October of each year a certified abstract from his/her assessment books of all property within the corporate limits of the City made taxable by law for State purposes and the assessed valuation thereof as agreed upon by the Board of Equalization, which abstract shall be immediately transmitted to the Board of Aldermen, and it shall be the duty of the Board of Aldermen to establish by ordinance the rate of taxes for the year.
Section 140.090. Clerk to Prepare Tax Books.
When the Board of Aldermen shall have fixed the rate of taxation for any given year, it shall be the duty of the City Clerk to cause to be prepared appropriate and accurate tax books and shall therein set out in suitable columns, opposite the name of each person and the item of taxable property, as returned by the Assessor and Board of Equalization, the amount of taxes, whether general or special, due thereon and shall charge the City Collector with the full amount of taxes levied and to be collected.
Section 140.100. Taxes Delinquent — When.
A. On the first (1st) day of January of each year, all unpaid City taxes shall become delinquent and the taxes on real estate are hereby made a lien thereon.
B. Each tract of land in the back tax book, in addition to the amount of tax delinquent, shall be charged with a penalty of eighteen percent (18%) of each year's delinquency except that the penalty on lands redeemed prior to sale shall not exceed two percent (2%) per month or fractional part thereof.
ARTICLE IV
Taxation
Section 140.110. Sales Tax Imposed.
[CC 1995 §140.120; CC 1974 §54.500; Ord. No. 031715, 3-17-2015]
From and after October 1, 1971, pursuant to the authority granted by and subject to the provisions of Sections 94.500 to 94.577, RSMo., a tax for general purposes is hereby imposed upon all sellers for the privilege of engaging in the business of selling tangible personal property or rendering taxable services at retail to the extent and in the manner provided in Sections 144.010 to 144.525, RSMo., and the rules and regulations of the Director of Revenue issued pursuant thereto. The rate of the tax shall be one percent (1%) on the receipts from the sales at retail of all tangible personal property or taxable services at retail within the City of Anderson, Missouri, if such property and taxable services are subject to taxation by the State of Missouri under the provisions of Sections 144.010 to 144.525, RSMo., and Subsection (4) of Section 94.510, RSMo., and shall be collected pursuant to the provisions of Sections 94.500 to 94.577, RSMo.
Section 140.120. Utility Tax.
[Ord. No. 031715, 3-17-2015]
The sales tax also applies to all sales of metered water services, electricity, electrical current, natural, artificial or propane gas, wood, coal or home heating oil for domestic use.
Section 140.130. One-Half Cent Sales Tax for Streets and Transportation.
There is hereby imposed a transportation sales tax of one-half of one percent (0.5%) as authorized by Section 94.700, et seq., RSMo. Said transportation sales tax of one-half of one percent (0.5%) shall be imposed upon all receipts from sale at retail of all tangible personal property or taxable services. Said tax shall be in the manner and to the extent provided in Sections 144.010 to 144.525, RSMo., and the rules and regulations of the Director of Revenue issued pursuant thereto. The said sales tax shall be imposed for transportation purposes, including but not limited to, the planning, acquisition of rights-of-way, and construction, reconstruction, repair and maintenance of streets, roads and bridges.
Chapter 145
CONFLICTS OF INTEREST
Section 145.010. Declaration of Policy.
The proper operation of municipal government requires that public officials and employees be independent, impartial and responsible to the people; that public office not be used for personal gain; and that the public have confidence in the integrity of its government. In recognition of these goals, there is hereby established a procedure for disclosure by certain officials of the City of Anderson (the "City") of personal or private interests in matters affecting the City.
Section 145.020. Conflicts of Interest.
A. The Mayor or any member of the Board of Aldermen who has a substantial personal or private interest in any bill introduced to the Board of Aldermen shall disclose to the Board of Aldermen the nature of such interest which disclosure shall be recorded in the minutes of the proceedings. Such person shall disqualify himself or herself from voting on any bill relating to such interest. "Substantial or private interest" is defined as ownership by the individual, his or her spouse, or his or her dependent children, where singularly or collectively, directly, or indirectly of:
1. Ten percent (10%) or more of any business entity; or
2. An interest having a value of ten thousand dollars ($10,000.00) or more; or
3. The receipt of a salary, gratuity, or other compensation or remuneration of five thousand dollars ($5,000.00) or more per year from any individual, partnership, organization or association within any calendar year.
Section 145.030. Disclosure Reports of Each Alderman, the Mayor and City Clerk.
A. Each Alderman, the Mayor and the City Clerk shall disclose in writing the following information by May first (1st) if any such transactions were engaged in during the previous calendar year:
1. For such person, and all persons within the first degree of consanguinity or affinity of such person, the date and the identities of the parties to each transaction with a total value in excess of five hundred dollars ($500.00), if any, that such person had with the City, other than compensation received as a fee or salary or payment of any tax, fee or penalty due to the City, and other than transfers for no consideration to the City; and
2. The date and the identities of the parties to each transaction known to such person with a total value in excess of five hundred dollars ($500.00), if any, that any business entity in which such person had a substantial interest, had with the City, other than payment of any tax, fee or penalty due to the City or transactions involving payment for providing utility service to the City, and other than transfers for no consideration to the City.
Section 145.040. Additional Disclosures by the Mayor and City Clerk.
A. The Mayor and the City Clerk also shall disclose in writing by May first (1st) for the previous calendar year the following information:
1. The name and address of each of the employers of such person from whom income of one thousand dollars ($1,000.00) or more was received during the year covered by the statement.
2. The name and address of each sole proprietorship that he or she owned; the name, address and the general nature of the business conducted of each general partnership and joint venture in which he or she was a partner or participant; the name and address of each partner or coparticipant for each partnership or joint venture with the Secretary of State; the name, address and general nature of the business conducted of any closely held corporation or limited partnership in which the person owned ten percent (10%) or more of any class of the outstanding stock or limited partnership units; and the name of any publicly traded corporation or limited partnership that is listed in a regulated stock exchange or automated quotation system in which the person owned two percent (2%) or more of any class of outstanding stock, limited partnership units or other equity interests.
3. The name and address of each corporation for which such person served in the capacity of a director, officer or receiver.
Section 145.050. Filing of Disclosure Reports.
Disclosure reports shall be filed with the City Clerk and with the Missouri Ethics Commission on forms prescribed by the City Attorney. The reports shall be available for public inspection and copying during normal business hours.
Section 145.060. Financial Interest Statement — When Filed.
A. The financial interest statements shall be filed at the following times, but no person is required to file more than one (1) financial interest statement in any calendar year:
1. Each person required to file a financial interest statement under this Section shall file such statement within thirty (30) days of appointment or election;
2. Every other person required to file a financial interest statement shall file the statement annually not later than May first (1st) and the statement shall cover the calendar year ending the immediately preceding December thirty-first (31st); provided that any member of the Board of Aldermen may supplement the financial interest statement to report additional interests acquired after December thirty-first (31st) of the covered year until the date of filing of the financial interest statement.
|
KILLER KUTS
(See Consensus Cuts on P. 2)
Crosby, Stills, Nash - "Dark Star"
"Shadow Captain"
"Cathedral"
"Just a Song Before I Go"
Roger Daltrey - "Avenging Annie"
"Say It Ain't So Joe"
Alan Parsons - "I Wouldn't Wanna Be..."
"I Robot"
Steve Winwood - "Time is Running Out"
"Hold On"
"Vacant Chair"
CONCERT RAVES
(See Concertizing on P. 26)
Doobie Bros./Burlington, Wisc.
Fleetwood Mac/Binghamton, N.Y.
Hall & Oates/Chattanooga, Tenn.
Herbie Hancock/West Hartford, Ct.
Judas Priest/Ft. Lauderdale, Fla.
Pink Floyd/New York, N.Y.
Leon Redbone/Anaheim, Ca.
Carole Bayer Sager/Santa Barbara
Boz Scaggs-S.S. Johnny/St. Louis, Mo.
REGIONALS
RADIO EMPHASIS (P. 3)
Northeast
576 C.S.&N.
565 Peter Frampton
512 Geils
476 Heart
471 Dan Fogelberg
465 Steve Miller
429 James Taylor
365 Little River Bd.
353 Cat Stevens
341 Supertramp
South
654 C.S.&N.
526 Peter Frampton
474 Dan Fogelberg
451 James Taylor
436 Steve Miller
414 Heart
406 Little River Bd.
391 Geils
376 Neil Young
368 Alan Parsons
Midwest
699 C.S.&N.
660 Steve Miller
635 Peter Frampton
558 Heart
519 Dan Fogelberg
442 James Taylor
436 Foreigner
436 Ted Nugent
378 Cat Stevens
372 Fleetwood Mac
West
712 C.S.&N.
628 Heart
586 Peter Frampton
545 Steve Miller
471 Dan Fogelberg
450 Cat Stevens
398 Supertramp
387 James Taylor
382 Little Feat
366 Geils
366 Alan Parsons
366 Neil Young
RADIO FUTURES (P. 3)
353 Steve Winwood
318 Yes
224 Strawbs
165 Kiss
165 Johnny Winter
331 Steve Winwood
248 Yes
180 Johnny Winter
143 Strawbs
135 M. Manchester
218 Yes
205 Steve Winwood
115 Strawbs
096 Styx
077 Johnny Winter
319 Steve Winwood
178 Johnny Winter
162 Strawbs
152 Yes
136 M. Manchester
PROGRESSIVE RETAIL (P. 19)
778 Peter Frampton
618 Fleetwood Mac
465 C.S.&N.
403 Dan Fogelberg
382 Steve Miller
368 Bee Gees
347 Eagles
333 Cat Stevens
333 James Taylor
319 Bro. Johnson
961 Fleetwood Mac
902 C.S.&N.
804 Peter Frampton
804 James Taylor
667 Eagles
549 Kiss
510 Dan Fogelberg
471 Jimmy Buffett
471 Emotions
412 Commodores
928 Fleetwood Mac
711 Heart
606 Steve Miller
544 Peter Frampton
533 C.S.&N.
467 Kiss
406 Ted Nugent
400 Foreigner
289 Boston
261 Commodores
947 Fleetwood Mac
840 C.S.&N.
653 Peter Frampton
400 Jimmy Buffett
387 Heart
387 Kiss
387 Steve Miller
333 Commodores
333 James Taylor
293 Neil Young
RETAIL FUTURES (P. 19)
458 Steve Winwood
396 Alan Parsons
375 Floaters
271 Crusaders
271 Al Jarreau
271 Lonnie L. Smith
412 Roger Daltrey
294 Alan Parsons
235 Little River Bd.
235 Steve Winwood
267 UFO
250 Roger Daltrey
550 Alan Parsons
350 Steve Winwood
267 Little River Bd.
267 UFO
250 Roger Daltrey
440 Rainbow
400 Steve Winwood
360 Floaters
240 Rita Coolidge
240 Derringer
## Consensus Cuts, Etc.
Consensus cuts are derived from information obtained by special survey of our contributors. The albums spotlighted are selected from available new releases. Tracks are listed in order of preference. An underline denotes an overwhelming choice. The top new tracks are listed in percentage order to the left.
### In Order of Preference
| Rank | Artist | Title |
|------|----------------------|--------------------------------|
| 1 | Roger Daltrey | "Avenging Annie" |
| 2 | Crosby, Stills, Nash | "Dark Star" |
| 3 | Roger Daltrey | "Say It Ain't So Joe" |
| 4 | Steve Winwood | "Time Is Running Out" |
| 5 | Crosby, Stills, Nash | "Shadow Captain" |
| 6 | Crosby, Stills, Nash | "Cathedral" |
| 7 | Alan Parsons | "I Wouldn't Wanna" |
| 8 | Steve Winwood | "Hold On" |
| 9 | Crosby, Stills, Nash | "Just a Song" |
| 10 | Alan Parsons | "I Robot" |
| 11 | Steve Winwood | "Vacant Chair" |
| 12 | Crosby, Stills, Nash | "Fair Game" |
| 13 | Roger Daltrey | "One Of The Boys" |
| 14 | Steve Winwood | "Luck's In" |
| 15 | Crosby, Stills, Nash | "I Give... Blind" |
| 16 | Crosby, Stills, Nash | "Run From Tears" |
| 17 | Strawbs | "Burning For Me" |
| 18 | Fools Gold | "Mr. Lucky" |
| 19 | Fools Gold | "Sweet Country Aire" |
| 20 | Crosby, Stills, Nash | "In My Dreams" |
| 21 | Rainbow | "Man On the Silver" |
| 22 | Burton Cummings | "My Own Way to..." |
| 23 | Burton Cummings | "Never Had a Lady..." |
| 24 | Alan Parsons | "Day After Day" |
### Significant Singles
- Alice Cooper
- Doobie Brothers
- Carole King
- Ramones
- Sanford-Townsend
- Suzy & The Red Stripes War
### Important Imports
- Beatles - Hamburg
- 801 - Live
- Genesis EP
- Sex Pistols EP
### Table of Contents
- Consensus Cuts, Etc. P. 2
- Radio Stats P. 3
- Radio P. 4
- Retail Stats P. 19
- Retail P. 20
- Concertizing P. 26
- New Releases P. 30
- Jobs, Misc. Info. P. 29
- Concepts P. 32
### Box Score
- Album Count: 65
- Year to Now: 1201
- Last Year: 1079
- At This Time: 1079
## RADIO STATS
Our relative charting, as seen below, resembles baseball's batting averages. It establishes a ratio between the ideal (heavy airplay at every station which reported), and the weighted count that a recording has actually earned. This mathematical formula is applied to each record—first regionally, then nationally. A dual weighting system is used to derive the count. It adjusts for market size and for heavy or moderate airplay. These charts show the relative intensity of radio airplay for both new and established recordings.
### RADIO EMPHASIS
| 2 ISSUES BACK | LAST ISSUE | CURRENT NATIONALS | ARTIST | TRACKS | N.E. | S. | MW | W |
|---------------|------------|-------------------|-----------------|----------------------|------|------|-----|-----|
| --- | .465 | .662 | C.S. & N. | Dark Star/Shadow | .576 | .654 | .699| .712|
| .454 | .697 | .580 | PETER FRAMPTON | Signed/Roadrunner | .565 | .526 | .635| .586|
| .688 | .676 | .529 | STEVE MILLER | Airline/Sacrifice | .465 | .436 | .660| .545|
| .571 | .651 | .528 | HEART | Barracuda/Queen | .476 | .414 | .558| .628|
| .554 | .574 | .483 | D. FOGELBERG | Promises/Love | .471 | .474 | .519| .471|
| --- | .247 | .425 | JAMES TAYLOR | Handyman/Smiling | .429 | .451 | .442| .387|
| .154 | .304 | .406 | GEILS | Monkey/Surrender | .512 | .391 | .353| .366|
| .563 | .533 | .380 | CAT STEVENS | A Star/Schoolyard | .353 | .316 | .378| .450|
| .275 | .443 | .358 | NEIL YOUNG | Hurricane/Bullet | .324 | .376 | .372| .366|
| .189 | .308 | .348 | LITTLE RIVER BD | Help/Days on Road | .365 | .406 | .282| .346|
| .383 | .353 | .326 | SUPERTRAMP | Give a Little/Quiet | .341 | .203 | .327| .398|
| .489 | .398 | .318 | FLEETWOOD MAC | Dreams/The Chain | .212 | .353 | .372| .346|
| .583 | .512 | .317 | LITTLE FEAT | A Hero/Hii Roller | .276 | .263 | .327| .382|
| --- | .229 | .294 | ALAN PARSONS | Be Like You/Robot | .159 | .368 | .288| .366|
| --- | .268 | .282 | ROGER DALTREY | Annie/Say It Ain't | .276 | .233 | .295| .309|
| --- | .365 | .262 | MARLEY/WAILERS | Exodus/Jammin' | .271 | .301 | .237| .246|
| .121 | .334 | .257 | TED NUGENT | Cat Scratch/Control | .135 | .226 | .436| .241|
| .231 | .244 | .255 | CHARLIE | Johnny Hold Back | .229 | .241 | .301| .251|
| .383 | .423 | .251 | GREGG ALLMAN | Come & Go/Sweet | .147 | .346 | .250| .277|
| 3/1 | .361 | .246 | FOREIGNER | Feels Like/Coldice | .206 | .180 | .436| .173|
| --- | --- | .216 | JOAN BAEZ | Time Rag/Freedom | .200 | .218 | .173| .272|
| .431 | .331 | .198 | BONNIE RAITT | Runaway/Forgive | .182 | .083 | .250| .251|
| .204 | .254 | .194 | H. WINCHESTER | Breeze/Twigs | .147 | .316 | .096| .230|
| --- | .211 | .182 | MINK DEVILLE | Cadillac/Mixed Up | .324 | .090 | .096| .188|
| .239 | .263 | .155 | OUTLAWS | Sinadown/Gassmoke | .135 | .075 | .237| .162|
| .253 | .207 | .151 | KENNY LOGGINS | Lady Luck/Believe | .088 | .165 | .186| .168|
| --- | --- | .142 | UFO | Handle/Love to... | .118 | .038 | .212| .178|
| .347 | .259 | .132 | DAVE MASON | So High/Let It Go | .129 | .143 | .147| .115|
| .415 | .313 | .117 | 10 CC | Judge/Benefit | .082 | .098 | .128| .152|
| --- | --- | .095 | PAT TRAVERS | Statesboro Blues | .035 | .053 | .115| .162|
### RADIO FUTURES
| NATIONAL % | ARTIST | TRACKS | N.E. | S. | MW | W |
|------------|-----------------|----------------------|------|------|-----|-----|
| .303 | STEVE WINWOOD | Time/Hold On | .353 | .331 | .205| .319|
| .231 | YES | --- | .318 | .248 | .218| .152|
| .163 | STRAWBS | Burning For Me | .224 | .143 | .115| .162|
| .151 | J. WINTER | Tired of Trying | .165 | .180 | .077| .178|
| .098 | M. MANCHESTER | Where You Are | .118 | .135 | .000| .136|
| .083 | STYX | --- | .076 | .060 | .096| .094|
| .071 | KISS | Christine Sixteen | .165 | .045 | .019| .047|
| .057 | O. KITAJIMA | Frost Flowers | .041 | .105 | .013| .073|
| .054 | PIERCE ARROW | Hot Summer/Believe | .076 | .068 | .064| .016|
| .049 | A. W. B. | --- | .041 | .030 | .038| .079|
| .049 | COUNTRY JOE | Martha/Reunion | .041 | .038 | .051| .063|
## BOSTON
**WBCN-FM**
**JOHN BRODEY**
**HEAVY AIRPLAY:**
- Charley
- Crosby/Stills/Nash
- Fleetwood Mac
- Foreigner
- Peter Frampton
- Geils
- Horslips
- Heart
- Garland Jeffreys
- Little Feat
- Steve Miller
- Mink DeVille
- Graham Parker
- Pierce Arrow
- Bonnie Raitt
- Cliff Richard
- Southside Johnny/Jukes
- *James Taylor*
- Wailers
**MODERATE AIRPLAY:**
- Greg Allman
- Bee Gees
- Dickey Betts
- Jimmy Buffett
- Crusaders
- Ringo Culbrey
- Kiki Dee
- Walter Egan
- Dan Fogelberg
- Illusion
- Joshua Tull
- Paul Kelly
- Greg Kihn
- Little River Band
- Dave Mason
- Frankie Miller
- Van Morrison
- Danny O'Keefe
- Poco
- Leon & Mary Russell
- Bob Seger
- Cat Stevens
- Supertramp
- Richard Torrance
- Ultravox
- Johnny Guitar Watson
- Rusty Wier
- Jesse Winchester
- Steve Winwood*
**SINGLES:**
- Cars
- Ry Cooder
- Michael Case
- Climax
- Hall & Oates
- Marvin Gaye
- Carly Simon
- Sparks
- Symbol 8
- Trinidad
- Ramones
**SPECIAL PROGRAMMING:**
- Broadcast Danny O'Keefe live last week and did the Little River Band this week.
**ADDS:**
- *Kiss
- John Klemmer
- Melissa Manchester
- Dictators
- Aalon
- James Winter
- Emotionals
- Rhead Brothers
- Pakalameridith
- Strawbs
- Hughie Hancock
- Ann Peebles
- Keith Jarrett
**NEWS/PUBLIC AFFAIRS:**
- Danny O'Keefe will be around until Sept. 1st, officially, however for the next two weeks, his typewriter is on vacation.
- J. B.
## BOSTON
**WCZO-FM**
**BEGONIA MIRE**
**HEAVY AIRPLAY:**
- Steve Miller
- Heart
- Bonnie Raitt
- Peter Frampton
- Cat Stevens
- Boston
- Fleetwood Mac
- Crosby/Stills/Nash
- Pousette-Dart Band
- James Taylor
- Geils
- *Yes
**MODERATE AIRPLAY:**
- Teddy Pendergrass
- Eagles
- Supertramp
- Jimi Hendriff
- Neil Young
- Dan Fogelberg
- Mink DeVille
- Foreigner
- Southern Johnny
- Donna Summer
- Ted Nugent
- Bros. Johnson
- Leon & Mary Russell
- Little River Band
- Roger Daltry
- Kiss
- Alice Cooper
- Steve Winwood
**SINGLES:**
- Suzy & The Red Stripes
- Sanford-Townsend
- Sparks
- Tim Morse
- Bruce Foster
- Trinidad
- "Sweet Soca Song"
- Chris "Way Down"
- Rusty Wier
- Doobie Bros.
- *Yes
**COMMENTS:**
- A while back, while deciding how to distribute our new WCZO T-shirts, we foisted around the idea of selling them and giving the money to a non-profit organization. Well, we set the idea in motion and decided to charge our listeners $3.50 per shirt, and give the proceeds to NORML. The response has been overwhelming that we're going to have to re-order.
## HARTFORD
**WHCN-FM**
**BARRY NEAL**
**HEAVY AIRPLAY:**
- James Taylor
- "Smiling Face"
- "Bartender's Blues"
- "Love on Broadway"
- "Terra Nova"
- "Handyman"
- Crosby/Stills/Nash
- "Some Before I Go"
- "Dark Star"
- "Cold Rain"
- "Shadow Capt."
- + Dan Fogelberg
- "Night Train"
- "Love Gone By"
- "Lessons Learned"
- + Cat Stevens
- "Sweet Jamaica"
- "Life"
- "Killing Time"
- "Bonfire"
- Heart
- "Little Queen"
- "Barracuda"
- "Love Alive"
- "Dream of the Archer"
- Yes - "Wonderous..."
- "Turn of the Century"
- "Parallel"
- "Going for the One"
**MODERATE AIRPLAY:**
- Charlie
- "Johnny Hold Back"
- "Don't Look Back"
**Hartford Continued...**
**WHCN-FM**
- Little River Band
- "Another Runaway"
- "Happy Anniversary"
- "Help is on Its Way"
- Kenny Loggins
- "I've Got the Melody"
- "Lady Luck"
- Patti Griffin
- "Watcha Gonna Do"
**COMMENTS:**
- Despite a short rain delay for our concert at Willowbrook Park in New Britain, Conn., was a great success. Our thanks go out to WEA and Perry Cooper, Bill Beamish, Pat Russell and WLP. Shalamar Performing before over 6,000 enthusiastic fans were Larry Coryell, Scarlet Rivera and Tim Morse.
- Peter Gabriel (Atlantic) dropped by the other day, whipped open his briefcase and on a Fairchild machine played us AC/DC "Back In My Arms" an imaginative clever and an attention grabber. Something to ponder for the future?????
## NEW HAVEN
**WPLR-FM**
**GORDON WEINGARTH**
**HEAVY AIRPLAY:**
- Johnny Winter
- Steve Winwood
- Neil Young
- Crosby/Stills/Nash
- Geils
- Fleetwood Mac
- Steve Miller
- *Yes
- Peter Frampton
- Mink DeVille
- Dan Fogelberg
- Foreigner
- Joan Baez
- John Klemmer
- Richard Torrance
- Stephen Bishop
- Lakes
- Peter Gabriel
- Weather Report
- Little Village
**MODERATE AIRPLAY:**
- Poco
- *Elton Deluxe
- Illusion
**AC/DC**
**Coryell/Mouzon**
**Melissa Manchester**
**Bruce Dalgler**
**Kevin Coyne**
**James Taylor**
**Ultravox**
**North Buffalo**
**Supertrump**
**Dictators**
**Capt. Beyond**
**Scarlet Rivera**
**Laurindo**
**Danny O'Keefe**
**Dixie Dregs**
**John Blair**
**Dirty Angels**
**SINGLES:**
- Eric Ellsworth Band
- On Spunout Records
**IMPORTS:**
- Kolovovits
- Genesis EP
**COMMENTS:**
- We were very proud and gratified that April-May AIR showings. Despite some problems with our signal during the rating period itself, we were #18-34 in New Haven/Hartford. ADDs also showed a general increase in hinter areas as well (e.g. Long Island). A pause for celebration, then back to creative worry.
- Not to forget - John Griffin joined us as production ace and airperson a couple of months ago.
## NEW YORK
**WRVR-FM**
**DONNA L. HALPER**
**HEAVY AIRPLAY:**
- Al Jarreau
- Eric Gale
- John Klemmer
- Crusaders
- Noel Pointer
- Ronnie Laws
- Weather Report
- Idris Muhammad
- Earl Klugh
**MODERATE AIRPLAY:**
- George Benson
- Chuck Mangione
- Hank Crawford
- Stuff
- Bobbi Humphrey
- Bob James
- Chico Hamilton
- Payne - Levin Band
- Maynard Ferguson
- David Sanborn
- Ramsey Lewis
- Pat Metheny
- Lonnie Liston Smith
- Houston Person
- John Klemmer
- Balletto Ballet
- Bill Evans
- McCoy Tyner
- Coryell/Mouzon
**SPECIAL PROGRAMMING:**
- Charlie Parker
- John Klemmer
- Art Pepper
- Herbie Hancock
- Keith Jarrett
(of ELP)
**COMMENTS:**
- Well the dust is gradually settling and boxes are being unpacked. Our new studios are looking very nice, but all is still sort of dis-oriented. Note our new address:
**WRVR**
41-30 58th Street
Weedside, N.Y. 11377
Phone: 212-335-1700
More news soon. Love and music -- Donna
## NEW YORK
**WPLJ-FM**
**CORINNE BALDASSANO**
**HEAVY AIRPLAY:**
- *Kiss
- *James Taylor
**SINGLES:**
- *Alice Cooper
## LONG ISLAND
**WLIR-FM**
**DENIS McNAMARA**
**HEAVY AIRPLAY:**
- *Kiss
- Crosby/Stills/Nash
- Roger Daltry
- Peter Frampton
- Strawbs
- Little Feat
- Outlaws
- *James Taylor
- Alan Parsons
- Greg Kihn
- Steve Howe
- Dave Mason
- Rubinoos
- U.F.O.
- Section
- *Pierce Arrow
- Pat Travers
- Charlie
- Supertramp
- Neil Young
- *Yes
Continued on top of next page.
## NORTHEAST
**Long Island Continued...**
WLIR-FM
**MODERATE AIRPLAY:**
* Crusaders
* John Klemmer
* Lake
* Horalsips
* Nilsson
* Burton Cummings
* Fools Gold
* Clover
* Fountains
* Earth Quake
* Mac McAnaui
* Rick Derringer
* Rainbow
* Johnny Winter
* Melissa Manchester
* Kiss
* Country Joe & The Fish
* Travis & Kirwan
* Les McCann
* Judas Priest
* Norton Buffalo
* Stuff
* Blue
* Al Jarreau
* Average White Band with Ben E. King
**SINGLES:**
* Doobie Bros.
* Carly Simon
* Carole King
**SPECIAL PROGRAMMING:**
Radio Concerts:
6th Annual 4th of July Jamboree
- Santana
- Jefferson Starship
- Marshall Tucker Band
- Billy Joel
- Atlanta Rhythm Section
- Dickey Betts
- David Bromberg
- Average Step
**Additions Radio Concerts:**
NRBG
38 Special
Interviews:
Denny Laine
**IMPORTS:**
* Hudson Ford
* David Bradstreet
* Genesis EP
**COMMENTS:**
Joe Bonadonna has left us to air his talks at WMMR in Philadelphia, we all wish him the best of luck.
**Current On-Air Schedule:**
6 - 10 am - Larry Kleinman
10 - 2 pm - Denis McNamara
2 - 6 pm - Joel Moss/PD
6 - 10 pm - Earle Bailey
10 - 12 pm - Suzy & White
2 - 6 am - Armin Lazlo
**Weekends - Michael Ross, John DeBella, Pam Merly, Ben Manilla**
Our space is short to thank individually all the people who have helped strengthen our programming over the last few weeks. So, you know who you are and thank you. We appreciate your time, effort and concern.
- Denis
---
## PHILADELPHIA
**WIQO-FM**
ALEX DEMERS/BD
HELEN LEIGHT/MD
**HEAVY AIRPLAY:**
* Yes
Foreigner
Steve Winwood
Roger Daltrey
Crosby/Stills/Nash
James Taylor
Little River Band
Dan Fogelberg
Peter Frampton
Steve Miller
---
## PHILADELPHIA Continued...
WIQO-FM
Fleetwood Mac
Heart
Kinks
Little Feat
Bonnie Raitt
Supertramp
10cc
Detective
Charlie
Chilliwack
Ultravox
Lake
Iguana
Strawbs
Bad Company
Dave Mason
Grateful Kihn
Rubinols
Frankie Miller
Neil Young
Joan Baez
Bob Marley/Wailers
Cat Stevens
Pink Floyd
Johnny Winter
Burton Cummings
Alan Parsons
Les Dudek
Moody Blues
Mink DeVille
Ted Nugent
Head East
LeBlanc & Carr
Isley Bros.
Nils Lofgren
Pablo Cruise
Tim Moore
Tom Petty
Southside Johnny/Jukes
Rough Diamond
Emerson, Lake, Palmer
Starr
Geils
UFO
Poco
Alexis
**LIGHT AIRPLAY:**
* Styx
Coryell/Mouzon
* War Platinum Jazz
Osamu Kitajima
Allman Bros.
Driver
Country Joe & The Fish Section
Kiss
Max Webster
Grateful Dead Band
Gary Burton
Fools Gold
Vassar Clements
Ruby Starr
John Klemmer
Van Morrison
Noel Pointer
Jerry Jeff Walker
Trooper
Illusion
Leon & Mary Russell
Alice Cooper
38 Special
Peter Tosh
Mahogany Rush
Gale Force
Rainbow
Derringer
Laura Nyro
Nazareth
Carly Simon
Doobie Bros.
Suzy & The Red Stripes
Ram Jam
Ronnie Spector
ELP
'Brain Salad Surgery'
Soake
**IMPORTS:**
Genesis EP
Slade
Hudson Ford
Medium Rare
Ian Gillian Band
Stiff Sampler
The Stranglers
**COMMAND:**
'Q' is continuing its series of free concerts for the summer.
---
## PHILADELPHIA Continued...
WIQO-FM
We are working with "The Other Side" in Wilmington, Del. and with the city of Philadelphia to bring lots of free music. Thanks to all the record company folks for the guests, promotions etc., etc.
We are also pleased to announce that well known Philadelphia air personality Ed Sciky will become a "Q" person on August 1st. More later...
Alex
---
## PHILADELPHIA
**WYSP-FM**
DENNY SOMACH
**HEAVY AIRPLAY:**
* Supertramp
Fleetwood Mac
Pete - Frampton
Little Feat
James Taylor
Dan Fogelberg
Cat Stevens
Steve Miller
Neil Young
Heart
Little River Band
Foreigner
Kiss
Crosby/Stills/Nash
Jimmy Buffett
Rita Coolidge
**MODERATE AIRPLAY:**
* Yes
Rainbow
Charlie
Nazareth
Jesse Winchester
Clover
Peter Arrow
Crusaders
Roger Daltrey
Mink DeVille
Bob Marley/Wailers
Geils
Graeme Edge Band
Steve Winwood
Ted Nugent
Alan Parsons
Garland Jeffreys
Weather Report
Mac McAnally
Derringer
Greg Kihn
UFO
Country Joe & The Fish
*John Payne/
Lewie Levin Band
*Strawbs
Roy Buchanan
Sanford-Townsend
Tangerine Dream
*John Winter
**SINGLES:**
Ram Jam
**SPECIAL PROGRAMMING:**
Jazz Program - hosted by Marcus Flaherty - featuring albums by:
Herbie Hancock
McCoy Tyner
Flora Purim
Al Jarreau
Jan Garbarek
Enrica Rova
John Coltrane
**IMPORTS:**
Genesis EP
---
## WASHINGTON/BETHESDA
**WHFS-FM**
DAVE EINSTEIN
**HEAVY AIRPLAY:**
Gregg Allman
Bob Azten
Joan Baez
Crosby/Stills/Nash
Roger Daltrey
Mink DeVille
Fools Gold
Peter Frampton
Aretha Franklin
Geils
---
## Wash./Bethesda Continued
WHFS-FM
Ialey Bros.
Denny Laine
Meters
Steve Miller
Melissa Manchester
New Grass Revival
Willie Nelson
Outlaw
Gram Parsons
Red, White & Bluegrass
Burning Spear
Stuff
38 Special
Peter Tosh
Bob Marley/Wailers
James Taylor
Doc & Merle Watson
Steve Winwood
Johnny Winter
Neil Young
Al Jarreau
Carl Burien/
Everard Weber
Herbie Hancock
Tim Everman
Raul de Sousa
Brand X
Balancing Fault
Bobby Bland
Eric Burdon/
Jim Witherspoon
Alvin Lee
Coal Kitchen
Vassar Clements
Country Gazette
Burton Cummings
Dirty Tricks
Rick Derringer
Dillard's/Hartford
Coon Elders
Eastern Heritage
Jeffrey Frericks
Robin Ford
Bruce Foster
David Grisman
Huey Lewis
Hollies
Horslips
Illusion
Greg Kihn
Kiss
Lake
Chuck McDermott
Modern Lovers
Mad Acres
Laura Nyro
O'Jays
Omaha Sheriff
Pierce Arrow
Dick Sharry
The Section
Swamp Dog
Cat Stevens
Strawbs
Tubes
Coryell/Mouzon
Noel Pointer
Kevin Ayers
Pat Metheny
Keith Jarrett
Jan Garbarek
John Klemmer
John Payne
Don Fulks
Dollar Brand
McCoy Tyner
Flora Purim
Earl Klugh
Lenny Williams
Lonnie Liston Smith
Brent Maglia
Country Joe & The Fish
Nilsson
Sonny Stitt
**SINGLES:**
Suzy & The Red Stripes
**SPECIAL PROGRAMMING:**
Johnny Guitar Watson
Sunnyside Slim
Ben Sidran
Continued on top of next page.
## RADIO
(PLUS (+) INDICATES AUDIENCE REACTION & ASTERISK (*) INDICATES NEW ADDITION)
### NORTHEAST
**Wash./Bethesda Continued**
- WHFS-FM
**IMPORTS:**
- Sandy Denny
- Roy Harper
- Bob Johnson/Pete Knight
- Dave Swarbrick
- Steeleye Span
**ALLENTOWN, PA.**
- WSAN
- **RICK HARVEY**
- HEAVY AIRPLAY:
- Steve Miller
- Peter Frampton
- Supertramp
- Ted Nugent
- Dan Fogelberg
- Little Feat
- Heart
- Charlie
- Kenny Loggins
- American Flyer
- Pablo Cruise
- UFO
- James Taylor
- Roger Daltrey
- Steve Winwood
- MODERATE AIRPLAY:
- Kiss
- Melissa Manchester
- Straws
- AC/DC
- Grasme Edge Band
- John Payne
- Omaha Sheriff
- Johnny Winter
- Bob Marley & Wailers
- LeBlanc & Carr
**NEW BRUNSWICK, N.J.**
- WRSU-FM
- **IRV LURIN**
- HEAVY AIRPLAY:
- *Straws
- +Yes
- Gregg Allman
- Steelye Span
- Demons
- Crosby, Stills & Nash
- Dan Fogelberg
- Peter Frampton
- Horslips
- Bob Johnson/
- Peter Knight
- Lake
- Moody Blues
- Laura Nyro
- Outlaws
- Pierce Arrow
- Turner & Kirwan
- Cat Stevens
- Jerry Jeff Walker
- MODERATE AIRPLAY:
- Johnny's Dance Band
- Norton Buffalo
- Dillard-Hartford-
- Dillard
- Kevin Coyne
- Bruce Foster
- Geils
- Mink DeVille
- Jonathan Richman
**PRINCETON, N.J.**
- WPRB-FM
- **GEORGE KORVAL**
- HEAVY AIRPLAY:
- Geils
- Bob Marley & Wailers
- Crusaders
- John Payne
- Mink DeVille
**WPRB-FM Continued...**
**MODERATE AIRPLAY:**
- Ornette Coleman
- Steve Winwood
- Pierce Arrow
- Fools Gold
- Charlie
- Focus
- Roger Daltrey
- Straws
- Steeleye Span
- Blev/Peacock/Altschul
- Gabor Szabo
- Kevin Coyne
- Donna Summer
- Primal Scream
- Coryell/Mouzon
- Chico Hamilton
- David Sanborn
- Muse Releases
- Chicago City Releases
- Savoy Releases
**SINGLES:**
- Genesis - EP
- Ramones
### TROY, N.Y.
**WRPI**
- **LOU TETREAULT**
- HEAVY AIRPLAY:
- *Joan Baez
- Cat Stevens
- Al DiMeola
- Horslips
- Straws
- Supertramp
- MODERATE AIRPLAY:
- Dickey Betts
- Brand X
- Roger Daltrey
- Focus
- Foreigner
- Peter Gabriel
- Keith Jarrett
- Bob Marley & Wailers
- McCoy Tyner
- Mink DeVille
- Moxy
- NRBB
- Laura Nyro
- Omaha Sheriff
- Outlaws
- Pierce Arrow
- Carole Bayer Sager
- The Section
- James Taylor
### SYRACUSE/UTICA, N.Y.
**WOUR**
- **TOM STARR**
- HEAVY AIRPLAY:
- Jesse Winchester
- Supertramp
- Mink DeVille
- Geils
- Bob Marley & Wailers
- Roger Daltrey
- Neil Young
- Lake
- Blue
- Steve Winwood
- Straws
- James Taylor
- Little River Band
- Yes
- Country Joe & Fish
- Crosby, Stills & Nash
- Dixie Dregs
- Clover
- Johnny Winter
- Horslips
- Alan Parsons Project
- Ben E. King
**SINGLES:**
- Doobie Bros.
- Carly Simon
**IMPORTS:**
- Hudson-Ford
### YOR/HARRISBURG/LANCASTER, PA.
**WRHY/STARVIEW**
- **JEFF KAUFFMAN**
- HEAVY AIRPLAY:
- ****Crosby, Stills & Nash!!
- Steve Miller
- Peter Frampton
- Dan Fogelberg
- Heart
- James Taylor
- Neil Young
- Ted Nugent
- Steve Winwood
- Cat Stevens
- Foreigner
- Alan Parsons Project
- Little River Band
- Supertramp
- Roger Daltrey
- UFO
- Charlie
- Moody Blues
- Geils
- Johnny Winter
- Jess Winchester
- Steve Miller
- Heart
- Bonnie Raitt
- Lew London
- Illusion
- Cat Stevens
- Geils
**MODERATE AIRPLAY:**
- Danny Kirwan
- Tubes
- Neil Young
- Laura Nyro
- Steve Winwood
- Dan Fogelberg
- Dave Mason
- Peter Frampton
- Straws
- Pierce Arrow
- Mink DeVille
- Fools Gold
- Little River Band
- Norton Buffalo
### ITHACA, N.Y.
**WVRB-FM**
- **ANDY DENEMARK**
- HEAVY AIRPLAY:
- Crosby, Stills & Nash
- Roger Daltrey
- James Taylor
- Steve Miller
- Heart
- Bonnie Raitt
- Lew London
- Illusion
- Cat Stevens
- Geils
**MODERATE AIRPLAY:**
- Danny Kirwan
- Tubes
- Neil Young
- Laura Nyro
- Steve Winwood
- Dan Fogelberg
- Dave Mason
- Peter Frampton
- Straws
- Pierce Arrow
- Mink DeVille
- Fools Gold
- Little River Band
- Norton Buffalo
### SYRACUSE, N.Y.
**WAER**
- **BOB SHEPHERDSON**
- HEAVY AIRPLAY:
- Geils
- Alan Parsons Project
- Laura Nyro
- Roger Daltrey
- Joan Baez
- Cat Stevens
- McCoy Tyner!!
- Heart
- John Payne -
- Louis Levin Band
- Earl Klugh
- Peter Frampton
- Frankie Miller
- James Taylor
- Grasme Edge Band
- PFM
- Crosby, Stills & Nash
- Dan Fogelberg
**MODERATE AIRPLAY:**
- Aalon
- Dave Van Ronk
- Roy Buchanan
- Dexter Gordon
- Roy Ayers
- Gary Burton Quartet
- Milt Jackson
### NEW YORK, N.Y.
**WNYU-FM**
- **CHRIS LONG**
- HEAVY AIRPLAY:
- American Flyer
- Crosby, Stills & Nash
- Kevin Coyne
- Roger Daltrey
- Fools Gold
- Peter Frampton
- Geils
- Heart
- Horslips
- Billy Joel
- Bob Johnson
- Peter Knight
- Little Feat
- Little River Band
- Straws
- James Taylor
- Peter Frampton
- The Section
- +Yes
- +Grasme Edge Band
- +John Payne/Louis Levin Band
- Woodstock Mountains
- Horslips
- Laura Nyro
### PITTSBURGH, PA.
**WYPE-FM**
- **DANIEL BARNETT**
- HEAVY AIRPLAY:
- Otis Rush
- Brand X
- Steeleye Span - Story...
- Ray Plazi
- Roots of R&B
- Carmen McRae
- Earth Wind
- Guitar Player
- McCoy Tyner
- Dillard-Hartford-
- Dillard
- Sun Ra
- Chris Williamson
- Arild Anderson
- David Grisman Quintet
- Clifton Chenier
- Slim Gaillard
- Taj Mahal
**MODERATE AIRPLAY:**
- Ray Bryant
- John Klemmer
- Melissa Manchester
- NRBB
- Poco
- Maddox Bros.
- Tanya Tucker
- J. S. Bach
## RADIO
(PLUS (+) INDICATES AUDIENCE REACTION & ASTERISK (*) INDICATES NEW ADDITION)
### NORTHEAST
**LEWISTON/PORTLAND, ME**
**WBLM**
JOSE DIAZ
**HEAVY AIRPLAY:**
- James Taylor
- Crosby, Stills & Nash
- Heart
- Jesse Winchester
- Geils
- Steve Winwood
- Dan Fogelberg
- Bob Marley & Wailers
- Greg Kihn
- Danny O'Keefe
**MODERATE AIRPLAY:**
- Fleetwood Mac
- Paynes/Levin Band
- Little River Band
- Reggae Knights
- Blue Öyster Cult
- Supertramp
- Charlie
- Jerry Jeff Walker
- Peter Frampton
- Mink Deville
- Leen & Mary Russell
- Norton Buffalo
- Pat Metheny
**BOSTON, MASS.**
**TK-101**
L.A.
**HEAVY AIRPLAY:**
- Crosby, Stills & Nash
- Stevie Miller
- Neil Young
- Gregg Allman
- Outlaws
- Dan Fogelberg
- Jesse Winchester
- James Taylor
- Laura Nyro
- Little Feat
- Heart
- Randy Rhoads
- Rusty Wier
- Little River Band
- Jerry Jeff Walker
- Sanford/Townsend Band
- American Flyer
- Rigo Coolidge
**MODERATE AIRPLAY:**
- Cliff Richard
- Dusty Springfield
- Peter Arrow
- Fools Gold
- Kenny Loggins
- Geils
- Cat Stevens
- Van Morrison
**CAMBRIDGE, MASS.**
**WCAS-AM**
DON COHEN/M.D.
MOE SIGNORE/P.D.
**EXTRA HEAVY AIRPLAY:**
- Crosby, Stills & Nash
- Bert Jansch (import)
- Chuck McDermott
- W. Sherman
- Laura Nyro
- Peter Arrow!
- James Taylor
**HEAVY AIRPLAY:**
- Joan Baez
- Jaime Brockett
- Norton Buffalo
- Dillard/Hartford-Burlington
- Dan Fogelberg
- Al Jarreau!!
- Little River Band
- Bob Marley & Wailers
- Dick Fosbury
- Magic Mountain
- Steve Winwood
**MODERATE AIRPLAY:**
- Gregg Allman Band
- American Flyer
- Keith Jarrett
### WORCESTER, MASS.
**WAAF**
TOM DANIELS/P.D.
**HEAVY AIRPLAY:**
- Beatles - Hollywood...
- Boston
- *Crosby, Stills & Nash
- Eagles
- Fleetwood Mac
- Dan Fogelberg
- Peter Frampton
- Heart
- Steve Miller
- Bonnie Raitt
- Poussette-Dart Band
- Bob Seger
- Cat Stevens
- Stevie Wonder
- *James Taylor
- *Yes
**MODERATE AIRPLAY:**
- Foreigner
- *Kiss
- Little River Band
- Bob Marley & Wailers
- 10cc
- *Attitudes
- *Grateful
- *Steve Winwood
- *Lake
### NEW HAVEN, CONN.
**WYBC**
DOUG KEOGH
**HEAVY AIRPLAY:**
- Osamu Kitajima
- Ben Sidran
- George Duke
- McCoy Tyner
- Passport
- Opa
- *Keith Jarrett
- *Eddie Jefferson
- Gilco Hamilton
**MODERATE AIRPLAY:**
- Herbie Hancock
- Randy Rhoads
- Noel Pointer
- Payne/Levin Band
- Carla Bley
- Dexter Gordon
- Little Gong
- Flora Purim
- Al DiMeola
- Ronnie Laws
- Gary Burton
### ALBANY, N.Y.
**WBKB-FM**
JACK HOPKE
**HEAVY AIRPLAY:**
- Crosby, Stills & Nash
- Peter Frampton
- Geils
- Little Feat
- Steve Miller Band
- Van Morrison
- Mink Deville
- Bonnie Raitt
- Supertramp
- James Taylor
- *Kiss
- *Johnny Winter
- *Steve Winwood
**MODERATE AIRPLAY:**
- Gregg Allman
- Bad Company
- Roy Buchanan
- Dan Fogelberg
- Heart
- *Yes
- Geils
### BINGHAMTON, N.Y.
**WQBK-FM Continued...**
- Jonathan Richman
- Bob Marley & Wailers
- Neil Young
- *Strawbs
### SOUTH
**DALLAS/FT. WORTH**
**KZEW**
MIKE HEDGES
**HEAVY AIRPLAY:**
- Crosby, Stills & Nash
- James Taylor
- Ted Nugent
**MODERATE AIRPLAY:**
- *Jerry Jeff Walker
- ****Al Jarreau
- Alan Parsons Project
- SINGLETS
- Carly Simon
- Doobie Bros.
**COMMENTS:**
- Great news. The ARB was kind to us. The ZOO pulled #1 18-24 adults, #2 18-34 adults, and #2 12-24. Bob Marley came out on a morning ZOO plug. Bob comes to us from WBCN, where he was a P. D. Our brand new afternoon dude is Dave Lee Austin, masquerading as Dave Scott. He has been production director and DJ at WGCC and WLIB. He's "real" excited to have Bob & Dave with us. Here's our current lineup... 1-6 a.m., LG Richardson
- 6-10 a.m., Bob Shannon's
- 10-2 p.m., Doug Keogh
- 10-2 p.m., Mark Christopher
- 2-6 p.m., Dave Lee Austin
- 6-10 p.m., Michael Brown
- 10-1 a.m., Mike Hedges MD
- Paul Timmons, Beverly Beesley, Jack Robinson,
- Jon Dillon, & Charlie Jones.
In conjunction with London Records, & the Moody Blues, we're Live album, the ZOO is giving away a deep sea fishing trip to the Gulf of Mexico. ZOO Safari is co-sponsored with lots of 93 FM listeners trekking across the country to rock concerts. Recent Safaris have trekked to see Heart, Lynyrd Skynyrd, The O, Spedweeds, & Dan Fogelberg. We're using a traveling custom lounge that has recently transported America and Japan on their tours.
Our ZOO FREE SUNDAY concerts are still going. Mink Deville on July 17, & America's Tears on July 31. THE GULF OF SAFARI on the air each Wednesday night at 1, with an early Sunday morning repeat. Satellite broadcasts on the ZOO from the East Coast are Alan, Bonnie Raitt, & Hall & Oates. Hope to run into some Walrus buddies at the Billboard convention in August. I think I'm talking about "the future of radio" on a panel there.
Peaceful ways,
Ira Lipson, Zoologist
## SOUTH
### NASHVILLE
- WKDF-FM
- JACK CRAWFORD
- HEAVY AIRPLAY:
- Crosby/Still/Nash
- Steve Miller
- Peter Frampton
- James Taylor
- Heart
- Dan Fogelberg
- Neil Young
- Ted Nugent
- Joan Baez
- Fleetwood Mac
- Cat Stevens
- Gregg Allman
- Dixie Dregs
- Yes
- Kiss
- MODERATE AIRPLAY:
- Kenny Loggins
- Roy Buchanan
- LaBlanc Carr
- Alan Parsons
- Dave Mason
- Little River Band
- Billy Joel
- Jesse Winchester
- Charlie
- Johnny Guitar Watson
- Les Dudek
- American Flyer
- Little Feat
- Roger Daltrey
- Pat Travers
- Steve Winwood
- Journey Winter
- Calla
- Jaleo Bros.
- Waylon Jennings
- Al Jarreau
- Ben E. King
- Rainbow
- Grassey Edge Band
- Clovis
- Melvin Manchester
- Scarlet Rivera
- SINGLES:
- Carly Simon
- Doobie Bros.
- Carole King
- Mike Conldige
- Star Wars - Smtrk.
- Johnny Rivers
- Suzy & The Red Stripes
- Three Dog Night
- Attitudes
- Sanford-Twensend
- Jimmy Webb
- Michael Stanley
- COMMERCIAL:
- Dave Walton has departed as music director. I am looking for a replacement. (See Jobs Section for info)
### ATLANTA
- WRAS-FM
- JOHN WHIN
- HEAVY AIRPLAY:
- Yes
- Charlie
- Dixie Dregs
- Little Feat
- + Grassey Edge Band
- Michael Stanley
- Heart
- Mindless
- Wes Willis
- Ian Matthews
- Dickie Betts
- Miller Cross
- Jesse Winchester
- * Neil Young
- Dan Fogelberg
- Garland Jeffreys
- The Band
- Richard Torrance
- The Band
- Peter Frampton
- Bruce Cockburn
- Passport
- Atlanta Rhythm Aces
- UFO
- MODERATE AIRPLAY:
- Kate/Anna McGarrigle
- Flamingos
- Steve Hunter
- Al DiMeola
- Jess Roden
- Bouquet & Dart Band
- Dave Edmunds
- Jeff Beck
- Gong
- Dave Grisman
- Brand X
- Mellard
- + Ultravox
- Television
- Crosby/Still/Nash
- Les Dudek
- * Alan Parsons
- SPECIAL PROGRAMMING:
- Saturday morning 33-1/2 is a new program at FM-107 --where we feature live concerts and new LP's overnight from midnight Friday.
### ORLANDO
- WORJ-FM
- BILL McCATHY
- HEAVY AIRPLAY:
- Steve Miller
- ++ Crosby/Still/Nash
- Peter Frampton
- + Heart
- Fleetwood Mac
- Jimi Hendrix
- Dan Fogelberg
- + Cat Stevens
- Foreigner
- Supertramp
- Kenny Loggins
- Graege Allman
- Emerson, Lake, Palmer
- + Little River Band
- MODERATE AIRPLAY:
- Meter
- Boston
- Cat Stevens
- James Taylor
- Joan Baez
- Marshall Tucker
- Moody Blues
- Bad Co.
- Atlanta Rhythm Section
- Bob Seger
- Heart
- Crosby, Stills & Nash
- Foreigner
- Kinks
- Jimmy Buffett
- Bonnie Raitt
- Climax Blues Band
- LIGHT AIRPLAY:
- *Steve Winwood
- Charlie
### NEW ORLEANS
- WRNO
- MIKE COSTELLO/P.D.
- TOM OWENS/M.D.
- HEAVY AIRPLAY:
- Peter Frampton
- Steve Miller
- Fleetwood Mac
- MODERATE AIRPLAY:
- Meter
- Boston
- Cat Stevens
- James Taylor
- Joan Baez
- Marshall Tucker
- Moody Blues
- Bad Co.
- Atlanta Rhythm Section
- Bob Seger
- Heart
- Crosby, Stills & Nash
- Foreigner
- Kinks
- Jimmy Buffett
- Bonnie Raitt
- Climax Blues Band
- LIGHT AIRPLAY:
- *Steve Winwood
- Charlie
### NEW ORLEANS Continued...
- WRNO
- Dan Fogelberg
- Poco
- Stephen Sinclair
- UFO
- Jesse Winchester
- Mink DeVille
- Outlaws
- Mac McAnally
- Bob Marley & Wailers
- American Flyer
- Melissa Manchester
- Roger Daltrey
- Electric Light Orch.
- Neil Young
- Jerry Jeff Walker
- Emerson, Lakes&Palmer
- Little River Band
- Santana
- Dickey Betts
- Al Stewart
- Geils
- Detective
- Gregg Allman
- Kiss
- Rocky - Smtrk.
- Yes
- SINGLES:
- Cat Stevens
- *Merilee Rush
- Andrew Gold
- Bob Seger
- Stevie Bishop
- Eagles
- Fleetwood Mac
- *Isley Bros.
- Foreigner
- Climax Blues Band
- Rita Coolidge
- Jimmy Buffett
- Steve Miller
- Ruth
- Atlanta Rhythm Section
- *Sanford Townsend Band
- Hall & Oates
- Peter Frampton
- *Supertramp
- Alice Cooper
- Crosby, Stills & Nash
- Pablo Cruise
- Marshall Tucker
- James Taylor
- Bonnie Raitt
### JACKSONVILLE
- WAIV-FM
- JAMIE BROOKS
- HEAVY AIRPLAY:
- Geils
- *Steve Winwood
- +Jerry Jeff Walker
- +Ted Nugent
- +Alan Parsons Project
- Lake
- MODERATE AIRPLAY:
- Kenny Loggins
- James Taylor
- Bill Summerville
- Yes
- Detective
- *Geils
- +Michael Stanley
- Neil Young
- +Foreigner
- SPECIAL PROGRAMMING:
- Jan. 1 - 2 hours 7-9 p.m. Sunday.
- Special Woodstock LP feature soon.
- NEWS & PUBLIC AFFAIRS
- Week length 6-6:30 a.m.
- Monday - Friday.
- Two-way stereo talk show.
- COMMENTS:
- 6-10 a.m. Mike McHale
- 12M-6 a.m. Dan Quitter
### HOUSTON
- KLOL-FM
- SANDY MATHIS
- HEAVY AIRPLAY:
- Heart
- Dan Fogelberg
- Crosby, Stills & Nash
- James Taylor
- Steve Miller
- Steve Winwood
- Peter Frampton
- Fleetwood Mac
- MODERATE AIRPLAY:
- Cat Stevens
- Gregg Allman
- Alice Cooper
- Roger Daltrey
- Graege Edge Band
- Fleetwood Mac
- Geils
- Bob Marley & Wailers
- Alan Parsons Project
- Leon & Mary Russell
- SINGLES:
- Ry Cooder
- "School's Out"
- Genesis
- "The Match"
## SOUTH
### WABB-FM Continued...
**MODERATE AIRPLAY:**
- Yes
- Steve Winwood
- PFM
- Dictators
- Mink DeVille
- Charlie
- Melissa Manchester
- Alan Parsons Project
- Ted Nugent
- Johnny Guitar Watson
### JACKSON, MISS.
#### WZZQ
**WAYNE HARRISON**
**HEAVY AIRPLAY:**
- Kiss
- R.E. O. Speedwagon
- Rickie Lee Jones
- Waylon Jennings
- Fleetwood Mac
- Crosby, Stills & Nash
- Dan Fogelberg
- Kenny Loggins
**MODERATE AIRPLAY:**
- Ted Nugent
- James Taylor
- Boz Scaggs
- Mac McAnally
- Bad Company
- Foreigner
- Steve Miller
- Peter Frampton
- Heart
- Jesse Winchester
- Alan Parsons Project
- Supertramp
- Pablo Cruise
**LIGHT AIRPLAY:**
- Brownsville Station
- Charlie
- Gregg Allman
- Neil Young
**SINGLES:**
- Sanford/Townsend
### LAFAYETTE, LA.
#### KSMB-FM
**MIKE MITCHELL**
**HEAVY AIRPLAY:**
- Marshall Tucker Band
- James Taylor
- David Crosby
- Dave Mason
- Johnny Guitar Watson
- Cat Stevens
- Peter Frampton
- The Eagles
- Jimmy Buffett
- Waylon Jennings
- Dan Fogelberg
- Little River Band
- Crosby, Stills & Nash
- Heart
- Dickey Betts
- Sam Leval
- Steve Miller
- Quicksilver
- Rusty Wier
**MODERATE AIRPLAY:**
- Mink DeVille
- Alan Parsons Project
- Roger Daltrey
- Steve Winwood
- Alexis
- Jesse Winchester
### NEW ORLEANS, LA.
#### WTUL
**SHEPARD SAMUELS**
**HEAVY AIRPLAY:**
- Yes
- Crosby, Stills & Nash
- Jesse Winchester
- Heart
- Bob Marley & Wailers
- Steve Miller
### BATON ROUGE, LA.
#### WFMF
**JIMMY BEYER**
**HEAVY AIRPLAY:**
- Peter Tosh
- Gale Force
- Pierce Arrow
- Keith Sykes
- Jimmie, Novi & Ernie
- Alexis
- Bob Marley & Wailers
- The Crusaders
**SINGLES:**
- Seawind
- Dixie Dregs
- The Meters
- Roger Daltrey
- Al Jarreau
- Steve Winwood
- James Taylor
- Jesse Winchester
- Stephen Sinclair
- Little River Band
- Grateful Dead
- Fools Gold
- Melissa Manchester
- Mink DeVille
- Rusty Wier
- Reggie Knighton
- Burton Cummings
### LAKE CHARLES, LA.
#### NOVA 104
**SKY**
**HEAVY AIRPLAY:**
- Gregg Allman
- Joan Baez
- Jimmy Buffett
- Crosby, Stills & Nash
- Little River Band
- John Lennon
- LeBlanc & Carr
- Cat Stevens
- Supertramp
- Neil Young
**MODERATE AIRPLAY:**
- Roy Buchanan
- Chick Corea
- Quicksilver
- Geils
- Ted Nugent!!
- Bob Marley & Wailers
- Cat Stevens
- Strawbs
- 38 Special
- Tubes
- UFO
- Joe Cocker Band
- Steve Winwood
- Jesse Winchester
### LITTLE ROCK, ARK.
#### KAAY
**STUART MCRAE**
**HEAVY AIRPLAY:**
- Gregg Allman
- Jeff Beck
- Crosby, Stills & Nash
- Emerson, Lake & Palmer
- Fleetwood Mac
- Peter Frampton
- Geils
- Heart
- Stevie Miller
- Ted Nugent
- Pink Floyd
- R.E. O. Speedwagon
- Cat Stevens
- Jerry Jeff Walker
- Yes
**MODERATE AIRPLAY:**
- Clover
- Dan Fogelberg
- Styx
- James Taylor
- Wes Willis
- Steve Winwood
- John Denver
**SINGLES:**
- Carole King
- Ram Jam
- Bachman-Turner Overdrive
### OKLAHOMA CITY, OKLA.
#### KATT
**JIM STAFFORD**
**HEAVY AIRPLAY:**
- Crosby, Stills & Nash
- Waylon Jennings
- Foreigner
- Jimmy Buffett
- Oklahoma
- James Taylor
**MODERATE AIRPLAY:**
- Alan Parsons Project
- Steve Winwood
- Jimmy Webb
- Dixie Dregs
- Lab
- Average White Band/Albert King
- Melissa Manchester
- Crosby, Stills & Nash
- Pierce Arrow
- Laura Nyro
- Captain Beyond
- Osamu Kitajima
- Al Jarreau
- Roger Daltrey
- Rainbow
- John Denver
**SINGLES:**
- Doobie Bros.
### AUSTIN, TEX.
#### KOKE-FM
**JOE GRACEY/P.D.**
**CAROLYN ALLEN/M.D.**
**HEAVY AIRPLAY:**
- Johnny Winter
- Crosby, Stills & Nash
- Willie Nelson
- Geils
- Bob Marley & Wailers
- Peter Frampton
- Alvin Lee
- Waylon Jennings
- Jerry Jeff Walker
- Steve Miller
- Rusty Wier
- Neil Young
**MODERATE AIRPLAY:**
- Townes Van Zandt
- James Taylor
- Jesse Winchester
- Burton Cummings
- Dan Fogelberg
- Wes Willis
- Danny O'Keefe
- Ben Sidran
- Clifton Chenier
- Heart
- Steve Winwood
- Scarlet Rivera
**SINGLES:**
- Willie Nelson
## MIDWEST
### KANSAS CITY
#### KYIO2
**MAX FLOYD**
**HEAVY AIRPLAY:**
- Steve Miller
- Ted Nugent
- REO Speedwagon
- Dan Fogelberg
- Peter Frampton
- Crosby/Stills/Nash
- Kiss
- James Taylor
- Little River Band
- Alan Parsons
- Steve Winwood
**MODERATE AIRPLAY:**
- UFO
- Roger Daltrey
- Charlie
- Pat Travers
- Graeme Edge Band
- Horslips
- Greg Kihn
- Lake
- Jesse Winchester
- Tangerine Dream
- Pierce Arrow
- Roy Buchanan
- Geils
- Star Wars - Sntrk.
## MIDWEST
### Chicago Continued...
**WXRT-FM**
- Ram Jam
- Ramones
- Ramones Factory
- Sex Pistols
**SPECIAL PROGRAMMING:**
- Guests:
- Herbie Hancock
- Frankie Miller
- Phil Magg (UFO)
- Les Morgenstein (Chicago)
4th of July:
- Rock 'n' Roll America
**IMPORTS:**
- Stiff
- Genesis
- Kolovovits
- Turner & Kirwan
**COMMENTS:**
- Harvey Wells is leaving us to join the air staff at WQKS. While we're sorry to see him go, we understand his priorities and wish him well. Until we can thoroughly check out our own staff and the outside world to find a replacement, we will do without a music director and have Sam Mason fill in his air shift. See the Jobs Section of this issue for a want ad.
Saturday July 9th was WXRT's "Playing Richard's Faire," a return to the fun and games of the Renaissance era, in addition to getting our listeners in character back on the jocks crown the best in the best period costumes the king & queen of the fair. The winners are to receive a free library from ABC Records, WXRT-shirts, and passes to all the summerstage passes for the coming season.
### CHICAGO
**WDAL-FM**
**KURT HANSON**
**HEAVY AIRPLAY:**
- Fleetwood Mac
- Foreigner
- Peter Frampton
- Ted Nugent
- Steve Miller Band
- Heart
- Crosby/Stills/Nash
- Emerson, Lake, Palmer
- REO Speedwagon
- Yes
**MODERATE AIRPLAY:**
- Dan Fogelberg
- Elk
- Kenny Loggins
**SINGLES:**
- Alice Cooper
- Rod Stewart
- Ram Jam
- Doobie Bros.
- Supertramp
- Boston
- Rickie Lee
**SPECIAL PROGRAMMING:**
We have begun featuring a "Classic Album" in its entirety at 11:55 pm each week night with Dave Alexander. The first albums included "Led Zeppelin IV," "Every Picture Tells a Story," "Abbey Road," "Talkin' It to the Streets."
**COMMENTS:**
After over three-and-a-half years, Bill Todd is no longer program director of WDAL.
Our new program director, Roger Sholnik, was formerly program director of WDAI in Detroit, and more recently has been teaching in the communications dept. of the University of Illinois at Circle Campus while writing a textbook on broadcasting.
Evening jock Chip Hobart spent his recent vacation in Alaska where he gained both weight and broke one arm as a result of falling off a mountain while being chased by an angry mother moose.
### CHICAGO/ELGIN
**WJKL/TH FOX**
**TOM MARKER/P.D.**
**HEAVY AIRPLAY:**
- Steve Winwood
- Strawbs
- Delbert McClinton
- Johnny Winter
- Stuff
- Alan Parsons
- Bonnie Raitt
- Neil Young
- Dillard/Hartford-Dillard
- Little Feat
- Crosby/Stills/Nash
- Southside Johnny/Jukes
- Peter Tosh
- Country Joe & The Fish
- Cat Stevens
- Brand X
- Pat Travers
- Charlie
- Ben Sidran
- Rough Diamond
**MODERATE AIRPLAY:**
- Cathy Chamberlain
- Crusaders
- Deaf School
- Date Drive
- Al DiMeola
- New Grass Revival
- Ted Nugent
- Laura Nyro
- Petula Clark
- Scarlet Rivera
- Average White Band
- Lonnie Liston Smith
- REO Speedwagon
- Joan Baez
- Balcones Fault
- Roy Buchanan
- James Schafer
- Harpers
- Sonny Criss
- Coryell/Mouzon
- Marino/Mahogany Rush
- Graeme Edge Band
- Poco
- Roger McGuinn
- Bob Marley/Wailers
- Frankie Miller
- Steve Hillage
- Van Morrison
- Dave Mason
- Jack Lancaster/
Robert Lamm
- LeBlanc & Carr
- Little River Band
- Dickey Betts
- Bobby Bland
- Jack Bruce Band
- Les Dudek
- Dave Edmunds
- Ethos
- Roger Daltry
- Mink DeVille
- Art Farmer
- Greg Kihn
- Dan Fogelberg
- Peter Frampton
- Geils
- Vassar Clements
- Elkie Brooks
- Derringer
- Earth Quake
- Dirty Tricks
- John Klemmer
- Meters
- Florid Parlim
- Sons of Champlin
- Teresa Tull
- McCoy Tyner
- Steely Dan
- Phil Woods Six
- Teruo Nakamura
- Jess Roden Band
- Supertramp
- Tangerine Dream
- James Taylor
- 38 Special
- UFO
- Weather Report
- Wet Willie
- Jesse Winchester
- David Grisman Quintet
- Herbie Hancock
- Heart
- Al Jarreau
- Kingfish
**COMMENTS:**
Things are looking and sounding real good again here at The Fox. Some internal problems a couple of months ago led to losing some key people including our program director (Armand Chianti), half our music department (Trudy Fisher), and mid-day co-operson (Shel Laszig). A new program director who pledged to maintain our "format" was hired. He then proceeded to tighten up the format, change the commercial policy and in general cause the spirit of the station to plummet to new lows.
Then came the recent turnaround (6/22/77). Our new P. D. was replaced, a couple of our very qualified part-timers were moved into full-time positions, and our ideas, philosophies and morals are once again in good shape. We plan to continue to provide the Chicago market with a true progressive radio, as we have done now for nearly 3 years.
Current line-up...
**Mornings:** Glen Moberg
**Mid-days:** Wally Leisering
**Afternoons:** Tom Marker
**Evenings:** Gary Wagner
**Nights:** Chris Heim
- Tom
### ST. LOUIS
**KSHE-FM**
**TED HABECK**
**HEAVY AIRPLAY:**
- Steve Winwood
- Johnny Winter
- Stuff
- Alan Parsons
- Geils
- Fools Gold
- Burton Cummings
- Crosby/Stills/Nash
- Kiss
- Roger Daltry
- Steve Miller
- Fleetwood Mac
- Eagles
- Dave Mason
- Marshall Tucker Band
- Bonnie Raitt
- Portishead
- Cat Stevens
- Bob Seger
- Heart
- Peter Frampton
- Bad Co.
- Ted Nugent
- Rusty Wier
- Neil Young
- Little River Band
- Trooper
- Roy Buchanan
- Outlaws
- Kingfish
- Status Quo
- Moody Blues
- UFO
- Gregg Allman
**ST. LOUIS Continued...**
**KSHE-FM**
- Judas Priest
- Gale Force
- Charlie
- Poco
- Widowmaker
- Alice Cooper
- Golden Earring
- Little Feat
- Detective
- Frankie Miller
- Brownsville Station
- 10cc
- Dickey Betts
- Uriah Heep
- Supertramp
- Les Dudek
- Head East
- Pink Floyd
- Chilliwack
**REO**
- Justin Hayward
- Flame
- Lake
- Dan Fogelberg
**MODERATE AIRPLAY:**
- James Taylor
- Omaha Sheriff
- Pierce Arrow
- Derringer
- Paradise Edge Band
- Clover
- Rainbow
- Mink DeVille
- Southside Johnny/Jukes
- Joan Baez
- Earth Quake
- Pat Travers
- Capt. Beyond
- Placebo
- Lord Kenny Loggins
- Sons of Champlin
- Procol Harum
- Steve Gibbons
**SPECIAL PROGRAMMING:**
- Ram Jam
- Double Bros.
**IMPORTS:**
- Pink Floyd
- Masters of Rock
### ST. LOUIS
**KADI-FM & AM**
**P. PARSI**
**HEAVY AIRPLAY:**
- Yes
- Peter Frampton
- Crosby/Stills/Nash
- Little River Band
- Fleetwood Mac
- Supertramp
- Heart
- Jimmy Buffett
- REO Speedwagon
- Rita Coolidge
- Pabst Blue Ribbon
- Brownsville Station
- Steve Miller
- Alice Cooper
- Ted Nugent
- Kiss
- Dan Fogelberg
- James Taylor
- Cat Stevens
- Steve Winwood
**MODERATE AIRPLAY:**
- Styx
- Alan Parsons
- Sanford-Townsend
- Bonnie Raitt
- Pat Travers
- Gregg Allman
- Geils
- Bob Marley/Wailers
- Bonzo's
- Pat Travers
- Greg Allman
- UFO
- Outlaws
- Flame
- Bee Gees
- Lake
- Alexis
- Rainbow
- Bob Marley/Wailers
- Strawbs
- Brian Johnson
- Judas Priest
- Neil Young
- Moody Blues
**SINGLES:**
- Ram Jam
- Doobie Bros.
## Midwest
### Columbus, Ohio
**WCOL-FM**
**Guy Evans**
**Heavy Airplay:**
- Crosby, Stills & Nash
- Neil Young
- Cat Stevens
- Dan Fogelberg
- Neil Diamond
- Roger Daltrey
- AC/DC
- Charlie
- Foreigner
- Heart
- Peter Frampton
- Geffen
- Alan Parsons Project
- Detective
- Jerry Jeff Walker
- David Bowie
- Bob Marley & Wailers
- James Taylor
- Neil Young
- Roy Buchanan
- Jimi Hendrix
**Moderate Airplay:**
- Steve Winwood
- Frankie Miller
- Dictators
- Ruby
- Mink DeVille
### Detroit, Mich.
**WDET-FM**
**Judy Adams**
**Judy Donlin**
**Heavy Airplay:**
- Zeidman/Marsh/Harris
- 360 Degree Music Experience
- Don J. Allen (Black Saint)
- Dollar Brand
- Positive Force
- Don Cherry
- Laura Nyro
- John Coltrane
- Claus Ossmann Orch.
**Medium Airplay:**
- David Murray
- Steelye Span
- New Grass Revival
- David Grisman Quintet
- Moody Blues
- Al Green
- The Awakening
- Stone Alliance
- Anthony Braxton
- Randy Weston
- Captain Beefheart
- Julius Humphill
- Eric Anderson
- Ornette Coleman
- Don Pullen
- Roy Buchanan
### Ann Arbor, Mich.
**WQUB-FM**
**Neil Lasher/John Gleis**
**Heavy Airplay:**
- Steve Miller
- Peter Frampton
- Crosby, Stills & Nash
- Little Feat
- James Taylor
- Steve Winwood
- Dan Fogelberg
- James Winchester
- Bob Marley & Wailers
**Moderate Airplay:**
- Supertramp
- Neil Young
- Johnny Winter
- Cat Stevens
- Kenny Loggins
- Ben Sidran
- Ill Special
- Heart
- Philo Cruise
---
### Evanston/Chicago, Ill.
**WNUR**
**Chuck Schwartz/Bill Cochran**
**Heavy Airplay:**
- James Taylor
- Genesis - EP
- McCoy Tyner
- Rush
- Roger Daltrey
- Dictators
- Crosby, Stills & Nash
- Earth Quake
- Midnight Johnny/Jukes Charlie
**Moderate Airplay:**
- Gramma
- Barbara Streisand
- Attitudes
- Ray Pharo
- Perigoso
- Just Water
- Arctic Traum
- Jelly
- John Payne/Louis Levin
- Judas Priest
- Guitar Player
- Food
- Barney Kessel/Herb Ellis
**Imports:**
- **+**Seals
**Special Programming:**
- Jim Post - Live at Amazing Grace
### Rockford, Ill.
**Y-95**
**Brad Hoffman**
**Heavy Airplay:**
- Peter Frampton
- Dan Fogelberg
- Steve Miller
- Fleetwood Mac
- Ted Nugent
- Foreigner
- Crosby, Stills & Nash
- Kris Kristofferson
- Little Feat
- Boston
- Paul Horn
- Emerson, Lake & Palmer
- Hiss
- Marshall Tucker Band
- Outlaws
- Climax Blues Band
- Jimmy Buffett
- Weather Report
- Neil Young
- Straws
- Rainbow
- Singles:
- Ram Jam
### Urbana/Champaign, Ill.
**WPGU-FM**
**Dean Moss**
**Heavy Airplay:**
- Straws
- Steve Winwood
- Crosby, Stills & Nash
- Dan Fogelberg
- Alan Parsons Project
- Genesis Edge Band
- Timberrlane
- James Taylor
- Judas Priest
- Geffen
- American Flyer
- Joan Baez
- Outlaws
**Moderate Airplay:**
- Little Feat
- Greg Kihn
---
### Madison, Wisc.
**WORT-FM**
**Jerry Zeiger**
**Heavy Airplay:**
- Dollar Brand
- Daniel Hecht
- McCoy Tyner
**Moderate Airplay:**
- Chuck Hamilton
- Roy Ayers
- Cecil Taylor
- Richie Cole/Eric Kloss
- Bob Dylan
- Doug & Jean Carn
- Ornette Coleman
- Paul Horn
- Steve Lacy/Michael Smith
- Ornette
- Arild Andersen
- Don Cherry
- Jean-Pierre Mas
- The Mutha (Pablo)
- Gregory Perry
- James Taylor
- Townes Van Zandt
- Clifton Chenier
- William Nelson
- Steelye Span
- Paul Horn
- Cleveland Eaton
### Madison, Wisc.
**WIBA-FM**
**Peter Bolger**
**Heavy Airplay:**
- Heart
- Ben Sidran
- Crosby, Stills & Nash
- Bonnie Raitt
- Steve Miller
- Al Jarreau
- Little River Band
- Steve Winwood
- Norton Buffalo
- Bruce Cockburn
- Little Feat
- James Taylor
- John Klemmer
---
### Milwaukee, Wis.
**WZMP**
**Don Rockwell**
**Heavy Airplay:**
- Bob Marley & Wailers
- Crosby, Stills & Nash
- Straws
- James Taylor
- Heart
- Alan Parsons Project
- Ted Nugent
- Supertramp
- Steve Miller
- Neil Young
- Foreigner
- U.F.O.
- Cat Stevens
- Charlie
- Bad Co.
- Chilliwack
**Moderate Airplay:**
- Ruby
- Rough Diamond
- Widowspeak
- Orisha Sheriff
- Johnny Cash
- Puerto Ares
- Country Joe & Fish
- Mahogany Rush
- Stephen Sinclair
### Lincoln, Neb.
**KFMO**
**Rich Meyer**
**Heavy Airplay:**
- **+**Straws
- Crosby, Stills & Nash
- Neil Young
- James Taylor
- Heart
- Peter Frampton
- Eric Fosberg
- Kenny Loggins
- Fleetwood Mac
- Eagles
- Steve Miller Band
- Foreigner
**Moderate Airplay:**
- Bad Co.
- Rita Coolidge
- Chilliwack
- Emerson, Lake & Palmer
- Little Feat
- Little River Band
- Dave Mason
- Ted Nugent
- Outlaws
- Poco
- Bonnie Raitt
- Cat Stevens
- Alan Parsons Project
### Omaha, Neb.
**KQ98**
**Barry Taft**
**Heavy Airplay:**
- **+**Average White Band
- Eagles
- Marshall Tucker Band
- Thunderlane
- Pink Floyd
- Ted Nugent
- Jerry Jeff Walker
- Dan Fogelberg
- R.E.O. So Speedwagon
- Fleetwood Mac
Continued on top of next page.
## MIDWEST
**KQ8 Continued...**
- Stevie Wonder
- Gregg Allman Band
- Duane Allman
- Peter Frampton
- Foreigner
- Steve Miller Band
- Little Feat
**MODERATE AIRPLAY:**
- Bluz
- Clover
- Rainbow
- Pierce-Arrow
- Stephen Sinclair
**Seattle Continued...**
**KZOK**
**SPECIAL PROGRAMMING:**
- Midnight Album Feature:
- Kiss
- Tangerine Dream
- Country Joe & The Fish
- Steve Winwood
- Rainbow - Live
- Johnny Winter
- Roy Buchanan
- Greg Kihn
- Styx
**COMMENTS:**
- Greg Kihn - air interview with the members of the Gells Group here in town for the Frampton concert -
Magic Dick even played some harmonica. Also had live music from the Frampton concert at the Kingdome -
every hour reports starting in the morning - covered traffic, lines, backstage, etc.
**Seattle/Bellevue Continued**
**KZAM-FM&AM**
- Jimmy Buffett
- Atlanta Rhythm Aces
- Joan Baez
- Ian Matthews
- Bruce Cockburn
- David Grisman
- Van Morrison
- Little Feat
- Fats Gold
- Peter Tosh
- Jesse Winchester
- Emmylou Harris
- Keith Jarrett
**MODERATE AIRPLAY:**
- Hoyt Axton
- Band
- Marshall Tucker Band
- Steve Miller
- Johnny Winter
- Horslips
- Jonathan Edwards
- Danny O'Keefe
- Poco
- Pousette-Dart Band
- Weather Report
- Jesse Colin Young
- Country Joe & The Fish
- Roger Daltrey
- Peter Frampton
- New York, N.Y. - Sndtrk.
- John Payne
- Lou Martin
- Melissa Manchester
- Pierce Arrow
- Joe Turner
- Cathy Chamberlain
- Rita Coolidge
- Dean Friedman
- Al DiMeola
- John Klemmer
- Crusaders
- Laura Nyro
- Duni & Minanzi
- Marimba Ensemble
- (local)
- Ben Sidran
- Persuasions
**OTHER NEW ADDITIONS:**
(6/25-7/8)
- Yes
- Ben E. King & A.W.B.
- Dollar Brand
- Eric Schoenberg
- Stuji
- Ray Bryant
- LeLanc & Carr
- Osamu Kitajima
- Omaha Sheriff
- Wavy Gravy
- Lonnie Liston Smith
- Roland Prince
- Carl Adams (local)
- Strayrbs
- Robert Ford
- Paul Horn
- Jaime Brockett
- Tony Rice
- Blues
- Sunny jazz!
**SINGLES:**
- Doobie Bros.
- Carl Simon
**SPECIAL PROGRAMMING:**
- Interviews with Barry Melton, the Fish, and the Outlaws.
**IMPORTS:**
- 801 - Live
- Genesis EP
## WEST
### LOS ANGELES
**KLOS**
**DABAR HOORELBEKE**
**HEAVY AIRPLAY:**
- Fleetwood Mac
- Cat Stevens
- Peter Frampton
- Heart
- Bee Gees
- Jimmy Buffett
- Dan Fogelberg
- Crosby, Stills & Nash
- Kenny Loggins
- James Taylor
**SINGLES:**
- Carole King
- Doobie Bros.
- Foreigner
- Rita Coolidge
- England Dan/J.F. Coley
**SEATTLE**
**KZOK-102-1/2**
**MAYE MACCKFF**
**HEAVY AIRPLAY:**
- Fleetwood Mac
- Heart
- Peter Frampton
- Foreigner
- Crosby/Stills/Nash
- Eagles
- Neil Young
- Boston
- **James Taylor**
- Roger Daltrey
- Bonnie Raitt
- Bob Seger
- Alan Parsons
- Steve Miller
- Jethro Tull
- Bob Seger
**MODERATE AIRPLAY:**
- Marshall Tucker Band
- Al Stewart
- Jimmy Buffett
- Crusaders
- John Klemmer
- Little River Band
- Joan Baez
- Cat Stevens
- Rita Coolidge
- Gells
**SINGLES:**
- Carole King
- Doobie Bros.
**SEATTLE**
**KISW-FM100**
**BILL BARTLETT**
**HEAVY AIRPLAY:**
- Supertramp
- Foreigner
- Heart
- Ted Nugent
- Peter Frampton
- Bob Seger
- Al DiMeola
- Fleetwood Mac
- Crosby/Stills/Nash
- Stevie Miller
**MODERATE AIRPLAY:**
- Little River Band
- Kiss
- James Taylor
- Little Feat
- Greg Kihn
- Pat Travers
- Gells
- Bob Seger
**SINGLES:**
- Ringo Jean
**SPECIAL PROGRAMMING:**
July 4th - FM 100 played Seattle's top 100 album of all time - yes, "Stairway to Heaven" still came out #1.
**NEWS/PUBLIC AFFAIRS:**
The FM 100 program Advisory Board is in town today. Each month we select 12 listeners from the Seattle area who will meet once a month to discuss our programming procedures at FM 100. We will give them records to listen to along with having the board members do street interviews. Our first meeting is coming up in late July.
**COMMENTS:**
- Announces Line-up...
5 - 10 am - Tom McDonald
10 - 3 pm - Steve Nickolas
3 - 7 pm - Bill Bartlett/PD
7 - midnite - Chris Taylor
12 - 6 am - Bob Cardenas
**SEATTLE/BELLEVUE**
**KZAM-FM&AM**
**TOM CORRYDOR**
**JON KERTZER**
**HEAVY AIRPLAY:**
- James Taylor
- Steve Winwood
- Dan Fogelberg
- Crosby/Stills/Nash
- Fleetwood Mac
- Little Feat
- Greg Kihn
- Bonnie Raitt
- Cat Stevens
- Norton Bufalo
- Gells
- Willie Nelson
- Bob Marley
- Kinks
**MODERATE AIRPLAY:**
- Marshall Tucker Band
- Al Stewart
- Jimmy Buffett
- Crusaders
- John Klemmer
- Little River Band
- Joan Baez
- Cat Stevens
- Rita Coolidge
- Gells
**SINGLES:**
- Carole King
- Doobie Bros.
**SEATTLE/Bellevue Continued**
**KZAM-FM&AM**
We've been busy building a new facility, enjoying our best ARB yet.
Newly wrapped up in energy politics - Alaskan crude, hydro power, and in poison disposal.
Come see us if you pass through.
- Tom
## SACRAMENTO
**KZAP-FM**
**BRUCE MEIER**
**HEAVY AIRPLAY:**
- Crosby/Stills/Nash
- Steve Winwood
- Steve Miller
- Neil Young
- Little Feat
- Gregg Allman
- Heart
- Bonnie Raitt
- Mink DeVille
- Laura Nyro
- Fleetwood Mac
- Cat Stevens
- Charlie
- Dan Fogelberg
- Jesse Winchester
- Peter Frampton
- Poco
**MODERATE AIRPLAY:**
- Greg Kihn
- Lake
- Waylon Jennings
- Norton Buffalo
- Bette Midler
- Supertramp
- Wallers
- Alan Parsons
- John Martyn
- Roger Daltrey
- Country Joe & The Fish
- Strawbs
- Johnny Winter
- Little River Band
- Dixie Dregs
- Gells
- Townes Van Zandt
- Clover
**SINGLES:**
- Rockford Falconer
- Doobie Bros.
- Sparks
- Graham Central Station
**SPECIAL PROGRAMMING:**
- Album Features: Crosby, Stills, & Nash and Steve Winwood.
- 4th of July Special: Rock and Roll America.
**IMPORTS:**
- 801 - Live
- Genesis EP
## SAN FRANCISCO
**KSAN-FM**
**BONNIE SIMMONS**
**HEAVY AIRPLAY:**
- Mink DeVille
- Steve Winwood
- Crosby/Stills/Nash
- Gells
**COMMENTS:**
We've finally had our first staff departure - after 2-1/2 years, Shelley Morrison 9-noon Anderson, promotion director, show-business Frisbee champion, and head camp counselor, will be moving to London to study, play, write and eat. He will miss her....taking over as promotion director, Patricia Silverman. Moving up from part-time to full-time airperson, Steve Suplin (nee KMYR).
Continued on top of next page
## WEST
**San Francisco Continued...**
**KSAN-FM**
- Al Jarreau
- Bryan Ferry
- Johnny Winter
- Isley Bros.
- Norton Buffalo
- Clifton Chenier
- UFO
- Little Feat
- Kiss
- Mott's
- Heart
- Credibility Gap
- Horslips
- Charlie
- Soundside Johnny/Jukes
- Alice Cooper
- Dave Lewis
- Dictators
- Dianne O'Keefe
**SINGLES:**
- *Doobie Bros.
- *Norton Buffalo
- *Little Feat
- *Kiss
- *Heart
- *Credibility Gap
- *Horslips
- *Charlie
- *Soundside Johnny/Jukes
- *Alice Cooper
- *Dave Lewis
- *Dictators
- *Dianne O'Keefe
**SPECIAL PROGRAMMING:**
- Interviews with Norton Buffalo, Gale Force, and Ronnie Van Zandt.
**IMPORTS:**
- Genesis
---
## SAN JOSE
**KSJO**
**KATE INGRAM**
**HEAVY AIRPLAY:**
- Tubes
- Nutz
- Peter Frampton
- Alan Parsons Project
- Geils
- Greg Kihn
- UFO
- Frankie Miller
- Ted Nugent
- Little River Band
- Steve Winwood
- Charlie
- Widowmaker
- Roger Daltrey
- Pat Travers
- Heart
- Neil Young
- Little Feat
- Alice Cooper
**NEW ADDS:**
- Styx
- Melissa Manchester
- Average White Band
- Yes
- Stravas
- Waves
**MODERATE AIRPLAY:**
- Crosby, Stills & Nash
- Mink DeVille
- Dan Fogelberg
- Geils
- *Ted Nugent
- Alan Parsons Project
- James Taylor
- UFO
- Bob Marley & Wailers
- *Steve Winwood
- *Yes
**MODERATE AIRPLAY:**
- Styx
- Gregg Allman
- *Norton Buffalo
- Country Joe & The Fish
- *Crusaders
- Roger Daltrey
- Alice Cooper
- *Steve Miller
- Pat Travers
- *Neil Young
- Clovis
- Dixie Dregs
- Focus
- Peter Frampton
- Horslips
- Mott's
- John Flannery
- Lake
- Little River Band
- Laura Nyro
- Outlaws
- Lee Ritenour
- Leon & Mary Russell
- *The Section
- *Gorham - Snider
- *Cat Stevens
- *Strawbs
- *Johnny Winter
- *AC/DC
- Average White Band
- *Cosyall/Mauron
- *Kevin Coyne
- Burton Cummings
- Rick Dancer
- Distinct
- Earth Quake
- *Graeme Edge Band
- Illusion
- *Al Jarreau
- *Kiss
- *Melissa Manchester
- *Osaka Sheriff
- *Osamu Ritajima
- *Pioneer Arrow
- *Flora Purim
- Rainbow
- Scarlet Rivera
- David Babson
---
## PORTLAND
**KVAN-AM**
**BOB ANCHETA**
**HEAVY AIRPLAY:**
- *Nazareth
- *Gregg Allman
- Peter Frampton
- *Lake
- **Nils Lofgren
- *Dixie Dregs
- *LeBlanc & Carr
- *Johnny Winter
- Neil Young
- *Little Feat
- *Judas Priest
- *Leo Dudek
- *Lee Ritenour
- *Reggie Knighton
- Steve Winwood
- *Tubes
- *3R Special
- *Little River Band
- Pablo Cruise
- Heart
- Jimmy Buffett
- *Crosby, Stills & Nash
- *Roy Buchanan
- *Earth Quake
- *Charlie
- *10cc
- *Moxy
- Kingfish
- Supertramp
- Gold Earring
- Burton Cummings
- Dan Fogelberg
- **Pat Travers
- *Graeme Edge
- MODERATE AIRPLAY:**
- Outlaws
- Poco
- Frankie Miller
- Mink DeVille
- Widowmaker
- Tom Petty
- Mahogany Rush
- Ted Nugent
- Kansas
- Loggins
- Jess Roden Band
- Crusaders
- Climax Blues Band
- Gale Force
- Sonny Champlin
- Rainbow
- *Country Joe & The Fish
- *Horslips
- *Pierces Arrow
- *Leon & Mary Russell
- *Norton Buffalo
- Cliff Richard
- Noel Pointer
- Masters
- Phillip Walker
- Max Webster
- Roger Daltrey
- Greg Kihn
- James Taylor
- Clover
- Fools Gold
- Ronnie Laws
- Horslips
- Derringar
- Ultravox
- Bob Marley & Wailers
- Country Joe & The Fish
- George Carlin
- Graeme Edge Band
- Jerry Jeff Walker
- Troopers
- Bruce Foster
- Leon & Mary Russell
- Dirty Tricks
- Joan Jett
- Kiki Dee
- American Flyer
- Bonnie Raitt
- Al Jarreau
**SINGLES:**
- *Ram Jam
- *Doobie Bros.
- SPECIAL PROGRAMMING: Rock Around the World Innerview
- Susan Berkley's Natural Food Restaurant
- Side Salad
- Classic Wax
---
## NEWS & PUBLIC AFFAIRS:
KVAN has begun a New Magazine for the Ears titled What It Is. This is featured Monday at 5:15pm. What It Is features almost anybody who has done something we think our listeners would like to hear about. Recently featured: The Paramount Theatre - Portland's rock ball.
- A Groove Digest
- A Collection Agency
- A Greg Dealer
- A Train Engineer
- And that's What It Is.
- Nancy Jackson
**COMMENT:**
KVAN is co-sponsoring the following events - July 21 & 22 - Bobby Bland; August 3 - Little River Band, Nils Lofgren; August 13 - Jerry Jeff Walker, Pura Prairie League.
Due to changes at KVAN I am now handling programming and music airplay reports will be given noon to 4 p.m. weekdays. Steve Lewis joins up ll a.m. to 3 p.m.
Be sure and get your KVAN "Back To Mono" buttons. Small quantity is available upon request.
Bob Ancheta
---
## MESA / PHOENIX
**KDKB-FM**
**DWIGHT TINDLE**
**HEAVY AIRPLAY:**
- Crusaders
- Cat Stevens
- Bob Seger
- Dickie Betts
- Crosby/Stills/Nash
- Fleetwood Mac
- Stevie Wonder
- Marvin Gaye
- Heart
- Pablo Cruise
- Bee Gees
- Supertramp
- Walter Trout
- Waylon Jennings
- Jerry Ropelle
- Emerson, Lake, Palmer
- Alice Cooper
- Roger Daltrey
- Garland Jeffreys
- Jesse Winchester
- Bonnie Raitt
- James Taylor
- Peter Frampton
- Geils
- Jimmy Buffett
- Bros. Johnson
- Steve Miller
- Dan Fogelberg
**MODERATE AIRPLAY:**
- Little Feat
- Poco
- Rita Coolidge
- Tubes
- Gregg Allman
- Kenny Loggins
- Jethro Tull
- Dave Mason
- Neil Young
- Joan Baez
- Blue
- Mink DeVille
- Clover
- American Flyer
- Attitudes
- Bobby Bland
- War
- *Johnny Winter
- *Styx
- *Fools Gold
- *Lake
- *Waves
- *Sunshine
- *Stuff
## WEST
### FT. COLLINS, COLO.
**KTCI-FM**
**CHARLIE WROBBEL**
- **HEAVY AIRPLAY:**
- Alan Parsons Project
- Crosby, Stills & Nash
- Neil Young
- Joan Baez
- Dan Fogelberg
- Steve Winwood
- Grateful Allman Band
- **MODERATE AIRPLAY:**
- Doc & Merle Watson
- Steelye Span - Story
- Norten Buffalo
- Blackwater
- Strawbs
- Lonnie Liston Smith
- Scarlet Rivera
- Steve Winwood
- James Taylor
- Jim Post
- Jaime Brockett
- Crusaders
- Celia
- John Klemmer
- Laura Nyro
- Ben Sidran
- Townes Van Zandt
- Reggie Knighton
### EUGENE, ORE.
**KZEL-FM**
**STAN GARRETT**
- **HEAVY AIRPLAY:**
- Heart
- Alan Parsons Project
- Peter Frampton
- Pablo Cruise
- Crosby, Stills & Nash
- Joni Mitchell
- Rick Derringer
- Steve Winwood
- Ted Nugent
- Roger Daltrey
- John Lennon
- Barn Jam
- Waylon Jennings
- **MODERATE AIRPLAY:**
- Wreckingaker
- AC/DC
- Riki Dee
- James Taylor
- Neil Young
- Janis Joplin
- Bob Marley & Wailers
- Pat Travers
- Dan Fogelberg
- Mindy McCready
- LaMont Dozier
- Willie Hutch
### FRESNO, CALIF.
**KFJG**
**ART FARKAS**
- **HEAVY AIRPLAY:**
- Cat Stevens
- Rod Stewart
- James Taylor
- Crosby, Stills & Nash
- Linda Ronstadt
- Supertramp
- Dan Fogelberg
- Kenny Loggins
- Moody Blues
- Joan Baez
- **MODERATE AIRPLAY:**
- Roger Daltrey
- Waylon Jennings
- Michael McDonald
- Bob Seger
- Carole Bayer Sager
- Geils
- Norten Buffalo
- FM 85:
- Crosby, Stills & Nash
- England Dan/J.F. Coley
- Supertramp
- Cat Stevens
- Waylon Jennings
---
## KFIG Continued...
- Brothers Johnson
- Burton Cummings
---
## KTYD-AM/FM Continued...
- Greg Allman
- Bob Marley & Wailers
- Southside Johnny
- Paul Horn
---
## KUOP Continued...
- Bill Evans
- Paul Horn
- Horlups
- Jan Garbarek
- Tangerine Dream
---
## STOCKTON/MODESTO, CA.
### KSRT-FM
**MARK SHERONY**
- **HEAVY AIRPLAY:**
- David Bowie
- Charlie
- Crosby, Stills & Nash
- Heart
- Head East
- Little Feat
- Steve Miller
- The Outlaws
- Cat Stevens
- 10cc
- Pat Travers
- Tubes
- UFO
- Max Webster
- Steve Winwood
- Genesis (EP)
- **MODERATE AIRPLAY:**
- Joan Baez
- Dan Fogelberg
- Foreigner
- Peter Frampton
- Little River Band
- Dave Mason
- James Taylor
- Alan Parsons Project
- Johnny Winter
---
## CHICO, CALIF.
**KFMP**
**RON WOODWARD**
- **HEAVY AIRPLAY:**
- Crosby, Stills & Nash
- Janis Joplin
- Peter Frampton
- James Taylor
- Little River Band
- Geils
- Pablo Cruise
- Steve Miller
- Fleetwood Mac
- Little Feat
- Neil Young
- Gary Kihn
- Sea Level
- Steve Winwood
- Heart
- Bob Seger
- Meters
- Van Morrison
- Foreigner
- Supertramp
- Cat Stevens
- Roger Daltrey
- Bonnie Raitt
- Norton Buffalo
- Ben Sidran
---
## SAN BERNARDINO/RIVERSIDE/ONTARIO, CA.
**KCAL-FM**
**JOHN LESLIE/JIM JAMES**
- **HEAVY AIRPLAY:**
- Strasbs
- Blue
- Styx
- Johnny Winter
- REO Speedwagon
- Poco
- Les Dudek
- Geils
- Quits
- Kiss
- Peter Frampton
- Neil Young
- Derringer
- Roger Daltrey
- Rainbow
- Alan Parsons Project
- Steve Winwood
- **MODERATE AIRPLAY:**
- Eric Clapton
- James Taylor
- Dirty Tricks
- Melissa Manchester
- Pools Gold
- Friars Benefit
- Steppen Sinclair
- The Section
- Lee Ritenour
---
## FRESNO, CALIF.
**ROCK 96 FM**
**JEFF POLLACK**
- **HEAVY AIRPLAY:**
- Crosby, Stills & Nash
- Peter Frampton
- Steve Miller
- Fleetwood Mac
- Heart
- Supertramp
- Ted Nugent
- James Taylor
- **MODERATE AIRPLAY:**
- Charlie
- Roger Daltrey
- Foreigner
- Gregg Allman
- Neil Young
- Dan Fogelberg
- England Dan/J.F. Coley
- Kiss
- Little River Band
- Steve Winwood
- Pat Metheny
- Kenny Loggins
- LeBlanc & Carr
- Bonnie Raitt
---
## SANTA BARBARA, CALIF.
**KTYD-AM/FM**
**LAURIE COBB**
- **HEAVY AIRPLAY:**
- Melissa Manchester
- Steve Winwood
- Country Joe & The Fish
- Crosby, Stills & Nash
- Little Feat
- Quicksand
- Jesse Winchester
- Carol Bayer Sager
- James Taylor
- Little Feels
- Jerry Rigsbee
- Poco
- Bonnie Raitt
- **MODERATE AIRPLAY:**
- Alan Parsons Project
- Coryell/Mouzon
- Roger Daltrey
- Willie Nelson
- Dillard, Hartford, Dillard & Associates
- Kenny Loggins
---
## STOCKTON, CALIF.
**KUOP**
**JOHN SHAFFER**
- **HEAVY AIRPLAY:**
- Osamu Kitajima
- Genesis
- **MODERATE AIRPLAY:**
- Opal
- Alan Parsons Project
- Steve Kuhn
- Gary Burton
- Pat Metheny
- Focus
- Nutt
- Coryell/Mouzon
- Don Cherry
- John Coltrane
- Gary Bartz
- Steelye Span
- Lake
- Jeremy Steig
- FM 4
## WEST
### ALBUQUERQUE, N.M.
**KMYR**
**CHARLIE WEIR**
**HEAVY AIRPLAY:**
- Geils
- Heart
- Little Feat
- Neil Young
- Norton Buffalo
- Steve Miller
- Peter Frampton
- Crosby, Stills & Nash
- Dan Fogelberg
- *James Taylor
- Little River Band
- Bob Marley & Wallers
- Roger Daltrey
- *Yes
- Alan Parsons Project
- *Steve Winwood
- Lake
**MODERATE AIRPLAY:**
- Fleetwood Mac
- Bonnie Raitt
- *Melissa Manchester
- *Average White Band
- & Ben E. King
- *Country Joe & The Fish
- *Fools Gold
- *Strawbs
- *Perigo
### LA CRUCES, N.M.
**KNMS-AM CA FM**
**JEFF LYON**
**HEAVY AIRPLAY:**
- Cat Stevens
- Crosby, Stills & Nash
- American Flyer
- Fleetwood Mac
- Heart
- Peter Frampton
- Waylon Jennings
- The Greatest - Soundtrack
- Barry Manilow - Live
- Kiss
**MODERATE AIRPLAY:**
- Graeme Edge Band
- Willie Nelson
- Roger Daltrey
- Steve Winwood
- Moody Blues
- Outlaws
- Foreigner
- Poets
- Jimmy Buffett
- Dave Mason
- Simon & Garfunkel
- Barry Manilow
- Peter Frampton
- Steve Miller
- Jimmy Buffett
- Fleetwood Mac
---
## RENO, NEV.
**KGIR**
**PAUL EMERY**
**HEAVY AIRPLAY:**
- Outlaws
- Alan Parsons Project
- Heart
- Roger Daltrey
- Peter Frampton
- Dan Fogelberg
- Illusions
- Horslips
- Kenny Loggins
- Crosby, Stills & Nash
- Steve Winwood
- Norton Buffalo
- Peter Frampton
- Lake
- Graeme Edge Band
- Steve Miller Band
- Laura Nyro
- *Yes
- Neil Young
**MODERATE AIRPLAY:**
- Omaha Sheriff
- *Styx
- *Average White Band
- & Ben E. King
- *Melissa Manchester
- *Earth Quake
- *Pat Travers
---
## KAWY Continued...
- Pierce Arrow
- Artie Traum
- **Yes
- Greg Kihn
- +++Bob Daltrey
- **Lenny Williams
- *Average White Band
- *Lake
- +++Osamu Kitajima
- Danny Kirwan
- **Johnny Winter
---
## ALBUQUERQUE, N.M.
**KUNM**
**MIKE REGAN**
**HEAVY AIRPLAY:**
- *Steve Winwood
- *Paul Horn
- *Johnny Winter
- *Bruce Brockett
- *Carolina Payton
**MODERATE AIRPLAY:**
- *Earl Hines
- *With Marva Josie
- *Omaha Sheriff
- *Jessi Colter
- *Jim Ransom
- *Kevin Coyne
- *Joe Val & New England
- *Buckwass
- #Parigon
- *Ray Pizzi
- *Melissa Manchester
- *Enrico Rava
- *Coryell/Mourzon
- *Strawbs
- *Osamu Kitajima
- *Burton Speer
- *Wild Anderson
- *Ray Bryant
- *Country Joe & The Fish
- *Robben Ford
- *Bob Dorough
- *Peter Alsop
---
## EUGENE, ORE.
**KFMY**
**DAVID HARPER**
**HEAVY AIRPLAY:**
- Joan Baez
- Jim Croce
- Jimmy Buffett
- Crosby, Stills & Nash
- Roger Daltrey
- Dan Fogelberg
- Peter Frampton
- Lake
- Heart
- Dillard, Hartford, Dillard
- Waylon Jennings
- Alan Parsons Project
- Tom Petty
- Pink Floyd
- Supertramp
- Ben Sidran
- *Average White Band
- Gary Burton Quartet
- Crusaders
- Maynard Ferguson
- Urbie Green
- David Jacobs
- John Klemmer
- Flora Purim
- Enrico Rava
- Jeremy Steig
---
## CASPER, WYO.
**KAWY**
**FRED MOORE**
**HEAVY AIRPLAY:**
+++Strawbs
+++Steve Winwood
- Crosby, Stills & Nash
- James Taylor
- Cat Stevens
- **Geils
- Little River Band
- Laura Nyro
- +Jesse Winchester
- Steve Miller Band
- Dan Fogelberg
- +++Brothers Johnson
**MODERATE AIRPLAY:**
++Alan Parsons Project
++Scarlet Rivera
---
## TUSCON, ARIZ.
**KWFM**
**HEAVY AIRPLAY:**
- Charlie
- George Allman
- Crosby, Stills & Nash
- Lake
- Supertramp
- Alan Parsons Project
- Dixie Dregs
- 10cc
- Steve Miller
- Sea Level
- Joan Baez
- Brothers Johnson
- Sons of Champlin
- Fools Gold
- Little River Band
**MODERATE AIRPLAY:**
- American Flyer
- Kenny Loggins
- Laura Nyro
- Don O'Keefe
- Heart
- Roy Buchanan
- Steve Winwood
- James Taylor
- Mink DeVille
## WEST
### ANAHEIM, CALIF.
**KEZY-FM**
**RON BURNSTEIN**
**HEAVY AIRPLAY:**
- Crosby, Stills & Nash
- Peter Frampton
- Steve Miller
- Dan Fogelberg
- James Taylor
- Heart
- Fleetwood Mac
- Frankie Miller
- Southside Johnny/Jukes
- Neil Young
- Steve Winwood
- Cat Stevens
- Alan Parsons
- Supertramp
- Mike Oldfield
- American Flyer
- Bob Marley & Wailers
- Roger Daltrey
- Chic
**MODERATE AIRPLAY:**
- Little River Band
- Kenny Loggins
- Melissa Manchester
- Roderick Falconer
- Outlaws
- Johnny Winter
### SAN RAFAEL, CALIF.
**KTIM-AM/FM**
**TONY BERARDINI**
**HEAVY AIRPLAY:**
- +Norton Buffalo
- +Mother's Finest
- Greg Kihn
- AC/DC
- Peter Frampton
- Brothers Johnson
- +Ted Nugent
- Girls
- Steve Winwood
- +Osamu Kitajima
- Yes
- Alan Parsons
- UFO
- Johnny Winter
- Heart
- Earth Quake
- Crosby, Stills & Nash
- Steve Miller
- Supertramp
- Steve Miller
- Little River Band
- +Sex Pistols
- +Genesis
- IMPORTS:
- +Jean Michele Jarre
- AC/DC
### ALBUQUERQUE, N.M.
**KRST-FM**
**BILL STAMBAUGH**
**HEAVY AIRPLAY:**
- James Taylor
- Steve Winwood
- Fools Gold
- Yes
- Styx
- Average White Band
- Steve Miller
- Crosby, Stills & Nash
- Peter Frampton
- Dan Fogelberg
- Foreigner
- Heart
- Cat Stevens
- Fleetwood Mac
- Little Feat
- Roger Daltry
**MODERATE AIRPLAY:**
- Alan Parsons Project
- Little Feat
- Gregg Allman
- Supertramp
- Kenny Loggins
- Little River Band
- Outlaws
- Ted Nugent
- Bonnie Raitt
### DENVER, COLO.
**KCFR**
**BOB STECKER**
**REGULAR AIRPLAY:**
- Stephen Alcicus (import)
- Osamu Kitajima
- Paul Horn
- Cano (import)
- Alan Parsons Project
- Star Wars - Smtrk.
- Star Wars - Smtrk.
- Scarlet Rivera
- Robben Ford
- Bob Marley & Wailers
- Steeleye Span
## KCFR-FM Continued...
**Dick Pinney**
Newest releases on:
- ECM
- Pablo
- Inner City
- Muse
- Arista/Freedom
- Asylum
- Adelphi
- Flying Fish
- Rounder
- Columbia Masterworks
- H. N. H. (classical)
### BAKERSFIELD, CALIF.
**KXXX-FM**
**TEARY GAISER**
**HEAVY AIRPLAY:**
- +Crosby, Stills & Nash
- Fleetwood Mac
- Peter Frampton
- Heart
- +Dave Mason
- Steve Miller
- Supertramp
- Cat Stevens
- James Taylor
**MODERATE AIRPLAY:**
- +American Flyer
- Burton Cummings
- Geils
- Gregg Allman Band
- Joan Baez
- Charlie
- Dan Fogelberg
- Brothers Johnson
- Little River Band
- Ted Nugent
- Alan Parsons Project
- *Sanford-Townsend Band
- *Tubes
- Steve Winwood
**SINGLES:**
- Sanford-Townsend Band
- Brothers Johnson
## POWER AND/OR MARKET LIMITED CONTRIBUTORS
(Record listings provided by the following stations are used, along with all other stations, in our compilations.)
- ACRN/Athens, Ga.
- KASF/Alamosa, Colo.
- KBLE/Des Moines, Iowa
- KBSU/Boise, Idaho
- KCR/San Diego, Calif.
- KCSN/Northridge, Calif.
- KSU/Ft. Collins, Colo.
- KFTC/Los Altos Hills, Ca.
- KFTR/Fresno, Calif.
- KLCT/Brookhaven, Ohio
- KOHL/Fremont, Calif.
- KMR/Sun Valley, Idaho
- KOSU/Roseville Park, Calif.
- KZSU/Sanford, Calif.
- WBCR/Brooklyn, N.Y.
- WBSD/Burlington, Wisc.
- WBNN/Ann Arbor, Mich.
- WCRW/Chester, Pa.
- WCUD/Worcester, Mass.
- WEMU/Ypsilanti, Mich.
- WIDR/Kalamazoo, Mich.
- WJUR/Dowell, Mass.
- WJHW/WI Pleasant, Mich.
- WMIR/Lake Geneva, Wisc.
- WMMR/Minneapolis, Minn.
- WNHU/West Haven, Conn.
- WQAX/Bloomington, Ind.
- WRNW/Westchester-NYC
- WRPL/Charlotte, N.C.
- WRUC/Schenectady, N.Y.
- WSPU/Murphysboro, Ill.
- WUMB/Boston, Mass.
- WVPE/Elkhart, Ind.
- WWUH/W. Hartford, Conn.
- WZLY/Wellesley, Mass.
- RUBY LASER
- MICHAEL T. DENNIS
- RON SORENSON
- ROBB CAMPBELL/
CARL SCHNEIDER
- BARRY WEILLMAN
- KEVIN STERN
- PAT BARRY
- FRED LEVY
- PETER STEEN
- DEBBIE GREENSPAN
- PAMELA PASSANEI
- C. GATES
- MARK ANDERSON
- JAMES JONES
- LLOYD JASSIN
- JEFF WEST
- MICHAEL KREMEN
- RICH BOHS
- GWEN MERCERKS
- DENNIS HARTFORD
- VAL ELLETT
- RICH GINGRAS
- DAVID MALLEN
- FERRY HAYEL
- CULLEN ENGLAND
- TOM SABELLICO
- NEIL SHARROW
- BRADLEY GRIFFIN/
"JOE FROM CHICAGO"
- DEBBI CALTON
- RANDY MEYERS
- TODD CAVE/JOE HALPIN
- CHRIS HOLDT
- TIM EBY
- STU LOVEJOY
- SARA BEDROSIAN
## RETAIL STATS
Our relative charting, as seen below, resembles baseball's batting averages. It establishes a ratio between the ideal (top 5 sales at every reporting retailer), and the weighted count that a recording has actually earned. This mathematical formula is applied to each record—first regionally, then nationally. A dual weighting system is used to derive the count on the hits. It adjusts for store chain size and reporting location in the top 15. A single weighting system which adjusts for store chain size is applied to the breakout reports. These charts show the relative intensity of retail interest for both new and established recordings.
### PROGRESSIVE RETAIL
| 2 ISSUES BACK | LAST ISSUE | CURRENT NATIONALS | ARTIST | N.E. | S. | M.W. | W. |
|---------------|------------|-------------------|-----------------|------|------|------|------|
| .856 | .880 | .836 | FLEETWOOD MAC | .618 | .961 | .928 | .947 |
| .322 | .510 | .667 | PETER FRAMPTON | .778 | .804 | .544 | .653 |
| --- | .161 | .504 | C.S. & N. | .465 | .902 | .533 | .840 |
| .411 | .426 | .444 | HEART | .236 | .176 | .711 | .387 |
| .626 | .574 | .444 | STEVE MILLER | .382 | .137 | .606 | .387 |
| --- | .226 | .362 | KISS | .153 | .549 | .467 | .387 |
| --- | --- | .329 | JAMES TAYLOR | .333 | .804 | .189 | .333 |
| .543 | .525 | .296 | EAGLES | .347 | .667 | .217 | .133 |
| .290 | .206 | .293 | DAN FOGELBERG | .403 | .510 | .233 | .080 |
| .253 | .366 | .289 | FOREIGNER | .243 | .020 | .400 | .293 |
| .178 | .215 | .269 | JIMMY BUFFETT | .285 | .471 | .144 | .400 |
| .292 | .297 | .251 | COMMODORES | .139 | .412 | .261 | .333 |
| .226 | .209 | .222 | TED NUGENT | .042 | .353 | .406 | .040 |
| .274 | .239 | .211 | BOSTON | .257 | .000 | .289 | .080 |
| .201 | .224 | .191 | BROS. JOHNSON | .319 | .353 | .078 | .107 |
| .226 | .228 | .182 | CAT STEVENS | .333 | .118 | .139 | .040 |
| .132 | .133 | .169 | BEE GEES | .368 | .039 | .117 | .000 |
| --- | .185 | .164 | EMOTIONS | .125 | .471 | .078 | .240 |
| --- | .355 | .156 | NEIL YOUNG | .104 | .039 | .172 | .293 |
| --- | --- | .100 | W. JENNINGS | .104 | .098 | .106 | .080 |
| --- | --- | .096 | MARLEY/WAILERS | .194 | .059 | .056 | .027 |
| .372 | .183 | .087 | MARVIN GAYE | .215 | .000 | .000 | .107 |
| --- | --- | .087 | PABLO CRUISE | .000 | .000 | .139 | .187 |
| .171 | .127 | .078 | M. TUCKER BAND | .000 | .039 | .144 | .093 |
### RETAIL FUTURES
| NATIONAL % | ARTIST | N.E. | S. | M.W. | W. |
|---------------|-----------------|------|------|------|------|
| .400 | ALAN PARSONS | .396 | .294 | .550 | .120 |
| .380 | STEVE WINWOOD | .458 | .235 | .350 | .400 |
| .287 | FLOATERS | .375 | .118 | .233 | .360 |
| .247 | ROGER DALTREY | .229 | .412 | .250 | .160 |
| .207 | RITA COOLIDGE | .188 | .118 | .233 | .240 |
| .200 | LITTLE RIVER BD | .208 | .235 | .267 | .000 |
| .180 | RAINBOW | .146 | .118 | .117 | .440 |
| .160 | STUFF | .146 | .176 | .233 | .000 |
| .160 | UFO | .146 | .059 | .267 | .000 |
| .140 | LONNIE L. SMITH | .271 | .000 | .100 | .080 |
## RETAIL
(ISP) INDICATES SALES FROM IN-STORE PLAY)
### NORTHEAST
**BOSTON, MASS.**
- **N.E. MUSIC CITY**
- **TONY MARINO**
- NEW REACTION:
- Ted Nugent
- (ISP) Alan Parsons
- Dan Fogelberg
- Little River Band
- Illusion
- Heart
- Roger Daltrey
- (ISP) Peter Frampton
- (ISP) Eric Clapton
- (ISP) Grasme Edge Band
- TOP 15:
- Crosby/Stills/Nash
- Fleetwood Mac
- Star Wars - Sndrk.
- Peter Frampton
- Beatles
- Bee Gees - Live
- Steve Miller
- James Taylor
- Stevie Winwood
- Eagles
- Foreigner
- Bob Marley/Wailers
- Moody Blues
- Paul Horn
### PROVIDENCE, R.I.
**BEAGON RECORDS**
- **STEVE PERRY**
- NEW REACTION:
- Alan Parsons
- (ISP) Pat Travers
- Grasme Edge Band
- Rain
- Pablo Cruise
- Roger Daltrey
- Melissa Manchester
- (ISP) James Taylor
- Liston Smith
- TOP 15:
- Barbra Streisand
- James Taylor
- Steve Miller
- Crosby/Stills/Nash
- Kiss
- Barry Manilow
- Marvin Gaye
- Neil Young
- Bee Gees
- Steve Winwood
- Star Wars - Sndrk.
- Pousseur-Dart Band
- Peter Frampton
- Moody Blues
- Kenny Rogers
- Bill Conti
- Bob Marley/Wailers
- SELLING IMPORTS:
- Hudson-Ford
- Nutz - Hard Nutz
- Stones (all)
- Gong - Caenese
- Herd
- From the Underground
- Tangerine Dream (all)
- Genesis - Lamb
- Rolling Stones
- Greatest Hits
- No Stone Unturned
- Beatles - Hamburg
### NEW YORK, N.Y.
**KORVETTES**
- **BERNARD H. OSHIN**
- (Representing 58 stores...)
- NEW REACTION:
- Barbra Streisand
- Neil Young
- Crosby/Stills/Nash
- Floater
- Emotions
- Crusaders
- Donna Summer
- Kiss
- James Taylor
- Annie - Sndrk.
### KORVETTES Con't.
- TOP 15:
- Barry Manilow - Live
- Peter Frampton
- Fleetwood Mac
- Foreigner
- Marvin Gaye - Live
- Rocky - Sndrk.
- Bros. Johnson
- Cat Stevens
- Crusaders
- Boz Scaggs
- Boston
- Eagles
- Bee Gees - Live
- Dan Fogelberg
- Star Wars - Sndrk.
### NEW YORK, N.Y.
**JIMMY'S MUSIC WORLD**
- **JEFF TEITELBAUM**
- (Representing 38 stores...)
- NEW REACTION:
- Lonnie Liston Smith
- Perenders
- Carol Douglas
- Kiss
- Love & Kisses
- The Deep - Sndrk.
- Adriisi Bros.
- Roy Ayers
- Al Jarreau
- Joan Baez
- TOP 15:
- Teddy Pendergrass
- Peter Frampton
- James Taylor
- Crosby/Stills/Nash
- Silver Convention
- Cat Stevens
- Eagles
- Dan Fogelberg
- Salson Orchestra
- Aretha Franklin
- Barbra Streisand
- Neil Young
- Bros. Johnson
- Kraftwerk
### MOORESTOWN, N.J.
**W E THREE RECORDS**
- **F. B KONDROSKY**
- NEW REACTION:
- James Taylor
- Olivia Newton-John
- Emotions
- Wiz
- Rainbow
- Rita Coolidge
- Brainstorm
- Crusaders
- Neil Young
- New York, N.Y. - Sndrk.
- TOP 15:
- Barry Manilow - Live
- Fleetwood Mac
- Eagles
- Peter Frampton
- Kiss
- Pink Floyd
- Steve Miller
- Lynyrd Skynyrd - Live
- Crosby/Stills/Nash
- Barbra Streisand
- Star Wars - Sndrk.
- Bob Marley/Wailers
- Eat Stevens
- Rocky - Sndrk.
- Shaun Cassidy
### WASHINGTON, D.C.
**WAXIE MAXIE'S**
- **KENNY DOBBIN**
- (Representing 15 stores...)
- NEW REACTION:
- James Taylor
- War
- Crusaders
- Barbra Streisand
- Pablo Cruise
- Roger Daltrey
- Gary Bartz
- Steve Winwood
- Sunbear
### WAXIE MAXIE'S Con't.
- Alan Parsons
- Little River Band
- Lonnie Liston Smith
- Stuff
- C.I.A. & Co.
- Charlie
- Floaters
- UFO
- TOP 15:
- Barry Manilow
- Jimmy Buffett
- Bros. Johnson
- Peter Frampton
- Bee Gees
- Emotions
- Steve Miller
- Star Wars - Sndrk.
- Heart
- Roy Ayers
- Crosby/Stills/Nash
- Rita Coolidge
- Kiss
- Brainstorm
- Bob Marley/Wailers
- SELLING IMPORTS:
- Rolling Stones
(5Lp Set)
- Beatles (all)
- Iggy Pop
- Raw Power
- Pink Floyd - Animals
(pink vinyl)
### RICHMOND, VA.
**GRAMOPHONE**
- **BILL TROUT**
- (Representing 3 stores...)
- NEW REACTION:
- (ISP) Attitudes
- Roger Daltrey
- Little River Band
- Steve Winwood
- Stuff
- Alan Parsons
- TOP 15:
- Pablo Cruise
- Peter Frampton
- James Taylor
- Crosby/Stills/Nash
- Kiss
- Caruso
- Olivia Newton-John
- SELLING IMPORTS:
- Hugh Hopper
- Hippopotamus Box
- Steve Winwood
- "God Save the Queen"
- Bert Jansch
- A Rare...
### LONG ISLAND, N.Y.
**RECORD WORLD/TSS**
- **IRA ROHSTEIN**
- (Representing 17 stores...)
- NEW REACTION:
- Steve Winwood
- Jerry Jeff Walker
- (ISP) James Taylor
- (ISP) James Taylor
- Elvis Presley
- Crosby/Stills/Nash
- Star Wars - Sndrk.
- Barbra Streisand
- Rita Coolidge
- Neil Young
- TOP 15:
- Fleetwood Mac
- Barry Manilow
- Peter Frampton
- Steve Miller Band
- Heart
- Bee Gees
- Cat Stevens
- Eagles
- Boston
- Dan Fogelberg
- Beatles
- Supertramp
- Bob Marley/Wailers
- Waylon Jennings
### RECORD WORLD Con't.
- Jimmy Buffett
- SELLING IMPORTS:
- Rock Folliés '77
- Nat King - Live in N.Y.
- The Damned
- Hollies - Live
- 801 - Live
- ELV - The Light Shines
- Bevilaqua - Hamburg
- Elvis Presley - Tapes
- Bay City Rollers
- Rollin'
(5 Lp set)
### ROCHESTER, N.Y.
**RECORD THEATRE**
- **BOB SLIGAR**
- (Representing 5 stores...)
- NEW REACTION:
- (ISP) Peter McCann
- (ISP) Roger Daltrey
- Willie Nelson
- Johnny Winter
- Rainbow
- AC/DC
- (ISP) Stevie Winwood
- Bay City Rollers
- Geils
- (ISP) Olivia Newton-John
- TOP 15:
- Barbra Streisand
- Fleetwood Mac
- Eagles
- Dan Fogelberg
- Lake
- Boston
- Ted Nugent
- Crosby/Stills/Nash
- James Taylor
- Bob Marley
- Horslips
- Heart
- Kiss
- Shaun Cassidy
### HARTFORD, CT.
**RECORDS UNLIMITED**
- **GLENN MASON**
- (Representing 3 stores...)
- NEW REACTION:
- Rainbow
- Star Wars - Sndrk.
- UFO
- Tim Weisberg's Band
- Kiss
- Geils
- Little River Band
- Lou Rawls
- Supertramp
- Rita Coolidge
- Geils
- Alan Parsons
- James Taylor
- Eagles
- Neil Young
- Foreigner
- Heart
- Derringer - Live
- SELLING IMPORTS:
- Coliseum II
- Electric Savage
- Chicago Symphony
- Ash Ra Temple
- Genesis EP
## Retail
(ISP indicates sales from in-store play)
### Northeast
**Cambridge, Mass.**
- Harvard Coop
- Brink's Jackson (Representing 3 stores...)
**New Reaction:**
- Mink DeVille
- Emotions
- Danny O'Keefe
- Jamie Brockett
- Alan Parsons Project
- Dictators
- Laura Nyro
- Dave Edmunds
- John Martyn
- Bob Marley/Wailers
**Top 15:**
- Fleetwood Mac
- Crosby/Stills/Nash
- Barbra Streisand - Live
- Bonnie Raitt
- Bee Gees - Live
- Dan Fogelberg
- Stevie Wonder
- Keith Jarrett
- Staircase
- Beatles - Hollywood
- Jimmy Buffett
- Marvin Gaye
- Jessi Winchester
- James Taylor
- Star Wars - Sndtrk.
- Cat Stevens
**Selling Imports:**
- Stooges
- Raw Power
- The Stranglers
- The Saints
- I'm Stranded
- The Band
- Turner & Kirwan
- Sex Pistols (single)
- "God Save the Queen"
- Bruce Springsteen (all)
- The Who - Story of Tim Buckley
- Greetings from L.A.
**Comments:**
It is interesting to have so many top-selling soundtracks at once. Most notably, *Star Wars*, *The Sorcerer* and *New York, N.Y.* The music soundtrack has improved to the point where it stands on its own. On the other hand, many new rock, folk & jazz releases sound bland what we used to expect from soundtracks - bland.
### Philadelphia, Pa.
**Third Street Jazz**
**Jerry Gordon**
**New Reaction:**
- Horace Silver
- Charlie Parker
- Lester Young
- Al Jarreau
- David Bowie
- Carole King
- Mary Lou Williams
- (ISP) Alan Parsons (disco cuts)
- (ISP) Julian Priester
- Hilton Ruiz
- Sun Ra - Cosmos
**Top 15:**
- The Greatest - Sndtrk.
- Roy Ayers
- War
- Ronnie Laws
- Crusaders
- Earl Klugh
- Commodores
- Flutes
- Brahms
- Lonnie Liston Smith
- NCCU
- Enronos
- Marvin Gaye
- Bob Marley/Wailers
- Teddy Pendergrass
- Selling Imports:
- Roxy London WC2
- Dr. Feelgood
- Heartbreakers
- Vibrators
- Dexter Gordon/
Sonny Gray
- Don Cherry
- Old & New Dreams
- Woody Shaw
- Ichii Ban
### Allentown, Pa.
**Phantasmagoria**
**Rick**
**New Reaction:**
- (ISP) Steve Winwood
- Strapps
- Rainbo
- John Martyn
- Neil Young
- (ISP) Karko Krog/
Archie Shepp
- Jimmy Buffett
**Top 15:**
- Supertramp
- AC/DC
- Alan Parsons
- Foreigner
- Phil Lynott
- Fleetwood Mac
- Steve Miller
- Ted Nugent
- Judas Priest
- Crosby/Stills/Nash
- Chuck Berry
- Supertramp - Crime
- Kiss
- Charisma
- Little River Band
- Selling Imports:
- Hawkwind - Quark
- SAMB - Four Play
- 801 - Live
- Budgie - Best of
### Alexandria, Va.
**Rainbow Tree**
**Rick Miller**
(Representing 3 stores...)
**New Reaction:**
- Steve Winwood
- Star Wars - Sndtrk.
- Kiss
- Mink DeVille
- Jaime Brockett
- Outlaws
- Barbra Streisand
- Brand X
- Roger Daltrey
- Little River Band
- Rita Coolidge
- Average White Band
- Bee Gees - Live
- Poco
**Top 15:**
- Crosby/Stills/Nash
- James Taylor
- Fleetwood Mac
- Peter Frampton
- Geils
- Jimmy Buffett
- Eagles
- Ted Nugent
- Heart
- Dan Fogelberg
- UFO
- Alan Parsons
- Kenny Loggins
- Commodores
- Marshall Tucker Band
**Selling Imports:**
- Stooges (all)
- Klaus Schulze - Mirage
- Beatles (all)
### Titus Oaks Cont.
**Top 15:**
- Fleetwood Mac
- Crosby/Stills/Nash
- Bob Marley/Wailers
- Peter Frampton
- Teddy Pendergrass
- Marshall Tucker Band
- Bros. Johnson
- James Taylor
- Neil Young
- The Chieftains - Sndtrk.
- Alan Parsons
- Supertramp
- C.J. & Co.
- Roger Daltrey
- Edwin Starr
### New York, N.Y.
**Sam Goody, Inc.**
(Representing 17 stores...)
**New Reaction:**
- James Taylor
- Jerry Jeff Walker - Live
- Annie - Sndtrk.
- Salsoul Orchestra
- Emotions
- Flamingos
- Steve Winwood
- Moody Blues - Live 45
- Olivia Newton-John
- Al Jarreau
**Top 15:**
- Fleetwood Mac
- Barbra Streisand
- Peter Frampton
- Steve Miller
- Barry Manilow - Live
- Boston
- Bee Gees - Live
- Dan Fogelberg
- Star Wars - Sndtrk.
- Waylon Jennings
- Heart
- Shaun Cassidy
- Jimmy Buffett
- Bob Marley & Wailers
### Dallas, Texas
**Sound Town**
**Denny Moeseman**
(Representing 6 stores...)
**New Reaction:**
- Alan Parsons
- Roger Daltrey
- Bob Marley/Wailers
- Laura Nyro
**Top 15:**
- Crosby/Stills/Nash
- Barbra Streisand
- James Taylor
- Kiss
- Peter Frampton
- Dan Fogelberg
- Waylon Jennings
- Cat Stevens
- Fleetwood Mac
- Eagles
- Bros. Johnson
- Kenny Loggins
- Commodores
- Steve Winwood
- Pat Travers
### South
**Houston, Texas**
**Record Rack**
**New Reaction:**
- Cat Stevens
- Roger Daltrey
- Neil Young
- Laura Nyro
- C.J. & Co.
- Bee Gees
- Maze
**Selling Imports:**
- Beatles - Hamburg
- Nektar - Live in N.Y.
- 801 - Live
- MC5 - Kick Out...
- Electric Light Orch.
- Night the Lights...
- Hollies - Live
- Tangerine Dream
- Rubicon
- Saints - I'm Stranded
- Stooges - Fun House
- Downliners Sect
- The Sect
## SOUTH
**FT. LAUDERDALE, FLA.**
- **PEACHES**
- Dave Flaherty
- **NEW REACTION:**
- Graeme Edge Band
- Hot
- Judas Priest
- Pat Travers
- **(ESP) Larry Coryell/Mouzon**
- TOP 15:
- Fleetwood Mac
- Crosby/Stills/Nash
- Jimmy Buffett
- Barry Manilow - Live
- Stevie Wonder
- Kenny Loggins
- Eagles
- Peter Frampton
- Dan Fogelberg
- Steve Miller
- Kiss
- Cat Stevens
- Barbra Streisand
- Commodores
- Emotions
- **SELLING IMPORTS:**
- Jean Michel Jarre
- Oxygen
- Absolute Elsewhere
- In Search of Ancient...
- Allman Brothers
- Doing a Moonlight
- England
- Garden Shed
- Duncan Mackay - Score
**AUSTIN, TEXAS**
- **INNER SANCTUM**
- James Cooper
- **NEW REACTION:**
- New York, N.Y., - Sndrk.
- Billy Idol/Hartford/
- Dillard
- Crystal Gayle
- Coryell/Mouzon
- McCoy Tyner
- Burning Spear
- Rainbow
- Brand X
- Moroccan Roll
- Silvertones
- Alan Parsons Project
- **TOP 15:**
- Townes Van Zandt
- Crosby/Stills/Nash
- Wailers
- Ted Nugent
- Willie Nelson
- Barbra Streisand
- Little Feat
- Jerry Jeff Walker
- Dan Fogelberg
- Neil Young
- Waylon Jennings
- Steve Miller
- Johnny Winter
- Mink DeVille
- Rusty Wier
- **SELLING IMPORTS:**
- by Neil Rutenberg
- Rocky Horror Picture Show - Sndrk.
- Keith Jarrett
- Staircase
- The Clash
- The City
- Singles & EP's:
- Sex Pistols
- "God Save the Queen"
- Nick Lowe - "Bowl"
- The Vibrators
- "Baby Baby"
## NEW ORLEANS, LA.
- **GRAMAPHONE**
- Gladin Scott
- (Representing 2 stores...)
- **NEW REACTION:**
- Crusaders
- Geils
- Meters
- Al Jarreau
- Olivia Newton-John
- Steve Winwood
- **TOP 15:**
- Fleetwood Mac
- Steve Miller
- Peter Frampton
- Barry Manilow
- Crosby/Stills/Nash
- Bros. Johnson
- James Taylor
- Commodores
- Heart
- Emotions
- Electric Light Orch.
- Joan Baez
- Jerry Jeff Walker
- Foreigner
- Eagles
## FRANKLIN MUSIC Con't.
- LeBlanc & Carr
- Mother's Finest
- SELLING IMPORTS:
- Hollies - Live
## PENNYLANE RECORDS
- Weather Report
- Oregon
- Earl Klugh
- Ronnie Laws
- Dexter Gordon
- Humble Pie
- David Grisman Quintet
- Jan Garbarek
## LIBERTYVILLE, ILL.
- **DOG EAR RECORDS**
- Rick Johnson
- (Representing 3 stores...)
- **NEW REACTION:**
- Floaters
- Geils
- Roger Daltry
- Steve Winwood
- (ESP) Ultravox
- Earth Quake
- Rainbow - Live
- (ESP) Hollywood Television
- **(ESP) Charlie**
- TOP 15:
- Fleetwood Mac
- Steve Miller
- Crosby/Stills/Nash
- REO
- Heart
- UFO
- Pink Floyd
- James Taylor
- Alan Parsons
- Dan Fogelberg
- Ted Nugent
- Foreigner
- Dave Mason
- Neil Young
- Supertramp
- **SELLING IMPORTS:**
- 801 - Live
- Novalis - Konzerte
- The Jam - In the City
- The Damned
- Klaus Schulze
- Ody Love
- Weather Report
- Live in Tokyo
- Rolling Stones
- 5 Lp Set
- Beatles - Hamburg
## MIDWEST
## MINNEAPOLIS, MN.
- **WAX MUSEUM**
- Kate Drike
- (Representing 5 stores...)
- **NEW REACTION:**
- Floaters
- Rita Coolidge
- Geils
- Ted Nugent
- Alan Parsons
- New York, N.Y., - Sndrk.
- John Klemmer
- Frankie Miller
- **TOP 15:**
- Crosby/Stills/Nash
- James Taylor
- Fleetwood Mac
- Emotions
- Heart
- Weather Report
- Pablo Cruise
- Wylton Jennings
- Wizzard
- Supertramp
- Barbra Streisand
- Dan Fogelberg
- Jimmy Buffett
- Herbie Hancock
- Marvin Gaye
## INDIANAPOLIS, IND.
- **KARMA**
- Dave Curtis
- (Representing 6 stores...)
- **NEW REACTION:**
- Peter
- Caroline Peyton (!)
- Lonnie Liston Smith
- Mother's Finest
- Coal Kitchen
- Manchild
- Pakalameredith
- Strawbs
- Dimi Dreg
- **TOP 15:**
- Crosby/Stills/Nash
- James Taylor
- Kiss
- Stuff
- Fleetwood Mac
- Barry Manilow
- Peter Frampton
- Alan Parsons
- Barbra Streisand
- Foreigner
- Rainbow - Live
- Steve Winwood
- Neil Young
- Brooks & Dunn
- Dan Fogelberg
- **SELLING IMPORTS:**
- Colloseum II
- George Hatcher
- Genesis EP
## RETAIL
(ISP) INDICATES SALES FROM IN-STORE PLAY
### MIDWEST
#### DAYTON, OHIO
- GOLDEN ROD
- RORY MAYS (Representing 3 stores...)
**NEW REACTION:**
- Geils
- Lonnie Liston Smith
- UFO
- 38 Special
- Little River Band
- Amazing Rhythm Aces
- TOP 15:
- Crosby/Stills/Nash
- Peter Frampton
- James Taylor
- Neil Young
- Leon & Mary Russell
- Poco
- Heart
- Star Wars - Sndtrk.
- Crusaders
- Ramsey Lewis
- Kiss
- Cat Stevens
- Fleetwood Mac
- Eagles
- Alan Parsons Project
- SELLING IMPORTS:
- Jimi Hendrix
- Loose Ends
- ELO
- Lights Went Out
- Jimi Hendrix
- Box French Import
- Pink Floyd - Animals
- Wings - 1st single
#### CINCINNATI, OHIO
- SIGHT IN SOUND
- GLENN LINDLAHL (Representing 2 stores...)
**NEW REACTION:**
- Roger Daltrey
- TOP 15:
- Jerry Jeff Walker
- Barry Manilow
- Crosby/Stills/Nash
- Crusaders
- Bob Marley/Wailers
- Eagles
- Kiss
- Jimmy Buffett
- Bee Gees
- Heart
- Sorcerer - Sndtrk.
- Neil Young
- Fleetwood Mac
- Pablo Cruise
- Roy Ayers
#### ST. LOUIS, MO.
- STREET SIDE RECORDS
- STEVE FERZACCA
**NEW REACTION:**
- (ISP) Alexis
- Flame
- Roger Daltrey
- UFO
- Alan Parsons Project
- Steve Winwood
- Brainstorm
- American Flyer
- Melissa Manchester
- Stuff
- Roy Ayers
- Slave
- Jimmy Buffett
- John Klemmer
- Gladys Knight
- TOP 15:
- Fleetwood Mac
- Bros. Johnson
- Crosby/Stills/Nash
- Emotions
- Floaters
- Dan Fogelberg
- Supertramp
- Little Feat
- Crusaders
- Commodores
- Shalamar
- Bob Marley/Wailers
- James Taylor
---
### STREET SIDE Cont.
- Barry Manilow
- Waylon Jennings
- SELLING IMPORTS:
- Genesis EP
- Pete Gabriel (single)
- Sex Pistols
- Stooges (all)
- Jack Larcaster &
Robin Lumley
- Moroscape
- MC 5 (all)
### HARMONY HOUSE Cont.
- Al DiMeola
- TOP 15:
- Fleetwood Mac
- Peter Frampton
- Marshall Tucker Band
- Steve Miller
- Commodores
- Foreigner
- Ted Nugent
- Shaun Cassidy
- Kiss
- Crosby/Stills/Nash
- Emotions
- Love Asla
- James Taylor
- Brainstorm
- Heart
### NATIONAL
#### PICKWICK/MUSICLAND
- GREG HAGGLUND (Representing 190 stores...)
- TOP 15:
- Fleetwood Mac
- Barry Manilow - Live
- Eagles - Hotel Calif., Boston
- Peter Frampton
- Star is Born - Sndtrk.
- Barbra Streisand
- Barry Manilow
- This One's For You
- Steve Müller
- Rocky - Sndtrk.
- Eagles - Hits
- Kiss
- Heart
- Bee Gees - Live
- Stevie Wonder
- SELLING IMPORTS:
- Beatles - Hamburg
- Rolling Stones (5 LP set)
- Bay City Rollers
- Rollin'
- Snoges - Raw Power
- Denny Laine
- Ahh Laine
- Styx - Pieces #1
- Tim Buckley
- Greetings from L.A.
- Snoges - Fun House
#### MINNEAPOLIS, MN.
- LIEBERMAN ENT.
- REX HULT (Representing 150 stores...)
**NEW REACTION:**
- James Taylor
- Neil Young
- Barbra Streisand
- Melissa Manchester
- Steve Winwood
- Little River Band
- Peter Frampton
- Roger Daltrey
- Alan Parsons
- Stuff
- TOP 15:
- Crosby/Stills/Nash
- Fleetwood Mac
- Kiss
- Heart
- Steve Miller
- Marshall Tucker Band
- Pablo Cruise
- Boston
- Ted Nugent
- Dan Fogelberg
- Weather Report
- Foreigner
- Heart
- Waylon Jennings
- Bob Seger
#### CHICAGO, ILL.
- FLIP SIDE, INC.
- RICHARD S. CURRIER (Representing 7 stores...)
**NEW REACTION:**
- UFO
- Waylon Jennings
- Crosby/Stills/Nash
- James Taylor
- Les Dudek
- Alan Parsons
- Johnny Winter
- Steve Winwood
- Rainbow
- Earth Quake
- TOP 15:
- Bee Gees
- Barry Manilow
- Heart
- Steve Miller
- Fleetwood Mac
- Foreigner
- Ted Nugent
- Neil Young
- Supertramp - Crime...
- Barry Manilow
- Barbra Streisand
- Boston
- Bob Marley/Wailers
- Cat Stevens
- Maynard Ferguson
- Star Wars - Sndtrk.
## Retail
(199) indicates sales from in-store play.
### Chicago, Ill.
**Hear Here**
Bob Criz (Representing 3 stores...)
**New Reaction:**
- Coal Kitchen
- Steve Winwood
- New York, N.Y. - Sadtrk
- Kiss
- Melissa Manchester
- Alan Parsons
- Earl Klugh
- Roy Buchanan
- Geils
- Little River Band
**Top 15:**
- Fleetwood Mac
- Crosby/Stills/Nash
- Barbra Streisand
- Ted Nugent
- Bros. Johnson
- Foreigner
- Kenny Loggins
- Barry Manilow
- REO Speedwagon
- Commodores
- Pink Floyd
- Emerson, Lake, Palmer
- Heart
- Supertramp
- James Taylor
**Comments:**
Sales were up 20% in June. It seems like a lot of excitement is being created in our market through performances. I would like to see more support from the labels in the form of in-store promotions coordinated with artist appearances. Let's get some artist support at the point of purchase and create even more sales and positive energy.
### Chicago, Ill.
**Jazz Record Mart**
(Representing 2 stores...)
**New Reaction:**
- McCoy Tyner
- Super Trio
- Joe Zawinul - Main Man
- Barry Altschul
- You Can't
- Vanilli/Tarna
- Supertrio
- Charlie Parker
- Bird At The Roost
- Lester Young
- Prest Lives
- Richard Bona & Hall
- AH Pepper - The Trip
- Grante Coleman
- Count Basie
- Prime Time
**Top 15:**
- Herbie Hancock
- V.S.O.P.
- Dexter Gordon
- Horace Silver
- John Coltrane
- Lifetyle
- Don Pullen
- Don Cherry
- Herbie Hancock
- Paul Baxcomb
- Bad Bacchus
- Art Farmer
- Houston Person
- Sonny Stitt
- Anthony Braxton
- Montreux
- Clifford Brown - Vol. 2
- Bill Evans
- Charles Mingus
- Crusaders
- From As The Wind
- John Coltrane
- Africa Blue
- Mel Torme
- A Florida
### Skokie, Ill.
**Sound Unlimited**
Frederick Michaels (Representing 200 stores...)
**New Reaction:**
- Star Wars - Sadtrk.
- UFO
- Eliza Coolidge
- (ESP) Flinders
- Emotions
- REO Speedwagon
- James Taylor
- Alcatraz Express Project
- (ESP) Crosby/Stills/Nash
- (ESP) Bay City Rollers
- (ESP) Miki Dee
- C.J. & Co.
- Supertramp
**Top 15:**
- Fleetwood Mac
- Peter Frampton
- Steve Miller
- Commodores
- Heart
- Barry Manilow
- Cat Stevens
- Bad Company
- Foreigner
- Ted Nugent
- Dan Fogelberg
- Neil Young
- Barbra Streisand
- Kiss
- Jimmy Buffett
**Selling Imports:**
- Beatles - Hamburg
- Weather Report
- Live In Tokyo
- Beck, Bogart, Appice
- Fleetwood Mac
- Humble Pie
- ELP - Works
- King Crimson - Guide...
- Rory Gallagher
- Eric Clapton
- Jimi Hendrix
- Rainbow Bridge
- The Who
- Quadraphonic
- MX
- Kick Out the Jam...
### Midwest
**Record Bar**
Barbara Toomer (Representing 17 stores...)
**Top 15:**
- Fleetwood Mac
- Barbra Streisand
- Barry Manilow
- Shaun Cassidy
- Foreigner
- Peter Frampton
- Kiss
- Crosby, Stills & Nash
- Ted Nugent
- Eagles
- Heart
- James Taylor
- R. E. O. Speedwagon
- Jimmy Buffett
### Lakewood/Denver
**Independent Records**
John Broholm (Representing 2 stores...)
**New Reaction:**
- Roger Daltrey
- Steve Winwood
- Kiss
- Hank Crawford
- Alan Parsons
- Roy Ayers
- Heart Live
- James Taylor
- Neil Young
- Crosby/Stills/Nash
**Top 15:**
- Fleetwood Mac
- Heart
- Emotions
- Steve Miller
- Bruce Johnston
- Marshall Tucker Band
- Barry Manilow
- Dan Fogelberg
- Supertramp
- Florents
- Commodores
- Bob Seger
- Isley Bros.
- Fitfall
- Boz Scaggs
### Tucson Area
**Zip's Records**
Jim Hardy (Representing 2 stores...)
**New Reaction:**
- Steve Winwood
- Elton John
- Gabo Barbieri
- Crosby/Stills/Nash
- James Taylor
- The Sorcerer - Sadtrk.
- Flippers
- Rainbow
- Marianne Rush
- Bob Marley/Wailers
**Top 15:**
- David Ruggberg
- Fleetwood Mac
- Marvin Gaye
- Jimmy Buffett
- Peter Frampton
- Pablo Cruise
- Isley Bros.
- Eagles
- Supertramp
- Heart
- Bonnie Raitt
- Barry Manilow
- Barbra Streisand
- Peter Frampton
### Seattle/Bellevue
**Everybody's Records**
John Hananger (Representing 2 stores...)
**New Reaction:**
- (ESP) John Mayall
- (ESP) Joan Baez
- (ESP) Rubinson
- Roger Daltrey
- Alan Parsons
- Wailers
- (ESP) Upra
- Steve Winwood
- Willie Nelson
- Halflife
**Top 15:**
- Crosby/Stills/Nash
- Fleetwood Mac
- Heart
- John Klemmer
- James Taylor
- Supertramp
- Boston
- Steve Miller Band
- Jimmy Buffett
- Peter Frampton
- Barbra Streisand
- Neil Young
### Everybody's Rec. Cont'd.
- Bonnie Raitt
- Alan Parsons
- Little River Band
**Selling Imports:**
- Hollies - Live
- Al Stewart - (catalog)
- Damned
- Bunch of Stiffs (various)
- 801 - Live
### West
**Record Bar**
Barbara Toomer (Representing 6 stores...)
**Top 15:**
- Fleetwood Mac
- Kiss
- Waylon Jennings
- Peter Frampton
- Barry Manilow
- Crosby, Stills & Nash
- Shaun Cassidy
- Boston
- Barbra Streisand
- Eagles
- Steve Miller Band
- Star Wars - Sadtrk.
- Jimmy Buffett
- Heart
- Linda Ronstadt
### Eugene, Ore.
**Chrystal Ship**
David Odell
**New Reaction:**
- Olivia Newton-John
- Trooper
- (ESP) Galil
- Kiss
- Paul Horn
- (ESP) Rick Derringer
- Steve Miller
- Keith Jarrett
- Melissa Manchester
- James Taylor
**Top 15:**
- Slade - Warz - Sadtrk.
- Peter Frampton
- Crosby, Stills & Nash
- John Klemmer
- Maynard Ferguson
- Marshall Tucker
- Marvin Gaye
- Jimmy Buffett
## WEST
### SAN FRANCISCO, CA.
**TOWER RECORDS**
**MATHIE KONING**
**NEW REACTION:**
- Norton Buffalo
- Sylvester
- Mike DeVille
- Floater
- Roy Ayers
- Peter Frampton
- Kiss
- Andy Gibb
- Stevie Winwood
**TOP 15:**
- Fleetwood Mac
- Barbra Streisand
- Emotions
- Crosby/Still/Nash
- Star Wars - Sndtrk.
- Love
- Bonnie Raitt
- Al Jarreau
- Barry Manilow
- Meco
- Commodores
- Marvin Gaye
- Eagles
- Cat Stevens
- Donna Summer
**SELLING SINGLES:**
- Bob Seeger
- Frankie Miller
- Emotions
- Marvin Gaye
- Kiss
- Donna Summer
- Fleetwood Mac
- Kiss
- Allee Cooper
---
## LICORICE PIZZA Co.'s
- Bennie Raft
- New York, N.Y. - Sndtrk.
- Kiki Dee
- Emotions
- Bro.A. Johnson
**TOP 15:**
- Fleetwood Mac
- Crosby/Still/Nash
- Peter Frampton
- Kiss
- Barbra Streisand
- Neil Young
- Star Wars - Sndtrk.
- Peter Frampton
- James Taylor
- Jimmy Buffett
- Eagles
- Heart
- Supertramp
- Barry Manilow - Live
- Commodores
---
## SOUTHERN CALIFORNIA
**MUSIC PLUS**
**STEVE BOUDREAU**
(Representing 15 stores...)
**NEW REACTION:**
- Johnny Winter
- Rainbow
- Kiss
- Rockpile
- The Sorcerer - Sndtrk.
**TOP 15:**
- Fleetwood Mac
- Crosby/Still/Nash
- Barbra Streisand
- Star Wars - Sndtrk.
- Peter Frampton
- James Taylor
- Neil Young
- Commodores
- Alan Parsons Project
- Steve Winwood
- Kiss
- Foreigner
- Supertramp
- Kenny Loggins
- Steve Miller
**SELLING IMPORTS:**
- Clearlight Orch.
- Les Cortes
- Colosseum
- Electric Savage
- Groucho's
- Rockpennell's Land
- Gryphon - Treason
- Peter Hammill - Over
- Hawkwind - Quark
- Iggy & Stooges
- The Jam - In the City
- MC 5 (all)
- Feckin' Mathematics
- Bronch Following
- Rolling Stones
- (French Box)
- Saints - Stranded
- Klaus Schulze - Mirage
- Christian Vander
---
## VAN NUYS, CA.
**MOBY DISC**
**BOB CALTON**
**NEW REACTION:**
- Live at The Bat (various)
- Roger Daltry
- James Taylor
- Perigo
- Madness Manchester
- Earl Klugh
- Lake
- Laura Nyro
**TOP 15:**
- Crosby/Still/Nash
- Fleetwood Mac
- Supertramp
- Sorcerer - Sndtrk.
- Barbra Streisand
- Alan Parsons Project
- Foreigner
- Star Wars - Sndtrk.
- Steve Winwood
- Jimmy Buffett
- Dan Fogelberg
- Kiss
- Charlie
- UFO
- Al Stewart
**SELLING IMPORTS:**
- Peter Hammill
- Pink Floyd - Animals
- (all)
- Colosseum II
- Electric Savage
- Rose - Taste of Neptune
- Christian Vander
- Trixie Whitley
- Hawkwind - Quark
- Motor Head - 12" single
- Roxy London - Various
---
## LICORICE PIZZA
**BOB ADELS**
(Representing 19 stores...)
**NEW REACTION:**
- Kenny Loggins
- Ted Nugent
- Charlie
- Derringer
- Floater
---
## TOWER RECORDS Comp't.
- Crosby/Still/Nash
- Peter Frampton
**HOT SINGLES:**
- Alanis
- Garfield Jeffreys
- "That's What Friends Are For"
- "Telegram"
- War
- "A Real Mother for ya - 12" Singles"
- Vicki Sue Robinson
- Ronnie Smith & Jones
- Metal Words
- Cleveland Eaton
**COMMENTS:**
Where's Mickey Thomas... good record give him a listen.
---
## MUSIC MILLENNIUM
- Tangwino Dream
- Rubycop
- Sex Pistols (single)
- Scorpions (orig. cover)
- Virgin Killer
- Schiller - Mirage
- Body Love
- Hawkwind - Quark
- Colosseum II
**COMMENTS:**
Walrus Inclusion Shitik... Thanks be to GRT Canada for turning us onto the Ian Thomas collection of LP's. Ian is just great!
The only drawback to his not being a large person, as far as I can see, would be fear on the part of Neil Young America, Eagles, etc. stc., etc. "management gang" to surprise him and attack him in rage, and for the most part, their delivery are right there, and solid success, sales-wise.
Wash up you hit-seekers, go back to your guy and you'll get rich or, just leave some on the table for ol' Mother Earth.
Music is better than ever, with tons of pure art from Sweden, Finland, Poland, Austria, Canada, the Arab Amblia and France. Thanks to Archie Patterson and John Salts.
- Den MacLurk
---
## OREGON
### EVERYBODY'S RECORDS
**TOM**
(Representing 4 stores...)
**NEW REACTION:**
- Neil Young
- John Klemmer
- Keith Barrett
- (ISP) Supertramp
- Kiss
- (ISP) Northern Buffalo
- Steve Winwood
- (ISP) Geila
- (ISP) Rainbow
**TOP 15:**
- Crosby/Still/Nash
- Fleetwood Mac
- Heart
- Barbra Streisand
- Peter Frampton
- Marshall Tucker Band
- Jimmy Buffett
- Pablo Cruise
- Steve Miller
- Little River Band
- Star Wars - Sndtrk.
- Ted Nugent
- James Taylor
- Crusaders
- Alan Parsons
**SELLING IMPORTS:**
- Tangerine Dream (all)
- 801 - Live
- Hollies - Live
- Judas Priest
- B.J. Harvist - Live
**Singles:**
- Sex Pistols
"God Save the Queen!"
**COMMENTS:**
Now that the price of LP's are going up, the companies are cutting down on tickets to concerts and the promo LP's. This has been quite erratic at times.
The only way to promote certain LP's is in-store play by having store personnel "into" the music - don't forget the ones who do promote!
- T.X.
WJUL/Lowell, Mass. - Rich Gingras
Artist(s): RAMSEY RAYNE
Place & Date: Boston City Hall Plaza - July 3
Ticket Price: Free concert put on by the city of Boston.
Attendance: About 4,000
Production Values: Good
Reviewer's Reaction: Reacting well to the beautiful weather and enthusiastic audience, Ramsey had everyone shakin' and tappin' to his unique style of funky jazz. Backed up by a strong, talented band, Ramsey performed tunes from his new album, "New World" as well as old favorites such as "Brasilica" and "Sun Goddess." Ramsey outdid his performance three weeks earlier at Paul's Mall. He never fails to please.
JIMMY'S MUSIC WORLD/New York, N.Y. - Jeff Teitelbaum
Artist(s): PINK FLOYD
Place & Date: Madison Square Garden
Ticket Price: $10.00
Capacity: 20,000
Attendance: Full
Production Values: Good
Reviewer's Reaction: A group that must be seen to be fully appreciated! Be prepared for the audio-visual show of a lifetime! They weave music into their films in a way that must be seen to be believed! They played cuts from Animals, Wish You Were Here, and Dark Side of the Moon. After two 15 minute standing ovations they played "Money" and "Us and Them" from Dark Side... the audience loved it!
PEACHES/Ft. Lauderdale, Fla. - Dave Flaherty
Artist(s): JUDAS PRIEST
Place & Date: Fox Theatre, Atlanta, Ga.
Type of Place: Auditorium
Attendance: Almost full
Production Values: Excellent
Reviewer's Reaction: Judas Priest have to be the most dynamic, aggressive, and talented hard rock band working today. While their performance was limited to only 25 minutes, the crowd and many other people in the Theatre were in awe. Whether it was the blistering guitar solos from Glenn Tipton & K.K. Downing, or the amazing vocal range of one Rob Halford, Judas Priest gave a brilliant performance of their material and it should be played. Other so called rock and roll bands or hard rock bands that have no talent and get undeserved radio exposure should listen to Judas Priest. Then these mediocre bands might learn something.
INNER SANCTUM/Austin, Texas - James Cooper
Artist(s): ALBERT LEE & THE 10-4THS; ASLEEP AT THE WHEEL
Place & Date: Armory World - July 4th
Type of Place: Old national guard armory
Ticket Price: $5.00
Capacity: 1,500
Attendance: 1,000
Production Values: Excellent
Reviewer's Reaction: Great concert! Actually just a band put together for one show. A fantastic jam, Albert Lee, lead guitar, vocals (Emmylou Harris' Hot Band) - Guy Clark, John Conlee, Floyd Dunning, and the great Kenny Yeast (Asleep At The Wheel). Rodne Crowell, rhythm & vocals (Emmylou Harris' Hot Band) - Karen Brooks, rhythm & vocals (Lost Gonzo Band) - Mickey Rapheal, harmonica; Bee Spears, bass (Asleep At The Wheel) - Darrell Nellis (Buddy Blue). All the good stuff of fantastic instrumental solos and beautiful vocal harmonies. Need I say more.
ACRN/Athens, Ohio - Larry Schmeler
Artist(s): FRANK MARINO & MAHOGANY RUSH
Place & Date: Chippewa Lake Park - June 19
Type of Place: Once active amusement park
Ticket Price: Free, thanks to WKDD
Attendance: 20,000
Production Values: Good
Reviewer's Reaction: Production values could have been excellent but we've got to take off some points for the sound problems that delayed the start of the show. Frank Marino is a hell of a singer and player. He also showed how to use his Hendrutzied guitar in giving the crowd some great rock and roll in the form of "Talkin' 'Bout a Feeling," "A New Rock & Roll," "The Answer" and even "Johnny B. Goode." See 'em it's fun, Not Jimi but...
WIBA/Madison, Wis. - Dave Benson
Artist(s): CHEAP TRICK
Place & Date: Stone Hearth - June 27
Type of Place: Big Bar
Ticket Price: $3.00
Capacity: 1,000
Attendance: 1,000
Production Values: Fair
Reviewer's Reaction: Cheap Trick's return to Madison and The Hearth provided the band with an excellent opportunity to expose some of their new material to a loyal and loving audience. Whenever CT hits Madison it's a guaranteed party. Continued above...
STREET SIDE RECORDS/St. Louis Mo. - Steve Ferracca
Artist(s): BOZ SCAGGS & SOUTHSIDE JOHNNY
Place & Date: Mississippi River Festival - June 29
Type of Place: Festival site (tent, etc.)
Ticket Price: $3.50 lawn seats - under the tent
$4.00, $5.00, $6.00
Attendance: 10,000
Production Values: Excellent
Reviewer's Reaction: Whether we like it or not rock and roll is not the devilish, boogie wuthering music once professed to be and if you don't believe it check out a Boz Scaggs concert. But probably the best example in the business of the type of concert that is becoming more and more the norm. The evening kicked off to a hot and humid beginning with the rocking sons of Southside Johnny. This is one of the best live bands around today and they were a perfect lead in for Boz's show which to say the least was slick. Even though the Boz Scaggs act tends to be a bit like a Vegas艳rocity Boz's spontaneity keeps you on your feet waiting for heat every time no matter how many times you've heard them on the radio. The show included just about every tune on the Silk Degrees LP mixed with songs from his not yet released LP and older tunes for the old folks in the crowd. The concert was held at the Mississippi River Festival grounds and fit in the 10,000 person concert site in the country with fantastic sound and more than enough room to stretch out. Besides being a good look at what to expect from the future the evening with Southside Johnny and Boz Scaggs was a well spent one.
KZEL/Eugene, Ore. - Stan Garrett
Artist(s): PABLO CRUISE
Place & Date: Lane County Fairgrounds
Type of Place: Auditorium
Ticket Price: $5.00 & $8.00
Capacity: 1,700
Attendance: SRO
Production Values: Good
Reviewer's Reaction: Pablo Cruise is just about ready to break into bigness. They're very professional, don't mess around and are one of the most complete 4-piece bands you'll ever see/hear. The crowd was enthusiasm-plus.
KZOK-102-1/2/Seattle, Wash. - Mavis Mackoff
Artist(s): PETER FRAMPTON
Place & Date: Kingdome - June 27
Type of Place: Multipurpose facility, holds around 60,000+ depending on event
Ticket Price: $10.00
Capacity: 60,000+
Attendance: Approx. 40,000
Production Values: Good
Reviewer's Reaction: Kingdome drawbacks aside - Frampton did manage (with the help of about 40,000 people) to come alive. Let's face it, besides being just another rock star, he's extremely talented - and the small fragile guy has got an attracting force - something called charisma I believe... that draws that kind of a crowd (the Geils Band and Foghat were merely additions) and has them screaming and flailing their arms to every gesture he makes, they couldn't hear from the stage. The giant video screen was helpful - nice to get a closer look at that famous open shirted bare-chested Goldilocks-but aside from the thousands sitting along side - it could have been a Mini-Mike-Songfest. The 3 track sound system, however, rendered a remarkably distorted sound as Pete belted out all the biggies - at least a dozen - but to answer that famous question - did I feel like you did? Not quite, Pete, not quite.
KCSN/Northridge, Calif. - Kevin Stern
Artist(s): PETER FRAMPTON
Place & Date: Anaheim, Calif. - July 6
Type of Place: Baseball stadium
Ticket Price: $9.50 & $10.50; day of event $12.50
Capacity: 55,000
Attendance: According to promoters 55,000 - however, there were many open seats.
Production Values: Fair
Reviewer's Reaction: Concert was very disappointing for a long time Peter Frampton fan. Frampton seems to have fallen into the "Top 40" syndrome. Throughout the concert Frampton seemed to be trying to back and play for the teeny boppers in the audience. Reaction to this concert by Frampton fans and this writer was very disappointing which caused a large section of the crowd to leave well before the concert was over.
KSUN/Robert Park, Calif. - Mark Anderson
Artist(s): ROBIN WILLIAMSON
Title: HIS MERRY BAND
Place & Date: Inn of the Beginning, Cotati - 6/26
Type of Place: Club
Ticket Prices: $5.50
Capacity: 300 seated
Attendance: 150
Production Values: Good
Reviewer's Reaction: Robin & His Merry Band (Sybil Woods, Carol Ann Campbell, and Jerry McMillan) made the three quite at home in the fun's intimate setting, performing 2 warmly received sets of Celtic-flavored folk music. Musically speaking, the band is excellent, fitting together like a well-oiled machine with all the right parts. Both sets featured selections from Robin's latest album, Journey's Edge, including the thoughtful "Mythic Times," and "The Tune I Hear No Well." Bits and pieces of the concert were aired over KSUN after the concert, along with excerpts from an interview with Robin from earlier in the day.
KUSP-FM/Santa Cruz, Calif. - Lance Linsner
Artist(s): THE PERSUASIONS
Place & Date: The Backroom, Santa Cruz - June 22
Type of Place: Club
Ticket Prices: $5.00
Capacity: 300
Attendance: T.H.
Production Values: Good
Reviewer's Reaction: Despite the lack of truly lead singer Jerry Lawson, The Persuasions tore up the Backroom and our live radio audience with their non-stop, magnetic group. A real thrill to listen to their intricately arranged I.D. which we now feature on the board! If you're lucky... they'll come to your town.
KFMG/Lincoln, Neb. - Michael F. Famer
Artist(s): STALLION
Production Values: Good
Reviewer's Reaction: The summer seems to be the right time to catch the Indians in full force. It was typical after a week of sunshine the day of the concert looking pretty bleak. Despite a constant threat of rain a good crowd of kite-flying music hungry Lincolners showed up to hear Stallion's music. After two hours based on an already ambitious program, Stallion took on stage aided by an excellent sound system. Stallion played a mixture of their music, album cuts and some new tunes. The music was as good as the setting - located in a heavily wooded area, out of town. Stallion's strong showing enhanced their famous following.
WBSD-FM/Burlington, Wis. - Jeff Weis
Artist(s): CHARLIE DOOBIE BROTHERS
Place & Date: Milwaukee Arena - July 7
Ticket Prices: $5.50; $6.50; $7.50
Capacity: 9000
Attendance: 7,500
Production Values: Excellent
Reviewer's Reaction: Charlie opened with "Turning To You" and continued to play material from their latest LP No Second Chance. (Two days from Britain are something to watch for in the summer, especially when the drummer Steve Gadd is around, suspenders and all.) The Doobies' music couldn't have been better. Starting off with "Cain's Grave" and lifting the audience to their feet. The lighting and special effects were terrific. Christine Baxter (the right hand of Jim Dunlop and Charlie's producer) had her own not another ordinary guitarist. His solos made 7,500 people go insane and at one point the crowd was as loud, you couldn't even hear Shuki. The Doobies, doing a 10 minute version of "Without Love" had the crowd dancing in the aisles. The Doobes came back for two encores that made this concert one of the best Milwaukee will see this year.
WXRO/Wausauch, Ill. - Rick Ralston
Artist(s): BOB SCAGGS
Place & Date: Alpine Valley, Wisc. - July 1
Type of Place: Outdoor music theater
Ticket Prices: $4.00 reserved and $7.00 general admission
Capacity: 5,200 seated, 20,400 total
Production Values: Good
Reviewer's Reaction: Bob Scaggs opened the summer concert series at the Herb Alpert Valley outdoor music theater. Seating and sound were good whether you were under the roof or under the stars. Bob had good rapport with the audience; he must have felt right at home, since in Wisconsin, where he plays with Stevie Miller in the '60s, Bob, during his time with the band, was known as his versatility, from singing to playing the guitar and electric piano. Most of the show consisted of songs from his Degrees, opening with a great rendition of "Lawrence" and "What Can I Do." He also did a few numbers from his new album which has been released recently. Bob and his 11 piece back up group moved smoothly from the mellows ("Clover Lights") to some Chicago style blues ("Runnin' Roomi, Runnin' Blue") to the rockin' "1440 Waffles" which led to a curtain. It was a great show and looks like a good summer of music at Alpine Valley.
KARMA/Indianapolis, Ind. - Dave Curtis
Artist(s): BRAND X
Place & Date: Boomerang in Cincinnati - July 3
Type of Place: Club
Ticket Prices: $5.00
Capacity: 3-400
Attendance: Near capacity
Production Values: Fair
Reviewer's Reaction: A bad mix and a too short set hampered Brand X's first area appearance. Admittedly the band's rhythm section is its mainstay but John Goodall and Robby Krieger's guitar work kept the set reasonably audible. End result: lots of heat but not much light. Still, Brand X on record is a formidable fusion band. One hopes that with so much time, effort and money going into a tour, the sound and production should be of top priority. With some minor changes, Brand X could have been incredible. Maybe next time...
WDR/Kalamazoo, Mich. - Valerie Elliott
Artist(s): AMERICA: POCO
Place & Date: Wings Stadium
Ticket Prices: $7.50
Capacity: 7500
Attendance: 5800
Production Values: Good
Reviewer's Reaction: Poco opened to a warm reception from a usually tough to break crowd. They played a long set strung out material, trying hard to compensate for the lack of a front man. Their enthusiasm and appreciation for the audience was very refreshing. America, going without Dan Peek, put on a similar culmination of their material. However, the enthusiasm was totally absent. Their stage presence was poor and they seemed lacking the audience. It was obvious they weren't having a good time and would have preferred being somewhere else. This is understandable but to convey that, is a dip in the professional attitude one would imagine a group like America to have. Hopefully, once America readjusts from the loss of Peek - they will get their live act together.
HHR/Curran Christi, Tex. - Jay Stevens
Artist(s): CLARK TERRY; FRANK ROSOLINO, SOI & MANY OTHERS; 11TH ANNUAL TEXAS JAZZ FESTIVAL
Place & Date: Municipal Auditorium - July 3
Type of Place: Arena
Ticket Prices: Free
Capacity: 5600
Attendance: 3500
Production Values: Good
Reviewer's Reaction: The people of Corpus Christi got treated to over 6 hours of free, almost uninterrupted jazz. Music spanned all phases of the idiom from Dixieland - to jazz-rock. Three stars were C-101 air personalities Jay Stevens, George Peters and Charlie the Piano Man, bringing right along. Highlights included Soi, a Latin jazz-rock group from Houston led by Joe Gallardo, ace trombonist Frank Rosolino (the man responsible for the great trombone solo on Quincy Jones' "Stompin"), and Clark Terry who brought the house down with a wild version of "Mumbles" to close out the show. Jazz is alive and well in South Texas!
WSM/Chattanooga, Tenn. - Don Mathisen
Artist(s): DARYL HALL & JOHN OATES; GENE COTTON
Place & Date: Memorial Auditorium, Chattanooga, Tenn. - June 28
Ticket Prices: $4.00 advance, $7.00 day of show
Capacity: 5000
Attendance: 3/4 of capacity
Production Values: Good
Reviewer's Reaction: The music and showmanship of Daryl Hall & John Oates was superb. They captured Chattanoogans with their energy and urban blue-eyed soul style. After the concert I taped responses for broadcast on WSM. "It was great, I loved it. Good, but it was outslight. Fantastic, I thought it was great, but I liked Hall & Oates. I liked the drummer man, he was putting it out. It was fine, but The Little River Band wasn't there." Originally, the bill was the Little River Band and Hall & Oates. L. R. B. cancelled the day before because of their problems with equipment transportation. Gene Cotton, a singer song writer from Nashville opened. His band was in California so he went on as a solo. His material was homey. Talking and singing about his two children and the problems of a six year old. Everyone enjoyed the evening of music.
WRAS/Atlanta, Ga. - Carl Jackson
Artist(s): R.E.O. SPEEDWAGON; STARZ; JUDAS PRIEST
Place & Date: Fox Theater - Atlanta - July 3
Type of Place: Old movie theater - now concert hall
Capacity: 3600
Attendance: 3000
Production Values: Good
Reviewer's Reaction: R.E.O. Speedwagon demonstrated their appeal by driving the audience to their feet more than once. The crowd reaction to these two well-known acts are a professional and dynamic hard rockin' group. It's unfortunate that the other two opening acts were incredibly overloud.
WAAL-FM/Binghamton, N.Y. - Steve Becker
Artist(s): PEZHAND; FLEETWOOD MAC
Place & Date: Broome County Arena - June 27
Ticket Price: Reserved seating $8.00 & $9.00
Capacity: 6200 (scaled down from 7000 with no back stage seating)
Attendance: Sold out
Production Values: Excellent
Reviewer's Reaction: The Pezhand warmed up the audience receiving mild reaction from a hard core "Mac" following. Though a bit loud, the Pezhand gave a polished and energized performance. However, this was far from their night to "steal" the show. In fact, I seriously doubt if anybody on this bill could have topped Fleetwood Mac. Aside from the opening "Say You Love Me," Fleetwood Mac routinely showed why they're the hottest act, Chris McVie's vocals shined on this number, as well as the other hits. She seems to be the Jayhawks of the group, keeping everyone in line with McVie's bass lines. She even brought out the congas and Mick Fleetwood's drumming (the best I've heard from him) kept the songs lively and hot. Of course, the concert centered around the most recent additions to the group, Buckingham/Nickelback. Mick is a fine player on guitar, bringing the audience on their feet on his sole in "I'm So Afraid" and belting out in fine voice on "Monday Morning," "Second Hand News" and excelling on Peter Green's, "Oh Well." But the highlight of the evening was Stevie. Though the voice was tired, he broke a couple of times, she stole the audience's heart (and mind). Her stage presence is captivating. By far the best concert in Binghamton for this year. (Sorry Bruce.)
WRNW/Westchester-NYC - Megless Griffin
Artist(s): PATTI SMITH GROUP
Place & Date: My Father's Place, Roslyn, Long Island, N.Y.
Type of Place: Club
Ticket Price: $5.00
Capacity: 400?
Attendance: Sold out
Production Values: Fair
Reviewer's Reaction: Despite a consistent buzz and warm feedback, the Patti Smith Group did it well. As usual, tributes to the other rock 'n' roll crusaders were made by performances of their songs. Beatles, Stones, Lou Reed, The Who, etc. As a group I ask you to consider... for depending on whether or not you do, they could, as Patti said..."end up at Madison Square...or the Poconos..."! Radio Ethiopia.
WWUH/West Hartford, Conn. - Stu Lovejoy
Artist(s): JOHN KLEMMER; HERBIE HANCOCK V.S.O.P.
Place & Date: The Music Inn, Latham, Mass. - 7/4
Type of Place: Outdoor amphitheatre type
Ticket Price: $8.50 person
Capacity: 2000-2500
Attendance: 1500 approx.
Production Values: Good
Reviewer's Reaction: The Quintet of Hancock, Tony Williams, Wayne Shorter, Ron Carter, and Freddie Hubbard are a legend. Together they played some of the purest tightest jazz I've ever heard. Herbie Hancock uses only an acoustic piano. Nothing better. John Klemmer and his band played very strong, but slightly sloppy. "Touch" went over real nice with the crowd, on this nice sunny day in the Berkshires. All in all, a musically fulfilled day.
WAAF/Worcester, Mass. - Lynn Gottlieb
Artist(s): ORLEANS; POUSSETTE-DART BAND; NILS LOFGREN; DUKES OF THE DRIVERS; BOOKER T. & M.G.'S; BBC ALLSTARS; Leon Fleisher, Paul Butterfield, Dr. John, Saturday Nite Live Horns
Place of Type: Outdoor Fairgrounds
Ticket Price: $3.00 in advance, $12.00 day of show
Capacity: 20,000
Attendance: 20,000
Production Values: Good excellent
Reviewer's Reaction: Supersession 77 was absolutely fantastic! In addition to very fine music and an incredibly well-behaved crowd, the weather couldn't have been beat! This show was a gold for several reasons: After the performance, John Hall left Orleans, and it was a reunion show for Duke & The Drivers and Booker T. (featuring Steve Cropper.) Two local based bands (Zonkerz and The Continued above...
**CONCERTIZING**
**WPRB-FM/Princeton, N.J. - George Korvel**
- **Artist(s):** ORNETTE COLEMAN
- **Place & Date:** Avery Fisher Hall, N.Y.C. - June 30
- **Type of Place:** Concert hall
- **Ticket Price:** $8.50; $7.50; $6.50
- **Capacity:** 2800
- **Attendance:** 2800
- **Production Values:** Good
- **Reviewer's Reaction:** Ornette Coleman's first N.Y.C. appearance in a year - and first concert in over 4 years - was the highlight of the Newport-New York Jazz Festival. In the first half Ornette was reunited with his sidemen from the '60's, including Don Cherry, Charlie Haden, and Ron Carter. Don Cherry Redman did, but rather than simply recreating the past (a la VSOP), Ornette wrote five new compositions for the occasion which showed why he continues to be an innovator. In part two Ornette introduced his new band, Peace Time, featuring two new members in addition to the personnel on his new Horizon LP. It's electric but thankfully it's not funk: Ornette maintains his musical standards and has more room to solo. This music promises to be quite influential. And let's hope the tape of the concert is released as a 2 LP set soon.
**KALX-FM/Berkeley, Calif - Tim Devine**
- **Artist(s):** COUNTRY PORN
- **Place & Date:** The Boarding House - July 8
- **Attendance:** 350
- **Production Values:** Excellent
- **Reviewer's Reaction:** Ever have the feeling that you've seen it all before? Such was the case with Chinga Chavin and his Country Porn Revue. It's not that Chavin's approach is interesting but simply that the Country Porn presents is neither as fulfilling nor as well executed as an act like the Tubes or National Lampoon. Even Chinga's "Grand Old Opry" Show had no match for his "Gumbo World Tour." The "grand" performances quickly loses its "grandness" as it becomes increasingly predictable so that by the end of the show, tunes like "Jailbait" and "Pin-Up Boy" make the band seem almost like a parody of itself. This is not to say that the band is bad, just that they are in need of some effective reworking of some of their material. After all, how interesting is a toilet seat guitar once you have seen Fee Waybill's "O" and Ian Hunter's iron cross axe? While certainly not dried up, Country Porn seems to be getting, dare I say, crusty.
**RATHER RIPPED/Berkeley, Calif - Tim Gray**
- **Artist(s):** PSYCHOTIC PINEAPPLE
- **Place & Date:** La Salamandra
- **Type of Place:** Small club
- **Ticket Price:** 99¢
- **Capacity:** 100
- **Attendance:** 100
- **Production Values:** Good
- **Reviewer's Reaction:** This newly activated band has come on as one of the hottest rock sensations to hit Berkeley in recent years. Their format touches on all levels of psychedelic rock rooted from the middle and late 60's. While they play various psych-rock classics such as "Pushing Too Hard," "Little Red Book" and The Kinks' "I Need You" they also have some originals including "I Want Her So Bad" and "Rock & Roll Is Dead." It's refreshing to see this music making a comeback in person because this is the 10th anniversary of the "Summer of Love." The stage control was strikingly professional as they went through some psychedelic classics leading up to a well put together medley of Jimi-Gadda-Gadda-Da-Vida mixed into "Interstellar Overdrive" and back to "Da-Vida." Crowd response was somewhat subdued at first but then developed well to dancing and several encores were demanded.
**KTVD-AM/FM/Santa Barbara, Calif - Laurie Cobb**
- **Artist(s):** CAROLE BAYER SAGER
- **Place & Date:** Roxy, Los Angeles
- **Type of Place:** Showcase
- **Ticket Price:** Promotion
- **Capacity:** 500
- **Attendance:** Full
- **Production Values:** Excellent
- **Reviewer's Reaction:** After countless audience reaction to albums and videos, finally, I was able to experience her in person. Carole's sincerity poured over the audience in a wave of comfort and delight...she paced herself to please even the tuffest expectations from this star filled crowd in Hollywood. The highlight of the evening was towards the end when Natalie Imbruglia joined Carole to sing one of the many songs they wrote together, "Home To Myself." This showcase revealed a subtle glow that's soon to blossom into a bright new career for Carole Bayer Sager!! How lucky we are..(I am also pleased to see a record company believe in their artist...smiles to Elektra!).
**KEZY-FM/Anaheim, Calif - Ron Burnstein/M.D.**
- **Artist(s):** LEON REDBONE
- **Place & Date:** Golden Bear - May 25
- **Ticket Price:** $5.00
- **Capacity:** 500
- **Attendance:** Sold out
- **Production Values:** Excellent +
- **Reviewer's Reaction:** To say it was just another great concert is not enough. Leon (The Eccentric Gentleman) Redbone is one of the craziest men in his own style of cool that only he can achieve. It was a Leon crowd with all of its members participating in the hilarity of Mr. Redbone's "yells" which contain the best combination of grunts, warps and other throat sounds I could find. No wonder Leon Redbone is in charge. It's about time more and more people are appreciating the quality and craftsmanship of a true performer, even though Leon is a quiet, almost shy fellow who talks to nobody (except maybe himself). Comedic humor type is much his forte which all were written early this century. Leon certainly couldn't be charged with plagiarism, since I sure can't think of any performer, past or present, who sings and performs with the type of class as Leon Redbone.
**JOBS & MISC. INFO**
**JOBS**
WKDF, Nashville is looking for a Music Director. Must be strong on-the-air, and be into music research. Send tapes and resumes to Jack Crawford at WKDF, 1202 Stahlman Building, Nashville, Tenn. 37201.
WJKL/The Fox, Elgin/Chicago is looking to fill an immediate part-time opening and possible future full-time openings. Needed is a person with a smooth but natural delivery, a working knowledge of a broad range of music, and the ability to do good non-inpe production. Contact: Tom Mark at WJKL/The Fox, 18-1/2 Douglas Avenue, Elgin, Illinois 60126.
TX-101 is soliciting air-checks for future positions. Excellent in all work, production, initiative, and imagination is imperative. If you don't have commercial on-air experience, we're not interested. Tapes and resumes to: Bill Heiser, WTTK, 234 Clarendon Street, Boston, Mass. 02116.
STARVIEW '92 hopes to shorten shifts with staff expansion. Now accepting tapes for probable future opening ~ 8-midnight. Aircheck must include production. Include salary requirements. No calls, please. Contact: Hal German, STARVIEW Radio, R. D. 4, York, Pa. 17404, EOE
Music Director and/or production job needed. Music director of KFCSN for the past 2 years, production engineer for K-100 for the last 3 years and worked at KGRI for 6 months (All L.A. studios.) First class tenor wants job as music director and/or production engineer at a good station. Will work hard for decent salary. Contact: Kevin Stern, 13111 Bassett Street, North Hollywood, Ca. 91605.
**JOBS CONTINUED**
Seeking news/public affairs and/or announcing-production position. Strong writing, announcing, production experience, 3rd endorsed. Presently news director at D. C. area's progressive WHFS-FM. For resume and tape, contact Suzanne Gordon, 103 Croydon Court #4, Silver Spring, Md. 20901. Or call 301-536-0600.
**MISCELLANEOUS**
Effective immediately, "Hot News!" a syndication produced by the Earth News audio people will include:
1. Firsing Theatre - doing topical satirical comments, twice each week.
2. Leon Chandler - singing the news, once a week.
3. Plus other young "turned-on" reporters of the nature of Paul Crosswhite doing alternative news.
Decent public affairs programs wanted for inspirational purposes and commitment. Contact Joe "From Chicago" Prater at WRNW, 55 Woodside Avenue, Briarcliff Manor, New York 10510.
Wanted: Live tapes for concert series with large audience - send to WTTK, 234 Clarendon Street, Boston, Mass. 02116. Attn: Bill Heiser 617-257-0121.
WORJ is interested in doing interviews with any willing artists. Please contact Bill McGathy at WORJ, Orlando, Fla. by calling 305-298-5510.
Live tapes wanted for concert series. The series has extensive advertising and a large audience. Send to WRSU-FM 126 College Avenue, New Brunswick, N. J. 08903. Attention: Irv Linkin.
NEW RELEASES
BELOW IS A CATALOGUE OF NEW RELEASES. THEY ARE DIVIDED INTO CATEGORIES DESIGNED TO GIVE YOU AN IDEA OF THEIR RELATIVE MUSICAL WORTH. THESE FIRST IMPRESSIONS ARE NOT MEANT TO RELATE IN ANY WAY TO PAST, PRESENT OR FUTURE SALES, NOR SHOULD THEY BE TAKEN AS THE WORD OF GOD.
SPECIAL MENTION ALBUMS
(These records which, in our opinion, deserve special mention because they come closest to perfecting their musical genre regardless of progressive radio application.)
None.
STRONGLY MERITORIOUS
(We feel that these LP's have an extra edge.)
None.
MERIT PLUS ALBUMS
(All Merit and Merit Plus Albums are recommended for serious progressive radio consideration, recognizing that not all of these recommendations are suitable for all stations. The Merit Plus Albums distinguish themselves with an extra vitality.)
The Clifton Chenier Band/CAJUN SWAMP MUSIC LIVE/ Tomo TCD 2-7002 (Lawrence Welk would have apoplexy if he heard Clifton's accordion. Recorded at Morehouse, the LP has so much music to be absorbed, it should not have been a 2 record set. Simple out the blues tracks and the rocker, Chenier adds some musical spice to life.)
The Dingoes/FIVE FOR THE SUN/Atlantic 4356 (As that acrobatic lead voice edges its way through Dingos man, the heart rate on listening value. Those tunes are real catchy in a very basic way. The rockers which open each side are camouflage.)
Roderick McDowell/THAT'S MY ROCK CITY/United Artists UA LA 777G (Falcon has dropped the phony, sterilized approach moving his music back to the rudiments. His quality writing, even in his first LP, did not and does not require the charade. This time, formal content are made clear. This album should have included Carole King/SIMPLE THINGS/Capitol SMAS-U667 (There are no surprises here. Carole's LP is essentially what it claims to be: a fairly down to earth representation of good tunes. Simple Things doesn't ever get untartrated from its goal.)
Lonnie Liston Smith/LIVE!/RCA APL 1-2433 (Passable sound quality fades into the background as the energetic, joyful music of Lonnie Liston Smith surges forward. Forced into musical, his live album captures the wholesome attitude which should be embodied in his studio LPs. Favor.)
Styx/THE GRAND ILLUSION/A&M SP-4637 (Producing a rather refined brand of power rock puts Styx in an enviable position. They are respected on a musical level while they get to push air with the best of them. Finally, their tunes deserve the effort.)
War/PLATINUM JAZZ/Blue Note BN-LA 690-72 (As far as we can tell, the first side of this two record set are new while the last side reissues old material. In any case, the music of War flows organically with tight rhythm and ear appealing melodies. Superior talents.)
MERIT ALBUMS
Average White Band & Ben E. King/BENNY AND US/Atlantic ST 1905 (A comfortable R&B album results from this creative union. Ben E. King becomes the dominant force as AWB's sound is shaped by his. It's really his LP. Well chosen material in seven track-to-track quality.)
Blue/ANOTHER NIGHT TIME FLIGHT/MCA PG-2209 (Mild rocking, smooth harmonies and nice songs should result in wearing bannars. Yet, Blue never quite gets there. Their LP is solid on all levels but it lacks a boil over the top. Edible personality is the missing element.)
Tom Brosseau/KICKED BACK/A-BC Dot-DG-2084 (Placed in a lighted rockabilly setting, Brosseau's entry into contemporary music, he can't really be called a country artist, since there isn't a pedal steel to be found on this LP. Nice tunes and an easy going style satisfy.)
Burning Spear/HARD & HEAVY/Mango MLPS-101 (Though these melodies are good, the timidity of the reggae rhythm requires more inspired treatment. Burning is a legitimate voice of the struggle but, less effective in the larger world than a Marley has a chance to be.)
Buzz Cason/BUZZ/OJIM DILEMA-9 (Professional bland music which you can't get tired of listening to. While few mistakes are made the music feels anonymous. Your thirst isn't quenched. Another studio musician and another competent LP.)
Richard and Eric Lloyd/SHATTLE OF THE SAXES VOL II/Muss M-1001 (The battle setting is sound for both of these players. It forces them to aim attack to their music. Further, the competition seems to generate creativity as one really tries to out do the other. Look forward to volume III.)
Billy Gray/CRAZY CRADLE/LIVE!/ABC Dot DG-2062 (Billy is a rockabilly soul. This live LP captures his loose, raucous essence and the fun that it must be to see Crash work.)
MERIT ALBUMS CONTINUED
Driver/SO ACCIDENT/As M SP 46-45 (Driver delivers what his name implies: a powerful brand of rock and roll. There being nothing new in the genre but, they do master it. No disappointments.)
Elvin Jones/TIME CAPSULE/Vanguard VSD 79389 (You expect drummer albums to be punchy but, you don't expect them to be so satisfactory. Elvin has combined a neat combination of the beat, his vast contemporary mind respects the avant garde without boggling the mind.)
The Bernie Leadon-Michael Georgiades Band/NATURAL PROGRESSIONS/Elektro TE-1105 (Soft harmonizing and interplay between the horns is a natural marriage. This understated Gly Dohen production that don't bore you over but, it never lets you down either. Easy.)
Dave Matthews' Big Band/NIGHT FLIGHT/Music MR 5098 (Matthews' smartly updates the big band by emphasizing the horns. The result is a blend of the horns and of assembling playing in the horn section then results in a big band which sounds more like an enhanced combo. And they cook too.)
Christopher Morris/Music Band/MCA 2287 (Christopher Morris' singing is a series of good intentions which he is not really able to carry off. His voice should be harmonized or doubled. All kinds of production jive manages to keep the band's music aloft but, that too runs the risk of wearing thin. Passable.)
Ann and the Gift of Gab/HEAVEN/HI MLP 6002 (The Ann Peebles - Willie Mitchell connection is marvelous. Always good songs and a solid production to bolster them. This LP tempts me somewhat heavier handed on the strings than I would prefer.)
Smokie Robinson/SOUNDTRACK BIG TIME/Tamla TS-3558 (Smokie is always a delight. He engenders a warm feeling in the audience which is a rare occurrence. His music for this film comes in too degrees of on-allow. The same song spans through the sides while the title dominates two. The Big Time theme is notable.)
The Stranglers/IV RATTUS NOVUS/Epic/ECSAE M SP-46-48 (Previously reviewed as a bonus. From their chosen material, the Stranglers are today, a kind of term as a culturally deformed Roxy Music. Their songs are good enough and they manage to sustain the attitude throughout. Merit.)
Town Zandt/END/LIVE AT GOLD QUARTER/Tomato TCD 7001 (The real Van Zandt talent lies in his ability to weave a potent lyric. His singing and his melody writing are merely acceptable. Hence, his talking blues tends to be the form which uses his talents best. But you can't do that all of the time. Van Zandt requires vocal skill more than he does lyrics.)
The Tim Weisburg Band/TWB/United Artists UA LA 773-G (We have been arguing for years that Tim had to manifest his full sound to balance himself properly with his rocking acid trip. Despite his clever production, he just the opposite with the same guitar effect. He tightened the back-up, opening up the rhythm to leave musical lines available to the smooth Weisburg chime sound. A melodic step in the right direction. "Cascade" and "Mercy, Mercy, Mercy" are standouts.)
Knobs & Linda Williams/SHINE AND/OR MADON/Symposium SYS 4007 (A sable's delight. The Williams', who don't give in to electrification, are content to entertain in a lindy acoustic setting. Their voices, chosen and original, create a solid album moving easily.)
O.V. Wright/INTO SOMETHING.../HI HLP 6001 (Supported by the tasty, understated Willie Mitchell style C. O. Wright does all right. Good songs help to make it even stronger. "I'm Going To Live My Life" and "I'm Living My Life.")
Yes/LOOKING FOR THE GAME/Atlantic SD 19106 (The return of Rick Wakeman should put new enthusiasm into Yes fans, if the record doesn't take it out again. All of the busy, bombastic grooves are crammed into the high midrange. Anderson's vocals are buried and distorted and is deflated. Wakeman fills in every possible whole in the music giving in no chance to breath. Just the lack of dynamic range reduces the album to a superficial haze. If I could have been; so much better had it been mixed differently.)
SATISFACTORY ALBUMS
(Albums of acceptable quality, which, of themselves, require only secondary progressive radio consideration.)
Hall & Danielsen/GENTLEMEN OF LEISURE/Flight FR 5001 (A warm natural setting is always appealing but, there is no way around the undeveloped nature of Hall & Danielsen. The ballads are their forte and are like immature first efforts which normally would never make it past a home tape recorder. A minority of two tunes are of recording quality and they are the LP's first two tracks. They provide hope.)
NEW RELEASES
BELOW IS A CATALOGUE OF NEW RELEASES. THEY ARE DIVIDED INTO CATEGORIES DESIGNED TO GIVE YOU AN IDEA OF THEIR RELATIVE MUSICAL WORTH. THESE FIRST IMPRESSIONS ARE NOT MEANT TO RELATE IN ANY WAY TO PAST, PRESENT OR FUTURE SALES, NOR SHOULD THEY BE TAKEN AS THE WORD OF GOD.
UNDISTINGUISHED ALBUMS
(In our opinion, below standard Lps except where noted.)
Cain/STINGER/ASL 214 (Back in 1968 we heard lots of stuff like this. Most every element is weak. The writing especially and recording quality vie for weakest links. From the opening "I Can't Stop" to the end, Glimmers of hope come from "Gimme Some Lovin'" and "All I Need Is You.")
FRINGE ALBUMS
(Good albums which are not progressive, yet hold some interest for progressive audiences.)
Allspice/At-Home AH-401 (Classy. Beautifully mounted and packaged, this album maintains presence in spite of its bigness. Silky voices transmit a fine pop R&B message ala Wayne Henderson. He inquires that there is an undercurrent of rhythm intensity always present.) Babyface/ASL 213 (Faddy on a conceptual level. Half of the LP is schoolboy MOR, the other half is hard rock. There is little relation between these two facets of Babyface. Therein lies the communications problem they make for themselves. Who are they?) Bay City Rollers/IT'S A GAME/Arista AL 7004 (An appropriate title. It suits the quality of this production. The all out pop approach and the tactical advantages of the studio make B.C.R. sound good.)
Steve Cauthen/SINGS TOO!/Barbarek BB 3334 (Though Steve Cauthen is not an extraordinary singer, this record represents the schlockier side of the music business. His voice, embellished with many overdubs, sings the provided tunes accurately and the production has only touches of childishness. It's a shame, this 16 year old singer's voice ought to be left to the oval.) Lesley Duncan/MAYBE IT'S LOST/MCA 2274 (An excellent pop singer when she has written good material, Lesley has always been close to real acceptability. The production context in which she operates is always a little too facelifted and too showy. Folk-MOR.)
Honeyeye/IN CONCERT/Myrrh MSB-6584 (Honeyeye is a female John Denver who is infected with Christianity. Her music is light folksy stuff with a touch of country and it is more than bad. Some of her lyrics are memorable.) Willis Jackson/D'THE ALLEY/Muse MR 5100 (In the annals of funny jazz Willis Jackson is a notable. As the liner notes rightly suggest, Willis is a rhythm and blues player, emphasizes the blues in his jazz context. Check into "Little Red," "The Alley," "Playin' againme," L.T.D., /SOMETHING TO LOVE/ & M SP 4646 (You won't be transfixed by the songs which L.T.D. sings. Still, their music is enjoyable moving from a fairly smooth pop style to funk and soul. It's Deliberate and finished.) Peter McCann/20th Century T-544 (The singer/songwriter, MOR formula-crossover city. A tenor, some decent songs, standard charts played with automation perfection and sweetening. The latter are the elements of the formula. He so it is called flat flatteringly.)
Dorothy Moore/Malaco 6353 (Her album is too snazzy but its underpinning is basic rhythm and blues. Tunes like "Love Me," "Make It So," and "Old Time's Sake" and "Tone Blues" are far apart from the most basic launching pads before they reach overdone.)
Sonny Phillips/MY BLACK FLOWER/Muse MR 5118 (Funky or cool Sonny rarely establishes himself as the leader. Not that he can't, the title tune proves he is quite capable, but he doesn't go strongly to the front. The LP is OK, though no discernible Phillips personality emerges.) Hargus "Pig" Robbins/COUNTRY INSTRUMENTALIST OF THE YEAR/Elektra (Pig Robbins has been in the studio for so long that he has forgotten how to lead a band. Those who made you are still waiting for the vocalist to come in, it never happens.)
Ronnie Sessions/MCA 2285 (Unreined, raucous rockabilly music spews from Ronnie Sessions. His exuberance and his ability to incorporate seemingly alien instruments like the moog, endear him in spite of the production sloppiness.)
Staff/MORE STUFF/Warner Bros. BS 3061 (More Stuff means more of the same. Nothing new. The studio players are incapable of stepping out. The same was true of Area Code 615 and is true for The Section. Glen Campbell may have been the last studio player to establish himself. Try "More Of Mine" and "I'm Nobody's Clark Terry/FIVE GLITTERS/Vanguard VSD 3393 (Living in the past, Clark's aggregate strides coolly into the setting sun. Their presentation is smooth and graceful however, exemplary of another age.)
Joe Venuti/JN MIDIAN/Vanguard VSD 3396 (Venuti's violin playing is great. The rhythm section accompanies do too. He doesn't develop the melancholy tone which is so attractive in Stephane Grappelli's playing.)
SPECIAL PROGRAMMING
(Spoken, poetry - comedy and extraneous musics.)
Barry Altschul/YOU CAN'T NAME YOUR OWN TUNE/Muse MR 5124 (Mostly spaced, sometimes strikingly linear [the first tune] and sometimes rocking [Finish Chart, "I'm a King Korn"], Barry has gathered around him several of the acknowledged leaders in avant jazz for what sounds like a foray into our musical nerve ends.) Maurice Andre/JOURDE II/RCA FRL 1-3504 (Andre, a trumpeter, has become a musical adventurer in jazz. Rhythms to come up with a sound on the classical side of Brubeck. It is typically French in concept especially if you think of The Swingin' Singers.)
The Credibilities/THE BRONZE AGE OF RADIO/Warner 1 (If this LP had merely gave us "Who's On First?" it would be worth the time it takes to listen to the rest of the LP. The balance is more an exercise in clever ideas than a barrel of laughs.)
Marcelli/Melody & FANTASY/NEW VILLAGE ON THE L.I./Black Oak BSR 0012 (An interesting LP. It juxtaposes jazz with an avant slant and primitive vocal harmonizing. The instrumental is spaced while the vocal sounds like a more guttural Irish folk music harmony. Strange, exotic stuff.)
Tete Montoliu/MUSIC FOR PERLA/Inner City IC 2021 (Montoliu's piano improvisations on side two are more expressions of a technical ability than of musical inspiration. Side one are piano pieces based on a pan alley tune. They are pleasing to the ear rather than technically. Worth a listen especially for the Jarrett-Corea fans. Write: 43 W. 61st St., New York, N.Y. 10023.)
Various Artists/A TRIBUTE TO LEADBELLY/Tomato TOM 2-7002 (This is a collection of extremely important figures in the musical past. Gathered for the honors are Arlo, Pete Seeger and Sonny and Brownie. The music is good and loose as it give us a peek at our roots.)
Various Artists/OLD AND NEW DREAMS/Black Saint BSB 2013 (Don Cherry, Dewey Redman, Charlie Haden and Eddie Blackwell are the various artists joined in this excursion. All musical descendent of Ornette Coleman, they move around the outside of the melody without ever straying off course. Haden's "Quirnman Mao" is the most accessible melody. Available through New Music Dist. Service, 6 W. 95th St., NYC 10025.)
REISSUES
David Bowie/STARTING POINT/London LC 50007 Judy Collins/SO EARLY IN THE SPRING.../Elektra ELS 2002 John Coltrane-Wilbur Hardin/DIAL AFRICA/Savoy SJL1110 Electric Light Orchestra/SOUNTRACK-JOYRIDE/United Artists UA LA 784-H (About half of the music is done by Jimmie Haskell who contributes the filler.) Genesis/IN THE BEGINNING/London LC 50006 Howard McGhee/MAGGEDON/Blue Note SLJ 2219 Roger McGuinn/AIRPLAY ANTHOLOGY/Columbia AS 353 Charlie Parker/BIRD AT THE ROOST/Savoy S JL 1108 Sha Na Na/ALL HERE AT STAY/Buddah BDS 5692 Stephen Stills/OFF STAGE/Warner Bros. BWL 1-330 Ten Years After/GREATEST HITS/London LC 50008 Various Artists/THE ROOTS OF ROCK 'N' ROLL/Savoy S JL 2221 Various Artists/THE TENOR SAX ALBUM/Savoy SJL 2220 Lester Young/PRES LIVES/Savoy S JL 109
NOTABLE SINGLES
The Doobie Brothers/LITTLE DARLING/Warner Bros. WB 8408 Stanley Frank/SCHOOL DAYS/attic AT 130 (Import.) London/EVERYONE'S A WINNER/MCA 305 (Import.) Models/FREEZE/A Step-Forward (Import.) The Pioneers/FADE OUT/EMI PSLP 211 (Import.) Rheal Brothers/DEDICATE/EMI PSLP 218 (This LP, 12 inch, 45 rpm disc introduces the Rheal Brothers in a most favorable way. Smooth, lovely tunes delivered in a like manner build real anticipation for their LP.) Sparks/OVER THE SUMMER/Columbia 3-10579
IMPORTS
(Our imports are provided with the help of Intergalactic Trading Co., Portland, Ore.; Jem Records, P. O. Box 362, South Plainfield, N.J. 07080 - (201) 753-6100.)
The Clash/CBS 82000 (Audacious and deliberately bratish, The Clash seems to have left themselves nothing to regret. The title track, "White Riot" and the title, conveys the message "I'm So Bored With the U.S.A.," "White Riot," "Hate & War" - that's on side one, in spite of the alienation it's "Janie Jones" which has the superior melody. Merit.)
NEW RELEASES
BELOW IS A CATALOGUE OF NEW RELEASES. THEY ARE DIVIDED INTO CATEGORIES DESIGNED TO GIVE YOU AN IDEA OF THEIR RELATIVE MUSICAL WORTH. THESE FIRST IMPRESSIONS ARE NOT MEANT TO RELATE IN ANY WAY TO PAST, PRESENT OR FUTURE SALES, NOR SHOULD THEY BE TAKEN AS THE WORD OF GOD.
IMPORTS CONTINUED
Omega/ON TOUR/Bellaphon BBS 2527 (These journeyman rockers don't give away their Germanness through their music. They just fail to move you the album with their song "Adequacy in the watch room." Merit.)
Alan Stivell/BEFORE LANDING/Fontana 9286 999 BD (A Brittany ethnic, Alan Stivell attempts through his music to educate us about his country. Sung in Celtic tongues, you'll have to learn a bit before he picks up. He weaves contemporary folk tales about the past and the present. The language makes it a little esoteric. Special Programming.)
Michal Urbaniak's Fusion/SMILES AHEAD/MPS 63-165 (If there is a flaw in Urbaniak's formulation it is that his band only displays purposefulness when he is out front. The other players, including Urszula, are rarely able to transcend their technique, whereas Michal's solos ring. Merit.)
Various Artists/THE ROXY LONDON W.C. 2/EMI SHSP 4069 (This LP is the London equivalent to the CBGB's album. The LP tasters groups called Slaughter & The Dogs, The Unwanted, Wire, The Adverts, Buzzcocks, X-Ray Spex, Easteer, and Johnny Moped. All of the music is raw street music made to be raw and tough to take and much of it is. Merit.)
The Vibrators/PURE MANIA/Epic EPC 82097 (New Wave opponents dismiss it as all bad just as its proponents tend to dismiss it as all good. In truth, probably it comes down to the tunes, it always does. Out of 15 songs The Vibrators need only "Baby Baby" to justify themselves. There are several more good tracks, though nothing else of that caliber. Merit.)
IMPORTS CONTINUED
Larry Coryell-Philip Catherine/TWIN-HOUSE/Atlantic ATL 50342 (Why is it that so much of the music which is recorded for art's sake never borders Europe? Here is a case in point. These lovely guitar duets between two enormous talents, needed to be done in spite of any commercial consideration. At times they attain the spiritual level of the better piano improvisation LPs by Jarrett and Coryell. Special Programming.)
RECORD SERVICE
KCSN/Northridge, Calif. - Polydor
KOHL/Fremont, Calif. - RCA; Bluebird; Capitol;
KRMR/Sun Valley, Idaho - Kudu; Motown; United
WBSD/Burlington, Wisc. - Artists; All country labels
WMHW/Mt. Pleasant Mich. - MCA; Mercury
WMIR/Lake Geneva, Wisc. - Capitol; MCA; United Artists
WTAO/Maryphsboro, Ill. - Warner City
WUMB/Boston, Mass. - Warner Bros.
WXRD/Woodstock, Ill. - Casablanca
WYCI/Philadelphia, Pa. - Mercury; Polydor
- CTI; Catalyst
CONCEPTS
GONE ARE THE POLITICS OF THE 60'S
We have been watching the progress of the Gregg Allman LP in the Radio Emphasis chart since its release. If any album might be able to measure the changing political winds as they have blown through album radio in the last few years, we felt that this LP could.
Gregg's legal entanglements and marital involvements are little understood by the outside world but, in both cases they operate to the negative as regards his musical career. Especially, the drug business which had the potential to strip him of any and all hip media support forever. But, not today.
As of the last issue of Walrus, Gregg's album was still moving up the Emphasis chart in an individual ascent. He had advanced 100 percentage points in four weeks to the eighth national airplay position. Of course, he was substantially (about 25%) stronger in the South than anywhere else but, had that region not been used in the calculation he would have dropped only a few positions.
The significance of these observations is the apparent absence of political revenge on a radio level. This may be the final evidence that the consciousness of the sixties no longer exists for it is hard to imagine this particular Gregg Allman album getting any greater airplay emphasis than it is currently.
Rock and roll is here to stay as the politics just fade away.
|
CONTENTS
Editorial_______________________________________________________ 1
The Rose Cross and Holy Grail, Costello ___________________________ 2
Reasons for Bacon’s Participation, Brameld ________________________ 4
Alchemy and The Holy Grail, Bayley _______________________________ 26
Francis Bacon: A Forerunner, Srigley ______________________________ 39
Shake-speares Sonnets, Bokenham _________________________________ 46
Ovid cum Shakespeare, Begley ___________________________________ 51
The Baconian 33 and The Archetypal 28, Hollenbach ________________ 58
Correspondence _________________________________________________ 69
Publications____________________________________________________ 75
In the East ____________________________________________________ 79
Sir Francis Bacon: A Biography, Fuller ____________________________ 80
Francis Bacon, Shakespeare and The Rosicrucians, Bokenham _______ 80
The Francis Bacon Society ___________________ Inside Back Cover
© Published periodically by THE FRANCIS BACON SOCIETY INCORPORATED at Canonbury Tower, Islington, London N1 2NQ, and printed by Woolnough Bookbinding Ltd., Irthlingborough, Northants NN9 5SE
ISSN 0961-2173
THE FRANCIS BACON SOCIETY
(INCORPORATED)
Among the Objects for which the Society is established, as expressed in the Memorandum of Association, are the following:
1. To encourage, for the benefit of the public, the study of the works of Francis Bacon as philosopher, statesman and poet; also his character, genius and life, his influence on his own and succeeding times, and the tendencies and results of his writing.
2. To encourage for the benefit of the public, the general study of the evidence in favour of Francis Bacon’s authorship of the plays commonly ascribed to Shakespeare, and to investigate his connection with other works of the Elizabethan period.
OFFICERS AND COUNCIL
Hon. President
Sir George Trevelyan, Bt.
Hon. Vice President
Miss Mary Brameld
Council:
Chairman
T. D. Bokenham, Esq.
Francis Carr, Esq. Gerald Salway, Esq.
Robert Cowley, Esq. Peter Welsford, Esq.
Clifford Hall, Esq.
We have to report the much regretted retirement as our Editor of Mr Clifford Hall, who has now left Buckingham University as a Senior Lecturer in Law and who may shortly be moving to other climes on the far side of the Atlantic. We much appreciate his work not only as Editor but also for his valuable advice and co-operation on the Council. Mr Peter Welsford has offered to take over as Editor at least for the time being.
At the end of his Editorial in our last issue of *Baconiana* Clifford mentioned some exciting developments involving our Chairman. It seems that in the Autumn there will be some interest shown on the Shakespeare works and the B.B.C. is planning to produce a Television programme on those heretical claims concerning their authorship. "On the 23rd of October, B.B.C.2 broadcast a well produced programme on the Shakespeare authorship question in which Thomas Bokenham and Gwyn Richards battled for Bacon. Other claimants included Lord Burford for Oxford and Dolly Wraight for Marlow."
We are publishing in this issue a few articles concerning the esoteric side of Francis Bacon's teachings which may enlighten those who believe that he and his Rosicrucian Fraternity were attempting to pervert our Christian beliefs.
In 1993 the Society was delighted to be in receipt of two unexpected legacies amounting to £6,000 from Mrs Irene Rowland, the stepdaughter of Alfred Dodd and the widow of a former Council member, Grieves, for which we are very grateful.
At the recent Annual General Meeting of the Society, it was proposed by our Chairman that we should contribute a sum of £100 to the Star & Garter Home for the Disabled at Richmond towards their D-Day Anniversary Appeal Fund which has been gratefully received.
THE ROSE, THE CROSS AND THE HOLY GRAIL
David Costello
London 1993
The creative tension that exists in the emblem of the Rosicrucians, the Rose and the Cross, is echoed at several levels in the image of the Grail.
Its primary manifestation is in the shape of the Grail itself. The image of the stemmed cup is clearly emblematic of the conjunction of the male and female principles — the basis of continuing life but at the same time representing the androgyne, potentially the end of the cycle of birth and rebirth. "In my end is my beginning..." and the first paradox, the paradox of the wine, brings new life from the certain end of life.
The second paradox is in the essential basis of the Grail legend. This could be described as the paradox of the bread, since just as the breaking of bread — "the staff of life" — destroys its form so the attainment of the Grail destroys the element of the Grail Quest. Without the Quest the Grail is just another cup, without the cup the Quest is just another journey.
The journey of the Grail Legend echoes the journey in the Rose Cross Ritual* itself and also recalls the phrase used as a recognition code by Freemasons to describing a Freemason as a "traveller from the East". One legend has it that the Rosicrucians came from the East bringing the knowledge of their tradition and then returned there, retreating from the growing materialism of the Christian world.
There is though a more profound tension in the concept of the lost cup, holder of the creative wine, which is amongst us and yet invisible.
Two statements recur in writings about Rosicrucians. The first is that there is a distinction between Rosicrucians and people who are Rose-Croix. Rosicrucians are members of a Society which, like the Freemasons and Alchemists uses more or less ancient rituals in an attempt to uncover esoteric knowledge of man's position in the Universe. Someone who is Rose-Croix however is someone already in possession of that knowledge or perhaps in "state of grace" of which possession of such knowledge is either a part only or an irrelevance.
* Please note: "Rose Cross Ritual" refers to the Ritual used in 18th Degree "Scottish Rite" masonry, not any Rosicrucian Ritual.
The second statement made about people who are Rose-Croix is that they can never reveal themselves as such: anyone who claims to be a Rose-Croix is, by definition, not one. This poses a further paradox, fairly straightforwardly expressed thus: ultimately there is no such thing as a really secret society. There will always be someone at whatever level who will out of spite or disillusion or as part of a genuine moral imperative ultimately reveal the secrets. Therefore if this statement is true — that a Rose-Croix will never reveal himself or herself as such — then the only explanation for this must be that he or she does not know it: that a genuine Rose-Croix is ignorant of the fact that he is one — the Grail does not know of its own existence. Presumably if the Rose-Croix attains the knowledge that he is one then he must immediately cease to be one — the immortal who falls in love with a human loses the attributes of immortality.
This fits with the Christian version of the Grail legend. The Grail — the androgyne without the power of birth and rebirth (innocence) — nonetheless contains the life force represented as wine (experience) which becomes the blood of Christ (knowledge — the “invisible sphere” of the Kabbalists). God made man loses his godhood and can be killed. He can only regain his immortality by dying as a man and rising as a spirit.
Thus the journey of the Grail becomes the journey of the soul, the Rosicrucian traveller with his staff coming from the East of dawn and returning there. The Eagle of the Scottish Rite flying into the rising Sun.
There is though one other underlying paradox. We have noted that a true Rose-Croix can never reveal he or she is one. Perhaps he cannot know it or perhaps by revealing himself he loses the knowledge of being Rose-Croix. But if this is the case it is impossible that we should know that the condition of being Rose-Croix actually exists.
There is no answer to this but an act of faith. To paraphrase Casanova “The secret of the Grail cannot be imparted: he who knows where it is cannot find the words to explain its location, he who has not found it will never understand the directions to it.”
The general public know that Francis Bacon was the principal founder of empirical science and its inductive method, a writer of philosophical treatises and moral essays, as well as being a parliamentarian, a statesman and an eminent solicitor, ultimately rising to the top of his legal career by being given the office of Lord Chancellor of England in 1618 in the reign of King James I.
Some might suppose that the latter was a profession he chose because legal matters really interested him. Baconians know that in point of fact other subjects were much nearer to his heart, and occupied his time and attention to a larger extent than the laws of his country during his early manhood.
Cultural pursuits provided him with a greater stimulus. He passionately loved poetry, drama, Greek mythology and symbology, and the Mystery Teachings, and he threw himself into the creation of dramatic entertainments with enormous enthusiasm.
The study of the Law was imposed upon him in order that he could earn a living and become self-reliant, but it was not congenial to him, whereas the writing and production of Masques, pageants and plays was to him a source of amusement, relaxation and enjoyment.
In her book 'Francis Bacon and his Secret Society' Mrs. Pott (the founder of the Francis Bacon Society) tells us:
There is reason to think that, Francis, in childhood, showed great talent for acting, and that he took leading parts in the Latin Plays which were performed at college. At home, such doings were checked by Lady Anne's Puritan prejudices. The strong tendency which Anthony and Francis evinced for the theatre, and for "numming and masquing" with their companions, was a source of great anxiety.
* Script written in collaboration with Elizabeth Brameld.
and displeasure to this good lady. She bewailed it as a falling-off from grace, and prayed that it might not be accounted a sin that she shold permit her dear son Francis to amuse himself at home in getting up such entertainments, with the help of the domestics.\(^1\)
The one big compensation for commencing legal studies at Gray’s Inn was the opportunity this afforded him in the creation of Masques — a unique form of entertainment put on by the students for presentation to the élite.
Masques comprised a fusion of many different arts, such as poetry, acting, disguising, presentation of symbolic gifts, vocal and instrumental music, dancing, specialised costume, pageantry and splendid scenic decoration.
These entertainments were divided into sections. The main masque was performed by the nobility followed by the anti-masque, the latter being performed by professional actors, acrobats, dancers, and were of a much coarser type, even grotesque. In his essay, Bacon said: — ‘let anti-masque not be long’. . . In the last section of the evening’s entertainment at the conclusion of the masque there followed the dancing, referred to as Revelling or Commoning. When the wizards or masks which had disguised the aristocratic performers were finally removed, the Masquers came down from the stage and took the audience to dance with them in the main hall. The intermingling of players and spectators was a characteristic feature of Masques.
They were devised for the purpose of celebrating birthdays, or weddings, or recovery from illness, or other important events such as the anniversary of Queen Elizabeth’s accession to the throne.
I would now like to quote an extract from a slim volume, entitled ‘Did Francis Bacon write Shakespeare?’ which gives Mrs. Pott’s description of a masque called the Prince of Purpoole at Gray’s Inn at the end of 1594. On pages 19/20 she writes:
The Christmas revels in which the students at Gray’s Inn had formerly prided themselves had been intermitted for three or four years, and they were resolved to redeem the time by producing this season something out of the common way. A device, or elaborate burlesque, which turned Gray’s Inn into a mimic court, was arranged. A Prince
---
1. Francis Bacon and his Secret Society — Mrs. Henry Pott.
of Purpoole and a Master of the Revels chosen, and the sports were to last twelve days.
The Prince, with all his state, proceeded to the Great Hall of Gray’s Inn, on December 20, and the entertainment was so gorgeous, so skilfully managed, and so hit the taste of the times, that the players were encouraged to enlarge their plan and to raise their style. They resolved, therefore (besides all this court pomp and their daily sport amongst themselves) to have certain “grand nights”, in which something special should be performed for the entertainment of strangers. But the excitement produced on the first grand night, and the throng of people, which was beyond everything which had been expected, crowded the hall so that the actors were driven from the stage. The performers had to retire and when the tumult partly subsided, they were obliged (in default of those “very good inventions and conceits” which had been intended) to content themselves with ordinary dancing and revelling, and when that was over with A Comedy of Errors (like to Plautus his Menechums), which was played by the players. This was on Dec. 28.
The next night was taken up with a legal inquiry into the causes of those disorders, and after this (which was a broad parody upon the administration of justice by the Crown in Council), they held “a great consultation for the recovery of their lost honour,” which ended in a resolution that the Prince’s Council should be reformed, and some graver conceits should have their places.” It is most probable that one of these “Graver conceits” was by Bacon himself. It is certain that an entertainment of a very superior kind was produced a few days after, in which he took a principal part. This entertainment, “one of the most elegant that was ever presented to an audience of statesmen and courtiers,” was performed on Friday, January 3, 1595. It was called the “Order of the Helmet”
In 1612 King James I’s daughter Elizabeth was married to Frederick V, the Count Palatine, and to celebrate this marriage certain members of Gray’s Inn and the Inner Temple jointly presented a masque which they called ‘The Marriage of the Rhine and Thames.’ Francis Bacon was
2. ‘Did Francis Bacon Write Shakespeare?’ by Mrs. Henry Pott, pp. 19-20, Parts 1 and 2.
alluded to as the chief contriver. The Lord Chamberlain gives a vivid account of it:
"On Tuesday," writes Chamberlain, February 18, 1612 "it came to Gray's Inn and the Inner Temple's turn to come with their masque, whereof Sir Francis Bacon was the chief contriver; and because the former came on horseback and in open chariots, they made choice to come by water from Winchester Place, in Southwark; which suited well with their device, which was the marriage of the River Thames to the Rhine; and their show by water was very gallant, by reason of infinite store of lights, very curiously set and placed, and many boats and barges with devices of lights and lamps, with three peals of ordnance, one at their taking water, another in the Temple Garden, and the last at their landing; which passage by water cost them better than £300. They were received at the privy stairs, and great expectation there was that they should every way excell their competitors that went before them, both in device, daintiness of apparel, and above all in dancing, wherein they are held excellent, and esteemed for the properer men.
"But by what ill planet it fell out I know not, they came home as they went, without doing anything: the reason whereof I cannot yet learn thoroughly, but only that the hall was so full that it was not possible to avoid³ it, or make room for them; besides that, most of the ladies were in the galleries to see them land, and could not get in. But the worst of all was that the king was so wearied and sleepy with sitting almost two whole nights before, that he had no edge to it, whereupon Sir Francis Bacon adventured to entreat of His Majesty that by this difference he would not, as it were, bury them quick; and I hear the King should answer that then they must bury him quick, for he could last no longer; but withal gave them very good words and appointed them to come again on Saturday.
But the grace of their Masque is quite gone, when their apparel hath been already showed, and their devices vented, so that how it will fall out, God knows, for they are much discouraged and out of countenance. Their devices, however, went much beyond the mere exhibition of themselves and their apparel, and there was novelty
3. Clear it. Francis Bacon and his Secret Society by Mrs. Henry Pott., pp. 133-134.
enough behind the curtain to make a sufficient entertainment by itself, without the water business for overture."
Beaumont is said to have written this Masque, but since the Lord Chamberlain refers to Francis Bacon as being the chief contriver, and when the Masque was printed the dedication began with an acknowledgement that Bacon, with the gentleman of Gray's Inn and the Inner Temple had "spared no pain nor travail in the setting forth, ordering, and furnishing" of this Masque", it becomes fairly obvious that he must have been a good producer and stage-manager. The following reference in the dedication — "And you, Sir Francis Bacon, especially, did by your countenances and loving affections advance it" — points to his dramatic skills being recognised and followed with enthusiasm, trust, and goodwill.
The next year, 1613, when Bacon was created Attorney-General, we hear of Francis once again preparing a Masque to honour another marriage — this time the betrothed couple were the Earl of Somerset and Lady Essex. The bride's father was the Earl of Suffolk, then the Lord Chamberlain. Mrs. Pott in her book 'Francis Bacon and His Secret Society' explains this masque as follows:
It was proposed that during the week of festivities which celebrated this marriage the four Inns of Court (the Middle and Inner Temple, Gray's Inn and Lincoln's Inn) should join in getting up a masque, but they could not manage it. Although Ben Jonson and Shakespeare were at the height of their fame we find it was Francis Bacon, the Attorney-General, who, once more was called upon to supply their dramatic deficiencies. Surely this is another indication of his knowledge and aptitude, his versatility, practicality and artistry? It appears that Bacon considered that he owed Somerset some complimentary offering, because Somerset claimed (though Bacon doubted it) to have used his influence with the King to secure Bacon's promotion. While all the world were making presents — one of plate, another of furniture, a third of horses, a fourth of gold — he chose a Masque for which an accident supplied him with an excellent opportunity. When the united efforts of the four inns of court failed to produce the required entertainment Bacon offered, on the part
4. Advance to completion. Bloomsbury Dictionary of Word Origins, J. Ayo, p.244
5. Behaviour, Ibid., p.140.
of Gray's Inn, to supply the place of it by a masque of their own. We learn from a letter of the Lord Chamberlain that Sir Francis Bacon prepared a masque to honour this marriage, which will stand him in above £2,000.
The nature of the obligations considered, as Mrs. Potts explains: "there was judgement as well as magnificence in the choice of retribution. The obligation being for assistance in obtaining an office, to repay it by any present which could be turned into money would have been objectionable, as tending to countenance the great abuse of the times (from which Bacon stands clear) — the sale of offices for money. There was no such objection to a masque. As a compliment it was splendid, according to the taste and magnificence of the times; costly to the giver, not negotiable to the receiver; valuable as a compliment but as nothing else. Also it conferred great distinction upon Gray's Inn."4
The masque in question was called 'The Masque of Flowers'. It was published shortly after its performance with a dedication to Bacon as "'the principal and in effect the only person that doth encourage and warrant the gentlemen to show their good affection in a time of such magnificence; . . . wherein you have made a notable demonstration thereof in the lighter and less serious kind, by this, that one Inn of Court by itself, in time of a vacation, and in the space of three weeks, could perform that which hath been performed; which could not have been done but that every man's exceeding love and respect to you gave him wings to overtake time, which is the swiftest of things.'"5
Usually a group of people were engaged in the presentation of Masques, and honours were not normally given to one person because teamwork was essential. It is evident from this dedication that Bacon's personality was such that he engendered love and respect among his colleagues, and galvanised them into continued action. When we remember that a masque consisted of a fusion of many of the arts with Dancing, Singing, Ceremony, and Spectacle, Disguising and the Spoken Word, the fact that this masque was created in the
6. Francis Bacon and His Secret Society. Mrs. Henry Pott. pp. 133-134.
7. Francis Bacon and His Secret Society — Mrs. Henry Pott. p. 137.
space of three weeks, is a clear indication that Bacon knew exactly what he was about, and that he achieved team work.
Indeed we only have to read Bacon's Essay 'Of Masques and Triumphs' (especially after a study of the history of Masques) to realise that he must have had much personal experience in their preparation and production. He discusses with great accuracy therein technical details such as gestures, costume design, apparatus for scenery, colours that show best by candlelights, the placings of the singers, and the fitness of the music. Different ways of introducing variety concerned him. That he was very practical as well as imaginative is therefore evident.
In Bacon's essay 'Of Masques and Triumphs' he concludes with a brief reference to jousts, tourneys, and barriers, with these words:
"For jousts, and tourneys, and barriers, the glories of them are chiefly in the chariots, wherein the challengers make their entry; especially if they be drawn with strange beasts: as lions, bears, camels, and the like; or in the devices of their entrance, or in the bravery of their liveries, or in the goodly furniture of their horses and armour. But enough of these toys."8
Jousting and tilting were favourite pastimes with the nobility, and at the accession of Queen Elizabeth pageantry was introduced. They were often dramatised within an allegorical frame and became known as Accession Tilts.
Like Masques these were often spectacles on a lavish scale and in both, dramatic speeches were required. The main differences between the two genres, so it would seem, were that a Masque was performed indoors with music and dancing as an essential part of it, whereas the main feature of a tilt, apart from dramatic speeches spoken by characters in the device was a combat by two armed men on horseback which took place out of doors in the tiltyard, during daylight hours.
Sir Philip Sydney, and Sir Fulke Greville are known to have been champions of the Tiltyard and together with the Earl of Arundel — all friends of Francis Bacon — took part in Tournaments and triumphs.
In 1592 and again in 1595 Francis wrote, for the benefit of the Earl
8. Essay 'Of Masques and triumphs', Francis Bacon.
of Essex, the dramatic speeches for two Accession Tilts organised by the latter, and sometimes referred to as the 'Conferance of Pleasure' and the 'Philautia Device' respectively. In 1597 he again wrote speeches for another tilt.
Jousts, tourneys, and barriers were slightly differing though related forms of entertainments for celebrating special events, where festivities were required to take place on subsequent days, spanning perhaps a whole week.
As I have already mentioned the main feature of these was a fight on horseback between two armed combatants, or challengers.
Tourney was derived from the Latin word tornus, signifying combatants turning or wheeling round to face each other for battle. Barriers indicated the combatants coming toward a bar or rail, or partition. This term was also used by lawyers, signifying a partition separating qualified versus unqualified lawyers in hall who were 'called to the bar'.
From what Francis Bacon wrote at the conclusion of his essay 'Of Masques and Triumphs' and how he refrained from elaborating upon jousts and triumphs in detail but tossed the subject aside with the ejaculation 'but enough of such toys' suggests that armed combats did not interest him. Nevertheless, although tilts and jousts being a martial type of amusement, did not appeal to Francis Bacon himself they did so to the Earl of Essex who, from time to time liked to present to the Queen and her retinue a show of this kind. However, the contrivance of a Device and the writing of dramatic speeches, which preceded a joust or tilt, was, it would seem, not one of the talents of the Earl of Essex whereas it was one of Bacon's accomplishments. Therefore we can assume that Francis was asked by Robert Devereux to provide the first part of the spectacle to enable him to make such occasional offerings to the Queen, and obviously Francis acceded to the requests.
Thus we can perceive that this must have been the reason for Francis Bacon's involvement in this particular pastime which called for dramatic speeches, even though he did not much care for this form of recreation and festivity.
Similarly, it was because Bacon was either asked or he offered to give a helping hand on more than one occasion, and a to a lesser or greater degree, in the contrivance of masques at Gray's Inn, initially as a student and thereafter as a respected Member of the Inn, that Francis participated in that type of dramatic entertainment. However, Masques and Revels
were to his taste, even though these also were designed mainly for recreational purposes for the nobility and aristocracy during times of celebration.
This brings to a close information regarding the subject of Masques and Revels, held at Gray's Inn (and some of the other Inns of Court), and of Devices, culminating in Jousts and Triumphs which took place in tiltyards.
For the sake of avoiding confusion I have purposely discussed these differing types of dramatic entertainment separately. Now I am going to turn my attention to the other category of dramatic art with which Francis Bacon was intimately concerned and which occupied so much of this thought and time from 1594 until 1623. But before embarking upon this subject I must state that his participation in all these dramatic pursuits were, in fact, (as all Baconians know) being pursued concurrently, but not for the same reasons. Initially my next remarks may seem like a digression, but my comments will turn out to be relevant, I can assure you.
It must be remembered that both Sir Nicholas and Lady Ann Bacon were keen classical scholars, proficient in Latin and Greek, possessing a good library in their home at Gorhambury, and they encouraged Anthony and Francis to read books. Moreover the latter had access to other libraries and sources of information not available to most people. Francis, in particular, was an avid reader, and made extensive researches into all manner of subjects. He also imbibed specialised knowledges from two of his tutors, Sir Amyas Paulet and Dr John Dee.
Branches of learning which were of particular interest to Francis were etymology and languages, occult philosophy, human philosophy, natural philosophy, metaphysics, Greek mythology, classical drama, morality plays, symbolism, and the mystical tradition.
These studies into the wisdom of the ancients made a great impact upon this precocious teenager. So too did his two and a half year stay in France, October 1576—March 1579 under the guardianship of Sir Amyas Paulet, who was not only Bacon's French tutor, but also had been created English Ambassador Elect by Queen Elizabeth, giving him the privilege of visiting the French Court. There the young Francis Bacon had the opportunity, amongst other things, of meeting Pierre de Ronsard, of becoming acquainted with the other members of 'La Pléiade', and learning about the French Renaissance and enjoying the beauty of their enriched language. Francis also witnessed French theatricals.
Bacon’s inherent philosophical turn of mind; his Christian faith; his awareness of Humanistic thought and the ideals of the Reformation; together with the inspiration gained from Sir Nicholas Bacon’s example, and ideas for the advancement of learning; his deep love of and reverence for Nature’s beauties, bounties, and processes; his phenomenal knowledges, imbibed from the vast quantity of books he had read in a number of languages; from life’s experiences during his journeys in England and on the Continent; his keen and perceptive observation of people in all walks of life from the humblest to the highest; his participation in classical drama at college; and in Masques at Gray’s Inn; these were all factors of influence which acted as a springboard for the burning desire he had, to create a different form of dramatic entertainment which would not only give to him greater scope for his creativity, but also yield a far higher purpose than that of mere celebration of a special event or only for amusement. His imagination was also fired in the direction of trying to bring about and produce an English Renaissance.
This new literary project would, he realised, need on the one hand to be well organised, and on the other hand would require helpers to give him practical assistance in its outworking. Everything would have to be unfolded in stages, systematically and with perseverance. Not surprisingly Anthony Bacon was in agreement with Francis Bacon and was a willing collaborator. Also Philip Sidney and a few other friends and poets.
This small band of literary colleagues, whom Francis referred to as his “‘good pens who forsake me not,’” recognised and acknowledged his superior abilities and they regarded Francis as the initiator and director of a vast scheme which would gradually enlarge the scope of culture and learning. They viewed themselves as members of the “‘choir of the Muses’” with Francis Bacon as their much loved and inspired leader.
Just as the member of ‘La Pléiade’ in France had laboured for years to enlarge and beautify their language, as a needful requisite for the French Renaissance, so too did Fancis Bacon and his ‘good pens’ resolve to follow their example and amplify the English tongue to an even greater extent, as an important first stage in the creation of an English Renaissance.
When this task was accomplished, Francis and his collaborators incorporated this new and flexible vocabulary into all manner of writings in prose, poetry and drama. The task of providing English translations of many of the classics was also undertaken. But English dramatic works
had to be done in secret and printed either anonymously, or using initials, or pen-names.
Although poets and men of letters were held in high esteem in France by all people, including the Kings of France, and Marguerite of Navarre attracted poets and writers to her Court, this policy had not been adopted by the English monarchs, as you know. How frustrating and bewildering it must have been to Francis Bacon and his confederates to be confronted by the huge contrast of opinions and conventions which still existed in 1576, the year he went to France and experienced the marvel of the labours of 'La Pléiade' and their beautiful new language, and to find that this had evoked the admiration of their countrymen. Yet in England it was considered discreditable for a gentleman to be amorous of any learned art, and many writers felt obliged not to use their own names.
The second half of the decade of the 1570's was indeed an important period for Francis Bacon, for these years marked the conception and commencement of a major aspect of his life's work.
Amongst my researches I have been reading some of Peter Dawkins' writings, especially his "Commentary on Bacon's Great Instauration". I have found some of his ideas so thought-provoking that I would like to share them with you. He maintains that having been inspired with the idea, which Bacon attributed to divine revelation, of creating a scheme for a total regeneration of the Arts and Sciences, which, as you know, was eventually referred to as The Great Instauration, Francis started in earnest to put it into operation, after he had left Cambridge University.
Because he believed this plan for Enlightenment was divinely revealed, as a mark of respect to his Creator and in imitation of divine Law Francis Bacon decided to make the scheme of the Great Instauration fall into six parts, echoing the Six Days Work of Creation. In addition to this, throughout the scheme runs a triple principle, again imitating the Law of the Holy Trinity.
Thus his aim was to erect, metaphorically, a true Pyramid of Philosophy which could then find expression in action. It was his conviction that, just as the stone pyramids of ancient Egypt were three-faced or aspected, so should his intellectual Pyramid of Philosophy deal with the three basic knowledges or aspects of Philosophy: Divine Philosophy, Natural Philosophy, Human Philosophy. As he later wrote in the 1605 edition of his Advancement of Learning:—
"‘In Philosophy, the contemplations of man do either penetrate unto God,—or are circumferred to nature—or are reflected or reverted upon himself. Out of which several enquiries there do arise three knowledges, divine philosophy, natural philosophy and human philosophy or humanity. For all things are marked and stamped with this triple character of the power of God, the difference of nature, and the use of man.’"
The Pyramid of Philosophy, besides being three-faced, was also planned to be three-tiered: the base or first stage was to be History (i.e. well ordered and digested experience), the second or middle stage was to be physics, and the third and final stage that of metaphysics. As Francis explained:
"For knowledge are as pyramids, whereof History is the basis. So of Natural Philosophy the basis is Natural History; the stage next the basis is Physique; the stage next the vertical point is Metaphysique. As for the vertical point (Opus quod operatur Deus a principio usque ad finem) the Summary Law of Nature, we do not know whether man's enquiry can attain unto it . . ."
Peter Dawkins goes on to explain that the second and third stages of the Pyramid of Philosophy deal with the knowledges relating to physical and metaphysical laws respectively. The fourth part is concerned with the presentation of the History to the mind using the imaginative arts, that is to say, combining a structure of drama and poetry, and referred to as Tables of Invention or Discovery.
From many years of study as well as from intuitive perception and reasoning, some of the past and current Baconian researchers have put two and two together and cognized and 'discovered' that the Tables of Invention and Discovery were none other than the poetic dramas, brought forth anonymously at first but later under the pseudonym of William Shakespeare, and given to the world as examples of these Tables of Invention, relating to Human and Divine Philosophy.
Peter Dawkins is also of the opinion that
"‘Francis Bacon perceived that divine Emotion (or Love) was the
9. Advancement of Learning. 1605. Francis Bacon.
10. Advancement of Learning. Francis Bacon.
fundamental life force or motivating power of the universe, which forms and animates all things. He therefore considered that the study of emotion, in all its manifestations, was (or should be) the primary study of man, so as to discover and learn the causes of all motions and things, and so eventually to discover the supreme cause, the ultimate truth, divine Love."11
If Peter Dawkins' theory is correct (and I think it is a plausible one), and if Francis Bacon did set out to write in detail about the study of human emotion, in all its manifestations, and record a history of human natures, one can understand why he decided to present this History as a series of plays — comedies, histories, tragedies. He obviously realised that if each play was carefully written, possessing the correct structure for such a purpose, it would be possible to show people the causes of human attitudes, motivations and behaviour.
Thus in the Shakespeare plays he planned to record a collection of observations of human emotions, thoughts and actions. Francis Bacon gave hints about his intentions in his De Augmentis Scientiarum.
"Let a full and careful treatise be constructed. Not, however, that I would have their characters presented in ethics (as we find them in history, or poetry, or even in common discourse) in the shape of complete individual portraits, but rather the several features and simple lineaments of which they are composed, and by the various combinations and arrangements of which all characters whatever are made up, showing how many, and of what nature these are, and how connected and subordinated one to another; that so we may have a scientific and accurate dissection of minds and characters, and the secret dispositions or particular men may be revealed; and that from a knowledge thereof better rules may be framed for the treatment of the mind. And not only should the characters of dispositions which are impressed by nature be received into this treatise, but those also which are imposed upon the mind by sex, by age, by region, by health and sickness, by beauty and deformity and the like; and again, those which are caused by fortune, as
11. Bacon, Shakespeare, and Fra Christian Rose Cross. p.27. Peter Dawkins.
12. The knowledge touching the affections and perturbations which are the diseases of the mind.
sovereignty, nobility, obscure birth, riches, want, magistracy, privateness, prosperity, adversity and the like." . . .
"But to speak the truth the poets and writers of history are the best doctors of this knowledge,\textsuperscript{12} where we may find painted forth with great life and dissected, how affections are kindled and excited, and how pacified and restrained, and how again contained from act and further degree; how they disclose themselves, though repressed and concealed; how they work; how they vary; how they are enwrapped one within another; how they fight and encounter one with another; and many more particulars of this kind;" . . .\textsuperscript{13}
Here is another hint regarding Bacon's intentions relating to poetic drama.
In his "Distributio Operis" he writes thus:
"I do not speak of these precepts and rules by way of illustration (for of these I have given plenty in the second part of the work); but I mean actual types and models, by which the entire process of the mind and the whole fabric and order of invention from the beginning to the end in certain subjects, and those various and remarkable, should be set as it were, before the eyes. . . . To examples of this kind, being in fact, nothing more than an application of the second part in detail, and at large, the fourth part of the work is devoted."\textsuperscript{13}
If Bacon had envisaged constructing plays to act as mirrors reflecting to the spectators the inner processes of the minds and emotions of the characters, then we can perceive the reasons for writing this new type of psychological drama as well as for choosing the novel idea of introducing soliloquies into the structure of each play. The employment of soliloquies was one of the means he used to reveal the inner processes of the thoughts and motivations of the character who was speaking the soliloquy. Of course, the latter were also expressed in the dialogue in conversation between two or more of the characters.
The last two quotations alone show what a master Bacon became in
\textsuperscript{13} De Augmentis Scientiarum. Francis Bacon.
\textsuperscript{13} Distributio Operis. Francis Bacon.
the art of veiling truth, and wrapping up his meaning and yet, at the same time, of hinting at it, with carefully selected words and phrases, offered as signposts to discerning seekers-after-truth, who are able on the one hand to use reason, logic and analysis, and on the other hand to exercise their powers of intuitive perception to guide them on the treasure trail. It was these two extracts, as well as one or two other quotations from Francis Bacon's writings, together with the introduction of so many legal words and phrases in the Plays, which caused the Baconian pioneers, as well as current researchers, to arrive at the conclusion that the psychological dramas (which had been ascribed to William Shakespeare) must surely have constituted the supposedly missing fourth part of Bacon's Great Instauration, which he referred to as "'a full and careful treatise", in which should be presented in detail, examples of the internal workings of the minds and affections-of characters.
In my opinion two of the 'certain subjects' to which he referred in the last quotation, would seem to have been the Wisdom Teachings of past ages, and what we now call psychology. He makes this clear by stating that this treatise should 'set, as it were, before the eyes the entire process of the mind, from the beginning to the end'. It is obvious that he recognised the potential power of human thoughts and feelings, and how these were the motivating impulses to actions, good or bad. He would no doubt have remembered that above the entrance to the Greek and Egyptian temples were the words 'Man, Know Thyself, and thou shalt know God and the Universe.'
These mystery teachings of the ageless wisdom revealed to the student, in gradual stages, knowledge concerning the sevenfold constitution of man: of his earthly, reasoning, rational, conscious mind, and of his spiritual, intuitive, super-conscious mind; of his soul and psyche; and the control and development of these faculties. It was a long and arduous training but led ultimately to the acquisition of wisdom, enlightenment, peace of mind, and kind and virtuous behaviour.
Undoubtedly it would have been the ancient wisdom teachings which would have brought into sharp focus Bacon's recognition of the fact that a man was a composite being comprising body, soul and spirit, possessing tangible and mortal, as well as intangible and divine vehicle of expression; negative and positive attributes and qualities; and personalities which could be trained and disciplined, and thus changed from manifesting only the baser, unrefined and often unpleasing human natures and responses
towards expressing more pleasant, virtuous, and divine reactions and attributes.
The inter-related concepts of the evolution of consciousness, and of the metamorphosis of human natures, as the aspirant treads the Path of Attainment under invisible direction, was regarded by Francis Bacon as being of supreme importance. These subjects often constituted the underlying theme of a number of the Shakespeare Plays.
Beryl Pogson and Mabel Sennett, both past, staunch Members of the Francis Bacon Society, who studied psychology and the mystical tradition, maintained that many of the Plays were allegorical and symbolic at one level. In 1950 Beryl Pogson wrote a book entitled "In The East my Pleasure Lies" presenting esoteric interpretations of nine of the Shakespeare dramas.
In 1949 the Francis Bacon Society published a book called "His Erring Pilgrimage" by Mabel Sennett. This provided an in-depth esoteric interpretation of the play 'As You Like It' as portraying the development of human consciousness. She regards the characters as symbolising different aspects of human consciousness, some characters expressing the level of the ordinary human self, referred to as the Natural Man who is unawakened spiritually, and others who are more advanced, having disciplined their human nature and developed their mind and will, to become the Regenerate Being, the spiritual man.
Act by act through the play we are shown the gradual transformation of the self as it grows and matures psychologically, or spiritually. Some characters really are transformed and changed for the better, while others do not have the capacity to grow in wisdom so they remain the same all through the play.
When I myself went to see a production of 'As You Like It' performed by Rudolf Steiner-trained actors and actresses who, of course do study levels of consciousness, I noted with interest how well they brought out this viewpoint.
Alongside the many philosophical discussions which are deftly woven into the dialogue, especially between characters such as Jacques, Orlando, Rosalind, Celia and Touchstone, where there are references to the tenets
14. 'In the East my Pleasure Lies' has now been reprinted and can be bought from Watkins bookshop in London.
15. 'His Erring Pilgrimage', Mabel Sennett.
of the Path of Attainment, there is also another level of wisdom and teaching which is much easier to comprehend. The subject of ethics and morals interested Bacon and apart from discussing them in his book of moral Essays there are many incidents in the Plays which reflect to the audience clearly recognised examples of good or bad behaviour in human beings, moral instruction given but in a way which was simple to understand, often portraying the kind of reactions which many people have. But although the subject of morality and ethics is present it is never overdone, the dramatist always remembered that the function of the play was to entertain as well as to instruct, and to cater for different levels of comprehension. The spectator was given the choice to aspire to deeper perception, or to shun it, as he pleased.
Of course being a philosopher as well as a dramatist, coupled with the knowledge of the esoteric tradition and the potential it gave for widening man's understanding and of gaining new insights, I think that he hoped that many people, current and future generations, would accept and make use of the wisdom offered. He perceived the possibility of inculcating a gradual change of attitude and better behaviour in men and women in all walks of life and that the new psychological dramas might be a helpful way of dropping new seeds of thought via the technique of reflection, and of showing up negative traits in human beings in contrast to positive, charitable and kindly expressions of thought and feeling in the living of life, aiming to inculcate the desire to develop virtue, and demonstrating too the need to bridle and control sensuous instincts, negative impulses and passions.
What daring innovations his new concepts regarding the use of drama must have introduced in Elizabethan times. These Shakespeare Plays certainly did provide a new type of dramatic entertainment which had most unusual and startling strands woven into the weft and warp of the fabric of each drama.
Reflecting on the history of drama, we can recognise that the introduction of morals and ethics into the dialogue and plot was reminiscent of morality plays of former times, but with additions which were more subtle, and introducing teachings expounded by pre-Christian teachers such as Pythagoras and other Sages.
In the Middle Ages when morality plays were allowed to be acted in churches, moral instruction was made obvious to the spectators because each character was given the name of the passion he was portraying.
The action and dialogue revealed to the audience the temptations of the soul of every man during his pilgrimage through life.
It portrayed visibly an interior problem as to whether the soul (which the leading character in the play represented) was strong enough morally to overcome the gravitational pull of earthiness and expression of the baser negative aspects of his human personality, or whether the soul was not able to be victorious, either in resisting temptations or in acknowledging his own shortcomings.
Positive or negative attributes were easily recognised by the spectators because each character was given the name of a virtue or a strong passion, such as Love, Goodness, Lust, Pride etc. But in the Shakespeare plays there was far greater subtlety. Characters were given more ordinary names, e.g. Orlando, Adam, Oliver, Audrey. Shakespeare had a talent for character delineation. I agree with William Smedley who, on page 186 of his book "The Mystery of Francis Bacon" wrote:
If Shakespeare deserves our admiration for his characters he is equally deserving of it for his exhibition of passion, taking this word in its widest signification, as including every mental condition, every tone from indifference or familiar mirth to the wildest rage and despair.
... He paints, in a most inimitable manner, the gradual progress from the first origin. He gives, as Lessing says, 'a living picture of all the most minute and secret artifices by which a feeling steals into our souls; of all the imperceptible advantages which it there gains, of all the stratagems by which every other passion is made subservient to it, till it becomes the sole tyrant of our desires and our aversions.'
Francis Bacon recognised that although he sought to 'cleanse the foul body of the infected world' showing the way of the soul from darkness to light, from inner conflict, through illusions and temptations of the darker side to harmony and joy and the light, he must convey his teaching subtly without arousing antagonism amongst the public of his times to whom the subject of morals and of self-discipline would have no appeal.
Leaving the subject of character delineation and returning briefly to the passages of philosophising which formed another feature, it is
16. Page 186, 'The Mystery of Francis Bacon', William Smedley.
interesting to note that although different aspects of human philosophy were discussed more often, there are a number of references to nature and natural philosophy, and also to divine philosophy. The latter was alluded to sometimes in the actual theme of the play which often centres upon a cosmic law or principle. The play 'Romeo and Juliet' displays examples of the cosmic law of opposites such as love and hate, strife and friendship, life and death. So many of the plays touch on great spiritual truths, and show especially the need to tread the middle way so as to harmonise opposing opinions and drastic and harmful actions. In the comedies especially the opening act usually introduces the disagreement, misunderstanding or other negative half of a polarity, while in the final act one sees the unravelling of complexities. So where there had been disagreement or antagonism, or unfair rejection, banishment, separation, by the end of the play the misunderstandings have become understood and resolved, and harmony, reconciliation and reunion have taken their place. And this is what Francis would refer to as divine philosophy, which is something quite different to orthodox religion. The three philosophies are a closely-linked triad and Bacon realised this truth.
As most of the plays incorporate poetic passages I think it is significant and interesting to find a few pages on the subject of poesy in *The Advancement and Proficience of Learning* under Francis Bacon's own name, and to note that this topic also embraced three aspects. Thus he wrote about narrative Poesy, Dramatical or Representative Poesy, and also what he termed Parabolic, Allegoric and Allusive Poesy. These references to the three different categories of poesy serve as a broad hint, taking our minds at once to the Shakespeare Plays which do indeed possess these different types of poesy in their structure.
Francis regarded poetry as the science and art of the imagination and perceived that poetry (or poesy as he called it) should have a vital place in his scheme for presenting human philosophy in the form of the imaginative arts, which could simultaneously appeal and speak to the feelings and heart of man's soul and intuitive mind as well as to the outer, intellectual mind.
This concept of playing upon man's mind and feelings is subtly hinted at by Bacon in another passage in book 2 of his *De Augmentis Scientarium* relating to the action of the theatre and the highest use of drama when he wrote:—
Dramatic poesy, which has the theatre for its world, would be of excellent use if well directed. For the stage is capable of no small influence both of discipline and of corruption. Now of corruption in this kind we have enough, but the discipline has in our time been plainly neglected. The action of the theatre, though modern states esteem it but ludicrous unless it be satirical and biting, was carefully watched by the Ancients that it might improve mankind in virtue; and indeed many wise men and great philosophers have thought it to the mind as the bow to the fiddle'.\textsuperscript{17}
Via a musical analogy Francis inferred that just as the bow was employed to play upon the fiddle to create musical harmony, so could the action of the theatre have a similar function and influence upon the spectators, as they watched and listened to a well ordered and inter-related stage play, possessing carefully placed moral and spiritual instruction concealed within the play. If some of the audience were able to finally draw conclusions and perceive flashes of wisdom and truth from what had been spoken and enacted whilst they were being entertained, then the highest use of drama would be achieved.
It is significant that the word ‘discipline’ was used in this extract since the latter had a Latin derivation meaning ‘instruction’ and this he remarked ‘had been plainly neglected’.
However instruction would need to be veiled and therefore Bacon followed the example of the Ancients and incorporated into the play parables, analogies, allusions and symbology, as part of his system of teaching. Hence the introduction of parabolic and allegoric poesy in conjunction with narrative poesy and dramatic poesy.
From the wisdom of the ancient sages Francis had learnt that the triad of symbology, imagery and poetry, constituting a three-fold sacred language as they did, were important requisites for raising the minds and souls of the audience to a higher plane of consciousness, and also for heightening the intensity of feeling and of dramatic situations in important scenes and speeches.
There is no doubt that Francis Bacon from an early stage and all through his life was no low grade poet and dramatist, and no ordinary philosopher, and that the fusion of his inherent talents, under the aspiration of his
\textsuperscript{17} De Augmentis Scientiarum, Book II, Chapter 13. Francis Bacon.
Muse, produced a genius who was capable in due time of creating such dramatic masterpieces as the poetic psychological plays ascribed to his symbolic pen-name William Shakespeare.
Bacon never regarded these plays as mere box-office attractions. He viewed their creation from the angle of a philosopher and also as an ethical and spiritual teacher would, that of providing the great potential for education of the ancient wisdom in a disguised and acceptable way. This could be achieved by inserting in the structure of each play a didactic stratum of parable, allegory, analogy, and spiritual truth, concealed under the outer garb of easily recognised entertainment such as a comedy, or a tragedy, or a history play.
From all that has been discussed I hope that the following comparisons have emerged:
The cycle of psychological stage plays known to the world as the Shakespeare plays, were totally different types of dramatic entertainment to the more aristocratic genre called Masques and Revels, the latter being performed in the legal Inns of Court as well as in palaces and stately homes and whose performers included the nobility, while the former were acted mainly in theatres, or in courtyards of certain Inns such as the Bull Inn, in Bishopsgate Street, and whose actors were largely drawn from the uncultured classes, the commoners. Also the purpose for Bacon’s participation in each category was for different reasons.
His involvement in Masques and Revels was for current use and outwardly for diplomatic and social purposes, as a customary form of amusement and celebration.
In contrast to this, Bacon’s preoccupation with the construction of psychological stage plays, which took up the biggest proportion of his time had of course a two-fold intention, partly as a gift to posterity of Comedies, Histories, and Tragedies in the great Folio edition printed in 1623 under his symbolic pen-name and also as an important, but secret, fourth part of Bacon’s ‘Great Instauration’ which was introduced to the world, as we all know, under his own name.
Surely, it must have given Francis and his collaborators a sense of satisfaction that they had all managed to make worthwhile contributions to the creation of an English Renaissance, Bacon with his own acknowledged and pseudonymous writings, and they with their own individual work, as well as their group endeavour of enlarging and enriching the English language, which had been widely circulated.
especially in the Shakespeare Plays, together with the translations into English of many of the classics of antiquity.
Perhaps the success of the anonymity and pseudonymity of much of this labour had also made them smile, and gain pleasure and a sense of reward for their willingness to work so hard without recognition. Francis Bacon and his 'good-pens' certainly did make a valuable gift to the culture of England, and made their mark on the literature of their country.
Bacon must also have felt pleased to recall that with the creation of the Shakespeare Plays he had really achieved his goal of inventing a new form of dramatic art, in which he had found a way of incorporating a unique blend of moral and spiritual wisdom as well as a detailed study of human natures and the motivations to actions, woven together in two triads of philosophies and poesies, coupled with wonderful entertainment, for current and future generations.
All in all I think it is true to say that Francis Bacon's participation in all these dramatic entertainments constituted the most creative, rewarding, and enjoyable aspect of his whole life's activities.
... I invite you to examine some of the facsimiles of paper-marks shown herewith. They are representative of thousands and thousands of similar character, which any seeker may find for himself. It is generally assumed by orthodox bibliographers that paper-marks were the trade signs of paper-makers, but an investigation of this theory proves it to bristle with technical difficulties which render it untenable. Driven into the corner by the logic of facts which I need not here discuss, but shall be happy to do should anyone care to challenge my conclusions. Orthodoxy has fatuously suggested that the "curious shapes" which are assumed by these mysterious marks were due to the workmen having varied them for their own amusement. As a matter of fact, the great majority of paper-marks in mediaeval literature are not trade signs at all, but symbols of the many secret and invisible associations which were at work sowing seeds of sweetness and light, and risking their existence in the attempt to shake off the nightmare of ecclesiasticism. For the purpose of illustration I have selected merely two or three of the hundreds of philosophical symbols which prove the reality and the extent of ethical Alchemy. Just as the fossil is a positive proof of former life, so these curious marks in paper seem to me tangible evidence of the energy and virility of the "mystical trash" condemned by Hallam. The first object that I have chosen to point this suggestion is the paper-mark of a jug or pot. There are two ideas underlying these figures, both springing from the Legend of the Holy Grail. "We cannot be sure," says Mr. Alfred Nutt, "how this or that writer conceived the story as a whole, or in what sense he figured the Grail to himself." It is true we cannot define the exact meanings which were attached by particular writers, but the forms in which these writers figured the Grail are depicted in infinite variety in the paper-marks of the period. Of these many are necessarily crude, but you must bear in mind that they were impressed upon wet paper pulp by being designed in wire and then stitched to the paper-maker's tray or mould.
Most of us are familiar with the Legend of the Grail in the form which
was grafted on to it by Christian writers, but as a symbol it is very much older than Christianity. The Rev. Baring Gould describes it as "the mysterious relic of a past heathen rite," and Mr. Alfred Nutt terms it "a mythical talisman of increase and plenty."
The Rev. Baring Gould quotes an old British poet as his authority for the claim that the St. Grail "inspires poetic genius, gives wisdom, discovers the knowledge of futurity, the mysteries of the world, the whole treasure of human sciences. That this vessel of the liquor of wisdom," continues Mr. Gould, "held a prominent place in British mythology is certain from the allusions made to it by the bards." "Taliesin, in the description of his initiation into the mysteries of the basin, cries out. 'I have lost my speech,' because on all who had been admitted to the privileges of full membership secrecy was imposed. This initiation (continues Mr. Gould) was regarded as a new birth, and those who had once become members were regarded as elect, regenerate, separate from the rest of mankind, who lay in darkness and ignorance."
Jacob Behme leads off in Chapter I. of *De Mysterio Magno* with the words:—"If we would understand what the New Birth is and how it is brought to pass, then we must first know what Man is."
Now Hitchcock, who derived his opinions from the study of upwards of 200 works on Alchemy, sums up his conclusions as to their *real* object by saying he could liken it to nothing better expressive than the experience known in religion as THE NEW BIRTH. He adds: "There are many signs in Alchemical volumes of a secret society in which possibly the language used was conventionally determined. I have at times thought that some members of the Masonic fraternity might have found the secret language of the Alchemists a convenient mode of publishing, or rather circulating, among the initiated doctrines which they had taken an oath not to speak directly or to make known except to a brother."
In these independent extracts we thus find correlated the ideas of the exhaustless vase of wisdom, secrecy, and the New Birth. It is my conviction that the vast movement which, when it appeared above the surface of History was known, or, at any rate, is to-day known, as the RENAISSANCE or New Birth, was merely the effect of which the secret and unrecognised efforts of the Alchemists and other kindred reformers were the direct cause.
You will find a great deal of information on the Holy Grail in Mr. Alfred Nutt's book, and I would also refer you to Mrs. Cooper Oakley's *Traces of a Hidden Tradition in Masonry and Mediaeval Mysticism*. Mrs.
Oakley says: "Gathered round the Holy Grail are the Knights and Guardians of the Grail Kingdom, led by Titurel the mystic King, to whom is entrusted the Holy teaching. Then later we find the Knights Templar taking up the sacred mission. But everywhere and always is there the inner doctrine for the few who seek the Holy Grail, for it is invisible to all but those who form the inner circle. The chief function of the Grail Kingdom was to supply a constant type of a divinely governed Society, a Society ruled from the inner and spiritual planes, and to train in the Kingly art of ruling leaders for such communities as needed them. It was destined to be a practical civilising power as well as a Palace spiritual; not a passive force only, but active and powerful for the suppression of all evil on earth."
Who that is at all familiar with the works of Francis Bacon can doubt that he was a leader among the many very perfect Grail Knights of his period?
Mr. Alfred Nutt tells us that, "although caught up to very Heaven, though filled with the essence of Divinity, still the Grail retains the material characteristics of an increase and plenty talisman." Mrs. Cooper Oakley, who approaches her subject from a totally different aspect, sums it up as her conclusion that in the Grail myth "we are face to face with a symbol of man; man who is the Temple of the Holy Spirit. The chalice or cup is but another way," says she, "of denoting the coats of skin, the veils or vestures which garment man on earth, robes woven by the Nature powers in which and through which the divine spark has to dwell, until in process of time the vestures or chalice become permeated through by the divine light within."
Now Hitchcock and other ethical interpreters of the Alchemists are convinced that it was MICROCOSMOS, the little world of Man, which was the real stone upon which they were experimenting and working to transmute into gold. In his Lives of the Alchemystical Philosophers, Mr. A. E. Waite quotes an anonymous writer who states that "the publication of the writings of Jacob Boehmer caused the Alchemists who were his contemporaries to fear that their art could not much longer remain a secret, and that the mystic vase in particular would be shortly revealed to all. This vase is the vas insigne electionis, namely, Man, who is the only all-containing subject, and who alone has need to be investigated for the eventual discovery of all." We thus see that in all probability the pot
water-marks which you have before you emblemise the very essence of Alchemy.
Before passing from these Grail water-marks I would invite you to consider the almost infinite variety with which they are ornamented. Time does not permit me to linger over the symbolism of these decorations, but I should just like to draw your attention to the handles arranged in the form of the double SS standing for Sanctus Spiritus. Also to the variety of initials which appear upon them. These, almost without doubt, are the first letters of the words of certain phrases. They form part of the mystic system of the Cabala known as "notaricon." By this system certain initials came to be perfectly well understood, conveying profound meanings. Rossetti mentions that Dante made frequent use of the method, and there are even today many relics of it among us. I might mention the expression AMEN, which is a composition word, and the Italian secret society known as the MAFIA, said to be so named from the initials of the sentence, MAZZINI AUTORIZZA FURTI INCENDI AVVELENAMENTI.
The remembrance of the paper-mark we have been discussing has lingered until the present day, and is the origin of the modern technical term "pott," used to denote a certain size of paper. The term foolscap, with which you are all familiar, is likewise the survival of an old paper-mark. The facsimiles which you have before you are typical representatives of it. You will notice the curious sort of pigtail, with a cross on the end. Some years ago I was told that this pigtail was the badge of a jester in the service of an ecclesiastic, but it is only recently that I have struck on what is, I think, probably the explanation. Everyone has heard of the Troubadours, but it is not generally realised that they were heretics under the ban of the Church and driven hither and thither by that relentless antagonist. Their mission, Aroux tell us, was to redress the wrongs of Rome, to take up the defence of the weak and oppressed. They were also represented and celebrated as the true soldiers of the Christ, the exponents of celestial chivalry, and the champions of the poor, attacking under all their forms the monstrous abuses of the Priesthood. It is said that great number of the higher classes became Troubadors, wandering from Court to Court and castle to castle, spreading the doctrine of the organisation for which they were acting as emissaries.
This uncanonical "Church of the Grail" as it has been called, was extraordinarily methodical and extended in its operations. It claimed a higher authority than the official Church of Christendom. Aroux tells
us that it had its Priests, Bishops, and Deacons, who wandered far and wide disguised under the hoods of Troubadours. It is said that the frightful persecutions which scattered the Templars were due to the belief that they were Knights of the secret Church of the Holy Grail. You will now see why these Troubadour-jester emblems are distinguished by the clerical badge, and you will appreciate that among the Troubadours were exponents of the same unseen movement to which some of the so-called Alchemists were undoubtedly allied.
Nearly all foolscap water-marks have pending from them a figure of Four, and three circles. The figure Four was held sacred by the Pythagoreans, being the perfect square. It was the emblem of Moral Justice and Divine Equity geometrically expressed. The ineffable Name of The Deity was expressed by this sacred number Four, which was regarded as a most binding and solemn oath by the ancient mystics. The three circles, I think, probably denote the three great principles of Alchemistic Philosophy — Sulphur, Mercury, and Salt, or in other words — Body, Soul, and Spirit.
It is said that Dante was a Troubadour, and that St. Francis of Assissi had been one. Aroux mentions many eminent names and includes among them King Richard of England. Some time about 150 years ago the Troubadour’s head disappeared from paper and in its place there appeared the design you have before you. This emblem has survived until the present day, and enshrines a world of romance and suffering. It is, I think, without doubt a counterfeit presentment of the Troubadour’s mysterious and perfect Lady, in other words the Virgin Sophia eulogised by Dante, Petrarch, and a host of poets, among them our Elizabethan Sonneteers.
To introduce this perfect, mysterious Lady to you, I cannot, I think do better than quote some passages from a letter written by that saint of rationalism — Giordano Bruno. He writes: “I am displeased with the bulk of mankind; I hate the vulgar rout; I despise the authority of the multitude and am enamoured with one particular lady. ’Tis for her that I am free in servitude, content in pain, rich in necessity, and alive in death. . . . Hence it is even for my passion for this beauty that, as being weary, I draw not back my feet from the difficult road, nor, as being lazy, hang down my hands from the work that is before me; I turn not my shoulders, as grown desperate, to the enemy that contends with me, nor, as dazzled, divert my eyes from the divine object. . . . ’Tis for the love of true Wisdom and by the studious admiration of this Mistress
that I fatigue, that I disquiet, that I torment myself."
This, ladies and gentlemen, was the spirit of the Grail which the Inquisition and all the power of the governments of Europe was engaged for many centuries in endeavouring to crush. The result of the suppression was that the Spirit of Truth was idolized and discussed under the protective veils of "Beatrice," "Laura," and a variety of other names to which the imperceptive have more or less vainly endeavoured to fit physical women. I chanced the other day on a coincidence with regard to Dante's "Beatrice" to which I am not aware that attention has hitherto been drawn. We are told in the Vita Nuova (I need hardly draw your attention to the significance of this Alchemistic title) that "Beatrice" was nine years of age when Dante first met her. He remarks, "Many times the number nine hath appeared among the preceding words whereby it appeareth that it is not without reason." He then says that he will assign the reason "why this number was so friendly to her," and explains that three being the root of nine Beatrice was accompanied by the number nine to give to understand that she was a nine, that is a miracle whose root is the wondrous Trinity alone. Then he gives us permission to speculate a little by adding: "Perchance a more subtle person might see in it a yet more subtle reason." A few weeks before reading this passage I happened to have pasted into my commonplace book the following newspaper cutting:
Sir William Huggins, at the dinner of the Maccabees, referred to the curious properties of the Hebrew word for Truth. It comes from a root signifying strength — that which could not be moved. The letters of the word are equivalent to the mysterious number nine. When multiplied that figure frequently gives figures so true to each that when added together they again prove the figure nine; thus twice 9 are 18, thrice 9 are 27, and so on.
In England this same spirit of Truth, or spirit of Nature, was invoked under the veils of Phoebe, Idea, Licia, Cynthia, Elizabeth, etc., and numerous sonnets were written in her honour. It think it unquestionable that much of the adulation which is supposed to have been lavished upon Queen Elizabeth by servile poets was never in reality intended for her at all, but for that more mystic Elizabeth of whom we catch a glimpse in Spenser.
BACONIANA
32
ALCHEMY AND THE HOLY GRAIL
33
BACONIANA
34
ALCHEMY AND THE HOLY GRAIL
35
The third my Love my lifes last ornament
By whom my spirit out of dust was raised
To speak her praise and glory excellent
Of all alive most worthy to be praised.
Ye three Elizabeths for ever live
That three such graces did unto me give.
One of the lesser-known English sonneteers — Richard Smith — dedicates his sonnet sequence DIANA “Unto Her Majesties Sacred Honourable Maids.”
It is obvious that it is not the maids of honour of Elizabeth Tudor that are in the poet’s eye, for he leads off:
Eternal twins that conquer Death and Time
Perpetual advocates in Heaven and Earth
Fair, chaste, immaculate, and all divine
Glorious alone before the first man’s birth.
Rossetti comments at length upon the double and sometimes triple meanings which were placed by the secret schools upon apparently innocent and orthodox words and phrases. I have but little doubt that many of the works dedicated in what is apparently the most fulsome flattery were in reality addressed in a spirit of religious ecstasy to a mystic Elizabeth. I am told that the name El-izza-beth is practically the same as Beth-el of the Old Testament, and means The House of God. This is synonymous with the Temple of the Holy Spirit, and that, as we have seen, was symbolised by the St. Grail. Please take this suggestion for what it may be worth!
The sister figure which now appears upon our coinage as Britannia was I think also originally intended to suggest the same mysterious and perfect Lady of the middle ages. She appeared suddenly about 1676, just when the anti-Papal storm was brewing, which, when it burst, cost James II, his throne. If you will refer to the coin collection at the British Museum you will notice that Britannia has only comparatively recently donned the trident and helmet. On her first appearance she held an olive branch in one hand and a spear in the other. The spear was the attribute of Pallas, the Goddess of Wisdom, and if it is a bad guess on my part it is at any rate an agreeable fancy to believe that a variant of the Virgin Sophia is one of the everyday symbols of our Nation. You will notice
that the lady in paper-mark bears a trefoil instead of an olive branch, again the emblem of the three Alchemical principles — Sulphur, Mercury, and Salt.
It is becoming daily more recognised that Elizabethan literature was not a spontaneous and national home growth; rather it was an exotic imported from the continent. Especially was this so in the case of the sonnet literature which was so luxuriant a weed in the reign of Elizabeth. Much of this was directly borrowed; so directly, in fact, that Mr. Sidney Lee holds up his hands in pained perplexity. He denounces Lodge as a scandalous example of the literary thief, and it was in a sonnet sequence entitled "Phyllis" that, according to Mr. Lee, Lodge sank most deeply into "the mire of deceit and mystification." Now I have shown elsewhere that practically the whole of Elizabethan poetry is a symphony and that there must have existed at that time a very abnormal system of collaboration; further, that the facts point unmistakably to Francis Bacon as the hinge upon which that system turned. It is significant that the birth of English literature coincides with the birth and career of Francis Bacon. In Europe, letters had been flourishing for many centuries, but they had awakened no answering enthusiasm from our semi-barbarous island. I therefore infer that it was Bacon who introduced into this country, and cherished with his influence, the vast literature of Europe. With this must have come the methods of the mystic schools to which much of it was due, and I regard, therefore, all these alleged pilferings and plagiarisms not as proof of bad faith but of the existence here of the same system as was at work elsewhere. The beautiful and mystic theories figured in the book emblems and paper emblems of the period seem, so far as I can gather, to have sprung originally from the East, whence they were revived by Pythagoras. Of the Troubadours, the Templars, the Alchemists, the Rosicrucians and other Idealist schools, the philosophy of Pythagoras was undoubtedly the nursing mother. "Virgil," says W. F. C. Wigston, "takes up the lighted torch of Homer and hands it on to Dante, who passes it to the genius behind the Shakespeare mask — Francis Bacon. Thus the handing of the 'Lamp for Posterity' has been kept going by a chain of giant poets, who, like the distant peaks of some mighty range of Alps, beckon and nod to each other o'er the cloudland of ignorance and above the mists of the ages."
Through these mists I have sometimes thought to have perceived glimpses of the Palace of Wisdom upon which these great master masons
were at work, but I feel to-night very like the poor fool who brought a brick as a sample of the house he wished to show. I thank you very much for lending my remarks such patient attention.
HAROLD BAYLEY.
FRANCIS BACON: A Forerunner
by Michael Srigley
'Believing that I was born for the service of mankind . . . I set myself to consider in what way mankind might be best served and what service I was myself best fitted by nature to perform.'
(Interpretation of Nature, 1603)
What has Francis Bacon to say to us today? It is now over three and a half centuries since he died, and yet his reputation is still obscured by misunderstanding, and the work to which he devoted his life is not yet fully understood. The belief that he betrayed the Earl of Essex has not died, and it is still widely accepted that he was guilty of the charge of corruption that led to his fall as Lord Chancellor in 1621.
Misunderstanding also surrounds the nature of his life's work. He is generally regarded as one of the founders of the scientific method, and today his writings are studied almost exclusively from this angle. But, for Bacon, the scientific revolution he planned was part of a vast plan of restoration for the benefit of humanity. If mankind was to come into its promised heritage, this involved the freeing of man from physical want and to this end Bacon believed that the powers of the human mind should be harnessed to the task of investigating and controlling nature. In this respect he is a forerunner of such modern thinkers as Alice A. Bailey and the Tibetan, Teilhard de Chardin and Sri Aurobindo. In all of these there is a refusal to avoid the problem of the material world, to achieve a spirituality based on flight from the world, and a striving to anchor man's highest intuitions in matter. Bacon, along with a group of kindred spirits spread throughout Europe in the late 16th and early 17th century, paved the way towards what might be called this material spirituality, and much of what is being done today is foreshadowed in their endeavours.
The closing decade of the 16th century witnessed a European movement embracing such people as John Dee, Giordano Bruno, Francis Bacon, Campanella and Robert Fludd, that only in recent years has caught the attention of such scholars as Francis A. Yates. It lasted from 1575 to about 1625, and was a loose association of scholars, thinkers and artists bound, like men and women of goodwill today, by a common ideal: to
bring about a fundamental revolution in man's way of life. Its aims included religious tolerance, social reform and the ending of material want by encouraging science and technology. The first twenty-five years were preparative, the second twenty-five witnessed the attempt to carry out the revolution. Throughout the entire fifty years, and giving the period its peculiar intensity, there was a belief that a new era was opening, ushering in a golden age and marking the reappearance of the Christ. In a very real sense it is as though those fifty years, four hundred years ago, were a preview or dress-rehearsal for what is happening now. This may justify the following brief account of what unfolded then.
In August, 1572, in the constellation Cassiopeia, a new star made a sudden and dramatic appearance. It was as bright as Venus, and to an astonished world taught to believe that the heavens were immutable, it shone as a portent of some convulsive change in the order of the world. To Theodore Beza, the Calvinist Biblical scholar, it heralded the Second Coming, while Paracelsus saw it as "the sign and harbinger of the approaching revolution: there is nothing concealed which shall not be revealed, for which cause a marvellous being shall come after me, who as yet lives not, and who shall reveal many things". Tycho Brahe wrote a small tract on the "new and much admired star" of 1572, which eventually appeared in 1602 dedicated to that royal occultist Rudolph II, Holy Roman Emperor and part model for Shakespeare's Prospero. In it Brahe writes that for him the star marked the entrance of the world into the seventh revolution that would inaugurate the golden age: "Some great Light is now at hand which shall enlighten and by degrees expell the former darkness." He goes on to say that the star would be followed by a major conjunction when the actual fulfilment of the prophecies of the star of 1572 would be realised. Such then was the reaction to the new star which remained visible until 1574; it was the Star of Bethlehem inaugurating the Second Coming and the restoration of the Golden Age. In the reference to the coming major conjunction of the planets we find a hint as to the timetable followed by those actively striving to bring about the restoration, including Francis Bacon.
As James Spedding, Bacon's biographer, put it, the star of 1572 "shone with full lustre" on Bacon's freshmanship at Trinity College, Cambridge, which he entered in 1573 with his brother, Anthony, and left toward the end of 1575. It was in this latter year that Bacon laid the foundation for his life's work, based on a deep dissatisfaction with the prevailing
Aristotelianism of the time and its failure as far as "the production of works for the benefit of the life of man" was concerned. Instead of abstract scholastic philosophy, Bacon conceived in his fifteenth year a way of investigating nature that would result in concrete benefits for mankind. This was in 1575.
Readers of Volume I of *Esoteric Psychology* will recall that it was in that self-same year that the second ray of Love-Wisdom came back into manifestation. Among the names of the Ray-Lord given are "the Displayer of Glory", "the Master Builder", "the Great Geometrician", and "the Light Bringer". The keynote in the associated aphorisms is revelation of the hidden: "Send forth the Word and speak the radiant Love of God. Make all men hear... Pull forth into the light of day from out the night of time the one thou lovest... When light and love are shown forth then let the power within produce the perfect flower... Send forth the Word, and lead the sons of men from off the path of knowledge onto the path of understanding."
What a flood of light these words throw on the endeavours of Francis Bacon and his associates. One of the most frequent images to be met with in the literature and emblems of the period in any way connected with the movement is that of light emerging from darkness, a spark emerging from flint, the dark and light Aleph of contemporary Cabalism incorporated in the printer's device of the so-called "double-A" found in books from 1575 onwards, and the morning "that steales upon the night, melting the darknesse", as Shakespeare expresses it in that dramatic enigma, the *Tempest*. The task of leading "the sons of men from off the path of knowledge onto the path of understanding" was peculiarly Bacon's own in that he set himself to woo his contemporaries from barren speculation towards a systematic understanding of Nature and her laws for the eventual benefit of the human race. He continued what John Dee and, before him, Roger Bacon had prefigured, and laid the foundation for the scientific and industrial revolution that has matured in the 20th century. It remains for us to temper with light and love the enormous power thus gained.
**The Major Conjunction**
Between the formulation of his life's work, in 1575, and 1605, Francis Bacon published no philosophical works. In the winter of 1603-4, after
the death of Elizabeth, Bacon started to put into words the ideas first conceived in 1575, and two years later *The Two Books of the Proficience and Advancement of Learning* was published. In the same winter of 1603-4 occurred one of the rare conjunctions of Saturn and Jupiter. It is Robert Fludd, the English Rosicrucian and follower of Paracelsus, who provides the details of this important event:
"In the year 1603, the 29th of December, following the New Style, at midday, I perceived Saturn enter $8^\circ 39'$ Sagittarius. Likewise Jupiter nearly at the same time entered the same sign Saggittarius at $8^\circ 39'$" (*Tractatus Apologetici*, ch. V, p.1).
Mars also was in conjunction, while the Sun and Venus were in the same sign. Fludd, who was defending the Rosicrucians in this tract, interpreted the conjunction as a sign to the Brotherhood to expand their membership and begin the restoration of the world. It marked the emergence of the Brotherhood into the open, after a period of secrecy that began according to Fludd at the time of the new star of 1572. In 1603-4 then, began the *externalisation* of the Rosicrucian Brotherhood on the one hand, and the implementation in practical form of Bacon’s life work on the other. There is no doubt that Bacon was aware of the major conjunction observed by Fludd and forecast by Brahe. A mysterious handwritten note on the title-page of his *Valerius Terminus* gives the year 1603 and a number of astrological signs identified as Mercury, Jupiter, probably Saturn, Sagittarius, Aquarius, possibly Capricorn. It seems most likely that this refers to the same rare line-up of planets that Fludd observed, and that it was in Bacon’s mind at this time is shown by these words on the significance of such a conjunction:
"But this is that which will dignify and exalt knowledge: if contemplation and action may be more nearly and straitly conjoined and united together than they have been; a conjunction like unto that of the highest planets, Saturn, the planet of rest and contemplation, and Jupiter, the planet of civil society and action."
These words from *The Advancement of Learning* must have been written shortly after the actual conjunction of these planets. The contemplative years stretching from 1575 to 1603 now issued in activity, and the remainder of Francis Bacon’s life was devoted to the writing of what he called the *Great Instauration*.
Renewal
The word *instauration* means restoration or renewal of something that has been lost. Throughout his writings there are scattered hints of a belief in the existence of a civilisation in the remote past that was higher than our own, and which Bacon believed could be restored if man applied his brains to the secrets of Nature "For the Glorie of the Creator and the reliefe of man's estate". The same belief is implicit in the title of his *New Atlantis*. In harmony with Bacon, the Rosicrucians in a series of pamphlets and tracts that reached a climax about 1623, also called for the restoration of a lost golden age. In the *Universal Reformation*, for example, we hear a call for a "Perfect Method of all Arts" to be used for the benefit of all men, and in their manifestos they appealed to the thinkers and writers of Europe not to "keep their secrets close only to themselves". The same note of fervour and expectation called forth by the star of 1572 rings in their publications:
"One thing should here, O mortals, be established by us, that God hath decreed to the world before her end, which presently thereupon shall ensue, an influx of truth, light and grandeur, such as he commanded should accompany Adam from Paradise and sweeten the misery of man." (Waite: *History of the Rosicrucians*, p. 92).
This fervour and expectation is abroad in the world today, and the same call for renewal has been sounded. Now it embraces the world, then only Europe. It is as though what happened between 1575 and 1625 was an anticipation of what is about to happen now. We are told that from 1975 the potency of three major constellations that have produced much of the upheaval of this century "will greatly lessen until it fades out", and that "around the year 2025" the teaching on astrology contained in the writings of Alice A. Bailey and the Tibetan will be continued, based on the firm footing already established. (*Esoteric Astrology*, pp. 537 and 589).
When Bacon died in 1626 his work was incomplete. Parts of the *Great Instauration* were either never written, which is the normally accepted view, or Bacon followed the plan expressed in *Of The Interpretation of Nature* (Spedding, vol. iii, p. 248) "of publishing part and reserving part to a private succession". Many Baconians believe that the missing
parts of the *Great Instauration* are the works of Shakespeare. It seems more likely that either there were, and are, manuscripts preserved in "a private succession", or that the missing parts are locked in the body of his published work in the form of a cipher. If there is a cipher, it is possible that part of the key to it is connected with the fact that the capital letters in the second book of the *Advancement of Learning* "have several magnitudes, or sizes", although "therein is meant no difference". (Errata page to 1605 edition of *The Advancement of Learning*). It is quite possible that Bacon's true significance lies hidden in his public works awaiting revelation.
His work is incomplete in another sense. "The commerce between the mind of man and the nature of things" which Bacon hoped might be "restored to its perfect and original condition" has been started and yet there is still more of exploitation in it than of exchange. The next stages in this commerce have been outlined in the Tibetan's "Letter to a Scientist" (*Esoteric Healing*, pp. 376-9), and the science of Bionics based on the discoveries of Viktor Schauberger (1885-1958) and his principle of implosive energy, points to a future cooperation of man and nature that may bring the relief of man's estate that Bacon dreamt of. The man who "Mourned the divisions of thy Church", and whose most intimate friend was a Roman Catholic in an age when such friends were dangerous, would also still find much to mourn as well as signs that the divisions were slowly being healed. As to the alleviation of man's physical lot, Bacon's immediate goal, this is far from complete. The inequality between the rich and poor countries increases, and distribution has not yet undone excess, to use Lear's words.
It was Bacon's genius to have foreseen the major trends the world would follow in the centuries succeeding his own, and we are reaping what he and his fellow-workers sowed four centuries ago. Bacon clearly foresaw that power divorced from love and light would only bring destruction. He was the victim of this in others in his own life. As the Tibetan has said repeatedly, service is the safeguard, and in Bacon we have an exemplar of what such service could mean. Nowhere is this sense of dedication and service more memorably expressed than in the prayer penned by Bacon on the eve of his trial for a crime of which he was innocent, but to which he pleaded guilty out of a sense of duty:
"Remember how thy servant hath walked before thee: remember what I have first sought, and what hath been principal in my intentions. I have
loved thy Assemblies, I have mourned the diversions of thy Church, I have delighted in the brightness of thy Sanctuary. This Vine, which thy Right Hand hath planted in this nation, I have ever prayed unto thee that it might have the first and the latter rain; and that it might stretch her branches to the seas and the floods. The state and the bread of the poor and oppressed have been precious in my eyes: I have hated all cruelty and hardness of heart: I have — though in a despised weed — procured the good of all men. If any have been my enemies, I thought not of them; neither hath the sun set on my displeasure: but I have been as a dove, free from superfluity of maliciousness. They creatures have been my books, but thy Scriptures much more. I have sought thee in the courts, fields and gardens, but I have found thee in thy temples."
SHAKE-SPEARES
SONNETS.
Neuer before Imprinted.
AT LONDON
By G. Eld for T. T. and are
to be solde by John Wright, dwelling
at Christ Church gate.
1609.
"SHAKE-SPEARES SONNETS"
T. D. Bokenham
Baconiana numbers 37-40 of 1912 contain no less than six articles on these enigmatic poems. These include one by W. T. Smedley, the author of "The Mystery of Francis Bacon", who believed that the key to the sonnets will be found in Sonnet 62 which starts:
"Sinne of selfe-love possesseth al mine eie
And all my soule, and al my every part:"
Smedley added, "He has been picturing himself as he was when a young man. He turns to his glass and sees himself beated and chopt with tanned antiquity; forty summers have passed over his brow." This remark is a misquotation from Sonnet 2. "When fortie Winters shall besiege thy brow".
Another writer, Parker Woodward, believed that the sonnets are sometimes soliloquies and sometimes pleas with the far-off decipherer. However, he adds, "my hypothesis is that the first twenty five of them are addressed by Francis to himself. Numbers 26-32 were written to his decipherer, while No. 33 being Bacon's name sonnet, is naturally very beautiful and reminiscent. It recounts how,
"Even so my sunne one early morn did shine
But out alack, he was but one hour mine".
Other writers in this series of articles were the American, J. E. Roe, whose book "The Mortal Moon or Bacon and his Masks" had been published in 1891, John Hutchinson and R. M. Theobald, who believed that the sonnets were written for the young Earl of Southampton.
Clearly, these articles contain some element of truth, but it is obvious that they were based on only a few of the sonnets. My studies of a cipher system used by Bacon and demonstrated in the cipher book "Cryptomenytices et Cryptographiae" of 1624 which I own, show that a number of the Shakespeare sonnets were designed for the eventual discovery of many of Bacon's well kept secrets. They confirm his royal birth as a son of Queen Elizabeth and Robert Dudley, his youthful love for Marguerite of Navarre, his Muse Pallas Athene, the story of the ring
given as "a troth" by Queen Elizabeth to her other son, Robert Earl of Essex, his Rose Fraternity, his "Rose Sonnets", which he called "Times best Jewell", and his great admiration for Dante, the so-called "rival-poet". It should be noted that the first English translation of his "Divine Comedy" was published in 1802, though part of it, the "Inferno", appeared some twenty years earlier. Some rival!
Some of these encipherments also deal with Bacon's ethical and Rosicrucian teachings which are contained in the "beautious and lovely youth" and "Dark Lady" sonnets who turn out to be Adonis, who anciently represented the Sun, or Light and Artemis, the Mother Goddess of Nature whose statue at Ephesus was made of a black substance, probably ebony, representing her earthly origins. Sonnet 20, the "Master-Mistris" sonnet reveals that those mythical beings represent what Pythagoras called the Light and Dark sides of our natures, the one, the spiritual, to be developed and the other to be controlled or cleansed. Pythagoras travelled to Egypt and other Middle East lands and became a member of the Essene Brotherhood whom Jesus contacted some five hundred and fifty years later, and this teaching illustrates the age old precept "From Darkness to Light". Some of our dark sides include our prejudices, our ignorance, our intolerance and the divisions in the Christian Church which have been the cause of so many wars between nations. Bacon believed, as did Shakespeare, that Ignorance is the chief of those shortcomings. I believe that the wealthier members of his Rosicrucian Fraternity were the patrons and sponsors of most of the translations from the Classics and other great books published during Bacon's life-time, including the Shakespeare Folio of 1623. Those plays were not only designed as "Histories, Comedies and Tragedies" but also to teach mankind about himself, his failings and his otherwise noble character.
Other encipherments found in the sonnets include messages concerning Bacon's innocence over taking bribes in the Chancery Court and one sonnet, number 125, actually includes the words "Impeacht" and "subborn'd informer" in the open text. The subborned informer was John Churchill who, for rewards, gave false evidence against Francis St Alban during his indictment in the House of Commons in 1621. This sonnet is enciphered "Francis St Alban Author" which proves that these sonnets were not published in 1609 as seen on their first edition. I must add that the words SHAKE-SPEARES SONNETS on their title page add, in Bacon's simple cipher, to 221 the count of FR ST ALBAN (88)
THINE AUTHOR (133). Three of these sonnets, Numbers 48, 71 and 74, all suggest that Francis St Alban left this country in 1626 when he is supposed to have died. Sonnet 71 starts:
"Noe longer mourne for me when I am dead,
Then you shall heare the surly sullen bell
Give warning to the world that I am fled
From this vile world with vilest wormes to dwell:"
Under the words "when I am dead" is an enciphered message "fled from this land". Sonnet 74 starts:
"But be contented when that fell arest,
With out all bayle shall carry me away",
And under those last three words is an encipherment giving "to other shores".
Sonnet 48 however, tells us a little more. It starts:
"How carefull was I when I tooke my way,
Each trifle vnder truest barres to thrust",
Under the words "my way" are the letters R E S T O T H which, with shared letters can spell "to other shores". There are four other enciphered messages, one spelling FRA TUDOR AUTHOR, another, starting in line 9 which refers to those "trifles", spelling THE GORHAMBURY VAULT. Above this is another message A LONELY GRAVE and beside this, starting with line 5 which mentions "my jewels trifles" gives MY PLAIES AND SONNETS - FRA TUDOR. The lines and columns which contain this symmetrical group of letters, plus their initial letters, number 274 the count of HID (21) IN (22) GORHAMBURY (121) APSE (39) VAULT (71), the expression used in the Bacon monument encipherment.
John Aubrey, in his biography of Bacon of 1681 told us that Sir Harbottle Grimston, the then owner of the Gorhambury estate, removed Bacon's coffin from that vault to make room for his own. Grimston had married secondly the widow of Sir Thomas Meautys, Bacon's former Secretary, who was responsible for erecting that monument with its enciphered epitaph, "manuscripts in apse vault". It is obvious that the Grimstons, and possibly Aubrey, were aware that Bacon had left this country and that those precious manuscripts were concealed in that coffin. It is
interesting that Aubrey did not say where that coffin was reburied, but it is certain that those manuscripts were in existence in 1681 and not lost or destroyed as is generally thought. I cannot say any more but I could give a shrewd guess where they now are.
T. D. BOKENHAM
The motto which Shakespeare chose for "the first heir of his invention," *Venus and Adonis*, is the following:—
"Vilia miretur vulgus; mihi flavus Apollo
Pocula Castalia plena ministret aqua."
This couplet is taken from Ovid's *Amores*, Book I, xv.36; and it is worth notice that this poem is one of those translated by Marlowe, and known as Ovid's Elegies. The translation is very skilful, very accurate, generally very closely literal, so that a struggling student might use Marlowe as a crib in grinding through the *Amores*. These verses are thus rendered by Marlowe:—
"Let base conceited wits admire vile things,
Fair Phoebus lead me to the Muse's springs."
Following Marlowe's translation is one by "B. J." and it appears complete in Ben Jonson's *Poetaster*. Jonson's translation of the same couplet is:—
"Kneel hinds to trash: me let bright Phoebus swell,
With cups full flowing from the Muse's well."
It is curious and interesting to see Shakespeare and Marlowe and Ben Jonson and Ovid all harnessed together in one team. There are many indications in the Marlowe version of Ovid's *Amores* that Marlowe and Shakespeare are inextricably coupled together; and that when Marlowe paraphrases, instead of translating literally, Shakespeare uses the same words. Thus, Marlowe translates *longas hiemes* (I. viii.) by "winter's lasting rage." "Winter's rages" are heard in *Cymbeline* IV. ii. 260; and a close approximation to it in *Sonnet* xiii.:—
"Against the stormy gusts of winter's day
And barren rage of death's eternal cold."
And again in Marlowe's *Edward II*. II. i. 61, —
"The shepherd nipt with biting winter's rage."
*Rage* is a very favourite word with Shakespeare.
In the 13th Elegy Ovid writes, "Adspice quot somnos juveni donarit amato,—Luna!" Literally, "See what slumbers the moon bestows on the youth she loves." Marlowe names the youth—
"The moon sleeps with Endymion every day."
And the same joint slumbers of the moon and Endymion are referred to in Merch. Ven. V. i. 109.
The bitter question put by Shylock — "Hates any man the thing he would not kill?" — is evidently a reflection of the line in Ovid's Amores II. ii. 10, "Quem metuit quisque, perrise cupit," which "Marlowe" renders —
"Believe me whom we fear we wish to perish."
The comparison between death and its "cold image," sleep, so frequently repeated, in varied modes of expression, in Shakespeare (see Promus 1204) is derived from Ovid's Amores II. ix. 41. Stulte (the Promus, as printed, has FALSA: perhaps a misreading of the MS.) Quid est somnus gelidae nisi mortis imago? — and "Marlowe" translates it, very literally,
"Fool! What is sleep but image of cold death?"
In the last Elegy of the second book, line 35, we find the words "Quod sequitur, fugio; Quod fugit, ipse sequor," translated by "Marlowe,"
"What flies, I follow, what follows me I shun."
This recalls the speech of the fourth Counsellor in the Gesta Grayorum: "The proverb is a country proverb, but significative, 'Milk the cow that standeth still, why follow you her that flieh away?' " And this, again, recalls the lines in Sonnet 143:
"Whilst her neglected child holds her in chase,
Cries to catch her whose busy care is bent
To follow that which flies before her face,
Not prizing her poor infant's discontent.
So run'st thou after that which flies from thee,
Whilst I, thy babe, chase thee from far behind."
And,
"Love like a shadow flies, when substance love pursues,
Pursuing that which flies and flying what pursues."
—Mer. V. II. ii. 187.
These comparisons are pointed out in the translation of the Promus 553. Unfortunately two of the references are wrong; the "SECOND COUNSELLOR" should be the fourth; and the last quotation is given as Mer. V. II. 3, instead of II. 2.
The 15th Elegy of the first book also anticipates the sentiment which closes Bacon's Essay of Death: — "Death hath this also: that it openeth the gate to good fame, and extinguisheth envy." Mr. Reynolds, in his edition of the Essays, quotes the passage from this Elegy:
"Pascitur in vivis Livor, post fata quiescit,
Cum suus ex merito quemque tuetur honos."
Translated nearly alike in the two versions — Marlowe's and B. J.'s —
"Envy the living, not the dead doth bite,
For after death all men receive their right."
There is another very striking anticipation of Shakespeare in the 14th Elegy of the third book. The subject is described as "Ad amicam, si peccatura est, ut occulte peccet," and the whole drift of it, repeatedly expressed with ingenious variations and reiterations, is this: — The poet tells his mistress to hide her frailties if she cannot avoid them. She is to wear modesty as a garment even if it is not a native quality of her character — she is to "Put it on" when she dresses, — "Indue cum tunicis metuentem crimina vultum." The whole Elegy is well reflected in the speech of Luciana to Antipholus of Syracuse — Com. Er. III. ii. 7-28. Both poets tell their wooer to "Muffle your false love with some show of blindness." Both give the counsel that virtue should be assumed even if it is not possessed: Sit tibi mens melior, saltemve imitare pudicas," Teque probam, quamvis non eris, esse putem (13, 14).
"Apparel vice like virtue's harbinger;
Bear a fair presence though your heart be tainted;
Teach sin the carriage of a holy saint."
Both Ovid's Latin and Marlowe's translation are too naked and unabashed
for entire quotation. The same advice is given by Hamlet to his mother:—
"Assume a virtue if you have it not."—Ham. III. iv. 160.
And he tells her of custom,
That to the use of actions fair and good,
He likewise gives a frock or livery
That aptly is put on."
Obviously Bacon's philosophy of behaviour, as a dress that can be put on or taken off, is reflected in both these plays, as it is in about fifty passages in Shakspeare (See Chapter VIII. in my "Shakespeare Studies in Baconian Light"). And it is curiously anticipated by the one word "Indue" in Ovid.
As a sequel to these references to Marlowe's translation of Ovid's Elegies, I am glad to be able to add what Rev. Walter Begley wrote on the same subject. This is the chapter in his uncompleted work on Marlowe.
R. M. THEOBALD
OVID'S ELEGIES
Translated by C.M.
These have been universally attributed to Marlowe, and I should not have thought of raising a doubt if it had not been for my researches into Ben Jonson's Poetaster.*
I will only say that Ben Jonson, when he brings Bacon on the stage, at the very beginning of the Poetaster, as Ovid Junior, composing a new revised edition of Ovid's fifteenth Elegy, does certainly seem to suggest the Baconian author of these translations, or anyhow of the latter and revised one.
It is against commonsense to suppose that any author intending to turn one of Ovid's Elegies into English verse, should at once take the previous translation of the same Elegy by another author, and, changing only a word
* Cf. Is it Shakespere? Index, &c.
or two here and there, put it forth as his own. Nothing can induce belief that Ben Jonson, or B. J., or anyone else except the original author, is responsible for the second revised version of the first. Whoever wrote the first, wrote the revised also. We have not here two independent attempts by different authors. Whatever, then, was the reason that those two versions should be presented to the public in the second edition of *Hero and Leander* with the title put over the second one, "The same by B. J." — when it must be clear to the common sense of even the uncritical reader that the two versions were from the same pen — only the second had a few verbal and metrical improvements.
It is most amusing to see how the critics are nonplussed by these two Elegies, and at what extraordinary and contradictory conclusions they arrive. Mr. Bullen, the last editor, having had the advantage of perusing his predecessor's amazing attempts, gives us the following footnote to the title of the second version: — "The same by B. J., i.e., Ben Jonson, who afterwards introduced it into the *Poetaster*. This version is merely a revision of the preceding, which must also have been written by Ben Jonson. (Not in Isham copy or Ed.A.)." Notice the "must," which I have italicised. How categorically and imperatively it settles the question! But Elegy XV., in its first version, was in the original edition and all succeeding ones, and Mr. Bullen will find it rather hard to show how Ben Jonson got this particular Elegy mixed up and printed with Marlowe's when they were first published. Is it supposed that Ben Jonson had any more of his work among Marlowe's Elegies? Ben Jonson and Marlowe never wrote in collaboration, for Jonson was unknown to fame at Marlowe's death and for some little time afterwards.
It really seems as if the Bacon theory is the only one to explain these versions. The matter is simple enough on this supposition, and could be thus explained. Bacon (or Ovid Junior, as Ben Jonson called him), wrote some English translations of a few of Ovid's *Amores*, and they were printed "at Middleborough," under the title of *Epigrammes and Elegies by J. D. and C. M.* J. D. was the Sir John Davies to whom Bacon, in 1603, wrote the famous letter referring to "concealed poets," and C. M. was supposed to stand for Christopher Marlowe. Not long afterwards another edition appeared — also printed "at Middlesbrough" — containing "All Ovid's Elegies, 3 Books by C. M., Epigrammes by J. D." (There is no date on these books. Col. Cunningham suggests *circa* 1597.) Now we get for the first time the second version of Elegy I. xv.,
and the initials B. J. given as the author. My suggestion is that both are Bacon's composition, and as he had improved his original version and wished to print it, he was obliged to attribute it to someone, for everyone would know that it could not be Marlowe's, who had been in his grave for several years. Therefore Bacon put some initials to the new production, viz., B. J. (perhaps John Bodenham), which a casual reader might take for Ben Jonson and think no more of it. But on my supposition Ben Jonson would not thus pass it over, for he would know well enough that it was not his, and so I suggest that he made use of his knowledge in the Poetaster when he brings before us Ovid Junior, correcting this very Elegy, and saying, "Yes! it shall go so," and it goes into the Poetaster, as it had before in the volume. If Jonson had really made the second translation he would not have given it to Ovid Junior, but if he knew the real author of both, then the Poetaster makes things perfectly clear, and the way Jonson puts the matter meets all the difficulties.
But with regard to these Ovidian Elegies I can offer what seems to be a stronger proof still, hitherto unnoticed. Referring to the two title-pages, we see that the Elegies, some years after Marlowe's death, were apparently first published in part, and then, when Marlowe had been a little longer in the grave, they were completed in three books. Now if they were written by Marlowe and left behind him at his death, why was the first edition incomplete? If the reason was that another version of Marlowe's translation had been found, why was not this much required explanation given? For it surely required a little explaining why a man's poems or translations should grow in size after his death.
To say the least this is suspicious; but I have referred to a stronger proof, which comes from John Stow's Annales, 1615, p. 811, as follows: — "Our moderne and present excellent poets, which worthely florish in their owne workes, and all of them in their very owne knowledge lived together in this Queenes raigne, according to their priorities as neere as I could. I have orderly set down." Then follows a long list, beginning with George Gascoigne, Esquire, and ending with M. George Withers. In this list — and here is my proof — only two poets are coupled together, and they are Sir Francis Bacon and Sir John Davies!
Now why should these two be thus coupled, and why should Bacon, of all men, be put among the illustrious poets, when, so far as we know, he had never published a line of poetry in his own name of any kind in 1615? I think the answer is obvious. John Stow coupled these two
because he had reason to know that the Epigrammes and Elegies of J. D. and C. M. were really written by Sir John Davies and Francis Bacon, and as these poems were published together he did not make a separate entry to the two, but wrote them down as joint authors, thus: — Sir Francis Bacon, Knight, & Sir John Davies, Knight; this ampersand being the only one in the whole list. When Sir John Davies published his collected works in 1622, he did not include these Epigrammes. Why? We may conjecture; we do not know.
It should also be noticed that the several early editions of the Elegies, which came in pretty rapid succession, had all, including the first, the imprint Middlebough. Why Middlebough? Simply, I think, as a great joke. They were probably all printed in London; and as Mr. Edmunds found a copy of one of the Middlebough editions of the year 1599 in the Lamport cupboard, Jaggard would probably be the printer. The joke about the imprint would arise from the fact that such lascivious poems should issue from Middlebrough, the very place which the Brownists and rabid Puritans put as a blind on their own very different productions. In fact, it was as amusingly deceptive as the somewhat parallel case of Mirabeau, two centuries later, who published the Erotica Biblion as emanating from the press of the Vatican itself (à Rome de l’Imprimerie du Vatican, 1783).
Rev. Walter Begley
THE BACONIAN 33 AND THE ARCHETYPAL 28
Hollenbach
In the last issue of *Baconiana* the author of "The Prerogative Instances And The Shakespeare Plays" presented examples to support his thesis of an archetypal twenty-eight that manifests synchronistically across cultures in history, architecture, literature, and myths as twenty-eight separate and distinct phases, personalities, and characteristics.
One example were pages 3 and 4 of *THE GREAT ASSIZES* by George Withers, which is duplicated in CHART 1. On the left of CHART 1 (page 3 of The Great Assizes) under the ornate box containing the name "APOLLO" are listed twenty-eight names beginning with The Lord Verulam (Francis Bacon). On the right of CHART 1 (page 4 of The Great Assizes) four names are listed.
One of the codes used by Bacon was a simple number code in which A is 1, B is 2, etc (I and J being the same). In this code "Bacon" is number 33. The twenty-eight names on the left of CHART 1, the four names on the right, and the name Apollo in the ornately trimmed box at the top total 33, Bacon's cipher name.
The combination of 33 and 28 as the Baconian 33 and the Archetypal 28 are discovered in three other publications that esoterically are attributable to Francis Bacon: The Catalogue of Shakespeare plays, the Dedication to the Shakespeare Sonnets, and The Chymical Wedding of Christian Rosenkreutz.
THE CATALOGUE OF SHAKESPEARE PLAYS
The Catalogue from the 1623 First Folio of Shakespeare Plays is shown in CHART 2. Of the thirty-six plays, "Troilus and Cressida" is not listed in the Catalogue. Two of the listed plays are the first and second parts of Henry Fourth, and three of the plays are listed as the first, second, and third parts of Henry Sixth.
These five listings as "parts" are essentially two *lengthy* plays of Henry the Fourth and Henry the Sixth. The thirty-six plays, then, are actually thirty-three, Bacon's cipher name. In CHART 2 these thirty-three plays are numbered with large numerals, and the twenty-eight characters
mentioned in the play titles (for example, 1 and 2 in "The Two Gentlemen of Verona" and 16 and 17 in "Romeo and Juliet") are numbered with small numerals.
THE DEDICATION FROM SHAKESPEARE'S SONNETS
The dedication from Shakespeare's sonnets appears in CHART 3. The entire dedication consists of twenty-eight words each of which is followed by a period. Five of these twenty-eight words are the abbreviation Mr., the two single letters "W" and "H", and the two hyphenations "ever-living" and "well-wishing". In CHART 3 small numerals appear under each of the twenty-eight periods that follow each of the twenty-eight words of the dedication.
Five other markings are numbered with a large numeral above each marking: The small raised "r", the two hyphens, and the two periods of the "T.T." at the end of the dedication. These five markings plus the twenty-eight total thirty-three, Bacon's cipher number.
THE CHYMICAL WEDDING OF CHRISTIAN ROSENKREUTZ
The puzzling periods at the end of each word in the dedication have baffled readers and Baconians, who more readily assume the periods indicate a cipher. Penn Leary in THE. SECOND. CRYPTOGRAPHIC. SHAKESPEARE. asks what are all those periods doing there, and were they stuck in for no reason except for someone's attempt at decoration? Cavalierly, Leary places periods at the end of each word in the title of his book in which he discovers a cipher based on four, the last word in the dedication ("Forth").
This profusion of unnecessary periods appears also in the Chymical Marriage or Chemical Wedding of Christian Rosenkreutz, the third of three Rosicrucian manifestoes that appeared in the second decade of the seventeenth century. The first two were the FAMA FRATERNITATIS and the COMFESSION FRATERNITATIS. Esoterically Francis Bacon is considered to have been involved with each. Dr. H. Spencer Lewis, Imperator of the Rosicrucian Order, AMORC during the first half of this century, was certain that Bacon was the author of the FAMA.
On the sixth day of the Chemical Wedding Christian Rosenkreutz discovers a great square Copper Kettle with inscriptions on each of its four sides. The complete inscription from A CHRISTIAN ROSENKREUTZ ANTHOLOGY, edited by Paul M. Allen, is shown in CHART 4.
For the forty-one words comprising the inscription twenty-eight are followed by a period (including the five periods that are the bottom period of a colon). CHART 4 numbers these twenty-eight periods with small numerals below each period. There are five periods that are the top period of each of the five colons in the inscription, and large numerals appear over each in CHART 4. These five plus the twenty-eight total thirty-three, Bacon’s cipher name.
Some German editions omit the “I” in the second line of the inscription. Except for punctuation, however, the inscription in THE CHEMICAL WEDDING OF CHRISTIAN ROSENKREUTZ, translated by Joscelyn Godwin, and the inscription in Arthur Edward Waite’s REAL HISTORY OF THE ROSICRUCIANS are the same as in A CHRISTIAN ROSENKREUTZ ANTHOLOGY. In the Godwin translation there are 30 periods and in Waite’s book 26 periods. These two are shown in CHART 5. Interestingly, their average (30 and 26) is 28.
THE CEILING OF THE SISTENE CHAPEL
The numbers 28 and 33, which seem a synchronicity in Baconian lore, appear coincidentally on the ceiling of the Sistine Chapel.
Michelangelo considered himself a sculptor and rebelled when Pope Julius II ordered him to decorate the ceiling of the Sistine Chapel. In a letter Michelangelo said that the Pope told him to do what he liked. The scheme of decoration, a vast complex of humanity thundering down upon the viewer, was largely his own idea.
The ceiling consisted of thirty-three panels, which are numbered on the sketch of the ceiling in CHART 6. Twenty-eight names of human figures are titles for twenty-seven panels; the remaining six panels have titles of biblical events. The panels, the twenty-eight names, and six biblical events are listed numerically on CHART 6.
The twenty-eight names within the titles of the thirty-three panels are simplified in the following:
PANELS
23 panels with single names produce 23 names.
2 panels with 2 names each produce 4 names.
2 panels with the same name produce 1 name.
6 panels of biblical events produce 0 name.
33 28
The appearance of the numbers twenty-eight and thirty-three within each of these examples (four from literature and one painting) may be only a coincidence. However, from the perspective and understanding of the archetypal 28 and the Baconian 33 (which may symbolise the thirty-two mysterious paths of wisdom in the Kablistic Tree of Life) the coincidence becomes meaningful, becomes a synchronicity.
BACONIANA
CHART 1
PAGES 3 AND 4 OF THE GREAT ASSIZES BY GEORGE WITHERS
Hollenbach
A. Page 3 of The Great Assizes
B. Page 4 of The Great Assizes
C. Number of persons in each section
The 1623 First Folio of Shakespeare Plays lists thirty-five under three categories. A thirty-sixth play "Troilus and Cressida" is not listed but is printed in the Folio.
TO. THE. ONLIE. BEGETTER. OF.
1 2 3 4 5
THESE. INSVING. SONNETS.
6 7 8
1
Mr. W. H. ALL. HAPPINESSE.
9 10 11 12 13
AND. THAT. ETERNITIE.
1 15 16
PROMISED.
17
BY.
18
OVR. EVER-LIVING. POET.
19 20 21
WISHETH.
22
THE. WELL-WISHING.
23 24
ADVENTVRER. IN.
25 26
SETTING.
27
FORTH.
28
T. T.
CHART 3
THE DEDICATION FROM SHAKE-SPEARE'S SONNETS
THE BACONIAN 33 AND THE ARCHETYPAL 28
O. BLI. TO. BIT. MI. LI.
1 2 3 4 5 6
KANT. I. VOLT. BIT. TO. GOLT.
7 8 9 10 11 12
SANITAS. NIX. HASTA.
13 14 15
F. I. A. T.
16 17 18 19
QUOD.
gnis: Aer: Aqua: Terra:
21 22 23 24
SANCTIS REGUM ET REGI—
5
NARUM NOSTR:
25
Cineribus
Eripere non potuerunt.
26
Fidelis Chumicorum Turba
IN HANC URNAM
Contulit.
27
A.
28
CHART 4
THE CHYMICAL WEDDING
A CHRISTIAN ROSENKREUTZ ANTHOLOGY
Edited by Paul M. Allen
65
O. BLI. TO. BIT. MI. LI.
KANT. I. VOLT. BIT. TO. GOLT.
SANITAS. NIX. HASTA.
F.I.A.T.
QUOD
Ignis : Aer : Aqua : Terra :
SANCTIS REGUM ET REGI
NARUM NOSTR:
Cineribus
Eripere non potuerunt.
Fidelis Chymicorum Turba
IN HANC URNAM
Contulit.
Ad.
A
CHART 5
The inscriptions from (A) THE CHYMICAL MARRIAGE OF CHRISTIAN ROSENKREUTZ in REAL HISTORY OF THE ROSICRUCIANS BY Arthur Edward Waite and . . .
P.BLI.TO.BIT.MI.LI.
KANT.I.VOLT.BIT.TO.GOLT.
SANITAS.NIX.HASTA.
F.I.A.T.
QUOD.
Ignis: Aër: Aqua: Terra:
SANCTIS REGUM ET REGINARIUM NOSTR:
Cineribus.
Eripere non potuerunt.
Fidelis Chymicorum Turba.
IN HANC URNAM
Contulit.
Ad.
B
... (B) THE CHEMICAL WEDDING OF CHRISTIAN ROSENKREUTZ, Translated by Joscelyn Godwin and Introduced by Adam McLean.
| PANEL | NAME |
|-------|-------------------------------------------|
| 1 | DAVID and GOLIATH |
| 2 | JOEL |
| 3 | ZOROBABEL |
| 4 | ERYTHRAEAN SIBYL |
| 5 | OZIAS |
| 6 | EZEKIEL |
| 7 | ROBOAM |
| 8 | PERSIAN SIBYL |
| 9 | SALMON |
| 10 | JEREMIAH |
| 11 | HAMAN, The death of |
| 12 | JONAH |
| 13 | I The Brazen Serpent |
| 14 | LIBYAN SIBYL |
| 15 | JESSIE |
| 16 | DANIEL |
| 17 | ASA |
| 18 | CUMAEAN SIBYL |
| 19 | EZEKIAS |
| 20 | ISAIAH |
| 21 | JOSIAS |
| 22 | DELPHIC SIBYL |
| 23 | JUDITH and HOLOFERNES |
| 24 | ZECHARIAH |
| 25 | NOAH. Drunkenness of |
| 26 | NOAH. Sacrifice of |
| 27 | II The Flood |
| 29 | EVE, Creation of |
| 30 | ADAM, Creation of |
| 31 | III Temptation and Expulsion |
| 32 | IV Congregation of the Waters |
| 33 | V Creation of the sun-moon-planets |
| | VI Separation of light from dark |
**CHART 6**
THE CEILING OF THE SISTINE CHAPEL
68
Dear Mr. Bokenham,
I enclose my cheque for £10 in respect of my subscription. I am sending it early in case I forget — elderly people sometimes are forgetful. On February 13th next year I shall be 86 years old, so you will understand my caution.
I would like to make a few comments to you about Gwynne’s article, acknowledging at the same time that I can by no means match his standard of scholarship.
(a) It seems obvious to me (as I suppose it does to you) that Gwynne is not an Initiate into the Universal mysteries. He therefore does not realise that Masonry (with all its faults) is a modern School of the Mysteries. As such, its aims are necessarily spiritual and motivated by benevolence towards mankind.
(b) John Dee was NOT a black magician! What he did was to obtain information from certain spirits, always putting himself and Kelly under Divine Protection first. Whether the vast system of tabulated invocations that emerged would ever be of general interest is a matter of some doubt. I myself think the whole thing is much too cumbersome for general use. But the invocations are effective and genuine. But any system of magic which seeks to balance evil and good spirits (and this is one) is properly the work of an Adept. Anyone of lower grade than that is likely to get into trouble.
(c) Whether Bacon intended to weaken the Catholic religion I do not know. The Catholic religion stands self-condemned by its rejection of the mysteries. Christianity was originally a mystical religion. This is clearly shown in “Esoteric Christianity” by Annie Besant. The Doctrine of Reincarnation is central in the
Mysteries, and originally formed part of the Christian Faith. This doctrine was condemned in the Eastern Church by the council of Constantinople, in A.D. 553, and in the Western Church by the council of Lyons in 1274 and the Council of Florence in 1493. This was done to safeguard the church's finances by the sale of pardons, by the founding of Chantry Chapels and Masses for the dead. If men believed the Reincarnation gave them a "second chance" and subsequent chances, the Church would lose out.
The Catholic Church was — and is — a totalitarian institution, and devout Catholics are told what to believe. They have been brainwashed for centuries. This is the reason why — if you tell a Catholic — anything that does not agree with the party line, it simply does not register!
A great deal more might be said about these matters. Jesus said "the tree is known by its fruits". The fruits of the Catholic Religion in the past have included massacres, tortures, and the horrors of the Spanish Inquisition, and the much milder Papal Inquisition.
I hope an adequate reply will be made to Gwynne's article either by yourself or some other learned Baconian.
With best wishes
Very Sincerely Yours,
H. T. HOWARD
Telephone: 0273 509460
9 Clermont Court
Clermont Road, Brighton BN1 6SS
November 26, 1993
The Editor *Baconiana*,
*The Parentage of Francis Bacon*
In the article by George Stronach, written in 1902, and re-published in the 1992 issue of *Baconiana*, the author has no answer to the facts which I stated in my article on this subject in the 1987 *Baconiana*. Parker Woodward in fact gave him an easy time.
Mr. Stronach has nothing to say about Dr. William Rawley’s statement that Bacon was born in “York House or York Place”, i.e. Whitehall Palace. He has nothing to say about Bacon’s residence in Twickenham Park, a fine house with 87 acres of parkland opposite Richmond Palace, the reference to “ffrauncis, your Soveraign” in the Northumberland Manuscript, the word between Elizabeth and James that has been erased from the inscription in Canonbury Tower, Bacon’s peremptory demand that he should be released from the Tower of London, after his trial in 1621, the Hilliard drawing of ‘a Queen and her Son’, or the amazing knowledge of the language of the 27 kings and queens displayed by the author of the Shakespeare plays.
I raised other points in my article, none of which were answered by Mr. Stronach.
Francis Carr
29 November, 1993
Steep House
6 Church Lane, Wymington, Rushden
Northants NN10 9LW
The Editor Baconiana,
Sir,
The late Mrs. Hilda Brameld told me she regarded as far more important than proving Bacon wrote Shakespeare the defence of his character from vile slander, and I had always imagined this to be one of the objects of the Francis Bacon Society. It was therefore with considerable surprise and distaste that I found myself reading in the issue of its formal organ which has just reached me, such an attack upon his moral worth and aims as could only have been made by an enemy.
What was the motivation of the writer? The word “Satanist” used by him more than once is employed mainly by Biblical fundamentalists, who regard all forms of esoteric teaching as directed to the worship of Satan, a being not believed in outside of the fundamentalist churches. The writer plainly hated all the Tudors; he also hated Rosicrucians and Freemasons. What kind of person hated both Tudors, Rosicrucians and Freemasons? The answer flashed on me: Catholics – the writer must
be a Roman. Turning to the thumbnail biography on the inside cover, I see my hunch confirmed.
Further comment is perhaps superfluous, yet I will point out that so far from perverting the doctrines of Plato and Aristotle, Bacon in his *New Atlantis* sought, as Plato in his *Republic*, and Thomas Moore in his *Utopia*, to design an ideal society; and if one has been reading Aristotle's *Metaphysics*, one finds the transition from that to Bacon's *Novum Organum* so natural and easy it could be imagined they came from the same pen.
Yours sincerely,
Jean Overton Fuller
*(Author: Sir Francis Bacon, A Biography)*
Telephone: 0892 513278
Wroxton Lodge
134 Upper Grosvenor Road,
Tunbridge Wells, Kent TN1 2EX
March 28, 1994
The Editor *Baconiana*,
Sir,
As a newcomer to *Baconiana* I was initially impressed to see N. M. Gwynne’s article *The Occult Life, Works and Importance of Francis Bacon*. With most pressure groups and societies, tolerance of contrary views is not one of their strong points. Any opinion which fails to support or — heaven forfend — dares to challenge their basic precept is usually characterised as part of some Great Conspiracy and unhesitatingly suppressed.
However, while I am impressed by your broad-mindedness I am far from convinced that intrinsically the article was worth publishing. Warning bells started to ring when I reached the phrase ‘true religion.’ The assertion of the existence of a ‘true religion’ is usually the death knell of reasoned argument. Only a line or two later Bacon was — according to your writer — instigating a ‘revolution . . . against a properly ordered society’ and within a paragraph Bacon was also intending to destroy ‘the true philosophy’. By this point the warning bells had become a full set of changes. Finally Bacon was revealed as intent on destroying the whole
world order and replacing it with — horror of horrors — Freemasonry.
Now unlike Gwynne I will declare an interest and say that I am a Freemason. Furthermore the idea of moderating the present world order with even the smallest amount of universal brotherhood and tolerance does not strike me as so terrible. Nonetheless I can quite accept that Freemasonry is not everyone’s cup of tea.
However, the organisation which has most consistently and violently opposed Freemasonry is the Roman Catholic Church and sure enough, in Gwynne’s next paragraph it is the ‘wholly Catholic influence’ that the evil — and later Satanic — Bacon was intent on overthrowing.
So a letter — which could have presented a reasoned argument against the idea that Bacon wrote pretty well everything published or performed in his lifetime, turns out to be no more than unsubstantiated bigotry. Worse it is inspired by the writer’s undeclared adherence to archaic tenets of the Roman Catholic Church which few present day Catholic intellectuals would seriously countenance.
But this is not my reason for writing: Gwynne is entitled to his opinions, such as they are, and you are to be congratulated on allowing them to be presented. My real objection to his article is this: How can a Society which advances the possibility that Francis Bacon was responsible for some of the greatest literature in the English language publish something so badly written?
Yours faithfully
DAVID COSTELLO
January 5, 1994
Dear Mr. Bokenham,
Enclosed is a cheque for my subscription to *Baconiana* for 1994, plus a bit extra to help with expenses. My daughter and I were very much interested in your article, *The Money Pit*. I think you are right that this is Raleigh’s money. I was appalled by that dreadful article by N. M. Gwynne, but I suppose your editor feels that we should hear all sides.
I hope for you and the Francis Bacon Society the best of New Years.
With kind regards
OLIVE DRIVER
The Editor *Baconiana*,
I feel I must comment on Martin Gwynne’s article in *Baconiana* 191 in which he tells us that he agrees with the opinions of most of the Francis Bacon Society members regarding Francis Bacon’s authorship of the Shakespeare and other works and of his connection with the Rosicrucian and Freemason Brotherhoods. He also included a number of other works whom he thinks were by Bacon.
He then supplies us with further allegations regarding Bacon’s character and amplified this in a repulsive and extremely biased way. He writes about Bacon’s attempts to overthrow Christian beliefs “which had existed almost universally up till that time, with a ‘shell’ religion which has the appearance of traditional Christianity but was emptied of its meaning and a reversion to paganism”.
This sort of abject nonsense has two main purposes,
1. To try and split the Society into two groups based on religious prejudice.
2. To use *Baconiana* as a vehicle for presenting his own personal views on the divisions in the Christian Church. In the past, these divisions were the cause of unholy wars, due to the intolerance and autocratic doctrines of that Church, a typical example being “The Thirty Years War” which laid waste most of central Europe. Not a particularly Christian way of settling differences. It is well known that Francis Bacon deplored these divisions in Christianity and condemned intolerance on both sides.
During the last hundred years the Francis Bacon Society has done its best to clear Bacon’s name from the malice of the Macaulays of this world. Moreover, those religious differences should be forgotten at this time when the Churches should unite in order to combat the warring factions which now disgrace civilisation.
I would also remind Martin Gwynne that *Baconiana* is not a vehicle for discussing religious differences or a member’s views on that subject.
Yours faithfully
THOMAS BOKENHAM
All the following publications are available from the Francis Bacon Society. Enquiries should be made to the Chairman, T.D. Bokenham, at 56 Westbury Road, New Malden, Surrey KT3 5AX, from whom an up-to-date price list may be obtained.
Baker, H. Kendra
*The Persecution of Francis Bacon*
A story of great wrong. This important book presents lucidly the events and intrigue leading up to the impeachment of Francis Bacon, Lord Chancellor. (Paperback – 1978).
Barker, Richard
*How to Crack the Secret of Westminster Abbey*
A step by step guide to one of the key ciphers concealed in the Shakespeare Monument, and a signpost to what it implies.
Bokenham, T. D.
*A Brief History of the Bacon—Shakespeare Controversy*
A concise and clear summary, concluding with some new cipher evidence. Illustrated. (Paperback – 1982).
Dawkins, A. P.
*Faithful Sayings and Ancient Wisdom*
A personal selection of Francis Bacon's *Essays* and *Fables* from the Wisdom of the Ancients, chosen for the teachings that Bacon gives in these concerning the fundamental laws of Creation and Redemption. Illustrated. (Paperback – 1982).
*Journal 3: Dedication to the Light*
The Bardic Mysteries. The secret marriage of Elizabeth I and Leicester: the birth, adoption and upbringing of Francis Bacon in Bardic and Platonic fashion. (Bacon's life: 1561–2).
*Journal 5: Arcadia*
The Egyptian Mysteries and Hemeticism. The mystery of Arcadia. The secret Arcadian Academy of English alchemical poets & beginnings of modern Freemasonry. (Bacon's life: 1579–85).
*Francis Bacon — Herald of the New Age*
An introductory essay to the genius and hidden nature of Sir Francis Bacon, and to the nature of his vast philanthropic work for mankind.
*Bacon, Shakespeare & Fra. Christian Rose Cross*
Three essays: Francis Bacon, Father of the Rosicrucians / Celestial
Timing – The Virgin Queen and the Rose Cross Knight / Shakespeare: The Sons of the Virgin.
Dodd, Alfred
*Francis Bacon’s Personal Life–Story*
A revealing account of Bacon’s secret as well as public life, revealing his genius and role as poet, author, playwright and director of the English Renaissance, as ‘Shakespeare’, as ‘Solomon’ of English Freemasonry, and as Francis Tudor, son of Queen Elizabeth I. (Hardback – 1986).
Gundry, W. G. C.
*Francis Bacon — A Guide to his Homes and Haunts*
This little book includes some interesting information and many illustrations. (Hardback – 1946).
*Manes Verulamiani*
A facsimile of the 1626 edition of the elegiac tributes to Francis Bacon by the scholars and poets of his day, showing Francis Bacon to have been considered a scholar and a poet of the very highest calibre although ‘concealed’. With translations and commentary, this is a most valuable book. (Hardback – 1950).
Johnson, Edward D.
*Francis Bacon’s Maze*
*The Bilateral Cipher of Francis Bacon*
Durning-Lawrence, Sir Edwin
*Bacon is Shakespeare*
With Bacon’s *Promus*.
Macduff, Ewen
*The Sixty-Seventh Inquisition*
*The Dancing Horse Will Tell You*
These two books demonstrate by means of diagrams and photofacsimiles that a cipher, brilliantly conceived, but simple in execution, exists in the 1623 Shakespeare Folio. The messages revealed, and the method of finding them, form a fascinating study and an unanswerable challenge to disbelievers. The books are the result of many years’ careful research. (Hardbacks – 1972 & 1973).
Melsome, W. S.
*Bacon—Shakespeare Anatomy*
Dr. Melsome anatomises the 'mind' of Shakespeare, showing its exact counterpart in the mind of Francis Bacon. (Hardback – 1945).
Pares, Martin
*Mortuary Marbles*
A collection of six essays in which the author pays tribute to the greatness of Francis Bacon. (Paperback).
*A Pioneer*
A tribute to Delia Bacon. (Hardback – 1958).
*Knights of the Helmet*
Useful notes on the Baconian background. (Paperback – 1964).
Sennett, Mabel
*His Erring Pilgrimage*
An interpretation of *As You Like It*. (Paperback – 1949).
Woodward, Frank
*Francis Bacon's Cipher Signatures*
A well presented commentary on many of the 'Baconian' cipher signatures in text and emblem, with a large number of photofacsimiles. (Hardback – 1923).
IN THE EAST
MY PLEASURE LIES
and other esoteric interpretations
of plays by
WILLIAM SHAKESPEARE
BERYL POGSON
This important book, first published in 1953, has been republished with additional material by Lewis Creed, one of Beryl Pogson’s former pupils.
Obtainable from: Watkins Books Ltd., 19 Cecil Court, London WC2N 4EZ (Tel. 071 836 2182) and from Quacks Books, Petergate, York YO1 2HT (Tel. 0904 635967).
SIR FRANCIS BACON
A BIOGRAPHY
Jean Overton Fuller
Reprint in paperback £10.95, obtainable from George Mann Books,
P.O. Box 22, Maidstone, Kent ME14 1AW.
FRANCIS BACON, SHAKESPEARE
AND THE ROSICRUCIANS
Booklet by T. D. Bokenham, 56 Westbury Road, New Malden,
Surrey KT3 5AX, £5.00.
THE FRANCIS BACON SOCIETY
(INCORPORATED)
The subscription for membership is £7.50 payable on election and on the first day of each succeeding January. Bankers Orders can be provided on request.
Members receive a copy of each issue of *BACONIANA* without further payment, and are entitled to vote at Annual General Meetings. They will also receive invitations whenever possible to lectures and discussions organised by or on behalf of the Society.
Members would assist the Society greatly by forwarding additional donations whenever possible, and by recommending friends for election. Application forms for membership are obtainable from the Chairman, T. D. Bokenham, 56 Westbury Road, New Malden. Surrey. KT3 5AX.
*BACONIANA*
(COPYRIGHT RESERVED)
The official *Journal* of the Francis Bacon Society (Inc.) is published periodically. Back numbers can be supplied. When enquiry is made for particular copies the date should be specified. Some are now scarce, and, in the case of early issues, difficult to obtain unless from members of the Society who may have spare ones. Enquiries for back copies should be made to the Chairman at the address above.
THE FRANCIS BACON SOCIETY LIBRARY
The Society owns a unique collection of some 2000 works relating to Bacon’s life and times, some of which are very rare. Details about the books and where they may be studied are available from the Chairman.
SUBMISSION OF MANUSCRIPTS
The Editor will be glad to receive manuscripts with a view to their publication in a future issue of *Baconiana*. They should be sent to P. A. Welsford, 34 Hartslock Court, Shooter’s Hill, Pangbourne, Berks. RG87 7BJ.
|
Protective Effect of Phloroglucinol on Oxidative Stress-Induced DNA Damage and Apoptosis through Activation of the Nrf2/HO-1 Signaling Pathway in HaCaT Human Keratinocytes
Cheol Park 1,†, Hee-Jae Cha 2,†, Su Hyun Hong 3,4, Gi-Young Kim 5, Suhkmann Kim 6, Heui-Soo Kim 7, Byung Woo Kim 8, You-Jin Jeon 5* and Yung Hyun Choi 3,4,*†
1 Department of Molecular Biology, College of Natural Sciences, Dong-eui University, Busan 47340, Korea; email@example.com
2 Department of Parasitology and Genetics, College of Medicine, Kosin University, Busan 49267, Korea; firstname.lastname@example.org
3 Department of Biochemistry, Dong-eui University College of Korean Medicine, Busan 47227, Korea; email@example.com
4 Anti-Aging Research Center, Dong-eui University, Busan 47227, Korea
5 Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju 63243, Korea; firstname.lastname@example.org (G.-Y.K.); email@example.com (Y.-J.J.)
6 Department of Chemistry, College of Natural Sciences, Pusan National University, Busan 46241, Korea; firstname.lastname@example.org
7 Department of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 46241, Korea; email@example.com
8 Biopharmaceutical Engineering Major, Division of Applied Bioengineering, College of Engineering, Dong-eui University, Busan 47340, Korea; firstname.lastname@example.org
* Correspondence: email@example.com; Tel.: +82-51-850-7413
† These authors equally contributed to this work.
Received: 13 March 2019; Accepted: 11 April 2019; Published: 13 April 2019
Abstract: Phloroglucinol (PG) is a component of phlorotannins, which are abundant in marine brown alga species. Recent studies have shown that PG is beneficial in protecting cells from oxidative stress. In this study, we evaluated the protective efficacy of PG in HaCaT human skin keratinocytes stimulated with oxidative stress (hydrogen peroxide, H$_2$O$_2$). The results showed that PG significantly inhibited the H$_2$O$_2$-induced growth inhibition in HaCaT cells, which was associated with increased expression of heme oxygenase-1 (HO-1) by the activation of nuclear factor erythroid 2-related factor-2 (Nrf2). PG remarkably reversed H$_2$O$_2$-induced excessive ROS production, DNA damage, and apoptosis. Additionally, H$_2$O$_2$-induced mitochondrial dysfunction was related to a decrease in ATP levels, and in the presence of PG, these changes were significantly impaired. Furthermore, the increases of cytosolic release of cytochrome c and ratio of Bax to Bcl-2, and the activation of caspase-9 and caspase-3 by the H$_2$O$_2$ were markedly abolished under the condition of PG pretreatment. However, the inhibition of HO-1 function using zinc protoporphyrin, a HO-1 inhibitor, markedly attenuated these protective effects of PG against H$_2$O$_2$. Overall, our results suggest that PG is able to protect HaCaT keratinocytes against oxidative stress-induced DNA damage and apoptosis through activating the Nrf2/HO-1 signaling pathway.
Keywords: phloroglucinol; keratinocytes; oxidative stress; ROS; Nrf2/HO-1
1. Introduction
Reactive oxygen species (ROS) act as important regulatory molecules of the intracellular signaling system for physiological responses. However, excessive production of ROS beyond the antioxidant capacity of cells can cause irreversible severe oxidative damage to the cells [1,2]. Such damage has been reported to correlate with the incidence of various diseases, such as inflammation, aging, and even cancer [3,4]. Despite the fact that skin has efficient mechanisms to defend against oxidative stress, accumulation of ROS overwhelming the protection from ultraviolet B (UVB) exposure has been shown to have a decisive influence on skin cell damage, including keratinocytes. The skin cells exposed to this injury should induce apoptosis in order to remove damaged cells, and there is much evidence that various enzymatic and non-enzymatic antioxidants reduce ROS-mediated apoptosis [5–7]. Among the intracellular organelles, mitochondria are the main source of ROS production, and are also the most vulnerable targets of ROS. Therefore, inaccurate accumulation of ROS by oxidative stress has been recognized as one of the mechanisms that lead to apoptosis following DNA damage associated with mitochondrial dysfunction [8,9]. These observations suggest that antioxidant supplementation can maintain normal skin cell function by preventing free radical accumulation through the activation of signaling pathways that block ROS production.
There is increasing evidence that various phenolic compounds derived from marine organisms can protect cells from oxidative stress by eliminating free radicals [10,11]. Among them, phloroglucinol (1,3,5-trihydroxybenzene, PG) is one of the components of phlorotannin, and is found in abundance as a polyphenol compound in *Ecklonia cava*, an edible brown alga belonging to the Laminariaceae family [12,13]. PG has been shown to have a variety of pharmacological properties, based on its potent antioxidant activity. For example, PG has been reported to inhibit the apoptosis of blood lymphocytes and splenocytes, and of lung fibroblasts induced by ionizing radiation, by inhibiting the production of intracellular ROS [14,15]. It has also been suggested that PG blocks DNA damage and the apoptosis of lung fibroblasts, keratinocytes and mouse skin by UVB, restoring the activity of antioxidant enzymes, which are associated with the elimination of ROS production [16,17]. In addition, Park and Han [18] found that PG increased the viability and insulin secretion of pancreatic β-cells, by inhibiting the oxidative stress caused by high glucose. In addition to these observations, several previous reports have been published on the in vivo and in vitro protective effects of PG on oxidative stress induced by hydrogen peroxide (H$_2$O$_2$), which has proven the strong antioxidative effects of PG [19–21]. Moreover, the antioxidant effect of PG in Parkinson’s disease model was associated with the increased expression of nuclear factor erythroid 2-related factor (Nrf2), and the activation of Nrf2-mediated antioxidant systems [22]; similarly, Nrf2 was found to be involved in the inhibition of osteoclastogenesis by this substance [23].
Nrf2 is a leucine zipper redox-sensitive transcriptional regulator that regulates the expression of antioxidant and detoxification genes, after translocation from the cytoplasm into the nucleus in response to oxidative stress [24,25]. Among the Nrf2-dependent cytoprotective enzymes, heme oxygenase-1 (HO-1) plays a critical role in heme catabolism, and produces biliverdin, ferrous iron, and carbon monoxide. This rate-limiting enzyme is activated in response to various oxidative signals, and provides adaptive and beneficial cellular responses to oxidative damage, not limited to the degradation of toxic heme released by hemoproteins [26,27]. Although there is diverse evidence that Nrf2/HO-1 signaling protects against cell death by preventing excessive ROS generation under various oxidative stress conditions, studies to date on the direct evidence that its signaling is involved in overcoming oxidative stress by PG have not been well reported. Therefore, in the present study, we investigated whether PG ameliorates oxidative stress (H$_2$O$_2$)-induced DNA damage and apoptosis, and whether the Nrf2/HO-1 signaling pathway is involved in this process in HaCaT human skin keratinocytes.
2. Results
2.1. PG Inhibits $H_2O_2$-Induced Cytotoxicity in HaCaT Keratinocytes
HaCaT cells were treated with various concentrations of $H_2O_2$ for 24 h, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to establish the experimental conditions. Figure 1A shows that HaCaT cells treated with $H_2O_2$ showed significant decrease in cell viability in a concentration-dependent manner. Therefore, the $H_2O_2$ concentration for inducing oxidative stress was selected to be 1 mM, which showed a survival rate of about 60% or less, compared to untreated control cells. To evaluate the protective effect of PG on $H_2O_2$-induced cytotoxicity, HaCaT cells were pretreated with 25 and 50 $\mu$M PG at concentrations that did not show cytotoxicity for 1 h before treatment with 1 mM $H_2O_2$, and cultured for 24 h. Figure 1B shows that the pretreatment with PG significantly restored cell viability in a concentration-dependent manner, compared to $H_2O_2$ alone.
2.2. PG Activates the Nrf2/HO-1 Signaling Pathway in HaCaT Keratinocytes
To investigate whether the anti-cytotoxic effect of PG correlates with the activation of Nrf2/HO-1 signaling, the effect of PG on the expression of Nrf2 and its downstream gene HO-1 was determined. Immunoblotting results showed that the expression of HO-1 protein was slightly increased in HaCaT cells treated with $H_2O_2$, as well as PG alone (Figure 1C,D). However, the expression of HO-1 in $H_2O_2$-treated cells was significantly increased by pretreatment with PG. In addition, the enhanced expression of HO-1 by PG was associated with an increase in total protein expression of Nrf2 and its phosphorylation (p-Nrf2) at serine 40, the active form of Nrf2, whereas, the expression of Kelch-like ECH-associated protein 1 (Keap1) was reduced in a concentration-dependent manner. To test whether the protective effect of PG on $H_2O_2$-induced cytotoxicity is due to the blockade of oxidative stress associated with increased HO-1 expression, N-acetyl cysteine (NAC), a potent antioxidant, and zinc protoporphyrin IX (ZnPP), a selective inhibitor of HO-1, were pretreated. Figure 1E shows that the decrease in cell viability by $H_2O_2$ was markedly attenuated in the cells pretreated with NAC. However, when HO-1 activity was blocked using ZnPP, the inhibitory effect of PG on the cytotoxicity induced by $H_2O_2$ was significantly abolished.
Figure 1. The protective effects of PG against H$_2$O$_2$-induced cytotoxicity in HaCaT cells. Cells were treated with various concentrations of H$_2$O$_2$ for 24 h (A) or treated with the indicated concentrations of PG for 24 h, or treated with 1 mM H$_2$O$_2$ for 24 h after pre-treatment with PG, 10 mM N-acetyl cysteine (NAC), and/or 10 µM zinc protoporphyrin IX (ZnPP) for 1 h (B–E). (A,B,E) After treatment, cell viability was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data are expressed as the mean ± standard deviation (SD) of three independent experiments. Statistical significance analysis was carried out using Student’s $t$-test or ANOVA (* $p < 0.05$ compared with the control group, # $p < 0.05$ compared with the H$_2$O$_2$-treated group, & $p < 0.05$ compared with the PG- and H$_2$O$_2$-treated group). (C) The cellular proteins were prepared, and the levels of HO-1, Nrf2, p-Nrf2 and Keap1 expression were assayed by Western blot analysis using an enhanced chemiluminescence (ECL) detection system. Actin was used as an internal control. These images are representative of at least three independent experiments. (D) Quantification of the ratios of band intensity of HO-1, Nrf2, p-Nrf2, and Keap1 relative to actin. The data were shown as mean ± SD obtained from three independent experiments. Statistical significance analysis was carried out using Student’s $t$-test or ANOVA (* $p < 0.05$ compared with the control group, # $p < 0.05$ compared with the H$_2$O$_2$-treated group, & $p < 0.05$ compared with the PG- and H$_2$O$_2$-treated group).
2.3. PG Inhibits H$_2$O$_2$-Induced ROS Generation in HaCaT Keratinocytes
We investigated whether mitigation of the cytotoxic protective effect of PG on H$_2$O$_2$ in the presence of ZnPP was related to the changes in ROS production. Figure 2A,B show that the production of ROS was markedly increased in H$_2$O$_2$-exposed HaCaT cells; however, the accumulation of ROS in the cells pretreated with PG was significantly reduced, compared to H$_2$O$_2$ alone treatment. The effect of preventing ROS formation was reconfirmed using a fluorescence microscope. Consistent with the results from the flow cytometry, the increase in the DCF-DA fluorescence intensity observed in the cells treated with H$_2$O$_2$ was weakened by the pretreatment of PG, as shown in Figure 2C. Furthermore, in order to determine whether PG-induced HO-1 activation was involved in its inhibitory effect of ROS generation, cells were pretreated with ZnPP and PG, and then exposed to H$_2$O$_2$. Figure 2 shows that ZnPP abrogated the protective effect of PG on H$_2$O$_2$-induced ROS production; but the production of ROS was restrained under the condition that NAC was present at the same time.
Figure 2. Cont.
Figure 2. Inhibition of H$_2$O$_2$-induced ROS generation by PG in HaCaT cells. Cells were pretreated with 50 µM PG, 10 µM ZnPP, and/or 10 mM NAC for 1 h, and then treated with 1 mM H$_2$O$_2$ for 1 h. (A) After staining with 2',7'-dichlorofluorescein diacetate (DCF-DA) fluorescent dye, DCF fluorescence was monitored by a flow cytometer. (B) The data were shown as mean ± SD obtained from three independent experiments. Statistical significance analysis was carried out using Student’s $t$-test or ANOVA (* $p < 0.05$ compared with the H$_2$O$_2$-treated group, # $p < 0.05$ compared with the PG- and H$_2$O$_2$-treated group). (C) The fluorescent images were obtained by fluorescence microscope (original magnification, ×200). These images are representative of at least three independent experiments.
2.4. PG Blocks H$_2$O$_2$-Induced DNA Damage in HaCaT Keratinocytes
We subsequently performed the following three assays to determine whether PG prevents DNA damage. The immunoblotting results in Figure 3A,B show a marked increase in γH2AX phosphorylation (p-γH2AX, at serine 139), one of the DNA strand break markers, in H$_2$O$_2$-stimulated cells, compared to untreated control cells; however, the increased levels of p-γH2AX by H$_2$O$_2$ were almost suppressed to the control level in the presence of PG. In addition, H$_2$O$_2$ treatment significantly increased the production of 8-hydroxy-2′-deoxyguanosine (8-OHdG) adduct, a specific marker of DNA oxidative damage, compared to the control group, but pretreatment of PG significantly reduced the production of 8-OHdG by H$_2$O$_2$ (Figure 3C). Furthermore, in the comet assay, another method for detecting DNA strand breaks, there was no smeared pattern of nuclear DNA in the untreated control and PG alone treated cells. However, in the H$_2$O$_2$-treated cells, the length of the comet tail clearly increased, which means DNA damage occurred, and in the PG pretreated cells, tail length was obviously shorter than in the H$_2$O$_2$-treated cells (Figure 3D). On the other hand, ZnPP abrogated some, but not all, of the protective effects of PG on the H$_2$O$_2$-induced DNA damages.
Figure 3. Cont.
Figure 3. Protection of H$_2$O$_2$-induced DNA damage by PG in HaCaT cells. Cells were pretreated with 50 µM PG and/or 10 µM ZnPP for 1 h, and then stimulated with or without 1 mM H$_2$O$_2$ for 24 h. (A) The cellular proteins were prepared, and p-γH2AX and γH2AX protein levels were assayed by Western blot analysis, using an ECL detection system. Actin was used as an internal control. (B) Quantification of the ratios of band intensity of p-γH2AX relative to γH2AX. (C) The amount of 8-hydroxy-2′-deoxyguanosine (8-OHdG) in DNA was determined using an 8-OHdG-enzyme immunoassay (EIA) kit. The measurements were made in triplicate, and values are expressed as the mean ± SD. Statistical significance analysis was carried out using Student’s t-test or ANOVA (* $p < 0.05$ compared with the control group, # $p < 0.05$ compared with the H$_2$O$_2$-treated group, & $p < 0.05$ compared with the PG- and H$_2$O$_2$-treated group). (D) The comet assay was performed, and representative images of comet assay were taken by fluorescence microscope (original magnification, ×200). Representative photographs are shown.
2.5. PG Suppresses H$_2$O$_2$-Induced Apoptosis in HaCaT Keratinocytes
4,6-diamidino-2-phenylindole (DAPI) staining, flow cytometry analysis, and agarose gel electrophoresis were performed, to investigate whether the cytoprotective effect of PG against H$_2$O$_2$ on HaCaT cells was related to apoptosis suppression. The fluorescent images in Figure 4A reveal that the control cells have intact nuclei, while the H$_2$O$_2$-treated cells show significant chromatin condensation, which is observed in the apoptosis-induced cells. However, the morphological changes are markedly attenuated in the cells pretreated with PG before the treatment with H$_2$O$_2$. The results of Annexin V/propidium iodide (PI) double staining also show that the pretreatment of PG significantly decreased the frequency of apoptotic cells in H$_2$O$_2$-stimulated cells (Figure 4B,C). In addition, in the cells treated with PG prior to H$_2$O$_2$ stimulation, the inhibition of colony formation by H$_2$O$_2$ is markedly reduced in a concentration-dependent manner (Figure 4D). Furthermore, H$_2$O$_2$-induced DNA fragmentation is markedly blocked in the presence of PG (Figure 4D).
Figure 4. Cont.
Figure 4. The inhibitory effects of PG against H$_2$O$_2$-induced apoptosis in HaCaT cells. Cells were pretreated with 50 μM PG and/or 10 μM ZnPP for 1 h, and then stimulated with or without 1 mM H$_2$O$_2$ for 24 h. (A) The cells were fixed and stained with 4,6-diamidino-2-phenylindole (DAPI) solution. The stained nuclei are pictured under fluorescence microscope (original magnification, ×400). Representative photographs are shown. (B) The cells were stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (PI) for flow cytometry analysis. The percentages of apoptotic cells were determined by counting the percentage of Annexin V-positive cells. (C) Data are expressed as the mean ± SD of three independent experiments. Statistical significance analysis was carried out using Student’s t-test or ANOVA (* $p < 0.05$ compared with the control group, # $p < 0.05$ compared with the H$_2$O$_2$-treated group, & $p < 0.05$ compared with the PG- and H$_2$O$_2$-treated group). (D) DNA fragmentation was analyzed by extracting genomic DNA, electrophoresis in 1.5% agarose gel, and then visualizing by ethidium bromide (EtBr) staining.
2.6. PG Reduces H$_2$O$_2$-Induced Mitochondrial Dysfunction in HaCaT Keratinocytes
To examine the protective effect of PG on mitochondrial dysfunction by H$_2$O$_2$, mitochondrial membrane potential (MMP) and intracellular ATP levels were evaluated. According to the results of 5,5′/6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) staining shown in Figure 5A,B, changes in the ratio of polarized and depolarized cell populations were observed in HaCaT cells treated with H$_2$O$_2$. Along with the results, the concentration of ATP in cells exposed to H$_2$O$_2$ was significantly decreased, compared with cells cultured in normal medium (Figure 5C). However, PG was able to prevent these changes, and the protective effects of PG were significantly abrogated in the presence of ZnPP.
Figure 5. Attenuation of H$_2$O$_2$-induced mitochondrial dysfunction by PG in HaCaT cells. Cells were pretreated with 50 μM PG and/or 10 μM ZnPP for 1 h, and then stimulated with or without 1 mM H$_2$O$_2$
for 24 h. (A) The cells were collected and incubated with 10 μM of 5,5′6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1), and the values of mitochondrial membrane potential (MMP) were evaluated by flow cytometer. (B) The results are the mean ± SD obtained from three independent experiments. (C) A commercially available kit was used to monitor the ATP production using a luminometer. Each point represents the mean ± SD of three independent experiments. Statistical significance analysis was carried out using Student’s *t*-test or ANOVA (* $p < 0.05$ compared with the control group, # $p < 0.05$ compared with the H$_2$O$_2$-treated group, & $p < 0.05$ compared with the PG- and H$_2$O$_2$-treated group).
### 2.7. PG Restores H$_2$O$_2$-Induced Alteration of the Apoptosis Regulatory Genes in HaCaT Keratinocytes
To further investigate the mechanisms of the anti-apoptotic effect of PG and the role of HO-1, we examined the effects of PG and ZnPP on the H$_2$O$_2$-induced changes of expression of apoptosis regulatory genes. The immunoblotting results of Figure 6A,B show that anti-apoptotic Bcl-2 protein was significantly down-regulated in H$_2$O$_2$-treated HaCaT cells, while the pro-apoptotic Bax protein was up-regulated. Additionally, the expression of pro-caspase-9 and -3 was markedly reduced in H$_2$O$_2$-treated cells as compared with the control, and the expression of cleaved poly (ADP-ribose) polymerase (PARP), a representative substrate protein degraded by activated caspase-3, was also increased. Furthermore, the expression of cytochrome *c* in H$_2$O$_2$-stimulated cells increased in the cytoplasmic fraction, compared to the mitochondrial fraction, indicating that cytochrome *c* was released from the mitochondria into the cytoplasm (Figure 6C,D). However, these changes by H$_2$O$_2$ treatment were relatively conservative in the PG-pretreated cells, and the protective potentials of PG disappeared under the condition in which the activation of HO-1 was suppressed.
**Figure 6.** Effect of PG on the expression of apoptosis regulatory genes in H$_2$O$_2$-treated HaCaT cells. (A) After treatment with 50 μM PG and/or 10 μM ZnPP in the presence or absence of 1 mM H$_2$O$_2$ for 24 h, the cellular proteins were prepared, and the protein levels were assayed by Western blot analysis using an ECL detection system. Actin was used as an internal control. (B) Quantification of the ratios of band intensity of Bcl-2, Bax, caspase-9, caspase-3, and PARP relative to actin. (C) The mitochondrial and cytosolic proteins isolated from cells cultured under the same conditions were separated by sodium-dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and transferred to the membranes. The membranes were probed with anti-cytochrome *c* antibody. Equal protein loading was...
confirmed by the analysis of oxidase subunit IV (COX IV) and actin in each protein extract (M.F., mitochondrial fraction; C.F., cytosolic fraction). Representative photographs are shown.
(D) Quantification of the ratios of band intensity of cytochrome c relative to COX IV or and actin, respectively. Statistical significance analysis was carried out using Student’s t-test or ANOVA (* p < 0.05 compared with the control group, # p < 0.05 compared with the H$_2$O$_2$-treated group, & p < 0.05 compared with the PG- and H$_2$O$_2$-treated group).
3. Discussion
In the present study, we investigated whether PG is effective in preventing oxidative stress-induced cytotoxicity through Nrf2-mediated HO-1 activation in HaCaT keratinocytes. The results of the present study demonstrate that PG prevented H$_2$O$_2$-induced DNA damage and apoptosis through the rescue of mitochondrial function by blocking ROS accumulation. We also found that PG promoted activation of the Nrf2/HO-1 signaling pathway, and inhibition of HO-1 activity eliminated the protective effect of PG, suggesting that the protective effects of PG in HaCaT cells were at least HO-1 dependent.
The mechanism of Nrf2 induction depends on the inducers and cell types, but Nrf2 plays a central role in protecting cells from oxidative damage by regulating the transcriptional activity of antioxidant genes, including HO-1 [24,27,28]. Under normal physiological conditions, Nrf2 binds to the repressor protein Keap1, and is thereby sequestered in the cytoplasm. However, in order to protect against oxidative stress, phosphorylation by several kinases is necessary for the nuclear translocation of Nrf2 liberated by the degradation of Keap1 through the ubiquitin proteasome pathway [28,29]. In our results, H$_2$O$_2$ alone partially increases the expression of HO-1, as well as the activation of Nrf2, but their expression and activity were further markedly increased by the co-treatment with PG, compared to cells treated with H$_2$O$_2$ alone. Similar to the results of this study, previous studies have shown that H$_2$O$_2$ can enhance the expression of HO-1 [30,31], and up-regulation of HO-1 has been identified as a defense mechanism against H$_2$O$_2$-induced apoptosis in a variety of cell types [24,25]. Therefore, we hypothesized that the induction of HO-1 by PG could block H$_2$O$_2$-induced DNA damage and apoptosis, through blocking ROS generation. In this study, we investigated the effect of ZnPP on the inhibitory effect of ROS production by PG in H$_2$O$_2$-treated cells, and observed that ZnPP treatment weakened the ROS scavenging activity by PG. These results suggest that the protective effect of PG on H$_2$O$_2$-induced oxidative stress in HaCaT keratinocytes is mediated through the activation of Nrf2/HO-1 signaling. Furthermore, the beneficial effect of PG on H$_2$O$_2$-induced DNA damage was significantly reduced by ZnPP, which also implied that the protective role of PG on H$_2$O$_2$-induced cytotoxicity was dependent on HO-1. These results are in good agreement with previous results that HO-1 confers resistance to DNA damage and apoptosis induced by oxidative stress [26,27,30], and inhibition of HO-1 activity increases oxidative stress-induced cytotoxicity, and decreases the efficacy of antioxidants [31,32].
Much experimental evidence supports that excessive ROS accumulation beyond the antioxidant function of cells is one of the mechanisms leading to apoptosis associated with mitochondrial injury [1,2]. In the induction of ROS-mediated apoptosis, ROS overload causes free radical attack of the membrane phospholipid, which in turn leads to mitochondrial membrane depolarization, resulting in the loss of MMP. Subsequently, the apoptogenic factors are released into the cytoplasm from the mitochondrial intermembrane space, and the caspase cascade is activated, which could eventually trigger apoptosis. This is considered to be the onset of the intrinsic apoptosis pathway [33,34]. At the same time, mitochondrial dysfunction promotes abnormalities in the mitochondrial respiratory chain’s electron transport pathways, ultimately interfering with intracellular ATP production [33,35]. Therefore, the intracellular ATP levels can also be used as an important index to assess the homeostasis of mitochondrial energy metabolism associated with oxidative stress [36,37]. However, HO-1 activation is recognized as a cell protection mechanism against oxidative stress-mediated mitochondrial dysfunction through inactivation of the mitochondria-mediated intrinsic apoptosis pathway [26,27]. Consistent
with previous studies [38–40], the present study shows that when cells were exposed to H$_2$O$_2$, MMP levels and ATP contents were significantly reduced compared to controls, whereas PG significantly reversed the H$_2$O$_2$-induced loss of MMP and APT. However, in the presence of ZnPP, PG-mediated repair of mitochondrial dysfunction and the decreased production of ATP were significantly abolished. These results also match well with previous results showing that the protective effects of apoptosis against oxidative stress are related to the maintenance of ATP production by the preservation of mitochondrial function [41,42]. We therefore consider that the conservation of ATP production due to the retention of mitochondrial function is one possible mechanism by which PG can preserve the cell survival pathway from oxidative stress in HaCaT keratinocytes through HO-1 activation.
Activation of caspase-9 by cytosolic release of apoptotic factors including cytochrome c due to the loss of MMP is a major initial step in the initiation of a caspase-dependent intrinsic apoptosis pathway [43,44]. Activation of caspase-9 sequentially activates downstream effector caspases, including caspase-3 and -7, eventually leading to cell death. This process is accompanied by degradation of the substrate proteins of effector caspases, such as PARP, as evidence that caspase-dependent apoptosis is induced [43,45]. The activation of caspase is regulated by various proteins, including Bcl-2 family members consisting of anti-apoptotic and pro-apoptotic proteins. Among the Bcl-2 family members, anti-apoptotic proteins, such as Bcl-2, are located on the outer mitochondrial membrane to prevent the release of apoptogenic factors [45,46]. On the other hand, pro-apoptotic proteins, including Bax, antagonize anti-apoptotic proteins, or translocate to mitochondrial membranes to form membrane-integrated homo oligomers that induce mitochondrial pore formation, leading to the loss of MMP, and resulting in the cytosolic release of apoptotic factors [47,48]. Therefore, the balance of apoptotic Bax family proteins to the anti-apoptotic Bcl-2 family proteins serves as a determinant to induce or inhibit the activation of the caspase cascade for the initiation of the intrinsic apoptosis pathway. Many previous studies have shown that the induction of apoptosis by H$_2$O$_2$ was associated with a decrease in the Bcl-2/Bax ratio and/or activation of caspases [49,50]. However, several antioxidant natural products that can protect H$_2$O$_2$-mediated cytotoxicity have altered this tendency [51,52]. Furthermore, it has been reported that these phenomena are also involved in the defense mechanism against oxidative stress of PG [14,18,53]. Consistent with previous findings, our results show that the decreased expression of Bcl-2 and increased expression of Bax observed in H$_2$O$_2$-treated HaCaT cells reverted in the presence of PG. In addition, PG administration also blocked the H$_2$O$_2$-induced activation of caspase-9 and -3, and the degradation of PARP. In this respect, it is suggested that PG can rescue H$_2$O$_2$-induced cytotoxic injury in HaCaT cells by blocking mitochondria-mediated oxidative stress and apoptosis. However, these protective effects of PG against H$_2$O$_2$ were markedly hindered by the inhibition of HO-1 function using an HO-1 inhibitor, consistent with other studies [38,54,55]. Therefore, further studies on the relevance of various Nrf2-dependent enzymes besides HO-1 should be attempted, but the current results suggest that the cellular protective potential of PG against oxidative stress in HaCaT cells is at least dependent on the activation of Nrf2/HO-1 signaling.
4. Materials and Methods
4.1. Cell Culture and PG Treatment
HaCaT keratinocyte cell line was obtained from the American Type Culture Collection (Manassas, MD, USA). The cells were maintained at 37 °C in an incubator with a humidified atmosphere of 5% CO$_2$ and cultured in RPMI 1640 medium (WelGENE Inc., Daegu, Korea) containing 10% (v/v) heat-inactivated fetal bovine serum (WelGENE Inc.), streptomycin (100 µg/mL), and penicillin (100 Units/mL). PG was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich Chemical Co.), and diluted with cell culture medium to adjust the final treatment concentrations, prior to use in the experiments.
4.2. Cell Viability Assay
For the cell viability study, HaCaT cells were seeded in 96-well plates at a density of $5 \times 10^3$ cells per well. After 24 h incubation, the cells were incubated with PG at 25 and 50 $\mu$M or 1 mM H$_2$O$_2$ for another 24 h, or pre-incubated with 25 and 50 $\mu$M PG for 1 h, before 1 mM H$_2$O$_2$ treatment for 24 h. The cells were also treated with 10 $\mu$M ZnPP (Sigma-Aldrich Chemical Co.), a well-established HO-1 inhibitor, or 10 mM NAC (Sigma-Aldrich Chemical Co.), a ROS scavenger, for 1 h in the presence or absence of H$_2$O$_2$. Subsequently, the medium was removed, and 0.5 mg/ml MTT (Sigma-Aldrich Chemical Co.) was added to each well and incubated at 37 °C for 3 h. The supernatant was then replaced with an equal volume of DMSO, to dissolve the blue formazan crystals for 10 min. Optical density was measured at a wavelength of 540 nm with a microplate reader (Dynatech Laboratories, Chantilly, VA, USA). All experiments were performed in triplicate, and the results were expressed as the percentage of MTT reduction, assuming that the absorbance of control was 100%.
4.3. Western Blot Analysis
After being subjected to the necessary experimental treatments, the cells were harvested, washed with phosphate buffered saline (PBS), and lysed with lysis buffer (25 mM Tris-Cl (pH 7.5), 250 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 1% Nonidet-P40, 1 mM phenylmethylsulfonyl fluoride, 5 mM dithiothreitol) for 30 min to extract whole-cell proteins. In a parallel experiment, mitochondrial and cytosolic proteins were extracted using a mitochondria isolation kit purchased from Active Motif (Carlsbad, CA, USA), in accordance with the instructions of the manufacturer for detecting the cytosolic release of cytochrome c from mitochondria. Equal amounts of protein samples (40 $\mu$g) were subjected to SDS-polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% bovine serum albumin (Sigma-Aldrich Chemical Co.) for 1 h in a mixture of Tris-Buffered Saline and Tween-20 (TBST), and probed with primary antibodies (Table 1) overnight at 4 °C. Immunoreactive bands were detected using horseradish peroxidase (HRP)-conjugated secondary antibodies (GE Healthcare Pharmacia, Uppsala, Sweden), and visualized using an ECL reagent (Amersham Biosciences, Westborough, MA, USA), according to the manufacturer’s instructions. The immunoreactive bands were detected and exposed to X-ray film. Bands of Western blotting were quantified using ImageJ (Ver. 1.46; NIH, Bethesda, MD, USA) and the ratio was determined.
| Antibody | Manufacturer | Item No. | Dilution |
|--------------|-------------------------------|------------|----------|
| HO-1 | Merck Millipore | 374090 | 1:1000 |
| Nrf2 | Santa Cruz Biotechnology, Inc.| sc-13032 | 1:1000 |
| p-Nrf2 | Abcam, Inc. | ab76026 | 1:1000 |
| Keap1 | Santa Cruz Biotechnology, Inc.| sc-15246 | 1:1000 |
| p-$\gamma$H2AX | Cell Signaling Technology, Inc. | 9718 | 1:500 |
| $\gamma$H2AX | Cell Signaling Technology, Inc. | 7631 | 1:500 |
| Bcl-2 | Cell Signaling Technology, Inc.| sc-509 | 1:1000 |
| Bax | Cell Signaling Technology, Inc.| sc-493 | 1:1000 |
| Caspase-9 | Santa Cruz Biotechnology, Inc.| sc-7885 | 1:1000 |
| Caspase-3 | Santa Cruz Biotechnology, Inc.| sc-7272 | 1:1000 |
| Cytochrome c | Santa Cruz Biotechnology, Inc.| sc-7159 | 1:500 |
| COX IV | Santa Cruz Biotechnology, Inc.| sc-376731 | 1:1000 |
| Actin | Santa Cruz Biotechnology, Inc.| sc-47778 | 1:1000 |
4.4. Measurement of ROS Level
To measure the formation of intracellular ROS, cells were seeded onto 6-well plates with a density of $3 \times 10^5$ cells per well for 24 h, and treated with or without PG, ZnPP, or NAC for 1 h, before adding H$_2$O$_2$ for a further 1 h. The cells were washed twice with PBS, suspended in PBS, and stained with
10 μM DCF–DA (Sigma-Aldrich Chemical Co.) for 20 min at 37 °C. The relative fluorescence intensity of the cell suspensions was measured by flow cytometer (Becton Dickinson, Becton Dickinson, San Jose, CA, USA). For image analysis for intracellular ROS production, the cells were seeded on a coverslip loaded 6-well plate. After 24 h of plating, cells were treated with PG, ZnPP, or NAC; and 1 h later, H₂O₂ was added to the plate for 1 h. After washing with PBS, 10 μM DCF–DA was added to the well, and incubated at 37 °C for an additional 20 min. The fluorescence of DCF was visualized by fluorescence microscopy (Carl Zeiss, Oberkochen, Germany).
4.5. Comet Assay for DNA Damage
The comet assay was performed to assess the oxidative DNA damage in individual cells. After the respective treatment, the cells were detached from the culture surface, mixed with 0.75% low-melting agarose (LMA), and then spread on a fully frosted microscopic slide that was pre-coated with 0.75% normal melting agarose. After solidification of the agarose, the slides were covered with LMA, and immersed in lysis solution (2.5 M NaCl, 100 mM Na-EDTA, 10 mM Tris, 1% Triton X-100, and 10% DMSO, pH 10) for 1 h at 4 °C, to remove proteins. The slides were then placed in a gel-electrophoresis apparatus containing electrophoresis buffer (300 mM NaOH, 10 mM Na-EDTA, pH 10) for 20 min, to allow for DNA unwinding and the expression of the alkali labile damage. Thereafter, an electrical field was applied in the same buffer for 20 min at 4°C, to draw the negatively charged DNA towards the anode. After electrophoresis, the slides were rinsed gently three times with the neutralization buffer (0.4 M Tris-HCl, pH 7.5) for 10 min at 25 °C, followed by staining with 40 μg/mL EtBr (Sigma-Aldrich Chemical Co.). The slides were observed under fluorescence microscopy.
4.6. Determination of 8-OHdG
The BIOXYTECH® 8-OHdG-EIA™ kit (OXIS Health Products Inc., Portland, OR, USA) was used for quantitative measurement of oxidative DNA damage according to the manufacturer’s instructions. Briefly, after the necessary experimental treatment, the cellular DNA was isolated using the DNA Extraction Kit (inTRON Biotechnology Inc., Sungnam, Korea) following the manufacturer’s protocol, and quantified. The amount of 8-OHdG, a deoxyriboside form of 8-oxoGuanine, in the DNA was determined by calculation on a standard curve measured at 450 nm absorbance, using a microplate reader, according to the manufacturer’s instructions.
4.7. Apoptosis Assay Using Fluorescence Microscopy
To evaluate the induction of apoptosis by observing the morphological changes of nuclei, the cells were harvested and washed with PBS, and then fixed with 4% paraformaldehyde (Sigma-Aldrich Chemical Co.) in PBS for 30 min at room temperature (RT). Next, the cells were stained with 1.0 mg/ml DAPI (Sigma-Aldrich Chemical Co.) solution, a DNA-specific fluorescent dye, for 10 min at RT in the dark, and then washed twice with PBS. The degree of nuclear condensation was evaluated in the stained cells under fluorescence microscopy.
4.8. Apoptosis Analysis Using Flow Cytometry
The extent of apoptosis was determined by flow cytometer using Annexin V/PI double staining. In brief, the cells were re-suspended in supplied binding buffer, and then stained with FITC-conjugated annexin V and PI (BD Pharmingen, San Diego, CA, USA) at RT for 20 min in darkness, according to the manufacturer’s protocol. The fluorescence intensities of the cells were detected by flow cytometer (Becton Dickinson), and acquisition was performed using the Cell Quest Pro software (Becton Dickinson). The annexin⁻/PI⁻ cell population was considered as normal, while the annexin V-FITC⁺/PI⁻ and annexin⁺/PI⁺ cell populations were considered as indicators of apoptotic cells.
4.9. Detection of DNA Fragmentation
The collected cells were dissolved in lysis buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, and 0.1 mg/mL proteinase K) for 30 min at RT. DNA from the supernatant was extracted by chloroform/phenol/isoamyl alcohol (24/25/1, v/v/v; Sigma-Aldrich Chemical Co.), and was precipitated by ethanol. The extracted DNA was then transferred to 1.5% agarose gel containing 0.1 μg/mL EtBr, and electrophoresis was carried out at 70 V, using Tris-acetate-EDTA (pH 8.0) running buffer. DNA aliquots were observed and photographed under UV illumination (260 nm) in a BioRad gel documentation system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
4.10. Analysis of MMP
To measure the loss of MMP, the cells were collected, and incubated in media containing 10 μM JC-1 (Sigma-Aldrich Chemical Co.), which is a mitochondria-specific fluorescent dye, at 37 °C for 20 min at RT in darkness. After washing twice with PBS to remove unbound dye, the green (JC-1 monomers) and red (JC-1 aggregates) fluorescence ratio that monitored the proportion of mitochondrial depolarization was immediately acquired on flow cytometer, by following the manufacturer’s instructions.
4.11. Detection of ATP Levels
The levels of intracellular ATP were determined using a firefly luciferase-based ATP Bioluminescence Assay Kit (Roche Applied Science, Indianapolis, IN, USA), according to the manufacturer’s instructions. Briefly, cells cultured under various conditions were lysed with the provided lysis buffer, and the collected supernatants were mixed with an equal amount of luciferase agent, which catalyzed the light production from ATP and luciferin. The emitted light was immediately measured using a microplate luminometer, and the ATP level was calculated according to the ATP standard curve. Intracellular ATP levels were calculated as a percentage of the untreated control.
4.12. Colorimetric Assay of Caspase-3 Activity
The activity of the caspase-3 was determined using a colorimetric assay kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s protocol. Briefly, at the end of treatment, the collected cells were lysed, and equal amounts of proteins were incubated with the supplied reaction buffer, containing dithiothreitol and tetrapeptides (Asp-Glu-Val-Asp (DEAD)-p-nitroaniline (pNA)) as caspase-3 substrates at 37 °C in the dark. After 2 h of reaction, the reactions were measured by changing the absorbance at 405 nm using a microplate luminometer, as per the manufacturer’s protocols. The results were represented as multiples of untreated control cells.
4.13. Statistical Analysis
Data were expressed as the means ± SD from at least three independent experiments. Statistical significance analysis was carried out using Student’s t-test or ANOVA. A p-value less than 0.05 were considered to indicate statistical significance.
5. Conclusions
In summary, the present study demonstrates that PG can effectively protect HaCaT keratinocytes from H$_2$O$_2$-induced cytotoxicity, by blocking oxidative stress-mediated DNA damage and mitochondria-dependent apoptotic pathway through the activation of Nrf2/HO-1 signaling. Although studies of mitochondrial damage-associated energy metabolism and PG downstream signal molecules are needed, these findings may be presented as evidence that PG can alter the redox state of cells, and thereby regulate cellular antioxidant signaling pathways. Therefore, additional studies should be conducted to determine whether pathways other than the mitochondria-mediated pathway may be involved in inducing apoptosis by oxidative stress, including in vivo animal experiments.
**Author Contributions:** Conceptualization, data analysis, methodology, writing and review of manuscript, funding acquisition, project administration, Y.-J.J. and Y.H.C.; performing experiments, data analysis, writing and initial draft preparation, C.P., H.-J.C. and H.-S.K.; performing experiments, data analysis, S.H.H., G.-Y.K., S.K. and B.W.K.; set up first experiments, C.P. and H.-J.C.; manuscript review, Y.H.C.
**Funding:** This research was a part of the project titled ‘Development of functional food products with natural materials derived from marine resources (20170285)’, and ‘Omics based on fishery disease control technology development and industrialization (20150242)’, funded by the Ministry of Oceans and Fisheries, Korea.
**Conflicts of Interest:** The authors declare no conflict of interest.
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18. Park, M.H.; Han, J.S. Phloroglucinol protects INS-1 pancreatic β-cells against glucotoxicity-induced apoptosis. *Phytother. Res.* **2015**, *29*, 1700–1706. [CrossRef]
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I LOVE YOUR SMILE
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THOUGHT I'D DIED AND GONE TO HEAVEN
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HEAD OVER HEELS
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UNTIL YOUR LOVE COMES AROUND
RTZ
YOUR SONG
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CHURCH OF YOUR HEART
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SAVE THE BEST FOR LAST
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MASTERPIECE
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HITS TO WATCH
YOU'RE ALL THAT MATTERS TO ME
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Big House
SHE RUNS HOT
Little Village
SHAKE THIS TOWN
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Tina Turner
ALL WOMAN
Lisa Stansfield
AIN'T IT HEAVY
Melissa Etheridge
IF YOU COULD SEE ME NOW
Devonsquare
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WAYNE'S WORLD
Soundtrack
AWESOME HITS
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Little Village
NO GRAPHIC AVAILABLE
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Mr. Big
Atlantic
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THERE AIN'T NOTHIN' WRONG WITH THE RADIO
Aaron Tippin
EVERY SECOND
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THE TIPS OF MY FINGERS
Steve Wariner
THE ROCK
Lee Roy Parnell
FAMILIAR PAIN
Restless Heart
PLAY, RUBY, PLAY
Clinton Gregory
LOVER NOT A FIGHTER
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Mark Chesnutt
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No. 1 ALBUM
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DGCD-24425-J
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COLOR ME BADD
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Brent Lee and the Outsiders must own some kind of record. They've been on MuchMusic 15 times and they don't even have a video out yet. -Page 7
CRTC clarifies ownership of MAPL logo and system
The Canadian Radio-Television and Communications Commission (CRTC) has issued a press release (Feb. 25/92) which addresses some misunderstandings about the use of the MAPL system logo.
An article in the Toronto Star (Feb. 8/92) featuring CBC Entertainment's Karen Gordon confused many people in the industry when she suggested that a Canadian producer would qualify for P (production) in the system. The fact sheet from the CRTC shows that, in fact, only the geographical location of the recording qualifies, and that the recording must be produced in Canada to qualify for P. The fact sheet also clarifies that the MAPL logo was created by and is owned by Stan Klees and used in RPM. The logo is available for use by the industry in both the English and French sectors through permission from RPM.
RPM is reprinting this press release to ensure that the industry has a full understanding of the MAPL system and the use of the MAPL logo.
The MAPL System
What is it?
The MAPL* system refers to the four elements in the Radio Regulations used to identify musical selections as Canadian. These elements were selected as part of the Canadian content regulations, following an extensive public hearing process.
Objectives
The primary objective of the regulation -- a cultural one -- is to encourage increased exposure of Canadian musical performers, lyricists and composers to Canadian audiences.
The secondary objective -- an industrial one -- is to strengthen the Canadian music industry, including both the creative and production components.
How does it work?
Basically, this is a point system. To qualify as "Canadian content" a recording must fulfill at least two of the following four conditions:
Music (M) - the music must be composed by a Canadian.
Artist (A) - the music or lyrics must be principally performed by a Canadian.
Production (P) - the musical selection consists of a live performance that is (i) recorded wholly in Canada, or (ii) performed wholly in Canada and broadcast live in Canada.
Lyrics (L) - the lyrics must be written by a Canadian.
This Canadian content regulation is designed to stimulate all components of the Canadian music industry and to be as simple as possible for the industry to implement and regulate.
*The MAPL acronym and logo were created by Stan Klees for RPM Weekly and are used by both the English and French sectors of the Canadian music industry. The logo is made available for use by the industry upon request to RPM Weekly.
Sony’s new Mini Disc set for August launch
Sony’s Mini Disc, a breakthrough in the evolution of digital audio technology, was introduced to members of the Canadian music industry last Friday.
The 64 millimetre (2.56 inch) Mini Disc, and its player, are both smaller than compact discs and players. Another Mini Disc advantage is its ability to digitally record up to 74 minutes per disc while still offering the same noiseless and distortion-free digital technology as CDs during playback.
The Mini Disc offers quick random access like CDs and can’t stretch, break or tangle like cassette tapes. The disc is housed in a caddy and new technology using an advanced semiconductor memory provides almost total shock resistance so there is no skipping even while jogging or driving.
Pre-recorded Mini Discs will be sold in record stores similarly to CDs and cassettes. Sony hopes to price Mini Discs on par with CDs and have them on the market by August. Recorder players will be in the $500 range while basic players will be around $200.
Sony plans on manufacturing three million Mini Discs by the end of the year.
Veteran Wilf Gillmeister dead after lengthy illness
Industry pioneer Wilf Gillmeister passed away Feb. 12 at his home in Ajax, Ont. after a lengthy illness. Mr. Gillmeister, who was 78 at the time of his death, joined RCA Victor in Montreal in 1939. He joined the RCAF when war broke out and went overseas as a navigator. He was captured after being shot down over France and remained a prisoner of war for 13 months.
Mr. Gillmeister returned to Canada after the war and rejoined RCA, working as an assistant to the late Hugh Josephs. He was heavily involved in A&R and royalties for the company, contributing much to the growing importance of these fairly new functions at RCA.
Mr. Gillmeister was also a well known trumpeter and, with Oscar Peterson on keyboards, toured with one of Montreal’s most popular big bands. He also played with many of the top visiting international big bands and quite often shared the spotlight with Maynard Ferguson.
Through his 37 year association with RCA, Mr. Gillmeister contributed much to the label’s evolution as one of the most successful record manufacturing companies in Canada. He was very dedicated to popularizing the Camden label, and was instrumental in launching Stereo 8. He served in managerial capacities in both the marketing and merchandising operations of the company, and also served as a product manager and manager of planning scheduling when the Smiths Falls plant was in operation.
Mr. Gillmeister is survived by his wife Phyllis, who also worked for RCA at the Don Mills, Ontario location and two daughters, Dorothy Gillmeister in Sault Ste. Marie, and Lorraine Krause of Huntingdon, Quebec.
Memorial services and burial will be held in Hudson, Quebec at a later date.
EMI Music Publishing signs Toronto’s Soil
EMI Music Publishing Canada has signed Vadim Rodomar and Matthew DeMatteo, primary songwriters for Soil, a Toronto-based alternative and dance band.
Hank Medress, president of EMI Music Publishing Canada (centre) with Vadim Rodomar and Matthew DeMatteo, primary songwriters for Toronto based Soil, recently signed to the publishing firm.
Maritime recording artists share awards spotlight
Cape Breton sweep of East Coast Music Awards
The East Coast Music Awards, held at the 1,100 seat Rebecca Cohn Auditorium in Halifax (Feb. 16) drew a packed house to honour the best from Canada's Atlantic provinces. There were some surprises and a lot of encouragement for independent recording artists at the awards show, which capped to the annual three-day music conference.
Cape Breton acts won eight of the 15 awards. The Rankin Family, from Mabou, a traditional/original music band, won the phone-in poll as Entertainer of the Year. The group also won two industry-judged awards -- as Live Act of the Year, and Jimmy Rankin's penning of Orangedale Whistle, which took the Song of the Year award.
Also from "the right side of the causeway," Barra MacNeils, a family of three brothers and a sister from Sydney Mines, took the Recording Band of the Year award and won the Album of the Year category for their Time Frame release. Sydney-based Real World won in the Pop-Rock Artist category.
In keeping with the Celtic treasures that often dominate music from the Atlantic Provinces, two fiddlers from Cape Breton didn't go unnoticed. Natalie MacMaster won for Best Roots-Traditional Artist and Howie MacDonald as Top Traditional Instrumentalist.
Cape Breton could also be credited with the Lennie Gallant win as Vocalist of the Year and for winning the Video of the Year for Man Of Steel. The Prince Edward Island Acadian singer/songwriter obviously touched a politico/social nerve with his song about unemployed Cape Breton steelworkers.
One of the surprise nominees on hand for the awards presentations was Nettwerk (Capitol) recording artist Sarah McLachlan, who also performed. A former Haligonian now living in Vancouver, McLachlan -- who has gained international recognition -- flew in from San Francisco to accept her award as Female Vocalist of the Year. She had been nominated in six other categories, but was obviously delighted with this important solo win.
New Brunswick's Joan Kennedy, no stranger to the charts, was voted Country Artist of the Year. The Dr. Helen Creighton Lifetime Achievement Award was awarded posthumously to the late Newfoundland fiddler, Rufus Guinchard. He was honoured him for preserving much of Newfoundland's traditional music.
Other award winners were Nova Scotia's (Sydney Mines) John Campbelljohn, who took the Unrecorded Artist of the Year Award, and Nova Scotia's Basil Donovan of Blue Rodeo who took the Goin' Down The Road Award, honouring an artist who has left the Maritimes and succeeded elsewhere.
The awards evening was hosted by Denis Ryan and Raylene Rankin. Performing along with McLachlan were Figgy Duff, Lennie Gallant, Brett Ryan, Expresso SVP, the fiddle duo of Howie MacDonald and Natalie MacMaster, and the Thomas Trio and the Red Albino. The performers returned to the stage for an all-star finale jam with Sam Moon.
Cochrane Makes US gains with Life Is A Highway
Tom Cochrane is effectively paving his way to his six Juno nominations. After wrapping a highly successful 11 date Atlantic Canada tour the Capitol recording artist is now poised to break across the US with Life Is A Highway. The single, the second taken from his Mad Mad World album has made impressive moves up the AOR charts of R&R and the Gavin Report and Album Network's Power Cuts chart.
The album, now five times platinum in Canada, was released in the US on Feb. 25. No Regrets, the second single taken from the album, is now nudging the top slot of the RPM 100 Hit Tracks chart and the accompanying video scored a No. 1 on MuchMusic's national video chart.
Cochrane has been nominated for six Juno Awards: Album of the Year for Mad Mad World, Single of the Year for Life Is A Highway, Male Vocalist and Songwriter of the Year, Best Video for Life Is A Highway, and producer for All The King's Men from Mad Mad World.
Songwriter Wright gets break through ARIA
Jason Wright, a native of Wilkie, Sask., now living in Olds, Alberta, was a finalist in this year's Alberta Recording Industry Association's (ARIA) Songwriters Competition. The 29 year old has been writing and performing for some time, playing local weddings and working the club circuit. But Wright's main focus centres around performing original material.
After making the semi-finals in the 1991 Gold Fever Festival and the finals in the Alberta music project, Wright and his band Slice Of Life came out the winners in a televised variety talent show sponsored by a Vancouver television station.
ARIA member and Olds resident Elaine Wilson profiled Wright and his songwriting ambitions as follows:
Jason Wright's entry entitled Distant Thunder is a haunting glimpse at teenage suicide. "It relates to kids not getting enough attention at an important time," Wright explained.
The song evolved slowly. "In this case, the music came before the lyrics. I'll try to fit the words in with the music if it's appropriate. Some songs come in a couple of days. This song, before it was finally finished, took a couple of months. I heard a program on CBC Radio about girls in this asylum. They had a terrible problem, but they were nice people who had been through terrible trauma."
A member of the Saskatchewan Recording Industry Association, Wright joined ARIA to familiarize himself with the business end of Alberta's music industry. Learning he was a finalist was "very exciting, there's no question. The band is very excited. The people I play with have been playing music for a long time. We believe in what we're doing."
Wright applauds the encouragement ARIA's 1992 Songwriters Competition offers. "There's a lot of good songwriters out there. It takes them a long time to get recognized. It's not that they're not writing good stuff. Timing is everything. I think it's important that there are contests like this, places where people can showcase their material. It helps the guy who is struggling."
WALT SAYS!
with Elvira Capreese
It's a real mess!!! Bruce and Bryan have been going compared to a situation where a Canadian artist does a Canadian song and takes a Canadian producer to Los Angeles to record the session and says it is AP in the MAPL system. That record wouldn't qualify, but why would you sue??? (See page 2 for the correct ruling). If Karen and Bryan didn't do it . . . why should anyone else? (EC: Don't!!!)
Industry backlash . . . ? Would an industry that votes for itself get involved in politics? I would hope not. But apparently there are some who believe that the Bryan Adams controversy will result in CARAS members finding themselves in political slop by voting against rather than for Adams. No matter what company they're with or how they might regard his opinions on the industry, if they feel he was the top male vocalist, the songwriter and producer of the year, that his single and album were the best of the year, and that he was the Canadian entertainer of the year, they should vote that way. Personally, I think Adams brought more attention to Canada's entertainment/recording industry in a week or so of heavy HEAVY news coverage than the industry has scored in 30 years. (EC: Most of them have to be shown how to vote anyway . . . you know, put your X here . . . !)
Peter Howell's my hero . . . ! Never, in all the years that I've been around (EC: Nobody can count that high . . . !) have I seen a major daily allow so much space to one newsmaker. The Feb. 24 issue of the Toronto Star, front-paged Peter Howell's piece on Bryan Adams . . . right across from the week's most dismal news story -- GM's closing of its St. Catharines plant. All of page 2 was then taken up with photos of Adams and his family and the most entertaining piece of investigative reporting I've seen in a long time. Howell left no stone unturned. He even prepared his subjects with faxed questions that he would like answered, and got some pretty good, not to mention important revelations. I particularly liked the comments from Jim Vallance, Adams' former songwriting partner. No matter how you look at it, this has been Bryan Adams' year . . . thanks, in part, to Howell's coining of the "un-Canadian" tag that got the whole thing started. (EC: Well, a little credit PLEASE for grizzly Allen . . . !)
New and improved . . . ! This year's Big Country Awards Banquet will be new and improved. We have a few new angles that will bring this event back to its historic prominence. The big event will be held June 7 in the Harbour Ballroom of Toronto's Harbour Castle Westin. Jot this date down in your diary because it will be one of the most tasteful, enjoyable and fun-filled evenings you'll ever have. You don't have to be in the country music end of the business. This will be an evening of style, elegance and panache. (EC: Do they serve the panache as a dip????) Not only do you get the show . . . but it's a sit down, catered affair!
Two days prior . . . ! On the Friday before the awards banquet, the Variety Club will hold its luncheon tribute to country music. For those who want to semi-schmooze, this is the event for all the country music wannabees. Mark that date down as June 5 in the Frontenac Ballroom of the Harbour Castle Westin. It's the largest head table (EC: A special table just for that???) in country music and the preamble to the big awards night on Sunday. Details coming soon in RPM.
Relax Junos!!!!!! After watching the Grammies, I'm sure the Junos will come off better and I'm still saying two million. This will be "The night Canadian music comes home," which either refers to Vancouver being in a foreign country (EC: Not yet!) or the fact that there will be no foreign acts on the variety show. (EC: It's an awards show!!!) You could have fooled me!!! Boy, were the Grammies long and boring. After the first 20 minutes they were long. I watched every minute to see if Bryan Adams would be changed back into a Canadian . . . but all he did was share one Grammy with two other guys. (EC: Were they Canadians???) One of them is rumoured to have written the title to the song. (EC: Don't upset Bruce!!!) Now I'm really confused by the pronunciation of Celine's last name. These foreign names are so hard to pronounce. Go get 'em Junos!!!
When it comes to dailies . . . ! Watch for the Globe and Mail to pump life back into its entertainment (The Arts) section. It took a Peter Howell to make the Star's entertainment section entertaining . . . and informative. Now watch Chris Dafoe, ex of the Star, to do the same for the Globe through their Vancouver bureau. (EC: A real reporter from Vancouver . . . ?) As for the other Toronto daily . . . who knows what goes on down there? If you're a good hockey player, you just might score a column . . . or keep a job. The only breath of fresh air is Diane Francis and we're eagerly awaiting her expose on Toronto's SkyDome. Perhaps when she gets back from Russia. (EC: That expose is a long time coming wouldn't you say . . . ?)
Distinct society . . . Eh!!! Who won the majority of the medals for Canada at the Olympics . . . and who won that crucial industry hockey game . . . 17 WHAT? The Quebeckers, that's who. (EC: So why do you have a photo of the losers . . . ?) See page 15.
The business is up 32 per cent . . . ! What great news. January showed a 32 per cent increase over last year . . . but nobody's happy. (EC: Everybody wants to pay their bills with product . . . !) But our visitors were happy. (EC: Wouldn't you be happy calling on RPM . . . ?)
Chuck Page - Page Two/Rainbow Records
Rick Wharton - RCA Records
Peggy Lee - MGM/Blue Moon
Dave Daeley - Epic Records
Julian Tuck - A&M Records
Lorne Lichtman - EMI International
Rick Campbell - Atlantic/Through
Fahim Durrani - A&M Records
Leslie Howe - One 2 One/A&M Records
Louise Reny - One 2 One/A&M Records
David Lindores - BMG Music
Doug Caldwell - Virgin Music
Roger Bartel - Capitol Records
Dale Kotyk - Warner Music
MAKE A WORLD OF DIFFERENCE
What a World of difference means:
• more than 20 years experience
• we can guide you through your custom project
• quality & service
When it's time to make a difference with the quality of your CD's & Cassettes - call
World Records
1712 Baseline Rd., W.
Courtice, Ontario L1E 2S8
416-433-0250 666-2828
Fax 416-433-1868
LARGER THAN LIFE...
THE NEW ALBUM FROM VANCOUVER'S
skywalk
From BOWIE to FREDDIE HUBBARD, and PAUL HORN; From HEART to CHET BAKER, and AEROSMITH;
SKYWALK HAVE PLAYED WITH THE BEST!
In 1992 they are back and BETTER THAN EVER!
EXCHANGE
Canada's foremost instrumental duo have teamed up with one of Canada's finest voices MARC JORDAN and acclaimed session singer AMY SKY — the result is the HIT SINGLE
"LOVE CALLED OUT MY NAME"
from the Original Recording of
Prince Valiant
EXCHANGE — already on
CHFI, CHUM-FM, MIX 99.9, DC103, CJCB, CHTN, CHEZ-106, FM96
© 1992 Mars/Bluemoon Recordings, Ltd.
Distributed in the USA by Rhino Records, Inc., in Canada by BMG Music Canada, Inc.
COVER STORY - by Steve McLean
Brent Lee and his band are Outsiders looking in
When Brent Lee first met Naoise Sheridan in 1988, they drank heavily, talked about AC/DC and Merle Haggard, and traded song ideas.
Today these two, along with bassist Mike Lynch and drummer Ron Desjarlais, express their fondness for rock, country and alcohol through their band, Brent Lee and the Outsiders. Lee and Sheridan dropped by RPM for an often irreverent chat about their burgeoning careers.
The group started in Vancouver but when things got stale they decided to leave their friends and family to seek out a new market in Toronto. The band’s first break came when they did an interview for MuchMusic’s country show, Outlaws & Heroes. Since then the band has made 15 appearances on MuchMusic -- all without the benefit of a video.
Their other bit of good fortune was meeting Dale Murray of Justin Entertainment. Lee, 28, remembers the night at a Toronto nightclub when the two sides first negotiated working together:
“By the end of the night Dale was lying on the front stairs with a wine glass in his hand and his tongue sticking out the side of his mouth. We were all standing around looking at him and saying, ‘This is the guy who’s going to be handling our careers.’”
Murray received four enthusiastic thumbs up and he has been dedicated to the band ever since.
Opening on tours by Dwight Yoakam and The Tragically Hip exposed the band to a wider audience and they would like to get similar gigs in the future. But for the next six weeks, the band will be doing their own tour through western Canada.
“We used to play any town big enough to have a gas station. Now they have to be large enough to have a McDonald’s before we play there,” says the 30-year-old Sheridan.
Lee and Sheridan often get together and work on songs while on the road, though only the person with the original idea is usually credited with writing it. They hope to write 10 songs on the upcoming tour if they’re not too busy selling T-shirts, CDs and tapes in between sets of their shows.
The band released their MCA-distributed debut album, Rose Tattoo, in December. The catchy first single, Would You Love Me, has achieved moderate chart success on a variety of radio formats. They’re hoping the next single, Where I’m Going, will bring the group to the attention of those with the financial backing they need to finally produce a video.
A tentative title for the band’s next album came to Lee at a recent Valentine’s Day show, where a scrawny and obviously inebriated man with seven roses approached the stage. He asked Lee to call a woman named Cher (no not that one) to the front, and he insisted that the singer propose to her over the microphone on his behalf.
“She never did answer yes, but she cried and parts of her clothing fell off,” Lee says. “Then everyone applauded. It was a beautiful thing.”
Weasel and Cher, the album, could be hitting record stores by the end of this year or in early 1993.
Anne Murray to Florida to begin southern tour
Anne Murray begins her 10 city concert tour of the southern United States with a March 4 date in Jacksonville, Fla. Murray began her ’92 concert season at the beginning of February. She played for one week at Bally’s in Las Vegas, chalking 94 per cent capacity houses. Weekend dates at Bally’s in Reno were sold out.
Murray’s Jacksonville date will be followed by Melbourne (5), Clearwater (6), Sunrise (7), Plant City for the Florida Strawberry Festival (8), and Fort Myers (9), all in Florida. She is also scheduled to play Memphis, Tenn. (12), Birmingham, Ala. (13), and two dates in Louisiana: Shreveport (14) and New Orleans (15).
Touring plans carry Murray into the late fall. These include two days at Westbury in New York, a concert at the Kennedy Centre in Washington, D.C., a May date with the Boston Pops Orchestra and three nights at Trump Plaza in Atlantic City.
A greatest hits album, Fifteen Of The Best, will be released April 24 on the Liberty label (formerly Capitol Nashville). I Can See Arkansas will be taken from the album and released as a CD single. Since the track does not qualify as Cancon, Murray’s “live” version of the song, taped during her command performance date in Toronto, has been included on the CD single which ships to radio April 2. This version qualifies as two-parts Cancon (AP). Murray was in Nashville at the end of February for the taping of the video for Arkansas. The video is being produced by Scene Three’s Cynthia Biedermann with Canadian Steven Goldmann as director.
1991 was a good year for songwriter Jordan
Marc Jordan’s 1991 Juno nomination for songwriter of the year caps off his most successful year ever.
Rhythm of My Heart, the lead single from Rod Stewart’s Vagabond Heart, propelled the album’s sales to 4.25 million units worldwide and triple platinum status in Canada. The song was the second most performed song on European radio, according to ASCAP. The song’s success spurred new interest in Jordan’s original catalogue, prompting WEA Canada and several other labels worldwide to reissue Mannequin, Blue Desert, and Live at the El Mocambo on compact disc.
Other international artists who have recorded Jordan’s songs include Diana Ross, Chicago, Kansas, and the Manhattan Transfer, who recently recorded their fifth Jordan cover, Gentleman with a Family.
Following several live performances last summer, Jordan began writing material for his latest album, which he calls “a collection of raw live jazz-oriented songs.” The album will be produced by Steely Dan producer Gary Katz, who also produced Jordan’s 1978 Juno-nominated debut album, Mannequin.
Late last year, Jordan co-wrote two songs with Exchange for the soundtrack of the animated American television show, The Legend of Prince Valiant. Love Called Out My Name, a duet with his wife, singer/songwriter Amy Sky, and Where The Truth Lies, are both receiving Canadian airplay.
Sarah McLachlan down home for concert dates
Nettwerk recording artist Sarah McLachlan will launch her first ever east coast tour on March 11 at Charlottetown’s Confederation Centre. McLachlan returned home to Halifax (Feb. 16) for the East Coast Music Awards where she won Female Vocalist of the Year. After Charlottetown McLachlan will play the Rebecca Cohn Theatre in Halifax (12), the Acadia University Hall in Wolfville (13), Corner Brook’s Arts & Cultural Centre (15), the Arts & Cultural Centre in St. John’s (16), Saint John’s High School Auditorium (19), and Fredericton’s Playhouse (20).
McLachlan was signed to the Nettwerk label in 1987 at age 19. After that, she moved to Vancouver. A year later she released Touch, her debut album which gained her a gold certification.
Solace, her second album, garnered critical acclaim on the international front. The album has enjoyed 30 weeks of charting on the RPM 100, and has now surpassed gold.
A&A rises from red and keeps up with competition
Mark Twain quipped, "Reports of my death have been greatly exaggerated."
Today he could be speaking for A&A Music and Entertainment, which industry wags are quick to eulogize.
Once, the record and tape giant was mired in bankruptcy just a year ago and more than a dozen of the stores were closed. True, steep discounts were made in select locations last fall from survivalist tactics, these moves were part of a calculated effort to reposition both the music and home entertainment outlet.
A&A is alive and well and we're right on track with our profit projects. But the industry is changing, and we're taking some bold steps to recapture lost market share and secure our viability for the future.
According to statistics released by the Canadian recording industry, dollar sales for recorded music in 1991 were up seven percent while unit sales were down five percent. The growth area was compact discs.
Recorders is a mature industry. Any growth is coming from new formats. What we're doing now is bringing related product lines to build upon our existing base.
Now with 147 stores coast-to-coast, A&A is in the process of expanding its inventory.
Additional steps will include adding sell-through movies and video games to its library of compact discs, audio cassettes and accessories. Records, for which consumer interest has fallen dramatically, were dropped last year. New consumer services and state-of-the-art retailing technology are rapidly being set in place.
The man with the vision to turn the once ailing company is Cliff Horwitz, president of Jumbo Video and chief executive officer of both Jumbo Video and A&A. Since a consortium of investors led by Lincoln Capitol Corporation rescued A&A from bankruptcy last fall, Cliff initially manned the helm of both firms.
Contrary to popular opinion that the closing of almost 100 stores is a symptom of impending doom, the new owners purchased only the most profitable ones in the chain and thus began a venture on a healthy footing. A corporate name change from A&A Records and Tapes to A&A Music and Entertainment, more accurately reflects the new corporate mission.
What I feel I bring to the A&A team is industry experience, product knowledge and background and a shared vision for where we are going. Last year, prices were dropped in four downtown Toronto and Montreal locations. The move was undertaken to increase market share in these areas and the consumer response was overwhelming.
In November, the company instituted a toll-free telephone number (1-800-387-6060) which enables consumers to find out if a selection is in stock or to place special orders. There are 20,000 releases out there and we can't possibly carry them all. But if we don't have something that someone wants, we'll get it for them.
Another enhancement is the installation of a computerized management information system which tracks inventory-by sku-at the point of sale. Now in the testing stages, the system will be rolled out to all stores this year.
What this means to the consumer is that we will have the right inventory mix for each store according to what that demographic is looking for. One store might have greater call for heavy metal and another for classical music. We'll be able to build each inventory based upon the buying patterns of that store's clientele.
A pro-active, consumer-driven marketing strategy is not the only prescription for success in a recession economy. A&A has strong nationalistic roots and a team-oriented approach to human resources. These are assets every bit as valuable as real estate and financial backing.
We're a Canadian-owned company with the largest number of retail music outlets in the country. We're a team of 800 employees, each working toward a common goal. All of our profits are reinvested in this country to expand our business and create even more opportunities for us all. And that's something you don't hear about much in the headlines these days.
1992: a time for being more selective
Frank Davies - President
TMP - The Music Publisher
1991 was a year that saw several important changes domestically and internationally for TMP, with the acquisition of TMP's assets from Canada Publishing Corporation and the subsequent formation of a joint venture with MCA Music worldwide.
Since its formation in 1986, TMP has sought to sign, develop and promote its songwriters, artists and their productions alongside the representation of several key foreign catalogues in Canada.
The joint venture with MCA Music has enabled TMP to solidify its long term goals of taking its Canadian songs and songwriters to the world.
1992 is off to a good start thanks to the massive Canadian success of Tom Cochrane's wonderful Mad Mad World album. TMP has two songs on the album including the upcoming single, Annette Ducharme's Sinking Like A Sunset. Also creating serious sales action in Germany is Ian Thomas' Boomers album What We Do, now scheduled for release in several other countries.
TMP is concentrating its efforts in 1992 on several artist/writer development deals including Annette Ducharme, John Cody, Ron Hynes, The Mozz and Rick Braun. We are about to announce the worldwide signing of a major songwriter.
For music publishers, who are still awaiting the completion of the new mechanical licensing agreement between the CMRRA and CRIA and who continue to face the ongoing attempt by "users" to reduce the value of their copyrights (as reflected by the opposition to SOCAN's tariff proposals), 1992 is a time for being more selective and expanding horizons while continuing to create and develop for the future. TMP plans to achieve these goals.
FACTOR's New Talent Demo Award has changes
FACTOR's New Talent Demo Award Program applicants have been fully committed for their deadline, March 31. As a result, FACTOR will no longer be accepting applications to the program for their remaining year.
The New Talent Demo Award Program provides assistance for the production of high-quality demo tapes which are essential to any recording or distribution deal. Only one applicant may apply to the program. FACTOR defines new talent as artists or performers who have commercially released fewer than 10 songs.
FACTOR's Board of Directors have confirmed the following changes to the program effective April 1, 1992:
- FACTOR will no longer place successful applicants in the studio where the recording is to take place. The choice of the studio will be up to the applicant.
- FACTOR will recognize a maximum of 25 hours studio and will provide a cash award of up to 50% of eligible award production costs to a maximum of $1,500.
- The applicant will be responsible for all costs incurred for the production and will be reimbursed the eligible award amount by FACTOR upon completion of the project.
Due to these changes new application forms must be used by those wishing to apply for the program and will be available in early March. Next deadline for submitting applications to the program in all musical categories is May 29, 1992. Last year the New Talent Demo Award Program provided assistance to approximately 129 artists across Canada.
Vancouver's Crusoe set Toronto listening date
John Palmer, who heads up Vancouver's Palmer & Associates, has scheduled a (6 p.m.) listening session at Toronto's Venture Sound for Crusoe, a new west coast singer.
Palmer is on a shopping trip with Crusoe's manager. Besides the Toronto session, he will attend the South by Southwest convention, an alternative music conference held in Austin.
Crusoe's album was produced by Stefano Bortoluzzi of Toronto and recorded at Venture's Venture Sound Studio.
Buffy Sainte-Marie
"The Big Ones Get Away"
Already Top 10 Music Week
From The Forthcoming Album
"Coincidence And Likely Stories"
"If the bad guys don't get you baby, then the good guys will."
EMI
**ONE**
**2 ONE**
- Pop
- 75021 5364 2-Q
One It...a new One 2 One album that forsakes its synth pop predecessors and focuses on both acoustic and electric guitars to carry out 10 tunes along. At times you may hear you've heard brief riffs or melodies somewhere else, but for the most part One 2 One manage to leave a fairly distinctive identity. The album's first single, Peace Of Mind (Love Goes On) is the band's most heartfelt and maybe best experience the 1980s. The rest of the songs primarily deal with every band's favourite subject: love. Bassist musician Frank Levin contributes some nice piano and keyboard work on Tough To Tame, while Gypsy Girl mirrors southeastern Canadian sounds before picking up the tempo and getting a bit atmospheric. Remy's voice stands out on Hobo's Haunt and Tryin' To Start A Cleaning, a country song that begins with the sound of gently falling rain and includes some good acoustic guitar work. The album was produced, engineered and arranged by band member Leslie Howe in his Ottawa studio. (CD reviewed)
**BOOTSAUCE**
- Alternative rock
- 101/512027-2-Q
A follow-up to Bootsaucce's gold-selling debut, The Brown Album, was the highest placing new entry (#49) last week's RPM Albums chart. With funky rhythms, Pere Fume's electric guitar, and lead singer King's decision that "the music has to continue to give this Montreal band a unique sound. You probably hear too much of this on AOR or FM because sordid sex is a wonderland theme throughout. The exception is the first single, Love Me Baby #9, which deals with the cruelty of lab animal testing. Bootsaucce's fondness for disco surfaces with Rollercoaster's Child, an adaptation of the Ohio Players' Love Rollercoaster. It's a logical progression from last year's club hit cover of Hot Chocolate's Every 1's A Winner. Motorhead's Lemmy and Wizzo guest on Hold Tight, so Bootsaucce is obviously in no danger of losing it's grungy edge. (CD reviewed)
**WILLIE P. BENNETT**
- Folk
Collectibles
Dark Light Music - DL-12001
Too often, critics and the industry alike rush to recognize new talent, forgetting some of the real troupers who have been bordering on success for more years than they want to admit. Willie P. Bennett is one of the enduring heroes of this industry. There's no pretentiousness here. He's not a Nashville wannabe, What you get from Bennett is realism in both lyrics and music. All the songs are Bennett originals, recorded in Canada - 100 per cent Canon, and all taken from three albums: Tryin' To Start Out Clean (1975), Here's Haunting You (1976) and Blackie And The Rodeo King (1979). Bennett's session people are superbly patched into the sensitive and whisper smooth Bennett vocals, particularly on You which features Rick Taylor on high string guitar, Ken Whiteley on lead acoustic guitar, Mike Gardner on bass and tinkle and drummer Steve Taylor. Also key are Country Squall, with Dennis LaPage on banjo, and Me And Molly with the late Ruth Dannon on pedal steel. Bassist David Petty with his keyboard, Ken Whiteley, Bennett supplies harmonica, acoustic guitar and high string guitar on various tracks. David Essig produced the tracks from Hobo's Haunt and Tryin' To Start Out Clean, while Bennett joined him to produce Blackie And The Rodeo King. Classic material, should be enjoyed by an international audience. (CD reviewed)
**SKYWALK**
- Instrumental/Jazz
Larger Than Life
Bluemoon - R 79173-N
This is Skywalk's fifth album, but their first on Bluemoon. On it, these six talented Vancouver session musicians, songwriters and producers continue to play self-composed, jazz influenced, contemporary instrumental music. When they're not playing in Skywalk, many of these players may be familiar to you through other projects. Bassist Rene Worst has recorded with Dave Bowie and Cher, Baker, Perussionist Jim Keenlyside is a former member of Spirit of the West. And sax player Tom Keenlyside has performed and recorded with Aerosmith and Motley Crue -- two of the last bands you'd associate him with judging by his soothing contributions to this album. The production by Bill Buckingham and the band is very clean and precise. Each of Skywalk's 10 songs average about five minutes in length and are velvety grooves throughout should have you relaxed before the album's even half completed. This music would be perfect background for Night Ride, Global TV's mellow video tour of downtown Toronto which nightly enraptures insomniacs at 3 a.m. (CD reviewed)
**VARIOUS ARTISTS**
- Rock/Dance
Gladiator - Soundtrack
Sony Music - CT 52434-H
This is an inconsistent album in terms of both music quality and selection. First, the good news. Civillies and Co. and contemporary dance and C&C Music Factory, do an interesting take of U2's Pride (In The Name Of Love). The song's combination of techno and a gospel choir-influenced chorus has taken it to #1 on the RPM Dance chart. Rap music from different perspectives comes via 3RD Bass' swagging Gladiator and P.M. Dawn's For The Love Of Peace. Latin Science combine English and Spanish lyrics, Latin and calypso rhythms, scratching, and an excerpt from David Bowie's Fame in their danceable Da Me La (Fama). After that, it's all downhill. Tony Terry makes an innocuous attempt at soulful dance music with Hold On Tight. Martin Page supplied a dull power ballad in Count On Me. Guy Ozo (the guy to blame for last year's Rice Cooker) banters with Sancho's Oye Como Va, while rapping about his prowess as a Latin lover in Latin Till I Die (Oye Como Va). For some reason, Warrant gets two songs: a heavy version of Queen's We Will Rock You that thankfully doesn't go into We Are The Champions; and, a song called The Power which is supposed to inspire but merely bores. What Is Cheap Trick still doing around and playing on this soundtrack? Answer: Sounding a little too much like Warrant. (Cassette reviewed)
**TEENAGE FANCLUB**
- Rock
Bandwagonesque
Geffen - DGCD-24461-J
"She wears denim wherever she goes, says she's gonna get some records by the Status Quo." The opening line of this album, from the song, The Concept, neatly summarizes what these Glaswegians are all about: a scruffy, no frills appearance and a guitar-oriented sound which often harkens back to early '70s rock and pop. The band received great press in Britain's New Spin magazine and Bandwagonesque the best album of 1991. I wouldn't go that far, but What You Do To Me, Star Sign and Sidewinder are all melodic, classic-style pop songs that could gain airplay. Satan is 80 seconds of feedback and guitar chaos which shows the band's darker side. Their current North American tour and recent appearance on Saturday Night Live should gain this quartet a wider audience. (CD reviewed)
NIRVANA
"Nevermind" (DGC) (CA) DGC2-34425 (CD) DGD2-34425-J
BRYAN ADAMS
"Waking Up In Neighbours" (A&M) (CA) 7801 5367-1 (CD) 75021 5367-2-Q
TOM COCHRANE
"Mud Maid World" (Capitol) (CA) CA-97723 (CD) C2 97723-F
U2
"Achingly Blue" (Island) (CA) 615 347-4 (CD) 510 347-2-Q
GEOFFREY GIBB
"We Can't Dance" - Atlantic (CA) 78-23444 (CD) C2-82344-P
CODY CHESNUTTE
"C'mon Get Happy" (MCA) (CA) 92-44294 (CD) C2-44294-P
MICHAEL JACKSON
"Bad" (Epic) (CA) ET-45400 (CD) ET-45400-H
TWO ROOMS
"Vertigo/Two Rooms" (Mercury) (CA) 845 749-2 (CD) BMS 749-2-Q
ENYA
"Silent Moons" (WEA U.K.) (CA) 17-45704 (CD) 75572-P
COWBOY JUNKIES
"black eyes" (RCA) (CA) 7863 61049-4 (CD) 07863 61049-2-N
BOYZ I'M MEN
"Coolyhoopy" (Motown) (CA) 845 6320-2 (CD) 07863 6320-2-Q
BARENAKED LADIES
"Barenaked Ladies" (Indie/dan) (CA) 0002 (CD) N/A
BLACK SHEEP
"A Wolf In Sheep's Clothing" (Mercury) (CA) 848 368-4 (CD) 848 368-2-Q
NAUGHTY BY NATURE
"Naughty By Nature" (MCA) (CA) IST-2032 (CD) SCD-2-32-H
METALLICA
"Load" (Elektra) (CA) 96-1138 (CD) C2-61133-P
MICHAEL BOLTON
"Time, Love & Tenderness" (Columbia) (CA) 75021 4877-1 (CD) C2-48771-H
ERIC CLAPTON
"More From The Rumour Soundtrack" (Reprise) (CA) 75774-H (CD) CD-26794-P
GUNS N' ROSES
"Use Your Illusion II" (Geffen) (CA) GEFCD-24415 (CD) GEFSD-24415-J
WAYNE'S WORLD
"Wayne's World" (MCA) (CA) 92-49854 (CD) CD-26805-P
CE CE PENISTON
"Finally" (MCA) (CA) 75021 5381-4 (CD) 75021 5381-2-Q
DIRE STRAITS
"Brothers In Arms" (Mercury) (CA) 510 416-2 (CD) 510 160-2-Q
RICHARD MARX
"Rush Street" (Capitol) (CA) 7801 53687-1 (CD) C2-53687-F
JOHN MELLENCAMP
"Warm And Wonderful" (Mercury) (CA) 810 151-2 (CD) 510 151-2-Q
GUNS N' ROSES
"Use Your Illusion II" (Geffen) (CA) GEFCD-24420 (CD) GEFSD-24420-J
M. R. BIG
"Lean Into It" (Atlantic) (CA) 7801 53678-1 (CD) C2-53678-P
MARK MAREY
"Emotions" (Columbia) (CA) CT-47860 (CD) C2-70986-H
CHUCK BERRY
"THE TEN COMMANDMENTS"
"The Glory Of These Me" (Merita) (CA) AC 8677 (CD) ARCD 8677-N
ROBBIE ROBERTSON
"THE LAST OF THE HURRAHS" (MCA) GEFCD-24303 (CD) GEFSD-24303-J
SASS JORDAN
"Rendezvous" (MCA) (CA) 92-6564 (CD) C2-566-F
GARTH BROOKS
"From The Wind" (Capitol) (CA) 845 68330 (CD) C2-96330-F
JUICE
"Spinning" (MCA) (CA) MCA-10462 (CD) MCA-10462-J
C + C MUSIC FACTORY
"Genoa Make You Sweat" (Columbia) (CA) 845 749-2 (CD) 75572-H
OZZY OSBOURNE
"No More Tears" (Epic) (CA) ZT-47678 (CD) ZK-47678-H
ROD STUART
"Highway To Heaven" (Warner Bros)
LISA STANSFIELD
"Real" (Arista) (CA) 7801 53679-4 (CD) 07862 18679-2-N
STEVIE RAY VAUGHAN
"The Sky Is Crying" (Epic) (CA) 78-23444 (CD) EK-47390-H
ALT 'N' PEPA
"The Hits Remixed" (Next Plateau) (CA) 828 249-4 (CD) 828 249-2-Q
SIMPLY RED
"Love Is All" (Mute U.K.) (CA) 17-58944 (CD) C2-75284-P
BOOTSAAUCE
"Boots" (Mercury) (CA) 512 027-4 (CD) 512 027-3-Q
HARRY CONNICK JR.
"Burnin', Red Light" (Columbia) (CA) 75088 (CD) C2-44668-H
PRINCE
"Diamonds & Pearls" (Purple Park) (CA) 845 6320-2 (CD) 07863 6320-3-P
RED HOT CHILI PEPPERS
"Blood Sugar Sex Magik" (Warner Bros) (CA) 845 6811 (CD) 848 6811-P
THE GRAPES OF WRATH
"These Days" (Capitol) (CA) 78-23444 (CD) C2-96431-F
SUPERTRAMP
"The Very Best Of Supertramp (Polyd) (CA) 365 329-2 (CD) 393 329-3-Q
BON JOVI
"Nothing But A Burning Light" (True North) (CA) TNT-77 (CD) TNK-77-H
BON JOVI
"Rock Of The Hard Way" (Capitol) (CA) C4-91111 (CD) C2-96111-F
BOB SEGER
"Night First Date" (Capitol) (CA) C4-91134 (CD) C2-91134-F
TOM PETTY & THE HEARTBREAKERS
"Into The Great Wide Open" (MCA) (CA) 92-49854 (CD) C2-53678-J
PAULA ABDUL
"Spettacular" (MCA) (CA) 92-49854 (CD) C2-53678-Q
LORENA McKENNITT
"The Visit" (WEA) (CA) 75-1514 (CD) C2-75151-P
RUSH
"Roll The Blues" (Arthium) (CA) 845 6811 (CD) ANK-1064-H
NATALIE COLE
"Underneath With Love" (Elektra) (CA) 96-10494 (CD) C2-61049-F
R.E.M.
"One Time" (Warner Bros) (CA) 92-49564 (CD) CD-26496-P
SARAH McLACHLAN
"Building A Rocket" (MCA) (CA) W-30055 (CD) W-30055-F
THE COMMITMENTS
"Soundtrack" (MCA) (CA) MCA-10286 (CD) MCAD-10286-J
HAMMER
"Took My Car To Capetown" (CA) 92-68151 (CD) C2-58151-F
BEAUTY & THE BEAST
"Soundtrack" (Walt Disney Records) (CA) 845 6811 (CD) 06168-2
EDDIE MONEY
"Right Here" (Columbia) (CA) 74-4756 (CD) C2-46756-H
THE CURE
"Ceremony" (Vertigo) (CA) 510 419-4 (CD) 510 419-2-Q
SHAGDAUGHS
"Badmouthed" (A&M) (CA) 75021 5374-2 (CD) 75021 5374-4-Q
PHANTOM OF THE OPERA
"Concerto" (Mercury) (CA) 847 699-2 (CD) 847 699-2-Q
VAN MORRISON
"Return To The Silkenveil" (Polydor) (CA) 845 6811 (CD) C2-61049-Q
PM DAWN
"Of The Heart" - The Utopian Experience (Island) (CA) 75021 5374-2 (CD) 75021 5374-Q
AWESOME HITS
"Awesome" (Various artists) (Polyd) (CA) 845 6811 (CD) 510 419-2-Q
LOU REED
"Magic And Loss" (Sire) (CA) 845 6811 (CD) 06168-2
THE TRAGICALLY HIP
"Rippled Apples" (MCA) (CA) MCAD-10173 (CD) MCAD-10173-J
SHAKIRA FRANKS
"As Real As It Gets" (Epic) (CA) ET-47310 (CD) EK-47310-H
COUNTRY HEAT
"Country Heat" (Warner Bros) (BMG) (CA) 91901 7220-4 (CD) 0161901 7220-2-N
PUBLIC ENEMY
"Apocalypse 91: The Enemy Strikes Back" (Columbia) (CA) C2-47310 (CD) C2-47310-H
HOLLY COLE TRIO
"Blame It On My Youth" (A&M) (CA) ZT-48108 (CD) ZK-221-9108-F
PAUL SIMON
"Concert In The Park" (Warner Bros) (CA) 91901 7220-4 (CD) 26737-P
PAUL YOUNG
"From Time To Time" (Columbia) (CA) 845 6811 (CD) C2-90866-H
TROOPER
"Ten" (WEA) (CA) C2-47304 (CD) C2-74703-P
EDSURE
"Chorus" (Vertigo) (CA) 845 66684 (CD) C2-36668-P
REBA McENTIRE
"For My Broken Heart" (MCA) (CA) MCAD-10400 (CD) MCAD-10400-J
Cindi Cain has just come through a pretty disappointing year. But the new year is beginning to look up. Cottage Records in London, England, have picked up her next album, scheduled to be released sometime in June. Don Grashey, who heads up Golden Eagles of Thunder Bay, Ont., produced. A past multi-nominee in her home province's country music awards, and a multi-winner over two years, Cain only managed a Female Vocalist nomination this year. Two other Manitobans who have made national news, Errol Ranville and Ray St. Germain, were also left hanging this year. CBC-TV's The Country Beat, which originates from Edmonton, will feature an interview with Cain and play the video Field Of Dreams on March 28. Top 10 Country Video out of Pickering has informed Grashey that the video will be televised by 14 stations throughout March.
Garth Brooks maintains the No. 1 slot on the RPM Country 100 with What She's Doing Now. The track was taken from his Capitol album Ropin' The Wind. No sooner had he hit No. 1 when Liberty, rushed out a anew CD, Papa Loved Mama. Brooks wrote this song with Kim Williams.
Wynonna Judd's She Is His Only Need makes the biggest jump on the chart this week, up to No. 34 from No. 67. The CD single is Wynonna's solo debut for MCA. The track was taken from her upcoming Wynonna album.
Paul Weber's new single, First Second Thoughts, the follow-up to Growing Pains, is included on the new Tennessee Star Trak CD compilation. The song was written by Cyril Rawson, David Woodward and Don King. Rawson and Tony Migliore produced the track which was recorded in Nashville.
Sylvia Tyson switches labels to Silver City (Sony) with the release of a CD single, I Walk These Rails. The song was written by Sylvia and Shirley Eikhart. Sylvia produced the session with Tom Russell. The single qualifies as four parts Cancon.
1270 CHAT Medicine Hat attracted listeners "in droves" to trade food for free concert tickets to Hal Ketchum and Sawyer Brown. The promotion was considered a "huge" success. Two van loads of non-perishable food items were collected for the Medicine Hat Food Bank.
Royalty's CD Sampler #6 has been shipped to radio. The CD contains a track each from four of the label's artists: Only A Cowboy Understands by Momentum Band, written by Dave Zan, self-produced and recorded at Sundae Sound in Calgary; As If I Didn't Know by Deserite, written by Marshall Gardner, produced by R. Harlan Smith and recorded at Edmonton's Damon/Soundtrek studios; Greg Paul's Texas On My Mind, written by Paul Hotchkiss and R. Harlan Smith, produced by Smith and recorded in Nashville; and Slippin' Away by Tinetta, written by Tineta Couturier and Denis Charney, produced by R. Harlan Smith and recorded at Damon/Soundtrek. Paul is making good chart gains with Feelin' Guilty which is included on Royalty Sampler #5.
CKRC Winnipeg is looking for record product and biographical material from Cancon artists. We'll have more details next week. In the meantime, send your records with a little background material to the station's Music Director Greg Mack, P.O. Box 9700, Winnipeg, Manitoba R3C 3E5. The station's new Program Director is Dauphin native Doug Anderson, who was recently with CJGX Yorkton.
CFCW's Star Search win for Edmonton songwriter
Former Dryden, Ont. native, Buzz Gardner, now living in Edmonton, has won the 1992 CFCW Canadian Country Star Search. Prizes include the production of a complete ten-song compact disc, plus a one-song video. Noted producer R. Harlan Smith, who heads up the Edmonton-based Royalty label, is in charge of production.
New, unpublished songs are being sought to audition for the final recording. Last year, approximately 200 songs were submitted from across Canada. Those interested should submit song material to R. Harlan Smith, Royalty Records Productions Ltd., P.O. Box 8768, Edmonton, Alta. T6C 4J5. Deadline for auditioning new material has been set as March 31, 1992.
Last year's winner, Tineta, has just released her second single from the Star Search package. Released on Royalty CD Sampler #6, the song was written by Tineta and veteran Canadian songwriter Dennis Charney. A video of this track has also been shipped and is scheduled for showing on CBC-TV's Country Beat.
**40AC (Adult Contemporary)**
March 7, 1992
| # | Title | Artist/Composer |
|---|--------------------------------------------|---------------------------------------------------------------------------------|
| 21| WHAT BECOMES OF THE BROKENHEARTED | Paul Young - Fried Green Tomatoes Soundtrack (A&M comp. # 368) |
| 22| BEAUTY AND THE BEAST | Elaine Dion & Peabo Bryson - Beauty & Beast Soundtrack (A&M comp. # 365) |
| 23| MISSING YOU NOW | Michael Bolton - Time, Love & Tenderness (Sony comp. # 393-H) |
| 24| LET GO | Justin Currie - Emotions (Jumbo/47880 (Sony comp. # 393-H) |
| 25| DON'T LET THE SUN GO DOWN ON ME | Michael Bolton - Time, Love & Tenderness (Sony comp. # 393-H) |
| 26| QOD FOR ME | Michael Jackson - Dangerous (Capitol comp. # 371-P) |
| 27| GRAVITY - Heart In Motion | Michael Jackson - Dangerous (Capitol comp. # 371-P) |
| 28| DON'T TURN AROUND | Michael Diamond - Loverscape (Sony comp. # 371-H) |
| 29| ANGEL | Lisa Stansfield - Real Love (BMG pop comp. # 257-N) |
| 30| OUTHER RAIN | Black Eyed Peas - Out Here (BMG pop comp. # 257-N) |
| 31| ARUBIAN BLUE | Black Eyed Peas - Out Here (BMG pop comp. # 257-N) |
| 32| DREAM COMING BACK | Black Eyed Peas - Out Here (BMG pop comp. # 257-N) |
| 33| MASTERPIECE | Black Eyed Peas - Out Here (BMG pop comp. # 257-N) |
| 34| ONE LITTLE WORD | Black Eyed Peas - Out Here (BMG pop comp. # 257-N) |
| 35| TEARS IN HEAVEN | Black Eyed Peas - Out Here (BMG pop comp. # 257-N) |
| 36| SAVE THE BEST FOR LAST | Black Eyed Peas - Out Here (BMG pop comp. # 257-N) |
| 37| TO BE WITH YOU | Black Eyed Peas - Out Here (BMG pop comp. # 257-N) |
| 38| UNTIL YOUR LOVE COMES AROUND | Black Eyed Peas - Out Here (BMG pop comp. # 257-N) |
| 39| PLACE THAT BELONGS TO YOU | Black Eyed Peas - Out Here (BMG pop comp. # 257-N) |
| 40| EVER CHANGING TIMES | Black Eyed Peas - Out Here (BMG pop comp. # 257-N) |
**Variety Club salutes BIG COUNTRY LUNCHEON**
Friday June 5th, 1992
HARBOUR CASTLE WESTIN
**CHARTS by Tim Evans**
No small move! This move is so big, they called it Mister. Going all the way from No. 7 to the top of the Hit Tracks chart is Mr. Big’s To Be With You. The last song to make a bigger jump to No. 1 was Elton John’s I Don’t Wanna Go On With You Like That. It hit the top all the way from No. 8 in August of 1988.
Let’s test your skill. Mr. Big’s To Be With You becomes the third song to hit No. 1 from an artist beginning with Mr., Mrs. or Miss. Name the other two songs.
Back and forth . . . Nirvana’s Nevermind edges back into the top spot on the Albums chart. Bryan Adams’ Waking Up The Neighbours had replaced Nirvana at No. 1 just last week. The two are very close and we could see them flip-flop again.
Party time . . . excellent! There’s often a strong relationship between the success of a movie and the success of a soundtrack. Such is the case with Wayne’s World. The soundtrack to the No. 1 box office hit blasts onto the chart at No. 19. The last albums to debut higher both came on Nov. 30 of last year. That’s when Genesis’ We Can’t Dance entered at No. 10 and U2’s Achtung Baby bowed at No. 14.
A one-hit wonder? The group with the No. 1 song also has this week’s biggest jump on the Albums chart. Mr. Big’s Lean Into It climbs from No. 78 to No. 25. Also making a move of note is the Cowboy Junkies. The group hits No. 10 with black eyed man. That’s one spot higher than their previous album peaked.
A change out west. There is a new music director at K-97 in Kamloops, B.C.. Brant Zwicker has left and is being replaced by Donovan Richards.
A little sneaky. Maybe it was a bit sneaky, but it would have been too easy otherwise. The only two songs to hit No. 1 from an artist beginning with Mr., Mrs. or Miss are Mr. Mister’s Kyrie (1985) and Broken Wings, also from Mr. Mister (1986).
ATI’s 4X4 idea was given the nod in Calgary where it was introduced by ATI president Scoot Irwin. The Quadrapak CD contains four songs by four ATI artists. “The response from radio was fantastic,” says Irwin. “We immediately added CHRB High River, the CBC and Country 105 in Calgary. Stations are keying in on Karen Cunningham’s Photograph, Floyd Tolfman’s Alberta Cowboy, If Nothing Ever Changes by Janan Frances, and The Loneliest Man by Hod Pharis. The ATI artists were shown people responding to the company’s new Associat
Norm B Sing You Three Roses on his record And My Mansion scheduled for release.
The Country Music Association of Canada is to the 28th Annual Canadian Country Music Awards what the Kennedy Center is to the Grammys. Videos submitted for the Country Music Awards, Rodeo, Kenny Rogers, Petty, Jir, And The Band, McDowell, Doug Ian and many others at 403-414-1111.
D.J. Honeysuckle Life, is the new album and record by The Honeysuckle Life. The album and record includes the single, I Want You and the single, I Want You.
The charge per word for Minimum is $1.00. The charge for a total. Ads. Send to Toronto, Ontario.
MO "DC 10" the exp morrin record. If this c is see succes all the t positio Please
Presti thelux. CPlay Cele
Calgary
Calgary Country Music Association president Mike McLean has announced four new members to the house from the Calgary area. "We have a lot of talent here, the new members' contributions are going to be a big help," he said. "Nothing But The Truth" and "The Wings" are two of the new artists.
The Country Beat television series out of Edmonton had its March 21 edition pre-empted to the 28th. Featured are interviews with Joan Kennedy, Alex Green (stuntman), Sawyer Brown and Brent Lee and The Outsiders. Videos scheduled are by Family Brown, Blue Rodeo, Kathy Mattea, Sawyer Brown, Tom Petty, Jim Grey and The Fender Kings, Baillie And The Boys, Vern Gosdin and Ronnie McDowell. For further information contact Doug Ianson of CBC Alberta Communications at 403-468-2325.
D.J. Hopson's CD debut, A Day In The Life, is now available on the Wellcraft label. The album was produced by Tony Migliore and recorded at Nashville's Chelsea Studio. Included on the album is Hopson's current charter, Have A Heart, written by Cyril Rawson and Terry Brown. This track is also included on RDR Countrypak 6.
CLASSIFIED
The charge for classified ads in RPM is $1.00 per word, $2.00 per word for upper case body copy, $3.00 per headline word. Minimum charge for an ad is $20.00. There is a $10.00 service charge for resending a business card. Please add 7% GST to the total. Ads containing more than 50 words will be run as display ads. Send ad copy to RPM Weekly, 6 Brencliffe Road, Toronto, Ontario M4G 3Y2. FAX: 416-425-8629.
OFFICE SPACE FOR RENT
One or two privates offices, plus two work stations are available for sublet in Attic's renovated premises in downtown west Toronto. Most appropriate for compatible non-competitive music industry company. For further information contact Alexander Mair 416-532-4487.
MODERN ROCK TALENT
CIMX-FM Windsor/Detroit's Modern Rock station is looking for a talented on-air performer who has solid product knowledge. If you love Modern Rock send tape and resume, in confidence, to:
Greg St. James
Program Director, CIMX-FM
300 Cabana Road East,
Windsor, Ontario
N9G 1A3
No calls please!
DO YOU HAVE A GREAT IDEA FOR A SYNDICATED RADIO SHOW?
CAPS can help you get your idea off the ground and onto the airwaves. For more information write:
Bob Mackowycz
CAPS
c/o KEY Radio Ltd.,
1 Yonge St., Suite 2416,
Toronto, Ont.
M5E 1E5
ROW's Vito Ierullo, proudly accepts his Falling Star Award from Warner's Helen Chung, while Warner's Ken Dion (l) and Franz Bruck look on.
HOW TO SUBSCRIBE TO RPM WEEKLY
Send us your name, address (with postal code) and a cheque or credit card information (Visa or MC). The rates for Canada are as follows:
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Creatively colour The Grapes Of Wrath "A Fishing Tale" illustration and you could win a trip for 2 to Langara Fishing Lodge in the remote wilderness of the Queen Charlotte Islands, British Columbia. Langara offers unsurpassed fishing for all 5 species of salmon as well as huge halibut, ling cod and snapper. You'll enjoy first class hospitality, unbelievable wildlife and the ultimate in sport fishing. The trip includes return airfare from a major Canadian gateway, chartered air transportation from Vancouver to Sandspit in the Queen Charlotte Islands, float plane to and from the Lodge, meals, accommodation, boats, tackle, fuel, bait, rain gear, survival suits, catch-care and Langara hat.
CONTEST RULES AND REGULATIONS
1. Colour The Grapes Of Wrath "A Fishing Tale" illustration. Entries will be judged on the basis of creativity, by Capitol Records solely at its discretion.
2. Send to: "SPIN A FISHING TALE" CONTEST c/o Capitol Records - EMI of Canada 309 Lawrence Drive Mississauga, Ontario L4V 1B6
3. Entrants may use the attached entry form or a photocopy.
4. No purchase necessary.
5. There is no cash equivalent for the prize. Prize will be awarded as described. Trip must be taken July 11 - 14, 1992. Prize is non-redeemable and non-transferable.
6. The Grapes Of Wrath "Spin A Fishing Tale" contest entry deadline is: 5:00 pm E.S.T. - April 10, 1992.
7. The winner will be notified by telephone April 16, 1992.
8. The winning entrant will be required to correctly answer a skill testing question in order to qualify for the grand prize.
9. The chance of winning will depend on the total number of entries received.
10. The winner must sign a release form from Capitol Records - EMI of Canada confirming compliance with the contest rules and acceptance of the prize awarded.
11. The Grapes Of Wrath "Spin A Fishing Tale Contest" is open to Canadian residents who are readers of The Record and/or RPM magazine, 19 years and over, excluding the province of Quebec, and employees of Capitol Records - EMI of Canada, Network Records, MuchMusic, The Record, RPM and members of their immediate families.
Approximate Value: $5,700.00
|
BPM
Optimism marks first quarter after X-MAS sell-through
by Sean LaRose
As reported last week in RPM, 1996 for the most part had the Canadian music industry on edge with soft sales at retail. Ironically, Soundscan and CRIA statistics for '96 didn't fair too badly in regards to numbers and certifications. Some record execs insist one contributing factor in the decline was lack of catalogue sales, which they say was due to the fact that the consumer has finally completed the transition from LPs to CDs.
Warner Music Canada has finished many a year at the top, making a great year hard to determine. However, the spotlight hit more than a few of their artists throughout the year, in particular double diamond selling Alanis Morissette. Warner's Senior vice-president, managing director Garry Newman was, despite his trademark vagueness, genuinely pleased with the final results of 1996.
The combination of top calibre artists Warner had in the Top 20 on RPM's Top 100 Albums of '96 chart, including Morissette (who held the #1 album of the year), Metallica, Hootie And The Blowfish and Tracy Chapman, and their obvious monopolization of the Top 100 Year End Hit Tracks chart with an incredible 50% take (four in the top five), clearly indicate that Warner experienced more than just a good year.
Morissette held the #1 and 2 positions with You Learn and Ironic, Eric Clapton's Change The World grabbed the number three spot followed by Tracy Chapman's come back single Give Me One Reason. As well, Morissette closed out the Top 10 with Head Over Feet. With those numbers under their belt, a modest Newman calmly reflects on the past year: "Yeah I think it was slow, although the CRIA stats for the month of December were very good, the industry was up big. It was a field day."
Newman apparently was pounding the pavement visiting retailers to get a more realistic representation of what was happening in the stores over the holidays.
"Going into stores and talking to store managers, they are the ones that tell the whole truth," he said. "The week between Christmas and New Years, from my understanding, was very successful."
Overall, Newman feels Christmas was reasonable.
"I wouldn't say we had a great Christmas," he says. "Space Jam did fine, the big one was Evita and I think it's going to be even bigger now. Rod Stewart is flying, that's doing really well; he is going right by our expectations, it's absolutely wonderful. The first single is still doing well and we're going to hold off the next single because this one is still going strong.
"Jewel is still continuing and we're on the second single from that release. Phil Collins did good; I think the next single will really get the fan base back. If I had to pick the three big movers they'd be Space Jam, Evita and Rod Stewart."
The label is optimistic as it heads into the new year with a priority release for Collective Soul, who managed 6X platinum on their last record. Country act Regina Regina will also be putting out a new album and there are high anticipations for the Howard Stern Soundtrack. Newman says he also expects the first quarter to be marked by strong returns from Greg Keelor's solo effort, Greg Fox's Greatest Hits (with two new singles) and Mark Morrison's newest.
EMI Music Canada released some notable product in '96, particularly on the domestic front. Anne Murray's latest self-titled release was a huge priority for EMI and rightfully so with powerful duets with Bryan Adams and Aaron Neville. Also Kim Stockwood strengthened her impact on the industry with her single Jerk.
The Beatles have also proven to be a valuable commodity for EMI with Anthology I & II selling through the roof and III enjoying some successful retail life as well. On the country side, LeAnn Rimes took the industry by storm with her first single Blue and continues to make waves with her follow-ups. As well Denna Carter is on her way to platinum with her latest release.
MAJORS continued on page 3
Diemer says that other strengths for EMI included The Beatles Anthology which "sold quite well" and children's product, particularly Sailor Moon.
The company's other big sellers included The Rankin Family, who are nearing double platinum and Leann Rimes' latest album.
WALT SAYS . . .
Deflowering Canada!!! Mickey (Shoo Shoo) Kantor is gone, but the melody lingers on, and so does the erosion of Canadian nationalism. During WWII, there was much talk about a fifth column and fifth columnists, the latter being a group of people who secretly aid and support the enemy from within their own country. It's no secret that the "Americanization" of Canada is underway, and it's a general malaise within the country that's allowing it to happen. Living next to this giant with our fragile cultural mosaic and the few flag-wavers we have left, doesn't leave too much room to manoeuvre. Just look at the problems Quebecers have in maintaining their unique society. The Americans are offering their culture "to go", like a fast-food chain, and there are enough Canadian wafflers to believe it is the way to go. Canadian culture is a threat to the U.S. cultural industry, and they have the power to put us in the back of the bus, sort of like an ethnic cleansing without the bloodshed. We can't shut out American television, nor should we. We have to live with their restrictions on our film industry, and if Canadian recording artists continue to be successful internationally, we'll feel the weight of American protectionism, if, in fact, we aren't already. Now that radio has opened its doors a little wider to foreign partners, we could see some muscle flexing there as well. Well, like one American friend told me: "It's inevitable. So why not accept American domination?" (EC: Nice attitude!!!)
Get ready for V2!!! RB is coming back into the business with, what else, V2. Not too blatant what? He's bypassing his former friends and is going with a competitor. He even hired DB to make sure he sails into his new partnership with ease. Don't know how legal it is, but with V2 he obviously wants to keep the brand name alive. (EC: Biting the hand, or biting off too much, comes to mind!!!)
The indies are angry!!! It was mentioned here in the dying weeks of last year that 1997 could be disastrous for the independents. Well, the purge is on. But all is not lost. There's still RPM, the alternative, which can be used to your advantage. Don't roll over and die. Fight! Fight! fight! (EC: Shades of 1968!!!)
Clean underwear??? I remember when I was a kid, my mother always reminded me to wear clean underwear. "You never know when you might get in an accident." Thinking it over, she would probably be more embarrassed than I would. Anyway, back to the 20th century. There's an underwear outfit that's wooing the country music world, not knowing, of course, that most country people, guys and gals, don't wear any. They're going to sponsor a tour and a music festival, along with merchandise giveaways. (EC: Will that be boxer, bikini, G-string (EC: Anal floss!!!), brief or longjohn???) You'll also see lots of television sponsored specials, radio syndication specials, plus retail promotions. (EC: Calvin Klein must be really hurting their sales!!!)
A new Juno??? Nothing official yet, but all the dailies have reported there will be a new Juno category this year and the people's choice category will undergo a name change. The new category, the International Achievement Award, will honour Canada's top-selling musical women. And, they have a corporate sponsor for the Entertainer of the Year award. The sponsor is North Star, the shoe people. We should have some fun with that one. We'll go into more detail if, and when, we get the official press release. (EC: I thought you were taken off their list!!!) I know I won't be invited to the Canadian Music Hall of Fame banquet this year.
Hockey night!!! Getting the chance to spend an hour or two in the Hockey Hall of Fame always gives me a rush of Cancon adrenalin. After all, this is visual proof of where it all began. Rumour have other American city that epitomizes this great sport, that was once the proud national sport of Canada. And, now that news has hit the American fan of plans of a gold coin honouring Alexander Graham Bell as being the father of the telephone, which he gave birth to in Brantford, Ontario, there are veiled threats of a U.S. takeover of Bell Canada, which I thought was underway already. Even the dumb Webster Dictionary claims Bell was "the U.S. inventor of the telephone." Anyway, back to the Hockey Hall of Fame, where RPM was one of the sponsors. I took in the All Star Game Party which was in support of the Canadian Mental Health Association. Lots of great food and the stuff to wash it down, all for $65.00, plus a silent auction of some great hockey memorabilia and prizes, prizes, prizes. In the crush of the crowd I couldn't see everybody who was there, but knowing how fanatic the record and radio industries are about hockey, I imagine there was quite a bunch of them. I did spot Vince Parr of Tower Records, BMG's Rick Fox and Sony's Gregg Danielli. There was a little side-bet going on as to who would win and by how much, the NHL All-Star Game. Having spent most of his life in the Pacific Rim countries where ice is seen only on the tallest mountains peaks and in drinks, Vince noisily predicted that the West would win by a score of 11-7. Guess what? They did. How much did he win? Nothing! He didn't bet. (EC: He's probably the only one who came up with a score like that!!!)
Another Hall of Fame!!! Warren Cosford sent along the winners of The (unofficial) Rock Radio Hall of Fame (1986-1997). There weren't any Canadian winners until this year and it's appropriately titled (Cancon), and correctly spelled. The winners are Roger Ashby, Norman Blakely "Norman B", Al Boliska, Don Daynard, Dave Marsden, Jay Nelson, Red Robinson, Brian Skinner, Terry Steele and (non-performer) Walter Grealis. The radio station is CHUM, Toronto. Interesting enough, the first award, made in 1986, went to "The father of Rock 'n Roll radio", Alan Freed "The Moon Dog" and the radio station was WJW Cleveland. Dick Clark, Wolfman Jack and Murray "The K" Kaufmann were 1988 Movers and Shakers. By the way, radio historian Dale Patterson is the administer of this hall of fame, and he can be reached at the following E-Mail address: email@example.com
The Stanley Cup is the centre piece with the Book Cellar Yorkville's Lori Bruner, Sun Life's Doug Lounsbury and RPM's Stan Klees and Walt Grealis.
Top Selling Albums
The top-selling albums compiled from a national survey conducted by Nielsen SoundScan.
Third album garners Warner artist Justuss
Snow gets cleared
A quick glance over the list of contributors for the new album should allay any doubts about the kind of respect Snow achieved in Jamaica: among them are the likes of Toots Hibbert, Al Anderson, and the legendary Lee "Scratch" Perry. The album is called *Justuss* and it's due out on February 17th.
Tito Puente Jr.'s inexhaustible Oye Como Va and contemporary hits from Proyecto Uno (El Tiburon) and Wil Veloz (A Mover La Colita, Guantanamera). The disc is an original within Latin compilations simply because it avails itself of the real thing -- a sampling of its heralding from Central and South America and the Caribbean. For those who have always wondered where the true stuff is to be got and for the aficionados who don't need to be told, Oro Latino will go far in heating the Latin club scene even on the coldest of January nights. -RG
MORTAL KOMBAT: -Alternative
MORE KOMBAT - Various Artists
TVT Soundtrax / A&M 8030-Q
Part of the reason the first instalment of Mortal Kombat did so well at retail and remained high on Billboard charts for well nigh a year was because it captured what life might sound like from within a video game. The incredible response to the album has occasioned this sequel, which should recapture the buying dollar of the gaming freak still immersed enough in the video to not miss the fact that there is no accompanying movie. TVT Records have demonstrated an inherent understanding for the underground collective's desire for techno, metal and industrial, and have gone as far as to commission many of the acts exclusively for the project. They have concocted a mighty serving of bad guy guys featuring Psychosonic (It Has Begun), Sepultura (Chaos B.C.), Killing Joke (Drug), Sister Machine Gun (Deeper Down), Babylon Zoo (Space Gun), Gudrun Gut (Firethirst), Cherubab (Exile), Scream Of Grief (Outburst), among others. An irresistible offer for instigators of last year's most lucrative youth phenomenon and a great way to introduce the kids to the dark side of modern music. -RG
ORO LATINO -Latin Dance
Various Artists
Quality QRSPD 1247-M
The commercially proven, geographically privileged Latino dance music craze continues with a selection of recent hits, hand-picked by Toronto's own Latin music expert at Energy 108 FM, Producer Magda de la Torre (Super Latin Hits), Radames Nieves and Quality have distilled a revealing cross-section from the wide array offered by the genre without missing the night and the wistful serenades the Macarena, Clave, Conggo and brass driven hits like Raul Ruiz' Luna Negra, Tito Rojas' Senora de Madrugada and Willie Colon's Idilio reside alongside modern remixes of Barbara Ruiz' El Bimbo (El Amor), Leap year benefits from Gryner's brother Frank Gryner's engineering prowess (John Paul Jones, 54.40, Filter, Mike Watt) to make an album that is focused in its intent and bang on its mark. Look for the first single, Hello Aquarius, to be followed by any of Fetching Decay, Wisdom Bus or 69 Days Of Alcatraz. -RG
LEN
Get Your Legs Broke
Funtrip Trip004
When a band has the wherewithal to start their own record label and obtain relevant industry info with the clever use of a hold button and a few mythical board execs, chances are there's going to be music to follow that is just as creative and gutsy. Which is definitely the case with Halifax-transplants Len and their second release Get Your Legs Broke. The twelve tracks on this indie effort are an eclectic mix of alternative and experimental mixtures of trippy guitar chords and powerful and oftentimes bizarre vocal combinations -- bassist/vocalist Sharon Castanza's sweet, bird-like voice gets pulverized by guitarist/vocalist and brother Marc's harsh and powerful harmonics. It's worth noting that a track may be bordering on the sugary side. Fans of candy pop-grunge and indie rock will enjoy the quirky cleverness of songs like Granny Love, Grand Theft Snowboard and the ominous title track which cleverly melds ballad-like music and sweetly-sung lyrics that are essentially a threat to kick the hell out of someone who has wronged a friend. Check this one out. After all -- this is the band who released their first album on eight-track and wrapped in fun fur. -LT
EMM GRYNER -Pop/Alternative
The Original Leap Year - Dead Daisy Records - EMM 001-Outside Music
This twenty-one year old native from Forest, Ontario has quickly established herself as a songwriting talent to be reckoned with and scooped up several awards while on her trek to authenticate the claim. A multi-instrumentalist by trade and a pianist by choice, Emm Gryner demonstrates amazing dexterity on her inauspicious debut, much of which derives its strength from a novel mixture of sweet-as-she-balls ballads with an alternative crunch. The result? A very effective, very modern stab at the contemporary pop artery which should have folks at the labels thinking along the lines of the recent industry interest in artists like Chantal Kreviazuk, Fiona Apple and Tori Amos. Independent at heart, The Original
Ashley MacIsaac leads the pack in ECMA nominations
Nominees for Canadian Music Industry Awards
Nominees for Canadian Music Industry Awards in a total of 35 different categories were announced today (January 6). The categories cover every area of Canada's growing music industry—from artists' managers to radio personalities, from music retailers to music talent buyers, from record company executives and promotion staffers to agents and publicists.
The awards will be presented at the Industry Awards Dinner, set for the Metro Convention Centre on Thursday, March 6. A highlight of the evening will be a presentation of the Canadian Music Week Lifetime Achievement Award to Gerry Lacoursiere, the recently-retired chairman of PolyGram Group Canada, one of the six major multi-national record companies operating in this country.
PERSONAL MANAGER
BRUCE ALLEN - Anne Murray, Bryan Adams
NEIL BELL - Neil Young, Celine Dion
TOM BERRY - Eric Clapton, Cole Kim Mitchell
RAY DANNIELS - Rush, Van Halen, Tea Party
JAKE GOLD - The Watchmen, The Tragically Hip
SHERRI JONES - Ashley Macisaac, Laura Smith, Frank Wiepert (Courage)
INDIE PROMOTION PUBLICITY
SHELLY BRESLAW (Shelly Breslaw Promotion)
RICHARD FLOHL (Richard Flohl & Associates)
BOBBY GALE (Plug Enterprises)
SHIRLEY HUGHES (Hughes Creative)
LAURA HOCROFT (Boundless)
MIRA LAUFER (Ethan Russell Promotions)
LINDA NASH (Cascovier Promotions)
JOANNE PEARSON (Laurie Smae Productions)
GREG SIMPSON (Mindstream)
YVONNE VALNEA (Last Tango)
ANYA WILSON (Anya Wilson Productions)
DANCE POOL
BAD VIBE
BOMBS N' BASS
CHERIE
CLUB SERVICE INTERNATIONAL
KEYSTONE
NRG RUSH
POOL FIST
POOF WEST
SPINNERS
MUSIC PUBLISHER
ATTIC/PONDWATER
BALMUR MUSIC
BMG
EMI
POLYGRAM
PEER
TMP
UNIVERSAL
WARNER/CHAPPELL
RECORDING STUDIO
CHARLIE CITTILOS - Claremont
GRANT AVENUE - Hamilton
LE STUDIO - Morin Heights
MANTA EASTERN - Toronto
MCGREGOR STUDIO - Toronto
METALWORKS - Mississauga
MUSHROOM - Vancouver
NUMBER NINE - Toronto
ORCA RECORDS - Vancouver
REACTION STUDIO - Toronto
SOLAR - Halifax
SUNSHINE - Winnipeg
CAMPUS STATION OF THE YEAR
CFMU (McMaster) - Hamilton
CFRC (Queens) - Kingston
CTIIS - Vancouver
CIUT (U of T) - Toronto
CJAM (UofW) - Windsor
CJSF (Simon Fraser) - Burnaby
CJSN (University) - Calgary
CKCU (Carleton), Ottawa
CKDU (Dalhousie) - Halifax
CKUT (McGill) - Montreal
SECONDARY MARKET MUSIC DIRECTOR
(Pop, under 300,000)
SEAN BARRETTE (Q93) - Sudbury
DEBRA BELL (CHUM) (QZ-FM) - St John's
CASEY CLARK (SILK) - Kelowna
DAWN GALLANT (KHJ) - Fredericton
GEORG GREGORY (CJCJ) - Sydney
JEFF HOLMES (CJCB) - Cambridge
KRISTY KNIGHT (HTZ-FM) - St Catharines
RICH NICKEL (CKGY) - Red Deer
MIKE ROSE (The River) - Brockville
JIM SCANLON (CKLZ) - Kelowna
SECONDARY MARKET PROGRAM DIRECTOR
(Pop, under 300,000)
TOM BERRY (KHJ) - Fredericton
MURRAY BROOKS (Q92) (Radio 4) Windsor
MIKE BROOKS (Q93) Charlottetown
DR. BUD (CKLZ) Kelowna
JOHN CALEY (CJCY) Medicine Hat
DAVE FAROUGH (Q101.03) Victoria
GREG HINTON (The River) Brockville
BOYD LEADER (CKGY) Red Deer
RICH NICKEL (CKGY) Red Deer
MALCOM SINCLAIR (WOLF) Peterborough
RANDY TAYLOR (HTZ-FM) St Catharines
JEFF WALTER (B101) Barrie
SECONDARY MARKET STATION
(Pop, under 300,000)
BT Barrie
CHAT - Medicine Hat
CHOI - Quebec
CIOC (The Ocean) - Victoria
CICY - Medicine Hat
CIGY - Medicine Hat
CKGY - Red Deer
CKLZ - Kelowna
HTZ-FM - St Catharines
OZ-FM - Sudbury's
Q100 - Victoria
Q93 - Charlottetown
Q92 - Sudbury
Rock 86 - Barrie The River - Brockville
WOLF - Peterborough
NETWORK SYNTH/SYNDICATOR
CHUM Satellite Network
Pelmexor Radio Network
Rock Radio Network
Sound Source
Telemedia Network
RADIO PERSONALITY
BEARDY M & FRAZIER (Q94)
BRUCE BOYD (Q93)
ROB CHRISTIE (Mix 99)
DON DAYNARD (CHF)
TERRY DENTON (Mix 98)
CLIVE DUMAS (CISS)
GERRY FORBES (CJAY)
JESSE & GENE (Q107)
KIM HUNT (The Edge)
HUMPLE & FRED (The Edge)
LARRY & WILLY (CFOX)
MAJOR MARKET MUSIC DIRECTOR
GLY BOURRIAUD (CKOI) - Montreal
PHIL KALLSEN (CKRY) - Calgary
KNEALE MANN (The Edge) - Toronto
MATTHEW MUELLER (The Pearl) - Edmonton
ROB ROBINSON (CFOX) - Vancouver
ANDY ROSS (Q93) - Winnipeg
ÉRIC STAFFORD (Q104 FM) - Halifax
BARRY STEWART (Q104 FM) - Toronto
CURTIS STONE (Q92) - Vancouver
BARRY TAYLOR (CITI) - Winnipeg
JANET TRECATREN (CISS) - Toronto
SCOTT WINTER (Energy 108) - Toronto
WAYNE WEBER (Mix 98) - Toronto
BILLY WILLIAMS (CISN) - Edmonton
MAJOR MARKET PROGRAM DIRECTOR
Steve Colwell (CHEZ FM) - Ottawa
Paul Fisher (CHF) - Toronto
Ford Gardner (CITI) - Winnipeg
Greg Haridasan (Q92) - Calgary
J.J. Johnson (Mix 99.9) - Toronto
Howard Kroeger (Q94) - Winnipeg
Stu Meyers (The Edge) - Toronto
Bob Mills (CFOS) - Vancouver
Eric Sanders (Z95) - Vancouver
Bob Spitzer (CISN) - Edmonton
Scott Turner (Energy 108) - Toronto
Ross Winters (CFMI) - Vancouver
STATION (News/Talk/Sports)
CFRA - Ottawa
CFRB - Toronto
CHED 630 - Edmonton
CHRL - Hamilton
CIAD - Montreal
CJBK - London
CJOB - Winnipeg
CKAC - Montreal
CKNW - Vancouver
STATION (Country)
CFCD - Saskatoon/Winnipeg
CHAM - Hamilton
CHFX - Halifax
CISN - Edmonton
CJSX - Sudbury
CJUR - Vancouver
CKBY - Ottawa
CKQM - Peterborough
CKRY - Calgary
CXKM - Victoria
STATION (Dance /CHR)
CHHR - Winnipeg
CKQJ - Montreal
CKMF - Montreal
Energy 105 - Toronto
Hot 105 - Toronto
Kool Fm - Ottawa
Power 92 - Edmonton
Mix 96 - Montreal
Z95.3 - Vancouver
STATION (AC)
C100 - Halifax
C101 - Toronto
CHFM - Calgary
CHUM FM - Toronto
CION - London
CITE - Montreal
CKIS - Calgary
CKKS - Vancouver
K-lite - Hamilton
Majic - Ottawa
Mix 98.9 - Toronto
STATION (Rock)
CFOR - Vancouver
CHEZ - Ottawa
CHOM-FM - Montreal
CITI - Winnipeg
CJAY - Calgary
OKIK - Calgary
HTZ FM - St Catharines
Q104 - Halifax
The Bear - Edmonton
The Bear - Ottawa
The Edge - Toronto
BROADCAST EXECUTIVE
Hal Blackadar (Shaw)
Bill Brady (Blackburn)
Ross Davies (Chum Ltd.)
Dennis Donlon (Citi/Bravo)
Doug Pringle (Ravico)
Duff Roman (Chum Ltd.)
Sandy Sanderson (Rogers)
Don Schreiber (Rogers)
Allan Slaight (Standard)
Gary Slaight (Standard)
Terry Strain (Shaw)
Bob Templin (Newcap)
Jim Watson (Chum Ltd.)
RETAIL STORE
A&B Sound - Calgary
Archambault Music - Laval
CD Warehouse - Ottawa
HMV - Toronto (Yonge St)
HMV - Vancouver (Robson)
Music Plus - Winnipeg
Music World - Toronto (Yorkdale)
Sam The Record Man - Toronto (Yonge St)
Summit Records (Yonge St)
Tower Records - Toronto
Virgin - Vancouver
RACK JOBBER/SUB DISTRIBUTOR
Handman
Pindoff Record Sales
Roblans
Saturn Distributors
Total Sound
INDEPENDENT DISTRIBUTOR
Carsip
Duckworth
Festival
Koch
Page
Select
RETAILER
A&B Sound
Archambault Music
CD Plus
HMV Canada
Music World
Sam The Record Man
Sunrise
RETAIL EXECUTIVE
ROSAIRE ARCHAMBAULT (Archambault)
TIM BAKER (Sunrise Records)
DAVID BELL (CD Plus)
LEONARD KELLY (Saturn Distributors)
RICK LOTMAN (Pindoff Record Sales)
PETE LUCKHURST (HMV Canada)
LANE ORR (A&B Sound)
KRIS PINKERTON (Robloff Record Sales)
JASON SNIDERMAN (Sam The Record Man)
SAM SNIDERMAN (Sam The Record Man)
ROGER WHITEMAN (HMV Canada)
RECORD SALES TEAM
BMG
EMI
PGS
Sony
Universal
Warner
MARKETING TEAM
A&M/Island/Motown
BMG
EMI
Mercury/Polydor
Quality Music & Video
Sony
Universal
Virgin
Warner
PROMOTION TEAM
A&M/Island/Motown
BMG
EMI
Mercury/Polydor
Sony
Universal
Virgin
Warner
LABEL PUBLICIST
CAR CARPENTER (BMG)
CORI FERGUSON (Universal)
ANGELA HERNS (Koch)
LIZ MCEHLERAN (EMI)
SAMANTHA MURPHY (Mercury/Polydor)
LORRAINE QUATUAR (Columbia)
ELENA RABINOVITCH (A&M/Island/Motown)
STEPHANIE ROBERTSON (Anthem)
KEVIN SMITH (EMI)
STEVE WAXMAN (Warner)
NANCY YU (Virgin)
INDEPENDENT LABEL
Alert
Anthem
Atlantica
Antic
Handsome Boy
Iron Music
Murder
Netterk
Numuzik
Pirate
Quality
Sonic Unyon
SPG
Star
Stony Plain
Stubble Jumper
True North
MAJOR RECORD COMPANY
BMG
EMI/Virgin
Polygram
Sony
Universal
Warner
MUSIC EXECUTIVE
Paul Alofs
Deane Cameron
Rick Camilleri
Doug Campbell
Sam Feldman
Stan Kulin
Randy Lennox
Alex McFarlar
Garry Newman
John Reid
Ross Reynolds
Joe Summers
From Broadcast to Narrowcast (07)
Iron Music enters US market with Is Music
Iron Music along with Start Records has recently inaugurated a new independent label based out of L.A. to channel product into the US marketplace. Is Music is the brainchild of André DiCesare and Anthony Muscolo of Start Records and Iron Music’s Aubrey Winfield, who have partnered for presence out of L.A.
The arrangement evolved out of the suggestion from Winfield, who had licensed Iron Music acts to Start in the past year, and had been considering the possibility of stateside representation. Winfield was especially intrigued by the promotional prowess of Muscolo, who has boasted at least one act on Billboard’s Top 100 chart for the past twenty-five years and whose exhaustive repertoire includes or has at one time included Garbage, The Offspring, Sheryl Crow, Poe, Enigma and Dave Matthews Band.
“We realized how much of a powerful tool he was to have on our side,” explains Winfield, “so we decided the best thing would be a joint venture between the companies where we would provide the repertoire and Tony would handle the product.”
Iron Music’s first licensed agreement will be for Bug!, a Toronto-based independent act that has elicited considerable international attention despite a lukewarm reception at home.
“When we started shopping the record we were really taken back by the type of positive response we were getting out of Europe and out of Southeast Asia,” says Winfield, adding that he expects the record’s resistance to category not to pose as big a problem south of the border.
“I think in the US there are more formats to go after, especially mix shows which we really don’t have here,” he says.
Apple to sponsor CMW’s Music/Multimedia show
Canadian Music Week’s Music & Multimedia show will be sponsored this year by Apple Canada. The annual conference, exhibit and trade show will take place at Toronto’s Metro Convention Centre (March 7-9).
The Apple Music & Multimedia Show, billed as “the largest show of its kind,” is designed to attract musicians and others interested in the latest technology and services available in the music, multimedia, virtual reality and home entertainment markets.
Neil Buchanan, manager of Apple Canada’s market development, points to the “long and mutually beneficial relationship” Apple has had with the music industry. “For thousands of musicians, Apple is the key to successful and powerful creative breakthroughs.”
Besides Apple, exhibitors will include instrument manufacturers and distributors, music schools, recording studios, consumer electronics companies and a variety of other specialist firms.
Though Iron Music plans to initially concentrate on channelling domestic releases to the label, they will eventually turn their attention to the “tremendous amount of material” from UK artists discovered by a rep in Bristol, England.
“Deals are being negotiated for further releases with three more by the first quarter and probably six records by the end of the year,” says Winfield.
Jodie Ferneyhough named to peermusic Canada post
Jodie Ferneyhough has been appointed creative manager of peermusic Canada. He replaces David Baxter.
Ferneyhough joins peermusic from The Alternative Management Group where he represented such artists as Monoxides, Adam Faux and pigfarm. He was previously involved with Canadian Music Week as talent coordinator from 1993 through 1996.
Founded more than 65 years ago, peermusic boasts 27 offices in 23 countries.
Toronto’s Cinram acquires U.K. video duplicator
One of the world’s leading independent manufacturers of pre-recorded multimedia products has added a new component to its vast base of facilities with the recent acquisition of Videoprint Ltd. of London, England.
Isidore Philisophe, chairman and CEO of Cinram Ltd. announced that through a wholly-owned U.K. subsidiary, Cinram has entered into an agreement with Videoprint Ltd to purchase the London-based company’s video cassette duplication and distribution business for a purchase price the equivalent to $14.7 million Canadian.
“This acquisition provides us with an important beachhead in the U.K.,” says Philisophe, “and, when added to our existing operations in France, represents a further step in our strategic objective of enhancing Cinram’s presence in the growing European community video market place.”
This year’s projected sales for Videoprint are near the $45 million mark. Completion of the transaction is scheduled for later this month.
In addition to this purchase, Cinram also recently acquired DMI Inc. in the U.S. and has an extensive broad base of multi-media facilities in North America, Latin America and Europe.
Bruce Allen takes Kim Stockwood under management
Vancouver-based Bruce Allen Talent kicked the year off with a new signing, Kim Stockwood. The EMI/Curb recording artist is the newest management client, joining a roster that boasts Anne Murray, Bryan Adams and Martina McBride.
Stockwood’s debut album, Bonavista, is now close to gold (50,000 units) in Canada. The album was recently revised to include two new songs, Jerk, which became a hit as a single, and You Won’t Remember This. Jerk has been released in the US on the Curb label.
Kim’s spirit, enthusiasm and drive attracted me immediately to the project,” beams Allen. “We hope to kick down some doors with Jerk and build some lasting relationships with radio in the states to set up the next album.”
Newfoundland-born Stockwood holds a BA in English and Business from St. John’s Memorial University and with this signing, jokes, “Now I have two BA’s,” the other being Bruce Allen as a manager.
Stockwood has been nominated for three East Coast Music Awards: Album for Bonavista, Song for Jerk, and with Naoise Sheridan, as SOCAN Songwriter for Jerk. She will be performing at East Coast Music Awards in Moncton on Feb. 16.
Major changes for EMI marketing
Deana Cameron, president of EMI Music Publishing, will be named president of EMI's music publishing division and will report directly to EMI chairman and CEO John K. Kimmel.
Cameron, who has been with EMI since 1984, will be responsible for overseeing the company's music publishing business in North America, Europe, Australia and Asia. She will report directly to Kimmel, who will continue to oversee the company's worldwide music publishing operations.
Cameron has been with EMI since 1984, serving in various capacities including vice president of A&R and president of EMI Music Publishing. She is a member of the board of directors of the National Association of Music Publishers (NAMM) and the Recording Industry Association of America (RIAA).
Cameron is a graduate of the University of California at Berkeley and holds a master's degree in music from the University of Southern California. She is a member of the board of directors of the National Association of Music Publishers (NAMM) and the Recording Industry Association of America (RIAA).
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Michelle Wright has a new single, The Answer, off her debut album, which was released in autumn 1996.
Carter USM's latest single, I'm Not Your Baby, is up from #65. Carter has been making some pretty effective TV appearances, particularly on the David Letterman show.
George Strait's latest release, King Of The Road, has been a huge success and he returns to talk about his new album, Where I Stand.
Ryan Peters is getting a large-sized promotion with his new album, The Best Of Ryan Peters.
### Adult Contemporary Tracks
| TW | LW | WD | JANUARY 27, 1997 |
|----|----|----|------------------|
| 1 | 7 | 6 | TO LOVE YOU MORE
| | | - Usher
| | | 580 Music/4-67641 (32 track) N |
| 2 | 2 | 8 | I FINALLY FOUND SOMEONE
| | | Sheryl Crow
| | | Warner Bros-48342 (comp 24) P |
| 3 | 5 | 10 | UNBREAK MY HEART
| | | Seal - Stacey
| | | Atlantic-82489 (comp 30) P |
| 4 | 8 | 8 | FLY LIKE AN EAGLE
| | | Stevie Nicks
| | | Warner Bros-48341 (comp 20) P |
| 5 | 10 | 5 | BETCHA BY GOLLY WOW
| | | The Artist - emancipation
| | | Warner Bros-48342 (comp 24) P |
| 6 | 4 | 9 | IF WE FALL IN LOVE TONIGHT
| | | Rod Stewart - little track
| | | Warner Bros-48452 (comp 24) P |
| 7 | 1 | 12 | WHEN YOU LOVE A WOMAN
| | | Janet Jackson - By My Side
| | | Columbia-87514 (pro single) H |
| 8 | 3 | 7 | KISSING RAIN
| | | Marillion - little track
| | | RV International/BMI-43978 (pro single) N |
| 9 | 12 | 9 | JUST ANOTHER DAY
| | | John Mellencamp - Mr. Happy Go Lucky
| | | MCA-1-4326 (comp 11) H |
| 10 | 11 | 9 | 100 YEARS FROM NOW
| | | Huey Lewis And The News - the Best Of
| | | Epic-61918 (comp 30) H |
| 11 | 9 | 10 | YOU MUST LOVE ME
| | | Madonna - Livin' USA
| | | Warner Bros-48348 (pro single) P |
| 12 | 14 | 6 | YOU WON'T REMEMBER THIS
| | | Michael Jackson - Remember
| | | EMI-52296 (comp 24) H |
| 13 | 6 | 13 | IF IT MAKES YOU HAPPY
| | | Sheryl Crow - Self-titled
| | | MCA-1-11825 (comp 8) U |
| 14 | 17 | 3 | DARK HORSE
| | | Amanda Marshall - Self-titled
| | | tpc-800-8237 (comp 15) H |
| 15 | 18 | 9 | DOING FINE
| | | Jan Hill - little track
| | | MCA-81012 (pro single) J |
| 16 | 21 | 5 | DON'T SPEAK
| | | Nicole Scherzinger - Kingdom
| | | Interscope-82680 (comp 18) J |
| 17 | 24 | 5 | STILL IN LOVE
| | | Ute Lemper - Louder Than Words
| | | Warner Bros-48348 (32 track) U |
| 18 | 25 | 7 | THE TEARS I CRY
| | | Gavin Hope - No album
| | | Warner Bros-48348 |
| 19 | 19 | 16 | YOU CAN MAKE SONGS
| | | Btton John - Love Songs
| | | MCA-1-11481 (comp 16) J |
| 20 | 13 | 14 | BLACK CLOUD RAIN
| | | Good Heart - Rainy Days
| | | Columbia-80240 (pro single) H |
### Adult Contemporary Tracks
| TW | LW | WD | JANUARY 27, 1997 |
|----|----|----|------------------|
| 21 | 23 | 6 | HURT BY LOVE
| | | 3odanes - blend
| | | Warner Bros-48216 (comp 30) P |
| 22 | 15 | 12 | MOUTH
| | | Merrii Blankenge - the Garden
| | | MCA-53109 (comp 14) U |
| 23 | 27 | 23 | IT'S ALL COMING BACK TO ME NOW
| | | Calypso - Going Into Your
| | | 550 Music/pc-87541 (pro single) H |
| 24 | 38 | 3 | WITHOUT LOVE
| | | Lonna Lewis - How It's Minute
| | | Atlantic-82489 (comp 30) P |
| 25 | 20 | 21 | LET'S MAKE A NIGHT TO REMEMBER
| | | Bryan Adams - 18 In 11ue
| | | Warner Bros-48342 (pro single) U |
| 26 | 16 | 10 | SAD CAPER
| | | Hookie & the Blowfish - Harweather Johnson
| | | Atlantic-82680 (comp 30) H |
| 27 | 26 | 12 | LIVE IS THE POWER
| | | Michael Jackson - This Is the Time
| | | Columbia-87621 (pro single) H |
| 28 | 35 | 5 | LEAVIN'
| | | Tony Rich Project - Words
| | | Warner Bros-48348 (comp 10) N |
| 29 | 30 | 3 | I BELIEVE IN YOU AND ME
| | | Whitney Houston - Proacher's Wife US1
| | | Avanti-80241 (comp 11) N |
| 30 | 31 | 5 | THE FLAME
| | | Fine Young Cannibals - Finest
| | | MCA-1-11825 (comp 20) U |
| 31 | 36 | 3 | FOR THE FIRST TIME
| | | Kenny Loggins - For the First Day US1
| | | Columbia-87514 (pro single) H |
| 32 | NEW | 3 | THROUGH YOUR HANDS
| | | The Artist - Emancipation
| | | Warner-24988 (12) track+P |
| 33 | 37 | 3 | I'M NOT GIVING UP ON YOU
| | | Ulrica Isakson - Iastea
| | | Epic-61918 (comp 10) H |
| 34 | 39 | 3 | SEIZE THE DAY
| | | Carolyn Arends - I Can Hear You
| | | Warner Bros-48348 (32 track) U-N |
| 35 | 32 | 14 | DANCE INTO THE NIGHT
| | | Pitbull - little track
| | | Atlantic-82489 (comp 30) P |
| 36 | NEW | 3 | DON'T CRY FOR ME ARGENTINA
| | | Madonna - Like A Virgin
| | | Warner Bros-48348 (12) track-P |
| 37 | 41 | 2 | JUMPING THE SHADOWS
| | | Ummahit Lovley - Shadows Wake Me
| | | Universal-82489 (comp 10) H |
| 38 | 44 | 2 | THE GREASE MEGAMIX
| | | John Travolta w/Lilivia Newton-John - Pure Disco
| | | Polydor-7897-877 (comp 49) U |
| 39 | 45 | 2 | LET IT RAIN
| | | Megan Mitchell - little track
| | | HM-36800 (pro single)+ |
| 40 | 33 | 14 | ALL OVER FEET
| | | Alana Mancuso - Little Big Little Hi
| | | Maverick-45901 (UK track) P |
### Dance Tracks
| TW | LW | WD | JANUARY 27, 1997 |
|----|----|----|------------------|
| 1 | 6 | 6 | JELLYFISH
| | | Chuck
| | | Arista-Koch |
| 2 | 7 | 6 | UP TO NO GOOD
| | | Pom Kings
| | | Popular-P |
| 3 | 5 | 5 | WANNABE
| | | Spice Girls - Virgin-P |
| 4 | 4 | 6 | THA WILD STYLE
| | | Salt-N-Pepa - Supergroup
| | | Distinct/Duality-M |
| 5 | 3 | 10 | FIRED UP
| | | Funky Green Dogs
| | | Warner-BMG-M |
| 6 | 2 | 10 | MY BOO
| | | Ghost Tower DJs
| | | Sony-H |
| 7 | 13 | 3 | 4 YOUR LOVE
| | | 4 Your Love
| | | Peeta-Quality-M |
| 8 | 18 | 3 | I WANT CANDY
| | | The Candy Girls
| | | Epic-61918 |
| 9 | 19 | 3 | SEVEN DAYS AND ONE WEEK
| | | B.B.E.
| | | Sony-H |
| 10 | 24 | 2 | ONE & ONE
| | | Robert Miles
| | | Arista-N |
### Dance Tracks
| TW | LW | WD | JANUARY 27, 1997 |
|----|----|----|------------------|
| 11 | 20 | 4 | ONLY YOU
| | | 112
| | | BMG-N |
| 12 | 23 | 2 | SUGAR IS SWEETER
| | | C.J. Rolland
| | | Mercury/Polygram-Q |
| 13 | 6 | 9 | FALLIN' IN LOVE
| | | Moby
| | | BMG-N |
| 14 | 9 | 14 | NO DIGGITY
| | | Blackstreet
| | | MCA-N |
| 15 | 10 | 10 | GET DOWN
| | | Backstreet Boys
| | | BMG-N |
| 16 | 25 | 2 | BRUNCH
| | | France Joli
| | | Popular-P |
| 17 | 8 | 11 | STAND UP
| | | Eve & Ibiza
| | | A&M-A |
| 18 | 11 | 4 | GINUWINE
| | | Poni
| | | Warner Bros-H |
| 19 | 22 | 5 | WATCHING THE WORLD GO BY
| | | Maxi Priest
| | | Virgin-P |
| 20 | 12 | 8 | OUNGIN'
| | | LL Cool J
| | | Polygram-Q |
### Dance Tracks
| TW | LW | WD | JANUARY 27, 1997 |
|----|----|----|------------------|
| 21 | 14 | 7 | JAZZ IT UP
| | | Real 2 Reel
| | | Quality-M |
| 22 | 16 | 9 | KEEP ON JUMPIN'
| | | The Dirty Beat, Martha Wash
| | | BMG-N |
| 23 | 21 | 4 | STAMP!
| | | Heavy & Amos
| | | Capitol-P |
| 24 | NEW | 4 | DO YOU MISS ME
| | | Jocelyn Enriquez
| | | Tommy Boy-Denon |
| 25 | NEW | 4 | HAPPY DAYS
| | | PJ
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COUNTRY continued from page 17
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Large deflections of a clamped-slider-pinned rod with uniform intrinsic curvature
V.G.A. Goss*, P. Singh, R. Chaouki
School of Engineering, London South Bank University, London SE10 0AA, United Kingdom
ARTICLE INFO
Article history:
Received 4 May 2017
Revised 20 July 2017
Available online 12 September 2017
Keywords:
Intrinsic curvature
Elastica
Rod
Elliptic integrals
Experiments
Loop formation
ABSTRACT
This paper examines large planar deflections of a slender elastic rod with uniform intrinsic curvature, which is clamped at one end and the other end is pinned to a slider allowing it to move freely in the vertical direction as the ends are displaced horizontally in a straight line. The analysis encompasses force-displacement loading paths and corresponding configurations, with a particular focus on the formation of loops. The analysis is accompanied with data obtained from experiments on nickel titanium alloy strips, demonstrating a good match with theoretical predictions.
© 2017 Elsevier Ltd. All rights reserved.
1. Introduction
Whilst the planar equilibrium of intrinsically straight rods have been subjected to considerable analysis there have been very few studies of intrinsically curved rods. Consequently, our understanding of the effects of intrinsic curvature remains relatively poor. This paper aims to contribute towards redressing that.
We remark here that few rod-like structures, whether artificial or naturally occurring, are actually straight, they nearly all contain a certain amount of intrinsic curvature. Indeed, preliminary studies of animal vibrissae suggest that intrinsic curvature may be advantageous to animals because it generates correspondingly large forces and bending moments at the vibrissal base (Quist and Hartmann, 2012). Deeper understanding of the effects of curvature can assist engineers in devising new technologies that are inspired by nature, for example robotics engineers developing robots that use flexible rods to gather information and explore spaces where light is restricted or too bright (HEvans et al., 2014; Pearson et al., 2007). Problems involving intrinsically curved rods arise in a variety of real world scenarios: for example when unwinding cable or wire from a spool. It also arises when intrinsically curved rod-like structures, such as textile fibres are bent (González and Lorca, 2004).
The problem of an intrinsically curved rod has been analysed by Love (1892), Frisch-Fay (1962), Snowdon (1963), Fichter and Pinson (1989), and González and Lorca (2004). Love examines the problem of an intrinsically curved rod that is loaded by an end moment. He concludes that the only way in which it affects the problem, compared to the intrinsically straight rod is that the terminal couple required to hold the wire in the form of a given elastica is diminished by the terminal couple that would be required to bend a naturally straight wire into the given originally circular form” (Love, 1892). However, he does not discuss the problem of an intrinsically curved rod loaded solely by an applied end force. That problem was examined by Frisch-Fay who writes the solutions to the problem in terms of elliptic integrals (Frisch-Fay, 1962). He alludes to an issue that crops up with respect to the elliptic parameter. He noticed that small adjustments in the value of the end force can cause dramatic changes in the value of the elliptic parameter. The net effect is that the distinction between inflectional and noninflectional elastica, which is clear in the case of an intrinsically straight rod, become muddled. Snowdon also analyses the problem in terms of elliptic integrals (Snowdon, 1963). He classifies the solutions into three ‘types’ according to the value of the elliptic parameter. His paper contains some plots of the shapes of the elastica for the three types. However, his classification has little bearing on how the end load affects those shapes. Fichter and Pinson examine the problem of an eccentrically applied end force (Fichter and Pinson, 1989). Rather than elliptic integrals, they express the solutions in ‘raw’ integral form (not elliptic integrals) and use a numerical scheme to obtain a relation between the applied end force and the end displacement. González and Lorca also examine the problem (González and Lorca, 2004). They solve the problem in terms of incomplete elliptic integrals numerically for...
certain pairs of values of end force and end displacement and then determine a polynomial fit for the force as a function of the end displacement. However they only consider the configuration of the rod under action of an axially applied end force acting in one direction and they do not examine loop formation.
Although our concern here is restricted to planar equilibrium states, we mention studies involving elastic rods with intrinsic curvature that deform spatially i.e., where twist plays a role. For example, Clampeyns et al describe a twisted equilibrium deformation whereby a one two-period helix wave configuration arises (Clampeyns et al, 2007). The authors additionally state that, in the case of spatial deformations, initial curvature breaks complete integrability (though that does not carry across to the planar case). Starotisin et al consider the problem of successively removing the loops in a helical spring under an end load (Starotin and van der Heijden, 2009). They find that the loops tend not to unwind when pulled, but deform into localised ‘hockles’ that pop-out one after the other as the end load is increased. Their analysis encompasses planar configurations as well as three-dimensional ones. The planar solutions are given in terms of elliptic integrals and elliptic functions, and arise from zero-moment boundary conditions.
Just as the simplicity of an intrinsically straight rod establishes a basis for practically all analytical studies of rod-like structures, this paper examines the particular case of an intrinsically uniformly curved rod. The rod is clamped at one end and the other end is pinned to a slider allowing it to move freely in the vertical direction as the ends are displaced horizontally in a straight line i.e., rigid loading along the horizontal axis only. That procedure may be considered as semi-rigid loading. We present a formulation of analysis and numerical methods to compute force-displacement diagrams and equilibrium shapes of the rod. The results are compared to experimental data obtained from a series of experiments on nickel titanium strips with appropriate intrinsic curvature. Our analysis encompasses the phenomenon of loop formation, which arises in a range of engineering applications: in his classic book on cable-buoy systems, Berteaux remarked that kinks are the ‘mortal enemy of wire ropes’ and noted that they are initiated by loop formation (Berteaux, 1976). They also arise in the textile industry (Rangamul, 2011) and in cable laying activities (Coyne, 1990). However, all the aforementioned studies are restricted to intrinsically straight rods.
2. Formulation of the boundary value problem
2.1. Geometry of the unstressed rod
Consider a rod that has uniform intrinsic curvature $\frac{1}{R}$ i.e., it is an arc of a circle with radius $R$, see the left hand illustration in Fig. 1. The rod has length $L = R\gamma_i$, where $\gamma_i$ is the centre angle of the arc. The arc length is parameterised by the variable $S$, with end points $S = 0$ and $S = L$, i.e., $0 \leq S \leq L$, where $S = 0$ is the origin of a Cartesian coordinate system $(X, Y)$.
In the case of the unstressed rod, the angle measured anticlockwise from the X axis is denoted $\Psi_i(S)$, as shown in the right hand figure in Fig. 1, where the suffix ‘i’ denotes the initial unstressed state. In this paper, the intrinsic curvature is positive. Inspection of Fig. 1 indicates that the coordinates $(X_i(S), Y_i(S))$ are given as $X_i(S) = R \sin \Psi_i(S)$, $Y_i(S) = R(1 - \cos \Psi_i(S))$ and that
$$\frac{dX_i(S)}{dS} = \frac{X_i(S)}{R - Y_i(S)} = \tan \Psi_i(S)$$
and
$$\gamma_i = \arctan \left( \frac{X_i(L)}{R - Y_i(L)} \right) = \arcsin \left( \frac{X_i(L)}{R} \right).$$
2.2. Geometry of the bent rod and experimental set-up
The configuration of the unstressed rod is its reference configuration. The end $S = 0$ is clamped and the end $S = L$ is pinned in a vertical slider, see the experimental set-up shown in Fig. 2. We consider the configuration of the rod when the end $S = L$ is displaced in a straight horizontal line along the X axis by amount $D$. For each rigid displacement $D$, the end $S = L$ is free to move vertically along the Y axis. Displacement by amount $D$ can either be in the positive X direction whereby the force in the rod is positive, or in the negative X direction whereby the force is negative, see Fig. 3. In the experiment the end force $T$ is recorded at intervals of $D$. All measurements of forces reported in this paper are in the range from $-0.5$ to $1$ N. The transducer used to measure the forces has a sensitivity of 0.0001 N. This semi-rigid loading arrangement is specified by the following boundary conditions
$$X(0) = 0,$$
$$Y(0) = 0,$$
$$\Psi(0) = 0,$$
$$X(L) = R \sin \gamma_i + D,$$
$$\frac{d\Psi(L)}{dS} = \frac{1}{R},$$
where $-L - R \sin \gamma_i \leq D \leq L - R \sin \gamma_i$. Note that Eqs. (3)-(5) state that the rod is anchored at the origin by clamps. Eq. (6) specifies the rigid loading with respect to the horizontal axis and Eq. (7) states that the curvature at the end $S = L$ is the intrinsic curvature $\frac{1}{R}$. For the purpose of experiment we use uniformly intrinsically curved nickel titanium rods of length $L$, ranging between 300–400mm and flexural rigidity $EI = 0.0034$ Nm$^2$, where the constants $E$ and $I$ are the modulus of elasticity and the second moment of area respectively. Each rod has a rectangular cross section with a width of 3mm, a thickness of 0.5mm. We assume that those rods (strips) are homogeneous, incompressible, inextensible and that the following linear constitutive relation between bending moment $M(S)$ and curvature $\frac{d\Psi}{dS}$ applies:
$$M = EI \left( \frac{d\Psi}{dS} - \frac{1}{R} \right).$$
During the aforementioned semi-rigid loading sequence the force $T$ acts in the line of action parallel with the X axis. Note that the force is conserved throughout the rod, i.e.,
$$\frac{dT}{dS} = 0.$$
The application of this force sets up a bending moment $M$, such that
$$\frac{dM}{dS} = T \frac{dY}{dS}.$$
The geometry of deformation of the bent rod is given by the following first order ordinary differential equations
$$\frac{dX}{dS} = \cos \Psi,$$
$$\frac{dY}{dS} = \sin \Psi.$$
Finally, using Eqs. (10), (12) and (8) we obtain the following second order ordinary differential equation (ODE),
\[ EI \frac{d^2 \psi}{dS^2} - T \sin \Psi = 0. \]
Eq. (13) is the equation for the elastica.
### 2.3. The non-dimensional form of the problem
For the purposes of analysis it is convenient to non-dimensionalise the variables, as follows;
\[
s = \frac{S}{L}, \quad x = \frac{X}{L}, \quad y = \frac{Y}{L}, \quad r = \frac{R}{L}, \quad d = \frac{D}{L}, \quad t = \frac{T L^2}{EI}, \quad m = \frac{ML}{EI},
\]
where \(0 \leq s \leq 1\). Accordingly, in non-dimensional form, the system of Eqs. (13), (9), (11) and (12) are respectively as follows:
\[
\frac{d^2 \psi}{ds^2} = t \sin \psi,
\]
\[
\frac{dt}{ds} = 0,
\]
\[
\frac{dx}{ds} = \cos \psi,
\]
\[
\frac{dy}{ds} = \sin \psi.
\]
### 2.4. Boundary conditions
In non-dimensional form, the boundary conditions Eqs. (3)–(7) are as follows:
\[
x(0) = 0,
\]
\[
y(0) = 0,
\]
\[
\psi(0) = 0,
\]
\[
x(1) = \frac{\sin \gamma_1}{\gamma_1} + d,
\]
\[
\frac{d\psi(1)}{ds} = \gamma_1,
\]
where \(-1 - \frac{\sin \gamma_1}{\gamma_1} \leq d \leq 1 - \frac{\sin \gamma_1}{\gamma_1}\) and
\[
\gamma_1 = \frac{1}{r}.
\]
The second order ODE Eq. (15) and the three first order odes Eqs. (16)–(18), together with the boundary conditions Eqs. (19)–(23) constitute a well posed system of equations.
3. Solution of the boundary value problem
3.1. First integral
Integrating Eq. (15) we obtain:
\[ \frac{1}{2} \left( \frac{d\psi}{ds} \right)^2 + t \cos \psi = \text{constant}. \]
(25)
To evaluate the constant in Eq. (25), we apply the boundary condition given by Eq. (23), and we find
\[ \text{constant} = \frac{1}{2} \gamma_i^2 + t \cos \gamma \]
(26)
where the angle \( \gamma \) is defined as
\[ \gamma := \psi(1). \]
(27)
It follows from Eqs. (25) and (26) that
\[ \left( \frac{d\psi}{ds} \right)^2 = \gamma_i^2 - 2t(\cos \psi - \cos \gamma). \]
(28)
Note that, with respect to Eq. (28), we only consider the \( +ve \) square root because a rod which has positive initial curvature \( \gamma_i \) (intrinsically curved upwards \( \sim \) ‘sagging’), cannot be bent into a configuration with negative curvature (downwards \( \sim \) ‘hogging’) under the semi-rigid loading sequence specified for this problem; i.e., under the action of a rigid displacement of the end \( s = 1 \) by amount \( d \) applied horizontally from the reference configuration and in accordance with the boundary conditions Eqs. (19)–(23).
Following rearrangement of Eq. (28) we obtain
\[ \frac{d\psi}{ds} = \left( \gamma_i^2 - 4tk^2 + 4t \sin^2 \frac{\psi}{2} \right)^{\frac{1}{2}}, \]
(29)
where we have used \( \cos \psi = 1 - 2 \sin^2 \frac{\psi}{2} \) and where
\[ k := \sin \frac{\gamma}{2}. \]
(30)
3.2. Expressions for \( s, x \) and \( y \)
From Eq. (29) we obtain the following integral
\[ s = \int_0^\psi \frac{d\theta}{\left( \gamma_i^2 - 4tk^2 + 4t \sin^2 \frac{\theta}{2} \right)^{\frac{1}{2}}}. \]
(31)
Rewriting Eq. (17) as \( dx/ds = \cos \psi = 2 \cos^2 \frac{\psi}{2} - 1 \), we obtain
\[ x = 2 \int_0^\psi \frac{\cos^2 \frac{\theta}{2} d\theta}{\left( \gamma_i^2 - 4tk^2 + 4t \sin^2 \frac{\theta}{2} \right)^{\frac{1}{2}}} - s. \]
(32)
Similarly, expressing Eq. (18) as \( dy/ds = \sin \psi = 2 \sin \frac{\psi}{2} \cos \frac{\psi}{2} \), we obtain
\[ y = \frac{1}{t} \left( \left( \gamma_i^2 - 4tk^2 + 4t \sin^2 \frac{\psi}{2} \right)^{\frac{1}{2}} - \left( \gamma_i^2 - 4tk^2 \right)^{\frac{1}{2}} \right). \]
(33)
3.3. The elliptic parameter
When formulating Eqs. (31)–(33) in terms of elliptic integrals, as listed in the Appendix, the elliptic parameter \( p^2 \) needs to be introduced. This is defined as:
\[ p^2 := k^2 - \frac{\gamma_i^2}{4t}. \]
(34)
At an inflection point \( \frac{d\psi}{ds} = 0 \) must hold. This condition together with equation Eq. (29) implies that
\[ \sin^2 \frac{\psi}{2} = p^2. \]
(35)
Clearly Eq. (35) is only true for \( 0 \leq p^2 < 1 \). When \( p^2 = 0 \) or \( p^2 > 1 \) we have a contradiction in Eq. (35) and therefore conclude that \( \frac{d\psi}{ds} \neq 0 \) i.e., an inflection point does not exist. Thus, the existence of inflection points can be determined from the value of the elliptic parameter \( p^2 \). For the case \( t = 0 \), both \( p^2 \leq 1 \) and \( p^2 > 1 \) are allowed in Eq. (34). Therefore, for \( t < 0 \) we can have both inflectional (\( p^2 < 1 \)) and noninflectional (\( p^2 > 1 \)) solutions. These correspond to Snowdon’s solutions of the “First Type” and “Second Type”, respectively. In the case of \( t = 0 \) we have \( p^2 < 0 \). Thus Eq. (35) breaks down and therefore the condition it is derived from (\( \frac{d\psi}{ds} = 0 \)) cannot be true. In which case \( \frac{d\psi}{ds} \neq 0 \) must hold and solutions are noninflectional. These correspond to Snowdon’s solutions of the “Third Type” (Snowdon, 1965). We remark here that Snowdon describes his Types of solutions in terms of compression (First and Second Types) and tension (Third Type). However, whether or not a rod is under compression or tension depends on the values of \( \gamma_i \) and \( d \) as well as \( t \).
We make some remarks on the elliptic parameter \( p^2 \) given by Eq. (34) and elliptic integrals.
- For a given \( \gamma_i \neq 0 \), \( p^2 \) depends explicitly on the force \( t \). This is not the case for the straight rod.
- The force \( t \) does not factor out neatly from the elliptic integrals as is the case for the straight rod. This means that the solutions, whether in our raw integral form (Eqs. (31)–(33)) or elliptic integral form (see Appendix), must be solved numerically to determine \( t \) and \( \gamma \).
- In the case \( t < 0 \), for a given \( \gamma_i \neq 0 \), we don’t know if \( p^2 \leq 1 \) or \( p^2 > 1 \), since we need to know \( t \) and \( \gamma \) to determine \( p^2 \), and these are not known yet. This makes it difficult to decide which form of the solutions in terms of elliptic integrals (given in the Appendix) to work with to determine \( t \) and \( \gamma \).
- Finally, for \( t = 0 \), since both \( p^2 \leq 1 \) and \( p^2 > 1 \) are allowed, there is no reason why not for a given \( \gamma_i \neq 0 \) both \( p^2 \leq 1 \) and \( p^2 > 1 \) can occur. In fact, for \( \gamma_i = \frac{\pi}{4} \), Fig. 4 shows this to be the case.
These observations indicate that it is problematic to work with the solutions in elliptic integral form to plot \( td \) diagrams and equilibrium shapes. We therefore proceed to work with the solutions to this problem given by Eqs. (31)–(33). Nevertheless, for completeness, the elliptic integral form for the solutions is given in the Appendix.
3.4. Solution for \( t \) and \( \gamma \)
Eqs. (31)–(33) can be solved numerically once \( t \) and \( \gamma \) are known. Setting \( s = 1 \), whereby \( \psi(1) = \gamma \), Eqs. (31)–(33) become
\[ 1 = \int_0^\gamma \frac{d\theta}{\left( \gamma_i^2 - 4tk^2 + 4t \sin^2 \frac{\theta}{2} \right)^{\frac{1}{2}}}. \]
(36)
Fig. 4. Plots of $p^2$ versus the magnitude of the force $t$, for the cases $t < 0$ (top) and $t > 0$ (bottom) using Eq. (34) with $\gamma = \frac{5}{4}$. In the case of $t < 0$ it can be seen that $p^2$ is initially greater than unity corresponding to noninflectional configurations, then drops below unity for a certain domain of $t$ where there are inflectional solutions and then rises above it rendering noninflectional solutions again, but is asymptotic to unity as $|t|$ increases i.e., $p^2 \to 1$ as $|t| \to \infty$. In the case of $t > 0$ the rod is always noninflectional because $p^2 < 0$, but is asymptotic to zero as $t$ increases i.e., $p^2 \to 0$ as $t \to \infty$.
Fig. 5. Configurations of two rods with $\gamma_1 = 2.4\pi$ (dashed curves top) and $\gamma_1 = 2.5\pi$ (solid curves bottom), deformed with $t < 0$ from the natural state (bold curve). Since $2.4\pi > \gamma_1^*$ no loops form and note that $\gamma$ decreases as the end force is applied. However, since $2.5\pi > \gamma_1^*$ loop formation arises and note that $\gamma$ increases as the end force is applied.
To find $t$ and $\gamma$ we need to simultaneously solve Eqs. (36), (37) together with Eq. (22) i.e.,
$$x(1) = 2 \int_0^\gamma \frac{\cos^2 \frac{\theta}{2} d\theta}{\left(\gamma_1^2 - 4tk^2 + \sin^2 \frac{\theta}{2}\right)^{\frac{1}{2}}} - 1,$$
$$y(1) = \frac{1}{t} \left(\gamma_1 - \left(\gamma_1^2 - 4tk^2\right)^{\frac{1}{2}}\right),$$
$$f(\gamma_1, \gamma, t) := \int_0^\gamma \frac{d\theta}{\left(\gamma_1^2 - 4tk^2 + 4r \sin^2 \frac{\theta}{2}\right)^{\frac{1}{2}}} - 1 = 0.$$
Fig. 6. Configurations of two rods with $\gamma_1 = 1.4\pi$ (dashed curves top) and $\gamma_1 = 1.5\pi$ (solid curves bottom), deformed with $t > 0$ from the natural state (bold curve). Since $1.4\pi < \gamma_1'$ no loops form and note that $\gamma$ decreases as the end force is applied. However, since $1.5\pi > \gamma_1'$ loop formation arises and note that $\gamma$ increases as the end force is applied.
and
$$g(\gamma_1, \gamma, t) := 2 \int_0^{\gamma} \frac{\cos^2 \frac{\theta}{2} d\theta}{\left( \gamma_1^2 - 4th^2 + 4t \sin^2 \frac{\theta}{2} \right)^{\frac{3}{2}}} - 1 - \frac{\sin \gamma_1}{\gamma_1} - d = 0,$$
where we input $d$ and solve for $(\gamma, t)$ pairs.
We remark here that for the case $\gamma_1 = 0$, a straight (inextensible) rod, solving Eqs. (39) and (40) is straightforward because $t$ factorises neatly out of the integrals such that Eqs. (39) and (40) become respectively:
$$f(0, \gamma, t) = \frac{1}{\sqrt{-t}} K(p) - 1 = 0,$$
(41)
and
$$g(0, \gamma, t) = \frac{2}{\sqrt{-t}} E(p) - 2 - d = 0,$$
(42)
where $p = \sin \frac{\gamma}{2}$ and $K(p)$ and $E(p)$ are the complete elliptic integrals of the first and second kind respectively. For the case $p = 0$ in Eq. (41) we obtain the magnitude of applied end force, denoted $t_E$, required to buckle a straight rod:
$$t_E := \frac{\pi^2}{4}.$$
Note $t_E$ is also known as the Euler buckling load.
4. Loop formation
4.1. Conditions for loop formation
Careful observations of experiments indicate that, under the semi-rigid loading sequence described by the boundary conditions Eqs. (19)–(23), whereby the end $s = 1$ is displaced by amount $d$ in a continuous line, a loop forms when the angle at the end $\gamma$ tends to increase, see Figs. 5 and 6. We note here that from
Fig. 7. Plots of $\dot{\gamma}$ and $\ddot{\gamma}$, i.e., Eq. (51) and Eq. (52) respectively. These plots can be used with Eqs. (46) and (47) to determine the critical angles $\gamma_c^i$.
Fig. 8. A rod with $\gamma_i = 12\frac{1}{2}\pi$ deformed into a configuration containing six small knots with $t = -50,000$.
Fig. 9. A rod with $\gamma_i = 11\frac{1}{2}\pi$ deformed into a configuration containing six small knots with $t = 35,000$.
Fig. 10. Plots of td loading paths for a range of $\gamma_i$. Note that the straight rod, which buckles at $t/t_E = -1$, has no solutions for $d > 0$ because the rod is inextensible.
Eq. (36) changes in $t$ correspond with changes in $\gamma$. Consequently, we are justified in stating that $\gamma = \gamma(t)$. For infinitesimally small $t$, applying a Taylor Series expansion and noting that $\gamma(0) = \gamma_i$, we obtain
$$\dot{\gamma}(t) = \gamma'(0) + \left. \frac{d\gamma(t)}{dt} \right|_{t=0} t + \frac{1}{2} \left. \frac{d^2\gamma(t)}{dt^2} \right|_{t=0} t^2 + O(t^3). \quad (44)$$
For convenience we can write
$$\dot{\gamma} := \left. \frac{d\gamma(t)}{dt} \right|_{t=0} \quad \text{and} \quad \ddot{\gamma} := \left. \frac{d^2\gamma(t)}{dt^2} \right|_{t=0}. \quad (45)$$
We consider Eq. (44) under rigid loading of $d$ from the unstressed state. For $t < 0$ Eq. (44) indicates that if $\dot{\gamma} > 0$ with $t$ decreasing, $\gamma(t)$ decreases and the rod is pulled straight. For $t > 0$ Eq. (44) indicates that if $\dot{\gamma} < 0$ with $t$ increasing, $\gamma(t)$ decreases and the rod is also pulled straight. For $t < 0$ and $\dot{\gamma} < 0$ with $t$ decreasing, $\gamma(t)$ increases and loops form. Similarly, for $t > 0$ and $\dot{\gamma} > 0$ with $t$ increasing, $\gamma(t)$ increases and loops form.
For the case that $\dot{\gamma} = 0$, we need to examine the term $\ddot{\gamma}$ in Eq. (44). If $\ddot{\gamma} < 0$ with $t^2$ increasing, then $\gamma(t)$ decreases for all $t$, in which case the rod is pulled straight. When $\ddot{\gamma} > 0$ with $t^2$ increasing, then $\gamma(t)$ increases, in which case loops form.
It follows, the conditions for loop formation are:
$$\dot{\gamma} < 0 \quad (t < 0), \quad \dot{\gamma} > 0 \quad (t > 0) \quad (46)$$
and for all $t$ we have
$$\ddot{\gamma} > 0. \quad (47)$$
### 4.2. Critical angles for loop formation
Physical reasoning leads us to expect that a loop will only form if the rod is sufficiently ‘long’ (has large enough $\gamma_i$) such that as $|d|$ increases, the end of the rod will self-intersect another point on the rod and form a loop i.e., loop formation can only arise once $\gamma_i$ exceeds a critical value. More generally, $n$ loops form at critical values denoted by $\gamma_n^c$. We now proceed to determine $\gamma_2^c$.
If there is a function $f(\theta, t)$ that is a continuous and differentiable in both $t$ and $\theta$, then according to Leibniz’s Integral Rule
$$\frac{d}{dt} \int_0^{\gamma(t)} f(\theta, t) \, d\theta = \int_0^{\gamma(t)} \frac{\partial f(\theta, t)}{\partial t} \, d\theta + f(\gamma(t), t) \frac{d\gamma(t)}{dt}. \quad (48)$$
Applying Eqs. (48)–(36) (and using $\cos \theta = 1 - 2 \sin^2 \frac{\theta}{2}$) we obtain
$$\frac{1}{\gamma_t} \frac{d\gamma}{dt} = - \int_0^\gamma \left( \frac{\cos \theta - \cos \gamma + t \sin \gamma \frac{d\gamma}{dt}}{\left[ \gamma_t^2 - 2t (\cos \theta - \cos \gamma) \right]^{\frac{3}{2}}} \right) d\theta. \quad (49)$$
Additionally, applying Eqs. (48) and (49), we obtain:
$$\frac{d^2\gamma}{dt^2} = -\gamma_t \int_0^\gamma d\theta \left[ \left( 2 \sin \gamma \frac{d\gamma}{dt} + t \cos \gamma \left( \frac{d\gamma}{dt} \right)^2 + t \sin \gamma \frac{d^2\gamma}{dt^2} \right) \right.$$ $$\left. + \frac{3 \left( \cos \theta - \cos \gamma + t \sin \gamma \frac{d\gamma}{dt} \right)^2}{\left( \gamma_t^2 - 2t (\cos \theta - \cos \gamma) \right)^{\frac{5}{2}}} \right] - \frac{t}{\gamma_t^2} \sin \gamma \left( \frac{d\gamma}{dt} \right)^2. \quad (50)$$
Setting $t = 0$ in Eq. (49) and integrating, we obtain
$$\dot{\gamma} = -\frac{1}{\gamma_t} \left( \frac{1}{\gamma_t} \sin \gamma_t - \cos \gamma_t \right). \quad (51)$$
Similarly, setting $t = 0$ in Eq. (50) and integrating, we obtain the following
$$\ddot{\gamma} = \frac{1}{\gamma_t^2} \left( \frac{9}{4\gamma_t^2} - 1 \right) \sin 2\gamma_t - \frac{5}{2\gamma_t^3} \cos 2\gamma_t - \frac{2}{\gamma_t^3}. \quad (52)$$
Plots of Eqs. (51) and (52) are shown in Fig. 7.
For $t < 0$, applying the condition in Eqs. (46)–(51), we find from Fig. 7 that $\gamma_t > 2.4590\pi = 442.63^\circ$. Then applying the condition Eqs. (47)–(52), we find from Fig. 7 that $\gamma_t > 2.4895\pi = 448.12^\circ$. To satisfy both conditions, take $\gamma_t^c = 2.4895\pi = 448.12^\circ$. We remark here that this result is in accordance with what one might expect from physical reasoning i.e., under the action of a negative force one would expect loops to form somewhere in the region $2\pi < \gamma_t^c < 5\pi/2$ i.e., when the rod is sufficiently ‘long’, as shown in Fig. 5.
For $t > 0$, applying the condition in Eqs. (46)–(51), we find from Fig. 7 that $\gamma_t > 1.4303\pi = 257.45^\circ$. Then applying the condition Eqs. (47)–(52), we find from Fig. 7 that $\gamma_t > 1.4815\pi = 266.67^\circ$. In this case, to satisfy both conditions, take $\gamma_t^c = 1.4815\pi = 266.67^\circ$.
### 4.3. Multi-loop formation
Analysis similar to that of Section 4.2 gives the critical angles for higher numbers of loops, shown in Table 1.
It may be observed that the difference between consecutive critical angles for loop formation is very close to $2\pi$. For $t < 0$:
$$\gamma_t^2 - \gamma_t^1 = 360.86^\circ$$
$$\gamma_t^3 - \gamma_t^2 = 360.32^\circ$$
$$\vdots$$
and for $t > 0$:
$$\gamma_t^2 - \gamma_t^1 = 362.01^\circ$$
$$\gamma_t^3 - \gamma_t^2 = 360.49^\circ$$
$$\vdots$$
For large $t$ the loops take the form of localised knots, see Figs. 8 and 9.
### 5. Loading paths: theory and experiments
A set of loading paths for a range of values of $d$, (where $-1 - \frac{\sin 2\gamma_t}{\gamma_t} \leq d \leq 1 - \frac{\sin 2\gamma_t}{\gamma_t}$), is plotted in Fig. 10 where it can be seen that the end force $t$ has been normalised by dividing by $t_E$, given by Eq. (43). Note that large forces arise when $d$ approaches...
its lower and upper limits. We additionally provide plots of data obtained from experiments for comparison, see Figs. 11–14. Those plots show the loading paths and accompanying photographs of the configurations of nitinol strips.
6. Conclusions
As pointed out in this paper, the solutions to this boundary value problem, as expressed in terms of elliptic integrals, which are set out in the Appendix, are unwieldy. Nevertheless using Eqs. (31)–(33) we have managed to carry out useful analysis of loop formation. It remains to be seen what can be done with respect to other boundary conditions, different loading sequences and cases where the intrinsic curvature is a function of arc length $s$. Indeed, this paper throws up new questions regarding the loading sequences required to attain inflectional and noninflectional equilibrium configurations; for it is evident that small changes in the boundary conditions and loading sequences can have significant effects on the configuration of the rod, encompassing both inflectional and noninflectional forms.
The experimental data presented here correlates well with that predicted by the theory. That is not a trivial point, for although there is a substantial body of literature and research on rod theory that has contributed to our understanding of the mechanics, perhaps rather too much of that remains of restricted interest due to its failure to demonstrate (rather than simply mention) connections with real world problems. Carefully conducted experiments serve to bridge that gap. We add here that our experiments involve nitinol strips i.e., long slender rods of rectangular cross section with a thickness considerably smaller than the width. These do not deform out of the plane under the experimental procedure followed here. However, for experiments on rods with circular cross sections, out of plane configurations can be observed. The analysis of those requires a three-dimensional spatial elastica model, as opposed to the two-dimensional planar elastica model used here.
Acknowledgement
This research was funded by London South Bank University.
Appendix A. Solutions in terms of Elliptic Integrals
This appendix sets out the solutions for this boundary value problem expressed as Elliptic Integrals. There are three cases for $t < 0$ and one case of $t > 0$.
Elliptic Integral Solutions for $t < 0$
Case 1: $0 < p^2 < 1$
In this case the elliptic argument $\phi$ is given as:
$$\phi = \arcsin \left( \frac{\sin \frac{\psi}{2}}{p} \right)$$
and
$$s = \frac{1}{\sqrt{-t}} F(\phi, p),$$
$$x = \frac{1}{\sqrt{-t}} (2E(\phi, p) - F(\phi, p)),$$
$$y = \frac{2p}{\sqrt{-t}} (1 - \cos \phi).$$
At $s = 1$, $\psi(1) = \gamma$ and we find the following
$$\phi_\gamma = \arcsin \left( \frac{\sin \frac{\gamma}{2}}{p} \right),$$
$$1 = \frac{1}{\sqrt{-t}} F(\phi_\gamma, p),$$
$$x(1) = \frac{1}{\sqrt{-t}} (2E(\phi_\gamma, p) - F(\phi_\gamma, p)),$$
$$y(1) = \frac{2p}{\sqrt{-t}} (1 - \cos \phi_\gamma).$$
Case 2: $p^2 > 1$
In this case the elliptic argument $\phi$ is given as:
$$\phi = \frac{\psi}{2}$$
and
\[ s = \frac{1}{\sqrt{-t}p} F\left(\phi, \frac{1}{p}\right). \]
\[ x = \frac{1}{\sqrt{-t}} \left(2pE\left(\phi, \frac{1}{p}\right) - \frac{2p^2 - 1}{p} F\left(\phi, \frac{1}{p}\right)\right). \]
\[ y = \frac{2p}{\sqrt{-t}} \left(1 - \left(1 - \frac{1}{p^2} \sin^2 \phi\right)^{\frac{1}{2}}\right). \]
At \( s = 1 \), \( \psi(1) = \gamma \) and we find the following
\[ \phi_\gamma = \frac{\gamma}{2}. \]
\[ 1 = \frac{1}{\sqrt{-t}p} F\left(\phi_\gamma, \frac{1}{p}\right). \]
\[ x(1) = \frac{1}{\sqrt{-t}} \left(2pE\left(\phi_\gamma, \frac{1}{p}\right) - \frac{2p^2 - 1}{p} F\left(\phi_\gamma, \frac{1}{p}\right)\right). \]
\[ y(1) = \frac{2p}{\sqrt{-t}} \left(1 - \left(1 - \frac{1}{p^2} \sin^2 \phi_\gamma\right)^{\frac{1}{2}}\right). \]
**Case 3: \( p^2 = 1 \)**
In this case the elliptic argument \( \phi \) is given as:
\[ \phi = \frac{\psi}{2} \]
and
\[ s = \frac{1}{\sqrt{-t}} \tanh^{-1} \sin \phi. \]
\[ x = \frac{2}{\sqrt{-t}} \left(\sin \phi - \frac{1}{2} \tanh^{-1} \sin \phi\right). \]
\[ y = \frac{2}{\sqrt{-t}} (1 - \sin \phi). \]
At \( s = 1 \), \( \psi(1) = \gamma \) and we find the following
\[ \phi_\gamma = \frac{\gamma}{2}. \]
\[ 1 = \frac{1}{\sqrt{-t}} \tanh^{-1} \sin \phi_\gamma. \]
\[ x(1) = \frac{2}{\sqrt{-t}} \left(\sin \phi_\gamma - \frac{1}{2} \tanh^{-1} \sin \phi_\gamma\right). \]
\[ y(1) = \frac{2}{\sqrt{-t}} (1 - \cos \phi_\gamma). \]
**Elliptic Integral Solutions for \( t > 0 \)**
In this case the elliptic parameter is given as:
\[ p^2 = k^2 - \frac{\gamma^2}{4t} < 0 \]
and for all \( p^2 \), the elliptic argument is given as
\[ \phi = \frac{\psi}{2} \]
such that
\[ s = \frac{1}{\sqrt{t(-p^2)}} F\left(\phi, \frac{1}{p}\right). \]
\[ x = \frac{2\sqrt{(-p^2)}}{\sqrt{t}} \left[2p^2 - 1 F\left(\phi, \frac{1}{p}\right) - E\left(\phi, \frac{1}{p}\right)\right]. \]
\[ y = \frac{2\sqrt{(-p^2)}}{\sqrt{t}} \left[\left(1 - \frac{\sin^2 \phi}{p^2}\right)^{\frac{1}{2}} - 1\right]. \]
At \( s = 1 \), \( \psi(1) = \gamma \) and we find the following
\[ \phi_\gamma = \frac{\gamma}{2}. \]
\[ 1 = \frac{1}{\sqrt{t(-p^2)}} F\left(\phi_\gamma, \frac{1}{p}\right) \]
\[ x(1) = \frac{2\sqrt{(-p^2)}}{\sqrt{t}} \left[2p^2 - 1 F\left(\phi_\gamma, \frac{1}{p}\right) - E\left(\phi_\gamma, \frac{1}{p}\right)\right]. \]
\[ y(1) = \frac{2\sqrt{(-p^2)}}{\sqrt{t}} \left[\left(1 - \frac{\sin^2 \phi_\gamma}{p^2}\right)^{\frac{1}{2}} - 1\right]. \]
We note that solutions are inflectional for \( t < 0 \) with \( p^2 < 1 \) and noninflectional for \( p^2 > 1 \). For \( t = 0 \) all solutions are noninflectional.
**References**
Bertseaux, H., 1976. Buoy Engineering. New York; John Wiley and Sons.
Champneys, A.R., Thompson, J., van der Heijden, G., 1997. Spatially complex localization after one-twist-per-wave equilibria in twisted circular rods with initial curvature. Proc. R. Soc. A Math. Phys. Eng. Sci. 355 (1732), 2298–2312.
Coyne, J., 1996. Analysis of the formation and elimination of loops in twisted cable. IEEE J. Oceanic Eng. 15, 72–83.
Fichthe, W., Pismen, M., 1989. Load shortening behavior of an initially curved eccentrically loaded column. NASA Technical Memorandum 101643 42, 1–21.
Friedrich, J., 1962. Curvilinear Waves. Washington, D.C.: Bureau of Mines.
González, C., Laroc, J., 2004. Stiffness of a curved beam subjected to axial load and large deflections. Int. J. Solids Struct. 42, 1537–1545.
Hobbs, M., Rice, C., Lepine, S., Kucharski, M., Sullivan, J., Prescott, T., 2014. The effect of whisker movement on radial distance estimation: a case study in comparative robotics. Active Touch Sens. 106, 108–118.
Love, A.E.H., 1882. A Treatise on the Mathematical Theory of Elasticity. Volume II. Cambridge University Press.
Pearson, M.J., A. G. Pipe, C.M., Mitchinson, B., Prescott, T.J., 2007. Whiskerbot: a robotic active touch system modeled on the rat whisker sensory system. Adapt. Behav. 15 (3), 223–240.
Quist, R., Vickers, M., 2012. Mechanical signals at the base of a rat vibrissa: the effect of intrinsic vibrissa curvature and implications for tactile exploration. J. Neurophysiol. 9, 2298–2312.
Rangarajan, G.B., 2010. The science of knitting. In: Au, K. (Ed.), Advances in Knitting Technology. The Textile Institute: Woodhead Publishing, Oxford, pp. 48–85, chapter 4.
Snowdon, W., 1963. The bending of a thin rod having initial uniform curvature. Int. J. Mech. Sci. 5, 213–221.
Starostin, E., van der Heijden, G., 2009. Cascade uncoiling of a low-pitch helical spring under tension. J. Mech. Phys. Solids 57, 959–969.
|
Crime Breaking Open
INSIDE
Banking Sector
NRNs: Making Differences
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We heartily wish for peace, prosperity and happiness to all Nepalese on the occasion of Bada Dashain and Deepawali 2066 B.S.
Cover Story: Crime and Punishment
NEWSNOTES 2
NRN MOVEMENT: Challenges Ahead 4
REAL ESTATE: Boom Of Concern 6
FORUM : SB Pun 14
SOCIAL INCLUSION 18
BOOK REVIEW 22
VIEWPOINT : Asi Mamanee 23
ART 24
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India Foreign Secretary’s Visit
Indian foreign secretary Nirupama Rao paid two days visit to Nepal. Before leaving India she organized a press conference. “It is an honour and a privilege to be in this beautiful city, the capital of Nepal. It brought back fond memories of my previous visits. It has been a very busy but most useful visit to Kathmandu. This is also my first visit to Kathmandu after assuming the charge of the Foreign Secretary,” Rao said.
During her stay she met President Dr. Ram Baran Yadav and Prime Minister Madhav Kumar Nepal, Deputy Prime Minister Bijay Kumar Gachhadhar, Foreign Minister Bhim Bahadur Rawal and Defence Minister Bidya Devi Bhandari.
“The purpose of the visit was to exchange views on the further improvement of our bilateral relations and to focus on implementation of the rich and vast agenda agreed upon during the visit of the Prime Minister of Nepal to India as contained in the joint press statement. As you know, the civil aviation secretary level talks have already taken place in New Delhi during which both sides have agreed to increase the number of seats from 6000 to 30000 between Kathmandu and the Indian metros. This will promote tourism, people-to-people contact and economic cooperation between the two countries. The Home Secretary level talks will take place on November 6-7 in Kathmandu during which the entire gamut of bilateral security issues will be addressed. I reiterated India’s security concerns and the use of Nepalese territory for anti-India activities. The Nepalese side unequivocally reiterated their commitment that such activities will not be allowed,” said Rao.
“India has an abiding interest in the success of Nepal’s transition to multiparty democracy and the completion of the peace process. A peaceful, democratic and prosperous Nepal is in the interest of the Nepali people, of India and of our region. India is committed to assist the government and people of Nepal in these processes of historic change in Nepal.”
Indian Technical and Economic Cooperation
The Indian Technical and Economic Cooperation (ITEC) Day was celebrated at India House, Embassy of India on September 17, 2009. H.E. Foreign Minister of Nepal Ms. Sujata Koirala, graced the occasion as the chief guest. Ganesh Nepali, Minister for Youth & Sports; Ram Chandra Kushwaha, Minister for Education; Gyan Chandra Acharya, Foreign Secretary of Nepal, and other senior bureaucrats were also present at the occasion.
Indian Technical and Economic Cooperation program, popularly known as “ITEC”, was launched in 1964 by the Indian government as a bilateral program of assistance. Under ITEC and its corollary Special Common Wealth Assistance for Africa Program (SCAAP), 156 countries in Asia, Eastern Europe, Central Asia, Africa and Latin America are invited to share in the Indian development experience.
The first batch of 12 Nepali participants traveled to India in the year 2000. The number of seats allocated to Nepal under ITEC has increased to 80 in 2009.
Law Commission Starts web site
Nepal Law Commission (NLC) has launched a new website—www.lawcommission.gov.np—with the aim to provide access to information about the Nepalese legal system to the world community.
According to the secretary of NLC Mohan Banjade, the commission is planning to translate all legal documents into English. “This will be done within few months,” said Banjade, although he warns that the English translation is unofficial and that the Nepali version published in Nepal Gazette will prevail in case of translation error. Currently there are 43 laws available in English and 200 in Nepali.
NLC was first constituted by an executive decision in 1953, immediately after the dawn of multiparty democracy. The NLC was given permanent status in 1984 and was restructured in 1990 after the restoration of multiparty democracy. Now, the Commission is a statutory body under the Nepal Law Commission Act, which was promulgated on January 14, 2007.
Pakistan Embassy Celebrates 44th Defense Day
Pakistani diplomatic mission in Kathmandu celebrated the 44th Defense Day on September 15 to commemorate the sacrifices rendered by the Pakistan Armed Forces for guarding the country against aggression. The reception, held in Hotel Soaltree Crowne, was attended by people from all segments of society including army, police, armed police, diplomats, government officials, civil servants, businessmen, academia, scholars, and media representatives attended the reception. Chief of Army Staff, Nepalese Army General Chhatra Man Singh Gurung was the honored guest.
On September 6, 1965, the Pakistani Armed Forces and the entire nation stood united to foil the nefarious designs of the adversary that had challenged the integrity and sovereignty of Pakistan. The celebration honors those who sacrificed their lives for the defense of the country. On this date Pakistan and its armed forces also renew their pledge to make sacrifices in order to defend the country against internal
Correction: The September 11 issue of the New Spotlight published a photograph of Chief of Army Staff General Chhatraman Singh Gurung with Chief of Army Staff of US Pacific Command Maj. General Stephan D. Tomi. The caption for the photograph on page 3 appeared with an inadvertent error in CoAS Gurung’s name. We regret the error.
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and external threats.
Tara Air Launched
This past month Tara Air launched a soft operation of charter flights by a fleet of two brand new Pilatus Porter aircraft (PC-6). They now have purchased six aircrafts and leased two. With the acquisition of eight STOL aircraft, operations of flights are scheduled to begin from mid September, 2009.
Tara Air will take over all schedule STOL flights that were earlier operated and now discontinued by Yeti Airlines Domestic. Included in the schedule are 15 daily flights from Kathmandu to Lukla, as well as daily flights from Kathmandu to Tumlingtar and from Pokhara to Jonson. Tara Air will also fly to Bharatpur, Meghauli and other routes.
Tara will operate specialty flights by PC-6 on a charter basis. Charter fights will go to unique destinations, such as Syangboche, Langtang, Dhorpatan and Jiri. They will also operate flights for special purposes such as skydiving, photography, and aerial survey.
According to group managing director Ang Tshiring Sherpa, and group ED and CEO Vijay Shrestha, Tara Air envisions providing access to all of Nepal. “We will help develop generally inaccessible regions of the country by connecting the local population, which will be instrumental for their economic development and social wellbeing. We aim to develop tourism in rural Nepal by providing reliable and safe air transportation.” ■
We heartily wish peace, prosperity and happiness to all Nepalese on the occasion of BADA DASHAIN AND TIHAR 2066 B.S.
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NRN MOVEMENT
Challenges Ahead
Six years after its establishment, challenges for the new leadership of the NRNA will be no less daunting
By BHAGIRATH YOGI IN LONDON
On September 6, expatriate Nepalese living in the UK had assembled at the Oak Farm Community School at Farnborough, outside London, to inaugurate a new team of the Non-resident Nepalese Association UK chapter. Surya Gurung—a UK-based entrepreneur—along with his team took over from Major (Retd.) Damer Ghale and his team. “The NRN UK has set an example by electing its leadership unanimously. All of us need to learn from them,” declared Ram Pratap Thapa, vice president of the NRN Association, who had arrived from Germany to take part in the ceremony. Thapa, however, is competing for the top job of the Association, including two other senior NRN personalities Bhim Udas and Dev Man Hirachan.
According to the NRN constitution, its founding president Dr Upendra Mahato can’t contest for the third term thereby opening room for free competition. Thapa and Hirachan are vice presidents of the NRNA International Coordination Council (ICC) while Udas is its Secretary-General. Thanks to the dynamic leadership of Dr Mahato, NRNA is now a well-known brand among the expat Nepalese around the world. It already has got 48 chapters known as National Coordination Councils (NCCs). Both NCCs and ICC have a tenure of two years.
Immediately after Dashain festival, hundreds of NRN leaders from around the globe will descend in Kathmandu to take part in the fourth global conference of the NRNA (October 13-15). The conference will elect its new leadership and will lobby with political leaders and lawmakers to get a formal recognition in the new constitution which is being formulated by the 601-member Assembly.
“The NRNs have recommended that the constitution should state that those who are Nepalese nationals by descent will not have to give up their Nepalese nationality if they acquire a foreign nationality. The children and grandchildren of Nepali nationals who become foreign nationals should have the right to acquire Nepali nationality while keeping their foreign nationality. The Election Commission should make provisions for NRNs to cast their votes in the presidential election of the country and in major referendums by registering with the Nepalese embassies and consulates in the country of their residence. There should be a provision for a multi-year, multi-entry long-term visa for the people of Nepali origin,” said Prof. Surya P Subedi, advisor to NRNA and coordinator of the Constitution Recommendation Committee constituted by the Association. The NRNA has already submitted its suggestions to the government of Nepal.
CA demanding, among others, provision of dual citizenship for NRNs.
“The (provision of) dual citizenship will inspire NRNs to contribute to Nepal’s socio-economic development,” said Dr Mahato. “We don’t want our second generation, born and brought up in foreign countries, to forget the roots of their ancestors,” he added.
Critics, however, say the provision of dual citizenship could prove detrimental to Nepal’s nationhood when the country is passing through a rollercoaster period of political transition, mainly influenced by foreign actors. “Dual citizenship is an issue that needs to be considered carefully and thoroughly. As a matter of policy, it may not be entirely consistent with the values we try to nurture for the growth of our nationhood,” said Dr Bipin Adhikari, a constitutional lawyer.
The government of Nepal recognises any Nepali, who has lived continuously for more than 183 days in countries other than SAARC nations, as an NRN. The government also passed NRN bill in August 2007 and established an NRN cell at the Ministry of Foreign Affairs.
The government of Nepal has drafted a bill that proposes to issue 10-year multiple entry visa to NRNs and also allow them to buy up to two ropanis of land in Kathmandu and up to eight katthas of land outside Kathmandu for residence.
While NRNs have been contributing in the socio-economic development of Nepal in various ways, analysts say as Nepali diaspora mainly comprises of first generation immigrants, it still has limited resources to invest back in Nepal compared to the Indian and Chinese diaspora. The Nepali diaspora could, however, play an important role as ‘unofficial ambassadors’ to promote Nepali culture and preserve Nepal’s interests abroad as well as in the transfer of skills and expertise.
Networking of Ideas
“In the global context, networking of ideas and knowledge has become a necessity for any country’s economic development,” said Dr. Raju Adhikari, a Nepali scientist based in Australia, who is also advisor to NRN Australia. “There is huge competition among many developing countries to retain, attract and generate science and technology manpower. But we do not seem bothered about it. All countries but Nepal have realised that a knowledge-based nation is the most prosperous nation in the long run,” he added.
As Nepal lagged behind in terms of prosperity, many Nepalese left the country looking for better opportunity—in terms of education and career. It is estimated that the number of expatriate Nepalese in countries, other than SAARC, has reached around 2.5 million. As the Nepali diaspora continues to expand, there is a need to channelize its resources and forge a stronger bond with the motherland.
“NRNA has its work cut out for many years to come if we dedicate ourselves to its mission; to be a partner in Nepal’s development,” said Naresh Koirala, NRNA’s regional coordinator for North America. Much will depend on how the new leadership of NRNA moves ahead by combining idealism with pragmatism.
NEPALI ARMY
Command Control
The centuries-old institution proves the Doubting Thomasses wrong under the first commoner supreme commander, but bigger challenges remain
By SUSHIL SHARMA
The commander-in-chief, Rookmangud Katwal, gracefully retired this week. His deputy, Chhatra Man Singh Gurung, took over the command.
It was a natural succession. Role command had not been breached. The traditional discipline has been kept in tact.
The centuries-old national institution remains united.
In his farewell speech at the office he bade goodbye, Katwal defended what did over the past three years. Mostly taking head on – single handedly — the challenge posed by a political party with a private army.
Gurung praised his predecessor’s role during the country’s difficult political transition from a monarchy to a republic.
The fear that commander Katwal was up to some mischief has been proved unfounded. That the supreme commander, President Ram Baran Yadav, was into a grand design has been proved hollow.
But some questions remain unanswered. Would the result have been the same, if the Maoist-led government’s sacking of Katwal had not been reversed through a late-night intervention by the head of the state four months before the army chief retired?
If President Ram Baran Yadav had not resisted internal pressure and external persuasion to go beyond and sack prime minister Prachanda, instead?
And later, if the President and the supreme commander, Ram Baran Yadav, had not successfully discouraged moves to extend the tenure of Katwal.
Any other developments could have disastrous consequences.
Disaster is not ruled out yet, with rival internal forces squabbling for power and external forces competing with each other for a larger space to play.
This will mean even bigger challenge ahead for the Nepali army and its civilian and uniformed bosses alike.
As constitution-making and the peace process face an uncertain future, acid test still awaits the 100,000-strong institution and the hands that handle it.
REAL ESTATE
Boom Of Concern
Officials of the central bank and IMF are worried as banks go on a lending spree in real estate
By SANJAYA DHAKAL
In the last one year, as banks in the United States faced felt the heat from the lending in subprime real estate triggering global economic recession, their counterparts in Nepal went on a lending spree riding the expanding bubble of property market.
The senior officials of Nepal Rastra Bank (NRB) and even International Monetary Fund (IMF) have warned about the ‘risk’ of investing in property market. They have said that the investments in real estate is not ‘reliable.’
Basking in the glory of historic profits they earned last year, the banks have turned the deaf ear.
The big US investment banks went bankrupt last year after the real estate crashed but at the same time, the Nepali banks have made lots of moolah from the investment in property market.
A recent report stated that out of 26 commercial banks, 23 made huge profits – the cumulative amount totaling over Rs 14 billion.
But the officials have asked the banks not to get over-excited.
Alexandar Pitt, the chief of IMF-Nepal office, feels that Nepali banks ought to look at what happened in the west in the last one year.
“Banks are all very profitable but at the same time the vulnerabilities are building up. And once, I would say, the speculative bubble in the real estate pops, then that will expose the
"Lending In Real Estate Not Too Much"
Shashin Joshi, president, Nepal Bankers' Association (NBA).
How has been the last one year for Nepali banks?
Nepali banks were relatively insulated from global recession. We were scared that the recession could bring down the inward remittances. But, fortunately, that did not happen.
But, the officials of IMF and NRB have said that Nepali banks' heavy investment in real estate is worrisome?
Only some banks have made heavy lending in real estate sector. In totality, only 14 to 15 percent of total lending is in real estate. Of the total lending amounting to Rs 450 billion, only Rs 50 to 60 billion have been lent to this sector. Relatively speaking, this is not too much.
What about NRB's concerns, then?
Well, in the last six months, most banks have started to become alert and not concentrate on real estate alone. Because investing in the sector when property prices have skyrocketed could be risky.
What are lessons from global financial crisis?
Surely, it is foolish to invest blindly in property market without properly assessing the risk factor.
weaknesses of the banks or the financial system in general," he said.
Many believe that it is due to the continued increase in the inward remittances from Nepalese workers overseas that the consumption level has soared in the country.
A sizeable chunk of the inward remittances has gone into the real estate sector, which has led to the current boom.
Unnatural
Most economists and officials agree that the present boom in the real estate sector in unnatural.
"The real estate boom looks unnatural. I think that once the current investment in housing complexes, condominiums and apartments leads to the situation of over-supply, the property prices could come down," said economist and former governor of NRB Dr. Tilak Rawal.
The NRB officials are worried that once the real estate prices come down, the banks could suffer a serious shock.
According to NRB governor Bijaya Nath Bhattarai, the real estate prices have become like a bubble that can burst any time.
He said that the banks were currently sitting atop huge volume of liquidity fueled by remittances.
"Once there is decrease in remittance, that will lead to liquidity crisis. And once there is liquidity crisis, the real estate bubble can burst sending shockwave to the banking and financial system," he said.
Governor Bhattarai said that the NRB has already informally requested the banks not to concentrate on property market.
The bankers, however, do not feel that they have invested heavily in the real estate.
"Except a couple of banks, most banks have remained within appropriate limits while lending to the property market," said Shashin Joshi, president of Nepal Bankers' Association (NBA).
"Even now, in totality, banks have lent only up to 15 percent of their total lending in the property sector," he said.
Joshi said that of the total lending of Rs 450 billion, around Rs 50 to 60 billion have gone to the real estate sector. "This is not too much," he added.
Lessons
Alexandar Pitt of IMF-Nepal, believes that Nepali banks must not forget to learn the lesson how banks in western countries suffered huge losses leading them to bankruptcy once their investment in real estate went all wrong.
"I think most important is to become vigilant even when times are good. The Nepali banking and Nepali financial system needs to learn the lesson that complacency should be avoided. Things were very well in the United States right up and until the crisis," he cautioned.
It is not clear how seriously the banks will take the words of cautions from the officials at a time when they are raking in moolahs big time.
"Remittance Increase Has Helped Avoid Disaster"
Bijayanath Bhattarai, governor, Nepal Rastra Bank
What steps did NRB take to make oversight on banks more stringent?
First of all, we have regulated the margin lending on stocks. We have informally requested the banks not to aggressively lend in the real estate sector.
Why are you advocating restraint on lending in real estate?
The inward remittances have increased liquidity in the market. The banks and financial institutions have huge deposits. The banks, FIs and even cooperatives – which are not under the purview of NRB – have invested heavily in real estate. Once the remittances drop, it might cause liquidity crisis. And once there is liquidity crisis, the bubble in the asset prices could burst anytime. If the property bubble bursts, it can invite shock in financial system. The central bank is closely watching this sector to see if this calls for further regulation.
Even during global recession, Nepali banks have shown huge profits. Why?
Our banks are not much integrated globally.
How did Nepal manage to steer clear of pitfalls of global crisis?
We had presumed that inward remittance would decrease. But they continued to increase.
Any advice to the banks?
They must remain cautious while lending. In the US, the banks there were found to have lent up to $ 58 for $ 1 worth security as the banks went on multiple lending spree.
But it is clear that recalling the situation the western banks found themselves in, it could be a blunder on the part of Nepali banks not to take their suggestions seriously.
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CRIME AND NO PUNISHMENT?
With no strong central government in place and no local government for years, the metropolitan Kathmandu has become increasingly vulnerable to deadly crimes. Despite the claims of the authority to the contrary, the culprits largely go unpunished.
By BHAGIRATH YOGI with SAROJ DAHAL
Nabin Dhungana, a resident of Balkot, Bhaktapur, teaches computing at a school at Baneswore in Kathmandu. His daily routine included leaving home for school early in the morning, spend the whole day with students and computers and return home late in the evening. But for the last 15 days, his routine has changed. Instead of going to school, he is busy visiting different police stations, making frantic calls to his relatives and scanning newspapers. Reason: his brother Pradip Dhungana, has gone missing.
As always, 26-year-old Pradip, a student, left for his college on the morning of July 1st on his motorbike. But he hasn’t returned since and hasn’t left any message. Nobody picks up his mobile phone, thereby giving room for suspicion. “Though he was three year younger than me, I treated him as a friend. He would never spend night outside without telling me,” said Nabin adding, “I have filed petitions at five different police stations requesting them to help locate my brother, but I am increasingly frustrated and becoming angry looking at the behaviour of policemen.”
When Pradip filed a written petition at the Kathmandu Metropolitan Office, he was told that Bhaktapur police hadn’t informed them about the missing person. “I can’t believe that we reside in the capital valley when police in Kathmandu don’t know what is happening in Bhaktapur. How can I knock doors of all 75 district police offices in the country looking for my brother?” he asked.
Ngamindra Dahal, who rents out a flat at New Baneswore, also has similar story to tell. When he visited New Baneswore police post to report about the incident of burglary in his flat, he was asked to report to the Crime branch
After taking the Home Ministry portfolio, Minister BHIM RAWAL has been making desperate efforts to maintain a normal law and order situation in the country. In three months, Minister Rawal travelled extensively across Nepal attending meetings of Chief District Officers and Police officers to implement a new security program. In this context, Minister Rawal spoke to KESHAB POUDEL, SAROJ DAHAL and SHARMINI on various issues related to Nepal’s law and order at his office in Singha Durbar on Monday afternoon.
Excerpts:
Three months have already elapsed since your appointment as the Home Minister, how do assess the present law and orders situation?
In the last three months, I have made efforts to make the law and order situation better, improve human rights conditions and end impunity by implementing special security program-2009. Under this program, security and administration personnel are moving ahead firmly. We have already arrested some criminals involved in terrorizing people and we have already made some gains in preventing road blockades.
Are you satisfied with the situation?
It is the people, like you, who should say whether there are certain improvements or not. So far as my observation is concerned, I have been doing my level best to maintain the law and order situation in the country. In the last few months, police have started to act. They have recently interrupted more than 40 incidents in which some miscreants were trying to block the road. Police have been responding very positively. This is a good beginning.
How about the situation in Terai?
The situation in Terai is returning to normalcy too. We need to separate criminal elements from political elements. If we isolate the criminal elements from the rest of the political forces, the law and order issue will be automatically settled. I have already enforced a special security work plan for Kathmandu and special work plan to control serious crimes. We have implemented work plans to control road blockades. We have also made programs to contain armed groups in eastern Nepal, central Nepal and far western regions. We have been making efforts to control criminal acts. All the programs are now being implemented. In the last few months, police have immediately acted against those who tried to block the road. We have also arrested a number of criminals, and seized arms and ammunitions. We have already arrested the person involved in Ranbir Sena and Nepal Defense Army. These are some results. We have already recovered huge amounts of ammunitions.
What is the status of your program?
After implementation of these programs, people will feel safe and the security environment will change. We have already organized six seminars in six different parts of the region where we intensively discussed the security related issues. We have already collected suggestions and we are analyzing them now.
How do you see the support of main political parties?
We have full backing of all parties. We have already discussed our plans with the political parties represented in the Constituent Assembly, human rights groups and journalists and all of them have backed us. We even discussed it with foreign envoys based in Nepal. The legislature parliament also backed us. When I met the leaders, they supported me. This proposal was passed by the districts as well as in the capital valley itself showing that people are losing faith on the law enforcement agencies.
Spokesman of Nepal Police, DIG Binod Singh, doesn’t agree. “People want instant justice and sometimes even take law unto their own hands. But we have to deal with crime in accordance with law,” said Singh. He, however, agreed that people’s trust on police seemed declining.
Some parties are raising suspicions but nobody has told us that we should not control crime. Our security is not directed to any political parties, regional or ethnic groups but this program is there to protect the rule of law by discouraging criminal activities. There is no place for criminals. We will not tolerate any efforts to weaken Nepal’s sovereignty, integrity and communal harmony. We will bring criminals before justice.
But it is reported that political parties are putting pressure on you not to execute the special security plan?
It is not true. No political party or leader has said that they support criminals. All the leaders have been stressing maintenance of law and order. There is consensus among all of us that peace is needed in the society. Nobody wants to see criminals walking scot free. Many criminals, affiliated with political parties, are also in prison. Police administration is now working impartially in maintaining law and order and rule of law. Police are now very much active and encouraged. Our security program is yielding results.
There are also reports that police are violating human rights. What do you say on this?
Police are violating human rights and rule of law. I am very much concerned about violation of human rights. If anything goes wrong, we will not tolerate it. We will not tolerate any cases of human rights violation. We will take immediate actions against those who commit such acts.
In her recent visit, Indian foreign secretary also mentioned about the worsening law and order situation. How was her response about the growing violence in Terai?
I met the Indian home minister and Indian foreign secretary recently and we held discussions about the criminal activities in Terai. We have made it clear that Nepal will not allow its soil to be used against any country and Nepal too wants support and cooperation from neighbors to control criminal activities. Indian foreign secretary clearly said that India will support Nepal to contain criminal activities inside Nepal. These kinds of understanding between two countries will help to prevent human trafficking, armed smuggling and criminal activities.
Recently, your police team also visited Lhasa and Chinese security officials reportedly expressed their concern over deteriorating law and order situation. How do you look at this?
Like in our southern border, we also want peace in our northern border. We have a clear cut policy that Nepal is ready to work with northern neighbor China to control criminal activity, if there is any. Our security officials discussed security issues with Chinese counterparts. We are very much concerned not to allow anti-Chinese activities in Nepalese soil. We are ready to provide necessary support.
What is the overall security scenario?
There are 109 armed groups throughout the country. We are now analyzing the modus operandi of such groups. We will not allow any group to violate the constitution and the rule of law. The government will respect human rights and rule of law. We will not tolerate any party which is involved in extortion and kidnapping.
How do you distinguish between criminal and political activities?
Under our special security program, we have a clear vision about what constitute criminal activities and what are political activities. Our program is not directed to peaceful political activities and this is directed to criminal activities. Nepalese people want to end impunity. No political party has ever asked me not to punish the criminals. I cannot allow killing, extortion and kidnapping in the name of political parties.
How do you assess the efficiency of the police administration?
The morale of police administration needs boosting. I have already directed police officials to work for the protection of the rule of law. We need to give leadership to police and administration. We have already started to encourage them. Police are performing their role quite well.
Do you believe that the law and order situation will improve?
If political parties support me like now, we can make our situation much better. Since law and order is everybody’s agenda, all of them will back me to maintain it.
Greetings and Happy Holidays on the occasion of the BADA DASHAIN and DIPAWALI 2066 B.S.
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(Under the management of V.S. Education Foundation)
Phone: 4482715/4495073, Fax 977-1-4478379
Email: firstname.lastname@example.org/ Website: www.vsniketan.edu.np
Incidences of Crime in the Capital
Burglary tops among the incidences of crime in the capital. Police say they receive complaints about burglary almost every day. According to the data compiled by Police Headquarters, 227 incidences of burglary had been registered in the capital in the last fiscal year while the number had reached 174 in the first nine months of this fiscal year. Police, however, say incidences of armed robbery have declined significantly this year. Last year, 50 such incidences were recorded while this year only around 20 incidences of armed robbery have been registered, according to the police.
Kathmandu residents complain that police is not paying due attention to incidences of burglary. Said Dahal, “Police tend to pay attention only if the crime involves murder or abduction.”
They seem to undermine incidences of burglary.”
Spokesperson of Nepal Police, Singh, however, claims that rise in incidences of burglary should not be linked directly with the overall situation of law and order. “It is up to households themselves to take good care of their house and property. How can police totally stop burglary?”
Criminologist Madhav Acharya doesn’t agree with DIG Singh. “It is the duty of the police to provide security to ordinary people. If they say they can’t control burglary, then what is the use of the department of police,” said Acharya adding, “Police can demand more manpower or resources for effective policing, but they must ensure that people feel safe.”
Police, on their part, classify crime in 17 different categories. It includes public crime, murder, armed robbery, burglary, suicide, hit and run, fraud, abduction and extortion, drug peddling and dealing as well as use of illegal arms. They say incidences of public crime top the list of crime in the valley.
**WHAT IS PUBLIC CRIME?**
**The Terror of Abduction**
The abduction of Khyati Shrestha, a 19-year-old student who was studying at a college in Kathmandu on 5th of June and her subsequent murder has sent tremor waves among the valley residents. Police say the incidences of abduction have gone up at once over the last few months. There used to be incidences of abduction of people due to various motives in the terai districts bordering India. But police say sudden rise of abduction for the purpose of extortion in the capital valley of late looks ‘unnatural.’
Police spokesman Singh insists that police have been able to rescue people successfully in most of the abduction cases. But there are concerns regarding the identification of people involved in abduction and legal action against them. There is the growing tendency among people to lynch people on suspicion of being involved in abduction rather than handing them over to police. Not only could innocent people fall victim of such a tendency, it could also unravel the very social fabric that makes the Nepali society.
Police say main motivation behind abduction remains extortion. People with criminal minds see abduction the easiest way to make quick money without major risk. DIG Singh, however, is worried to see that ‘organised criminal gangs’—that have links or even operate from outside the country—are found involved in many cases of abduction and extortion. They usually target leading businessmen and collect the extortion money through ‘hundi.’ Police say disputes related to foreign employment and transaction of money at individual level have also resulted into abduction.
Parents and guardians are more worried after incidences of child abduction have gone up. Now, many schools have advised parents to drop or escort their child up to the school and collect their children themselves.
According to police, just two incidences of abduction (with the motive of extortion) were registered with them in 2064 BS. A child, Rohit Gupta, was later murdered by his abductors while Mahesh Murarka, a leading businessman, was freed after a week. In 2065 BS, police have registered eight incidences of abduction in the Kathmandu valley in which two children were murdered and other six people were freed after paying ransom. But over the last three months of 2066 BS alone, there have been five incidences of abduction in which Khyati Shrestha, a student, was murdered.
According to data compiled by the Police Headquarters, 99 incidences of abduction were reported in the year
2064/65 DS while a total of 129 incidences of abduction have been reported by mid-June this year. DIG Singh admits that the actual incidences of abduction could be even higher since many people don’t report about such incidences owing the threat to life of the victim and their family members.
Criminologist Acharya believes that dwindling confidence of people upon police is one of the reasons leading to increase in the incidences of crime. “Politicalisation of the police organisation is rampant which had led into decline in faith of general public towards police. They usually refrain from reporting to police, which in turn encourages criminals,” he added.
In recent weeks, residents of Chapagaon, Gongabu and Thimi within the Kathmandu valley have either lynched or tried to lynch people on suspicion that they were trying to abduct children. Three persons have been killed while eight others have been injured seriously. Across the country, at least 16 people have been lynched on suspicion of being abductors. Such incidences show that people seem to be accepting violence as a legitimate means of expressing their grievance. Criminologist Acharya says if such a tendency is not discouraged or stopped, it will push our society towards further criminalisation and anarchy.
When contacted, spokesman at the Home Ministry, Nabir Ghimire, said the government was committed to control such activities. “We are strengthening the organisation of police and will take strong action against any unlawful activities,” he added.
**The Capability of Police**
Chief of the Police Circle (??) at Baneswore, DSP Biswo Raj Pokhrel, claims that there has been significant improvement in the performance of police compared to the past. He also dismisses allegations that criminal activities have gone up in the capital valley. In fact, five people who were detained pending further investigation, had escaped a few days back right under his nose. Pokhrel, however, refers to the incident as a ‘technical error.’ He said the strength of 150 personnel under him was inadequate for effective policing.
Crime Division: How vigilant?
Chief of Police Circle at Kalimati, DSP Sushil Singh Rathour, however, said he was satisfied with the manpower and resources under his command. “We can now reach the site of incident within 7 to 10 minutes. It is a great success,” said Rathour adding, “Number of criminal activities has almost declined by half after we have intensified patrolling.” He was quick to add that police force must be committed and honest.
Criminologist Acharya, however, says police have lost their effectiveness to a great extent mainly due to politicisation of their organisation. “Unless political parties make a joint commitment saying that they will no more intervene in the functioning of police and assist them whole-heartedly to control crime, incidences of crime will not decline,” he added.
Spokesman of Nepal Police, DIG Binod Singh, claims that Nepal Police is as competent as any other police organisation in the world. “We have nabbed criminals who had fled to Mumbai and resolved most of the abduction and murder cases,” he added. He, however, admitted that there have been some ‘rotten eggs’ within the police department. “We should take care not to blame the entire police department due to activities of few individual officers. Action has been taken against those found abusing their authority,” said Singh.
Policing a society that has just emerged from a decade-old armed conflict was never going to be easy. Police officials say they are now launching a new approach of ‘Police-People Collaboration’ to fight with crime. A Metropolitan Police office has already been set up to maintain law and order and fight crime within the capital. There are three Police PARISAR (???) in three districts and six Police Circles under the command of a Police Commissioner. The ratio of police to police in the capital is 600:1.
It is but obvious that there is acute lack of manpower and resources within the police department. But corruption and lack of rapport with general public are equally to blame. The involvement of some cadres of certain political parties in some of the incidences of crime point towards the complex situation.
But experts say the main challenge is to restore confidence of people upon the institution of police. Unless that can be done, police won’t get information and cooperation from members of the public. It could also undermine the very credibility of the state thereby engendering systemic crisis. Time is running out but a fragile coalition government at the centre is least likely to help the matter.
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Executive Chairman of Nepal Airlines SUGAT RATNA KANSAKAR is known for his vision and action. Kansakar proved his mettle during his stint at Nepal Telecom as its Chief Executive Officer. He is credited with doing the virtually impossible — expanding the CDMA network to the remotest corners of Nepal. This was a kind of revolution that provided access to the internet across the country. When this same man is at the helm of the country’s oldest airlines, there are high expectations of him. Kansakar is now working to transform the ailing Nepal Airlines, with ageing fleet of aircraft, from its lowest ebb to a new peak of success. In the last ten months since he took up the job, Kansakar has been trying to buy two new wide bodied aircraft to add to the fleet of Nepal Airlines. He desperately wants them to make the airline’s services reliable. Kansakar spoke to KESHAB POUDEL about his plans and progress.
Many look at two decades after Nepal Airlines first purchased two Boeing 757 aircraft, you are trying to buy new wide-bodied aircraft again. Will your efforts materialize?
It is almost certain that Nepal Airlines (NA) will have new aircraft within ten months. My target is that by June/July 2010 or the end of the current fiscal year, NA should have new aircraft in its fleet.
Given the history, many people have suspicions about this happening soon? How do you answer critics?
Many people say similar things to me. Some even say that the government will collapse if NA buys new aircraft. I am surprised by such comments. If NA needs to fly its fleet and compete with new airlines, it should have new aircraft. That is it.
Why then is there hue and cry about aircraft deals?
This is basically due to the lack of knowledge about the aircraft business. Even a senior politician and journalist recently said that an aircraft might cost Rs 40 to 50 billion. The aircraft I have been talking about costs no more than Rs 4 billion. Another reason may be because nobody has made determined efforts to buy a new aircraft for more than two decades. Many look at the ongoing debate on buying new aircraft like in the past when every management gave it up at the last minute. Yes, you have options too. You must either shut down the airlines or sell it out. How can you operate airlines without aircraft? It is silly to talk about managing NA without aircraft.
Don’t you have two aircraft?
Yes, we have two Boeing 757 aircraft. But they are too old now to compete with new wide-bodied aircraft of international airlines.
During your tenure as the CEO of Nepal Telecom, you transformed it into a modern and competitive organization. You have raised hopes about transformation of NA. Don’t you think it will have devastating effects, in case you failed to bring the new aircraft?
It will be psychologically devastating, yes. When I was the CEO, I faced similar challenges and criticisms from all sectors while introducing the CDMA system. Some lawyers even filed public litigation to stop it. However, when I convinced employees, ministers and others, I was able to introduce it. Thanks to CDMA, you can see internet accessed even in the remote parts of Nepal. CDMA also transformed Nepal Telecom. Some even make it a laughing stuff. Now you can link up with internet from all the 75 districts. This is a grand success. That system was much more expensive than buying new aircraft for NA. I think introduction of new aircraft will transform the NA. Some of my colleagues even accused me in Nepal Telecom that I sold out Telecom. If we cannot buy new aircraft, NA should be locked up.
You had a vision to build a 100-storey tower for Nepal Telecom. That did not materialize. How confident are you now that your vision of buying new aircraft will work?
My vision to build the tower was shattered at the last minute thanks to some unknown reasons. This vision will materialize since there is no option left at all. In the globally competitive airlines business, you need to compete with airlines. For instance, NA needs to compete with Gulf Air, Qatar, Thai, Jet Air, Air India, Bangladesh Biman and Singapore Airlines which have most sophisticated and new fleets of aircraft. Because of lack of aircrafts, more than 85 per cent of tourists and Nepalese are flying through foreign airlines. When NA has its aircraft, it will carry all those passengers. My aim is to increase the carrying capacity of airlines by bringing new aircraft. The situation now is that our share of market is about Rs. 4 billion whereas other airlines are collecting over 50 billion rupees.
Don’t you have other options than buying new aircraft?
We have very little options. Either we have to say that we are incompetent, incapable and timid people or to show to the world that we are the country of talented, competent and capable people with strong determination. Nepalese have all things with them. In the airlines sector, Nepalese pilots and technicians have a prestige and reputation that they are capable and efficient. This is very much true with Nepal Airlines, which has big pool of talents, determined and courageous employees, pilots and technical manpower. If Bhutan and Bangladesh can successfully fly their airlines, despite starting their airlines business much later than us, we cannot say that we are incapable.
What other basis do you have for saying this?
If you look up the records, NA is the world’s safest airline in terms of safety issues. If we have a new aircraft, we can fly much safer and better. I have a plan to fly our aircraft to Australia and New York. When Air India bought hundreds of aircraft and Sri Lankan air has done similar things, there was no hue and cry. I don’t understand why only our country has to face this kind of reaction.
You were recently summoned by the Public Accounts Committee of the Legislature Parliament. How do you look at it?
It was a very positive step. Members of parliament gave me enough time to explain the present situation and defend the case of purchasing new aircraft. After listening to my argument, they are very positive. Their concern not to allow corruption and irregularities to happen is genuine. We always need to pay utmost importance to such comments of our parliamentarians. Their concern is about the loan we are getting from the Provident Fund. There are more than 200 countries where national flag carriers are making profit. Why can we not do it? We must give up our negative thinking that Nepalese cannot do anything.
Looking at the past experience, whenever a general manager tried to bring a new aircraft, he was either fired or put on trial! Aren’t you afraid about it?
Others too ask me such things. When I have taken the initiative to move the country forward, I don’t think anybody will harm me. Everybody wants to see Nepal as a prosperous country and helps develop it and I have been doing what is required to translate this into reality. If you have clear intention and vision, you don’t need to worry. If everybody is scared of doing things, nothing will be done.
What are your experiences of working with Nepal Airlines?
I feel very proud to work with the country’s most competent and professional people. Nepal Airlines has all kinds of capability. What is lacking is just an aircraft. Once NA has new aircraft, the situation will be changed.
Why are you stressing on a new plane. Don’t you see any possibility of utilizing the current fleet of aircraft to make profit?
I have a plan for it. This fiscal year we are targeting to make profits of over 340 million rupees. You cannot increase the profit margin without adequate aircraft to fly.
Out of two aircraft, you have been unable to fly one for a long time. How can you say that the new plane will bring change?
If you have just two ageing aircraft, this scenario will appear again. This is the reason I am stressing purchase of a new aircraft which will make things different. Give me a new aircraft, I will give you change. I am an optimistic person with a positive thinking. I hope that every one supports our efforts.
There are more than 200 countries where national flag carriers are making profit. Why can we not do it? We must give up our negative thinking that Nepalese cannot do anything.
Whither Bijulee Adda – 4
Kulekhani: Disaster of Shrawan 4, 2050 (July 19, 1993) Through My Eyes
A. First Information Report
On the night of July 19, 1993 (Shrawan 4, 2050) I was preparing to go to bed around 10 PM when Govinda KC (GKC), Director, Kulekhani Disaster Prevention Project telephoned me from his quarter at Nibuwatar near Bhaanse. He reported extremely heavy rainfall that whole day (Nibuwatar gauged 267 mm of rain that day – around one foot of rainfall) and stated that an emergency situation has developed at Kulekhani-I Powerhouse (KL-I) with the turbine hall operators shutting down the machines and evacuating to the safer control room at a higher vicinity after hearing a loud noise and seeing sludge oozing out of the sump tank. Nima Tshering Blutia (NTB), Director-in-Chief, Operation and Maintenance Directorate had already been informed; so for easier communication I told him that I will head for the Load Dispatch Center (LDC) at NEA office, Ratna Park. I rang NTB and he said that Ajit Narayan Singh Thapa, Managing Director, was already informed and as NTB had no vehicle in that odd hour I told him that I will come to pick him up. My driver, Hem Bahadur Ghale, fortunately lived very close and he drove us to LDC by about 11:00 PM.
B. Control from Load Dispatch Center
Fortunately at LDC, communication through NEA’s power line carrier is intact. Contact is established with GKC at Nibuwatar. From his quarter, he could hear the roaring of the Rapti river with the tumbling and crashing of the boulders as well. He believes that the Bhaanse bridge has been washed out by the flood. At Dhorsing quarter is Raiendra Narisingh Pradhan (RNP), Chief of KL-I powerhouse. Communication between KL-I and the Damsite at Markhu is, however, disrupted. KL-2 Chief, Shamim Shah, is in his quarter at Nibuwatar with GKC. GKC suggests that the Army Headquarter may have its link with its 7th Brigade at Chanban which in turn could be in contact with its unit posted at Damsite and Bhaanse (KL-2 powerhouse). GKC believes that there is the faint possibility of the wash-out of the KL-I’s penstock pipe at BL-10 where it bridges over the small Jurikhet stream. But this is ruled out as the powerhouse water pressure gauge indicated the normal 60 kg/sq. cm. RNP from Dhorsing reports that the electricity feed-back to KL-I powerhouse is intact. He asks his staff and army guards to inspect the powerhouse and they report all is well inside including the pressure gauge reading of 60 kg/sq. cm. Accordingly, I talk with Brigadier General Digambar SJB Rana (DSR), Director Military Operation, who despite the uncertainly hours is very cooperative. He says that the army in that area has already been put on alert and he will instruct his officers on duty at the Headquarter to make any communication NEA needs through Army channel.
C. Confirmation of Penstock Pipe Wash-out
NTB is busy communicating the findings and the plan of action to the Managing Director and other authorities. It is only at 01:25 AM of July 20, 1993 (Shrawan 5, 2050) that the most dreaded and unthinkable picture emerges. Both GKC and RNP report that the water pressure at the powerhouse is actually 20 kg/sq. cm and not 60 kg/sq. cm as reported earlier. This confirmed our worst fear that the penstock pipe at BL-10 is washed out by the Jurikhet stream. The 20 kg/sq. cm pressure indicated is the pressure of water remaining between the powerhouse at Dhorsing and BL-10 i.e. a head of about 200 meters. GKC and RNP are asked to send our staffs again and reconfirm the pressure gauge reading. RNP confirms the reading and with the weight of the responsibility of the washout bearing on his shoulders and by nature an easily excitable person as he is, RNP with all the night exhaustion completely breaks down into a demoralized person. RNP says that the Japanese were requested to completely cover the exposed portion of the penstock pipe, but only Sabo check dams were provided. This bridging structure, designed by one of the top Japanese consultant, Nippon Koei, and approved by institutions like the World Bank and Japan’s OECF could not withstand the wrath of mother nature – only 11 years after its commissioning in 1982. That is why experts say, ‘Never fight Nature directly, go along with it.’ Sitting comfortably at LDC far away from the scene of disaster, the magnitude of destruction and its consequences took some time to seep into us. I tried to take RNP’s demoralized mind off by suggesting that he should plan the next line of action and not worry about the BL-10 washout. Suryanath Upadhyaya, Secretary/Ministry of Water Resources, is informed of this washout. Brigadier General DSR is also informed and requested to instruct our Markhu Damsite Supervisor Dambar to immediately close the Intake Gate of the tunnel at the Damsite and also open the spillway gates of the Dam. It then dawned to us that the water from the dam will be emerging from the broken BL-10 penstock pipe with a 400 meter head and the force of about 40 MW of power. The safety of the dam must receive full attention by opening both the spillway...
gates fully. NEA’s Engineer Hemanta Joshi at Hetauda substation is asked to immediately inform the CDO of Makwanpur at Hetauda. Sagar Narasingh Pradhan, NEA Board member and former Kulekhani-I Project Chief when informed of the disaster around 2:00 AM exclaimed, ‘Impossible! I don’t believe it.’ Though he refuses to believe the washout, he, however, asks that the local Nippon Koei office at Thapatthali be informed as someone always stays there. Message is given to a Mrs. Inoue to pass on the penstock washout message to Mr. Nonaka, former Nippon Koei KL-1 Deputy project manager and KL-2 Project Manager. The Brigadier General is again requested to pass on the very important ‘close the intake gate of the tunnel at the Damsite’ information to NEA staff at Markhu. The General says that this will take more than 3 hours as the Brigade at Chabban and the army unit guarding the dam site are far off.
D. Remedial Efforts
Because of the disruption of communication with the Damsite, NTB calls in his officers Jeevan Kumar Pradhan (JKP), Director, Generation Department and Engineer Chandra Bahadur Shrestha (CBS) to send them by vehicle to the Dam site to close the tunnel’s Intake gate. The opposition party, CPN-UML, had declared a 3 days’ Chakka Jam on 3rd, 4th and 5th of Sharwan so the police at the DIG office is asked to provide an armed escort. Interestingly this Chakka Jam was a blessing in disguise to all the people who could have traveled by buses to Kathmandu because the highway was so extensively damaged by the rain that there would have been many bus accidents and thousands would have been stranded and suffered for many days on the highway. At 3:00 AM in the morning there are no drivers available; so my driver Hem Bahadur Ghale, who has not slept at all, opts to take JKP and CBS with an armed Police Escort along the old Tribhuvan Highway to Markhu. The Brigadier General is informed about the dispatch of the engineers and is requested to provide a helicopter early in the morning to visit the dam site to close the intake gate. As he expressed his inability, Ajit Narayan Thapa requested the concerned Brigadier General, Tejendra Jung Thapa. Unfortunately, at 4:30 AM JKP and CBS returned back to LDC because the highway just a little ahead of Nag Dhunga is totally blocked by a huge landslide. GKC reports through the Army channel a very dubious report ‘the dumper truck is on the way to the dam site.’ We later decoded this message as Supervisor ‘Dambar is on the way to the dam site’ and not the dumper truck! As the Markhu-damsite motor road is also damaged, Dambar has to go on foot.
E. Efforts Fail
GKC, RNP, NTB, myself and many others get not a wink of sleep the whole night. Around 6:00 AM LDC surprisingly gets the telephone call from the Nippon Koei’s Nonaka in Tokyo. NTB talks with him and abruptly tells him to come here to see the situation for himself. He says he will come in the first available flight. Talked with the Army HQ, DMO who informs that NEA’s Supervisor Dambar Shrestha is attempting to close the intake at the dam site. At this point in time, this closure of the gate is the only saviour that we see at Jurikhet’s BL 10 to stop the 40 MW kinetic energy hammering away relentlessly. Hemant Joshi confirms relaying the penstock pipe burst to the Makwanpur CDO at night and also confirms that the 66 kV double circuit tower number 132 at Baghoru is washed out. At 11:00 AM the helicopter with the Minister LP Ghimire, Secretary SN Upadhyaya, NEA’s MD and JKP take off but could not proceed beyond the Bagmati river due to very bad weather. At 11:30 AM army HQ reports water spilling from the Dam’s spillway. This is a great relief for us as we are now certain that the spillway gate is open and the threat to the Kulekhani dam through overtopping is reduced. This means the water level is over 1520 meters, up from the previous day’s 1498.63 meter i.e an incredible 22 meter rise in one day!
F. Die is Cast
So with Nagdhunga access blocked, helicopter grounded due to bad weather and Dambar not Dumper trying to close the intake gate at dam site, other avenues as well are being explored to stop the water flowing from the broken penstock at BL 10. GKC, RNP, Okazi and Nishino of Nippon Koei at Nibuwatar see only one last option i.e close the emergency butterfly valve at the Valve House located at the junction of the headrace tunnel with the penstock pipe. There are fears that the vacuum created inside the penstock pipe when the valve is closed may buckle the pipe and there are cases where such collapsing of the pipes have occurred. But with about half of Kathmandu peak power (40 MW) hammering away at BL 10, there are no other options at all. After a long debate, the die is cast and the decision to close the butterfly valve at the valve house is taken. RNP sends two groups of technicians to the valve house located nearly 2,000 ft above the powerhouse through different routes with instructions on how to close the emergency butterfly valve.
G. Game of Wait and See
There is hardly anything that can be done on any front any more. All cards are played: the Tribhuvan Highway access blocked, helicopter waiting for the weather to clear, Dambar Shrestha to open the spillway gates and close the intake to the headrace tunnel, water spilling from the dam’s spillway, the 40 MW kinetic energy being dissipated at BL 10, two groups dispatched to the valve house for closing the emergency butterfly valve. We just wait, discuss and speculate. At 14:30 hours the Army DMO relays exact dam site water level as 1524.68 m at 12:00 hours – an incredible rise of 26.05 meter in 15 hours! Our attention is then diverted to load shedding and management of the power system with the loss of the 92 MW KL1 and KL2 power plants in a system of about 220 MW. Attention is also diverted on how to repair and finance the damages. The rehabilitation of the penstock at BL 10 could be done by:
(i) reconstructing the original bridge and embedding with Sabo concrete dam;
(ii) constructing a siphon across the Jurikhet stream and putting the concrete cover or
(iii) making a tunnel from BL 9 to BL 10.
The finance could come from the World Bank, the OECF who were the
original financiers or even NEA’s own pocket. The crucial guiding principle, we realize, is Time and that which ensures No Repetition of the disaster. KL1 Chief, RNP, late in the evening reports that one of the party returns without being able to reach the valve house as their route is too dangerous due to innumerable landslides. There is no news at all of the other party and RNP, the worrying man that he is, worries and frets very much for their safety due to the landslides that were occurring all over the hills.
**H. All’s Well that Ends Well**
The next day on July 21, 1993 at 7:15 AM GKC in consultation with Okazi of Nippon Koei believes the penstock pipe at BL 10 must go underground and be embedded with concrete. This may take a minimum of four to five months. At 7:45 AM the helicopter is preparing to take-off with the Water Resources Minister from the airport. At exactly 7:50 AM RNP from Dhorsing reports that the other party sent to the valve house has returned and they have successfully closed the butterfly valve at 5:00 PM the previous day. Due to bad weather they spend a night at the valve house itself and return just then only. At 8:00 AM GKC confirms through the Bhaise Army Major Bista that the intake gate at the damsite has been successfully closed. Also at 8:05 AM Captain Raghu Bhandari at Army DMO confirms closure of the dam site intake gate. At 8:15 AM this closure of the water flow is given to Minister of Water Resources, LP Ghimire, already airborne in the helicopter and he decides to return back to Kathmandu airport. This good news comes a full 15 hours after the successful closing of the butterfly valve. The intake gate to the headrace tunnel at the dam site was successfully closed at 02:00 AM of July 21, 1993 (Shrawan 6, 2050) i.e. a full 9 hours after the butterfly valve was closed. With the closure of the butterfly valve, the closure of the intake gate at the dam site and the opening of the dam’s spillway gates, the emergency is thus successfully tackled.
**1. Conclusion – An Emergency Successfully Tackled**
Reports start pouring about the damages caused by this ‘Cloud-burst’: rainfall gauged at Palung 540 mm in 24 hours, extensive damages and the loss of human lives at the Bagmati Barrage, Malekhu, Mahadev Besi and Bhaise bridges washed out. Nepal Telecommunication Corporation’s microwave tower at Simbhanjyang knocked out. Kulekhani river’s normal drainage prior to the construction the dam was into Bagmati. The dam thus mitigated the force in downstream Bagmati but at a tremendous cost to the life of the dam? – this we are to know later. The importance of totally reliable communication lines for such emergency operations is well illustrated above. NEA’s power line carrier, NTC’s microwave networks as well as the Army’s communication served the purpose well. It will not be justified to conclude this episode of the Kulekhani disaster without mentioning a few names of those who performed their duties in this crisis very well. First, of course, comes the five NEA employees who closed the butterfly valve: Basudev Gharti Magar, Chetra Bahadur Thapa, Bal Bahadur Thapa, Tul Bahadur Rana and Bachchu Bahadur Thapa. The others are Ramraj Shah and Shyamji Shrestha from NEA with Jamared Hari Bahadur Ghale and Havildar Jagnath Pandey who entered the turbine hall despite the pungent odour to take the penstock pipe pressure gauge reading; Havildar Dil Bahadur Karki, Sepoy Jaya Bahadur Ayer, Sepoy Krishna Bahadur Basnet and Piuith Ramesh Karki of the BL 10 guard house who stood by their duty; the operators on duty at 21:20 hours both at the KL1 and KL2 who immediately took necessary measures to shut the turbines, and of course the KL1, KL2, KDPP chiefs with the Japanese Okazi and Nishino as well as the officers manning the DMO at army HQ and Major Bista of Bhaise garrison who were 24 hours on duty. There are the LDC staffs with their Chief Buddha Narayan Manandhar and many many others who performed their duties but remained silent and unnoticed like driver Hem Bahadur Ghale. This is a classic example of the successful tackling of an emergency with the full cooperation and dedication of the personnel of various organizations: the Army, Police, CDO, the Japanese consultants, NEA and many others. They proved that the Nepalese, when confronted with an emergency, can perform over and above their normal call of duties.
**J. Postscript:**
This Postscript has been added with the kind inputs of Nima Tshering Bhutia, the then Director-in-Chief of Operation and Maintenance Directorate that operated and maintained NEA’s power houses.
**1. Extent of Damages:**
a) At KL 1 – (i) about 100 meters long penstock pipe with a diameter of 1.6 meter and 22 mm thickness (ii) some equipments submerged in water at the power house and (iii) about 200 meters of the tailrace outlet filled with sand, mud and stones.
b) At KL 2 – (i) Mandu’s Inlet headworks (diversion weir, sand flushway, inlet and sand settling basin) including a part of Head Pond all damaged or washed away (ii) about 1500 meter of the headrace tunnel filled with sand, mud and stones (iii) power house switchyard filled with mudslides. Major parts of the road between Bhaise, Nibiwatar, Dhorsing, BL 10 and Markhu damaged.
**2. Contract Award:**
a) General – 11 km road between Bhaise and KL 1 made motorable by Bhadra 17, 2050 (September, 1993); as Nippon Koei was consultant for ongoing Kulekhani Disaster Prevention Project, it was also made consultant of Kulekhani Rehabilitation Project; decision taken to go underground at Jurikhet BL 10 and embed the penstock in concrete.
b) Penstock pipes – On Bhadra 22, 2060 (Sept. 7, 1993) order for 118 meter long 1.6 meter diameter 22 mm thick penstock pipes weighing 130 metric tons awarded to Austria’s Voest-Alpine company; first delivery through Lufthansa’s normal passenger flights arrived on October 12, 1993 with last delivery on October 26, 1993 (Kartik 10, 2050) with a cost at Kathmandu of 1.4 million German Mark – total cost of Rs 5.50 crores inclusive of Rs 1.33 crores as Nepalese custom duties.
c) Civil works – Civil works (excavation, concreting, tunneling and penstock pipe installation) worth Rs 10.55 crores awarded to China International Water and Electric Corporation (CWE) also on September...
7, 1993; works to be completed by January 31, 1994 (Magh 18, 2050).
3. Construction Works:
Excavation to install the penstock pipes at EL 1090.10 meter 10 meter below the ground level; concreting of penstock pipes started on November 12 (Kartik 27) and finished on December 18, 1993 (Poush 3, 2050); excavation of 3.25 meter diameter tunnel of length 33.5 meter; welding of 129 meter long penstock pipe that need to be verified with X-rays completed in 22 days by December 9, 1993 (Mangsir 24, 2050); welding done by 4 Chinese, 4 Nepalese (3 from Nepal Hydro & Electric Pvt Ltd) and 3 Indian welders; plus the complicated task of coupling the old penstock pipe with reducer and expansion joint.
4. Commissioning of KL1 & KL2:
Water filling in the penstock pipe started on December 18, 1993; testing of the entire electrical and mechanical equipments (that idled for five months) in both the power houses done; 60 MW KL 1 successfully commissioned and inaugurated on December 27, 1993 (Poush 12, 2050) by Congress leader Ganesh Mani Singh; water filling in the tunnel of KL 2 from December 28, 1993; 32 MW KL 2 successfully commissioned on December 30, 1993 (Poush 15, 2050).
5. Remarks:
i) Expenditure & Revenue: Rs 5.50 crores for penstock pipes came from NEA and OECF/Japan picked up the Rs 10.55 crores for civil works and Rs 3 crores for consultancy – a total expenditure of Rs 19.05 crores. Whereas, average annual generation of KL1 211 GWh and KL2 105 GWh, that totals to 316 GWh which at NEA’s 1993 tariff of Rs 2.54 per unit meant a revenue of Rs 80.3 crores. An expenditure worth spending for!
ii) Reconstruction Period: The Kulekhani penstock pipe at Jurikhet BL 10 got washed out on July 19, 1993 (Shrawan 4, 2050). With Nippon Koei’s Nonaka telling Minister LP Ghimire that it would take at least 9 months, the power house was commissioned in December 28, 1993, within a record 162 days (5 months and 12 days).
iii) Working Environment: This was possible due to very quick decisions of NEA Board, very good working relationship within NEA directorates and at the Project site; Nippon Koei’s sincere Okazi and Nishino; capable civil contractor CWE and reputable penstock pipe supplier Voest-Alpine. Though the civil work contract completion target was January 31 1994, Voest-Alpine’s Austrian site-supervisor pushed the target ahead as he had to be home for the December Christmas. This was very much in NEA’s interest, to remove the heavy load shedding. The Austrian finished his penstock pipe installation supervision by third week of December and left for Austria. It was then up to the CWE contractor and NEA staffs to commission KL1 & KL2 by December 28 and 30, 1993 respectively.
iv) Nepalese Can Do: That Nepalese can also perform, when the nation wants, is illustrated by this example. If we apply the same Kulekhani disaster spirit of 2050 to the present 2066 dilemma of load shedding, Nepalese can perform insurmountable feats – provided, of course, our political leaders set the examples first by foregoing their selfish motives in the larger interest of the people and the country!
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1 Former Managing Director/NEA and former Officer on Special Duty/MOWR.
2 The 50ft mm in 24 hours ‘cloudburst’ that rushed down a valley left 26.05 meter in 15 hours also brought along huge debris (mud, sand, pebbles and rocks) crashing into the Kulekhani storage dam. The life of a storage dam, designed to last over 50 years, is dependent on the ‘dead storage’ capacity. When all of that capacity got filled up, Japan & OECF came to the rescue immediately by funding the construction of the sloping intake.
Introduction: Micro-finance, a mixed financial product of finance and social intermediation, has evolved as a global concern because it is the financial services of the poor. Specifically, the micro-finance initiatives have economic goals of reducing the poverty and tackling the marginalization of the poor particularly the deprived section of the society. Specifically, microfinance is distinct from Rural finance and Agriculture finance and micro-finance cooperatives operate saving and credit as its main cooperative business.
Micro finance commenced in United Kingdom in mid 19th Century through cooperative saving of 28 pounds from factory workers of ‘Rochdale Society of Equitable Pioneers’. In Nepal, the first credit cooperative society was formed in Chitwan district of central Nepal in 1956 with an aim to provide the agricultural credit to the flood-stricken people resettled in the Rapti Valley. Nevertheless, the formal micro-finance programme began in Nepal in 1970.
Social exclusion discourse developed in response to poverty describes the process that leads the individuals or groups from being wholly or partly excluded from the full participation in the society. Nepal, a Hindu kingdom for a long time; has the caste hierarchical system that dominate the most social and cultural value. The age long caste-based practices have made “Dalits” – the most deprived group in the society and the representation of Dalits in all socio-economic sectors is negligible. However, there is always debate on social inclusions spoils the sustainability. In this regard, this article, based on the SIRF funded research study; focus on the inclusion of Dalits in micro finance. In the research study, the researcher has used an unique measuring rod term as “Social Performance Measurement” to measure the social inclusion.
Microfinance Models: In 1970s and early 80s, the provision of cheap money to poor farmers and rural micro-entrepreneurs was considered key to stimulate the farm and non-farm investment, to boost the use of modern inputs and to augment production in the rural areas. By the mid-1980s, the results achieved by the traditional credit programmes turned out to be disastrous and the critics argued that only few poor people have access to the credit subsidies. It is now generally accepted that the cheap credit was ineffective in stimulating the agricultural investments and alleviating rural poverty. The cheap credit undermines the development of a viable rural financial market so it led to the formulation of a new paradigm of financial market. Under this approach, even small farmers are seen as potential source of savings and they are generally considered as able to pay interest. The subsidies are considered as transitory. Since the system has high risks and costs, a specific financial technology has been developed, tested and adjusted to local needs throughout the last decade of twenty century. This financial technology includes: Social control mechanisms; Intensive credit monitoring and stringent loan recovery efforts; Loan portfolio risk diversification measures; Character-based ending strategy; Acceptance of non-traditional collateral – joint liability/group guarantee; and Priority on saving mobilization.
Approach for Inclusive Microfinance: The different Global Summits on Micro Finance have envisaged various procedures to make Micro Finance inclusive. The micro finance institution can build institutional capacity to reach the poorest families by strengthening network and exchange practice in existing micro-credit institutions and preparing new practitioners through trainings. The regulation for micro financing systems needs to be revised with more focus to empower the poor. Likewise, these institutions can use of cost-effective poverty assessment tools to chart the movement of clients above the US$ 1 a day threshold. Further, the micro finance institutions can conduct a media campaign to expand awareness and support the promotion of Campaign’s learning agenda.
Micro Finance Sector in Nepal: Regarding micro finance sector of Nepal, the models widely practiced in Nepal include: Grameen Bank, Village Bank, Swablamban (Self-reliance), informal groups and Cooperative. Some of the Microfinance Development Banks which are active in microfinance sectors are: Nirdhan Utthan Bank, DEPROSC Bikas Bank, Chhimek Bikas Bank, Swablamban Bikas Bank, Grameen Bikas Bank in regional level promoted by Nepal Rastra Bank and Sana Kisan Bikas Bank promoted by ADB/N. Further, ADB/N, the largest rural credit provider, has been transformed into Small Farmer Cooperative Ltd (SFCL).
In term of Nepal’s legal and regulatory framework, there are two legal options to facilitate the establishment of Micro Finance Institution. First, Community Based Organizations include Small Credit Cooperatives established under Cooperative Act 2048 and NGOs established under Social Registration
The caste hierarchy system is one of the tragic facts of the Hindu religion which placed certain caste group in lowest level favoring the culture based hegemony of the high caste group so there persist rampant inequality between high and low caste in the Hindu society since ancient past. Where, the high caste groups in Hindu are: i) Brahmin who govern the civic rule of Hindu society and administer the traditional socio culture rites, and ii) the Chhetri who are warrior and ruler by kinship. On the other hand, the lowest caste groups are Dalits whose work and way of living is regarded as impure in Hindu society. So, the Dalits, being impure, are regarded as untouchable caste group and this traditional system of untouchable has nurture in our society so deeply that the socio-politico and state structure is unseeingly adopting this system despite of the assumed changes in cultural norm and value system.
It has been conceived that a touch of Dalits make high caste people impure so such high caste people should be sprinkle with Gold Water to make pure. Likewise, if Dalits touch water or public water source (viz. Ground well, tap, lake) located in the high caste localities then such water become impure and useless and the Dalits who committed such act are severely punished. Further, the Dalits are also untouchable to the traditional market economies as their work, pre defined by high caste, are regarded as non-tradable because their service should only provide economic welfare to high caste. The Dalits can only perform their work with the permission of high caste otherwise such activities are regarded as sin. In essence various range of discrimination against Dalits, the untouchable practices in society can be observed via two layers. First, the Dalits are untouchable to market which means as discussed earlier the Dalits can only perform their pre-define work with the permission of high caste and again for the welfare of high caste. Second, the Dalits are untouchable to overall culture – specifically Pani Na Chaline (Impurity of Water after the touch of Dalits) – which also include first one i.e. untouchable to market. In another word, the main agenda of Dalits is untouchable in culture but it won’t encompass all Dalits as some Dalits are also observed to be Pani Chalne (i.e. Touchable by Culture) but they are untouchable to market system. In socio-economic approach, the untouchable to market means limited access to resource which causes downturn so the competing inequality among Dalits is only possible if the untouchable in society is minimize.
Nepal is home to various caste groups of Dalits accounting 13 percent of the country’s population. As per these meetings, together with the opportunity cost of attendance can be a barrier for poor people to participate in microfinance programs. In term of sociocultural barrier, poor are socially intimidated, believing that the services offered by micro finance is not suited to their needs.
Recommendations: In order to make micro finance inclusive, the micro finance policy that is unfriendly to poor should be revised. Specifically, the Government needs to revise the existing laws and regulations and enhance its supervisory framework. Further, Micro Finance Institution should establish working relationship with FNCCI and its district chapters to access different facilities and support that may be available. This also develops the efficiency in networking and advocacy which supports to share knowledge, information and experience. Additionally, the civil societies need to educate people against caste-based discrimination as the ethno-caste-based discrimination is the root cause of exclusion of deprived communities in every sector of public life.
1. Mr BK is the Research Fellow of Social Inclusion Research Fund (SIRF) in 2006. This article is based on his SIRF funded research study.
geo political and social reality of the country, the Dalits of Nepal are generally grouped on the basis of ecological belts. First, Hill Dalits from hill ecological belt are untouchable to overall culture. Second, Terai Dalits from Terai ecological belt are also untouchable to overall culture but some caste groups of Terai Dalits (viz. Lohar, Shonar and Hajam) are only untouchable to market. The researches funded by Social Inclusion Research Fund (SIRF) have substantially raised the issues of untouchable in Dalits.
The SIRF researchers – Amar Bahadur BK, Yam Bahadur Charmakar and Ram Sundhar Nepali – highlighted on a board picture of Dalits’ untouchable in culture which is the corner stone of Hindu based civilization. It is a deliberate fact that the art, culture and knowledge of Dalits is the part of Hindu civilization but they are tagged as impure, untouchable and low graded so the SIRF researcher – Dhan Bahadur Sunar – argues that there is a trend of change in religion, culture and occupation among Dalits in Nepal. This implies that the Dalits feel suffocation in the traditional Hindu civilization as they are not treated humanely.
Likewise, the SIRF researchers - Bharat Gotame and Khadga Bahadur Ramtel - presented a comprehensive account of untouchable practice against Dalits in Hali system. Under this system the Dalits plough and work in the field of the high caste in the agriculture session and they get some cultivation as a reward in return. The social system of Hindu has defined that the high caste landowner can exchange his share of cultivation in the market regardless of who have produced them. But, the Dalits, on the other hand, cannot exchange his share in the traditional market and he can use them just for his own purpose. The refusal of food product from Dalits exists even today in some of the urban area of Nepal and it is rampant in rural area. The recent case of such instance dated five years back and was happened in Dhading district. The Dalits of the district were not allowed to sell their milk in the village claiming them and their milk to be impure.
Furthermore, the traditional social system has not provided any economic right to Dalits to defend their work. They can’t charge the value of their work like the business men price their goods in the market. Rather, they have to accept what their high caste clients reward them. Likewise, there is no culture of formal education in Dalits so they follow oral tradition for ingeneration transfer of their art and skill. Even today, in some rural areas, the Dalits children are not allowed to attend the school by sitting together in class. Either, they are provision separate seat or they have to stay outside class and peep from windows and door. This signifies that the Dalits have very less access to resource so they are downturn. Specifically, the poverty situation is highest in Dalits than any other caste group of Nepal; the Dalits women accounts the five out ten women died due to pregnancy related diseases in Nepal; and the 60 out of 100 Dalits children has never attended the school. In order to compete the inequity in Hindu based society like Nepal, the state needs to address the growing up untouchable in the society and state.
There is raising voice against inequity in Dalits from civil society, donors and other stakeholders. T. R. Bishowkarma is regarded as the initiator of the Dalits revolution of Nepal. The SIRF researcher – Dil Bahadur Bishowkarma – postulates the contribution of this man in the Dalit revolution. TR. Bishowkarma initiated this revolution by bringing the Dalits of Nepal with various backgrounds together and making untouchable as common agenda for their revolution. His initiation was later geared by the various activists and the political leaders resulting an intense discourse on untouchable culture. These discourse compelled the government to form National Dalits Commission for the welfare of Dalits and the political parties gradually started to address the Dalits inclusion issues in their manifesto. Further, Nepali Communist party Maoist made the issues of Dalits inclusion a high preference political agenda as they legitimize the inclusion as one of the main agenda of the decade long Maoist armed revolution.
SIRF has also contributed on discourse of Dalits inclusion which in later days focuses on the agenda for Dalits inclusion in new constitution building process. In this regard, SIRF is fortune to invite then Union Cabinet Minister of India from Dalit community, Mr Ram Bilash Paswan in March 2008. This visit was fruitful to SIRF in the sense Hira Bishowkarma, SIRF Interim Screening Committee Member, adjoin Mr. Paswan during his meeting with various high level personal of Nepal which include President, Prime Minister, Foreign Minister, President of Nepali Congress and other major political parties. In these meeting, Mr Bishowkarma placed the issues on Dalit’s inclusion identified by SIRF and its importance in present constitution making process. This diplomacy of SIRF was the unique means to disseminate the SIRF’s activity to high level personal.
The untouchable is the main issues of Dalit exclusion which evolve an inequality in society. The existence of untouchable culture roots in the family value system and shadowy entire state structure. The state should be cognizant on this deliberate fact of Dalits exclusion while addressing the social inclusion issues. So far, it seems that the government has addressed the issues of Dalits exclusion only by revising law and enhancing the service delivery mechanism (viz. free education, free health and others). But, it seems that the government has completely ignored the issues of how to wipe out the untouchable approach from mindset of high caste, family structure, culture and state structure.
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1 Mr. Manmohan and Mr. Rajacharya are associated with Social Inclusion Research Fund. The views presented are that of writer’s own and do not reflect that of authors’ affiliation.
“Dalits are one of the most deprived communities in Nepal”
-BISWENDRA PASWAN
Leader of Dalit Janajati Party BISHWENDRA PASWAN is the only member of his party in the Constituent Assembly. He became the CA member under the proportional representation system. Paswan, who has completed his intermediate level in law from Rajbiraj, spoke to NEW SPOTLIGHT on the state of dalits in the country. Excerpts:
What is the economic status of dalits?
Dalits are one of the most deprived communities in Nepal. Economically, they are the poorest and an overwhelming number of them are living below the poverty line. Madheshi dalits are more affected than dalits from the hills. Out of the 12 percent economically active population, only a small number get employment opportunities.
Do you think the new constitution will provide equal political and economic rights to dalits compared to any other community?
Given the present state, it is easier said than done. Despite their claims, political parties have yet to recognize the rights of dalits and work to bring dalits into the mainstream. If we look at the present structure of political organizations, there is a very nominal representation of dalits. Of course, they speak for the rights of dalits in public but their mindset is yet to change.
What do you suggest to improve the economic situation of dalits?
Dalits need protection and reservation for jobs in all sectors. Dalits need special economic packages and incentives to start their professions. Along with education opportunities, they also need economic incentives.
How do you compare the overall situations between dalits living in the hills and the Terai?
The status of Madhesis dalits is worse than the dalits of the hills. Our situation is much worse than all other communities among janajatis and dalits. From education to per capita income, the situation of dalits of Madhesh is more pathetic.
Nepalese political leaders have been drafting a new constitution with inclusion as a major slogan. How hopeful are you that the new constitution will be truly inclusive?
If you look at the present political trend, nobody seems to be serious about drafting a constitution to include dalits. Our voices are unheard and our views are ignored in the CA committees. Political leaders are just paying lip service in the name of inclusion.
What do you suggest would make the new constitution inclusive?
If this Constituent Assembly fails to address the problems of dalits, they will revolt against the state. This will create a more chaotic situation. We have to fight to establish our rights.
Don’t you think the proportional representation system has brought about certain changes in the kind of representation in CA?
Our experiences have shown that the equitable representation alone cannot resolve problems of exclusion—unless and until we are able to wield influence through direct and active participation. Dalits were excluded from the mainstream politics for a long time and I don’t think they are in a position to compete now with advantageous groups who have a long political influence and economically sound background.
What is the situation now?
Of course, dalits are able to send their representatives to the CA, but this alone is not enough. What we need is some kind of reservation in education and employment sectors.
Along with political rights, what do you prefer?
Political rights are important. After formulation of the new constitution, our political rights will be ensured. Economically, we need a lot of time to enhance our status at par with advantageous communities.
The government is providing special low interest rates loans to marginalized communities, how do you see this move?
These kinds of systems were there in the past but they failed to deliver the results. Giving access to the banking sector is not enough. What is required is the process which will ensure their reach. To increase the involvement of dalits in financial sectors, community approach is needed.
As Nepal is in the process of restructuring of the state, can it make any difference to the status of dalits in Terai?
Nepal Human Development Report (2009) has already shown the pathetic situation of dalits in Madhesh. Our experiences have shown that the transformation of the state is alone not enough to guarantee the rights of the dalits who have been exploited and excluded by the state for quite a long period of time.
How do you propose to make the society inclusive to all?
First of all, the new constitution must declare all discriminatory practices as illegal and unconstitutional. The new constitution needs to guarantee the representation of dalits in the political process, in economy, education and all other sectors. For the time being, we need a special package for reservation.
What is your impression of the recent trend of social inclusion?
Frankly speaking, one can see certain changes in terms of representation at the CA, but the situation is not like the same at all levels. As long as we are unable to end the rampant prevalence of discriminations against dalits in society, we cannot bring about any changes. The trends are once again no more than a political slogan to woo the voters.
What kinds of changes do you want?
I want the change where dalits are given economic as well as political opportunities. This is a must for the dalits living in Madhesh who are denied even the basic fundamental human right to live with dignity.
Contemporary Themes
Peace and Conflict
By BIPIN ADHIKARI
Hira Bahadur Thapa, Selected Essays on Foreign Relations (Kathmandu: Asia Publications Pvt Ltd, 2009) (Soft Cover Price Rs 175)
Hira Bahadur Thapa’s *Selected Essays on Foreign Relations* is the latest book in town on diplomacy, peace process and contemporary international issues. It is a collection of 54 newspaper essays published by Thapa between July 2007 and June 2009.
As the author notes, this period has been of historical significance because it covers the time when Nepal entered into UN facilitated peace process by ending a decade-long Maoist People’s War. Between August 2008 and May 2009A, career diplomat Thapa also served as the foreign policy advisor to Prime Minister Pushpa Kamal Dahal. This further afforded him a vantage from where to discuss issues important to Nepal in its peace process and foreign relations.
The subjects covered in this compilation range from preventing the peace process from collapse, the challenges of ongoing recruitment of national army and the combatants of the Communist Party of Nepal (Maoists), to the extension of the mandate of the United Nations Mission in Nepal (UNMIN). Some articles defend human rights, plea for establishing a culture of accountability, and stress healing the wounds of the Maoist conflict. Many of his articles have analysed the UN peacekeeping efforts, expansion of the UN Security Council membership, nuclear disarmament and non-proliferation and the theme of collective security.
Some articles deal with the problems of South Asia. There is no common theme, but they include expectations in the fifteenth summit of SAARC (South Asian Association for Regional Cooperation) nations, deepening Nepal Sri Lanka ties, enhancing Nepal China economic relations, fighting terrorism, and cultivating relations with neighbours. He has also devoted two articles on Bhutanese refugees in Nepal.
The book is useful to anybody who wants to understand Nepal’s diplomacy, peace process and other contemporary international issues over the period covered by the author. Thapa is a moderate analyst in most of his opinion pieces. Almost every essay has certain prescriptions to the concerned sector. He is futuristic in his outlook. This makes his approach valuable.
At places, the author does not hide his sympathy for the Maoist movement, and its leaders. One can also note that he is not critical about the Maoist “People’s war” which is said to have claimed approximately fifteen thousand lives, and derailed the democratic process by threatening parliamentary elections. Notwithstanding this criticism, the author has matured understanding of the issues that he has picked up for op-ed contributions.
The author could have considered writing an introductory chapter with a view to provide top up to each article. He could also have divided all these 54 articles in four or five broad themes, giving each theme a defined perspective. A reader who is not aware of the chronological development of Nepal’s politics may at times find difficulties in putting some of the essays in perspective.
Bishnu Prasad Poudel & Hari Bansh Jha (eds), The New Dynamics of Conflict in Nepal (Kathmandu: NAC-SSA, CETS & FES, 2009) (soft cover, price not disclosed)
This is a new book on the topical issue of conflict. It got this shape following a two-day national seminar held in November 2009 on “The New Dynamics of Conflict in Nepal: Challenges and Opportunities.” The seminar focussed on the post-conflict situation in the context of new armed groups creating law and order problems in the country, particularly in the Terai region, where killings, abductions, and extortions have become very common. It includes papers presented by Dev Raj Dahal, Bishnu P. Poudel, Hari Bansha Jha, Manish Thapa, Chandra Kishore, Som P. Pudasaini, and Dinesh Tripathi, in the area of their specialization, and has a brief report of the seminar at the end. The editors pinpoint in the preface of the book that unless the conflict is not handled carefully, the country might plunge into a long term civil war.
The book has a Foreword from the Rt. Hon’ble Vice-President of Nepal, Mr Paramanand Jha. It is a handy, useful work for anybody on the theme. None of the papers, however, adequately covers the India factor in the conflict dynamics of Nepal.
English-Nepali Glossary of Federalism Terms (Kathmandu: IDEA/Forum of Federations, 2009) (Soft cover, price not disclosed)
This is the first Glossary of basic terms used in the federalism discourse produced in Nepal. It offers definitions for some 300 federal terms and their translation into Nepali. The experts involved in developing it, have tried to give not just the basic understanding of each term, but also contextualize it in ways that help in the understanding of the Nepalese readers. Federalism as a form of government in which power is constitutionally divided between a central (national) government and sub-national (state, provincial, regional) governments is very new to Nepal. In this concept, both levels exercise some powers (rather than power being exclusive to the central government or the sub-national governments), and produce a shared government based on a written constitution. While Nepalese are debating a possible constitutional arrangement in this regard, this Glossary, as the publishers have pointed out, can help them understand and use the terms in proper perspective. It tries to address the need for clarity in the use of words or terms that have been central to any political discourse in this regard. By default, it will also help in the standardization of Nepali constitutional terminology. Finally, this Glossary may also prompt the critiques to suggest appropriate alternative translation for some terms in this Glossary which are still difficult to understand by all (for example, *agatikata*, *aritibhā*, for economics of scale, and *bharī pratīnīdhītwā* for weighted representation).
Suitable books for review may be sent to: Bipin Adhikari
Email: lawyers inc email@example.com
Thailand-Nepal Face Common Concerns
-By Asi Mamane
This year is a special year for Thailand and Nepal, as it marks the 50th anniversary of the establishment of our diplomatic relations. Throughout the past 50 years, the relations between Thailand and Nepal have always been based on mutual trust, respect, goodwill and understanding. Both our countries have worked hard to enhance our relations, to deepen and broaden our cooperation in various fields. The frequent exchanges of visits at different levels between the two countries have significantly contributed to the strengthening of relations.
Religious and cultural cooperation is one of the most important aspects of our long-standing relationship. Ninety percent of the Thai people profess Buddhism as their main religion, while Nepal is the birthplace of Lord Buddha. Therefore, the Thai people have special respect and feel very close to Nepal and the Nepalese people.
We are witnessing advances in the area of cultural cooperation. The links between our respective cultures are centuries-old, as evidenced in many similarities between various aspects of our cultures and traditions which have been inspired by Hinduism and Buddhism. They are testimony to the long history of contacts amongst peoples, of exchanges of ideas and values. And from such cultural cooperation comes enhanced understanding amongst our peoples.
The Royal Thai Government has continuously supported the Human Resource Development of Nepal by providing scholarships for higher education and short-course trainings for civil servants and personnel from the private sector in many subject areas.
Thailand reaffirms its continued support for technical assistance and cooperation with Nepal in the areas of Thailand’s expertise and according to Nepal’s development needs during its transitional period.
In the current year (2009), Thai Government provided 11 Master Degree scholarships and 49 short-course training fellowships to the Government of Nepal.
Thailand is willing to render its full support to Nepal’s peace process and its democratic transition. In this connection, the Royal Thai Government has dispatched military officers to join the United Nations Mission in Nepal or UNMIN since 2007 and also sent a team of election observers to observe Nepal’s CA election in April last year.
In the economic front, at present, the volume of two-way trade between Thailand and Nepal is relatively small. However, it is increasing year by year. There is a great potential for our trade and investment cooperation to further develop, particularly in the areas of tourism business, agricultural and consumer products, food processing, textiles, machineries, as well as infrastructure development.
Thai Airways International has been operating in Nepal for the past 40 years. Regular flights by the Thai Airways have been facilitating increasing numbers of tourists between the two countries. In the year 2008, approximately twenty thousand Nepalese tourists and businessmen visited Thailand. Likewise, large number of Thai tourists also visited Nepal last year. The number of Thai tourists coming to Nepal is on the verge of increasing, despite the global economic crisis. Now, Nepal has become one of the favorite destinations for the Thai tourists.
In this globalized world, it is important to strengthen our cooperation not only in the bilateral sphere but also in the regional and global arena.
I am pleased to note that our two countries are working closely together in many international and regional forums including the United Nations as well as BIMSTEC.
On all these fronts, let such close cooperation continue and indeed expand, in areas where we face common concerns, on issues where we have shared interests.
Excerpts of the remarks of Mr. Mamanee, Charge d’affaires a.i., Royal Thai Embassy given at the function organized to celebrate 50th anniversary of Diplomatic relations between Thailand and Nepal.
Happy greetings on the Auspicious Occasion of BADA DASHAIN AND TIHAR 2066 B.S.
Office: Radhakuti Arcade, 1st floor, Putalisadak P.O. Box 623, Kathmandu, Nepal Tel: 4411187, 4412268, Fax: 977-1-4412961
The Tibet tale, once again
Another photo exhibition shows the changing face of women in today’s Tibet
By PRADIPTI BHATTA
A photo exhibition on ‘Women of Tibet, China’, held at Shangri-la Hotel, Kathmandu, between September 9 and 15, showed how women are playing an important role in the modern transformation of the Tibet Autonomous Region (TAR) of China. The exhibition, organized by Women’s Association of TAR of China and the Chinese Embassy in Nepal, was inaugurated by Purna Kumari Subedi, Vice Chair of Nepal’s Constituent Assembly and Can Muqun, president of Women’s Association of Tibet Autonomous Region.
The exhibition focused on the women of Tibet in its display of a total of 111 photographs. Nevertheless, the pictures reflected the essence of Tibetan culture. Just like the previous exhibition, held last month, this one also richly portrayed the development of Tibet in facilitating cultural exchange and understanding between China and Nepal.
Most photographs showed the state of women in Tibet, women who were contributing to the development of Tibet. The exhibit clearly depicted how actively the women in this part of China have been participating in various aspects to help improve not only their households but also the society and the country as a whole. The pictures told a tale of their involvement as active members of the society.
Some of them also showed the beauty of the Tibetan terrains. Various scenarios were also part of the pictures. Tibet can certainly be seen as a land gifted by nature.
The pictures featured women of all ages involved in various sectors like business, health, education, mountaineering, farming etc. The women of Tibet no longer seemed limited to certain activities. They have caught up with their male counterparts in spreading out their wings and getting involved in all sectors.
Each picture, like it is regarded, was telling something, a lot more than written words, would be able to express. The photos showed smile and confidence in the face of the Tibetan women that words would fail to describe. In the least, they were happy about whatever they were doing and also looked independent.
The photographs also showed the rich culture and tradition of Tibet. Just like Nepal, the main activity of Tibet also is agriculture, so most of the photographs were related to this sector. Photos of women involved in agriculture were among the most eye-catching ones.
This was the second exhibition on Tibet of China held in Nepal. It has certainly allowed two different cultures know more about each other. Through this event, the Nepalese have been able to know more about the Chinese culture. This exhibition can be regarded as having given a window for the Nepalese to look across the Himalayas for a rich culture and a modernizing tradition. Especially the Nepalese women can take cues from the Tibetan women as an example and work in a manner similar to them in the development of the society.
As a whole, the seven-day event was a success. Hopefully, some aspects of the Nepali society and culture will also be put on a photo show in the neighboring country some day as well.
Happy greetings on the auspicious occasion of Bada Dashain and Tihar 2066 B.S.
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We extend the heartiest felicitation for peace, progress and prosperities to all our well wishers on the auspicious occasion of DASHAIN & DEEPAWALI 2066.
Suk Bahadur Gurung
Chairman
Pioneer Overseas Services (P) Ltd.
P.O.Box 21120, Baluwatar, Near Chinese Embassy, Kathmandu, Nepal
Phone: 00977-1-4433203, 4424946, 4439874, Fax: 00977-1-4428750
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Cert. No. 13794
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OUR MISSION STATEMENT:
"Saint Francis Xavier Parish is a Christian Family dedicated to the mission of Jesus and the teachings of the Catholic Church. We are committed to fostering spiritual growth and to responding to the human needs of both our faith community and the larger community by sharing our gifts and resources."
Liturgy Schedule
Weekend Masses: Holy Day Masses: See schedule inside.
Saturday Evening Vigil: 4:30 pm
Sunday Masses: 8:30 am & 10:45 am Daily Mass: 6:45 am Mon, Wed, Thur, Fri
Weekend Mass Schedule in our area
| St. Michael-St. Peter | St. Mary's of the Lake | St. Patrick's Jordan |
|-----------------------|------------------------|---------------------|
| Sat. Evening: 5:00 pm | Sat. Evening: 4:30 pm | Sat. Evening: 5:00 pm |
| Sunday: 7:30, 9:00 & 11:15 am | Sunday: 8:00 & 10:15 am | Sunday: 8:00 & 10:00 am |
PASTORAL STAFF:
Pastor: Rev. Daniel C. Muscalino*
Pastoral Associate: Deacon John J. Falge*
Religious Ed. Director: Renee O'Connor*
Parish Secretaries: Mary Jo Osterman Katherine Boos
Social Service Coordinators: Rosalie Durand Patty White
Music Director: Robin Schott, Jr.
Buildings and Grounds: Ron Schneider
Housekeeper: Kathy Welsh
Parish Historian: John P. Curtin
PARISH TRUSTEES:
Diane Foster*
Joseph E. Durand*
* Also on Parish Council
SACRAMENTS:
Baptism: Class required.
Call rectory for arrangements.
Reconciliation: Sat. 3:30 - 4:00
Marriage: Arrangements at least 6 months in advance.
NEW PARISHIONERS are most welcome. See Fr. Muscalino after Mass or call the Rectory.
PARISH COUNCIL
Jay Austin Roxanne Taylor
Terry Hall Kim Flood
Pen Anderson Marty McClusky
Cheryl Hunt Terry Kotlarz
Beth Cusick
FINANCE COMMITTEE:
Rick Reagan
Chris Hunt
Diane Foster
Rev. Daniel C. Muscalino
Joseph E. Durand
Jay Austin
http://sfxmarcellus.com
WEEKEND LITURGY SCHEDULE:
Saturday Vigil - 4:30 PM
Sunday Masses - 8:30 & 10:45 AM
Sacrament of Penance - Saturday 3:30pm
TELEPHONES:
Rectory: 673-2531
1 West Main St., P.O. Box 177
Rel. Ed. Center: 673-4107
53 North Street
**Mass Intentions FOR THE WEEK**
**Saturday August 22**
VIGIL TWENTY-FIRST SUNDAY IN ORDINARY TIME
4:30 PM- Henry Gooden- Vernon & Marilyn Bush
**Sunday August 23**
TWENTY-FIRST SUNDAY IN ORDINARY TIME
8:30 AM- People of St. Francis Xavier
10:45 AM- Owen Corrigan- Corrigan Family
**Monday August 24**
SAINT BARTHOLOMEW
6:45 AM- Carol A. McGrath- Family
**Tuesday August 25**
SAINT LOUIS AND SAINT JOSEPH CALASANZ
6:00 PM- Art Peterson- Marilyn Duffy
**Wednesday August 26**
No Mass
**Thursday August 27**
SAINT MONICA
6:45 AM- Robert Warnock, Jr- Marilyn
**Friday August 28**
SAINT AUGUSTINE
6:45 AM- Mary & Howard Harrington- Casey & Eileen Smith
**Saturday August 29**
VIGIL TWENTY-SECOND SUNDAY IN ORDINARY TIME
4:30 PM- Tomas Eisenberg- Maureen & John Curtin
**Sunday August 30**
TWENTY-SECOND SUNDAY IN ORDINARY TIME
8:30 AM- Fran Thorpe- Family
10:45 AM- People of St. Francis Xavier
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**What’s Happening This Week at St. Francis**
St. Francis is open for public Mass for **50 people**. In addition to weekend Mass, we will also have Monday, Tuesday, Thursday & Friday Mass. Parishioners need to sign up prior to the weekend and review safety instructions.
sfxmarcellus.com/events
*(please sign up several days prior to weekend Mass so we can print the list ahead of time)*
If you have any questions, call the rectory: 673-2531.
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**Our Weekly Collection**
Weekly Goal: $7,900 Last Week: $5,964
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**Please Pray For...**
Adeline Fagan, Robert Hammond, Richard Hammond
Isabelle Callahan, Dennis McEnery, Ed Dillon,
Riley Christine Perkins, Nick Olenych,
Roger Schott, Danielle Painter, Genie Barnes,
Nicole DeMore, Lisa Pikarski,
Michelle Fleegel, Jo-Ann Foertch, Ellie Hauver,
Justin MacLachlan, Jack Bragger,
Florence M. Linda, Peggy Paradise, Missy Fitz,
Austin Marlowe, Brian Stonelake, Brett Kresco,
Lucienne Henneberry, Allan Stonecipher,
Donna Martin, Kelly Cunningham Suberts,
Jo’Ann F., J.E.L., Sr. Mary Brendan Wilson
**Please Pray for Our Homebound...**
Isabelle LaClair, Theresa Holmes, Kay Wilson,
Jim Reagan, Helen Volecko, Mike Berish, Ethel Cole,
Marie Patterson, Mary Martin, Patricia Welch
Please call the Church Office to add anyone to our Prayer List.
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**Sacrament of Confession** - To receive the Sacrament of Confession, please call the rectory and make an appointment.
**Church Bulletin** - The weekly bulletin is available on our website. sfxmarcellus.com/weekly-bulletins
**Elizabeth Ministry** - The Elizabeth Ministry is here at St. Francis. This ministry is extremely important for it affirms that every member of the church has gifts and talents to build up the entire community.
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**SPIRITUAL COMMUNION PRAYER**
My Jesus, I believe that You are present in the Most Holy Sacrament. I love You above all things, and I desire to receive You into my soul. Since I cannot at this moment receive You sacramentally, come at least spiritually into my heart. I embrace You as if You were already there and unite myself wholly to You. Never permit me to be separated from You. Amen.
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**Holy Father’s Prayer Intentions** - Universal
*The Maritime World* – We pray for all those who work and live from the sea, among them sailors, fishermen and their families.
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**A Volunteer Opportunity**
Would you like to make a difference for others and feel that difference within yourself? Francis House is a home for people who are terminally ill. Our volunteers support our residents by helping in the kitchen, with phones, maintenance and light cleaning. Our house is safe. We sanitize every four hours and keep our volunteers in a protective environment. There are volunteer shifts available ranging from 8:30am to 12 noon, 12:00 pm to 4:00 pm and 4:00 pm to 7:00 pm. If you would like to give a few hours a month to help, please contact Rea Carver – 315-475-5422. Thank you.
SACRAMENT OF FIRST COMMUNION
Saturday, August 22nd at 9 & 11 a.m. Mass
Please remember these little ones in your prayers:
Blake Benz
Joshua Borst
Abigail Cullen
Elyse Donnelly
Logan Doshna
Anna Eriksson
Jack Lindgren
Addie Mahardy
Nicolina Masterpole
Eva Plew
Zoe Poquadeck
Sophie Smorol
Catherine Weber
Jaydin Weston
Ciara Wierbinski
Brystol Young
Dear St. Francis Xavier family:
We have all made a lot of adjustments to living and worshiping in the covid19 world. I would like to thank everyone who comes to Mass wearing their mask and practicing social distancing; both of which are so very important to keep everyone healthy and safe and to be able to continue celebrating the Mass publicly. Even though, as many of you know, I am not the most gifted when it comes to technology, I would like to remind us all that is necessary to sign up online for Mass; we usually print out the sign up on Friday for the weekend; this gives us the information necessary if, God forbid, we have to do some contact tracing. As we continue to move forward, we do so with faith, patience, love and a sense of humor.
God Bless,
Fr. Dan
RCIA - Rite of Christian Initiation
If you’re thinking about becoming a Catholic or maybe you still need Confirmation or First Communion Sacraments, please join us!
There is a new set of classes starting for RCIA. If you have any questions or would like to join us, please call the rectory.
Please remember to reach out to your church if you need assistance during these difficult times.
St. Francis Xavier – (315) 673-2531
August 23, 2020
21st Sunday in Ordinary Time
Scripture Readings
Isaiah 22:19-23
Psalm 138
Romans 11:33-36
Matthew 16:13-20
A Family Perspective by Bud Ozar – Like Jesus in today’s gospel, we are seen by others in different ways. Perhaps the name our family gives us is closest to our true identity. They know us best. Who do they say you are?
Question of the Week- Representatives of Christ
For Adults: If you were asked, how would you explain Jesus to a person who had not heard of him?
For Children: Who do you tell people Jesus is?
FRANCISCAN NORTHSIDE MINISTRIES, located at 808 North Salina St. hosts a free legal assistance clinic on Tuesday afternoons from 2-4pm. Consultation is free and confidential. For an appointment, call 315-423-9961.
Poverello Health Center also located at 808 North Salina St. is open on Wednesday evenings from 5-7pm. Services are for the uninsured. Appointments are necessary…to make an appointment call 315-423-9961.
The brick & mortar repair work on the steeple is winding down this week. While some of the localized damage was severe with multiple crumbling brick zones encountered, overall, following the re-pointing work, the tower will be in excellent condition. The upper wood-framed work is tentatively scheduled to begin the week of August 24th with a crew from the diocese performing the bulk of the construction. Based on current estimates, we are currently $75,000 short of our fund-raising goal. Thank you for your contributions to date. Your continued support is really appreciated! We understand these are difficult times and you may not be able to make a financial donation so please support us by praying for the success of our capital campaign.
Easier Giving Online Donations:
Visit www.sfxmarcellus.com
Light our Future tab
Make a Gift button
You may make a one-time contribution or initiate recurring gifts.
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RADIO AT WAR
IN THIS ISSUE
Serving Uncle Sam
War Communications
Walkie-Talkies
The Army Hour
Stars in Uniform
Women's Uniforms
Entertainment in Camps
Army, Navy, Marine, Coast Guard and Civilian Insignias
How to Display the Flag
V-Mail
Honor Roll
TOPEKA WIBW KANSAS
COLUMBIA BROADCASTING SYSTEM
Dear Folks:
This booklet, which we have called "Radio at War," was designed to show how radio communication is playing a vital role in winning the war. On the pages that follow are pictures not only of radio in service in military classrooms and on fighting fronts, but also on the home front, including staff pictures of Station WIBW.
Each individual station, the national and international networks and the radio industry as a whole, are dedicated to winning this great war. Cooperating with the many Government war agencies which have vital messages for our people and keeping the public properly informed and entertained are just two of the ways in which radio serves at home.
We have always dedicated WIBW to the service of its listeners, and in war this policy takes on added importance. We re-dedicate WIBW to this service and we Pledge ourselves without reservations to the job which lies ahead.
Cordially yours,
Ben Ludy
General Manager
Sen. Arthur Capper
In the U.S. Senate 24 years. Owner and Operator of WIBW.
Ben Ludy
General Manager of WIBW
Serving Uncle Sam
Public Service
Behind our war effort is a vast organization known as "Our Government". This government is composed of many federal agencies and officials reflecting the policies of the President and Congress. These various agencies and individuals have important missions to accomplish which require widespread understanding and cooperation.
In a Democracy - even at war - there is a limit to the effectiveness of regulations. In most instances, public acceptance must be secured. To reach our large population of 130,000,000, no medium is more effective than radio.
And radio, alert to its vital role in this part of the war effort, is generously contributing its facilities, its time, and its trained personnel to serve the government and our people.
How Radio Helps
The Record:
NATIONAL
U. S. Army Recruiting for Armed Forces
U. S. Navy Recruiting for Navy, Marines, Merchant Marine and Coast Guard
U. S. Civil Service Recruiting for War Production Workers
Maritime Commission Recruiting for Shipyards Workers
U. S. Employment Service Recruiting for War Factory Specialists
American Red Cross Recruiting for Nurses, Nurses Aids, etc.
War Production Board Production Drive Information
Office of Price Administration Price Control Information
U. S. Treasury Sale of War Bonds & Stamps
U. S. O. Campaigns for Funds
Department of Agriculture Food Conservationing, Rationing
Office of Price Administration Gas Rationing
War Production Board Rubber and Scrap Salvage
Federal Security Agency National Nutrition Drive
Office of Civilian Defense Air Raid Precautions
Department of Labor Child Welfare in Wartime
Office of Coordinator of Inter-American Affairs Information on other American Republics
War Production Board Conservation of Electric Power
Department of Agriculture Conservation of Household Equipment
REGIONAL
Department of Agriculture Grain Storage
Department of the Interior Reclamation Campaign
Department of Agriculture Relief for Farm Labor Shortage
Department of Interior Promotion of Power Programs
Department of Agriculture Promotion of supply of farm products vital to war
National Park Service Forest Fire Prevention
Department of Interior Mine Service
Each local area can add scores of items to this imposing list.
Orders from headquarters by radio as troops leave bivouac area.
Marine uses portable radio in landing operation.
Portable Army radio outfit operates on maneuvers. Note hand generator.
Reporting by radio from concealed command car. Note transmitter key on radio operator’s thigh.
Sergeant in foreground is tank crew member plotting attack on basis of information radioed from outpost.
Report on enemy aircraft is radioed to concealed artillery at rear.
OUR fighting forces throughout the world are linked to Command Headquarters in Washington by a vast network of military communication. Messages are necessarily sent in code -- for in them are the secrets of our future military operations. Our system of radio stations in the United States has been a reservoir which provided our Army and Navy with thousands of skilled specialists who now maintain our important lines of military radio communication. Meanwhile, the services are training thousands of additional men for radio duty on land, on the sea, and in the air. Today, radio is the nerve system of our military might. Crackling messages over the airways will carry the signal of the last great offensive and the first news of the final defeat of our enemies.
ON THE SEA
Some of the delicate radio equipment in a Navy radio room.
Radioman receiving message on U.S. Battleship.
Navy radio operators help to guard the sea lanes.
In the operational radio control of a Naval Air Station.
The Watch Below! Firemen report burner control readings.
Every one a radio operator.
Radio operator on Navy patrol blimp on anti-submarine duty.
Duty officer checking flight board after flight.
Radio operator on Navy bomber.
Coast patrol radio man keeps tabs on weather and directs surface ships to scene of disasters; keeps wary eye for enemy aircraft signals.
"Blind flying" by radio in ground school trainer.
School dismissed. The "desks" in a radio classroom.
Recruits receive instructions at Signal Corps Training school.
Diagram on wall aids Army Radio instruction.
Aviation cadets and student officers attend "buzzer" class.
Another class explores intricacies of radio code.
Flight instructor corrects students' errors after formation flying.
Here’s the famous walkie-talkie… “talk as you walk”.
Above: In landing operation, soldier reports back to ship.
Left: Lone sailor communicates from beach.
Lower left: The Marines have landed! ‘Nuf said.
Walkie Talkie on skis going up hill.
Two-way hook-up at message center.
Pack radio. Can be removed and operated on ground.
1. General View Field Transmitter, Power Unit and Antenna.
2. Battery of code keys at message center.
3. Radio-equipped Army Command Car.
4. Close-up of Army Field transmitter.
5. Motorcycle and side-car equipped with Radio.
A MILITARY MISSION
On April 5, 1942, the United States Army started a new kind of military operation. For the first time in history, the War Department was directly sponsoring and producing a radio program with a definite military objective -- "The Army Hour". Since then, the official "Army Hour" has established itself as an integral part of the global fight of the United Nations against the Axis.
Through the "Army Hour", which is broadcast each Sunday, America and the whole world is getting a weekly view of the progress of the war and how it is being fought. The program reaches to all parts of the globe to tell the story of the United Nations fight, with buck privates telling their important role as prominently as the top-ranking military chiefs.
When listeners heard the chatter of machine guns, they were hearing live bullets fired by the gun crew in this photo. Radio microphones enabled the listener to hear also, the bullets striking the target.
From the West Point air training field, the Army Hour introduced J. H. Weikert, Captain Donald Thurmar and Cadet Vincente Lim.
Bill Stern, famous sports commentator, describes how it feels to look through a bomb sight and pull the release that will send bombs from U.S. planes to blast the enemy.
Behind the scenes in any Army Hour broadcast is Art Feldman, the man who gives the signals and makes the check-ups on as high as 25 "switches" on a single program. He is in touch with each remote point, foreign or domestic, until each is off the air.
A message by Secretary of War Henry Stimson inaugurated the first official War Department radio program.
Lt. Gen. Hugh A. Drum, commander of the First Army, addressed an Army Hour audience. On the same program were: Lt. Generals McNair, Lear, Krueger, and DeWitt.
Instrumental in planning and arranging each War Department program is Lt. Col. E. M. Kirby, Col. Ernest R. DuPuy, Major General A. D. Surles and Col. R. B. Lovett.
Private Joe Louis, heavyweight champion, addressed Army Hour listeners, with Col. Ned J. O'Brien, Art Flynn and James Braddock.
The first Filipino Battalion in the United States Army staged a demonstration for Army Hour listeners.
WAR INFORMATION
With news, roundtables, speeches, forums, special events and dramatic programs, radio is keeping Americans the most informed people in the world. Today, more than ever before, Americans demand all the facts except those which will give aid and comfort to the enemy. From these truths come American unity and decision. Radio's task is to bring this information to our people as quickly and as clearly as possible.
ON THE AIR
When they are not too busy learning the methods of warfare, the Army, Navy and Marine Corps can present radio programs with a professional flavor, designed to entertain and inform the folks back home. Hundreds of radio entertainers, musicians, writers, announcers, production men and specialists are in the service, and they welcome the opportunity to resume association with their former civilian pursuits. Meanwhile, radio listeners, they make it possible for friends and relatives to visit camps and training stations, without moving away from the loud-speaker. Through this medium, radio can claim special distinction for building and maintaining our strong morale.
A portable organ, makeshift stage and soldiers entertain during maneuver rest periods.
Hawaiian soldiers find time for broadcast.
Trained Army Public Relations officers proved they could operate a radio station they "captured" during a 1941 maneuver.
Sailors compete in a quiz broadcast while buddies listen in audience.
Microphone catches formal guard mount at West Point.
West Point Band plays for radio in Cullom Hall.
Tyrone Power, of screen and radio, is sworn in as a private in the Marine Corps by Maj. Wm. Howard, U.S.M.C.
Wayne Morris, called to active duty with the Naval Aviation Cadet Selection Board, interviews flying cadet applicants.
Clark Gable is now serving with the United States Army Air Forces.
Rudy Vallee, of the Coast Guard, reporting to Lieut. M.A. Sturges.
After his driving chores, Robert Young joins the chow lines and loads up his plate.
This Army Air Corps looks on as Sgt. (honorary) McCarthy greets his friend James Stewart (right). Behind Charley is Edgar Bergen.
Becoming an army officer doesn't keep Glenn Miller from being a favorite with autograph seekers.
WIBW HONOR ROLL
FORMER EMPLOYEES
NOW IN THE SERVICE OF THEIR COUNTRY
Bill Stotts, Radio Technician, Navy (Operator)
Pvt. Dean Bailey Air Force (Salesman)
Lt. (jg) Hilton Hodges Navy (Announcer)
Merwin Startup Radio Technician, Navy (Operator)
Capt. Marion Beatty Army (Salesman)
Cpl. Victor Jacobs Army (Engineer)
Lt. Dave (Everette) Wisner Army (Announcer)
Coxswain Art Holbrook Coast Guard (Special Events)
Lt. Col. Joe Nickell Army (News Commentator)
Pvt. Jim Reed Army (Announcer)
Elmer H. Curtis, announcer, was influenced to get behind a "mike" after winning an international oratorical contest in 1931. He keeps making records--now on the way to his 2,500th consecutive Lee Food "Noon News" broadcast.
Iowa, Nebraska and Kansas have been home states for Trajan Clane "Gene" Shipley, former theatre owner, "ham" radio amateur, saxophonist and banjoist. Gene is one of the Midwest's most widely-heard market reporters.
"Henry and Jerome," those two masters of pleasing harmony, started in radio when the industry was a mere infant. They complete their 15th year on the air as a team November 11, 1943. Singly, Henry Peters has been broadcasting for 17 years and Jerome DeBord 21 years.
That happy cowboy, Clyde Mason, wears the fanciest horse-ridin' duds! His career as a radio warbler has taken him to the networks; also throughout Oklahoma on Defense Bond sales with Singer Gene Autry.
Starting the day out with a bang, Mondays thru Saturdays, is the "Daybreak Jamboree" gang. Here they are: left to right is Colonel Alex Zander Combs, champ war-bond salesman; Earl Bledsoe, Ralph Hunt, Ezra Hawkins and Bob Dick. Down front is Glenn Osborn. Morning after morning they're as wide awake as the rooster's crow.
Music and Program Director of WIBW, as well as pianist and good sport, is Maudie Shreffler. She is a member of the National Academy of Music; goes to bat for her talent staff whenever she can; and, here's the feminine side, likes to crochet. Had two offers from Columbia Network, but turned them down to remain with WIBW.
Fresh from the mornin' mail is a newspaper that Ezra Hawkins reads to his "Bar Nothing Ranch" hands. Ezra, with paper, pipe and whiskers, is surrounded by (left to right) Glenn Osborn, Bob Dick and Clyde Mason. Ole Livgren, busy as usual, had to miss the picture. Ezra's "Bar O" is one of the gayest and liveliest morning shows you ever "heerd" of.
Above: M. A. "Bill" Bryan broke into radio at WIBW. Announces special events. Served as "House Reader" at 1943 regular session of Kansas Legislature.
Above Left: Merle Housh was familiar to WIBW listeners way back in 1928, as one of the two "Barnyard Songsters." He left for a time but is now back as "Henry Hornsbuckle," proprietor of "Henry's Exchange."
Left: He lived on a farm until his college days, then worked in the harvest fields during summer vacations, and was a 4-H Club member. He's Don Hopkins, the man behind the microphone for the 7 a.m. news.
Below left: From Kentucky (but not a colonel as yet) is Bill Haley, singer, announcer, master-of-ceremonies. He and his wife, Hazel, are comparative newcomers to WIBW, and mighty popular right off!
Below: Actor, Comedian, Singer, Announcer Homer Cunningham was one of the first to originate a radio black-face act, working as many as five voice-characters by himself. How does he get along so well with everyone? His motto: "I don't hate nooo-body."
Bushels of hints--a very special and helpful kind--are broadcast Mondays thru Fridays on popular "Henry's Exchange." The gang's all here, and left to right they are Smiling Merle "Henry Hornsbuckle" Housh (proprietor), Clyde Mason, Fred "Axelbender" Warren sitting in front, Amato "Blackie" Guariglia, Auburn-haired Virginia Lee, Clark Wayne, Col. Combs, Alice Joyce Hensroth and Heine Haynes. Tune in and help yourself to helpful hints. Henry's new "Hint Book" is pictured at the left.
Thirty-five years of newspaper work is part of the experience of Olaf Soward, WIBW's afternoon and evening news commentator. Independent radio surveys in the middle-west have turned a bright spotlight on Soward. Has an unusual hobby of studying history of modern languages. A well-received public speaker, too.
One of the nation's best-known sports authorities, recognized coast-to-coast, is E.C. "Ernie" Quigley, who broadcasts sports four times weekly. He is public relations director of the National Baseball League; has won a mile of ribbons with his champion hogs.
Here's the little man with the great big name--Amato Guariglia, who is known as "Blackie" to all of us at the studio and on the air. In this shot, he has surrounded himself with his working tools--the organ, vibraphone and accordion.
Out of the heart of the Missouri Ozarks came Ralph Hume to join with St. Louis-born Earl Bledsoe to form one of radio's most talented folk-singer teams. "Ralph and Earl" have the hill country rhythm that give their numbers a genuine fascination.
Dude Hank peeks into the radiator to stock up against government water rationing. His real name, of course, is Roy Carlson, WIBW music arranger and trumpeter who has been 'round and 'round in music circles, including the networks, dance bands, and theaters.
Oct. 12, 1935, is the starting date at the "Voice of Kansas" station for Edmund Denney, tenor. An amateur contest one year earlier won him a birth in radio, and his star has risen continuously ever since.
In action with that smooth baritone voice of his is "The Shepherd of the Hills," whose career has led to Europe's radio stations and back. He got his start at WIBW. Name's William Wilhite.
Clark Wayne's first ambition was to be a musician, and today he's ranked with the best on the guitar. His second ambition must have been to be versatile, for he also performs great on the violin, bass, and mandolin. His hobby: automobile mechanics.
All set for another song and (by the looks of the dresses) the next dance, "Sally and Sue" smile at the photographer from above the curling stair rail in WIBW's home-studio. Sisters, the two are announced as those "sweet-singing Ozark sweethearts."
He's got a lotta brass! None other than Fred "Axelbender" Warren, who is seen cooking up a good excuse for missing a program. Axelbender's comic quips and musical versatility give him steady popularity with fans.
"The Colonel" is as jovial as this smile he's smiling. It's hard to best Colonel Combs, champ fiddler, with a joke, but the boys kid him for being hired on "April Fool's Day" 1934. It was no joke, though, for he proved himself to be a mighty popular guy with dialers.
When a piano fell on Ardis Charles, she decided then and there to take up music! It actually happened. As a contralto, she is winning honors. Pet like: her mom's banana-cream pie.
Tamed long enough to get a photo snapped are the "Corn County Wildcats," revealed in their Sunday finery. Reading to the right we find Amato "Blackie" Guariglia and his classic washboard noise-makers; Henry "Heinis" Haynes, the tuba wrestler; Fred "Axelbender" Warren, sending out on the "licorice stick"; and Roy "Dude Hank" Carlson, trumpet toter. With the villain's trade-mark (moustache) and the banjo down front is Clark Wayne Arbogast.
Right: Otho "Hoppi" Corbin is a Missouri lad who has played in opera companies, theatres, and the 7th Regiment Cavalry Band. His cello is always played with great skill. Also plays (but never seems to get around to them) the trombone, saxophone and melophone.
A violinist par excellence is Jud Miller, poised for action atop the studio-home stair rail. Jud's career is one of theater pits, dance orchestras, and radio. Has a keen sincerity in what he does and in what he believes.
All aglow is one of science's latest creations: Ole Livgren's "Olebox." He invented it himself. It is a combination Solovox and accordion, enabling him to play the Solovox with the Accordion keys or play them separately.
To the left above is his songbook, containing "On the Isle of Pine," "In My Dear Old Kansas Home," and "To the Red, White and Blue."
This is the entire staff, with the exception of a few, of Station WIBW. All of these folks are behind the programs heard over "The Voice of Kansas" at 580 kilocycles, Topeka. Unless the person is otherwise identified, the department to which he belongs is indicated as follows: (A) Announcing, (E) Engineering, (S) Secretarial, (T) Talent.
Left to right: 1st row--Jerome DeBord (T); Henry Peters (T); Ralph Hunt (T); Earl Bledsoe (T); Glenn Osborn (T); Sally Greeno (T); Sue MacLane (T); Alice Joyce Hensroth (T); Ole Livegren (T); Bob Dick (T); Edna Hann (S); Elsie Shideler (S); Maudie Shreffler, Music Director; Clark Wayne (T); Amato Guariglia (T); and Bill Haley (A) and (T).
2nd row--Virginia Lee (T); Rosemary Hannah (S); Zelda Kappelman (E); Ananora Shields (E); Barbara Colvin (E); Marjorie Mitchell (S); Mildred Rankin, Head of Mail Room; Frankie Anderson (S); Lucile
Macleod (S); Marjorie Carlson (S); Louise Zimmerman (S); Kathryn Young, War Program Manager; Ethel Alexander (S); Opal Hartley (S), Myrtle Denney (S); Margaret Hitz (S), and Germaine Ravese (S).
3rd row--Mason Coppinger (E); Bill Bryan (A); James McGinnis (T); Gene Shipley (A); Ernie Quigley, Sports Editor; Ed Keene (T); Homer Cunningham (A) and (T); Don Hopkins (A); Bill Smith and Courtney Irving, Maintenance Department; Hoppi Corbin, Music Librarian; Bill Wilhite (T); and Jud Miller (T).
4th row--Harold Ruff, in charge of Engineering; Roy Carlson (T); Heinie Haynes, (T); Olaf Soward (A); Col. Alex Zander Combs (T); Fred Warren (T); Ben Ludy, General Manager; Edmund Denney (T); Clyde Mason (T); Ardis Charles (T); Maureen Dawdy (T); Elsa Schlangen (T); Lois Kalish (S); Irvin Lehman (E); and Charles Hill, Publicity Director.
"Presenting: the organ artistry of Elsa!" Elsa Schlangen is at home equally well with the piano or organ. Likes to play for listeners and we know they like to hear Elsa play.
Three swell girls--and hear how they play--are Maureen Dawdy, violinist at left; Hazel Haley, center, vocalist-guitarist formerly of network programs; and Alice Joyce Hensroth, accordionist.
Busy with the bills is Ernest Thorn, auditor for WIBW. Here's one man who knows his figures! Has plenty of friends at the station, in spite of the fact that he makes out the paychecks.
Right--Smiling as though he's glad the job is over is Charles Hill, who is publicity director of WIBW and editor of this booklet.
For distinguished services rendered in behalf of the War Savings Program this citation is awarded to
Ben Ludy
Given under my hand and seal on January 29, 1943
Henry Morgenthau Jr.
Bob Dick, left, and Glenn Osborn put away their guitars after a session on the air. They're whizzes on those strings, too. Bob, red-haired, was born near Allen, Kansas, and Glenn's birthplace is Garnett.
Left: This certificate was a gift from the United States Treasury to our General Manager, Ben Ludy, who heads the station's bond selling campaigns.
1 Secretary Lois Kalish, left, buys war stamps from Receptionists Barbara Long, center, and Rosemary Hannah in lobby.
2 Bottom to top: Clark Wayne, Edmund Denney and the Shepherd of the Hills--the "Harmony Hicks."
3 "Ah's done been framed!" exclaims "Hambones," blackface comic, as he grins away others cares.
4 Alan Sells, staff photographer responsible for most of the shots in this booklet.
5 One to make good measure: the photographer snapped the "Crossroads Sociable" crowd.
6 Secretaries off duty! Lower row: Louise Zimmerman, Elsie Shideler, Edna Hann. Top row, Kathryn Young, Marjorie Carlson.
7 Birthplace: Alabama. Voice rating: Low Alto. Hair: Red like Flaming Sun. She's Virginia Lee!
8 Hoppi Corbin, music librarian, picks out a number for one of the performers.
AT THE WIBW CONTROLS
Lower: These girl operators are hard at work feeding programs to the transmitter, riding gain, running disks. Left is Barbara Collins at the controls, then Anantra Shields setting up a transcription, and Zelda Kappelman, checking with the transmitter engineer. Right: The engineers who keep WIBW on the air hours daily. Left to right are Mason Coppinger, Harold Ruff (engineer in charge) and Irvin Lehman in front of the modern transmitters.
FROM THE LISTENERS
Below left: 519,018 pieces of mail in 1942! Top row, left to right, Margaret Hitz, Mildred Rankin and Frankie Anderson. Lower row, Myrtle Denney and Marjorie Mitchell. Picture at Right Below: They helped, too! Left to right are Ethel Alexander, Alice Kaspar, Germaine Ravese, Opal Hartley, and Lucile Macleod. Inset: Mildred Rankin, head of Mail Room.
Master of the quick quip, Fred Allen is curtain-raiser for "The Texaco Star Theatre."
"Take It or Leave It" suggests Phil Baker, when he presents the quiz of that title Sunday nights.
Original interview-type program, "Vox Pop" is still headed by one of its two creators, Parks Johnson.
Left--Star of radio concert and opera is Gladys Swarthout, vocalovey of "The Family Hour."
Right -- "No, George, you're wrong. It's this way," corrects Gracie as she checks hubby on the "Burns and Allen" series.
The lovable "River's End" medico, "Dr. Christian," is portrayed by one of Hollywood's most famous, Jean Hersholt.
"When the Moon Comes Over the Mountain" we'll still be listening to Kate Smith, who made the song of that title stick in our hearts.
Left--Whoops! Lester Damon and Claudia Morgan are sleuths in "The Adventures of the Thin Man."
Right--Foreign correspondent, author of "Berlin Diary", and now news analyst Sunday afternoons --William L. Shirer.
Left--Idol of millions! Radio-ranging, broncho-busting troubadour of wide open spaces! Sergeant in the Army Air Forces--Gene Autry.
Right--Eddie Firestone, Jr., is ornery enough as Joey Brewster of "The Brewster Family."
A MISS who is a HIT of "Your Hit Parade" is Joan Edwards, warbler and pianist.
Andre Kostelanetz conducts the versatile classical orchestra heard on "The Pause That Refreshes on the Air."
No. 1 starter of the nation's toe-tapping is Harry James, whose No. 1 dance band is heard thrice-weekly.
Conrad Nagel is master-of-ceremonies of radio's newest "publications"—the "Radio Reader's Digest."
Dagwood and Mr. Dithers mix and "Blondie" unmixes 'em. She's Hollywood's Penny Singleton.
"Mayor of the Town," and a great one, is Lionel Barrymore.
Producer extraordinary! Cecil B. DeMille casts and directs the dramatic "Lux Radio Theatre."
Al Jolson (left) comes to the point of a story he is telling his Tuesday-night co-star, Monty Woolley.
Milton Berle's comicalities are wrapped up in all sizes on "The Milton Berle Show."
Possible! So exclaims Harmonica-Playing Herb Shriner after one of his tall ones on the "Camel Comedy Caravan."
EVERY SATURDAY NIGHT
"ON THE AIR"
WIBW'S FAMOUS "KANSAS ROUNDUP"
A FAVORITE OF MIDDLE WESTERNERS FOR NINE YEARS
HERE'S THE TALENT STAFF RESPONSIBLE
Robert Montgomery, U.S. Naval attache in London, salutes 8 year old bomb victim, Alan Locke.
Gene Autry (second from left) finds time to entertain his buddies while training for the Air Corps.
Wayne King now directs army activities instead of "The Waltz You Saved For Me".
Dave Breger (Left), creator of the "Private Breger" cartoons, cuts a piece of birthday cake for his buddy, Ezra Stone ("Henry Aldrich").
Robert Allen (left) gives Drew Pearson the real inside information on army life.
Charles (Buddy) Rogers (right) reports to Lt. Comdr. C.C. McCauley prior to taking up his flying duties.
Jimmy Fidler looks on as Joan Davis (heard with Rudy Vallee) bids goodbye to Jimmy Henaghan, Fidler's ace reporter.
Walter Winchell of the United States Naval Reserve is "back in a flash with a flash".
Naval cadets and sailors sing and play for radio audience.
A corner of the barracks serves as rehearsal room for this "jive" group.
All sergeants are not "hard-boiled". This one burlesques a "home-makers' hour", discussing a topic of child apparel that doesn't seem to impress the young admirers.
Soldiers fresh from field duty accompany Service Club worker in broadcast.
Radio network correspondents, wearing prescribed uniforms, report from maneuver areas.
Aviation cadets at Randolph Field have organized this Glee club for radio appearances.
Soldiers on duty in Washington, D.C. boast this Glee club.
Buddies gather 'round to enjoy some boogie woogie on a Service Club piano.
Maj. General Hugh Drum faces a battery of microphones.
No spot is too tough for radio special events men. Here's one following the Army engineers during a river crossing.
Entertainment aboard ship enroute to Australia.
Sailors at Pensacola rehearse before broadcast.
A soldier audience at an open air broadcast as seen by performing artists.
The Famous U.S. Marine Corps Band heard on many broadcasts.
KEEPING IN TOUCH
Wherever they are, in training or in action, U.S. fighting forces look to radio to maintain their association with "home" -- it may be the voice of a friend, word from the home town or news from the good, old U.S.A. It all serves the same purpose for the service men who have no intentions of losing contact with things that were familiar before the war interrupted their lives. Radio does this job, too.
Winter-clad soldiers anchor cable for antenna in far northern base.
Radio and games in the barracks at a Naval Air Station.
Concentrated listening by Army men at a railroad siding.
Short-wave listening at an outpost in Puerto Rico.
U.S. NAVY INSIGNIA OF RANK AND SERVICE DIVISION
OFFICERS' SHOULDER AND SLEEVE INSIGNIA
ADMIRAL VICE-ADMIRAL REAR ADMIRAL CAPTAIN COMMANDER LIEUT COMMANDER LIEUT LIEUT (JG) ENSIGN CHIEF WARRANT WARRANT OFFICER
OFFICERS' CORPS DEVICES
LINE MEDICAL DENTAL SUPPLY CHAPLAIN (Christian) CONSTRUCTION CIVIL ENGINEERING BOATSWAIN MACHINIST PAY CLERK
ELECTRICIAN GUNNER CARPENTER RADIO ELECTRICIAN PHARMACIST
SERVICE STRIPES
PETTY OFFICERS' RATING MARKS
CHIEF FIRST CLASS SECOND CLASS THIRD CLASS CHIEF
Each service stripe worn on the left sleeve below the elbow indicates completion of a four-year enlistment. After serving 15 years with good conduct, gold stripes are worn. Enlisted men's rating insignia become gold after completing three enlistments with good conduct. Petty officers' rating marks are worn on dress uniforms; chief petty officers' are worn on white.
ENLISTED MEN'S SPECIALTY MARKS
BOATSWAIN'S GUNNER'S TURRET CAPTAIN QUARTER-MASTER PAINTER PHOTOGRAPHER AVIATION ORDNANCE TORPEDOMAN CARPENTER'S MATES AVIATION METALS SMITH COOK FIRE CONTROL MAN YEOMAN PHARMACIST'S MATES GENERAL UTILITY ELECT. COMMISSARY MATE STEWARD RADIO MAN SIGNAL MAN BLACKSMITH'S MATE MACHINIST'S MATE BOMBSIGHT MECHANIC GUN CAPTAIN SEAMAN GUNNER RIFLE NAVY GUNNER EX-APPRENTICE RIFLE SHARPSHOOTER PARACHUTE MAN MASTER DIVER DIVING FIRST CLASS OFFICERS STEWARD SUBMARINE RIGID AIRSHIP GUN POINTER FIRST CLASS MASTER HORIZONTAL BOMBER
CHIEF PETTY OFFICERS AND PETTY OFFICERS
The rating badges are worn on the sleeve between shoulder and elbow. Petty officers, seaman branch, wear rating badges on the right arm; other petty officers wear them on the left arm.
CAP DEVICES
OFFICER WARRANT MIDSHIPMAN CHIEF PETTY OFFICER NAVY NURSE STORE KEEPER MUSICIAN BUGLER AEROGRAPHER
POCKET OR BREAST INSIGNIA
NAVAL AVIATOR AVIATION OBSERVER SUBMARINE MERCHANT MARINE PARACHUTIST
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U.S. ARMY INSIGNIA OF RANK AND SERVICE BRANCH
OFFICERS' INSIGNIA OF RANK
GENERAL LIEUT. GENERAL MAJOR GENERAL BRIG. GEN. COLONEL LIEUT. COLONEL MAJOR CAPTAIN FIRST LIEUT. SECOND LIEUT. CHIEF WARRANT OFFICER WARRANT OFFICER
CAP DEVICES
OFFICERS' LAPEL OR COLLAR INSIGNIA
U.S. OFFICERS WAAC ADJUTANT GENERAL AIDE TO GENERAL AIR FORCES CAVALRY CHAPLAINS (CHRISTIAN) CHAPLAINS (JEWISH)
GENERAL STAFF INFANTRY INSPECTOR GEN. JUDGE ADVOCATE COAST ARTILLERY ENGINEERS FIELD ARTILLERY FINANCE DEPARTMENT
CHEMICAL WARFARE SERVICE MEDICAL CORPS MEDICAL ADM. NURSE CORPS MILITARY POLICE NAT'L GUARD BUREAU ORDNANCE QUARTERMASTER CORPS
ARMORED FORCE SIGNAL CORPS OFFICERS NOT MEMBERS OF A BRANCH WARRANT OFFICERS U.S. MILITARY ACADEMY ARMY TRANSP. CORPS ARMY BAND ARMY SPECIALIST CORPS
NON-COMMISSIONED OFFICERS' INSIGNIA
MASTER SERGEANT FIRST SERGEANT TECHNICAL SERGEANT STAFF SERGEANT TECHNICIAN 3RD GRADE SERGEANT TECHNICIAN 4TH GRADE CORPORAL TECHNICIAN 5TH GRADE PRIVATE FIRST CLASS
BREAST INSIGNIA
COMMAND PILOT SENIOR PILOT PILOT SERVICE PILOT
GLIDER PILOT LIAISON PILOT NAVIGATOR BOMBARDIER
AIR CREW MEMBER COMBAT OBSERVER FLIGHT SURGEON PARATROOPS
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U.S. ARMY
INSIGNIA OF
CORPS AND DIVISION
THE GENERAL COMMANDS
HEADQUARTERS
AIR FORCES GROUND FORCES SERVICE OF SUPPLY
THE ARMIES
FIRST ARMY, SECOND ARMY, THIRD ARMY, FOURTH ARMY
SERVICE COMMANDS
FIRST SECOND THIRD FOURTH FIFTH SIXTH SEVENTH EIGHTH NINTH
ARMY CORPS
FIRST SECOND THIRD FOURTH FIFTH SIXTH SEVENTH EIGHTH NINTH
DIVISIONS
ELEVENTH TWELFTH THIRTEENTH FOURTEENTH 1st 2nd 3rd 4th 5th
6th 7th 8th 9th 26th 27th 28th 29th 30th
31st 32nd 33rd 34th 35th 36th 37th 38th 39th 40th 41st
42nd 44th 45th 76th 77th 78th 79th 80th 81st 82nd
83rd 84th 85th 88th 89th 90th 91st 92nd 93rd 94th
96th 98th 99th 100th 100th FRONTIER DEFENSE SECTORS 102nd 103rd 104th HAWAIIAN DIV. FIRST CAVALRY DIVISION
ARMORED FORCE
HQ & HQ COMPANY 1st CORPS 4th DIV. NEW ENGLAND NEW YORK PHILADELPHIA CHESAPEAKE BAY SOUTHERN COASTAL PACIFIC COASTAL PANAMA CANAL DEPT. HAWAIIAN DEPT.
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U.S. MARINES
INSIGNIA OF RANK AND SERVICE
OFFICERS' INSIGNIA OF RANK
LIEUTENANT GENERAL MAJOR GENERAL BRIGADIER GENERAL COLONEL LIEUTENANT COLONEL MAJOR CAPTAIN FIRST LIEUTENANT SECOND LIEUTENANT WARRANT OFFICER
CAP DEVICES
OFFICER SERGEANT MAJOR FIRST SERGEANT PLATOON SERGEANT MASTER TECHNICAL SERGEANT TECHNICAL SERGEANT STAFF SERGEANT SERGEANT CORPORAL PRIVATE FIRST CLASS
ENLISTED MAN
NON-COMMISSIONED OFFICERS' INSIGNIA
DEPARTMENTAL INSIGNIA
ADJUTANT & INSPECTOR'S DEPT. QUARTERMASTER'S DEPT. PAYMASTER'S DEPT. BRIG. GENERAL'S AIDE AVIATION CADET CHIEF GUNNER BAND LEADER
COAST GUARD
RANK AND SERVICE
CAP DEVICES
CADET OFFICER WARRANT OFFICER U.S.C.G.
OFFICER'S SHOULDER INSIGNIA
REAR ADMIRAL CAPTAIN COMMANDER LIEUT. COMMANDER LIEUT. COMMANDER LIEUT. (JG) ENSIGN CHIEF WARRANT WARRANT
OFFICER'S SLEEVE INSIGNIA
REAR ADMIRAL CAPTAIN COMMANDER LIEUT. COMMANDER LIEUT. COMMANDER LIEUT. (JG) ENSIGN CHIEF WARRANT WARRANT FIRST CLASS CADET SECOND CLASS CADET
ENLISTED MEN'S SPECIALTY MARKS
AVIATION MACHINIST'S MATES AVIATION PILOTS AVIATION METALSMITHS BUGLERS RADIO MEN PHOTOGRAPHERS PRINTERS COOKS CARPENTER'S MATES QUARTER-MASTERS SIGNAL MEN GUNNER'S MATES SWAIN'S MATES, COXSWAINS COMMIS- SARY STEWARDS PHARMACIST'S MATES YEOMEN MACHINIST'S MATES, WATER TENDERS BAND MASTER MUSICIANS FIRST CLASS SECOND CLASS
CADETS' SHOULDER INSIGNIA
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AMERICAN WOMEN IN UNIFORM
The figures presented on this page show American women in the uniforms authorized for their various types of war work. Never before in the history of the country have women played such important parts on the war front and the home front and enlisted in such numbers as today. This wholesale volunteering for war work releases large numbers of men for the actual business of fighting.
Member of the WAAC - Women's Army Auxiliary Corps.
Left: Member of Women's Auxiliary Ferrying Squadron (WAFS)
Right: Member of the WAVES - Women Appointed for Volunteer Emergency Service (Women's Reserve of the Naval Reserve)
Member of American Women's Voluntary Services.
Red Cross Worker
Right: Army Nurse
Left: Navy Nurse
Civilian defense worker.
Member of Red Cross Production Corps.
Member of Red Cross Motor Corps.
Red Cross Nurse
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Protecting Life and Liberty: The Constitutionality and Necessity of Civil Commitment of Sexual Predators
Jennifer Ann Smulin
Follow this and additional works at: https://via.library.depaul.edu/law-review
Recommended Citation
Jennifer A. Smulin, Protecting Life and Liberty: The Constitutionality and Necessity of Civil Commitment of Sexual Predators, 52 DePaul L. Rev. 1245 (2003)
Available at: https://via.library.depaul.edu/law-review/vol52/iss4/11
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PROTECTING LIFE AND LIBERTY:
THE CONSTITUTIONALITY AND NECESSITY OF
CIVIL COMMITMENT OF SEXUAL PREDATORS
INTRODUCTION
Before the revival of “sexual psychopath” laws less than a decade ago, the State of Washington was forced to release Earl Shriner from prison.\(^1\) Shriner’s criminal tirade spanned more than two decades.\(^2\) At the age of sixteen, he killed a schoolmate.\(^3\) He was later released from a mental institution, only to kidnap and rape two teenage girls.\(^4\) While incarcerated for those acts, Shriner told an inmate of his desire to own a van with cages in order to molest, torture, and murder children.\(^5\) Anxiously awaiting his release from prison, Shriner kept a diary detailing his criminal plans of torture and murder.\(^6\)
Shriner was not eligible for civil commitment under Washington’s general civil commitment law, which required mental illness and a recent overt act.\(^7\) So, after serving a ten-year sentence, the state released Shriner, freeing him to commit his most brutal crime yet.\(^8\) He kidnapped, raped, strangled, and then sexually mutilated a seven-year-old boy who was riding his bike around the neighborhood.\(^9\)
Shortly after the citizens of Washington were shocked by this unthinkable crime, the government acted to ensure this type of brutality never happened to one of its children again.\(^{10}\) Washington enacted the first modern law providing for the involuntary civil commitment of sexual predators.\(^{11}\) Several state legislatures have followed suit, motivated largely by the public’s concern for the protection of innocent children.\(^{12}\)
---
1. Barry Siegel, *Locking Up Sexual Predators*, L.A. Times, May 10, 1990, at A1.
2. Deborah L. Morris, *Constitutional Implications of the Involuntary Commitment of Sexually Violent Predators: A Due Process Analysis*, 82 Cornell L. Rev. 594, 611 (1997).
3. *Id.*
4. *Id.*
5. Siegel, *supra* note 1, at A1.
6. *Id.*
7. *Id.*
8. *Id.*
9. *Id.*
10. *See* Seling v. Young, 531 U.S. 250, 254 (2001).
11. Wash. Rev. Code Ann. §§ 71.09.010 - 79.09.902 (1992 & Supp. 2003).
12. *See* Seling, 531 U.S. at 254 (noting that the Washington State Community Protection Act was enacted in response to citizens’ concerns about laws dealing with sexually violent offenders).
Several scholars have sharply criticized these laws as unconstitutional.\textsuperscript{13} In fact, the majority of commentary available on the subject asserts that these laws are fundamentally opposed to the American notion of rights to “life and liberty.”\textsuperscript{14} This Comment argues that the contrary is true: these laws protect and defend the rights of society to life and liberty. These laws, though imperfect, are a necessary step toward preventing brutal sexual crimes from being repeated by individuals who escape the system.\textsuperscript{15}
Part II of this Comment begins by describing the problem of sexual violence in America.\textsuperscript{16} Part II also examines the solution of civil commitment posed by many states in response to this group of predators\textsuperscript{17} and provides the United States Supreme Court’s position on the constitutionality of these sexual predator laws.\textsuperscript{18} This section also includes a discussion of the Kansas Supreme Court’s and United States Supreme Court’s decisions in a recent sexual predator case, \textit{In re Crane}. Part III offers an analysis of judicial decisions in this area of law and addresses common arguments opposing civil commitment.\textsuperscript{19} Finally, Part IV explores the impact of the United States Supreme Court’s recent decisions on the future of civil commitment of sexually violent predators.\textsuperscript{20}
\section*{II. Background}
In order to understand why special laws are needed to address the problem of sexually violent predators, some explanation is required. This section will briefly discuss the problem of sexual violence in
\begin{footnotesize}
\begin{enumerate}
\item See Dawn J. Post, \textit{Preventative Victimization: Assessing Future Dangerousness in Sexual Predators for Purposes of Indeterminate Civil Commitment}, 21 \textsc{Hamline J. Pub. L. \& Pol’y} 177, 180 (1999).
\item \textit{Id.} at 180 (noting that over 150 articles have been published opposing this type of commitment legislation). For opposing arguments, see generally Kimberly A. Dorsett, \textit{Kansas v. Hendricks: Marking the Beginning of a Dangerous New Era in Civil Commitment}, 48 \textsc{DePaul L. Rev.} 113 (1998); Grant H. Morris, \textit{The Evil That Men Do: Perverting Justice to Punish Perverts}, 2000 \textsc{U. Ill. L. Rev.} 1199; Eric S. Janus, \textit{Foreshadowing the Future of Kansas v. Hendricks: Lessons From Minnesota’s Sex Offender Commitment Litigation}, 92 \textsc{Nw. U. L. Rev.} 1279 (1998); Madelyn J. Daley, Comment, \textit{Do Sexually Violent Predators Deserve Constitutional Protections?}, 23 \textsc{S. Ill. U. L.J.} 715 (1999); Cynthia A. King, \textit{Fighting the Devil We Don’t Know: Kansas v. Hendricks, A Case Study Exploring the Civilization of Criminal Punishment and Its Ineffectiveness in Preventing Child Abuse}, 40 \textsc{Wm. \& Mary L. Rev.} 1427 (1999).
\item See Katie Isaac, \textit{Kansas v. Hendricks: A Perilous Step Forward in the Fight Against Child Molestation}, 35 \textsc{Hous. L. Rev.} 1295, 1330 (1998).
\item \textit{See infra} notes 21-34 and accompanying text.
\item \textit{See infra} notes 35-53 and accompanying text.
\item \textit{See infra} notes 54-180 and accompanying text.
\item \textit{See infra} notes 181-251 and accompanying text.
\item \textit{See infra} notes 252-265 and accompanying text.
\end{enumerate}
\end{footnotesize}
America and will examine the sexually violent predator laws in detail. Supreme Court decisions on the subject will also be discussed in order to provide a background upon which to proceed with the legal discussion of these laws.
A. The Problem of Sexual Violence in America
The problem of sexual violence in our society has grown to alarming numbers. In 1995 alone, almost 355,000 rapes and sexual assaults were reported in America.\textsuperscript{21} Surprisingly, this number only includes those reports by victims over the age of twelve\textsuperscript{22} and is estimated to be significantly smaller than the number of actual assaults, given the vast underreporting unique to sexual crimes.\textsuperscript{23} In 1994, nearly 100,000 inmates were incarcerated for rape or sexual assault, almost half of them for victimizing children.\textsuperscript{24} In addition, 134,000 other sex offenders were either on probation or parole that year.\textsuperscript{25} From 1980 to 1994, sex offenders was the fastest-growing category of violent criminal.\textsuperscript{26}
Aside from the severity of the problem, sex offenders as a group pose particular problems for the traditional criminal justice system.\textsuperscript{27} For example, although imprisoning sex offenders may satisfy the retribution goal of incarceration, the goal of deterrence is often not achieved by time served in prison.\textsuperscript{28} This is because many sex offenders are affected by a mental abnormality or illness, which inhibits their self-control and thus makes deterrence unlikely.\textsuperscript{29}
Upon re-entering society after incarceration, sex offenders are much more likely to repeat a sexual crime than any other type of felon
\begin{footnotes}
\item U.S. Dep't of Justice, Bureau of Justice Statistics, Sex Offenses and Offenders 109 (1997) [hereinafter Sex Offenses and Offenders]; see also U.S. Dep't of Justice, FBI, Uniform Crime Reports 24 (1999).
\item See Sex Offenses and Offenders, supra note 21.
\item Michael G. Planty & Louise van der Does, Megan's Laws Aren't Enough, Wall St. J., July 17, 1997, at A22.
\item See Sex Offenses and Offenders, supra note 21, at 15; Lawrence A. Greenfield, U.S. Dep't of Justice, Bureau of Justice Statistics, Child Victimizerz: Violent Offenders and Their Victims 2 (1996).
\item See Sex Offenses and Offenders, supra note 21, at 15.
\item Id. at 18.
\item See id. at 15. It is clear from the statistics offered by the Department of Justice that sex offenders repeatedly find themselves within the criminal justice system, given their high rate of recidivism. Id.
\item Id. This discussion refers to specific deterrence (when a punishment has the effect of preventing a specific criminal from committing another crime himself) rather than general deterrence (when a punishment for one criminal has the effect of preventing others from committing the same crime), which is not addressed in this Comment.
\item Id.
\end{footnotes}
is to repeat his or her original crime.\textsuperscript{30} In fact, estimates of recidivism for untreated sex offenders have been as high as eighty percent.\textsuperscript{31} Although eighty percent is on the high end of estimates, the rate consistently reached in studies hovers between fifty and sixty percent.\textsuperscript{32} These numbers must also be considered in context: there is only a 3% chance of being apprehended for child molestation, and each adult homosexual pedophile has an average of 150 victims.\textsuperscript{33}
Before modern sexual predator laws were enacted to civilly commit such individuals, state correctional facilities released these predators into society.\textsuperscript{34} States stood by helplessly, waiting for the predators to re-offend so the criminal justice system could intervene again. The previous system was ineffective at handling the unique problem of sexual predators, and several states searched for an answer.
\textit{B. The Most Recent Solution}
That answer came in the form of resurrected “sexual psychopath” laws, now labeled “sexually violent predator” laws.\textsuperscript{35} These laws, enacted largely in the 1990s, sought to commit sexually dangerous criminals to mental health facilities for treatment and rehabilitation
\textsuperscript{30} See \textit{Sex Offenses and Offenders}, supra note 21, at 27; A.J. Beck et al., U.S. Dep’t of Justice, Bureau of Justice Statistics, \textit{Recidivism of Prisoners Released in 1983} at 6 (1997) (noting that rapists are ten and one half times more likely than non-rapists to be re-arrested for a similar crime, while other sex offenders were seven and one half times more likely than non-sex offenders to be re-arrested). It should be noted, however, that even these shocking numbers are presumed to be significant underestimates, given the lack of reporting often associated with sexual crimes.
\textsuperscript{31} U.S. Dep’t of Justice, National Institute of Corrections, \textit{A Practitioner’s Guide to Treating the Incarcerated Male Sex Offender} xiii (1988) (hereinafter \textit{Practitioner’s Guide}). The recidivism rate for treated sex offenders falls dramatically, however, to approximately fifteen percent. \textit{Id.}
\textsuperscript{32} M.E. Rice et al., \textit{Sexual Recidivism Among Child Molesters Released From a Maximum Security Psychiatric Institution}, 59 J. Consult. & Clinical Psychol. 381, 386 (1991); R.A. Pretky, \textit{Sexual Offender Recidivism Revisited: A Meta-analysis of Recent Treatment Studies}, 1997 J. Consult. & Clinical Psychol. 635, 659.
\textsuperscript{33} Gene G. Abel et al., \textit{Self-Reported Sex Crimes of Non-incarcerated Paraphiliacs}, 2 J. Interpersonal Violence 3 (1987).
\textsuperscript{34} See Siegel, supra note 1.
\textsuperscript{35} Sexual psychopath statutes, which were enacted by thirty states and the District of Columbia by 1958, sought to protect society and rehabilitate sex offenders. See Alan H. Swanson, \textit{Sexual Psychopath Statutes: Summary and Analysis}, 51 J. Crim. L. & Criminology Sci. 215, 215 (1960). The statutes, which viewed sex offenders as neither normal nor legally insane, attempted to remedy the problem of sexual crimes by committing sex offenders for indeterminate periods of time, until either treatment was complete, or the patient was safe to be at large. \textit{Id.} See also J. Harper Cook, \textit{Civil Commitment of Sex Offenders: South Carolina’s Sexually Violent Predator Act}, 50 S.C. L. Rev. 543 (1999). Courts, though often criticized, never struck down these statutes on constitutional grounds. See, e.g., Minnesota \textit{ex rel.} Pearson v. Probate Court, 309 U.S. 270 (1940); People v. Sims, 47 N.E.2d 703 (Ill. 1943); People v. Chapman, 4 N.W.2d 18 (Mich. 1942). Nonetheless, most were repealed by the 1970s for political reasons.
and to protect society from the dangers posed by these individuals.\textsuperscript{36} The period of confinement under such laws is indefinite, as the progress of treatment cannot be determined with specificity at the outset.\textsuperscript{37}
Washington was the first state to enact a sexually violent predator law, and many other states soon followed.\textsuperscript{38} Kansas's statute, the Kansas Sexually Violent Predator Act (Kansas Act), was closely patterned after Washington's and will be discussed throughout this Comment. Under the Kansas Act, a trial must be held in order to determine if a person is a sexually violent predator; that is, whether that person "has been convicted of or charged with a sexually violent offense and who suffers from a mental abnormality or personality disorder which makes the person likely to engage in repeat acts of sexual violence."\textsuperscript{39} Throughout the trial, the person being examined is afforded many of the rights associated with a criminal proceeding.\textsuperscript{40} These rights are in place to ensure the presence of as many safeguards as possible during a proceeding that has far-reaching consequences.\textsuperscript{41}
\begin{itemize}
\item \textsuperscript{36} Kan. Stat. Ann. § 59-29a01 (Supp. 2002).
\item \textsuperscript{37} Jones v. United States, 463 U.S. 354, 368 (1983) (noting “because it is impossible to predict how long it will take for any given individual to recover—or indeed whether he ever will recover—Congress has chosen . . . to leave the length of commitment indeterminate, subject to periodic review of the patient’s suitability for release). See also Kan. Stat. Ann. § 59-29a07.
\item \textsuperscript{38} National Ass'n of Attorneys General, Criminal Law, Juvenile Justice & Crime Prevention Survey, Sixth Annual NAAG Self-Report from the States A-9 (1999) (noting that, as of 1999, at least seventeen states and the District of Columbia had statutes providing for the civil commitment of sexually violent predators).
\item \textsuperscript{39} Kan. Stat. Ann. § 59-29a02. The definition of sexually violent predator includes people who have been charged with a sexual offense. \textit{Id}. However, this only applies if the person has been determined to be incompetent to stand trial pursuant to Kansas state law. \textit{Id}. Other persons eligible for commitment proceedings include those found not guilty of a sexually violent offense by reason of insanity and persons found not guilty of a sexually violent offense, but where the jury answered in the affirmative the special question asked pursuant to section 22-3221 of the Kansas Code (asserting special jury question, “Do you find the defendant not guilty solely because the defendant, at the time of the alleged crime, was suffering from a mental disease or defect which rendered the defendant incapable of possessing the required criminal intent?”). \textit{Id}. § 59-29a03. The Act defines “mental abnormality” as “a congenital or acquired condition affecting the emotional or volitional capacity which predisposes the person to commit sexually violent offenses in a degree constituting such person a menace to the health and safety of others.” \textit{Id}. § 59-29a02. “‘Likely to engage in repeat acts of sexual violence’ means the person’s propensity to commit acts of sexual violence is of such a degree as to pose a menace to the health and safety of others.” \textit{Id}. “‘Sexually motivated’ means that one of the purposes for which the defendant committed the crime was for the purpose of the defendant’s sexual gratification.” Kan. Stat. Ann. § 59-29a02.
\item \textsuperscript{40} Kan. Stat. Ann. §§ 59-29a01 – 59-29a20.
\item \textsuperscript{41} The controversy surrounding the fact that so many rights are afforded defendants in these proceedings will be discussed in Part III, infra notes 181-251 and accompanying text.
\end{itemize}
1. Procedure
When the State wishes to have a person determined a sexually violent predator, several procedural hurdles must be cleared before commitment can be ordered.\textsuperscript{42} A petition must first be filed with the court.\textsuperscript{43} A judge then determines whether there is probable cause to believe that the named person is a sexually violent predator.\textsuperscript{44} If probable cause is present, the court will order that the person be taken into custody.\textsuperscript{45} Within seventy-two hours, the person in custody will be notified of a hearing where he or she may contest the finding of probable cause.\textsuperscript{46} If probable cause is confirmed, the person will be subjected to a professional evaluation prior to trial.\textsuperscript{47} A trial follows, and if the fact-finder determines the person to be a sexually violent predator beyond a reasonable doubt, the person will be taken into custody by the Secretary of Social and Rehabilitative Services for “control, care and treatment until such time as the person’s mental abnormality or personality disorder has so changed that the person is safe to be at large.”\textsuperscript{48}
\textsuperscript{42} Samuel Jan Brakel, J.D. & James L. Cavanaugh Jr., M.D., \textit{Of Psychopaths and Pendulums: Legal and Psychiatric Treatment of Sex Offenders in the United States}, 30 N.M. L. Rev. 69, 70 (2000).
\textsuperscript{43} Kan. Stat. Ann. § 59-29a04.
\textsuperscript{44} \textit{Id.} § 59-29a05. Note that only certain sexual offenses make a person eligible for commitment under the Sexually Violent Predators Act. \textit{Id.} §59-29a02(e). These crimes are:
(1) rape; (2) indecent liberties with a child; (3) aggravated indecent liberties with a child; (4) criminal sodomy; (5) aggravated criminal sodomy; (6) indecent solicitation of a child; (7) aggravated indecent solicitation of a child; (8) sexual exploitation of a child; (9) aggravated sexual battery; (10) aggravated incest; (11) any conviction for a felony offense in effect at any time prior to the effective date of this act, that is comparable to a sexually violent offense as defined in subparagraphs (1) through (11) or any federal or other state conviction for a felony offense that under the laws of this state would be a sexually violent offense as defined in this section; (12) an attempt, conspiracy or criminal solicitation of a sexually violent offense; or (13) any act which either at the time of sentencing for the offense or subsequently during civil commitment proceedings pursuant to this act, has been determined beyond a reasonable doubt to have been sexually motivated.
\textit{Id.}
\textsuperscript{45} \textit{Id.} § 59-29a05.
\textsuperscript{46} \textit{Id.} At the probable cause hearing, the detained person has the following rights: (1) to be represented by counsel; (2) to present evidence on his or her own behalf; (3) to cross-examine witnesses testifying against him or her; and (4) to view and copy all reports and petitions in the court file. \textit{Id.}
\textsuperscript{47} Kan. Stat. Ann. § 59-29a05.
\textsuperscript{48} \textit{Id.} § 59-29a06. The defendant shall be examined by a professional of his or her choosing upon request. At trial, either party may request a jury. If a jury is chosen, the determination as to the defendant's status as a sexual predator must be unanimous. \textit{Id.} § 59-29a07. See generally Brakel & Cavanaugh, \textit{supra} note 42; Donna Cropp Bechman, \textit{Sex Offender Civil Commitments: Scientists or Psychics?}, 16-SUM Crim. Just. 24 (2001) (explaining modern methods of determining likelihood of re-offending).
2. The Number of Individuals Committed
The Kansas legislature, as well as the legislatures of other states, proclaimed its resolve to protect society from a small but "extremely dangerous group of sexually violent predators."49 The Kansas Act was written to address a very specific population of predator, and, as discussed above, several procedural safeguards were put in place to ensure the law be implemented as intended.50 Preliminary figures indicate that the goal of committing only the "worst of the worst" is being achieved.51 In fact, less than two percent of sex offenders incarcerated in Kansas have been civilly committed.52 The numbers vary among other states with similar laws, but all states have committed between just one and ten percent of their sex offenders since these laws were enacted several years ago.53 Although not determinative, these figures tend to show that the states are respecting the heavy burden the law requires for the involuntary civil commitment of sexual predators.
C. The United States Supreme Court Upholds Civil Commitment for Sexual Predators
On numerous occasions, the United States Supreme Court has addressed civil commitment generally, as well as commitment of sexual predators specifically, and has consistently held the concept of civil commitment constitutionally sound.54 The Supreme Court has identified two primary purposes or justifications for the involuntary civil commitment of citizens.55 First, under parens patriae powers, states have a legitimate interest in treating mentally deficient citizens.56 Second, states have a responsibility to protect society from dangerously ill individuals pursuant to state police power.57 Both of these purposes are implicated within the context of dealing with sexually violent predators, as all such predators suffer from mental illness or personality disorders and their violent acts certainly pose a danger to society.58
---
49. Kan. Stat. Ann. § 59-29a01.
50. Id.
51. See Brakel & Cavanaugh, supra note 42, at 81.
52. Id. at 79-81.
53. Id.
54. See Addington v. Texas, 441 U.S. 418 (1979); Allen v. Illinois, 478 U.S. 364 (1986); Kansas v. Hendricks, 521 U.S. 346 (1997); Seling, 531 U.S. 250.
55. Addington, 441 U.S. at 426; Jones, 463 U.S. at 361.
56. Addington, 441 U.S. at 426.
57. Id.
58. All sexually violent predators suffer from such disorders, as a disorder must be found before an individual fits the definition of a sexually violent predator under the statute. Kan. Stat. Ann. §§ 59-29a01 - 59-29a20.
Although the Court recognizes these compelling justifications, the possible loss of liberty these individuals face requires strict constitutional safeguards to prevent unnecessary commitment.\textsuperscript{59} A discussion of the following Supreme Court decisions outlines the constitutional requirements the Court has set forth for civil commitment of sex offenders.
\textit{1. Addington v. Texas}
\textit{Addington} was critical in shaping modern requirements for involuntary civil commitment. This 1979 decision determined that the “clear and convincing” standard of proof was appropriate for indefinite commitment proceedings.\textsuperscript{60} Although not a sexual predator, \textit{Addington} was committed several times due to mental illness.\textsuperscript{61} He was found to be mentally ill and a potential danger to himself or others by “clear, unequivocal, and convincing” evidence.\textsuperscript{62} The Court held that a standard above “preponderance of the evidence” was required for commitment, even though the proceeding was civil in nature, due to the significant liberty interest at stake.\textsuperscript{63} The Court noted that “loss of liberty calls for a showing that the individual suffers from something more serious than is demonstrated by idiosyncratic behavior,” and increasing the standard of proof guards against inappropriate commitments.\textsuperscript{64} However, the “reasonable doubt” standard invoked in criminal proceedings was likewise inappropriate, given that commitment was not punitive in nature, but rather focused on providing care for ill individuals.\textsuperscript{65} Further, the Court expressed concern that a burden so high might “completely undercut its efforts to further the legitimate interests of both the state and the patient that are served by civil commitments.”\textsuperscript{66} In balancing the individual’s interest in not being
\begin{itemize}
\item[59.] \textit{Addington}, 441 U.S. at 426.
\item[60.] \textit{Id.} at 418.
\item[61.] \textit{Id.} at 420.
\item[62.] \textit{Id.} at 421.
\item[63.] \textit{Id.} at 427.
\item[64.] \textit{Id.} Note that in \textit{Jones v. United States}, the Court explained that when a person is proven to have committed a criminal act due to mental illness, any civil commitment thereafter would ensure that the individual was not being confined based on “idiosyncratic behavior,” as a “criminal act by definition is not ‘within a range of conduct that is generally acceptable.’” \textit{Jones}, 463 U.S. at 367 (quoting \textit{Addington}, 441 U.S. at 427).
\item[65.] \textit{Addington}, 441 U.S. at 428-30. “The State of Texas confines only for the purpose of providing care designed to treat the individual.” \textit{Id.} at 428 n.4.
\item[66.] \textit{Id.} at 429. “Given the lack of certainty and the fallibility of psychiatric diagnosis, there is a serious question as to whether a state could ever prove beyond a reasonable doubt that an individual is both mentally ill and likely to be dangerous.” \textit{Id.} A reasonable doubt standard may “impose a burden the state cannot meet and thereby erect an unreasonable barrier to needed medical treatment.” \textit{Id.} at 432.
\end{itemize}
involuntarily confined for an indefinite period with the state's interest in providing care for the individual and protecting other citizens, the Court concluded that an intermediate standard adequately encompassed the relevant concerns.\textsuperscript{67} This opinion is often cited for support of the position that civil confinement of sexual offenders is not a criminal sanction.\textsuperscript{68}
2. Allen v. Illinois
In an early case concerning the civil commitment of sex offenders, the Supreme Court upheld the Illinois Sexually Dangerous Persons Act (Illinois Act) as civil in nature for purposes of the Fifth Amendment's protection against compulsory self-incrimination.\textsuperscript{69} Petitioner Terry B. Allen was charged with committing the crimes of unlawful restraint and deviate sexual assault.\textsuperscript{70} After his indictment, the State petitioned to have him declared a sexually dangerous person under the Illinois Act.\textsuperscript{71} Both examining psychiatrists testified that Allen was mentally ill, possessed criminal propensities to commit sexual assaults, and demonstrated such propensities through his prior criminal acts.\textsuperscript{72} Upon commitment, Allen appealed, claiming he was forced to answer incriminating questions during his psychiatric evaluations in violation of his Fifth Amendment rights.\textsuperscript{73} The Court held that because the Illinois Act was civil in nature, as demonstrated by a variety of criteria, the Fifth Amendment did not apply to the proceeding for commitment.\textsuperscript{74}
The legislature explicitly characterized the Illinois Act as civil in nature and, therefore, only upon the "clearest proof that the statutory scheme is so punitive . . . as to negate the State’s intention" that the proceeding be civil will it be deemed criminal.\textsuperscript{75} Given that the statute obligated the State to provide "care and treatment for persons
\begin{itemize}
\item \textsuperscript{67} Id. at 431.
\item \textsuperscript{68} See generally Hendricks, 521 U.S. 346.
\item \textsuperscript{69} Allen, 478 U.S. at 364.
\item \textsuperscript{70} Id. at 365.
\item \textsuperscript{71} Id. The Illinois Act defines sexually dangerous persons as: "All persons suffering from a mental disorder, which . . . has existed for a period of not less than one year. . . . coupled with criminal propensities to the commission of sex offenses, and who have demonstrated propensities toward acts of sexual assault or acts of sexual molestation of children." Illinois Sexually Dangerous Persons Act, ILL. COMP. STAT. ANN., ch. 205, 205/1.01 (1998).
\item \textsuperscript{72} Allen, 478 U.S. at 366.
\item \textsuperscript{73} Id. at 367.
\item \textsuperscript{74} Id. The Court observed that "the State’s interest in treating, and protecting the public from, sexually dangerous persons would be ‘almost totally thwarted’ by allowing those persons to refuse to answer questions posed in psychiatric interviews . . . ." Id.
\item \textsuperscript{75} Id. at 369.
\end{itemize}
adjudged sexually dangerous designed to effect recovery in a facility set aside to provide psychiatric care" and provided that upon recovery, the patient shall be discharged, Allen failed to prove the Illinois Act was criminal in nature.\textsuperscript{76} The Court also held that, although the Illinois Act afforded persons many rights available in criminal proceedings, the provision of these rights did not automatically convert the statute into one that was punitive in nature.\textsuperscript{77}
The dissent, written by Justice John Paul Stevens, and joined by Justices William Brennan, Thurgood Marshall, and Harold Blackmun, argued simply that the statute was criminal in nature.\textsuperscript{78} Justice Stevens urged that the law required an examination of the entire statute in context, and that such an examination led to the conclusion that the statute was so similar to a criminal statute that it should be labeled criminal as well.\textsuperscript{79}
Once the Supreme Court established the civil nature of these statutes, the next step for opponents was to argue that civil commitment was unconstitutional as a matter of due process, which was the heart of the argument in \textit{Kansas v. Hendricks}.\textsuperscript{80}
3. Kansas v. Hendricks
In 1997, the Supreme Court upheld the constitutionality of the Kansas Act, which provided for the civil commitment of certain sex offenders upon their release from prison.\textsuperscript{81} Leroy Hendricks was the first inmate committed under the Kansas Act and appealed on grounds of substantive due process, double jeopardy, and \textit{ex post facto} law-making.\textsuperscript{82}
Hendricks's criminal history of sexually abusing children spanned three decades, beginning in 1955 when he was found guilty of indecent exposure.\textsuperscript{83} In 1957, he was convicted of lewdness with a young girl and went to jail for a brief period.\textsuperscript{84} In 1960, while working for a
\begin{itemize}
\item \textsuperscript{76} \textit{Id.}
\item \textsuperscript{77} \textit{Allen}, 478 U.S. at 378.
\item \textsuperscript{78} \textit{Id.} at 376.
\item \textsuperscript{79} \textit{Id.}
\item \textsuperscript{80} \textit{See Hendricks}, 521 U.S. 346.
\item \textsuperscript{81} \textit{Id.} The Court quotes a portion of the Act's preamble: "'[S]exually violent predators generally have anti-social personality features which are unamenable to existing mental illness treatment modalities and those features render them likely to engage in sexually violent behavior.'" \textit{Id.} at 351 (quoting \textit{Kan. Stat. Ann.} § 59-29a01 (1994)).
\item \textsuperscript{82} \textit{Id.} at 350. \textit{See supra} notes 35-53 and accompanying text (discussing the Sexually Violent Predator Act).
\item \textsuperscript{83} \textit{Hendricks}, 521 U.S. at 354.
\item \textsuperscript{84} \textit{Id.}
\end{itemize}
carnival, he molested two young boys.\textsuperscript{85} He was paroled after serving just two years of that sentence, only to be re-arrested for molesting a seven-year-old girl.\textsuperscript{86} After a brief time in treatment for sexual deviance, he was deemed safe and released into society once again.\textsuperscript{87} But, shortly after, he sexually assaulted a young boy and girl.\textsuperscript{88} For these crimes he served less than five years and refused to participate in sex offender treatment while incarcerated.\textsuperscript{89} Hendricks was paroled in 1972 and abandoned treatment for pedophilia, the disorder with which he was diagnosed.\textsuperscript{90} Just after his latest parole, Hendricks began sexually abusing his stepdaughter and stepson, forcing them to engage in sexual activity with him over a period of four years.\textsuperscript{91} Finally, in 1984, Hendricks was convicted for taking indecent liberties with two thirteen-year-old boys and served a ten-year sentence in prison.\textsuperscript{92} At the end of the last prison sentence, the State of Kansas sought to have Hendricks committed under the Kansas Act.\textsuperscript{93} At trial, Hendricks admitted to being unable to control his urges to molest children and informed the court that the only way he could stop was “to die.”\textsuperscript{94} After the trial, Hendricks was deemed a sexually violent predator and committed for an indefinite period to a mental institution.\textsuperscript{95}
Hendricks claimed that the Kansas Act was violative of substantive due process because it only required a finding of a “mental abnormality” rather than demanding proof of “mental illness.”\textsuperscript{96} In response, the Supreme Court held that the term “mental abnormality” sufficed to classify a subclass of persons subject to civil confinement.\textsuperscript{97} The
\textsuperscript{85.} \textit{Id.}
\textsuperscript{86.} \textit{Id.}
\textsuperscript{87.} \textit{Id.}
\textsuperscript{88.} \textit{Id.}
\textsuperscript{89.} \textit{Hendricks}, 521 U.S. at 355. In fact, Hendricks made a point to tell a physician that “treatment is bull——.” \textit{Id.}
\textsuperscript{90.} \textit{Id.} at 354.
\textsuperscript{91.} \textit{Id.}
\textsuperscript{92.} \textit{Id.}
\textsuperscript{93.} \textit{Id.}
\textsuperscript{94.} \textit{Hendricks}, 521 U.S. at 354. Hendricks was diagnosed with pedophilia (a diagnosis with which he “readily agrees”), personality trait disturbance, and passive-aggressive personality. \textit{Id.} at 355 n.2.
\textsuperscript{95.} \textit{Id.} at 356-57.
\textsuperscript{96.} \textit{Id.}
\textsuperscript{97.} \textit{Id.} The Court stated:
The liberty secured by the Constitution of the United States to every person within its jurisdiction does not import an absolute right in each person to be, at all times and in all circumstances, wholly free from restraint. There are manifold restraints to which every person is necessarily subject for the common good. On any other basis organized society could not exist with safety to its members.
Court noted that it had historically sustained civil commitment statutes when they "have coupled proof of dangerousness with the proof of some additional factor, such as 'mental illness' or 'mental abnormality.'"98 These additional requirements served to limit the class of committed persons to a narrow group of dangerous persons.99 Finally, the Court held:
[t]he Kansas Act is plainly of a kind with these other civil commitment statutes: It requires a finding of dangerousness, and then links that finding to the existence of a "mental abnormality" or "personality disorder" that makes it difficult, if not impossible, for the person to control his dangerous behavior.100
In sum, the Court, unanimously and without qualification, held the Kansas Act comported with substantive due process requirements.101
Hendricks also claimed that the Kansas Act was criminal in nature and thus violated the Double Jeopardy and *Ex Post Facto* Clauses of the United States Constitution.102 As in *Allen*, the Court required a party questioning the nature of an explicitly marked civil statute to satisfy a heavy burden in order to persuade the Court to treat the statute as criminal.103 Hendricks failed to convince a majority of the Court of the merits of this argument.104 First, the Court held the Kansas Act did not implicate retribution or deterrence, the two primary goals of criminal punishment.105 No criminal culpability was attached to the civil commitment, so retribution was necessarily excluded as an objective.106 Further, the Court noted that since those committed persons were suffering from an abnormality or illness, they were prevented from exercising adequate control over themselves and were, therefore, unlikely to be deterred by the civil commitment statute.107
---
98. *Id.* at 357.
99. *Id.* at 357. The Court stated: "States have in certain narrow circumstances provided for the forcible civil detainment of people who are unable to control their behavior and who thereby pose a danger to the public health and safety." *Id.* Note, though, that the Court recalled this historical civil commitment justification, but did not hold that a person must be unable to control his behavior before commitment is warranted. Rather, it upheld the Kansas Act as it was, without qualifying the statutory criteria. This point will be further explored in Part V.
100. *Id.*
101. *Id.*
102. *Hendricks*, 521 U.S. at 350.
103. *Id.* at 361.
104. *Id.*
105. *Id.*
106. *Id.*
107. *Id.*
Moreover, those committed were treated in the same manner as other involuntarily committed persons, not as criminals in prison.\textsuperscript{108}
The fact that the program may fail to offer adequate treatment to some patients did not convince the Court that it was a punitive law.\textsuperscript{109} The Court noted that while some dangerous persons may not be treatable, that fact alone did not obligate their release into society.\textsuperscript{110} The majority further stated that “[u]nder appropriate circumstances and when accompanied by proper procedures, incapacitation may be a legitimate end of the civil law.”\textsuperscript{111} The fact that Kansas’s “overriding concern,” segregation of sexually violent offenders, was consistent with civil law and bolstered by the State’s ancillary goal of providing treatment to offenders when it was possible.\textsuperscript{112} With all of his arguments defeated, Hendricks’s civil commitment was reinstated, and the United States Supreme Court reversed the judgment of the Kansas Supreme Court.\textsuperscript{113}
Justice Anthony Kennedy, who noted his full agreement with the majority, entered a short concurrence.\textsuperscript{114} His agreement was based largely on the specific facts of this case.\textsuperscript{115} He offered a cautionary note: “If . . . civil confinement were to become a mechanism for retribution or general deterrence, or if it were shown that mental abnormality is too imprecise a category to offer a solid basis for concluding that civil detention is justified, our precedents would not suffice to validate it.”\textsuperscript{116}
A dissent, written by Justice Stephen Breyer, argued as Justice Stevens did in \textit{Allen}, that the statute was punitive in nature and should be recognized as such.\textsuperscript{117} Because of this categorical distinction, Justice
\begin{itemize}
\item \textsuperscript{108}. \textit{Hendricks}. 521 U.S. at 367-68.
\item \textsuperscript{109}. \textit{Id}. However, now persons committed under the Act receive approximately thirty-two hours of treatment per week. \textit{Id}. at 368.
\item \textsuperscript{110}. \textit{Id}. at 355-56.
\item \textsuperscript{111}. \textit{Id}.
\item \textsuperscript{112}. \textit{Id}.
\item \textsuperscript{113}. \textit{Hendricks}. 521 U.S. at 371. Specifically, the Court stated: “We hold that the Kansas Sexually Violent Predator Act comports with due process requirements and neither runs afoul of double jeopardy principles nor constitutes an exercise in impermissible \textit{ex post facto} lawmaking. Accordingly, the judgment of the Kansas Supreme Court is reversed.” \textit{Id}.
\item \textsuperscript{114}. \textit{Id}. at 371 (Kennedy, J., concurring).
\item \textsuperscript{115}. \textit{Id}. (Kennedy, J., concurring).
\item \textsuperscript{116}. \textit{Id}. at 373 (Kennedy, J., concurring). Justice Kennedy also noted that incapacitation was a goal common to both the civil and criminal systems of confinement. \textit{Id}. (Kennedy, J., concurring).
\item \textsuperscript{117}. \textit{Hendricks}. 251 U.S. at 379 (Breyer, J., dissenting). Specifically, Justice Breyer stated:
\[ \text{The Act before us involves an affirmative restraint historically regarded as punishment: imposed upon behavior already a crime after a finding of scienter; which restraint, namely, confinement, serves a traditional aim of punishment, does not primarily} \]
\end{itemize}
Breyer insisted the Court should have invalidated the commitment of Hendricks on *ex post facto* grounds.\textsuperscript{118} However, the majority of the Court upheld the statute with no qualifications as constitutionally sound.\textsuperscript{119}
A popular argument, the Court again revisited the civil versus punitive issue in 2001 in *Seling v. Young*, discussed below. However, before addressing that case, the following section will discuss *In re Crane*, a case decided at the state supreme court level before *Seling* and then by the United States Supreme Court after *Seling*.
4. *In re Crane*
The State of Kansas filed a petition seeking to have Michael T. Crane adjudicated as a sexually violent predator and committed pursuant to the Kansas Act.\textsuperscript{120} The State was successful and Crane appealed from his commitment, arguing that it was unconstitutional because the State did not prove that he was unable to control his behavior.\textsuperscript{121} The Kansas Supreme Court agreed and reversed Crane’s commitment, declaring that the United States Supreme Court read a volitional impairment requirement into the Kansas Act when it decided *Kansas v. Hendricks* in 1997.\textsuperscript{122} The State of Kansas petitioned the United States Supreme Court for certiorari, which was granted. The Supreme Court, for the second time in five years, considered the constitutionality of the same state law it upheld in *Kansas v. Hendricks*. This section discusses both court decisions regarding Crane.
a. Kansas Changed the Requirements in *In re Crane*
Crane was convicted of lewd and lascivious behavior for exposing himself to a tanning salon attendant in 1993.\textsuperscript{123} Crane also pleaded guilty to aggravated sexual battery, related to an incident that occurred after Crane left the tanning salon.\textsuperscript{124} He entered a video store, waited until he was alone in the store with the clerk, and then grabbed her from behind.\textsuperscript{125} With himself exposed, he lifted and pushed her
\textsuperscript{118.} *Id.* at 379 (Breyer, J., dissenting).
\textsuperscript{119.} *Id.* at 346.
\textsuperscript{120.} *In re Crane*, 7 P.3d 285, 285 (Kan. 2000).
\textsuperscript{121.} *Id.*
\textsuperscript{122.} *Id.*
\textsuperscript{123.} *Id.* at 286.
\textsuperscript{124.} *Id.*
\textsuperscript{125.} *Id.*
and squeezed her neck while ordering her three times to perform oral sex and telling her he was going to rape her.\textsuperscript{126} Then, suddenly, Crane stopped and fled the store, ending the attack.\textsuperscript{127}
At the commitment trial, two psychologists and a psychiatrist testified to the results of their evaluations of Crane.\textsuperscript{128} One psychologist, Douglas Hippe, concluded that Crane suffered from antisocial personality disorder, consistently exhibiting six out of seven criteria listed in the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV).\textsuperscript{129} Further, Hippe found that Crane suffered from exhibitionism.\textsuperscript{130} He expressed the opinion that Crane was a sexual predator due to both disorders.\textsuperscript{131} Hippe also noted that Crane's criminal incidents had increased in frequency and intensity, his actions became more daring and aggressive, and his disregard for the rights of others increased as well.\textsuperscript{132} Psychologist Robert Huerter testified that there was no merit to Crane's claim that he had been in a blackout state during the attack.\textsuperscript{133} Finally, psychiatrist Leonardo Mabugat testified to Crane's antisocial personality disorder, noting that he displayed "a pervasive pattern of disregard for and violation of the rights of
\begin{itemize}
\item \textsuperscript{126} In re Crane, 7 P.3d at 286.
\item \textsuperscript{127} Id.
\item \textsuperscript{128} Id.
\item \textsuperscript{129} Id. at 287. The Diagnostic and Statistical Manual of Mental Disorders offers the following criteria for antisocial personality disorder:
A. There is a pervasive pattern of disregard for and violation of the rights of others occurring since age 15 years, as indicated by three (or more) of the following: (1) failure to conform to social norms with respect to lawful behaviors as indicated by repeatedly performing acts that are grounds for arrest; (2) deceitfulness, as indicated by repeated lying, use of aliases, or conning others for personal profit or pleasure; (3) impulsivity or failure to plan ahead; (4) irritability and aggressiveness, as indicated by repeated physical fights or assaults; (5) reckless disregard for the safety of self or others; (6) consistent irresponsibility, as indicated by repeated failure to sustain consistent work behavior or honor financial obligations; (7) lack of remorse, as indicated by being indifferent to or rationalizing having hurt, mistreated, or stolen from another.
\item \textsuperscript{130} American Psychiatric Association, Diagnostic and Statistical Manual of Mental Disorders § 301.7 (1994) [hereinafter DSM-IV]. Items B through D are irrelevant to this discussion.
\item \textsuperscript{131} In re Crane, 7 P.3d at 287. Exhibitionism is defined as "[o]ver a period of at least six months, recurrent, intense sexually arousing fantasies, sexual urges, or behaviors involving the exposure of one's genitals to an unsuspecting stranger. The person has acted on these sexual urges, or the sexual urges or fantasies cause marked distress or interpersonal difficulty." DSM-IV, supra note 129, § 302.4.
\item \textsuperscript{132} Id.
\item \textsuperscript{133} Id.
\end{itemize}
Dr. Mabugat also stated that Crane’s behavior was a combination of both willful and uncontrollable behavior.\textsuperscript{135}
The lower court held that the State must prove “the existence of a mental disorder that makes [Crane] likely to reoffend.”\textsuperscript{136} On appeal, however, the Kansas Supreme Court pointed to several sentences from the \textit{Hendricks} opinion, which allegedly supported the inclusion of a requirement that an individual be completely unable to control himself.\textsuperscript{137} Therefore, the Kansas Supreme Court held that, despite evidence that Crane suffered from a volitional impairment, his commitment was unconstitutional absent a specific finding that Crane was \textit{unable} to control his behavior.\textsuperscript{138}
b. The United States Supreme Court Vacated the Kansas Decision
In a 7-2 decision announced on January 22, 2002, the United States Supreme Court vacated the Kansas Supreme Court’s reversal of Crane’s commitment under the Kansas Act.\textsuperscript{139} The Court held that \textit{Hendricks} did not require \textit{total or complete} lack of control, but that the Constitution did not permit commitment of the type of dangerous sexual offender considered in \textit{Hendricks} without \textit{any} lack-of-control determination.\textsuperscript{140} The Court instead said “[i]t is enough to say that there must be proof of serious difficulty in controlling behavior.”\textsuperscript{141} Recognizing the lack of a bright-line rule in this area of law, the Court stressed that the “safeguards of human liberty in the area of mental
\textsuperscript{134} \textit{Id.} Dr. Mabugat testified that Crane’s behavior included the following: failure to conform to social norms with respect to lawful behaviors as indicated by repeatedly performing acts that were grounds for arrest; impulsivity or failure to plan ahead; irritability; and aggressiveness, as indicated by repeated physical fights or assaults. \textit{Id.} at 290.
\textsuperscript{135} \textit{Id.} at 287.
\textsuperscript{136} \textit{In re Crane}, 7 P.3d at 288. Note that this standard corresponds to the Kansas statute, which provides treatment for “sexually violent predators who have a mental abnormality or personality disorder and who are \textit{likely} to engage in repeat acts of sexual violence if not treated” \textit{Kan. Stat. Ann.} § 59-29a01 (emphasis added).
\textsuperscript{137} \textit{In re Crane}, 7 P.3d at 288. Specifically, the Kansas Supreme Court cited the United States Supreme Court’s statement: “These added statutory requirements serve to limit involuntary civil confinement to those \textit{who suffer from a volitional impairment rendering them dangerous beyond their control}.” \textit{Id.} (quoting \textit{Kan. Stat. Ann.} § 59-29a02(b)) (emphasis in original). Further, the Kansas Supreme Court noted the statement, “‘it requires a finding of future dangerousness, and then links that finding to the existence of a mental abnormality or personality disorder that makes it \textit{difficult, if not impossible, for the person to control his dangerous behavior.’” \textit{Id.}
\textsuperscript{138} \textit{Id.} at 290.
\textsuperscript{139} \textit{Kansas v. Crane}, 534 U.S. 407, 415 (2002).
\textsuperscript{140} \textit{Id.}
\textsuperscript{141} \textit{Id.} at 413.
illness and the law” are not always best served by black and white rules.\textsuperscript{142}
Kansas raised the question of how imposing such a control requirement would impact the part of the statute allowing individuals with emotional impairments to be committed.\textsuperscript{143} Kansas noted that the state supreme court’s reading of \textit{Hendricks} would absolutely forbid the commitment of an individual with a mental abnormality affecting emotional capacity while volitional capacity remains intact.\textsuperscript{144} The Court acknowledged that the opinion in \textit{Hendricks} was limited to a discussion of volitional impairment, as the facts of that case necessitated.\textsuperscript{145} At that point, however, the Court specifically declined to answer the question whether commitment based on emotional impairment alone would be constitutional.\textsuperscript{146} The Court seemed to believe that this question was not very important, stating “our cases suggest that civil commitment of dangerous sexual offenders will normally involve individuals who find it particularly difficult to control their behavior.”\textsuperscript{147} However, the Court did note that it usually did not distinguish between volitional, emotional and cognitive impairments for constitutional purposes within the context of civil commitment.\textsuperscript{148}
The dissent, authored by Justice Antonin Scalia, and joined by Justice Clarence Thomas, argued for a reversal of the Kansas Supreme Court decision.\textsuperscript{149} Justice Scalia noted the Court’s departure from its own precedent in \textit{Hendricks} by stating:
\begin{quote}
We agree that \textit{Hendricks} limited its discussion to volitional disabilities. And that fact is not surprising. The case involved an individual suffering from pedophilia—a mental abnormality that critically involves what a lay person might describe as a lack of control. Hendricks himself stated that he could not “control the urge” to molest children. In addition, our cases suggest that civil commitment of dangerous sexual offenders will normally involve individuals who find it particularly difficult to control their behavior—in the general sense described above.
\textit{Id.}
Regardless, \textit{Hendricks} must be read in context. The Court did not draw a clear distinction between the purely “emotional” sexually related mental abnormality and the “volitional.” Here, as in other areas of psychiatry, there may be “considerable overlap between a . . . defective understanding or appreciation and . . . [an] ability to control . . . behavior.”
\textit{Id.}
\textit{Crane}, 534 U.S. at 415. No member of the Court voted to affirm the Kansas Supreme Court’s decision.
\end{quote}
Today the Court holds that the Kansas Sexually Violent Predator Act (SVPA) cannot, consistent with so-called substantive due process, be applied as written. It does so even though, less than five years ago, we upheld the very same statute against the very same contention in an appeal by the very same petitioner (the State of Kansas) from the judgment of the very same court.\textsuperscript{150}
Justice Scalia asserted that the requirements of the Kansas Act ensured that only individuals with mental abnormalities that impaired their control would be committed.\textsuperscript{151} Thus, the extra requirement added by the majority for a finding of inability to control behavior was unnecessary.\textsuperscript{152} Justice Scalia also took issue with the Court’s reopening of a question “closed by \textit{Hendricks}: whether the [Kansas Act] also cannot be applied as written because it allows for the commitment of people who have mental illnesses other than volitional impairments.”\textsuperscript{153} He observed that distinguishing between various types of impairments in the civil commitment setting lacked common sense.\textsuperscript{154} He noted that “[i]t is obvious that a person may be able to exercise volition and yet be unfit to turn loose upon society. The man who has a will of steel, but who delusionally believes that every woman he meets is inviting crude sexual advances, is surely a dangerous sexual predator.”\textsuperscript{155}
Moreover, Justice Scalia expressed concern over the vague test for constitutionality advanced by the majority.\textsuperscript{156} He argued that trial courts would have “not a clue” about how to determine if an individual possesses the requisite lack of control over himself to qualify for commitment.\textsuperscript{157} Finally, the dissent asserted that the majority opinion degraded the authority of the Court by revoking a decision made less than five years earlier.\textsuperscript{158} Justice Scalia stated that “[t]here is an obvi-
\textsuperscript{150} \textit{Id.} (Scalia, J., dissenting).
\textsuperscript{151} \textit{Id.} at 419-20 (Scalia, J., dissenting).
\textsuperscript{152} \textit{Id.} (Scalia, J., dissenting). Justice Scalia stated: “[T]he SVPA’s required finding of a causal connection between the likelihood of repeat acts of sexual violence and the existence of a ‘mental abnormality’ or ‘personality disorder’ necessarily establishes ‘difficulty if not impossibility’ in controlling behavior.” \textit{Id.} at 419 (Scalia, J., dissenting) (emphasis in original).
\textsuperscript{153} \textit{Id.} at 421 (Scalia, J., dissenting).
\textsuperscript{154} \textit{Crane}, 534 U.S. at 422 (Scalia, J., dissenting).
\textsuperscript{155} \textit{Id.} (Scalia, J., dissenting).
\textsuperscript{156} \textit{Id.} at 422-23 (Scalia, J., dissenting).
\textsuperscript{157} \textit{Id.} at 423 (Scalia, J. dissenting).
\textsuperscript{158} \textit{Id.} at 424 (Scalia, J., dissenting). Justice Scalia asserted:
The Kansas Supreme Court still did not like the law and prevented its operation, on substantive due process grounds, once again. The State of Kansas again sought certiorari, asking nothing more than reaffirmation of our 5-year-old opinion—only to be told that what we said then we now unsay.
\textit{Id.} (Scalia, J., dissenting).
ous lesson here for state supreme courts that do not agree with our jurisprudence: ignoring it is worth a try."159
5. Seling v. Young
After the Kansas Supreme Court decided *In re Crane*, but before the United States Supreme Court issued an opinion in *Crane*, *Seling v. Young* was decided by the United States Supreme Court.
The Supreme Court upheld Washington State’s Community Protection Act (Washington Act), a civil commitment statute for sexual predators.160 Andre Brigham Young appealed from a unanimous jury determination that he was a sexually violent predator, subject to confinement under the Washington Act.161 Young argued that the Washington Act was unconstitutional because it was punitive in application to him.162
Young was convicted of six rapes over three decades.163 It was upon his latest release from incarceration that the State of Washington sought to have him committed as a sexually violent predator.164 An expert testified that Young suffered from a severe personality disorder with primarily paranoid and antisocial features.165 Young was also diagnosed with severe paraphilia,166 classified as either
---
159. *Crane*, 534 U.S. at 424 (Scalia, J., dissenting).
160. *Seling*, 531 U.S. at 253.
161. *Id.*
162. *Id.* at 253-54.
163. *Id.* at 255.
164. *Id.*
165. *Id.* at 255-56.
166. A paraphilia is a condition whereby a person’s ability to become sexually aroused and/or gratified is dependant upon either fantasizing about or performing certain atypical or extreme sexual behavior. *The Sinclair Intimacy Institute Website*, at http://www.intimacyinstitute.com/sex_data/topics/parahilia.html (last visited Feb. 18, 2003) (on file with DePaul Law Review). Paraphilias are much more prevalent among men than women, and the focus of such paraphilias is usually constant and specific. *Id.* Examples of paraphilias include: fetishism (fixation on an object or body part that is not considered sexual in nature); transvestism (males’ need to dress in women’s clothing in order to become aroused); voyeurism (becoming sexually aroused by watching people undress or engaged in sexual activity, without their knowledge or consent); exhibitionism (compulsively exposing one’s genitals to unsuspecting victims in order to shock or frighten them); sadism (deriving sexual gratification from inflicting pain on one’s partner); masochism (deriving sexual gratification from being subjected to pain); pedophilia (fixation on fantasy or engagement in sexual acts with children); and bestiality (engaging in sexual activity with animals). *Id.* See also DSM-IV, *supra* note 129, at 522. Definition of paraphilia is as follows: recurrent, intense sexually arousing fantasies, sexual urges, or behaviors generally involving 1) non-human objects, 2) the suffering or humiliation of oneself or one’s partner, or 3) children or other non-consenting persons, that occur over a period of at least 6 months. The behavior, sexual urges, or fantasies cause clinically significant distress or impairment in social, occupational, or other important areas of functioning. *Id.*
paraphilia sexual sadism\textsuperscript{167} or paraphilia not otherwise specified (rape).\textsuperscript{168}
The Washington Supreme Court concluded that the Washington Act was “concerned with treating committed persons for a current mental abnormality, and protecting society from the sexually violent acts associated with that abnormality, rather than being concerned with criminal culpability.”\textsuperscript{169} The United States Supreme Court agreed with this finding and, as it did in \textit{Allen} and \textit{Hendricks}, found the Washington Act civil on its face.\textsuperscript{170} Because the Washington Act was determined to be facially civil, Young’s “as applied” challenge had to fail.\textsuperscript{171} To allow such challenges when the statute was civil, concluded the Court, would prove “unworkable.”\textsuperscript{172} “The civil nature of a confinement scheme cannot be altered based merely on vagaries in the implementation of the authorizing statute.”\textsuperscript{173} The Court reserved the question of whether specific circumstances might be considered in the primary inquiry into the nature of such a statute.\textsuperscript{174}
Concurring opinions by Justices Scalia and Thomas primarily focused on answering the question that the majority reserved.\textsuperscript{175} Both concurrences concluded that there was no difference between a primary inquiry and subsequent ones for purposes of as applied challenges to a statute.\textsuperscript{176} Justice Scalia stated, “We repeated, to be sure, the principle that the statutory scheme would be criminal if it was suf-
\begin{itemize}
\item Where, over a period of at least six months, a person experiences “recurrent, intense sexually arousing fantasies, sexual urges, or behaviors involving acts (real, not simulated) in which the psychological or physical suffering (including humiliation) of the victim is sexually exciting to the person.” DSM-IV, \textit{supra} note 129, § 302.84. The definition also includes the requirement that the “person has acted on these sexual urges with a non-consenting person, or the sexual urges or fantasies cause marked distress or interpersonal difficulty.” \textit{Id.} (emphasis added).
\item \textit{Seling}, 531 U.S. at 256. “The State’s expert concluded that Young’s condition, in combination with the personality disorder, the span of time during which Young committed his crimes, his recidivism, his persistent denial, and his lack of empathy or remorse, made it more likely than not that he would commit further sexually violent acts.” \textit{Id.}
\item \textit{Id.} at 257.
\item \textit{Id.} at 260-61.
\item \textit{Id.} at 263.
\item \textit{Id.} In his concurrence, Justice Thomas stated that not only are as-applied challenges “unworkable,” they are prohibited by \textit{Hudson v. United States}. \textit{Seling}, 531 U.S. at 272 (Thomas, J., concurring) (citing \textit{Hudson v. United States}, 522 U.S. 93 (1997)).
\item \textit{Id.} at 263.
\item \textit{Id.} at 265-66.
\item \textit{Id.} at 267-74 (Scalia & Thomas, J.J., concurring).
\item \textit{Id.} Justice Scalia noted that “harsh executive implementation cannot ‘transfor[m] what was clearly intended as a civil remedy into a criminal penalty,’ any more than compassionate executive implementation can transform a criminal penalty into a civil remedy.” \textit{Id.} at 269 (Scalia, J., concurring). Justice Scalia further noted the Court’s “sound and traditional reluctance to be the initial interpreter of state law.” \textit{Seling}, 531 U.S. at 270 (Scalia, J., concurring).
\end{itemize}
ficiently punitive ‘either in purpose or effect,’ but it was clear from the opinion [Hudson v. United States] that this referred to effects apparent upon the face of the statute.”\textsuperscript{177}
The sole dissenter, Justice Stevens, urged that Young should have been allowed to prove that the statute was criminal in nature as applied to him and thus agreed with the Ninth Circuit’s holding.\textsuperscript{178} Despite this dissent, Young’s constitutional challenge to the law failed, and his commitment under the Washington Act was reinstated.\textsuperscript{179} The Ninth Circuit’s judgment was reversed.\textsuperscript{180}
III. Analysis of Judicial Treatment and Popular Arguments
The analysis in this section focuses primarily on four cases: \textit{Kansas v. Hendricks}, \textit{In re Crane}, as decided by the Kansas Supreme Court, \textit{Seling v. Young}, and \textit{Kansas v. Crane}, the most recent United States Supreme Court decision on the subject of civil commitment for sexual predators. This Comment asserts that \textit{Hendricks} was properly decided, \textit{In re Crane} was improperly decided in light of \textit{Hendricks} and \textit{Young}, and the United States Supreme Court failed to follow its own precedent when deciding \textit{Kansas v. Crane}. This section also explores common arguments against civil commitment for sexual predators and why they fail to be persuasive.
A. Kansas v. Hendricks was Properly Decided
In \textit{Hendricks}, the Supreme Court faced several constitutional arguments, and properly rejected each of them. This section discusses why the Court was correct in its ruling on substantive due process and explains why it was logical, rather than determinative, that the Court addressed \textit{Hendricks} in terms of volitional control specifically. Finally, this section addresses the public policy interests upheld by the Court’s decision.
\textsuperscript{177} \textit{Id.} at 269 (Scalia, J., concurring) (citing Hudson v. United States, 522 U.S. 93, 99 (1997) (quoting United States v. Ward, 448 U.S. 242, 248-49 (1980)) (emphasis in original)).
\textsuperscript{178} \textit{Id.} at 275 (Stevens, J., dissenting).
\textsuperscript{179} \textit{Id.} at 267.
\textsuperscript{180} \textit{Id.} It is worth noting that nowhere in the Court’s opinion do the phrases “volitional impairment” or “unable to control” appear, despite the fact that the Court reproduced the part of the Washington statute that defined those persons eligible for commitment.
1. Civil Commitment Statutes for Sexual Predators Do Not Violate Due Process
The Supreme Court held in *Hendricks* that the Kansas Act did not violate substantive due process.\(^{181}\) "Substantive" due process is the constitutional requirement that legislation must be fair and reasonable in content and that it must further a legitimate government interest.\(^{182}\) Here, according to the Court, Kansas's use of the term "mental abnormality" easily satisfied substantive due process as fair and reasonable in content.\(^{183}\) The use of this term allows the legislature and the medical community necessary flexibility in identifying and treating sex offenders with a myriad of mental problems, many of them not readily diagnosable as illnesses or diseases. Such mental problems, although not fitting neatly into ready-made medical pigeonholes, cause the patient to experience severe social and personal distress,\(^{184}\) as evidenced by repeated criminal behavior. This distress may manifest in several ways, including, as the Kansas Act recognizes, emotional impairment of the individual.\(^{185}\)
Critics argue that these sex offenders should not be committed because they could not be committed under general civil commitment laws and, thus, the new laws are violative of substantive due process.\(^{186}\) This argument fails to acknowledge that states have authority to devise more than one commitment law to treat more than one category of ill individual.\(^{187}\) In fact, it was necessary for states to design these sexual predator laws to handle a growing problem in society that was not adequately addressed by then-existing legislation.\(^{188}\) Under current sexual predator laws, persons with mental abnormalities pose the requisite danger to society and possess a need for treatment such
\(^{181}\) *Hendricks*, 521 U.S. at 346.
\(^{182}\) *Black's Law Dictionary* 517 (7th ed. 1999).
\(^{183}\) *Hendricks*, 521 U.S. at 346. The Court stated that "Hendricks' diagnosis as a pedophile, which qualifies as a 'mental abnormality' under the Act, thus *plainly* suffices for due process purposes." *Id.* at 360 (emphasis added).
\(^{184}\) *See supra* note 166 (defining paraphilia, per DSM-IV).
\(^{185}\) The definition of "mental abnormality" in the Kansas Sexually Violent Predator Act reads: "congenital or acquired condition affecting emotional or volitional capacity which predisposes the person to commit sexually violent offenses." *Kan. Stat. Ann.* § 59-29a02(b).
\(^{186}\) This was argued by the defendant in *Kansas v. Hendricks*, who claimed civil commitment required a specific finding of mental illness, ignoring the distinction between general civil commitment laws and sexual predator commitment laws. *Hendricks*, 521 U.S. at 361-62.
\(^{187}\) *Id.* at 359. "[W]e have never required state legislatures to adopt any particular nomenclature in drafting civil commitment statutes. Rather, we have traditionally left to legislators the task of defining terms of a medical nature that have legal significance." *Id.*
\(^{188}\) Legislation prior to the sexual predator commitment laws consisted only of general commitment laws, which required a finding of mental illness as a predicate for confinement and treatment. *See Kan. Stat. Ann.* § 59-29a01.
that commitment would be proper. The Court in *Hendricks* properly dismissed the argument the petitioner tried to assert.\textsuperscript{189} No due process consideration was offended by the addition of another, more specialized, commitment statute.\textsuperscript{190}
Having established the governmental interests of protecting its citizens and treating the ill, the Kansas Act was deemed fair and reasonable in content, and thus not violative of substantive due process.\textsuperscript{191} This decision by the Supreme Court was a necessary step in allowing states to protect their citizens from new dangers, namely sexually violent predators.
2. \textit{The Court’s Opinion in Kansas v. Hendricks was Necessarily Replete with References to Volitional Control}
The specific facts of *Hendricks* lent themselves to the Court’s repetitive language concerning volitional control. Several statements throughout the opinion made reference to Hendricks’s admitted lack of control over his behavior.\textsuperscript{192} This is to be expected where a defendant testified before the court that he would not stop molesting children until he dies.\textsuperscript{193} Such clear and unequivocal admission of lack of control makes a commitment decision easier than when a defendant professes, even if falsely, remorse and a promise never to do it again. Here, Hendricks gave the district court a clear reason to commit him, as he even agreed with the diagnosing physician that he was a
\textsuperscript{189} The Court stated that “mental abnormality” is equally sufficient, for substantive due process purposes, as “mental illness.” \textit{See supra} notes 80-119 and accompanying text (discussing *Hendricks*).
\textsuperscript{190} *Hendricks*, 521 U.S. at 357.
\textsuperscript{191} \textit{Id.} at 350.
\textsuperscript{192} Such statements by Justice Thomas included: “States have in certain narrow circumstances provided for the forcible civil detention of people who are unable to control their behavior and who thereby pose a danger to the public health and safety.” \textit{Id.} at 357. “These added statutory requirements serve to limit involuntary civil confinement to those who suffer from a volitional impairment rendering them dangerous beyond their control.” \textit{Id.} at 358.
The Kansas act is plainly of a kind with these other civil commitment statutes: It requires a finding of future dangerousness, and then links that finding to the existence of a “mental abnormality” or “personality disorder” that makes it difficult, if not impossible, for the person to control his dangerous behavior.
\textit{Id.} “[I]t narrows the class of persons eligible for confinement to those who are unable to control their dangerousness.” \textit{Id.} “This admitted lack of volitional control, coupled with a prediction of future dangerousness, adequately distinguishes Hendricks from other dangerous persons who are perhaps more properly dealt with exclusively through criminal proceedings.” *Hendricks*. 521 U.S. at 360. “Those persons committed under the Act are, by definition, suffering from a ‘mental abnormality’ or a ‘personality disorder’ that prevents them from exercising adequate control over their behavior.” \textit{Id.} at 362.
\textsuperscript{193} \textit{Id.} at 355. “He stated that the only sure way he could keep from sexually abusing children in the future was ‘to die.’” \textit{Id.}
pedophile.\textsuperscript{194} Such facts contributed largely to the Supreme Court’s language, but despite all the references to volitional control, the holding of the case merely upheld the Kansas Act and noted that the criteria contained therein would make it clear that a defendant would have difficulty controlling his behavior.\textsuperscript{195} That is, in order to satisfy the remainder of the statute, some impairment had to be present, though the Court did not specify that the impairment must be volitional. Under the statute, and therefore presumably under the Court’s holding, an emotional impairment would suffice. The Court’s volitional language should not be read to require the presence of volitional impairment as a predicate, as the Court should have been explicit in the holding if it intended such a requirement be added.
3. \textit{The Supreme Court Reaffirmed Sound Policy by Upholding the Kansas Act}
The \textit{Hendricks} decision was based on sound policy considerations that have been long recognized by the Supreme Court. Civil commitment has historically been approved as proper for a specific group of persons in order to protect society from them, and them from themselves.\textsuperscript{196} The Court affords the Kansas legislature and other state legislatures the latitude they need when dealing with modern problems. This latitude is granted in spite of, in fact, \textit{because of}, disagreement within the medical community.\textsuperscript{197} When society wants to enact a law, but medicine and science do not offer certainty as to the course, the legislature is granted broad power.\textsuperscript{198} This is a necessary policy of the Court, given the lack of uniformity in the area of psychology.
Moreover, the Court gave the protection of society due weight in this decision. Often, the rights of the offender take precedence over
\textsuperscript{194} \textit{Id.} “Hendricks readily agreed with the state physician’s diagnosis that he suffers from pedophilia and that he is not cured of the condition.” \textit{Id.}
\textsuperscript{195} Specifically, the Court stated: “We hold that the Kansas Sexually Violent Predator Act comports with due process requirements and neither runs afoul of double jeopardy principles nor constitutes an exercise in impermissible \textit{ex post facto} lawmaking.” \textit{Hendricks}, 521 U.S. at 371. During oral arguments in \textit{Kansas v. Crane}, the Court noted that it also held the following: “The Kansas Act is plainly of a kind with these other civil commitment statutes: It requires a finding of future dangerousness, and then links that finding to the existence of a ‘mental abnormality’ or ‘personality disorder’ that makes it difficult, if not impossible, for the person to control his dangerous behavior.” \textit{Id.} at 358.
\textsuperscript{196} \textit{See id.} at 357; \textit{Addington}, 441 U.S. at 426-27; Foucha v. Louisiana, 504 U.S. 71, 80 (1992).
\textsuperscript{197} \textit{See Jones}, 463 U.S. at 365.
\textsuperscript{198} “[R]egarding congressional enactments, when a legislature ‘undertakes to act in areas fraught with medical and scientific uncertainties, legislative options must be especially broad and courts should be cautious not to rewrite legislation.’” \textit{Hendricks}, 521 U.S. at 360 n.3 (citing \textit{Jones}, 463 U.S. at 370) (internal quotation marks and citation omitted).
the rights of society, and this opinion offered a proper balance. The State has a heavy burden to prove—beyond a reasonable doubt in the minds of twelve jurors or a judge—that a sex offender is in fact a sexual predator in need of treatment and confinement.\textsuperscript{199} Once this is proven, however, society is protected from mentally deficient offenders until such time as the person is no longer a danger to others.\textsuperscript{200} This is especially important for protecting children, which has historically been deemed a compelling interest by the Supreme Court.\textsuperscript{201}
Finally, the Court allowed states to compel an ill person to undergo treatment where it was necessary.\textsuperscript{202} Although not always successful, studies have shown drastically decreased recidivism rates for sex offenders who have received treatment.\textsuperscript{203} However, when sex offenders are offered treatment during incarceration, it is very often refused for a number of reasons.\textsuperscript{204} The Kansas Act provides for treatment that may benefit the individual, when he otherwise would not accept it. This protects society as well as the treated individual, as repeated incarceration, not surprisingly, has been shown to have negative effects on inmates.\textsuperscript{205}
\textit{B. In re Crane was Wrongly Decided by the Kansas Supreme Court and Only Partially Redeemed by the United States Supreme Court}
The Kansas Supreme Court improperly read a requirement of complete lack of control into the \textit{Hendricks} decision. Although the United States Supreme Court, in \textit{Kansas v. Crane}, acknowledged this error, it added a lack of control requirement that was seemingly absent in \textit{Hendricks} and never even mentioned in \textit{Seling v. Young}. While the general statute was upheld, the Court amended its earlier decision in a break from precedent. This section discusses why the
\begin{itemize}
\item \textsuperscript{199} See \textit{Hendricks}, 521 U.S. at 357-59.
\item \textsuperscript{200} See Kan. Stat. Ann. § 59-29a07.
\item \textsuperscript{201} See Isaac, \textit{supra} note 15, at 1314-15. Isaac discusses the Supreme Court’s opinion in \textit{New York v. Ferber}, 458 U.S. 747 (1982), where the Court stated:
A democratic society rests, for its continuance, upon the healthy, well-rounded growth of young people into full maturity as citizens. Accordingly, we have sustained legislation aimed at protecting the physical and emotional well-being of youth even when the laws have operated in the sensitive area of constitutionally protected rights.
\item \textsuperscript{202} See Kan. Stat. Ann. § 59-29a07.
\item \textsuperscript{203} See \textit{Practitioner’s Guide}, \textit{supra} note 31; John W. Parry, \textit{Shrinking Civil Rights of Alleged Sexual Predators}, 25 Mental & Physical Disability L. Rep. 318, 320 (2001).
\item \textsuperscript{204} See Daley, \textit{supra} note 14, at 720 (noting that sex offenders often refuse treatment in prison because they do not want other inmates to know about their crimes, as rapists and child molesters are hated by other inmates).
\item \textsuperscript{205} See generally Dorsett, \textit{supra} note 14.
\end{itemize}
Kansas Supreme Court’s analysis was flawed and why the United States Supreme Court, albeit to a lesser degree, was also incorrect in its analysis of prior case law.
1. The Supreme Court Did Not Explicitly Add a Volitional Impairment Requirement to the Kansas Act in Its Holding in Kansas v. Hendricks
The explicit holding of *Kansas v. Hendricks* was “that the Kansas Sexually Violent Predator Act comports with due process requirements and neither runs afoul of double jeopardy principles nor constitutes an exercise in impermissible *ex post facto* lawmaking.”\(^{206}\) Nowhere in that holding did the Court add another constitutional requirement to the Kansas Act. When the Supreme Court heard oral arguments on this case on October 30, 2001, Justice Scalia stated the holding as including the following statement:
The Kansas Act is plainly of a kind with these other civil commitment statutes: It requires a finding of future dangerousness, and then links that finding to the existence of a “mental abnormality” or “personality disorder” that makes it difficult, if not impossible, for the person to control his dangerous behavior.\(^{207}\)
Even given this version of the holding, there is no language to support a separate requirement of volitional control specifically. In fact, Justice Scalia, in his dissent in *Kansas v. Crane*, explained that the above holding recognized that some lack of control was inherently present in individuals who qualified under the Kansas Act.\(^{208}\) Thus, no separate finding should have been required. The fact that the Kansas Supreme Court required one demonstrates a lack of understanding of *Hendricks* and an improper reading of the Kansas Act. The United States Supreme Court now requires a separate finding of some lack of control, which is confusing given its own precedent in *Hendricks* and *Young*.
2. Kansas v. Crane Conflicts with Seling v. Young
The decision in *Young*, coming down after *In re Crane* and before the Supreme Court heard arguments on *Kansas v. Crane*, offers clarification for many of the arguments the Kansas court endorsed in *In re Crane*. The Kansas court focused primarily on the Supreme Court’s
---
\(^{206}\) *Hendricks*, 521 U.S. at 371.
\(^{207}\) See Oral Argument of Carla J. Stovall on Behalf of Petitioner, *Kansas v. Crane*, 2001 WL 1398618, at *28 (statement by Justice Scalia) (hereinafter Oral Argument).
\(^{208}\) See supra notes 150-159 and accompanying text.
references to volitional control when it decided *Hendricks*.\textsuperscript{209} Notably, the *Young* decision made absolutely no reference to volitional control, or lack thereof, as being a requirement for a statute to be constitutional on its face.\textsuperscript{210} In fact, Justice O’Connor, writing for the majority, recited the definition of a sexual predator under the Washington Act, and the jury’s findings at Young’s trial.\textsuperscript{211} Both statements are completely void of any language regarding volitional control. Yet never does the majority, the concurrences, or the dissenting opinion even mention the “added requirement” that was supposedly asserted in *Hendricks*. One would think that if the Supreme Court had altered the Kansas Act to require an additional finding, it would certainly have altered the Washington Act similarly, as the two are practically identical in wording. It reasonably follows that failure to indicate the additional requirement means there is no such additional constitutional requirement at all. Although the Washington Act came before the Court under a presumption that it was facially civil,\textsuperscript{212} surely the Court would have pointed out that the omission of the new requirement makes the statute patently unconstitutional on its face if that were the case. The Court even compared the Washington Act with the Kansas Act explored in *Hendricks*, calling the two “strikingly similar.”\textsuperscript{213} Nor was the point of volitional control moot, because the lower court did not make a specific finding that Young was unable to control his behavior.\textsuperscript{214}
The United States Supreme Court’s decision in *Kansas v. Crane* is puzzling given this recent case. Fortunately, however, the Supreme Court did not endorse the Kansas court’s complete lack of control standard. Rather, the majority agreed with Kansas that such a stan-
\textsuperscript{209} See generally *In re Crane*, 7 P.3d 285.
\textsuperscript{210} See *Seling*, 531 U.S. 250.
\textsuperscript{211} Id. at 254.
The Act defines a sexually violent predator as someone who has been convicted of, or charged with, a crime of sexual violence and who suffers from a mental abnormality or personality disorder that makes the person more likely to engage in predatory acts of sexual violence if not confined in a secure facility.
\textit{Id.}
In the state expert’s opinion, severe paraphilia constituted a mental abnormality under the Act. The State’s expert concluded that Young’s condition, in combination with the personality disorder, the span of time during which Young committed his crimes, his recidivism, his persistent denial, and his lack of empathy and remorse, made it more likely than not that he would commit further sexually violent acts.
\textit{Id.} at 256.
\textsuperscript{212} \textit{Id.} at 260-61. “As the Washington Supreme Court held and the Ninth Circuit acknowledged, we proceed on the understanding that the Washington Act is civil in nature.” \textit{Id.}
\textsuperscript{213} *Seling*, 531 U.S. at 260.
\textsuperscript{214} \textit{Id.}
dard was unworkable and unnecessary. This new standard, however, may present application problems. Just how little control must a person have over himself before he may be committed under the Kansas Act?
3. **How Much and What Type of an Impairment Should Be Required? The United States Supreme Court Gives No Clear Answer**
The United States Supreme Court seems to have made *any* type of impairment of ability to control behavior and decisions—not merely physical inability to control—grounds for commitment. This is the most logical and workable standard, because differentiating between types of impairment could create problems in the application of the statute. Because such a standard has been adopted, there really is no need for an additional factual finding of such impairment, as impairment of some type is required by the DSM-IV in order to diagnose a disorder or abnormality in the first place.\(^{215}\) Therefore, the Supreme Court's requirement of a separate finding seems redundant, but is hopefully harmless from a practical standpoint.
The potentially larger problems arise when courts must decide just how much control over oneself is enough to escape commitment. Not only is the *type* of impairment needed unclear, but the *extent* of the impairment is also vague. Justice Scalia, in his dissent, highlighted the subjectivity of the majority's chosen standard.\(^{216}\) He questioned whether a percentage value, frequency ratio, or merely an adverb should be attached when charging a jury with what they must find in order to commit.\(^{217}\)
---
\(^{215}\) See Oral Argument, *supra* note 207, at *6-31*. Ms. Stovall made several references to this requirement being “part and parcel” to a psychiatric diagnosis and, therefore, there was no need for a separate finding of something inherent in an initial diagnosis. *Id.*
\(^{216}\) *Crane*, 534 U.S. at 422-24.
\(^{217}\) *Id.* “How is one to frame for a jury the degree of ‘inability to control’ which, in the particular case, ‘the nature of the psychiatric diagnosis, and the severity of the mental abnormality’ require?” *Id.* (emphasis in original).
Will it be a percentage (“Ladies and gentleman of the jury, you may commit Mr. Crane under the SVPA only if you find, beyond a reasonable doubt, that he is 42% unable to control his penchant for sexual violence”)? Or a frequency ratio (“Ladies and gentlemen of the jury, you may commit Mr. Crane under the SVPA only if you find, beyond a reasonable doubt, that he is unable to control his penchant for sexual violence 3 times out of 10”)? Or merely an adverb (“Ladies and gentlemen of the jury, you may commit Mr. Crane under the SVPA only if you find, beyond a reasonable doubt, that he is appreciably—or moderately, or substantially, or almost totally—unable to control his penchant for sexual violence”)? None of these seems to me satisfactory. *Id.*
It should be recognized that a disorder that impairs the ability to make a decision, because it severely distorts the person’s view of the situation, is just as dangerous as a disorder that compels a person’s behavior. Although the Supreme Court declined to rule on whether an emotional impairment would suffice under constitutional standards, presumably it would if the state could show it affected behavior and/or decision-making abilities to the extent required. After all, the Court noted in its opinion that it has historically declined to distinguish between certain types of impairments and, as Justice Scalia noted, doing so would not make sense.
In sum, the Kansas legislature included both emotional and volitional impairments in order to cover the range of disordered individuals who are both dangerous to society and sexually violent. The Kansas Supreme Court did not have the authority to overrule the United States Supreme Court’s earlier unqualified endorsement of the Kansas legislature’s enactment of the Kansas Act. It may not couch its disfavor of commitment legislation for sexual offenders in constitutional language and expect that language to legitimize its own personal objections to a law properly enacted by the state government. On the other hand, the United States Supreme Court does have the authority to overrule its earlier unqualified endorsement of the Kansas Act, and it seems to have done so here.
Having explored the judicial opinions on the subject, the following section of this Comment will address several arguments often embraced by scholars in opposition to civil commitment of sexual predators.
C. Common Arguments Opposing Civil Commitment of Sexual Predators
Scholars and commentators have advanced several arguments as to why civil commitment of sexual predators is unconstitutional. The most common arguments include the following: (1) the “slippery slope” argument; (2) these laws are examples of “reactionary legislation;” (3) the statutes are truly punitive in nature because treatment is delayed until the end of a prisoner’s incarceration; (4) civil commitment of sexual predators equals a life sentence for them; and (5) civil commitment statutes do not address the majority of offenders, who are never apprehended and convicted. This section discusses these arguments and why they fail to be persuasive.
1. The "Slippery Slope" Argument is Inadequate to Challenge Sexual Predator Commitment Laws
An argument often advanced by critics of sexual predator commitment laws is the familiar "slippery slope:"\textsuperscript{218} If we allow commitment of sexual predators, what will follow, commitment of robbers and drunk drivers?\textsuperscript{219} All criminals have relatively high recidivism rates, and many have antisocial personality traits, so the argument goes. However, this argument is flawed in four significant ways.
First, sexual predators as a class are distinguishable from general criminals. Their recidivism rates are dramatically higher than are those of other types of crimes.\textsuperscript{220} They are driven to criminality by serious mental conditions, often stemming from their own childhood abuse. They are not motivated by monetary need, as are many who rob. Even the Federal Rules of Evidence recognize sexual offenses as different from other violent crimes.\textsuperscript{221} As one commentator explained, "[w]here rape is involved, the rules of the game are simply different."\textsuperscript{222} Specifically, Rules 413-415 allow for admission of evidence in sex offense cases, both criminal and civil, that would otherwise not be admissible.\textsuperscript{223} The rules themselves, and the congressional discussion of them, clearly indicate the legislature's recognition of sex crimes as a category in need of and deserving special considerations.\textsuperscript{224}
\textsuperscript{218.} See Daley, \textit{supra} note 14, at 731-32; Dorsett, \textit{supra} note 14, at 144 (suggesting that drug addicts who commit robberies to obtain money or alcoholics may be committed under the Kansas statute).
\textsuperscript{219.} See Dorsett, \textit{supra} note 14.
\textsuperscript{220.} Barbara K. Schwartz & Henry R. Celini, \textit{Sex Offender Recidivism and Risk Factors in the Involuntary Commitment Process}, in \textit{The Sex Offender: Theoretical Advances Treating Special Populations and Legal Developments} 8-16 (Barbara K. Schwartz ed., 1997).
\textsuperscript{221.} See Fed. R. Evid. 412-415. For agreement with this argument, as well as further elaboration, see Isaac, \textit{supra} note 15, at 1314-15.
\textsuperscript{222.} Vivian Berger, \textit{Man's Trial, Woman's Tribulation: Rape Cases in the Courtroom}, 77 Colum. L. Rev. 1, 10 (1977).
\textsuperscript{223.} Federal Rule of Evidence 413 provides, in part: "In a criminal case in which the defendant is accused of an offense of sexual assault, evidence of the defendant's commission of another offense or offenses of sexual assault is admissible, and may be considered for its bearing on any matter to which it is relevant." \textit{Fed. R. Evid.} 413. Federal Rule of Evidence 414 provides, in part: "In a criminal case in which the defendant is accused of an offense of child molestation, evidence of the defendant's commission of another offense or offenses of child molestation is admissible, and may be considered for its bearing on any matter to which it is relevant." \textit{Fed. R. Evid.} 414. Federal Rule of Evidence 415 provides for admission of evidence of prior acts in civil trials. \textit{Fed. R. Evid.} 415.
\textsuperscript{224.} 140 Cong. Rec. H8991-H8992 (daily ed. Aug. 21, 1994) (statement of Rep. Molinari): The enactment of this reform is first and foremost a triumph for the public—for the women who will not be raped and the children who will not be molested because we have strengthened the legal system's tools for bringing the perpetrators of these atroMoreover, the harm inflicted by these offenders is primarily aimed at children, who are in special need of protection.\textsuperscript{225} Aside from murder, sexual abuse is possibly the most traumatic and horrible crime a victim could endure, with effects that last a lifetime. The harm is so great in these crimes, especially when the victim is a child, that these laws are necessary to protect the public from a different kind of criminal and a different kind of harm.
Second, although the concern over defining the proper candidates for involuntary commitment is extremely valid, there are statutory and procedural safeguards in place. For example, although many inmates may have antisocial personality traits, far fewer have antisocial personality disorders provable beyond a reasonable doubt.\textsuperscript{226} Some studies estimate seventy-five percent of incarcerated individuals exhibit antisocial personality traits.\textsuperscript{227} However, of these that are violent sex offenders in the first place, the mere exhibition of antisocial personality traits is not enough to warrant commitment under such a statute.\textsuperscript{228} There must be a finding of antisocial (or other) personality \textit{disorder}, not merely traits, and this disorder must be proven beyond a reasonable doubt.\textsuperscript{229} Further, all jurors must be unanimous in their decision.\textsuperscript{230} Then, the State has the burden of proving that this disorder is what causes the person to be likely to repeat his violent sexual crimes.\textsuperscript{231} This must also be proven beyond a reasonable doubt.\textsuperscript{232} All of these safeguards ensure that the government cannot become overzealous and seek to commit all sex offenders indefinitely.
Third, the evidence simply does not support a finding that prosecutors are abusing their discretion to continue to “punish” all sex offend-
\textsuperscript{225} The Supreme Court has upheld the protection of children as compelling, even when constitutional rights are at stake. \textit{See Ferber}, 458 U.S. 747 (upholding a conviction, despite a First Amendment challenge, under a law that banned the knowing promotion of a sexual performance by a child under age sixteen).
\textsuperscript{226} \textit{See Oral Argument, supra} note 207, at *9-14.
\textsuperscript{227} \textit{See id.} at *15.
\textsuperscript{228} \textit{See Kan. Stat. Ann.} §§ 59-29a01 - 59-29a20.
\textsuperscript{229} \textit{Id.} These diagnoses are based on the opinions of medical professionals, experts in their field. Further, the statutes provide for a defendant to have his own expert evaluation by a professional of his choosing, for which the State will pay if the defendant is indigent. \textit{Id.}
\textsuperscript{230} \textit{Id.}
\textsuperscript{231} \textit{Id.}
\textsuperscript{232} \textit{Id.}
ers, much less going after other categories of criminals.\textsuperscript{233} In Kansas, under two percent of sex offenders incarcerated for sexually violent crimes satisfying the statute have been committed pursuant to this law.\textsuperscript{234} In fact, of the few who have been committed, six are now in the process of release from the program.\textsuperscript{235} None of the six, though not completely discharged yet, has repeated their crimes.\textsuperscript{236} The statistics in other states are similar to Kansas.\textsuperscript{237} Prosecutors are only using this law in its intended capacity: to protect citizens from a “small but extremely dangerous group” of criminals.
Finally, the argument ignores the power of the courts to prevent the legislature from extending beyond constitutional bounds. The slippery slope argument cannot prevail as a reason to repeal these laws because it improperly dismisses the power of the courts. If the legislature began passing laws allowing for the civil commitment of burglars, for example, there is no question the courts would fulfill their duty and put a stop to the unconstitutional lawmaking.\textsuperscript{238} There is no need to prematurely assume the worst of the legislature and government when no evidence exists to support such a belief. The Supreme Court has often acknowledged its power to stop abuse of discretion if and when it arises.\textsuperscript{239} There is no occasion to doubt that power now.
2. \textit{The Validity of Legislation is Not Diminished Because the Public Requested It}
It is true, as many scholars assert, that this legislation was prompted largely by societal pressure.\textsuperscript{240} However, societal need is a perfectly legitimate reason to enact new legislation. After all, this pressure came from parents of young children who ride their bikes around their neighborhoods, much like Earl Shriner’s seven-year-old victim did before he was savagely attacked and left to die. It came from sisters
\begin{itemize}
\item \textsuperscript{233} See Brakel & Cavanaugh, \textit{supra} note 42, at 79-81.
\item \textsuperscript{234} \textit{Id.}
\item \textsuperscript{235} See Oral Argument, \textit{supra} note 207, at *20.
\item \textsuperscript{236} \textit{Id.}
\item \textsuperscript{237} See Brakel & Cavanaugh, \textit{supra} note 42, at 79-81.
\item \textsuperscript{238} The Court in Hendricks noted that most criminals, non-sex offenders, are better dealt with by the criminal justice system. \textit{See Hendricks}, 521 U.S. at 360.
\item \textsuperscript{239} \textit{See, e.g., Commodity Futures Trading Comm’n v. Schor}, 478 U.S. 833 (1986) (declining to place an absolute prohibition on particular types of jurisdiction to avoid where “some hypothetical ‘slippery slope’ may deposit us”); \textit{see also Ballew v. Georgia}, 435 U.S. 223 (1978) (exercising the Court’s authority to limit the number of jurors required to no fewer than six, despite an earlier ruling leaving the question open).
\item \textsuperscript{240} \textit{See generally Sarah E. Spierling, Lock Them Up and Throw Away the Key: How Washington’s Violent Sexual Predator Law Will Shape the Future Balance Between Punishment and Prevention}, 9 J.L. & POL’Y 879 (2001); Dorsett, \textit{supra} note 14; Brakel & Cavanaugh, \textit{supra} note 42; King, \textit{supra} note 14; Daley, \textit{supra} note 14.
\end{itemize}
and brothers of those women who had been brutally raped by men like David Burdette.\textsuperscript{241} It came from outraged and frightened citizens who were being forced to share their neighborhoods with sexually violent child molesters like Kevin Haenchchen,\textsuperscript{242} who should have been in a mental hospital rather than a suburban townhouse.
This pressure on legislatures to enact laws that would protect innocent victims, women and children who fell prey to these sex offenders, was not the result of a conspiracy to permanently imprison all the men in the country who had an occasional impure thought.\textsuperscript{243} It was the desire of a democracy to require a law to protect itself from the most dangerous and mentally ill recidivist criminals out there. Legislatures should be commended for responding, rather than condemned for doing so. It is their responsibility to act in the people’s best interests, and often the people know what is in their best interests and are capable of communicating it to their government. Chief Justice William Rehnquist has written:
[A] presumption of constitutionality [for Congressional enactments] makes eminent good sense. If the Supreme Court wrongly decides that a law enacted by Congress is constitutional, it has made a mistake, but the result of its mistake is only to leave the nation with a law duly enacted by the popularly chosen member of the House of Representatives and the Senate and signed into law by the popularly chosen President. But if the Court wrongly decides that a law enacted by Congress is not constitutional, it has made a mistake of considerably greater consequence; it has struck down a law duly enacted by the popularly elected branches of government, not because of any principle in the Constitution but because of the individual views of desirable policy held by a majority of the nine justices at that time. Every time an individual or a group asserts a claim of constitutional right against a legislative act, the principle of majority rule and self-government is placed on one side of the judicial scale, and the principle of the individual right is placed on the other side of the scale. The function of the Supreme Court is, indeed, to hold
\textsuperscript{241} David Burdette was already a serial rapist in 1982, when he then began raping women he saw in a magazine article describing them as Omaha’s ten most “eligible women.” He served less than six years for those rapes, was released, and began raping recently widowed women he discovered through the obituary columns in local newspapers. One woman was a young widow with two daughters, ages seven and eight. Burdette tied one child to each of the woman’s arms and forced them to watch their mother’s rape. \textit{See} Don Sternberg, \textit{Why Nebraska Needs Civil Commitment Proceedings for Sex Offenders}, 33 \textit{Creighton L. Rev.} 721, 721 (2000).
\textsuperscript{242} Kevin Haenchchen sexually assaulted twenty-seven children before being arrested in 1987. \textit{See} Daley, \textit{supra} note 14. He received only a ten-year sentence. \textit{Id.} at 715. Before being released from prison, Haenchchen promised to continue molesting children and decided to torture and kill them as part of a new fantasy he had developed. \textit{Id.} He stated, “I know what will happen when I see a child. There’s nothing a parent can do.” \textit{Id.}
\textsuperscript{243} \textit{See Kan. Stat. Ann.} § 59-29a01. The statute specifically targets “a small but extremely dangerous group.”
the balance true between these weights in the scale, and not to consciously elevate one at the expense of the other.\textsuperscript{244}
3. \textit{A Delay in Treatment Does Not Render the Statute Criminal in Nature}
A particularly compelling argument that the commitment laws for sex offenders are truly punitive lies in the fact that treatment is often delayed until such time that the offender is going to be released from prison.\textsuperscript{245} This situation may appear to prove that the commitment law is truly criminal in nature, and not civil, as it has been deemed. Although in the author's opinion, treatment should begin earlier, absence of such a provision does not render the law punitive.
First, the criminal laws do provide for incarceration when a person commits a violent sexual crime.\textsuperscript{246} There is no reason to revoke the state's right to punish merely because a provision to treat later is available. Criminal law is used as a method to achieve goals of deterrence, incapacitation, rehabilitation, and retribution.\textsuperscript{247} In fact, treatment for sex offenders is universally available for prisoners while incarcerated, but many refuse to accept it, as Hendricks refused during his prison career.\textsuperscript{248} Civil commitment laws are used to incapacitate and rehabilitate.\textsuperscript{249} They do not serve all of the purposes of
\textsuperscript{244} William H. Rehnquist, The Supreme Court 279 (2001). The analogy between state government enactments and federal governmental enactments can be easily drawn, especially given the Court's historic respect of and deference to a state's interpretation of its own laws. Seling, 531 U.S. at 270 (Scalia, J., concurring); see R.R. Comm'n of Tex. v. Pullman Co., 312 U.S. 496, 500-01 (1941).
\textsuperscript{245} See Hendricks, 521 U.S. at 385 (Breyer, J., dissenting).
\textsuperscript{246} See, e.g., 720 Ill. Comp. Stat. 5/12-13 (2002) (defining and describing Criminal Sexual Assault and providing sentencing requirements for repeat offenders); 720 Ill. Comp. Stat. 5/12-14 (defining Aggravated Criminal Sexual Assault); 720 Ill. Comp. Stat. 5/12-14.1 (defining Predatory Criminal Sexual Assault of a Child); 720 Ill. Comp. Stat. 5/12-15 (defining Criminal Sexual Abuse); 720 Ill. Comp. Stat. 5/12-16 (defining Aggravated Criminal Sexual Assault).
\textsuperscript{247} See Sandy Kadish & Stephen J. Schulhofer, Criminal Law and Its Processes 97-167 (6th ed. 1995).
\textsuperscript{248} Leroy Hendricks refused treatment several times during his ten-year sentence in Kansas. See John Douglas & Mark Olshaker, Obsession 346 (Lisa Drew ed., 1998). Treatment in prison is usually offered on a voluntary basis, and is often unsuccessful. "One reason for this is that, even among the numerous murderers and other felons, sexual offenders, especially child molesters, are despised by other inmates." See Daley, supra note 14, at 719-20. Because of this hatred, sex offenders will often not accept treatment, for fear that other inmates will learn of their crimes. Id.
\textsuperscript{249} See Hendricks, 521 U.S. at 363-66. The Court recognized that confinement of dangerously mentally ill persons for the protection of society "is a legitimate non-punitive governmental objective and has been historically so regarded." Id. at 363. Further, the Court noted that "incapacitation may be a legitimate end of the civil law." Id. at 366. See also Kan. Stat. Ann. §§ 59-29a01 - 59-29a20 (establishing civil commitment procedure for "the long-term care and treatment of the sexually violent predator").
criminal laws and thus should not be substituted for them. Further, civil commitment procedures allow for the Department of Corrections to perform its duties before the more expensive treatment provided by the statutes is imposed. It is the hope that civil commitment will not be necessary after incarceration, as is true in over ninety-eight percent of the Kansas cases involving sex offenders.\textsuperscript{250} Civil commitment is the last resort, the final hope for treating the severely ill, to be utilized only after efforts during incarceration have failed.
4. \textit{Civil Commitment of a Sexual Predator is Not Necessarily a Life Sentence}
The Kansas Act’s indefinite period of confinement is seen as a life sentence for many offenders. This may be true, but commitment will not result in lifetime confinement for those who accept and participate in their own treatment and who actively engage in their own recovery. Those offenders will be reintegrated into society as healthier citizens. Offenders who refuse to participate in the therapy will not rejoin society as soon, but is this undesirable from society’s standpoint? Of course it is undesirable from the offenders’ standpoint, but so is incarceration in the first place. The criminal justice system does not release criminals into society simply because they do not enjoy prison. Why should the mental health system release dangerous, untreated individuals into society just because they want to go? Violent sexual predators who deny they have a problem and refuse to take responsibility for their actions or participate in treatment have no place near our children. These are not the rehabilitated citizens ready to re-enter society, they are instead sick individuals in need of help, even if they cannot admit it.
The only criminals “harmed” by the civil commitment system are those completely untreatable predators who were fortunate enough to obtain a plea bargain for less than life imprisonment because, but for the statute, they would escape without treatment. Offenders who received life sentences, treatable or not, will not encounter the program, so it is irrelevant to them. Treatable offenders will receive help and be reintegrated into society. If mandatory life sentences were handed out for all those offenders described by the predator statute, many treatable offenders would never regain their liberty.
\textsuperscript{250} See Brakel & Cavanaugh, \textit{supra} note 42, at 79-81.
5. The Fact That These Statutes Do Not Prevent All Sexual Offenders from Victimizing Does Not Diminish Their Value
Civil commitment statutes keep some dangerous predators away from children. However, it has been argued that the “real” sex criminals are still at large, as many sex offenders are relatives of their victims and are never apprehended, and these statutes are ineffective at handling the problem.\textsuperscript{251} While many sex offenders are never caught, these statutes address a very dangerous group of criminals and are essential to preventing further crimes from being committed by the ones that are caught. Simply because these statutes do not address every type of sex offender does not diminish their appropriateness or justify their repeal. In fact, if they help reduce violent sex crimes at all, they should be applauded, not attacked.
Despite the many arguments in opposition of civil commitment for sexual predators, the courts, including the Supreme Court, continue to uphold its constitutionality. However, the Kansas Supreme Court’s decision could have had disastrous consequences had it been upheld. The following section addresses that issue as well as the impact the current requirements may have on the system.
IV. Impact of \textit{Kansas v. Crane}
The impact of the Kansas Supreme Court’s decision would have been frightening. The impossible standard would have completely undermined the Kansas Act, making it unenforceable. Fortunately, the United States Supreme Court prevented that result, but the impact of its decision is uncertain. If other courts interpret the decision as permission to require almost total lack of control, the result may be similar. On the other hand, as this Comment argues and Justice Scalia has asserted, the additional finding of a lack of control is unnecessary because it was already incorporated into the Kansas Act’s requirements. Based on this, the practical impact may be minimal, because if the State can prove a mental abnormality in the first place, some degree of inability to control will accompany the diagnosis and should be
\textsuperscript{251} See King, \textit{supra} note 14, at 1429.
The incarceration of a handful of individuals is insufficient to address the widespread problem of sexual abuse. Legislators may propose and enact sexually violent predator statutes and claim to solve the problem, yet the implementation of such laws protects but a small minority of victims. By assuming that incarceration is the appropriate remedy, lawmakers and the Supreme Court, which endorsed the legislation, ignore the need to delve deeper into the “silent epidemic” of the sexual abuse problem in the United States and to think creatively about methods of prevention.
\textit{Id.}
readily apparent to the fact finder. Thus, the burden of an additional finding may be small.
There will be courts, though, like the Kansas Supreme Court, that disfavor this type of legislation and will interpret the holding to mean an almost total lack of control must be proved. The potential impact in those jurisdictions is explored below. The effects discussed could also result if courts disallow emotional or cognitive impairments to function as a basis for commitment.
A. Danger to Society
If courts endorse an unreasonably high standard of proof, as the Supreme Court’s decision may allow them to do, many sex offenders will be free to commit violent sex crimes upon new victims. Although only between one and ten percent of sex offenders are subsequently committed under sexual predator statutes,\textsuperscript{252} that one or two or ten percent could be released into America’s neighborhoods rather than being detained in a treatment facility. A man like Earl Shriner, for example, could move in next door to a family with a seven-year-old boy, unhindered to continue his criminal plan.\textsuperscript{253} Children and women across the nation would again be subjected to convicted and violent sexual predators who served time in prison and were likely to have rejected therapy while incarcerated.\textsuperscript{254} These laws exist to protect society from the worst type of violent criminal, and their purpose is circumvented by insistence on an improvable standard.
B. Economic Impact
Although the physical and psychological harm done to victims of sex offenders is of course the foremost concern, impact on the economy is always a consideration when evaluating government legisla-
\textsuperscript{252} See Bracket & Cavanaugh, \textit{supra} note 42, at 81.
\textsuperscript{253} For a discussion of Earl Shriner’s crimes, see \textit{supra} INTRODUCTION.
\textsuperscript{254} It has been repeatedly noted that prisoners have a right to refuse treatment in prison, and sex offenders very often do refuse such treatment. Leroy Hendricks was one such prisoner who refused treatment for pedophilia while incarcerated. \textit{See} Hendricks, 521 U.S. at 354-55; \textit{see also} Robert M. Wettstein, \textit{Predators and Politics: A Psychiatric Perspective on Washington’s Sexually Violent Predators Statute}, 15 U. PUGET SOUND L. REV. 597, 614 (1992) (discussing how the “violent, threatening atmosphere of a correctional facility socializes an offender into never showing weakness or vulnerability and dissuades him from discussing his crimes, especially those involving the rape and murders of children”). This atmosphere apparently discourages many inmates from obtaining treatment while in prison. However, should society have to accept an untreated criminal because he refused treatment based on a fear of inmate aggression at the disclosure of his crimes? If an offender is not even willing to undergo treatment to ensure he does not re-offend when released, he is clearly not ready for release in the first place.
tion. Both the cost to victims for treatment and the government for providing therapy to the offenders must be considered.
Victims of the most violent and traumatic crimes may need inpatient psychiatric care for a period following the attack. The cost of this treatment begins at approximately $1000 per day and increases with the need for prescription drugs or other physical care.\textsuperscript{255} Victims without the money or need for inpatient care may opt for outpatient care; specifically, hour-long sessions with a psychiatrist or psychologist on a regular basis. Although it is impossible to determine the costs for the “average victim,” costs for this type of treatment can range from $2000 per year for monthly sessions in a midwestern suburban clinic, to $9000 per year for weekly sessions in the same clinic.\textsuperscript{256} These estimates vary by geographical location and do not include any prescription medications that may be needed. It is important to keep in mind that the average salary for an American is less than $30,000.\textsuperscript{257} Many victims of sex crimes cannot afford this expensive treatment, and turn to support groups or clergy members for counseling. These estimates provide a rough picture of the financial cost of therapy for victims, but cannot begin to encompass the other costs to a victim, including the possible loss of employment, deterioration of romantic relationships, and constant fear of another attack. These life-altering costs cannot be quantified.
The cost of treating sex offenders, which can be quantified, is also quite high. In 1998 and 1999, California spent just over $31 million on the sex offender program, about 2% of its mental health services budget.\textsuperscript{258} The cost per inmate treated was about $107,000.\textsuperscript{259} As more offenders are committed, obviously the total program cost will increase. Proportionately, however, only 2% of the California budget goes toward treating predators and, thus, possibly preventing future horrible crimes. Society’s safety from predators is surely worth such a small fraction of the government’s resources. Further, it is estimated that pedophiles have an average of 150 victims each,\textsuperscript{260} making the
\textsuperscript{255} Estimate obtained from St. James Hospital, Olympia Fields, Illinois.
\textsuperscript{256} Estimate obtained from Suburban Heights Medical Center, Psychiatry Department, Chicago Heights, Illinois. Specifically, for a victim of a sex trauma, a psychiatrist will meet with the patient for an initial consultation, at a cost of $237.00. The following week, the patient will attend another session, at a cost of $171.00. All subsequent visits will be billed at $171.00.
\textsuperscript{257} See Salary Too Small? Try Moving to Connecticut, U.S. News & World Rep., May 7, 2001, available at http://www.usnews.com (last visited Feb. 18, 2003) (reporting that average American salary in 2000 was $29,676).
\textsuperscript{258} See Brackel & Cavanaugh, \textit{supra} note 42. at 89.
\textsuperscript{259} Id.
\textsuperscript{260} See \textit{supra} note 33 and accompanying text.
cost of treating the victims of just one pedophile between $300,000 and $1.35 million annually.\textsuperscript{261}
If, however, courts are concerned about high treatment costs for offenders, awareness of these amounts may encourage imposing longer prison sentences for those offenders the courts deem “untreatable.” In fact, in the wake of the widespread enactment of sexual predator laws, many states have increased prison sentences for repeat sex offenders.\textsuperscript{262} More repeat sex offenders are now serving longer sentences, and some states have passed mandatory life sentences for certain sex offenders.\textsuperscript{263} Longer prison sentences clearly cut costs for treatment programs, as incarceration (including voluntary therapy) is about one-fifth the cost of formal sex offender treatment.\textsuperscript{264} If a certain offender is truly thought to be untreatable, then a life sentence may be the only way to protect society. However, many sex offenders are treatable, and should be given the opportunity to rehabilitate and make a positive contribution to society. The sexual predator laws allow for both scenarios, as offenders are released when and if they are deemed fit to rejoin society.\textsuperscript{265}
V. Conclusion
The advantages of allowing civil commitment for sexually violent predators pursuant to the Kansas Act are compelling and warrant the government’s exercise in restraining offenders’ liberty. Criminals who commit violent sexual crimes are among the most dangerous type of criminal. These offenders differ from other criminals in many ways, necessitating that they be considered in a category of their own for purposes of confinement, punishment, and treatment.
The civil commitment of sexual predators protects society from these dangerous individuals. Commitment also provides violent sex offenders with desperately needed treatment. Studies show that although treatment will not obviate the problem of recidivism entirely,
\textsuperscript{261}. See \textit{supra} note 256 and accompanying text (approximating costs for treatment of victims).
\textsuperscript{262}. \textit{See, e.g., Colo. Rev. Stat.} § 16-19-203 (1996).
\textsuperscript{263}. Lawrence A. Greenfeld, U.S. Dept of Justice, \textit{Sex Offenses and Offenders, An Analysis of Data on Rape and Sexual Assault} (1997). \textit{See, e.g., Cal. Penal Code} § 1170.12 (1998); 1996 Wash. Laws ch. 288 (amending Wash. Rev. Code § 9.94A.030 and § 9.94A.120(4) to authorize a mandatory life sentence for a second time serious sex offender). The Washington law is also known as the “two strikes and you’re out law.” Rachel Zimmerman, \textit{Two-Strikes Sex Crime Law Getting Second Test: Lake Forest Park Man is Charged}, Seattle Post-Intelligencer, July 10, 1997, at B1.
\textsuperscript{264}. \textit{See} Brackel & Cavanaugh, \textit{supra} note 42, at 88.
\textsuperscript{265}. \textit{See supra} notes 41-53 and accompanying text.
it does help.\textsuperscript{266} In fact, it may reduce recidivism by as much as thirty percent.\textsuperscript{267} States that have enacted sexual predator commitment laws have worked diligently to design programs that benefit the patient. The laws work toward achieving the goals they were designed to achieve—sexually violent offenders are kept away from society so they cannot harm more victims and treatment is provided to reduce or eliminate their recidivism upon release.\textsuperscript{268} These laws are constitutional, as evidenced by prior Supreme Court decisions, and necessary, as evidenced by tragedies such as one that occurred after Earl Shriner was released from prison.
The Supreme Court has consistently upheld civil commitment statutes for sexually violent offenders, and fortunately, is not discontinuing the practice now. However, the Kansas Act should have been enforced as written, without the modification required by the United States Supreme Court. Given the \textit{Selig} decision, as well as the \textit{Hendricks} decision, the Supreme Court had ample precedent to reverse the Kansas Supreme Court’s misinterpretation of \textit{Hendricks} and enforce the Kansas Sexually Violent Predator Act as it was written.
\textit{Jennifer Ann Smulin*}
\textsuperscript{266}. See Robert Prentky & Ann W. Burgess, \textit{Rehabilitation of Child Molesters: A Cost-Benefit Analysis}, 60 \textit{Am. J. of Orthopsychiatry}, 108, 108-17 (1992); William L. Marshall & William D. Pithers, \textit{A Reconsideration of Treatment Outcome with Sex Offenders}, 21 \textit{Crim. Just. & Behav.} 10, 10-27 (1994); G.C.N. Hall, \textit{Sexual Offender Recidivism Revisited: A Meta-Analysis of Recent Treatment Studies}, 63 \textit{J. Consulting & Clinical Psychol.} 802, 802-09 (1995).
\textsuperscript{267}. \textit{Id.}
\textsuperscript{268}. See \textit{supra} notes 40-64 and accompanying text. For discussion of treatment methods used in several states with sexual predator laws, see John Kip Cornwell et al., \textit{The New Jersey Sexually Violent Predator Act: Analysis and Recommendations for the Treatment of Sexual Offenders in New Jersey}, 24 \textit{Seton Hall Legis. J.} 1 (1999). The article describes the phase method employed by many states that is based on a cognitive-behavioral/relapse prevention model. \textit{Id.} at 16. “Increasing victim empathy, developing relapse prevention skills, acquiring interpersonal skills, managing negative emotions, and decreasing deviant sexual arousal are common elements of almost all programs reviewed.” \textit{Id.} at 19.
* Thank you to my grandparents and parents, and to Emily and Brian—for always believing in me. And special thanks to my mom, whose unconditional love and support have made me who I am.
|
OUR MISSION STATEMENT:
"Saint Francis Xavier Parish is a Christian Family dedicated to the mission of Jesus and the teachings of the Catholic Church. We are committed to fostering spiritual growth and to responding to the human needs of both our faith community and the larger community by sharing our gifts and resources."
Liturgy Schedule
Weekend Masses:
Saturday Evening Vigil: 4:30 pm
Sunday Masses: 8:30 am & 10:45 am
Holy Day Masses: See schedule inside.
Daily Mass: 6:45 am Mon, Thur, Fri
Tuesday 6:00pm
Weekend Mass Schedule in our area
| St. Michael-St. Peter | St. Mary's of the Lake | St. Patrick's Jordan |
|-----------------------|------------------------|----------------------|
| Sat. Evening: 5:30 pm | Sat. Evening: 4:30 pm | Sat. Evening: 5:00 pm|
| Sunday: 9:30 & 11:00 am | Sunday: 8:00 & 10:15 am | Sunday: 8:00 & 10:00 am |
PASTORAL STAFF:
Pastor: Rev. Daniel C. Muscalino*
Pastoral Associate: Deacon John J. Falge*
Religious Ed. Director: Renee O'Connor*
firstname.lastname@example.org
Parish Secretary:
Katherine Boos email@example.com
Social Service Coordinators:
Rosalie Durand, Patty White
Music Director:
Buildings and Grounds: Ron Schneider
Housekeeper: Kathy Welsh
Parish Historian: John P. Curtin
Confirmation Coordinators:
Laurie LeFever, Laura Carver
firstname.lastname@example.org
PARISH TRUSTEES:
Diane Foster*
Joseph E. Durand*
* Also on Parish Council
SACRAMENTS:
Baptism:
Class required.
Call rectory for arrangements.
Reconciliation: Sat. 3:30 - 4:00
Marriage: Arrangements at least 6 months in advance.
NEW PARISHIONERS are most welcome. See Fr. Muscalino after Mass or call the Rectory.
PARISH COUNCIL
Jay Austin Roxanne Taylor
Terry Hall Kim Flood
Pen Anderson Marty McClusky
Terry Kotlarz Carter Austin
Beth Cusick John Ketterer
Patty Gooden
FINANCE COMMITTEE:
Rick Reagan
Chris Hunt
Diane Foster
Rev. Daniel C. Muscalino
Joseph E. Durand
Jay Austin
Mass Intentions FOR THE WEEK
Saturday July 23
VIGIL SEVENTEENTH SUNDAY IN ORDINARY TIME
4:30 PM- James & Mary Quinn- Family
Sunday July 24
SEVENTEENTH SUNDAY IN ORDINARY TIME
8:30 AM- Carol Anne McGrath- The McGrath Family
10:45 AM- People of St. Francis Xavier
Monday July 25
SAINT JAMES
6:45 AM- Gerry Joyce- Friends
Tuesday July 26
SAINTS JOACHIM AND ANNE
6:00 PM- Elizabeth Donnelly- Mary McGreal
Wednesday July 27
No Mass
Thursday July 28
No Mass
Friday July 29
SAINT MARTHA
No Mass
Saturday July 30
VIGIL EIGHTEENTH SUNDAY IN ORDINARY TIME
4:30 PM- People of St. Francis Xavier
Sunday July 31
EIGHTEENTH SUNDAY IN ORDINARY TIME
8:30 AM- Tim Foster- Friends
10:45 AM- Nick Olenych- Kitty Reich and Luke Cole
Ministry Schedule for St. Francis Xavier
Saturday, July 30 Sunday, July 31
4:30 8:30 10:45
Lector Lector Lector
Mary Dailey Pat Canny Mike McAuliff
Altar Server Altar Server Altar Server
Blake LaPlant Mac Ciras Anna Carver
Jesse LaPlant Cecelia Carver
Holy Father’s Prayer Intentions
For the elderly - We pray for the elderly, who represent the roots and memory of a people; may their experience and wisdom help young people to look towards the future with hope and responsibility.
“What can there be too many children? That is like saying there are too many flowers.”
— Mother Teresa
What’s Happening This Week at St. Francis
Monday, July 25
Lectio Divina- Ch Hall- 6:30pm
Thursday, July 28
Rosary- Church- 7:00pm
Our Weekly Collection
Weekly Goal $7,900 Last Week: $7,485
Please Pray for...
Tom Palmer, Andy Nagen, Martin Long, J.E.L., Robert Darling, J. Michael Kelly, Barb Stapleton, Peyton James Perkins, Mary Dorsch, Msgr. Bob Yeazel, Gretta Petterson, Donna Martin, Robert Hammond, Richard Hammond, Isabelle Callahan, Riley Christine Perkins, Roger Schott, Danielle Painter, Genie Barnes, Nicole DeMore, Lisa Pikarski, Michelle Fleegeel, Justin MacLachlan, Jack Bragger, Missy Fitz, Austin Marlowe, Brett Kresco, Lucienne Henneberry, Allan Stonecipher, Kelly Cunningham Suberts, Sr. Mary Brendan Wilson
Please Pray for our Homebound...
Frieda Stopyro, Isabelle LaClair, Helen Volcko, Mike Berish, Marie Patterson, Mary Martin, Ethel Cole
Please call the Parish Office to add anyone to our Prayer List.
(315) 673-2531
Save the Date: Church of St. Michael and St. Peter's Doug's Fish Fry To Go fundraiser in support of their Twin Parish in Haiti. The Doug's To Go Truck will be in their parking lot on the corner of Velasko Road and West Seneca Turnpike on Wednesday, August 24 from 11 am until 6 pm. There will be cold drinks available for purchase and a bake sale! Indoor seating will be available. Don't miss it!
Adoration is the First Friday of each month. If you are interested in sitting for an hour with our Lord, please email Beth at email@example.com
Senior Lunch in Marcellus
As many of you remember, Peace, Inc. served a hot meal at the Marcellus Methodist Church for several years. Peace, Inc. as well as St. Francis Xavier are in discussion as to the possible return of these social gatherings. If it's possible to bring back the senior lunch, we need to have an idea of how many people would be interested. If you're interested, call Katherine at the rectory and give her your name & number?
Dear parish family,
The vestments that are worn at Mass date back centuries. The basic white under-vestment that altar servers, deacons, priests and bishops wear is called an Alb. The Alb is secured by a cord that is called a cincture. Over the Alb a deacon wears a stole over his left shoulder, across his chest and secured on his right side; the priest wears a stole over both shoulders; the stole is a symbol of the office, either of a deacon or a priest (a bishop also wears a stole in the same manner as does priest). The outer garment for a deacon is a Dalmatic, this has sides and sleeves, the priest and bishop’s outer garment, a Chasuble, is worn over the head without sleeves or sides. You can tell what liturgical season we are in, or what type of Feast or Solemnity we are celebrating by the color of the outer vestment.
Green: this is for Ordinary Time, after the Easter Season, until Advent and the Christmas Season and again from the end of the Christmas Season until the beginning of Lent.
Purple is worn for both Advent and Lent.
White (or Gold) is worn for the Christmas and Easter Season and all major Feasts, Solemnities and Holy Days. White is also the appropriate color for any time for a Feast of the Eucharist.
Red is worn for Passion (Palm Sunday), Good Friday, the Feast of the Triumph of the Holy Cross and any commemoration of Martyrs, Pentecost and any celebration of the Holy Spirit.
Rose (Pink) is worn twice a Liturgical Year: once during Advent and once during Lent: Gaudate and Lortari Sundays.
God Bless,
Fr. Dan
St. Joe’s Trunk Sale
Saturday, July 30th 9:00am – 2:30 pm
St. Joseph’s Church of Camillus
SELLERS WANTED! $20 Fee Per Space
For info, call St. Joe’s rectory office at 315-488-8490
CHARLEES Ice Cream Will Be Onsite!
Office of Family/Respect Life:
The Third Option- Building Better Marriages
Ending the Blame Game: It Feels Good
Online, Sunday, August 14th from 7 to 9pm
Contact Stephanie Stewart at firstname.lastname@example.org
Hope Appeal funded programs. All are welcome.
A Family Perspective by Bud Ozar – Today’s gospel guarantees if we “ask” we will “receive,” perhaps not what we wanted but what our Heavenly Father knows is best for us, His children. All the love we have for our children, God has for us and more. As our children trust us, we must trust “Our Father.”
Question of the Week - Daily Prayer
For Adults: Have your most recent prayers been prayers of praise, petition, or sorrow? Which kind of prayer do you need to practice more often?
For Children: What are you most thankful for right now?
Please visit our Facebook page or website to view the summary of the Synod Listening Sessions for the Syracuse Diocese. www.sfxmarcellus.com
Prayer for the Synod
We stand here before You, Holy Spirit, as we gather together in Your name.
With You alone to guide us, make Yourself at home in our hearts;
Teach us the way we must go and how we are to pursue it.
We are weak and sinful; do not let us promote disorder.
Do not let ignorance lead us down the wrong path nor partiality influence our actions.
Let us find in You our unity so that we may journey together to eternal life and not stray from the way of truth and what is right.
All this we ask of You, who are at work in every place and time, in the communion of the Father and the Son, forever and ever. Amen
Annual Parish Picnic: Sunday, August 7th from Noon - 4:00 pm
Come and enjoy a delicious lunch at Marcellus Park (in the pavilion across the parking lot from the playground) and mingle with other members of your parish family!!
Caring
Courteous
Confidential
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Mary Ryan Carlton
44 East Main Street
Marcellus, NY
315-673-2345
Reagan and Dailey
Attorney at Law
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Reagan Companies
Risk Management and Investment Services
3 E. Main Street • P.O. Box 191, Marcellus, New York 13108
315.673.2094 800.777.2094
ReaganCompanies.com
McClurg
DESIGN BUILD REMODEL REPAIR
(315) 673-2051
www.McClurgTeam.com
Trim • Take Down • Removal • Firewood • Mulch
Senior Citizen Discount • Insured
BUDGET TREE SERVICE
We Go Out On A Limb To Save You Money!!
6347 Newport Rd. 672-3417
Warners, NY 13164 - Free Estimate -
PAINTING
interior
Mitch Rudy
over 30 years exp.
315-254-0355
free estimates
email@example.com
Plis Funeral Home, Inc.
“Continuing the Commitment”
33 North Street
Marcellus, New York 13108
315-673-2017
www.plisfuneralhome.com
For Advertising Information
Call 463-1923
Renewal by Andersen
WINDOW REPLACEMENT an Andersen Company
RBAofCNY.com firstname.lastname@example.org
315-391-4762
ELITE PROPERTY MAINTENANCE, LLC
Driveway Sealing • Lawn & Landscaping
Snow Plowing • Interior Painting
FULLY INSURED FREE ESTIMATES
www.elitesyracuse.com 315-882-7430
SUPERIOR PRINTING
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Steve Rudy 315-463-1923 2025 Teall Avenue
|
Northside Middle School
JETS
Title I Handbook
2024-2025
Dr. Alma Mundy, Principal
500 Johnson Road
Warner Robins, GA 31088
Ms. Tiffany Davis, Family Engagement Liaison
Revised: July 30, 2024
A Parent’s Guide to Title I
What is Title I?
Title I, Part A is a federally funded program under the Every Student Succeeds Act (ESSA). The purpose of Title I under ESSA is to ensure that all children have a fair, equal, and significant opportunity to obtain a high-quality education and reach, at a minimum, proficiency on challenging state academic achievement standards and state academic assessments.
Which Houston County schools are Title I schools?
| Elementary Schools | Middle Schools | High Schools |
|-----------------------------|------------------------|--------------------|
| CB Watson Primary | Northside High | Tucker Elementary |
| Centerville Elementary | Northside Middle | Warner Robins Middle |
| Eagle Springs Elementary | Parkwood Elementary | Warner Robins High |
| Huntington Middle | Pearl Stephens Elementary | Westside Elementary |
| Miller Elementary | Russell Elementary | |
| Morning Elementary | Shirley Hills Elementary | |
| Northside Elementary | Thomson Middle | |
What supports are provided through Title I?
The Title I Program offers a variety of supports, which may include, but are not limited to, additional teachers, support staff, instructional materials and supplies, technology to support student learning, tutoring, professional development for school staff, and capacity building events for families.
What role does family engagement play in Title I?
Family engagement is an integral part of the ESSA law. Districts and schools receiving Title I funding must:
Ensure that high-quality academic assessments, accountability systems, teacher preparation and training, curriculum, and instructional materials are aligned with challenging State academic standards so that students, teachers, parents and administrators can measure progress against common expectations for student academic achievement.
Afford parents substantial and meaningful opportunities to participate in the education of their children.
Families,
You can have a tremendous influence on your child’s success in school. By partnering with the school and participating in the Title I program, you will:
- show your child that you support and value his/her education.
- be able to closely monitor your student’s progress.
- build stronger relationships between home and school.
- provide valuable input about schoolwide programs.
Research shows that students whose families are engaged perform better academically, socially and behaviorally. Be sure to become involved in your child’s school by:
- communicating regularly with your child’s teacher.
- attending academic-based family events and parent-teacher conferences.
- volunteering at the school.
- joining the Parent Action Team or School Council.
- providing input concerning the Title I program at the school.
Northside Middle School
THE CONNECTION
Family-School Engagement Plan and Compact
2024-2025
Dr. Alma Mundy, Principal
500 Johnson Road
Warner Robbins, GA 31088
www.nms.hcbe.net
Revision Date: 07/30/2024
What is Title I?
Northside Middle School is identified as a Title I school as a part of Every Student Succeeds Act (ESSA). Title I is designed to support State and local school reform efforts tied to the challenging State academic standards to improve teaching and learning for students. Title I programs must be based on effective means of improving student achievement and include strategies to support family engagement. All Title I schools must jointly develop with parents and family members a written parent and family engagement plan and compact.
What is it?
The Connection is a comprehensive document joining our School-Family Engagement Plan and Compact together with the purpose of strengthening the bond between school and home. The Engagement Plan describes how our school will provide opportunities to improve family engagement that will support student learning at school and at home. All students and their families are invited and encouraged to fully participate in the opportunities described in this plan. The school will provide information and as many opportunities as possible for the participation of all family members. The Compact explains what teachers, parents and students each will do in an effort to work together to make sure that our students reach grade level standards. The Compact is discussed with families during parent-teacher conferences throughout the school year.
How is it revised?
The Connection is jointly developed and revised by our school’s stakeholders. All families are invited to attend our annual Shared Decision-Making Meeting (SDM) held each spring. Families are asked to review and give feedback on the Engagement Plan, Compact, and budget including the 1% set aside. All feedback forms from the SDM Meetings are collected, reviewed and used to revise this document and to improve program planning for the next school year. Families who are not able to attend the SDM Meeting have the opportunity to provide input by completing an online feedback form and completing the Title I Parent Satisfaction Survey that seeks suggestions regarding the plan, compact and budget.
Who is it for?
The Connection is for all students attending a Title I school and their families. We encourage and invite families to fully participate in the opportunities described in this document. Our school will provide full opportunity for the participation of parents and family members with limited English, with disabilities, of migratory children and caretakers of students served in Neglected and Delinquent Centers.
Where is it available?
The Connection is included in our Title I Handbook that is provided to all parents and families at the beginning of the year or whenever a new student enrolls. The Connection is also available on our school website and in our Parent Resource Center. Families can request a copy at any time during the school year.
Let’s Stay Connected
Northside Middle School believes that family engagement means the participation of parents and family members in regular two-way, and meaningful communication involving student academic learning and other school activities. Here are the ways to stay connected and informed.
- Progress Reports and Report Cards
- Infinite Campus
- School Website
- Social Media
- School Messenger callouts
- Emails
- Parent-Teacher Conferences
- Newsletters
Access to Staff
- Our school has an open-door policy.
- All teachers and staff are available through email. See the school webpage for contact information.
- Conferences may be scheduled directly with your child’s teacher or through the Main office at (478) 929-7845.
Northside Middle School wants to help all our families participate in our family engagement activities. If you need assistance with childcare or transportation to attend the events listed in this plan, contact our Family Engagement Liaison for more information and assistance.
Tiffany Davis
(478) 988-6200 ext. 2771
firstname.lastname@example.org
2024-2025 District Goals
Increase by at least 2 percentage points the number of students performing at or above proficiency in core content areas in grades 6-8 as measured by Milestones assessment scores.
2024-2025 School Goals
Northside Middle School will increase our 2024 CCRI content mastery score by 3% in Language Arts, Math, Science, and Social Studies. Northside Middle School will also implement, with fidelity, research-based programs designed to address positive behaviors.
In addition, by the end of the 2024-2025 school year, 50% of the student population will meet or exceed their projected growth in reading and mathematics as measured by MAP (Measure of Academic Progress).
Grade Level: 6-8
Focus Areas
- Literacy
- Reading fluency and comprehension
- Math
- Operations with rational numbers
NMS will...
- Provide parents with their student’s MAP scores and encourage use of IXL for additional support in reading and math.
- Provide families with resources such as websites, newsletters, and videos related to Literacy and Math.
- Offer a variety of opportunities for parents to be involved in their student’s education, such as academic-based workshops, and Shared Decision-Making Meetings.
Families will...
- Set goals for students to help improve their Lexile score through working with IXL.
- Encourage students to work on skill plans using IXL.
- Utilize the school resources available, such as websites, newsletters and videos related to Literacy and Math to assist their student.
- Be an active participant in student’s education by taking advantage of academic-based workshops, meetings, and surveys initiated by the school.
Students will...
- Read daily and strive to reach the goal set to increase their Lexile score through IXL.
- Be diligent in accessing IXL to improve Math skills.
- Utilize school resources provided on websites, newsletter, and videos related to Literacy and Math.
- Attend academic-based workshops with parents.
July 31, 2024
Right to Know Professional Qualifications of Teachers and Paraprofessionals
Dear Parent(s) or Legal Guardian(s),
In compliance with the requirements of the Every Student Succeeds Act, the Houston County School System would like to inform you that you may request information about the professional qualifications of your student’s teacher(s) and/or paraprofessional(s). The following information may be requested:
• Whether the student’s teacher —
• has met State qualification and licensing criteria for the grade levels and subject areas in which the teacher provides instruction;
• is teaching under emergency or other provisional status through which State qualification or licensing criteria have been waived; and
• is teaching in the field of discipline of the certification of the teacher.
• Whether the child is provided services by paraprofessionals and, if so, their qualifications.
If you wish to request information concerning your child’s teacher’s and/or paraprofessional’s qualifications, please contact your child’s school or you may contact Dana Morris, Director of Federal Programs, at the Houston County Board of Education at (478) 988-6200 ext. 3449 or at email email@example.com.
Thank you for your interest and involvement in your child’s education.
Sincerely,
Dana Morris, Director of Federal Programs
Copyright Piracy Awareness Notification
Title I, Part A, Section 1116 of the Every Student Succeeds Act (ESSA) states that Title I schools shall provide information and resources to help parents and families work with their children to understand the harms of copyright piracy. In keeping with this requirement, definitions of relevant terms as well as links to websites that may be helpful in educating children concerning copyright piracy regulations are included below.
Copyright-a form of protection given to creators and authors of literary, dramatic, musical and artistic works. A copyright means that the author has the right to do or let other do any of the following things:
- Make copies
- Distribute copies
- Perform work publicly
- Display work publicly
- Make modifications or adaptations
Generally, it is illegal for anyone to do any of the things listed above without the creator’s permission. However, there are some exceptions and limitations to this right. One major limitation is the Doctrine of Fair Use.
Copyright Piracy-Unauthorized reproduction for sale or use of a copyrighted work such as a book, lyric or software.
Online Resources:
https://www.commonsensemedia.org/videos/teaching-kids-about-copyright-piracy
http://www.copyrightkids.org/
If additional assistance or more information is needed, the media specialist at your child’s school can help.
Title I Complaint Procedures
Although the below steps are recommended for the most efficient resolution at the lowest level, the parent/student has the right to by-pass these steps at any time and request an impartial due process hearing related to decisions or actions regarding your child’s identification, evaluation, educational program or placement. The parent and the student may take part in the hearing and have an attorney represent you at your own expense. The impartial Hearing Officer will be selected by the district. Hearing requests must be made to the Title Coordinator identified in Step II below.
Step I
The complaint shall be presented orally or in writing to the school principal within ten (10) calendar days after the most recent incident upon which the complaint is based. Any witness or other evidence should be provided at this time. The administrator will conduct an investigation and render a written decision within ten (10) calendar days of the filing of the complaint.
Step II
A complainant dissatisfied with the decision of the school principal may appeal to the System Title I/Title II Coordinator by submitting a written statement of complaint to the System Title I/Title II Coordinator. This statement must be filed within ten (10) calendar days after the complainant receives the decision from the school principal. The complaint should be mailed to:
Mrs. Dana Morris, Federal Programs Director
Houston County Board of Education
Post Office Box 1850
Perry, GA 31069
Phone: (478) 988-6200
firstname.lastname@example.org
Upon receipt of the written statement, the Title I/Title II Coordinator will schedule a meeting to attempt resolution of the concerns. The System Title I/Title II Coordinator will render a written decision within ten (10) calendar days after the meeting.
Step III
A complainant dissatisfied with the decision of the System Title I/Title II Coordinator may appeal to the Houston County Board of Education by filing a written request to the Office of the Superintendent. The complaint should be mailed to:
Dr. Mark Scott
Office of the Superintendent
Houston County Board of Education
Post Office Box 1850
Perry, GA 31069
The appeal must be filed within ten (10) calendar days after the complainant receives the decision from the System Title I/Title II Coordinator. The Board of Education will act on the complaint at the next scheduled BOE meeting.
PBIS
NMS PBIS Mission
The mission of Northside Middle School is to establish a strong foundation for life-long learning by preparing students to achieve their maximum potential.
| EXPECTATION | CLASSROOM | BATHROOM | CAFETERIA | HALLWAYS | GYMNASIUM |
|-------------|-----------|----------|-----------|----------|-----------|
| I AM POSITIVE | Use positive words | Give personal space | Use positive words | Use positive words | Use positive words |
| | Have positive interactions | | Use school language | Move peacefully to your next location | Move peacefully to your designated area |
| | Apply good manners | Be friendly | Exhibit appropriate behaviors | Include others in activities | Line up quickly when signal is given |
| I AM RESPONSIBLE | Be prepared with materials & supplies | Flush then wash hands | Use good manners | Gather food items and utensils the first time | Follow activity or assembly rules |
| | Complete all assignments | Wait your turn | Wait your turn | Move in a quiet orderly manner | |
| | Keep areas clean & safe | Avoid hanging out | Keep areas clean & safe | Keep areas clean & safe | Keep areas clean & safe |
| I HAVE INTEGRITY | Be honest | Respect privacy | Be honest | Report to designated area | Use equipment properly |
| | Stay in designated area | Use manners | Line up properly | Report concerning matters | Be a good audience member (assemblies) |
| | Report concerning matters | | | Listen watch | |
| I AM DISCIPLINED | Prompt | Move quietly, appropriately | Stay in your seat | Have a pass | Participate appropriately |
| | Follow instruction | Use time wisely | Talk quietly | Move like line in one direction | Remain with assigned teacher |
| | Enter/exit quietly | Enter/exit quietly | Refrain from throwing items | Use inside voices | |
| | Stay organized | | | Stay out of other classrooms | |
| I AM ENGAGED | Participate actively | Get in and get out quickly | Follow instruction | Walk on the right side | Show good sportsmanship |
| | Be productive | | Use inside voices | | Be productive/participate daily |
| | Protect resources | | | | |
What is PBIS?
Positive Behavioral Interventions and Supports (PBIS), is an evidence-based, data-driven framework proven to reduce disciplinary incidents, increase a school’s sense of safety and support academic outcomes.
The premise of PBIS is that continual teaching, combined with acknowledgement or feedback of positive student behavior will reduce unnecessary discipline and promote a climate of greater productivity, safety and learning.
PBIS schools apply a multi-tiered approach to prevention, using disciplinary data and principles of behavior analysis to develop school-wide, targeted and individualized interventions and supports to improve school climate for all students. (OSEP Technical Assistance Center on Positive Behavior Interventions & Supports). Excerpt from Middle Georgia RESA website.
What is PBIS at Northside Middle School?
Northside Middle’s School-Wide Expectations are for students to have PRIDE by:
- Being Positive
- Being Respectful
- Having Integrity
- Being Disciplined
- Being Engaged
These expectations, along with the appropriate positive behaviors, are displayed in different areas of the school as reminders for students. Students will receive ongoing instruction from staff on our school-wide expectations in all areas of our school. The classroom, restroom, cafeteria, hallways, bus, computer lab, media center, gym, and after-school activities are all settings where students will be expected to act in a prideful manner.
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IMPACT OF THE COVID-19 PANDEMIC ON SMEs IN CHINA, CROATIA, NORTH MACEDONIA AND SLOVENIA
Recommendations for improvement of policy measures
ISBN 978-953-7520-15-1 (e-book)
CEPOR – SMEs & Entrepreneurship Policy Centre (Croatia)
The Centre for Polish and Central and Eastern European Studies, School of International Studies, Sichuan University (China)
International Cooperation Centre, National Development and Reform Commission (China)
ESTIMA – Association for Strategy Creation, Research, Education and Promotion and International Values (North Macedonia)
Authors:
Mirela Alpeza
Ana Krstinosvska
Zhenyan Xi 席珍彦
Maja Has
Siyu Ang 昂思
Li Lei 李蕾
Editor:
Mirela Alpeza
Reviewers:
Ružica Šimić Banović, PhD, Associate Professor at Faculty of Law, University Zagreb, Croatia
Anamarija Delić, PhD, Associate Professor at Faculty of Economics in Osijek, J.J. Strossmayer University of Osijek, Croatia
English proofreading:
Ana Krstinosvska
Chinese translation:
Xinman Li 李欣蔓
Publisher:
CEPOR – SMEs and Entrepreneurship Policy Center
Trg J.F. Kennedy-a 7
10000 Zagreb, Croatia
www.cepor.hr
Copyright:
Global Partnership Center of Central Eastern European Countries and China
Financial support for the research and publication of the report was provided by
Global Partnership Center of Central Eastern European Countries and China
Parts of this publication may be reproduced unaltered without authors’ authorisation, on condition that the source is indicated.
Layout:
ACT PRINTLAB Čakovec, Croatia
Zagreb, June 2022
ISBN: 978-953-7520-15-1 (e-book)
# CONTENTS
1. **INTRODUCTION AND METHODOLOGY DESCRIPTION** ........................................... 5
2. **COUNTRY REPORT – CHINA** ............................................................................. 8
2.1. SME sector in China in 2020 ............................................................................ 8
2.2. Development difficulties and opportunities for Chinese SMEs in the post-pandemic era ......................................................................................................................... 9
2.3. Policy measures supporting SMEs in China during the COVID-19 pandemic .......................................................................................................................... 10
2.4. Recommendations for the improvement of policy support for SMEs during the pandemic .................................................................................................................. 17
2.5. Entrepreneurial response to opportunities emerged during the crisis caused by the COVID-19 pandemic – case studies from China .............................................................................. 19
3. **COUNTRY REPORT – CROATIA** ....................................................................... 24
3.1. Quantification of the impact of pandemic on SME sector in Croatia .................. 24
3.2. Policy measures supporting Croatian SMEs during the COVID-19 pandemic .... 36
3.3. Review of the adopted government measures for supporting SMEs during the pandemic in Croatia ........................................................................................................ 40
3.4. Entrepreneurial response to opportunities emerged during the crisis caused by COVID-19 pandemic – case studies from Croatia .............................................................................. 41
4. **COUNTRY REPORT – NORTH MACEDONIA** ..................................................... 45
4.1. Quantification of the impact of pandemic on SME sector .................................. 45
4.2. Policy measures supporting SMEs in North Macedonia during the COVID-19 pandemic .................................................................................................................. 51
4.3. Impact of the adopted government measures and recommendations for a successful economic recovery ........................................................................................................ 53
4.4. Entrepreneurial response to opportunities emerged during the crisis caused by COVID-19 pandemic – case studies from North Macedonia .............................................................................. 56
5. **COUNTRY REPORT – SLOVENIA** ..................................................................... 60
5.1. Quantification of the impact of pandemic on SME sector in Slovenia ............... 60
5.2. Policy measures supporting SMEs during the COVID-19 pandemic .................. 68
5.3. Review of the adopted government measures for supporting SMEs during the pandemic in Slovenia ........................................................................................................ 72
5.4. Entrepreneurial response to opportunities emerged during the crisis caused by COVID-19 pandemic – case studies from Slovenia .............................................................................. 74
6. **CONCLUSION AND RECOMMENDATIONS** ....................................................... 77
6.1. Impact of the pandemic on the small and medium enterprise sector in China, Croatia, North Macedonia and Slovenia .................................................................................. 77
6.2. Government measures to support the economy during the pandemic .............. 78
6.3. Review of the policy measures to combat the negative effects of pandemic on SMEs and recommendations .................................................................................. 80
7. **BIBLIOGRAPHY** .................................................................................................. 84
8. **LIST OF TABLES** .................................................................................................. 88
9. **LIST OF FIGURES** ............................................................................................... 89
INTRODUCTION AND METHODOLOGY DESCRIPTION
1. INTRODUCTION AND METHODOLOGY DESCRIPTION
Global Partnership Center of Central and Eastern European Countries and China (GPC) is a non-profit think tank association founded in 2019 that brings together organizations representing 17 Central and Eastern European Countries (CEEC) and China. The aim of the association is to encourage the exchange of information, implement joint research and education projects for the benefit of the countries involved, through cooperation of the organizations that are signatories to the Cooperation Agreement. GPC activities include support for the implementation of research focusing on key issues in the pragmatic cooperation between CEECs and China, which aims to promote the quality and efficiency through in-depth research on policies, laws and regulations, investment, business environment, etc.
In August 2021, an internal tender process was conducted among GPC member organizations for research projects aimed at researching:
1. China-CEECs Economic Cooperation in the Post-Pandemic Era;
2. Current Business Policies of CEECs and China, and Improvement Measures.
In October 2021, with the support of the GPC, a consortium composed of four organizations: CEPOR – SMEs & Entrepreneurship Policy Centre from Croatia, The Centre for Polish and Central and Eastern European Studies, School of International Studies, Sichuan University from China, International Cooperation Centre – National Development and Reform Commission from China and ESTIMA – Association for Strategy Creation, Research, Education and Promotion of International Values from North Macedonia, started the implementation of the project “Impact of the COVID-19 pandemic on SMEs in China, Croatia, North Macedonia and Slovenia – recommendations for improvement of policy measures”.
The aim of the research project was to provide a deeper insight into the impact of the pandemic on the small and medium enterprise (SME) sector in the four observed countries of focus (China, Croatia, North Macedonia and Slovenia), to provide an overview and critical review of the measures taken by governments to support the sustainability of small and medium enterprises during the pandemic, and point to examples of good practice in entrepreneurial responses to recognized pandemic opportunities leading into innovative products, services, processes or business models. The conducted research generated conclusions and recommendations based on good practice in supporting the small and medium enterprise sector in times of crisis.
The research conducted within the project included primary and secondary sources of information. Secondary sources of information relate to the collection of quantitative data on the performance of the SME sector in the pre-pandemic 2019 and pandemic 2020, and the comparison of indicators to identify the impact of the pandemic on sector performance. Sources of data for quantifying the impact of the pandemic on the SME sector included public institutions in the four observed countries. Publicly released (secondary) sources were also used to identify measures taken by governments to support the sustainability of the SME sector. Primary sources in the form of interviews with entrepreneurs were used to present examples of good practice related to innovative products, services, processes or business models created as a result of the perceived opportunity during the crisis. Innovative examples were identified through
media records and, in accordance with needs and capabilities, information was further collected by conducting semi-structured interviews with entrepreneurs. Entrepreneurs, together with identified economic analysts, were also the primary source of information for a critical review of government measures to support small and medium enterprises during the pandemic and to identify recommendations for better sizing and tailoring thereof, according to the needs of the sector. In addition to interviews with entrepreneurs and economic experts, data and reflections on the adequacy of measures and their effectiveness were collected through media statements by the representatives of entrepreneurs’ associations as well as data from published research on the subject.
Through discussion, the concluding part of the study compares the impact of the crisis on the SME sector in the four observed countries, identifies positive aspects of government measures to support the SME sector during the crisis, and identifies recommendations in the form of lessons learned that can be useful to policy makers in situations where support for the sustainability of the sector is necessary due to the impact of external adverse conditions.
COUNTRY REPORT
CHINA
Zhenyan Xi 席珍彦
Siyu Ang 昂思
Li Lei 李蕾
2. COUNTRY REPORT – CHINA
2.1. SME sector in China in 2020
On March 1st 2020, the Center for Urban and Competitiveness Research of the Chinese Academy of Social Sciences and the Enterprise Chacha Big Data Institute jointly released the “2020 China Enterprise Development Data Annual Report” which showed that as of February 2021, there were 144 million market entities in operation in China, including 44.572 million enterprises and 96.046 million individual industrial and commercial households.
The China Household Finance Survey and Research Center of Southwest University of Finance and Economics conducted a nationwide survey on the impact of the Covid-19 pandemic on SMEs in August 2020 and December 2020. The survey data showed that among SMEs\(^1\), there were 11 percent micro, 17 percent small and 72 percent medium companies. The number of micro, small and medium enterprises in China, and the share of employment in micro, small and medium companies is indicated in Table 1.
Table 1: Number of enterprises and employees with regard to size
| Economic criterion | Enterprise size |
|--------------------|-----------------|
| | Micro | Small | Medium |
| Number of enterprises (approximation) | 93.6 million | 22.1 million | 14.3 million |
| Median number of employees | 5 | 11 | 29 |
| Proportion of employed persons | 41% | 22% | 37% |
Sources: China Household Finance Survey and Research Center of Southwest University of Finance and Economics
On January 27, 2020, the Ministry of Finance released the publication on “Economic Operation of State-owned and State-controlled Enterprises Nationwide from January to December 2020”. The report showed that the total business income of the state-owned enterprises in 2020 amounted to 8,283.61 billion EUR. According to the official website of the Central Government of the People’s Republic of China, the total business revenue of the top 500 private enterprises in 2020 is 4.6 trillion EUR. According to the same source, there are more than 750 million employees in China, with almost half of them working in the tertiary sector (Table 2).
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\(^1\) According to the VAT threshold and the criteria for identifying small-scale taxpayers, SMEs can be classified into three categories according to their annual business income: medium (annual business income of over $5 million), small (annual business income of $1.2 million to $5 million) and micro (annual business income of less than $1.2 million).
Table 2: Number of employees in China in 2020
| 2020 Employed persons | Numbers |
|-----------------------|---------|
| 2020 Employed persons (10,000) | 75,064 |
| Employed in the primary sector (10,000) | 17,715 |
| Employed persons in the secondary sector (10,000) | 21,543 |
| Employed in the tertiary sector (10,000) | 35,806 |
Sources: The Central People's Government of the People's Republic of China. http://www.gov.cn/
2.2. Development difficulties and opportunities for Chinese SMEs in the post-pandemic era
The sudden COVID-19 pandemic had significant impact on many private enterprises, especially SMEs. Data show that in the first quarter of 2020, more than 460,000 private small and micro enterprises were closed and bankrupt. While the SMEs have encountered difficulties, the pandemic led to the emergence of new business models. Therefore, the post-pandemic era brings both difficulties and opportunities for SMEs.
Difficulties
(1) **Economic downward pressure**: In recent years, with the slowdown of the global economy and the decline in export growth caused by trade frictions, the downward pressure on China’s economy has intensified. SMEs were already facing great difficulties and the pandemic further amplified the downward pressure on the economy.
(2) **Global economic changes**: The pandemic further emphasized the need for companies to embrace digitalization in order to survive. The financial dilemma of ultra-low interest rates and high leverage appeared. The breakage and restructuring of global industry chains affected SME development. The pandemic also affected SMEs imports and exports, as well as cross-border investment.
(3) **Cash flow management dilemma**: The pandemic has led to a reduction or transfer of orders. The flow of raw materials, labor and other production factors required by enterprises is not smooth, the upstream and downstream supply chains are not connected and production cannot be carried out normally. Sales channels are blocked, and finished products cannot be sold as usual. Rigid expenses cannot be avoided.
(4) **Difficulties in financing**: The enterprises’ either lack or have imperfect risk mechanisms to cope with the pandemic.; Low loan approval rates and low quotas make it difficult to meet financing needs in the post-pandemic era. The lack of matching financial institutions further affects SMEs which face an inability to raise funds and insufficient financing under the impact of the pandemic.
(5) **Difficulty in implementing government policies**: According to relevant studies, the State promulgated many measures to help SMEs during the pandemic, but 2/3 of SMEs
still indicated that they did not enjoy preferential subsidies. Formalities in the process of implementing government policies at all levels need to be improved, and the gap between policy expectations and implementation needs to be reduced.
**Opportunities**
1. **Strong government policy support**: Measures have been targeted to help SMEs reduce tax burden, lower operating costs, reduce expenses and increase job stabilization efforts.
2. **Digital transformation presents opportunities**: The companies’ own awareness of digital transformation has increased significantly. The foundations for digital transformation are more solid.
3. **The high-tech industry has seen opportunities**: It has promoted the use of 5G, big data and artificial intelligence; the development of medical equipment, medical materials and other pharmaceutical industries; the rapid development of industrial Internet, artificial intelligence, biomedicine, medical equipment and other emerging industrial technologies; and the development of the Internet economy.
4. **International trade opportunities**: The post-pandemic era has facilitated the development of digital trade and lowered the entry barriers to international trade. This will provide a convenient platform and valuable opportunities for product innovation and brand creation for the SMEs, potentially reshape the status of the global industrial division of labor and promote the industry in the middle and high end of the value chain.
### 2.3. Policy measures supporting SMEs in China during the COVID-19 pandemic
In order to strengthen the capacity of small and medium enterprises to ensure business sustainability despite the changed business environment affected by the pandemic, support measures for entrepreneurs were introduced at the national and local level, which primarily focused on four key areas:
1. Enhancing financial support
2. Reducing the burden of taxes and fees
3. Reducing operating costs
4. Increasing efforts to stabilize jobs.
**Increasing financial support**
For small, medium and micro enterprises in cultural and creative industries that have suffered large losses in project investment during the pandemic and have difficulty to resume normal operations within a short period of time, provinces opened green channels for financial services, helping them to overcome economic difficulties with measures such as lowering loan interest rates, extending loan terms, reducing or waiving financial service fees, and extending the collection of financial lease rentals.
Implementing and improve tax incentives
The State Taxation Administration issued the Notice on Optimizing Tax Payment Services in line with the Prevention and Control of the New Coronavirus Infection Pneumonia Epidemic. They proposed that the deadline for filing tax returns be extended according to the needs to ensure epidemic prevention and control. Taxpayers and withholding agents affected by the pandemic could also apply for further extensions in accordance with the law if they still have difficulties to process the tax returns after the deadline for filing tax returns was extended in February 2020.
Further reducing the social burden on SMEs
The Ministry of Information Technology's "Notice on Measures Aimed to Help SMEs Resume Work and Production to Overcome the Difficult Period in Response to the New Coronavirus Pneumonia Epidemic" proposes 20 measures in terms of tax reduction, fiscal support, financial support, innovation and public services, requiring relevant departments and local authorities to actively introduce policies to support SMEs from various aspects.
Policy provisions to support SMEs to accelerate technological transformation
Shanghai’s Opinions on “Actively Responding to the Epidemic to Promote the Steady and Healthy Development of Cultural Enterprises” vigorously develop new cultural industries such as digital publishing, digital reading, animation and games, short video, e-sports, intelligent broadcasting, digital entertainment, online education, and digital cultural blogs, and promote the digitalization and intelligent transformation of traditional cultural industries.
In order to support the sustainability of the small and medium enterprise sector during the COVID-19 pandemic, in addition to government measures at the national level, measures at the level of provinces / municipalities have also been created. Table 3 shows the Chinese local government policies and measures to support the SMEs’ deal with the effects of the COVID-19 pandemic in 2020. The description of measures is quoted directly from original government documents, found at official websites of China’s local governments.
| Provinces/municipality | Documents | Content | Date |
|------------------------|---------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------|
| Beijing | Measures to Promote the Sustainable and Healthy Development of the SMEs in Response to the Impact of the Covid Outbreak | 16 measures have been formulated to effectively mitigate the impact of the epidemic on the SME production and operations, to help them overcome the difficult times and stabilize their development. | 5 Feb., 2020 |
| Shanghai | Several Policy Measures for Supporting the Stable and Healthy Development of Service Enterprises in Shanghai by Making Every Effort to Prevent and Control the Epidemic | In order to support small and medium-sized enterprises which rent operating properties of state-owned enterprises in the city to engage in production and business activities, they will initially be exempted from paying two months’ rent for February and March (this includes various development zones, industrial parks, technology business incubators, etc.). | 8 Feb., 2020 |
| Shandong Province | Several Opinions on Supporting the Stable and Healthy Development of SMEs in Response to the COVID-19 | Twenty suggestions have been made to support the stable and healthy development of SMEs by strengthening financial support, reducing the burden of taxes and fees, lowering operating costs and increasing efforts to stabilize jobs. Measures were also taken to alleviate the cost pressure on enterprises through rent reductions for SMEs, extension of contract performance periods, establishment of additional business incubation bases, subsidies for park operations, increased support for guaranteed loans for business start-ups, reduction of logistics costs for enterprises and “non-stop supply of unpaid fees”, etc. | 4 Feb., 2020 |
| Zhejiang Province | Opinions of the Leading Group for Prevention and Control of Pneumonia Infected by the Coronavirus in Zhejiang Province on Supporting Small and Micro Enterprises to Overcome Difficult Times | Increased support for SMEs foreign exports. | 5 Feb., 2020 |
| Province | Policy Title | Summary | Date |
|---------------|------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------|
| Guangdong | Several Policy Measures on Supporting Enterprises to Resume Work and Production to Promote Stable Economic Operation in Response to the COVID Outbreak | A total of 20 policy measures in 5 areas have been proposed to increase the efforts related to resuming work and production, reducing employment costs, alleviating the burden of business operation, increasing financial and fiscal support and optimizing government services, targeting various enterprises affected by the epidemic. | 6 Feb., 2020 |
| Jiangsu | Policy Measures on Supporting SMEs to Mitigate the Impact of the Epidemic and Maintain Stable and Healthy Development | A total of 22 initiatives in four areas, including reducing the burden on enterprises, strengthening financial support, stabilizing employment protection and coordinating protection services, will actively support and help SMEs in the province to resume work and production as soon as possible, alleviate the impact of the epidemic, overcome operational difficulties, enhance coping capacity, boost confidence in development and maintain stable and healthy development. | 12 Feb., 2020 |
| Qinghai | Policy Measures to Support the Development of SMEs and Promote the Commencement and Resumption of Major Projects in Response to the Epidemic | Six chapters and 27 specific measures are proposed to coordinate the prevention and control of the epidemic with the restoration of economic and social order, stabilize growth, expectations and supply, minimize the impact of the epidemic to the greatest extent possible, maintain smooth economic operation, social harmony and stability, provide support and protection for winning the battle against the epidemic, and strive to achieve the objectives and tasks. | 17 Feb., 2020 |
| Gansu | Several Measures to Support the Stable and Healthy Development of SMEs in Response to the Epidemic | The document proposes six aspects, including ensuring the orderly resumption of production, improving financing services, stabilizing employment, financial and taxation support, reducing operating costs and strengthening service protection. It aims to effectively solve the production and operation difficulties of SMEs and support their stable and healthy development. | 14 Feb., 2020 |
| Henan | Several Policy Measures to Support the Stable and Healthy Development of SMEs in Henan Province in Response to the Impact of the Epidemic | A total of 20 specific measures have been introduced to support the stable and healthy development of SMEs in five areas, including making every effort to ensure the orderly resumption of work and production, strengthening financial support, enhancing financial and taxation support, reducing the burden on enterprises and increasing support for job stability. | 13 Feb., 2020 |
| Province | Document Title | Description | Date |
|---------------------------------|---------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------|
| Yunnan Province | Opinions of the People’s Government of Yunnan Province on 22 Measures to Stabilize Economic Operations in Response to the Epidemic | The goal is to do a good job in the prevention and control of the epidemic, while solving the outstanding difficulties and problems in terms of economic operation, and promoting the stable operation and healthy development of the economy. | 12 Feb., 2020 |
| Shaanxi Province | Opinions on Resolutely Winning the Battle of Epidemic Prevention and Control and Promoting Stable and Healthy Economic Development | The document consists of 22 initiatives in seven areas aimed to ensure the orderly resumption of production by reducing the burden of taxes and fees, deferring payment of social security fees, strengthening financial support, increasing financial subsidies, and supporting enterprises to employ and stabilize jobs. | 10 Feb., 2020 |
| Guizhou Province | Circular on Response to the Epidemic for Smooth and Healthy Development of SMEs | 15 measures have been launched in five major areas, including effectively reducing the tax burden on enterprises, further reducing the operating costs of enterprises, helping enterprises to relieve financial pressure, making every effort to ensure normal production of enterprises, and improving and optimizing service protection, to support SMEs to actively cope with the impact of the epidemic and promote the stable and healthy development of SMEs. | 8 Feb., 2020 |
| Ningxia Autonomous Region | Measures to Address the Impact of the COVID to Promote the Healthy Development of SMEs | The document puts forward 18 “hardcore” measures in five areas to support SME development. They include increasing credit support, stabilizing the workforce, reducing the burden of enterprises, increasing financial support and strengthening service protection, in order to help boost the confidence of SMEs and stabilize their operations. | 8 Feb., 2020 |
| Guangxi Zhuang Autonomous Region| Several Measures to Support Winning the Epidemic Prevention and Control Battle and Promoting Smooth Economic Operation | 30 specific measures were launched to help enterprises resume work, prepare for the spring plowing and boost consumption, while at the same time do a good job in preventing and controlling the epidemic, and coordinate the work of reform, development and stability to achieve the annual economic and social development goals and tasks. | 7 Feb., 2020 |
| Province/Region | Policy Title | Description | Date |
|--------------------------|------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------|
| Inner Mongolia Autonomous Region | Policy Measures on Supporting the Healthy Development of Enterprises Focused on the Prevention and Control of the Epidemic and SMEs Affected by the Epidemic and Having Difficulties in Production and Operation | Measures are taken in four areas: increasing financial support, reducing the burden on enterprises, optimizing approval services and providing good business services to help enterprises overcome the difficult times. | 7 Feb., 2020 |
| Hainan Province | Eight Measures to Support SMEs to Tide Over the Difficulties in Response to the Covid in Hainan Province | Eight specific support measures have been introduced in four areas, with specified responsible units and implementation deadlines. They include stabilizing enterprise employment, reducing operating costs, easing the burden of taxes and fees, and strengthening financial support, with a focus on supporting SMEs affected by the pneumonia epidemic (caused by the new coronavirus infection) and experiencing difficulties in production and operation. | 5 Feb., 2020 |
| Sichuan Province | Policy Measures to Address the COVID to Alleviate the Production and Business Difficulties of SMEs | 13 specific policy measures are proposed in four areas: increasing support for burden reduction, increasing financial support, increasing financial and taxation support and increasing support for job stability, effectively alleviating the production and operation difficulties of SMEs, and resolutely winning the battle against epidemic prevention and control. | 5 Feb., 2020 |
| Hunan Province | Several Policy Measures to Address the Impact of the COVID and Promote Healthy Business Development | The measures provide real support for enterprises to deal with the impact of the epidemic and promote healthy development. They include financial subsidies, social security contributions, tax support, to ensure stable employment, stable foreign trade and foreign investment, to ensure smooth logistics and optimize the business environment. | 18 Feb., 2020 |
| Hubei Province | Circular on the Issuance of Policy Measures to Support SMEs to Overcome the Difficulties in Response to the Covid | Support policies are proposed in various aspects, such as reducing water and gas costs, reducing rent, increasing credit support, reducing financing costs, reducing or waiving relevant taxes and fees, deferring social insurance contributions and increasing employment subsidies. | 9 Feb., 2020 |
| Province | Policy Title | Description | Date |
|---------------|------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------|
| Jiangxi Province | 20 Policy Measures on Effective Response to the Epidemic to Stabilize Economic Growth | In order to further strengthen support for enterprises producing epidemic prevention and control materials and essential goods, help real enterprises overcome difficulties, and increase efforts to stabilize jobs and promote employment, a total of 20 policy measures in 4 areas have been proposed to fully support and promote enterprises and employees affected by the epidemic to resume work and production as soon as possible. | 4 Feb., 2020 |
| Tianjin | Notice of the General Office of the Tianjin Municipal People's Government on the Issuance of Certain Measures for Winning the Prevention and Control of the COVID in Tianjin to Further Promote Sustainable and Healthy Economic and Social Development | On the basis of continuous tax reduction and fee reduction for enterprises, the measures have been upgraded and strengthened again, by adopting 21 measures in 6 areas, fully reflecting the three characteristics of providing relief for enterprises, focusing on the practicality of policies and playing a market-oriented role, and truly helping enterprises to "lighten up" with "real money and silver". | 7 Feb., 2020 |
| Jilin Province | Measures to Support SMEs to Maintain Business and Stable Development in Response to the COVID | The four initiatives revolve around efforts to reduce labor costs and stabilize the workforce; promote tax cuts and fee reductions to reduce the burden on enterprises; increase financial support and properly address financing for enterprises in difficulty; and improve policy implementation and ensure a strong policy effect. | 7 Feb., 2020 |
| Liaoning Province | Several Policy Measures to Support the Production and Operation of SMEs in Liaoning Province in Response to the COVID | Twenty-five initiatives, such as financial subsidies, tax breaks, rebates on social security contributions and rent reductions, promote the role of SMEs in epidemic prevention, healthy operation and smooth development. | 6 Feb., 2020 |
| Fujian Province | Measures to Combat the COVID Outbreak in Fujian Province and to Ensure “Six Steady” Efforts | All departments are required to conscientiously implement the work of prevention and control of the epidemic, and at the same time, to coordinate the work of reform, development and stability, to fully support and organize the activities for various types of production enterprises to resume work and production, and to promote sustainable and healthy economic and social development. | 6 Feb., 2020 |
| Province | Document Title | Summary | Date |
|-------------------|---------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------|
| Heilongjiang | Policy Opinions of the General Office of the People's Government of Heilongjiang Province on Supporting the Healthy Development of SMEs in Response to the COVID Outbreak | It is important to support the healthy development of small, medium and micro enterprises affected by the new coronavirus-infected pneumonia epidemic and experiencing difficulties in production and operation, as well as to provide corresponding policy support in areas such as enterprise fiscal and tax policy, stabilizing the workforce of enterprises, increasing financing support, helping enterprises stabilize production, and reducing the burden of production and operation of enterprises. | 5 Feb., 2020 |
| Chongqing | Twenty Policy Measures of the General Office of Chongqing Municipal People's Government on Responding to the COVID and Supporting SMEs to Tide Over the Difficulties | The burden on enterprises is further reduced through tax relief, tax deferment, deferral of social insurance premiums, providing aid to enterprises to stabilize jobs during the epidemic period, reduction of the burden of housing fund contributions and rent relief. | 4 Feb., 2020 |
| Hebei Province | Measures to Combat the Prevention and Control of the COVID and Promote Stable and Healthy Economic and Social Development | The document envisages 30 powerful measures in seven areas, aimed to continue to promote the prevention and control, to upgrade, strengthen, and promote the province’s stable and healthy economic and social development, to protect the supply of materials during the epidemic, to support enterprises to resume work and production, and to increase fiscal and financial support. | 4 Feb., 2020 |
Sources: the official websites of China’s local governments
### 2.4. Recommendations for the improvement of policy support for SMEs during the pandemic
Small and medium enterprises (SMEs) are the foundation of people’s livelihood. An SME in operation solves the employment of a few, a dozen, dozens or even hundreds of people, and fulfils people’s basic livelihood needs. But the SMEs were most affected during the pandemic. The necessary quarantine measures taken to prevent and control the pandemic led to the stagnation of economic operations, with SMEs bearing the brunt of the impact. As foreign pandemics are not controlled and imported pandemics occur from time to time, local quarantine measures are taken to restrict the movement of people and economic operations. Support for SMEs is also the basis of the “Six Stabilities” and “Six Guarantees”, especially in terms of employment, ensuring people’s livelihood and protecting market players are the foundation of the “Six Guarantees”. In this regard, the government should introduce some effective policies to support SMEs\(^2\).
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\(^2\) The policy recommendations were identified based on the interview conducted with Chinese economist Zhu Fangming.
First, providing fiscal support. It is important to provide SMEs with tax cuts, fee reductions, financial subsidies and related subsidies to reduce the burden of enterprises, as well as to improve relevant laws and regulations, speed up the establishment of a comprehensive disaster management system and financial emergency mechanism, so that various support policies can be put in place quickly. As the pandemic disaster is a force majeure, enterprises should be allowed to use the extension of tax declaration procedures in accordance with the regulations, and the taxation department should not fine for overdue tax, impose administrative penalties, or adjust the tax credit evaluation. There should be increased support for re-employment through fiscal policies, unemployment insurance stabilization rebates, vocational training subsidies and support for the SMEs human resources, so that those unemployed due to the pandemic can quickly resume employment.
Second, offering financial support. Active financial policies should be adopted, alongside an increase in the financial support for the enterprise production after the disaster. In coordination with commercial banks, the Central Bank has adopted favorable policies for enterprises severely affected by the pandemic, service industries, labor-intensive enterprises that absorb a large number of jobs and relevant high-tech enterprises, and has implemented liberal financial policies to support them. It opened a “green channel” for post-disaster reconstruction loans, so that those enterprises with good credit records and in urgent need of funds can obtain loans as soon as possible, due to the “force majeure” nature of the pandemic disaster. SMEs that are affected by the pandemic and have difficulties to repay loans should not be withdrawn, cut off or penalized, and should be supported by renewing loans on expiry and given appropriate support in the form of lower interest rates.
Third, establishing a good insurance payout mechanism. It is essential to provide timely insurance claims to give enterprises the financial compensation they need to recover and develop, as well as to promote innovation in the insurance system and introduce new varieties suitable for pandemic disasters to provide insurance support to enterprises and people for post-disaster reconstruction., They could include adding insurance to cover business interruption caused by the pandemic, thus solving the problem of widespread work stoppage, long downtime and serious losses caused by the pandemic.
Fourth, sustaining demand and creating a good macroeconomic operating environment. It is necessary to strengthen demand-side management, open up blockages, make up for shortcomings, cut through production, distribution, circulation and consumption, and form a good macroeconomic operating environment in which demand pulls supply and supply creates demand. Expanding domestic demand is closely related to and complementary to livelihood projects and the development of the SMEs. Improving people’s livelihood is an important focus point for expanding consumer demand, hedging the impact of the pandemic and promoting high-quality domestic economic development. In the post-pandemic period, apart from providing support to the SMEs through various fiscal and financial instruments, expanding domestic demand is one of the most sustainable relief measures. By expanding domestic demand, enterprises can gain access to production opportunities in the market and provide people with employment opportunities, hence it is more dynamic and sustainable than fiscal subsidies and funding.
Effective policies of Japan – an example of good practice
It is particularly interesting to analyze the approach in supporting the sustainability of the small and medium enterprise sector in other countries.
(1) In April 2020, the Japanese government issued emergency economic measures to increase financial support for the pandemic and proposed the first supplementary budget to provide significant subsidies to industries that were significantly affected by the pandemic, while focusing on financial support for companies that strengthen the medical system, accelerate the development of new vaccines, and provide support for economic recovery. In order to expand economic support, the government proposed and established the 2nd supplementary budget in June, 2020. The breakdown of the revised accounting budget shows that the largest number of support items are intended for financing SMEs. In addition, support for business operators includes subsidizing rent for setting up offices, support for continued employment, etc. Building on the 1st supplementary budget, financial support for strengthening the medical system, securing vaccine production, developing business ventures, and supporting ICT development is included as well.
(2) Issuing “Tax Reform 2021” to provide tax incentives for SME development
(3) Issuing “the Basic Law on Building a Digital Society” to promote the digital transformation of SMEs.
2.5. Entrepreneurial response to opportunities emerged during the crisis caused by the COVID-19 pandemic – case studies from China
Seizing opportunities to advance digital transformation and develop digital trade
Throughout the pandemic, the companies that managed to surmount the crisis share some common characteristics. They all fall within the online, digital and intelligent trends, showing the new economy industry traits. Some examples include AI temperature measurement and robot food delivery to support the front line of pandemic prevention; or cloud video conferencing tools and cloud document collaboration tools to provide tools for enterprises to resume work remotely.
Joint production with foreign enterprises (i.e. collaboration in the production of anti-pandemic materials, etc.) to open up overseas markets
After the pandemic, the business world will enter the Open 2.0 phase. Hema Fresh has "shared staff" with restaurants such as Yun Hai Yao and Youth Restaurant (Beijing) to meet its own surging demand for orders and to help restaurants absorb costs. Meituan also quickly followed
this cooperation manner, reaching “shared staff” cooperation with Putian Restaurant, Lugang Town, Xiao Nan Guo and other catering companies, while Walmart, Fresh Legend, Jingdong, Suning, Lenovo, 58 Tongcheng and many other companies launched similar “shared staff” measures. It can be said that openness is the best “moat”.
**Upgrading and improving the industry chain to adapt to the pandemic and move to the middle and high end of the value chain**
Starting from the supply chain system “Internet of Everything”, the application of 5G, IoT, big data, AR/VR and other emerging technologies with great potential can help enterprises collaborate remotely and empower them to match the dynamic changes in supply and demand and scientific scheduling.
During the pandemic, considerable number of SMEs went bankrupt, especially those which mainly do offline business such as restaurants and hotels. However, there were many companies which survived the pandemic owing to their type of business and the creative measures they undertook during the pandemic. Here are two companies in Chengdu which ran their business well throughout the pandemic.
**CHENGDU LIGENG CULTURE COMMUNICATION CO. LIMITED**
*CHENGDU LIGENG CULTURE COMMUNICATION CO. LIMITED* is a small and medium-sized enterprise, established in April 2018, with a shareholding structure jointly funded by two private person shareholders, with a shareholding of 70% and 30% respectively. The company belongs to the cultural communication industry and its main business direction is to provide public cultural services, the publication and distribution of books and the organization and planning of cultural activities for the society.
The outbreak of the Covid left the vast majority of companies facing practical difficulties in terms of declining business performance, which was inevitable, and *CHENGDU LIGENG CULTURE COMMUNICATION CO. LIMITED* company was no exception. Especially in the early stages of the outbreak in the first half of 2020, the lack of adequate knowledge and scientific understanding of the coronavirus inevitably caused a psychological panic, a panic that made people feel that the only safe place to be was at home. Any activities related to consumption were suspended except those related to the life necessities at that time. But when the whole society pressed the pause button on consumption, it was inevitable that companies experienced a sharp decline in business performance. In the case of *CHENGDU LIGENG CULTURE COMMUNICATION CO. LIMITED* company, the impact on business performance in terms of public cultural service projects was relatively small, mainly because their service contracts were signed before the outbreak and the company actively adjusted its service and organized some cultural activities online, such as reading and lectures. That allowed people staying at home during the pandemic to enjoy digital public cultural services through their computers or mobile phones and to continue the implementation of the service contracts. However, the company was basically unable to conduct its business or organize any cultural activities offline. The biggest challenge the company normally faces is the fierce competition in the industry, both in terms of event ideas and in terms of service quality and price. But these challenges seem to pale into insignificance in the face of the pandemic since after all, “*even fierce competition is better than no competition at all*”.
The challenge for all companies during the pandemic was the dramatic contraction of the consumer market. CHENGDU LIGENG CULTURE COMMUNICATION CO. LIMITED is a relatively small company. However, when faced with these difficulties, in addition to actively adjusting and organizing offline activities to be carried out online to the extent possible, in order to maintain the continuity of the business operations, the company management calmly observed and thought about the opportunities which had emerged on the market under the impact of the pandemic and whether the company could take advantage of them to develop some new cultural service projects.
At the beginning of the pandemic, the company was more passive in waiting or expecting an early end of the pandemic. But, as the pandemic continued to spread globally and as the management gained a new understanding of the pandemic, they started planning to open up a new scenario of cultural services in June 2021. The company already opened a new model of cultural services in the community – Yumei Space. It is expected to be officially start in January 2022. They expect this new model of cultural services to be accepted by the market and to better serve every societal group.
There is no doubt about the positive impact of the company’s innovations on its performance, notably through the shift from offline to online activities. During the pandemic, in order to meet the obligations stemming from the service contracts, the company had to carry out their activities online in order to ensure that the contracts were effectively implemented and the income was protected. They are also confident in their new cultural service business, “Yumei Space”, which will be launched in January 2022.
**CHENGDU LONG YUAN NETWORK TECHNOLOGY CO.**
Longyuan Network Technology Co. Limited was founded in 2013 and currently employs over 600 people. The company is a leading mobile game developer and operator in China, and has launched a number of products popular with players since its inception. In 2019, Longyuan Network created Duo Duo Auto Chess, the first Chinese original and globally popular e-game. Duo Duo Auto Chess has shown phenomenal popularity worldwide since its launch. It won the Google App Store 2019 Most Innovative Game of the Year award in 17 countries around the world. In addition, the company launched games such as Gathering Explosion, Wanxiang Story and Music World Cytus 2. The company has won 40 industry awards and honors.
The pandemic affects industries and company operations in two ways: first, employees are unable to work together, which affects productivity. From time to time, employees are quarantined due to the recurring COVID. The gaming industry is intelligence-intensive and requires cooperation and collaboration from many sides, thus the inability to work together has a significant impact on efficiency. Second, the uninterrupted risk in multiple regions of the country has an impact on the possibility to travel, thus communication with the upstream and downstream of the industry is not as convenient as before.
The company introduced protection measures where possible, with staff temperature testing, ledgers, going to work with masks, regular daily disinfection and active attention to health and travel. Second, they actively promoted the widespread use of teleconferencing and building a good VPN for the company’s intranet to facilitate employees to do their work remotely or from home. Because the company’s product format is primarily geared towards Internet users, there
is very little offline delivery. So, gradually adapting to teleconferencing and teleworking was an innovative way of operating in response to the pandemic. As a result of the pandemic, the company paid even more attention to long-term development and accelerated the renewal of its own strategy to adapt to this era of rapid change.
The use of teleconferencing and telecommuting may not have a direct impact on a company’s performance, but it does make it easier for people to work and interact with each other. Things that were previously difficult to explain clearly by telephone could be better explained by teleconferencing, while looking at the presentation material over and over again. It has a positive impact on productivity.
COUNTRY REPORT
CROATIA
Maja Has
3. COUNTRY REPORT – CROATIA
3.1. Quantification of the impact of pandemic on SME sector in Croatia
In Croatia, as in most of the world, the small and medium enterprise sector has by far the largest share in the total number of enterprises (99.7 percent). In 2020, almost 140,000 enterprises operated in Croatia, and as many as 89.5 percent of the total number were micro enterprises. Despite significant changes in business conditions as a result of the COVID-19 pandemic, the growth trend in the total number of enterprises did not stop even in 2020, when 2,749 new companies were registered, which represents a growth of slightly more than 2 percent (Figure 1).
**Figure 1:** Enterprise structure in Croatia with regard to size in 2019 and 2020
Sources: Adapted from “Results of Croatian entrepreneurs in 2019 classified by size”, FINA, 2020
“Financial results of entrepreneurs in 2020 – classified by size”, FINA, 2021
In 2020, under the influence of the pandemic, there was a decrease in the total income\(^3\) of all categories of business entities in Croatia. The largest decrease in total income was recorded by micro enterprises (23 percent), and the smallest by large enterprises, whose income decreased by 1.2 percent. Total income in small enterprises decreased by 7 percent, and in medium enterprises by 4 percent compared to 2019 (Figure 2).
**Figure 2:** Enterprise size and total income (million EUR) in 2019 and 2020 in Croatia
*Sources: Adapted from: “Results of Croatian entrepreneurs in 2019 classified by size”, FINA, 2020
“Financial results of entrepreneurs in 2020 – classified by size”, FINA, 2021*
Due to significant restrictions on business activities in the conditions of the pandemic, there was a decrease in export activities in 2020. The largest decline in exports was recorded by micro enterprises (19.5 percent). In small and large enterprises, export activities fell by about 7 percent, while decline in exports was the least pronounced in medium enterprises, where it was 2 percent (Figure 3).
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\(^3\) Original data related to the amounts of total income and exports expressed in HRK were obtained from the Financial Agency – FINA. For the purposes of preparing this report the exchange rate of 1 EUR=7.55 HRK was used for currency conversion and the amounts were rounded without decimal places. This methodology was applied in the entire chapter Country report - Croatia when converting HRK into EUR.
As a consequence of the pandemic and the reduction of income, in 2020 the number of employees in the micro, small and medium enterprise sector decreased by 5 percent compared to 2019, which reduced their share in the total number of employees in business entities in Croatia by 2 percentage points (Figure 4).
Compared to 2019, almost 28,000 jobs were lost in the micro enterprise sector in 2020, about 5,400 jobs in small enterprises, about 3,000 jobs in medium enterprises, while almost 14,500 new jobs were created in large enterprises, which partially mitigated the decline in total employment in 2020 compared to 2019, which amounted to 2.2 percent or almost 22,000 jobs (Table 4).
Table 4: Enterprise size and employment, total income and exports in 2019 and 2020 in Croatia
| Economic criterion for sector evaluation | Micro | Small | Medium | Large |
|-----------------------------------------|-------|-------|--------|-------|
| | 2019 | 2020 | 2019 | 2020 | 2019 | 2020 | 2019 | 2020 |
| Number of employees | | | | | | | | |
| | 286,603 | 258,644 | 249,826 | 244,445 | 183,189 | 180,166 | 250,158 | 264,619 |
| Employment (share)\(^4\) | 29.6% | 27.3% | 25.8% | 25.8% | 18.9% | 19 % | 25.8% | 27.9% |
| Total income (million EUR) | 16,689 | 12,897 | 25,058 | 23,361 | 21,877 | 20,933 | 41,823 | 41,332 |
| Total income (share) | 15.8% | 13.1% | 23.8% | 23.7% | 20.7% | 21.2% | 39.7% | 42% |
| Exports (million EUR) | 1,640 | 1,320 | 4,114 | 3,812 | 4,866 | 4,765 | 9,440 | 8,778 |
| Exports (share) | 8.2% | 7.1% | 20.5% | 20.4% | 24.3% | 25.5% | 47.1% | 47% |
Sources: Adapted from “Results of Croatian entrepreneurs in 2019 classified by size”, FINA, 2020
“Financial results of entrepreneurs in 2020 – classified by size”, FINA, 2021
Based on aggregate data related to operations of the small and medium enterprise sector, it can be seen that the pandemic affected a decrease in the number of employees by 5 percent, i.e. that there were 36,363 fewer jobs in the small and medium enterprise sector in 2020 compared to 2019. Total income generated at the sector level in 2020 was 10 percent lower than in 2019, which is EUR 6.4 billion less, while the value of exports decreased by EUR 723 million, or almost 7 percent. (Figure 5).
\(^4\) Slight deviations in the sum of the total share (100%) are possible due to rounding of amounts.
Figure 5: Number of employees, total income and exports in the small and medium enterprise sector in 2019 and 2020 in Croatia
Sources: Adapted from “Results of Croatian entrepreneurs in 2019 classified by size”, FINA, 2020
“Financial results of entrepreneurs in 2020 – classified by size”, FINA, 2021
Figure 6 shows the differences between micro, small, medium and large enterprises in terms of their contribution to employment, total income and exports in 2020. In 2020, the small and medium enterprise sector employed 72 percent of the total number of employees and generated 51 percent of total income and 53 percent of the total Croatian exports.
In 2020, the Croatian business sector achieved a positive consolidated result, but lower by almost 33 percent compared to 2019. The decrease in this indicator is the result of a decrease in total profit in 2020 by 6 percent with a simultaneous increase in total loss by 42 percent compared to the previous year (Table 5). As with previous indicators, after two consecutive years of growth in the amount of total net profit at the level of all Croatian companies, in 2020, under the influence of the pandemic, there is a change in the financial performance of enterprises. Despite the reduction in net profit, in 2020 all categories of enterprises achieved a positive financial result, which was not the case in 2019, when micro enterprises reported a loss that was higher than the generated profit.
Table 5: Financial efficiency of enterprises in 2019 and 2020
| Enterprises | 2019 | 2020 |
|-------------|------|------|
| | Million EUR | % | Million EUR | % |
| **Micro** | | | | |
| Profit | 1,372 | 21.2 | 1,296 | 21.3 |
| Loss | 1,392 | 59.7 | 1,290 | 39.0 |
| Consolidated result – Net profit/loss | -20 | 0.5 | 6 | 0.2 |
| **Small** | | | | |
| Profit | 1,522 | 23.5 | 1,544 | 25.4 |
| Loss | 383 | 16.4 | 537 | 16.3 |
| Consolidated result – Net profit/loss | 1,139 | 27.5 | 1,007 | 36.3 |
| **Medium** | | | | |
| Profit | 1,244 | 19.2 | 1,246 | 20.5 |
| Loss | 301 | 12.9 | 529 | 16.0 |
| Consolidated result – Net profit/loss | 943 | 22.8 | 716 | 25.8 |
| **Large** | | | | |
| Profit | 2,335 | 36.1 | 1,996 | 32.8 |
| Loss | 254 | 10.9 | 949 | 28.7 |
| Consolidated result – Net profit/loss | 2,081 | 50.2 | 1,048 | 37.7 |
| **Total** | | | | |
| Profit | 6,473 | | 6,082 | |
| Loss | 2,330 | | 3,305 | |
| Consolidated result – Net profit/loss | 4,143 | | 2,777 | |
Sources: Adapted from “Results of Croatian entrepreneurs in 2019 classified by size”, FINA, 2020
“Financial results of entrepreneurs in 2020 – classified by size”, FINA, 2021
Micro enterprises made a profit of EUR 1.3 billion in 2020, which is a decrease of 5.5 percent compared to the profit made in 2019. In addition to the reduction in profits, in 2020 the business of micro enterprises was marked by a reduction in losses by 7.4 percent. Unlike the year before, in 2020 micro enterprises showed a positive consolidated result, and their share in total losses of the business sector decreased by 20.7 percentage points.
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5 Slight deviations in the sum of the total share (100%) are possible due to rounding of amounts.
Small enterprises generated EUR 1.5 billion in profit in 2020, which is an increase of 1.5 percent compared to the result achieved in 2019. At the same time, they reported losses in the amount of EUR 537 million, which is 40 percent more than the previous year. With these results, the share of small enterprises in the total profit of enterprises in Croatia in 2020 increased by 1.9 percentage points, while the share in total losses remained almost at the level of 2019 (16 percent).
Medium enterprises made EUR 1.2 billion in profit in 2020, which is approximately at the level of the results from the previous year. Operations of medium enterprises in 2020 were marked by a significant increase in losses amounting to EUR 529 million, which is as much as 76 percent more than in 2019. With this increase in reported losses, medium enterprises also increased their share in total reported losses of the economy by 3 percentage points.
Large enterprises generated a profit in the amount of almost EUR 2 billion in 2020, which is a decrease of 14.5 percent compared to 2019. At the same time, in addition to the decrease in profits, operations of large enterprises were marked by a significant increase in losses. In 2020, large enterprises reported a loss of EUR 949 million, which is 3.7 times more than in 2019. Although large enterprises accounted for the largest share of profits at the level of the economy in 2020 (33 percent of the total generated profit), their share in total reported losses at the level of the economy increased by 17.8 percentage points.
Based on the presented data, it can be concluded that the negative effects of the pandemic had the greatest impact on financial operations of large enterprises. Their consolidated result in 2020 is 50 percent lower compared to 2019. On the other hand, according to the presented data, the negative effects of the pandemic had the least impact on the operations of micro and small enterprises. These results can be explained by the agility and flexibility of micro and small enterprises, which are reflected in the rapid adaptation of their business to changes, but the Government's economic support measures certainly also mitigated the negative economic consequences of the pandemic, at least in the short term, as presented in this analysis.
In the small and medium enterprise sector, out of the total number of enterprises that made a profit in 2020, the largest share of enterprises was engaged in wholesale and retail trade, repair of motor vehicles and motorcycles (22 percent), professional, scientific and technical activities (19 percent), and construction (13 percent). The largest number of enterprises that reported losses in 2020 also operated in wholesale and retail trade, repair of motor vehicles and motorcycles (19 percent) and accommodation and food service activities (15 percent) (Figure 7). The difference between “profit-makers” and “loss-makers” was greatest in the manufacturing industry, construction, wholesale and retail trade, and repair of motor vehicles and motorcycles in which the number of “profit-makers” greatly exceeded the number of “loss-makers”. On the other hand, activities in which the number of “loss-makers” was higher than the number of “profit-makers” were accommodation and food service activities, real estate activities, administrative and support service activities, arts, entertainment and recreation, and other service activities. These results can be explained by the changes that have marked operations of companies in 2020 in several areas. The consequences of the earthquake in March 2020 in Zagreb and the surrounding area affected the work of companies in the construction sector and indirectly the real estate companies. Epidemiological measures in the fight against the COVID-19 pandemic and the suspension of operation of catering facilities, cultural activities, service activities and
sports competitions, as well as the ban on holding public events and gatherings, affected the work of companies providing accommodation and food service activities, administrative and other service activities, and arts, entertainment and recreation activities.
In 2020, according to the Croatian Bureau of Statistics (2021) data, 12,948 legal entities were established in Croatia, i.e. 22 percent less compared to 2019. In the same period, 656 legal entities were terminated, which is 51 percent less than in 2019 (Table 6).
**Table 6:** Establishment and termination of registered legal entities in 2019 and 2020
| Establishment | 2019 | 2020 | Difference |
|---------------|--------|--------|------------|
| Trade companies | 14,805 | 11,518 | - 22.2% |
| Other | 1,766 | 1,430 | - 19% |
| **Total** | **16,571** | **12,948** | **- 21.9%** |
| Termination | 2019 | 2020 | Difference |
|-------------|------|------|------------|
| Enterprises | 6 | 7 | 16.7% |
| Trade companies | 939 | 444 | - 52.7% |
| Other | 389 | 205 | - 47.3% |
| **Total** | **1,334** | **656** | **- 50.8%** |
*Source:* Number and structure of business entities, December 2020, First Release no. 11.1.1/4., 2021, Croatian Bureau of Statistics
The Central Bureau of Statistics collected and analyzed data related to the effects of the COVID-19 disease pandemic on socio-economic indicators. In the context of business entities, it is possible to analyze statistics of registrations and bankruptcies\(^6\) that includes business entities operating on a market basis. According to existing data, there is a significant decline in the number of registrations of business entities in the first and second quarters of 2020, which can be explained by a decrease in normal business activities and increased measures to combat the contagion that marked the period. Although the number of registrations of business entities increased in the third and fourth quarters of 2020, the number was still lower than in 2019 due to uncertain economic situation that is still ongoing. Regarding the number of open bankruptcy proceedings, the first quarter of 2020 saw an increase, while other quarters were marked by a decrease in the number of bankruptcies. In 2021, that number approached the 2019 level (Figure 8).
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\(^6\) The number of registrations also includes crafts, while the number of bankruptcies applies only to trading companies.
In the observed period, the highest number of registrations of business entities was in the areas of financial activities and insurance activities, real estate, professional, scientific and technical activities, and administrative and support service activities, while the lowest number of registrations was in the activity of transport and storage. The highest number of open bankruptcies was in wholesale and retail trade, repair of motor vehicles and motorcycles, and accommodation and food service activities, while the lowest was in education, health and social welfare, arts, entertainment and recreation, and other service activities.
3.2. Policy measures supporting Croatian SMEs during the COVID-19 pandemic
In Croatia, State aid measures for entrepreneurs affected by the COVID-19 pandemic\(^7\) were prescribed on the basis of the “Temporary Framework for State aid measures to support the economy in the current COVID-19 outbreak” adopted by the European Commission on March 19, 2020. Its further amendments enabled the continuation of allocation of state aid until December 31, 2021. The types of state aid defined by the provisions of the “Temporary Framework” are as follows\(^8\):
1) Aid totaling up to EUR 1,800,000 per entrepreneur in the form of direct grants, tax reliefs and more favorable payment terms, or in some other form;
2) Aid in the form of loan guarantees and in the form of subsidized interest rates on loans exceeding EUR 1,800,000 per entrepreneur;
3) Aid in the form of guarantees and loans through credit institutions or other financial intermediaries;
4) Short-term loan insurance;
5) Aid for research and development in the field of COVID-19;
6) Aid for investment in construction or upgrading of testing infrastructures and for the improvement of processes from the laboratory to the production level;
7) Aid for investments in the production of products essential for the control of the spread of COVID-19;
8) Aid in the form of deferrals of taxes and / or social security contributions;
9) Aid in the form of subsidies for employees’ salaries or the income of self-employed persons equivalent to salary;
10) Recapitalization measures for non-financial entrepreneurs;
11) Aid in the form of aid for uncovered fixed costs.
Following these conditions, the Government of the Republic of Croatia adopted its own package to help the economy. On March 20, 2020, the Croatian Employment Service adopted measures of direct financial support to employers with the aim of retaining jobs with employers whose economic and business activity has been disrupted or reduced due to extraordinary circumstances.
\(^7\) Measures to help the economy due to the COVID-19 epidemic, available at: https://mingor.gov.hr/mjere-za-pomoc-gospodarstvu-uslijed-epidemije-covid-19-7731/7731 and Tax information related to the state of emergency caused by the spread of the COVID-19 virus, available at: https://www.porezna-uprava.hr/Stranice/COVID_19_informacije.aspx
\(^8\) Categories of state aid to support the economy during the COVID-19 pandemic, available at: https://infin.gov.hr/istaknute-teme/koncesije-i-drzavne-potpore/drzavne-potpore/drzavne-potpore-za-podrsku-gospodarstvu-tijekom-pandemije-covid-a-19/3044
The application for support in the first quarter (March–May 2020) could be submitted by: (a) entrepreneurs prevented from performing their activity by decisions of the Civil Protection Headquarters, (b) entrepreneurs in accommodation and food and beverage service activities, and transport and storage activities, and labor-intensive activities within the manufacturing industry (textiles, clothing, footwear, leather, wood and furniture), and (c) other entrepreneurs that can demonstrate the impact of “special circumstances”. Already in the first month of aid allocation, March 2020, the number of recipients exceeded 500,000 employees, and their growth continued in April and May 2020.
Despite significant pressure on the state budget to meet these obligations (it was calculated that the net support for the period will amount to EUR 821 million, with another EUR 371 million for mandatory contributions), social actors linked to the economy assessed the whole change package as extremely positive. It is considered that these interventions successfully prevented numerous bankruptcies of business entities, a large number of layoffs and generally eliminated the ultimate scenario of economic collapse and social instability.
After that, the Government of the Republic of Croatia extended the period of application of these measures, and in the period from September to December 2020, they were divided into:
1) Aid to micro-entrepreneurs intended for all economic entities (including craftsmen) employing up to 10 workers, if their income was reduced by 50% in the comparative period. The aid amounts to EUR 265 per employee, but they had to be employed prior to August 31, 2020.
2) Aid to employers from explicitly listed activities for the sectors most at risk, such as transport and storage (with special emphasis on air transport) and a wide range of activities related to the service and catering sector. The condition for receiving financial assistance was a drop in turnover of at least 60% for the previous month and it amounted to EUR 530 per worker.
3) Aid to employers who are organised for the employment of persons with disabilities, regardless of the registered activity, which amounted to EUR 530 per worker, provided that the turnover of the related business entity fell by at least 50% for the previous month.
4) Aid for shortening work hours for employers employing 10 or more workers, regardless of the registered activity, due to a temporary reduction in the scope of work. The precondition was a drop in turnover of at least 20% of the full-time fund if they employ 10 to 50 workers, and at least 10% if they employ 51 or more workers. The amount of aid is EUR 265 per worker.
The only exception in this case are those micro-entrepreneurs and employers who have been prohibited or prevented from working by the provisions of the Civil Protection Headquarters at the local, regional or national level. Also, some of the defined measures can be implemented by self-employed persons (including craftsmen) if they meet the prescribed conditions. For the
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9 Aid for preservation of jobs (Ivica Urban), available at: http://www.ijf.hr/hr/publikacije/casopisi/12/osvrti-institutaza-javne-financije/109/potpora-za-ocuvanje-radnih-mjesta/159/, http://www.ijf.hr/upload/files/1131.pdf
10 Review of financial assistance measures to employers for preservation of jobs until the end of 2020, available at: https://www.rif.hr/pregled-mjera-novcanih-pomoci-poslodavcima-za-ocuvanje-radnih-mjesta-do-kraja-2020-godine/
period until December 31, 2021, the amount of aid was EUR 530 per worker (in the period from January to February 2021, this included aid for the preservation of jobs for areas affected by the earthquake). As part of aid to employers, in cases when there had been a temporary reduction in the scope of work, a subsidy in the amount of EUR 477 per worker was paid.
In addition to financial aid for job preservation, other measures to help entrepreneurs in activities affected by the coronavirus include approval of loans with favorable interest rates for micro and small entrepreneurs under the auspices of the Croatian Agency for SMEs, Innovations and Investments (HAMAG BICRO). This type of aid is divided into “COVID-19 loans” for amounts up to EUR 50,331 (including the group of medium-sized entrepreneurs) and “Micro loans for working capital for rural development” and “ESIF Micro loans for working capital” for amounts from EUR 1,000 to 25,000. The Croatian Bank for Reconstruction and Development also enables the use of various types of loans as temporary measures within the lending program for small, medium and large entrepreneurs\(^{11}\).
*The National Recovery and Resilience Plan 2021–2026 of the Republic of Croatia was adopted in July 2021, based on the establishment of the Recovery and Resilience Facility at the European Union level. Its measures and activities are aimed at helping the national economy recover after the crisis caused by the COVID-19 disease pandemic and creating a more resilient and sustainable society in the event of new economic shocks*\(^{12}\). By the end of 2021, Croatia expects a disbursement of about EUR 808 million, which is an advance of 13% of the total amount of grants provided under the Plan.
The National Recovery Plan is structured through 6 components:
1) Economy
2) Public administration, judiciary and public property
3) Education, science and research
4) Labour market and social protection
5) Health care
6) Initiative: Renovation of buildings
With regard to strengthening the productivity and competitiveness of the economy, special attention has been paid to reforms and investments aimed at “green” and digital transformation and achieving smart, sustainable and inclusive growth in general. In order to achieve optimal performance when implementing the Plan and spending the available funds, specific projects within these broadly defined goals should be aimed at increasing employment, growth and development of micro, small and medium enterprises, encouraging innovation and technological development, i.e. high-return investments that will significantly contribute to strong economic growth in the medium and long term, thus annulling the initial negative impact on the budget.
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\(^{11}\) Measures to help the economy due to the COVID-19 epidemic, available at: https://mingor.gov.hr/mjere-za-pomoc-gospodarstvu-uslijed-epidemije-covid-19-7731/7731
\(^{12}\) Recovery Plan: About the plan, available at: https://planoporavka.gov.hr/o-planu/9
CITY OF ZADAR - an example of a local measure for entrepreneurs in dealing with the crisis caused by the COVID–19 pandemic\(^{13}\)
In the City of Zadar, the city budget directly provided around EUR 1.3 million in aid by April 2021 through measures of price reduction or exemption of payment for lease of business premises and public space, and waiving a portion of utility fees, and in order to provide additional support to entrepreneurs and craftsmen, additional funds have been secured for the future period. In the fight against the negative consequences of the pandemic, Zadar has created two financial instruments for lending to entrepreneurs and craftsmen: “COVID–19 – loan for preservation of liquidity” and “COVID–19 – loan for investment”, through which the City subsidises interest on loans. The total credit potential for both programmes is EUR 26.5 million, the interest rate of seven contracted banks ranges from 2.5 to 3 percent, of which 1 percent is covered by the city’s programme. The grace period for liquidity loans is up to one year, and up to two years for investments. Beneficiaries of the Programme can be micro, small and medium business entities that are registered and operate in the area of the City of Zadar, operate throughout the year, have at least one full-time employee and who meet the conditions for disbursement of loans in accordance with the rules of commercial banks that have concluded an Agreement on the implementation of the Programme with the City of Zadar. The specifics of these programmes for lending to entrepreneurs and craftsmen are as follows:
1. **Lending programme “COVID–19 – loan for preservation of liquidity”**
Loan purpose: loan for preservation of liquidity – “kuna loan” for financing current operations, which includes procurement of raw materials, intermediate goods, semi-finished products, small inventory, settlement of liabilities towards suppliers, labour costs, general operating expenses, refinancing of existing adverse loans, settlement of short-term liabilities towards financial institutions and the state;
Loan amount: from EUR 13,245 to EUR 132,450 (HRK 100,000 to HRK 1,000,000) (maximum one loan per entrepreneur);
Loan repayment period: up to 3 years, including the term of use up to 6 months and a grace period of up to 1 year.
2. **Lending programme “COVID–19 – loan for investment”**
Loan purpose: kuna loan for purchase, construction, arrangement, reconstruction or expansion of business facilities and purchase of new equipment or new part of equipment;
Loan amount: from EUR 26,490 to EUR 264,901 EUR (HRK 200,000 to HRK 2,000,000) (maximum one loan per entrepreneur);
Loan repayment period: up to 10 years, including the term of use up to 12 months and a grace period of up to 2 years.
In the budget of the City of Zadar for 2021, funds in the amount of EUR 198,675 were planned for subsiding interest on loans for preservation of liquidity, that is, EUR 66,225 HRK 500,000 for subsidising interest on loans for investment.
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\(^{13}\) https://www.grad-zadar.hr/vijest/opce-vijesti-28/gradonacelnik-dukic-predstavio-nove-covid-mjere-grada-zadra-za-poduzetnike-kredit-do-200-milijuna-kuna-za-likvidnost-i-investicije-poduzetnika-6721.html
3.3. Review of the adopted government measures for supporting SMEs during the pandemic in Croatia
One of the most used measures during the COVID–19 pandemic is aid for job preservation. Entrepreneurs who used these measures believe that the measure was an adequate response of the Government to the new situation in March 2020, and that they saved many jobs or at least delayed layoffs. But, in the long run, entrepreneurs believe that measures will not help them save businesses from collapsing. Even if they don’t have a single employee or if they lay off everyone, entrepreneurs have fixed costs that they have to pay and that they won’t be able to cover. For this reason, in the opinion of entrepreneurs, these measures alone were not sufficient to prevent companies from going into bankruptcy. In the national budget for 2021, not enough funds are allocated for investments in the economy and new projects that would stimulate the economy and the consumption.
Regarding the adoption of measures related to the prevention of the disease spread and accompanying measures related to operations of business entities from certain industries, it is noticeable that some entrepreneurs were dissatisfied by inconsistency, incoordination and delays with the implementation regulations, as well as the omission of certain activities when defining measures, which further confused entrepreneurs.
Employers associated in the Croatian Employers’ Association – CEA, believe that the National Recovery and Resilience Plan should provide for much more investment in the real sector, manufacturing, and the introduction of new technologies in order to create added value and new jobs. Most of the investments envisaged in the National Recovery and Resilience Plan are primarily intended for public projects, from sewerage, water management to transport investments. These investments benefit the lives of citizens but will not create enough jobs in the long run and increase the domestic product as much as possible.
The Economic Council of the Voice of Entrepreneurs association, which brings together micro, small and medium entrepreneurs, self-employed craftsmen and employees in the private sector, has a similar opinion. They believe that the National Recovery and Resilience Plan will not distribute sufficient funds to small and medium enterprises, which will have a negative impact on the economy as a whole and its recovery from the effects of the COVID–19 pandemic. The National Recovery and Resilience Plan envisages the distribution of funds so that 54% of the funds are directed to the economy and 46% to “reforms” in the public sector. However, the 54% of aid to the economy include wastewater, waste management, road construction and transport infrastructure projects, which means investing in public companies that have shown low efficiency thus far. Therefore, they conclude that the National Recovery and Resilience Plan is not aimed at helping the private sector and encouraging the competitiveness of the Croatian economy. In order for the National Recovery and Resilience Plan to have a greater effect, it is necessary to prioritize investments in the private sector, focus on high return projects and monitor the return on investment and fiscal effects. In addition, it is necessary to disable corrupt distribution channels of funds and politically preferred projects.
Following the above, the Economic Council of the Voice of Entrepreneurs association proposed the following proposals for a successful economic recovery:
- A higher relative ratio of funds in the National Recovery and Resilience Plan should be directed to the private sector and citizens to recover demand, in line with the plans of other comparable countries in the European Union;
- Create models to help citizens who lost their jobs due to the pandemic;
- Implement aid to private companies;
- Tax relief for the affected sectors;
- Investment in renewable energy;
- Full transparency of the allocation process.
3.4. Entrepreneurial response to opportunities emerged during the crisis caused by COVID-19 pandemic – case studies from Croatia
By innovating, companies bring new products to the market, achieve growth and ensure long-term competitiveness. During normal functioning of the economy, companies try to fight the competition by innovating, but in the conditions of crisis and unpredictable market movements, innovation becomes even more important. The global health crisis caused by the COVID-19 epidemic in the first wave prompted many economies to close, and consequently many companies were unable to continue their normal operations. Some of the companies recognized the opportunity and created new products, services and processes that meet the customers’ needs resulting from the changed circumstances of living and working in a pandemic.
**ALARM AUTOMATIKA**
*(www.alarmautomatika.com)*
Through more than 30 years of market presence, since its foundation in 1989, Alarm Automatika has been developing its business operations in 11 countries, covering at the end of 2021 the whole Southeast Europe region. Alarm Automatika has more than 150 employees that provide support for more than 2,000 regular customers. The offer includes products from more than 200 global manufacturers, but also the company’s own brands, with their characteristics tuned for the specific markets.
As stated on company’s website: “The company’s mission is to create a value proposition that includes high quality, innovative products, services and complete solutions which fulfil individual customer needs in order to reduce risk to customers and business partners, to support smooth business operations and faster growth by using the common platform.”
The position of regional market leader is built by continuous investment in technical, economic, organizational, engineering, IT and management competencies of employees. In order for the company’s portfolio to be maximally competitive and technologically advanced, integrated and adapted to the users’ specific needs, the entire team of engineers is constantly monitoring new
technologies, developing new products, services and software, as well as its own brands. The company has been repeatedly awarded for the developed solutions and realized projects.
Both the company and the employees have all the necessary authorizations in the field of technical security, construction and informatics, as well as the Business Security Certificate. Operations are in accordance with the ISO 9001: 2015 certificate for business management and the ISO 27001 certificate for the information security. The success of the work has been verified by the 3A Creditworthiness certificate from Company Wall Business for more than 15 years.
In 2020, Alarm Automatika saw the coronavirus pandemic as an opportunity to look for solutions that they can offer on the market and thus fight the upcoming crisis. The company has created several products to help its clients cope more successfully with the challenges that the pandemic has brought to everyone. The ACC terminal is a unified solution for access control, temperature measurement and recognition of wearing a face mask, which enables contactless control of the entrance to the premises. Integration of systems that limit the number of people in a space by counting entrances and exits in, for example, shopping centers, but also in public transport, has been designed.
Furthermore, a practical solution that enables contactless communication between people in separate rooms (e.g. banks, public buildings) has been devised. The company also offers a medical thermal camera for body temperature detection, which also enables video recording at a distance of up to 12 meters, as well as real-time analytics, which can be of significant benefit to hospitals and airports.
One of the latest innovations was the solution that was implemented in November 2021 for the needs of organizing the Interliber book fair at the Zagreb Fair. Limiting the number of people is an essential measure to prevent the spread of coronavirus and a condition for larger gatherings to take place at all. In order to be able to organize fairs, the Zagreb Fair has implemented an Alarm Automatika’s solution for determining the number of people in its exhibition pavilions. Alarm Automatika designed the solution, delivered the necessary equipment and implemented the solution. Special Vivotek 3D cameras and recorders are used for counting people, as well as software for calculating and displaying the current and maximum allowed number of people in the space. The solution was implemented on the eve of Interliber, one of the most visited events at the Zagreb Fair, and will continue to be used at other fairs. Thus, the Zagreb Fair has increased the level of safety of visitors and provided a permanent tool for accurate collection and analysis of data on visitors, which will enable them to better organize the events.
Through a quick response and the use of its innovation capacity, the company continued to successfully operate and grow even in difficult times.
Company Grad-Export Ltd. was founded in 1992 with its headquarters in Vinkovci, in the Eastern part of Croatia. Since its establishment, the company has been continuously developing and growing by investing in process and product innovation. Its main business operations are focused on the production of decorative ALU, PVC and HPL panels for entrance doors. Manufacturing facilities are located in the Industrial zone of Vinkovci on 38,000 m².
Grad-Export is exporting its products to 26 countries all over Europe and beyond, and is one of the leading European manufacturers of decorative front door panels. Their product portfolio consists of 13 lines of decorative panels with over 250 different models that are the result of many years of work, 180 experienced and trained employees as well as modern technologies used in the production.
At the very beginning of the COVID-19 disease pandemic in 2020, the company saw the emerging crisis as an opportunity to seek new solutions that they could offer in the market and keep the business stable. The company has created the DS barrier, i.e. a disinfection barrier that is made in different dimensions and can be attached to a variety of surfaces. By creating a new product using the existing resources and technology, the company has recognized the trend of production of protective equipment and has adapted to the market situation. Successful sales of the new product continued in 2021 and, in cooperation with the existing foreign partners, the company exports disinfection barriers to foreign markets such as Spain, Ireland and Italy.
Through a quick reaction to the new situation of the global pandemic and using the company’s existing resources and innovation capacity, the company continued to successfully operate and grow even in difficult times.
COUNTRY REPORT
NORTH MACEDONIA
Ana Krstinovska
4. COUNTRY REPORT – NORTH MACEDONIA
4.1. Quantification of the impact of pandemic on SME sector
SMEs in North Macedonia represent the lynchpin of the economy. In 2019, they constituted 99.7% of all business entities, employed 74% of the private sector workforce and accounted for 65.4% of the income (turnover).
**Figure 9:** Share of the SMEs in the economy of North Macedonia in 2019
*Source: State Statistical Office of North Macedonia (MAKSTAT database https://makstat.stat.gov.mk/)*
North Macedonia was one of the most affected European countries by the pandemic in terms of sanitary and economic impact, while at the same time the fiscal response provided by the Government was one of the weakest, amounting to only 3.5% of GDP\(^{14}\). The drop of the GDP in the second quarter of 2020 of almost 15% is unprecedented in the country’s history. While the growth picked up by the end of 2020 as a result of the government measures, it has been largely contingent upon the state support. Hence, as the support was discontinued in the beginning of 2021, there was another severe drop in the GDP.
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\(^{14}\) Jovanovik, B. et al., 2021. “Inequality in times of corona: The effects of the COVID-19 pandemic on the Macedonian economy”. Westminster Foundation for Democracy in North Macedonia. 4.
The pandemic affected 3/4 of the companies in North Macedonia and most of the employers resorted to reduction in salaries and all additional payments to their employees, as well as laying off staff in the more serious cases\textsuperscript{15}. Given that unemployment has continuously been the most serious socio-economic challenge for the country since its independence in 1991, most of the government measures were designed to support the companies not to reduce their number of employees. However, despite the measures, the unemployment rate which had been constantly declining from a record 38% in 2005 to around 17% in 2019 increased again by 53% between January 2020 and February 2021 (Figure 11).
\textsuperscript{15} Finance Think. 2020. “Economic effects of the corona-crisis”.
The biggest portion of SMEs in North Macedonia operate in retail trade, processing industry, hospitality, tourism and transport, sectors which, by the very nature of their activities, were mostly affected both by the global implications of the pandemic and by the national sanitary measures aimed to prevent the spread of the virus. In addition, according to a study by the European Central Bank, small companies have significantly less access to external financing, compared to medium and large enterprises, which makes them particularly vulnerable to external shocks.\textsuperscript{16} Hence, around 4% of all registered business entities in North Macedonia, or a total of 2,846, had to cease their activities as a result of the pandemic and all of them fall within the category of micro and small enterprises.
\textsuperscript{16} Moder, Isabella, Bonifai, Niccolò. September 2017. “Access to finance in the Western Balkans”. European Central Bank, Occasional Policy Paper.
The downward economic tendencies have been also reflected in the declining number of employees across all company sizes. While the private sector workforce shrank by 3.2%, micro and small enterprises saw a decline of 1.6% and 2.9% respectively, while medium and big companies laid off 4.8% and 4% of the workforce respectively.
When it comes to income, while the overall drop in the turnover amounts to 7.8%, micro and small companies lost 5 and 5.8% in comparison to 2019, medium companies faced minor losses of an average of 0.5%, while large enterprises seem to be worst hit with a 12.7% drop in their income.
Figure 14: Turnover per company size in North Macedonia, 2019-2021, in million EUR
Source: State Statistical Office of North Macedonia (Structural business statistics published for 2019 and 2020)
4.2. Policy measures supporting SMEs in North Macedonia during the COVID-19 pandemic
The first COVID-19 cases in North Macedonia were registered in the first half of March 2020. A general state of emergency was declared by the President on March 18 and it was extended several times throughout the following one-year period. In a state of emergency and as the Assembly was dissolved, the Government initiated the adoption of decrees with legal force.
The first introduced measures included a ban on movement from March 21 onwards, which, depending on the development of the pandemic in North Macedonia, covered different time intervals throughout 2020. A complete ban on movement on weekends was introduced during the contagion peaks. The border crossings and the airports were closed, except for strictly defined categories of passengers, for transit and freight vehicles. Besides that, additional occupational safety measures were introduced which demand the maintenance of distance between workers, wearing personal protective equipment – disposable masks and gloves, providing higher hygiene standards (access to water, soap, waste management, disinfectants, etc.) Given that schools and childcare facilities were closed between March and the end of the school year in June 2020, companies had to allow to some of their employees who had children at a young age to either stay at home or work from home whenever possible, in order to be able to take care of their children.
On one hand, the measures contained in the decrees introduced restrictions related to the movement of people and the operations of different non-essential businesses. On the other hand, they introduced different types of assistance targeting the most affected businesses and citizens in order to mitigate the economic repercussions of the restrictions. A total of 6 emergency assistance packages have been adopted so far amounting to around 1.2 billion EUR.\(^{17}\)
The first set of measures announced at the end of March targeted the tourism, hospitality and transport sectors since those companies were ordered to cease all their economic activities. An interest-free credit line amounting to a total of EUR 5.7 million was put at the disposal of SMEs through North Macedonia's Development Bank. The loans ranged between EUR 3,000 and 30,000, depending on the number of employees, with a grace period of 6 months and repayment over 2 years. Subsidies were offered for the most affected companies, in the amount of up to 50% of the average salary paid in 2019 (or around EUR 240 net), provided that the company does not lay off staff at least until September 2020, does not pay dividend or bonuses. The Central Bank reduced the basic interest rate to 1.75% and the mandatory interest rate on late payments was halved. Additional measures were taken related to freezing the prices and the reduction of custom duties for certain important commodities and products (flour, oil, dairy, eggs, some medicines, medical products and equipment).
The second set of measures extended the assistance and other measures until May 2020 and introduced several novelties. Some liberal professions and freelancing activities (sports, arts etc.) were included in the most affected categories eligible for assistance, as well as the media. As an alternative to salary subsidies, companies were allowed to ask for subsidies only to cover
\(^{17}\) This report focuses only on the most important measures targeting companies on the basis of the financial implications and outreach (number of SMEs concerned).
fringe benefits in the amount of up to 50%. Monthly unemployment fee of 50% of their average salary in the past 2 years was introduced for people who lost their jobs as a result of COVID-19, during a period contingent upon their paid work experience. The Law on forceful execution of payments was suspended until the end of June 2020.
With a landmark decision, the Central Bank amended its methodology on loan risk management in order to allow the banks and other financial enterprises to postpone or extend the maturity of the loans issued to physical and moral persons and to introduce more favorable terms for loan reprogramming, lowering interest rates and extending grace periods of the loans issued to companies. Additional EUR 8 million were put at the disposal of the most affected SMEs in the form of interest-free loans and EUR 50 million were disbursed through the commercial banks at an interest rate of around 1.5%, targeting SMEs willing to launch new projects, open new jobs or increase exports. The Government also banned the initiation of bankruptcy procedures during the state of emergency and 6 months after its end (announced for 31 December 2021).
The third set of measures (May 2020) directly targeted natural persons in socially disadvantaged categories. However, indirectly, it was supposed to help domestic companies by stimulating the consumption of their products and services. It provided citizens with EUR 100 vouchers they could use in hotels in North Macedonia and EUR 50 debit cards which could only be used to purchase domestic products. Moreover, a VAT-free weekend was organized during which all citizens were able to buy domestic products exempt of VAT charges in pre-determined sectors.
The fourth set of measures (September 2020) re-introduced the option for subsidizing salaries (which applied between March and May), for additional three months October–December 2020. This time, the amount of the subsidy was contingent upon the decrease of the company’s turnover and could reach up to EUR 350. The estimate was that a total of 250,000 employees would benefit and 83,000 jobs would be saved, given that the condition was for companies not to lay off staff until 31 July 2021. The European Investment Bank supplied the Government with EUR 100 million in low-interest rates loans to be disbursed through the commercial banks. The grace period for the previously issued interest-free loans was extended for additional 3 months. The state also issued a guarantee of EUR 10 million as collateral aimed to help the companies secure additional EUR 65 million in loans from the commercial banks.
Additional support was provided to the tourism, hospitality and transport sectors through grants for the travel agencies amounting between EUR 3000–7000, return of the tourist tax paid by accommodation capacities for 2019, 3 months of minimum wage for the tour guides, grants for specialized wedding reception facilities, extension of the operating licenses for night clubs and transport companies. VAT of artisans’ products was reduced from 18% to 5% in order to strengthen their competitiveness, and VAT for restaurant services was reduced from 18% to 10%. VAT exemption was also announced for donations of public character. SMEs were also able to use exemption from payment of profit tax advance payment for a period of 3 months, deferral of VAT payment by 1 week and an increase of the threshold to enter the regime of total income taxation from EUR 50,000 to 80,000.
The fifth set of measures (February 2021) included subsidies to the salaries for February and March 2021; interest-free loans with a potential grant component of 30–50%, in the total amount of EUR 10 million, targeting sectors such as tourism, event industry, hospitality, transport and private healthcare providers; specific financial support for media and broadcasters, crafts, liberal professions and companies with a reduction in turnover exceeding 50% in the previous
year; state guarantee scheme of EUR 5 million to support the disbursement of EUR 30 million in loans from the commercial banks and another extension of the grace period for the previous interest-free loans. The government also created a EUR 10 million fund to support export-oriented companies, EUR 6 million fund to support the technological development of SMEs and reduced or suspended custom duties on around 100 raw materials.
The sixth set of measures was announced in April 2021 and amounts to EUR 17,8 million. It specifically targets the most affected companies with grants and loans, such as the event industry and the hospitality sector, fitness centers, photo studios and travel agencies.
4.3. Impact of the adopted government measures and recommendations for a successful economic recovery
There seems to be a consensus among national experts that the value of the government’s response was adequate compared to the overall size of the budget, represented an important boost and helped many SMEs survive the initial wave of the pandemic. However, there is also a consensus that had the design and implementation of the measures been better, its impact could have been much stronger.
According to university professor Tamara Jovanov,¹⁸ the measures should have been adopted in a timelier manner in order to mitigate the initial and strongest impact of the pandemic. One of the more serious shortcomings is related to the high level of “red tape” (forms and documents) that companies were supposed to collect and submit in order to be eligible for assistance, as well as the often unclear and/or changing procedures and guidelines. This turned out to be a significant administrative burden for SMEs who generally have few employees and often lack the knowledge to prepare complex applications. Furthermore, the criteria for receiving assistance initially excluded companies which had been profitable before the pandemic, leaving some of the most vulnerable sectors ineligible. At the same time, there were also allegations of “informal corruption”, with some companies close to the ruling government coalition receiving government assistance despite the fact that they were profitable throughout the pandemic.
For the president of the Business Confederation of Macedonia,¹⁹ the support measures had predominantly a social component and did not take into full account the longer-term needs of the SMEs. While on the short term the subsidies, loans and tax relief measures served as a way to relieve “the headache” of many entrepreneurs who faced both a decrease in the demand and reduced liquidity, the uncertainty whether, when and how (much) they will need to return the received funds left a strong feeling of bitterness among entrepreneurs who had been paying taxes and sustaining the economy for years, hence expected more support in return. Moreover, the measures did not help to restore the much-needed trust in the economy and stimulate domestic consumption. Instead, faced with the imminent threat to their health and life, consumers prefer to save money and not to purchase non-essential goods and services.
¹⁸ Personal interview with professor Tamara Jovanov (PhD), Economic Faculty, University Goce Delchev – Shtip, who is also a member of the advisory board of the National Platform for Women Entrepreneurship, 26.11.2021.
¹⁹ Personal interview with Mr. Mile Boshkov, President of the Business Confederation of Macedonia, 25.11.2021
But according to professor Jovanov, the crisis mostly affected the middle and low classes, while the higher-end of the society maintained its purchasing power. Hence, the strong decrease in domestic consumption was partly due to the lack of adaptability of SMEs and their lack of skills and know-how to reinvent their offer and target new consumers and markets. Namely, the specific features of SMEs in North Macedonia as a combination of internal factors (entrepreneurs out of necessity and not out of opportunity, underdeveloped business skillset and digital competences among both managers and staff, not diversified company portfolios) and external factors (highly dependent on both imports and exports, limited access to finance opportunities) acted as an impediment for many of them to swiftly adapt and find a way out of the crisis.
A survey conducted by ILO at the end of 2020\textsuperscript{20} revealed that slightly more than half of all enterprises (52%) offered new products or services, or modified the existing ones. About a third decreased the price of selected products/services with a view to increasing sales and revenues (assuming customers were highly price sensitive). A quarter of the enterprises reached out to customers by providing delivery and/or online purchasing. According to the same survey, 61% of the enterprises were not satisfied with the government measures and suggest there could have been improvements in terms of their criteria, inclusiveness and targeting.\textsuperscript{21} Around 1/3 of all active companies used the government measures, especially those supporting jobs which benefited a total of 31% of the private sector workforce. In terms of registered companies 44% of all small companies used the government support, 41% of medium, 36% of micro and 31% of large companies, but when it comes to supporting jobs, the statistics go largely in favor of large enterprises where 50% of the jobs were supported, as opposed to 29–32% of the salaries in SMEs.\textsuperscript{22}
Job subsidies were the most popular support measure among SMEs and largely contributed to prevent an even bigger increase in the unemployment rate, followed by the subsidized loans. However, due to the fact that salaries were paid to the companies, and not to the workers directly, there were 181 cases of abuse when employers received state aid, but did not pay salaries to their employees. Until present day, no sanctions have been undertaken against those employers.
**Conclusions and recommendations**
- It is important to increase consumers’ trust and target domestic consumption as a component which can be easily stimulated by policy makers on the short term. This was done to a certain extent, although it could have been more efficient had the scope of products and the criteria for support to citizen categories been broader.
- The criteria to obtain financial support should have been better designed to ensure that it will go into the hands of those entities which need it most and are unable to secure other funding to overcome the crisis, while taking into account the profitability as well.
- Applications for recovery and long-term support measures should bear in mind the administrative capacity of SMEs and include only the strict minimum of bureaucracy.
\textsuperscript{20} Mojoska Blazhevski, Nikica. 2021. “Evolving challenges and expectations facing Macedonian enterprises during the COVID-19 pandemic (Second edition).” International Labour Organization. 19.
\textsuperscript{21} Mojoska. 2021. 8.
\textsuperscript{22} Jovanovik. 2021. 14.
• There should be an oversight mechanism to ensure that the financial support is spent for the right purpose and there is no abuse.
• The State should help SMEs develop their flexibility and adaptability to external shocks by envisaging continuous support for their investments in equipment, research and innovation, digitalization, staff training and re-qualification.
**Best practice – Vilnius turned into an open-air café**
The hospitality sector was one of the most adversely affected during COVID-19, with restaurants, bars and cafes in many countries either entirely closed or operating at significantly reduced capacities and with great losses at different stages of the pandemic. In order to help these SMEs recover, in April 2020 the city of Vilnius decided to allow them to use much of the public spaces in the old town to set up tables during the summer season. A total of 18 squares, piazzas and streets were initially designated as places that restaurants and bars could use to expand their outdoor seating and 160 SMEs applied to use that opportunity. The measure was warmly welcomed by the Lithuanian Association of Hotels and Restaurants as a great balance to “accommodate more visitors and bring life back to the city streets, but without violating security requirements”. At the same time, the city authorities provided a total of 400,000 EUR worth of restaurant vouchers to public health workers, as a way both to reward them for their services during the pandemic and to stimulate the hospitality sector.
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23 Henley, John. April 2020. “Lithuanian capital to be turned into vast open-air café”. The Guardian. https://www.theguardian.com/world/2020/apr/28/lithuanian-capital-to-be-turned-into-vast-open-air-cafe-vilnius
4.4. Entrepreneurial response to opportunities emerged during the crisis caused by COVID-19 pandemic – case studies from North Macedonia
GALINA
GALINA\(^{24}\) is a small enterprise established in Bitola in 2016 as a clothing manufacturer-subcontractor for foreign textile brands. Its products, predominantly tops and shirts, have been exported to a number of EU Member States, mostly Germany, Sweden and France. Although the restrictive sanitary measures did have an impact on enterprises because of the requirements related to distancing, wearing masks, providing higher sanitary conditions and restrictions on shifts, the apparel sector was once of the worst hit economic activities in the country mostly due to the closure of borders, the disruption of supply chains and the lower global demand for apparel products. At the outbreak of the pandemic, GALINA faced a decrease in its orders and serious difficulties to supply the necessary production materials. Hence, its manager decided to explore other options in order to find intermediary short-term solutions to overcome the crisis without laying off staff. Having extensive experience in the textile industry, she familiarized herself with the fabric specifications necessary to produce face masks and quickly re-oriented the production. She also sought to establish new distribution channels and offered Galina’s face masks to public entities in the Bitola region who at the time needed large quantities of face masks.
Given the uncertainty regarding the duration of the crisis, Galina’s manager conducted market research in order to identify textile products which demand is likely to remain despite the pandemic. She decided to start a production line of casual underwear multipacks and seized an opportunity offered through the USAID Business Ecosystem Project to obtain a donation of 3 sewing machines for this particular purpose. Having trained the employees to manufacture the new products herself, she contacted the companies she used to work with in the Western European countries to offer them the new products.
\(^{24}\) Personal interview with Roza Vasilevska, general manager, 11.11.2021.
**Figure 15:** Number of employees working at GALINA, per semester, 2019–2021
*Source: Personal interview with GALINA’s general manager*
**Figure 16:** GALINA’s cumulative turnover, per semester, 2019–2021, in EUR
*Source: Personal interview with GALINA’s general manager*
Introducing new products was the key to the company’s survival at a time when most companies in the apparel sector faced huge losses. In 2020, at the peak of the pandemic, the number of its employees increased by 80% and the turnover doubled, with the same tendency continuing in 2021. The company did not use any of the measures provided by the Government because they were not targeted to meet its needs. Instead, it leveraged the flexibility, adaptability and team spirit both of the manager and the staff as the key assets not only to overcome the crisis, but also to transform it into a growth opportunity.
GRADITEL-B\textsuperscript{25} is a small enterprise established in Ohrid in 1996 in the hospitality sector. They operate the restaurant Sajgija with a capacity of around 500 people which is open on regular basis and also hosts wedding receptions as the most important share of its usual income. The number of employees in the past several years has been constantly 13, despite the fact that the hospitality sector was one of the worst hit with the pandemic. Furthermore, the very fact that the restaurant is located in Ohrid, the Macedonian “tourism capital”, it was additionally adversely affected with the border closure and the lack of foreign tourists.
With the restrictions on movement and assembly, between March and May 2020, all restaurants, bars, night clubs and coffee shops were completely closed. As the first wave of the pandemic weakened and the weather improved, the restrictions were partially lifted and restaurants were allowed to open their terraces at the end of May 2020, but only up to 30% of the capacity. They were also operating at reduced working hours and were not allowed to host any celebrations (no more than 4 people could be seated at one table, initially only from the same family). The full restrictions returned during the fall 2020 and in the spring of 2021 at the peak of the second and the third wave of the pandemic when the restaurant was again closed for several weeks. But, even in the meantime the entire hospitality sector was operating at significantly reduced capacities and most of the time only in the outdoor parts (terraces). Moreover, given the looming risk of the contagion, many citizens did not feel comfortable eating out, which also affected the consumption/demand.
In order to save the business and not to lay off any staff, Graditel-B introduced a take-away and delivery service in May 2020. Moreover, in August 2020, they introduced a variety of home-made winter food products – which could be purchased in the restaurant. Macedonian people have a long tradition of preparing winter preserves – pickled vegetables, pepper paste (so-called ajvar and pindjur) and smoked meat. But, in the past years, as the pace of life accelerated and given that the winter preserves require time and skills (canning, preserving etc.), the new generations have been increasingly reluctant to prepare winter stock themselves. At the same time, the market offer for such products is usually higher in prices and lower in quality compared to the customer expectations. Hence, these products turned out to be a big success. After the initial sale of 20 jars the first week, the sale increased to 300 jars per week, allowing the restaurant to fully cover the operation costs and survive.
At the same time, the enterprise used three types of State assistance: salary subsidies during the months when they were available, a subsidized loan and 2 grants targeting wedding reception facilities. While such measures were helpful, they were much lower than the turnover generated through normal operations and insufficient to cover all the expenses, especially those related to ensuring decent salaries. In order to stimulate domestic consumption and increase the turnover, the VAT on restaurant services was reduced from 18% to 10%.
The hospitality sector and especially wedding reception facilities only resumed work at full capacity in the summer of 2021. However, with the fourth wave of the pandemic and the new restrictions in terms of vaccine certificates introduced in September 2021 (whereas only fully vaccinated people are able to go to restaurants and bars), it remains in a precarious position. To illustrate, as the restaurants’ turnover picked up during the summer of 2021, increasing from EUR 6,800 in July to EUR 10,000 in August, it marked a severe drop in September to EUR 3,300.
\textsuperscript{25} Personal interview with Slagjana Kotevska, general manager, 24.11.2021.
COUNTRY REPORT
SLOVENIA
Maja Has
Mirela Alpeza
5. COUNTRY REPORT – SLOVENIA
5.1. Quantification of the impact of pandemic on SME sector in Slovenia
In 2020, there were 68,125 enterprises operating in Slovenia (Table 7). As is the case in most world economies, 99.4 percent of the total number of enterprises belonged to the Slovenian small and medium enterprise sector. A total of 67,683 enterprises operated in the small and medium enterprise sector, and the largest share belonged to micro enterprises (84.7 percent).
Table 7: Enterprise structure, employment and total income with regard to size in 2019 and 2020 in Slovenia
| Economic criterion for sector evaluation | Micro | Small | Medium | Large |
|----------------------------------------|-------|-------|--------|-------|
| | 2019 | 2020 | 2019 | 2020 | 2019 | 2020 | 2019 | 2020 |
| Number of entities | 57,146| 57,722| 8,521 | 8,846 | 1,095 | 1,115 | 416 | 442 |
| Number of entities (share) | 85.1% | 84.7% | 12.7% | 13% | 1.6% | 1.6% | 0.6% | 0.6% |
| Number of employees | 94,886| 93,204| 141,399| 140,171| 105,023| 101,221| 178,197| 175,105|
| Employment (share) | 18.3% | 18.3% | 27.2% | 27.5% | 20.2% | 19.9% | 34.3% | 34.4% |
| Total income (000 EUR) | 9,963,457| 9,364,247| 22,644,057| 21,839,493| 19,201,471| 17,946,912| 52,083,463| 48,371,512|
| Total income (share) | 9.6% | 9.6% | 21.8% | 22.4% | 18.5% | 18.4% | 50.1% | 49.6% |
Sources: Adapted from “INFORMATION ON BUSINESS OPERATIONS OF COMPANIES IN THE REPUBLIC OF SLOVENIA IN 2019”, Agency of the Republic of Slovenia for Public Legal Records and Related Services, Ljubljana, 2020 & “INFORMATION ON BUSINESS OPERATIONS OF COMPANIES IN THE REPUBLIC OF SLOVENIA IN 2020”, Agency of the Republic of Slovenia for Public Legal Records and Related Services, Ljubljana, 2021
Although 2020 was marked by the pandemic and its characteristics that affected business conditions, 947 new enterprises were registered in Slovenia, which is an increase of 1.4 percent compared to the previous year.
26 Slight deviations in the sum of the total share (100%) are possible due to rounding of amounts.
27 Slight deviations in the sum of the total share (100%) are possible due to rounding of amounts.
**Figure 17:** Enterprise structure with regard to size in 2019 and 2020 in Slovenia
Sources: Adapted from “INFORMATION ON BUSINESS OPERATIONS OF COMPANIES IN THE REPUBLIC OF SLOVENIA IN 2019”, Agency of the Republic of Slovenia for Public Legal Records and Related Services, Ljubljana, 2020
“INFORMATION ON BUSINESS OPERATIONS OF COMPANIES IN THE REPUBLIC OF SLOVENIA IN 2020”, Agency of the Republic of Slovenia for Public Legal Records and Related Services, Ljubljana, 2021
Nevertheless, the negative impact of the pandemic is visible in 2020, which resulted in the reduction of the number of employees in the entire business sector of Slovenia by almost 2 percent compared to 2019. In 2020, 1,682 jobs were lost in the micro enterprise sector, 1,228 jobs in small enterprises, 3,802 jobs in medium enterprise, and 3,092 jobs were lost in large enterprises (Figure 18).
**Figure 18:** Enterprise size and employment in 2019 and 2020 in Slovenia
| Enterprise Size | 2019 | 2020 |
|-----------------|------|------|
| Micro | 94,886 | 93,204 |
| Small | 141,399 | 140,171 |
| Medium | 105,023 | 101,221 |
| Large | 178,197 | 175,105 |
*Sources:* Adapted from “INFORMATION ON BUSINESS OPERATIONS OF COMPANIES IN THE REPUBLIC OF SLOVENIA IN 2019”, Agency of the Republic of Slovenia for Public Legal Records and Related Services, Ljubljana, 2020
“INFORMATION ON BUSINESS OPERATIONS OF COMPANIES IN THE REPUBLIC OF SLOVENIA IN 2020”, Agency of the Republic of Slovenia for Public Legal Records and Related Services, Ljubljana, 2021
Due to significant restrictions on business activities in the conditions of the pandemic, there was a decrease in the total income for all categories of business entities in 2020. The decrease in income was most pronounced in large enterprises (7.1 percent), and least in small enterprises (3.6 percent). Total income in micro enterprises decreased by 6 percent, and in medium enterprises by 6.5 percent compared to 2019.
Figure 19 shows the differences between micro, small, medium and large enterprises in terms of their contribution to employment and total income. In 2020, enterprises in the small and medium enterprise sector employed 65.7 percent of the total number of employees, and they accounted for 50.4 percent of total income generated at the level of the entire business sector. Within the small and medium enterprise sector, small enterprises stand out with a share in total employment of 27.5 percent and a share in total income of 22.4 percent.
Sources: Adapted from “INFORMATION ON BUSINESS OPERATIONS OF COMPANIES IN THE REPUBLIC OF SLOVENIA IN 2019”, Agency of the Republic of Slovenia for Public Legal Records and Related Services, Ljubljana, 2020
“INFORMATION ON BUSINESS OPERATIONS OF COMPANIES IN THE REPUBLIC OF SLOVENIA IN 2020”, Agency of the Republic of Slovenia for Public Legal Records and Related Services, Ljubljana, 2021
The consequences of the pandemic also affected the enterprises’ financial results. In 2020, 41,666 enterprises operated with a profit, which is 8.6 percent less than in 2019. On the other hand, 24,469 operated with a loss, or 25.4 percent more than in 2019.
In 2020, the Slovenian business sector generated a profit of EUR 4.6 billion, which is a decrease of 15 percent compared to 2019. On the other hand, the total reported losses of the entire business sector amounted to EUR 1.8 billion, which is 2.3 times higher than the amount reported in 2019. These results also affected the total consolidated result, i.e. net profit, which was 39.3 percent lower than in 2019, and amounted to EUR 2.8 billion.
Table 8: Financial efficiency of enterprises in 2019 and 2020
| Enterprises | 2019 | 2020 |
|-------------|------|------|
| | Thousand EUR | % | Thousand EUR | % |
| **Micro** | | | | |
| Profit | 818,675 | 15.2% | 705,157 | 15.4% |
| Loss | 344,653 | 44.8% | 472,883 | 26.6% |
| Consolidated result – Net profit/loss | 474,022 | 10.2% | 232,273 | 8.3% |
| **Small** | | | | |
| Profit | 1,165,782 | 21.6% | 1,094,117 | 23.9% |
| Loss | 116,259 | 15.1% | 221,509 | 12.4% |
| Consolidated result – Net profit/loss | 1,049,523 | 22.7% | 872,608 | 31.1% |
| **Medium** | | | | |
| Profit | 893,265 | 16.6% | 836,963 | 18.3% |
| Loss | 95,886 | 12.5% | 156,612 | 8.8% |
| Consolidated result – Net profit/loss | 797,379 | 17.2% | 680,351 | 24.2% |
| **Large** | | | | |
| Profit | 2,515,934 | 46.6% | 1,949,578 | 42.5% |
| Loss | 212,062 | 27.6% | 928,894 | 52.2% |
| Consolidated result – Net profit/loss | 2,303,871 | 49.8% | 1,020,684 | 36.4% |
| **Total** | | | | |
| Profit | 5,393,656 | | 4,585,815 | |
| Loss | 768,860 | | 1,779,899 | |
| Consolidated result – Net profit/loss | 4,624,796 | | 2,805,916 | |
Sources: Adapted from “INFORMATION ON BUSINESS OPERATIONS OF COMPANIES IN THE REPUBLIC OF SLOVENIA IN 2019”, Agency of the Republic of Slovenia for Public Legal Records and Related Services, Ljubljana, 2020 & “INFORMATION ON BUSINESS OPERATIONS OF COMPANIES IN THE REPUBLIC OF SLOVENIA IN 2020”, Agency of the Republic of Slovenia for Public Legal Records and Related Services, Ljubljana, 2021
28 Slight deviations in the sum of the total share (100%) are possible due to rounding of amounts.
Numerous challenges and changes that marked operations of enterprises during 2020 affected the financial performance of the entire business sector and resulted in a simultaneous reduction in the amount of generated profit and an increase in the amount of reported losses in enterprises of all sizes. Nevertheless, regardless of the mentioned changes, in 2020 all categories of enterprises achieved a positive consolidated result, i.e. net profit.
Micro enterprises generated EUR 705.2 million in profit and reported losses in the amount of EUR 472.9 million in 2020. Compared to 2019, profits decreased by almost 14 percent, while losses of micro enterprises increased by 37.2 percent. The small enterprise sector generated a profit of EUR 1.1 million in 2020, which is a decrease of 6.1 percent compared to the result achieved in 2019. Losses of small enterprises amounted to EUR 221.5 million, which is as much as 90.5 percent more than the previous year. Medium enterprises made a profit of EUR 837 million during 2020, which is 6.3 percent less than in 2019. The amount of reported losses in the medium enterprise sector amounted to EUR 156.6 million, which is 63.3 percent more than in 2019. Large enterprises made a profit of more than EUR 1.9 billion in 2020, and this result represents a decrease of 22.5 percent compared to the previous year. In addition to the decrease in profits, operations of large enterprises in 2020 were marked by a significant increase in losses. Large enterprises reported a loss of EUR 928.9 million, which is almost 4.4 times more than in 2019.
These financial results affected changes in the structure of the share of individual enterprises in total profit and loss at the level of the entire Slovenian business sector. Micro, small and medium enterprises in 2020 increased their shares in total profit with their business results and reduced their shares in total losses at the level of the economy. The most significant reduction is visible in the category of micro enterprises, whose share in total losses decreased by 18.2 percentage points compared to 2019. With their results, large enterprises took the biggest share in the profits at the level of the economy in 2020, although this share decreased by 4.1 percentage points compared to 2019. Unlike the previous year, when micro enterprises participated with the largest share (44.8 percent) in total losses, in 2020 the share of large enterprises in total losses of the economy increased by 24.6 percentage points, whereby large enterprises took the majority share (52.2 percent).
Although the consequences of the pandemic affected the operations of all business entities in Slovenia during 2020, it can be seen, based on the above data, that they had a more pronounced impact on the operations of large enterprises than on the small and medium enterprise sector.
5.2. Policy measures supporting SMEs during the COVID-19 pandemic
Slovenia’s Recovery and Resilience Plan (RRP)\textsuperscript{29} pursues the general objective of the Recovery and Resilience Facility to promote economic, social and territorial cohesion within the European Union by introducing reforms and investments focused on attaining digital and green transition. Slovenia will thus improve and strengthen the resilience of the economy and achieve the level of economic growth, recorded before the pandemic, at a faster rate. This will be achieved through the digital transformation of certain sectors of the economy and society, reduced administrative burdens and greater openness and flexibility of the Slovenian economy, and by strengthening the innovation potential of all relevant stakeholders. Slovenia will also invest in environmental, transport, energy, education, healthcare, social and other infrastructures which can make a significant contribution to economic growth in the coming years and help pursue the objective of climate neutrality by 2050. The recovery instrument NextGenerationEU makes a total of EUR 5.7 billion available to Slovenia, of which EUR 3.6 billion loans and 2.1 billion grants (EUR 312 million from REACT-EU, EUR 134 million from the Just Transition Fund, EUR 1.6 billion from the Recovery and Resilience Facility and EUR 68 million for Rural Development). Slovenia’s RRP allocates the available funding to the following four key development areas: 1) Green Transition, 2) Digital transformation, 3) Smart, sustainable and inclusive growth and 4) Healthcare and social security. Each component within an individual development area includes interrelated reforms and investments. The relevant milestones and objectives are defined for each measure, which will enable implementation monitoring.
Due to the severe negative shock that the restrictions pose for the economy and the income of the population, the Government adopted and drafted an array of measures to limit negative consequences. To this end, the Act Determining the Intervention Measures to Contain the COVID-19 Epidemic and Mitigate its Consequences for Citizens and the Economy (ZIUZEOP) or the so-called anti-corona package 1 and its amendments, and the Act on Additional Liquidity to the Economy to Mitigate the Effects of the COVID-19 Infectious Disease Epidemic (the so-called anti-corona package 2 were adopted.\textsuperscript{30} The specific objectives of the measures proposed are the preservation of jobs and business operations; improvement of the social security of individuals, particularly of those affected the most by the COVID-19 disease; extraordinary assistance for the self-employed; improvement of corporate liquidity; assistance to science and research projects to combat the COVID-19 epidemic; reduction of attendance fees and salaries of public officials at the state level; exemption from payment of distribution services and assistance to agriculture. Package 2 included key measures that enabled companies to revive investment activities and thus preserve jobs.
To support measures to mitigate consequences of the COVID-19 pandemic, the Government of the Republic of Slovenia also adopted decisions relating to the implementation of the cohesion policy on 24 March 2020. The budget users involved in the implementation of the cohesion policy had to submit proposals to redistribute funds from the European Social Fund (ESF) and the European Regional Development Fund (ERDF) into measures to assist companies and preserve jobs and health care, and were invited to draft new or adjusted measures due to the epidemic.
\textsuperscript{29} Republic of Slovenia – Government Office for Development and European Cohesion Policy (2021). \textit{Recovery and Resilience Plan}. Available at: https://eu-skladi.si/en/post-2020-1/recovery-and-resilience-plan
\textsuperscript{30} Republic of Slovenia (2020). 2020 Stability Programme – April 2020, the COVID – 19 scenario
As an urgent and speedy measure, the Government of the Republic of Slovenia also enhanced commodity reserves and purchased special equipment to fight COVID-19.
An advisory group has been established to assist ministries in the preparation of the first legislative package with the following orientations that are part of the Anti-Corona Package 1 guidelines\(^{31}\):
1) Measures to preserve jobs – a new scheme for co-financing wage compensations, providing for the rewarding of persons employed and activated in critical sectors, sick pay and additional funds for already subsidized employment;
2) Measures to improve the social situation of people – the status of employees who were unable to work due to force majeure, unemployment benefit from the first day of unemployment, waiving the payment of fees for public services that are not provided, solidarity bonus for pensioners;
3) Measures to provide emergency assistance to self-employed persons – emergency assistance, basic monthly income amounting to 70% of the net minimum wage, writing off contributions while maintaining rights, suspending the prepayment of income tax;
4) Measures to keep businesses in operation – all contributions for pension insurance of employees who work to be covered by the state;
5) Measures to improve the liquidity of businesses and provide assistance to scientific research projects aimed at fighting the epidemic – purchasing claims against Slovenian companies, suspending the prepayment of corporate income tax and the payment of self-employment income tax, reducing deadlines for payments to private suppliers in the public sector, redirecting unused ESF funds;
6) Reduction of meeting fees and wages and exemption from paying fees for distribution services;
7) Providing aid to the agriculture sector; and
8) Measures related to public procurement.
The second legislative package included measures to ensure the liquidity of the economy and adjustments to the first package. By December 2021, the Government adopted eight packages of measures to mitigate the consequences of the pandemic for citizens and the economy.\(^{32}\) Those packages include provisional measures in the areas of labor, public finance, the economy, agriculture, forestry and food, scholarships, subsidized student meals, higher education, infrastructure, and public procurement. Table 9 presents the relevant support measures in response to COVID -19 related to the small and medium enterprise sector in Slovenia.
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\(^{31}\) OECD (2020). Inclusive Entrepreneurship Policies, Country Assessment Notes – Slovenia, page 26
\(^{32}\) https://www.gov.si/teme/koronavirus-sars-cov-2/odpravljanje-posledic-epidemije/osmi-paket-ukrepov-zamolitev-posledic-epidemije-pkp8/
| Type of support | Instrument | Description |
|-----------------|------------|-------------|
| Business costs | Reduction or waiver of administrative and government fees | Credit payments to the state has been deferred by 12 months. State suppliers were paid within 8 days instead of a minimum of 30 days. |
| Debt finance | New lending – under concessional terms | The state-owned export and development bank made additional funding, totaling 2% of GDP, available. Liquidity release in the amount of EUR million 115. Legislation passed allowing a 12-month loan holiday to businesses and farmers. |
| Provide wage as alternative to direct payments to individuals | Wage subsidies for suspended workers due to pandemic-related closures and quarantined people (about €50 million). Persons employed and activated in sectors that are key in overcoming the epidemic were rewarded with an increase in their basic salary between 10% and 200%, based on the decision of their superiors, in the form of hazard pay and higher workload bonuses. The Government will pay all social security contributions for firms that continue operations during the crisis. Moreover, the state will co-finance 20% of employees’ net compensation. In case of quarantine, the state covers 80% of wages. All contributions for temporarily laid-off employees paid into the healthcare and pension insurance schemes were covered by the state, and the insured persons’ rights were preserved. The measure was in place until May 31, 2020, with the possibility of an extension. |
| Employment support | Subsidies for employee sick leave | The national health insurance fund financed employers’ obligation to pay sick leave for the first 30 days of sickness for people falling sick during the crisis. Sick pay for all workers during the pandemic from the first day onwards was covered by the Health Insurance Institute of Slovenia, not the employer. Parents who stayed home to care for their children were entitled to 50% wage compensation. |
| Support for informal or self-employed workers | Self-employed workers who were unable to operate due to the crisis or whose operations have been significantly reduced were entitled to a monthly cash transfer in the amount of 70% of the minimum net wage. Self-employed workers were guaranteed a minimum income of 70 percent of the minimum net wage and no longer subject to social security contributions. |
| Unemployment benefits | Unemployed persons were entitled to benefits from the first day of unemployment. |
| Tax | Corporate tax rate reductions, credits, waivers, and/or deferrals | On 18/3/2020, the tax burden on businesses was eased with a 12-month deferral of credit payments. Corporate income tax payments have been deferred for up to 24 months without incurring interest. |
Source: Adapted from https://www.worldbank.org/en/data/interactive/2020/04/14/map-of-sme-support-measures-in-response-to-covid-19
In addition to the Government’s measures and laws, the Slovenian Export and Development Bank (SID Banka) and the Slovene Enterprise Fund (SPS) also adopted measures to provide support to the companies. Together with the Ministry of Economic Development and Technology, SID Banka offers financial products to small and medium enterprises and large companies in the total amount of EUR 800 million. The funds are intended to address liquidity problems, including liquidity in the supply of services and products, problems due to a drop in demand, production fall-out, supply chain difficulties and investment difficulties. Measures also include insurance and bank loan refinancing. Furthermore, the Slovene Enterprise Fund in cooperation with the Ministry of Economic Development and Technology, offers a package of measures for micro companies and SME sector related to liquidity loans, guarantees for bank loans with interest rate subsidy, microloans for companies in problematic areas and other measures intended to support the SME sector during the COVID – 19 pandemic.\(^{33}\)
**MUNICIPALITY OF PTUJ – an example of local support measure for supporting SMEs during the pandemic\(^{34}\)**
*The Municipality of Ptuj, with its activities, is a good example of local support measures in the fight against the economic consequences of the COVID-19 pandemic. Specifically, the measures refer to the allocation of financial aid to economic entities in the municipality of Ptuj which have suffered business damage due to the pandemic. The total amount of financial aid was EUR 50,000. The aid was intended for craftsmen, micro enterprises with less than 10 employees or whose annual turnover and / or assets do not exceed two million euros, and small enterprises with less than 50 employees whose annual turnover and / or assets do not exceed ten million euros. Requests for financial assistance could be submitted by enterprises based in the municipality of Ptuj and those that operate in the municipality, but due to the pandemic did not operate for at least a month or faced restrictions. The measures excluded enterprises owned by the state or municipalities. Eligible costs included material costs such as accounting costs, proportional share of property insurance, rent of business premises, waste management, electricity, drainage and treatment of municipal water and drinking water supply, and the aid was divided into two application phases.*
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\(^{33}\) https://selih.si/en/covid-19-en/measures-to-support-slovenian-economy/
\(^{34}\) https://ptujinfo.com/novica/politika-gospodarstvo/ptujska-obcina-bo-s-50000-evri-pomagala-podjetnikom-ki-so-v-epidemiji
5.3. Review of the adopted government measures for supporting SMEs during the pandemic in Slovenia
In the fall of 2020, research\textsuperscript{35} on the effectiveness of anti-corona aid packages (PKP) was conducted among members of the Association of Employers of Slovenia. The survey was answered by 204 employers operating in various industries, with the majority coming from the manufacturing industry (32 percent), followed by the catering industry (8 percent), professional activities (7.3 percent) and trade (7 percent).
The employers projected that the pandemic will have a very negative impact on both, the European and Slovenian economies, and that their revenues will decrease by at least 17 percent, with some even reporting a 100% drop in revenue. Most employers were convinced that the economic crisis triggered by the pandemic will last for more than two years. According to the survey results, employers carried out a number of activities to combat the negative effects of the pandemic, but still, the crisis was expected to reduce investment in tangible assets (by 75 percent), intangible capital (by 67 percent) and investment in new product development (by 53 percent). Furthermore, the pandemic seriously affected the labor market. More than 60 percent of employers reported a reduction in the number of employees and companies reduced their investment in training. Adjustments to the pandemic are reflected in the introduction of work from home (remote work), the use of protective equipment, the payment of crisis allowances with an exemption from pension and disability insurance, restrictions on business travel, the introduction of new means of communication and the like. The biggest challenge for employers in 2020 was managing insecurity, mental health of employees and maintaining/increasing employee productivity.
Regarding anti-corona government measures, it was found that employers mostly benefited from measures related to the payment of wage compensation and social security contributions, including reimbursement for workers whose enterprises were closed due to the sanitary restrictions and reimbursement of 100 percent of compensation in case of quarantine due to contact with an infected person in the workplace. From the viewpoint of employers, the most appropriate measures in the first anti-corona aid package included the entitlement to compensation for temporary absence from work at the expense of compulsory health insurance and reimbursement of labor costs for workers on forced leave and quarantined workers, which 90% of employers rated as useful or very useful. The least useful measures, according to employers, were those related to court proceedings, the establishment of additional credit lines through public funds and SID Bank and the postponement of self-employment contributions for two years. Many respondents did not benefit from any measure because, in their view, they were not eligible for aid.
Employers also provided their suggestions related to the improvement in government measures to help their company cope more effectively with the COVID-19 pandemic: measures should have been simpler, faster and include as few exceptions as possible. Furthermore, the most common suggestions were related to reduction in the payment of taxes or refunds of salary compensations. Employers also emphasized the need to help companies provide liquidity.
\textsuperscript{35} The research was conducted within the project “School of Negotiation – Training of Employers for Social Dialogue (Negotiating School 2)” in the period from 15th October – 8th November 2020. Source: Franca, V., Domadenik, P. i Redek, T. (2020). \textit{Research on the effectiveness of anti-corona measures – PKP (slo. Raziskava o učinkovitosti protikoronskih ukrepov – PKP)}. Ljubljana
through unconditional grants, facilitate access to credit, defer contributions and taxes and, above all, ensure a transparent system of all applicable measures without excessive bureaucracy.
Based on the research results, authors Franca et al (2020) provided a set of recommendations for improvement of the government response in supporting the sustainability of businesses in a time of crisis:
- resources need to be directed to funding smart containment strategies, which will include intensive testing and tracking, restrictions on mobility in case of infections and real-time risk assessment;
- incentive schemes should be created for both start-ups and companies that have good business models and identified products or services, but do not have sufficient financial resources for further development;
- employment of young people and the elderly needs to be encouraged and better matched with the needs of the labor market through retraining and upskilling programs.
Finally, it was concluded that too rapid reduction in fiscal measures aimed at helping businesses would prolong the recession and exacerbate poverty and inequality.
5.4. Entrepreneurial response to opportunities emerged during the crisis caused by COVID-19 pandemic – case studies from Slovenia
LOTRIČ Metrology (www.latric.si/si)
LOTRIČ Metrology, a family business in the Selška Valley, has been operating in the area of measurement, calibration, product development and implementation of metrological solutions for three decades. As an entrepreneur, the current owner and general manager Marko Lotrič started the craft in 1991, working mainly in the field of calibration of scales, weights and pipettes. Today, his group unites over 170 experts in the field of metrology, employed in seven countries. In addition to the parent company LOTRIČ Metrology, which operates since its beginnings in Selška valley, there are four companies in Slovenia which operate in Železniki - LOTRIČ Certificiranje, Ljubljana - PSM merilni sistemi and Puconci - Mikro Medica. Subsidiaries are operating in Austria, Germany, Croatia, Bosnia and Herzegovina, Serbia and North Macedonia, where they are established as accredited laboratories.
During the coronavirus epidemic, the company demonstrated traditionally strong attributes of family businesses that respond well to crises by displaying their flexibility and resilience. At the announcement of the epidemic, the company immediately set up a test laboratory and certification body to help start the Slovenian production and self-sufficiency in terms of face masks. In parallel, together with the companies Domel, EKWB, BPMC, Technology Park Ljubljana and Zavod 404, they developed a new, Slovenian medical ventilator called DIHAM. They joined forces with the goal of developing a prototype of a life-saving respirator that could be completely self-sufficiently produced in Slovenia, with Slovenian components and Slovenian knowledge. Along the way, they have developed a culture of cooperation, sacrifice, agility and help and finally, they managed to bring this initiative from complete chaos to a working prototype. It is a so-called turbine pressure respirator (without inflatable bellows). The device is designed in such a way that it can operate both in a hospital environment and in mobile hospitals or even independently. Furthermore, in response to the pandemic, company started testing protective masks and measuring CO2 in indoor areas. Together with the National Institute of Biology, they started testing protective masks, conducting over 150 tests for 50 various models of masks and materials and became a certified body for FFP protective masks. In view of the pandemic, they also decided to implement a project related to the measurement of carbon dioxide levels indoors, which is one of the main indicators of air quality. In cooperation with the local school in Selca, the Exactum system was installed, which gives teachers insight into real-time measurements and thus more efficient ventilation of the premises.
LOTRIČ Metrology was recipient of the Excellence Award for Family Business in 2020, awarded by EY Slovenia to the best Slovenian family businesses. Looking ahead, as it celebrated its 30th anniversary in 2021, LOTRIČ Metrology wants to become a company with a zero negative impact on the environment by 2031. In the future, it will continue to focus on the development of its own metrology products and customer-tailored solutions which allow for a higher degree of production and business automation.
Optifarm Smart is an IT managing system for short food-chain optimization, which connects all the participants in the market. Farmers, who sell agricultural products and buyers, who seeking transparency of their demands, ease of use and orderly distribution, with all the logistics already covered. Optifarm Smart connects those different factors and unites them in a common point. Farmers produce the products and buyers buy them, the management is in charge of orderly management, local stores provide support through the logistics department. Optifarm Smart supports transparency, traceability and safe purchase of high-quality local and ‘eco’ fruit, vegetables and other products. Short food chains and optimization of farmers/producers’ businesses are the company’s primary focus, as well as providing farmers with modern sales channels in terms of IT support to sell their products.
The company, founded in 2016, and was originally intended to connect local suppliers of agricultural products with public institutions. But once it was developed, they realized that the stakeholders already operating in the public procurement market do not need this system. After this experience, the company decided to switch to retail, and so they developed the Optifarm Smart app. The coronavirus epidemic greatly accelerated its debut. They originally planned to put it into use in September 2020, but when having realized in March last year how quickly food shopping had moved online, they were in a hurry. Hence, the app became operational as early as April.
The Optifarm Smart application enables an overview of providers and their products. The user puts products in the cart, selects the method of payment and transport, and pays. The application takes care of the delivery to the last kilometer, using different types of services, connecting with transport service providers and products, enabling pick-up at local markets, home delivery, and supporting traveling shopping. In the spirit of promoting short delivery routes, it offers the user products close to him, depending on his location.
In general, the business model is based on the payment of sales commission and their approach is comprehensive: at the regional level or at the state level. By partnering with providers and presenting their individual stories, they are also trying to influence people’s mindset about where to get food and who produces it. Given their contribution to entrepreneurship and innovation in the field of food self-sufficiency, Optifarm won the award in the category for the best example of integration and cooperation in the Agrobiznis competition 2021. The long-term goal of the company is to connect all the stakeholders in local markets with local customers, support the local economy and participate in developing solutions for a smart society.
CONCLUSION AND RECOMMENDATIONS
6. CONCLUSION AND RECOMMENDATIONS
The concluding part of the study compares the impact of the crisis on the SME sector in the four observed countries, identifies positive aspects of government measures to support the SME sector during the crisis, and recommendations in the form of lessons learned that can be useful to policy makers in situations where support for the sustainability of the sector is necessary, due to the impact of external adverse conditions.
6.1. Impact of the pandemic on the small and medium enterprise sector in China, Croatia, North Macedonia and Slovenia
The COVID-19 pandemic has left a significant mark on the health, social and economic picture of most countries, including China, Croatia, North Macedonia and Slovenia, which are the focus of this study. As a result of the pandemic, by the end of 2021 in China 4,636 people had lost their lives, 12,538 people in Croatia, 7,960 people in North Macedonia, and 5,589 people in Slovenia.\textsuperscript{36} In 2020, the GDP in Croatia decreased by 8.1% compared to 2019, in North Macedonia by 5.2% and in Slovenia by 4.2%, while in China it increased by 2.3%\textsuperscript{37}.
The small and medium enterprise sector is an important part of the economy in all the four observed countries. In China, the SME sector provides jobs for 130 million people. In Croatia, 72% of employees work in small and medium enterprises, 74% in North Macedonia, and 66% in Slovenia.
In all the four countries, the companies faced a significant disruption in business activity. The flow of raw materials, labor and other production factors required by enterprises was interrupted and production could not be carried out smoothly. Sales channels were blocked and finished products could not be sold. The pandemic revealed an absence of SMEs’ own risk mechanisms to cope with the pandemic, but it also increased the need for an entrepreneurial answer to newly emerged opportunities coming from changed environment and needs. The awareness on the importance of digital transformation grew significantly and, in some industries, like health and IT, the barriers for international trade were lowered due to increased need for the products. This study presents 8 cases\textsuperscript{38} of companies from all four observed countries which identified an opportunity and successfully created new products, services and processes that meet the customers’ needs, resulting from the changed circumstances of living and working in a pandemic.
In China, in the first quarter of 2020, more than 460,000 private small and micro enterprises went bankrupt. With the slowdown of the global economy and the decline in export growth caused by trade frictions, the pressure on China’s economy has intensified in the last couple of years. SMEs
\textsuperscript{36} https://www.worldometers.info/
\textsuperscript{37} https://data.worldbank.org/indicator/NY.GDP.MKTP.KD.ZG?name_desc=false
\textsuperscript{38} Companies that are presented in the report as an example of good practice are: CHENGDU LIGENG CULTURE COMMUNICATION CO. LIMITED, CHENGDU LONG YUAN NETWORK TECHNOLOGY CO., ALARM AUTOMATIKA, GRAD EXPORT, GALINA, GRADITEL – B, LOTRČI METROLOGY, OPTIFARM SMART
were facing greater difficulties while the pandemic has been adding to the downward pressure on the economy.
In 2020, the Croatian business sector achieved a positive consolidated result, but lower by almost 33% compared to 2019. The decrease in this indicator is the result of a decrease in total profit in 2020 by 6% with a simultaneous increase in total loss by 42% compared to the previous year. In Croatia, the negative effects of the pandemic had the greatest impact on financial operations of large enterprises and the least impact on operations of micro and small enterprises. These results can be explained by the agility and flexibility of micro and small enterprises, which are reflected in the rapid adaptation of the business to changes, but also to the government’s economic support measures.
The pandemic affected three quarters of companies in North Macedonia, and most employers resorted to reduction in salaries and all additional payments to their employees, as well as laying off staff in more serious cases. Around 4% of all registered business entities in North Macedonia, or a total of 2,846 businesses, had to cease their activities as a result of the pandemic and all of them fall within the category of micro and small enterprises.
The negative impact of the pandemic is visible also in Slovenia, resulting in the reduction of the number of employees in the entire business sector in 2020 by almost 2% compared to 2019. Due to significant restrictions on business activities during the pandemic, there was a decrease in the total income for all the categories of business entities in 2020. In 2020, the Slovenian business sector generated a profit of EUR 4.6 billion, which is a decrease of 15% compared to 2019. On the other hand, the total reported losses of the entire business sector amounted to EUR 1.8 billion, which is 2.3 times higher loss than the amount reported in 2019.
Due to this pressure and the negative impact of the pandemic on the economy, especially on the small and medium enterprise sector, governments around the world, including China, Croatia, North Macedonia and Slovenia, recognized the economic and social importance of the small and medium enterprise sector. Soon after the pandemic broke out, special packages of measures to help the economy were created in all the four countries, with a special focus on small and medium enterprises.
### 6.2. Government measures to support the economy during the pandemic
Epidemiological measures to contain the pandemic have led to a reduction of economic activity and a decline in sales in companies that were directly or indirectly subjected to these measures. In order to overcome the challenges, many countries implemented measures to mitigate the economic consequences of the pandemic, with a special focus on small and medium enterprises (SMEs), which make up a dominant part of national economies of most countries.
The measures introduced by governments shortly after the outbreak of the pandemic included direct grants, tax reliefs and more favorable payment terms; loan guarantees and subsidized interest rates on loans; short-term loan insurance; deferrals of taxes and / or social security contributions; subsidies for employees’ salaries or the income of self-employed persons equivalent to salary; interest free loans, etc.
Some of the measures were aimed directly at companies that were helping to find the answers to the health aspects of the COVID-19 pandemic. In Croatia, the government prescribed measures to provide aid for research and development in the field of COVID-19; aid for investment in construction or upgrading of testing infrastructures and for the improvement of processes from the laboratory to the production level; and aid for investments in the production of products essential for the control of the spread of COVID-19.
In Slovenia, too, the government introduced measures to improve the liquidity of businesses and provide assistance to scientific research projects aimed at fighting the epidemic – purchasing claims against Slovenian companies, suspending the prepayment of corporate income tax and the payment of self-employment income tax, reducing deadlines for payments to private suppliers in the public sector, redirecting unused ESF funds.
Other government measures in Slovenia included support to preserve jobs, measures to improve the social situation of people (e.g. employees who were unable to work due to force majeure), measures to provide emergency assistance to self-employed persons – emergency assistance, basic monthly income amounting to 70% of net minimum wage, writing off contributions while maintaining rights, suspending the prepayment of income tax; measures to keep businesses in operation – all contributions for pension insurance of employees who work to be covered by the state, etc.
Some of the measures in North Macedonia related to direct support to the population, especially vulnerable groups in society, with indirect support to domestic companies by stimulating the consumption of their products and services (e.g. a VAT-free weekend was organized during which all citizens were able to buy domestic products exempt of VAT charges in pre-determined sectors).
In addition to measures at the national level, additional measures to support the sustainability of business and jobs have been created at the regional and local levels. In China, for example, some provinces introduced measures aimed at:
- Increasing financial support (such as lowering loan interest rates, extending loan terms, reducing or waiving financial service fees, and extending the collection of financial lease rentals);
- Implementing and improving tax incentives;
- Reducing the social burden on SMEs (tax reduction, fiscal support, financial support, innovation and public services, requiring relevant departments and local authorities to actively introduce policies to support SMEs from various aspects);
- Policy provisions to support SMEs to accelerate technological transformation (promotion of digitalization and intelligent transformation of traditional industries).
The support for EU Member States was insured through EU funds and mechanisms, and the European Investment Bank supplied the Government of North Macedonia with 100 million EUR in low-interest rates loans to be disbursed through the commercial banks.
Based on the establishment of the Recovery and Resilience Facility at the European Union level (whose measures and activities are aimed at helping the national economy recover after the crisis caused by the COVID-19 disease pandemic and creating a more resilient and sustainable society in the event of new economic shocks), Croatia and Slovenia, as EU Member States adopted the National Recovery and Resilience Plan 2021–2026 in Croatia and the Recovery and Resilience Plan in Slovenia respectively.
Slovenia’s Recovery and Resilience Plan (RRP) responds to the urgent need of fostering a strong recovery. The reforms and investments in the plan will help Slovenia become more sustainable, resilient and better prepared for the challenges and opportunities of the green and digital transitions, with 42% of the plan intended to support climate objectives and 21% of the plan to foster the digital transition. The plan consists of 55 investments and 33 reforms. They will be supported by EUR 1.8 billion in grants and EUR 0.7 billion in loans. Slovenia’s RRP allocates the available funding to the following four key development areas: 1) Green Transition, 2) Digital transformation, 3) Smart, sustainable and inclusive growth and 4) Healthcare and social security.
For its National Recovery and Resilience Plan 2021–2026 under the Facility, Croatia was provided with financial resources in the amount of almost EUR 9.9 billion, of which EUR 6.3 billion are grants, and about EUR 3.6 billion are soft loans. Croatia will allocate its recovery funds on 1) Economy, 2) Public administration, judiciary and public property, 3) Education, science and research, 4) Labor market and social protection, 5) Health care 6) Initiative: Renovation of buildings.
### 6.3. Review of the policy measures to combat the negative effects of pandemic on SMEs and recommendations
There seems to be a consensus among experts and entrepreneurs in the four observed countries that the governments’ response represented an important boost for the economy and helped many SMEs survive the initial wave of the pandemic. However, there is also a consensus that if the design and implementation of the measures were better, its impact could have been much stronger.
Most of the objections to the measures adopted by the governments were not related to the content of support measures, but to the definition and implementation of epidemiological measures that affected various activities. Because of their inconsistency, incoordination and delays with the implementation regulations, as well as the omission of certain activities when defining the measures, entrepreneurs experienced significant difficulties in adapting their operations to the changing conditions.
An important objection in Croatia relates to the decision to provide a more significant allocation of the funds from the National Recovery and Resilience Plan 2021–2026 to the public, instead of the private sector. There is a high level of agreement among employers in Croatia about the need for much more investment in the real sector, manufacturing, and the introduction of new technologies in order to create added value and new jobs. Most of the investments envisaged in the National Recovery and Resilience Plan are primarily intended for public projects, from sewerage, water management to transport investments. These investments, according to the Croatian Employers’ Association, benefit the lives of citizens but will not create enough jobs in the long run and increase the domestic product as much as possible.
It is also important to keep in mind that one of the serious shortcomings of policy measures is related to the high level of “red tape” (forms and documents) that companies were supposed to collect and submit in order to be eligible for assistance, as it was the case in North Macedonia, as well as the often unclear and/or changing procedures and guidelines. This turned out to be a significant administrative burden for SMEs who generally have few employees and often lack the knowledge to prepare complex applications.
While in the short-term the subsidies, loans and tax relief measures served as a way to help the entrepreneurs who faced both a decrease in the demand and reduced liquidity, the long-term needs of SMEs should be taken into account since the recovery of consumption is dependent on their recovery (SMEs as an important, and in most countries the largest employer).
The following are the key recommendations for improvement of policy response for SMEs in crisis, especially in the forthcoming period:
**Recommendation 1:**
**Fiscal support for SMEs and its implementation mechanisms**
*The main measures of government support for SMEs in crisis should include tax cuts, fee reductions, financial subsidies and related subsidies to reduce the burden of enterprises. When a crisis breaks out, a comprehensive disaster management system and financial emergency mechanism with various support policies should be established as fast as possible.*
**Recommendation 2:**
**Creating good macroeconomic operating environment to stimulate domestic demand**
*Apart from providing support to SMEs through various fiscal and financial instruments, expanding domestic demand is one of the most sustainable relief measures. By expanding domestic demand, enterprises can gain access to production opportunities in the market and people can gain access to employment opportunities, which is more dynamic and sustainable than fiscal subsidies and funding.*
**Recommendation 3:**
**Cutting the “red tape” in policy support measures**
*Applications for recovery and long-term support measures should bear in mind the administrative capacity of SMEs and include only the strict minimum of bureaucracy. However, there should be an oversight mechanism to ensure that the financial support is spent for the right purpose and there is no abuse. Measures should be simple, fast and include as few exceptions as possible.*
Recommendation 4:
**Principle of fairness in implementation of policy support measures**
The criteria to obtain financial support should be designed in a way to ensure that it will go into the hands of those entities which need it the most and are unable to secure other funding to overcome the crisis, while also taking profitability into account.
Recommendation 5:
**Establishing a good insurance payout mechanism**
Innovation in the insurance system should be promoted so that new varieties of insurance products suitable for pandemic disasters are introduced.
Recommendation 6:
**Support for unemployed due to the crisis**
Increasing the support of fiscal policies for re-employment and vocational training subsidies so that those unemployed due to the pandemic can quickly resume employment.
Recommendation 7:
**Developing SMEs’ own risk mechanisms to cope with external shocks**
The Government should help SMEs develop their flexibility and adaptability to external shocks by envisaging continuous support for their investments in equipment, research and innovation, digitalization, staff training and re-qualification.
Recommendation 8:
**Support for SMEs after the pandemic: post-pandemic reconstruction loans**
Too rapid reduction in fiscal measures related to SME support would prolong the recession and exacerbate poverty and inequality. Financial support for SMEs after the disaster should be prolonged, especially for those severely affected by the pandemic (post-disaster reconstruction loans).
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# LIST OF TABLES
| Table | Description | Page |
|-------|-----------------------------------------------------------------------------|------|
| 1 | Number of enterprises and employees with regard to size | 8 |
| 2 | Number of employees in China in 2020 | 9 |
| 3 | SME-Support Measures in Response to COVID-19 adopted by Chinese provinces/municipalities | 12 |
| 4 | Enterprise size and employment, total income and exports in 2019 and 2020 in Croatia | 28 |
| 5 | Financial efficiency of enterprises in 2019 and 2020 | 31 |
| 6 | Establishment and termination of registered legal entities in 2019 and 2020 | 34 |
| 7 | Enterprise structure, employment and total income with regard to size in 2019 and 2020 in Slovenia | 60 |
| 8 | Financial efficiency of enterprises in 2019 and 2020 | 66 |
| 9 | SME-Support Measures in Response to COVID-19 implemented by the Slovenian Government | 70 |
LIST OF FIGURES
Figure 1: Enterprise structure in Croatia with regard to size in 2019 and 2020 ........... 24
Figure 2: Enterprise size and total income (million EUR) in 2019 and 2020 in Croatia .... 25
Figure 3: Enterprise size and exports (million EUR) in 2019 and 2020 in Croatia .......... 26
Figure 4: Enterprise size and number of employees in 2019 and 2020 in Croatia ........... 27
Figure 5: Number of employees, total income and exports in the small and medium enterprise sector in 2019 and 2020 in Croatia ................................................. 29
Figure 6: Total employment, income and exports by enterprise size in 2020 ............. 30
Figure 7: Number of “profit-makers” and “loss-makers” in the small and medium enterprise sector in 2020, by area of activity ......................................................... 33
Figure 8: Number of registrations and bankruptcies of business entities in the period from 2019 to 2021, by quarters, in Croatia ......................................................... 35
Figure 9: Share of the SMEs in the economy of North Macedonia in 2019 ............... 45
Figure 10: Quarterly GDP in the period 2019-2021, in million EUR ......................... 46
Figure 11: Monthly unemployment rate, 2020–2021 in North Macedonia .................. 47
Figure 12: Number of active companies per size in North Macedonia, 2019–2020 ..... 48
Figure 13: Number of employees per company size in North Macedonia, 2019–2021 .... 49
Figure 14: Turnover per company size in North Macedonia, 2019–2021, in million EUR .. 50
Figure 15: Number of employees working at GALINA, per semester, 2019–2021 ........ 57
Figure 16: GALINA’s cumulative turnover, per semester, 2019–2021, in EUR ............ 57
Figure 17: Enterprise structure with regard to size in 2019 and 2020 in Slovenia ........ 61
Figure 18: Enterprise size and employment in 2019 and 2020 in Slovenia ............... 62
Figure 19: Enterprise size and total income (thousand EUR) in 2019 and 2020 in Slovenia ................................................................. 63
Figure 20: Total employment and income by enterprise size in 2020 in Slovenia ........ 64
Figure 21: Number of enterprises that made a profit and reported a loss in 2019 and 2020 ................................................................. 65
"The study provides an overview and a critical review of the measures taken by the governments of the observed countries with the aim of sustaining small and medium-sized enterprises during the pandemic and points to examples of good practice in entrepreneurial responses to recognised pandemic opportunities transformed into innovative products, services, processes or business models. On the basis of the conducted research, recommendations based on good practice in supporting the small and medium-sized enterprise sector were made.
Presenting short case studios further illustrated the specifics and trends in small and medium-sized entrepreneurship in the time of crisis. The identified lessons learned are a valuable contribution to future debates on the responsiveness of governments in times of external shocks in which small and medium-sized enterprises are particularly vulnerable."
Ružica Šimić Banović, PhD
Associate Professor at Faculty of Law, University Zagreb, Croatia
"The COVID-19 virus pandemic caused one of the biggest crises in economic history, and it certainly represents a valuable research area, which is greatly helped by this study, which provides a clear and detailed overview of the state of the small and medium-sized enterprise sector, as well as the implemented measures aimed at mitigating the negative consequences – unemployment growth, insolvency, losses, bankruptcies and liquidations. The special value of the study is reflected in the comparative analysis of countries from the environment with the world's leading economy. The study is certainly a valuable document for all participants in the process of adopting policies and regulations, as well as for lobbyists focused on policies and laws that would strengthen the small and medium-sized enterprise sector."
Anamarija Delić, PhD
Associate Professor at Faculty of Economics in Osijek
J.J. Strossmayer University of Osijek, Croatia
“The International Cooperation Center (ICC) of the National Development and Reform Commission (NDRC) is a directly affiliated institution of NDRC and a major institution of international cooperation.
As a decision-making advisory body serving the NDRC to study major strategies, coordinate major policies, promote large projects, analyze major trends, and cooperate with the NDRC to implement the “Belt and Road” construction and international capacity cooperation, the main implementation of a series of international cooperation mechanisms the organization has provided macro-decision support around the work of the Party Central Committee, the State Council, and the NDRC. It has played an important supporting role in providing policy advisory services to domestic and foreign enterprises and governments at all levels, and has formed comprehensive advantages such as policy research, international cooperation, planning consulting, forum training, and public diplomacy.
Facing the future, under the guidance of Xi Jinping Thought on Socialism with Chinese Characteristics for a New Era, under the correct leadership of the Party committee of the NDRC, the ICC closely focuses on the overall work of the Party and the country’s opening up and the work of the NDRC, and always insists on down-to-earth, enterprising and innovative work principles. It is committed to building thinkers, explorers, and practitioners who work together to build the “Belt and Road”, and promote the high-quality development of local economies and enterprises. We have always been committed to being a thinker, practitioner and promoter of making a more open China positively engaged with the world.”
The Centre for Polish and Central and Eastern European Studies, School of International Studies, Sichuan University (China)
“The Center for Polish, Central and Eastern European Studies of School of International Relations, Sichuan University, was established in 2016. In 2017, the center was registered as a base of region and country-specific studies of the Department of International Cooperation and Exchanges of the Ministry of Education. It is an important unit for research and policy consultation on Poland, Central and Eastern Europe. Since its establishment, the Center has concentrated its resources and actively carried out academic research, decision-making consultation and international exchanges.
The center aims to serve the country’s “Belt and Road” construction, and serve national diplomacy, economy and humanities cooperation as its own responsibility. Based in Sichuan, facing the world, oriented to major national and local needs, we strive to become an important base for research and talent training in Poland and Central and Eastern Europe and a high-end platform for exchanges between China and Central and Eastern Europe. Since its establishment, the center has pooled its superior resources and actively carried out academic research, decision-making consultation and external exchanges. The center will strengthen contacts with research institutions and think tanks of world-class universities, and jointly build an international cooperation network.
In recent years, our school has established a close cooperative relationship on teaching and scientific research with the University of Warsaw, Jagiellonian University, Lodz University in Poland; Charles University in Czech Republic; University of Pecs and Corvinus University of Budapest in Hungary; University of Ljubljana in Slovenia; University of Latvia in Latvia and other renowned universities in Central and Eastern Europe.
The Center holds the “Sichuan University-University of Warsaw International Relations Research Roundtable” twice a year to promote mutual recognition and academic cooperation between China and Central and Eastern European countries. So far, five sessions have been held.”
CEPOR – SMEs and Entrepreneurship Policy Center is an independent policy center which deals with issues of small and medium enterprises in Croatia. CEPOR was established in 2001, based on an agreement between the Government of the Republic of Croatia and Open Society Institute Croatia. Institutional founders of CEPOR are: Institute of Economics, Zagreb, Josip Juraj Strossmayer University of Osijek – Faculty of Economics in Osijek, Institute for International Relations Zagreb, Croatian Chamber of Economy, Croatian Chamber of Crafts and Trades, REDEA Development Agency Medimurje – Čakovec, IDA Istrian Development Agency – Pula, Center for Entrepreneurship Osijek, Open Society Institute Croatia and UHIPP, Croatian Institutions for Promotion of Entrepreneurship Association. CEPOR’s mission is to influence the public-political environment emphasizing the role of entrepreneurship and small and medium enterprises in the development of Croatian economy. CEPOR wants to contribute to the shaping of entrepreneurial culture and to creation of stimulating institutional and regulatory framework for entrepreneurial activity.
ESTIMA is a civic association established in 2016, specialized in policy research, analysis and advocacy, with a geographic focus on the Western Balkans, the EU and China. Founded by knowledge enthusiasts in North Macedonia, it strives to challenge the existing regional standards in the area of knowledge creation and exchange and to define new, higher ones. Its mission is to establish and develop the first globally recognized knowledge-brand in the country and the broader region.
Its analysts have a background in economic and development issues, focusing on private sector, particularly SMEs and women entrepreneurship. Their recent publications include the first Women Entrepreneurship Barometer in North Macedonia (November 2021), a set of 3 policy briefs related to the EU’s Single Market (Digital Single Market, Access to Finance for SMEs and Protection of Intellectual Property Rights, November 2021), The Sofia Summit – Takeaways and Lessons Learnt (assessment of the Western Balkans Common Regional Market, November 2020), The impact of COVID-19 on the construction industry in North Macedonia (June 2020), Macedonian companies in the Belt and Road Initiative (April 2020), etc.
|
Implications of anomalous relative sea-level rise for the peopling of Remote Oceania
Juliet P. Sefton\textsuperscript{1,2*}, Andrew C. Kemp\textsuperscript{1}, Simon E. Engelhart\textsuperscript{3}, Joanna C. Ellison\textsuperscript{4}, Makan A. Karegar\textsuperscript{5}, Blair Charley\textsuperscript{6}, and Mark D. McCoy\textsuperscript{7}
\textsuperscript{1} Department of Earth and Ocean Sciences, Tufts University, Medford, MA 02155, USA
\textsuperscript{2} School of Earth, Atmosphere, and Environment, Monash University, Clayton VIC 3800, Australia
\textsuperscript{3} Department of Geography, Durham University, South Road, Durham DH1 3LE, UK
\textsuperscript{4} School of Geography, Planning, and Spatial Sciences, University of Tasmania, Launceston 7520, Australia
\textsuperscript{5} Institute of Geodesy and Geoinformation, University of Bonn, Bonn, Germany
\textsuperscript{6} Kosrae Island Resource Management Authority, Kosrae State Government, Tofol, Kosrae, Federated States of Micronesia
\textsuperscript{7} Department of Anthropology, Southern Methodist University, Dallas, TX 76275, USA
*Juliet P. Sefton
Email: firstname.lastname@example.org
Competing Interest Statement: The authors declare no competing interest as defined by PNAS policy.
Classification: Physical Sciences/Earth, Atmospheric, and Planetary Sciences; Social Sciences/Anthropology
Keywords: sea level, mangrove, archaeology, Micronesia, Oceania
This PDF file includes:
Main Text
Figures 1 to 3
Beginning ~3500–3300 yrs BP humans voyaged into Remote Oceania. Radiocarbon-dated archaeological evidence coupled with cultural, linguistic, and genetic traits indicates two primary migration routes: a Southern Hemisphere and a Northern Hemisphere route. These routes are separated by low-lying, equatorial atolls that were settled during secondary migrations ~1000 years later after their exposure by relative sea-level fall from a mid-Holocene highstand. High volcanic islands in the Federated States of Micronesia (Pohnpei and Kosrae) also lie between the migration routes and settlement is thought to have occurred during the secondary migrations despite having been above sea level during the initial settlement of Remote Oceania. We reconstruct relative sea level at multiple sites on Pohnpei and Kosrae using radiocarbon-dated mangrove sediment and show that, rather than falling, there was a ~4.3-m rise over the past ~5700 yrs. This rise, likely driven by subsidence, implies that evidence for early settlement could lie undiscovered below present sea level. The potential for earlier settlement invites reinterpretation of migration pathways into Remote Oceania and monument building. The UNESCO World Heritage sites of Nan Madol (Pohnpei) and Leluh (Kosrae) were constructed when relative sea level was ~0.94 m (~770–750 yrs BP) and ~0.77 m (~640–560 yrs BP) lower than present, respectively. Therefore, it is unlikely that they were originally constructed as islets separated by canals filled with ocean water, which is their prevailing interpretation. Due to subsidence, we propose that these islands and monuments are more vulnerable to future relative sea-level rise than previously identified.
Settlement of Remote Oceania began ~3500–3300 years ago and coincided with falling sea level across the equatorial Pacific Ocean. Archaeological evidence suggests that people arrived on Pohnpei and Kosrae (high islands in Eastern Micronesia) ~1000 years later than on other high islands. We reconstruct sea level on Pohnpei and Kosrae using mangrove sediment and find that rather than falling, sea level rose by ~4.3 meters over the past ~5700 years because of subsidence. This rise likely submerged coastal evidence for the initial settlement and current estimates of when people arrived are therefore biased young. Our results allow reconsideration of the pathways and interactions between voyaging groups across Remote Oceania, and the interpretation of the Nan Madol and Leluh monuments.
INTRODUCTION
Settlement of remote Pacific islands began ~3500–3300 years before present (yrs BP) and marked one of the final major phases of pre-modern human migration into uninhabited regions. This migration required formidable long-distance ocean voyaging and the geographic pattern and timing of settlement has long fascinated Western scholars (1). Models for the migration of people into ‘Remote Oceania’ (2), and the relationship between these voyagers and modern people, are built upon absolute dating of archaeological remains (3, 4), artifact analyses (5), historical linguistics (6), oral histories (7), human genetics (8, 9), computer simulations of voyaging (10, 11), and the history of commensal plants and animals that accompanied humans (12, 13). These data indicate that settlement progressed in two simultaneous, but largely independent, expansions (Figure 1). The Southern Hemisphere route saw migration from Taiwan via New Guinea into island groups south of the equator. This route is marked by a shared Lapita-styled pottery and was foundational to settling of parts of Melanesia, Polynesia, and Eastern Micronesia. The Northern Hemisphere route saw migration out of the Philippines, or other nearby island groups, and was foundational to settlement of islands in Western Micronesia (including Palau and the Mariana Islands).
Separation of the two migrations routes is inferred from the apparent delayed settlement of low-lying, equatorial atolls and high volcanic islands (e.g., Pohnpei and Kosrae) in the region between them. People appear to have arrived in this region at least ~1000 years after settlement of high islands to the west (~3300 yrs BP) and south (~3500 yrs BP) as part of a secondary migration (likely from the south; 14). This interpretation assumes that relative sea level (RSL) fell from a mid-Holocene highstand caused by the spatially-variable response of Earth’s crust to the transfer of mass from high-latitude continents to the global ocean through ice melt (15, 16). The occurrence of the mid-Holocene highstand and subsequent RSL fall in the equatorial Pacific
Ocean is a robust feature of Earth-ice model predictions (16) and is supported by proxy evidence such as raised coral reefs and beach sediments (17, 18). RSL fall increases the likelihood that archaeological evidence of initial coastal settlement (19) is preserved and accessible on land. Delayed human settlement of the low-lying equatorial atolls is consistent with their exposure by RSL fall (20–21). The rapid discovery and settlement of low-lying atolls after exposure is sometimes offered as an example of a natural ‘autocatalysis’ (23, 24:13) for exploration of the unoccupied regions: in this case the area between the migration routes. However, the late settlement of the high volcanic islands of Pohnpei and Kosrae is puzzling. Their elevation means they were not just habitable but likely desirable for settlement long before they were inhabited. Thus, while people on the Southern and Northern Hemisphere migration routes possessed the long-distance voyaging capacity to reach the islands (25, 26), the absence of evidence for earlier settlement has been taken as evidence for their settlement as part of the secondary migrations onto equatorial atolls.
We reconstruct RSL on Pohnpei and Kosrae using radiocarbon-dated mangrove sediments. In contrast to other islands in Remote Oceania, RSL at Pohnpei and Kosrae did not fall but rather rose steadily over the past ~5000 years. This sustained rise submerged coastal areas that may hold archaeological evidence for the initial settlement of the islands. The resulting bias in the visibility of the archaeological record may explain the apparent delay in settling these high islands and suggests reconsideration of the degree of separation between the Southern and Northern Hemisphere migration routes. The reconstructed RSL rise also has implications for the interpretation of coastal monumental architecture. We propose that the UNESCO World Heritage Site of Nan Madol (on Pohnpei; Figure 2) and Leluh (on Kosrae) were originally built on land, rather than on islets separated by hallmark canals filled with ocean water.
RESULTS
Mangroves inhabit low-energy coastlines in the (sub)tropics and are widespread on Pohnpei and Kosrae (Figure 1B–C). The elevation range of mangrove environments is intrinsically linked to the tides, which allows RSL to be reconstructed by dating mangrove sediment preserved in the stratigraphic record (27–29). We measured the modern elevation range of mangroves on Pohnpei and Kosrae using field surveys along transects and combined our results with existing survey data (30) to create a regional-scale dataset. Tidal datums were calculated from water-level measurements made by tide gauges (see Materials and Methods). Survey data were linked to the local tidal datums by correlating water-level measurements from loggers deployed during fieldwork to tide-gauge measurements. The elevation of mangroves is $0.12 \pm 0.62$ m MTL (mean tide level; 95% confidence interval) on Pohnpei ($n = 233$) and $-0.04 \pm 0.63$ m MTL on Kosrae ($n = 31$; Figure S2).
RSL rise creates accommodation space that is filled in by *in-situ* accretion of mangrove sediment, which allows the mangrove sediment surface to maintain its elevation in the tidal frame. This process can result in thick sequences of mangrove sediment (31) that record the position of RSL over time. Reports from the literature (32–34) and our own field observations show that Holocene sequences of mangrove sediment up to 6-m thick are preserved on Pohnpei and Kosrae. In the absence of RSL rise, the thickness of mangrove sediment is limited to approximately one half of the tidal range (35, 36). Given (great diurnal) tidal ranges of 0.88 m on Pohnpei and 1.17 m on Kosrae (see Materials and Methods), this stratigraphic observation suggests that sustained and substantial late Holocene RSL rise occurred (37, 38).
We compiled new and existing radiocarbon ages from mangrove sediments on Pohnpei and Kosrae (see Materials and Methods) following recommendations made by the HOLSEA working group (Dataset S3; 39). We used the modern distribution of mangroves on Pohnpei and Kosrae as analogs for interpreting mangrove sediment preserved in the stratigraphic record, which along with the radiocarbon age measurements enabled us to produce ‘sea-level index points’ that constrain the unique position of RSL in time and space with vertical and chronological
uncertainty. In total, we generated 68 sea-level index points from nine sites on Pohnpei and six sites on Kosrae (Figure 1B–C).
Given the similarity of sea-level index points from multiple sites on Pohnpei and Kosrae (Figure 3A), we combined results from all sites and both islands into a single dataset. This decision is supported by Earth-ice models which predict almost indistinguishable Holocene RSL histories for these islands (Figure 2A; 16). To provide a quantitative RSL history with a probabilistic assessment of uncertainties, we used a statistical model (40) that fits 2000, equally-likely RSL histories to the sea-level index points and decadally-averaged tide-gauge observations from Pohnpei for 1970 to 2019 CE (Figure 3A). Reported values are the mean ($\pm 1\sigma$) of the 2000 individual members in the model ensemble. RSL rose by $4.3 \pm 0.4$ m from $\sim5700$ yrs BP to present (Figure 3A), at a mean rate of $\sim0.7$ mm/yr (Figure 3B). The consistency of RSL reconstructions across multiple sites and both islands indicates that sustained RSL rise is not the result of local-scale processes (Figure S3). Comparison of basal (unlikely to be compacted) and non-basal (susceptible to compaction) sea-level index points (Figure S3A) demonstrates that the rise cannot be attributed to post-depositional lowering of the samples used to reconstruct RSL (41).
We also compiled radiocarbon ages on samples that were interpreted (at the time of their publication) to represent human activity on islands throughout Remote Oceania (Datasets S4–5). The median calibrated age for the oldest archaeological samples on Pohnpei and Kosrae is $\sim2500$ yrs BP, and our analysis indicates that RSL rose by $2.5 \pm 0.4$ m since this time (Figure 3A). RSL rose by $0.94 \pm 0.3$ m and $0.77 \pm 0.3$ m since the construction of Nan Madol (770–750 yrs BP; 42) and Leluh (640–560 yrs BP; 43), respectively.
**DISCUSSION**
**Absence of a mid-Holocene highstand**
All reasonable combinations of Earth models and ice-melt histories predict a mid-Holocene RSL highstand in the equatorial Pacific Ocean (15, 16), although its timing (approximately 6000–2500 yrs BP) and magnitude (approximately 0.6–2.5 m above present) varies among models. Geological proxies (e.g., elevated coral reefs) across the region record the occurrence of the mid-Holocene highstand (20, 44). In contrast, we reconstruct sustained late Holocene RSL rise on Pohnpei and Kosrae (Figure 3A), which we attribute to ongoing subsidence. Five additional lines of evidence support our interpretation that Holocene RSL did not exceed present on Pohnpei and Kosrae. First, several studies (37, 38, 45) and our own field observations failed to find convincing geomorphic or sedimentary evidence of a RSL highstand despite explicitly searching for it. There is no proposed evidence for a highstand on Pohnpei in the literature. On Kosrae, the proposed evidence is radiocarbon-dated allochthonous coral fragments in beach rock (46, 47). Beach rock can form quickly through transport of allochthonous material during storms and rapid cementation (48). For example, (38) identified cemented beach rock above contemporary sea level in Micronesia during fieldwork in the 1960s and noted that it contained material from World War II alongside older allochthonous coral, which indicates that dating the coral would return unreliable ages of formation. In a wider context, the only proposed evidence for a highstand at Chuuk (a high island archipelago ~700 km west of Pohnpei) is an undated notch cut into resistant basalt (49), that may not be above the influence of present storm surges and sea spray (38). Furthermore, there is no evidence of a Last Interglacial (~125 kyrs BP) shoreline preserved above present sea level, in contrast to other islands in Remote Oceania (50, 51).
Second, a GPS CORS (Continuously Operating Reference Station) on Pohnpei measured subsidence of $1.0 \pm 0.2$ mm/yr since 2003 CE ($1\sigma$ error; Figure 4). There is no GPS CORS on Kosrae. Although a short time series, this rate is approximately the difference between RSL predicted by Earth-ice models (-0.4 mm/yr, i.e., RSL fall) and reconstructed from mangrove sediments (~0.7 mm/yr; Figure 3B). This subsidence trend is anomalous in the wider context of equatorial Pacific islands that are not on active tectonic margins (52), which suggests that the mechanism of subsidence is unique to the high islands.
Third, analysis of RSL measurements made by the Pohnpei tide-gauge reveals subsidence. Decomposition of the observed trend following (53) estimates the temporally-linear contribution to be 0.59 mm/yr (0.1–1.0 mm/yr; 66% credible interval; 54; Figure S6A). This contribution includes Earth-ice processes and subsidence and is therefore comparable in composition and magnitude to our long-term estimate of RSL rise at ~0.7 mm/yr derived from mangrove sediment. It is notably fast in the context of the equatorial Pacific Ocean outside of islands experiencing active tectonism and/or volcanism (Figure S6A). For example, at Kapingamarangi atoll (~750 km away) where Earth-ice models predict that RSL is almost indistinguishable from that at Pohnpei and Kosrae, there is modest net emergence of -0.1 mm/yr (-0.7–0.5 mm/yr; 66% credible interval). This pattern suggests that subsidence is likely restricted to high islands rather than being regional in scale. The difference between the rate of sea-level change measured at one location by satellite altimetry and a tide gauge is proportional to the rate of vertical land motion (55). The difference (1.7 mm/yr; 56; Figure S6B) at Pohnpei indicates subsidence and is anomalously large for an island in the equatorial Pacific not undergoing active tectonic forcing, although quantifying an absolute rate of subsidence is challenging using this approach (55). For comparison, the difference at Kapingamarangi is 0.2 mm/yr indicating relative stability.
Fourth, the distribution of U/Th ages on coral used to construct mortuary buildings at Leluh is skewed towards younger ages (43). This distribution may support RSL rise because the most accessible construction material is likely young coral in shallow water, while older material is less accessible since it is in deeper water. RSL fall from a highstand would have left relatively old coral on land as accessible construction material resulting in older ages for fill. This interpretation assumes that the people building Leluh consciously chose the most conveniently located material to use as fill. However, the columnar basalts used to construct the outward-facing structures at Nan Madol are evidence of the willingness and ability to move large volumes of heavy material considerable distances (57). The presence of coral fragments from a range of species representative of a complete coral colony, their unweathered condition, and oral histories indicate that coral living in shallow water (rather than dead coral that was, for example, left exposed by
RSL fall) was harvested for construction material at Leluh (43). We propose that the age, type, and condition of construction materials used at Leluh are compatible with RSL rise on Kosrae.
Fifth, much of the earliest archaeological evidence on Pohnpei and Kosrae was excavated from intertidal or fully submerged sites (58–60). These diverse and independent lines of evidence support our result that late Holocene RSL rose on Pohnpei and Kosrae due to island-scale subsidence out-pacing the regional-scale RSL fall from a mid-Holocene highstand. As part of the Caroline Seamount Chain, Pohnpei and Kosrae are islands formed by hotspot volcanism (61, 62) that ceased when plate motion moved the islands away from the upwelling mantle reservoir. Subsidence of hotspot volcanic islands is well documented globally, although at many islands — including Pohnpei and Kosrae — observed subsidence rates are greater than would be expected given hotspot swell bathymetry, overriding plate motion, and the age of the lavas (which for Pohnpei and Kosrae are ~5.2 and 1.4 million years, respectively; 61, 62, 63). High subsidence rates specific to Pohnpei and Kosrae may point to localized subsidence processes resulting from, for example, coral loading, or the viscoelastic relaxation of the lithosphere in response to volcanic loading (63, 64), although an exact mechanism has not yet been determined for these islands.
Archaeological evidence for human arrival on Pohnpei and Kosrae
On Pohnpei, the oldest age (median of calibrated range) associated with pottery, a definitive marker of human settlement across Remote Oceania, is 1863 yrs BP (65). The oldest age for ceramic-bearing cultural layers on Kosrae is 1895 yrs BP (58; Beta-30787, Dataset S4). Recent assessment of these earliest ages indicates that Pohnpei and Kosrae were continuously settled since 2000–1800 yrs BP (66). However, on Pohnpei, a charcoal fragment in a sediment core (interpreted to have an anthropogenic origin since the wet climate makes wildfire rare; 58, 67) yielded an age of 2540 yrs BP (Gak-7647; Dataset S4), but the sample was not recovered from an artifact-bearing horizon. On Kosrae, charcoal fragments and changes in pollen assemblages in sediment cores (with an age of 2430 yrs BP; Beta-31733, Dataset S4) are interpreted as
evidence of human modification of natural forest including cultivation of breadfruit and use of fire (34). These ages point to settlement of Pohnpei and Kosrae at least 1000 years (and possibly more than 1500 years) after high islands to the west and south (Figure 1A). There are ample radiocarbon ages from archaeological sites on Pohnpei \((n = 16)\) to suggest that these apparent young ages are unlikely caused by insufficient sampling. While there are fewer ages from archaeological sites on Kosrae \((n = 5)\), based on this suite of ages, Pohnpei and Kosrae appear to have been settled at the same time as the atolls between the Southern and Northern Hemisphere migration routes in a secondary pulse of migration within Remote Ocean at ~2000 yrs BP (Figure 1A).
This proposed timing and geographic pattern of settlement of Pohnpei and Kosrae is puzzling. If these high islands were settled at the same time as the equatorial atolls, then atoll emergence was indeed an autocatalysis for the discovery of Pohnpei and Kosrae (23, 24:13) and earlier voyagers did not find (or at least chose not to settle) these islands. However, we argue that it is unlikely that two high volcanic islands (separated by ~500 km) were missed by people on either the Southern or Northern Hemisphere routes during the initial phase of settlement beginning ~3500–3300 yrs BP only to be settled near-simultaneously. People migrating along both routes possessed the long-distance voyaging technologies and skills required to locate Pohnpei and Kosrae (25, 26), although we note that these technologies and strategies changed during the period of migration. Although proximity is not always a good predictor of settlement of small islands in the vastness of the Pacific Ocean, the central location of Pohnpei and Kosrae places them on shortest-voyage trajectories between several island groups and prevailing winds or ocean currents would not make them less likely to be discovered (10, 11), depending on where voyages began from. Equatorial atolls were settled during a later, secondary migration not only because atoll existence relied on RSL fall from a mid-Holocene highstand (68), but because they are less desirable for settlement than high islands. Atolls have poor access to freshwater, poor-quality coralline soils for agriculture, limited building material (in amount and variety), and
are vulnerable to storm surges compared to high islands (69). Therefore, the high islands of Pohnpei and Kosrae were desirable targets for settlement.
Implications for human migration into Remote Oceania
On Pohnpei and Kosrae, archaeological deposits bearing ceramics were recovered below present sea level (66), and there are two possibilities to explain the submerged archaeological material. Under an assumption that a mid-Holocene RSL highstand (and subsequent RSL fall) occurred, some researchers proposed that the high islands were settled by people living in houses built on stilts over the shallow coral reef (66, 70). However, there is no direct evidence of stilted houses on Pohnpei or Kosrae. Our results support an alternative explanation, where people lived on the coast as they did elsewhere in Remote Oceania (19, 71), particularly given the steep terrain that characterizes the interior of both islands. Therefore, submerged archaeological evidence is the result of RSL rise (Figure 3A). We argue that evidence of earlier settlements was submerged by this anomalous RSL rise, and that the current estimates of initial settlement are systematically skewed young because of the difficulty accessing submerged and buried evidence, coupled with land above sea level being the target of previous archaeological investigations because of the assumption of RSL fall.
There is another example of local RSL trends obscuring evidence for human settlement in Remote Oceania. In Sāmoa, the earliest evidence for settlement (at ~2750 yrs BP) is a pottery-bearing horizon discovered nearly 2 m below present sea level during construction of a dock (72). Sāmoa’s location on an active tectonic boundary makes it prone to vertical land motion, including episodic subsidence during earthquakes (73). Although the mechanism for subsidence is likely different to Pohnpei and Kosrae (which are located far from active plate boundaries and show no evidence for episodic vertical motion), the impact on the archaeological record (submergence of evidence) is similar. Sāmoa also has multiple, independent lines of evidence for anomalous, subsidence-driven RSL rise including direct measurement by a GPS
CORS (52, 74) and the presence of thick sequences of mangrove sediment (75). IPCC AR6 sea-level projections for Sāmoa include 1.3 mm/yr of subsidence estimated from tide-gauge measurements (54).
Evidence of earlier settlement on Pohnpei and Kosrae may yet be uncovered in shallow marine environments (59). The age and nature of such evidence could provide new information on the interactions between Lapita voyagers on the Southern Hemisphere route and their Northern Hemisphere counterparts who settled the Palau and Mariana archipelagos. Ancient DNA of commensal animals, notably pigs (76), dogs (77), and chickens (78), suggest these began as largely independent, parallel migrations into Remote Oceania. At present, the only artifact indicating a direct link between the islands of the Federated States of Micronesia and those settled during the Southern Hemisphere migration is a stone adze uncovered through reef dredging on Pohnpei (59, 79).
Studies of human genetics offer insight into the long-term history of human migration across Remote Oceania (9, 80). Newly reported DNA from people living on Pohnpei and the Chuuk archipelago coupled with ancient DNA from eight burials at Nan Madol defined a ‘Central Micronesian’ population (80). Comparison of this Central Micronesia population to others in Remote Oceania suggests a genetic origin rooted in migration of people from the south, although a genetic contribution from the west cannot be excluded (80). The DNA of individuals buried at the Nan Madol mortuary complex at 500–300 yrs BP may, or may not, be representative of a population that arrived earlier on Pohnpei, but whose remains were submerged along with other archaeological evidence by sustained RSL rise. Thus, it is possible that the high islands in Central Micronesia were part of the earlier initial migration into Remote Oceania from the west, prior to major migration from the south.
More directly dated material evidence (including human remains) is required to parse between competing models of interaction between the Northern Hemisphere and Southern Hemisphere migrations. This evidence may lie submerged around Pohnpei and Kosrae.
Implications for monumental architecture
Nan Madol (sometimes described as the ‘Venice of the Pacific’) is an administrative and mortuary architectural complex on Pohnpei (Figure 2). It is characterized by artificial islets constructed from columnar basalt, boulders, and corals, and surrounded by narrow ‘canals’ currently filled with sea water and shallow coral reef (81). Nan Madol’s massive sea break walls are interpreted as being constructed to shield the site from storm surges (66). Columnar basalt was sourced from locations across Pohnpei and U-Th ages on coral fill places construction of the tomb of the island’s first rulers at ~770–750 yrs BP (42). The prevailing interpretation is that Nan Madol was intentionally constructed in a shallow marine environment to allow the political elite to isolate themselves from mainstream society on Pohnpei (66), and that this isolation was greater at the time of construction than observed today because of subsequent RSL fall. Modern flooding of the lowest-lying buildings at Nan Madol during high tides was thought to indicate that the islets subsided since construction (82). Leluh is a monument on Kosrae that shares many architectural characteristics with Nan Madol. U-Th ages on coral from three mortuary buildings show construction began at ~640–560 yrs BP (43).
Based on our RSL reconstruction, we propose that Nan Madol and Leluh were built on land above the reach of the high tides (and likely most storm surges). We estimate that RSL rose by 0.94 ± 0.3 m and 0.77 ± 0.3 m since the construction of Nan Madol and Leluh, respectively (Figure 3A). We surveyed the bottom of the canal adjacent to one of the main mortuary buildings at Nan Madol (islet H113; 81) to be approximately -0.2 m MTL (-0.68 m relative to mean higher high water, MHHW). Assuming a stationary tidal regime and no sedimentation of canals, this location was at ~0.26 m above MHHW when Nan Madol was constructed. Over the 1983–2001 tidal epoch the 1% inundation level for the Pohnpei tide gauge is 0.23 m above MHHW and the single highest water level was 0.63 m above MHHW. Therefore, the canals at Nan Madol were likely dry when constructed, except during rare and transitory events when water depths of just a few centimeters could have occurred. Due to ongoing RSL rise, low points surrounding buildings
at Nan Madol would have become increasingly flooded, leading to the formation of individual buildings as ‘islets’ and the famous ‘canals’ separating them. However, this was a slow process on human timescales. When the rulers of Nan Madol were overthrown at ~350 yrs BP (83), and the site was no longer the primary seat of political power over the island, it is unlikely that the islet we examined would have been surrounded by a permanent canal as it exists today (although sections of it were likely flooded at high tide).
Subsidence-driven RSL rise through the late Holocene indicates that these sites (and modern socio-economic activity on Pohnpei and Kosrae) may be more vulnerable to future rise than previously anticipated (39, 36, 84), since current projections underestimate the contribution from subsidence (54). Thus, our results have implications for both the interpretation and ongoing care of these World Heritage Sites, and more broadly for the island nation of the Federated States of Micronesia.
CONCLUSIONS
Models for human migration into Remote Oceania beginning ~3500–3300 yrs BP assume that the equatorial Pacific Ocean experienced RSL fall from a mid-Holocene highstand. Separation and isolation of Southern and Northern Hemisphere migration routes is inferred from the delayed (by at least ~1000 years) settlement of equatorial atolls and high volcanic islands (including Pohnpei and Kosrae) in the region between them. While emergence of atolls in response to RSL fall made them more habitable through time, the apparent late settlement of high islands is puzzling because they represent desirable, available, and reachable targets for settlement. It is possible that Pohnpei and Kosrae, like more distant targets such as the Hawaiian Islands, were beyond the seafaring strategies and technologies of people during the initial stages of settlement in Remote Oceania. However, given the similar settlement timing for Pohnpei and Kosrae, we suggest that this shared pattern is due to a shared RSL history. Using mangrove sediment, we find that Pohnpei and Kosrae experienced sustained RSL rise of ~4.3 m during the past ~5700
years at a rate of $\sim0.7$ mm/yr because of island-scale subsidence. We suggest that estimates of when these high islands were settled are systematically biased young because RSL rise submerged evidence for the initial occupation of low-lying coastal sites. This finding invites reexamination of the degree of separation between the Southern and Northern Hemisphere migration routes. The new RSL history also has implications for interpreting the long-term socio-political and architectural histories of monuments. In contrast to prevailing interpretations, the administrative centers of Nan Madol and Leluh were originally built on land only to become inundated over subsequent generations due to ongoing RSL rise. Our study highlights the importance of site-specific reconstructions of past environments and relative sea level, as local environmental changes can depart from those predicted by broad-scale models.
**MATERIALS AND METHODS**
**Tide analysis**
*Pohnpei*
We downloaded hourly water-level measurements (expressed relative to station datum) made by the Pohnpei-B tide gauge from the University of Hawaii Sea Level Center (85; last accessed May 12, 2022). From these measurements, we calculated tidal datums following the National Oceanic and Atmospheric Administration definitions and using the national tidal datum epoch (currently 1983–2001). We isolated high and low tides from using the VulnToolKit package (86) for R (Figure S1A, Dataset S1). In this step, each tide was identified as a higher high, lower high, higher low, or lower low and means for these groups provide the elevation of tidal datums (relative to station datum) for the location of the Pohnpei tide gauge (in Kolonia; Figure 1B).
To determine if tides vary around Pohnpei, we deployed pressure transducer water loggers at five sites across two field seasons (two sites in 2016 between July 4–8th, and three sites in 2019
between July 19–28th; Figure 1C). Each logger was deployed in open water immediately adjacent to the seaward edge of the mangrove at low tide to ensure that all subsequent low and high tides were measured by the loggers. Water-level measurements at six-minute intervals were corrected for atmospheric pressure changes measured by an additional logger that was simultaneously deployed close by. Comparison of water levels among sites and with those made by the Pohnpei-C tide gauge (hourly measurements) indicates that the timing and magnitude of tides does not vary between them, hence tidal datums established at the tide gauge (Dataset S1) are applicable elsewhere on Pohnpei. By correcting for the difference between water-level measured by the tide gauge (relative to station datum) and each water-level logger (depth), we established the elevation of each water logger relative to tidal datums.
The United States Geological Survey also deployed water-level loggers at two sites on Pohnpei from July 2016 to March 2017 (30). Water-level measurements made by these instruments are reported relative to the EGM2008 geoid. The uncertainty of the EGM2008 geoid is typically ±0.05–0.1 m (87). The two water loggers were deployed at elevations where they could only measure high tides, and alignment of the two time series shows no spatial difference in water level variability and that measurements showed near-identical variability to the Pohnpei tide gauge. This further confirms our conclusion that tides are invariable around Pohnpei, and that the relationship among tidal datums established at the tide gauge are applicable elsewhere. Through differencing these water-level measurements with those made simultaneously by the Pohnpei-C tide gauge, we estimate that EGM2008 lies at -0.92 m MTL (0.18 below station datum for the Pohnpei-B tide gauge).
Kosrae
There are no published and accessible tide-gauge data for Kosrae. However, the National Institute of Water & Atmospheric Research/Taihoro Nukurangi installed a tide gauge at Leluh which collected observations between 2011 and 2016 (88). We used these data from Leluh
(which is measured relative to the Kosrae Local Datum) to calculate tidal datums as for Pohnpei, except that 2011–2016 rather than 1983–2001 was used as the tidal epoch out of necessity (Figure S1B; Dataset S1). To determine any spatial variability in tides around Kosrae, we deployed water-level loggers at three mangrove sites around Kosrae (July 8–15th 2019). Water-level measurements were corrected for atmospheric pressure changes measured by an additional logger that was simultaneously deployed close by. We observed no changes in timing and magnitude of tides around Kosrae. Therefore, tidal datums established at the site of the Kosrae tide gauge can be applied at other sites on the island, with the caveat that the 2011-2016 window of measurements is shorter than the 19-year window typically used to calculate datums.
We established the elevation of each water logger relative to tidal datums by applying a correction to measured depths to remove the difference to simultaneous water-level measurements made by the tide gauge.
**Elevation of the mangrove modern analogue**
We used two sources of data to estimate the elevation occupied by mangroves in Micronesia. First, surveys by the United States Geological Survey (30, 36) investigated the elevations of mangroves at seven sites around Pohnpei. Mangrove surfaces were surveyed using several methods and elevations are reported relative to EGM2008 and converted to orthometric elevation. We applied the 0.92 m adjustment (established empirically from water-level logger and tide-gauge measurements; see Tidal Analysis) to each EGM2008 elevation observation to express it relative to MTL (from which correction to other tidal datums is achieved using the relationships determined over the tidal epoch, Dataset S1). Our analysis is limited to observations from 2019 that are noted to have mangrove plant species present at the measurement site (and therefore are measurements of sediment surfaces within a mangrove).
The USGS survey is inherently a conservative dataset to use for estimating the paleo-elevation at which mangrove sediment preserved in the stratigraphic record accumulated. In Micronesian
mangroves (and elsewhere), site geomorphology dictates that a large proportion of the change in elevation between the lower and upper limits occurs in a spatially-narrow band close to the landward edge of the mangrove. Consequently, the majority of mangrove surface area lies within a smaller subset of elevational range (see for example Figure 7 in 30 and Figure 5 in 36) and the probability that a sample of mangrove sediment recovered in a core accumulated within the narrow band at the seaward or most landward edge of the mangrove is low (in the absence of additional proxy evidence to refine the environment of deposition).
Second, we conducted new surveys of modern mangrove distribution at five sites on Pohnpei and two sites on Kosrae (Figure 1B–C). We used an autolevel and staff to measure surface elevation at points located along transect through each site. Transects began inland of the upper limit of mangroves and extended to the seaward edge of the mangrove where shallow, sub-tidal environments occur. The elevation of each point surface station was established relative to tidal datums by autolevel survey to water-loggers or by taking timed water-level measurements using the autolevel and staff, which were compared to simultaneous measurements made by the Pohnpei-C tide gauge.
We combined these two sources of data into a single dataset (n = 233 for Pohnpei and n = 31 for Kosrae). Since tidal range is different on Pohnpei and Kosrae, we expressed elevation as a standardized water level index (SWLI) value:
\[
\text{SWLI} = \left( \frac{\text{sample elevation (m MTL)}}{\text{MHHW elevation (m MTL)}} \right) \times 100 + 100
\]
Where a value of 100 SWLI is equivalent to local mean tide level (MTL) and a value of 200 SWLI corresponds to local mean higher high water (MHHW). Mangroves on Pohnpei and Kosrae occur at elevations of 7-249 SWLI (95% confidence interval). For Pohnpei this corresponds to elevations of \(0.12 \pm 0.62\) m MTL. For Kosrae, this corresponds to elevations of \(-0.04 \pm 0.63\) m MTL (Figure S2).
**New mangrove radiocarbon ages**
Mangrove sites were selected based on existing literature that describes the coastal stratigraphy around both Pohnpei and Kosrae (e.g., 32). We selected sites that showed the deepest mangrove sequences (to potentially obtain the longest, highest resolution records), as well as those that showed mangrove sediments overlying incompressible substrate (e.g., carbonate reefs) to assess any post-depositional compaction on the sediment sequence. One mangrove sediment core (Pwok) was collected using a Eijelkamp peat sampler, and core top elevations were surveyed using differential levelling from timed water level measurements. Three mangrove sediment cores were collected using a Hiller corer and elevation obtained using a Trimble differential GPS (30, 36). Bulk mangrove sediment samples were prepared for radiocarbon analyses by pre-treatment with acid to remove carbonate and were measured at the U.S. National Ocean Sciences Accelerator Mass Spectrometry Facility and Beta Analytic. Reported radiocarbon ages were calibrated individually using the Intcal20 calibration curve (89) using the BChron package for R (90). Data is available in Dataset S2.
**RSL database construction and analysis**
To reconstruct RSL, we collated and standardized published radiocarbon ages from coastal sediment samples on Pohnpei and Kosrae into a database following the protocols developed by the HOLSEA program (39), including our new radiocarbon ages. Inclusion in the database required each sample to have: 1) a geographic location; 2) an age (with uncertainty) of sample formation (in this case a radiocarbon age with a unique identifier); 3) an estimate (based on sample type) of tidal elevation at the time of formation with uncertainty (termed the reference water level); and 4) a modern altitude relative to sea level. Samples were excluded from the database if any of these attributes could not be estimated. Radiocarbon ages were individually calibrated using the IntCal20 dataset for terrestrial material (which includes mangrove sediment). The susceptibility of each sample to physical compaction was categorized based on its stratigraphic context (91); base of basal (sample overlies, within 10 cm, an incompressible layer,
compaction unlikely), basal (sample is from lower stratigraphic unit, compaction possible), intercalated (sample is from intercalated stratigraphic unit, compaction possible), surface peat (sample is from upper stratigraphic unit, compaction possible), and unknown (no suitable stratigraphic details were available to assign sample a compaction class).
For each sample in the database relative sea level (RSL) is reconstructed as:
\[ \text{RSL} = \text{Altitude} - \text{Reference Water Level} \]
Where altitude is measured directly as depth in core, and core top elevation is expressed relative to tidal datums. If the sample had a thickness, we used the mid-point in our calculation of RSL and treated the thickness as a source of uncertainty. Frequently, core-top elevation was not reported, but rather a core top was qualitatively described as being in a mangrove. In those instances, we used the elevation range of modern mangrove environments established from the modern surveys as the core top elevation (with uncertainty). The reference water level for samples determined to have formed in a mangrove is also the range of modern mangroves established from modern surveys (see Elevation of the mangrove modern analogue). The proxies used to confirm that a sample formed in a mangrove environment are principally descriptions of sediment texture, although a small subset of samples present other evidence (e.g., pollen assemblages). The age of each sample in calendar years is from calibration of the radiocarbon age. The result of this approach is the creation of individual sea-level index points which constrain the unique position of RSL in time and space, with vertical and chronological uncertainty. The creation of individual sea-level index points assumes a stationary tidal prism over time. The database and references are available in Dataset S3 and SI References, and the general locality of relative sea-level data is presented in Figure 1B–C.
A RSL history was generated by applying the errors-in-variables integrated Gaussian process (EIV-IGP) model (40) to the database of 68 sea-level index points (Dataset S3). The model was developed specifically to analyze late Holocene RSL reconstructions characterized by vertical and temporal uncertainties, and an uneven distribution of observations through time. Based on the coherence in RSL histories between Pohnpei and Kosrae, among sites on each island, and
stratigraphic context (susceptibility to compaction), we analyzed all sea-level index points as a single dataset. In addition, we calculated decadal average RSL from the Pohnpei tide gauge measurements to include in the EIV-IGP model. Hourly tide-gauge measurements were binned into 10-year increments (starting in 1970) and averaged. The temporal position for each 10-year average is the mid-point (e.g., 1975) with an age error of ±5 years. The vertical error is the 95% confidence interval of the measurements. The EIV-IGP model estimated RSL and the rate of RSL change with uncertainty (95% credible intervals) at timesteps of 100 years from ~5730 yrs BP to present.
Acknowledgements
This work was supported by National Science Foundation (NSF) awards OCE-1831382 and OCE-1831405 to Kemp and Engelhart, respectively. We thank Byron Halavik and Madeline Varney for their assistance in surveying and collecting the mangrove cores from Pohnpei and Kosrae. Access to field sites on Pohnpei was made possible by Saimon Lihpai (Department of Land and Natural Resources, Pohnpei State Government). We are especially grateful to Maxson Nithan (Kosrae Island Resource Management Authority) and Yosta Hirata (Department of Land and Natural Resources) for their expertise in navigating the mangrove sites and helping with sample collection. We thank Fred Taylor for his help on fieldwork, expertise on the geology of the Pacific Islands, mentoring and friendship. The Beta Analytic radiocarbon ages presented in Dataset S2 and associated fieldwork were enabled by the Micronesia Conservation Trust and the Conservation Society of Pohnpei. We thank two anonymous reviewers for their comments and suggestions that helped improve the manuscript. This paper is a contribution to PALSEA (Palaeo-Constraints on Sea-Level Rise) and the International Geoscience Programme (IGCP) Projects 639 “Sea-Level Change from Minutes to Millennia” and 725 “Forecasting Coastal Change: From Cores to Code”.
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**Figure Legends**
**Figure 1.** (A) Distribution of calibrated radiocarbon ages on archaeological samples across Remote Oceania (excluding eastern margins; see Dataset S4). Oldest ages and number of observations at an island scale (circles = atolls, triangles = high islands) are represented by color and size. The green squares highlight the high volcanic islands of the Federated States of Micronesia (FSM) (Pohnpei and Kosrae, west to east) of focus in this study. Arrows represent the approximate route taken by the Southern Hemisphere and Northern Hemisphere migrations into Remote Oceania. The black dotted line denotes the boundary between Near Oceania and Remote Oceania. (B, C) Location of modern mangrove surveys (from this study) and sites (numbered) with RSL data compiled in the database of sea-level index points for Pohnpei and Kosrae (Dataset S3).
**Figure 2.** (A) Satellite imagery of Nan Madol (6.84° N, 158.34° E) with polygons showing the position and shape of building foundations (81). Sometimes called the ‘Venice of the Pacific,’ prevailing interpretation has been that monumental architecture found at this former island capital were built as artificial islets connected by a series of canals. (B) Image of a ‘canal’ next to the tomb of the island’s first rulers a structure called Nandowas, identified as feature #113 in (93, 94). We present new evidence that demonstrates that 800-600 years ago when Nan Madol, and a similar site called Leluh on the neighboring island of Kosrae, were constructed, relative sea level was significantly lower. Both sites were originally built on dry land only to become submerged by rising relative sea levels. The impacts of anomalous sea level rise described in this study, and
encroaching mangrove, continue to threaten the integrity of architecture across the site, as seen at (C) Lelou (#120), (D) Lemenkau (#129), (E) Peitaup (#44), and (F) Peiniot (#118). Photo credit: © Osamu Kataoka (images reused with attribution).
**Figure 3.** (A) Relative sea level reconstructed for Pohnpei (purple) and Kosrae (orange) using radiocarbon-dated mangrove sediment. Each of the 68 sea-level index points is presented as a box that captures vertical and chronological uncertainty. Decadally-averaged tide gauge data (green) are also presented as boxes. Application of the errors-in-variables integrated Gaussian process (EIV-IGP) statistical model (40) was applied to this dataset to generate a relative sea-level history (shaded envelope represents the 95% credible interval; CI). The dashed line is relative sea level predicted by the ICE-7G_NA (VM7) Earth-ice model (16) for Pohnpei and Kosrae. For reference, the vertical dashed lines show the approximate timing of the initial settlement on high islands Pohnpei and Kosrae, the construction of Nan Madol (Pohnpei) and Leluh (Kosrae) (B) Rate of RSL change at Pohnpei and Kosrae as calculated by the EIV-IGP model (50th percentile and 95% credible interval).
**Figure 4.** Vertical land motion measured by a GPS Continuously Operating Reference Station (POHN) on Pohnpei. Data points are vertical positions and the red line is a least-squares (LSQ) model fit to the time series. The LSQ rate is -0.9 mm/yr and the MIDAS rate is -1.0 mm/yr (see Supplementary Text for LSQ and MIDAS rates definitions; Dataset S6).
(A) n observations
Island type
- Atoll
- High
Oldest possible age (cal BP, 95% CI)
- 0
- 1000
- 2000
- 3000
Taiwan
Philippines
Mariana Islands
Palau
Chuuk
FSM
Marshall Islands
Bismark Islands
Solomon Islands
Vanuatu
New Caledonia
Fiji
Tonga
Kiribati
Tokelau
Sāmoa
Equatorial atolls
1000 km
(B) Pohnpei
1 Nanitipw
2 Oegono
3 Sapwalap
4 Madolenihmw
5 Rohi
6 Pwok
7 Palikir
8 Lewetik
9 Ipwal
Pohnpei
782 m
km
(C) Kosrae
1 Pukusruk
2 Malem
3 Utwe
4 Walung
5 Yela
6 Okat
Kosrae
619 m
km
Mangrove
Tide gauge location (existing or past)
Modern mangrove survey site and/or sediment core site
Water loggers (this study)
POHN GPS site
(A) Aerial view of the site showing the location of the archaeological structures (B–F).
(B) View of the stone wall and steps.
(C) View of the stone wall from the sea.
(D) View of the stone wall and steps from the sea.
(E) Close-up view of the stone wall and steps.
(F) View of the mangrove forest near the stone wall.
(A) Relative sea level (m)
- Arrival at high islands
- Nan Madol construction
- Leluh construction
ICE-7G_NA(VM7) model
- Kosrae
- Pohnpei
EIV-IGP model
95% CI
mean
Tide Gauge
Pohnpei
Kosrae
(B) Rate of RSL change (mm/yr)
Mean rate
Subsidence
ICE-7G_NA(VM7) model rate
Calibrated Years Before Present
Site POHN: RMS 4.4 mm
Vertical displacement (cm)
Daily GPS vertical displacement
Least-squares model fit
Year Common Era
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OUR MISSION STATEMENT:
"Saint Francis Xavier Parish is a Christian Family dedicated to the mission of Jesus and the teachings of the Catholic Church. We are committed to fostering spiritual growth and to responding to the human needs of both our faith community and the larger community by sharing our gifts and resources."
WEEKEND LITURGY SCHEDULE:
Saturday Vigil - 4:30 PM
Sunday Masses - 8:30 & 10:45 AM
Sacrament of Penance - Saturday 3:30pm
TELEPHONES:
Rectory: 673-2531
1 West Main St., P.O. Box 177
Rel. Ed. Center: 673-4107
53 North Street
Liturgy Schedule
Weekend Masses:
Saturday Evening Vigil: 4:30 pm
Sunday Masses: 8:30 am & 10:45 am
Holy Day Masses: See schedule inside.
Daily Mass: 6:45 am Mon, Wed, Thur, Fri
Weekend Mass Schedule in our area
St. Michael-St. Peter
Sat. Evening: 5:00 pm
Sunday: 7:30, 9:00 & 11:15 am
St. Mary's of the Lake
Sat. Evening: 4:30 pm
Sunday: 8:00 & 10:15 am
St. Patrick's Jordan
Sat. Evening: 5:00 pm
Sunday: 8:00 & 10:00 am
PASTORAL STAFF:
Pastor: Rev. Daniel C. Muscalino*
Pastoral Associate: Deacon John J. Falge*
Religious Ed. Director: Renee O'Connor*
Parish Secretaries: Mary Jo Osterman
Katherine Boos
Social Service Coordinators:
Rosalie Durand
Patty White
Music Director: Robin Schott, Jr.
Buildings and Grounds: Ron Schneider
Housekeeper: Kathy Welsh
Parish Historian: John P. Curtin
PARISH TRUSTEES:
Diane Foster*
Joseph E. Durand*
* Also on Parish Council
SACRAMENTS:
Baptism:
Class required.
Call rectory for arrangements.
Reconciliation: Sat. 3:30 - 4:00
Marriage: Arrangements at least 6 months in advance.
NEW PARISHIONERS are most welcome.
See Fr. Muscalino after Mass or call the Rectory.
PARISH COUNCIL
Jay Austin
Terry Hall
Pen Anderson
Cheryl Hunt
Beth Cusick
Roxanne Taylor
Kim Flood
Marty McClusky
Terry Kotlarz
FINANCE COMMITTEE:
Rick Reagan
Chris Hunt
Diane Foster
Rev. Daniel C. Muscalino
Dc. John J. Falge
John P. Curtin
Joseph E. Durand
Mass Intentions FOR THE WEEK
Saturday August 1 – First Saturday
VIGIL EIGHTEENTH SUNDAY IN ORDINARY TIME
4:30 PM- Henry Smith- Family
Sunday August 2
EIGHTEENTH SUNDAY IN ORDINARY TIME
8:30 AM- Barbara Grzasko- Bob & Amy Mann
10:45 AM- People of St. Francis Xavier
Monday August 3
No Mass
Tuesday August 4
SAINT JOHN VIANNEY
No Mass
Wednesday August 5
No Mass
Thursday August 6
THE TRANSFIGURATION OF THE LORD
6: 45 AM- Jonathan E. Clark- Family
Friday August 7 – First Friday
SAINT SIXTUS II AND SAINT CAJETAN
6:45 AM- Peter Jula- Family
Saturday August 8
VIGIL NINETEENTH SUNDAY IN ORDINARY TIME
4:30 PM- Paul & Elizabeth Curtin- Maureen & John Curtin
Sunday August 9
NINETEENTH SUNDAY IN ORDINARY TIME
8:30 AM- People of St. Francis Xavier
10:45 AM- James & Mary Quinn
The Assumption of the Blessed Virgin Mary – Vigil Mass on Friday, August 14th at 6pm and Mass on Saturday, August 15th at 9:00am and 4:30pm. This is not a Holy day of Obligation this year. (See the sign-up app)
Sacrament of Confession - To receive the Sacrament of Confession, please call the rectory and make an appointment.
Church Picnic - We have decided to hold off on our annual church picnic this year and look forward to a wonderful picnic next year!
Elizabeth Ministry - The Elizabeth Ministry is here at St. Francis. This ministry is extremely important for it affirms that every member of the church has gifts and talents to build up the entire community.
Syracuse Diocese - For information regarding the Syracuse Diocese filing Chapter 11, please visit our website under announcements: sfxmarcellus.com
What’s Happening This Week at St. Francis
St. Francis is open for public Mass on August 1 & 2 for 50 people. In addition to weekend Mass, we will also have Thursday & Friday Mass. Parishioners need to sign up prior to the weekend and review safety instructions.
sfxmarcellus.com/events/event/mass-schedule
(please sign up several days ahead for printing)
Our Weekly Collection
Weekly Goal: $7,900 Last Week: $5,296
Please Pray For...
Isabelle Callahan, Dennis McEnery, Ed Dillon,
Riley Christine Perkins, Nick Olenych,
Roger Schott, Danielle Painter, Genie Barnes,
Nicole DeMore, Lisa Pikarski,
Michelle Fleegeel, Jo-Ann Foerth, Ellie Hauver,
Justin MacLachlan, Jack Bragger,
Florence M. Linda, Peggy Paradise, Missy Fitz,
Austin Marlowe, Brian Stonelake, Brett Kresco,
Lucienne Henneberry, Allan Stonecipher,
Donna Martin, Kelly Cunningham Suberts,
Jo’Ann F., J.E.L., Sr. Mary Brendan Wilson
Please Pray for Our Homebound...
Isabelle LaClair, Theresa Holmes, Kay Wilson,
Jim Reagan, Helen Volcko, Mike Berish, Ethel Cole,
Marie Patterson, Mary Martin, Patricia Welch
Please call the Church Office to add anyone to our Prayer List.
Holy Father’s Prayer Intentions - Universal
The Maritime World – We pray for all those who work and live from the sea, among them sailors, fishermen and their families.
Good News: The steeple reconstruction program has begun with an initial focus on repointing the damaged brick and mortar section of the tower.
Thank you to all that have given so graciously to this project – prayers and money. If you’re able to make a donation and have been putting it off, please consider donating today. And if you are not able to make a donation, that is perfectly OK. Prayers are appreciated. Our goal is $375,000 and we have collected $156,963 (with future pledges: $211,653).
Easier Giving Online Donations:
Visit www.sfxmarcellus.com / Light our Future tab
Make a Gift button - You may make a one-time contribution or initiate recurring gifts.
Confirmation
We will be celebrating the Sacrament of Confirmation with our 37 candidates on Tuesday, August 11 and Wednesday, August 12 at 6:00pm. Due to limited seating, only the candidate, sponsor and parents will be present but we will livestream both celebrations!
Please pray for our Confirmation Candidates who will be Confirmed by Bishop Cunningham.
Christopher Barbaro Daniel Mitchell
John Buff Emily Moses
Quinn Burnett Kate O’leary
Hannah Card Lucy Powell
Emma Carver Seamus Powell
Amanda Castellino Adison Roberts
Eve Chapman Emma Roseboom
Gavin Ciota Carl Santariello
Marcus Darminio Holly Southern
Michael Flynn Anna Spitzer
Ryan Flynn Heidi Steigerwald
Grady Germain Adam Sullivan
Harry Hoey Kadie Tierney
Zachary Kisil Madelyn Vetsch
Emma Maclachlan Mitchell Weeks
Kathryn MacLachlan John White
Mary Mahoney Aidan Wiley
Alyssa McShane Kaitlyn Worden
Brielle McShane
Dear St. Francis family,
Fr. Abe Esper, a good friend of mine and a good priest, will be back at St. Francis this weekend as I will be spending some time with my sister and her family. As some of you know, Fr. Abe has some interesting stories that he likes to tell. There are two very important events that are coming up in August. On August 11 and 12, we will celebrate the Sacrament of Confirmation for 37 young men and women, please continue to keep them in your prayers. On August 22, the feast of the Queenship of Mary, we will celebrate First Holy Communion; again, please keep these children in your prayers. Both events are great blessings for our parish and continuing signs of the Holy Spirit active and alive in our midst.
God Bless you, Fr. Dan
SPIRITUAL COMMUNION PRAYER
My Jesus, I believe that You are present in the Most Holy Sacrament. I love You above all things, and I desire to receive You into my soul. Since I cannot at this moment receive You sacramentally, come at least spiritually into my heart. I embrace You as if You were already there and unite myself wholly to You. Never permit me to be separated from You. Amen.
August 2, 2020
18th Sunday in Ordinary Time
Scripture Readings
Isaiah 55:1-3
Psalm 145
Romans 8:35, 37-39
Matthew 14:13-21
A Family Perspective by Bud Ozar – The apostles were just being practical when they asked Jesus to “dismiss the crowds to go buy food.” Jesus knew there is always enough when we place our trust in the Father, even with just 5 loaves and fishes. Trust is the bridge to miracles.
Question of the Week - Acting with Courage
For Adults: To whom could you bring the assurance of Christ’s love this week?
For Children: Jesus fed the crowds today. How does he offer you spiritual nourishment?
We would like to welcome Fr. Abe Esper to St. Francis this weekend!
Information Regarding Mass
We would like to remind everyone that we are dispensed from the obligation to attend Mass until further notice. If you are uncomfortable with attending any group gathering or have a preexisting medical condition that would make you more susceptible; or are ill or have any symptoms of covid19; please stay home and take care of yourself. You may view the weekend Mass on our Facebook page or through the link on our website.
We welcome the newest member of our faith family:
Gina McGivern
who received the Sacraments of Initiation on Sunday, July 26.
Summer Prayer
Long warm days...
The pace of life slows...
A time for picnics and rest in the shade...
Lord,
help me to rest awhile
in the cooling shade of your presence.
Slow down my restless heart
and fill me with gentle compassion
for all your people.
Amen.
-author unknown
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ATTENDEES:
Mayor L. Kelly Jones
Council Member Phillip Poole
Council Member Brian Libbey
Council Member Michael Dingman
Council Member Robert Fitzgerald
Council Member Halden Griffith
City Administrator/Sec Brandy Barrett
Police Chief Kevin Reaves
Deputy City Secretary Elisa Hickey
Interim City Attorney Tracie Kenan
ABSENT:
CALL TO ORDER by Mayor Jones at 7:14 PM
INVOCATION was given by Councilman Griffith.
PLEDGE OF ALLEGIANCE was led by Mayor Jones.
REGULAR SESSION:
1. **MOTION to approve the agenda with the removal of Items 7D-F as the petitioner withdrew their request.**
- MADE BY: Halden Griffith. SECOND: Phillip Poole.
- Motion passed by a vote of 5 Ayes and 0 Nays.
2. **Approval of the Consent Agenda:**
*All matters listed as Consent Agenda are considered to be routine by the City Council and will be enacted by one motion. There will not be a separate discussion of these items. If discussion is desired, that item will be removed from the consent agenda and will be considered separately.*
A. **Approval of the Minutes:**
- Council Meeting – March 12, 2024
B. **Approval of the Financial Reports:**
- TexPool Report
- TexStar Report
- A/P Disbursements
| BALANCES Mar-24 | GENERAL | WATER | CRIME CONTROL | CAPITAL PROJECTS | DEBT SERVICE | STREET | WRA | HCGC | GAS ROYALTIES |
|-----------------|-------------|-------------|---------------|------------------|--------------|-----------|------------|------------|--------------|
| Revenue * | $640,868 | $121,822 | $77,814 | $6,100 | $221 | $38,907 | $40,470 | $179,211 | $0 |
| Disbursement * | $191,234 | $97,998 | $37,855 | $64,360 | $108,771 | $18,522 | $49,823 | $115,630 | $0 |
| Cash on Hand | $206,977 | $216,151 | $545,618 | $267,099 | $3,349 | $344,283 | $220,255 | $406,060 | $51,853 |
| TexPool | $5,123,516 | $128,674 | $0 | $262,332 | $52,633 | 0 | $372,605 | 0 | 0 |
| TexStar | $756,930 | $1,630,156 | $157,516 | $420,042 | $135,500 | 0 | $227,895 | $1,028,934 | $2,100,294 |
* Month end closings/Jes and bank reconciliation pending.
**MOTION to approve the Consent Agenda.**
- MADE BY: Halden Griffith. SECOND: Mike Dingman.
- Motion passed by a vote of 5 Ayes and 0 Nays.
3. **STAFF UPDATES:** The Department Directors recapped their monthly reports.
4. **MAYOR’S REPORT:** The mayor presented a recap of the prior 30 days in office.
5. **COMMITTEE UPDATES:** The Councilmen provided updates for what took place during each of the below meetings that occurred in the prior month:
- There were no meetings held during the prior month.
6. **PUBLIC INFORMATION/ANNOUNCEMENTS**
A. **Announcements and Proclamations**
- TRWD has partnered with NASJRB volunteers to do a Trash Bash at Airfield Falls and local citizens are invited to participate. The event will be April 11th from 8:00am-noon.
- Blue Angel pilot and mechanic will be speaking at city hall, April 12th from 8:30-9:30am.
- The League of Women Voters will be moderating the general election candidate forum on April 18th at 6:30pm in the council chambers.
- Early voting begins on April 22nd at any Tarrant County polling location.
- Election day is May 4th and our community room will be one of the county’s polling places.
- Spring Picnic will be held in Melva Campbell Park on May 10th from 5:30 – 7:30pm.
B. **Meetings**
- Planning and Zoning Commission, April 2nd at 6:00pm
- Regular Council meeting, April 9th at 7:00pm
C. **Citizen Comments**
- The following spoke:
- Matthew Norris, 6644 Fairway Drive
- Richard Cervenka, 6604 Fairway Drive
- Joni Boulware, 6604 Fairway Drive
- Doug Drexler, 6617 Fairway Drive
- Emily Maas, 6600 Fairway Drive
- Susan Akhtar, 6613 Fairway Drive
- Jerry Wood, 6608 Fairway Drive
- Richard Meszaros, 6625 Fairway Drive
- Teddy Berdan, 5702 Popken Drive
7. **Public Hearings, Briefings and Action Items:**
A. **Mayor Jones**
Discuss and take action to adopt an ethics policy. *(On June 13, 2023, Mayor Jones appointed an Ad hoc committee, naming Lance Rahn, Barbara Deakins and Judge Russell Nelms as members, chaired by Councilman Fitzgerald, to write a Code of Ethics. Following a public hearing and discussion on December 12th the council has postponed action to the next meeting at each subsequent council meeting. In February, the committee was asked to make a final review and change it to a policy.)*
Councilman Griffith read a statement into the record, “I am a massive proponent of ethical standards. Show me an ethical standard, I will vote for it. It is the behavior we cannot legislate, the political maneuvering, vindictive targeting, and abuse of power that I disagree with. It does not belong in Westworth Village.”
MOTION to adopt an ethics policy with all the highlighted language that is in this policy and expand all that highlighted language to include the section of the Local Government Code, and that will be our policy; anything not highlighted in yellow in this policy will be scrapped.
- MADE BY: Halden Griffith. SECOND:
Councilman Griffith stated he “wanted it added to the record that the ethical concern was when the city mailed misinformation in a letter to every single citizen in Westworth Village.”
- Motion died due to lack of a second.
Motion to adopt the ethics policy removing “Should a violation of this Policy occur”, in the middle of the first paragraph, capitalizing “It” as the start of the sentence; removing all items starting on page 8, beginning with “Open Government” through page 11, item D, just before “Conclusion”.
- MADE BY: Phillip Poole. SECOND: Michael Dingman
Motion to amend by also removing the first paragraph on page 3 and the entire section of “Substantial Interest Standards” on page 4.
- MADE BY: Halden Griffith. SECOND: Brian Libbey
- Motion to amend the original motion passed by a vote of 5 Ayes and 0 Nays
- Motion as amended passed by a vote of 5 Ayes and 0 Nays
B. Mayor Jones
Discuss and take action on Resolution 2024-06 adopting the city’s written investment policy as required by the Public Investment Act. (This is the annual review of the investment policy. The council postponed action in February, requesting the city attorney highlight the state laws, review any comments submitted by citizens and report changes from prior years.)
Motion to adopt Resolution 2024-06 the city’s written investment policy as required by the Public Investment Act.
- MADE BY: Phillip Poole. SECOND: Robert Fitzgerald
- Motion passed by a vote of 4 Ayes and 1 Nays (Griffith)
C. Mayor Jones
Discuss and take action on the Golf and Parks Advisory Board recommendation to paint the T-33 in the livery of its last active-duty station in Point Magoo and review options to finish the project. (The current livery is accurate for its last active-duty station. No quotes have been submitted after multiple attempts; the local aviation museum’s referral has not responded to repeated request and is currently on vacation out of state.)
- No action was taken, the council requested the item be on the May agenda.
D. Mayor Jones
Public Hearing to receive citizen comments and input on citizen petition to privatize a section of Leonard Trail. (A public hearing is not required by law on this item.)
- This item was removed from the agenda.
E. Mayor Jones
Discuss and take action on the petition filed by the residents to privatize a section of Leonard Trail. (The ordinance in Item G will only be considered if the council accepts the citizen’s petition.)
- This item was removed from the agenda.
F. Mayor Jones
Discuss and take action on Ordinance 508 regarding the citizen petition to privatize a section of Leonard Trail. (In accordance with Texas Transportation Code 311.008, if the council rejects the citizen’s petition in Item F, no action on this item will be taken.)
- This item was removed from the agenda.
G. Mayor Jones
Discuss and take action on the request to publish audio files online and upgrade the audio/video system in the council chamber. *(Staff worked with the website host and found a way to link the audio files for 30 days following a meeting. The link is currently active on the Minutes and Agenda’s page of the website.)*
Motion to accept the staffs action putting audio files online for 30 days.
- MADE BY: Michael Dingman. SECOND: Phillip Poole
- Motion passed by a vote of 5 Ayes and 0 Nays
H. Mayor Jones
Discuss and take action on the IT services recommendation to upgrade two servers at a cost not to exceed $87,000. *(Staff received notice from the IT contractor that two servers that are nearing end of life need to be replaced for security reasons.)*
After a brief discussion, on the item, it was staff recommendations that for cyber security reasons the council consider the item in executive session.
EXECUTIVE SESSION:
Mayor Jones convened in closed executive session at 9:01 PM to deliberate the following items:
A. Consultation with attorney pursuant to Texas Government Code Section 551.071.
- Receive legal advice and discuss status of the Burgess property condemnation.
- Receive legal advice and discuss status of the Blue Atlantic tax refund litigation.
- Receive legal advice and discuss status of the St. Anne & St. Joachim, LLC litigation.
- Receive legal advice and discuss status of the MedStar Interlocal Agreement & potential litigation in anticipation of Fort Worth’s withdrawal from the agreement.
Mayor Jones re-convened in open session at 9:42 PM, where the following action was taken:
Motion to authorize the expenditure of up to $87,000 to upgrade two servers.
- MADE BY: Phillip Poole. SECOND: Michael Dingman
- Motion passed by a vote of 5 Ayes and 0 Nays
The meeting was adjourned at 9:43 PM by Mayor Jones.
MINUTES APPROVED BY:
L. Kelly Jones, Mayor
SIGNATURE ATTESTED BY:
Brandy G. Barrett, TRMC
City Administrator/City Secretary
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La mer
うみ
1990年2月
日仏海洋学会
La Société franco-japonaise d'océanographie
Tokyo, Japon
SOCIETE FRANCO-JAPONAISE D'OCEANOGRAPHIE
Comité de Rédaction
(de l'exercice des années de 1988 et 1989)
Directeur et rédacteur: Y. ARUGA
Comité de lecture: S. AOKI, M. HANZAWA, M. HORIKOSHI, A. KAMATANI, M. MAEDA,
M. MURANO, M. OGURA, S. OKABE, H. SUDO, T. YANAGI
Rédacteurs étrangers: H.J. CECCALDI (France), E.D. GOLDBERG (Etats-Unis), T. ICHIYE (Etats-Unis),
T.R. PARSONS (Canada)
Services de rédaction et d'édition: S. WATANABE, Y. YAMAGUCHI
Note pour la présentation des manuscrits
La mer, organe de la Société franco-japonaise d'océanographie, publie des articles et notes originaux,
des articles de synthèse, des analyses d'ouvrages et des informations intéressant les membres de la
société. Les sujets traités doivent avoir un rapport direct avec l'océanographie générale, ainsi qu'avec
les sciences halieutiques.
Les manuscrits doivent être présentés avec un double, et dactylographiés, en double interligne,
et au recto exclusivement, sur du papier blanc de format A4 (21×29,7 cm). Les tableaux et les
légendes des figures seront regroupés respectivement sur des feuilles séparées à la fin du manuscrit.
Le manuscrit devra être présenté sous la forme suivante:
1° Il sera écrit en japonais, français ou anglais. Dans le cadre des articles originaux, il comprendra
toujours le résumé en anglais ou français de 200 mots environ. Pour les textes en langues euro-
péennes, il faudra joindre en plus le résumé en japonais de 500 lettres environ. Si le manuscrit est
envoyé par un non-japonophone, le comité sera responsable de la rédaction de ce résumé.
2° La présentation des articles devra être la même que dans les numéros récents; le nom de l'auteur
précédé du prénom en entier, en minuscules; les symboles et abréviations standards autorisés par le
comité; les citations bibliographiques seront faites selon le mode de publication: article dans une revue,
partie d'un livre, livre entier, etc.
3° Les figures ou dessins originaux devront être parfaitement nettes en vue de la réduction nécessaire.
La réduction sera faite dans le format 14,5×20,0 cm.
La première épreuve seule sera envoyée à l'auteur pour la correction.
Les membres de la Société peuvent publier 7 pages imprimées sans frais d'impression dans la
mesure à leur manuscrit qui ne demande pas de frais d'impression excessifs (pour des photos couleurs,
par exemple). Dans les autres cas, y compris la présentation d'un non-membre, tous les frais seront
à la charge de l'auteur.
Cinquante tirés-à-part peuvent être fournis par article aux auteurs à titre gratuit. On peut en
fournir aussi un plus grand nombre sur demande, par 50 exemplaires.
Les manuscrits devront être adressés directement au directeur de publication de la Société: Y.
ARUGA, Université des Pêches de Tokyo, Konan 4-5-7, Minato-ku, Tokyo, 108 Japon; ou bien au
rédacteur étranger le plus proche: H. J. CECCALDI, EPHE, Station marine d'Endoume, rue Batterie-
des-Lions, 13007 Marseille, France; E. D. GOLDBERG, Scripps Institution of Oceanography, La Jolla,
California 92093, Etats-Unis; T. ICHIYE, Department of Oceanography, Texas A & M University,
College Station, Texas 77843, Etats-Unis; ou T. R. PARSONS, Department of Oceanography,
University of British Columbia, Vancouver, B. C. V6T 1W5, Canada.
The role of cryptozoology in achieving an exhaustive inventory of the marine fauna*
Paul H. LeBlond**
The Sea has played a vital role in the development of many areas of Science. Its rich and varied fauna has provided naturalists with clues from which they have been able to make sense of the diversity of life. The shape of the ocean basins and of the great ridges which traverse them has led to the development of plate tectonics and to a new understanding of the Earth's crust.
In recent years, there has been increased emphasis on the exploitation of the Ocean, with concerns focusing on the quantitative aspects of marine biology. Questions such as "How many?" rather than "What kind?" of marine organisms have come to the fore.
Have we now perhaps left behind the Age of Exploration to enter that of Exploitation of the Sea? Not entirely. The ocean depths retain much of their mystery and continue to attract daring explorers and broad public interest. Millions thrill with Jacques Cousteau in watching an undersea world where there always seems to be something new to be discovered.
But, is there really anything worthwhile left to be discovered in the ocean? In this age of electronic in-situ sampling, deep-diving submersibles, remote sensing and computer modelling, can there be anything significant left to find? Have we not by now explored the oceans widely and thoroughly enough that only matters of detail or of complexity need be settled?
Most oceanographers would argue that our knowledge of the oceans is still incomplete and superficial, that much remains to be discovered and clarified in flow dynamics, fish behaviour, ecological relationships, tectonic processes, climatic mechanisms, etc... Thousands of marine scientists around the world wouldn't be exploring and studying the ocean with such dedication if they did not believe that something important remains to be discovered!
One should thus have little difficulty in convincing a scientific audience that great discoveries are still to be made in many fields of marine sciences. To explore the broader aspects of future discovery in marine sciences is however a challenge which I will leave for some other occasion. Rather, I have selected a more specific, and more controversial question which exemplifies many of the points related to the anticipation of potential discovery.
The question asked here is the following: Are there any important elements of the marine fauna left to be discovered? In other words, are there still any large, or new, or unexpected animals in the oceans which remain to this day unknown to science? And if so, how many?
Many have answered the general question in the affirmative, and some, like Heuvelmans (1983), have even ventured to extrapolate from historical rates of discovery.
to obtain an estimate of the number of future discoveries to be expected. Let us refrain from searching for a definite answer to the question: we cannot positively know that there are undiscovered animals until they are actually found, identified and categorized to the general satisfaction of the scientific community. Asking the question nevertheless forces us to consider the process whereby new elements of the marine fauna are become known to science.
Most, if not all, marine animals have been discovered by accident, through man's efforts at fishing. As its diversity has revealed itself, the marine fauna has been catalogued and apportioned in families, classes and phyla. As in other areas of science, improvements in sampling and harvesting methods have led to unsuspected discoveries: new animals have been added to the marine bestiary. Deep submersibles, for example, have made detailed visual exploration of the ocean floor possible, leading to the discovery of a completely unexpected fauna near deep sea thermal vents. PARSONS (1985) has emphasized the need to explore for the sake of discovery in order to broaden our knowledge of the oceans. His remarks apply directly to the discovery of new marine creatures.
Occasionally, there have also occurred significant and surprising discoveries unrelated to the development of new observational methods. The coelacanth, *Latimeria chalumnae*, had already been fished up many times from the mid-depths of the Indian Ocean by Comoro Island natives before a specimen came to the attention of Marjorie Courtenary-Latimer in 1938 (SMITH, 1956; COURTENARY-LATIMER, 1979). Because of its kinship to primitive fishes known only from the fossil record, its modern discovery attracted considerable attention. The megamouth shark, *Megachasma pelagios*, (TAYLOR et al., 1983) on the other hand did not have a fossil pedigree. The first specimen was brought up in 1976 tangled in the anchor chain of a US Navy ship in Hawaii. Two more specimens have since been found, one off California, the other off western Australia, suggesting a global distribution (ISC, 1988).
In the face of such unexpected discoveries, and considering the prospects for improvement in techniques of observing and sampling the oceans, it would seem presumptuous to declare that no further discoveries are to be expected. We are thus back to the question: "How many?"
One might argue that on the time scale of modern scientific discovery, there is a fixed and finite number of animal species in the oceans. The more have been discovered, the less there remains to be found. That is however of little use since the total remains unknown, and hence so does the residue. Beyond the arguments of plausibility based on expectations of better sampling and on past accidental discoveries, there are however observations which point towards the possibility of additional discoveries and give hints as to their nature.
A definite addition to the fauna is made by catching an animal. But what if the animal is only seen, not caught? How do we account for those that get away? Most zoologists would rather discard near misses and wait until a tangible type-specimen is available to pronounce on species and genus. Until caught, a creature remains a rumour, an unconfirmed visual observation, not yet the object of zoology, but rather that of cryptozoology.
The term cryptozoology was coined by the French zoologist Bernard Heuvelmans in the 1950's (HEUVELMANS, 1982) to describe the study of creatures which are known only through visual or incomplete material evidence. Such animals are also called cryptids, a more appropriate term than monsters; the term is usually reserved for rather large creatures, observable at sea without instrumentation. Cryptids are often well known to natives before attracting the attention of science: such was the case for the coelacanth. Sometimes scientists are awarded a fleeting glimpse of an animal which they can only describe without being able to collect a specimen. This is what happened to MEADE-WALDO and NICOLL (1906) as they saw an unidentified marine animal with a long neck
and a large dorsal fin swim past their ship off the coast of Brazil.
Cryptids provide a clue of what is to be expected in terms of future discoveries, at least for rather large animals. Indeed some erstwhile cryptids have already graduated to zoological acceptance. There are many examples on land: the gorilla, the okapi, the platypus. A marine example is the giant squid *Architeuthis*, the Kraken of Norse mythology, now known from many strandings.
So, what does cryptozoology tell us? HEUVELMANS (1986) has brought together a checklist of unknown animals, the result of three decades of research on eye-witness reports. Marine cryptids figure prominently. What follows is drawn from HEUVELMANS' (1986) list; further documentation if found in HEUVELMANS (1968).
Unconfirmed cetaceans lead the list of marine animals for which no specimens have been captured. A high-finned sperm-whale, 60 feet in length, is said to have been frequently seen around the Shetland Islands in the 17th century. It is reported by Sir Robert Sibbald, and described by him as *Physeter tursio* (HEUVELMANS, 1986). Another long-finned whale, 20–30 feet long, was reported by WILSON (1907) who saw three specimens in January 1902 and four more the following month while on Robert Scott's *Discovery* expedition in the Antarctic.
It is not surprising to find a possible new species of beaked whales in the list. A number of beaked whales, the Mesoplodons in particular (WATSON, 1981), are known only from a few strandings and are difficult to identify at sea. HEUVELMANS (1968) quotes P.H. Gosse as having watched a school of 30 ft long, yet unidentified whales, black above and white below, in the North Atlantic over a century ago.
HEUVELMANS also includes a new kind of killer whale, entirely sepia brown with star-shaped scars, sighted several times in the Gulf of Aden by MORZER BRUYNS (1971). The same author mentions three dolphins, one from the Mediterranean, one from the coast of Senegal, and one from the Philippines. A black and white spotted dolphin with two dorsal fins has also been reported from the Mediterranean and from the Southern Ocean (HEUVELMANS, 1968).
Tales of less conventional creatures are also to be expected: the undiscovered is bound to be strange. "Mermaids" and "mermen" are mentioned by HEUVELMANS (1986) as having been frequently reported from areas where no recent species of sirenians are known to have lived during historical times. Rumours of survival of Steller's sea-cow (*Hydrodamalis gigas*) continue to be heard along the Bering Sea shores. Both MACKAL (1980) and HEUVELMANS mention the "sea-ape", a creature seen off the Aleutian Islands by Steller in 1741.
Reports of large, mostly elongated marine animals usually referred to as sea-serpents have been classified by HEUVELMANS (1968) into seven categories corresponding to their morphological characteristics: the super-otter, the many-humped sea-serpent, the long-necked sea-lion, the merhorse and a many finned, armoured archeocete—all mammals—with a huge ocean-dwelling saurian and a large eel-like fish in addition. HEUVELMANS examined over 600 reports to arrive at his classification. A more localized survey on the western coast of North America (LEBLOND and SIBERT, 1973) identified three types of marine cryptids: two of them variants of Heuvelmans' merhorse, the other an elongated creature, all three apparently air breathers and mammalian.
Finally, there are the reports of giant octopus, focusing on a mysterious standing on a Florida beach at the end of the last century (see also MACKAL, 1980).
If only a fraction of these reports is based on fact, there remains a rich harvest of discovery of major faunistic elements in the world ocean. However, because all these observations are based only on eye-witness reports and cannot be backed by tangible specimens, their status remains doubtful. Cryptozoological evidence is subjective and non-reproducible. While there is no a priori reason to reject as fanciful every shred of visual information, the scientific method has
gained respect as a method of arriving at universally accepted truths precisely because it deals with objective and reproducible evidence. Cryptozoology should thus perhaps be seen as a precursor of zoology, arriving at its conclusions earlier, but with less confidence. One should thus not conclude that the various cryptids glimpsed in the sea must all be discovered some day; some may, others may not. Cryptozoology only provides clues as to what may yet be confirmed; it is like a road map based on hear-say: better than nothing, but not entirely to be trusted.
Cryptozoological information should thus be seen as an adjunct to zoological discovery; not a body of information which is to be believed or disbelieved, but a collection of indices which should not be ignored and which may inspire and guide marine scientists in their effort to complete the inventory of the larger elements of the marine fauna. There is no such guide for small or microscopic creatures, where the need for exploratory research is even more acute.
References
COURTENARY-LATIMER, M. (1979): My story of the first coelacanth. p. 6–10. In: J.E. MCCOSHER and M.D. LAGIOS, ed., The Biology and Physiology of the Living Coelacanth. Calif. Acad. Sci. Occas. Paper No. 134.
HEUVELMANS, B. (1968): In the Wake of the Sea-Serpents. Hill and Wang, New York. 645 pp.
HEUVELMANS, B. (1982): What is cryptozoology. Cryptozoology, 1, 1–12.
HEUVELMANS, B. (1983): How many animal species remain to be discovered? Cryptozoology, 2, 1–24.
HEUVELMANS, B. (1986): Annotated check-list of apparently unknown animals with which cryptozoology is concerned. Cryptozoology, 5, 1–26.
ISC (1988): Third Megamouth Found. Internat. Soc. of Cryptozoology Newsletter, 7 (4), 4.
LEBLOND, P.H. and J.R. SIBERT (1973): Observations of large unidentified marine animals in British Columbia and adjacent waters. Dept. of Oceanogr., Univ. of British Columbia, M.S. Report 28. 64 pp.
MACKAL, R. (1980): Searching for Hidden Animals: an Inquiry into Zoological Mysteries. Doubleday, New York.
MEADE-WALDO, E.G.B. and M.J. NICOLL (1906): Description of an unknown animal seen at sea off the coast of Brazil. Proc. Zool. Soc. Lond., 2 (68), 719.
MORZER BRUYN, W.F.J. (1971): Field Guide of Whales and Dolphins. C.A. Mees, Amsterdam.
PARSONS, T.R. (1985): Hypothesis testing and rigorous statistics as criteria for marine research proposals. La mer, 23, 109–110.
SMITH, J.L.B. (1956): Old Fourlegs. Longmans, Green and Co. 260 pp.
TAYLOR, L.R., L.J.V. COMPAGNO and P.J. STRUHSAKER (1983): Megamouth – A new species, genus, and family of lamnoid sharks (Megachasma pelagios, Family Megachasmidae) from the Hawaiian Islands. Proc. Calif. Acad. Sci., 43, 87–110.
WATSON, L. (1981): Sea Guide to Whales of the World. Dutton, New York. 302 pp.
WILSON, E.A. (1907): Antarctic Mammalia. In: National Antarctic Expedition, Natural History. London, 2, 4–5.
Internal tidal waves and internal long period waves in the Sanriku coastal seas, eastern coast of northern Japan*
Moriyoshi OKAZAKI**
Abstract: The study of internal tidal waves in a bay was carried out on the basis of observed records in Toni Bay (located in the northeastern area of Honshu) in autumn 1984.
a) It was found that the internal tidal waves were predominant and appeared intermittently with a duration of several days in the bay. On the basis of T-S diagram analyses, it was suggested that the intermittency of the internal tidal waves in the bay was closely related to the intermittent vertical displacement of the offshore thermocline.
b) The cause of the intermittent vertical displacement of the thermocline was investigated by analyzing subsurface water temperature at two bays and the variation of sea surface level anomaly (local difference of tidal deviations) among three bays adjacent to Toni Bay. From the analysis of the variation of the thermocline depth, some evidence was obtained for the existence of internal long period waves propagating southward along the coast. The intermittent appearance of the internal tidal wave in the bay occurred from the superposition of the internal tidal wave propagating onshore and the internal long period wave propagating alongshore. Moreover, it is suggested that the internal long period waves are related to the behaviour of an offshore density front at the sea surface in Sanriku coastal seas.
c) The internal tidal waves of semidiurnal period in the bay were likely related to the 2nd mode of the offshore internal tidal waves from comparison of the dispersion relations of the internal tidal waves in the bay with those out of the bay and from the simple model estimation of an energy efficiency of the offshore internal tidal waves propagating into the bay.
1. Introduction
In reports on observations of internal tidal waves, the intermittency of occurrence of internal tidal waves has been noted only recently. In the open ocean, the internal tidal waves are found as predominant semidiurnal fluctuations of the current velocity in several subsurface depths. For example, WUNSCH (1975) quoted from MAGAARD and MCKEE (1973) and indicated that the intermittency of about 10 days was remarkable in the variation of the amplitude of the internal tidal waves.
In coastal sea areas, there are reports on the propagations of semidiurnal internal waves and their breaking at the shelf break in the northwestern coastal sea area of Australia (HOLLOWAY, 1984, 1985). The intermittency of about 10 days was noticeable in his records of water temperature and velocity components, though he describes nothing about it. In Japan, the reports of the internal tidal waves have increased in recent years; there are only two reports indicating the intermittency of the internal tidal waves for the present. INABA (1981) reported that in the subsurface layer of Suruga Bay, a diurnal period was dominant in the tidal current in contrast to the predominance of a semidiurnal period in the sea surface tide and this was due to the existence of an internal wave in the subsurface layer. MATSUYAMA (1987) suggested the intermittency of 7-10 days in the records of the velocity components in INABA (1981). In Uchiura Bay located at the head of Suruga Bay, the record of sea water temperature in the subsurface layer showed a predominant semidiurnal fluctuation, and this was due to resonance of the semidiurnal tide with the internal seiche in Uchiura Bay (MATSUYAMA, 1985). In addition, he de-
* Received January 8, 1988
Revised and accepted December 15, 1989
** Earth Science Laboratory, Institute of Physical and Chemical Research, Wako, Saitama, 351-01 Japan
scribed that the semidiurnal fluctuations intermittently disappeared in 3-5 days due to the passing of a typhoon near Suruga Bay. In the coastal sea of Joban, southern neighbour of the Sanriku sea area, the internal tidal waves were dominant in semidiurnal period and propagated onshore, and the intermittent behavior of the semidiurnal internal tide was noticeable in his record of the subsurface water temperature (MATSUNO, 1989).
Sanriku coast is one of the suitable fields for studying the phenomena of the internal mode, since the Tsugaru Warm Water (TWW) is lighter than the Oyashio Cold Water (OCW) (in spite of higher salinity, smaller water density than that of OCW is attributed to higher temperature of TWW), the strong thermocline is usually kept offshore. The lighter and warmer water flowing through the Tsugaru Strait to the Pacific Ocean from the Japan Sea is the origin of TWW, which is generally flowing southwards along Sanriku coast in a surface layer with thickness of 150-200 m (HANAWA, 1984). In autumn, TWW spreads sometimes about 100km to the east of the Tsugaru Strait before flows southward along the Sanriku coast. A remarkable pycnocline front (or thermocline front) at the sea surface is observed at the eastern boundary of TWW, because the heavier OCW is found under and off TWW. The depth of the thermocline under TWW becomes deeper shoreward as an extension of the surface front in contrast with the Kuroshio front which is shallower shoreward in the south sea area of Honshu. Thus, the thermocline is also clear near the coast, and the internal wave is easy to generate in such a sea area.
A phenomenon of "Sakashio", named by Japanese fishermen, occurs sometimes in many bays of the Sanriku coast. "Sakashio" means the subsurface tidal flow opposite to the surface flow. When "Sakashio" occurs in a bay, it is difficult to wind up "the Set Net", which is a kind of fish net widely used in Sanriku coast. This phenomenon is closely related to the occurrence of the internal waves in the bay. Therefore the internal waves are one of the important oceanographic phenomena for coastal fishery, and they are also important for cultural fishery, since the internal tidal waves force the exchange of coastal sea waters in the bay for offshore sea waters.
In order to clarify the behavior of the internal tidal waves in Sanriku coastal seas, the observations were carried out in one of the bays in Sanriku coast (Toni Bay, located in Kamaishi) from October to December 1984.
In this paper, several properties of the internal tidal waves become clear, especially their intermittent appearance in the bay. In chapter 2, the outline of observations will be given. In chapter 3, some typical examples of records are shown, and the properties of the internal tidal waves observed in Toni Bay and the intermittency in the internal tidal waves with duration of several days are explained. In addition, the intermittent property is described with relation to the vertical displacement of the thermocline. In section 1 of chapter 4, some evidence for the existence of the internal long period wave will be shown. Discussion is extended to the role of such waves as a cause of the intermittent vertical displacement of the thermocline and the relation between such waves and the oceanic front. In section 2 of chapter 4, the properties for propagation of the internal tidal waves in the bay will be stated in relation to the properties of the offshore internal tidal waves, and the shoaling up of the internal tidal waves into the bay will be discussed concerning the nature of the exchange of sea water in the coastal seas.
2. Observations and data
The Sanriku coast is called a Rias type in geology, and has a complicated coast line. The configuration of sea bottom in this sea area is also complicated in areas shallower than 130 m depth, and the bottom contours run almost straight north to south in deeper seas. The sea floor increases gradually in depth toward the Japan Trench. The complicated coast line is conspicuous, especially southward of Miyako Bay in the Sanriku coast. Otsuchi Bay is 33km southward, Kamaishi Bay is 45km southward of Miyako
Bay, and Ofunato Bay is 35 km southward of Kamaishi Bay. Toni Bay is 8 km southward of Kamaishi Bay (Fig. 1). Its bay mouth faces eastwards to the Pacific Ocean. The sea valley runs into the center of Toni Bay, and the breadth of the bay mouth is about 4 km. The depth is about 90 m at the center of the bay mouth and about 40 m on the average; the length of the bay is 5-6 km.
Previous physical oceanographic research in Toni Bay showed that there was a counterclockwise circulation in the bay and the flow in the bottom layer differed from that in the surface layer (TSUJITA, 1974).
Two observations were carried out in Toni Bay from 1 October to 15 December 1984. The first observation was made at 14 stations in Toni Bay (Sta.0 - Sta.13) every three days on a ship-contained STD (portable) which measured water temperature and salinity at 1 m intervals from the sea surface to the bottom (or maximum 80 m deep) (Fig. 1). The second observation was the continual observation by means of current meters which were fixed to the bottom by anchors and subsurface buoys of the mooring system. Sta.A (33 m depth) was settled near the head of the bay, Sta.D (65 m depth) was north of the center of the bay mouth, Sta.C (63 m depth) was south of the center of the bay mouth and Sta.B (44 m depth) was at the midpoint of the north side of the bay (Fig. 1). At all stations, Aanderaa current meters were set in the surface layer (10 m below sea surface) and in the bottom layer (5 m above bottom) to measure current speed, current direction, water temperature and salinity every 10 minutes. The record of a current meter was averaged over an hour before analysis. The continual record was divided into two parts, 1 October - 8 November and 9 November - 15 December. Unfortunately, the record of the current meter in the bottom layer at Sta.D was not obtained because of
Fig. 2. An example of the time series of Aanderaa current meter record in the bottom layer at Sta. C (Oct. 1-31, 1984). Wind, vector expression of wind every 3 hours. U, eastward component of velocity (thick line) and its 25 hour running mean (thin line). V, northward component of velocity (thick line) and its 25 hour running mean (thin line). Temp., sea water temperature.
mechanical trouble.
In addition to those records, the following data of the routine observations by governmental offices were available to the present paper:
1) The monthly data of routine coastal observations in 4 lines off the Sanriku coast by Iwate Prefecture Fishery Experimental Station (Fig. 1).
2) The hourly data of tidal observations in Miyako Bay, Kamaishi Bay and Ofunato Bay (the forecasted data and the observed data) by Branches of Meteorological Agency and others.
3) The meteorological data every three hours at Miyako, Kamaishi and Ofunato (air pressure, wind speed and wind direction).
4) Simultaneous data of temporal deep sea observations far off Sanriku, by Meteorological Agency.
The Iwate Prefecture Fishery Experimental Station has carried on the monthly observation of water temperature and salinity in several depths shallower than 300 m at each station in 4 observation lines off the Sanriku coast. In each observation line, 8 stations are set, for example, Sta. 1 is 0.7 km and Sta. 8 is about 130 km from the coast in the Osaki line. Details of 4 coastal routine observation lines are:
Kurosaki Line (KI - KVIII) (40°00'N, 141°52' - 143°30'E);
Todosaki Line (TD I - TD VIII) (39°32'N, 142°06' - 143°35'E);
Osaki Line (OS I - OS VIII) (39°15'N, 142°04' - 143°30'E);
Tsubakijima Line (TJI - TJ VIII) (38°56'N, 141°44' - 143°14'E).
Those routine data were very useful to the present work as indicators of information in the offshore sea area, especially the Osaki line located about 3 km from Toni Bay (Fig. 1).
3. Analysis and results
1) An example of records
a) Time series of current meter records
Time series of several components of the current meter record in the bottom layer at Sta. C are shown in Fig. 2. The variation of water temperature is apparently different from that in the surface layer (refer to Fig. 6). Water temperature fluctuated periodically and its range was 6°C in maximum value. Before 15 October, the period of the fluctuations was semidiurnal, but it was nearFig. 3. Vertical distribution of temperature (°C), salinity (permill) and specific volum anomaly (cl/ton) of sea water in the mouth of Toni Bay (14:43 - 15:46, Oct. 1, 1984).
Diurnal in 16-24 October, though its fluctuation was considerably disturbed. It is of interest that the days of steady and high temperature were 5-8 and 25-29 October. After 29 October, the record is obscure because of some loss of record from troubles with the water temperature sensor. On the other hand, in the surface layer, water temperature tends to decrease gradually, and sometimes drops 1-2°C per day (Fig. 6). This change corresponded to the duration of fluctuation of water temperature in the bottom layer, which suggested that the dominant fluctuation of bottom water temperature was relevant to the lowering of surface water temperature.
The eastward component of velocity (U) in the bottom layer mostly fluctuated in the positive area, which meant that the flow was generally eastward (Fig. 2). The basic flow in the bottom layer at Sta.C was eastward and it seemed to be related to counterclockwise circulation in Toni Bay (TSUJITA, 1974). The magnitude of fluctuation of the U or V component in the surface layer was equal to that in the bottom layer (figure of the record in the surface layer at Sta.C is not shown). U and V fluctuated periodically, but they were rather disturbed by noise compared with the fluctuation of water temperature. The fluctuation of semidiurnal period prevailed in the beginning of October and the fluctuation of diurnal period was found with that of semidiurnal period in the second half of October. When the tidal fluctuation of bottom water temperature was large, fluctuation of U reached to 40 cm/sec in range of U (in the term of about 6 hours).
The vector expression of wind was composed of records of wind speed and direction every 3 hours at Kamaishi. During the observations, the wind blew weaker than the usual condition in late autumn. For example, the wind speed was usually 3-4 m/sec and sometimes 7-8 m/sec.
b) Data of STD measurement
Fig. 3 is an example of the vertical distribution of water temperature, salinity and specific volume anomaly at six stations near the bay mouth from STD measurements during the afternoon on 1 October. A very clear thermocline was found at a depth of 30-40 m
Fig. 4. Correlations between velocity components in the surface and in the bottom layers, and vertical distributions of sea water temperature. X-axis, $U_s$ (velocity component in the surface layer); Y-axis, $U_b$ (velocity component in the bottom layer). a) strong density stratification (Oct. 1-5, 1984); b) transient density stratification (Oct. 6-10, 1984); c) vertically homogeneous density (Nov. 13-17, 1984).
in Toni Bay. The thickness of the thermocline was about 10 m and the temperature difference between upper and lower layers was 6°C (20-14°C) in maximum value. On the other hand, salinity was almost homogeneous ($\text{Sal.} > 34.0 \text{ permill}$) at all depths in Toni Bay, hence, colder sea water in the bottom layer of Toni Bay was not the Oyashio Water ($\text{Sal.} < 33.8 \text{ permill}$) but the cooled Tsugaru Warm Water.
The strong density stratification in Toni Bay depended on the vertical distribution of water temperature.
2) Properties of the internal tidal waves
*Vertical distribution of specific volume anomaly of sea water and currents in stratified sea*
The thermocline of about 10 m thickness between the upper warmer sea waters about 20°C and the lower colder sea waters less than 14°C has a very large vertical gradient of water temperature (0.6°C/m, Fig. 3). When stratification occurs in Toni Bay, an inverse correlation between the velocity components in the surface and the bottom layers is shown at top of Fig. 4a. This correlation of currents between both layers indicates a dominant internal mode.
On the other hand, the density distribution in the bay was homogeneous on 13 November at the bottom of Fig. 4c. The vertical distribution of currents was also homogeneous in 13-17 November (top of Fig. 4c).
The density stratification was weak on 8 October, and the correlation between the velocity components in the surface and the
bottom layers seemed to be zero (Fig. 4b). This shows that the vertical distribution of currents in the bay was in a transient state between the strong density stratification state (Fig. 4a) and the homogeneous distribution state (Fig. 4c).
**Spectral property of fluctuations**
As the fluctuation of bottom temperature was different between the terms of 1-4 and 16-24 October (Fig. 2), power spectra of water temperature in the bottom layer at Sta.C were calculated for each of the terms (Fig. 5a). Power spectra of water temperature for the former had predominant period of 13 hours, which nearly equaled the semidiurnal period (left of Fig. 5a). Power spectra in the bottom layer at Sta.A and Sta.B had a prevailing semidiurnal period in this term.
On the other hand, for 16-24 October, the predominant period was about 35 hours in the bottom layer at Sta.B and Sta.C (right of Fig. 5a), and there was no dominant period in density distribution of power spectra at Sta.A in the bay head (refer to Fig. 6).
Comparing eastward components of velocity (U) for 1-4 October with those for 16-24 October at Sta.C (Fig. 2), the amplitude of fluctuation in the former was larger than that in the latter, and those seemed to have a similar period. The power spectral density of U for 1-4 October had a dominant period at about 11 hours near the semidiurnal period (left of Fig. 5b). The density of low frequency was large; this was shown in a gradient of the smoothed curve (running mean velocity) in the fluctuation of U in Fig. 2. In the bottom and surface at Sta.A and Sta.B, and in the surface at Sta.D, the predominant period of the semidiurnal was obtained, but it was about 14 hours in the surface at Sta.C.
For 16-24 October, the power spectra of U had a dominant period of about 31 hours, similar to the case of water temperature (right of Fig. 5b), and in the bottom at Sta.A and Sta.B the dominant periods were about 22 and 19 hours, respectively.
As mentioned above, there was apparently a difference of period between the two terms in both water temperature and velocity component (U).
**The characteristics in fluctuation of water temperature in Toni Bay**
In Fig. 6, water temperature in the surface layer varies similar to an envelope of water temperature in the bottom layer at each station. The patterns of bottom water temperature at the three stations are similar to each other, and their fluctuations seem to decrease gradually from Sta.C at the bay mouth to Sta.A at the bay head. The other noticeable feature of the fluctuation of water temperature in the bottom layer is that there are several calm days or the days of no periodicity. The internal tidal waves seem to be strongly intermittent (Fig. 6). It should be noted that for 8-15 October, the maximum of water temperature at the bottom layer at Sta.C was nearly equal to that at the surface throughout the term. This suggests that the
Fig. 6. Comparison of sea water temperature variations in the surface with those in the bottom layer at Stations A, B and C. At the bottom of the figure, the term of the internal wave type is shown: single line, the term of the semidiurnal fluctuation of sea water temperature at Sta. C in Fig. 2; double line, the term of diurnal fluctuation; chain line, the term of stable and high temperature (no fluctuation).
thermocline disappeared in the bay; the thermocline sank deeper than the depth at the bay mouth and the warm surface water filled the whole depth in the bay. The colder sea water at the bay bottom repeated to flow in and to flow out every tidal period for 8–15 October. This is an interesting mechanism for the exchange of sea water in a shallow bay.
3) Intermittency of the internal tidal waves and vertical displacement of pycnocline
a) Intermittency of the internal tidal waves
The pattern of fluctuations of water temperature in Fig. 2 suggests the intermittency of the internal tidal waves. In the record of water temperature, semidiurnal internal tidal waves were predominant for 1–4 and 9–15 October, near diurnal were dominant for 16–24 October, and the terms of the stable and higher water temperature were for 5–8 and 25–29 October. In fluctuations of velocity component (U), amplitudes of U seemed to be diminished in the terms of stable and higher water temperature and this was due to the intermittency of the internal tidal waves in the bay.
To confirm the intermittency, “the figures of the day by day variations of density of power spectra” from records in the bottom layer at Sta.C are shown in Fig. 7. The figure has frequency of fluctuations on the X-axis and the elapsed days of 3–21 October are marked on the Y-axis. Isosteric lines of the power spectra of fluctuation are shown in the figure. Power spectra of fluctuations were calculated at terms of every 5 days from 1 to 23 October. For example, the power spectra of 3 October at bottom in the figure were calculated on data in 1–5 October, next power spectra of 4 October were calculated from data in 2–6 October. Those calculations were carried out to 21 for data in 19–23 October day by day. The dominant periods for fluctuations of water temperature are at 11 hours near the semidiurnal period and at long period component (above 50 hours) on 4 October (the representative day of 2–6 October, Fig. 7a). The density of power spectra decreases to lower levels at all frequency fields in 5–8 October. The semidiurnal period component increases in 10–14 October. After 15 October, the components of frequency shorter than 25 hours disappear and those of frequency longer than 25 hours
Fig. 7. Day by day variations of power spectra at Sta. C bottom layer. Power spectra were calculated every 5 days sliding a day (Oct. 1-23, 1984). a) Sea water temperature (Temp.). b) Eastward component of velocity (U).
become more dominant.
Another figure of day by day variation in density of power spectra is shown in the eastward component of velocity (U, Fig. 7b) in a similar manner to water temperature. Distributions of isosteric lines differ from those of water temperature, and the peaks at each frequency in each elapsed day are more obscure than those of water temperature. The components of near semidiurnal period and long period (above 50 hours) are dominant for the first 5 days, and the components at all frequencies are at lower levels for 7-13 October. The predominant component is a semidiurnal period for 13-14 October and a diurnal period is prevailing for 16-18 October.
To conclude, the figures were very suggestive of the intermittency of the internal tidal waves in the bay.
b) Vertical displacement of pycnocline (thermocline)
The inflowing of colder sea water to the bottom of Toni Bay from offshore was confirmed from the result that the lowering of water temperature generally took place earlier at Sta. C than at Sta. A (for example, Fig. 17).
Next problems were where the origin of colder waters offshore was and how deep the layer of colder waters was. In order to solve those problems, T-S diagram analyses of sea waters for the term of density stratification were made (Fig. 8). The waters in the hatched area (13-15°C, about 34.2 permill) in Fig. 8a were the bottom water in the bay, the waters in area enclosed with broken lines (19-21°C, 33.9-34.2 permill) were the surface water. The waters of similar property to the bottom waters in the bay were found in 70-100 m layer at nearshore stations in three lines north of the Osaki line (Fig. 8b, c, d). An offshore thermocline of 13-17°C (near Sta.OSV) was found at 40 m depth, increased in depth towards the coast, and was found at 75-100 m depth at Sta.OSII (8 km off coast, Fig. 14c). This deepening of the thermocline near the coast is usual in the vertical distribution of water temperature in this season. The bottom sea water in the bay was different from the offshore sea water at the correspondent depth; therefore, it might be the offshore sea water rising up from a slightly deeper thermocline by an unknown external force. When the thermocline nearshore rose intermittently the density stratification appeared in Toni Bay and the internal tidal waves propagated into the bay.
The sea waters similar in T-S property to
Fig. 8. Comparisons of the sea water property in T-S diagram in Toni Bay with that in the offshore sea area. Hatched area in T-S diagram: T-S property of the colder sea water at the bottom in Toni Bay. Area bounded by broken line in T-S diagram: T-S property of the warmer sea water at the surface in Toni Bay. I, II, III·····: number of the offshore station. 10, 50, 75····: observed depth (m). a) T-S diagram of Stations 8-13 in Toni Bay. b) T-S diagram of Stations I-III in Osaki line. c) T-S diagram of Stations I-III in Todosaki line. d) T-S diagram of Stations I-IV in Kurosaki line. e) T-S diagram of Stations IV-VIII in Osaki line. f) T-S diagram of Stations I-IV in Tsubakijima line.
the bottom sea water in the bay were not found at stations offshore from Sta.OSV and at all southern stations in the Tsubakijima line (Fig. 8e, f). Therefore, the intermittent vertical displacement of the thermocline occurred only in the northern sea area of Toni Bay.
It is confirmed from observations that the intermittency of the internal tidal waves in Toni Bay was due to the vertical displacement of the thermocline on a time scale of several days. Remarkable meteorological disturbances were not found in the term under investigation since wind was usually weaker than 5 m/sec in October 1984 (Fig. 2). Therefore, the long period waves were of interest for the cause of vertical displacement of the thermocline on a time scale of several days, if those waves existed in the Sanriku coastal seas.
4. Discussions and conclusions
1) Some evidence on the internal long period waves
In chapter 3, it was pointed out that the internal tidal waves appeared intermittently in the time scale of several days, and the T-S property of colder sea waters in Toni Bay coincided with colder sea waters under the thermocline in a slightly deeper layer outside of Toni Bay.
It is only recently that the intermittency in the internal tidal waves attracted scientist's attention (e.g. WUNSCH, 1975). However, there are very few reports on this problem. In Japan, a few researchers have noticed such an intermittency in the continual records of sea water temperature and current velocity in the subsurface layer. They concluded that this resulted from the disappearance of stratification of the ocean caused by a change in the wind system, e.g. access of a typhoon (INABA, 1981; MATSUYAMA, 1985). The intermittent variations were clearly found in the record of bottom water temperature in the Joban coastal sea area, southern neighbour of the Sanriku sea area, though he described nothing about it (MATSUNO, 1989). Similar intermittent variation was found in the record of bottom water temperature and current velocity components in subsurface layers in the northwestern coastal sea of Australia (HOLLOWAY, 1985). Thus the intermittency in the internal mode occurred generally in every sea area in the world, though its origin and structure were different in each sea area.
In the Sanriku coastal seas, it was possible that the space scale of the intermittency of the internal tidal waves had a larger horizontal scale than that of Toni Bay (Fig. 8). Similar variations of temperature were expected in the bay adjacent to Toni Bay. It was very convenient that a continuous measurement of sea water temperature had been carried out in several subsurface layers above 15 m deep at Otsuchi Bay to the north of Toni Bay (Fig. 1, SHIKAMA et al., 1985). A variation of sea water temperature (1 hour running mean values of every 10 minutes records) at 10 m depth in Otsuchi Bay (observing station was located at about 6 km inward from the bay mouth) was very similar to that in the surface layer at Sta.D in Toni Bay (Sta.D was located at about 1 km inward from the bay mouth). The records in Fig. 9 are the subsurface water temperature deviations of 25 hours running mean values from mean value of full data in Toni Bay and Otsuchi Bay. A temporal decrease of water temperature appeared in Otsuchi Bay on 3 and 17 October, and a similar temporal decrease appeared in Toni Bay a day later. Those variations of water temperature in the two bays were in high correlation with each other and the cross correlation coefficient ($\alpha$) was 0.89. The propagating speed of this decrease was estimated from the difference of the propagating distances for the two stations in the bays and the lag (22 hours) of the time when $\alpha$ was maximum in the cross correlation. Therefore, it corresponded to the length of 15 km (=20 - 6 + 1), and the propagation speed of a long period wave was estimated about 19 cm/sec and from north to south in direction.
This propagating speed was compared with the phase speed of the coastal trapped wave (internal Kelvin type wave) calculated from observed data. The dispersion relation of the coastal trapped wave was calculated by means of a reduced gravity model taking into account the details of bottom configuration. The estimated phase speed of the coastal trapped wave was more than 30 cm/sec; this was considerably larger than the propagating speed of the above long period wave (19 cm/
Fig. 10. Comparison of sea surface levels in three bays (cm). Terms of internal wave type in d) and e) are the same as those in Fig. 6. a) $\zeta_M$ is value of 25 hour running mean of the tidal deviation in Miyako Bay. b) The same as in a) except for in Kamaishi Bay. c) The same as in a) except for in Ofunato Bay. d) $\Delta \zeta_{KM}$ is the sea surface level anomaly (local difference of tidal deviations) between Kamaishi Bay and Miyako Bay ($\Delta \zeta_{KM} = \zeta_K - \zeta_M$). e) The same as in d) except for between Ofunato and Kamaishi Bays ($\Delta \zeta_{OK} = \zeta_O - \zeta_K$).
The small magnitude of the phase speed suggested that the long period wave was a kind of internal wave; the internal long period wave treated here was different from the coastal trapped wave.
In the coastal seas of Japan, some results on the long waves were reported. In the Joban coastal sea area (southward of the Sanriku coast), the periodical current fluctuation (about 100 hours) was dominant and it propagated southward with speed of 3-5 km/hour (83-139 cm/sec), and this corresponded to the second mode and the third mode of the shelf waves (KUBOTA et al., 1981; KUBOTA, 1982, 1985). These reports were related to the external long wave, and the propagation speed was larger than that of the internal long period wave in the present study.
On the other hand, there are few reports in Japan on observations of the internal long period wave as yet. When the internal long period waves propagate from north to south along the coast, it is expected that the sea surface level changes out of phase to the depth of the thermocline, even if it is very small. The deviations of the observed tide (after correction by atmospheric pressure) from the predicted tide were obtained in each of Miyako Bay, Kamaishi Bay and Ofunato Bay (Fig. 10a, b, c). These sea surface level anomalies are shown in Fig. 10d, e. Although $\Delta \zeta_{KM}$ contains some noise, $\Delta \zeta_{KM}$ is evidently negative with its magnitude of 3-4 cm for 1-5 October and 14-24 October when the internal tidal waves were dominant in Toni Bay (except 9-13 October). The negative $\Delta \zeta_{KM}$ means that sea surface level at Kamaishi Bay was lower than that at Miyako Bay. Because the sea surface level variation caused by the internal wave changes out of phase with that of the thermocline as mentioned above, the negative sea surface level anomaly ($-\Delta \zeta_{KM}$) in Fig. 10d probably indicates upward displacement of the thermocline at Kamaishi, and the dominant fluctuation of the thermocline may appear in the shallow bay in those terms (Fig. 10d and Fig. 6). The time variation of $\Delta \zeta_{OK}$ (Fig. 10e) is out of phase with that of $\Delta \zeta_{KM}$ and the amplitude of $\Delta \zeta_{OK}$ seems to be a little smaller than that of $\Delta \zeta_{KM}$. $\Delta \zeta_{KM}$ is inversely proportional to $\Delta \zeta_{OK}$ (Fig. 11), and the variation of sea surface level near Miyako Bay was in phase with that near Ofunato Bay. Therefore, the tendencies of $\Delta \zeta_{KM}$ and $\Delta \zeta_{OK}$ indicated the existence of the internal long period wave along the Sanriku coast.
Those results suggested the existence of internal long period waves propagating alongshore with an appropriate space scale (about 100 km) that was nearly twice the distance between Miyako Bay and Kamaishi Bay.
(about 45 km). The tendency that the amplitude of $\Delta \xi_{OK}$ was a little smaller than that of $\Delta \xi_{KM}$ was speculated to occur because the distance between Ofunato Bay and Kamaishi Bay was about 35 km which was fairly shorter than a half of the space scale of the internal long period waves. Also, the amplitude of $\Delta \xi_{KM}$ (3-4 cm) in Fig. 10d was a reasonable order in relation to the magnitude in the density difference of sea water ($\Delta \rho = 0.0011 - 0.0017$) in the surface and bottom layers, compared with the vertical displacement of the thermocline ($H_L$: about 20 m, estimated later) caused by propagation of the internal long period waves ($\Delta \xi_{KM} / H_L = \Delta \rho / \rho$, $\Delta \xi_{KM} = 0.0017 \times 20 \text{m} = 3.4 \text{cm}$).
Next, the relation of the time changes in $\Delta \xi_{KM}$ (or $\Delta \xi_{OK}$) and in the vertical displacement of the thermocline will be examined. Unfortunately, there was no continual record of the vertical displacement of the thermocline during this observation. Hence, the record of sea water temperature (after 25 hours running mean) in the bottom layer at Sta. C in Toni Bay was treated instead of the vertical displacement of the thermocline. When the thermocline becomes shallower (deeper), the deviation of sea water temperature in the bottom ($\Delta T$, °C) will decrease (increase). The correlation between $\Delta \xi_{KM}$ and $\Delta T$ was positive and the correlation coefficient ($\alpha$) was 0.68 (Fig. 12). This meant that negative $\Delta \xi_{KM}$ corresponded to the lower sea surface level near Kamaishi than that near Miyako, and the lower sea surface level corresponded to a shallower thermocline near Kamaishi in the internal mode. Consequently, a shallower thermocline corresponded to the lower water temperature in the bottom layer of Toni Bay near Kamaishi. The positive correlation between $\Delta \xi_{KM}$ and $\Delta T$ in Fig. 12 was very reasonable. These
results suggested propagation of a wave from Miyako Bay to Ofunato Bay, and supported statistically the existence of the internal long period waves.
However, there was another possibility of high correlation between $\Delta \xi_{KM}$ and the deviation of sea water temperature; the time change of $\Delta \xi_{KM}$ was possible to depend on the variation of the local difference of sea surface water temperature between Kamaishi Bay and Miyako Bay ($\Delta T_{KM}$, °C). NISHI and KUNISHI (1985) investigated the influence of the local difference of sea water temperature in the surface layer upon the difference of sea levels in two distant stations off Shikoku Island. In the present study, there was no relationship between $\Delta \xi_{KM}$ and $\Delta T_{KM}$ (the latter was only one observation each day, Fig. 13). Therefore, the variation of $\Delta \xi_{KM}$ and $\Delta \xi_{OK}$ was possibly due to the internal long period waves, and the existence of the internal long period waves was highly possible.
The space scale (about 100 km) was a match for the time scale (several days) of the internal long period waves. Thus, the space scale through the local difference of sea surface levels in the two bays also verified the propagation of the internal long period waves along the Sanriku coast.
As mentioned above, the intermittency of the internal tidal waves in Toni Bay was attributed to the propagation of the internal long period waves accompanied by the vertical displacement of the thermocline. The internal long period waves were independent of the internal tidal waves in their mechanism of generation and propagation. The superposition of these two kinds of waves created a
Fig. 14. Time series record and vertical distributions of sea water temperature. a) Time series record of the sea water temperature at Sta. C bottom (Oct. 1-7, 1984). Arrow corresponds to the observation time of vertical distribution of sea water in c). b) Vertical distribution of the sea water temperature at Sta. C (Oct. 1, 1984). c) Vertical distribution of the sea water temperature in Osaki line (Oct. 5, 1984). d) Vertical distribution of the sea water temperature at Sta. C (Oct. 8, 1984).
new phenomenon (the intermittency of the internal tidal waves) in the bay.
The wave heights of the internal tidal waves and the internal long period waves
From two kinds of records of water temperature in Fig. 14, wave heights of the internal tidal waves ($H_T$) and the internal long period waves ($H_L$) were estimated. From the continual record of the sea water temperature (Fig. 14a) in the bottom layer (58 m deep) at Sta.C, the sea water temperature fluctuated semidiurnally between 19 and 13°C on 1 October. As the magnitude of the semidiurnal fluctuation is related to the height of the internal tidal wave ($H_T$), $H_T$ can be estimated when the vertical distribution of the sea water temperature is simultaneously obtained. Fig. 14b is the vertical distribution of the sea water temperature at Sta.C in the afternoon on 1 October and shows a very conspicuous thermocline in the bay. The isotherm of 19°C was 30 m deep and that of 13°C was 60 m deep, and the difference of these depths was about 30 m. From (a) and (b) of Fig. 14, the wave height of the internal tidal wave ($H_T$) was estimated to be about 30 m (on 1 October).
On the other hand, the continual record of sea water temperature in Fig. 14a shows a fluctuationless period of water temperature higher than 18°C at the bottom in several days after 5 October. This shows that the warmer sea water in surface layer extended all layers and the thermal stratification of sea water was weak in the bay (Fig. 14d). The thermocline existed in the bay on 1-4 October and then it sank deeper and disappeared in the bay on 5-8 October (Fig. 14a, d). The vertical distribution of offshore water temperature (in the Osaki line) was obtained in the morning of 5 October and the sea water of 18°C was distributed from the sea surface to about 60 m deep (Fig. 14c), and the corresponding sea water temperature at 58 m deep (the bottom layer) in the bay was about 18°C (indicated by the arrow in Fig. 14a). The sea water of 13°C found at about 60 m deep in the bay on 1 October (Fig. 14a, b) descended about 40 m from 60 m deep to 100 m deep on 5 October (Fig. 14c). This vertical movement of the isotherm probably corresponded to the vertical displacement of the thermocline by the superposition of the internal tidal wave ($H_T$) and the internal long period wave ($H_L$). Therefore, the wave height of the internal long period wave ($H_L$) of about 10 m was obtained as the difference of 40 m ($H_T + H_L$) and 30 m ($H_T$). In addition, the sea water temperature of 18°C in the morning of 5 October was not the maximum temperature of the sea water yet, but the maximum temperature (about 19°C) was found on 7-8 October (Fig. 14a). This meant that, for example, the maximum depth of the isotherms on 7 October in phase with the deepest thermocline might be found about 10 m deeper than that on 5 October (Fig. 14a, c). Accordingly, the vertical displacement of waves ($H_L + H_T$) of about 50 m corresponded to the height of the internal long period wave ($H_L$) of about 20 m. Ultimately, the height of the internal long period wave in the beginning of October 1984 was estimated about 20 m in the Sanriku coastal seas.
The internal long period waves and the behavior of offshore front at sea surface
Considering the characteristic ocean structures off Sanriku containing the Tsugaru Warm Current (coastal boundary current), the internal long period waves are very possibly the internal coastal boundary wave.
In some satellite images, there is often a solitary wavelike behavior of a clear front at the sea surface between the Tsugaru Warm Water (TWW) and the Oyashio Cold Water (OCW) off the Sanriku coast. In autumn 1984, TWW spread over the eastern sea area of the Tsugaru Strait. For example, it was found in satellite picture images that the offshore convex-shaped front moved southward with a phase speed of about 16 cm/sec (Fig. 15, YASUDA et al., 1987). This propagation speed of the offshore convex-shaped front was quite similar to the propagation speed (19 cm/sec) of the internal long period waves described above and this suggested some relationships between them. The offshore front between TWW and OCW extended the thermocline in the coastal sea area, and the thermocline became deeper
approaching the coast (Fig. 14c). From this relation between them, the vertical displacement of the thermocline nearshore was possibly related to the offshore convex-shaped front.
There are many examples of the coastal boundary currents with the sea surface front and those flow generally along the coast on the right-hand side (in the northern hemisphere) in the world ocean. The wavelike behavior of those oceanic fronts is clearly seen in recent picture images of satellites.
Theoretical research on the wavelike behavior of oceanic fronts has been activated in the 1980's.
The semigeostrophic coastal current has two waves in a reduced gravity model in which a little lighter sea water (of uniform potential vorticity) in the upper layer flows along the coast as quasi-geostrophic current over a heavier sea water (of no flow) in the lower layer which has a thickness much larger than that of the upper layer (KUBOKAWA and HANAWA, 1984; KUBOKAWA, 1986). The first wave is a Semigeostrophic Coastal Wave (SCW, Kelvin wave type) and is characterized by the variation in the depth of the upper layer near the coast without change in the breadth of the current. The second wave is a Semigeostrophic Frontal Wave (SFW) characterized by the variation in the breadth of the current and propagates upstream. This was a very simple model neglecting the flow in the lower layer and the sea bottom configuration.
Following these papers, KUBOKAWA (1987, 1988a, b) studied a two layer model with a finite depth of the lower layer, taking into account the effects of shallow depth nearshore and the flow in the lower layer. When there is a flow in the upper layer and the mean flow is zero in the lower layer, a pycnocline between the two layers inclines and a vorticity gradient arises in the lower layer, and the wave of vorticity mode appears in the lower layer. When the wave of vorticity mode in the lower layer couples with SFW in the upper layer, the baroclinic instability is generated and the disturbance of the density front propagates at the speed of the wave of vorticity mode in the lower layer. This disturbance of the front from the baroclinic instability exists when the basic current in the upper layer is weak near the coast or the breadth of the current is large. These conditions were satisfied in the Sanriku sea area in autumn 1984. Furthermore, KUBOKAWA showed that it was possible to grow for small amplitudes of the initial disturbance of the front by coupling the waves of two types in the upper and lower layers as mentioned above, if its initial amplitude was larger than the critical amplitude, even though the basic flow was linearly stable in the upper layer or the basic flow was not small near the coast.
This process suggests some possibility that the frontal disturbance propagates southward along the Sanriku coast with a phase speed of shelf waves, after warmer sea water spread in the eastern sea area of the Tsugaru Strait. The frontal disturbance traveling southward
Fig. 16. Schematic diagram of circulation of cold sea water in the lower layer and growing solitary disturbance (wavelike front). The shadow indicates the warm water, and the solid lines represent the stream lines in the lower layer. This indicates that the cold sea water in the lower layer flows ashore at downstream side (southward) of wavelike front and the pycnocline becomes shallower (by KUBOKAWA, 1988b).
is accompanied by clockwise circulation in the lower layer at the downstream side (southward) of the peak of the frontal disturbance (the convex-shaped front) and counterclockwise circulation in the lower layer at the upstream side (northward) of the peak (Fig. 16). For this reason, the sea water in the lower layer tends to flow ashore and then the pycnocline becomes shallower in the southern coastal sea area of the peak of the frontal disturbance. Therefore, when the frontal disturbance approaches the northeastern sea area of Toni Bay, the pycnocline (thermocline) becomes shallower in the coastal sea area near Toni Bay (KUBOKAWA, 1987, 1988a, b).
It was speculated that the internal long period waves were related to the behavior of the frontal disturbance offshore of Sanriku. It is important that some evidence for the existence and the propagation of the internal long period waves was obtained from the observation record in the coastal seas and was supported by a theoretical model study.
2) Propagation of the internal tidal waves into shallow bay
a) Phase speed and direction in propagation of the internal tidal waves
The propagation speed of the internal tidal waves was in the range of 35–50 cm/sec from the phase lag in sea water temperature records at the bottom between Sta.C and Sta.A and the distance between them (Fig. 17). If the median value, 42 cm/sec, was a typical propagation speed, the wave length of the semidiurnal internal tidal wave (L) was estimated about 20 km (16–23 km, L = phase velocity × period (12.5 hours)). Those waves traveled from the northeast as estimated by the cross correlations of velocity components (U) at Stations B, C, D in Toni Bay (Fig. 1). MATSUNO (1989) reported that the propagating direction of the internal tidal wave in his observation was from the east in the Joban coastal seas. This difference in the direction
Fig. 18. Records of sea surface tide and the internal tide (sea water temperature) (Oct. 1-31, 1984). D, term of diurnal period fluctuation. SD, term of semidiurnal period fluctuation. a) Sea surface tide at Kamaishi Bay. b) Sea water temperature at Sta. C bottom in Toni Bay.
of propagation might be due to the offshore bottom configuration as a source area of the internal tidal wave.
The relation of the surface tide and the internal tidal wave
The period type is shown as D or SD in Fig. 18; "the day of diurnal period type (D)" was defined as the day when one in two peaks of fluctuation in a day was less than half of the other, and "the day of semidiurnal period type (SD)" was defined as two peaks of fluctuation in a day were comparable in amplitude. The surface tide was compared with the internal tide (subsurface water temperature); the internal tide had a semidiurnal period but the surface tide had a diurnal period for 1-3 October. Both had a semidiurnal period for 9-12 October, and the surface tide had a diurnal period but the internal one had a semidiurnal period for 13-15 October. For 16-21 October, both of them had a diurnal period. The surface tide was different in the period type from the internal tide for 22-24 October. INABA (1981) reported that in Suruga Bay the internal tide (tidal current) was predominant in the diurnal fluctuation from the observations of currents in the subsurface layer but the surface tide was prevailing in the semidiurnal fluctuation. The relation between them in Toni Bay was more complicated than that in Suruga Bay.
The surface tide already had a diurnal period on 1 October and the beginning day of a diurnal period was unknown, but the internal tide changed to the diurnal period on 4 October. The surface tide changed to the diurnal period on 13 October, and the internal one changed on 16 October. The beginning day of the diurnal period in the internal tide seemed to be 3-4 days behind that in the surface tide. If the internal tidal waves were generated far offshore by obtaining energy from the surface tide, the internal tidal waves with lower propagation speed than that of the surface tide must arrive at the coast behind the surface tide and both of the period types did not coincide at the coast. If the delay time of the internal tidal wave was 3-4 days and its propagation speed was 42 cm/sec, the generation area of the internal tidal wave was estimated at about 110-150 km offshore. The northeast sea area at 110-150 km from Toni Bay was the edge zone where the sea bottom configuration changed from the contiFig. 19. Comparison of the dispersion relations of the internal tidal waves in Toni Bay with those offshore. a) Comparison of the dispersion relations; thick line, 1st mode; broken line, 2nd mode; thin line, wave length range at semidiurnal period. b) Vertical distribution of specific volume anomaly (cl/ton) of sea water at Sta. OSIV offshore. c) Comparison of vertical distributions of the vertical velocity at Sta. 8 with those at Sta. OSIV. Abscissa, ratio to maximum velocity.
The internal tidal waves are able to propagate into a shallow bay only in the case of a thermocline formed initially in a bay. Fig. 19a shows the dispersion relations of the internal waves calculated from the vertical distribution of sea water density observed at stations inside and outside of Toni Bay. The dispersion relations of the internal wave at Sta.8 and Sta.10 were calculated as the representative data in the bay, and Sta.OSIV (930 m depth, 36 km offshore) was a representative offshore station and the vertical distribution of the specific volume anomaly of sea water at Sta.OSIV was shown in Fig. 19b. If the period was semidiurnal, the wave length of the 1st mode at Sta.OSIV was 35-40 km, and this was inconsistent with the estimated wave length (about 20 km) from observations in Toni Bay (Fig. 19a). The dispersion relation of the 2nd mode at offshore Sta.OSIV was very similar to that of the 1st mode at Sta.8 and Sta.10 in the bay. In this semidiurnal internal wave, the wave length was 16-23km from the estimated speed of 35-50 cm/sec from observations in the bay (thin lines in Fig. 19a show this range). This wave length (16-23km) was easy to resonate with the length of Toni Bay (about 5 km; length of area deeper than 20 m depth). The properties of the 1st mode of the internal tidal waves in the bay were nearly equal to those of the 2nd mode of the internal tidal waves outside of the bay, and the vertical distribution of vertical velocity (W, cm/sec) was also similar in the internal waves inside and outside of the bay (Fig. 19c).
In conclusion, these results showed that the 2nd mode of the internal tidal wave offshore was well related to the 1st mode of that in Toni Bay.
Comparison of propagation ratio of the energy of the internal wave into shallow bay
The probability that the 1st or the 2nd mode of the internal waves offshore propagates into a bay was studied by analytical calculation in a simple model in which the depth in the bay was 50% of the depth outside of the bay and the basic condition of the vertical distribution of sea water density ($\rho$) outside of the bay was a constant Brunt-Väisälä Frequency, $N^2 = -\frac{g}{\rho} \times (\frac{\partial \rho}{\partial z})$ (CRAIG, 1987). The left in Fig. 20a is the case of initially given the 1st mode of the internal wave outside of the bay, and the density of stream lines indicates the energy
Fig. 20. Transport efficiency of the internal wave energy propagating into the shallow bay from the offshore sea. Solid lines represent stream lines. a) The case that the incident wave is the 1st mode of the internal wave. b) The case that the incident wave is the 2nd mode of the internal wave.
The right in Fig. 20a is the distribution of stream lines after the internal wave propagated into a shallow bay, and it shows that the wave energy transported into the bay is 25% of the offshore incident wave energy. The case of initially given the 2nd mode of the internal wave outside of the bay is shown at left in Fig. 20b. In this case, it is indicated that 39% of the wave energy of the 2nd mode of the offshore incident wave propagate into the bay. It is sure that more energy of the 2nd mode of the offshore internal wave propagates into the bay than the energy of the 1st mode of that wave.
Shoaling up of the internal tidal waves into the bay
As mentioned in chapter 3, the record of the internal tidal waves as fluctuations of the sea water temperature in the bottom layer suggested that there were two cases for the propagation of the internal tidal waves into a bay. The first was the general case of the internal wave, and the internal tidal waves propagated through the thermocline which was already formed in the bay. The second was the case of no thermocline initially in the bay and the thermocline was formed after colder sea water flowed into the bottom layer of the bay. In the second case, therefore, the thermocline in the bay disappeared in every cycle of the tidal period.
Semidiurnal periods for 1-3 and 9-15 October corresponded to the second case, because the sea water temperature in the bottom layer rose to near the sea surface water temperature in every tidal cycle (Fig. 6). After the colder sea water in the bottom flowed out of the bay, the thermocline disappeared in the bay. If the thermocline remained always in the bay and the internal tidal wave was dominant, a thermometer (contained in the current meter) in the bottom layer stayed always in the colder sea water under the thermocline (the general case), and the sea water temperature in the bottom layer should remain at 15-16°C (Fig. 6). However, the sea water temperature in the bottom layer was found at 19°C, near that in the surface layer (about 20°C) in the record. In particular, for 9-15 October, the maximum sea water temperature in the bottom layer agreed almost with that in the surface layer. This suggested that "a colder sea water front" shoaled up on the bottom of the bay every semidiurnal tidal cycle.
The raw records of velocity (eastward component (U), in the bottom layer at Sta.
Fig. 22. Schematic pictures of shoaling up of the colder sea water front, and the corresponding figure of variations of velocity and sea water temperature at the bottom in the bay. a) Schematic pictures of the moving cold front: (1) cold front starts to flow into bay, (2) cold front passes the station into bay, (3) cold front stops at head of bay, (4) cold front flows out of bay, (5) cold front passes the station out of bay, (6) cold front stops out of bay. b) Schematic figure of variations of the velocity component and sea water temperature at the bottom in the bay. (1)–(6) correspond to (1)–(6) in figure (a).
A) and the sea water temperature (in bottom layer at Stations A, B, C) for 1–4 October are shown in Fig. 21. Fluctuation curves of the sea water temperature are not a sinusoidal pattern but have a peculiar pattern. On 1 October, the sea water temperature fell and rose suddenly at all stations and they were different from a sinusoidal wave. If it was the general case (the first case), the changes of the sea water temperature and that of direction of velocity (U) must occur simultaneously in the time when the thermocline passed the station of the thermometer (as shown by arrows 1 in Fig. 21). But, Fig. 21 showed that the sea water temperature changed at a different time from the change in the direction of velocity (as shown by arrows 2 in Fig. 21). At Sta.A, at 14:00 on 1 October when the flow changed suddenly from outward (U>0) to inward (U<0), the sea water temperature changed simultaneously to colder suddenly (arrows 1), but at 19:00 when the flow changed to outward, the sea water temperature was steady (arrows 2), and when the speed of the flow reached positive maximum, the sea water temperature began to be warmer (arrows 3).
The time interval of the minimum temperature on 1 October in the bay mouth (Sta.C) was longer than that in the bay head (Sta.A). This suggested that the flow in the bottom of the bay was not the internal wave case with steady thermocline in the bay, but was the shoaling up and down of the colder sea water front on the bottom of the bay.
The process of the shoaling up and down of the colder sea water front in the bay will be inferred in the schematic picture (Fig. 22).
At the beginning, the colder sea water front starts shoaling up from the bay mouth into the bay which is filled with warmer sea water only, and the flow at a station of the thermometer is outward (U>0), (1) in Fig. 22. When the colder front passes the station toward the bay head, the direction of velocity changes to flow inward (U<0) and the sea water temperature falls abruptly to the minimum, (2). The colder sea water front keeps
on flowing inward and then stops at the bay head, (3). When the internal tide starts to ebb, the colder front at the bay head starts to shoal down and the direction of the flow at the station changes to outward of the bay \((U > 0)\), but the sea water temperature remains at almost the minimum, (4). When the colder front passes the station toward the bay mouth, the direction of velocity changes inward \((U < 0)\) and the sea water temperature rises suddenly up to the maximum, (5). Afterward, the colder front continues to flow outward and stops outside of the bay, (6). At this time, there is only the warmer water in the bay. Those variation patterns were similar to the variation patterns of the observed record of 1-2 October (Fig. 21).
In the case of the semidiurnal fluctuations in the beginning of October, it was concluded that the occurrence of the stratified structure in Toni Bay was temporal and cyclic, and the propagation of the semidiurnal internal tidal wave into the bay must be the shoaling up of the cold sea water front.
CAIRNS (1967) reported on the internal tidal bore in the shallow sea off south California, from the variation pattern of the water temperature observed by thermister arrays. His variation pattern was similar to the shoaling up in Toni Bay, and the shoaling phenomenon seemed to be considerably important in the exchange of the sea water in a shallow bay.
5. Summary
A study of the internal tidal waves in a small bay was carried out on the basis of the observed records in Toni Bay, autumn 1984.
1. The internal tidal waves in the bay were predominant and had the characteristics of intermittent occurrence in several days. From T-S diagram analyses of the sea water property at stations inside and outside of the bay, the property of the sea water in the bottom layer was closely related to that of offshore sea water in the layer (70-100 m deep, this was the depth under a thermocline) a little deeper than the bottom layer in the bay. It was found that the intermittency of the internal tidal waves was related to the vertical displacement of the thermocline outside of the bay, namely the internal tidal waves appeared in the bay predominantly when the thermocline became shallower.
2. The cause of the intermittent vertical displacement of the thermocline was investigated through the analyses of (a) variations in subsurface water temperature and (b) variations of the local difference of tidal deviations (sea surface level anomaly) at three stations along the Sanriku coast.
a) The cross correlation of variations of the sea water temperature in the surface layer between in Toni Bay and in Otsuchi Bay was very high with a correlation coefficient \((\alpha)\) of 0.89, and the phase of the variation in Otsuchi Bay was in advance of that in Toni Bay. It was found that the variation of the sea water temperature (variation of the thermocline depth) propagated southward at a speed of about 19 cm/sec. This was considerably lower than the wave speed of a coastal trapped wave (internal Kelvin wave, more than 30 cm/sec). The variation of the thermocline depth seemed to be due to a kind of the internal long period wave propagating southward alongshore.
This propagation speed was similar to the southward propagation speed (16 cm/sec) of the pycnocline front with a convex shape at the offshore edge of the Tsugaru Warm Waters from satellite images. It was suggested that the internal long period waves were related to the behavior of the pycnocline front. Recent theoretical research showed that the frontal disturbance propagated as a coupled wave of Semigeostrophic Frontal Wave in the upper layer (KUBOKAWA and HANAWA, 1984) with the wave to be restored by potential vorticity in the lower layer. This frontal disturbance travels southward accompanied by clockwise circulation in the lower layer at the downstream side of the peak of the front (KUBOKAWA, 1987, 1988a, b), where the sea water in the lower layer tends to flow onshore and the thermocline becomes shallower. These features and the lower propagation speed of the frontal distribution were in good agreement qualitatively with those of the internal long period waves.
b) When the internal long waves propagate along the coast, it is expected that the sea surface level changes out of phase to the depth of the thermocline, even if it is very small. When the sea level at Kamaishi minus that at Miyako ($\Delta \xi_{KM}$) was negative, the thermocline off Toni Bay (near Kamaishi Bay) must become shallower. Accordingly, the cold sea water offshore was able to intrude into the bottom of the bay, and the duration of negative $\Delta \xi_{KM}$ corresponded to that of the predominant internal tide in the bay near Kamaishi. $\Delta \xi_{KM}$ was highly correlated with the variation of the sea water temperature in the bottom layer of Toni Bay; the correlation coefficient ($\alpha$) was 0.68. Another local difference ($\Delta \xi_{OK}$) between Ofunato and Kamaishi was out of phase with $\Delta \xi_{KM}$, which supported also the alongshore propagation of the internal long period waves. The results indicated the existence of the internal long period waves propagating alongshore with a suitable space scale to their time scale, because the distance between Miyako and Kamaishi (45 km) was nearly a half wave length of the wave with time scale of several days and wave speed of 19 cm/sec.
The internal long period waves were independent of the internal tidal waves in their generation and propagation. The internal tidal waves appearing intermittently in the bay occurred from the superposition of the internal tidal waves propagating onshore and the internal long period waves propagating alongshore.
3. The propagation speed of the internal tidal waves was estimated about 42 cm/sec (35-50 cm/sec) from the observations. The source area of the internal tidal waves was roughly estimated at 110-150 km northeast from Toni Bay. The dispersion relation of the 1st mode of the internal waves in the bay was similar to that of the 2nd mode offshore. Moreover, the vertical distribution of the vertical velocity of the 1st mode of the internal waves in the bay was quite similar to that of the 2nd mode offshore. From the dispersion relations, the phase speed of the semidiurnal internal tidal waves was calculated about 40 cm/sec which was consistent with the phase speed estimated from the time lag of observed records mentioned above.
The energy flux transported into the bay due to the 2nd mode of the offshore internal wave was 1.6 times greater than that due to the 1st mode of the wave in an analytical calculation in a simple model. The semidiurnal internal waves in Toni Bay were closely related to the 2nd mode of the internal waves offshore.
In addition, it was suggested that the semidiurnal internal tidal waves shoaled up with the intrusion of a cold sea water front from offshore into the bottom layer of the bay.
Acknowledgements
The author would like to express his hearty thanks to Prof. Hideaki KUNISHI and Prof. Norihisa IMASATO, Kyoto University, for their valuable advice and comments on the present study. The author wishes to express his sincere thanks to Dr. Hideki NAGASHIMA, a colleague in the laboratory, for his continuous collaboration and helpful advice during the present study, and to Dr. Sanae UNOKI, Chief of the laboratory (presently Tokai University), for his encouragement. The author is also grateful to Mr. Sachio NAGAHORA and Mr. Kyoichi ISHIDA, Iwate Prefecture Fishery Experimental Station, for cooperation in observation and collection of routine data, and to Dr. Atsushi KUBOKAWA, Tohoku University, for helpful suggestions on the oceanic fronts. Thanks are extended to Dr. Nobuyuki SHIKAMA, Otsuchi Marine Research Center, University of Tokyo, for offering routine data in Otsuchi Bay for processing of time series data in Toni Bay, to Mr. Yukiyasu SATOU, the computation center in Institute of Physical and Chemical Research, for his help in computer processing, and to Mrs. Hiroko INOUE for typing and drafting. This study was supported in part by the Association of the Set Net Fishery in Iwate Prefecture.
References
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KUBOKAWA, A. (1987): Instability and nonlinear evolution of a density-driven coastal current with a surface front in a two-layer ocean. Kaiyou Kagaku (Marine Science), 19, 48–52. (in Japanese)
KUBOKAWA, A. (1988a): Instability and nonlinear evolution of a density-driven coastal current with a surface front in a two-layer ocean. Geophys. Astrophys. Fluid Dyn., 40, 195–223.
KUBOKAWA, A. (1988b): Theoretical models of the variations of surface density fronts and possible application to the Tsugaru Warm Current. Bull. Tohoku Reg. Fish Res. Lab., 50, 131–136. (in Japanese)
KUBOKAWA, A. and K. HANAWA (1984): A theory of semigeostrophic gravity waves and its application to the intrusion of a density current along a coast. Part 1 Semigeostrophic gravity waves. J. Oceanogr. Soc. Japan, 40, 247–259.
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三陸沿岸における内部潮汐波と内部長周期波
岡 勝 守 良
要旨: 内湾における内部潮汐波の特性について、1984年秋季、三陸沿岸の唐丹湾において行われた観測を基に、解析を行い次の事がわかった。
i) 湾内の顕著な内部潮汐波は長期間連続的に現れるのではなく、数日程度の間欠性がみられた。湾内外の海水のT-S図解析の結果、この間欠性が水温躍層の深さの変動に関係していることが判明した。その間欠的上昇に応じて湾内底層に冷海水が流入して成層構造が形成され、内部潮汐波が間欠的に湾内に出現するものと考えられた。
ii) このような間欠的な水温躍層の深さの変動の原因を究めるため、唐丹湾近傍の2つの湾の水温変動の相関を調べ、また3つの湾の海面水位の変動と水温躍層の鉛直変動との関連を調べた。その結果、水温躍層は海岸に沿って北から南へ伝わる内部長周期波によって上昇することが示唆された。この内部長周期波は約19cm/secで南進し、約100km程度の空間規模を持っていることが示された。これは北東から岸向きに伝播する内部潮汐波とは独立な波であり、これら2つの波が重畳したときに、湾内の内部潮汐波に間欠性が現れる。この他、この内部長周期波は津軽暖水域東端の密度フロントの南への波動的伝播に関連のあることが示唆された。
iii) 湾内外の観測データから求めた内部潮汐波の分散関係や波速等の比較、及び簡単なモデルによる沖合の内部波エネルギーの湾内への伝播効率の比較等の結果、湾内の卓越した半日周期の内部潮汐波は沖合のそれの第2モードに関連の深いことが示唆された。
Density fluctuation of caprellid amphipods (Crustacea) inhabiting the red alga *Gelidium amansii* (LAMOUROUX) LAMOUROUX, with emphasis on *Caprella okadai* ARIMOTO*
Ichiro TAKEUCHI**, Hiroshi YAMAKAWA*** and Masamu FUJIWARA****
**Abstract:** Species composition and density fluctuation of caprellid amphipods inhabiting a red alga *Gelidium amansii*, which is a true-perennial alga, were investigated in a small inlet of the rocky shore facing the Pacific Ocean, at Amatsu-Kominato, Chiba Prefecture. Six caprellid species were found to be present. Among them *Caprella okadai* was the most dominant species. Its share in the caprellid fauna was more than 67% throughout the year. The density during May to August was 10 times higher than that during September to March, and mature females were found almost all the time. Several species of *Caprella* which inhabit *Sargassum* spp. have been reported to exhibit different occurrence pattern, suggesting that life forms of algae influence the occurrence of caprellids. Review of distribution of *C. okadai* reveals that this species is restricted to calmer zones of rocky shore facing the open sea.
1. **Introduction**
Caprellid amphipods, as well as gammarid amphipods, represent an important component in the ecosystem of “Garamo-ba” (*Sargassum* zone) which flourishes on rocky shores of southern Japan, as these crustaceans are the major predated forms by rocky shore fishes (FUSE, 1962; HIRAYAMA, 1978; KANAMOTO, 1977, 1979; OMORI, 1980). Ecological studies on the caprellids on the “Garamo-ba” have mostly dealt with either substrate selection (HIRAYAMA and KIKUCHI, 1980; IMADA et al., 1981; NORTON and BENSON, 1983; TAKEUCHI et al., 1987; TAKEUCHI et al., MS) or population dynamics (IMADA and KIKUCHI, 1984; AOKI, 1988). These studies, however, are usually restricted to animals collected from the *Sargassum* species which are larger algae among various kinds of algae found in “Garamo-ba”. Only HIRAYAMA and KIKUCHI (1980) and TAKEUCHI et al. (1987) reported on caprellid species compositions on several species of smaller algae on the basis of seasonal collections.
In this study, we focus on species composition and density fluctuation of the Caprellidea inhabiting the red alga *Gelidium amansii* (LAMOUROUX) LAMOUROUX, which is a true perennial alga (KATADA, 1963).
2. **Materials and Methods**
This study was conducted in the innermost part of an inlet called “Jizogi” (Fig. 1) in the vicinity of the Kominato Marine Biological Laboratory of the Tokyo University of Fisheries (KMBL) (now the Kominato Marine Laboratory, Faculty of Science, Chiba University). This inlet is situated on the southwest of Uchiura Bay facing the Pacific Ocean. Sampling was carried out from the center of a thick community of *Gelidium amansii* at the depth of 1.3 m.
Duplicate samples of *G. amansii* from a quadrat of $20 \times 20$ cm were collected by skin diving at monthly intervals from April 1978 to March 1979. Algae were gently removed and quickly placed in a vinyl bag. In the laboratory, the algae were shaken in freshwater for dislodging free-living epifauna. Caprellids were carefully sorted out and preserved in 10
Fig. 1. Maps showing the study site. A. Boso Peninsula. Marked area (arrow) indicates the location of Uchiura Bay. B. Uchiura Bay. KMBL: Kominato Marine Biological Laboratory, Tokyo University of Fisheries. C. Map of Jizogi showing the sampling point. Broken lines indicate the highest low water during the neap tide.
Fig. 2. Seasonal change of seawater temperature based on the daily observation records of the former Kominato Marine Biological Laboratory, Tokyo University of Fisheries. Each solid circle indicates the average temperature for the first, second and last 10 days in every month.
% buffered formalin. Caprellid species identification was conducted under a binocular microscope. Sex of *Caprella okadai*, the most dominant species, was determined from the development of gnathopod in males and the presence of oostegites in females. The other small individuals which did not possess the sexual characters were classified as juveniles. Furthermore, females were separated into two categories, i.e. immatures in which oostegites are without setae, and matures in which oostegites with long setae form a brood pouch. Wet weight of algae was measured to the nearest g after leaving them on papers for two hours.
Seawater temperature data based on the daily observation record of KMBL have been used in the present study. It ranged from 13.2 to 25.5°C (Fig. 2). Salinity variation was restricted to 33.1–34.0‰ during the period of
the investigation (HAYASHI, 1989).
3. Results
The wet weight of *Gelidium amansii* per 400 cm$^2$ varied from 80 to 175 g and no seasonal fluctuation was recognized.
Six species of the Caprellidae were collected. All these six species belong to the genus *Caprella*: *C. okadai* ARIMOTO, 1930, *C. simia* MAYER, 1903, *C. kominatoensis* TAKEUCHI, 1986, *C. penantis* LEACH, 1814, *C. decipiens* MAYER, 1903 and *C. generosa* ARIMOTO, 1977. The last species is the same as *Caprella* sp. III reported from Amatsu-Kominato by TAKEUCHI et al. (1987).
*C. okadai* was the most dominant species throughout the year. The share of *C. okadai* to total number of caprellids varied from 67 to 100 % (Fig. 3). Its individual density per 100 g wet weight of the alga increased rapidly from April to May and was kept high until August (> 150 ind./ 100 g wet weight) (Fig. 4). In September, it decreased to 16.2 ind./ 100 g wet weight, and such a low density (< 50 ind./ 100 g wet weight) extended up to March. The density pattern based on their abundance per 400 cm$^2$ bottom area in the quadrat sample also showed a similar projection (Fig. 4).
Juveniles, i.e. those individuals which had a body length less than 4.0 mm, constituted the bulk of the population throughout the year except in August and November (Table 1). Mature females were also collected throughout the year, except during January 1979. This indicates that *C. okadai* could mature and produce eggs all the year round in this area, where seawater temperature varied between 13 and 26°C (Fig. 2).
The second dominant species was *C. simia*. Its maximum share was only 33 % and was encountered in October 1978 (Fig. 3). In July and August, its density was relatively high (15.4 and 5.9 ind./ 100 g wet weight, respectively), but in other months it was less than 2.0 ind./ 100 g wet weight. Mature females were not found.
4. Discussion
In this study, *Caprella okadai* was found to be exclusively dominant throughout the year on the red alga *Gelidium amansii* in the inner part of the inlet (Fig. 3). Although we did not measure the length of algae, it is estimated to range from ca. 15 to 20 cm throughout the year. TAKEUCHI et al. (1987) report-
Fig. 4. Seasonal fluctuation in the density of *Caprella okadai* ARIMOTO and *C. simia* MAYER. Solid circle: the number of individuals per 100 g wet weight of *Gelidium amansii* (LAMOUROUX) LAMOUROUX. Solid triangle: the number of individuals per 400 cm$^2$ of bottom area.
Table 1. Seasonal variations in proportion (%) of juveniles, males, immature and mature females to the total individuals and sex ratio (males/females) of *Caprella okadai* ARIMOTO.
| | 1978 | 1979 |
|----------------|---------------|---------------|
| | 12 Apr. 18 May 11 June 7 July 21 Aug. 12 Sept. 19 Oct. 16 Nov. 19 Dec. 24 Jan. 28 Feb. 17 Mar. | |
| Juveniles | 76.4 84.8 67.1 51.3 30.5 47.1 50.0 16.7 60.0 52.2 55.2 46.2 | |
| Males | 11.8 5.7 14.0 17.6 28.6 26.4 25.0 41.7 25.7 21.7 15.2 28.8 | |
| Females | | |
| immatures | 10.0 4.9 10.2 20.3 17.9 14.7 0.0 33.3 2.9 26.1 18.4 17.3 | |
| matures | 1.8 4.6 8.7 10.8 23.0 11.8 25.0 8.3 11.4 0.0 11.2 7.7 | |
| Sex ratio | 1.00 0.60 0.74 0.57 0.70 1.00 1.00 1.00 1.80 0.83 0.51 1.15 | |
Juveniles 1.5 - 4.0mm, males 4.0 - 8.5mm, immature females 3.5 - 6.0mm and mature females 4.5 - 8.0mm in body length.
ed that *C. okadai* was the dominant species on such large algae (>10cm) as *G. amansii*, *Cladophora wrightiana* HARVEY and *Sargassum piliferum* (TURNER) C. AGARDH, which were growing at the zones protected from wave exposure in the inlet "Heito". This inlet is located to the north of "Jizogi" (Fig. 1). The present results support their conclusion.
Year-round breeding in different species of *Caprella*, similar to the present observation on *C. okadai*, has been reported in studies carried out in the temperate region; *C. penantis* (BYNUM, 1978; CAINE, 1983), *C. laeviuscula* MAYER (CAINE, 1979), *C. californica* STIMPSON (KEITH, 1971), *C. equilibra* SAY (KEITH, 1971), *C. gorgonia* LAUBITZ and LEWBEL (LEWBEL, 1978), *C. tsugarensis* UTINOMI (IMADA and KIKUCHI, 1984), *C. decipiens* (IMADA and KIKUCHI, 1984) and *C. verrucosa* BOECK (AOKI, 1988).
The occurrence of caprellids is influenced by the life forms of algae with which caprellids are associated. In this study, *C. okadai*
was collected throughout the year, and the high density was recorded during May to August. However, the occurrence of *C. danilevskii* CZERNIAVSKI, *C. tsugarensis* and *C. decipiens*, which were associated with the brown alga *Sargassum horneri* (TURNER) C. AGARDH, was restricted from fall to early summer (IMADA and KIKUCHI, 1984). Similar seasonal fluctuation was also observed in the case of *C. verrucosa* on *S. patens* C. AGARDH (AOKI, 1988). *S. horneri* is an annual alga (TERAWAKI et al., 1983b; TERAWAKI, 1986), and the entire thalli flowed away during spring to early summer (IMADA and KIKUCHI, 1984). The larger parts of *S. patens* thalli, which is a stem-survived perennial species (KATADA, 1963; TERAWAKI et al., 1983a), also flowed away during late spring to early summer, leaving behind short round stems only (AOKI, 1988). TAKEUCHI et al. (1987) reported that *C. okadai*, *C. danilevskii*, *C. tsugarensis* and *C. decipiens* were mostly collected from large algae (> 10 cm) which possess filamentous or long thalli. All these four species of *Caprella* are typically similar in having an elongate body with a sharp and short basis of gnathopod II. They observed that these caprellids, except *C. decipiens*, clung the substrata by gnathopod I and pereopods V–VII with body straightened. This reveals a possibility that morphological characteristics of caprellids might have adapted to the feature of algae. Hence, the presence of suitable feature of algae for clinging is one of the most important factors which have influence on the occurrence of caprellid amphipods. Therefore, these species of *Caprella* associated with *S. horneri* and *S. patens* could not be collected during the summer.
This conclusion is also supported by the result of a comparative study on populations of *Caprella penantis* collected from two sites along the Atlantic Ocean of North Carolina (BYNUM, 1978). He compared the population structure of this caprellid from the estuarine site where hydroids and bryozoans were the most common substrate and that from the coastal site where red and green algae predominated. Population at each site peaked during July. During late August, the caprellid density at estuarine site rapidly decreased with disappearance of *Tubularia crocea*, a common hydroid, while the high density continued to prevail at the coastal site.
*Caprella okadai* was reported first from Tateyama, Chiba Prefecture (ARIMOTO, 1930, 1976) and then from Tomioka, Amakusa-Shimoshima Island, Kumamoto Prefecture (IMADA et al., 1981) and Amatsu-Kominato (TAKEUCHI et al., 1987). Besides these records, one of the authors (IT) has also found it on several occasions in coastal waters of Japan, i.e. Nabeta Bay of Izu Peninsula, Shizuoka Prefecture, Unose on Amakusa-Shimoshima Island and Magari on Notojima Island, Ishikawa Prefecture. In all these instances, *C. okadai* was collected from the rocky shore facing the ocean influenced by either the Kuroshio Current or Tsushima Current. Since this species of *Caprella* has never been found in eutrophic bays such as the Seto Inland Sea, Ise Bay, Tokyo Bay, etc. until now, it can be presumed that the distribution of *C. okadai* is restricted to the areas protected from wave exposure of rocky shore facing the open sea.
**Acknowledgments**
We are grateful to Prof. J.-S HO, California State University, Long Beach and Dr. A. C. ANIL, The University of Tokyo, for careful reading and useful comments of the manuscript, Dr. Y. NAITO, Kominato Marine Biological Laboratory, Tokyo University of Fisheries (now the National Institute of Polar Research, Tokyo), for use of the facilities, and Prof. R. HIRANO of The University of Tokyo for advice and encouragement. The senior author (IT) wishes to thank the following persons for cooperation of collecting *Caprella okadai*; Dr. Y. YOKOHAMA, Shimoda Marine Research Center of Tsukuba University, Dr. T. KIKUCHI and Mr. M. AOKI, Amakusa Marine Biological Laboratory of Kyushu University, and Mr. S. ISHIMARU, Rokusei High School.
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TAKEUCHI, I., R. KUWABARA, R. HIRANO and H. YAMAKAWA 1987. Species compositions of the Caprellidea (Crustacea: Amphipoda) on the *Sargassum* zone of the Pacific coast of Japan. Bull. Mar. Sci. 41: 253–267.
TAKEUCHI, I., H. YAMAKAWA and M. FUJIWARA (MS). The Caprellidea (Crustacea: Amphipoda) on the green alga *Cladophora*
マクサ上のワレカラ類，特にオカダワレカラの密度の周年変動
竹内一郎・山川 紘・藤原正夢
要旨: 千葉県天津小湊町地先のガラモ場にて、真多年生海藻であるマクサに棲息するワレカラ類の種組成および密度の周年変動を調査した。オカダワレカラは、一年中、全個体の67%以上を占める優占種であり、密度は5月から8月に特に高く他の季節より10倍以上高い値を示した。成熟個体がほぼ一年中採集されたことから、繁殖期が周年にわたるものと推察された。従来のガラモ場のワレカラ類の研究によると、一年生あるいは基部多年生海藻であるホンダワラ類の上では、ホソワレカラなどの出現は秋から初夏に限られることが報告されている。これらのことから、海藻の生活形がワレカラ類の出現に影響を及ぼすと考えられる。また、オカダワレカラの分布に関する知見を整理すると、本種の棲息域は外洋に面した岩礁域の波浪の影響の少ないところに限られるのではないかと推察される。
Seasonal changes of the Secchi disc depth and suspended solid at six stations along the main channel of the High Dam Lake, Egypt*
OLFAT Anwar Habib**, IBRAHIM Omar Mohamed**,
MOHAMED Shehata Mohamed** and Yusho ARUGA***
Abstract: Seasonal changes of the Secchi disc depth, suspended solid, chlorophyll $a$, ignition loss and particulate organic matter were investigated at six stations (Stns. 1-6) along the main channel of the High Dam Lake in Egypt during the period from September 1986 to December 1988. Water samples were collected from the surface and 2 m layer at each station and analyzed. Patterns of seasonal changes of the parameters and the ratios of chlorophyll $a$ to suspended solid and to particulate organic matter were similar mostly among Stns. 1-3 in the northern part and among Stns. 4-6 in the southern part of the lake. The Secchi disc depth was significantly correlated with suspended solid in a hyperbolic manner when the two parameters were plotted on linear scales. It is shown that both the ratios of chlorophyll $a$ to suspended solid and to particulate organic matter are regionally and seasonally quite variable.
1. Introduction
The High Dam Lake is one of the largest artificial lakes in Africa. It was filled in 1960 after the construction of Aswan High Dam (ENTZ, 1974). In order to know the environmental conditions of the High Dam Lake, the Fishery Management Center (FMC) of the High Dam Lake Development Authority, Egypt, set up stations in the main channel and in Khor El Ramla in 1982, and has been conducting monthly field surveys. The distributions of chlorophyll $a$ in Khor El Ramla and adjacent water in the main channel were partly reported in a previous paper (OLFAT et al., 1987). The results obtained in the main channel were reported by OLFAT and ARUGA (1988). In these reports the relationships between chlorophyll $a$ and Secchi disc depth were presented with rather scattered data, suggesting that the proportion of chlorophyll $a$ in suspended solid might be variable presumably according to the seasons and regions of the lake.
The present paper describes the seasonal changes of the Secchi disc depth and suspended solid at six stations along the main channel of the High Dam Lake in relation to chlorophyll $a$ concentration and ignition loss, mainly focusing on the relationship between the Secchi disc depth and the suspended solid.
2. Material and methods
Water samples were collected from the surface and 2 m depth with a Van Dorn type water sampler at six stations (Stns. 1-6) from September 1986 to December 1988 (Fig. 1). Each water sample was filtered through a glass fiber filter (Whatman GF/C, 47 mm) which was precombusted at 450°C for 3 hrs in a Muffle furnace and weighed, and the filter with suspended solid on it was weighed after drying overnight (or for 24 hrs) in a drying oven at 85°C. The amount of suspended solid (SS) was determined as the difference of dry weights before and after filtration of water sample. The filter was ignited again in a Muffle furnace at 450°C for 3 hrs and weighed after cooling, and the percent decrease of dry weight was regarded as the ignition loss.
The amount of particulate organic matter (POM) was calculated by multiplying SS with IL.
The transparency of water was measured with a Secchi disc (30 cm in diameter) at each station. The chlorophyll $a$ concentration was determined in the same way as described in previous papers (OLFAT et al., 1987; OLFAT and ARUGA, 1988).
Fig. 1. Map of the High Dam Lake showing the locations of stations (1-6).
3. Results
The Secchi disc depth was highest, 5.7 m, in February 1987 at Stn. 4 and lowest, 0.2 m, in September 1988 at Stn. 6. Seasonal changes of the Secchi disc depth at Stns. 1-6 are illustrated in Fig. 2. In average, the Secchi disc depth was highest in February and lowest in August. It is noticeable that differences of the readings at six stations were small from April to June but large from August to March. Among the six stations, the Secchi disc depth was mostly lowest at Stn. 6, whereas at Stn. 1 it was highest except in January-March 1987 and February 1988. This suggests the decrease of suspended solid with flowing water along the main channel. Stns. 1 and 2 showed similar seasonal changes of the Secchi disc depth, and Stns. 5 and 6 also showed similar changes. Stn. 3 showed quite similar changes to the average of the six stations. Seasonal pattern at Stn. 4 was different from those at other stations in having big differences between the maximum and the minimum values.
Seasonal changes of the amount of suspended solid in the surface water and 2 m layer at Stns. 1-6 are illustrated in Fig. 3. The level of suspended solid was generally higher (1.0 - 59.0 g/m$^3$) at Stns. 4-6 in the southern part and lower (0.5 - 10.0 g/m$^3$) at Stns. 1-3 in the northern part of the lake. The patterns of seasonal changes of suspended solid were similar among Stns. 1-3 and among Stns. 4-6, but the patterns at the latter stations were...
Fig. 3. Seasonal changes of the suspended solid at Stns. 1-6 in the main channel of the High Dam Lake. Lines are for averages of the surface (○) and 2 m (●) samples.
Fig. 4. Seasonal changes of the chlorophyll $a$ at Stns. 1–6 in the main channel of the High Dam Lake. Lines are for averages of the surface (○) and 2 m (●) samples.
clearly different from those at the former stations. The range of variations was quite big at Stns. 4–6 as compared with that at Stns. 1–3. Rapid increases in suspended solid were observed in April and May at Stns. 1–3 in 1987 and 1988, whereas at Stns. 4–6 rapid increases were observed in August and September in 1987 and 1988. The suspended solid was clearly high during the flood season (August–October) at Stns. 4–6.
Figure 4 illustrates seasonal changes of chlorophyll $a$ concentration in the surface water and 2 m layer at Stns. 1–6. Average chlorophyll $a$ concentrations were slightly higher at Stns. 4–6 (1–26 mg/m$^3$) than at Stns. 1–3 (1–21 mg/m$^3$), if the higher values of 28 mg/m$^3$ at Stn. 2 in April 1988 and 30 mg/m$^3$ at Stn. 3 in November 1986 were excluded. The seasonal patterns of chlorophyll $a$ concentration were similar among Stns. 1–3 and among Stns. 4–6, even though the patterns at Stns. 1 and 6 were somewhat obscure as compared with those at other stations. The patterns at Stns. 1–3 were different from those at Stns. 4–6. The range of seasonal variations of chlorophyll $a$ concentration was big at Stns. 4–6 as compared with that at Stns. 1–3. The patterns of seasonal changes of chlorophyll $a$ concentration (Fig. 4) were different from those of the suspended solid (Fig. 3) at each station, even though the corresponding peaks were sometimes observed.
Figure 5 shows seasonal changes of the ignition loss of suspended solid in the surface water and 2 m layer at Stns. 1–6. The ignition loss was generally higher (25–98%) at Stns. 1–3 and lower (6–92%) at Stns. 4–6. The patterns of seasonal changes of ignition loss were similar among Stns. 1–3 and among Stns. 4–6. However, the patterns at the latter stations were clearly different from those at the former stations. The pattern at Stns. 4–6 were characterized by the lower levels of ignition loss mainly in 1988. Variations in ignition loss were big at Stns. 1–3 as compared with those at Stns. 4–6. Rapid increases in ignition loss were observed in August and September at Stns. 1–3 in 1987 and 1988, whereas at Stns. 4–6 rapid decreases were observed in the same period in 1987 and 1988. These differences could be due to the differences of variations in the proportion of inorganic suspended solid to the particulate organic matter or to the total suspended solid in the lake water. The patterns of seasonal changes of the ignition loss (Fig. 5) were quite different from those of the suspended solid (Fig. 3). This suggests that there had been big seasonal variations in the proportion of inorganic suspended solid to the total suspended solid or to the particulate organic matter.
Seasonal changes of the amount of particulate organic matter in the surface water and 2 m layer at Stns. 1–6 are illustrated in Fig. 6. The levels of particulate organic matter at Stns. 1 and 3 were comparatively low as compared with those at other stations. The average amount of particulate organic matter ranged from 0.4 to 7.5 g/m$^3$. Similar seasonal patterns were observed with the maximum in July–September and the minimum in December–February at Stns. 4–6. The seasonal patterns were similar at Stns. 1–3 if the two sharp peaks at Stn. 2 in April 1987 and 1988 were excluded. The range of variation was quite big at Stns. 4–6 as compared with that at Stns. 1–3. Peaks of particulate organic matter appeared a little earlier at Stns. 1–3 (April–July) than at Stns. 4–6 (July–August). The seasonal patterns of particulate organic matter (Fig. 6) were similar to those of suspended solid (Fig. 3) except that at Stn. 6.
Figure 7 shows seasonal variations of the percentage of chlorophyll $a$ in suspended solid in the surface water and 2 m layer at Stns. 1–6. The average percentage of chlorophyll $a$ was higher at Stns. 1–3 (0.1–0.6%) than at Stns. 4–6 (0.01–0.3%). The patterns of seasonal changes of the percent chlorophyll $a$ in suspended solid were similar among Stns. 1–3 with higher values during November–January and lower values during May–July, and between Stns. 5 and 6 with higher values in January or February and lower values in September and October. The seasonal patterns at the former stations were clearly different from those at the latter stations. The patterns at Stns. 4–6 were characterized
Fig. 5. Seasonal changes of the ignition loss of suspended solid at Stns.1–6 in the main channel of the High Dam Lake. Lines are for averages of the surface (○) and 2 m (●) samples.
Fig. 6. Seasonal changes of the particulate organic matter at Stns. 1–6 in the main channel of the High Dam Lake. Lines are for averages of the surface (○) and 2 m (●) samples.
Fig. 7. Seasonal changes of the ratio of chlorophyll $a$ to suspended solid at Stns.1-6 in the main channel of the High Dam Lake. Lines are for averages of the surface (○) and 2 m (●) samples.
by frequent changes with lower levels especially in 1988.
Relationships were investigated between the chlorophyll $a$ concentration and the particulate organic matter for the surface water and 2 m layer separately (Fig. 8). For both cases, there was positive correlation with considerably scattered data points between the two parameters, and no significant difference was observed between the two cases.
Figure 9 illustrates the relationship between the Secchi disc depth and the amount of suspended solid for all the data of suspended solid in the surface water and 2 m layer from September 1986 to December 1988 at Stns. 1-6. A clear hyperbolic relationship was obtained on a normal diagram and an exponential relationship on a semilogarithmic diagram. The results indicate that the Secchi disc depth was almost directly dependent on the amount of suspended solid in the lake water.
The relationship between chlorophyll $a$ and suspended solid was examined separately for the surface water and for the 2 m layer as illustrated in Fig. 10. Positive correlations can be seen between the two parameters, however the data points were scattered very much. About 90% of the data were in the ranges of 1-30 mg/m$^3$ in chlorophyll $a$ and 1-
Fig. 8. Relationships between the chlorophyll $a$ and the particulate organic matter at Stns.1-6 in the main channel of the High Dam Lake.
Fig. 9. Relationships of the Secchi disc depth to the suspended solid at Stns.1-6 in the main channel of the High Dam Lake. $\bigcirc$, 0 m; $\bullet$, 2 m.
15 g/m$^3$ in suspended solid. There is no significant difference between the relationships in the two layers.
4. Discussion
The seasonal patterns of the Secchi disc depth were similar to those previously reported (OLFAT et al., 1987; OLFAT and ARUGA, 1988) with higher values during the low temperature period and with lower values during the high temperature period. There seems to be a tendency for the Secchi disc depth to be lower in the southern part and higher in the northern part of the lake, which may be correlated with the decrease of suspended solid with water flow along the main channel (Fig. 3).
The levels of suspended solid were generally high during the flood season at Stns. 4-6 in the southern part of the lake. The seasonal patterns of suspended solid were quite similar with a very high peak in August or September at Stns. 5 and 6, and the peaks became lower at Stn. 4. On the other hand,
Fig. 10. Relationships between the suspended solid and the chlorophyll $a$ at Stns.1-6 in the main channel of the High Dam Lake. $\bigcirc$, Stn.1; $\bullet$, Stn.2; $\times$, Stn.3; +, Stn.4; $\square$, Stn.5; $\blacksquare$, Stn.6.
At Stns. 1-3 the seasonal patterns of suspended solid were quite different from those at Stns. 4-6 (Fig. 3) possibly due to changes in the composition of suspended solid, e.g. the ratio of particulate organic matter to particulate inorganic matter.
In the present investigation a similar trend was obtained in the relationship between the Secchi disc depth and the suspended solid as reported in previous papers (OLFAT et al., 1987; OLFAT and ARUGA, 1988) between the Secchi disc depth and the chlorophyll $a$ concentration, in the latter case the data points being much dispersed. This suggests that the Secchi disc depth was more directly dependent on the amount of suspended solid in the lake water.
According to HURST (1957) about 100 million tons of suspended sediments, composed of 30% fine sand, 40% silt and 30% clay, are carried annually with the Nile water on entering Egypt. The quantities of these sediments greatly increase at the beginning of the Nile flood. ELSTER and VOLLENWEIDER (1961) pointed out that the average value of the suspended matter in the Nile at the Egyptian borders during the flood period (August–October) amounted to 1.6 kg/m$^3$. However, after construction of the Aswan High Dam and creation of the High Dam Lake these features were basically changed. In this respect, ENTZ (1980) mentioned that suspended material in Lake Nasser (High Dam Lake) does not exceed a few milligrams per litre and that is mostly organic matter of planktonic origin. This means that the main bulk of suspended clay was sedimented in Lake Nubia. According to the High Dam and Aswan Dam Authority (personal communication) no clay reached the Egyptian water only with high turbidity in the southern part of the lake. EL-OTIFY (1985) reported that the total suspended matter fluctuated between a minimum of 10 mg/l and a maximum of 132 mg/l, and a gradual increase in total suspended matter was recorded along the main body of the High Dam Lake from north to south in autumn 1982 and summer 1983. The present results for suspended solid are in agreement with EL-OTIFY’s (1985) range of fluctuation.
It should be noted that Stns.. 1-3 showed similar patterns of seasonal changes in suspended solid, chlorophyll $a$, ignition loss, particulate organic matter and the ratios of chlorophyll $a$ to suspended solid and to particulate organic matter, and Stns. 4-6 showed other similar patterns of seasonal changes in
these parameters, even though sometimes Stn. 4 had seasonal patterns somewhat different from Stns. 5 and 6. It is clearly shown that both the ratio of chlorophyll $a$ to suspended solid and the ratio of chlorophyll $a$ to particulate organic matter are quite variable with seasons and regions in the High Dam Lake. This suggests that particulate organic matter is composed of phytoplankton and other variable organic materials, or at least the chlorophyll $a$ content in a cell is quite variable, with seasons and regions, and that the total suspended solid rather than phytoplankton generally plays an important role in determining transparency of water (the Secchi disc depth) of the lake.
Acknowledgements
The authors are grateful to the Captain and the crew of the research boat "El Sadaka" for their great help while collecting the samples. Helpful cooperation of Mr. Abu Wafa Hassen and Mr. Rabi Said are gratefully acknowledged.
References
EL-OTIFY, A. M. A. M. 1985. Studies on phytoplankton of Aswan High Dam Lake. A thesis submitted for the degree of M. Sc. in Botany. Bot. Dept., Fac. Sci., Aswan Assiut Univ. 140+ ivpp.
ELSTER, H. J. and R. VOLLENWEIDER 1961. Beiträge zur Limnologie Ägyptens. Arch. Hydrobiol., 57: 241–343. (cited in SAAD, 1980)
ENTZ, B. 1974. The morphometry of Lake Nasser and Lake Nubia. Lake Nasser Development Center Project (RPA, UNDP/SE, FAO) Working Paper No. 5. Aswan. 90pp. + 5 figs.
ENTZ, B. A. G. 1980. Sedimentation processes in the reservoir Lake Nasser-Nubia during 1965–1974 and future aspects. In A. K. BISWAS (ed.): Water Supply & Management, Vol. 4. Pergamon Press, Oxford. p. 63–66.
HURST, H. E. 1957. The Nile. Constable, London. (cited in SAAD, 1980)
OLFAT A. H., T. IORIYA and Y. ARUGA 1987. The distribution of chlorophyll $a$ as an index of primary productivity of phytoplankton in Khor El Ramla of the High Dam Lake, Egypt. J. Tokyo Univ. Fish., 74: 145–157.
OLFAT A. H. and Y. ARUGA 1988. Changes of the distribution of phytoplankton chlorophyll $a$ in the main channel of the High Dam Lake, Egypt. J. Tokyo Univ. Fish., 75: 343–352.
SAAD, M. A. H. 1980. A limnological study on Lake Nasser and the Nile in Egypt (Records of February 1970). In A. K. BISWAS (ed.): Water Supply & Management, Vol. 4. Pergamon Press, Oxford. p. 81–92.
Characterization of the environment in the Beppu Bay, Iyo Nada and Bungo Channel*
Naoji KUBOTA**, Tadashi HANO***, Yusaku TAKITA***, Takashi SHIMAZAKI** and Fumiaki HORI***
Abstract: Environmental characteristics of the sea area around Oita Prefecture were investigated by employing the monthly observational data (1965-1988) at 54 stations in Beppu Bay, Iyo Nada and Bungo Channel. Analysis of the horizontal and vertical distributions of water temperature and salinity revealed that the sea area was subdivided into several small areas. The characteristic of the Seto Inland Sea was different from that of the Bungo Channel. There was the isolated mixing zone in the northern and southern parts of the Hayasui Strait where water temperature and salinity were uniform by the strong tidal currents, and the tidal fronts were observed. Beppu Bay seemed to be semiclosed area, which was characterized by large seasonal variations in water temperature and salinity. MEM spectra emerged dominant periods of one year and 2-4 years in water temperature and in salinity. These periods can be related to those of atmospheric temperature and precipitation, respectively. The characteristics of the Seto Inland Sea appeared to depend on the climate of the area. The Kuroshio branch strongly affected the characteristics of the Bungo Channel.
1. 緒言
海洋が環境に及ぼす影響は非常に大きなものであり、わずか2-3℃の海水温の変化が全地球的異常気象を引き起こすことさえある。このような地球規模の海洋と大気の相互作用ばかりでなく、小さな内湾や沿岸における環境も、四方を海に囲まれた日本にとっては重要な研究対象である。たとえば、瀬戸内海は我が国最大の内海であり、豊後水道や紀伊水道などのわずかな部分で太平洋とつながっているため、これらの水道付近の海況について研究することは、瀬戸内海の特性を把握する上でも役にたつものと考えられる。
大分県は、豊予海峡を境にしてその北側が瀬戸内海に、南側は豊後水道に面しており、豊後水道はさらに日本の南西海域に接している。また、瀬戸内海からは豊後水道へ恒流として内海系冷水が南下している。そのため、この海域の海況はきわめて複雑であり、漁況に与える影響も小さくない。特に大分県沿岸に来遊するイワシ、アジ、サバ類は、主として春から夏にかけて北上し、秋から冬にかけては成魚が南下する傾向にあり、この付近の海況について明らかにすることは漁業にとっても大きく貢献できるものと思われる。これらの海域の個々の特性については、既に柳ら（YANAGI, 1980, 1983; 柳・大庭, 1985; YANAGI and KOIKE, 1987）の報告があるが、広範囲にわたる総括的な報告はあまりなされていない。
以上のことを考慮しながら、主に統計的手法で、伊予灘、別府湾および豊後水道の海況特性の解析を試みた。
2. データおよび解析方法
(1) データ
大分県水産試験場（1965-1988）は、毎月1回伊予灘、別府湾および豊後水道の54定点を選んで海洋観測を行っている。これらの観測点は、海底地形や潮流を考慮して選定されている。Fig.1に観測点（Stn. 1-54）を示した。1965年4月から1988年3月までの23年間の各観測点における水温（0, 5, 10, 20, 30, 50, 75m深）、塩分（0, 5, 10, 20, 30, 50, 75m深）、天候、気温および瀬戸内海側におけるDO、COD、窒素、リンなどの栄養塩類についてデータベースを構築し、解析に用いた。
また、大分地方気象台（1965-1988）が毎日観測している気温および降水量データ、さらに海上保安庁水路部（1965-1988）が年間24回観測を行っている黒潮本流の水温データも同様に処理した。
(2) T-S解析
水塊分布を把握するのに有効なT-S（水温-塩分）ダイアグラムを作成した。まず、各観測点ごとに海面、5, 10, 20, 30, 50, および75m深における水温と塩分の23年間のデータを月別に平均した。得られた各観測点における全水深の値を月別にグラフ上にプロットした。また、海面における全観測点の値を月別にプロットした。水深10, 20, 30および50m深における値についても同様に処理した。
(3) 水温および塩分の鉛直分布
鉛直方向の水塊分布より海況特性を検討するために、伊予灘北端から速吸瀬戸を通り豊後水道南端まで南下する線（縦断線と呼ぶ）上の海域を考え、月別平均水温および月別平均塩分から、水温および塩分の鉛直分布図を作成した。選ばれた観測点は、伊予灘のStn. 3, 7, 13, 16, 豊後水道のStn. 34, 38, 41, 43, 45, 48, 51および54である。同様に、別府湾のStn. 18, 20, 22, 26および30を選び、別府湾についても考察した。それぞれの観測点をFig.1に黒丸で示した。
(4) スペクトル解析
各観測点の水温および塩分データに欠測があるので、その欠測月の前後数か月にn次補間多項式を当てはめて補間し（Aitken-Neville法）、サンプル間隔1か月の時
系列データとした。スペクトルの計算にはMEMを用い、ARモデルの次数は、最大モデル次数を（サンプル数）/3とし、赤池の方法（南，1986）によって決定した。また、12か月周期を消去するために$1-2\cos(2\pi 3/12)B+B^2$なる調和フィルター（岩井ら，1980）を用いた。また、水温および塩分測定期間に相当する大分市の毎日の平均気温および降水量についても、MEMスペクトルを計算した。さらに、海上保安庁水路部より得た黒潮本流の水温観測データについても、MEMスペクトルを計算した。黒潮水温データは、豊後水道に近い宮崎県都井岬沖および高知県足摺岬沖におけるものを選んだ。
Fig. 2. Representative observation stations of sea area classified with water temperature-salinity diagrams.
3. 結果と考察
（1）データ
観測データは、毎月ほぼ10日前後に得られたものであり、水温は1965年から、塩分は1969年からのものであった。また、各観測点の海面における平均欠測数は水温で21個（最大35個、最小7個）、塩分で21個（最大38個、最小11個）あった。ただし、水深が増すにつれて欠測数は増加した。
（2）T-S解析
54の観測点すべてについて、各月ごとに（すなわち
54 × 12 = 648 個の）海面から最深観測深度にいたるまでの T-S ダイアグラムを作成した。全般的に、春季から夏季にかけて海面から最深観測深度までの変化が大きく、秋季から冬季にかけてはそれが小さかった。つまり、春季から夏季にかけては、成層が発達していることがわかった。ただし、瀬戸内海は塩分の変化の大きい成層であるのに対し、豊後水道は等密度線に対し垂直に変化している成層であった。また、豊後水道における Stn.33-39 では、年間を通じて非常に変動が小さかった。
つぎに、得られた T-S ダイアグラムを類似の季節変動をする海域ごとに分類し、それぞれの中から代表点を選んだ（Fig.2）。選ばれた観測点の T-S ダイアグラムのうち、2月、5月、8月および11月をそれぞれ冬季、春季、夏季、および秋季の代表として Fig.3 に示した。また、海面における T-S ダイアグラムのうち2月、5月、8月および11月のものを Fig.4 に示した。Fig.3 から、国東半島沿岸の Stn.10 では、冬季に著しい低温傾向を示していることがわかる。国東半島沿岸は、冬季に特有の冷水塊を形成することが知られており（YANAGI, 1980），このことは Fig.4 の2月の T-S ダイアグラムにもはっきり現われている。つまり、伊予灘の Stn.1, 8-11 および21だけが9℃前後の独立した水塊分布を示しており、国東半島沖との水温差が著しい沿岸フロントを形成していることが予想される。これは、冬季の強い北西季節風（河村, 1977）によって国東半島沿岸を周防灘系冷水塊が吹送流となって南下しているためと考えられる。この傾向は11月に国東半島北部沿岸から始まり、3月まで続いた。また Fig.3 から、速吸瀬戸北側に位置する Stn.16 では、秋季から冬季に瀬戸内海としては比較的高温高塩分を示し、夏季に期待される成層の程度も小さかった。一方、別府湾内の Stn.30 では、夏季に海面の低塩分と非常に安定した成層状態を示した。つまり、夏季における浅海暖水の典型である。しかしながら、Fig.5 に見られるようにこの高温低塩分も海面のみであり、10, 20, 30m と水深が増すにつれてほぼ一定の低温高塩分に近づいて行くのがわかる。この別府湾の極端な例が、別府湾内の大野川河口付近の Stn.23 であり、Fig.3 からわかるように、春季から夏季にかけて海面における著しい低塩分を示し、成層が発達していた。また、秋季でも塩分の差が大きかった。ここでは、梅雨および秋雨期の降雨による河川水流
Fig. 4. Mean water temperature-salinity diagrams at sea surface from 1965 to 1988. ○, Iyo Nada; △, Beppu Bay; and □, Bungo Channel. Numerals denote station numbers.
入増に起因する激しい塩分の変化が特徴であるが、この希釈効果はたかだか5 m ぐらいまでであり、日本のように水量の少ない河川に特有の現象である。Fig.6 に、この解析期間と同じ期間の大分市における月別平均降水量を示した。塩分低下と降水量との関係がよく対応している。さらに Fig.3 より、速吸瀬戸南側の Stn.35 は、冬季比較的高温高塩分であり、夏季にも水温および塩分の変化が小さい。また、速吸瀬戸北側の Stn.16 によく似た挙動を示している。豊後水道の Stn.51 では、夏季に成層が発達するものの、それ以外の季節では変化が小さかった。豊後水道を南下するにつれて、高温高塩分で変化の小さい T-S ダイアグラムが得られ、より黒潮本流の季節変動に近づいていると考えられる。
また Fig.4 から、豊後水道ではいずれの季節でも、
Stn.54から33へ北上するにつれて水温のみが大きく低下し、塩分の変化は小さかった。一方、伊予灘および別府湾では塩分が大きく変化しており、水温も季節ごとに特徴ある変化を示した。春季および夏季には伊予灘北部、別府湾および豊後水道南部ともに水温が高く、速吸瀬戸の南北のStn.13-16およびStn.33-39の狭い海域だけ低温になっていることがわかる。この様子をFig.7の水平分布図に示した。これは、速吸瀬戸の南北の海域以外では夏季の表面加熱によって成層しているのに対して、ここでは最大4.6ktにも達する1日4回の潮汐流（海上保安庁水路部，1987）による水平および鉛直方向の活発な混合により、海面から最深観測深度まで均一な水温および塩分の水塊を形成しているためと考えられる。これが、緒方ら（1985）の言う夏季における速吸瀬戸付近での冷水塊に相当しているものと考えられる。
(3) 水温および塩分の鉛直分布
2月、5月、8月および11月の縦断線上の各観測点における水温および塩分の鉛直分布をFig.8およびFig.9に示した。
2月には、海面冷却による鉛直混合が起こり、海面と最深観測深度との水温差が全くない。この傾向は、豊後
Fig. 8. Vertical distribution of water temperature in Iyo Nada and Bungo Channel.
Fig. 9. Vertical distribution of salinity in Iyo Nada and Bungo Channel.
同様に豊後水道のStn.41以南では34.5と差がないものの、そこから北上するにつれて海面と最深観測深度の差が生じた。また、南高北低の塩分分布を示し、かつその差は2月より大きく1.5になっており、内海への陸水の流入量が増加しはじめることを表わしている。
8月には、海面の水温上昇が著しく、海面と最深観測深度との温度差も非常に大きいことがわかる。海面と50m深の水温差は、伊予灘で5度、豊後水道南部で6度にも達した。しかしながら、豊後水道のStn.34では2度ほどしかなく、この傾向は、速吸瀬戸の北と南に隣接する伊予灘のStn.15-17および豊後水道のStn.33-35でもほとんど同じであった。塩分も同様に、豊後水道のStn.34ではほとんど差がなく、速吸瀬戸からはなれるにしたがって海面と最深観測深度の差が大きかった。このように、夏季には縦断線上で水温および塩分ともに速吸瀬戸付近で鉛直方向の強い混合が起こっていることが明らかであるが、これは、藤原・早川（1978）の瀬戸内海の海峡部における速い潮流による鉛直混合発生と同じメカニズムと考えられる。夏季には瀬戸内海の水温に鉛直方向の大きな分布が現われるので、この主な原因は潮汐流によると思われるが、強い混合は年間を通して生じているものと推定される。
9月には海面と最深観測深度の水温差が小さくなりはじめ、11月になるとその温度差が全くなくなり、さらに伊予灘側と豊後水道側との間にも温度差がなくなっていることがわかる。つまり、水平方向にも鉛直方向にも水温差がない。詳細に見ると豊後水道の北部と南部で1-2度の水温差があるが、この差も年間を通して最も小さい。塩分の分布は2月と同様に、南高北低でその差は1程度になっているものの、海面と最深観測深度との塩分の差
Fig. 10. Vertical distribution of water temperature in Beppu Bay.
Fig. 11. Vertical distribution of salinity in Beppu Bay.
はほとんど見られなかった。これが、秋季の典型的な海況である。
以上のように、内海ほど気温と陸水の影響による水温および塩分の季節変動が大きく、一方、豊後水道ではあまり影響を受けていない。特に、豊後水道のStn.41以南の塩分は降水量の多い夏季を除いてほぼ34.3-34.7のせまい範囲に納まっていた。また、これらの図を見ると、特に冬季に伊予灘のStn.7と13との間および豊後水道のStn.38と41との間で等温線または等塩分線の間隔が狭くなっており、この部分でフロントを形成している（柳・大庭，1985）ことを示している。これは、速吸瀬戸付近の混合域の広さを表しており、速吸瀬戸の南北の混合海域のStn.13-18およびStn.33-39に相当するものであろう。速吸瀬戸南側のフロントは、吉岡（1971, 1984）による紀伊水道のフロントに対応するものと考えられる。
以上のような伊予灘—豊後水道で行った解析を、別府湾内でも行った。Fig.10およびFig.11にその結果を示した。選択された観測点は、湾中央部を東西に横断している。いずれの観測点においても最低水温を示すのは3月で、冬季には海面から最深観測深度まではほぼ同じ水温と塩分を示した。4月には水温上昇が始まるが、海面の方が上昇速度も大きく、成層が始まる。さらに夏季には、湾奥部に行くほど海面と最深観測深度の水温差、塩分差が大きく、湾口部には潮汐流の混合効果があると考えられる。冬季の別府湾のStn.20と18の間の等温線および等塩分線の間隔が狭く、ここにもフロントが形成されているものと考えられる。
（4）スペクトル解析
Fig.12に、観測期間の長い代表的な観測点の水温と塩分の時系列データを示した。瀬戸内海では水温および塩分の振幅が大きく、逆に豊後水道では小さいことがわかる。また、例としてFig.13およびFig.14に伊予灘のStn.1における海面水温および塩分のMEMスペクトルを示した。予想されたように、どちらも1年周期が卓越していた。しかしながら、それぞれのスペクトル強度は水温で$10^6$、塩分では$10^4$程度にすぎなかった。同様
Fig. 12. Time series of sea surface water temperature and salinity at representative observation stations. Open circles denote the interpolated data.
Fig. 13. MEM spectra of sea surface water temperature at Stn. 1 in Iyo Nada. Solid line represents the spectrum for the raw data and broken line for the data through the harmonic filter.
Fig. 14. MEM spectra of sea surface salinity at Stn. 1 in Iyo Nada. Solid line represents the spectrum for the raw data and broken line for the data through the harmonic filter.
Table 1. Dominant periods of MEM spectra of water temperature, salinity, atmospheric temperature and precipitation.
| Station no. | Dominant period of water temperature (Year) | Dominant period of salinity (Year) |
|-------------|---------------------------------------------|-----------------------------------|
| 1 | 1.0 3.2 | 1.0 4.3 |
| 2 | 1.0 1.8 3.4 | 1.0 3.6 |
| 3 | 1.0 2.8 | 1.0 3.8 |
| 4 | 1.0 2.1* | 1.0 4.7 |
| 5 | 1.0 2.8 | 1.0 4.2 |
| 6 | 1.0 2.7 | 1.0 4.0 |
| 7 | 1.0 2.3 | 1.0 3.5 |
| 8 | 1.0 2.2 5.5 | 1.0 3.5 |
| 9 | 1.0 3.4 | — 4.7* 2.7* |
| 10 | 1.0 3.2 | 1.0 3.8 |
| 11 | 1.0 3.3 | 1.0 3.8 |
| 12 | 1.0 3.6 | 1.0 4.0 |
| 13 | 1.0 4.1 | 1.0 3.9 |
| 14 | 1.0 4.7 | 1.0 3.5 |
| 15 | 1.0 3.6 | 1.0 4.1 |
| 16 | 1.0 4.6 | 1.0 4.1 |
| 17 | 1.0 4.5 | 1.0 4.1 |
| 18 | 1.0 4.0 | 1.0 3.5* |
| 19 | 1.0 4.7 1.4 | 1.0 3.4* |
| 20 | 1.0 4.3 1.8 | 1.0 5.1* |
| 21 | 1.0 3.5 | 1.0 4.1 |
| 22 | 1.0 4.0 2.3 | 1.0 4.0 |
| 23 | 1.0 4.2 | — 4.0* |
| 24 | 1.0 2.8 | 1.0 3.0 |
| 25 | 1.0 2.4 | 1.0 2.7* |
| 26 | 1.0 3.3 | 1.0 3.5 |
| 27 | 1.0 3.3 | 1.0 4.0 |
| 28 | 1.0 3.4 | 1.0 4.1 |
| 29 | 1.0 3.6 | 1.0 4.5 |
| 30 | 1.0 3.4 | 1.0 3.7 |
| Station no. | Dominant period of water temperature (Year) | Dominant period of salinity (Year) |
|-------------|---------------------------------------------|-----------------------------------|
| 31 | 1.0 3.2 | — 4.5* 2.7* 1.7* |
| 32 | — | — |
| 33 | 1.0 3.7 | 1.0 2.7 1.4 |
| 34 | 1.0 4.8 2.5 1.3 | 1.0 3.5 |
| 35 | 1.0 5.2 1.3 2.3 | 1.0 6.8* |
| 36 | 1.0 1.5 4.4 | 1.0 2.6* 6.5* |
| 37 | 1.0 1.6 4.2 | 1.0 6.5* |
| 38 | 1.0 2.0 3.6 | 1.0 3.0* |
| 39 | 1.0 3.7 1.4 | 1.0 2.3* |
| 40 | 1.0 3.1 | 1.0 3.9* |
| 41 | 1.0 3.7 | 1.0 1.2 4.2 2.6 |
| 42 | 1.0 3.5* | 1.0 4.1 |
| 43 | 1.0 3.6* | 1.0 |
| 44 | 1.0 1.7 | 1.0 |
| 45 | 1.0 4.3 1.6 | 1.0 3.0 |
| 46 | 1.0 1.9 | 1.0 3.0* |
| 47 | 1.0 2.8 | 1.0 5.4* |
| 48 | 1.0 2.1 | 1.0 3.6 1.6 |
| 49 | 1.0 5.1 1.4 1.9 | 1.0 5.4* 2.0* |
| 50 | 1.0 4.4 1.6 | 1.0 4.0 1.8 1.3 |
| 51 | 1.0 4.6 | 1.0 1.7 3.7 |
| 52 | 1.0 3.9 1.6 | 1.0 1.8 1.2 3.8 |
| 53 | 1.0 3.7 1.6 | 1.0 3.8* |
| 54 | 1.0 3.0 | 1.0 2.3* |
| Toi | 1.0 2.7* | |
| Ashizuri | 1.0 2.6* | |
| Atmospheric temperature | 1.0 2.8* | |
| Precipitation | — 3.1* | |
*Spectra for the data through the harmonic filter.
に、各観測点の表面水温および塩分の MEM スペクトル解析を行った。各水深の測定データからも MEM スペクトルを計算したが、海面と周期の違いは見られなかったので、海面について考察した。いずれの観測点でも 1 年周期が卓越していた。また、気温および黒潮水温も 1 年周期が卓越していた。しかし、河口付近の塩分および降水量の生データは確率過程に含まれず、スペクトルは得られなかったため、調和フィルターを通じて 1 年以外の周期性があるかどうかを検討した。また、1 年周期しかみられない観測点の水温および塩分についても同様に処理した。このようにして得られた結果を Table 1 にまとめた。
水温に関しては、1 年以外に国東半島沿岸の Stn. 1, 9-11, 21 で 3.2-3.5 年、速吸瀬戸北側の Stn. 12-20 で 3.6-4.7 年、別府湾の Stn. 26-31 で 3.2-3.6 年、別府湾河口域の Stn. 24 と 25 で 2.4-2.8 年の周期性が見られた。これらは、先の T-S 解析から明らかになった海域にはほぼ一致している。一方、豊後水道では、Stn. 39-43 に 3.1-3.7 年、Stn. 50-53 に 3.7-4.6 年の周期性が見られたが、T-S 解析の結果とはそれほどよい対応を示さなかった。大分市の気温には 2.8 年、都井岬沖および足摺岬沖の黒潮本流にそれぞれ 2.7 年および 2.6 年の周期があり、いずれも伊予灘および別府湾の水温における周期性とはほぼ一致しており、互いに強い相関がある。また、塩分に関しては T-S 解析とそれほどよい一致を示さなかったが、速吸瀬戸北側の Stn. 10-17 で 3.5-4.1 年、別府湾の Stn. 27-31 で 3.7-4.5 年、別府湾河口域の Stn. 24 と 25 で 2.7-3.0 年の周期性が見られ、豊後水道では 2-4 年の
種々の周期性が見られた。大分市の降水量については3.1年の周期があり、伊予灘および別府湾の塩分、特に別府湾の河口域の塩分における周期性とよく一致している。YANAGI（1983）は、同じ豊後水道の愛媛県側の水温および塩分のスペクトル解析により、3〜5年の水温周期性と、3年の塩分の周期性を認めている。しかしながら、本研究における豊後水道の大分県側では種々の周期性が得られた。これは、秋山・柳（1989）の日向灘定点観測データの解析でも明らかにされているように、豊後水道の愛媛県側は黒潮分岐流の影響を直接受けるが、大分県側では瀬戸内海からの南下流と黒潮分岐流の影響を受けるためと考えられる。そのため、黒潮勢力の変化により2年から4年にわたる比較的幅広い周期性が観測されたものと思われる。
4. 結論
本報の検討結果により、伊予灘、別府湾および豊後水道ではその海況特性がそれぞれ異なっていることが明らかになった。特に、瀬戸内海と豊後水道では挙動が大きく異なっていた。水温および塩分の解析から、速吸瀬戸の南北の狭い海域で潮汐によると思われる強い混合が起こっていることが確認された。さらに、この混合域は、北部で国東半島沖の海域と、また南部では豊後水道南部の海域とのあいだにフロントを形成していることが明らかになった。一方では、別府湾の閉鎖性があらためて確認された。水深が浅いため、年間を通しての水温および塩分の変化の大きいことが特徴で、この閉鎖性は別府湾奥部に行くにしたがって著しかった。
水温および塩分には、1年周期以外に2〜4年の弱い周期性がみられ、気温、降水量および黒潮の影響を強く受けていることがわかった。
謝辞
本研究を進めるにあたり、貴重な資料を提供いただいた大分県水産試験場および有益なる助言をいただいた宮澤正化学科長に深謝いたします。また、スペクトル解析にあたり、討論していただいた大分大学工学部電気工学科杉坂政典教授、同じく組織工学科永井武昭教授、越智義道助教授に感謝いたします。なお、本研究の中の黒潮本流の水温データは、海上保安庁水路部の海洋速報を使用させていただいたことを付記いたします。
文献
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YANAGI, T. (1983): Variability of the oceanic condition in the Bungo Channel. La mer, 21, 21-28.
柳哲雄、大庭哲哉（1985）：豊後水道のTidal Front. 沿岸海洋研究ノート，23，19-25。
YANAGI, T. and T. KOIKE (1987): Seasonal variation in thermohaline and tidal fronts, Seto Inland Sea, Japan. Continental Shelf Res., 7, 149-160.
吉岡洋（1971）：冬期紀伊水道のOceanic Frontについて。海と空，46，31-44。
吉岡洋（1984）：沿岸海域のフロント。沿岸海洋研究ノート，21，110-117。
Two patterns of typhoon-induced storm surges around the Japanese Islands*
Shigehisa NAKAMURA**
Abstract: Two recently observed patterns of typhoon-induced storm surges around the Japanese Islands are introduced. Meteorological anomaly of the sea level is obtained after eliminating the predicted tides out of the mareogram at each tide station for the typhoons 8506 and 8719. The meteorological anomaly is separated into two parts, i.e. barometric and non-barometric effects. The local winds are considered in relation to a part of the non-meteorological effects, though only a qualitative notice is given. A discussion is made to know whether any hazardous storm surge is a type of the Kelvin-like waves.
1. 緒言
台風は北太平洋西部沿岸域で高潮をひきおこし、水災害の要因のひとつである。この台風や高潮については、伊勢湾台風以来、気象庁（1961）によって予・警報に関連した問題としての調査報告が出されている。一方、DEACON and DEACON (1982) は、北海の高潮を論するにあたり、NOMITSU（たとえば、1934）の気圧に対する海面の応答に関心を示している。北海の高潮の数値計算（1953年1月31日-2月1日）の例も HANSEN（1956）により発表されている。また、FANDRY et al.（1984）は、サイクロンによって生じた Kelvin 型の高潮を考えた。台風高潮については、中村（NAKAMURA, 1981; 1988; 中村, 1988）の検討例がある。
本論文では、最近、日本列島に強い影響を及ぼした台風高潮の2例について、検潮記録によってとらえられたパターンを示す。これによって、台風の経路が異なるとき、台風による潮位偏差にも顕著な差異があることを示すことができる。気圧低下の効果のほかに、検潮所の位置によっては、風の効果が大きいこともある。具体的観測例によって再確認するとともに、高潮災害と Kelvin 型高潮との関連についても考察する。
2. 台風の経路
ここで検討の対象とする台風高潮については、中村（1988）が海面水温と関連づけて調べている。本論文では、それと別の面から検討を試みる。便宜上、2つの台風例をえらび、その台風の日本列島周辺における経路を Fig.1 に示した。すなわち、台風 8506（実線）と台風 8719（破線）である。台風 8506 は、九州南方洋上から紀伊半島沖を経て静岡県沿岸に上陸し北東進した。台風 8719 は、四国南方洋上から加古川市付近へ北上し日本海へ出た後に北東進した（cf. Fig. 2）。
3. 台風高潮の特徴
これらの台風高潮の特徴をとらえるため、気象庁所管の検潮記録を利用することとした。台風 8506 に関連して利用した検潮記録は、宮古、大船渡、小名浜、岡田、御前崎、尾鷲、浦神、潮岬、南部、室戸岬、土佐清水、油津である（ただし、南部は京都大学の圧力式潮位計による）。上に示した検潮所の位置を記号化し、Fig.1 に示した。すなわち、それぞれ MIY, OFU, ONA, OKA,
Fig. 1. The tracks of Typhoons 8506 and 8719. Solid line: the track of Typhoon 8506 from 29 June to 1 July 1985. Dotted line: the track of Typhoon 8719 from 16 to 17 October 1987. Dots: tide stations where the mareograms of Typhoon 8506 were obtained.
Fig. 2. The track (solid line) of Typhoon 8719 from 20h on 16 October to 8h on 17 October 1987. Dots: tide stations where the mareograms of Typhoon 8719 were obtained.
Fig. 3. Meteorological anomaly (solid line) of the sea level and barometric effects (dotted line) of Typhoon 8506.
OMA, OWA, URA, SIO, MIN, MUR, TOS, ABU である。
台風 8719 については、土佐清水、高知、室戸岬、潮岬、白浜、和歌山、徳島、高松、洲本、神戸、舞鶴、浜田の検潮記録を対象とした。これらの検潮所の位置を記号化し、Fig. 2 に示した。すなわち、それぞれ TOS, KTI, MUR, SIO, SIR, WAK, TKU, TAK, SUM, KBE, MAI, HAM である。
（1）台風 8506 の例
各検潮記録から予想潮（主として天文潮）を差引いて潮位偏差を求める。台風 8506 の場合、1985 年 6 月 30 日～7 月 1 日を対象として、潮位偏差を Fig. 3 の実線のように示した。また、気圧低下の効果を静水圧近似によって推定した結果を、Fig. 3 の破線によって示した。一般に、外洋では、台風高潮の潮位偏差は気圧低下の効果
Fig. 4. Meteorological anomaly of the sea level for Typhoon 8506. Solid line: non-barometric effects. Arrows: local winds.
として考えてよいとされているし、その理論的根拠も与えられている（たとえば NAKAMURA, 1981）。ただ、Fig. 3 では、実線と破線とはかならずしも一致してはいない。
台風高潮の潮位偏差が非線型現象の場合には検討が複雑になる。簡単のため、線型的なデータ処理が可能なものと考えよう。このとき、Fig. 3 に示した潮位偏差から気圧低下の効果を差引いた偏差は、Fig. 4 の実線のようになる。Fig. 4 には、各検測所に対応した地点の風速を矢印によって示した。これによって、たとえば南部では、風向の変化が偏差の変化によく対応していることがわかる。これは、いわゆる風による吹き寄せ効果である。小名浜でも短時間ながらこの風の効果が認められる。
御前崎では、風が変わって、西風となると偏差が大となり潮位低下の原因となっている。油津、室戸岬、潮岬、岡田では、この偏差は比較的小さく、気圧低下の効果がとくに顕著なことがわかる。これは外洋性高潮の特徴とみてよいであろう。また、尾鷲と浦神とでは、台風が最も接近した位置にある時に偏差も最大である。これには、気圧低下の効果の増幅や検測所周辺の地形条件も考慮すべきであろう。土佐清水では台風通過後に偏差の負値が異常に大きくなっている。
ここでは定性的検討にとどめたが、Fig. 3 および Fig. 4 では、偏差のさらに短時間の変動はわからない。
（2）台風 8719 の例
上述の台風 8506 の例と同様にして、1987 年 10 月 16-17 日の台風 8719 の例を示す。潮位偏差は Fig. 5 に実線で示した。また、気圧低下の効果は Fig. 5 の破線のように推定される。Fig. 5 の実線と破線のパターンは、Fig. 3 にみられたパターンと対照的である。これは台風の経路の相異によるものである。
Fig. 6. Meteorological anomaly of the sea level for Typhoon 8719. Solid Line: non-barometric effects. Arrows: local winds.
潮位偏差から気圧低下の効果を除いた偏差を Fig. 6 に実線で示した。風は矢印によって示した。高知と土佐清水とでは気圧低下の効果を除いた偏差は小さい。舞鶴と浜田とでは台風通過後に偏差が顕著に大きくなっていることがわかる。これは日本海沿岸の高潮として検討すべきかもしれない。太平洋側の室戸岬、潮岬、白浜では偏差は最大約 0.5m である。これには増幅作用が関連しているようにみえる。さらに、紀伊水道・大阪湾の海域では、和歌山、高松、洲本、神戸の各検潮所で、風による吹き寄せ効果が非常に顕著である。とくに、大阪湾奥に位置する神戸では、偏差の最大は約 1.5m に達している。周辺の海底地形・海岸形状も考慮すべき因子であろう。
4. 外洋に面した沿岸での高潮
高潮が岸沿いに移動する場合、これは、外力によって生じた Kelvin 波の問題として処理される（たとえば GILL, 1982, とくに p.398-403）。この場合、Rossby の変形半径と岸沿いの高潮のスケールとの大小関係が重要になる。Rossby の変形半径の決定には対象とする水深が与えられなくてはならないが、水深の値に任意性があると問題は力学的意義を失ってしまうことになる。しかし、時間のスケールが $f^{-1}$ より小さいときは、このようなことは問題の対象ではなくなる。ここで、着目する緯度を $\xi = 30^\circ N$ とすると、
$$f^{-1} = \frac{1}{2\Omega \sin \xi} \sim \Omega^{-1} \approx 2.4 \text{ hours}.$$
ただし、$\Omega$ は地球自転角速度である。
台風 8506 および台風 8719 の例では、高潮偏差の時間スケールは約 20 時間であり、上の $f^{-1}$ の値の約 10 倍とみてよい。台風の経路からみて、台風 8506 の場合には高潮偏差が Kelvin 型に近い挙動をするようであったとしても、台風 8719 の場合はそれほど現象は簡単ではない。
GILL (1982) は、1953 年 1 月 30 日 - 2 月 2 日の北海における高潮の例 (ROSSITER, 1954) を引用している。FANDRY et al. (1984) は、岸に平行な風や気圧勾配は Kelvin 波の特性をもった変動をひきおこす要因であるとして KAJIURA (1962) や THOMSON (1970) の例を示している。
ところで、日本列島周辺で高潮災害をともなった台風の経路は、1934 年の室戸台風や、1954 年の伊勢湾台風、その他の多くの場合、台風 8719 の経路に近い。台風 8506 のような経路の場合には Kelvin 型の高潮があらわれるにちがいないが、かならずしもこれが高潮災害につながらない。高潮の力学的機構や予測についてなお今後の検討をまつ必要がある。
謝辞
本研究をすすめるにあたって、気象庁の御好意により気象資料・検潮記録などの利用ができた。
文献
DEACON, G.E.R. and M.B. DEACON (1982): Modern Concepts of Oceanography. Hutchinson Ross Publ. Co., Stroudsburg, Pennsylvania. 385pp.
FANDRY, C. B., L.M. LESLIE and R.K. STEEDMAN (1984): Kelvin-type coastal surges generated by tropical cyclones. J. Phys. Oceanography, 14, 582-593.
GILL, A.E. (1982): Atmosphere-Ocean Dynamics. Int. Geophys. Ser., Vol. 30. Academic Press, N.Y. 662pp.
HANSEN, W. (1956): Theorie zur Errechnung des Wasserstandes und der Stroemungen in Randmeeren nebst Anwendungen. Tellus, 8, 287-300.
KAJIURA, K. (1962): A note on the generation of boundary waves of Kelvin-type. J. Oceanogr. Soc. Japan, 18, 49-58.
気象庁（1961）: 伊勢湾高潮の総合調査報告. 気象庁技術報告 No.4. 286pp.
NAKAMURA, S. (1981): On factors magnifying a storm surge. Proc. XIX Congress IAHR, New Delhi, Subject B(a), Paper No.5, 47-54.
NAKAMURA, S. (1988): An observation of factors related to typhoon. Proc. VI Congress IAHR-APD, Kyoto, Vol.4, 273-280.
中村重久(1988)：近畿圏沿岸の高潮災害の要因としての黑潮について. 京都大学防災研究所年報, No.31 B-2, 753-773.
NOMITSU, T. (1934): Coast effect upon the ocean current and the sea level. II. Changing state. Mem. Coll. Sci., Univ. Kyoto, Ser. A, 17, 249-280.
ROSSITER, J.R. (1954): The North Sea surge of 31 January and 1 February 1953. Phil. Trans. R. Soc. London, Ser. A, 246, 371-400.
THOMSON, R.E. (1970): On the generation of Kelvin-type waves by atmospheric disturbances. J. Fluid Mech., 42, 657-670.
1. 1989年12月1日 東京水産大学において平成2年度学会賞受賞候補者推薦委員会（第1回）が開かれ、委員長に鎌谷明善氏を選出し、推薦の方法及び次回の日程を決めた。
2. 1989年12月25日 東京水産大学において学会賞受賞候補者推薦委員会（第2回）が開かれ、評議員から推薦のあった候補者6名について審議し、2名が合格者として残された。この2名の代表的な論文リスト等を委員全員に送付し検討してもらうこととし、第3回委員会の日程を決めた。
3. 1990年1月12日 東京水産大学において学会賞受賞候補者推薦委員会（第3回）が開かれ、研究業績について審議の結果、落合正宏氏（都立大・理）が最適格者との結論に達し、この結論を会長に報告することとした。
4. 1990年2月14日 東京水産大学において平成2・3年度評議員選挙の開票が行われた。
5. 新入会員
| 氏名 | 所属 | 紹介者 |
|------|------|--------|
| 小池孝知 | 東京水産大学海洋生産学科漁業工学講座 | 高橋正 |
| エリック・シャルルリー | 東京都港区南青山2-24-15 | 日本スリオ電子㈱ |
6. 退会
近藤恵一，斉藤行正
7. 受贈図書
日本学術会議月報 31(2)
なつしま (104)
海洋産業研究資料 20(10)
海産研ニュース (16)
航海 (102)
日本鯨類研究所年報 昭63
1987/88年南水洋鯨類資源予備調査の成果
外国人の指摘する捕鯨禁止の不当性
東海大学記要 (29)
養殖研ニュース (18)
養殖研究所研究報告 (16)
イカ類資源・漁海況検討会議研究報告
日本海における資源解析
総産卵量による浮魚類の資源量推定法
—北米カタクチイワシへの適用—
NTT R & D 38(11, 12), 39(1)
Preliminary Report of the Hakuho Maru Cruise KH-88-4
Bull. Ocean Res. Inst., Univ. of Tokyo (27)
科学通報 34(16〜24)
水産学報 13(3)
韓国海洋学会誌 24(3)
青島海洋大学学報 18(2), 19(3, 4)
海洋与湖沼 20(3, 4)
Aquatic Living Resources 2(4)
Israel Oceanogr. & Limnol. Res. 12
Definicaoae classificacao dos stpos de navios de pesca (13)
日仏海洋学会役員・評議員
(1988〜1989年度)
顧問 ユベール・ブロシェ ジャン・デルサルト
ジャック・ロペール アレクシス・ドランデール
ベルナール・フランク ミシェル・ルサージュ ロベール・ゲルムール ジャック・マゴー レオン・ヴァンデルメルシュ オーギュスタン・ベルク
名誉会長 ユベール・セカルディ
会長 宇野寛
副会長 高木和徳
幹事（庶務）須藤英雄，有元貴文；（会計）松生洽，高橋正；（涉外）有賀祐勝，佐伯和昭；（研究）関文威，小池勉夫；（編集）山口征矢，渡辺精一
監事 久保田穣，辻田時美
評議員 青山恒雄，阿部友三郎，有賀祐勝，石井丈夫
石野誠，磯舜也，井上実，岩井保，岩宮浩，宇野寛，大塚一志，岡市友利，岡部史郎，小倉通男，梶浦欣二郎，鎌谷明善
川合英夫，国司秀明，黒木敏郎，西条八束，佐伯和昭，坂本市太郎，坂本亘，佐藤孫七
杉森康宏，須藤英雄，関文威，平啓介，高木和徳，隆島史夫，高橋正，高橋正征，多紀保彦，谷口旭，辻田時美，寺本俊彦，鳥羽良明，冨永政英，中村重久，永田豊，奈須敬二，奈須紀宰，根本敬久，野村正，畑幸彦，平野敏行，松生治，松山優治，丸茂隆三，三浦昭雄，宮本悟，村野正昭，森田良美，柳哲雄
(54名，会長推薦評議員を含む)
編集委員長 有賀祐勝
日本学術会議だより
公開講演会盛会裡に終了
平成2年2月 日本学術会議広報委員会
日本学術会議は、平成元年度に主催の公開講演会を3回開催しました。今回の日本学術会議だよりでは、その公開講演会の概要に加えて、本会議が実施している国際的活動などについて、お知らせいたします。
日本学術会議主催公開講演会
本会議では、科学の向上発達を図り、行政、産業及び国民生活に科学を反映浸透させるという本会議の設置目的に沿うための活動の一環として、毎年、公開講演会を開催している。この講演会は、本会議員が講師となり、学術的香気が高く、かつ、時宜にかなったテーマを選定して開催している。
今年度も3回の公開講演会を開催したので、その概要を以下に紹介する。
Ⅰ．公開講演会「人間は地球とともに生きられるか」
標記講演会は、去る平成元年10月27日（金）13時30分～17時に、本会議講堂で約280人の参加を得て開催された。
最初に、吉野正敏・第2部会員（筑波大学地圏科学系教授）が、「地球の温暖化とその影響」と題して、大気中の二酸化炭素とフロン・メタンなどの増加による気温の上昇に伴う、農林水産業をはじめ人間の社会経済にもたらす大きな影響などについて述べた。
続いて、久馬一剛・第6部会員（京都大学農学部教授）が、「地球環境と農業のかかわり」と題して、人口の増加、消費水準の向上などが世界で農業そのものの変貌を余儀なくしており、その中で土壌侵食や塩類化などによる生産力の退化（砂漠化）や、水質汚濁・土壌汚染などを引き起こしていることを指摘し、農業のあるべき姿について述べた。
最後に、藤井隆・第3部会員（名古屋大学経済学部教授）が、「地球環境の経営と人間社会の発展」と題して、地球環境の経営は、地球環境の科学的研究に加えて、人間社会の運行との相関についての研究が必要であることを指摘し、人間社会の持続的発展を考えていくための視点について述べた。
Ⅱ．公開講演会「*人権の歩み*から何を学ぶか—フランス人権宣言200年を記念して—」
標記講演会は、去る平成元年11月18日（土）13時30分～17時に、本会議講堂で約250人の参加を得て開催された。
最初に、弓削達・第1部会員（フェリス女学院大学長）が、「*人権*以前の世界」と題して、人権思想のなかったギリシャ・ローマ時代における人権思想の萌芽とも言うべきものについて、同時代の一哲学者の奴隷をめぐる精神的苦闘を例にとって述べた。
次いで、大石嘉一郎・第3部会員（明治学院大学経済学部教授）が、「近代日本の人権思想—自由民権運動の人権論を中心に—」と題して、主として、明治時代の自由民権運動における人権論について、その特徴、特徴の起因となった当時の社会的条件、それが与えた影響などについて述べた。
続いて、杉本大一郎・第4部会員（東京大学教養学部教授）が、「科学技術と人権」と題して、近年の著しい科学技術の発展によってもたらされた、広い意味での「人権と自由」の様々な様相について、それらの事情と問題点などについて述べた。
最後に、南博方・第2部会員（一橋大学法学部教授）が、「人権の進化と創造」と題して、現在までの人権の進化の跡を回顧するとともに、最近における人権立法や人権思想を紹介しながら、今日生成しつつある現代型の新しい人権について述べた。
Ⅲ．公開講演会「くらしと学問の近未来—これからのくらしと学問—」
標記講演会は、去る平成元年12月8日（金）13時～17時に、仙台市の勾当台会館で約70人の参加を得て開催された。
この講演会の開催に当たっては、東北大学、宮城県教育委員会、仙台市教育委員会、河北新報社の支援を受けた。
最初に、藤沢温・第3部会員（東北大学名誉教授）が、「長寿社会の条件—長生きを支える医学と医療」と題して、健康を阻害する危険や長生きできるための条件を充足するには厳しい現実があることを指摘し、長生きを支える医学と医療にとっての問題点を指摘し、それらの解決策について述べた。
続いて、澤登俊雄・第2部会員（国学院大学法学部教授）が、「犯罪現象への多様な接近」と題して、犯罪抑止や犯罪者の社会復帰などのための施策の推進には犯罪や犯罪者をつくりあげていくプロセスの分析など多様な視点からの検討が不可欠であることなどについて述べた。
最後に、上飯坂貴・第6部会員（東京農業大学農学部教授）が、「森とむらと都市の共生」と題して、健全な森林づくりのために都市の住民と山村の住民が連帯することは、新しい人間尺度の文化の創造につながることを、日本と外国との場合を対比させ述べた。
（なお、これらの講演会の講演要旨は、「日学双書」として、（財）日本学術協力財団から出版されます。）
地球環境問題における工学研究の在り方について—第5部報告
このたび、本会議の第5部は、標記報告をとりまとめ、本会議運営審議会の承認を得て公表した。
（要旨）
近年における人間活動の大規模化は、地球環境に急速な変化をもたらし、その変化が人類の生存基盤そのものさえも脅かすようになってきた。そのため、次の世紀に向けて人類が総力を上げて取り組むべき、極めて重要かつ緊急の課題として地球環境問題が広く国際的な関心を集めているのは周知のとおりである。
元来、この問題は、時間的にも空間的にも極めてスケールが大きく、かつ、気候、水圏、地圏及び生物圏全体にわたる相互干渉の結果として現れるものだけに、対応策を選択するに当たって、従来とは異なる長期的、学術的かつ国際的視野に立ち、持続的に取り組む必要がある。また、局所的な現象に目を奪われた一時的な対応や規制ではなく、地球全体における持続的な発展という観点から、地球を一つのシステムとして捉え、自然環境と人間活動とをいかなる価値観に基づいて調和させていくべきかという視点から、総合的に取り組むべき問題である。
地球環境問題が人間活動の結果として現出したことを考えると、今後すべきことは、地球環境に配慮した健全な人間活動のために新しい工学的基準を導入するなどにより、人間活動と地球環境の調和に役立つ広く新しい学問領域を創出することである。
それは、地球環境と人間活動をと結合して一つのシステムとして捉え、そこにおける物質・エネルギー循環・エネルギー収支のあるべき姿を定量的に評価できる手法を開発し、人類が生存・得る新しい技術体系を構築する学問領域である。このような地球システムの工学を「地球システム工学」と呼ぶことにする。
このような新たな学問領域創出と新たな技術体系確立のためには、既往の学問分野からの協力により速やかに研究体制を構築する必要がある。このためには、当面、研究プロジェクトを設定することにより効力な研究推進を行う必要がある。我が国がかかる学問領域を率先して創造し、国内外の研究者とともに地球環境問題解決に取り組むことは、研究開発力、技術力に対する海外の期待というものを考えた時、我が国の責務と考えられる。
平成2年（1990年）度共同主催国際会議
本会議は、昭和28年以降おおむね4件の学術関係国際会議を関係学術研究団体と共同主催してきたが、平成2年（1990年）度には、2件増えて、次の6国際会議を開催する。
■国際土壌科学会議
開催期間 平成2年8月12日～18日
開催場所 国立京都国際会館（京都市）
参加者数 国外1,000人、国内500人、計1,500人
共催団体 （社）日本土壌肥料科学会
■第15回国際微生物学会議
開催期間 平成2年9月13日～22日
開催場所 大阪城ホール外（大阪市）
参加者数 国外2,000人、国内3,500人、計5,500人
共催団体 日本微生物学協会
■第11回国際数学連合総会及び第21回国際数学者会議
開催期間 平成2年8月18日～29日
開催場所 神戸国際会議場（神戸市）外
参加者数 国外1,500人、国内2,000人、計3,500人
共催団体 （社）日本数学会外6学会
■第11回国際神経病理学会議
開催期間 平成2年9月1日～8日
開催場所 国立京都国際会館（京都市）
参加者数 国外600人、国内900人、計1,500人
共催団体 日本神経病理学会
■第5回国際生態学会議
開催期間 平成2年8月23日～30日
開催場所 横浜プリンスホテル（横浜市）
参加者数 国外900人、国内1,000人、計1,900人
共催団体 日本生態学会
二国間学術交流事業
日本学術会議では、二国間学術交流事業として、毎年2つの代表団を外国に派遣し、各訪問国の科学者等と学術上の諸問題について意見交換を行って、相互理解の促進を図る事業を行っている。
平成元年度には、①10月25日から11月4日まで、イタリア及びスイスへ、渡邊剛副会長以下5名の会員から成る代表団を、②12月4日から12日まで、インドへ、白石泰彦副会長以下7名の会員から成る代表団をそれぞれ派遣した。イタリア及びスイス派遣代表団は、イタリアでは、イタリア学術研究会議、大学・科学技術研究省、ローマ大学、ローマ日本文化会館など、スイスでは、スイス学術会議、連邦内務省教育・科学局、スイス科学財団、ベルン大学、チューリヒ大学などを訪問した。
各訪問先では、関係者との間で、それぞれの国の学術研究体制や科学技術政策などをめぐって意見交換が行われた。特に、イタリアでは、研究行政の一本化を図るために、大学を文部省の管轄からはずして、大学・科学技術研究省を設置していることについて、また、スイスでは、連邦政府とともに、強い権限を持つ州政府が存在する同国の行政の仕組みと学術行政との関連について、それぞれ熱心に意見の交換がなされた。
インド派遣代表団は、インド国家科学アカデミー、科学産業研究会議、計画委員会、科学技術庁、ネル大学、デリー大学、タタ基礎研究所などを訪問した。
各訪問先では、関係者との間で、科学技術振興方策及び両国間の今後の積極的な学術交流などをめぐって意見交換が行われ、また、政変の直後ということもあって、選挙の話から、経済力の向上、中産階級の躍進などの政治、経済の問題などについて熱心に意見の交換がなされた。
御意見・お問い合わせ等がありましたら、下記までお寄せください。
〒106 東京都港区六本木7-22-34
日本学術会議広報委員会 電話03(403)6291
| 賛助会員 | 住所 |
|----------|------|
| 旭化成工業株式会社 | 東京都千代田区有楽町1-1-2 三井ビル |
| 株式会社旭潜研 | 東京都北区栄町9-2 |
| 阿部嘉方 | 東京都練馬区春日町2-15-6 |
| 株式会社内田老鶴園内田悟 | 東京都文京区大塚3-34-3 |
| 有限会社英和出版印刷社 | 東京都豊島区駒込1-7-10 |
| 海上電機株式会社 | 東京都西多摩郡羽村町栄町3-1-5 |
| 倫海洋生物環境研究所 | 東京都千代田区内神田1-18-12 北原ビル内 |
| 株式会社川合海苔店 | 東京都大田区大森本町2-31-8 |
| 三信船舶電具株式会社 | 東京都千代田区神田1-16-8 |
| 昭和電装株式会社 | 高松市寺井町1079 |
| 新日本気象海洋株式会社 | 東京都世田谷区玉川3-14-5 |
| 全日本爬虫類皮革産業連合会 | 東京都足立区梅田4-3-18 |
| 株式会社高岡屋 | 東京都台東区上野6-7-22 |
| 株式会社鶴見精機 | 横浜市鶴見区鶴見中央2-2-20 |
| 株式会社東京久栄技術センター | 埼玉県川口市柴鶴ケ丸6906-10 |
| 株式会社東急フーズミート | 東京都品川区東品川4-10-21 |
| 株式会社西日本流体技研 | 長崎県佐世保市朔方町283 |
| 日本アクアラング株式会社 | 神奈川県厚木市温水2229-4 |
| 株式会社日立造船技術研究所バイオ研究センター宮本悟 | 大阪市此花区桜島1-3-22 |
| 株式会社読売広告社 | 東京都中央区かきどき3-3-5 かきどきビル 神本地郷 |
| 渡辺機開工業株式会社 | 東京都中央区銀座1-8-14 |
| 株式会社渡部計器製作所 | 愛知県渥美郡田原町神戸大坪230 |
| | 東京都文京区向丘1-7-17 |
スライド式高速曳航体
Underwater Sliding Vehicle System (USV)
○本システムは海洋科学技術センター殿の御指導によって開発されました。
○USVは小型かつ軽量で極めて優れた水中運動性能を有しております。
○電磁誘導伝送方式を使うことにより船上からのUSVの昇降運動制御及びリアルタイム信号モニターリングを行うことができます。
○取得データはFD及びハードディスクに記録し2次電算機処理に供します。
SPECIFICATIONS
Towing Speed 0～8knots
Operation Depth: Max 400m
Tow Cable 8mm hydrodynamically fared stainless steel wire rope with polyurethane coating
Sensor Range Accuracy
Conductivity 20～70ms ±0.05ms
Temperature −2～35℃ ±0.05℃
Depth 0～400dbar 0.5%FS
Data Transport Inductive Coupling Data Communication System
Sampling Rate 5times per second
Sensor Battery 50hours
Life
T.Sスライド式高速曳航体によって得られた三陸沖の水温鉛直分布のカラー画像。
（海洋科学技術センター殿から資料を御提供頂きました。）
Trajectory of the USV
本社 〒230 神奈川県横浜市鶴見区鶴見中央2-2-20
TEL.(045) 521-5252 FAX.(045) 521-1717
白河工場 〒969-03 福島県西白河郡大信村大字中新城字弥平田
TEL.(0248) 46-3131 FAX.(0248) 46-2288
株式会社 鶴見精機 TSK AMERICA INC.
828 MILLS PL.N.E. NORTH BEND, WA. 98045, U.S.A.
TEL.206-888-3404 TLX.230754235 TSKA SEA UD
応援します。
良い海苔づくり—
生海苔活性調整機
RS-2型
海苔の等級が数段あがる
※生海苔を活性化し、海苔製品の表面をなめらかに光沢よく仕上げます。
渡辺機開工業株式会社
愛知県渥美郡田原町神戸大坪230
電話 05312（2）1121（代表）
当社は環境アセスメントを始め環境の質を把握するため、水域及び大気環境調査から分析・予測・解析まで一貫してユーザーの要望に応える環境総合コンサルタントです。
★海洋，河川，ダム湖，湖沼，道路，鉄道の環境実態調査
水質・底質・プランクトン・底生生物・魚類・鳥類・哺乳動物・植生
★海域，河川，ダム湖，湖沼の水質予測解析
潮流・恒流・吹送流解析，COD拡散解析，SS沈降拡散，富栄養化予測解析，ダム湖の水温・濁度予測解析
★環境アセスメント調査
港湾・空港・大規模工業団地・石油精製・石油基地・海の公園・人工海浜造成計画等の環境アセスメント調査
河川・ダム湖・河口堰・鉄道計画等の環境アセスメント調査
★分析・実験
水質分析，底質分析，土壌分析，産業廃棄物分析，生物分析，天気分析，水質汚濁機構解析のための生産量，分解量，溶出量，酸素消費量実験およびAGP試験，土砂の沈降試験，ノリの成育実験，魚類室内実験，土壌中の有害物質の植物検定
★気象海象観測，予報，解析
天気予報，気象観測整理解析，降雨污水解析，大気・騒音・振動の環境調査，波浪推算調査，波浪予報，漂砂調査，大気拡散シミュレーション
新日本気象海洋株式会社
本社 東京都世田谷区玉川3-14-5 TEL 03-708-1161
環境分析研究所 東京都目黒区上目黒4-17-18 TEL 03-793-0591
大阪支店 大阪市西区江戸堀3-2-23 TEL 06-448-2551
九州事務所 福岡県北九州市小倉区片野新町1-15 TEL 093-922-2214
事業所 釜石（岩手県），小名浜（福島県），金沢（石川県），沖縄
Pearl & Jewelry
JEWELER miwa
No.7-2, 6-CHOME, GINZA,
TOKYO Phone(03)572-5011
営業案内
○科学魚探SIMRAD
○理研式GEK
○曳航式水温計
D. B. T. 水中テレビジョン 採泥器類
C / S T D 自記流向流速計 電気流速計
水中照度計 比重計、水色計、標準海水 船用機器模型及標本類
水中濁度計 アクアラング 標識票類
溶存酸素測定器 プランクトンネット類
サリノメーター 採水器類
株式会社 本地郷
東京都中央区勝どき3丁目3番5号 かちどきビル内 〒104 TEL 533-7771（代）
TELEFAX 533-4094
代表取締役 宮本悟 取締役 大塚昌治
海苔の養殖から販売までの専門会社
まごころプラスワン 幸福のり TAKAOKAYA
株式会社 高岡屋
東京都台東区上野6丁目7番22号
Takaokaya Company Limited.
7-22, Ueno 6-chome, Taito-ku, Tokyo, 110 JAPAN
日仏海洋学会入会申込書
（正会員）
| 氏名 | 年度より入会 | 年 月 日申込 |
|------|--------------|-------------|
| ローマ字 | 年 月 日生 |
| 住所 | 勤務先機関名 | 電話 |
|------|--------------|------|
| 自宅住所 | 電話 |
|---------|------|
| 紹介会員氏名 |
|--------------|
| 送付金額 | 円 | 送金方法 |
|----------|----|----------|
| 会誌の送り先（希望する方に○をつける） | 勤務先 | 自宅 |
|---------------------------------|--------|------|
（以下は学会事務局用）
| 受付 | 名簿原簿 | 会費原簿 | あて名カード | 学会記事 |
|------|----------|----------|-------------|----------|
入会申込書送付先：〒101 東京都千代田区神田駿河台2-3
（財）日仏会館内
日仏海洋学会
郵便振替番号：東京5-96503
日仏海洋学会編集委員会（1988-1989）
委員長：有賀祐勝
委員：青木三郎，半沢正男，堀越増興，鎌谷明善，前田昌調，村野正昭，小倉通男，岡部史郎，須藤英雄，
柳哲雄
海外委員：H. J. CECCALDI（フランス），E. D. GOLDBERG（アメリカ），T. ICHIYE（アメリカ），T. R. PARSONS（カナダ）
幹事：渡辺精一，山口征矢
投稿の手引
1. 「うみ」（日仏海洋学会機関誌；欧文誌名 La mer）は、日仏海洋学会正会員およびそれに準ずる非会員からの投稿（依頼稿を含む）を、委員会の審査により掲載する。
2. 原稿は海洋学および水産学両分野の原著論文、原著短報、総説、書評、資料などとする。すべての投稿は、本文、原図とも正副2通とする。副本は複写でよい。本文原稿用紙はすべてA4判とし、400字詰原稿用紙（和文）に、または厚手白紙にダブル・スペース（和文ワープロでは相当間隔）で記入する。表原稿および図説明原稿は、それぞれ本文原稿とは別紙とする。
3. 用語は日、仏、英3カ国語の何れかとする。ただし、表および図説明の用語は仏文または英文に限る。原著論文（前項）には約200語の英文または仏文の要旨を、別紙として必ず添える。なお、欧文論文には、上記要旨の外に、約500字の和文要旨をも添える。ただし、日本語圏外からの投稿の和文要旨については編集委員会の責任とする。
4. 投稿原稿の体裁形式は最近号掲載記事のそれに従う。著者名は略記しない。記号略号の表記は委員会の基準に従う。引用文献の提示形式は、雑誌論文、単行本分載論文（単行本の一部引用を含む）、単行本などの別による基準に従う。
5. 原図は版下用として鮮明で、縮尺（版幅または1/2版幅）に耐えられるものとする。
6. 初校に限り著者の校正を受ける。
7. 正会員に対しては7印刷ページまでの掲載を無料とする。ただし、この範囲内であっても色彩印刷を含む場合などには、別に所定の費用を著者負担することがある。正会員の投稿で上記限度を超える分および非会員投稿の印刷美費はすべて著者負担とする。
8. すべての投稿記事について、1篇あたり別刷50部を無料で請求できる。50部を超える分は請求により、50部単位で作製される。別刷請求用紙は初校と同時に配布される。
9. 原稿の送り先は下記の通り。
〒108 東京都港区港南4-5-7 東京水産大学 有賀祐勝 気付
日仏海洋学会編集委員会
1990年2月25日印刷
1990年2月28日発行
定価 ¥1,600
編集者 有賀祐勝
発行所 日仏海洋学会
財団法人 日仏会館内
東京都千代田区神田駿河台2-3
郵便番号:101
電話:03(291)1141
振替番号:東京5-96503
印刷者 佐藤一
印刷所 有限会社英和出版印刷社
東京都豊島区駒込1-7-10
郵便番号:170
電話:03(941)6500
Tome 28 N°1
SOMMAIRE
Article spécial
The role of cryptozoology in achieving an exhaustive inventory of the marine fauna .......................................................... Paul H. LEBLOND 1
Notes originales
Internal tidal waves and internal long period waves in the Sanriku coastal seas, eastern coast of northern Japan ................................................. Moriyoshi OKAZAKI 5
Density fluctuation of caprellid amphipods (Crustacea) inhabiting the red alga Gelidium amansii (LAMOUROUX) LAMOUROUX, with emphasis on Caprella okadai ARIMOTO .................................................. Ichiro TAKEUCHI, Hiroshi YAMAKAWA and Masamu FUJIWARA 30
Seasonal changes of the Secchi disc depth and suspended solid at six stations along the main channel of the High Dam Lake, Egypt ........................................... OLFAT Anwar Habib, IBRAHIM Omar Mohamed, MOHAMED Shehata Mohamed and Yusho ARUGA 37
Characterization of the environment in the Beppu Bay, Iyo Nada and Bungo Channel (in Japanese) .................................................. Naoji KUBOTA, Tadashi HANO, Yusaku TAKITA, Takashi SHIMAZAKI and Fumiaki HORI 48
Faits divers
Two patterns of typhoon-induced storm surges around the Japanese Islands (in Japanese) .......................................................... Shigehisa NAKAMURA 58
Procès-verbaux ............................................................................................................. 63
第28巻 第1号
目次
特別寄稿
未確認動物学の海産動物完全目録完成上の役割（英文） ........................................... Paul H. LEBLOND 1
原 著
三陸沿岸における内部潮汐波と内部長周期波（英文） ........................................... 岡崎守良 5
マクサ上のワレカラ類、特にオカダワレカラの密度の周年変動（英文） ............ 竹内一郎・山川絵・藤原正夢 30
ハイダム湖（エジプト）の主水路に沿う6測点における透明度と懸濁物の季節的変動（英文） ........................................... OLFAT Anwar Habib, IBRAHIM Omar Mohamed, MOHAMED Shehata Mohamed and Yusho ARUGA 37
別府湾を含む伊予灘および豊後水道海域における海況特性の解析 .................. 久保田直治・羽野忠・滝田祐作・島崎孝・堀文昭 48
資 料
日本列島周辺の台風高潮パターン2例 .......................................................... 中村重久 58
学会記事 ............................................................................................................. 63
|
OUR MISSION STATEMENT:
"Saint Francis Xavier Parish is a Christian Family dedicated to the mission of Jesus and the teachings of the Catholic Church. We are committed to fostering spiritual growth and to responding to the human needs of both our faith community and the larger community by sharing our gifts and resources."
Liturgy Schedule
Weekend Masses:
Saturday Evening Vigil: 4:30 pm
Sunday Masses: 8:30 am & 10:45 am
Holy Day Masses: See schedule inside.
Daily Mass: 6:45 am Mon, Wed, Thur, Fri
Weekend Mass Schedule in our area
| St. Michael-St. Peter | St. Mary's of the Lake | St. Patrick's Jordan |
|-----------------------|------------------------|---------------------|
| Sat. Evening: 5:00 pm | Sat. Evening: 4:30 pm | Sat. Evening: 5:00 pm |
| Sunday: 7:30, 9:00 & 11:15 am | Sunday: 8:00 & 10:15 am | Sunday: 8:00 & 10:00 am |
PASTORAL STAFF:
Pastor: Rev. Daniel C. Muscalino*
Pastoral Associate: Deacon John J. Falge*
Religious Ed. Director: Renee O'Connor*
Parish Secretaries: Mary Jo Osterman
Katherine Boos
Social Service Coordinators:
Rosalie Durand
Patty White
Music Director: Robin Schott, Jr.
Buildings and Grounds: Ron Schneider
Housekeeper: Kathy Welsh
Parish Historian: John P. Curtin
Confirmation Coordinators:
Laurie LeFever
Laura Carver
PARISH TRUSTEES:
Diane Foster*
Joseph E. Durand*
* Also on Parish Council
SACRAMENTS:
Baptism:
Class required.
Call rectory for arrangements.
Reconciliation: Sat. 3:30 - 4:00
Marriage: Arrangements at least 6 months in advance.
NEW PARISHIONERS are most welcome.
See Fr. Muscalino after Mass or call the Rectory.
PARISH COUNCIL
Jay Austin
Terry Hall
Pen Anderson
Cheryl Hunt
Beth Cusick
Roxanne Taylor
Kim Flood
Marty McClusky
Terry Kotlarz
Carter Austin
FINANCE COMMITTEE:
Rick Reagan
Chris Hunt
Diane Foster
Rev. Daniel C. Muscalino
Joseph E. Durand
Jay Austin
Mass Intentions FOR THE WEEK
Saturday May 15
VIGIL, SEVENTH SUNDAY OF EASTER
4:30 PM- People of St. Francis Xavier Parish
Sunday May 16
SEVENTH SUNDAY OF EASTER
8:30 AM- Jeanette Santangelo- Anita, Doug & Shirley Salerno
10:45 AM- James Reagan- John & Maureen Curtin
Monday May 17
6:45 AM- Fran Ciaccio- John & Lynda Quinn & Family
Tuesday May 18
VIGIL, SAINT JOHN I
6:00 PM- Elizabeth & Fred Siddall – Skvarch Family
Wednesday May 19
No Mass
Thursday May 20
SAINT BERNARDINE OF SIENA
6:45 AM- Joe Fenlon (anniversary & birthday)- Terry Kotlarz
Friday May 21
SAINT CHRISTOPHER MAGALLANES
6:45 AM- Helen & John Steele- Skvarch Family
Saturday May 22
VIGIL, PENTECOST SUNDAY
4:30 PM- Clint Tallman- John & Maureen Curtin
Sunday May 23
PENTECOST SUNDAY
8:30 AM- Joseph Delaney- Gloria Weeks and Family
10:45 AM- People of St. Francis Xavier Parish
Information Regarding Mass & Confession
**Mass:** The dispensation from the obligation of attending Mass on Sundays and Holy days of Obligation is still in effect, but only for those who need it because of health or age. You may view the weekend Mass on our Facebook page or through the link on our website. sfxmarcellus.com under events
**Confession:** We have confessions each Saturday in the Church from 3:30-4:00 PM. Fr. Dan will hear confessions in the Sacristy. Please wear a mask and maintain social distancing while waiting in the pews. Please do NOT line up outside of the Sacristy, when the door nearest St. Francis Xavier's statue is closed you will know that Fr. Dan is not free, when it is open, you are welcome to come in for confession. Thank you.
Armed Forces Day - Saturday, May 15th
Thank you to all the men and women serving in the Armed Forces.
What’s Happening This Week at St. Francis
SIGN UP online if you plan on attending Mass & review safety measures.
Note: We print the weekend Mass schedule Friday at noon. If you sign up online after sign-up sheets are printed or if you do not sign-up online, we will simply write down your information when you arrive for Mass. Please do NOT let this prevent you from coming to Mass. www.sfxmarcellus.com/events
Wednesday May 19
Parish Council Meeting- Ch Hall- 7:00pm
Our Weekly Collection
Weekly Goal: $7,900 Last Week: $7,467
Please Pray For...
Eileen Smith, Msgr. Bob Yeazel, Greta Petterson, Robert Hammond, Richard Hammond, Isabelle Callahan, Dennis McEnery, Riley Christine Perkins, Nick Olenych, Roger Schott, Danielle Painter, Genie Barnes, Nicole DeMore, Lisa Pikarski, Michelle Fleegeel, Jo-Ann Foercht, Justin MacLachlan, Jack Bragger, Missy Fitz, Austin Marlowe, Brian Stonelake, Brett Kresco, Lucienne Henneberry, Allan Stonecipher, Donna Martin, J.E.L., Kelly Cunningham Suberts, Sr. Mary Brendan Wilson
Please Pray for Our Homebound...
Isabelle LaClair, Helen Volcko, Mike Berish, Marie Patterson, Mary Martin, Ethel Cole
Welcome New Parishioners!
Are you a new parishioner or visiting for the 1st time? Welcome! Please introduce yourself to Fr. Dan & Dc. John. We invite you to become an active participant of our parish community.
St. Francis Xavier New Parishioner Registration
Name ____________________________
Address ____________________________
_______________________________
Phone ____________________________
Email ____________________________
Fill out and drop in collection basket. We will mail you a registration form.
Young people of our parish: WE NEED YOU!
Any boy or girl who made First Communion; please consider becoming an Altar Server. There will be a training: Sunday, May 23rd at 1:00pm.
Rite of Christian Initiation of Adults
Are you or do you know someone interested in becoming Catholic?
Were you baptized in another Christian denomination?
Were you baptized Catholic, but have not celebrated the Sacraments of Eucharist and Confirmation?
Through the RCIA process, you have the opportunity of becoming fully initiated Catholics. Please call the church office for more info: (315) 673-2531
Bishop Lucia has decided to declare a Year of Vocations in the Diocese of Syracuse under the patronage of St. Joseph. St. Francis Xavier is hosting the Icon of St. Joseph from Saturday, June 5th - June 12th. The Icon is traveling around the diocese promoting Vocations. We are encouraged to intercede to St. Joseph for all discerning their vocations.
PACE CNY (Program of All-inclusive Care for the Elderly) is a part of the Loretto Family of Care. PACE CNY, based on a national model known as PACE, provides an alternative to placement in a nursing home. PACE CNY is particularly helpful to families desiring to keep their aging loved ones at home. At two Loretto PACE Centers (one in North Syracuse and one in East Syracuse) and their own place of residence, participants receive the home care, medical, rehabilitative, social, transportation, and support services needed to remain at home. For more information or to make a referral, please call Ginny Turley, Intake Specialist, at (315) 452-5800 and visit our website at www.pacecny.org.
Holy Father’s Prayer Intentions
Universal – The world of finance
Let us pray that those in charge of finance will work with governments to regulate the financial sphere and protect citizens from its dangers.
St. Francis Website
The weekly bulletin is available on our website: sfxmarcellus.com. Look under Our Parish-Weekly Bulletins. The new bulletin is available on Wednesday for the upcoming weekend.
May 16, 2021
Seventh Sunday of Easter
Scripture Readings
Acts 1:15-17, 20a, 20c-26
Ps 103:1-2, 11-12, 19-20
1 Jn 4:11-16
Jn 17:11b-19
A Family Perspective by Bud Ozar – Jesus was worried about His disciples and He turned to prayer. No matter how much time we spent trying to “guard and protect” our children, each day they become less dependent on us and we must let go and let God. Prayer and parenting go together.
Question of the Week – Ascension of the Lord
Who do you know who has passed away yet whose spirit is still alive and making an impact on others?
OFFICIAL PRAYER FOR THE
Year of Vocations
GOD, MY FATHER,
YOU CREATED ME WITH A SPECIFIC PURPOSE
FOR MY LIFE; THIS IS MY VOCATION.
BY FOLLOWING YOUR PLAN,
I WILL BE HAPPY ON EARTH,
EARN THE REWARD OF HEAVEN,
AND HELP OTHERS TO DO THE SAME.
PLEASE HELP ME TO HEAR,
UNDERSTAND, AND FOLLOW
YOUR CALL WITH MY WHOLE HEART,
ESPECIALLY WHEN IT SEEMS MOST DIFFICULT.
SAINT JOSEPH, PATRON OF THE
UNIVERSAL CHURCH,
PRAY FOR ME TO KNOW AND ACCEPT,
GOD’S WILL FOR MY LIFE.
AMEN.
Vocation Corner – Mark 16:15-20
“But they went forth and preached everywhere, while the Lord worked with them.” Christ called his disciples to go out and preach the gospel to “every creature”. As His disciples, how are you being called to go and spread the gospel to all the world?
Each week we will print one name of our Seminarians so that we can remember them specifically, by name, in prayer throughout the week. This week we remember in prayer: Gianni Verginio.
Caring
Courteous
Confidential
Ryan Funeral Home
Mary Ryan Carlton
44 East Main Street
Marcellus, NY
315-673-2345
Reagan and Dailey
Attorney at Law
18 East Main Street • Marcellus, NY
ph: 673-4864 • Mary Reagan Dailey
General Practice of Law, including Estate Planning, Probate, Real Estate
DEPENDABLE DISPOSAL
472-7455
52 Weeks of reliable Service, locally owned & operated
The Best Coverage at the Best Price!
We've been a faithful member of your community for 90 years.
Our clients are our friends and neighbors.
We're dedicated to providing the best customer care you'd expect from someone you know.
Reagan Companies
Risk Management and Investment Services
R.E. Main Street • P.O. Box 191, Marcellus, New York 13108
315.673.2094 800.777.2094
Auto • Home • Boat • Umbrella ReaganCompanies.com
McClurg
DESIGN BUILD REMODEL REPAIR
(315) 673-2051
www.McClurgTeam.com
ELITE PROPERTY MAINTENANCE, LLC
Driveway Sealing • Lawn & Landscaping
Snow Plowing • Interior Painting
FULLY INSURED FREE ESTIMATES
www.elitesyracuse.com 315-882-7430
PAINTING
interior/exterior
Mitch Rudy
315-254-0355
over 30 years exp. free estimates
email@example.com
Plis Funeral Home, Inc.
“Continuing the Commitment”
33 North Street
Marcellus, New York 13108
315-673-2017
www.plisfuneralhome.com
For Advertising Information
Call 463-1923
GEDDES FEDERAL SAVINGS
and Loan Association
2208 W. Genesee St., Syracuse
240 W. Seneca St., Manlius
(315) 468-6281
MAIN STREET PHARMACY
of Marcellus
OPEN M-F
8:30am-6pm
Sat 8:30am-12:30pm
Many thanks to our wonderful community for your continued support of our local business.
11 E. MAIN ST. MARCELLUS
(315) 673-2410
‘CUSE CARD GUYS
FOOTBALL • BASEBALL • BASKETBALL • POKEMON
We’ll Pay CASH For Your Cards & Collections
firstname.lastname@example.org 315-416-9565
SKINNER ASSOCIATES REALTY
Ready to meet your buying or selling needs
Owner & Broker
Stephen Skinner
315.373.6896
email@example.com
MICHAEL P. KACZOR, D.D.S.
Office Hours by Appointment
Phone: 315-487-2828 5109 W. Genesee St.
Fax: 315-487-2825 Camillus, NY 13031
SUPERIOR SP PRINTING
“Your Full Service Offset Printer Since 1980”
★ Business Cards
★ Newsletters
★ Fax Service
★ Raffle Tickets
★ Letterheads
★ Laminating
★ Business Forms
★ Envelopes
★ Complete Bindery Services
★ Color Copies
★ Black & White Copies
Steve Rudy 315-463-1923 2025 Teall Avenue
|
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About Us
2008
Edumilestones.com is a pioneer in online career counselling platform industry
Business Partners
1500+ career counsellors, Educationists, professional across 80+ location in India
Career Assessment
Most comprehensive assessment, considered as a benchmark in the industry
Reports
60k+ reports are generated by our counselling partners
Location
We are based out of Bangalore
Team
Career counsellors, psychologists, writers, developers, research associates
Branded Login Page and Career Library Snapshot
Explore and Find out your Most Suitable Career Path Out of 18 Career Clusters, 150+ Career Paths and 3000+ Occupations
Start Career Assessment
Career Counselling
1. Five critical factors while making career choices are:
- Individual Interests
- Career Values
- Personality
- Learning Style
- Skills & Abilities
2. Career counselling helps to identify these critical factors and help the students.
About Us
The mission of evolve is to be there for the kids, parents, teachers, Principals and all those join hands to grow for a better tomorrow. As evolve we envision a future wherein your child is able to transit through each phase of life growing as an independent and emotionally strong individual who is looking forward to be a contributing member of this society.
About Counsellor
I have been working with children as Special Educator for the last 16 years with various organisations under different setups.
I was associated for 11 years with Bombay Cambridge Group of Schools as a Special Ed and later as the Resource Coordinator. Apart from regular remedial sessions, I planned and executed the Career Awareness Program for the students each year where we discussed their Aptitude test results and professions.
evolve
I was attached with the Learning Disability Clinic at LTMG Hospital for 3 years after which I started my own setup under the name ‘evolve’ where I can cater to the needs of students, parents and teachers with services like Career Counseling, Arts Based Therapy, Occupational Therapy and Counseling.
Career Reports & Recommendations Snapshot
Your Career Clusters
Career Clusters are groups of similar occupations and industries that require similar skills. It provides a career roadmap for pursuing further education and career opportunities. They help you connect your Education with your Career Planning. Career Cluster helps you narrow down your occupation choices based on your assessment responses. Results show which career clusters would be best to explore. A simple graph report shows how you have scored on each of the career clusters.
Your Career Paths
| Career Cluster 1: Government Services |
|--------------------------------------|
| **Interest Cluster** | **Career Interest** | **Skill and Abilities** | **Comment** |
| Civil Administrative Services | Very High:70 | High:62 | Top Choice |
| Indian Economic Services (IES) | High:62 | Average:54 | Good Choice |
| Staff Selection Commission (SSC) | Average:51 | High:62 | Optional |
| Career Cluster 2: Business Management |
|---------------------------------------|
| **Interest Cluster** | **Career Interest** | **Skill and Abilities** | **Comment** |
| Human Resource | High:69 | High:67 | Good Choice |
| Administrative And Operations Support | High:68 | Average:58 | Good Choice |
| Business Financial Management | Average:58 | Average:53 | Optional |
| BPO | Average:55 | High:67 | Optional |
Business page and reviews of career counsellors powered by edumilestones
Career Counselling in Faridabad - Calibre connect
4.9 ★★★★★ 13 Google reviews
Career consultant in Faridabad, India
Address: B-410, 411 Nehru Ground., Faridabad, Haryana 121007
Hours: Open today · 9AM–7PM
Phone: 098100 59933
Career Counselling in Mumbai By Snehal Vyas - CNA VEDSHALA
5.0 ★★★★★ 1 Google review
Career guidance service in Mumbai, India
Address: B/7, 1st Floor, Ehanu Park, Adukla Road, Near Station Kandiwalli West, Mumbai, Maharashtra 400067
Hours: Open today · 9:30AM–6:30PM
Phone: 096993 58444
Jacqueline Career Counselling Center Mumbai
Website Directions
Career guidance service in Mumbai, India
Address: Oasis, No.7, Yagnik Nagar, Amboli, Andheri west, Mumbai, Andheri West, Maharashtra 400058
Hours: Open today · 12AM–7:30PM
Phone: 093224 07617
Best Career Counselling in Chennai- Kriya Career Counseling Centre
4.9 ★★★★★ 17 Google reviews
Student career counseling office in Chennai, India
Address: 12, I Main Road, Sivagami Nagar., Chitlapakkam, Chennai, Tamil Nadu 600064
Hours: Open today · 8AM–8PM
Phone: 089396 21119
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Course Content
- Psychometric Theories and Career Analysis
- Module: 1
- Personality: 2min 3Sec
- Personality Types: 4min 52Sec
Career Assessment
4 Dimensional Career Assessment + 8 skills and Abilities
- Career Interest
- Personality
- Career Values
- Learning Style
Explore Out of 16+ Career Clusters, 120+ Career Paths and 3000+ Occupations
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Set Your Child’s Biological Clock for Better Sleep
**DIM THE LIGHTS AT NIGHT**
Darkness causes a release of melatonin, the body's natural sleep hormone. Help your child feel tired by dimming the lights the hour before bedtime, and keeping the room dark all night.
**HAVE BRIGHT DAYS**
Light signals the brain to be alert. Natural daylight is the best, particularly in the morning. Aim for time outside or near a window every day. Getting outside is best, whenever you can do it!
**KEEP SLEEP HOURS HUSHED**
If your child wakes for feedings, diaper changes or potty runs - keep the lights low, your voices quiet and movements slow and relaxed to maintain the sleepy mood.
**GIVE PLENTY OF DAYTIME FOOD**
Babies and young children are prone to shorting daytime calories and then waking at night hungry. Focus on providing plenty of breastfeeding, or healthy meals and snacks throughout the day.
**STICK TO A BED TIME**
A staggered bed time is like creating jet lag every day. Putting your child to bed at the same time every night sets an internal clock. If your child is a finicky sleeper this tip might really do the trick.
**KEEP A GOOD NAP ROUTINE**
Children who don't nap get overtired and wired. They find it hard to fall asleep at night. Their fatigue interferes with their body's natural rhythms.
**HAVE A JOYFUL SEND-OFF**
Your bedtime routine should be relaxed and happy. Book reading, lullabies, massage, positive conversation and quiet connection. A peaceful mind lets a child fall asleep easier.
Signs that your child needs a DAILY NAP
RESISTS TAKING A NAP
... but eventually does fall asleep - and then sleeps an hour or longer.
WAKES UP IN A GOOD MOOD
... but gets cranky, whiny or short-tempered as the day goes on.
IS TYPICALLY PATIENT
... but is much more easily aggravated or frustrated later on.
IS COORDINATED IN THE MORNING
... but later falls down, or can't manage a puzzle, or has trouble tying their shoes.
HAS AN AFTER-DINNER SLUMP IN ENERGY
... but after the lull gets a second wind, maybe even too much wind!
HAS A NORMAL PACE
... but later in the day becomes wild, wired up or hyper-active.
CHATS & PLAYS IN THE CAR
... but later in the afternoon or early evening often falls asleep during a drive.
WAKES UP EASY WHEN WELL-RESTED
... but the day after missing a nap has a hard time waking up in the morning.
The No-Cry Sleep Solution ~ NoCrySolution.com
Does separation anxiety hit when you drop your child off? Here are some ideas to help make good-byes easier.
1. **Create very specific routines**
Do and say the same things for every drop-off and pick-up. Include a special kiss/hug/handshake/good-bye phrase: See ya later alligator!
2. **Set an arrival cue each day**
Link your return to a certain activity, such as after nap, playground or snack time. Let your child know when to expect you back. "I’ll be back right after your snack time."
3. **Provide an adjustment period**
At drop-off & pick-up allow a 5 minute adjustment period. Ask your kid to show you something fun. When it’s time to go, follow your good-bye routine.
4. **Build friendships at playdates**
Organize play sessions away from school to allow kids to develop more personal friendships. The connection between them gives security when they are away from home.
5. **Plan arrival with other kids**
Walk or ride to school with another family. Having friends to walk into the building with can change the dynamics of the drop off routine.
6. **Have fun on the journey**
Bring a snack bag every day. Play a game, such as I-spy, or count all the red cars you see. Discuss things your child can plan for, or share memories about fun times they’ve had.
7. **Stay calm if your child isn’t**
Your child desperately needs your calm and peaceful reassurance. Tune out the other kids and parents so that you can focus on your child only for the moment.
How to Build a Better Bedtime Routine
MAKE A RITUAL TO ENJOY
... convey a feeling of value. This is a great way for your family to connect at the end of every day.
MAKE THE FIRST THING FUN
... make the first step easy and nice so it won't be avoided. Be creative - the routine doesn't have to be boring.
MAKE IT DARK AND COZY
... dimmer lighting brings on sleepiness. Use battery-operated candles, twinkle lights, or night lights with dim bulbs.
LISTEN TO AN AUDIO BOOK
... turn off the lights and listen to a story. This keeps kids in bed and builds a happy, relaxing and enriching habit to end each day.
PICK A TIME & STICK TO IT
... begin your routine at the same time every night. Set an alarm with a funny sound (dog bark!) to signal it's time.
CREATE A WALL CHART
... make a poster that shows all the steps in your routine. Hang it up and let your child guide the process.
DON'T RUSH THE PACE
... If you try to get it done quickly it will take longer! Take a breath. Find a way to enjoy this part of the night.
USE SOFT SOUNDS FOR RESTFUL AMBIENCE
... gentle background music or sounds of rain or ocean waves can help kids and parents to relax.
The No-Cry Sleep Solution ~ NoCrySolution.com
How to Get your Picky Eater to Try New Foods
FIRST, PRIORITIZE
... Is your child’s overall diet healthy? They shouldn’t have to taste everything. Relax – take the pressure off.
TEENY-TINY BITS
... Start small – two chickpeas! Let your kid poke it, smell it, and smash it. Experimentation is the first step.
MAKE IT FAMILIAR
... Put a bit on your child's plate three times a week. Exposure promotes comfort – so it’s no longer new.
MONKEY SEE – MONKEY DO
... Let your child see you eat the new food. If YOU actually enjoy it, your kid might decide it's something to try.
SHOW AN EXAMPLE
... Offer another adult a taste. When they declare it tasty your child may be more willing to take a bite.
MAKE IT AN APPETIZER
... Try putting it out before dinner is served. If it's the first thing offered, it might be more tempting.
BE PATIENT & PERSISTENT
... It may take a while. This isn’t something you fix in a day, but will improve gradually one food at a time.
Your child may not seem overtired, because kids don’t always act tired — at least not in the ways we expect. But chronic overtiredness can affect daytime behavior, learning and growth. Here are the signs. If you see a few of these then work hard to improve your child's sleep.
1. **TENDS TO BE HYPERACTIVE**
- Is wired or amped up, especially at times when it makes more sense to be tired
2. **RESISTS SLEEP AT BEDTIME**
- Is restless and has difficulty settling down and falling asleep when put into bed
3. **IS STUBBORN & DAWDLES**
- Is often sluggish or slow-to-start and requires prodding to get going, particularly in the morning
4. **NODS OFF DURING THE DAY**
- Falls asleep often when in the car, bus or train, or when reading or watching TV
5. **IS NOT AN EARLY BIRD**
- Sleeps later in the morning – or takes a longer nap – on those days when the house is quiet
6. **WAKES UP SLOWLY**
- Is grumpy when awoken or takes a long time to become fully alert in the morning
Get Your Child to Happily Cooperate!
OFFER CHOICES
Let your child choose between two or three things. It sets a tone of collaboration and respect.
SET PRIORITIES
“When/Then” is clear and polite: “WHEN you finish your homework, THEN you may play your game.”
PICK YOUR BATTLES
... BUT DON'T PICK EVERY ONE. Every single issue doesn't have to be a struggle. Let some things go for now - or for good.
STATE A FACT
Make an observation, “There are dirty dishes in your room. What do you think needs to be done?”
HAVE SOME FUN
Children aren't serious like we are. Almost any task can be made fun with a race, contest or a silly prize.
MAKE IT TALK
Give a voice to a green bean, a sock, the trash can, or even your hand. It's MAGIC with little kids and will make big kids laugh!
How to Help Siblings Become Friends
Appreciate your children's differences... and their similarities, too. Teach them to do the same. They are different people and can learn to respect each other.
Give them time to play... Together and alone, both organized and unstructured, with and without you around: it's all important.
Find interests they share... and arrange ways for them to pursue those interests. If they like different things, encourage them to support each other.
Catch them being good... and reward them for getting along with positive attention. Don't wait until there's a problem to step in -- jump in with compliments, too.
Let them complain to each other about things that make them mad (including you)... It builds their bond and gives them someone to talk to about big emotions.
Read & eat dinner as a family... Having these two activities all together as a family on a regular basis creates connection and unity.
Don't referee every skirmish... You shouldn't dictate all the answers. Teach the kids how to negotiate and compromise with each other to find their best solution.
Teach that 'fair' doesn't always mean 'equal.'... Help each child focus on their own personal needs rather than what a sibling has.
Fix sticking points... Don't let them continue to fight over the same things day after day. Solve repeat issues by helping them come up with solutions.
Think of lifetime goals... Don't focus on this one moment only, think long term. Aim towards having them be friends when they become adults.
GENTLE No-Cry WAYS TO IMPROVE YOUR BABY’S SLEEP
You desperately need sleep.
You don’t want to make your baby cry.
Here’s how to help your baby sleep better.
1. KNOW THAT YOU DON’T HAVE TO FOLLOW UNWANTED ADVICE
Your baby is unique. Your family is unique. There are no one-size-fits-all answers. Find what works best for your baby and for YOU.
2. LEARN TO READ YOUR BABY’S OWN TIRED SIGNALS
Watch your child for signs of tiredness, then put your baby immediately to bed. Missing the signs creates over-tiredness.
3. PAY ATTENTION TO YOUR BABY’S SPAN OF AWAKE TIME
Newborns can be happily awake for only an hour or so. By 3 months it’s two hours. At 6 months it’s three hours. Tired = cranky.
4. WORK WITH BIOLOGY
Darkness increases the body’s sleep hormone – a biological “stop” button. So dim the lights the hour before bedtime.
5. LET YOUR SLEEPING BABY SLEEP
Babies are noisy, active sleepers. These sounds and motions don’t mean they need your attention. Quietly watch and wait.
6. USE THE MAGIC OF LULLABIES OR WHITE NOISE
Soft background sounds soothe babies to sleep. It also keeps them sleeping by blocking out loud, sharp noises.
7. PROVIDE GENTLE MOTION FOR RELAXATION
A rigid, unmoving crib can make it hard for some babies to settle. Gentle rocking can help Baby get tired & fall asleep easier.
The No-Cry Sleep Solution ~ NoCrySolution.com
Does your child have a sleep disorder?
Nightwaking and resisting sleep are normal, so how can you tell if it’s more?
Here is a list of symptoms associated with common children’s sleep disorders. If you have a feeling something isn’t right, and some of these apply to your child, give your doctor a call.
1. Snores loudly most nights, breathes through the mouth
2. Is a noisy sleeper; snorts, gasps, wheezes, coughs
3. Sweats heavily; restless; can’t get comfortable
4. Frequent or intense night terrors or nightmares
5. Sleeps in odd, contorted positions
6. Often wakes up with a headache or sore throat
7. Tired, inattentive, irritable or hyperactive during the day
The No-Cry Sleep Solution ~ NoCrySolution.com
|
AUGSA Minutes
Date: July 14, 6:00pm MDT
Place: Web conference
Attendance:
Executive Committee: Mary-Anne Parker (President), Ashley Ravenscroft (VP Operations & Finance), Judd Asoyuf (VP Academic), Crys Vincent (VP External)
Faculty of Business Representatives: Margaret Clappison, David Newman, Brandon Simmons
Faculty of Health Disciplines Representatives: Julia Cornester, Lindsay McNena
Faculty of Humanities and Social Sciences Representative: Kelli Buckreus, David Cloutier, Heather McGilvary, Bernard Kikechi
Faculty of Science and Technology: Philip Kirkbride, Liliana Quyen Tang
Staff: Meaghan Sullivan (Executive Director), Bob Cole (Communications Coordinator)
Guests: Christine Cao, Tanille Shandro (abGPAC Representatives)
Absent: Nicole Klix, Scott Howell
| | PRESENTER | ACTION | TIME |
|---|-----------|------------------------------------------------------------------------|------|
| 1.0 | President | The meeting was called to order at 6:02 pm MDT. | 6:02 pm |
| 2.0 | President | **Motion 2.0 To approve the agenda as distributed.**
Moved: Heather McGilvary
Seconded: Julia Cornester
Motion carried without opposition. | 6:05 pm |
| 3.0 | Approval of Previous Minutes | President | Motion 3.0 To approve the minutes of June 9, 2020.
Moved: Julia Cornester
Seconded: Heather McGilvary
Motion carried without opposition. | 6:07 pm |
|-----|-----------------------------|-----------|---------------------------------------------------------------------------------|--------|
| 4.0 | Reports and Presentations | | | |
| | 4.1 abGPAC Presentation | Tanille Shandro and Christina Cao | Crys Vincent introduced Tanille Shandro and Christine Cao from abGPAC. Tanille and Christine co-presented an overview of abGPAC, its structure, members, roles, diversity, and advocacy priorities.
2020-2021 Advocacy Priorities:
1. Graduate Student Wellbeing & Safety
2. Graduate Student Opportunities
3. A Strong/Sustainable/Accessible Post-Secondary System | 6:08 pm |
| | 4.2 Executive Reports | Executives | As per attached reports. | 6:38 pm |
| | 4.3 Financial Report | VP Operations & Finance | Ashley noted a drop in revenue and enrollment has been noticed for March/April that could be due to COVID-19. Margaret noted this could be a normal dip. Ashley is awaiting follow up with the University. | |
| 5.0 | Bookkeeper Proposal | VP Operations & Finance | Motion 5.0: To approve the hiring of a bookkeeper with amendments to the responsibilities to include payroll, and reflect a sliding scale for hourly commitment.
Moved: Margaret Clappison
Seconded: Brandon Simmons
Motion carried without opposition. | 7:00 pm |
| 6.0 | Council Reports | | Student inquiries and contacts come through the website to the ED email and inquiries are forwarded onward to Faculty Reps and Executives depending the portfolio. | 7:13 pm |
| Section | Committee | Role | Report |
|---------|-----------|------|--------|
| 6.1 | Faculty of Science and Technology | Faculty Representation | No reports at this time. |
| 6.2 | Faculty of Humanities and Social Sciences | Faculty Representation | No reports at this time. |
| 6.3 | Faculty of Health | Faculty Representation | No reports at this time. |
| 6.4 | Faculty of Business | Faculty Representation | No reports at this time. |
| 7.0 | Communications Strategy | Communications Coordinator | Bob Cole presented the Communications – 2020 Social Media Strategy |
| 8.0 | Old Business | President | |
| 9.0 | New Business | President | |
| 10.0 | Committees | President | |
| 10.1 | Governance Committee | Chair, Governance | The committee has met and reviewed bylaw objectives. The revised bylaws will be presented at AGM and include revisions such as inclusion of a Finance Committee. The Committee has re-written some of the committee descriptions and included updates as minor revisions. |
| 10.2 | Awards Committee | Chair, Awards | The committee met on June 17th with multiple applications for review. The committee is continuing to build in supports for adjudicating applications (such as a threshold) and revamping the application to be bursary/awards specific. |
| 10.3 | Engagement Committee | Chair, Engagement | The committee has met with the intention to finalize work plan. They reviewed the Convocation 2020 plan and discussed how to best represent AUGSA. The committee is considering purchasing swag, supporting local artists, or finding other creative strategies to engage. Goals for an external communications strategy are being worked on with Crys Vincent and Bob Cole. |
| 10.4 | Strategic Planning Committee | Chair, Strategic Planning | The committee has met and discussed the plans around the annual membership satisfaction survey. Questions on mental |
| | | | | |
|---|---|---|---|---|
| 11.0 | Upcoming Events | President | health and COVID-19 will be added to the survey. | 7:42 pm |
| | | | 1. AGM: We require quorum, which is 15 students. Please ensure your attendance and bring a fellow student. | |
| | | | 2. Grad Research Conference on Oct 16-17. | |
| | | | 3. Convocation. | |
| 12.0 | Other Business | President | GCAP: Lyndsay McNena inquired on a GCAP concern related to Convocation. She will send a follow-up to Mary-Anne via e-mail. | |
| 13.0 | Adjournment | President | **Motion 13.0:** To adjourn the meeting at 7:54 pm.
Moved: Crys Vincent
Seconded: Bernard Kikechi
Carried without opposition. | 7:54 pm |
__________________________
Mary-Anne Parker, President
__________________________
Meaghan Sullivan, Executive Director
__________________________
Date of Approval
President Report
July 2020
AUGSA Executive/Council Meetings
- June 1, 8th, 15th, 22nd, & 29th – President/ED weekly check ins.
- June 2nd – AUGSA executive meeting
- June 9th – council
- June 15th – External team meeting
- June 22 – engagement cmte meeting
- June 26th – Joint ED/VPOF catch up
AU Meetings
- June 1 – University Relations – Covid Bursary follow up
- June 2nd – FHSS Faculty Council (2.5 hrs)
- June 4th – FGS Council (3 hours)
- June 4th – AUSU/AUGSA executive meet and greet
- June 5th, 12th, 19th – weekly president check up with AUSU
- June 8th-12th – mtg with hiring candidates
- June 15th – mtg w VP Provost – tuition and fees/ILE
- June 15th – Advisory Committee – Dean FST
- June 16th – AVPR and FGS candidates’ recap
- June 16th – National strategy session with AUSU
- June 18th – AUSU/AUGSA & University relations communications vis a vis covid bursaries
- June 22nd – FGS – Grad lounge line up
- June 25th – Joint GFC/B of G session
Alberta Graduate Provincial Advocacy Council (abGPAC)
- June 1st – BMO – financial transfer (treasurer)
- June 2nd – weekly catchup
- June 3 – Board mtg. x’
- June 4 – mtg with new Board chair.
- June 16th – GSA Labour relations advisory committee
- June 16th – Executive Cmte meeting
- June 17th – Board meeting
- June 23rd – meeting with Advanced Ed department
June 24th – Day 1, retreat, overview and intro.
June 24th – governance cmt meeting
June 24th – SAGE overview
June 25th – Government relations principles and practice – training - retreat
June 25th – advocacy principles discussion - retreat
June 26th – swot and pestle analysis – retreat
June 26th – strategic plan – retreat
June 29th – advocacy and outreach – retreat
June 30th – resource summary & next steps – retreat
June 30th – special board meeting.
Canadian Alliance of Student Associations
Other Meetings/Events
- June 5th, 19th, 26th Grad Lounge – weekly co-hosting duties
- Weekly “president’s coffee” with Natasha Donaghue
General/Other
- Approval of Expenses
- Reading reports/developing presentations
- Responding to Email/Text/Phone Communications
- Administrative/Miscellaneous
HIGHLIGHTS
1. Role determination – Internal and External
After the initial May start up and inundation of meetings it was imperative that I look for redundancies within our teams. Having three people at meetings seems an ineffective way to use our time. This has come about due to the necessary shift, each year, of executive roles that fit the individuals in the role and the interests/personalities of those occupying the positions. My mantra is to build capacity within the council in order to ensure AUGSA’s ongoing success. June was the month for me to re-assess when my presence is needed and when I can step back so others can step up. I’m hoping that by the end of summer there will be a breadth of expertise in key roles.
2. Grad Lounge
This was initiative co-formed by AUGSA and FGS. It has incredible potential. Each week is
assigned a theme and I have been working closely with FGS to deliver a variety of formal and informal topics. We have also encouraged the participation of grad students. This is a fledgling imitative that has proven invaluable to those who have spent time with us and for me to hear the challenges and success of grad students. It has allowed me to advocate both formally and informally. We hope to keep building on the positive experience. I am hoping that we can have an AUGSA contingent there every week.
3. Collaboration
I have been invited to more than 10 meetings in which internal AU departments have wanted a student voice as part of both planning processes and recruitment of staff. This bodes well for AUGSA in that we are being positioned as the consultative voice within AU and being given a chance to question candidates about their views on student experience and research needs.
4. Ab-GPAC
A strategic retreat combined with start-up committee meetings have provided a strong base for AUGSA’s position within this organization. We are viewed as the experts in online education and have provided important input in terms of future directions and research needs. We have a strong external team that is well on its way to promoting AUGSA student interests.
VP Academic’s Report
June 2020
AUGSA Executive/Council Meetings
- June 2nd – Executive Check In.
- June 9th – Council meeting
Faculty of Graduate Studies (FGS)
- June 4th – Faculty Council meeting
- June 17th – Faculty Council meeting
Awards Committee
- June 17th - Reviewed 1 computer bursary and 1 emergency bursary
- Awarded 1 computer bursary
General Faculty Council
- June 25th – GFC meeting.
General/Other
- June 8th – AVPR candidate meeting with AUSU and AUGSA Executive
- June 9th – AVPR candidate meeting with AUSU and AUGSA Executive
- June 10th – Dean of FGS candidate presentation to advisory committee
- June 10th – Dean of FGS candidate interview with advisory committee
- June 10th – Dean of FGS candidate meeting with AUSU and AUGSA Executive
- June 10th – Dean of FGS candidate open forum meeting with advisory committee
- June 11th – Dean of FGS candidate presentation to advisory committee
- June 11th – Dean of FGS candidate interview with advisory committee
- June 11th – Dean of FGS candidate meeting with AUSU and AUGSA Executive
- June 11th – Dean of FGS candidate open forum meeting with committee
- June 12th – Dean of FGS candidate presentation to advisory committee
- June 12th – Dean of FGS candidate interview with advisory committee
- June 12th – Dean of FGS candidate meeting with AUSU and AUGSA Executive
- June 12th – Dean of FGS candidate open forum meeting with advisory committee
- June 15th – Meeting with Dr Matthew Prineas regarding fees consultation
• June 15th – Meeting with CUPE/AUSU/AUGSA
• June 16th – AVPR / Dean of FGS Candidate recap
• June 18th – Meeting #4 Advisory committee Dean of FGS.
VP External’s Report
June 2020
AUGSA Executive/Council Meetings
- June 9th – Regular council meeting
- June 11th - Touchbase with Kristine Williamson on MLA meetings in AB
- June 12th - Hosted the grad lounge with FGS
- June 15th - External team meeting (chair)
- June 16th - Conversation with Finance department and debrief with President and Exec
- June 18th - Meeting with AU on Reimagined Convocation plan
- June 22nd - Engagement Committee Meeting
- June 24th - Conversation with Chris Bell (abGPAC) Evan Wong (UofA) AUGSA President on the SAGE program and Supporting Indigenous students
- June 25th - Check in with President
- June 26th - Meeting with AUSU VPX and Kristine Williamson Minister strategy planning
- June 29th - Meeting with Minister Sawhney, Kristine, and AUSU (rescheduled - no show)
Alberta Graduate Provincial Advocacy Council (abGPAC)
- June 9th – Exec meeting
- June 10th - Agenda planning meeting with ED (A&O Committee)
- June 11th - Advocacy and Outreach Committee Meeting (chair)
- June 16th - Executive Meeting
- June 17th - Regular Board Meeting
- June 23rd - Executive Meeting
- June 24th - Opening session of Board Retreat
- June 25th - Government Relations Training with Keill and Co
- June 25th - Land Acknowledgment Workshop
- June 25th - Advocacy Principles Planning Session
- June 26th - SWOT and PESTEL Analysis Session
- June 26th - Strategic Plan Review
- June 27th - Advocacy Priorities Session
- June 27th - Research Priorities Session
- June 29th - Advocacy and Outreach Topic Discussion
- June 29th - Governance Topic Discussion
- June 30th - Board Retreat Wrap up session
- June 30th - Regular Exec Meeting
Canadian Alliance of Student Associations
- Responded to Doodle polls on first committee meetings
Other Meetings/Events
General/Other
- Responding to Email/Text/Phone Communications
- Created info graphic for outreach
- Administrative/Miscellaneous
VPOF Report
June 2020
AUGSA Internal/External Meetings
- Executive Meeting (June 2)
- Council Meeting (June 9)
- TD Meeting with AUGSA (June 10)
- TD CMS Meeting (June 11th)
- ED/VPOF Meeting with Alumni Relations (June 12)
- ED/VPOF Meeting (June 4, June 26) and about three times a week (via text message, and phone calls, THANK YOU MEAGHAN!)
- ED/VPOF/President Meeting (June 26)
AUGSA ACTION ITEM UPDATE:
- UPDATE ON INQUIRIES
- What server does the university utilize, and could AUGSA use this in future, if requested?
- Would AU be willing to extend/host/provide use of their internal security systems, Office 365 (their is a potential for savings here), and/or hosting the website on their server.
- With new Credit Card fee being collected when students pay tuition with a credit card, will the University considering payment plans, and also, as Business were previously told they could ONLY use credit cards (meaning they are forced to pay the associated fee), or are they going to allow them to pay via an debit?
VPFO GOALS:
July through August, I plan to focus on:
- Continuing to transfer data into quickbooks, to ensure all entries have back-up embedded.
- Continuing to streamline financial processes and continue to add to the Standard Operating Procedure/processes for future VPOF's.
- Hire a bookkeeper/akin to continue entering transactions and updating the books (target hire date September 1st 2020).
- Follow-up on 'lost,' revenue from the March-onward period (AU)
• Issuing T4A to individuals missed in 2019
|
Deglaciation of the Eurasian ice sheet complex
Henry Patton\textsuperscript{a, *}, Alun Hubbard\textsuperscript{a}, Karin Andreassen\textsuperscript{a}, Amandine Auriac\textsuperscript{b}, Pippa L. Whitehouse\textsuperscript{b}, Arjen P. Stroeven\textsuperscript{c, d}, Calvin Shackleton\textsuperscript{a}, Monica Winsborrow\textsuperscript{a}, Jakob Heyman\textsuperscript{e}, Adrian M. Hall\textsuperscript{c}
\textsuperscript{a} CAGE—Centre for Arctic Gas Hydrate, Environment and Climate, Department of Geosciences, UiT The Arctic University of Norway, 9037 Tromsø, Norway
\textsuperscript{b} Department of Geography, Durham University, South Road, Durham, DH1 3LE, UK
\textsuperscript{c} Geomorphology and Glaciology, Department of Physical Geography, Stockholm University, Stockholm, Sweden
\textsuperscript{d} Bolin Centre for Climate Research, Stockholm University, Sweden
\textsuperscript{e} Department of Earth Sciences, University of Gothenburg, Sweden
\textbf{Article history:}
Received 11 March 2017
Received in revised form 17 May 2017
Accepted 22 May 2017
Available online 14 June 2017
\textbf{Keywords:}
Eurasian ice sheet complex
Barents sea
Fennoscandian ice sheet
Late Weichselian
Deglaciation
Glacio-isostatic adjustment
Subglacial lakes
Proglacial hydrology
Younger Dryas
Fleuve Manche
\textbf{Abstract}
The Eurasian ice sheet complex (EISC) was the third largest ice mass during the Last Glacial Maximum with a span of over 4500 km and responsible for around 20 m of eustatic sea-level lowering. Whilst recent terrestrial and marine empirical insights have improved understanding of the chronology, pattern and rates of retreat of this vast ice sheet, a concerted attempt to model the deglaciation of the EISC honouring these new constraints is conspicuously lacking. Here, we apply a first-order, thermo-mechanical ice sheet model, validated against a diverse suite of empirical data, to investigate the retreat of the EISC after 23 ka BP, directly extending the work of Patton et al. (2016) who modelled the build-up to its maximum extent. Retreat of the ice sheet complex was highly asynchronous, reflecting contrasting regional sensitivities to climate forcing, oceanic influence, and internal dynamics. Most rapid retreat was experienced across the Barents Sea sector after 17.8 ka BP when this marine-based ice sheet disintegrated at a rate of $\sim$670 gigatonnes per year (Gt a$^{-1}$) through enhanced calving and interior dynamic thinning, driven by oceanic/atmospheric warming and exacerbated by eustatic sea-level rise. From 14.9 to 12.9 ka BP the EISC lost on average 750 Gt a$^{-1}$, peaking at rates $>3000$ Gt a$^{-1}$, roughly equally partitioned between surface melt and dynamic losses, and potentially contributing up to 2.5 m to global sea-level rise during Meltwater Pulse 1A. Independent glacio-isostatic modelling constrained by an extensive inventory of relative sea-level change corroborates our ice sheet loading history of the Barents Sea sector. Subglacial conditions were predominately temperate during deglaciation, with over 6000 subglacial lakes predicted along with an extensive subglacial drainage network. Moreover, the maximum EISC and its isostatic footprint had a profound impact on the proglacial hydrological network, forming the \textit{Fleuve Manche} mega-catchment which had an area of $\sim$2.5 $\times$ 10$^6$ km$^2$ and drained the present day Vistula, Elbe, Rhine and Thames rivers through the Seine Estuary. During the Bolling/Allerød oscillation after c. 14.6 ka BP, two major proglacial lakes formed in the Baltic and White seas, buffering meltwater pulses from eastern Fennoscandia through to the Younger Dryas when these massive proglacial freshwater lakes flooded into the North Atlantic Ocean. Deglaciation temporarily abated during the Younger Dryas stadial at 12.9 ka BP, when remnant ice across Svalbard, Franz Josef Land, Novaya Zemlya, Fennoscandia and Scotland experienced a short-lived but dynamic re-advance. The final stage of deglaciation converged on present day ice cover around the Scandes mountains and the Barents Sea by 8.7 ka BP, although the phase-lagged isostatic recovery still continues today.
© 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (\url{http://creativecommons.org/licenses/by-nc-nd/4.0/}).
\section*{1. Introduction}
Northern Eurasia was covered by three semi-independent ice sheets that between 26 and 19 ka BP (Clark et al., 2009) coalesced to form a single Eurasian ice sheet complex (EISC) during the last...
glacial maximum (LGM) (Svendsen et al., 2004). This complex had an impressive latitudinal and longitudinal coverage, with continuous ice cover spanning over 4500 km extending southwest of the Isles of Scilly (50°N, 6°W) on the Atlantic seaboard to beyond Franz Josef Land (81°N, 56°E) in the Russian High Arctic (Fig. 1). It was the third largest ice mass after the North American and Antarctic ice sheets, and, with a combined volume three times the present Greenland ice sheet, accounted for at least 20 m of eustatic sea-level lowering (Patton et al., 2016). Growth of the EISC initiated from three main nucleation centres located over Britain and Ireland, Fennoscandia, and the Barents–Kara seas, with contrasting styles of glaciation and associated conditions and processes reflecting these settings, from marine-terminating, fast-flowing ice streams in maritime regions to extensive frozen-based glaciation in inter-ice-stream and upland areas.
Knowledge of the maximum extent, chronology, and patterns of retreat of the EISC has improved greatly in the last decade, particularly in offshore sectors where marine geophysical surveys have addressed a number of notable gaps in understanding (e.g., Landvik et al., 2005; Ottesen et al., 2005; Bradwell et al., 2008; Dunlop et al., 2010; Winsborrow et al., 2010; Andreassen et al., 2014; Sejrup et al., 2016). Moreover, developments in cosmogenic exposure dating, as well as refinement of radiocarbon and other dating techniques, has enabled detailed onshore deglaciation chronologies to be developed (Rinterknecht et al., 2006; Linge et al., 2007; Ballantyne, 2010; Stroeven et al., 2011; Briner et al., 2016). Subsequent publication of data-rich compilations and review studies has thus set in place a strengthened empirical framework against which modelling investigations of the EISC can be made and tested (Napieralski et al., 2007; Clark et al., 2012; Hormes et al., 2013; Hughes et al., 2014, 2016; Patton et al., 2015; Auricai et al., 2016; Cuzzone et al., 2016; Stroeven et al., 2016), although the utility of such empirical datasets for modelled reconstruction comparisons must be considered in light of the quality of legacy data they incorporate (e.g., Small et al., 2017).
Since the early numerical modelling undertaken as part of the QUEEN programme (cf. Siegert and Dowdeswell, 2004), progress on modelling the Late Glacial retreat of the EISC has been limited to a number of regional reconstructions (Holmlund and Fastook, 1993; Boulton et al., 2001; Boulton and Hagdorn, 2006; Hubbard et al., 2009) or otherwise focussed primarily on process dynamics (Arnold and Sharp, 2002; Forström and Greve, 2004; Nølsrud et al., 2005; van den Berg et al., 2008a, 2008b, Clason et al., 2014, 2016). An alternative to these process-based models are EISC reconstructions developed through glacial isostatic adjustment modelling (Peltier, 2004; Lambeck et al., 2010; Peltier et al., 2015). These inverse models are calibrated using empirically determined ice extents and relative sea-level data, but the resulting reconstructions are static and do not provide insight into the dynamics of ice sheet retreat, nor do they inform the climatic/oceanic forcing that drove it.
In this paper, we apply a first-order, thermomechanical ice sheet model to investigate the dynamic retreat of the EISC after 23 ka BP. The primary aims are twofold: i) to present a robust, 4D high-resolution, synoptic reconstruction of EISC deglaciation from 23 to 8 ka BP, from its local LGM extent, through the Younger Dryas stadial (12.9–11.7 ka BP), and into the Early Holocene; and, ii) to validate and discuss model output against a suite of empirical data that constrain both the pattern and rate of retreat of the EISC, including its glacial-isostatic footprint, chronological data for the timing of deglaciation, flowset vectors, and its sub- and pro-glacial hydrological legacy.
The study extends the work of Patton et al. (2016) who previously explored the asynchronous and asymmetric growth of the EISC to its maximum LGM extent from 37 to 19 ka BP. A variety of geomorphological, geophysical and geochronological data are used to constrain and validate the broad-scale dynamics of the retreating ice mass. In particular though, where terrestrial constraints are notably lacking across the relatively data-sparse Barents and Kara seas, we utilise glacial isostatic adjustment modelling to test transient ice loading and retreat rates.
2. Methods
2.1. The ice flow model
The 3D thermomechanical model and associated initial boundary condition data applied are of the same derivation as that used to previously model the pre-LGM build-up of the EISC by Patton et al. (2016), where a more complete description of the model setup and implementation can be found. In brief, the ice flow model is a first-order approximation of the Stokes equations, adapted from Blatter (1995), Hubbard (1999, 2000), Marshall et al. (2005), and Pollard and DeConto (2007). The approach to solving the three dimensional stress/strain field equates to the L1L2 classification of higher-order models defined by Hindmarsh (2004), and includes longitudinal (membrane) stresses that become increasingly important across steep gradients in topography and motion. The model is integrated forward through time on a finite-difference grid with a resolution of 10 km through perturbations in climate and eustatic sea level (Fig. 2A–B). Isostatic loading is implemented using an elastic lithosphere/relaxed asthenosphere scheme described by Le Meur and Huybrechts (1996), which provides a computationally pragmatic solution in the absence of a full spherical earth model. Gridded output is projected under an equal area Lambert Azimuthal projection, with a central meridian of 73°E.
Surface mass balance is determined by a positive degree-day scheme, applied according to Laumann and Reeh (1993), and derives total melt from integrated monthly positive temperatures. Both temperature and precipitation adjust to the evolving ice sheet surface through applied lapse rates derived from multiple-regression analyses of meteorological observations at a resolution of 1 km from the WorldClim database (Hijmans et al., 2005; Version 1.4). To account for the large variations in climate regime across the Eurasian domain, regional reference climates and associated forcing are tuned independently for each of the three major accumulation centres (Fig. 2C–D). An additional mass balance term incorporated is the net water vapour flux to and from the ice sheet surface – a predominant component of ablation in cold continental settings where humidity can be very low (e.g., Fujii and Kusunoki, 1982; Kameda et al., 1997).
Calving losses at marine-terminating margins are coupled to relative sea level (RSL) (Waelbroeck et al., 2002) using a standard empirical function relating the calving flux to ice thickness and water depth (Brown et al., 1982; van der Veen, 1999). The sensitivity of calving to, for example, variations in ocean temperature (Luckman et al., 2015) and sea-ice buttressing (Hoff et al., 2016) is controlled spatially and temporally through a depth-scaled calving parameterisation (Hubbard, 2006) (Fig. 2D). In the absence of explicit calculations of such external feedbacks, this depth-related calving coefficient provides a pragmatic and computationally efficient parameterisation for determining mass loss at marine terminating margins of the ice complex. The model is applied to a 10 km finite-difference mesh with the inclusion of grounding-line dynamics based on the analytical boundary-treatment of Schoof (2007) and adapted in 2D by Pollard and DeConto (2007), which defines the ice flux at the grounding line as a function of ice thickness linearly interpolated between the adjacent node that bracket floating and grounded ice (Hubbard et al., 2009).
Fig. 1. Major drainage routes of the Eurasian ice sheet complex, adapted from Stokes and Clark (2001), Ottesen et al. (2005) and Clark et al. (2012). Locations of major trough mouth fans (brown) adapted from Dahlgen et al. (2005) and Batchelor and Dowdeswell (2014). PB: Porcupine bank; BDF: Barra and Donegal Fans; RB: Rosemary Bank; NSF: North Sea Fan; Bj: Bjornegrenna Fan. Glacial limits are compiled from Ehlers and Gibbard (2007), Patton et al. (2015) and Stroeve et al. (2016). Recent evidence for the extension of the Celtic ice sheet onto Porcupine Bank (PB) (Peters et al., 2015) and into the southern Celtic Sea (Praeg et al., 2015) is also incorporated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
2.2. Experiment setup and empirical constraints
The deglaciation scenarios presented in this study follow on directly from the suite of EISC build-up experiments carried out by Patton et al. (2016), and thus forms part of a continuous reconstruction for the full Late Weichselian (37–8 ka BP). With a greater abundance of empirical evidence constraining the deglaciation of the ice sheet, the prescribing of model parameters in order to yield a transient experiment that follows the broad pattern of ice retreat becomes increasingly challenging. This challenge is greatly aided by the implementation of three sub-domains that span each of the major nucleation centres of the complex, within which climatic and oceanographic forcings can be tuned independently. During the reconstructed build-up of the ice complex, major climatic differences between these three regions included a general enhancement of climate cooling and a decrease in precipitation across southern latitudes. To encourage growth of the marine-based Barents Sea ice sheet (BSIS), sensitivity to calving losses was also reduced relative to that of the Fennoscandian and Celtic ice sheets, simulating the influence of colder ocean temperatures and buttressing effects from perennial sea ice (Patton et al., 2016).
Various lines of geological and geophysical data were used to guide the broad retreat patterns of the modelled ice sheet, including key dating constraints, geomorphological flow sets, notable moraine/grounding zone wedge positions, and patterns of isostatic loading. The most important datasets in this respect are those that incorporate a holistic approach to the ice-complex reconstruction, for example, the DATED-1 1 ka-interval timeslice reconstructions made using an extensive database of chronological constraints (Hughes et al., 2016). The value of this comprehensive approach becomes apparent where conflicting or competing regional interpretations occur, as the data can be rigorously examined within a broader and glaciologically consistent context, and thus provide a sense of probability or even highlight unreliable data points. To this end, ice sheet modelling fulfils a useful and independent tool for comparison, exploring the feasibility and probability of competing hypotheses for ice evolution and dynamic behaviour.
A useful distinction can be made between the pattern and timing of deglaciation in constraining and testing the deglaciation scenario. Except for the Barents and Kara seas, coverage of geomorphological data has expanded hugely in recent years through interpretation of terrestrial LiDAR elevation data and seafloor multibeam mapping (e.g., Winsborrow et al., 2010; Hughes et al., 2014; Greenwood et al., 2015). This wealth of data provides assurances that the broad pattern of deglaciation of the EISC has been identified correctly, including the main stillstands and readvances. However, the time sequence of deglaciation of the EISC remains less certain. This deficit is partly a product of the limited availability of dating controls for Russian territories and the floor of the Arctic seas. Yet existing dates elsewhere currently often lack precision at millennial time scales. A comprehensive compilation of radiocarbon, optically-stimulated luminescence and $^{10}$Be cosmogenic isotope ages for the last Fennoscandian ice sheet (FIS) has
shown differences of several thousand years in dates for major stillstands, underlining the greater precision of the established varve chronology (Stroeven et al., 2016). Large $^{10}$Be datasets for individual moraines (Rinterknecht et al., 2006) and for specific sites (Ballantyne, 2010) also show wide date ranges. Maximising the utility of legacy geochronological data for the purposes of informing numerical modelling studies has only recently come to the fore following the compilation of datasets that span entire ice sheet domains (e.g., Dyke et al., 2002; Hughes et al., 2011, 2016; Bentley et al., 2014; Stroeven et al., 2016), driving standards for sampling strategies and criteria for data reporting (e.g., Small et al., 2017). In this respect, the current chronology of EISC deglaciation is insufficiently robust, particularly early in deglaciation, to pin down the timings of millennial-scale events. Indeed part of the value of this simulation lies in the independent constraints that it provides on the likely timings of major events during EISC decay. An assessment on the conformity of the optimum deglaciation scenario against the empirical datasets used is presented in section 4, following the description of the modelled deglaciation.
2.3. Glacial isostatic adjustment modelling
Ice thickness predictions derived from the ice model above were incorporated into an independent glacial isostatic adjustment model to examine the overall fit of ice loading to sea-level histories across the Barents Sea. The sea-level equation (first derived by Farrell and Clark (1976)) is solved using the implementation from Mitrovica and Milne (2003) and Kendall et al. (2005). Gravitationally self-consistent sea-level changes are computed, taking into account shoreline evolution as well as the time-dependent evolution of marine-based ice margins. The sea-level equation is solved iteratively using an extended pseudo-spectral algorithm. This numerical code assumes a spherically symmetric Earth, whose properties are based on the Preliminary Reference Earth Model (PREM; Dziewonski and Anderson, 1981). The Earth model is implemented as an input with three variables: lithosphere thickness and upper and lower mantle viscosity. We use 300 different Earth models, where the lithosphere thickness ranges from 46 to 120 km and the upper and lower mantle viscosities range from $0.05 \times 10^{21}$ to $5 \times 10^{21}$ Pa s and $1 \times 10^{21}$ to $50 \times 10^{21}$ Pa s, respectively. These Earth models cover the range of Earth parameters generally found or inferred for this area from a range of geophysical techniques (e.g., Kaufmann and Wolf, 1996; Steffen and Kaufmann, 2005; Klitzke et al., 2015). After solving the sea-level equation, an estimate of the time evolution of relative sea level at specific locations is determined.
3. Results
3.1. Ice sheet build-up
The deglaciation scenario presented in this paper forms a direct continuation of the optimum build-up experiment (37–19 ka BP) presented by Patton et al. (2016). The progression and dynamic behaviour of the retreating ice complex is therefore, at least initially, predetermined by this pre-LGM growth trajectory. For example, the eastwards migration of the central ice divide of the complex that forces asynchronous maximum extension between 25 and 20 ka BP plays a dominant role in the subsequent timing and direction of ice retreat. In order to place the new experiment results in context, a brief description of the modelled build-up of the EISC by is provided here; areal and volumetric changes for each semi-independent ice sheet through the full Late Weichselian are provided in Fig. 3.
From a distribution of limited ice cover, widespread ice growth initiates across all three sectors in response to climate deterioration during the latter stages of marine oxygen isotope stage (MIS) 3 (<37 ka BP). Continuous net accumulation sees the emergence of fast flowing outlet glaciers that reach the shelf break west of Svalbard, Norway, Britain, and Ireland around 30-29 ka BP, indicative of extensive and thick terrestrial ice sheets. By 24.5 ka BP the expanding Barents Sea and Fennoscandia ice sheets coalesce, forming a major ice divide running northwards over Finnmark and the central banks of the Barents Sea. The Celtic and Fennoscandia ice sheets merge in the North Sea 1.5 ka later, completing the formation of a coherent Eurasian ice sheet complex by 23 ka BP. With stable western margins between 25 and 23 ka BP, continuing ice divide migrations force an ice incursion into eastern European sectors as late as 20 ka BP. A weak coalescence between the Celtic and Fennoscandian ice sheets sees the two ice sheets split shortly after 22.4 ka BP, with the major Norwegian Channel ice stream persisting in the North Sea until at least 19 ka BP. Shortcomings of this build-up scenario that impinge on the accuracy of the deglaciation scenario presented include the limited expansion of the Norwegian Channel ice stream ~400 km short from the continental shelf edge at 22.7 ka BP, the failure to reproduce the extended outlet lobes of the eastern FIS (Dvina, Vologda and Rybinsk basins), and the shortfall of the maximum modelled Fennoscandian ice margin in Germany and Poland.
3.2. Eurasian ice sheet complex deglaciation
3.2.1. 23.0–17.8 ka BP
A period of climate stability c. 23.0–19.5 ka BP (Fig. 2B) sees relatively minor volume and areal fluctuations across the entire complex (Fig. 3). The furthest extent of the EISC through the White Sea and into northwest Russia occurs early in the deglaciation sequence at 20.4 ka BP (Fig. 4A). The asynchronous development of the LGM complex resonates with empirical observations (Hughes et al., 2016; Stroeven et al., 2016), with maximum extension of the north-eastern sectors of the FIS possibly occurring as late as 18–16 ka BP (Larsen et al., 1999b, 2016). Elsewhere, retreat of westward draining ice along much of the western continental shelf edge is interrupted, stabilising at positions upslope from the shelf break. A phase of warming after 19.5 ka BP is most severely felt across Celtic sectors, the North Sea Basin, and the south eastern FIS. Ice retreat occurs throughout much of Ireland, Wales, northern England, with substantial ice drainage occurring through the Malin Sea, and southern Scandinavia. However, the pace of retreat is stepwise, with a brief respite linked to mean annual air temperature (MAAT) cooling at 17.8 ka BP (Fig. 4B). Surface melting across Fennoscandia and the Barents Sea at this time has a limited impact on ice extent, with major outlet glaciers undergoing minimal retreat.
A manually forced reduction of the calving coefficient along the Arctic Ocean margin (>78°N), resulting in negligible calving losses, enforces a largely stationary margin throughout this period, and was found to be a necessary forcing in order to satisfy relative sea-level constraints on Franz Josef Land and Novaya Zemlya (cf. section 3.3). However, fluctuations in MAAT force at least three phases of stillstand/readvance within Bjørnøyrenna (Fig. 4B) before 17.8 ka BP, some 100 km away from the shelf break.
3.2.2. 17.8–14.9 ka BP
The minor standstill at 17.8 ka BP for some margins of EISC is interrupted by a phase of rapid retreat at 17.5 ka BP, for example, through Bjørnøyrenna and the coast parallel troughs of northern Fennoscandia (Fig. 4C), with only a minor re-advance c. 16 ka BP interrupting continuous retreat (Fig. 4D). Vigorous draw-down of ice through the various troughs of the central Barents Sea forces an effective collapse of this ice saddle, with the BSIS and FIS eventually
separating at 15.5 ka BP (Fig. 4D–E). Chronological control on this separation is poor, and is constrained here by a single radiocarbon date of 14.9 cal ka BP from glaciomarine sediments in the outer Pechora Basin (Polyak et al., 1995). Further calving losses continue to force rapid withdrawal of the ice margin northwards through the Barents Sea, with little topographic relief available to provide stable pinning points.
Despite widespread ice losses from all margins of the FIS after 17.5 ka BP, the ice sheet stabilises c. 1 ka later, with various outlet glaciers, including those in the Norwegian Channel, Baltic Sea and White Sea, even undergoing a phase of readvance after c. 16 ka BP until the end of Greenland Stadial-2.1a at 14.7 ka BP (Fig. 4E).
For the diminishing, yet sensitive, Celtic ice sheet (CIS), the Late Glacial warming is critical. By 16.5 ka BP the CIS is all but pinned to the Scottish coastline until the end of GS-2.1a. During this period, active drainage routes include the Moray Firth, Minch, and Sea of the Hebrides (Fig. 4E).
3.2.3. 14.9–12.9 ka BP
The climate warming that marks the start of the Bølling-Allerød oscillation inflicts a period of widespread melt, at times with mass losses >3000 Gt a$^{-1}$ occurring across the entire complex (Fig. 3B). During this period (14.9–12.9 ka BP) the EISC loses on average 750 Gt a$^{-1}$, partitioned roughly equally between surface melt and dynamic losses, with the EISC contribution to Meltwater Pulse 1A (Deschamps et al., 2012) on the order of ~2.5 m of sea-level equivalent ice volume. Within 500 years of the initiation of the Bølling, the remnants of the CIS are but completely disappeared (Fig. 4F). Combined with continued calving losses, this period is equally catastrophic for the BSIS – by the start of the Younger Dryas at 12.9 ka BP all that is left of the ice sheet are isolated ice caps across the islands, and an ice saddle between eastern Svalbard and Franz Josef Land (Fig. 4G). For the FIS the greatest losses occur along the southern and eastern margins furthest away from the central ice divide, forcing a westward migration of the ice divide. Although the western ice margin continues to be pinned along the Norwegian coast throughout this period, retreat of ice through Sweden, Finland and northwest Russia continues unabated until 12.9 ka BP, when substantial drops in MAAT halt retreat.
Although proto versions of the Baltic and White Sea proglacial lakes are in place prior to the Bølling-Allerød oscillation (Fig. 4B–E), their capacity only dramatically expands with the onset of ice retreat through the respective basins during this interstadial, as suggested by Björck (2008).
3.2.4. 12.9–8.0 ka BP
The initiation of the Younger Dryas at 12.9 ka BP sees a re-emergence of cold-based ice cover over the Scottish uplands, though this is short-lived and disappears soon after the end of the Younger Dryas by 11.4 ka BP (Fig. 4G). The response of the FIS is somewhat different, with the Bothnian Sea ice stream continuing to retreat through the beginning of this stadial despite thickening of ice at the divide. The FIS eventually purges mass at 12.3 ka BP, undergoing a rapid readvance mainly across its southern margin, but also offshore into the Norwegian Sea. The readvance culminates at 11.7 ka BP, after which warming during the early Holocene forces terminal retreat of ice into two separate ice caps across the Scandes. The smaller of the two is focussed over the southern mountains around Jotunheimen, while the larger retreats to the northern Swedish mountains (Fig. 4H). The death of the Bothnian Sea ice stream is marked by a phase of rapid flow with an onset zone close to the present-day coastline at c. 10 ka BP. The northern FIS centre finally disappears at 8.7 ka BP.
Despite the climate deterioration during the Younger Dryas, marine-based sectors of the BSIS finally disappear during this period, with the ice saddle connecting Svalbard and Franz Josef Land collapsing at 12.2 ka BP. Reduced moisture supply at this time is insufficient to drive a readvance of the remaining ice caps, and warming through the Holocene continues the overall trend of margin retreat.
3.3. Glacial isostatic adjustment modelling
Results from the comparison between our optimum deglacial scenario and 46 (RSL) observations from around the Barents Sea are given in Table 1. These are subdivided into the four main terrestrial
areas bordering the domain: Svalbard, Franz Josef Land, Novaya Zemlya and northern Fennoscandia. RSL curves, selected as being representative of the full array of results are presented in Fig. 5. The full set of RSL plots can be found as supplementary material in Figure S1. Table 1 presents the best-fit earth model parameters for each region of the Barents Sea, as well as the corresponding value of $\chi^2$ – a measure of data fit derived from the mean weighted residual sum of squares between the modelled and empirical data points. These values are compared to an earlier published iteration of the modelled deglaciation wherein ice retreat was more loosely constrained (Auriac et al., 2016).
While the global best fit ($\chi^2_g$) is still well above 1, indicating that the ice load scenario fails to reproduce the RSL observations simultaneously at all sites, work to better constrain the 3D geometrical evolution of the BSIS has yielded improvements if regional variations to the Earth model are assumed (Table 1). Northern Fennoscandia shows the most clear improvement, with a decrease in overall misfit ($\chi^2$) from 51.9 to 8.13. Across Franz Josef
Land and Novaya Zemlya misfit values remain low at 3.1 and 0.3, respectively. However, model results for the Svalbard region show a clear decrease in accuracy, with no single earth model able to provide a consistently good fit for all RSL sites. Just considering RSL observations in eastern Svalbard yields some improvement, but matching the rapid, early Holocene RSL fall recorded on the western coast (e.g., Sv10 and Sv11; Figure S1A) remains elusive with the current ice load scenario.
4. Evaluation of the optimum deglaciation scenario against field data
The robustness and conformity of the optimum deglaciation scenario (section 3.2) is assessed through comparison with current compilations and syntheses of the empirical record. Prominent datasets that record the large-scale footprint of the ice sheet include time-transgressive flowsets that record ice-flow direction (e.g., Kleman et al., 1997; Winsborrow et al., 2010; Hughes et al., 2014), ice margin positions inferred from sediment distributions and ice-marginal landforms (e.g., Clark et al., 2012; Stroeven et al., 2016), and chronological data that can be used to infer the pace of ice retreat (e.g., Hughes et al., 2016; Stroeven et al., 2016). Details regarding the level of correspondence of the maximum ice-sheet with regards to ice thickness, extent, flow direction and asynchrony have been discussed previously by Patton et al. (2016), and hence the focus here is on the subsequent patterns and rates of retreat.
Ice masses have a profound impact on their substrates, through isostatic depression, enhanced glacial and hydraulic erosion, and sediment and precipitate deposition, resulting in long-term landscape evolution (e.g., Suggen and John, 1976; Hubbard and Hubbard, 1998; van der Veen, 1999). Assessing affinity with the empirical record in the Barents Sea is challenging as data collected from across this vast marine domain is still fragmentary and with poor chronological control, particularly in Russian sectors (Grosswald and Hughes, 2002; Svendsen et al., 2004; Patton et al., 2015; Hughes et al., 2016). This is reflected in the major discrepancies in the pattern of retreat interpreted and modelled around Novaya Zemlya (Fig. 6B–C). For this reason, the RSL dataset (Fig. 5A) represents a crucial constraint in our experiments for tuning the spatial distribution of ice volumes and the broad timing of retreat across the Eurasian Arctic. Aspects of the marine-based collapse of the BSIS can be inferred from sediments adjacent to the continental shelf break. For example, the onset of hemipelagic sedimentation 35 km east of the shelf break in Storfjordrenna at c. 20 ka BP (Rasmussen et al., 2007), combined with ice-rafter debris concentration peaks dated to c. 20.5–20 ka BP found in nearby cores (Jessen et al., 2010) provide good evidence for the regional commencement of deglaciation. The timing of this ice-sheet perturbation is enforced in the optimum model experiment through a dramatic rise in calving sensitivity across this sub-domain (Fig. 2D; Fig. 4A–B), that triggers its collapse and separation from the FIS. Compared to previous ice-sheet modelling experiments where this retreat initiates much later (e.g., <15 ka BP – Siegert and Dowdeswell, 2004), poorly reproducing RSL observations (Auriac et al., 2016), this experiment yields a notable improvement. The pace of subsequent retreat is unconstrained across the Barents Sea, though radiocarbon ages between 16.9 and 17.5 cal. ka BP (Rüther et al., 2011) and 11.1–11.6 cal. ka BP (Salvigsen, 1981) provide a timeframe for the complete retreat of the Bjørnøya renna ice stream from the shelf break to Kong Karls Land (Fig. 4B–G; Andreassen et al., 2014). Affinity with the interpreted ice stream dynamics associated with the Fennoscandian-Barents Sea separation in the western Barents Sea is reviewed in further detail in the discussion (section 5.1).
The rich archive of palaeo glaciological studies across the Fennoscandian domain has produced an abundant record of ice-sheet history. The resulting conceptual models that describe the detailed evolution of ice-sheet configuration and flow pattern in Fennoscandia based on geomorphological and chronological data (e.g., Kleman et al., 1997; Hughes et al., 2016; Stroeven et al., 2016) provide an accessible tool by which to compare the modelled deglaciation to the broad empirical footprint of the FIS. While the general pattern and sequence of the ice sheet aligns well with the time-transgressive formation of flow traces during deglaciation, a weakness of the reconstruction is the timing and pace of retreat across southern margins of the FIS, particularly through the Baltic Sea and Gulf of Bothnia (Fig. 6; Fig. 7). The warming associated with the Bolling/Allerød oscillation after c. 14.6 ka BP (Fig. 2B) induces a major collapse of this ice stream not recognized in an empirical record that is precisely dated in southern Sweden through the varve chronology (Strömberg, 1985). This discrepancy occurs despite imposed increases to the bulk precipitation over the FIS sub-domain at this time (Fig. 2C). Cooling during the Younger Dryas allows the FIS to stabilise and eventually advance up to c. 150 km across its southern margins, matching well most of the empirically defined margins during this stadial (green surge fans – Fig. 7A–B). An exception is in southern Sweden where the modelled margin is up to 200 km north of reconstructed limits, a direct result of the prior retreat during the Bolling-Allerød.
Our optimum numerical experiment reveals an early separation of the CIS and FIS at c. 22.4 ka BP across the Norwegian Channel (Patton et al., 2016) compared with recent reconstructions (Sejrup et al., 2016). Flow of the CIS across Caithness (Hall and Riding, 2016) and Orkney (Hall et al., 2016) after the LGM conforms to simulated flow paths. However, complete deglaciation of the northern North
Fig. 5. Comparisons between relative sea level (RSL) data (red dots) and model predictions for 5 locations (green stars) representative of the four regions – Franz Josef Land (FJL), Novaya Zemlya (NZ), northern Fennoscandia (Fe), and Svalbard (Sv). Black circles and error bars are empirical observations, with the black dashed line giving the elevation of the highest marine limit. The UiT_2016 model data (blue dashed line) refers to an earlier published iteration of the modelled deglaciation wherein ice retreat was more loosely constrained (Auriac et al., 2016). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Sea before 18 ka BP (Fig. 4B) is incompatible with radiocarbon dates of 17.5–16.2 cal ka BP for large moraines on the sea bed (Sejrup et al., 2015). The position of the Shetland ice cap and its coalescence with the CIS and FIS would have allowed the ice margin to persist later near the shelf edge and slowed its subsequent break up.
The timing of retreat of the Norwegian Channel ice stream from the shelf is now better constrained. Glacigenic debris flow sediments from the North Sea fan indicate that its calving front was close to the channel mouth until c. 19 ka BP (King et al., 1998). Cosmogenic ages from Karmøy and Utsira 300 km upstream have been interpreted as indicating an early retreat to this position by c. 20 ka BP (Svendsen et al., 2015), although a subsequent reanalysis of the cosmogenic data suggested that this age may be 10% too old.
due to prior exposure, with the area probably deglaciating at 18.5–18.0 ka BP (Briner et al., 2016). This timing fits the position onshore of the modelled outlet glacier after c. 17 ka BP (Fig. 4C; Fig. 7B). Its further retreat is also in line with dating of the Halland Coastal Moraines in southwest Sweden to c. 17.4–16.0 ka BP (Lundqvist and Wohlfarth, 2000; Larsen et al., 2012; Stroeven et al., 2016).
The final phase of FIS deglaciation is characterised by topographically controlled retreat to a northerly positioned ice cap over the Scandes hinterland. While this pattern of retreat is replicated in the optimum experiment, the persistence of ice over northern Finland allows the onset zone of the Bothnian Sea ice stream to remain over the northern Bothnian coastline (Fig. 4G–H). In reality, an onset zone has been clearly identified further southwest over the Angermanland-Västerbotten coastline (Greenwood et al., 2015). While this may have been a tributary to the main trunk of the ice stream, the contemporaneous flow sets suggest the ice cap was more westerly focussed by 10 ka BP than predicted in the model (Fig. 7).
The 10 km horizontal resolution of the model precludes detailed comparison with specific empirical evidence of the CIS beyond the broadest patterns of ice sheet volume and extent. For example, poor topographic resolution prevents the emergence of the Welsh ice cap despite climatic conditions conducive for its growth (cf. Patton et al., 2013a, Fig. 6). Similarly a Shetland based ice cap (Hall, 2013) is also conspicuously missing. The sensitivity of this ice sheet and its margins to dynamic behavioural responses, such as migrating ice-divides, potential surging and binge-purge cycles (Greenwood and Clark, 2009; Evans and Thomson, 2010; Clark et al., 2012; Hughes et al., 2014) therefore necessitates a more focussed, kilometre-scale approach to the model setup (e.g., Hubbard et al., 2009). The experiment does though provide support for an independent Younger Dryas glaciation of Highland Scotland, a matter of recent controversy (Bromley et al., 2014; Small and Fabel, 2016).
5. Discussion
The deglaciation of the EISC has attracted workers from a wide spectrum of geophysical and modelling disciplines (cf., Elverhøi et al., 1998; Larsen et al., 1999a; Svendsen et al., 2004; Evans et al., 2005; Clark et al., 2012; Ingólfsson and Landvik, 2013; Patton et al., 2015). A full discussion of our modelling results within the entire context of this history of research is overly ambitious, and beyond the purposes of this study. Various themes within this discussion have thus been chosen based on the more recent advances in our understanding of the ice complex development, as well as future directions where modelled output may prove useful in deciphering the complexities of a rapid, dynamic deglaciation.
5.1. Ice sheet collapse
The separation of the three semi-independent ice sheet centres represents an area of major uncertainty with regards to the deglaciation of the EISC. In the North Sea, recent interpretations from seismic (Graham et al., 2010; Sejrup et al., 2016), seafloor bathymetry (Bradwell et al., 2008; Ottesen et al., 2016) and chronological (Svendsen et al., 2015; Briner et al., 2016) data have narrowed down the timing and events that led to the breakup of ice from the shelf edge in this sector, although consensus is still to be accomplished. Due to the complexities of ice interaction in the region and the simplifications of the stress balance within the model, an optimum experiment was chosen whereby coalescence west of the Norwegian Channel was minimal (Fig. 6B; Patton et al., 2016). In experiments where continued expansion of North Sea ice to the shelf edge does occur, a stable ice-divide structure develops between Scotland and southern Norway that persists well into the Late Glacial, providing a major disruption to the modelled patterns of retreat for the southern Fennoscandian and Celtic ice sheets.
Less focus has been given in the literature towards the separation of the Fennoscandian and Barents Sea ice sheets, which in the optimum model run represents a much wider and more stable coalescent zone within the complex. During maximum conditions, the pivot point of this central ice divide lay north of the Baltic Sea ice stream in central Fennoscandia, with the position of the northward branch into the Barents Sea adjusting eastwards in response to vigorous drainage through the large troughs of the western shelf break (Patton et al., 2016). Surface glacial geomorphology both on- and offshore constrain the relative timing of glacier flows through deglaciation in this region (Hättestrand and Clark, 2006; Winsborrow et al., 2010; Bjarnadóttir et al., 2014), although knowledge of retreat sequences are limited in Russian waters.
A clear observation in the landform record is the phases of fast-flowing outlet glaciers parallel to the Norwegian coastline during deglaciation. Winsborrow et al. (2010) identified three active ice streams from flowsets of megascale glacial lineations and associated grounding zone wedges — the Coast-parallel Trough, Djuprenna, and Nordkapppbanken-east — all fed by catchment areas lying over Finnmark and the Kola Peninsula (Fig. 8A). Model results confirm that a number of “coast-parallel” ice streams operated north of Finnmark once Barents Sea and Fennoscandian ice merged, and likely persisted until the disintegration of this ice saddle c. 15 ka BP (Fig. 4C–D).
Not identified by previous workers is the mirrored eastwards flow of another “coast-parallel” ice stream flowing towards the Kanin Peninsula, as well as a further discharge route from the central ice dome through Sentraldjupet (Fig. 4C–D). Limited descriptions of sub-marine geomorphology from the White and Pechora seas places large uncertainties on ice flow patterns in this area of northwest Russia, though model results suggest that fast-flowing outlet glaciers persisted here from the LGM up until ice sheet separation at c. 15 ka BP. The Murmansk Bank moraine — a large acoustically transparent body — is the only prominent feature to have been mapped south of Sentraldjupet. It is composed of glaciomarine sediments overlying a normally consolidated till, speculated to have been deposited beneath warm-based ice coincident with large volumes of basal meltwater (Epshtein et al., 2011). Various interpretations have been placed on this moraine, including a standstill position (Svendsen et al., 2004; Bjarnadóttir et al., 2014) and an inter-ice stream ridge for ice flowing west (Winsborrow et al., 2010). Based on the predicted position of the collapsing ice saddle c. 15 ka BP in the optimum model experiment, neither of these interpretations appear viable. An alternate interpretation is that the Murmansk Bank moraine represents the amalgamation of an inter-ice stream ridge between ice streams flowing east and a lateral shear margin moraine (cf. Hindmarsh and Stokes, 2008) between ice exiting in Sentraldjupet and the cold-based central ice divide.
To test the accuracy of predicted model vectors in this region, flow direction analyses were carried out using the automated techniques described by Li et al. (2007), providing a systematic and quantitative comparison between modelled flow patterns and field observations. For each flowset, flow correspondence is measured through temporal variations of the mean difference (residual) and the residual variance. Detailed mapping of seafloor glacial lineations across the southern Barents Sea has revealed a comprehensive palimpsest of switching flow sets (Ottesen et al., 2005; Winsborrow et al., 2010; Rüther et al., 2011; Bjarnadóttir et al., 2014; Piasecka et al., 2016). Ice drainage in this region is
dominated by the Bjørnøyrenna ice stream, which drained extensive portions of the central and southern Barents Sea shelf (Patton et al., 2015), the retreat of which is documented by a series of grounding zone wedges that are overprinted with MSGLs (Winsborrow et al., 2010; Rüther et al., 2011). At least 4 major episodes of standstill/readvance have been identified (flowsets 12–16; Fig. 8A) related to topographically confined retreat northwards, with the first readvance dated to c. 17.1 cal ka BP (Rüther et al., 2011). Correspondence to mapped lineations is best during the initial advance of the ice sheet, after which coalescence with Scandinavian ice at 24 ka BP deviates ice sheet flow by c. 50°. Although the timing of retreat of this ice stream closely matches empirical estimates (Winsborrow et al., 2010), flow correspondence tends to decrease upstream (Fig. 8B), possibly related to over-estimations of ice thicknesses within the trough.
Flowset patterns around the Finnmark coast are characterised...
by topographically confined flow within the fjords (flowsets 2, 4) as well as coast-parallel ice streams (flowsets 5, 6, 17). Sharp deviations in the modelled flow reveal that the latter flowsets aligned perpendicular to the current coastline were most likely imprinted while the Barents and Fennoscandian ice sheets were still coalesced. Conversely, ice flow within the coastal fjords (flowsets 2, 4) aligns only once this saddle collapses and ice thicknesses begin to decrease c. 15 ka BP (Fig. 8C).
This pattern is similarly true for the offshore troughs draining the northwest Norwegian coast. In most cases the effects of ice thinning during deglaciation serve to better align model flow with the mapped lineations (Fig. 8D). The northernmost flowset of this group in Håkjerringdjupet (flowset 8) experiences some deviation imposed by the advancing BSIS, suggesting this ice stream was not fully constrained by the basal relief. The north-eastwards direction of flowset 10, although confined within a small trough (Sorøydjupet), appears to contradict the majority of flow patterns recorded along this coast, though it has been attributed with an LGM age (Winsborrow et al., 2010). Model data show that once the ice sheets merged in this sector, the weak relief of this trough was not enough to resist the general ice sheet flow perpendicular to this trough. Only during the latter stages of deglaciation does model flow begin to align with this trough (Fig. 8D).
5.2. Retreat, still-stands and readvances
The retreat of the EISC and its outlet glaciers has been typically shown to be non-linear and asynchronous, undergoing temporary pauses, or even readvances, in response to a number of external or internal responses (Chiverrell et al., 2013; Patton et al., 2013b; Andreassen et al., 2014; Bjarnadóttir et al., 2014; Clason et al., 2016; Stroeven et al., 2016). The ability of the model to reproduce these dynamics and recreate the observable geomorphological signature can be examined by calculating the relative positioning of ice margins at each experiment timeslice and locating margin standstills that are not subsequently overrun during deglaciation – effectively producing a map of potential moraine systems (Fig. 9). The pattern of potential moraines generally matches mapped moraines (Fig. 7; Stroeven et al., 2016), with multiple moraine lines along the southern and eastern margin of the FIS, clear Younger Dryas moraines, and an absence of post-Younger Dryas moraines.
Some of the identified standstill positions relate to major readvances associated with downturns in the climate forcing record, for example the Younger Dryas interstadial, while others can be attributed to stable positions associated with the retreat of ice into topographic constrictions. Prominent examples of the latter include the deglaciation of ice in the Kandalaksha Gulf, the Irish Sea ice stream, and between Franz Josef Land and Novaya Zemlya. Conversely, areas of rapid and catastrophic ice retreat are also evident by large gaps between standstill positions. Examples of such pronounced retreat occur along the southern margins of the CIS after 23 ka BP, and the FIS after 15 ka BP through the Baltic Sea. However, both instances are overshadowed by the disintegration of the BSIS after 17.8 ka BP, which collapsed at a peak rate of 669 Gt a\(^{-1}\) (Fig. 3B).
Partitioning of ice losses clearly shows calving processes, exacerbated by limited topographic relief, were the dominant drivers of widespread collapse of grounded ice across the southern Barents Sea, with ice cover only stabilising once back into terrestrial sectors by c. 12.5 ka BP (Fig. 10D). The discharge of large fluxes of iceberg into the Polar North Atlantic, peaking at c. 15.2 ka BP and coincident with Heinrich Event 1 (Hoff et al., 2016), correlates with pronounced ice rafted debris (IRD) spikes along the western continental shelf break (Bischof, 1994; Jessen et al., 2010). Although the collapse of the BSIS is semi-conditioned within the optimum model experiment by increasing the sensitivity to ice-calving in this sector after 22 ka BP (Fig. 2D), the growing length of the calving margin during separation of the FIS and BSIS provides a clear and simple mechanism for amplifying rapid collapse of this marine-based sector through to the Bolling (Fig. 4C–E).
The timing of turbiditic sedimentation and IRD fluxes obtained from the continental slope adjacent to the CIS (Scourse et al., 2009) is also well correlated in most respects with the modelled evolution of this marine-terminating ice sheet. A pronounced increase in IRD flux at the Rosemary Bank and the Barra-Donegal Fan at c. 29 ka BP is reflected by the early growth offshore of the CIS (Fig. 10B). By 24 ka BP, all cores record a peak IRD flux coincident with Heinrich Event 2. Although a specific peak in the calving flux record is not observed from the optimum model experiment, the rate of mass loss at the marine margins in consistently high over this time period, and periodically exceeds surface melt driven by rising air temperatures, likely enhanced by localised faunal factors that potentially played a role in the retreat of the Laurentide ice sheet (Squeak and Diddelsworth, 1987). Post-LGM warming forced the rapid retreat northwards of the CIS (Fig. 9), although persistent ice across northern Britain during deglaciation would have contributed to the IRD peaks observed on Rosemary Bank up to c. 17 ka BP (Scourse et al., 2009). After 15 ka BP, all records show the IRD flux close to zero (Kroon et al., 1997; Austin and Kroon, 2001; Scourse et al., 2009), indicative of retreat back onto land (Fig. 10B), except for a minor IRD spike during the Younger Dryas, possibly related to the expansion of marine terminating glaciers in Scotland.
5.3. Glacial isostatic adjustment modelling
The limited presence of coastlines in the Barents-Kara Sea domain represents a fundamental physical challenge for attempting to procure a comprehensive history of RSL change. However, a long history of research initiated during the 1960s (e.g. Blake, 1961; Hoppe et al., 1969) has produced a sizable database of RSL data that covers most peripheral areas of the former Late Weichselian BSIS (cf. Forman et al., 2004). Previous inter-comparison between available ice-load scenarios of the Barents Sea deglaciation and empirical RSL data reveal wide discrepancies between reconstructions, with none able to reproduce a good fit to the 46 RSL observation sites simultaneously around the domain (Auricà et al., 2016). However, all scenarios tended to support the hypothesis of a single ice dome centred on the Barents Sea during the LGM.
The isostatic footprint associated with the optimum experiment presented here achieves a best fit to the RSL observations when using Earth models optimised across the four sub regions of the Barents and Kara seas. Spatial heterogeneity in the preferred Earth properties may reflect real differences in Earth structure across the region; for example, the lithosphere is observed to thicken from ~80 to ~150 km beneath the eastern Barents Sea/Kara Sea region (Ritzmann and Faleide, 2009; Klitzke et al., 2015). However, it may also reflect biases inherited from an incomplete RSL observation record or incorrectly tuned ice load history. For example, across Novaya Zemlya, empirical data relating to relative sea level during the Early Holocene is largely missing, making it difficult to quantify the change in sea-level rate since the Late Glacial, and hence the relaxation time of the upper mantle. If there are inaccuracies in the ice history model this can, to some degree, be compensated for by the choice of Earth model. For example, in the case of a too-thick LGM ice sheet the model will prefer an unrealistically low value for upper mantle viscosity in an attempt to force the rebound to take place relatively quickly in order to fit the data. Some coherence between the various regional Earth models is observed though, with a relatively thick lithosphere of 120 km predicted throughout the majority of the region, and a relative insensitivity to lower
mantle viscosity observed at all sites, with the latter result being in agreement with earlier studies (e.g., Lambeck et al., 1998; Steffen and Kaufmann, 2005).
Svalbard represents the region of greatest mismatch; in particular, no single Earth model is able to reproduce the RSL data along the west coast. The most notable discrepancy is related to the rapid RSL fall of 15—30 m ka$^{-1}$, observed at Sv8-Sv11 and Sv26 (Forman, 1990) (Fig. 5) during the earliest Holocene, which is speculated to have coincided with rapid retreat of fjord glaciers on western Svalbard prior to 10.5 ka BP (Forman, 1989). By ignoring these sites a reasonable fit can otherwise be obtained for sites further east using a weak upper mantle viscosity ($0.3 \times 10^{21}$ Pa s) (Table 1), in agreement with the findings of Steffen and Kaufmann (2005). RSL data across central and eastern Svalbard indicate large magnitude (up to $\sim$100 m), exponentially-decaying sea-level fall across the region throughout the Holocene (Forman et al., 1997), consistent with these sites being in close proximity to the location of greatest loading (Forman et al., 2004).
One implication of the large isostatic footprint across marine sectors of the Eurasian Arctic is the potential for changes in oceanographic circulation between the Atlantic and High Arctic. It has been previously hypothesized that bathymetric depression in the Barents Sea during the last deglaciation permitted subsurface Atlantic Water to traverse topographic barriers and enter the northern Franz Victoria and Saint Anna troughs (e.g., Lubinski et al., 2001). Increased Atlantic Water inflow during the Bølling-Allerød
has also been recorded east of Svalbard, suggested also to have been facilitated by isostatic depression at this time (Kristensen et al., 2013).
Although a number of other factors could explain the changing fluxes of Atlantic Water into the northern Barents Sea, for example, atmospheric circulation patterns or high insolation seasonality,
isostatic modelling indicates that postglacial bathymetric changes remains a viable hypothesis (Fig. 11). Sills southwest of Franz Victoria Trough that can be found at depths of c. 50–150 m today were >200 m deeper around 12.0 ka BP. Similarly, the upper reaches of Bjørnøya renna, large areas of Sentraldjupet, and the eastern Barents Sea were still >350 m deep during the Late Glacial.
The heterogeneous effects of glacial isostasy across the western Eurasian continental shelf, where extensive hydrocarbon reserves exist, also has important ramifications on the patterns and timing of subglacial gas hydrate sequestration, subsequent dissociation and potentially abrupt methane seepage that occurred post deglaciation (Hovland et al., 2005; Rise et al., 2015; Crémière et al., 2016; Portnov et al., 2016; Serov et al., in press; Andreassen et al., 2017). Directly pinpointing the timing of past methane release events is difficult, though this can be obtained empirically through U-Th dating of methane-derived authigenic carbonates found close to the seafloor seeps (e.g., Teichert et al., 2003; Crémière et al., 2016). A more readily available solution is to model the evolution of the gas-hydrate stability zone and its response to fluctuations in ice overburden, basal temperatures and isostatic loading, constrained by observations of the gas flare composition (e.g., Andreassen et al., 2017; Serov et al., in press; Portnov et al., 2016). Given a well-constrained reconstruction of the EISC through the last glacial cycle, this modelling output can provide insights into the evolution of the gas hydrate system through glacial-interglacial cycles with potentially important consequences on atmospheric composition and climate (Andreassen et al., 2017).
5.4. LGM hydrology
5.4.1. Subglacial lakes
Subglacial lakes are a common feature of the Antarctic hydrological system, occurring in a variety of topographic, thermal and ice-dynamical settings, and at a range of scales (e.g., Wright and Siegert, 2012), with many more postulated to exist (Livingstone et al., 2013b). However, the identification of palaeo subglacial lakes remains sporadic and controversial (e.g., McCabe and Ó Cofaigh, 1994; Munro-Stasiuk, 2003; Sutinen et al., 2007; Walker et al., 2007; Christoffersen et al., 2008; Livingstone et al., 2013a), limited by a lack of distinguishable geological identifiers (cf. Livingstone et al., 2012).
To examine the potential locations of lakes beneath the LGM EISC a three-dimensional basal hydraulic potential surface ($\phi$) was created following the approach of Livingstone et al. (2013a, 2013b) using the following equation (Shreve, 1972; Clarke, 2005):
$$\phi = \rho_w g h_b + F \rho_i g H,$$
where $\rho_w$ is the density of water (1000 kg m$^{-3}$), $\rho_i$ is the density of ice (917 kg m$^{-3}$), $g$ is the acceleration due to gravity, $h_b$ is the bed elevation, and $H$ is the ice thickness. $F$ is the flotation fraction and represents the ratio between ice overburden pressure and subglacial water pressure. Where $F = 1$, the pressure in subglacial conduits is at the ice overburden pressure; where $F = 0$ it is at atmospheric pressure. This value is spatially and temporally variable according to drainage system character, basal ice temperature, and basal geology (Clarke, 2005). Here, we use a value of 0.925 for the flotation fraction, based from empirically constrained hydrological modelling of subglacial catchments in Greenland (e.g., Banwell et al., 2013; Lindbäck et al., 2015). In this calculation we also assume that the bed was warm based, and that basal melting and effective pressure were uniform. In reality, ice sheets are in fact polythermal and so basal melting will vary across the bed, with basal ice temperatures below the pressure melting point restricting basal melt.
Meltwater routing along the maximum gradient of the hydraulic potential surface was determined using Arc Hydro Tools. Lakes were identified from hydraulic potential lows that were subsequently filled to their lip. The elevation model used is an isostatically adjusted and resampled version of the GEBCO_2014 Grid at 500 m resolution, with glacial isostatic effects derived from the coupled elastic lithosphere/relaxed asthenosphere scheme of the ice model. Upsampling of $H$ to the same resolution was carried out using a 500 m bilinear resampled version of the modelled ice surface.
The relatively simplistic nature of the approach has some
obvious limitations, including using an elevation model that is already filled with post-glacial sediments and bodies of freshwater, the potential inaccurate representation of ice features during the upscaling process, and a lack of dynamic coupling between ice and subglacial meltwater. As such, these factors will tend to influence the under-prediction of lake locations. Conversely, the bed is also considered to be entirely warm-based. Given that large areas of the LGM bed were permanently frozen (e.g., Kleman and Glasser, 2007) this factor will lead to an over-prediction of lakes.
For the LGM timeslice used (22.73 ka BP), a total of 6143 individual potential subglacial lakes are predicted (Fig. 12). A threshold surface area of 2 km$^2$ was used to remove probable interpolation artefacts. Although only one timeslice is analysed here, this snapshot shows that lake formation beneath the EISC was potentially widespread. However, the size distribution is heavily skewed towards small features — 71% of the lakes have a surface area $\leq 10$ km$^2$, and only 106 of them are $\geq 50$ km$^2$. A similar size distribution skew exists for lakes identified beneath the Antarctic Ice Sheet, with a large majority shorter than 10 km (Wright and Siegert, 2012).
Potential lake locations are varied, with lakes found beneath ice divides, fast-flowing outlet glaciers and ice sheet convergence zones (Fig. 12). Rugged topography attracts a greater proliferation of smaller lakes, in particular across Fennoscandia and the United Kingdom. In particular, the greater relief of Fennoscandia forces ponding to occur along the major subglacial drainage pathways. The collection of lakes west of Novaya Zemlya is also another example of where strong relief is able to force subglacial ponding. The relative absence of lakes throughout offshore sectors is in stark contrast, potentially exacerbated by a number of factors including the generally smooth relief of the continental shelf, poor coverage of multibeam echosounder data, and subsequent glacial/marine sedimentation. The central high of Sentralbanken and Storbanken, beneath the central ice divide of the BSIS, appears to be the most likely location for lake formation in marine sectors of the Eurasian Arctic during the glacial maximum, in support of recent observations of extensive networks of tunnel valleys in this region (Bjarnadóttir et al., 2016). Similarly, lakes in the North Sea tend to occupy pre-existing tunnel valleys (e.g., Kristensen et al., 2007). Finally, the topographic narrowing of the Gulf of Bothnia into the Baltic Sea represents a distinct area of concentrated lake formation that includes the largest lake of the domain (514 km$^2$).
Distinguishing palaeo subglacial lakes from proglacial water bodies in the sedimentological record is a challenging task that has led to limited reporting from the palaeo record (Livingstone et al., 2012). Subglacial lake locations have been speculated upon in northern Germany (Livingstone et al., 2015) and eastern Ireland (McCabe and Ó Cofaigh, 1994), but while lakes are potentially present in both these regions during the LGM, a positive match at both sites is absent. It is envisaged that further examination into the persistence of predicted lake locations throughout the glacial cycle will identify sites prime for further investigation.
The proliferation of potential subglacial lakes and low relief within the Gulf of Bothnia and Baltic Sea indicate lakes here were probably connected, and may have formed a major control on the dynamic behaviour of the Baltic Sea ice stream, similar to observations from Antarctica (Bell et al., 2007; Fricker and Scambos, 2009; Kim et al., 2016). The importance of hydrological feedbacks on ice stream behaviour in the Baltic sector has been highlighted by previous process modelling, which showed that surface meltwater-enhanced basal sliding during deglaciation could significantly quicken ice sheet drawdown and prompt earlier ice stream
**Fig. 12.** The distribution of 6143 potential subglacial lake locations beneath the LGM EISC. A number of key assumptions will affect the under- and over-prediction of these lake locations, including use of an initial elevation model draped with (post-) glacial sediments and bodies of freshwater, a lack of dynamic coupling between ice and subglacial meltwater, and the treatment of the bed as being entirely warm-based. Water routing and filling of the basal hydraulic potential surface was carried out using Arc Hydro Tools for ArcGIS 10.3. The elevation model used is an isostatically adjusted and re-sampled version of the GERCO_2014 Grid at 500 m resolution, with glacial isostatic effects derived from the coupled elastic lithosphere/relaxed asthenosphere scheme of the ice model. Lakes with a surface area <2 km$^2$ were discarded from this analysis. Acronyms used: Se – Sentralbanken; St – Storbanken.
cessation (Clason et al., 2014). Extensive areas of De Geer moraines across southern Finland also point to the importance of marginal glacial lakes for glacier retreat dynamics (Ojala, 2016). However, the true influence of the hydrological feedbacks on ice sheet dynamics will remain a source of uncertainty until physically based coupling of the subglacial hydrology system is implemented in ice sheet models (cf. Stokes et al., 2015).
5.4.2. Paraglacial river network
One of the greatest impacts that ice loading and eustatic sea level lowering had on western European palaeo geography was the modification of the paraglacial river network (Toucanne et al., 2015). During glacial times, the Channel/Fleuve Manche palaeoriver routed substantial volumes of water to the North Atlantic via the Celtic Sea, resulting in the largest river system to have ever drained the European continent (Gibbard, 1988; Lericolais et al., 2003; Ménot et al., 2006). By melding isostatically adjusted topography with a subglacial hydraulic potential surface, the probable routing and potential catchment reach of this river system across Eurasia and beneath the EISC can be reconstructed (Fig. 13). When the ice complex was at its maximum size at 22.73 ka BP (Patton et al., 2016), the Fleuve Manche mega-catchment covered an area of $2.56 \times 10^6$ km$^2$ (Table 2) – 50% larger than the Mackenzie River in Canada today – incorporating drainage via the Seine, Rhine, Thames, Elbe, and Vistula. Over half of its catchment was ice-covered ($1.36 \times 10^5$ km$^2$), capturing subglacial meltwater drainage from large portions of the coupled English-Welsh ice domes, the southern North Sea and the Gulf of Bothnia/Baltic Sea (Fig. 13). Many of these areas were drained by relatively large, warm-based ice streams, including the North Sea and Baltic Sea ice streams (Fig. 1; Stokes and Clark, 2001). Added to this the surface melting fluxes from across 33 degrees of longitude, the glacial system thus exerted a major control on this catchment during the LGM and early deglaciation. The vast reach of the catchment, combined with the sensitivity of the system to periodic meltwater injections triggered by ice sheet fluctuations, meant this palaeoriver likely perturbed regional North Atlantic hydrography, possibly even influencing the timing of Heinrich Events that punctuated past glacial periods (Nesje et al., 2004; Eynaud et al., 2007; Toucanne et al., 2009, 2015). In this respect, modelling the evolution of the glacial-paraglacial hydrological system across Eurasia through a full glacial cycle will provide an important step forward.
5.5. Younger Dryas
The deglaciation of the EISC was interrupted by a pronounced climate deterioration and renewed glacier growth c. 12.9–11.7 ka BP, referred to as the Younger Dryas stadial. The legacy of this readvance is reflected in the glacial landform assemblage by prominent sediment and moraine systems across Fennoscandia and the Scottish Highlands (Lundqvist, 1990; Bennett and Boulton, 1993; Andersen et al., 1995; Lundqvist and Saarnisto, 1995; Mangerud, 2004; Golledge, 2010; Stroeven et al., 2016).
Within the Eurasian Arctic, evidence for a major glacier advance at this time is relatively limited (cf. Patton et al., 2015). Relatively low IRD concentrations on the Yermak Plateau (Birgel and Hass, 2004; Chauhan et al., 2014), and at the western (Slubowska-Woldengen et al., 2007) and northern (Koc et al., 2002) Svalbard shelf indicate that ice rafting was reduced at this time even though waters here were seasonally ice free. Furthermore, reports that glaciers on Spitsbergen were smaller during the Younger Dryas than during the Little Ice Age (1900 CE) appear to corroborate overall minimal glacier advance during this cold interval (Mangerud and Svendsen, 1990; Mangerud and Landvik, 2007; Reusche et al., 2014). Limited empirical evidence appears to indicate that the subdued glacier growth here was climatically controlled, driven by prevailing easterly winds (Renssen et al., 2001; Birgel and Hass, 2004) isolated from a source of moisture by near-perennial sea ice (Kristensen et al., 2013; Müller and Stein, 2014). The starvation of precipitation was also exacerbated by relatively high summer insolation, with values at this latitude about 10% higher than today (Berger and Loutre, 1991). In the absence of distinct geomorphology, defining distinct marginal limits for the Younger Dryas ice sheet remnant over Svalbard has therefore become a task of probability based on the locations of deglaciation dates (e.g., Hormes et al., 2013).
While relatively little is known of the Younger Dryas ice sheet over Svalbard, even less is known of ice cover further east at this time. Similar negative glacier mass balance relationships have been inferred across Franz Josef Land during the Holocene, with radio-carbon dates indicating that glaciers were either near or behind present limits at the start of the Holocene c. 11.5 cal ka B.P. (Lubinski et al., 1999). Elsewhere, pollen data indicate that some coastal areas of western Novaya Zemlya were probably ice free during the Younger Dryas (Serebryanny et al., 1998).
Although the resolution of the model reconstruction precludes any detailed insights for the relatively small Eurasian Younger Dryas ice caps, it is clear from the prescribed forcing (Fig. 2C) that the distribution of precipitation is key for determining the rapid regrowth of ice cover across more southern latitudes. Across Celtic, and to a lesser extent Fennoscandian, sectors an (asynchronous) increase in bulk precipitation is required post-LGM in order to prevent runaway losses in the mass balance budget and eventually yield realistic Younger Dryas extents. The importance of precipitation distribution has been further illustrated through high-resolution modelling of the Scottish Younger Dryas ice cap, wherein strong rain-shadow and precipitation seasonality effects are necessary to match the empirically constrained ice expansion (Golledge and Hubbard, 2005; Golledge et al., 2008, 2010).
The onset of climate cooling in the Younger Dryas halts the overall negative mass balance of the FIS, and eventually leads to a slight net gain in ice volume during the course of the stadial (Fig. 3B). A major glacial advance during this period appears to be triggered by a short-lived warming oscillation (0.9 °C MAAT) at 12.5 ka BP, that induces the spread of subglacial sliding and the draw-down of ice via outlet glaciers across the southern margin 200 years later, reaching a maximum extension c. 11.7 ka BP. The oscillatory nature of the FIS at this time is well preserved in proglacial sediments, with evidence for approximately 10 climatically induced ice margin recession/readvances of the southeastern FIS during the Younger Dryas (Boden et al., 1997). A major topographic pinning point between Finland and Sweden, the Aland Islands, proves to be a stable margin position for the large Bothnian Sea ice stream (Lundqvist, 2007), which was wider at this time due to delayed glacial isostatic uplift (Fig. 4G–H). The largest discrepancy between modelled and observed limits is in southern Sweden. Ice that occupied this region would have been sourced from the mountains of mid-south Norway (e.g., Skarvan and Trollheimen — as seen in earlier timeslices). The absence of ice here from as early as the Bølling-Allerød oscillation therefore may reflect poor predictions in the distribution of the precipitation/snowfall budget in this typically maritime region of the domain, or a dynamically driven advance not accounted for by the model.
Where ice margins block the natural drainage of ice-free catchments, proglacial lake formation can occur. In the case of the retreating FIS, two major lakes formed (cf. Fig. 3 in Stroeven et al., 2016), the largest being the Baltic Ice Lake (349,000 km$^2$; Jakobsson et al., 2007) (Fig. 14). The other was the “White Sea Ice Lake” (e.g., Larsen et al., 2006; Lunkka et al., 2012), dammed at the Kola Peninsula by a prominent 100 km wide, sub-aerially exposed
sill. At the peak of the Younger Dryas glacial extension, glacial isostatic adjustment effects left both lakes separated from the adjacent seas by topographically controlled spill points at elevations of 24 and 23 m above contemporaneous sea level, respectively. For the Baltic Ice Lake this predicted spill point position deviates away from the commonly attributed drainage site north of Mount Billingen, 1 m higher in elevation (Fig. 13; O'Regan et al., 2016).
The combined volume of the two lakes, assuming each was filled to spill-point, was 27,635 km$^3$ – a volume 20% larger than that of the present Laurentian Great Lakes combined (Table 2). Varved clay records and cosmogenic nuclide exposure dating from the Baltic Ice Lake drainage region indicate that the final drainage of the Baltic Ice Lake occurred c. 11.62 ka BP, releasing c. 7800–9400 km$^3$ of fresh water into the North Atlantic in under a year (Andrén et al., 2002; Jakobsson et al., 2007; Stroeven et al., 2015), shortly after the period of maximum extension modelled during this stadial at 11.73 ka BP. It is likely that the catastrophic release of such vast volumes of fresh water from both proglacial lakes would have played a role in disturbing North Atlantic circulation, as well as the remaining marine-terminating margins of the EISC (Clark et al., 2001; Andrén et al., 2002; Lunkka et al., 2012).
These two lakes formed a major part of the proglacial surface hydrological network during the latter stages of Eurasian
Fig. 14. The peak glacial extent during the Younger Dryas stadial compared to empirically derived limits (red line; Stroeven et al., 2016). During this brief interstadial the Baltic and White Sea ice lakes were major components of the proglacial hydrological system, damming Eurasian river drainage and surface melt fluxes from the Fennoscandian ice sheet. Their outlets were c. 24 and 23 m above contemporaneous sea level, respectively. Mount Billingen (MB) is the commonly attributed spill point for the Baltic Ice Lake at c. 25 m above contemporaneous sea level (e.g., Stroeven et al., 2015). Surface areas of the two lakes total 321,715 and 55,610 km$^2$, respectively (Table 2). Catchment areas (not including glacial sources) total $4.97 \times 10^5$ km$^2$ for the Fleuve Manche River in the English Channel (orange), $9.26 \times 10^5$ km$^2$ for the Baltic Ice Lake (yellow), and $3.61 \times 10^5$ km$^2$ for the White Sea Ice Lake (green; Table 2). The contemporary continental river network (blue lines) was calculated using Arc Hydro Tools for ArcGIS 10.3. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 2
Dimensions and capacities of major hydrographic features of the Eurasian domain during the last glacial cycle. Younger Dryas catchment areas for the Baltic Ice Lake and White Sea Ice Lake concern their paraglacial portions only.
| | Lake area km$^2$ | Lake volume km$^3$ | Basin area $10^5$ km$^2$ |
|--------------------------------|------------------|--------------------|--------------------------|
| **Last Glacial Maximum** | | | |
| Fleuve Manche palaeoriver | – | – | 25.58 |
| (of which subglacial) | – | – | 13.62 |
| (of which paraglacial) | – | – | 11.96 |
| **Younger Dryas** | | | |
| Fleuve Manche palaeoriver | – | – | 4.97 |
| Baltic Ice Lake | 321,715 | 23,464 | 9.26 |
| White Sea Ice Lake | 55,610 | 4171 | 3.61 |
deglaciation, with both capturing the drainage of most eastern European and western Russian river systems, as well as meltwater from the diminishing FIS (Fig. 14). The combined effects of isostatic adjustment and reduced eustatic sea-level position c. 11.7 ka BP also still had a far-reaching impact on the drainage catchments of many western European rivers, particularly those feeding towards the North Sea. With a land bridge still in place between England and continental Europe during the Younger Dryas, the Fleuve Manche in the English Channel captured surface drainage from a number of major river systems including the Thames, Rhine, and Seine. The drainage area of this palaeoriver during the Younger Dryas of c. $4.97 \times 10^5$ km$^2$ is estimated to have been much reduced from its earlier configuration at the LGM (Fig. 13; Table 2).
6. Conclusions
A first-order ice sheet model was used to reconstruct the deglaciation of the last Eurasian ice sheet complex (EISC; 23–8 ka BP), in a direct continuation of previous work by Patton et al. (2016) who modelled the asynchronous growth of the ice complex to its Last Glacial Maximum (LGM) extent. Various lines of geophysical data were used to guide the broad retreat patterns of the modelled ice sheet, including key dating constraints, geomorphological flow sets, notable moraine/grounding zone wedge positions, and patterns of isostatic loading. An evaluation into the robustness of the optimum deglaciation experiment against these empirical constraints, as well as a detailed analyses of the results, reveals the following insights into the retreat of the EISC:
- Retreat of the ice complex was asynchronous, reflecting regional sensitivities to climate change and various drivers of retreat. Even with increases in bulk precipitation, the Celtic ice sheet diminished rapidly in response to even modest increases in mean annual air temperature, receding to the Scottish Highlands by 16 ka BP. However, retreat of the marine-based Barents Sea ice sheet was not markedly affected by surface melt, and was instead driven by a combination of reduced mass input (precipitation) and increased rates of calving.
- Independent glacial isostatic modelling shows that the predicted pattern of ice loading matches reasonably well with Late
Glacial relative sea level records from across the Eurasian Arctic, particularly over Franz Josef Land, Novaya Zemlya and northern Fennoscandia. Model-empirical misfits are best resolved through regional optimisations of the Earth model. There is support for a low-viscosity zone beneath some regions of the Barents Sea, as proposed in earlier work, but spatial differences in the optimum Earth model may also reflect shortcomings in the ice sheet reconstruction.
- Periodic discharges of icebergs from different regions through the Late Weichselian resonate with numerous observations from the ice-rafted debris record. These include pronounced fluxes at c. 29 ka BP along the western Celtic margin associated with early and rapid expansion of ice in this sector, but also the collapse of the marine-based Barents Sea ice sheet which peaked at 15.2 ka BP with a mass loss rate equivalent to 669 Gt a\(^{-1}\). The total EISC contribution to Meltwater Pulse 1A, a short-lived period of dramatic global eustatic sea-level rise coeval with the Bølling warming that exceeded 40 mm per year (14.65–14.31 ka BP; Deschamps et al., 2012), was c. 2.5 m of sea-level equivalent ice volume.
- Ice caps reacted differently across the domain during the Younger Dryas. Increased accumulation of ice across the central sectors of the Fennoscandian ice sheet eventually forced a purge of ice through the Gulf of Bothnia at 12.3 ka BP. Reduced bulk precipitation across the Arctic limits any expansion of the remaining ice caps across Svalbard and Russian territories, while a cold-based Scottish ice cap grew rapidly from ice-free conditions within 1 ka.
- The chronology of retreat of Fennoscandian ice through the Baltic and Bothnian seas is a major shortcoming of the model reconstruction, despite an otherwise sound match to the pattern of ice flow history recorded by numerous flowsets. Coupling of the ice model to climate modelling output, for example, via PMIP4 (Kageyama et al., 2016), may potentially resolve such discrepancies in the deglacial mass balance regime, and is an area worthy of future inspection.
- The large Baltic and White Sea proglacial lakes expanded rapidly after retreat of the FIS during the Bølling/Allerød transition, and persisted through the Younger Dryas stadial capturing surface runoff from a combined coverage of \(12.9 \times 10^5\) km\(^2\) of the Eurasian continent at this time.
- Hydrological routing beneath the LGM ice complex reveals 6143 potential subglacial lake locations across a variety of glaciological settings, with the distribution heavily skewed to surface areas \(\leq 10\) km\(^2\). The largest lake lies in the Gulf of Bothnia, beneath a zone of prominent ice streaming from the Fennoscandian ice sheet, and in combination with a proliferation of smaller lakes thus likely formed a major control on its dynamic behaviour.
- Catchment areas for the major Eurasian rivers were heavily affected by the presence of the EISC as well as eustatic sea-level lowering, with the Fleuve Manche river mega-catchment dominating the LGM European geography. Its catchment area exceeded \(2.5 \times 10^6\) km\(^2\) during the Last Glacial Maximum, including the drainage of meltwater runoff from one quarter of the ice complex. The discharge for such a river system, sensitive to periodic meltwater injections triggered by ice sheet fluctuations, was a probable driver for North Atlantic circulation perturbations for much of the glaciation.
**Acknowledgements**
The research is part of the Centre for Arctic Gas Hydrate, Environment and Climate (CAGE), and the GLANAM (GLAciated North Atlantic Margins) Initial Training Network. It was supported by the Research Council of Norway through its Centres of Excellence funding scheme (grant 223259), the PetroMaks project “Glaciations in the Barents Sea area (GlaciBar)” (grant 200672), the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007-2013/ (REA grant agreement 317217), and a Stockholm Uni SUCLIM consortium grant (to Stroeven), that supported Hubbard in the early development of the model code. We thank two anonymous reviewers for their constructive feedback to improve the manuscript.
**Appendix A. Supplementary data**
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.quascirev.2017.05.019.
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A method, system, and computer program product in a data processing system are disclosed for dynamically selecting software buffers for aggregation in order to optimize system performance. Data to be transferred to a device is received. The data is stored in a chain of software buffers. Current characteristics of the system are determined. Software buffers to be combined are then dynamically selected. This selection is made according to the characteristics of the system in order to maximize performance of the system.
15 Claims, 4 Drawing Sheets
FIG. 1
300 START
302 DETERMINE NUMBER OF CPUs IN SYSTEM
304 DETERMINE SPEED OF EACH CPU
306 DETERMINE ACCESS LATENCY OF MEMORY SUBSYSTEM
308 DETERMINE DMA CAPACITY FOR EACH I/O ADAPTER USING SLOT SIZE AND SPEED OF I/O ADAPTER
310 DETERMINE A THRESHOLD FOR EACH I/O ADAPTER USING NUMBER AND SPEED OF EACH CPU, MEMORY ACCESS LATENCY, AND DMA CAPACITY OF THE I/O ADAPTER
312 STOP
FIG. 3
START 400
TRANSFER DATA? 402
NO
DETERMINE THRESHOLD FOR I/O ADAPTER WHICH IS TO RECEIVE THE DATA 404
MAKE FIRST BUFFER OF BUFFER CHAIN OF DATA TO BE TRANSFERRED THE CURRENT BUFFER 406
DETERMINE SIZE OF CURRENT BUFFER 408
LAST BUFFER IN BUFFER CHAIN? 410
YES
SIZE OF LAST BUFFER > THRESHOLD? 412
NO
CREATE A NEW BUFFER BY COPYING THE DATA FROM THE LAST BUFFER AND THE DATA FROM THE BUFFER IMMEDIATELY PREVIOUS TO THE LAST BUFFER IN THE BUFFER CHAIN INTO THE NEW BUFFER 414
REPLACE COMBINED BUFFERS IN BUFFER CHAIN WITH NEW BUFFER 416
TRANSFER DATA USING BUFFERS CURRENTLY IN BUFFER CHAIN 418
STOP 420
NO
MAKE NEXT BUFFER IN THE BUFFER CHAIN THE CURRENT BUFFER 426
LEAVE BUFFER ALONE IN BUFFER CHAIN 424
SIZE OF CURRENT BUFFER > THRESHOLD? 422
NO
CREATE A NEW BUFFER BY COPYING THE DATA FROM THE CURRENT BUFFER AND THE DATA FROM THE NEXT BUFFER IN THE BUFFER CHAIN INTO THE NEW BUFFER 428
REPLACE COMBINED BUFFERS IN BUFFER CHAIN WITH NEW BUFFER 430
MAKE THE NEW BUFFER THE CURRENT BUFFER 432
FIG. 4
| CPU | MEMORY ACCESS LATENCY | DMA CAPABILITY | AGGREGATION THRESHOLD |
|-----|-----------------------|----------------|-----------------------|
| NUMBER | SPEED | LATENCY | WIDTH | SPEED | THRESHOLD |
| 4 | 1.45 MHz | 5 MICROSECONDS | 32 BITS | 33 MHz | 2049 BYTES |
| | | | 66 MHz | 1792 BYTES |
| | | | 133 MHz | 1408 BYTES |
| | | | 64 BITS | 33 MHz | 1792 BYTES |
| | | | 66 MHz | 1408 BYTES |
| | | | 133 MHz | 1025 BYTES |
**FIG. 5**
**FIG. 6a**
**FIG. 6b**
**FIG. 6c**
METHOD, SYSTEM, AND COMPUTER PROGRAM PRODUCT FOR DYNAMICALLY SELECTING SOFTWARE BUFFERS FOR AGGREGATION ACCORDING TO CURRENT SYSTEM CHARACTERISTICS
This application is a divisional of application Ser. No. 10/798,934, filed Mar. 11, 2004, status pending.
BACKGROUND OF THE INVENTION
1. Technical Field
The present invention is directed to an improved data processing system. More specifically, the present invention is directed to a method, system, and computer program product for dynamically determining, prior to transferring data using a chain of buffers in which the data is stored, whether to aggregate buffers by combining selected buffers according to current system capabilities to create a new chain of buffers.
2. Description of Related Art
For high speed I/O adapters, data is typically transferred from the system memory to the adapter using the Direct Memory Access (DMA) transfer method because it usually minimizes CPU utilization and increases the I/O bus bandwidth, generally resulting in overall operating efficiencies. However, the overhead required to set up the DMA makes this method better suited for large contiguous data transfers. If the data is scattered across several small buffers, the efficiency of the DMA decreases due to the necessity of having to physically program the DMA engine for each individual buffer.
To illustrate this point, consider that during normal TCP/IP communications, the protocol stack delivers a packet to the networking adapter driver for transfer out to the network. To avoid a memory copy from the TCP/IP buffer to the driver buffer, the packet is constructed into more than one buffer (i.e., one for the TCP/IP header and at least one for the payload). In some cases, the packet can be further scattered across multiple buffers. As stated above, the DMA setup is CPU consuming, which can vary from system to system, depending on the number of bridges and I/O bus bandwidth. This results in diminished memory and I/O bus performance that can be exacerbated if the data is not aligned to the cache line on each of these buffers.
A solution to this problem is to reduce the number of DMA transactions by aggregating the data from these small buffers into a big contiguous system buffer through copying. However, the additional work to aggregate the data into a single buffer results in the use of extra CPU cycles. This may not be tolerable if the system does not have enough available CPU cycles.
Clearly, the method to aggregate by copying all the small buffers into one big contiguous buffer, and the method that delivers multiple small buffers packets to the adapter for DMA transfers are not always good for all systems. In the former case, the I/O and memory latencies may not be tolerable due to insufficient memory and I/O bandwidth. In the latter case, additional CPU cycles may not be available in some systems.
Therefore, a need exists for a method, system, and computer program product for dynamically selecting, prior to transferring data stored in a chain of separate software buffers, ones of these buffers in the buffer chain to aggregate by combining the selected buffers to form a new buffer chain of buffers according to current system capabilities.
SUMMARY OF THE INVENTION
A method, system, and computer program product in a data processing system are disclosed for dynamically selecting software buffers for aggregation in order to optimize system performance. Data to be transferred to a device is received. The data is stored in a chain of software buffers. Current characteristics of the system are determined. Software buffers to be combined are then dynamically selected. This selection is made according to the characteristics of the system in order to maximize performance of the system.
The above as well as additional objectives, features, and advantages of the present invention will become apparent in the following detailed written description.
BRIEF DESCRIPTION OF THE DRAWINGS
The novel features believed characteristic of the invention are set forth in the appended claims. The invention itself, however, as well as a preferred mode of use, further objectives and advantages thereof, will best be understood by reference to the following detailed description of an illustrative embodiment when read in conjunction with the accompanying drawings, wherein:
FIG. 1 is a pictorial representation which depicts a network of data processing systems that includes the present invention in accordance with the present invention;
FIG. 2 is a more detailed illustration of a computer system that includes the present invention that may be used to implement any of the computer systems of FIG. 1 in accordance with the present invention;
FIG. 3 illustrates a high level flow chart that depicts determining system characteristics and setting an aggregation threshold for each I/O adapter in accordance with the present invention;
FIG. 4 depicts optimizing I/O throughput and CPU utilization by dynamically configuring software buffers considering the performance impact of buffer aggregation in accordance with the present invention;
FIG. 5 illustrates a block diagram of system and I/O adapter characteristics and the resulting thresholds that are assigned to each combination of these system and I/O adapter characteristics in accordance with the present invention;
FIG. 6a illustrates a chain of original buffers that store data that is to be transmitted to an I/O adapter in accordance with the present invention;
FIG. 6b depicts a chain of modified buffers that store data that is to be transmitted to a first I/O adapter in accordance with the present invention; and
FIG. 6c illustrates a chain of modified buffers that store data that is to be transmitted to a second I/O adapter in accordance with the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
A preferred embodiment of the present invention and its advantages are better understood by referring to the figures, like numerals being used for like and corresponding parts of the accompanying figures.
The present invention is a method, system, and computer program product for dynamically determining according to current system capabilities, prior to transferring a chain of buffers in which data to be transferred is stored, whether to
aggregate buffers in the buffer chain by combining selected buffers to form a chain of modified buffers in which the data is stored.
When the system performance is limited by the capabilities of the I/O subsystem, i.e. I/O adapter and/or size of the slot into which the adapter is inserted, smaller buffers will be aggregated to create larger buffers that are used in order to avoid the DMA overhead on the I/O bus. When the system performance is limited by the processor capacity, aggregation is restricted so that the original, smaller buffers are used to transfer the data.
A threshold is assigned to each possible combination of I/O adapter and slot size. Thus, a threshold is determined for each speed of I/O adapter in each width of I/O slot for a given set of system characteristics. Thereafter, during processing, each time data is to be transferred to an I/O adapter, the threshold that is assigned to that speed of adapter and the slot size in which that adapter resides is used to dynamically select software buffers to be combined.
The data to be transferred to an I/O adapter will typically reside in one or more software buffers that may vary in size. The combination of the software buffers that contain the data to be transferred are referred to as a buffer chain. This is a chain of software buffers in which is stored all of the data that is to be transferred.
For each buffer in the buffer chain, if a buffer is smaller or equal to the threshold, the contents of the buffer will be combined with the contents of one or more other buffers in the chain until the threshold is reached. This is referred to as aggregating these buffers.
Buffers that are greater than the threshold are left alone in the buffer chain. In this manner, a new buffer chain is created that will include buffers from the original buffer chain that exceeded the threshold as well as the newly created aggregated buffers.
A threshold is set for each combination of I/O adapter speed and slot size in combination with particular system performance characteristics. The following factors are used to set a threshold: (1) the number of CPUs in the system and their respective speeds; (2) the access latency of the memory subsystem; and (3) the DMA capability of each I/O adapter.
Those skilled in the art will recognize that any method may be used to set a threshold using these factors. A threshold may first be assigned and then later adjusted in order to fine tune the performance of the system. Typically, the thresholds are set according to the best judgment of a design engineer.
The number of CPUs, the speed of the CPUs, and the access latency of the memory subsystem are posted by the system's firmware and operating system during system Initial Program Load (IPL). The system firmware and operating system updates the number and speed of CPUs in the system and the memory access latency of the system based on the system model. The I/O adapter DMA capability is determined when the I/O adapter is initialized.
FIG. 1 depicts a pictorial representation of a network of data processing systems in which the present invention may be implemented. Network data processing system 10 is a network of computers in which the present invention may be implemented. Network data processing system 10 contains a network 12, which is the medium used to provide communications links between various devices and computers connected together within network data processing system 10. Network 12 may include connections, such as wire, wireless communication links, or fiber optic cables.
In the depicted example, a server 14 is connected to network 12 along with storage unit 16. In addition, clients 18, 20, and 22 also are connected to network 12. Network 12 may include permanent connections, such as wire or fiber optic cables, or temporary connections made through telephone connections. The communications network 12 also can include other public and/or private wide area networks, local area networks, wireless networks, data communication networks or connections, intranets, routers, satellite links, microwave links, cellular or telephone networks, radio links, fiber optic transmission lines, ISDN lines, T1 lines, DSL, etc. In some embodiments, a user device may be connected directly to a server 14 without departing from the scope of the present invention. Moreover, as used herein, communications include those enabled by wired or wireless technology.
Clients 18, 20, and 22 may be, for example, personal computers, portable computers, mobile or fixed user stations, workstations, network terminals or servers, cellular telephones, kiosks, dumb terminals, personal digital assistants, two-way pagers, smart phones, information appliances, or network computers. For purposes of this application, a network computer is any computer, coupled to a network, which receives a program or other application from another computer coupled to the network.
In the depicted example, server 14 provides data, such as boot files, operating system images, and applications to clients 18-22. Clients 18, 20, and 22 are clients to server 14. Network data processing system 10 may include additional servers, clients, and other devices not shown. In the depicted example, network data processing system 10 is the Internet with network 12 representing a worldwide collection of networks and gateways that use the TCP/IP suite of protocols to communicate with one another. At the heart of the Internet is a backbone of high-speed data communication lines between major nodes or host computers, consisting of thousands of commercial, government, educational and other computer systems that route data and messages. Of course, network data processing system 10 also may be implemented as a number of different types of networks, such as for example, an intranet, a local area network (LAN), or a wide area network (WAN). FIG. 1 is intended as an example, and not as an architectural limitation for the present invention.
FIG. 2 is a more detailed block diagram of a central electronic complex (CEC) within a data processing system in which the present invention may be implemented. CEC 100 may include a plurality of processors 101, 102, 103, and 104 connected to system bus 106. Alternatively, a single processor system may be employed. Also connected to system bus 106 is memory controller/cache 108, which provides an interface to a plurality of system memories 160-163. I/O bus bridge 110 is connected to system bus 106 and provides an interface to I/O bus 112. Memory controller/cache 108 and I/O bus bridge 110 may be integrated as depicted.
Each system memory 160-163 may include one or more software buffers that are used to store data. For example, system memory 162 includes a buffer A 202 and a buffer B 204. According to the present invention, these buffers may be combined by copying the data from each buffer into a new, larger buffer. For example, the data from buffer A 202 may be copied into a larger buffer 206. In addition, the data from buffer B 204 may also be copied into buffer 206. Thus, buffer A 202 and buffer B 204 are aggregated by combining the contents of the original buffers into a new buffer A+B 206. Buffer 206 is the size of buffer A 202 plus the size of buffer B 204.
CEC 100 is a logically partitioned data processing system. Thus, CEC 100 may have multiple heterogeneous operating systems (or multiple instances of a single operating system) running simultaneously. Each of these multiple operating systems may have any number of software programs executing within it. CEC 100 is logically partitioned such that different I/O adapters 120-121, 128-129, 136, and 148-149 may be assigned to different logical partitions.
Thus, for example, suppose CEC 100 is divided into three logical partitions, P1, P2, and P3. Each of I/O adapters 120-121, 128-129, 136, and 148-149, each of processors 101-104, and each of system memories 160-163 is assigned to one of the three partitions. For example, processor 101, memory 160, and I/O adapters 120, 128, and 129 may be assigned to logical partition P1; processors 102-103, memory 161, and I/O adapters 121 and 136 may be assigned to partition P2; and processor 104, memories 162-163, and I/O adapters 148-149 may be assigned to logical partition P3.
Each operating system executing within CEC 100 is assigned to a different logical partition. Thus, each operating system executing within CEC 100 may access only those I/O units that are within its logical partition.
Peripheral component interconnect (PCI) Host bridge 114 connected to I/O bus 112 provides an interface to PCI local bus 115. A number of Input/Output adapters 120-121 may be connected to PCI bus 115. Typical PCI bus implementations will support between four and eight I/O adapters (i.e. expansion slots for add-in connectors). Each I/O Adapter 120-121 provides an interface between CEC 100 and input/output devices such as, for example, other network computers.
An additional PCI host bridge 122 provides an interface for an additional PCI bus 123. PCI bus 123 is connected to a plurality of PCI I/O adapters 128-129 by a PCI bus 126-127. Thus, additional I/O devices, such as, for example, modems or network adapters may be supported through each of PCI I/O adapters 128-129. In this manner, CEC 100 allows connections to multiple network computers.
A memory mapped graphics adapter 148 may be connected to I/O bus 112 through PCI Host Bridge 140 and PCI-PCI bridge 142 via PCI buses 144 and 145 as depicted.
A PCI host bridge 130 provides an interface for a PCI bus 131 to connect to I/O bus 112. PCI bus 131 connects PCI host bridge 130 to the service processor mailbox interface and ISA bus access pass-through logic 194 and PCI-PCI bridge 132. The ISA bus access pass-through logic 194 forwards PCI accesses destined to the PCI/ISA bridge 193. The NVRAM storage is connected to the ISA bus 196. The Service processor 135 is coupled to the service processor mailbox interface 194 through its local PCI bus 195. Service processor 135 is also connected to processors 101-104 via a plurality of JTAG/I²C buses 134. JTAG/I²C buses 134 are a combination of JTAG/scan busses (see IEEE 1149.1) and Phillips I²C busses. However, alternatively, JTAG/I²C buses 134 may be replaced by only Phillips I²C busses or only JTAG/scan busses. All SP-ATTN signals of the host processors 101, 102, 103, and 104 are connected together to an interrupt input signal of the service processor. The service processor 135 has its own local memory 191, and has access to the hardware op-panel 190.
When CEC 100 is initially powered up, service processor 135 uses the JTAG/scan buses 134 to interrogate the system (Host) processors 101-104, memory controller 108, and I/O bridge 110. At completion of this step, service processor 135 has an inventory and topology understanding of CEC 100. Service processor 135 also executes Built-In-Self-Tests (BISTs), Basic Assurance Tests (BATs), and memory tests on all elements found by interrogating the system processors 101-104, memory controller 108, and I/O bridge 110. Any error information for failures detected during the BISTs, BATs, and memory tests are gathered and reported by service processor 135.
Service processor 135 is responsible for saving and reporting error information related to all the monitored items in CEC 100. Service processor 135 also takes action based on the type of errors and defined thresholds. For example, service processor 135 may take note of excessive recoverable errors on a processor's cache memory and decide that this is predictive of a hard failure. Based on this determination, service processor 135 may mark that resource for reconfiguration during the current running session and future Initial Program Loads (IPLs). IPLs are also sometimes referred to as a "boot" or "bootstrap".
Those of ordinary skill in the art will appreciate that the hardware depicted in FIG. 2 may vary. For example, other peripheral devices, such as optical disk drives and the like, also may be used in addition to or in place of the hardware depicted. The depicted example is not meant to imply architectural limitations with respect to the present invention.
FIG. 3 depicts a high level flow chart which illustrates determining system characteristics and setting an aggregation threshold for each I/O adapter in accordance with the present invention. The process starts as depicted by block 300 and thereafter passes to block 302 which illustrates determining the number of processors, also called CPUs, in the data processing system. Next, block 304 depicts determining the speed of each processor (CPU). Thereafter, block 306 illustrates determining the access latency of the memory subsystem. The memory subsystem includes memory controller/cache 108 and system memory 162.
Block 308, then, depicts determining the DMA capacity for each I/O adapter. This determination is made using the slot size and the speed of each I/O adapter that may be inserted into each slot. Thereafter, block 310 illustrates determining a threshold for each I/O adapter using the number and speed of each processor, the memory access latency, and the DMA capacity of the I/O adapter considering the size of the slot into which the I/O adapter is inserted. The process then terminates as depicted by block 312.
FIG. 4 illustrates a high level flow chart which depicts optimizing I/O throughput and CPU utilization by dynamically configuring software buffers considering the performance impact of buffer aggregation in accordance with the present invention. The process starts as depicted by block 400 and thereafter passes to block 402 which illustrates a determination of whether or not data is to be transferred. If a determination is made that no data is to be transferred, the process passes back to block 402. If a determination is made that data is to be transferred, the process passes to block 404 which depicts determining the threshold for the I/O adapter that is to receive the data. Next, block 406 illustrates making the first buffer of the chain of buffers to be transferred the current buffer.
The process then passes to block 408 which depicts determining the size of the current buffer. Thereafter, block 410 illustrates a determination of whether or not this is the last buffer in the buffer chain that holds data to be transferred. If a determination is made that this is the last buffer in the buffer chain, the process passes to block 412 which depicts a determination of whether or not the size of the last buffer is greater than the threshold. If a determination is made that the size of the last buffer is greater than the threshold, the process passes to block 418.
Referring again to block 412, if a determination is made that the size of the last buffer is not greater than the threshold, the process passes to block 414 which illustrates creating a new buffer by copying the data stored in the last buffer and the data stored in the next to last buffer, i.e. the immediately previous buffer in the buffer chain, to form the new buffer. Thus, the last buffer and the next to last buffer in the buffer chain are selected and are aggregated. The process then
passes to block 416 which depicts replacing in the buffer chain the buffers selected for aggregation with the new buffer. The process then passes to block 418 which illustrates transferring the data using the buffers that are currently in the buffer chain. The process then terminates as illustrated by block 420.
Referring again to block 410, if a determination is made that this is not the last buffer in the buffer chain, the process passes to block 422 which illustrates a determination of whether the size of the current buffer is greater than the threshold that was determined for this I/O adapter. If a determination is made that the size of the current buffer is greater than the threshold determined for this I/O adapter, the process passes to block 424 which depicts leaving this current buffer alone in the buffer chain. Thus, the data stored in this buffer is not aggregated with another buffer, and this buffer remains unchanged in the buffer chain. Block 426, then, illustrates making the next buffer in the buffer chain the current buffer. The process then passes back to block 408.
Referring again to block 422, if a determination is made that the size of the current buffer is not greater than the threshold, the process passes to block 428 which depicts creating a new buffer by copying the contents of, i.e. the data stored in, the current buffer and the contents of, i.e. the data stored in, the next buffer in the buffer chain into a new, bigger buffer. Thus, these two buffers are selected for aggregation and their contents are aggregated by being copied into a new buffer.
Block 430, then, illustrates replacing, in the buffer chain, the buffers selected for aggregation with the new, larger buffer. Thus, those buffers that were combined are no longer in the buffer chain. Instead of the combined buffers, the buffer chain now contains the new buffer. Thereafter, block 432 depicts making the new buffer the current buffer. The process then passes to block 408.
FIG. 5 depicts a block diagram of system and I/O adapter characteristics and the resulting thresholds that are assigned to each combination of these system and I/O adapter characteristics in accordance with the present invention. For example, the system includes four processors, each operating at 1.45 MHz. The memory access latency for the memory subsystem is 5 microseconds.
An I/O adapter may be inserted into either a 32 bit wide slot or a 64 bit wide slot. When an I/O adapter that is capable of transferring data at 33 MHz is inserted into a 32 bit-wide slot, a threshold of 2049 bytes has been assigned. A threshold of 1792 bytes has been assigned when an I/O adapter that is capable of transferring data at 66 MHz is inserted into a 32 bit wide slot. When an I/O adapter that is capable of transferring data at 133 MHz is inserted into a 33-bit wide slot, a threshold of 1408 bytes has been assigned.
When an I/O adapter that is capable of transferring data at 33 MHz is inserted into a 64-bit wide slot, a threshold of 1792 bytes has been assigned. A threshold of 1408 bytes has been assigned when an I/O adapter that is capable of transferring data at 64 MHz is inserted into a 64-bit wide slot. When an I/O adapter that is transferring data at 133 MHz is inserted into a 64-bit wide slot, a threshold of 1025 bytes has been assigned.
Those skilled in the art will recognize that FIG. 5 depicts just one example of threshold values that may be assigned. Any other threshold value may be used for these combinations of I/O adapter and system characteristics.
FIG. 6a illustrates a chain of buffers that store data that is to be transmitted to an I/O adapter in accordance with the present invention. The entire 12 Kbytes of data that is to be transferred, according to this example, is stored in a buffer chain 600. Buffer chain 600 currently includes five buffers. Thus, the 12 Kbytes of data are distributed throughout the five buffers. A total of 4096 bytes are stored in a first buffer 602 in chain 600. A total of 1024 bytes are stored in a second buffer 604 in chain 600. A total of 2048 bytes are stored in a third buffer 606 in chain 600. A total of 3072 bytes are stored in a fourth buffer 608 in chain 600. And, a total of 2048 bytes are stored in a fifth buffer 610 in chain 600.
FIG. 6b depicts a chain of modified buffers that store data that is to be transmitted to a first I/O adapter in accordance with a first example of the present invention.
The following is an example of determining whether one or more, and which ones, of the buffers in the buffer chain 600 should be aggregated in order to optimize the performance of the system when the 12 Kbytes of data are to be transferred to a particular I/O adapter. When buffers are aggregated, the contents of the selected buffers are copied into a new buffer that is the size of that total of both of the original selected buffers.
For the first example, the 12 Kbytes of data are to be transferred to an adapter A that is capable of transferring data at 133 MHz and that is inserted in a 64 bit wide slot. The buffer aggregation threshold assigned to this I/O adapter in the system having the characteristics depicted by FIG. 5 is 1025 bytes.
The present invention may be used to dynamically determine whether it would optimize the system and I/O adapter to combine two or more of the buffers in buffer chain 600. In addition, the present invention may be used to dynamically select the buffers to combine in order to optimize the system and adapters.
Thus, following the example, the 12 Kbytes of data are to be transferred to I/O adapter A. The 12 Kbytes of data are originally stored in buffer chain 600. After completion of the present invention, a new buffer chain 612, as depicted by FIG. 6b, is created. Buffer chain 612 will then be used when transferring the 12 Kbytes of data to adapter A.
The first buffer 602 is evaluated. Buffer 602 stores 4096 bytes of data. The threshold of adapter A is 1025 bytes. Therefore, because the contents of buffer 602 are greater than the threshold for adapter A, the contents of buffer 602 will not be combined, or aggregated, with the contents of any other buffer. Buffer 602 is then left alone in the new buffer chain 612 as depicted by FIG. 6b.
Next, buffer 604 is considered. The contents of buffer 604 require 1024 bytes of storage. Because the contents of buffer 604 are less than the threshold for adapter A, the contents of buffer 604 will be combined with the contents of the next buffer in the chain, buffer 606. Therefore, the contents of buffer 604 and the contents of buffer 606 will be copied into a new buffer, buffer 614. Buffer chain 612, as depicted by FIG. 6b, now includes buffer 614 and does not include buffers 604 or 606. Buffer 614 replaces buffers 604 and 606 in the new buffer chain 612. Buffer 614 stores the contents of buffers 604 and 606, and is thus 3182 bytes.
Next, buffer 608 is considered. Buffer 608 stores 3072 bytes of data. Because buffer 608 contains 3072 bytes which is greater than the threshold for adapter A, the contents of buffer 608 are not combined with another buffer and remain in buffer 608. Thus, buffer chain 612 includes original buffer 608.
Buffer 610 is considered next. Buffer 610 contains 2048 bytes which is greater than the threshold for adapter A. Therefore, buffer 610 remains unchanged. The contents of buffer 610 are not combined with another buffer and remain in buffer 610. Thus, buffer chain 612 includes original buffer 610.
The data is then transferred to adapter A using buffer chain 612 which now includes four buffers instead of the original five buffers.
For the second example, the 12 Kbytes of data are to be transferred to an adapter B that is capable of transferring data at a rate of 33 MHz and that is inserted in a 32 bit-wide slot. The buffer aggregation threshold assigned to this I/O adapter in the system having the characteristics depicted by FIG. 5 is 2049 bytes. The 12 Kbytes of data are originally stored in buffer chain 600. After completion of the present invention, a new buffer chain 620, as depicted by FIG. 6c, is created. Buffer chain 620 will then be used when transferring the 12 Kbytes of data to adapter B.
The threshold of adapter B is 2049 bytes. Thus, the data stored in buffer 602 will not be copied, or aggregated, because the size of the data, 4096 bytes, is greater than the threshold of 2049 bytes. Therefore, buffer 602 remains unchanged. The contents of buffer 602 are not combined with another buffer and remain in buffer 602. Thus, buffer chain 620 includes original buffer 602.
Next, buffer 604 is considered. The data stored in buffer 604 requires 1024 bytes. The contents of buffer 604 should be combined with another buffer. Buffer 606 is then considered. Buffer 606 stores 2048 bytes. The combination of buffers 604 and 606 is now 3072 bytes which is greater than the threshold. Therefore, buffer 622 is created into which is copied the contents stored in original buffers 604 and 606. Buffer chain 620 now includes buffer 622 which replaces buffers 604 and 606. Buffer chain 620 does not include buffers 604 or 606.
Next, buffer 608 is considered. The data stored in buffer 608 requires 3072 bytes, and, thus, does not need to be combined. Buffer 610 is then considered. The contents of buffer 610 require only 2048 bytes. Because this is less than the threshold for adapter B and because buffer 610 is the last buffer in chain 600, buffer 610 is combined with the previous buffer, buffer 608, to create a new buffer, buffer 624, which stores the 5120 bytes of original buffers 608 and 610.
The data is then transferred to adapter B using buffer chain 620 which now includes three buffers instead of the original five buffers.
It is important to note that while the present invention has been described in the context of a fully functioning data processing system. Those of ordinary skill in the art will appreciate that the processes of the present invention are capable of being distributed in the form of a computer readable medium of instructions and a variety of forms and that the present invention applies equally regardless of the particular type of signal bearing media actually used to carry out the distribution. Examples of computer readable media include recordable-type media, such as a floppy disk, a hard disk drive, a RAM, CD-ROMs, DVD-ROMs, and transmission-type media, such as digital and analog communications links, wired or wireless communications links using transmission forms, such as, for example, radio frequency and light wave transmissions. The computer readable media may take the form of coded formats that are decoded for actual use in a particular data processing system.
The description of the present invention has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiment was chosen and described in order to best explain the principles of the invention, the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated.
The invention claimed is:
1. A method in a data processing system for dynamically selecting software buffers for aggregation in order to optimize system performance, wherein said data processing system comprises a processor and an I/O subsystem and wherein said I/O subsystem comprises I/O adapters and I/O slots, said method comprising:
- assigning a threshold for each possible combination of I/O adapter speed and width of I/O slot;
- receiving data, which is stored in a chain of software buffers, to be transferred to a particular I/O adapter that resides in a particular I/O slot;
- determining a speed of said particular I/O adapter and a width of said particular I/O slot;
- determining a particular threshold assigned for a combination of said speed of said particular I/O adapter and said width of said particular I/O slot;
- determining current characteristics of said system; and
- using said particular threshold to dynamically select software buffers of said chain of software buffers to aggregate to maximize performance of said system while said data is being transferred.
2. The method according to claim 1, further comprising:
- determining whether said system is more limited by its I/O subsystem capacity or its processor capacity;
- in response to a determination that said system is more limited by its I/O subsystem capacity, increasing a level of aggregation of software buffers; and
- in response to a determination that said system is more limited by its processor capacity, restricting said level of aggregation of software buffers.
3. A data processing system for dynamically selecting software buffers for aggregation in order to optimize system performance, said system comprising:
- a processor;
- an I/O subsystem comprising I/O adapters and I/O slots;
- a threshold assigned for each possible combination of I/O adapter speed and width of I/O slot;
- a device for receiving data to be transferred to a particular I/O adapter that resides in a particular I/O slot, said data being stored in a chain of software buffers;
- a CPU executing code for determining a speed of said particular I/O adapter and a width of said particular I/O slot;
- said CPU executing code for determining a particular threshold assigned for a combination of said speed of said particular I/O adapter and said width of said particular I/O slot;
- said CPU executing code for determining current characteristics of said system; and
- said CPU executing code for using said particular threshold to dynamically select software buffers of said chain of software buffers to aggregate to maximize performance of said system while said data is being transferred.
4. The system according to claim 3, further comprising:
- said CPU executing code for determining whether said system is more limited by its I/O subsystem capacity or its processor capacity;
- in response to a determination that said system is more limited by its I/O subsystem capacity, increasing a level of aggregation of software buffers; and
- in response to a determination that said system is more limited by its processor capacity, restricting said level of aggregation of software buffers.
5. A computer program product, which is stored in a computer recordable-type medium, for dynamically selecting software buffers for aggregation in a data processing system in order to optimize system performance, wherein said data processing system comprises a processor and an I/O subsystem and wherein said I/O subsystem comprises I/O adapters and I/O slots, said product comprising:
instruction means for assigning a threshold for each possible combination of I/O adapter speed and width of I/O slot;
instruction means for receiving data to be transferred to a particular I/O adapter that resides in a particular I/O slot, said data being stored in a chain of software buffers;
instruction means for determining a speed of said particular I/O adapter and a width of said particular I/O slot;
instruction means for determining a particular threshold assigned for a combination of said speed of said particular I/O adapter and said width of said particular I/O slot;
instruction means for determining current characteristics of said system; and
instruction means for using said particular threshold to dynamically select software buffers of said chain of software buffers to aggregate to maximize performance of said system while said data is being transferred.
6. The product according to claim 5, further comprising:
instruction means for determining whether said system is more limited by its I/O subsystem capacity or its processor capacity;
in response to a determination that said system is more limited by its I/O subsystem capacity, instruction means for increasing a level of aggregation of software buffers; and
in response to a determination that said system is more limited by its processor capacity, instruction means for restricting said level of aggregation of software buffers.
7. The method according to claim 1, further comprising:
assigning the threshold for each possible combination of I/O adapter speed and width of I/O slot using the number of CPUs in the system and their speeds, an access latency of a memory subsystem, and a DMA capability of each I/O adapter.
8. The method according to claim 1, further comprising:
evaluating a first buffer in said chain of software buffers; if a size of said first buffer does not exceed said particular threshold, copying contents of said first buffer and contents of a second buffer in said software chain into a first new buffer;
evaluating said first new buffer;
if a size of said first new buffer does not exceed said particular threshold, copying contents of said first new buffer and contents of a third buffer in said software chain into a second new buffer.
9. The method according to claim 8, further comprising:
if a size of said first buffer does exceed said particular threshold, leaving said first buffer unchanged and evaluating said second buffer;
if a size of said second buffer does not exceed said particular threshold, copying contents of said second buffer and contents of said third buffer in said software chain into a third new buffer;
evaluating said third new buffer;
if a size of said third new buffer does not exceed said particular threshold, copying contents of said third new buffer and contents of a fourth buffer in said chain into a fourth new buffer.
10. The system according to claim 3, further comprising: said CPU executing code for assigning the threshold for each possible combination of I/O adapter speed and width of I/O slot using the number of CPUs in the system and their speeds, an access latency of a memory subsystem, and a DMA capability of each I/O adapter.
11. The system according to claim 3, further comprising: said CPU executing code for evaluating a first buffer in said chain of software buffers;
said CPU executing code for if a size of said first buffer does not exceed said particular threshold, copying contents of said first buffer and contents of a second buffer in said software chain into a first new buffer;
said CPU executing code for evaluating said first new buffer;
said CPU executing code for if a size of said first new buffer does not exceed said particular threshold, copying contents of said first new buffer and contents of a third buffer in said software chain into a second new buffer.
12. The system according to claim 11, further comprising: said CPU executing code for if a size of said first buffer does exceed said particular threshold, leaving said first buffer unchanged and evaluating said second buffer;
said CPU executing code for if a size of said second buffer does not exceed said particular threshold, copying contents of said second buffer and contents of said third buffer in said software chain into a third new buffer;
said CPU executing code for evaluating said third new buffer;
said CPU executing code for if a size of said third new buffer does not exceed said particular threshold, copying contents of said third new buffer and contents of a fourth buffer in said chain into a fourth new buffer.
13. The product according to claim 5, further comprising: instruction means for assigning the threshold for each possible combination of I/O adapter speed and width of I/O slot using the number of CPUs in the system and their speeds, an access latency of a memory subsystem, and a DMA capability of each I/O adapter.
14. The product according to claim 5, further comprising: instruction means for evaluating a first buffer in said chain of software buffers;
instruction means for if a size of said first buffer does not exceed said particular threshold, copying contents of said first buffer and contents of a second buffer in said software chain into a first new buffer;
instruction means for evaluating said first new buffer;
instruction means for if a size of said first new buffer does not exceed said particular threshold, copying contents of said first new buffer and contents of a third buffer in said software chain into a second new buffer.
15. The product according to claim 14, further comprising: instruction means for if a size of said first buffer does exceed said particular threshold, leaving said first buffer unchanged and evaluating said second buffer;
instruction means for if a size of said second buffer does not exceed said particular threshold, copying contents of said second buffer and contents of said third buffer in said software chain into a third new buffer;
instruction means for evaluating said third new buffer;
instruction means for if a size of said third new buffer does not exceed said particular threshold, copying contents of said third new buffer and contents of a fourth buffer in said chain into a fourth new buffer.
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A genome wide transcriptional model of the complex response to pre-TCR signalling during thymocyte differentiation
Hemant Sahni¹, Susan Ross¹, Alessandro Barbarulo¹, Anisha Solanki¹, Ching-In Lau¹, Anna Furmanski¹, José Ignacio Saldaña¹, Masahiro Ono¹, Mike Hubank¹, Martino Barencro¹,* and Tessa Crompton¹,*
¹ Institute of Child Health, University College London, London WC1N 1EH, UK
* These authors are co-senior authors and contributed equally to this work
Correspondence to: Tessa Crompton, email: firstname.lastname@example.org
Keywords: pre-TCR, thymus, foetal thymic organ cultures, DP, genome wide transcriptional modelling, Immunology Section, Immune response, Immunity
Received: July 07, 2015 Accepted: September 08, 2015 Published: September 22, 2015
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
ABSTRACT
Developing thymocytes require pre-TCR signalling to differentiate from CD4-CD8- double negative to CD4+CD8+ double positive cell. Here we followed the transcriptional response to pre-TCR signalling in a synchronised population of differentiating double negative thymocytes. This time series analysis revealed a complex transcriptional response, in which thousands of genes were up and down-regulated before changes in cell surface phenotype were detected. Genome-wide measurement of RNA degradation of individual genes showed great heterogeneity in the rate of degradation between different genes. We therefore used time course expression and degradation data and a genome wide transcriptional modelling (GWTM) strategy to model the transcriptional response of genes up-regulated on pre-TCR signal transduction. This analysis revealed five major temporally distinct transcriptional activities that up regulate transcription through time, whereas down-regulation of expression occurred in three waves. Our model thus placed known regulators in a temporal perspective, and in addition identified novel candidate regulators of thymocyte differentiation.
INTRODUCTION
In this study we investigate the whole genome changes in gene transcription in response to a developmental stimulus through time, in a synchronized population of differentiating cells, in their physiological environment. Many studies have used analysis of the steady-state transcriptome to investigate the changes in gene expression that take place during development and differentiation. In general such work has been limited to taking ‘snap-shots’ of changes in transcription at a particular time point in development, or has examined gene expression in populations of cells developing along a particular cell-lineage by comparing expression in sequential populations defined by the cell-surface expression of developmentally regulated markers. In this study, we followed the transcriptomes of developing thymocytes in response to pre-TCR signal transduction, under conditions where differentiation is synchronous. To capture the changes in transcription in the physiological microenvironment of the thymus, we used foetal thymic organ cultures (FTOCs) and measured the heterogeneity in gene expression and degradation using microarrays.
T lymphocytes develop in the thymus, which provides an essential environment for T cell fate specification, and for the differentiation of multipotent progenitor cells into functional T cells. This cellular developmental programme has been well defined in terms of stepwise changes in cell surface markers and rearrangement of TCR gene loci, as the developing thymocytes move through different thymic microenvironments. Briefly, the most immature progenitors lack expression of both the CD4 or CD8 co-receptors, and are termed DN (Double Negative) thymocytes. The DN cells differentiate to become CD4+CD8+ double positive (DP) cells, which give rise
to single positive (SP) CD4 or CD8 $\alpha\beta$ T cells [1, 2]. The DN population can be subdivided into four major subsets by the ordered expression of CD44 and CD25: CD44+CD25- (DN1); CD44+CD25+ (DN2); CD44-CD25+ (DN3); CD44-CD25-(DN4) [3]. Notch signalling is essential for this developmental programme. Notch proteins promote T cell fate in BM precursors entering the thymus, and in mice deficient in Notch1 T-cell lineage specification/commitment fails in the thymus, while forced expression of Notch1 leads to generation of T cells in the bone marrow [2, 4, 5]. Notch signals maintain survival of early DN cells until the DN3 stage, but following pre-TCR signalling, they are abruptly down-regulated as Id3 protein rapidly rises and suppresses E2A-mediated Notch [6].
The TCR-$\beta$ gene locus is in the germ line configuration until the DN2 stage, when its rearrangement begins, requiring the Recombinase activating genes (Rag)1 and Rag2. The expression of a rearranged TCR-$\beta$ chain together with pre-Tg and CD3 molecules to form a pre-TCR complex represents a critical check point in T cell development [7]. Signals from the pre-TCR complex are essential to initiate the developmental transition, and thus to cease further recombination of the TCR-$\beta$ loci, for differentiation from DN3 to subsequent stages of development, for expansion, and to rescue developing T cells from apoptosis. Despite identification of several molecules that regulate this process [2, 6, 8-12], an understanding of the temporal regulation of the genome-wide molecular events and the molecular regulators underpinning this transition remains restricted, and new experimental approaches that allow the identification of potential novel developmental regulators are required.
Here, we used time series analysis of the synthesis and degradation data with a genome-wide transcriptional modelling strategy, which has previously been validated in cell lines [13]. This approach dissected the transcriptional response to the pre-TCR signal into five principal activities, whereas down-regulation of gene expression occurred in three main waves. The analysis temporally integrated all molecular mediators downstream of the pre-TCR, including those already known by genetic approaches.
T acute lymphoblastic leukaemia (T-ALL) arises from the malignant transformation of thymocytes at early stages of their development, and frequently involves dysregulated Notch1 signalling and dysregulation of the developmental processes associated with pre-TCR signalling [14-20]. Our study is thus important to our understanding of T-ALL, in addition to the information it provides about the molecular regulators of physiological T cell development, and it places genes previously identified to be functionally important in T-ALL in the temporal context of pre-TCR signal transduction.
**RESULTS**
**Pre-TCR signal transduction in Rag1/- FTOC**
The pre-TCR complex can oligomerize and signal in the absence of a ligand [21], but its signal transduction is dependent on the CD3-$\varepsilon$ and CD3-$\gamma$ chains [22]. Rag-deficient thymocytes arrested at DN3 can be induced to differentiate to DP cell by anti-CD3 treatment, and this has been used extensively experimentally to investigate pre-TCR function and study the transition of developing cells in a synchronous manner [23]. We treated E17.5 Rag1/- FTOCs with anti-CD3 antibody and followed their differentiation for 2 days (Figure 1A). RNA was isolated every 3.5 hours for 21 hours from sorted Thy1.2+ cells and microarrays performed to obtain whole genome transcriptomic data. At 21 hours post treatment ~18% of cells were DN4, and none had progressed to the DP stage, while ~38% of cells were DP after 2 days. Although such cultures have been widely used to study this developmental transition, we first validated our experimental system, and tested if the changes in transcription measured by microarray through time, mirrored those observed in phenotypically defined differentiated thymocyte populations (DN3, DN4, DP). To do this we used Canonical Correspondence Analysis (CCA) [24-26] to compare our microarray data to external publically available microarray data sets from sorted DN3, DN4 and DP thymocyte populations (Immgen database [27]). CCA is a mathematical methodology that has been developed by ecologists and sociologists for multivariate analysis, and it allows comparison of microarray data from a given experiment to externally generated microarray data sets. We generated a scale of DN3$\rightarrow$DN4 (Figure 1B) and DN3$\rightarrow$DP (Figure 1C) using the Immgen microarray data, and used this as a gradient for CCA of our own microarray data sets through time. The transcriptome of the anti-CD3-treated thymocytes increased in similarity to the defined DN4 and DP transcriptomes through time. During the first 10 hours after treatment, the transcriptional signature of the thymocytes changed rapidly from that of DN3 cells towards that of DN4 cells, and plateaued in the transcriptional signature of ‘DN4-ness’ at around 12 hours (Figure 1B). In contrast, on the scale of DN3 to DP, the gene expression scored closer to DN3 for the first 10 hours, and then increased steeply in the transcriptional signature of ‘DP-ness’ until the end of the experiment (Figure 1C). This analysis thus demonstrated that anti-CD3 stimulation of Rag-/- thymocytes reproduces the physiological transcriptional response to pre-TCR signalling, as measured in sorted populations of DN4 and DP thymocytes. Interestingly, the CCA showed that the changes in transcription induced by the stimulus occurred rapidly and were detectable long before the cell surface
phenotypes of DN4 or DP populations were apparent.
**Pre-TCR signal transduction initiates a complex molecular process**
In order to understand the magnitude of changes downstream of the pre-TCR signal, we measured the number of transcripts induced or repressed following the anti-CD3 stimulus (Figure 2A-2B). Compared to control (t=0), 925 transcripts had a fold change of greater than two at the end of the time course. Assessment of the differentially regulated transcript number over time revealed a complex picture in which the up-regulated genes follow a biphasic time course, and an early response is followed by a second flux in the later time points (Figure 2A-2B). Within the first 7 hours following stimulation, there was a two-fold up-regulation of *Ild3*, known to interact with the E2A transcription factor and decrease its occupancy from its target enhancer elements (Figure 2C-2D) [28, 29]. Other transcripts up-regulated at 7 hours included those for the immune-modulatory cell surface protein CD5, the transcription factor Nab2 and the tyrosine kinase Zap-70, while transcript levels for CD25 protein were found to be down-regulated; all previously reported to be downstream of pre-TCR signal transduction [30]. Analysis of *Cd4* and *Cd8* expression, the defining markers of the DP population, revealed up-regulation
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**Figure 1: Time course analysis of transcriptional changes in Rag1-/- FTOC on anti-CD3 treatment.**
**A.** Differentiation of thymocytes from E17.5 Rag1-/- embryos in FTOCs after anti-CD3ε stimulation analysed by flow cytometry through time, at time points stated. Contour plots show staining for CD44 and CD25 on cells gated to be CD4 CD8 (upper panel) and staining for CD4 and CD8 (lower panel). Th1.2+ cells were purified from these cultures at the time points shown, RNA extracted, and gene expression measured by microarray. **B.** and **C.** Canonical Correspondence Analysis (CCA) of the transcriptome of anti-CD3 treated Rag1-/- thymocytes through time (in hours): **B.** plotted against a scale of DN3 to DN4, generated from transcriptome data from sorted DN3 and DN4 thymocytes from the Immgen database; **C.** plotted on a scale of DN3 to DP generated from transcriptome data from sorted DN3 and DP thymocytes from the Immgen database. Dotted lines join the values at each time point, and solid lines show the best fitting curve.
of *Cd8β* and *Cd8α* from 17 hours onwards, while *Cd4* expression remained very low (not shown) throughout the time-course. The expression pattern of *CD8α* and *Cd8β* is distinct from other known up-regulated genes such as *Zap70* or *Id3*, which were up-regulated earlier (Figure 2D).
**Expression analysis of down-regulated genes**
There were 1311 genes down-regulated by at least 1.5 fold on one or more time points, and we clustered these genes according to their expression patterns by Pearson correlation (at threshold=0.8). This revealed down-regulation in three major waves (Figure 3A-
**Figure 2:** **The pre-TCR initiates a complex molecular response.**
**A.** Number of genes with fold change >2 with respect to time zero, plotted through the time course of the experiment, showing up- and down-regulated genes. The number of up-regulated genes are shown in red, and are shown graphically in the positive half of the x-axis, whereas the number of down-regulated genes are shown in blue, and are illustrated graphically in the negative half of the x-axis. Dotted lines join the values at each time point, and solid lines show the best fitting curve.
**B.** Number of genes with fold change >2 at transitions between successive measurements. The number of up-regulated genes between successive measurements are shown in red, and are shown graphically in the positive half of the x-axis, whereas the number of down-regulated genes between successive measurements are shown in blue, and are illustrated graphically in the negative half of the x-axis. Dotted lines join the values at each time point, and solid lines show the best fitting curve.
**C.** Genes with fold change >2 with respect to time zero at seven hours after anti-CD3 stimulation. Selected genes have been marked on the plot. Up-regulated genes are shown in red, and down-regulated genes are shown in blue. Light red and light blue dots represent those not annotated to any gene on the current Affymetrix 1.0 ST definitions.
**D.** The expression pattern of selected transcripts through time in the experiment. Up-regulated genes are shown in red, and down-regulated genes are shown in blue.
The first wave of down regulation included some genes which have been previously well defined at this transition and are known to be repressed by pre-TCR signalling, and have E2A binding-sites, such as *Notch1*, *Notch3* and their canonical target *Hes1* [6, 31]. *Runx1*, and the Ets family member *SpiB*, whose forced expression have been shown to block pre-TCR induced differentiation, were also grouped in this expression cluster [32, 33] (Figure 3B). Thus, in the first wave of down-regulation, there is rapid reduction in expression of regulatory genes (>50 transcription factors), including genes that must be down-regulated to allow pre-TCR induced differentiation, and several genes associated with leukaemia/lymphoma. Down-regulation of *Notch1*, *Notch3* and the canonical Notch target *Hes1*, underlines the importance of suppression of Notch signalling at this developmental transition (Figure 3C). The second wave of downregulation included the Wnt-responsive transcription factor Hnf1 (Tcf1), with a well characterised role in T cell development and T-ALL [34, 35], and Smad7, also involved in T-ALL [36], whereas the third wave contained genes that have been less extensively studied in thymocytes.
**Measurement of degradation of transcripts following pre-TCR signal transduction**
In order to understand correctly the kinetics of transcription of up-regulated genes, it is necessary to take into account both degradation and accumulation of transcripts [37, 13]. We decided to use a previously validated Genome Wide Transcriptional Modelling (GWTM) strategy to model the kinetics of transcription of up-regulated genes. GWTM has been previously validated experimentally in the transcriptional response of a cell line to irradiation, but not to our knowledge used previously.
**Figure 3: Down regulation of expression of genes following pre-TCR signal transduction.**
A. Expression based analysis of down-regulated genes. Clustering was performed on expression profiles of 1311 down-regulated genes, which fell into three groups. The number of genes in each group is given in white, within the dark circles, and the number of transcription factors in each group is given in black, below the circles. B. The normalized mean expression profiles for each of the three clusters are shown as graphs, with normalised expression plotted against time. C. Transcription factors that fell within the down-regulated clusters are listed.
**Figure 4:** Measurement of transcript degradation and influence of degradation on interpretation of expression data.
A.-C. Genome-wide RNA degradation was measured by microarray at stated time points after addition of Actinomycin D to Rag1-/-FTOC after 10 hours of anti-CD3 stimulation. Time zero represents the time of addition of Actinomycin D, and time is shown on the x-axis in all plots. **A.** The decay pattern of transcripts U15b and H3f2 through time in the degradation dataset. **B.** The number of transcripts with fold change >1.5 with respect to time zero in the degradation dataset, shown in the negative part of the x-axis to graphically illustrate down-regulation. **C.** The number of probes with fold change >1.5 at transitions between successive measurements in the degradation dataset, shown in the negative part of the x-axis to graphically illustrate down-regulation. **D.-G.** The hypothetical effects of transcript degradation on modelling transcriptional activities, illustrated graphically. **D.** The activity profile, $F$, of a hypothetical transcription factor with **E.** the corresponding normalized responses of three hypothetical target gene transcripts (green, brown and red) with increasing degradation rates. At a higher degradation rate (0.95 for red), the initial response and the subsequent decay are quick such that the transcript expression profile closely resembles the profile of the activity it is driven under. In contrast, transcripts with smaller rates of degradation (0.02 for green, 0.15 for brown) show expression profiles that are delayed. **F.** Three different hypothetical activity profiles, $F1$, $F2$ and $F3$ of different transcription factors with **G.** the expression profiles of their hypothetical corresponding targets, in red, brown and green respectively. The variations in degradation rates of these transcripts (0.95 for red, 0.15 for brown, and 0.02 for green) influence the shapes of their net expression profiles such that all the shapes are highly correlated with each. This can be misleading in cases of clustering solely on expression, and can lead to the false assumption that the transcripts are under one common transcriptional activity.
to investigate a physiological developmental process. For this analysis, we selected genes whose expression was up-regulated by >50% on at least one time point, and was not significantly down-regulated during the time course: 3375 genes met these criteria.
We first measured genome-wide degradation profiles, by treatment of anti-CD3 stimulated Rag1-/-FTOCs with Actinomycin D to arrest transcription at 10 hours after anti-CD3 treatment. Global gene expression was measured by microarray on addition of Actinomycin D (t=0), through time. Degradation profiles varied greatly among the individual up-regulated transcripts, with
**Figure 5:** Visualization of the effects of rate of RNA degradation on modelling transcriptional activites, giving examples of specific genes. **A.** Known targets of transcription factor E2F4 activity, *Dot1l* and *Cl15k*, display expression profiles (i) which have low correlation. The consideration of the expression data (ii, iii) with the degradation data (iv,v) of both genes simultaneously by GWTM shows a relatively low RSS of 125. This suggests that it is likely that both genes are driven by the same transcriptional activity, since *Cl15k* shows a much higher degradation rate than *Dot1l*, leading to the differences in their expression profiles. **B.** Two transcripts *Ptpn7* and *Tango9* showed highly correlated expression profiles (i). However, on consideration of their expression (ii, iii) and degradation (iv, v) profiles simultaneously by GWTM, it is clear that both transcripts are unlikely to be driven under the same transcriptional activity as the pair has a high RSS of 470. Thus, the high rate of degradation of *Ptpn7* compared to *Tango9*, makes it unlikely that both genes will fall under the same transcription regulatory activity, despite displaying similar expression profiles. Dashed lines represent the corresponding values obtained by GWTM using optimized parameters for expression and degradation.
some showing no decay until two hours after blocking, and others degrading more than 50% within the first hour (Figure 4A-4C). For example, *H3f2* degraded by more than half in the first hour, whereas *U15b* showed no decay in the first two hours (Figure 4A). Fold change analysis of the decay of 3375 transcripts up-regulated during the expression time-course revealed that 1153 (or 34%) of these rapidly showed 1.5 fold decay in the first hour on the addition of Actinomycin D and 572 of these showed a further decrease by another 1.5 folds in the next hour (Figure 4B-4C). By 5.5 hours, 3275 (or 97%) of the 3375 up-regulated transcripts decayed by 1.5 fold. We incorporated these differences in the rate of degradation of transcripts in the GWTM model to dissect the transcriptional activities downstream of the pre-TCR.
**Genome wide transcriptional modelling**
Exploration of the effects of degradation on network modelling, using the pre-validated Genome Wide Transcriptional Modelling (GWTM) approach, revealed that the targets of the same transcriptional activity can generate very different expression profiles (Figure 4D-4E). Alternatively, significantly different transcriptional activities may lead to an indistinguishable expression pattern of the targets (Figure 4F-4G), misleading us to believe that they are controlled by the same transcriptional activity. For example, the E2F4 targets *Dot1l* and *Cl15k* [38] showed expression profiles with low correlation, but GWTM modelling correctly accommodates the effect of the rapid degradation rate of *Cl15k* to group both genes together under the same transcriptional activity (Figure 5A).
**Figure 6:** Genome wide transcriptional modelling reveals five principal groups of genes that are up regulated by pre-TCR signal transduction. **A.** There are five principal transcriptional activities downstream of the pre-TCR signal. RSS were calculated, followed by hierarchical clustering by Ward’s minimum variance method using square root RSS as the distance measure. The resulting cluster dendrogram is composed of five principal transcriptional clusters (a-e) obtained under two major transcriptional responses. **B.** Compound production values, G, were calculated for all genes. The normalized mean G profiles for each of the five clusters are shown to visualise the transcriptional activity profile. Dotted lines join the calculated points and coloured lines show the best fitting curve.
model fitting is shown in Figure 5A. In contrast, despite being very well correlated in expression profile, *Ptpn7* and *Tango9*, cannot be driven by the same activity, given the differences in their degradation rates (Figure 5B).
Simultaneous consideration of the differences in the expression and degradation of the transcripts using GWTM[13] thus allowed us to dissect the transcriptional activities downstream of the pre-TCR and to cluster the up-regulated genes according to the shared transcriptional activities that control them, in order to place them in a dynamic temporal context. To separate transcriptional clusters, we determined residual sum of squares (RSS) between all possible 5693625 pairs of the 3375 genes and then applied hierarchical clustering by Ward’s minimum variance criterion (Figure 6A). We identified two major transcriptional responses, the first controlled by a transcriptional activity that peaked immediately after the pre-TCR signal and the second by a regulatory activity that built up slowly and peaked later. These transcriptional phases gave rise to five principal transcriptional activities downstream of the pre-TCR signal, based on the decay kinetics of regulatory activity. To visualise the dynamics of transcription in these principal activity clusters, we calculated for each of the five clusters, the mean production profile (G profile), which represents an affine transformation of the active transcription regulator activity controlling the genes (Figure 6B). These profiles (Figure 6B) thus represent the average pattern of transcription through time of the genes in each of the five clusters. The genes in each of these activity clusters are provided in
**Figure 7: Transcription factors affected by the principal transcriptional activities.** The figure shows that the 3375 transcripts modelled using GWTM clustered into five clusters of 561, 401, 602, 890 and 921 genes, named ‘early maintained’, ‘early short’, ‘early intermediate’, ‘late continuous’ and ‘late short’ clusters respectively. Within each of these clusters controlled by the five principal transcriptional activities were transcription factors (TFs), the numbers for which are shown in black. The transcription factors varying with a range (R) >200 units (non-log RMA) in each of the five clusters are shown.
Supplementary Tables 1-5. Consistent with the observation of a second gene flux in the second half of the time-course, we found that 85% of the transcripts newly up-regulated greater than 2-fold at 14.5 hours (Figure 1D) were under the control of the slower transcriptional activities (late continuous and late short).
**Identification of transcription factors in the five principal clusters**
We found that 356 of the up-regulated genes encoded transcription factors (>10%), which were divided as 66, 35, 69, 92 and 95 within the five activity clusters, and those with range greater than 200 units (RMA normalized non-log values) are shown in Figure 7. A large number of these transcription factors (>50) have not to our knowledge been previously associated with any specific function in lymphocytes, or T cell development. Among those previously characterized in lymphocytes, several regulators such as the *Nfats*, *Gata3* and *Id3* are already known to influence pre-TCR induced development [6, 9, 39]. For these factors, the transcriptional model is useful to reconcile the existing literature with a temporal perspective. For example, Nfat is known to be downstream of pre-TCR signalling and involved in early thymocyte development [40–42]. The model revealed that the pre-TCR signal first triggers the principal transcriptional activity controlling Nfat isoform *Nfatc1*, and maintains it throughout the transition. In contrast, the isoform *Nfatc3*, is controlled by a late activity which builds steadily over time, similar to that of *Gata3* and *Id3*. This is consistent with the recently observed effects of short-term mRNA knockdown of different Nfat isoforms on peripheral T cell activation [43], suggesting specific roles of the isoforms through differential regulatory activities. Several of the transcription factors controlled by the various activities are involved in patho-biology of leukaemia and lymphomas. For example, transcription factors Dot1l and Nrip1 which are part of the ‘early-maintained’ response have been implicated in mixed lineage leukaemia [44] and acute lymphoblastic leukaemia [45] respectively.
In order to select novel transcription factors, important for differentiation from DN3 to DP, the 356 transcription factors were intersected with the 1000 genes from the expression dataset, which contributed most to the DN3→DP scores in the CCA analysis. This intersection highlighted 19 transcription factors, distributed between the five major clusters, that are likely to be important at the transition (Figure 8). These could be divided into three functional sets: (a) those with generic roles in cellular differentiation, such as the modifiers of chromatin structure Chd3 and Satb1 [46, 47]; Polr1a [48], the catalytic unit of RNA polymerase, and Rbl, the regulator of cell cycle; (b) those known to regulate T cell development in the thymus, such as Klf13 [49], Id3 [6], Nfatc3 [39]; and (c) those with defined roles in differentiation of cells outside the thymus but still uncharacterized in thymocyte development.
**Figure 8:** Intersection of modelled transcription factors with high scoring CCA genes. The intersection of the 356 transcription factors with the top 1000 genes of the expression dataset contributing to the DN3->DP CCA score, revealed 19 important transcription factors, which are shown distributed between the five principle clusters.
DISCUSSION
This time series analysis of the transcriptional changes induced by pre-TCR signalling in a synchronised population of developing thymocytes in their thymic environment uncovered a complex transcriptional response in the first 21 hours following the stimulus, at time points when cell surface changes were not yet apparent. The analysis revealed many transcription factors that have not previously been investigated in thymocyte development to be transcriptionally regulated by pre-TCR signal transduction. Intersection of these factors with genes that contribute highly to the DN3→DP CCA scores highlighted genes that will be candidates for future functional studies. Analysis of down-regulated transcripts revealed that down regulation of transcription following pre-TCR signalling occurs in three main waves.
Our study measured genome-wide RNA degradation of individual genes and found great heterogeneity in rates of RNA degradation of individual genes. This finding underscores the importance of examining both expression and degradation profiles in investigating up-regulation of transcription in response to a stimulus. GWTM has previously been validated in experiments to test the response of a cell-line to irradiation. Here, we applied GWTM to a biological system, and refined the methodology to take into account both expression and degradation in a single step, thus improving fine-clustering (see Supplementary Information).
Our GWTM model is useful to place both known regulators of this stage of differentiation and genes that have previously been shown to be involved in T-ALL, in a temporal perspective. The model revealed five main transcriptional activities downstream of pre-TCR signalling, and in the future it will be interesting and important to identify the transcriptional regulators that control these main activities.
In addition, our time-series expression and degradation data will provide a valuable publically available resource to the research community, which complements microarray datasets from Facs sorted developmentally defined thymocyte populations.
The meaningful implementation of GWTM in response to the pre-TCR signal required us to measure the transcriptome in a synchronized population of cells. We therefore used the Rag1-/- FTOC+ anti-CD3 system, as it allowed thymocytes to remain in their normal cellular environment, but to receive the pre-TCR signal simultaneously. This culture system has been used for many years to investigate pre-TCR induced development from DN to DP thymocyte, but in order to validate the experimental system, we used CCA to compare the time series transcriptome to those of sorted DN3, DN4 and DP populations from the Immgen database. CCA showed here that the transitions over time in the experimental system mirror the molecular changes on the genomic level in the pre-characterized highly purified T-cell precursors DN3, DN4 and DP, such that the molecular profiles of the stimulated cells came to resemble more and more the profile of the more differentiated cell product population over time. Thus, the transcriptional changes that take place in Rag-deficient thymocytes when treated with anti-CD3 monoclonal antibody mirror those that are measured in sorted developmentally defined thymocyte populations from the adult, validating this experimental system.
In summary, our study highlights the benefits of time course analysis in the investigation of the regulation of differentiation and allows us to visualise the complexity of the transcriptional response to a single developmental stimulus, in a way that would not have been possible from a simple comparison of the transcriptomes of sorted thymocyte populations.
MATERIALS AND METHODS
Animals
Rag1-/- mice, purchased from The Jackson Laboratory (Maine, USA), and C57BL/6 were bred and maintained at UCL under UK Home Office regulations.
FTOC
E17.5 Foetal thymi were cultured on 0.8μm Millipore filters (Millipore, Massachusetts, US) on 1ml AIM-V serum free medium (Invitrogen, US) in 24-well plates for one day and then stimulated with 1μg/ml anti-CD3ε (BD Pharmingen, US). Actinomycin D (Sigma-Aldrich, US) at 10μg/ml was added to the culture wells to arrest transcription where stated.
Antibodies and flow cytometry
Cell suspensions were prepared and stained using combinations of directly conjugated antibodies supplied by EBioscience (San Diego, US) and analysed using Flowjo 10.6 (Tree Star, US) as described [25].
Microarray time course with anti-CD3-treated Rag1-/- thymocytes
Thymocytes were extracted by crushing thymus lobes between 2 pieces of ground glass. For expression arrays Rag1-/- FTOCs were used at the time points 0, 3.5, 7, 10.5, 14, 17.5, 21 hours following anti-CD3 stimulation; while for degradation arrays Rag1-/- FTOCs were used at 0, 1, 2, 5.5 hours following Actinomycin D addition into the 10 hour anti-CD3 stimulated cultures. Thy1.2+
cells with a purity of >90% were magnetically separated by positive selection using the EasySep system (Stem Cell Technologies, BC) with DynaMag-PCR-Magnet (Invitrogen, US) following manufacturer’s instructions. RNA for microarray analysis was extracted using Arcturus PicoPure RNA Isolation kit (Applied Biosystems, US) following manufacturer’s instructions and quantity and quality determined by Nanodrop spectrophotometer and Bioanalyser 2100 (Agilent). Microrarrays were performed by UCL Genomics on the Affymetrix Mouse Gene 1.0 ST Platform (GPL6246) using standard Ambion (Invitrogen, US) chemistry. All microarrays are publically available on ArrayExpress (E-MTAB-3088).
**Transcript level prediction in degradation arrays**
An iterative procedure was used to normalize the degradation arrays [13].
**GWTM and the simultaneous consideration of expression and degradation**
GWTM considers the rate of expression as well as degradation for identification of transcriptional clusters and in-principle could be applied to both upregulated and downregulated genes. However, in this study, only the upregulated genes have been considered for GWTM. GWTM was not applied to down-regulated genes in this study because in the case of down-regulated genes there is no way to measure and distinguish between the decrease in mRNA due to transcriptional down-regulation on stimulation of the cell from the down-regulation due to rate of degradation.
The GWTM equation can be re-arranged in a discrete vector-time representation to predict the transcript expression:
$$x_k = (A + D_k I)^{-1} (B_k + S_k f)$$
where A is a differential operator approximate. For two transcripts that are co-regulated, all above parameters are specific to the individual transcripts other than the time varying transcriptional factor activity $f$ which is shared. Both the equations predicting transcript concentrations in degradation and in expression arrays were simultaneously considered for a transcript pair and compared with the empirically observed values to calculate a residual function i.e. the squared difference between transcript concentrations predicted by the model equations and the experimentally measured concentrations at each data point divided by variance of the data value. For every transcript pair possible, the transcript specific parameters for each transcript along with the shared $f$ were optimized until the minimal value of the sum of squared residuals was achieved using Levenberg-Marquardt algorithm (LMA). These RSS were used as a measure of distance between all pairs of transcripts to which the model was fitted. This allowed us to take into account uncertainties in empirical degradation rates and allow for these in subsequent stages of the model to deliver a better grouping at sub-cluster level than a two-step approach of first calculating degradation rates empirically, and then using them to calculate compound production for clustering by shape (See Supplementary information for implementation and comparison on an experimentally validated data set).
**First derivative estimation from data**
The entries of the differential operator $A$ are obtained by rational functions of the time intervals between observation points, thereby implementing Lagrange interpolation as described[37]. In this study, we arrived at the following matrix, which is applicable to the case of seven equally spaced measurements between 0 and 21 hours.
$$A = \begin{bmatrix}
0 & 0 & 0 & 0 & 0 & 0 & 0 \\
-1/84 & 1/7 & 1/7 & -1/84 & 0 & 0 & 0 \\
1/1251 & -4/21 & 1/7 & 8/89 & -1/84 & 0 & 0 \\
0 & 1/42 & -4/21 & 0 & 4/21 & -1/42 & 0 \\
0 & 0 & 1/42 & -4/21 & 0 & 4/21 & -1/42 \\
0 & 0 & 0 & 1/21 & -2/7 & 1/7 & 2/21 \\
0 & 0 & 0 & 0 & 1/7 & -4/7 & 3/7
\end{bmatrix}$$
All matrix rows sum up to zero and the first row is filled with zeros as it is assumed that the net rate of change for every transcript is nil at time zero at which the stimulus is applied.
**Clustering and pre-clustering filtering**
To select up-regulated genes to analyse, we used transcripts with an RMA normalized expression range of 1.5 or greater, that were up-regulated in at least one time point during the expression time course and were not lower than ten percent of the time zero value at any time. RSS for every possible transcript pair was calculated and agglomerative clustering performed based on the square root of RSS as a distance measure using the hclust clustering function in R. Genes were grouped according to Ward’s minimum variance to minimize total within-cluster variance. Transcription factors were identified by intersection with the 1838 murine transcription factors in the Riken Genomics Transcription factor database (version 2013) [50].
**Canonical correspondence analysis**
Canonical correspondence analysis on microarray data (CCAM) was carried out as previously described to analyse CD4+ T cell populations in the context of helper T cell subsets and to analyse disease samples in the context of hematopoietic cell differentiation [24-26]. To represent the environmental variables of interest, the top differentially expressed genes (based on p-values)
between the respective starting and ending precursor population were used. The experimental time-course microarray samples were then linearly regressed onto the environmental variables. The CCA function of the CRAN package library ‘vegan’ [51] was used for the calculations. Briefly, the CCA first regresses the dataset for experimental samples onto differentiation variables (also called environmental variables) i.e. the gene expression gradient obtained from well characterized datasets. The differentially expressed genes used for the well characterized datasets were obtained using eBayes moderated t-statistics. CCA finds new axes by assigning numerical values to samples and genes in order to maximize the dispersion. This is algebraically equivalent to performing singular value decomposition of the matrix that is standardized in the $\chi^2$ metric. In CCA, the weighted variance (or inertia) is equivalent to the eigenvalues of PCA. Selection of genes is fixed if inertias are to be compared between different differentiation variables. However, when comparisons are made between the inertias of different differentiation variables, different number of genes are selected for different variables. Further details on CCA can be read here[52, 53]. To represent the environmental variables of interest, the top two thousand differentially expressed genes (based on p-values) between the respective starting and ending precursor populations were used. The p-values after adjusting for false positives were calculated by moderated eBayes using three different microarray samples deposited in the Immgen Database[27] (Geo: GSE15907) for each of the DN3a, DN4 and DP T-cell precursor populations with >99% purity.
**FUNDING**
This work was funded by the Wellcome Trust, MRC, BBSRC and UCL Graduate School; HS was supported by UCL-ORS/CHRAT Studentship; AB by an Instituto Pasteur/Cenci Bolognetti Foundation fellowship; MO by BBSRC David Phillips fellowship; AF by Asthma UK fellowship; MB by MRC Methodology fellowship; and AS and TC by Great Ormond Street Hospital Children’s Charity.
**CONFLICTS OF INTEREST**
The authors have no conflicts of interest.
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A Home-made Automatic Noise-Figure Measuring System
A Publication for the Radio-Amateur Especially Covering VHF, UHF and Microwaves
VHF communications
Volume No. 15 · Spring · 1/1983 · DM 6.50
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In the Focus
It is with special pleasure and some pride that I publish a Home-Made Noise-Figure Measuring System. As a UHF-minded radio amateur I struggled to obtain "the very best" noise figure for my equipment and optimum measuring techniques for over two decades now, always wishing I had an instrument like this.
Martin Dohlus (26) is the son of the company founder Hans Dohlus, DJ3QC. Martin is an engineer, doing post-graduate studies at present. He worked in our laboratory in his vacations in the last years. I was able to pass on some of my experience to him and to also discuss measuring problems with him. This eventually led to the development and publication of this noise-figure system.
In our material price list at the end of this edition, you will find an offer of a kit for the Noise-Figure Measuring System (although the concluding part of the description is still to come). Of course, the price may not be in reach of a number of amateurs but, how about considering the construction of such an instrument as a club project?
When the system is completed, and you are about to start measuring noise figures, don't forget to refer to the article "Some Pitfalls in Noise Figure Measurement" (VHF COMMUNICATIONS 1/1982), and check-off the various points in a similar way to a pilot consulting his check-list before take-off.
With kind 73s
your's
Robert E. Lentz
DL3WR
A Home-Made Automatic Noise-Figure Measuring System
This two-part article describes an automatic noise-figure measuring system that allows rapid alignment for minimum noise figure. If the noise source is calibrated, it is also possible for absolute noise-figure measurements to be made. The frequency range of the measuring system is determined by the noise source. The described measuring system possesses a 144 MHz input, but it is easily possible for it to be changed to other frequency ranges such as 29 MHz, 10.7 MHz or even AF. The noise figure is read off directly in dB on the meter, as can be seen in the photograph of the author's prototype in Figure 1.
In contrast to most noise-figure measurements such as the 3 dB method of Y-factor method, that only allow a time-consuming alignment process, the measuring system described here allows automatic measurements to be made in a rapid sequence, which means that the results of the alignment can be seen immediately. In practice, such a measuring system is very useful, especially in conjunction with passive mixers; however, also in conjunction with converters and preamplifiers using expensive input transistors, one will soon see that the costs of such devices are only worthwhile if they can be aligned to optimum on a noise-figure measuring system. In order not to bore our practical readers, we are going to keep the theory of noise-figure measurements to a minimum.
Fig. 1: Photograph of the author's prototype of the noise-figure measuring system
1. MEASURING TECHNOLOGY
The noise figure is a criterion for the sensitivity of a system. However, it does not exist in the same manner as voltage, current, or power. This means that it can only be measured indirectly. The measuring technology has not changed considerably subsequent to the IEEE-standardization in 1957.
During the measurement, the test object is excited by two known noise levels one after another, and the output power is determined. These two levels then allow the noise figure to be calculated (1).
The term "Excessive Noise Ratio" - ENR - specifies the noise source with which the system is to be driven.
\[ \text{ENR}_{\log} = 10 \lg \left( \frac{P_2}{P_1} - 1 \right) \]
\( P_1 \): Available output power from the source in state 1 (passive)
\( P_2 \): Available output power from the source in state 2 (active)
Both power levels are mean noise powers and are referred to the bandwidth \( B \).
If \( P_1 \) corresponds to the power that a resistor provides at ambient temperature (\( 1 \ kT_0 \)), the noise figure can be calculated according to the following equation:
\[ \text{NF}_{\log} = \text{ENR}_{\log} - 10 \lg \left( \frac{P_{2M}}{P_{1M}} - 1 \right) \]
\( P_{1M} \): Output power of the test object when driven with \( P_1 \)
\( P_{2M} \): Output power of the test object when driven with \( P_2 \)
The measuring technology that can be derived from this equation is used in virtually all automatic noise-figure measuring systems. The best systems achieve accuracies of \( \pm 0.2 \ \text{dB} \) using this system.
1.1. SPECIAL FEATURES OF THE DESCRIBED METHOD
The block diagram of the system is shown in Figure 2; Figure 3 shows the time-plan of the measurement. The "clock" switches the noise source at a speed of 50 ms. The source is passive during the period \( t_1 \), and is active during \( t_2 \). The output noise powers of the test object are amplified and converted to AF-level. It is now possible for the noise levels to be determined using the mean value of the noise voltages.
not have an adverse effect on the measuring results, the "mean values" are only formed during the periods $t_3$ and $t_4$. The formation of the mean value is achieved using a lowpass filter and an I-control.
The proportionality factor "const" is determined via the variable amplifier so that $U_{1M}$ has a defined value. This means that a fixed relationship exists between $U_{2M}$ and $NF_{\log}$ (equation 5) with the exception of the term $ENR_{\log}$.
This relationship is taken into consideration in the evaluation electronics so that the noise-figure can be directly indicated.
It is difficult to give the accuracy of the described system as a single number. The accuracy of the noise source has a considerable influence. In the case of the following typical values, the maximum error caused by stages subsequent to the test object is less than 0.4 dB:
$$
\begin{align*}
ENR_{\log} &= 17.0 \text{ dB} \\
NF_{\log} &= 2.0 \text{ dB} \\
V &= 20.0 \text{ dB} \\
V &= \text{Gain of the test object}
\end{align*}
$$
A detailed discussion of possible errors, and measures which can be taken to increase the measuring accuracy are to be discussed in an appendix.
2. BRIEF DESCRIPTION OF THE CONSTRUCTION
As can be seen in Figure 4, the measuring system can be split into six modules:
- Noise source
- Receive converter
- Demodulator and variable amplifier (RMG 03)
- Oscillators
- Control electronics (RMG 02)
- Read-out electronic and reference-voltage generator (RMG 01)
Figures 5 and 6 show the prototype from above and below. The individual modules are to be described individually in the following sections.
3. NOISE SOURCE
The noise source can be built up in a similar manner to that described in (2). It is only necessary for it to be extended so that the switching between $P_1$ and $P_2$ can be made electronically (Figure 7).
It is advisable for the noise source to be installed in a separate case so that it can be directly connected to the test object. This ensures that measuring errors due to attenuation, reflection, and transformation in the additional connectors and cables will not falsify the measuring results.
The knowledge of the ENR-value is very important for absolute noise-figure measurements. It can be determined by comparing it with calibrated noise sources; for instance at a university, technical college or even other amateurs with calibrated sources.
Fig. 5: Plug-in boards RMG 01 and 02 and power supply
Fig. 6: The screened modules converter, RMG 03 and 04 on the lower side of the system
The noise-source is operated with a switched voltage of at least 12 V.
4. RECEIVE CONVERTER
The input module DK 1 OF 034 described by J. Kestler in (3) is suitable for use in the noise-figure measuring system after carrying out a few modifications. Figure 8 shows the modified circuit.
4.1. BANDWIDTH
The highest possible measuring bandwidth is required in order to measure the characteristic value of the noise in a fast, and accurate manner. For this reason, measuring bandwidths of several MHz are often used for professional applications. If radio communications are made in this frequency range, usually screened cages are used. Since considerable radio communication is made on the amateur bands, and the radio amateur will hardly have a screened Faraday cage available, the author has chosen an RF-bandwidth of only 30 kHz.
The crystal filter XF-107B of the original description has been replaced by a 30 kHz crystal filter. Due to the different impedance of the filter, it is necessary to increase the values of resistors R 1, R 2 to 2 kOhm, whereas C 1 and C 2 are replaced by 30 pF trimmers.
Fig. 8: A part of the circuit DK1OF 034 is used as receive converter
4.2.
OUTPUT COUPLING
The FM-demodulator CA 3089 is not used during the noise measurements. The 10.7 MHz noise-signal is therefore coupled out after L 8 and the 47 Ohm resistor.
4.3.
CONTROL
The gain of the input module DK 1 OF 034 can be influenced greatly with the aid of the control voltage $U_C$. However, module RMG 03 is responsible for the gain control in order to allow the use of other receive converters and to avoid the higher noise-figure that would be present when the gain was controlled down. For this reason, $U_C$ is kept at a constant value via a voltage divider.
Although the noise figure of the input transistor was given to be 2 dB, we were only able to obtain a value of 4.5 dB after carefully aligning this converter.
5.
THE DEMODULATOR AND AGC AMPLIFIER
The demodulator and control module is designated RMG 03. It is designed so that it can also be used with other receive modules. The noiseband (10.685 – 10.715 MHz) is converted to an AF-band of 5-35 kHz with the aid of the described receive converter and a 10.720 MHz oscillator.
5.1.
CIRCUIT DESCRIPTION
Figure 9 shows the circuit diagram of the demodulator and AGC amplifier. The mixer IE 500 converts the noise band to AF-level. It is provided with two 50 Ohm matching filters connected in series at the IF-output which are provided to ensure that no RF-signals can be fed to the connected audio amplifier. The first stage of the AF-amplifier is also designed so that it represents a 50 Ohm termination. Since the noise voltage is in the order of several $\mu$V under unfavourable conditions, a considerable amount of filtering is required for the voltage supply and operating point adjustment. For this reason, an additional voltage of approximately 10 V is stabilized and passed through two RC-links which are connected in series.
The lower cut-off frequency of the amplifier is designed to be 1 kHz in order to ensure that no measuring errors are caused by the 1/f noise of the transistor. The FET T2 is used as a variable resistor and allows the overall gain to be varied by approximately 40 dB.
Fig. 9: Demodulator and variable amplifier RMG 03
5.2. CONSTRUCTION OF MODULE RMG 03
A PC-board of 135 mm x 50 mm was designed for accommodating this module. The component locations are given in Figure 10.
L 301: Special coil set D 41-2165; 13 turns of 0.3 mm enamelled copper wire
L 302: 120 µH choke
R 302: 220 kOhm
This resistor is reduced in value after the noise alignment of the receive converter.
The PC-board can be enclosed in a standard metal box and should be provided with the screening panels shown in the circuit diagram and in Figure 11.
Fig. 11:
The photograph shows the receive converter (above), and module RMG 03 (below)
5.3.
SWITCHING ON AND ALIGNMENT
Before commencing alignment, one requires a voltmeter (approx. 10 kOhm/V), an oscilloscope, and an AF-generator (at least a 10 kHz square-wave generator with variable voltage divider).
The input $U_C$ should be connected to a variable, negative voltage (e.g. via a 100 kOhm potentiometer between ground and $-15$ V). The internally stabilized voltage of 10.6 V should be checked after connecting the operating voltage. Resistor R 1 should be adjusted so that a voltage of $-5.8$ V is present at TP 1. Due to the large time constant of the filtering for the operating point voltage, the alignment can only be carried out slowly. For this reason, it takes several seconds after switching on for the stage to operate. The AF-generator is connected to TP 2 via a 1 MOhm resistor, and aligned to approximately 10 kHz. The voltmeter is now connected to $U_C$. A voltage of approx. $-10$ V is fed to $U_C$. The output voltage of the AF-generator is now set so that $U_{AF}$ is not distorted. The gain between TP 2 and $U_{AF}$ should be approximately $10^5$. On slowly reducing the value of $U_C$, the gain will at first remain fairly constant, after which it will drop suddenly (Fig. 12). This voltage threshold $U_{CT}$ should be noted. It is required for the alignment of the control circuit. A gain range of approximately 40 dB should be possible by varying the bias voltage.
**Fig. 12:**
Gain as a function of the control voltage
6.
OSCILLATORS
A carrier of 133.400 MHz at 0 dBm is required for driving the receive converter. This can be achieved using a local oscillator circuit such as provided on module DJ 7 VY 002 by M. Martin in (4). Very few modifications are required:
| Q : | 66.700 MHz |
|-----|------------|
| L 7: | 7 turns (otherwise as described) |
| L 9: | 5 turns (otherwise as described) |
The signal is coupled out after F 4.
The mixer of the RMG 03 is driven with a 10.720 MHz signal at a level of 7 dBm. The circuit described by F. Krug, DJ 3 RV in (5) can be used for this. Of course, one will not require the components for switching the oscillator.
Part 2 or this article is to describe the following modules:
Control module RMG 02
Indicator electronics, and reference voltage generator RMG 01
REFERENCES to part 1
1) William E. Pastori
Figuring Noise Figure
December 1979 Issue of Quality
1979 Hitchcock Publishing Company
2) Michael Ulbricht, DB 2 GM:
A Noise Generator for VHF and UHF
VHF COMMUNICATION 14,
Edition 1/1982, pages 38 - 43
3) J. Kestler, DK 1 OF
An FM Transceiver for the 2 m Band
Part 1: The Receiver
VHF COMMUNICATIONS 11,
Edition 1/1979, pages 44 - 53
4) M. Martin, DJ 7 VY:
A Modern Receive Converter for 2 m Receivers Having a Large Dynamic Range and Low Intermodulation Distortions
VHF COMMUNICATIONS 10,
Edition 4/1978, pages 218 - 229
5) F. Krug, DJ 3 RV:
A Versatile IF-module Suitable for 2 m Receivers or as an IF-Module for the SHF-Bands. Part 3: Controlled RF-amplifier, Notch filter, Demodulators, BFO, and AF-amplifier.
VHF COMMUNICATIONS 14,
Edition 3/1982, pages 172 - 189
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A digital storage and scan converter for weather satellite images
Part 2: Storage Module
Edition 4/1982 of VHF COMMUNICATIONS described the electronic module board YU3UMV 001. Part 2 is now to describe the storage board YU3UMV 002. As was mentioned in the appendix to part 1 on page 208, the original design has been equipped with the latest 64 kBit memories in order to obtain a 384 kBit storage that provides a spatial resolution of 256 lines of 256 pixels, and 64 grey levels.
Part 3 of this article will describe a colour modulator with VHF oscillator that allows the images to be displayed on a CCIR PAL colour TV-receiver.
If there is sufficient interest among our readers, the author would be willing to discuss how this video storage module can be used for SSTV.
The serial data generated by YU3UMV 001, or another source, are firstly converted to a parallel format. In order to avoid disturbing the read operations from the frame memory, data is written into the frame memory during the horizontal fly-back period. The frame memory is scanned in both write and read modes by generating the addresses using a series of binary counters. It should be mentioned immediately that it is only important to read the data out of the memory exactly in the same sequence that it has been written into it; the physical position of the data within the memory is not important from the user's point of view. It is thus important to immediately distinguish between external memory scanning (corresponding to the image scanning), and internal memory organization, which is also a bi-dimensional matrix. These two organizations have no relationship to another (at least not in theory), which means that all address lines are equivalent from the user's point of view, except for some technical constraints in the case of dynamic RAMs, which we shall discuss later.
The same circuits that generate the scanning addresses in the read mode, also generate the TV-blanking and synchronizing pulses. These are combined with the digital video information in the D/A-converter to obtain a standard, analog TV video signal.
2.1. CIRCUIT DESCRIPTION
In order to simplify the description of the circuit, the actual circuit diagram of the frame memory YU3UMV 002 was divided into four parts as shown in the block diagram given in Figure 12.
2.1.1. Write Logic
The write logic shown in Figure 13 comprises the serial-to-parallel conversion, the write-cycle synchronizing circuit, and the write pixel counter.
The serial data originating from YU3UMV 001 (or other source) are clocked into the shift register I 201. I 202 counts the number of bits clocked into I 201. After all six data bits of a pixel (grey levels) have been accepted by I 201, I 202 will generate a strobe pulse to transfer the data into the latch of I 201. A delayed strobe pulse also resets the SR-flipflop comprising two NOR gates (I/2 I 203), which in turn resets the I 202 counter and generates a request for a write cycle to the write-cycle synchronizing flipflops (I 204).
The write command is synchronized with the rise slope of the RAS clock (Row Address Strobe) at the end of a TV-line in order to perform the write cycle during the horizontal fly-back. The write command also advances the write pixel counter to generate the write horizontal addresses WHA 0 to WHA 7.
The next pixel clock pulse sets the SR flipflop enabling I 202 to count the bits once more and to repeat the write cycle. After 256 pixels have been written into the memory, the pixel counter reaches its final state, after which pin 7 (CO = Carry Out) of I 206 will go low thus inhibiting further write cycles, and will generate a scroll clock pulse, which sets a new line in the image.
The following line can only be written when the write pixel counter has been reset by the line clock pulse (compare the time plan given in Figure 9 of part 1 of this article).
The pixel counter can count up or down, according to the scanning direction of the image to be displayed. A front panel switch is provided to select either right (West-to-East) or left (East-to-West) scanning, according to whether the polar-orbiting satellite is in its ascending or descending mode: (North of South going).
Fig. 13: Write logic circuit
Fig. 14: TV-pulse and horizontal scanning generator
2.1.2. TV Clocks and Horizontal Scan Oscillator
This part of the circuit is given in Figure 14 and includes a divider and a series of mono-stables to generate the various synchronizing pulses that are all derived from the 1 MHz clock on module YU3UMV 001. In addition to this, it also includes the horizontal pixel oscillator and the read pixel counter.
The 1 MHz clock pulse is fed to I 207 which divides it by 64 to obtain a frequency of 15.625 kHz corresponding to the TV line frequency. This frequency is divided further by 32 in I 207 and by 10 in I 208 to provide a total division factor of 320 to obtain the 48.8 Hz frame (vertical) frequency. Two simple monostable circuits obtained using C-MOS gates (1/2 I 209), provide the composite TV synchronizing pulses, designated TV SYNC. The horizontal blanking pulses are obtained in one half of I 215 whereas vertical blanking pulses are obtained by delaying the pulses present at pin 2 (Q 4) of I 208 using a flipflop (1/2 I 215). The composite TV blanking pulses are then designated TV BLK.
The clock and other signals required to drive the dynamic memories are best explained together with Figure 17.
At the commencement of a TV-line, a pulse generated in one half of I 210 (pin 4) commences the horizontal pixel oscillator built up using the two mono-stable circuits of I 212. The period of the horizontal pixel oscillator can be adjusted with the aid of C 201. This determines the width of the required image. Since there are 256 pixels per line, the useful line length is in the order of 51.2 μs out of the 64 μs of a complete TV-line, including fly-back.
The most difficult task during the design of this scan converter was to retrieve the data at a sufficiently high rate from the relatively slow 64 kBit dynamic memories available. The parameter of interest is the read cycle time, which is specified as 230 ns even for the fastest 150 ns access time devices. A "page mode" reading process is the only possible choice.
The RAS clock pulse is generated in the second, retriggerable mono-stable circuit of I 210 (pin 12). Since the associated RC-constant is quite high, the mono-stable circuit is continuously in the active state when triggered by the fast pixel oscillator. In this manner, a long RAS read pulse is generated. The CAS (Column Address Strobe) clock pulse is generated by delaying (and amplifying) the pixel oscillator pulses. An SR-flipflop (1/2 I 211) generates the ASEL (Address Select) signal that controls the address multiplexer.
Each pulse of the pixel oscillator advances the pixel counter by one, generating the read horizontal addresses RHA 0 to RHA 7. As soon as the 256 pixels have been counted, a flipflop is triggered (1/2 I 215) that disconnects the feedback path of the oscillator. Any write request is synchronized with the rise slope of the RAS clock pulse. Since the feedback of the pixel oscillator is interrupted, the write command produces a single CAS pulse. The counters, flipflop, and mono-stable circuits are then reset by the next line start pulse generated in the first half of I 210 (pin 4).
During the vertical fly-back period, the page mode cycles are shortened to a few CAS cycles in order to decrease the memory power dissipation, since their only function is to enable write cycles.
2.1.3. Vertical Scan and Address Multiplexer
The vertical scan generator comprises the scroll counter and the line counter. These circuits are shown together with the address multiplexer in Figure 15.
The most obvious solution is to scan the memory in exactly the same way during the write and read operations. Unfortunately, this is quite unsatisfactory when receiving radiometer, scanned images from polar orbiting satellites, since the displayed images are usually divided into two sectors, one containing old image data, and the other new information being written into the memory. A much more elegant method is to scroll the image: New data lines are introduced to the top or bottom of the image field and the previous information is shifted down or up respectively. In this way, the display acts in a similar manner to a window moving along a radiometer-scanned image. Fortunately, scrolling is quite easy to implement in practice.
The scroll counter I 218 provides the vertical addresses during the write process. The scroll counter is incremented after writing a complete line into the frame memory. The scroll counter also supplies the start address for the line counter that generates the vertical addresses during the read process. In this way, data is always written into the first scanned line of the TV frame memory. This line acts like a buffer memory. Since the content of this line is continuously changing, it is blanked by the TV BLK signal. Whether the previous image information is shifted up or down is dependent on whether the line counter is counting up or down respectively. A front-panel switch is provided to select the scroll up for North-to-South scanned images, and scroll down for South-to-North images.
The 64 kBit memories require 16 address Bits to select the desired location in the memory. Most 64 kBit dynamic RAMs have 8 address lines. The 16 Bit address is then provided sequentially as two consecutive 8 Bit words. Since the delay between supplying the first part of the address, and the second part is quite short (down to 20 ns), a fast electronic switch (low-power Schottky TTL), designated as address multiplexer, has to be used. The address multiplexer shown in Figure 15 has the additional function of switching between read and write addresses during write cycles. The address multiplexer includes eight 4-to-1 switches controlled by the two select lines: ASEL for the selection between the first and second part of the address, and R/W for the selection between read and write addresses. The eight outputs MA 0 to MA 7 drive the memory address lines directly.
Fig. 16: Video storage and digital/analog converter with output video amplifier
2.1.4. Frame Memory and D/A Converter
The frame memory and the D/A converter are shown in Figure 16 together with the associated logic circuitry. The six 64 kBit dynamic memories are operated in parallel: Each memory stores one Bit of the 6 Bit data word which represents one individual pixel of the image. The data word at the output of the memory is valid only for a short period (see Figure 17). A data latch (I 231) is triggered by the rise slope of the CAS clock pulse in order to extend the data to the whole cycle. The TV BLK signal resets the latch and thus forces the outputs to zero (black level).
Integrated circuit type DAC 0800 is a current-multiplying D/A converter. The output current is a fraction of the current flowing to the + REF pin 14, and the fraction is determined by the digital word present at the B 1 to B 8 inputs. B 1 is the most significant Bit and is driven by the TV SYNC signal. The synchronizing pulses are somewhat greater than prescribed by the TV-standard, however, no disadvantage has been noticed in practice. The data word containing the video information is applied to the six subsequent inputs. The last input, representing the least significant Bit, is not used.
The DAC 0800 is a very fast digital/analog converter, and the fall time of the output current is in the order of 100 ns. For this application, it is much more important that it does not generate large switching transients as was the case with older integrated D/A converters, such as MC 1408. This means that a fast (and critical) sample-and-hold stage could be eliminated at the video output.
The output level of I 232 is, however, quite low. For this reason, a video amplifier μA 733 (I 233) is used to increase the signal level to approximately 1.5 V_{pp}/75 \text{ Ohm}, which is suitable for most TV-monitors.
2.1.5. Interfacing to YU3UMV 001
The frame memory module YU3UMV 002 requires exactly the same control signals as the previously designed 16 k x 6 Bit storage module, which means that no modifications are required to any circuits on module YU3UMV 001. However, there are some slight differences in the connection of the resolution-zooming selector (Figure 4 of part 1). Now, only a three-position selector is used (instead of four) since the 2.4 kHz signal is no longer required and the corresponding connection to Pt 107 is deleted.
Table 1 of part 1 is thus no longer valid and is now replaced by Table 2 below.
| Zooming resolution switch | Sampling frequency | METEOSAT WEFAX | NOAA APT 1) | METEOR 240 lines/min. | METEOR 120 lines/min. 2) |
|---------------------------|--------------------|----------------|-------------|-----------------------|--------------------------|
| Bit clock | | | | | |
| 19.2 kHz | 2400 Hz | x 2 | x 2 | x 2 | -- |
| 9.6 kHz | 1200 Hz | x 1 | x 1 | x 1 | x 2 |
| 4.8 kHz | 600 Hz | -- | VIS + IR | -- | x 2 |
| 2.4 kHz | 300 Hz | -- | -- | -- | x 1 |
1) Meteo/NOAA switch (Pt 11) in NOAA position
2) External 2400 Hz synchronisation required
Table 2: Possible image formats of the 64 kByte storage module
2.2. CONSTRUCTION
The frame memory module YU3UMV 002 is accommodated on a double-coated PC-board having the same dimensions as the control board YU3UMV 001 (190 x 85 mm). The conductor side of this PC-board is given in Figure 19 together with the components location plan. The connection pin density of the integrated circuits demands a very close geometry on the board; for this reason, extreme concentration is required when soldering the components into place in order to ensure that no unwanted solder bridges are made. Special attention must be paid to the condition of the through-contacts, which are provided on the component side below the integrated circuits. Remember, that it is not possible to check or correct these after soldering the ICs into place!
The MOS-ICs of the B-series are protected, which means that there are no advantages of using sockets. However, if sockets are preferred, it is important to only use the very best quality available. Low-cost sockets easily cause poor contacts, which are very difficult to locate in such a complex circuit.
Similar to YU3UMV 001, all connections to switches, connectors, and the electronic module are made via connection pins. The only connection that requires a certain amount of care is the video output. Although the video signal level is high (1.5 V\(_{pp}\)), it is a wideband signal having a wide dynamic range, and is thus very sensitive to interference generated by the logic circuits. Experience has shown that such interference is usually obtained by ground loops, which underlines the importance of good grounding.
2.3. SELECTION OF THE COMPONENTS
The most important components of module YU3UMV 002 are listed later. It is recommended that C-MOS ICs of the new B-series are used. All TTL circuits are low-power Schottky types, since only these are fast enough, and can be driven by C-MOS logic.
Time-determining components always cause problems due to their wide tolerances. A spread of 100% was noticed for the dual mono-stables type 74LS123 made by different manufacturers! This means that it is important for the two 74LS123 to be from the same manufacturer and, if possible, have the same date of manufacture printed on the case.
There are a number of different 64 kBit dynamic memories available on the market. The major differences between 16 pin-DIL RAMs are in the function of pin 1. This pin enables a self-refresh mechanism in some memories, whereas others do not have this pin connected. Both types can be marked 4164! The circuit of module YU3UMV 002 was designed for memories not having this self-refresh feature.
The 64 kBit memories can have different internal organizations, for example, the TMS 4164 (Texas Instruments) is organized as 256 rows x 256 columns, whereas the HM 4864 (Hitachi) has 128 rows x 512 columns. This is important in practice, since the HM 4864 requires only half as many cycles as the TMS 4164 for refresh. Finally, each manufacturer selects his memories into various speed groups after production. Memries having a row access time of 150 ns or less are required for this circuit.
The maximum time between two refresh cycles is usually specified as 2 ms for 128 row memories, and 4 ms for 256 row memories. Experiments have shown, however, that dynamic memories are able to maintain the data without refresh for many seconds at normal ambient (room) temperatures! Probably, the above mentioned limits only apply for operation at the maximum permissible ambient temperature, and maximum device dissipation power. Furthermore, in contrast to computer application, 100 percent reliability is not required here, and it is possible for a complete refresh to be performed only once every TV-frame (20 ms) in the case of 256 row memories, or twice in the case of 128 row memories.
It is also possible for the "page-mode" read cycle to be increased longer than the maximum value specified in the data sheet (usually 10 μs). The power dissipation and thus the operating temperature of the memories are very low in this circuit, since they are dependent mainly on the number of RAS cycles per second, which is very low in our application due to the page-mode addressing.
Modern dynamic memories are usually specified in the data sheets as being fully TTL compatible. Unfortunately, this does not necessarily mean that low-power TTL circuits can directly drive highly capacitive memory clock and address lines. In particular, the active pull-up of the low-power Schottky ICs may not be sufficient, and an external pull-up resistor (680 Ohm – 1.5 kOhm) may be required. The author has had no such problems in conjunction with the TMS 4164, however, he had many such problems in conjunction with earlier circuits. Both low-power Schottky TTL circuits and dynamic memories exhibit the large variations of these parameters from manufacturer to manufacturer.
| Type | Manufacturer |
|------|--------------|
| TMS 4164 | Texas Instruments |
| MCM 6665 | Motorola |
| UPD 4164 D | NEC |
| HM 4864 | Hitachi |
| MB 8264 | Fujitsu |
| IMS 2600 | INMOS |
| MK 4564 | Mostek |
| I 2164 | Intel |
| NMC 4164 | National Semiconductor |
| TMM 4164 C | Toshiba |
| MSM 3764 | Oki Semi |
| F 4164 | Fairchild |
| HYB 4164 | Siemens |
All these 16-pin DIL ICs should be usable without modifications in module YU3UMV 002. However, the author has only been able to check types TMS 4164 and HM 4864 in the actual circuit.
Fig. 19:
The video storage module is accommodated on a double-coated PCB board with through-contacts.
Fig. 20:
A photograph of the author's prototype YU3UMV 002
Connection diagrams of the less-known integrated circuits
2.4.
ALIGNMENT
Since module YU3UMV 002 obtains the power supply, clock pulses, and control signals from module YU3UMV 001, it is obvious that the latter module must have been already tested before connection, at least the power supply and the 1 MHz oscillator. There are only two trimmers on the board of the second module. It is recommended that the alignment be commenced with R 201 (horizontal position) at minimum and with C 201 (horizontal width) at a center position.
After switching on the modules, a pattern showing the random initial memory content should appear on the TV-monitor, which will probably be shifted to the left on the TV-screen. It is now possible for the two trimmers to be aligned for correct position and width of the image.
It should be noted that the adjustment of the image width has precise limits. If C 201 is completely misaligned, one may not able to obtain any image at all! After adjusting R 201 and C 201, it may be necessary for L 101 on module YU3UMV 001 to be realigned to suppress any interference and/or difficulties in the synchronization of the TV-monitor.
3.
APPENDIX
Operation of Dynamic RAM Memories
The actual storage elements in dynamic RAMs are capacitors (reverse polarized PN-junctions) that are internally organized as a two-dimensional array of rows and columns. Each storage capacitor has an associated MOS-transistor switch, which can connect it to the common column line.
Each memory access cycle (read, write, page-mode operation, or only refresh) starts with a negative transition of the \(\overline{\text{RAS}}\) clock pulse; Both \(\overline{\text{RAS}}\) and \(\overline{\text{CAS}}\) clocks are high during standby. This \(\overline{\text{RAS}}\) clock pulse strolls the first part of the address, designated Row Address, into the corresponding latch of the memory. The content of the latch is immediately decoded, and all the capacitors of the selected row are connected to the corresponding common-column lines. Each common-column line is terminated in a sensitive amplifier to detect whether the small storage capacitor was charged or not.
Since each actual capacitor is leaky, it cannot store the information for an indefinitely long period of time. For this reason, the logic automatically restores the charges on the capacitors after the column-sense amplifiers have detected the states of the corresponding capacitors: The capacitors found charged are charged to the full supply voltage, whereas the capacitors found discharged are completely discharged. This process is called "Refresh", and it must be performed sufficiently frequently to maintain the information in the memory. Since each \(\overline{\text{RAS}}\) clock cycle only refreshes a single row of the memory capacitor array, a certain number of \(\overline{\text{RAS}}\) cycles is required for the full refresh of the complete array.
\[
\begin{align*}
C & \triangleq \text{Column} \\
A & \triangleq \text{Address} \\
R & \triangleq \text{Row} \\
S & \triangleq \text{Strobe}
\end{align*}
\]
In order to communicate with the memory, the second part of the address has to be provided together with the negative transition of the \(\overline{\text{CAS}}\) clock pulse, usually 20 to 100 ns after the \(\overline{\text{RAS}}\) transition. The column address is stored in a latch, and decoded by selecting the desired column-sense amplifier/refresh logic. During a read operation, the column decoder simply connects the output of the selected column-sense amplifier to the output pin of the memory IC.
In order to write the data into the desired memory cell, the selected column-refresh logic is forced to copy the data present at the data input pin, instead of the data at the output of the column-sense amplifier. This is achieved by charging or discharging the selected cell capacitor correspondingly.
For some particular applications, like video displays, the exact order in which data will be read from, or written into the memory is known. If cells from a single row are to be accessed, it is not necessary to repeat the same row address each time. More than one \(\overline{\text{CAS}}\) cycle may be performed during a single \(\overline{\text{RAS}}\) cycle, accessing many different columns. This mode of operation is called "Page Mode". It provides a considerably higher data flow since a number of operations need not be repeated!
A Mini-SSB-Transceiver for 144 MHz
Part 1
A large number of descriptions have been published for transmit and receive converters for the higher frequency bands 70 cm, 23 cm, and higher. These are usually driven with available (commercial) 10 m or 2 m transceivers. If these transceivers are also to be used for operation on the 2 m or 10 m band, this means that they must be disconnected and reconnected, and how often it is that one wishes to have a separate transceiver for use as driver.
For this reason, the author has designed a small, inexpensive 144 MHz transceiver whose technical specifications are at least equal to, if not much better than the commercial equipment available on the market.
On the transmit side, the output power has been limited to values that are suitable for driving the transverter. This allows the power transistors to be deleted, and thus to construct a very compact unit having dimensions of only 148 mm x 74 mm x 30 mm. According to the application, it can be constructed for a frequency range of 144 to 146 MHz, or 135 to 137 MHz.
1. CONCEPTION AND CONSTRUCTION
Single-conversion with an intermediate frequency of 9 MHz is used for both transmit and receive.
Fig. 1: All components shown outside of the dashed case belong to module DC6HL 011
Expensive and larger components such as crystal filters are used both in the transmit and receive path (see Figure 1).
All components are accommodated on a small, double-coated PC-board with through-contacts, and the 9 MHz crystal filter is used to interconnect the AF/IF-board to the RF-board.
In order to achieve ideal electrical conditions, the crystal filter is mounted outside of the metal case, and only its connections protrude into both chambers, in which the PC-boards are accommodated. This allows the high selectivity of the filter to be utilized to the full (see Figure 2).
The operating voltage of the miniature transceiver is between 12 V and 16 V. A voltage stabilizer circuit is provided on the RF-board to ensure that the internal operating voltage is maintained at 11.5 V, which can also be used for external accessories.
The transmit/receive switching is made via a PTT-contact which is grounded. The stabilized voltages + $U_{TX}$ and + $U_{RX}$ are also available for external applications.
**CIRCUIT DESCRIPTION**
**2.1. AF/IF Board DC 6 HL 011**
This part of the circuit is shown in Figure 3. All component numbers are higher than 50.
**2.1.1. Transmit Path**
The AF-signal is fed from the microphone via Pt51 to the input of the integrated mixer I 51, which is built up symmetrically. The drive is made, however, in an unbalanced manner, in order to save using an AF-transformer. For this reason, it is important that inputs 7 and 8 are connected symmetrically, in other words short-circuited for the 9 MHz local oscillator frequency. The RF-signal is injected in a balanced manner (non-grounded). A DSB-signal is available at the push-pull output circuit comprising L 52, whose carrier suppression is excellent, providing that R 51 has been aligned for correct balance. The signal is passed via a buffer (T 51) to the monolithic crystal filter 9M22D1. The termination of the filter is made using the 680 $\Omega$ load resistor of the buffer together with the parallel capacitor connected to Pt54.
Fig. 3: Circuit diagram of the AF/IF board of the transceiver.
Fig. 4: Circuit diagram of the VHF-module DC6HL 010
2.1.2. Receive Path
After passing through the crystal filter, the IF-signal is passed through the two AGC amplifier stages comprising T 52 and T 53, and is fed via the resonant circuit comprising L 54 to the integrated mixer I 52. The mixer is also provided with the 9 MHz local oscillator frequency via L 55. The demodulated AF-signal is available at the load resistor of 2.2 kΩ, and is subsequently amplified by 11 times in a part of the quadruple operational amplifier I 54. The signal path is separated at the output of this amplifier.
The required oscillators are actuated by shorting or opening a ground contact, and their output signals are combined at a further gate.
The squarewave signal of 11 V (peak-to-peak) drives transformers L 51 and L 55 via a voltage divider. In order to generate the operating voltage for the carrier oscillator, diodes D 51 and D 52 combine the transmit and receive voltage so that an additional, continuously available voltage is not required.
2.2. VHF-BOARD DC 6 HL 010
The circuit diagram of this module is shown in Figure 4; all component numbers are less than 50.
2.2.1. Transmit Path
The operating voltage + U\textsubscript{TX} switches diodes D 02, D 04, D 06, and D 08 to conduct. The transformation link comprising 100 pF and L 01 matches the crystal filter to the input of the 9 MHz amplifier equipped with T 01. At the output of the crystal filter, the impedance is transformed to 50 Ω with the aid of the 39 pF coupling capacitor.
The ring mixer Mx01 converts the signal to 144 MHz. The mixer is followed by a two-stage bandpass filter and two amplifier stages with intermediate bandpass filter and PIN-attenuator. The PIN-attenuator is opened in the transmit-mode with the aid of diode D 12.
The signal is passed via a resonant circuit comprising L 10 and connection point Pt 105 and leaves the transmit amplifier at output HF-TX.
2.2.2. Receive Path
The operating voltage + U\textsubscript{RX} causes diodes D 01, D 03, D 05, and D 07 to conduct so that the VHF-signal from input HF-RX can pass through the two-stage 144 MHz amplifier with PIN-attenuator. This is followed by the bandpass filter comprising L 04 and L 05 and the mixer that converts the signal to 9 MHz. The output of the mixer is terminated by the input impedance of the 9 MHz amplifier.
After the signal has passed through the amplifier, it is fed via D 03 and the transformation link, comprising L 01 and the 100 pF capacitor, to the crystal filter.
2.2.3. Voltage Stabilization and PTT-Switching
Transistors T 04 to T 06 form a voltage stabilizer circuit that is driven with a voltage of +12 V to +16 V. The internal operating voltage of +11.5 V is provided at the output. The circuit can be short-circuited but requires a diode (D 15) to actuate. The operating voltages for transmit and receive are selected by the relay, which is controlled from the PTT-connection.
All other capacitors: Ceramic disk types for 2.5 mm spacing.
All resistors for 10 mm spacing or smaller, (some resistors are mounted vertically).
Relay: RS-12 V or RHD-12 V (National)
T 51: J310 (Siliconix) or BF 246
T 52, T 53: 40841 or similar DG-MOS FET
I 51, I 52: S042P (Siemens)
I 53: HEF 4011 (due to speed, no alternative possible)
I 54: LM324 (National Semiconductor)
I 55: LM386 (National Semiconductor)
D 51, D 52: 1N4148, 1N4151
D 53: C8V2 zener diode
D 54: C3V3 zener diode
D 55, D 56: 1N4148, 1N4151
Crystal filter: SSB, with carrier crystals type 9M22DI (Nikko Denshi) or XFM-9B (KVG)
L 51, L 55: 2 x 6 turns double-wound, approx. 0.3 mm dia. enamelled copper wire on toroid core R6, 3N30 (Siemens)
L 52 – L 54: Special coil set type 5138 (blue/red/white)
Ceramic disk capacitors: 2.5 mm spacing 100 nF capacitors (2 pcs.): 5 mm or 7.5 mm spacing
Polarized capacitors: Tantalum electrolytics, drop type, 16 V
2 pcs. trimmer potentiometers 20 kOhm: round types, approx. 6 mm dia.
15 pcs. feedthrough capacitors for solder mounting, approx. 2.2 nF (value uncritical), short construction;
4 pcs. PTFE feedthroughs
1 metal case, 74 x 148 x 30 mm
3. COMPONENTS
| Component | Description |
|-----------|-------------|
| T 01 – T 03 | J310 (Siliconix) possibly also BF246 |
| T 04 | BD 138 (Siemens) |
| T 05 | BC 415 (PNP) |
| T 06 | BC 413 (NPN) |
| D 01 – D 08 | BA 282, BA 244 (switching diodes) |
| D 09 – D 11 | BA 379 (PIN, Philips) |
| D 12 – D 14 | 1N 4148, 1N 4151 |
| D 15, D 16 | C6V3 (zener diodes) |
| Mx01 | IE-500, SRA-1 or better: SRA-1H or even better: SRA-3H |
| L 01, L 03 | Special coil set, type 5138 (blue/red/white) |
| L 02 | 120 µH miniature choke |
| L 04, L 05 | Air-spaced coils from 0.8 mm dia. silver-plated copper wire (see drawing). 7.5 turns on 6 mm former, coil tap: 1.25 turns from the cold end |
| L 06, L 09 | 2.7 µH miniature choke |
| L 07, L 08, L 10 | Special coil set, type 5118 (silver-plated) |
| C 01, C 02 | Ceramic miniature spindle trimmer 6 pF (Philips) |
| C 03 | 16 mm piece of 120 Ohm balanced cable (see Fig. 4) |
A VFO with frequency-locked loop
The most critical part of a frequency-locked loop is the frequency-to-voltage converter. Conventional circuits such as (1), and (2) use RC or LC links for frequency discrimination and exhibit considerable disadvantages because of this with respect to temperature stability and/or characteristic slope. Based on an article in (3), experiments were made using a PAL-delay line as frequency discriminator. The result of these experiments is a continuously variable VFO of simple construction, whose stability is sufficient for applications in FM-systems up to and including the 70 cm band.
is now provided in the keying ratio of the output voltage of the flipflop; if this squarewave voltage is integrated, one will obtain a DC-voltage that is linear-proportional to the frequency. Since the saturation voltage of the TTL-output transistors is very temperature-dependent, a C-MOS analog switch I 5 is provided before integration.
The integrated squarewave voltage is compared to a (variable) DC-voltage. This is provided by operational amplifier I 6, which is used as comparator and integrator. The output voltage of this OP-amp. provides the control signal for the varactor diode D 1 of the VCO after further filtering. The antiparallel diodes D 2 and D 3 at the input of the OP-amp. reduce the transient time.
1. Circuit
As can be seen in Figure 1, the output signal of the VCO (constructed according to details given in Figure [4]), is generated directly at the required frequency, after which it passes through a two-stage buffer amplifier. Approximately 20 mW (into 50 Ohm) are available at the output; a small part of the output power is tapped off and used to drive divider I 1. The division ratio of the subsequent dividers I 2 and I 4a can be programmed by disconnecting conductor lanes. The reset signals for the dividers, steep, negative pulses of the NAND-gate I 3 set the flipflop I 4b and also drive the delay line VZL. The signals at the output of the delay line are amplified in transistor T 4 and reset flipflop I 4b. The frequency information
1.1. Operation of the Frequency/Voltage Converter
The frequency/voltage converter comprises the delay line (VZL), transistor T 4, and flipflop I 4b. Pulses at the input of the VZL appear at the output with a delay of 63.943 μs. Further pulses can be fed into the delay line before the first pulse has reached the output, and these are delayed by the same value. This principle can be used up to pulse frequencies of approximately 1 MHz.
Figure 2 shows the time plan at output Q of the flipflop for various input frequencies. In the case of frequencies whose even multiples are 1/63.943 μs = 15.64 kHz, the keying ratio will
jump from 0 to 1 (or from 1 to 0). The variation range in which the keying ratio is clearly defined, thus amounts to 15.64 kHz; in practice, however, it is necessary for maximum and minimum keying ratios to be avoided for stability reasons (jumping into another frequency range). The theoretical variation range of the required frequency results from $\Delta f = 15.64 \times n$ (kHz) $n \ldots$ division ratio. If a certain output frequency is divided by $\Delta f$, i.e. $f / \Delta f = a, b$, one will usually obtain a rational number. The figures behind the comma, when multiplied with the operating voltage $b \times U_{op} = U_{int}$, will correspond to the DC-voltage from the frequency/voltage converter. The two subsequent final frequencies with which the division $f / \Delta f$ results in a whole number, are the theoretical corner frequencies:
$$f_i / \Delta f = a, 0; f_o / \Delta f = (a + 1), 0$$
An example for the 144 MHz band:
$n = 170; U_{op} = 5 V$, therefore
$\Delta f = 170 \times 15.64$ kHz $= 2658.62$ kHz and
$f / \Delta f = 145000/2658.62 = 54.539$
$U_{int} = 0.539 \times 5 V = 2.69$ V
The corner frequencies can then be calculated as:
$f_i = 54 \times 2658.62$ kHz $= 143565$ kHz and
$f_o = 55 \times 2658.62$ kHz $= 146224$ kHz
The frequency/voltage converter operates exclusively in the time domain, which means that it receives its information directly from the time plan of the input signal. For this reason, the steepness of the rise and fall slopes has a direct effect on the stability, as shown in Figure 3. A good compromise between slope and current drain is provided by the use of low-power ECL, and low-power Schottky ICs. However, this limits the maximum VCO-frequency that can be processed to 180 MHz. Transistor T4 is critical with respect to its temperature behaviour; since it is operated with a low collector quiescent current (90 µA), temperature variations affect a shift of the quiescent current by shifting the base-emitter voltage, which has drastic effects on the operating point: With increasing temperature, the pulse will become wider, the switching slope will be shifted in the time domain, and the frequency will drift upwards. This can be avoided by using a faster switching transistor, and using a PTC-resistor thermally coupled to the transistor in the base-current line.
This means that they have sufficient spacing to the limits of the 144 MHz amateur bands. The optimum division ratios for a number of common frequency ranges have been calculated with the aid of a computer (Table 1):
| Band (MHz) | I1 | I2 + 4a | Corner Frequencies (MHz) |
|------------|----|---------|--------------------------|
| 144.0 – 146.0 | :10 | :17 | 143.565 – 146.224 |
| 133.3 – 135.3 | :10 | :17 | 132.930 – 135.589 |
| 135.0 – 137.0 | :16 | :11 | 134.870 – 137.622 |
2. CONSTRUCTION
The circuits of the VCO, buffer amplifier, and frequency/voltage converter shown in Figure 1 are accommodated in three chambers formed by the required screening panels; the VCO is built up without PC-board, and has therefore not been designated. The VCO and the two boards can be mounted in a metal case of $74 \times 111 \times 30$ mm, see Figure 4.
The holes for the feedthroughs, or feedthrough capacitors are drilled on the side of the case, and in the two intermediate panels of $72 \times 28$ and $60 \times 28$ mm which are constructed from 0.5 mm tin plate. These holes have a diameter of 3.5 mm, and should be drilled with a spacing of 10 mm from the cover. A hole of 6 mm diameter should be drilled in the center of the other cover for alignment of trimmer C 2. The position of all holes can be seen in Figure 4 and in the photograph of the author's prototype given in Figure 5. After these preparations, the feedthrough capacitors are soldered into place and the case in fitted together.
Fig. 4:
Component locations for VCO, buffer (OE7WMI 002) and frequency/voltage converter (OE7WMI 003)
The buffer amplifier is soldered into position with a spacing of 5 mm, and the digital part with a spacing of 12 mm from the base panel, and they are then soldered all around the board to the case. The construction of the VCO can be made easily by comparing Fig. 4 and Fig. 5.
The previously calculated division ratio is programmed according to Table 2 on PC-board OE7WMI 003.
| | e | d | c | b | a |
|---|---|---|---|---|---|
| 1 | * | * | * | * | |
| 2 | * | * | * | | |
| etc. in dual code | | | | | |
| 29 | | | | | |
| 30 | | | | | |
The * means that the appropriate conductor lane must be disconnected.
Finally, the four adjacent connections of the delay line should be bent carefully close to the plastic case, (the two other connections are not required and can be disconnected), and the delay line can be soldered to the PC-board directly.
Fig. 5: Photograph of the author's prototype constructed according to Fig. 1 and Fig. 4. The delay line is to be found on the conductor side of the board.
### 2.1. Special Components
| Component | Description |
|-----------|-------------|
| T 1 | U310 |
| T 2, T 3 | BF961 |
| T 4 | BFX59 or BFY90 |
| D 1 | BB 105 |
| D 2, D 3 | 1 N 914 or 1 N 4148 |
| I 1 | According to frequency range or division ratio: |
| | SP 8655 B | 1:32 |
| | SP 8657 B | 1:20 |
| | SP 8659 B | 1:16 |
| | SP 8660 B | 1:10 |
| I 2 | 74LS197 |
| I 3 | 74LS30 |
| I 4 | 74LS73 |
| I 5 | 4066 in a socket |
| I 6 | 3140 in a socket |
| I 7 | 78L05 |
| I 8 | 78L08 |
| L 1 | Aged ceramic coil, 6 turns of 10 mm dia. or silverplated copper wire of 1 mm dia. wound on a glass rod of 8 mm dia. |
| L 2 | 3.5 turns of 0.4 mm dia. enameled copper wire, using special coil set 5140500000, orange core |
L 3: 3.5 turns of 0.8 mm dia. silver-plated copper wire, wound on a 6.5 mm former
VZL PAL delay line, e.g. Siemens AZ 1702, or AZ 1706
PTC: Siemens P 270-C11, colour black
R 1, R 2: see text, standard each 680 kOhm
C 1: see text, for 144-146 MHz: standard 1 nF
Tr 1: Double-hole core (Siemens B 62152-A7-X17) primary and secondary 3 turns, each of 0.2 mm dia. enameled copper wire
output power (20 to 30 mW). The value of capacitor C 1 and the coupling point of the varactor diode determine the variation range of the VCO. It must be less than the theoretical variation range, since otherwise the frequency in the vicinity of the corner frequencies could jump into another frequency range. For this reason, C 1 should be varied while observing the frequency counter until one is within the variation range even at maximum variation of the diode voltage (0 V to $U_{op}$).
After connecting the control loop (varactor diode is connected to the output Pt 1 of the digital portion), the VFO will be ready for operation if the previously mentioned adjustments had been carried out correctly. However, it is advisable to check with a frequency counter to see whether the VFO jumps to another frequency range on varying the reference voltage.
Now a word regarding the temperature compensation of transistor T 4: The PTC-resistor is bent over transistor T 4, and thermically coupled to it with the aid of heat-conductive paste. The increase of the parallel (R 1), and decrease of the
---
**Fig. 6:** The FLL-VFO can be used as FM-transmitter using this simple modulator and calling-tone generator
series resistance (R 2) operate against the temperature drift of the transistor. A value of 680 kOhm has been found as optimum value for R 1 and R 2 in several prototypes.
The author would like to point out to a difficulty that was encountered with one prototype: This prototype exhibited a noise as interference on the modulation. The cause of this was a strong noise generated in the stabilizer 78L08.
4. ACCESSORY MODULES
4.1. Modulator
It is possible to use the VFO both as local oscillator in a receiver and as an oscillator in FM-transmitters. Figure 6 shows a modulator and calling-tone generator for the latter application. Both circuits, as well as 8 trimmer potentiometers for programmable frequencies are accommodated on board OE7WMI 004 (Figure 7). The circuit does not offer any special features, both construction and alignment are easy; it is only necessary to align the frequency deviation limiter and calling-tone level. If the microphone gain is too low, this can be increased by reducing the value of R 1.
Fig. 7: The single-coated PC-board for modulator and calling-tone generator OE7WMI 004
Fig. 8: This VHF-amplifier with lowpass filter amplifies the transmit power to 1 W
4.2. Transmit Amplifier
The circuit of a VHF-transmit amplifier (OE7WMI 005) with lowpass filter and an output power of 1 W is given in Figure 8. This PC-board can be accommodated in an additional metal case. The construction of this module can be seen in Figure 9. The alignment is simple: The five trimmer capacitors should be aligned for maximum power at the center of the band.
5. OPERATING AT HIGHER FREQUENCIES
It is not possible to use the described design without change for higher frequencies, such as 430 to 440 MHz. The metal case is not stable enough, which means that frequency variations due to thermal or mechanical variations of the case cannot be completely compensated for due to the finite loop gain. Frequency errors will remain that are not permissible, even in FM-systems.
There are two ways of using the VFO for higher frequencies:
Either one can use a case with thicker side panels such as cast aluminium cases which allow the VFO to oscillate directly at the required frequency, or one can use a frequency multiplication. If the 2 m-band is to be avoided, one can use the frequency range 215 to 220 MHz as VFO-frequency.
Both types require a divider having a higher cutoff frequency (unfortunately also higher current drain) in the digital circuit. For this reason, the divider SP 8515 is used in the circuit of OE7WMI 006 (see Figure 10). The programming and construction are, otherwise, identical to that of OE7WMI 003, and it is only necessary to use a stabilizer 7805 with its higher rating. This can be directly connected to the case next to the delay line to provide better cooling, or it can be soldered into position there. Figure 11 shows PC-board OE7WMI 006 that was developed for this application instead of board 003.
| Component | Description |
|-----------|-------------|
| L 1 | 3 turns of 1 mm dia. silver-plated copper wire, wound on a 6 mm former, coil tap at the center; |
| L 2 | 3 turns of 1 mm dia. silver-plated copper wire, wound on a 6 mm former; |
| L 3 | 4 turns, otherwise as L 2 |
| L 4, L 5 | 4 turns of 0.8 mm dia. enameled copper wire, wound on a 5 mm former |
| Metal case| 3711130 |
6. FURTHER APPLICATIONS
The FLL-VFO is also suitable as inexpensive solution for stabilizing frequencies in many applications. Several possibilities that have been examined with success by the author are to be mentioned briefly:
One can use the same VFO without large modifications for transmit and receive if the same division ratio results for both frequencies (e.g. 144 to 146 MHz and 133.3 to 135.3 MHz). The switching of the frequency ranges can be obtained using...
an additional capacitance and an RF-switching diode in the VCO. If the reference voltages are also switched at the same time (with the aid of a C-MOS analog multiplexer 4066), one will have obtained the whole frequency processing for both transmit and receive, which will form the basis of an inexpensive FM-transceiver (Fig. 12).
If different division ratios are required for transmit and receive, one can use an additional digital switching (AND/OR) at the inputs of I 3 as a simple solution.
Or course, one can also use reconversion methods even with a delay line-VFO. In this manner, one could save the ECL-divider and could use available crystals, since one is relatively versatile in the selection of the conversion frequency. Reconversion will also improve the overall stability, providing that one uses a stable crystal.
7. REFERENCES
1) G. Hoffschmidt, DL9FX:
The AFC Loop – A Simple and Cheap Method of Obtaining Stable VHF Frequencies
2) T. Krieg, DK8GY:
A Frequency Control Loop for a 433 MHz VCO
VHF COMMUNICATIONS 10, Edition 3/1978, pages 186-190
3) W. Kassenbrock, DK8BH:
Zweit VFO für FT 277/101
CQ-DL 52 (1981), Edition 3
4) M. Martin, DJ7VY:
Low-Noise VHF Oscillator with Diode Tuning,
Digital Frequency Control, and Frequency Indicator
VHF COMMUNICATIONS 13, Edition 2/1981, pages 66-82
5) J. Kestler, DK10F:
9 MHz FM Exciter Matching the 80-Channel Synthesizer
VHF COMMUNICATIONS 5, Edition 4/1973, pages 194-202
6) E. Schmitzer, DJ4BG:
Active Audio Filters
VHF COMMUNICATIONS 1 (1969), Edition 4, pages 218-235
A Stripline GaAs-FET Preamplifier and Mixer for the 10 GHz Band complete with IF-Preamplifier, Image Frequency Filter, and Power Supply.
Part I
GaAs-FET preamplifier and mixer for the 10 GHz band in stripline technology complete with IF-preamplifier, image frequency filter, and power supply.
The following article is to describe the result of a large number of experiments with components for the 3 cm band in order to provide suggestions for stripline preamplifiers and mixers, thus allowing an alternative to straight-through mixers. The article is not to describe the hardware down to the last detail, but is more a collection of experiment results and will include many tips and aids to design. In order to indicate the information that is to be given, the article is to be commenced with an index.
1. Stripline Hybrid Mixer for 10 GHz
1.1. General Details
1.2. Noise Measurements
1.3. Mixer Design
1.4. Measured Values
2. IF-Preamplifier
3. Two-Stage Waveguide Filter
4. Stripline Amplifiers for 10 GHz
4.1. Circuit Diagram and Layout
4.2. Construction Details for the Preamplifier Board and Mixer
4.3. Determining the Constructional Values
4.4. Setting up the Amplifier
4.5. Measuring Results on Integrated Preamplifier/Mixer
4.6. Measuring Results with Preamplifier and Image Frequency Filter in Front of the Mixer
4.7. Instructions for Mounting onto the Waveguide
5. Power Supply
6. References
STRIPLINE HYBRID MIXER FOR 10 GHz
1.1. General Details
The idea for this article was based on publications describing such equipment for lower frequencies (1.2 and 2.3 GHz) in VHF COMMUNICATIONS, and whether these circuits could also be used on 10 GHz now that suitable mixer diodes having low noise figures are available for this application (4). The experiments were made exclusively using Schottky diodes type BAT 14-073, 14-083, and 14-093 (see Figure 1), which are manufactured by Siemens. According to the data sheets, single-sideband noise figures of between 5.5 and 6.5 dB are possible. These values require an IF-noise figure of approximately 1.5 dB.
The main component is a conventional detector probe in a waveguide type R 100 (WR 90) where a diode 1 N 23C is driven into its blocked range via a resistor of approximately 10 kOhm and a stabilized voltage of approximately 10 V. The diode was selected for maximum noise power. A circuit as described in (9) and (11) (current injection via FET) would be more suitable. The calibrated noise power is 14 dB ENR.
All noise generators using this technology have one thing in common and that is that the generated noise power will vary when an RF-power of more than 0.5 mW is injected to the noise diode. This means that the calibration will no longer be correct. It is possible that an additional directional coupler or circulator may be able to suppress this effect. Absolute measurements on straight-through mixers, or mixers with a low suppression of the oscillator frequency can be problematic when not using the previously mentioned aids, and can only be used for orientation.
By the way, the noise source constructed by the author is also used in an automatic noise figure measuring system (9) with success. It allows the ratio \((S+N)/N\) to be determined clearly.
The results of the experiments confirmed the values given in the data sheets. With the exception of a calibrated, professional semiconductor noise source, no special measuring equipment was required. Only those aids were used that any serious microwave amateur will usually have available, or will have access to. With the exception of the noise generator, the author did not need to loan out any equipment (QRL).
1.2 Noise Figure Measurements
By the way, the author also uses a home-made noise source for the 10 GHz band, that was calibrated by comparing it to a professional noise source. Since this may be of interest to other amateurs, the main features are now to be given:
Fig. 1:
Construction of the 10 GHz mixer diode BAT 14-073
Fig. 2:
Hybrid ring for 10.25 GHz
1.3 Design of the Mixer
The design of the ring hybrid mixer was made in a similar manner to (4) and (10). The principle of construction is shown in Fig. 2. The following material was used for the PC-board:
RT/Duroid 5870 having:
\[
E_r = 2.35 \quad \text{and} \\
d = 0.79 \text{ mm}
\]
This results in a conductor lane width of approx. 2.3 mm for the two 50 Ohm branches and a conductor lane width of approx. 3.5 mm for the two 50 Ohm/1/2 branches. According to the equation given in (7)
\[ E_{\text{eff}} = 1 + (E_r - 1) \left[ \frac{1}{2} \left( 1 + \frac{1}{\sqrt{1 + \left( \frac{10}{w/h} \right)}} \right) \right] \]
the velocity factor is:
\[ V_p = \frac{1}{\sqrt{E_{\text{eff}}}} \approx 0.7 \]
The length of the square ring results as 5.1 mm with \( \lambda/4 \) at 10.25 GHz after taking \( E_{\text{eff}} \) and \( V_p \) into consideration. However, 4.5 mm was selected in order to extend the \( \lambda/4 \)-length somewhat into the conductor lane (bandwidth).
**Figure 3** shows all dimensions of the PC-board, and **Figure 4** shows two different versions according to whether the receive signal is to be extracted using a coupling pin directly from the waveguide, or via semirigid cable. The oscillator frequency is fed in using semirigid cable in both cases. This cable can be soldered to the board virtually without mismatch when soldered as shown in **Figure 5**.
The design of the pin probe and the mounting of the mixer board on the waveguide are indicated in **Figure 6**.
**Fig. 4:**
PC-board of the hybrid ring mixer;
Left: for connection to semirigid cable,
Right: for waveguide mounting
**Fig. 5:**
Mounting of semirigid cable (RG-141)
to microstrip boards
The two mixer diodes are soldered into place quickly, without using too high a soldering-iron temperature. The lowpass filter for the IF is obtained as capacitance on the PC-board, and it also represents a \( \lambda/4 \)-line of low impedance. The IF-preamplifier is directly, capacitively coupled to the output of the mixer.
**Fig. 6:**
Mounting and coupling of a mixer board to R 100 (WR 90) waveguide
The crystal-controlled oscillator frequency of 10.368 GHz is obtained by multiplying the output frequency of a 1152 MHz oscillator by nine. A 3-stage filter is provided at the output. The signal purity is monitored during the experiments using a 20 dB coupler connected to a spectrum analyzer, since frequency multipliers having a high frequency modulation factor can be critical. The local oscillator power amounts to approximately 10 mW, and can be adjusted with the aid of an attenuator.
A home-made IF-amplifier is used for the input circuit of the intermediate frequency at 144 MHz. This amplifier must have a good linearity range of at least 20 dB with the AVC switched off. The AF-output power is measured with a thermometric power meter (HD 434 A). The noise generator has already been mentioned.
1.4. Measured Values
1.4.1. Receive Mixer
\[ f_0 = 10.368 \text{ GHz} \]
\[ \text{IF} = 144 \text{ MHz} \]
\[ f_{\text{in}} = 10.224 \text{ GHz} \]
Oscillator power saturation from approximately 3 mW; sensitivity loss below approx. 2.5 mW.
Noise figure:
3–3.5 dB (double-sideband)
6–6.5 dB (SSB with 10.224 GHz filter, see 3.)
Suppression of the oscillator frequency approx. 12 dB, whereby any unpaired diodes of the given type were used; (Figure 7).
The given measuring values were achieved with both types of construction (board 50/50 Ohm, and with probe coupling to the waveguide).
When using the waveguide construction, the minimum noise figure could only be obtained after alignment of the matching screws on the signal side.
1.4.2. Transmit Mixer
The local oscillator signal was fed in with the power of approximately 10 mW at 10.368 GHz. The IF-power at 144 MHz amounted to approximately 5 mW. Approximately 0.5 mW SSB-power was measured at 10.224 GHz.
The efficiency is most certainly not optimized. No further experiments were made to further optimize the circuit, however, later experiments have indicated that it is possible to reduce the size of the previously large, narrowband frequency oscillator chains.
2. IF-PREAMPLIFIER
In order to ensure that the noise figure of the mixer is not deteriorated after taking the conversion loss into consideration, it is necessary for the subsequent IF-preamplifier to possess a high gain and a low noise figure.
A wideband amplifier described in a Siemens application sheet (Figure 8) equipped with a low-cost BFT 66 is able to fulfill these demands. The
Fig. 7: A prototype 10 GHz hybrid ring mixer using two BAT 14-083 F
Fig. 8:
Low-noise preamplifier for 30–150 MHz
L 1: 4 turns of 0.3 mm enamelled copper wire wound on a 4 mm former, self-supporting
RFC 1: Ferrite choke 2–3 µH, e.g. 5 turns of enamelled copper wire on a ferrite bead
The capacitance and inductance values have been changed somewhat in order to limit the frequency range. The electrical values obtained are:
Gain = 15–20 dB
Frequency range: = 30–150 MHz
Noise figure: approx. 1.5 dB
The given operating frequency range allows the common intermediate frequencies of 30 MHz and 100 MHz often used for the X-band to be covered.
The output impedance of the mixer is in the order of 150 to 300 Ohm. This is listed as a common value for Schottky diodes, which will also be affected by the oscillator power. It is necessary that the signal level is small with respect to the oscillator power.
The direct connection of the IF-preamplifier to the mixer provided good measuring results which means that an input matching was not required. An attempt to improve matching using a variable transformation link and taking the Sparameter of the BFT 66 into consideration, resulted in a hardly noticeable noise figure improvement. However, if a coaxial cable is to be connected between mixer and preamplifier, the noise figure will be deteriorated considerably even if this is only a few centimeters in length.
The amplifiers are built up on a 1.5 mm thick PC-board of 40 mm x 25 mm. This double-coated PC-board is designated DL 3 ER 002, and all components are mounted on the ground side of the board (Figure 9). Suitable counter-sinks should be made – using a drill – around the holes for insulation of non-grounded components. The transistor BFT 66 is soldered into position on the conductor side of the board. This facilitates removal later, if required; no tendency to oscillation was observed. The case and the emitter connection of the BFT 66 were through-contacted to the ground side.
3. TWO-STAGE WAVEGUIDE FILTER FOR 10 GHz
Several filters were required for the experimental construction and for carrying out the measurements. They should be easy to construct and are easily reproducible. The construction of this filter originates from Max Münich, DJ 1 CR, and is based on a professional prototype.
Fig. 13: Photograph of a completed two-stage filter
Figure 11 shows virtually all important details. The waveguide (R 100, WR 90) is cut with a circular or hand-saw to the given dimensions. The width of the cut should correspond exactly to that of the panels to be inserted in order to ensure that these fit tightly. They are finally soft-soldered together with the two connection flanges (heat up on an electric cooker, or Butan-gas flame). The author used a conventional soldering fluid comprising hydrochloric acid/zink filing solution. This flux can be removed more easily by intensive washing with hot water than the flux of conventional solder.
The measured values are shown in Figure 12, which were obtained using a simple power measurement (Gunn oscillator, absorption wave-meter, directional coupler at the input, as well as microwattmeter at the output).
The insertion loss in the passband range is in the order of approximately 0.5 dB. The filter (Figure 13) has not been plated galvanically. The filter is easily tunable with the aid of the tuning screws and their counter-nuts. The input and output of the filter are provided with the usual three screws for tuning and have springs to maintain the adjustment; when higher demands are to be placed on the stability, counter-nuts can be used. The filter has satisfied all demands placed on it in practice.
Colour ATV-Transmissions are no problem for our new ATV-7011
The **ATV-7011** is a professional quality ATV transmitter for the 70 cm band. It is only necessary to connect a camera (monochrome or colour), antenna and microphone. Can be operated from 220 V AC or 12 V DC. The standard unit operates according to CCIR, but other standards are available on request.
The **ATV-7011** is a further development of our reliable ATV-7010 with better specifications, newer design, and smaller dimensions. It uses a new system of video-sound combination and modulation. It is also suitable for mobile operation from 12 V DC or for fixed operation on 220 V AC.
**Price** .......................................................... DM 2750.00
The ATV-7011 is also available for broadcasting use between 470 MHz and 500 MHz, and a number of such units are in continuous operation in Africa.
**Specifications:**
Frequencies, crystal-controlled:
Video 434.25 MHz, Sound 439.75 MHz
IM-products (3rd order): better than – 30 dB
Suppression of osc.freq. and image: better than – 55 dB
Power-output, unmodulated: typ. 10 W
Delivery: ex. stock to 8 weeks (standard model)
UKWtechnik Terry D. Bittan · Jahnstr. 14 · Postfach 80 · D-8523 Baiersdorf
Tel. 09133/855 (Tag und Nacht)
Friedrich Krug, DJ 3 RV
Versatile IF-module suitable for 2 m Receivers, or as an IF-module for the SHF Bands
Part V: Description of the IF-amplifier module DJ3RV 002 and BFO-module DJ3RV 004
This part of the description describes the heart of the IF-module. This IF-amplifier is able to handle signals at low distortion over a dynamic range of more than 100 dB.
It is advisable to describe the BFO-module at this point since its signals can be used for alignment of the modules that have been described already.
The next edition will describe the demodulator module, and give details regarding the interconnection of all modules, as well as recommendations of a suitable power supply and AF-amplifier.
Circuit Description
Figure 44 gives the circuit diagram of the module, whose operation was described in detail in part 3 (VHF-COMMUNICATIONS 3/1982). The input signal is fed in via a coaxial connector from the crystal filter module and is passed via a relay to the notch filter, or directly to point Pt 20 on the PIN diode attenuator of the IF-amplifier.
The notch filter possesses an amplifier at the input equipped with T 1 and T2b, which compensates for the insertion loss of the filter, and the source follower T 2a and T 3. The filters can be aligned separately with the aid of varactor diodes D 4 and D 5. This has the advantage that two different interference signals can be filtered out. Furthermore, it is very difficult to achieve an exact tracking even when using selected components.
Fig. 44: Controlled IF-amplifier with double notch filter
The IF-amplifier corresponds to the circuit diagram given in Figure 13 with the exception of three modifications. In order to achieve a better decoupling, an additional $22 \mu H$ choke was placed in the power supply line between T 7 and T 8. The $22 k\Omega$ resistor parallel to L 3 can be deleted. The critical stability in the control behaviour encountered with the first prototype when not using this resistor was not noticed when constructing later prototypes. The third modification is due to a drawing error in Figure 13: The 1 nF bypass capacitor between the emitter of T 5 and diode 1 N 4148 to ground is drawn at the wrong position. It should be between diode 1 N 4148 and the 560 $\Omega$ resistor, otherwise large intermodulation distortions will be caused. The value of the bypass capacitor was also increased to 10 nF. The author would like to thank DK 5 LV here for the extensive measurements.
**Components**
The details given in section 4.2.1. are also valid for the selection of components for this and for the subsequent modules.
The inductances are all built up using the special filter set D 41-2165, colour: orange, after removing the center pins that do not fit into the holes on the board. They can be removed easily with the aid of tweezers.
| Component | Description |
|-----------|-------------|
| L 1 + L 2 | 3.5 turns + 17.5 turns, approx. 0.2 mm dia. enameled copper wire |
| L 3 + L 4 | 17.5 turns + 4.5 turns, approx. 0.2 mm dia. enameled copper wire |
| L 5 | 17 turns, approx. 0.2 mm dia. enameled copper wire |
| L 6 | 17.5 turns, approx. 0.2 mm dia. enameled copper wire |
| L 7 | 17 turns, approx. 0.2 mm dia. enameled copper wire |
| Tr 1, Tr 2| 2 x 16 turns of 0.2 mm dia. enameled copper wire twisted together and wound on a toroid core Siemens R 6.3 K 1 |
**Mounting the Components**
*Figures 45 and 46 give the components location plan, and a photograph of the author's prototype.*
The prototype board is double-coated, but does not possess through-contacts, which means that the ground points of the components marked in the component location plan must be soldered at both sides of the board.
The mechanical work should be made firstly, and should be executed according to the details given already in Section 4.2.1. When installing the board into a 30 mm high case, the spacing between base and surface of the PC-board should amount to approx. 4.5 mm.
After this, it is possible for all components to be mounted on the PC-board with the exception of the connection of the input connector. The source resistors of the FETs T 1, T 8, and T 12 should be selected so that the transistors are operated at a slope of approx. 20 mS. The values given in the circuit diagram for the source-resistors only serve as orientation values. In order to measure the transistors, the details given in the previous section should be observed. The author used all those FETs for modules DJ 3 RV 002 to 004 that were not paired together for module DJ 3 RV 001.
If the transistors type BF 910 (T 6 and T 7) are sunk into the board, it is necessary for the drilled-out ground connection to be replaced by a wire bridge on the conductor side, and this is indicated as a dashed line in the component location plan. Otherwise, the IF-amplifier will oscillate!
**Preparations and Alignment**
The author has used the following "check list" for the preparations and alignment:
Firstly switch on the IF-amplifier without notch filter, and continue in the following sequence:
- Dampen the input of the PIN-attenuator (Pt 20) with a 56 $\Omega$ resistor to ground with no signal fed to the input
- Place the wipers of potentiometers P 1 and P 3 to ground. Adjust P 2 to null $\Omega$.
- Terminate the demodulator output with 50 $\Omega$.
- It is now possible for the operating voltage of + 15 V to be connected to Pt 21. It is advisable to use a power supply with an adjustable current limiting and to increase this slowly towards higher current values. The current drain should amount to approximately 75 mA.
If it differs greatly from this, a fault will be present in the circuit, or an incorrect operating point will exist for T 8 or T 12. It is easy to establish whether the amplifier is oscillating with the aid of an oscilloscope or millivolt-meter connected at the output to the demodulator, and at the IF-output. If this is the case, a ground fault will exist such as a forgotten feedthrough connection.
Fig. 45:
Double-coated PC-board DJ 3 RV 002 with component locations. The positions marked with crosses show points where through-contacts must be made. The PC-board allows various circuits to be made, which will be discussed at the end of this article.
- Check that approx. 6.8 V are present at Pt 22 and that Pt 22 is connected to Pt 25 using a wire bridge outside of the case.
- Adjust P 3 so that a voltage + 5.6 V is present at the wiper (10 µF capacitor). All voltages should be measured at high impedance using a voltmeter with ≥ 1 MΩ!
- P 1 should be adjusted so that a voltage of + 4.8 V is present at the collector of T 5. Approximately + 3.1 V will then be present at the emitter of T 5. The operating current at Pt 21 will then amount to approx. 100 mA.
Fig. 46:
Photograph of the author's prototype module DJ 3 RV 002 showing the component layout
- Remove the 56 $\Omega$ resistor connected to Pt 20 and directly connect this point via a wire bridge, or direct to the input socket. Feed a 9 MHz signal at a level of $-100$ dBm, and align inductances L 1 to L 6 for maximum level at the demodulator output. The cover on the conductor side of the board should be in place during this alignment.
- The level at the input should now be increased to approximately $-60$ dBm, after which the level at the output to the demodulator is aligned with P 2 to 10 mV$_{rms}$ (28 mV$_{pp}$). The level behaviour shown in Figure 16 should be present when the input signal level is varied.
- The alignment of filter XF 910 is made with the aid of L 7 to obtain the best passband curve. This is made by sweeping the input signal (frequency deviation < 100 Hz). In order to ensure that the control operation does not falsify the shape of curve, the signal should be below the level of the control threshold, or the control should be slowed down by connecting a tantalum electrolytic of 10 $\mu$F in parallel to C$_T$.
After this, the IF-amplifier is ready for operation with the exception of the S-meter. This can be aligned only together with the complete receiver as was described in section 3.3.4.
In order to align the notch filter, it is necessary for the IF-amplifier to be switched off and the input connector connected according to the wiring plan. The output of the filter in front of point Pt 20 should be terminated with 50 $\Omega$.
It is necessary for the notch filter to be slowly swept during the alignment process so that the transient time is known. An oscilloscope with storage tube is very suitable for displaying the output signal. Since most amateurs do not have such an instrument, it is only possible to carry out this measurement using a point-for-point measurement. The alignment signal can be taken from the crystal oscillators of the BFO-module DJ 3 RV 004, by pulling the frequency with the aid of the trimmers. This board also provides a reference voltage for Pt 27 and Pt 28.
It is necessary for each filter to be aligned individually. This is achieved by removing the coupling capacitors and bridging each stage. The alignment should be made at the center of the band with the aid of the 20 pF trimmer, and the 40 pF trimmers are used to align a symmetrical notch. A voltage variation range from +1 V to +20 V at Pt 27, or Pt 28 resulted in a pulling range of 2.2 kHz in conjunction with the crystal XF-901, however, the symmetric behaviour of the notch deteriorates at the band limits.
The current drain at Pt 26 should amount to approx. 100 mA.
### 4.2.3. BFO-Module DJ 3 RV 004
PC-board DJ 3 RV 004 accommodates the BFO with three switchable crystal oscillator circuits, and the output stage. This module also provides a swept oscillator for 9 MHz equipped with a low-frequency sawtooth generator designed for slow sweeping. This swept-frequency generator is an aid for alignment of the crystal filter circuits and is especially provided for those readers that do not have such measuring equipment. This part of the board can be cut off later and the BFO will then fit into a metal case having the dimensions 74 mm x 111 mm.
#### Circuit Description
The operation of the BFO-circuit was already described in part 3 (VHF COMMUNICATIONS 3/1982). An output level of max. 0 dBm is required when using it together with the demodulator board DJ 3 RV 003. As can be seen in the circuit diagram given in Figure 47, the Schottky diode at the gate of the oscillator transistor can be deleted, and the output amplitude aligned with the aid of C 3. The value of C 3 was in the order of 33 pF to 150 pF in the various prototypes. It is also possible for the voltage stabilizer REF 01 to be replaced by a pass transistor with zener diode. However, even these component-saving measures do not deteriorate the quality of the signal. However, if the circuit is to be used for intermodulation measurements, these measures should not be used since the carrier noise would then be too great.
Fig. 47a: A swept-frequency oscillator as alignment aid for module DJ 3 RV 004
DJ3RV 004/1 Wobbeloszillator / Sweep osc.
Fig. 47b: Circuit diagram of BFO-module DJ 3 RV 004 (only basic circuit of the swept-oscillator is indicated)
The circuit of the swept-frequency oscillator does not possess any special features, and is very similar to that of the crystal oscillators.
The 9 MHz signal is fed via a source follower and an attenuator and is coupled out at a level of approx. $-20$ dBm into $50 \Omega$. This level will not overdrive the crystal filter stages.
The sawtooth generator is built up with discrete components. Capacitor C is charged from a current source via R, zener diode C5V1, and transistor T4 until transistor T3 conducts, which then discharges capacitor C via T1 and the Darlington transistor T2. Transistors T3, T1, and T2 are then blocked again, and capacitor C will be charged again. The sawtooth voltage is coupled out at low impedance with the aid of transistors T5 and T6. The sweep deviation can be varied using the 47 kΩ potentiometer.
**Components**
| Component | Description |
|-----------|-------------|
| L1: | 7 µH, 18 + 6 turns of approx. 0.2 mm dia. enamelled copper wire in special coil set D 41-2165, colour/orange |
| L2: | 7 µH, 25 turns, otherwise as L1 |
**Construction Details**
As can be seen in Figure 48, the PC-board can be installed in a 30 mm high metal case. This means that the crystals must be directly soldered into the circuit, or be provided with a socket for horizontal mounting.
The heat sink of transistor P 8002 must be slightly shortened, or bent.
The rest of the components can be mounted after completing the mechanical work, with the exception of C3. The component locations are shown in Figure 49.
**Connection and Alignment**
The BFO and the swept-frequency generator are completely separate circuits that must be aligned separately.
For those readers that intend to construct all boards of this series of articles and who do not possess a swept-frequency generator, it is advisable to firstly complete the BFO-board. This allows one to have suitable signals for alignment.
One will then only require a calibrated attenuator having a maximum attenuation of 100 dB.
Fig. 49:
Component location plan of the double-coated PC-board DJ 3 RV 004; the positions marked with crosses on the PC-board should be provided with through-contacts.
Fig. 50:
Four different attenuation values of 100 dB, 80 dB, 60 dB, and 40 dB can be realized using wire bridges.
Fig. 51:
Single-coated PC-board DJ 3 RV 005 with component plan
Fig. 52: Photograph of the author's prototype 100 dB-attenuator; impedance 50 Ω
Fig. 53: The wire bridges and compensating capacitors can be seen on the conductor side of the board. This allows the attenuator to also be used in the 145 MHz range. The value of the four capacitors is in the order of 1.5 to 8.2 pF; this depends on the type.
Section 4.2.4. is to describe a matching homemade attenuator for this.
The swept-frequency generator does not offer any problems and was equipped with $C = 4.7 \mu F$ and $R = 100 k\Omega$ in the author's prototype. The alignment and measurements are made as follows:
- Connect the operating voltage of + 15 V to Pt 48. The current drain should amount to approx. 40 mA.
- A sawtooth voltage of 4 $V_{pp}$ and approx. 1.8 Hz should be present at Pt 49 (x-deflection).
- Set deviation control (47 k$\Omega$ pot.) to zero and adjust the 40 pF trimmer to 2/3 of the maximum value.
- Adjust the frequency of the output signal to 9 MHz with the aid of L 2. The output level should then amount to approximately - 20 dBm into 50 $\Omega$.
A maximum sweep deviation of ± 40 kHz can be adjusted with the aid of the deviation control, and the center frequency should be aligned with the aid of the 40 pF trimmer.
The BFO is just as simple to align:
- Connect the operating voltage of + 15 V to Pt 41. The current drain should amount to approx. 40 mA with the crystal oscillator switched on; (connect Pt 42, Pt 43, or Pt 44 to ground as required).
- 10 to 11 V should be present at Pt 46.
- Align the crystals to their nominal frequency with the aid of the series trimmer. In this case, approximately + 5 V must be connected to Pt 45.
- Align L 1 for maximum output level using the CW-crystal XF 903.
- Align the output level with the aid of C 3 to approximately - 5 dBm (75 mV$_{rms}$).
### 4.2.4. Home-Made Attenuator
Figure 50 shows the circuit diagram of a five-stage attenuator using a $\pi$-circuit of 20 dB per stage. The switching is made by providing bridges on the board. The insertion loss amounts to 40 dB, and 3 x 20 dB can be switched in additionally. The component locations are shown in Figure 51.
As can be seen in the photographs given in Figures 52 and 53, the 74 mm x 148 mm single-coated PC-board DJ 3 RV 005 is also enclosed in a metal case. Only two resistance values of 120 $\Omega$ and 1 k$\Omega$ are required. All resistors should be low-inductance and low-capacitance types. Capless, composite carbon resistors have been found to be very suitable.
When higher demands are placed on the accuracy, it will be necessary for the resistors to be selected. When using a 50 $\Omega$ attenuator in a $\pi$-circuit having 20 dB attenuation, the exact value of the series resistor amounts to 247.5 $\Omega$, and that of the resistors to ground to 61.1 $\Omega$. When using a capacitive compensation, the attenuator can be used up to approximately 150 MHz.
#### Monolithic Crystal Filters from KVG for the DJ3RV IF-Module (for instance)
| Model | Value | Price |
|---------|-------|-------|
| XFM-9B02 | 6809 | DM 148.— |
| XFM-9S01 | 6810 | DM 148.— |
| XFM-9S02 | 6811 | DM 148.— |
| XFM-9S03 | 6812 | DM 148.— |
| XFM-9S04 | 6813 | DM 148.— |
| XFM-9S05 | 6814 | DM 148.— |
| XFM-9S06 | 6815 | DM 148.— |
| XFM-9S07 | 6816 | DM 148.— |
| XFM-9S08 | 6817 | DM 212.— |
For Technical Data see rear cover page of VHF COMMUNICATIONS
## MATERIAL PRICE LIST OF EQUIPMENT
described in Edition 1/83 of VHF COMMUNICATIONS
### A home-made automatic noise-figure measuring system
| Item | Description | Art. No. | Ed. 1 + 2/83 |
|------|-------------|----------|--------------|
| **1. Noise source** | | | |
| Parts | noise source | 1 metal case, 1 N connector, 1 BNC socket, 2 transistors, 2 diodes, 1 chip capacitor, 1 disc cap., 2 carbon- and 4 metal film resistors, 1 RF choke | 6750 | DM 35.00 |
| **2. Receive converter** | | | |
| PC-board | DK10F 034 | single coated, with printed component location plan | 6442 | DM 12.00 |
| Parts | RMG E.K. | 3 transistors, 1 crystal filter 10.7/30, 3 coil formers with core, 1 Neosid coil, 2 RF chokes, 1 ferrite bead, 1 m each of 0.8 and 0.12 mm dia. as well as 1 mm dia. silver plated wire, 2 foil trimmers, 3 feed-through and 24 ceram. disc capacitors, 23 resistors, 1 N connector, 1 metal case | 6751 | DM 105.00 |
| Kit | Receive converter, complete with above parts | 6752 | DM 115.00 |
| **3. Indication electronics/Reference voltage generator** | | | |
| PC-board | RMG 01 | single coated, drilled, with component location plan | 6753 | DM 31.50 |
| Parts | RMG 01 | 9 OpAmps, 1 temp. compensated Z-diode, 12 switching diodes, 9 ceramic and 2 tantalum capacitors, 18 trimmer potentiometers, 44 carbon and 15 metal-film resistors | 6754 | DM 65.00 |
| Kit | Indication electronics/Reference voltage generator, complete | 6755 | DM 95.00 |
| **4. Control module** | | | |
| PC-board | RMG 02 | Double coated with through-contacts | 6756 | DM 46.00 |
| Parts | RMG 02 | 4 transistors, 19 switching and 2 Z-diodes, 6 FET-OpAmps, 2 OpAmps, 2 C-MOS ICs, 19 ceram. disc. caps., 5 tantalum and 3 aluminium electrolytic caps., 3 foil caps., 6 trimmer potentiometers, 40 carbon resistors | 6757 | DM 120.00 |
| Kit | Control module, complete with above parts | 6758 | DM 165.00 |
| **5. Mixer and variable amplifier** | | | |
| PC-board | RMG 03 | single coated, drilled, with component location plan | 6759 | DM 23.00 |
| Parts | RMG 03 | 1 ring mixer, 8 transistors, 2 Z-diodes, 1 coil kit, 1 m wire 0.3 mm diam. 3 RF chokes, 12 ceram. disc caps., 6 tantalum and 5 aluminium electrolytic caps., 4 feed-through caps., 1 trimmer potentiometer, 19 resistors, 1 metal case | 6760 | DM 130.00 |
| Kit | Mixer and variable amplifier, complete | 6761 | DM 150.00 |
6. Dual crystal oscillator
| PC-board | RMG 04 | single coated, drilled, with component location plan |
| Parts | RMG 04 | 5 transistors, 3 Schottky-diodes, 1 precision voltage regulator, 3 coil kits, 1 coil former with core, 1 two-hole core, 1 m wire each: 0.12 mm dia., 0.3 mm dia., 0.8 mm dia. silver plated, 7 RF chokes, 2 foil trimmers, 22 ceramic disc caps, 2 feed-through caps., 1 tantalum electrolytic cap., 13 resistors, 1 crystal each: 66.7 MHz and 10.72 MHz, 1 metal case |
| Art. No. | Ed. 1 + 2/83 |
|----------|--------------|
| 6762 | DM 23.00 |
| 6763 | DM 206.00 |
| 6764 | DM 225.00 |
Kit Dual crystal oscillator, complete
7. Remaining parts for the noise-measuring system
| Parts | RMG E1 | 2 each: 31-pole connectors, male and female, 1 potentiometer 5 kΩ/10 turns, 2 switching- and 2 LEDs (red), 1 toggle switch |
|-------|--------|------------------------------------------------------------------------------------------------------------------|
| Parts | RMG E2 | 1 mains transformer 220 V/2 x 15 V/2 A, 2 bridge rectifiers, 1 PC-board DK10F 028, 2 electrolytic caps. 1000 µF/40 V |
| Art. No. | Ed. 1/83 |
|----------|-----------|
| 6765 | DM 75.00 |
| 6766 | DM 67.00 |
| 6767 | DM 899.00 |
Kit Automatic Noise-figure measuring system, all parts from 1 to 7
OE 7 WMI VFO with frequency-locked loop
PC-board OE7WMI 002, 003 and VCO: single coated, with component location plan
PC-board OE7WMI 004: single coated, with component location plan
PC-board OE7WMI 005: single coated, with component location plan
PC-board OE7WMI 006: single coated, with component location plan
Metal case 7411130
Other parts on request!
DJ 3 RV Versatile IF-Module
| PC-board | DJ 3 RV 001a | double-coated, drilled |
|----------|-------------|------------------------|
| | DJ 3 RV 001b | double-coated, drilled |
| | DJ 3 RV 002 | double-coated, drilled |
| | DJ 3 RV 003 | double-coated, drilled |
| | DJ 3 RV 004 | double-coated, drilled |
| | DJ 3 RV 005 | single-coated, drilled |
Editions 4/81; 2/82; 3/82; 4/82; 1/83; 2/83
| Art. No. | Ed. 1/83 |
|----------|-----------|
| 6729 | DM 28.— |
| 6730 | DM 28.— |
| 6749 | DM 28.— |
| 6746 | DM 28.— |
| 6748 | DM 28.— |
| 6747 | DM 18.— |
| 9069 | DM 89.— |
| 9577 | DM 8.50 |
| 9501 | DM 6.50 |
| 9159 | DM 1.50 |
| 9166 | DM 2.50 |
P 8002 Power-FETs
BFQ 69 UHF transistor
Tinned-metal case 74 x 148 x 30
Toroid core R6,3K1
Coil set D41 – 2165 orange
An Amateur-Television Transmitter for Home Construction
A television transmitter built from modules described in VHF COMMUNICATIONS is shown in the above block diagram. Each function is realised on an individual PC-board. Each PC-board is built into its own tinned-metal box, which leads to a very clean operation without unwanted stray coupling and without problems caused by radiation. Each module may be aligned and tested on its own. All this encourages the home constructor since it makes it easy to understand the different functions, and it finally leads to a high-value ATV transmitter to which all possible video sources (black/white or color) may be connected.
For an amplification of the transmit power, a variety of linear amplifiers for the 70 cm band may be used (not FM «linears»!), whereby care should be taken to adjust the drive so that the output power does not exceed half the PEP value of the SSB mode.
The ATV modules listed have been published by three authors. The descriptions are detailed and will enable successful duplication. They are to be found in the following editions of VHF COMMUNICATIONS:
VHF COMMUNICATIONS 1/1973
VHF COMMUNICATIONS 2/1973
VHF COMMUNICATIONS 2/1976
VHF COMMUNICATIONS 1/1977
Individual kits:
DJ 4 LB 001a kit, complete DM 98.—
DJ 4 LB 002a kit, complete DM 99.—
DJ 4 LB 007 kit, complete DM 90.—
DJ 6 PI 003 kit, complete DM 50.—
DJ 6 PI 004 ready-to-operate DM 115.—
DJ 4 LB 003 kit, complete DM 92.—
DJ 1 JZ 002 kit, complete DM 131.50
Set of complete kits for the 70 cm ATV transmitter (without power amplifier)
comprising DJ 4 LB 001a, DJ 4 LB 002a, DJ 4 LB 007, DJ 6 PI 003,
DJ 6 PI 004, DJ 4 LB 003, DJ 1 JZ 002
DM 650.00
UKWberichte Terry D. Bittan · Jahnstr. 14 · Postfach 80 · D-8523 Baiersdorf
Tel. West Germany 9133-855. Representatives see cover page 2
VHF communications
offers ...
A System for Reception and Display of Weather-Satellite Images using a digital scan converter/storage module with 256 lines, 256 pixels and 64 grey levels
As a modern replacement for the DC3NT video processor, VHF COMMUNICATIONS is now able to offer a digital scan converter with the above mentioned features. The 2400 Hz subcarrier is fed from the VHF receiver (DC3NT 003/004), processed and stored in the scan converter, and the CCIR video output can be displayed on any suitable monitor. The scan converter itself consists of two PC-board modules and will be published in editions 4/1982 and 1/1983. We can, however already offer these modules since they are available since February 1983.
All these modules are available as kits, ready-to-operate aligned modules, or as complete equipment in cabinets (see next page).
A) A complete system as kits
| Part in the system | Described in Edition | Kit designation | Art. No. | Price DM |
|------------------------------------------------------------------------------------|-----------------------|----------------------------------|----------|----------|
| 1. Parabolic antenna, 1.1 m diam., 12 segm. to be screwed or riveted together, 3 plastic supports for radiator | 3/1979 | Set of 12 segm. + supports | 0098 | 180.00 |
| | | Riveting machine + rivets | 0105 | 93.00 |
| | | 1.7 GHz Cavity radiator kit | 0091 | 90.00 |
| 2. Low-noise amplifier for 1.7 GHz (Originally described for use at 2.4 GHz, this unit is tuned to 1.7 GHz) | 1/1980 | DJ6PI 010 | 6565 | 225.00 |
| 3. METEOSAT Converter, consisting of two modules – Output first IF = 137.5 MHz | 4/1981 1/1982 | DJ1JZ 003 DJ1JZ 004 | 6705 6714| 189.00 185.00 |
| 4. VHF Receiver, frequency range: 136 – 138 MHz; Output: 2400 Hz sub-carrier | 4/1979 1/1980 | DC3NT 003 DC3NT 004 | 6141 6145| 225.00 80.00 |
| 5. Digital scan converter (256 x 256 x 6 Bit) | 4/1982 1/1983 | YU3UMV 001 YU3UMV 002 | 6736 | 675.00 |
| 6. PAL-Color module with VHF mod. | 2/1983 | YU3UMV 003 | 6739 | 150.00 |
B) Aligned ready-to-operate PCB-modules and equipment
| Equipment | Art. No. | Price DM |
|---------------------------------------------------------------------------|----------|----------|
| Cavity radiator for above parabolic antenna | 0092 | 150.00 |
| VHF receiver for 136 – 138 MHz, DC3NT 003 | 6731 | 395.00 |
| Oscillator for VHF receiver, DC3NT 004 | 6732 | 168.00 |
| Digital scan converter (256 x 256 x 6 Bit) YU3UMV 001 + 002 | 6734 | 1150.00 |
| PAL-Color module with VHF oscillator YU3UMV 003 | 6738 | 285.00 |
C) A complete system, ready-to-operate in cabinets
| Equipment | Art. No. | Price DM |
|---------------------------------------------------------------------------|----------|----------|
| Parabolic antenna, 12 segments, riveting machine and rivets, cavity radiator, supports | | 423.00 |
| METEOSAT converter with GaAs-FET preamplifier and mixer, 2 channels, in casing | 3026 | 645.00 |
| Antenna for orbiting satellites, DJØBQ-137 (VHF COMMUNICATIONS 4/1981) | 0101 | 198.00 |
| Power combiner for above, AT-137 | 0306 | 98.00 |
| 6-channel VHF receiver in cabinet, programmed for: 137,130 / 137,300 / 137,400 / 137,500 / 137,620 / 137,850 MHz | 3300 | 1298.00 |
| Digital scan converter, 256 x 256 x 6 Bit, with control electronic, in cabinet | 6735 | 1980.00 |
| PAL-Color module with VHF oscillator and power supply, in cabinet | 6737 | 650.00 |
| Video monitor, black/white, with 31 cm C.R.T. | 3301 | 550.00 |
A new service of UKW-BERICHTE/VHF COMMUNICATIONS:
| Service | Art. No. | Price DM |
|-------------------------------------------------------------------------|----------|----------|
| All 10 editions of VHF COMMUNICATIONS containing information on weather satellite reception | 6742 | 49.00 |
| Dissemination Schedule of METEOSAT, incl. surface mail | 005D | 3.00 |
| Audio Compact Cassette with 2 x 30 minutes of selected subcarrier recordings of METEOSAT and NOAA, resp. | 6740 | 25.80 |
OUR GREATEST now with reduced dimensions!
| DISCRETE CRYSTAL FILTER | Application | MONOLITHIC EQUIVALENT with impedance transformation | MONOLITHIC EQUIVALENT without impedance transformation |
|-------------------------|-------------|--------------------------------------------------|--------------------------------------------------|
| | | Type | Termination | Case | Type | Termination | Case |
| XF-9A | SSB | XFM-9A | 500 Ω | 30 pF | 15 | XFM-9S02 | 1.8 kΩ | 3 pF | 13 |
| XF-9B | SSB | XFM-9B | 500 Ω | 30 pF | 15 | XFM-9S03 | 1.8 kΩ | 3 pF | 14 |
| XF-9C | AM | XFM-9C | 500 Ω | 30 pF | 15 | XFM-9S04 | 2.7 kΩ | 2 pF | 14 |
| XF-9D | AM | XFM-9D | 500 Ω | 30 pF | 15 | XFM-9S01 | 3.3 kΩ | 2 pF | 14 |
| XF-9E | FM | XFM-9E | 1.2 kΩ | 30 pF | 15 | XFM-9S05 | 8.2 kΩ | 0 pF | 14 |
| XF-9B01 | LSB | XFM-9B01 | 500 Ω | 30 pF | 15 | XFM-9S06 | 1.8 kΩ | 3 pF | 14 |
| XF-9B02 | USB | XFM-9B02 | 500 Ω | 30 pF | 15 | XFM-9S07 | 1.8 kΩ | 3 pF | 14 |
| XF-9B10* | SSB | — | — | — | — | XFM-9S08 | 1.8 kΩ | 3 pF | 15 |
* New: 10-Pole SSB-filter, shape factor 60 dB : 6 dB 1.5
Dual (monolithic twopole) XF-910; Bandwidth 15 kHz, $R_T = 6$ kΩ, Case 17
Matched dual pair (four pole) XF-920; Bandwidth 15 kHz, $R_T = 6$ kΩ, Case 2 x 17
DISCRIMINATOR DUALS (see VHF COMMUNICATIONS 1/1979, page 45)
for NBFM XF-909 Peak separation 28 kHz
for FSK/RTTY XF-919 Peak separation 2 kHz
CW-Filters – still in discrete technology:
| Type | 6 dB Bandwidth | Crystals | Shape-Factor | Termination | Case |
|------|----------------|----------|--------------|-------------|------|
| XF-9M | 500 Hz | 4 | 60 dB : 6 dB 4.4 | 500 Ω || 30 pF | 2 |
| XF-9NB | 500 Hz | 8 | 60 dB : 6 dB 2.2 | 500 Ω || 30 pF | 1 |
| XF-9P * | 250 Hz | 8 | 60 dB : 6 dB 2.2 | 500 Ω || 30 pF | 1 |
* New !
KRISTALLVERARBEITUNG NECKARBISCHOFSEHM GMBH
D-6924 Neckarbischofsheim · Postfach 61 · Tel. 07263/6301
|
Negative regulation of transcription of the *Saccharomyces cerevisiae* catalase T (*CTT1*) gene by cAMP is mediated by a positive control element
T.Belazzi, A.Wagner, R.Wieser, M.Schanz, G.Adam, A.Hartig and H.Ruis
Institut für Allgemeine Biochemie der Universität Wien and Ludwig Boltzmann-Forschungsstelle für Biochemie, A-1090 Wien, Austria
Communicated by G.Kreil
Transcription of the *CTT1* (catalase T) gene of *Saccharomyces cerevisiae* is controlled by oxygen via heme, by nutrients via cAMP and by heat shock. Nitrogen limitation triggers a rapid, cycloheximide-insensitive derepression of the gene. Residual derepression in a cAMP-nonresponsive mutant with attenuated protein kinase activity (*bcy1 tpk1\textsuperscript{W} tpk2 tpk3*) demonstrates the existence of an alternative, cAMP-independent nutrient signaling mechanism. Deletion analysis using *CTT1-lacZ* fusion genes revealed the contribution of multiple control elements to derepression, not all of which respond to the cAMP signal. A positive promoter element responding to negative control by cAMP was inactivated by deletion of a DNA region between base pairs $-340$ and $-364$. Upstream fragments including this element confer negative cAMP control to a *LEU2-lacZ* fusion gene. Northern analysis of *CTT1* expression in the presence or absence of heme, in *RAS2\textsuperscript{+}* (high cAMP) and *ras2* mutant (low cAMP) strains and in cells grown at low temperature (23°C) and in heat-shocked cells (37°C) shows that *CTT1* is only induced to an appreciable extent when at least two of the three factors contributing to its expression (oxidative stress signaled by heme, nutrient starvation (low cAMP) and heat stress) activate the *CTT1* promoter.
**Key words:** cAMP control element/cytosolic catalase/nutrient control/transcriptional regulation/yeast
**Introduction**
*Saccharomyces cerevisiae* cells are able to adjust their metabolic activity, their growth rate and the decision between the initiation of a new round of mitotic cell division and entry into a $G_0$-like state or into sporulation (in the case of $\alpha/\alpha$ diploid cells) to the nutritional conditions (Pringle and Hartwell, 1981). Cyclic AMP plays a crucial role in the transduction of the nutrient signal to various intracellular sites (Matsumoto et al., 1985). Whereas the nutrient sensing mechanisms of yeast cells are still unclear it has been demonstrated that *S.cerevisiae* RAS1 and RAS2 proteins are activated by a complete set of nutrients (Tatchell, 1986). These RAS proteins in turn activate adenylate cyclase and trigger an increase in cellular cAMP level when nutrients are available in sufficient amounts. As in other eukaryotes, cAMP activates protein kinase A (encoded by three genes, *TPK1*, *TPK2* and *TPK3*) by binding to its regulatory subunit, which is encoded by the *BCY1* (*SRA1*) gene. Protein kinase A-catalyzed phosphorylation of a number of proteins, most of them yet unknown, will then trigger a variety of responses on the transcriptional and metabolic level, stimulating cell growth and, at least indirectly, cell division. On the other hand, a low cAMP level signaling nutrient starvation causes an alternate pattern of transcription and metabolism and triggers entry of cells into the resting state.
As part of this pleiotropic response to nutrient conditions, a number of yeast genes encoding various stress proteins are under negative cAMP control. Their expression is low in the presence of a complete set of nutrients and is derepressed during nutrient starvation. Examples for such genes are *UBI4* encoding polyubiquitin (Tanaka et al., 1988), *CTT1* (Bissinger et al., 1989), which encodes the cytosolic catalase T (Spevak et al., 1983, Hartig and Ruis, 1986), *SSA3*, one of the HSP70 genes of yeast (Werner-Washburne et al., 1989) and *HSP12*, which encodes a 14.4 kd heat shock protein (Praekelt and Meacock, 1990).
The yeast *S.cerevisiae* produces two catalase proteins, the peroxisomal catalase A and the cytosolic catalase T (Seah and Kaplan, 1973, Seah et al., 1973, Skoneczny et al., 1988). In contrast to the *CTT1* gene, *CTA1*, the gene encoding catalase A (Cohen et al., 1985), has been shown to be under glucose repression and to be induced by fatty acids (Hörtner et al., 1982, Skoneczny et al., 1988). Transcription of both catalase genes is controlled by heme (Hörtner et al., 1982). Like other yeast genes demonstrated to be under negative cAMP control, *CTT1* was recently shown to be under heat shock control (Wieser,R., Adam,G., Wagner,A., Schüller,C., Marchler,G., Ruis,H., Krawiec,Z. and Bilinski,T., submitted for publication).
At present, the mechanisms involved in negative control of transcription by cAMP and in derepression by nutrient starvation are only poorly understood. Therefore, it is the aim of our studies to identify the factors involved in the transfer of a nutrient signal from cAMP-dependent protein kinases to the *CTT1* gene. These studies led us to the identification of a cAMP control element of this gene. The localization of this control element and its synergistic interaction with other elements of the promoter of the catalase T gene are the main topic of this paper. While these studies were in progress, a cAMP responsive transcriptional control element of the *SSA3* gene was described by Boorstein and Craig (1990).
**Results**
*CTT1* expression during nitrogen starvation
Expression of the *CTT1* gene is repressed when yeast cells are grown on complete medium (YPD). Its transcription is derepressed when cells are transferred to a medium containing limiting amounts of nitrogen (STMD). This derepression is a consequence of low cAMP-dependent protein kinase activity (Bissinger et al., 1989). In a characterization of control regions mediating this response...
Fig. 1. Plasmid pTB3 used for single copy genomic integration of \( CTTI-lacZ \) genes. pTB3 was obtained by cloning a \( CTTI-lacZ \) fusion gene (Bissinger et al., 1989) into the unique \( Sall \) site of a derivative of plasmid YIp5 (Struhl et al., 1979) obtained by deletion of a 0.4 kb EcoRI–BamHI fragment. The variable region modified in various deletion derivatives (see Figures 4 and 5) is indicated by hatching. The \( Sall \) site present in the deletion derivatives produced by linker insertion is indicated in parenthesis. Deletion derivatives were produced by replacing the small EcoRI–BamHI fragment of pTB3 by corresponding deletion fragments. Genomic integration into the \( URA3 \) locus was carried out after linearization of the plasmids with NcoI. B: BamHI; E: EcoRI; N: NcoI; P: PstI; S: Sall.
Fig. 2. Kinetics of derepression of \( CTTI-lacZ \) fusion gene after transfer from YPD to nitrogen starvation medium (STMD). Strain: TB3; circles: specific \( \beta \)-galactosidase activity; squares: absorbance of culture at 600 nm.
It was necessary to analyze whether the derepression observed is a fairly direct process or whether it is perhaps triggered by arrest of cells in a G\(_0\)-like state caused by nitrogen limitation. Furthermore, it was interesting to analyze whether derepression is entirely dependent on the RAS-cAMP pathway or whether there exists a cAMP-independent alternative signaling mechanism as in the case of other processes mediated by cAMP (Cameron et al., 1988).
\( CTTI \) expression was analyzed by single copy chromosomal integration of a \( CTTI-lacZ \) fusion gene available in plasmid pTB3 (Figure 1) or by Northern analysis of \( CTTI \) transcripts. As documented in Figure 2, derepression of \( CTTI \) expression occurs rapidly. Levels of \( \beta \)-galactosidase produced by expression of the fusion gene reach a maximum after 3 h, prior to arrest by nitrogen starvation. In agreement with this observation, high levels of \( CTTI \) transcript are observed after derepression for 30 min (Figure 3). Further experiments (data not shown) demonstrated that \( CTTI \) mRNA levels reach a saturation level after this time period. No inhibition of accumulation of \( CTTI \) transcripts by cycloheximide is observed under these conditions (Figure 3). These results indicate that derepression of the \( CTTI \) gene is not a consequence of G\(_0\) arrest, but is one of the early and immediate events occurring under conditions where yeast cells enter a resting state.
It has previously been demonstrated that the characteristic pleiotropic response of yeast cells to changing nutrient levels (Pringle and Hartwell, 1981) is not exclusively mediated by the RAS-cAMP pathway. Cameron et al. (1988) have shown that cells lacking a functional regulatory protein kinase A subunit (\( bcy1 \) mutants) can still respond to environmental changes. This became apparent when the protein kinase A activity of the mutants, which is constitutive because of the \( bcy1 \) mutation, is reduced by mutations in protein kinase A structural genes (e.g. in a \( bcy1 \) \( tpk1^{w} \) \( tpk2 \) \( tpk3 \) mutant with two protein kinase A genes inactivated by gene disruption and with a third \( TPK \) gene (\( tpk1^{w} \)) producing only low levels of protein kinase A activity). To test whether this cAMP-independent nutrient signaling pathway affects \( CTTI \) expression, \( CTTI \) mRNA levels of strains bearing the wild type allele of the \( TPK1 \) gene as sole catalytic subunit of protein kinase A and of a \( tpk1^{w} \) \( bcy1 \) mutant strain were compared (Figure 3). The results obtained show that \( CTTI \) expression on YPD medium is increased in the \( tpk1^{w} \) \( bcy1 \) mutant, apparently because of the decrease in protein kinase A activity. Nevertheless, the gene still responds significantly to nutrient starvation in the absence of a functional regulatory subunit of protein kinase A. It can be concluded therefore that \( CTTI \) expression is partly controlled by nutrient levels via a cAMP-independent signaling pathway.
**Localization of upstream sequences responding to nitrogen starvation**
To localize control regions important for cAMP-mediated or cAMP-independent nutrient control of \( CTTI \) expression, a set of deletion derivatives of pTB3 (Figure 1) was constructed. Deletions tested for their effect on transcription cover the entire region previously demonstrated to be important for expression of the gene (Spevak et al., 1986). Single copy chromosomal integrants were tested for expression of the \( CTTI-lacZ \) fusion gene on complete medium and after derepression on nitrogen starvation medium. The results summarized in Figure 4 demonstrate that a number of DNA elements contribute to derepression.
These fall into two categories: deletion of positive elements (e.g. in strains TB301, TB305 or TB350) reduces expression on nitrogen starvation medium (STMD), deletion of negative regions (e.g. in strains TB321 or TB308) mainly enhances expression on complete medium (YPD). Not all of these elements do necessarily directly respond to cAMP levels. Some might be targets of the cAMP-independent signaling pathway. Others, like the HAP1 (heme control) element (Winkler et al., 1988) deleted in strains TB301 and TB304 act synergistically with those elements mediating nutrient control (see results presented in Figure 8).
Because of the apparent complexity of the CTT1 promoter, further experiments described in this paper concentrated mainly on the localization of the DNA element(s) responding directly to the signal mediated by cAMP-dependent protein kinase. To assay for such elements, the RAS2 gene of selected strains containing pTB3 or one of its derivatives was inactivated by gene disruption. The ras2 disruption mutation reduces the cellular cAMP level on YPD medium. This leads to a (partial) derepression of CTT1 transcription in ras2 mutant strains compared to isogenic wild type cells on YPD medium (Bissinger et al., 1989). It is therefore possible to use the extent of derepression of CTT1 on YPD by a ras2 gene disruption as a fairly direct indicator for negative cAMP control of transcription of the gene. Deletion mutants, which partly or completely loose the 'up'-phenotype of ras2 strains characteristic for the wild type CTT1 gene should contain a pTB3 derivative, in which a cAMP responsive DNA element of the CTT1 promoter has been deleted. In these experiments, derepression by nitrogen starvation was also assayed. Initial experiments with ras2 disruption strains (results not shown) demonstrated that these strains arrest growth on STMD medium much later than wild type cells. Derepression on STMD for 16 h as in earlier experiments
| Strain | EcoRI | PstI |
|--------|-------|------|
| TB3 | - | 0.3 | 3.1 |
| TB358 | -497/-482 | 0.2 | 2.4 |
| TB301 | -497/-455 | 0.3 | 0.8 |
| TB304 | -471/-431 | 0.3 | 0.8 |
| TB305 | -430/-387 | 0.3 | 0.7 |
| TB347 | -379/-331 | 0.1 | 0.5 |
| TB314 | -379/-260 | 3.7 | 3.5 |
| TB350 | -364/-340 | 0.1 | 0.5 |
| TB354 | -335/-289 | 0.4 | 4.1 |
| TB352 | -320/-289 | 0.3 | 2.4 |
| TB310 | -317/-231 | 1.8 | 7.5 |
| TB321 | -313/-250 | 1.0 | 3.7 |
| TB356 | -279/-250 | 0.7 | 4.0 |
| TB308 | -279/-218 | 1.3 | 15.0 |
| TB312 | -217/-190 | 0.1 | 0.9 |
**Fig. 4.** Derepression of CTT1-lacZ fusion genes under nitrogen starvation conditions. After growth on YPD, cells were incubated on STMD (nitrogen starvation medium) for 16 h.
| Strain | EcoRI | PstI |
|--------|-------|------|
| TB3 | - | 0.2 | 1.0 |
| TB301 | -497/-455 | 0.1 | 0.4 |
| TB304 | -471/-431 | 0.2 | 0.7 |
| TB347 | -379/-331 | 0.1 | 0.5 |
| TB314 | -379/-260 | 3.7 | 3.4 |
| TB350 | -364/-340 | 0.1 | - |
| TB310 | -217/-231 | 1.1 | 4.5 |
**Fig. 5.** Effect of ras2 mutation on expression of CTT1-lacZ fusion genes. After growth on YPD, cells were incubated on STMD (nitrogen starvation medium) for 3 h.
(Figure 4) would therefore not allow a valid comparison of CTT1 expression in RAS2+ and ras2 mutant strains. Thus, in further experiments derepression was carried out for a time period of 3 h. At this time point neither RAS2+ nor ras2 mutant strains had arrested growth and were still growing at a similar rate.
A comparison of β-galactosidase expression in wild type and ras2 strains is presented in Figure 5. A group of deletion mutants exhibits a pronounced decrease in the ras2:RAS2+ ratio of β-galactosidase activities on YPD. The largest of these deletions (TB314) is characterized by an increased expression apparently caused by elimination of a negative region also detected previously (Spevak et al., 1986). The smaller two deletions (TB347, TB350) have very similar 'down'-phenotypes. This indicates the presence of one or more positive elements in the corresponding regions of the wild type gene. It can be concluded from these results that at least one positive element located within or overlapping with base pairs −340 to −364 (see deletion mutant TB350) is inactivated by cAMP.
In strain TB347, most of the ras2 response of CTT1 gene expression characteristic for the wild type gene (strain TB3) is lost. A CTT1 promoter lacking base pairs −331 to −379 is therefore not significantly controlled by cAMP. Nevertheless, the derepression of CTT1 expression by nitrogen starvation is still quite pronounced in this strain. This result demonstrates the existence of one or more DNA-elements which respond to cAMP-independent nutrient signaling and are separate from those responding to cAMP. Since derepression by nitrogen starvation in a RAS2+ background is lost completely when base pairs −260 to −379 are deleted (TB314) it appears likely that cAMP-independent nutrient responsive elements are located between base pairs −260 and −330. However, this interpretation does not explain why strain TB314 does respond to nitrogen starvation after disruption of the RAS2 gene. Further experiments are therefore necessary to locate cAMP-independent nutrient responsive elements.
Although the analysis of deletion mutants has led to the detection of a region of the CTT1 promoter important for cAMP control it does not clarify whether this region is sufficient for a response to this signal. Therefore, CTT1 upstream elements were tested in combination with a S.cerevisiae LEU2 promoter lacking its own UAS element. The LEU2-lacZ integration vector pLS9 (Sarokin and
Carlson, 1985) was used for this purpose (see Figure 6). Single copy pLS9 derivatives were integrated into the *URA3* locus and β-galactosidase was assayed in extracts from the transformed cells. UAS activities of *CTT1* upstream regions and their response to nitrogen starvation and to the *ras2* genotype were tested (Table I). The *LEU2* promoter itself does not provide any cryptic sequences mediating negative cAMP control to UAS elements tested in this context, as was shown with a synthetic heat shock element (strain AW3). It exhibits temperature-dependent UAS activity, but is equally active in the isogenic *RAS2*⁺ and *ras2* mutant strains. In contrast, those upstream fragments or synthetic oligonucleotides including the region deleted in strains TB347 and TB350 are sufficient for mediating the response to nitrogen starvation and to the *ras2* mutation to a heterologous promoter. Like other yeast UAS elements, the cAMP-responsive element is active in both orientations. Remarkably, strain MS226N, which contains the promoter region corresponding to base pairs −224 to −384 and should therefore activate transcription and respond to cAMP control, is almost entirely inactive. It is tempting to speculate that positive and negative elements in this construct cancel each other in their effects, whereas such constructs lacking the negative region of the *CTT1* promoter (e.g. AW1N) or those possessing additional positive elements (MS903) are active.
In summary, deletion analysis and sufficiency experiments have demonstrated that the *CTT1* promoter contains a positive element, which is inactivated by cAMP control. They are consistent with the assumption that such an element is located within or at least overlapping with the region characterized by the smallest deletion tested (−364/−340).
**Synergism between different types of UAS elements of the *CTT1* gene**
*CTT1* expression is induced by heat shock (Wieser et al., submitted for publication) and a pronounced interdependence of stimulation of transcription of this gene by low cAMP levels and by heat stress was noticed. This phenomenon was therefore analyzed in greater detail. The isogenic strains JC482 (wild type), JC302-26B (*ras2*) and JC303-79 (*ras2 sra1-13*) were used. The *sra1* mutation (Cannon et al., 1990) present in strain JC303-79 is one of a number of suppressors of the growth defect of *ras2* mutants on non-fermentable carbon sources (Cannon et al., 1986). It is allelic to *bcyl* mutations (protein kinase A regulatory subunit gene) (Matsumoto et al., 1985). A defect in the regulatory subunit of protein kinase A has previously been shown to repress *CTT1* transcription almost completely compared to wild type or *ras2* mutants when cells are grown at 30°C (Bissinger et al., 1989). When cells are grown at 23°C, however, catalase T levels are generally low and are virtually independent of the genotype of the three strains (Figure 7). Heat shock treatment of cells grown at 23°C restores their response to cAMP levels. Heat inducibility of catalase T is enhanced by the *ras2* mutation and is almost entirely abolished by the *sra1* mutation. Therefore, heat shock and cAMP control of *CTT1* expression are mutually interdependent: at 23°C, virtually no cAMP control is observed and heat shock induction is prevented by high protein kinase A activity. However, canonical heat shock elements (HSEs) are apparently not the targets of cAMP control: a number of genes controlled via HSEs are not under cAMP control (Werner-Washburne et al., 1989), a synthetic HSE does not respond to cAMP (Table I) and HSEs and cAMP responsive elements have been shown to be separable in the case of the *SSA3* gene (Boorstein and Craig, 1990).
---
**Table I.** UAS activities of *CTT1* upstream elements
| Strain | Insert | RAS2⁺ | ras2 | ras2/RAS2⁺ (YPD) |
|----------|-----------------|-------|------|------------------|
| | | YPD | STMD | YPD | STMD | |
| MS903N | −522/−142 | 1.4 | 15.7 | 4.7 | 25.7 | 3.4 |
| AW1N | −382/−325 | 3.1 | 24.4 | 17.0 | 37.5 | 5.5 |
| AW1I | −325/−382 | 2.2 | 9.3 | 13.7 | 19.4 | 6.2 |
| AW2X | −382/−325 | 6.3 | 65.7 | 28.9 | 111.1| 4.6 |
| MS226N | −384/−224 | 0.4 | 1.2 | 0.4 | 1.6 | 1.0 |
| | 23°C | 2.8 | – | 2.6 | – | 0.9 |
| AW3 | 30°C | HSE | 4.3 | – | 4.7 | 1.1 |
| | 37°C | 55.6 | – | 56.4 | – | 1.0 |
---
*a* After growth on YPD, cells were incubated on STMD (nitrogen starvation medium) for 3 h.
*b* Reverted orientation.
*c* Two tandem copies.
*d* Synthetic heat shock element.
*e* Cells were grown at 23°C and 30°C, respectively; one half of the culture grown at 23°C was heat shocked at 37°C for 60 min.
The mechanistic basis of the synergistic response to the two regulatory signals will have to be investigated in more detail.
In a further experiment, we have extended our investigation to include the positive regulator heme. It has previously been shown to control \( CTT1 \) expression at least partly via a HAP1 binding site located between base pairs \(-450\) and \(-470\) (Winkler et al., 1988). A second HAP1 binding site has been localized between base pairs \(-200\) and \(-220\) (unpublished results). Deletions in both regions reduce expression of the gene under nitrogen starvation conditions (Figure 4). It was therefore attractive to test whether heme control, which is at least partly exerted via HAP1, also exhibits synergism with the two other mechanisms controlling \( CTT1 \) expression. Levels of \( CTT1 \) mRNA were analyzed in two isogenic strains (\( heml \ RAS2^+ \) and \( heml \ ras2 \)). The \( HEM1 \) gene encodes \( \delta \)-aminolevulinate synthase, the enzyme catalyzing the first specific step in heme biosynthesis. The two strains were grown in the presence of concentrations of the heme precursor \( \delta \)-aminolevulinate sufficient to compensate for the defect of the \( heml \) mutant ('Heme\(^+\)') or, alternatively, under severe \( \delta \)-aminolevulinate limitation ('Heme\(^-\)'). Cells were grown at 23°C and were tested for \( CTT1 \) mRNA levels at this temperature and after a subsequent heat shock (37°C) (Figure 8). The Northern results obtained with heme-positive strains are consistent with the results obtained with another set of strains by assaying catalase T activities (Figure 7). Further, no positive heme control of the promoter is observed at 23°C and heat shock control is insignificant in the absence of heme in a \( RAS2^+ \) strain. However, the heme requirement of the promoter is lost in a \( ras2 \) strain subjected to heat shock treatment. The results presented in Figures 7 and 8 demonstrate a pronounced synergism of heme control of \( CTT1 \) expression with heat shock induction and derepression at low cAMP levels.
**Discussion**
*Mechanisms involved in signaling of nutrient levels*
Within cells of the yeast *S.cerevisiae*, nutrient levels are signaled by at least two different mechanisms. One of them, the RAS–cAMP pathway (Matsumoto et al., 1985) has been studied extensively, an alternate, cAMP-independent signal transduction pathway (Cameron et al., 1988) is presently only poorly understood. The transcription of several yeast genes has been demonstrated to be under negative control by cAMP (\( UBI4 \) (Tanaka et al., 1988); \( CTT1 \) (Bissinger et al., 1989); \( ADH2 \) (Cherry et al., 1989); \( SSA3 \) (Werner-Washburne et al., 1989); \( HSP12 \) (Praekelt and Meacock, 1990); \( IME1 \) (Matsuura et al., 1990)). In contrast, no published information exists concerning yeast genes that are under positive transcriptional control by cAMP.
The experiments described in this publication demonstrate that derepression of \( CTT1 \) transcription by nitrogen starvation is a rapid and fairly direct process. They do not necessarily imply, however, that protein kinase A directly inactivates a positive transcription factor by phosphorylation. The results obtained further demonstrate that the cAMP-independent alternative nutrient signaling pathway also contributes to transcription control of \( CTT1 \) by nutrient levels and that this cAMP-independent control occurs at least partly via (a) DNA element(s) separate from the cAMP responsive element. No
published information is available concerning cAMP-independent nutrient control of transcription of any other yeast gene.
**The cAMP responsive element of the CTT1 gene**
The results of this investigation demonstrate that a positive element of the *CTT1* promoter responding to negative control by cAMP is located between or is at least overlapping with base pairs $-340$ and $-364$ (see Figure 9). This region, which also mediates heat shock control of *CTT1* (Wieser *et al.*, submitted for publication) contains three sequence motifs that could be involved in transcriptional control: (i) a sequence similar to the cAMP-responsive PDS element consensus of the *SSA3* gene (Boorstein and Craig, 1990) is located between base pairs $-360$ and $-365$ (see Figure 9). In accordance with the assumption that this type of DNA sequence is not only active in the *SSA3* promoter, but has more general importance as a cAMP responsive UAS element, regions with sequence similarity can also be detected in the *UBI4* (Özkaynak *et al.*, 1987) and *HSP12* (Prakelt and Meacock, 1990) upstream regions. (ii) A sequence element with limited similarity to heat shock elements (HSEs) binding heat shock transcription factor is located between base pairs $-342$ and $-349$. (iii) A further region that might be important for expression of *CTT1* and other genes, which are under similar control, is the 10 bp sequence immediately downstream of the HSE-like element (see Figure 9). An identical sequence flanks the heat shock element of the *UBI4* gene at its upstream side. The relevance of the similarities of these three elements with sequences detected in other promoters remains to be investigated. In any case, cAMP control of *CTT1* is not mediated by the *ADR1* protein, the activator of the *ADH2* gene shown to be controlled by cAMP-dependent protein phosphorylation (Cherry *et al.*, 1989). In contrast to the expression of *CTT1*, *SSA3* and *UBI4*, the expression of *ADH2*, which is mainly mediated by *ADR1*, strongly responds to carbon catabolite repression. *CTT1* is not controlled by *ADR1* (G.Adam and M.Simon, unpublished results) and the region sufficient for cAMP control does not contain sequence elements with any similarity to the ADR1 binding site of the *ADH2* gene (Eisen *et al.*, 1988).
**Synergism between promoter elements of CTT1**
Several yeast genes have been demonstrated to be under transcriptional control by heat shock and by cAMP. Whereas there is no evidence for a direct mechanistic link between the two modes of control, it is plausible to postulate a functional connection, since all genes affected encode proteins, which (could) have functions under stress conditions. If the effects of heat shock and nutrient starvation on transcription are independent of each other without any positive or negative interaction, dual control probably indicates that the gene product affected is needed by the cell under two different sets of physiological conditions. *UBI4* seems to be an example of a gene induced by heat shock and derepressed when cAMP levels are low without evidence for synergism between the two regulatory mechanisms (Tanaka *et al.*, 1988). However, a different regulatory pattern should arise if a gene product is important only when a combination of two physiological conditions (e.g. heat stress combined with nutrient starvation) occurs. Synergistic action of the regulatory factors or elements involved would then be appropriate. Such a pronounced synergism is evident in the case of *CTT1*. A similar effect has also been described for the *SSA3* gene (Boorstein and Craig, 1990), but the coupling between heat shock and cAMP control appears to be much less tight for the *SSA3* gene than for *CTT1*. Other heat shock genes appear to be also under this type of synergistic control (Shin *et al.*, 1987).
Among genes controlled by both heat shock and cAMP *CTT1* appears to be a special case not so much because of the extent of synergism observed, but because of the fact that a third regulatory factor, heme, which functions as an oxygen signal and acts via the transcription activator HAP1, also participates in this synergism. A negative region of the promoter located between base pairs $-220$ and $-280$ and probably consisting of several functional elements also contributes to the synergism exhibited by the promoter. It should be mentioned that a part of this negative region corresponding to base pairs $-222$ to $-230$ of *CTT1* has been demonstrated by Luche *et al.* (1990) to be similar in sequence to a negative element present in the promoter of the yeast *CARI* gene and of a number of other yeast genes with a wide spectrum of functions. The element of the *CTT1* gene has been demonstrated by these authors to decrease expression of a heterologous yeast promoter and to form a complex with a factor also binding to the *CARI* negative element. Results of our deletion experiments show that elimination of DNA elements of the negative region causes increased expression under repressed and derepressed conditions, but has a greater effect on the repressed promoter.
At the present stage it can only be speculated why different promoters apparently sharing the same types of regulatory elements differ in the extent of synergism between these elements. It has been demonstrated by Boorstein and Craig (1990) that the positions of these elements relative to each other are not important for synergism. Therefore, a direct interaction of the transcription factors binding to these elements seems an unlikely explanation. The quantity and the quality of the different types of positive elements and their interaction with negative elements attenuating their effects on transcription initiation could be more relevant. It seems worthwhile to point out that the HSE-like element of *CTT1* deviates considerably from the classical heat shock consensus (Bienz, 1985) as well as from the sequences defined more recently as functional HSEs of *Drosophila melanogaster* (Amin *et al.*, 1988, Xiao and Lis, 1988). It has to be tested by further experiments whether this element requires synergistic interaction with other positive elements to be functional.
Independent of the mechanisms determining the observed synergism of promoter elements this effect is probably relevant for the function of the cytosolic catalase encoded by the *CTT1* gene. In many eukaryotic organisms and tissues the existence of an extraperoxisomal catalase has been controversial. Therefore, there was hardly a basis for speculation about a specific function of such an enzyme. In *S.cerevisiae*, there is not only a separate gene encoding this extraperoxisomal catalase, but control of expression of this gene differs dramatically from that of the *CTA1* gene encoding the peroxisomal catalase A, which is induced by fatty acids, is not under heat shock control and is only moderately affected by cAMP levels (Simon,M., Adam,G., Rapatz,W., Spevak,W. and Ruis,H., in press). High level expression of the *CTT1* gene requires not only the presence of oxygen (heme), which causes oxidative stress, at least
partly by formation of hydrogen peroxide, but the combination of oxidative stress with other types of stress (heat shock, nutrient starvation). Our recent studies have demonstrated that catalase T provides some protection to yeast cells under 'lethal heat shock' conditions (50°C) and that pretreatment of cells containing a functional \( CTT1 \) gene by a mild heat shock (37°C) protects these cells against exogenous hydrogen peroxide (Wieser et al., submitted for publication). These data illustrate the functional importance of the cytosolic catalase T and of its mode of regulation.
**Materials and methods**
**Yeast strains, media, growth conditions**
*S.cerevisiae* strain WS17-5D (\( \alpha \) leu2 trp1 ura3 arg1) was used as recipient for transformation with plasmids pTB3, pLS9 and their derivatives (see Figures 1 and 4) by integration into the *URA3* locus. Designations for the constructs integrated (e.g. TB3) are also used as designations for the corresponding yeast strains. Strains S7-1A (\( \alpha \) leu2 ura3 his3 trp1 ade8 TPK1\(^{+}\) tpk2::HIS3 tpk3::TRP1) and S13-58ArA (\( \alpha \) leu2 ura3 his3 trp1 ade8 bcy1::LEU2 tpk1\(^{+}\) tpk2::HIS3 tpk3::TRP1) were obtained from C. Denis. The isogenic strains JC482 (\( \alpha \) ura3 leu2 his4), JC302 – 26B (\( \alpha \) ura3 leu2 his4 ras2::LEU2) and JC303-79 (\( \alpha \) ura3 leu2 his4 ras2::LEU2 sra1-13) were obtained from K. Tatchell. Strain HP52 (\( \alpha \) leu2 trp1 ura3 his3 pep4 hem1) was converted into the isogenic strain RW13-41 (\( \alpha \) leu2 trp1 ura3 his3 pep4 hem1 ras2::LEU2) by gene disruption.
Strains were routinely grown on YPD medium (Fink, 1970) at 23°C or 30°C so an absorbance at 600 nm of 1.5 (for transfer to STMD medium) or 3 (for heat shock experiments). In nitrogen starvation experiments, cells were transferred to STMD medium (0.17% yeast nitrogen base without amino acids and ammonium sulfate, 2% glucose and limiting amounts of auxotrophic requirements (1 mg/l in the case of tryptophan, 5 mg/l in all other cases)) for the time periods indicated under 'Results'. In heat shock experiments, cells were incubated at 37°C or 39°C after growth at 23°C.
**Plasmids, DNA constructions and oligonucleotides**
The structure of plasmid pTB3 used for single copy genomic integration of \( CTT1-lacZ \) genes is illustrated in Figure 1. The mutated region of one of the plasmids constructed for deletion analysis of the \( CTT1 \) upstream region (pTB310) was derived from a corresponding multicopy plasmid of the pFI-1/1 series used in a previous analysis (Spevak et al., 1986). All other deletions are derived from products of Sall linker (5'-GGTGCACC-3'; Amersham) insertion (Tatchell et al., 1981) into a plasmid containing the \( CTT1 \) upstream region in vector pUC13. Positions of the inserted linkers and size and position of deletions created during linker insertion were assayed by DNA sequencing. Linker insertion derivatives with appropriate deletions were used directly, other deletions were produced by recombining Sall linker fragments with suitable end points. For this purpose, the small \( SalI-BamHI \) fragment of one pUC13 linker insertion derivative was replaced by another one with appropriate end point. In all cases, the small \( EcoRI-BamHI \) fragment of plasmid pTB3 was then replaced by the corresponding deletion fragment isolated from the appropriate pUC13 derivative.
Vector pLS9 (Figure 4; Sarokin and Carlson, 1985) was donated to us by M. Carlson. Fragments inserted into its unique \( EcoRI \) site were synthetic oligonucleotides with \( EcoRI \) ends in the case of AW1 and AW2 plasmids. Oligonucleotides used were kindly supplied by R. Hauptmann, Ernst Boehringer Institut für Arzneimittelforschung, Vienna. For construction of the pMS plasmids, \( CTT1 \) upstream fragments derived from linker insertion mutants were used. In the case of pMS903N, \( SalI \) ends (position –142) were treated with Klenow polymerase, \( EcoRI \) linkers (5'-GGAATTC-3'; Boehringer Mannheim) were added and the small fragment produced by cutting the resulting pUC13 derivative at the natural and at the artificial \( EcoRI \) site was cloned into pLS9. pMS226N was similarly constructed by converting a \( SalI \) site (position –384) into an \( EcoRI \) site and by adding a \( PstI-EcoRI \) adaptor (5'-CGAAATTCTGCA-3'; donated by H. Dorndey) to the free end produced by cutting the insert with \( PstI \). A derivative of plasmid pUC19 containing a synthetic heat shock element, which was donated by P. Sorger, was cut with \( PstI \), a \( PstI-EcoRI \) adaptor was again used to provide \( EcoRI \) ends and the fragment produced after cutting with \( EcoRI \) was cloned into pLS9 to obtain plasmid pAW3.
Plasmid pRa530 obtained from K. Tatchell (Tatchell et al., 1984) was used for \( LEU2 \) disruption of the \( RAS2 \) gene.
**Yeast transformation**
Yeast transformation with linear fragments (plasmids linearized with \( NcoI \) in the case of pTB3 and pLS9 derivatives, a \( HindIII-XbaI \) fragment of plasmid pRa530 in the case of \( RAS2 \) disruptions) was carried out by the method described by Beggs (1978) or by the procedure described by Ito et al. (1983). Transformants obtained by integration of derivatives of pTB3 or pLS9 into the \( URA3 \) locus were tested by hybridization (Southern, 1975) and single copy integrants were used for further analysis.
**RNA isolation and Northern analysis**
Yeast poly (A)\(^+\) RNA isolated as previously described (Richter et al., 1980) was fractionated by electrophoresis, blotted and hybridized essentially as described by Thomas (1980). Band intensity on autoradiograms was evaluated using an Elscript-400 (Hirschmann) densitometer.
**Enzyme activities**
\( \beta \)-Galactosidase activity of extracts prepared by breakage of yeast transformants with glass beads (Rose and Botstein, 1983) was assayed using o-nitrophenyl-\( \beta \)-D-galactoside as substrate (Miller, 1972). Catalase activity of extracts was assayed spectrophotometrically at 240 nm (Beers and Sizer, 1952). Protein concentrations were assayed according to Bradford (1976).
**Acknowledgements**
The authors thank Bettina Dekrout, Harald Nierlich, Adriana Oancea and Hannelore Wrba for excellent technical assistance, Barbara Hamilton for design, drawing and photography of Figures, Manuel Simon for carrying out the densitometric evaluation of autoradiograms, Christoph Schüller for valuable help during some experiments and for important discussions, and Andrea Barta for critical review of this manuscript. Financial support by grants (S29/08, P7233) from the Fonds zur Förderung der wissenschaftlichen Forschung, Vienna, Austria, and from the Bundesministerium für Wissenschaft und Forschung is gratefully acknowledged.
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|
Diamond Firetail Finch
by Matthew M. Vriends, Ph.D.
The Netherlands
The Diamond Fire-tailed Finch comes from many parts of Australia. It is commonly known to U.S. aviculturists as the Diamond Sparrow. It is not, however, related to sparrow-like birds.
Scientific names:
Emblema guttata, Stagonopleura guttata, and Zonaegnibus guttatus. The first stated name is the most recent.
Other names:
Diamond Firefinch, Diamond Finch; also frequently Diamond Sparrow (a name that is not recommended as this bird is no close relation to sparrow-like birds), and Spotted-sided Finch.
Subspecies:
None. According to J.A. Keast (1958), there are no geographical races; the species philordi, mentioned by G.M. Matthews in his book, "The Birds of Australia" (London, 1910-27) must therefore be regarded as a synonym of guttata.
Description of Wild Bird
Cock: The head and neck are grayish-blue; back and wings olive-brown; rump and upper tail coverts crimson; throat white; breast with a wide, black band that runs along the flanks and is bordered by the wings; underside whitish; flanks, black with white spots "diamonds"; the second part of the scientific name guttata means "spotted" and zona, in the already from 1796 dated name Zonaegnibus, is the Latin word for "band": the Greek aigintha is a "kind of bird." Beak maroon; the lores are black. The eyes are bordered with a conspicuous red eye-ring, the same color as the iris. The legs and feet are grayish-brown.
Hen: Is usually difficult to distinguish from the male; with enough comparative material the hen's head and body seems generally slighter in structure. The lores are brown instead of black; this is a good distinguishing mark in older birds. The eye-ring is generally lighter in color, as is the beak (pinkish-red).
Immature: Generally less colorful, with greenish-brown wings and back; the rump and upper tail coverts are carmine-red; the tail feathers are not black, but brownish-black; unlike the conspicuous ash-blue head of the adults, in juveniles it has a greenish wash, as has the sides of the head. The flanks are greenish-brown with large gray-white bars and spots; the underside of hens is light gray, in young cocks, however, much lighter, almost white. The beak of young hens at about three
months of age is generally light red; that of young cocks is dark red with a violet tinge. The rump and upper tail coverts of young hens are dull red; that of young cocks are lighter red with a pale sheen. These color differences can sometimes be seen in older birds, especially when not in breeding condition. A cock bird ready for mating, however, is easy to distinguish by his frequent singing, and his vivid-red eyerings. Should the bird moreover take a grass stem in the beak, stand high on the legs, and press the beak down against the breast, it is definitely a male. During this performance he will also let his song be heard. The cock sometimes sings outside the breeding season, but much less frequently. Also, outside the breeding season, the hen takes no notice of the male's singing; she just keeps to herself.
Apart from the male song, sexes of young birds are difficult to determine outwardly. Even the white spots on the flanks – which are sometimes larger in hens – are no sure indication. The only sure way, as we have said, is the song of the male.
**Size:** 4.5 inches (11.5 cm), sometimes a little smaller, sometimes to 4.75 inches (12 cm); tail 1.7 in (4.3 cm); wings 2.5-2.75 inches (6-7 cm).
**Distribution:** Central and southern Queensland, and via the Great Dividing Range in New South Wales, through Victoria to western South Australia (Eyre Peninsula); also on Kangaroo Island (large island south of Adelaide, South Australia).
**Field Biology:** Diamond finches live in open terrain, including grasslands, mallee thickets, gardens, parks, and open woodland, generally in the immediate neighborhood of water. I have seen these birds on Kangaroo Island close to towns, especially in gardens and parks. In Queensland I have also frequently seen them close to human habitations. In Adelaide I observed them especially in the sparsely wooded hills, where they had plenty of space to move about in. By leg-banding numbers of birds over a two year period, I ascertained that they were largely sedentary and almost never left the area in which they were hatched and reared. It seems that most pairs build a new nest in the same shrub in which they nested the previous year. I observed some pairs even repairing or rebuilding their old original nests and reusing them. It is therefore understandable that in some areas, where the habitat is in their favor, they remain all year round. Juveniles usually also stay in the area in which they were reared, breeding themselves when the time is ripe. In spite of this, continual urbanization is gradually forcing populations of the birds further inland. I observed a typical example of this when, on Kangaroo Island in 1983, Diamond Finches were forced more than 1,100 yards (about 1 km) further inland in a single year.
Diamond Finches are very strong on the wing; they fly powerfully with flowing, light undulations – this is a characteristic of birds that live in open terrain and must be quick on the wing to escape from predators. This typical kind of flight can be seen with many Australian Finches, but especially in all firetail species. However, it has to be said that the flight of the Diamond Finch is the most beautiful of all.
**Food:** Diamond Finches are mainly seedeaters. Like sparrows, the finches hop on the ground in search of seeds, but they also jump up to the heads of seeding grasses with some success. During the breeding season, the birds supplement their menu with insects which they also find on the ground or "pluck" from the foliage of plants. In captivity, Diamond Finches will be content with a commercial seed mixture as the main part of the diet, but they show preference for white and Japanese millets, especially outside the breeding season. During the breeding season sprouted seeds and seeding grass heads are taken avidly; millet sprays and bunches of seeding weeds can also be given in the aviary. It is recommended that such seed heads are offered throughout the year; if they are offered only in the breeding season it is possible that the birds will eat too much of them and become too fat. If they are given all year round it will discourage the birds from eating these seeds exclusively and they will also seek out other food. In this connection it should be noted that hemp is also one of their favorite seeds. But this should be given only sparingly, even when the
birds are in a large aviary with plenty of exercise space.
In the wild various termites, other insects and their larvae are taken by the breeding adults and fed to the nestlings. In the cage or aviary, small mealworms, or maggots can be given; these larvae should first be killed by immersing them in boiling water (use a net or a piece of old nylon pantyhose). Fruit flies and white worms (*Enchytrae*) can also be given, as can commercial egg food (Cédé, L/M's Universal Plus etc.). Grit must be available throughout the year, and a mineral block should also be available. In the wild I have seen the birds eating pieces of charcoal. In captivity I would therefore also recommend that charcoal is made available (grains of charcoal are present in most commercial grit mixtures). However, charcoal should be given very sparingly and cautiously. The necessity of a daily supply of charcoal is questionable as it is suspected of absorbing vitamins A, B2, and K from the intestinal tract. If this is correct, it can mean that charcoal can cause vitamin-deficiency disease.
During the breeding season I also like to give my birds some water-soaked brown bread, although it must be removed after a few hours as it will sour rapidly. Put egg food, soaked bread, and other quickly perishable foods in the sheltered part of the aviary so that they don't spoil too quickly. Never use milk in which to soak bread as many birds cannot tolerate it.
As in the wild, captive birds drink regularly but especially in the early hours of the morning. Unlike most birds but like Zebra Finches, Gouldian Finches, doves and pigeons, they suck the water up. This "method" works very well early in the morning, or after light rain, when dewdrops hang from the foliage; the birds can suck up the water directly from the leaves, or from a concave stone, etc. Birds that live in areas subject to drought are often able to suck up water in such a way. You may often see this in an outdoor aviary after a shower of rain; the birds gently hopping along twigs and sucking up drops of water that hang from them. I have also seen both aviary inmates and wild finches jumping up to hanging dewdrops, catching them on the tip of the beak, and swallowing as they land. In the aviary, the birds are very partial to running water; a little fountain with fresh running water is indeed a luxury for most fanciers but it will not only be good for the health and influence behavior strongly and positively, it also cuts out the danger of spoiled, infected water. In spite of this I give my Diamond Finches (and other birds) a fresh dish of water in which a dose of vitamin/mineral supplement is dissolved, twice per day.
**Calls:** Before the breeding season actually gets underway, the cock is already charming his spouse with a simple song. The song is somewhat harsh sounding and consists of a series of simple notes; I can best describe it as "qweat, qweat, qweat-the-qweat-the-qwheat" (ea as in sea); sometimes one can hear variations. In the wild, as well as in the aviary, one can hear the contact calls, not only when a group is together (usually 20-30 birds including the current year's offspring), but also when one or more birds have strayed too far away from the group and are being warned to return. Such contact calls have a nasal character, are fairly consistent but quite urgent beginning with a high tone and then gradually descending into the second syllable; "who-o-o-o-o-hee-ee-ee" (oo as in wood; ee as in heat). A similar somewhat softer call is used for contact between cock and hen. During the breeding season the call (especially near the nest), especially that of the cock, is higher and louder. The cock identifies himself with such a call when he approaches the nest, so that the hen is not alarmed. Normal contact calls are used when the birds seek shelter from the midday heat, and also evenings when they assemble to roost.
In this connection it is worth mentioning the "snoring call" first described by the ornithologist J. Welschke. This is a call uttered by one of a pair before flying from the food hopper to the nest in order to relieve his/her mate. This "snoring call" is actually uttered *before* the bird takes to the wing and the bird in the nest answers in a similar fashion. I have observed this behavior, together with the "snoring call" several times in large cages, especially in outdoor aviaries, and even once in the wild (in Queensland) whereby one of the birds, about 5 ft. from the nest (and about 12 ft. from where I was concealed) was acrobatically removing some seeds from grass heads. Once the bird finished eating, it sat dead still on the ground, and uttered a low toned, somewhat drawn-out "chrou-chrou-chrou" (ou as in ought) which was answered, almost immediately by the bird in the nest.
Welschke, as well as Immelmann, is convinced that the answer from the bird in the nest means that all is safe and well, and that the relief can take place without danger. This behavior can also be seen in the Red-eared Firetail Finch. I agree with both of these ornithologists, having observed the behavior myself several times. Immelmann is right in pointing out that this form of communication (that we know from several other species of Australian finch, including the Zebra Finch and the earlier mentioned Red-eared Firetail Finch) has evolved as a result of the long entrance tunnel to the nest, in which a bird in the nest would have absolutely no visual contact with the outside world.
In the case of a nest not yet fully completed, the bird can look outside and see possible danger in time, whereas a bird in a fully completed nest with tunnel cannot see such danger itself and requires assistance. The tunnel is thus constructed first, before the nest, and we must note further that the birds continue to add to the nest even when they have eggs or youngsters.
The so-named snoring call, is something like the somewhat harsh song of the cock, only it is not so long, and is interspersed with an "ee" (as in deep) sound: Kwee-ou-kwee-ou, kwee-ou-thee-ou-thee-kwee, and the necessary variations on this.
The lure call (which the birds use on approaching each other) of the cock is higher than that of the hen, and is held longer; it sounds like: Woo-ooh-ee-ee, woo-oh-ee-ee (oo as in too; eee as in sea).
**Courtship and Mating:** Diamond Finches are very temperamental, and this shows during courtship. In Australia they can sometimes breed throughout the year, when suitable conditions prevail. However, all year breeding of captive birds is not advised, as sooner or later you will be confronted with egg-binding, weak youngsters or other problems.
It has been determined that Diamond Finches pair up early in life and that this pair bond is sustained for life. I consider the courtship display of the Diamond Finch to be exceedingly pronounced: the cock repeatedly makes a spectacular display. Before beginning his dance, he seeks out a long stalk of grass which he then holds in his beak. Thus "kitted-out", he flies to a high, thin branch. Once there he sits bolt-upright and bobs up and down with the stem...
in his beak. (I have observed birds with stems over 4.5 ft. [135 cm] in the beak trying to balance, and even falling off the perch.) The head is stretched up as far as it will go, but with the beak pointing down towards the breast, and all the time trying not to lose the stem! Additionally, he spreads out his flank and belly feathers imposingly, and appears almost doubled in size. Once in this position he begins to make bobbing movements as his legs and feet are bent and stretched in turn, like a “bouncing ball” as described by Immelmann. His toes always hang tightly onto the twig during this performance; in other words the whole display takes place in one spot. While performing he lets out his harsh “kweet, kweet, kweet, kweet” calls. As soon as the hen reacts and comes into his sight, the cock makes the last deep movement and stretches his neck like a youngster begging for food (compare this with the well-known begging behavior of young Zebra Finches!). He also lets out his “kweet, kweet” sound while doing this. This behavior can be best observed with birds in a large outdoor aviary.
**Nest:** The Diamond Finch often builds its rough nest under the huge nests of eagles and other birds of prey. I have also found their nests among the thickly packed twigs of crows’ nests. Twice I have found nests of Diamond Finches under the eaves of a derelict barn used by cattle at night; five times in a lemon tree in a friend’s garden; eight times in a rose bush, three times in an orange tree, once in the nest of a crested hawk, *Aviceda subcristata*, and many times in eucalyptus trees infested with mistletoe. You can thus imagine that these birds are not particularly fussy with regard to nesting sites.
Generally, however, the bottle-shaped nests are found in thick shrubs or other undergrowth; sometimes in high spots, even in trees (I have found nests sometimes as high as 25 m up in trees). The nest itself has a long, tunnel-like entrance up to 3 in. (8 cm) long and the main nest is about 6 in. (15 cm), with the nest chamber about 3 in. (8 cm) in diameter. The nest is made from grass stalks and strips and similar items. Sometimes very long stalks (I have found stalks used up to 45 cm long) are used, but also thin twigs and strips of bark are used. The inner chamber is lined with soft grass and feathers. Both cock and hen continue to strengthen and repair the nest, even when there are eggs or young in it. From this you can well imagine that Diamond Finches are very active birds and in captivity must have an aviary or at least a very roomy cage otherwise they are likely to pine away. Outside the breeding season the birds are kept busy building dormitory nests or practice nests; once a number are built, the birds will unceasingly restore, repair, rebuild, strengthen, lengthen tunnels, etc. I saw several sleep/breeding nests that had an extra entrance/exit tunnel (often opposite the main entrance but sometimes in the roof or one of the sides); these were probably used as emergency exits. While Australia has a large population of snakes and lizards, these emergency exits are no luxury; a meaning held by many ornithologists. The extra tunnel is, at first, kept closed; the opening in the wall or roof is first made when the young have hatched. It is interesting to note that many African waxbills, Cordon Bleus, etc. build a similar nest (Immelmann).
**Incubation:** The clutch normally consists of 5-8, but may range from 4-9 eggs, each about 18 x 13 mm. The birds start to incubate after the second egg has been laid. The sexes take it in turn to incubate, but usually both parents incubate at night. The period of incubation is two weeks.
In their native Australia the main natural breeding season stretches from August to well into January (spring and summer), but sometimes additionally in autumn from March to the end of May, and exceptionally in winter during June and July depending on weather conditions and availability of food.
It is thus possible for a pair to rear three broods in the year. The young leave the nest at about one month of age, but sometimes as young as 21-26 days. However, they are still fed by the parents for a considerable time after fledging. They attain full adult plumage in one to three months and are then difficult to distinguish from their parents.
**Social Behavior:** As we have already said, the birds live in groups of about 30 outside the breeding season, though odd isolated pairs may still be seen occasionally. They inhabit mallee scrub, eucalyptus woodland, farmland and riverine areas. During the breeding season the groups remain in loose contact, often building their nests in close proximity, sometimes even in the same shrub or tree!
In the aviary, as we will see, the situation is quite different. Pairs are best kept singly, as males are prone to fight and we cannot afford such risks with breeding birds. It would only take two cocks to fight to destroy the harmony of all birds in the aviary.
In the wild I saw the birds in groups repeatedly drinking together, foraging for food, even helping each other out in nest building and feeding the fledglings.
---
**The Diamond Finch in Aviculture**
**Introduction:** The Diamond Finch is not really suitable as a cage bird, being a very active bird it would soon get too fat in a confined space. Trying to breed with obese birds will not be very successful. Small aviaries with numbers of other finch-like birds are also not very suitable as Diamond Finches have the nasty habit of vandalizing nests, disturbing eggs and nestlings, and generally interfering with breeding. However, I must honestly say that I have come across several pairs of Diamond Finches that were quite friendly and sociable; but strong behavioral differences mean that fanciers must keep a close eye on Diamond Finches when they are kept in community aviaries, so that any potential problems can be swiftly nipped in the bud.
Many breeders, including myself, like to place several Diamond Finches of both sexes together in a large aviary and allow them to pair up themselves. This is recommended because Diamond Finches are strongly monogamous; wild birds often pair bond, even before they have their full adult plumage. It is quite easy to follow this method if you have leg banded your birds with rings of various colors. As soon as a pair has formed it must be immediately removed to a large cage, placed out of hearing of the other Diamond Finches. The pairs can be returned to aviaries when they are completely rested and at least one year old. If you want to breed selectively (breeding mutations for example) and must place chosen pairs together, this is quite easy as long as you keep them out of sight and sound of the other birds until the pair is in breeding condition.
We will of course be using only acclimatized birds, for example, those bred in Europe (Australia has banned all exports of native birds since 1960)
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must be kept for the first 30 days in large, dry cages, observed, and gradually accustomed to the USA available seed menu before being used for breeding. Diamond Finches are not particularly difficult birds to breed and, with a little insight and knowledge, even the beginner can be successful. Incidentally, the first captive breeding results in Europe were in France about 1855, and The Netherlands from 1865, with Mr. Cornelly being the first breeder.
**Housing:** As we have said, the most ideal housing for Diamond Finches is a large, well designed aviary, so that the birds can take cover if necessary. You can keep other finches in such an aviary, but bear in mind that certain individuals can “cause trouble.” Some breeders who have an aviary at least 8 m long (26 ft.) use already formed pairs to form a colony. This will be successful, as long as we remove any “trouble-makers.” Such an aviary will house seven or eight pairs of Diamond Finches along with other finch species.
The aviary must always be dry and draft-free, protected from cold winds. In areas with prolonged rainy periods it is recommended that at least half of the outside flight is roofed over with transparent roofing material, so that the floor stays dry. The length of the aviary must always be enough to allow the birds adequate flying room, without hitting something once in flight. Medium sized shrubs, clumps of grass, and similar low plantings are recommended, so that birds can take shelter if there is any trouble.
Furthermore, it is important, as we have mentioned above, to provide a fountain with running water so that the birds always have fresh water. It would be ideal to install a sprinkler system in the open part of the flight, not only for the benefit of the plants; the birds will also use it. It is a wonderful sight seeing the little finches have a shower!
However, the floor of the aviary must not be permanently wet. Many fanciers have the sprinkler system only in the open part of the flight and set it to work in the morning say from 10-12 AM, allowing the midday sun to dry everything up again before evening, so that the birds don’t have to go to roost in a wet environment. If for some reason or other you cannot supply running water or a sprinkler system, you must give water in large, shallow, earthenware dishes so that there is no danger of the birds drowning. Such dishes must naturally be cleaned and refilled with fresh water several times per day.
An adequate temperature is important for the well-being of the birds. Temperatures below 60° F (15° C) are not well tolerated. Birds housed in outdoor quarters must therefore be taken indoors as soon as temperatures start to drop in early fall.
**Diet:** Diamond Finches may be fed on various kinds of millet and sorghum seeds, but they prefer white and Japanese millet, especially during the breeding season and in the colder months of the year. Canary grass seed, seeding grass heads both ripe and unripe, and weed seeds can all be offered. Ripe seed heads will be taken greedily if you place them in bottles of water set into the ground. Millet and canary seed can also be given in soaked or sprouted form, as well as greenfood such as lettuce, spinach, chick weed, endive, chicory and so on. Although not all birds will take them, insects are an important part of the diet; commercial rearing and eggfoods for canaries can also be given, not only in the breeding season, but throughout the year. Yet, as I have already said, I have known pairs of Diamond Finches that ignored insects as long as they had seeding grass heads available. In spite of this I recommend that insects such as the following are made available: ant pupae, small mealworms, enchytrae, maggots, spiders and similar. Fruit flies (*Drosophila*) are taken avidly; you can attract these (and other insects) with a piece of banana (or other fruit) laid in the sun and covered with mesh to stop the birds coming into contact with it. The birds will catch the insects hovering around the fruit or sitting on the mesh. The fruit should be changed every couple of days. Finally, the birds must have a mineral block, salt wheel (available commercially), cuttle fish bone, oyster grit, and some charcoal (but not too much of the latter). I give my birds stale wholegrain bread soaked in water twice a week (but beware of mold), and a good brand of vitamin/mineral supplement in powder form that I sprinkle over the bread or greenfood.
**The Breeding Period:** It is very important to provide adequate nesting material at breeding time. You must keep a good eye on the birds, who will steal materials from other nests if they don’t have enough! Such behavior can be detrimental to other broods as the thieves don’t make any distinction between empty nests or those containing eggs or young!
Cocks in breeding condition will perform their courtship dance and partners will be continually preening each other. Preening is a very important activity in the life of the Diamond Finch, and is carried out between partners at all times of the day. Newly fledged young start preening themselves and each other almost immediately. Preening is a family affair, pairs or siblings rarely carrying out with “unrelated” members of the group.
In Australia, well cared for captive Diamond Finches will breed throughout the year, but in our area we should stick to no more than three broods per annum, beginning in April or May. Many fanciers start breeding only when they are sure they have a constant supply of grass and weed seeds plus insects. The birds will show they are in breeding condition by courtship dances (by the cock) and the collecting of nesting materials. A large amount of nest material is used by Diamond Finches, often three to four times more than other Australian finches! So make sure they have enough. Experience has shown that long, pliable, grass stems are favorite for building the nest chamber, interwoven with small down feathers (chick feathers are prized). Hay, small twigs, and wool may also be offered.
The nest is bulky and usually bottle-shaped, with one entrance. Frequently one or two extra nests are built on top or on the sides for roosting purposes. Both partners build the nest. Make sure you have thick shrubs in the aviary (2-2.5 m high) (6 ft.); these will be used as nesting sites in preference to nest boxes. However, it is advisable to also offer a number of nest boxes. However, it is advisable to also offer a number of nest boxes at various sites, minimal measurements 15 x 15 x 15 cm (6 x 6
x 6 in.) – you never know!
**Clutch:** The hen lays usually 4-6 eggs (in the wild 7-9), but four young are usually reared. Both parents take turns at incubating. Diamond Finches are regarded as excellent parents, but are not tolerant of too many nest inspections, especially after the young have hatched. It is therefore recommended that youngsters are first leg banded just before they fledge. Young that leave the nest too early rarely return for the night and there is a great risk that they will succumb to the cold.
Incubation starts after the second egg is laid, and takes about 12 days. It may be prudent to point out that the eggs of first breeding pairs will do better if fostered out to Society Finches (Bengalese). I have noticed that if allowed to rear their own chicks, young pairs often abandon them after only a couple of days feeding.
In Australia, I noticed that many breeders divided a brood of Diamond Finches among two or three pairs of Society Finches (or placed them in the nests of Diamond Finches that had only one or two young), as it is impossible for these foster parents to rear a whole brood of Diamond Finches; young Diamond Finches eat a lot more than young Society Finches, so the food supply of the foster parents is inadequate to feed them all properly.
**Young:** The young are fed by both parents, and are never left alone in the nest! So, when one parent is foraging for food the other stays in the nest, only leaving when the partner has returned. Only after 12-14 days will it happen that both parents leave the young alone for short periods. After foraging, they always stay awhile in the nest, but this period also shortens and when the young are about 20 days old both parents will be busy going backwards and forwards keeping them fed! This behavior encourages the young themselves to leave the nest, especially when one of the parents sits outside with food in its beak, luring the youngster out! The strongest youngster makes the first break into the wide world. As soon as the other youngsters see this – especially when they are also hungry – they will follow suit.
Shortly after the youngsters have fledged the parents will become extremely agitated, as the fledglings do not stay easily together, but seem to disappear into every corner of the aviary. It is interesting to watch (with binoculars) how the parents try to get the young together where they will have more control over them. After a few hours they usually give up these attempts and feed their brood in a somewhat calmer manner; they continue to feed the young a variety of foods for about three weeks, before they become independent. Evenings and nights the parents and young return to the old nest or to a sleeping nest to roost. After leaving the nest early in the morning, they have a mutual preening session before going to eat and drink.
Diamond Finches remain fertile for a relatively long time; five years is about the average. They should not, however, be allowed to breed until they are at least 12 months old. Average life expectancy for a Diamond Finch is seven to eight years, providing it receives optimum housing, care and feeding.
Three color mutations are known at the present time: the recessive yellow (the conspicuous red rump of the normal form is replaced by orange), the sex-linked fawn, and obviously, the yellow split for fawn (yellow/fawn).
---
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A STUDY OF FUEL INJECTION AND MIXTURE FORMATION FOR A GASOLINE DIRECT INJECTION ENGINE
2005.
SCHOOL OF ENGINEERING
PhD Thesis
A STUDY OF FUEL INJECTION AND MIXTURE FORMATION FOR A GASOLINE DIRECT INJECTION ENGINE
Supervisor: Professor D A Greenhalgh
September 2005
This thesis is submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy
© Cranfield University, 2005. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright owner
Future requirements for lower automotive emissions have led to the development of new internal combustion (IC) engine technologies. Gasoline Direct Injection (GDI), for example, is one of these promising new IC engine concepts. It offers the opportunity of increased efficiency through unthrottled operation. However, the realization of this concept is critically dependent on the in-cylinder mixture formation, especially in the late injection/lean operation mode. Ideally, this would require a precise stratification of the in-cylinder fuel-air mixture in 3 distinct zones: an ignitable pocket located at the spark plug, surrounded by a stoichiometric mixture of fuel and air, encompassed by air. To enable this stratification, the GDI concept utilizes advanced injector technology.
Phase Doppler Anemometry (PDA), Planar Laser-Induced Fluorescence (PLIF) and the combination of PLIF and Mie scattering in the Laser-Sheet Dropsizing (LSD) technique, have been applied to sprays in the past to obtain dropsize information and study the mixture formation process. These new GDI sprays are denser, their droplet sizes are smaller and they evaporate faster, and as such, place us at the limit of the validity of these measurements techniques.
The diagnostics were applied to a GDI spray in a pressure vessel for realistic in-cylinder conditions, ranging from supercooled to superheated environments. Tracer evaporation issues in the PLIF technique were resolved by using a dual tracer system. The study showed that the LSD technique provided good quantitative data in low evaporation regimes. In highly evaporating regimes, the technique still gave reliable dropsize data for the early stages of the injection, but was limited afterwards by vapour-phase contribution to the fluorescence signal. Variations between PDA data and LSD results also suggested a deviation of the Mie scattering signal from the assumed $d^2$ dependence. This was further investigated and was found to be true for small droplets ($d/\lambda < 0.2$). This source of error might be improved by using a different observation angle. High density seriously compromises the accuracy of PDA, whilst its effect through multiple scattering is of second order for the LSD technique.
In low evaporating regimes, LSD has the overall advantage of being a 2-D measurement technique, and will yield data with a maximum error of 30% in dense parts of the spray where PDA data is totally unreliable. If the spray evaporates quickly, PLIF by itself is an appropriate tool for following the air-fuel mixture, because short droplet lifetimes limit the 2-phase flow behaviour of the spray.
Particle Image Velocimetry (PIV), the LSD technique and equivalence ratio LIF measurements were applied to a BMW single cylinder optical GDI engine. The early injection operation showed no particular issues. However, the results obtained in the late injection highlighted the poor mixing and inappropriate stratification.
Acknowledgements
First, I would like to thank Professor Douglas A. Greenhalgh for giving me the opportunity to work on this project. His scientific knowledge and insight on industry’s needs are at the source of this study. This project was made all the more interesting by the diversity of scientific fields it involved. I am truly grateful.
Dr. James Kelman was an endless source of inspiration, both scientific and humanistic. For the discussions, the suggestions, the support and his constant availability, I am eternally grateful.
I would also like to thank Bob Wilson for being a great technician and friend, and for putting up with my Triumph Spitfire. Bob sadly died on December 1st 2003, and laboratory work was never quite the same afterwards. This thesis is dedicated to him.
Many thanks also to Dr. Glenn Sherwood for being a great experimentalist and having the patience to share an office with me.
Thanks to my friends and colleagues for making life and work in Cranfield such a pleasure. A special thanks to Adam for the 3-D drawings of the pressure vessel.
Many thanks also to Dr. Joachim Höffner’s team at BMW AG in Munich. Particular thanks to Robert Eigenschenk with whom I spent most of the time in the lab. Also thanks to Dr. Matthias Hartmann for the discussions and the “nasty questions” and Dr. Martin Schenk for his knowledge of PIV and DaVis. Thanks to Peter Steil as well for arranging an evening out in ultimate driving machines for each of my stays in Munich.
The EPSRC provided the funding for this project. BMW AG supplied the optical engine, a state-of-the-art laboratory and many man-hours. I am grateful for all this financial and technical support.
Merci à mes amis, ma famille, et surtout à ma p’tite Maman, de m’avoir supporté pendant ces quelques années. Cette thèse est aussi dédicacée à Papé, mon grand-père.
And a special thanks to Bill Gates for making such wonderful operating systems…
iv
# Table of Content
Abstract .................................................................................................................. i
Acknowledgements ............................................................................................ ii
Table of Content ................................................................................................ v
Abbreviations ..................................................................................................... xi
List of Figures .................................................................................................... xiii
List of Tables ...................................................................................................... xxii
## PART I OVERVIEW AND BACKGROUND
1. Introduction ........................................................................................................ 3
1.1. Engine Basics ............................................................................................... 3
1.1.1. Emissions ............................................................................................. 3
1.1.2. Combustion Basics .............................................................................. 4
1.1.3. Engine Thermodynamics ..................................................................... 5
1.2. Lean Burn Combustion in SI engines ......................................................... 7
1.2.1. Lean Operation ..................................................................................... 7
1.2.2. Extending the Lean Operation Limit ................................................... 7
1.2.3. Stratification .......................................................................................... 9
1.3. Gasoline Direct Injection ............................................................................. 11
1.3.1. Development ......................................................................................... 11
1.3.2. Advantages of GDI .............................................................................. 12
1.3.3. GDI Combustion Chamber Concepts .................................................. 13
1.3.4. Issues .................................................................................................. 16
126.96.36.199. Emissions ...................................................................................... 16
188.8.131.52. Catalysts for GDI engines ............................................................. 17
1.4. Summary ..................................................................................................... 18
1.5. Aims of this Thesis ..................................................................................... 19
1.6. Outline of this Thesis .................................................................................. 19
2. Laser Diagnostics in Spark Ignition Engines .................................................. 21
2.1. Light Scattering Techniques ....................................................................... 21
2.1.1. Laser Rayleigh Scattering (LRS) ....................................................... 22
2.1.2. Spontaneous Raman Scattering (SRS) .......................................................... 23
2.1.3. Mie scattering .................................................................................................. 25
184.108.40.206. Total scattered signal .............................................................................. 26
220.127.116.11. Mie scattering as a function of angle ....................................................... 29
2.1.4. Planar Laser-Induced Fluorescence (PLIF) .................................................... 32
18.104.22.168. Description ................................................................................................. 32
22.214.171.124. PLIF applied to IC engines ..................................................................... 35
2.1.5. Morphology Dependent Resonances (MDR) ................................................ 39
2.2. Velocity Measurements ....................................................................................... 40
2.2.1. Laser Doppler Anemometry (LDA) ............................................................. 40
2.2.2. Particle Image Velocimetry (PIV) ................................................................ 41
2.3. Size Measurement ............................................................................................... 46
2.3.1. Laser diffraction ............................................................................................. 46
2.3.2. Interferometric Particle Imaging ................................................................. 47
2.3.3. Phase Doppler Anemometry (PDA) ............................................................ 49
2.3.4. Laser Sheet Dropsizing (LSD) .................................................................... 54
PART II DEVELOPMENT OF THE LASER SHEET DROPSIZING (LSD) TECHNIQUE 57
3. Development of Laser Sheet Dropsizing for Evaporative Sprays ......................... 59
3.1. Principle ............................................................................................................. 59
3.1.1. Early development ..................................................................................... 60
3.1.2. The Laser Sheet Dropsizing principle ....................................................... 62
3.2. Laser-Induced Fluorescence for the LSD technique ........................................ 65
3.2.1. Fluorescent Tracers .................................................................................... 65
3.2.2. Absorption measurements .......................................................................... 68
3.2.3. Temperature dependence .......................................................................... 73
3.2.4. Pressure dependence .................................................................................. 77
3.2.5. Tracer evaporation ...................................................................................... 78
126.96.36.199. Background ........................................................................................... 78
188.8.131.52. Vapour-Liquid Equilibrium Calculations ........................................... 79
184.108.40.206.1. The model ..................................................................................... 79
220.127.116.11.2. The parameters ............................................................................. 83
18.104.22.168.3. The programme ................................................................. 85
22.214.171.124. Results for single tracer systems ........................................... 88
126.96.36.199.1. 3-Pentanone ............................................................... 88
188.8.131.52.2. 2-Hexanone .............................................................. 90
184.108.40.206.3. Toluene ................................................................. 91
220.127.116.11.4. TEA ................................................................. 92
18.104.22.168.5. Conclusion .......................................................... 93
22.214.171.124. The 3-Pentanone / 2-Hexanone tracer system ....................... 93
3.3. Imaging for the LSD technique .................................................. 97
3.3.1. Imaging Optics ............................................................. 97
126.96.36.199. The stereoscopic imager ............................................ 97
188.8.131.52. The prism set-up .................................................... 98
184.108.40.206. The 2 lens set-up .................................................. 99
220.127.116.11. The 5 mirror set-up ............................................... 100
18.104.22.168. The 4 mirror set-up ............................................... 101
3.3.2. Geometric Calibration .................................................. 102
3.3.3. Calibration procedure .................................................. 102
4. Experimental Setup for LSD Validation ........................................ 105
4.1. The test rig ........................................................................ 106
4.1.1. Vessel design ............................................................ 106
4.1.2. Optical Access .......................................................... 106
4.1.3. Vessel Conditioning .................................................. 107
4.1.4. Injector ................................................................. 108
4.1.5. Fuel ........................................................................ 109
4.2. The “Mie/LIF” Pressure Vessel .............................................. 109
4.2.1. Fuel .......................................................................... 109
4.2.2. Optical access .......................................................... 110
4.2.3. Laser and sheet forming optics .................................... 110
4.2.4. Imaging system .......................................................... 111
4.2.5. Timing and Synchronisation ....................................... 112
4.2.6. Spatial Calibration .................................................... 114
4.2.7. Traverse system ....................................................... 115
4.3. PDA pressure vessel ................................................................. 116
4.3.1. Optical Access ............................................................... 116
4.3.2. Laser and beam splitter .................................................. 116
4.3.3. Measurement volumes ................................................... 117
4.3.4. Timing ............................................................................ 118
4.3.5. Traversing system ............................................................ 119
4.3.6. Tuning ............................................................................. 119
4.4. Test rig operation ................................................................. 119
4.4.1. Manual mode ................................................................. 119
4.4.2. Automatic mode .............................................................. 121
5. Measurements and Results .................................................. 125
5.1. Introduction ....................................................................... 125
5.2. LIF/Mie scatter measurements for the LSD technique ........ 127
5.2.1. Fuel/tracer systems ...................................................... 127
5.2.2. Experimental Settings .................................................. 129
5.2.3. Experimental Procedure ............................................... 130
5.2.4. Image Processing ......................................................... 130
5.3. PDA Measurements .......................................................... 132
5.3.1. Measurement grid ........................................................... 132
5.3.2. Downmix / Sampling frequency settings ...................... 134
5.3.3. Timing ........................................................................... 134
5.3.4. Measurement procedure ................................................ 135
5.3.5. Time selection ............................................................... 136
5.3.6. Data processing ............................................................. 137
5.3.7. PDA accuracy ............................................................... 139
22.214.171.124. Filtering using 2 data sets ....................................... 140
126.96.36.199. Filtering using 1 data set ......................................... 145
5.4. LSD - PDA Comparison .................................................... 147
5.4.1. Calibration Constant ..................................................... 147
5.4.2. Results .......................................................................... 149
5.5. Analysis ............................................................................. 156
5.5.1. Fluorescence yield ......................................................... 156
5.5.2. Vapour phase fluorescence ................................................................. 158
5.5.3. Evaporation regime ........................................................................... 159
5.5.4. Conclusion ......................................................................................... 161
5.5.5. PLIEF to remove the vapour contribution ........................................ 161
5.5.6. PDA – LSD discrepancies .................................................................. 163
5.6. Summary ............................................................................................... 165
PART III ENGINE SPRAY VISUALISATION MEASUREMENTS 167
6. Engine Measurements .............................................................................. 169
6.1. Experimental Set-Up ............................................................................ 169
6.1.1. Engine configuration ..................................................................... 169
6.1.2. Laser and Light Sheet Optics ....................................................... 174
6.1.3. Spatial Calibration ........................................................................ 175
6.1.4. Timing and Synchronising for the Experiment ............................ 176
6.1.5. Engine Operation Procedure .......................................................... 179
6.1.6. Image Acquisition ........................................................................ 179
6.2. LIF measurements for Equivalence ratio ............................................. 180
6.2.1. Measuring equivalence ratio using LIF ....................................... 180
6.2.2. Experimental setup ....................................................................... 183
6.2.3. Measurement procedure ............................................................... 184
6.2.4. Fluorescence yield correction ...................................................... 185
188.8.131.52. Early injection case ............................................................... 186
184.108.40.206. Late injection strategy ........................................................... 187
6.2.5. Image Processing ........................................................................ 189
6.2.6. Results ......................................................................................... 195
220.127.116.11. Early Injection ..................................................................... 195
18.104.22.168. Late Injection ....................................................................... 195
6.3. LSD measurements ............................................................................ 197
6.3.1. Experimental Setup ..................................................................... 197
6.3.2. Spatial Calibration ....................................................................... 198
6.3.3. Measurement procedure ............................................................... 198
6.3.4. Image Processing ........................................................................ 199
6.3.5. Results ......................................................................................... 205
22.214.171.124. Early Injection ................................................................. 206
126.96.36.199. Late injection ................................................................. 206
6.4. PIV Measurements .................................................................. 207
6.4.1. Experimental Setup ............................................................ 207
6.4.2. Spatial Calibration ............................................................. 208
6.4.3. PIV Settings ...................................................................... 209
6.4.4. Results ............................................................................. 210
6.4.5. Discussion ....................................................................... 215
188.8.131.52. Early injection ............................................................ 215
184.108.40.206. Late Injection ............................................................. 215
6.5. Summary .................................................................................. 216
PART IV CONCLUSION, REFERENCES AND APPENDICES ........... 217
7. Conclusion .................................................................................. 219
References ..................................................................................... 223
APPENDIX A .................................................................................. 235
APPENDIX B .................................................................................. 239
APPENDIX C .................................................................................. 241
| Abbreviation | Description |
|--------------|-------------|
| AFR | Air-to-Fuel Ratio |
| ASOI | After Start Of Injection |
| AESOI | After Electronic Start Of Injection |
| AOSOI | After Optical Start Of Injection |
| a.u. | arbitrary unit |
| CA | Crank Angle |
| cc | cubic centimetre |
| EGR | Exhaust Gas Recirculation |
| FAR | Fuel-to-Air Ratio |
| GDI | Gasoline Direct Injection |
| HC | Hydrocarbon |
| IC | Internal Combustion |
| imep | indicated mean effective pressure |
| IRO | Intensified Relay Optics |
| LDA | Laser Doppler Anemometry |
| LDV | Laser Doppler Velocimetry |
| LIF | Laser-Induced Fluorescence |
| LNT | Lean NOx Trap |
| LRS | Laser Rayleigh Scattering |
| LSD | Laser-Sheet Dropsizing |
| MDRs | Morphology Dependent Resonances |
| MP | Measurement Point |
| Nd:YAG | Neodymium-Yttrium-Aluminium-Garnet |
| NOx | Nitrous Oxyde |
| PDA | Phase Doppler Anemometry |
| PDF | Probability Density Function |
| PDS | Planar Droplet Sizing |
| PFI | Port Fuel Injection |
| PIV | Particle-Image Velocimetry |
| PLIEF | Planar Laser-Induced Exciplex Fluorescence |
| Abbreviation | Description |
|--------------|-------------|
| PLIF | Planar Laser-Induced Fluorescence |
| ppm | parts per million |
| PTU | Programmable Timing Unit |
| RPM | revolutions per minute |
| SCR | Selective Catalytic Reduction |
| SCV | Swirl Control Valve |
| SI | Spark Ignition |
| SMD | Sauter Mean Diameter |
| SRS | Stimulated Raman Scattering |
| TDC | Top Dead Centre |
| TWC | Three-Way Catalyst |
| UV | Ultra Violet |
| 2-D | Two-Dimensional |
List of Figures
Figure 1-1: Clapeyron diagram of a theoretical 4-stroke SI engine cycle 6
Figure 1-2: Clapeyron diagram of a theoretical throttled cycle 6
Figure 1-3: Swirl and Tumble flows 8
Figure 1-4: Mitsubishi MVV 3-valve tumble-guided stratified lean-burn engine 10
Figure 1-5: Cutaway section of the 1954 Mercedes 300SL direct injection engine 12
Figure 1-6: Mitsubishi GDI wall-guided system 14
Figure 1-7: Audi FSI air-guided system 15
Figure 1-8: Spray-guided system with Piezo-electric injector 15
Figure 2-1: Raman scattering energy levels: Stokes (red) and anti-Stokes (blue) 24
Figure 2-2: Scattered light from a droplet 25
Figure 2-3: Scattering efficiency for droplets up to 5µm, λ=532nm, m=1.44 (Le Gal 1999) 27
Figure 2-4: Scattering efficiency, λ=266nm, m=1.4 28
Figure 2-5: Polar diagram (log scale) of Mie scattering for a 10µm droplet 29
Figure 2-6: Mie scattering observed at an angle 30
Figure 2-7: Computed variation of scattered light intensity with diameter for a 90° collection angle and no absorption (Sankar et al. 1997) 31
Figure 2-8: Computed variation of scattered light intensity with diameter for a 90° collection angle and weak absorption (Sankar et al. 1997) 31
Figure 2-9: Two energy level diagram for molecules excited by a laser source (Seitzman and Hanson 1993) 33
Figure 2-10: Spectra of 10% Naphtalene, 1% TMPD, 89% cetane mixtures in the ligand and vapour phases - T=220°C (Melton 1983) 38
Figure 2-11: Spatial distribution of the internal intensity in a microsphere (Serpengüzel et al. 1992) 39
Figure 2-12: Interference fringes 40
Figure 2-13: Light modulation from a droplet crossing the fringe pattern 41
Figure 2-14 – Single frame - double exposure and Auto-correlation analysis 43
Figure 2-15 – Double frame – double exposure and Cross-correlation analysis 43
Figure 2-16: Measurement volume for the line-of-sight scattering technique 46
| Figure Description | Page |
|------------------------------------------------------------------------------------|------|
| Figure 2-17: Interference pattern in out-of-focus imaging of droplets | 47 |
| Figure 2-18: Interferometric image (Maeda et al. 2000) using | 49 |
| Figure 2-19: PDA sizing principle (figure is not to scale: in practice, R>>f) | 50 |
| Figure 2-20: Calibration curves for detectors' phase shifts (1-2 and 1-3) | 52 |
| Figure 2-21: Trajectory effect due to beam profile | 52 |
| Figure 2-22: The slit effect | 53 |
| Figure 2-23: Dantec Dual-mode PDA | 54 |
| Figure 3-1: Light scattering from an absorbing droplet | 59 |
| Figure 3-2: Experiment schematics - Yeh et al. (1993a). | 60 |
| Figure 3-3: Droplet distribution of the spray, as seen by the CCD chip | 63 |
| Figure 3-4: Iso-octane | 67 |
| Figure 3-5: Toluene | 67 |
| Figure 3-6: Fluorobenzene | 67 |
| Figure 3-7: 3-Pentanone | 68 |
| Figure 3-8: TEA | 68 |
| Figure 3-9: 2-Hexanone | 68 |
| Figure 3-10: Light extinction for different absorbances | 69 |
| Figure 3-11: Droplet illumination for increasing absorptions | 70 |
| Figure 3-12: Absorption measurement for 1% 3-Pentanone in Iso-Octane | 70 |
| Figure 3-13 - Absorption measurements for 0.3% Fluorobenzene in Iso-Octane | 71 |
| Figure 3-14 - Absorption measurements for 0.2% Toluene in Iso-Octane | 71 |
| Figure 3-15: Absorption measurements for 2-Hexanone in Iso-Octane | 71 |
| Figure 3-16: Absorption measurements for TEA in Iso-Octane | 72 |
| Figure 3-17: Brass container, heaters and cuvette | 73 |
| Figure 3-18: Experimental set-up for temperature dependence of LIF | 73 |
| Figure 3-19: Transmittance curve for the BG37 filter | 74 |
| Figure 3-20: Fluorescence in the cuvette - rectangle | 74 |
| Figure 3-21: Intensity variation in 2 different rectangles | 75 |
| Figure 3-22: 3-Pentanone - temperature dependence of fluorescence | 75 |
| Figure 3-23: 2-Hexanone - temperature dependence of fluorescence | 75 |
| Figure 3-24: Toluene - temperature dependence of fluorescence | 76 |
| Figure 3-25: Fluorobenzene - temperature dependence of fluorescence | 76 |
| Figure 3-26: TEA - temperature dependence of fluorescence | 76 |
| Figure 3-27: Fluorescence intensity profile across the cuvette for 3-Pentanone | 77 |
| Figure | Description | Page |
|--------|-----------------------------------------------------------------------------|------|
| 3-28 | Saturation curve for Toluene | 84 |
| 3-29 | Saturation curves for various compounds | 85 |
| 3-30 | Programme structure for the distillation calculations. | 86 |
| 3-31 | Vapour-liquid equilibrium diagram for the 3-Pentanone/Iso-Octane system at 50°C | 86 |
| 3-32 | Programme structure for the calculation of fluorescence variation in an evaporating droplet | 87 |
| 3-33 | Liquid-vapour equilibrium of 3-Pentanone in Iso-Octane for different temperatures | 88 |
| 3-34 | Fluorescence vs. Volume for an evaporating droplet containing 2% 3-Pentanone | 89 |
| 3-35 | Evaporation of 2-Hexanone in Iso-Octane for different temperatures | 90 |
| 3-36 | Fluorescence vs. Volume for an evaporating droplet containing 2% 2-Hexanone | 90 |
| 3-37 | Evaporation of Toluene in Iso-Octane for different temperatures | 91 |
| 3-38 | Fluorescence vs. Volume for an evaporating droplet containing 0.3% Toluene | 91 |
| 3-39 | Evaporation of TEA in Iso-Octane for different temperatures | 92 |
| 3-40 | Fluorescence vs. Volume for an evaporating droplet containing 3% TEA | 92 |
| 3-41 | Fluorescence vs. Volume for an evaporating droplet at 50°C containing various 3-Pentanone / 2-Hexanone mixtures in 98% Iso-octane | 94 |
| 3-42 | Fluorescence vs. Volume for an evaporating droplet at 100°C containing various 3-Pentanone / 2-Hexanone mixtures in 98% Iso-octane | 95 |
| 3-43 | Fluorescence vs. Volume for an evaporating droplet at 150°C containing various 3-Pentanone / 2-Hexanone mixtures in 98% Iso-octane | 96 |
| 3-44 | Fluorescence vs. Volume for an evaporating droplet at 200°C containing various 3-Pentanone / 2-Hexanone mixtures in 98% Iso-octane | 96 |
| 3-45 | The stereoscopic beam splitter with its filters | 97 |
| Figure Description | Page |
|------------------------------------------------------------------------------------|------|
| Figure 3-46: The stereoscopic imager, lens, intensifier and camera set-up | 98 |
| Figure 3-47: The prism separation | 98 |
| Figure 3-48: The 2-lens set-up | 99 |
| Figure 3-49: Stereoscopic imaging - Different light paths in dense sprays | 100 |
| Figure 3-50: The 5-mirror set-up | 100 |
| Figure 3-51: The 4-mirror set-up | 101 |
| Figure 3-52: Original image and separated images on the two parts of the chip | 102 |
| Figure 4-1: View of the 2 Pressure Vessel rigs (Photo: BMW AG) | 105 |
| Figure 4-2: Cutaway section of the Mie/LIF Pressure vessel - inner/outer cylinder, windows and window holders | 107 |
| Figure 4-3: Schematics of the pressure vessel | 108 |
| Figure 4-4: The “Mie/LIF” pressure vessel and its 3 side windows | 109 |
| Figure 4-5: The laser set-up | 111 |
| Figure 4-6: Imaging Set-up (Photo: BMW AG) | 112 |
| Figure 4-7: Window for the imaging timings | 113 |
| Figure 4-8: PTU synchronisation sequence (not to scale) | 113 |
| Figure 4-9: Calibration plate with the adjustable aluminium diffuser plate | 114 |
| Figure 4-10: Calibration images for the Mie path (left) and the LIF path (right) | 114 |
| Figure 4-11: Schematics of the LSD setup | 115 |
| Figure 4-12: View of the PDA pressure vessel | 116 |
| Figure 4-13: Schematics of the PDA vessel | 118 |
| Figure 4-14: Injector Trigger | 120 |
| Figure 4-15: Pressure and temperature settings in the manual mode | 120 |
| Figure 4-16: Fuel Pump schematics and parameters in the manual mode | 121 |
| Figure 4-17: Automatic mode - vessel parameters | 122 |
| Figure 4-18: Schematics of the Master-Slave computer communication in the automatic mode | 123 |
| Figure 5-1: Saturated Pressures of various compounds - Engine conditions | 125 |
| Figure 5-2: Injector $\alpha$ and $\gamma$ angles | 126 |
| Figure 5-3: Transmittance of the BG4 and GG400 filters and their combination | 128 |
| Figure 5-4: Image processing of the Mie scatter (left) and LIF (right) images | 131 |
| Figure 5-5: Measurement half planes (red) for the LSD and PDA techniques | 132 |
| Figure 5-6: Measurement points in the grid | 133 |
| Figure 5-7: Trigger and Temporal filter (blue) for the PDA data acquisition | 134 |
| Figure | Description | Page |
|--------|-----------------------------------------------------------------------------|------|
| 5-8 | Example of a PDA data file for 1 measurement point | 135 |
| 5-9 | PDA measurement volume | 136 |
| 5-10 | Cutaway section of the measurement volumes | 137 |
| 5-11 | Program Structure for PDA data Processing | 138 |
| 5-12 | TSI-Dantec SMD variations for all measurement points | 139 |
| 5-13 | TSI-Dantec SMD variations for all measurement points with $\Delta \phi < 15\%$ | 140 |
| 5-14 | PDF for MP 19 (y=12, z=20) at 0.7ms ASOI | 141 |
| 5-15 | PDF for MP 49 (y=16, z=30) at 1.2ms ASOI | 141 |
| 5-16 | PDF for MP 2 (y=0, z=10) at 2.2ms ASOI | 142 |
| 5-17 | PDF for MP 62 (y=8, z=60) at 2.7ms ASOI | 142 |
| 5-18 | PDF for MP 1 (y=0, z=5) at 2.2ms ASOI | 142 |
| 5-19 | PDF for MP 31 (y=12, z=35) at 1.7ms ASOI | 143 |
| 5-20 | PDF for MP 14 (y=12, z=25) at 2.7ms ASOI | 143 |
| 5-21 | PDF for MP 60 (y=12, z=55) at 2.7ms ASOI | 144 |
| 5-22 | PDF for MP 12 (y=4, z=25) at 2.7ms ASOI | 144 |
| 5-23 | TSI-Dantec SMD comparison | |
| | $\Delta SMD < 15\% - Samples > 60 droplets - \Delta \phi < 15\%$ | 145 |
| 5-24 | TSI-Dantec SMD comparison | |
| | Samples > 60 droplets - $\Delta \phi < 15\%$ | 145 |
| 5-25 | PDF for MP 32 (y=16, z=35) at 2.7ms ASOI | 146 |
| 5-26 | PDA-LSD comparison at 1bar – 25°C | 150 |
| 5-27 | PDA-LSD comparison at 3bar – 135°C | 151 |
| 5-28 | PDA-LSD comparison at 5bar – 195°C | 152 |
| 5-29 | PDA-LSD comparison at 10bar – 295°C | 153 |
| 5-30 | PDA-LSD comparison at 15bar – 360°C | 154 |
| 5-31 | Temperature dependence of 3-Pentanone Fluorescence in atmospheric Nitrogen - Koch and Hanson (2003) | 157 |
| 5-32 | Fluorescence and Temperature for a 266nm excitation wavelength Han and Steeper (2002) | 157 |
| 5-33 | LIF and Mie scatter images at 5bar and 195°C | 158 |
| 5-34 | Vapour contribution in the estimation of the SMD | 159 |
| 5-35 | Differential evaporation and vapour phase contribution | 160 |
| Figure | Description | Page |
|--------|-----------------------------------------------------------------------------|------|
| 5-36 | Liquid-vapour emission spectra for the LIEF - Fröba et al. (1998) | 162 |
| 5-37 | Relative fluorescence intensities of the TEA/Benzene exciplex and monomer in Iso-Octane (Kornmesser et al. 2001) | 162 |
| 5-38 | Fluorescence and Mie scatter intensity as a function of diameter (Domann and Hardalupas 2003) | 164 |
| 5-39 | Calibration constant for monodisperse droplets (Domann and Hardalupas 2003) | 164 |
| 5-40 | Error in Measured SMD for different size distributions (Domann and Hardalupas 2003) | 165 |
| 6-1 | Single cylinder optical research engine | 170 |
| 6-2 | Complete Optical Engine | 171 |
| 6-3 | Elongated and hollow piston | 171 |
| 6-4 | Engine block and fused silica crown | 171 |
| 6-5 | Piston head | 171 |
| 6-6 | A view of the cylinder head configuration | 172 |
| 6-7 | Fuel Pump and Pressure Regulator | 173 |
| 6-8 | Air filter, intake, plenum chamber and calibration injector | 173 |
| 6-9 | Sheet-forming Optics | 174 |
| 6-10 | Laser sheet target and illumination | 175 |
| 6-11 | Positioning plate and Calibration plate | 175 |
| 6-12 | The cam sensor and single tooth | 176 |
| 6-13 | Schematic of the instrumentation for laser diagnostics in the engine | 177 |
| 6-14 | Synchronising the Laser with the Engine trigger signal | 178 |
| 6-15 | Timing sequence for the laser | 178 |
| 6-16 | User-PC interface for the measurements | 179 |
| 6-17 | Content of fuel and air in the local measurement volume | 181 |
| 6-18 | Imaging Setup for the Equivalence Ratio measurement | 183 |
| 6-19 | Enthalpy change for early injection | 186 |
| 6-20 | Change in enthalpy for late injection | 187 |
| 6-21 | In-cylinder Temperatures during the compression stroke | 188 |
| 6-22 | Ratio of fluorescence yield during the compression stroke | 189 |
| 6-23 | Equivalence ratio and its fluctuations (in%) in the vertical plane in the centre of the combustion chamber. Early Injection (SOI at |
420°CA) and Late Injection (SOI at 670°CA). 360°CA is the exhaust/inlet TDC and 0°/720°CA is the compression/combustion TDC.
Figure 6-24: Imaging Setup for the LSD technique
Figure 6-25: LIF (left) and Mie scatter (right) images of the calibration plate
Figure 6-26: SMD image from the 70°/0° Bosch Swirl Injector
Figure 6-27: Early Injection (SOI: 420°CA) - LIF and Mie scatter images (Average and Rms) presented in a logarithmic scale. The SMD image is a calibrated LSD image presented in linear scale.
Figure 6-28: Late Injection (SOI: 670°CA) - LIF and Mie scatter images (Average and Rms) presented in a logarithmic scale. The SMD image is a calibrated LSD image presented in linear scale.
Figure 6-29: Injector Opening and Spray Regime
Figure 6-30: PIV Imaging Setup
Figure 6-31: Triggering scheme for cross-correlation PIV
Figure 6-32: Interrogation regions of the spray – non dense (a) and dense (b)
Figure 6-33: Calibration Image
Figure 6-34: Single Shot PIV Images (32×32), 2-pass
Figure 6-35: Velocities and corresponding Average Mie scatter images for the Early and Late injection cases. The vector fields are presented with a grey scale Mie scatter image in the background.
| Table | Description | Page |
|-------|-----------------------------------------------------------------------------|------|
| 3-1 | Properties of Iso-Octane and various tracers | 67 |
| 3-2 | Tracer concentrations for an optimal absorbance | 72 |
| 3-3 | MOSCED parameters for various compounds at 20°C | 83 |
| 3-4 | Vapour pressures (in bar) at 4 different temperatures | 85 |
| 4-1 | PDA parameters | 117 |
| 5-1 | Filters for the different tracer systems | 128 |
| 5-2 | Injection and imaging parameters | 129 |
| 5-3 | Grid size and measurement points | 132 |
| 5-4 | PDA data points for Samples > 60 droplets and $\Delta \phi < 15\%$ | 146 |
| 5-5 | Calibration Constant for the different tracer systems | 149 |
| 6-1 | Operating points for the GDI engine | 173 |
| 6-2 | Operating Parameters for the equivalence ratio measurements | 185 |
| 6-3 | Thermodynamic values at 30°C for the early injection case | 187 |
| 6-4 | Thermodynamic values for the late injection case | 188 |
| 6-5 | Displacements in pixels for various velocities and $dt$ | 209 |
PART I
OVERVIEW AND BACKGROUND
Chapter 1
Introduction
In this chapter, the concept of direct-injection spark-ignition engines is introduced, along with its potential to increase efficiency and therefore reduce emissions, especially in the stratified/lean burn mode. First, a brief review of thermodynamics and combustion fundamentals relevant to lean burn and direct-injection are given. This is followed by the description of several combustion concepts for direct injection and the issues that this technology faces. The objectives of the work and an outline of the thesis complete the chapter.
1.1. Engine Basics
1.1.1. Emissions
Irremediably, the exhaust gases of a gasoline engine contain mainly water (H$_2$O) and carbon dioxide (CO$_2$), with small amounts of nitrogen oxides (NOx), carbon monoxide (CO) and unburned hydrocarbons (UHC).
Before 1970, no legislation limited the amount of any of these emissions. However, as CO and UHC became known as a risk to human health, the USA was the first to limit these sources of pollution through legislation. More accurate control of the fuel was sufficient to avoid these emissions.
Later on, NOx was discovered to be the source of smog when it interacts with sunlight and HC. Exhaust gas after-treatment with 3-way catalysts (TWC) allowed to meet the new NOx and HC regulations.
More recently, CO$_2$ has been regarded as a source of pollution, because it is a greenhouse gas which is considered to contribute to global warming. The European Commission has undertaken to reduce CO$_2$ emissions by 8% between 1990 and 2012, in compliance with the Kyoto protocol. With this in mind, the main automotive companies are aiming to limit average CO$_2$ emissions from vehicles sold in Europe to 140 g/km in 2008 (compared with 164 g/km in 2002) and a further reduction to 120 g/km by 2012 is also being considered. For a petrol engine, CO$_2$ emissions can only be reduced by improving the fuel consumption. Because the engine manufacturers do not want to compromise on engine performance, they have only one solution: increase the gasoline engine’s efficiency.
### 1.1.2. Combustion Basics
By definition, an air/fuel mixture is stoichiometric when the exact amount of air is supplied to burn the fuel completely. In theory, the end products of a stoichiometric combustion of a hydrocarbon fuel in air will be CO$_2$, H$_2$O and N$_2$.
$$C_xH_y + \left(\frac{x}{2} + \frac{y}{4}\right)(O_2 + 3.76 \cdot N_2) \rightarrow xCO_2 + \frac{y}{2}H_2O + 3.76 \cdot \left(\frac{x}{2} + \frac{y}{4}\right)N_2$$
**Equation 1-1: Ideal stoichiometric combustion of a hydrocarbon fuel**
In practise, the fuel might not have sufficient time to burn completely before the exhaust valve(s) open, especially in the transient conditions, when air-to-fuel ratio (AFR), spark timing and Exhaust Gas Recirculation (EGR) may not be properly matched. Quenching of the flame in the later phase of the expansion stroke - when the combustion is especially slow - is a source of unburned hydrocarbon and carbon monoxide emissions. Another source of UHC emissions is engine oil left in a thin film on the cylinder wall, piston and cylinder head. These oil layers can absorb and desorb hydrocarbons before and after combustion thus permitting a fraction of fuel to escape the combustion process unburned. Fuel trapped in crevices too narrow for the flame is also a source of UHC (Heywood 1988).
Also, for combustion temperatures above 2000K, CO$_2$, O$_2$ and H$_2$O molecules are dissociated to CO, O and H$_2$. The molecular oxygen then reacts with the nitrogen of the
air. This leads to the formation of small quantities of nitrogen oxides, mainly of nitric oxide (NO) and small amounts of nitrogen dioxide (NO₂). The formation of NO increases with the temperature of the burnt gases.
Catalytic converters can remove NO from the burnt gases by reduction using CO and H₂. They also oxidise CO and hydrocarbons into CO₂ and H₂O.
Today’s engines are all equipped with 3-way catalysts to reduce the unwanted CO, UHC and NOx. However, these catalysts require a stoichiometric mixture for optimal conversion. If an engine is going to be run lean, the oxygen-rich environment will not allow an efficient reduction of NOx. Therefore, the engine has to run sufficiently lean in order to form only acceptable quantities of nitrous oxides.
### 1.1.3. Engine Thermodynamics
In 1862, French engineer Alphonse Beau de Rochas patented the principle of a 4-stroke internal combustion engine with a cycle consisting of 2 isentropic and 2 isochoric curves. His idea was that more work could be retrieved if the air/fuel mixture was compressed before the combustion. 14 years later, German engineer Nicolaus Otto built the engine, known as the spark ignition engine.
The cycle is illustrated in Figure 1-1:
- 1 → 2 Constant pressure intake
- 2 → 3 Adiabatic and reversible compression
- 3 → 4 Adiabatic combustion at constant volume
- 4 → 5 Adiabatic and reversible expansion
- 5 → 6 → 7 Exhaust
The efficiency of this theoretical cycle is:
\[
\eta = \frac{W_e}{m_f \cdot Q} = 1 - \frac{1}{r_c^{\gamma-1}}
\]
where \( r_c \) is the compression ratio and \( \gamma \) is the specific heat ratio. \( W_e \) is the recovered work, and is represented by the blue area.
Equation 1-2 states that the efficiency of the cycle can be improved by increasing the compression ratio, and by decreasing the specific heat ratio.
Figure 1-1: Clapeyron diagram of a theoretical 4-stroke SI engine cycle
Most petrol engines operate with homogeneous, near stoichiometric air/fuel mixture in order to perform the complete combustion of the pre-mixed charge. To run such an engine at part load, the amount of fuel is reduced. The amount of air must also be reduced to keep the mixture stoichiometric. That is usually done by throttling with a butterfly valve. This throttling induces pumping losses - represented by the green area of the PV diagram of a throttled cycle (see Figure 1-2).
Figure 1-2: Clapeyron diagram of a theoretical throttled cycle
If an engine could be operated at part load without throttling the air, its efficiency would be increased. However, not throttling the air implies that the engine must run on an overall lean air/fuel mixture.
1.2. Lean Burn Combustion in SI engines
1.2.1. Lean Operation
Typically, the stoichiometric air-to-fuel ratio (AFR) is 14.6 for petrol engines. An engine will run with a homogeneous lean charge, within the lean operation limit. Beyond that limit, the mixture will fail to ignite, leading to unacceptable UHC emissions, and cycle-to-cycle fluctuations in torque which will affect the driveability and smoothness.
However, even within that limit, as the mixture is leaned out, the time to establish the flame kernel, the flame propagation period and the fluctuations in indicated mean effective pressure (imep) all increase (Young 1981). For slow burning cycles, the mixture might not be completely burnt before the exhaust valve opens, leading to a rapid increase in UHC emissions and fuel consumption (Quader 1976). Therefore, the slowest burning cycle actually defines the practical (or stable) lean operation limit for a homogeneously lean operating engine.
Furthermore, in order to optimise torque in SI engines, the bulk of the heat release must occur at a specific time in the cycle. The variations in flame propagation implies that for faster burning cycles, the heat release will occur too soon, and for the slower burning cycles it will occur too late. This leads to a reduction of torque and power, thus a reduction in efficiency.
Further dilution may cause flame extinction before the exhaust valve is open, or before the flame has propagated across the chamber.
1.2.2. Extending the Lean Operation Limit
As seen previously, leaning out the mixture reduces flame propagation speed: the slower burning rates limit the engine’s lean operation. The factors that influence the flame development and propagation are:
- compression ratio
- spark location
- mixture formation
- turbulence level
They have the potential to extend the lean operation limit.
Increasing the compression ratio increases the charge’s temperature, thus helping combustion. It also reduces the fraction of residual gases, which limit the lean operation (Quader 1974). However, the compression ratio can only be increased to the knock limit. This is determined by the engine’s geometry and the fuel’s octane rating.
Rapid combustion can also be helped by placing the spark plug in a central position, or adopting a twin sparkplug strategy.
Increasing the turbulence level will increase the burning rate, provided that the flame is not extinguished through too much stretching or increased heat transfer. A practical way of creating turbulence in the cylinder is by introducing kinetic energy during the intake stroke, which will be converted to turbulence during the compression stroke.
Swirl, or air that rotates around the cylinder axis, will result in turbulence generation throughout the cycle (Liou and Santavicca 1983, Hall and Bracco 1987). Swirl can be generated by helical inlet ports (like the ones in the Honda V-TEC engine), by shrouded valves or by asymmetric inlet (Pajot 2000). Above a certain level, swirl does not significantly improve the lean limit (Inoue et al. 1993).
Tumble, or air that rotates round a horizontal axis, generates turbulence in the second part of the compression stroke (Haddad and Denbratt 1991, Kiyota et al. 1992). Tumble can be obtained with 4-valve configurations.
 
**Figure 1-3: Swirl and Tumble flows**
Squish is also a source of turbulence which helps the development of the flame, as long as the spark plug is located in the squish flow (Gatowski and Heywood 1985). Tumble
ratios above 2.5 tend to increase misfires and imep fluctuations, for part load and ultra lean mixtures (Iwamoto et al. 1992).
For a fixed valve timing and lift, turbulence is a function of engine speed. If adequate levels of turbulence are generated at low engine speeds, this may lead to excessive turbulence levels at high RPM. High fluctuations in flow might then cause flame extinction due to excessive stretch.
The issue with lean burning is the flame development. The variations in flame development are mainly caused by variations in laminar flame speed (Keck et al. 1987). Flame speed is a function of the ignition process and mixture strength influences the ignition (Pischinger and Heywood 1988, Le Coz 1992).
The lean operation for homogeneous mixtures is therefore limited by the weak mixture at ignition. This leads to the concept of fuel stratification in order to produce an ignitable pocket near the spark plug at the time of ignition.
### 1.2.3. Stratification
Three Japanese manufacturers developed lean burn engines. All used the same strategy: at low RPM and load, the engine runs with a lean air/fuel ratio for maximum fuel economy. As the combustion temperature is low, NOx output is also low: the catalyst just oxidises HC and CO. At higher load, the engine reverts to homogeneous stoichiometric mixing for increased power and the catalyst also reduces the NOx.
Toyota’s lean-burn engine operates with an AFR of 25 below 4800 RPM and below 75% of maximum torque. At higher speed and load, it reverts back to a stoichiometric mixture, and runs at full load with an AFR of 12.5. The cylinder head features a 4-valve per cylinder pent-roof configuration with separated dual intake ports. One is a helical port shape, and the other is straight, containing a swirl control valve (SCV). To increase volumetric efficiency, a small passage between the two ports allows a small air flow to the straight when the SCV is closed. A twin spray fuel injector injects into both ports. It is claimed that the fuel is finely atomised by the swirling flow in the helical port and the high speed flow in the straight port, resulting in a homogeneous charge.
The Honda V-TEC engine follows the same concept: it operates with an AFR of 22 below 3000 RPM and at low load. At high load or above 3000 RPM, the engine reverts to homogeneous stoichiometric combustion. The engine also has a 4-valve pent-roof head with a central spark plug. One of the inlet valve controls a high swirl port, in order to insure high turbulence. In the lean mode, only this inlet valve opens fully, while the other opens about 1mm briefly to avoid fuel build-up. At full load, both valves open fully for good volumetric efficiency. With double injection in the lean operation mode, it is claimed that the charge is stratified near the spark plug at ignition (Horie et al. 1993, Hardalupas et al. 1995, Berckmüller 1996).
Although both lean burn port-injection engines follow the same strategy (i.e. swirl intake and sequential injection), it is noteworthy that Toyota’s engine is claimed to be a homogeneous charge engine (Shimuzu et al. 1992) while Honda’s engine is stratified charged. However, Berckmüller (1996) signals that the measurements on the Toyota engine can be deceptive as only 1 region was measured.
In 1992, Mitsubishi began producing the Mitsubishi MVV (Mitsubishi Vertical Vortex) 3-valve per cylinder engine. It used the combination of tumble and injection of fuel through one port to stratify the mixture. It is claimed that the absence of swirl maintains the stratification during the compression stroke. The exhaust valve situated opposite the fuel intake valve is replaced by the spark plug, thus positioning the latter near the axially stratified charge (see Figure 1-4). Using tumble instead of swirl reduces the pumping losses. The lean-burn MVV engine can achieve complete combustion with an air-fuel ratio as high as 25.
**Figure 1-4:** Mitsubishi MVV 3-valve tumble-guided stratified lean-burn engine
One of port fuel-injection’s (PFI) limitations is that the fuel can only enter the cylinder when the intake valves are open. By directly injecting fuel into the cylinder, the injection process is no longer limited to valve opening, but can be extended through the whole cycle, and more interestingly, during the compression stroke. The advantage of direct injection is that the fuel can be placed in the combustion space in a more controlled manner than with conventional port injection systems: this gives it a greater potential for charge stratification.
1.3. Gasoline Direct Injection
1.3.1. Development
Since the 1920’s, attempts have been made to develop hybrid internal combustion engines that would combine the best features of petrol and diesel engines.
During World War II, Daimler-Benz, in conjunction with Bosch and the Reich Air Ministry experimented with direct injection. The primary aim was to eliminate fuel starvation during steep dives and improve performance for air combat. The result was the DB 601 family of engines, which equipped various fighters and bombers of the Luftwaffe, its most famous association being with the Messerschmitt Me 109 in the Battle of Britain. Direct fuel injection into the cylinders was provided by means of a twelve-unit high-pressure pump mounted between the cylinder blocks where it was fed by a Graitzin transfer pump. The high-pressure leads between the pump and the cylinders were of equal length. This fuel injection system proved to be very smooth and accelerated rapidly when the throttle was opened.
In 1954, Mercedes-Benz introduced the world's first four-stroke direct injection gasoline engine in a production car. It had a 3-litre, in-line 6-cylinder engine with a Bosch direct mechanical fuel injection. The engine produced 215 horsepower, and gave the car a top speed of 160 mph.
However, these DI engines operated with homogeneous stoichiometric mixtures. The idea behind GDI today is that directly injecting into the cylinder allows a better control of the fuel location within the cylinder, therefore giving it a greater potential for mixture
stratification. This should allow GDI to extend the AFR of lean-burn gasoline engine to values higher than 25, and reach equivalence ratios similar to those of Diesel engines. If the gasoline engine can be run unthrottled, then the reduction of the pumping losses will enable it to increase the efficiency and therefore reduce fuel consumption: theoretically, GDI has the potential to reduce the fuel consumption by 20 to 25% at low speeds and loads compared to homogeneous stoichiometric operation (Karl et al. 1996).
In 1996, Mitsubishi launched the first stratified spark-ignition direct injection engine, called GDI (Gasoline Direct Injection). This engine was said to reduce fuel consumption by over 15%, without compromising on driveability.
### 1.3.2. Advantages of GDI
The GDI concept is meant to increase the efficiency of the petrol engine and therefore reduce the fuel consumption. In turn, this reduces the CO₂ emissions. This increase in efficiency is possible through 4 main features:
- Higher compression ratio
- Increased ratio of specific heat (\( \gamma \))
- Reduction of pumping losses
- Less heat losses
By injecting the fuel directly inside the chamber, the charge is cooled inside the cylinder as it evaporates. In PFI engines, the cooling starts in the intake manifold, but the charge is then warmed up as it travels through the port before reaching the cylinder. Therefore, with GDI, the temperature of the charge at the time of compression is lower than that of a PFI engine. Because its temperature is lower than that of a PFI charge, it can be compressed more before it spontaneously ignites. This enables to increase the compression ratio, thus increasing the cycle’s efficiency.
Because the charge is leaned out, the mixture contains a higher ratio of diatomic molecules (N₂ and O₂). This increases the ratio of specific heats, which increases the efficiency (see Equation 1-2).
Running lean with very high AFRs enables to control the engine power output by varying the amount of fuel without having to throttle the air. This reduces the pumping losses, increasing to the cycle’s efficiency at part load.
The increase in efficiency also comes from the presence of excess fresh air in the stratified mode. This reduces the heat and exhaust losses due to lower temperature in the cylinder during the combustion and expansion stroke.
Direct injection can also improve cold-start hydrocarbon emissions and engine transient response because it avoids the deposition and build-up of fuel in the intake ports of conventional PFI engines. However, for most engine operating conditions, direct-injection has higher engine-out smoke and HC emissions than their PFI counterparts.
### 1.3.3. GDI Combustion Chamber Concepts
The idea of direct injection is to create a perfect, stratified charge by forming a richer cloud in the vicinity of the spark plug. The challenge is to control precisely the amount, size and distribution of the fuel droplets to suit varied driving conditions, and to do this reliably over the life of the vehicle.
Within a few milliseconds, the fuel spray must be atomised in very fine droplets, and be completely evaporated before ignition. At the same time, it is absolutely crucial that the
mixture at the spark location is optimal at the time of ignition. Otherwise, the combustion is not efficient, and the pollutant output increases.
Three different concepts have been developed to obtain the stratified mixture for direct injection combustion systems:
- Wall-guided
- Air-guided
- Spray-guided
In reality, for the three concepts, the stratification is achieved by a combination of these mechanisms.
In the wall and air-guided systems, the spark plug is usually located centrally, and the injector is to the side. Fuel is injected at a distance from the spark location at pressures typically around 100 bar, and is redirected towards the spark plug by a secondary mechanism. In the case of wall-guided systems, the piston crown has a bowl shape that redirects the fuel towards the spark plug. Mitsubishi’s GDI engine (see Figure 1-6) features upright straight intake ports rather than horizontal intake ports used in conventional engines. The upright straight intake ports direct the airflow down at the curved-top piston, which redirects the airflow into a strong reverse tumble for optimal fuel injection.
**Figure 1-6: Mitsubishi GDI wall-guided system**
Audi’s FSI (Fuel Stratified Injection) technology (see Figure 1-7) is an air-guided system. A vortex is created inside the combustion chamber by a tumble flap in the
intake manifold combined with a specially-shaped piston crown. The injected fuel is redirected by the air motion towards the centrally placed spark plug. The FSI engines range from a 1.6 Litre in-line 4 cylinder engine with a compression ratio of 12:1 to the latest 3.2 Litre V6 engine with a compression ratio of 12.5.
**Figure 1-7: Audi FSI air-guided system**
The spray-guided system, where the fuel is injected directly towards the spark gap location was investigated as early as 1972 (Simko et al.). Such systems suffered from two main problems: spark plug fouling and poor combustion robustness. However, with the progress in injector technology, and the introduction of Piezo-electric injectors, spray-guided systems are now knowing renewed interest (Matsumoto 2004). These injectors operate at higher pressures than the 1\textsuperscript{st} generation GDI injectors. Their response time is quicker, more precise and the droplet sizes are much smaller.
**Figure 1-8: Spray-guided system with Piezo-electric injector**
1.3.4. Issues
220.127.116.11. Emissions
The difficulty in GDI is that the charge must remain stratified while the mixing process occurs. Paradoxically, the presence of the fuel must be short enough not to diffuse and long enough to mix properly.
The stratified charge preparation relies on mixing of the air and the spray just enough to obtain an ignitable mixture underneath the spark plug, surrounded by air or an extremely lean mixture. This inevitably results in rich regions inside the ignitable pocket and leaner regions at the periphery. Both regions are subject to incomplete combustion, which is the first source of HC emissions.
The first generation of GDI designs (wall-guided systems) used a bowl to direct the spray towards the spark plug. The impingement of the fuel on the piston results in the formation of a liquid fuel film that constitutes the second source of HC emissions (Karlson and Heywood 2001). Frank and Heywood (1991) had found little changes in HC emissions with changes in piston temperature, and concluded that fuel wetting of the piston surface was not a significant source of HC emissions. However, they acknowledged that they used a high volatility fuel. Stanglmaier et al. (1999) studied the influence of wall wetting location for HC emissions and found that the wetting of the cylinder liner on the exhaust side was the strongest contributor, followed by the wetting on the piston top, and finally the wetting of the liner under the intake valves being the best case. Depositing 10% of the total fuel mass on the top of the piston results in a 30 to 70% increase in HC emissions for idle and part load conditions (Li et al. 1999).
Sandquist et al. (2000) investigated these hydrocarbon emissions and confirmed that over-mixing at the boundaries of the air/fuel mixture cloud and under-mixing both in the spray centre and on the surface of the piston bowl are the dominant mechanisms. There is a fuel injection timing optimum that minimises the sum of HC from over-mixing and under-mixing. As the mixing time is reduced, carbon monoxide (CO) and soot emissions increase. Drake et al. (2003) confirmed this and found that wall wetting was only responsible for 15% of UHC emissions for a swirl injector and 2% for a multihole injector in a wall-guided system. They found that most of the fuel-film mass
evaporates during the cycle and burns as pool fires. The fuel-films are the dominant source of smoke emission with 10% of the fuel-film mass converted to emitted smoke for the swirl injector. Smoke emissions are very small when using the multihole injector, which is consistent with the decrease in fuel-film.
With stratified-charge combustion, the rich regions produce high temperatures which are favourable for NOx formation in the presence of excess air. Replacing the excess air with EGR can reduce NOx, but is limited by the fact that EGR reduces laminar flame speed (Metghalchi and Keck 1982, Rhodes and Keck 1985) and is therefore detrimental to the combustion process.
### 18.104.22.168. Catalysts for GDI engines
The idea behind GDI is to decrease CO₂ emissions, but this must not compromise on HC or NOx emissions. The 3-way catalysts equipped on PFI engines are only efficient at reducing NOx under stoichiometric operation. Therefore, an adequate system must be developed for GDI engines in order to run under both homogeneous and lean burn conditions.
Lean NOx catalytic converters currently come in two varieties: the Selective Catalytic Reduction (SCR) and the Lean NOx Trap (LNT).
The Lean NOx Trap is equipped of a 3-way catalyst and a NO₂ trap. This trap is made of an alkaline earth compound (such as barium or strontium) or an alkali metal compound (such as potassium) which adsorb the NO₂ and form a stable metal nitrate. Under lean conditions, the catalyst oxidises NO to produce NO₂ which is adsorbed by the trap along with the NO₂ resulting from the in-cylinder combustion. When the device is full, the engine reverts to a stoichiometric combustion and the NO₂ is reduced to N₂. The storage-type converter is controlled by a mapped operating characteristic and by temperature. When the storage device is saturated, the engine's mixture is made richer for a short time (typically for 2-3 seconds each minute). This raises the temperature of the exhaust gas, so that the NO₂ is released and reduced in the 3-way catalyst.
Originally, the Selective Catalytic Reduction concept was used on stationary sources using ammonia or urea as the reducing agent. The Selective Catalytic Reduction using
hydrocarbons (SRC-HC) has the advantage that the catalyst utilizes hydrocarbon species present in the exhaust stream for NOx reduction. The reaction can be supplemented with additional hydrocarbons either via secondary injection into the cylinder or direct injection into the exhaust stream.
Lean NOx traps (LNTs) are currently the technology of choice for treating the NOx emissions from lean-burn SI engines because of their higher efficiency. However, the sulphur tolerance of aged LNTs is much lower than that of the fresh LNTs. Moreover, the aged LNTs are more difficult to desulphate relative to the fresh ones. Long-term use of this type of NOx catalytic converter in Europe, where the sulphur content of petrol is 200ppm, would result in almost total loss in effectiveness. The robustness of the LNT still needs to be improved. With the help of the newly established low sulphur fuel to be introduced in the European and the U.S. markets, the widespread application of LNT technology will become more promising (Li et al. 2001).
### 1.4. Summary
One method to reduce CO$_2$ emissions from SI engines is to improve their efficiency. This can be achieved by operating with a lean mixture. In the late 1980’s Japanese manufacturers launched stratified charge port-injection engines. It was clear that direct injection could extend the stratification by allowing injection during the compression stroke. The improvements in fuel injection technology as well as advances in the understanding of the processes involved in mixture preparation and stratified charge combustion have allowed the realisation of the concept. In 1996, the technology became commercially available with Mitsubishi’s GDI engine: the first generation of stratified charge direct injection SI engines with a wall-guided system.
Compared to port injection SI engines, HC emissions were thought to be improved because of the absence of wall wetting in inlet ports. However, the wetting of the piston and the cylinder walls during the injection and mixing process are important issues. Also, the stratification in the late injection, lean burn mode requires refinement, because the mixing time is poor, which irremediably creates inhomogeneous regions prone to bad combustion. By re-introducing spray-guided direct injection and using the lean burn concept in the future, the gasoline engine fuel consumption will come even closer to the supreme economy of a modern diesel.
1.5. Aims of this Thesis
The mixture formation process for the lean operation mode is therefore a key issue to its further development. It is essential to obtain a perfectly stratified charge with an optimal stoichiometry in a localised mixture near the spark plug. If zones of the mixture are too rich, soot will be produced and if too lean, misfire or hydrocarbon (HC) emissions will occur. Further, it is critical to avoid the mixture having excessive wall contact or impacting crevices which can enhance HC emissions. Understanding the mixture formation process in detail requires an accurate knowledge of the spray formation (i.e., droplet sizes and their evolution during injection as they evaporate). The Laser Sheet Dropsizing (LSD) technique is an important new laser diagnostics tool with the potential to study such a system. Combined with other 2-dimensional, non-intrusive diagnostics to measure droplet speed and local AFR, this would give development engineers a powerful tool for rapidly characterising sprays. The aim of the thesis was to develop such a tool. This requires the improvement and validation of the LSD technique for highly evaporative sprays. The diagnostic is combined with Particle Image Velocimetry (PIV) and Planar Laser-Induced Fluorescence (PLIF) to measure drop size, speed and fuel distribution in a BMW GDI optical research engine.
1.6. Outline of this Thesis
This thesis is organised in 4 parts. Part I includes Chapter 1 and 2 to provide background information on GDI’s potential to meet future legislation and presents the different laser techniques used in SI engines to better understand and improve the mixing process. Part II (Chapters 3-5) concerns the development of the Laser Sheet Dropsizing (LSD) technique for an evaporating GDI spray. Part III (Chapter 6) is the application of the LSD technique to an optical engine, along with other laser techniques for a complete characterisation of the spray. Part IV contains the conclusion and discussion of this work, along with the references and appendices.
Chapter 2 reviews the various laser diagnostics used to study and characterise spray development and mixture formation inside engines and gives examples of applications. An intensive study of fluorescence for the development of the LSD technique is found in Chapter 3. This includes a study of liquid-phase fluorescence and the determination
of the best fuel/tracer system regarding absorption, temperature and evaporation issues. Chapter 4 describes the experimental set-up that enabled to study a GDI spray in realistic engine conditions using both Phase Doppler Anemometry (PDA) and the LSD technique. Chapter 5 presents and compares the results obtained from the LSD and the PDA technique in order to validate and find the limitations of the LSD technique for different realistic conditions. In Chapter 6, the LSD technique is applied to an optical engine, along with PIV and AFR measurements for a complete 2-dimensional study of the spray development. The conclusions of the thesis work are discussed in Chapter 7.
Chapter 2
Laser Diagnostics in Spark Ignition Engines
The importance of in-cylinder fluid motion and the need for visualisation and measurement has been recognised since the early days of engine research. In-cylinder flow was studied by Clerk as early as 1921 using a low speed optical engine designed by Ricardo.
The combustion phenomenon is a direct result of the in-cylinder air-fuel mixing and the ignition, therefore any information on mixture strength distribution, spray size distribution and velocities could prove useful. Combustion chambers are harsh environments, with usually limited access, which makes measurements difficult. Laser-based diagnostics allow for non-intrusive measurements within these systems. With high power lasers, improved imaging systems, imaging techniques and optical engines, non-intrusive measurements methods have developed greatly. This has allowed a greater understanding of the in-cylinder phenomenon.
This chapter first presents light scattering theory, followed by a review of various laser diagnostics that are used to study and characterise sprays and the air-fuel mixing process.
2.1. Light Scattering Techniques
Light scattering is a term referring to physical processes involving the interaction of light and matter. This interaction partially “deflects” the incident light in directions deviated from the incident direction.
The passage of light through a medium induces vibrations which give rise to secondary waves. In a perfectly homogeneous medium, the secondary waves are equally scattered.
in all directions: their sum is a wave that has the same propagation direction as the incident light. Molecules in a liquid or a gas induce small variations in refractive index, therefore creating a non homogeneous medium. As a result, the secondary waves do not cancel each other out, and the resulting light is scattered in different directions to that of the incoming light.
A droplet can be seen as a collection of antennas that emit (scatter) an incident electric field. This scattering depends on the size and shape, the observation angle (scattering angle), the response of each antenna (composition), and the polarisation state and wavelength of the incident wave.
Scattered signals can be either elastic or inelastic. The elastic scattering is an interaction without an energy loss (or exchange) between the light and the matter within a droplet: after the interaction, the liquid drop is at the same state of energy as it was initially, and the energy in the light exiting the interaction point is equal to the energy of the incident light. This restriction of equal energy does not prohibit a change in direction, but according to Planck’s law, it prohibits a change in frequency.
\[ E = h \cdot v \Rightarrow \Delta E = h \cdot \Delta v \]
**Equation 2-1: Planck’s law**
where \( E \) is the energy and \( \Delta E \) is the energy variation, \( v \) is the frequency and \( \Delta v \) is the frequency variation and \( h \) is Planck’s constant.
In the case of an elastic scatter (i.e. when there is no energy exchange - \( \Delta E = 0 \)) there is no frequency change (\( \Delta v = 0 \)). On the other hand, an inelastic process describes a permanent energy exchange and involves a frequency shift. In practice, the two types of scattering may take place simultaneously.
### 2.1.1. Laser Rayleigh Scattering (LRS)
LRS is the elastic scattering of incident laser light with particles smaller than the wavelength of the light. From a quantum mechanics point of view, LRS occurs when a photon collides with a molecule, exciting it to a higher energy state. The molecule then relaxes back to the original state, emitting a photon of the same wavelength.
For a sphere of radius $r$ much smaller than the wavelength of the light ($r << \lambda$), Rayleigh deduced that the intensity of scattered light is proportional to $r^6 / \lambda^4$. Usually, Rayleigh scatter is detected at 90°, and allows to measure species concentration. If the pressure and species are known, the density measurement can be converted to temperature (Dibble and Hollenbach 1981).
Since LRS is an elastic process it suffers from background light of the same wavelength. That includes Mie scatter from particles, which, for a 1 $\mu$m droplet, is about 20 orders of magnitude higher than the Rayleigh scattering. Scattering from optics and other surfaces are also an issue.
Arcoumanis and Enotiadis (1991) used LRS and showed that the fuel concentration in the combustion chamber of a port-injected spark-ignition engine was affected by the injection time and duration.
Zhao et al. (1993) used LRS with a 532nm laser light sheet in a motored optical SI engine. They used Freon-12 and propane to study the mixing process, and found that the concentration distribution was non-homogeneous at the time of ignition.
Espey et al. (1997) obtained quantitative 2-D images of fuel vapour concentrations in an evaporating and combusting diesel spray, using a 532nm laser light sheet in an optical single cylinder DI Diesel engine. They estimated the error to be around 20%.
### 2.1.2. Spontaneous Raman Scattering (SRS)
When a photon collides with a molecule, it transfers its energy and excites the molecule to a higher energy state. Generally, both rotational and vibrational energies of the molecule are affected through the collision with the photon. In the case of SRS, the energy of the photon is more than can be stored by rotation or vibration in the molecule, but not enough to allow the molecule to reach the first electronic state. The molecule is therefore excited to a highly unstable virtual state and drops back almost immediately to a stable energy level, emitting a photon in the process. If the final level is the same as the original level, Rayleigh scatter is observed. If the final level is higher than the original level, the energy transfer is called Stokes Raman, and is red shifted. If the final level is lower than the original level, the result is the emission of a photon of lower wavelength, referred to as anti-Stokes Raman.
Raman scattering is an inelastic process which allows species and temperature measurements. The scattered light is collected and its intensity and spectral characteristics measured with a spectrometer. The species concentration is obtained from the intensity and the temperature from the spectral distribution.
SRS was first applied to IC engines in 1979 when Johnston obtained AFR measurements in a DI stratified charge engine from the ratio of signals of the fuel (Propane) and N₂ at different crank angles.
Because of the small Raman scattering section, interference from other light scattering processes can be a serious problem to the technique. For this reason, little progress was made in the development of SRS during the 1980’s (Zhao and Ladommatos 1998).
More recently, Miles and Dilligan (1996) measured major species (H₂O, CO₂, O₂ and fuel (C₃H₈)) in the Sandia transparent research engine. The residual gas mole fraction was obtained.
**Figure 2-1: Raman scattering energy levels: Stokes (red) and anti-Stokes (blue)**
Knapp et al. (1997) performed SRS in one of the cylinders of a 4-cylinder SI engine. They measured single-cycle and cycle-averaged concentrations of H₂O, O₂ and Iso-Octane (C₈H₁₈) relative to N₂ at different temperatures to take into account the temperature dependence of the Raman cross sections. By normalising the fuel and air
signals, the AFR measurement obtained from single shots eliminated pulse-to-pulse laser variation and window fouling. A precision better than 4% was obtained in the low load case and better than 3% in the high load case.
Miles (1999) describes a simultaneous multipoint single laser shot SRS measurement system applied to an optical engine.
Since SRS measures AFR directly, it can be applied to situations where EGR gas exists and has been accorded renewed interest. The drawback of the technique has been the low signal strength, but with the new high power UV lasers, this is less of a problem.
2.1.3. Mie scattering
Mie scattering is an elastic process which appears if the scattered particles are large compared with the incident wavelength (i.e. $d >> \lambda$). If light is scattered by particles that are smaller than the light’s wavelength, the phenomenon is Rayleigh scattering.
The Mie scattering process is based on a change of the electrical and magnetic properties inside and in the vicinity of the scatterer. Due to the large size of the particles, the scattering process is due to reflections, refraction and diffraction. The exact solution for scattering of electromagnetic waves from a spherical dielectric body was first obtained by Gustav Mie in 1908. The field of radiation resulting from the interaction between a single droplet and an incident light beam can therefore be calculated using the Mie-Lorenz theory. The resulting effects on the emitted light are changes in phase, amplitude and polarisation.
For Mie scattering, the mathematical solution of the Lorentz-Mie equation is a function of droplet diameter, scattering angle, refractive index of the droplet, polarisation and wavelength of the incident beam (Bohren and Huffman 1983).
22.214.171.124. Total scattered signal
A droplet redirects (scatters) light proportionally to its scattering cross section ($\sigma_{scatt}$):
$$I_{s, total} = C \cdot I_{laser} \cdot \sigma_{scatt}$$
**Equation 2-2**
where $I_{laser}$ is the intensity of the incoming light and $C$ is a proportionality constant.
The Mie scattering efficiency for a droplet is its ability to redirect power. It is defined as the scattering cross section divided by the cross-sectional area:
$$Q_{scatt} = \frac{\sigma_{scatt}}{\sigma} = \frac{4}{\pi} \cdot \frac{\sigma_{scatt}}{D^2}$$
**Equation 2-3**
Combining **Equation 2-2** and **Equation 2-3** gives an expression of the total scattered intensity as a function of diameter:
$$I_{s, total} = C' \cdot I_{laser} \cdot Q_{scatt} \cdot D^2$$
**Equation 2-4**
The scattering and extinction efficiencies are provided by the Lorenz-Mie theory (Bohren and Huffman 1983):
$$Q_{scatt}(m, x) = \frac{2}{x^2} \cdot \sum_{n=1}^{\infty} \left\{ (2n+1) \cdot |a_n|^2 + |b_n|^2 \right\}$$
**Equation 2-5**
$$Q_{ext}(m, x) = \frac{2}{x^2} \cdot \sum_{n=1}^{\infty} \left\{ (2n+1) \cdot \text{Re}(a_n + b_n) \right\}$$
**Equation 2-6**
where $x$ is the sizing parameter defined by $x = \pi \cdot D/\lambda$ with $D$ the droplet diameter. $m$ is the refractive index and coefficients $a_n$ and $b_n$ are the Mie scattering coefficients, and can be found explicitly in Van de Hulst (1957) or Bohren and Huffman (1983).
The absorption efficiency is then defined by:
$$Q_{abs} = Q_{ext} - Q_{scatt}$$
Equation 2-7
In case of absorption, the refractive index must be used in its complex form:
$$m = m_{Re} - im_{Im}$$
Equation 2-8
The real part ($m_{Re}$) corresponds to the refractive index (i.e. the ratio of the speed of light to the phase velocity in the medium). The imaginary part ($m_{Im}$) expresses attenuation or absorption of the light as it propagates through the medium.
Le Gal (1999) computed the scattering efficiency as a function of droplet diameter for a 532nm incident wavelength and non absorbing droplet of refractive index $m=1.44$. His results (see Figure 2-3) demonstrated the features of Mie scatter found in Conwell et al. (1984):
- $Q_{scatt}$ sharply increases as the droplet diameter increases from the Rayleigh limit ($D<\lambda$)
- $Q_{scatt}$ approaches the geometrical optics limit of 2 as the diameter becomes large
- The spectrum consists of rapid oscillations superimposed on a slowly varying background.
Figure 2-3: Scattering efficiency for droplets up to 5µm, $\lambda=532$nm, $m=1.44$
(Le Gal 1999)
These low frequency oscillations are due to interferences between refracted and diffracted light (Knight et al. 1992). Each peak occurs at regular intervals and can be associated with a resonance which correspond to Morphology Dependent Resonances (MDRs) and are discussed further in this chapter.
The result also suggests that for diameters above a few microns, the Mie scatter intensity could vary as a function of the diameter squared.
The FarField Mie scattering LightLab software developed by Valley Scientific, Inc. was used to determine the scattering efficiency as a function of droplet diameter for a 266nm incident wavelength. A refractive index $m=1.4$ was used to simulate scattering from non absorbing Iso-Octane droplets.
**Figure 2-4: Scattering efficiency, $\lambda=266\text{nm}, m=1.4$**
As expected, when the wavelength is halved, the characteristics of the Mie scattered intensity occur at half the diameter sizes. This suggests that the total scattered intensity will deviate from surface dependence by a maximum of 5% for droplets larger than 6µm.
126.96.36.199. Mie scattering as a function of angle
As seen previously, the total scattered light is proportional to the surface area. However, the scattered intensity is not evenly distributed. *Figure 2-5* is the polar diagram (in a logarithmic scale) for the scattering intensity of 266nm light by a 10µm droplet. The figure shows three important results:
- The intensity of the forward scattered light (\(\theta=0^\circ\)) is much larger than the backscatter or any other direction.
- The characteristics of the intensity distribution changes very strongly with observation angle.
- The intensity distribution from two polarisations can be very different.
**Figure 2-5:** Polar diagram (log scale) of Mie scattering for a 10µm droplet
If the scattered light from a droplet of diameter D is observed at an angle \(\theta\) with a collection angle of \(\Delta\theta\) (see *Figure 2-6*), the intensity will be:
\[
I_s(D,\theta,\Delta\theta) = C \cdot I_{laser} \cdot F(D,\theta,\Delta\theta) \cdot Q_{scatt} \cdot D^2
\]
*Equation 2-9*
where \(F\) represents the proportion of light scattered within the collection angle. At a given wavelength, \(F\) is a function of droplet diameter, observation angle and collection angle.
In their notations, Yeh et al. (1993a, 1993b) combine $F$ and $Q_{scatt}$ in a single term and write Equation 2-9 as:
$$I_s(D,\theta,\Delta\theta) = C \cdot I_{laser} \cdot Q_{scatt}(D,\theta,\Delta\theta) \cdot D^2$$
where $Q_{scatt}(D, \theta, \Delta\theta)$ can be assimilated to the scattering efficiency for the specific observation and collection angles.
Using the geometrical approach, Glantschnig and Chen (1981) demonstrated that the average measured scattered intensity is proportional to the diameter of the spherical scatterer:
$$I_{scatt}(D,m,\theta) = K(m,\theta) \cdot D^2$$
where $K(m,\theta)$ is a constant depending on the refractive index ($m$), the collection angle ($\theta$) and the geometry of the scattering set-up.
Equation 2-11 is correct, as long as the receiver’s f-number is small enough to average over the angular fluctuations resulting from the interference between the reflected and refracted light (Bachalo and Houser (1984)).
Sankar et al. (1997) modelled the scattering intensity based on the Mie-Lorenz theory in order to predict the variation of scattered signal with droplet size.
Figure 2-7: Computed variation of scattered light intensity with diameter for a $90^\circ$ collection angle and no absorption (Sankar et al. 1997)
The authors found that the scattered intensity is lower at $90^\circ$ compared to $45^\circ$ or $145^\circ$ angles. Also the variations around a $D^2$ dependence were higher at $90^\circ$ than for the two other angles.
By adding an absorption coefficient to the refractive index ($m=1.334-0.001i$) the oscillations seen in Figure 2-7 were reduced (see Figure 2-8): for weak absorbing droplets, the Mie signal is very well approximated by a $D^2$ relationship.
Figure 2-8: Computed variation of scattered light intensity with diameter for a $90^\circ$ collection angle and weak absorption (Sankar et al. 1997)
2.1.4. Planar Laser-Induced Fluorescence (PLIF)
188.8.131.52. Description
In fluorescence scattering (Demtröder 1982), a molecule absorbs a photon of the incident light. This energy allows the molecule to “jump” to a higher electronic level. The states of these upper levels are metastable with characteristic lifetimes of about $0.1\text{ns}$ to $10\mu\text{s}$. Therefore, very shortly after the transition to the upper level, the molecule drops back to a stable energy level with the ground electronic state. Fluorescence occurs when a molecule relaxes radiatively after having been excited to a higher electronic state.
For a simple two level energy system, following the excitation from state 1 to state 2 ($B_{12} \cdot I_\nu$), a molecule can undergo 5 important processes:
- The molecule returns to its original quantum state by laser-induced stimulated emission $B_{21} \cdot I_\nu$
- The molecule can absorb another photon and reach a higher state, eventually reaching an ionized level $B_{2i} \cdot I_\nu$
- Inelastic collisions with other molecules can produce vibrational and rotational transfers $Q_{\text{vib,rot}}$. In many cases, these collisions result in electronic energy transfers $Q_{\text{elec}}$, also referred to as quenching
- Interactions between the individual atoms of the molecule can produce internal energy transfer and dissociation of the molecule $Q_{\text{pre}}$
- The originally populated state and nearby states indirectly populated through collisions return to a lower state through the emission of light $A_{21}$, producing the laser induced fluorescence
Considering a two energy level system, where $A_{21}$ is the Einstein coefficient for spontaneous emission (fluorescence), $B_{12}$ and $B_{21}$ the Einstein coefficients for stimulated excitation and emission respectively and $Q_{21}$ is the total quenching factor (i.e. a combination of the pre-dissociation quenching factor $Q_{pre}$, the collisional or electronic quenching factor $Q_{elec}$, and the intermolecular collisional quenching $Q_{vib,rot}$), the rate of change of the population at the excited level is given by:
$$\frac{dN_2}{dt} = N_1 \cdot B_{12} \cdot I_\nu - N_2 \cdot (B_{12} \cdot I_\nu + A_{21} + Q_{21})$$
where $N_1$ and $N_2$ are the lower and upper level population respectively.
As the laser pulse ($\sim 10^{-8}$ s) is generally longer than the fluorescence lifetime ($\sim 10^{-9}$ s), it can be assumed that steady state conditions are reached, and therefore Equation 2-12 is equal to zero. With the population conservation, where $N_0$ is the initial total population at the lower level, $N_0 = N_1 + N_2$, the population at the excited state can be written as:
$$N_2 = N_0 \cdot \frac{B_{12} \cdot I_\nu}{A_{21} + Q_{21} + (B_{12} + B_{21}) \cdot I_\nu}$$
By defining the saturation intensity as:
\[
I_{sat} = \frac{A_{21} + Q_{21}}{B_{12} + B_{21}}
\]
Equation 2-14
\(N_2\) can be expressed as:
\[
N_2 = N_0 \cdot B_{12} \cdot I_\nu \frac{1}{A_{21} + Q_{21}} \cdot \frac{1}{1 + \frac{I_\nu}{I_{sat}}}
\]
Equation 2-15
The fluorescence is proportional to the upper state population multiplied by its probability to fluoresce:
\[
F \propto N_2 \cdot A_{21}
\]
Equation 2-16
The fluorescence can therefore be expressed as:
\[
F \propto N_0 \cdot B_{12} \cdot I_\nu \cdot \frac{A_{21}}{A_{21} + Q_{21}} \cdot \frac{1}{1 + \frac{I_\nu}{I_{sat}}}
\]
Equation 2-17
In the saturation regime (\(I_\nu >> I_{sat}\)), the fluorescence intensity is:
\[
F \propto N_0 \cdot B_{12} \cdot \frac{A_{21}}{B_{12} + B_{21}}
\]
Equation 2-18
The fluorescence signal is independent of both the laser fluence and the quenching: the signal is directly related to the species concentration. However, it is very difficult to reach saturation when performing PLIF.
At low laser excitation regimes ($I_v << I_{sat}$), Equation 2-17 becomes:
$$F \propto N_0 \cdot B_{12} \cdot I_v \cdot \frac{A_{21}}{A_{21} + Q_{21}}$$
Equation 2-19
This is the linear regime: the fluorescence is proportional to the laser intensity. In this regime, the signal is also proportional to the species concentration, and inversely proportional to the quenching.
In the linear regime, if the quenching rate is low ($Q_{21} << A_{21}$), the fluorescence signal becomes:
$$F \propto N_0 \cdot B_{12} \cdot I_v$$
Equation 2-20
If the quenching rate is high ($Q_{21} >> A_{21}$), Equation 2-15 becomes:
$$N_2 = N_0 \cdot B_{12} \cdot I_v \cdot \frac{1}{Q_{21}}$$
Equation 2-21
and the fluorescence signal becomes:
$$F \propto N_0 \cdot B_{12} \cdot I_v \cdot \frac{A_{21}}{Q_{21}}$$
Equation 2-22
In these conditions, the fluorescence signal is proportional to the laser intensity and the species concentration.
184.108.40.206. PLIF applied to IC engines
The wide application of PLIF in IC engines is mainly due to the strength of the fluorescence process, relative to those of Raman or Rayleigh scattering. Also, PLIF can
be used to image fuel location in both the liquid and vapour phase (Tait and Greenhalgh 1992).
For in-cylinder measurements, two different strategies have been adapted: natural fluorescence from the species of interest or fluorescence obtained from an added dopant (also called tracer).
Most of the early fluorescence work carried out in IC engines was performed in the linear non-quenching regime. In these conditions, the fluorescence signal is proportional to the laser intensity and the species concentration.
Naturally fluorescing species like OH were excited to look at flame front (Felton et al. 1988, Suntz et al. 1988). For fuel visualisation in IC engines, gasoline was first used because it contains fluorescing compounds. However, these different compounds each have individual fluorescence properties and for that reason, quantitative measurements are not possible. For quantitative measurements, Arnold et al. (1990) used a specific tracer in the fuel (acetaldehyde). Lawrenz et al. (1992) studied various tracers to replace the gasoline fuel by a single compound fluorescent fuel. They found Ethylmethylketone (EMK – or 2-Butanone) to be the best and measured AFR using throughout the intake and compression stroke and found appreciable inhomogeneities in fuel distribution. Baritaud and Heinze (1992) introduced Iso-Octane as a single component fuel, doped with biacetylene to obtain the fluorescence. Since, many fuel studies in IC engines have used various tracers with Iso-Octane. The choice of added tracers to visualise the fuel is discussed in Chapter 3. Berckmüller et al. (1994) used 3-Pentanone to visualise the fuel vapour and measure crank-angle resolved AFR in a lean burn Honda V-TEC transparent engine and found that there was substantial cycle-to-cycle variations in local fuel concentration at the spark electrodes.
LIF has also been used to measure oil film thickness, as early as 1974 by Smart and Ford. Shaw et al. (1992) developed an optical fibre based LIF measurement system and determined oil film thickness at the piston ring / cylinder liner interface.
Until this point, tracers had been selected for their non-quenching behaviour in oxygen. If the tracer was quenched by oxygen (e.g. TriEthylAmine -TEA- which has a high fluorescence yield but is strongly quenched), experiments were carried out in a Nitrogen environment.
Reboux et al. (1994) showed that quenching in the vapour phase, which had been considered as an unwanted effect could, on the contrary, be used for quantitative determination of AFR. If the quenching rate is directly proportional to the concentration of oxygen \((Q_{21} = k \cdot [O_2])\), a linear relation exists between the fluorescence intensity and the fuel-to-air ratio (FAR):
\[
F \propto \frac{N_0}{Q_{21}} \propto \left[ \frac{\text{fuel}}{[O_2]} \right] \propto \Phi
\]
Equation 2-23
Frieden and Sick (2003) measured Oxygen distribution using toluene and 3-pentanone tracers. Because the fluorescence spectra of the two tracers are distinct, their signals can be easily separated. In addition, toluene’s fluorescence is quenched in oxygen, whereas 3-pentanone’s is not. The ratio of their signals can be used to extract \(O_2\) concentration. By knowing the amount of fuel and the amount of air, the distribution of the 3\(^{rd}\) species (EGR) can then be obtained.
\[
\begin{align*}
F_{\text{pent}} & \propto \left[ \text{fuel} \right] \\
F_{\text{tol}} & \propto \left[ \frac{\text{fuel}}{[O_2]} \right]
\end{align*}
\]
\[
\Rightarrow \frac{F_{\text{pent}}}{F_{\text{tol}}} \propto [O_2]
\]
One issue with this technique is that the signal is inversely proportional to the species that is being measured.
One of the issues with PLIF is that the fluorescence from the liquid or vapour phase of a single component cannot be spectrally separated. This is because the fluorescence spectra of organic molecules dissolved in non-polar solvents are virtually identical to the spectra of the same molecules in the vapour phase. To overcome this, Melton (1983) introduced Laser Induced Exciplex Fluorescence (LIEF). The technique uses a mixture of two monomers, one serving as an electron donor (\(D\)) and the other as an electron acceptor (\(A\)). When \(A\) and \(D\) are excited (to \(A^*\) and \(D^*\) respectively) and fluoresce, they can bind with the opposite ground state monomer to form a third fluorescent species \((A-D)^*\) which is bound in the excited state but not in the ground state. The newly formed
species is called the excited state complex or exciplex. The probability that $A$ and $D$ form an exciplex is influenced by temperature and concentrations of the donor and acceptor. By adequately choosing the concentration of the donor and the acceptor, it can be assured that the liquid-phase fluorescence is predominantly characterised by the red-shifted emission from the exciplex, whereas the vapour phase fluorescence is dominated by the emission of the monomers $A$ and $D$.
**Figure 2-10: Spectra of 10% Naphtalene, 1% TMPD, 89% cetane mixtures in the ligand and vapour phases - T=220°C (Melton 1983)**
The first exciplex system for LIEF was developed by Melton (1983) and applied to droplets. The mixture consisted of 10 to 15% naphthalene combined with 1% tetramethyl-p-phenylene diamine (TMPD) in a synthetic diesel fuel (hexadecane). Melton and Verdieck (1985) later used a 2.5% Naphtalene / 1% TMPD exciplex mixture in 96.5% hexadecane (cetane) to study the vapour and liquid phase mixing of a Delavan hollow cone injector. *Figure 2-10* shows the spectral separation of the liquid phase fluorescence of the exciplex with the vapour phase fluorescence.
Melton (1993) proposed exciplex systems with boiling points matching those of gasoline fuel. They were based on tertiary alkyl amines like TriEthylAmine (TEA) and $n$-PropylDiEthylAmine (NPDEA) mixed with electronegatively substituted benzenes like Fluorobenzene (FBZ) and 4-FluoroToluene (4FT). Fröba et al. (1998) introduced TriEthylAmine (TEA) and Benzene as a new fluorescent tracer system for LIEF. A mixture of 2% TEA and 3.4% Benzene was used in Iso-Octane. This tracer mixture has two advantages: it predominantly forms an exciplex in the liquid phase, and it is also
quenched by oxygen. This allowed to simultaneously separate the liquid and vapour phase fluorescence whilst using the quenching behaviour to determine FAR.
2.1.5. Morphology Dependent Resonances (MDR)
Morphology Dependent Resonances (MDRs) of spherical and smooth non-spherical particles are reviewed by Hill and Benner (1988). MDRs in microspheres are responsible for the ripple structure observed in Mie scattering (see Figure 2-3). They are also responsible for the large optical feedback necessary for the lasing (Serpengüzel et al. 1992).
(Serpengüzel et al. 1992)
An interpretation of MDRs is that of rays propagating around the droplet’s surface. MDRs are usually treated as standing waves that can be decomposed into two counter-propagating waves that are contained by total internal reflection and travel around the droplet rim (see Figure 2-11). For the correct droplet size, the waves may return to their starting point with their initial phase and constructive interference takes place. The structure due to the constructive interference is correlated to the morphology of the drops, hence the usage of the term morphology dependent resonance.
Resonance peaks have been observed in elastic and inelastic scattering, and can modify the emission from a micro-droplet. Resonant effects are only likely to be important if the spacing between MDRs exceeds the emission line width of the fluorescent molecules (Hill et al. 1996). In practice, resonance peaks are dampened by surface imperfections and droplet shape. Inhomogeneities in refractive index near the surface of an evaporating droplet will also dampen resonance peaks (Knight et al. 1992).
2.2. Velocity Measurements
2.2.1. Laser Doppler Anemometry (LDA)
Laser Doppler Anemometry (LDA) - or Laser Doppler Velocimetry (LDV) - was pioneered by Yeh and Cummins (1964) and is described in detail by Durst et al. (1981). It is a point measurement technique.
A probe volume is created by splitting the laser beams in two equal intensity beams and crossing them at an angle. At first, beam splitters were used, and the interference pattern of the coherent light resulted in fringes. Later, a Bragg cell was used as a beam splitter. It is a glass crystal with a vibrating piezo crystal attached. The vibration generates acoustical waves acting like an optical grid. The output of the Bragg cell is two beams of equal intensity and one at the same wavelength as the input beam, the other slightly shifted (a 40MHz shift on a 600THz frequency).
**Figure 2-12: Interference fringes**
The fringe spacing $d_f$ is given by:
$$d_f = \frac{\lambda}{2 \cdot \sin \theta}$$
**Equation 2-24**
The frequency shift obtained by the Bragg cell makes the fringe pattern move at a constant velocity ($V_{\text{shift}}$). Particles which are not moving will generate a signal of the shift frequency $f_{\text{shift}}$. The velocities $V_{\text{pos}}$ and $V_{\text{neg}}$ will generate signal frequencies $f_{\text{pos}}$ and $f_{\text{neg}}$, respectively.
When a single droplet crosses the interference fringes in the probe volume, it scatters intensity-modulated light. The rate of intensity variation (i.e. the Doppler frequency $f_D$) indicates the time the droplet takes to travel the distance between two fringes. The droplet’s velocity component normal to the fringes and relative to the fringe velocity is:
$$V_r = d_f \cdot f_D$$
**Equation 2-25**
The actual velocity of the particle is:
$$V = V_{shift} + V_r$$
**Equation 2-26**
### 2.2.2. Particle Image Velocimetry (PIV)
In 1921, in-cylinder flow was studied by Clerk using a low speed optical engine designed by Ricardo. At this early stage the flow was visualised using smoke, and allowed Clerk to confirm the turbulent behaviour of the in-cylinder flow and also to disprove Otto’s theory that the charge was stratified.
In 1937, studies, undertaken by Lee, employed a number of innovations. In particular, pieces of feather were introduced in the flow as tracers and the motion was recorded using a high-speed cine camera and an intense light source. By manually matching the location of the seeds in consecutive frames, velocity was determined. It was the early form of Particulate Tracking Velocimetry (PTV). With CCD cameras, photographs are now digitised and tracking algorithms automate the process.
Correlation-based Particle Image Velocimetry (PIV) does not require the matching of individual droplets. Instead, it determines average motion of groups of particles contained within a small area called the interrogation region. The overall frame is divided into regions and the correlation function is computed over each region, yielding a velocity vector per interrogation region. It is possible to calculate average velocity maps, instantaneous or average vorticity maps, turbulence intensity maps and spatial correlation maps.
Compared to PTV, the averaging over multiple particle pairs within an interrogation region makes the measurement noise-tolerant and robust. This measurement technique gives access to quantitative information that is very useful for the characterisation of flows. It can be used both in aerodynamics and hydrodynamics, and the measurable velocity range extends from very low velocities to the supersonic regime.
While LDA is limited to the point measurements, PIV is used to extract 2-dimensional velocity fields. Due to the short laser pulse duration, each image contains the instantaneous position of the particles. The local velocity in the flow is measured by determining the displacement of the particles between two laser pulses (images). If the seeding density is sufficiently high, a two-dimensional velocity map of the illuminated region can be generated.
When the pairs of images are recorded on the same frame, the analysis is carried out by auto-correlation: the displacement is determined by shifting the image relative to itself and summing the product of the corresponding pixels for each shift. This sum goes through a maximum when the shift matches the displacement. For each square interrogation region of width $w$, the correlation function for each shift $(i,j)$ is given by:
$$R(i,j) = \sum_{x=-\frac{w}{2}}^{\frac{w}{2}} \sum_{y=-\frac{w}{2}}^{\frac{w}{2}} I(x,y) \times I(x+i,y+j)$$
Equation 2-27
Figure 2-14 – Single frame - double exposure and Auto-correlation analysis
The auto-correlation function shows a central peak (i.e. $R(0,0)$ is the maximum) and two symmetric secondary peaks. The position of these secondary peaks with respect to the centre of the analysis window gives the displacement of the particles. The symmetry of the correlation function prevents knowledge of the direction of displacement, which has to be determined from the flow.
To eliminate the ambiguity, the two particle images can be recorded on different frames. The analysis is then carried out by cross-correlation: the determination of the displacement is done by shifting the second image relative to the first image and summing the product of the corresponding pixels for each shift. This sum reaches its maximum when the shift is equal to the displacement. In discrete terms, the cross-correlation is given by:
$$R(i,j) = \sum_{x=-\frac{w}{2}}^{\frac{w}{2}} \sum_{y=-\frac{w}{2}}^{\frac{w}{2}} I_1(x,y) \times I_2(x+i,y+j)$$
Equation 2-28
Figure 2-15 – Double frame – double exposure and Cross-correlation analysis
In this case, the cross-correlation function shows only a single intense peak whose position with respect to the centre gives access to the direction (with no ambiguity) and length of the displacement of the particles in the analysis window (see Figure 2-15).
Evaluating the correlations in the direct way is not efficient as regards computing time. However, the Wiener-Khinchin theorem states that the correlation of two signals $f$ and $g$ ($f \otimes g$) in the temporal domain can be computed as a multiplication in the frequency domain:
$$f \otimes g = F^{-1}\left(\tilde{f}^* \times \tilde{g}\right)$$
Equation 2-29
where $\tilde{f}$ and $\tilde{g}$ are the Fourier transforms of $f$ and $g$ respectively, $\tilde{f}^*$ is the complex conjugate of $\tilde{f}$ and $F$ is the inverse Fourier transform.
Using the Fast Fourier Transform (FFT) is far more efficient: the auto-correlation (where $f=g=I$) can be quickly calculated by 1 forward transform and 1 inverse transform:
$$R(i,j) = F^{-1}\left[\tilde{I}(u,v)\right]^2$$
Equation 2-30
The cross-correlation ($f=I_1$ and $g=I_2$) can be obtained quickly with 2 forward transforms and 1 inverse transform:
$$R(i,j) = F^{-1}\left[\tilde{I}_1^*(u,v) \times \tilde{I}_2(u,v)\right]$$
Equation 2-31
PIV was first applied to in cylinder measurements by Reuss et al. (1989) in a motored engine. The velocity of the swirling bulk flow could be measured in a portion of the engine cylinder, parallel to the piston crown. Vorticity structures and strain rates were derived from instantaneous flow fields. It was suggested that the magnitude of the observed strain rates and the random distribution of high strain regions could affect the early flame growth and therefore contribute to cyclic variations in engine performance.
Nino et al. (1993) demonstrated PIV in a fired 2-stroke engine with high swirl inlet configuration. Instantaneous and ensemble averaged velocity fields parallel to the piston crown and velocity fluctuations could be obtained in the unburned gas.
Reeves (1994) measured the instantaneous velocity field in a motored 4-valve engine. Illumination of a plane parallel to the cylinder axis through a piston window allowed the study of large scale tumble vortices.
Pajot (2000) made turbulence velocity measurements in a port injection RENAULT engine and also showed that the axis of tumble and swirl movement were not respectively exactly horizontal and vertical.
Hentschel et al. (1999) studied droplet PIV of a swirl injector. They used 2 Excimer lasers (308nm) for the measurements in a pressure chamber, and a double pulse frequency-doubled Nd:YAG laser (532nm) for the engine experiments. They found that in some cases, the droplet velocities could only be obtained on the edge area due to the high density inside the spray.
Yamakawa et al. (2001) studied simultaneously the spray and air motion around the spray. They injected a solution of water and Rhodamine into the air and used a 532nm light source. The scattered light from the spray droplets was separated from the fluorescence of the Rhodamine in the air. Spray velocities were obtained by Mie scatter PIV and air motion with LIF PIV.
2.3. Size Measurement
The droplet size distribution is directly related to the spray’s atomisation and vaporisation process. The quality of the air-fuel mixing is critically dependent on these factors. Early development of sizing techniques include freezing drops (Longwell 1943) or molten wax techniques (Joyce 1949). The first technique used an alcohol bath kept at the temperature of dry ice, which was cold enough to freeze the drops. This enabled to sample the droplets. The second technique used the fact that molten wax has the same properties as kerosene, and was fed through aero injectors, and would solidify at the exit. Since then, non intrusive methods have greatly developed.
2.3.1. Laser diffraction
Low-angle laser light scattering (LALLS, commonly called laser diffraction) systems typically pass a laser beam of known wavelength through a suspension of the material to be analyzed and measure the angular distribution and intensity of the forward-scattered (diffracted) light by the particles in suspension. A theoretical model, based on diffraction of particles with particular properties and grain-size distribution, is then fitted to the actual diffraction results. When the first laser diffraction instruments were launched, the constraints of most commonly available computers meant that the instruments used an approximation of Mie light-scattering theory known as the Fraunhofer approximation to produce particle size analyses. However the full solution of the light scattering of spheres as predicted by Gustav Mie can now be applied.
Figure 2-16: Measurement volume for the line-of-sight scattering technique
This line-of sight technique uses forward scattered light from moving particles. The detected light is not sensitive to refractive index or shape, and therefore is only a function of size (Swithenbank 1976). The measurement of this angular light distribution is the basis of the optical particle sizer manufactured by Malvern Instruments Ltd.
The technique measures the forward scattered light of a cloud of particles in a laser beam. Not only is the measurement averaged over the light path (line-of-sight technique), it is also averaged over the beam diameter, which gives this method limited spatial resolution. However, if the spray is axisymmetric, the averaged measurements of optical extinction and droplet size distribution can be deconvoluted into radial variations of drop size distribution and number density (Hammond 1981).
2.3.2. Interferometric Particle Imaging
This technique is based on the principle of defocused imaging of Mie scattering from transparent droplets. The interference of reflected and refracted light results in fringe patterns (see Figure 2-17) which contain the information about the size of the droplet in the spacing of the fringes.
It goes by various names: PMSI - Planar Mie Scattering Interferometry, PII – Planar Interferometric Imaging, PPIA – Planar Particle Image Analysis, ILIDS – Interferometric Light Imaging for Droplet Sizing.
Figure 2-17: Interference pattern in out-of-focus imaging of droplets
The technique was pioneered in 1986 by König et al. They focused a laser beam onto a stream of monodisperse droplets and measured the resulting fringe pattern in the far field. They recognised the potential for accurate drop size measurements.
Van de Hulst and Wang (1991) proposed that the droplets give two glare points in the focussed plane, and that it is possible to determine the diameter from the distance between the 2 glow spots. However, this would require high magnification to resolve them, and this technique would offer no advantage over direct particle imaging.
These two points act as 2 spherical emission sources. Their overlap creates parallel fringes, and can be seen in the defocused plane.
Using geometrical optics, Hesselbacher et al. (1991) demonstrated that the diameter and the fringe spacing were linked, and described the relationship between particle diameter $d$ and number of fringes $N$ by:
$$d = \frac{2 \cdot \lambda \cdot N}{\alpha} \cdot \frac{1}{\cos(\theta/2) + \sqrt{m^2 - 2 \cdot m \cdot \cos(\theta/2) + 1}}$$
where $m$ is the refractive index, $\lambda$ is the laser wavelength, $\theta$ and $\alpha$ are the scattering and collecting angle respectively.
Glover et al. (1995) extended the technique to a light sheet and applied it in an engine to a spatially sparse spray. Pajot and Mounaïm (1998) successfully obtained dropsize data from the non-dense part of a spray in an SI engine, but the denser part of the spray was not measurable due to the overlapping of the circular interferogram.
Koboyashi et al. (2000) further improved the technique by optically compressing the circular fringe images using an anamorphic system consisting of two cylindrical lenses. This not only avoids signal overlap but also increases signal-to-noise ratio of the captured interferential image. They obtained an error of less than 3% for arithmetic mean diameter.
Figure 2-18: Interferometric image (Maeda et al. 2000) using
(A) Conventional technique – 15 droplets
(B) Compression Optics - >100 droplets
Maeda et al. (2000) combined the compression technique with double-pulsed images and simultaneously measured drop size and velocity.
The technique shows great promise but only manages to capture parts of the spray; typical imaging is $10\text{mm} \times 10\text{mm}$ on a $1008 \times 1018$ pixel camera (Maeda et al. 2003). Commercial instruments are available from Dantec (FlowMap Particle Sizing System) and LaVision (SizeMaster).
2.3.3. Phase Doppler Anemometry (PDA)
The basic principle of this method was proposed by Durst and Zaré in 1975 and by Bachalo in 1980 as an extension of Laser Doppler Anemometry (LDA). The technique uses the phase angle between adjacent Doppler signals for drop size measurement.
When a droplet crosses a fringe pattern formed at the intersection of two focused laser beams, it acts as a lens and forms a magnified version of the fringes at the two detectors (see Figure 2-19).
A stationary sphere of diameter $D$ will project magnified fringes at a distance $R$ from the centre of the droplet. If $f$ is the focal length of the sphere, considered as a thick lens, and $d_f$ is the object fringe spacing, then the measured spacing of the image fringes $d_{mag}$ is given by:
$$d_{mag} = (R - f) \cdot \frac{d_f}{f}$$
As $R >> f$, Equation 2-31 can be simplified to:
$$d_{mag} \approx R \cdot \frac{d_f}{f}$$
For a sphere of refractive index $n$, considered as a thick lens for small scattering angles:
$$f = \frac{n}{n-1} \cdot \frac{D}{4}$$
If the particle moves, the fringes will appear to rotate about the focal point of the thick lens. A detector at a given point will therefore see an oscillating pattern of light and dark fringes. If another detector is separated by the distance $a$, the signal will be out of phase from the first detector by an angle:
$$\Phi = 2\pi \cdot \frac{a}{d_{mag}}$$
Combining Equation 2-34, Equation 2-35 and Equation 2-36 we obtain:
$$\Phi \approx \pi \cdot a \cdot \frac{n}{n-1} \cdot \frac{\sin \theta}{R \cdot \lambda} \cdot D \propto D$$
One complication with PDA is that the light from a droplet is both refracted and reflected and the phase difference from these two scattering mechanisms is not necessarily the same. The solution is to use polarised laser light and to arrange for the detectors to be close to Brewster’s angle $\Phi_b = 2 \cdot \text{Arctan}(1/n)$ at which refraction dominates reflection. In practice the phase-diameter relationship is near linear between $30^\circ$ to $80^\circ$, with the best linearity at Brewster’s angle normally a maximum in the scattering at the angle of $1^{st}$ refraction often referred to as the rainbow angle.
A second issue concerns the compromise between accuracy and range afforded by a total phase angle of $2\pi$. To overcome this limitation, normally 3 detectors are used, yielding two different separations between pairs so that the wider-separated pair phase angles greater than $2\pi$ can be measured without ambiguity. This arrangement also increases the redundancy of the measurement and permits validation of the measurement.
Signals are typically processed using a dedicated signal processor and in particular a covariance processor which can simultaneously measure phase and frequency from a Doppler burst. More recently, commercial manufacturers have developed discrete Fourier transform processors. These offer higher bandwidth and hence velocity range, higher accuracy, and reduced dead-time.
A major uncertainty with the PDA is caused by the measurement volume effect (MVE). This is due to the beams’ Gaussian profile and is also referred to as the trajectory effect: if a large particle penetrates the probe volume, the intensity profile of the incident beams causes a non-uniform illumination of the particle and induces reflected and refracted rays which may have roughly the same intensities on the detectors (see Figure 2-21). For dominating reflected light, this effect results in considerable errors in size measurement.
Figure 2-21: Trajectory effect due to beam profile
Using geometric optics, Sankar et al. (1992) showed that the measurement of a 45 μm droplet placed in a measurement volume with a 80 μm waist might result in an underestimation as large as 67% of the true diameter. One way to minimise the distribution effect is to increase the size of the measurement volume. However, this increases the probability of multiple occupancy. Another method is to use an additional detector (Gréhan et al. 1992).
The slit aperture can also suppress completely the light scattering property selected for calculating the particle diameter. If a particle crosses the right part of the volume (see Figure 2-22), the refracted light will be suppressed. If this was the chosen scattering mechanism, the equipment will determine a wrong drop size.
Durst et al. (1994) showed that the non uniform illumination effect due to the image boundary of the spatial slit filter in the receiving optics is even more critical than the Gaussian beam defect.
In spray applications, the PDA system is usually set up to receive refracted light. Due to the trajectory and slit effect, reflected light can damage the measurement. Aizu et al. (1993) proposed the Planar PDA (PPDA) system which consists in a 90° rotation of the transmitting and receiving optics.
Tropea et al. (1994) proposed the dual-mode PDA. This four beam system basically combines two detectors of the standard PDA (SPDA) with two detectors of the Planar PDA in a single receiving unit (see Figure 2-23). The comparison of the droplet size obtained with the SPDA and PPDA allows to validate the measurement.
PDA in dense spray cannot provide reliable data (Wigley 1994). This is partly due to the fact that PDA cannot make reliable measurements when more than one drop enters the measurement volume. Also, in dense sprays, random scattering and attenuation due to numerous droplets crossing the incident and reflected/refracted light is also an issue.
Wigley et al. (1999) improved the PDA system by reducing the measurement volume and increasing the power (120mW and 250mW per beam at 488 and 514nm respectively) and measured velocities and drop sizes in a GDI injector (Wigley et al. (2002).
### 2.3.4. Laser Sheet Dropsizing (LSD)
For a given number ($N$) of droplets, the Sauter Mean Diameter (SMD or $D_{32}$) corresponds to the diameter of a drop that would have the same ratio of volume-to-surface area as that of the $N$ droplets. This diameter is frequently specified because it allows the calculation of the total area of an atomised volume of liquid. It is also the most useful value for determining the rate of evaporation. Therefore, lower SMDs indicate better atomisation of the fuel.
\[
D_{32} = \frac{\int_0^\infty \frac{dn}{dD} \cdot D^3 \cdot dD}{\int_0^\infty \frac{dn}{dD} \cdot D^2 \cdot dD}
\]
\[
D_{32} = \frac{\sum_i n_i \cdot D_i^3}{\sum_i n_i \cdot D_i^2}
\]
**Equation 2-38: Definition of the Sauter Mean diameter (continuous – discrete)**
Theoretically, the ratio between the LIF and Mie scatter signal from liquid droplets can be used to extract the SMD. The technique originated in 1993 from Yeh et al. and was applied on a diesel injector. Sankar et al. (1996) developed a similar technique - Planar Drop Sizing (PDS) - which was applied to a Delavan swirl injector. Le Gal (1999) developed the Laser Sheet Dropsizing (LSD) technique which uses the same principle as Sankar et al. (1996) with a UV light source from a Nd:YAG laser. An in depth review of the principle can be found in Chapter 3.
The basic principle of the LSD technique uses the property that the LIF and Mie scatter signals from a low absorbing droplet \((k)\) are volume and surface dependent respectively. The local ratio of the two signals will therefore be proportional to SMD:
\[
D_{32}(i,j) \propto \frac{I_{LIF}(i,j)}{I_{Mie}(i,j)}
\]
Equation 2-39
Le Gal (1999) verified the volume and surface dependence of the LIF and Mie scattering signals respectively using a TSI droplet generator. He later applied the technique to a Delavan pressure-swirl atomiser spray and found good agreement with results obtained with PDA.
Park et al. (2002) verified the technique with a droplet generator using unleaded gasoline and also found good agreement for the \(d^3\) and \(d^2\) dependence of the LIF and Mie scatter signals respectively. They applied the technique to a GDI swirl type injector and determined the calibration constant using PDA. They found that the characteristics of the injector varied with injection pressure.
Zimmer et al. (2002) applied the technique to an industrial oil burner and found that droplet clusters form in high density regions. They identified flow structures using PIV, possibly explaining this cluster formation.
Domann and Hardalupas (2003) and Charalampous et al. (2004) have proposed recent improvements to the technique, which are discussed in Chapter 5.
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3. **Desktop Application Development**: This includes creating desktop applications that run on Windows, macOS, or Linux operating systems. It typically uses languages like Java, C#, or Python.
4. **Game Development**: This involves creating video games. It can be for consoles, PC, mobile devices, or virtual reality platforms. Popular game engines include Unity and Unreal Engine.
5. **IoT (Internet of Things) Development**: This involves creating devices that connect to the internet and perform tasks autonomously. It often requires knowledge of embedded systems and wireless communication protocols.
6. **AI/ML (Artificial Intelligence/Machine Learning) Development**: This involves developing algorithms and models that enable machines to learn from data and make predictions or decisions without being explicitly programmed.
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Each type of project has its own set of challenges and requirements, so it's important to choose a project that aligns with your skills and interests.
PART II
DEVELOPMENT OF THE LASER SHEET DROPSIZING (LSD) TECHNIQUE
Chapter 3
Development of Laser Sheet Dropsizing for Evaporative Sprays
The Laser Sheet Dropsizing (LSD) technique is based upon the combination of elastic Mie scatter and inelastic scatter (LIF) of illuminated droplets. Mie scattering is a very robust technique for liquid-phase imaging, but the behaviour of fluorescence in an evaporative spray is less obvious. The feasibility of the LSD technique is strongly dependent on the accuracy of PLIF in the evaporating liquid phase. In this chapter, the requirements and selection of an appropriate tracer/fuel system are discussed. Temperature and Pressure dependence are investigated along with evaporation calculations. This is followed by the description of a dual imaging set-up along with the calibration procedures required for the LSD technique.
3.1. Principle
Because the fluorescence and the Mie scatter are volume and surface effects respectively, theoretically, the ratio between the LIF and Mie scatter signal from liquid droplets can be used to extract the Sauter Mean Diameter.
**Figure 3-1: Light scattering from an absorbing droplet**
3.1.1. Early development
Yeh et al. (1993a, 1993b) were the first to use the intensity ratio of simultaneous LIF and Mie scatter signals to measure Sauter Mean Diameter (SMD). They applied the technique on an axisymmetric spray.
They assumed that the LIF intensity was proportional to droplet volume for droplets smaller than $80\mu m$. At location $(r,z)$ the fluorescence is given by:
$$I_f(r,z) = I_0 \cdot e^{-kx} \cdot C_1 \cdot \int_0^\infty D^3 \frac{dn}{dD} dD$$
**Equation 3-1**
where $I_o$ is the incident laser intensity and will be attenuated by $kx$ according to Beer-Lambert’s law, $x$ is the distance from the incident spray edge and $k$ the attenuation coefficient. $C_1$ is a constant value that includes the absorption coefficient, the quantum yield and the detection system’s characteristics.
They defined the Mie scattered signal as:
$$I_s(r,z) = I_0 \cdot e^{-kx} \cdot C_2 \cdot \int_0^\infty Q_{scatt}(\alpha,\theta,\Delta\theta) \cdot D^2 \frac{dn}{dD} dD$$
**Equation 3-2**
where $C_2$ is the constant relating to the incident light intensity and the detection system characteristics. $Q_{scatt}(\alpha,\theta,\Delta\theta)$ is a function of the droplet parameter $\alpha = \pi \cdot D/\lambda$.
By assuming a particle size distribution, they defined an average scattering coefficient:
\[
\frac{Q_{scatt}(\alpha_{32}, \theta, \Delta \theta)}{\alpha_{32}} = \frac{\int_0^\infty Q_{scatt}(\alpha, \theta, \Delta \theta) \cdot D^2 \frac{dn}{dD} dD}{\int_0^\infty D^2 \frac{dn}{dD} dD}
\]
Equation 3-3
where \( \alpha_{32} = \pi \cdot D_{32}(r, z)/\lambda \) and \( D_{32}(r, z) \) is the local Sauter Mean Diameter and is given by:
\[
D_{32} = \frac{\int_0^\infty D^3 \frac{dn}{dD} dD}{\int_0^\infty D^2 \frac{dn}{dD} dD}
\]
Equation 3-4
By combining Equation 3-1, Equation 3-2, Equation 3-3 and Equation 3-4 they obtained:
\[
D_{32}(r, z) = \frac{Q_{scatt}(\alpha_{32}, \theta, \Delta \theta)}{C} \cdot \frac{I_f(r, z)}{I_s(r, z)}
\]
Equation 3-5
where \( C = C_1/C_2 \).
By assuming that the spray was axisymmetric, they integrated Equation 3-5 cylindrically over the spray to extract a value of the constant \( C \):
\[
C = \frac{Q_{scatt}(\alpha_{32}, \theta, \Delta \theta)}{D_{32}} \cdot \frac{\int_0^Z \int_0^{R(z)} I_f(r, z) \cdot 2\pi r \cdot dr \cdot dz}{\int_0^Z \int_0^{R(z)} I_s(r, z) \cdot 2\pi r \cdot dr \cdot dz}
\]
Equation 3-6
where \( D_{32} \) is the SMD for the whole spray, \( Z \) is the height of the spray and \( R(z) \) is the radius of the spray at the height \( z \).
They then used an empirical equation to determine the SMD for the whole spray ($D_{32}$) and, which enabled the calculation of $\overline{Q_{scatt}}(\alpha_{32}, \theta, \Delta \theta)$.
The local SMD ($D_{32}(r,z)$) was then determined using an iterative process on Equation 3-5 as $\overline{Q_{scatt}}(\alpha_{32}, \theta, \Delta \theta)$ is a function of ($D_{32}(r,z)$).
They applied the technique using TMPD in a base fuel and the fluorescence was generated using a Nd:YAG laser at a wavelength of 355nm. They collected the light at 90° and separated the LIF from the Mie scatter using a doubling prism and filters.
This technique is self-calibrating if the spray’s overall SMD is known and the spray can be assumed axisymmetric. The determination of the local SMD also requires the local droplet size distribution and in many cases, it is unknown.
### 3.1.2. The Laser Sheet Dropsizing principle
Le Gal (1999) developed the Laser Sheet Dropsizing (LSD) technique based on the same principle. However, whereas the approach by Yeh et al. (1993) used the exact Mie scatter signal - which takes into account the variation of the scattering coefficient ($Q_{scatt}$) for different droplet sizes - the LSD technique simplifies this by assuming that the scattering coefficient is constant (i.e. the Mie scatter signal collected at 90° is simply a function of the diameter squared).
Under low absorption conditions, fluorescence is expected to be proportional to liquid volume or liquid mass distribution (Talley et al. 1996, Domann and Hardalupas 2000) and the LIF signal from a droplet $k$ can therefore be expressed as:
$$I_{f,k} = C_1 \cdot I_{laser,k} \cdot d_k^3$$
where $I_{laser,k}$ is the laser power which excites droplet $k$.
Sankar et al. (1997) showed that for weak absorption, the Mie scatter signal can be well approximated by:
\[
I_{s,k} = C_2 \cdot I_{laser,k} \cdot d_k^2
\]
Equation 3-8
where \(C_1\) and \(C_2\) are constants independent of the droplet size or laser power.
If both images are captured on a CCD, the spray image is digitised into small windows, as seen in Figure 3-3.
**Figure 3-3: Droplet distribution of the spray, as seen by the CCD chip**
The intensity of pixel \((i,j)\) corresponding to the fluorescence of \(N(i,j)\) droplets can be expressed as:
\[
I_f(i,j) = \sum_{k=1}^{N(i,j)} I_{f,k} = \sum_{k=1}^{N(i,j)} \left( C_1 \cdot I_{laser,k} \cdot d_k^3 \right) = C_1 \cdot \sum_{k=1}^{N(i,j)} \left( I_{laser,k} \cdot d_k^3 \right)
\]
Equation 3-9
Equation 3-9 corresponds to the discretisation of Equation 3-1.
Because the droplets within a pixel are randomly placed, the scattered light lacks coherence and will not interfere (Sankar et al. 1996). The intensity of pixel \((i,j)\) from the Mie scattering of \(N(i,j)\) droplets can be written as:
\[
I_s(i,j) = \sum_{k=1}^{N(i,j)} I_{s,k} = \sum_{k=1}^{N(i,j)} \left( C_2 \cdot I_{laser,k} \cdot d_k^2 \right) = C_2 \cdot \sum_{k=1}^{N(i,j)} \left( I_{laser,k} \cdot d_k^2 \right)
\]
Equation 3-10
Equation 3-10 is a discrete version of Equation 3-2, where the scattering coefficient is independent of the diameter.
If the LIF and Mie scatter signals follow Equation 3-9 and Equation 3-10 respectively, for each pixel \((i,j)\) of the CCD array imaging \(N(i,j)\) number of droplets, the intensity ratio of the signals will be:
\[
\frac{I_f(i,j)}{I_s(i,j)} = \frac{C_1 \cdot \sum_{k=1}^{N(i,j)} \left( I_{laser,k} \cdot d_k^3 \right)}{C_2 \cdot \sum_{k=1}^{N(i,j)} \left( I_{laser,k} \cdot d_k^2 \right)}
\]
Equation 3-11
For low absorption and small pixel sizes (typically a pixel represents \(100\mu m \times 100\mu m\)), the laser absorption is negligible: the laser power can be assumed homogeneous within the pixel (i.e. \(\forall k \in [1,N(i,j)] \quad I_{laser,k} = cst\)). Also, if the images are recorded simultaneously, the laser intensity is the same for both scatterings. Therefore, the ratio can be written as:
\[
\frac{I_f(i,j)}{I_s(i,j)} = \frac{C_1}{C_2} \cdot \frac{\sum_{k=1}^{N(i,j)} d_k^3}{\sum_{k=1}^{N(i,j)} d_k^2} = C \cdot D_{32}(i,j)
\]
Equation 3-12
where $C = C_1 / C_2$
The local SMD is given by:
$$D_{32}(i, j) = \frac{1}{C} \cdot \frac{I_f(r, z)}{I_s(r, z)}$$
Equation 3-13
Equation 3-13 shows that the uneven distribution of laser fluence across the CCD chip is eliminated by the ratio of the signals pixel by pixel, and that the result is proportional to Sauter Mean Diameter.
Theoretically, the proportionality constant ($C$) is not a function of the pixel. Therefore, the known SMD in any pixel could be used to calibrate the data on the whole CCD chip. Furthermore, the assumption that the scattering coefficient is constant eliminates the iterative process in the local calculation of the SMD.
Le Gal (1999) demonstrated the LSD technique at ambient pressure and temperature conditions. This work will try to demonstrate the technique in a GDI spray under realistic engine conditions, i.e. higher pressures and temperatures. The critical issue with the LSD technique is the accuracy of the fluorescence signal, and its ability to continuously yield a volume-dependent signal for evaporating droplets. The following subchapter discusses this issue.
### 3.2. Laser-Induced Fluorescence for the LSD technique
#### 3.2.1. Fluorescent Tracers
Since the introduction of Iso-Octane as a single component fuel, the application of LIF has seen the use of various fluorescing tracers for fuel distribution measurements in transparent engines. Tracers such as Acetaldehyde (Arnold et al. 1990), Biacetyl (Baritaud and Heinze 1992) and Acetone (Wolff et al. 1994) were used. Recent work suggests the widespread use of 3-Pentanone. This tracer’s popularity is due to its almost matching boiling point with Iso-Octane (101.7°C and 99.2°C respectively). Qualitative work using 3-Pentanone is reported amongst others by Neij et al. (1994), Ossler and Aldén (1997), Steeper and Stevens (2000). Berkmüller et al. (1994) and Stojkovic and
Sick (2001) performed quantitative measurement of local AFR measurements. Stojkovic and Sick (2001) also used the ratio of LIF and Mie scattering signals to determine SMD. However, recent study by Davy et al. (2003) has demonstrated that 3-Pentanone’s boiling point is deceptive, and that under engine evaporating regimes, the fluorophore does not perform its required task: tracing the fuel.
In the engine, the fuel/tracer system is subject to evaporation, rises in pressure and temperature and finally combustion. In order for the fluorescent dye to properly act as a tracer for the Laser Sheet Dropsizing technique, it must follow certain requirements.
The tracer should:
- Absorb at the laser wavelength
- Co-evaporate with the fuel
- Be soluble in the fuel / Consumed during combustion
The tracer’s fluorescence should:
- Yield sufficient signal
- Be volume-dependent
- Be independent of temperature / pressure
- Not quench
Of the commonly used fluorophores, the first 3 properties enabled to select 5 potential tracers. The likelihood of co-evaporation was done by selecting tracers with boiling points close to that of iso-octane:
- 3-Pentanone
- 2-Hexanone
- Toluene
- Fluorobenzene
- TEA
| Compound | Formula | Molecular Weight (g/mol) | Boiling Point (°C) | Density (g/cm³) | Heat of vaporisation (J/g) |
|--------------|-----------|--------------------------|--------------------|-----------------|-----------------------------|
| Iso-Octane | C₈H₁₈ | 114.23 | 99.2 | 0.6919 | 269 |
| 3-Pentanone | C₅H₁₀O | 86.14 | 101.7 | 0.8138 | 388 |
| 2-Hexanone | C₆H₁₂O | 100.16 | 127.6 | 0.812 | 363 |
| Fluorobenzene| C₆H₅F | 96.1 | 84.7 | 1.02 | 324 |
| Toluene | C₇H₈ | 92.14 | 110.6 | 0.867 | 360 |
| TEA | C₆H₁₅N | 101.19 | 89.5 | 0.725 | 306 |
Table 3-1: Properties of Iso-Octane and various tracers
Figure 3-4: Iso-octane
Figure 3-5: Toluene
Figure 3-6: Fluorobenzene
3.2.2. Absorption measurements
In order to fluoresce, the tracer must absorb at the excitation wavelength. In order for the fluorescence to be volume dependent, the entire volume of the droplet must be homogeneously illuminated. This requires an optimal absorption.
Beer-Lambert law gives the theoretical behaviour of light as it passes through an absorbing medium:
\[ I = I_0 \cdot e^{-\varepsilon \cdot c \cdot d} \]
where \( I_0 \) is the incident light energy, \( I \) is the energy after travelling the distance \( d \), \( c \) is the concentration and \( \varepsilon \) is the molecular absorptivity (or extinction coefficient).
Parker (1964) uses a more practical form of this equation with a base 10 logarithm and defines the optical density (or absorbance) as:
\[ A = \log \left( \frac{I}{I_0} \right) = \varepsilon \cdot c \cdot d \]
where \( c \) is expressed in mol.L\(^{-1}\), \( d \) in cm and \( \varepsilon \) in L.mol\(^{-1}\).cm\(^{-1}\).
Using a droplet generator and different dye concentrations, Le Gal (1999) showed that the LIF signal is proportional to $d^n$, where $n$ varied from 3.2 to 2 and found that an absorbance of 2.7 gave a diameter cubed LIF signal.
In the extreme case of a highly absorbing droplet, the incoming light will be completely absorbed near the surface (see Figure 3-11(c)). The bulk of the fluorescence will appear to come from the surface, thus yielding a fluorescence signal proportional to surface area, hence $n=2$.
In the case of low absorption, the droplet’s volume is homogeneously illuminated and the fluorescence signal is volume dependent (see Figure 3-11(a)). However, if the tracer concentration is too low, the droplet may exhibit amplified stimulated emission (ASE), where the droplet acts as a cavity with gain greater than 1. Fluorescing systems with low quantum gain, for example low quantum efficiency or less efficient internal reflection (lower refractive index relative to medium) are likely to show less pronounced ASE and have $n$ nearer 3.
Domann and Hardalupas (2000) computed the LIF signal in a droplet and showed that it was unevenly distributed and that the $d^3$ dependency was accurate for low tracer concentrations.
In their study, Yeh et al. (1996) refer to work done by Felton et al. (1993) to justify that fluorescence intensity is proportional to the third power if the droplet diameter is smaller than 80 μm. They used 1% NNN’N’-tetramethyl-p-phenylenediamine (TMPD) in their base fuel (O-solvent). Sankar et al. (1996) used water as a test liquid and added small amounts of fluorescein-di-sodium salt to obtain a low absorbing tracer system. These small concentrations will typically give low absorption mediums. *Figure 3-10* shows that such small mediums will only absorb a fraction of the incoming light, and the illumination within the droplet will be quasi-homogeneous.
In order to accurately determine the appropriate tracer concentrations, the absorbances of various fuel/tracer systems for different dye concentrations were measured using a PERKINS-ELMER Lambda 7 UV/VIS Spectrophotometer. The absorbance measurements were done in a 1 cm path-length fused silica cuvette. The following graphs represent absorbance as a function of wavelength. The red dot represents the ideal point (i.e. $A=2.7$ at $\lambda=266$ nm).
*Figure 3-12: Absorption measurement for 1% 3-Pentanone in Iso-Octane*
Figure 3-13 - Absorption measurements for 0.3% Fluorobenzene in Iso-Octane
Figure 3-14 - Absorption measurements for 0.2% Toluene in Iso-Octane
Figure 3-15: Absorption measurements for 2-Hexanone in Iso-Octane
Figure 3-16: Absorption measurements for TEA in Iso-Octane
The tracer concentrations which give a 2.7 absorbance at 266nm (the excitation wavelength of this study) are summarised in Table 3-2. These values give an indication of the maximum tracer concentration. Concentrations below these figures will still satisfy the diameter cubed dependence, as long as the tracer does not exhibit ASE.
Furthermore, the absorption spectrums of the two ketones (3-Pentanone and 2-Hexanone) peak at 277nm, so their variation of absorption around 266nm (the excitation wavelength for this work) is relatively small. As the absorption spectrum shifts with temperature, this is an indication that the temperature dependence of the fluorescence of these two compounds is likely to be weak.
| Tracer in Iso-Octane | % Volume in Iso-Octane | Slope (nm$^{-1}$) |
|----------------------|------------------------|------------------|
| 3-Pentanone | 1.5 | 6% |
| 2-Hexanone | 1.5 | 6% |
| Toluene | 0.2 | 25% |
| Fluorobenzene | 0.3 | 50% |
| TEA | 12 | 25% |
Table 3-2: Tracer concentrations for an optimal absorbance
3.2.3. Temperature dependence
Another requirement for the tracer is that its fluorescence is independent of temperature. Experiments were carried out to investigate the temperature dependence of liquid-phase fluorescence for an excitation wavelength of 266nm.
A fused silica cuvette, containing Iso-octane and the fluorophore, was inserted in a rectangular brass container heated by four cylindrical heaters in each corner (see Figure 3-17).
**Figure 3-17: Brass container, heaters and cuvette**
The Spectra Physics PIV400 Nd:YAG laser was used. It generated 532 and 266nm wavelengths. The beams were separated using a Calcium Fluoride (CaFl) prism. The 532nm beam was then reflected at 90 degrees and dumped (see Figure 3-18).
A Flowmaster 3 CCD Camera coupled to a UV 105mm Nikon lens recorded the LIF images. A BG37 filter (see Figure 3-19) cut out the Mie scatter occurring at 266nm.
**Figure 3-18: Experimental set-up for temperature dependence of LIF**
The computer was the experiment master and the Davis imaging software from LaVision used to trigger the laser and the camera. The image processing was also done with Davis.
**Figure 3-19: Transmittance curve for the BG37 filter**
The heaters were turned on and off with a switch, and the temperature of the brass container was measured by a thermocouple. The container was heated and maintained at a stable temperature for 1 minute before the images were recorded, in order to allow the fuel/tracer mixture to reach that temperature. 60 images were recorded. To eliminate laser fluctuations while the laser warms up, only the last 30 images were averaged to give a final image. 2 rectangles within the image were selected and the average intensity within each rectangle was extracted.
**Figure 3-20: Fluorescence in the cuvette - rectangle**
A sensitivity study showed that the variations in intensity from one rectangle to the other were identical (see *Figure 3-21*).
Figure 3-21: Intensity variation in 2 different rectangles
The following set of graphs present the variation of fluorescence intensity at various temperatures.
Figure 3-22: 3-Pentanone - temperature dependence of fluorescence
Figure 3-23: 2-Hexanone - temperature dependence of fluorescence
Figure 3-24: Toluene - temperature dependence of fluorescence
Figure 3-25: Fluorobenzene - temperature dependence of fluorescence
Figure 3-26: TEA - temperature dependence of fluorescence
For all tracers, except Fluorobenzene, the liquid phase fluorescence showed little change below boiling point, the worst case being 10%. Part of that variation can be attributed to experimental error, due to laser fluctuations from shot to shot, and deviation in power as the experiment progressed. The measurement points closer to 100°C show a positive deviation. This deviation is the smallest for 2-Hexanone, whose boiling point is 25°C higher than that of Iso-Octane.
The increase of the intensity around and beyond the boiling point can be attributed to the formation of bubbles in the cuvette. The resulting layer of gas-phase decreases the absorption. This in turn increases the laser power at the measurement volume and therefore the fluorescence intensity.
### 3.2.4. Pressure dependence
Because the liquid phase is quasi-incompressible, the distribution of the tracer within the liquid fuel should remain unchanged with pressure. To investigate the pressure dependence of the liquid phase fluorescence, the same type of experiment as the temperature study was carried out in a high pressure vessel. The fluorescence of the fuel tracer within a quartz cuvette was recorded on an intensified CCD camera. 5 Pressures were investigated: 1, 5, 10, 15, and 20 bar. When the vessel had reached the desired pressure, 200 images were recorded, and an average of the last 100 was calculated. The fluorescence intensity profile of the average image was plotted.
**Figure 3-27:** Fluorescence intensity profile across the cuvette for 3-Pentanone
As expected, the variation of the profile was weak. A first experiment had consisted in taking images at increasing pressures (1, 5, 10, 15 and 20bar). It was observed that the profiles were very similar, but decreasing with pressure. The experiment was reiterated by choosing another order for the pressures (1, 10, 20, 15 and 5bar), and it was found that the decrease of the profile’s intensity was actually a function of time. This decrease is due to the breaking down of the ketone bond (C=O) from the repetitive UV excitation (Tait 1994).
The deviation of the profile from the Beer-Lambert’s law can be attributed to the honeycomb structure which was clearly visible with the intensifier used.
All the tracers tested verified that the liquid phase fluorescence is independent of pressure.
### 3.2.5. Tracer evaporation
#### 220.127.116.11. Background
The fluorescence signal of a droplet is a function of the tracer volume (concentration) within the droplet. In order to maintain the diameter cubed dependence of the LIF signal, the tracer’s relative concentration should not change during evaporation. It is absolutely crucial that the tracer evaporates at the same rate as the fuel.
Two extreme regimes of droplet evaporation must be considered (Law 1982). In the rapid evaporation (also referred to as diffusion-limited evaporation), the rate of surface regression is one or two orders of magnitude greater than the rate of liquid-phase mass diffusion. This “freezes” the composition of the droplet, and the evaporation is homogeneous. On the other hand, in the slow evaporation regime, the compounds at the surface of the droplet will evaporate at different rates and the diffusion will redistribute the compounds. This is also known as the distillation regime.
For conditions below the saturation curve (e.g. a spray injected in ambient conditions -25°C and 1 bar) the evaporation will occur in the slow regime. Around boiling conditions, the vaporisation can be either slow or fast, depending on the initial size of the droplets. In the superheated regime (i.e. for conditions above the saturation curve), when the spray is injected in the late part of the compression stroke (e.g. 300°C and 10bar), rapid evaporation will take place.
3-Pentanone has been the preferred tracer for AFR measurements because of its close matching boiling point with Iso-Octane (99.2°C vs. 101.7°C at 1 bar) and because its fluorescence is well characterised (Ghandi and Felton (1996), Grossman et al. (1996). However, Le Coz et al. (1994) reported that the molar fraction of 3-Pentanone in the vapour phase was twice that of the liquid phase for a 5/95 mixture in Iso-octane, and suggested the use of 2-Hexanone because of its lower volatility. Davy et al. (2003) also indicated that 3-Pentanone is not a suitable tracer at low concentrations due to its preferential evaporation when mixed with Iso-octane, even though it’s boiling point is higher. This is because 3-Pentanone and Iso-Octane form an azeotrope.
An azeotrope is a mixture of liquids that has a constant boiling point and thus cannot be separated by distillation. Ethanol (boiling point 78.5°C) and water (boiling point 100°C) form a binary azeotrope having a boiling point of 78.2°C and a composition that is 95.6% ethanol. Therefore, a mixture of 95% ethanol and 5% water will co-evaporate.
In the case of non-azeotropic mixture, a close matching boiling point will be beneficial. However, for mixtures which form an azeotrope, the concentrations should match the azeotropic mixture. However, if that mixture has a too high absorbance, than it will not be suitable.
### 18.104.22.168. Vapour-Liquid Equilibrium Calculations
#### 22.214.171.124.1. The model
To characterise and calculate the differential evaporation of various tracer systems in Iso-Octane, a model using the gamma-phi method following the guidelines from Malanowski and Anderko (1992) was developed for flash calculations at a fixed temperature.
The liquid vapour equilibrium (VLE) of any component within a multi-component mixture is given by:
\[
y_i \cdot P \cdot \Phi_i = x_i \cdot \gamma_i \cdot P_i^o
\]
Equation 3-16
where:
\[ y_i: \quad \text{vapour mole fraction of component } i \]
\[ P: \quad \text{equilibrium pressure} \]
\[ \Phi_i: \quad \text{fugacity coefficient/correction factor for the non-ideality of the vapour phase of compound } i \]
\[ x_i: \quad \text{liquid mole fraction of component } i \]
\[ \gamma_i: \quad \text{activity coefficient of component } i \]
\[ P_i^o: \quad \text{vapour pressure of pure component } i \]
For low pressures, the fugacity coefficient can be considered equal to 1.
The activity coefficients can be estimated using the Wilson (1964) equation:
\[
\ln \gamma_i = -\ln \left[ \sum_{j=1}^{n} x_j \cdot \Lambda_{i,j} \right] + 1 - \sum_{k=1}^{n} \frac{x_k \cdot \Lambda_{k,i}}{\sum_{l=1}^{n} x_l \cdot \Lambda_{k,l}}
\]
Equation 3-17
At infinite dilution, the activity of component \( i \) over component \( j \) is extrapolated from the case where the molar fraction of component \( i \) is 1 and the molar fraction of \( j \) is 0.
This leads to:
\[
\ln \gamma_{i,j}^\infty = -\ln \Lambda_{i,j} - \Lambda_{j,i} + 1
\]
Equation 3-18
\[
\ln \gamma_{j,i}^\infty = -\ln \Lambda_{j,i} - \Lambda_{i,j} + 1
\]
Equation 3-19
Parameters \( \Lambda_{ij} \) are found from simultaneous solution of the above relations for two components at infinite dilution.
The activity at infinite dilution can be found using the Modified Separation of Cohesive Energy Density model (MOSCED) proposed by Thomas and Eckert (1984). It is based on the assumption that forces contributing to the cohesive energy density are additive. Forces included are dispersion, orientation, induction and hydrogen bonding. The five parameters associated to these forces are the dispersion parameter $\lambda$, the induction parameter $q$, the polar parameter $\tau$, and the acidity and basicity parameters (respectively $\alpha$ and $\beta$). These modifications affect the activity coefficients in a symmetric way, contrary to experiment. Thomas and Eckert introduce two more parameters ($\psi$ and $\xi$) to account for asymmetry effects resulting from differences in polarity and degree of hydrogen bonding respectively. These two are functions of other parameters.
The activities at infinite dilution are given by:
$$\ln \gamma_{i,j}^\infty = \frac{\nu_i}{RT} \cdot \left[ (\lambda_i - \lambda_j)^2 + \frac{q_i^2 \cdot q_j^2 \cdot (\tau_i - \tau_j)^2}{\psi_j} + \frac{(\alpha_i - \alpha_j) \cdot (\beta_i - \beta_j)}{\xi_j} \right] + d_{j,i}$$
Equation 3-20
where $\nu_i$ is the liquid molar volume at 20°C in cm$^3$.mol$^{-1}$ and is given by:
$$\nu = \frac{M}{\rho}$$
Equation 3-21
with $M$ the molecular weight in g.mol$^{-1}$ and $\rho$ the density in g.cm$^{-3}$.
$d_{ij}$ is the Flory-Huggins combinatorial term to account for differences in molecular size:
$$d_{ij} = \ln \left( \frac{\nu_j}{\nu_i} \right)^{aa_j} + 1 - \left( \frac{\nu_j}{\nu_i} \right)^{aa_j}$$
Equation 3-22
Parameters $\alpha$, $\beta$, $\tau$, $\psi$, $\xi$ and $aa$ are temperature dependent:
$$\alpha = \alpha_0 \cdot t^{0.8}$$
Equation 3-23
\[ \beta = \beta_0 \cdot t^{0.8} \]
\[ \tau = \tau_0 \cdot t^{0.4} \]
The subscript 0 refers to 20°C.
\[ \psi = POL + 0.011 \cdot \alpha \cdot \beta \]
\[ \xi = 0.68 \cdot (POL - 1) + \left\{3.4 - 2.4 \cdot \exp\left[(-0.023) \cdot (\alpha_0 \cdot \beta_0)^{1.5}\right]\right\}^2 \]
\[ POL = q^4 \cdot \left[1.15 - 1.15 \cdot \exp(-0.02 \cdot \tau^3)\right] + 1 \]
\[ aa = 0.953 - 0.00968 \cdot \left(\tau^2 + \alpha \cdot \beta\right) \]
Having determined the activities and the saturated pressures (for a fixed liquid fraction), the equilibrium pressure is:
\[ P = \left( \sum_{i=1}^{n} y_i \right) \cdot P = \sum_{i=1}^{n} (y_i \cdot P) = \sum_{i=1}^{n} (x_i \cdot \gamma_i \cdot P_i^o) \]
The vapour fraction of a component \( i \) can then be calculated as:
\[ y_i = \frac{x_i \cdot \gamma_i \cdot P_i^o}{P} \]
126.96.36.199.2. The parameters
The parameters required for the MOSCED calculation were taken from the literature (Reid et al. 1987). All but one compound were found in a table. The parameters for 2-Hexanone were estimated by the following equations:
\[
\alpha_0 = C_a \cdot \left( \frac{4.5}{3.5 + N_C} \right) \cdot \left( 1 + \frac{N_C - 1}{100} \right)
\]
Equation 3-32
\[
\beta_0 = C_\beta \cdot \left( \frac{4.5}{3.5 + N_C} \right) \cdot \left( 1 + \frac{N_C - 1}{100} \right)
\]
Equation 3-33
\[
\tau_0 = C_r \cdot \left( \frac{4.5}{3.5 + N_C} \right) \cdot \left( 1 + \frac{N_C - 1}{100} \right)
\]
Equation 3-34
where \(N_C\) is the number of carbon atoms in the molecule. For ketones, \(C_a = 0\), \(C_\beta = 4.87\) and \(C_r = 3.93\).
For saturated compounds, \(q_0 = 1\).
The values of the different parameters are found in Table 3-3.
| Compound | \(v_0\) | \(\lambda_0\) | \(\tau_0\) | \(q_0\) | \(\alpha_0\) | \(\beta_0\) |
|--------------|---------|---------------|------------|---------|-------------|-------------|
| Iso-Octane | 165.1 | 7.84 | 0.00 | 1.00 | 0.00 | 0.00 |
| 3-Pentanone | 105.8 | 7.86 | 2.77 | 1.00 | 0.00 | 3.43 |
| 2-Hexanone | 123.3 | 7.85 | 1.95 | 1.00 | 0.00 | 2.42 |
| Toluene | 106.3 | 8.45 | 1.56 | 0.90 | 0.15 | 1.60 |
| TEA | 139.0 | 7.52 | 0.53 | 1.00 | 0.00 | 4.98 |
Table 3-3: MOSCED parameters for various compounds at 20°C
The vapour pressures are usually not well documented for values above the boiling point at atmospheric conditions. However, the data for Toluene is almost complete for temperatures ranging from 0°C to the critical point (318°C). Figure 3-28 represents the
saturation curve of Toluene. The values for the different series are obtained using the Antoine equation which expresses the vapour pressure $P$ (in bar) as function of the temperature $T$ (in Kelvin):
$$\log(P) = A - \frac{B}{T + C}$$
Equation 3-35
where $A$, $B$ and $C$ are the Antoine parameters determined empirically. These parameters are usually defined for a specific temperature range.
The extrapolation of the data points between Series 1 and 2 and Series 2 and 3 fit well. Also, Series 3 can be extrapolated to the critical point. These features were used to approximate the saturation curves for the other compounds.
The critical points can be found in the CRC Handbook of Chemistry and Physics and the Antoine parameters for some temperature ranges were found in the NIST Chemistry WebBook. The other saturation points were determined using the generalised enthalpy diagram. The saturation curves are represented in Figure 3-29, and the vapour pressures at 4 different temperatures can be found in Table 3-4.
Figure 3-29: Saturation curves for various compounds
| Compound | 50°C | 100°C | 150°C | 200°C |
|--------------|------|-------|-------|-------|
| Iso-Octane | 0.19 | 1.13 | 3.97 | 9.80 |
| 3-Pentanone | 0.15 | 0.95 | 3.56 | 9.79 |
| 2-Hexanone | 0.03 | 0.34 | 2.03 | 6.49 |
| Toluene | 0.12 | 0.74 | 2.75 | 7.46 |
| TEA | 0.26 | 1.38 | 5.03 | 13.24 |
Table 3-4: Vapour pressures (in bar) at 4 different temperatures
188.8.131.52.3. The programme
A programme was written in C++ to calculate the equilibrium pressure and vapour molar fraction as a function of liquid molar fraction of the tracer system (see Figure 3-30). The calculation was done for liquid molar fractions ranging from 0 to 1 in step of 0.001. This allowed to build the vapour-liquid equilibrium diagram and understand the evaporative behaviours of the tracers in Iso-Octane.
The programme was validated by tracing the 3-Pentanone boiling and condensation curves for 50°C (see Figure 3-31) and comparing them with the ones from Davy et al. (2003). In their work, Davy et al. (2003) used empirical data from Fuchs et al. (1984) which use slightly different vapour pressures than the ones obtained from the literature. For the purpose of the validation, those vapour pressures were used.
Figure 3-30: Programme structure for the distillation calculations.
Figure 3-31: Vapour-liquid equilibrium diagram for the 3-Pentanone/Iso-Octane system at $50^\circ C$
A second programme was used to determine the evolution of the fluorescence intensity relative to the volume of a droplet (see Figure 3-32).
**Figure 3-32: Programme structure for the calculation of fluorescence variation in an evaporating droplet**
The calculation consisted of 1000 steps, corresponding to the removal of 1 mole from a 1000 mole droplet. For each numerical step, the vapour molar fraction of the removed mole was determined to recalculate the composition of the liquid-phase mixture. Assuming that the fluorescence is not quenched and that the droplet is homogeneously
illuminated droplet (volume dependent signal), the fluorescence intensity was directly proportional to the tracer concentration in the remaining volume.
184.108.40.206. Results for single tracer systems
For each tracer, two graphs are plotted.
The first graph presents the vapour molar fraction as a function of the liquid molar fraction. This indicates whether the tracer or the Iso-Octane is preferentially expelled. It also allows to notice if the tracer/fuel mixture forms an azeotrope, and if so, at which mixture concentration.
The second graph presents the fluorescence of a droplet as a function of the volume, as the droplet evaporates. For the purpose of this programme, the fluorescence is proportional to the tracer concentration (i.e. there is no quenching). The fluorescence and the volume have initial values of 100[a.u.].
220.127.116.11.1. 3-Pentanone
Figure 3-33: Liquid-vapour equilibrium of 3-Pentanone in Iso-Octane for different temperatures
Figure 3-31 showed that the boiling and condensation curves go through an extremum (a maximum in the case of 3-Pentanone and Iso-Octane), characteristic of a (positive) azeotrope. Figure 3-33 shows the azeotrope’s shift with temperature: at $50^\circ$C, the azeotropy is for a 40/60 mixture whilst at $200^\circ$C the azeotrope corresponds to a 60/40 mixture.
However, the azeotrope occurs at high concentrations of 3-Pentanone which are not suitable for the volume dependence fluorescence of the droplets studied.
At low tracer concentrations, the mixture will preferentially evaporate the 3-Pentanone, even though its boiling point is higher.
A 2% 3-Pentanone liquid composition will have a vapour composition of 4.1 % at $50^\circ$C and 3.1 % at $200^\circ$C.
**Figure 3-34:** Fluorescence vs. Volume for an evaporating droplet containing 2% 3-Pentanone
*Figure 3-34* demonstrates the importance of differential evaporation. When the droplet has half evaporated, the fluorescence intensity has decreased by 80%, already yielding an error greater than 50%. The differential evaporation decreases with temperature, but would still be unsatisfactory for slow evaporation regimes.
18.104.22.168.2. 2-Hexanone
Figure 3-35: Evaporation of 2-Hexanone in Iso-Octane for different temperatures
Hexanone and Iso-Octane do not form an azeotrope. Because 2-Hexanone’s saturation curve is higher than Iso-Octane’s, there is preferential evaporation of the Iso-Octane: the fluorescence signal will increase relative to the volume as the droplet evaporates (see Figure 3-35). A 2% liquid mixture will have a vapour composition of 1.1% at 50°C and 1.7% at 200°C.
Figure 3-36: Fluorescence vs. Volume for an evaporating droplet containing 2% 2-Hexanone
22.214.171.124.3. Toluene
Figure 3-37: Evaporation of Toluene in Iso-Octane for different temperatures
Like the 2-Hexanone/Iso-Octane mixture, the Toluene/Iso-Octane mixture will preferentially evaporate the fuel. A mixture of 0.3% liquid Toluene will have a vapour composition of 0.44% tracer at 50°C and 0.4% at 200°C. Because the Iso-Octane is preferentially evaporated and is in much higher quantity than the Toluene, the relative change in concentration should be negligible. Figure 3-38 demonstrates this feature: the differential evaporation is weak, and becomes even weaker at higher temperatures.
Figure 3-38: Fluorescence vs. Volume for an evaporating droplet containing 0.3% Toluene
126.96.36.199.4. TEA
Figure 3-39: Evaporation of TEA in Iso-Octane for different temperatures
TEA does not form an azeotrope in Iso-Octane (see Figure 3-39). Because of its lower saturation curve relative to Iso-Octane, TEA will be preferentially evaporated. A 3% TEA liquid mixture will have a vapour composition of 4.3% and 4% at 50°C and 200°C respectively. The differential evaporation is weaker than for 3-Pentanone (see Figure 3-40).
Figure 3-40: Fluorescence vs. Volume for an evaporating droplet containing 3% TEA
188.8.131.52.5. Conclusion
The tracers can be categorised either as expellers (Toluene and 2-Hexanone) or expulsed (TEA and 3-Pentanone). In the case of non azeotropic mixtures, this can be predicted by the saturation curve, and the closer matching curves are optimal. In the case of azeotropic mixtures like 3-Pentanone in Iso-Octane, quasi-identical saturation curves are deceptive in 2 ways: they give a false impression of co-evaporation, and their relative position can mislead as to which of the two compounds will be preferentially expelled.
Toluene and TEA evaporation characteristics are the closest to co-evaporation. Their boiling points are within Iso-Octane’s ± 10°C. However, their quenching behaviour is the limiting factor for measurements in a fired engine.
On the other hand, ketones show very little quenching in oxygen (Tait 1994), so they are more appropriate from that point of view for the LSD technique. However, as has just been shown, 3-Pentanone and 2-Hexanone do not satisfy co-evaporation behaviours. Nevertheless, because they exhibit opposite expulsion behaviours, the combination of these 2 tracers could balance out the differential evaporation.
However, the two tracers don’t have the same quantum yield: even if a dual tracer system evaporates homogeneously, the fluorescence will not be an adequate indication of the volume. A compromising tracer composition should be possible to optimise the tracer system’s fluorescence with an evaporating droplet.
The programmes described previously were adapted for 3-component mixtures.
184.108.40.206. The 3-Pentanone / 2-Hexanone tracer system
At the time of this work, Han and Steeper (2002) suggested a mixture of 3-Pentanone and 3-Hexanone to balance the differential evaporation. They found their ratio of quantum yields to be 5. While imaging a spray in the pressure vessel at BMW AG, it was found that changing the tracer from 2% 3-Pentanone to 2% 2-Hexanone necessitated an increase of the intensifier’s gain by 10 (68 to 78) in order to match the fluorescence intensity. From the intensifier’s data sheet, the ratio of quantum yields was calculated to be 4.3.
The two ketones have similar absorption/absorbance spectrums. As seen previously, both tracers could be used individually at a concentration of 2% in Iso-Octane. Therefore, the dual tracer system could also have a concentration of 2%. The evaporation of a droplet was simulated for 5 mixtures of 3-Pentanone and 2-Hexanone in 98% Iso-Octane. These mixtures ranged from 3-Pentanone to pure 2-Hexanone.
For convenience, a mixture of $x\%$ 3-Pentanone, $y\%$ 2-Hexanone $z\%$ Iso-Octane is referred to as $x/y/z$.
**Figure 3-41:** Fluorescence vs. Volume for an evaporating droplet at 50°C containing various 3-Pentanone / 2-Hexanone mixtures in 98% Iso-octane
At 50°C, the 0.25/1.75/98 mixture balances perfectly the co-evaporation and the quantum ratio for the first 40% of the volume evaporation, but yields an increasing discrepancy afterwards.
With a 0.5/1.5/98 mixture composition, at the start of the evaporation, the fluorescence signal is underestimating the droplet size by a maximum error of 13%. At the lower end of the evaporation, the signal is overestimated by a maximum of 33%.
The LIF from a 1/1/98 mixture will underestimate the volume during most of the evaporation with a maximum error of 42% and will overestimate the rest of the evaporation.
Figure 3-42: Fluorescence vs. Volume for an evaporating droplet at 100°C containing various 3-Pentanone / 2-Hexanone mixtures in 98% Iso-octane
As the temperature is increased to 100°C, the curves for the single tracers get closer to the $y=x$ line, signifying that the compromise for the tracer system is much more open (see Figure 3-42).
The 1/1/98 mixture still exhibits a 39% underestimation of the volume, but the 0.5/1.5/98 mixture is precise within 11% for 85% of the volume evaporation. Once again the 0.25/1.75/98 mixture is perfect for the first half of the evaporation, but the excess 2-Hexanone makes this mixture’s behaviour overestimate the volume by a factor of 3.6 for the rest of the evaporation.
As the temperature is further increased, (see Figure 3-43 and Figure 3-44), the narrowing of the curves around the $y=x$ line is still improved. At 150°C, the 1/1/98 and 0.5/1.5/98 mixtures underestimate the volume throughout the whole evaporation, with maximum errors of 51% and 22% respectively. The error with the 0.25/1.75/98 mixture is within 4% for most of the evaporation and only increases within the last 5% of the remaining volume.
At 200°C, the 0.25/1.75/98 mixture is optimal, with a worst-case underestimation and overestimation error of 17% and 1.6% respectively.
Figure 3-43: Fluorescence vs. Volume for an evaporating droplet at 150°C containing various 3-Pentanone / 2-Hexanone mixtures in 98% Iso-octane
Figure 3-44: Fluorescence vs. Volume for an evaporating droplet at 200°C containing various 3-Pentanone / 2-Hexanone mixtures in 98% Iso-octane
In the case of a non-quenching environment, Toluene would be the obvious simple choice as regards tracer evaporation. In the presence of oxygen, depending on the temperature, the optimal 3-Pentanone / 2-Hexanone tracer system will vary slightly. Below 100°C, the 0.5/1.5/98 mixture is the best compromise between the fluorescence’s
initial underestimation and final overestimation of the volume. For temperatures above 150°C, the most adequate would tend to be 0.25/1.75/98.
But for more specific use such as in engines, for temperatures or pressures above 200°C and 5bar, the fuel and tracer(s) will be in the superheated regime, where the evaporation is diffusion limited, and the differential evaporation should be minimal.
3.3. Imaging for the LSD technique
3.3.1. Imaging Optics
There are several methods to collect the Mie scatter and Fluorescence images. The most appropriate is to collect them simultaneously. The easiest – and most expensive – method requires the use of 2 intensified cameras, one for each image. A cheaper option is to use one camera onto which both images can be recorded. This single camera technique requires image separation optics. The following paragraphs presents several ways to achieve this.
220.127.116.11. The stereoscopic imager
The stereoscopic imager is a very compact design that consists of two front apertures with mirrors to redirect the incoming light out a third aperture situated in the back (see Figure 3-45)
On the front left aperture, a BG 4 band pass filter can be fitted to select the LIF and reject the Mie scatter.
On the other front aperture, a 266nm interference filter selects the Mie scatter signal only. A neutral density (ND) filter is added to the interference filter to attenuate the Mie signal, in order to obtain LIF and Mie intensities of the same order of magnitude on the CCD chip.
However, one of the drawbacks of this imaging system is the small size of its mirrors, which gives a weak light collection (10% efficiency). As the LIF signal is already weak, this type of imager is not suitable for our experiments.
### 18.104.22.168. The prism set-up
The following optical set-up by Park et al. (2002) uses a doubling prism. On one half, a band pass filter and a ND filter selects and attenuates the Mie scatter signal. On the other half, two long wavelength pass filters select the LIF and reject the Mie scatter (see Figure 3-47).
In the presence of 532nm light, an appropriate filter can be added to the two long-pass filters.
The stereoscopic imager and the prism share a common drawback: they both reject half of the intensity from each signal. This is an issue, considering the weak fluorescence intensity of 2-Hexanone and 3-Pentanone.
### 22.214.171.124. The 2 lens set-up
This set-up is adapted from the separating optics used by Kelman and Masri (1994). It uses two lenses. A glass lens focuses the fluorescence on the intensifier and rejects the Mie scatter. The Mie scatter signal is collected on a different path. It is redirected into the intensifier using dichroic mirrors and focused using a quartz lens of longer focal length (because the optical path is longer than that of the fluorescence).
**Figure 3-48: The 2-lens set-up**
In the presence of additional 532nm light, an appropriate filter (e.g. BG4) can be located in front of the glass lens.
The light collection angle is increased. However, this set-up, like the two previous others, still suffers from a major drawback: the stereoscopic view of the object. Because the two light paths are not the same, in a dense spray, the signal collected through the Mie path does not go through the same parts of the spray as the signal collected for the LIF path (see *Figure 3-49*). This induces error that cannot be quantified.
126.96.36.199. The 5 mirror set-up
Le Gal (1999) used a 5-mirror system to separate the Mie scatter from the fluorescence. It consists of 2 dichroic mirrors and 3 broadband mirrors, all 2 inch in diameter and positioned at an angle of $22.5^\circ$ (see Figure 3-50).
In this setup, the light is reflected off a broadband mirror. It comes onto a 266nm dichroic mirror (quartz glass with a 266nm coating) which reflects the Mie signal and lets the LIF through. Both signals are then reflected on two different broadband mirrors on adjustable mounts. The signals rejoin at the second dichroic mirror where the Mie signal is reflected and the LIF signal goes through. Both signals have been separated using the adjustable mirrors, but they originate from the same light path. Another
advantage of the set-up is that the camera is positioned parallel to the incoming light path.
A neutral density filter is in the Mie signal path to attenuate the signal’s intensity in order to match the weaker LIF intensity.
A BG4 filter is in the LIF path to cut out the eventual Mie scatter that could have been transmitted through the dichroic mirrors. It also cuts out 532nm Mie scattering because the laser sheet contains both second and fourth harmonics (532 and 266nm).
However, this set-up is too long: the mirrors represent increasingly smaller apertures as they are further away from the camera.
### 188.8.131.52. The 4 mirror set-up
This setup is based on Le Gal (1999) 5-mirror imaging system. It abandons the first broadband mirror, and the other mirrors are mounted at $45^\circ$. The camera is therefore mounted orthogonally to the incoming light (see Figure 3-51). Because this set-up is more compact and contains fewer mirrors, the aperture phenomenon is improved.
However, because the mirrors are mounted at $45^\circ$ instead of the previous $22.5^\circ$, their effective surface is narrower, and the set-up must be finely tuned for imaging. For this reason, the same short set-up was designed with 3-inch mirrors.
3.3.2. Geometric Calibration
The LSD technique requires the ratio of fluorescence and scattered light from the same droplets. From an imaging point of view, the technique requires that the intensity from a pixel in the LIF image is divided by the intensity of a geometrically corresponding pixel in the Mie scatter image. In other words, the two scattering images must be superimposed. However, the use of a single camera with a 4 mirror set-up will rotate, translate and project the images in the separation process. Therefore, a geometric calibration of the camera is required to superimpose the LIF and Mie scatter image perfectly: each part of the CCD chip must have its own calibration to rotate, de-warp and translate the image so that the two will superimpose in order to match for the division in the LSD technique.
The simplest method requires the illumination of three non-aligned points. By finding their pixel co-ordinates, the rotation, relative warping and translation can be obtained.
Instead of using the 3-point calibration, the DaVis software from LaVision has a calibration procedure which requires the image of regularly spaced crosses (or dots). The software finds the position of each cross and maps the correction so that the corrected image looks like the original image.
3.3.3. Calibration procedure
In a first step, the optical system must be set up. This requires the adjustment of the 4-mirror system, the focusing of the lens and the camera should have a resolution of approximately $100\mu m/pixel$.
This can be done by the following sequence:
Install the system
- Install the calibration plate in the imaging plane.
- Install the 4-mirror and camera system
- Check that all mirrors are at $45^\circ$
Calibration plate illumination
- Illuminate the calibration plate with a lamp
- Take images
- Adjust the LIF mirror to position the calibration image on one half of the camera
Camera and lens rough adjustment (using the LIF path)
- Adjust the camera’s distance and focusing to obtain a resolution of approximately $100\mu m / \text{pixel}$
- Stop taking images
- Decrease the Intensifier Gain back to 0
Mie scatter path mirror adjustment
- Block off the LIF path
- Start Taking Images
- Remove all filters in the Mie scatter path.
- Illuminate the calibration plate with 266nm light and adjust Intensifier Gain
- Adjust mirror to position the Mie scatter calibration image on the other half of the camera
- Fine adjust the focusing of the lens using the Mie scatter image.
Following the adjustment of the mirrors, the following step consists in imaging the spray in realistic conditions in order to determine the ND filters required for the attenuation of the Mie scatter signal. The adjustment mirrors can also be tuned to eliminate overlap of noise or reflections from one image onto the other’s spray image.
With the correct filters in place and the mirrors adjusted, the calibration plate should be re-inserted and calibration images for the Mie scatter and LIF path must be recorded. It was found that illuminating the calibration plate with the laser light sheet provided both
a Mie scatter and fluorescence image of the calibration plate. The fine adjustment of the light sheet at an incoming small angle was absolutely crucial, as was the quality of the calibration plates. An aluminium plate or sheet was used at the other end of the calibration plate to reflect and diffuse the light for optimal illumination.
Chapter 4
Experimental Setup for LSD Validation
To investigate the validity of the LSD technique for a highly evaporating GDI spray, measurements were carried out in Munich in the Benzin-Direkt-Einspritzung Optischer Prüfstand (BDE-OP – Gasoline Direct Injection Optical Test Bed) at BMW AG. This chapter presents and describes the test rig. It consists of two pressure vessels: the first vessel enabled simultaneous measurements of Mie scatter and LIF images on a GDI spray and to apply the LSD technique. The second vessel allowed acquisition of PDA data for the same spray in order to compare the two techniques.
Figure 4-1: View of the 2 Pressure Vessel rigs (Photo: BMW AG)
4.1. The test rig
The test rig is a 1.5 million Euro facility composed of 2 fully automated pressure vessels for laser diagnostics on injectors: one allows for 2-D imaging of LIF and Mie scatter, the other is for PDA.
The design of the vessel is by RWTH Aachen’s ITM department headed by Professor Peters. The automation of the test rig is by Sonplas. The imaging system and software for the Mie scatter/LIF vessel is from LaVision and the system for the PDA vessel was originally from TSI and was later replaced by Dantec.
The vessels enable the simulation of various temperature and pressure conditions under which injectors have to operate. To do so, the inside of the vessels are pressurised and heated with continuous flow of Nitrogen. The maximum operating conditions are $400^\circ\text{C}$ and 35bar.
4.1.1. Vessel design
The basic design of each vessel consists of two concentric cylinders (see Figure 4-2). The outer cylinder is 60mm thick to resist to pressures up to 35bar. It is closed at the top and bottom. The pressure tightness is ensured by rubber O-rings which are water cooled. The inner cylinder is 105mm in diameter and 260mm high. It contains the Nitrogen flow and the injector spray. It is open to the outer cylinder, so that it is not subject to pressure differentials. Its main role is to insulate the thick outer cylinder in order to reduce the thermal inertia of the system: this allows the experiment to proceed from one set of Pressure and Temperature conditions to the next without having to cool down or warm up the bulk of the pressure vessel.
4.1.2. Optical Access
Optical access inside the pressure vessel is possible through windows. Both vessels have inserts in the outer cylinder to accommodate for 35mm thick outer side windows. Window holders are bolted to the vessel with 12 nuts, and allow an apparent window aperture of 60mm×140mm. Each outer window has an inner window counterpart inserted in the inner cylinder. Both vessels also have a bottom window to allow for imaging from below.
4.1.3. Vessel Conditioning
Liquid Nitrogen is stored in a pressurised 3000 Litre tank. It is delivered to the vessels by evaporation, thus avoiding using an expensive and high maintenance pump. In order to operate the rig continuously, a valve and a throttle (one upstream and one downstream of the vessel) control the pressure inside the chamber while continuously purging the gas flow. For chamber pressures below 2.5bar, a suction pump is activated in order to produce a sufficient pressure difference between the inlet and the outlet of the vessel to maintain a permanent purging gas flow.
The temperature conditioning is done by passing the Nitrogen flow through heaters before it enters the vessel. A first set of heaters is used for the 25-150°C temperature range. For temperatures above 150°C, a second set of heaters is used, and the Nitrogen flow has to be at a minimum of 60Nm$^3$/h to avoid damaging them.
To allow for space around the top of the vessel, the nitrogen enters the chamber through 3 pipes which connect to the side of the head (see Figure 4-3).
4.1.4. Injector
The injector is inserted and held in the injector mount. Originally, the pressure vessel’s design only allowed positioning of the injector mount on top of the chamber, injecting downwards. The vessel design was modified to have the possibility to operate with a motorised side mount, allowing automatic rotation of the injector. Therefore, the injector can sit either on the top of the chamber and inject downwards, or can be installed on the side, injecting towards the side windows. Whichever position is not occupied by the injector is replaced by a blank.
The injector temperature is conditioned by a silicon oil conditioned heating/cooling unit. The injection is triggered by the Test-rig computer. A timing signal is sent to the injector driver, which delivers the appropriate power signal to the injector.
Figure 4-3: Schematics of the pressure vessel
4.1.5. Fuel
Fuel is drawn from the fuel tank by a piston pump. Once the pump is full, it can pressurise the fuel up to a maximum pressure of 200bar. The pressure is maintained by moving the pump piston with a fast stepping motor. This enables the delivery of fuel at a constant pressure - therefore pulsation-free, contrary to a rotating pump system.
Before entering the injector, the fuel is conditioned by another silicon oil bath in the pump and in a cross-flow heat exchanger near the injector.
At the exit of the vessel, the nitrogen/fuel mixture passes through heat exchangers, condensers and an active carbon filter to retrieve the UHC.
4.2. The “Mie/LIF” Pressure Vessel
This vessel allows for laser sheet diagnostics.
4.2.1. Fuel
This vessel has two separate fuel lines: one for pure iso-octane only and another for an iso-octane/tracer mixture. The non-traced and traced fuel are contained in 2 different tanks. In order to minimise contamination, each tank has a separate fuel line containing its individual pump and pipes. The pipes only rejoin 10cm upstream of the injector. Excess fuel or purged fuel is dumped in a third tank.
Figure 4-4: The “Mie/LIF” pressure vessel and its 3 side windows
4.2.2. Optical access
Optical access inside the pressure vessel is possible through 4 windows (3 rectangular side windows and 1 round bottom window). The two diametrically opposed side windows allow two opposed laser sheets to enter and exit the chamber. The third side window and the bottom window allow for the imaging.
4.2.3. Laser and sheet forming optics
The laser used is a PIV 400 Nd:YAG from Spectra Physics. It is a twin cavity laser having independent control between cavities. Each cavity consists of an oscillator and amplifier mounted in series. Both are made up of a flashlamp, rod and reflective cavity. The flashlamps are triggered at a fixed rate of 10Hz (with an allowed tolerance of ±10%) to build up the energy. This energy is released by a Q-switch: this changes the polarisation of the back plate and allows the light to pass through. For maximum power output, the laser requires an optimal delay between the flashlamp trigger signal and the Q-switch signal - typically around 180µs for this laser. Therefore, the laser cannot fire on demand.
The fundamental output beams (1064nm) are combined with beam steering optics and fed through two doubling crystals to form 532nm and 266nm beams. Typical energies are round 400mJ/pulse at 532nm and 60mJ/pulse at 266nm.
In the laser head, a first mirror allows the dumping of the fundamental immediately, reflecting the 2nd and 4th harmonics. The 532 and 266nm beams are then redirected through a 532nm beam attenuator.
The beam is then divided with a 50/50 beam splitter, to form two opposing laser sheets. This allows to obtain a more even laser power through the spray, as opposed to an attenuated sheet that traverses only one way. Because the coating had degraded and the ratio was of the order of 90/10, the beam splitter was replaced by a 266 mirror, and the laser sheet entered the vessel through the left side window only. This was possible because the laser attenuation in the LSD technique is not critical, as the ratio of two scattering images cancels it out.
The 266nm mirror directs the beam upwards. A finely adjustable $45^\circ$ mirror then redirects it into the sheet forming optics. This consists of 2 pairs of concave and convex fused silica cylindrical lenses: 2 to focus the sheet horizontally at the centre of the pressure vessel, and 2 to expand the sheet vertically. The lenses are located on a slider which is encased in a black cylinder. Circular knobs slide the lenses backwards and forwards for the adjustment of the laser sheet. The sheet-forming optics can be rotated $90^\circ$ to form either a vertical or horizontal light sheet, and are mounted on a motorised vertical translation stage, allowing to move the sheet up and down.
The laser sheet was positioned at the centre of the chamber, below the injector tip. The sheet was focused at the middle of the vessel by looking at the sheet profile at the entry and exit of the chamber. The sheet was centred on the windows, so that it was perpendicular to the chamber.
### 4.2.4. Imaging system
The separation of the Mie scatter and LIF was done using the 4-mirror set-up with 3 inch mirrors (see *Chapter 3*, §184.108.40.206). The 4-mirror set up was positioned on a sliding rail located in the axis of 2nd side window. Because the camera needed to be mounted orthogonal to the incoming light, a sliding rail was mounted on a vertical translation stage, for the positioning and adjustment of the camera (see *Figure 4-6*).
Imaging was done using the DaVis software from LaVision. The images were recorded using a 12 bit FlowMaster 3 CCD camera coupled to a gated IRO image intensifier with a Halle $f/2$ 100mm achromatic fused silica lens.
4.2.5. Timing and Synchronisation
The injection is triggered by the Test-rig computer. The Imaging computer uses this signal to trigger its Programmable Timing Unit (PTU). The PTU sequence synchronises the laser (Flashlamp and Q-switch), Intensifier and Camera exposure, so that the images occur at a specific time after the start of injection (see Figure 4-8). The user sets the imaging times in the window illustrated in Figure 4-7.
Each injector has a specific “dead time” which corresponds to the delay between the triggering - or electronic start of injection (ESOI) - and the actual start of fuel injection - also called optical start of injection (OSOI). This “dead time” is a combination of an electronic delay between the trigger signal input to the injector driver and the signal it sends to the injector and a mechanical delay due to the needle lift.
The DaVis software requires the input of this parameter. This allows it to determine the PTU delay so that the imaging can correspond to timings after the actual start of injection (i.e. OSOI).
Figure 4-7: Window for the imaging timings
Figure 4-8: PTU synchronisation sequence (not to scale)
4.2.6. Spatial Calibration
The set-up of the 4-mirror separation optics requires the selection of band-pass and neutral density filters and the adjustment of the mirrors so that the LIF and the Mie images are on two parts of the CCD chip. It is then crucial to have a spatial calibration of the two halves of the chip.
The test rig in Munich has an anodised aluminium plate where crosses have been marked out with a laser, the distance between each cross being 6mm (see Figure 4-9).
Figure 4-9: Calibration plate with the adjustable aluminium diffuser plate
Figure 4-10: Calibration images for the Mie path (left) and the LIF path (right)
The right side window was removed and the calibration plate was inserted in its place. The plate was designed to be located exactly underneath the injector tip, in the middle of the bomb. A 266nm dichroic mirror was laid over the beam splitter in order to redirect the 266nm laser light through the left sheet-forming optics. The adjustment
knobs on the top $45^\circ$ mirror situated before the sheet-forming optics allowed to direct the sheet onto the plate at a very low angle. An aluminium plate was used to scatter the light. It was found that this illumination of the plate gave a good 266nm Mie scatter image of the plate, as well as a good fluorescence image. Images were taken by running the DaVis imaging software on internal trigger (i.e. the PTU is triggered by the computer at a rate of 10Hz).
### 4.2.7. Traverse system
The laser optics and camera system are mounted on a common motorised translation stage, so that the sheet can be moved inside the chamber without requiring re-focusing or re-calibrating. To ensure positional accuracy, each time the rig is switched on, the translation stages must be referenced. The Test-rig computer performs this by moving the traverses to the end of the stage. Only afterwards can the traverse be moved freely. For these experiments, measurements were done in a single plane, so the traverse was only adjusted for the laser sheet positioning.
**Figure 4-11: Schematics of the LSD setup**
4.3. PDA pressure vessel
This vessel is actually equipped with a Dual PDA enabling to measure 2 component velocities and diameter. Originally, the diagnostics system was from TSI, consisting of a Fibre Coupler, transmitting and receiving optics, a Photodetector Module (PDM 100) and the Multibit Digital Processor (FSA 4000) controlled by the PC-based software package called Flow Sizer. The experiments were carried with this system. Later, a DualPDA system from Dantec was installed, with a new fibre coupler, transmitting and receiving optics, Photodetector Module (detector Unit), Processor (BSA P80) and software (BSA Flow Software) which enabled to renew part of the experiment with a different system.
4.3.1. Optical Access
The PDA pressure vessel has two side windows, at an angle of $115^\circ$ (i.e. PDA is done using refraction at an off-axis angle of $65^\circ$. The transmitting (or sending) and receiving optics are each located in front of a window.
4.3.2. Laser and beam splitter
The laser is an Innova Model 305C 5W Argon Ion Laser from Coherent. The laser light goes through a Bragg cell. This device splits in the beams in equally intense beams and adds a 40MHz optical shift frequency onto the 1st order beam. The beams of orders 0 and 1 are extracted and fall onto a prism. This splits the light into the
discrete Argon ion wavelengths. The most intense green (514nm) and blue (488nm) beams are redirected with mirrors into the fibre coupling. From there, they reach the Transmitting optics.
### 4.3.3. Measurement volumes
The 2 pairs of beams interfere and form 2 measurement volumes which must be superimposed. This requires the beams to intersect at the same point. To adjust the intersection of the beams, the sending optics were removed from the mount and pointed towards a wall. A 4mm focal length spherical lens was placed at the intersection to magnify the image of the beams and allow optical adjustment.
The sizes of the measurement volumes for the TSI and Dantec system are found in Table 4-1.
| PDA system | TSI | Dantec |
|---------------------|----------------------|----------------------|
| **Transmitter** | XRV 208-6.3 | 112mm Fiber |
| Beam distance | 60mm | 75mm |
| Beam diameter | 2mm | 4.5mm |
| Off-axis angle | 65° | 65° |
| Focal length (front lens) | 300mm | 310mm |
| Measurement volume diameter | 98μm | 47μm |
| Measurement volume length | 980μm | 390μm |
| Fringe distance | 2.58μm | 2.16μm |
| Number of static fringes in MV | 38 | 10 |
| Calibration factor | 2.58 m/s/MHz | 2.16 m/s/MHz |
| **Receiver** | RCV 208 | 112mm |
| Aperture | 72mm | 90mm |
| Focal length (front lens) | 300mm | 310mm |
| Slit width | 50μm | 25μm |
**Table 4-1: PDA parameters**
4.3.4. Timing
For each new injection, the PDA system requires a temporal reference to determine the time at which an event occurred after the start of injection. This reference is the injector trigger from the Test-rig computer, i.e. the electronic start of injection.
The user can also set a window at which the events are validated, (e.g. from 1ms ASOI to 3ms ASOI).
Figure 4-13: Schematics of the PDA vessel
4.3.5. Traversing system
The transmitting and receiving optics are mounted on the same horizontal aluminium beam. This beam is mounted on a three-way translation stage to allow for displacement in all directions. The stage is motorised and the coordinates are input to the Test-rig computer. In the same way as the other vessel, the stage must go to a reference point before being operated. The point of origin is set manually at the injector tip. This is done by inserting a specific blank with a small needle in replacement of the injector mount. The design is such that the tip of the needle is at the location of the injector tip. The traverse system is adjusted so that the measurement volume hits the tip of the needle. The precision is ±250μm.
4.3.6. Tuning
A monodisperse spray from a water nebuliser was used for the fine adjustment of the system. It allowed an accurate positioning of the receiving optics, by checking the collected data along with the collection rate (typically 90% and more).
4.4. Test rig operation
The test-rig can be operated either manually or in automatic mode.
Each vessel is controlled by its individual Test-rig computer. This computer controls the conditioning of the vessel and the fuel, the movement of the traverses and also triggers the injector: it is the experiment master.
Each rig then has a specific computer for the laser diagnostics which run as slaves.
4.4.1. Manual mode
The manual mode allows set-up of the experiment and adjustment of the optics for the injection measurements. In this mode, the user sets all the parameters remotely. The conditioning of the vessel (Pressure, Temperature and Flux) is done using the windows shown in Figure 4-15. The user fills the fuel pump by opening and closing a series of valves and inputs the pressure (see Figure 4-16). The injection is triggered using the window illustrated in Figure 4-14 in which the user inputs the injection duration and repetition rate.
Figure 4-14: Injector Trigger
Figure 4-15: Pressure and temperature settings in the manual mode
Figure 4-16: Fuel Pump schematics and parameters in the manual mode
4.4.2. Automatic mode
Once the setup is complete, the rig can be operated in automatic mode.
In this mode, the user inputs the vessel’s conditioning parameters in a line on the Test-rig computer (see Figure 4-17). Each line represents one set of measurements.
The parameters are:
- the vessel’s Pressure and Temperature
- the fuel’s Pressure and Temperature
- the translation stage’s position
- the injector voltage, injection time and frequency
There is a serial communication protocol to exchange data between the two PCs, consisting of 4 commands (status, start, break, end) and 3 statii (ready, busy, error): the Test-rig computer (master) continuously sends commands to the diagnostics computer (slave) and checks the slave’s status. Each command has to be answered within a few seconds, otherwise the test-rig PC (master) will switch the rig off.
Once the experimental parameters have been set, the diagnostics computer (slave) is set to a Standby mode (status “ready”), waiting for the Test-rig computer (master) to reach the conditioning parameters of the vessel. The conditioning sequence starts with the release of Nitrogen into the chamber. First, the valves stabilise the pressure and the purging flow. The heaters then heat up the Nitrogen whilst the valves are continuously adjusted to maintain the pressure and the flow. When the nitrogen has reached a stable temperature, the fuel is conditioned to the desired temperature. Once the conditioning is stabilised ($\pm 3\%$), the fuel pump is filled. When the pump has reached the desired pressure, the master computer tells the slave computer it is ready to start. As soon as the slave is ready, it gets a new command, and measurements can begin. At this point the slave status becomes “busy”. The injection proceeds until the diagnostics computer has finished the data acquisition for this conditioning line. At this point, the slave computer changes its status to “ready” and the Test-rig computer moves to the next conditioning parameters. The end of the measurements is indicated by a $0$ at the beginning of the next line. At that point, the rig releases the pressure and maintains a flux until the heaters are cooled down.
In the meantime the rig-PC has 15 threads running to control the rigs hardware and is surveying 6 safety loops. The rig-PC is also connected to an I/O-profibus delivering data from sensors (temperature, pressure, fuel levels, water flow, etc.), pneumatic valves, actors and pumps. In addition to the profibus, the rig-PC is connected by TCP/IP with 10 units controlled with a serial RS232 or RS485 interface such as gas flow valves and throttles, silicon oil conditioners. Timings and the pump control is done by a separate controller connected via a CAN bus to the PC. Above all there is a SPS acting as profibus master. Safety features are all SPS controlled.
Chapter 5
Measurements and Results
This chapter presents the results obtained from the experiments carried out in BMW AG’s BDE-OP test-rig in Munich. The aim was to find the best tracer mixture for the LSD technique applied to an evaporating GDI spray. First, the GDI injector and the Pressures and Temperatures conditions for the measurements are introduced. This is followed by the presentation of the experimental procedure and data processing for the LSD and the time-resolved PDA techniques. Finally a comparison of the two is presented and the validity and limitations of the techniques are discussed.
5.1. Introduction
A GDI spray from a swirl injector was investigated at 5 different Pressures and Temperatures. These conditions were chosen to represent both realistic in-cylinder conditions and different boiling regimes (see Figure 5-1).
These conditions are:
- 01bar - 25°C
- 03bar - 135°C
- 05bar - 195°C
- 10bar - 295°C
- 15bar - 360°C
The injector used in this work was the Bosch 70°/0° high pressure swirl injector. It is a needle-type injector, with an axisymmetric spray (γ=0°) and a cone-angle of α=70°.
**Figure 5-2: Injector α and γ angles**
The delay between the electronic injection signal (or Electronic Start Of Injection – ESOI) and the Optical Start Of Injection (OSOI) was measured using Mie scatter images. The “Offset” parameter was set to 0ms and images were iteratively taken at various times after OSOI (in this case ESOI=OSOI), until two consecutive images showed the absence and the presence of fuel respectively. For this Bosh swirl injector, the “dead-time” was measured to be 0.68ms ± 0.025ms (i.e. 4%).
The injection duration was set to 1.5ms. Five different times After Start Of Injection (ASOI) were chosen: 0.7ms, 1.2 ms, 1.7ms, 2.2ms and 2.7ms. These times represent the different phases in the development of the spray.
5.2. LIF/Mie scatter measurements for the LSD technique
5.2.1. Fuel/tracer systems
5 different tracer systems were tested, 4 of which consisted of different mixtures of 3-Pentanone and 2-Hexanone in Iso-Octane:
- 2.0% Pentanone – 98% Iso-Octane
- 1.0% Pentanone – 1.0% Hexanone - 98% Iso-Octane
- 0.5% Pentanone – 1.5% Hexanone - 98% Iso-Octane
- 2.0% Hexanone - 98% Iso-Octane
As will be discussed at the end of the chapter, these 4 tracer systems were limited by vapour contribution to the fluorescence signal. An Exciplex system developed by Fröba et al. (1998) was used as the 5th tracer mixture:
- 2% TEA – 3.4% Benzene – 94.6% Iso-octane
To change the fuel/tracer mixture, the tank must be completely emptied and washed to avoid contamination. The tank was not designed specifically for this. Instead, the mixtures were prepared in glass bottles, and the pipe from which the mixture is drawn from the tank was inserted into the bottle.
For each change in the tracer system, a sequence of operations was carried out to ensure that the next measurement campaign did not contain residuals from the previous mixture. This consisted of:
- Removing the fuel pipe from the glass bottle
- Letting the previous tracer/fuel mixture run out from the pipe
- Inserting the pipe in the next glass bottle
- Filling the fuel pump
- Emptying the fuel pump directly into the waste tank
- Filling the fuel pump a second time
- Injecting the fuel into the rig until the pump was empty
- The pump was filled a third time and the fuel was injected until the pump was empty.
The images of the injection after the 3rd pump filling showed a stable fluorescence signal, suggesting that the previous fuel was completely evacuated from the system.
With the new fuel in place, images were recorded. The LIF intensity varied from the previous mixture, due to the difference in fluorescence yield and tracer concentrations. The LIF path contained a BG4 filter to attenuate any 266nm light and Mie scatter from the 532nm light present in the pressure vessel. In the case of the Exciplex mixture, the liquid phase fluorescence was selected using the WG335 long-pass filter, rejecting the spectrum below 335nm. The Mie scatter light path was accordingly attenuated so that the Mie scatter intensity matched that of the LIF signal. The filters used are found in Table 5-1.
**Figure 5-3: Transmittance of the BG4 and GG400 filters and their combination**
| Tracer composition | ND filter | Filter in LIF path |
|--------------------|-----------|--------------------|
| 2.0% Pentanone | $3 + 0.2 \sim 3.3$ | BG4 |
| 1.0% Pentanone | $3 + 0.6 \sim 3.6$ | BG4 |
| 1.0% Hexanone | | |
| 0.5% Pentanone | $3 + 0.6 \sim 3.6$ | BG4 |
| 1.5% Hexanone | | |
| 2.0% Hexanone | $3 + 1 \sim 4$ | BG4 |
| 2.0% TEA | | |
| 3.4% Benzene | $3 + 1 + 0.3 \sim 4.3$ | BG4, WG335 |
**Table 5-1: Filters for the different tracer systems**
The alignment of the 4-mirror setup and the intensified camera was done for the first tracer mixture. For the other mixtures, only the filters had to be changed. Each filter change modified the light paths. Therefore, a new spatial calibration was carried out for each new mixture.
### 5.2.2. Experimental Settings
Because of the limited capacity of the fuel pump, 80 images were recorded for each of the 5 timings. This allowed the experiment to proceed with the recording of the 5 different sets in a single run, without having to refill the pump and interrupt the experiment.
An injection frequency of 1Hz was chosen. A frequency of 2Hz was tested, but the fluorescence image suffered from contamination by vapour remains from the previous injection.
The calibration images enable the positioning of the Mie scatter and LIF images in identical locations in two separate images. It also determines the resolution of the image. The injector tip was set as the reference point. This procedure involved determining the pixel co-ordinates of the tip, and its accuracy was within 1 pixel, i.e. 100µm.
| Parameter | Value |
|----------------------------|----------------|
| Fuel Pressure | 80 bar |
| Injection duration | 1.5ms |
| Injection Frequency | 1 Hz |
| Start of imaging | 0.7ms ASOI |
| End of imaging | 2.7ms ASOI |
| Increment | 0.5ms |
| Number of timings | 5 |
| Injector dead-time | 0.68ms |
| Images recorded | 80 |
**Table 5-2: Injection and imaging parameters**
5.2.3. Experimental Procedure
With the calibrated optical system in place and the laser sheet adjusted, the parameters for the test-rig computer and the imaging computer were set. The first step required the adjustment of the laser. This consisted in:
- Warming up the laser for 20 minutes
- Optimising the laser power by adjusting the doubling crystals
- Letting the laser run for another 15 minutes
- Checking the laser power and adjusting if necessary
Once the laser power was stable, the imaging computer was set to the “Ready” status by setting the software to “External trigger” and pressing the “Start” button.
The measurement was then initiated by pressing the “Start” button on the test-rig software.
The laser power was monitored on-line. If the case of large laser fluctuations, the laser power could be readjusted.
Between two sets of measurements (i.e. when the pressure vessel was conditioned to the next set of Pressure and Temperature), the laser power was checked and adjusted if necessary.
5.2.4. Image Processing
For each measurement condition, the intensifier was set to a fixed gain. A background image was recorded with the laser firing inside the pressure vessel, without injection. This background image was automatically subtracted to the images.
For each time step, the dual images were then averaged, separated and corrected for geometrical alignment.
An additional background value was subtracted to account for spray-induced background. This value was determined by averaging the intensity of a selected region around the spray for each image.
The images were then masked by setting all intensities below a threshold (typically 10 counts) to 0.
Because the spray was nearly axisymmetrical, the data from one half of the spray was extracted. Due to the interference of side reflections with the left part of the fluorescence image, the right half of both images was chosen. However, the mirrors in the system invert the images from right to left. Therefore, the data extracted in the right half of each image corresponded to the half spray, left of the injector tip.
The average LIF and Mie scatter images can be found in Appendix A.
5.3. PDA Measurements
The aim of the PDA experiments was to obtain local SMDs in the spray for the same five Pressure and Temperature conditions, in order to calibrate and analyse the data obtained from the LSD measurements.
5.3.1. Measurement grid
Measurements were carried out along a grid, with a vertical spacing of 5mm. The horizontal spacing varied in order to obtain a minimum of 50 measurement points for each condition.
To minimise the distance travelled through the spray, the measurement volume was moved in the “$(0,y,z), y<0$” vertical half plane, located below the injector tip and closest to the receiving optics. However, the measurements in the “Mie/LIF” pressure vessel were performed in the “$(0,x,z), x<0$” half plane. Therefore, the injector was rotated $90^\circ$ anti-clockwise for the PDA measurement.
| | 1bar 25°C | 3bar 135°C | 5bar 195°C | 10bar 295°C | 15bar 360°C |
|------------------|-----------|------------|------------|-------------|--------------|
| Grid - V×H (mm×mm) | 4 × 5 | 3 × 5 | 3 × 5 | 2.5 × 5 | 2 × 5 |
| Measurement points | 64 | 74 | 56 | 54 | 49 |
Table 5-3: Grid size and measurement points
The grid size intervals and the measurement points were determined using the Mie scatter images previously obtained for the same spray.
Figure 5-6: Measurement points in the grid
5.3.2. Downmix / Sampling frequency settings
The photodetectors transfer the light into an electrical signal which is read out by the digital processor. The incoming raw signal from the PMs is first high-pass-filtered to remove the pedestal. It is then log-amplified to obtain a nearly squared signal from the Gaussian signal. The signal’s frequency is proportional to the velocity of the detected droplet, offset by 40MHz due to the moving fringes.
To increase the signal-to-noise ratio (SNR), the user sets a downmix and a sampling frequency. This allows a quadrature mixer to downmix the signal to a lower frequency, after which a band-pass filter is applied.
Before running the rig in automatic mode, the optimal setting for each measurement point was determined. For this, the rig was operated in manual mode. At each Pressure and Temperature condition, the measurement volume was positioned at the various locations of the grid. At each location, PDA measurements were performed with the widest filter possible until sufficient data was plotted on the real-time diameter vs. frequency graph to determine the smallest filter width applicable.
For each conditioning, the points were grouped by downmix frequency setting.
5.3.3. Timing
To reduce the file sizes, the PDA software can filter the data temporally. The LSD technique was applied at timings ranging from 0.7 to 2.7ms after optical start of injection (OSOI). The PDA data was therefore filtered around that range, with a tolerance of 0.2ms. Because the PDA reference point corresponds to the electronic start of injection (ESOI), the PDA temporal window was set between 1.18 and 3.58ms.
\[ t=0 \text{ for PDA} \]
\[ t=0 \text{ for LSD} \]
Figure 5-7: Trigger and Temporal filter (blue) for the PDA data acquisition
5.3.4. Measurement procedure
The Argon-Ion laser was first warmed up and the power was then adjusted. The measurement positions were input in the test-rig computer’s conditioning table. For each Pressure and Temperature setting, the measurement points with the same downmix/sampling frequency setting were regrouped in tables. The PDA software was run in coincidental mode, which compares the 2 diameters measured by the dual PDA system, and only validates the events where both diameters are similar.
The rig was then operated in automatic mode. From one line to the next, the conditioning was released at the end of the data acquisition. This allowed sufficient time to check and adjust laser power between two runs.
At the end of the run, the software creates a file. A 5-line header indicates the position of the measurement volume in the spray as well as the parameters measured. Following the header, each line contains the information about each validated PDA event.
Each line contains 4 parameters:
- the time $t$ after ESOI at which the droplet was detected (in ms)
- the vertical speed $v_z$ (in m/s)
- the horizontal speed $v_y$ (in m/s)
- the diameter $D$ of the droplet (in $\mu$m)
**Figure 5-8: Example of a PDA data file for 1 measurement point**
The PDA data was used to calculate the SMD of droplets for the five timings at each measurement point.
5.3.5. Time selection
The LSD technique freezes the spray and allows the measurement of the SMD of droplets present in one pixel at a specific time (e.g. $T = 1.2\text{ms AOSOI}$). On the other hand, the PDA measurement technique does not freeze the spray: it analyses the passage of a droplet through the measurement volume, and indicates the time at which the event occurred. These two techniques are intrinsically different. Therefore, the PDA data must be carefully selected to match the LSD measurement. One method of freezing the PDA data in time could consist of selecting the events which occurred specifically at that time. However, a more appropriate way of matching the PDA data to the LSD experiment is to select the droplets which would be present in the LSD measurement volume at the time of interest. Because the pixels are binned 2 by 2, the actual LSD measurement volume is larger than the PDA’s. Therefore, droplets detected by the PDA system just before or after the time of interest (e.g. 1.204ms or 1.997ms) might be located within that volume at the time of interest (1.2ms).
The measurement volume has the shape of an elongated rugby ball, of horizontal and vertical axis measuring $\sim 900\mu\text{m}$ and $\sim 100\mu\text{m}$ respectively. Due to the slit in the receiving optics (50$\mu\text{m}$ for the TSI system), the effective measurement volume is a cylinder of diameter $\sim 100\mu\text{m}$ and a height of 50$\mu\text{m}$ (see Figure 5-9).
For each event, the PDA data provides the horizontal and vertical speeds of the droplet ($v_y$ and $v_z$ respectively). Assuming that the droplet’s speed remains constant over small distances, the location of a droplet shortly before and after the event can be determined (see Figure 5-10). If a droplet is spotted before or after our time of interest (at $T' = T \pm \delta t$), between $T$ and $T'$, the droplet will have had a horizontal and vertical displacement $\delta y = v_y \cdot \delta t$ and $\delta z = v_z \cdot \delta t$ respectively. If these displacements are both smaller than 100$\mu\text{m}$,
the droplet was present at the time of interest in the measurement volume used for the LSD experiment.
However, the distance criterion chosen for the time-resolved data can be an issue. In a highly evaporating environment, or for very small droplets, the diameter may evolve within the allowed displacement. Also, as the sampling size is changed, it is possible that erroneous diameters appear or disappear, thus creating large variations in SMD. For this reason, the SMD data was time-resolved using 3 displacement tolerances: 50, 100 and 150μm. The 100μm displacement was used as the reference, the two others served as indicators of the accuracy/stability of that reference. The variation in SMD was used as a criterion for selecting the final data and was defined as:
\[
\Delta \phi = \max \left( \left| \frac{SMD_{50\mu m} - SMD_{100\mu m}}{SMD_{100\mu m}} \right|, \left| \frac{SMD_{150\mu m} - SMD_{100\mu m}}{SMD_{100\mu m}} \right| \right)
\]
Equation 5-1
### 5.3.6. Data processing
A programme was written in C++ in order to extract time-resolved SMD from the PDA data. The programme consists of reading out a line, verifying that the line is valid, and separating it in the 4 parameters: time, vertical speed, horizontal speed and diameter).
The velocity data then allows to check whether the distance travelled between the event and a time of interest is within the distance criteria. If so, the diameter is stacked in an array. At the end of the file, this array then allows the calculation of the time-resolved SMD and PDF of each measurement point.
**Figure 5-11: Program Structure for PDA data Processing**
The SMD values and corresponding sample sizes were transferred to a spread sheet and were placed in a table, representing their position in the half spray (see Appendix B).
5.3.7. PDA accuracy
Because the spray was very dense, the validation rate and therefore the number of samples obtained was low. The time selection further reduced the samples. Typically, the time-resolved SMDs were calculated with samples ranging from 1 to 1000 droplets, most samples containing less than 100 droplets. Therefore, the validity of the PDA data must be studied closely to ensure statistical accuracy with low samples. This is crucial, as the SMDs obtained with this technique were used as the reference to validate the LSD technique.
For comparison purposes, measurements were performed with 2 different PDA systems for the 1bar-25°C case. This enabled to study the validity of the PDA data. In Figure 5-12, all the time-resolved SMDs from the TSI system are compared to the SMDs obtained at the same point with the Dantec system. The SMDs were calculated with an allowed displacement of 100μm. The diagonal indicates is the $y=x$ line, and the dotted lines above and below indicate +15% and -15% variation respectively. The colours indicate the time ASOI.
The comparison shows that out of the 166 points compared, only 75 (~½) agree within 15% variation. This raises 3 issues: is the PDA data reliable? When both TSI and Dantec data are available, can they be filtered to ensure reasonable accuracy? How can one ensure accuracy when only TSI data is available?
By selecting the data where both SMDs showed less than 15% variation for the three distance criteria (i.e. $\Delta \phi < 15\%$), most of the points outside the 15% deviation are eliminated (see Figure 5-13).
This process also eliminated 18 points which previously agreed within 15%. This suggests that the remaining points might still contain errors, and that further filtering is required.
To better understand the discrepancies between the two measurements, the droplet size distribution were studied at each measurement point and for each timing. These distributions can be found in Appendix C.
### 220.127.116.11. Filtering using 2 data sets
The TSI and Dantec data can be considered reliable if both measurement systems give similar droplet size distributions for the same point. Because of the number of distributions, a study was carried out to determine the parameters which could be used to automatically filter/eliminate the measurements which yielded different size distributions. A few distributions at different measurement points (MP) are represented below to illustrate the different cases encountered. The blue distributions are obtained from TSI data and the red distributions from the Dantec system.
Three types of cases were found:
1. the distributions and SMDs both do not match
2. both distributions and SMDs match
3. the SMDs match but the distributions are different
A variable representing the variation of SMD was defined as:
\[
\Delta SMD = \frac{SMD_{Daniec} - SMD_{TSI}}{SMD_{TSI}}
\]
Equation 5-2
In case n°1, illustrated by MP 19 (see Figure 5-14) and MP 49 (see Figure 5-15), the $\Delta SMD$ are 31% and 33%. These measurements can be eliminated by setting a maximum allowed value for $\Delta SMD$.
Figure 5-14: PDF for MP 19 ($y=12$, $z=20$) at 0.7ms ASOI
Figure 5-15: PDF for MP 49 ($y=16$, $z=30$) at 1.2ms ASOI
An issue arises when the SMDs are similar. For this case, another variable must be used to separate the measurement points with different distributions from those with similar distributions.
It was found that matching SMDs which had both been calculated from higher sample sizes showed similar distributions (see Figure 5-16, Figure 5-17 and Figure 5-18).
**Figure 5-16: PDF for MP 2 (y=0, z=10) at 2.2ms ASOI**
**Figure 5-17: PDF for MP 62 (y=8, z=60) at 2.7ms ASOI**
**Figure 5-18: PDF for MP 1 (y=0, z=5) at 2.2ms ASOI**
On the other hand, when at least one of the two sample sizes was low, the distributions were different (see Figure 5-19 and Figure 5-20).
**Figure 5-19: PDF for MP 31 (y=12, z=35) at 1.7ms ASOI**
**Figure 5-20: PDF for MP 14 (y=12, z=25) at 2.7ms ASOI**
For both these cases, the SMDs were similar within 9%, and the $\Delta \phi$ were both low. The dissimilarities in distributions can be attributed to the low number of droplets used to determine the SMD. In the case of MP 31, the TSI data is low, whereas in the case of MP 14 the Dantec data is low.
These examples illustrate that the sample size should also be used to filter the data. They also demonstrate that one system does not systematically detect more droplets than the other. Therefore, a minimum sample sizes for both distributions must be considered simultaneously.
Measurement point MP 60 (see Figure 5-21) showed the case where the distributions were different for high sample sizes (>100) with a $\Delta SMD$ of 17%.
On the other hand, MP 12 (see Figure 5-22) showed the case of different distributions for a $\Delta SMD$ of 15% but where the SMD had been calculated with 58 droplets.
**Figure 5-21: PDF for MP 60 (y=12, z=55) at 2.7ms ASOI**
**Figure 5-22: PDF for MP 12 (y=4, z=25) at 2.7ms ASOI**
By setting the highest allowable value of $\Delta SMD$ and $\Delta \phi$ to 15% and the minimum sample size to 60 droplets, the remaining data (21 points) had matching SMDs and distributions.
18.104.22.168. Filtering using 1 data set
Figure 5-23 illustrates the PDA points validated with $\Delta SMD < 15\%$, $\Delta \phi < 15\%$ and a minimum sample size of 60 droplets.
**Figure 5-23: TSI-Dantec SMD comparison**
$\Delta SMD < 15\% - Samples > 60$ droplets $- \Delta \phi < 15\%$
By keeping the same minimum number of samples and $\Delta \phi$, but without imposing a $\Delta SMD$ (see Figure 5-24), 16 new points appeared.
**Figure 5-24: TSI-Dantec SMD comparison**
Samples $> 60$ droplets $- \Delta \phi < 15\%$
These points were identified as:
- 0.7ms: MP 29, 34, 23, 24, 14
- 1.2ms: MP 30, 49
- 1.7ms: MP 35, 36,
- 2.2ms: MP 3, 55, 58
- 2.7ms: MP 62, 30, 31, 32
Their variation and distribution were studied (see Table 5-4). It was found that 78% had resembling distributions. The standard deviation for these 37 points was 27%. However, the only point showing a variation well over 35% (MP 32 – see Figure 5-25) displayed the unique case of nearly resembling distributions, one of which contained isolated samples bigger than 26μm. If this point was eliminated, the deviation of the 36 points dropped to 15%, which is very acceptable considering the density of the spray.
| ΔSMD | 0-15% | 15-20% | 20-25% | 25-35% | 30-35% | +35% |
|------------|-------|--------|--------|--------|--------|------|
| Number of points | 21 | 8 | 3 | 2 | 2 | 1 |
| Matching Distribution | 21 | 4 | 3 | 0 | 1 | ? |
Table 5-4: PDA data points for Samples > 60 droplets and Δϕ < 15%
Figure 5-25: PDF for MP 32 (y=16, z=35) at 2.7ms ASOI
It was concluded that in the case where only one set of PDA data was available, the SMDs selected as references would be the ones calculated with a minimum of 60 droplets, and with a Δϕ < 15%.
5.4. LSD - PDA Comparison
5.4.1. Calibration Constant
Theoretically, the division of the LIF and Mie scatter intensity is proportional to SMD. This can be expressed as:
\[
SMD_{LSD}(i,j) = C \cdot \frac{I_{LIF}(i,j)}{I_{Mie}(i,j)}
\]
Equation 5-3
where \(C\) is the calibration constant which needs to be determined.
Using the SMD obtained with PDA as a reference, the local relative error in SMD measured by LSD can therefore be defined as:
\[
\varepsilon(i,j) = \frac{SMD_{LSD}(i,j) - SMD_{PDA}(i,j)}{SMD_{PDA}(i,j)}
\]
Equation 5-4
For \(N\) points, the most appropriate calibration constant is obtained by minimising the relative errors. This can be done by minimising the error’s deviation from zero:
\[
\sigma_\varepsilon = \sqrt{\frac{\sum_{i=1}^{N} \varepsilon(i,j)^2}{N}}
\]
Equation 5-5
For every pressure and temperature setting and for each tracer system, a spread sheet was created to calculate the deviation.
The sheets consisted of:
- SMD and sample size for a distance criteria of 50µm from TSI data
- SMD and sample size for a distance criteria of 100µm from TSI data
- SMD and sample size for a distance criteria of 150µm from TSI data
- SMD and sample size for a distance criteria of 50µm from Dantec data (only at 1bar-25°C)
- SMD and sample size for a distance criteria of 100µm from Dantec data (only at 1bar-25°C)
- SMD and sample size for a distance criteria of 150µm from Dantec data (only at 1bar-25°C)
- LIF data
- Mie scatter data
The spreadsheet was structured so that the data could be filtered by selecting
- the minimum number of droplets in the PDA samples
- a maximum value for $\Delta SMD$ (only at 1bar-25°C)
- a maximum value for $\Delta \phi$
For the 1bar-25°C case, the reference SMDs were calculated by averaging the SMDs obtained from the TSI and Dantec systems:
$$SMD_{PDA}(i,j) = \frac{SMD_{TSI}(i,j) + SMD_{Dantec}(i,j)}{2}$$
Equation 5-6
For a given calibration constant, the spread sheet would give:
- The deviation ($\sigma_e$)
- The number of points compared
- A plot of the filtered SMDs obtained with PDA versus the SMDs obtained with the LSD technique.
Calibration constants were determined by minimising the deviation.
For a given tracer system, the calibration constant should be constant. Due to the confidence in the PDA data for the 1bar-25°C case, it was obvious that the calibration constant should be determined using the LSD data obtained in the 1bar-25°C case.
| Tracer composition | Calibration Constant (C) | PDA-LSD Deviation ($\sigma_d$) |
|--------------------|--------------------------|-------------------------------|
| 2.0% Pentanone | 18 | 25% |
| 1.0% Pentanone | 23 | 23% |
| 1.0% Hexanone | | |
| 0.5% Pentanone | 34 | 22% |
| 1.5% Hexanone | | |
| 2.0% Hexanone | 27 | 26% |
| 2.0% TEA | 78 | 32% |
| 3.4% Benzene | | |
Table 5-5: Calibration Constant for the different tracer systems
5.4.2. Results
The results are presented as a comparison of the SMD obtained from the PDA data plotted against the SMD obtained with the LSD technique. The continuous grey line represents the $y=x$ line (i.e. where PDA and LSD agree). The dotted lines around the grey line are variations. In the 1 bar-25°C case, these lines represent ±15%. For all other cases, they represent ±20%.
The colours indicate the time ASOI.
Figure 5-26: PDA-LSD comparison at 1bar – 25°C
\[ x / y / z \iff x\% \text{3-Pentanone} - y\% \text{2-Hexanone} - z\% \text{Iso-Octane} \]
Exciplex \( \iff 2\% \text{TEA} - 3.4\% \text{Benzene} - 94.6\% \text{Iso-Octane} \]
Figure 5-27: PDA-LSD comparison at 3bar – 135°C
\[ x / y / z \iff x\% \text{ 3-Pentanone} - y\% \text{ 2-Hexanone} - z\% \text{ Iso-Octane} \]
Exciplex \( \iff 2\% \text{ TEA} - 3.4\% \text{ Benzene} - 94.6\% \text{ Iso-Octane} \]
Figure 5-28: PDA-LSD comparison at 5bar – 195°C
\[ x / y / z \iff x\% \text{3-Pentanone} - y\% \text{2-Hexanone} - z\% \text{Iso-Octane} \]
Exciplex \( \iff 2\% \text{TEA} - 3.4\% \text{Benzene} - 94.6\% \text{Iso-Octane} \]
Figure 5-29: PDA-LSD comparison at 10bar – 295°C
\[ x / y / z \iff x\% \text{3-Pentanone} - y\% \text{2-Hexanone} - z\% \text{Iso-Octane} \]
Exciplex \( \iff 2\% \text{TEA} - 3.4\% \text{Benzene} - 94.6\% \text{Iso-Octane} \]
Figure 5-30: PDA-LSD comparison at 15bar – 360°C
\[ x / y / z \iff x\% \text{ 3-Pentanone} - y\% \text{ 2-Hexanone} - z\% \text{ Iso-Octane} \]
Exciplex \( \iff 2\% \text{ TEA} - 3.4\% \text{ Benzene} - 94.6\% \text{ Iso-Octane} \]
The tracer systems composed of a mixture of the two ketones displayed two types of behaviours which enabled to categorise them into 2 groups:
1. the 3 tracer systems which contain 2-Hexanone
2. the 3-Pentanone/Iso-Octane mixture
The Exciplex system was expected to eliminate the overestimation of the SMD obtained with the LSD technique. The results show this was not achieved. This behaviour is explained further in the analysis of the results.
The analysis is carried out by looking at two issues:
- the discrepancies between the PDA and the LSD results
- the variations of LSD relative to the tracer systems.
Generally, the 5 following behaviours were noticed:
- Good agreement for all tracers at atmospheric conditions, within 30% variation.
- Above atmospheric conditions, the LSD technique showed an increasing overestimation of the SMD for later stages of the injection.
- The LSD technique showed an increase of the overestimation of the SMD as the environment became more superheated.
- From 3bar-135°C to 10bar-295°C, the SMD obtained were higher from the Exciplex and 3-Pentanone/Iso-Octane mixtures at the later stages.
- At 15bar and 360°C, the overestimation of the SMD level out.
For all tracer mixtures, the results obtained at 1bar and 25°C do not show extreme variations between the PDA and LSD technique. The best tracer systems are the 1.0/1.0/98 and 0.5/1.5/98 mixtures.
At 3bar and 135°C, the 1st group matches the PDA data much better than the 2nd, especially in the later phases of the injection. The best results are still obtained with the 1.0/1.0/98 and 0.5/1.5/98 mixtures. As the conditions evolve toward the superheated regime, the discrepancies between PDA and LSD results worsen as the injection progresses. Also, for the 5bar – 195°C and 10bar – 295°C cases, the variations in SMD between group 1 and 2 increase. However, at 15bar and 360°C, all systems show the same order of variation.
5.5. Analysis
The discrepancies between the results obtained with the LSD technique and the PDA technique can be caused by three factors:
- The LIF signal
- The Mie scatter signal
- The PDA data
The variation of LSD data relative to the different mixtures can only be explained by the LIF or Mie scatter signals. Because the tracer concentrations in Iso-Octane were very weak (only a total of 2% for the 3-Pentanone / 2-Hexanone mixtures), the spray (i.e. the droplets’ evolution) was expected to be independent of the fuel mixture. Therefore the variation in SMD from one mixture to another can be attributed predominantly to the fluorescence.
To better understand these variations in fluorescence, 3 issues must be examined:
- fluorescence yield
- vapour phase contribution to the LIF signal
- evaporation regime
5.5.1. Fluorescence yield
The fluorescence is proportional to the amount of tracer and also to the fluorescence yield. The behaviour of 3-Pentanone’s fluorescence in various environments is well documented for several UV excitation wavelengths (Wolff et al. (1995), Grossman et al. (1996), Fujikawa et al. (1997), Ossler and Aldén (1997), Koch and Hanson (2003) - see Figure 5-31).
However, 2-Hexanone’s fluorescence behaviour is not documented. Han and Steeper (2002) have also looked at mixtures of ketones (3-Pentanone and 3-Hexanone) to produce co-evaporating tracer systems. They studied the fluorescence of two tracer systems for a 266nm excitation wavelength and found that the normalised fluorescence of the mixture containing only 3-Pentanone decreased less than the fluorescence of the tracer system containing a mixture of the two ketones (see Figure 5-32). This suggests that the fluorescence of 3-Hexanone decreases more with temperature than with 3-Pentanone. The same can be assumed between 2-Hexanone and 3-Pentanone.
Figure 5-31: Temperature dependence of 3-Pentanone Fluorescence in atmospheric Nitrogen - Koch and Hanson (2003)
Figure 5-32: Fluorescence and Temperature for a 266nm excitation wavelength Han and Steeper (2002)
IO/3P $\iff$ Iso-Octane / 3-Pentanone
Ternary $\iff$ 90% Iso-Octane / 10% 3-Hexanone / 1% 3-Pentanone
These graphs suggest a decrease of fluorescence with temperature. They do not justify the increase of the LIF signal.
5.5.2. Vapour phase fluorescence
At atmospheric conditions, 3-Pentanone’s liquid phase is 210 times denser than the vapour phase. 2-Hexanone’s liquid phase is 180 times denser. Prior to the experiments, the vapour phase fluorescence was therefore assumed to be negligible compared to the liquid-phase fluorescence.
However, for conditions above the saturation curve, all tracer systems displayed an increase of the fluorescence signal in time, relative to the Mie scatter signal. As the conditions evolved towards more superheated regimes, this increase became larger. This suggests a significant vapour contribution to the signal.
Figure 5-33: LIF and Mie scatter images at 5bar and 195°C
If the local fluorescence signal is separated in a liquid phase and a vapour phase signal, the local ratio of the fluorescence and the Mie scattering intensities in the LSD technique is:
The desired SMD would be the ratio of the liquid-phase fluorescence and the Mie scatter intensities. Defined as $SMD'$, Equation 5-7 can be written as:
\[
\frac{LIF(i,j)}{Mie(i,j)} = \frac{LIF_l(i,j) + LIF_v(i,j)}{Mie(i,j)} = \frac{LIF_l(i,j)}{Mie(i,j)} + \frac{LIF_v(i,j)}{Mie(i,j)}
\]
Equation 5-7
\[
\frac{LIF(i,j)}{Mie(i,j)} = SMD'(i,j) + \frac{LIF_v(i,j)}{Mie(i,j)}
\]
Equation 5-8
Figure 5-34 is an illustration of Equation 5-8. At 1bar and 25°C (i.e. low evaporation conditions), the vapour fluorescence is negligible, and the ratio of scattering intensities is close to the SMD determined by PDA.
**Figure 5-34:** Vapour contribution in the estimation of the SMD
### 5.5.3. Evaporation regime
When the vapour contribution to the fluorescence signal is at least of the same order as the liquid LIF, the second term of Equation 5-8 cannot be neglected. In the case of a 2-Hexanone/Iso-Octane mixture, for slow evaporation, the relative tracer concentration increases in the droplet. Therefore $SMD'$ is expected to be
overestimated. Any additional fluorescence intensity from the presence of vapour in the measurement volume will increase the overestimation (see Figure 5-35).
**Figure 5-35: Differential evaporation and vapour phase contribution**
With the 3-Pentanone/Iso-Octane mixture, in the case of slow evaporation, the tracer is preferentially expelled from the droplet: $SMD'$ is underestimated. At the later stage of the injection, the measurement volume contains not only vapour from the evaporating droplets present, but also vapour originating from the previous passage of “older” droplets. This may result in the mass of vapour being larger than the mass of the liquid: the tracer system which contains more fluorophore in the vapour phase will therefore give a larger apparent SMD.
As the conditions evolve towards a homogenous evaporation regime, the contents of vapour will contain similar amounts of tracer. At that point the values of the SMDs should become independent of the tracer system. This is the case at 15bar and 360°C. However, at 10bar and 295°C, there were still noticeable differences between the tracer systems containing 2-Hexanone and the 3-Pentanone/Iso-Octane mixture. This suggests that even though the environment is superheated, the droplet’s warming-up phase is not negligible, which limits the evaporation rate and allows for diffusion/differential evaporation.
5.5.4. Conclusion
The work undertaken with tracer systems composed of various mixtures of 3-Pentanone and 2-Hexanone (i.e. 2 ketones with opposite vaporisation characteristics) showed that the LSD technique works well at atmospheric conditions. This is because the evaporation is weak, and the vapour can diffuse. This allows the amount of tracer in the vapour phase to be sufficiently low not to contribute significantly to the fluorescence signal. In this case, the ratio of LIF to Mie scatter signal is proportional to the ratio of Volume to Surface of the liquid phase, thus yielding close SMD, within 30% variation.
With the increase in pressure and temperature, the technique still gives reasonable results for the early stage of the injection because the vapour content of the spray is not significant enough. However, the technique fails to deliver accurate SMD at the later phases of the injection. The increase in pressure causes the spray to collapse and limits the diffusion of the vapour. The rise in temperature generates vapour more quickly. These two phenomena explain the increase of the presence of tracer in the vapour phase, which increases the fluorescence signal relative to the Mie scattering signal.
In a homogeneous evaporation regime, all tracers will yield the same SMD values. In the case of differential evaporation, the tracer with the lowest saturation pressure will generate more vapour phase fluorescence, and may give higher SMD than less evaporating tracers due to the presence of vapour from previous droplets.
5.5.5. PLIEF to remove the vapour contribution
The vapour phase contributes significantly to the fluorescence signal for conditions above atmospheric. To eliminate the vapour contribution to the LIF signal, an exciplex system was tested. The system showed similar results for the 1bar – 25°C, where evaporation is not an issue. However, at 3 bar and above, the values of SMD increased in a similar way as the 3-Pentanone/Iso-Octane mixture.
As the temperature is increased, the monomers’ - i.e. the vapour phase’s - emission spectrum is red-shifted. Furthermore, Kornmesser et al. (2001) showed that the fluorescence yield of the monomer increases by a factor of 4 between 20 and 100°C (see Figure 5-37). Also, both TEA and Benzene have much lower boiling points than the
other tracers and Iso-Octane itself (85 and 80°C respectively). The vapour phase will therefore contain much more tracer than with the other mixtures.
These 3 effects contribute to amplifying the fluorescence in a spectral range that is not eliminated because the filter was not sufficiently selective. Therefore, the experiments should be carried out by selecting the fluorescence which occurs above 400nm.
**Figure 5-36:** Liquid-vapour emission spectra for the LIEF - Fröba et al. (1998)
**Figure 5-37:** Relative fluorescence intensities of the TEA/Benzene exciplex and monomer in Iso-Octane (Kornmesser et al. 2001)
5.5.6. PDA – LSD discrepancies
The work shows that at atmospheric conditions, the evaporation is low and the tracer systems behave in a similar way. These conditions were expected to give matching results between the LSD and the PDA techniques. However, there are variations between the SMD obtained with the PDA and LSD technique.
Le Gal (1999) suggested that for dense sprays, the multiple scattering effects between the laser sheet and collecting optics was responsible for the deviations of the LIF and Mie scatter signals from their $d^3$ and $d^2$ dependence respectively. It is believed that the multiple scattering is of second order, because it affects both signals: the effect of additional intensity caused by multiple scattering is attenuated in the ratio of the fluorescence and Mie scattering signals.
Other problems lie in the fact that PDA is not an accurate tool for measuring dense sprays. The best measurements are done in parts of the spray where the number of droplets is sufficiently low to limit multiple occupancy. However, in these parts of the spray, the fluorescence and Mie scattering signals are low, and their SNR is high, yielding erroneous SMD. Therefore, where the PDA data provides reliable data, the LSD technique can not, and vice-versa.
The variations in the results can be attributed to imperfections in the fluorescence signal, multiple scattering and the PDA technique. The validity of the surface dependence of the Mie scatter signal, however, was never questioned following the findings of Bachalo (1984) and the work by Sankar et al. (1996) where numerical work had showed that “for absorbing droplets, the scattered light intensity is proportional to the droplet diameter, especially for droplets greater than about 1μm in diameter”.
Figure 5-38 shows numerical simulations by Domann and Hardalupas (2003) of Fluorescence and Mie scatter signal intensities using an excitation wavelength of 514.5nm (Argon-Ion), scattering at 90° and a collection angle of 3.8°. The graphs show large variations of the Mie scatter signal from the $d^2$ dependence (in red) for droplets smaller than 100μm in diameter. This suggests at best a good agreement for droplet diameters smaller than 50μm for an excitation wavelength of 266nm. On the other hand, the fluorescence signal agrees well with a $d^n$ law. They found that $n=2.996$ for an absorbing droplet, which implies that the fluorescence signal will deviate from the droplet volume by a maximum of 2.5% for droplet diameters smaller than 50μm.
Figure 5-38: Fluorescence and Mie scatter intensity as a function of diameter (Domann and Hardalupas 2003)
Following this, they numerically calculated the SMD obtained for single droplets using the ratio of the LIF and Mie scatter signals. By comparing it to the real SMD (in this case, the diameter), they obtained the calibration constant (K). Figure 5-39 displays the calibration constant as a function of size. $K_{fit}$ represents the calibration constant if both signals followed the ideal volume and surface relationship. In the 20-50 $\mu$m range, the error will be as high as 30%. The plot also suggests that the discrepancies worsen as the diameters get smaller.
Figure 5-39: Calibration constant for monodisperse droplets (Domann and Hardalupas 2003)
Figure 5-40 illustrates the errors that these variations create when the SMD is measured for different distribution of droplets. The error is smaller for distributions containing large droplets. In the case of the Bosch Swirl injector in this study, the distributions were usually of the Rosin-Rammler or Log-normal type, with a maximum droplet diameter of $50\mu m$, similar to distribution (a) in Figure 5-40. This distribution gives an error of 31%.
**Figure 5-40: Error in Measured SMD for different size distributions**
(Domann and Hardalupas 2003)
The comparison of the SMDs obtained with the LSD and the PDA techniques showed variations of this order of magnitude.
### 5.6. Summary
PDA measurements were carried out on a spray to study the validity of the LSD technique for in-cylinder conditions. Even though the PDA data was not necessarily reliable because of the nature of the spray, several issues were identified.
The most important issue arises from the previously assumed result that the Mie scattering signal follows a $d^2$ dependence. However, as was discussed, for the range of droplet diameters in this particular spray, the Mie scatter signal will deviate strongly
from that surface dependence, because the proportion of light emitted towards the collection optics varies strongly from one size droplet to the other. The integration of the signal over a droplet size distribution will attenuate these variations. Determining the calibration constant using PDA data from a similar droplet distribution will further reduce the discrepancies. Charalampous et al. (2004) have carried out such work using the geometrical approach and found the best accuracy was obtained for a $60^\circ$ observation angle. Further studies could include computational work using the Mie scatter theory to simulate the intensities of the Mie scatter signal and determine the optimal observation and collection angles.
The influence of multiple scattering and its contribution to measurement error is currently under investigation by Berrocal et al. (2005) for inhomogeneous polydisperse turbid media.
In the case of high evaporation, discrepancies as high as 1000% were found, due to the contribution of vapour phase fluorescence. This can be dealt with by using an Exciplex system and by selecting an appropriate high pass filter to completely eliminate the monomers’ fluorescence.
In the case of high evaporation at high pressures, the increase in the SMD obtained with the LSD technique is actually an indication of vapour apparition, and can be used to compare evaporation rates between injectors.
Experimentally, the best tracer system was the 3-Pentanone / 2-Hexanone / Iso-Octane mixture at proportions of 0.5 / 1.5 / 98 by volume. The maximum error was 30% up to 5bar and 195°C. Above these conditions, the vapour phase contribution to the fluorescence signal is non negligible. However, in highly evaporating conditions, the droplet life-times are short because of their small sizes. In this case, the location of the vapour phase is more relevant to the combustion process.
PART III
ENGINE SPRAY VISUALISATION MEASUREMENTS
Chapter 6
Engine Measurements
Several laser diagnostics techniques were applied to a GDI optical engine to characterise the spray for two injection strategies: early/homogenous injection and late/stratified injection. These diagnostics include PLIF measurements to determine equivalence ratio, simultaneous Mie scatter / LIF images in the LSD technique for calculating SMD, and PIV measurements using Mie scattering to determine droplet velocities. This chapter presents the optical engine and the experimental set-up. The setup for each technique is described, and the results are analysed.
6.1. Experimental Set-Up
6.1.1. Engine configuration
These experiments were carried out on a BMW single-cylinder research engine with optical access. The engine is composed of a Ricardo crank casing, an elongated engine block, an elongated piston, a fused silica annulus and the cylinder head (see Figure 6-1) and has a displacement of 499cc.
The block has a $45^\circ$ mirror mounted inside. The piston is elongated and hollow with intake and exhaust side walls removed, enabling the positioning of the $45^\circ$ mirror directly under the cylinder axis. This allows optical access of the combustion chamber through a fused silica window located in the piston top. Previously, a bowl-shaped piston with a small centred window was used for wall/air-guided mixing. For these experiments, the piston is flat, allowing a large diameter window (50mm) and therefore favouring optical access rather than in-cylinder mixing (see Figure 6-2, Figure 6-3, Figure 6-4 and Figure 6-5).
Figure 6-1: Single cylinder optical research engine
The engine has a 4-valve asymmetric pent-roof cylinder-head configuration to accommodate bigger inlet valves. One of the inlet valves is deactivated in order to create a swirl motion inside the cylinder. A spark plug is located at the centre of the roof line. A Bosch 70°/10° Swirl injector is located between the two intake valves (see Figure 6-6).
Optical access in the cylinder is also possible through the fused silica annulus located between the engine block and the cylinder head. The 2 triangular sections of the cylinder-head also have fused silica inserts for optical access in the pent-roof region of the combustion chamber.
Air enters the engine by passing through an air filter, an intake duct, a throttle body and finally an inlet plenum (see Figure 6-8). The entire length of the intake manifold is heated to 40°C using heating mats. The manifold pressure is measured in the inlet plenum. An additional injector (the calibration injector) is mounted approximately 1m upstream of the intake. This allows to create a charge that is homogenously mixed before entering the cylinder. This system was designed by Berckmüller and O’Young, in 1996, but was not published before 2000 (Kelman et al.). Ipp et al. (1999) used the same air intake system for their FAR-LIF experiments.
The tracer/fuel mixture is stored in a tank and is pressurised in two stages before reaching the injector. First, the tanks are pressurised to 3bar using Nitrogen. The fuel is then delivered to the high pressure fuel pump. The 3-piston type pump is mounted on the top of the cylinder head and is belt-driven from the intake cam-shaft. The pressurised fuel is then fed into a pressure regulator before being sent to the injector (see Figure 6-7).
The engine is mounted on an English Electric dynamometer regulated by a Safrronics controller to provide constant speed, which was 1000 rpm for this set of experiments. The engine speed is regulated to a pre-set value by the controller of the dynamometer on which the engine is mounted.
The engine’s cylinder head is water cooled and its coolant is preheated to 70°C. The engine block is cooled by cold water. The oil is circulated from the crank-case to the cylinder-head via an independent electrical pump. The operating pressure is 40psi.
The engine was designed for early GDI development work and was adapted here for good optical access. This piston geometry compromised the mixing process. The first task consisted in finding 2 operating points: one in the early injection/homogeneous mode, and another in the late injection/lean/stratified mode. The conditions are summarised in Table 6-1. The crank angle reference (0°CA) is the compression/combustion top dead centre. 360°CA is the exchange TDC.
| | Early injection (Homogeneous) | Late injection (Stratified) |
|----------------------|-------------------------------|-----------------------------|
| SOI | 420°CA | 670°CA |
| Injection duration | 1.85ms | 1.80ms |
| Ignition | 702°CA | 702°CA |
| ΔP | 340mbar | 20mbar |
| λ | 1 | ~3 |
Table 6-1: Operating points for the GDI engine
6.1.2. Laser and Light Sheet Optics
A Spectra Physics PIV 400 Nd:YAG laser was used as the illumination source. The laser emits at 1064nm. Doubling crystals allow to generate 532nm and 266nm light. For the LSD and AFR-LIF experiments, the fuel was excited using the $4^{th}$ harmonic (266nm) whilst the $2^{nd}$ harmonic (532nm) was chosen to carry out the PIV measurements.
A laser sheet was formed inside the cylinder to image the fuel in the central plane of the combustion chamber. A vertical sheet coming through the piston was used to illuminate the whole chamber, including the pent-roof, in order to visualise the fuel around the sparkplug at time of ignition.
At the exit of the laser, the beam was redirected with two dichroic mirrors (266nm or 532nm depending on the experiment) so that it was perpendicular to the engine, horizontal and pointing at the middle of the $45^\circ$ mirror located under the cylinder axis. The laser sheet was then formed using a combination of 3 fused silica lenses: a spherical lens of 2m focal length thinned the beam to less than 500µm inside the engine. Two cylindrical lenses then expanded the beam in one direction to form a sheet of approximately 5cm in width. The sheet was then directed into the cylinder via the $45^\circ$ mirror.
**Figure 6-9: Sheet-forming Optics**
The laser sheet’s adjustment required the removal of the cylinder head. A target was positioned over the annulus window. It was set against the two cylinder head guides, used as reference, and had markings for several positions left and right of the central plane. The laser sheet position was fine adjusted using the 2\textsuperscript{nd} dichroic mirror and the rotary mount of the 2\textsuperscript{nd} cylindrical lens.
**Figure 6-10: Laser sheet target and illumination**
### 6.1.3. Spatial Calibration
To determine the resolution and the position of the images, a calibration plate containing regularly spaced white crosses on a black background was used to calibrate the CCD chip. A plate similar to the laser target, with a circular hole cut out, enabled the insertion of the plate inside the cylinder and to position it at the same location as the laser sheet (see *Figure 6-11*).
**Figure 6-11: Positioning plate and Calibration plate**
6.1.4. Timing and Synchronising for the Experiment
The timing and synchronisation is schematically represented in Figure 6-13. The engine is the experiment master. The dynamometer keeps the engine at a constant speed. A Kistler type 2612B sensor situated at the end of the crankshaft provides a 1 pulse / °CA signal, and an optical sensor with a single tooth disk on the exhaust camshaft provides a 1 pulse / 2 rev signal (see Figure 6-12).
**Figure 6-12: The cam sensor and single tooth**
These signals are fed into the engine management box. This box allows the user to set:
- the crank angle for start of injection (SOI)
- the injection duration (in ms)
- the crank angle for ignition
- the crank angle for the laser trigger
Every 4-stroke cycle, the engine management box outputs an injection, an ignition and a laser-trigger signal.
The injection signal is fed into the injector driver to provide the signal for the injector whilst the ignition signal is amplified. Both signals are then sent into a Control Box which allows the user to activate the injection and ignition. A 2-way switch controls the spark for the ignition. A 3-way switch allows the user to start injecting the fuel. Pushing the switch forwards will activate the DI injector located in the head. Pushing the switch backwards will activate the calibration injector located upstream of the air intake.
The laser-trigger signal supplied by the engine management box is amplified before being used to trigger the computer’s Programmable Timing Unit (PTU). The PTU runs the laser, intensifier (IRO) and camera (CCD). The imaging software used was DaVis6.2 by LaVision.
Figure 6-13: Schematic of the instrumentation for laser diagnostics in the engine
At 1000 rpm, the management box is sending the computer a signal at 8.33Hz (1 signal per 4-stroke cycle i.e. every 120ms). The laser runs efficiently between 9.5 and 10.5 Hz. The camera’s maximum repetition rate is 8Hz. Taking these frequencies into account, the software calculates the laser frequency for optimal image recording. For these experiments, the laser ran at 10.41667 Hz (i.e. every 96ms), enabling the capturing of an image every 5 4-stroke cycles.
Figure 6-14: Synchronising the Laser with the Engine trigger signal
Because the laser must be Q-switched 186µs after the Flashlamp trigger, the laser cannot be triggered on demand. The software allows to set a delay (at least equal to the Q-switch delay) between the PTU trigger and the Q-switch trigger. This delay was set to the smallest interval corresponding to an integer number of degree crank angle: 333µs, i.e. 2°CA at 1000rpm. The lasing occurred 2°CA after the management box’s trigger.
Figure 6-15: Timing sequence for the laser
The repetition rate of the camera (8Hz), combined with the laser frequency (10Hz) do not allow high-speed imaging. Instead, many images were recorded at the same crank angle over many cycles. This was repeated at different crank angle positions.
6.1.5. Engine Operation Procedure
The procedure of a typical test run was as follows:
- Warm the cylinder head cooling water to 70°C using a heater in the coolant system
- Switch on the oil pump to circulate and warm up the oil to 70°C.
- Get the engine up to speed (1000rpm)
- Pressurise the fuel tank to 3bar using Nitrogen
- Open the fuel line
- Set the voltage to the Pressure regulator
- Turn the ignition switch on and check for the spark
- Set the throttle position
- Dial the desired settings in the engine management box
- Start injection
6.1.6. Image Acquisition
A User/PC interface was programmed to automate the image acquisition, storage and nomenclature.
Figure 6-16: User-PC interface for the measurements
6.2. LIF measurements for Equivalence ratio
Equivalence ratio measurements were performed to compare the evolution of fuel distribution and stoichiometry for the early and late injection modes. A mixture of 20% Pentanone in Iso-Octane was used. The stoichiometric equation for the combustion of 1 mole in air is:
\[
\left( \frac{4}{5} \cdot C_8H_{18} + \frac{1}{5} \cdot C_5H_{10}O \right) + \frac{115}{10} \cdot (O_2 + 3.76 \cdot N_2) \rightarrow \frac{37}{5} \cdot CO_2 + \frac{41}{5} \cdot H_2O + 3.76 \cdot \frac{115}{10} \cdot N_2
\]
**Equation 6-1: Combustion of 20% 3-Pentanone – 80% Iso-Octane in air**
The stoichiometric AFR for this fuel mixture is \( AFR_{st} = 14.5 \).
6.2.1. Measuring equivalence ratio using LIF
The idea of the experiments is to perform two sets of LIF measurements. The 1\textsuperscript{st} set, referred to as DI (Direct Injection) consists in recording (at a specific crank angle) the fluorescence of fuel that is directly injected into the cylinder using the injector located in the cylinder head. In the 2\textsuperscript{nd} measurement set, referred to as Cal (Calibration), fuel is injected in the intake duct using the injector located upstream of the air-intake and the fluorescence images of this homogeneous air/fuel mixture are recorded.
In both cases, a local measurement volume will have an equivalence ratio of:
\[
\Phi = \frac{AFR_{\text{fl}}}{AFR} = AFR_{st} \cdot \frac{m_{\text{fuel}}}{m_{\text{air}}}
\]
**Equation 6-2**
where \( m_{\text{fuel}} \) and \( m_{\text{air}} \) are the local masses of fuel and air respectively.
Assuming that the cylinder is filled exclusively with fuel and air, the measurement volume (\( V_T \)) can be written as:
\[
V_T = V_{\text{fuel}} + V_{\text{air}} = \frac{m_{\text{fuel}}}{\rho_{\text{fuel}}} + \frac{m_{\text{air}}}{\rho_{\text{air}}}
\]
**Equation 6-3**
Combining Equation 6-2 and Equation 6-3, the measurement volume is:
\[
V_T = m_{fuel} \cdot \left( \frac{1}{\rho_{fuel}} + \frac{1}{\rho_{air}} \cdot \frac{AFR_{st}}{\Phi} \right) = \frac{1}{\Phi} \cdot m_{fuel} \cdot \frac{AFR_{st}}{\rho_{air}} + \frac{m_{fuel}}{\rho_{fuel}}
\]
Equation 6-4
This measurement volume for the Calibration and DI experiments are identical. This can be written as:
\[
\frac{1}{\Phi^{Cal}} \cdot m_{fuel}^{Cal} \cdot \frac{AFR_{st}}{\rho_{air}} + \frac{m_{fuel}^{Cal}}{\rho_{fuel}} = \frac{1}{\Phi^{DI}} \cdot m_{fuel}^{DI} \cdot \frac{AFR_{st}}{\rho_{air}} + \frac{m_{fuel}^{DI}}{\rho_{fuel}}
\]
Equation 6-5
Figure 6-17: Content of fuel and air in the local measurement volume
Rearranging Equation 6-5 gives:
\[
\Phi^{DI} = \Phi^{Cal} \cdot \frac{m_{fuel}^{DI}}{m_{fuel}^{Cal}} \cdot \frac{1}{1 + \frac{1}{AFR_{st}} \cdot \frac{\rho_{air}}{\rho_{fuel}} \cdot \left( 1 - \frac{m_{fuel}^{DI}}{m_{fuel}^{Cal}} \right)}
\]
Equation 6-6
If a fluorophore that is not quenched by Oxygen is added to the fuel, its fluorescence signal is given by:
\[
I_f = C \cdot I_{laser} \cdot \eta \cdot f(P, T) \cdot n_{tracer}
\]
**Equation 6-7**
where \(C\) is a proportionality constant, \(I_{laser}\) is the local laser intensity, \(\eta\) is the collection efficiency of the detection system, \(n_{tracer}\) is the amount of tracer and \(f(P, T)\) is the fluorescence yield of the tracer at a given pressure \((P)\) and temperature \((T)\).
When the fuel is fully vaporised, the amount of tracer is proportional to the amount of fuel. In terms of mass of fuel \((m_{fuel})\), the fluorescent intensity can be written as:
\[
I_f = C' \cdot I_{laser} \cdot \eta \cdot f(P, T) \cdot m_{fuel}
\]
**Equation 6-8**
If the LIF measurements are performed using the DI injector, the local fluorescent intensity will be:
\[
I_f^{DI} = C' \cdot I_{laser} \cdot \eta \cdot f(P^{DI}, T^{DI}) \cdot m_{fuel}^{DI}
\]
**Equation 6-9**
If calibration measurements are performed by injecting a homogenous charge using the calibration injector, the local fluorescent intensity can be expressed as:
\[
I_f^{Cal} = C' \cdot I_{laser} \cdot \eta \cdot f(P^{Cal}, T^{Cal}) \cdot m_{fuel}^{Cal}
\]
**Equation 6-10**
Dividing **Equation 6-9** by **Equation 6-10** gives the local ratio of mass:
\[
\frac{m_{fuel}^{DI}}{m_{fuel}^{Cal}} = \frac{I_f^{DI}}{I_f^{Cal}} \cdot \frac{f(P^{Cal}, T^{Cal})}{f(P^{DI}, T^{DI})}
\]
**Equation 6-11**
Combining Equation 6-6 and Equation 6-11 gives the equivalence ratio as a function of the fluorescent intensities, the calibration stoichiometry and the fluorescence yields:
\[
\Phi^{DI} = \Phi^{Cal} \cdot \frac{I_f^{DI}}{I_f^{Cal}} \cdot \frac{f(P^{Cal}, T^{Cal})}{f(P^{DI}, T^{DI})} \cdot \frac{1}{1 + \frac{1}{AFR_{st}} \cdot \frac{\rho_{air}}{\rho_{fuel}} \cdot \left(1 - \frac{I_f^{DI}}{I_f^{Cal}} \cdot \frac{f(P^{Cal}, T^{Cal})}{f(P^{DI}, T^{DI})}\right)}
\]
Equation 6-12
### 6.2.2. Experimental setup
The fluorescence images were recorded using a 12bit Flowmaster3 CCD camera, coupled to a gated IRO image intensifier with a Halle \( f/2 \) 100mm achromatic fused silica lens. A BG4 filter in front of the lens selected the fluorescence. To avoid damage to the lens/intensifier/camera system in case the quartz annulus exploded, a broadband (BB) UV-enhanced mirror was used to reflect the fluorescence at 90° before entering the imaging system.
**Figure 6-18: Imaging Setup for the Equivalence Ratio measurement**
6.2.3. Measurement procedure
The first task consisted in determining the fuel injection duration of the calibration injector for the two throttle positions. The stoichiometry setting for the DI and Calibration run was matched as best as possible in order to obtain similar flame and therefore wall temperatures.
The calibration run requires the mixture to ignite in order to obtain a correct reading from the Lambda sensor. In the late injection case, the engine operated with a lean stratified mixture with an overall equivalence ratio $\Phi = 0.33$ (AFR of $\sim 45$). A homogeneous mixture will not ignite at this value of AFR. Thus, the mixture was leaned out as much as possible without reaching the misfire limit.
For the early direct injection case, the engine operated with a stoichiometric throttled air-fuel mixture. The calibration injection duration was adjusted to meet the same stoichiometry.
At each crank angle of interest, 2 sets of 80 LIF images were recorded. The first set consisted of images of the direct injection (referred to as DI images), the second set consisted of images from a homogeneous mixture of known equivalence ratio. (referred to as Calibration images).
For each crank angle measurement, the procedure consisted in:
- Dial the desired Crank Angle (-2°CA due to PTU delay)
- Take background images (no injection)
- Dial the desired Direct Injection duration
- Switch DI injector on
- Take images
- Switch DI injector off
- Dial the desired Calibration injection duration
- Switch on the Calibration injector
- Take images
- Switch Calibration injector off
- Repeat for other Crank Angles
Equation 6-12 is obtained by assuming that the laser intensities in the DI measurement and its corresponding Calibration measurement are identical. By alternating a DI and a Calibration measurement, the fluctuation of laser power with time was minimised.
| Injection duration | Early Injection | Late Injection |
|--------------------|-----------------|---------------|
| | DI | Calibration | DI | Calibration |
| λ | 1 | 1 | ~3 | 1.55 |
| ΔP | 340mbar | 20mbar |
Table 6-2: Operating Parameters for the equivalence ratio measurements
6.2.4. Fluorescence yield correction
Obtaining equivalence ratio images requires crank-angle resolved determination of the thermodynamic in-cylinder conditions, in order to account for variations in fluorescence yield for the DI and Calibration image.
The differences between the DI and Calibration cycle is due to the charge cooling effect caused by the vaporisation of the fuel that is directly injected into the cylinder. The energy exchange between the air and the fuel can be written as:
\[ m_{air} \cdot \Delta H_{air} = -m_{fuel} \cdot \Delta H_{fuel} \]
where \( \Delta H_{air} \) and \( \Delta H_{fuel} \) are the variation of enthalpy for the air and the fuel respectively.
If the initial temperature of the air is \( T_{air}^i \) and the temperature at the end of the charge cooling is \( T^f \), assuming that the cooling process occurs and constant pressure, the air’s change in enthalpy is:
\[ \Delta H_{air} = c_{p,air} \cdot (T^f - T_{air}^i) \]
where \( c_{p,air} \) is the specific heat of the air.
22.214.171.124. Early injection case
When direct injection occurs during the intake stroke, even though the injected fuel is in the super cooled regime, it will evaporate until it saturates the air. The partial pressure of the fuel at 25°C is a $10^{th}$ of the total pressure ($P_{fuel}^{sat} = 0.06\text{bar}$). Because the volume of air is 50 times that of the fuel when running at stoichiometric conditions, the fuel can therefore totally vaporise. The initial and final pressure and temperature conditions of the fuel are $(P_{fuel}^i, T_{fuel}^i)$ and $(P_{fuel}^{sat}, T^f)$ respectively.
The change in the enthalpy of the fuel can be decomposed as shown in Figure 6-19. This enables to write it as:
$$\Delta H_{fuel} = \Delta^v H_{fuel} + c_{p,fuel_{gas}} \cdot (T^f - T_{fuel}^i)$$
Equation 6-15
where $c_{p,fuel}$ is the specific heat of the fuel and $\Delta^v H_{fuel}$ is the heat of vaporisation of the fuel.
By introducing the AFR, the final temperature is given by:
$$T^f = \frac{AFR \cdot c_{p,air} \cdot T_{air}^i + c_{p,fuel_{gas}} \cdot T_{fuel}^i - \Delta^v H_{fuel}}{AFR \cdot c_{p,air} + c_{p,fuel_{gas}}}$$
Equation 6-16
The charge cooling was found to be 20°C and the thermodynamic values were taken at 30°C (see Table 6-3). The temperature of the charge was calculated assuming an isentropic compression (see Figure 6-21).
| | Iso-Octane | 3-Pentanone | Fuel |
|----------------|------------|-------------|------|
| $c_{p,gas}$ (J/g.K) | 2.3 | 1.6 | 2.2 |
| $\Delta^vH$ (J/g) | 300 | 440 | 328 |
Table 6-3: Thermodynamic values at 30°C for the early injection case
126.96.36.199. Late injection strategy
In the case of late injection (670°CA, i.e., 50° BTDC), the in-cylinder pressure and temperature at the end of the intake stroke are identical for the DI and Calibration cases: 0.980 bar and 40°C.
The variation between the cycles occurs at 670°CA when the direct injection of fuel causes the charge cooling in the compression stroke. The change in enthalpy can be decomposed as shown in Figure 6-20:
$$\Delta H_{fuel} = m_{fuel} \cdot [c_{p,fuel_{liq}} \cdot (T^i_{fuel} - T^{sat}_{fuel}) + \Delta^vH_{fuel} + c_{p,fuel_{gas}} \cdot (T^{sat}_{fuel} - T^f)]$$
Equation 6-17
where $c_{p,fuel_{liq}}$ and $c_{p,fuel_{gas}}$ are the specific heats of the fuel in the liquid phase and gas phase respectively.
Introducing the AFR, the final temperature is:
\[
T^f = \frac{AFR \cdot c_{p,air} \cdot T^i_{air} - c_{p,fuel_{liq}} \cdot T^i_{fuel} + (c_{p,fuel_{gas}} - c_{p,fuel_{liq}}) \cdot T^{\text{sat}}_{fuel} - \Delta^v H_{fuel}}{AFR \cdot c_{p,air} + c_{p,fuel_{gas}}}
\]
**Equation 6-18**
Pressure and temperature were plotted against Degree Crank Angle (°CA) to determine the in-cylinder conditions. At the start of injection, they are \(P=5\)bar and \(T=220\)°C. The temperature drop was 17°C. The thermodynamic values were therefore taken at 210°C and can be found in *Table 6-4*. The injection lasted approximately 15°CA: the fuel imposed a temperature drop of 1°C per crank angle. This temperature drop was implemented in the isentropic compression for each crank angle.
| \(c_{p,liq}\) (J/g.K) | Iso-Octane | 3-Pentanone | Fuel |
|------------------------|------------|-------------|------|
| \(c_{p,gas}\) (J/g.K) | 2.9 | 2.0 | 2.75 |
| \(\Delta^v H\) (J/g) | 170 | 250 | 183 |
| \(T^{\text{sat}}\) (°C)| 160 | 160 | 160 |
**Table 6-4: Thermodynamic values for the late injection case**
**Figure 6-21: In-cylinder Temperatures during the compression stroke**
Figure 6-22: Ratio of fluorescence yield during the compression stroke
Figure 6-22 shows a maximum increase of the fluorescence yield ratio by 10% for the ignition crank angle. However, the calculations do not take into account heat input from the cylinder walls, therefore the charge cooling was overestimated. This suggests an overestimation of the increase of the fluorescence yield ratio. For the image processing, the ratio was therefore assumed constant and set to unity.
6.2.5. Image Processing
For each crank angle, the average background image was calculated as:
\[
\overline{B(i,j)} = \frac{\sum_{k=1}^{N} B_k(i,j)}{N}
\]
Equation 6-19
where \( N \) is number of images per set, and \( B_k(i,j) \) is the \( k^{th} \) single shot background image.
The raw images at a specific crank angle were “average background” subtracted:
\[
I_{f,k}(i,j) = I_{raw,k}(i,j) - \overline{B(i,j)}
\]
Equation 6-20
The local average DI and Calibration fluorescent intensity in pixel \((i,j)\) are respectively:
\[
\overline{I_f^{DI}(i,j)} = \frac{\sum_{k=1}^{N} I_{f,k}^{DI}(i,j)}{N}
\]
Equation 6-21
\[
\overline{I_f^{Cal}(i,j)} = \frac{\sum_{k=1}^{N} I_{f,k}^{Cal}(i,j)}{N}
\]
Equation 6-22
The local single-shot equivalence ratio is given by:
\[
\Phi_k^{DI}(i,j) = \Phi_k^{Cal} \cdot \frac{I_{f,k}^{DI}(i,j)}{I_f^{Cal}(i,j)} \cdot \frac{1}{1 + \frac{1}{AFR_{st}} \cdot \frac{\rho_{air}}{\rho_{fuel}} \cdot \left(1 - \frac{I_{f,k}^{DI}(i,j)}{I_f^{Cal}(i,j)}\right)}
\]
Equation 6-23
The average equivalence ratio is expressed as an average of mass, and therefore as an average of intensities:
\[
\overline{\Phi^{DI}(i,j)} = \Phi_k^{Cal} \cdot \frac{\overline{I_f^{DI}(i,j)}}{\overline{I_f^{Cal}(i,j)}} \cdot \frac{1}{1 + \frac{1}{AFR_{st}} \cdot \frac{\rho_{air}}{\rho_{fuel}} \cdot \left(1 - \frac{\overline{I_f^{DI}(i,j)}}{\overline{I_f^{Cal}(i,j)}}\right)}
\]
Equation 6-24
The standard deviation is then defined as:
\[
\sigma_{\Phi^{DI}}(i,j) = \sqrt{\frac{\sum_{k=1}^{N} (\overline{\Phi^{DI}(i,j)} - \Phi_k^{DI}(i,j))^2}{N}}
\]
Equation 6-25
The relative fluctuation is:
$$F(i, j) = \frac{\sigma_{\Phi_D}(i, j)}{\Phi^{DI}(i, j)}$$
Equation 6-26
The final equivalence ratio images have an intensity of 1 for an equivalence ratio of 1. The images were multiplied by 64, and presented in a logarithmic scale ranging from 0 to 512, thus allowing to illustrate equivalence ratios ranging from 0 to 8 and maintaining good colour resolution around stoichiometry.
The images for the equivalence ratio fluctuation were multiplied by 64 and were presented on a linear scale ranging from 0 to 124, to illustrate variations up to 200%.
Following Pages:
**Figure 6-23:** Equivalence ratio and its fluctuations (in%) in the vertical plane in the centre of the combustion chamber. Early Injection (SOI at $420^\circ$CA) and Late Injection (SOI at $670^\circ$CA). $360^\circ$CA is the exhaust/inlet TDC and $0^\circ/720^\circ$CA is the compression/combustion TDC.
| Injection Timing | Crank Angle | Equivalence Ratio |
|------------------|-------------|-------------------|
| Early Injection | 580° | |
| | 620° | |
| | 650° | |
| | 670° | |
| | 680° | |
| Late Injection | 675° | |
| | 678° | |
| | 681° | |
| | 684° | |
| | 687° | |
Equivalence Ratio Scale:
- Lean: 0.05 - 0.1
- Average: 0.2 - 0.5
- Rich: 1 - 8
Average and Fluctuation images show the distribution of equivalence ratio at different crank angles for early and late injection timings.
Equivalence Ratio
Crank Angle
Average
Fluctuation
Early Injection
SOI 420°CA
Late Injection
SOI 670°CA
0 20% 40% 60% 80% 100% 120% 140% 160% 180% 200%
lean rich
6.2.6. Results
188.8.131.52. Early Injection
The images show vertical structure caused by low laser power regions in the laser sheet (and therefore high SNR). This was due to combustion material deposits on the piston crown window.
In the case of the early injection, the images show a homogeneous and stoichiometric mixture after 650°CA.
The equivalence ratio images obtained at 580°CA (40° ABDC) and 640°CA indicate rich regions. However, at these piston positions, the fluorescence signal was low (20 counts) because the amount of fuel per unit volume is low.
The homogeneity observed at 650°CA (i.e. ~50°CA before ignition) suggests that the fuel and air will still have sufficient time to mix at full load (6 to 7 times the speed). This would also be helped by the increase in turbulence at higher rpm. The cycle-to-cycle fluctuation is of the order of 60%. This value is slightly high, and can be explained by the low signals throughout the experiment.
In this early injection mode, there were no misfires and the flame was blue indicating a stoichiometric, well premixed flame. Therefore, the images were expected to be that of a homogeneous stoichiometric mixture. Had the fluorescence yield correction been applied, the results would have become inconsistent. This further agrees with the idea that the energy input to vaporise the fuel is not only supplied through cooling of the air but also by the cylinder walls.
184.108.40.206. Late Injection
The late injection images show a collapsed spray shooting across the combustion chamber. The early part of the injection shows a very rich mixture ($\Phi > 5$) with very little cycle-to-cycle fluctuation. This indicates that the early part of the injection is very stable and can be explained by the high kinetic energy of the ballistic liquid fuel. At the end of the injection (687°CA) a stoichiometric mixture appears at the bottom edge of the spray. This region corresponds to the location in the spray where the
fuel/air interface is the largest. The mixing in that region is also the highest and is indicated by the higher fluctuations (although the higher fluctuations can also be attributed to low signals and high signal-to-noise ratio). After the end of the injection, the cloud of fuel is entrained towards the cylinder wall on the exhaust side. The cycle-to-cycle fluctuations increase, indicating that the mixture is in the gas phase and is prone to fluctuations from in-cylinder air motion. The cloud is stratified: it has very fuel-rich region nearest to the exhaust side, and an increasingly diluted mixture towards the sparkplug.
At time of ignition, the rich fuel cloud has travelled beyond the spark plug, and has disappeared from the optical access. A small stoichiometric mixture is present at the top left of the spark plug, but the mixture around the spark plug is mainly lean. The part of the combustion chamber closest to the injector is ultra lean.
Advancing the time of ignition or retarding the start of injection would locate the ignitable mixture closer to the spark plug. However, in the ideal stratified mixture, the ignitable pocket is the richest region, and the neighbouring air/fuel cloud should be stoichiometric. In this late injection strategy, the ignitable pocket is surrounded by an even richer mixture on one side and a leaner mixture on the other. This results in the production of a sooty flame in the rich region and unburnt hydrocarbons in the other. Another solution could consist in putting the spark plug between the exhaust valves, opposite to the injector and retarding the time of ignition. This would give the rich cloud some additional mixing time, and reduce the equivalence ratio to a more appropriate value (~1.1-1.2) for ignition.
In these conditions, the engine misfired and the flame was yellow, indicating rich regions and that the charge was not well premixed.
The excess air played an important role in keeping the temperature of the burnt gases down: the exhaust temperature stabilised at around 300°C. This allowed to run the engine continuously, whereas in the early/homogenous injection, the exhaust temperatures would exceed 400°C, and injection had to be stopped after 40 images in order not to damage the engine.
6.3. LSD measurements
6.3.1. Experimental Setup
The LSD technique was applied to the engine in the early and late injection modes. The tracer system that gave the best results in the previous study was used: 3-Pentanone (0.5%) mixed with 3-Hexanone (1.5%) in Iso-Octane (98%).
Simultaneous LIF and Mie scatter images were recorded using the same optical set-up as the measurements performed in the pressure vessels: a 4-mirror system and a 12 bit Flowmaster3 CCD camera, coupled to a gated IRO image intensifier with a Halle $f/2$ 100mm achromatic fused silica lens. A combination of two ND filters (3 and 0.3) was in the Mie scatter path to attenuate the signal to the same order of magnitude as the fluorescence signal. A BG4 filter was in the LIF path to eliminate residual 266nm and 532nm light. Because the images section was wider than it was high, the camera was mounted at 90° to the IRO to optimise the separation of the PLIF and Mie Scatter image on the two halves of the CCD chip (see Figure 6-24).
**Figure 6-24:** Imaging Setup for the LSD technique
6.3.2. Spatial Calibration
A calibration plate with crosses placed at 4mm intervals was used. The ND filter in the Mie scatter path attenuated the intensity of the Mie scatter signal by a factor of $10^3$. Because the laser power was low, an acceptable Mie scattering image of the calibration plate was difficult to obtain. This proved to be the major difficulty in applying the technique. The light sheet used to illuminate the calibration plate required extremely fine adjustments and an average of 2000 images was necessary to obtain a suitable calibration image for the image-correction algorithm.
6.3.3. Measurement procedure
For both the early and late injection case, 80 images were recorded at each crank angle during the injection duration. In the Early Injection mode, this required 2 runs of 40 images due to the temperature of the exhaust gases. Only 1 run was necessary in the Late Injection mode. For each crank angle, a background image was averaged from 80 single shot images where the laser was fired in the engine without injecting fuel.
The laser sheet was positioned at the centre of the combustion chamber. The triggering scheme for the laser, intensifier and camera was the same as the equivalence ratio measurements.
6.3.4. Image Processing
The images were processed to obtain average and rms data for the PLIF and Mie scatter images.
The images were background subtracted. An additional spray-induced background was subtracted (typically 2 to 5 counts). The images were then corrected for position. The LSD images were obtained by dividing the corrected average image of PLIF by the corrected average image of the Mie scatter.
SMD images were then determined by multiplying the LSD image by a Calibration constant. Due to the similarities between the injector present in the engine (Bosch swirl $70^\circ/10^\circ$) and the injector used in the pressure vessels (Bosch swirl $70^\circ/0^\circ$), it was assumed that both would yield similar SMDs. The LSD images were therefore calibrated using the SMD values of from the $70^\circ/0^\circ$.
Following pages:
**Figure 6-27:** Early Injection (SOI: $420^\circ$CA) - LIF and Mie scatter images (Average and Rms) presented in a logarithmic scale. The SMD image is a calibrated LSD image presented in linear scale.
**Figure 6-28:** Late Injection (SOI: $670^\circ$CA) - LIF and Mie scatter images (Average and Rms) presented in a logarithmic scale. The SMD image is a calibrated LSD image presented in linear scale.
Crank Angle
422° 423° 424° 425° 426° 427° 428°
LIF
Rms
Avg
Mie scatter
Avg
Rms
LSD
0 10µm 20µm 30µm 40µm
0 200 400 600 800 1000
0 200 400 600 800 1000
Crank Angle
429° 430° 431° 432° 433° 434° 435°
Rms
LIF
Avg
Mie scatter
Rms
LSD
0 10µm 20µm 30µm 40µm
Crank Angle
672° 673° 674° 675° 676° 677° 678°
LIF
Rms
Avg
Mie scatter
Avg
Rms
LSD
0 10µm 20µm 30µm 40µm
0 200 400 600 800 1000 1200 1400 1600 1800 2000
0 200 400 600 800 1000 1200 1400 1600 1800 2000
| Crank Angle | 679° | 680° | 681° | 682° | 683° | 684° | 685° |
|-------------|------|------|------|------|------|------|------|
| Rms | | | | | | | |
| LIF | | | | | | | |
| Avg | | | | | | | |
| Mie scatter | | | | | | | |
| LSD | | | | | | | |
Color scale:
- 0 to 200: Blue to Yellow
- 200 to 500: Yellow to Orange
- 500 to 1000: Orange to Red
- 1000 to 1500: Red to Pink
- 1500 to 2000: Pink to White
Crank Angle: 679°, 680°, 681°, 682°, 683°, 684°, 685°
LSD: 0 to 40 μm
6.3.5. Results
The images show an injector delay corresponding to $2^\circ$CA (i.e. 0.33$\mu$s) between the injector trigger signal and the actual start of injection (or Optical Start Of Injection – OSOI).
At 1000rpm, an injection duration of 1.8ms represents $11^\circ$CA. However, the intensities in the PLIF and Mie scatter images only start reducing $12^\circ$CA after OSOI. At $13^\circ$CA after OSOI, there is no more fuel at the injector tip, indicating that the injector closes within $2^\circ$CA. That suggests that the injector opens fully within $2^\circ$CA.
The injection process for both the early and late strategies shows 3 distinct phases. During the first $4^\circ$CA after OSOI, the spray is in a transient phase. This transient phase is twice as long as the needle lift. The variation of the fuel passage causes the fuel pressure to oscillate and these oscillations are not immediately attenuated when the injector is fully open. The fuel exiting the injector at this early phase has no common characteristics with the later part of the spray. It is referred to as “pre-spray”. It takes approximately 0.66ms for the spray to fully develop. From $4^\circ$CA to $11^\circ$CA after OSOI, the injection is in steady state and the spray geometry is unchanged: as the injector closes, the cone angle is unchanged.
**Figure 6-29: Injector Opening and Spray Regime**
When the spray is fully developed, the fluorescence signal remains constant even though there is an increasing amount of fuel present inside the cylinder. This can be explained by the conical nature of the spray, whereby the number of droplets per unit volume decreases as they travel away from the injector tip. The evaporation of these droplets also diffuses mass outside the sheet. Eventually, the fluorescence from the diluted tracer is below the detection threshold of the intensifier/camera system.
220.127.116.11. Early Injection
For this injection strategy, the cone angle of the fully developed spray is $70^\circ$.
At the start of the injection ($422^\circ$CA), the SMDs are very high. These values are inaccurate, because the fuel is likely to be present in ligaments and the LSD technique only applies to spherical droplets.
In these conditions, the fuel temperature is below boiling point. Therefore the evaporation process requires the presence of unsaturated air.
At $423^\circ$CA, most of the pre-spray has SMD values around $20\mu m$. The droplet sizes at the edge of the spray in the near nozzle region are twice as high.
At $424^\circ$CA, the spray has started to open up, therefore enhancing evaporation: the droplet sizes have decreased to around $15\mu m$.
Beyond $425^\circ$CA, when the spray is established, the droplet sizes around the nozzle are $30\mu m$). The proximity of the cylinder head limits the motion and availability of air for the top part of the spray, therefore compromising the mixing and evaporation. On the other hand, the bottom part of the spray benefits from almost unlimited amounts of unsaturated air, and no boundary conditions. This results in higher SMDs at the top ($15\mu m$) compared to the bottom ($10\mu m$). Furthermore, the swirling motion of the spray drives the larger droplets towards the edge of the spray. This further contributes to the high SMDs in the top edge ($18\mu m$).
The SMD values for the last part of the injection ($434$ and $435^\circ$CA) are unfortunately unreliable, because the intensity counts of the fluorescence signal are too low to yield accurate data.
18.104.22.168. Late injection
As the spray is injected into higher pressures (approximately 5bar at $670^\circ$CA), the cone angle collapses to $30^\circ$. This feature is common to swirl injectors and is explained by the breaking down of the swirling motion due to the increased density of the air. The reduced centrifugal force homogenises the droplet distribution and the SMDs within the spray are approximately $13\mu m$. This collapse of the spray increases the density of the droplets and therefore reduces the air/fuel mixing.
The droplet sizes around the nozzle ($35\mu m$) are identical to the early injection case.
6.4. PIV Measurements
6.4.1. Experimental Setup
Mie scatter PIV was applied to the optical engine using a 532nm light sheet. The fuel consisted of Iso-Octane only.
The images were captured on a FlowMaster3 Camera coupled to a Nikon $f/4$ 50mm lens. A 532nm filter was installed on the end of the lens to select the Mie scattering. A broadband mirror reflected the signal at $90^\circ$.
**Figure 6-30: PIV Imaging Setup**
The images were recorded in the Cross-correlation mode, i.e. 2 images per frame. The triggering scheme for the laser and camera for this type of measurement is illustrated in *Figure 6-31*. In this mode, the camera exposure is fixed to 10µs for the 1st frame and 125ms for the 2nd frame at the end of which both frames are read out to the computer. The camera trigger is such that the first lasing occurs at the end of the 1st frame.
6.4.2. Spatial Calibration
Usually, PIV is applied to flow measurements, where the flow is seeded with low density particles. The cross-correlation is then accurate because the intensities in the interrogation window have distinct intensity peaks. In the case of fuel velocity measurements, the fuel acts as the seed and can be extremely dense (see Figure 6-32).
Figure 6-32: Interrogation regions of the spray – non dense (a) and dense (b)
For the cross-correlation to be successful, the image resolution must be such that structure can be resolved. The spray was magnified as much as possible whilst maintaining it entirely within the camera frame. The focusing of the camera and the spatial calibration of the combustion chamber was performed by illuminating the calibration plate with a flashlamp. The resolution was 30µm per pixel.
**Figure 6-33: Calibration Image**
### 6.4.3. PIV Settings
For a fixed resolution, the crucial variable is the time interval between the two laser shots (dt), because it defines the displacement. The expected velocities of the spray ranged from 1m/s to 80m/s (these velocities were obtained from the PDA measurements with the 70°/0° Bosch swirl injector). *Table 6-5* shows the pixel displacement for various speeds, at various values of dt.
| Velocity | 1m/s | 5m/s | 10m/s | 20m/s | 50m/s | 80m/s |
|----------|------|------|-------|-------|-------|-------|
| **05µs** | 0.2 | 0.8 | 1.7 | 3.3 | 8.3 | 13.3 |
| **10µs** | 0.3 | 1.7 | 3.3 | 6.7 | 16.7 | 26.7 |
| **15µs** | 0.5 | 2.5 | 5.0 | 10.0 | 25.0 | 40.0 |
| **20µs** | 0.7 | 3.3 | 6.7 | 13.3 | 33.3 | 53.3 |
**Table 6-5:** Displacements in pixels for various velocities and $dt$
For each crank angle, 100 double-frame images were recorded for 3 different $dts$: 5, 10 and 20 $\mu$s. Before each set of 100 images, a background image was averaged from 100 single shot images where the laser was fired in the engine without injecting fuel.
The images were background subtracted and corrected. To reduce computing time, the images were masked, so that vectors would only be calculated in the region where the spray was present.
The individual images were then processed with different interrogation window sizes with single passes (64×64 and 32×32), constant window-size multipasses (64×64 and 32×32) and adaptive multipasses (64×64 then 32×32).
### 6.4.4. Results
The Mie scatter images were used to determine the displacement of the pre-spray. Its velocity was calculated to be approximately 80 m/s. The results that gave the closest velocity values were obtained with a $dt$ of 5 $\mu$s.
The spray density is the main reason for this result. Many droplets are travelling in slightly different directions. By setting a short time interval, their displacement is kept low and similar: the peak in the cross-correlation indicates this displacement. For longer time intervals, the displacements of the numerous droplets will be different and the peak observed in the cross-correlation will not correspond to any physical displacement.
Amongst the results obtained with $dt=5\mu s$, the best match was obtained using a constant window size multipass with a 32×32 interrogation region and a 50% overlap.
The single shot vector fields obtained in Figure 6-34 illustrate the problems caused by high density: in both the early and late injection cases, the vectors obtained in the very dense region around the nozzle are incorrect. Also, some regions of the image have patches where velocity data are incorrect, or could not be determined at all. These patches with incorrect data vary from one image to another. Therefore, the averaging of the single shot vector fields gives an ensemble idea of the spray velocities, but the velocities obtained in the denser parts of the spray, where the data are ambiguous, are likely to be underestimated.
Figure 6-34: Single Shot PIV Images (32×32), 2-pass
Following pages:
Figure 6-35: Velocities and corresponding Average Mie scatter images for the Early and Late injection cases. The vector fields are presented with a grey scale Mie scatter image in the background.
| Crank Angle | 423° | 424° | 425° | 426° | 427° | 428° |
|-------------|------|------|------|------|------|------|
| **Early Injection** | **Velocity** |  |  |  |  |  |  |
| | **Mie Scatter** |  |  |  |  |  |  |
| Crank Angle | 673° | 674° | 675° | 676° | 677° | 678° |
|-------------|------|------|------|------|------|------|
| **Late Injection** | **Velocity** |  |  |  |  |  |  |
| | **Mie Scatter** |  |  |  |  |  |  |
| Angle | Speed (m/s) |
|-------|------------|
| 429° | 0, 10, 20 |
| 430° | 30, 40, 50 |
| 433° | 60, 70, 80 |
| 434° | |
| 435° | |
| 436° | |
| 437° | |
| 679° | 0, 5, 10 |
| 680° | 15, 20, 25 |
| 683° | 30, 35, 40 |
| 684° | |
| 685° | |
| 686° | |
| 687° | |
Color scale:
- Dark blue: 0 m/s
- Light blue: 250 m/s
- Medium blue: 500 m/s
- Green: 750 m/s
- Yellow: 1000 m/s
- Orange: 1250 m/s
- Red: 1500 m/s
- Bright red: 1750 m/s
- Dark red: 2000 m/s
6.4.5. Discussion
Even though different regions of the instantaneous vector fields show ambiguous results, the averaged vector fields in the less dense regions agree with the evolution of the spray given by the crank-angle resolved Mie scatter images. This can be attributed to the high number of images used in the calculation.
The lens aperture is set so that the high density (therefore brightest) regions of the spray illuminate the CCD to the maximum of 4000 counts. However, this limits the resolution in the less dense part of the spray, where the intensity can be 500 times lower.
22.214.171.124. Early injection
At $423^\circ$CA, the spray is very dense and the resolution is too low to obtain reliable PIV data. As the cone angle starts to widen, the spray becomes more dilute and the structure can be resolved in the less dense regions. At $424^\circ$CA, the pre-spray is the least dense part of the spray, and its velocity is $80 \text{ m/s}$. The velocities of the droplets following the pre-spray are lower, typically 30 to $40 \text{ m/s}$. This phenomenon is expected because the effective fuel passage is smaller as the needle just begins to lift, therefore giving higher velocities.
The density of the fuel present beyond the spark plug is too low to obtain a sufficiently high Mie scatter signal and the velocities are therefore inaccurate.
126.96.36.199. Late Injection
In the late injection mode, the collapsing of the spray increases the density, and the PIV measurements cannot provide accurate data until the cone angle has opened sufficiently ($675^\circ$CA). The velocities throughout the injection range typically between 20 and $30 \text{ m/s}$. They are lower than the ones obtained for the early injection because the higher pressure increases the collision rate between air and fuel molecules. This induces a lower spray penetration which is clearly visible on the Mie scatter image at $676^\circ$CA.
However, the fuel speeds are still high, and the fuel is projected past the spark plug.
6.5. Summary
Three measurement techniques were applied to the optical engine to characterise the spray and mixture formation process for two different injection strategies.
These results showed that direct injection during the intake stroke gave a wide spray which facilitated the air/fuel mixing to give a homogeneous premixed charge at time of ignition, as a PFI engine would.
When the fuel was injected in the later phase of the compression stroke, the spray gave similar droplet sizes. However, the collapse of the cone angle reduced the air/fuel mixing. This poor mixing created a fuel stratification. The low mixing time resulted in an inadequate stratified charge for "clean" combustion: at the time of ignition, there remained a rich region next to an ignitable pocket, and the latter was not at the appropriate location. The reduced evaporation also implies that more fuel was in the liquid phase and was able to maintain a high velocity. One method to reduce the penetration and avoid the fuel going past the spark plug could consist in injecting smaller droplets. They would evaporate faster, thus reducing the speed of the fuel cloud. This would also increase the mixing. The optimal start of injection would likely be a function of load (i.e. injection duration) and engine speed, and could be determined with LIF imaging.
PART IV
CONCLUSION, REFERENCES AND APPENDICES
Chapter 7
Conclusion
In order to meet the new emissions legislation, the automotive industry is having to refine the operation of its petrol engines. The GDI concept is a promising technology: it offers the opportunity of increased efficiency through unthrottled operation. However, the realisation of this concept is critically dependent on the in-cylinder mixture formation, especially in the late injection/lean operation mode. Ideally, this would require a precise stratification of the in-cylinder fuel-air mixture in 3 distinct zones: an ignitable pocket located at the spark plug, surrounded by a stoichiometric mixture of fuel and air, encompassed by air. High pressure injection and piston bowl geometries were early attempts to redirect the spray and stratify the air-fuel mixture. Air-guided systems improved the system by limiting wall wetting. However, the future seems to be in the spray-guided systems: the realisation of GDI concept strongly depends on the advances in injector technology.
The GDI sprays have become very refined. Due to the elevated injection pressures, the atomisation is improved, the particle sizes are much smaller and the mass flux is increased. These sprays are very fine and dense. The idea behind this project was to develop a tool that could be applied to these sprays for rapid 2-D characterisation. This involved applying Phase Doppler Anemometry (PDA), Planar Laser-Induced Fluorescence (PLIF) and the Laser-Sheet Dropsizing (LSD) technique to denser sprays in harsher environments, where these tools are at the limit of their validity.
The study lead to three important results.
Firstly, an optimal tracer system was determined for the application of the PLIF technique for liquid-phase measurements. It uses the combination of two ketones (3-
Pentanone and 2-Hexanone) which have opposite evaporative behaviours when mixed with Iso-Octane. This limits the differential evaporation. The low concentrations of these tracers give a good volume-dependent fluorescence signal in the liquid phase, whilst retaining reasonable signal intensities. Ketones also have the advantage of not being quenched by Oxygen, therefore enabling the use of this tracer system for realistic IC engine studies.
Secondly, the LSD and PDA techniques were applied to a GDI spray in a pressure vessel for realistic in-cylinder conditions, ranging from supercooled to superheated environments. The comparison of the results demonstrated several problems which can be improved.
The PDA technique suffers from multiple occupancy of droplets in these very dense sprays. Higher laser power and smaller measurement volume can increase the accuracy. The study also showed that the LSD technique provided good quantitative data at atmospheric conditions. In highly evaporative conditions, the technique still gave reliable SMD data for the early stages of the injection where evaporation was not dominant, but was limited afterwards by vapour-phase contribution to the fluorescence signal. This could be resolved in non-quenching environments by using Exciplex tracer systems and the appropriate filter to eliminate entirely the vapour phase from the collected fluorescence. On the other hand, the comparison of a two-phase LIF signal and liquid-phase Mie scatter signal can provide an indication of the evaporation rate.
Discrepancies between the results obtained with the PDA and LSD techniques were also attributed to 2 issues. The first is the $d^2$ dependence assumption of the Mie scattering signal. For small droplets, the proportion of scattered light at 90° can vary substantially for different diameters. Perhaps another observation angle would yield a better surface dependence of the Mie scatter signal. Second, multiple scattering will contribute additional intensity to both fluorescence and Mie scattering signals. This surplus of light is not related to the local dropsize distribution. However, this effect is probably of second order as the LSD technique uses a ratio of signals. The quantification of multiple scattering is currently under investigation.
In low evaporating regimes, the LSD technique therefore has the overall advantage of being a 2-D measurement technique, and will yield data with a maximum error of 30% in dense parts of the spray where PDA data is totally unreliable. If the spray evaporates
quickly, PLIF by itself is an appropriate tool for following the air-fuel mixture, because short droplet lifetimes limit the 2-phase flow behaviour of the spray.
Lastly, 3 diagnostics (PIV, PLIF and LSD technique) were applied in an optical GDI engine. First, it demonstrated the application of the LSD technique in an engine. The major difficulty proved to be the spatial calibration of the two images when using a single camera. Secondly, the study of the spray’s behaviour and the location of the fuel highlighted the important issues that the GDI concept faces: the stratification was at the wrong location because the fuel maintained ballistic speed for too long. By reducing the droplet diameters, the fuel would vaporise and therefore slow down much faster, enabling the formation of the cloud at the spark plug. Most importantly, the equivalence ratio measurements allowed the determination of the location and stoichiometry of the air-fuel mixture. This suggests that stratification can be achieved with small droplet spray-guided systems. It is now up to the injector manufacturers to provide such technology.
222
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Zhao, H. and Ladommatos, N. (1998) *Optical Diagnostics for In-Cylinder Mixture Formation Measurements in IC Engines*, Progress in Energy and Combustion Science, vol. 24, pp. 297-336.
Zimmer, L., Ikeda, Y., Domann, R. and Hardalupas, Y. (2002) *Simultaneous LIF and Mie scattering measurements for branch-like spray cluster in industrial oil burner*, AIAA 02-0340.
APPENDIX A
LIF and Mie Scatter images
Average fluorescence (LIF) and Mie scatter images obtained in the Pressure Vessel. For each (Pressure-Temperature) condition, the images at the five times after Start of Injection are illustrated.
| Pressure | Temperature | LIF | Mie |
|----------|-------------|-----|-----|
| 01 bar | 25°C | | |
| 03 bar | 135°C | | |
| 05 bar | 195°C | | |
| 10 bar | 295°C | | |
| 15 bar | 360°C | | |
| Time (ms) | LIF | Mie |
|-----------|-----|-----|
| 0.7 | | |
| 1.2 | | |
| 1.7 | | |
| 2.2 | | |
| 2.7 | | |
APPENDIX B
Time-resolved PDA results
| SMD (µm) | 0.7ms | 1.2ms | 1.7ms | 2.2ms | 2.7ms |
|----------|-------|-------|-------|-------|-------|
| | 0 | 4 | 8 | 12 | 16 | 20 | 24 |
| 5 | 0.0 | 0.0 | 0.0 | 6.3 | 7.1 |
| 10 | 10.5 | 2.9 | 0.0 | 13.3 | 7.2 |
| 15 | 9.4 | 0.0 | 0.0 | 13.9 | 10.7 |
| 20 | 13.6 | 0.0 | 0.0 | 16.8 | 10.4 |
| 25 | 19.8 | 0.0 | 0.0 | 16.9 | 12.0 |
| 30 | 17.7 | 0.0 | 0.0 | 18.1 | 14.4 |
| 35 | 16.2 | 0.0 | 0.0 | 18.5 | 14.5 |
| 40 | 15.2 | 0.0 | 0.0 | 18.6 | 15.2 |
| 45 | 15.6 | 0.0 | 0.0 | 18.0 | 15.4 |
| 50 | 16.4 | 0.0 | 0.0 | 18.0 | 15.0 |
| 55 | 16.8 | 0.0 | 0.0 | 18.1 | 16.6 |
| 60 | 16.9 | 0.0 | 0.0 | 18.8 | 16.9 |
| 65 | 16.1 | 0.0 | 0.0 | 18.1 | 16.1 |
| 70 | 16.1 | 0.0 | 0.0 | 18.1 | 16.1 |
| Number of Droplets | 0.7ms | 1.2ms | 1.7ms | 2.2ms | 2.7ms |
|--------------------|-------|-------|-------|-------|-------|
| | 0 | 4 | 8 | 12 | 16 | 20 | 24 |
| 5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 10 | 1 | 5 | 72 | 0 | 0 | 0 | 0 |
| 15 | 2 | 0 | 16 | 20 | 0 | 0 | 0 |
| 20 | 1 | 0 | 1 | 146 | 117 | 0 | 0 |
| 25 | 22 | 19 | 117 | 100 | 20 | 0 | 0 |
| 30 | 70 | 257 | 36 | 7 | 12 | 0 | 0 |
| 35 | 84 | 224 | 23 | 1 | 0 | 0 | 0 |
| 40 | 108 | 125 | 4 | 0 | 0 | 0 | 0 |
| 45 | 73 | 52 | 5 | 0 | 0 | 0 | 0 |
| 50 | 24 | 7 | 5 | 0 | 0 | 0 | 0 |
| 55 | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| 60 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 65 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 70 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| | 0 | 4 | 8 | 12 | 16 | 20 | 24 |
| 5 | 0 | 25 | 0 | 0 | 0 | 0 | 0 |
| 10 | 0 | 1 | 60 | 0 | 0 | 0 | 0 |
| 15 | 0 | 0 | 27 | 147 | 0 | 0 | 0 |
| 20 | 0 | 0 | 0 | 69 | 7 | 1 | 0 |
| 25 | 0 | 0 | 6 | 18 | 22 | 30 | 0 |
| 30 | 0 | 1 | 10 | 53 | 93 | 240 | 0 |
| 35 | 45 | 47 | 90 | 37 | 38 | 33 | 0 |
| 40 | 87 | 140 | 170 | 2 | 0 | 0 | 0 |
| 45 | 114 | 176 | 102 | 0 | 2 | 0 | 0 |
| 50 | 107 | 138 | 24 | 3 | 0 | 0 | 0 |
| 55 | 75 | 75 | 19 | 1 | 0 | 0 | 0 |
| 60 | 37 | 34 | 5 | 0 | 0 | 0 | 0 |
| 65 | 11 | 0 | 0 | 0 | 0 | 0 | 0 |
| 70 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| | 0 | 4 | 8 | 12 | 16 | 20 | 24 |
| 5 | 0 | 5 | 0 | 0 | 0 | 0 | 0 |
| 10 | 0 | 0 | 37 | 0 | 0 | 0 | 0 |
| 15 | 0 | 0 | 30 | 8 | 0 | 0 | 0 |
| 20 | 0 | 0 | 0 | 71 | 120 | 3 | 0 |
| 25 | 0 | 0 | 13 | 35 | 91 | 32 | 0 |
| 30 | 0 | 0 | 36 | 38 | 15 | 3 | 0 |
| 35 | 0 | 0 | 1 | 33 | 68 | 161 | 3 |
| 40 | 2 | 2 | 30 | 130 | 91 | 77 | 1 |
| 45 | 76 | 103 | 200 | 41 | 1 | 1 | 0 |
| 50 | 138 | 154 | 153 | 10 | 0 | 0 | 0 |
| 55 | 133 | 156 | 116 | 4 | 0 | 0 | 0 |
| 60 | 77 | 102 | 35 | 0 | 0 | 0 | 0 |
| 65 | 40 | 0 | 0 | 0 | 0 | 0 | 0 |
| 70 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| | 0 | 4 | 8 | 12 | 16 | 20 | 24 |
| 5 | 370 | 5 | 0 | 0 | 0 | 0 | 0 |
| 10 | 168 | 68 | 1 | 0 | 0 | 0 | 0 |
| 15 | 118 | 40 | 19 | 1 | 0 | 0 | 0 |
| 20 | 28 | 14 | 0 | 98 | 91 | 14 | 0 |
| 25 | 12 | 27 | 91 | 116 | 96 | 106 | 0 |
| 30 | 0 | 5 | 36 | 65 | 114 | 22 | 0 |
| 35 | 0 | 1 | 26 | 41 | 12 | 3 | 1 |
| 40 | 0 | 1 | 17 | 27 | 56 | 117 | 1 |
| 45 | 0 | 0 | 1 | 79 | 313 | 188 | 0 |
| 50 | 10 | 38 | 106 | 175 | 69 | 0 | 0 |
| 55 | 92 | 120 | 201 | 90 | 0 | 0 | 0 |
| 60 | 158 | 158 | 174 | 0 | 0 | 0 | 0 |
| 65 | 130 | 0 | 0 | 0 | 0 | 0 | 0 |
| 70 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| | 0 | 4 | 8 | 12 | 16 | 20 | 24 |
| 5 | 510 | 1 | 0 | 0 | 0 | 0 | 0 |
| 10 | 292 | 15 | 0 | 0 | 0 | 0 | 0 |
| 15 | 278 | 8 | 3 | 0 | 0 | 0 | 0 |
| 20 | 119 | 27 | 0 | 19 | 20 | 9 | 0 |
| 25 | 143 | 170 | 141 | 130 | 108 | 138 | 0 |
| 30 | 92 | 87 | 121 | 138 | 105 | 84 | 1 |
| 35 | 50 | 70 | 67 | 100 | 180 | 8 | 0 |
| 40 | 32 | 33 | 61 | 40 | 66 | 1 | 0 |
| 45 | 14 | 29 | 52 | 41 | 46 | 34 | 0 |
| 50 | 1 | 4 | 10 | 26 | 131 | 0 | 0 |
| 55 | 1 | 3 | 19 | 165 | 0 | 0 | 0 |
| 60 | 32 | 112 | 220 | 0 | 0 | 0 | 0 |
| 65 | 128 | 0 | 0 | 0 | 0 | 0 | 0 |
| 70 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
APPENDIX C
Time-resolved PDF of TSI and Dantec data
Each line represents a measurement point (MP).
The PDF from TSI data is on the left and the PDF from Dantec data is on the right.
The colours indicate the time ASOI.
MP 1
MP 2
MP 3
MP 4
MP 5
MP 6
MP 7
MP 8
MP 9
MP 10
MP 11
MP 12
MP 13
MP 14
MP 15
MP 16
MP 17
MP 19
MP 20
MP 23
MP 24
MP 25
MP 26
MP 29
MP 30
MP 31
MP 32
MP 34
MP 35
MP 36
MP 39
MP 40
MP 41
MP 43
MP 44
MP 45
MP 46
MP 47
MP 48
MP 49
MP 50
MP 52
MP 53
MP 54
MP 55
MP 61
Counts
diameter (µm)
MP 62
Counts
diameter (µm)
|
Rational Design of a GFP-Based Fluorogenic Caspase Reporter for Imaging Apoptosis In Vivo
Highlights
- Split GFP is “zipped” or redesigned to sense protease activity
- ZipGFP-based caspase reporter detects apoptosis in human cells
- ZipGFP caspase reporter enables imaging physiological apoptosis in zebrafish
Authors
Tsz-Leung To, Antonino Schepis, Rubén Ruiz-González, ..., Zhiqiang Dong, Shaun R. Coughlin, Xiaokun Shu
Correspondence
email@example.com
In Brief
Shu et al. develop a fluorogenic protease reporter by “zipping up” each fragment of split GFP in a manner that prevents their associate and fluorophore formation until release by specific proteolytic cleavage. The large signal enables imaging of protease activity in vivo.
To et al., 2016, Cell Chemical Biology 23, 875–882
July 21, 2016 © 2016 Elsevier Ltd.
http://dx.doi.org/10.1016/j.chembiol.2016.06.007
Rational Design of a GFP-Based Fluorogenic Caspase Reporter for Imaging Apoptosis In Vivo
Tsz-Leung To,1,2 Antonino Schepis,2 Rubén Ruiz-González,1,2,3 Qiang Zhang,1,2 Dan Yu,1,2 Zhiqiang Dong,4 Shaun R. Coughlin,2 and Xiaokun Shu1,2,*
1Department of Pharmaceutical Chemistry
2Cardiovascular Research Institute
University of California, San Francisco, San Francisco, CA 94158, USA
3Institut Quimic de Sarrià, Universitat Ramon Llull, Via Augusta 390, 08017 Barcelona, Spain
4College of Life Sciences and Technology, Huazhong Agricultural University, Wuhan 430070, China
*Correspondence: firstname.lastname@example.org
http://dx.doi.org/10.1016/j.chembiol.2016.06.007
SUMMARY
Fluorescence resonance energy transfer-based executioner caspase reporters using GFP are important tools for imaging apoptosis. While these reporters are useful for imaging apoptosis in cultured cells, their in vivo application has been handicapped by poor signal to noise. Here, we report the design and characterization of a GFP-based fluorogenic protease reporter, dubbed ZipGFP. ZipGFP-based TEV protease reporter increased fluorescence 10-fold after activation by protease. A ZipGFP-based executioner caspase reporter visualized apoptosis in live zebrafish embryos with spatiotemporal resolution. Thus, the ZipGFP-based caspase reporter may be useful for monitoring apoptosis during animal development and for designing reporters of proteases beyond the executioner caspases.
INTRODUCTION
Förster resonance energy transfer (FRET)-based reporters using GFP are valuable tools in dissecting signaling pathways in living cells. Further understanding of molecular and cell biology requires investigation of cell signaling in living animals. However, in vivo use of FRET reporters is difficult for two main reasons (Kardash et al., 2011). First, the signal of FRET reporters is weak due to small fluorescence change in the donor and acceptor fluorophores. Second, fluorescence imaging of living animals is challenging because of tissue autofluorescence, cell heterogeneity, and rapid shape and position changes. It is thus not surprising that use of FRET-based executioner caspase reporters in animals is limited (Campbell and Okamoto, 2013; Takemoto, 2003; Takemoto et al., 2007; Yamaguchi et al., 2011).
To overcome the limitations of FRET-based executioner caspase reporters, we sought to design a GFP-based fluorogenic protease reporter that achieves several-fold fluorescence increase upon caspase activation. Very recently, an infrared fluorogenic caspase reporter (iCasper) based on an infrared fluorescent protein (mIFP) was reported for imaging apoptosis in Drosophila (To et al., 2015). However, the bacterial phytochrome-based iCasper requires the cofactor biliverdin, which seems to be limiting in Drosophila and zebrafish (Yu et al., 2015). Although expression of heme oxygenase, which converts heme into biliverdin, overcomes this problem, a GFP-based fluorogenic reporter would be preferred since its fluorescence requires no cofactor in animals. Two GFP-based fluorogenic reporters, CA-GFP (Nicholls et al., 2011) and VC3AI (Zhang et al., 2013), have been used to image apoptosis in cultured cells. However, neither has been shown to detect apoptosis in animals such as zebrafish. Here we report the rational design of a GFP-based fluorogenic caspase reporter and its application to imaging apoptosis in the zebrafish embryo with spatiotemporal resolution.
RESULTS AND DISCUSSION
Design of a Protease Reporter by Caging GFP with mIFP
To design a GFP-based fluorogenic protease reporter, we modified self-assembling split GFP so that its self-assembly is regulated by protease activity. GFP contains 11 β strands (Movie S1) (Ormö et al., 1996; Yang et al., 1996). One part of split GFP contains ten β strands (β1–10); the other part contains the 11th β strand (β11) (Cabantous et al., 2005). β1–10 contain three amino acids that form the chromophore (Tsien, 1998, 2009), whereas β11 contains the highly conserved Glu222 that catalyzes chromophore maturation (Sniegowski, 2005). It has been shown that β11 rapidly binds to β1–10 and green fluorescence develops within several tens of minutes (Cabantous et al., 2005). The crystal structure of GFP suggests a compact fit of β1–10 and β11, which presumably provides the structural basis of the high binding affinity between the two parts (Movie S2). Based on this, we sought to prevent β11 binding to β1–10 by inserting β11, a short peptide (16 residues), into another protein in a manner such that β11 could be hidden until made available by proteolysis.
To achieve this, we first used iProtease (the protein scaffold of iCasper) as a vehicle to cage β11 rather than as a fluorogenic reporter (Figure S1A). We inserted β11 between the PAS and GAF domains of iProtease together with a TEV protease cleavage sequence (TevS) at a location previously suggested to be constrained until released by protease (Figure S1A) (To et al., 2015). The length of the insert plus TevS was 23 amino acids.
Because insertions of >11 residues at this site led to potentially decreased constraint based on the appearance of weak infrared fluorescence of the iProtease reporter in the absence of the protease activity, we truncated the PAS domain so that β11 would likely be tightened or distorted. We made four constructs with various truncations of the PAS domain (Figure S1A). In addition, we used heterodimerizing coiled coils (E5 and K5) (De Crescenzo et al., 2003) to lock the PAS and GAF domains together. In the original iProtease design, the self-assembling split GFP was used to lock the PAS and GAF domains together. We next co-expressed the β11–mIFP and β1–10 in HEK293 cells (and mCherry as a reference), with or without the TEV protease (Figures S1B and S1C). However, we observed constitutive fluorescence without TEV dependence. We hypothesized that this might be due to β1–10 binding to β11 before the translation or folding of the PAS domain and/or that β11 was not sufficiently constrained in the fully folded β11–mIFP.
To solve this problem, we decided to occlude the binding cavity of β1–10 so that it would not accommodate β11, with the occlusion being released by protease activity. We flanked the N and C termini of β1–10 with the heterodimerizing E5 and K5 coiled coils (Figure S1B), such that the leucine zipper-like E5/K5 coiled coils might block or “zip” the binding cavity and prevent β11 binding. Indeed, the new design obtained ~5-fold fluorescence increase upon TEV protease cleavage (Figure S1D). However, we noticed punctate structures of green fluorescence when employing β11–mIFP with truncations to Cys18 and Ile23, both in the absence and presence of TEV protease. The number of punctate structures was significantly reduced for β11–mIFP with truncations to Gly27 and Ile29. We considered further truncating the PAS domain to possibly reduce this potential aggregation problem and increase the signal gain to >5-fold. However, Ile29 is already at the knot region of mIFP (Figure S1A and Movie S3) and further truncations would be expected to either prevent protease cleaving or block β11 from binding to β1–10 after protease cleavage by pulling β11 into the knot region. Therefore, the mIFP caging approach was abandoned, but the principle of caging both β11 and β1–10 was applied and turned out to be important (see below).
**Design of a Protease Reporter by Zipping GFP with Coiled Coils**
In order to solve the aggregation problem and to further increase the signal, we decided to simply flank β11 with E5 and K5 since the coiled coils appear to successfully block β1–10. Based on this new design, the E5/K5 heterodimer (with protease cleavage sequence) zips both β11 and β1–10 in order to prevent self-assembly of the split GFP (Figure 1A). Upon protease cleavage, both β11 and β1–10 are unzipped, which enables self-assembly and leads to an increase in fluorescence. We named this reporter ZipGFP. As a proof of concept, we designed a TEV protease reporter by inserting a TEV cleavage sequence into ZipGFP (both the zipped β11 and β1–10). This ZipGFP-based TEV reporter achieved 10-fold fluorescence increase upon TEV cleavage in HEK293 cells with no punctate structures of green fluorescence (Figures 1B and 1C). The fluorescence quantum yield of the reassembled ZipGFP was determined to be 0.25. Zipping either β11 or β1–10 alone led to bright fluorescence even in the absence of TEV protease, and the fluorescence did not increase upon protease cleavage (Figure 1B). Thus, zipping of both β11 and β1–10 was required to adequately prevent reconstitution of split GFP in this system.
We next measured the kinetics of the ZipGFP-based protease reporter to determine its temporal resolution. First, we separately purified two parts of ZipGFP in the presence of TEV protease. Then we mixed the cleaved ZipGFP and monitored green fluorescence over time. The green fluorescence increased over time with time to half-maximal fluorescence ($T_{1/2}$) of ~40 min (Figures 1D and 1E), which is similar to the previously reported kinetics for split GFP self-assembly (Cabantous et al., 2005). We also measured the kinetics of ZipGFP in cells with a rapamycin-activatable TEV system, in which the two parts of split TEV were fused to FKBP and Frb (Gray et al., 2010; Wehr et al., 2006). The addition of rapamycin induces a strong interaction between FKBP and Frb, which leads to reconstitution of split TEV. We co-expressed ZipGFP, mCherry, and the rapamycin-activatable TEV system in HEK293 cells and monitored green and red fluorescence over time upon addition of rapamycin, which revealed $T_{1/2}$ of ~100 min (Figure S2 and Movie S4).
**ZipGFP Caspase Reporter Detects Apoptosis in Mammalian Cells**
To design a ZipGFP-based caspase reporter for imaging apoptosis, we inserted the consensus cleavage sequence (DEVD) of caspase-3 into both parts of ZipGFP. The well-characterized signaling pathways of apoptosis are composed of extrinsic and intrinsic pathways, both of which lead to activation of the executioner caspases that include caspase-3 (Crawford and Wells, 2011). Detection of activated caspase-3 using antibody has been proved to be useful in identifying apoptotic cells in animals including zebrafish (Sorrells et al., 2013). And the conserved cleavage sequence (DEVD) of caspase-3 has been used previously in caspase reporters (Ding et al., 2015; Nicholls et al., 2011; Takemoto, 2003; To et al., 2015; Zhang et al., 2013).
We expressed ZipGFP in human glioblastoma LN229 cells. Upon addition of staurosporine, which induces apoptosis by activating caspase-3 (Tee and Proud, 2001), ZipGFP’s green fluorescence increased significantly in cells treated with staurosporine (Figure 2A and Movie S5), whereas the fluorescence signal of ZipGFP showed no or little change in cells not treated with staurosporine (Movie S6). To account for a potential increase in the concentration of reporter with time after transient transfection, we co-expressed mCherry. The ratio of green fluorescence to mCherry’s red fluorescence revealed a 5- to 10-fold increase in ZipGFP’s fluorescence upon apoptosis. Activation of caspase-3 in ZipGFP fluorescing cells was confirmed using a commercially available small-molecule active-site modifier for far-red fluorescent imaging of caspase-3 activity (Figure 2B). And the onset of apoptosis in single cells as detected by ZipGFP varied between 2 and 5 hr after addition of staurosporine (Figure 2C), consistent with previous results (Albeck et al., 2008). In addition, replacing the caspase cleavage site in ZipGFP β11 with TevS abolished the increase in fluorescence upon apoptosis (Figure S3), suggesting that the increased fluorescence requires caspase cleavage.
We next compared ZipGFP with a previous GFP-based fluorogenic caspase reporter CA-GFP. CA-GFP was reported to show
a 45-fold fluorescence increase after caspase activation in bacteria and a 3-fold increase in mammalian cells upon staurosporine-induced apoptosis (Nicholls et al., 2011). However, we observed less than 1-fold fluorescence increase after normalization by a co-expressed red fluorescent protein in LN229 cells upon addition of staurosporine (Figure 2D). We also compared ZipGFP with VC3AI, another previous GFP-based caspase reporter. Green fluorescence of VC3AI normalized to co-expressed mCherry in LN229 cells showed 2- to 6-fold fluorescence increase upon addition of staurosporine (Figure 2E), which is slightly smaller than ZipGFP. Together, these data suggest that the newly designed ZipGFP is one of the best choices for genetically encoded caspase detection in mammalian cells.
It should be pointed out that compared with iCasper, the fluorescence increase of ZipGFP is slower after triggering apoptosis. This is consistent with the fact that the in vitro $T_{1/2}$ of ZipGFP ($\sim$40 min) is significantly slower than that of iCasper (<10 s). The different kinetics is likely due to different mechanisms of fluorescence development: for iCasper, the chromophore is an endogenous molecule and rapidly binds to the activated reporter, and thus quickly becomes fluorescent (To et al., 2015). On the other hand, the chromophore of GFP is formed from three amino acids in a reaction that requires tens of minutes (Tsien, 1998). Nevertheless, our results demonstrated that ZipGFP develops fluorescence sufficiently fast to visualize caspase-3 activity and apoptosis in cultured mammalian cells.
**ZipGFP Visualizes Caspase Activity and Apoptosis in Live Zebrafish**
To determine whether the fluorescence signal and the temporal resolution of ZipGFP are sufficient for imaging apoptosis in animals, we expressed the ZipGFP-based caspase reporter in zebrafish. Zebrafish was chosen based on optical transparency and its importance as a model organism among vertebrate animals (Grunwald and Eisen, 2002). We injected mRNA encoding ZipGFP caspase reporter and mCherry linked via a T2A site into the embryo at the one-cell stage. T2A is a self-cleaving peptide used in co-expression of multiple genes (Szymczak et al., 2004).
We exposed the zebrafish embryo at the 50% epiboly stage (i.e., the blastoderm covers 50% of the entire distance between...
the animal and vegetal poles) to 100 μg/ml cycloheximide (Figure 3A), which was previously shown to induce apoptosis in the early zebrafish embryo (Negron and Lockshin, 2004). The embryo continued to develop with epiboly continuing for 2 hr after exposure to cycloheximide (Figure 3B and Movie S7), but cell movement became arrested at 2 hr and cells started to retract at 3 hr. During this time, the embryo showed bright mCherry fluorescence, indicating that the reporter was well expressed, but green fluorescence was absent or dim, suggesting that the caspase was inactive in the embryo. However, by 4 hr, green fluorescence became obvious in a few cells, and after 5–6 hr of treatment, many cells showed strong green fluorescence, suggesting caspase activation and apoptosis. Indeed, many of these cells were also fragmented or were round in shape, consistent with cellular morphological change during apoptosis. In the absence of cycloheximide treatment, embryonic development continued normally with evident somite formation as expected; red fluorescence was easily detected but green fluorescence was negligible, consistent with little apoptosis occurring during this early stage of development (Figure 3C). Caspase activation using antibody staining against the cleaved caspase-3 was confirmed in the zebrafish embryo treated with cycloheximide (Figure S4), and the observed effects of cycloheximide treatment on the zebrafish embryo are consistent with a previous demonstration of cycloheximide-induced apoptosis in this setting (Negron and Lockshin, 2004).
After demonstrating that the ZipGFP caspase reporter could be used to image cycloheximide-induced apoptosis in early zebrafish embryo during the gastrulation stage, we next demonstrated that this system could be used to visualize the spatiotemporal dynamics of apoptosis during normal embryo development after the gastrulation stage. We again expressed the ZipGFP caspase reporter (and mCherry) in zebrafish embryos by injection of mRNA at the one-cell stage. Time-lapse imaging of the head was started at 24 hr postfertilization (hpf) (Figure 3D). At this time, little green fluorescence was observed (Figure 3D and Movie S8). Ten hours later, green fluorescence was detected in a couple of cells. After 15–20 hr, more cells showed strong green fluorescence. By contrast, mCherry fluorescence was weak and decreased over time (Movie S8), presumably because its mRNA injected at the one-cell stage was degraded over time and/or diluted as development progressed. Green, presumed apoptotic cells were distributed around the developing retina and the forebrain. In particular, time-lapse imaging first detected green fluorescence on the rostral aspect of the forebrain. The number of green cells increased over time, and they appeared to form clusters. These cells appeared to have neuron-like long extensions or processes (Figure 3D, inset). Z section imaging indicated that many of them appeared to be located on the outer layer of the forebrain and the retina (Movie S9). Caspase activation in the brain region of zebrafish embryo at 48 hpf was confirmed using antibody staining against the cleaved
Figure 3. ZipGFP Caspase Reporter in Imaging Apoptosis in Zebrafish
(A) Confocal images of the zebrafish embryo at the 50% epiboly stage expressing ZipGFP caspase reporter and mCherry (see Movie S7).
(B) Time-lapse fluorescence images of zebrafish embryo in the zoomed area in (A) after addition of cycloheximide.
(C) Confocal images of the control zebrafish embryo (no addition of cycloheximide) expressing ZipGFP caspase reporter at 12 hpf. Arrow points to one of the somites.
(D) Time-lapse images of the brain of the zebrafish embryo expressing ZipGFP caspase reporter and mCherry. Imaging started at 24 hpf (see Movies S8, S9, and S10). Fb, forebrain; R, retina; L, lens. The dashed lines outline the retina.
(E) Images around the trunk of the zebrafish embryo expressing ZipGFP caspase reporter and mCherry at 48 hpf. Arrow points to the tail bud.
Scale bars, 200 μm (A); 50 μm (B); 200 μm (C); 80 μm (D and E).
caspase-3, which overlapped with ZipGFP fluorescence (Figure S5). Imaging of the trunk region of the zebrafish embryo also revealed green cells around the dorsal part of the somites and in the tail bud (Figure 3E). Some of them extended into the intersomitic regions (inset of Figure 3E and Movie S10).
The spatial pattern of ZipGFP fluorescence that we observed in the zebrafish embryo is consistent with a previous study based on TUNEL staining that showed clusters of apoptotic cells in the developing brain as well as in the trunk, including the tail bud (Cole, 2001). While TUNEL staining lacks temporal resolution, the temporal pattern revealed by ZipGFP indicated that apoptosis first occurred at the rostral part of the forebrain (Movie S6). It has been suggested that apoptosis provides a mechanism of removing neurons that failed to form appropriate connections during early neural circuit formation. Although our data suggest that the apoptotic cells in the brain are morphologically like neurons, detailed studies will be required to identify the cell type(s) and to reveal the molecular mechanisms. For example, specific promoters may be used to express the ZipGFP caspase reporter in the neurons or other cell types in the transgenic zebrafish, or zebrafish that express ZipGFP ubiquitously may be bred with existing lines that express fluorescent markers specifically in cell types of interest. And genetic manipulation can be used to investigate specific mechanisms of apoptosis.
SIGNIFICANCE
We have designed a fluorogenic protease reporter by “zipping” both parts of the self-assembling split GFP. ZipGFP-based protease reporter achieves ~10-fold fluorescence increase upon proteolytic cleavage, which is significantly larger than FRET-based fluorescence changes in donor and acceptor fluorophores and is thus ideal for imaging apoptosis in vivo. We have demonstrated that ZipGFP-based executioner caspase reporter is able to visualize apoptosis in the living embryos of zebrafish, which is an important model vertebrate for understanding embryonic development (Grunwald and Eisen, 2002). The ZipGFP caspase reporter will thus be an important tool in imaging apoptosis in vivo, which plays essential roles in animal development, maintenance of tissue homeostasis, and disease (Fuchs and Steller, 2011; Jacobson et al., 1997). It will be useful to dissect signaling networks that lead to apoptosis by hypothesis-driven studies as well as unbiased high-throughput screening in live animals such as zebrafish. ZipGFP may be further used to design reporters of many other proteases, which are essential for many biological processes (Lopez-Otin and Bond, 2008). These fluorogenic reporters overcome the sensitivity limitation of FRET-based reporters and will be suitable for imaging protease activity in physiological environments.
EXPERIMENTAL PROCEDURES
DNA Constructs
The details of all constructs in this study are listed in Table S1. All plasmid constructs were created by standard molecular biology techniques and confirmed by exhaustively sequencing the cloned fragments. To create the ZipGFP, we flanked split GFP 1–10 (β1–10) and split GFP 11 (β11) with the K5/E5 heterodimerizing coiled-coil system. Both K5 and E5 coils are 35 amino acids in length. In earlier designs, the PAS and GAF domains from mFP were also incorporated as scaffolding elements. In the original design, the E5-mFP(GAF)-protease cleavage sequence was fused to the N terminus of the split GFP 11, whereas mFP(PAS)-K5 was fused to the C terminus. In the later design, K5 was fused to the N terminus of split GFP 11, and protease cleavage sequence E5 to the C terminus. For split GFP 1–10, E5 was fused to the N terminus, whereas protease cleavage sequence-K5 was fused to the C terminus. To create a TEV protease reporter, we used the TEV consensus cleavage sequence ENLYFQS in both zipped split GFP 1–10 and split GFP 11. To create a caspase-3 reporter, we used the Caspase-3/7 cleavage sequence DEVDG (an additional glycine was added before the cleavage sequence as a flexible linker) in a manner similar to the TEV reporter. The two parts of the ZipGFP were cloned into the pcDNA3.1 vector (Life Technologies) for mammalian cell expression. The part containing the split GFP 1–10 was co-expressed with the mCherry fluorescent marker using a Thosea asigna virus 2A cleavage site (T2A). The two parts of the ZipGFP were either co-transfected as two plasmids (e.g., TEV reporter) or co-expressed in a single plasmid using an additional T2A (e.g., caspase-3 reporter). We also created unzipped controls in which either split GFP 1–10 or split GFP 11 was not zipped by K5/E5. As unzipped control, the split GFP 11 was fused to the C terminus of the FKBP protein. The CA-GFP construct pT-CA-GFP-IRES-mLumin was a gift from Jeanne Hardy at UMass Amherst (Addgene plasmid no. 32749). To create the construct for in vitro transcription of mRNA, ZipGFP caspase reporter (with T2A mCherry) was cloned into the pCS+ vector. DNA of VC3Al was synthesized with codon optimization for expression in mammalian cells.
Protein Purification and Characterization
Zipped split GFP 1–10 and split GFP 11 containing the TEV cleavage sequence (ENLYFQS) were individually expressed with a C-terminal polyhistidine tag on pBAD expression vector (Invitrogen). The TEV protease was co-expressed with each ZipGFP subunit using a ribosomal binding site in order to generate the cleaved form of each subunit in Escherichia coli. The pBAD constructs used for protein expression in E. coli are listed in Table S1. Proteins were purified with the Ni-NTA purification system (QIAGEN). Protein concentration was measured by the BCA method (Pierce). The protein solutions were assayed by LDS-PAGE using NuPAGE Novex 4%–12% Bis-Tris protein gels (Life Technologies). All spectroscopic and kinetic measurements of the protein solutions were performed in protein elution buffer (50 mM Tris, 300 mM NaCl, 350 mM imidazole [pH 7.9]). For the absorbance and fluorescence measurements, 35 μl of 0.25 mg/ml spGFP1–10 was mixed with 65 μl of 0.10 mg/ml spGFP11. The absorbance and fluorescence emission spectra were obtained using the Tecan Infinite M1000 microplate reader. Measurement of complementation kinetics was done in protein elution buffer in a total volume of 100 μl (spGFP1–10, 2.7 μM; spGFP11, 6.3 μM) at 37°C as maintained by the Tecan microplate reader. The quantum yield of re-assembled ZipGFP was measured using a fluorometer LS-55 (PerkinElmer) with fluorescein as the standard. The two parts of ZipGFP were purified in the presence of TEV and were then mixed. The absorbance of fluorescein and the re-assembled ZipGFP was matched and their fluorescence spectra were taken by the fluorometer. The areas of the emission spectra of both samples were then compared, and the quantum yield was calculated.
Mammalian Cell Cultures
The HEK293T/17 (ATCC CRL-11268) and LN229 (ATCC CRL-2611) were obtained from ATCC. Cells were passaged in DMEM supplemented with 10% fetal bovine serum, non-essential amino acids, penicillin (100 units/ml), and streptomycin (100 μg/ml). All culture supplies were obtained from the UCSF Cell-Culture Facility.
HEK293T/17 and LN229 cells were transiently transfected with the protease reporter (and the protease of interest if applicable) with the calcium phosphate method. Cells were grown in 35 mm glass-bottom microwell (14 mm) dishes (MatTek Corporation). Transfection was performed when cells were cultured to ~50% confluence. For each transfection, 4.3 μg of plasmid DNA was mixed with 71 μl of 1× Hanks’ balanced salts buffer and 4.3 μl of 2.5 M CaCl₂. Cells were imaged 24 hr after transient transfection.
Confocal Microscopy
For characterization of the protease reporters in cultured mammalian cells, transfected HEK293T/17 or LN229 cells were imaged in 35 mm glass-bottom...
microwell dishes on a Nikon Eclipse Ti inverted microscope equipped with a Yokogawa CSU-W1 confocal scanner unit (Andor), a digital CMOS camera ORCA-Flash4.0 (Hamamatsu), an ASI MS-2000XYZ automated stage (Applied Scientific Instrumentation), and a Nikon Plan Apo × 20X air (NA 0.75) objective. Laser inputs were provided by an integrated laser engine (Spectral Applied Research) equipped with laser lines (Coherent) 488 nm (6.3 mW) for GFP imaging and 561 nm (3.5 mW) for mCherry/mLumin imaging. The confocal scanning unit was equipped with the following emission filters: 525/50 nm for GFP imaging and 610/60 nm for mCherry/mLumin imaging. For the characterization of ZipGFP-based reporters in mammalian cells, images were acquired with an exposure time of 100 ms for both GFP and mCherry. For CA-GFP, an exposure time of 500 ms was used for both GFP and mLumin. For the characterization of ZipGFP caspase-3 reporter in zebrafish embryos, images were acquired with an exposure time of 500 ms for GFP and 500 ms (for embryo at the 50% epiboly stage) or 1 s (for embryo at 24 hpf) for mCherry. For the time-lapse imaging of embryo at the 50% epiboly stage, a z stack spanning 115 μm at an interval 5 μm was acquired every 8 min for ~7 hr. For the time-lapse imaging of embryo at 24 hpf, a z stack spanning 225 μm at an interval 5 μm was acquired every 8 min for ~24 hr. Image acquisition was controlled by the NIS-Elements Ar microscope imaging software (Nikon). Images were processed using NIS-Elements and ImageJ (NIH).
**Characterization in Cultured Mammalian Cells**
HEK293T/17 cells transiently transfected with TEV reporter or TEV reporter + TEV were imaged in 35 mm glass-bottom dishes ~24 hr after transfection. For time-lapse imaging of the caspase-3 reporter during apoptosis, LN229 cells at ~24 hr after transient transfection with the reporter were grown to ~90% confluence in 35 mm glass-bottom dishes. Time-lapse microscopy was performed using the confocal microscope described above with the aid of an environmental control unit incubation chamber (InVivo Scientific), which was maintained at 37 °C and 5% CO₂. To induce apoptosis, 1 μM of staurosporine was added to cells in PBS. The imaging dish was then quickly transferred to the incubation chamber for imaging. The time-lapse imaging continued for 6–8 hr with image acquisition every 8 min. For caspase-3 staining of cells treated with staurosporine, the 1× FLICA reagent from the FLICA 680 caspase-3/-7 assay kit (Immunochemistry Technologies) was added to the cell-culture medium. Cells were incubated for 60 min under existing culture conditions. Afterward, cells were washed twice with the apoptosis wash buffer provided in the detection kit and imaged in PBS (without fixation) using the confocal microscope describe above. For characterization of the kinetics of ZipGFP in HEK293 cells, the cells expressing ZipGFP, mCherry, and the split TEV system were treated with 10 nM rapamycin.
**Characterization in Zebrafish Embryos**
Zebrafish were handled in compliance with local animal welfare regulations and were maintained according to standard protocols ([http://zfin.org](http://zfin.org)). The culture was conducted in conformity with UCSF IACUC and AAALAC guidelines. In order to make mRNA for injection, the plasmid pCS2+ ZipGFP-T2a-mCherry was linearized with KpnI. In vitro transcription was performed using the mMachine mMessage Kit (Ambion). mRNA (100 pg) was injected at the one-cell stage. To perform live imaging, embryos at the 50% epiboly stage were manually dechorionated and mounted in 0.5% agar; 100 μg/ml cycloheximide in embryo medium was added shortly before recording. For the normal embryonic development experiment, embryos at 24 hpf were mounted in 1% agar. Time-lapse microscopy was performed using the confocal microscope described above with the aid of an environmental control unit incubation chamber (InVivo Scientific), which was maintained at 28 °C. For whole-mount immunostaining, embryos were fixed at 12 hpt with 4% paraformaldehyde (PFA) in 1× PBS. After several washes with PBS, the embryos were permeabilized with 0.5% Triton for 1 hr and then incubated in blocking solution (PBS, 10% goat serum, 1% DMSO, 0.1% Triton) for 2 hr. The embryos were then incubated with anti-activated caspase-3 antibody (1:100; BD Pharmingen, catalog no. 559565), followed by several washes in PBS 0.1% Triton and incubation with secondary Alexa Fluor 647 conjugated antibody (1:500; Invitrogen) in blocking solution. Finally, the embryos were mounted in agar and imaged with a Zeiss 710 confocal microscope. For antibody staining of the zebrafish brain, embryos at 48 hpf were fixed with 4% PFA in PBS for 2 days and post-fixed in 100% methanol for 2 hr. The embryos were then treated with 1% Triton in PBS for 1 hr and incubated in the blocking solution. The embryos were then incubated with anti-activated caspase-3 antibody (1:100; BD Pharmingen, catalog no. 559565), followed by several washes in PBS 0.1% Triton and incubation with secondary Alexa Fluor 647 conjugated antibody (1:500; Invitrogen) in blocking solution. Embryos were mounted in agar and imaged with the confocal microscope.
**SUPPLEMENTAL INFORMATION**
Supplemental Information includes five figures, one table, and ten movies and can be found with this article online at [http://dx.doi.org/10.1016/j.chembiol.2016.06.007](http://dx.doi.org/10.1016/j.chembiol.2016.06.007).
**AUTHOR CONTRIBUTIONS**
X.S. and S.R.C. initiated the project. X.S. and T.-L.T designed the reporters. S.R.C. and A.S. designed the zebrafish experiments. T.-L.T., A.S., R.R., Q.Z., D.Y., and Z.D. performed the experiments. X.S. and T.-L.T. wrote the manuscript. All the authors contributed to the final draft.
**ACKNOWLEDGMENTS**
This work was supported by National Institute of General Medical Sciences (NIGMS) R01 GM115399 (to X.S.), R01 HL054737 and R01 HL121387 (to S.R.C.), and EMBO fellowship ref. 306-2015 (to R.R.-G). We thank Dr. Binghui Li for sharing the coding sequence of VC3AI.
Received: March 13, 2016
Revised: May 31, 2016
Accepted: June 8, 2016
Published: July 21, 2016
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Katariina Heikkilä
SUSTAINABILITY STUDIES IN UNIVERSITIES
– Review on Study Modules of Sustainable Development and Responsible Business in Finnish and Some European Universities
FINLAND FUTURES RESEARCH CENTRE
FFRC eBook 13/2015
Katariina Heikkilä
Project Manager
Finland Futures Research Centre, University of Turku
firstname.lastname@example.org
Copyright © 2015 Heikkilä & Finland Futures Research Centre, University of Turku
Second revisited edition
ISBN 978-952-249-408-5 (First edition)
ISBN 978-952-249-411-5
ISSN 1797-1322
Finland Futures Research Centre
Turku School of Economics
FI-20014 University of Turku
Visiting address: Rehtorinpellonkatu 3, 20500 Turku
Korkeavuorenkatu 25 A 2, FI-00130 Helsinki
Åkerlundinkatu 2, FI-33100 Tampere
Tel. +358 2 333 9530
utu.fi/ffrc
email@example.com, firstname.lastname@example.org
1. THE AIM AND PROCESS OF ANALYSIS ................................................................. 4
2. STUDY MODULES AND PROGRAMMES AT THE INTERNATIONAL LEVEL .................. 8
Common Remarks on Study Programmes ........................................................................... 8
Learning Methods .................................................................................................................. 13
Research Centres and Institutes ............................................................................................ 22
3. STUDY MODULES AND PROGRAMMES AT THE FINNISH LEVEL ............................. 23
Minor Study Modules ........................................................................................................... 23
Master’s Programmes .......................................................................................................... 26
4. DISCUSSION .................................................................................................................. 30
Discussion of the Contents of Sustainable Development, Sustainability and Responsible Business .......................................................................................................................... 30
Importance of the visibility of the offerings of teaching and research ............................... 32
The Role of Interdisciplinarity, Interaction and Cooperation with Stakeholders in Teaching .............................................................................................................................................. 34
The Role of Education for Sustainable Development (ESD) and PRME in Universities ........ 35
REFERENCES ....................................................................................................................... 38
APPENDIXES ......................................................................................................................... 45
1. THE AIM AND PROCESS OF ANALYSIS
The task of this report was to gather and analyse material about Finnish and additionally some international, in practice European, higher education programmes that offer minor or major subject in sustainable development or responsible business/corporate social responsibility (CSR).
Benchmarking was done in order to understand how the education is connected with research or practical projects, what educational methods are used and what kind of role is given to multidisciplinarity in study modules. The purpose was to achieve comprehension whether Sustainable Development Studies in Turku University has unique characteristics and what might be the most interesting lines of development in the future.
The main focus of this benchmark report is on sustainable development. Sustainable development and responsible business/corporate social responsibility are subjects that intertwine together in many ways. In the Turku University Sustainable Development Studies are offered at the university level and Responsible Business at the level of the Turku School of Economics. However, they have clear connection and cooperation and Sustainable Development Studies are administered in the Finland Futures Research Centre that is situated at the Turku School of Economics.
Information retrieval was done through Internet search. Different actors working in the field of sustainable development and responsibility gather information about study programmes concerning sustainability issues. For this report, the portals of The Forum on Science and Innovation for Sustainable Development, Association for the Advancement of Sustainability in Higher Education (AASHE), University Leaders for a Sustainable Future (ULSF), The Higher Education for Sustainable Development (HESD) and Network for Business Sustainability (NBS) were used. The amount of information included in these portals varies. The AASHE’s Academic Program database for example contained at the time of the retrieval 1436 sustainability-focused academic programs at 475 campuses in 65 states and provinces but the emphasis was in the North America. Some portals are kept updated while the information of some others seems to be collected for some specific purpose and they are not necessarily updated afterwards.
Besides using the ready-made portals and lists of study programmes, information retrieval was done in websites in order to explore the phenomenon at a more general level. The following words in different combinations were used as search words: Education for Sustainable Development; Sustainability; Sustainable Development; Responsible Business; Corporate Responsibility and Corporate Social Responsibility /+ research /+ education.
Several scientific journals publish articles concerning sustainable development and responsible business. Some journals concentrate on the pedagogic side and Education for Sustainable Development (ESD). Journals have been used sporadically. Articles especially from following journals have given insight to the benchmark process: International Journal of Sustainability in Higher Education, The Journal of Sustainability Education, Journal of Cleaner Production, Sustainability Science, and Journal of Business Ethics. Several reports describing pedagogic features and outcomes related to courses in Sustainable Development have been explored.
Material available was vast and time resources were limited. The benchmarking process was carried out using qualitative hermeneutic analysis: exploring the web sites and reading articles by turns and analyzing characteristics that were revealed in dialogue with these two material sources.
Numerous international study programmes in different universities were explored. Sometimes it was easy to find exact course programmes while in other cases the information available was more general by character. All the material that has been gone through has had an effect on the understanding about the phenomenon and the chapter describing international perspective includes even sporadic remarks. However, to be able to describe some modules in more detail, a selection of cases has been made and the tables of those cases are available in the appendixes. As the case descriptions rely on the information that was easily achieved in the Internet the tables occasionally lack some details. Nordic countries create a natural point of interest as neighboring states and they are also acknowledged to have a good reputation in both education and research in the field of sustainable development.\(^1\) In addition there are some cases from the Netherlands, Germany and the United Kingdom and one consortium case. The cases are chosen as the examples of typical study programmes or they have some interesting feature be it a studying method or research institute etc.
International material used in this report deals mainly with international Master’s programmes where teaching is given in English. This has been a deliberate choice keeping in mind that present-day students have a variety of options where to pursue their Master’s degree after Bachelor studies. Exploring the Master’s programmes gives an overview how universities value the education related to sustainable development and responsible business/CSR in their international study programme supply. However, some cases of minor studies on Bachelor level or a separate course have been added to the report because of their specific pedagogic aims and solutions.
At the national level, the aim was to go through minor studies and Master’s programmes that concentrate on sustainable development or responsible business/corporate social responsibility at the university level. However, it is quite likely that not all the study modules were registered through
\(^1\) E.g. Matten and Moon 2004.
the search and as universities develop their study supplies constantly, new courses might have emerged or courses mentioned here may have been ceased after this report has been finished. Study modules in the universities of applied sciences were not taken into consideration in this report. The description of national cases is in chapter 3 and the tables of national minor and master study modules are available in the appendix.
The benchmarking report to internal education development use within sustainable development studies was ready at the beginning of January 2015. A slight check of information was done and some remarks and corrections were made to the original text at the end of 2015 before the report was published in FFRC series.
Higher Education units are worldwide facing tough competition for the economic resources and best students. The working life relevance of education, cooperation with business life and academic entrepreneurship, quality of education and internationalization are issues that are raised on the agenda in order to get along the competition and in some universities sustainability is included among the agenda.\(^2\) However, Ryan and Tilbury note that it has to compete with other priorities in the field of universities development strategies.\(^3\)
Enhancing sustainability skills among students has been a core issue in the United Nation’s Decade on Education for Sustainable Development (DESD) since its launch in 2005. In the United Kingdom the National Union of Students and Higher Education Academy have commissioned research on how first and second year students take the sustainability skills. Surveys made in 2011 and 2012 show that over two-thirds of both groups viewed universities as key players in the delivery of sustainability skills. Students also thought that future employers value sustainability skills.\(^4\) Jones Christensen et al. in their turn discovered in 2006 that the MBA students in the business schools with top rankings showed growing interest in ethics, corporate social responsibility and sustainability topics.\(^5\)
A large-scale project “Rio+20 implementation in the Nordic HEIs: inventorying and exploring new steering measures to integrate sustainable development and ESD into all operations” was granted two years funding by the Nordic Council of Ministers in 2014. The project includes also a benchmarking process where educational aspects will be measured on the number of available sustainability
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\(^2\) E.g. Anglia Ruskin University was named UK Entrepreneurial University of the Year at the Times Higher Education Awards in November 2014 and its sustainability research in Global Sustainability Institute (GSI) got excellent results in Research Excellence Framework (REF) in 2014. [http://www.anglia.ac.uk/entrepreneurial-university](http://www.anglia.ac.uk/entrepreneurial-university)
\(^3\) Ryan & Tilbury 2013, 278.
\(^4\) Ryan and Tilbury 2013, 278
\(^5\) Jones Christensen et al. 2007, 359.
courses and diplomas, combined with an evaluation of the teachers’ qualifications and skills in Nordic universities. The results of this benchmarking process will enrich the overview that is given in this report.\textsuperscript{6}
The definition of sustainable development used in this report is based on the definition presented in WCED-report in 1987 according to which sustainable development is development which meets the needs of the present without compromising the ability of future generations to meet their own needs.\textsuperscript{7} Sustainable development includes ecological, economic, cultural and social aspects and the focus in this report has been on sustainable development study programmes that have somehow multi- or interdisciplinary character.
Matten and Moon’s research on the CSR education uses CSR as an umbrella term that encompasses ethics, CSR and sustainability.\textsuperscript{8} In this benchmarking report, distinction has not been made between them either. Basic definitions used are: Corporate Social Responsibility is the voluntary actions taken by a company to address the economic, social, and environmental impacts of its business operations and the concerns of its principal stakeholders. Responsible or Sustainable Business is business that contributes to an equitable and ecologically sustainable economy. It offers products and services that fulfill society’s needs while contributing to the well-being of the inhabitants of the earth.\textsuperscript{9}
The results of the benchmarking process are described in chapters 2 and 3. According to the given task, results are grasping the question of multi- and interdisciplinarity, learning methods and the connection with research and practical orientation. Chapter 4 recapitulates the findings and basic elements of case examples are gathered together in appendixes.
\textsuperscript{6} Finnsson 2014; NSCN 2014.
\textsuperscript{7} World Commission on Environment and Development 1987, 41. About notions of sustainable development and sustainability see e.g. Coleman 2011; Kagan 2011, in the connection of business studies e.g. Brammer et al. 2012.
\textsuperscript{8} Matten and Moon 2004.
\textsuperscript{9} Jones Christensen et al. 2007, 351; Nicholls et al. 2013, 130; Crane et al. 2013.
2. STUDY MODULES AND PROGRAMMES AT THE INTERNATIONAL LEVEL
Common Remarks on Study Programmes
Several concepts are used when discussing different aspects of integration of sciences. In multidisciplinary research different disciplines each from their own premises and methods gather to treat a research problem in common. The integrative element is the acceptance of the complex nature of the research problem and the recognition of separate discipline’s insufficiency to solve the task alone. Interdisciplinarity adds in the former deeper dialogue between disciplines and strives to seek solutions together. Transdisciplinarity represents the strongest phase of integration of sciences and aims to achieve even conceptual and methodological cohesion.\(^{10}\) Interdisciplinarity and transdisciplinarity are discussed at a broad level in the scientific arenas at the moment.\(^{11}\)
The understanding that the sustainable development includes ecological, economic, social and cultural perspectives, makes scientific cooperation and dialogue essential part of research and education of sustainable development and responsible business/CSR. Also the global nature of the so called wicked problems and challenges related to sustainable development worldwide calls for research that goes cross the disciplinary boundaries. In many occasions transdisciplinary research nowadays means combining interdisciplinarity and participatory approaches and considering even non-scientific knowledge in the research processes.\(^{12}\)
Aspects of interdisciplinarity and transdisciplinarity are raised often when the subject of sustainability is discussed. This can be detected in the descriptions of study programmes addressing sustainable development or corporate social responsibility. At the same time, though, the importance of the essential disciplinary content and clear formulation of the transfer points to neighboring scientific fields is maintained. New combinations and cross-disciplinary endeavors do not lessen the importance of the content and skills of separate disciplines. This is expressed e.g. in the strategies of Leuphana University. The overarching aim in Leuphana’s Sustainability Science is to provide theoretical, methodological, organizational, and communicative skills needed to develop, realize, and reflect
\(^{10}\) E.g. Klein 1990 and 2010; Cantell et al. 2009; Godemann 2006.
\(^{11}\) E.g. Aalto et al. 2012; Barry and Born 2013; Frodeman et al. 2010; Huutoniemi & Tapio 2014; Myllyntaus et al. 2011.
\(^{12}\) United Nations 2015, 26; Stock & Burton 2011.
on one’s own disciplinary and interdisciplinary research work and projects. This holistic way of thinking in sustainability research requires a close connection between sustainability studies on the humanities and the natural sciences through a transdisciplinary approach in research and teaching.\textsuperscript{13} Prerequisites for Master in Sustainability Science in Leuphana University include a distinguished bachelor’s degree and basic knowledge on at least one of the fields of sustainability sciences which include subjects both in natural and human sciences.\textsuperscript{14}
Departments of science or environment seem to be typical bases for the Master’s programmes that concentrate on sustainability issues. Social science’s perspective forms one part of most course structures but there were far less examples of social sciences as a base department. However, many programmes are practically carried out in collaboration with several disciplines even though administration is held in one department. There are e.g. several examples where the study programme is organized together with Faculty of Science or Environment and Business School, e.g. the University of Plymouth, the University of Leeds and Royal Holloway, the University of London.\textsuperscript{15} Sustainable development study programmes administered in humanities are scarce. In the University of Oslo there is Master’s Programme Culture, Environment and Sustainability producing the academic degree of Master of Philosophy.\textsuperscript{16} Case Master’s programmes are introduced in the table 1.
\textsuperscript{13} \url{http://www.leuphana.de/en/graduate-school/master/course-offerings/sustainability-science.html}
\textsuperscript{14} These subjects include in Leuphana: Biological chemistry, Biology, Chemistry, Ecology, Physics, Geology, Geoecology, Environmental Communication, Environmental Planning, Environmental Politics, Environmental Management, Environmental Economics, Environmental Law, New Media (Media Sciences), Environmental Informatics, Participation and Sustainability and Sustainability Ethics.
\textsuperscript{15} University of Plymouth: Faculty of Science and Environment and Plymouth Business School, University of Leeds: Faculty of Environment and Leeds University Business School, and Royal Holloway, University of London: Department of Geography and School of Management.
\textsuperscript{16} \url{http://www.uio.no/english/studies/programmes/ces-master/}
| University | Master’s Programme | Students |
|---------------------------------------------------------------------------|------------------------------------------------------------------------------------|----------|
| Blekinge Institute of Technology BTH, Department of Strategic Sustainable Development | Master’s in Strategic Leadership towards Sustainability (MSLS), 60 ECTS, 10 months | ? |
| Erasmus University in Rotterdam, Department of Business-Society Management | MSc in Global Business & Stakeholder Management (GBSM), 60 cr, one year | 38 |
| Leuphana University Lüneburg, Faculty of Sustainability Science | Master’s Program Sustainability Science (MSc), 120 ECTS, 4 semesters | 38 |
| Lund University, Faculty of Social Sciences, Lund University Centre for Sustainability Studies, LUCSUS | Master’s Programme in Environmental Studies and Sustainability Science (LUMES) (MSc), 120 ECTS two years | 40 |
| Nottingham University Business School, International Centre for Corporate Social Responsibility (ICCSR) | MSc in CSR, 12 months full-time, 180 credits | max 20 |
| Royal Holloway, University of London, jointly by the School of Management and the Department of Geography | MSc Sustainability and Management, one year full-time, 180 credits | ? |
| Stockholm University, Department of Biology Education, Stockholm Resilience Centre | Master’s Programme Social-Ecological Resilience for Sustainable Development (SERSD), 120 ECTS, two years | 15 |
| Uppsala University, Department of Earth Sciences in collaboration with the Swedish University of Agricultural Sciences (SLU) | Master’s Programme in Sustainable Development (MSc), 120 ECTS, two years (alternative 60 ECTS one year) | 60 |
| University of Exeter, College of Life and Environmental Sciences | MSc Sustainable Development, 180 credits\(^{17}\), 12 months | ? |
| Universities of Graz, Leipzig, Utrecht, Basel, Hiroshima and Ca’ Foscari University of Venice | International Joint Master’s Programme in Sustainable Development (MSc), two years, 120 ECTS | students’ number varies annually |
| University of Oslo, Centre for Development and the Environment (SUM) | Master’s Programme in Development, Environment and Cultural Change (DEC), (MA in Philosophy), 120 ECTS, two years | 20 |
**Table 1.** Selection of Master’s Programmes related to Sustainable Development and/or Responsible Business.
---
\(^{17}\) When converted, 180 credits is equivalent to 90 ECTS and that is completed in 12 months in Exeter and other corresponding cases in the UK. [http://admin.exeter.ac.uk/academic/tls/tqa/Part%202013/13CQF.pdf](http://admin.exeter.ac.uk/academic/tls/tqa/Part%202013/13CQF.pdf)
Master’s programmes are typically two years’ full time programmes demanding 120 ECTS for the degree. Another quite typical option is one year full time programme with 60 ECTS particularly in the Business schools. Especially in the United Kingdom many universities and business schools also offer part time programmes and options with blended learning.
Master of Science (MSc) seems to be the common degree granted and much used also in the context of business schools. In some cases the name of the degree has been deliberately changed from MA to MSc and that is mentioned in the introduction of the programme, e.g. International Centre for Corporate Social Responsibility (ICCSR) that arranges responsibility studies in Nottingham University Business School since 2002, informs that their MA in CSR was reclassified to MSc in CSR in 2010.\footnote{http://www.nottingham.ac.uk/business/ICCSR/teaching.php} On the other hand, the degree can vary inside the same programme depending on the specialization track or orientation of former Bachelor studies towards natural or social sciences or humanities. Names of the Master’s programmes related to sustainable development and responsible business are many as can be seen in the Table number 1.
After finishing this benchmark report in January, the Rotterdam School of Management has decided to change the name of its MSc in Global Business & Stakeholder Management to MSc in Global Business & Sustainability. The name change will take place in the next year and all students graduating after 1 February 2016 will receive the new degree title.\footnote{http://www.rsm.nl/master/msc-programmes/msc-global-business-sustainability/overview/}
Master’s programmes in general make a distinction between natural and social sciences and humanities in their study modules in some way. In some programmes, like in Leuphana MSc Sustainability Science the distinction is clear and students choose either the in-depth perspectives of natural or human sciences while in some other cases the distinction is not that strict. Issues of sustainable development and CSR stimulate collaboration between universities and research institutes in different countries. This becomes evident both in the field of research and education when surfing through the websites of universities.
One example is the Joint International Master in Sustainable Development which was established in 2008. The Institute of Systems Science, Innovation & Sustainability Research of the University of Graz is the co-ordinating university and other degree awarding consortium members are Ca’Foscari University of Venice, Leipzig University and Utrecht University. Basel University and Hiroshima University function as associated mobility partners. University of Stellenbosch and TERI University in New Delhi joined the consortium as mobility partners in 2013. Emphasis of this two-year full time
programme (120 ECTS) is on interdisciplinary perspective. The focus is set on a comprehensive approach to the question of sustainable development and the needs and possibilities of societal transformation. The strengths and specializations in teaching and research of six prestigious partner universities are combined to offer a high profile programme to be concluded with a Master of Science joint or multiple degree. Curriculum includes 30 ECTS studies at one of the partner universities and at least 60 ECTS at home university.\textsuperscript{20}
Issues related to sustainable development also call for new perspectives and generate new interdisciplinary combinations. One example of the new fields of research that crosses the scientific boundaries and has sustainability issues in its core might be Industrial Ecology. This interdisciplinary field has been developing quickly since late 1980’s. It combines natural, technical and social sciences in a systems view at the scale levels from the global to the local. The new field has inspired even new higher education studies.\textsuperscript{21} Leiden University for instance offers Master’s programme in Industrial Ecology together with Delft University of Technology. Industrial Ecology is described in the web pages of Leiden University “as an emergent scientific discipline that promotes a systemic approach to human problems, integrating technical, environmental and social aspects. It is argued that this approach will show the way to sustainable development. For that reason Industrial Ecology is considered the toolbox for sustainable development or the science of sustainability.”\textsuperscript{22}
Also a new Erasmus Mundus Master’s programme called MIND has evolved round Industrial Ecology. This two-year programme (120 ECTS) for up to 25 students is the first international master programme in Industrial Ecology worldwide. Consortium includes seven universities.\textsuperscript{23} The MIND programme trains the students to conduct industrial ecology analyses of complex sustainability problems, to design industrial ecology solutions for these problems and to develop implementation strategies for those solutions identified.\textsuperscript{24}
\begin{footnotesize}
\begin{itemize}
\item[\textsuperscript{20}] \url{http://www.jointdegree.eu/sd/}
\item[\textsuperscript{21}] International Society for Industrial Ecology (ISIE) made a list of some course offerings related to industrial ecology and it was updated in 2009: \url{http://www.is4ie.org/Resources/Documents/IE%20Curriculums.pdf}
\item[\textsuperscript{22}] \url{http://www.cml.leiden.edu/education/master/industrial/about-master-ie.html}
\item[\textsuperscript{23}] University of Graz (co-ordinator), Chalmers University of Technology, Delft University of Technology, Leiden University, Asian Institute of Technology, Rochester Institute of Technology and Waseda University.
\item[\textsuperscript{24}] \url{http://www.emmind.eu/}
\end{itemize}
\end{footnotesize}
Learning Methods
Master’s Programmes describe their learning methods in the web pages at a general level and they are quite similar. Throughout the study programme descriptions concerning the sustainability it is emphasized that in order to understand and deal with the so-called wicked problems of the present-day world it is indispensable to get used to interdisciplinary dialogue and working in teams. The following learning methods are typically listed: lectures, group work, case studies, simulation games, individual assignments, presentations, workshops, seminars and field trips. Additionally an open and interactive environment, peer learning and a collegial coaching relationship between instructors and learners are mentioned in one way or another. Group works and case studies are in the focus when interdisciplinarity is emphasized.
In the following three examples of study courses are given and thereafter the attention is directed to distance learning and MOOCs, and student-led initiatives.
Three Case Examples of Study Modules
Case courses presented here are chosen in order to give some examples of different ways and practices to teach sustainable development issues in the university level. Case examples are all interdisciplinary by character and they each have their own profile. Related to all cases, sustainable development studies are seen important in the university level and interdisciplinarity is encouraged. Furthermore, two of these courses are offered at the Bachelor level. They have been chosen keeping in mind the learning method of the course KEK01 in the University of Turku. The KEK01 course is open to both undergraduate and Master’s students and therefore the examples of team work courses also in the Bachelor level are seen as an interesting point of comparison.
Leuphana University Lüneburg, Germany
| Organizational Context | Study Module | Special Course | Form of Study |
|------------------------|--------------|----------------|---------------|
| Leuphana University Lüneburg, 4 Faculties: Education, Business, Culture and Sustainability | All Bachelor programme students start with the so called Leuphana Semester (30 cr) | Leuphana Semester includes an interdisciplinary course Science bears responsibility (10 cr) | All students get an introduction to research, sustainability issues and interdisciplinarity through project work and Conference Week |
In the Leuphana University Lüneburg, all students participate so called Leuphana Semester as they start their Bachelor programme studies. Leuphana Semester includes 30 credit points of which a module called ‘Science bears responsibility’ gives 10 credit points. During the Leuphana Semester students
come together in interdisciplinary learning communities and develop inter- and transdisciplinary discursive competence, which is firmly rooted in disciplinary competence. Later on after Leuphana Semester students can also take part in the Complementary Studies programme, which offers interdisciplinary seminars.\textsuperscript{25}
‘Science bears responsibility’ module consists of four elements: lecture series, tutorials, project seminar and Conference Week. During the lecture series and accompanying tutorials students are introduced to the complexity of sustainable development and provided with a preliminary set of tools so that they can orientate themselves in the subsequent transdisciplinary debates about sustainability. In roughly 60 project seminars, each with 25 participants, students start to gain an in-depth look at a single topic in sustainable development. Students carry out small-scale projects where they test their own hypotheses, which are then presented to the general public at the end of the semester in the Conference Week. There will be guests from politics, science and civil society and Conference Week is an opportunity to enter into discussions with guests about the opportunities and limits of shaping the future.\textsuperscript{26}
Whatever academic specialization students later choose, the module ‘Science bears sustainability’ offers students the opportunity in their first semester to explore an interdisciplinary topic. This happens by using the normative concept of sustainable development to investigate fundamental issues related to the responsibility of science in society. Students analyse research questions in interdisciplinary project seminars and present their results during the Conference Week. The module gives students both an interdisciplinary introduction to science, and formats for confronting issues in sustainable development.\textsuperscript{27}
The analogy to the academic research process and the project character of the seminars are the two core didactic elements of the module ‘Science bears responsibility’. The lecture series and tutorials represent the traditional form of knowledge acquisition while the project seminars are closer to the actual way knowledge is generated. The Conference Week in its turn resembles the communicative phase of the research process. The project-oriented profile is achieved through the high degree of independent group work embedded in a real problem context associated with the challenges of sustainable development. Project work includes the planning of the research project from identifying a specific research question to the research design, to creating a work schedule and the implementation of the project together with presentation of the results and critical reflection on the research
\textsuperscript{25} Michelsen 2013; see also: \url{http://www.leuphana.de/en/college/first-semester.html}
\textsuperscript{26} Michelsen 2013, 1508-9; \url{http://www.leuphana.de/college/studienstart/konferenzwoche.html}
\textsuperscript{27} Michelsen 2013, 1507.
results. During the Conference Week, students learn both about a common format of critical reflection in science and the production of knowledge and its influence on society. It also allows an assessment of student work in quasi-realistic contexts. According to Michelsen, the role of the teacher in this study module is more like a moderator of independent learning processes than a traditional teacher.\textsuperscript{28}
\textbf{University of Gothenburg, Sweden}
| Organizational Context | Study Module | Target Group | Form of Study |
|------------------------|--------------|--------------|---------------|
| University of Gothenburg, Faculty of science, cooperation with Chalmers and Centre for the Development and the Environment (GMV) | Separate course in Masters level: Project Management for Sustainable Development, 15 cr | Offered to students both in University of Gothenburg and Chalmers University of Technology since 2002. | An interdisciplinary course with large project task done in groups, includes field work in nearby region. |
A course called Project Management for Sustainable Development (Fallstudiekurs i hållbar utveckling in Swedish) is arranged in the University of Gothenburg in the faculty of science. This 15 credits course covers ten weeks of full-time studies and includes a large project assignment supported by seminars and lectures. Previous to the course, a term of reference for the project is formulated by the teachers and stakeholders in a specific local context in the Gothenburg region. The course proceeds through seven phases that are: 1. Planning, 2. Mapping, 3. Modeling, 4. Scenario identification and formulation, 5. Indicator identification and formulation, 6. Evaluation and 7. Report and the presentation phase. The aim of the course is to give theoretical and empirical knowledge about complex social and environmental problems from interdisciplinary perspectives. It also gives methodological training and learns students to plan, carry out and report work done individually and in a group. Both qualitative and quantitative methods are used.
The course is interdisciplinary by character and it is offered to students both in the University of Gothenburg and Chalmers University of Technology. The first course was arranged in 2002 and an evaluation report was made of the course in 2010.\textsuperscript{29} Until recently the course was organized together with the Centre for Environment and Sustainability (GMV Göteborgs Miljövetenskapliga Centrum)
\textsuperscript{28} Michelsen 2013, 1509–1510.
\textsuperscript{29} Undén 2010; Undén and Forshufvud 2010.
which is a joint centre of the University of Gothenburg and Chalmers. Some of the older reports produced in the course are found in the web page of the Centre for Environment and Sustainability.\textsuperscript{30} The course plan is available in the web pages.\textsuperscript{31}
\textbf{Leiden University, the Netherlands}
| Organizational Context | Study Module | Target Group | Form of Study |
|------------------------|--------------|--------------|---------------|
| Leiden University,Faculty of Science, the Institute of Environmental Sciences (CML) | Minor in Sustainable Development (30 EC) in Bachelor programme | Half year full-time intensive programme with three courses, designed for all degree programmes | An interdisciplinary character that is carried out by working in groups |
Faculty of Science in Leiden University in the Netherlands organises Minor in Sustainable Development. Minor is available to apply for all third year Bachelor students in different programmes and yearly intake is 50 student maximum.\textsuperscript{32} Half year full-time Minor is shortly presented here as an example of minor studies that has strong interdisciplinary character and combines academic and practical skills. The teaching is offered by the Institute of Environmental Sciences (CML) and the key educational feature of the institute is to bring together students from different disciplinary backgrounds and e.g. during this study year 19 disciplines are represented.
The minor consists of three courses that are closely coordinated. Courses are not a combination of existing courses but they are planned to form a coherent whole. Minor starts with a course ‘Sustainable Development: Big Issues New Answers’ (15 ETCS) that takes 11 weeks.\textsuperscript{33} The course includes teaching three days a week between 9.30–17 hours. Studying methods include lectures, debates, seminars, writing and presenting short papers and a longer paper. Two days a week are for self-study reading and writing. Professor Arnold Tukker, the Director of the CML, is among teachers.
Second course is ‘Project group: Design of European Research’ (10 ETCS). Students work in groups and they have as a task to plan, write and defend a full proposal for a research study on a sustainability theme in the context of the 7\textsuperscript{th} Framework Programme of the European Union
\textsuperscript{30} \url{http://gmv.gu.se/samverkan/samverkan-inom-utbildning/fallstudiekurs-i-hallbar-utveckling}. One example: \url{http://gmv.gu.se/digitalAssets/1447/1447740_rapport_fallst_2012_hisingen_2050_12-05-26.pdf}
\textsuperscript{31} \url{http://utbildning.gu.se/kurser/hitta-kursplan/kursplan/?courseId=ES2415}
\textsuperscript{32} \url{http://www.cml.leiden.edu/education/minor/minor_overview.html}
\textsuperscript{33} \url{http://media.leidenuniv.nl/legacy/schedule-course-big-issues-new-answers-cml.pdf}. For study year 2015-2016 see: \url{https://studiegids.leidenuniv.nl/en/courses/show/46935/Duurzame_ontwikkeling_tp}
During this process, students apply their knowledge and skills gained in the previous course (Big Issues New Answers). Many of the topics in EU/FP7 relate to the interdisciplinary problems concerning sustainability. Students imitate the submission procedure by finding partners from other countries, presenting an extensive state-of-the-art report and submitting a detailed schedule. They will even write a rebuttal to the evaluation of their proposal done by the CML tutors and fellow students. The course instructs students to use the vocabulary of project management and to work in an interdisciplinary team to address a complex sustainability problem. The course gives a valuable learning experience of making an international research grant application.
Third course is an area study and there are two options which to choose, either a) Waste not Want not: A Future without Waste (7 ECTS)\textsuperscript{34} or b) International course on water issues and water management in the Philippines (10 ECTS). The latter one includes field work in Philippines in January 2015. Since 2011 the international course on water and water management has been organized in the Philippines. 28 students participate in this one month course: 14 European students through Leiden University and 14 Philippine students through Isabela State University. Intercultural teams conduct a short field study on a theme related to water management in Isabela Province. The aim of the course is to learn about water issues and to gather practical experience with fieldwork and working in interdisciplinary, international teams. The diverse background of the students includes anthropology, biology, forestry, agriculture and civil engineering among others.\textsuperscript{36}
\textbf{Distance Online Learning}
The growth of distance learning courses can be seen as part of more general phenomenon where higher education is developing more fluid boundaries with many areas of further education, professional practice and community learning.\textsuperscript{37} Many universities and business schools offer distance learning possibilities as well in the realm of sustainability and responsible business. Distance learning is seen as one competitive feature in the supply of teaching and it is used also in the marketing. The University of Edinburgh for example, advertises its distance learning possibilities as follows: “Our online learning courses also offer flexible exit routes, allowing you to shape your academic journey to
\begin{itemize}
\item[34] For the study year 2015–2016 the context has been updated and it concerns the Horizon 2020 Program. \url{https://studiegids.leidenuniv.nl/courses/show/46937/Projectgroep_Ontwerp_van_Europees_Onderzoek}
\item[35] At the study year 2015–2016 there will be an area study ‘2030: sustainable Leiden?’: \url{https://studiegids.leidenuniv.nl/courses/show/46939/Area_study_sustainability}
\item[36] Watercourse Philippines – Leiden University. \url{https://www.facebook.com/pages/Watercourse-Philippines/312676025504087}; for year 2016 see \url{https://studiegids.leidenuniv.nl/en/courses/show/45903/Fieldschool_Water_Management_in_the_Philippines_Winter}
\item[37] e.g. Ryan & Tilbury 2013, 273–274.
\end{itemize}
suit your needs.\textsuperscript{38} There is a quickly growing trend to develop study modules providing more flexible study opportunities instead of strict programmes throughout the universities. This is linked with the changing values and demands of education among young people and the increasing demand to combine studies and working life.\textsuperscript{39}
There are plenty of opportunities to finish separate courses or whole degrees through either complete distance learning or by combining workshop days at the campus with online learning in different universities as the following two examples illustrate:
- Bournemouth University, the School of Applied Studies offers possibility to conduct the whole MSc Green Economy through distance learning. The annual intake of students varies from 10–15. The duration is one year full time and two years part time. It is also possible to conduct the degree in parts which helps with the payments.\textsuperscript{40}
- The Distance and Independent Studies Center (DISC) at TU Kaiserslautern advertises itself as “one of the leading post-grad distance learning programmes in Germany”. It offers ‘Sustainable Development Cooperation’ distance learning Master’s degree held in German.\textsuperscript{41}
\textit{Massive Open Online Courses (MOOCs)}
Massive Open Online Course, MOOC, started as an innovative experiment to strengthen the co-learning aspect and since the first experimental course in Canada in 2008 the development has got many directions.\textsuperscript{42} MOOC is a study course that is offered online to anyone interested worldwide and participation has been free of charge so far. Courses include in average 5–12 weekly online sessions. In addition to online lectures, students participate in online discussions and perform individual assignments that demand varying number of independent study hours per week. Attending a MOOC requires an enrollment and study materials are available only during the course.
According to some studies, the profitability of MOOCs is not an easy task as they require a lot of planning work before the course and human work resources during the course to sustain web discussions and feedback.\textsuperscript{43} Also the quite low completion rate of MOOCs has raised discussion among
\textsuperscript{38} \url{http://www.ed.ac.uk/studying/postgraduate/degree-guide/online-learning/programmes}
\textsuperscript{39} Bisoux 2014. See e.g. University of Exeter, Distance Learning and Modular Programmes. \url{http://www.exeter.ac.uk/postgraduate/degrees/distance/}
\textsuperscript{40} \url{http://courses.bournemouth.ac.uk/courses/postgraduate-degree/green-economy/none/3126/}
\textsuperscript{41} \url{http://www.zfuw.uni-kl.de/en/distance-learning-courses/management-law/sustainable-development-cooperation/}
\textsuperscript{42} Cormier & Siemens 2010; Hollands & Tirthali 2014.
\textsuperscript{43} Hollands & Tirthali 2014; Ruth 2012.
educators. One obvious reason is related with the custom: as registering for a MOOC is often a precondition to seeing its content and so evaluating its worthiness, students might shop multiple courses before settling on which to commit to.\textsuperscript{44}
MOOCs are one possibility among different virtual teaching methods and not an end in itself. The strategy that the University of Bath has expressed related to MOOCs puts the phenomenon quite well into words as it says:
“\textit{MOOCs have been available for some years but are still a highly experimental area of study. The University of Bath is committed to providing a wide range of opportunities for independent learning, which is why we are excited by the potential of MOOCs. The experience of running our three current courses will influence the future MOOCs we offer. As well as providing courses, we are keen to play a leading role in shaping future developments in MOOCs and other approaches to online learning. Our current courses are available on FutureLearn.com. The University of Bath is a member of the FutureLearn partnership, which includes some of the best UK and international universities, as well as institutions with a huge archive of cultural and educational material.}\textsuperscript{45}
As this quote describes, MOOCs are part of a larger phenomenon, that is to say distance online learning and many universities are at the phase of experimenting MOOCs and their value by the cost. According to an extensive literature review by Haggard (2013) MOOCs might act as a tipping point for Higher Education even though their meaning may have been over hyped so far.\textsuperscript{46}
MOOCs offer a possibility to view lectures given by well-known experts in the field but they do not necessarily train students to the challenges of multidisciplinary dialogue. The number of participants in a MOOC may run to thousands and that challenges the way how web-based interaction succeeds in assignments related to lectures. Several universities worldwide offer MOOCs related to sustainable development issues as well, e.g. Copenhagen Business School launched its first MOOC on Social Entrepreneurship in September 2014 and more than 22.000 people signed up. The course was re-run already in April 2015.\textsuperscript{47} From the case universities, e.g. Stockholm Resilience Centre offered fall
\textsuperscript{44} Reich 2014.
\textsuperscript{45} \url{http://www.bath.ac.uk/study/moocs/}
\textsuperscript{46} Haggard 2013.
\textsuperscript{47} \url{https://www.coursera.org/course/socialentrepreneur}
2014 MOOC Planetary Boundaries and Human Opportunities: The Quest for Safe and Just Development on a Resilient Planet together with the Sustainable Development Solutions Network (SDSN) and it was re-run in September 2015.\textsuperscript{48}
After completing a MOOC course, the student may get a verified certificate or Statement of Participation that may cost or be free of charge depending on the platform where the course is organized. At the moment, students may include earned MOOC certificates in their degree studies with varying degree but a trend towards granting credits might be evolving. Above mentioned Stockholm Resilience Centre for instance, mentions that while their course is not credit granting, they encourage students to work with their own institutions to explore the option of granting credit for online coursework.
Below in table 2. are some examples of MOOCs offered at the beginning of 2015.
| Selection of MOOCS at the beginning of 2015 | Make an Impact: Sustainability for Professionals | 12.1.–16.2. 2015, 3 hours work/week | https://www.future-learn.com/courses/sustainability-for-professionals, Statement of Participation to purchase |
|--------------------------------------------|--------------------------------------------------|-----------------------------------|----------------------------------------------------------------------------------------------------------------|
| The University of Bath | Greening the Economy: Lessons from Scandinavia | 19.1.–20.2. 2015, 5-8 hours work/week | https://www.coursera.org/course/greeningtheconomy verified certificate 49 dollars |
| The Lund University, IIIEE – International Institute for Industrial Environmental Economics | Critical thinking in global challenges | 19.1.–23.2. 2015, 1–2 hours work/week | Not a course on global challenges themselves; instead it uses the context of these thought-provoking challenges to practice critical thinking. https://www.coursera.org/course/critical-thinking verified certificate 49 dollars |
Table 2. Selection of MOOCs which started in January 2015.
\textsuperscript{48} \url{https://www.sdsn.edu.org/learn/planetary-boundaries-and-human-opportunities-fall-2014}; \url{https://www.sdsn.edu.org/learn/planetary-boundaries-and-human-opportunities-september-2015}
The MOOC organized by the International Institute for Industrial Environmental Economics (IIIE) in the Lund University got positive feedback and a new course with some new materials and format improvements started already in September 2015 and will be relaunched again in January and February 2016. The same organizers have even created a new course Greening the Economy: Sustainable Cities that starts in January 2016. Richter et al. presented in the Global Cleaner Production and Sustainable Consumption conference 2015 their practical experience with the curriculum design, production and delivery of the MOOC Greening the Economy: Lessons from Scandinavia and analysed how the combination of different learning methods succeeded in a course with about 20 000 participants all over the world.\textsuperscript{49}
\textbf{Student-led courses}
Students’ active role in teaching related to sustainable development issues creates quite an unique example in Uppsala in Sweden. Centre for Environment and Development Studies, CEMUS, was created in 1995 as a joint centre between Uppsala University and the Swedish University of Agricultural Sciences in Uppsala and it is a student initiated and student-run university centre. Today it is a part of the Uppsala Center for Sustainable Development, CSD Uppsala.\textsuperscript{50}
Student-led initiative has proved to be fruitful and CEMUS has given dozens of courses during over 20 years of activities. The teaching language has been English already many years since exchange students are eager to participate in the courses. It is said that CEMUS has affected the way how teaching and learning are carried out in Uppsala.\textsuperscript{51} Students are employed by CEMUS as course coordinators. They plan, run, and evaluate university courses at the undergraduate and graduate level. The syllabus and learning outcomes are developed and solidified through a collaborative process involving students and university staff. At the moment, e.g. the following courses include in the course offerings:\textsuperscript{52}
- Hållbar utveckling B (Sustainable development) 30 cr
- Hållbar utveckling (Sustainable development) 15 cr
- Livsfilosofi och det moderna samhället (Philosophy of live and the modern society) 7,5 cr
\textsuperscript{49} The information is gathered from the official page for Greening the Economy – A Massive Open Online Course (MOOC) at Lund University in Sweden \url{https://www.facebook.com/iiieemooc} and the course page in the Coursera \url{https://www.coursera.org/learn/greening-the-economy/}; Richter et al. 2015.
\textsuperscript{50} The Baltic University Programme (BUP), a university collaboration based in sustainable development is also a part of CSD Uppsala. Student driven education at CEMUS: \url{http://www.cemus.uu.se/dokument/sustainable_dev_uppsala.pdf}
\textsuperscript{51} Hald 2011.
\textsuperscript{52} CEMUS education see \url{http://www.csduppsala.uu.se/education/}
- Applied Sustainability Science 30 cr
- Actors and Strategies for Change – Towards Global Sustainabilities 7,5 cr
- Sustainable Design 7,5 cr
- Climate Change Leadership 15 cr
- Sustainable Development Project Management and Communication 15 cr
**Research Centres and Institutes**
According to the benchmarking process versatile study programmes and research centres often go hand in hand. Research feeds teaching and vice versa. Active research institutes give food for thought to students in many ways as they organize seminars and international conferences and publish their scientific results in journals. Research institutes invite visiting scholars and students become acquainted with research projects with international partners. Part of the research centres and institutes have an active role in education and offering study courses and part of them act more like a hub of information and contacts and concentrate on research. They may offer scientific support for students in the phase of writing the thesis or PhD dissertation. In Stockholm University for example, the courses of Master’s Programme Social-Ecological Resilience for Sustainable Development are firmly embedded in the research of Stockholm Resilience Centre. Theses are mainly incorporated in on-going research projects and are all related to one of the research themes at the Stockholm Resilience Centre. Graduates are encouraged to publish their research in the peer reviewed journals.\(^{53}\)
There are other forms of research networks as well which promote the visibility of expertise related to sustainable development issues. For instance sustainable environments and societies are one of the Plymouth University’s priority subject areas for research and university has compiled together into one web page its eight research centres addressing sustainability questions from different perspectives.\(^{54}\) Plymouth has much sustainability research expertise and another web page gathers together more centres and gives an overview of the variety of university staffs’ and students’ activities related to sustainability.\(^{55}\)
\(^{53}\) Stockholm Recilience Centre. [http://www.stockholmresilience.org/21/education.html](http://www.stockholmresilience.org/21/education.html)
\(^{54}\) Plymouth University. Research themes: sustainable environments and societies: [http://www1.plymouth.ac.uk/research/ourresearch/themes/Pages/environment.aspx](http://www1.plymouth.ac.uk/research/ourresearch/themes/Pages/environment.aspx)
\(^{55}\) Plymouth University. Research expertise: [http://www1.plymouth.ac.uk/research/issr/centres/Pages/default.aspx](http://www1.plymouth.ac.uk/research/issr/centres/Pages/default.aspx)
3. STUDY MODULES AND PROGRAMMES AT THE FINNISH LEVEL
In the following two chapters, some features of minor studies and Master’s programmes in Finnish universities are presented.
Minor Study Modules
According to this benchmarking process study modules related to sustainable development, responsible business/CSR and environment have been increasing recently. At least ten universities offer some minor studies in this field.\(^{56}\) Finnish universities and their study programme opportunities related to sustainable development and responsible business/CSR can be found in different portals and databases with varying success. Minor study modules gain less visibility but the case is the same with minors in the international context.
The information retrieval showed that instruction and research related to sustainable development can be scattered in different disciplines and research institutes and the fragmented pieces of information do not get as good visibility and coverage as separate minor and major studies or entities focused on sustainability. In the University of Oulu for example, there are individual courses in both masters and PhD-level related to sustainable development in several disciplines and in the spring 2015 Oulu Business School piloted a new course Globally Responsible Business.\(^{57}\) In December 2014 there was a doctoral defense related to Education for Sustainable Development (ESD) in the Faculty of Education.\(^{58}\) Additionally, The Martti Ahtisaari Institute’s research activities focus on the challenges of sustainable and responsible business, as well as functioning of the global markets and economy as a whole. The Institute is part of the Oulu Innovation Alliance and it offers an international doctoral programme related to the research focus areas.\(^{59}\) All this interesting information has to be picked up separately from the university’s different web pages.
\(^{56}\) Numbers of the units offering teaching must not be taken exact as gaps may easily have been left in the information retrieval. However, the numbers give an overview of the scale of the teaching related to sustainable development minor study modules in Finnish universities in 2015.
\(^{57}\) [http://www.oulu.fi/blogs/node/29750](http://www.oulu.fi/blogs/node/29750)
\(^{58}\) Villanen 2014.
\(^{59}\) [http://www.maigbe.com/martti_ahtisaari_institute](http://www.maigbe.com/martti_ahtisaari_institute)
Minors included to this report are presented in Table 3.
| University | Minor | Credits | To whom |
|-------------------------------------------------|----------------------------------------------------------------------|------------------|------------------------------------------------------------------------|
| Aalto University | The Creative Sustainability (CS) | 15–25 ECTS | master’s students in Aalto |
| Hanken School of Economics (Helsinki and Vaasa) | Corporate Responsibility (CR) | 25 cr | all students + 25 stud. in U. of Helsinki |
| Lappeenranta University of Technology | Sustainability | approx. 24 ECTS | all degree programme students |
| Swedish School of Social Science | Minor in Environment | 25 cr | all students, also in U. of Helsinki |
| UniPID network | Virtual Minor: Sustainability in Development | 25 cr | students at member universities |
| University of Eastern Finland | Multidisciplinary studies in Environment | 25 or 60cr | all students |
| University of Helsinki | Multidisciplinary minor in Environment (YMS) | 25 cr | all students |
| University of Jyväskylä | Environment and Society | 25–40 cr | all students |
| University of Lapland | Environmental Studies | 25–60 ECTS cr | all students |
| University of Tampere, School of Management + open university | Responsible Business | 15 ECTS | many student groups |
| University of Tampere + open university | The Sustainable Development Study Programme | 25–35 ECTS | all students |
| University of Turku, School of Economics | Responsible Business | 25 cr | all students |
| University of Turku | Sustainable Development Studies | 25 cr | all students |
| University of Vaasa | Minor in Environment | 26 cr | all students |
| Åbo Akademi University + open university | Environmental knowledge | 25–60 cr | all students |
Table 3. Minor studies related to sustainable development and environment or responsible business offered in Finnish universities 2015.
According to the titles of the minors three of them concentrate on responsible business perspective while the rest are named after sustainable development, sustainability or environment. Most minors include course options from different disciplines in order to give a versatile view of the issues related to sustainable development and environment. Distance learning opportunity is offered by UniPID-programme where it is possible to complete virtual minor in Sustainability in Development. In some cases (e.g. in the University of Turku and Åbo Akademi) students can include separate courses from UniPID virtual minor to their minor in the home university.
A distinctive pedagogic feature of some minors related to sustainable development, in the universities of Helsinki and Turku for instance, is that they include a joint introduction course with a group work. Project courses related to real-life cases are used in a few study modules in Responsible Business as well, e.g. in Hanken, the Aalto University and the University of Tampere. In the following, one example of such a course is given.
**University of Helsinki**
| Organizational Context | Study Module | Target Group | Form of Study |
|------------------------|--------------|--------------|---------------|
| Helsinki University Centre for Environment (HENVI), minor in Environment | Mandatory course: Introduction to multidisciplinary environmental studies, 5 cr | Offered to students in the University of Helsinki. | An interdisciplinary course with lectures and a group work. |
Helsinki University Centre for Environment (HENVI) organizes since 2006 Multidisciplinary minor in Environment (25 credits). It includes a mandatory course called Introduction to multidisciplinary environmental studies (5 credits) and elective courses which are chosen freely from a selection of study courses in different disciplines. However, students have to take courses equally from natural sciences and humanities and social sciences. The overall aim of the mandatory course is to introduce students to environmental studies and teaching personnel in different faculties. The Introduction course runs the fall semester. It consists of lectures, individual assignments and a group work that is done during the course. The groups are formed of students from different backgrounds so that students can practice interaction in a multidisciplinary environment and even get a touch of interdisciplinarity during the work. Students carry out multidisciplinary group works that deal with some phenomenon related to environment. A separate panel discussion addressed with one of the group themes is organized at the end of the course. The panel is open to a larger audience and it has some visiting experts. In the
course web page it is possible to see what kind of themes students have been addressing during recent years and read student evaluations of previous courses.\textsuperscript{60}
\textbf{Master’s Programmes}
Most master’s programmes included in this report concentrate on the connections of sustainable development and business. The Master’s Degree Programme in Environmental Policy and Law in the University of Eastern Finland has two courses related straight to sustainable development issues: Sustainability and Natural Resources (5 cr) and Corporate Social Responsibility in Natural Resources Management (5 cr) but otherwise it consists of the advanced study of law and policy in relation to the environment, climate change and natural resources. Master of Science in Technology programmes, except the joint programme in Creative Sustainability in the Aalto University, are not included in this report as the information retrieval concentrated more on programmes in other faculties than technology or engineering. It is acknowledged that studies on environmental engineering for instance often have sustainable development issues among their core learning targets and it would be useful to go through them as well.\textsuperscript{61}
\begin{footnotesize}
\textsuperscript{60} Cantell et al. 2009; \url{http://www.helsinki.fi/henvi/opetus/johdantokurssi.htm}
\textsuperscript{61} See e.g. Study Guide of Lappeenranta University of Technology \url{https://uni.lut.fi/en/web/lut.fi-eng/study-guides9} or the Tampere University of Technology, Department of Chemistry and Bioengineering. \url{http://www.tut.fi/en/about-tut/departments/chemistry-and-bioengineering/index.htm}
\end{footnotesize}
| University | Master’s Programme | Students |
|---------------------------------------------------------------------------|------------------------------------------------------------------------------------|---------------------------|
| Aalto University: Schools of Business, and Arts, Design and Architecture, and Engineering | Master’s Programme in Creative Sustainability (CS), 120 ECTS | varies, 2014 8 2015 16 |
| Lappeenranta University of Technology | Master’s Programme in Strategy, Innovation and Sustainability (MSIS), 120 ECTS | 20 * |
| | *+ graduate students of the School of Business who choose programme for their Master studies | |
| University of Jyväskylä, School of Business and Economics | Master’s Degree Programme in Corporate Environmental Management (CEM), 120 ECTS | 20–25 |
| University of Tampere, School of Management | Master’s Programme in Business Studies – Specialization Responsible Business, 120 ECTS | 10 |
| Hanken School of Economics | Master’s Programme in Business Management with the specialization track in International Strategy and Sustainability, 120 ECTS | 10–15 |
| Aalto University, Department of Management Studies | Master’s Programme in Management and International Business (M&IB), specialization in Sustainability Management, 120 ECTS | varies, 2014 29 2015 40 |
| University of Eastern Finland | Master’s Degree Programme in Environmental Policy and Law, 120 ECTS | 20 |
Table 4. Master’s programmes related to sustainable development or responsible business/CSR in Finnish universities in 2015.
Discussion about the meaning and role of MOOCs is under way also in Finnish Universities. Although the contents of MOOCs are reviewed often very general in character it is suggested that they might function as introductory courses to different fields of interest.\(^{62}\) In the Master’s Programme in Strategy, Innovation and Sustainability (MSIS) in the Lappeenranta University of Technology students may include MOOCs in the MSIS degree, but they must be agreed beforehand with the Academic Director by submitting an informal application letter. The maximum of 12 ECTS of MOOCs can be accepted in the degree. These courses can be located to replace elective courses in core studies (strategy, innovation or sustainability).\(^{63}\)
\(^{62}\) Törnänen 2014.
\(^{63}\) Lappeenranta University of Technology Study guide 2014-2015, page 259; https://uni.lut.fi/en/web/lut-fi-eng/study-guides9
Encouraging students to keep track of ongoing research can be put into practice by inviting them to participate in seminars and conferences, inviting guest speakers and visiting lecturers and by organizing special courses. One example is Cases in Organizational Ethics -course that was a joint course of BI Norwegian Business School, Estonian Business School, the Kaunas University of Technology, the Riga International School of Economics and Business Administration, the University of Jyväskylä, the University of Tampere, and the Vilnius University. The master students in business or economics could enroll to this course that was a practical result of a joint project financed by Nordplus Higher Education. The aim was to create and coordinate an intensive course for the Master students of business and economics that stimulate them to act in a morally responsible way and deepen their knowledge and skills how to create organizations which contribute to sustainable development. The project partners took problem-based learning and specifically case studies as the basic teaching method tackling environmental and ethical issues in the Nordic-Baltic context and applied it in the course that was kept in one of the partner universities in turn. Students from Master’s Programmes both in Tampere and Jyväskylä had the possibility to enroll this course. According to the evaluation of implemented courses students have valued the course especially because it included real-life cases and exercises. The course also offered a natural starting point for a dialogue in an international context.\(^{64}\)
The International Master’s Degree Programme in Corporate Environmental Management in the University of Jyväskylä is shortly described here as an example of the development of Master’s studies on corporate social responsibility in Finland.
**The School of Business and Economics of the University of Jyväskylä**
| **Organizational Context** | **Master Degree** | **History** | **Interdisciplinary character** |
|---------------------------|-------------------|------------|-------------------------------|
| The School of Business and Economics of the University of Jyväskylä, The Corporate Environmental Management (CEM) | International Master’s Degree Programme in Corporate Environmental Management | Has its origins at the beginning of 1990’s | Interdisciplinary is emphasized and some compulsory basic studies on environmental science are included |
The School of Business and Economics of the University of Jyväskylä was the first university in the Nordic region to offer a complete Master’s and doctoral programme in Corporate Environmental
\(^{64}\) Puč ‘e ‘taite ‘ 2012; See also Development of Moral Competence in Leadership and Management (DECOM) project: [https://www.jyu.fi/jsbe/en/research/groups/leadership/ethos/projects](https://www.jyu.fi/jsbe/en/research/groups/leadership/ethos/projects).
Management. International Master’s Degree Programme in Corporate Environmental Management has its origins already at the beginning of 1990’s when environmental issues started to emerge in both research and teaching at the School of Business and Economics. In 1995 a new master’s programme dedicated to environmental issues was launched. The programme got a new name Corporate Environmental Management in 2001. The focus of the orientation toward environmental sustainability issues in organizational and business context is stressed meanwhile integration with the other two dimensions of corporate social responsibility is also implemented into the curriculum.\(^{65}\)
The structure of the teaching stems from the early start in 1990’s and the curriculum stresses understanding at three levels: 1. identifying environmental impacts of organizations and products 2. managing environmental impacts, that is to improve the environmental performance of the company and 3. creating eco-competitiveness through excellence in the previous levels.\(^{66}\) Interdisciplinarity is considered important because environmental issues are interdisciplinary by character and they do not respect the traditional boundaries of disciplines. It is taken for essential that the students of environmental management have basic knowledge of physical environmental impacts and there is a vivid cooperation with the Department of Environmental Sciences. All students make some compulsory basic studies on environmental science and technology.
The Corporate Environmental Management (CEM) in the School of Business and Economics has as a central pedagogic principle to value continuing dialogue between theory and practice during the entire learning process and to encourage social interaction between the students and the teachers. Emphasis is also put on the active connection to the scientific research done within the discipline. Current research interest includes: material flow management, sustainable production and consumption, sustainability strategies and marketing, environmental values and cultures, and strategies and business concepts for sustainable energy solutions.\(^{67}\)
\(^{65}\) Pesonen 2003.
\(^{66}\) Pesonen 2003, 166.
\(^{67}\) Pesonen 2003, 168; https://www.jyu.fi/jsbe/en/cem
4. DISCUSSION
The purpose of this report was to achieve comprehension whether Sustainable Development Studies in the University of Turku has unique characteristics and what might be the most interesting lines of development in the future. Based on the benchmarking process some general observations were made. In many ways, these remarks are intertwined together even though they are here described separately.
1. Discussion about the contents of sustainable development, sustainability and responsibility
2. Importance of the visibility of the offerings of teaching and research
3. The role of interdisciplinarity, interaction and cooperation with stakeholders in teaching
4. The role of Education for Sustainable Development (ESD) and PRME in universities
Discussion of the Contents of Sustainable Development, Sustainability and Responsible Business
There are a growing number of study modules worldwide, both in the undergraduate and the postgraduate level as well in the PhD-level that concentrate on issues of sustainable development and responsible business. The meaning and contents of these concepts are discussed at every turn and different perspectives are emphasized.\(^{68}\)
The questions related to sustainable development require to be studied from different viewpoints and disciplines and therefore multidisciplinary and interdisciplinary research and education are seen important. Benchmarking also showed that issues related to sustainable development attract the new openings and new fields of research to emerge. Industrial ecology is one example that was described in the report. Different aspects of innovation are in many occasions combined with sustainability, for example the Aalto University, the Copenhagen Business School, the University of Graz and the Nottingham University Business School have education in this field.
The notion that sustainable development is development which meets the needs of the present without compromising the ability of future generations to meet their own needs includes the perspective of the future and many study modules and courses bring this forth in their study objectives at the general level. It would be another task to figure out how much actual futures research methods are
\(^{68}\) e.g. O’Byrne et al. 2015.
taught and used in the courses. The student led courses and initiatives occasionally integrate futures thinking and perspective in their course and event offerings.\textsuperscript{69}
Some universities that offer Master’s programmes on sustainable development have specific key concepts that they focus on throughout the studies. In the Stockholm University Master’s Programme concentrates on the notion of social-ecological resilience for example. In Blekinge Institute of Technology the students concentrate on the Framework for Strategic Sustainable Development (FSSD) that is a research based generic framework for planning in complex systems, focus either on research or more practical orientation.\textsuperscript{70} The research perspective of humanities is expressed e.g. in the research of the Centre for Development and the Environment (SUM), that organizes the Master’s Programme Development, Environment and Cultural Change (DEC) in Norway.\textsuperscript{71} It seems, however, that cultural sustainability is an area that would deserve more explicit attention in majors and minors. That might be a study area that could be highlighted more in the context of sustainable development studies in the University of Turku as the cultural dimension is one part of the study course KEKOI.\textsuperscript{72}
Godemann et al. note that some business schools have first integrated sustainability issues to their MBA programmes, and rankings such as the Aspen Institute Ranking which evaluates the integration of sustainability into the MBA programmes may have influenced on this trend. According to Godemann et al. this may lead to an increase in the integration of sustainability issues into BA/BSc, MA/MSc and PhD programmes in the near future.\textsuperscript{73} Rasche et al., however, point out that there is much to do also in the field of MBA programmes. As a result of research based on data from the Beyond Grey Pinstripes survey they conclude that even though elective courses on business ethics have increased more effort should be put on getting business ethics integrated into all disciplines.\textsuperscript{74}
There are discussions what are the differences between the notions of sustainable development, sustainability and sustainability science.\textsuperscript{75} According to O’Byrne et al., there should be even more
\textsuperscript{69} E.g. In teaching of CEMUS or in the activities of an international non-profit student-driven organization on sustainable economics and management, oikos, that launched in 2011 the oikos FutureLab as its new annual flagship event to convene student members from all chapters together with alumni, faculty, advisors, and partners and share perspectives on the future. See e.g. CEMUS Sustainable Development a Course: Why sustainability needs fiction. Imagination, Future Worlds, Design Fiction, and Diegetic Prototypes. \url{http://cemusstudent.se/wp-content/uploads/2012/02/141210-Lakin-Anderson-Why-Sustainability-Needs-Fiction.pdf}; Oikos FutureLab: \url{http://oikos-international.org/programmes/conferences/futurelab/}
\textsuperscript{70} \url{http://www.bth.se/ste/tmslm.nsf/pages/0f88849955f94f01c1257dac002e10cf?OpenDocument}
\textsuperscript{71} \url{http://www.sum.uio.no/english/research/}
\textsuperscript{72} About the study structure see: \url{http://www.utu.fi/fi/sivustot/kestava-kehitys-ja-vastuullinen-liiketoiminta/Sivut/home.aspx}
\textsuperscript{73} Godemann et al. 2011, 41.
\textsuperscript{74} Rasche et al. 2013, 83.
\textsuperscript{75} E.g. Komiyama & Takeuchi 2006.
discussion about what the new emerging field called sustainability science should include, in order to achieve more understanding in common to the content of sustainability science studies.\textsuperscript{76} Some universities have started to use the term sustainability science, e.g. the Leuphana University, the Lund University and the Tokyo University. It is interesting to notice that at the beginning of 2015 when this report was being written the University of Helsinki opened a new position of a Sustainability Science Fellow who “will be working within a context of sustainability science, an emerging field that facilitates the design and implementation of sustainability through a critical framework.”\textsuperscript{77}
New initiatives of collaboration arises round the education and research related with sustainability. One of the recent examples is the Joint Center for Global Sustainability and Cultural Transformation (CGSC) that is established by Leuphana University of Lüneburg (LUL) and Arizona State University (ASU). Their aim in common is to pursue excellence in sustainability research and education and they will develop further their joint transdisciplinary learning environment the Global Classroom and a new joint Master’s programme Global Sustainability Science.\textsuperscript{78} In order to strengthen the cooperation between those universities that give sustainability degree education even a Network of Programs in Transformational Sustainability (NEPS) has been established in 2015 and its partners are at the moment from Europe (Maastricht University, Lund University, UPC Barcelona, Leuphana University of Lüneburg), Asia (Tokyo University), Africa (Stellenbosch University) and North America (Arizona State University).\textsuperscript{79}
\textbf{Importance of the Visibility of the Offerings of Teaching and Research}
In the era of overwhelming information parade it is a challenge to get coverage and stand out from the rest in order to get the best students and funding. Universities are in such a competitive situation that they use all possible arguments for marketing their study programmes. Ranking lists, accreditations and awards are all carefully mentioned in the web pages worldwide and annual reports describe
\textsuperscript{76} O’Byrne et al. 2015.
\textsuperscript{77} http://www.helsinki.fi/henvi/news/150109_sustainabilityScienceFellow.html
\textsuperscript{78} https://global.asu.edu/center-global-sustainability-and-cultural-transformation; http://www.leuphana.de/zentren/cgsc.htm; http://www.leuphana.de/graduate-school/master/studienangebot/global-sustainability-science.html
\textsuperscript{79} https://global.asu.edu/center-global-sustainability-and-cultural-transformation; http://www.leuphana.de/zentren/cgsc.htm; http://www.leuphana.de/graduate-school/master/studienangebot/global-sustainability-science.html
in words, numbers and, figures the activities of schools, departments and institutes. The use of social media and short videos in the universities’ web pages is common.\textsuperscript{80}
International case examples include several research centres and institutes of varied size. Many of them coordinate or take part into teaching besides research activities. Examples can be found also in Finland. The RESP MAN Research Group in the University of Tampere for instance, organizes and gives the teaching of responsible business.\textsuperscript{81} In Joensuu the Institute for Natural Resources, Environment and Society (LYY), a network organization based at the University of Eastern Finland (UEF), combines the social and cultural research expertise for application in the analysis of the environment and natural resource uses in several faculties and shares information about seminars and other activities through its web pages.\textsuperscript{82} The Arctic Centre in the University of Lapland is specialized in the Arctic region multidisciplinary research and education, and sustainable development issues are among its expertise areas.\textsuperscript{83}
The decision of the Sustainable Development Studies in the University of Turku in the fall of 2014 to launch an informal and interactive umbrella seminar for researchers and teachers interested in topics related to sustainable development and corporate responsibility is a step forward from the perspective of visibility. The information about seminar gatherings is given in the web page bulletin of the University of Turku and so acts for the visibility of research activities related to sustainable development and corporate responsibility even outside the academia. The high relevance of publicity of research seminars can be seen in other universities as well, the Helsinki University Centre for Environment (Henvi) for example informs actively about its research and teaching in the public web pages and aims to develop as a Sustainability Science Centre in coming years. Henvi offers the minor subject, Environmental multidisciplinary studies, that resemble the Sustainable Development Studies in the University of Turku, and also Doctoral programme in interdisciplinary environmental sciences (DENVI).\textsuperscript{84}
Visibility can be viewed from yet another angle, namely the working life relevance of the studies. Working life relevance is highly topical in all education in universities at the moment, and in the web pages that describe the study programmes the working life relevance of the studies is described in
\textsuperscript{80} E.g. Annual report of Centre for Research into Sustainability (CRIS). \url{https://www.royalholloway.ac.uk/Management/Cris/documents/CRIS-Annual-Report-2013-14.pdf} and an example of communicating rankings in the same university: \url{https://www.royalholloway.ac.uk/management/aboutus/whymanagement.aspx}
\textsuperscript{81} \url{http://www.uta.fi/jkk/en/research/themes/respmann/introduction.html}
\textsuperscript{82} \url{http://www2.uef.fi/en/lyy/about-us}
\textsuperscript{83} \url{http://www.arcticcentre.org/EN/ABOUT-US}
\textsuperscript{84} \url{http://www.helsinki.fi/henvi/}; \url{http://www.helsinki.fi/henvi/denvi/index.html}
detail.\textsuperscript{85} Studies of Sustainable development are seen as relevant for the future working life both among the students and employers.\textsuperscript{86} From this point of view it is of importance to mediate the content of studies, whether it is minor or major, in such a way that employers for instance can easily access the information about the study structure even outside the university.
\textbf{The Role of Interdisciplinarity, Interaction and Cooperation with Stakeholders in Teaching}
There are case examples in the material where students have an active role in the implementation of the course. In Finland the Helsinki University Centre for Environment (Henvi) offered in 2015 for the first time Master’s degree students an opportunity to join a multidisciplinary team to plan a course Sustainable development in teaching. Students are also in charge of the teaching in the course.\textsuperscript{87} This resembles student led course offerings in CEMUS in Uppsala.
Cooperation and dialogue between students and teaching staff (and possibly stakeholders outside the university) strengthens the learning process and may produce interesting and high-quality results. Study modules with interdisciplinary team work include in several study programmes. The aim is to instruct dialogue and encourage for cooperation between disciplines in teams. The learning objectives are twofold: learn to apply suitable methods for solving problems related to issues of sustainability and to do that in an interdisciplinary team context. The goal is to find a balance between theory and practicality as the problems of sustainable development are complex and solutions demand integrative in-depth knowledge from many perspectives. As a result of the team work students produce a scientific paper, a research report or a research grant proposal for example.
The studies of sustainable development draw multidisciplinarity and interactive learning methods. The Master’s Programme Sustainability Science and Policy (SSP) organized by the International Center for Integrated Assessment and Sustainable development (ICIS) in the Maastricht University for instance describes that problem based learning is used as an educational format throughout the studies and it is one example of how the learning methods are gradually gaining more attention in the study programme descriptions.\textsuperscript{88} Multi- and interdisciplinary cooperation is put forward through new
\begin{footnotesize}
\textsuperscript{85} Ryan 2011; National Co-ordinating Centre for Public Engagement 2014; e.g. \url{http://www.nottingham.ac.uk/business/programmes/msc/index.aspx}
\textsuperscript{86} See e.g. Undén & Forshufvud 2010; Pesonen 2003.
\textsuperscript{87} \url{http://www.helsinki.fi/henvi/uutiset/150924_keke_opetuksessa_kurssi.html}; \url{https://kekeop.wordpress.com/}
\textsuperscript{88} \url{http://www.maastrichtuniversity.nl/web/Schools/ICIS/TargetGroup/MScSustainabilityScienceAndPolicy/SustainabilityScienceAndPolicy.htm}
\end{footnotesize}
combinations of disciplines in joint Master programmes. One example is The Erasmus Mundus Joint Master Degree programme in Pervasive Computing and Communications for Sustainable Development (PERCCOM) which combines advanced Information and Communication Technologies (ICT) with environmental awareness and sustainability, and organizes teaching in different universities and private companies during four semesters.\textsuperscript{89}
The observation that one has to join forces in order to be able to solve the wicked problems in the society encourages transdisciplinary cooperation with organizations outside the academia. Transdisciplinary efforts are put into practice in different ways, in May 2015 in the Rotterdam University established the Wicked Problems Plaza being one example.\textsuperscript{90}
It is suggested that new learning and teaching methods, e.g. the student led courses and different forms of virtual learning, are challenging the traditional way to carry out higher education in universities.\textsuperscript{91} The benchmarking process gives some support to these trends as it showed the growing flexibility of studies and the expanding amount of online learning possibilities in the explored universities. The complex character of sustainable development issues calls for active participation and discussion between representatives of different disciplines. This kind of active participation is modelled in distant online courses through virtual group discussion. In MOOCs like Greening the Economy: Lessons from Scandinavia (organized by the IIIEE in Lund) there can be thousands of students discussing the challenges of sustainability in different societies. At the same time, however, in many study modules and Master’s programmes the intensive interdisciplinary face to face interaction and problem solving in the campus area is mentioned to be the strength of the studies.
\textbf{The Role of Education for Sustainable Development (ESD) and PRME in Universities}
Themes of sustainable development and responsible business/CSR have got more attention in many universities during recent years. It has been noticed that students show increasing enthusiasm toward sustainability issues and the complex challenges that present-day societies face demand new solutions and interdisciplinary mindset.\textsuperscript{92} The question at the moment seems to be whether the sustainability and CSR should be integrated into curricula at all levels and disciplines or whether to develop
\textsuperscript{89} \url{http://perccom.blog.univ-lorraine.fr/}
\textsuperscript{90} \url{http://www.rsm.nl/about-rsm/news/detail/4819-centre-for-resolving-wicked-problems-opens-in-the-hague/}
\textsuperscript{91} Johnson et al. 2014; Bisoux 2014.
\textsuperscript{92} E.g. Hanken School of Economics 2014, 9; Jones Christensen et al. 2007.
separate study programmes.\textsuperscript{93} The UN related activities like Education for Sustainable Development (ESD)\textsuperscript{94} and the PRME initiative\textsuperscript{95} aim to promote the gradual integration aspect. Some case universities have actively adopted this line of the procedure in their common strategies. Gothenburg University for instance, adds a certain sustainability label to all courses that with varying degrees include the elements of sustainable development in their curriculum and so helps students to seek and find that type of courses.\textsuperscript{96} Gothenburg University Library also offers a Toolbox in Education for Sustainable Development (ESD). It is meant as an aid to educational planners and teachers at the University of Gothenburg in the process of integrating sustainable development in teaching.\textsuperscript{97} Also in the University of Plymouth and Leuphana University Lüneburg sustainability integration in education and research is among the core issues in the development strategies.\textsuperscript{98}
The ideas of ESD and Green Campus are developed in universities in Finland as well. Lappeenranta University of Technology (LUT) for instance has included sustainability at the core of the university’s strategy and its endeavors have been awarded with environmental certification in accordance with the ISO 14001 standard. LUT is the first university in Finland where the environmental system covers
\textsuperscript{93} In some case universities, both ways are used: e.g. all undergraduate students in Nottingham University Business School participate the core course Business Ethics in their third year of studies and there are two Master’s programmes and one MBA related to CSR available as well.
\textsuperscript{94} E.g. Ryan 2011.
\textsuperscript{95} Launched in 2007 the Principles for Responsible Management Education (PRME) initiative provides an engagement structure for academic institutions to advance social responsibility through incorporating internationally accepted values into curricula and research gradually. The PRME is the first organized relationship between the United Nations and business schools. So far Aalto University, School of Business; Hanken School of Economics; HAAGA-HELIÁ University of Applied Sciences, JAMK University of Applied Sciences, Lappeenranta University of Technology and Turku School of Economics from Finland have joined the initiative. Altogether over 500 business schools and management-related academic institutions from over 80 countries across the world are signatories to the PRME. \url{http://www.unprme.org/about-prme/index.php}. See also Godemann et al. 2014.
\textsuperscript{96} For Sustainability Labeling in University of Gothenburg see: \url{http://utbildning.gu.se/kurser/hallbarutveckling}
\textsuperscript{97} The Toolbox includes a wealth of material from various sources like scientific literature, reports, links of institutions and NGOs with different initiatives and films related to sustainable development issues. Gothenburg University Library. \url{http://libguides.ub.gu.se/ESDverktyg}
\textsuperscript{98} Plymouth University pioneered in 1973 one of the UK’s earliest interdisciplinary programmes in environmental sciences. In 2005 the UK government funded the establishment of a national Centre of Excellence called the Centre for Sustainable Futures (CSF) in Plymouth University. The CSF leads the university’s ongoing commitment to embed environmental, sustainability and ethical education across the subject spectrum, with the result that some 49% of all programmes reflect sustainability principles. \url{https://www.plymouth.ac.uk/your-university/sustainability/sustainability-education}
the operations of the institution as a whole: research, teaching, social interaction and support functions. The Green Campus project of LUT was awarded in the International Sustainable Campus Network competition as the best campus in Excellence in Campus category in 2013.\textsuperscript{99}
Projects related to Education for Sustainable Development (ESD) work actively for the implementation of sustainability issues at different levels in the universities and they produce reports of the achievements and challenges. Recent project reports for instance study the development of Education for Sustainable Development in Nordic countries and highlight good practices in the European Higher Education Institutions.\textsuperscript{100}
\textsuperscript{99} \url{http://www.lut.fi/web/en/news/-/asset_publisher/IJh4SAywhcPu/content/id/355865}; \url{http://www.lut.fi/web/en/green-campus?utm_source=frontpage-big-banners&utm_medium=banner&utm_content=green-campus&utm_campaign=internal-links-en}
\textsuperscript{100} Karvinen et al. 2015; UESD 2015.
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**Internet links in footnotes according to the university**
Anglia Ruskin University
http://www.anglia.ac.uk/entrepreneurial-university
http://www.anglia.ac.uk/global-sustainability-institute-gsi
Arizona State University
https://global.asu.edu/center-global-sustainability-and-cultural-transformation
Blekinge Institute of Technology
http://www.bth.se/ste/tmslm.nsf/pages/0f88849955f94f01c1257dac002e10cf?OpenDocument
Bournemouth University
http://courses.bournemouth.ac.uk/courses/postgraduate-degree/green-economy/none/3126/
Copenhagen Business School
https://www.coursera.org/course/socialentrepeneur
Erasmus Mundus Master’s programme MIND, University of Graz as co-ordinator
http://www.emmind.eu/
Joint International Master in Sustainable Development, University of Graz as co-ordinator
http://www.jointdegree.eu/sd/
Lappeenranta University of Technology
https://uni.lut.fi/en/web/lut.fi-eng/study-guides9
http://perccom.blog.univ-lorraine.fr/
http://www.lut.fi/web/en/news/-/asset_publisher/IGh4SAywhcPu/content/id/355865
http://www.lut.fi/web/en/green-campus?utm_source=frontpage-big-banners&utm_medium=banner&utm_content=green-campus&utm_campaign=internal-links-en
Leiden University
http://www.cml.leiden.edu/education/master/industrial/about-master-ie.html
http://www.cml.leiden.edu/education/minor/minor_overview.html
http://media.leidenuniv.nl/legacy/schedule-course-big-issues-new-answers-cml.pdf
https://studiegids.leidenuniv.nl/en/courses/show/46935/Duurzame_ontwikkeling_tp
https://studiegids.leidenuniv.nl/courses/show/46937/Projectgroep_Ontwerp_van_Europees_Onderzoek
https://studiegids.leidenuniv.nl/courses/show/46939/Area_study_sustainability
https://www.facebook.com/pages/Watercourse-Philippines/312676025504087
https://studiegids.leidenuniv.nl/en/courses/show/45903/Fieldschool_Water_Management_in_the_Philippines_Winter
Leuphana University Lüneburg
http://www.leuphana.de/en/graduate-school/master/course-offerings/sustainability-science.html
http://www.leuphana.de/en/college/first-semester.html
http://www.leuphana.de/college/studienstart/konferenzwoche.html
http://www.leuphana.de/zentren/cgsc.html
http://www.leuphana.de/graduate-school/master/studienangebot/global-sustainability-science.html
http://www.leuphana.de/zentren/cgsc.html
Lund University
https://www.facebook.com/iiieemoc
https://www.coursera.org/learn/greening-the-economy/
Maastricht University
http://www.maastrichtuniversity.nl/web/Schools/ICIS/TargetGroup/MScSustainabilityScience-AndPolicy/SustainabilityScienceAndPolicy.htm
Nottingham University Business School
http://www.nottingham.ac.uk/business/ICCSR/teaching.php
http://www.nottingham.ac.uk/business/programmes/msc/index.aspx
Oikos
http://oikos-international.org/programmes/conferences/futurelab/
Plymouth University
http://www1.plymouth.ac.uk/research/ourresearch/themes/Pages/environment.aspx
http://www1.plymouth.ac.uk/research/issr/centres/Pages/default.aspx
https://www.plymouth.ac.uk/your-university/sustainability/sustainability-education
Rotterdam School of Management
http://www.rsm.nl/master/msc-programmes/msc-global-business-sustainability/overview/
http://www.rsm.nl/about-rsm/news/detail/4819-centre-for-resolving-wicked-problems-opens-in-the-hague/
Royal Holloway, University of London
https://www.royalholloway.ac.uk/Management/Cris/documents/CRIS-Annual-Report-2013-14.pdf
https://www.royalholloway.ac.uk/management/aboutus/whymanagement.aspx
Stockholm University
https://www.sdsnedu.org/learn/planetary-boundaries-and-human-opportunities-fall-2014
https://www.sdsnedu.org/learn/planetary-boundaries-and-human-opportunities-september-2015
http://www.stockholmrésilience.org/21/education.html
Tampere University of Technology
http://www.tut.fi/en/about-tut/departments/chemistry-and-bioengineering/index.htm
https://uni.lut.fi/en/web/lut.fi-eng/study-guides9
Technische Universität Kaiserslautern
http://www.zfuw.uni-kl.de/en/distance-learning-courses/management-law/sustainable-development-cooperation/
University of Bath
http://www.bath.ac.uk/study/moocs/
University of Eastern Finland
http://www2.uef.fi/en/lyy/about-us
University of Edinburgh
http://www.ed.ac.uk/studying/postgraduate/degree-guide/online-learning/programmes
University of Exeter
http://admin.exeter.ac.uk/academic/tls/tqa/Part%2013/13CQF.pdf
http://www.exeter.ac.uk/postgraduate/degrees/distance/
University of Gothenburg
http://gmv.gu.se/samverkan/samverkan-inom-utbildning/fallstudiiekurs-i-hallbar-utveckling
http://gmv.gu.se/digitalAssets/1447/1447740_rapport_fallst_2012_hisingen_2050_12-05-26.pdf
http://utbildning.gu.se/kurser/hitta-kursplan/kursplan/?courseld=ES2415
http://utbildning.gu.se/kurser/hallbarutveckling
http://libguides.ub.gu.se/ESDverktyg
University of Helsinki
http://www.helsinki.fi/henvi/opetus/johdantokurssi.htm
http://www.helsinki.fi/henvi/news/150109_sustainabilityScienceFellow.html
http://www.helsinki.fi/henvi/denvi/index.html
http://www.helsinki.fi/henvi/utiset/150924_keke_opetuksessa_kurssi.html
https://kekeop.wordpress.com/
University of Jyväskylä
https://www.jyu.fi/jsbe/en/cem
https://www.jyu.fi/jsbe/en/research/groups/leadership/ethos/projects
University of Lapland
http://www.arcticcentre.org/EN/ABOUT-US
University of Oslo
http://www.uio.no/english/studies/programmes/ces-master/
http://www.sum.uio.no/english/research/
University of Oulu
http://www.oulu.fi/blogs/node/29750
http://www.maigbe.com/martti_ahtisaari_institute
University of Tampere
http://www.uta.fi/jkk/en/research/themes/respman/introduction.html
University of Turku
http://www.utu.fi/fi/sivustot/kestava-kehitys-ja-vastuullinen-liiketoiminta/Sivut/home.aspx
University of Uppsala, Uppsala Center for Sustainable Development, CSD Uppsala
http://www.cemus.uu.se/dokument/sustainable_dev_uppsala.pdf
http://www.csduppsala.uu.se/education/
http://cemusstudent.se/wp-content/uploads/2012/02/141210-Lakin-Anderson-Why-Sustainability-Needs-Fiction.pdf
International Networks related to Education and Research of Sustainable Development and Responsible Business/Corporate Social Responsibility:
AASHE Association for the Advancement of Sustainability in Higher Education
http://www.aashe.org/
ABIS The Academy of Business in Society
http://www.abis-global.org/
The Aspen Institute Centre for Business Education. Beyond Grey Pinstripes – an Aspen CBE Initiative.
http://www.beyondgreypinstripes.org/content/corporate-environmental-strategy-and-marketing
BUP The Baltic University Programme
http://www.balticuniv.uu.se/
COPERNICUS Alliance – European Network on Higher Education for Sustainable Development
http://www.copernicus-alliance.org/
GRLI Globally Responsible Leadership Initiative
http://www.grli.org/
HESD The Higher Education for Sustainable Development
http://www.iau-hesd.net/en
ISCN International Sustainable Campus Network
http://www.international-sustainable-campus-network.org/
ISSS International Society for Sustainability Science
http://sussci.org/
ISDRS International Sustainable Development Research Society
http://isdrs.org/
NBS Network for Business Sustainability
http://nbs.net/
NSCN Nordic Sustainable Campus Network
http://nordicsustainablecampusnetwork.wordpress.com/
PRME Principles for Responsible Management Education
http://www.unprme.org/index.php
UE4SD University Educators for Sustainable Development
http://www.ue4sd.eu/
ULSF University Leaders for a Sustainable Future
http://www.ulsf.org/about.html
APPENDIXES
Appendix 1. The minor and major studies on sustainable development or responsible business in Finnish universities. Page 46
Appendix 2. The international case examples of major and one minor studies on sustainable development in Sweden and Norway. Page 58
Appendix 3. The international case examples of minor and major studies on sustainable development in United Kingdom, Germany, the Netherlands and one Joint programme. Page 63
Appendix 4. The International case examples of minor and major studies in CSR/responsible business in Denmark, the Netherlands and the United Kingdom. Page 67
## Appendix 1. The minor and major studies on sustainable development or responsible business in Finnish universities.
### MINORS 1.
| Faculty or school where the education is organized? | Aalto University | Hanken School of Economics | University of Tampere School of Management | University of Turku Turku School of Economics | UniPID |
|-----------------------------------------------------|------------------|----------------------------|-------------------------------------------|---------------------------------------------|-------|
| Joint programme of the three Aalto University schools: School of Business, School of Arts, Design and Architecture and School of Engineering. [http://acs.aalto.fi/minor-study/](http://acs.aalto.fi/minor-study/) | Administered by the department for Supply Chain Management and Social Responsibility [http://www.hanken.fi/en/node/34518](http://www.hanken.fi/en/node/34518) | School of Management, Specializing studies in responsible management [http://www.utu.fi/jkk](http://www.utu.fi/jkk) [https://www10.utu.fi/opas/opintoko-konaisuus.htm?rid=7211&lang=en&uilang=en&lv=2014](https://www10.utu.fi/opas/opintoko-konaisuus.htm?rid=7211&lang=en&uilang=en&lv=2014) | Administered by Management and Organization discipline [http://www.utu.fi/fi/sivustot/kestava-kehitys-ja-vastuullinen-liiketoiminta/vastuullinen-liiketoiminta/Sivut/home.aspx](http://www.utu.fi/fi/sivustot/kestava-kehitys-ja-vastuullinen-liiketoiminta/vastuullinen-liiketoiminta/Sivut/home.aspx) | the Finnish University Partnership for International Development - UniPID, a partnership network between 10 Finnish universities, coordinated by University of Jyväskylä [http://www.unipid.fi/en/page/262/unipid_minor_sustainability_in_development/](http://www.unipid.fi/en/page/262/unipid_minor_sustainability_in_development/) |
| Type of education: Study module/Minor subject or Bachelor/Master’s programme? | Minor: The Creative Sustainability (CS) Programme 15–25 ECTS Offered to all master’s student in Aalto University in order to help broaden sustainability knowledge and get acquainted with multidisciplinary approach during one’s own master degree studies. | Minor: Corporate Responsibility (CR) minimum 25 cr. 25 students who are not degree students at Hanken are offered the opportunity to complete the Corporate Responsibility study module, targeted mainly to students at the University of Helsinki, popular among students. | Responsible Business 15 ECTS, Open for degree programme students, other students, doctoral students and exchange students | Minor: Responsible Business, 25 cr | Virtual Minor: Sustainability in Development, 25 ECTS The registration for the Minor is continuous and the programme is free of charge for all students at member universities. |
| Any research group/institute/centre in the background? | Collaborates with NODUS and CESR in teaching and research. [http://acs.aalto.fi/research/](http://acs.aalto.fi/research/) | - | RESPMAN Research Group [http://www.utu.fi/jkk/en/research/themes/respmann/introduction.html](http://www.utu.fi/jkk/en/research/themes/respmann/introduction.html) | - | - |
| Core elements | A course palette from which students choose relevant courses to make an individual study plan. [http://acs.aalto.fi/minor-study/course-list/](http://acs.aalto.fi/minor-study/course-list/) CS brings together students from different fields to study in multidisciplinary teams that increases understanding of different disciplines and enables adapting a holistic approach.
The pedagogical approach is based on integrating teaching and research, problem-based learning, blended learning and strong connection to practical outcomes.
Studies combine insights and courses from systems thinking, sustainable urban and building design, responsible business, sustainable product and service design, environmental impact assessment of landscape planning, real estate and global development issues. |
| --- | --- |
| Study plan for Study Module in Corporate Social Responsibility 2014-2015: at least 25 credits among the following: The Corporation and its Employees 6-8 ECTS Gender, Management and Organisation 8 ECTS Business Ethics 8 ECTS Literature Course 8 ECTS Business, Government and Society 8 ECTS Work and Industrial Relations 8 ECTS Literature course 8 ECTS Corporate Social Responsibility: From Principles to Practice 8 ECTS Humanitarian Logistics 8 ECTS Sustainable Supply Chain Management 8 ECTS Project Course in Societal and Environmental Responsibility 8 ECTS Corporate Sustainability 8 ECTS Introduction to Corporate Responsibility 1 ECTS, students external to Hanken. |
| Business Ethics 5 ECTS Corporate Responsibility and Accounting 5 ECTS CSR in Different Business Models and Contexts 5 ECTS |
This table represents studies in study year 2014–2015, it has been partly renewed for years 2015-2018. The new Responsible business and sustainability study module is larger (20-35 ECTS) and it can be seen here: [http://www.uta.fi/jkk/kat/opintosuunnat/vlt/eteneminen.html](http://www.uta.fi/jkk/kat/opintosuunnat/vlt/eteneminen.html)
You can check the differences in this pdf: [http://www.uta.fi/jkk/kat/opintosuunnat/vlt/siirrym%C3%A4ns%C3%A4n%C3%A4nn%C3%B6kset_2015.pdf](http://www.uta.fi/jkk/kat/opintosuunnat/vlt/siirrym%C3%A4ns%C3%A4n%C3%A4nn%C3%B6kset_2015.pdf)
| Responsible Business Introduction Course 6 cr Selection of optional courses in the School of Economics, together 19 cr Examples of optional courses: Corporate Responsibility Reporting 2 cr Ethical Issues in Finance and Responsible Investing 3 cr New Challenges of Global Business 6 cr Globalisation and Corporate Responsibility 5 cr Global responsible business 6cr Information Technology and Ethics 5 cr Trade and Transport Facilitation 2-6 cr Global Challenges and Sustainable Futures 6 cr |
| --- | --- |
| The Minor is comprised of at least five (5) courses (worth 5 ECTS credits each) for a minimum total of twenty-five (25) ECTS credits. Students are free to choose courses from the list of courses offered to tailor an individual study programme but at least two courses should be general level. [http://www.unipid.fi/en/courses/](http://www.unipid.fi/en/courses/) |
| The role of multidisciplinarity in teaching? | Multidisciplinarity is in the core of studies. | A cross-disciplinary approach | - | - | Teaching is given by many disciplines in several universities |
|---------------------------------------------|-------------------------------------------------|-------------------------------|-----------------|-----------------|--------------------------------------------------|
| Additional information | - | - | Courses offered also in open university teaching for restricted number of students | - | - |
**MINORS 2.**
| Faculty or school where the education is organized? | Lappeenranta University of Technology | Åbo Akademi University | Swedish School of Social Science | University of Vaasa | University of Helsinki |
|-----------------------------------------------------|--------------------------------------|------------------------|---------------------------------|--------------------|-----------------------|
| The module of Sustainability is coordinated by the Degree Programme in Environmental Technology. | Faculty of Science and Engineering [http://www.abo.fi/fakultet/miljokunskap](http://www.abo.fi/fakultet/miljokunskap) | Swedish School of Social Science [https://weboodi.helsinki.fi/hy/vl_kehys.jsp?MDsavain=&Kiel=2&Org=75334693&vl_tila=1&Opas=4489](https://weboodi.helsinki.fi/hy/vl_kehys.jsp?MDsavain=&Kiel=2&Org=75334693&vl_tila=1&Opas=4489) | Joint minor organized by regional studies, public law, marketing, economics, commercial law, industrial management, accounting and financing [http://www.uva.fi/fi/for/student/studies/study/minor/philosophy/](http://www.uva.fi/fi/for/student/studies/study/minor/philosophy/) | Minor is administered by Faculty of Agriculture and Forestry [http://www.helsinki.fi/henvi/opetus/vaatimukset.htm](http://www.helsinki.fi/henvi/opetus/vaatimukset.htm) [http://www.helsinki.fi/henvi/opetus/johdantokurssi.htm](http://www.helsinki.fi/henvi/opetus/johdantokurssi.htm) |
| Type of education: Study module/Minor subject or Bachelor/Master’s programme? | Minor: Sustainability, targeted for third-year Bachelor’s students and Master’s students in all degree programmes. | Minor in Environmental knowledge, 25-60 cr, open to all students, some courses offered in cooperation with the open university of ÅA | Minor in Environment 25 open to all students in Swedish S. of SS and University of Helsinki. | Minor in Environment, 26 cr, open to all students in University of Vaasa | Multidisciplinary minor in Environment (YMS) 25 cr, started 2006. Open to all students, with the Flexible Study Rights Scheme (JOO) to other students as well. |
| Any research group/centre in the background? | - | - | - | - | HENVI = Helsinki University Centre for Environment |
| Core elements | The minor subject consists of elective courses in sustainable development and the compulsory course Introduction to Sustainability. The degree programmes will adapt the module to their own curriculum. [http://www.lut.fi/web/en/green-campus/measures/sustainability-minor-subject](http://www.lut.fi/web/en/green-campus/measures/sustainability-minor-subject) Minor is a joint multidisciplinary module that consists of courses from the three different Schools. See Master’s Programme MSIS for description of Sustainability minor courses adapted by the School of Business |
| --- | --- |
| Mandatory course: Project Work 5 cr or Ecology and conservation 5 cr Elective courses can be chosen from 25 different courses and from three different faculties, e.g. Environment and ethics 5 cr Environmental Law and Administrative Structure 10 cr Basics of Environmental economics 5 cr Cultural Ecology 5 cr | Focus on environmental governance and regional policy. Small minor consists of 5 electives. Large minor can include also courses outside the Swedish S. of SS. Minor strengthens the skills and employability of those students who study environment from natural sciences perspective. |
| The aim of the study module is to give students comprehensive view of the interaction between man, nature, and business both in global and local level seen from the scientific framework of University of Vaasa. Comprises of mandatory study courses that are offered by different faculties: 1. Nature, society and environmental protection 5 cr 2. Environmental philosophy 5 cr 3. Energy Economics 5 cr 4. Sustainable energy business 5 cr 5. Environmental law 6 cr | Mandatory introduction course (5 cr) and elective courses which are chosen freely from a selection of study courses in different disciplines. [http://www.helsinki.fi/henvi/opetus/valinnaisetopinnot.htm](http://www.helsinki.fi/henvi/opetus/valinnaisetopinnot.htm) Introduction to multidisciplinary environmental studies introduces students to environmental studies and teachers in different faculties, includes lectures and a multidisciplinary group work that deals with some phenomenon related to environment, panel discussion with visiting experts open to larger audience in the end of the course. Former panel discussion themes have been e.g. Use of oil shale and shale gas and their environmental effects, plastic waste in oceans. Students’ feedback from former courses: [http://www.helsinki.fi/henvi/opetus/2012_palaute_JMY12.pdf](http://www.helsinki.fi/henvi/opetus/2012_palaute_JMY12.pdf) [http://www.helsinki.fi/henvi/opetus/2011_palaute.pdf](http://www.helsinki.fi/henvi/opetus/2011_palaute.pdf) |
| The role of multidisciplinarity in teaching? | - | Multidisciplinarity is in the core as the learning target is to give better ability to consider sustainability issues multidisciplinary |
| --- | --- | Minor includes studies from different sciences in order to add knowledge of different perspectives, interdisciplinarity is not emphasized |
| The module is defined as multidisciplinary as it gives an overview of environment from several disciplines perspective. | The aim of the minor is to make it easier to study courses that go beyond one’s own faculty. Multidisciplinarity feeds the understanding of the multifaceted nature of environmental questions. |
| Additional information | - | - | - | - | - |
| Faculty or school where the education is organized? | University of Tampere | University of Turku | University of Lapland | University of Eastern Finland | University of Jyväskylä |
|---------------------------------------------------|-----------------------|--------------------|----------------------|-------------------------------|------------------------|
| Coordinated by the School of Social Sciences and Humanities [http://www.uta.fi/kyk/opiskelu/vaalinaisetopinnot/kestava_kehitys.html](http://www.uta.fi/kyk/opiskelu/vaalinaisetopinnot/kestava_kehitys.html) | Coordinated through Finland Futures Research Centre [http://www.utu.fi/fi/sivustot/kestava-kehitys-ja-vastuullinen-liiketoiminta/sustainable-development-studies/Sivut/home.aspx](http://www.utu.fi/fi/sivustot/kestava-kehitys-ja-vastuullinen-liiketoiminta/sustainable-development-studies/Sivut/home.aspx) | Organized by the Faculty of Social Sciences [http://www.ualapland.fi/Suomeneki/Yksikot/Yhteiskunta-tiedekunta/Opsiskelu/Oppiaineet-ja-maisteriohjemat/Siivuaineet/Ymparistoopinnot](http://www.ualapland.fi/Suomeneki/Yksikot/Yhteiskunta-tiedekunta/Opsiskelu/Oppiaineet-ja-maisteriohjemat/Siivuaineet/Ymparistoopinnot) | Organized in the campus of Joensuu, teaching from all faculties [http://www2.uelf.fi/fi/ymparisto](http://www2.uelf.fi/fi/ymparisto) | Organized by Faculty of Social Sciences |
| Type of education: Study module/Minor subject or Bachelor/Master’s programme? | Minor: Sustainable Development Study Programme, 25-35 ECTS, open for all students, offered also in the open university | Minor: Sustainable Development Studies, 25 cr | Environmental Studies, basic and intermediate module 25-60 cr, open for all students | Multidisciplinary Environmental Studies, free to all students in U. of Eastern Finland Basic studies 25 cr and intermediate level 35 cr | Environment and Society study module 25-40 cr, free to all students |
| Any research institute/centre? | - | Coordinated through Finland Futures Research Centre | - | - | - |
| Core elements | Choose at least 25 ECTS, at least from two different themes: **Sustainable development study module, theme 1** Global Risks and Environmental Security 5 ECTS Ecosocial Dynamics 5 ECTS Climate Change and Health 5 ECTS Health & Illness in a Global Context 5 ECTS | KEKO1: Implementing the principles of sustainable development 10 cr KEKO2 Selection of optional courses of sustainable development, together 15 cr Optional courses are chosen from the selection offered by different faculties of University of Turku. Also some online courses are available e.g. from UniPID offerings. The studies cover a broad variety of issues relating to the | Basic module mandatory course: Introduction to Environmental Studies 3 ECTS cr Other courses selected from: Natural Resources and Environmental Economics 5 cr Sustainability in Tourism 5cr Ecology and Nature Conservation 4cr Society and Environment 4cr Environmental Philosophy I 2-6 cr Environmental Studies Theory 3 cr | Mandatory courses in basic studies: Introduction to environmental sciences 3 cr Basics of Ecology 4 cr Basics of Environmental policy 3cr Introduction to Environmental history 3 cr Rest of the study modules in basic studies can be chosen freely. The aim is to consider environmental problems and | Kestävä Kehitys (Sustainable development) 5cr Ympäristösosiologia (Environmental Sociology) 15 cr Ympäristösosiologia II 5 cr Ruoka (Food) 5cr Energia (Energy) 5 cr Ympäristöetikka (Environmental Ethics) 5 cr |
| Human Mobility and Health 5 ECTS
**Sustainable development study module, theme 2**
History of Modern World 5 ECTS
Sociology of Globalization 5 ECTS
International Society 5 ECTS
Political Institutions & Processes 5 ECTS
Consumption and the Market 5 ECTS
Critical Perspectives on Accounting 5 ECTS
Peace, Security and International Institutions 5 ECTS
**Sustainable development study module, theme 3**
Current Issues in Social Sciences 5 ECTS
Introduction to the Politics of Environment and Regions 5 ECTS
Comparative European Politics and the Finnish Political System 5 ECTS
Citizenship & Social Movements 5 ECTS
Russian Society 5 ECTS
**Sustainable development study module, theme 4** | four dimensions of sustainable development: ecological, social, cultural and economic. Examples of course offerings: Ethical Thinking and Society Introduction to Population Culture in East and Southeast Asia (online course) Religions of East and Southeast Asia (online course) Resources of the Earth Environment and Development Sustainable urban development and living What Food Crisis (online course) Public Health Challenges and Scenarios in South Asia (online course) Corporate Responsibility Reporting Responsible Business: an introduction Globalisation and corporate responsibility Information and Ethics Foundation of Futures Studies Systems thinking | Environmental Movements 3cr
Northern Environmental Questions 3 cr
**Intermediate module selection:**
Environmental Politics 10cr
Current Trends of Environmental Studies 5cr
Nature and Culture 5cr
Seminar 5cr
Environmental Philosophy II 3cr
Art, Environment and Aesthetics 3cr
Environmental Law 6cr
Environmental Conflicts 3cr
Environmental Economy 3cr
Environmental Impacts and Sustainable Development of Tourist Destinations 3cr | conservation from multidisciplinary perspective as a process of interaction between man and nature. The target is to produce new knowledge through interaction between different disciplines and multidisciplinary cooperation. | Sociology of Consumption and Lifestyles 5 cr
Kulutuksen ja elämäntavan sosiologia II 5 cr
https://www.jyu.fi/ytk/opiskelu/opetussuunnitelmat/opsit/manual/ops2014-2017/ytk-sivuaineet/opintokokonaisuudet/ymy
| **Agency & Relationships with Media 5 ECTS** | **Multicultural Relationships with Media and Citizenship 5 ECTS** | **Classics of Translation Studies 5 ECTS** | **Information Systems and Society 5ECTS** | **Sustainable development study module, theme 5** | **Ethics and Social Philosophy 5 ECTS** | **Business Ethics 5 ECTS** |
|---------------------------------------------|---------------------------------------------------------------|------------------------------------------|------------------------------------------|---------------------------------------------|------------------------------------------|------------------------------------------|
| **The role of multidisciplinarity in teaching?** | Minor is multidisciplinary by character, dimensions of social and cultural sustainability are stressed | Interdisciplinarity is in the core of the KEK01 course | Multidisciplinary approach | Multidisciplinarity is mentioned to be central element of studies | - | - |
| **Additional information** | - | - | Even a multidisciplinary doctoral programme Northern cultures and sustainable natural resource politics is available [http://www.ualpland.fi/InEnglish/Research/Graduate-School/Doctoral-programmes/Northern-cultures-and-natural-resource-politics](http://www.ualpland.fi/InEnglish/Research/Graduate-School/Doctoral-programmes/Northern-cultures-and-natural-resource-politics) | - | - | - |
| Any research group/institute/centre in the background? | No own research group, collaborates with NODUS and CESR in both teaching and research. [http://acs.aalto.fi/research/](http://acs.aalto.fi/research/) |
|--------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------|
| Core elements | Programme studies 84 cr
**Mandatory studies at the School of Business 48 cr:**
Doing Qualitative Research 6 cr
Capstone in Creative Sustainability 6 cr
Master’s Thesis 30 ECTS
Master’s Thesis Seminar 6 ECTS
**Compulsory Joint Studies in CS 10 cr:**
Creative Teamwork 2 cr
Creating the Mindset of Sustainable Societies 2 cr
Continuous Transformation 2 cr
Systems Thinking 1, 2 cr
Systems Thinking 2, 2 cr
**Elective CS-courses from the School of Business, together 18 cr:**
How to Change the World: Innovation toward Sustainability 6 cr
Corporate Responsibility in Global Economy 6 cr
Sustainability Politics and CSR – Reading Seminar 6 cr
Sustainable Business and Consumption 6 cr
**Elective courses in CS 8 cr,** may be chosen freely from different schools |
| Core studies 48 ECTS of which compulsory 33ECTS and electives 15 ECTS; here only compulsory courses:
Empirical Strategy Research 6 ECTS
Business Models & Strategy 6 ECTS
Technology and Innovation Management 6 ECTS
Contemporary Issues in Strategic Management & Innovation 3 ECTS
Sustainable Strategy and Business Ethics 3 ECTS
Corporate Responsibility and Management 1, 3 ECTS
Master’s Transferable Skills 3 ECTS
Master’s Thesis Seminar 3 ECTS
**Specialisation Studies 42 ECTS compulsory:**
Strategy Consulting 6 ECTS
Knowledge-based Networks 6 ECTS
Master’s Thesis 30 ECTS
**Minor studies 24 ECTS,** here only Sustainability minor:
Compulsory courses 13 ECTS |
| Research group of Corporate Environmental Management (CEM) is proactive in integrating sustainability issues in its research and curriculum [https://www.jyu.fi/jsbe/en/research/groups/cem](https://www.jyu.fi/jsbe/en/research/groups/cem) |
| RESP MAN Research Group, organizes and gives teaching [http://www.uta.fi/jkk/en/research/themes/respmanteaching.html](http://www.uta.fi/jkk/en/research/themes/respmanteaching.html) |
| Compulsory major studies 72 ECTS
Orientation to JSBW Master’s Programmes 1 ECTS
Material Flow Management 5 ECTS
Material Flow Management, Computer Demonstrations 3 ECTS
Managing a Green Organisation 5 ECTS
Corporate Environmental Management, Project Work or Internship 5 ECTS
Corporate Environmental Strategy and Marketing 5 ECTS
Environmental Management in Networks 5 ECTS
Climate Business 5 ECTS
Master Level Research Tutorial 3 ECTS
Master’s Thesis 35 ECTS
**Optional major studies 10 ECTS,** e.g.:
Introduction to International Environmental Law 4 ECTS
Motivating Sustainable Consumption 4 ECTS
Stakeholder Management 6 ECTS
Environmental Economics 6 ECTS
Social and Environmental Accounting 6 ECTS |
| Responsible Management, 10–15 ECTS:
Ethical Leadership 5 ECTS
Stakeholder Theory and Management 5 ECTS
Management Communication 5 ECTS
**Social and Environmental accounting,** 10–15 ECTS:
Social and Environmental Accounting and Reporting 10 ECTS
Critical Perspectives on Accounting 5 ECTS
**Business Law,** 5 ECTS:
Applied Law and Economics 5 ECTS
**Insurance and risk management,** 5-10 ECTS:
Current Issues in Social Security 5 ECTS
Enterprise Risk Management 5 ECTS
**Research seminar and master’s thesis,** 40 ECTS
Optional Studies 15-30 ECTS
Language studies 10 ECTS |
| **Minor Studies 24 cr**
Aalto Elective Courses 12 cr
http://study-guides.aalto.fi/biz/2014-13/en/master-of-science-degree-studies/study-programmes/creative-sustainability.html
Study structure varies according to each CS discipline: Architecture, Business, Design, and Real Estate. Study structure in CS in Business | Introduction to Sustainability 3 ECTS
Cleaner Technologies and Markets 5 ECTS
Social Responsibility 5 ECTS
Electives, choose at least 11 ECTS:
Sustainable Strategy & Business Ethics 3 ECTS
Corporate Responsibility Management 1, 3 ECTS
Wind Power and Solar Energy Technology and Business 5 ECTS
Basic Course on Environmental Management and Economics 5 ECTS
Independent Study in English 1-4 ECTS
https://uni.lut.fi/en/web/lut.fi-eng/study-guides9 | Course descriptions: https://www.jyu.fi/en/studywituus/programmes/cem/course-descriptions | |
| --- | --- | --- | --- |
| **The role of multidisciplinarity in teaching?** | Multidisciplinarity is in the core of studies. Competence areas covered in CS programme are: multidisciplinary approach, systems thinking, business management, design thinking and project management. | Multidisciplinarity is in the selection of courses. Students can include maximum of 12 ECTS of MOOCs in the MSIS degree, but they must be agreed beforehand with the Academic Director by submitting an informal application letter (course details and ECTS, suitability to the programme). These courses can be located to replace elective courses in core studies (strategy, innovation or sustainability). | An integrated and interdisciplinary approach to environmental issues. Students also do some compulsory basic studies in environmental science and technology. | - |
| **Additional information** | - | The MSIS programme offers a double degree option with the Graduate School of Management | In the Beyond Grey Pinstripes survey of 2011 the School of Business and Economics was positioned | This table represents studies in study year 2014-2015, it has been partly renewed for years |
| Faculty or school where the education is organized? | Hanken School of Economics
http://www.hanken.fi/en/studies/apply-hanken/apply-masters-degree-programmes/masters-degree-programmes/masters-business-and | Aalto University
http://www.aalto.fi/en/studies/education/programme/management-international_business_master/ | University of Eastern Finland
Administrated through the UEF Law School, organized between two disciplines: environmental law and environmental policy.
http://www.uef.fi/en/envlawandpolicy/home |
| --- | --- | --- | --- |
| Type of education: Study module/Minor subject or Bachelor/Master’s programme? | Master’s programme in Business Management with the specialisation track in International Strategy and Sustainability, 2 years full-time, 120 cr, 10-15 students | Master’s Programme in Management and International Business (M&IB), two years, 120 ECTS | The Master’s Degree Programme in Environmental Policy and Law, multidisciplinary, 2 years, 120 cr MSocSc / Master of International and Comparative Law (MICL) , 20 students |
| Any research group/institute/centre in the background? | - | - | Centre for Climate Change, Energy and Environmental Law (CCEEL) established in 2013. http://www.uef.fi/en/cceel Also connection to the Institute for Natural Resources, Environment and Society (LYY) http://www.uef.fi/en/lyy |
| Core elements | Integrated compulsory studies 30 ECTS: Corporate Sustainability 8 ECTS Academic writing 6 ECTS Strategic Management 8 ECTS Service and Relationship marketing 8 ECTS | In the M&IB Master’s Programme one can specialise in one or two of the following fields: Human Resource Management, International Business, Strategy Work and Sustainability Management. The studies on sustainability management give insight | Courses related to Sustainability and CSR: Sustainability and Natural Resources 5 cp, Corporate Social Responsibility in Natural Resources Management 5 cp Eligible for students holding a Bachelor’s degree in environmental sciences, social-scientific environmental studies, natural sciences, environmental engineering, social |
| **Obligatory core courses in track specialisation** 24 ECTS: Sustainability or International Strategy
**Elective studies**, at Hanken or on an exchange abroad 24 ECTS
Research Seminar part1,6ECTS Research Seminar,part2 6ECTS Master’s Thesis (30 ECTS)
Programme helps the student to identify and manage the challenges related to running an organization that spans multiple economic, social, environmental, geographic, institutional, cultural and linguistic contexts.
Studies during 2015-2016 [https://hanken-weboodi.it.helsinki.fi/hanken/](https://hanken-weboodi.it.helsinki.fi/hanken/) | into responsible management of global businesses.
**Common studies** 54 cr
**Specialisation studies** 30 cr:
Capstone: Management and International Business 6 cr
Alternative mandatory course duo 12 cr includes Creative sustainability option: Sustainable Business and Consumption 6 cr + one other from following: How to Change the World: Innovation toward Sustainability 6 cr Corporate Responsibility in Global Economy 6 cr Sustainability Politics and CSR – Reading Seminar 6 cr Sustainable Business and Consumption 6 cr Responsibility Management, book exam 6 cr
**Elective studies** 12 cr
**Minor studies** 24 cr
**Other electives** 12 cr [http://study-guides.aalto.fi/biz/2014-13/en/master-of-science-degree-studies/study-programmes/management-and-international-business.html](http://study-guides.aalto.fi/biz/2014-13/en/master-of-science-degree-studies/study-programmes/management-and-international-business.html) | sciences or law. Programme combines group teaching and independent research with the objective of providing participants with professional skills in environmental law and policy. The programme consists of advanced study of law and policy in relation to the environment, climate change and natural resources.
**Two alternative majors:** Natural Resources Governance or Environmental and Climate Change Law. [http://www.uelf.fi/en/web/envlawandpolicy/structure-of-the-programme-2014-2016](http://www.uelf.fi/en/web/envlawandpolicy/structure-of-the-programme-2014-2016) |
| **The role of multidisciplinarity in teaching?** | Multidisciplinarity is integrated into some Corporate Sustainability courses | Encourages students to cross disciplinary boundaries and promotes interdisciplinarity | Multidisciplinary programme |
| **Additional information** | Students applying for the specialisation track International Strategy and Sustainability within the Master’s Degree Programme in Business and Management have the option to apply for a Double Degree with EM Lyon Business School. | - | - |
## Appendix 2. The international case examples of major and one minor studies on sustainable development in Sweden and Norway
| Theme | Lund University | Stockholm University, Stockholm Resilience Centre | Uppsala University & the Swedish Univ. of Agricultural Sciences | University of Oslo |
|----------------------------------------------------------------------|---------------------------------------------------------------------------------|---------------------------------------------------|------------------------------------------------------------------|-------------------|
| **Under which faculty or school is the education organized?** | Lund University, Faculty of Social Sciences, teaching is situated within the Lund University Centre for Sustainability Studies, LUCSUS [http://www.lumes.lu.se/home](http://www.lumes.lu.se/home) | BIG – Department of Biology Education, Stockholm University [http://www.big.su.se/_html/utbildningar/master/index_master_egg.shtml#serds](http://www.big.su.se/_html/utbildningar/master/index_master_egg.shtml#serds) | The master programme is hosted by the Department of Earth Sciences at Uppsala University in collaboration with the Swedish University of Agricultural Sciences, SLU [http://www.geo.uu.se/education/programmes/programmepage?pKod=THU2M&lsar=15/16](http://www.geo.uu.se/education/programmes/programmepage?pKod=THU2M&lsar=15/16) | Teaching is given by Centre for Development and the Environment (SUM) [http://www.sum.uio.no/english/studies/programmes/ces-master/](http://www.sum.uio.no/english/studies/programmes/ces-master/) |
| **Type of education: Study module/Minor subject or Bachelor/ Master’s programme?** | International Master’s Programme in Environmental Studies and Sustainability Science (LUMES), 40 students, two years, 120 ECTS, MSc
The programme began in 1997 and was then one of the first interdisciplinary programs of its kind in Sweden, graduation rate 90%. | Master’s Programme Social-Ecological Resilience for Sustainable Development (SERSD), 2 years, 120 ECTS, 15 students | International Master’s Programme in Sustainable Development
Master of Science, two years, 120 cr (one year option 60 cr), 60 students.
Smaller units by CEMUS (7,5-30 credits) in undergraduate and master’s level | Master’s Programme Development, Environment and Cultural Change (DEC) (formerly Culture, Environment and Sustainability CES), MA in Philosophy, 2 years, 20 students, 120 credits: 30 cr compulsory courses, 30 cr electives, 60 cr thesis |
| **Does there exist a research institute or centre in the background?** | the Lund University Centre for Sustainability Studies, LUCSUS ([www.lucusus.lu.se](http://www.lucusus.lu.se))
LUCSUS is a multidisciplinary centre that works together with all seven faculties at the university and is a platform for education, research and cooperation with the society outside the academia, in questions related to environment and sustainable development. | Stockholm Resilience Centre
Courses are firmly embedded in the research at the centre. [http://www.stockholmresilience.org/21/education.html](http://www.stockholmresilience.org/21/education.html) | Uppsala Centre for Sustainable Development (CSD Uppsala), an interdisciplinary centre, consists of CEMUS, the Baltic University Programme and Uppsala Water Centre [http://www.csduppsala.uu.se/](http://www.csduppsala.uu.se/)
CEMUS, Center for Environment and Development Studies, began as an initiative by stu | Centre for Development and the Environment (SUM), an interdisciplinary research centre at UiO [http://www.sum.uio.no/english/](http://www.sum.uio.no/english/) |
| Core elements | The first term: 3 compulsory full-time courses:
Earth System Science 10 cr
Social Theory & Sustainability 10cr
Sustainability Science, 10 cr
The second term: 4 compulsory full-time courses:
Governance of Sustainability 7.5 cr
Urban and Rural Systems and Sustainability 10 cr
Economy and Sustainability 7.5 cr
Knowledge to Action 5 cr
The third term elective courses totalling 30 credits, the students choose two 7.5 credits courses from each block, courses offered are closely connected to the research conducted by the staff at LUCSUS and may vary yearly:
Block 1:
Water and Sustainability
Health Systems, Community Resilience and Sustainability
Gender and Sustainability in Theory and Everyday Life
Societal Resilience
Block 2:
Social Movements and Sustainability
Energy and Sustainability
Capacity Development | First year:
Social-Ecological Systems: Challenges & Approaches 15 ECTS
Systems Theory and Resilience Thinking 15 ECTS
Governance and Management of Social-Ecological Systems 15 ECTS
Resilience Reflections and Applications 7.5 ECTS
Optional courses or a traineeship 7.5 ECTS
Second year:
Master’s thesis 60 ECTS.
Trains students to conduct problem-driven transdisciplinary environmental research.
The 4 mandatory courses are designed with an emphasis on resilience - the ability to deal with change and continue to develop. Programme aims to enhance students’ knowledge of the complex interactions between ecological and social systems.
Theses are mainly incorporated in on-going research projects and are all related to one of the research themes at the Stockholm Resilience Centre. Graduates are encouraged to publish their research in peer-reviewed journals. | Semester 1:
Our Natural Resources 10 cr
Sustainable Development – Worldviews and Visions – Seminar Series 5 cr
Introduction to Interdisciplinary Science 5 cr
Man, society and the environment 10 cr (in Swedish university of agricultural sciences SLU)
Semester 2:
Environmental Assessment 5 cr
Sustainable Development – Worldviews and Discourses – a Seminar Series 5 cr
Systems analysis for sustainable development 5 cr (at SLU)
Interdisciplinary Practice 15 cr (at SLU)
Degree Project D in Sustainable Development 15 cr for those who wish to do a one year masters
Semester 3 variety of options:
Advanced courses in special fields 15-30 cr
Courses at lower level outside your special field, 15-30 cr
Practical experience for sustainable development 15-30 cr
Semester 4:
Degree Project E in Sustainable Development 30 credits | Compulsory courses:
Research Methods and Project Design 15 cr
Key Issues in Development and Environment 15 cr
Electives:
Consumption, Sustainability and Social Change 10 cr
What Works? Success Stories in International Development 10cr
Food, Health and Sustainability 10 cr
Emerging Economies and Challenges to Sustainability 10cr
Global Governance for Sustainable Development 10 cr
Master’s Thesis 60 cr
Students will get advanced knowledge within the interdisciplinary academic field of sustainable development, with a particularly thorough knowledge of political, anthropological, socio-economic and cultural-historical perspectives. Programme takes up a number of important topics related to sustainability, including ethics, culture, development theory, poverty, business and consumption. |
| **Fourth semester** | Master’s thesis 30 credits
http://www.lumes.lu.se/about/programme-outline |
|---|---|
| **Special features** | The Lund University offers MOOCs that are open to anyone with an interest, the courses are free of charge and are taught in English. Attendants receive a certificate upon completion of a course. However, MOOC course cannot be included as a part of a degree or receive university credits for it. The International Institute for Industrial Environmental Economics (IIIEE) at Lund University offers MOOC: Greening the Economy: Lessons and Experiences from Scandinavia (starting 19.1.2015) https://www.coursera.org/course/greeningtheconomy |
| | Stockholm Resilience Centre has offered fall 2014 MOOC "Planetary Boundaries and Human Opportunities: The Quest for Safe and Just Development on a Resilient Planet" together with the Sustainable Development Solutions Network (SDSN). https://www.sds-nedu.org/learn/planetary-boundaries-and-human-opportunities-fall-2014 |
| | Active and versatile student-led teaching by CEMUS |
| | - |
| **The role of multidisciplinarity in teaching?** | LUMES emphasizes knowledge integration, strives to make international and national connections. The programme is based on systems analysis as a unifying and common means of communication between disciplines. The programme uses critical and system thinking approaches, rather than focusing expertise in particular methods or tools. Attention is also placed on students’ understanding of both natural and social dynamics of complex sustainability challenges, the theoretical underpin- |
| | SRC wants to attract top international and Swedish students and ensure that they are embedded in the research life at the Centre. Integrating students, post docs and senior researchers is important as this provides professional role models, develops students’ practical research skills and builds important social networks. |
| | Aim for a highly interdisciplinary approach, where classes are focused around problems and solutions rather than classical subject areas. The core idea is that environmental issues are best discussed with a multidisciplinary approach. |
| | An important objective of the program is to introduce students to the complexities of interdisciplinary research. |
| **Core elements** | Strategic Sustainable Development 12,5 ECTS
Innovation for Sustainability 5 ECTS
Research Methodology for Sustainability 5 ECTS
Strategic Management for Sustainability 7,5 ECTS
Leadership in Complexity 10 ECTS
The Master’s Thesis in Strategic Leadership towards Sustainability 20 ECTS
Focus on Complexity, a systems thinking approach, leadership in complexity, diversity and cross-system connection.
Programme starts with assessing and understanding society’s current sustainability challenges. Curriculum then introduces complementary skills in strategic management, organizational learning and change, economics, engineering, and effective collaboration.
A Spiral learning approach, beginning with an overview of core concepts and followed by application of the concepts later on through a self-directed learning approach. The programme is structured to provide leadership opportunities as year progresses. Periods 3 and 4 are devoted to a major project, where students work in small groups and produce a final thesis.
The programme is delivered in a non-traditional educational setting with experiential and holistic learning methods.
A couple of field trips include the programme and they are mandatory. Their aim is for students to learn about applications of SD. | An interdisciplinary course with large project task done in groups, includes field work in nearby region, the course proceeds through seven phases that are:
1. Planning phase
2. Mapping phase
3. Modeling phase
4. Scenario identification and formulation phase
5. Indicator identification and formulation phase
6. Evaluation phase
7. Report and presentation phase |
| **Special features** | The Framework for Strategic Sustainable Development FSSD (also known The Natural Step Framework) and its application is introduced to build a shared and comprehensive understanding of sustainability. | This is description of one separate course, other instruction related to Sustainable Development in Gothenburg and Chalmers is not explored in this table. |
| **The role of interdisciplinarity in teaching?** | The programme is founded on the basic premise that a “whole-system”, transdisciplinary approach is needed to deal with the sustainability challenge of meeting our society’s needs today and into the future.
2 integrated themes; strategic sustainable development (SSD) and organizational learning and leadership. From these principles, one can “backcast” to the present and begin to take action. | Interdisciplinarity is part of the project course as participants represent different disciplines |
| **Additional information** | - | Some former years project reports are found through GMV’s web pages: [http://gmv.gu.se/samverkan/samverkan-inom-utbildning/fallstudiekurs-i-hallbar-utveckling](http://gmv.gu.se/samverkan/samverkan-inom-utbildning/fallstudiekurs-i-hallbar-utveckling)
The library of Gothenburg University has a study guide related to Education for Sustainable Development issues: [http://libguides.ub.gu.se/ESD](http://libguides.ub.gu.se/ESD) |
## Appendix 3. The international case examples of minor and major studies on sustainable development in United Kingdom, Germany, the Netherlands and one Joint programme.
| Question | Leuphana University of Lüneburg | University of Exeter | Joint Master of 6 universities | Leiden University |
|-------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------|
| **Under which faculty or school is the education organized?** | Faculty of Sustainability Science [http://www.leuphana.de/en/graduate-school/master/course-offerings/sustainability-science.html](http://www.leuphana.de/en/graduate-school/master/course-offerings/sustainability-science.html) | College of Life and Environmental Sciences, Disciplines: Geography, Development & Social Policy, Sustainable Management, Policy and Governance [http://www.exeter.ac.uk/postgraduate/taught/geography/sustdev/](http://www.exeter.ac.uk/postgraduate/taught/geography/sustdev/) | Universities of Graz, Leipzig, Utrecht, Basel and Hiroshima and Ca’ Foscari University of Venice; additional mobility partners University of Stellenbosch (South-Africa) and TERI University in New Delhi [http://www.jointdegree.eu/sd/](http://www.jointdegree.eu/sd/) | Faculty of Science, Minor taught by CML but contributions from many others at Leiden University. [http://www.cml.leiden.edu/education/minor/minor-overview.html](http://www.cml.leiden.edu/education/minor/minor-overview.html) [https://www.facebook.com/minduurzameontwikkeling](https://www.facebook.com/minduurzameontwikkeling) |
| **Type of education: Study module/Minor subject or Bachelor/ Master’s programme?** | Master’s in Sustainability Sciences (MSc)
120 ECTS, 4 semesters (two years), 48 students | MSc Sustainable Development,
1 year full time or 2 years part time, 180 credits. When converted, 180 credits is equivalent to 90 ECTS and that is completed in 12 months. [http://admin.exeter.ac.uk/academic/its/tqa/Part%2013/13CQF.pdf](http://admin.exeter.ac.uk/academic/its/tqa/Part%2013/13CQF.pdf) | International Joint Master’s Programme in Sustainable Development, 120 ECTS, two years, MSc joint or multiple degree, number of students vary annually | Minor in Sustainable Development (30 EC) during Bachelor programme, half year full-time intensive programme (usually 3 days per week 9.30-16.30), started fall 2014 for the 6th time: 35 students, 19 different major degree studies represented), taught in English |
| **Does there exist a research institute or centre in the background?** | Part of teaching is given by the Centre for Sustainability Management (CSM) [http://www.leuphana.de/en/institutes/csm/teaching.html](http://www.leuphana.de/en/institutes/csm/teaching.html) | Programme has a close relationship with the Environment and Sustainability Institute (ESI) [http://www.exeter.ac.uk/esi/](http://www.exeter.ac.uk/esi/) | Institute of Systems Sciences, Innovation and Sustainability Research (ISIS) in University of Graz holds the academic co-ordination and offers teaching in several modules [http://isis.uni-graz.at/en/institute/](http://isis.uni-graz.at/en/institute/) | The Institute of Environmental Sciences (CML), bringing together students from different disciplinary backgrounds is a key feature of CML’s educational philosophy. [https://www.universiteitleiden.nl/en/science/environmental-sciences](https://www.universiteitleiden.nl/en/science/environmental-sciences) |
| Core elements | Interdisciplinary and transdisciplinary, research-oriented, and problem-focused. Optional Mandatory Modules 1st semester
**In-depth Perspectives of Natural Sciences**
Ecosystem Responses to Chemical Pollution and Biogeochemical Processes
Earth Systems and Climate Change
Geochemical Aspects of Sources and Sinks of Compounds in the Environment
**In-depth Perspectives of Human Sciences**
Sustainability Communication
Sustainability Management
Sustainability Governance 2nd semester
**In-depth Perspectives of Natural Sciences**
Conservation Biology
Progress made in Ecosystem and Biodiversity Research
Environmental Analytics
Practical Exercise in Environmental Analytics
**In-depth Perspectives of Human Sciences**
Theories and Perspectives of Sustainability Communication
Sustainability Performance Measurement, Management and Communication
Sustainability Economics
Sustainability, Digital Media, and the Information Society
Sustainability, Governance, and Law 3rd semester
**In-depth Perspectives of Natural Sciences** | **Compulsory modules 135 cr.**
Key Skills 15 cr
Dissertation 90 cr
Understanding Environmental Change 15 cr
Governing Sustainability 15 cr
**Optional modules (45cr)**
15cr each:
Statistical Modelling
Environmental Sustainability in Practice
Independent Study or Independent Work-based learning
Introduction to Energy Policy and Sustainability
Climate, Hazards and Risk Assessment
Themes in Climate Change Assessment is through a range of methods including: essays; oral, written and web presentations; project work; learning journals; and a 10,000 word dissertation. [http://geography.exeter.ac.uk/current-students/postgraduate/modules_cornwall/](http://geography.exeter.ac.uk/current-students/postgraduate/modules_cornwall/) Specification of intended learning outcomes (ILOs) | Compulsory part consists of following tracks:
Basics in Sustainable Development Integration Module
The programme consists of two general modules (1st and 3rd semester) which follow the same structure at all consortium universities, and of specialisation tracks (2nd semester). Academic year 2014-2015 the consortium offers 12 specialisation tracks:
Energy and Materials
Global Change and Ecosystems
Environmental Governance
Sustainable Business Management
Sustainable Urban and Regional Development
Global Environmental Change
Environmental Technology Management
Resources Management
Sustainability: The Social Dimension
Sustainable Development Science and Technology
Sustainable Development Planning | Interdisciplinary, combines academic and practical skills
Consists of 3 courses that are closely coordinated (they are not a combination of existing courses):
Sustainable Development: Big Issues New Answers 15 EC
Project group: Design of European Research 10 EC
Area study: one of the two options:
Waste not Want not: A Future without Waste 7 EC (year 2016 there will be instead an Area Study ‘2030: sustainable Leiden?’)
International course on water issues and water management in the Philippines 10 EC (field course)
[http://www.cml.leiden.edu/education/minor/minor_overview.html#courses-and-schedules](http://www.cml.leiden.edu/education/minor/minor_overview.html#courses-and-schedules) |
| **Additional information** | Centre for Sustainability Management (CSM) at Leuphana University Lüneburg offers a distance learning MBA in Sustainability Management, the first Green MBA worldwide, started 2003.
http://www.leuphana.de/en/institutes/csm/about-the-csm.html
Master’s Program in Global Sustainability Science is a dual degree study programme with Arizona State University in Phoenix/USA
http://www.leuphana.de/graduateschool/master/studienangebot/global-sustainability-science.html | Offers also: MSc Sustainable Development (Climate Change and Risk Management), it has a different emphasis of compulsory modules focusing more on the science of climate change and its influence on climate, environment and energy policy.
http://geography.exeter.ac.uk/postgraduate/taught/sustdevccrm/ | The contents of the studies can be found through:
http://www.jointdegree.eu/sd/index.php?option=com_content&view=article&id=23:outlineprogramme&catid=37:catprogramme&Itemid=11 | CML organizes also MSc Industrial ecology concerning tools and practices for sustainable production and consumption; MSc started in September 2004 and is organised together with Delft Technical University.
http://www.cml.leiden.edu/education/master/industrial/about-master-ie.html |
## Appendix 4. The International case examples of minor and major studies in CSR/responsible business in Denmark, the Netherlands and the United Kingdom.
| Question | Copenhagen Business School | Erasmus University in Rotterdam | Royal Holloway, University of London | Nottingham University Business School |
|--------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| **Under which faculty or school is the education organized?** | Centre for Corporate Social Responsibility [http://www.cbs.dk/en/research/departments-and-centres/department-of-intercultural-communication-and-management/centre-corporate-social-responsibility](http://www.cbs.dk/en/research/departments-and-centres/department-of-intercultural-communication-and-management/centre-corporate-social-responsibility) | Department of Business- Society Management [http://www.rsm.nl/master/msc-programmes/msc-global-business-stakeholder-management/](http://www.rsm.nl/master/msc-programmes/msc-global-business-stakeholder-management/) | Organized jointly by the School of Management and the Department of Geography [https://www.royalholloway.ac.uk/Management/Cris/teaching.html](https://www.royalholloway.ac.uk/Management/Cris/teaching.html) | Administered as part of Nottingham University Business School, teaching by ICCSR and Haydn Green Institute for Innovation and Entrepreneurship [http://www.nottingham.ac.uk/business/ICCSR/teaching.php?c=12](http://www.nottingham.ac.uk/business/ICCSR/teaching.php?c=12) |
| **Type of education: Study module/Minor subject or Bachelor/Master’s programme?** | The Minor Sustainable Business 22.5 ECTS, free to all students | The RSM MSc in Global Business & Stakeholder Management (GBSM), one year, 60 credits (ECTS), 38 students | The MSc Sustainability and Management, one year full time | The MSc in CSR 180 cr, 12 months full time, intake max 20 students |
| **Does there exist a research institute or centre in the background?** | Centre for Corporate Social Responsibility (cbsCSR) Centre is actively involved in the teaching and supervision of numerous CSR-related elective courses and minors offered at CBS at all educational levels | Erasmus Research Institute of Management (ERIM) is in the background as RSM teaching faculty are all active researchers at ERIM, but it does not organize the Master [http://www.erim.eur.nl/](http://www.erim.eur.nl/) | The Centre for Research into Sustainability CRIS [https://www.royalholloway.ac.uk/Management/Cris/https://www.royalholloway.ac.uk/Management/Cris/documents/CRIS-Annual-Report-2013-14.pdf](https://www.royalholloway.ac.uk/Management/Cris/https://www.royalholloway.ac.uk/Management/Cris/documents/CRIS-Annual-Report-2013-14.pdf) | International Centre for Corporate Social Responsibility (ICCSR), founded 2002, [http://www.nottingham.ac.uk/business/ICCSR/index.php](http://www.nottingham.ac.uk/business/ICCSR/index.php) |
| **Core elements** | The minor consists of three 7.5 ECTS elective courses: Corporate Social Responsibility in Global Supply Chains Sustainable Business Strategy and Innovation Creating markets for sustainable products [https://e-campus.dk/studium/stud-](https://e-campus.dk/studium/stud-) | Mandatory courses (22 ECTS): Sustainability Leadership & Planetary boundaries Global Business Strategies Personal Narrative, Communication and Career Corporate Integrity Sustainability Grand Challenges Boot Camp Elective courses (18 ECTS): Managing NGO’s | Core Taught Courses Sustainability and Governance (20 cr) Sustainability & Society 20cr Corporate Social Responsibility: Conceptual Foundations (20 cr) Corporate Social Responsibility: Applications and Tools (20 cr) Core Research Courses Community Volunteer Project (10 cr) Research Training (10cr) | Semester 1 Introduction to Research Methods Strategies for Responsible Business Business Ethics One from: Further Quantitative Research Methods Further Qualitative Research Methods Semester 2 Corporations, Power and Society |
| **Minors in CBS consist normally of 3 courses (22.5 ECTS) concluded with a certificate to be added to the Masters diploma.** | Corporations & Justice Climate Change Strategy Role-play which is a joint elective with MSc International Management/CEMS; GBSM Research Project Companies in Ecologies Cradle to Cradle Thesis 20 ECTS, includes Research Methods (4 ECTS) Programme focuses on real-world business relevance. Students take a practical and critical approach to sustainability issues and examine and understand the strategies of companies, governments and non-governmental organisations. [http://www.rsm.nl/master/msc-programmes/msc-global-business-sustainability/curriculum/](http://www.rsm.nl/master/msc-programmes/msc-global-business-sustainability/curriculum/) | Dissertation (60 credits) Elective units (choose 1 from the following or another 20 cr course): Practices of Sustainability in Developing Areas 20 cr Corporate Governance 20cr Global Business Strategy20cr [https://www.royalholloway.ac.uk/management/coursefinder/mscsustainabilityandmanagement.aspx](https://www.royalholloway.ac.uk/management/coursefinder/mscsustainabilityandmanagement.aspx) The programme is characterised by blending together subject matter from the sciences, (notably environmental sustainability) and the more value driven corporate social responsibility. | Globalization, Business and Development Two from: Corporate Strategy Evaluating Sustainability Innovation and Policy Supply Chain and Operations Strategy and Practice Tourism and Sustainability Summer 12,000-15,000 word Corporate Social Responsibility dissertation [http://www.nottingham.ac.uk/business/programmes/msc/courses/corporate-social-responsibility.aspx](http://www.nottingham.ac.uk/business/programmes/msc/courses/corporate-social-responsibility.aspx) |
| --- | --- | --- | --- |
| **The role of multidisciplinarity in teaching?** | - | - | The programme gives an interdisciplinary education and it enables students to understand connectivities beyond the borders of just one discipline |
| **Additional information** | There are many electives in CBS related with issues of sustainability and CSR and students can incorporate them into their degree studies. | - | Teaching in CSR started with MBA in CSR in 2003 and with MA in 2004. The MA programme was reclassified as an MSc programme in 2010. In 2015, MSc offering was revamped with the launch of the MSc in Sustainability. ICCSR offers now 3 programmes: MBA in CSR, MSc in CSR and MSc in Sustainability. |
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SUSTAINABILITY STUDIES IN UNIVERSITIES
– Review on Study Modules of Sustainable Development and Responsible Business in Finnish and Some European Universities
ISBN 978-952-249-411-5 (Second edition)
ISSN 1797-1322
|
STUDIES OF THE DROSOPHILA BRAIN USING P[GAL4] ENHANCER TRAP LINES
A Thesis Submitted for the Degree of Doctor of Philosophy at the University of Glasgow
by
Ming Yao Yang
Division of Molecular Genetics, University of Glasgow
Glasgow G11 5JS, Scotland, UK.
January 1996
All rights reserved
INFORMATION TO ALL USERS
The quality of this reproduction is dependent upon the quality of the copy submitted.
In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion.
Published by ProQuest LLC (2019). Copyright of the Dissertation is held by the Author.
All rights reserved.
This work is protected against unauthorized copying under Title 17, United States Code
Microform Edition © ProQuest LLC.
ProQuest LLC,
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This
10463
Cv21
The research reported in this thesis is my own original work, except where otherwise stated, and has not been submitted for any other degree.
Ming Yao Yang
This thesis is dedicated to my wife and my daughter.
# CONTENTS
| Contents | Page |
|-------------------------------------------------------------------------|------|
| List of contents | i |
| List of figures and tables | vii |
| Abbreviations | ix |
| Acknowledgements | x |
| Summary | xi |
## Chapter 1 Introduction
1.1 The P element as a tool for the study of *Drosophila* genetics ........... 2
- 1.1.1 The P-element ................................................................................. 2
- 1.1.2 Germ-line transformation .......................................................... 5
- 1.1.3 Controlled mutagenesis .................................................................... 6
- 1.1.4 Other applications .......................................................................... 8
1.2 The enhancer trap approach .................................................................. 9
- 1.2.1 what is an enhancer ? ..................................................................... 9
- 1.2.2 An enhancer trap element .............................................................. 9
- 1.2.3 β-gal expression in the nervous system ....................................... 12
- 1.2.4 Staining patterns represent the expression patterns
of neighbouring genes ............................................................................. 13
- 1.2.5 "Second Generation" enhancer trap system .................................. 16
1.3 The structures of the *Drosophila* brain ........................................ 18
- 1.3.1 The mushroom body ........................................................................ 19
- 1.3.2 The central complex ....................................................................... 22
- 1.3.3 The antennal lobe .......................................................................... 23
- 1.3.4 The optic lobe .............................................................................. 25
1.4 Aim of the project ............................................................................... 26
## Chapter 2 Materials and Methods
| Section | Page |
|------------------------------------------------------------------------|------|
| Basic materials | 29 |
| Drosophila Strains | 29 |
| Bacterial strains, plasmids and phage vectors | 29 |
| Culture media for *E.coli* and *Drosophila* | 30 |
| Enzymes | 30 |
| Chemicals and Reagents | 30 |
| buffers and solutions | 31 |
| Microscopy and films | 32 |
| Histochemistry and Immunohistochemistry | 32 |
| X-gal staining of frozen cryostat sections | 32 |
| Embedding of adult fly heads | 32 |
| Embedding of whole fly | 33 |
| Sectioning and staining | 33 |
| X-gal staining of embryo, larval and adults brains | 34 |
| X-gal staining of embryo | 34 |
| X-gal staining of larval, pupal and adults brains | 34 |
| Immunohistochemistry and confocal microscopy | 35 |
| Anti-β-gal antibody staining of adult brains | 35 |
| Confocal microscopy | 35 |
| In situ hybridisation | 35 |
| in situ hybridisation to tissue sections | 35 |
| in situ hybridisation to whole brains and embryos | 37 |
| in situ hybridisation to polytene chromosomes | 37 |
| General methods for molecular biology | 38 |
| Isolation of Genomic DNA | 39 |
| Isolation of bacteriophage DNA | 39 |
| Host cell preparation | 39 |
| Isolation of phage DNA | 40 |
| Section | Title | Page |
|---------|----------------------------------------------------------------------|------|
| 2.4.3 | Isolation of plasmid DNA | 40 |
| 22.214.171.124 | Large scale plasmid preparation | 40 |
| 126.96.36.199 | Small scale plasmid preparation | 41 |
| 2.4.4 | Transformation of *E. coli* | 41 |
| 188.8.131.52 | Electrotroptransformation | 41 |
| 184.108.40.206 | Chemical transformation | 42 |
| 2.4.5 | Growth of *E. coli* | 43 |
| 2.4.6 | Plasmid rescue techniques | 43 |
| 220.127.116.11 | Isolation of genomic DNA from flies | 43 |
| 18.104.22.168 | Digestion of genomic DNA | 43 |
| 22.214.171.124 | Ligation of DNA fragments | 44 |
| 126.96.36.199 | Transformation of *E. coli* | 44 |
| 188.8.131.52 | Isolation of plasmid DNA | 44 |
| 2.4.7 | Isolation of RNA | 44 |
| 184.108.40.206 | Isolation of total RNA | 42 |
| 220.127.116.11 | Isolation of poly (A)⁺ mRNA | 45 |
| 2.4.8 | Quantification of nucleic acids | 46 |
| 2.4.9 | Labelling of nucleic acids | 46 |
| 18.104.22.168 | First stand cDNA probe | 46 |
| 22.214.171.124 | Random primed DNA labelling with $^{32}$P | 47 |
| 126.96.36.199 | Nick translated Biotin probe | 47 |
| 188.8.131.52 | DIG RNA labelling probe | 48 |
| 2.4.10 | Electrophoreses and blotting | 48 |
| 184.108.40.206| DNA electrophoresis and Southern blots | 48 |
| 220.127.116.11| Reverse Northern | 49 |
| 18.104.22.168| RNA electrophoresis and Northern blots | 49 |
| 2.4.11 | Screening lambda genomic DNA and cDNA libraries | 50 |
| 22.214.171.124| First round screening | 50 |
| 126.96.36.199| Secondary screening | 50 |
| Section | Page |
|------------------------------------------------------------------------|------|
| 2.4.12 DNA sequencing techniques | 51 |
| 188.8.131.52 Preparation of polyacrylamide gel | 51 |
| 184.108.40.206 Preparation of glass plates and pouring the gel | 51 |
| 220.127.116.11 Preparation of DNA sequencing samples | 51 |
| 18.104.22.168 Electrophoresis of sequencing gel | 52 |
| 22.214.171.124 Computer-assisted sequence analysis | 52 |
| **Chapter 3 Screening of P[GAL4] enhancer trap lines** | 53 |
| 3.1 Introduction | 54 |
| 3.2 Generation of new P[GAL4] insertion lines | 56 |
| 3.3 Screening of new P[GAL4] insertion lines | 60 |
| 3.4 Examples of interesting patterns in the brain | 63 |
| 3.4.1 The mushroom body | 63 |
| 3.4.2 The central complex | 67 |
| 3.4.3 The antennal lobe | 67 |
| 3.4.4 The optic lobe and eye | 69 |
| 3.4.5 Other interesting patterns | 72 |
| 3.5 Chromosomal locations of P[GAL4] insertion lines | 72 |
| **Chapter 4 Characterisation of GAL4-directed expression patterns in the central complex** | 80 |
| 4.1 Introduction | 81 |
| 4.2 Specific staining patterns in the ellipsoid bodies | 84 |
| 4.3 Expression patterns in the fan-shaped bodies | 89 |
| 4.4 Expression patterns in the other sub-structures | |
| of the central complex | 91 |
| 4.5 Developmental study in the central complex | 93 |
| Chapter 5 | Plasmid rescue of the flanking genomic DNA | 99 |
|----------|------------------------------------------|----|
| 5.1 | Introduction | 100 |
| 5.2 | GAL4-directed expression patterns of the central complex lines for plasmid rescue | 103 |
| 5.3 | Plasmid rescue of flanking genomic DNA of the central complex | 105 |
| 5.4 | "Reverse Northern" analyses | 112 |
| 5.5 | Isolation of genomic clones corresponding to lines c507 and c161 | 115 |
| Chapter 6 | Cloning of Genes Neighbouring the P[GAL4] Insertion in Lines c507 and c232 | 118 |
|----------|-------------------------------------------------------------------------|----|
| 6.1 | Introduction | 119 |
| 6.2 | Isolation of cDNA clones related to c507 flanking DNA | 119 |
| 6.2.1 | cDNA clones from the "downstream" side of the P[GAL4] element in line c507 | 119 |
| 6.2.2 | Isolation of a full length cDNA clone related to pMY5 | 121 |
| 6.2.3 | cDNA clones from the "upstream" side of the P[GAL4] element in line c507 | 122 |
| 6.2.4 | Genomic Southern for three different groups of cDNA clones | 123 |
| 6.3 | Analysis of sequence | 123 |
| 6.3.1 | General sequence features of the pkMY4 cDNA clone | 125 |
| 6.3.2 | General sequence features of the pMY51 cDNA clone | 127 |
| 6.3.3 | General sequence features of the pMY8 cDNA clone | 133 |
| 6.4 | Northern analyses | 135 |
| 6.4.1 | Northern blotting using head and body mRNA | 135 |
| 6.4.2 | A developmental Northern blot for line c507 | 137 |
| Section | Title | Page |
|---------|----------------------------------------------------------------------|------|
| 6.4.3 | Northern analysis for line c507 and wild type flies | 137 |
| 6.5 | *in situ* hybridisation | 138 |
| 6.5.1 | Localisation of the pMY51, pMY8 and pkMY4 cDNA clones to polytene chromosomes | 138 |
| 6.5.2 | Co-localisation of *LacZ* and alkaline phosphatase gene expression | 140 |
| 6.6 | Conclusion | 142 |
| Chapter 7 | Discussion | 145 |
| 7.1 | Introduction | 146 |
| 7.2 | General discussion | 146 |
| 7.3 | Future work | 151 |
| References | | 153 |
| Appendices | | 171 |
| Appendix 1 | Summary of DNA clones | 171 |
| A 1.1 | Rescued plasmid clones | 171 |
| A 1.2 | Genomic lambda DNA clones | 171 |
| A 1.3 | cDNA clones | 172 |
| Appendix 2 | Publications relevant to this thesis | 173 |
| A 2.1 | Conditional cell ablation in *Drosophila*. | |
| A 2.2 | Reverse genetics of *Drosophila* brain structure and function. | |
| A 2.3 | Subdivision of the *Drosophila* mushroom bodies by enhancer-trap expression patterns | |
| A 2.4 | Functional dissection of the *Drosophila* mushroom bodies by selective feminisation of genetically defined subcompartments. | |
# List of Figures and Tables
## Chapter 1
- Figure 1.1 The structure of the full length 2.9 kb P-element ........................................... 4
- Figure 1.2 A controlled P-element mutagenesis strategy .................................................. 7
- Figure 1.3 Enhancer trap strategies ..................................................................................... 11
- Figure 1.4 Diagram of plasmid rescue technique ............................................................... 15
- Figure 1.5 Schematic diagram of the *Drosophila* brain .................................................... 18
- Figure 1.6 Schematic drawing of an opened head capsule of *Drosophila* ...................... 20
## Chapter 3
- Figure 3.1 Crossing scheme (1) .......................................................................................... 57
- Figure 3.2 Crossing scheme (2) .......................................................................................... 59
- Figure 3.3 Mushroom body structures ............................................................................... 64
- Figure 3.4 X-Gal staining in the mushroom bodies of six P[GAL4] lines ..................... 66
- Figure 3.5 Central complex structures .............................................................................. 68
- Figure 3.6 Antennal complex staining ................................................................................. 70
- Figure 3.7 The optic lobe and eye staining ........................................................................ 71
- Figure 3.8 Other staining patterns ....................................................................................... 73
- Figure 3.9 Polytenic chromosomal *in situ* using a pBluescript probe .......................... 75
- Table 3.1 GAL4 expression patterns in the brain ............................................................... 61
- Table 3.2 Cytological locations of P[GAL4] insertions .................................................... 78
## Chapter 4
- Figure 4.1 The schematic diagram of the *Drosophila* central complex .......................... 82
- Figure 4.2 The staining patterns in the ellipsoid bodies .................................................. 85
- Figure 4.3 GAL4-directed staining patterns in the subdivision of the ellipsoid bodies .... 87
- Figure 4.4 *LacZ* expression in the fan-shaped bodies ..................................................... 90
- Figure 4.5 Expression patterns in the other substructures of the central complex ........ 92
- Figure 4.6 *LacZ* expression in the *eb* line c232 at the different developmental stages .... 94
- Figure 4.7 *LacZ* expression in the *eb* line c561a at the different developmental stages .... 96
- Figure 4.8 *LacZ* expression in the *fb* line c5 at the different developmental stages ........ 97
- Table 4.1 Comparison of the ring structures and ring neurons of the *eb* ....................... 88
Chapter 5
Figure 5.1 GAL4-directed β-gal expression patterns of the central complex lines used for plasmid rescue ................................................................. 104
Figure 5.2 Map of the P[GAL4] construct .................................................. 106
Figure 5.3 Genomic southern analysis of rescued plasmids .......................... 109
Figure 5.4 Genomic southern analysis showing the relationship between lines c105 and c561 ................................................................. 109
Figure 5.5 The relationship between the lines c232 and c507 ...................... 111
Figure 5.6 "Reverse Northern" analysis ..................................................... 114
Figure 5.7 Maps of the λ genomic DNA clones ........................................... 117
Table 5.1 The result of plasmid rescue from seven P[GAL4] enhancer trap lines ................................................................. 108
Chapter 6
Figure 6.1 The organisation of the region surrounding the P[GAL4] element in lines c507 and c232 ................................................................. 120
Figure 6.2 A Northern blot using a insert probe from pMY5 ....................... 122
Figure 6.3 Southern blot of *Drosophila* genomic DNA ............................... 124
Figure 6.4 pkMY4 is a cDNA clone of a calcineurin A1 transcript ................. 126
Figure 6.5 The nucleotide and deduced amino acid sequence of the pMY51 cDNA clone ................................................................. 128
Figure 6.6 Multiple sequences alignment for alkaline phosphatase ............... 130
Figure 6.7 Schematic tree of alkaline phosphatase genes ............................ 132
Figure 6.8 The nucleotide and derived amino acids sequence of the cDNA clone pMY8 ................................................................. 134
Figure 6.9 Northern blots analysis ............................................................. 136
Figure 6.10 Polytene chromosomal *in situ* using different probes from pBluescript, pMY51, pMY8 and pkMY4 cDNA clones ....................... 139
Figure 6.11 Co-localisation of expression patterns in the tissue sections by *in situ* hybridisation ................................................................. 141
| Abbreviation | Description |
|--------------|-------------|
| amp | ampicillin |
| BCIP,X-phosphate | 5-bromo-4-chloro-3-indolyl-phosphate |
| BSA | bovine serum albumin |
| bp | base pairs |
| bis | N, N'-methylenebisacrylamide |
| cDNA | complementary DNA |
| Ci | Curies |
| cpm | counts per minute |
| DAB | diaminobenzidine |
| DEPC | Diethylpyrocarbonate |
| DMSO | dimethylsulphoxide |
| DNase I | deoxyribonuclease I |
| d(N₆) | Random Hexanucleotides |
| dNTP | deoxyribonuleotide triphosphate |
| DTT | dithiothreitol |
| EDTA | ethylenediamine tetra-acetic acid |
| EtBr | ethidium bromide |
| IPTG | isopropyl-β-D-thiogalactopyranoside |
| kb | Kilobases/kilobasepairs |
| LMP | Low melting point |
| MOPS | morpholino propane sulphonic acid |
| mRNA | messenger ribonucleic acid |
| NaPPi | sodium pyrophosphate |
| NTB | 4-nitrobluetetrazoliumchloride |
| OD | optical density |
| PEG | polyethylene glycol |
| pfu | plaque forming units |
| RF | RNase Free |
| RNase A | ribonuclease A |
| rpm | revolutions per minute |
| SDS | sodium dodecylsulphate |
| TEMED | N, N, N' N', -tetramethylenediamine |
| tRNA | transfer RNA |
| μci | microcuries |
| UV | ultraviolet light |
| X-Gal | 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside |
ACKNOWLEDGEMENTS
Many thanks to my supervisor, Kim Kaiser, for his helpful advice during the lab work of this project and extra special thanks for him for critically reading and correcting this thesis.
I am grateful to Andrea Brand and Norbert Perrimon, and Tim Tully who kindly provide us original P[GAL4] strains.
I would like to thank Alan Paterson, Douglas Armstrong, Christian Hehn, Ali Sozen for some help in generating P[GAL4] lines, and Douglas Armstrong for the screening work. Special thanks go to my wife, Zongsheng Wang, for most chromosomal localisations.
Thanks also goes to Kevin O’Dell, Douglas Armstrong and Andy Mounsey for reading and correcting some chapters of the thesis. James Clark has been most generous in giving his time reading and correcting all chapters of this thesis.
Thanks to everyone in the lab, in past and present, for their friendship, enthusiasm, helpfulness and moral support. My appreciation also goes to the prep-room ladies for their excellent work.
Finally, I thank my mum and dad for their encouragement and love.
SUMMARY
This thesis is concerned with the application of enhancer trap technology to illustrate the structures of *Drosophila* brain and identify the genes relevant to the central complex function in the brain.
Over 1400 novel enhancer trap lines bearing P[GAL4] insertions were generated by genetic crosses and they were then screened for GAL4-directed β-gal expression in cryostat sections of the *Drosophila* head. More than 300 lines display interesting patterns in the brain from an anatomical perspective. Of these, as many as 100 are more or less restricted to specific regions or neuronal sub-populations of brain. Particularly exciting are lines that express GAL4 in the mushroom bodies and the central complex, structures that have been implicated in associative learning and memory. For most of lines, the chromosomal locations of P[GAL4] insertions were identified by *in situ* hybridisation to polytene chromosomes.
P[GAL4] expression patterns have suggested multiple roles for certain *Drosophila* brain structures in integration of signals. In the mushroom bodies, the blue staining patterns have revealed axonal processes corresponding to Kenyon cells and showed that the *Drosophila* mushroom bodies are compound neuropils in which parallel sub-components exhibit discrete patterns of gene expression. A strong prediction that different sub-sets of Kenyon cells perform different functional roles is made. In the ellipsoid body of the central complex, four different ring structures are revealed by P[GAL4] expression patterns. Developmental analysis indicates that the *lacZ* expression in the central complex lines begins at early pupal stages to the adults.
The central complex of the *Drosophila* brain has been shown to act as a higher centre for locomotor activity and other behaviours. To identify the genes relevant to central complex function, seven P[GAL4] enhancer trap lines with staining patterns specific to the central complex were selected. Genomic DNAs flanking each insertion site
were cloned by plasmid rescue. Rescued genomic DNAs from some of lines were used as probes for screening a cDNA head library. Corresponding cDNA clones were isolated.
In the case of line c507, P[GAL4] staining is restricted to the ellipsoid body of the central complex of brain and to the Malpighian tubules in the *Drosophila*. Three genes, two located downstream and one upstream of the P[GAL4] element, are identified. Sequencing of a 1.8 kb cDNA clone from pMY51 which is located downstream of the P[GAL4] reveals a protein with significant homology to the alkaline phosphatase gene family in other organisms. The other cDNA clone, closely linked with pMY51, represented by pMY8 is sequenced and a full length predicted amino acid sequence identified. It has head-elevated expression as judged by Northern blot analysis. The gene which is located upstream of the P[GAL4] element is identified as calcineurin A1, the Ca$^{2+}$/calmodulin-stimulated protein phosphatase.
*in situ* hybridisation to tissue sections using cDNA probe generated from a whole insert of pMY51 reveals that the gene has expression patterns in the cell bodies of the ellipsoid body and the Malpighian tubules consistent with the X-Gal staining. Results indicate that the *Drosophila* "alkaline phosphatase" enhancer is trapped and the corresponding gene has been cloned by enhancer trap approach.
Chapter 1
Introduction
1.1 The P element as a tool for the study of *Drosophila* genetics
1.2 The enhancer trap approach
1.3 The structure of brain
1.4 Aims of the project
This chapter provides a background to some of the topics discussed in this thesis. A general introduction to P elements and their applications is presented first. This is followed by a review of the "enhancer trap" system. Then, the structure of the *Drosophila* brain is described. The final section of this chapter describes the initial aims of this project and the strategies used to achieve them.
### 1.1 The P element As A Tool for the Study of *Drosophila* Genetics
#### 1.1.1 The P Element
Transposable genetic elements are segments of DNA with the special ability to jump from place to place on the chromosomes. They are typically found in many copies scattered about the genome, and their position is widely variable between individuals of *Drosophila melanogaster*. Of these, the P element family is the most extensively studied and widely used in the molecular biology of *Drosophila*. The P elements are of particular interest because their transpositional activity is potentially high but under strict genetic control (Engels, 1989).
P elements have been shown to be the causal agents of P-M hybrid dysgenesis, a syndrome whose traits include high rates of sterility, mutation, and chromosomal rearrangements (Engels, 1989; ). P element transposition is genetically regulated, occurring at very high frequency only in the progeny from a cross in which P element containing males (P strains) are mated to females lacking P elements (M strain). No dysgenic traits are observed in the progeny of the reciprocal M male by P female cross or in the progeny from PxP or MxM crosses. Moreover, transposition is restricted to cells of the germ-line. Thus, P element transposition is regulated in two ways: genetically and tissue specifically. The distinguishing characteristic of P strains is that they contain full-length autonomous P elements, encoding their own transposase. P strains also contain defective, generally internally deleted, P elements. Transposition in a P strain is tightly regulated. It is repressed by a product of the full-length P element itself. This condition is known as a "P cytotype". M
strains, in comparison, lack autonomous P elements and are permissive for P element transposition ("M cytotype"). Thus, transposition and hybrid dysgenesis are induced when potentially active P elements are introduced into the permissive cellular environment of M cytotype which is transmitted by the female parent.
Molecular analysis indicated that the complete P element is 2.9 kb in length (Fig. 1.1). Four long open reading frames encode an 87 kD transposase, activity of which is restricted to the germ-line as the result of differential splicing (Rio, 1991). Inverted 31 bp terminal repeats, together with as much as 138 bp and 216 bp at the both ends respectively, are the cis acting determinants of transposition (Mullins et al., 1989). In addition to full-length P elements, most P strains contain a range of internally deleted elements varying in length from 500 to 2500 bp. These P elements are non-autonomous because they are unable to produce functional transposase. But many such elements retain cis-acting determinants that allow their mobilisation in the presence of full-length elements (Engels, 1989). It is noted that an engineered P element with the third intron removed ($\Delta 2$-3), as shown in Fig. 1.1, can produce transposase in both somatic and germ line cells (Laski et al., 1986). It is often used to mobilise internally deleted P elements in *Drosophila* genetics.
When P elements transpose they leave behind a double-stranded break that can be the subject of widening by exonucleases and the repair of which appears to require a template. Usually the template is provided by a sister chromatid, in which case P element sequences are restored at the site. Occasionally it is provided by an homologous chromosome. Where this carries a wild-type allele the impression will be given of precise excision from locus. Remobilisation can also result in imprecise excision, presumably reflecting failure of the repair process (Engels, 1992).
The applications of P elements will be briefly discussed below.
Figure 1.1. Structure of the full length 2.9 kb P-element, internally deleted P-element and Δ 2-3 element. The terminal 31 bp inverted repeat and the direct 8 bp target site duplication are denoted by arrow heads (black and shaded, respectively). Translation initiation and termination codons are shown for the full length P-element and the Δ 2-3 element. (Diagram taken from Sentry and Kaiser, 1993)
1.1.2. Germ-line Transformation
DNA-mediated germline transformation is an indispensable tool for analysing many problems in *Drosophila* molecular genetics. Germ-line transformation of *Drosophila* is achieved by the injection of cloned and suitably manipulated P element DNA (as a component of a bacterial plasmid) into embryos undergoing the transition between nuclear syncitium and cellular blastoderm (Rubin and Spradling, 1982; Spradling and Rubin, 1982).
P element vectors, carrying a marker, are the defective P elements which lack a functional transposase gene but have all of the cis-acting determinants necessary for transposition. Transposase can be supplied by co-injection of a plasmid that encodes it, by co-injection of purified protein, or by injection of Δ2-3 embryos. DNA injected at the posterior pole prior to cellularisation will become incorporated into germ-line precursors, and occasional transposition will occur from the injected plasmid to the *Drosophila* genome. Adults that develop from injected embryos are genetic mosaics with respect to the presence of the transposon in their germ-line. True transformed individuals can be recovered in the next generation, usually the transposon of interest carries a phenotypic marker to allow identification of transformants (Sentry and Kaiser, 1993).
Germ-line transformation experiments have had a major impact on *Drosophila* molecular genetics for obvious reasons. P element vectors can be used to transform flies with cloned genes to rescue a mutant phenotype, to prove that a DNA fragment carries the corresponding gene, and can be used to identify cis-acting regulatory sequences involved in its correct spatial and temporal expression (Bargiello et al., 1984; Bourois and Richards, 1985; Haenlin et al., 1985; Fischer and Maniatis, 1986). In addition, genes manipulated *in vitro* can be reintroduced into the animal and their biological consequences assayed *in vivo*.
1.1.3. Controlled Mutagenesis
In order to control P-element insertional mutagenesis, flies containing two types of defective element are used; "mutator" element, which is defective in both transposase and P-repressor function while retaining intact 31bp P element end and other cis-acting sequences required for transposition, so it can be mobilised when provided with a source of transposase in trans. Appropriate phenotypic markers such as Adh (Goldberg et al., 1983), ry (Rubin and Spradling, 1982) and w (Bier et al., 1989) are included on the mutator element in order to trace their integration. Transposase is supplied by a "jumpstarter" element whose terminal repeat structures have been mutated to eliminate self-mobilisation. In other words, jumpstarter elements produce transposase and can therefore catalyse transposition of mutator elements but not themselves. Both elements can be maintained in stock, in isolation, and brought together in a dysgenic cross. Mobilisation then occurs within a proportion of the germ cells from F₁ individuals. Novel insertions are stabilised in the next generation by segregation of the jumpstarter chromosome from the target chromosomes, facilitated by selection against genetic markers closely linked to the jumpstarter element (Fig. 1.2).
The jumpstarter element, Js-1 (Cooley et al., 1988) can produce low levels of germline restricted transposase. This element, however, has the disadvantage that at low frequencies (1-2%) it will self-mobilise, requiring outcrossing at each generation and occasional confirmation of genomic position. Currently, the most efficient jumpstarter element available appears to be P[ry⁺ Δ2-3] (99B), reported by Robertson et al., (1988) lying at position 99B7-10 on the third chromosome. The Δ2-3 element can cause mobilisation of other elements at unusually high frequencies, yet is itself remarkably stable. It produces very high levels of active transposase.
Depending on the application, multiple or single insert lines can be produced by a combination of different mutator and jumpstarter chromosomes to control the rate and frequency of transposition (Cooley et al., 1988; Robertson et al., 1988).
Figure 1.2. A controlled P-element mutagenesis strategy. An enhancer trap P element (mutator) is mobilised by the Δ 2-3 transposase (jumpstarter) in the germ line cells of F₁ males. Each sperm carries a different spectrum of new insertions. Selection against the transposase source in the F₂ generation ensures that new insertions remain stable.
The generation of lines containing only a single marked mutator element has many advantages as a method of mutagenesis. Phenotypic and molecular analyses of new mutations are greatly simplified. The mutant gene can be mapped by identifying the transposon insertion site using *in situ* hybridisation to polytene chromosomes. DNA flanking the insertion site can be cloned simply by screening a library for P element homology. Revertants, including new alleles generated by imperfect excision, can be recovered by reintroducing a jumpstarter element and scoring for loss of the phenotype specified by the mutator element's marker gene. In addition, single-insert lines have intrinsic long-term value for manipulating the *Drosophila* genome (Cooley, *et al.* 1988; Sentry and Kaiser, 1995).
### 1.1.4. Other Applications
The P element has also been applied to other areas. For example, it can be used to clone genes by transposon tagging (Bingham *et al.*, 1981; Searles *et al.*, 1982), for precise and imprecise excision (Tsubota and Schedl, 1986; Salz *et al.*, 1987) and local jumping (Tower *et al.*, 1993; Zhang and Spradling, 1993). It can be also used to create double-strand DNA breaks, in experiments where the flanking DNA is replaced with modified sequences (Gloor *et al.*, 1991).
"Site-selected" P element mutagenesis is a PCR-based screen for P-element insertion events. It allows the detection and isolation of a P element transposon into or near a cloned gene of interest (Ballinger and Benzer, 1989; Kaiser and Goodwin, 1990; Sentry and Kaiser, 1994). The corresponding mutants can be used to analyse the function of the gene (S. Goodwin, per com.).
P elements can be also engineered with reporter genes and used to identify the timing and tissue distribution of the expression of genes that happen to lie near the insertion site (O'Kane and Gehring 1987). For this point, I will review in more detail in the next section.
1.2 The Enhancer Trap Approach
1.2.1. What Is An Enhancer?
Enhancers are regulatory sequences which stimulate transcription from eukaryotic promoters with which they are associated. They are probably the major mechanism for regulating gene expression in eukaryotes. They are activated by the binding of a specific protein and then act as sites for the assembly of transcriptional initiation complexes. They differ from most regulatory sequences due to the following reasons: (1) They may be located either upstream or downstream of the gene and several kilobases away from the gene whose transcription they control. (2) They act in either orientation and so can simultaneously influence the expression of two genes, one on each side of the enhancer sequence. (3) They must be located on the same molecule of DNA as the regulated gene, but the sequence can be on either DNA strand. (4) Enhancers are not gene-specific but they are tissue-specific. Preferentially enhancers stimulate transcription from the nearest promoter (Smith-Keary, 1991).
1.2.2. An Enhancer Trap Element
In *Drosophila*, the "enhancer trap" method was initially described by O'Kane and Gehring (1987) and has since been modified in various ways (Bellen *et al.*, 1989; Bire *et al.*, 1989; Brand and Perrimon 1993). The major advantage of the enhancer trap is that instead of identifying genes by means of the phenotype caused by a mutation, they are identified by their pattern of expression. The underlying assumption is that a developmentally important gene will show a specific temporal and spatial expression pattern related to its function. This notion is supported by many examples (see review, Freeman, 1991).
Enhancer trap constructs are defective P-elements within boundaries of intact 31 bp terminal repeats and other essential *cis*-acting sequences. Such constructs can be introduced
into the *Drosophila* genome by coinjection with a helper element into embryos, as in the germline transformation technique mentioned as section 1.1.2. The reporter gene in the enhancer trap is a translational fusion that brings the *E. coli* β-galactosidase gene under the control of a weak promoter (usually the P-transposase promoter). The coding sequence of the β-galactosidase gene is fused in frame to a sequence in the second exon of the P transposase gene. Following chromosomal integration of the P element, the transposase promoter may be influenced by nearby genomic enhancers, leading to developmental regulation of β-galactosidase expression (Fig. 1.3a). Of the first generation enhancer trap element, report gene is *lacZ*. In addition, the element carries a dominant eye colour gene such as *w*⁺ or *ry*⁺ to identify flies that contain it, and plasmid sequences that facilitate the cloning of flanking genomic DNA.
The presence of β-galactosidase expression pattern can be easily visualised *in situ* by staining whole-mount embryos, larvae or adult flies with a chromogenic substrate for the enzyme. Generally, staining is localised to cell nuclei, by virtue of the nuclear localisation signal present on the N-terminal fraction of the P-transposase gene to which the reporter is fused (Grossniklaus et al 1989). Although nuclear staining allows visualisation of the location of the neuronal cell-body, cytoplasmic localisation is favourable for the analyse of cells with extensive processes, such as axons and dendrites of neurons. Some efforts have been made to address this problem (Smith and O’Kane, 1991; Giniger *et al.*, 1993; Callahan and Thomas, 1994).
Since P element transposition is, to a first approximation, a random process, lines in which enhancer trap elements have become integrated at new locations in the genome can be generated easily by following a P-element mutagenesis strategy described as section 1.1.3. For example, the *lacZ* reporter gene is used as “mutator” element and P\[ry⁺ Δ2-3\] as the “jumpstarter” element, the number of new enhancer trap lines can be produced by genetic crosses instead of by embryo microinjection.
Figure 1.3 Enhancer trap strategies
a). First generation enhancer trap element. After integration into the genome, the reporter may be influenced by nearby genomic enhancers, leading to developmental regulation of *lacZ* expression. Enhancer trap elements have a dominant eye colour gene (w+) which allows flies with insertions to be recognised. The ampicillin resistance (*ampR*) determinant and *E.coli* origin of replication (*ori*) facilitate plasmid rescue of flanking sequences.
b). A GAL4 enhancer trap element. As above, except that GAL4 expression is influenced by a nearby enhancer. GAL4 activates transcription from a second reporter gene linked to the GAL4-responsive promoter, UAS<sub>G</sub>. (Diagram slightly modified from Sentry et al., 1994)
The insertion of an element may or may not be mutagenic itself (approximately 10% of enhancer trap lines contain insertions that are mutagenic themselves), depending upon its precise location with respect to the gene. Even if the insertion of the element does not itself disrupt gene function, then the element can be remobilised to generate imprecise excisions. This approach is facilitated by a dominant eye-colour marker carried by the enhancer trap element, whose loss can be easily scored in an appropriate excision screen.
### 1.2.3. β-gal Expression in the Nervous System
The enhancer trap method has been used to observe β-gal expression specifically in the cells of the central nervous system (CNS) and peripheral nervous system (PNS). (Bellen *et al.*, 1989; Bier *et al.*, 1989; Wilson *et al.*, 1989; Ghysen and O’Kane, 1989; Klambt *et al.*, 1991; Smith and O’Kane, 1991; Han *et al.*, 1992; Hartenstein and Jan, 1992; Skoulakis *et al.*, 1993; Giniger *et al.*, 1993; Callahan and Thomas, 1994).
Bier *et al.* (1989) found that about 31% of their lines were expressed in the embryonic nervous system. Bellen *et al* (1989) described approximately 50% of all lines stained specifically in the embryonic nervous system with around 20% of these showing either CNS or PNS specific staining. Nose *et al.* (1992) reported that they screened about 11000 enhancer trap lines and found a number of lines stained in the nervous system.
Besides the embryonic nervous system, the enhancer trap method has been recently applied to the adult nervous system by several groups. Schneuli and Heisenberg (Wurzberg, personal com.) found that as many as 70% of the lines showed some degree of staining in the brain, with a smaller though significant subset of these showing more restricted patterns of expression, specific to certain anatomical domains or groups of cells. Han *et al.* (1992) screened approximately 5300 enhancer trap lines and found about 90 of these lines showed preferred or exclusive expression of *lacZ* in mushroom body cells. Skoulakis *et al.* (1993) have isolated a *DCO* (the catalytic subunit of protein kinase A) mutant by an enhancer trap
screen for genes preferentially expressed in the mushroom bodies. In our lab, Paterson and Kaiser (1993) generated and screened about 350 new enhancer trap lines. They found that approximately 65% of the lines showed lacZ expression within the adult brain.
Enhancer trapping has become a widely used approach for the generation of cell and tissue makers in *Drosophila*. For example, Klamt *et al.*, (1991) isolated enhancer trap lines as markers for specific midline lineages of CNS. Hartenstein and Jan (1992) analysed enhancer trap marker lines for the embryonic development and specific subdivision of the embryo. In the CNS, for example, perineurial cells can be clearly divided into two layers, an inner layer and an outer layer. Nelson and Laughon (1993) used enhancer trap lines as a marker to demonstrate the glial architecture and development. Using BrdU incorporation and enhancer trap lines, Prokop and Technau (1994) identified a reproducible spatial and temporal pattern of DNA replicating cells in the abdominal larval CNS (A3-7 neuromeres) of *Drosophila*. Some of marker lines are not only useful for studying normal development, but they can also be employed in the analysis of defects in mutant flies (Han *et al.*, 1992; Skoulakis *et al.*, 1993).
**1.2.4. Staining Patterns Represent the Expression Patterns of Neighbouring Genes**
The enhancer trap system can be used to analyse many different genes in *Drosophila*, including those that cannot be identified or characterised initially by classical genetics. More evidence has accumulated to show that known P element location lie close to genes with known expression patterns which match, closely or exactly, the observed β-galactosidase pattern of the construct (Bellen *et al.*, 1989; Bier *et al.*, 1989; Wilson *et al.*, 1989; Ghysen and O’Kane 1989; Fasano *et al.*, 1991; Han *et al.*, 1992; Bier *et al.*, 1992; Mlodzik and Hiromi, 1992; Nose *et al.*, 1992; Skoulakis *et al.*, 1993; Callahan *et al.*, 1995).
By an enhancer trap screen, seven lines were isolated with P element insertions in the cytogenetic vicinity of the learning and memory gene, *rutabaga*. (Han *et al.*, 1992). For another learning gene *DC0*, mutations have also been isolated in a such screen. (Lane and Kalderon, 1993; Skoulakis *et al.*, 1993). Their expression patterns of *lacZ* are the same as those of genes which are predominantly elevated in the mushroom bodies. Mlodzik and Hiromi (1992) showed examples that two insertion lines displayed an identified subset of the expression patterns observed for the endogenous genes (*Toll* and *fasIII*) in the embryonic CNS. Bellen *et al.* (1989) reported that six lines were identified in which the staining pattern seemed to reflect the expression pattern of a gene adjacent to the insertion by mapping 68 insertion lines cytologically. Wilson *et al.* (1989) confirmed this at the molecular level in two cases. In addition, they tested whether the cloned genomic fragments encode transcripts expressed in the pattern predicted by the embryonic β-galactosidase staining of the corresponding insertion line. Therefore, they estimated that at least 25% of enhancer trap lines will reflect the expression of a neighbouring gene. Since the expression pattern of the vast majority of *Drosophila* genes is not yet known, it is difficult to assess the efficiency of gene detection from this data. However, genes may be identified and cloned solely on the basis of their expression pattern by this method, with no requirement for information regarding associated function.
As the enhancer trap element carries "plasmid sequences", it is possible to facilitate the plasmid rescue of flanking genomic DNA. For example, "designer element", P[ArB] (Wilson *et al.*, 1989) contains a bacterial origin of replication (*ori*) and antibiotic resistance gene (*ampR*). They allow production of a clonable plasmid which contains *Drosophila* DNA from adjacent to the site of P element insertion. Figure 1.4 demonstrates the procedure of plasmid rescue. Firstly, genomic DNA from flies carrying an enhancer trap insertion is digested with a restriction enzyme that cuts in one of the polylinker sequences in the construct. This produces many fragments including one that contains the *ori* and *ampR* sequences and adjacent genomic sequences extending to the next restriction site. Secondly, dilution of the digested DNA and subsequent ligation leads to intramolecular ligation of the
Figure 1.4. Diagram of plasmid rescue technique. PL=polylinker, enz=enzyme. See text for full description. (Diagram was redrawn and slightly modified from Bellen et al., 1990). See text for further details.
different fragments. If these circular molecules are introduced into *E coli*, only the cells containing the bacterial origin of replication and the resistance gene will grow on plates containing ampicillin. The surviving colonies carry a plasmid which contains genomic sequences directly adjacent to the enhancer trap element. Digestion with a suitable restriction enzyme can then produce a fragment containing 3' (or 5') P-element sequences and all of the adjacent cloned genomic DNA. The genomic fragment generated from such a digest of plasmid DNA can be isolated on a gel and labelled as a probe for Southern and Northern analysis or *in situ* hybridisation to tissue. On the other hand, the rescued plasmid can be used to probe a wild type library to identify clones of interest. Following this method, Wilson *et al* (1989) have cloned genomic regions flanking the insertion sites in 23 lines. Within our lab, Y. Guo, and A. Gillan (personal comm.) have obtained the rescued plasmids from about 2000 lines.
### 1.2.5 “Second Generation” Enhancer Trap System
More recently, to exploit a method for turning on genes in a tissue-specific manner during any stage of development, “second generation” enhancer trap systems have been developed (Brand and Perrimon 1993, Kaiser 1993). This method separates the activator from its target gene in distinct lines, to ensure that the individual parent lines are viable: in one line the activator protein is present but has no target gene to activate, in the second line the target gene is silent. When the two lines are crossed, the target gene is turned on only in the progeny of the cross, allowing dominant phenotypes (including lethality) to be conveniently studied. This two part system consists of the yeast transcriptional activator, GAL4, and a gene of interest, which is transcriptionally controlled by the GAL4 upstream activator sequence (UAS$_G$).
To assay transactivation by GAL4, for example, flies that express GAL4 are crossed to those bearing a *lacZ* gene whose transcription is driven by GAL4 binding sites. Their progeny will contain both the GAL4 enhancer trap element and the UAS$_G$-*lacZ* gene and will,
therefore, express β-galactosidase only in those cells in which GAL4 is first expressed (Fig. 1.3b). The β-galactosidase encoded by the UAS$_G$-lacZ construct is localised in the cytoplasm rather than in the nuclei (Brand and Perrimon 1993). It will be very useful for one to visualise the neuropil structures in the brain and axons for neuronal pathfinding and synaptic connectivity.
The advantage of this system is that different markers can be tested in a single GAL4 enhancer trap line if the suitable marker becomes available (Brand and Perrimon 1993; Greig and Akam 1993; Ferveur et al., 1995; Sweeney et al., 1995; Yeh et al., 1995). For instance, it might be useful to cross a cell-surface marker into an enhancer trap line that expresses GAL4 in neurones, thereby allowing the axonal processes to be traced. More specifically, fusion between lacZ and genes encoding the microtubule-associated proteins kinesin (Giniger et al., 1993) and tau (Callaghan and Thomas, 1994) may allow complete mapping of long, microtubule-rich neuronal projections. Furthermore, the gene attached to the UAS$_G$ need not simple encode a marker. By linking a UAS$_G$ to a gene involved in development, the consequences of ectopically expressing that gene in the cells in which the enhancer trap is active can be assayed.
One of the most powerful uses of the second generation enhancer trap system will be the ability for cell ablation. In *Drosophila*, cell ablation experiments can be done by transactivation of ricin or diphteria toxin expressing constructs (Kunes and Steller, 1991; Moffat et al., 1992; Bellen et al., 1992). Recently Sweeney et al., (1995) reported that targeted expression of tetanus toxin light chain in *Drosophila* specifically eliminates synaptic transmission and causes behavioural defects; in one case, the olfactory escape response is reduced. Hidalgo et al., (1995) showed that targeted ablation of glia disrupts axon tract formation in the *Drosophila* CNS. Such a strategy could be used to address a range of questions concerning the development and function of specific groups of cells (O’Kane and Moffat, 1992; Sentry et al., 1993)
1.3 The Structure of the *Drosophila* Brain
P[GAL4] enhancer trap lines are not only suitable to facilitate the cloning of flanking genes as mentioned before, but also useful as neuronal markers for anatomical analysis (Yang et al., 1995; Smith and Shepherd, 1995; S. Renn, personal com.; R. Stocker, personal com.) and for functional studies in *Drosophila* brain (Ferveur et al., 1995; Sweeney et al., 1995; O’Dell et al., 1995). In this section I will briefly summarise the structures of the *Drosophila* adult brain.
**Fig 1.5** Schematic diagram of the *Drosophila* brain.
As shown in Figure 1.5, the brain of the *Drosophila*, in common with that of other insects, can be divided into two regions: the supra-oesophageal and sub-oesophageal ganglion (SOG). The supra-oesophageal ganglion is made up of two hemispheres. They can be divided into three regions, namely the protocerebrum, the deutocerebrum and the
tritocerebrum. The protocerebrum occupies most of the ganglion mass. The deutocerebrum lies under it, just above the oesophageal foramen (A hole through which the oesophagus passes is called the oesophageal foramen). The tritocerebrum is a region at the side of the oesophageal foramen. The sub-oesophageal ganglion lies under the oesophageal foramen. Its neuropils are linked to the upper brain by a pair of connectives situated either side of the oesophageal foramen (Power, 1943; Strausfeld, 1976; Mobbs, 1987).
The *Drosophila* brain has two layers. The inner region of the brain is called the neuropil. The peripheral layer that surrounds the neuropil is called the cortex. The neural cell bodies situated on the cortical rind and the processes extend into the neuropil, which is the site of synaptic interactions. In the underlying neuropil, at least four conspicuous main structures can be observed. They are (1) the mushroom body, (2) the central complex, (3) the antennal lobe and (4) the optic lobe. Figure 1.6 shows the schematic picture of the whole brain. In the following sections, more details are described.
The *Drosophila* brain is highly variable in size. Most neuropil regions such as calyx, lobule are continuously reorganised throughout life in response to specific living conditions (Technau, 1984; Heisenberg et al., 1995).
### 1.3.1. The Mushroom Body
The structural organisation and connectivity of the mushroom bodies (MBs) in the *Drosophila* brain has been investigated by Power (1943), Heisenberg et al. (1985), Davis (1993) and Yang et al. (1995).
The mushroom bodies comprise two symmetrically arranged parts of characteristic shape in the protocerebral brain hemispheres. They can be divided into three major parts: (1) an upper cap-shaped part called the calyx; (2) a stalk-like structure, the pedunculus; and (3) three lobes named the alpha, beta and gamma lobes respectively (Fig. 1.6).
Figure 1.6. Schematic drawing of an opened head capsule of *Drosophila* showing the most prominent parts (enlarged) of its central brain (Taken from Heisenberg et al., 1985).
The MBs are composed of two main types of neurons: extrinsic and intrinsic. The extrinsic neurons can be divided into three classes: input neurons projecting from sensory neuropils to the calyxes, output neurons projecting from the lobes of the MBs to other parts of the brain, and feedback neurons connecting the lobes of the MBs with the calyxes. The intrinsic neurons, or Kenyon cells, are restricted to the MBs. They arise from dense clusters of cell bodies dorsal and posterior to each brain lobe. Their dendrites form the mushroom bodies calyces, large regions of input from the antennal lobes and other central, while beneath each calyx the Kenyon cell axons converge to form the pedunculus. The pedunculus extends almost to the front of the brain, at which point it gives rise to three lobes: a dorsally-projecting $\alpha$ lobe, and $\beta/\gamma$ lobe complex projecting towards the mid-line.
The MBs have been implicated in associative learning and memory, and in controlling a variety of complex behavioural repertoires (Davis, 1993; de Belle, 1995). In the case of *Drosophila*, single gene mutations that cause defective mushroom body anatomies have been shown to interfere significantly with olfactory associative learning (Heisenberg *et al.*, 1985; Heisenberg, 1989). Ablation of mushroom body neuroblasts at an early stage of development, selectively causing mushroom body absence from the adult brain, has an even more profound effect (de Belle and Heisenberg, 1994). Three *Drosophila* learning/memory genes *dunce, rutabaga* and *DC0*, all of which encode components of the cAMP signalling pathway, have a significant component of their expression in the MBs (Nighorn *et al.*, 1991; Han *et al.*, 1992; Skoulakis *et al.*, 1993). Gynandromorph analysis implicates MBs, or adjacent neuropils, in control of the male courtship repertoire (Hall, 1979), a behaviour that relies heavily on olfaction. Taken together, the picture that emerges is of a specialised neuropil involved in associating and storing multimodal sensory information, thereby providing the organism with memory, and predictive behaviour.
More recently, enhancer trap expression patterns have revealed subdivision of the *Drosophila* MBs (Yang *et al.*, 1995). Rather than being homogenous, MBs are compound neuropils in which parallel sub-components exhibit discrete patterns of gene expression.
Different patterns correspond to hitherto unobserved differences in Kenyon cell trajectory, and placement. It is possible that different sub-sets of Kenyon cells perform different functional roles. This notion is supported by selective feminisation of genetically defined subdivision of MB in terms of sex-specific behaviour (O'Dell et al., 1995).
1.3.2. The Central Complex
Along with the mushroom bodies, the central complex (CC) is one of the most fascinating structures in the insect brain. It is a compact neuropil and highly symmetrically organised. A similar structure and location were found in all the insects (Williams, 1975; Strausfeld, 1976; Hanesch et al., 1989). The CC resides in the middle between the two protocerebral hemispheres just above the oesophagus, flanked on either side by the pedunculi of the mushroom bodies. In *Drosophila*, the CC consists of four interconnected main neuronal region or substructures: (1) the protocerebral bridge; (2) the fan-shaped body; (3) the ellipsoid body; and (4) the paired noduli (Fig. 1.6). In addition, two accessory structures are closely associated with the CC, the ventral bodies and the lateral triangles. The structural organisation of the CC may be characterised as columnar small-field elements linking different substructures, or regions in the same substructure and tangential large-field neurons forming strata perpendicular to the columns (Hanesch et al., 1989).
In insects, the CC has connections to many other parts of the brain. There are three main systems of tracts entering or leaving the CC. The first tracts are ones entering at the end or along the protocerebral bridge. The second tracts connect the central complex to the lateral accessory lobes (the ventral bodies in *Drosophila*) and the posterior lateral protocerebrum. The third tracts are called the anterior bundles, connecting the central complex to the anterolateral protocerebrum (Homberg, 1987). In *Drosophila* the main input to the CC is through large-field neurons such as ring neurons and fan-shaped neurons and the main candidate for outputs from the central complex is the class of small-field neurons projecting with clublike terminal to the ventral bodies (Hanesch et al., 1989).
For the possible function of the CC, early experimental data from surgery and electrical stimulation have suggested a role in the regulation of behavioural activity (Homberg, 1987). Recently more systematic studies on genetic lesions in *Drosophila* have led to similar conclusions. Mutants with defects in the CC show a variety of behavioural impairments in locomotion, vision and learning (Heisenberg *et al.*, 1985, Strauß *et al.*, 1992, Strauß and Heisenberg, 1993, Bouhouche *et al.*, 1993, Illus *et al.*, 1994 and Bausenwein *et al.*, 1994).
### 1.3.3. The Antennal Lobes
Another prominent structure is called the antennal lobe (AL). The AL is situated in the anterior part of the *Drosophila* brain, at the level of the oesophagus, as a pair of protrusions (Fig. 1.6). The ALs are the first order neuropils of the olfactory chemosensory pathway. They are divided into a series of spherical neuropil compartments called glomeruli. There are 35 glomeruli in each antennal lobe of the *Drosophila* brain, of which 30 are located in the periphery of the lobe and 5 in its centre. No obvious sexual dimorphism regarding the number, size or location of glomeruli has been observed although there is sexual dimorphism in other insects, e.g. moths (Rospars, 1983). The glomerular organisation is a reflection of the architecture of the olfactory and gustatory receptor terminals, the intrinsic interneurons, and the extrinsic output and feedback cells within the deutocerebrum. The ALs are thought to be a centre of topographic, multimodal and numerical convergence (Stocker *et al.*, 1990; Stocker, 1994).
The major input and output tracts of the ALs are the antennal nerve (about 1800 axons) as a principal input tract, the antennal commissure (about 2500 fibres) as the connection of the two lobes and three tracts extending into higher brain centres as output.
The antennal lobe receives afferents from antennal, maxillary, and pharyngeal sensilla and connects a considerable number of afferents with a smaller number of relay interneurons.
The three output tracts of the ALs were described by Stocker *et al.* (1990): (1) The inner antenno-cerebral tract (iACT; synonyms: antenno glomerular tract ATG). It runs from the postero-dorsal end of the lobe straight up to the median dorso-posterior protocerebrum, then turns laterally towards the calyx of the mushroom bodies and extends further into the lateral protocerebrum. (2) The middle antenno-cerebral tract (mACT). This tract branches off the iACT laterally some distance behind the antennal lobe and runs straight into the lateral protocerebrum. Occasionally, fibres leave the mACT halfway and extend along the pedunculus of the mushroom bodies to the calyx. (3) The outer antenno-cerebral tract (oACT), emerges from the antennal lobe lateral to the iACT and extends into the lateral protocerebrum.
Three major types of interneurons have been reported in the ALs of flies (Stocker *et al.*, 1990). They are local interneurons, relay interneurons and the giant bilateral neurons. The local interneurons branch in many of the glomeruli of one antennal lobe and appear to connect glomeruli. The relay interneurons densely arborize in a single glomerulus or more than one glomerulus and extend into the ipsilateral calyx of the mushroom bodies and the lateral protocerebrum or exclusively into the lateral protocerebrum. The giant bilateral neurons are characterised by extensive mirror-symmetric arborizations in both antennal lobes, a pair of giant processes leading towards a second arborization region in the posterior brain, and a cell body located in the ventral midline of the SOG.
Based on data from *Drosophila* and other insects, Stocker (1994) further described the connectivity of the antennal lobe in *Drosophila*. The antennal lobe appears to be constructed of four types of glomeruli: monosensillar type-1 glomeruli, which are targets of specialised sensilla; monosensillar type-2 glomeruli, receiving a wider spectrum of information from a single type of olfactory sensillum; polysensillar type-1 glomeruli, receiving olfactory input from different types of antennal sensilla; and polysensillar type-2 glomeruli, which are targets of antennal and nonantennal sensilla. Each glomerulus occupies
a specific and constant position and is associated with particular groups of receptor fibres. In other word, individual glomeruli are functionally specialised and represents the convergence of inputs with some similarity of response.
1.3.4. The Optic Lobes
It is well known that the optic lobes, flanking lateral to the fly midbrain, comprise four pairs of neuropils, namely the lamina, medulla, lobula and lobula plate (Fig. 1.6). Distally (farthest from the centre of the brain) lies the lamina beneath the compound eye, and proximal to this are the medulla and lobula and lobula plate. Lamina and medulla are connected by the first (outer) optic chiasm and medulla, lobula, and lobula plate by the second (inner) optic chiasm (Fischbach and Dittrich, 1989).
The lamina receives input from the retinal terminal of short visual fibres and *en-passant* input from the long visual fibres that run on into the medulla. In the lamina the neurological map is retinotopic. This map is reversed in the horizontal axis by the first optic chiasm, the medulla receiving a mirror image of the retinal map. This image is subsequently re-reversed by the second optic chiasm by fibres running into the lobula. As the output neuropil, there are various tracts connecting the optic lobe with the central brain, such as the anterior optic tract and the optic foci. Of the visual neuropils, the lobula is most intimately connected to the central brain (Strausfeld, 1976; Fischbach and Dittrich, 1989).
According to the different neuronal cell types in optic lobes, they can be classified as either columnar or tangential elements. Fischbach and Dittrich (1989) have demonstrated that the columnar neurons establish multiple and stacked retinotopic maps in the optic lobe. They connect the distinct cellular regions, such as retina, lamina, medulla, lobula, lobula plate, and optic foci with the central brain. The number of columns in each neuropil corresponds to the number of ommatidia in the compound eye. In the fly, sets of eight photoreceptors lie beneath the lenslet of each ommatidium. Six of these cells project to the first order neuropil,
the lamina, and the remaining pair (R7 and R8) projects directly to the medulla via the first optic chiasm. There are many neurons connecting medulla with lobula and lobula plate, such as transmedullary neurons (Tm), transmedullary Y cells (TmY) and T2-4 cells.
It is clear that different lamina monopolar cells communicate with different set of Tm and TmY. The larger number of columnar neurons reflects the segregation of many paralleled functional pathways, which are all retinotopically organised.
The tangential elements are oriented perpendicularly to the columns. The arborizations of different tangential neurons are restricted to different layers of the optic neuropils, within such layers their dentritic fields may span the entire retinotopic field or only part of it. The medulla neuropile can be subdivided into ten layers (M1-M10), the lobula plate into four layers (Lop1-Lop4), and the lobula into six layers (Lo1-Lo6). Strikingly, the serpentine layer is formed by tangential neurons entering the medulla anteriorly via Cuccatti's bundle. The cell bodies of these tangential neurons lie clustered in front of the medulla neuropile. Some send their axons via the posterior optic tract into the contralateral medulla (Fischbach and Dittrich, 1989).
Other neuropil structures, such as suboesophageal ganglion, thoracic ganglia and giant commissure were not described in this chapter.
1.4 Aims of the Project
The structure and function of the brain is one of the major outstanding problems in biology. A study of brain structure-function relationships at the molecular and genetic level is a big challenge. The complexity of the nervous system of most organisms make them refractory to detailed analysis. For this reason, much work has concentrated on invertebrate model systems. The fruitfly *Drosophila melanogaster* has proved to be an invaluable model system in the field of neurobiology, since it is particular suited to genetic and molecular
analyses of neural development and function (Rubin, 1988). Though vastly simpler than the human brain, the fly brain is still immeasurably more sophisticated than the best man-made computational device. The research on the fly brain will illuminate many of the basic mechanisms which can be applied to other organisms.
Currently several new approaches have been developed in the field of *Drosophila* neurobiology. A powerful new tool, "enhancer trapping", has been used successfully to identify and clone genes expressed more or less specifically in the nervous system of the fly (Bier et al., 1989; Bellen et al., 1989; Wilson et al., 1989; Ghysen and O'Kane, 1989; Klambt et al., 1991; Bier et al., 1992; Nose et al., 1992). In the main this has involved looking for staining of the embryonic nervous system, at least in part because of the ease with which whole-mount of embryos can be prepared. Searching for staining of the adults brain is more labour intensive because it requires the generation of cryostat sections from each independent line. However, investigation of region-specific staining in the adult brain has great significance for an elucidation of relationships between structure and function because sections of adult flies can show clearly brain structures in greater detail. In addition, some adult specific genes required in the CNS can be identified by enhancer trap expression patterns.
In order to study the structure-function relationships in the *Drosophila* brain and identify some genes required in the adults central nervous system, I have embarked upon such studies by using an "enhancer trapping" approach mentioned as section 1.2.
Chapter 2
Material and Methods
2.1 Basic materials
2.2 Histochemistry and immunohistochemistry
2.3 *in situ* hybridisation
2.4 General methods for molecular biology
This chapter contains the general procedures used in the experiments which make up the basis of this thesis. This chapter is divided into 4 main sections for convenience. Section 2.1 is about basic materials used, section 2.2 is involved in the techniques of histochemistry and immunohistochemistry, section 2.3 contains the methods for *in situ* hybridisation and the final section 2.4 describes the methods for molecular biology.
### 2.1 BASIC MATERIAL
#### 2.1.1 *Drosophila* Strains
The *Drosophila* stocks used were listed as follows. Some of them were described by Lindsley and Grell (1968) or Lindsley and Zimm (1992). Others were given for references. The stocks were maintained at either 25°C or 18°C.
**Wild types strain:** (1) Canton S, (2) Oregon R.
**White eye strains:** (1) $w^{1118}$ (2) $w$(SC10) (Dura *et al.*, 1993)
**Transposase strains:** (1) Dr, $\Delta$ 2.3/TM6B, (2) CyO/Sp; Dr, $\Delta$2-3/TM6, (Robertson *et al.*, 1988).
**P[GAL4] strains:** (1) GAL4/FM7, (2) GAL4,CyO, (Brand and Perrimon 1993; Tully, personal comm.).
**lacZ strain:** UAS$_G$-lacZ on the second chromosome (Brand and Perrimon 1993).
#### 2.1.2 Bacterial Strains, Plasmids and Phage Vectors
**XL1-Blue:** rec A$,^-$ a derivative of *Escherichia coli* K-12. Bullock (1987); Stratagene.
**NM621:** $hsdR$ $mcrA$ $mcrB$ $suppE44$ $recD$ 1009 $thy+$. Whittaker *et al.*, (1983).
**MC1061:** $hsdR$ $mcrB$ ara D139 (*ara ABC-leu*) 7679 Meissner *et al.*, (1987).
**pBluescript II (KS+/- and SK+/-): amp$^r$** Short *et al.*, (1988); Stratagene, USA.
**pBRrp49:** The *Drosophila* ribosomal protein 49 gene. O'Connell & Rosbash (1984).
**pST41:** A cDNA clone of *nina E* gene (Zucker *et al.*, 1985), major opsin of *D. melanogaster*. Gift from S.Tomlinson (Glasgow Genetics Department).
**α-tublin:** Kalfayan and Wensink, (1982).
**The Genomic DNA libraries:** One was constructed using the bacteriophage lambda GEM-11 replacement vector. The other was constructed using the bacteriophage lambda vector EMBL3 (Russell and Kaiser, unpublished)
**The male head cDNA library:** It was constructed using the lambda NM1149 replacement vector supplied. (S. Russell, unpublished)
### 2.1.3 Culture Media for *E. coli* and *Drosophila*
**L-Broth:** 10g Bacto-tryptone (Difco), 5g yeast extract (Difco), 5g NaCl, 1g glucose made up to 1 litre in distilled water and adjusted to pH 7.0 with NaOH.
**0.7% (w/v) Top Agarose:** 0.7g agarose added to 100ml of L-broth, containing 10mM MgSO₄ heated to dissolve the agarose and left to cool to 50°C before use.
**Glasgow fly food:** 10g Bacto-agar (Difco), 15g sucrose, 30g glucose, 35g yeast, 15g maize meal, 10g wheatgerm, 30g treacle, 10g Soya flour, 0.1% (v/v) Nipagen and 0.5% (v/v) propionic acid per litre of water.
**Fly food for egg laying plate:** 20g Agar, 52.5g Glucose, 26g Sucrose, 7g Yeast, 9ml Red grape juice and 6ml Nipagin per litre of water.
**Rich Larval media:** 100g glucose, 100g yeast, 20g agar, 0.1% (v/v) Nipagen per litre of water.
### 2.1.4 Enzymes
Restriction enzymes, T4 DNA ligase and 10X buffers supplied by BRL and Promega.
### 2.1.5 Chemicals and Reagents
The chemicals and reagents used in this thesis were supplied by one of following companies: BDH, Sigma, Boehringer Mannheim, BRL, Pharmacia, Amersham, United States Biochemical, Vector, Capell, Whatman.
2.1.6 Buffers and Solutions
**X-Gal Staining buffer:** (FeNaP buffer) 10mM NaH$_2$PO$_4$.H$_2$O, 10mM Na$_2$HPO$_4$.2H$_2$O, 150mM NaCl, 1mM MgCl$_2$.6H$_2$O, 3.1mM K$_4$(Fe$^{2+}$CN)$_6$, 3.1mM K$_3$(Fe$^{3+}$CN)$_6$, 0.3% Triton X-100, pH 7.8.
**Biotin Prehybridisation solution:** 0.6M NaCl, 50mM Sodium Phosphate pH 6.8, 1xDenhardts, 0.5mM MgCl$_2$.
**10mg/ml RNase/DNase solution:** DNase I and RNase A were mixed to give a 10mg/ml stock solution. Stored at -20°C.
**RNA Denaturing solution:** 4M guanidinium thiocyanate, 0.1M Tris.HCl pH 8.0, 10µl antifoam A, made up to 100ml with DEPC treated water. Added 0.1M β-mercaptoethanol immediately before use.
**DNA Homogenisation buffer:** 0.03M Tris.HCl pH 8.0, 0.01M EDTA, 0.1M NaCl, 10mM β-mercaptoethanol.
**Nuclear lysis buffer:** 0.1M Tris.HCl pH 8.0, 0.1M EDTA, 0.1M NaCl, 0.5 mg/ml Proteinase K.
**Oligo (dT) cellulose binding buffer:** 0.5M NaCl, 10mM Tris.HCl pH 8.0, 1.0% (v/v) Sarcosine, 1mM EDTA. Made up to 500ml for 2x binding buffer, 1 litre for 1x binding buffer.
**5x 1st strand cDNA buffer:** 250mM Tris.HCl pH 8.3, 700mM KCl, 50mM MgCl$_2$, 50mM DDT.
**4x Random Priming buffer:** 250mM Tris.HCl, pH 8.0, 25mM MgCl$_2$, 100µM dNTPs, 1M Hepes pH 6.6, 27 A$_{260}$ units/ml random hexanucleotides.
**10x Nick Translation Buffer:** 0.5M Tris.HCl pH 7.5, 0.1M MgSO$_4$, 1mM DDT, 500µg/ml BSA (Fraction V).
**Spermidine:** 1M stock solution, dissolve 2.54g in 10ml water. stored at -20°C.
**Ampicillin:** 50mg/ml stock solution in sterile distilled water. Stored at -20°C.
**Tetracycline:** 12.5mg/ml stock solution in absolute ethanol. Stored at -20°C.
**X-Gal:** X-Gal was dissolved in dimethylformamide to give a 20mg/ml stock solution, and stored at -20°C.
**IPTG:** It was dissolved in sterile distilled water as 20mg/ml stock solution. Stored at -20°C.
**4% (w/v) Paraformaldehyde:** 40g Paraformaldehyde added to 800ml warm PBS, made up to 1L with PBS.
**5x Agarose gel loading buffer:** 0.025% (w/v) bromophenol blue, 0.025% (w/v) xylene cyanol, 25% (w/v) ficoll, 0.5% (w/v) SDS, 50mM EDTA
**PBS:** 8g NaCl, 0.2g KCl, 1.44g NaP₂HPO₄ and 0.24g KH₂PO₄, made up to 1 litre (pH adjusted to 7.4 using HCl).
**PBT:** PBS containing 0.1% (v/v) Tween 20.
**PAT:** 1% (v/v) Triton X-100, 1% (w/v) BSA (fraction V) in PBS.
Other solutions used in this thesis were made as described by Sambrook *et al.*, (1989).
### 2.1.7. Micosopes and Films
Nikon stereoscopic zoom microscope; Zeiss inverted microsocope, Leica Orthoplan microsocope; Molecular Dynamics Multiprobe laser scanning confocal microscope. Kodak film (ASA 100 or 25); Fuji film (ASA 100); Polaroid 545 land film; Fuji NIF RX X-ray film.
### 2.2 HISTOCHEMISTRY AND IMMUNOHISTOCHEMISTRY
#### 2.2.1. X-Gal staining of frozen cryostat sections
#### 126.96.36.199. Embedding of Adult Fly Heads
**For frontal section:**
Flies are anaesthetised with CO₂ and threaded up side by side into a fly collar by their necks, and a well-known staining pattern fly is set beside them as a control (the fly collar was modified from Heisenberg and Böhl 1979). A drop of freezing medium OCT 4583 (TISSUE-TEK, USA) is added onto the fly heads and left to soak for 5-10 min. The fly heads are embedded on a Cryocassetter of Cryotme 620 (Anglia Scientific). Firstly, a drop of OCT is put onto the Cryocassetter and the Cryocassetter is placed on the Peltier Element.
When OCT is almost frozen, more drops of OCT are added and the fly collar is placed onto the Cryocassetter immediately (fly heads are embedded into OCT). The fly collar is then pressed down with the copper 'heat sink'. When the OCT is frozen the fly collar is taken away. Only fly heads are left in the frozen block. Another drop of OCT is added on the top of block and the copper 'heat sink' is immediately applied again. The Cryocassetter with the block is placed on the Cryocassetter Holder after a few minutes and block is cut into rectangle shape and trimmed.
**For horizontal section:**
The procedure of embedding is the same as above Frontal Section except re-embed the frozen block for the fly proboscis faces down.
**For sagittal section:**
The procedure of embedding is the same as the Frontal Section except flies should be mounted in a fly collar with different orientation.
### 188.8.131.52. Embedding of Whole Fly
After the fly is anaesthetised with CO\textsubscript{2}, a drop of OCT is added onto the Cryocassetter on the Peltier element. When the OCT is almost frozen, more drops of OCT are added and a whole fly is placed into OCT with the needed orientation. Then, a drop of OCT is added on the top of block and the copper 'heat sink' is applied immediately. The Cryocassetter with the block is placed on the Cryocassetter Holder and block is cut into rectangle shape and trimmed.
### 184.108.40.206. Sectioning and Staining
The frozen block with flies is cut in thickness of 12μm using Cryotome 620 (Anglia scientific) at -18°C. The ribbon of sections are collected on gelatinised slides and placed directly on a slide warmer at about 35°C for 1 min. Serial sections may be collected onto one slide provided the slide is not heated for more than 5 minutes in total. Then, they are fixed in
a coplin jar containing 1% glutaraldehyde in PBS and left for 15 min. at 4°C. After washing three times in PBS for 10 minutes, the slides are rinsed in the FeNaP staining buffer (see 2.1.6). Slides are then placed in a humid box and added 200 µl of pre-warmed staining solution with 0.2% X-gal (diluted from an 8% stock solution in DMSO). A coverslip is carefully placed on top to spread out the solution over the specimen. The box is left at 37°C for 1-2 hrs. After rinsing in PBS for 5-10 min., the slides are dehydrated by immersing them in solutions of increasing concentrations of ethanol at room temperature, i.e. 30%, 70%, 95%, 2X100%, and mounted in glycerol gelatin (Sigma).
### 2.2.2. X-gal Staining of Embryo, Larval, Pupal and Adult Brains
#### 220.127.116.11. X-gal Staining of Embryo
Embryos are collected from yeasted apple/grape juice agar plates into a container with a nylon mesh screen at the bottom, dechorionated by dipping into 50% bleach for 4 minutes and washed thoroughly with water. Embryos are placed into an Eppendorf tube containing a mixture of 0.35ml fix solution (1% glutaraldehyde in PBS) and 0.7ml n-heptane and fixed for 15 minutes at room temperature on a rotating mixer. After removing heptane and fix solution from tube, embryos are washed three times for 10 min. in PBS and 0.1% Triton X-100, and resuspended in staining buffer with 0.2% X-gal for 1-2 hours at 37°C. After staining, staining solution are removed and about 400µl of mixture solution (Glycerol : staining buffer = 2:1) are replaced. Embryos can then be mounted on a slide in a coverslip chamber.
#### 18.104.22.168. X-gal Staining of Larval, Pupal and Adult Brains
Brains are dissected in PBS, and fixed in 4% paraformaldehyde for 30-60 minutes. They are then washed three times for 20 minutes in PBS, and stained with staining buffer and 2% X-gal for 1-2 hours at 37°C. They are then washed for 20 minutes in PBS, cleared overnight at 4°C with PBS/12.5% hydrogen peroxide, washed for 10 min. with PBS, dehydrated through graded ethanol, and mounted in glycerol gelatin.
2.2.3. Immunohistochemistry and Confocal Microscopy
22.214.171.124. Anti-β-gal Antibody Staining of Adult Brains
Intact adult brains are dissected under PBS, fixed in 4% paraformaldehyde for 30 minutes and washed twice for 1 hour in PAT (1 X PBS; 1% bovine serum albumin; 1% Triton X-100). They are then incubated overnight with rabbit polyclonal anti-β-gal antibody (Cappel) diluted 1:2000 in PAT, 3% normal goat serum (SAPU); washed three times in PAT for 1 hour; incubated overnight with secondary antibody (fluorescein-labelled goat anti-rabbit IgG; Vector Labs) diluted 1:250 in PAT; washed twice for 1 hour in PAT, and once for 5 minutes with PBS. All of the above procedures are carried out at room temperature. Stained brains are mounted in VectaShield (Vector).
126.96.36.199. Confocal Microscopy
Intact brains are examined with a Molecular Dynamics Multiprobe laser scanning confocal microscope. Excitation (480nm) and emission (530±15nm) filters are appropriate to the fluorescein-based labels of the goat antibody used. Three dimensional reconstructions are performed using the programme 'ImageSpace' (Molecular Dynamics). Pseudo colour is added using the programme 'NIH-Image' (National Institutes of Health, Washington).
2.3 in situ HYBRIDISATION
2.3.1. in situ Hybridisation to Tissue Sections
The method is essentially described by Nighorn et al., (1991), but some modifications are made. Wild type flies (Canton S) are cut as 12 μm sections in a cryostat (Anglia Scientific, Cryotome 620) described as section 2.2.1., placed on gelatinised slides, and postfixed in freshly made PLP fixative (4% (w/v) paraformaldehyde, 0.01M sodium metaperiodate, 0.075 M lysine, 0.044M NaCl, 0.037M phosphate buffer, pH 7.2) for 10 min. After two washes in PBS, they are treated with Proteinase K (10μg/ml) at 37°C for 15 min. After wash in PBS, slides are re-fixed in 4% paraformaldehyde in PBS for 20 min, and
acetylated with 0.25% (v/v) acetic anhydride and 0.1M triethanolamine in PBS for 10 min. After the sections are dehydrated though graded methanol, they are incubated with 200 µl of prehybridisation solution at 55°C for 1-2 hrs. They are then incubated with DIG RNA labelling probes at 55°C. After overnight incubation, the sections are washed in 50% formamide, 2XSSC, then in the following mixes of 50% formamide/ 2XSSC: 3:1; 2:2; 1:3 and finally in 2xSSC at 55°C. After washes once in 1 X SSC and once in 0.5 X SSC at room temperature, a final wash is performed in low salt buffer (2mM NaPPi, 1mM NaPO4, 1mM EDTA, pH 7.2) at 45°C.
**Colorimetric Detection with NBT and X-phosphate**
For immunological detection of the hybridised probes, the sections are washed in PBT and incubated with 200 µl of 5% (v/v) sheep serum in PAT for 2-3hr. The sections are then incubated with 150-200 µl of anti-digoxigenin antibody conjugated with alkaline phosphatase (Boehringer Mannheim) diluted at 1:500 PAT for 2-3 hr at room temperature or overnight at 4°C. The sections are washed twice in PBT. After extensive washes in 100mM Tris-HCl, pH 9.5, 100mM NaCl, 50mM MgCl2, 2mM levamisole, the sections are placed with 200 µl of diluted chromogenic substrate solutions (NTB and BCIP, X-phosphate) following the manufacturer's instructions (Boehringer Mannheim), incubated in the dark at room temperature for 2-4 hrs. The reaction is stopped by washing in PBT for 20 min and mounted with glycerol gelatin (Sigma).
**Detection with DAB and H₂O₂**
The sections are washed in PBT and incubated with 200 µl of 5% (v/v) sheep serum in PAT for 1-2 hr. They are then incubated with 150-200 µl of anti-digoxigenin-horse radish peroxidase (anti-DIG-HRP) Fab fragments (Boehringer Mannheim) diluted at 1:500 PAT for 2-3 hr at room temperature or overnight at 4°C. After extensive washes, the sections are detected with diaminobenzidine and hydrogen peroxide according to condition recommended by manufacturer (Boehringer Mannheim). The reaction is stopped by washing in PBT for 20 min and mounted with glycerol gelatin (Sigma).
2.3.2 *in situ* Hybridisation to Embryos and Whole Brain
The method is adapted from Jowett and Lettice (1994) and employs a Boeringer Mannheim Digoxigenin (DIG) labelling and detection kit.
2.3.3. *in situ* Hybridisation to Polytene Chromosome using Biotin Labelled Probes.
The procedure for *in situ* hybridisation to polytene chromosomes is essentially as described by Pardue (1986).
**Preparation of chromosome squashes**
Larvae are grown at 18°C using food with extra yeast up to 3rd instar, washed in 0.7% NaCl solution to remove food and dissected in 45% glacial acetic acid on a clean slide. 4-8 glands are transferred onto a drop (10µl) of 1:2:3 (Lactic Acid:dH$_2$O:GAA) on a siliconised coverslip (18 mm$^2$) and fixed for 5 minutes. It is covered by a clean subbed slide. To spread out chromosomes, the coverslip is tapped with a pencil for at least one minute. The slides are left for a week at 4°C. After that, slides are placed on dry ice for 10 minutes for flipping off the coverslips with a razor blade, immersed immediately into freshly made ethanol:acetic acid (3:1) for 10 minutes and dehydrated in 100% ethanol for 10 minutes. After drying, the slides are examined using a phase contrast microscope. The slides with well spread chromosomes are selected for hybridisation.
**Pretreatment of chromosomes for hybridisation**
The slides are incubated in preheated 2xSSC for 30 minutes at 65°C, washed in 2xSSC for 2 min. at room temperature and acetylated in 0.1M triethanolamine-HCl (pH 8.0) and 0.125% Acetic Anhydride for 10 minutes. After twice washes in 2xSSC for 2 min., slides are dehydrated twice in 70% ethanol and in 95% ethanol for 5 minutes respectively. Then, they are denatured in 0.07N NaOH for 3 min. (exactly), washed in 2xSSC for 5 min.
and dehydrated again in 70% and 95% ethanol for 5 min, respectively. After drying, slides are ready for hybridisation.
**Hybridisation and washes**
The biotinylated DNA probe prepared as described in section 188.8.131.52. is denatured. Then, 20µl probe is applied to per slide with a 18x18mm coverslip. The edges of coverslips are sealed with Cow Gum thinned with diEthyl Ether. The slides are incubated in a moist chamber at 58°C for 12-18 hours. Cow Gum and coverslips are peeled off in 2xSSC with fine forceps at 53°C. The slides are washed three times in 2xSSC for 20 minutes each at 53°C.
**Signal detection**
After twice washes in 1xPBS for 5 minutes, once in PBT (1xPBS and 0.1% Triton X-100) for 2 minutes and rinse in 1xPBS at room temperature, slides are placed in a humid box, added 300µl Vectastain mix (Vector Lab) and incubated at 37°C for 45 min. Again after twice washes in 1xPBS, once in PBT and rinse in 1xPBS at the room temperature, slides are placed in a tray and covered with 250µl-500µl DAB solution (0.5mg/ml DAB and 1/100 volume of 1% H₂O₂). They are then incubated in dark at room temperature for 1 hour. The slides are washed in 1x PBS and stained with Giemsa (2.5ml Giemsa in 50ml 10mM Na buffer) for 12 min. After staining, they are washed in water and mounted with a drop of DPX mountant.
**2.4. GENERAL METHODS FOR MOLECULAR BIOLOGY**
Generally, molecular biology techniques are performed as described by Sambrook *et al.*, (1989) unless otherwise described.
2.4.1 Isolation of Genomic DNA
Approximately 1g of flies are added to a mortar and pestle which have been precooled in liquid Nitrogen. Before the liquid Nitrogen evaporated completely, the flies are ground to a fine powder. Using a small paint brush, (cooled in N₂) the powder is transferred from the mortar into a 15ml Wheaton homogeniser (on ice) containing 9 ml of ice cold homogenisation buffer (HB) and 0.5 ml of 10% (v/v) Triton X-100. This is homogenised thoroughly and the resulting homogenate decanted through gauze into a sterile 15ml tube on ice. They are spun immediately at 4000 K for 10 min at 4°C in a cooled rotor. The supernatant is decanted and the nuclear pellet is resuspended in 1ml of ice cold HB. 5ml of nuclear lysis buffer and 200μl of 30% (v/v) Sarkosyl are added to this solution. This is mixed by swirling until lysis had occurred. The lysate is incubated overnight at 37°C. After centrifugation to remove the debris, the supernatant is decanted into a preweighed Falcon Tube and 1.25g CsCl per ml of homogenate is added. The solution is loaded into Polyallomer tubes which are filled up with 1.25g/ml CsCl/ Water. The tubes are ultracentrifuged at 45K for 24 hours in Ti70 rotor at 25°C. Samples were collected by dripping the gradient through an 18 gauge needle at the bottom of the centrifuge tube. 1.5 ml fractions were collected initially, and then 0.5 ml fractions once it appeared more viscose. The concentration of the DNA samples is roughly estimated using EthBr plates. The best fractions are pooled and dialysed against TE. The yield of genomic DNA obtained are generally 100-200μg/g of starting material.
2.4.2 Isolation of Bacteriophage DNA
184.108.40.206 Host Cell Preparation
1.0 ml of overnight culture of NM621 is added into 100 ml L Broth with 1ml 20% Maltose, 100μl 1M MgSO₄ and grown at 37°C with shaking to OD₆₀₀ of 0.3. After centrifugation, cells are resuspended in 10mM MgSO₄ to a final A₆₀₀ of 1.0.
220.127.116.11. Isolation of Phage DNA
This protocol is adapted from Chisholm (1989). About $2 \times 10^6$ eluted phage are incubated with 500µl ($4 \times 10^8$) of NM621 plating cells for 30 min. at 37°C. Then they are transferred into a flask with 37ml of L-broth and are grown with shaking at 37°C for 15 hours. After lysising, the mixture is transferred into 50 ml Falcon tube containing 100 µl chloroform. After adding 370 µl of nuclease solution (50mg DNase I, 50mg RNase A, in 10 ml of 50% glycerol, 30mM NaoAc), the mixture is incubated at 37°C for 30 min. Then the mixture 2.1g NaCl is added and then centrifuged at 4K for 20 min. at 4°C. The supernatant is transferred to a new tube containing 3.7g PEG 8000. It is left on a 'rock and roller' until all the PEG had dissolved. The solution is left for one hour at 4°C to precipitate the phage. In order to pellet the phage, the solution is spun at 10K for 20 minutes. when the pellet is resuspended in 500 µl phage buffer, 500µl chloroform is mixed and centrifuged for 10 min.. The phage are decanted into a centrifuge tube, then EDTA is added to give a final concentration of 20mM, Proteinase K to a final concentration of 50µg/ml and SDS to a final concentration of 0.2% (v/v). This is incubated at 65°C for 1 hour. The solution is then extracted by phenol and chloroform and precipitated by isopropranol. The pellet is washed with 70% ethanol and resuspend in 200 µl of TE. This usually gives a yield of 50-100µg bacteriophage DNA.
2.4.3 Isolation of Plasmid DNA
18.104.22.168 Large Scale Plasmid Preparation
Large scale preparations of plasmid DNA are prepared by alkaline lysis method. The plasmid containing bacteria are inoculated into L-broth containing the appropriate antibiotic. This is grown with shaking at 37°C. The culture is spun down and the bacterial pellet resuspended in 20ml solution I (50mM glucose, 25mM tris.HCl pH 8.0, 10mM EDTA), to this suspension is added 20ml of freshly prepared solution II (0.2N NaOH, 1% SDS). The contents are mixed by inversion and incubated at room temperature for 5-10 min.. 20ml of ice cold solution III (5M KOAc pH4.8) is added and the solution shaken vigorously before
incubating on ice for 10 min. The white precipitate is removed by centrifugation at 4000 rpm for 15 minutes at 4°C. The supernatant is mixed with 0.6 vol. of isopropanol and left at 4°C for 10 minutes. After centrifugation, the pellet is washed with 70% ethanol, left to dry and resuspended in 10ml of TE pH 8.0. The plasmid DNA is purified using CsCl gradients. 1g CsCl is added per ml of DNA solution. In addition, 0.5ml of EthBr (10mg/ml) is added. The solution is transferred to a polyalomer tube and ultracentrifuged at 49K for 18 hour in a Ti70 rotor at 20°C. The resulting plasmid band is removed from the tube, extracted with water saturated butanol to remove the EthBr and dialysed against 1xTE. Yields of 1-2mg are obtained.
22.214.171.124 Small Scale Plasmid Preparation
The above protocol is also used to produce small scale plasmid preparation. In this instance, 10ml of the overnight culture is used, 300µl of solution I, 300µl of solution II, 300µl of solution III and 600µl isopropanol. The resulting plasmid DNA is resuspended in 100µl of TE pH 8.0 containing RNase A. Yields of 20µg are obtained. In some cases, the Promega wizard™ preparations are used to isolate small amounts of DNA for checking rescued plasmids or for sequencing reactions. Procedure is followed as described by the manufacturer.
2.4.4. Transformation of *E.coli*.
126.96.36.199. Electrotransformation
Electrocompetent cell preparation.
1.0 ml of overnight culture of MC1061 is added into 400 ml L Broth and grown at 37°C with shaking to O.D$_{600}$ of 0.4-0.5 (approx. 3 hrs.). After chilling on ice for 30 min., they are centrifuged in Jouan at 4000rpm and 4°C for 10 min. Then cells are washed twice in 200 ml ice-cold distilled water and resuspend in 100ml ice-cold 10% glycerol. Then, cells are centrifuged again and resuspend in the final volume 1.0ml 10% glycerol. They are stored at -70°C.
Electrotransformation
When electrocompetent cells are thaw, *E. coli* Pulser cuvettes are placed on ice. 5µl of DNA (DNA should be salt free) and 40µl of cell suspension are mixed in a cold eppendorf tube and allowed to sit on ice for one min. The mixture is subsequently transferred to 0.1 cm cuvettes. When the cuvette is placed in pulsing chamber, pulse are applied (set E.coli Pulser to 1.8kV). 1.0ml of LB is added immediately. They are transferred to glass tube and incubated at 37°C for 30 min. in shaking water bath. After centrifugation, 200µl of cells are plated onto LB plates containing the appropriate antibiotics (i.e. 100µm/ml ampicillin for plasmid-rescue purposes) and incubated overnight at 37°C.
188.8.131.52. Chemical Transformation
Competent cell preparation
1.0 ml of overnight culture of XL1 Blue cells is added into 100 ml L Broth and grown at 37°C with shaking to O.D$_{600}$ of 0.5. After centrifugation, cells are resuspended in 10ml of ice-cold 50mM CaCl$_2$ solution. The cells are pelleted again, resuspended in 1ml of ice-cold 50mM CaCl$_2$.solution. Competent cells are either used fresh, or stored at -70°C.
Chemical Transformation
Transformations are carried out in sterile 1.5 ml microfuge tube. An aliquot of ligation mixture (or plasmid) containing up to 100ng plasmid DNA is added to 200µl aliquots of competent cells and they are incubated on ice for at least 30 min.. The DNA/cell mixture is then heat shock at 42°C for 90 seconds chilled on the ice for 2 min. 800µl of L-broth is added to the tube and incubated at 37°C for 30 min.. The cells are then plated onto LB plates containing the appropriate antibiotic/chromogenic substances and incubated overnight at 37°C to select for transformants.
2.4.5. Growth of *E. coli*
Liquid cultures of *E. coli* strains from which plasmids are to be isolated are grown in L broth with the appropriate antibiotic selection (usually ampicillin at 50µg/ml). The volume of L broth inoculated depended on the quantity of plasmid required. Routinely, 1.5 ml and 400 ml cultures are used for mini and large scale plasmid preparations respectively (section 2.4.3). The cultures are incubated at 37°C in an orbital shaker at ca. 250 rpm.
2.4.6. Plasmid Rescue Techniques
The basic methods for plasmid rescue is described by Pirrotta (1986). However, some modifications are made.
184.108.40.206. Isolation of Genomic DNA from Flies
In addition to the method described as section 2.4.1., the following method is applied for obtaining a small amount of genomic DNA. About 100 flies are ground in 1ml cold Homogenisation buffer (5% sucrose, 80mM NaCl, 0.1M Tris pH8.0, 0.5%SDS, 50mM EDTA) in 15ml Wheaton homogeniser. The samples are transferred to a tube and frozen for 10 minutes. They then are incubated at 70°C for 30 minutes. When KAc (pH4.8) is added to final concentration of 160mM, the samples are placed on ice for 30 minutes. After centrifugation, the supernatant is removed to a fresh tube and extracted twice with an equal volume of phenol/choroform and chloroform. DNA solution is precipitated by the addition of 0.75 volumes of isopropanol. After mixing, the DNA is pelleted by centrifugation. The pellet is washed in 70% (v/v) ethanol and dry. Genomic DNA is then resuspended in 0.5ml of TE containing RNase A(20µg/ml).
220.127.116.11. Digestion of Genomic DNA
1µg Genomic DNA from the insertion line is digested using appropriate enzyme and buffer at 37°C for 3 hours. Digestion is checked on a mini-gel.
18.104.22.168 Ligation of DNA Fragments
Ligations are performed usually in 20µl of 1x ligation buffer provided by BRL, containing 1U of T4 ligase per µg of DNA. The reactions are incubated for 4 hours at room temperature or overnight at 16°C.
22.214.171.124 Transformation of *E. coli*
Transformation of *E.coli* is described in section 2.4.4. Afterward a single colony is grown in 10ml LB of the glass tube with ampicillin at 50µg/ml described as section 2.4.6.
126.96.36.199 Isolation of Plasmid DNA
The mini prep of plasmid DNA isolation is described in section 188.8.131.52. Then the rescued plasmid containing the flanking DNA is analysed by restriction enzyme mapping.
2.4.7 Isolation of RNA
184.108.40.206 Isolation Total RNA
Separation of fly heads from bodies
About 15 ml of stunned flies is placed into a Falcon tube. Liquid nitrogen is slowly poured in, a perforated cap screwed on and vortexed for 30-60 seconds until all the N₂ boils off. More N₂ are added and the vortex is repeated. Then, the contents of the tube are poured into a 710 µm sieve already immersed in N₂ with porcelain basin. The preparation is checked that all heads have detached from the bodies. When flies from about 10 Falcon tubes are poured into the same sieve, the cool nylon bristled brush is used to force heads through the sieve using a circular "grinding" motion. The body preparation on this sieve are knocked into a Falcon tube and stored in liquid Nitrogen until needed. The mixture containing heads, appendages and some bodies in the basin are transferred into a 600µm sieve which will retain all the remaining bodies. Most of the heads and appendages will go through the sieve into the basin below. To separate heads from appendages, this mixture are put through the 425µm
sieve, all heads are retained and stored into a pre-cooled Falcon tube as a pure head preparation.
**Isolation of fly head or body RNA**
This procedure is adapted from the protocols described in Chomczynski and Sacchi (1987). 4g of fly heads or bodies are homogenised in 25ml of RNA Denaturing solution (4M Guanidine thiocyanate, 0.1M Tris.HCl. pH8.0, 2% β-mercaptoethanol and 0.1μl/ml antifoam A.) using a Kinematica Polytron. After spinning, the homogenate is transferred to RF 50ml Falcon tube. Then, 1/10 vol. 2M Na acetate pH 4.0, an equal volume phenol and 1/5 vol. chloroform are added. This is shaken vigorously before being incubated on ice for 15 min.. The solution is centrifuged for 15 minutes at 15,000 rpm. The clear upper phase is removed into a fresh tube. An equal volume of isopropanol is added and the RNA precipitated at -20°C for an hour. After this time, the solution is centrifuged for 10 minutes at 10,000 rpm. The supernatant is discarded and the RNA pellet resuspended in 5ml denaturing solution. The RNA is again precipitated using an equal volume of isopropanol followed by incubation for an hour at -20°C. After centrifugation the RNA is resuspended in 5 ml of DEPC treated water. The RNA is stored as an ethanol precipitate at -20°C. Yield is 1mg/gram flies.
**220.127.116.11 Isolation of Poly (A)+ mRNA**
5g of oligo (dT) are equilibrated in 10ml of 1x binding buffer (BB). This is left to swell for 1 hour at 4°C. A 5ml syringe is plugged with RF glass wool and filled up with oligo dT cellulose to give a bed volume of 1ml. The column is washed with 10ml of 0.1M NaOH and rinsed with several volumes of RF water until the pH of the effluent is less than pH 8.0. The column is washed with 10-20 volumes of 2xBB. The RNA is dissolved in 2xBB, heated to 65°C, cooled on ice and added to the column. The effluent is collected, reheated and re-applied to the column, this procedure is repeated so that the effluent is applied to the column three times. The column is now washed with 10-20 volumes of 1xBB. The bound RNA is eluted from the column using RF water that had been heated to
65°C (usually 3ml of water were used). An equal volume of 2xBB is added to the eluted RNA. The column is treated with NaOH as before and the whole procedure repeated. Once the RNA has been eluted, it is precipitated using 1/10 vol. of NaOAc pH 5.2 and 2.5 vol. of ethanol and left at -20°C overnight. Generally, yields of 20-50µg of poly(A)+ mRNA/g of tissue are obtained.
2.4.8. Quantification of Nucleic Acids
In order to determine the concentration of DNA or RNA in a sample, readings are taken at wavelengths of 260nm. An OD$_{260}$ = 1 corresponds to 50µg/ml for double-stranded DNA, 40µg/ml for RNA and 33µg/ml for oligonucleotides. In other instances, the concentration of DNA is estimated by spotting the sample and known standards onto the surface of a 1% (w/v) agarose gel containing EtBr (0.5µg/ml). The gel is photographed using short-wavelength UV illumination (254nm) and the concentration of the DNA sample is estimated by comparing the intensity of fluorescence in the sample with those of known DNA concentration standards.
2.4.9. Labelling of Nucleic Acids
18.104.22.168. First Stand cDNA Probe for Reverse Northern
In order to produce high specific activity 1st strand cDNA probes, 150µCi 600 Ci/mmole of [α-32P]dCTP is dried down in a siliconised microfuge tube, to this is added 4µl 5x 1st strand buffer, 1µl 80mM NaPPi, 30U RNAGUARD, 2µl Oligo (dT)$_{12-18}$, 1µg Poly A+ RNA, 20U AMV reverse transcriptase and made up to a final volume of 20µl using RF water. The reaction is incubated at 42°C for 30 min, at which time 1µl of 10mM dCTP is added and the incubation continued for a further 30 min. Hydrolysis of the RNA is achieved by the addition of 1 volume of 0.6M NaOH, 20mM EDTA followed by incubation at 65°C for 30 min. Unincorporated nucleotides are separated using Sephadex G-50 columns (Sambrook et al., 1989). Incorporation is assessed in a scintillation counter using
Cherenkov counting on a small aliquot of the probe. Specific activities of $1 \times 10^8$ cpm/μg RNA are normally obtained.
### 22.214.171.124. Random Primed DNA Labelling with $^{32}$P
DNA fragments are purified from 1% (w/v) LMP (Low Melting Point agarose, BRL) agarose gels in 1x TAE or using the Magic™ DNA purification system from Promega, using the condition recommended by the manufacturers.
The DNA labelling procedure is essentially as described in Feinberg and Vogelstein (1983). Between 25-50ng of denatured DNA sample in 12μl of water is mixed with 6μl 4x Random priming Buffer, 30μCi 600 Ci/mmole of [α-$^{32}$P]dCTP and 5U of Klenow enzyme (Promega). The mixture is usually incubated for at least 12hr at room temperature. Probes are purified using Sephadex G-50 columns prepared in 1ml syringes. Incorporation of radioactive precursor is calculated using the scintillation counter and Cherenkov counting. For random primed probes specific activities of $10^8$-$10^9$ cpm/μg are normally obtained.
In some cases, "ready-to-go™ DNA labelling kit" is used as labelling DNA, using the condition recommended by the manufacturers (Pharmacia Biotech).
### 126.96.36.199. Nick Translated Biotin Probe
In order to produce biotin labelled probes for *in situ* hybridisation, 0.5μg of plasmid DNA, 2.5μl of 10x Nick translation buffer, 2.5μl of dNTP mix (0.3mM of each base), 2μl of Bio-16-dUTP (Boehringer), 1μl of $^{32}$P (trace), 1.5μl of DNase I diluted (1:1000 in 10mM Tris pH7.5, 10mM MgCl$_2$), made up to 25μl with distilled water and 10U of DNA Polymerase I are mixed in an eppendorf. This is incubated at room temperature for 60 min. The probe is separated by the addition of 1μl of 0.2M spermidine followed by incubation for 30 min on ice and spun down at 4°C for 30 min. When the supernatant is removed and stored, the pellet is resuspended in 75μl Hybridisation buffer. Incorporation is roughly
estimated by comparing the resuspended pellet with the supernatant. An incorporation of between 25-30% is found to produce optimal probes.
188.8.131.52. DIG RNA Labelling Probe
RNA probe synthesis is according to the method recommended by Boehringer Mannheim.
2.4.10 Electrophoreses and Blotting
184.108.40.206 DNA Electrophoresis and Southern Blots
DNA is electrophoresed on agarose gels which are made and run in 1xTBE. A range of agarose concentrations (0.8-1.2%, w/v) is used depending on the sizes of fragments to be resolved (Sambrook et al., 1989). Applied voltages varies between 2 and 10V/cm, depending on the time of running. Markers are usually the 1kb ladder supplied by the manufacturers (BRL). Gels are photographed on the UV transilluminator after staining agarose gels with EtBr or adding EtBr to the sample (0.5µg/ml) using a Polaroid camera loaded with 667- land film and fitted with a Kodak Wratten filter No. 23A. The DNA is transferred onto Hybond N membrane following the procedure of Southern (1975). After electrophoresis, the DNA is immersed in Denaturing solution for 20 min, followed by soaking in two changes of Neutralising solution for 20 min each on a 'rock-and-roller'. The gel is gently agitated during this time. It is left to transfer onto the membrane overnight. DNA is fixed to the membrane by baking at 80°C for two hours or by UV irradiation using a Stratalinker™ (Stratagene).
Hybridisation Conditions.
All hybridisations are carried out in hybridisation tubes in a Hybaid oven at 65°C. The membrane is prehybridised in SSC prehybridisation solution for at least two hours before the addition of the probe. The probe (described as section 2.4.9) is boiled for 5 min before being added to the filter and left to hybridise for at least 10 hrs. Filters are washed in
2xSSC at room temperature followed by wash for 15 min in low stringency wash at 65°C, and two washes for 15 min in the high stringency wash also at 65°C. The filters are blotted dry and covered in Saran Wrap™. Autoradiography of probed filters is carried out at -70°C, using intensifying screens and Fuji NIF RX X-ray film. Films are developed using a Kodak X-Omat film processor.
220.127.116.11. Reverse Northern
1μg of plasmid DNA restricted with the appropriate enzymes are run in duplicate on a 0.8% (w/v) TBE agarose gel. The gel is blotted as described above. The filter is cut in half, one half probed with the head cDNA probe, the other with the body cDNA probe (section 18.104.22.168). Filters hybridisation and washing conditions are as described above.
22.214.171.124. RNA Electrophoreses and Northern Blots
5μg mRNA and RNA ladder (BRL) in a volume of 5μl are loaded onto a 1.0% (w/v) MOPS/Formaldehyde denaturing gel. Before being loaded onto the gel the samples are denatured by the addition of 10μl of formamide, 2μl of 5xMOPS buffer, 3.5μl of formaldehyde, 1μl of EtBr (1mg/ml stock), and heated to 70°C for 15 min, cooled immediately on ice. Prior to loading 2μl of formaldehyde loading buffer are added to each sample (Sambrook et al., 1989). The gels are run in 1xMOPS, with constant circulation from anode to cathode chambers in order to maintain a constant pH. The gels are photographed as before. After photography, the gel is soaked in 0.2M NaOH for 20 min followed by soaking for 30 min in 20xSSC. The gels are transferred to Hybond C+. RNA is fixed to the membrane by baking for 2hr at 80°C.
Hybridisation conditions
DNA/RNA hybridisations are carried out at 42°C in Formaldehyde prehybridisation solution. Filters are pre-hybridised at 42°C for at least 3hr before addition of the denatured radioactive probe and hybridised for a minimum of 16hr. After hybridisation, the filters are washed in 2xSSC at room temperature followed by two 20 min washes in low stringency
wash solution at 65°C, followed by one 15 min. wash in high stringency wash solution at 65°C. The filters are blotted dry and covered with Saran Wrap™ before autoradiography.
2.4.11. Screening Lambda Genomic DNA and cDNA Libraries
126.96.36.199. First Round Screening
Screening of lambda Genomic DNA and cDNA libraries is essentially described by Sambrook et al., (1989). Briefly, cells from a prepared bacterial suspension (as described in section 188.8.131.52) are infected with phage from the bacteriophage lambda libraries. Then, about $2 \times 10^3$ pfu are plated onto 10x10 cm prtri dishes using 8ml of 7% (w/v) top agarose in LB. The plates are incubated at 37°C for about 8 hours or until the phage are just visible. Replica filters are lifted from each plate, denatured for 3 min, neutralised twice for 5 min and washed in 2xSSC for 15 min. They are air-dried before using crosslinking. The prehybridised and hybridisations described in section 184.108.40.206. The positive plaques are stabbed out of the plates using the big end of a sterile glass Pasteur pipette. The plug is left in 1ml of phage buffer with 100µl chloroform for two hours at room temperature (or overnight at 4°C) to allow bacteriophage particles to diffuse out of the agar.
220.127.116.11. Secondary Screening
Similarly, the host cells are infected with phage from the first round screening bacteriophage. Then, they are plated onto a 9mm circular petri dishes. The following steps are as same as the first round screening.
An individual plaque of interest is picked from the plate using the narrow end of a sterile glass Pasteur pipette. The plug is left in 1ml of phage buffer with 100µl chloroform for two hours at room temperature (or overnight at 4°C). Then the protocol of isolation of bacteriophage DNA is followed in section 2.4.2.
2.4.12. DNA Sequencing Techniques
18.104.22.168 Preparation of Polyacrylamide Gel
6% (w/v) denaturing polyacrylamide gel are used for sequencing. The gel are prepared from the following stock solution:
For 100 ml:
| Component | Amount |
|--------------------|--------|
| 40% (w/v) acrylamide stock | 15 ml |
| urea | 55 g |
| 10x TBE | 10 ml |
| dH$_2$O | 35 ml |
The urea is dissolved by heating the mix to 37°C and then cooled to room temperature. Before pouring the gel, 1ml of freshly prepared 10% (w/v) Ammonium persulphate and 40µl of TEMED are added to the gel solution.
22.214.171.124. Preparation of Glass Plates and Pouring the Gel
The sequencing plates are cleaned thoroughly with SDS, alcohol and water and assembled using two spacers along the vertical sides and 3MM Whatman paper (cut as a spacer) along the bottom of the gel. The entire assembly is held in place by clamps. The gel solution is applied using a 50ml syringe on one edge of the plates while tilting the plates at an angle of about 30°. The plates are then laid at an angle of 5° and the sharks tooth combs inserted on the flat side to provide an even surface of the top of the gel. The gel is usually left to polymerise overnight before use.
126.96.36.199. Preparation of DNA Sequencing Samples
Double stranded sequencing reactions using the dideoxy chain termination method (Sanger et al., 1977) are carried out as described in the Sequenase Version 2.0 manual (United States Biochemical Corporation).
188.8.131.52. Electrophoresis of Sequencing Gel
The gel is pre-electrophoresed for 1hr in 1xTBE at a constant power of 100W, after which time, the gel temperature is normally 50°C. Before loading, the samples are denatured for 2 min at 75°C. Gels are run for 2 hours for a short run (150 bp) or for 5 hours for a long run (300 bp) on constant power, and then dried for 1-2hr at 80°C onto Whatman 3MM paper under vacuum. Autoradiography is carried out without intensifying screens at room temperature.
184.108.40.206. Computer-assisted Sequence Analysis
The DNA sequences are input into MacVector™ 4.1.4. programme using IBI Gel Reader. The DNA sequences are then analysed using the following programmes.
MacVector™ 4.1.4. and AssemblyLIGN™ (Sequence Analysis Software); Fasta, Pileup. Distances and Growtree (GCG, Program Manual see the Wisconsin Package); Blast searches, Prosite search (National Centre of Biotechnology Information, National Institutes of Health).
Chapter 3
Screening of P[GAL4] Enhancer Trap Lines
3.1 Introduction
3.2 Generation of new P[GAL4] lines
3.3 Screening of new P[GAL4] lines
3.4 Examples of interesting patterns in the brain
3.5 Chromosomal locations of P[GAL4] insertion lines
This chapter describes the results of generating and screening novel P[GAL4] lines for patterns of expression in the adult *Drosophila* brain. The chapter then details some specific staining patterns in the brain. Finally, the chapter lists the chromosomal locations of P[GAL4] insertions.
### 3.1 Introduction
As mentioned in section 1.2., one disadvantage of the first generation enhancer trap elements is that they express β-gal fused to the N-terminal nuclear localisation signal of the P element transposase. Nuclear staining has its uses but precludes visualisation of cellular geometry, an important requirement in the study of cells with long processes such as neurons. In *Drosophila*, the cell bodies of the central brain neurons lie in a thin rind just beneath the cuticle, whereas the structural elements (neuropil) described by conventional anatomy consist of axonal and dendritic projections and the synapses between them. In order to visualise neuropil it is therefore necessary to use a reporter gene that directs expression to the cytoplasm and whose product will be actively transported.
A second generation enhancer trap element has now been developed (Brand and Perrimon, 1993; Kaiser, 1993) that provides for expression of a cytoplasmically-localised reporter and that in addition can be used to target expression of any desired gene product to the marked cells. The reporter of P[GAL4] is a yeast transcription factor that is functional in *Drosophila* (Fisher *et al.*, 1988), and that can be used to direct expression of other transgenes placed under the control of a GAL4 -dependent promoter (UAS$_G$). A cross between a fly with a new GAL4 insertion and a fly containing UAS$_G$-lacZ, for example, causes β-gal to be expressed in a pattern that reflects GAL4 activity (Fig. 1.3.). Any number of other transgenes can then substitute for lacZ (Greig and Akam, 1993; Ferveur *et al.*, 1995; Sweeney *et al.*, 1995; O'Dell *et al.*, 1995; Yeh *et al.*, 1995; Brand, 1995).
P[GAL4] was created by modification of plwB, in which p, l, w, and B stand for plasmid, lacZ, $w^+$ eye marker and Bluescript respectively (Wilson et al., 1989). Briefly, the vector plwB was first digested with HindIII to remove the lacZ gene and the N-terminus of the P-transposase gene. These were replaced with the entire GAL4 coding region behind the TATA box of the P-transposase gene (Brand and Perrimon 1993). Although the frequency of transposition of the GAL4 element is less than that of first-generation elements, it is not prohibitively so. Therefore, a large number of new P[GAL4] insertion lines can be created by controlled P-element mutagenesis as described in section 1.1.3.
The reporter construct, the UAS$_G$-lacZ, was constructed as follows (Brand and Perrimon 1993): First, pUAST was designed to direct GAL4-dependent transcription of a gene of choice. It contains five tandemly arrayed, optimised GAL4 binding sites, followed by the hsp70 TATA box and transcriptional start site, a polylinker with unique restriction sites, and the SV40 small t intron (SV40 small t plays a supplementary role in both the establishment and maintenance of transformation) and polyadenylation site. Then, an Adh-lacZ fusion gene was removed from pCaSpeR-AUG-$\beta$-gal (Thummel et al., 1988) and subcloned in pUAST between the TATA box and SV40 transcriptional terminator.
Investigation of region-specific staining in the adult brain has great significance for an elucidation of relationship between structure and function because adult flies can show clearly brain structures in greater detail compared with larva and pupae. As discussion before, the GAL4 enhancer trap system provides us novel means to do this. When specific expression pattern or individual cell staining pattern in particular structure of the brain are found, they can be used to further analyse for brain anatomy and flanking genes cloning. Furthermore, these specific expression patterns can be used for ablation for function and behavioural studies. In order to pursue such a study, I began a search for specific expression patterns in adult brain using the GAL4 enhancer trap system. In the following sections, the results of screening 1400 newly generated P[GAL4] lines are reported.
3.2 Generation of New P[GAL4] Insertion Lines
We used two different strains as P[GAL4] donors. One had a recessive lethal insertion on the X chromosome (Brand and Perrimon 1993). Another P[GAL4] insertion strain has a single insertion on the second chromosome balancer CyO (kindly provided by Tim Tully, Cold Spring Harbour). To mobilise the insertions from the above strains to new chromosomal locations, the "jumpstart technique" (Cooley et al., 1988) for P element mutagenesis was employed. Two controlled P element mutagenesis schemes are shown in Figure 3.1 and 3.2.
In the first crossing scheme (Fig. 3.1), females carrying the P[GAL4] insertion on the X chromosome over the balancer FM7 were mated *en masse* with males carrying a stable genomic transposase, the P\[ry^+\Delta2-3\] element on the third chromosome with a dominant marker *Drop* (*Dr*), over the TM6, *Ubx* balancer (Robertson et al 1988). This cross yields F1 "jumpstart" females carrying both the P[GAL4] insertion and the transposase gene. Therefore, virgin females with flies red eye colour and *Dr* bar eye shape were selected for the next cross. The Δ2-3 transposase in the germ-line cells of these females can excise the P[GAL4] element and, in some cases, insert it at new locations. When a single jumpstart female was mated with *white* males (*w*<sup>1118</sup>), the male progeny bearing new P[GAL4] insertion were selected by their coloured eyes (the original P[GAL4] insertion is male lethal). Then, males carrying new insertions were individually crossed to *w*<sup>1118</sup> females to establish a set of 280 independent lines which were labelled from line 1Y to 280Y.
For the above crossing scheme, It is found that the frequencies with which new P[GAL4] insertion were recovered was very low. This might be attributed to the alterations in the pGawB that allow GAL4 to be expressed from its own AUG start codon, rather than as a P-transposase-GAL4 fusion protein (Brand and Perrimon 1993). In addition, when recovering F<sub>2</sub> progeny, only males can be selected because it is impossible
Crossing Scheme (1)
mutator
\[ P \quad Q \quad \frac{P[GAL4]}{FM7} : + ; + \quad \otimes \quad \frac{w}{Y} : \frac{CyO}{Sp} : \frac{Dr, \Delta 2-3}{TM6,Ubx} \quad \sigma \]
en masse
\[ F_1 \quad Q \quad \frac{P[GAL4]}{w} : \frac{CyO/Sp}{+} : \frac{Dr, \Delta 2-3}{+} \quad \otimes \quad \frac{w^{118}}{Y} : + ; + \quad \sigma \]
single cross
mobilisation
\[ F_2 \quad Q \quad \frac{w^{118}}{w} : + ; + \quad \otimes \quad \frac{w}{Y} : \frac{P[GAL4]}{\frac{CyO/Sp/+}{+}} : + \quad \sigma \]
single cross
\[ \frac{w}{Y} : \frac{+}{\frac{CyO/Sp/+}{+}} : \frac{P[GAL4]}{+} \quad \sigma \]
new GAL4 lines
Figure 3.1. Genetic scheme for mobilization of a recessive lethal P[GAL4] on an X-chromosome. For details see text in section 3.2.
to distinguish between the original P[GAL4] insertion and new P[GAL4] insertion in females at this stage.
Due to above reasons, I set up a second crossing scheme (Fig. 3.2) when a P[GAL4], CyO strain became available (Tully, et al., personal comm.). In the second crossing scheme, similarly, flies carrying a stable genomic P-transposase source P[ry\textsuperscript{+}; \Delta 2-3] were crossed to the P[GAL4], CyO insertion strain. When males that carried both GAL4 and \Delta 2-3 P elements were mated with w (CS10) females (Dura et al., 1993), F\textsubscript{2} males and females with red but normally shaped eyes and wild-type wings could be selected against CyO or Dr, \Delta 2-3 carrying progeny. Then, these transformed flies were backcrossed to w (CS10) again and 1125 new P[GAL4] lines were established (line c1 to line c856). Sometimes, more than one transformed fly came from one individual vial in F\textsubscript{1} cross, they were also used to establish lines. These sublines were sub-marked as a, b, c, etc. For example c123a, c123b and c123c.
Finally, in order to make the novel P[GAL4] lines into the Cantonised background for further analysis, they were crossed with w (CS10) at least two generations. The w (CS10) strain was derived by backcrossing w\textsuperscript{1118} flies to wild-type (Canton-S) flies for ten generations (Dura et al., 1993).
During the establishment of new P[GAL4] lines, it was estimated that about 8% insertion lines were sterile in F\textsubscript{2} stage. This was probably due to P-element insertion.
Insertions segregating with the X chromosome were detected by examining the progeny of crosses between the P[GAL4] line and flies for homozygous UAS\textsubscript{G}-lacZ. About one-fifth of the GAL4 lines have insertions on the X chromosome. This event is in accord with assumption that P-element insertion occur randomly on a chromosomal scale (Spradling 1986).
Figure 3.2. Genetic scheme for mobilization of P[GAL4] from the balancer, CyO on the second chromosome. For details see text in section 3.2.
3.3 Screening of New P[GAL4] Insertion Lines
To screen a large number of lines for β-gal expression in fly heads, it is preferable to perform serial cryostat sections and use a histochemical detection method. It is quick and convenient compared with whole-mount staining and antibody *in situ*.
In screening, rather than to visualise GAL4 expression directly, it is used to drive a secondary reporter gene linked to a GAL4-dependent promoter, UAS$_G$-lacZ, and thus β-galactosidase is expressed in a pattern that reflects GAL4 activity. β-gal activity is readily detected as a blue stain produced by conversion of the chromogenic substrate X-Gal. Each of the 1405 P[GAL4] lines was crossed to flies homozygous for a UAS$_G$-lacZ transgene on the second chromosome (Brand and Perrimon 1993) and their progeny were examined for GAL4-directed β-gal expression in the fly head.
The recombinant flies (males and females) carrying both P[GAL4] and P[UAS$_G$-lacZ] were mounted side by side in "fly collars" and soaked in OCT for 10 minutes. They were then frozen and embedded heads separated from bodies. 12μm serial frontal head sections were cut in a cryostat (Anglia Scientific) at -18°C. The sections were then placed on slides and stained according to the methods described in section 220.127.116.11. When an interesting staining pattern was found, it was always checked again from a new cross.
A very large proportion of the lines showed some β-galactosidase expression in the brain. A summary of the β-gal expression patterns is given in Table 3.1.
As can be seen from the results, about 10% of GAL4 lines had no staining pattern in the brain within four hours of staining. However, the other 90% of insertion lines exhibited a wide range of different expression patterns. In the "general staining" group, as many as 80% of the lines showed some degree staining in the brain. The staining patterns vary from a little blue staining to "all over" staining, and from weak to strong staining. Some of lines are stained in complex pattern that mark many different tissues and cells.
Table 3.1 GAL4-directed Expression Patterns in the Brain
| STAINING REGION | NUMBER OF LINES | PERCENTAGE |
|--------------------------|-----------------|------------|
| no brain staining | 139 | 9.9% |
| general staining | 1132 | 80.6% |
| mushroom body (MB) | 24 | 1.7% |
| central complex (CC) | 20 | 1.4% |
| antennal complex (AC) | 18 | 1.3% |
| optic lobe (OL) & eye | 17 | 1.2% |
| MB+CC | 7 | 0.5% |
| MB+AC | 7 | 0.5% |
| MB+OL | 10 | 0.7% |
| CC+AC | 8 | 0.6% |
| CC+OL | 7 | 0.5% |
| specific tracts+others | 16 | 1.1% |
| TOTAL | 1405 | 100% |
Such patterns might reflect the presence of multiple regulatory elements near the P[GAL4] insertion, each one of which controls a different gene (Wilson et al., 1989). Often, we observed quite complicated patterns which were difficult to interpret.
A small proportion of staining patterns showed a specific staining in one or more substructure of the brain. Approximately 1.7% had β-gal expression principally in the mushroom bodies, around 1.4% had specific staining patterns in the central complex, about 1.3% had staining patterns more or less restricted to the antennal complex (antennal lobes and antennal nerves). Another 1.2% had staining patterns restricted to the optic lobe and eye, and nearly 3% showed staining patterns in more than one substructure of the brain. About 1.1% of GAL4 lines showed the blue staining in specific tracts or other
regions of the brain. In these groups, the percentage includes staining patterns that stain weakly all over but that still have a strong superimposed tissue-specific pattern. These interesting expression patterns will be useful for further analysis.
As mentioned above, when more transformed flies came from an individual vial in $F_1$ cross of the second crossing scheme, they were sub-marked. During the screening, it was found that about 60% of the sub-marked progeny showed the same $\beta$-gal expression patterns as a sibling P[GAL4] line. In such cases it was inferred that each of these lines carried an insertion at the same location and probably was the result of the same premeiotic transposition event in the germ line of the male parent as its sibling. The rest of sub-marked lines showed different staining patterns and were considered as independent new insertions. Such lines were obtained due to the following results. (1) the P[GAL4] was inserted at the different chromosomal location as the sibling line and (2) P[GAL4] insertion came from different "jumpstart male" in the same vial, in which there were three "jumpstart males" together.
In some cases, the same or very similar patterns of GAL4-directed expression are observed in different independent lines. This is probably due to the same chromosomal location of the inserted P[GAL4] element and to staining resolution which is not high enough to distinguish these patterns. On the other hand, it is also possible that the same or a similar pattern could arise from insertion at different sites (see section 3.5).
An attention was given to the choice of staining conditions that minimise the fly's endogenous $\beta$-galactosidase activity. The endogenous $\beta$-galactosidase activity could be eliminated at higher pH values and in a shorter staining time allowing visualisation of enhancer-trap specific lacZ expression. Staining solutions at pH 7.8 and staining times of 1-4 hours were used routinely. An alternative way to remove the endogenous $\beta$-gal staining would be to delete the endogenous $\beta$-gal gene in flies (Fargnoli et al., 1985). On the other hand, It was also found that flies bearing both P[GAL4] and UASG-lacZ were
more sensitive to β-gal staining than those bearing P[LacW]. This is probably due to the high level expression of GAL4 in those flies.
After further observation of the staining patterns, more than 300 lines were kept which display interesting patterns in the brain from an anatomical perspective. Of these, as many as 100 lines are restricted to specific regions or neuronal sub-populations of brain. The examples of these patterns will be given in the next section.
### 3.4 Examples of Interesting Patterns in the Brain
As mentioned in section 1.3, at least four conspicuous main structures can be observed in the *Drosophila* brain. They are (1) the mushroom body, (2) the central complex, (3) the antennal lobe and (4) the optic lobe. In some of P[GAL4] lines, the β-gal expression was more or less restricted to these regions.
#### 3.4.1 The Mushroom Bodies
As has been mentioned in Chapter One, the mushroom bodies are the most fascinating structure in the *Drosophila* brain, a region thought to play a central role in learning and memory. As can be seen from the screening result, a number of P[GAL4] lines showed the staining patterns in the mushroom bodies. Figure 3.3 showed a series of sections through the brain of line 30Y, in which β-gal expression was a good match for the classical view of *Drosophila* mushroom body architecture (Heisenberg, 1980). Note that the staining extended from the cell body layer to the tips of the lobes, i.e. the Kenyon cell body layer, the calyx, the pedunculus and α, β, and γ lobes of the mushroom body. Only Kenyon cells have such a trajectory project out of the calyx to form the pedunculus and lobes (Pearson, 1971) thus, the β-gal staining patterns must represent Kenyon cells expression in the mushroom bodies.
More blue staining patterns in the mushroom bodies are depicted in Figure 3.4 (a-f).
Figure 3.3. Mushroom body structures
(a). Schematic representation of the *D. melanogaster* mushroom bodies. The sagittal view of dense clusters of Kenyon cell bodies (cb), above and behind the calyx (ca). The calyx is formed by Kenyon cell dendrites and afferents from the olfactory lobes. Beneath each calyx, Kenyon cell axons converge to form a stalk-like structure, the pedunculus (ped). This extends almost to the front of the brain, where it divides into a dorsally-projecting $\alpha$ lobe, and a $\beta/\gamma$ complex projecting towards the mid-line. (b) to (o) showing $\beta$-gal expression revealed by X-Gal staining in representative 12$\mu$m cryostat frontal sections through the head of P[GAL4] line 30Y. (b-d) show blue staining in the cell bodies and the calyx; (e-j) show blue staining in the pedunculus; and (k-o) show expression in the three lobes of the mushroom bodies. Scale bar: 10$\mu$m.
One line, 201Y, has a very restricted neuronal expression pattern. Staining is almost exclusively in Kenyon cells belonging to core elements of the $\alpha$ and $\beta$ lobes, and to most of fibres within the $\gamma$ lobe. Similarly, line 103Y showed the dark blue staining in the $\alpha$ and $\beta$ lobes corresponding to the same neurones as 201Y and relative weak staining in other parts of the mushroom bodies. On the other hand, line c772 showed staining in the $\alpha$ and $\beta$ lobes with an unstained core, and in all the $\gamma$ lobe. Line 11Y showed strong staining patterns only in $\alpha$ and $\beta$ lobes but no staining of the $\gamma$ and the spur. In line c532, all the mushroom bodies pattern can be seen, but the intensity differences between $\gamma$ lobe with a spur and other lobes of the mushroom body are observed. But line c253 has a strong staining in part of $\alpha$ lobe, all the $\beta$ and $\gamma$ lobes of mushroom bodies.
Based on observations of staining patterns, it is found that groups of Kenyon cell axons disposed at "characteristic", often concentric, positions within the pedunculus, beyond which they segregate to specific regions of the lobes. A spur-like structure is observed at the branch point of the pedunculus and the lobes. The $\alpha$ and $\beta$ lobes are organised concentrically and $\lambda$ lobe that is correlated with a spur seems to be different in its organisation.
For most of MB lines, P[GAL4] insertions are localised by *in situ* (see the next section). Different lines have a P[GAL4] insertion at a different chromosomal location and display quite different patterns even though these lines have wild-type mushroom bodies as judged by interference phase-contrast microscopy or autofluorescence. Therefore, different staining patterns represent GAL4 expression in different, genetically specified, subsets of Kenyon cells in the mushroom bodies.
Based on further examination of whole-mount brain preparations stained by X-Gal (data not shown) and confocal sections stained by the anti-$\beta$-gal antibody (JD. Armstrong, personal comm.), the covert anatomical subdivision of the mushroom bodies is confirmed. More details about subdivision of the mushroom bodies by expression patterns can be seen in Appendix 2 (Yang *et al.*, 1995).
Figure 3.4. Cryostat frontal sections (12 µm) showing X-Gal staining in the mushroom body of six P[GAL4] lines. (a) line 201Y, (b) line 103Y, (c) line c772, (d) line 11Y, (e) line c532, and (f) line c253. Abbreviations are as in Fig. 3.4. See text for full description.
3.4.2 The Central Complex
The structure and the connection of the central complex have been described in Chapter One. In a number of P[GAL4] lines the different substructures of the central complex are clearly revealed by expression patterns.
Figure 3.5 shows some sections through the brain of one such line, c161. The blue staining can be seen in the protocerebral bridge, the fan-shaped body, the ellipsoid body and the noduli. More than one kind of neuron may be responsible for this staining pattern (Hanesch et al., 1989). In this line, connection fibres between the substructures of the central complex, such as the “vertical fibre system” (VFS) (Hanesch et al., 1989), are stained. The cell bodies of the VFS lies in the dorso-caudal cortex. Its main fibre passes the protocerebral bridge, sending spiny arborization into one glomerulus, enters the fan-shaped body ipsilaterally in layer 4 and takes a downward turn to the contralateral noduli. The β-gal expression of this line is a good match for the classical view of *Drosophila* central complex structure (Fig. 3.5a, Hanesch et al., 1989). The further characterisation of expression patterns in the central complex will present in Chapter Four.
3.4.3 The Antennal Lobes
The antennal lobe is the major brain neuropil that receives olfactory input in *Drosophila*. (Fig. 1.6) The olfactory information from the antennae is conveyed to the antennal lobe via the antennal nerve. Relay neurons from the antennal lobe project via a large tract, (antennno-glomerular tract AGT), to synapse on the dendrites of mushroom body cells in the calyx (Stocker et al., 1990). More output tracts from the antennal lobes to the other parts of the central brain were described in Chapter One.
As can be seen in Table 3.1, about 1.2% of GAL4 lines has expression patterns in the antennal complex, representing a substantial fraction of its cellular components.
Fig. 3.5. Central complex structures.
A: Schematic diagram of the *Drosophila* central complex (taken from Hanesch *et al.*, 1989). The four substructures: the protocerebral bridge (pb), the fan-shaped body (fb), the ellipsoid body (eb) and the paired noduli (no). B(a-d): β-gal expression revealed by X-Gal staining in the main neuronal region of the central complex in representative 12μm cryostat frontal sections through the central complex of line c161. (a) Blue staining in the protocerebral bridge; (b) in the fan-shaped body; (c) in the paired noduli and (d) in the ellipsoid body.
Examples of lines which have a staining pattern in the antennal lobes and antennal nerve are given (Figure 3.6).
Line 253Y shows stained subsets of fibres in the antennal nerve, in which about two thirds of the volume of axons is stained, whereas, in lines c588 the whole antennal nerve appears labelled. A strong staining in the glomeruli of the antennal lobe is observed in line 59Y. Line c503a shows a staining pattern in the antennal lobes, the antennal nerve and in the antennal commissure. However, line c133 has an expression pattern only in the periphery of the antennal lobes. Interestingly, line c492b showed intense staining in the antennal lobes and the mushroom bodies. Staining in the AGT connection between these substructures is also observed in this line.
It is well known that the antennal complex (lobes and nerve) plays an important role in passing and organising olfactory and gustatory information (Stocker, 1994). Recently, Ferveur et al., (1995) and O'Dell et al., (1995) used P[GAL4] antennal complex lines to study courtship behaviour. They were able to feminize specific regions of the male antennal complex by targeted expression of the female form of the *transformer* sex determination gene. They found that these males can court both males and females, suggesting a role for these regions of antennal complex in sexual orientation. For analysis of structure-function in antennal lobes and antennal nerve, these P[GAL4] lines are indeed good markers.
### 3.4.4 The Optic Lobe and Eye
Among the lines investigated, a number of lines have shown specific GAL-directed expression patterns in the optic lobes and eye. More specifically, Figure 3.7 showed examples of the *lacZ* expression in the compound eyes (line 54Y), glia cells between the retina and the lamina (c340), lamina (line c246), the first optic chiasm (line c829), medulla (line c577), the second optic chiasm (line c637a) and lobula complex (line c469) These classes of lines can be used as marker for further analysis.
Fig. 3.6 Antennal complex staining
The β-gal expression patterns in the antennal lobes (AL), the antennal nerves (AN) and the antennal commissure (AC), and the mushroom bodies (Mb). (a) line 253Y, (b) line c588, (c) line 59Y, (d) line c503a, (e) line c133, and (f) line c492b. For a description of these lines, see the text.
Fig. 3.7. The optic lobe and eye staining
(a) A semithin horizontal section (1.5μm) for the optic lobe and eye of a wild type fly (method see Tix and Fischbach, 1992). Eye (E), Lamina (La), the first optic chiasm (X1), medulla (Me), the second optic chiasm (X2), lobula complex (Lo) are marked.
(b-h) showing the β-gal expression patterns in the optic lobe and eye. (b) line 54Y showing the staining in the eyes, (c) line c340 showing lacZ expression in the glia cells between the retina and the lamina. Blue staining patterns expressing in (d) lamina (line c246), (e) the first optic chiasm (line c829), (f) medulla (line c577), (g) the second optic chiasm (line c637a) and (h) lobula complex (line c469).
3.4.5 Other Interesting Patterns
Apart from the staining patterns described as above, there are also other interesting patterns in the brain. Although some tracts and dots stained have not been identified, some examples are showed in Figure 3.8. Line 18Y has a staining pattern in the Great Commissure that connects both hemisphere of the protocerebrum and in line c215 a tract is stained within deutocerebrum which is possibly related to the antennal lobe. Lines c518 and c600 show dots staining patterns in the brain that have not been identified yet. In the frontal sections, some dots are stained in the brain. These dots seem to be a part of tracts or neurons.
In a summary, GAL4-directed expression patterns are indeed a unique window for anatomy. They can be used to analyse the mushroom bodies (Yang et al., 1995), the central complex (Armstrong et al., submitted), antennal lobes (R. Stocker, personal comm.) and also to study brain developmental (S. Renn, personal comm.; JD. Armstrong, personal comm.). In addition, they have been used to study the structure-function roles in the *Drosophila* brain by GAL4-directed expression patterns (Ferveur et al., 1995; Sweeney et al., 1995; O'Dell et al., 1995; deBelle, 1995)
More staining patterns of P[GAL4] lines have been described in "Flybrain", an online atlas and database of the *Drosophila* nervous system (Armstrong et al., 1995). Flybrain can be accessed via the World Wide Web from servers in Glasgow (http://flybrain.gla.ac.uk).
3.5 Chromosomal Locations of P[GAL4] Insertion Lines
*Drosophila* are able to over-replicate their chromosomes in some tissues such as the salivary glands. This over replication enables the visualisation of the chromosomes, allowing the determination of the chromosomal location of the particular clone. As P[GAL4] element contains the pBluescript (pBS), its location can be determined by
Figure 3.8 Other staining patterns
(a) Line 18Y showing a staining pattern in the Great Commissure. (b) Line c215 has a staining in a tract.
(c) Line c518 and (d) line c600 showing the blue staining dots in the brain. More detail see text.
probing pBS to the polytene chromosomes of the salivary glands. The pBS probe is hybridised to the chromosome spreads and the signal detected as described in Section 2.3.3.
P[GAL4] insertions were localised by *in situ* hybridisation to polytene chromosomes for 70 lines showed interesting GAL4 expression patterns in the brain. Two examples are given in Figure 3.9. These insertions mapped to 63 distinct chromosomal locations. Cytological mapping of insertions is shown in Table 3.2.
Only one line (c492b) is showed to carry two independent insertions and these are on the same chromosome. The rest of lines contained a single P element insertion. Therefore, their simple or complex staining patterns result from the effects of genomic regulatory elements on a single P element. Certain lines which give very similar staining patterns have the same P element locations (e.g. line c105 and line c561). But only a few lines which give the different staining patterns have similar location (e.g. line c61 and line 277Y). This is because P[GAL4] was not inserted into the exact same position in the genome. The polytene chromosomal location does not provide precise information on the site. This problem can be solved by mapping the rescued flanking genomic DNA (see chapter 5). On the other hand, the different locations give different staining patterns in the brain although some similar patterns were observed. All the staining pattern is reproducible among individuals of the same insertion line. No apparent sexual dimorphisms are found.
Enhancer-trap elements are not mere anatomical markers. Besides revealing intriguing cellular arrangements within brain structures, each staining pattern implicates a flanking gene in its function. In Table 3.2, the possible genes related to P element insertions are listed and named as candidate genes. These genes are selected by virtue of their functions in the nervous system. From P element locations, we may predict that some P[GAL4] elements were inserted into or near to the interesting genes related to their functions. For example, in the case of line 43Y, P[GAL4] at the location 2C was
Figure 3.9 Polytene chromosomal *in situ* using a pBluescript probe.
(A) shows a set of polytene chromosomes displaying the chromosomal arms radiating from the chromocentre. Arrow indicates the position of the hybridisation signal for P[GAL4] insertion (line 277Y). (B) shows the P[GAL4] insertion in the polytene chromosome of line c831. Arrow indicates the position of the hybridisation signal.
supposed to insert into or near to the known genes named *usp* (ultraspiracle) or *Actn* (*α*-actinin). When sequencing the flanking DNA of this line, it proved that the P[GAL4] was indeed inserted between genes of "usp" and "Actn". When the corresponding antibodies were applied to determine the expression pattern of the protein in fly heads, "Actn" expression was largely restricted to the mushroom bodies in a manner very similar to that of enhancer trap expression in line 43Y. In other words, GAL4 expression pattern reflects the true expression pattern of nearby gene, in this case, "Actn" which could play a key role in regulating neuronal plasticity in the mushroom bodies (A. Mounsey, Glasgow, personal comm.)
Another line 189Y, P[GAL4] at the location of 24A, has an insertion in the gene named "foraging" which is related to locomotor activity of the central complex function (Varnam and Sokolowski, 1994; M. Sokolowski, personal comm.). Another example is provided by line c481 whose insertion is located at 18A. This region has a candidate gene called Neural Conserved at 18A (Nc18A), whose RNA *in situ* distribution mainly in the brain and thoracic ganglia (Perelygina *et al.*, 1992). The X-Gal staining in the brain has a similar distribution (data not shown).
A mutation can be caused by P[GAL4] itself, if it inserted into the gene and disrupt gene function. For example, line c161 is a recessive lethal mutation. P[GAL4] is inserted within an essential gene at 66A, leading homozygous lethal at 2nd instar larva stage. (D. Shepherd, Southampton, personal comm.). Line c549 exhibits the "sn" phenotype which gives severe defect in "bristle morphology" when homozygous. For this line, P[GAL4] was situated at chromosomal band of 7D1, the position of the *singed* bristle locus thought to be hot spot of P element insertion (Roiha *et al.*, 1988).
Over 1400 novel P[GAL4] enhancer trap lines were generated and screened for GAL4-directed β-gal expression in cryostat sections of the *Drosophila* head. More than 100 display interesting patterns which are more or less restricted to specific regions or neuronal sub-populations of brain. They are unique markers for anatomy analysis and
developmental study. For most of these lines, the chromosomal locations of P[GAL4] insertions were identified by *in situ* hybridisation to polytene chromosomes. These insertion lines carrying a single P element are readily amenable to a genetic and molecular analysis (Wilson *et al.*, 1989, Bier *et al.*, 1989). The studies at the molecular level for some of these lines will be presented in Chapter 5 and 6.
| line | staining pattern | location (arm) | candidate genes |
|-------|------------------|----------------|-----------------|
| 11Y | MB | 71C (3L) | |
| 30Y | MB | 70E (3L) | gnu |
| 43Y | MB | 2C (X) | usp, Actn |
| 46Y | MB | 29E (2L) | |
| 52Y | CC | 69E (3L) | |
| 59Y | AL | 11E (X) | |
| 64Y | CC | 70B (3L) | |
| 72Y | MB | 21B (2L) | mbm, plc21C |
| 78Y | CC | 84D (3R) | ids |
| 93Y | CC | 11E (X) | |
| 103Y | MB | 2D (X) | aperB |
| 104Y | CC | 26D (2L) | |
| 117Y | MB | 34C (2L) | |
| 121Y | MB | 71B (3L) | |
| 154Y | MB | 61C (3L) | |
| 181Y | MB | 57B (2R) | |
| 187Y | CC | 97D (3R) | |
| 188Y | CC | 54D (2R) | klp 54D |
| 189Y | CC | 24A (2L) | for, Pkg24A |
| 201Y | MB | 56D (2R) | |
| 203Y | MB | 1C (X) | |
| 210Y | CC | 70B (3L) | |
| 227Y | MB | 1A (X) | ewg |
| 238Y | MB | 48C (2R) | |
| 252Y | CC | 42B (2R) | |
| 253Y | AL | 4D (X) | |
| 259Y | CC | 57F (2L) | |
| 277Y | MB | 11B (X) | |
| C5 | CC | 88B (3R) | |
| C35 | MB | 44A (2R) | Myc |
| C61 | CC | 1A (X) | ewg |
| C62 | CC | 48A (2R) | inv |
| C105 | CC | 12F (X) | rut, ste |
| C107 | CC | 19F (X) | slgA, sol |
| C115 | MB | 77A (3L) | polo |
| C123a | AL | 68A (3L) | |
| C133 | AL | 11D (X) | rad |
| C161 | CC | 66A (3L) | Tph |
| C232 | CC | 100B(3R) | chp |
| C245 | MB | 82D (3R) | |
| C253 | CC | 49D (2R) | |
| C263 | AL | 34F (2L) | |
| C271 | CC | 70C (3L) | Gl |
| C302 | MB | 18C (X) | |
| C401 | CC | 39E (2L) | |
| C481 | CC | 18A (X) | Nc18A |
| C486 | CC | 1A (X) | ewg |
| C492b | AL | 49C/30B (2R) | |
| C502 | MB | 3A (X) | hypo A |
| | | | |
|---|---|---|---|
| C502b | MB | 12C (X) | rdgB |
| C503a | AL | 95F (3R) | Esm |
| C507 | CC | 100B(3R) | chp |
| C522 | CC | 64D (3L) | KlP64D |
| C532 | MB | 47E (2R) | |
| C547 | CC | 61B (3L) | |
| C561 | CC | 12F (X) | rut, ste |
| C681 | AL | 45A (2R) | pk45C |
| C737 | MB | 49D (2R) | |
| C739 | MB | 40A (2L) | |
| C742 | AL | 8C (X) | |
| C747 | MB | 42A (2R) | EcR |
| C753 | CC | 78A (3R) | |
| C758 | MB | 42A (2R) | EcR |
| C761 | AL | 93D (3R) | |
| C767 | CC | 86D (3R) | |
| C772 | MB | 42A (2R) | neuro |
| C819 | CC | 93C (3R) | gustM |
| C831 | MB | 42B (2R) | |
| C855a | CC | 79E (3L) | Csp |
| C549 | CC | 7D (X) | sn |
MB = Mushroom bodies, CC = Central complex, AL = Antennal lobes.
X = X-chromosome. 2L = the left arm of the second chromosome, 2R = the right arm of the second chromosome. 3L = the left arm of the third chromosome, 3R = The right arm of the third chromosome.
Chapter 4
Characterisation of GAL4-directed Expression Patterns in the Central Complex
4.1 Introduction
4.2 Specific staining patterns in the ellipsoid bodies
4.3 Expression patterns in the fan-shaped bodies
4.4 Expression patterns in the other sub-structures of the central complex
4.5 Developmental study of the central complex
This chapter will further detail GAL4-directed β-gal expression patterns in the different substructures of the central complex of adult brain. Then, the chapter will describe the central complex expression patterns at different developmental stages.
4.1 Introduction
As mentioned above, the central complex (CC) refers to a series of intimately related neuropils in the midbrain. Even though different terminology is used to describe them, the CC has a similar structure and location in all insects (Williams, 1975; Strausfeld, 1976; Mobbs, 1982; Homberg, 1985; Hanesch et al., 1989).
In *Drosophila* the CC is located in the centre of the brain (Fig. 1.6) and receives projections from both hemispheres (Power, 1924; Hanesch et al., 1989). Using silver staining, Hanesch et al. (1989) revealed the general neuropil architecture of the CC. A schematic diagram of the *Drosophila* central complex is given in Figure 4.1. It includes four substructures: the protocerebral bridge (*pb*), the fan-shaped body (*fb*), the ellipsoid body (*eb*) and the paired noduli (*no*). In addition, there are two accessory structures: the ventral bodies (*vbo*) and the lateral triangles (*ltr*).
By Golgi staining, about 30 single neurons of the CC were observed (Hanesch et al., 1989). They were classified as either small-field or large-field elements. The small-field neurons connect different substructures or regions within a single substructure. The majority of these neurons are intrinsic to the CC. In contrast to the small-field elements, the large-field neurons (e.g. Ring neurons and Fan-shaped neurons) reside only in a single substructure and link it to one or two central brain regions outside the CC. These neurons are thought to be the main input to the central complex.
The CC is the only unpaired neuropil in the central area of the insect brain. Therefore, its function is probably related to an integration of information from the right and left halves of the brain (Homberg, 1987). This notion was supported by early
Figure 4.1 Schematic diagram of the *Drosophila* central complex (taken from Hanesch et al., 1989). The four substructures: the protocerebral bridge (pb), the fan-shaped body (fb), the ellipsoid body (eb) and the paired noduli (no). Two accessory structures: the ventral bodies(vbo) and the lateral triangles (ltr).
experimental evidence from surgery and electrical stimulation. In these experiments, stridulation, walking, escape, respiration, and feeding behaviour were affected. Both inhibitory and excitatory influences, depending on the position of the electrode, were observed (Homberg, 1987, for a review). Recently more experiments in *Drosophila* have demonstrated the similar conclusion that the central complex is a control centre for behavioural activity. Mutants with defects in the central complex anatomy show a variety of behavioural impairments such as locomotor (Strauss and Heisenberg 1993) and learning (Bouhouche *et al.*, 1993).
In the Heisenberg laboratory, they have identified and characterised structural mutants of eight independent genes with behavioural phenotypes. The apparent specificity of the structural phenotypes and experimental results derived from the use of mosaics, point to the central complex as the major brain centre for motor control. All mutants show slow initiating activity, slow walking, and disturbed leg coordination during turns and start-stop manoeuvres (Strauss and Heisenberg 1993). One of the mutants, *no-bridge* (*nob*) showed frequent spasmodic attacks and withdrawal from sensory stimulation, the protocerebral bridge apparently being the only affected component of the central complex. In a walking test, consisting of tracking the fly’s path back and forth between two stripes (Buridan’s test), the mutant flies tested perform quite abnormally in terms of trajectory and speed (Strauss *et al.*, 1992).
In addition, most of mutant flies learn poorly in olfactory and visual discrimination tasks, and they show complex abnormalities in visual flight control (Heisenberg *et al.*, 1985; Bouhouche *et al.*, 1993; Ilius *et al.*, 1994; Bausenwein *et al.*, 1994). These data further support earlier notions that the central complex is involved in the initiation and organisation of behaviour and that it integrates visual data of the two brain hemispheres.
The behavioural complexity implies a more complex neuronal organisation and function. To study the relationship between structure and function, it is important to find the specific expression patterns in the substructure, or subdivision, of the central complex.
Then, further manipulation can be carried out, such as specific cell marking, targeted gene expression and gene cloning.
Among a large collection of P[GAL4] lines, certain lines have shown the staining patterns more or less restricted to the substructure of the central complex (Table 3.1). Here more details will be described.
### 4.2 Specific Staining Patterns in the Ellipsoid Bodies
The large-field Ring neurones innervate the ellipsoid body (*eb*). The ringlike arborizations fill in the *eb* structure. Figure 4.2 shows the staining patterns in the ring neurones (R-neurones) of the *eb* and in the lateral triangles, presumably input region to ring neurones. The cell body clusters of ring neurones are located at the rostral cellular cortex and the axons run in a prominent fibre tract, the lateral ellipsoid body tract, to the ipsilateral triangles. Then it extends further towards the midline and ends in the *eb*.
Three types of ring neurones (R-neurones, ExR1-neurones and ExR2-neurones) can be distinguished based on their cell body locations and their arborizations according to Hanesch *et al.* (1989). Among them, R-neurones were the most abundant and can be subdivided into four types of ring structures. R1-R3 arborize from the *eb* canal outward and R4 arborizes from the periphery of the ring inward. The branches of R1 terminate within the inner region of the *eb*. R2 and R4 -neurones innervate the outer ring of the *eb* and R3-neuron branch in the inner and outer ring. ExR1 has a large cell body in the pars intercerebralis, and spiny arborizations in the *eb*, the dorsal *fb*, the *vbo*, and other parts of the medial protocerebrum ipsilateral to the cell body. ExR2 has its branches in the median protocerebrum and most likely in the *vbo*.
On the basis of our observation, the ellipsoid body ring structure can be divided into four layers. They are inner-ring (A), inner mid-ring (B), outer mid-ring (C), and outer-ring (D) (Fig. 4.3a). The different ring neurones arborize into different parts of ring structures.
Figure 4.2 The staining patterns in the ellipsoid body (eb).
(a) shows a paraffin section from a wild type fly. (b) shows a cryostat section of line c507 stained by X-Gal. (c) shows the whole mount staining by X-Gal for the same line. (d) shows a confocal image stained by the anti-β-gal antibody for the same line (provided by J.D. Armstrong). The ellipsoid body (eb), cell bodies (cb) of the eb, the lateral ellipsoid body tract (lebt) and the lateral triangles (ltr) are marked.
In the preparations, the staining patterns can be seen in different layers of the *eb* ring structure. As seen in Fig. 4.3b-l, the blue staining in line 198Y occurs in layer A and line 189Y has a dark blue pattern in A region and light staining in B layer. But in line c105, GAL4-directed expressed only in the B ring layer of the *eb*. These expression patterns correspond to R1 neurones described by Hanesch *et al.* (1989). In this type of line, besides cell bodies having their branches in the *eb*, they also send a small branch with bleblike terminals into the *vbo* area in median protocerebrum.
In addition, line 64Y showed staining in all A, B and C layers of the *eb*. And line 78Y showed a strong staining in A and B regions. Line c547 has a strong staining in C layer and a light blue staining in B and D layers. These patterns correspond to R2 and R3 neurons.
Lines c93, c819 and c42 showed the specific staining in outer ring layer of the *eb*. However line c42 has a slight difference in staining density because less cells were stained. These patterns match for R2 or R4 neurones described by Hanesch *et al.*, (1989).
Moreover, extensive staining in the whole ring structure was found in different lines. For example, line c107 showed the strong blue pattern in the layer D and light staining in the rest of layers. But line c232, the staining pattern was seen in all ring structure including the centre of ring, in which A, B and D layers had a strong staining and C layer had a relative weaker staining. Comparing these types of lines with other *eb* lines, these lines have more cell bodies stained. In case of c232, about 30 cells can be seen in each hemisphere.
Using an enhancer trap approach, we can subdivide the *eb* ring structure into four different layers rather than two rings (Hanesch *et al.*, 1989). By comparing the classification of ring neurones from Hanesch *et al* (1989) and ring structures from us, it is obvious that there are some overlapping each other (Table 4.1). Our results, combined with theirs, can provide more detailed substructures of the *eb*. Furthermore, staining patterns by β-gal
Figure 4.3 GAL4-directed staining patterns in the subdivision of the ellipsoid body (eb).
(a) Schematic diagram for 4 layers eb ring structure: the inner-ring (A), inner mid-ring (B), outer mid-ring (C), and outer-ring (D). (b) Line 198Y showing the staining in the layer A. (c) 189Y showing the staining in the layers A and B. (d) c105 showing the staining in B layer. (e) 64Y showing the staining in the layers A, B and C. (f) 78Y showing the staining in the layers A and B. (g) Line c547 showing the staining in the layers B to D. (h-j) Lines c93, c819 and c42 showing the staining in the layer D. (k) Line c107 showing the strong staining in the layer D and faint staining in other layers. (l) Line c232 showing the staining in all the layers of the eb. For full description, see text.
expression reveal genetically specified subdivision of the ring structures. They also suggest that there is a functional difference between the R-neurons as they express different genes.
**Table 4.1 Comparison of the ring structures and ring neurones of the eb**
| line | The layer of the ring structure | R-neurones Hanesch et al., (1989) |
|--------|---------------------------------|-----------------------------------|
| 198Y | A | R1+R3 |
| 189Y | A+B | R1+R3 |
| c105 | B | R1 |
| 64Y | A+B+C | R1+R2+R3 |
| 78Y | A+B | R1+R3 |
| c547 | B+C+D | R2+R3 |
| c93 | D | R2+R4 |
| c819 | D | R2+R4 |
| c42 | D | R2 or R4 |
| c107 | All layers | All R-neurones |
| c232 | All layers | All R-neurones |
The ellipsoid body seems to be specific in dipterans. It is a perfectly round "doughnut with hole" shape. In other insects such as *Schistocerca gregaria* its homologue is a half circular structure (Williams, 1975). In the ellipsoid body mutants of *Drosophila*, the *eb* is opened up ventrally to varying degrees and may appear as a flat glomerulus (Strauss and Heisenberg, 1993). GAL4-directed staining patterns selectively expressed in the different ring structures provide further insight into the neuroarchitecture of the *eb*. Different ring structure of the *eb* may have different functional roles. The main role of the *eb* is thought to be inhibitory control of behaviour due to most of the ring neurones showing dense GABA immunocytochemical staining which is known as an inhibitory neurotransmitter (Hanesch et al. 1989; Bausenwein et al. 1994). However, octopamine immunoreactivity in the *eb* was observed recently (Monastirioti et al. 1995). This neurotransmitter has excitatory modulatory actions in some muscles e.g. locust flight muscle (Malamud et al. 1988). It is likely that this biogenic amine has an excitatory role in the *eb*. Therefore, we presume that different ring structure or ring neurones could give rise
to the different integrative function of the \( eb \). Targeted toxin ablation of different ring neurones may eventually help to elucidate their role in behaviour.
### 4.3 Expression Patterns in the Fan-shaped Bodies
The fan-shaped body (\( fb \)) is the largest substructure in the central complex. The \( fb \) is saucer-shaped with the convex side pointing dorso-posteriorly (Fig. 4.1). It is a regular structure of horizontal layers and vertical segments. Its long axis is perpendicular to the plane of symmetry of the brain. The \( fb \) can be divided into six roughly horizontal layers. They run parallel to each other and are the consequence of stratification of large-field neurones. Along the transverse axis the \( fb \) is divided into eight segments according to the regular arrangement of bundles of medium-sized fibre from the protocerebral bridge (Hanesch et al., 1989).
In the collection of P[GAL4] lines, a number of expression patterns representing a substantial fraction of its cellular components in the \( fb \) were found. Some type of neurones can be attributed to certain horizontal layers or vertical segments of the \( fb \) (Fig 4.4a-h). Line c5 only showed staining pattern in the first layer of the \( fb \). Line 121Y showed a strong staining in the first two layers corresponding to "superior arch" described by Strausfeld (1976). In line 210Y, a strong staining pattern was found in the 1st, 2nd and the 6th layers of the \( fb \). In addition, line c522 showed the staining in the 4th layer of the \( fb \) and 13Y has the staining pattern in 5th layer region. Line c61 showed the staining in the lowest division of the fan-shaped body. On the other hand, a number of lines have staining patterns in different segments longitudinally. Such a line 188Y showed the dense staining in both outer segments and pale staining in the rest segments longitudinally. And line 34Y showed the blue staining only in 3 and 4 segments.
Above \( lacZ \) expression patterns in the \( fb \) are consistent with the \( fb \) neuronal architecture described by Hanesch et al. (1989).
Figure 4.4 *lacZ* expression in the fan-shaped bodies (*fb*).
Staining patterns were observed in different layers of the *fb* in (a) line c5, (b) line 121Y, (c) line 210Y, (d) line c522, (e) line 13Y, and (f) line c61. Staining patterns were observed in different segments of the *eb* in (g) line 188Y and (h) line 34Y. See text for full description.
4.4. Expression Patterns in Other Substructures of the Central Complex
A pair of noduli lie ventrally to the fb. These are roughly spherical glomeruli each segmented into two subunits along the anteroposterior axis (Fig. 4.1). As seen in Figure 4.5 (a-c), line c767 showed a strong staining in the paired noduli and the lower division of fb. The linking fibres (Hanesch et al. 1989) from the fb and the contralateral noduli were also observed. In line c252, upper parts of the noduli have a faint staining compared to lower parts which have a strong blue staining. Line 78Y also stains in the noduli.
The protocerebral bridge is composed of 16 glomeruli in a row, 8 on each side of the midline. It looks like the handlebar of a bicycle. Figure 4.5d shows the protocerebral bridge of wild type fly using autofluorescent (Heisenberg and Boehl, 1979). In P[GAL4] lines, 78Y expressed the β-gal staining in all rows of the protocerebral bridge (Fig. 4.5e) and c161 showed staining in the protocerebral bridge which was restricted to some of the glomeruli (Fig. 4.5f).
The two ventral bodies were thought to be the main output areas from the CC (Hanesch et al., 1989). But we have not found any specific staining pattern in these substructure of the CC so far.
GAL4-directed expression was observed in the fan-shaped body, noduli and the protocerebral bridge and their connecting fibres. These regions were thought to be the excitatory control centre in the fly because acetylcholine, an excitatory neurotransmitter, was found in these substructures (Buchner et al., 1986).
The confocal examination of expression patterns in the central complex revealed that some types of neurones indeed show morphological specialisation which had not been noted previously (JD. Armstrong, Glasgow, personal comm.) It will be helpful for us to interpret the behavioural complexity.
Figure 4-5 Expression patterns in other substructures of the central complex
(a) Line c767 showing strong staining in the paired noduli (no) and the lower division of the \( fb \). (b) Line c252 showing the staining in the lower parts of the no and a faint staining in upper parts. The pedunculus (ped) of the mushroom body are also marked. (c) Line 78Y showing the staining in the paired no and the lower division of the \( fb \).
(d) showing the protocerebral bridge (\( pb \)) in the wild type fly, (e) and (f) showing the staining in the protocerebral bridge in lines 78Y and c161.
4.5 Developmental Study of the Central Complex
The central complex of the *Drosophila* brain appears late in development (Hanesch *et al.*, 1989). At the third instar larvae stage, only few cell bodies were observed in some of lines. For most of lines, the *lacZ* expression began at early pupal stages and their levels varied during metamorphosis. Based on the whole-mount *lacZ* expression at different developmental stages, the blue staining pattern in the ellipsoid body and the fan-shaped body will be described in detail.
**eb line, c232:** (Fig 4.6)
**Embryo stage:** No staining except in salivary glands was found. In P[GAL4] lines, it seems to be quite a common feature that the salivary glands were stained. Brand and Perrimon (1993) examined 220 P[GAL4] lines and found about 80% of lines had staining in the salivary glands. This suggests that in constructing the GAL4 vector, a position-dependent salivary gland enhancer was generated.
**Larva stage:** No staining is seen in the brain at the 1st instar. At the 2nd and 3rd instar, one pair of large cell bodies are stained in the pars intercerebralis of the brain. Their axonal branches extended into the ventral ganglion can be seen faintly. There are lots of cell bodies stained in the ventral ganglion.
**During metamorphosis:** Cell bodies of the *eb* are stained at about 30 hr after puparium formation (APF) and inner ring pattern (pale staining) can be seen at 41 hr APF. Whole ring patterns and the lateral triangles can be seen at 45 hr. The number of cell bodies increases onwards to about 25 in each hemispheres at 48 hr. After that, the pattern of the *eb* is almost same as that in adult although it is still growing and enlarging.
**Adult:** The staining only in the *eb* and in the lateral triangles can be seen. It has a very strong staining in inner ring and strong staining in outer ring. About 30 cells can be counted in each hemisphere.
Figure 4.6 *lacZ* expression in the *eb* line c232 at the different developmental stages (a) showing staining in salivary glands of the embryo (*sg*), (b) showing *lacZ* expression in the central brain and the ventral ganglion (*vg*) of the second instar larva, (c) showing *lacZ* expression in the central brain and the *vg* of the third instar larva, (d) showing the cell bodies (*cb*) of the *eb* ring neurons at about 30 hr after puparium formation (APF), (e) showing the *eb* ring neurons structure appeared at 41 hr APF, (f) at 45 hr APF. (g) at 48 hr APF, (h) 66 hr APF, (i) 70 hr APF and (j) adult. See text for full description.
On the other hand, the staining of the cell bodies in ventral ganglion begins at 2nd instar larvae. At 3rd instar, most staining disappeared, staining is restricted to ventral ganglion (T3 region) and abdominal neuromeres region. This staining gradually disappeared during metamorphosis. It possibly reflects an activity of relevant gene.
**eb line, c561a:** (Fig 4.7)
The staining in embryo is the same as line c232. From the 1st instar larvae onwards, more cells are stained than those seen in line c232, which do not belong to the *eb* cell. The *eb* pattern can be seen at about 43 hr APF. Up to adult, about 6-8 cell bodies can be counted in each hemispheres. In the ventral ganglion, discrete clusters staining can be seen. Interestingly, the staining pattern of a motor neuron in T1 region of thoracic ganglion lasts from 40 hr APF up to adult. This is probably related to locomotor activity.
**fb line, c5:** (Fig 4.8)
At the third instar larvae, staining is found in the brain but it is not sure that these cell bodies are a component of the *fb*. On the other hand, an extensive staining in ventral ganglion can be seen. At about 16 hr APF few cell bodies can be seen which is likely to be cell bodies of the fan-shaped body. More other cell bodies are stained during after 24 hr. At about 80 hr APF, the staining in the fan-shaped body can be clearly seen. This staining continues into adulthood. With respect to the ventral CNS, the staining is found from the larvae onwards through adulthood although some of the staining disappears later.
In *Drosophila*, metamorphosis is accompanied by the degeneration of most larval tissues and the construction of adult structures from imaginal discs and clusters of imaginal precursor cells. However, in the central nervous system (CNS), most neurones of the larvae do not die, but rather they persist through metamorphosis and join with groups of new adult-specific neurones to form the CNS of the adult. (Truman *et al.*, 1993). In other words, the neurones of the adult consist of "old neurones" from larvae and "new adult-specific" neurones formed during metamorphosis. Based on the observation of the *lacZ* expression
Figure 4.7 *LacZ* expression in the *eb* line c561a at the different developmental stages. (a) showing staining in salivary glands of the embryo, (b) showing the staining in the central brain and the ventral ganglion (vg) of the first instar larva, (c) showing the staining in the central brain and the vg of the second instar larva, (d) showing the staining in the central brain and the vg at 8 hr APF, (e) showing the staining in the cell bodies (cb) of the *eb* ring neurons at about 40 hr APF. A motor neuron in the T1 region of the thoracic ganglion is marked. (f) showing the *eb* staining at 43 hr APF, (g) at 60 hr APF, (h) at 74 hr APF and (i) in the adult. See text for full description.
Figure 4.8 *lacZ* expression of the *fb* line c5 at the different developmental stages
(a) showing the staining in the central brain and the ventral ganglion of the third instar larva. (b) showing stained cell body (*cb*) of the fan-shaped body (*fb*) at 24 hr APF. (c) showing the staining in the part of the *fb* at 66 hr APF. (d) and (e) showing the staining in the part of the *fb* and other regions in the brain at 80 hr APF and in the adult.
patterns at different developmental stages, it is suggested that the neurones of the central complex belong to new adult-specific neurones which appear during metamorphosis.
We observed that the *lacZ* expression started at early pupal stages in the *eb* and the *fb* lines. However, there is a possibility that some cells in the central complex may pre-exist but *lacZ* has not been activated until a later stage due to the delay of GAL4-directed β-gal expression by the two-step promoter interaction (Ito et al. 1995). The staining of antibody raised against GAL4 protein can solve this problem when the antibodies become available in *Drosophila*.
It is interesting that all the central complex lines have expression patterns not only in the substructures of the central complex but also in motor neurones of ventral or thoracic ganglion. It seems to be that these two classes of neurones have some degree of connection functionally. These staining patterns may provide more evidence for the notion that the central complex is related to locomotor control.
Using the P[GAL4] system, we observed that specific expression patterns in the CC of the *Drosophila* brain. Furthermore, the *eb* can be divided into four different ring structures genetically by P[GAL4] expression patterns. Developmental analysis indicates that the *lacZ* expression in the central complex lines begins at early pupal stages although there is a possibility that some cellular components of the CC already exist at earlier stage. As discussed before, P[GAL4] lines are not only used for anatomical analyse, but are also used for the cloning of flanking genes. In the next two chapters, I will present the results of analysis of the central complex lines at the molecular level.
Chapter 5
Plasmid Rescue of the Flanking Genomic DNA
5.1 Introduction
5.2 GAL4-directed expression patterns of the central complex lines for plasmid rescue
5.3 Plasmid rescue of flanking genomic DNA of the central complex lines
5.4 "Reverse Northern" analysis
5.5 Isolation of genomic DNA clones corresponding to lines c507 and c161
This chapter describes the plasmid rescue experiments for the central complex lines. Then, "reverse Northern" data and genomic clones of some lines are presented.
5.1 Introduction
Understanding the molecular and genetic control of brain function remains one of the most challenging problems of modern biology. Although a number of brain structural and functional mutants have been isolated, their number and diversity is small compared to the complexity of the *Drosophila* nervous system and is also small compared with the large number of transcription units expressed in the brain (Flybase, 1994).
In *Drosophila*, several strategies may be used to identify genes involved in brain structure and function. The classical approach has been to utilise mutagenic screens to identify genes whose mutant phenotype results in perturbation of the developmental pathway being studied. This approach has been used successfully to identify genes involved in brain structure and function (Delaney *et al.*, 1991; Miyamoto *et al.*, 1995). The pioneering work of Heisenberg and Böhl (1979) used the mass histological technique, following chemical mutagenesis, to isolate structural brain mutants. There are more than 40 mutant strains with structural defects in the mushroom bodies, the central complex and the optic lobes (Fichbach and Heisenberg, 1981; Heisenberg *et al.*, 1985). Subsequently, Delaney *et al.*, (1991) cloned one of structural brain genes, the small optic lobes (*sol*). They found that two major transcripts were produced from this locus and present throughout the entire life cycle. Miyamoto *et al.*, (1995) recently reported they have characterised the mushroom body deranged (*mbd*) locus. A putative *mbd* transcriptional unit, which is transcribed at various developmental stages, has been identified and isolated as a cDNA clone. However, with the classical mutagenesis screen, it is easy to miss some mutations with subtle phenotypes. So screens based on mutant phenotype alone may lead to an underestimation of the number of genes required for brain development.
On the other hand, due to the availability of molecular techniques a large number of novel genes relevant to brain function have been cloned in *Drosophila* by virtue of their sequence conservation to already known genes or on the basis of an interesting expression pattern.
One method for cloning genes is by screening with a mammalian probe. This technique requires the use of a variety of methods of homology screening. It makes the assumption that structural features of the functionally defined locus e.g. DNA or amino acid sequence are sufficiently conserved between *Drosophila* and mammals. It also makes the assumption that should a motif be conserved at the polypeptide level, it will perform the same function in the target organism. By homology screening a wide variety of genes have been successfully cloned. These include transmitter related genes such as acetylcholine receptor, *ard*, (Hermanns-bergmeyer et al., 1989); second messenger genes such cAMP dependent protein kinase (PK-A) (Foster et al., 1988), and genes involved in physiological functions including Na+/K+ATPase $\alpha$-subunit (Lebovitz et al., 1989). Another similar method involves a screen for gene expression patterns using panels of monoclonal antibodies raised against specific parts or whole of the organism (Fujita et al., 1982). These screens based on homology, however, have their own limitations. These include the nature of the DNA or the protein probes used and the fact that these depend, *a priori*, on functional conservation of the genes in question. In addition, most genes that are cloned are generally not amenable to immediate genetic analysis.
An alternative way is to use 'Reverse Genetic' approaches such as differential screening or enhancer-trapping to isolate genes expressed in a particular tissues.
Differential (+/-) hybridisation screening is a method that allows the isolation of genes based solely on their pattern of expression. The technique is able to detect messages that comprise of as low as 0.1% of the mRNA population (Sambrook et al., 1989). It has
been used successfully in various systems to identify tissue or stage-specific transcription units including those involved in the nervous system. For example, head-specific genes such as *ninaE* (Levy et al., 1982) and Na+/K+ATPase β-subunit (Tomlinson et al., submit). This technique relies on generating two different mRNA populations, one of which is enriched in the sequences of interest, the other, in which these transcripts are reduced or lacking. A differential screening approach can be used to identify genes whose transcription is tissue-specific or is highly regulated in a spatial or temporal fashion. It does not however, facilitate the detection of transcripts which show differential splicing.
A powerful approach to identify new genes has been the use of the technique of enhancer trapping, pioneered in *Drosophila* by O’Kane and Gehring in 1987. A major advantage of this technique is its ability to detect genes which do not give an obvious morphological defect when mutated but are required for normal development. Enhancer-trapping has been used to identify transcription units which are expressed in a particular tissue of interest, in this case, identifying genes that are expressed in the brain. The details of enhancer trapping has been discussed in Chapter One.
So, in the case of a head/body differential screen, when one gets genes whose transcription is tissue-specific, it would still be necessary to perform *in situ* hybridisation to head sections to see which, if any, had restricted expression patterns. The enhancer-trapping technology, by comparison, gives us immediate access to tissue-specific patterns which represent gene expression patterns. Even these may not be 'gene' patterns, rather they may just represent a fraction of the gene total expression pattern (i.e. just the enhancer). It is still useful for expressing something under GAL4 control to manipulate specific cells. So, the enhancer-trapping technique is indeed a novel tool for identification and developmental characterisation of *Drosophila* genes.
As discussed before, in the enhancer trap system, the P[GAL4] element contains a bacterial origin of replication (ori) and the \( \text{amp}^R \) gene conferring resistance to the ampicillin (see Fig. 1.3). Therefore, it provides the simplest and quickest way for cloning of adjacent genomic DNA and then of adjacent genes.
One of the aims of this project is to identify and clone the genes relevant to the structure-functional in \textit{Drosophila} brain by enhancer trap approach. When the interesting expression patterns become available, cloning of genes will be pursued.
\subsection*{5.2 GAL4-directed Expression Patterns of the Central Complex Lines for Plasmid Rescue}
Seven P[GAL4] enhancer trap lines, which specifically stained in the substructures of central complex, were selected for plasmid rescue. Examples of the patterns revealed by X-Gal staining in frontal sections of the adult brain are shown in Figure 5.1. Line c561a and c105 appear to have the same staining pattern in the inner ring region of the ellipsoid body. In addition, these two lines have the same chromosomal locations of the P[GAL4] insertion. Line c819 has a blue staining almost exclusively in the outer ring neurons of the ellipsoid body. Line c232 and c507 have the same chromosomal locations of the P[GAL4] and the same staining patterns in almost all neurons of the ellipsoid body. In addition, all lines also showed the blue staining in the lateral triangles, a presumed input region to the ring neurons (Hanesch et al., 1989).
Staining in line c61 is restricted to the lower part of the fan-shaped body. However, line c161 showed staining in all the substructures of the central complex, e.g. in the protocerebral bridge, the fan-shaped body, the ellipsoid body and the noduli. This insert is homozygous lethal at the second instar larvae (D. Shepherd, personal comm.) and thus P[GAL4] is probably inserted in an essential gene. More detail concerning the
Figure 5.1. GAL4-directed β-gal expression patterns of the central complex lines used for plasmid rescue. Each panel is a 12μm frontal cryostat section. (a) A schematic diagram of the *Drosophila* central complex (taken from Strauß and Heisenberg, 1993). The four substructures are the protocerebral bridge, the fan-shaped body, the ellipsoid body and the paired noduli. (b) line c561a and (c) line c105 show the same staining patterns in the inner ring region of the ellipsoid body. (d) line c819 showing blue staining in the outer ring neurons of the ellipsoid body. (e) c232 and (f) line c507 showing staining patterns in almost all neuronal sub-type of the ellipsoid body. (g) line c61 exhibits staining in the lower part of the fan-shaped body. (h) line c161 showing staining in all the substructures of the central complex (in this section only the ellipsoid body and parts of noduli can be seen. Other substructures of this line were described in Figure 3.5).
the characterisation of P[GAL4] expression patterns in the central complex can be seen in Chapter 4.
5.3 Plasmid Rescue of Flanking Genomic DNA of the Central Complex Lines
First of all, the P[GAL4] element construct is drawn in Figure 5.2. on the basis of the plwB (Wilson et al., 1989) and the pGawB (Brand and Perrimon, 1993) vectors. As can be seen in this diagram, whole P[GAL4] construct contains a number of sites, e.g. *Pst I*, in polylinker 3 which are not found 3' of this polylinker. Therefore, these sites can be used for plasmid rescue experiments (described as section 2.4.6.) to clone genomic sequence downstream of the P element. Similarly, polylinker 4 also contains unique sites, e.g. *Kpn I*, which can be used to subclone sequences upstream of the P[GAL4] element.
To clone genomic sequence downstream of the P[GAL4] element, genomic DNAs from seven P[GAL4] lines were digested with *Pst I* that cut in PL3 and other sites of genomic DNA. This produced many fragments including one that contained the *ori* and *ampR* sequences and adjacent genomic sequences extending to the next *Pst I* site. After dilution and ligation, transformation of competent *E.coli* cells was performed. In this experiment, the electro-transformation technique (described as section 18.104.22.168) was used because of its high efficiency. By selection on Amp-plates, only the cells containing the Bluescript can replicate and confer ampicillin resistance. Therefore, the surviving colonies carry a plasmid which contained genomic sequences directly adjacent to P[GAL4] element. These rescued plasmids were named pPC507, pPC819, etc.
In order to cut out the vector and isolate the genomic sequences flanking the insertion site, double digestion with *Pst I* and *Bam HI* was performed to produce a fragment containing just the 3' P-element sequence (vector) and all of the adjacent cloned genomic
Figure 5.2. Map of pIGAL41 construct, based on the plwB (Wilson et al., 1989) and pGawB (Brand and Perrimon, 1993) vectors.
5' (3') P= the 5' (3') end of the construct. T7 (T3)= T7 (T3) direction of pBluescript. PL = polylinker. B= BamHI, BX=BstXI, E=EcoRI, ES=EcoRV, V=HindIII, K=KpnI, N=NcoI, P=PstI, S=Sall, S2=SacII, Sf=SfiI, Sm=Smal, Sp=SpeI, X=XbaI, Xi=MboI. Restriction sites in bold are unique.
DNA. Of seven P[GAL4] lines, the size of rescued plasmids and genomic DNA fragments were summarised in Table 5.1. The longest genomic DNA fragment was 4.6 kb from pPC507. The shortest one was 0.3 kb from pPC819. From line c105 and c561a, rescued fragments of the same size were obtained.
Genomic DNA fragments generated from such digests of plasmid DNA were isolated on a gel and labelled as a probe for further analysis, such as Southern and Northern blots and the screening of genomic and cDNA libraries.
In order to check that each of the rescued plasmids was identical in size to the expected fragment, all the isolated plasmids downstream of the P[GAL4] element were analysed by the genomic Southern blots. Genomic DNAs from seven P[GAL4] lines and corresponding plasmid DNAs from rescue experiment were run on the agarose gel side by side. Before loading the gel, DNAs were adjusted to approximately equivalent amounts. After they were transferred onto Hybond N membrane, they were hybridised with the pBluescript probe. As seen in Figure 5.3, the hybridisation bands of the genomic DNA were same sizes as those in resulting plasmids. This indicated that these rescued plasmids were as expected. Moreover, the genomic Southern showed only one band in each P[GAL4] line, indicating only one insertion in each line and also confirming the chromosomal *in situ* hybridisation data.
On the other hand, it is possible that genes related to enhancer trap element were located at upstream of the P[GAL4] element. Therefore, *Kpn I* was used to digestion for cloning genomic sequence upstream of the P[GAL4] element. The results can be seen in Table 5.1. Plasmid rescue from the 5' end in five lines were successful. These rescued plasmids were named as pKC507, pKC161, etc. The genomic sequences adjacent to the 5' end of the P[GAL4] insertion can be obtained by digestion of above plasmids with *Kpn I* and *HindIII*. For the other two lines genomic clones flanking insertion were not obtained with
| line | c61 | c105 | c161 | c232 | c507 | c561a | c819 |
|------|------|------|------|------|------|-------|------|
| staining pattern | lower fan-shaped body | inner ring ellipsoid body | all central complex | whole the ring ellipsoid body | whole the ring ellipsoid body | inner ring ellipsoid body | outer ring ellipsoid body |
| chromosomal location | IA | 12F | 66A | 100B | 100B | 12F | 93B |
| name of 3' plasmid | pPC61 | pPC105 | pPC161 | pPC232 | pPC507 | pPC561a | pPC819 |
| size of 3' plasmid | 4.8 kb | 4.3 kb | 4.3 kb | 3.8 kb | 7.6 kb | 4.3 kb | 3.3 kb |
| size of genomic fragments (3') | 1.8 kb | 1.3 kb | 1.3 kb | 0.8 kb | 4.6 kb | 1.3 kb | 0.3 kb |
| name of 5' plasmid | pKC61 | pKC105 | pKC161 | | pKC507 | pKC561a | |
| size of 5' plasmid | 14.3 kb | 25.9 kb | 14.5 kb | | 18.2 kb | 25.9 kb | |
| size of genomic fragments (5') | 3.3 kb | 14.9 kb | 3.5 kb | | 7.2kb | 14.9 kb | |
Table 5.1. The result of plasmid rescue from seven P[Gal4] enhancer trap lines
The size of 3' plasmids includes pBluescript and the adjacent genomic fragment. The size of 5' plasmids includes pBluescript, mini-white, GAL4 and the adjacent genomic fragments. The genomic fragments from (3') direction were obtained by digestion with *Pst*I and *Bam*HI. The genomic fragments from (5') direction were obtained by digestion with *Kpn*I and *Hind*III.
Figure 5.3 Genomic Southern analysis of rescued plasmids.
Of seven different GAL4 lines, genomic DNAs (G) and rescued plasmids (P) from 3' ends were digested with *PstI* and run on gel side by side. They were hybridised by pBluscript probe.
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Figure 5.4 Genomic Southern analysis showing the relationship between lines c105 and line c561a.
The genomic DNA from wild type (OR) flies and from P[GAL4] line c561a and c105 were digested with *PstI* and run on duplicate gels. Two filters from duplicate gels were probed with the insert from pPC561a (A) and pPC105(B) respectively.
*Kpn*I enzyme. It seems likely that this is due to an unusual distribution of enzyme sites near the insert. Perhaps the *Kpn* I site is too far away from the insertion site. Even though corresponding larger plasmids were obtained, it was difficult to achieve transformation due to low transformation efficiency.
As can be seen in Table 5.1, line c105 and c561a give rescued plasmids of the same size, in addition to having at the same chromosomal locations and with the same blue staining patterns. In order to check that these rescued plasmids were identical, Southern blots were employed. Duplicate genomic DNA blots were prepared from wild type flies and P[GAL4] line c105 and line c561a and probed with both rescued fragments from the 3' ends. The result showed they had an identical hybridisation signals and did cross-hybridise with each other (Fig. 5.4). Thus it proved that these two lines not only had same position of P[GAL4] insertion, but also had same captured fragments.
Interestingly, line c232 and c507 give a same chromosomal location and similar blue staining patterns, but they had different sized rescued fragments from the 3' end. In order to examine further the relationship between those two lines, Southern analysis was performed. In the case of line c507, three genomic fragments (3.3, 0.9 and 0.45 kb) from the 3' end (pPC507) were released by a *Pst I/BamHI* double digest. And three genomic fragments (0.4, 4.5 and 2.2 kb) from the 5' end (pKC507) were obtained by a double digest with *Kpn* I and *Hind III* (Fig. 5.5a). When they were probed with a rescued genomic fragment from line c232, hybridisation signals can be seen in bands of 3.3 kb from the 3' direction, and 0.4 kb and 4.5 kb from the 5' direction (Fig. 5.5b). From this information and the sequencing data surrounding two P elements (data not shown), the map of relationship between line c232 and c507 was drawn in Figure 5.5c. Two P[GAL4] elements were inserted into the *Drosophila* genome only 46 bp away from each other but in different orientation. Their expression patterns were probably activated by the same enhancer. This also implied that the enhancer could act throughout the entire length of the P[GAL4] element. On the other hand, it is noted
Figure 5.5. The relationship between the line c232 and c507.
(A) shows an ethidium bromides stained gel prior to blotting. In track 1, the 1kb ladder is a size marker from Gibco-BRL. In track 2 a genomic fragment released by a $PstI/BamHI$ double digest from pPC232. In track 3, genomic fragments released by a $PstI/BamHI$ double digest from pPC507; The track 4 shows the genomic fragments digested by $KpnI/HindIII$ from pKC507. (B) shows the blot hybridised with a probe from a whole insert of pPC232. (C) shows the restriction map of the genomic region encompassing the P[GAL4] insertion of line c507 & c232. Restriction sites shown are B-$BamHI$, P-$PstI$, K-$KpnI$, H-$HindIII$. See text for more details.
that although the polytene chromosomal location looks like same, the precise genomic site of P element insertion is slightly different.
5.4 "Reverse Northern" Analyses
As a first step towards determining whether rescued plasmids contained transcribed sequences, "Reverse Northern" analysis was performed. The mRNA was used as a probe to simultaneously screen DNA clones encoding transcribed sequences, a process called reverse Northern blot analysis (Fryxell and Meyerowitz, 1987). In this case, head mRNA and body mRNA were transcribed into cDNA and then made into the cDNA probes described in Section 22.214.171.124.
A Reverse Northern does not assess the range or stage specificity of different transcripts, what is assessed here are all transcripts that hybridise to a particular DNA fragments. Each plasmid from the 3' end were double digested with *Pst I* and *BamHI*, while plasmids from the 5' end were double digested with *Kpn I* and *HindIII* (Fig. 5.6a, d). The digested plasmid DNAs were run in duplicate on a 0.8% agarose gel and transferred to nylon filters. The filters were hybridised in parallel with cDNA probes produced by reverse transcription of head and body mRNA. The autoradiographs were compared to determine which fragments exhibited a head-elevated pattern of hybridisation. In order to check the efficiency of probes, two controls were included on each filter. One was λST41, a clone of *NinaE* gene (Zucker *et al.*, 1985) that only expressed in the head and other gene was α1-tublin (Kalfayan and Wensink, 1982) which was expressed in head and body (Kindly provided by S. R. Tomlinson, Glasgow).
All of the plasmids from the 3' end when digested with *Pst I* and *BamHI* released a fragment of 2.968 kb that was the pBluescript and genomic flanking sequences. The plasmids from the 5' end when digested with *Kpn I* and *HindIII* released two fragments of 8
and 3 kb corresponding to the pBluescript plus the mini-*white* gene and the GAL4 element in addition to genomic flanking sequences. As seen in Figure 5.6, pPC507 from line c507 releases three fragments of about 3.3, 0.9 and 0.45 kb. Two fragments of 0.9 and 0.45 kb hybridise strongly to head cDNA and body cDNA probes (Fig. 5.7b-c). And pPC61 from line c61 had a fragment of 1.8 kb. This fragment was identified (Fig. 5.7b-c) which showed hybridisation to the head and body probes. Other lines did not show any hybridisation signal in the plasmid rescued fragments. This suggested either that the transcript was at too low a level to be detected, or that transcribed sequences are further away. If the reason is the former, these fragments should be checked by Northern blots because Northern blotting is a more sensitive technique than reverse Northern (Sambrook, *et al.*, 1989). If rescued fragments are too short to contain the transcribed sequences, this problem can be solved by following ways. First, digestion with a different unique enzyme (e.g. *XhoI*) for genomic DNA of P[GAL4] lines will possibly produce longer genomic fragments which contain the transcribed sequences. The second way is to screen genomic library using rescued fragments as probes to get longer genomic DNA clones and then find the transcribed sequences.
As can be seen in Figure 5.6, about 3 kb common bands of pBluescript had a hybridisation signal as well. This phenomena was observed by other researchers in our laboratory. It is probably due to a few basepairs DNA homology between probes and pBluescript, or other unknown reasons.
Unfortunately, when the 5' end rescued fragments were run on the gel for “Reverse Northern”, they were not digested well enough to identify the size of fragments (Fig. 5.6.d). Therefore, it is difficult to identify whether these fragments include transcribed sequences or not when they were hybridised by head and body cDNA probes (data not shown).
Figure 5.6. “Reverse Northern” analysis
(a) shows an ethidium bromide stained gel prior to blotting. Each plasmid from the 3' end is restricted with *PstI* and *BamHI* to release the insert, except λST41 and α tubulin which are taken as positive controls. Blot filters are probed with head (b) and body (c) cDNA probes of almost equivalent activity. The arrows indicate the hybridised fragments that are transcribed regions. (d) shows an ethidium bromides stained gel prior to blotting. Each plasmid from 5' end restricted with *KpnI* and *HindIII* to release the insert. Again blot filters are probed with same head and body cDNA probes (data not shown).
5.5 Isolation of Genomic DNA Clones Corresponding to Lines c507 and c161
As "reverse Northern blot" analysis, only two rescued plasmids pPC507 and pPC61 contain transcribed sequences. In order to find more transcribed regions for these lines and other lines, it would certainly be useful to obtain genomic lambda DNA clones that span the site of insertion and extends further 3' and 5' ends.
Two $\lambda$ libraries of *Drosophila* genomic DNA have been constructed. One library was constructed in the lambda vector EMBL3 and contains 9-22 kb inserts derived from Oregon-R DNA partially digestion with *Sau3A* (K. Kaiser, unpublished). Another library was constructed in the lambda GEM-11 (Russell and Kaiser, unpublished). The screening was performed as described in section 2.4.11. The rescued fragments from the 3' end were used as probes to clone the longer genomic DNA fragments.
For line c507, four identical positive clones of 14 kb ($\lambda_1$-$\lambda_4$) were recovered from the genomic library in the lambda GEM-11 after screening twice, but these clones only extend the 3' end. Therefore I decided to screen the different library. When the EMBL3 genomic library was used, two positive clones ($\lambda_5=13$kb; $\lambda_6=14.5$kb) were purified. In order to map genomic lambda clones, the phage DNAs were restricted with a combination of the different enzymes. Comparing restriction map of rescued fragments (see section 5.3) and their cDNA clones (see section 6.2), they provided sufficient information to construct a restriction map for line c507 which spans the site of insertion and extends further 3' and 5' ends. The resulting restriction map is shown in Figure 5.7a.
A similar screen has been performed for lines c161, c105, c61 and c819. In the case of c161, when the EMBL3 genomic library was used, four genomic lambda DNA clones were obtained. Two of them were mapped and showed in Figure 5.7b. For other lines some
"positives" plaques were identified which, due to lack of time, have not yet been resolved to single plaques or isolated to lambda phage DNAs.
When the longer genomic clones were obtained from these lines, they should also be analysed by "reverse Northern" blot analyses to find transcribed sequences in both sites of the P element and search for the head specific or head elevated genes. But due to lack of time I have not done that yet. As two lines (c507 and c61) have already shown the transcribed sequences, I decided to screen the cDNA library to find corresponding cDNAs for further analysis. The further characterisation of these lines at the molecular level will be discussed in the subsequent chapters.
Figure 5.7. Maps of the $\lambda$ genomic DNA clones
(A) The restriction map of the $\lambda$ genomic DNA clones of line c507.
(B) The restriction map of the $\lambda$ genomic DNA clones of line c61.
Restriction sites shown are B-BamHI, P-PstI, S-SstI, X-XhoI, E-EcoRI, K-KpnI, H-HindIII, SI-SalI.
Chapter 6
Cloning of Genes Neighbouring the P[GAL4] insertion in lines c507 and c232
6.1 Introduction
6.2 Isolation of cDNA clones related to c507 flanking DNA
6.3 Analysis of sequence
6.4 Northern analyses
6.5 *in situ* hybridisation
6.6 Conclusion
6.1 Introduction
As has been described in Chapter Five, genomic DNAs flanking P[GAL4] insertions have been obtained from seven central complex lines. They have been further analysed at the molecular level. So far, I have obtained and sequenced a number of cDNA clones for these lines, however, I do not intend to present all the data in this thesis. I will focus on the analysis of line c507 and c232, in which their P elements were identified to insert in the genome about 500 bp away from each other but in different orientation (see Fig. 6.1a). This chapter will first describe isolation of cDNA clones for line c507 and c232. The chapter will then report the sequencing analysis. Finally, the Northern analysis and coexpression patterns will be presented and the conclusion will be made.
6.2 Isolation of cDNA Clones Related to c507 Flanking DNA
To isolate cDNA clones of both sides of the P[GAL4], a conventional cDNA library screening approach was chosen. A male head cDNA library in λ-phage NM1149 (which was made by S. Russell 1989) was available for use in our laboratory.
6.2.1 cDNA clones from the "downstream" side of the P[GAL4] element in line c507
As discussed in section 5.4, the rescued plasmid from pPC507 produced a 0.9 kb BamHI fragment which contains a transcribed sequence judged by "reverse Northern" analysis. This fragment was used as a probe for isolation of cDNA clones related to the flanking genes downstream of the P[GAL4] element. Four independent cDNA clones were obtained and designated pMY3, pMY5, pMY8, and pMY10 (Fig. 6.1).
In order to determine the relationships between cDNA clones, they were digested with the enzymes KpnI, PstI, BamHI, XhoI, SalI and SstII. The result has provided sufficient information to construct a restriction map for these cDNA clones. It appears to
Figure 6.1 The organisation of the region surrounding the PIGAL-4J elements in lines c507 and c232.
(A) shows the restriction map of the genomic region encompassing the PIGAL-4J insertion of line c507 and c232. Two P[IGAL-4J] elements were inserted into the *Drosophila* genome only 46 bp away from each other but in different orientation. (B) shows two probes from the genomic DNA fragments used for screening cDNA library. (C) shows one group cDNAs matched upstream of the P[IGAL-4J] and two groups of transcript units in the downstream of the P[IGAL-4J] insertion in line c507. (D) shows the names of genes. The restriction sites shown are K-KpnI, B-BamHI, P-PstI, H-HindIII, E-EcoRI. NB: It should not be assumed that the cDNAs are necessarily co-linear with the genomic DNAs.
divide these cDNA into two groups. Group one consists of the pMY3 and pMY8, pMY10. The longest clone pMY8 hybridised with the other two clones of this group. So, they were thought to be same transcribed sequences but different length. This result was also supported by sequence data (data not shown). But the pMY5 belongs to a different group of cDNA. It had no restriction cleavage site for the above enzymes and did not hybridise with other cDNA clones.
Meanwhile, genomic DNA was probed with the cDNAs from pMY8 and pMY5. The two groups of cDNA hybridised to the closely linked positions in the genome. The region of hybridising unit from pMY5 is nearer to the P[GAL4] element while that of hybridising unit from pMY8 is located further away. A schematic diagram of the organisation of the transcription units relative to the genomic DNA is shown in Figure 6.1. In this diagram, however, it should not be assumed that the cDNAs are necessarily co-linear with the genomic DNAs because the mapping of potential intron/exon boundaries has not been carried out completely.
### 6.2.2 Isolation of A "Full Length" cDNA Clone Related to pMY5
Using a $^{32}$P labelled DNA probe generated from the insert sequence of pMY5, a preliminary Northern blot from head poly (A) mRNA demonstrated the presence of two transcripts of about 2.0 kb and 3.7 kb. The 2.0 kb band had a strong signal which indicates the size of main transcript of this gene (Fig. 6.2). This result also suggested that pMY5 (0.8 kb) was not the full length of the cDNA. Therefore it is necessary to find longer cDNA clones related to pMY5.
When the screening of the cDNA library again, the longer cDNA clone (1.8 kb) was obtained and named as pMY51. It is presumed that the 1.8 kb clone is a full length of cDNA because this gene has a abundant transcript in about 2.0 kb (Fig. 6.2). In fact, the longer clone is an extended version of the clone pMY5 which has a internal EcoRI site (Fig. 6.1). This suggests that when the cDNA library was constructed, the methylation used to protect against restriction of internal EcoRI site was not fully functional.
Figure 6.2 A Northern blot using an insert probe from pMY5.
A Northern filter with the heads poly(A)$^+$ RNA prepared from the wild type adults (Canton S) was hybridised to a cDNA probe from pMY5.
6.2.3 cDNA clones from the "upstream" side of the P[GAL4] element in line c507
It is possible that a gene relevant to enhancer trap expression patterns is located upstream side of the P[GAL4] insertion. Therefore, it was necessary to get cDNA clones from this side. A 4.5 kb HindIII fragment from the rescued plasmid, pkC507, was used as a probe to screen a head cDNA library. Five "positive" plaques were identified. Three independent cDNA clones were purified and designated pkMY2, pkMY3 and pkMY4. The longest clone pkMY4 showed hybridisation with others, suggesting that these three cDNA clones come from the same transcribed sequence but are different versions (Fig. 6.1c).
In order to determine the corresponding position of the transcribed region within the genome, the genomic DNA clones were restricted with different enzymes and hybridised with the cDNA probe generated from the whole insert of pkMY4. The approximate corresponding position of the cDNA is illustrated in Figure 6.1 as well.
6.2.4 Genomic Southern for three different groups of cDNA clones
In order to determine if a gene has a single copy in *Drosophila*, genomic DNA derived from Canton-S was probed with representative cDNAs. Figure 6.3B showed that genomic DNA was restricted with *Pst*I and hybridised with the cDNA probe of pMY51. A strong hybridisation signal in band of about 5 kb was found which was expected, and two faint bands were seen in 7 kb and 9.5 kb. This result could be explained in two ways: Either this gene possibly has two more copies in the genome or has a homology with other genes of their family.
When whole insert from pMY8 was probed for genomic DNA restricted with *Bam*HI, one strongly hybridising band of 2 kb and one weaker band of 0.9 kb were seen from Figure 6.3C. This is because the main insert of the pMY8 cDNA clone corresponds to the 2 kb of *Bam*HI fragment and a few base pairs DNA are homologous to the 0.9 kb band in the genome. Similarly, when the genomic DNA was digested with *Hind*III and probed with the insert of pkMY4, one band of 4.5 kb was seen (Fig. 6.3A). Above results suggest that these two clones represent single copy genes in *Drosophila*. In addition, the number and size of bands observed in these cases agree with the restriction map derived earlier.
6.3 Analysis of Sequence
Three representative cDNAs (pMY8, pMY51 and pkMY4) were identified to locate at both sides of the P[GAL4] insertion (Fig. 6.1). They all have been sequenced. Then, their open reading frames, putative terminators and protein sequence obtained from translation of the DNA sequence were also analysed.
Figure 6.3 Southern blot of *Drosophila* genomic DNA (wild type CS).
Upper diagram shows the genomic map and corresponding cDNA clones surrounding the [PGAL4] element of line c507 & c232. H (*HindIII*); P (*PstI*); B (*BamHI*).
A Genomic DNA was digested with *HindIII* and probed with whole insert of pkMY4 (probe A).
B Genomic DNA was restricted with *PstI* and probed with whole insert of pMY51 (probe B).
C Genomic DNA was restricted with *BamHI* and probed with whole insert of pMY51 (probe C).
6.3.1 General Sequence Features Of the pkMY4 cDNA clone
When sequencing clone pkMY4, the partial sequences were put through the GenEMBL database as a DNA search using Blastn. The result (Fig. 6.4) shows the alignment of the published sequence of the *Drosophila* Calcineurin A1 and the 5' sequence from pkMY4. Clearly pkMY4 is a cDNA clone of a Calcineurin A1 transcript (Guerini et al., 1992). When comparing the amino acids between the calcineurin A1 and the pkMY4, they are also identical. The amino acids derived from the pkMY4 are parts of the coding region of the calcineurin A1. (Fig. 6.4).
Calcineurin, Ca$^{2+}$/calmodulin-stimulated protein phosphatase, has been implicated in synaptic transmission, cytoskeletal dynamics and other calcium-regulated events (Tallant and Cheung, 1986; Klee et al., 1987). It is composed of two subunit: a catalytic subunit, calcineurin A and a Ca$^{2+}$-binding regulatory subunit, calcineurin B. The Ca$^{2+}$stimulation of calcineurin is mediated by two different Ca$^{2+}$-binding proteins, calcineurin B and calmodulin; Ca$^{2+}$ binding to calcineurin B promotes a small basal activity, whereas Ca$^{2+}$ binding to calmodulin facilitates calmodulin's interaction with calcineurin A and results in a ten-fold further activation of the enzyme (Klee et al., 1987). Calcineurin A can be divided into two classes of clones, calcineurin A1 and calcineurin A2, based on the restriction mapping, sequence analysis and the Northern blot (Guerini and Klee, 1989).
In *Drosophila*, calcineurin A was originally cloned by homology to the human sequence. The cDNA sequence of calcineurin A1 contains a 1704 bp open reading frame (ORF) coding for a 568 amino acid protein. The putative ATG initiator codon is preceded by a 60-bp open reading frame without initiator codon. The coding region consists of 12 exons. The 263-bp 3'-noncoding sequence lacks a poly(A) tail and the polyadenylation signal (Guerini et al., 1992). However, from my sequencing data of the clone pkMY4, a poly(A) tail and the polyadenylation signal **AATAAA** were found, in addition to the sequence overlapped with the calcineurin A1 (data not shown). This success resulted from the different cDNA library used.
6.3.2 General Sequence Features Of the pMY51 cDNA Clone
Figure 6.5 shows the full length sequence of the pMY51 cDNA clone which has 1888 bp long including 22 poly A$^+$ tail remnant. This cDNA contains a 547 amino acids ORF identified by the MacVector™. The long ORF of pMY51 is translated in the first frame, beginning from nucleotide 43 and ending at nucleotide 1758. It is followed by a TGA stop codon encoded by DNA residues 1759-1861. By searching the database, this predicted polypeptide has a significant homology to the alkaline phosphatase (ALP) gene family in other organisms at protein level (Fig. 6.6). No *Drosophila* homologue has so far been described. Like most ALP, the predicted polypeptide from pMY51 has five potential N-linked glycosylation signals. These sites vary with different species (Manes *et al.*, 1990; Itoh *et al.*, 1991). By the Prosite search (Fuchs, 1994), pMY51 has a matching pattern with the APL, which is "VPDSAGTAT". Unexpectedly, the normal polyadenylation site is not found in the 3' untranslated region of cDNA.
Alkaline phosphatase (EC 126.96.36.199) (ALP) is a zinc and magnesium-containing metalloenzyme which hydrolyses phosphate esters, optimally at high pH. It is found in nearly all living organisms, with the exception of some plants. In *E. coli*, ALP (gene *phoA*) is found in the periplasmic space. In yeast it (gene *PHO8*) is found in lysosome-like vacuoles and in mammals, it is a glycoprotein attached to the membrane by a GPI-anchor (McComb *et al.*, 1979; Trowsdale *et al.*, 1990).
In mammals, four different isozymes are currently known. Three of them are tissue-specific: the placental, placental-like (germ cell) and intestinal isozymes. The fourth form is tissue non-specific and was previously known as the liver/bone/kidney isozyme. Each isozyme is encoded by a separate gene (Harris, 1989; Manes *et al.*, 1990). In *Drosophila*, at least four allelic forms of the ALP were reported by histochemical studies (Yao, 1950; Beckman and Johnson, 1964; Schneiderman *et al.*, 1966; Harper and Armstrong, 1972; 1973), but there is no idea about relationship between mammals and *Drosophila*.
Figure 6.5 The nucleotide and deduced amino acid sequence of the pMY51 cDNA clone. The nucleotide sequence of the full-length alkaline phosphatase cDNA is shown along with the amino acid sequence of the longest deduced open reading frame starting with a methionine, numbering is in bold. Numbering of the DNA starts at the most 5' end base of the cDNA. Sites of putative N-glycosylation are underlined. The stop codon TGA is marked by asterisks.
TGAATGAGAGTGAGCGAGATGGGAGAAGTGATCGTTGCACAATTAAACAAATAGATACTACAAAAAATAAAAAAAAAAA
AAAAAAA
Figure 6.5 The nucleotide and deduced amino acid sequence of the pMY51 cDNA clone. The nucleotide sequence of the full-length alkaline phosphatase cDNA is shown along with the amino acid sequence of the longest deduced open reading frame starting with a methionine, numbering is in bold. Numbering of the DNA starts at the most 5' end base of the cDNA. Sites of putative N-glycosylation are underlined. The stop codon TGA is marked by asterisks.
In order to compare the pMY51 clone, presumably encoding a ALP in *Drosophila*, to the ALP from other species, the putative amino acids sequence is aligned with those deduced for the ALP from different species, using the PILEUP programme in GCG package (see section 188.8.131.52). The result from the pileup's can be seen in Figure 6.6. Amino acid positions that are identical in all eight proteins, or in at least six proteins (one is *Drosophila*) are marked by stars and quotation marks respectively. Among the *Drosophila* and other species, there is about 40% homology (24% amino acid identity and 15% partial homology) in 533 overlap amino acids. There is over 70% similarity between *Drosophila* ALP and any one individual ALP in question by FastA search (data not shown).
It is accepted that knowledge of the intron-exon organisation of a gene helps in identifying possible functional domains in the protein molecule. However, in the case of ALPs, the intron-exon junctions do not correspond to anything that could loosely be defined as a catalytic domain. Instead, the residues that constitute the "active site pocket" are spread throughout the molecular (Millan *et al.*, 1988). The metal and substrate phosphate binding sites determined in the *E. coli* ALP by X-ray crystallography (Sowadski *et al.*, 1985) are shown and marked with "@" in Figure 6.6. These conserved sites in *Drosophila* are identical to other species, reflecting essential functional domains. A significant conservation in residues coordinating the active pocket suggested that *Drosophila* ALP has a common ancestral gene with other species APL genes.
In order to analysis the evolutionary relationships among the ALPs (Human, Mouse, Bovine, Rat, *Drosophila*, *Bombyx* and *E coli*) at protein level, "growtree and distances" programmes from GCG package were used (see section 184.108.40.206). Based on the above result, a phylogenetic tree of parts of ALPs is constructed and shown in Figure 6.7. The diagram shows that mammalian ALPs are more closely related than insects or *E coli*; two insects (*Drosophila* and *Bombyx*) are more related than others; *E coli* APL seems to be separate from others, although they all have a common ancestral root. These data are consistent with previous homologue analysis. There is more homology among the mammalian ALPs (Millan, 1988; Manes *et al.*, 1990). There is higher homology between
Figure 6.7 Schematic tree of alkaline phosphatase genes.
Diagram showing the postulated evolutionary relationships among Mouse ALP, tissue-nonspecific isozyme precursor (Terao and Mintz, 1987); Rat ALP, tissue-nonspecific isozyme precursor (Thiede et al., 1988); Bovine ALP, tissue-nonspecific isozyme precursor (Garattini et al., 1987); Human ALP, tissue-nonspecific isozyme precursor (Weiss et al., 1988); Silkmoth (*Bombyx*) membrane-bound ALP precursor (Itoh et al., 1991); *Drosophila* ALP (pMY51); *E. coli* ALP (Bradshaw et al., 1981). (diagram is not to scale)
insects than mammalian ALPs (this section) and less homology between *E. coli* ALP and mammalian or insects ALPs (Millan, 1988; Itoh *et al.*, 1991). This may reflect the evolutionary history of the ALP gene family. They appear to have evolved from a common ancestral gene by a series of successive gene duplications.
The strong sequence homology between this locus in *Drosophila* and in other organisms is overwhelming evidence that this cDNA (pMY51) encodes an alkaline phosphatase in *Drosophila*, which can be classified as tissue non-specific isozyme.
### 6.3.3 General Sequence Features Of the pMY8 cDNA clone
The pMY8 cDNA was sequenced on both strands. The full length sequence is 887 bp long excluding the 3' 20 bp polyA tail remnant as shown in figure 6.8. Eighty two nucleotides upstream of 3' poly (A) tracts in the cDNA clone is the sequence TATAAA. This is not the most common of polyadenylation signals but is used occasionally (Manley, 1988). When the MacVector™ map program was used to predict the restriction map of the cDNA, it showed very close correspondence to that obtained using restriction enzymes.
The MacVector™ was also used to identify ORF and the predicted polypeptide. Figure 6.8 also shows that this cDNA contained a long ORF with a hypothetical translation product of 223 amino acids. This ORF is bounded at the 5' end by a potential ATG start site at DNA residues 64-66 and terminated at the 3' end by a TAG stop codon encoded by DNA residues 733-735. The remainder of the cDNA is presumed to be the 3' untranslated region. But the deduced amino acid sequence data available does not identify pMY8 as being related to other proteins in the BLAST, Swiss-Protein, or Pro-Site database. The gene, therefore, appears to encode a novel protein possibly involved in neural function because the Northern blot shows that this clone has a head-elevated expression (see section 6.4.1).
Figure 6.8 The nucleotide and derived amino acids sequence of the cDNA clone pMY8
Shown is the complete sequence of cDNA clone pMY8 with its hypothetical 223 amino acid translation product. Numbering of the DNA starts at the most 5' end base of the cDNA and numbering (in bold) of the protein starts at the first methionine of the open reading frame. The putative polyadenylation signal sequences is underlined.
6.4 Northern Analyses
In order to investigate the above genes expression in the *Drosophila*, Northern blots are performed to analysis these cDNA clones.
6.4.1 Northern Blotting Using Head and Body mRNA
The whole insert from pMY8 was excised using *Eco*RI and *Hind*III and used to probe a Northern blot made with both head and body poly(A)+ mRNA. The resulting autoradiograph (Fig. 6.9a) shows hybridising transcripts to the pMY8 probe were found abundantly in the head but relatively weakly in the body. By comparison to the *rp*49 (O'Connell and Rosbach, 1984) load control, the head RNA was approximately evenly loaded relative to that of the body, suggesting pMY8 has a head-elevated expression. From the Northern blot estimates of the band size is around 0.9 kb. As the cDNA pMY8 insert is about 0.9 kb, it is very likely that this clone contains a full length copy of the message. This is also supported by sequence data (Fig. 6.8).
Figure 6.9b shows a Northern blot hybridised with a cDNA probe generated from the whole insert sequence of pMY51 (ALP). The probe clearly identifies at least two transcripts in both head and body. They are approximately 2.0 kb and 3.7 kb. It can be seen from blot that there are similar transcripts expression in the head and body. This blot was reprobed with *rp*49 as a loading control. By comparison to the *rp*49 load control the head RNA was approximately evenly loaded relative to that of the body, suggesting that "alkaline phosphates" gene in *Drosophila* has expression in both head and body. This agrees with the result of *in situ* hybridisation to tissue sections (see section 6.5.2).
The head and body Northern blots probed with the insert from pkMY4 of calcineurin A1 failed to detect a clearly discernible band (data not shown). Guerini *et al.*, (1992) reported the same result. Presumably this gene is expressed at very low levels. It may be a specialised form required in only a few cell type at specific time during
Figure 6.9 Northern blots analysis
(a) RNA blot of adult head and body poly(A)+ RNA (10 µg per lane) were hybridised to a cDNA probe from pMY8. (b) RNA blot of adult head and body poly(A)+ RNA (10 µg per lane) were hybridised to a whole insert probe generated from the pMY51 clone. (c) A Northern filter containing 10 µl of mRNA from each of the stages indicated was hybridised to a whole insert probe from pMY51. The mRNA was prepared from *Drosophila* Canton-S mixed embryo (E), mixed third instar larvae (L), mid-pupal (P), adult head (H) and adult body (B). Subsequent these filters were reprobed with a ribosomal protein probe, *rp49 EcoRI-HindIII fragment*, as a loading control. (d) The poly(A)+ RNAs (10 µg per lane) prepared from the P{GAL4} line c507 and wild type strain (CS) were hybridised to a whole insert probe from pMY51.
6.4.2 A Developmental Northern Blot
The cDNA clone pMY51 was used to probe Northern blot of the mRNA prepared from various developmental stages, e.g. the mixed embryo, mixed third instar larvae, mid-pupal, adult head and adult body of the Canton-S wild type. The blot result is shown as figure 6.9c, together with an autoradiograph produced after the blot was re-probed using rp49 as a loading control. The developmental RNA blotting experiment indicated that the abundance of alkaline phosphatase RNA was expressed during the larvae and adult stages. But no detectable transcription was observed in embryo and pupal stages. This may imply that the gene has not switched on during the embryonic stage and thus it will start to express at the larvae stage. The dramatic increase of alkaline phosphatase activity in third instar larvae before the secretion of pupal cuticle suggested a role for this enzyme in cuticle formation and possibility of its regulation by the ring gland (Schneiderman et al., 1966). Afterwards, the gene expression reduces to undetectable level by Northern blot during pupal stages. Then the gene has abundant expression in the adults. It is important for adults to have transport function in the brain and the Malpighian tubules (see section 6.6). The developmental Northern observation is in good agreement with the observation of the development study using X-Gal staining in section 4.5. It also supports the earlier data that alkaline phosphatase activities in insect, are highest during the active larval and early adult stages and lowest during pupation (McComb et al., 1979).
6.4.3 Northern Analysis for Line c507 and Wild Type Flies
To determine whether insertion of P element has disturbed the gene expression, Northern blot was performed. In this experiment, the poly(A)+ RNA prepared from wild type (Canton-S) and P[GAL4] line c507 flies was hybridised with cDNA probe generated from a whole insert of pYM51. The result (Fig. 6.9d) shows that these lanes have hybridisation signals at about 2 kb. No difference between wild type and line c507 was
observed. This is because that the P[GAL4] was not inserted into the gene of this line, so insertion didn't disturb the gene expression and its function. But the imprecise excision can be used to generate total loss-of-function alleles (Engels, 1989). The imprecise excision experiment is being carried out.
6.5 in situ Hybridisation
in situ hybridisation techniques are used to localise specific nucleic acids sequences in morphologically preserved chromosomes, cells or tissue sections. In combination with immunocytochemistry, in situ hybridisation is capable of relating microscopic topological information to gene activity at the DNA, mRNA and protein level.
6.5.1 Localisation of the pMY51, pMY8 and pkMY4 cDNA clones to Polytene Chromosomes
In order to ensure the cDNA fragments corresponding to the flanking regions of P[GAL4] insertion, polytene in situ s were performed as described in section 2.3.3 using the whole inserts of pMY51, pMY8 and pkMY4 as probes, respectively. When these probes were hybridised to polytene chromosome of wild type flies, the hybridisation signals were seen in the band 100B on the third chromosome (Fig. 6.10). This band is the identical region as the P[GAL4] element insertion (section 3.5).
Guerini et al., (1992) localised the calcineurin A1 gene into 21EF on the second chromosome. However we found this gene located at the third chromosome. A more likely explanation is that they may have made wrong chromosomal localisation. By comparison to the sequence from both of us, we cloned the same gene. It should be at the same position on the polytene salivary gland chromosome because the genomic southern has shown only one copy of this cDNA in the Drosophila (see section 6.2.4.) Another evidence that the P[GAL4] was inserted into the third chromosome came also from genetic crosses for line 507. Furthermore, both cDNA pMY51 and pkMY4 hybridised genomic DNA clone λ6
Figure 6.10 Polyene chromosomal *in situ* using different probes from pBluescript, pMY51, pMY8 and pkMY4 cDNA clones.
Arrows indicate the position of the hybridisation signals at 100B on the third chromosomes. A shows a set of chromosomes from line c507 displaying the hybridisation signal using PBS probe, which hybridises the PGAL4 element. B, C and D show a different set of polyene chromosomes from wild type strain displaying the hybridisation signals as same position as A when probes from the pMY51, pMY8 and pkMY4 cDNA clones are used respectively.
Therefore, we can say positively that calcineurin A1 gene is located at 100B on the third chromosome rather than at 21EF.
6.5.2 Co-localisation of LacZ and Alkaline Phosphatase Gene Expression
With respect to the enhancer trap system, it makes the assumption that expression pattern of the reporter gene will reflect that of a nearby *Drosophila* gene. In other words, a relevant endogenous gene should have same expression pattern as that of the reporter. In the case of line c507, GAL4-directed β-gal expression patterns were found in the ellipsoid body of the brain and Malpighian tubules in body. In order to determine whether the alkaline phosphates gene cloned by plasmid rescue has same expression patterns in wild type fly, *in situ* hybridisation to the head and body using digoxigenin labelled RNA probes was performed. The DIG RNA probes were generated from pMY51 according to Boeringer Mannheim Digoxigenin (DIG) labelling kit and hybridised to 14μm cryostat head and body sections (described as in section 2.3.1) The hybridisation patterns were detected using the NBT/X-Phosphate colour reaction. Positive and negative controls are performed along side the pMY51 *in situ* and for this other clones (as negative) and pST41 insert (a Opsin clone as a positive control) were used.
Figure 6.11 shows that co-expression of alkaline phosphatase gene in the cell bodies of the ellipsoid body and the cell bodies of the Malpighian tubules with X-gal staining by *in situ* hybridisation to tissue sections. Therefore, it is reasonable to say that the gene relevant to enhancer trap element has been found.
As above *in situ* hybridisation using RNA or DNA probe only allows the reaction to occur in the nuclear of the cell bodies, it would not be expected to see the ring shape (axonal projections) of the ellipsoid body. Hybridisation signals can only be seen in the cell bodies of the ellipsoid body and the Malpighian tubules. If antibodies against relevant genes are available, the antibody staining can be used to improve result.
Figure 6.11 Co-localisation of expression patterns in the tissue sections by *in situ* hybridisation.
(A) In P[GAL4] line c507, the cell bodies of the ellipsoid body (*eb*) are stained by X-Gal. (B) For wild type fly, the coexpression of pMY51 RNA is detected in the same cell bodies of the *eb* by DIG-RNA labelling probe. The hybridisation signals are arrowed. (C) In P[GAL4] line c507, the Malpighian is stained by X-Gal. (D) For wild type fly, the coexpression of pMY51 RNA is detected in the same tissue by DIG-RNA labelling probe. The hybridisation signals are arrowed. The closer view of hybridisation regions are stuck in the corners of photos. The open arrow indicates the hybridisation signals with different colour detection system.
During the colour detection of this experiment, the fresh lavamisole was used to block endogenous phosphatase expression (McComb et al., 1979), even though residual alkaline phosphatase activity was usually lost during hybridisation. On the other hand, in order to avoid directly staining by the NBT and X-phosphate (J. Dow, per comm.), different colour detection system with diaminobenzidine (DAB)/H$_2$O$_2$ (as described method in section 2.3.1) was used to confirm the result (Fig. 6.11).
Whole mount embryonic *in situ* was performed using DIG labelled RNA probe from pMY51. No expression patterns were detected. This result was consistent with that of X-Gal staining and developmental Northern blot.
On the other hand, when whole insert from pkMY4 (calcineurin A1) was used as a DIG probe for *in situ* hybridisation to the adult head and body sections, no staining patterns were matched with the patterns stained by X-Gal. In the head sections, staining was found in all the cortex layer of the brain but pale staining pattern was observed in the body (data not shown).
Taken together these data suggest that a "correct" enhancer was trapped and "correct" gene, *Drosophila* alkaline phosphatase, was identified by an enhancer trap approach.
### 6.6 Conclusion
We have successfully identified and cloned the genes flanking P[GAL4] element of line c507 by enhancer trap system. Three different genes located at 100B were cloned and characterised. One of them is called calcineurin A1, the Ca$^{2+}$/calmodulin-stimulated protein phosphatase (Guerini et al., 1992) which is located at the upstream of the P[GAL4] element. Other two closely linked genes are localised at the downstream of the P[GAL4] element. The cDNA clone pMY51 near to the P[GAL4] has a significant homology to alkaline phosphatase from a variety of organisms. And other cDNA clone pMY8 which sits
further away from P[GAL4] has a head-elevated expression judged by Northern blot, but it is yet to be identified as being a member of a known gene family. *in situ* hybridisation to tissues sections of flies reveals that the cDNA clone pMY51 is from a nearby gene which has same expression patterns with those stained by X-Gal. This gene, *Drosophila* alkaline phosphatase, has expression patterns in the ellipsoid body of the brain and in the Malpighian tubules of *Drosophila*. What is its possible function in the *Drosophila* ellipsoid body and Malpighian tubules?
Although the function of alkaline phosphatase is not well understood, a large number of experiments demonstrate its possible functions, such as hydrolysis of phosphate esters at high pH, transfer of phosphates from a high-energy to a lower-energy state, transport of a number of substances (e.g. Na\(^+\), K\(^+\), inorganic phosphate,) across membranes (McComb *et al.*, 1979).
Alkaline phosphatase activities in the brain are generally lower than those in liver and kidney, however, its distribution in the brain has been reported in different species (McComb *et al.*, 1979). More specific to the nervous tissue, intense alkaline phosphatase activity has been located in nerve cell bodies and processes in some parts of central and peripheral nervous systems of various animals such as mouse (Sood and Mulchandani, 1977), rat (Nandy and Bourne, 1963), guinea-pig (Song *et al.*, 1994), monkey (Friede, 1966) and fish (Sood and Sinha, 1983). At the ultrastructural level, the enzyme has been localised on the outer surface of plasma membranes of nerve cell bodies and dendrites (Mayahara *et al.*, 1967; Mori and Nagano, 1985), postsynaptic membrane and synaptic vesicles (Sugimura and Mizutani, 1979). Biochemical studies also suggested that alkaline phosphatase is associated with synaptic vesicles isolated from bovine cerebral cortex (Zisapel and Haklai, 1980). In view of these facts that the ALP reactivity has been localised to discrete subsets of neurones in different species, it is suggested that the ALP has a function related to particular features of the reactive neurones. From all available evidence, it appears that the ALP may play some role in transmembrane transport and cell differentiation or proliferation in the nervous tissue. Moreover, the synaptic alkaline phosphatase may play
an important role in the storage and release of transmitter, postsynaptic reception of chemical stimuli, and Ca$^{2+}$ transport following stimulatory excitation of the membrane.
In our preparations, X-Gal and "alkaline phosphatase" cDNA clone have co-expression patterns in the ellipsoid body of the brain and the Malpighian tubules of the abdomen in *Drosophila* (see section 6.5.2). Therefore, it is reasonable to say that the ellipsoid body neurones are alkaline phosphatase-reactive neurones which have the same function as discussion above. More specifically, it is possible that the ALP is functional in the storage and release of transmitter in neurones of the ellipsoid body. On the other hand, in vertebrates, evidences show that alkaline phosphatase is related to absorption, transport and secretion in intestine and kidney (McComb *et al.*, 1979). It is therefore not surprising that the alkaline phosphatase distributes in the Malpighian tubules of *Drosophila* abdomen because this tissue has similar functions as vertebrates, e.g. transport, secretion and absorption (Maddrell and O'Donnell, 1992; Dow *et al*, 1994).
Chapter 7
Discussion
7.1 Introduction
7.2 General discussion
7.3 Future work
7.1 Introduction
Although most of the results were discussed within their own chapters, this general discussion aims to link relevant findings from different chapters and to assess their significance. It also attempts to sum up the questions that have arisen in light of the data presented and suggest future work which would address these questions.
7.2 General Discussion
The enhancer trap technique has allowed us to study structures of the *Drosophila* brain and identify some genes relevant to the central complex function in the brain.
Over 1400 of P[GAL4] enhancer trap lines have been analysed. They show a great variety of β-galactosidase expression patterns, reflecting the influence of many different genomic regulatory elements on the P[GAL4] reporter gene. More than 300 display interesting patterns in the brain from an anatomical perspective. Of these, as many as 100 are more or less restricted to specific regions or neuronal sub-populations of brain. It is obvious that these P[GAL4] lines are a resource that can be exploited in a number of different contexts including: domain specific expression of toxins or inhibitors, domain specific rescue of behavioural mutations, neuro-anatomical studies etc. (Sweeney et al., 1995; Hidalgo et al., 1995; Yang et al., 1995; Ferveur et al., 1995; O’Dell et al., 1995; Smith and Shepherd, 1995)
Many P[GAL4] lines we isolated can be used for various purposes as marker lines for anatomical analysis. The expression patterns of P[GAL4] lines do reveal novel substructures of the neuropil most of which are not apparent in silver stained material. We have been able to observe the subdivision of the *Drosophila* mushroom bodies (Yang et al., 1995) and subdivide the ellipsoid bodies of the central complex into the four genetically distinct regions (see chapter four). Stocker (pers. comm.) also found novel interneurons in
the antennal lobe of the *Drosophila* brain. Smith and Shepherd (1995) reported that they used one of our P[GAL4] line to visualise the central projections of proprioceptive sensory neurons of the thorax and abdomen. In the abdomen, they found GAL4 was expressed in Wheeler's organ and a segmentally repeated array of internal sensory neurons that have not been previously described. In addition to adults, Ito *et al.* (1995) used GAL4 as a marker to reclassify the glial cells during late embryogenesis and generate a three-dimensional map of the glia in the embryonic nervous system. More recently, the green fluorescent protein (GFP) from the jelly fish has been used as a cell marker, combined with P[GAL4] system, to label neurons in living embryos, larvae, pupae and adults for developmental study *in vivo* without invasive manipulation (Yet *et al.*, 1995; Brand, 1995; Y. Yu, Glasow). Taken together, P[GAL4] enhancer trap lines are indeed excellent neuronal markers for anatomical and developmental study.
The GAL4 expression patterns were used to analysis the substructures and development in the *Drosophila* central complex (see chapter four). The expression patterns in neuronal organisation may implicate the multiple integrative role for the central complex. Developmental study revealed that the central complex in *Drosophila* appears from pupal stage to adults. It seems that the central complex has not formed in early stages such as embryo and larvae. This result is consistent with that of other researchers (Hanesch, 1989; S. Renn, personal comm.).
In *Drosophila*, the "central complex" is a term used to describe a series of intimately related neuropsils in the midbrain (Hanesch *et al.*, 1989). However, in most insects the "central body" is termed to refer to main neuropsils in the midbrain (Williams, 1975; Strausfeld, 1976; Mobbs, 1982; Homberg, 1987). In order to be consistent with other insects, it is suggested that the term of "central body complex" for *Drosophila* should be used in future. It includes four main substructures: the protocerebral bridge (*pb*), the fan-shaped body (*fb*), the ellipsoid body (*eb*) and the paired noduli (*no*) and two accessory structures: the ventral bodies (*vbo*) and the lateral triangles (*ltr*).
Enhancer-trap elements are not mere anatomical markers. Besides revealing intriguing cellular arrangements within the brain, each staining pattern implicates a flanking gene in their functions. A large number of staining patterns provide an abundance of target site for brain function-specific gene actions. To identify the genes relevant to central complex function, seven P[GAL4] enhancer trap lines with staining patterns specific to the central complex were selected. Genomic DNAs flanking each insertion site were cloned by plasmid rescue (Pirrotta, 1986) "Reverse Northern" analysis were performed to identify the transcribed units. Rescued genomic DNAs were used as probes for screening a cDNA head library. Corresponding cDNA clones were isolated.
In case of line c507, three different genes around P[GAL4] were cloned and characterised. Insertion site of P[GAL4] was mapped to between genes of the calcineurin A1 and the alkaline phosphatase (see Fig. 6.1).
One of gene located at the upstream side of the P[GAL4] is called calcineurin A1, the Ca$^{2+}$/calmodulin-stimulated protein phosphatase (Guerini et al., 1992). Calcineurin is a brain enriched phosphatase that plays an important role in the regulation of brain physiology, including synaptic transmission, cytoskeletal dynamics and other calcium-regulated events (Tallant and Cheung, 1986; Klee et al.; 1987). This gene is very interesting for our group for further analysis even if it is not relevant to the enhancer trap expression pattern.
Other two closely linked genes are localised at the downstream side of the P[GAL4] element. The 1.8 kb pMY51 cDNA near to the P[GAL4] contains a 356 amino acids ORF producing a predicted polypeptide that shows homology to the alkaline phosphatase from other organisms at protein level (see section 6.3.3). *in situ* hybridisation to tissues sections of flies reveals that this cDNA clone represents a nearby gene which has same expression patterns in the ellipsoid body and the Malpighian tubules with those stained by X-Gal. Northern blot also supports this result. The other cDNA clone pMY8, which sits further away from P[GAL4], has a head-elevated expression judged by Northern blot, but it is yet
to be identified as being a member of a known gene family.
The ellipsoid body of the central complex is mainly thought to have an inhibitory role in its function because of distribution of inhibitory neurotransmitter in these neurons (Hanesch et al., 1989; Bausenwein et al., 1994). Localisation of alkaline phosphatase activity in the ellipsoid body also supported this notion as alkaline phosphatase activity was confined to inhibitory motor neurons of the guinea-pig as well (Song et al., 1994).
Alkaline phosphatase (ALP) are glycoproteins and there are at least four isozyme forms of APL in human. They are tissue nonspecific ALP, also known as "liver/bone/kidney' ALP, intestinal ALP, placental ALP and placental-like APL. Each isozyme is encoded by a separate genes. Expression of the intestinal, placental, and placental-like ALPs is limited to a few specific tissues as their names indicate, whereas tissue nonspecific ALP is present in many cell types (McComb et al., 1979; Harris, 1989; Manes et al., 1990). Therefore, the *Drosophila* alkaline phosphatase encoded by pMY51 cDNA can be grouped into "tissue nonspecific ALP". It is not surprising that this ALP express in the ellipsoid body of the brain and the Malpighian tubules of the gut in *Drosophila*. It is controlled by the third chromosome locus, 100B, corresponding to one of *Drosophila* alkaline phosphatase described by Beckman and Johnson (1964). This type ALP is different from other ALP, from adult hindgut, which is controlled by the second chromosome locus Aph-2 (Schneiderman et al., 1966).
It is interesting to find that Alkaline Phosphatase (EC. 220.127.116.11) and Protein Phosphatase (also called calcineurin) (EC. 18.104.22.168) are located in the same region of polytene chromosome at 100B. They are only a few kb away each other. Another kind of phosphatase, Acid Phosphatase (EC. 22.214.171.124) is located at 99C of the same chromosome based on deletion mapping (Frisardi and MacIntyre, 1984). All the above three phosphatases are classified as "phosphoric monoester hydrolases" by IUB for Enzyme Nomenclature in 1978. It is likely that these three similar functional genes are clustered in the same region of the chromosome. A similar phenomenon was observed in human
alkaline phosphatases (Harris, 1989). If this is assumed to be the case, it is possible for us to predict that more "phosphoric monoester hydrolases" genes will be found near that region.
As mentioned before, the P[GAL4] system has allowed to ablate a selected cells by expressing cell autonomous toxin genes under the control of a UAS. In *Drosophila* nervous system, for example, targeted expression of tetanus toxin light chain specifically eliminated synaptic transmission and caused behavioural defects; in one case, the olfactory escape response was reduced (Sweeney *et al.*, 1995). Again, ablation of the interface glia early in development led to a complete loss of the longitudinal axon tracts, and ablation of the glia later in embryonic development resulted in defects comprising of weakening and loss of axon fascicles within the connectives (Hidalgo *et al.*, 1995). For us, such a strategy could be used to ablate the specific cells in the mushroom bodies or central complex of the brain to test their functions and other roles. This system is a simple and efficient method of targeted cell ablation because cell killing is rapid due to the expression of the wild-type toxin, no invasive manipulation of the animal is required and cell ablation is autonomous (O'Kane and Moffat, 1992; Sentry *et al.*, 1993; Hidalgo *et al.*, 1995).
Enhancer trapping, however, is not without problems. It is generally accepted that P elements do not insert randomly in the genome (Engels, 1989; Smith *et al.*, 1993). Some genes are "hot spots" for P element insertion, whereas others are "cold spots". It is, therefore, easy to miss some important genes which are "cold spots" for P element insertion during our screening. On the other hand, the insertion site may also be close to more than one enhancer element and give a false pattern of expression. Consequently, the gene that is isolated may have a pattern of expression that bears little relation to that seen in the enhancer-trap line (Bolwig, *et al.*, 1995).
It is also noted that an interesting expression pattern may not always be indicative of the true domains of genetic function of the endogenous gene. For instance, in the many other eye photoreceptor cells in addition to R7 photoreceptor express *sevenless* even after
their determination (Banerjee *et al.*, 1987). In addition, in an enhancer trap screen one may easily miss or overlook the genes such as *Notch* that are expressed ubiquitously but play important developmental roles in specific regions or cell types.
Nevertheless, the advantages and utilities of the enhancer trap technique outweigh some of its disadvantages. It is, therefore, widely used in *Drosophila* biology.
### 7.3 Future Work
Using P[GAL4] enhancer trap system, we have successfully analysed some of the lines for their expression patterns in mushroom bodies and central body complex of *Drosophila* brain (Yang *et al.*, 1995; Armstrong *et al.*, submit; also see chapter four). However, a large collection of P[GALA] lines still need to be characterised in details for expression patterns in other substructures of the brain when our anatomical knowledge accumulates in further.
As mentioned above, P[GAL4] enhancer trap system allows us to cross different UASg-marker for function analysis. For example, the next thing I would like to do is to cross these P[GAL4] lines with UASg-toxin for cell ablation to address range of questions concerning the function and development of specific groups of cells (O’Kane and Moffat, 1992; Sentry *et al.*, 1993; Sweeney *et al.*, 1995; Hidalgo *et al.*, 1995). More evidence will be added to support the hypothesise that different genetic identities reflect different functional roles in the brain.
Now that genes have been successful cloned and sequenced (see chapter six), there are many further experiments which need to be carried out.
In order to map the precise position of these genes to genome and find the potential intron/exon boundaries, corresponding genomic DNA will be sequenced completely.
A cDNA clone pMY51 encoded alkaline phosphatase in *Drosophila* and other cDNA clone pkMY4 encoded calcineurin A1 gene were identified by P element insertion. However, we have not observed any abnormalities phenotype in the homozygotes as the direct result of transposon insertion. The reason is that the P[GAL4] was not inserted into the genes themselves but just flanking these genes. In order to study the function of these genes in *Drosophila*, it is useful to generate flanking deletions so as to produce true nulls (Tsubota and Schedl, 1986; Salz *et al.*, 1987). Candidate lines will be analysed genetically, by Southern blot and Northern blot or PCR approach analysis, and where the deletions are non-lethal for anatomical and behavioural abnormalities. This imprecise excision experiment is being carried out.
On the other hand, to make new mutants, P[GAL4] insertion can be used for the local jumping (Tower *et al.*, 1993; Zhang and Spradling, 1993) to re-mobilise P element into the alkaline phosphatase gene or calcineurin A1 gene. These mutants will be analysed further. Now the mutational studies are facilitated by the tag that the gene carries.
The clone pMY8 is yet to be identified as having homologies to known *Drosophila* genes or to genes from other organism. But its expression pattern in the Northern blot is of continuing interest in the laboratory. It would be very informative to do a more complete study of the expression of this locus.
In short, P[GAL4] enhancer trap lines are a rich resource and can be used for *Drosophila* research in many aspects in the future.
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Appendix 1. Summary of DNA Clones
Listed are the DNA clones including the rescued plasmid clones, λ Genomic clones and cDNA clones used during this thesis.
A 1.1. Rescued plasmid clones
| line | clone number | approx. size | description & comments |
|--------|--------------|--------------|------------------------|
| c61 | pPC61 | 4.8 kb | pBS + DNA insert |
| c61 | pKC61 | 14.3 kb | pBS+w+GAL4 + DNA insert|
| c105 | pPC105 | 4.3 kb | pBS + DNA insert |
| c105 | pKC105 | 25.9 kb | pBS+w+GAL4 + DNA insert|
| c161 | pPC161 | 4.3 kb | pBS + DNA insert |
| c161 | pKC161 | 14.5 kb | pBS+w+GAL4 + DNA insert|
| c232 | pPC232 | 3.8 kb | pBS + DNA insert |
| c507 | pPC507 | 7.6 kb | pBS + DNA insert |
| c507 | pKC507 | 17.9 kb | pBS+w+GAL4 + DNA insert|
| c561a | pPC561a | 4.3 kb | pBS + DNA insert |
| c561a | pKC561a | 25.9 kb | pBS+w+GAL4 + DNA insert|
| c819 | pPC819 | 3.3 kb | pBS + DNA insert |
A 1.2. Genomic lambda DNA clones
| line | clone number | approx. size of insert | description & comments |
|--------|--------------|------------------------|------------------------|
| c507 | λ1(c507) | 14 kb | isolated from GEM-11 |
| c507 | λ2(c507) | 14 kb | isolated from GEM-11 |
| c507 | λ3(c507) | 14 kb | isolated from GEM-11 |
| c507 | λ4(c507) | 14 kb | isolated from GEM-11 |
| c507 | λ5(c507) | 13.5 kb | isolated from EMBL3 |
| c507 | λ6(c507) | 14.5 kb | isolated from EMBL3 |
| c161 | λ3(c161) | 12.4 kb | isolated from EMBL3 |
| c161 | λ4(c161) | 16 kb | isolated from EMBL3 |
### A 1.3. cDNA clones
| line | clone number | approx. size of insert | description & comments |
|------|--------------|------------------------|------------------------|
| c507 | pMY3 | 0.75 kb | E/H frag. in pBluescript |
| c507 | pMY5 | 0.8 kb | E/H frag. in pBluescript |
| c507 | pMY8 | 0.9 kb | E/H frag. in pBluescript |
| c507 | pMY10 | 0.6 kb | E/H frag. in pBluescript |
| c507 | pMY51 | 1.8 kb | E/H frag. in pBluescript |
| c507 | pkMY2 | 0.55 kb | E/H frag. in pBluescript |
| c507 | pkMY3 | 0.6 kb | E/H frag. in pBluescript |
| c507 | pkMY4 | 0.7 kb | E/H frag. in pBluescript |
| | rp49 | 0.6 kb | E/H frag. in pBluescript |
| | λ41 | 1.5 kb | in NM1149 |
| | α1 tubulin | 1.5 kb | α1 3' in pBR322 |
|
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As Canada's premier publicist, Gino Empry has literally met every major star in the entertainment world of the last 30 years. - Page 7
INXS' 1987 Kick album diamond after four years
Music Canada has certified Kick, the 1987 album from Australia's INXS, as a "diamond," signifying sales of more than 50,000 units in Canada.
The album was released in October of 1987 and proved to be what is described as a "breakthrough" for the band, generating four chart-topping sized hits: Need You Tonight, New Sensation, Never Tear Us Apart and Devil Inside.
Canada became the band's favourite and most successful territory. They toured the country extensively and released several well-received videos.
Through Ken Derrett of Labatt Breweries of Canada and, in conjunction with Warner Music Canada, a major Labatt's Blue Superticket competition was mounted in 23 markets across Canada, sending the winner and a guest from each market to Australia to meet the band and sit in on a recording session with INXS.
New Christmas song from Roger Whittaker
Edward Preston, President of Tembo Music Canada, is anticipating "healthy" sales of Roger Whittaker's Family Christmas release. The album is distributed in Canada by BMG Music.
The album contains Ken Pritchett's arrangement of Shadows Of Blue, which Preston describes as "a beautiful new Christmas song, that should become an evergreen."
Shadows Of Blue was produced in Canada by Eric Robertson and Hayward Parrott and features Whittaker with the Junior Choir from the Seventh Avenue Public School in Richmond Hill, Ontario.
Also of note is the Christmas Dreams suite, with accompaniment by internationally-acclaimed classical guitarist Liona Boyd and Whittaker on the pan flute. This track was also produced by Robertson and Parrott.
Other Christmas fare included on the album are the traditional carols, Have Yourself A Merry Little Christmas, O Come All Ye Faithful, Away In A Manager, plus Winter Wonderland, Happy Everything and others.
Bryan Adams sweeps Australian music awards
Canadian superstar, Bryan Adams has taken home top honours in the International category at the Australian Music Awards (Nov. 23) in Melbourne.
The A&M recording star won Most Popular International Male, Most Popular Song for (Everything I Do) I Do It For You and Most Popular International album for Wakin' Up The Neighbours.
Adams has also been honoured as Best Male Vocalist of 1991 by three German magazines: Popcorn, Bravo and Pop Rocky. He is currently playing to sold-out audiences in Europe and plans to play Australia in February of next year.
Dangerous "makes street date" at Zellers - Kennedy
Several skids of product were spirited away from Saturn's Toronto warehouse on Nov. 22. Included were Michael Jackson's new Dangerous CD, plus Genesis and Prince product among others, "A little bit of everything," says Saturn President Leonard Kennedy. It's expected however that the shipment was heavy on Jackson's much talked-about new release. An audit is underway to determine the dollar value, which will also reveal the number of Jackson CDs that are missing. All the product was apparently stickered with Zellers logos.
In view of the investigation that is underway by Metro Toronto Police and insurance investigators, Kennedy was unable to even suggest an estimate of the loss. It has been rumoured by those in the industry however, that the dollar value could range as high as $250,000.
Contrary to news reports, which indicated that Zellers stores would not have the Jackson CD, Kennedy assures that "Zellers definitely had Jackson CD product in their stores. We worked through the weekend to get the stores back in stock position, which we accomplished. We made the street date (Nov. 25)." The shipment that went missing was destined for Zellers stores in Ontario and Quebec, the last two markets to be serviced for the pre-release date.
Rush doesn't crap out on Roll The Bones tour
Rush continues to come up with anything but "snake eyes" on their Roll The Bones tour. The band has been packing arenas throughout North America, including recent dates in Ottawa, Montreal and Quebec City.
The bones will temporarily stop rolling for a Christmas break with a homecoming date at Toronto's Maple Leaf Gardens on Dec. 16th. The band, along with the Tragically Hip, will donate proceeds from the performance to the United Way. A minimum of $7.00 per ticket will be donated. The Daily Bread Food Bank will also be a recipient of the band's good will as concert-goers are encouraged to bring a bag of canned goods to the Gardens.
In the new year, the band will hit the road again with a tentative date for Vancouver scheduled for February. The band then plans to tour Europe beginning in April.
Guitarist Sambora sings praises of Vancouver
A broad smile creased across the face of Bon Jovi guitarist, Richie Sambora, when a reporter mentioned Vancouver to him backstage, after his recent outstanding solo show he performed at Toronto's Concert Hall.
"Vancouver has been mucho good to me and I'll be back for sure," the handsome axeman said, looking like he just stepped off his solo album cover, dressed in leather pants and broad brim hat.
Indeed, Vancouver has been very good to Bon Jovi and a lot of other bands that headed to Vancouver in the wake of Jovi's monster platinum sales. "But we were the first," Sambora proudly stated.
An hour before, Sambora had climaxed his show with a jam that included Canada's Aldo Nova on guitar and Gowan on vocals. "That was a lot of fun, Jon and I have known Aldo for years."
Backstage, Sambora took the time to sign autographs and offer a hug to some younger girls who were near tears at meeting their idol. Of course, the perennial long legged backstage ladies were in attendance with the skirts that went up to here and the tops that plunged down to there.
Bon Jovi drummer, Tico Torres, along for Sambora's solo tour, stood looking on with an amused smile but turned serious when asked if they were going to have Canada's Bruce Fairbairn on board as producer for the next Bon Jovi album.
"We'd love to have him again, but we understand he's committed to producing Aerosmith in January. We're hoping to get Bob Rock to do it."
Asked to explain the magic, multi-platinum formula that Fairbairn has come up with every band from AC/DC to Bon Jovi to Aerosmith, Torres said there was no one special thing. "Bruce has just got a very good ear and can bring out the best in a band, which is what a good producer is all about."
Snow and Wiseman set to play Toronto Gallery date
Toronto's Music Gallery (1087 Queen St. West) will be the setting for a unique concert by well-known visual artist Michael Snow and Blue Rodeo's keyboardist Bob Wiseman. The concert will be performed solely on two grand pianos and include music by Thelonious Monk, Duke Ellington and Otis Spann as well as their own original works.
Aside from playing the pianos conventionally, Snow and Wiseman will also play on the inside together at one piano while simultaneously playing both pianos.
Snow also performs on the guitar and trumpet. During the fifties and sixties he toured Toronto clubs with his own group as well as with Mike White's Dixieland Band and other well-known jazz musicians including Pee Wee Russell, Rex Stewart, Jimmy Rushing, Cootie Williams and Edmond Hall. He also participated in the Free Jazz revolution of the early sixties in New York. He returned to Toronto where he played and recorded with the Artists Jazz Band and was a founding member of the Category Crunching Music Creators (CCMC) in 1974.
Wiseman, besides being Blue Rodeo's keyboard player and heading a solo folk/rock and indie record producing career, has an obvious strong connection to improvisational piano. He has independently released Hits Of The '60s and '70s and WEA just released an album entitled, Presented by Lake Michigan Soda, that features special guests Eddie Brickell, Jane Siberry and Eugene Chadbourne.
Kenny MacLean's Don't Look Back won a SOCAN award for co-writers Claude Desjardins, Paul Henderson and Maclean (lto r) Desjardins, presenter Ian Thomas, publisher Tony Tsavdaralis, awards emcee CFRB's Ted Woloshyn and Maclean.
BMI VP Ekke Schnabel (l) presented a BMI Special Citation of Achievement to Toronto composer Johnny Cowell, whose instrumental Our Winter Love has reached the one million air performance level.
WALT SAYS!
with Elvira Capreese
were those shadowy figures...? I wondered what the building at 4881 Yonge St had in common when I noticed the Juno CARAS people in the vicinity of the office. I checked the directory and there are a couple of restaurants, one being the Golden Mile, but it was too late for breakfast. There were also offices for the Consulate General of Pakistan and the Jamaican Liaison Office, the Internal Audit Bureau, The Employment and Immigration Services and the Royal Bank. Now, that really makes for an intriguing scenario. Are they thinking of taking Junos to Jamaica... or Pakistan? Maybe CMA is extending an open door to country music from Pakistan and/or Jamaica. Could the Internal Audit Bureau be arranging plea bargaining for next year's tickets? Then again, they might have been looking for jobs at the Employment and Immigration Services... or just banking their money. Oh, Yes! Balmur's offices are in that building. But they couldn't be meeting with Leonard Rambeau... could they? (EC: I wouldn't never have guessed it. Say it's not true...!)
Bryan Adams Un-Canadian??? Well, I've heard everything. Bruce Allen sure stirred up a whole country when he took on the CRTC regulations... the very regulations that made him rich and infamous. He also took on Anne Murray about not showing up at the Junos and he was wrong that time too. Allen likes to stir up shit and some people take him seriously. He knows how to manipulate the media... hold them in the palm of his hand and make them react to his every whim. Now, I hear that the CRTC have taken him seriously and even Keith (The Phantom) Spicer is ready to do something stupid. Brucel A joke is a joke, but this has gone too far. Phone the CRTC and apologize. Bryan doesn't need a handout. (EC: Bruce Allen apologize? But that does take a load off my mind!!!)
Only SOCAN, you say...? What folly is this. A government-funded songwriting contest that's restricted to SOCAN members? Is this a government-sponsored membership drive? We'll be hearing from the others, I'm sure. Does that mean that Brian Mulroney can't enter? (EC: He can't sing either... but David Foster does wonders for him...)
When Paul's away...? Well, our very public retail figure, Paul Alofs missed all the fun. But when he gets back he'll have the opportunity to explain away in a column or letter or two about the shenanigans at HMV's Scarborough Town Centre store. Was there enough security? Did the mall people know about the concert? Better still, did they know it was a rap group (Naughty By Nature)? The name alone would have even been enough warning for a United Church minister. If the mall was closed for two hours... are other merchants, not to mention a very large retailer, considering some kind of legal action? Being closed for two hours, someone said, could have resulted in a $200,000 loss for some, or at least one. (EC: A tempest in a teapot...)
The manure and mink set...? Thanks to Dyanne and David Halliday, Fran and Mel Shaw, I had the opportunity to walk among the mink and manure at the Royal Horse Show. We also saw Don Jerulla's newly-bought jumper in action. She scored a record time, but unfortunately lost out by just a fraction of a second. A great looking horse... but $2.5 million...? (EC: Didn't you read the denial in RPM...?)
Tommy Hunter... going, going? The Sun's Gary Dunford has picked up on that hurtin' thorn in the side of some of the CBC hierarchy, the Tommy Hunter Show. The voices in the hall have been getting louder. Besides, the charts indicate that country is looking for a younger image. So, maybe Dunf's onto something this time. If you wait long enough, most rumours do come to pass. If the Hunter show goes, maybe the CRTC will take that country channel away from the Nashville crowd, who, for reasons, I think I'm one of the few to know, won out against a very reliable Canadian contender. The Hunter Show, its token Cancon entry, really didn't say too much for our homegrown acts, who had to fight for recognition against the Nashville mob. (EC: Country's about to come out of the closet...!)
The ink is dry...? I've heard it from a couple of sources, not too reliable, both of them, that the ink is almost dry and corks are about to be popped on the champagne with the announcement that Capitol has taken Virgin under its wing. Another source told me that Virgin had taken over Capitol, PolyGram, MCA and BMG... just to be safe. (EC: I read it right here, so it must be true...!)
Good news and more good news!!! The stores are loaded, the crowds are waiting for a little more snow... and ZOWIE... we should have a great holiday sales season. The other good news is that the promotion people are happy once more.
Alan Fletcher - Sony Music
Peter Burge - Musa/Bluemoon Records
Gordon Bell - Raw Energy Records
John Stewart - Raw Energy Records
Dave Dealey - Epic Records
Faisal Durrani - A&M Records
David Gieseman - Warner/WEA artist
Jan Crafford - Warner Music
Don Christensen - Bob Wiseman publicist
Michael Peters - WEA artist
Wayne Patton - Patton Pending Publishing
David Pierson - BMG Music
Doug Caldwell - Virgin Records
Roger Bartel - Capitol Records
Rick Wharton - MCA Records
Joe Wood - RDR Promotions
A few of the many who dropped by to say Happy 50th anniversary to Sam's Adelyne Freifeld; Warner Music's Garry Newman, Herb Forgie, Carroll Warner and Brian Irwin, Sony's Alan Fletcher and Ross Ferris, Capitol's John Toner and Michael Pollock, Rick DeLapp and Laura Hunter, MCA's Wesley Hyden and Joel Pye, A&M's Hank Koch, BMG's Doug MacDonald, and Sam's Thom McKercher.
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1712 Baseline Rd., W.
Courtice, Ontario L1E 2S8
416-433-0250 686-2828
Fax 416-433-1868
CMPA SONG OF THE YEAR AWARDS
AWARDED NOVEMBER 25, 1991
Adult Contemporary/Pop
Award: EVERY LITTLE TEAR
Written By: Paul Janz
Performed By: Paul Janz
Zoological Musik/Irving Music
First Runner-Up: DON'T WANNA FALL IN LOVE
Written By: Jane Child
Performed By: Jane Child
Warner-Tamerlane/Canadiana/Brett & Peter Publishing
Second Runner-Up: GIRL WITH A PROBLEM
Written By: Jay Semko
Performed By: Northern Pikes
Northern Pike Songs
Alternative
Award: LET YOUR BACKBONE SLIDE
Written By: Anthony Davis, Peter Davis and Wes Williams
Performed By: Maestro Fresh Wes
Warner/Chappell Music Canada Ltd./Tasting Music/D.S.G. Publishing
First Runner-Up: DROP THE NEEDLE
Written By: Anthony Davis, Peter Davis and Wes Williams
Performed By: Maestro Fresh Wes
Warner/Chappell Music Canada Ltd./Tasting Music/D.S.G. Publishing
Second Runner-Up: SHE'S SO YOUNG
Written By: Moe Berg
Performed By: Pursuit of Happiness
EMI April Music (Canada) Ltd./Pursuit of Tunes
Country
Award: LIME RICKEY
Written By: George Fox
Performed By: George Fox
Warner/Chappell Music Canada Ltd./Balmur Music
First Runner-Up: BACHELOR GIRL
Written By: George Fox
Performed By: George Fox
Warner/Chappell Music Canada Ltd./Balmur Music
Second Runner-Up: HELLO AGAIN
Written By: Tim Taylor
Performed By: Anita Perras
Prehensile Publishing Co.
Rock
Award: ALL THE LOVERS IN THE WORLD
Written By: Lawrence Gowan and Eddie Schwartz
Performed By: Gowan
Mark-Cain Music/EMI Music (Canada) Ltd./High Frontier Music
First Runner-Up: SHE AIN'T PRETTY
Written By: Bryan Potvin
Performed By: Northern Pikes
Northern Pike Songs
Second Runner-Up: WHATCHA DO TO MY BODY
Written By: Lee Aaron, John Albani
Performed By: Lee Aaron
Abovewater Music/Pondwater Music
CMPA 1991 Songwriter Scholarships
Jennifer Lord, Fanshawe College
Cam MacInnes, Harris Institute for the Arts
The Canadian Music Publishers Association is the largest association of music publishers in Canada. Formed in 1949, it promotes and protects the interests of composers and publishers of popular and serious music in Canada, and has taken a leading role in the ongoing process of reform to Canada's copyright legislation. CMPA encourages the growth of the Canadian industry through more effective copyright and broadcasting legislation, and by public events such as the CMPA Song of the Year Awards, which heighten the profile of Canada's songwriters.
For further information about the Celebration of Songwriters and CMPA Song of the Year Awards or CMPA, please contact Fran Shaw at (416) 367-2070.
## HIT TRACKS & where to find them
### Canada's Only National 100 Hit Track Survey
| Rank | Artist | Title | Label | Catalog # | Date |
|------|--------|-------|-------|-----------|------|
| 1 | LIFE IS A HIGHWAY | Tom Cochrane - Mad Mad World | Capitol-97724 (Capitol comp. # 45)-F | 14 |
| 2 | BEAUTY AND THE BEAST | Collie Dawg/P. Bryson - Beauty & The Beast soundtrack | BMG | 68 |
| 3 | CAN'T STOP THE THING WE STARTED | Billy Adams - Wake Up The Neighbours | A&M-79021 5367 (A&M/Virgin comp. # 1090)-Q | 35 |
| 4 | THE BEASTIE BOYS | Bob Sieger - The Fire Inside | Capitol-81134-F | 36 |
| 5 | SAY YOU DON'T KNOW ME | New Jersey - Built - Monsters Under The Bed | WEA-75538-P | 39 |
| 6 | STAND BY MY WOMAN | Bryan Kenny - Magic Seed | Virgin-3073 (A&M/Virgin comp. # 1091)-Q | 38 |
| 7 | NO SON OF MINE | George - We Can't Dance | Atlantic-48265 (A&M comp. # 115)-P | 34 |
| 8 | I CAN'T LET GO | Matt Cardle - Emotions | Columbia-47968 (Promo CD single)-H | 39 |
| 9 | BROKEN ARROW | Rod Stewart - Vagabond Heart | Epic-46988 (Sony comp. # 111)-P | 40 |
| 10 | I CAN'T MAKE YOU LOVE ME | Bonnie Raitt - Luck Of The Draw | Capitol-81119 (A&M CD single)-F | 42 |
| 11 | WHEN A MAN LOVES A WOMAN | Michael Bolton - Time, Love & Tenderness | Capitol-81118 (A&M comp. # 38)-H | 41 |
| 12 | THAT'S WHAT LOVE IS FOR | Amy Grant - Heart In Motion | Atlantic-48264 (A&M/Virgin comp. # 1091)-Q | 42 |
| 13 | GET A LEG UP | John Mellencamp - Whenever We Wanted | Mercury-510 151 (PolyGram comp. late Sept'91)-Q | 43 |
| 14 | KEEP COMIN' BACK | Richard Marx - Rudderless | Capitol-95874 (Capitol comp. # 50)-F | 44 |
| 15 | SOMETHING GOT ME STARTED | The Rock - I'm Gonna Be (What I Want) | Capitol-81108 west U.K.-75284 (Warner comp. # 106)-P | 45 |
| 16 | SET THE NIGHT TO MUSIC | The Beach Boys - Puttin' The Night To Music | Atlantic-82321 (Warner comp. # 111)-P | 46 |
| 17 | SHOT OF POISON | The Fixx - Love Curves | Warner-810 625 (BMG pop comp. # 25)-N | 73 |
| 18 | BLACK OR WHITE | Michael Jackson - Dangerous | Epic-46987 (Sony CD single)-H | 47 |
| 19 | I'LL FALL ALL OVER AGAIN | The Bangles - Love | Quality-2001 (Promo CD single) | 48 |
| 20 | I WONDER WHY | Curtis Stigers - Curtis Stigers | Atlantic-7822 (PolyGram comp. # 23)-N | 49 |
| 21 | DO ANYTHING | Natural Selection - eastward | Capitol comp. # 106)-P | 50 |
| 22 | CALLING ELVIS | Dire Straits - On Every Street | Virgin-3072 (PolyGram comp. early Sept'91)-Q | 51 |
| 23 | ALL I LOVE | Color Me Bad - C.B. | MCA-10517 (MCA comp. # 117)-P | 52 |
| 24 | INTO THE GREAT WIDE OPEN | Tom Petty - Into The Great Wide Open | MCA-10517 (MCA comp. # 1951)-J | 53 |
| 25 | LET'S TALK ABOUT SEX | Self - Pegs - The Hits Remixed | New Planet-810 249 (PolyGram comp. late Sept'91)-Q | 54 |
| 26 | WALKAWAY | Allan - Aladdin | MCA-10518 (MCA comp. # 2001)-J | 55 |
| 27 | SATURDAY NIGHT'S ALL RIGHT ... | The Who - Two Rooms | Mercury-510 150 (PolyGram comp. # 45)-F | 56 |
| 28 | ONE LITTLE WORD | The Boogaloo - We Do | WEA-75516 (Warner comp. # 117)-P | 57 |
| 29 | THE SKY IS CRYING | Steve Ray Vaughan - The Sky Is Crying | Epic-46987 (Sony comp. # 45)-F | 58 |
| 30 | LOVE DON'T LAST FOREVER | Chrissie Hynde - Magnet To Steele | Capitol-35683 (Capitol comp. # 48)-F | 59 |
| 31 | I DON'T WANT TO BE A FOOL | Luther Vandross - Love Power | Epic-46789 (Sony comp. # 31)-H | 60 |
| 32 | WILDSIDE | Mokey Mark & The Funky Bunch - Music For The People | Epic-91737 (Warner comp. # 118)-P | 61 |
| 33 | O.P.P. | Naughty By Nature - Naughty By Nature | Atlantic-48263 (A&M comp. # 109)-P | 62 |
| 34 | NEVER LOOK BACK | Darby Mills - Never Look Back | Atlantic-48263 (A&M comp. # 1291)-Q | 63 |
| 35 | THERE WILL NEVER BE ANOTHER ... | Billy Adams - Wake Up The Neighbours | A&M-79021 5367 (A&M/Virgin comp. # 1291)-Q | 64 |
| 36 | NO MORE TEARS | Ozzy Osbourne - No More Tears | Atlantic-48263 (A&M comp. # 115)-P | 65 |
| 37 | SALTWATER | Julian Lennon - Help Yourself | Atlantic-82280 (Warner comp. # 115)-P | 66 |
| 38 | THE COMMITTED SON | The Cocteau Twins | Vertigo-510 419 (PolyGram comp. late Sept'91)-Q | 67 |
| 39 | YOU'RE THE VOICE | Heart - Rock The House "Live" | Capitol-95797 (Capitol comp. # 47)-F | 68 |
| 40 | POWERDRIVE | Longfellow - Sacrifice To Sassy | EKE-001 (Promo CD single) | 69 |
| 41 | THE FIRE INSIDE | Bob Sieger - The Fire Inside | Capitol-81134-F | 70 |
| 42 | SPY FANTASY | The Beach Boys - Puttin' The Night To Music | Atlantic-82321 (Warner comp. # 1091)-Q | 71 |
| 43 | THE ROAD OF HOPE | Spy Fantasy - The Kite | A&M-79021 5367 (A&M/Virgin comp. # 1091)-Q | 72 |
| 44 | HEY DONNA | Rhythm Syndicate - Pyten In Paradise | Capitol-95874 (Capitol comp. # 1091)-J | 73 |
| 45 | SHOT OF POISON | The Fixx - Love Curves | Warner-810 625 (BMG pop comp. # 25)-N | 74 |
| 46 | 2 LEGIT 2 QUIT | Hammer - Too Legit To Quit | Capitol-81151 (Capitol comp. # 50)-F | 75 |
| 47 | WAITING | Rich Vosine - Rich Vosine | Sun-8026 (Sun-8026 (Promo CD single) | 76 |
| 48 | NORTHERN RAINY NIGHT (Without You) | Queenstown - Friends | Capitol-EM-92006 (Capitol comp. # 47)-F | 77 |
| 49 | ON THE VERGE | The Beach Boys - Road Apples | MCA-10517 (MCA comp. # 1991)-J | 78 |
| 50 | LOVE REACTION | The Beach Boys - Road Apples | WEA-75109 (Warner comp. # 116)-P | 79 |
| 51 | HOLY HEARTED | Extremes - Holy Hearted | A&M-79021 5313 (A&M/Virgin comp. # 591)-Q | 80 |
| 52 | RESCUED (By The Arms Of Love) | The Beach Boys - Road Apples | MCA-10517 (MCA comp. # 1991)-J | 81 |
| 53 | CONVICTIONS OF THE HEART | Kenny Loggins - Leap Of Faith | Capitol-95874 (Capitol comp. # 36)-H | 82 |
| 54 | STANDING PUSH AND FALL | World On Edge - World On Edge | Virgin-1010 (Promo CD single) | 83 |
| 55 | I'LL BE IN THE WORLD | Blue Train - The Beach Boys Dreams | Zod-72445 1 0007 (BMG pop comp. # 24)-N | 84 |
| 56 | IT'S ON AND IT'S ON | Chasney Hutcherson - Chasney Hutcherson | Chrysalis-41861 (MCA comp. # 1391)-J | 85 |
| 57 | DO YOU FEEL LIKE I FEEL? | Belinda Carlisle - Live Your Life Be Free | MCA-10517 (MCA comp. # 2009)-J | 86 |
| 58 | FALL AT YOUR FEET | The Beach Boys - Road Apples | WEA-75109 (Warner comp. # 116)-P | 87 |
| 59 | ONE LITTLE WORD | The Boogaloo - We Do | WEA-75516 (Warner comp. # 117)-P | 88 |
| 60 | THE SKY IS CRYING | Steve Ray Vaughan - The Sky Is Crying | Epic-46987 (Sony comp. # 45)-F | 89 |
| 61 | LOVE DON'T LAST FOREVER | Chrissie Hynde - Magnet To Steele | Capitol-35683 (Capitol comp. # 48)-F | 90 |
| 62 | I DON'T WANT TO BE A FOOL | Luther Vandross - Love Power | Epic-46789 (Sony comp. # 31)-H | 91 |
| 63 | WILDSIDE | Mokey Mark & The Funky Bunch - Music For The People | Epic-91737 (Warner comp. # 118)-P | 92 |
| 64 | O.P.P. | Naughty By Nature - Naughty By Nature | Atlantic-48263 (A&M comp. # 109)-P | 93 |
| 65 | NEVER LOOK BACK | Darby Mills - Never Look Back | Atlantic-48263 (A&M comp. # 1291)-Q | 94 |
| 66 | THERE WILL NEVER BE ANOTHER ... | Billy Adams - Wake Up The Neighbours | A&M-79021 5367 (A&M/Virgin comp. # 1291)-Q | 95 |
| 67 | NO MORE TEARS | Ozzy Osbourne - No More Tears | Atlantic-48263 (A&M comp. # 115)-P | 96 |
| 68 | SALTWATER | Julian Lennon - Help Yourself | Atlantic-82280 (Warner comp. # 115)-P | 97 |
| 69 | THE COMMITTED SON | The Cocteau Twins | Vertigo-510 419 (PolyGram comp. late Sept'91)-Q | 98 |
| 70 | YOU'RE THE VOICE | Heart - Rock The House "Live" | Capitol-95797 (Capitol comp. # 47)-F | 99 |
---
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COVER STORY - by David Pierson
Canada's premier publicist has no plans to retire
Closing in on 30 years as Canada's premier publicist, Gino Empry has certainly got a lot of wonderful memories of stars, shows and events to draw on when recounting his career. But his all-time favourite was when he brought the actress Glenda Jackson and jazz great Ella Fitzgerald together.
"Over the years, I've found stars are notoriously shy people out of the limelight and those two are no exception. But Glenda told me that Ella was her favourite singer and she was so disappointed that she was going to miss her show since she had to be onstage in a play herself that night.
"So I said, 'no problem, let's go to her rehearsal.' So I took her down and knocked on Ella's door and Glenda held back but Ella was thrilled and said, 'oh, bring her in, she's my favorite actress.' And they hit it off fabulously."
Getting Empry to talk about himself is rather hard as he admits to a terrible shyness when not working. However, mention the names of the stars which cover his office walls with personally autographed photos, then the anecdotes start flowing.
Elvis Presley?
"I met him in '72 at the Niagara Falls Convention Centre. They couldn't come into Canada because of all the guns he and his entourage carried. Spoke with him for five minutes, very shy and polite, but very charismatic."
Ann Margaret?
"A very down-to-earth lady. Totally professional and a terrific entertainer."
Frank Sinatra?
"I don't know why he's got a reputation as being difficult. I've always found him to be a gentleman."
Peggy Lee?
"She's one of my oldest and best friends. I stay at her place in Bel Air when I visit Los Angeles. When she calls she always sings her opening greeting."
Tina Turner?
"When I used to book Tina at the Imperial Room, the management complained that she didn't wear enough clothes. Of course they didn't complain that the house was always packed. I knew she was going to be big and was very happy for her when she broke through."
Hal Linden, who quipped after his opening song at the Imperial Room, "what the hell is Barney Miller doing singing in a night club?"
"People forget that Hal was an actor/singer on Broadway for years before his television show."
Born and raised in Toronto, Empry founded his public relations agency in 1963, the same year he signed his first client, the Royal Alexandra Theatre, commencing a career-long relationship with the flamboyant entrepreneur Ed Mirvish.
When the sod was recently turned on Mirvish's new theatre to be built in downtown Toronto, Empry managed to get the past, present and future Lieutenant Governors of Ontario to attend. "I like doing deals and promotions that people say can't be done."
Empry was Tony Bennett's manager for 10 years and currently represents Peggy Lee, Roger Whittaker, figure skating champion Brian Pockar, actor/director William Hutt, ballet star Karen Kain, comedian Andre-Philippe Gagnon, actors Ken James, Craig Russell and Robin Ward, plus Playboy Magazine.
Despite the countless offers to go to the US, Empry said that he prefers being the big fish in the small pond, which really isn't all that small a pond, considering Toronto is the third largest entertainment centre in North America.
"I love the hustle and bustle of L.A. and New York but I always like coming back to Toronto. People always ask me if I'm ready to retire? Retire to what? I love this business and don't ever want to leave it. I'm just getting my second wind."
Valleyfield's KOD 137 sets industry first for Canada
A first in Canada's broadcasting industry took place on Nov. 26 at 5 pm at Brasserie Labatt in Montreal. At that time Jean-Pierre Major, President of Radio Express Inc. announced that KOD 137 Valleyfield was the first radio station in Canada to adopt a 100 percent Cancon only format, 24 hours a day.
"No foreign artists on KOD 137," boasts Major, "no francophones from France, Belgium, no anglophones from England or the US, just 100 percent of our own artists. On November 26th, the record industry has a new partner."
Many recording artists plus officials from SOCAN, Musicaction, Gens de la radio and officials from the federal and provincial communications ministries assisted in the Montreal premiere.
Major concludes with "what we wish now is that other Canadian broadcasters will follow KOD 137's example."
BARRY MANILOW - Pop
Showstoppers
Arista - 0782-18687-2-N
The time for this Manilow, Eddie Arkin production couldn't be better, and the material right down Manilow's Tin Pan Alley vocal magic. He is more at home with this type of material than most, and the tribute he pays to some of these modern day masters is superb. Priceless tracks like I'll Be Seeing You and Bring Him Home. This is one of those good for giving packages with 18 tracks. Try on I'm Sorry And Dolls Baby featuring Manilow, Michael Crawford and Hinton Battle. Great stuff. Worth the price of the CD alone. The Kid Inside, written by Craig Carnelia, has been taken as the priority track and is included on the BMG Pop Promo CD No. 26. Should garner AC play. Front-rack this item. A must for Santa's stocking. (CD reviewed) - WG
RUSS IRWIN - Pop
Russ Irwin
SBK K2-96915-F
A left-fielder songwriter with a delivery and vocal magic to match. Irwin has a laid-back, but 'hey we're in a hurry' approach to his music which draws on rock and jazz with more than a bit of classical overtones. To say he gets into the lyrical content of the material is an understatement. There seems to be a personal yet compassionate edge to his writing vocal style that has no bounds, probably the influence of producer Phil Ramone, no slouch when it comes to this kind of talent. Key, Is My Heart Belongs To You, taken as the first single, and Get Outta Town, a very poignant message ballad. All songs were written by Irwin with the exception of Don't Like The Way You Look At My Love, which was written by Holly Knight and Gregory Darling and She's Part Of Me, penned by Russ Irwin and Randy Goodrum, which should be the next single. (CD reviewed) - WG
BOB WISEMAN - Alternative
Presented By Lake Michigan Soda
WEA - CD 75782-P
In between recording and gigs as Blue Rodeo's keyboardist, Bob Wiseman likes to keep busy. So, in between producing other artists and other shows, Wiseman has found time to release his third solo album. The title pokes fun at corporate rock sponsorship and there are numerous other humourous spots throughout the album. But beneath the humour, Wiseman takes a searing look at the plight of the North American Indian on three of the tracks. Etude #10 opus 668 has a fellow friend musician playing the electric rake and yes, the 668 is a joke for all those looking for hidden devil messages in songs. Notan easy listen, but an interesting one. (CD reviewed) - DP
ETTA JAMES - R&B
R&B Dynamite
Flair /Virgin - 2-91695-Q
Etta James has got a voice that could curl the hairs of her blonde wig. Often regarded as the queen of R&B, this best of collection is a must for anyone who likes their lady singers with brass and balls. Alternately asserting herself on tracks like Woman, Hey Henry, Tough Love and then demurring her legendary track like I'm No Fool, How Big A Fool and Hope You're Satisfied, James delivers the R&B goods with style. (CD reviewed) - DP
B.B. KING - Blues
The Best of B.B. King Vol. 1
Flair /Virgin - 2-91691-Q
B.B. King said recently that his latest release was his best album, but he'd be hard pressed to top this best of collection. One classic after another, starting with his first hit, Three O'Clock Blues, continuing on into Sweet Little Angel, Every Day (I Have The Blues), Sweet Sixteen and closing with the rocking Please Love Me. No dross amongst these 20 gems and a perfect sampling to show the debt that all those British blues boys owe the King of the Blues. (CD reviewed) - DP
JERMAINE JACKSON - Funk
You Said, You Said
LaFace - LFPCD-4003-N
Too bad the critics are only interested in what Jermaine says about his brother, and too bad Jermaine is such a big mouth. He's got talent, lots of talent, as displayed on this fat produced album by LaFace Family. You Said is now beginning to take hold with AC programmers and his album should command chart action shortly. This Jackson has a way with ballads, and they appear to be tailor-made for him, probably because of his writing cronies: L.A. Reid, Babyface and Daryl Simmons. He does a duet with Babyface on I'm Not Ya Right and that's Vernon Reid of Living Colour providing the guitar fire on Rebel (With A Cause). Lots of great stuff here if you can just dig through the brother battle bullshit. (CD reviewed) - WG
BIG IDEA - Rock
Big Idea
Artiste Records CD-108
The Big Idea are Montreal guitarist Robert Martins and Peterborough organ/bassist Tony Collins. A strange, but nonetheless obviously compatible team. Good fare for the campus crowd that should spill over to the mainstream, if they get the right break. Welcome To The World, taken as the first single, should command both AOR and CHR radio play and should open the door to even more spunk pop and rock, like Everyone's Man, Face To The Ground, Blue Of The Water and a very strange, but likeable Paperback Writer. The vocal, and, for that matter, the writing approach is quite refreshing. Martins and Collins work off each other and the tussle evolves into a finely-crafted package of solid entertainment. (CD reviewed) - WG
## Funk
The King of Rock and Roll is interested in talking about his new album, but he's not talking about the record itself. He's focused on his new album, which will be released on March 12th.
### The Commitments
The Commitments are a band from Dublin, Ireland, who have been playing together since 1984. They are known for their energetic performances and their ability to play a variety of genres, including rock, soul, and funk.
### The Eurythmics
The Eurythmics are an English pop duo consisting of Annie Lennox and Dave Stewart. They have been active since the late 1970s and have sold over 100 million records worldwide.
### The House Party
The House Party is a compilation album featuring songs by various artists, including The Beatles, The Rolling Stones, and The Beach Boys. It was released in 1965 and has become a classic of the era.
### The Tragically Hip
The Tragically Hip are a Canadian rock band formed in 1983. They are known for their unique sound, which blends elements of folk, country, and rock. Their music often deals with themes of love, loss, and social issues.
### The White Stripes
The White Stripes are an American rock band formed in Detroit, Michigan in 1997. They are known for their raw, garage rock sound and their intense live performances. Their music often deals with themes of anger, frustration, and rebellion.
### The Who
The Who are an English rock band formed in 1964. They are known for their energetic stage shows and their powerful vocals. Their music often deals with themes of rebellion, freedom, and social issues.
### The Eagles
The Eagles are an American rock band formed in 1971. They are known for their harmonies, guitar solos, and powerful vocals. Their music often deals with themes of love, loss, and social issues.
### The Doors
The Doors are an American rock band formed in Los Angeles, California in 1965. They are known for their psychedelic sound and their powerful vocals. Their music often deals with themes of rebellion, freedom, and social issues.
### The Beatles
The Beatles are an English rock band formed in Liverpool, England in 1960. They are known for their innovative sound, their powerful vocals, and their impact on popular culture. Their music often deals with themes of love, loss, and social issues.
### The Rolling Stones
The Rolling Stones are an English rock band formed in London, England in 1962. They are known for their powerful vocals, their innovative sound, and their impact on popular culture. Their music often deals with themes of love, loss, and social issues.
### The Beach Boys
The Beach Boys are an American rock band formed in Los Angeles, California in 1961. They are known for their harmonies, guitar solos, and their impact on popular culture. Their music often deals with themes of love, loss, and social issues.
Retail Council honours to HMV's Yonge Street store
The Retail Council of Canada recently bestowed its Retail Advertising and Marketing Communications Award of Merit on HMV Canada. The award was in recognition of HMV's campaign to launch its superstore at Yonge Street, which began in December 1990 and continued beyond its May 3, 1991 opening.
The campaign was tagged as "unique", and it "successfully combined the need to create awareness for the new store with a strong product focus."
On being informed of the award, Paul Alofs, President, HMV Canada noted, "we are really excited about the marketing programme that launched our Toronto superstore. It was truly a team effort, including extraordinary support from the record label suppliers. The commitment from our staff, agencies and suppliers resulted in excellent value for the money spent. The marketing campaign was extremely successful on a number of dimensions and we appreciate everyone's contribution."
Alofs went on to point out, "the launch programme for the superstore successfully put music in front of a lot of people, which is extremely beneficial for the music industry overall. We retailers have to use the co-op funds available to us to get the public excited about music. Unfortunately, due to the lack of creativity and vision, much of the co-op funds spent by retailers are basically wasted."
Since its opening, the Yonge Street store has reportedly performed "well above original expectations," and is now positioned as one of the leading record retailing outlets in North America.
McKennitt's WEA debut reaches gold sales status
The Visit, Loreena McKennitt's debut release for WEA, has reached sales of gold status in Canada. Fueled by the single release of Greensleeves, which is No. 7 on the RPM AC chart, the album has made good gains up the RPM 100 where it is at No. 28 this week after only five weeks of charting.
The success of McKennitt's debut heralds the rush release of three more of her albums that were previously released but are now available via Warner Music Canada.
Parallel Dreams was first released on McKennitt's Quinlan Road label in 1989. The album sold more than 25,000 units, primarily through word of mouth. Producer Brian Hughes, who worked on The Visit, also collaborated on this album as guitarist and composer. However, the majority of the material on this album was written by McKennitt.
Elemental was first released by McKennitt in 1985 and also released on her Quinlan Road label. The material here is described as a "blend of the traditional with the experimental and Celtic harp. McKennitt, as with The Visit, borrows from some of the well-known poets of yesteryear including W.B. Yeats (Stolen Child) and William Black (Lullaby). Also included are popular Celtic folk songs She Moved Through The Fair and Carrighfergus.
To Drive The Cold Winter Away was released in 1987 and was previously available only on cassette. The release is timely in that if offers what is described as "a different look" at Christmas and the winter season. Included are little-known carols and seasonal songs with a modern audience in mind. The album was recorded in a variety of locations, from a Canadian church to an artists' retreat, to a Benedictine monastery near Limerick, Ireland.
Pharis targets charity for Littlest Christmas Angel
Hod Pharis, regarded as an Alberta legend, has returned to radio in time for Christmas (RPM - Nov. 2/91). His vehicle is The Littlest Christmas Angel, a narrative with sisters Heather and Paula Fleming, native Calgarians, now living in Edmonton, supplying background vocals. Composer/arranger Jo Hanson wrote the music for the track. He is best known for his work on 15 Calgary Stampede Grandstand Shows as well as the Medal Ceremonies at the 1988 Calgary Winter Olympics.
Lyricist Dawna Roskies, also a Calgarian, reportedly wrote the song in a scorching August heatwave three years ago. Former Calgarian Gary Kollger produced the track, which was recorded at Edmonton's Beta Sound Recorders.
The Littlest Angel is available on cassette single as A Christmas Card from Heaven - to the Heart of an Old Cowboy, complete with envelope, Christmas Card, story, sheet music and song.
ATI's Scoot Irwin, the Toronto and Calgary-based independent, on which the cassette has been released, reports that a deal has been struck with Trend Records for the release of the evergreen Christmas song card for next year. Additional copies may be obtained from Applause in Mount Royal Village (a project of the Calgary Centre for Performing Arts) or through ATI Records in Toronto or Calgary.
Pharis was born in Pincher Creek, Alberta and lived in Calgary during most of his career. He retired to B.C.'s Okanagan in the late eighties. He first gained fame in country music in 1951 when he wrote and recorded Call Of The Mountain. An interesting aside to the recording is that to obtain the "echo effect" on the record, Ann Little performed the background vocals in the men's washroom.
I Heard The Bluebird Sing was written by Pharis in 1952 and recorded by Hal Lone Pine and Betty Cody. He claims that at the time he "came awful close to throwing it away." However, in 1957 the song was recorded and became a hit for Jim, Ed Maxine and Bonnie Brown, and today is in the classic gold category in country music.
To date, the song has been recorded by the late Marty Robbins, Kris Kristofferson and Rita Coolidge to name just a few. It has also been recorded in many different languages and went to No. 1 in Ireland when released by an Irish duo.
After several years as a Calgary Police officer and later as a government trapper, Pharis returned to Calgary radio as an on-air personality and released two albums with his own band, Hod Pharis and the Plainsmen.
Earlier this year and prior to the release of The Littlest Christmas Angel in 13 countries on Oct. 24, Pharis gained a western foothold on the charts with another of his originals, The Loneliest Man, released on the ATI label.
Plans are now underway to put a family show together for a tour.
Prairie Oyster to No. 1 on Country 59's Top 40
Toronto's Country 59 moves Prairie Oyster's current single, Did You Fall In Love With Me, from No. 5 into the coveted top of the chart. Greg Stevens, the station's Promotions and Marketing Director, points out that "this is the first time a Canadian recording has made it to number one."
Says Program Director Bill Anderson, "it's another indication of the growing popularity of our homegrown artists. Those of us in the business have long known that the talent existed here to compete with the Nashville acts. Now, it's clear that the public feels the same way."
Anderson hosts the Country 59 Countdown show Saturdays from 9 to noon. The program is repeated Sunday evenings from 6 to 9.
Prairie Oyster's Russell deCarle was scheduled to make a special guest appearance on the show on Dec. 1.
COUNTRY
Tim Taylor, the second runner-up in the recent Canadian Music Publishers Association's (CMPA) Country Song of the Year awards, for his writing of Hello Again, recorded by Anita Perras, has been busy with his own recording endeavours. He recorded his second solo project at German Town Studios, scheduled for a spring '92 release. He has also taped two television shows, The Joan Kennedy Show and That Country Feeling.
Lou Paul is stepping up promotion on the release of Running South On A Northbound Train. Mike Durrell, of CKRW Whitehorse, was one of the first to suggest Running South as a single release. Ritch Nickle of CKGY Red Deer also gave the track the nod. Mike Monroe of CIVH Vanderhoof, B.C. had playlisted the track immediately on receiving the CD. He calls it "an excellent toe-tapper. If you're not tapping your toes, you're dead!"
Saddlestone's Gerry King is not only receiving very impressive national and international support on his Heartwreck release, the accompanying video is on heavy rotation as well. The video is being aired on the Nashville Network's Video PM Show and
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experience and a strong desire to help maintain the momentum of growth underway on the Avalon. Hicks replaces Brendon McCarthy, who has left to work with one of the newspapers in the market.
Larry and Willie morning show hosts of 99.5 The Fox have made a pledge to meet all their listeners, all 350,000 of them, in one year. Each week, the popular pair are set to go out and meet and greet their listeners. They will have meeting booths set up at trade fairs, concerts and wherever possible, complete with an automatic LED display reading "Larry & Willy Meet Every Listener" which will also supply the number of listeners they have met at the time. A budget of $10,000 has been allocated for the project. As big-hearted Program Director JJ Johnston points out, "unlike all those other stations, who give away one big prize and everyone else gets shafted, Larry & Willy will share the budget with everyone. That works out to 3 cents each." Each person met will get a suitably emblazoned business card indicating what number they were on the list. The card is redeemable for 3 cents cash at 99.5 The Fox.
Oldies 990 and Classy Car Wash raised almost $6,000 for Starlight Canada during a series of benefit car washes. Oldies 990 personalities were on hand for the four Sundays in July and August to help wash cars and raise money. Every dollar collected was donated to the Starlight Foundation which grants wishes to critically, chronically and terminally ill children.
Gary James and his Oldies 990 James Gang morning show was visited (Nov. 14) by Justine Bateman. The star of Family Ties talked about her new film Dead Bolt, currently filming in Montreal, and took calls from listeners.
DeLilla feels the key to CJEZ's success is working together as a team with the key people in different departments. "Bob Wood, the general manager here, has put together a bunch of fantastic people and it's been a joy to work with pros like John Majhor."
Regarding CJEZ's new format of easy listening without the ultra-mellow mood music, DeLilla says that the station went from 13th to 7th in the last rating sweeps and the station is hoping their audience will keep on growing.
"We're targeting that older 35 plus age group who've mellowed a little, but still want something with a little kick. Jailhouse Rock might be a little jarring but we'll play something by Elvis like Teddy Bear or Love Me. I mean even my dad still likes to rock and roll and tap his foot."
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Tomahawk, April 12, 1932
College of the Holy Cross
Follow this and additional works at: https://crossworks.holycross.edu/crusader
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Recommended Citation
College of the Holy Cross, "Tomahawk, April 12, 1932" (1932). Student Newspapers. 214.
https://crossworks.holycross.edu/crusader/214
This Newspaper is brought to you for free and open access by the College Archives at CrossWorks. It has been accepted for inclusion in Student Newspapers by an authorized administrator of CrossWorks.
Archbishop Murray Will Be Honored
Testimonial Dinner Planned at Waldorf-Astoria in New York
LARGE ATTENDANCE IS EXPECTED AT AFFAIR
Holy Cross College Alumni from many parts of the country will gather at the Hotel Waldorf-Astoria in New York City tomorrow night, April 13, for a testimonial banquet to Archbishop John Gregory Murray of the class of 1897. Archbishop Murray, who was recently named to the See of St. Paul, is the first graduate of the college to have the honor of being elevated to an Archibishopric.
Among the invited guests are the Rt. Rev. Andrew J. Brennan, '00, Bishop of Richmond, and the Rt. Rev. William J. Hafey, '09, Bishop of Raleigh. Numerous other Church and civil dignitaries will attend.
Speakers for the evening will be Bishop Brennan; Dr. George Henry Derry, '98 President of Marygrove College, Detroit; James A. Crotty, '11, of Worcester, President of the General Alumni Associations; Rev. M. F. O'Sullivan, S.J., President of Holy Cross; and John J. O'Flahna, '25, President of the Greater New York Chapter of the Alumni. Hon. William E. Leahy, '07, of
(Continued on Page 2, Col. 5)
SENIORS TO DEBATE JUNIORS; FRESHMEN WILL OPPOSE SOPHOMORES IN INTERCLASS DEBATES
The contest to select the speakers for Commencement Day debate will be held this year in Fenwick Hall on Sunday, May 8th, at 9 a.m. Names of the contestants must be submitted to the Office of the Dean before Thursday, May 6th.
PRIZES WILL BE GIVEN TO VICTORIOUS TEAMS
Sophomores and Freshmen to Discuss Unemployment Insurance
Announcement has been made concerning the arrangements for the inter-class debates which hold the interest of the students so keenly each year. The Seniors will debate the Juniors and the Freshmen will oppose the Sophomores, both debates to take place in Fenwick Hall.
The subject for the Senior-Junior debate, as decided upon by both sides, is "Resolved: That the United States should cancel all war debts in the interest of International welfare." This question presents a great opportunity for discussion as it has many different angles and is a very prominent question in international circles and national affairs today. Although the exact date for this discussion is not yet arranged, it is expected to take place during the week of April the 14th. The Seniors will uphold the affirmative side of the question, while the Juniors will defend the negative.
The Sophomore-Freshman debate will be on the subject of unemployment and will be stated thus, "Resolved: That several states should enact legislation providing for compulsory unemployment insurance." The Sophomores will maintain the affirmative side of this discussion and the Freshmen will argue the negative. This debate will also take place in Fenwick Hall but the date, as yet, has not been decided upon.
These inter-class debates are
(Continued on Page 7, Col. 1)
Modern Views On Molecules Given
SCIENCE CLUB HEARS DANTE'S DISCUSSION
Last night in the Physics Lecture Hall in Alumni, a meeting of the Scientific Society was held at which Lee F. Dante, '33 discussed "The Liquid State of Molecules." In his discussion he gave proof of the actual as well as the potential existence of molecules in liquids. He put forth "statements" of the various phenomena of the liquid state and showed that the postulation of the actual existence of molecules is the only hypothesis that will adequately explain all these phenomena.
The rest of the dissertation was consumed with a treatment of formulae that directly or indirectly pertain to molecules. The discourse revealed that Dante had a thorough understanding of his subject; while his expression was clear enough to make his listeners the idea which he wished.
A fairly large audience attended to hear the paper read. It is hoped that more students will in the near future come to these meetings as they are very beneficial in a better comprehension of the sciences.
(Continued on Page 2, Col. 5)
COLLEGE HOST TO EDUCATORS
Dinand Library is Scene of Classical Association Convention
Educators from many parts of New England assembled at Holy Cross on Friday and Saturday, April 1 and 2, when the Classical Association of New England held its twenty-second annual meeting. Arrangements for the meeting were under the direction of Prof. J. Leo O'Gorman of Holy Cross. The meeting was held in the Memorial Library.
At the commencement of the session Friday morning, Rev. Francis Dolan, S.J., Dean, gave an address of welcome. Miss Mary Kendall Stark, retiring president of the Association, responded.
The following were elected to office: President, Dr. Harry M. Rabbell of Yale; vice-president, Miss Adele Arey of Holyoke High School; secretary-treasurer, Williams; Dr. Monroe N. Wetmore of Williams College; executive
(Continued on Page 5, Col. 5)
Cancellation of War Debts to be Junior-Senior Debate Topic
Musical Club Concert in Fenwick Hall
Faculty and students will have the opportunity of hearing the combined Musical Clubs of Holy Cross College give their annual concert in Fenwick Hall on Thursday evening, April 14, at 7:30. The regular concert as presented during the clubs' tour will be offered.
History Society Discusses Peace
ANTI-WAR MEASURES MET WITH APPROVAL
On last Friday evening the History Society held its regular weekly meeting and occupied itself with a continuation of the peace conference introduced at a previous gathering. Various methods for attaining peace were discussed and voted upon.
Several motions were rejected by the society after consideration. The entry of the United States into the League of Nations and the World Court was opposed as was also the institution of an international police force. Recognition of Bolshevism was generally resisted and the production of a United States of Europe was not considered practical.
Among the measures favored were progressive disarmament, limitation of war debts, free trade, a Danube league, the use of plebiscites to determine the status of small race units. The Monroe Doctrine was upheld and Papal participation in peace negotiations was strongly urged. Rev. Fr. Higgins, S.J., pointed out that accord-
(Continued on Page 2, Col. 3)
Cheerleaders Have Trials
Final Selection of Leaders for Next Year to be Made in May
Aspirants for the vacancies which will occur in the ranks of the cheer leaders, because of graduation, are now going through a series of tryouts, according to Tom Vareda, '32, head cheer leader.
The new men, who have reported for tryouts to date are: Thomas McKeon, '33; Frank Rooney, '33; Paul Hintelmann, '33; Joseph Walsh, '33, Daniel Murdoch, '33; Herbert Riley, '33; Thomas Delehue, '33; Thomas McNally, '33; and Richard Healy, '33.
The outgoing leaders are: song leader, Joseph Reynolds, '32; Donald Woods, '32; William Moffitt, '32; William Endres, '32; and Frank Vareda, '32.
Practice sessions are being held at present under the direction of Frank Vareda. Final selections will not take place until the week.
(Continued on Page 2, Col. 2)
Philomath Debates Unusual Subject
Social Development of Holy Cross Students Topic of Discussion
HORGAN AND KENNEDY WIN CLOSE DECISION
In the first post-Easter debate of the Philomath Debating Society, Charles Horgan, '33 and Edward Kennedy, '34 for the negative, defeated John Quirk, '34, and Frederick Moriarty, '34, who defended the question, Resolved: That Holy Cross men possess social development equal to that possessed by undergraduates of other Eastern non-sectarian colleges and universities.
Quirk, who opened for the affirmative, began his speech with a definition of terms and then insisted that the opportunities for social development had been considered before a comparison could be made with other colleges.
Speaking for the negative, Horgan began with a refutation of previous remarks. He stated that social development was accidental, not an essential effect of education. As he said "Though the education at Holy Cross might be far superior to that offered at other institutions, nevertheless the opportunities for acquiring the veneer, poise and social development are inferior."
The concluding affirmative speaker, Moriarty, proved the statement of his colleague that social development depended on fundamental knowledge, ability to press oneself well and social experience in dealing with our fellow men. As these requirements were met, Horgan said, Holy Cross equaled as well as any other school, he concluded that our social development is equal to that possessed by students in other schools.
Kennedy, the final speaker for the negative, returned to the argument that has been begun by his colleague and quoted particular instances to show that the opportunities for social development were not as great in Worcester as in other places. The system in effect in Holy Cross was also mentioned as being detrimental to social development.
The debate was concluded by a rebuttal that was noteworthy for the number of distinctions that were made.
Next week's question was announced as Resolved: That this House goes on record as approving the emergence of American women into public life. John Matthews, and Joseph Kelly, '33 will argue affirmatively. They will be opposed by John Linahan, '34 and Edwin Moline, '34.
Tryouts Held To Replace Graduating Cheerleaders
(Continued from Page 1)
preceding the junior orals. Thus all "tryouts" will have a chance to show their wares during spring football practice.
Selections will be based entirely on the ability and talent demonstrated. "Tumblers" are in demand and all interested are urged to report to Varela. All are encouraged to enter into the competition.
SENIOR BALL PLANS ADVANCE
Ticket and Music Committees Report Progress Made for Dance
Robert E. Dillon, '32, chairman of the ticket committee for the senior ball, has announced that tickets will be available in the latter part of May for underclassmen and for seniors at any time up to the evening of the affair.
During the Easter holidays Charles Murphy, '32, general chair-
Musicians Enjoy Successful Tour
Concerts Given at Albany, Waterbury, Paterson, Hartford
WELL RECEIVED BY CAPACITY AUDIENCES
The members of the Holy Cross Musical Club convened in Albany on Easter Monday to begin their annual Easter tour. Before an audience which braved a heavy storm to be present the Orchestra and Glee Club gave their first concert in the auditorium of the Albany High School. After they closed with the playing of the Alma Mater, Fred Mirilani's Crusaders played for the dancing which continued for the rest of the evening.
Tuesday evening brought the musicians to Waterbury where they were enthusiastically received by a large and colorful audience. Buckingham Hall proved to be acoustically perfect, and as a result the presentation was particularly effective, the "Echo Song" featuring the program.
With the last farewell in Waterbury, the motor coaches sped through lower Connecticut to White Plains where a stop was made for lunch. The trip was resumed and soon the buses were crossing the new span of the Hudson, the Washington Bridge.
Beneath the Palisades the club was greeted by an escort of motorcycle police who led them without a halt to the Paterson City Hall. There they were welcomed by Mayor Hinchliffe, who presented a key to the city of Paterson to each member of the club.
Needless to say the concert was a success from a financial as well as a musical standpoint. Old friendships were renewed, and new ones made; and the Holy Cross Musical Club had again won acclaim for the Garden State.
History Club Has Peace Conference
(Continued from Page 1)
ing to Pope Benedict XIII the basis of peace negotiations was mutual free concessions without hope of selfish profit.
Representatives of various nations were appointed to present a list of their country's demands, concessions, and propositions at the meeting scheduled for Friday, April 15, when the same subject will be discussed in a more formal manner by the appointed delegates of the various nations represented in the society's discussion.
SODALITY HEARS INFORMAL TALK
Literature Committee Reports Progress in Choosing Books
The regular Monday evening meeting of the B. V. M. sodality presented Father Fair, S.J., a speaker on topics of a varied nature. First he read to the large gathering the letter that Sister Mary Teresa, superior of the convent in Jamaica, recently sent to Holy Cross to express her gratitude for the money sent her as a gift of the sodality.
Next the plentiful indulgence granted for the making of the work of the Cross were explained, an explanation also given to all applicants for splendid enthusiasm they had shown for this devout practice during Lent.
Books of interest to sodalists, John M. Ducey, '32, chairman of the Sodality Committee on Catholic Literature, endeavored to make known to be found in that section of the library reserved for the sodality, directly under the new bookshelf, and it is hoped that all sodalists will take advantage of this splendid opportunity for selected Catholic reading.
Alumni Gather To Honor Archbishop
(Continued from Page 1)
Washington, D. C., will officiate a toastmaster.
The banquet is expected to prove the most distinguished affair ever undertaken by the graduate organization of the college. A very considerable gathering is expected, since more than four hundred alumni and guests have already signified their intentions of being present.
Complete arrangements for the dinner are in the efficient hands of Mr. Charles Bowman Stroma, '32, Executive Secretary of the General Alumni.
Frosh Debaters Await Mock Trial
PREPARATIONS MADE FOR PRIZE DEBATE
With a Mock Trial, a Prize Debate, and several Section Debates scheduled for the remaining weeks, the Freshman Debating Society plans to round out a successful year. On April 20 the Section Debate will be resumed when Section F opens on Section B.S. The Mock Trial of Aaron Burr will take place early in May, while the Prize Debate will come later in May.
For the Section Debate on April 20 the question is Resolved: That a change to a Democratic Administration would be for the best interests of the United States. Charles Connor and Edward Sugre will represent Section F, and John Barry and Charles Cuneo will debate for Section B.S. The debaters of the winning Section will receive charms.
The Mock Trial of Aaron Burr, an innovation promising to arouse interest, will take place early in May. Those taking part are: Aaron Burr, the prisoner, Thomas Gilligan; the judge, James Desmond; clerk of court, John Nyhoff; prosecuting attorney, Frank Scalfi and John Scanlon, defending attorneys. Russell Olsen and George Brannan. The jury will be picked from the members of the club.
Six members will be chosen to exhibit their most eloquent argumentation for the Freshman Prize Debate to be held the middle of May. Ten dollars in gold is the prize given to the best individual debater. The judges are picked from members of the faculty.
H. C. - B. C. JOIN IN COMBINED CONCERT
(Continued from Page 1)
chestra presented "Londonderry Air," "Naila," and "Japanese Sunset." "Naila," by Delibes was the best received. Two different numbers, "Turkey in the Straw" and "Manhattan Serenade," were presented by the Holy Cross Philharmonic orchestra. Of these the latter was more skillfully executed.
The excellent performance of two instrumental soloists lent a note of variety to the program. The first, Mr. Joseph G. Brennan '32, of Boston College, presented a group of violo-cello solos. "Ave Maria" was very effective due to the sweetness and softness of performance.
George J. Brennan, '35, received tremendous approval for his finished performance of "Bolero" by Moskowksi. An encore, "Intermezzo" followed.
Boston College can well be proud of its baritone soloist, Mr. Paul J. Mahoney, '32. Mr. Mahoney upheld his fine reputation with his singing of "Kingdom by the Sea" and "Give a Man a Horse He Can Ride."
Both glee clubs were well received and gave an unusual performance. The Holy Cross Glee Club excelled in its presentation of "O'er in the Twilight Glow" by Drake-Merwin. The singing of both Alma Mater's concluded the concert.
All supervisors, students and student organizations are requested to avail themselves of the opportunity for free scholarships made possible through the courtesy of the leading magazine publishers again this year. It is requested to reply to the National Organization.
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MEET THE CROWD AT MANNIX'S
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College Bookstore
Now is the time to secure some new stationery at the Bookstore. Make your letter distinctive by writing on the beautiful stationery of the college. Stationery with the Dinand Memorial Library seal is very popular. Come in and we will show you how complete your college store is at the present time.
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Chesterfield Radio Program
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COLUMBIA NETWORK
© 1932, Liggett & Myers Tobacco Co.
THE TOMAHAWK
Published Weekly at Holy Cross College, Worcester, Mass.
Subscription $2.00 Yearly
EDWARD J. HIDALGO, '33
Editor-in-Chief
John M. Joy, '33
Anthony J. Wenzel, '33
John F. Regan, '33
John Cahill, '33
Louis E. O'Connell, '33
L. Domnelli Bowskowi, '34
Lawrence J. O'Brien, '34
Edward A. Kennedy, Jr., '34
Peter J. O'Brien, '34
James F. Mathias, '34
John J. O'Brien, '34
Edwin G. Molina, '34
EDITORIAL BOARD
Edward B. Hanify, '33, Chairman
Raymond G. Leddy, '33
Richard J. McCabe, '33
Christopher J. Reynolds, '33
Lewis R. Wheelock, '33
Frank P. Cronin, '34
SPORTS STAFF
J. Frank Morris, '33, Sports Editor
Robert J. Woods, '34
Joseph F. Mulready, '34
Assistant Sports Editors
BUSINESS BOARD
Charles R. Callahan, '33, Business Manager
Advertising Manager
Circulation Manager
Assistant Business Managers
Vol. VIII. APRIL 12, 1932. No. 23.
LIBERALS AND MEDDLERS
The press lately has been filled with tales of college students revolting, college students rioting, college students investigating. If the press accounts had any significance it was lost in the ridiculous elements of flying caps, hair rising, temper tantrums and lowered stature in argument speeches. That in the different episodes were traces of the workings of the same so-called "liberal" influences which are capitalizing on discontent in the country is a fact lost sight of by many writers, who must be witty at any price.
The sociologically minded students who were advised "to go elsewhere" by Southern officials have figured prominently in the daily papers. The leading meddlers were probably headed as they deserved. Anyone who has looked in a locality where a strike has been declared and who has witnessed the advent of Communists into the neighborhood to foment more trouble and prevent any adequate or peaceable agreement between local labor units and management realizes the evils of outside interference by people with an axe to grind. Anyone who has lived in a town where the orderly processes of justice have been assailed by radical poseurs and misinformed outsiders looks skeptically on "disinterested" reformers sent by liberal organizations.
The perambulating college sociologists may have meant well, the conditions they came to investigate may have been noxious, but what good ultimately would these immature, hasty investigators have done? Their effects really began at home and the very students investigated have their own laws and their own offenses. When the ejection of the students is followed by a simultaneous announcement that certain advocates of the Russian Five-Year Plan intend to follow them up and test the right of American citizens "to come and go as they please" the directing force of the investigation grows clearer. It was bad enough to sense an atmosphere of the whole trip which was associated with the shamming tours of Junior League girls. When the slow hoof of radicalism appears matters are not improved. The connivance with which may satirize the local Southern authorities as they will, but they should be careful that they do not create sympathy for interests hostile to our institutions when they do so.
Moving back to the North and leaving for a while those students who "made a noise" and the administration of a large Eastern university embarrassed by the advocates of an expelled student-editor who riot and strike to preserve liberalism in the university. According to the Dean of the school, the editor was expelled for continuous persistence in innuendoes which he could not support. In the language of a non-partisan Boston paper he was "insane" about the premises. It would have been a much deeper move to publicly emblazon in Alma Mater than any single influence in her history. The capacity of the modern student-editor to get "drunk on ink" is of course notorious. We do not presume to know the inside situation at this large university. If things in its interior organism needed action it does strike us that a more moderate, tactful and less notorious-seeking an attitude might have gone further. The editor of the "Lincolnian" defense of the "Yale Alumni Weekly" wisely remarks, "Free speech is too dynamic a thing to be used without tact or intelligence." But what interests us most in the whole episode is that the very same organization which is going to follow up the student investigators into Kentucky has one of its attorneys representing the expelled editor in a suit against the university. It is also interesting to note that the rioting students had hoped that the very prominent socialists would address them. Yes, the "liberals" are at work today in colleges and other places, hammering away at the established order.
The college liberal, the youthful apostle of Karl Marx is usually very clever. He is well-educated if you look at education from its effect on the intellect alone. The fact is that you can't. Education which centers only on the intellect produces as one sided a creature as the trained athlete with an untrained brain. The latter might just as well be termed "educated" as the man whose intellect alone has known cultivation.
He hum! So sorry, it must be the weather. "In the spring a young man's fancy turns to thoughts"—Ugh, what an ugly word! Have you ever tried typing it out twenty times? Well, type down, if you must be a precisionist. However, the vibration on the bony thorax, chest to you, is very effective in keeping one awake. Rather a paradox, that putting something on your chest to get something off it. Speaking of paradoxes, you have heard of the beggar on horseback haven't you? Well, we found one top that, a beggar in spats no less. He stepped on our toes in a sudden rush during the holidays—ah, those holidays—and literally impressed upon us the necessity of buying a little tin monkey. We gave him a dime and reached for the monkey, but it must be aaux-rat to take a monkey from a man in spats dying of consumption for he was so astounded that he forgot to cough. Consumption? Yes, he had it bad. In fact it seemed that everyone in his family except the canary, were racing so we who could die first. In his exordium he mentioned so many relations past and present, that for a while we were afraid that he might turn out to be seventy-seven years old or something. He left the train at the next station and as he jauntily strade away, spats and all, looking better dressed than we were, we began to feel sorry. It was like a nice little tin monkey. However, to get back to dear old Worcester What is that strange noise?—have you noticed the latest advertising methods employed by one of the local restaurants? They have launched a symbol—no demerits if spelled with the "ol"—gracefully in a box in the window directly beneath a sign reading "Fresh Killed Lamb." The idea being, we suppose, that one would enjoy it perhaps more if one knew the little lamb personally. The innovation also calls for a revised Mother Goose:
"Little lamb, little lamb, have you a chop?"
"Yes sir, yes sir, see it as I hop."
With apologies to Tennyson, Millard (Continued on Page 7.)
The absent-minded professor again. This time he walked into a well filled classroom at Stanford and set about calling roll. Not a single syllable was uttered in response to his careful pronunciation of a long list of names. At the end of the roll, the "prof" looked up and discovered he had entered the wrong classroom. His retreat was hasty and disorderly.
At the University of Minnesota, chemists use five miles of rubber tubing, six tons of acid, fifty thousand bottles, and a half million matches yearly.
American collegians have a hundred ninety-three names for intoxication, sixty-two appellations for automobiles, and one hundred seventy-four ways of telling undesired personages to remove themselves.
College students in Budapest, Hungary, are seeking the job of public hangman in order to defray their expenses.
The double standard appears to be in vogue at Syracuse. A few weeks ago, the co-eds were permitted to stay in bed and eat classes since many caught colds. The unfortunate men had to attend class.
From the Manhattan "Quarant" comes this bit of old New York: A drunk standing in front of a huge building dropped a penny into the mailbox and looking up at the clock tower we heard to mutter, "M'gosh. I've lost eleven pounds."
General Ethics - Special Ethics
Outline of Lectures on Ethics
By JOSEPH F. SULLIVAN, S.J.
Professor of Ethics, Holy Cross College, Worcester, Mass.
A Text Book Suitable for Classes in Ethics
Adoptions:
Fordham University, New York City.
Georgetown University, Washington, D. C.
St. Joseph's College, Philadelphia, Pa.
Loyola College, Baltimore, Md.
Catholic University, Buffalo, N. Y.
Holy Cross College, Worcester, Mass.
Boston College Graduate School, Boston, Mass.
University of Detroit, Detroit, Mich.
Loyola University, Chicago, Ill.
John Carroll University, Cleveland, Ohio.
St. Louis University, St. Louis, Mo.
Creighton University, Omaha, Neb.
Loyola University, Venice, Calif.
University of California, San Francisco, Calif.
Seattle College, Seattle, Wash.
St. Xavier College, Cincinnati, Ohio.
Spring Hill College, Mobile, Ala.
St. John's University, Collegeville, Minn.
Georgetown Visitation Convent, Washington, D. C.
Drexel Institute, Philadelphia, Pa.
College of St. Elizabeth, Convent Station, N. J.
Emmanuel College, Boston, Mass.
Mundelein College, Sheridan Road, Chicago, Ill.
Notre Dame College, South Euclid, Ohio.
Convent of Mercy, Mohawk, Ala.
The Spalding College, Lawrence, Kan.
Loyola College, Montreal, Canada.
St. Mary's College, Halifax, Nova Scotia.
Duquesne University, Pittsburgh, Pa.
Niagara University, Niagara Falls, N. Y.
Columbia College, Davenport, Iowa.
College of the Holy Family, Syracuse.
The College of St. Rose, Albany, N. Y.
Address:
HOLY CROSS COLLEGE PRESS
MR. FRANK MILLER
Worcester, Mass.
General Ethics—$2.00
Special Ethics—$2.00
25% discount to students
Cross Campus
By Peter J. Kennedy, '34
Yes, the worst part of any vacation is coming back to school at the end of it. . . . Bull sessions on the recent big doin's at home help to prolong the agony. . . . But a few things are worth mentioning, for instance. . . .
At the Waterbury Dance. . . . While the Glee Club and orchestra entertained, the four managers gathered in the balcony to view prospects for the coming dance. . . . Joe Misset put in an appearance, as per usual, in heavy formal. Ah well, that's the way with these impressionable lads who see a lot of movies. . . .
Down Paterson way. . . . Ted Keegan thoroughly enjoyed that long awaited reunion. . . . Ye unfortunate scrabe brought along a date, but, due to deprivations of purse-lipped musical club men, spent the dancing time holding down a chair. . . .
Bostonian high spots. . . . Frank Duane crashing in street clothes at eleven o'clock. . . . Henry Hayward obliging all present with a specialty number reputed to be a combination of the Charleston, the ragtime gavotte, hinky-doo, New Bedford buckwack, and a few private inventions attributed to the artist. . . .
Some notes matched at Holyoke. . . . Much of the success of the night can be attributed to the presence of Clarence Marshall, Edward Garvey and Howard Tucker. . . . Charlie Connors was noteworthy, first for stopping in the middle of a number, thinking it was the end, and later for offering an intermission tap dance. . . .
In the Metropolitan area. . . . Ed Halliwel was out for a visit, got lost in Bay Ridge. Well, who wouldn't? . . . It is still contested that there were a few Cross men among the crowd at the Met dance. . . . The absence of Ray Howe left a space difficult to fill. . . .
Out in Pittsburgh, two seniors who go under the names of Walsh and Gallagher held an impromptu soirée. . . . Weather reports indicate that the affair made up in intensity whatever it may have lacked in numbers. . . .
Just at random. . . . Frank Hanify and Ben McGrath are supposed to possess the unique distinction of owning the only window shade on Fourth Fenwick. . . . Has anyone, either for the sake of sanitation or of esthetic satisfaction, considered the possibilities of washing corduroy trousers? . . .
Martin Healy sincerely bawled out returning after the holidays by bus. . . . Bob Woods, Tom Tony Woods and a few other fellows lost their traditional tendency so far as to sink to accepting motor car seats. . . . The sixty-three out for spring football practice are expected to last until the first vigorous grass drill. . . .
Honor Roll for Third Quarter
(Continued from Page 1)
A J. Leo O'Gorman, Jr.
B Edward B. Hanify
C Peter S. Zarecki
E Edward J. O'Brien
E Richard J. McCarthy
B.S. Joseph E. Keating
B.S. Charles J. McGee
A Thomas J. Carlin
D John A. Dill
D Francis H. O'Brien
D Albert J. O'Connor
D Edward L. William
E Paul E. Fleming
E Rowland K. Hazard
E John F. Scannell
G George C. Hoyt
G Michael J. McDonald
B.S. John A. Shea
B.S. Charles A. Fetscher
A Charles F. Fity
A Thomas J. Gilligan
A Thomas F. Grogan
A Franklin V. Murphy
A Cornelius K. Roche
R Robert F. Devoy
B Lawrence S. Riley
C James W. Carney, Jr.
C Michael D. Meehan
C Russell C. Allen
D John P. B. Hunt
F George S. DePrisio
G John J. Caulfield
G Robert E. Courtney, Jr.
G John J. Galvin
G John D. O'Connell
B.S. Henry J. Puchalsky
B.S. Charles W. Smith
St. Peter's High School
B. M. C. Durfee High School
Lawrence High School
Cheverus High School
Cathedral High School
Gorham High School
Xavier High School
SOPHOMORE CLASS
St. Peter's High School
Crocker High School
Senior High School
St. Aloysius Academy
Poughkeepsie High School
St. John's High School
Wickford High School
South High School
William H. Hall High School
St. Leo's High School
Edward Little High School
Far Rockaway High School
FRESHMAN CLASS
Regis High School
Regis High School
Public Latin School
B. M. C. Durfee High School
Xavier High School
St. John's High School
St. Thomas' Seminary
B. M. C. Durfee High School
Keily Academy
St. Ignatius High School
St. Bernard's High School
Manfield High School
St. Bernard's High School
Bulkeley High School
De La Salle Academy
Senior High School
Barre High School
Abitong High School
Quarterly Average of 85% or Over
SENIOR CLASS
Classical High School
Campion Preparatory School
Cheverus Classical High School
Boston College High School
Regis High School
Cathedral Preparatory School
Chicago Military School
Jamaica High School
De la Salle Academy
B. M. C. Durfee High School
Victor High School
Brooklyn Preparatory School
Fitchburg High School
Xavier High School
St. Peter's High School
Worcester, Mass.
Prairie du Chien, Wis.
Portland, Me.
Boston, Mass.
New York, N. Y.
Erie, Pa.
New Hope, Mass.
Jamaica, N. Y.
Newport, R. P.
Fall River, Mass.
Victor, N. Y.
Brooklyn, N. Y.
Fitchburg, Mass.
New York, N. Y.
Worcester, Mass.
JUNIOR CLASS
Boston College High School
St. Mary's High School
Maury High School
Xavier High School
North High School
Boston College High School
St. John's High School
Brookfield High School
Cheverus Classical High School
South High School
Xavier High School
Xavier High School
B. M. C. Durfee High School
Cushing High School
Wakefield High School
Regis High School
Newtown High School
St. Jerome High School
Leominster High School
Cathedral High School
Brooklyn Preparatory School
St. Joseph's High School
Regis High School
St. John's High School
Presentation Mary High School
Clinton High School
All Hallow's Institute
St. John's High School
Worcester, Mass.
Fall River, Mass.
Lawrence, Mass.
Portland, Maine.
Springfield, Mass.
Gorham, Maine.
New York, N. Y.
Jersey City, N. J.
Waterbury, Conn.
New Britain, Conn.
Rome, N. Y.
Poughkeepsie, N. Y.
North Cambridge, Mass.
Wickford, R. I.
Westfield, Mass.
West Hartford, Conn.
Ashley, Pa.
Auburn, Me.
Far Rockaway, N. Y.
New York, N. Y.
New York, N. Y.
Bristol, Mass.
Fall River, Mass.
New York, N. Y.
Worcester, Mass.
Hartford, Conn.
Fall River, Mass.
Lowell, Mass.
Chicago, Ill.
Fitchburg, Mass.
Manfield, Mass.
Fitchburg, Mass.
Hartford, Conn.
Newport, R. I.
New Britain, Conn.
Barre, Mass.
Abitong, Mass.
Classicists Convene In Dinand Library
(Continued from Page 1)
Committee for two years, Miss Helen Fairbanks Hill of Rogers Fall School, Lowell, and William D. Goodwin of Pittsfield High School, and executive committee for one year, Prof. Thomas Means of Bowdoin College and Miss Mary Marlett of Manchester High School.
The paper, "A Little Voyage of Discovery Through the Manuscripts," by Dr. John Savage of Winchester, was then presented.
In the afternoon the first paper was by Mr. Irving T. McDonald, Professor of English and Librarian of the Dinand Library at Holy Cross. His subject was, "Rene Rapin, S.J., Seventeenth Century Virgilian." To call Rapin a Virgilian is not so much a matter of praise as of accuracy in description," Mr. McDonald stated.
Friday evening, "The Janus Faced Art of Propertius," by Miss Elizabeth Haight of Vassar and "A Visit to Cyrene and Cyrenaica," by Prof. Vladimir Rostovtzeff of Yale were delivered.
Proceedings on Saturday began with a paper presented by Prof. Horace Poynter of Phillips Andover Academy on "Latin as a Fetish." This was followed by "Magistrate Names on the Coins of Aragon" by Mrs. Newell Couch of Providence; "Monning a Poet," by Prof. Robert of Amherst; "Methods of Accounting in the Zenon Papryri," by Miss Grier of Columbia; and "The Descent of Grammar," by Prof. Caro Lynn of Wheaton.
Talks on "The Real Cicero," by Prof. Rice of Boston University; "Gleanings in Etruscan Fields," by Prof. Gray of Smith; and "Justin XIII and the Ephemerides of Alexander's Expedition," by Prof. Robinson of Brown were also given.
That the Association is unalterably opposed to any step on the part of the College Board on Entrance Examinations that will discriminate against Latin and Greek was clearly shown when the period for general discussion of papers which had been given was held.
During the general discussion, Dr. John T. Kirtland of Phillips Academy, Exeter, brought the question of possible discrimination by the College Board on Entrance Examinations against Latin and Greek before the Association. He said: "Although the fact is not generally known, the College Board contemplates changes in entrance examinations which have far-reaching and hostile effects upon Latin and Greek." Agreeing with Dr. Kirtland that the matter demanded immediate action, the meeting, on motion of Dr. Henry M. Hubbel of Yale voted that a committee be named to present vigorously the attitude of the association. Dr. Kirtland was named chairman of the committee with power to appoint his fellow members.
Considerable discussion developed over Dr. Kirtland's disclosure. It was concluded that the proposed changes in tests for college entrance would be inimical to proper higher education.
Communication
The Editor of The Tomahawk:
In the issue of Feb. 23rd, there appeared a criticism of the Purple magazine "Songs of the American Indian," in the following temper:
"A tomahawk should be used on all writers who attempt to glorify the 'Red Man' and his ways, and the most cruel of all Mongolians. The Indians had no music, their ears are tone deaf, and soul deaf to the language of the white man. They have woven about them romance without truth. They have attempted to cull from a language of grunts and crude sounds a melody that is fragrant with a beauty that would arouse a noble emotion. They always have the fad followers who put their faith in such nonsense."
Rather handicapped by space, an outlay of proofs in substantiation of the truth that the Indians did have songs, may seem inadequate. However, I shall attempt to treat this criticism from demonstration of the preserved and intact legends which the Indians themselves today possess, and the historical truth whether or not the first or early contacts with the Indians evidenced such possessions.
There have been hundreds of volumes edited on the Indians of America, but there is no finer work than "The Indians' Book," which was published in 1907. In it we find the authentic and legendary songs which have been handed down through the centuries among the Indians. This edition was not a mere endeavor by several students, but was the gathering and accumulation of the Indian tribes in America, made, it is true by one Nannie Curtiss. The reference reads: "The Indians are the authors of this volume. The songs and stories are theirs; the drawings, cover design, and title pages were made by them." The work of recorder has been but the editing and the arranging of the Indians' contributions.
A copy of these songs and legends, taken from the Indians of the Plains, the Southwestern Indians, the Northwest, the Eastern, and the Middle, was sent to Theodore Roosevelt, when he was President, and his animated response in handwriting was: "These songs cast a wholly new light on the depth and dignity of Indian thought, the simple beauty, and the strange charm—the charm of a vanished elder world—of Indian poetry."
For added information, the reader may find the following works extremely useful: "Indian Music" by Edwin Bazar, American Naturalist, Vol. XVIII, pp. 267-274; "Aboriginal American Poetry," by John Reade, American Roy. Soc. Proc.; "Music of North American Indians," by Albert Gotschel, in the American Naturalist. Besides, one cannot pass over the works of Alice Fletcher, Francis Densmore, and James Fillmore, nor the many articles to be found in the Smithsonian Institute and the Peabody Museum on Archaeological and Ethnological studies in America. The gist of the music and song of the Indian, due to a study, might be best summed in these words of a student of Indian music: "Of the music of the Indians, what do they differ from that of civilization? Music is a trinity: rhythm, melody, and harmony. The Indian music has rhythm and melody, not harmony. Next, let us turn to history and read from these pages about the early contacts with the Indians, and what they revealed in regard to the Indian and song. We shall confine ourselves to a set of books, known to the historian as the fountain source of facts, called the Jesuit Relations and Allied Documents." Certain things are the truths of the lives of the Jesuit missionaries in the New World between the years 1610 and 1791. The present form of the editions which consists of seventy volumes edited in 1901 by Reuben Gold Thwaites, a Protestant and Secretary of the Historical Society of Wisconsin.
Joseph Marie Chaumont, in 1640, writing about the Hurons: "Now hear then, Savages children come to Mass every day with the greatest reverence as they hear them sing during the elevation, they have remembered one of the motets so well that they sang it finely at St. Josephs on Christmas Day. Later on in his relation he states: "They made also sing the Irinikit, a song composed in their own language." Father Bressani in his writings, recorded the sentences: "In France people have believed that their speeches and addresses which we reported were fictitious; but I can assert that most of these when translated into the Indian language are in their own language. They have an excellent ear for music, but their music is different from, and in some degree, more martial than ours."
Le Jeune's relation in 1688, states: "We sing the Apostles' Creed in their native rhymes." And then again, Rale, in a letter to his brother in 1716, wrote: "Here is the O Salutarius in the native Abnakis tongue:
O salutarius Dei genitrix
Spem kik kipollu go i danek
Nemouk kipollu danan ghaberk
Taha soii grihre."
There are many more references, but space does not permit a further research. Hence, I hope this article will serve as a light to the path toward truth, and toward relieving the notion that the "Indian has no Song" from our midst.
William Charles Zeller, '33.
Honor Roll for Third Quarter
(Continued from Page 5)
C Carl J. Graf
C Alvin G. Seelman
D Raymond J. Byron
D Lawrence F. Gillarer
D John G. Kelleher
D John R. Linehan
D John W. Moore, Jr.
D Harry J. Walsh
E William A. Carroll
E Edward C. Harold
E Richard T. Harrity
E Martin J. Hickey
E Bernard E. Hobson
E John W. McCormick
E Charles P. Read
F Anthony R. DeFranco
F Charles H. Hoye
F James M. Kennedy
F Paul L. Morley
F James C. O'Shea
G Arthur M. Carrellas
G William J. Findlan
G Joseph L. Hammond
G Joseph L. Henneke
G Albert W. McCarthy
Ph.B. James P. Bradley
Ph.B. Gabriel P. Ferrazzano
Ph.B. Paul H. Holmberg
Ph.B. Stanley J. Koceera
Ph.B. Joseph L. Lotulidge
Ph.B. Thomas A. Shea
B.S. William E. Lyons
St. Gregory Seminary
Marquette Univ., High School
Hudson High School
Blackstone High School
Greenfield High School
New Haven High School
Roxbury Latin School
St. Francis High School
Uxbridge High School
West High School
Clinton, Mass.
All Hollows Institute
Mt. Carmel High School
Wyoming Seminary
St. Mary's High School
Cathedral Academy
St. Mary's High School
North High School
Concord High School
St. Aloysius Academy
De la Salle Academy
Campion Preparatory School
St. Joseph's High School
Gardner High School
Rockland High School
Ware High School
Warren High School
Brooklyn Preparatory School
New Bedford High School
Milton Technical School
Mt. St. Charles Academy
Brockton High School
Freshman Class
B. M. C. Durfee High School
St. John's High School
Xavier High School
La Salle Academy
Public School
St. John's Preparatory School
Pittsfield High School
Perth Amboy High School
St. John's High School
South Portland High School
East Boston High School
St. Patrick's High School
North High School
William H. Hall High School
Beverly High School
Ridgewood High School
St. Joseph's High School
De la Salle Academy
Loomis School
Norwood High School
Danbury High School
Sacred Heart High School
St. John Preparatory School
Fitchburg High School
Gardner High School
Cambridge Latin School
English High School
Classical High School
Worcester High School
Arlington High School
Classical High School
Mt. Carmel High School
North Brookfield High School
Crusader Council Will Hold Dance
On Friday, April 22, the Crusader Council of the K. of C. will hold its annual dance in the main ballroom of the Bancroft Hotel. Dancing will last from nine until two. Twenty-thirtieth permissions may be obtained by those students who have paid. The admission bids are three dollars and a half.
The dance will feature the well-known Smith Ballew orchestra which has become increasingly popular in collegiate circles. This group has played for the important social functions of such schools as Princeton, Lehigh, Lafayette, and St. Joseph's.
Those who wish to become members of the Crusader Council this year still have an opportunity as another degree will be conferred soon.
Mock Trial in Leonard Hall
Departing from its usual procedure, the B. J. F. Debating society will hold no meeting tonight. Instead a mock trial has been planned and will be held in the Leonard Debating Room in the basement.
As previously announced in the Tomahawk, the trial will be held for the purpose of determining whether the Allied nations or the Imperial German government is responsible for the destruction of World War II. The wide appeal and present pertinence of the subject selected justifies its choice. James M. Beck, assistant attorney general of the United States, expressed the present stand on the matter by the following words: "No consideration either of sympathy admiration or pity can in any manner affect the determination of the great ethical question as to the moral responsibility for the great crime against civilization... To determine whether this moral responsibility lies should be the purpose of immediate generations."
International Court proceeding to be in order at the trial. The men chosen to participate are: Chief Justice, Peter J. Kennedy '34; Counsel for the Allies, James P. Bradley, '34; Witnesses for the Allies, Edward J. O'Brien, '32; Lewis H. Whipple, '29; and Edward J. McCarthy, '33; Counsel for the Imperial German Government, Vernon B. Santen, '32; Witness for the Imperial German Government, Matthew J. Ashe, '33; Francis J. Costigan, '33, and Francis Murphy, '33. There will also be a number of court officials.
At the conclusion of the proceedings the Chief Justice will deliver a decision in favor of either one of the contending parties. A special prize will then be awarded to the man esteemed by the jury judges to be the best speaker of the evening for case presentation. Every participant is eligible for this award.
— TRY OUR —
Expert Shoe Repairing and Hat Cleaning Service
Central Shoe Repair Shop
Opposite Slater Building
Dance in the Robin Hood Room at
The Bancroft
Special Dinner 5.30 to 8.30
$1.50
Dancing each evening from 8 to 12
Music by the Robin Hood Orchestra
A la Carte Service
No Cover Charge
Paul Mac-Hale's Lobster Garden
375 MAIN STREET
WORCESTER, MASS.
Booth and Table Service
Banquet Rooms for Parties up to two hundred
Class Debates Are Announced
(Continued from Page 1)
growing steadily in popularity, judging from the great number of applicants responding to the call for volunteers. The faculty encourages these debates because they reach out to more students and give them greater opportunity for experience and practice than the varsity debates. There has always been keen and close rivalry between the classes in the matter of debate, and this year the rivalry as class spirit is more noticeable than ever.
The winner of the Senior-Junior debate and the winner of the Sophomore-Freshman debate will be given awards from the O'Connor purse, which was established for this purpose.
Pillar to Post (Cont.)
(Continued from Page 4)
ton, Burns and you, Dear Reader. The mention of Tennyson et al. reminds us that this is the open season for poets and we appeal to your sense of justice and ask that you adopt our slogan, "Think before you shoot!" Be a sportsman! Poets are such easy game; these days that the bagging of them is not an accomplishment of which to boast. Look deep into those dunces' pleading eyes—accent your own adjectives—and shoot if you can! What does it matter if their song disturbs your slumber? What boots it if in their rapture they trudged upon your toes and see you not? Do you believe that you have the right to stop the flights of genius or muffe the mouths of these sweet singers of song? Do you? Well, adopt our slogan: "Think Before You Shoot"—and your aim will be surer.
ORATORICAL CONTEST RULES ANNOUNCED
Thursday evening, April 28th, will be the date of the oratorical contest in Fenwick Hall. Preparatory to its presentation tryouts are to be conducted April 17th in Fenwick Hall. The subject may be of the contestant's own choosing and the time limit is to be restricted to five minutes. All prospective competitors must submit their names to the Dean's office before April 14th.
In the contest conducted last year, first honors were awarded to Owen P. McGivern '32. The honors for second and third place in the same contest were given to Edward J. Hidalgo, '33, and to James P. Bradley, '34.
BART STARTING TWENTIETH YEAR
Has Notable Career as Track Coach During Past Two Decades
By Joseph F. Mulready, '34
What a glorious panorama of brilliant performances would pass in review before the eyes of track sportsporn if it were possible to recall that select group of immortals of Holy Cross track history during the past two decades. Surely the spectacle would be an awe-inspiring and thrilling one for all Purist well-wishers. These great runners have had their day of glory and eventually joined the ranks of the "has-beens;" but the man who guided them has been content to remain in the background while his boys received the plaudits of their fans.
Bart has raised many an unheralded youth to the heights in rapid fashion. Walter Mulvihill, of Worcester, became a star of no mean repute when he came under Bart's tutelage. The late Leo Lariviere became a formidable opponent in the mile and gave the great trackmen of his day plenty of think about. When Nurni made his first appearance in this country, Lariviere gave a great exhibition and received high praise from Nurni himself after the completion of their gruelling duel.
Whenever the talk turns to great quarter-milers, Joe Tierney's name is immediately brought up. Tierney was the peak of his class and was seldom defeated when in race. He learned to keep his pace running when he left the magic fold presided over by Bart. One of the most formidable relay teams to ever represent the Cross was that famous combination of Maher, Roche, Burns and Mulvihill. That quartet won the class of the mile relay teams in its heyday.
Bart has attained the greatest success in his first love and hobby—relay racing. In this field the simple mentor has been declared a legend in the competency with which he turned out winning teams. When his combinations seem to reach the peak of fine running the proverbial jinx appears on the scene to ruin all hope of Bart's most cherished dreams being realized. This fate has been verified for the past four years. In 1929 the team ran an undefeated record in six starts only to have illness smash up the lineup when the Intercollegiates came around. With such a situation confronting him, Bart drafted George Mann from among the ranks of the spares. Morin ran a beautiful race, but Hal Klumback couldn't quite make up the deficit and was nosed out at the tape by a Yale man. Dartmouth won the finals; but the Purple should have been out front on the conclusion of the best time.
Bart wielded together a fast quartet in 1930, but Harvard came out with their finest team in years. An idea of the thrills furnished in the finals may be imagined when the two men of the two high class teams broke the world's record in the same race. Bart was bitterly disappointed as he saw Record cross the tape barely a stride in front of Bernie. Last season Bart produced what is considered one of the greatest arrays of Cookley, Hollister, Mann and McCafferty. However, Penn brought out a quartet that was forced by the Crusaders to break the world's record once again. For two successive years the Purple teams were in there forcing another relay team to gain the rec-
(Continued on Page 8)
Conceding Fitch Field to the latest whim of Worcester weather, the Braves were forced to cancel their game with the Crusaders yesterday.
Besides missing the first opportunity of seeing Jack Barry's 1932 model in action, the fans missed what would have been a memorable moment—when Bill Keating would have indulged in a public bull session with scrappy Charlie Shires, one of Bill's pals on the Milwaukee Brewers.
So the Crusaders will have to open up next Saturday against Springfield of the Eastern League over at Fitch Field. Jim Sullivan, last year's Holy Cross captain, has a pitching berth on this Eastern League ball club and might be given the chance to face his old teammates.
Jim Cavalieri became the fourth member of the pitching staff after facing the batters on Sunday afternoon. Mahoney, Quinn and Mulligan had been selected when the uniforms were given out last week.
So if Jack Barry is to have another five-man pitching staff, he has not made much time to make from the remaining hurlers. Make your guess from Lee, Droftar, Keating and Leach. Only one will get a suit.
Over on Freshman Field Jack Reed has been besieged by a horde of ambitious freshmen. Judging by numbers he has enough candidates for the nine positions to conduct a league of his own.
Sharing the same field is Capt. McEwan's spring football squad. This turnout is another maximum capacity affair. That is until scrimmages start after a week or so.
Of course track and baseball attracted most of the football lettermen who were capable enough to make either squad, and you cannot blame them. Three months of football seems to be enough for every New England college excepting Yale and Holy Cross.
Yet you'll agree that the more trying out for next year's eleven the better. Well, it will be Holy Cross next fall. There are just a few important places on the team to be awarded to new men. And some of them are working out now.
Up on the terraces the tennis courts are still taking sun treatments between showers. But soon there will be nets and straight white lines to attract the raquetters.
---
**BASEBALL**
In one of the most desperately fought contests of the entire basketball season, the class of '33 managed to carry off the titular honors in the inter-class final down at St. John's Gym last Saturday afternoon by the score of 29 to 20. Despite the fact that the sophs went down to defeat is their great ability to dominate from the vicious onslaught made by the juniors in the first half stands out as the real feature of the game.
With the opening whistle the Beaventors jumped off to an amazing lead which they kept piling up until the end of the second period. Speed and accuracy characterized their attack, so much so, that the younger Alumni dwellers were completely baffled by the rapidity with which the Junior team tore through the hoop. Tom and Jack McCarthy led their forces in this half and they were responsible for the lead of 15 to 4 over the second-year men. Bob McMahon kept his team in the game by his valiant efforts to score.
At the beginning of the last half of this championship game, the outcome looked very much like a Junior runaway victory, but it was then that the Sophomores just began to play the game. They opened up with a fierce and overwhelming offensive from which the men from Beacon cringed and were barely able to hold advantage in gaining the precious points. Jim Sullivan and the inevitable McMahon began the assault, combining 14 points between them, most of which were attained in the exciting fourth period. The game ended over 100 in this final stanza and only a determining stand by the subsequent victors held that diminished lead until the final blast of the seniors' whistle echoed throughout the gym.
The rivalry between these two competition has met with success and some schools have gone so far as to make participation a school academic requirement.
"Northeastern has taken a definite step in this direction in organizing inter-class sports. Football between classes will form an important part of the sports program next fall under the direction of the student activities department. However, minor sports facilities have been too long on the enthusiasm of a single member of the class or faculty to organize teams and arrange for competition. Most students will agree that the present system is inadequate and should be replaced."
"The organization of a committee on inter-class sports made up of representatives from each of the classes would provide a possible solution to the problem. The group could arrange schedules and provide all necessary equipment for the games at a minimum of expense. They could provide a program sufficiently diversified and intense to accommodate the athletic inclinations of the student body. Such a program would go far toward creating the interest in sports which has been lacking in recent years."
(Signed) Sport.
The fact that intra-mural activities have been stressed by other colleges and universities throughout this country is due almost entirely to the increasing tendency of the modern institutions towards the physical as well as the mental development of the students.
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**Bart Sullivan In Twentieth Year**
(Continued from Page 7)
ord and the glory. This year the mystic jinx was still a step ahead of the Cross. In the second period, with the play going their heat in the fastest trial of the night and appeared to be the team to beat for the title. McCafferty received the stick some five yards behind the leaders. He made a valiant effort; but cut down the inside of the team. He seemed ready to unleash his famous last lap spurt when he was knocked out of stride on the last turn. He fought back to within two yards of the winner at the tape, thereby forcing N. Y. U. to retain the record.
For years now the Holy Cross coach has been taking certain men who had attained a degree of success in their specialty and turned them into star performers without detracting from their worth. Bernie McCafferty never ran a quarter in his life before Bart decided to make the change. Joe Tierney came to Saint St. John's as a high jumper. George Morin was a champion sprinter. Yet all three became champion quarter-milers.
There are three men who will join the ranks of the Holy Cross track immortals next year. It is a great privilege to Bart. Last season, George Morin and Ned Flanagan joined such stars as Kelley, Tierney, Higgins, Larivee, Mulhivill, Roche, Quinn, Maher, Burke, Durkin, McDonough, Wylie, and Klineback. McCafferty, Johnny Holland, and Art McDonnell will become members of that select group as they close their champion quarter-milers.
It would be a rather hard problem to figure out just who will replace the stars that will leave this year. Bart knows the worth of his boys, everyone of them, and the plodder of today will be made the star of tomorrow by his careful training. There will be great Holy Cross teams as long as Bart Sullivan is at the helm of track on the Hill.
---
**Sportsgraphs**
By Joe Mandry, '33
Since it is commonly surmised that basketball is one of the fastest of games, the Joint Basketball Committee, at its annual meeting, designed a few rules to speed up the game. The motto is if the game is going to be fast, let it be good and fast.
Yale's swimming team, which has captured the league championship for the eighth successive year, will lose many veterans through graduation. Just to discourage pretenders to the crown, however, there is an undefeated Freshman team from which to choose players.
The tennis team at St. Thomas College is starting a six month tour of the world. The team will play in England, Spain, China, Germany and France. It is probably a good-will tour.
Word comes from my southern correspondent, Gethell Kerrah, that Tulane University, runner-up in last year's national championships, decided to abolish football as a major sport and adopt Greek letter in its place.
A bit of news about the above mentioned correspondent. Gethell used to act as pallbearer for his dearest of friends, but that was only a matter to an end. Knowing his capabilities, Mr. Morris (he's our sports editor), sent him south it kept us posted on sports.
Pundruch Popitski of Amherst University sent in this, "don't blame me. Oliver, the captain of the Lacrosse team, asked me in an inquiring voice: 'Who was that woman I saw you going down the street with the other day?'"
Pound answered: "That was my sister, that was an alley."
Boston College's hopes of staging a comeback in the two-mile relay, the next event, Cornell next month suffered a severe blow when Don Fleet was forced to withdraw from competition for two months due to serious sickness.
The California Bears have exercised their superiority over St. Mary's on the diamond for four long years. The Gaels have lost eleven out of twelve contests, the last time they won being in 1926.
Keene Fitzpatrick, track coach at Princeton, has organized four track and team tracks for forty-five years. He is also the one who taught Alonzo Stagg the fundamentals of track and other sports.
Purdue, Big Ten basketball champions, scored 718 points in 18 games. How could they average 40 points per game?
---
**Inter-Class Basketball Champions**
The following members of the junior class basketball team have been awarded the intramural basketball championship:
Charles Callahan, Westfield, Mass.; Maurice B. Martin, Holyoke, Mass.; Thomas W. McCarthy, Springfield, Mass.; John F. McDonough, Naugatuck Conn.; Francis J. Quine, Akron, Ohio; George S. Quinn, Chicopee, Mass.; Thomas P. Scannell, Brooklyn, N. Y.; Paul F. Schoenrock, Westfield, Mass.
---
**Downyflake Shop**
Sandwiches, Doughnuts
COFFEE
Look for the Doughnut
413 MAIN STREET
Imported White Buckskin
Sport Shoes - $3.00
All High Grade Shoes. Cannot advertise the names.
J. J. MORAN
135 Front Street
---
**Save Money by Letting Us Do Your Work**
GEORGE CHIN LAUNDRY
Experienced Laundering
Lowest Prices
Corner
Cambridge and Southbridge Sts.
---
**Always ready with those Famous Meals that Satisfy Any Appetite**
THE CANTEEN
GEO. F. GRANGER
|
June 20, 2024
Ms. MARIA IRISH ALOHA CUBILLAN
PIO, Navotas City
Mr. RENATO REYES
ICTO, Navotas City
Dear Sir / Madam,
Respectfully forwarded herewith are copies of Invitation to Bid for the procurement proceeding of the City Government of Navotas, to wit;
1. PROCUREMENT OF SECURITY SERVICES FOR THE USE OF NAVOTAS CITY HOSPITAL / ABC PHP 1,925,700.00
2. PROCUREMENT OF ICT EQUIPMENT FOR THE USE OF CITY GENERAL SERVICES OFFICE / ABC PHP 2,120,000.00
3. PROCUREMENT OF FOOD SUPPLIES EXPENSES FOR THE USE OF NAVOTAS CITY HOSPITAL / ABC PHP 5,499,820.14
4. NEGOTIATED PROCUREMENT FOR THE SUMMARY PROCUREMENT OF GARBAGE MINI DUMP TRUCKS AND GARBAGE DUMP TRUCK FOR THE USE OF CENRO / ABC PHP 7,775,000.00
Pre-Bidding Conference June 27, 2024 1:00 pm
Public Bidding July 11, 2024 1:00 pm
Relative thereto, pursuant to the Revised IRR of Republic Act 9184, we would like to request your respective offices to post the same in the Official Website (MIS) and designated bulletin boards on conspicuous places within the City Government (PIO) for public information continuously for seven calendar days from receipt of this endorsement in aid of transparency and accountability.
We would like to further request your offices to provide us a Certificate of Posting after the lapse of the posting period. The said certificate shall be used by the Committee in issuing a consolidated Certificate of Posting for the said procurement.
Your immediate preferential consideration of this request will be highly appreciated.
Thank you very much.
Respectfully yours,
Atty. JOEL JOSELITO DG. PARONG
BAC Chairperson
INVITATION TO BID FOR
PROCUREMENT OF SECURITY SERVICES FOR THE USE OF NAVOTAS CITY HOSPITAL
1. The City Government of Navotas, through 2024 Supplemental Budget No. 1, intends to apply the sum ONE MILLION NINE HUNDRED TWENTY FIVE THOUSAND SEVEN HUNDRED PESOS (PHP 1,925,700.00), being the ABC to payments under the contract for PROCUREMENT OF SECURITY SERVICES FOR THE USE OF NAVOTAS CITY HOSPITAL (PIN 2024-07-095). Bids received in excess of the ABC shall be automatically rejected at bid opening.
2. The City Government of Navotas now invites bids for the above Procurement Project. Delivery of the Goods is required TWENTY FOUR (24) HOURS DAILY, INCLUDING SATURDAYS, SUNDAYS AND HOLIDAYS. Bidders should have completed, within Two (2) Years from the date of submission and receipt of bids, a contract similar to the Project. The description of an eligible bidder is contained in the Bidding Documents, particularly, in Section II [Instructions to Bidders].
3. Bidding will be conducted through open competitive bidding procedures using a non-discretionary "pass/fail" criterion as specified in the 2016 revised Implementing Rules and Regulations (IRR) of Republic Act (RA) No. 9184.
Bidding is open to all interested bidders, whether local or foreign, subject to the conditions for eligibility provided in the 2016 revised IRR of RA No. 9184.
4. Prospective Bidders may obtain further information from City Government of Navotas – Bids and Awards Committee and inspect the Bidding Documents at the address given below during Monday to Friday, 8:00 AM to 5:00 PM.
5. A complete set of Bidding Documents may be acquired by interested Bidders on June 20, 2024 from the given address and website(s) below and upon payment of the applicable fee for the Bidding Documents, pursuant to the latest Guidelines issued by the GPPB, in the amount of Five Thousand Pesos (PHP 5,000.00). The Procuring Entity shall allow the bidder to present its proof of payment for the fees, in person, by facsimile, or through electronic means.
6. The City Government of Navotas – Bids and Awards Committee will hold a Pre-Bid Conference on June 27, 2024, 1:00 PM at the Mayor’s Conference Room, Fourth Floor, Navotas City Hall Building, which shall be open to prospective bidders.
7. Bids must be duly received by the BAC Secretariat through manual submission at the office address indicated below on or before July 11, 2024, 12:00 noon. Late bids shall not be accepted.
8. All Bids must be accompanied by a bid security in any of the acceptable forms and in the amount stated in ITB Clause 14.
9. Bid opening shall be on July 11, 2024, 1:00 PM at the Mayor’s Conference Room, Fourth Floor Navotas City Hall Building. Bids will be opened in the presence of the bidders’ representatives who choose to attend the activity.
10. All interested bidders should submit, upon request of the Bidding Documents a Letter of Intent, in person, by facsimile, or through electronic
INVITATION TO BID FOR
PROCUREMENT OF ICT EQUIPMENT FOR THE USE OF CITY GENERAL SERVICES OFFICE
1. The City Government of Navotas, through 2024 Supplemental Budget No. 1, intends to apply the sum TWO MILLION ONE HUNDRED TWENTY THOUSAND PESOS (Php 2,120,000.00), being the ABC to payments under the contract for PROCUREMENT OF ICT EQUIPMENT FOR THE USE OF CITY GENERAL SERVICES OFFICE (PIN 2024-07-096). Bids received in excess of the ABC shall be automatically rejected at bid opening.
2. The City Government of Navotas now invites bids for the above Procurement Project. Delivery of the Goods is required 30 CALENDAR DAYS UPON RECEIPT OF APPROVED PURCHASE ORDER. Bidders should have completed, within two (2) Years from the date of submission and receipt of bids, a contract similar to the Project. The description of an eligible bidder is contained in the Bidding Documents, particularly, in Section II (Instructions to Bidders).
3. Bidding will be conducted through open competitive bidding procedures using a non-discretionary "pass/fail" criterion as specified in the 2016 revised Implementing Rules and Regulations (IRR) of Republic Act (RA) No. 9184.
Bidding is open to all interested bidders, whether local or foreign, subject to the conditions for eligibility provided in the 2016 revised IRR of RA No. 9184.
4. Prospective Bidders may obtain further information from City Government of Navotas – Bids and Awards Committee and inspect the Bidding Documents at the address given below during Monday to Friday, 8:00 AM to 5:00 PM.
5. A complete set of Bidding Documents may be acquired by interested Bidders on June 20, 2024 from the given address and website(s) below and upon payment of the applicable fee for the Bidding Documents, pursuant to the latest Guidelines issued by the GPPB, in the amount of Five Thousand Pesos (Php 5,000.00). The Procuring Entity shall allow the bidder to present its proof of payment for the fees, in person, by facsimile, or through electronic means.
6. The City Government of Navotas – Bids and Awards Committee will hold a Pre-Bid Conference on June 27, 2024, 1:00 PM at the Mayor's Conference Room, Fourth Floor, Navotas City Hall Building, which shall be open to prospective bidders.
7. Bids must be duly received by the BAC Secretariat through manual submission at the office address indicated below on or before July 11, 2024, 12:00 noon. Late bids shall not be accepted.
8. All Bids must be accompanied by a bid security in any of the acceptable forms and in the amount stated in ITB Clause 14.
9. Bid opening shall be on July 11, 2024, 1:00 PM at the Mayor's Conference Room, Fourth Floor Navotas City Hall Building. Bids will be opened in the presence of the bidders' representatives who choose to attend the activity.
10. All interested bidders should submit, upon request of the Bidding Documents a Letter of Intent, in person, by facsimile, or through electronic means.
INVITATION TO BID FOR
PROCUREMENT OF FOOD SUPPLIES EXPENSES FOR THE USE OF NAVOTAS CITY HOSPITAL
1. The City Government of Navotas, through 2024 Supplemental Budget No. 1, intends to apply the sum FIVE MILLION FOUR HUNDRED NINETY NINE THOUSAND EIGHT HUNDRED TWENTY PESOS AND 14/100 CENTAVOS (PHP 5,499,820.14), being the ABC to payments under the contract for PROCUREMENT OF FOOD SUPPLIES EXPENSES FOR THE USE OF NAVOTAS CITY HOSPITAL (PIN 2024-07-097). Bids received in excess of the ABC shall be automatically rejected at bid opening.
2. The City Government of Navotas now invites bids for the above Procurement Project. Delivery of the Goods is required BASED ON ORDERING AGREEMENT. Bidders should have completed, within Two (2) Years from the date of submission and receipt of bids, a contract similar to the Project. The description of an eligible bidder is contained in the Bidding Documents, particularly, in Section II (Instructions to Bidders).
3. Bidding will be conducted through open competitive bidding procedures using a non-discretionary "pass/fail" criterion as specified in the 2016 revised Implementing Rules and Regulations (IRR) of Republic Act (RA) No. 9184.
Bidding is open to all interested bidders, whether local or foreign, subject to the conditions for eligibility provided in the 2016 revised IRR of RA No. 9184.
4. Prospective Bidders may obtain further information from City Government of Navotas – Bids and Awards Committee and inspect the Bidding Documents at the address given below during Monday to Friday, 8:00 AM to 5:00 PM.
5. A complete set of Bidding Documents may be acquired by interested Bidders on June 20, 2024 from the given address and website(s) below and upon payment of the applicable fee for the Bidding Documents, pursuant to the latest Guidelines issued by the GPPB, in the amount of Ten Thousand Pesos (PHP 10,000.00). The Procuring Entity shall allow the bidder to present its proof of payment for the fees, in person, by facsimile, or through electronic means.
6. The City Government of Navotas – Bids and Awards Committee will hold a Pre-Bid Conference on June 27, 2024, 1:00 PM at the Mayor’s Conference Room, Fourth Floor, Navotas City Hall Building, which shall be open to prospective bidders.
7. Bids must be duly received by the BAC Secretariat through manual submission at the office address indicated below on or before July 11, 2024, 12:00 noon. Late bids shall not be accepted.
8. All Bids must be accompanied by a bid security in any of the acceptable forms and in the amount stated in ITB Clause 14.
9. Bid opening shall be on July 11, 2024, 1:00 PM at the Mayor’s Conference Room, Fourth Floor Navotas City Hall Building. Bids will be opened in the presence of the bidders’ representatives who choose to attend the activity.
10. All interested bidders should submit, upon request of the Bidding Documents a Letter of Intent, in person, by facsimile, or through electronic means.
11. The City Government of Navotas – Bids and Awards Committee reserves the right to reject any and all bids, declare a failure of bidding, or not
Notice for Negotiated Procurement
Reference Number: 10968647
Procuring Entity: CITY GOVERNMENT OF NAVOTAS
Title: NEGOTIATED PROCUREMENT FOR THE SUMMARY PROCUREMENT OF GARBAGE MINI DUMP TRUCKS AND GARBAGE DUMP TRUCK FOR THE USE OF CENRO
Area of Delivery: Metro Manila
| Solicitation Number: | NEGOTIATED PROCUREMENT FOR THE SUMMARY PROCUREMENT |
|----------------------|--------------------------------------------------|
| Trade Agreement: | Implementing Rules and Regulations |
| Procurement Mode: | Negotiated Procurement - Two Failed Biddings (Sec. 53.1) |
| Classification: | Goods |
| Category: | Vehicles |
| Approved Budget for the Contract: | PHP 7,775,000.00 |
| Delivery Period: | |
| Client Agency: | |
Contact Person:
MARITES LARA
Member - BAC Secretariat
1052 M. Naval Street Brgy. Sipac-Almacen
Navotas City
Metro Manila
Philippines 1485
63-2-82837415 Ext.401
63-2-82818861
firstname.lastname@example.org
Description
NOTICE OF NEGOTIATED PROCUREMENT
SUMMARY PROCUREMENT OF GARBAGE MINI DUMP TRUCKS AND GARBAGE DUMP TRUCK FOR THE USE OF CENRO
The City Government of Navotas, through its Bids and Awards Committee, will conduct a NEGOTIATED PROCUREMENT for SUMMARY PROCUREMENT OF GARBAGE MINI DUMP TRUCKS AND GARBAGE DUMP TRUCK FOR THE USE OF CENRO (PIN 2024-05-074), with an approved budget for the contract amounting SEVEN MILLION SEVEN HUNDRED SEVENTY FIVE THOUSAND PESOS (PHP 7,775,000.00), pursuant to Section 53.1 of Revised IRR – R.A. 9184.
The Committee through the BAC Secretariat have invited three (3) suppliers for the purpose namely:
1. ALPHATRUCK PHILIPPINES INC.
Ms. Kim Joan Rivera
Authorized Representative
Address: North Diversion Road Meycauayan, Bulacan
2. SHACMAN MOTORS INCORPORATED
Mr. Carl Kayne Nakanishi
Authorized Representative
Address: #89 Ninoy Aquino Ave. cor. Pelaez St. Barangay San Dionisio, Parañaque
3. FIRECOACH TRADING
Mr. Artie D. Guillen
Authorized Representative
Address: #54 Don Basilio Bautista Blvd., Brgy. Dampalit, Malabon City
Relative thereto, we would like to invite you to join the said proceeding and to submit to the committee on June 27, 2024, on or before 12:00 noon, the certified copies of the following documents (1 Original and 1 Photocopy):
1. Certificate of PhilGEPS Registration
2. Current Tax Clearance
3. Business/ Mayor's Permit
4. DTI / SEC Registration
5. Audited Financial Statement stamped received by the BIR
6. Annual Income Tax Return
7. Bid Proposal
Bid opening will be on JUNE 27, 2024 at 1:00 pm at the Mayor's Conference Room, 4th floor, Navotas City Hall Building.
|
CHAPTER 6
Disaster in the Mediterranean
It was in September, 1943, while I was on the second day of a course at Brighton where German was being taught that I was hastily called back to Goodwood because my request for an overseas posting had been granted. I was sent to a tropical medicine course in London, being billeted in the Great Central Hotel at Marylebone Station where we were quite unaware of the fact that the floor below us was occupied by MI9b which debriefed escapers returning from Europe. Then came embarkation leave in Edinburgh where my mother, with memories of the losses amongst her acquaintances in the First World War, no doubt wondered whether I would ever return. I had no further direct contact with my former unit at Goodwood but learned later that it landed in Europe on June 26th 1944 (D Day being June 6th), going on to Weert, south east of Eindhoven, and that it was involved in the Rhine crossing in March 1945, ending up near Lunen in Germany. There had been a large turn-over in personnel during the inactive days after I left.
We moved northwards in our troop train, bound for an unknown destination, and found ourselves at the Waverley station in Edinburgh. This, however, was only a transient stop while the local WVS supplied us with tea and sandwiches. I did not see anybody I knew amongst those kind ladies and resisted the temptation to phone my home. On we went to Greenock, discussing amongst ourselves whether we had been issued with pith helmets to confuse spies or whether we were indeed bound for a hot country. It certainly seemed unlikely that we were heading for Italy which had surrendered unconditionally on September 8th: on October 13th Italy declared war on Germany. One of the officers whom I knew well told me that he had been appointed to be a consultant neurologist in India but it did not follow that we were all bound for the same destination.
When I had been home on leave I had arranged a code based on our telephone number and carried an old Canadian passport which had belonged to an aunt who was not Canadian but was confused and had somehow obtained it when she was visiting there. Perhaps I had been reading too many spy stories, but was taking precautions in case I became a prisoner of war, and thought that this passport, suitably
doctored, might be useful in an escape effort, and that the ability to send coded messages might be even more useful.
We embarked at Greenock on the troopship HMT G6 (in fact the Highland Princess) and were told that the senior officer on board was Lieut.-Col. Lord Stratheden. Our convoy, when finally assembled, was a large one, the largest vessel being the Orion: the official designation of this convoy was KMF 26. We set sail past the Tail of the Bank and made our way around the North of Scotland then, because a pack of submarines was known to be ahead of us, zig-zagged across the Atlantic almost to the mouth of the St Lawrence and back to the Straits of Gibraltar, having observed with interest that there was no blackout in Portugal.
We were six RAMC officers in a cabin designed for two, but three of us were old acquaintances from Edinburgh and we managed to get along together without friction. The men in the lower deck accommodation were in very cramped conditions but concerts and lectures were put on to keep spirits raised and time was taken up with Captain’s inspection, alarm drills and training sessions. We were still in the dark as to our immediate or ultimate destination. Were we bound for the Middle East or was it to be the Far East? Was Turkey about to enter the war? Was it likely that we would be going there? We heard from the BBC on the wireless that German aircraft were renewing their attacks on shipping in the Mediterranean and we had all been given stations to go to in case of attack, but gave little or no thought to any such danger except that we slept in our uniforms so as to be ready for action.
We had on board a ‘Stars in Battledress’ company and the dance orchestra of the RAMC. There were a number of nursing sisters of the QAIMNS and some FANYs (First Aid Nursing Yeomanry). We were rather puzzled about their function but understood from them that they drove ambulances. It was very much later that it was revealed that many of them were involved in Secret Service work.
Off Oran we were joined by a few more ships, the vessel nearest to us on the port side now being one filled with American troops. It was after the war that we learned that this was the Rohna, an 8,600 ton vessel of the British India Steam Navigation Company, and it was coming with our convoy to carry the Americans to India. Soon after it joined us on 26th November we were enjoying a concert being given by the RAMC band as we sailed off Jidelli in Algeria when the alarm bells started to ring and we all rushed to our emergency stations. Mine was on the top deck aft on the port side and with me at this post was a sergeant from
the RAMC band; he was Oscar Grasser, the leader in peace time of Victor Sylvester’s dance orchestra. As we dashed to our stations we saw some thirty enemy planes approaching from the north. I was tempted to rush to our cabin to get my camera but thought that I might be accused of deserting my post, so unfortunately there is no visual record of the events that took place. In later emergency situations I have always grabbed my camera immediately to avoid such a mistake.
Guns blazed from the escorting naval craft and from the troopships that had this capacity. We had Oerlikon guns which were fired, but we succeeded only in shooting down our own barrage balloon. Bombs were falling all around the convoy and of course most of those on board our ship were below decks, unable to see what was happening. From our vantage but exposed position on the top deck we saw an object with flames trailing behind it apparently coming from one of the planes and heading towards us. Instead, however, it scored a direct hit on the unfortunate *Rohna* which immediately seemed to explode into engulfing flames. The next bomb appeared to be heading for my sergeant and me but it fell into the water close to us and failed to explode; we were drenched with sea water but alive. This was the first attack on a convoy by a glider bomb (Hs 293) and as darkness fell we sailed on, leaving the *Rohna* blazing behind us. Apparently, despite efforts to rescue survivors, 1115 of approximately 2000 Americans on board lost their lives. The captain was a survivor but 120 of his crew were lost. This was one of the worst troop transport disasters of the war; more were lost in the *Lancastria* in the evacuation at St Nazaire in June 1940 and a large number of Allied prisoners were drowned in September 1944 when American submarines attacked the *Rakuyo Maru* and other Japanese transports, not having been informed that they were carrying prisoners from Singapore to Japan. Correctly the BBC news bulletin minimised our incident, merely stating that an attack had taken place and giving the number of German planes that had been shot down. We sailed on but were attacked again as a concert was taking place while we sailed near Crete. All the guns went into action again and I thought I had seen a direct hit on the *Orion* as the bombs fell around the convoy; in fact it failed to explode and seemed to bounce across the ship to land in the Mediterranean on its port side. We liked to think that the enemy bombs were being sabotaged by workers in occupied Europe. Later, too, depth charges were dropped as submarines were in the vicinity.
We did not know that Churchill had been sailing ahead of us and even now it does not seem likely that the attack on our convoy could have been intended for him. We were both very glad to reach Alexandria the next day, although the dangers were still very real. The good advice of the British and Alexandrian authorities to the Egyptian authorities was appreciated.
We spent the next few days with various officials, by train, motor car and boat, in the Lakes district where we had successfully tried to find out how the aircraft had been shot down; the army commander-in-Chief, General Montgomery, found the evidence and made a decision to send a suitable force to the area. I received a letter dated 16 December 1944 from Churchill saying that he may stay in Egypt.
We were back in England the week after Christmas and on 27 November 1944 I was called up again for the second time. Thereafter I was in hospital for several weeks feeling unwell and having to have his phlebitis treated. My machine was sent to work with Mr. H. J. B. Smith’s synthetic rubber plant, suspiciously enough, and he might have used it for his new drug.
We were all very happy in our part of the world, but we were also very sad that so many of our friends had been killed.
have been related to his presence as he had reached Cairo before we were bombed. On 16th November he had been in Algiers and on the next day in Malta, where he had been advised that it would be too dangerous to sail to Cairo, which in the light of our experience was good advice. Ignoring this he continued his journey on the 19th to Alexandria in the Renown, arriving on the 21st and flying to the Egyptian capital.
We sailed into Port Said and were quickly surrounded by small boats with various goods and fruit on offer, then disembarked to travel by train, rejoining our ship near Ismailia and passing through the Bitter Lakes where there were anchored some of the Italian battleships which had surrendered to Admiral Cunningham in September. We did not know that some ships of the Italian Navy had been attacked by German aircraft as they sailed to the agreed rendezvous when the Italians signed the armistice and that the Roma, flagship of the Italian Commander-in-Chief, had been sunk in the attack. At Suez we disembarked and found that we were to be stationed temporarily at Port Tewfik pending a decision about the final destination of the six of us. We were given a suitably large tent and told to erect it, not a difficult task in this desert area. I sent a cable home and it arrived on 9th December. Air mail letter cards could be sent only once a week and one posted on 6th December arrived a week later. In it I wrote 'I see that Roosevelt, Churchill, Stalin and Chang Kai Shek have been meeting, so things may start happening soon'.
We were on the doorstep of the most important Allied meetings of the war without knowing it. They took place in Cairo from 22-26th November, Teheran from 28th November to 1st December and Cairo again 4th-6th December. On the 11th Churchill left for Tunis by plane thence to be driven to stay with Eisenhower in Carthage. He was feeling ill and was febrile and Lord Moran who travelled with him as his physician did not know what was wrong but obtained an X-ray machine from Tunis. Pneumonia was found and treated successfully with M&B 693 (sulphapyridine), a drug which had so recently been synthesised that some of my teachers had regarded it with doubt and suspicion. Lord Moran was afraid of a fatal outcome and his pessimism might well have been justified had not May and Baker introduced this new drug in 1938.
We were aware of course that something important was going on in our part of the world and that the leaders of the countries were meeting but we only suspected that it might be in Cairo. The headquarters of that session of the conference was Mena House beside the Pyramids,
and preliminary talks there focused on the need for a Supreme Commander of all operations against Germany; it was in Teheran that Stalin joined Churchill and Roosevelt in the discussions. There it was agreed that 'Operation Overlord,' the invasion of northern Europe would be launched in May or June 1944 subject to moon and tides, that there would be an operation against the Axis in southern France and that efforts should be made to persuade Turkey to enter the war. At the second Cairo conference the Turkish President was a participant but could not be persuaded to bring his country into the conflict. Here, too, Churchill learned that Eisenhower was to be in charge of the 'Overlord' operation rather than Marshall whom he had expected.
We did not know that the likelihood of us going to Turkey was vanishing even as we sat in our tent at the corner of the desert. We swatted flies, blessed the fact that the temperature was only 70 degrees, took precautions against thieves, bargained with optimistic Egyptian traders and watched with interest the trains on a nearby railway line with more passengers travelling on the roof or hanging from the windows and doorways than were in the carriages. Malaria was not yet a serious problem in Egypt so we did not have nets and were not issued with antimalarials. The food supplied to us was terrible so we ate in the nearby YWCA and on occasion we were invited to a British tented hospital sited close to our patch of desert. Nevertheless we were bored and Capt Alastair Wright, RAMC (from Edinburgh) and I decided to go to Cairo. Technically we were deserters but we arranged to give our telephone number to one of our group when we had found a hotel, saying that we would return if anybody wanted us. Along the sandy road there were army pick up points and one just queued up there until a vehicle with available space came along and got on board.
Thus we travelled the 85 miles to Cairo and had lunch at Shepheard's Hotel which at that time was in Ibrahim Pasha Street, well back from its present site by the river. On its terrace were wicker tables and chairs and inside was the Moorish Hall with a great staircase leading up from it. The bar was full of officers and, it was rumoured, spies. It was on 26th January 1952 that this original Shepheard's Hotel, together with other properties associated with the British, were burned down by angry mobs at a time when there was a mixture of Islamic fundamentalism, revolutionary fervour and hatred of foreign domination. Six months later the Egyptian Army was to seize power and King Farouk had to leave his country. All this was to be in the future, but in 1942 we had heard strange rumours about King Farouk. He had abdicated, was under arrest, was keeping out of the way because of his health and had been seen to have crashed his car. He was so very much longer time ago.
We managed to get Shepheard's Hotel key cards, had been issued to us in Cairo, Egypt in case of emergency. We had also been warned that we did not pay our bill and demanded that we swear words of oath were pursued by the police to possess copies of the End of the World. In 1942 the German and Egyptians had fought in Cairo was now turned to the British. Some action was taken by General Kreipe who had planned in advance to take whole a party including Wright and myself to visit the YWCA in Alexandria and to the Egyptian Museum encountered.
Next we went to see the Pyramids of Giza, visited upon by the Turks before Turkey and Egypt had come to see the end of the World. This was not made this possible by Mena House, but we had much later to take extra precautions.
The next day, two days later, we were told that
because of annoyance about the Cairo conference, was in hospital or had been seriously injured. The fact was that on 15th November he had crashed his car and been admitted to a British military hospital where he was so well satisfied with his treatment that he stayed for rather a longer time than had been expected.
We managed to obtain accommodation in a less expensive hotel than Shepheard’s (the Hotel Trianon) and set out to see the sights. We had been issued with revolvers but had been advised not to carry them in Egypt in case we might be attacked by thieves wanting to steal them. We had also been warned about shoe blacks who might attack us if we did not pay to have our boots cleaned at the pavement edge. Sure enough one of them dabbed black polish on my brown boots and demanded payment to remove it again. I had learned some Arabic swear words so used them and wiped my boots with his shirt. I was pursued by shoe blacks and took refuge in a book shop which is why I possess copies of *De Montmarte à Tripoli* by André Gnarner and *The End of the Beginning*, the third volume of Churchill’s speeches. In June 1942 the Germans had been within sixty miles of Alexandria and many Egyptians has been prepared to welcome them. All this was over and Cairo was no longer a centre of military activity, all eyes now being turned to the continued fighting against the German troops in Italy. Some actions in the Mediterranean area, such as the kidnapping of General Kreipe, Commander of the Sevastopol Division in Crete were planned in Cairo about the time we were there, but there was on the whole a party atmosphere after years of stress and strain. Even Alastair Wright and I, not renowned for our social activities, went to a dance at the YWCA and were just restrained in time from attempting to fox-trot to the Egyptian national anthem which we had not previously encountered.
Next we made our way to the Pyramids and the Sphinx. The three Pyramids of Gaza date back to around 2600 BC and have been gazed upon by the troops of Greece, Rome, various Moslem peoples, France, Turkey and Britain. Now fresh British servicemen and their Allies had come to see the last remaining example of the Seven Wonders of the World. This was most impressive and we were pleased that we had made this unauthorised journey from our camp. We looked, too, at Mena House but now there was no evidence of great activity; it was much later that we discovered what had been going on there. Security precautions had been excellent.
The next day we thought we should return to Port Tewfik and a few days later, on 20th December, all six of us, having been informed that
CHAPTER 7
Settling down in India
I had heard so much about India during my early childhood days in Dundee because of connections through the jute trade that it did not feel that I was entering a completely foreign land when we sailed into harbour at Bombay on 31st December, 1943. The possible cultural shock was somewhat diluted by our short stay in Egypt, but the few lectures based on previous peace time service that we had heard on board had been of little value; we had more to think about than the season for pig-sticking and little valued descriptions of the best polo grounds. We were interested to learn however how few Europeans there were in India in peacetime, the total being less than the population of Aberdeen, whereas the population of the whole country was about 390,000,000. I was puzzled by the term Anglo-Indian because I knew a few of them in Dundee and they were British born people who had worked in India whereas people of mixed race were known as Eurasians. Here, however, a person of mixed race was an Anglo-Indian. One thing I had heard something about was the incredible snobbery of the English in peacetime India, manifesting itself in such a way that somebody who had been in retail business in England was likely to be turned down for membership of a golf club in India. Professional people did not dine with those in trade. If the English did this to each other what on earth did they do to the Indians? Did the peacetime Scots do this to each other too? Certainly our troopship was not bringing further snobbery to India.
Bombay is the finest port in the country; it derives its name from the Portuguese words *bom*, meaning good, and *baia*, or bay. Stimulated by Henry the Navigator, fourth son of King Joao I of Portugal and his English wife Phillipa, daughter of John of Gaunt, there was established in Portugal a school of navigation which led its mariners to travel widely throughout the world. Bombay included a group of islands which were acquired by the Portuguese in 1509 but given to Charles II as part of the dowry of his Portuguese bride Catherine of Braganza in 1662 and seven years later it was leased to the East India Company. With land reclamation it was developed as an entity and became prominent in the cotton industry and as a major shipbuilding centre.
From our ship we gazed with interest at this country of teeming millions and wondered where we might be sent. The possibilities were very numerous and it was likely that once we had been give our postings we would not meet again until the war was over.
In August 1943 the British and United States governments had formed a South-East Asia Command to control the Allied forces in Burma, Malaya, Ceylon, Siam, Indo-China and the Dutch East Indies. The Supreme Commander was Admiral Mountbatten. India Command, however, was separate and came under General Sir Claude Auchinleck. A new 14th Army had been formed and came under SEAC, not India Command and it was deployed on a seven hundred mile front from the Chinese border to the Bay of Bengal. We were curious about where each of us would be fitted into this overall picture but untroubled about possible future developments since we all anticipated a complete Allied victory and the personal survival of all of us. We knew that, unlike what happened on other fighting fronts, medical casualties would heavily outnumber patients with battle injuries even in Burma and none of the six of us who had voyaged together from Greenock was a surgeon. For the first time we must be prepared to diagnose and treat all manner of tropical illnesses in addition to the diseases we were accustomed to see at home.
We went ashore and were taken by Army transport to dormitory accommodation on the south west side of Bombay. One of our number, having discovered that the races were on, disappeared for a few hours and then returned, happily clutching a fistful of rupees. Then we went off to see the sights of the city; the absence of a blackout was striking and the crowded streets, bustling with people in all manners of garb, occasioned no surprise. We were of course disturbed to see the obvious poverty that was all around and this troubled us throughout our stay in the country no matter where we went. This was a time of famine which was occurring particularly in Bengal where it has been said that over three million died of undernutrition in 1943–1944. It was clear in Bombay that many of the population, despite their cheerfulness, were badly nourished and in some instances obviously ill. Some of the beggars or their children had the most appalling deformities. We learned that children, often as soon as they could walk, were employed in all manners of labour with no possibility of protection of their health. Indeed it was said that the number of children at work in India was equal to the total population of England and Wales. At first we were alarmed by the red patches in the streets but it was not quite the colour of blood and it was soon clear that this was produced by the habit of chewing betel nut which remains; this is now illegal.
Some of the most beautiful nature, are to be found in the desert, prophet Zoroaster said. It was a good thought in the nineteenth century when the deserts were still for their charm and the only survivors of the great drought. Silence which was not broken, were not created by the noise of the vultures and the jackals, but there wandered the wind and the transmigration of the stars and turned a little.
The shops were interesting, how much worse than those of medical men. We had tried out a few words which were not the local vernacular but in India and we were confused with those of Marathi and Hindi which are complicated. The word however is interesting. The name ‘Modern’ is derived from satisfaction. The term ‘Devanagari’ describes the Devanagari script, the words written in Sanskrit, Persian–Arabic and Urdu known as Urdu. The Roman–Urdu alphabet, it could have been said, had confused the British using Pimm’s and had an additional character.
We looked at the houses built following the British, interested to see the
habit of chewing betel nut with leaves and spices and spitting out the remains; this also explained the red Indian teeth.
Some of the richest Indians in Bombay, because of their industrious nature, are the Parsees who follow a religion, founded in Persia by the prophet Zoroaster, which includes fire worship and the practice of good thought, word and deed. They had fled to India in the 7th century when the Arabs conquered Persia and had now become noted for their charitable works. They have been described as the last survivors of the old Iranian race. We passed the Parsee Towers of Silence which were shielded from our view, but knew that the bodies were not cremated but laid out within the towers to be picked clean by the vultures which we could see circling above. Around the streets there wandered the many bulls, sacred to the Hindus who believe in the transmigration of souls. Some are bought when young by the pious and turned adrift in the street.
The shops were well stocked and I was particularly gratified to find how much was available in the book shops, including the latest editions of medical textbooks. The shopkeepers all spoke English, but when I tried out a few words of Urdu this was well received even although it is not the local language. There are over two hundred languages spoken in India and indeed some inhabitants of the south cannot communicate with those from the north. The languages spoken in Bombay are Marathi and Gujerati whose scripts are similar to those of Hindi. There are complications — I bought two Urdu manuals, one of which however is entitled ‘Hindustani Manual’ while the other carries the name ‘Modern Colloquial Hindustani’. Each of them includes letters from satisfied customers referring to this language as Roman–Urdu. The term Hindi (rather than Hindustani) is usually employed to describe the language of much of the north and it is written in Devanagari characters; there is a strong Sanskrit influence. The same words written by Muslims and now the language of Pakistan, using the Persian–Arabic alphabet with some added Persian and Arabic words is known as Urdu. However my manuals were in what had been called Roman–Urdu because the words were written using our own alphabet; it could have been called Roman–Hindi, but this term is never used. I had confused things even further on the ship coming from Egypt by using Pitman’s shorthand and had quickly to forget about this additional complication.
We looked at the archway usually known as the ‘Gateway to India’, built following the visit to India of King George V in 1911 and were interested to see close by it Bombay’s best known hotel the Taj Mahal.
have I eaten a salad in an Eastern country. The arrival of a train at a wayside station in India is regarded as a great event and into a third class compartment there are carried children, tiny babies and all manner of possessions including food for the journey. The scene is a most colourful one as the crowds jostle with each other to get aboard and sometimes, as in Egypt, there are passengers on the roof or hanging from the doors and windows.
It is always necessary to be on one's guard against possible theft both on the trains and elsewhere. On one occasion I had to struggle with a thief as the train was leaving a station but he fell off and I hope was not hurt. Another time I was lying in a sleeper but wearing my army boots when a thief tried to break in but, seeing that I was awake and wearing boots which might have been traumatic to him, he fled. On this journey from Bombay to Delhi, however, the only theft was of a bunch of bananas by monkeys which invaded our compartment at Sawai Madhopur. We had been warned always to be prepared for anything, being told of an occasion when bandits had entered a dining car and not only robbed those having a meal but had shot two who had shown resistance. Another story was of four soldiers who were sleeping in a tent with their valuables under their pillows. When they awoke in the morning not only had their possessions gone but the tent had disappeared as well. An officer wearing spectacles as he walked along the Bombay pavement was astonished when a passing thief neatly flicked the spectacles from his nose. My officer's 'swagger stick' was in fact a swordstick which I always carried for defence purposes.
In New Delhi we were accommodated at the British Military Hospital and after three days, mainly devoted to sightseeing, we were informed about our postings. New Delhi, a city of great splendour with wide avenues, impressive buildings of red sandstone and splendid vistas was designed as an Imperial capital by Sir Edwin Lutyens and Sir Herbert Baker and there can be no doubt that they succeeded in their endeavours. In the centre is Connaught Place with radiating spokes to Connaught Circus from which Parliament Street leads past the ancient and spectacular Jantar Mantar observatory to the Parliament Buildings and near them what was in those days the Viceroy's House, but is now Rashtrapati Bhawan, the official residence of the President of India. We were aware that this was the eighth city of Delhi and in our horse-drawn tongas went on to see something of the previous cities that were nearby. We were taken to the Purana Quila (old fort) of Emperor Sher Shah from the sixth city, the Red Fort constructed at the behest of the Moghul Emperor Shah Jahan in 1639, and went on to see Humayun's
Tomb where, in 1857 during the siege of Delhi at the time of the Indian mutiny, Lieutenant William Hodson captured the aged King of Delhi and his sons. The visitor to Delhi must surely be impressed by the atmosphere of history that is all around.
But now we were told, after an interview, where we were all going. I had an MRCPEd. diploma because of an examination I had taken while awaiting call up and so was sent as medical specialist to 57 IGH (C) at Lahore. The initials indicate that it was an Indian General Hospital and it was combined in that it took both Indian and British patients. When I reached it I found that it did not yet exist but consisted of a number of tents in the railway yard in the centre of Lahore. The officers, who were most pleasant colleagues, were regretting the fact that they had left the hospital’s previous site in Ceylon and wondered where they were bound for next. I found that my duties would involve the supervision of 14 medical officers; the Commanding Officer, Col. Craig, IMS, thought that I should be promoted to Major but discovered that at the age of 26 this was not possible. I found that I had a batman, Shah Mohamed Khan, who could speak Urdu but not English, and very helpful he was throughout my period of stay at this hospital.
During our days of waiting a few of us took the opportunity to visit Amritsar, the central focus of the Sikh religion and went into the Golden Temple where holy men read continuously from books of the world’s religions including the Bible and there we were received and shown round with great courtesy. Close by, however, is an open space, the Jallianwala Bagh where in 1919 Brigadier-General Dyer astonishingly gave his troops the order to open fire upon those taking part in a mass demonstration, the result being the death of more than 300 protestors; bullet marks could still be seen.
Three days later with the aid of camel carts our equipment was loaded on to a train and we made our way to a field at Deolali, near the holy city of Nasik which was out of bounds to us. Nasik is on the Godavari River, just over a hundred miles north-east of Bombay but for us the important point was that we were very close to the British Base Reinforcement Camp (BBRC) through which passed most of the troops arriving in or departing from India. The term ‘Deolali tap’ is well known as a description of a form of madness that could affect British troops in India. By a strange chance the medical officer in charge of troops at the BBRC when we arrived was Major Charles Robertson who had been my closest friend as a medical student. Together with other students, we were billeted in Hostels in the town.
We obtained accommodation in large tents which were heavily infested with lice, part of a common purpose; sleeping bags were such a common sight everywhere that we had no beds. Flea-borne typhus and poliomyelitis were rife amongst the troops rather than good health and we discovered that we were in all directions of the area intended for the long period of the Burma Americans.
In fact at the time of Myitkyina, the American forces were in battle while we were in some new recruits for General Wingate’s force into a hilltop.
On March 2nd the force had moved into Burma near the river and mules and horses were the American means of transport. The main force had already secured the road leading from the river to the area undertaken.
The British had planned the attack on P.C. Cooke’s force and almost parallel to General Wingate’s
students, one of whom he had married, we had toured the Youth Hostels in our vacations.
We obtained our equipment from the railway siding and erected our large tented hospital, but found that the cracks in the ground were heavily infested with fleas. I found it difficult to direct the unloading of part of a train when I had to use my newly acquired Urdu for this purpose; somehow I did not seem to have learned the right words for such a complex operation. Mosquitoes were a problem, but here as everywhere else in India we had to erect mosquito nets over our camp beds. Flies, too, were a nuisance and we soon discovered that poliomyelitis was one of the diseases which they commonly spread amongst troops in India at a time when no method of prevention other than good hygiene was known and no satisfactory treatment had been discovered. The area of our hospital was large and there were tents in all directions, the very large ones serving as wards, some being intended for Indian troops and some for British; at no stage during my period of service in India did I encounter an American patient as the Americans were active in areas in the far north-east.
In fact at about this time in March the Japanese control of the town of Myitkyina in the north of Burma was about to be challenged by the American General Stilwell with his Chinese and American troops in a battle which eventually went on until August. All this was two thousand miles away on the other side of the subcontinent but we had some news of what was happening and learned on March 25th that General Wingate had been killed the previous day when his plane flew into a hillside near Imphal.
On March 19th it had been publicly announced that a large Allied force had been landed by gliders 200 miles behind the Japanese lines in Burma near Indaw on the Rangoon – Myitkyina railway line. Men, mules and bulldozers had been flown over the 7000 foot Chin Hills; American engineers used the bulldozers to clear the ground for the main forces to land. It was stated that men of a north country regiment secured the perimeter of the landing ground; they were, in fact, men from the 2nd Leicestershire Regiment who, with others, had undertaken the incredibly difficult march from Ledo in India.
The British, including Slim and his eccentric subordinate Wingate planned the mission, and American pilots under their commander, Col. P.C. Cochrane towed the gliders. A major problem was Stilwell’s almost pathological hatred of the British. It was well known too that Wingate was usually at odds with army administrators in Delhi and
that his views about suitable rations for his men were quite unacceptable to experts in the field of nutrition. However, this deep penetration of troops behind the Japanese lines was intended amongst other objectives to cut the supply route to the north along the railway from Rangoon and thus help Stilwell and his Chinese and American forces.
We ourselves thought that we were only to be temporarily at Deolali and were uncertain as to whether we were bound for Burma or the Middle East, which was just about as near, or perhaps we would even be sent to Europe. In February 1944 we had a sweepstake to predict the date of the Second Front in Europe and I plumped for June 6th, thus in due course winning the 'sweep.'
Our numbers grew and we were joined by additional QA nursing sisters (still in short supply in India) and by Anglo-Indian nurses. Conditions were primitive but I shared my tent with Captain Ingram, the Quartermaster and a valuable ally. Screened latrines had been built and showers were constructed from old petrol tins. There were a few flimsy buildings used as office accommodation but everything else was tented. Until the hospital was in operation it was agreed that I could help out in the British Military Hospital in Deolali town; we were stationed on a flat plain with distant low hills and the BMH was about two miles away, down a very steep hill. The only transport that could be provided for me was a bicycle which was alright for the downhill journey but it was hot work cycling back up the dusty hillside path in the tropical sun.
I had heard that the London MRCP examination was to be held overseas for the first time in history, and that the examination centres were to be at Poona and Cairo. I entered at once and joined 355 candidates at Poona on 17th March to sit a preliminary screening examination. It was clear that the forces in India were temporarily badly depleted of medical officers by this examination, but not all candidates were from the services. In due course I was notified that I was one of the 140 who would be permitted to sit the true examination and on the appointed date I returned to Poona where the arrangements were being organised by one of my former teachers, Brigadier J.D.S. Cameron who could not however examine as he was not a Fellow of the London College. The examiners were Sir Henry Letheby Tidy and Dr G.E.S. Ward who had come out from London and Brigadier H.L. Marriot and Col. B. Schlesinger who were already in India. There were written papers on 15th and 16th May, clinical examinations from 17th to 22nd May and orals between the 23rd and 25th of the month, a very thorough test indeed. I passed with flying colours but there was a slight hitch in that I knew that some of the candidates could not be attending the next day in 1945 that we were to be Physicians of India.
Once our examination was over the months of July and August saw many outpatients and patients who had escaped from the hundred mile radius of the hospital from disease. In the hospital the outpatients were examined medically every day and nearly 800 men in regular attendance had detected tuberculosis in their chests and treated.
All manner of ailments were commonest being malaria and dysentery. As July approached the heat admitted 27 men suffering from heat exhaustion and dysentery from a group of sailors on troopship and the heat was so bad that judgement became difficult in the station in the afternoon, regulating myself with a fan but punkhhas.
On 13th July there was lightning, and the rain flooded the hospital tents and blocked the drainage channels during the night and continued until the 17th. Then it rained around our tents and the cows, bulls, buffaloes and shoes upside down. The grass in Deolali however was so short that the grass outside the tents was used to sleep on most nights.
this men were quite different. However this deep feeling was intended amongst the British troops north along the railway line to the Chinese and American forces.
We were temporarily at Deolali waiting for Burma or the Indian campaign, perhaps we would even win the sweepstake to predict the date of D-day, slumped for June 6th, 1944.
There were additional QA nursing officers and some Anglo-Indian nurses. The latrines with Captain Ingram, were built and latrines had been built by the Indians. There were a few problems but everything else was going well. It was agreed that I could stay in Deolali town; we were told that the BMH was about to be built and any transport that could be spared was right for the downhill journey on the dusty hillside path in the monsoon.
The examination was to be held in Bombay and the examination centres were to be held once and joined 355 men. The preliminary screening of all India were temporarily put off examination, but not all were successful. I was notified that I had passed the true examination and that there the arrangements were made. Others, Brigadier J.D.S. Letheby Tidy and Dr H.L. Smith and Brigadier H.L. Smith were in India. There were examinations from 17th July to 20th August of the month, a very thorough test of knowledge and experience. In all 24 candidates passed but there was a long period of silence and it was not until 20th August that I knew of my success. This was because two of the successful candidates could not be traced and there was then further delay pending the receipt of the fees in London. It was not until 25th January 1945 that we were admitted to Membership of the Royal College of Physicians of London.
Once our hospital was operative it became more and more busy as the months of 1944 went by and in addition to the inpatients there were many outpatients: as my orderly at the outpatient tent I had a sepoys who had escaped from the Japanese in Burma by walking for several hundred miles through the jungles. Very many of the refugees had died from disease or malnutrition as they attempted to reach India. Not all the outpatients were ill; all the new arrivals at the BBRC had to be medically examined and I assisted in this, on one occasion examining 800 men in two days. Xray films were not available but I thought that I had detected three men with pulmonary tuberculosis just by examining their chests and they were returned to Bombay.
All manner of diseases were to be seen in the wards, the three commonest being hepatitis, bacillary dysentery and amoebic dysentery. As July approached it became hotter and hotter and one day we admitted 27 men, some with heat stroke and some with the less serious heat exhaustion. Despite our best efforts two of them died. They were from a group which had gone ashore at Bombay from an overcrowded troopship and been brought straight from there to Deolali, the error of judgement being that they had then been marched for three miles from the station in the heat of the day. This was more than their temperature regulating mechanism could deal with. There were no fans in our wards but punkhas had been rigged up and ice was available.
On 13th July the monsoon broke after days with spectacular sheet lightning, and a niagara of water fell upon us bringing down five hospital tents on top of the patients. They were quickly rescued and channels dug around the tents to avoid serious flooding; the rains continued unabated by day and night, continuing in a less severe form until the 27th of August. After that we had problems with wild life around our tents; there were scavenging dogs, jackals, donkeys, ducks, cows, bulls, cats, snakes and scorpions. In hot climates I still turn my shoes upside down and tap them on the ground before I wear them. In Deolali however the biggest nuisance was that of cows noisily cropping the grass outside my tent only a few feet from where I was attempting to sleep on my camp bed.
Before this however we had heard on our radio set that landings had taken place in northern Europe on 6th June. We were able to pick up the General Forces programme, the Eastern service of the BBC, All India Radio and some Japanese stations, but the only reliable set was the one in the officer’s mess tent. Accordingly when the announcement of the opening of the Second Front was made I went off to tell the nursing sisters and arranged for them to join us to hear the 7.30 p.m. news bulletin. The Indian other ranks were paraded and given this news in Urdu; they all cheered dutifully but unfortunately many of them did not know which side Germany was on or where Europe was. One Indian officer told his batman who became much upset because he thought the Americans were invading Britain.
Daily we heard news of the fighting in the east and although many of the Indian troops knew nothing about Germany most certainly understood about the war against Japan. On 6th March the Japanese, having entered India, were on the march against the Kohima-Imphal road. From Kohima it was no distance to Dimapur and westwards from it was the Brahmaputra river; there the boats were destroyed so that they would not be available to the Japanese. The siege of Kohima by the enemy lasted from 5th to 20th April, but fighting continued until 21st June by which time the monsoon (which occurred earlier than in our area more than 1500 miles away) had broken. The British were now in pursuit of the Japanese there and daily we studied the map which hung beside the radio set. One strange incident was that two rather smart junior non-medical Indian officers were posted to our hospital, but soon disappeared again and we were told that it had been discovered that they were spying for the Japanese.
Once the monsoon was over there were signs of preparation for active warfare by the troops in the area of land just to the north of us where the 19th Indian Division was forming and some of our patients came from there; it was a privilege that I much appreciated to be the medical specialist to whom there were referred Indian troops, British army personnel and Gurkhas. This division which eventually consisted of about 1000 officers and 14,000 men was involved in the advance on Rangoon in May of the following year under the command of Major General ‘Pete’ Rees. An account of its adventures has been written by John Masters in his book *The Road Past Mandalay*.
Our hospital was so busy that we had little time for non-medical activities but we had a visit from the comedienne ‘Gert and Daisy’, an ENSA concert and an Indian entertainment (tamasha) with dancing girls, musical instruments playing tunes which were very strange to our ears, and a dance which almost everyone took part in. The different religions of Moslem, Hindu and Indian were very large in number while some of the musicians were Moslems and they were probably commemorating son Ishmael but I think that however much this incident was enjoyed.
There was a new hospital doctor who had been taken to England and had to return to India to shave every day. He was Brahmin and in charge of the water system and he always has to be shaved. He was unhappy at being in India where he could meantime indulge in orthodox habits.
The British like good food, but they have a method of eating it. Our mess sergeant was quite happy to be accustomed to the conflict. I watched him; there is no need to look at the food, the habit of feeding into it was a delicacy.
ears, and a liberal supply of tea and sweet-meats. Indian music is almost entirely a matter of improvisation and varies a great deal in different areas of the country. Towards the end of September the Moslem IORs (Indian Other Ranks) invited the officers, both British and Indian, to their celebration of the Festival of Eid-ul-Adha. In a very large decorated tent we were plied with various rice dishes and tea while some dancers in traditional costume gyrated and sang as the musicians, squatting on the floor, played their instruments. Our Moslems also squatted on the floor of the tent but for the officers chairs were provided. I was surprised to find that the festival was to commemorate Abraham’s test of faith when he was told to sacrifice his son Ishmael; my recollection of the Old Testament was somewhat hazy, but I thought that this test referred to his son Isaac. Apparently however most Moslems say that it was Ishmael and that the place of the incident was near to Mecca.
There was no friction between the Hindus and Moslems in our hospital despite the great difference in their beliefs, but great care was taken to ensure that suitable foodstuffs were provided for both. We also had to respect the caste system amongst Hindus and to be careful not to shave even the hairs on the chest of a Sikh. Some of the Indian officers were Brahmins, thus being of very high caste and the female officer in charge of the dysentery ward was not a Hindu but a Parsee. The caste system applied only to Hindus, not to Moslems, Sikhs or Parsees and it has to be admitted that we did not fully understand it or appreciate the unhappy lot and hopeless future of the millions of untouchables in India who could not improve their state until their next existence and meantime had to perform the most menial and degrading jobs. A very orthodox Brahmin would avoid even the shadow of an untouchable.
The British officers were willing to eat either Indian or Western food, but the latter usually consisted of corned beef; every possible method of preparing it was tried, the cooks showing much ingenuity. Our mess waiters were Italian former prisoners of war who were now quite happily carrying out duties to which many of them had been accustomed in peace time. Italy, of course was now on our side in the conflict. When soya sausages arrived we tried and quickly rejected them; the Italians shared our opinion. The Indians troops would not look at them and even the local waifs and strays whom we were in the habit of feeding recoiled so we arranged for a large hole to be dug and into it went the soya sausages; we returned to our corned beef delicacies.
CHAPTER 8
Searching India for a Disease
I had heard that anaemia was a problem in Indian troops at the fighting front to a much greater extent than elsewhere. It was a constant problem, particularly with new recruits, some of whom joined the Indian army in order to obtain food and very large numbers were suffering from hookworm infestation which inevitably led to iron deficiency. It was clear, however, from what I had heard that the problem in Burma was a different one and soon after I arrived in India I sent to Edinburgh for blood counting apparatus. At Deolali I was carrying out a small research project which included blood counts when a medical Brigadier came on a visit to the hospital and was most interested in my activities; the next thing I knew was that I received a posting order to leave the hospital and report to GHQ in Delhi. On October 1st 1944 I left Deolali, uncertain as to whether I was to return, but taking all my equipment with me in case I did not come back. My tin trunk was very heavy because of all my medical books and I was rather alarmed at the way the railway porters carried it on their heads as I feared it would damage their necks.
For a week I was stationed in a tent at the side of the Rajpath, near the Viceroy's House, close to the temporary two storey concrete buildings which constituted GHQ (New Delhi). The Brigadier who was most closely concerned with my activities was absent but I found that the Medical Directorate was staffed by most pleasant officers, all either Scottish or Irish. I was issued with GHQ badges to wear on my sleeve, each consisting or a rectangle which was half red and half black, but with a golden star in the centre, and informed that I would be investigating the mysterious epidemic of anaemia that was playing havoc with some units in the fighting area east of the Brahmaputra river. Meantime I was to go to Rawalpindi away up in the north-west hill area to visit Major Martin Hynes who had laboratories there which enabled him to study the common form of anaemia found in new recruits. When I first arrived in India I knew that my friend Charles Robertson was in the country and it had been a happy coincidence when I found myself stationed in the field next to his quarters at Deolali. However, I also had the address of Mr and Mrs Fairweather, parents of my wife's sister, and when I arrived at GHQ the officer's mess was full of encounters with their knowledge.
Three weeks later information was created for me, but finally the answer was to have to go to Hardakeri to study Malnutrition. There was no time to score a point against that we might have probably been right, so it had almost to be done.
Now I was posted and each day I went to camp on the outskirts of medical, where there were numerous tents instead of huts amongst which I carefully searched for available food, down unlabelled according to what they were carrying.
While there I was given an inappropriate job in hospital catering, cost, cooking, value, vitamins etc., by my Urdu speaking staff at GHQ. This was my first experience of a mixture which was in the bottle.
parents of another of my student-day companions, and to my surprise when I arrived at Rawalpindi I found that I was staying in a Gurkha officer's mess in the house next door to them. This was a most happy encounter because we had a lot to discuss although I had no recent knowledge of their son who was in the Royal Air Force.
Three days later I was on my way back to Delhi to receive information about my research duties. A new type of post was being created for me and the authorities were uncertain how best to arrange it but finally decided to give me the title of Nutrition Research Officer. I was to have a technician with me and eventually Sergeant Robert Hardaker, RAMC, arrived. I was told that we were to be called the Malnutrition Unit, but I said that it was bad for morale to suggest that there was malnutrition in the Services, and that the Japanese could score a propaganda point by referring to such a unit. I asked instead that we might be called the Marasmus Team, since this term would probably not be understood by many, including the Japanese, although it had almost the same meaning; this was accepted.
Now I was back in a tent in South Avenue awaiting further orders, and each morning I could see the Viceroy, Lord Wavell, riding past our camp on his horse. There were almost 500 officers, mainly non-medical, in this transit area and in the mess tent the flies were so numerous that the insides of the sugar bowls were black with flies instead of being white with sugar. Poliomyelitis was rife, particularly amongst officers, perhaps because officers' kitchens were usually less carefully supervised than those of the 'other ranks'; no vaccine was available and the risk was high. I threatened to have the camp closed down unless some effort was made to improve hygiene and reported accordingly to the relevant section at GHQ. Thorough improvements were carried out.
While the authorities waited for War Office approval of their scheme I was given various duties of a clerical type including, most inappropriately, the revision of the diets for British and Indian patients in hospital. This required consideration of the palatability, availability, cost, cooking facilities, religious prejudices, shipping problems, caloric value, vitamin content and practicability. I went to the food stalls with my Urdu dictionary and to the hospital kitchens; the Anglo-Indian staff at GHQ were particularly helpful, but all the time I remembered my first effort at prescribing when the local chemist said that the mixture which I had prescribed, if made up, would not come out of the bottle. At the same time, while waiting, I took a course in statistical
India until at the Durbar in 1911 King George V announced that Delhi was to replace it other than in commercial activities. Situated on the Hooghly river, greater Calcutta had a population of uncertain numbers but possibly about 4,500,000, equivalent almost to that of Scotland. I was stationed for the night at the Grand Hotel in the main street, at that time called Chowringhee, this being the most luxurious accommodation I had yet encountered in the army although admittedly there were so many people expecting tips before one obtained a room that a fistful of rupee notes was required. Across the road was a large grassy expanse, the maidan (which can mean plain or battlefield in Urdu) measuring about two square miles in area with the race course, the Eden Garden and Victoria Memorial each around the periphery. In the centre is Fort William, built in 1781 after the 'Black Hole of Calcutta' revolt, with the maidan now replacing jungle to give a clear line of fire for cannons.
Calcutta was swarming with people and everything was very dusty and dirty. There were tramcars in the main streets and traversing the maidan but as always animals wandered about freely. There was much evidence of poverty and malnutrition amongst the inhabitants and beggars were everywhere, many with gross deformities. It was not surprising that those who were starving were liable to attempt to steal from the newcomers who, to them, were so wealthy; the trouble was that if one gave money or anything else to a beggar one was immediately surrounded by others, particularly children, and escape was sometimes very difficult. European type of food was not really acceptable even by those who were malnourished. Buying anything in the streets, less westernised shops or in the bazaar involved concentrated bargaining and this was expected by the pedlars or small shopkeepers. What was purchased, particularly in the street, was liable to be a completely bogus copy. For example I purchased a splendid Parker pen which had no mechanism but gave good results if ink was poured down the empty barrel. Two 'hand painted miniatures on ivory' were printed copies pasted on to bone.
Calcutta was a leave centre for those serving further east and the Grand Hotel was indeed grand, particularly in the eyes of those who had just come from jungle areas. There were seven course breakfasts, nine course dinners and some two hundred waiters could be seen in the main dining hall. The menus were in French which the waiters did not understand but everything was numbered so it was necessary to order by numbers; unfortunately many of the waiters did not understand English so it was advisable to do this in Urdu even although the local
easy about the contrast in the light and the gloom of those sleeping quarters.
It was hallucinating when I arrived by the Calcutta Scottish Railway, the complicated business of customs, but once this was done I was in Sylhet, entering 14th Army Area, in North East Bengal, a part of India bordering on Pakistan and then, on the other side, with Burma after border disputes not known to us but we knew about them, the problem as we were told, was the difference between the two largest rivers in the area. In November December, the cold season, the temperature is 70 degrees, a night temperature of 50-80%. By March the temperature rises and the monsoon could be expected. The area is a low lying country and the floods are devastating, with cattle and people drowning in the surfaces above the water, and the local inhabitants are driven from their homes, living around them.
The Brahmaputra river and the Ganges are regarded as the administrative boundaries. The intersecting streets of this town consisted of basha huts serving as houses; they did not have walls above the ground level, to keep out the sun by mats and curtains, forming a bazaar area. A river ran through the bazaar which was close by; there were small houses and small shopping areas where one could walk about freely and without fear. Of course, there was no water supply and the river carried the main street traffic, a small river bed beside the road. The local populace were most friendly and were strange people from another world, coming to the local hospital which was
Lieut.-Col. Neill, the Commanding Officer, an Irishman who had been in the Indian Medical Service and was now in the Indian Army Medical Corps (IAMC). I was impressed by his friendliness, his pigs, his hens and even his goats. There were ten other officers including a padre, twelve QAs and six members of the Indian Military Nursing Service. Assisting them were a number of medical orderlies. The hospital covered a large part of the north east of the town with some of the wards looking straight into the jungle; there were about three hundred beds over a scattered area.
The officer’s mess was in a building, not a tent, and was separate from that of the sisters; each QA had an individual small room, a group of which formed a line along the west side of their mess and in front of this was a tennis court. It was clear that there was a tremendous corporate spirit in this remote outpost and I was made most welcome by all members of the staff, whether British, Irish or Indian. Both the CO and the O/C Medical Division, Lieut.-Col. Woods, gave me a free hand to undertake whatever work I thought necessary. This, of course, was a transit hospital with convoys of sick and wounded coming at irregular intervals from Burma and Assam by river transport followed by a short road journey to the hospital. Later I was intrigued by the American TV Series MASH which dealt with a similar type of unit.
I had studied the statistics at GHQ and knew that in 1943 one hundred and twenty men had been evacuated with a medical or psychiatric disorder for every one sent back because of bullet wounds or other surgical conditions. Field-Marshal Sir William Slim in his book ‘Defeat into Victory’ refers to malnutrition, affecting British, Indian and Gurkha troops and says, correctly, that it was not due to shortage of food. Instead, he says, he was informed that ‘the constant mental strain of fighting in the jungle had of itself reacted on the metabolism of the men’s bodies so that often food passed through them without the normal amount of nourishment being extracted from it.’
This was so except that it was unlikely that mental strain was the cause. I had been sent to carry out preliminary investigations into this unexpected disorder which was leading to severe anaemia, particularly in Indian troops. My equipment consisted of the simplest possible apparatus for carrying out blood counts, if necessary without electricity, a microscope and a hand centrifuge. For the information of a non-medical reader it should be explained that the red cells of the blood which were deficient in numbers in the victims of this disease are formed in the marrow cavity of the bones and the way to find out whether the cells that form them are abnormal is to put a needle in the sternum (breast bone) and suck out a small amount of the marrow then
look at it under a microscope. This is not as painful a procedure as it sounds. It involves shaving the chest before the needle is put into the bone but I soon found that for religious reasons Sikhs would not agree to any such shaving being done; very careful antiseptic procedures had to be carried out instead. Once the marrow particles had been examined one had a clue as to the type of anaemia, particularly whether it was due to the deficiency of a vitamin rather than to lack of iron and sometimes of course it would be both. It was widely believed that the deficiency that had been causing such serious problems in Burma and some areas of eastern India was due to lack of absorption of a vitamin but at the time our knowledge of numbers and sources of vitamins was not extensive and indeed it was not until after the war that knowledge advanced sufficiently for more comprehensive investigations to become possible and treatment to be put on a scientific basis. I did, however realize that I was looking for deficiency of more than one missing factor so arranged for crates of crude liver extract for injection to be sent to me from a pharmaceutical firm in England because it was likely that the vitamins or other factors that were not being absorbed would be present in liver as indeed turned out to be the case.
Col. O'Neill provided me with a ward and a laboratory and said that I should feel free to go into any other ward if I so wished. He would see to it that any patient whom I thought required fuller investigation would be transferred to the ward which he was providing for me. I much appreciated the extent of this co-operation.
At the time of my arrival the epidemic of this sudden severe anaemia, usually associated with diarrhoea and a painful tongue, had abated and I turned my attention to an assessment of the medical conditions affecting the British troops being evacuated from the fighting front in order to keep GHQ informed of the latest position. There were various forms of dysentery, scrub typhus, hepatitis, bronchitis, paralysis because leg ulcers had become infected with diphtheria and a wide variety of other conditions: malaria was now being prevented by the regular taking of mepacrine tablets and it was a serious offence to develop this illness. I was kept extremely busy working from 8 a.m. to 9 or 10 p.m. each day since I was doing my own history-taking, examination of the patients and laboratory work; the sergeant who was to join me had been delayed because of illness, but later on was a great help in the laboratory. At Sirajgunj every attempt was made to organize social events amongst the staff, joined on occasion by three civilians who lived nearby, but at this time I was too busy to attend the parties or picnics. Paperbacks were sent out to me from home. I read them, passed them round to the patients and from him I received a British 'other rank' ration of one shilling a week for the patients while in the hospital and it was nice to read a book.
I was attempting to solve mysterious and baffling cases because they were not written down was, for me, of far more value to the patients in British and Indian hospitals which the men visited.
Christmas was a time of friendliness of spirit and was assisted by presents that I could find. The hospital was in a brick building and scavenging was always necessary because of the danger of rats. The patients had to have a bath daily. However on Christmas Day all the wards and the kitchen were well head; there was a party amongst the patients and I joined in. On Christmas Day the doctors and nurses were not needed but a fresh crop of patients.
By the middle of January this time that the worst occurrence at the hospital would be prevented by dishonesty and theft. There were more than enough patients to collect their rations and what enraged me was the months. An American was shot in the leg and died howling and crying.
passed them to the quartermaster who in turn gave them to the padre and from him they went to the other officers, then the sister’s mess, the British ‘other ranks’ mess and finally the patients. Seldom can the sum of one shilling for a book have been so well expended; the reason why the patients were not given a high priority was that this was a transit hospital and it was not intended that patients should stay long enough to read a book.
I was attempting to move further forward to Imphal in search of the mysterious anaemia, but this did not seem sensible to those at GHQ because they had reports that the disease which I was trying to track down was, for the time being, dormant everywhere and they found it of more value to have my accounts of medical conditions and blood counts in British and Indian troops. I included information about the diets which the men had received.
Christmas came and increasingly I began to appreciate the friendliness of this strange hospital on the edge of the jungle. The staff, assisted by patients, made colourful lanterns from any materials they could find. The church service on the evening of 24th December, held in a brick building, was well attended but was interrupted by jackals and scavenger dogs howling outside like lost souls in torment; it was always necessary to keep well clear of those animals because of the danger of rabies and one nurse who was bitten by a dog just after this had to have an unpleasant series of injections into her abdominal wall. However on this evening nurses and doctors went carol singing around the wards and finished up in the bazaar singing in a circle around the well head; the eccentricities of the British no longer occasioned surprise amongst the local inhabitants and some of them even attempted to join in. On Christmas Day itself there was a combined dinner for doctors and nurses with special meals and some entertainment for the patients, but a fresh convoy arrived and the work intensified.
By the middle of January 1945 the cold spell was over, and it was at this time that an empty ward went up in flames; this was a common occurrence and was thought to be caused by local contractors who would be paid to rebuild the huts. There was certainly a lot of dishonesty about; a new hospital non-medical registrar discovered that more than twenty of the local inhabitants turned up each week to collect their pay although they were not working in the hospital, and what enraged him was they had been receiving this money for several months. Animals continued to be a problem; thirty stray dogs were shot in the hospital area in a week and two of the wounded animals ran howling and bleeding through a ward much to the distress and
indignation of nurses, patients and doctors alike. The CO, who was not directly responsible for this shooting, had a soft spot for the cats which constantly invaded the mess; they were put in a sack and sent surreptitiously by train to Calcutta, but, strangely enough, some of them returned. Vultures as large as turkeys hovered above us or squatted in the fields and if we ate food out of doors there was the danger that a kite hawk might come swooping down to remove it even from between the lips, scratching one’s face with its large wings. There were also many beautiful birds with bright plumage – hoopoes, bulbuls, parakeets, kingfishers and bee-eaters. Always, too, there were mynahs.
The work continued throughout January, with men from the 14th Army coming through on their way to base hospitals. They were beginning to feel that their efforts and sacrifices were being overlooked at home and the term ‘The Forgotten Army’ was being commonly heard. For the sake of morale it was important that mail to and from the UK should be transmitted rapidly and indeed when I wrote to Edinburgh for some specimen abnormal bone marrow slides (in case I began to forget what they looked like) they arrived just three weeks after I had written. The overseas service of the BBC, relayed by All India Radio was a valued link. It was in January, too, that I was notified that I had been promoted to Major.
At this point the effervescently cheerful and youthful Sister Mary Williams, QAIMNS (R), from the small village of Calstock in Cornwall came on night duty. Our first encounter in the wards had not perhaps been too harmonious in that she had come to assist me when I was doing a marrow puncture and the conversation went like this:-
‘Sister, is this needle sterile?’
‘Of course it is. I wouldn’t give you it if it wasn’t.’
‘It’s my responsibility to see that everything is done correctly.’
‘You don’t think I’d give you a non-sterile needle, do you?’
‘No, but I have to make sure that you don’t’.
‘I don’t hand out septic instruments.’
‘It’s not an instrument, it’s a needle’.
‘If I give you a needle, it’s sterile.’
‘All right, don’t get into such a tiz about it.’
Now, however my former sparring partner has taken pity on me with my late night working in the wards or offices attached to them and thoughtfully produced some Horlicks for me to drink. I, in turn, was rather worried about her safety as she walked in the darkness the quarter mile along the dusty paths between the wards with palm trees looming on either side. She insisted on carrying a torch and lamp and I followed behind her as she searched for the snake which had found a place under a bed in our night quarters and was now howling.
Before long we met on occasion at the local tennis court where we had spare time to play. This project was not a success as we were both too busy and we did not meet up again until hospital life was over. I was at other times told that ‘In every crisis there is immediate help’. The nursing staff were always working overtime.
One day I asked him to show me his master’s room. He took me to the top floor of the master’s house and showed me a week’s work.
On another occasion he bombarded me with questions which I could not answer.
One day he told me that hundreds of thousands had done their duty and that he looks forward to the day when wonder will no longer have been heard of. The 2nd Indian Division had been in Mandalay for two months later. The 3rd Division was going to be sent to strange
The CO, who was not a cat lover, had a soft spot for the cats which he would put in a sack and send home. Strangely enough, some of these cats always hovered above us or even, when we went out of doors there was the occasional cat flying down to remove it even from the roof with its large wings. There were also night plumage – hoopoes, owls and bats. Always, too, there were jackals.
I was joined with men from the 14th CCS who were in these hospitals. They were all young officers who were being overlooked for promotion. ‘Why’ was being commonly asked. I remember the amount that mail to and from India cost at this time; indeed when I wrote to my mother enclosing bone marrow slides (in case I needed them) they arrived just three weeks later. A news item on the BBC, relayed by All India Radio in January, too, that I was working in India.
The kind and youthful Sister Mary Anne, from the village of Calstock in Cornwall where she lived, and whose wards had not perhaps been so badly hit, came to assist me when I was in trouble. Our conversation went like this:-
‘It wasn’t.’
‘It is done correctly.’
‘Is the needle, do you?’
‘Yes.’
‘Thank you.’
She has taken pity on me with her kindnesses attached to them and has given me to drink. I, in turn, was given a drink in the darkness the light of the wards with palm trees looming over, accompanied only by a young Indian civilian who insisted on walking behind rather than in front as he carried his oil lamp on the end of a pole. Accordingly on some nights I accompanied her as she walked from ward to ward, always being on the look out for snakes, attackers and other hazards. At this time two murders took place beside the hospital, both involving the local population, and one night the wards shook in a minor earthquake. The jackals continued to howl.
Behind the administrative building was the tennis court and on a few occasions when we were both off duty Sister Williams and I played tennis together. Once a week the officers and nurses who could be spared from duty patronized the cinema in the bazaar. It had only one projector so there was a break at the end of each reel while the spools were being changed, but Sister Williams and I, now sitting together, did not mind this. At times there was disorder and a notice went up in hospital orders to the effect that ‘Officers will not throw monkey nuts at other ranks during cinema performances’. Another order had been ‘In extremely hot weather shirts need not be worn’, There was an immediate enquiry by the medical staff as to whether this applied to nursing officers. It was explained that this was intended only for men working out of doors.
One of the orderlies had a little pet mongrel dog which went with him to the cinema each Wednesday and sat on a seat by his side. His master was admitted to hospital and the dog accompanied the two of us to the pictures instead. On our return to the hospital he went into his master’s ward and slept at the bedside, repeating this performance each week until his owner was discharged.
On 14th February we heard that 130,000 had been killed in the bombing of Dresden by the RAF and US Air Force. The majority view which I shared was that surely this was not justifiable.
On the following day I performed on a severely anaemic patient my hundredth bone marrow puncture at Siraigunj; punctures had been done only on patients who might benefit from this procedure which looks slightly alarming but is safe and simple to do. On that day I wondered about the whereabouts of the units to which I had previously been attached because one of the patients said that the 19th Indian Division which I had known so well at Deolali was heading towards Mandalay. I was not to see the town myself until 1984, thirty nine years later. In February, 1945, 23 CCS which I had left at Goodwood House, was preparing for the crossing of the Rhine the following month and, strangely enough, the officer who replaced me in that unit was posted
to 67 IGH so it is likely that, even if I had not applied for an overseas posting, I would have been sent to the same hospital in India, although not as a research worker. On the very day that I was wondering about the activities of the South Lancashire Regiment to which I had been attached in early 1942 it was just recovering from a chaotic crossing of the Irrawaddy at Pagan, a task which required the assistance of both the RAF and the USAAF. When they got across they found that the opposition came, not from the Japanese, but from the Indian National Army (INA).
The INA was formed in 1942 after the fall of Singapore and was led by a Sikh officer who, aiming at having an independent India, raised a force of Indian troops which changed sides to fight with the Japanese against the British and Indian armies. He did not receive the whole hearted cooperation that he expected and vanished from the active scene. However in late 1943 a new Indian leader aiming to raise a force to establish an independent India using captured Indian troops proved to be more acceptable to the Japanese. This was Subhas Chandra Bose whose anti-British feelings had been conditioned in part by the Amritsar massacre. He came from Bengal but had studied in England; he had visited Hitler and was arrested for sedition by the British in 1940. He escaped, made his way north-west by train to Peshawar, and from there went to Kabul where he was given an Italian passport. He travelled via Moscow to Berlin and first attempted to enlist Indian POWs captured in North Africa to fight against the Allies. His success was but slight and he went on by submarine to Tokyo and began to recruit Indian POWs to form an army, again called the INA or Azad Hind Fauj, which, under overall Japanese command would fight against the British and march to Delhi. This time he was more successful (though not with the Gurkhas) and raised a force of some 20,000 men. They did not fare well as fighting troops, particularly at Kohima in April 1944; some had enlisted in the INA to get to the front so that they could surrender to the British or to loyal Indian units as soon as they could. They had considerable problems no matter what they did; for instance on February 28th 1945 four sepoys in the INA involved in the operation south of Mandalay attempted to rejoin the Indian Army but were caught and shot on the orders of the INA Command. On February 19th Captain Saghal of the INA had written in his diary ‘There is no discipline left and the morale is gone’. The term JIF was usually employed by the British when referring to the members of the INA and others, sometimes civilians, actively supporting the Japanese cause. It stood for Japanese India Forces.
As I was leaving the hospital I was told that the next day I should arrive at the office of the Director of Medical Services, who was, I was informed, preparing to leave for Rawalpindi. The Commanding Director, Major General Assistant Surgeon, myself, was to be in charge of the hospital.
The next day I was last seen by my patients, who were a strange mixture of a large number of a poor quality of revolutionary soldiers and the few British officers that remained.
For the next two weeks a red cross ambulance at Rawalpindi was my daily home. I had never been in a hospital ward, but I had been in an Indian Army hospital, and the guards were always ready to help me out at its entrance. There was no Agnew’s Law to continue to do what I wanted to do, and I did that.
One day I bought a bicycle and started to ride to the hospital.
At the time of the fall of Mandalay my preliminary work was finished and Sergeant Robert Hardaker who was now restored to full health had arrived to act as a laboratory technician. There was doubt as to what I should do next. I still thought I should go to Imphal, Col. O'Neill who scented a possible romance in the air thought I should stay where I was, the padre tried to persuade me to go to Kalimpong on holiday, the parents of my friend Alexander Fairweather whom I had visited in Rawalpindi wanted me to go there, the Consultant Physician, Eastern Command wanted me to attend a conference to Calcutta, the Assistant Director of Nutrition in GHQ wanted me to return to Delhi and the Assistant Director of Research wanted me to travel to Jalna. I wrote myself a posting order to Delhi, since the available work did not justify prolonging my stay at Siraigunj.
This was on a Wednesday and as Sister Williams and I set off on our last visit to the cinema we came across a group of excited Indian patients looking up at the roof of their ward. Somewhere amongst the straw there was a loud hissing noise which they said was being made by a large snake which they assured me was very poisonous. I climbed up a post to see the snake but failed and at this point an officer with a revolver arrived and fired five shots, none of which, however, affected the hissing sounds; then somebody arrived with a lantern and we saw that there was no snake — only two baby birds in a nest in the rafters.
From Delhi I received approval of my proposed return together with a request that I should go from there to speak at a medical conference at Ranchi, in the state of Bihar. There was a farewell party and the next day I set off, arriving three days later at 8 a.m. in Delhi. One night had been spent in the Grand Hotel in Calcutta and on the following night I was robbed in the train when I was asleep; only my bush shirt was stolen, but it contained my cheque book, fountain pen and spare spectacles. Under my pillow were the records of my research work together with my wallet and identity card. At the gate of GHQ I was asked by the guard for my pass. I did not have one, but assuming that he could not read, I produced a Calcutta tram ticket (first class). The guard looked at it, gave a crashing salute and permitted me to enter the compound. Then came another hazard; I was accosted by the formidable Sister Agnes MacGeary, the only person who had been able to manage the controversial Major-General Orde Wingate. Fortunately she only wanted me to buy a ticket for a raffle and in a moment of weakness I did this and won a thermos flask.
Once again I was in a tent in South Avenue and was able to hire a bicycle so that I could travel between two parts of the Medical
Directorate which were respectively in New and Old Delhi. I was asked to write various memoranda that would be sent to the many military hospitals and also gained further knowledge of medical statistical analysis. I was involved in the inspection of faulty articles of equipment; many articles manufactured in India were unusable or wrongly labelled. At the beginning of my work at Sirajgunj I had been surprised to find that everybody, myself included, was anaemic and suspected that there was something wrong with the apparatus, simple though it was. It is possible to get an idea as to whether or not anybody is bloodless by putting a drop of blood on a piece of blotting paper and, once it has dried, comparing the colour with that of a series of shades of red. I had done this and it did not confirm the results of the other test which is a simple one but involves the use of a few chemicals. It was a while before I discovered that a bottle labelled hydrochloric acid contained nitric acid instead, while another labelled distilled water contained hydrochloric acid and that this was the reason for my strange findings. All the tests had to be repeated with fresh chemicals from another source of supply; some of the staff at the hospital were slightly disappointed to learn that they would not be sent home on medical grounds. I visited three military hospitals around Delhi and found wrongly labelled bottles of chemicals in all of them. One serious problem for which a board of inquiry was established was a very large quantity of rum which was causing blindness. As expected we found that it contained methyl alcohol and it was all taken away to be dumped at sea.
Now came the conference at Ranchi but as I had lost two stones in weight, being down to seven and a half stones (47.6 kg), I was persuaded to follow this up with a holiday. I knew that Sisters Williams and Nolan from Sirajgunj were going to have a week's leave in Darjeeling, so arranged to go there. I understood too that Lieut.—Col. Scott Charlton, the Australian OC Surgical Division of the same hospital, was going to be in Darjeeling just after the nurses, so booked for two weeks. The trip consisted first of a twelve hour journey from Calcutta by the normal rail line to the crowded town of Siliguri, with a change there to the famous small scale railway which steams uphill on a single line track making four complete loops after having left the plains and jungles, bringing the traveller within sight of the massive Himalayan mountain range. Darjeeling (Dorje Ling or Place of Thunderbolts) at a height of 7000 feet is dominated by Kanchenjunga (28,146 feet), the third highest mountain in the world. In the valley below are the tea plantations and in the streets are to be seen the smiling faces of hill people from the highlands of Tibet. The roads are well supported by the local bullock cars.
I was billeted in the house of Major H. J. B. Smith who had accommodation for the convalescent officers becoming rather short of beds rather than of patients. The parachute troops were due to take over the area and the land was being cleared for intercommunication and workshops. There were some scenic views and the officers' mess was a fine day. My wife was not far away, but I was not sure how to impress her on the ice rink. I joined the Golf Club but was not allowed to live dangerously and had to wait to be introduced to her on the golf course. She accused me of attempting to kill her in situ and I had to admit that beastly habit of the hospital was to lie down and nurse the wounded, we never did.
The next day we were friends again and she carried me off to a restaurant sitting on my lap. I had a grip on her hand and the surgical scissors had broken through the thin plastic.
of hill people who look so like the inhabitants of nearby Nepal or Tibet. Women carried heavy loads using a band across the forehead to support the heavy weights on their backs. There were ponies but no cars.
I was staying in a private house named 'Rivershill', the residence of a Major Roberts whose wife provided officers on leave with accommodation at low cost. One of the fellow guests was an officer convalescing from an attack of the form of jaundice which was becoming increasingly prevalent and was now being called hepatitis rather than catarrhal jaundice. I later heard that subsequently on a parachute drop he was killed when the chute failed to open. It did not take long to discover that the two QAs from Sirajgunj were staying at the large Mount Everest Hotel. Darjeeling consists of a number of interconnecting roads up on the hillside above the railway station and shops. I had to walk about a mile to the hotel along one of the most scenic routes in the world; the three of us were soon joined by another officer, Capt Wylie, and prepared for a week of relaxation. On a clear day Mount Everest can be seen from Tiger Hill, about seven miles away, but throughout our stay we were advised that the weather was not sufficiently clear. However, in the hotel Sister Williams attempted to improve my dancing ability and I in turn took her to a roller skating rink. Her friend Sister Nolan, created consternation in the Planters Club by asking for coffee as she did not like tea; sometimes the Irish live dangerously! At the square known as Chowrasta where pony hirers wait to entice customers, Sister Williams persuaded me to accompany her on a pony ride; this was fine apart from the fact that she was accustomed to riding ponies and I was not. The animal galloped off and attempted to throw me over the hillside, but I managed to remain in situ and sped round and round the interconnecting streets until the beast was restrained. It may have been noticed that even in a jungle hospital or on leave, Christian names were not used between doctors and nurses in those days although I cannot guarantee that in Darjeeling we never departed from this accepted code of conduct.
The week soon passed and then I had to say goodbye to my two QA friends, probably, I thought, for ever. Sadly I watched them being carried away by the toy train as it puffed down the hill with a man sitting over the front wheels to pour sand down when they lost their grip on the track. During the remainder of this short holiday my surgical colleague failed to materialise and news came that cholera had broken out in Calcutta, several cases occurring in the Grand Hotel transit camp. I learned later that the orderly officer who suggested the
diagnosis was an Edinburgh graduate, Hugh Robson, then a naval medical officer; in the fullness of time he became Principal of Edinburgh University. I went to the local army medical centre in Darjeeling and gave myself a prophylactic anticholera innoculation. I then said farewell to this lovely hill station with its cheerful people and set off for Calcutta and then Delhi where I was put into the Grand Hotel in Old Delhi just in time to hear that British and Indian forces had reached Rangoon. On May 3rd war correspondents entered the city and on May 4th at Montgomery’s HQ at Luneburg Heath in Germany representatives of the German High Command surrendered.
The war in Europe ended on May 8th–9th but this caused surprisingly little excitement at GHQ in Delhi. There was, however, much concern about the increasing tension between the mainly Hindu Congress party and the Muslim League with the possibility of major trouble, if not conflict between the two in the future. Muhammad Ali Jinnah, a lawyer trained in London, was determined that there would be a homeland for the 70,000,000 Moslems and the name Pakistan had been put forward before the war began; it was perhaps derived from the initial letters of the mainly Moslem states in India but another explanation was that it came from two commonly used Urdu words, Pak (pure or holy) and stan which indicates place. The war in Europe might be over but the Japanese still had to be eradicated from the lands they had invaded, prisoners in their hands had to be released and, if anything, the possibility of major disagreement between Hindus and Moslems had become greater despite the knowledge that Britain would withdraw from India after hostilities ceased.
Two days after the war ceased in Europe I was sent back via Calcutta to Sirajgunj, just in time to receive two letters of personal interest from Edinburgh. One was from the Royal College of Physicians there telling me that I had been elected a Fellow. (I had taken the Membership examination while awaiting call up only as a basis for a post-war FRCS qualification which I never attempted as I had no surgical experience) and a note from Professor Stanley Davidson asking me to reply ‘Yes’ immediately by cable if I wanted a post as Lecturer in his Department after the war ended. I answered in the affirmative at once although I had no idea how much longer the war might last in the Far East, that day ending my projected surgical career before it began.
A major factor that was lowering morale in British troops in India was the uncertainty about the length of time they might have to stay either there or in Burma; when we first arrived we were thinking in terms of six or seven years, but on 7th June 1945 the Supreme Commander in Chief announced that the war would be reduced to a campaign and must be shortened. A date must be found. By this pronouncement many must have been tempted to postpone their return to Malaya.
However, there was a great celebration of the news which was broadcast on the radio with jubilation.
Having dinner with friends from the local hospital I was delighted to learn that that evening the Queen Mother was making a broadcast to the British soldiers overseas. Fortunately I had a torch and the opportunity arose, time, hesitation and all, to take her hand.
During my time in India patients who came to Sirajgunj were Hindu, Moslem, Sikh, Christian, Naga. Nagaland is the capital, Kohima, in north-eastern India. Now, the area is being investigated by the British and Indian (Burma) armies from Burma to Sirajgunj and north and south. The latter is the most completely devastated concentration camp area by the river which we arrive to Sirajgunj from the hospital. Amongst them are good Englishmen who have gone into the sisterhood to care for patients when they are ill.
Commander was told by the Secretary of State for War that the period would be reduced to three years four months and that the men affected must be sent home as soon as possible even if replacements could not be found. Even although I was at GHQ at the time I did not know of this pronouncement and fear that the possible psychological boost to many must have been lost by lack of publicity; the decision caused us to postpone to 9th September the target date for the Allied invasion of Malaya.
However, I was in Siraigunj on May 13th, the official day of the celebration of victory in Europe, and we joyfully heard Churchill’s broadcast on our radio. That day the patients were unrestrained in their jubilation.
Having disappeared from the hospital some had obtained Nasik gin from the local bazaar and were either very merry or fighting drunk. I was delighted to have this unexpected reunion with Sister Williams and that evening we went out together for a walk towards the little town; she was most alarmed when, in the darkness, one of the inebriated British soldiers was just about to hit me on the head with a bottle. Fortunately for me and for him, just at that moment somebody shone a torch and the patient, seeing the crown on my shoulder for the first time, hesitated long enough for me to remove the weapon from his hand.
During my previous periods of attachment I had investigated patients who were British, Irish (in the British army), Indian (whether Hindu, Moslem or Sikh), Gurkha, West African, and on one occasion, Naga. Nagaland is a remote hilly country north of Manipur and its capital, Kohima, was the furthest point of the Japanese advance into India. Now, however I had been sent for the specific purpose of investigating the nutritional state of prisoners of war, both Japanese and Indian (JIFs). There were two lines of evacuation from Assam and Burma to Siraigunj by river steamer, some coming from Gauhati in the north and some from the area around Dhaka (then spelt Dacca) in the south. The lines of evacuation by air, sea, river, road and train were most complex but with the fall of Mandalay and Meiktila there was a concentration of casualties at Comilla. Our prisoners had been brought by the river steamer *Kite* from Daudkandi, south east of Dhaka, up river to Siraigunj Ghat and then by the very short train journey to the hospital. Amongst them was a Japanese medical officer who spoke very good English and there were also two Japanese nurses who were put into the sisters’ quarters. So far as we were concerned, patients were patients whether or not they were prisoners and doctors were doctors:
it was most useful to have a Japanese medical officer who was willing and able to interpret and give information about the medical condition, blood groups, etc of his fellow countrymen, this, of course, being very much in their interests. Sergeant Hardaker was with me again so we were well placed to carry out our investigations.
There were 78 Japanese prisoners of whom 39 were in hospital because of surgical conditions, the remainder having medical disorders; there was no evidence of psychiatric problems. There were 228 Indian prisoners of war who for one reason or another had sided with the Japanese and 22 of them were surgical patients. The most common medical illness was malaria, something that had almost been eliminated amongst the Allies by the use of mepacrine. Of the Indian prisoners 204 had suffered or were currently infected with this disease whereas 21 Japanese had never had malaria. Gross malnutrition was not a feature although the POWs were on average more anaemic than our normal Indian sepoy; there were a few patients with severe anaemia to the extent that transfusion was required. I was still seeking the disease which caused diarrhoea, severe anaemia of a particular type, wasting and a sore tongue. In this month of May it was still dormant and the Japanese medical officer told me that he had never encountered the condition. It certainly did not seem to have been a feature at any time in the prisoners now in our hospital. The Japanese officer asked me whether he could join the RAMC, but I had to indicate that this was not possible.
So far as the morale of the prisoners was concerned, it appeared that when they were first captured they were fearful of their future prospects and thought that they might be tortured and executed; they had already discovered on the river steamer that they were being accepted by the medical and nursing staff merely as men who were ill and requiring treatment and now even the Japanese feeling of shame at being captured appeared to have vanished; the Indians, who had not been so fearful of the future were glad that they were still alive and back in their own country. Some wanted to rejoin their units in the Indian army. In fact at Calcutta and two other centres an attempt was made to sort out these and other members of the INA, some being permitted to return to the Indian army and others discharged depending upon what could be established about their activities. Their Supreme Commander, Subhas Chandra Bose, was in Burma in April but shortly afterwards returned to Tokyo and was killed in a plane crash so the question of a post-war trial for him did not arise. At the Red Fort in Delhi three officers in the INA were tried for murder and sentenced to death but the sentence was quashed the next day.
Our ordinary diet consisted of rice against the background of unspeakable cruelty to the Koreans and Chinese and the captured Allied statements in the House of Representatives in America about in December 1945 those forced to work on the Thailand railway. I was called to the ordnance depot to act as guard to a wounded prisoner that I would have to take on duty again soon. For some reason than I could not only asleep at the time but woke him up but slept through it.
I had two nurses available in India to help with the treatment of the prisoners. The other difficulty was using Japanese nurses as volunteers; there seemed to be none that anyone knew of.
The work was carried on which was under the control of Williams, and we hoped to meet again soon. My wife in Calcutta was very disappointed when she very diplomatically opened the window while I was trying to visualise my future in an open-backed chair and not until we could get away had it been permanently closed.
On this occasion I was sitting on the verandah with Ian Hill whom I had invited to set up a special camp for the
officer who was willing to look into the medical condition, this, of course, being very difficult with me again so we had to leave.
Of them 39 were in hospital suffering from various medical disorders; there were 228 Indian prisoners who had sided with the Japanese. The most common disease had almost been eliminated by the end of the war. Of the Indian prisoners 104 suffered from this disease whereas 21 had beri-beri. Malnutrition was not a feature and they were more anaemic than our normal patients, ranging from severe anaemia to the norm. I was still seeking the disease which was of a particular type, wasting disease, which was still dormant and the Japanese had never encountered the disease. It was then a feature at any time when a Japanese officer asked me to indicate that this was so.
Concerned, it appeared that they were fearful of their future and were tortured and executed; they were afraid that they were being treated merely as men who were ill and not as human beings. The Japanese feeling of shame at having killed these Indians, who had not died, was that they were still alive and would rejoin their units in the jungle. In the centres an attempt was made to form of the INA, some being discharged and others discharged without their activities. Their centre was in Burma in April 1945 and was killed in a plane crash but them did not arise. At the time they were tried for murder and sentenced to deportation for life but Field Marshal Auchinleck sensibly quashed the sentences; the officers were released.
Our ordinary British and Indian patients not only had been fighting against the Japanese but also were aware, as we were, of the unspeakable atrocities that had been perpetrated by the Japanese and Koreans and yet there was no evidence of any desire to retaliate against the captured enemies. On 17th November 1944 there had been statements in the House of Commons and in Australia's House of Representatives about the atrocities, some of which we already knew about in Delhi, and particular mention was made of the toll amongst those forced to work under appalling conditions on the Burma-Thailand railway. Naturally the prisoners were kept in different wards to the ordinary patients, rather to my surprise with only one armed guard to a ward. I did not carry my revolver as I did not think it likely that I would have to defend anyone, but Sister Williams was on night duty again so I accompanied her on her ward rounds with even more reason than before. We were annoyed one night to find the guard not only asleep at his post, but asleep on an empty bed; we merely wakened him up but should have taken more severe action.
I had two practical medical problems. Penicillin had just become available in limited quantities and I had to decide whether to use it for the treatment of prisoners of war; in fact I did so for severe infections. The other difficulty was that of blood transfusion, but I solved this by using Japanese blood for Japanese patients and there was no shortage of volunteers; the prisoners knew their blood groups to the limited extent that anyone knew about the groups at that time.
The work being finished, I returned to Delhi to make my report which was urgently required. Again I said 'Goodbye for ever' to Sister Williams, and this time it was much more likely that we would not meet again so it was a very sad farewell indeed. Travelling with me to Calcutta was another, older, QA, Sister Thom from Londonderry and she very diplomatically looked the other way as I hung out of the window while the train steamed out of Sirajgunj station. I can still visualise my QA friend in her tropical uniform standing inside an open-backed ambulance waving farewell with a large white hankerchief until we could no longer see each other. The friendship seemed to have been permanently brought to a close by the exigencies of the Service.
On this occasion I made my first flight ever, from Calcutta to Delhi, sitting on the floor of an old Dakota. At GHQ I learned from Brigadier Ian Hill whom I had known when I was a student that there were plans to set up a special research unit in which I might study blood disorders.
in the Army and that it would probably be at Bangalore; I did in fact reach Bangalore but not until twenty years later and was shown laboratories which it was thought might have been the ones intended for me. This particular project never took place. Since Brigadier Hill and I knew each other well and as he had in the past worked with Stanley Davidson in Aberdeen I told him about the full time but low paid academic post to which I had agreed to return after the war, now having abandoned my original thought of becoming a neurosurgeon. He gave me two pieces of advice — the first was to accept that I would never have a significant income, and the second that I could not contemplate marriage before the age of 32. I was 28, but the first piece of advice did not worry me whereas I was beginning to have profound new thoughts about the latter situation; perhaps I was being a bit slow about changing course.
I was given this information as we drove in a horse drawn tonga along the Rajpath with a temperature of 113 degrees in the shade and when I returned to the Grand Hotel in Old Delhi I learned that the officer in the room next to mine had just died of heatstroke. There was, of course, no air conditioning and indeed I was never in an air conditioned building until I went to the United States in 1948, but if I had known about the officer’s illness I might have been able to save his life.
A week later, to my surprise, I was on my way back to Calcutta and Sirajgunj. There I found that Col O’Neill in his romantic Irish way had been most upset when he received the signal that I was returning. He explained to me almost tearfully that Sister Williams had been posted to work on ambulance trains and was somewhere in central India. Immediately he heard that I was coming he had gone to Calcutta to see whether he could get her posted back but to no avail. It was fortunate that he had not succeeded because I was merely collecting equipment which I had to take back to Calcutta and there I remained for three days. Part of my time there was spent in writing letters about my pay because I had been attempting for six months to convince the Army Pay Corps at Meerut that I had been promoted to Major. To make matters worse I was being charged rental for accommodation of which I had never heard and certainly had not occupied. I had once met an officer who had been completely unable to obtain his pay and was becoming desperate; in view of a story that I had heard about the way that a problem was quickly resolved I suggested that he should write to the King. He did so and whether it was coincidence or not the back pay quickly arrived and he was able to retire in comfort.
I left Calcutta for Meerut by train and in the following year I was well enough to begin my new job as a neurologist at the Stanley Hospital. I was very happy to be back in the hospital where I had been so comfortable during the war, but I was not sure what I wanted to do when I got there. I had no idea what I could do.
Bangalore; I did in fact arrive there later and was shown the way to the ones intended for me. Since Brigadier Hill had in the past worked with me at the full time but low pay, he returned after the war, now as a neurosurgeon.
It was not easy to accept that I would be seconded that I could not be posted home when I was 28, but the first piece of advice I was beginning to have profound respect for was I was being a bit slow and stupid.
I travelled in a horse drawn tonga and was 10 degrees in the shade and in the heat of Delhi I learned that the British had heatstroke. There was, however, I was never in an air raid in the United States in 1948, but if I had been I might have been able to save his life.
On my way back to Calcutta and in the romantic Irish way had a telegram that I was returning. He said that Williams had been posted to a hospital somewhere in central India. I had gone to Calcutta to see if I could avail. It was fortunate that I was busy collecting equipment for the hospital where I remained for three months writing letters about my pay and was able to convince the Army that I needed to Major. To make matters worse accommodation of which I was in need. I had once met an Indian who obtain his pay and was able to convince him that he should write to the Government or not the back pay quickly arrived; I was not sufficiently inconvenienced to crave regal intervention.
However, Sergeant Hardaker and I were still the Forward Marasmus Team and now we were sent to Dhaka, east of the Brahmaputra and 26 years later to be the capital of Bangladesh. We arrived to find that we were attached to 76 IGH (IT), this name indicating that the hospital catered only for Indian patients. We were the sole British members of staff but had a ward of 25 beds, a laboratory and a diet kitchen. I had the assistance of an Indian medical officer, an Indian orderly and a batman. Apart from discussions with the medical staff the language of communication was Urdu which created a problem for a few patients who came from the South and spoke Tamil, Telegu or Malayalam but could not understand my Urdu.
The rainy season had arrived and so too at last had the disease which I was seeking to investigate. I was presented with 124 Indian patients with anaemia, weight loss, diarrhoea and, in many instances, a sore tongue. Marrow punctures were essential and it was found that half the patients, including those who were most severely anaemic, had the changes that I was looking for in the bone marrow cells. The medical reader will of course realise that this is a description of tropical sprue, something I had suspected from the start but it was the first time that I had encountered a group of patients with the expected (megaloblastic) changes in the marrow cells. Tropical sprue is a strange disease in that it can occur sporadically or in an epidemic form and my view was that the mysterious disease each year was an epidemic of tropical sprue and that it was probably of viral origin. The difficulty had been that although it was known as a condition which afflicted Europeans in the tropics and had affected the RAF at Chittagong it was said that it did not occur in Indian patients, but clearly this was untrue. There was no absolute test to confirm the diagnosis but I had crates of liver extract with me and the injected material supplied the as yet undiscovered missing vitamins which the patients temporarily could not absorb. With this and, where necessary, blood transfusions all my patients recovered. Forty five years later the viral theory still held but was unproven.
At Dhaka my sergeant and I were so busy that we were seldom out of the hospital; he was accommodated in a British sergeant's mess nearby. While we were there two events of note occurred. One was that, together with very many of the British service personnel I voted in the general election of July 5th by proxy. The projected arrangements for demobilisation were that it would be based on age and length of
service; it was widely believed that Winston Churchill was opposed to this and that he wanted those who were nearest home to be demobbed first. This led to an enormous proxy vote for the Labour party from India and Burma, and the troops became most unsettled. The other event was serious insurrection from an unexpected quarter in Dhaka. One night when I was busy in my hospital ward I heard the sound of shooting. This was a mutiny by West African troops in a nearby hospital: the acting Commanding Officer of the hospital, an eye specialist, was killed and there was great confusion as other troops were called out to round up the mutineers. It appears that the men wanted reassurance about how much longer it would be before they could return to Africa. Soon afterwards there was unrest amongst American troops in Calcutta who wanted to go home, but the real troubles were yet to come, particularly in the Indian Navy.
On June 6th we had heard on the radio that Russia would occupy half of post-war Germany including Leipzig, Chemnitz and Weimar and that Berlin would be divided into four zones, occupied individually by America, Britain, France and Russia, but encircled by the Soviet area of administration. Occupied as we were by the difficulties of the east we did not foresee Europe’s problems.
On 17th June I was called to see General Maitland Wilson, who said that in a few days he would be affected by the same problem I thought I had. He was concerned with the food supply, especially the religious orders, and asked me what that I could do about it. I suggested malnutrition and vitamin and multivitamin supplements.
Having been away from home to my relief I was not at Tollygunge. The early morning telephone call following the news of the death of Tollygunge’s beloved doctor, who told her that he was going to die, that I had no intention of ending my career, that I would probably be back soon. This was the first time I had pointed out to her that I was ‘Yes’, so I went to Tollygunge where, remarkably, I stayed (In 1982 I was invited to Tollygunge to be studied and I obtained a grant from the Wellcome Trust pointing out that I was not an Arab, but a native of South Africa).
When I returned to Tollygunge from all over the world I had done already. I was called to the ambulance station and I found a puzzled look on the face of the
CHAPTER 9
High Speed Matrimony
On 17th July I reported back to Calcutta at the request of Major-General Neil Cantlie and told him the results of my investigations and that in my opinion the troops whether Indian or British were being affected from time to time by epidemics of tropical sprue and that I thought this must be due to a virus. I had analysed the diets, discussed the food actually supplied, broken groups down according to their religious prejudices, considered the different castes of Hindus and felt that I could firmly say that the problem was not one of primary malnutrition. I recommended the giving of liver injections and multivitamin tablets: no pattern of treatment with diet had emerged.
Having done this I went in search of Sister Williams and discovered to my relief that she was working at 119 IGH (C) at Alipore in Calcutta. The earliest time she could meet me was when she came off duty the following morning, so we met and made our way to the Club at Tollygunj. There, sitting on the edge of the bunker at the 18th green I told her that I had accepted a full-time post at Edinburgh University, that I had virtually no interest in the possible financial side of a medical career, that I would soon be posted away to an unknown destination, probably further east and followed this up by asking her to marry me. This was not perhaps the most romantic of proposals but at least I had pointed out the known difficulties and to my great joy the answer was ‘Yes’, so we went to the fashionable Calcutta jewellers, Hamilton, where, rather to my surprise my cheque was taken without question. (In 1982 the stone fell out of the ring on a very hot day when a bus was stuck in the sand in Saudi Arabia but a replacement diamond was obtained without difficulty and I attempted to dispel any sadness by pointing out that not many ladies obtained a diamond in India, lost it in an Arabian desert and had it replaced in Scotland by a diamond from South Africa.)
When I told General Cantlie he congratulated me but added that from all the fuss Col. O’Neill was making he thought we were engaged already. Four days later I was back in Dhaka and Mary was back on ambulance train duty. When her train reached Lucknow it was met by a puzzled ADMS (Assistant Director of Medical Services) who had
received a message instructing him to send Sister Mary Williams back to Calcutta. This, it turned out, was a kindly gesture by a transport officer who had read of our engagement in the Statesman and, thinking I was in Calcutta, sent my fiancée back there. However, I was in Dhaka.
Meantime much was happening; on 26th July Winston Churchill failed to win the election in Britain and the possible consequences were not clear to those of us who were in India. General Slim had become Allied Land Forces Commander and had moved from Burma to Rangoon to plan the Malayan campaign, first going on leave for a month to England. A new 12th Army Headquarters was established in Rangoon. For my part I received a message which read ‘Army Signals 26/7/45; Major R.H. Girdwood c/o 76 IGH Dacca — Return Calcutta immediately preliminary to departure overseas. Await further orders at District Lab. Technician and all equipment should accompany you Calcutta. Notify total weight equipment.’
I reached Calcutta early on the morning of Sunday, 29th July and rushed to 119 IGH where to my relief Mary was off duty. The conversation was as follows:
‘Let’s get married. I’m on the move again.’
‘But I’ve nothing to wear!’
‘Don’t be silly. Get something. Let’s call a taxi.’
We dashed to St Andrew’s Church in Dalhousie Square and had the banns called that morning, something that was possible in the Church of Scotland under wartime regulations and arranged with the minister, the Rev James Mathieson, that our wedding would be two days later. One problem was that, because of the amount of bigamy that had occurred, it was necessary to get one’s CO’s certificate to say that one was not married already. I did not have a CO so wrote the certificate myself; my conscience was clear and I must have looked honest. On the Monday a ring was bought, a dress maker took measurements and arranged for the dress to be ready by Tuesday morning, the Grand Hotel made arrangements for a reception to be held on a first floor balcony area overlooking the main dining room, the chef undertook to prepare both a wedding cake and suitable menu for lunch and a photographer was engaged to take our wedding pictures. My wife-to-be obtained ten day’s leave although it was thought unlikely that I would be in Calcutta for such a length of time. We could only obtain accommodation for one night in the Grand Hotel, but by fortunate chance marriage was arranged to be on the same day. The time did not suit the matron and only a few people who had managed to get tickets were being married as we had received.
On the Tuesday when I dropped the Congress flag off for payment and picked up photographs and service dress, there were anything but.
That evening we were wakened somewhat excitedly by:
‘What’s the matter?’
‘Sir, a gentleman wants to see you.’
‘Well it’s got to be.’
We heard that the next day we moved to the hotel, but sooner had we arrived than asked the bellboy about Bengal, Mr Mathieson married quite a number of rooms.
Officially I was due posting to another unit, but such thwarted plans were so, receiving a telegram that I was to receive a Allowance for Mrs Reginald Passey, in the University.
On 1st August the paragraph went: ‘I know where I am.’
chance married quarters were opened in the YWCA that week and we arranged to be the first occupants after we left the hotel. Unfortunately time did not permit us to invite guests from Sirajgunj but between the matron and off duty nurses from the hospital at Alipore together with a few people whom I knew in the hotel dining room on the Monday we managed to raise nine guests. I wrote home to tell my mother that we were being married the next day, an event which took place before she had received the letter to say we were engaged.
On the Tuesday the wedding took place without a hitch and indeed when I drove to the church my taxi driver not only replaced the Congress flag on his vehicle with a Union Jack but refused to accept payment and gave us his blessings instead. My uniform in the photographs is a rather strange and unorthodox tropical version but my service dress was still in a tin trunk at GHQ in New Delhi. Uniforms were anything but standardised.
That evening we retired to our room but in the middle of the night were wakened by a loud knocking on the door. I opened it to find a somewhat excited Indian police inspector.
'What's the matter?'
'Sir, a gentleman has been murdered in the room next door.'
'Well it's got nothing to do with me. Goodnight.'
We heard nothing more about this strange incident. The following day we moved as arranged to the YWCA building near Park Street. No sooner had we settled in our room than there was a knock at our door. I asked the bearer what he wanted and was told that the Governor of Bengal, Mr Richard Casey, had arrived and wanted to inspect the married quarters; I firmly directed him to one of the unoccupied rooms.
Officially I was attached to the District Laboratory while awaiting posting to an as yet undetermined destination. I discovered that under such thwarted circumstances I could claim Hardship Allowance and did so, receiving it later when my tangled pay was sorted out. It may be that I was the only officer in the British Army to receive Hardship Allowance for his honeymoon. In the Laboratory I met Lt. Col. Reginald Passmore, IMS, with whom I later had a great deal of contact in the University and the Royal College of Physicians of Edinburgh.
On 1st August 1945 Mary wrote a letter to my mother including a paragraph which commenced 'There is so much to tell you that I hardly know where to begin.' On 13th July 1974 our newly acquired
daughter-in-law, Roberta, wrote a letter to Mary under rather similar circumstances concerning the arrangements for her wedding to our son in Australia and it started with ‘There is so much to tell you but I don’t quite know how to begin.’
Despite the fact that we had just been married I was anxious to know where I was going next and for what purpose; I had heard that ships were being assembled for the invasion of Malaya and the capture of Singapore and thought that I would be involved in that operation, code named Zipper. On the morning of August 7th while sitting in the recreation room of the YWCA we read that something new, an atom bomb, had been dropped on Hiroshima, but did not realise that this was so devastating a new type of weapon that it was to save many lives on both sides by quickly bringing the war to a close. Indeed our Allied Land Forces Commander, General Slim, who was on leave in England had just been told about atomic bombs. We knew that on 26th July a warning from the Allied leaders at Potsdam had told the Japanese to surrender or face prompt and utter destruction but did not realise that this referred to a horrific new weapon. Nor did we know that there had been complete disagreement between Churchill, Truman and Stalin about plans for the future of Europe. On 8th August the USSR declared war on Japan and the Red Army invaded Manchuria and Korea; Pu Yi, the last Emperor of China who had become the puppet Emperor of Manchukuo under Japanese rule was now a prisoner of the Russians.
In the midst of all this international turmoil it is surprising that anybody remembered about me but on 9th August I was instructed to move to Rangoon taking my technician, Sergeant Hardaker, and all my equipment with me. The reason for the move was not given but it appeared that I was going to a British General Hospital which meant that we would not be studying disease or deficiency states in Indian troops. I went immediately to the RAF to seek transport, but without success; I next tried the American Air Force, again in vain. On 10th August we read that a second atom bomb had been dropped, this time on Nagasaki.
That day I secured a berth for my sergeant and myself on the Karapara, a hospital ship which was sailing from Calcutta to Rangoon presumably for some duty connected with the coming invasion of Malaya. We sailed on 11th August and three days later Japan surrendered; this changed all the plans, whatever they had been, and our ship was ordered to return to Calcutta just as we were approaching Chittagong. On 17th August I was again in the Grand Hotel and Mary was back on leave. I shall never forget Malaya, but I was, however, very glad that Admiral Mountbatten, while General Slim, and the latter would have first be consulted, there should be no delay in the formalities. Admiral Mountbatten’s food and drink for the Japanese in Burma was excellent, but about when he would make a crowded table, and the statistics were added to the problem. On 12th that the ceremony was held and the continued to be dropped in the usual formal ceremony.
General MacArthur at Pearl Harbor was ready for war on the day the Japanese surrendered.
was back on duty at her hospital; my orders to go to Rangoon still held: I shall never know whether my final destination was to have been Malaya, but part of the invasion fleet had already set sail. The British, however, were now having their problems with General MacArthur. Admiral Mountbatten was Supreme Commander in South-East Asia while General MacArthur was Supreme Commander in the Pacific, but the latter insisted that the formal surrender of the Japanese should first be completed in the area under his command and that until then there should be no landings in Japanese-held territory. This stage of the formal surrender was delayed until September 2nd. Fortunately Admiral Mountbatten ignored this instruction and relief teams with food and medical supplies were immediately parachuted into the Japanese POW camps. Nevertheless the evacuation of our men to Burma was delayed. After having had a row with a transport officer about whether or not there was room for us, my sergeant and I boarded a crowded troopship bound for Rangoon. Once on board I continued the statistical analysis of my considerable data and listened on the radio to the progress of the Japanese surrender; it was not until September 12th that the formal surrender ceremony relating to South-East Asia was held and it took place in Singapore. Some Japanese troops continued to fight in Burma, Malaya and elsewhere and leaflets were dropped informing them of the surrender and showing pictures of the formal ceremony.
General Hideki Tojo, the prime minister who ordered the attack on Pearl Harbour, unsuccessfully attempted suicide; he was tried in Tokyo for war crimes and hanged by the American army with six other Japanese war leaders on 23rd December, 1948.
CHAPTER 10
Released Prisoners of War
As we sailed past Monkey Point and the Batataung Pagoda from the Gulf of Martaban up Rangoon River on 26th August it was very hot but one thing that struck us was that a blackout was still being observed, something that we had not seen in India. The hospital to which I was to be attached had recently been evacuated by the Japanese and was a solid building that had been a hospital in peace time. I found twenty five letters from the UK waiting for me together with an order cancelling my posting to Rangoon, this having been sent from Delhi the day after we sailed. My mother who had just heard of our engagement was ill with hypertension and my newly acquired wife was in hospital with dengue. Fortunately this painful condition, spread by the mosquito, *Aedes aegypti*, was self limiting and it was not until much later that a serious and sometimes fatal form appeared.
I was still under India Command administration which was unusual since I was in a South-East Asia Command area; my work obviously had to be adapted to medical problems that were occurring. There was no difficulty in deciding which disorders to tackle but the CO of the hospital objected to any activities being carried out by this small alien unit which had been sent to his hospital from Delhi. Prisoners released from the Japanese camps were being sent to Rangoon, but the CO would not agree to my having laboratory accommodation and put my sergeant on general duties. I found a large unused bathroom and turned it into a laboratory, locked it and removed the key. Next I went to 12th Army headquarters and complained about the lack of co-operation. I was told that I had their full backing and that I would have, as patients, men released from the POW camps. It was arranged that I would look after those whose vision had been most affected by malnutrition in the camps; I stressed that I would do nothing that might make the patients feel that they were being used as ‘guinea pigs’ and in particular that I would not carry out any marrow punctures. This, indeed had been my policy previously with the Japs and Jifs. I then consulted Major Harold Ridley, an eye specialist of distinction, and he agreed to test the patients’ vision before and after I had given injections of crude liver extract which of course I had been carrying with me in a crate for other uses.
Prisoners of war were classified into three categories: first, those definitely fit for duty; secondly, those who needed further examination; and thirdly, those who had been seriously ill or injured and were unfit for immediate return to the Army. The first group were sent back to their units definitely fit for duty, the second group were sent to hospitals in the interior and the third group were sent to the 12th Army base at Hainan Island. The majority of the prisoners were Irish and English reservists who had been captured in the fall of Singapore and briefly interned in Changi.
The majority of the prisoners were ex-servicemen who had been returned to the British Isles and could not be repatriated. The total number of prisoners was 250,000 and the average age was 34, if I remember correctly. They were going to be repatriated in groups or, as I said, in batches. This was to be done through the port of Rangoon.
I was told that there was an international agreement to strengthen the British Navy. But the British Navy was not capable of taking on the task which was to be undertaken, so that the job fell to the Royal Air Force. The RAF had 600 aircraft available for the delivery of supplies and the diet consisted of tinned meat, rice, biscuits and tea. The food was unpalatable and the diet was probably responsible for some of the diseases.
uses. It was known that the ex-prisoners (RAPWI or Recovered Allied Prisoners of War and Internees) were suffering from multiple vitamin deficiency states, but the cause of their blindness was unknown. Liver extract contains many vitamins, probably including some that had not been identified at the time, so it was at least possible that vision might improve in some of the men. The CO of the hospital wrote to 12th Army HQ saying that he would not permit the investigations unless definitely ordered to do so. I went to headquarters and discussed my intended work with the Deputy Director of Medical Services, Brigadier Harris (later Lieut General Sir Frederick Harris, KBE), a splendid Irishman from Dublin and told him that the CO had said he would resign his commission if I continued with my work. The answer was brief; ‘Good.’
The RAPWI whom I saw were British, Dutch and Australian; Indian ex-prisoners were in another hospital and the most seriously ill remained in Thailand. Medical teams had been parachuted into the countries where the camps were situated or arrived there by sea; the total number of prisoners is uncertain but may have been about 250,000 in South East Asia. They were removed as rapidly as possible if fit to travel by air transport, hospital ships and troopships, some going direct to the UK with others to Rangoon, South India, Colombo or, if found to be too ill to travel further, to hospital in Singapore. I was informed that 6484 British RAPWI passed through the hospitals in Rangoon in September.
Many had been working on the Burma–Thailand railway which was intended to connect Bangkok with Rangoon, with a new section to stretch for 250 miles from Ban Pong in Thailand to Thanbyuzayat in Burma. The scandalous treatment of the prisoners by the Japanese came as no surprise to those who knew of the murder and torture which they carried out on such a large scale in China after they invaded that unhappy country in July 1937. The treatment of prisoners was outrageous and in many instances barbarous. There were probably 60,000 Service prisoners employed in the building of the railway of death as we came to call it, being used as coolies, and probably 10,000 died. In addition captured Malays, Indians, Chinese and Javanese civilians were ruthlessly used in the project, and the total death toll is unknown. When I was in Rangoon the guess was 100,000. Some prisoners had been employed in building a road to Mergui in the far south of Burma and had been equally badly treated.
Many of the RAPWI whom I treated had been in Japanese hands for $3\frac{1}{2}$ years and had been starved, ill-treated and overworked; their
illnesses had been neglected except that captured Allied medical officers had usually done a splendid job but without equipment or drugs. One of the prisoners, who had been a fellow student, was Dr Stanley Pavillard and he became known as the Bamboo Doctor because of his skill in making needles from bamboo shoots and giving intravenous infusions using stethoscope tubing and jam jars; he used equally crude apparatus to distil water. When bullocks died the Japanese sometimes replaced them with prisoners of war, even if they were ill. Two bullocks were used to pull six large bags of rice, but six men were made to pull ten bags. Sick persons were given less food than healthy ones but some of the Thais managed to help, at considerable risk to themselves, by supplying duck eggs and more than one of the patients said that he owed his life to the help given by sympathetic Thai peasants. Apart from rice the rations supplied were usually a fraction of what was officially said to be given. The rice was often maggoty as was fish when it was supplied. I obtained accurate details of the food given since secret diaries had been kept and I passed the information to Delhi and to 12th Army HQ.
Two of the patients saw Australian nurses being lined up and shot and if not immediately killed in this way, bayoneted. This too I reported to both headquarters. I heard, too, of dead bodies being booby trapped. I was told that some men had been beaten to death for having a wireless set but a few sets survived and one patient showed me one that had been built into a standard Army water bottle; I understand that it is now in the Royal Signals Museum. When the bridges were built it was common practice for our men to make holes in the wooden supports and fill them with white ants to cause destruction as fast as possible. The Japanese put prisoners in the first trains to go along the new sections of line.
I looked after 180 ex-prisoners and 85% of them were known to have had malaria, sometimes to their knowledge on fifty or more occasions. Most had suffered from and many were still affected by the residues of a multiplicity of diseases apart from malnutrition including amoebic and bacillary dysentery, dengue, typhoid fever, scrub typhus, hepatitis, diphtheria (in the throat or in leg ulcers) and cholera. Psychological disturbances were not obvious at this stage; morale was high. There had been widespread illness from vitamin deficiencies which gave oedema, beri beri, pellagra, scrotal dermatitis, a painful tongue and the little understood ‘burning foot syndrome’ and, of course, various degrees of blindness. The worst year for vitamin deficiency features had been 1943 and of my patients 77 had some degree of blindness due to pellagra and 16 to beri beri.
In 1943. In 1943. In 1943. Improvements in the multivitamin tablets and quarters were made precise due to pellagra.
There were originally no medical officers and none could now be found.
There were several medical officers. The improvement in soldier health.
My camp was on grounds where the prisoners were repatriated. One morning I found I have been taken to the Japanese whisky bar in a jeep. At the end of RAPWELL road I felt feeling of Dagon. 99 feet (99 feet) is a large reclining figure when I thought destroyed.
To the extent enough were done nothing had been National changed no grucaptured Allied medical officers without equipment or supplies, my fellow student, was Dr John H. Smith, the Bamboo Doctor because he made bamboo shoots and giving them to the men jam jars; he used to say that when bullocks died the men ate the eyes. The prisoners of war, even if they were given large bags of rice, but six months later were given less food than before, had to help, at considerable risk, to bury more than one of the dead. The men by sympathetic Thai farmers were usually a fraction of what they had been often maggoty as was the rice. The details of the food given to the men were sent to Delhi.
The men were lined up and shot with bayonets. This too I saw, of dead bodies being seen beaten to death for no reason. One patient showed me his Army water bottle; I took it to the Museum. When the Japanese ordered our men to make holes in the railway line to cause destruction of the trains in the first trains to go through.
The men were known to have suffered fifty or more occasions. They were affected by the residues of the jungle infection including amoebic dysentery, scrub typhus, hepatitis, malaria and cholera. Psychological morale was high. There were deficiencies which gave rise to a painful tongue and the mouth, and, of course, various vitamin deficiency features such as the degree of blindness due to previous malnutrition, the onset usually having been in 1942 or 1943. In 15 of those the changes in the optic nerve were such that no improvement could be expected, but all 77 were given liver injections, multivitamin tablets and injections of vitamin B1 and nearly three quarters had some improvement in visual acuity when re-tested. The precise deficiency that caused the blindness was never established.
There was no evidence of tropical sprue, the condition which I had originally set out to investigate in Indian troops, and released medical officers told me that they had never encountered it. One suggestion made to me was that this was because of the rice diet and that the men could not have fatty diarrhoea if they were not eating fat.
There was contempt of the Japanese and in many instances hatred; several men were particularly bitter about the actions of the Koreans. The impression was given that occasionally a decent Japanese officer or soldier had been encountered. Little was known about the Jifs.
My camp bed was in a room in a bungalow within the hospital grounds and I borrowed another such bed and invited one of the ex-prisoners, an Infantry officer, to share the room while awaiting repatriation. It was at this time that I looked under my bed one morning and saw a deadly krait lurking there. My new colleague would have been really unlucky had he been killed by a snake after surviving the Japanese atrocities and dangers of the jungle. I had won a bottle of whisky in a raffle but was a teetotaller and exchanged it for the use of a jeep. Amongst other things I used the latter to take some of my RAPWI patients to see the sights of Rangoon and to increase their feeling of freedom; those who so wished were able to visit the Shwe Dagon Pagoda, said to be built over eight hairs of Buddha. It is 326 feet (99 metres) high, with much gold plating on its sides and around it is a large terrace with smaller pagodas, temples, statues and a striking reclining Buddha. Perhaps, though, the patients were more interested when I showed them the former Japanese headquarters which we had destroyed and certainly when we visited it they said explicitly what they thought about the Emperor of Japan and his military machine.
To those men who had suffered so much Rangoon was pleasant enough but to go home was the driving force and the administrators were doing everything possible to hasten their repatriation. The men knew nothing about the politics of Burma, but then neither did we. There had been a Burma Independence Army (later called the Burma National Army) under Aung San and it had supported the Japanese but changed sides to support the British in March 1945. Our patients had no grudge against the Burmese and admired the elegant ladies of
Rangoon as they walked along the roadside with their many-coloured parasols in the sunshine.
On 15th September Lord Louis Mountbatten arrived at the hospital to give a talk which was particularly intended for the released prisoners. The hospital had a large recreation room with a stage but he insisted on standing on a tea box. I was most impressed as he told the men about the terrific invasion fleet which was to have sailed to Singapore and Malaya that month and added that he had just been there and had found little evidence of Japanese defences. The Japanese commander who was there had sent a message to him saying that the forces would surrender ‘and if your Excellency can help me to locate my forces in the Northern area I shall tell them to surrender too.’
I had a discussion with Brigadier Harris and suggested that I might be able to help the Indian ex-prisoners but he told me that the evacuation of all RAPWI from Rangoon was now so efficient that this was not possible and that I might be better to return to Delhi to resume research work there. I started to use my Delhi address for all correspondence, but this was premature because with the turmoil of repatriation of ex-prisoners giving way to the problem of sending home those whose term of duty in the Far East was over I could really see little need for a research team to seek a new project in Burma or in India, particularly when the time for my repatriation was nearing. My view about this was accepted and I was transferred to South East Asia Command, becoming Medical Specialist to the hospital to which I had been temporarily attached. Meantime I continued my battle with the Pay Office in Meerut which, perhaps not surprisingly, had not been able to keep up with my posting orders; it was not until late in October that I received nine months arrears of pay including the Hardship Allowance for our wedding.
My wife and I were now living apart and I would have preferred to have a virtual break-up of our marriage. Having been given the job in charge of the hospital I was in the same position as my wife in London. I was in the hospital and she was in London for me. We had to find a way of solved the problem. I went to Edinburgh and we had an unexpected meeting with my mother in Calcutta.
Now the news of the outbreak of mutiny in the Indian Army was making headlines and the normal order of things was occupied by the problems because of the situation in which he must have been placed. The next day I was called to Sirajgunj and the reason for the call had been that I had heard from shell-shocked patients who progressed to the point of giving up with life. I had to try to put this into perspective. Perhaps I was being paranoid.
I was then called to the ward at the hospital where the hospital commander, Major General, was staying.
The major general was both to be my friend and my
CHAPTER 11
Problems after Victory
My wife Mary was again on ambulance train duties in India which itself would have made communication difficult but in addition there was a virtual breakdown in the delivery of mail between India and Burma. Having received no letters for a month I went to see the officer in charge of postal affairs about this only to learn that he was having the same problems himself and that he could not communicate with his wife in India. Some patients were sent from my ward in Rangoon to the hospital in Calcutta on which my wife was based and kindly took letters for me. Occasionally I found an RAF pilot who could help but I finally solved the problem by sending my letters for Calcutta to my mother in Edinburgh who, having recovered from the shock of having unexpectedly acquired a daughter-in-law, readressed them to Calcutta where they arrived within three weeks.
Now that the war was over morale was slumping and there was word of mutinies; indeed I knew of this even before I reached Rangoon. I was made Entertainments Officer at the hospital in addition to my normal duties and I fully realised the importance of keeping everybody occupied. The first dance I organised was not very successful, partly because the CO was seen to be dancing with his batman; I told him that he must leave the floor if he could not dance with one of the nurses. The next one was quite a success because I remembered that at Sirajgunj one evening there had been an elimination dance at which the call had been 'Would any lady wearing more than four garments apart from shoes please leave the floor' and had then been followed by a progressive scaling down of the number of garments permitted, ending up with two. I had been surprised by the results at the time but when I put this event on in Rangoon I was not surprised; I was astonished! Perhaps it was bravado.
I was asked by 12th Army Headquarters to write a report on morale at the hospital and so far as the members of staff of this particular hospital was concerned it made gloomy reading thus;
General Statement
The morale of this Unit can only be described as poor. This applies both to officers and to men. A variety of causes is responsible
particularly in relation to release and repatriation.
Officers. Those who have been out East for long periods are more lethargic and prone to follow a 'laissez-faire' policy.
Men. A deeply rooted cynicism and apathy is the rule amongst the BORs.
Standard of Discipline and Turnout. This is fair and is improving. Shirts are now worn.
Reaction to Army Education. Any efforts to educate the men are resented. An example of this was when the film 'King Henry V' was shown in the recreation room. Several of the men turned away even before the film was shown, muttering 'bloody education'. Within ten minutes of the commencement many, including senior NCOs, were walking out and only half the audience remained at the end of the show. Laurence Olivier was not a draw to this audience.
Response to a meeting of 'those interested in education' was:
Officers: One
Nursing Officers: Nil
Other Ranks: Two
This was only part of the report but it gives an idea of the general feelings at the time. Knowing the dangers of having discontented personnel I felt obliged to ensure the accuracy of the information so that headquarters might do what they could to improve morale at this time when the war effort was being scaled down and the majority were interested only in going home. The medical work was still considerable but it was equally important to attempt to keep everybody as happy as possible.
The Rangoon radio station had been destroyed by the Japanese, but British experts had brought sufficient equipment from India and the UK to start broadcasting again on September 10th with broadcasts in English, Burmese and Urdu. Entertainers, including Gracie Fields, came from Britain and the first issue of the bulletin giving programmes was issued by the Army Department of Psychological Warfare on 4th November. I was detailed by Headquarters to write a weekly medical broadcast and to deliver it at 21.15 hours each Wednesday in the 25 metre band, each broadcast to last for exactly 15 minutes. The wording had to be approved by Lord Louis Mountbatten's headquarters in Ceylon and delivered exactly as written without any deviation from the text; in the event no changes were introduced. I had a completely free hand to speak on any medical subject that I wished and, naturally, anti-Japanese propaganda was included. The Japanese had attempted to stop our troops taking mepacrine as an anti-malarial by spreading the
story that it caused sterility so I had to counter this propaganda. Unfortunately mepacrine made those who took it (which should have been all our Service personnel) turn an unpleasant shade of yellow; I remember unblushingly saying in one broadcast ‘every morning before I take my porridge I take my mepacrine tablet’, well aware that I never took porridge and that my skin was not yellow. In later years when in malarial countries I took anti-malarial precautions more seriously.
The subjects on which I chose to broadcast were:
- Nutrition in Wartime
- Recent Advances in Medical Treatment
- The Spread of Infection
- The Battle Against Infection
- The Endocrine Orchestra
In an attempt to ensure an audience the organizers put this type of programme on just after something that was sure to be a ‘draw’. For example on 19th December I followed Bing Crosby except that he was on record and I was ‘live’. Only once did I meet anybody who actually listened to any of the broadcasts and he became President of the Royal College of Surgeons of Edinburgh. The two events were not related.
Additional to this and my normal duties I was sent by South Burma District to report on the conditions in various ancillary medical units. In one hospital I found a ward holding British troops suffering from pulmonary tuberculosis, this, of course, being at a time when no drugs were available for the treatment of the condition. They had been there for some time and when I asked why they had not been sent home I was told that all the shipping was required for American personnel whose permitted time overseas was very limited. I could get no sense out of the transport officer concerned, so said that if they were not sent home at once I would write to a prominent Member of Parliament whose name I gave. They went home almost immediately.
The Officer in Charge of the medical division of the British General Hospital at which I was based was Lieut.-Col. John Stokes and we got on very well together. The Commanding Officer now was Col. Gilroy and he encouraged us in all that we were doing. Christmas was coming and John Stokes and I decided to put on a pantomime which we entitled ‘Christmas Crackers’. He wrote the music and I contributed most of the words. We found that we had in the hospital a carpenter who had worked in a circus, and could build suitable scenery for the stage: the tragedy of his life was that one day at Ealing he had not secured the lion’s cage properly so the animal escaped and killed a child.
Before the day of the performance, however, John Stokes was posted away and the Consultant Physician to South Burma Command, Brigadier Max Rosenheim, later Lord Rosenheim, President of the Royal College of Physicians of London, appointed me as O/C Medical Division in his place with the acting rank of Lieutenant Colonel. Our pantomime took place as planned on the 27th of December with John Stokes temporarily back to play the piano while I performed with others on the stage.
The following day I had a phone call from 12th Army Headquarters warning me that all leave was to be cancelled at midnight and that I had better get away fast if I wanted to rejoin my wife in Calcutta. The hospital was well provided with medical staff at the time and Col. Gilroy raised no objections so I went off in search of immediate transport that same day to Calcutta. Fortunately the RAF were able to take me sitting on the floor of an old Dakota which ran into such bad flying conditions that even the pilot was sick; the trouble was that the plane not only dipped from side to side and fore and aft but also dropped several hundred feet on occasion because of thermal currents. When we reached Dum Dum airport at Calcutta we could not land because of crashed planes on the runway so flew to another smaller landing strip from which we obtained transport to the city. Once again I was in the Grand Hotel.
I went to my wife’s hospital and she was truly astonished when I appeared at the nursing station of her ward. In the absence of the Matron the CO granted Mary leave and after discussion about the likely temperature in Simla in December we decided instead to seek a warmer venue so set off by train for the town of Puri in Orissa. This state is on the eastern seaboard of India to the south of Bengal and Puri, a Hindu holy town, is about three hundred miles down the coast from Calcutta and has a fine beach. We stayed at the Railway Hotel which is on the main road overlooking the sands; it was not luxurious but we had a big double bed with a mosquito net and in the roof there was a large electric fan. It was a quiet period in this resort and most of our time was spent on the beach under a straw umbrella. We went around the town either on foot or in a rickshaw and saw the outside of the main place of pilgrimage, which is the great Jagannath temple which non-Hindus cannot enter. At the hotel we were told about the summer festival when huge cars are dragged from the temple to commemorate the journey of Krishna from Gokul to Mathura. The cars are so big that the word ‘juggernaut’ is derived from them and at the time of the festival they are pulled by several thousand men. We were told that
very picturesque and the wheels of the carts are so large that after the carts have been constructed the wheels have had the same treatment.
The beach was full of ‘fishermen’ who were in their safe position of standing in swimming trunks and fishing nets in the sun.
At the end of the holiday her life was changed completely and she was told that she had almost certainly got a problem with her heart. She was sent for a check-up and in the process of this examination it was discovered that she had various problems which were picked up and diagnosed by the doctors. She was really surprised and expressed her thanks.
A private friend of mine recalling the trip asked me if I was asking for a holiday? I said yes, but, as usual, I was on holiday.
There was no background to this trip and other similar ones where one airline was cheaper than another, or where services were better in India and Simla than in the British Isles. I was impressed by the hospitality by Americans and Canadians, the novelty of the trip and the wonderful people.
very pious Hindus have been known to throw themselves under the wheels of the cars in order to die in sight of the gods of the temple and that after the ceremony the cars are broken up, new ones being constructed each year. Although we did not see a juggernaut at least we had the opportunity to follow a sacred elephant around the town.
The waves were dangerous and it was obligatory to accept a local ‘fisherman’ each, his job being to see that we did not get drowned. The safe procedure was to dive through the waves and go out beyond them, swimming in deeper but calmer water. Once on shore again our fishermen sold us coconut oil to protect our skin from the fierce rays of the sun; at this time of the year the beach was virtually deserted.
At the water’s edge we met the Hon. Mary Scott who was devoting her life to the running of the Kalimpong Mission up in the Himalayas and she attempted to persuade us to join her staff; I explained that I had already accepted a post in Edinburgh. This good lady had two problems when we met her. One was that she had knitted a bathing suit for herself and had not realized that it would stretch when she went in the water so that she was virtually topless. The other was that she was doing her best to be ‘with it’ in her speech and kept explaining that various things had ‘gone phut’. Unfortunately, however, she had picked up the wrong slang term and was using a word much favoured by the troops and rhyming with ‘muck’. On reflection I feel that I really should have explained to her the difference in meaning of the two expressions.
A problem arose in that the Matron of Mary’s hospital sent a signal recalling her, but since I outranked the Matron I sent a message back asking her to quote her authority. She did not need any such authority but, as I hoped, this confused matters sufficiently for us to finish our holiday and then we returned to Calcutta.
There we found that there was unrest about repatriation and that ground and maintenance crews in the RAF at Dum Dum and certain other stations in India were refusing to carry out their duties. Although one aircraftsman was charged it was decided to call this a strike rather than a mutiny and I returned to Rangoon in a plane which had been serviced by warrant officers. Apparently this ‘strike’ spread to Ceylon and Singapore. In Calcutta too there was considerable anger amongst British troops about a film ‘Objective Burma’ which gave the impression that the relief of Burma had been carried out almost entirely by Americans. As has already been said there were American troops in the north of Burma working with the Chinese and I had experience of the work of the US Army Air Force. However this film featured only
American troops who were assisted by two unlikely looking Gurkhas and finished with scenes showing Burma being relieved by hordes of paratroopers, all of whom were American. The British troops returning from jungle warfare, in most instances never having seen an American, wrecked the inside of the cinema and Lord Louis Mountbatten had to issue an order that the showing of the film must cease.
It was at this time that we learned from home that a new airport named Heathrow was being built to the west of London and that test flights had commenced. Little did we think that the time would come when I would have as many as fifty visits to Heathrow in the course of a year.
In Rangoon I returned to my duties as O/C Medical Division. It had been firmly established that release from military service would be based on age and length of service but there was distrust of those in authority in the UK to such an extent that Field Security Police attended any large gatherings of troops. However, in SEAC, the forces newspaper of South East Asia Command, the following announcement had been reported at the end of September 1945: “‘Over my dead body will the Age and Service scheme be scrapped,’ War Minister Jack Lawson told a cheering crowd of BORs at the Swimming Club, Rangoon, when he opened his SEAC tour”.
He added that he hoped the Government would be able to secure for this purpose three big ships, lent to the US under reverse Lease-Lend. One decision that had received universal acclaim was that the released prisoners must be given absolute priority as regards transport home but now it was 1946 and their repatriation had been completed. It was not encouraging to read in the same paper that the US was crippled by strikes and that there was a lightning protest strike of bus drivers and conductors in London. In October, too, there had been a dock strike in Britain.
There were some who could not necessarily be released on the due date under the general scheme and I was one of them because there was a delay in the release of officers in charge of divisions in hospitals. However I had now been claimed by Edinburgh University to return as a Lecturer in the Department of Medicine and although this was accepted by South Burma District HQ a new difficulty had arisen. I was in South East Asia Command whereas my wife was in India Command and it had been decreed that those in SEAC could not return home by India, possibly because of a log jam at the repatriation centre at Deolali. My replacement officer had arrived but I could not find anyone who was willing to write me a movement order to go home by Indian mail. I had become so used to this and to the other handshakes and handshakes and the other office work that I went on 8th November.
When I got back I had been told that HMG had decided to hand over the control of the Indian Army to the Indians themselves and, as a result, the fighting in Burma had already started. I went back to the Rangoon INA Club to hear the news again. The club was small and I was near the stage where a strike was going on. I might have been killed also.
The next day a mutiny broke out on a vessel which was identified as the INA Club. The mutineers again took over the armoury and asked for help. A number of them died and the rest were involved in the Indian League of National Strife and the British surrender.
At the same time
likely looking Gurkhas were relieved by hordes of British troops returning having seen an American, so Mountbatten had to intervene.
I knew that a new airport was in London and that test flights at the time would come about in the course of 1946.
The Medical Division. It had been my duty service would be with the disrust of those in the Field Security Police Corps, in SEAC, the forces following announcement 25- "Over my dead body," War Minister Jack Churchill the Swimming Club,
I should be able to secure for reverse Lease-Lend. It was that the released vessels transport home but not completed. It was not the US was crippled by strike of bus drivers and there had been a dock strike in
I was released on the due to them because there was divisions in hospitals. University to return as although this was difficulty had arisen. I my wife was in India EAC could not return the repatriation centre but I could not find order to go home by
India; fortunately when I had been an independent research unit I had become well experienced in writing my own movement orders so did this again, knowing, of course, that this was of doubtful validity. I then handed in all my Army equipment, ensured that I received a receipt, handed over my research apparatus to a scrub typhus research team and went in search of transport back to India. A puzzled transport officer accepted my dubious movement order and I sailed for Calcutta on 8th February, arriving five days later.
What I did not know was that the anti-British 'Quit India' movement had become active and that on the very day that I had sailed, a mutiny had occurred in a shore based Indian Naval establishment named HMIS Talwar in Bombay. This may have been precipitated by faulty handling of the naval personnel by their officers but, in a large section of the Indian population, there had been a reversal of views about the Indian National Army. At first most Indians had never even heard of it and, when they did, many felt a sense of shame about some of their fighting men deserting to the other side although, as has been said already, some did this merely with the thought that they could cross back again at the first opportunity. Later on the show trial of three INA officers at the Red Fort in Delhi made some public opinion swing again: however I saw one of the three officers, Shah Nawaz Khan, addressing his followers in Calcutta and the number present was very small. Another factor leading to unrest was widespread knowledge in the Services about the RAF mutiny which had been played down as a strike, a reasonable course of action when hostilities had ceased. It might have been reasonable to call the Indian Naval mutiny a strike also, but it was rapidly turning into a major 'Quit India' movement.
The discontent rapidly spread throughout much of India; there was mutiny on HMIS Hooghly in Calcutta and then on two Indian naval vessels in Bombay harbour. We knew at the time that one ship (later identified as the Narbarda) had trained its guns on the Bombay Yacht Club. Fighting took place with both British and Indian troops lined up against mutineers in the Indian Navy in Bombay where a British armoured regiment was sent to assist if necessary, but fortunately its help was not required. It is believed that two or three hundred Indians died in the fighting, which spread to the civilian population and involved armed police. Flags of the Congress Party and of the Muslim League multiplied and in addition there was some intercommunal strife, but by 23rd February the mutineers throughout the country had surrendered.
At the time of all this turmoil I sailed up the Hooghly river to
Calcutta, completely ignorant of the fact that there was a problem, and made my way as usual to the Grand Hotel. I had been surprised at the absence of troops on the crowded streets but, after I had secured a room, I bought a newspaper and learned something of what was going on. At the time telephones were not at all reliable so, instead of attempting to phone my wife, I stepped out on to the main street, Chowringhee, and found that there was nobody else there in uniform. Apparently troops were confined to their units to ensure that the passions of the populace were not inflamed and I had read in the paper about British civilians being attacked in the streets. However, mutiny or no mutiny I had to get to 119 IGH to tell Mary that we were on our way home; no transport either by taxi or tonga seemed to be available but at that moment a truckload of Gurkhas appeared. I had no difficulty in persuading the smiling occupants of this Army vehicle to take me to the hospital and once again astonished my wife by appearing on the ward where she was on duty.
There was little chance of the hospital being attacked but I remembered the shots that I had heard in Dhaka when the West Africans had mutinied so asked the CO if he had any arms in the hospital that could be used if necessary to protect the patients: I had left my own revolver in Rangoon when I signed off my army equipment. After a search somebody found the key for the hospital armoury but there was no need for any weapons.
The procedure for me to claim my wife's discharge was simple and straightforward and I well remember looking out at the Victoria Memorial for the last time as we drove to Howrah Station, both of us having a sense of sorrow at leaving the bustle, dirt, crowds and smell of this large city which we had come to know so well. Quite soon it would be unnecessary for us to shake our footwear in case of lurking scorpions or to regard all dogs as potentially rabid; no longer would we have to regard all water and uncooked foodstuffs as a likely source of infection, and even mosquito nets would soon be a thing of the past. Our train took three days to carry us the 1200 miles to Deolali and we had to share a four berth sleeper with a lady and her baby.
Deolali, which I had left fourteen busy months previously, was now processing service personnel due for repatriation at remarkably high speed. The overworked transport officer to whom I reported was taken aback to find that I had come from Burma, which was not surprising considering that I had written my own movement order, but our papers were in order and we had a marriage certificate so he tried to have us flown home, but this was not possible. While we were waiting we saw the film 'I Know Where I'm Going' and the scenery of the train was never bettered. We had a small boat journey and I felt that she was a good case. On seeing the house looking through the window which lies below the house stay at the railway station we were worried because the house had been damaged. Some of those who knew what might happen.
At last it was the Polish liner Batory which informed the naval officer on the train. The naval officer, on unrest, the train were the charge of operating station in a attack. For Batory when travelling he cabin. The northern before fell asleep.
We did not opened fire British property twenty Egyptian 4th March the troubled area going on so.
When we were not all being repatriated differentiated cases of sma
there was a problem, and I had been surprised at the result, after I had secured a something of what was going all reliable so, instead of went on to the main street, nobody else there in uniform. units to ensure that the and I had read in the paper streets. However, mutiny Mary that we were on our saga seemed to be available has appeared. I had no of this Army vehicle toished my wife by appearing
I being attacked but I Dhaka when the West he had any arms in the protect the patients: I had I signed off my army the key for the hospital ns.
discharge was simple and out at the Victoria owrah Station, both of us dirt, crowds and smell of well. Quite soon it would case of lurking scorpions longer would we have to likely source of infection, of the past. Our train Deolali and we had to baby.
months previously, was now ration at remarkably high whom I reported was taken which was not surprising ent order, but our papers ate so he tried to have us we were waiting we saw
the film ‘I Know Where I’m Going’ at the local cinema and admired the scenery of the west coast of Scotland which it featured. Mary had never been in Scotland and I hoped that the picture it showed of a small boat going into the Corryvreckan whirlpool would not make her feel that she was heading for a worse maelstrom than was in fact the case. On several occasions in later years we stood on the mainland looking through binoculars at the spray from this natural cauldron which lies between the islands of Scarba and Jura, and thought of our stay at the repatriation camp. For those who were returning home and were worried about possible accommodation in the UK a prefabricated house had been brought out from Britain and erected at the camp. Some of those whose homes had been blitzed were very pleased to see what might be at least a temporary replacement abode.
At last it was decided that we could sail home, our ship being the Polish liner Batory which had arrived at Bombay harbour. I was informed that as I was a Lieutenant-Colonel I would be the senior officer on the train to Bombay and warned that we might be attacked. The naval mutiny was not yet fully under control, there was civil unrest, the ‘Quit India’ movement was gathering strength and added to this was the possibility of friction between Hindus and Moslems. If the train were to be attacked or attempts made to derail it I had to take charge of operations. Accordingly Mary and I set off from Nasik Road station in a shuttered train with armed troops all prepared for an attack. Fortunately we encountered no problems and boarded the Batory where I found to my surprise that I was the most senior officer travelling home and that accordingly we had been given a splendid cabin. The voyage was without incident except that it was snowing at the northern end of the Red Sea and a child who had never seen snow before fell and broke his arm.
We did not know it but on February 21st British troops in Cairo had opened fire on angry rioting crowds of people destroying and looting British property as they demanded an end to foreign influence; about twenty Egyptians were killed. In Alexandria there was a violent riot on 4th March with the loss of British lives. We calmly sailed through this troubled area of the world, having no knowledge of the unrest that was going on so close to us.
When we arrived at Greenock there was an unexpected delay. We were not allowed to disembark because it was said that one of men being repatriated had developed smallpox. It was sometimes difficult to differentiate between this disease and chickenpox, but I had seen many cases of smallpox and asked if I could see the patient; the frosty repl y
was that I could not as this particular ship was under the control of the RAF, not the Army.
The Daily Express featured a report on our plight on March 17th including a picture which shows the two of us standing on the top deck gazing at the quay upon which we could not land. In the event the condition was chickenpox so we were all allowed ashore. I phoned Edinburgh and introduced my wife to my mother, thus establishing a happy relationship which was to last until my mother’s death six years later in March 1952.
Thus ended our experience in World War II, a conflict which, on a world wide scale, caused about 55 million deaths. Although 10 million were said to have died in the First World War, it seemed that almost every family in the country was affected directly or indirectly, whereas despite the bombing of civilian targets in Britain and the fact that the conflict lasted longer, fewer families in the UK lost loved ones in the Second World War. Of the 199 medical students who graduated in Edinburgh in 1939, seven were killed by enemy action.
‘Ye’
The next day we were invited to become citizens of the United States of America at the American Embassy in London. It was our first visit to the USA and it was certainly a beautiful country.
‘Pleasant people, but not enough to eat.’
From the Embassy we went to receive our degrees from Professor David Macintosh, the Dean of Medicine, at the University of Edinburgh. We had been told that we would be called about 4.30 p.m. and we arrived there on April 1st.
Our degrees were not yet ready, so we signed the register and waited for the demonstration of the graduation gown at Yester Hall. The gown which we wore was made from the old gown of Professor Macintosh, stained with blood from his sleepless nights during the war. When the gown was finally ready, we tried it on and found that it had been made for a tall man. The RAF had given us a height of 6 feet, the average height of a man in Tavistock, but we were both taller than those who had been drafted into the RAF. Even so, the gown was too short, although it was a good fit for the several years that we wore it.
CHAPTER 12
Touching down in Edinburgh
'Ye'll no get a taxi here tae tak aw that stuff'.
This was my wife's first introduction to the natural pessimism of a citizen of Edinburth when we left the Glasgow train and asked a porter at the Waverley Station to help us to obtain transport for ourselves and our luggage which consisted of two tin trunks, a folding camp bed and a bedding roll.
'Pay no attention. They're all like that here,' I explained, and, sure enough, there was no problem.
First I had to introduce Mary to my mother who had, of course, received wedding photographs and then I went in search of Professor Davidson who, true to form, wanted me to start work the following day. I explained firmly to him that it was the 19th of March, that we had to be 'demobbed', that my wife came from Cornwall, that we were about to visit her parents and that I would be available to start work on April 1st.
Off we went to Aldershot and the nearby RAMC depot where I could not find anybody to sign my certificate of medical examination so signed it myself, certifying that my medical category was A1. Mary was demobbed at Millbank and then we spent a night at the Station Hotel at York in which town I was measured for my demobilization suit which was to be sent to me within eight weeks. Next we made our way by way of London to Cornwall: before I went overseas the underground stations had been used as air raid shelters with many Londoners sleeping on the platforms overnight but all this had of course ceased. When we reached the small railway junction of Bere Alston in Devon we transferred to the tiny branch line for Calstock in Cornwall where I had the pleasure of meeting Mary's parents, brother (just out of the RAF) and three aunts. Calstock is a very small village on the bank of the River Tamar about two miles from the road that runs from Tavistock to Liskeard; in a pleasant rural atmosphere, the fields, in those days planted with daffodil bulbs, wound down to the river. Everybody there knew (and commonly was related to) everybody else although an occasional stranger came from the outside world and, after several years, was accepted. To some of the local inhabitants England
was almost a foreign country and indeed in 1480 Caxton, in describing Britain, had to make the point that although Cornwall was not one of the shires of England it was proper to add it to their number as it did not belong either to Wales or Scotland. This may have been a reference back to the seventh century when the Romano-Britons remained only in Cornwall, Wales, Strathclyde and Brittany, having been driven there by the Jutes, Angles and Saxons. However two world wars had inevitably broadened the outlook of the men from this rural area and even some of the fair sex from the village had been in the armed forces.
Probably Mary had travelled furthest, and her upbringing had been atypical. A tiny fee-paying school which aimed to educate girls to become young ladies occupied a house about half a mile from Calstock and one day the headmistress had noticed Mary, aged about six, attempting to help a butterfly that somehow had become trapped: she was struck by this thoughtful act and offered Mary’s parents a place for her in the school. Although the school could hardly have been smaller and was grossly deficient in equipment the training given there somehow combated the parochialism which otherwise would inevitably have occurred in a village where many of the womenfolk knew but little about the world outside. The farms, bulb fields and local gossip filled their lives. Although she appreciated and enjoyed her childhood Mary could not get away fast enough from this isolated community when she had grown up, and the first step was to train as a nurse in Plymouth. This might also have been the last step since Plymouth was being heavily bombed because of the proximity of Devonport dockyard and the hospital in which she was training was hit: fortunately she was not one of the casualties.
In Britain at this time food and clothes rationing continued. Bread and potatoes had been freely available but for other food-stuffs there was a points system. Meat, eggs, poultry, bacon, milk, sugar, cheese, butter, margarine and cooking fat were very strictly controlled; everyone who had suitable ground was urged to grow vegetables. The health of children improved during the period of rationing because there was a more even distribution of what was available and little evidence of a ‘black market’. At the time of our return to the UK there had to be cuts in the amount of food available and, from 1946 to 1948, bread, too, was rationed; in January 1947 the meat ration was cut to a shilling’s worth a week. In 1947 and 1948 even potatoes were rationed. Meantime ex-service men in large numbers were seeking divorces and many families and businesses in the blitzed areas had to seek new homes or premises. In Plymouth the devastation was considerable.
It is interesting to note that the British Government had planned a National Health Service before the war, but it was not until pre-war unemployment was a large problem that it was set up by introducing a tax on the population. It was intended to maintain the existing medical treatment and to provide free day surgery and out-patient services. Public health was to be freed from the control of the church and the provision of maternity similar to that in Germany was to be made available to anyone who needed it. The scheme was not implemented.
It is interesting to note that volunteers were recruited very quickly to fill the vacancies depriving the country of men who had been trained and were available to do the work. It seems to demonstrate that people know what they want and will consider working hard to achieve it, leading to the shortage of shop assistants and nurses regarding.
I should like to end this again with a quotation from a conversation I had with the Deputy Director of the Medical Lectureship Department who has been in charge of the Medical Stamps since 1952. He said recently:
It was just about the time we reached home that we learned that the British Medical Association had set up a fighting fund to oppose the plans which had been drawn up for the introduction of a National Health Service. I did not agree with the BMA about this, having seen the problems faced by middle class families to obtain medical care in pre-war Britain. The wealthy had no problem and the National Insurance Act of 1911 provided a means of insurance against illness for a large part of the working population, weekly contributions being paid by insured workers and their employers. However a large section of the population was not covered and there was an underlying fear amongst many about the possibility of ill health and inability to pay for treatment. In the *Edinburgh Evening News* dated 19th July 1939 (the day of my graduation) I found an advertisement for the Edinburgh Public Medical Service which provided medical attendance, with freedom to choose one's doctor, together with all necessary medicines and dressings for the dependents of insured persons and for others of similar means. For one subscriber the cost was sixpence a week, rising to one shilling if four people were covered. I never encountered anybody who had subscribed to this precursor of private medical care schemes, and it did not cover hospital treatment.
It is true that the Royal Infirmary of Edinburgh, then the largest voluntary hospital in the country, had no private beds other than in two very specialized areas but the pressure for accommodation for the more deprived citizens was great and it was for them that the hospital had been built in the first place. There was need for a service which was available to the whole population and it was time that hospitals ceased to depend on legacies and flag days for their funding. Moreover I had known poorly paid office workers and others who refrained from consulting a doctor at all because they might be entering on a path leading to expense which they could not afford. My father's chemist's shop had been a half way house for some, but he had not wished to be regarded as a health consultant.
I started another round of correspondence about my Army pay which again had failed to catch up with me (bringing this quest to a successful conclusion after three months), and reported for duty in the Department of Medicine on Monday, April 1st. I was now a full time Lecturer on a salary of £600 a year which was some £200 less than I had been promised, but in the Royal Infirmary I was the Clinical Tutor in Stanley Davidson's wards and this carried a payment of £70 a year. Stanley, who was always careful about money, decided that I could not receive the payment for this work as I was a full-time Lecturer but
hoped that I would carry out the duties. To make matters worse he suggested that I should buy a car in order to answer more speedily any calls to the wards; I explained that I could only just afford to buy a bicycle, and did so.
When he returned to Edinburgh as Professor of Medicine in 1938 Stanley Davidson decided to build up the municipal hospitals in the city (the Western, Northern and Eastern General Hospitals) as teaching hospitals, taking the pressure off the Royal Infirmary and now that the war was over he turned his attention to the building up of a Department of Medicine consisting of physicians with specialized interests whose beds would be in those three former municipal hospitals. None, he had decided, was to be an Edinburgh graduate since he wanted to avoid any parochialism and I was intended to be the exception but, unlike the others, was to work in the Royal Infirmary; moreover my first duties each morning were to be in the Department itself in Teviot Place and there I was expected to arrive before him and deal in a preliminary way with the mail in conjunction with his secretary, Mrs Mona Wilson. Gradually my duties in this respect increased until I was writing the replies to many of the letters and leaving them for his signature. The time came when he even asked me to sign some of the letters with his signature. I consulted a lawyer and was told that this was permissible if I had been instructed to do so and the person giving the instruction was prepared to affirm that this was his signature. I seldom had to do this until 1955 when Stanley Davidson set off for the United States, quite correctly without telling us that he was to receive a knighthood. In his absence I was in charge of his Department and had to send a cable both congratulating him and asking what he wanted done about the letters that were pouring in. I was instructed to have a printed reply prepared, to sign the copies of it with his signature and to send them off. I fixed a light bulb inside a box, put a ground glass screen at one end and laid a letter with his real signature on top of the screen. Using this as my guide I signed all the printed letters of thanks and had them posted.
When I first began those duties in 1946, however, I found that another Edinburgh graduate was attached to the Department and wards, this being Dr H. N. Robson who had been given a post in a somewhat unusual way. At the end of 1945 he had just returned from service as a Naval medical officer when he met Stanley Davidson in the New Quadrangle and was greeted with ‘You’re just the man I want, Forbes.’ and offered a post which he gratefully accepted. The government had initiated a scheme to pay returning service doctors so that they could continue their training. Norrie Robson was one of several occasions that Stanley Davidson, as Lecturer in Medicine, was distinguished by his presence in Edinburgh.
The Department had never had a library and various hospital libraries were in the way of the new university buildings when Stanley Davidson’s students should have been to be called upon. One of the members of the Department was to write a book and behind him was a man who, with my help, was the editor of the book. He was to correct proofs and complete it. He insisted on paying me for it, which was the best-kept secret of all, perhaps I should say, because I was dividing it with him.
However, I was not being a student but an academic with a salary. This was an appeal to the young pharmacists who wanted to know why there was a great shortage of their clinical colleagues in medical schools and why each hospital had only 207 places instead of 208 being expected. It was a good outlook; doctors had always been almost the only undergraduates in the University.
that they could be trained as specialists and this provided finance for Norrie Robson’s post. We discussed this strange meeting on several occasions but never discovered who Forbes was; later Norrie became a Lecturer in Medicine to the dental students and went on to a distinguished career in Aberdeen, Adelaide and Sheffield, returning to Edinburgh as Principal of the University.
The Department as such was a very loosely bound entity in that we never had departmental meetings and the members of staff in the various hospitals throughout the town were free to carry on their work in the way they thought most appropriate, without having to visit the university at all. Indeed the only staff meeting I can recall was the one when Stanley decided that the lecture notes which had been on sale to students should be published, suitably amended, as a book which was to be called the *Principles and Practice of Medicine*. He summoned the members of his staff and told them in turn which part of the book each was to write. At the time I was working on an MD thesis, so stood behind him so that he would miss me out and leave me free to get on with my real work. However, I emerged from the meeting as joint editor of the chapter on ‘Disorders of the Blood’. Later I was instructed to correct the English of some of the chapters and to prepare a complete index. Much to Stanley’s indignation the publishers insisted on paying me for having indexed the volume. This book became one of the best-selling textbooks of medicine in the English-speaking world; perhaps I should have stood in front of him rather than behind when he was dividing out the work.
However, the basic position was that I had given up all thought of being a surgeon and had achieved my aim of being in a full time academic post with considerable clinical work and I was on a fixed salary. The idea of having a financial motivation for work did not appeal to me hence I always refused payment from patients or pharmaceutical firms. I had no quarrel with private medicine but wanted to have nothing to do with it myself. On the teaching side there was a great deal to do because although the students no longer did all their clinical work in the Royal Infirmary, which was next door to the medical school, the number of students was very large and the staff in each hospital unit was small. In 1946 there were 1425 applications for 207 places in the University medical school, many of the applicants being ex-service men who were obviously more mature in their outlook; during the war years the annual intake of medical students had been almost as high. However, in addition to the University undergraduates there were many others to be taught in the wards and
by lectures. First there were the 'college' undergraduates, many of them American, who were taking the alternative non-university qualification of LRCP, LRCS, LRFP&S. This had been established by the Scottish Royal Colleges in 1884, and classes for it continued until 1948. Then there were the dental students who had some clinical training in the wards; next there were the postgraduate students from all over the world, the intake sometimes exceeding that of the Postgraduate School in London which, however, did not take undergraduates. Finally, but by no means least, was that unique institution, the Polish Medical School in exile.
The reorganisation of the Polish forces took place in Scotland in 1940 and in the Polish Forces there were not only university professors and lecturers but also medical students. At the time the Officer in Command of the military hospital at Edinburgh Castle was Brigadier Frank Crew, Professor of Animal Genetics at Edinburgh University, a man with great vision and imagination. He had the idea that there might be created in Edinburgh University for the period of the emergency a Polish Medical School, and Prof Sydney Smith, the Dean, gave him full support. The President of Poland, Wladyslaw Raczkiewicz formally opened this medical school on 22nd March, 1941 and it continued in being until 1949, having produced some 200 medical graduates, many of whom joined the Polish forces. Most of the clinical teaching was carried out in a special section of the Western General Hospital, but I had some contact with the Polish school on the wards, in lectures and in examinations. Some members of the Polish teaching staff and some of the new graduates remained in the United Kingdom after the school closed down; much later I met others in Warsaw. The rapport between the Scots and the Poles was always most excellent; it has to be remembered that the 'Old Pretender' (James III and VIII to the Jacobites) had married Clementina Sobieska of the Polish royal family in 1719. Early in the 19th century many Polish refugees came to Scotland after an unsuccessful uprising in their country.
In Stanley Davidson's ward unit there was one other consultant, two doctors funded by the government scheme for training specialists and Dr James Innes who was attached to the Postgraduate Board in Medicine. My official appointment in the wards was that of clinical tutor, a title which had a different meaning in England, there referring to those working in postgraduate teaching centres. Our staff and patients were faced with excessively large numbers of students, both undergraduate and postgraduate, who were attached for clinical tuition. Stanley did not like this situation and it was necessary to limit the size of the large group of students. When the illness and the patient were consulted and I was asked twice do I remember the patient asked what subject for teaching? This is a question that would have been an object to having to deal with emergencies and patients on weekends and in the evenings. The hospital had patients of all ages and accustomed to all scales.
In March 1946 we went to Pitlochry in Perthshire and then to Perth and we were very happy. Then, in Perth, we went to Pitlochry and when I was pregnant and to the amazement of all, I had not been eating any higher than the road, but with the roads being bad and taking a sharp turn, we went out into the rain. Despite all this, in 1947, I was at work and had promised to go away for a short period and was happening and happening.
In 1947 the hospital and the house were used for laboratory research and by several friends and research workers and become part of the involved growing of vitamins to be used
Stanley did not believe in planning the ward teaching programme so it was necessary to be prepared to set off at a moment's notice to gather a large group of students around a patient's bed and start teaching about the illness and resulting problems. First, of course, the patient was consulted and had to agree before any such teaching was done. Only twice do I remember a patient refusing, whereas on several occasions a patient asked why he or she was not given the chance of being used as a subject for teaching. 'Am I not sufficiently interesting?' was the sort of question that was asked. Stanley had said at a meeting that he did not object to having the weekend as his period for the admission of emergencies and this meant that I had always to be available at weekends and had to visit the ward on Saturday and Sunday mornings and evenings. The winter of 1946-47 was a very cold one and we even had patients developing hypothermia in the wards. I had been accustomed to treating heat stroke but this was at the other end of the scale.
In March 1947 we purchased a small second hand car and set off for Pitlochry in Perthshire; first we became stuck in the snow on the way to Perth and were helped out by Polish troops who lifted the car out. Then, in Perth, I asked the AA about conditions on the road to Pitlochry and was told that nothing had been able to get through; Mary was pregnant and I did not tell her what had been said but set off and, to the amazement of the hotel management, arrived safely; the journey had not been easy, the snow drifts at the roadside sometimes being higher than the roof of the car. Soon after we had returned home and with the roads now clearer I tried to teach Mary to drive but when taking a sharp turn she stood on the accelerator instead of the brake and we went over a wall, ending upside down in a field in the pouring rain. Despite all this our son, Richard, was born safely on 3rd May, 1947. I was at work in the hospital at the time and the obstetrician who had promised to tell me about the birth forgot to do so; Mary and I had a short period of anxiety until I phoned up to find out what was happening and received profuse apologies. It was still snowing that day.
In 1947 the University promoted me to a higher grade lectureship and the household came nearer to solvency. I was still engaged in laboratory research in addition to all my other duties and was advised by several friendly senior colleagues that if I seriously wanted to be a research worker in my field of interest I should go to the United States and become proficient in the new microbiological techniques which involved growing bacteria in a culture medium which required certain vitamins to be added in very small traces if growth was to occur. Thus
of those vitamins in time to go abroad and techniques that were available at the press. I did not feel that I was working as regards results or laboratory work myself. I arrived into 1948 with low tone was concerned with the world scene. It had been India in June 1948 but the partition of India did not happen. Moreover Cabinet accepted this; and there followed mass of the new India. Fierce with perhaps 400,000
Royal of Music and Drama of Rudol Bing who I listened to a concert happily reunited with there that I heard 'von der Erde', the first Bruno Walter in Munich
the wedding of Princess Edinburgh, as he was now city.
specialists in London, in the National proposed. There were this but the vote did not more one of 'wait and on me to go to the techniques had built up to and Bethell at the accepted, provided, of from elsewhere. My was successful and I by the University of
Edinburgh for period of a year from October, 1948. I was surprised that the University was not willing to pay its contribution to my superannuation during the period of absence but fortunately, as it turned out later, I paid both my own contribution and that of the University while I was away.
It could hardly have been a worse time to leave my family. There were dock strikes in June and fears of more to come; the 'cold war' was becoming graver from April onwards with a Soviet blockade of Berlin followed by an Allied airlift to that city. Rationing continued and currency restrictions made travel abroad almost impossible. In June 1947 George Marshall, the American Secretary of State, alarmed at the state of the economy of Europe, proposed an aid plan to help nations that were outside the Soviet block. Matters were serious in Britain. We had ended the war with 200,000 homes destroyed and four million damaged. Half our overseas investments had gone and we were paying back loans given by the United States and Canada. There was a shortage of manpower and National Service men were to continue to be called up until the last day of December 1960. We were not meeting our export targets and a vast amount of our shipping had been destroyed. The winter of 1946/7 had been the worst for sixty years and there was a lack of fuel and power. Power cuts had been necessary and might be required again in the event of severe weather conditions. The National Health Service came into being on July 5th which was a blessing but I did not know how it would affect my University post.
My bank manager informed me that I could not obtain any dollars and that I could take only enough sterling to cover incidental expenses during my voyage to New York. During the war separation of husbands and wives had been commonplace but not so now and it became apparent that the sum to be paid by the Rockefeller Foundation would not be sufficient to meet the cost of medical care in a maternity hospital in the United States; I could not add to this sum since the transfer of money out of Britain was not permitted. The Rockefeller Fellowship regulations made it quite clear that the travelling and other expenses of families was not covered and that it was expected that families would be left at home unless independent financial resources were available. Even if they had existed the money could not have been taken to the United States. Of the dollar equivalent of £800 paid to me I had to allow £350 for normal expenses at home, sufficient money to meet the maternity home bill and over £120 to maintain my superannuation payments. The Rockefeller regulations indicated that I would have to meet the expenses of
attending meetings and congresses in the United States from my stipend.
Academically the outlook for this year abroad was bright but otherwise gloom prevailed.
To visit the United States I had to obtain a visa from the British Embassy in Washington. This, like other things, was not without its blow. The visa was not taken up until after the university examinations.
Since I had to leave home before the examinations, I also had to get a ticket on the Queen Mary. This was booked two days later than the date of the boat's departure. The ticket was not taken up until the day during which the ship was due at the gate. The ship left on Tuesday, 10th June, three days later than the date shown on the Edinburgh University Fellowship certificate. This delay was due to the fact that the citizen of the United States had missed his flight. The ticket was invalid because he had taken a cab to the airport instead of some other means of transport off from the hotel where he recovered himself. The ship reported that the tickets were invalid and that we had to pay for them.
The ship was a large ocean liner, photographed by the state photographer.
CHAPTER 13
Experiences in North America
To visit the United States I had first to renew my passport and then obtain a visa: I was informed that I had to attend the American Embassy in London in person to obtain it. I had to affirm amongst other things that I was not a Communist and that I did not intend to blow up the President of the United States. Then my finger prints were taken, something that many people resented; my own view was that universal fingerprinting would be a good idea.
Since it was impossible to take my pregnant wife and infant son with me because of the currency restriction I sadly said farewell to them and also to my mother and set off for Southampton by train, boarding the *Queen Elizabeth* which first took us to Cherbourg on 16th September, two days before the Berlin airlift carried a record tonnage of supplies to the beleaguered inhabitants of that city; the blockade by the Soviets was not lifted until May 12th, 1949. Nothing of interest occurred during the voyage and I arrived at New York, filled with admiration for the grandeur of the skyline at daybreak. We docked at 7.30 a.m. on Tuesday 21st September and I met for the first time Kenneth Donald, later to be a close colleague when he came in 1959 from Birmingham to Edinburgh as Professor of Medicine. He, too, was on a Rockefeller Fellowship and his wife, Rethe, accompanied him. There was a long delay at immigration, no attention being paid to us until the American citizens were all ashore but on board there was delivered to me a message saying that I was to go to the Henry Hudson Hotel; enclosed, I was relieved to find, were some dollar notes. The Donalds and I shared a cab but found that one of Rethe’s suitcases has disappeared and somehow we knew which truck had gone off with it. Kenneth and I set off for the dock area where the trucks had gone and, surprisingly, recovered the suitcase after an argument. The next morning when we reported into the Rockefeller Foundation at 49 West 49th Street we were told that this was an extremely dangerous thing to have done, but we had realised that at the time.
The next day I went to the top of the Rockefeller Building to take photographs and then was on my way by train to Ann Arbor in the state of Michigan where I was made most welcome by Dr C.C. Sturgis,
the Director of the Department and by Dr Frank Bethell, his deputy, with whom I would be working closely. Indeed Dr Bethell was at the station to meet me and help me with my luggage. I very much appreciated the friendliness of all members of the staff in the Thomas Henry Simpson Memorial Institute in which I was to be working. This was a four storey building situated on a ridge in close proximity to the University Hospital; funds for it had been provided by the widow of Thomas Simpson in the hope that a cure might be found for pernicious anaemia and, ironically, the value of liver treatment was discovered by Minot and Murphy in Boston in 1926, just too late to save Mr Simpson who had died of the condition in 1923.
The first enquiry Dr Sturgis made was about my accommodation and it became clear to him that I would find it difficult to pay for any suitable lodgings so he very kindly said that I could have an attic room in the Institute free of charge. I was completely ‘boxed in’ financially since I could not have money sent from the UK, an Edinburgh medical degree was not accepted in the State of Michigan and in any case my visa did not permit me to earn anything in the United States: I was soon so busy in the laboratory (no technical assistance being available) that I could not have earned money even had it been permitted. Part of my work was with guinea pigs and it was sometimes after midnight before I had time to feed them; this was perhaps just as well as I used to speak to them in Urdu to keep up my knowledge of the language. Fortunately there was nobody else from Great Britain (or for that matter anywhere else) living in the Institute at the time as it might have been thought that I was ‘going peculiar’ had there been anybody else in the building to hear me talking in this strange way to the animals. Quite a number of outpatients came to the Institute and I was always doubtful about the wisdom of having patients and animals so close to each other.
The state of Michigan forms a peninsula virtually surrounded by the Great Lakes except on the south where it is bounded by Indiana and Ohio. Lake Michigan is to the west and Lakes Huron and Erie to the east with the main town, Detroit, sandwiched in between those last two. French exploration began in 1618 and the area became a centre for the fur trade; it was ceded by France to Britain in 1763 and then to the USA in 1796, becoming a state in 1837. In the north there are mineral deposits while the south is largely agricultural, an area in the south west being famed for its tulip fields, first established by a group of Dutch who arrived in 1846 seeking religious liberty. On the shores of Lake Michigan the sand dunes are driven into mounds more than 200 feet high.
Ann Arbor is the home of the University of Michigan at Detroit, the largest city in the country. The University is a university because it was founded by Ann Arbor and the people of the town had a broad vision of the kind of homes they wanted to live in with green lawns and trees. They were willing to pay taxes for weeks to make sure that from the top of the hill the open countryside was visible. Research was encouraged and Law Quarters were built in which the law students could study and sympathise with the Tower of London. It was nice to shine in the sun and see the colours of the flag flying despite the fact that it was particularly windy and I had been told that I would find that out.
Ann Arbor was named from Victoria, a ship named after the Queen in 1850 and was settled elsewhere in the North West of the state working its way south but none went to the Pantridge Institute. After six months I was told that there were no more occasions for radiophysics classes to be held.
feet high in places; this Nagow Wudjoo area is real Hiawatha country.
Ann Arbor itself is not close to the lakes, being 38 miles west of Detroit, situated in the Huron Valley, surrounded by rolling countryside. Its population was less than 50,000 and it was very much a university town with 19,000 students. I felt a sense of isolation partly because of the absence of any fellow countrymen but largely because Ann Arbor is itself in an isolated position, particularly to those who like me had no form of transport. It was a most pleasant little town with broad tree-lined streets dominated by the University buildings; the homes of the inhabitants varied in size but were detached buildings with grass and shrubs predominating in the gardens which, pleasingly, were without fences. The crime rate was so low that people went away for weekends leaving their front doors unlocked. A photograph taken from the air shows how spacious the town is and that its situation in open country is a most attractive one. Eye-catching is the Legal Research Library which looks like a cathedral, while the W.W. Cook Law Quadrangle is built like an Oxford College. There was a stadium which could seat 86,000, the Hill auditorium to which came concerts and symphony orchestras, and prominent was the Burton Memorial Tower with the Baird Carillon on top. Somehow the sun always seemed to shine and I have no recollection of rainy days while in the fall the colours of the leaves on the trees were breathtaking. Nevertheless despite the attractive surroundings and the natural beauty I felt cut off, particularly at weekends when there was nobody else in the building. I had been accustomed to working a seven day week and was surprised to find that the staff in the Institute worked for only five days.
Ann Arbor was settled in 1823 by John Allen and Elisha Rumsey from Virginia and so named, it is said, because both their wives were named Ann. The University of Michigan was founded in Detroit in 1817 and moved to Ann Arbor in 1837. The medical school was started in 1850 and attracted patients from Detroit, other parts of the State and elsewhere. During the period of my stay I met postgraduates from Northern Ireland, Dublin, Algiers and Lima, all of whom were working in the hospital and we usually had lunch together although none was there for the full period of my attachment: Dr Frank Pantridge from Queen's University, Belfast was at Ann Arbor for five months and I came to know him rather well. Every Thursday evening there was a get together of the medical staff of the hospital, a social occasion which ended up in a local café. I attended classes in nuclear radiophysics and in advanced protein chemistry, the usual fees for the classes being waived at Dr Bethell's request.
However, life here for me was very strange. I had been accustomed first to the busy poverty-stricken India in wartime and then to a largely shattered post-war Britain but I was now in a small town in which the war was seldom mentioned, although I was asked to give a talk at a local military hospital. The United States alone amongst the industrial powers finished the war richer than when it entered and its industrial plant was undamaged; meantime the economy was booming. It had a monopoly of nuclear capability although Russia was believed to have exploded an A-bomb and the development of nuclear power was continuing in Britain. I had been dealing with patients almost daily ever since I had entered my clinical years twelve years previously but now, of my own volition, I was to be a laboratory worker for a year. Above all I had absented myself from home, leaving my wife not only with an infant to look after but also with another due in just over three months time; it was an unhappy experience for all of us. I hoped to see something of the United States towards the end of my year so attempted to put money aside from my monthly pay cheque to cover the cost of travel. Accordingly I had to economise by eating lunch in the hospital canteen and having both breakfast and the evening meal in a nearby diner; I used to be pleased when baked heart appeared on the hospital menu because I could then have adequate protein.
In Britain rationing of food continued but I was able to arrange for CARE packages to be sent home and remember thinking how well the tins of Spam or York ham would be received. It was also possible to send clothing home and I paid embarrassed visits to the ladies underwear department of a store in downtown Ann Arbor. Nylon stockings were not obtainable in the UK so I sent them one at a time in air mail envelopes believing that there could not be a customs charge on single stockings. Clothes rationing came to an end in the UK on March 15th 1949 but I did not cease sending parcels; food rationing became more severe in 1949 and continued until July 1954.
Details of the work I was doing at Ann Arbor will not be of interest to most non-medical readers and will be summarised. I was provided with a large laboratory for my work and Dr Marian Swendseid showed me the microbiological assays which I had come to learn. Additionally I was advised to visit Lederle Laboratories at Pearl River in New York State and there Dr E.L.R. Stockstad gave me further information about the techniques. This was a good time to be studying the subject because it was in 1948 that it was discovered that the vitamin which was not being absorbed in patients with pernicious anaemia was vitamin B12; this was different from folic acid which was the main
vitamin that failed to be absorbed by the patients whom I had treated for sprue in India. Deficiency of either vitamin causes similar changes in those bone marrow cells which give rise to red blood cells; both vitamins were present in the liver extract that I had been using in India and Burma and indeed I was now learning a technique which could be utilised to measure the content of each in various brands of liver extract used for injection. The precise action of each vitamin in cellular function in the body are a matter of great complexity and I spent several later years studying this and writing papers on the subject; in 1950 a whole number of the *Edinburgh Medical Journal* was devoted to a very detailed analysis of the subject which I had written and in it I included 270 references from the literature. Unfortunately this did not attract any attention from other workers in this field of interest as the journal was not known to them. At Ann Arbor I also carried out experiments to produce similar changes in the blood of guinea-pigs. I do not like doing research work on animals and have carried it out only in the United States where it had occurred to me that if two drugs with totally different actions were used simultaneously the changes which I was trying to produce might occur in the guinea-pig and that this might lead to important applications in the treatment of disease in man; changes did occur in the guinea-pig cells and I published the paper in the *British Journal of Nutrition*. When I visited the Wellcome laboratories at Chapel Hill in North Carolina in 1974 the workers there produced a copy of this paper and told me that it had played a significant part in the development of Septrin and Bactrim, an antibacterial drug which is marketed under two different names and is a combination of the two types of drugs that I had been using in the animals except that at the time one of the pair I was using was too toxic to give to man but was all that was available. The combined preparation is widely used in the treatment of a large variety of infection. In all, this year of research led directly to the publication of four scientific papers but it was a totally different article that led to an avalanche of requests for reprints. This was one entitled *The Factors that Commonly Worry the Patient in Hospital* and it was based on lecture notes used by the co-author, the Rev Malcolm Ballinger, Protestant Chaplain to the University Hospital. During my stay at Ann Arbor I attended some of Dr Sturgis's ward rounds and he was usually accompanied by the chaplain, a very friendly person whom I grew to know well. We were both concerned that in such a scientific environment the real worries of the patients might be overlooked and this paper was designed to draw attention to the sorts of things that
patients were really concerned about. We had jointly decided that the paper, if accepted, was to be published in Britain and so it had to be modified since the problems in the two countries were not identical. One of the major worries in North America was the cost of treatment that had so often to be borne by the patient or relatives. For example a father brought a child with a high temperature to the hospital but was turned away because he could not confirm that he could pay the bill. Treatment in some patients ceased when the money ran out. One of the postgraduate doctors was in difficulties because his currency had been devalued; he fell off a bicycle and injured his arm but could not pay to have it X-rayed. A nurse in the hospital injured her wrist but was told that she could not be treated unless she produced evidence that she could pay; she was most upset until I suggested that she should point out that as she had injured it on duty she was going to sue the hospital. She was investigated and treated at once. I was accustomed to almoners in hospital being available to help patients who were in difficulties but had no experience of administrators who could form barriers between the potential patient and the doctor pending establishment of ability to pay. At the time there were a few aggrieved British doctors touring the country in order to give lectures about the bad features of a National Health Service. I offered to give a talk about the benefits of the new British Service; Dr Sturgis was horrified, explaining to me that he did not personally object but that it would be thought that he was harbouring a Communist in the Institute and that there would be objections from the American Medical Association and other consequences which would be detrimental to his department. He fully appreciated that I was not a supporter of communism but if I voiced support of the National Health Service it would be difficult to persuade his colleagues of this. At the time there was a great deal of adverse propaganda about the NHS being put out by the AMA and the country was paranoid about communism. Indeed in June 1949 President Truman had to express his anxiety about the activities of the House Committee on un-American Activities. The newspapers were full of speculation about the actions of a former State official, Alger Hiss, who was alleged to have passed secret documents to Whittaker Chambers, said to be a former Russian agent; in due course there were the absurd witch hunts by Senator Joseph McCarthy from 1950 until they were brought to a halt in 1954.
Some time after I had returned home we entertained to dinner a doctor whom I had met in the United States and he was accompanied by his wife; for some reason Senator McCarthy’s name was mentioned, whereupon the wife started shouting ‘The devil’ and the dancing attendants fled from the world from the embarrassed husband.
At the end of the year about events in China (Chiang Kai-shek’s forces were routed, Chiang fled to Formosa, Tsetung). Formosa was indeed the site of the Presidential election. An American friend went to the booth and discovered that the President’s name of a Republican was on the heading and that he was from one party, the Democratic Republican Party, the Socialist Party and the Socialist Party were voting together. This was considerably to my surprise and made me that the future was uncertain about the outcome. The trouble was that the Governor, the Attorney General and Auditor General and Representative were all Twelfth District of the County: President, County Treasurer, County Clerk and County Surrogate were all county offices. It was not easy to work out amongst them who was even possible to vote for the most competent person.
On the morning of the news-stand I read that the new President was Republican.
whereupon the lady got to her feet, waving her arms about and shouting 'The man's a saint. While your Guards in their fancy pants are dancing attendance on your Royal family this good man is saving the world from the greatest evil of our time.' Her husband looked embarrassed.
At the end of October, 1948 there was great concern in America about events in China. Just before the Presidential election day Marshal Chiang Kai-shek, despite being heavily backed by the United States, was routed by the Communist troops under Mao Zedong (Mao Tsetung). Peking fell in January 1949 and Shanghai in May; there was indeed cause for international concern. Meanwhile, however, the Presidential election took place on November 2nd 1948 and my American friends went out to vote. I got as far as the inside of a polling booth and obtained ballot forms which I kept as a souvenir. Voting for the President was relatively easy, there being seven parties with the name of a possible President and Vice President being printed under the heading of each party: it was not possible to vote for a President from one party and a Vice President from another. The parties were Republican, Democratic, Prohibition, Socialist Labor Party of America, Socialist Workers Party, Progressive Party and, finally, Socialist Party. The voters and the newspapers had no doubt that they were voting between Dewey and Truman. Apart from that there was considerable confusion and even some of the staff of the Institute told me that they either did not understand the voting procedure or were uncertain about the duties of those in the various posts that were listed. The trouble was that they were also voting for the following; State: Governor, Lieutenant Governor, Secretary of State, State Treasurer and Auditor General. Congressional: United States Senator, Representative in Congress Second District. Legislative: State Senator Twelfth District, Representative in State Legislature First District. County: Prosecuting Attorney, Sheriff, County Clerk, County Treasurer, Register of Deeds, Drain Commissioner, Coroner and County Surveyor. Not all parties had put forward candidates for all the offices. It was possible to vote a straight party ticket or to weave in and out amongst the parties putting a cross against various names. It was even possible to write in names that had not been listed. It all seemed most complicated.
On the morning of Wednesday November 3rd I read on the hospital news-stand the front page of the Chicago Daily Tribune and learned that the new President was to be Senator Thomas E. Dewey, a Republican who was born in Owosso, Michigan and had been an
outstanding student at Ann Arbor. However, members of the staff whom I knew told me that there was something far wrong about this report which seemed to be based on Gallup poll predictions rather than fact. Transistor radio sets had not yet been invented but somebody had lent me a small wireless set which needed an earth and aerial and on which I could receive programmes in my attic room because I had attached the aerial wire to a metal dustbin. I went back to listen to a news bulletin and learned that the newspaper report was quite false and that Harry S. Truman had been elected by a comfortable majority. The Democrats were in control in both Houses of Congress; I never grasped what were the fundamental differences between Democrats and Republicans. Incidentally the ‘S’ in Harry S. Truman’s name was not an abbreviation; it was the whole word. Occasionally American names seemed strange to me, particularly that of Dr First whose parents had given him the unlikely first name ‘Safety’; from time to time I read scientific papers written by Safety First.
Some unexpected subjects of discussion arose from time to time. One doctor who came from the South told me that he was twelve years of age before he discovered that ‘Damn Yankees’ was not the real name of the inhabitants in the north. Two doctors had never seen the sea and one did not know that there were tides; however, it has been said that Adolf Hitler was also unaware of this natural phenomenon. In a café a waitress who was uncertain about the geographical situation of Scotland, when told that we were situated to the north of England, asked whether we had a separate King and Queen. In fairness it has to be said that in 1964, 3% of British people who responded to a questionnaire thought that Eisenhower was the name of the British Prime Minister, while of ten people asked at Piccadilly Circus in a BBC television programme to name the capital of Scotland only one gave the correct answer; Carlisle, Newcastle, Glasgow and Dublin were mentioned.
I had problems about the meanings of certain words but my American friends were most understanding; I had never heard of a faucet, but discovered that it was a tap. I thought that suspenders were used to hold up socks but they hold up trousers in America. The last letter of the alphabet is ‘zee’ not ‘zed.’ A greater problem (and it was one that caused difficulties in Allied planning during the war) was that in North America the date 1/3/49 is the 3rd of January 1949 rather than the first of March and I had of course to use American spelling when writing scientific papers. I had difficulty with the meaning of the figure ‘one billion’ since in the UK it then meant a million million but in the USA and Canada it meant a thousand million.
One day I was invited to a party at the home of Dr and Mrs Keremina. I had not been to a dinner party for many years where I slept soundly and woke up feeling refreshed and not at all sleepy. It was a cold winter’s night; she had put on her dressing gown coat; she had taken off her slippers and pulled them over her feet and pulled them over her feet and they were warm and comfortable for some time after she had taken them off.
One year I was invited to a dinner party where I was much appreciated. The hostess had painted on the wall a picture of a man. He deserved to be remembered in modern times.
Christmas was a time of high temperature, high humidity, high noise and pleasure. There was a Christmas party, several of us were outside playing with bells and whistles. Haematologists are useful laboratory disorders.
However, in 1950, I was married and on the same day my eldest daughter was born. I was once again provided with a wife who continued to be interested in me with a view to furthering her researches into the field of scientific medicine. She accompanied me to a scientific meeting in London and then to another scientific meeting in New York. Knowing that I was booked for a long time, it was very difficult to persuade her to come to the USA.
USA and now the UK a thousand million.
One day at Ann Arbor I went to the local cinema to see Anna Kerenina. As the story unfolded an obese gentleman along the row from where I sat felt constrained by his trousers and undid his zip then fell asleep. It was a cold day and along the row came a lady wearing a fur coat; she had to pass the sleeping film viewer and, startled, he got up and pulled at the zip as the lady tried to sit down in the empty seat beside him. Most unfortunately the zip caught in her fur coat and there they were, locked together face to face. They both struggled in vain; for some reason the coat would not come off so as a twinned package they struggled to the side aisle and then the exit.
One youthful motorist at Ann Arbor heard of radar traps but did not appreciate how they functioned; determined not to be caught in one he painted on the side of his vehicle a car going in the opposite direction. He deserved credit for ingenuity but needed some instruction about modern technology.
Christmas came without the expected fall of snow and although the temperature fell the sun continued to shine; the atmosphere was crisp and pleasant. In the Institute there was a lunch-time Christmas day party, seventeen members of staff being present. On the sidewalks outside the larger shops the Father Christmas look-alikes rang their bells and cried 'Ho. Ho. Ho!' Dr Sturgis gave me a copy of the Haematology textbook which he had written and it proved to be very useful later particularly as regards developments in the study of blood disorders.
However, my concern was with what was happening in Edinburgh and on 6th January I received a cable telling me that I now had a daughter and that all was well; clearly the day when I could be home again predominated in my thoughts but that was nine months ahead. I continued with my various research projects as the months went by but continued to send food parcels and clothing home, meantime saving up with a view to spending my last six weeks or so in visiting various research centres. Dr Bethell arranged for me to be admitted to scientific conferences in Detroit, and suggested that I should accompany him to Atlantic City at the end of April first to attend a meeting where all the blood cells were likely to be given new names and then to be at the American Federation for Clinical Research national meeting at the Chalfonte–Haddon Hall and to give a paper there. Knowing of my financial problems he suggested that when he made the bookings he would arrange for us to share a room in the hotel, and I was very pleased to agree. We arrived on the 30th of April and I was
delighted to find that Professor Leslie Witts from Oxford whom I knew was also at the Federation meetings; because of the continuing currency restrictions in Britain he too was having financial problems and together we searched Atlantic City for a place where we could afford to purchase a meal. The hotel itself was a splendid one on the beach beside the boardwalk and had everything anyone could want for a high class seaside holiday. However, its clients were usually reasonably wealthy and even breakfast there was out of the question for penniless visitors from across the Atlantic. Atlantic City is in New Jersey and is on a sandbar some sixty miles south east of Philadelphia. It is claimed that its seven mile long beach is one of the finest in the world but when I went swimming at the end of April I was the only visitor to enter the sea; the Americans thought that it was too cold and that I was crazy. The boardwalk which is at the edge of the beach is flanked by very large hotels and some shops and there I saw for the first time a clothes washing machine and decided that as soon as they were available in Edinburgh I would buy one.
The attempt to rename all the cells in the blood came to nothing with some of the experts becoming very angry with each other. I was intrigued by the way the Federation's scientific meeting was organised since according to the programme 25 papers were to be presented but another 11 were listed under the heading 'To Be Read If Time Permits', while a further 53 including my own were listed under 'Read By Title'. When I asked what purpose this served I was told that under United States tax regulations this ensured tax relief for expenses incurred by Americans in attending the meeting. There was no doubt that the quality of scientific work in the United States was high but I felt that some of the speakers needed some training in the best way to deliver papers; there was no point in a lecturer standing with his back to the audience and galloping through information which completely covered the screen with columns of material which the audience did not have time to read. Fortunately this applied only to a few of the contributors to what was a most successful meeting.
At Atlantic City it was a great pleasure to meet two of my former classmates at Edinburgh University, one being Dr R.M. MacDonald, a Canadian from Halifax, Nova Scotia whom I met not only then but subsequently at medical congresses all over the world. The other was Dr Alan Hill, a rheumatologist who was holding a Nuffield Foundation Fellowship at Harvard Medical School. It had always been my intention to visit centres in the United States before returning home and Alan and I decided to travel around together when the time for this
came. First, however, I had to return to Ann Arbor in order to continue with my researches.
When I returned there I found that Stanley Davidson was coming to the States, his funding fortunately coming from an American source hence protecting him from the problems of other British visitors, and that he wanted me to meet him. Some weeks before he had visited my wife and asked her to let me know that I would have to choose between clinical and laboratory work since it was impossible to do both. She had politely suggested that if that was his message he had better tell me himself; I had replied that if that was the case I would look for a post in some University other than Edinburgh and had already made enquiries about one that I knew was about to be advertised. Stanley had answered to say that I should do nothing hasty and now he wanted me to come to the Mayo Clinic in Rochester, Minnesota. The cost of attending the meeting at Atlantic City had been such that I had to borrow some money from the Rockefeller Foundation but on 22nd May I set off by train to meet Stanley and his wife, Peggy. I had to change trains at Chicago and when I went outside the station I was just in time to see the police dealing with a murder call and then saw somebody who had been mugged being picked up from the sidewalk. Perhaps there was a lot to be said for the peace and quiet of Ann Arbor. On arriving at Rochester I looked around for somewhere to stay and on the side of one possible guest house I saw the words ‘7 Up.’ Not having heard that this was a beverage I sought somewhere cheaper since seven dollars was too much for a night’s stay.
This was a most interesting time to have gone to the Mayo Clinic because, in Rochester, E.C. Kendall to whom we were introduced had developed Compound E, and P.S. Hench had found that it was of great value in the treatment of rheumatoid arthritis. Dr Hench showed us a film which clearly showed the dramatic improvement that could occur. While we were having lunch together he told us of his concern about the way in which some companies were promoting vitamin E for the treatment of arthritis in place of this new substance and wondered whether we thought that cortisone would be a suitable name for it; we agreed and this indeed became its official name. In 1950 Hench, Kendall and Reichstein (a Swiss investigator who had worked on the basic chemical research that led to the synthesis of the steroid) were awarded the Nobel Prize in Medicine for their work. Stanley Davidson said no more about his suggestion that I could not combine clinical work with laboratory research and we parted good friends.
In July Alan Hill and I began our travels by train and Greyhound
Bus. First I met the great Dr W.B. Castle whose researches at an early stage in his career had shown what really was the defect in patients with pernicious anaemia; forty years later we were still corresponding with each other. He was born in Cambridge, Massachusetts in 1897 and remained there all his life apart from the occasions when he visited abroad, sometimes to accept a well deserved honour for his outstanding work on the causation of various anaemias. Tall and distinguished looking he remained modest and friendly and has been a source of inspiration to many; he declined the Nuffield Chair of Medicine at Oxford, preferring to stay in Boston.
Alan and I made our way to Washington, DC which we reached on July 14th. Here one of the most beautiful capitals in the world has been built in an area which had formerly been marshland. It has been the seat of national government since 1800 and was planned and designed by Major Pierre l’Enfant, a French engineer in the continental army. Central to the plan is the Capitol, a splendid structure at the east end of Pennsylvania Avenue; the foundation stone was laid by George Washington in 1793.
I have three old history books, two dating from my school days. In dealing with the 19th century the Scottish one fails to mention either Napoleon or the United States; the ‘British’ one says ‘England, meanwhile had become entangled in a new war. Our naval policy had caused the Americans, urged on by Napoleon, to declare war upon us in 1812.’ Half a page is devoted to this. The American book devotes 84 pages to an account of the war, caused in the view of the Americans at the time by ‘a series of acts by Great Britain, hostile to the United States.’ A bill declaring war was approved by the House of Representatives and the Senate and received the approbation of the President on 18th June, 1812. This war, which seems to have escaped my attention as a schoolboy, continued until a peace treaty was signed at Ghent on 24th December, 1814. It is mentioned here because the British General Ross captured Washington and burned the Capitol containing the Senate Chamber, Representative Hall, the Supreme Court Room, the Congressional Library, the Public Library, the Treasury, War Office, Naval Office, General Post Office and President’s House. The guide book of Washington which I purchased tactfully omits all of this; the erection of the present building was begun in 1818, there being a central block and two wings with a striking rotunda over the central section. Everywhere in Washington there is white stone and marble with green parks and wide tree-lined avenues. As we walked around I photographed the White House, the
Washington Monument, the Lincoln Memorial, the tomb of Abraham Lincoln, the Jefferson Memorial and the Capitol overlooking the Potomac.
We visited the Smithsonian Institute which is housed in a building as a link between the 16th and 17th centuries, the 16th being the century of Columbus and the 17th that of Galileo; it is under the direction of officers of the Navy and the machine shop is where the first touchstone was made. General Lafayette was once set off by a cannon shot from the west front of the capital city. The building has been restored and contains rooms which were occupied particularly by the men who had fought at Saratoga, Arbigbeg, Yorktown and Edinburgh. I was interested under the guidance of a graduate student in the Washington Monument, but it is under repair and the treatment of the stones is recommended.
Alan and I did not enjoy the hotels. Roosevelt was a project rather than a house, the Grand Hotel was close to the sea and the notable thing about the old hotel is that it cannot be called a French hotel in any words.
sensibly, a patate. We visited the church known as Notre Dame des Victoires. It was named Notre Dame de la Victoire in 1691 when the French defeated the British general, Sir William Phips, but after the British were again defeated in 1711 it was renamed so as to include this second victory; outside there is a large bust of Louis XIV. To the south west we visited the Plains of Abraham and saw the monument to General Wolfe who defeated Montcalm in 1759, both generals dying in the conflict. On 16th April 1746 Wolfe had been the officer in charge of the men on the left flank of Cumberland’s army at Culloden and is said to have refused to massacre a wounded man in the Prince’s army; now, thirteen years later, he was to be killed while leading the British assault on Quebec.
Next came Montreal, then Toronto, followed by a day at Niagara Falls where we sailed close to the torrent on the Maid of the Mist then, donning oilskins, joined a party which was being taken along a passage that led right under the falls from the Canadian side. Next we sailed on Lake Erie in a truly Victorian steamer, the City of Detroit III, and in Detroit paid a visit to Greenfield Village, part of the Edison Institute. Here there is much to see; in founding it Henry Ford’s intention was that there would be preserved part of the American history and tradition, and there are homes, schools, shops, historic structures and laboratories occupying an area of 220 acres. The stern–wheeler Suwanee floats on the waters of the Suwanee River in a valley to the northeast of the village green. I had never been to the Cotswolds in England but now the area had come to me because there had been imported a Cotswold group of houses and even a herd of Cotswold sheep. The reason for this was not clear to me as Edison’s family were of Dutch origin on his father’s side and Scottish on his mother’s; he was born in Milan, Ohio in 1847 and died in New Jersey in 1931, having moved up the ladders from being a boy selling newspapers on railway trains to being one of the greatest inventors of his time. Amongst his patents, more than a thousand in number, were those for systems of telegraphic communication, the gramophone, the typewriter, the thermionic valve, a system of wireless telegraphy from moving trains, the first practical incandescent lamp and the first carbon telephone transmitter, a modification of the instrument patented in 1876 by Alexander Graham Bell who had come from Edinburgh and taken a post in Boston. I was intrigued to visit Edison’s Laboratory which has been re–erected in its entirety and to see a train which he set on fire as a boy; he sold candles in addition to newspapers on the railway. We were told that the conductor of the train boxed his ears and that he became partially deaf as a result. There are many other interesting things to see, such as homesteads, a blacksmith shop, a printing press, a forge, a pioneer kitchen, Lincoln’s desk, a model of the first electric light bulb, and so on, which I cannot mention here.
It was in Detroit that I met a friend who was attending a meeting where a paper was to be read on the subject of medical research. He was a doctor and was interested in the work of the travelling doctors, and I was interested in the work of Dr. John Louis, who had been working for several years in a small town miles beyond the city limits of the river that runs through Detroit, one of the most polluted rivers in the same state. We talked about each other’s work and the visitor was very pleased with the several positive results.
In the evening we had a problem in getting home, trying to get the bus to run, and we omit a small town and had to walk off there. We had to wait a couple of hours for the next bus and were joined by some passengers who were hallucinating and were being persecuted by voices. This occurred in the middle of the round about and we were suggested to go back to the hotel and we did.
My first impression of the building was that it was not arriving at the right place again or that I was dreaming.
I had a room on the second floor and was given a key to the door.
known as Notre Dame des Victoires in 1691 when the English Admiral William Phips, but after the war it was renamed so as to include this victory over King Louis XIV. To the south of the church we saw the monument to the two generals, both generals dying in battle; one was the officer in charge of the Jacobite army at Culloden and is said to have been shot by the Prince's army; now, however, he is leading the British assault.
We were delayed by a day at Niagara Falls on the Maid of the Mist then, before sailing taken along a passage on the Canadian side. Next we sailed on the Great Lakes to Detroit III, and in the vicinity of the Edison Institute. Henry Ford’s intention was to preserve American history and industry, historic structures and machinery. The stern-wheeler Suwanee was built in a valley to the northeast of the Cotswolds in England but the breeders had been imported a number of Cotswold sheep. The Edison’s family were of Dutch descent from mother’s; he was born in 1847 and in 1931, having moved up to New York, he took up work on railway trains to develop the telephone. Amongst his patents, there were for systems of telegraphic communication, the thermionic valve, electric railway trains, the first practical telephone transmitter, a telephone set in 1876 by Alexander Graham Bell, and a post in Boston. I was told that he had been re-erected in its original form as a boy; he sold candles to pay for it. We were told that the Edison Institute, he became partially deaf as a result. Amongst the re-erected buildings were Edison’s original homestead, Henry Ford’s birthplace, the Wright Brother’s cycle shop, a pioneer log cabin, the Logan County Courthouse in which Abraham Lincoln occasionally practiced law, and the Scotch Settlement School which Henry Ford once attended.
It was all most instructive, but it was time to return to Ann Arbor, where Alan and I parted company (knowing that we would soon be meeting again at home). I completed my work and set off to visit research centres engaged in projects related to what I had been doing and which I intended to continue in Edinburgh. This involved travelling by bus to Cleveland, Ohio, then Columbus, Ohio and St. Louis, Missouri. This last city is on the west bank of the Mississippi 20 miles below its confluence with the Missouri; these two together give a river that is 3800 miles long, one of the longest rivers in the world. At one of the centres I found that two research workers were working on the same project unknown to each other, so put them in touch with each other. This was nothing compared with the discovery by another visitor in a different city who found that a research worker had spent several years on a project involving colour matching to obtain his results, not knowing that he was colour blind.
In the course of my various journeys by bus there were a few minor problems; one driver got lost and had to be told by the passengers how to get to his destination. Another, who was running late, decided to omit a stop if the passengers agreed and twice called out the name of a small town that he was about to enter, asking if anybody wanted to get off there; there was no reply, but twenty miles further on an elderly couple called out that he had passed their town and he had to turn the bus and go back with them. On one occasion it was clear that the passenger sitting next to me, a burly middle aged man, was having hallucinations and was paranoid; he was shouting to the driver to stop persecuting him, mentioning visual events that were not in fact occurring. I brushed against him to feel whether I could detect a gun round his waist or at his hip, then moved up to the driver and suggested that he should drive to the nearest police station. This he did and we unloaded our protesting but potentially dangerous passenger.
My final destination was New York where I saw the United Nations building in the course of construction and a group of Jewish refugees arriving, at last to have freedom. On September 4th I sailed for home, again on the Queen Elizabeth.
I had been asked to submit a report on my work, which I gladly did and was also told that general comments would be welcome. Since this
reflects the views of an individual British visitor about the United States after a major war it is perhaps of interest to record what I wrote. I had not, of course, visited the deep south and had not encountered racial problems.
The hospitality given to visitors to the United States is overwhelming. There appears to be a little difference between the cost of living in the two countries, but a great difference in the standard of living.
The cost of medical treatment to the unfortunate invalid in America is, frankly, staggering. This must have a detrimental effect in many instances in preventing a patient from going to his doctor at an early stage of his illness.
It is regrettable that the publicity campaign firm employed by the AMA to fight President Truman’s schemes for ‘socialised medicine’ should devote so much of its campaign to a biased attack on the alleged low standard of British medicine.
(I did not put it in my report but I had read in a magazine article that British doctors could not be expected to know about modern developments such as the use of penicillin. I told Prof. Fleming about this later and he was merely amused).
The American medical profession appears to be unaware of the fact that a basic difference between the two countries is that 84% of the population of Britain has a net income of less than £250 per annum, and that 60% of the population of the United States has a net income of more than £500 per annum. When informed of this basic difference and of the fact that the general practitioner in Britain cannot be contrasted directly with his colleague in America because the laboratory investigations done by the latter in his office are done at the hospital in Britain, most American doctors become quite sympathetic to the problems of the British medical profession.
The close collaboration between University research workers and the drug firms in America appears to have distinct advantages.
It is surprising what osteopaths, chiropractors and others without medical qualifications are permitted to do in the United States.
American medical students and recently qualified hospital doctors are better able to present their patients at meetings than are their British contemporaries.
Hospital records are better maintained in the United States.
In making my comments I also made the point that I had found American medical students to be more dependent on laboratory tests than their British counterparts and somewhat lacking in clinical ability. I had found, for example, that some senior medical students at Ann Arbor could not tell the difference between a case of pneumonia to be seen in the clinic and one which had been diagnosed by the laboratory. This is a serious matter and one which needs major comment in view of the increasing incidence of tuberculosis severity.
This is not the place to discuss the Suez Canal affair, but one that was of considerable importance to me in October 1956. It was the first time that the British Medical Association International Committee had met and I was asked to make a suggestion about the future of the International Committee. The research workers in the United States do this very well. The Department of Health and Social Security need to learn how to do this. The anaemic state of the International Committee remains unchanged. The holiday season is over, the school year is over, the depression is over, the building programme is over, the resumption of work is over, the week before Christmas is over, the liner is afloat, the liner is afloat, the liner again is afloat.
We are now faced with the deterioration of the Suez Canal. President Nasser of Egypt closed the Suez Canal (which is owned by the British) and the British Government froze all Egyptian assets in Britain. The British Government withdrew from Suez and the United States suggested that we should stay out of the area. In the meantime, the British had two days to negotiate and then on the 29th they announced that they would leave the Suez Canal. The British recalled their Ambassador.
Arbor did not know how to treat a sprained ankle; they considered this to be something that Boy Scouts should deal with. I was, however, impressed by their knowledge of scientific method and of the laboratory investigations that might be done in diseased states. So far as major post-war differences were concerned there was no need to comment on the financial problems of the British nation and the severity of our continued rationing.
This was the first of several visits to the United States and the only one that merits detailed mention since it deals with first impressions. In 1956 I was invited to give one of the main papers at the Sixth International Congress of Haematology in Boston and Stanley Davidson suggested that I should take the opportunity of working in a pure research laboratory again. The Carnegie Trust supplied funds for me to do this; for three months I was to work in Dr. Arnold Welch's Department in Yale University at New Haven, Connecticut continuing to learn new techniques applicable to the study of megaloblastic anaemias. This time I arranged for my wife to come with me and to remain for three weeks, the longest time possible because of school holidays; we sailed on the Queen Mary which I had first seen when, as a schoolboy, I had sailed on the Clyde in the 1930s during the days of the depression. Then it was merely a very large ship, Number 534, the building of which had been suspended. However its construction was resumed and the launching took place on September 26th, 1934 in the week before I became a medical student. Then it was the largest ship afloat, destined to be passenger liner, troop ship and then passenger liner again.
We sailed on 16th August 1956 at a time when there was a deteriorating international situation. We had come home through the Suez Canal ten years previously, but on July 26th Colonel Nasser, President of Egypt, had nationalized the Anglo-French controlled Suez Canal Company because Britain and the United States had refused to finance the building of the Aswan High Dam. Two days later Britain froze all Egyptian assets held in the United Kingdom. When we sailed from Southampton there was a concentration of shipping which suggested to me that war was about to break out in the Suez Canal area. In fact an aircraft carrier had already sailed from Portsmouth and two days after we reached New York a five-nation team went to Cairo to negotiate for an international body to control the Canal. By August 29th there was a considerable build up of French and British forces in the Eastern Mediterranean. We were concerned, but I was ready to be recalled for military service if necessary. However we could but
continue with our plans and together we saw something of New York and Washington, DC, then travelled by train to Boston, (with a side trip to my wife's cousin's home in New Rochelle).
The Congress lasted for a week and I was delighted to find that Dr. W. B. Castle and I were on the programme together on Friday, August 31st. The discussion was led by that well known haematologist, Maxwell M. Wintrrobe of Salt Lake City who later wrote a book giving details of blood specialists whom he had met. I was surprised to read in it that I had been born in West Africa when, in fact, I did not visit that part of the world until 1963 and there was no family connection. On the Wednesday a New England clam bake was laid on at Ipswich for the participants; there for the first time in her life my wife developed a severe allergy to shellfish and in later travels this became such a problem that I had to carry a syringe and ampoules of hydrocortisone. The other alteration in habits was that at a party given for delegates by Dr. Dameshek I changed my mind about taking alcohol and ceased to be a teetotaller; nothing in particular occurred to lead to this change and in a spirit of adventure I tried out all the seven beverages that were brought to me — from sherry to liqueurs with everything in between. Surprisingly there were no after effects the following day.
We went on to Halifax, Nova Scotia where we stayed with my friend Bob MacDonald and his wife, Kay. I was surprised to find that I was on the programme to speak at the Nova Scotia Division of the Canadian Medical Association and it was not on a subject that I knew much about. We liked Halifax and the surrounding area with its bays and inlets; we were particularly taken with Peggy's Cove and also admired the view from Citadel Hill. At one time it was the custom for those who wished to be doctors in Nova Scotia to graduate in Edinburgh. Of the fourteen doctors practising in Halifax in 1845, all but one had studied at the Scottish capital and a driving force behind the establishment of a Medical Faculty in Halifax was Dr. Charles Tupper (MD Edin, 1843). He played an important part in the Canadian Union of 1864 and was Prime Minister of Canada in 1896.
Next we visited the Niagara Falls, giving us something with which to compare the Victoria Falls when we visited Rhodesia in 1972, and then went on to Ann Arbor, where it was a pleasure to introduce my wife to members of the staff with whom I had worked; there we stayed at the home of Dr. Bethell who had taken over as Director of the Simpson Memorial Institute. Broad of build and jovial he was able to visit us later at our home but, regrettably, died in 1959 at the age of 56. Back we went to New York where Mary embarked on the Queen Elizabeth while I remained to attend the American Pharmaceutical Association meeting. This was held in the same hotel as the Congress and because of the proximity of the two events I was able to attend both. The Congress was held in the main hall on the ground floor while the Pharmaceutical Association was on the second floor. The Congress was involved in a number of activities and I was able to keep in touch with my wife by telephone. The Pharmaceutical Association with attendance of 10,000 was more formal and the problems of getting around the hall were greater than those outside the Congress. I was informed that the Congress was outside the building and that during the evening the hall was used for large business meetings. In this case I had to go through the hall to the one entrance and I was not so chanced upon by the rowdy hoodlums who were outside. In the evenings the hall was used by members of the American Department of State for hospitality.
In the meantime the Suez crisis was developing. The situation was less simple than the invasion of Hungary in 1956. The troops poured into Egypt and Israel swept in from the Sinai. The United States asked for a ceasefire in Egypt. The President had shown his support for General Eisenhower in 1956 and now more coolly supported Nasser. Egypt threatened to use its widespread nuclear weapons.
while I made my way to Arnold Welch’s Department at Yale. Small and bespectacled he was full of enthusiasm and gave me a comprehensive account of the work going on in his Department of Pharmacology. I had known in advance that University accommodation was available and this was on one of the lower floors of a large block of student rooms. I was told that the female students were all on the top two floors and that this was so that they could repel the male students involved in what were described as ‘panty raids’. I was somewhat out of touch with undergraduate frolics, and spent my time in the laboratories with attendance each week at ‘grand rounds’ where interesting clinical problems were shown. On one occasion when I was attending a lecture the hall was quickly emptied because somebody had run over a skunk outside the entrance. I was warned not to go out alone at night and informed that just before I arrived somebody had been murdered outside the front door of the hospital. I did in fact go out on my own in the evenings but kept my right hand in my jacket pocket grasping a large bunch of keys which could serve as knuckle duster and in any case I had been trained in ju-jitsu by the army for unarmed combat. On the one occasion when I thought I was about to be attacked a policeman chanced to come round the corner at the very moment when two hoodlums appeared to be about to close in on me. However my evenings were certainly not lonely as I was very frequently invited out by members of the University staff and when I left the members of the Department staff gave a farewell dinner at Mory’s, a famous old hostelry, and presented me with a tankard as a token of goodwill.
In the students’ block there was a pleasant sitting room with a large television set and there was considerable media coverage of the Suez crisis. There were, however three major events taking place more or less simultaneously. On October 26th Hungary rose in revolt against the Russians who occupied their country and on November 5th the Hungarian army was crushed by heavily armed Soviet forces; refugees poured across the Austrian border. On October 29th Israeli forces swept into Egypt whereupon Britain and France called on Egypt and Israel to pull their forces back from the Canal zone. Egypt refused and asked for American and Russian support; Britain and France attacked Egypt. There were angry debates at the United Nations, these being shown live on television, and it was very clear that Dulles and Eisenhower were furious about Britain’s actions. There seemed to be more condemnation at the United Nations of what Britain was doing in Egypt than of what Russia was doing in Hungary. There was a widespread belief that Russia would bomb Britain on the day of the
American Presidential election which, on November 6th, was the third major event at this difficult time; one newspaper report suggested that the Chinese would join the Russians in an attack on Britain and I was advised to consider sending for my family to come to the United States immediately. In an encyclopaedia I read up the history of the Panama Canal; on the very few occasions when anyone raised the subject of our actions, which I understood were to protect our interests in the Suez Canal, I asked ‘What about the Panama Canal?’ The answer was always the same — ‘That’s different. It’s necessary for America’s interests.’ However it was mainly American pressure that led the British and French to accept the United Nations call for a cease fire on November 8th; this was of interest to me when the United States invaded Panama at the end of 1989. I was in the sitting room with a group of American students in 1956 when Secretary of State Foster Dulles announced on television that he had just averted World War IV. A voice from the back row of students called out ‘Silly old bastard. He can’t count up to three!’ We had a lot of support amongst the people of America, many of whom thought that we had ceased our action only because of the fear of intervention by Russia. Soon afterwards I was visiting the Walter Reed Hospital in Washington and saw Foster Dulles who seemed to be having the preliminary investigations which showed the malignancy that caused his death in May, 1959.
During October there was much activity leading up to the Presidential election. It seemed unlikely that Adlai Stevenson, the Democratic candidate, would be able to prevent Eisenhower having a second term of office and the message that was being vigorously put out by the Democrats was ‘A vote for Eisenhower is a vote for Nixon.’ Nixon was standing as the Republican Vice-presidential candidate while Eisenhower had had a coronary thrombosis in September, 1955 and was on treatment with anticoagulants. Richard Nixon visited Yale one dark October evening and I saw him being pelted with toilet rolls by a group of students.
I sailed for home on November 21st just after Sir Anthony Eden, our Prime Minister, had flown to Jamaica because of ill health, diagnosed at the time as ‘severe overstrain’. His political career was finished but by April 1957 he was being treated in Boston, not for exhaustion, but for disease of his liver and biliary tract.
Nothing need be said concerning short trips that were made to the United States in 1974 and 1975 for scientific meetings or, at this stage, about later more official visits either as Dean of the Faculty of Medicine or as President of the Royal College of Physicians of Edinburgh. I was invited to the Commonwealth Scientific and Industrial Research Laboratories in Puerto Rico where the tropical scenery was very glad to accept the invitation. There we found that it was cheaper to undercarve the car than to use up fuel for air-conditioning controls. The buildings were abandoned and the connection with the outside world over the telephone was poor and is of course in marked contrast to the modern tin shack in which I accommodated my houses in the West Indies away from home for long hours during which I photographed and took rounds of golf. During this I had been absent from absence from home and travelled to the United States on the occasion of a meeting at which I was to take part.
Edinburgh. All the journeys now were by air. In 1962 I was invited by the Commanding Officer of the U.S. Army Tropical Research Medical Laboratory in New York to speak at a meeting in San Juan, Puerto Rico which was to be held from March 20-22nd, the subject being tropical sprue, the condition which I had been studying in India. I gladly accepted and set off on a BOAC plane on Sunday 18th March. There were very few passengers on board and when we reached Boston it was clear that there was a problem. The difficulty was that the undercarriage had jammed and we circled Boston for about an hour to use up fuel, the captain meantime struggling with his undercarriage controls. Eventually he managed to land but that plane had to be abandoned and we flew to New York in another; I had missed my connection but celebrated my 45th birthday in a Pan American plane over the Caribbean. Puerto Rico is a splendid tropical island to visit and is described as part of the American Commonwealth but the contrast between luxury hotels with private beaches for the visitors and tin shacks for the native inhabitants is great; we were all in Army accommodation. The Spanish influence is still obvious in churches and houses but once again I was advised not to wander about on my own away from the area of the main hotels; I disregarded this in the few hours of free time that we had as I wandered around taking photographs, but by the Saturday I was home again. In fact I did ward rounds on two consecutive Saturdays without my patients knowing that I had been away except that I usually did a daily ward round so my absence may have been noticed; what they did not know was that I had travelled 14,000 miles. I attended two further similar short conferences on the same subject in Puerto Rico in 1968 and 1973. On the second occasion I took a cine camera and was astonished when a water skier whom I was filming stood on his head on the skis and continued on his way without falling off. ‘He doesn’t often do that’ said a local.
In the early leading up to the election of Adlai Stevenson, the incumbent Eisenhower having a chance of being vigorously put out of office, ‘A war is a vote for Nixon.’ The vice-presidential candidate was in London in September, 1955 when Richard Nixon visited Yale University; pelted with toilet rolls and eggs.
Sir Anthony Eden, our Prime Minister of ill health, diagnosed as suffering from cancer, his career was finished but not for exhaustion, but for illness.
The invitations that were made to the meetings or, at this stage, the Dean of the Faculty of Medicine of the College of Physicians of
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This is Exhibit 5 referred to in the affidavit of Samuel Berg sworn before me, this 18th day of February, 2015.
A witness, after taking affidavits
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Rational and Constitutional Approaches to Airline Safety in the Face of Terrorist Threats
Jamie L. Rhee
Follow this and additional works at: https://via.library.depaul.edu/law-review
Recommended Citation
Jamie L. Rhee, Rational and Constitutional Approaches to Airline Safety in the Face of Terrorist Threats, 49 DePaul L. Rev. 847 (2000)
Available at: https://via.library.depaul.edu/law-review/vol49/iss3/7
This Comments is brought to you for free and open access by the College of Law at Digital Commons@DePaul. It has been accepted for inclusion in DePaul Law Review by an authorized editor of Digital Commons@DePaul. For more information, please contact firstname.lastname@example.org.
RATIONAL AND CONSTITUTIONAL APPROACHES TO AIRLINE SAFETY IN THE FACE OF TERRORIST THREATS
INTRODUCTION
"They are all targets... every day... they will receive a new corpse...."
—Osama bin Laden, multimillionaire terrorist, issuing a threat to Americans.
The modern threat of terrorism, coupled with recent attacks on United States embassies, has led to levels of federal intrusion into the airline industry not seen since before airline deregulation. In response to the explosion of TWA Flight 800 in 1996, President Bill Clinton summoned a commission under the leadership of Vice President Al Gore to assess the current problems with safety at United States airports and on United States aircraft, and to recommend solutions to these problems. This Comment will demonstrate that the policy approach of the Gore Commission was fundamentally flawed and that the solutions recommended were both inadequate to provide real safety, and threaten Fourth Amendment privacy rights, Fifth Amend-
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1. Hous. Chron., Nov. 8, 1998, at 4 (quoting bin Laden interview with ABC News, May 1998). Bin Laden also said that "[w]e do not differentiate between those dressed in military uniforms and civilians." Id. The continuing seriousness of this terrorist threat is emphasized by the United States State Department's pre-millenium warnings advising Americans to avoid large groups—especially overseas. Public Announcement (visited Dec. 21, 1999) <http://travel.state.gov/www_terrorist.html>. The United States Customs Service placed "an extra 300 guards on duty" and altered their border posts following two arrests of Algerians who allegedly attempted to smuggle explosives into the United States through Canada. John Donnelly, U.S. Scrambles to Unravel Terrorist Threat, Boston Globe, Dec. 22, 1999, at A1, available at 1999 WL 30401514.
2. Forty percent of worldwide terrorist incidents were directed toward Americans in 1992. See John Rogers, Bombs, Borders, and Boarding: Combating International Terrorism at United States Airports and the Fourth Amendment, 20 Suffolk Transnat'l L. Rev. 501, 502 (1997).
3. While the aviation industry has been deregulated for more than 20 years, many airline officials, as well as others in the industry, fear that current federal intervention amounts to nothing more than re-regulation. For a deeper analysis of this subject, see Brian F. Havel, In Search of Open Skies: Law and Policy for a New Era in International Aviation 222-29 (1997).
4. See Exec. Order No. 13,015, 61 Fed. Reg. 43937 (1996). For the findings of this commission, see generally White House Commission on Aviation Safety and Security, Final Report to President Clinton (Feb. 12, 1997) [hereinafter Final Report].
ment travel rights, and Fourteenth Amendment Equal Protection rights of airline passengers under the United States Constitution.
The Gore Commission appears to have accepted, without examination, the commonplace notion that an increased threat to safety necessarily creates a clash between our constitutional liberties and our need for personal safety.\(^5\) The position taken in this Comment is that this clash is illusory. The fundamental error of the Gore Commission was the paternalistic notion that government can and should make airline passengers safe.\(^6\) Both the Constitution and practical realities demand that the government help airline passengers keep themselves safe, just as the government works to help drivers keep themselves safe on the highways. Paternalistic approaches to airline safety are both foolish and un-American.
It is both unwise for individual members of the public to ignore responsibility for their own safety, and unconstitutional for the government to implement security plans presuming that the public will do so. Benjamin Franklin wrote, “[t]hey that can give up essential liberty to obtain a little temporary safety deserve neither liberty nor safety.”\(^7\) Experience shows that such cravens in fact generally experience what Franklin observed.\(^8\) The danger today is too great to tolerate a cowardly and irrational response. We cannot afford to lose either safety or liberty.\(^9\) The danger in the skies is real and serious, but it does not
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5. The assumption that there is a necessary clash between an individual’s constitutional rights and safety seems to be accepted both by the political left and right. Justice Thurgood Marshall criticized the political right when he wrote: “[h]istory teaches that grave threats to liberty often come in times of urgency, when constitutional rights seem too extravagant to endure.” Skinner v. Railway Labor Executives’ Ass’n, 489 U.S. 602, 635 (1989) (Marshall, J., dissenting). Judge Friendly, generally regarded as politically liberal, wrote: “[w]hen the risk is the jeopardy to hundreds of human lives and millions of dollars of property inherent in the pirating or blowing up of a large airplane, the danger alone meets the test of reasonableness” for a search. United States v. Bell, 464 F.2d 667, 675 (2d Cir. 1972) (Friendly, J., concurring).
6. The Gore Commission well illustrates the wisdom of Justice Brandeis, who wrote:
[e]xperience should teach us to be most on our guard to protect liberty when the Government’s purposes are beneficent. Men born to freedom are naturally alert to repel invasion of their liberty by evil-minded rulers. The greatest dangers to liberty lurk in insidious encroachment by men of zeal, well-meaning but without understanding.
Olmstead v. United States, 277 U.S. 438, 479 (1928) (Brandeis, J., dissenting).
7. Benjamin Franklin, Historical Review of Pennsylvania, in Bartlett’s Familiar Quotations 348 (Emily Morison Beck ed., 1980).
8. It was not in spite of our liberty, but because of it, that Americans in the 1930s and 1940s were far safer than were most people, such as the Germans. Heavy-handed Nazi security may have provoked, but could not prevent, the Hindenburg disaster. See infra notes 13-24 and accompanying text. Over the last two centuries, the prosperity and happiness enjoyed by Americans has been based, in large part, on the safety that we enjoy. The Constitution works now, and has consistently worked amazingly well, to assure our safety.
9. See generally Lawrence Lessig, Fidelity in Translation, 71 Tex. L. Rev. 1165 (1993) (arguing that the Constitution is antiquated and too inflexible to deal with modern dilemmas). However,
force us to choose between bomb-toting terrorists and jack-booted security officers.
This Comment will begin in Part I with a summary of the history of violent threats to airline safety and the responses by industry and government to these threats, and the Constitutional rights associated with travel.\textsuperscript{10} Part II will present a theoretical structure for effective and constitutionally sound government involvement in airline safety and will make specific alternative recommendations for steps needed to prevent terrorist attacks on air travel.\textsuperscript{11} In conclusion, Part III of this Comment will re-emphasize that an individual, not a bureaucracy, can ensure airline safety.\textsuperscript{12}
I. Background
A. Factual Background: The History of Terrorism
1. The Hindenburg
The first regular international air transit services were provided by Lufthansa’s dirigibles (“Zeppelins”) in the 1920s between Frankfurt and both New York and Rio de Janiero.\textsuperscript{13} This service came to a violent end on May 6, 1937, in Lakehurst, New Jersey, with the explosion
\textsuperscript{10} See infra notes 13-141 and accompanying text.
\textsuperscript{11} See infra notes 142-256 and accompanying text.
\textsuperscript{12} See infra note 257 and accompanying text.
\textsuperscript{13} See Michael M. Mooney, \textit{The Hindenburg} 54-56, 59, 63 (1972). Mooney’s account is based on interviews with survivors or examination of their diaries, and of voluminous official United States and German records of the investigations after the explosion and many other sources. See id. at 269-73. Many dirigible experts today reject Mooney’s account as sensationalized, but his was the only independent study done on the disaster when interviews with witnesses were still possible. See E-mail from John Dziadeck (Sept. 20, 1999) (on file with author) (maintaining a group of web-sites commemorating dirigibles).
of the world’s largest airship, the Hindenburg, on its maiden voyage.\textsuperscript{14} A private investigation found that a bomb placed by one of the German crewman exploded,\textsuperscript{15} setting the airship on fire and killing thirteen of the thirty-six passengers and twenty-two of the sixty-one crew members, including the bomber himself.\textsuperscript{16} The disaster is still shrouded in mystery, partly because both the United States and German governments avoided public disclosure of the explosion’s true cause due to the embarrassment of having failed to prevent terrorism.\textsuperscript{17} The bomber\textsuperscript{18} succeeded in destroying the airship even though German security was warned of a threat to bomb the Hindenburg when it arrived in Lakehurst\textsuperscript{19} and three German S.S. Officers were on board to ensure security.\textsuperscript{20} Security efforts failed because the S.S. Officers, who were not competent to find the bomb themselves,\textsuperscript{21} alienated the dirigible’s crew,\textsuperscript{22} who were unwilling to believe that one of their own would bomb the ship.\textsuperscript{23} German security efforts were also misdirected because of suspicion of involvement by passengers who were Jewish or had criticized the Nazi party.\textsuperscript{24} As will be discussed in this Comment, the response of the Gore Commission is hauntingly similar to that taken by the United States and Nazi Germany more than seventy years ago.
\textsuperscript{14} The Hindenburg explosion actually came seven years after the first recorded airplane hijacking, which was in 1930 in Peru. \textit{See} Sanford L. Dow, Comment, \textit{Airport Security, Terrorism, and the Fourth Amendment: A Look Back and a Step Forward}, 58 J. Air L. & Com. 1149, 1158 (1993). Revolutionaries hijacked a plane and used it to distribute leaflets. \textit{See id.}
\textsuperscript{15} \textit{See} Mooney, \textit{supra} note 13, at 216-18.
\textsuperscript{16} \textit{See id.} at 258-59.
\textsuperscript{17} The Secretaries of Commerce and Interior both wrote to the American investigator that “‘a finding of sabotage might be cause for an international incident . . . .’” \textit{Id.} at 271. Nonetheless, “almost every dirigible man (including Captain Pruss)” who testified in the investigation “in private agreed that sabotage was the cause.” \textit{Id.} During the investigation, “the German advisors and the American commissioners and advisors held meetings at night to discuss, off-the-record,” how to minimize the “inescapable evidence of sabotage . . . .” \textit{Id.} An American commissioner’s diary, now in the National Archives, expressed that the recollections of the German and American advisors were similar. \textit{See id.} at 271-72. A clearer case both for the bombing and for the governmental attempts at a cover-up could hardly be made, despite continued official insistence that the actual cause of the explosion is a mystery. \textit{See id.} at 271.
\textsuperscript{18} It appears that the bomber, Eric Spehl, was provoked by the torture of a friend by the Gestapo. \textit{See id.} at 217-18.
\textsuperscript{19} \textit{See id.} at 115-18.
\textsuperscript{20} \textit{See id.} at 110-16.
\textsuperscript{21} \textit{See} Mooney, \textit{supra} note 13, at 258-59.
\textsuperscript{22} \textit{See id.} at 152-56.
\textsuperscript{23} \textit{See id.} at 11-12.
\textsuperscript{24} \textit{See id.} at 123-26.
2. Early Airplane Hijackings
Between 1949 and 1985, there were 498 successful and 281 failed hijacking attempts worldwide\textsuperscript{25} and 1539 persons killed in eighty-seven aircraft bombings.\textsuperscript{26} Between 1948 and 1960 there were twenty-nine successful hijackings around the world.\textsuperscript{27} Between 1961 and 1967 there were sixteen hijackings.\textsuperscript{28} But the year 1960 alone saw a record thirty hijackings, seventeen of which were of United States-registered aircraft.\textsuperscript{29} In 1963, the international aviation community responded to this wave of hijackings with the Tokyo Convention,\textsuperscript{30} which outlawed dangerous acts on aircraft,\textsuperscript{31} and created the International Civil Aviation Organization ("ICAO").\textsuperscript{32} The Tokyo Convention preserved national criminal jurisdiction over such acts and gave airline commanders quasi-police authority.\textsuperscript{33} The United States has been an active signatory of the Tokyo Convention.\textsuperscript{34}
3. Establishment of the DOT and FAA
In 1967, the Department of Transportation ("DOT") was established as the "focal point in the Federal Government for the coordinated national Transportation Policy."\textsuperscript{35} Additionally, the DOT was to oversee "transportation safety improvements and enforcement."\textsuperscript{36}
\begin{itemize}
\item[25.] See Michael S. Simons, A Review of Issues Concerned With Aerial Hijacking and Terrorism: Implications for Australia's Security and the Sydney 2000 Olympics, 63 J. Air L. & Com. 731, 738 (1998).
\item[26.] See id.
\item[27.] See Paul Stephen Dempsey, Aerial Piracy and Terrorism: Unilateral and Multilateral Responses to Aircraft Hijacking, 2 Conn. J. Int'l. L. 427, 429 (1987).
\item[28.] See id.
\item[29.] See id.
\item[30.] See Convention on Offenses and Other Acts Committed on Board Aircraft, Sept. 14, 1963, U.S.T. 2941, 704 U.N.T.S. 219 (entered into force Dec. 4, 1969) [hereinafter Tokyo Convention].
\item[31.] See Haro F. Van Panhuys, Aircraft Hijacking and International Law, 9 Colum. J. Trans-Nat'l. L. 1, 1-2 (1970).
\item[32.] See Simons, supra note 25, at 740-41 n.43 (noting that the ICAO grew from 49 signatories in its first year to 86 by 1969).
\item[33.] See Tokyo Convention, supra note 30, at arts. 3, 6.
\item[34.] Earlier conventions chiefly gave international approval to actions already being taken by signatories. See generally Dionigi (Dan) M. Fiorita, Aviation Security: International Response, 3 Air. L.J. Sci. & Tech. 267 (1993) (describing the Tokyo Convention and other conventions dealing with aviation security). Later conventions strengthened sanctions against nations that did not take strong measures against terrorists. See id.; see also Simons, supra note 25, at 739-50; Heather E. Reser, Comment, Airline Terrorism: The Effect of Tightened Security on the Right to Travel, 63 J. Air L. & Com. 819, 823-28 (1998). The United States is also a signatory of the conventions that have continued the work of Tokyo Convention. See Fiorita, supra at 285-93.
\item[35.] About the Department of Transportation (visited Sept. 28, 1998) <http://www.dot.gov/general/aboutdot.html>.
\item[36.] Id.
\end{itemize}
Other areas currently under DOT’s control are international transportation agreements and the continuity of transportation services in the public interest.\textsuperscript{37}
The Federal Aviation Administration (“FAA”), an operating administration of the DOT, is responsible for “promoting safe air travel and enforcing security measures affecting aircraft and air terminals.”\textsuperscript{38} Traditionally, the FAA has placed emphasis on promoting air travel, but recently there has been a “paradigm shift” to focus on safety, and especially counterterrorism.\textsuperscript{39} Along with the independent National Transportation Safety Board (“NTSB”), the FAA conducts safety studies and investigations, usually after serious accidents.\textsuperscript{40} While the ultimate control over policies and regulations lies with the United States government, airlines, airport owners and operators play an integral part in the aviation industry. Moreover, the airlines are typically charged with funding security.\textsuperscript{41}
4. \textit{Early United States Responses to Terrorism in the Air}
In the late 1960s, the United States government reacted to the increasing number of terrorist attacks by creating a special FAA task force to implement strategies for detecting and deterring possible terrorists.\textsuperscript{42} The initial procedures called for heightened security measures against certain individuals by using profiling to try to identify terrorists.\textsuperscript{43} A “profile” is a list of personal attributes that, though when pinpointed individually may be legal and non-threatening, cumulatively suggests a person is statistically more likely to hijack an airplane.\textsuperscript{44} Unfortunately, despite implementation of this profiling system, the number of attacks continued to increase.\textsuperscript{45}
Congress responded to the increasing threat of hijacking with a statutory scheme that included the Anti-Hijacking Act of 1974\textsuperscript{46} and the
\begin{itemize}
\item[37.] See id.
\item[38.] Reser, \textit{supra} note 34, at 829 n.54 (citing 49 U.S.C. § 472(i)-(m), (o) (1994)).
\item[39.] See Anthony Fainberg, \textit{Aviation Security in the United States: Current and Future Trends}, 25 \textit{Transp. L.J.} 195, 196 (1998).
\item[40.] See Rudolf Kapustin, \textit{How to Prevent Major Accidents Effectively}, 10 \textit{Air & Space Law} 1, 12 (1996).
\item[41.] See Fainberg, \textit{supra} note 39, at 197; see also Diane Westwood Wilson, \textit{The Federal Aviation Reauthorization Act of 1996}, 11 \textit{Air & Space Law}. 1, 10 (1997).
\item[42.] See Rogers, \textit{supra} note 2, at 506.
\item[43.] See id.
\item[44.] See Jin Tai Choi, \textit{Aviation Terrorism: Regional Variations and Responses} 30 (1994) (discussing the passenger profiling system).
\item[45.] See id. at 24 (noting that between 1968 and 1973 there were 125 hijackings of American aircraft).
\item[46.] See H.R. Rep. No. 93-885, at 9 (1974).
\end{itemize}
Air Transportation Security Act of 1974.\textsuperscript{47} The Anti-Hijacking Act made it illegal to carry a concealed weapon aboard an aircraft.\textsuperscript{48} The Air Transportation Security Act called for a uniform mandate that all carry-on luggage undergo screening procedures.\textsuperscript{49} Instead of searching a handful of passengers targeted for suspicion, the new system would search everybody. These measures seem to have been effective because air terrorism in the United States almost came to a halt. In 1972, the last year in which the United States only used profiling to ensure safety, twenty-eight American airliners were hijacked.\textsuperscript{50} Between 1975 and 1986 only two such aircraft were so threatened, and only one of those, a TWA flight near Athens in 1986, originated in the United States.\textsuperscript{51} The other was Pan Am Flight 103, which exploded over Lockerbie, Scotland in 1988.\textsuperscript{52} The bomb that destroyed Pan Am Flight 103 seems to have been put on board the plane during a stopover in Frankfurt, Germany.\textsuperscript{53} Thus, it appears that not a single bomb has evaded airline security on American aircraft at United States airports since all airports began screening passengers.\textsuperscript{54}
\textit{5. Reaction to Pan Am Flight 103}
On December 21, 1988, somebody in Frankfurt, Germany loaded a portable radio packed with explosives into his checked baggage on Pan Am Flight 103.\textsuperscript{55} He did not board the plane himself.\textsuperscript{56} The plane exploded a few hours later over Lockerbie, Scotland, killing all 259 people on board and eleven residents of Lockerbie.\textsuperscript{57} As a result
\begin{itemize}
\item \textsuperscript{47} See 49 U.S.C. §§ 1356-1358 (1988) (repealed 1994).
\item \textsuperscript{48} H.R. REP. NO. 93-885, at 1-5.
\item \textsuperscript{49} 49 U.S.C. §§ 1356-1358. See also Shirlyce Manning, Comment, \textit{The United States' Response to International Safety}, 61 J. AIR L. & COM. 505, 510 (1996).
\item \textsuperscript{50} See ACLU Freedom Network, \textit{Profiling Endangers Privacy} (visited Sept. 4, 1998 <http://www.aclu.org/news/w100996b.html>) [hereinafter Profiling Endangers].
\item \textsuperscript{51} See Gregory T. Nojeim, \textit{Aviation Security Profiling and Passengers' Civil Liberties}, 13 AIR & SPACE LAW. 3, 6 (1998).
\item \textsuperscript{52} See \textit{In re Air Disaster at Lockerbie Scotland on Dec. 21, 1988}, 37 F.3d 804, 810 (2d Cir. 1994).
\item \textsuperscript{53} See id. at 811.
\item \textsuperscript{54} The moral of this experience is clear—low-level screening of everybody is effective. However, targeting a few people for intense heightened scrutiny is pointless. Those who are determined enough to construct an effective bomb are also almost inevitably also serious enough to avoid behavior likely to single themselves out for special scrutiny.
\item \textsuperscript{55} See President's Commission on Aviation Security and Terrorism, Report to the President 13 [hereinafter Report to the President] (discussing the bombing of Pan Am Flight 103); see also Rogers, supra note 2, at 509.
\item \textsuperscript{56} See Report to the President, supra note 55, at 13-14.
\item \textsuperscript{57} See id. at 14; see also Garret Hodes, \textit{Terrorist Threats: The Friendly Skies Aren't Too Friendly About Notification}, 46 U. KAN. L. REV. 365, 366 (1998); Steven Mirmina, \textit{Aviation Safety And Security—Legal Developments}, 63 J. AIR L. & COM. 547, 547-48 (1998).
\end{itemize}
of this incident, President George Bush created the President's Commission on Aviation Security and Terrorism.\textsuperscript{58} In response to the findings of this commission, Congress implemented the Aviation Security Improvement Act of 1990,\textsuperscript{59} requiring the FAA Administrator to counteract terrorism by adopting new security technology.\textsuperscript{60} United States air carriers instituted a strict bag matching policy to remove the baggage of any passenger who failed to actually board the flight. Currently, however, the policy applies only to international flights.\textsuperscript{61}
While an important step towards thwarting the efforts of the terrorist using "drop and run" tactics,\textsuperscript{62} the positive bag identification system provides no assistance in preventing those determined to give up their own lives for their causes.\textsuperscript{63} Bag matching also provides no protection against those who place explosives in bags belonging to other innocent passengers.\textsuperscript{64}
\section*{6. TWA Flight 800 and the Gore Commission}
The debate over aircraft and airport security became most intense in the aftermath of the TWA Flight 800 disaster. The mid-air explosion over Long Island, New York that killed everyone on board was initially blamed on terrorists.\textsuperscript{65} On July 17, 1996, a major change occurred in the aviation industry because the disaster forged a consensus regarding the need for drastic measures to stop terrorist activity.\textsuperscript{66} President Clinton promptly established the White House Commission on Aviation Safety and Security on August 22, 1996, led by Vice President Gore.\textsuperscript{67} The Commission's focus was to assess and recommend security measures to airlines and airports.\textsuperscript{68} The Commission's work reflected its "vision for the future," namely, "[t]o ensure greater safety and security for passengers, to restructure the relationships between government and industry into partnerships for progress, and to main-
\begin{itemize}
\item[58.] See Exec. Order No. 12,686, 54 Fed. Reg. 32,629 (1989).
\item[59.] Pub. L. No. 101-604, 104 Stat. 3066 (1990) (codified as amended in scattered sections of 49 U.S.C. and 22 U.S.C.).
\item[60.] See id.
\item[61.] See Robert W. Hahn, The Economics of Airline Safety and Security: An Analysis of the White House Commission's Recommendations, 20 Harv. J.L. & Pub. Pol'y 791, 799 (1997).
\item[62.] A "drop and run" occurs when a terrorist checks luggage onto an airplane, but the terrorist does not board the plane. Id. at 793.
\item[63.] See id. at 798-99.
\item[64.] See id. at 798.
\item[65.] See Fainberg, supra note 39, at 195-96.
\item[66.] See id.
\item[67.] See Exec. Order No. 13,015.
\item[68.] See Final Report, supra note 4, at 3.
\end{itemize}
tain global leadership in the aviation industry."69 Some of the recommendations focused on new technology to be utilized for surveillance and screening.70 Recommendations also included training additional bomb sniffing dogs, creating an automated passenger profiling system, and dramatically increasing the FBI's workforce on counterintelligence.71
President Clinton hastily signed the proposal into law with a total budget of $1.097 billion, of which the Commission was allocated $429.4 million.72 The Commission's initial report was issued just forty-
69. Id. at 24. Notably lacking from this vision is the active and informed participation of the general public and of passengers and crew. See id.
70. See id. at 31-47.
71. See id.
72. See The President's Counterterrorism Proposal (visited Jan. 29, 2000) <http://www.epic.org/privacy-terrorism/WH_fact_sheet_10_96.html> [hereinafter President's Proposal]. The proposal allocated (dollars in millions):
- Screen Checked Baggage, $91.1
- Screen Carry-on Baggage, $37.8
- Canine Teams, $8.9
- Augment FAA Security Research, $20
- Security Workforce, $18
- Vulnerability Assessments, $5.5
- Passenger Profiling, $10
- Screener Training, $5.3
- Screen Passengers (Portals) and Document Scanners, $1
- Anti-terrorism Assistance to Foreign Governments, $2
- Deploying Existing Technology to Inspect International Air Cargo, $31.4
- Provide Additional Air/Counterterrorism Security, $26.6
- Taggants Study, $21.3
- Explosives Detection Training, $1.8
- Capacity to Collect and Assemble Explosives Data, $2.1
- Improve Domestic Intelligence, $38.9
- Improve Forensic/Crisis Management Capabilities, $16
- Increased Staffing, $91.7
- Persian Gulf Force Protection, $122.6
- Overseas Physical Security Upgrades, $138.5
- Upgrade Overseas Security for the International Trade Administration, $9.4
- Enhance Security of Infectious Disease Laboratories, $23
- Department of Interior Facility Security and Training, $15.9
- Courts of Appeals, District Courts, and Other Judicial Services, $10
- FBI: Building Security, $7
- United States Attorneys, $15.6
- Drug Enforcement Administration, $7
- Diplomatic Security, $23.7
- ATF: Critical Incident Response Teams for Post Blast Deployment, $7.2
five days after the Commission was formed.\textsuperscript{73} However, the Commission allocation of dollars was misguided because much of the information needed to effectively counter terrorism simply does not exist.\textsuperscript{74} The profound lack of hard data that the Commission had to contend
\begin{itemize}
\item ATF: Additional Security for Federal Facilities, $6.7
\item United States Secret Service (“USSS”): Additional Security Equipment, $1.1
\item USSS: Equipment Replacement Related to Airplane Crash, $1.4
\item Customs Service: Explosives and Radiation Detection Equipment, $2.2
\item Overseas Building Security, $0.6
\item Personnel and Protective Measures, $3.3
\item Firefighter/Emergency Services Financial Assistance, $2.7
\item Headquarters Building Security, $0.2
\item Public Building and Museum Security, $7.3
\item Facility Security, $2.5
\item Other Treasury Department: Building Security, $14.7
\item Expanding the Bureau of Export Administration’s Efforts to Detect Illegal Exports, $3.9
\item Improve Technology to Prevent Nuclear Smuggling, $8
\item FBI: Critical Incident Response Facility, $2
\item Immigration and Naturalization Service (“INS”), $15
\item Other Justice Department Activities, $14.2
\item Counterterrorism Fund, $35
\item ATF: Inspecting Explosives Licensees and Permittees, $1.8
\item Federal Law Enforcement Training Center, $4.1
\item Salaries and Expenses, $6
\item Emergency Fund, $1
\item Enhance Nest Nuclear Counterterrorism Program, $15
\item Research and Special Projects Administration (“RSPA”), $2.5
\item RSPA: Advisory Committee on Surface Transportation Security, $0.5
\item ATF: Expand Canine Training and Certification Program, $7.5
\item ATF: Car Bomb Studies, $3
\item ATF: Emergency Contingency Funding, $15
\item ATF: Explosives Intelligence and Support Systems, $14.2
\item Departmental Offices: Office of Foreign Assets Control: Seize Foreign Assets of Terrorists and Terrorist-Sponsoring Organization, $0.3
\item Office of Emergency Preparedness, $5.8
\item Training, Awareness, and Information Programs, $93.1
\item Consequence Management Planning and Coordination, $9.3
\item Federal/State/Local Assessment, Training, and Exercises, $8.4
\item National Foreign Intelligence
\item Creation of a National-Level Foreign Terrorism Warning Group Within the Counterterrorism Center
\end{itemize}
\textit{Id.}
\textsuperscript{73} See Hahn, supra note 61, at 792.
\textsuperscript{74} See Nojeim, supra note 51, at 6.
with is illustrated by the fact that the TWA Flight 800 explosion, which had provoked the Commission's formation, was not actually the result of terrorism at all. Closer investigation showed that defects in the design of the central fuel tank appeared to be the probable cause of the TWA Flight 800 tragedy.\textsuperscript{75}
The Commission had invited the participation of the American Civil Liberties Union,\textsuperscript{76} airport safety representatives,\textsuperscript{77} and other aviation officials.\textsuperscript{78} President Clinton proclaimed that as a result of the new measures, "[n]ot only will the American people feel safer, they will be safer."\textsuperscript{79} The President announced that he endorsed "zero tolerance" for airline terrorism.\textsuperscript{80} However, as will be argued later in this Comment, the President's "zero tolerance" policy extends to a countenance of massive invasions of individual privacy and to huge monetary expenditures, but not to measures which would disrupt foreign policy or risk war.\textsuperscript{81}
a. High-Technology Security Equipment
The FAA Act of 1996 authorized the purchase of fifty-four CTX-5000 luggage scanners.\textsuperscript{82} These devices cost more than a million dollars each to be installed\textsuperscript{83} and are able to screen only 120 bags per hour.\textsuperscript{84} The proposed plan is to use these luggage scanners in tandem.\textsuperscript{85} However, even if two scanners are used, a rate of 240 bags per hour (under 3000 per twelve-hour daytime shift) is inadequate for the number of passengers that pass through any major airport every day. For example, in 1997, over 70 million passengers passed through Chi-
\textsuperscript{75} See Michael Grunwald, \textit{FBI Sought to Suppress Report on TWA Crash}, Wash. Post, May 9, 1999, at A01, available at 1999 WL 17002020.
\textsuperscript{76} See ACLU Freedom Network, \textit{Individual Rights Have 'Low Profile' at Aviation Security Conference} (visited Jan. 15, 1997) <http://www.aclu.org/news/n122597a.html>.
\textsuperscript{77} Interview with Richard Kunicki, Deputy Commissioner of Airport Security at Chicago O'Hare International Airport (Dec. 11, 1998).
\textsuperscript{78} A commentator has noted that "[t]he White House has neither given a clear indication of the effectiveness of these measures in preventing terrorist acts nor acknowledged the true cost of implementation," including flight delays, constitutional violations, and establishing a precedent for using taxpayer money to pay for airline security rather than requiring the airlines (who pass the cost along to passengers) to bear this responsibility. Hahn, \textit{supra} note 61, at 793.
\textsuperscript{79} The White House Office of the Press Secretary, \textit{Remarks By The President During White House Commission on Aviation Safety Announcement: The Oval Office} (visited Sept. 8, 1998) <http://www.nitea.or.kr/www.whitehouse.gov/WH/New/html/aviation.html>.
\textsuperscript{80} \textit{See id.}
\textsuperscript{81} \textit{See infra} notes 191-202 and accompanying text.
\textsuperscript{82} \textit{See} Nojeim, \textit{supra} note 51, at 5.
\textsuperscript{83} \textit{See} Hahn, \textit{supra} note 61, at 797.
\textsuperscript{84} \textit{See} Nojeim, \textit{supra} note 51, at 5.
\textsuperscript{85} \textit{See id.}
cago O’Hare International Airport, which translates into an average of 192,000 passengers each day. If each passenger averaged one or two checked bags, a pair of CTX-5000 scanners could screen fewer than one bag in a hundred. InVision, the company that manufactures the CTX-5000, has plans to produce faster models, but even if the machines were ten times as fast, a pair of machines would still be overwhelmed by the traffic at an airport such as O’Hare. Moreover, the estimated cost to supply seventy-five of the United States’ busiest airports is $2.2 billion. Critics argue that CTX-5000 scanners have the potential to cause false alarms and to scan a lower number of bags than the FAA’s intended amount.
Technology beyond the use of X-ray vision, such as machines that detect trace particles of explosives are currently being developed. With the introduction of the CTX-5000s into airports, the marketplace has experienced an increase in the number of companies interested in design, production, and manufacture of new technology. For example, BodySearch, developed by American Science and Engineering, and largely funded by the FAA, is an X-ray device capable of seeing through a person’s clothing. One commentator aptly analogized the BodySearch to Superman’s X-ray vision. It is conceivable that such devices would be highly effective in preventing contraband from boarding the aircraft. The Commission did call for the purchase of “upgraded X-rays, and other innovative systems.” Since November of 1999, United States Customs has introduced the use of BodySearch
86. See Chicago Department of Aviation, The Chicago Airport System: Annual Report 6-7 (1997).
87. See Fainberg, supra note 39, at 197.
88. See Hahn, supra note 61, at 798.
89. See id.; Fainberg, supra note 39, at 198-99.
90. See Fainberg, supra note 39, at 197. “[A]t least two other corporations, L3 Communications and Vivid Technologies, are seriously engaged in developing certifiable explosives detection systems.” Id.
91. See Robyn E. Blumner, Welcome to the Unfriendly Skies (visited Sept. 8, 1998) <http://www.aclufl.org/r-sky.htm>.
92. See id.
93. See id. Whether this type of search could withstand constitutional challenge remains to be seen. Current plans do not call for the purchase of such devices in the United States for use on passengers. See id.
94. However, improved technology on the governments’ part only encourages the same on the part of terrorist groups, and they have kept up thus far. Therefore, it leads one to wonder whether such provisions are merely means by which the government is trying to soothe Americans citizens’ fears, instead of being a . . . solution.
Reser, supra note 34, at 832.
95. Blumner, supra note 91.
for selected international passengers at many major American airports.\textsuperscript{96}
b. Computer Assisted Passenger Screening
The Gore Commission also called for the creation of a national database on passenger travel habits and history entitled the Computer Assisted Profiling System ("CAPS"), for which funding to begin work on this database has already been approved.\textsuperscript{97} A test program has been under development at Northwest Airlines for several years.\textsuperscript{98} The FAA, commenting on CAPS, revealed that "[s]oon, if not already, airline agents who enter a passenger's name at check-in will get either a red light or a green light" depending on whether the passenger fits targeted profiles.\textsuperscript{99} Safety officials concede that profiling is necessary because the existing technology is "inadequate in scanning 100% of the baggage."\textsuperscript{100}
Though the current proposed database includes only travel information, it could later be cross-indexed with FBI, CIA, or criminal records—or with vital statistics, family names, or credit histories.\textsuperscript{101} Civil libertarians fear the existence of a nationwide computer system filled with details about personal backgrounds and behavior.\textsuperscript{102} Such
\textsuperscript{96}. See Scanning Equipment for Customs Searches is Set for Six Airports, \textit{Wall St. J.}, Aug. 3, 1999, at B11E, available at 1999 WL-WSJ 5463051.
\textsuperscript{97}. See Federal Aviation Reauthorization Act of 1996, Pub. L. No. 104-264, 110 Stat. 3213 (1996).
\textsuperscript{98}. See Final Report, supra note 4, at 37.
\textsuperscript{99}. Interview with FAA Officer James Paget, a participant in the Gore Commission hearings (Nov. 6, 1998).
The use of criminal records is especially troubling, since such records include arrests for which no conviction followed and for situations in which the person involved is entitled to a presumption of innocence. The constitutional right to due process requires that the state prove guilt "beyond a reasonable doubt." \textit{In re Winship}, 397 U.S. 358, 364 (1970). But if a person was subject to heightened security on the basis of profiles including arrests, he would be punished for having been arrested—even if the arrests themselves were completely illegal. \textit{See} Gil Klein, \textit{FAA Steps up Airport Security}, \textit{Richmond Times Dispatch}, Aug. 24, 1997, at A11, available at 1997 WL 7627160.
\textsuperscript{100}. Interview with Richard Kunicki, Chicago Department of Aviation Deputy Commissioner for Safety (Nov. 18, 1998). \textit{See} Fiorita, supra note 34, at 312.
\textsuperscript{101}. See ACLU Freedom Network, \textit{Executive Summary of the House Terrorism Bill H.R. 2768} (visited Feb. 9, 1996) <http://www.aclu.org/congress/hr2768.html>.
\textsuperscript{102}. \textit{See generally} ACLU Freedom Network <http://www.aclu.org/> (chronicling cyberspace's affect on privacy interests). This fear is not confined to the ACLU.
Employing a combination of psychological, sociological, and physical sciences to screen, inspect and categorize unsuspecting citizens raises visions of abuse in our increasingly technological society. Proposals based upon statistical research designed to predict who might commit crimes and giving them the special attention of law enforcement agencies is particularly disturbing. United States v. Lopez, 328 F. Supp. 1077, 1100 (E.D.N.Y. 1971).
a system could eventually form the basis of a coordinated data system allowing officials to track almost every aspect of an individual's personal life and activities.\textsuperscript{103}
\section*{B. Constitutional Background}
Airport security risks running afoul of the constitutional rights to travel, to privacy, to protection from search and seizure, and to equal protection. The possibility that airport security measures may have already crossed these constitutional lines is demonstrated by class action suits brought by minority passengers against United States Customs and airlines.\textsuperscript{104}
\subsection*{1. The Right to Travel}
The right to travel under the Fourth Amendment is the most obvious right challenged by security measures that delay or potentially prevent a person's air travel plans. Generally, the right to intrastate or interstate travel has been regarded as fundamental and subject to very few restrictions, whereas the right to international travel is subject to a somewhat greater level of interference.\textsuperscript{105} For example, passports may be required for international but not for domestic travel.\textsuperscript{106}
Justice William Douglas, writing for the majority of the Supreme Court in \textit{Kent v. Dulles},\textsuperscript{107} stated that:
The right to travel is a part of the "liberty" of which the citizen cannot be deprived without due process of law under the Fifth Amendment... [D]eeply engrained in our history [is] this freedom of movement . . . [a]cross frontiers in either direction, and inside frontiers . . . . Freedom of movement is basic in our scheme of values.\textsuperscript{108}
However, the tone of the Supreme Court opinion in \textit{Haig v. Agee},\textsuperscript{109} which allowed the Secretary of State to withhold a passport from a renegade CIA agent, seems to suggest that the exceptions to the "right" to travel may be so broad as to almost consume the right at
\begin{itemize}
\item \textsuperscript{103} Abuse of such information would be almost inevitable, and the system itself could propel us further into a situation in which our "virtual" identities overshadow our real existences and behavior. \textit{See generally} Michael Higgins, \textit{Looking the Part}, A.B.A. J., Nov. 1997, at 48, 52 (stating that neutral criteria processed by automated security systems will affect minority travelers disproportionately).
\item \textsuperscript{104} \textit{See} Anderson v. Cornejo, No. 97-C7556 (N.D. Ill. 1998).
\item \textsuperscript{105} \textit{See} United States v. Laub, 385 U.S. 475, 481, 482 (1967); \textit{see also} Kent v. Dulles, 357 U.S. 116, 125 (1958).
\item \textsuperscript{106} \textit{See} Laub, 385 U.S. at 481; Kent, 357 U.S. at 121-22.
\item \textsuperscript{107} 357 U.S. 116 (1958).
\item \textsuperscript{108} \textit{Id.} at 125-26.
\item \textsuperscript{109} 453 U.S. 280 (1981).
\end{itemize}
least for international travel.\textsuperscript{110} The Court wrote, “[t]he history of passport controls since the earliest days of the Republic shows congressional recognition of Executive authority to withhold passports.”\textsuperscript{111} Authentic threats to national security justify restrictions on international travel,\textsuperscript{112} but even international travel rights cannot be restricted based on spurious national security risks, such as a bare claim that the traveler is a Communist.\textsuperscript{113} International travel to and from the United States is so common that it is impractical to closely scrutinize every traveler.
2. \textit{Fourth Amendment Administrative Search Doctrine}
It has been established that passengers have a Fourth Amendment privacy interest in their carry-ons and luggage.\textsuperscript{114} However, partly to accommodate the existing airport searches, an entire body of law has evolved which allows administrative searches\textsuperscript{115} with little or no individualized probable cause that the person being searched is dangerous or has committed a crime.\textsuperscript{116} Though common today, such administrative searches would have clearly clashed with the understanding of searches outside the authority of written warrants that existed at the time the Constitution was written.\textsuperscript{117} At that time, all searches required written judicial authorization except those directly incident to a felony arrest.\textsuperscript{118}
“[S]earches conducted as part of a general regulatory scheme, done in furtherance of administrative goals rather than to secure evidence of a crime, may be permissible under the Fourth Amendment without a particularized showing of probable cause.”\textsuperscript{119} The government must establish three elements. First, a compelling need for the intrusion
\begin{itemize}
\item \textsuperscript{110} See id.
\item \textsuperscript{111} Id. at 293.
\item \textsuperscript{112} See id. at 302.
\item \textsuperscript{113} See Kent, 357 U.S. at 118, 130.
\item \textsuperscript{114} See United States v. Chadwick, 433 U.S. 1, 11-13 (1977) (holding that individuals have a Fourth Amendment privacy interest in containers and bags).
\item \textsuperscript{115} See Committee on Commercial Aviation Security et al., Airline Passenger Security Screening: New Technologies and Implementation Issues 35 (1996).
\item \textsuperscript{116} See, e.g., Michigan Dept. of State Police v. Sitz, 496 U.S. 444, 455 (1990) (providing for random sobriety stops of auto drivers). See also Skinner v. Railway Labor Executives Ass’n, 489 U.S. 602, 633 (1989) (providing for routine drug testing of railway workers).
\item \textsuperscript{117} “[O]n its face, [the Fourth Amendment] states seizures of persons or their property require the issuance of a judicial warrant . . . .” John F. Decker, Revolution to the Right: Criminal Procedure Jurisprudence During the Burger-Rehnquist Court Era 35 (1992) (citing U.S. Const. amend. IV). “[W]arrantless searches and seizures are ‘per se’ unreasonable . . . .” Id. (citing Katz v. United States, 389 U.S. 347, 357 (1967)).
\item \textsuperscript{118} See United States v. Edwards, 498 F.2d 496, 503 (2d Cir. 1974) (Oakes, J., concurring).
\item \textsuperscript{119} United States v. Bulacan, 156 F.3d 963, 967 (9th Cir. 1998).
\end{itemize}
must be established.\textsuperscript{120} Second, it must be shown that the intrusion will be strictly limited to fulfilling that need.\textsuperscript{121} Third, it must be demonstrated that the decision to search a particular person is not subject to the discretion of the official in the field.\textsuperscript{122}
The power to conduct a search of another is a tremendous power that carries with it a vast potential for abuse.\textsuperscript{123} Thus, the courts must determine whether an administrative search has expanded into an unlawful one.\textsuperscript{124} Courts have stressed the importance of keeping administrative searches from becoming “‘infected by general law enforcement objectives, and the concomitant need for the courts to maintain vigilance.’”\textsuperscript{125} If the government is allowed to freely conduct discriminatory searches under the guise of an administrative search, then “officials [will] routinely invade the privacy and property of countless millions; hardly anyone [will escape] their clammy grasp.”\textsuperscript{126}
3. \textit{Equal Protection}
The Equal Protection Clauses of the Fifth and Fourteenth Amendments provide that the government shall not “make or enforce any law which shall . . . deny to any person within its jurisdiction equal protection of the Laws.”\textsuperscript{127} The courts have identified race, religion, and national origin as suspect classifications that trigger equal protection analysis.\textsuperscript{128} According to the FAA, the criteria used in profiling do not involve such classifications.\textsuperscript{129} However, equal protection analysis is proper regardless of whether the discrimination is directly apparent from the words of a challenged law, from its administration,\textsuperscript{130} or from its effect.\textsuperscript{131} Where a law has a discriminatory effect on per-
\begin{itemize}
\item \textsuperscript{120} See United States v. $124,570$ United States Currency, 873 F.2d 1240, 1244 (9th Cir. 1989).
\item \textsuperscript{121} See id. at 1244-45.
\item \textsuperscript{122} See Camara v. Municipal Court, 387 U.S. 523, 536-37 (1967).
\item \textsuperscript{123} See United States v. Soyland, 3 F.3d 1312, 1316 (9th Cir. 1993) (Kozinski, J., dissenting).
\item \textsuperscript{124} See United States v. Davis, 482 F.2d 893, 909 (9th Cir. 1973).
\item \textsuperscript{125} Soyland, 3 F.3d at 1316 (Kozinski, J., dissenting) (citing $124,570$ U.S. Currency, 873 F.2d at 1244).
\item \textsuperscript{126} Id.
\item \textsuperscript{127} U.S. Const. amends. V, XIV.
\item \textsuperscript{128} See Hernandez v. Texas, 347 U.S. 475, 482 (1954).
\item \textsuperscript{129} See Higgins, supra note 103, at 50 (reporting statement by FAA Spokeswoman Rebecca Trexler).
\item \textsuperscript{130} See Yick Wo v. Hopkins, 118 U.S. 356, 373 (1886).
\item \textsuperscript{131} See Rogers v. Lodge, 458 U.S. 613, 625 (1982). Under current procedures, profiled passengers are not themselves publicly searched. Instead, their baggage is subject to heightened inspection. Kunicki Interview, supra note 100. Profiled passengers often have their luggage tagged with a colored sticker, indicating to fellow passengers that they are marked individuals.
\end{itemize}
sons belonging to a suspect classification, the law is subject to strict scrutiny\textsuperscript{132} and can be upheld only if it is closely tailored\textsuperscript{133} to fulfill a compelling governmental purpose.\textsuperscript{134}
\textbf{4. State Action}
Most security procedures at airports are carried out by employees of the airlines. This presents two dangers. First, persons who are not trained in law enforcement, and thus are not familiar with constitutional rights of individual citizens, carry out activities that the courts often disfavor even when performed by those who do have proper training.\textsuperscript{135} Second, airlines are private companies not directly bound by due process requirements, even when acting on information provided by the government.\textsuperscript{136} However, the courts have ruled that private airline employees are no different from public officials when conducting a search.\textsuperscript{137} Constitutional restraints upon government would be “severely undercut if the government were allowed to actively encourage conduct [prohibited by the government] by ‘private’ persons or entities . . . .”\textsuperscript{138} In \textit{United States v. Davis},\textsuperscript{139} a passenger’s briefcase was searched prior to boarding the aircraft by a ticket agent.\textsuperscript{140} The court noted, “[t]he search was part of the overall, nationwide anti-hijacking effort, and constituted ‘state action’ for the purposes of the Fourth Amendment.”\textsuperscript{141}
\textit{Id.} If the same group is consistently subject to public scrutiny, it may reinforce negative public opinion about that group. Moreover, the group would suffer increased risk of prosecution for crimes other people could commit without fear of detection. In both of these ways, profiling can have a discriminatory effect.
\textsuperscript{132} See Adarand Constructors, Inc. v. Pena, 515 U.S. 200, 227 (1995).
\textsuperscript{133} See id.
\textsuperscript{134} See id.
\textsuperscript{135} For example, private security personnel perform personal searches on persons who fail metal-detector tests at airport security. Kunicki Interview, \textit{supra} note 100. West German authorities so severely questioned the qualifications of private American security officers that in the airport in Frankfurt, searches by American security were conducted out of sight of German officials. \textit{See Report to the President}, \textit{supra} note 55, at 32. French officials threatened to limit the number of American security firms at Charles de Gaulle Airport on grounds that personnel “could themselves present a security risk.” \textit{Id.} at 33.
\textsuperscript{136} See Williams v. Trans World Airlines, 509 F.2d 942, 948-49 (2d Cir. 1975).
\textsuperscript{137} See United States v. Davis, 482 F.2d 893, 904 (9th Cir. 1973).
\textsuperscript{138} \textit{Id.} See Reitman v. Mulky, 387 U.S. 369, 380 (1967); Lustig v. United States, 338 U.S. 74, 79 (1949).
\textsuperscript{139} 482 F.2d 893 (9th Cir. 1973).
\textsuperscript{140} See \textit{id.} at 896.
\textsuperscript{141} \textit{Id.} at 904.
II. ANALYSIS
A. Profiling and Limits on the Administrative Search Doctrine
Given the strict limits on the administrative search doctrine, it seems unlikely that the courts would approve of CAPS or the use of profiling, even though the doctrine was developed partly for airline searches. As to the first requirement of the administrative search doctrine—a compelling administrative need—profiling and follow-up searches resemble existing, constitutional, security searches because they serve the same administrative function as part of essentially the same regulatory scheme to keep dangerous people and items off of aircraft.\textsuperscript{142} This function, of course, has a very high priority and will justify far more intrusive behavior than would most administrative purposes.\textsuperscript{143}
However, by limiting the searches to only certain individuals, profiling approaches the forbidden line between administrative and criminal searches. Thus, it fails to satisfy either of the second two elements of the administrative search doctrine—strict limitation to non-criminal purposes and freedom from discretion of officers in the field. Profile-based searches are likely to be used to attempt to track down and provide evidence against suspected terrorists (and perhaps other criminals as well), so that heightened security against certain individuals almost inevitably infects the regulatory scheme with criminal investigative purposes which cannot be supported by the administrative search doctrine.
The profiling system was designed to remove some human subjectivity by eliminating the choices of the personnel who identify persons targeted for heightened security.\textsuperscript{144} After each passenger’s name is entered into a computer, the system makes a determination and flashes a green or red light.\textsuperscript{145} The passenger is not told of this event, and is subjected only to the same search as are all other passengers.\textsuperscript{146} The process focuses on checked baggage rather than individuals. The CTX-5000 is used on the bags, and if the bags are suspicious, they are set aside for further scrutiny. For example, equipment able to detect trace of explosives might be employed. In some cases, the bags would
\textsuperscript{142} See id. at 908.
\textsuperscript{143} See id.
\textsuperscript{144} Paget Interview, supra note 99.
\textsuperscript{145} See InVision Receives FAA Contract for Minimum of 54 and Up to 100 InVision CTX 5000 SP Explosives Detection Systems, Bus. Wire, Dec. 26, 1996, available in LEXIS-NEXIS Academic Universe, Wire Service Reports [hereinafter InVision Receives FAA Contract].
\textsuperscript{146} Paget Interview, supra note 99.
be opened. The intensity of the search would be based on the level of suspicion.
Though workers in the field appear to have no choice in initiating the process, because the computer makes this determination, field officers must decide whether to halt or continue the search at each of the additional steps. They examine the CTX-5000 images and decide whether what they see looks like a gun or another suspicious item. Thus, field officers have almost complete discretion to choose the intensity of heightened security, and nothing would prevent them from using such discretion to satisfy human curiosity or to look for contraband not dangerous to a flight, such as drugs or ivory. Moreover, the computer itself is analogous to a field officer. Even though it is not human, it poses a similar threat as does a human agent and its actions are not readily subject to judicial scrutiny.
Profiling is inherently impossible to monitor by the public or by either legislative or judicial authorities. The same information that the public or Congress would need to engage in an informed debate about profiling, and that courts would need in order to determine whether the profile used legal or illegal criteria, could be used by terrorists to evade the profiles. Making profiles public is necessary to make them legal, however, doing so would also destroy their usefulness.
It is impossible to determine whether the profiles now in use involve illegal criteria because the FAA has declined to publicize the nature of the criteria.\textsuperscript{147} The FAA has emphatically denied that race, ethnicity, religion, or gender play a role in the profiles.\textsuperscript{148} The FAA has merely noted that “it has to do with people’s travel patterns and how well they’re known in the system.”\textsuperscript{149} Such information is not helpful in determining whether constitutional claims arise. Allowing such profiling forces the American people and courts to take the FAA at its word.\textsuperscript{150} Government action with no opportunity for review is entirely outside our basic constitutional framework of independent checks and balances upon all government power.\textsuperscript{151}
\begin{itemize}
\item[147.] FAA spokeswoman Rebecca Trexler noted that making the profiles public “would be telling the terrorist what we’re looking for.” Higgins, \textit{supra} note 103, at 50.
\item[148.] \textit{See id.}
\item[149.] \textit{Id.} at 52.
\item[150.] The Gore Commission submitted its criteria for profiles to the Attorney General’s Office, which determined that they are constitutional. Paget Interview, \textit{supra} note 99. However, such a review cannot facilitate public debate or judicial review of their constitutionality.
\item[151.] The “separate and distinct exercise of the different powers of government . . . is admitted on all hands to be essential to the preservation of liberty . . . .” \textit{The Federalist} No. 51, at 321 (James Madison) (Clinton Rossiter ed., 1961).
\end{itemize}
The Fourth Amendment is notoriously confusing, and it is unreasonable to expect security officers who are untrained as police officers to exhibit the kind of sensitivity to individual privacy rights that the Constitution demands. Courts and police alike have been accused of inconsistently applying the doctrines underlying the Fourth Amendment. Courts have carved out so many exceptions that the exceptions have virtually swallowed the rule. Limited administrative searches have been authorized for borders and airports.
Professor Wayne R. LaFave, in analyzing the Fourth Amendment, attempted to untangle the nine search and seizure decisions issued during the Supreme Court's 1982-83 term. He proclaimed these cases as a group, to be "illogical, inconsistent with prior holdings and generally, hopelessly confusing." Such decisions offer poor guidance to those responsible for conducting administrative searches. Without understanding the individual Fourth Amendment protections at issue, how can society meaningfully participate in debates about the future of such protections in airline security?
While the Fourth Amendment does allow reasonable administrative searches, there are limitations. "To meet the test of reasonableness, an administrative screening search must be as limited in its intrusiveness as is consistent with satisfaction of the administrative need that justifies it." Due to long term ramifications of their decisions, courts must not merely consider just the facts of the case before them but must consider all "searches permissible under the scheme."
One of the greatest dangers of profiling is that it will expose certain persons to an unusually high risk of exposure to seizures unrelated to the purpose of keeping explosives off aircraft, for example under the plain view doctrine. "The interests protected by the Fourth Amendment are diminished when an object is found in plain view." The owner loses his expectation of privacy and "the owner’s remaining in-
---
152. See Craig M. Bradley, Two Models of the Fourth Amendment, 83 Mich. L. Rev. 1468, 1472 (1985).
153. See id. at 1468.
154. See id. at 1473-74.
155. See United States v. Martinez-Fuerte, 428 U.S. 543, 556-57 (1976).
156. See United States v. Davis, 482 F.2d 893, 908 (9th Cir. 1973).
157. Wayne R. LaFave, Fourth Amendment Vagaries (of Improbable Cause, Imperceptible Plain View, Notorious Privacy, and Balancing Askew), 74 J. Crim. L. & Criminology 1171, 1171 (1983).
158. Davis, 482 F.2d at 910.
159. See, e.g., United States v. Bulacan, 156 F.3d 963, 967 (9th Cir. 1998).
160. Id.
161. Id. at 968.
terests in the object are merely those of possession and ownership."162 "The problem with the 'plain view' doctrine has been to identify the circumstances in which plain view has legal significance rather than being simply the normal concomitant of any search, legal or illegal . . . ."163 The officer seizing objects in plain view must have a legal right to be in that location and have a "lawful right of access to the object itself."164 Since profiles provide such "lawful right of access," certain persons are at an unfair risk of detection of criminal activity in which others may engage with more impunity.
One federal court has noted a more serious problem with expanding the administrative search doctrine in that it gives those favoring more intensive searches leverage over the development of constitutional law.165 As the government increases the intensity and frequency of searches, the public's reasonable expectation of privacy diminishes—and with it diminishes the concomitant constitutional privacy right.166 Thus, in effect, investigators can use the administrative search doctrine to justify ever-greater intrusions into the privacy of individual citizens in areas totally unrelated to the original purpose of the doctrine. For example, it is at least arguable that twenty years ago a citizen had a reasonable expectation of privacy in closed luggage.167
---
162. Texas v. Brown, 460 U.S. 730, 739 (1983) (citation omitted).
163. Coolidge v. New Hampshire, 403 U.S. 443, 465 (1971).
164. Horton v. California, 496 U.S. 128, 137 (1990).
165. See United States v. Lopez, 328 F. Supp 1077, 1089 (E.D.N.Y. 1971).
166. In the end, "hardly anyone escapes [the] clammy grasp" of "government officials [who] routinely invade . . . privacy." United States v. Soyland, 3 F.3d 1312, 1316 (9th Cir. 1993) (Kozinski, J., dissenting).
167. See, e.g., United States v. Place, 462 U.S. 696, 703 (1983) (holding that the seizure of luggage for even 90 minutes outweighed law enforcement concerns). Judge Oakes' concurrence in United States v. Edwards implicitly assumes that there is such a reasonable expectation that would trigger Fourth-Amendment protections. 498 F.2d 496, 501 (2d Cir. 1974) (Oakes, J., concurring). That airport searches have already begun to erode such reasonable expectations is shown by the fact that Judge Friendly's majority decision completely avoided the discussion of such privacy expectations, and instead regarded the enormity of the danger implicit in air piracy as making a search implicitly reasonable, stating that "[n]othing in the history of the [Fourth] Amendment remotely suggests that the framers would have wished to prohibit reasonable measures to prevent the boarding of vessels by passengers intent on piracy." Id. at 498. This Comment suggests that this observation evades the issue of the privacy of passengers who are not pirates.
In Edwards, the defendant had been convicted of possession of heroin which could never have endangered the flight or have been used as an instrument of air piracy. See id. at 497. The real question, then, is whether the framers would have been troubled by the use of the danger of piracy to excuse enforcement of laws having no logical relation to that piracy in ways which would be otherwise impermissibly intrusive. One wonders whether Jefferson or Adams would have objected to a warrantless search of baggage he carried to Liberty Hall in Philadelphia by British officers intent on reducing the enormous Eighteenth century dangers of highway robbery and sea piracy. Judge Oakes' concurrence in Edwards suggested the obvious answer is that the
If, however, searches of such baggage can today be justified by the administrative search doctrine, any such expectation would be unreasonable. Thus, any search of luggage, whether or not related to the administrative purpose, would face far less constitutional restraint than such searches would have faced ten years ago. Widespread searches already seem to have intimidated even citizens with good reason to resist them. The United States Court of Appeals for the Fifth Circuit has noted that, “[w]e think it strikingly unusual that so many individuals stopped at airports consent to search while carrying drugs.”¹⁶⁸ Eventually, the administrative search doctrine could make any expectation of privacy in any place unreasonable and, thus, completely obliterate the purpose of the Fourth Amendment.¹⁶⁹
B. Practical Critique of Profiles
Only twice in the past twelve years has an American airliner faced a terrorist attack and only one of those incidents led to an arrest.¹⁷⁰ This leaves officials with one known airline bomber from which to make a profile.¹⁷¹ Profiles used in the test program at Northwest are not based on data about actual terrorists. The FAA developed the criteria based on “consultations with a large number of security and terrorism experts, who gave their assessments of the likely patterns of behavior of individuals intending to attack civil aviation.”¹⁷² In other words, the criteria are based on the opinions of experts rather than on actual data.
Though failure to use real data might seem irresponsible, there is a very good reason why no data was used—because none exists, and it would be almost impossible to obtain such data even if a concerted effort was made to do so. Most evidence of terrorist attacks is destroyed in an airline crash, so that officials do not have enough information to form a profile even if such a methodology could ever succeed.¹⁷³ Moreover, there have been no terrorist attacks on Ameri-
¹⁶⁸ United States v. Berry, 670 F.2d 583, 598 n.16 (5th Cir. 1982).
¹⁶⁹ It would effect a “systematic wrenching of the administrative search from its constitutional roots.” Soyland, 3 F.3d at 1318 (Kozinski, J., dissenting).
¹⁷⁰ See Nojeim, supra note 51, at 6.
¹⁷¹ See id.
¹⁷² Fainberg, supra note 39, at 200.
¹⁷³ Even if circumstances did permit information-gathering, there is little assurance that “experts” would utilize it rationally. For example, the FBI gathered information about feminist groups from 1969 to 1979. The FBI amassed a file of nearly 3,000 pages, including an account of a meeting at which, according to the agents assigned, “[t]he Women, in general, appeared to be hippies, lesbians or from far-out groups. Most of them were colorfully dressed, but the majority
can flights for more than twenty years.\textsuperscript{174} Even if data on terrorists from the 1960s or from overseas were collected, such data would tell little about behavior patterns in the United States today. Data about non-aviation terrorists might be extrapolated for airline-terrorist profiles, but a profile developed in this way would ultimately depend on the reasonable but unverifiable assumption that air terrorists are like other terrorists. Furthermore, such a profile would logically be two steps removed from the person against whom it is directed. Not only would the information used to justify searching a person not be particularized to him or her as an individual, it would not even be particularized to any group to which he or she actually belongs. Rather, it would be particularized only to a different group assumed to be similar to the one to which he or she belongs.
Profiling has been tried several times, always without success.\textsuperscript{175} The German S.S. profile for a potential bomber of the Hindenburg made Jewish and dissident passengers suspects, but the actual bomber proved to be a German patriot.\textsuperscript{176} Before the 1974 anti-terrorist legislation provided for screening of all passengers, the airlines used a manual profiling system similar to the automated system proposed by the Gore Commission, but despite this profiling, hijackings continued to increase in frequency.\textsuperscript{177}
Both of these profiling schemes, like that proposed by the Gore Commission, were based on poor information. Profiling has failed even when security officers had accumulated excellent information. Prior to the Pan Am Flight 103 bombing, Pan Am knew that an anonymous caller to the United States Embassy in Helsinki had claimed that a Finnish woman would bomb a Pan Am flight from Frankfurt during December of 1988.\textsuperscript{178} Pan Am merely used a profile identifying Finnish women for heightened security and took no other special security measures despite a number of other known similar threats.\textsuperscript{179}
\textsuperscript{174} See Nojeim, \textit{supra} note 51, at 6 (illustrating that in 1972, when profiling was used exclusively, there were 28 hijackings on American aircraft, but since 1972, when all luggage began to be X-rayed, there have been none).
\textsuperscript{175} See \textit{supra} notes 42-54 and accompanying text.
\textsuperscript{176} See \textit{supra} notes 15-24 and accompanying text.
\textsuperscript{177} See \textit{supra} notes 42-54 and accompanying text.
\textsuperscript{178} See \textit{Report to the President}, \textit{supra} note 55, at 8-9.
\textsuperscript{179} See \textit{id}.
The murderous failure of these procedures is well known.\textsuperscript{180} Perhaps knowing that plans to use a female Finnish bomber had been revealed, the terrorists employed a man of Arabic descent as a bomber instead.\textsuperscript{181} Thus, even under ideal circumstances, with inside information about the terrorists and their plans, profiling failed.
Common sense suggests that profiling will likely fail. Profiling has a deplorable record in attempts to halt drug traffic into the United States.\textsuperscript{182} In order for profiling to work, terrorists must be unaware of its use. Simply by planting a bomb on an unsuspecting passenger, a terrorist can use that passenger who does not meet the profile to circumvent the airport security system. Terrorists will continue to seek innovative ways to place bombs on an aircraft without having to take it on themselves. The FAA has tacitly conceded the ineffectiveness of its profiles by providing for some passengers to be subjected to heightened security by random selection.\textsuperscript{183} It is likely that the random checks will provide more deterrence than would profiles, since random checks cannot be evaded. However, the existence of profiling shows that the FAA is not content to rely on random checks alone, since doing so reduces security to mere chance.
One group that has been noticeably affected by enhanced airport security is the Arab-American population.\textsuperscript{184} Though there has never been evidence tying the Middle East to the bombing of the Oklahoma City Federal Building in 1996, public sources voiced suspicion of Middle Easterners immediately and Arab Americans faced increased detentions and suspicion.\textsuperscript{185} The Council on American Islamic Relations
\begin{itemize}
\item \textsuperscript{180} See supra notes 55-64 and accompanying text.
\item \textsuperscript{181} See REPORT TO THE PRESIDENT, supra note 55, at 8-9.
\item \textsuperscript{182} “Profiling techniques used by the Customs Service do not stop the drug trade. If they actually worked, drugs would not be coming into the United States.” Profiling Endangers, supra note 50. This does not reflect poorly on the personnel who are committed to protecting the citizens of the United States from overseas dangers. This author had the pleasure to work closely with immigration and customs officers for several years in O’Hare’s International Terminal, and can confirm that they are highly trained and experienced professionals. “Our inspectors constantly strive to show courtesy and respect to all those whom they encounter.” Interview with Patrick Noonan, O’Hare International Airport Port Inspector, United States Customs Service (Aug. 17, 1998). The problem inherent in profiling is not with the people who implement it. It might be argued that profiling has helped fill American prisons with drug offenders. However, it has not deterred the drug trade. Likewise, filling prisons with air terrorists caught by profiles might be emotionally satisfying, but it will not keep airlines safe.
\item \textsuperscript{183} See InVision Receives FAA Contract, supra note 145.
\item \textsuperscript{184} See ACLU Freedom Network, Profiling of Fliers Raises Racial Issue (visited Sept. 26, 1998) <http://www.aclu.org/news/w092697d.html> [hereinafter Profiling of Fliers].
\item \textsuperscript{185} For example, Mr. Abraham Ahmad, an American of Jordanian descent, left Oklahoma City at the time of the bombing. See Nojeim, supra note 51, at 8. In three United States cities he was strip searched and interrogated, and then, after he reached London, he was forced to return to the United States. See id. According to the ACLU account, other than the timing of his
\end{itemize}
("CAIR"), which assists profiled Islamic Americans who feel their constitutional rights have been violated, contends that profiling essentially boils down to stereotyping.\textsuperscript{186} CAIR reported a 1000% increase in complaints in one year by Islamic Americans who were detained, searched, and questioned in American airports.\textsuperscript{187} Even if the FAA does not use religion itself as a criterion, among the factors that seem to increase a person's likelihood of heightened security are place of birth and the number of trips to Middle Eastern countries.\textsuperscript{188} Naturally, such factors indirectly single out Arab-Americans more often than other Americans.
As discussed above, such discrimination could only be defensible if it serves a compelling governmental purpose.\textsuperscript{189} Though airline safety is arguably a compelling purpose, the only purpose served by profiling is cost-cutting, not airline safety. If the public purpose is zero-tolerance for terrorism,\textsuperscript{190} then everyone should be searched. This would inevitably curb potential terrorism better than the use of profiles. Targeting only certain individuals reflects a refusal to accept the monetary and political cost of universal searches—and such a refusal is not even an honest governmental purpose, let alone a compelling one.
\textbf{C. Constitutional Alternatives to Ends Sought by the Gore Commission}
\textbf{1. Theoretical Standpoint}
To discourage terrorism, society must take steps to prevent terrorists from achieving their objectives. Their long term goal, of course, is not to kill Americans but to intimidate and alter behavior by restricting freedom. Terrorists seek a world in which fear, not free-
\textsuperscript{186} See Council of American-Islamic Relations (visited Sept. 8, 1998) <http://www.cair-net.org>.
\textsuperscript{187} See id.
\textsuperscript{188} See Higgins, \textit{supra} note 103, at 52.
\textsuperscript{189} See \textit{supra} notes 105-141 and accompanying text.
\textsuperscript{190} See Aviation Security Improvement Act of 1990, Pub. L. No. 101-604, 104 Stat. 3066 (1990) (codified as amended in scattered sections of 49 U.S.C. and 29 U.S.C.).
dom, is the rule. When government restricts freedom to fight terrorism, it actually carries out the objectives of the terrorists. Such appeasement encourages terrorism.\textsuperscript{191}
A Chinese proverb recalls that the purpose of terror is “[t]o kill one and frighten 10,000 others.”\textsuperscript{192} The objective of terrorism is to create fear. To overcome terrorism, society simply must not become frightened. Terrorists crave fear, and it is this that we must always refuse to allow them. Thus, stopping terrorism is not chiefly a matter of security measures, but of social courage and education. It cannot be done by government, but must be done in the hearts and minds of American people who say to themselves “we will never fear that” and say it to the world loud enough for the terrorists to hear.
Real security measures can enhance our fearlessness,\textsuperscript{193} but measures such as those proposed by the Gore Commission are obviously motivated by fear, and as such they feed the craving of terrorists. If we are so fearful that we allow our government to restrict our freedom and carelessly spend our billions, we tell the world how frightened we are and we tell the terrorists that for every one they kill, they can frighten and intimidate ten thousand. A government band-aid presenting a façade of safety cannot be a substitute for individual Americans taking responsibility for our own safety and demonstrating our unwillingness to be intimidated. Such band-aid measures, when they impinge on individual freedoms, send the wrong message to terrorists and actually repress the exact kinds of personal courage which spark the only realistic hope of stopping terrorism at its roots.
World tensions and the increasing availability of weapons of mass destruction\textsuperscript{194} make terrorism as great a threat today as it has ever been in the United States. Current political sentiment suggests that drastic action is called for to address security concerns. Rather than
\textsuperscript{191}. One commentator has said:
It is wrong to allow yourself to be intimidated, because if you do so you encourage intimidation. If somebody is pointing a gun at your head, it is essential to consider the fact that death is perhaps unavoidable and that therefore avoiding death is no longer a rational objective, but that other objectives (especially the preservation of freedom and self-respect) may still be attainable. The only moral response to such violent threats is to say, “go ahead and shoot. I will never do what you want.”
Interview with Eirik Marr Johnson, Parliamentarian, self-confessed “freedom extremist,” and an attorney member of the Illinois ACLU (Aug. 12, 1998).
\textsuperscript{192}. Dow, \textit{supra} note 14, at 1152 n.12.
\textsuperscript{193}. Fear is not the same thing as an apprehension of danger and steps taken to reduce it. The law is careful to distinguish between fright, an emotion having no legal significance, and apprehension, which gives rise to the tort of assault and may justify self-defense. \textit{Restatement (Second) of Torts} § 24 cmt. b. Fear encourages terrorism, whereas rational action based on apprehension can reduce terrorism.
\textsuperscript{194}. See Dow, \textit{supra} note 14, at 1049-51.
exploiting such sentiment as an excuse to erode constitutional rights and fatten existing bureaucratic budgets, this sentiment must be harnessed to create a truly safe airline system.
Heavy-handed security measures provoked but could not prevent the most famous airline disaster in history—the destruction of the Hindenburg—and the consequent destruction of pre-war international air travel.\textsuperscript{195} The Gestapo was not concerned with American-style privacy rights.\textsuperscript{196} It was the paternalistic insensitivity of German security measures, not individual rights, which prevented the German S.S. from making the Hindenburg safe.\textsuperscript{197} Twenty-First Century America cannot afford to make the security mistakes of Depression-era Nazi Germany.\textsuperscript{198}
The Constitution is a blueprint for effective strategies whereby Americans may secure our personal safety, rather than a barrier to such strategies. There is no need to make a trade-off between safety and constitutional freedom. An unconstitutional reaction to terrorism is, by its nature, an ineffective reaction, both because it ignores the accumulated wisdom of the Constitution and because such an approach will face constant, inevitable, and rightful opposition.
The most powerful tool America has against terrorism is the willing assistance of American individuals. Heavy-handed government intrusion into the travel lives of Americans will serve chiefly to create a hostile relationship between travelers and the officials responsible for protecting them,\textsuperscript{199} and thus would squander our most powerful anti-terrorist asset—the goodwill and intelligence of American travelers.
\textsuperscript{195} See Mooney, \textit{supra} note 13, at 11-12.
\textsuperscript{196} See id.
\textsuperscript{197} See id.
\textsuperscript{198} See id.
\textsuperscript{199} Even existing security procedures have created an adversarial relationship between passengers and officials. Following the filing of a class action lawsuit by nearly 50 African American females against United States Customs at O'Hare International Airport in Chicago, both Senators from Illinois took notice. Letter from Sens. Carol Moseley-Braun and Richard Durbin to Acting Comptroller General James F. Hinchman, General Accounting Office (June 9, 1998). In that letter to the General Accounting Office ("GAO"), the Senators requested "review of the United States Customs Service search seizure procedures at O'Hare Airport." \textit{Id}. According to the letter, the Senators were concerned "about the consequences of using profiles that have the potential to invade the privacy of innocent Americans." \textit{Id}. Further, the letter asked that the strip search techniques also be "reviewed for their efficiency in relation to other means of finding contraband." \textit{Id}. The following day the Senators jointly released a press statement to voice their concern with the current practices. Press Release, Sens. Carol Moseley-Braun and Richard J. Durbin (June 10, 1998). When questioned about criteria used by airlines, the DOT reported that "no airline has been . . . cited for discriminating in its searches . . ." Higgins, \textit{supra} note 103, at 52. The FAA denies "emphatically" that criteria such as ethnicity, gender, or religion are included in its profiles. \textit{See id.} at 50. However, an American Airlines' document advised its emThe personal rights in the Constitution can and should serve to advance the cause of safety. Constitutional safeguards prohibit only paternalistic safety strategies based on the premise that individuals must be important pawns in a game played between terrorists and safety officers. The Constitution prohibits the government from using such a game-playing model in constructing safety strategies. Whatever the details of constitutional doctrine, the essence of the Constitution is individualistic. Americans cannot be deprived of an effective individual role in securing their own safety. Such an individual involvement in safety is both constitutionally required and the only sound basis for effective security measures. Unfortunately, the recommendations of the Gore Commission on aviation security and safety reflect an unquestioning acceptance of the game-playing model and largely ignore not only individual constitutional rights, but also the wiser strategies enjoined by the Constitution. The Commission has both failed to make us safe and to make acceptable inroads into our constitutional rights.
The Gore Commission’s acceptance of profiling betrays its lack of concern for public involvement in safety. The profile can only be effective if it is kept secret from the very people whom it is supposed to protect. There is no way for travelers to take any role in implementing a profile-based security structure. The Gore Commission’s reactionism is remarkably similar to that of officials during early hijacking days. As discussed above, in 1972, the FAA mandated air carriers create (within seventy-two hours) an “acceptable” screening system “to prevent . . . devices or weapons in carry-on baggage or on . . . the persons of passengers.” In 1972, passengers were subjected to a behavioral profile, a magnetometer, identification check, and physical search. Likewise in reaction to what was believed to be a terrorist attack, the Gore Commission hastily acted on legislation to take immediate measures to secure safety. In the 1970s, real safety—both from terrorists and from threats to the Constitution—was achieved only when profiling was abandoned and everybody was searched. Likewise, real security today requires evenhanded searches of everybody.
Real security procedures directed at real threats are both constitutionally more sound and practically more effective than are the steps proposed by the Clinton Administration and now enacted into law as
employees to give closer scrutiny to “those with Arab-sounding names who don’t carry U.S. passports.” Profiling of Fliers, supra note 184.
200. United States v. Davis, 482 F.2d 893, 900 (9th Cir. 1973) (quoting 37 Fed. Reg. 2500-01).
201. See FAA Press Release No. 72-26 (Feb. 6, 1972).
the 1996 FAA Reauthorization Act. Flying is still one of the safest forms of travel, but terrorist threats are likely to increase in the near future.\textsuperscript{202} Due to the recent terrorist threats and bombings of United States embassies, the need to improve existing security at airports is at an all time high. It is possible that a combination of the steps outlined below are likely to realistically improve airline security. Some of these proposals may seem utopian, but since large outlays of money have been authorized, it ought to be spent constitutionally and effectively.
2. Specific Safety Recommendations
a. Improve Existing Safeguards
Existing airline security measures have proven to be amazingly effective.\textsuperscript{203} Before gaining computer capabilities, airlines relied on manual screening procedures. In fact, El Al wages the most aggressive visual screening campaign in the industry. One of the most tragic aviation stories told is that of an Irish national who attempted to board an El Al flight at Heathrow airport on April 16, 1986.\textsuperscript{204} After repeated questioning by the ticket agent, the passenger was pulled to the side so a search could be conducted of her baggage.\textsuperscript{205} Inside the lining, her Palestinian fiancé had inserted a bomb, presumably to explode mid-air and kill his fiancée and unborn child.\textsuperscript{206} It is unlikely that any automated system would have identified this passenger, but an alert airline employee did. Alert passengers might be able to do the same if they were empowered and informed. The use of suspicious, even if innocent, behavior is less paternalistic than is profiling when used to intensify investigation because it is particularized to the individual who is then investigated, and the investigation is thus partly under his control.
Hence, the most reasonable step in improving security is to see what has worked and do it even better. One potential weakness in the existing system is that most security personnel are poorly paid and
\textsuperscript{202} See Nadine Strossen, \textit{Check Your Luggage and Liberties at the Gate} (visited Feb. 8, 2000) <http://www.intellectualcapital.com/issues/issue100/item4464.asp>.
\textsuperscript{203} See \textit{supra} note 54 and accompanying text.
\textsuperscript{204} See Faye Bowers, \textit{Ground Personnel: Gap in Airport Security System}, \textit{Christian Sci. Mon.}, Aug. 7, 1996, at 1.
\textsuperscript{205} See \textit{id}.
\textsuperscript{206} See \textit{Report to the President}, \textit{supra} note 55, at 169 (repeating testimony given by Billie H. Vincent before the Commission on November 17, 1989, and the subsequent letter of April 19, 1990).
trained, which increases the risk that they could be bribed, fooled, or infiltrated by determined terrorists. Thus, the most important thing we can do to make airports more secure is to improve the professionalism, training, and screening of security personnel. The Gore Commission recommended seven times as much funding to protect the FBI and DEA buildings as it allotted for training security workers in detecting explosives. Profiling received five times as much funding as did training for the whole country. In all, training airport security officers accounts for only 2% of the total package signed into law by President Clinton, and little of the other funds will likely to be devoted directly to improving the skills and circumstances of the front-line officers charged with airline security.
A second potential weakness of the existing system concerns the "secure" areas of many airports. These areas that can only be entered after submitting to security checks are very large, making them impossible to monitor and allowing non-passengers to enter. For example, it is not possible to enter many of the restaurants and other attractions at many airports without passing security. This means much of the security effort is devoted to people who never board airplanes. Either the secure areas should be made much smaller and access to them should be limited to passengers and those assisting them, or else small heightened security enclaves limited in these ways should be created within the existing larger secure areas of airports.
207. This problem presents danger not only to civil liberties, which may be ignored by unknowledgeable guards, but also to safety itself. This is an illustration of the central contention of this Comment—that proper respect for civil liberties often enhances rather than weakens security. Properly-trained guards demanded by the Constitution would also provide better security. The inability of existing guards to do their jobs is demonstrated by numerous breaches of airport security, such as the August 26, 1999 incident at the United Terminal at Chicago O'Hare International Airport, in which "[a] man carrying a tan canvas bag ran past an unarmed security guard . . . prompting evacuation of 6,000 people . . . during one of the busiest times of the day . . ." Gilbert Jimenez & Frank Main, Chaos at O'Hare, Chi. Sun-Times, Aug. 27, 1999, at 1.
208. The vulnerability of security personnel to security lapse is highlighted by the recent scandal at American Airlines involving a massive conspiracy to bring guns and drugs into the country from Latin America on American Airlines planes by 58 airline employees and Department of Agriculture employees, and officers of both the INS and local Sheriff's Department. See Editorial, Airline security lax, Chi. Sun-Times, Aug. 27, 1999, at N49.
209. See President's Proposal, supra note 72.
210. See id. Each bureaucracy received seven million dollars to defend its headquarters. Approximately two million was earmarked for training. See id. Ten million dollars was invested in profiling. See id.
211. See id. Training accounts for approximately two million dollars of a budget of about a billion dollars. See id.
212. The cases that authorized administrative searches date to the 1970s when searches were made only of passengers and only almost immediately before boarding. See United States v. Davis, 482 F.2d 893, 899 (9th Cir. 1973). These factors were considered by the courts that allowed such searches. "Near the entrance to the boarding gate were two large signs, plainly
curity checks could be made on the aircraft themselves after all passengers have boarded. Existing security equipment has been effective, but improved explosives technology could enable well-financed terrorists to evade the equipment, and therefore the technology of detection equipment must keep pace.
b. Resolve Problems With Tracking Checked Baggage
The age-old problem of lost or delayed checked baggage must also be solved. Fearing delays and lost baggage, many passengers chose to carry-on their bags. Such bags are not only security risks, but they can endanger the plane itself or the passengers in a crash. Bag matching to assure that passengers board the same flight as their baggage would both improve baggage service and prevent the tactics that led to the Lockerbie disaster. However, imposing bag matching on the current muddle of domestic bag handling would dramatically slow flights. Therefore, the current system must be improved, both to insure the safety of the checked baggage through bag matching as well as encourage passengers to use the checking system and reduce their
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213. American security efforts have been intensive and largely effective. An attorney and former FAA employee who has strong concerns for both security and the Constitution has expressed intense respect for the system currently in place to protect American air travelers. Interview with Ken Quinn, Attorney and Former FAA employee (Dec. 13, 1998). Mr. Quinn points out that the heavy hand of American power should descend upon those who threaten us, not upon American citizens. See id. In many cases, we know who threatens us and how, but fail to take action against them owing to other national agendas. See id. A true policy of zero tolerance for terrorism demands that we strike hard and fast when we know who threatens us, even at the cost of other important foreign-policy objectives. See id. Part of the price we must pay for security is not our own individual freedoms, but perhaps in a reduced flexibility in foreign policy. See id.; see also supra note 9 (discussing Jefferson's forceful response to the terrorism of his day, namely piracy).
214. This concern has led to strict controls on the size, number, and weight of carry-on bags.
215. See Kapustin, supra note 40, at 1-2.
216. See N.J. Strantz, From Technology to Teamwork: Aviation Security Reform Since Pan Am Flight 103, 3 ALB. L.J. SCI. & TECH. 235, 251 (1993).
217. One hundred percent bag matching using the current technology would "cripple the system." Kunicki Interview, supra note 77.
dependence on unsafe carry-on bags.\textsuperscript{218} Perhaps the computer resources that the Gore Commission would utilize to profile passengers could instead be employed to track checked baggage.
c. Reduce the Adversarial Nature of Security Procedures
Almost all passengers are deeply concerned about safety and would be anxious to actively assist in preventative measures. Systems which force passengers to submit against their will and which humiliate or otherwise offend them transform willing and able potential assets in a safety system into enemies.\textsuperscript{219} Actions which alienate passengers by violating their rights are, by nature, bad for security, because they prevent passengers from using their abilities to enhance security.
There is a famous story alleging that a group of terrorists caught before they had done any damage, said to their captors, “today, we were unlucky. But remember, we only have to be lucky once. You will have to be lucky always.”\textsuperscript{220} This story is used to justify extreme security measures, since being lucky every time is not an easy task.\textsuperscript{221} But conversely, consider if the story involved a victim who had just failed to prevent a terrorist from foiling his violent plot. The victim, too, could say, “we only have to be lucky once.” Part of the danger of terrorism is that there are always more potential terrorists than there are security officers. But it is equally true that there are always more passengers than there are hijackers. Relying on a handful of outnumbered “experts” for security is as hopeless as the famous story above suggests. However, were such experts to marshal the eyes, ears, and intelligence of every passenger against terrorism, the odds would be reversed in safety’s favor.\textsuperscript{222} There are many things passengers can and would do to enhance security if only they knew how. The most
\textsuperscript{218} It appears that some of the money already allocated for implementation of the Gore Commission recommendations could be earmarked for these purposes. \textit{See President's Proposal}, supra note 72. It ought to be.
\textsuperscript{219} For example, hundreds of young Japanese-American men who escaped relocation camps during World War II proved their American loyalty by joining the armed forces and becoming war heroes. Yet, authorities had found the task of individually assessing the threat posed by individual Japanese-Americans overwhelming and had lumped them all into a single “profile.” \textit{See Frank F. Chuman, The Bamboo People: The Law and Japanese-Americans} 143-81 (1976).
\textsuperscript{220} Thomas Strentz, \textit{A Terrorist Psychological Profile Past and Present}, 57 FBI L. ENFORCEMENT BULL. 13, 16 (1988) (quoting \textit{Wash. Post}, Oct. 12, 1984, at A1) (reporting the comments of an Irish Republican Army member).
\textsuperscript{221} \textit{See id.}
\textsuperscript{222} This author does not propose arming passengers and having them shoot it out with hijackers. Keeping guns out of passengers’ hands was part of the 1974 legislative anti-terrorist program. \textit{See supra} notes 46-49 and accompanying text.
important step would be to require airlines and the FAA to inform passengers and crew about known terrorist threats.\textsuperscript{223}
Just as public awareness of seat belt use and drunk driving has made auto travel safer\textsuperscript{224} in the United States and has allowed individuals to assume a greater degree of responsibility and control for their own safety and that of others. Similar programs could enhance airline safety. For example, most passengers are unaware that the chief danger of leaving baggage unattended is that the baggage could be tampered with by terrorists. Passengers fear only that it will be stolen, a risk which they feel it is their own right to take. Thus, law-abiding and conscientious citizens sometimes leave bags alone for a few moments and trigger an expensive and ultimately pointless utilization of security resources because airport personnel must report the bag, and both local police and federal officials must respond.\textsuperscript{225} Most airports already have television monitors installed that passengers watch while waiting or collecting baggage. Such monitors ought to provide practical information which passengers could use to help make themselves and their fellow passengers safer. Few of us would recognize what was happening even if we were watching a terrorist opening a suitcase and activating a bomb. We have a right, and a need, to know what to look for.
There is no convenient, standardized system whereby passengers can become proactively involved in their own safety, such as a mechanism for reporting behavior that seems dangerous or suspicious. Standard questions asked of passengers should be expanded to help them know what to look for, not only in their own baggage, but in their surroundings. If passengers were advised to be observant and believed that their observations would be valued and acted upon, people would take care to watch baggage and would report those who do not. This would both increase security and reduce the burden on security resources that could be more effectively used otherwise.
Part of the problem stems from the FAA's attempt to make people feel safe about air travel, which has resulted a rather paternalistic approach to air safety. The FAA has avoided public education as a means of enhancing travel safety since public discussion could make people feel that air travel is unsafe.\textsuperscript{226} Unfortunately, the Gore Com-
\begin{itemize}
\item \textsuperscript{223} Such a proposal is outlined in greater detail by Hodes, \textit{supra} note 57.
\item \textsuperscript{224} See generally National Highway Transportation Safety (visited Feb. 29, 2000) <http://www.nhtsa.dot.gov.html> (announcing results and additional grant funding for greater auto safety).
\item \textsuperscript{225} Kunicki Interview, \textit{supra} note 77.
\item \textsuperscript{226} The FAA received several warnings about threats to Pan Am Flight 103 up to two weeks before the bombing took place, but Pan Am allegedly refused to tell the pilots for fear the pilots
\end{itemize}
mission has uncritically adopted rather than challenged this approach.\textsuperscript{227}
The FAA's paternalistic attitude sometimes extends beyond the passengers even to air crew. For example, in November of 1974, TWA Flight 514 crashed into Mt. Weather as it approached Dulles International Airport, killing all ninety-two people on board.\textsuperscript{228} Just six weeks earlier, a United Airlines DC-8 had been cleared to land on the same flight path.\textsuperscript{229} It cleared the peak by approximately thirty feet.\textsuperscript{230} The FAA showed little interest, but the pilot and copilot were concerned when they discovered how close they had come to disaster.\textsuperscript{231} The FAA assured the pilots that air traffic controllers could and would prevent such an event from happening again.\textsuperscript{232} Nevertheless, the FAA did not distribute information about the danger.\textsuperscript{233} Had the FAA made a priority of informing the airlines and flight crews of the danger, it is likely that the pilots on Flight 514 would have known to avoid the danger and could have protected themselves and their
\textsuperscript{227} See generally Final Report, supra note 4 (neglecting to recommend public education as a means of improving security).
\textsuperscript{228} See Kapustin, supra note 40, at 12.
\textsuperscript{229} See id. at 13.
\textsuperscript{230} See id. at 12-13.
\textsuperscript{231} See id. at 13.
\textsuperscript{232} See id.
\textsuperscript{233} See id.
passengers. Rather than lose control of the situation or admit that danger existed, the FAA chose to keep everybody ignorant.\textsuperscript{234}
That is the same approach taken by the Gore Commission, and it will not work. Nobody would crash into a mountain known to be there, and nobody would fly on a plane known to be unsafe. Knowledge is the best security. The true balance that must be struck in airline security is not between danger and personal liberties, but it is between the risk of danger and costs to the airline industry of making threats known so that passengers and crew can avoid them.
d. Ombudsmen
One of the major obstacles a passenger faces in traveling is where to get information on how to log a complaint or report suspicious activity. With all of the different local, state, and federal agencies at work, it is often difficult to determine which office is the right office to handle the matter. Thus, a twenty-four hour central office, providing ombudsmen between the passengers and airport operators, federal agencies and airlines, would ease the flow of disseminating information and expedite the process of making a report.
The ACLU maintains an on-line complaint form that is accessible through its web-site.\textsuperscript{235} Using this form, the ACLU has attempted to remove some of the bureaucratic complexity of registering a complaint against an official. The ACLU uses the data from the complaints to determine whether discriminatory or abusive patterns exist.\textsuperscript{236} The web-site, however, is not intended for reporting suspicious behavior or for obtaining safety information, and so functions only to report unconstitutional behavior—not dangerous activity.
e. Enforceable Passenger Bill of Rights
Airport screening searches are valid only if they recognize the right of a person to avoid search by electing not to board the aircraft.\textsuperscript{237} Many passengers traveling through the airport do not understand their rights as passengers.\textsuperscript{238} Furthermore, even most lawyers today would not guess that complaints about security abuses should be filed
\textsuperscript{234} See Kapustin, \textit{supra} note 40, at 13.
\textsuperscript{235} ACLU Freedom Network, \textit{Passenger Profiling Complaint Form} (visited Feb. 7, 2000) <http://www.aclu.org/forms/complaint.aviation.html>.
\textsuperscript{236} See ACLU Freedom Network, \textit{As Airlines Debut Profiling System, ACLU Launches Web Complaint Form} (visited Sept. 4, 1998) <http://www.aclu.org/news/nl23197a.html>.
\textsuperscript{237} See United States v. Meulener, 351 F. Supp. 1284, 1289-1290 (C.D. Cal. 1972); United States v. Allen, 349 F. Supp. 749, 752 (N.D. Cal. 1972).
\textsuperscript{238} The Gore Commission apparently put low priority on passenger rights by completely neglecting the issue in the Commission's report. The Commission's report spoke in terms of
with an obscure branch of the FAA. Therefore, next to signs explaining prohibited activity in airports,\textsuperscript{239} there should be a comparable sign detailing your rights in an airport. For example, administrative searches in an airport should be publicly defined so that passengers can know if the searches to which they are subjected exceed legal bounds. Since X-ray security devices have been in place for twenty years, as travelers, we expect to place all carry-ons on the X-ray machine and to empty change from pockets and remove watches, but it is unclear where such actions cross the line and become a search.\textsuperscript{240} Such rights must be enforced by an independent agency similar to the United States Equal Opportunity Commission, whose agents should include the on-site ombudsmen described above,\textsuperscript{241} available to assist passengers in protecting themselves, to inform passengers of both their rights and duties, and to reduce friction by creating a safe travel environment.
f. Bifurcated Airline System
The essential constitutional objection to security procedures is the passenger's lack of choice and the basic practical objection is cost. Yet, given a choice most travelers would willingly submit to security procedures and would also be willing to pay for them. Thus, one security solution would be to create a bifurcated airline system giving passengers a constitutionally and economically meaningful choice. Some flights would have full security while remaining flights would not. This would allow passengers a choice of taking the cheaper flight and risk security, and asserting their constitutional rights, or taking a pricier flight, surrendering such rights, and obtaining greater security. Though such a system would solve almost all constitutional and practical security problems, at least on the secure part of the system, it would be expensive to create and maintain, and administratively difficult to double all flight opportunities as the system would require.
\textsuperscript{239} Airports are known for abundant and often confusing signs. In recent years, many airports have posted signs reading “No laughing matter,” and threatening arrest if passengers so much as mention bombs, pirates, or any other threatening words.
\textsuperscript{240} The administrative search doctrine evolved partly from concerns that searches under \textit{Terry v. Ohio}, 392 U.S. 1 (1967), might not suffice to allow effective airline searches. See Rogers, \textit{supra} note 2, at 512-13. Under \textit{Terry}, a peace officer who has a “reasonable suspicion” of the commission of a crime, supported by “articulable facts,” may stop and briefly detain a person and search for weapons and contraband. 392 U.S. at 21.
\textsuperscript{241} See \textit{supra} notes 235-236 and accompanying text.
g. Bureaucratic Reform
It is difficult to regulate the aviation industry given that it requires an interface of local and federal authorities with private industry. Each airport is unique in management, funding, and ownership. Many airports are run by municipalities and most are both enmeshed in political controversy and caught between competitive administrative systems. The proposal for a new Chicagoland airport in Peotone, Illinois caused friction between state officials who favored the proposal, and local and federal officials (and the airlines) who opposed it.\textsuperscript{242}
To implement any effective proposals for improved security, what the nation needs is a new, professional, and independent body insulated from political pressure and existing bureaucratic interests at all levels. Both the FAA and DOT have been in existence so long that they are the victims of entrenched interest groups both in the industry and within their own administration. Most local agencies also face the same issues. Society cannot afford to sustain the existing bureaucratic status quo. An examination of the distribution of funds under Gore Commission recommendations suggests that existing agencies with strong political clout, especially the Pentagon and the FBI, used the public panic about terrorism to justify massive increases in their budgets.\textsuperscript{243} By dismantling the existing administrative structures, we can free talented professionals in the field to utilize their skills, just as FAA and National Aeronautics and Space Administration did in the early days of their existence. Creation of this new body would allow representatives from all areas of aviation to assess and recommend guidelines and procedures for airports to follow. While there are organizations, such as Airports Council International and American Association Airport Executives, membership is voluntary.\textsuperscript{244} These organizations are special interest groups. Membership dues from the airport owners and operators fund these organizations.\textsuperscript{245} Therefore, the main interest represented is for themselves, not those of the public.
\textsuperscript{242} See Dennis Byrne, \textit{Area's future still up in air}, \textit{Chi. Sun-Times}, Aug. 14, 1997, at 33.
\textsuperscript{243} See supra notes 65-103 and accompanying text (discussing the Gore Commission).
\textsuperscript{244} For membership information about these organizations, see Airports Council International-North America, \textit{Membership and Benefits} (visited Feb. 7, 2000) <http://www.aci-na.org/new_website/membership/airport_membership/html>; American Association of Airport Executives, \textit{About AAAE} (visited Feb. 7, 2000) <http://www.airportnet.org/depts/membership/info.htm>.
\textsuperscript{245} See supra note 244.
h. Reporting Immunity
Another obstacle in discovering potential safety hazards at airports is the threat of punitive measures being taken against the officials or the airport when reported. In order to encourage employees, employers, and officials to report security recommendations or complaints, immunity should be granted to both those people reporting and to those against whom the complaint is directed. A review board could initially assess the situation and direct officials to make the necessary changes without taking punitive measures. Currently, this type of program is offered to pilots, encouraging them to report unsafe equipment on airplanes and to encourage pilots to do preliminary investigations prior to boarding the aircraft.\textsuperscript{246}
i. Increased Control of Access to Airfields
Access to airfields must be controlled at all levels. Stricter standards are needed to toughen the regulations on access to all areas airside and landside of the airport. At O’Hare, a revolutionary program is being implemented in the cargo area.\textsuperscript{247} The first system of its kind to be introduced into American airports, drivers bringing cargo to the airport will have to undergo a special fingerprint analysis to gain entrance.\textsuperscript{248} When the thumb is placed in a holder, information about that person will appear at a command center.\textsuperscript{249} This is a very high safety precaution, with only minor intrusion to the person, and places a security emphasis on employees rather than passengers. In addition to personal information (name, employer, etc.), a freight order would also appear so that in the event of an airline accident, the airport officials could verify all deliveries of cargo, as well as, the name of the driver and supplier.\textsuperscript{250}
All employees in the airport should have to undergo this process for gaining access to restricted areas. Currently, O’Hare uses a badging system, that, while effective, does not ensure whether the badge has been stolen and the individual’s PIN number memorized. The fingerprint detector measures not only the fingerprint but also measures the width of the finger and the temperature, making it nearly impossible for a terrorist to gain entry with a detached finger. While not without
\textsuperscript{246} See Chicago O’Hare International Airport Noise Office, Fly Quiet Program (June 17, 1997) (on file with author).
\textsuperscript{247} Videotape: Universal Air Cargo Security Access System (SecurCom, Inc. 1999) (on file with SecurCom, Inc. (888) 826-8401).
\textsuperscript{248} See id.
\textsuperscript{249} See id.
\textsuperscript{250} See id.
high costs, this type of employee information would limit the access of individuals to restricted areas and keep detailed computer records of people on the airfield at all times.
j. Passenger Security Charge ("PSC")
Under current law, each airline passenger pays a three-dollar Passenger Facility Charge ("PFC") for the use of the airport facilities\(^{251}\) and the costs associated with airport maintenance and development.\(^{252}\) This surcharge is collected by the airlines at the time the ticket is sold.\(^{253}\) Some industry professionals contend that the PFC current use should be expanded to include a portion for safety.\(^{254}\) Airline security measures, both those proposed by the Gore Commission and in this Comment, are expensive, but this cost could be manageably funded through a program similar to the PFC.\(^{255}\) An analogous surcharge placed on a ticket for security improvements could also be collected by the airlines.\(^{256}\) The surcharge would be paid by the very people whose safety it helps secure and should be mandated for use for research and technology needed for airport security.
III. Conclusion
[N]ew technology becomes available with increasing speed. However, as technology is not the only answer, human effort must be afforded the proper focus. Therefore, the bottom line is that until some of the new technology is available and in place, tightened security must continue with a watchful eye toward protection of the right to travel.\(^{257}\)
---
251. See 14 C.F.R. § 158 (1998).
252. See id.
253. See id.
254. FAA Reauthorization Bill, S. 82, 106th Cong. § 201 (1999). Currently, there are talks in Congress to raise the PFC charge from $3 to $5, however, the intended use for the increase is for airline competition and not security. PFCs are authorized for use for safety improvements, but not so mandated. This author contends that the increase of $2 should be used only for safety and security enhancements.
255. Chicago O'Hare International Airport has currently committed $20 million of its PFC funds for enhanced security. See Robert C. Herguth, *O'Hare to boost security against terrorists, thieves*, CHI. DAILY HERALD, Nov. 1, 1998, § 1, at 10.
256. Such a move would require federal authorization. "[S]uch charges were prohibited in the early 1970s . . . [and since they were restored in 1990] . . . have been subject to federal restriction." ACI HIGHLIGHTS (Airports Council International-North America, Washington, D.C.), Vol. 36, Jan. 1999, at 3. At $2, a PSC would raise almost 900 million dollars annually, almost 90% of the total Gore Commission recommended expenditures. See President's Proposal, supra note 72 (tabulating the Gore Commission total of $1.097 billion). PFCs currently generate more than $1.3 billion dollars a year (at $3). See id.
257. Reser, *supra* note 34, at 823.
Current political sentiment—fear of terrorism—has been used to justify massive budget expansions of the Pentagon, the FBI, and other agencies, and to justify actions which challenge constitutional rights to travel, privacy, and equal protection. Something surely should be done, but big-budget big-brother responses based on ignorance are no substitute for considered action. Profiling has already been tried, and has been shown to be ineffective. Profiling, and the personal data-gathering and management that it calls for, is the worst aspect of the new proposed security measures, the least likely to be effective, and the most likely to threaten the liberties upon which our nation is founded.
If drastic action is called for to address security concerns, the action should be taken to reorganize the bloated and ineffective bureaucracy. Such creative solutions would inevitably provoke intense resistance within the existing system. Only strong pressure could overcome such resistance. It is in the nation’s interest to harness our increasing concern for airline safety to break down bureaucratic obstacles to safety, not to justify massive uninformed expenditures and limits on personal freedom as the Gore Commission did.
Jamie L. Rhee
|
Diffusion of Ultra-Cold Neutrons in Randomly Rough Channels
Sarah Elizabeth Brent
University of Rhode Island, firstname.lastname@example.org
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DIFFUSION OF ULTRA-COLD NEUTRONS IN RANDOMLY ROUGH CHANNELS
BY
SARAH ELIZABETH BRENT
A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN PHYSICS
UNIVERSITY OF RHODE ISLAND 2018
DOCTOR OF PHILOSOPHY DISSERTATION
OF
SARAH ELIZABETH BRENT
APPROVED:
Dissertation Committee:
Major Professor Alexander Meyerovich
Leonard Kahn
David Freeman
Nasser H. Zawia
DEAN OF THE GRADUATE SCHOOL
UNIVERSITY OF RHODE ISLAND
2018
ABSTRACT
This thesis deals with ultra cold neutrons, or, more precisely, with beams of ultra-cold neutrons. Ultra-cold neutrons are longwave particles produced in a reactor from which they are coming to experimental cells through narrow channels. The beams are collimated so that the distribution of longitudinal and transverse velocities is narrow. The energies of the neutrons that we consider as ultra cold are somewhere around $100neV$.
Neutrons with such low energies have long wavelengths; $\lambda \sim 100nm$. Neutral particles with such large wavelengths exhibit nearly (locally) specular reflection when reflected by the solid surfaces at almost any angle of incidence.
The number of ultra-cold neutrons available for experiment is extremely small. Therefore, a major experimental challenge is not to lose any particles while they travel from the reactor to the lab. Some of the main losses occur in the channel junctions when the neutrons disappear into the gaps between the overlapping channels. We explore the possibility of recovering some of these otherwise "lost" neutrons by making the inside surfaces of the junctions rough: scattering by the surface roughness can send some of the neutrons back out of the gap. This practical goal made us to re-examine diffusion of neutrons through rough channels which is by itself an interesting problem. We assume that the correlation function of random surface roughness is either Gaussian or exponential and investigate the dependence of the mean free path on the correlation radius $R$ of the surface inhomogeneities. My results show that in order to ensure better recovery of the "lost" neutrons the walls of the junction should be made rough with the exponential correlation function of surface roughness with as small a correlation radius as possible. The results also show that the diffusion coefficient and the mean free path of UCN in rough channels exhibit a noticeable minimum at very small values...
of the correlation radius. This minimum sometimes has a complicated structure.
The second goal is the study of UCN in Earth’s gravitational field. One of the most interesting features of ultra-cold neutrons is a possible quantization of their vertical motion by the Earth’s gravitational field: the kinetic energies are so low that they become comparable to the energy of neutrons in Earth’s gravitational field. This results in quantization of neutron motion in the vertical direction. The energy discretization occurs on the scale of several peV.
In the first part of my thesis I ignore the presence of the gravitational field and look at the transport of neutrons through rough waveguides in the absence of gravity. The effects of gravity are be explored in the last part. To streamline the transition I use the common notations suitable for both types of problems.
More specifically, I am studying the diffusion of ultra-cold neutrons in the context of the experiments done at ILL in Grenoble in the frame of the multinational GRANIT collaboration. The parameters used in numerical calculations are the ones most common to ILL experiments. I will be calculating the diffusion coefficient and the mean-free path (MFP) under the conditions of the quantum size effect. Specifically I look at the dependence of the diffusion coefficient and the MFP on the correlation radius of surface inhomogeneities, $R$. In the second and third parts of the thesis I include the study of the neutron diffusion accompanied by slow continuous disappearing of neutrons as a result of penetration into the channel walls. This includes calculating the number of neutrons $N(t = \tau_{ex}, h, R)$, where $\tau_{ex}$ is the experimental value of the time of flight in GRANIT experiments and $h$ is the channel width. I look not only at the square well geometry, but will also include the effects of the Earth gravitational field. The results show that while the neutrons in the square well potential disappear almost immediately, the small perturbation near the bottom of the well caused by the presence of the Earth’s
gravitational field drastically changes the results and is solely responsible for the observed exit neutron count in GRANIT experiments. The shape of the curves describing the exit neutron count on the width of the waveguide is extremely robust. Out brute force calculations also confirms that the earlier biased diffusion approximation is quite accurate.
ACKNOWLEDGMENTS
I would like to acknowledge first and foremost my thesis adviser, Professor Alexander Meyerovich. I am deeply grateful for the guidance, advice, tremendous insight and immeasurable patience and support that you provided to me over the past 6 years.
I would also like to acknowledge Professor Leonard Kahn for the wonderful teacher that he is, for the encouragement that he provided even at times when I had doubts, and for the many informal conversations we had on a vast array of topics.
Sincere thanks go out to Professor Gerhard Muller, for all the physics tools he gave me over the past 6 years, as well as the many enjoyable and insightful conversations.
Thanks to Professor Peter Nightingale, for the crazy difficult classes that provided me with great challenges, as well as for his wit and generosity in sharing knowledge about music, history and other topics outside of physics.
Thank you to Professor Richard McCorkle for being a great support to me in my function as a TA, as well as always having a pleasant outlook on life.
Thank you to Linda Connell for always cleaning up my administrative messes, as well as being a great friend and support over the last 6 years.
Thank you to Steve Pellegrino for helping me with countless technology and computer problems, as well as for his friendship.
Thank you to David Notorianni for always fixing anything that went wrong in the labs or offices, as well as for having a kind heart behind the rough exterior.
Thank you to Professor Donna Meyer, for introducing me to engineering which has become another passion of mine, as well as for being a great role model for a woman in the hard sciences. I have great admiration for you.
Thank you to Professor Stephen Barber, who though not a disciple of the sciences, I have learned so much from, not only in English literature, theory and history but from who you are as a person. You are one of the most generous and loving people I know, thank you for sharing yourself with me, I’m a better person because of it.
Thank you to my dear friend Mauricio Escobar, who has always been a stellar friend as well as a confident and support, even after he himself was done with his Ph.D.
Thank you to Eden Uzrad and the entire Uzrad family in Israel, whom I got to know very well over the course of my Ph.D. Thank you for all the support you gave me, for your generosity, and for opening my eyes about your beautiful country.
Thank you to the woman of my life, Cecile Cres, whom I’ve been lucky to have known since high school. You always encouraged me to pursue my passions, and I thank you so much for being my greatest confident, as well as source of comfort and support in my life.
Lastly, but certainly not least, a huge thank you to my parents for the education they gave me, for always allowing me to follow my dreams, and providing me with endless support in all my endeavors. In particular I would like to thank my mom, who has always had my back 100 percent of the time, even at times when she didn’t agree with my decisions. I wouldn’t be the person I am today without you.
DEDICATION
This thesis is dedicated to the two most important women in my life who have loved and encouraged me always. To my incredibly strong and bright mother, Antoinette Duymovitsh, whom I admire so much, and to whom I owe everything. And to my partner Cécile who encourages and supports me unconditionally on this wild and fun ride called life, and from whom I learn daily.
## Contents
**ABSTRACT**
ii
**Acknowledgements**
vi
**List of Tables**
xii
**List of Figures**
xiii
Chapter 1. Introduction
1
1.1. Preliminary Comments
1
1.2. GRANIT Experiment
8
1.3. Notations and Dimensionless Variables
14
1.4. Theoretical Background
17
Chapter 2. Diffusion Coefficient and Mean Free Path in a Rough Waveguide
26
2.1. Introductory Comments
26
2.2. The Diffusion Coefficient
27
2.3. Mean Free Path
28
2.4. Numerical Results
29
2.5. Conclusions
37
Chapter 3. Neutron Beams between Absorbing Rough Walls: Square Well Approximation
3.1. Description of Problem 44
3.2. Wavefunction for the Square Well 48
3.3. Transition Probabilities and Neutron Count 50
3.4. Numerical Results 52
3.5. Conclusions 60
Chapter 4. Neutrons in the Rough Waveguide in the Presence of Gravity 67
4.1. Gravity-Imposed Changes: Similarities and Differences with the Previous Chapter 67
4.2. Results from the Preceding Work: the Biased Diffusion Approximation 70
4.3. Exact Calculation of the Absorption Time 75
4.4. Numerical Results 78
4.5. Conclusions 85
Chapter 5. Summary and Conclusions 96
5.1. Main Conclusions 96
5.2. Recommendations for Future Work 99
Appendix A. Dimensionless Transition Probabilities 101
Appendix B. Asymptotic Expansion for Transition Probabilities for Surfaces with Exponential Roughness Correlator 103
Appendix C. Diagonal Elements of Transition Probabilities for the Biased Diffusion Approximation 105
Bibliography 107
List of Figures
1.1 Sketch of the experimental cell with a neutron beam between two plates. The neutrons bounce between the "rough" ceiling and "smooth" floor. The neutrons with low vertical velocity, which do not reach the ceiling get to the detector.
1.2 The GRANIT experiment: a more technical sketch of the experiment as a whole. The experimental cell is on the right.
1.3 "Rough" ceiling mirror. The patches 1-5 represent the spots where the roughness has been measured using vertical scanning interferometry.
2.1 Minima of the diffusion coefficient $d(r)$ as a function of the correlation radius for the Gaussian inhomogeneities. The diffusion coefficient starts growing again at larger $r$.
2.2 Diffusion coefficient $d(r)$ for the Gaussian surface correlator over large range of $r$. The minima in $d(r)$ cannot be resolved on this scale.
| Section | Description | Page |
|---------|-----------------------------------------------------------------------------|------|
| 2.3 | The next figure (Fig.[2.4]) shows the diffusion coefficient for the exponential correlation function of surface roughness. | 34 |
| 2.4 | Diffusion for the surface inhomogeneities with the exponential correlation function over large range of $r$. | 35 |
| 2.5 | Mean free path $l(r)$ for the surface with Gaussian roughness over wider range of $r$. | 36 |
| 2.6 | Mean free path for the surface with exponential roughness over wider range of $r$. | 37 |
| 2.7 | Mean-free path $l(r)$ for both Gaussian and exponential correlation functions for small $r$. | 38 |
| 2.8 | MFP for Gaussian correlation function for various channel widths $h = 16, 8, 4$. | 39 |
| 2.9 | MFP for Gaussian (1) and exponential (2) surface correlation functions over a wide range of $r$. | 40 |
| 2.10 | Power law fitting for the MFP $l(r)$ surfaces with the Gaussian correlation function for $r$ from 20 to 40. | 41 |
| 2.11 | The power law fit for MFP $l(r)$ for Gaussian inhomogeneities over the range of $r$ from 40 to 60 | 42 |
| 2.12 | Power law fit for $d(r)$ the exponential surface correlation function over a large range of $r$. | 43 |
| Section | Description | Page |
|---------|-----------------------------------------------------------------------------|------|
| 3.1 | Sketch of neutron beam entering the experimental cell: the neutrons pass between rough "ceiling" and smooth "floor". | 45 |
| 3.2 | Square well levels. | 47 |
| 3.3 | Coefficients $b_j(h)$ Ref[3.3] as a function of the width of the channel $h$ for the square well. | 50 |
| 3.4 | $N_e$ as a function of the cutoff parameter $S1$ for $h = 8$ and $r = 0.65$. Here we can see the initial increase. | 54 |
| 3.5 | Neutron count as a function of the size of the matrix $S1$ for $h = 8$ and $r = 0.65$. We can see how it saturates nicely, at about $S1 = 300$. In this plot we are looking at $N_e$ over a larger scale. | 55 |
| 3.6 | $N_e$ as a function of the size of the matrix $S1$ for $h = 5$ and $r = 0.65$. We are looking at $N_e$ closer scale, so that we can see the initial increase and gradual saturation. | 55 |
| 3.7 | Saturation of the neutron count as a function of the size of the matrix $S1$, for $h = 5$ and $r = 0.65$. | 56 |
| 3.8 | $N_e$ as a function of the matrix size $S1$ for $h = 3$ and $r = 0.65$. We are looking at $N_e$ closer scale, so that we can see the initial increase and gradual saturation. | 56 |
| 3.9 | Neutron count as a function of the cutoff parameter $S1$ and $r = 0.65$ for $h = 3$. | 57 |
| Section | Description | Page |
|---------|-----------------------------------------------------------------------------|------|
| 3.10 | $N_e(h)$ as a function of $h$ for $r = 0.1$. | 58 |
| 3.11 | $N_e(h)$ as a function of $h$ for $r = 1$. | 59 |
| 3.12 | $N_e(h)$ as a function of $h$ for $r = 5$. | 60 |
| 3.13 | $N_e(h)$ as a function of $h$ for $r = 10$. | 61 |
| 3.14 | $N_e(h)$ as a function of $h$ for $r = 30$. | 62 |
| 3.15 | Total neutron count $N_e$ as a function of different correlation radii $r$ for small well width $h = 3$. | 63 |
| 3.16 | Total neutron count $N_e$ as a function of different correlation radii $r$ for well width $h = 5$. | 64 |
| 3.17 | Total neutron count $N_e$ as a function of different correlation radii $r$ for well width $h = 7$. | 65 |
| 3.18 | Total neutron count $N_e$ as a function of different correlation radii $r$ for well width $h = 9$. | 66 |
| 4.1 | Sketch of the gravitational well as a function of z. | 68 |
| 4.2 | The first three eigenvalues for both the square well and the gravitational well as a function of the well width $h$. For better comparison, the bottom of the square well is chosen in the middle of the bottom of the gravitational well, $mgh/2$, and is drifting with $h$. The eigenvalues for the gravitational well are the lower curves, for the SW-the upper. | 69 |
| Section | Description | Page |
|---------|-----------------------------------------------------------------------------|------|
| 4.3 | A few coefficients $b_j$'s as a function of well width size $h$ for both the square (lower curves) and the gravitational wells (upper curves). | 70 |
| 4.4 | The first nine coefficients $b_j$ as a function of the width of the channel $h$ for the gravitational well. The lowest curve is $b_1$ and the highest $b_9$. | 71 |
| 4.5 | The transition probabilities $W_{1j'}$ as a function of $j'$ exhibit the peak around $j' = 100$. | 78 |
| 4.6 | Potential with Earth’s gravity field. | 80 |
| 4.7 | Neutron exit count as a function of $h$, for experimental parameters. | 82 |
| 4.8 | Neutron count as a function of $h$ for correlation radius of roughness $r = 0.1$. | 83 |
| 4.9 | Neutron count as a function of $h$ for correlation radius of roughness $r = 1$. This is approximately twice the experimental value of $r$. | 84 |
| 4.10 | Neutron count as a function of $h$ for correlation radius of roughness $r = 5$. | 85 |
| 4.11 | Neutron count as a function of $h$ for correlation radius of roughness $r = 10$. | 86 |
| Section | Description | Page |
|---------|-----------------------------------------------------------------------------|------|
| 4.12 | Neutron count as a function of $h$ for correlation radius of roughness $r = 20$. | 87 |
| 4.13 | Neutron count as a function of $h$ for correlation radius of roughness $r = 30$. | 88 |
| 4.14 | Neutron count as a function of $h$ for correlation radius of roughness $r = 50$. | 89 |
| 4.15 | Neutron count as a function of $h$ for correlation radius of roughness $r = 100$. | 90 |
| 4.16 | Neutron count as a function of $h$ for correlation radius of roughness $r = 500$. | 91 |
| 4.17 | Neutron count as a function of $h$ for correlation radius of roughness $r = 1000$. | 92 |
| 4.18 | Neutron exit count for fixed well width at $h = 9$, over a large range of $r$. | 93 |
| 4.19 | Neutron exit count for fixed well width at $h = 5$, over a large range of $r$. | 94 |
| 4.20 | Neutron exit count for fixed well width at $h = 3$, over a large range of $r$. | 95 |
CHAPTER 1
Introduction
1.1. Preliminary Comments
The main goal of this thesis is to provide a rigorous theoretical description for the diffusion of ultra cold neutrons (UCN) through narrow rough channels, which is based on the theory of quantum transport in systems with rough boundaries formulated by Meyerovich et al.\cite{1}-\cite{12}. We look at two separate problems: diffusion of the neutrons through rough waveguides on the way from the reactor to the experimental cell and the neutron count for neutrons exiting experimental cell with absorbing walls.
We use numerical computations to investigate the effect of two types of random roughness on the diffusion coefficient and use numerical methods to evaluate the neutron count using the experimental values of input parameters. We analyze two types of potentials inside the cell: one the idealized square well potential (SW) and the other the SW potential with an addition of the gravitational field. The experimental parameters were provided for us by our experimental collaborators at the Institute Laue-Langevin (ILL) in Grenoble, France in the frame of the GRANIT project.
The purpose of this multinational collaborative experimental and theoretical work is two-fold: to investigate the quantization of the motion of UCN by the Earth gravitational field and to create UCN with well-defined energies in the peV range necessary for studies of fundamental forces in quantum field theory.
Typical UCN coming out of the reactor have large wavelengths, $\lambda \sim 100$ nm. Neutral particles with such large wavelengths exhibit nearly (locally) specular reflection when reflected by the solid surfaces at almost any angle of incidence. One of the most interesting features of ultra-cold neutrons is their quantization in the Earth gravitational field: the particle kinetic energies can be so low ($\sim 1$ peV) that they become comparable to the gravitational energy of neutrons in Earth’s gravitational field. This results in quantization of neutron motion in the vertical direction. This discretization is illustrated in the sketch below showing the discrete energy levels of neutrons in the Earth gravitational field. The energy discretization occurs on the scale of several peV. The first experimental observation of such a quantization was done by Nesvizhevsky et al. Ref.[14]–[24] by using the GRANIT spectrometer (see below).
Ultra-cold neutrons are longwave particles produced in a reactor from which they are coming to experimental cells through narrow channels containing various mirrors and collimators. The UCN beams are collimated so that the distribution of longitudinal and transverse velocities is narrow. The energies of the neutrons that we consider as ultra-cold are somewhere around 100 neV, and below.
The particles in the beam that reach the cell have a relatively large horizontal velocity and much smaller vertical velocities. Still standard collimation and cooling methods are insufficient to limit the vertical energies to the peV scale comparable to gravitational energies. The typical UCN beam brought to the experimental cell contains neutrons in thousands of occupied gravitational states making it virtually impossible to study the quantization of vertical motion.
The purpose of the GRANIT spectrometer is to eliminate the particles in higher gravitational states and leave only the ones in the few lowest states. This allows one to achieve both goals: to study the quantization of neutron motion in the gravitational field and to produce neutrons with well-defined energies in the peV range.
The lower surface of the spectrometer is as close as possible to being perfectly smooth, in order to make it to be a perfect reflector which specularly reflects the UCN. The upper surface of the GRANIT cell has microscale roughness. This "rough" ceiling scatters the UCN in higher gravitational states, which can reach it. The scattered neutrons from the higher gravitational states eventually acquire large vertical velocities sufficient to trigger penetration through the walls and disappearance from the system. Due to this setup, only the UCN in low gravitational states, which do not reach the rough ceiling, can continue bouncing along the flat floor and arrive at the exit neutron detector.
The use of rough mirrors as quantum state selectors is possible because the very large horizontal velocities in the beam and peculiarities of quantization of the
vertical motion in the gravitational field. This kind of state selector is used or is planned to be used, in numerous other applications not exclusive to GRANIT experiments or to UCN beams. Some examples of these potential applications include: the observation of quantum gravitational states for other ultra-cold particles and anti-particles in the context of the GBAR project at CERN [[28]-[32]]; the resolution of centrifugal quantum states in UCN in the "whispering gallery" [[26],[27]]; the search for fundamental forces at extra-short range as predicted by the grand unification theory [[33]-[41]]; the test of the weak equivalence principle [[39]-[42]]; the continual extension of understanding of quantum mechanics. Additionally, these GRANIT-like experiments could potentially be used to measure the electric dipole moment of a neutron [[46],[47]], if it exists, help to search for the potential neutron charge [[48],[49]], and make a precise measurement of neutron lifetime [[50]].
The resolution and the quality of the observed quantum gravitational states of UCN rely on the quality of the roughness of the upper surface of the GRANIT cell. Meyerovich et al.. developed a theoretical framework in which they analyzed the particle diffusion along the random rough walls and linked it to the roughness parameters of the rough mirror. The theory generally agrees with the experimental results despite uncertainty in certain parameters.
Additionally, Meyerovich et al. [[52]-[55]] discovered that the shape of the correlation function of surface inhomogeneities (CF) plays a very important role in the diffusion of UCN along rough walls. It turns out that the roughness-driven
transition probabilities between the states are directly proportional to the Fourier image (the so-called power spectrum) of the CF. In previous work, Escobar et al. [8] showed that within the biased diffusion approximation all the information about the surface imperfections can be accounted for in the neutron count as a single parameter $\Phi$, which is a complicated integral of the power spectrum. However, in practice, it is impossible to create imperfections with a predetermined CF on real surfaces, and even if it were possible it would be highly non-trivial to identify this CF.
In order to increase the resolution of the observed quantum gravitational states of the UCN in the GRANIT spectrometer, proper identification of the surface correlator is paramount. If one can establish a superior way to control the necessary random roughness of the scatterer and absorber mirror, it will contribute greatly to the optimization of results from the GRANIT experiment.
In the context of the theoretical background and numerical experiments, we designate the shape of the CF explicitly, and analyze its potential impact on physical variables. In previous papers, Meyerovich et al. [9] have analyzed the generated rough surface by measuring it with the computational analog of STM needle (scanning tunneling microscope). Unlike the CF used to generate the surface, the correlator was extracted by direct computation and analyzed using various fitting functions. Alternatively, the extracted correlator was fed directly into the equations for the observables without the fitting functions. The reason for the importance of the numerical experiments with the simulated surfaces lies in showing
how to avoid certain limitations that might stymie the dependable identification of a surface correlator for a real surface.
There is also a supplementary practical issue. The number of ultra-cold neutrons available for experiment is extremely small. Therefore, a major experimental challenge is not to lose many particles while they travel from the reactor to the lab. The main losses occur in the channel junctions when the neutrons get into the gaps between the overlapping channels. Therefore the minimization of losses in channel junctions becomes an important goal which will also be approached in this thesis.
This thesis is arranged as follows:
In the remainder of Chapter 1, we will provide a fairly detailed description of the experiment and its setup used by GRANIT to observe the quantum gravitational states of the ultra cold neutrons. In particular we will describe the GRANIT cell, and introduce the important parameters that are used to describe the roughness of the surfaces of the GRANIT mirror. In section 2 we will discuss the details of the mirror used in the newer experiments including the design and providing a description of how they made the roughness. In section 3 we introduce the main parameters and dimensionless variables. And, finally, in section 4, we will provide the main equations and the theoretical framework for the quantum transport equation and diffusion.
In Chapter 2, we will explore the possibility of recovering these "lost" neutrons that we discussed above by making the inside surfaces of the junctions rough:
scattering by the surface roughness can turn some of the neutrons back. This practical goal made us to re-examine diffusion of neutrons through rough channels which is by itself an interesting general problem. We assume that the correlation function of surface roughness is either Gaussian or exponential (see below) and investigate the dependence of the mean free path on the correlation radius $R$. Our conclusion is that if ideally we could create the type of roughness we want, it would be better to use exponential roughness.
In Chapter 3, we will be discussing the exit neutron count in an idealized condition, in the square-well potential without gravity. This involves solving large sets of equations with complicated coefficients which tie together neutrons in thousands of quantum states. We first look to investigate the exit neutron count as a function of matrix size, in order to assess the value of the possible cutoff. The matrix size here being the number of equations we are solving. In other words, to reduce the computation time, we deduce what size of the matrix is sufficient for our computations to be accurate. We then cut off the matrix at the cutoff parameter and proceed to extract the neutron count and its dependence on the width of well $H$. In the case of the square well potential we will see that the neutron count should go quickly to zero.
In Chapter 4, we will be doing something very similar to Chapter 3, except this time we take into account the gravitational potential. To simplify the computations, we assume that the matrix of the interstate transition probabilities has a block structure. The first block contains the transitions between the lowest
(gravitational) states. Since for the higher states there is practically no difference between the gravitational and square well states, the other three blocks describe the transitions between the square well states and between the gravitational and square well states. From this we derive the neutron count.
In the last chapter we will summarize the results presented earlier, and discuss some suggestions for what can be done looking towards the future.
1.2. GRANIT Experiment
1.2.1. Description of the Actual GRANIT Experiment
Neutrons are elementary particles with no charge and a relatively long lifetime ($\sim 900$ s) compared to many other elementary particles, such as mesons ($\sim 10^{-17} - 10^{-8}$ s). This makes neutrons quite a good candidate for experimental observation of quantum mechanical bound states in the weak Earth’s gravitational field [[59]]. The quasi-classical estimation of energy levels of bouncing quantum mechanical particles on an ideal horizontal surface in the Earth gravitational field gives a spectrum of a few peV for the lowest energy states of the neutrons [[56]-[58]]. Such low energies make the observation of gravitational quantum bound states very difficult. The primary reason for this is the weakness of the gravitational field compared to the electromagnetic and nuclear forces.
The first experimental observation of quantum mechanical bound states of neutrons in the Earth’s gravitational field was made by Nesvizhevsky et al. in 2002 [[14]–[24]] after a series of experiments in high precision neutron gravitational spectrometry (GRANIT). The GRANIT experiment uses the fact that neutrons have a relatively long lifetime by sending a collimated beam of UCN to the cell through a long complicated waveguide with reflective walls.
One can visualize in a simple way the observation of gravitationally induced quantum states of UCN experiment. There is a collimated beam entering an experimental cell (see Fig.1.1) consisting of a smooth "floor" and a rough "ceiling". More details of the GRANIT experiment will be discussed below [[14]-[24]].
More explicitly, we have a collimated beam of UCN with a large horizontal velocity on the order of $\sim (5 - 15)$ m/s and a small vertical velocity of a few cm/s.
propagating between two parallel horizontal sapphire mirrors. The bottom mirror or "floor" is made as close to perfect as possible. This ensures high probability of specular reflection for the bouncing neutrons. The upper mirror or "ceiling" has a rough surface which is made rough by simply scratching the surface [[16],[61]]. This rough mirror effectively serves as a selector for the vertical component of the velocity of the neutrons. The scattering by the rough ceiling makes the velocity vector turn, which increases the vertical component of velocity and, therefore, the probability of absorption of the neutrons by the wall material. When the vertical velocity exceeds a certain critical value ($\sim 4$ m/s) as a result of scattering by roughness, the neutrons penetrate the wall and disappear. Only the neutrons with a low vertical velocity do not reach the rough ceiling, do not scatter and, therefore, survive.
Figure 1.2. The GRANIT experiment: a more technical sketch of the experiment as a whole. The experimental cell is on the right.
The location of the waveguide is in the uppermost part of the spectrometer. The reason for this is to isolate it from the effects of external vibrations and from electromagnetic fields. The mirrors in the waveguide can be moved or even interchanged. The positions can be adjusted vertically and horizontally depending on what is needed in the experiment [[23]-[25]]. The configuration that we use in this thesis is the one shown in the figure above in which the edges of the mirrors are perfectly aligned and are of the order of 10cm long. The length represents the minimum horizontal distance covered by the UCN inside the cell; the estimated flight time is about 20 ms. Additionally, the vertical separation between the mirrors (the width of the waveguide $H$) can be changed. The minimal width of the waveguide is $\sim 15 \mu m$, which is comparable to the semi-classical amplitude of the bounces of UCN in the ground state. The quantization of the UCN by the Earth’s
gravitational field translates into the quantization of the amplitudes of the bounces from the floor mirror.
Ideally, the neutron count at the location of the detector should be a step function of the width $H$ of the waveguide. The reason why we should have a stepwise type function is because of the quantum size effect. The quantum size effect occurs from a gravity-induced perpendicular quantization of the motion to the bottom of the mirror, and leads to a split in the energy spectrum into mini-bands. It is interesting to note that the sharpness of the quantum size effect in neutron count is related to the increase in roughness of amplitude $l$ rather than the correlation radius of roughness $R$ (see below).
The roughness of the imperfections of the ceiling mixes the gravitational states and broadens the energy levels. Below, we provide a quantitative description of the roughness parameters governing the surface inhomogeneities.
1.2.2. Experimental Analysis of the Mirror Roughness
One of the main goals of the GRANIT project is to continuously refine the observation of the UCN spectrum. Since the first experiments in 2002, there have been improvements made to the GRANIT spectrometer in order to reduce uncertainties in the waveguide. Various parameters such as the correlation radius of roughness $R$, amplitude of roughness $l$, and the oscillation frequency for neutrons in the gravitational well $\tau_0$ have been adjusted and measured more accurately. The latest improvement was the installation of a new large rough mirror on the "ceiling".
The dimensions of the mirror are shown in the Fig.[1.3]. The UCN are propagated along the 90mm long edge. The five square patches in the figure represent
the areas where the mirror roughness has been measured. Each patch in the Figure represents $0.504 \times 0.504$ mm$^2$ and consists of a matrix of $\sim 2500 \times 2500$ experimental data points for which the surface position with respect to a mean reference plane was measured. The surface roughness was measured using Vertical Scanning Interferometry (VSI) technique. The surface was scanned using a light source that splits into two coherent light beams. One of the two beams is sent towards a mirror which is coupled with a different light beam that has been reflected from a sample (amplitude of roughness of 0.5 Å). The interference patterns are then analyzed using a CCD camera and provide a surface profile. Unfortunately however, this technique is not perfect. For example, the measurement fails if some peak is too sharp and therefore the beam doesn’t reflect back onto the detector. The experimental data on the surface profile were analyzed numerically. It was determined that the roughness correlation function most likely has an exponential shape. [9]
This technique though is more appropriate than other scanning techniques such as the Atomic Force Spectroscopy. One of the reasons that VIS is better is that the scanned surface is considerably larger than the correlation radius.
1.3. Notations and Dimensionless Variables
For the purpose of this work it is useful to introduce some uniform notations for the calculations in both presence and absence of gravitational field. Some of the parameters below will be used to make the equations dimensionless. We are looking at the effects of gravity on the transport of the UCN.
1. In this case it is useful to measure all lengths in units of:
\[
l_0 = \left( \frac{\hbar}{2m^2g} \right)^{1/3} \approx 5.87 \, \mu m,
\]
This is the amplitude of the particle bouncing in the lowest quantum state in the presence of the Earth gravitational field.
2. The energy scale is defined by:
\[
e_0 = mg l_0 \sim 0.602 \, \text{peV},
\]
This is the gravitational energy of the neutron in the ground state.
3. The velocity scale is defined by:
\[
v_0 = \sqrt{2gl_0} = \frac{\hbar}{ml_0} \sim 1.1 \times 10^{-2} \, \text{m/s}.
\]
4. The time scale is given by:
\[
\frac{1}{\tau_0} = \frac{\sqrt{2\pi}}{4m} \frac{\hbar}{l_0^2} \sim 1149 \, \text{s}^{-1}.
\]
This is roughly the frequency of bounces in the lowest state.
5. The width of the waveguide \( H \) in units of \( l_0 \) is:
\[
h = \frac{H}{l_0}.
\]
6. The roughness correlation radius $R$ expressed as a dimensionless variable is:
$$r = \frac{R}{l_0}.$$
7. Similarly, the amplitude of roughness $l$ as a dimensionless variable is expressed as:
$$\eta = \frac{l}{l_0}.$$
8. The quantized energy levels $E_j$ of the ultra-cold neutrons in the gravitational well are given by:
$$\lambda_j = \frac{E_j}{e_0},$$
9. The absorption threshold $U_c$ of the mirror material is given by:
$$u_c = \frac{U_c}{e_0},$$
where $U_c \approx 100$ neV, and, therefore, $u_c \approx 1.4 \times 10^5$.
10. The flight time for the ultra-cold neutrons through the waveguide of the length $L$ is given by:
$$\tau_L = \frac{L}{v_x},$$
In experimental conditions $\tau_L \approx 2 \times 10^{-2}$s. In dimensionless units $\tau_L/\tau_0 \approx 26$.
11. The neutron momenta are measured in units of:
\begin{equation}
q_0 = \frac{\hbar}{l_0}.
\end{equation}
### 1.4. Theoretical Background
#### 1.4.1. Quantum Size Effect (QSE)
Ultra-cold neutrons (UCN) are longwave particles. We are looking at UCN in narrow waveguides in which the width is comparable to the wavelength and the motion across the waveguide is quantized. This QSE automatically discretizes the initially continuous equations. This quantization turns out to be very fortuitous as it helps in numerical calculations: if we were working with a continuous system, we would need to discretize the problem anyway. QSE leads to a split of the energy spectrum $\epsilon(p)$ into a set of minibands $\epsilon_j(q)$ such that $\epsilon(p_x, q) \longrightarrow \epsilon_j(q)$, where $p$ is the 3D momentum, and $q$ is the 2D momentum in the plane of the surface.
More explicitly, an initially parabolic spectrum, $\epsilon(p) = p^2/2m$ becomes
\begin{equation}
\epsilon_j(q) = \frac{1}{2m} \left[ \left( \frac{\pi \hbar j}{H} \right)^2 + q_j^2 \right]
\end{equation}
and the 2D momentum for miniband $j$ becomes
\begin{equation}
q_j^2 = [2mE - \left( \frac{\pi \hbar j}{H} \right)^2]
\end{equation}
where $E$ is the overall kinetic energy of particles, $m$ is the mass of the neutrons, and $H$ is the width of the channel.
In an ideal waveguide, the quantum levels are well defined and the states are not mixing. Scattering by random surface inhomogeneities leads to inter- and intraband transitions and eventually mixes and broadens the quantum states.
Sometimes, as in experiments performed at ILL (Grenoble), the waveguides, or, more precisely, one of the neutron mirrors, are made rough on purpose.
### 1.4.2. Transport Equation
Studies on the effect of random surface roughness on wave or particle scattering describe the diffusion flows of UCN along a rough waveguide. Meyerovich et al. ([1]-[8]) developed a rigorous theoretical framework of quantum transport theory in system with random rough boundaries. This framework incorporates the boundary scattering directly into the bulk transport equation. It includes the roughness of the walls explicitly into the roughness-driven transition probabilities between quantum states. The transport equation for distribution functions $n_j(q)$ in a miniband $j$ has the form
\[
\frac{dn_j}{dt}(q) = 2\pi \sum_{j'} \int W_{jj'}(n_j - n_{j'}) \delta(\epsilon_{jq} - \epsilon_{j'q'}) \frac{d^2q'}{(2\pi)^2}
\]
where \( n_j(q) \) is the distribution function of the particles, \( \epsilon_{jq} \) is the energy spectrum, \( q \) is the momentum in the plane parallel to the surface, and \( W_{jj'}(q,q') \) are the scattering-driven probabilities of transitions between the states \( \epsilon_j(q) \) and \( \epsilon_{j'}(q') \).
The probabilities of direct transitions from the lowest states to the continuous spectrum above the threshold \( U_c \) are negligible and such transitions can be disregarded. After integration over the energies, the transport equation acquires the following form:
\[
\frac{\partial N_j}{\partial t} = \frac{m}{2\pi} \sum_{j'} \int d\theta W_{jj'}(|q_j - q_{j'}|)(N_{j'} - N_j)
\]
where \( N_j \) is the number of neutrons in the state \( j \), and \( \theta \) is the angle between \( q_j \) and \( q_{j'} \).
Our goal is to find the diffusion coefficient and the mean-free path, which is proportional to the diffusion coefficient. After standard transformations (a more detailed derivation can be found in the Appendices) the transport equation reduces to a set of linear equations for \( \nu_j(q_j) \):
\[
Q_j = -m \sum_{j'} \frac{\nu_{j'}(q_{j'})}{\tau_{jj'}}
\]
Here \( Q_j \) is the momentum, the transition times \( \tau_{jj'} \) are given below by Eq.(1.24), and \( \nu_j \) is the first angular harmonic of the distribution function \( n_j^{(1)} = \nu_j \delta(\epsilon - \epsilon_F) \).
at $q = q_j$, Ref.[4],[5]]. The equations can be made dimensionless using
$$q_j q_0 = -m \sum_{j'} \frac{\tilde{\nu}_{jj'} \nu_0}{\tilde{\tau}_{jj'} \tau_0}$$ \hspace{1cm} (1.17)
which leads to
$$q_j = -\frac{m \nu_0}{q_0 \tau_0} \sum_{j'} \frac{\tilde{\nu}_{j'}}{\tilde{\tau}_{jj'}},$$ \hspace{1cm} (1.18)
where
$$q_j = \frac{Q_j}{q_0}, \quad \tilde{\nu}_j = \frac{\nu_j}{\nu_0} \text{ and } \tilde{\tau}_{jj'} = \frac{\tau_{jj'}}{\tau_0}.$$ \hspace{1cm} (1.19)
Finally, the dimensionless transport equation acquires the form:
$$q_j = -\frac{m l_0^2}{h \tau_0} \sum_{j'} \frac{\tilde{\nu}_{j'}(q_{jj'})}{\tilde{\tau}_{jj'}}.$$ \hspace{1cm} (1.20)
### 1.4.3. Transition Probabilities
The roughness-driven transition probabilities between quantized states have the following form:
$$W_{jj'} = \zeta |\psi_j(h)|^2 |\psi_{j'}(h)|^2 U_c^2$$ \hspace{1cm} (1.21)
if the absorption threshold $U_c$ is finite. Alternatively,
$$W_{jj'} = \frac{1}{4m^2} \zeta |\psi'_j(h)|^2 |\psi'_{j'}(h)|^2$$ \hspace{1cm} (1.22)
when the absorption threshold $U_c \to \infty$. Here $j$ and $j'$ are the miniband indices, $\zeta$ is the correlation function of surface homogeneities (see below), $\psi_j(h)$ is the wavefunction at the surface.
In the case of the square well potential this equation becomes the following:
\begin{equation}
W_{jj'}(q,q') = \frac{\hbar}{m^2L^2} \zeta \left( \frac{\pi hj}{L} \right)^2 \left( \frac{\pi hj'}{L} \right)^2.
\end{equation}
The transitions times in the transport equation are directly related to the angular harmonics of these transition probabilities as follows:
\begin{equation}
\frac{2}{\tau_{jj'}} = m \sum_{j''} \left[ \delta_{jj'} W^{(0)}_{jj''} - \delta_{j'j''} W^{(1)}_{jj'} \right]
\end{equation}
### 188.8.131.52. Correlation Function of Roughness
The correlation function of surface roughness (CF) is defined as:
\begin{equation}
\zeta(|s|) = \langle \xi(s_1)\xi(s_1+s) \rangle \equiv A^{-1} \int \xi(s_1)\xi(s_1+s)ds_1,
\end{equation}
\begin{equation}
\zeta(|p|) = \int d^2se^{iq_s s}\zeta(|s|) = 2\pi \int_0^\infty \zeta(s) J_0(qs) s ds,
\end{equation}
where $\xi(|s|)$ is the exact profile of the wall and $A$ is the area over which the averaging is done. The mathematical form of the CF cannot be found theoretically except in very few instances in which we have exactly solvable models of surface
roughness. It is usually assumed that the CF has the following general form:
\[
\zeta(x) = l^2 \varphi(x/R)
\]
with some function \( \varphi(x/R) \), where \( l \) and \( R \) are the average amplitude and correlation radius. However, nothing prevents the CF to acquire a more complicated form, for example, with several correlation scales \( R_c \). In calculations we assume that we know the shape of the CF. The most commonly used correlation functions have either the Gaussian
\[
\zeta(s) = l^2 \exp(-s^2/2R^2),
\]
or exponential
\[
\zeta(s) = l^2 \exp(-s/R)
\]
forms. Sometimes people also use a CF with a power law shape. Here \( R \) is the correlation radius of surface inhomogeneities., \( r = R/l_0 \), and the dimensionless
amplitude is defined as $\eta = l/l_0$. There are reasons to believe that the correlation function in Grenoble experiments might be exponential, Ref.[9].
The angular harmonics of the Gaussian correlation function are
\begin{equation}
\zeta^{(0)} = 4\pi l^2 R^2 \left[ e^{-q q' \cdot r^2} I_0(q q' \cdot r^2) \right] e^{-r^2/2(q-q')^2}
\end{equation}
\begin{equation}
\zeta^{(1)} = 4\pi l^2 R^2 \left[ e^{-q q' \cdot r^2} I_1(q q' \cdot r^2) \right] e^{-r^2/2(q-q')^2}
\end{equation}
This means the transition probabilities $W$, Eq.(1.23), are equal to Ref.[4]:
\begin{equation}
W_{jj'}^{(0)} = \frac{\hbar}{m^2 L^2} \left( \frac{\pi j}{L} \right)^2 \left( \frac{\pi j'}{L} \right)^2 4\pi l^2 R^2 \left[ e^{-q q' \cdot r^2} I_0(q q' \cdot r^2) \right] e^{-r^2/2(q-q')^2}
\end{equation}
\begin{equation}
W_{jj'}^{(1)} = \frac{\hbar}{m^2 L^2} \left( \frac{\pi j}{L} \right)^2 \left( \frac{\pi j'}{L} \right)^2 4\pi l^2 R^2 \left[ e^{-q q' \cdot r^2} I_1(q q' \cdot r^2) \right] e^{-r^2/2(q-q')^2}
\end{equation}
In dimensionless variables,
\begin{equation}
w_{jj'}^{(0)} = \frac{8\pi r^2}{\sqrt{2\pi} h} \left( \frac{\pi j}{h} \right)^2 \left( \frac{\pi j'}{h} \right)^2 \left[ e^{-q q' \cdot r^2} I_0(q q' \cdot r^2) \right] e^{-r^2/2(q-q')^2}
\end{equation}
\begin{equation}
w_{jj'}^{(1)} = \frac{8\pi r^2}{\sqrt{2\pi} h} \left( \frac{\pi j}{h} \right)^2 \left( \frac{\pi j'}{h} \right)^2 \left[ e^{-q q' \cdot r^2} I_1(q q' \cdot r^2) \right] e^{-r^2/2(q-q')^2}
\end{equation}
where the dimensionless roughness parameters and $r$ and $\eta$ can be used as free parameters in the identification of surface correlations. These two parameters are often sufficient to describe the surface roughness.
The dimensionless transition probabilities for exponential roughness can be written as
\begin{equation}
\Omega = 2r \sqrt{\frac{qq'}{1 + r^2 (q + q')^2}}
\end{equation}
\begin{equation}
w_{jj'}^{(0)} = \frac{32r^2\eta^2}{\sqrt{2\pi}h^2} \left(\frac{\pi j}{h}\right)^2 \left(\frac{\pi j'}{h}\right)^2 \frac{E(\Omega)}{\left(1 + r^2 (q - q')^2 \sqrt{1 + r^2 (q + q')^2}\right)}
\end{equation}
\begin{equation}
w_{jj'}^{(1)} = \frac{32r^2\eta^2}{\sqrt{2\pi}h^2} \left(\frac{\pi j}{h}\right)^2 \left(\frac{\pi j'}{h}\right)^2 \frac{(1 + r^2 (q + q')^2) E(\Omega) - (1 + r^2 (q - q')^2 K(\Omega))}{\left(1 + r^2 (q - q')^2 \sqrt{1 + r^2 (q + q')^2}\right)}
\end{equation}
The diffusion of ultra-cold neutrons displays a strong directional upward bias in terms of the transitions between $j \rightarrow j'$. This bias is due to the rapid growth of the product of the wavefunctions on the boundary $\left|\psi_j(h)\right|^2 \left|\psi_{j'}(h)\right|^2$. This allows a growth of roughly as $j^2 j'^2$, see Eq.(1.23). There are two main consequences of this bias. The first one being that the strong upward bias may allow one to neglect particles returning back to the lowest states. And the second consequence is that the time necessary for a neutron in one of the lowest gravitational states to
diffuse upward towards the absorption barrier is spent almost entirely on the first transition.
In the next chapter we will examine more closely the process of diffusion, and expand upon and develop a more detailed theoretical approach.
CHAPTER 2
Diffusion Coefficient and Mean Free Path in a Rough Waveguide
2.1. Introductory Comments
Let us examine the transition probabilities which for the sake of the numerical computations need to be made dimensionless. Furthermore, we are going to define some parameters used in the numerical computations. In the context of our research, we want to look at both Gaussian and exponential roughness associated with the correlation functions $\zeta$, which together with the wavefunctions at the wall form the transition probabilities, Eq.(1.36)-Eq.(1.37), and Eq.(1.39)-Eq.(1.40).
The transition probabilities are proportional to the square of the amplitude of roughness $\eta$. Therefore, the scaling of the results with the roughness amplitude $\eta$ is trivial and in most of the computations we simply assume $\eta = 1$. The scaling of the results with the correlation radius $r = R/l_0$ is complicated and is not known beforehand. One of our main goals is to find out the dependence of the diffusion parameters on $r$.
In relevant experiments the width of the channels leading to the cell is $H = 50 \mu m$, and the particle energy is $E = 150 \text{ neV}$; this makes $h = 8.52$, and $e = 2.49 \cdot 10^5$. The highest occupied quantum level $j_{\text{max}}$ satisfies the inequality,
\begin{equation}
e - \frac{\pi^2 j_{\text{max}}^2}{h^2} \geq 0
\end{equation}
Solving for $j_{\text{max}}$ we get
\begin{equation}
j_{\text{max}} = \sqrt{\frac{eh^2}{\pi^2}} = 1352
\end{equation}
which means that the transport equation in this case reduces to a set of 1352 coupled equations.
### 2.2. The Diffusion Coefficient
The main purpose of this section of the work was to find the diffusion coefficient and the mean free path for UCN in rough channels. We are trying to examine how the diffusion coefficient changes under different conditions. More explicitly, we are interested in its dependence on $r$. Diffusion is a process that originates from random motion of particles when there is a net flow from one region to another. As a result, in our case, in the presence of a concentration gradient $\nabla \rho$ the diffusion equation reduces to
\begin{equation}
\frac{1}{Sm} \nabla \rho * Q_j = - \sum_{j'} \frac{v_{jj'}}{\tau_{jj'}},
\end{equation}
where $\rho$ is the particle density. Its concentration gradient $\nabla \rho$ is a simple scaling parameter, which, in the end, cancels out from the equation for the diffusion coefficient. After this cancellation, the diffusion coefficient $D$ becomes
$$D = -\frac{1}{m} \sum_{j'} Q_j \nu_{j'} \quad (2.4)$$
The dimensionless diffusion coefficient
$$d = D/d_0 = \sum_{j'} d_j,$$
$$d_j = q_j \tilde{\nu}_j,$$
$$d_0 = h/m = 6.3 \cdot 10^{-8} m/s^2.$$
The dimensionless distributions $\tilde{\nu}_j = \nu_j/l_0$ are obtained from numerically solving the transport equation.
### 2.3. Mean Free Path
We also want to calculate the particle mean-free path (MFP) in a rough waveguide. The mean free path in very basic terms is the average distance traveled between collisions. Here we define it with respect to the diffusion coefficient as
$$L = D/v, \quad (2.5)$$
where \( v \) is the velocity. The dimensionless velocity
\[
\tilde{v} = \frac{1}{v_0} \sqrt{\frac{2e_0}{m}} \sqrt{e}
\]
where \( v_0 = 1.5 \cdot 10^{-2} m/s \). The dimensionless mean free path \( \ell = L/l_0 \),
\[
\ell = \frac{2d_0}{l_0 v_0} \frac{d}{\tilde{v}} = \frac{2d_0}{l_0} \sqrt{\frac{m}{2e_0}} \frac{d}{\sqrt{e}}.
\]
As one can clearly see the MFP is intimately related to the diffusion coefficient.
### 2.4. Numerical Results
Before presenting the results, let us summarize the dimensionless equations from above. The transport equation,
\[
q_j = -\frac{ml_0^2}{h\tau_0} \sum_{j'} \frac{\tilde{\nu}_{j'}(q_{j'})}{\tilde{\tau}_{jj'}},
\]
contains the transition times
\[
\frac{2}{\tilde{\tau}_{jj'}} = m \sum_{j''} \left[ \delta_{jj'} w^{(0)}_{jj''} - \delta_{j'j''} w^{(1)}_{jj'} \right].
\]
The dimensionless harmonics of the transition probabilities for the exponential and Gaussian roughness correlators are given in explicit detail in the Appendix A.
For the overall and "partial" diffusion coefficients \( d \) and \( d_j \) the dimensionless equations are as follows
\[ d = \frac{D}{d_0} = \sum_j d_j, \]
\[ d_j = q_j \tilde{\nu}_j. \]
Finally, the MFP is
\[(2.8) \quad \ell = \frac{2d_0}{l_0} \sqrt{\frac{m}{2e_0}} \frac{d}{\sqrt{e}}.\]
Using the dimensionless equations for the transition probabilities from the previous section, we are now able to perform computations to get the diffusion coefficient and MFP. The computations are done in Mathematica, where we use the function `LinearSolve[m,b]` which finds an x that solves the matrix equation m.x==b. to get the \( \nu \) values. This is the part of the program that is computationally the longest as it essentially solves a system of 1352 linear equations with complicated coefficients and varied parameters. After that, we calculate the dimensionless partial diffusion coefficients,
\[(2.9) \quad d_j = \tilde{\nu}_j q_j\]
and sum them to get the overall diffusion coefficient. We easily get the MFP by using Eq. (2.8).
In our numerical simulations we were using a fixed channel width $h = 8.52$ (the number given to us by GRANIT experimentalists) and were changing the correlation radius of surface inhomogeneities $r$. Before we go into the descriptions of the various curves, it is important to note that all the curves for $d(r)$ are expected to have the minimum at, approximately, $qr \sim 1$ for both Gaussian and exponential surface correlators. The experimental value of the particle energy is $E = 150$ neV, i.e., $e \approx 2.5 \times 10^5$, which makes $q_1 \approx 500$. This means that the minimum corresponds to very small values of $r$, $r \sim 0.002$, and cannot be resolved on many of the curves below. The explanation for this minimum is rather simple. The scattering by surface inhomogeneities is most effective at $qr \sim 1$ leading to a minimum in the diffusion coefficient $d(r)$. There could be several small minima at $q_j r \sim 1$ but all corresponding values of $r$ are small. For this reason, below we will show mostly the results for noticeably larger values of $r$, i.e., to the right of the minimum.
Fig.[2.1] presents $d(r)$ around the minimum. The computation was done for Gaussian inhomogeneities; the figures for the exponential correlation function look similar (Fig.[2.2]). Note that the values of the correlation radius close to the minimum $r \sim 0.002$ are too small to be studied experimentally.
Fig.[2.3] shows the total diffusion coefficient $d$ as a function of the correlation radius $r$ of Gaussian surface inhomogeneities plotted up to $r = 15$. The minimum $d(r)$ is barely noticeable on this scale. We see that the diffusion coefficient rapidly increases as the correlation radius increases. This is understandable: with increasing $r$ the surface becomes smoother and the effective scattering cross-section decreases.
Figures [2.5] and [2.6] show the MFP $l(r)$ for Gaussian and exponential correlation functions of surface inhomogeneities. It is hard to plot the results for the exponential correlator on the same plot with the Gaussian one: $l(r)$ for the exponential correlator increases by orders of magnitude slower than for the Gaussian correlator due to the fact that the Gaussian function is much sharper than the exponential function. However $l(r)$ is increasing for both types of surface correlators. We tested this for several different values of $h$ Fig.[2.8]: the shapes of the curves and the difference between them remained qualitatively the same.
Figure 2.3. The next figure (Fig.[2.4]) shows the diffusion coefficient for the exponential correlation function of surface roughness.
Figure [2.7] compares the MFP for Gaussian and exponential surface correlation functions for a small range of r.
Similarly, Figure [2.9] illustrates the fact that the MFP $l(r)$ for the Gaussian and exponential correlation functions is more or less the same up to $r \sim 2$. Starting from this point the result for the Gaussian correlation function increases much faster than for the exponential function. We think the reason is that the Gaussian function decays much faster than the exponential which manifests itself at large values of $r$.
The next few curves, Figures.[2.10 – 2.12], illustrate fitting of the MFP curves for $l(r)$ by the power law functions. If we look at the Gaussian correlation function
Figure 2.5. Mean free path $l(r)$ for the surface with Gaussian roughness over wider range of $r$.
$d(r)$ in the ranges of $r$ from 1 to 20 we get a pretty good fit using the power function $d(r) \propto r^p$ with the index $p = 2.7$.
Looking at $d(r)$ in the range of $r \sim 20 - 60$, we also get a good fit using the power law, with the power $p = 3.5$. Looking at the exponential correlation function $d(r)$ in the ranges of $r$ from $1 - 40$, we see that we get a good fit using the power law, with the power $p = 2.95$.
### 2.5. Conclusions
- We calculated the diffusion coefficient and the mean free path for ultracold neutrons in narrow channels with random rough walls.
Figure 2.7. Mean-free path $l(r)$ for both Gaussian and exponential correlation functions for small r.
- We have concluded that there is a complicated minimum in $d(r)$ and $L(r)$ for small correlation radius $r \sim 2 \times 10^{-4}$.
- We have also concluded that the diffusion coefficient and the MFP rapidly increase as the correlation radius $r$ increases, though at different rates depending on the surface correlation function.
- The growth is not monotonic, there is more than one minimum at $q_j \sim 1/r$.
- We compared the behavior of $d(r)$ and $L(r)$ for surfaces with the Gaussian and the exponential correlation functions of surface roughness.
Figure 2.8. MFP for Gaussian correlation function for various channel widths $h = 16, 8, 4$.
- The function $d(r)$ behaves roughly as $r^3$, though the exponent slightly drifts with $r$. This seems to be an important conclusion, though we do not have an explanation for this functional dependence.
- The computations were done for realistic values of the channel width $h = 8.52$. At different values of $h$ the results were qualitatively the same.
- The growth of $d(r)$ and $\mathcal{L}(r)$ for the Gaussian surface correlation function is much slower than for the exponential correlation function.
- If one wants to effectively turn back the neutrons which got into the gaps in the channel junctions, one should make the correlation radius of surface
Figure 2.9. MFP for Gaussian (1) and exponential (2) surface correlation functions over a wide range of $r$.
roughness as small as possible, and, if possible, to have roughness with an exponential correlation function.
Figure 2.10. Power law fitting for the MFP $l(r)$ surfaces with the Gaussian correlation function for $r$ from 20 to 40.
Figure 2.11. The power law fit for MFP $l(r)$ for Gaussian inhomogeneities over the range of $r$ from 40 to 60
Figure 2.12. Power law fit for $d(r)$, the exponential surface correlation function over a large range of $r$.
Neutron Beams between Absorbing Rough Walls: Square Well Approximation
3.1. Description of Problem
In this Chapter we deal with a slightly different UCN diffusion problem which is more directly related to the GRANIT experiments in the ILL, Grenoble. In experiments the UCNs travel between rough absorbing walls and the number of UCNs exiting the cell is measured as a function of the distance between the walls. We start from discussing the case without gravity because it is simple and will serve as a good reference point. By comparing numerical results obtained with and without gravity we will understand what part of the experimental results should be directly attributed to the Earth’s gravitational field.
The neutrons in the cell are passing between the two mirrors, the perfectly smooth bottom mirror ("floor"), and the randomly rough upper mirror ("ceiling").
The UCNs entering the cell have a large horizontal velocity ($\sim 5 - 15$ m/s) and very small vertical velocities. When the neutrons scatter off the rough ceiling, the velocity vector with large horizontal component turns thus increasing the vertical component of the velocity. If the vertical velocity exceeds a certain velocity threshold (the critical velocity is $\sim 4$ m/s), the neutrons penetrate the wall, are absorbed, and do not reach the detector. The neutrons which manage to make it through the cell without reaching the critical vertical velocity are not absorbed and reach the neutron detector at the end of the cell.
The parameter that can be easily manipulated in experiment (and, of course, in calculations) is the cell width $H$. In computations we look at about 1000 values of dimensionless $h = H/l_0$ between 0 and 9. This problem differs from the setup mentioned in the section above where we are studying the diffusion coefficient $d(r)$
and the MFP not only in the fact that we are now measuring the exit neutron count, but also in the fact that we are now dealing with absorbing walls and time-dependent numbers of neutrons. Again, the main difference with regard to the previous chapter on diffusion is that previously we were letting the neutrons just bounce around without disappearing (they decay naturally at around 900 s). Now the neutrons disappear forever as the component of the velocity normal to the wall reaches a threshold value $\sqrt{2mU_c}$ and the number of neutrons becomes time-dependent as well.
The quantization of restricted motion is a well-known quantum phenomenon. In the absence of gravity we are dealing with the simplest square well potential with the energy levels
\begin{equation}
E_j = \frac{1}{2m} \left( \frac{\pi \hbar j}{H} \right)^2.
\end{equation}
If one adds weak gravity, the square well gets distorted by the appearance of a linear potential near the bottom, $mgz$ (see Fig.[4.1]). With the presence of a linear potential the problem still remains solvable, though there is no simple analytical expression for the energy levels.
To recap from the experiment briefly, we are dealing with a collimated beam being sent between two horizontal solid plates (one which is an almost ideal mirror and the other is rough) that are at a distance of several micrometers apart. We know that the neutrons hitting the wall with the normal velocity above 4 m/s get absorbed by the plates. Below this threshold velocity the neutrons get reflected. The reflection is specular locally.
First, we will neglect the presence of gravity. The effects of gravity will be introduced later, in the next chapter.
3.2. Wavefunction for the Square Well
We start by introducing the equation for the wavefunction on the wall in the square well,
\begin{equation}
\psi_j(H) = \sqrt{\frac{2}{H}} \sin \left( \frac{\pi j h}{H q_j} \right)
\end{equation}
where $H$ is the width of the waveguide. Below we will use the same dimensionless variables as in the previous chapter.
To determine the roughness-driven transition probabilities, Eq.(3.7), we need the value of the square of the wavefunction at the upper wall. The equations that we are using can be written in terms of $b_j$ Eq.(3.3) and since it is the quantity that has been used throughout the years in the papers by Meyerovich et al, Ref.[1]-[8] it is also the notation that we will be using from here on out in this thesis. Hence, we define the $b_j$ in dimensionless units as follows,
\begin{equation}
b_j(H) = 10^5 \frac{l_0 \psi^2(H)}{2}.
\end{equation}
In the case of the square well this reduces to
\begin{equation}
b_j = 10^5 \frac{\lambda_j}{h u_c},
\end{equation}
where $\lambda_j$ is defined as
\begin{equation}
\lambda_j = \left( \frac{\pi j}{h} \right)^2.
\end{equation}
The values of $b_j$'s in the gravitational potential we will get from the Airy functions, which will be discussed more in the next section. The constant $10^5$ is here merely as a scaling factor to avoid dealing with very small numbers.
In Fig.[3.3] we see the values of wavefunctions squared on the wall as a function of the width of the channel $h$.
### 3.3. Transition Probabilities and Neutron Count
As mentioned above, we are dealing with the same set of transport equations as in the previous Chapter of diffusion and mean-free path. Here again, we start
with the transport equation,
\[
\frac{\partial N_j}{\partial t} = \frac{m}{2\pi} \int d\theta \, W_{jj'} (|q_j - q_{j'}|) (N_{j'} - N_j).
\]
In this section we are looking only at the exponential correlation function of the surface inhomogeneities and are not interested in the potential Gaussian correlations. The reason for this is that recent analysis of the surface roughness of the new "rough" mirror have led us to believe that the surface roughness is exponential rather than Gaussian Ref.[9] We therefore use the transition probabilities with the exponential correlation function. Since we previously introduced the transition probabilities, we will write them directly in dimensionless variables,
\[
w^{(0)}_{jj'} = \frac{32r^2\eta^2}{\sqrt{2\pi}h^2} \left(\frac{\pi j}{h}\right)^2 \left(\frac{\pi j'}{h}\right)^2 \frac{E(\Omega)}{\left(1 + r^2(q - q')^2\right)\sqrt{1 + r^2(q + q')^2}},
\]
\[
w^{(1)}_{jj'} = \frac{32r^2\eta^2}{\sqrt{2\pi}h^2} \left(\frac{\pi j}{h}\right)^2 \left(\frac{\pi j'}{h}\right)^2 \frac{(1 + r^2(q + q')^2)E(\Omega) - (1 + r^2(q - q')^2)K(\Omega)}{\left(1 + r^2(q - q')^2\right)\sqrt{1 + r^2(q + q')^2}},
\]
where
\[
\Omega = 2r\sqrt{\frac{qq'}{(1 + r^2(q + q')^2)}},
\]
and \(E(\Omega)\) and \(K(\Omega)\) are elliptical integrals.
As above we use the transition probabilities to get the dimensionless transition frequencies $\tau_{jj'}^{-1}$,
\begin{equation}
\frac{1}{\tau_{jj'}} = \sum_{j''} \left[ \delta_{jj'} w^{(0)}_{jj''} - \delta_{j'j''} w^{(1)}_{jj''} \right]
\end{equation}
we can now write the neutron exit count in terms of these relaxation times in a simple form
\begin{equation}
\frac{N_e(L)}{N_0} = \sum_{jj'} \exp \left( -\frac{t_L}{\tau_j} \right),
\end{equation}
where $t_L$ is the time of flight of the UCN between the mirrors and $\tau_j$ are the eigenvalues of Eq. (3.10).
### 3.4. Numerical Results
Initially, we want to start off by defining and discussing the parameter that we introduce, $S1$. The $S1$ parameter is a cutoff parameter. If we were to numerically solve the above equations for the whole system, we would be solving more than $10^3$ coupled linear equations with complicated coefficients. Solving a system of that many equations is computationally very expensive time-wise even with modern computers, as each value takes approximately 30 min, and we do 900 iterations over values of $h$ from 9 to 1 for each $r$. Therefore, we wanted to examine if there were a reasonable cutoff, which would keep enough equations to not lose much accuracy in the calculations, while also being much less expensive time-wise.
computationally. To identify the $S1$ cutoff, we simply run the computations using more and more equations until we see that there is a saturation in the results. The saturation point becomes our cutoff point. We define that parameter as $S1$. In this section we are presenting some of our numerical results.
As we see in the figures below, Fig.[3.4]-Fig.[3.9], we are plotting the number of neutrons exiting the waveguide as a function of the size of the matrix $S1$. As one can see from all these figures presenting the exit neutron count as a function of $S1$ for various values of $r$ and $h$, in all the cases $S1 \sim 300$ can serve as a good cutoff parameter. From this point onward, we choose in all computations $S1 \sim 300$ and just occasionally check the results for larger matrices.
As a next step, we compute the dependence of the exit neutron count $N_e$ on $r$ and $h$. The following figures show $N_e(h)$ for $r = 1; 5; 10; 30$. The data in the figures show that, in principle, the neutron count is very sensitive to both $r$ and $h$. The common feature is that the exit neutron count is always extremely small except for very small values of $h$ and large values of $r$. For practical purposes this means that taking into account the small number of ultra-cold neutrons entering the waveguide, we should not expect any neutrons exiting at all. The obvious conclusion is that the existence of neutrons exiting the cell in the Grenoble experiments is due only to the Earth gravitational field (see the next section). Though this gravitational field is extremely weak, without it the neutron count would have shown zero neutrons exiting the cell with rough walls.
Figure 3.4. $N_e$ as a function of the cutoff parameter $S1$ for $h = 8$ and $r = 0.65$. Here we can see the initial increase.
As we can see from the plot, Fig.[3.10], representing the total neutron count as a function of $h$ for very small $r = 0.1$, already at large $h$ the number of surviving neutrons goes to zero almost immediately when we have such a small correlation radius.
For $r = 1$, Fig.[3.11], we see that the depletion of the total neutron count to zero is slightly less rapid, though it also goes to zero very quickly around $h = 9.4$. Though again, if one pays attention to the $N_e(h)$ axis, we see that the number starts from what is essentially zero to begin with.
Figure 3.5. Neutron count as a function of the size of the matrix $S1$ for $h = 8$ and $r = 0.65$. We can see how it saturates nicely, at about $S1 = 300$. In this plot we are looking at $N_e$ over a larger scale.
Figure 3.6. $N_e$ as a function of the size of the matrix $S1$ for $h = 5$ and $r = 0.65$. We are looking at $N_e$ closer scale, so that we can see the initial increase and gradual saturation.
Figure 3.7. Saturation of the neutron count as a function of the size of the matrix $S1$, for $h = 5$ and $r = 0.65$.
Figure 3.8. $N_e$ as a function of the matrix size $S1$ for $h = 3$ and $r = 0.65$. We are looking at $N_e$ closer scale, so that we can see the initial increase and gradual saturation.
The results for the total neutron count for $r = 5$, Fig.[3.12] are consistent with the above results, though now the neutron count goes to zero for the width size of $h = 8.7$.
In the last two plots, we are computing the total neutron count $N_e(h)$ over the full range of $h$ for $r = 10$ (Fig.[3.13]) and $r = 30$ (Fig.[3.14]). As with all the results that we presented previously for varying $r$, we observe again that as we increase the radius of roughness the total neutron count goes to zero slower which here means at a smaller width size $h$.
We see in Fig.[3.15] the total neutron count as a function of the radius of roughness $r$. It’s clear that only as the radius increases and becomes very large, the total neutron count becomes noticeable. The explanation is relatively simple. At very large $r$ the walls become essentially flat and reflection is practically specular. Under these conditions the normal component of velocity remains small and neutrons do not penetrate the walls.
Figure 3.11. $N_e(h)$ as a function of $h$ for $r = 1$.
Figure [3.16] similarly shows the total neutron count over various $r$, for $h = 5$. Again only for large $r$ does the neutron count become non-zero.
The last two figures, Fig.[3.17]. and Fig.[3.18]., show that, as in the previous ones, the neutron count becomes non-negligible only for large $r$ for $h = 7$ and $h = 9$, respectively. This is consistent with the theory that, for large enough $r$ or in the limit that $r \to \infty$ we will not have any roughness and therefore all the neutrons will make it to the detector.
### 3.5. Conclusions
The main conclusions for this section are as follows.
Irrespective of the well width and the correlation radius, a good cutoff parameter for all computations is around $S1 = 300$.
As we increase the radius of roughness $r$, the total neutron count goes to zero for at a slower rate, meaning for smaller and smaller values of $h$.
However, as we can see from the plots, the numbers that we get for $N_e(h)$ are always extremely small, so essentially all the neutrons die almost immediately.
As we will see in the following section, this is not the case for the gravitational well. Hence, we can say that the square well approximation is very
Figure 3.14. $N_e(h)$ as a function of $h$ for $r = 30$.
poor for this particular problem: though the weak Earth gravitational field introduces only a small distortion near the bottom of the potential well, its effect on the neutron survival rate is very profound.
Figure 3.15. Total neutron count $N_r$ as a function of different correlation radii $r$ for small well width $h = 3$.
Figure 3.16. Total neutron count $N_e$ as a function of different correlation radii $r$ for well width $h = 5$.
Figure 3.17. Total neutron count $N_e$ as a function of different correlation radii $r$ for well width $h = 7$.
Figure 3.18. Total neutron count $N_e$ as a function of different correlation radii $r$ for well width $h = 9$.
4.1. Gravity-Imposed Changes: Similarities and Differences with the Previous Chapter
In this Chapter we are looking at the same setup as in the previous Chapter but in the presence of the Earth’s gravitational field. The purpose is twofold: to give an accurate description of the experiments of the Grenoble group and to understand what part of the observed anomalies can be attributed to the gravitational field. In the case of the gravitational well, we are dealing with a slightly different geometry than the square well. Of course, this change is due to the effect of the gravitational field. A sketch of this potential well is provided below (Fig.[4.1]). For the sake of comparison, the next figure (Fig.[4.2]) shows the dependence of the lowest energy levels on $h$ for both the square well and the gravitational well.
Figure 4.1. Sketch of the gravitational well as a function of $z$.
As we can see in the Figure below, we are looking at the first three eigenvalues for both the square well and gravitational potentials as a function of the width $h$. It is clear from this figure that for small $h$ there is hardly any difference between
Figure 4.2. The first three eigenvalues for both the square well and the gravitational well as a function of the well width $h$. For better comparison, the bottom of the square well is chosen in the middle of the bottom of the gravitational well, $mgh/2$, and is drifting with $h$. The eigenvalues for the gravitational well are the lower curves, for the SW—the upper.
the lowest eigenvalues of these two potentials; for larger $h$ the differences become significant.
Formally the transport equations for the rough waveguides with and without gravity are the same as Eq.(1.14). However, the transition probabilities are different. This difference, though significant, is related mostly to the values of the wavefunctions on the walls, i.e., to the coefficients $b_j(h)$, Eq.(4.2).
Figure 4.3. A few coefficients $b_j$'s as a function of well width size $h$ for both the square (lower curves) and the gravitational wells (upper curves).
4.2. Results from the Preceding Work: the Biased Diffusion Approximation
The preceding work used what the authors called the biased diffusion approximation. Since the transitions $j \rightarrow j'$ show a strong upward bias due to the factor $b_j b_{j'}$ in the transition probabilities $W_{jj'}$ (essentially the factor $j^2 j'^2$ in Eq.(1.23)), the probabilities for the neutrons to return back to the lower states $j$ after they jump to a higher state $j'$ appear to be small and can be neglected. Then the
Figure 4.4. The first nine coefficients $b_j$ as a function of the width of the channel $h$ for the gravitational well. The lowest curve is $b_1$ and the highest $b_9$.
The absorption times $\tau_j$ are
$$\frac{1}{\tau_j} = m \sum_{j > j'} \int \frac{d\theta}{2\pi} W_{jj'}(|q_j - q_{j'}|),$$
where $\theta$ is the angle between $q_j$ and $q_{j'}$. Note, that since the absorption threshold $u_c$ is very high ($\sim 10^5$), direct transitions from the lower levels over the threshold are negligible.
What is more, the absorption times for neutrons that initially occupy some of the lowest minibands $j$ differ from each other mostly by the values of the coefficients $b_j$,
$$b_j = \frac{10^5 l_0 \psi_j^2 (H)}{2}$$
and therefore the above equation Eq.(4.1) becomes,
$$\frac{1}{\tau_j} = \frac{b_j}{b_1} \frac{1}{\tau_1}$$
where $\tau_1$ is the depletion time for the neutrons in the first (or lowest) gravitational state. Note that this equation loses its accuracy for large values of $j$. Here we also note that the $b_j$ are the dimensionless values of $\psi_j^2 (H)$.
The justification for biased diffusion is that the transition rates ($\tau_{jj'}^{-1}$) between the states ($j$ and $j'$) rapidly increase with both of these quantum numbers. Since the rates of the direct absorption processes also rapidly increase as $j$ gets larger, this means that the neutron lifetimes in the higher states are orders of magnitude shorter than the lifetimes in the lower states. Therefore the diffusion of a neutron between energy levels has a strong upward bias. The increase in the jump rate from $j$ to $j'$ is moderated only by the correlation function, which is determined by the correlation radius $r$ and starts rapidly decreasing at large $|j - j'| r$. This is why $w_{jj'}$ acquires a narrow peak centered at $j_1 >> j$ Fig.[4.5] The bias is so strong that almost all the time $\tau_j$ spent for the neutron in a low gravitational state to
transition up to higher states and over the absorption barrier is spent in the first transition upwards.
The values of $\tau_j$ determine the depletion time of each quantum state. The overall exit neutron count is
$$N_e = \sum N_j = \sum N_j(0) \exp\left(-L/v_j \tau_j\right),$$
where $N_j(0)$ is the number of neutrons in a state $j$ entering the waveguide of length $L$. Additionally, for the lowest levels the velocities $v_j$ are almost the same, $v_j \approx \sqrt{\varepsilon} v_0$. The equation above can be rewritten using $v_j \approx \sqrt{\varepsilon} v_0$ and we can directly get all the $\tau_j$. Then it is easy for us to get the total neutron count which is just a sum over all $j$. In the end, in the biased diffusion approximation all the pertinent parameters of roughness and the waveguide entering the exit neutron count collapse into a single variable $\Phi$ Ref[8] and we get an analytical solution,
$$\frac{N_e(h)}{N_0} = \sum_j \exp\left(-\Phi b_j(h)\right)$$
where $\Phi$ is a complicated weighted integral of the correlation function that is dependent on the correlation radius. If the roughness is two-dimensional,
$$\Phi = A_2 \eta^2 r^2 \int_0^1 z^2 \psi_2(y_1, y(z)) \, dz$$
and
\begin{equation}
A_2 = \left( \frac{2}{\pi} \right)^{3/2} \times 10^{-5} \frac{L}{\tau_0} u_c e^{3/2},
\end{equation}
where $\psi_2(y_1, y)$ is the dimensionless zeroth harmonic of the correlation function $\zeta(|q_j - q_{j'}|)$ over the angle between the vectors $q_j$ and $q_{j'}$, and $y_1 = r \sqrt{\varepsilon}$, $y(z) = y_1 \sqrt{1 - z^2}$.
We can write the relaxation time $\tau_1$ for the lowest gravitational state as
\begin{equation}
\frac{1}{\tau_1} = m \sum \int \frac{d\theta}{2\pi} W_{1j'}(|q_1 - q_{j'}|),
\end{equation}
where $\theta$ is the angle between the vectors $q_1$ and $q_{j'}$. Finally, after replacing the summation by the integration,
\begin{equation}
\frac{\tau_0}{\tau_1} = 2 \times 10^{-5} u_c^2 \left( \frac{\eta}{r} \right)^2 b_1(h) F_2(r, h),
\end{equation}
where
\begin{equation}
F_2(r, h) \simeq r^4 \sqrt{\frac{2u_c}{\pi \chi^3}} \int_0^1 dz \ z^2 \psi_2(y_1, y).
\end{equation}
When we combine the equations Eq.(4.9) and Eq.(4.10) we get the following equation,
\begin{equation}
\Phi(\eta, r) = A_2 \eta^2 r^2 \int_0^1 dz \ z^2 \psi_2(y_1, y)
\end{equation}
This concludes the description of the preceding work, which provides the approximate analytical expression to the exit neutron count. We will now describe how we deal with the same problem computationally without relying on the biased diffusion approximation.
4.3. Exact Calculation of the Absorption Time
In our case we are not using the biased diffusion approximation, but actually using the brute force technique to solve the full set of transport equations numerically.
As a result, we do not get a nice analytical solution to the problem. Instead we use the full matrix, meaning the matrix with transitions upwards and downwards, for which we can only get a numerical solution. The structure of the diagonal and off-diagonal elements in the matrix transport equations is different. The diagonal elements have the structure defined below in Eq.(4.12), where for an element in row $j$ we are summing over all the elements $j'$. The off-diagonal elements are simpler: these are simply $w_{jj'}$. We rewrite the transition probabilities $W_{jj'}$ defined in Eq.(1.22) that represent the diagonal elements of the matrix, in a notation that is closer to the one that was used in the most recent papers in this field. Therefore specifically for diagonal elements of our matrices, when $j = j'$, we write,
\begin{equation}
F_j(r,h) = \sqrt{\frac{2}{\pi}} \times 10^{-5} r^4 b_j \sum_{j' \neq j} b_{j'} \psi_2(y_1,y_{j'}) ,
\end{equation}
(4.12)
where $\psi_2(y_1, y_j)$ here also represents the dimensionless zeroth harmonic of the correlation function over the angles between the vectors $q_i$ and $q_{j'}$. Since we are not working under the context of biased diffusion, we can only perform our computations numerically.
We have off-diagonal elements in our matrix for square well are defined in dimensionless units as
\begin{equation}
w_{jj'} = \frac{32r^2\eta^2}{\sqrt{2\pi}h^2} \left(\frac{\pi j}{h}\right)^2 \left(\frac{\pi j'}{h}\right)^2 \frac{E(\Omega)}{\left(1 + r^2(q - q')^2\sqrt{1 + r^2(q + q')^2}\right)},
\end{equation}
where
\begin{equation}
\Omega = 2r\sqrt{\frac{qq'}{(1 + r^2(q + q')^2)}}.
\end{equation}
Hence our total matrix, which we will call $M_{jj'}$ looks like
\begin{equation}
M_{jj'} = \begin{pmatrix}
F_{11} & w_{12} & \ldots & \ldots & w_{1s \text{ max}} \\
w_{21} & \ldots & \ldots & \ldots & w_{2s \text{ max}} \\
\vdots & \ddots & \ddots & \ddots & \vdots \\
\vdots & \ddots & \ddots & \ddots & \vdots \\
w_{2s \text{ max}} & \ldots & \ldots & F_{s \text{ max}, s \text{ max}}
\end{pmatrix}.
\end{equation}
More detailed mathematics regarding the $F_{jj'} = F_j(r, h)$ for the discrete case will be available in Appendix C.
We do not have a simple analytical description of the gravitational states similar to that for the square well states in the previous chapter. However, for the states with high index $j$, and especially at small $\hbar$, the difference between the gravitational and square well states is negligible. Therefore, for higher states we can replace the gravitational states by the square well states.
As a result, our square matrix of transition probabilities acquires a block structure. One block representing the transitions between gravitational states, two of the blocks represent the transitions between the lower gravitational states and higher square well states, and the third block represents the transitions between higher square well states. Once we have the total matrix with all the block components, we are numerically computing the eigenvalues and eigenvectors of the total matrix. We can write the neutron exit count in terms of these absorption times $\tau_j$ in quite a simple form,
$$\frac{N_e(\hbar)}{N_0} = \sum_j \exp \left( -\frac{t_L}{\tau_j(\hbar)} \right),$$
where $t_L$ is the time of flight of the UCNs between the mirrors. Note that the above equation was introduced in the previous section and chapter as it is a general equation that can be used with and without the biased diffusion approximation.
It is interesting to note that the center of the peak of the transition probabilities $W_{jj'}$ for transitions from $j$ to $j'$ is located at some $j_1 >> j$, see Fig.[4.5] for example.
The peak is very high and relatively narrow.
### 4.4. Numerical Results
In this section we present the main results of the thesis as pertaining to the Grenoble experiments. As in the previous section regarding the square well potential, we are now looking at the exit neutron count in the gravitational potential. The gravitational potential was introduced in a previous chapter. However, we will reproduce a schematic figure here. In this figure, the particles on the lowest three levels "classically" do not reach the rough ceiling, do not scatter, and survive for a
long time. The particles from the higher levels are actively scattered on the rough ceiling, go rapidly upwards, and get absorbed by the walls. When the width of the waveguide $h$ becomes smaller, all the levels are rapidly squeezed up (see Fig. below).
Though here we do not present a figure to show the saturation of the total neutron count as a function of the cutoff parameter $S1$, after doing many numerical simulations, it was determined from the results that the cutoff parameter was more or less the same as in the case of the square well potential, meaning that $S1 = 300$ is a good cutoff size to maintain high accuracy in the calculations, while simultaneously keeping the computation time sufficiently short.
The series of figures below present the neutron count $N_e$ as a function of $h$ for several values of the correlation radius $r$. In all figures the roughness correlation function is assumed to be exponential with the average amplitude $\eta = 1.02$. The
current rough mirror used by the Grenoble group probably has $r = 0.65$ and $\eta = 1.02$. As one can see, all the figures are similar, though the curves slowly shift to the left with increasing $r$. This is understandable: the surface becomes flatter with increasing $r$ and the neutrons survive longer.
Figure 4.7. Neutron exit count as a function of $h$, for experimental parameters.
Figure [4.6] shows the neutron count $N_e(h)$ as a function of the width of the well $h$. The results are slightly different when you increase $r$, though we can see that for small $h$ the difference is insignificant. Even for large $h$ the difference is not huge, especially when you consider that the experimental $r = 0.65$ (Fig.[4.6]) is very small compared to $r = 5$ (Fig.[4.9]) or $r = 10$ (Fig.[4.10]). When we compare the figures of neutron count as a function of well width, we see one main feature; that is that as $r$ increases, the curves becomes increasingly smoother. From these figures we see, much like with the square well potential, when you increased the radius of roughness $r$, the neutrons lived longer and they can be detected even for very
small width $h$. This is happening as well in the case of the gravitational potential. We see that as the radius of roughness gets larger and larger, the neutrons survive for smaller and smaller well width sizes.
For very large $r$, for example in the figures where $r = 500$ (Fig.[4.15]) and $r = 1000$ (Fig.[4.16]), we see that the curve flattens and we don’t have the well defined steps that we see at smaller values of $r$, implying that the discrete energy levels cannot be detected so easily as with smaller $r$. However, it should be noted that this flattening happens only for unrealistically large $r$. In general the steps on the curves are very robust and the first bump remains detectable even for very
large $r$. This of course is due to the fact that the neutrons are not scattering and dying but instead can easily make it to the end of the cell and to the detector since they are not influenced by roughness for large $r$. The ideal conditions for scattering are at $qr \sim 1$. From this condition we know that as $r$ gets larger and larger and goes to infinity, we will have specular reflection.
The last figures, Fig.[18 – 20] show the neutron exit count for fixed widths $h = 9, 5, 3$ respectively. As we can see for all three widths, the neutron exit count increases as the correlation radius increases. This is because as we increase
the correlation radius, the ceiling becomes smoother, and therefore there is less scattering and absorption by the rough wall.
4.5. Conclusions
It is interesting to compare these results with the previous results without gravity in the square well section.
- The dependence of the exit neutron count on $r$ was much more significant in the case of the square well potential: $N_e(r)$ at fixed $h$ changed by many orders of magnitude.
Figure 4.11. Neutron count as a function of $h$ for correlation radius of roughness $r = 10$.
- It is interesting to note that the ratio of the relaxation times for the diagonal case (in which we do not take into account the transitions between the states, meaning the cases where $j \neq j'$) and the case described in this section (where we allow all the transitions, and not just the biased ones, to be taken into account) $\tau_{diag}/\tau_{full}$ is close to 1. This was rather unexpected.
- It is also interesting to note that the ratio of the results for the matrix in the square well potential and the matrix in the gravity potential goes to one as $h$ goes to 0, $\tau_{jSqwelt}/\tau_{jgrav} \to 1$ as $h \to 0$ since all the levels are
being squeezed up and the difference in potentials near the bottom of the well looses its significance.
- The major result of this section is the exit neutron count in the presence of gravity. It illustrates the total neutron count $N_e(h)$ as a function of the width of the channel $h$. When comparing this result to the work done previously using the biased diffusion approximation, we see that the curves are similar. This means that the biased diffusion approximation is indeed a very robust approximation.
Figure 4.13. Neutron count as a function of $h$ for correlation radius of roughness $r = 30$.
- In almost all computations the exit neutron count as a function of cell width $N_e(h)$ retained the step-wise nature. This may be considered as a unequivocal proof of quantization of neutron motion by the Earth gravitational field.
Figure 4.14. Neutron count as a function of $h$ for correlation radius of roughness $r = 50$.
Figure 4.15. Neutron count as a function of $h$ for correlation radius of roughness $r = 100$.
Figure 4.16. Neutron count as a function of $h$ for correlation radius of roughness $r = 500$.
Figure 4.17. Neutron count as a function of $h$ for correlation radius of roughness $r = 1000$.
Figure 4.18. Neutron exit count for fixed well width at $h = 9$, over a large range of $r$.
Figure 4.19. Neutron exit count for fixed well width at $h = 5$, over a large range of $r$.
Figure 4.20. Neutron exit count for fixed well width at $h = 3$, over a large range of $r$.
Summary and Conclusions
5.1. Main Conclusions
In summary, the main goal of this thesis was to provide a rigorous theoretical description for the diffusion of ultra cold neutrons (UCN) through narrow rough channels. We used the general transport theory of particles along rough surfaces to the gravitationally quantized diffusion of UCN in a rough waveguide. We looked at two separate problems: diffusion of the neutrons through rough waveguides on the way from the reactor to the experimental cell and the neutron count for neutrons exiting experimental cell with absorbing walls in the square well potential as well as the gravitational potential. We used numerical calculations to investigate the effect of two types of random roughness on the diffusion coefficient, as well as to evaluate the neutron count using the experimental input parameters in both potentials.
The main content of Chapter 2 is the calculations of the diffusion coefficient and the mean free path for ultra-cold neutrons in narrow channels with random rough walls. We determined that if one wants to effectively turn back the neutrons which got into the gaps in the channel junctions, one should make the correlation radius of surface roughness as small as possible. We compared the behavior of $d(r)$
and $l(r)$ for surfaces with the Gaussian and the exponential correlation functions of surface roughness. We found that there is a complicated minimum in $d(r)$ and $l(r)$ for small correlation radius $r \sim 2 \times 10^{-4}$. Additionally we found that the diffusion coefficient and the MFP rapidly increase as the correlation radius $r$ increases, though at different rates depending on the surface correlation function. The growth of the diffusion coefficient and MFP is not monotonic, there is more than one minimum at $q_j \sim 1/r$. We saw that at large $r$ the function $d(r)$ behaves roughly as $r^3$, where the exponent slightly drifts with $r$. The computations were done for realistic values of the channel width $h = 8.52$. At different values of $h$ the results were qualitatively the same. The growth of $d(r)$ and $l(r)$ for the Gaussian surface correlation function is much faster than for the exponential correlation function. As a result, it is preferable to have the junction walls with exponential correlation of inhomogeneities.
The conclusions for Chapter 3 regarding the UCN in the square well potential are as follows: Irrespective of the well width and the correlation radius, a good cutoff parameter for all computations is around $S1 = 300$. We see that as the radius of roughness $r$ increases, the total neutron count goes to zero for smaller and smaller values of $h$. However, as we can see from the plots, the numbers that we get for $N_e(h)$ are always extremely small, so essentially all the neutrons die almost immediately. As we will see in this Chapter, this is not the case for the gravitational well. Hence, we can say that the square well approximation is very poor for this particular problem: though the weak Earth gravitational field
introduces only a small distortion near the bottom of the potential well, its effect on the neutron survival rate is very large.
The main conclusion for Chapter 4 is the exit neutron count in the presence of gravity. It illustrates the total neutron count $N_e(h)$ as a function of the width of the channel $h$. When comparing this result to the work done previously using the biased diffusion approximation, we see that the curves are similar. This means that the biased diffusion approximation is a very robust approximation. This result was somewhat surprising. Other conclusions include the following. The dependence of the exit neutron count on $r$ was much more significant in the case of the square well potential where $N_e(r)$ at fixed $h$ changed by many orders of magnitude. It is interesting to note that the ratio of the relaxation times for the diagonal case (in which we do not take into account the transitions between the states, meaning the cases where $j \neq j'$) and the case described in this Chapter (where we allow all the transitions, and not just the biased ones to be taken into account) $\tau_{diag}/\tau_{full}$ is close to 1. This was rather unexpected. It is also interesting to note that the ratio of the results for the square well potential and the gravitational potential goes to one as $h$ goes to 0, $\tau_{jSwell}/\tau_{jgrav} \rightarrow 1$ as $h \rightarrow 0$ since all the levels are being squeezed up and the difference in potentials near the bottom of the well loses its significance, see Figure above.
5.2. Recommendations for Future Work
Using rough mirrors as a quantum state selector can be extended beyond this series of GRANIT experiments. Some of the more exciting experiments include the observation of quantum gravitational states for other ultra-cold particles and anti-particles in the context of the GBAR experiment at CERN.
One of the main goals of the GBAR experiments is to measure the acceleration in free fall of ultra-cold neutral anti hydrogen atoms in the Earth’s gravitational field. The experiment entails using anti hydrogen ions, which consist of one antiproton supplied by the ELENA deceleration ring at CERN and two positrons created by the linac, and cooling them below 10 $\mu$K with Beryllium plus ions. Their positive charge makes them easier to manipulate. Using lasers, their velocity can be reduced to half a meter per second. Once they are trapped by an electric field, one of their positrons will be removed with laser, which will make it neutral. Hence, at this point the Earth’s gravitational field will be the only force acting upon them, and they will be able to free fall a given distance, and their time of fall could be measured. The results of this experiment are much anticipated, because it could potentially mean that gravity might have a different effect on antimatter then it does on matter.
For the past 60 years, there has also been an ongoing search for the neutron electric dipole moment (nEDM). Over the course of the decades the accuracy of (negative) results has been improved by many orders of magnitude. The nEDM
potentially violates CP symmetry, meaning that it violates the presumption that if a particle and a respective anti particle are interchanged, while their spatial coordinates are inverted, then the laws of physics should remain the same. The goal of these ongoing and future nEDM experiments is to improve the sensitivity for detection nEDM by orders of magnitude. One of the experiments being done at Oak Ridge National Laboratory is to create a three-component fluid described as isotopically purified Helium-4, a trace amount of spin-polarized Helium-3, and spinpolarized ultra-cold neutrons. Then once that fluid has been created, it should be exposed to a small but homogeneous magnetic field and a large electric field. The nEDM could then be measured by looking at the neutron precession frequency which is linearly dependent on the magnitude of the electric field strength, and whose sign is dependent on the alignment between the magnetic and electric fields.
Above are just examples of the experiments that are ongoing or planned for the near future, however, our theoretical work on the use of rough mirrors as quantum state selectors are well suited for dealing with these and similar applications.
APPENDIX A
Dimensionless Transition Probabilities
In this Appendix we make the equations for the harmonics of the transition probabilities dimensionless. According to Ref.[2], the angular harmonics of the transition probabilities for surfaces with Gaussian correlations of surface inhomogeneities, are
\begin{equation}
W_{jj'}^{(0,1)} = \frac{\hbar}{m^2H^2} \left(\frac{\pi j}{H}\right)^2 \left(\frac{\pi j'}{H}\right)^2 4\pi l^2 R^2 I_0(QQ') Exp(-QQ') Exp(-\frac{1}{2}(Q-Q')^2).
\end{equation}
Then
\begin{equation}
W_{jj'}^{(0,1)} * w_0 = \frac{\hbar}{m^2H^2} \left(\frac{\pi j}{\hbar}\right)^2 \left(\frac{\pi j'}{\hbar}\right)^2 4\pi \eta^2 r^2 I_0(QQ') Exp(-QQ') Exp(-\frac{1}{2}(Q-Q')^2)
\end{equation}
where $w_0 = m\tau_0$, with $\tau_0 = (4ml_0^2)/(\sqrt{2\pi}\hbar)$. As a result the dimensionless equations become
\begin{equation}
w_{jj'}^{(0,1)} = \frac{\hbar}{m^2H^2} \left(\frac{\pi j}{\hbar}\right)^2 \left(\frac{\pi j'}{\hbar}\right)^2 4\pi \eta^2 r^2 \left(m \frac{4ml_0^2}{\sqrt{2\pi}\hbar}\right) I_0(QQ') Exp(-QQ') Exp(-\frac{1}{2}(Q-Q')^2)
\end{equation}
\begin{equation}
w_{jj'}^{(0,1)} = \frac{4l_0^2}{\sqrt{2\pi}H^2}(4\pi\eta^2r^2)\left(\frac{\pi j}{h}\right)^2\left(\frac{\pi j'}{h}\right)^2I_0(QQ')Exp(-QQ')Exp(-\frac{1}{2}(Q-Q')^2)
\end{equation}
\begin{equation}
w_{jj'}^{(0,1)} = \frac{8\pi\eta^2r^2}{\sqrt{2\pi}h^2}\left(\frac{\pi j}{h}\right)^2\left(\frac{\pi j'}{h}\right)^2I_0(QQ')Exp(-QQ')Exp(-\frac{1}{2}(Q-Q')^2)
\end{equation}
Therefore, our final equation for the transition probability for the Gaussian correlator becomes
\begin{equation}
w_{jj'}^{(0,1)} = \frac{8\pi}{\sqrt{2\pi}}\left(\frac{r}{h}\right)^2\left(\frac{\pi j}{h}\right)^2\left(\frac{\pi j'}{h}\right)^2I_{(0,1)}(QQ')Exp(-QQ')Exp(-\frac{1}{2}(Q-Q')^2)
\end{equation}
Note that in computations we assume the scaling parameter $\eta = 1$.
Additionally, in the same way, we derive the dimensionless transition probabilities for the exponential roughness,
\begin{equation}
w_{jj'}^{(0)} = \frac{32r^2}{\sqrt{2\pi}h^2}\left(\frac{\pi j}{h}\right)^2\left(\frac{\pi j'}{h}\right)^2\frac{E(2r\sqrt{\frac{q_jq_{j'}}{(1+r^2(q_j+q_{j'})^2)}})}{(1+r^2(q_j-q_{j'})^2)(\sqrt{1+r^2(q_j+q_{j'})^2})}
\end{equation}
\begin{equation}
w_{jj'}^{(1)} = \frac{16}{\sqrt{2\pi}h^2}\left(\frac{\pi j}{h}\right)^2\left(\frac{\pi j'}{h}\right)^2\times\frac{(1+r^2(q_j^2+q_{j'}^2))E(2r\sqrt{\frac{q_jq_{j'}}{(1+r^2(q_j+q_{j'})^2)}})-(1+r^2(q_j-q_{j'})^2)K(2r\sqrt{\frac{q_jq_{j'}}{(1+r^2(q_j+q_{j'})^2)}})}{(1+r^2(q_j-q_{j'})^2)(\sqrt{1+r^2(q_j+q_{j'})^2})}
\end{equation}
APPENDIX B
Asymptotic Expansion for Transition Probabilities for Surfaces with Exponential Roughness Correlator
Here we present an asymptotic expansion for the transition probabilities for the surfaces with the exponential roughness correlator.
We need an asymptotic expression for:
(B.1) \[ Exp(-x^2) * (I_0(x^2) - I_1(x^2)) \]
At large x,
(B.2) \[ I_0(x^2) \sim \frac{Exp(-x^2)}{\sqrt{2\pi}x^2} \left( 1 - \left( \frac{-1}{8x^2} \right) + \left( \frac{(-1)(-9)}{2!(8x)^2} \right) - ... \right) \]
since
(B.3) \[ I_0(x^2) \sim \frac{Exp(-x^2)}{\sqrt{2\pi}x^2} \left( 1 + \left( \frac{1}{8x^2} \right) \right) \]
and,
(B.4) \[ I_1(x^2) \sim \frac{Exp(-x^2)}{\sqrt{2\pi}x^2} \left( 1 - \left( \frac{4-1}{8x^2} \right) + \left( \frac{(4-1)(4-9)}{4*64*x^4} \right) - ... \right) \]
(B.5) \[ I_1(x^2) \sim \frac{Exp(-x^2)}{\sqrt{2\pi x^2}} \left( 1 - \left( \frac{3}{8x^2} \right) \right) \]
As a result,
(B.6) \[ u_{jj'}^{(0,1)} = \frac{8\pi}{\sqrt{2\pi}} \left( \frac{r}{h} \right)^2 \left( \frac{\pi j}{h} \right)^2 \left( \frac{\pi j'}{h} \right)^2 I_{(0,1)}(QQ') Exp(-QQ') Exp(-\frac{1}{2}(Q-Q')^2) \]
If we replace the exact transition probabilities by these asymptotic expansions, we get almost identical numerical results. We tried using this expansion to see if we could speed up computation time.
APPENDIX C
Diagonal Elements of Transition Probabilities for the Biased Diffusion Approximation
In this Appendix we try to replace the summation over discrete states in Eq.(4.12) by integration. Starting from the equation, Eq.(4.12) and using the following definitions,
\begin{align}
b_j &= 10^5 \frac{\lambda_j}{h u_c}, \\
\lambda_j &= \frac{\pi^2 j^2}{h^2},
\end{align}
\[ F_2(r,h) = \sqrt{\frac{2}{\pi}} \times 10^{-5} r^4 \sum_{j' > 1} b_{j'} \psi_2(y_1, y_{j'}) \]
we can rewrite this expression in the following way
\begin{align}
F_2(r,h) &= \sqrt{\frac{2}{\pi}} \times 10^{-5} r^4 \sum_{j' > 1} 10^5 \frac{\lambda_{j'}}{h u_c} \psi_2(y_1, y_{j'}) \\
&= \sqrt{\frac{2}{\pi}} r^4 \sum_{j' > 1} \frac{\lambda_{j'}}{h u_c} \psi_2(y_1, y_{j'}) \\
&= \sqrt{\frac{2}{\pi}} r^4 \left( \frac{\pi^2}{h^3 u_c} \right) \sum_{j' > 1} j'^2 \psi_2(y_1, y_{j'})
\end{align}
Now note that \( \mathcal{N} = \frac{\hbar}{\pi} \varepsilon^{\frac{1}{2}} \), and \( \chi = \frac{u_c}{\varepsilon} \), therefore we know that \( \mathcal{N} = \frac{\hbar}{\pi} \left( \frac{u_c}{\chi} \right)^{\frac{3}{2}} \). It should also be noted that we can write \( \left( \frac{\pi^2}{\hbar^3 u_c} \right) \) as follow,
\[
\left( \frac{\pi^2}{\hbar^3 u_c} \right) = \left( \frac{\pi \chi^{3/2}}{u_c^{1/2}} \right)^{-1} \left( \frac{\pi \chi^{3/2}}{u_c^{1/2}} \right) \left( \frac{\pi^2}{\hbar^3 u_c} \right)
\]
\[
= \frac{u_c^{1/2}}{\pi \chi^{3/2}} \left( \frac{\pi^3 \chi^{3/2}}{\hbar^3 u_c^{3/2}} \right)
\]
\[
= \frac{u_c^{1/2}}{\pi \chi^{3/2}} \left( \frac{1}{\mathcal{N}} \right)
\]
hence we can rewrite \( F_2(r, h) \) as,
\[
F_2(r, h) = \sqrt{\frac{2}{\pi}} r^4 \left( \frac{u_c^{1/2}}{\pi \chi^{3/2}} \right) \frac{1}{\mathcal{N}^3} \sum_{j' > 1} j'^2 \psi_2(y_1, y_{j'})
\]
Now to make this a continuous function we can replace the sum by an integral,
\[
F_2(r, h) = \sqrt{\frac{2}{\pi}} r^4 \left( \frac{u_c^{1/2}}{\pi \chi^{3/2}} \right) \frac{1}{\mathcal{N}^3} \int_1^\mathcal{N} j'^2 \psi_2(y_1, y_{j'})
\]
where we substitute \( z = \frac{j'}{\mathcal{N}} \) and \( \frac{dj'}{\mathcal{N}} = dz \) and then we can write \( z^2 = \frac{j'^2}{\mathcal{N}^2} \). The above equation now becomes,
\[
F_2(r, h) = \sqrt{\frac{2}{\pi}} r^4 \left( \frac{u_c^{1/2}}{\pi \chi^{3/2}} \right) \frac{1}{\mathcal{N}^3} \int_0^1 z^2 \mathcal{N}^2 \ dz \mathcal{N} \ \psi_2(y_1, y_{j'})
\]
\[
= \sqrt{\frac{2}{\pi}} r^4 \left( \frac{u_c^{1/2}}{\pi \chi^{3/2}} \right) \int_0^1 dz \ z^2 \psi_2(y_1, y_{j'}),
\]
which is Eq.(4.10) in the main text. This equation was used in Ref.
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|
High Temperature Oxidation Kinetics of Dysprosium Particles
Brian J. Jaques
*Boise State University*
Darryl P. Butt
*Center for Advanced Energy Studies*
**Publication Information**
Jaques, Brian J. and Butt, Darryl P.. (2014). "High Temperature Oxidation Kinetics of Dysprosium Particles". *Journal of Alloys and Compounds*, 644, 211-222. [https://doi.org/10.1016/j.jallcom.2015.04.174](https://doi.org/10.1016/j.jallcom.2015.04.174)
This document was originally published by Elsevier in *Journal of Alloys and Compounds*. This work is provided under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 license. Details regarding the use of this work can be found at: [http://creativecommons.org/licenses/by-nc-nd/4.0/](http://creativecommons.org/licenses/by-nc-nd/4.0/). doi: [10.1016/j.jallcom.2015.04.174](10.1016/j.jallcom.2015.04.174)
High temperature oxidation kinetics of dysprosium particles
Brian J. Jaques, Darryl P. Butt*
Department of Materials Science and Engineering, Boise State University, 1910 University Dr., Boise, ID 83725, USA
Center for Advanced Energy Studies, 995 University Blvd., Idaho Falls, ID 83415, USA
ARTICLE INFO
Article history:
Received 28 January 2015
Received in revised form 14 April 2015
Accepted 25 April 2015
Available online 1 May 2015
Keywords:
Dysprosium
Oxidation
Kinetics
Gas–solid reactions
Rare earth alloys and compounds
Corrosion
ABSTRACT
Rare earth elements have been recognized as critical materials for the advancement of many strategic and green technologies. Recently, the United States Department of Energy has invested many millions of dollars to enhance, protect, and forecast their production and management. The work presented here attempts to clarify the limited and contradictory literature on the oxidation behavior of the rare earth metal, dysprosium. Dysprosium particles were isothermally oxidized from 500 to 1000 °C in N₂–(2%, 20%, and 50%) O₂ and Ar–20% O₂ using simultaneous thermal analysis techniques. Two distinct oxidation regions were identified at each isothermal temperature in each oxidizing atmosphere. Initially, the oxidation kinetics are very fast until the reaction enters a slower, intermediate region of oxidation. The two regions are defined and the kinetics of each are assessed to show an apparent activation energy of 8–25 kJ/mol in the initial region and 80–95 kJ/mol in the intermediate oxidation reaction region. The effects of varying the oxygen partial pressure on the reaction rate constant are used to show that dysprosium oxide (Dy₂O₃) generally acts as a p-type semiconductor in both regions of oxidation (with an exception above 750 °C in the intermediate region).
Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
Global interest in rare earth element production and management has significantly increased in the past few years. In the year 2008, nearly $190 million worth of rare earth materials were imported and used in the U.S. alone. Such “strategic materials” are sought after for use in metallurgical applications and alloys, electronics, catalysts, and cathode ray tubes [1]. Compared to the light rare earth metals, relatively little published literature exists for praseodymium, promethium, thulium, lutetium, and dysprosium (also known as the heavy rare earths) although scientific interest in these metals is on the rise. In the next forty years, Hoenderdaal et al. [2] project that the demand for dysprosia (Dy₂O₃) and other dysprosium compounds will increase to between 7 and 25 times (to 14–50 ktons) from the 2010 demand. Modern studies suggest that dysprosium is more abundant than 44 other elements in the earth’s crust (5.2 mg/kg) contrary to earlier perception, making its utility more available for emerging materials technologies [3].
According to Hoenderdaal et al. [2], the proposed increase in demand is due to the future of magnetic applications (electric motors) due to dysprosium’s exceptional magnetic moment of 10.8 Bohr magnetrons, which is second only to holmium [3], and its ability to induce coercivity as an alloying agent or dopant [3,4]. However, the demand for dysprosium has become more prevalent in other applications due to its many other interesting properties. For instance, dysprosium’s isotopes strongly absorb neutrons which lead to its use in nuclear reactor control rod moderator materials [3,5–7] and Dy₂O₃ has been considered as a substitute for SiO₂ in high-k dielectric applications due to its large band gap (4.9 eV) and dielectric constant (14) [8,9]. Additionally, dysprosium mononitride (DyN) has been postulated as a suitable surrogate for americium mononitride (AmN) due to its similar physical and chemical attributes in studying its sintering and alloying effects in spent nuclear fuel reprocessing [10–15]. DyN has also been investigated as a material for ferromagnetic and semiconductor superlattice structures for spintronic applications [16–18].
Although a few manuscripts and reports were published in the late 1950s and 1960s on the oxidation behavior of dysprosium [19–22], the experiments were performed on monolithic samples (rather than powders or particles) and the results appeared to be contradictory. Of particular interest in this study is the high temperature oxidation kinetics of elemental dysprosium in the temperature range of 450–1000 °C in N₂–(2%, 20%, and 50%) O₂ and Ar–20% O₂ using simultaneous thermal analysis techniques. An understanding of the oxidation kinetics of dysprosium has a wide
range of implications ranging from semiconductor and electronics applications, to “green” energy and electric motor (magnetic) applications, to nuclear power generation applications.
2. Experimental details
The starting material used to study the high temperature oxidation kinetics of dysprosium was dysprosium particles (99.7 wt% purity, ESPI) sieved through a 40 mesh (<420 μm) sieve (Fig. 1). The particles had a surface area of $0.20 \pm 0.06$ m$^2$ g$^{-1}$, as determined by nitrogen adsorption Brunauer–Emmett–Teller (BET) techniques. The particles were characterized using X-ray diffraction (XRD) with a Cu Kα source, as shown in Fig. 2. Additionally, minor concentrations of iron, yttrium, terbium, and tungsten (<3 ppm each) were identified in the starting material using a Thermo XSeries II quadrupole inductively coupled plasma mass spectrometry (ICPMS).
Oxidation experiments were performed using a high resolution simultaneous thermal analyzer (Netzsch STA 449) which is capable of collecting thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) data simultaneously. Approximately 30 mg of the dysprosium particles (sealed and stored in an argon filled glovebox) was loaded into an 85 μL alumina combustion crucible which was immediately loaded in the STA furnace. In each case, a vacuum purge cycle was started using an oxygen-gettered ultra high purity (UHP) argon gas (<1 ppb oxygen). Once the chamber was fully purged, the thermal profile was started. The four process gases used for the oxidation studies presented in this manuscript were certified mixtures of N$_2$–(2%, 20%, and 50%) O$_2$ and Ar–20% O$_2$.
Non-isothermal oxidation data were first collected to determine suitable parameters to study the isothermal oxidation behavior. In the non-isothermal experiments, the furnace was ramped from 25 to 1100 °C at 20 °C/min in a gas flow rate of 100 mL/min while TGA and DSC data were collected. Each data set was replicated at least twice and the results were averaged. Isothermal oxidation experiments were completed in each of the atmospheres from 500 to 1000 °C. In each case, the temperature of the furnace was allowed to equilibrate for 5 min at the desired temperature prior to introducing the oxidizing atmosphere and the dysprosium particles were oxidized to completion. Each isothermal oxidation experiment was repeated 3–4 times. The isothermal oxidation data were averaged together and then the average mass gain was normalized by subtracting the initial mass gain (which was less than 0.1% in all cases). To account for geometry, the data was then divided by the maximum mass obtained and then multiplied through by the specific surface area (where the specific surface area is equal to the initial mass multiplied by the surface area of the particles obtained from BET, as described above). This allowed for a direct comparison of data and accurate kinetics analysis using the specific mass gain with units of μg/cm$^2$.
3. Results and discussion
Dysprosium is reported to have a hexagonal close packed (HCP) crystal structure and a melting temperature of 1412 °C [3], although there also exists two other allotropes; a low temperature ($<−187$ °C) orthorhombic phase (Cmcm) and a high temperature (>1381 °C) face centered cubic (FCC) phase (Im3m) [3]. As seen in Fig. 2, the starting material for the oxidation studies presented here is primarily a HCP dysprosium phase (P63/mmc) with a small fraction of the high temperature FCC phase. The HCP and FCC cubic phases were identified using X-ray diffraction data from the International Centre for Diffraction Data (ICDD) database provided by Spedding et al. [23] and Curzon et al. [24], respectively. Additionally, it is important to note that the diffraction peaks were slightly shifted from the reported positions (depicted by the diamonds and circles in Fig. 2), which is discussed in more detail later in this manuscript. Although the authors understand that the geometry and morphology of the particles are not ideal for an oxidation kinetics study, the authors were unable to identify a vendor to provide single phase dysprosium particles (with minimal oxide). However, the dysprosium particles from ESPI were nearly single phase and had a low surface area which reduced un-wanted oxidation during handling.
Fig. 2 also shows a typical diffraction pattern obtained from a fully oxidized dysprosium sample in each of the atmospheres and temperatures investigated. The particular diffraction pattern shown is the result of oxidizing the dysprosium particles completely in N$_2$–20% O$_2$ at 800 °C, which formed a cubic Dy$_2$O$_3$ phase (Ia3), as compared to data in the ICDD database provided by Swanson et al. [25]. Additionally, as shown by the inverted triangles, the oxide diffraction peaks agree well with the published values for Dy$_2$O$_3$. It is also worth pointing out that there are peaks in all of the oxide diffraction patterns that were not able to be indexed, as shown in Fig. 2.
TGA and DSC data were simultaneously collected during non-isothermal oxidation of dysprosium particles in each of the
Fig. 2. X-ray diffraction patterns of the dysprosium particles used as the starting material for the high temperature oxidation study and of a sample that was isothermally oxidized in N$_2$–20% O$_2$. As seen in the figure, the starting material is primarily a HCP dysprosium phase although there does exist a very small fraction of the FCC phase. The oxidized sample is a cubic Dy$_2$O$_3$ phase and is representative of all of the oxidized powder diffraction patterns (including all environments investigated here).
The results are plotted in Fig. 3. From this figure, it is seen that the oxidation behavior in N$_2$–20% O$_2$ and Ar–20% O$_2$ is very similar throughout the entire reaction although the onset of the reaction in the nitrogen carrier gas occurs at a slightly greater temperature (approximately 20 °C). Also apparent in Fig. 3 is that the TGA trace in the N$_2$–50% O$_2$ atmosphere begins very similar to the (Ar, N$_2$)–20% O$_2$ but the kinetics slow and the TGA trace begins to deviate at approximately 50% of the total mass gain and 680 °C, as indicated by the star in Fig. 3. The deviation increases with temperature and ultimately delays the complete oxidation of dysprosium in 50% O$_2$ to a temperature of 960 °C, which is approximately 100 °C greater than in 20% O$_2$. Additionally, the TGA trace from oxidation in N$_2$–2% O$_2$ follows similar behavior as the 20% O$_2$ atmospheres but the onset of the reaction occurs at a slightly higher temperature: the onset of oxidation in 2% O$_2$ occurs at 45 °C and 26 °C greater temperatures when compared to the argon and nitrogen carriers of 20% O$_2$, respectively. When comparing the DSC traces in each of the oxidizing atmospheres, there exists an exothermic peak at approximately 550 °C in all atmospheres except for the oxidation reaction in N$_2$–2% O$_2$.
The results of the non-isothermal experiments informed and subsequently lead to the region of study for the isothermal oxidation temperatures reported in the remainder of this manuscript. As previously stated, isothermal oxidation experiments of dysprosium particles were completed 3–4 times at each temperature and atmosphere to arrive at average behaviors, which was used for the kinetics analysis. Fig. 4 shows typical TGA data variability between duplicated oxidation experiments. In this figure, TGA data obtained from oxidizing dysprosium particles from 650 to 800 °C in N$_2$–20% O$_2$ are used to show the typical data variability, which is minimal. The variability that is present is due to the cumulative effects of several factors, including: irregular and non-uniform particle morphology (Fig. 1), the presence of a slight amount of secondary phase in the starting material (Fig. 2), the presumed presence of a native oxide and unquantified amounts of dissolved oxygen, the likelihood of residual stresses in the starting material due to vendor processing (as indicated by the slight leftward shift in the diffraction peaks when compared to the published values [23] shown in Fig. 2), as well as the sampling technique used (i.e. a scoop sampling technique was used whereas a more effective and representative sampling technique could have been used, such as powder riffling). However, since multiple data sets were averaged for all of the analysis, the assumption is that the results are representative. It should also be noted that although the effects of particle size distribution will influence the kinetics of thermal oxidation, no effort was made in this investigation to de-convolute such effects other than to account for the starting surface area and a nominal shrinking core.
Fig. 5 shows the isothermal oxidation behavior of dysprosium particles in N$_2$–20% O$_2$ from 500 to 950 °C. Two interesting observations from Fig. 5 are: there is a distinct inflection where the oxidation kinetics slow and the specific mass gain where the inflection occurs increases with isothermal oxidation temperature. The inflection, or transition from relatively rapid to slower oxidation kinetics, is seen more clearly in Fig. 5b, which focuses on the first two minutes of the oxidation reaction. Similarly, an inflection in the mass gain rate is observed in the TGA data of all of the oxidizing atmospheres investigated, as seen in Fig. 6 for each of the four oxidizing atmospheres at 700 and 800 °C. Note that all of the TGA traces reach the same terminal specific mass gain of $5.1 \cdot 10^{-10}$ μg/cm$^2$, which is based on the measured surface area and is nearly 4% less than the theoretical mass gain of the oxidation reaction. The cause of the difference is likely due to varying amounts of oxygen dissolution in the dysprosium prior to thermal treatments (predicted by Okabe et al. [26]) and variability in particle size distribution, as discussed later. Additionally, each of the TGA traces in the initial region of oxidation exhibits nearly identical mass gain.
Fig. 3. Non-isothermal oxidation traces of the oxidation of dysprosium particles. The simultaneous TGA and DSC behavior is shown for each of the oxidation atmospheres investigated (N\textsubscript{2}–(2%, 20%, or 50%) O\textsubscript{2} and Ar–20% O\textsubscript{2}). The stars indicate apparent locations where deviations in mass change occur due to different oxidizing atmospheres.
Fig. 4. Typical isothermal TGA traces used for the kinetics assessment of the oxidation of dysprosium particles in N\textsubscript{2}–20% O\textsubscript{2}. The traces shown are of multiple runs at the indicated temperatures to show the variability of the data. Average curves were calculated and used for the subsequent kinetics assessment. The inset details the initial region of oxidation and shows that a transition occurs in the first minute of the oxidation reaction in N\textsubscript{2}–20% O\textsubscript{2}.
rates for each atmosphere; the primary difference being that the inflection, as previously discussed, occurs at a larger specific mass gain with increasing isothermal oxidation temperatures.
As seen by the inflections illustrated in the insets of Figs. 4 and 6 as well as in Fig. 5b, the mechanism for oxidation appears to change after some specific mass gain, which is affected by temperature as well as the oxidation atmosphere. Accordingly, a single oxidation model is not capable of describing the entire oxidation reaction and thus, each of the two regions were defined for individual kinetics assessment in each atmosphere and at each isothermal oxidation temperature. The two regions of differing oxidation behavior in each of the four oxidizing atmospheres are
quantified in Fig. 7 and labeled as the initial and intermediate regions, respectively. The initial region is defined by the onset of mass gain to the onset of the inflection and the intermediate region begins immediately after the inflection through a reaction extent of 95%, or a specific mass gain of $4.85 \cdot 10^{-10} \mu g/cm^2$. Each data point in Fig. 7 corresponds to the average reaction extent, $x$, defining the end of the initial region of oxidation and the beginning of the intermediate region of oxidation (resulting in a point nearly centered on the inflection in the mass gain data shown in the insets of Figs. 4 and 6 as well as in Fig. 5b). The reaction extent ($x$) is defined as the ratio of mass gain at time $t$ divided by the total mass gain observed in the oxidation reaction.
An interesting observation from Fig. 7 is that the change in oxidation kinetics (from initial to intermediate reaction kinetics) occurs at lower reaction extents (i.e., the initial oxidation region is a smaller percentage of the overall reaction) in the Ar–20% O$_2$ oxidizing atmosphere when compared to the nitrogen carrier gases. Additionally, the oxidation reaction is nearly fully defined by the initial reaction region at temperatures equal to and greater than 900, 950, and 1000 °C in N$_2$–2% O$_2$, N$_2$–50% O$_2$, N$_2$–20% O$_2$,
Fig. 6. Isothermal thermogravimetric traces describing the oxidation of dysprosium particles at 700 and 800 °C in each of the atmospheres investigated (N₂–(2%, 20%, or 50%) O₂ and Ar–20% O₂). The inset details the initial region of oxidation and shows that the transition into the intermediate region is delayed in the N₂–2% O₂ atmosphere. The circles are intended to guide the reader to group similar temperature data together.
Fig. 7. Plot showing the effect of isothermal oxidation temperature on reaction extent where oxidation kinetics change for each of the oxidation atmospheres investigated (N₂–(2%, 20%, or 50%) O₂ and Ar–20% O₂). Each data point corresponds to the average reaction extent defining the end of the initial oxidation region and the beginning of the intermediate oxidation region (resulting in a point nearly centered on the inflection in the mass gain data shown in the insets of Figs. 4 and 6 as well as in Fig. 5b).
respectively, whereas the initial reaction region comprises only 30–40% of the oxidation reaction at the same temperatures in Ar–20% O₂.
As previously stated, the published literature on the oxidation kinetics of dysprosium is limited; very few publications were found that describe the oxidation kinetics of bulk materials and
none were found describing the behavior of powders or finely distributed particles. Of the literature found, the mechanism of the oxidation reaction is presumed to remain constant over the entire reaction at each temperature, contrary to the results presented here. In a report written by Love [20] in 1959, the rate of dysprosium oxidation was best fit to linear kinetics from 200 to 600 °C in clean air (nearly 20% O₂). However, in the late 1960s Pethe et al. [21] and Greene et al. [22] both reported that the oxidation kinetics follow a parabolic model. Greene et al. presents dysprosium oxidation in temperatures of 300–800 °C in clean air (N₂–20% O₂) and Pethe et al. from 500 to 800 °C in an atmosphere with an oxygen pressure of 100 Torr (13% O₂ at 1 atm total pressure).
In the work presented here, the isothermal oxidation (TGA) data in the initial and intermediate oxidation regions (as defined in Fig. 7) at each temperature and atmosphere were individually fit to many different kinetic models to best determine the kinetics rate constant (k). The models considered included various nucleation models, geometric contraction models, diffusion models, and reaction order models; many of which are described by Khawam et al. [27]. Each of the data sets were fit with each model and the model which best fit the majority of the isothermal oxidation data sets (as determined by a coefficient of determination, or R-squared parameter) was used for the kinetics analysis.
The initial regions of oxidation in each atmosphere was best fit using a contracting area model (also known as a contracting cylinder model), described by:
\[ k_{p,i} t = 1 - (1 - \alpha)^{\frac{2}{3}} \]
(1)
where \( k_{p,i} \) is the parabolic rate constant in μg/s cm², \( t \) is the isothermal oxidation time in seconds, and \( \alpha \) is the reaction extent. As previously discussed, the reaction extent (\( \alpha \)) was normalized to the maximum value observed after converting to a specific mass gain value with units of μg/cm². The contracting area model assumes that nucleation occurs rapidly at the surface of the dysprosium particles and the rate of oxidation is controlled by the resulting reaction interface progressing toward the center of the assumed cylinder [27]. This is a significant assumption considering the morphology of the starting material, as shown in Fig. 1.
As seen in Figs. 5–7, the initial region of the oxidation reaction is confined to a relatively short time period, early in the progression of the overall reaction (less than 1 min in all but the 2% O₂ atmosphere). Since the kinetics in this region are fast, some of the models could only be fit to a few data points, whereas other models (especially in the intermediate region) are defined by many thousands of data points. As seen in Table 1, this fact does not seem to effect the correlation coefficient in the N₂–2% O₂ and N₂–50% O₂ atmospheres, but the correlation coefficient begins to decrease below 700 °C in the N₂–20% O₂ atmosphere and above 700 °C in the Ar–20% O₂ atmosphere. This may suggest a breakdown of the contracting area kinetics model that was empirically chosen, as previously discussed. In the case of the initial region (below 700 °C) of oxidation in the N₂–20% O₂ atmosphere, the model that best fits the TGA data was a nucleation model (the Avrami–Erofeyev (AE2) [27]). However, in the case of the initial region (above 700 °C) of oxidation in the Ar–20% O₂, a better fit was not found.
In a similar manner as described above for the initial region kinetics, the best fit kinetics model in the intermediate region of the oxidation reaction in each of the atmospheres is the Ginstling–Brounshtein diffusion model (a deviation of the Jander model [27]), described by:
\[ k_{p,int} t = 1 - \frac{2}{3} \alpha - (1 - \alpha)^{\frac{2}{3}} \]
(2)
As seen in Table 2, the Ginstling–Brounshtein model correlates well to the oxidation data (TGA) in all atmospheres except for N₂–2% O₂, where geometric contraction models (in particular, a contracting area model) best fit the data.
Similar to the contracting area model used to describe the initial region of oxidation, the Ginstling–Brounshtein model is also a deceleratory model, suggesting that the rate of reaction slows as the reaction extent increases or the available area for reaction decreases. The reaction rate decreases due to the fact that the product layer increases with reaction extent and results in an increasing diffusion distance for cations and/or anions, and thus, slowing the reaction.
In each case, the parabolic rate constant (\( k_{p,i} \) and \( k_{p,int} \)) was determined by plotting the kinetics model (Eqs. (1) and (2)) versus the isothermal reaction time and extracting the slope from a linear relation. As noted previously, the parabolic rate constants for each temperature and atmosphere are listed in Table 1 for the initial oxidation region kinetics and Table 2 for the intermediate oxidation region kinetics.
In order to determine an activation energy for the oxidation reaction in each of the oxidizing atmospheres, the rate constants were plotted using the Arrhenius relation:
\[ k_{p,i}, k_{p,int} = A e^{-\frac{E_a}{RT}} \]
(3)
where \( A \) is a pre-exponential factor, \( E_a \) is the activation energy for the rate limiting mechanism(s) of the oxidation reaction, \( R \) is the universal gas constant (8.3145 J/mol K), and \( T \) is the absolute temperature in Kelvin. The Arrhenius behavior describing the oxidation of dysprosium particles in the four atmospheres investigated is shown in Fig. 8 for the initial region of oxidation and in Fig. 9 for the intermediate region. As shown in Fig. 9b, unusual oxidation behavior was observed in the N₂–50% O₂ oxidation atmosphere.
### Table 1
Parabolic rate constants (\( k_{p,i} \)) and correlation coefficients (R-squared values) for the fits of the oxidation kinetics in the initial oxidation region for all atmospheres using a contracting area model.
| Temp (°C) | N₂–2% O₂ | N₂–20% O₂ | N₂–50% O₂ | Ar–20% O₂ |
|-----------|----------|-----------|-----------|-----------|
| | \( k_{p,i} * 10^{-7} \text{ g}^2/\text{cm}^4\text{s} \) | \( k_{p,i} * 10^{-7} \text{ g}^2/\text{cm}^4\text{s} \) | \( k_{p,i} * 10^{-7} \text{ g}^2/\text{cm}^4\text{s} \) | \( k_{p,i} * 10^{-7} \text{ g}^2/\text{cm}^4\text{s} \) |
| 1000 | – | – | – | 58.9 |
| 950 | – | 36.6 | 0.999 | – |
| 900 | 55.1 | 34.0 | 0.999 | 71.7 |
| 850 | 3.96 | 38.6 | 0.999 | 75.1 |
| 800 | 3.77 | 32.8 | 0.996 | 60.2 |
| 750 | 3.81 | 29.8 | 0.988 | 53.6 |
| 700 | 3.40 | 21.8 | 0.956 | 48.0 |
| 650 | 3.60 | 16.4 | 0.986 | 45.2 |
| 600 | 2.72 | 15.8 | 0.976 | 30.2 |
| 550 | 3.15 | 12.6 | 0.970 | – |
| 500 | – | 10.5 | 0.969 | – |
| 450 | – | 6.96 | 0.979 | 9.27 |
R²: 0.994, 0.997, 0.999, 0.991, 0.990, 0.994, 0.998, 0.998, 0.990, 0.992, 0.993, 0.982, 0.980, 0.973, 0.957, 0.988, 0.982, 0.987, 0.980, 0.973, 0.957, 0.988
Table 2
Parabolic rate constants ($k_{p,\text{int}}$) and correlation coefficients (R-squared values) for the fits of the oxidation kinetics in the intermediate oxidation region for all atmospheres using the Ginstling–Brounshtein diffusion model.
| Temp (°C) | N$_2$–2% O$_2$ | N$_2$–20% O$_2$ | N$_2$–50% O$_2$ | Ar–20% O$_2$ |
|-----------|----------------|----------------|----------------|--------------|
| | $k_{p,\text{int}} \times 10^{-12}$ g$^2$/cm$^4$ s | R$^2$ | $k_{p,\text{int}} \times 10^{-12}$ g$^2$/cm$^4$ s | R$^2$ | $k_{p,\text{int}} \times 10^{-12}$ g$^2$/cm$^4$ s | R$^2$ | $k_{p,\text{int}} \times 10^{-12}$ g$^2$/cm$^4$ s | R$^2$ |
| 1000 | – | – | – | 96.8 | 0.992 |
| 950 | – | 76.8 | 0.982 | – | – |
| 900 | – | 38.8 | 0.989 | 22.9 | 0.970 | 46.0 | 1.000 |
| 850 | 31.5 | 0.978 | 23.2 | 0.990 | 16.3 | 0.971 | 41.0 | 0.995 |
| 800 | 15.5 | 0.972 | 17.2 | 0.997 | 9.87 | 0.980 | 33.2 | 0.995 |
| 750 | 1.02 | 0.975 | 10.7 | 0.977 | 8.40 | 0.988 | 21.5 | 0.995 |
| 700 | 6.04 | 0.959 | 6.73 | 0.992 | 8.37 | 0.997 | 16.6 | 0.999 |
| 650 | 3.42 | 0.966 | 4.72 | 0.976 | 6.59 | 0.996 | – | – |
| 600 | 1.84 | 0.984 | 3.40 | 0.992 | 3.87 | 0.995 | 5.00 | 0.995 |
| 550 | 0.93 | 0.994 | 1.54 | 0.992 | 1.79 | 0.995 | 1.39 | 0.991 |
| 500 | – | 0.528 | 0.990 | – | – | – | – | – |
| 450 | – | 0.237 | 0.996 | 0.231 | 0.993 | 0.290 | 0.998 |
Fig. 8. Arrhenius plot for the initial isothermal oxidation region of dysprosium particles in each of the atmospheres investigated using a contracting area kinetics model. The apparent activation energy is calculated from the linear relation and is labeled for each atmosphere.
where there exists a discontinuity in the reaction kinetics between 650 and 800 °C.
The oxidation rate constants in the initial region of the oxidation reaction were found to be much greater (5–6 orders of magnitude) with a lower apparent activation energy than those found for the intermediate region in each atmosphere. The activation energies in the initial region of oxidation for all of the atmospheres (except the N$_2$–2% O$_2$ atmosphere) were found to be approximately 24 kJ/mol (Fig. 8). The value might be compared to the 38 kJ/mol reported by Greene et al. [22], who reported the oxidation of monolithic dysprosium between 300 and 550 °C, but experimental details are lacking which make comparisons difficult. Another interesting trend in Fig. 8 is that samples in the N$_2$–50% O$_2$ atmosphere have a rate constant that is nearly twice that of those oxidized in the (Ar, N$_2$)–20% O$_2$ atmospheres and more than 10 times that of those oxidized in the N$_2$–2% O$_2$ atmosphere. However, the activation energy in the initial reaction region in the 2% oxygen atmosphere was significantly lower, at 8 kJ/mol.
Additionally, the oxidation kinetics in the intermediate region of the oxidation reaction were all found to have an apparent activation energy between 80 and 90 kJ/mol over the temperature range studied, with the exception of the discontinuity observed in the 50% oxygen atmosphere (Fig. 9). The fact that the activation energy in the 2% oxygen atmosphere is slightly higher (90 kJ/mol versus 80 kJ/mol) suggests that the mechanism for oxidation requires slightly more energy (e.g., temperature) than the higher oxygen concentration environments. In any case, these values are fairly similar to those presented by Greene et al. [22] and Pethe et al. [28] who described the overall kinetics of dysprosium oxidation using parabolic kinetics and determined an activation energy of approximately 105 kJ/mol. The discontinuity observed in the 50% oxygen atmosphere, which suggests an apparent activation energy of zero, is indicative of a spontaneous oxidation reaction but is suspected to be anomalous, but repeated, behavior.
In both the initial and intermediate regions of the isothermal oxidation reaction, it appears that the carrier gas (containing 20% O$_2$) has minimal effects the oxidation rate of dysprosium based on similar oxidation rate constants (argon results in a rate constant nearly double that observed in nitrogen) and apparent activation energies. However, by comparing the non-isothermal oxidation behavior (Fig. 3) and the selected isothermal behavior (Fig. 6), it is clear that the argon carrier gas promotes more rapid overall oxidation kinetics. This is likely due to the fact that the argon carrier gas induces a transition into the intermediate stage of oxidation at
an earlier reaction extent (Fig. 7) than nitrogen atmospheres. Being that the transition occurs at a lower reaction extent suggests that the initial oxidation layer is thinner, reducing the diffusion length of the oxygen ions, and resulting in faster kinetics in the intermediate region, as observed in Table 2.
A brief, additional study investigating the effects of gas flow rate on the oxidation reaction was performed. In the study, the gas flow rate was increased from 100 to 250 mL/min in an isothermal oxidation temperature range of 650–850 °C. The intermediate region reaction rate and the transition reaction extent (Fig. 7) remained constant, but an effect on the initial region oxidation rate was observed. Although the trends were similar for the initial region oxidation rates, the reaction onset occurred quicker and the rate of oxidation reaction increased with increased gas flow rate. The findings suggest that there is a time delay for the furnace reaction chamber to reach the nominal oxygen concentration of the gas stream. However, the fact that the transition reaction extent remained constant suggests that a critical volume or surface area must be consumed in order for the reaction mechanism to change.
To gain insight into the mechanism of dysprosium oxidation, it is helpful to understand the type of defects present in the oxide. Accordingly, Brouwer diagrams (also referred to as Kroeger–Vink plots [29]) were constructed in each of the identified oxidation...
regions using the three partial pressures of oxygen used to oxidize dysprosium. As seen in Fig. 10a, the log–log plot in the initial region of oxidation shows a nearly linear dependence of oxygen partial pressure on the rate constant for each temperature studied (600–800 °C). The slope gradually changes from +3/4 to +1 with increasing temperature. Due to the positive slopes over the entire range of temperatures, it is presumed that the oxide behaves as a p-type semiconductor and suggests that the oxidation mechanism is either due to diffusion of oxygen through interstitial sites or dysprosium through vacancies. However, the effects of oxygen pressure on the rate constant in the intermediate region of oxidation is more complicated. As seen in Fig. 10b, which also shows nearly linear correlations, the slope changes from +1/5 to −1/5 from 550 to 850 °C, respectively, with a near zero slope at 750 °C. As was
observed in the initial oxidation region, the positive slope suggests that the oxide acts as a p-type semiconductor in the lower temperature range and as an n-type semiconductor in the high temperature range, with competing defects counteracting each other’s charge near 750 °C. An n-type semiconductor suggests that the oxidation mechanism is either due to diffusion of dysprosium through interstitial sites or oxygen through vacancies. Oxygen diffusion is more likely due to the very large size of the dysprosium ions. The n-type behavior was also observed by Tare and Schmalzried [30] from 667 to 860 °C while a later report by Lal et al. [31] suggests that Dy$_2$O$_3$ is a p-type semiconductor at 627 °C with a 0.011 cm$^{-2}$ V$^{-1}$ s$^{-1}$ hole mobility and 0.010 cm$^{-2}$ V$^{-1}$ s$^{-1}$ electron mobility. Pethe et al. [21] also notes that the parabolic rate constant remains constant (within the experimental error) from 1% to 13% O$_2$ at 600 °C. However, it is known that minor concentrations of impurities in the starting materials can significantly alter the semiconducting behavior of the oxide. Since impurity concentrations were neither identified nor quantified in previously reported oxidation behavior, comparisons with the work presented here may not be valid. Further work in this area needs to be completed.
To provide further insight into the mechanism of oxidation and to eliminate possible rate limiting defect mechanisms, an inert marker experiment was performed. A thin gold wire was fixed to a nearly 2 mm thick monolithic dysprosium sample which was subsequently oxidized in N$_2$–20% O$_2$ at 650 °C for 45 h. The gold marker remained at the oxide/gas interface after oxidation, suggesting that growth occurs at the metal/oxide interface. This observation is consistent with the work of Pethe et al. who found similar behavior via an inert (silver) marker experiment at 800 °C. The results of the marker experiments suggest that when the oxide behaves as a p-type semiconductor, the primary defect mechanism is most likely the diffusion of oxygen via dysprosium vacancies in the oxide and when the oxide behaves as an n-type semiconductor, the primary defect mechanism is likely oxygen diffusion through oxygen vacancies in the oxide.
4. Conclusions
The high temperature oxidation of dysprosium particles in 2–50% oxygen atmospheres in both nitrogen and argon carrier gases was studied from 450 to 1000 °C using both non-isothermal and isothermal techniques. The results of this study have identified two distinct regions of oxidation in each of the four atmospheres investigated, termed the initial and intermediate stages. The reaction rates at 800 °C in the initial oxidation region in N$_2$–2% O$_2$, N$_2$–20% O$_2$, Ar–20% O$_2$, N$_2$–50% O$_2$ were found using a contracting area model as $10^{-7} \ast (3.77, 32.8, 31, \text{and } 60.2 \text{g}^2/\text{cm}^4 \text{s})$, respectively. The reaction rates at 800 °C in the intermediate oxidation region in N$_2$–2% O$_2$, N$_2$–20% O$_2$, Ar–20% O$_2$, N$_2$–50% O$_2$, were found using the Ginstling–Brounshtein diffusion model as $10^{-11} \ast (1.5, 17.2, 33.2, \text{and } 9.87) \text{g}^2/\text{cm}^3 \text{s}$, respectively. The apparent activation energy was found to be much lower in the initial stages (8 kJ/mol in 2% oxygen and 25 kJ/mol in the 20 and 50% oxygen atmospheres) than in the intermediate stages (80–90 kJ/mol).
Insight into the defect mechanism limiting the kinetics of the oxidation reaction was provided by an inert marker experiment as well as identifying the effects of oxygen pressure on the kinetics rate constant via Kroeger–Vink diagrams. It was determined that oxide growth occurs at the metal/oxide interface, most likely via oxygen diffusion. With the exception of above 750 °C in the intermediate oxidation region, the oxide behaves as a p-type semiconductor. The conclusion drawn from the evidence provided is that the rate limiting oxidation mechanism is oxygen diffusion via dysprosium vacancies to grow at the Dy/Dy$_2$O$_3$ interface. In the cases where the oxide behaves as an n-type semiconductor, oxidation is primarily rate limited by oxygen ion diffusion via oxygen vacancies to the Dy/Dy$_2$O$_3$ interface. However, further studies are needed to better understand the effects of impurity types and concentrations on the semiconducting properties of the oxide layer.
Acknowledgements
The authors would also like to acknowledge Nicole Leraas and Kelci Lester for their contributions and helpful discussions.
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Animal sexual signals:
Do they maximise or optimise information content?
Submitted by Iker Vaquero-Alba to the University of Exeter
as a thesis for the degree of
Doctor of Philosophy in Biological Sciences
In December 2011
This thesis is available for Library use on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement.
I certify that all material in this thesis which is not my own work has been identified and that no material has previously been submitted and approved for the award of a degree by this or any other University.
Signed: Iker Vaquero-Alba
Abstract
Traditional models of sexual selection based on the handicap principle assume a direct linkage between the degree of sexual signal exaggeration and the bearer’s quality, and set out a rather inflexible scenario where handicap exaggeration is maximised for sexual signalling purposes until it reaches the limit imposed by viability selection. Such a scenario makes it difficult to imagine the mechanisms by which multicomponent signalling systems can evolve and persist in time, given the costs of producing, disseminating and receiving signals. Based on non-equilibrium coevolutionary models, it has been suggested that variation in selection pressures derived from fluctuations in ecological and/or social conditions may lead to the emergence and maintenance of redundant and non-redundant multiple signals. Alternatively, the non-equilibrium dynamics to which coevolutionary systems are often subject can maintain multiple signals without environmental variability. Species with severe fitness constraints on costlier signal expression should be selected to utilize “cheaper” signals. And individuals not displaying at the maximum possible level might be selected to “compensate” their lack of fitness using phenotypically plastic traits, like behavioural ones. Here I investigate the effect of several potentially sexually selected barn swallow ornamental traits on several reproductive success indicators and on the habitat quality of foraging areas around breeding sites, and of several quality-indicating guppy traits on predator inspection activity, a behavioural character involved in mate choice. The findings presented here indicate ventral and throat plumage colouration, previously not studied for European barn swallows, to function as quality indicators and predict reproductive success and assortative mating patterns. Additionally, we found evidence for a “compensation mechanism” in both
species studied, for individuals investing in “cheaper” sexual signals or not displaying at the maximum possible level.
Acknowledgements
First of all, I would like to offer my deepest gratitude to Dr. Sasha Dall, the man who has made possible the dream of my entire life come true. During these four years, he has guided me through the process of becoming a scientist, and he has kindly shared his vast knowledge and his endless creativity with me, with a constant flow of new ideas and suggestions, which have undoubtedly and definitely improved my work in a significant way. He always had a kind word of support when I needed it, and he has always unshakably believed in me, even when my own faith in myself was not in the highest point. I will be indebted to him for the rest of my life. Equally, thank you very much to Professor Matthew Evans for his guidance and support, and for sharing with me his huge knowledge on barn swallows. His help throughout my research, especially during experimental design and data collection, was invaluable. I’m sincerely in debt with both of my supervisors and the time and effort they devoted to improving my research.
Thanks also to Dr. Andy McGowan, my “field mentor”, who captured the barn swallows in the field, allowing me to assess their colouration. He kindly let me use his barn swallow morphometric data, made me some interesting suggestions, and I had very interesting conversations with him during the long hours of swallow capturing. Thanks to Ian Blessley for showing me the field locations, introducing me to the landlords and letting me use some of the data he collected in the field.
Thanks to Dr. Rebecca Safran for allowing me to be part of her international barn swallow research group, and for her contribution to the study of these birds, which has been a huge inspiration for my work.
Thanks to Dr. Jon Blount for introducing me to the use of the spectrophotometer and for all the useful readings suggested. My gratitude to Dr. Tom Pike for kindly allowing me to use his Matlab equations for spectral data processing. Thanks to Dr. Joan Carles Senar for his invaluable help with repeatability-related issues.
Thank you very much to Dr. Darren Croft and Alessandro Macario for their interesting ideas and suggestions, and for sharing with me their expertise and knowledge in Trinidadian guppies. Thanks to Claire Canning for collecting the data and helping me in the planning and preparation of the fifth chapter. Thanks to Sonia Chapman for her invaluable help with guppies into the research facilities and for her kindness. Thank you guys for making the guppy chapter possible, and thanks to all the people in Exeter for making my time there an unforgettable period of my life.
I am very much indebted to all the landlords who kindly allowed me to enter their properties to have access to the nests of the barn swallows breeding on them. Not only would this research work have been impossible without their invaluable collaboration, but also they always had a kind smile and some time of friendly chat to share with me. Thanks to Julia Phillips, Brian Kessel, Michael and Nigel Vague, Max Chitty, Chris and Sarita Perkins, Ollie Bromley, Shirley Wainwright, Mrs. Tremaine, Anne Mattews and John Newton. And thanks to their lovely families, neighbours and pets.
Gracias a papá y mamá, a Javier y a la abuelita Rosa, por su amor incondicional y porque desde que era un mocoso fascinado por los bichos ya sabían que este momento llegaría tarde o temprano. Esta tesis doctoral está dedicada a ellos.
Thanks to Prof. José Luis Tellería and Dr. Ángeles Vázquez for making me love Zoology during my undergraduate course, and for helping me get the grant, which supported this PhD.
Thanks to Dr. MD Sharma, Alfredo Attisano, Eric Flores de Gracia, Dr. Iva Fukova, Dr. Jesús Tomás, Dr. Daniel Pincheira Donoso, Dr. Will Pitchers, Dr. Cristina López Chertudi, Alfonso Ramallo, Andrew Vickers, Aijan Martin, Ezgi and Alan Jenkins for their sincere friendship and continuous support. Thanks to Fran Tyler for all the celebration arrangements and for a cake that I do not have any doubt is going to be spectacular. Thanks to all the people in Centre for Ecology and Conservation and people in Falmouth for four unforgettable years in my life.
Thanks to Hannah “Spippy” Pearce, Laura Tucker and Philip Winslow for helping me not to lose my mind during the long writing-up seclusion period at home.
Thanks to Irene Palacios, Chelo Font and Javier Aragón. Thanks to all my friends in Ermua and Eibar (aun conservo la Tarjeta Ángel de la Guarda) and all the people who always believed in me.
This PhD studentship was sponsored by a grant from the “Programa de Formación de Personal Investigador, Departamento de Educación, Universidades e Investigación, Gobierno Vasco”.
Contents
Title Page: Animal sexual signals: Do they maximise or optimise the information content? ................................................................. 1
Abstract ........................................................................................................... 2
Acknowledgements ....................................................................................... 3
Contents ........................................................................................................... 6
Tables and Figures .......................................................................................... 7
Author’s Declarations .................................................................................... 11
Chapter One: Multicomponent signalling systems and the “compensation mechanism” ................................................................. 13
Chapter Two: Objective feather colour assessment using GLMMs and ANOVA: measurements in the lab are more reliable than in the field ...................................................... 22
Chapter Three: Multiple signalling, habitat quality and laying date in the European barn swallow *Hirundo rustica rustica* ............................................................................. 55
Chapter Four: Plumage colouration, as well as tail-streamer length, is a sexually selected trait in European barn swallows ............................................................................. 93
Chapter Five: Predator inspection activity in Trinidadian guppies (*Poecilia reticulata*): drab males are bolder than conspicuous males in the presence of Females .................................................................................................................. 144
Chapter Six: General discussion ........................................................................ 175
# Tables and Figures
**Chapter One** .................................................................................................................. 13
**Chapter Two** .................................................................................................................. 22
**Table 1**: ANOVA-derived Repeatabilities in 2009 plumage colouration measurements taken from live birds in the field, feather samples in the lab, and across both procedures (UV+Visible spectrum) ........................................................................... 46
**Table 2**: ANOVA-derived Repeatabilities in 2009 plumage colouration measurements taken from live birds in the field, feather samples in the lab, and across both procedures (Only Visible part of the spectrum) ........................................................................... 47
**Table 3**: ANOVA-derived Repeatabilities in 2010 plumage colouration measurements taken from live birds in the field, feather samples in the lab, and across both procedures (UV+Visible spectrum) ........................................................................... 48
**Table 4**: ANOVA-derived Repeatabilities in 2010 plumage colouration measurements taken from live birds in the field, feather samples in the lab, and across both procedures (Only Visible part of the spectrum) ........................................................................... 49
**Figure 1**: Location of the sampling area ............................................................................. 50
**Figure 2**: Topography of bird plumage regions: *thr* throat, *bre* breast, *bel* belly, *ven* vent (modified from Andersson & Prager, 2006) ........................................................................... 51
**Figure 3**: The avian tetrahedral colour space (from Endler & Mielke, 2005) ............... 52
**Figure 4**: Reflectance spectra for belly, breast, throat and vent patches of male and female barn swallow ........................................................................................................... 53
**Figure 5**: Repeatabilities (± 95% CI) in 2009-2010 plumage colouration measurements taken a) from live birds in the field, b) from feather samples in the
lab, and c) across both procedures, both when including the whole light spectrum or only the human-visible spectrum in the analyses ..................................................54
Chapter Three .............................................................................................................55
Table 1: GLM models within mated pairs, relating the intensity of expression of male ornaments (tail length, throat brightness and ventral brightness) and physical condition to those of their female partner (i.e. assortative mating), controlling for the effect of the year ........................................................................84
Table 2: Component loadings (unrotated) of first principal component (PC1) as quantified by principal component analysis ...................................................................84
Table 3: Summary of minimal adequate models ..........................................................85
Figure 1: The effect of throat and ventral brightness on large insect abundance (CV=coefficients of variation) .........................................................................................86
Figure 2: The effect of throat brightness and condition on large insect abundance (CV=coefficients of variation) .......................................................................................87
Figure 3: The effect of ventral brightness and condition on large insect abundance (CV=coefficients of variation) .......................................................................................88
Figure 4: The effect of tail length and ventral brightness on large insect abundance (CV=coefficients of variation) .......................................................................................89
Figure 5: Effect of male throat brightness on large insect abundance (CV=coefficients of variation) ........................................................................................................90
Figure 6: Effect of body condition on laying date ..........................................................91
Figure 7: Effect of throat brightness of males on laying date ........................................92
Chapter Four .............................................................................................................93
Table 1 Summary of minimal adequate linear mixed-effect models ......................125
Table 2: Description of the levels in the “plumage manipulation” factor ........127
Figure 1: Effect of throat brightness on hatching success .................................127
Figure 2: Effect of throat and ventral brightness on hatching success - 1st clutch (Model fit) .................................................................128
Figure 3: The effect of throat brightness and tail length on hatching success - 1st clutch (Model fit) .................................................................129
Figure 4: The effect of ventral brightness and large insect abundance on hatching success - 1st clutch (Model fit) ..................................................130
Figure 5: The effect of ventral brightness and large insect abundance on clutch size - 2nd clutch (Model fit) .......................................................131
Figure 6: Effect of plumage manipulation on clutch size (2nd clutch). (For a description of levels of “plumage modification”, see Table 2) ......................132
Figure 7: The effect of throat brightness and large insect abundance on hatching success - 2nd clutch (Model fit) .....................................................133
Figure 8: The effect of ventral brightness and large insect abundance on hatching success - 2nd clutch (Model fit) .....................................................134
Figure 9: The effect of throat brightness and tail length on hatching success - 2nd clutch (Model fit) .................................................................135
Figure 10: Effect of plumage manipulation on hatching success (2nd clutch). (For a description of levels of “plumage modification”, see Table 2) ............136
Figure 11: Effect of adult condition on chick physical condition (2nd clutch) ...........................................................................................................137
Figure 12: Effect of throat brightness on chick physical condition (2nd clutch) ...........................................................................................................138
Figure 13: Effect of tail streamer length on chick physical condition (2nd clutch) ...........................................................................................................139
Figure 14: The effect of ventral brightness and large insect abundance on chick physical condition - 2\textsuperscript{nd} clutch (Model fit) .................................................................140
Figure 15: The effect of tail length and large insect abundance on chick physical condition - 2\textsuperscript{nd} clutch (Model fit) .................................................................141
Figure 16: The effect of ventral brightness and tail length on chicks’ physical condition - 2\textsuperscript{nd} clutch (Model fit) ..................................................................................142
Figure 17: Effect of plumage manipulation on chicks’ physical condition (2\textsuperscript{nd} clutch) (For a description of levels of “plumage modification”, see Table 2).143
Chapter Five ...........................................................................................................144
Table 1 Summary of minimal adequate models .....................................................168
Figure 1: The effect of body length and carotenoid-based colouration area on shyness score (model fit) .................................................................169
Figure 2: The effect of black colouration area and carotenoid colouration area on shyness score (model fit) .................................................................170
Figure 3: Effect of male black colouration area on number of first inspections ......................................................................................................................171
Figure 4: Effect of male body length on number of first inspections ...............172
Figure 5: Effect of male carotenoid colouration area on number of first inspections ......................................................................................................................173
Figure 6: Effect of male black colouration area on time spent in risky zone ..174
Chapter Six ...........................................................................................................175
Author’s Declarations
Chapter One: Multicomponent signalling systems and the “compensation mechanism”
The views presented in this chapter are my own and were developed under the guidance of Dr. Sasha Dall and Prof. Matthew Evans.
Chapter Two: Objective feather colour assessment using GLMMs and ANOVA: measurements in the lab are more reliable than in the field
Dr. Sasha Dall and Prof. Matthew Evans provided guidance for planning and structure of all experimental procedures and in preparation of the manuscript. Dr. Andy McGowan and I collected the data. I conducted the analysis and am first author on the manuscript.
Chapter Three: Multiple signalling, habitat quality and laying date in the European barn swallow *Hirundo rustica rustica*
Dr. Sasha Dall, Prof. Matthew Evans and Dr. Andy McGowan provided guidance for planning and structure of all experimental procedures and in preparation of the manuscript. Dr. Andy McGowan, Ian Blessley and I collected the data. I conducted the analysis and am first author on the manuscript.
Chapter Four: Plumage colouration, as well as tail-streamer length, is a sexually selected trait in European barn swallows
Dr. Sasha Dall, Prof. Matthew Evans and Dr. Andy McGowan provided guidance for planning and structure of all experimental procedures and in preparation of the
manuscript. Dr. Andy McGowan, Ian Blessley and I collected the data. I conducted the analysis and am first author on the manuscript.
**Chapter Five: Predator inspection activity in Trinidadian guppies (*Poecilia reticulata*): drab males are bolder than conspicuous males in the presence of females**
Dr. Sasha Dall, Prof. Matthew Evans and Dr. Darren Croft provided guidance for planning and structure of all experimental procedures and in preparation of the manuscript. Claire Canning, Alessandro Macario and I collected the data. I conducted the analysis and am first author on the manuscript.
**Chapter Six: General discussion**
The general discussion, conclusions and future prospects presented in this chapter represent my own interpretation of the data presented in the previous chapters, under the guidance of Dr. Sasha Dall and Prof. Matthew Evans.
Chapter One
Multicomponent signalling systems and the “compensation mechanism”
Traditional models of sexual selection through the handicap principle assume a direct linkage between the degree of sexual signal exaggeration and the bearers’ quality (Zahavi, 1975). Likewise, they assume a rather inflexible scenario where handicap exaggeration is maximised for sexual signalling purposes, until the limit imposed by viability selection is reached. In such an scenario, it is difficult to imagine how multicomponent signalling systems can evolve and persist in time, especially when the signals involved are redundant, given there are costs associated with production, dissemination and reception of signals. These costs may manifest at the time of production and/or maintenance, and also continuously through increased risk of predation or parasitism (Magnhagen, 1991; Folstad & Karter, 1992).
Animals often experience fluctuating ecological and social conditions in their environment. The variability of such environments has important downstream consequences in a number of areas of their life, e.g. food availability, risk of predation or inter- and intraspecific competition etc. Transmission of information with sexual signalling purposes is also influenced by environmental fluctuations. Based on recent insights from non-equilibrium coevolutionary models, Bro-Jørgensen (2009), through his “fluctuating environments” hypothesis, suggested that variation in selection pressures derived from fluctuations in ecological conditions and/or social context may lead to the emergence and maintenance of both redundant and non-redundant multiple signals. When genotype-by-environment interactions (GEI) occur and environmental changes affect different genotypes in different ways, phenotypically fixed signals may become misleading, and new, more phenotypically plastic signals may evolve with a better quality-indicating value. Also, if environmental shifts make a signal too costly or too “cheap” to express for any individual, alternative condition-dependent signals, more able to reflect the variance in quality among signallers, may become selected.
Receiver preferences can also be influenced by environmental changes and these preferences themselves can act as a selective force for the evolution of alternative signals, favouring the evolution of multicomponent signalling systems. Sexual conflict over the reliability of condition-dependent indicators of genetic quality may keep the handicap process in a non-equilibrium situation (van Doorn & Weissing, 2006). Even in the absence of environmental variability the non-equilibrium dynamics, to which coevolutionary systems between signaller and receiver are often subject, can help maintain multiple signals.
Costs incurred by ornamental traits used for signalling purposes by animals can vary across a continuous range. At one end of the range, exaggeratedly expressed morphological traits, like extremely elongated tail feathers in birds, incur substantial production and/or maintenance costs, as well as increase the risk of predation via reductions in the aerodynamic and locomotive efficiency of their bearers. At the other end, behavioural signals incur significantly lower costs on a day-to-day basis, whereas pigment-based markings, still “cheaper” than the costlier morphological traits, would be located somewhere in the middle in terms of the costs they inflict to their bearers. Fitzpatrick (2000) points out that although handicap theory requires the handicap to be specific and act against the quality being signalled (Grafen, 1990; Zahavi & Zahavi, 1997), only a small proportion of Palaeartic birds have extremely elongated tails, a particularly costly ornamental trait in terms of aerodynamic efficiency present in some bird species. Species in which the day-to-day costs incurred by costlier signals have particularly severe fitness consequences might be selected to utilize “cheaper” signals such as markings to a greater extent and minimise elaboration of the more “expensive” traits.
Under non-equilibrium dynamics, not all the sexual signals may be expressed at the maximum possible level (see Saino et al., 2003). This may lead to unreliable communication, if we consider the quality-indicating value of certain ornamental signals alone, and highlight the quality-indicating value of more phenotypically plastic traits (e.g. those dependent on food intake or the behavioural context) for coping with dynamic variation in selection pressures. In accordance with the “fluctuating environments” or the “sexually antagonistic coevolution” hypotheses, discussed above, species with severe fitness constraints on costlier signal expression should be selected to utilize “cheaper” signals. Furthermore, we would also expect individuals with certain genotypic characteristics and/or under certain environmental circumstances to be selected to use less costly signals. Thus, high quality individuals might not be displaying at their maximum possible level, either due to not having invested all the available resources on an “expensive” trait or as a consequence of ecological and/or social fluctuations having rendered the trait less informative, altering the original information to cost ratio. These individuals might be selected to complement their sexual display using “cheaper”, phenotypically plastic signals, which might increase the amount of information signalled at a lower cost. Similarly, lower-quality individuals investing in less informative, “cheaper” traits, might be selected to “compensate” their lack of reproductive fitness using behavioural traits with implications for sexual selection. For example, European barn swallows defending higher habitat quality breeding sites (Vaquero-Alba et al., unpublished data; 3, 4) or guppies being bolder in front of predators in the presence of females (Vaquero-Alba et al., unpublished data; 5). This “compensation mechanism”, as part of new insights on multiple signalling, and unlike traditional view on costly signalling, might help explain the relatively high phenotypic variability which can be observed in certain species for traits apparently subject to a strong directional
selection. Furthermore, it might contribute to explain the persistence of individuals with lower sexual display intensity in natural populations, despite the presumed selective disadvantage and an impaired reproductive fitness.
Therefore, animals may have evolved sexual signalling systems that trade off the amount of information they signal to potential mates/rivals with their costs; thus, given their individual genotypic quality, their ability to express phenotypic plasticity and the way they are affected by environmental fluctuations, such sexual signalling systems may be selected to optimise rather than maximise their information content (i.e. they should maximise their information to cost ratio).
This thesis is an experimental investigation of the hypothesized “compensation mechanism” outlined above in two key species and the implications of this for the evolution of sexually selected multicomponent signalling systems. It integrates field and lab based research on multiple signalling for the selected species, with a focus on the interactions between the sexually selected traits studied. European barn swallow, *Hirundo rustica rustica*, was chosen for field work study, as it shows a highly developed multicomponent signalling system, comprising at least two types of sexually selected ornamental traits: an “expensive” sexual signal, i.e. the extremely elongated outermost tail feathers or tail streamers, which has been subject to numerous studies of sexual selection during the last few decades, including tail length manipulations (Möller, 1994; Buchanan & Evans, 2000; Turner, 2006); and a “cheaper” signal, i.e. throat and ventral colouration patches, main quality indicator in the North American subspecies (*H. rustica erythrogaster*; Safran, 2004), although not very well studied in the nominated subspecies (but see Ninni et al., 2004). The Trinidadian guppy, *Poecilia reticulata*, was chosen for lab based study as it offers the
possibility of exploring and experimenting with an interesting multiple signalling system which includes three different pigment-based colouration markings (melanin-based, carotenoid-based and structural) with different biochemical origins and, possibly, different production and maintenance costs, plus a sexually selected behavioural trait (predator inspection behaviour).
**A methodological approach: objective plumage colour assessment**
In the methodological chapter (Vaquero-Alba *et al.*, unpublished data; 2) the technique for objectively assessing plumage colouration of barn swallows in the lab that we use along the thesis, on feathers previously collected from live birds, is calibrated by calculating its repeatability, the repeatability of measurements taken directly on live birds in the field, and the comparability across data obtained with both techniques. Although we initially follow Quesada & Senar (2006), we also apply a novel statistical method for calculating repeatabilities using GLMMs (Nakagawa & Schielzeth, 2010), which allows us to control for the confounding effect of year variance by including it in the model as a random effect. Advantages of the new method over more traditional ones are discussed.
**Effect of ornamental traits on habitat quality, laying date and reproductive success**
The influence of several potentially sexually selected barn swallow ornamental traits on the date of laying of the first egg and on the quality of the foraging areas around breeding sites occupied by the barn swallow pairs is investigated (Vaquero-Alba *et al.*, unpublished data; 3). Also, tail length and ventral colouration manipulations are conducted and the influence of ornamental traits before manipulation and plumage
manipulations on several reproductive success indicators is investigated (Vaquero-Alba et al., unpublished data; 4). Sexual signalling function of pigment-based plumage markings is investigated, and evidence for supporting or rejecting the “compensation mechanism” through habitat quality hypothesis is gathered and discussed.
**Compensation through predator inspection behaviour in guppies**
We experimentally test the hypothesis that visually less conspicuous male guppies may compensate their lower quality-indicating value by being bolder than more conspicuous males in front of females through increased predator inspection activity (Vaquero-Alba et al., unpublished data; 5). The implications of the results for the evolution of sexual signalling systems are discussed.
**References**
- Andersson, M. (1994) *Sexual Selection*. Princeton University Press, Princeton, New Jersey.
- Bro-Jørgensen, J. (2009) Dynamics of multiple signalling systems: animal communication in a world in flux. *Trends in Ecology and Evolution* **25**, 292-300.
- Buchanan, K.L. & Evans, M.R. (2000) The effect of tail streamer length on aerodynamic performance in the barn swallow. *Behavioral Ecology* **11**, 228–238.
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- Quesada, J. & Senar, J.C. (2006) Comparing plumage colour measurements obtained directly from live birds and from collected feathers: the case of the great tit *Parus major*. *Journal of Avian Biology*, **37**, 609-616.
- Safran, R.J. & McGraw, K.J. (2004) Plumage coloration, not length or symmetry of tail-streamers, is a sexually selected trait in North American barn swallows. *Behavioral Ecology* **15**, 455-461.
- Saino, N., Romano, M., Sacchi, R., Ninni, P., Galeotti, P. & Møller, A.P. (2003) Do male barn swallows (*Hirundo rustica*) experience a trade-off between the expression of multiple sexual signals? *Behavioral Ecology and Sociobiology* **54**, 465-471.
- Turner, A.K. (2006) *The barn swallow*. T&A D Poyser, London.
- van Doorn, G.S. & Weissing, F.J. (2006) Sexual conflict and the evolution of female preferences for indicators of male quality. *The American Naturalist* **168**, 742-757.
- Vaquero-Alba, I., Evans, M.R. & Dall, S.R.X (unpublished) Objective feather colour assessment using GLMMs and ANOVA: measurements in the lab are more reliable than in the field.
- Vaquero-Alba, I., McGowan, A., Evans, M.R. & Dall, S.R.X. (unpublished) Multiple signalling, habitat quality and laying date in the European barn swallow *Hirundo rustica rustica*.
- Vaquero-Alba, I., McGowan, A., Evans, M.R. & Dall, S.R.X (unpublished) Plumage colouration, as well as tail-streamer length, is a sexually selected trait in European barn swallows.
- Vaquero-Alba, I., Canning, C., Macario, A., Croft, D.P., Endler, J.A., Evans, M.R. & Dall, S.R.X. (unpublished) Predator inspection activity in Trinidadian guppies (*Poecilia reticulata*): drab males are bolder than conspicuous males in the presence of females
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Chapter Two
Objective feather colour assessment using GLMMs and ANOVA: measurements in the lab are more reliable than in the field
Iker Vaquero-Alba, Matthew R. Evans & Sasha R.X. Dall
Centre for Ecology and Conservation, University of Exeter, Cornwall Campus
Penryn, Cornwall, TR10 9EZ, UK
Abstract
1. Colouration in birds and other animals can act as an important sexual signal for transmitting information to rivals and potential mates. Female birds can get direct or indirect benefits by choosing to mate with elaborately ornamented males, so birds with more colourful displays often have a selective advantage. Reflectance spectrometry has become a standard tool in behavioural ecology for measuring animal colouration, especially after the discovery of tetrachromacy in birds, which includes their ability to see in the UV and thus the existence of plumage patterns invisible to humans, necessitating a reliable and objective way of assessing colouration not dependent on human vision. Measurements of plumage colouration can be taken directly on live birds in the field or in the lab, or on feathers previously collected from the animals so it is important to compare the reliabilities of these different sampling methodologies.
2. Using a spectrophotometer, we assessed the repeatability of both methods separately, as well as comparing between them, for measurements of melanin-based colouration (brightness, chroma, hue and UV chroma) in different patches of the birds’ plumage. We used the ANOVA-based method for calculating the repeatability of measurements from two years separately, and the GLMM-based method to calculate the overall adjusted repeatability within each patch for data from both years. To our knowledge, this is the first time the latter, newly developed method has been used to assess the repeatabilities of colour measurements. We repeated the assessment for the whole reflectance spectrum range (300-700 nm) and only the human-visible spectrum range (400-700 nm) to assess the influence of the UV component on the reliabilities of the different plumage sampling methodologies.
3. Our results showed a very high repeatability for lab measurements and a lower but still moderate to high repeatability for field measurements, both of which
increased when limited to only the human-visible part of the reflectance spectrum, for all plumage patches except the throat, where we observed the opposite trend.
4. Repeatability between both sampling methods was quite low when including the whole spectrum in the analyses, but moderate when including only the human-visible part.
5. Our results suggest a higher reliability for measurements taken in the lab and a higher power and accuracy of the GLMM-based method. Also, they highlight the possibility of the capability to reflect UV light having different implications in different plumage patches.
**Introduction**
Colour vision involves the capacity to discriminate amongst different wavelengths of light independent of their intensity (Kelber *et al.*, 2003; Cuthill, 2006). Although humans have always been attracted to the colourful displays of birds, only relatively recently have scientists appreciated the importance of a systematic understanding of both function and evolution of bird colouration, as well as the mechanisms that underpin it (Hill and McGraw, 2006).
Mating with elaborately ornamented males can provide female birds with direct benefits (if ornamental traits reflect individual condition, useful individual attributes or somatic quality independent of condition) and/or indirect benefits (good genes or attractiveness for offspring – as conspicuous and costly male traits indicate highly heritable viability) (Pomiankowski, 1987; Andersson, 1994; Garamsziegi *et al.*, 2006). So, birds with more elaborated colourful displays are likely to enjoy a selective advantage, as they are often preferred as mates (Hill, 2006).
Due to the importance of studies of bird plumage colouration in behavioural and evolutionary ecology, methods for quantifying such colouration reliably and objectively are invaluable. Methods traditionally used for colour assessment include colour ranks on an arbitrary scale (Promislow et al., 1992), tristimulus colour models based on human vision, like the Munsell system (Peiponen, 1992), reference colour swatches (Hill, 1990) or digital photography (Loyau et al., 2005; Bortolotti et al., 2006; Stevens et al., 2007). All of these methods, despite being easy and affordable ways of getting colour measurements and useful in certain situations, in general lack reliability and objectivity (Andersson & Prager, 2006), and, more importantly, are tuned to the human visual system instead of the bird visual system.
Birds do not see colours in the same way we do (Bowmaker et al., 1997). They have a fourth cone type in their eyes, with a pigment that is sensitive to ultraviolet light. We are still far from understanding exactly how colours are perceived by birds (Hill and McGraw, 2006), but progress is being made towards understanding how colour vision works in general and how the spectral information is processed by birds and other non-human animals (Smith et al., 2002; Kelber et al., 2003; Kelber and Osorio, 2010). Indeed, methods have been developed for comparing colour patterns as birds see them, using known properties of bird eyes and generating detailed formal descriptions of colour spaces and the equations used to plot them (Endler and Mielke, 2005).
Since the 1990s, reflectance spectrometry has become the most widespread method for measuring animal colouration and a standard tool in behavioural ecology, and a wide range of methods for analyzing spectrophotometry data have emerged (Hill and McGraw, 2006). This development stems largely from the revival of interest in UV vision and tetrachromacy in birds and the fact that birds can see colours that humans cannot experience (Bennett et al., 1994; see Cuthill et al., 2000 for a review), with the
possibility of the existence of plumage patterns invisible to the human eye, and the discovery of mate choice based on the ultraviolet part of the bird reflectance spectrum (Maier, 1994; Bennett et al., 1997). Therefore, reliable and objective ways of quantifying bird coloration not dependent on human vision are at a premium at the moment. Indeed, the invention of miniature diode-array spectroradiometer systems, being lighter, more portable and affordable than previous spectrometry systems but as precise and objective in colour quantification, have provided very popular tools in any colour communication study (Hill and McGraw, 2006).
There are two main ways of assessing bird plumage colouration with spectrophotometers reported in the literature. Measurements may be taken either directly on the bird, applying the pointer of the spectrophotometer to plumage patches as they occur in situ (e.g. Senar et al., 2002; Bize et al., 2006; Herrera et al., 2008; Catoni et al., 2009; Doucet and Hill, 2009; del Cerro et al., 2010), or in the lab, with feather samples collected from the field, applying the pointer to “plumage patches” created by mounting these feathers on a flat surface in a way that mimics the original plumage structure (e.g. Cuthill et al., 1999; Keyser and Hill, 2000; Perrier et al., 2002; Safran and McGraw, 2004; Komdeur et al., 2005; McGraw et al., 2005; Safran, 2007). Despite the popularity of the use of spectrophotometers for colour assessment and the growing number of studies on bird colouration, few studies have rigorously assessed the consistency of both methods for measuring the colouration of plumage patches, and the repeatability of results obtained when using either one or the other (see Quesada and Senar, 2006 for such a comparison for carotenoid-based plumage coloration in great tits).
In contrast, there is little consensus about how to quantify the reliability, or repeatability, of spectral measurements. The most common measure of repeatability, or more precisely the coefficient of intraclass correlation ($r_i$), can be formally defined
as the proportion of the total variance explained by differences among groups (Nakagawa and Schielzeth, 2010; Sokal and Rohlf, 1995):
\[ r_t = \frac{\sigma_a^2}{\sigma_a^2 + \sigma_e^2} \]
(eqn 1)
where \( \sigma_a^2 \) is the between-group variance and \( \sigma_e^2 \) the within-group variance, whereas the sum of both comprises the total phenotypic variance (Nakagawa and Schielzeth, 2010). Until recently, the most common ways to estimate repeatabilities from data with Gaussian errors have employed the correlation-based method (Sokal and Rohlf, 1995) or the ANOVA-based method, commonly used by behavioural and evolutionary ecologists (Donner, 1986; see Lessells and Boag, 1987 for a complete review and description). However, Nakagawa and Schielzeth (2010) describe an innovative method for calculating GLMM-based repeatability estimates, which allows for confounding variables to be factored out and calculates the confidence intervals for each repeatability calculation, inferred from distributions of repeatabilities obtained by parametric bootstrapping.
In this study, using both ANOVA-based and GLMM-based methods, we test the consistency of measuring melanin-based plumage colouration in four different patches of the ventral plumage of the European barn swallow *Hirundo rustica rustica*, both directly on the bird and on feather samples in the laboratory, and we determine the repeatability of the results obtained with both methods. To our knowledge, this is the first time GLMM-based repeatability estimates have been used to assess the reliability of melanin-based plumage colouration measurements.
Materials and methods
Field work was carried out during May-August 2009 and March-August 2010 in several sites, mainly farms, located in the surroundings of the University of Exeter Campus, in Cornwall, UK (Fig. 1). 59 adult European barn swallows, *Hirundo rustica rustica* (21 in 2009, 38 in 2010), were caught using mist nets, ringed, morphometric measurements taken, plumage reflectance spectra quantified in the field and feather samples collected for subsequent assessment in the lab.
Colour was quantified based on Endler & Mielke (2005), using a USB2000 spectrometer (Ocean Optics, Dunedin, Florida), and a xenon flash lamp (Ocean Optics). We used a WS-1 SS Diffuse Reflectance Standard, a diffuse white plastic >98% reflective from 250-1500 nm, as the white reference (100% reflectance), and a piece of black velvet as the dark standard (0% reflectance) to correct for the noise when no light is reaching the sensor. At the far end of the reflection probe/light source, we put a non-reflective black pointer cut in a 45 degree angle, to avoid mismeasurement derived from the light reflected by the plumage reaching the sensor (Andersson and Prager, 2006; Pike, pers. comm.). Using the spectra acquisition software package OOIBase (Ocean Optics), we measured the reflectance of four body regions, namely the throat, breast, belly and vent of each bird (Fig. 2). For the measurements of feather samples in the lab, we collected enough feathers from live birds as to being able to mount them one on top of the other and simulate the original pattern found on live birds. We mounted the feathers on a piece of black velvet to avoid background noise. Once we had tested for the reliability of both methods separately, we averaged the three measurements for each method and used these average values to test the comparability between field and lab measurements.
We used the method described in Endler and Mielke (2005) to calculate brightness, chroma and hue, parameters generally used to specify a colour. Using their equations and the mathematical software Matlab (The MathWorks Inc., Natick, MA), we got the spectral sensitivity functions of the cones corrected for the cut points of oil droplets, calculated the quantal catch for each photoreceptor and converted those quantal catches into dimensional colour space coordinates in a tetrahedral colour space (Fig. 3). Chroma is defined as the strength of the colour signal or the degree of difference in stimulation among the cones, and it is proportional to the Euclidean distance from the origin, that is, the distance from the bird grey (achromatic) point to each point, specified by three space coordinates. Perception of hue depends on which cones are stimulated, and in tetrahedral colour space, it is defined by the angle that a point makes with the origin. As bird colour space is 3D, hue is defined by two angles, analogous to latitude and longitude in geography (Endler and Mielke, 2005).
Brightness is defined as the summed mean reflectance across the entire spectral range \((R_{300-700}\); Montgomerie, 2006; Galván and Möller, 2009\). As well as these parameters, we included UV chroma, a measure of spectral purity, into our analysis, which was calculated as the proportion of reflectance in the UV part of the spectrum \((R_{300-400})\) in relation to the total reflectance spectrum \((R_{300-700}\); Siefferman and Hill, 2005\). Cone sensitivities and oil droplet cut points were taken from Bowmaker *et al.* (1997), Hart (2001), Vorovyev *et al.* (1998), Govardovskii *et al.* (2000) and Hart and Vorovyev (2005).
Although all the avian families investigated show plumages reflecting significant amounts of UV light (see Eaton and Lanyon, 2003 for a review), in the particular case of barn swallows, ventral plumage shows a noisy reflectance pattern in the UV part of the spectrum, with some values even below 0, and does not exhibit a clear ultraviolet reflectance peak (Fig. 4; Safran and McGraw, 2004). For this reason, we calculated
the same colour variables both including and not including the UV part of the reflectance spectrum, and carried out a repeatability assessment using either the whole reflectance spectrum or only the visible part. When using only the visible part, we did not include UV chroma, for obvious reasons, nor hue, as values are identical in both cases.
**ANOVA-based method:** In order to test for the reliability of both procedures separately, we calculated the repeatability for colour variables in the four patches for the different procedures according to Lessells and Boag (1987), Senar (1999) and Quesada and Senar (2006). Repeatabilities were computed from the mean squares of ANOVA on three repeated measures per individual. Both in field and lab procedures, the second and third measurements were made after removing the reflection probe/light source and placing it again on the colouration patch. IV took all the measurements.
Once we calculated the repeatabilities for each method, we averaged the three measurements per patch per individual and assessed the repeatability of measurements across procedures, but this time the ANOVA was carried out on two repeated measures per individual, one from the field and another one from the lab.
We repeated this process for both 2009 and 2010 data separately. Because of the particular circumstances in which data were collected each year (Vaquero-Alba, Evans & Dall, unpublished), colour measurements in 2009 were taken in three different points within each patch, whereas in 2010 the three of them were taken in the same point.
**GLMM-based method:** We used a modified version of the R function *R.Anson*, which is itself a modification of *rpt.remlLMM* function (Nakagawa and Schielzeth,
We fitted two random-effect terms (individual identity and year) in our linear mixed-effects models, and calculated the adjusted repeatability estimate as:
\[ r_i = \frac{\sigma_a^2}{\sigma_a^2 + \sigma_v^2 + \sigma_r^2} \]
(eqn 2)
where \( \sigma_r^2 \) is the year variance.
In order to have a general idea of repeatability for each patch, we included all the colour variables in a principal component analysis (PCA) and calculated the repeatability (and confidence intervals) for the first component (PC1) within each plumage patch.
As we conducted multiple statistical tests on data subsets that are not likely to be biologically independent of each other (i.e. different components of the spectra, same metrics on different years, or same metrics in the lab and in the field), there was an increased probability of type I error rates. To control for this increased probability, we corrected our p-values for multiple testing based on the sequentially rejective Bonferroni procedure of Holm (Holm, 1979), using the `stats::p.adjust` function in R (R Development Core Team, 2010).
All the statistical analyses were carried out using R (Crawley, 2007; R Development Core Team, 2010).
**Results**
**ANOVA: 2009**
When including the whole spectrum in the analyses, measuring plumage colouration in the lab proved to be a reliable method, with brightness, UV chroma, chroma and
hue latitude and longitude being highly repeatable for all the patches, and hue latitude in the throat being less repeatable ($r_f=0.418$, $F_{21,44}=3.157$, $p=0.01$). The method of measuring the plumage colouration in the field was also quite consistent but with overall lower values of repeatability, although still reasonably high, for all the variables and patches, being especially low for hue latitude in breast ($r_f=0.382$, $F_{20,42}=2.856$, $p=0.025$) and vent ($r_f=0.394$, $F_{21,44}=2.955$, $p=0.017$; Table 1).
When including only the visible part of the spectrum in the analysis, overall repeatability was lower. The lab method again proved to be the most reliable, with high values of repeatability for brightness and chroma in all the patches. The field procedure was moderately repeatable for belly and throat, but showed low repeatability for brightness in the breast ($r_f=0.36$, $F_{20,42}=2.6885$, $p=0.038$) and for chroma in the vent ($r_f=0.428$, $F_{21,44}=3.241$, $p=0.007$; Table 2).
The values of repeatability across the field and laboratory procedures were very low for all the patches measured, both considering the whole reflectance spectrum or only the visible part ($r_f<0.35$ and $p>0.05$ in all cases), suggesting a lack of consistency across the two assessment methods for melanin-based plumage colouration. Repeatabilities of brightness measurements were slightly higher for the whole spectrum than for only the visible part, in all the patches except from the vent. However, in this case, including only the visible part of the spectrum yielded much more repeatable chroma values than including the whole range, sometimes even turning negative repeatability values into positive, e.g. for the belly (whole range: $r_f=-0.507$, $F_{20,21}=0.327$, $p=1$; only visible range: $r_f=0.346$, $F_{20,21}=2.056$, $p=0.599$), or the vent (whole range: $r_f=-0.758$, $F_{21,22}=0.138$, $p=1$; only visible range: $r_f=0.296$, $F_{21,22}=1.842$, $p=0.657$; Table 1 and Table 2).
ANOVA: 2010
When including the whole spectrum in the analyses, repeatability measurements in the field yielded considerably higher results than in 2009, with all the $r_i$ values above 0.60, except from hue latitude in the throat ($r_i=0.515$, $F_{37,75}=4.186$, $p<0.0001$), and with most of the values ranging from 0.74 to 0.91, except for brightness in the breast ($r_i=0.611$, $F_{37,76}=5.710$, $p<0.0001$), hue latitude in the belly ($r_i=0.63$, $F_{37,76}=6.1$, $p<0.0001$), hue latitude in the vent ($r_i=0.629$, $F_{37,76}=6.088$, $p<0.0001$) and hue longitude in the vent ($r_i=0.679$, $F_{37,76}=7.356$, $p<0.0001$). In the lab, repeatability values ranged from 0.71 to 0.95 in most of the patches, except from hue latitude in the throat ($r_i=0.65$, $F_{37,76}=6.569$, $p<0.0001$), and repeatability was overall higher than when measuring it on live birds, except from UV chroma in the belly ($r_i=0.857$, $F_{37,76}=18.986$, $p<0.0001$), breast ($r_i=0.788$, $F_{37,76}=12.117$, $p<0.0001$) and vent ($r_i=0.819$, $F_{37,76}=14.571$, $p<0.0001$) and hue latitude in the breast ($r_i=0.722$, $F_{37,76}=8.808$, $p<0.0001$), where it was slightly lower. Repeatability values were similar to the ones obtained in 2009 (Table 3).
When doing the analysis including only the visible part of the spectrum, measuring colouration in the lab was again the most reliable method of both, with all the $r_i$ values above 0.91, except for chroma in the throat ($r_i=0.881$, $F_{37,76}=22.964$, $p<0.0001$). Field procedure still yielded high repeatability values, with brightness in the breast ($r_i=0.569$, $F_{37,76}=4.959$, $p<0.0001$) and chroma in the vent ($r_i=0.696$, $F_{37,76}=7.839$, $p<0.0001$) being the only measurements with values below 0.81. For both methods, repeatability values were higher than in 2009 for all the variables within all the plumage patches (Table 4).
Repeatabilities across field and lab methods in 2010 were quite heterogeneous including the whole spectrum in the analyses: moderately high for hue longitude in the belly ($r_i=0.794$, $F_{37,38}=8.732$, $p<0.0001$) and breast ($r_i=0.657$, $F_{37,38}=4.818$,
p<0.0001), moderate for vent hue latitude ($r_i=0.463$, $F_{37,38}=2.723$, p=0.018) and longitude ($r_i=0.561$, $F_{37,38}=3.553$, p=0.001), belly hue latitude ($r_i=0.431$, $F_{37,38}=2.515$, p=0.034) and breast brightness ($r_i=0.482$, $F_{37,38}=2.861$, p=0.012), and low for breast chroma ($r_i=0.326$, $F_{37,38}=1.966$, p=0.21), throat UV chroma ($r_i=0.321$, $F_{37,38}=1.944$, p=0.312) and chroma ($r_i=0.274$, $F_{37,38}=1.755$, p=0.576) and vent brightness ($r_i=0.349$, $F_{37,38}=2.070$, p=0.141). For the rest of the cases, repeatabilities were very low ($r_i<0.23$ and p>0.05 for all the cases). When including only the visible part of the spectrum, repeatability was moderate to high and had a significant effect for both brightness and chroma in the breast, and for chroma in the vent and in the belly, whereas it was quite low and non-significant for brightness in the belly ($r_i=0.217$, $F_{37,38}=1.556$, p=0.899), and very low for both variables in the throat. Repeatability values for chroma in all the patches except for the throat were much higher than when we included the whole spectrum range, e.g. in the belly (whole range: $r_i=-0.049$, $F_{37,38}=0.906$, p=1; only visible range: $r_i=0.75$, $F_{37,38}=7.005$, p<0.0001) and in the vent (whole range: $r_i=0.224$, $F_{37,38}=1.579$, p=0.657; only visible range: $r_i=0.416$, $F_{37,38}=2.428$, p=0.047; Table 3 and Table 4).
**GLMM: 2009+2010**
When we included the whole spectrum in the analyses, for all the principal component analyses carried out within each patch for field and lab measurements, PC1 accounted for more than a 53% of the total variance, except for vent measurements in the field (where it explained a 49% of the total variance) and for measurements in the throat (where it explained between 44% and 47%). When including only the visible part of the spectrum, PC1 explained a 67% of the total variance for vent measurements in the field, and between 75% and 93% in the rest of the cases.
Repeatability of feather colour measurements was much higher and confidence intervals smaller when quantifying colouration in the lab than when doing it on live birds in the field, both including the whole spectrum in the analyses or only the visible part, being particularly high in the breast (whole range: $r_i=0.916$, 95%CI=[0.855,0.943], p<0.0001; visible range: $r_i=0.927$, 95%CI=[0.872,0.95], p<0.0001). All the repeatability values from lab measurements ranged between 0.71 and 0.93 and were highly significant (p<0.0001).
When using field measurements, repeatabilities were still moderately high (all $r_i$ values above 0.50) and higher when including only the visible spectrum range in the analyses than including the whole range, except in the throat (whole range: $r_i=0.629$, 95%CI=[0.303,0.874], p<0.0001; visible range: $r_i=0.564$, 95%CI=[0.266,0.816], p<0.0001; Fig. 5).
Repeatabilities across both field and lab methods yielded higher results when we included only the visible spectrum in the analyses than when we included the whole spectrum. Leaving the values for throat apart, as here repeatability was not significantly different from zero no matter the spectrum we included in the analyses, $r_i$ ranged between 0.19 and 0.41 when including the whole spectrum, and between 0.44 and 0.62 when including only the visible part (Fig.5). All the repeatabilities in belly, breast and vent were significant except for the belly when including the whole spectrum ($r_i=0.189$, 95%CI=[0,0.415], p=0.069), but it became significant and higher when only the visible spectrum was included in the analyses ($r_i=0.503$, 95%CI=[0.281,0.667], p<0.0001, Fig. 5).
Discussion
Measuring plumage colouration from feather samples in the lab proved to be a highly reliable method, with high values of repeatability in general for all the variables and patches in 2009, 2010 and when applying the GLMM-based method for both years. Measuring colouration directly on bird plumage in the field was also reliable, but with overall lower values of repeatability than the lab for most of the variables measured on different patches, with the exception of some variables in the throat in 2009 and UV chroma measurements in the belly, breast and vent in 2010 when considering the whole spectrum, which yielded higher values of repeatability when measured in the field. This may be due, on the one hand, to the fact that throat patch is smaller and much darker than the rest of the patches and the feathers that constitute it much smaller, so it is often quite difficult to obtain a reliable reflectance measurement with such a limited amount of photons reaching the spectrophotometer probe. Also, it is more difficult to create a “plumage patch” in the lab with a feather arrangement similar to the bird’s original one and big enough to be able to apply the spectrophotometer pointer to it. On the other hand, UV part of the spectrum shows a very noisy pattern in this species, so we would not necessarily expect highly consistent UV chroma repeatabilities across field or lab measurements, which may explain why repeatability values in the lab for UV chroma measurements are higher than in the field in 2010, whereas the rest of the repeatability values tend to be higher in the lab.
So, collecting feathers from birds and assessing their colouration in the lab, as well as being more convenient, minimising risk to a sensitive device like a spectrophotometer and reducing handling times of the animals (Quesada and Senar, 2006), is a more
reliable method for assessing melanin plumage colouration than doing so directly on live birds, according to our results.
When comparing both methods, the values obtained in 2009 for different variables measured in different plumage patches directly on the bird were poorly repeatable compared to the values obtained for the same variables measured from feather samples, and non-significant in all cases. In 2010, in contrast, repeatabilities were higher and significant for certain metrics in certain patches only. These results stand in marked contrast to the positive results of another study, which compared the repeatabilities between both colouration assessment procedures for carotenoid-based plumage (Quesada and Senar, 2006). There can be several reasons for this difference: first of all, due to the different characteristics of the two types of pigments, carotenoid-derived colouration is more variable among individuals than melanin-based colouration (Badyaev and Hill, 2000), and repeatability of a character increases with variability (Senar 1999). In order to increase the repeatability of some measurements, a possible solution could be to increase the number of measurements, for example from three to five, as it has already been done by several authors (e.g. Bennett et al., 1997; Perrier et al., 2002; Doucet and Hill, 2009). Unfortunately, this is not an option when working with live birds in the field, as we would be increasing the manipulation times and, consequently, the stress levels to an unacceptable degree, although it can be applied when assessing colouration in the lab on feather samples (Quesada and Senar, 2006).
Due to the way data were collected, the three plumage colouration measurements taken in the field in 2009 covered a wider area of each plumage patch than the measurements made on feather samples, which were restricted to the area covered by the bunch of feathers plucked from each patch on each individual. For that reason, repeatability of 2009 field measurements can be taken as an estimate of colouration
consistency within the plumage patches. Our results suggest a moderate to high within-patch consistency for melanin-based ventral colouration in the European barn swallow *Hirundo rustica rustica*. In 2010, however, the three field measurements were taken approximately in the same plumage area for each patch. As a result of this, the comparability of both procedures in 2009 may have been compromised, although the repeatability of the 2010 samples was higher even for lab measurements, especially when considering only the visible part of the spectrum, which may be indicative of higher patch colouration homogeneity in 2010.
The GLMM-based method (Nakagawa and Schielzeth, 2010), applied to data from both years, allowed us to control for year effects by adding the year variance into the total variance calculation, so that we could obtain the adjusted repeatability for data from both years. Thanks to the PCA, which allowed us to create composite variables accounting for almost 50% of the total variance in the metrics taken from each patch, we were able to estimate the overall repeatability within each patch for both methods separately and across methods. The possibility of calculating adjusted repeatabilities by including year as a random factor, together with the reduction in the number of variables accounting for a great proportion of the total variance achieved by the PCA, considerably reduced the amount of multiple tests necessary for repeatability calculation. Thus, the p-values obtained with this method were less affected by Bonferroni corrections than those obtained with the ANOVA-based method, reducing the probability of type II errors and increasing the power of this method for repeatability calculation. Repeatability was moderate to high within all the patches for field measurements, and considerably higher for lab measurements, suggesting that lab method is a more reliable way of assessing melanin-based colouration. The fact that almost all the repeatability measurements, and especially the repeatabilities across field and lab methods (in patches other than the throat), were higher when including
only the human-visible spectrum in the analyses, suggests that the noisy reflectance pattern in the UV part of the spectrum may be distorting the results and underestimating the comparability of the two methods. For throat plumage, however, we observed the opposite trend, with higher repeatability values when including the whole spectrum, which could be indicative that the UV part of the spectrum is more important in the throat than in the rest of the patches. We find support for this idea when looking at reflectance spectra plots for different patches (Fig. 4): throat reflectance spectra, although showing also quite a noisy pattern for the UV part, and unlike the rest of the patches’ spectra, tends to show UV reflectance peaks in both sexes. Further work is needed to find out whether there are UV reflectance differences amongst different plumage patches.
In conclusion, our results suggest that collecting feathers from live animals and assessing colouration in the lab is more reliable than taking measures on live birds directly, in the field. Moreover, since it is easier on equipment and minimises the length of time birds need to be handled (minimising the stress levels inflicted on them), feather sampling would appear to be the best method available. We also demonstrated the value of the GLMM-based method (Nakagawa and Schielzeth, 2010) for repeatability calculation, as it enables random factors to be accounted for and can calculate adjusted repeatability values, which are more accurate than those calculated using other (e.g. ANOVA) methods and increase the power of the tests. Finally, we have also shown that it is important to check for the effect that the UV part of the spectrum could be exerting on repeatability calculations, as the capability of the plumage to reflect the UV light could have different biological implications in different plumage patches.
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| | UV+Visible range |
|----------------------|------------------|
| | Belly | Breast | Throat | Vent |
| | F | r_i | F | r_i | F | r_i | F | r_i |
| Repeatability field | | | | | | | | |
| Brightness | 6.0446 | 0.627*** | 3.1183 | 0.414* | 12.273 | 0.79*** | 6.1868 | 0.634*** |
| UV Chroma | 10.203 | 0.754*** | 14.171 | 0.814*** | 13.861 | 0.811*** | 5.3196 | 0.59*** |
| Chroma | 9.0161 | 0.728** | 11.238 | 0.773** | 10.714 | 0.764*** | 4.5611 | 0.543*** |
| Hue latitude | 4.0921 | 0.508** | 2.8564 | 0.382* | 4.4146 | 0.532*** | 2.9545 | 0.394* |
| Hue longitude | 13.025 | 0.8*** | 9.1422 | 0.731*** | 24.348 | 0.886*** | 4.6771 | 0.55*** |
| Repeatability lab | | | | | | | | |
| Brightness | 13.188 | 0.802*** | 14.777 | 0.821*** | 8.2092 | 0.706*** | 20.212 | 0.865*** |
| UV Chroma | 46.493 | 0.938*** | 23.015 | 0.88*** | 34.895 | 0.919*** | 25.561 | 0.892*** |
| Chroma | 42.489 | 0.932*** | 26.975 | 0.896*** | 62.481 | 0.954*** | 29.052 | 0.903*** |
| Hue latitude | 11.986 | 0.785*** | 6.5101 | 0.647*** | 3.1573 | 0.418** | 23.024 | 0.88*** |
| Hue longitude | 25.986 | 0.893*** | 9.2833 | 0.734*** | 6.0119 | 0.625*** | 27.291 | 0.898*** |
| Comparison field-lab | | | | | | | | |
| Brightness | 1.4636 | 0.188 | 2.0702 | 0.349 | 0.865 | -0.072 | 1.8043 | 0.287 |
| UV Chroma | 1.3153 | 0.136 | 1.3591 | 0.152 | 0.7499 | -0.143 | 0.9006 | -0.052 |
| Chroma | 0.327 | -0.507 | 1.3716 | 0.157 | 1.0143 | 0.007 | 0.1376 | -0.758 |
| Hue latitude | 0.9084 | -0.048 | 0.8635 | -0.073 | 1.2523 | 0.112 | 0.706 | -0.172 |
| Hue longitude | 1.7364 | 0.269 | 1.7513 | 0.273 | 0.6709 | -0.197 | 1.1055 | 0.05 |
**** p<0.001; *** p<0.01; ** p<0.05 * p<0.1
Table 1: ANOVA-derived Repeatabilities in 2009 plumage colouration measurements taken from live birds in the field, feather samples in the lab, and across both procedures (UV+Visible spectrum).
| | Belly F | Belly $r_i$ | Breast F | Breast $r_i$ | Throat F | Throat $r_i$ | Vent F | Vent $r_i$ |
|----------------------|----------|-------------|-----------|--------------|-----------|--------------|---------|------------|
| **Repeatability field** | | | | | | | | |
| Brightness | 4.8958 | 0.565*** | 2.6885 | 0.36* | 11.221 | 0.773*** | 5.6072 | 0.606*** |
| Chroma | 6.7724 | 0.658*** | 6.4868 | 0.646*** | 6.6207 | 0.652*** | 3.2416 | 0.428** |
| **Repeatability lab** | | | | | | | | |
| Brightness | 11.818 | 0.783*** | 15.892 | 0.832*** | 6.9936 | 0.666*** | 19.326 | 0.859*** |
| Chroma | 28.865 | 0.903*** | 21.117 | 0.821*** | 29.402 | 0.873*** | 23.838 | 0.901*** |
| **Comparison field-lab** | | | | | | | | |
| Brightness | 1.461 | 0.187 | 2.0097 | 0.335 | 0.6804 | -0.19 | 1.8902 | 0.308 |
| Chroma | 2.0563 | 0.346 | 1.6683 | 0.25 | 1.1396 | 0.065 | 1.8415 | 0.296 |
**** p<0.001; *** p<0.01; ** p<0.05 * p<0.
Table 2: ANOVA-derived Repeatabilities in 2009 plumage colouration measurements taken from live birds in the field, feather samples in the lab, and across both procedures (Only Visible part of the spectrum).
| | UV+Visible range |
|----------------------|------------------|
| | Belly | Breast | Throat | Vent |
| | F | r₁ | F | r₁ | F | r₁ | F | r₁ |
| Repeatability field | | | | | | | | |
| Brightness | 16.422 | 0.837*** | 5.7104 | 0.611*** | 17.398 | 0.846*** | 14.534 | 0.819*** |
| UV Chroma | 25.333 | 0.89*** | 15.853 | 0.832*** | 12.357 | 0.791*** | 16.829 | 0.841*** |
| Chroma | 24.037 | 0.885*** | 14.749 | 0.821*** | 22.212 | 0.876*** | 12.377 | 0.792*** |
| Hue latitude | 6.0997 | 0.630*** | 9.7411 | 0.744*** | 4.1857 | 0.515*** | 6.0878 | 0.629*** |
| Hue longitude | 23.669 | 0.883*** | 31.363 | 0.910*** | 9.8914 | 0.748*** | 7.3556 | 0.679*** |
| Repeatability lab | | | | | | | | |
| Brightness | 55.197 | 0.947*** | 31.387 | 0.910*** | 30.9 | 0.909*** | 44.036 | 0.934*** |
| UV Chroma | 18.986 | 0.857*** | 12.117 | 0.788*** | 17.544 | 0.847*** | 14.571 | 0.819*** |
| Chroma | 25.375 | 0.890** | 25.936 | 0.893*** | 27.679 | 0.899*** | 39.854 | 0.928*** |
| Hue latitude | 8.3908 | 0.711*** | 8.8076 | 0.722*** | 6.5692 | 0.650*** | 9.1422 | 0.731*** |
| Hue longitude | 37.357 | 0.924*** | 31.683 | 0.911*** | 11.664 | 0.781*** | 37.517 | 0.924*** |
| Comparison field-lab | | | | | | | | |
| Brightness | 1.3041 | 0.131948 | 2.8612 | 0.482* | 0.7642 | -0.134 | 2.0703 | 0.349 |
| UV Chroma | 0.5572 | -0.284 | 1.059 | 0.029 | 1.9441 | 0.321 | 1.5873 | 0.227 |
| Chroma | 0.9059 | -0.049 | 1.9662 | 0.326 | 1.755 | 0.274 | 1.5788 | 0.224 |
| Hue latitude | 2.5152 | 0.431* | 1.2479 | 0.110243 | 0.7839 | -0.121 | 2.7232 | 0.463* |
| Hue longitude | 8.7322 | 0.794*** | 4.8175 | 0.657*** | 0.9731 | -0.014 | 3.5525 | 0.561** |
**** p<0.001; *** p<0.01; ** p<0.05 * p<0.1
Table 3: ANOVA-derived Repeatabilities in 2010 plumage colouration measurements taken from live birds in the field, feather samples in the lab, and across both procedures (UV+Visible spectrum).
| | Belly | Breast | Throat | Vent |
|----------------------|--------|--------|--------|--------|
| | F | r₁ | F | r₁ | F | r₁ | F | r₁ |
| **Repeatability field** | | | | | | | | |
| Brightness | 14.408 | 0.817*** | 4.959 | 0.569*** | 16.59 | 0.839*** | 13.527 | 0.806*** |
| Chroma | 22.566 | 0.878*** | 22.163 | 0.876*** | 20.007 | 0.864*** | 7.8386 | 0.696*** |
| **Repeatability lab** | | | | | | | | |
| Brightness | 59.713 | 0.951*** | 34.778 | 0.918*** | 34.133 | 0.917*** | 49.55 | 0.942*** |
| Chroma | 51.455 | 0.944*** | 57.897 | 0.950*** | 22.964 | 0.881*** | 72.314 | 0.959*** |
| **Comparison field-lab** | | | | | | | | |
| Brightness | 1.5559 | 0.217 | 3.1819 | 0.522** | 0.679 | -0.191 | 2.3696 | 0.406. |
| Chroma | 7.0047 | 0.750*** | 7.0261 | 0.751*** | 1.0876 | 0.042 | 2.4275 | 0.416* |
****, p<0.001; ***, p<0.01; ** p<0.05 *, p<0.1
Table 4: ANOVA-derived Repeatabilities in 2010 plumage colouration measurements taken from live birds in the field, feather samples in the lab, and across both procedures (Only Visible part of the spectrum).
Figure 1: Location of the sampling area.
Figure 2: Topography of bird plumage regions: $thr$ throat, $bre$ breast, $bel$ belly, $ven$ vent (modified from Andersson & Prager, 2006).
Figure 3: The avian tetrahedral colour space (from Endler & Mielke, 2005).
Figure 4: Reflectance spectra for belly, breast, throat and vent patches of male and female barn swallow.
Figure 5: Repeatabilities (± 95% CI) in 2009-2010 plumage colouration measurements taken a) from live birds in the field, b) from feather samples in the lab, and c) across both procedures, both when including the whole light spectrum or only the human-visible spectrum in the analyses.
Chapter Three
Multiple signalling, habitat quality and laying date in the European barn swallow *Hirundo rustica rustica*
Iker Vaquero-Alba, Andy McGowan, Matthew R. Evans & Sasha R.X. Dall
Centre for Ecology and Conservation, University of Exeter, Cornwall Campus
Penryn, Cornwall, TR10 9EZ, UK
Mating with elaborately ornamented males can provide female birds with direct and/or indirect benefits. Variation in selection pressures derived from fluctuations in the environment (ecological and social context) leads animals to use more than one signal to transmit information to others, in spite of the costs associated. We tested this idea, using the European barn swallow as our model species. We hypothesized that males arriving later to the breeding grounds should be selected to decrease the investment in tail streamer elongation in favour of “cheaper” ornaments, in order to balance the information transmitted and the costs associated. Also, we propose a “compensation mechanism” for individuals with a higher elaboration of “cheaper” sexual signals, according to which they should be selected to defend breeding sites associated with a higher quality foraging area, to outweigh their lack of fitness. As we predicted, individuals with darker throat and ventral plumage patches, or those with either brighter patches or in poor condition, occupied nesting sites next to areas richer in large insects. Also, pairs with individuals in better physical condition, and those with darker-throated males, started laying earlier. Plumage colouration seemed to be a more important sexual signal in our study population than previously thought for European barn swallow, suggesting a differential adaptation to dynamic selection pressures amongst populations.
Introduction
Mating with elaborately ornamented males can provide female birds with direct or indirect benefits, information about which is transmitted by inter-individual differences in the degree of exaggeration of the ornaments (Pomiankowski, 1987; Andersson, 1994; Garamsziegi et al., 2006). So, birds with more elaborate, colourful displays enjoy a selective advantage, as they are preferred as mates (Hill, 2006).
Such ornamental traits are also present in females of several species, and males can also benefit from mating with elaborately ornamented females, given that female ornamentation provides information on female reproductive capacity: older, more ornamented females breed earlier and lay larger clutches, so they can fledge more young (Komdeur et al., 2005). Thus, males and females do necessarily not gain fitness advantages from choosing mates with elaborate plumage in the same way (Komdeur et al., 2005).
Elaborate and colourful displays used for signalling purposes by birds are costly at evolutionary stability, according to models of sexual selection (Fisher, 1930; Zahavi, 1975; Iwasa et al., 1991). Costs are expressed during production and/or maintenance, and continuously through increased risk of predation or parasitism (Magnhagen, 1991; Folstad & Karter, 1992). More “expensive” signals, like elongated tail feathers, incur substantial production/maintenance costs and increase the risk of predation via reductions in the aerodynamic efficiency of their bearers. Other, “cheaper” signals, such as pigment-based plumage markings, or especially behavioural traits, incur substantially lower costs, particularly on a day-to-day basis. In the continuum of costs incurred and information transmitted by sexual signals, we would expect higher-quality individuals to benefit from using “expensive” signals such as tail elongation: for handicap signalling to work as an ESS, marginally better individuals should have
larger marginal fitness effects of advertising, because of lower costs, higher benefits or both (Getty, 1998).
Nevertheless, only a small proportion of Palearctic birds have extremely elongated tails. The marginal viability cost of advertising may increase with quality as long as the higher viability costs are compensated by higher fecundity benefits (Getty, 1998). Thus, in species in which the day-to-day costs incurred by elongated tails have particularly severe fitness consequences, or in circumstances in which it does not compensate to signal at maximal intensity (e.g. at low population densities when females cannot be as choosy), signalling individuals might be selected to utilize “cheaper” signals such as markings to a greater extent and minimise elaboration of the more “expensive” traits such as tail elongation (Fitzpatrick, 2000). Therefore, birds may have evolved plumage that trades off the amount of information it signals to potential mates/rivals with its costs; in other words, such traits may be selected to optimise rather than maximise their information content.
Animals often use more than one signal to transmit information to others (Bro-Jørgensen, 2009). For example, in the strut displays of the greater sage grouse (*Centrocercus urophasianus*) males combine wing and tail movements with popping vocalizations to attract females (Wiley, 1973); the male wolf spider *Schizocosacistridulans* uses its abdomen and pedipalp simultaneously to produce different components of its complex sexual display (Elias, 2006); and guppies (*Poecilia reticulata*) show complex colour patches based on carotenoids, pteridines and melanins (Houde, 1997) as well as predator ‘boldness’, which is the individual willingness to risk approaching predators to ‘inspect’ them (Dugatkin & Godin, 1992; Wilson *et al.*, 1994; Godin & Davies, 1995).
In the European barn swallow *Hirundo rustica rustica*, several ornamental traits potentially used for sexual signalling purposes have been studied. Barn swallow males preferred as social and extra-pair mates have been found to have longer tail streamers (Møller, 1988, 1994; Saino *et al.*, 1997), more symmetric outermost tail feathers (Møller, 1992, 1993b, 1994), larger white spots in the tail (Kose, 1999, Kose & Møller, 1999), a higher song rate (Møller *et al.*, 1998), and a more intense red facial plumage (Ninni *et al.*, 2004).
Amongst them, the one that has attracted most attention from researchers is an “expensive” signal, the elongation of tail streamers. Thus, males with longer tail streamers have been found to arrive earlier from wintering grounds in Africa (Møller, 1994), mate more often (Møller, 1994) and get more extra-pair copulations (Møller, 1988), have more broods (Møller, 1988, 1990, 1994), their females provide more parental care (Møller, 1991; de Lope & Møller, 1993), start breeding earlier and, overall, have a higher reproductive success (Møller, 1988, 1994). Furthermore, patterns of positive assortative mating by tail streamer length have been found (Møller, 1993b), suggesting a process of mutual sexual selection, and highlighting the quality-indicating value of such ornamental traits for European barn swallows. Also, some “cheaper” signals have been studied to figure out their relationship with tail length and with each other, and to see if they are having an effect in reproductive success. Perrier (2002), studied the dorsal plumage structural coloration, but he only found a weak and nonsignificant correlation with tail length and asymmetry and red facial colouration, as well as concluding that it was not condition dependent. Ninni *et al.* (2004) found a covariation between intense red facial plumage and the arrival date of barn swallows to breeding grounds, although the results were inconsistent: on first year of the study, early arriving animals had a duller red facial plumage than late
arriving, whereas in second year the observed trend was just the opposite. Kose *et al.* (1999) and Kose & Møller (1999) found a positive correlation between white spots on tail feathers and tail length. The size of the white spots was also related to an earlier start of breeding.
In the North American barn swallow, a different subspecies (*Hirundo rustica erythrogaster*), however, although some evidence was found that females mated to males with experimentally lengthened tails bred earlier (Smith *et al.*, 1991; Smith & Montgomerie, 1991) and that streamer length predicted fertilization success (although effect was confounded by age; Kleven *et al.*, 2006), no significantly greater social or extrapair reproductive success was found for swallows with longer tail streamers (Smith *et al.*, 1991; Smith & Montgomerie, 1991). Safran & McGraw (2004) did not observe any advantages for individuals with larger or/and more symmetrical tails. However, ventral plumage coloration was found to predict patterns of assortative mating and reproductive success, two characteristic features of sexually selected traits (Safran & McGraw, 2004). In a series of colour manipulations carried out in North American barn swallow males, Safran *et al.* (2005) found an increased social paternity for males with enhanced ventral plumage colour.
Thus, two barn swallow subspecies, which have diverged relatively recently, approximately 100,000 years ago (Zink *et al.*, 2006; Dor *et al.*, 2010) use very different ornamental characters as the main quality indicators in inter-sexual signalling. This may be due to adaptation to different environments. A possible explanation for this variability in the main sexual signal used during mate choice could be that North American barn swallow males may need to take part in the egg incubation due to their habit of nesting on exposed sites. In these exposed sites, it may be more difficult to keep eggs at a proper temperature (Smith & Montgomerie, 1992)
than in the case of the European barn swallows, which may prefer to nest in buildings in close association with humans, especially cowsheds (Møller, 1983), where temperature is typically warmer (Turner, 2006). For this reason, tail streamers in the North American barn swallow males may be more subject to breakage, and alternative mating systems and opportunities for sexual selection may have evolved as a consequence (Smith & Montgomerie, 1992).
In spite of the costs associated with production and reception of signals, animals often engage in multiple signalling systems. In a recent review, Bro-Jørgensen (2010), argues that variation in selection pressures derived from fluctuations in the environment (ecological conditions as well as social context) could be the factor which leads to the appearance and maintenance of multiple signals, both redundant and non-redundant, in animal systems. In the case of the barn swallow, this diversification of the sexual signals used to display individual quality could explain the fact that so many different ornamental traits have been found to have an effect in the reproductive success. Likewise, having multiple signals available with different production and maintenance costs (and thus differential information content) may explain why we still observe such a great variability in tail streamer length range (85-146 mm; Turner, 2006) in spite of this character being subjected to intense sexual selection by females. Actually, throat and ventral colouration might be more important ornaments in the European barn swallow than previously thought, and predict patterns of assortative mating and reproductive success, at least to a certain extent and in tune with the patterns predicted by tail streamer length.
Indeed, males with long tails take less time to acquire a mate. Furthermore, males which acquire a mate earlier will have a higher reproductive success than those pairing later in the season, due to decreasing probabilities of recruitment for nestlings.
and of producing a second clutch as the breeding season progresses (Møller, 1990b, 1994). Only males arriving earlier to the breeding grounds may be able to gain a reproductive success high enough as to balance the cost of an “expensive” signal such as elongated tail streamers. Thus, we hypothesize that males arriving later to the breeding grounds may be selected to decrease the investment in tail streamer elongation in favour of “cheaper” ornaments in order to tradeoff the amount of information signalled to mates with its costs. Also, we hypothesize that males with “cheaper” sexual signals may be selected to develop some sort of “compensation mechanism”, such as a greater willingness to defend a breeding site associated with a higher quality foraging area, to outweigh their lower signalling intensity.
In this study, we investigate the effect, on the date of the first egg laid and on the quality of the foraging areas associated with breeding sites, of physical condition and several feather ornaments potentially used with sexual signalling purposes in the European barn swallow *Hirundo rustica rustica*: one more “expensive”, the extremely elongated tail streamers, main quality indicator used by this subspecies, and another two “cheaper”, throat and ventral plumage colouration, major quality indicators in the North American subspecies. Providing the date of the first egg laid is correlated with the date of arrival of the barn swallows to the breeding grounds (Turner, 2006), we predict that earlier-breeding males (and females, if they mate assortatively), will preferently have longer tail streamers, whereas later-breeding ones will show a higher investment in pigment-based plumage markings. Likewise, we predict a better physical condition for earlier-breeding barn swallows. Finally, in accordance with the previously explained hypothesis, we predict that breeding sites related to foraging areas with higher large insect abundance, a measure of habitat quality, will be
preferently occupied by male (and female, if they mate assortatively) barn swallows with more elaborate (darker) pigment-based plumage displays.
**Materials and Methods**
Barn swallows are small- to medium-sized, socially monogamous semicolonial passerines. They are specialized on aerial-feeding, preying on insects caught on the wing, and they belong to the family of swallows and martins Hirundinidae (Møller, 1994; Turner, 2006).
Field work was carried out during March-September 2009 and 2010 in the surroundings of the University of Exeter Cornwall Campus, UK, in open farmland with scattered crop fields, pastures and hedgerows, where barn swallows breed in farms either solitarily or in colonies usually of up to about 10 pairs. Swallows were caught either during the day, using mist nets, or at night, using sweep nets, while they were roosting in their breeding sites. 112 adult barn swallows were caught across both years: 67 adults were caught in 2009 (33 males and 34 females) and 45 in 2010 (22 males and 23 females). Once caught, they were sexed according to the presence (females) or absence (males) of a brood patch in the belly area, ringed, several morphometric measurements taken (tail streamer length, tarsus and wing length), weighted and feather samples collected for subsequent objective colour assessment in the lab. Also, insect samples were collected in the barn swallows’ foraging areas using Malaise traps for subsequent calculation of the amount of prey available in different breeding sites throughout the breeding season, and several weather variables (temperature, humidity and wind speed) measured in those areas. Nests were individually monitored every day or every other day throughout the breeding season.
to find out the date of the first egg laid, as well as the clutch size and the total number of chicks hatched and fledged.
**Objective colour assessment**
Colour was quantified using a USB2000 spectrometer (Ocean Optics, Dunedin, Florida), and a xenon flash lamp (Ocean Optics). We used a WS-1 SS Diffuse Reflectance Standard, a diffuse white plastic >98% reflective from 250-1500 nm, as the white reference (100% reflectance), and a piece of black velvet as the dark standard (0% reflectance) to correct for the noise when no light is reaching the sensor. At the far end of the reflection probe/light source, a non-reflective black pointer cut in a 45 degree angle was placed to avoid mismeasurement derived from the light reflected by the plumage reaching the sensor (Andersson and Prager, 2006; Pike *et al.*, 2011). Using the spectra acquisition software package OOIBase (Ocean Optics), we measured the reflectance of feathers collected from four body regions: the throat, breast, belly and vent of each bird. The pointer was applied to plumage patches “created” mounting the feathers on a flat surface in a way that simulates the original plumage structure, following a method that has been widely used in bird colouration studies (e.g. Cuthill *et al.*, 1999; Keyser & Hill, 2000; Perrier *et al.*, 2002; Safran & McGraw, 2004; Komdeur *et al.*, 2005; McGraw *et al.*, 2005; Quesada & Senar, 2006; Safran, 2007; Vaquero-Alba, Evans & Dall, unpublished data). Feathers were mounted on a piece of black velvet to avoid background noise.
As we are interested in the signalling strength of ventral colouration as a sexual indicator, we used the brightness, which is defined as the summed mean reflectance across the entire spectral range ($R_{300-700}$; Montgomerie, 2006), and is the best predictor of melanin content in barn swallows’ feathers, with lower values, i.e. darker
colours, indicating a higher total content of melanin (McGraw et al., 2005; Galván and Møller, 2009).
**Insect collection**
Insect collection was carried out in Malaise traps, large and tent-like structures used for trapping flying insects, mainly diptera and hymenoptera. After hitting the central wall, insects tend to fly upwards and are then funnelled into a collecting vessel containing ethanol attached to the highest point.
Separated land patches or “fields” within an area of approximately 300 m around the nesting sites were located using aerial images of the sampling areas. Previous studies in English populations have found a greater number of foraging barn swallows within such ‘home ranges’ (Evans, 2001). After randomly assigning traps to these land patches, we placed them next to hedgerows, which are a good source of insects (Evans et al., 2003; Turner, 2006), and left them for approximately 3 or 4 hours. We sampled all the 14 sites four times during 2009 and twice during 2010, and spaced the sampling dates as evenly as possible across the breeding seasons. Between two and four sites were sampled every day during each sampling attempt, so that each of the attempts for the whole set of sites was carried out in up to four days. Two samples were taken at the same time in different locations in every site, as some of them were lost due to unstable ground, strong wind, curious cattle or other reasons.
Once collected, insects were counted, measured to the nearest millimetre, classified according to their size in small (<1-3mm), medium (4-7mm) and large (8->10mm) categories, abundance per hour calculated and quantities along the breeding season averaged for each size category within each site. Larger insects are the most
energetically efficient ones for barn swallows (Turner, 2006), so we used this variable as an estimator of the habitat quality within the different breeding sites.
**Statistical analyses**
A principal component analysis (PCA) was conducted on the weather variables measured in the foraging areas (temperature, humidity and wind speed), and first principal component (PC1) was used in subsequent analyses. Principal component analysis and extraction of component loadings were conducted using the *principal* function of *psych* package (Revelle, 2011) in R (Crawley, 2007; R Development Core Team, 2010).
To test the relationship between the physical condition and the intensity of expression of the plumage traits between males and females within the pairs (i.e. assortative mating), we implemented several GLM models, in which “male ornament” or “male condition” was our dependent variable, whereas “female ornament” or “female condition” was the covariate and “year” the independent factor. By testing the effect of the interaction between year and female ornament/condition on male ornament/condition, we could control for the possible influence of yearly variations on assortative mating patterns (Quesada & Senar, 2009).
We analysed the variables influencing the selection of breeding sites by barn swallows according to the habitat quality of the foraging areas within them, using generalized linear mixed effects model (GLMM; details explained below) implemented with the *lmer* function of *lme4* package (Bates & Sarkar, 2007) of R 2.13.0 (Crawley, 2007; R Development Core Team, 2010). Using GLMMs, it is possible to analyse the influences of both random and fixed effects on response variables (Calabuig *et al.*, 2010).
Factors affecting the abundance of large insects were analysed using a Gaussian error structure and identity link function. The throat brightness, ventral brightness - calculated as the average of belly, breast and vent brightness-, tail length and condition -calculated as the ratio of the weight to the tarsus length, a good estimator of avian body size (Senar & Pascual, 1997) - were included as covariates, and sex was included as a fixed factor, whereas year of sampling was included as a random factor to control for its potential influence on large insect abundance. As habitat quality was calculated for each of the breeding sites, where there were several individuals, we calculated the coefficients of variation of throat and ventral brightness, tail length and condition for all the individuals of the same sex present in each site on each year. The coefficient of variation is defined as the ratio of the standard deviation of several measurements to their arithmetic mean ($CV = \frac{s}{\bar{x}}$; Hendricks & Robey, 1936).
GLMMs were also used to analyse the effects of level of expression of sexual characters on the condition and reproductive performance of breeding pairs. Laying dates were transformed to Julian dates and analysed using a normal error structure and an identity link function, and so was condition. In all these analyses we included throat and ventral brightness, tail length and large insect abundance as covariates, to evaluate their effect in individual condition and reproductive performance. Weather was also included as a covariate to control for its potential confounding effects. As temperature was correlated with humidity (Pearson’s correlation test, $r = -0.77$, $t_{110} = -12.5621$, $p < 2.2e-16$) and in order to reduce the number of variables accounting for weather conditions, we included all the weather variables measured (temperature, humidity and windspeed) in a principal component analysis (PCA) and actually included the first component (PC1) in the model. Sex was included as a fixed factor, and site, nest ID and year were included as random effects to control for possible
differences in individual condition and breeding performance parameters between nest locations and years.
GLMMs were initially fitted with all explanatory terms included in them, including first-order interactions in all the models and second-order interactions in which several sexual ornaments (tail length, ventral or throat brightness) were involved for the insect abundance analysis. We chose our minimal adequate models after a stepwise deletion process of the least significant effects ($p>0.05$), starting with the highest-order interactions, providing the simplification did not significantly reduce the explanatory power of the model (Crawley, 2002; Schuett & Dall, 2010). Hypotheses were tested using chi-square ($\chi^2$) statistics. The residuals from the models were checked for normality and homocedasticity.
**Results**
We found a significant and positive covariation between male and female physical condition ($F_{1,51}=6.477$, $p=0.014$; Table 1), and also between male and female ventral plumage brightness ($F_{1,43}=24.774$, $p<0.0001$; Table 1), in our study population. There was no significant covariation between male and female tail streamer length ($F_{1,52}=2.1063$, $p=0.1527$; Table 1) or between male and female throat plumage brightness ($F_{1,43}=1.8024$, $p=0.1865$; Table 1). None of these relationships was confounded by year effect.
The first principal component (PC1) of the principal component analysis (PCA) conducted for weather variables (temperature, humidity and windspeed) measured in barn swallows’ foraging areas, explained more than a 71% of the overall amount of variance (Table 2).
The GLMMs showed a significant 3-way interaction between condition, ventral brightness and throat brightness on large insect abundance around breeding sites \( \chi^2_1 = 9.9225, p=0.002; \) Table 3).
Breeding sites surrounded by areas with highest large insect abundance were occupied by individuals with both darker throat plumage patches and darker ventral plumage patches. Barn swallows with either darker throat plumage or darker ventral plumage bred in sites associated with foraging areas with a low large insect abundance, whereas those with brighter throat and ventral patches foraged in sites with a slightly higher large insect abundance (Figure 1).
Barn swallows with brighter throat plumage and in better physical condition bred in sites next to foraging areas richer in large insects. Those swallows with darker throat plumage and in poorer physical condition also occupied sites surrounded by foraging areas with high large insect abundance, but slightly lower in this case. Darker-throated individuals in better physical condition and brighter-throated ones in poorer condition bred in sites surrounded by areas with much lower large insect abundance (Figure 2).
For the effect of the interaction between ventral brightness and condition on large insect abundance, the trends were similar to the above interaction, with greater amounts of large insects found for areas occupied by darker swallows in poorer condition, and less but still high abundance in areas where less dark individuals in better condition foraged (Figure 3).
Our analyses also showed a significant 2-way interaction between tail length and ventral brightness on large insect abundance around breeding sites \( \chi^2_1 = 8.306, p=0.004; \) Table 3, Figure 4). Individuals with brighter ventral areas bred in sites associated to foraging areas with greater amount of large insects, and this effect was more pronounced for swallows with shorter tail streamers. For swallows with darker
ventral areas, abundance of large insects in areas around breeding sites was very low, and slightly higher for longer-tailed swallows (Figure 4).
Finally for the habitat quality analysis, we found a 2-way interaction between throat brightness and sex on large insect abundance ($\chi^2 = 6.468$, $p=0.011$; Table 1). The graphical plot revealed that males with darker throat plumage patches bred in sites surrounded by foraging areas with greater large insect abundance (Figure 5), whereas there was no clear pattern for females.
When analysing the date of the first egg laid as the response variable, we found a significant effect of body condition on laying date ($\chi^2 = 8.359$, $p=0.004$; Table 3). Individuals in better physical condition started their first breeding attempt significantly earlier (Figure 6). We also found a weak but significant 2-way interaction between throat brightness and sex on the laying date of the first egg ($\chi^2 = 5.001$, $p=0.025$; Table 3). Males with darker throat colouration patches were mated to females laying their first eggs earlier (Figure 7). No such effect was observed for female throat colouration.
**Discussion**
As we predicted, barn swallows with further elaboration of “cheaper” sexual ornaments seem to actively seek higher habitat quality breeding sites: darker-throated males occupied better quality breeding sites according to large insect abundance in the surrounding areas. Males and females with simultaneously darker throat and ventral plumage patches also bred in sites associated with higher quality foraging areas. So did individuals with either poorer body condition and darker throat or ventral colouration, or better condition and less dark plumage. Thus, barn swallows investing in “cheaper” ornaments, but also those with some kind of “weakness” in their sexual
displaying performance or physical condition, seem to be selected to actively look for breeding sites close to higher-quality habitat patches. We observed patterns of positive assortative mating by ventral brightness and body condition, which highlight the condition-dependence and quality-indicating value of ventral brightness for European barn swallows (Safran & McGraw, 2004) and suggest the existence of mutual mate choice (Bitton, 2008). Females mated to darker-throated males laid significantly sooner, and so did females in better condition and/or mated to males in better physical condition. The results of our study suggest that throat and ventral plumage colouration function as quality-indicating traits in the European barn swallow, and suggest a “compensation mechanism” through habitat quality for individuals with impaired displaying ability. It’s the first time, to the best of our knowledge, that evidence for the quality-indicating value of pigment-based ornaments is found for the European bar swallow *Hirundo rustica rustica*.
We did not detect assortative mating patterns by tail streamer length in our population. The assortative mating pattern detected was more in accordance with the results of studies conducted in the North American barn swallow *Hirundo rustica erythrogaster*, where ventral colouration is the most important ornament used for sexual signalling purposes and is closely related to reproductive success (Safran & McGraw, 2004).
In other studies conducted on territorial species, territory attractiveness has been positively correlated with several reproductive success indicators (Hasselquist, 1998), and female choice was determined by an interaction of male and territory quality (Yasukawa, 1981). Even if barn swallows are not strictly territorial, males defend a breeding site of a few square metres (Turner, 2006). Quality of the foraging area surrounding it may be an important factor when selecting and defending a given
breeding site. In our population, nesting sites surrounded by areas with a higher large insect abundance are preferentially occupied by males with darker throats. Our results suggest that males with a higher investment in sexual cues related to colouration (like throat brightness), rather than tail streamers, may invest more in defending nesting sites associated with better foraging areas, as we predicted. If males and females assess the quality of nesting sites and foraging areas associated to them in a similar way, females may be using habitat quality as an indirect cue to select higher quality males. Alternatively, they may be selecting males with a higher investment in throat plumage colouration, in order to grant access to higher habitat quality sites.
In the tree swallow, a territorial species related to the barn swallow, males with territories were in better condition than floaters (Lozano, 1994). In our study, however, individuals with some sort of “weakness”, like poorer physical condition or brighter throat or ventral colouration, seemed to occupy breeding sites associated with a high quality foraging area, perhaps compensating for the lack of reproductive success derived from the aforementioned weakness. Swallows with both darker throat or ventral colouration and a good physical condition tended to occupy sites with a lower abundance of large insects. As these individuals were already indicating at a considerably high level, at least for colouration traits, they might not have any constraint to engage in a “compensation mechanism”.
Likewise, there were fewer large insects in sites occupied by brighter individuals in a poorer physical condition. Several possibilities have been proposed to explain the coexistence of multiple ornaments (Bro-Jorgensen, 2009; Freeman-Gallant, 2010): the “multiple messages” hypothesis (van Doorn & Weissing, 2004) suggests that all the signals might be honest quality indicators of male quality if each of them conveys information about different attributes or targets different receivers. Thus, our results
might suggest that plumage colouration in barn swallows is involved in agonistic male-male interactions rather than in mate choice: if darker throat and ventral plumage reflect a greater ability to defend a breeding site, we might expect to see individuals with darker plumage patches occupying sites associated with foraging areas richer in large insects, as we did. Also, individuals with brighter patches but in a better physical condition have much to win from getting access to a higher quality foraging area. It may be a way to compensate their low level of display to the females at a low cost (low ornament production/maintenance costs), saving themselves for future reproductive attempts. Dark individuals in a good condition may be able to naturally attract more females, so they might not have so much to win from escalating too much in an agonistic interaction for a breeding site. Bright individuals in poorer physical condition might suffer from an impaired ability to win agonistic interactions for breeding sites.
Although tail length alone was not found to have any effect on the quality of the foraging sites selected by barn swallows in our study, in interaction with ventral brightness it influenced the large insect abundance of the foraging areas associated with the breeding sites occupied by barn swallows. Individuals with brighter ventral patches selected breeding sites associated with foraging areas richer in large insect abundance, an effect that was more pronounced for individuals with shorter tail streamers. Tail length did not seem to have such a strong effect on the quality of the breeding site foraging areas occupied by barn swallows, and only for lower values of ventral brightness could we see a trend in shorter tailed individuals towards selecting areas with a greater abundance of large insects. So, individuals with a lower intensity in the expression of two different ornaments selected breeding sites associated with better quality foraging areas. This behaviour may compensate for the lack of
attractiveness of those ornaments with a lower signalling strength. For individuals with longer tail streamers, however, there was a less intense effect of ventral brightness on the quality of preferred areas. As male barn swallows with longer tail streamers have been found to have a higher overall reproductive success (Möller, 1988, 1994), they may not be selected to invest so much in compensating for the lack of intensity in the expression of ventral plumage colouration as the ones with shorter tail streamers.
Surprisingly, in interaction with tail length, the influence of ventral brightness in the quality of the selected foraging areas has the opposite effect to the one it has when it interacts with throat brightness. This may be because feather-size related and colouration-related ornaments impose different constraints in terms of production and/or maintenance costs and information content. Thus, throat and ventral colouration, when considered together, seemed to have a synergic effect on the quality of the foraging sites associated with the breeding sites selected by their bearers. When physical condition or tail length, a more “expensive” signal of individual viability, were considered, the effect of each of the “cheaper” plumage colouration ornaments in breeding site preferences varied, in order to satisfy the needs imposed by different combinations of costs and benefits.
Females mated to males with lower values of throat brightness (i.e. darker throat patches) tended to have an earlier laying date for their first egg. Likewise, physical condition also had an effect on laying date, so that individuals in better condition bred significantly earlier. As barn swallows in the population we studied mated assortatively by body condition, we did not detect any influence of sex in the relationship between laying date and condition, as we did for covariation between throat brightness and laying date (there was no assortative mating by throat brightness
in our population). These results are in concordance with previous studies. Ninni *et al.* (2004) found evidence of condition-dependence in the date of the first egg laid by barn swallows in a Spanish population, as well as an effect of the colour of the red facial feathers on the laying date, although results were not consistent for the two years studied. However, contrary to several studies that have found a strong negative relationship between date of arrival to the breeding grounds and laying date in barn swallows, and the length of the outermost tail feathers, i.e. the tail streamers (e.g. Möller, 1994, Ninni *et al.*, 2004, Teplitsky *et al.*, 2011), we did not observe any effect of tail streamer length on the laying date of the first egg, nor an assortative pairing pattern by tail length between males and females. Throat brightness, rather than tail streamer length, seemed to be the most important plumage ornament related to laying date, an indicator of reproductive success.
To sum up, throat and ventral plumage colouration seem to play an important role in the sexual signalling system of the European barn swallow. The results of our study suggest that they are part of a multicomponent signalling system which may have evolved as a response to dynamic selection pressures caused by fluctuations in ecological and social conditions (e.g. changes in farming activity, climate change and other factors may affect the signal expression in barn swallows). Future studies should be carried out to find out whether relative importance of each of the sexual signals taking part in multiple-signalling systems in the barn swallow might be changing as part of a dynamic process, as our results suggest. We also found evidence for a “compensation mechanism” by which individuals with lower displaying rates in one or more sexually selected traits should try to compensate their lack of reproductive fitness by defending higher quality breeding sites.
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Table 1: GLM models within mated pairs, relating the intensity of expression of male ornaments (tail length, throat brightness and ventral brightness) and physical condition to those of their female partner (i.e. assortative mating), controlling for the effect of the year
| Male ornament | Year:Female ornament | Year | Female ornament |
|---------------|----------------------|------|-----------------|
| | Test | p | Test | p | Test | p |
| Colouration | | | | | | |
| Throat brightness | $F_{1,41}=0.150$ | 0.701 | $F_{1,42}=0.0004$ | 0.984 | $F_{1,43}=1.802$ | 0.187 |
| Ventral brightness | $F_{1,41}=0.230$ | 0.634 | $F_{1,42}=0.017$ | 0.899 | $F_{1,43}=24.774$ | <0.0001 *** |
| Tail length | $F_{1,51}=3.249$ | 0.078 | $F_{1,51}=1.221$ | 0.274 | $F_{1,52}=2.106$ | 0.153 |
| Male condition | Year:Female condition | Year | Female condition |
|----------------|-----------------------|------|------------------|
| | Test | P | Test | P | Test | p |
| Condition | $F_{1,49}=0.783$ | 0.380 | $F_{1,50}=1.119$ | 0.295 | $F_{1,51}=6.477$ | 0.014* |
Table 2: Component loadings (unrotated) of first principal component (PC1) as quantified by principal component analysis
| Variable | Mean | SD | Principal Component PC1 |
|----------------|-------|------|--------------------------|
| Temperature | 24.596| 1.797| -0.90 |
| Humidity | 47.380| 6.961| 0.92 |
| Wind speed | 8.688 | 3.795| 0.70 |
| Eigenvalue | | | 2.137 |
| % variance explained | | | 71 |
Loadings over ±0.6 are marked in bold. $N = 112$
Table 3 Summary of minimal adequate models
| Response variable | N | ΔAIC | Explanatory variable | $X^2(df)$ | p |
|-------------------|-----|------|-------------------------------|-----------|------|
| Large insect abundance$^a$ | 31 | 6.03 | Tail length | 13.94 (1) | 0.0002 |
| | | | Ventral bright | 8.733 (1) | 0.003 |
| | | | Throat bright | 9.856 (1) | 0.002 |
| | | | Condition | 11.005 (1)| 0.0009|
| | | | Sex (male) | 3.051 (1) | 0.081 |
| | | | Ventral bright:throat bright | 14.565 (1)| 0.0001|
| | | | Ventral bright:condition | 14.016 (1)| 0.0002|
| | | | Throat bright:condition | 6.926 (1) | 0.008 |
| | | | Tail length:ventral bright | 8.306 (1) | 0.004 |
| | | | Throat bright:sex (male) | 6.468 (1) | 0.011 |
| | | | Ventral bright: throat bright:condition | 9.922 (1) | 0.002 |
Laying date$^b$ | 83 | 18.6 | Sex (male) | 0.286 (1) | 0.593 |
| | | | Throat bright | 92.415 (1)| <0.0001|
| | | | Condition | 8.3586 (1)| 0.004 |
| | | | Throat bright:sex (male) | 5.0198 (1)| 0.025 |
ΔAIC difference between AIC of maximal and minimal model, condition physical condition, covariate, large insects large insect abundance, covariate, nest:site nest location within each site, factor with 35 levels, sex factor with 2 levels, site geographic location of nesting site, factor with 12 levels, tail length tail streamer length, covariate, throat bright throat brightness, covariate, ventral bright ventral brightness, covariate, weather weather measurement, covariate, year year of study, factor with 2 levels
$^a$ Fixed effects and covariates included in maximal models: tail length, ventral bright, throat bright, condition, sex, tail length:ventral bright, tail length:throat bright, ventral bright:throat bright, tail length:condition, ventral bright:condition, throat bright:condition, tail length:sex, ventral bright:sex, throat bright:sex, condition:sex, tail length:ventral bright:throat bright, tail length:ventral bright:condition, tail length:throat bright:condition, ventral bright:throat bright:condition. Random effects: year.
$^b$ Fixed effects and covariates included in maximal models: sex, ventral bright, throat bright, tail length, condition, large insects, weather, ventral bright:sex, ventral bright:large insects, ventral bright:weather, ventral bright:tail length, ventral bright:throat bright, ventral bright:condition, throat bright:sex, throat bright:large insects, throat bright:weather, throat bright:tail length, throat bright:condition, tail length:sex, tail length:large insects, tail length:weather, tail length:condition, condition:weather, condition:large insects. Random effects: year, site/nest.
Figure 1: The effect of throat and ventral brightness on large insect abundance (CV=coefficients of variation)
Figure 2: The effect of throat brightness and condition on large insect abundance (CV=coefficients of variation)
Figure 3: The effect of ventral brightness and condition on large insect abundance (CV=coefficients of variation).
Figure 4: The effect of tail length and ventral brightness on large insect abundance (CV=coefficients of variation)
Figure 5: Effect of male throat brightness on large insect abundance (CV=coefficients of variation)
Figure 6: Effect of body condition on laying date
Figure 7: Effect of throat brightness of males on laying date
Plumage colouration, as well as tail-streamer length, is a sexually selected trait in European barn swallows
Iker Vaquero-Alba, Andy McGowan, Matthew R. Evans & Sasha R.X. Dall
Centre for Ecology and Conservation, University of Exeter, Cornwall Campus
Penryn, Cornwall, TR10 9EZ, UK
Choosing elaborately ornamented individuals as sexual partners can provide animals with direct and indirect benefits. As a result, birds with more elaborate and colourful displays often enjoy a selective advantage, being preferred as mates. Animals often use more than one signal to transmit information to others. The main reason for the emergence and maintenance of multiple signals, both redundant and non-redundant, is variation in selection pressures derived from fluctuations in the environment. We studied, in the European barn swallow, the effects of several potentially sexually selected ornaments, along with plumage manipulations, on a series of measures of reproductive success. It is the first time, to the best of our knowledge, that throat and ventral colouration as well as tail streamer length, have been investigated from a multi-component signalling perspective in this subspecies. Most significantly, we found that having darker plumage patches, alone or in interaction with other factors, was related to higher levels of reproductive success by male swallows. Given the well known impact of tail streamer length variation on male swallow mating success, this suggests the existence of a multi-component signalling system for the European barn swallow and highlights the important contribution of colouration-related sexual signals to this system.
Introduction
Models of sexual selection suggest that choosing elaborately ornamented individuals as sexual partners can provide animals with direct and indirect benefits. Inter-individual differences in the degree of exaggeration of the ornaments transmit useful information about the benefits involved (Pomiankowski, 1987; Andersson, 1994; Garamsziegi et al., 2006; Bro-Jorgensen et al., 2007). Birds with more elaborate and colourful displays enjoy a selective advantage, as they are preferred as mates (Hill, 2006). Theoretical models also predict that these ornamental characters will be costly at evolutionary stability (Fisher, 1930; Zahavi, 1975; Iwasa et al., 1991). This cost may be expressed at the moment of production and/or maintenance, and also continuously through increased risk of predation or parasitism (Magnhagen, 1991; Folstad & Karter, 1992). Costly signals are efficient quality-indicators, as the costs they inflict to their bearers make them likely to be general viability indicators, in accordance with the handicap principle (Zahavi, 1975). Sexual ornaments are quality-indicating signals kept honest (informative) by the handicap of their costs (Fitzpatrick, 2000).
Birds, as well as many other animals, often use more than one signal to transmit information to others (Bro-Jorgensen, 2009). For example, males of the greater sage grouse (*Centrocercus urophasianus*) combine wing and tail movements with popping vocalizations in their female-attracting strut displays (Wiley, 1973); and the male wolf spider (*Schizocosa stridulans*) produces different components of its complex sexual display by using its abdomen and pedipalp simultaneously (Elias, 2006). Animals often deploy multiple signalling systems, despite the costs associated with production and reception of signals. The main factor leading to the emergence and maintenance of such multiple signals, both redundant and non-redundant, is variation in selection pressures derived from fluctuations in the broader environment (including ecological conditions
and/or social context; Bro-Jørgensen, 2009). Animals might be selected to utilize “cheaper” signals when the day-to-day costs incurred by the costlier signals have particularly severe fitness consequences. Likewise, they might minimise the elaboration of the costliest traits, such as tail elongation (Fitzpatrick, 2000) in such contexts. Therefore, birds may have evolved plumage that trades off the amount of information it signals to potential mates/rivals with its costs; in other words, such traits may be selected to optimise rather than maximise their information content.
Several ornamental traits have been studied in the European barn swallow *Hirundo rustica rustica* and argued to have a role in sexual signalling. For example, males preferred as social and/or extra-pair mates have longer and more symmetric tail streamers (Møller, 1988, 1992a, 1993, 1994; Saino et al., 1997), larger white spots in the tail (Kose et al., 1999, Kose & Møller, 1999) or more intense red facial plumage (Ninni et al., 2004). Most of the studies of sexual selection in the European barn swallow have focused on variation in the elongated outermost tail streamers. Indeed, evidence is accumulating for a sexual function for these feathers over the last few decades (reviewed in Møller, 1994; Møller et al., 1998b). Although a mechanical and aerodynamic function of tail streamers in tight manoeuvres has been reported (Norberg, 1994), it is commonly agreed that they constitute a serious handicap in flight. Thus, sexual selection constitutes at least a partial explanation for exaggerated tail length in male barn swallows (Buchanan & Evans, 2000; Rowe et al., 2001, Møller, 1994). Males with longer tails mate more often, get more extra-pair copulations, have more broods and overall have higher reproductive success (Møller 1988, 1990, 1994). In addition, other types of signals have been studied to investigate their role in sexual selection and reproductive success, and their relationship with tail length. For example, Kose et al. (1999) and Kose & Møller (1999) studied white spots on tail feathers, finding a positive
correlation between their size and tail length. The size of the spots was also related to an earlier start of breeding. However, Perrier et al. (2002) did not find a significant correlation between the dorsal plumage structural coloration, tail length, tail asymmetry and red facial colouration. Saino et al. (2003) did not find a reduction in singing activity or song complexity of males with artificially elongated tail feathers, suggesting that males are not displaying multiple signals at their maximum possible levels.
Nevertheless, few studies have been conducted in the European barn swallow to study the possible role of sexual selection on ventral plumage colouration (but see Ninni et al., 2004 and Galván & Møller, 2009 for studies on the red throat patch). Indeed, ventral plumage colouration predicts pairing patterns and reproductive success in the North American barn swallow subspecies *erythrogaster*. Safran et al. (2005) found increased social paternity rates (i.e. paternity from their social mates, as opposed to extra-pair paternity) in males with experimentally enhanced ventral plumage darkness. Although evidence suggests that this ventral plumage colouration is the main quality indicator in inter-sexual signalling for the North American subspecies, tail length still plays a role in sexual signalling: females mated to males with experimentally lengthened tails breed earlier (Smith & Montgomerie, 1991; Smith et al., 1991) and streamer length predicts fertilization success (although this effect is confounded by age; Kleven et al., 2006). If both ornamental characters: tail streamer elongation and ventral plumage colouration, were already used by barn swallows as quality indicators before the European and the North American subspecies diverged, both ornaments may play roles in sexual signalling in both subspecies at present. Variation in the relative importance of different signal types on the different continents may be due to adaptation to distinct local environmental conditions.
We hypothesize that ventral plumage colouration, as well as elongated tail streamers, function as quality indicators and determine reproductive success in the European barn swallow *Hirundo rustica rustica*, as part of a multi-component signalling system, in response to variation in local selection pressures. In the present study, we investigate the relationship between several common measures of reproductive success (clutch size, hatching success, physical condition of the chicks and food provisioning of chicks) and several feather ornaments potentially used for sexual signalling purposes. The ornaments considered are likely to differ in their production and maintenance costs, and therefore information contents: the elongated tail streamers (the main quality indicator used by this subspecies) are likely to be costly to both produce and maintain and thus have a high quality-indicating value, while the throat and ventral plumage colouration (the dominant quality indicators in the North American subspecies) should be costly to produce but less costly to maintain and therefore offer less information to prospective mates. We included female ornaments in the study because it is still not clear whether they are also subject to sexual selection or just nonadaptive, correlated effects of selection on males (Møller, 1993; Cuervo et al., 1996; Amundsen, 2000; Safran & McGraw, 2004; Kraaijeveld et al., 2007). Before the beginning of the second clutch, we also conducted a series of manipulations of male tail streamer length and ventral plumage darkness and measured their impacts on the fitness measures. If these ornaments are part of a multi-component signalling system that has evolved via variable selection (Bro-Jørgensen, 2009), and males are not displaying multiple signals at maximum possible levels (Saino et al., 2003), we predict naturally longer-tailed and darker-coloured individuals will show higher reproductive success. Furthermore, we predict increased reproductive performance for pairs whose male’s ventral darkness was artificially enhanced, especially for those with shortened or unmodified tail streamers,
and decreased performance for couples in which male tail length, the costliest ornament, was lengthened.
**Materials and Methods**
Barn swallows are small (approx. 20 g), semicolonial, socially monogamous passerines, feeding on insect prey caught on the wing, belonging to the family of swallows and martins Hirundinidae (Møller, 1994; Turner, 2006). They have a U-shaped tail with extremely elongated outermost feathers (called tail streamers), and they display patches of rust-colored plumage in the throat and ventral region, this trait being much more exaggerated in the North American subspecies (*Hirundo rustica erythrogaster*), but still present in the European subspecies (*H. rustica rustica*; Møller, 1992b, Safran & McGraw, 2004).
Field work was carried out in the surroundings of the University of Exeter Cornwall Campus, UK, during March-September 2009 and 2010. The study area was open farmland with scattered crop fields, pastures and hedgerows, where barn swallows breed in farms either solitarily or in colonies usually of up to about 10 pairs. 112 adult barn swallows were caught across both years: 67 adults in 2009 (33 males and 34 females) and 45 in 2010 (22 males and 23 females). Most of the swallows were caught during the day, using mist nets placed into the buildings where they nest. The remaining ones were caught at night, using sweep nets, while they were roosting in their breeding sites. They were then sexed according to the presence (females) or absence (males) of a brood patch in the belly area, and provided with a numbered aluminium band. Several morphometric measurements were taken: tail streamer and wing length, using a ruler, and tarsus length, using an electronic caliper. They were weighted, using a digital precision balance, and feather samples collected for subsequent objective colour assessment in the
lab. Insect samples were collected in the barn swallow foraging areas using Malaise traps, for subsequent calculation of the amount of prey available in different breeding sites throughout the breeding season. Nests were individually monitored every day or every other day throughout the breeding season to record the size of the clutch and hatching success, calculated as the proportion of the eggs successfully hatched within one clutch. Within their first two weeks of life, chicks were weighted and their wing and tarsus length measured, using the same methodology described for adults. Physical condition of both adult barn swallows and chicks was calculated as the ratio of the weight to the tarsus length, a reliable estimator of avian body size (Senar & Pascual, 1997), and condition of the chicks within a nest was averaged for the statistical analyses.
In 2009, just after the end of the first successful breeding attempt and before the start of the second, 30 males were recaptured and their tail streamer length and ventral colouration brightness manipulated, whereas in 2010 males were not recaptured, and they were left unmanipulated. Tail manipulations were carried out as described by Möller and colleagues (reviewed in Möller, 1994), but elongating or reducing tail streamer length only by approximately 13 mm instead of 20 mm, to avoid interfering with the viability-selected part of the streamers (Buchanan & Evans, 2000; Rowe et al., 2001). After recapture, for tail manipulations, males were randomly assigned to one of two tail streamer manipulation groups (elongation or reduction) or to a control group, where feathers were cut and reglued without length modification, and for colour manipulations, they were randomly assigned to a colour enhancement (darkening) group or to a control group, where plumage colouration was not modified. See Table 2 for a summary of the levels within the manipulation factor. The handling and manipulation of
the adult barn swallows and chicks were made under ringing licenses from BTO and/or a license from Natural England.
**Objective colour assessment**
Plumage colour (before manipulation) was quantified using an Ocean Optics USB2000 spectrophotometer (range 250-800 nm) and a xenon flash lamp (Dunedin, FL). We used a “Spectralon” tablet (WS-1 SS Diffuse Reflectance Standard, Ocean Optics, Dunedin, FL), a diffuse white plastic >98% reflective from 250-1500 nm, as the white reference (100% reflectance), and a piece of black velvet as the dark standard (0% reflectance) to correct for the noise when no light is reaching the sensor. At the far end of the reflection probe/light source, we placed a non-reflective black pointer cut in a 45 degree angle to avoid mismeasurement derived from the light reflected by the plumage reaching the sensor (Andersson and Prager, 2006; Pike *et al.*, 2011). We measured the reflectance of feathers collected from four body regions: the throat, breast, belly and vent of each bird, using the spectra acquisition software package OOIBase (Ocean Optics). The pointer was placed on plumage patches “created” by mounting the feathers on a flat surface in a way that simulates the original plumage structure, following a method widely used in bird colouration studies (e.g. Cuthill *et al.*, 1999; Keyser & Hill, 2000; Perrier *et al.*, 2002; Safran & McGraw, 2004; Komdeur *et al.*, 2005; McGraw *et al.*, 2005; Quesada & Senar, 2006; Safran, 2007; Vaquero-Alba, Evans & Dall, unpublished data; 2). Feathers were mounted on a piece of black velvet to avoid background noise.
We used the brightness, defined as the summed mean reflectance across the entire spectral range ($R_{300-700}$; Montgomerie, 2006), for our analyses, as we are interested in the signalling strength of ventral colouration as a sexual indicator. Brightness is the best
predictor of melanin content in barn swallows’ feathers, with lower values, i.e. darker colours, indicating a higher total content of melanin (McGraw et al., 2005; Galván and Møller, 2009). We used two colour-related measures in our analyses: throat brightness and ventral brightness, calculated as the average of belly, breast and vent brightness.
**Insect collection**
Insects were collected in Malaise traps, which are large, tent-like structures used for trapping flying insects, mainly diptera and hymenoptera. When insects hit the central wall, they tend to fly upwards and are then funnelled into a collecting vessel containing ethanol, which is attached to the highest point.
Using aerial images of the sampling areas, separate land patches or “fields” within an area of approximately 300 m around the nesting sites were located. Previous studies in English populations have found a greater number of foraging barn swallows within such ‘home ranges’ (Evans, 2001). Traps were randomly assigned to these land patches, placed next to hedgerows, which are a good source of insects (Evans et al., 2003; Turner, 2006), and left for approximately 3 or 4 hours. All the 14 sites were sampled four times during 2009 and twice during 2010, spacing the sampling dates as evenly as possible across the breeding seasons. Each time, between two and four sites were sampled every day, so that each of the collection attempts for the whole set of sites was carried out in up to four days. In every site, two samples were taken at the same time in different locations, as some of them were lost due to unstable ground, strong wind, curious cattle or other reasons.
After collection, insects were counted, measured to the nearest millimetre, classified according to their size in small (<1-3mm), medium (4-7mm) and large (8->10mm) categories, abundance per hour calculated and quantities along the breeding season
averaged for each size category within each site. As larger insects are the most energetically efficient ones for barn swallows (Turner, 2006), we used their abundance as an estimator of the habitat quality within the different breeding sites chosen by different pairs.
**Statistical analyses**
We analysed the variables influencing several indicators of reproductive success in the barn swallows, using generalized linear mixed effects models (GLMM; details explained below) implemented with the `lmer` function of `lme4` package (Bates & Sarkar, 2007) of R 2.13.0 (Crawley, 2007; R Development Core Team, 2010). Using GLMMs, it is possible to analyse the influences of both random and fixed effects on response variables (Calabuig *et al.*, 2010). As plumage manipulations were carried out before the start of the second clutch in 2009, measurements of reproductive success were not comparable across both clutches. For that reason, we decided to analyse the 1\textsuperscript{st} and 2\textsuperscript{nd} clutches separately, instead of pooling data together.
Factors affecting the size of the clutch were analysed using a Poisson error structure and a log link function. The throat brightness, ventral brightness, tail length, physical condition and large insect abundance were included as covariates, and sex was included as a fixed factor. In the analyses corresponding to the second clutch, plumage modification was also included as a fixed factor. Factors affecting the hatching success and the physical condition of chicks were analysed using a Gaussian error structure and an identity link function. The same covariates and fixed factors as before were included in the analyses. Site identity, nest location and year were included as random effects to control for possible differences in reproductive investment and breeding performance parameters among sites, nest locations within sites and years. In the analyses of the
factors affecting physical condition of chicks, the age of the brood was included as a random factor as well, to account for the variance due to differences in growing speed amongst age periods (Turner, 2006).
GLMMs were initially fitted with all explanatory terms included in them, including first-order interactions in which sexual ornaments (tail length, ventral and throat brightness) and physical condition were involved. A summary of the maximal models fitted is given in Table 1. Minimal adequate models were chosen after a process of stepwise deletion of the least significant effects ($p > 0.05$), starting with the highest-order interactions, providing the simplification did not significantly reduce the explanatory power of the model (Crawley, 2002; Schuett & Dall, 2010). Consecutive models were compared using chi-square ($\chi^2$) statistics. If a factor containing several levels had a significant effect in the minimal model, we applied *post-hoc* pairwise comparisons between the levels of the factor using the `glht` function and Tukey’s test of additivity from the `multcomp` extension package (Hothorn, Bretz & Westfall, 2008) of R 2.13.0 (R Development Core Team, 2010). These pairwise comparisons allowed us to test the significance of the effect of each factor level compared to the rest of the levels (Table 1). The residuals from the models were checked for normality and homocedasticity; where appropriate, transformations of the response variables or standardizations of the explanatory variables were done following R Development Core Team (2010).
**Results**
No variable or interaction analysed was found to have a significant effect on clutch size for the first clutch, when this was tested as the response variable (Table 1).
Our analyses showed a significant two-way interaction between sex and throat brightness on hatching success, i.e. the proportion of eggs laid which successfully hatched, for the first clutch ($\chi^2_1=4.911$, $p=0.027$; Table 1). Eggs laid by females mated to males with darker throats showed a significantly higher hatching success than those of females mated to males with more brightly coloured throats. We could not find a similarly clear pattern in the case of females’ throat colouration (Figure 1). The analyses also showed a significant 2-way interaction between ventral brightness and throat brightness on hatching success ($\chi^2_1=9.450$, $p=0.002$, Table 1). In general, individuals with brighter throat plumage (males or females) had clutches with a higher hatching success for all the range of ventral brightness values. Hatching success amongst brighter-throated swallows was higher for those who also had brighter coloured ventral plumage. Amongst darker-throated swallows, however, hatching success was slightly higher for those with darker ventral colouration (Figure 2). Finally for hatching success in first clutch, we found a significant 2-way interaction between tail length and throat brightness on the proportion of successfully hatched eggs ($\chi^2_1=5.158$, $p=0.023$). Hatching was more successful for swallows with darker throats, especially for those with longer tail streamers. Amongst the swallows with brighter throat colouration, on the other hand, shorter-tailed individuals had a higher hatching success than longer-tailed ones (Figure 3).
When analysing the physical condition of the chicks raised in the first clutch by the barn swallows in our study population, we found it to be significantly determined by the 2-way interaction between ventral brightness and the abundance of large insects around breeding sites ($\chi^2_1=4.882$, $p=0.027$; Table 1). For swallows nesting in places next to foraging areas with a lower large insect abundance, ventral colouration of the parents did not seem to have any effect on physical condition of the chicks. In places related to
foraging areas richer in large insects, however, chicks raised by parents with a brighter ventral plumage colouration were in much better physical condition than those raised by parents with darker ventral plumages (Figure 4).
The analyses carried out for the second clutch yielded a significant effect of the 2-way interaction between ventral plumage colouration (before manipulation) and large insect abundance on clutch size ($\chi^2 = 4.019$, $p=0.045$). Clutches were bigger for couples nesting in sites associated with foraging areas richer in large insects. Females of pairs in which barn swallows had a brighter ventral plumage laid more eggs than those of couples with darker ventral plumage colouration, this difference being much more pronounced for individuals nesting in sites with foraging areas more abundant in large insects (Figure 5). Plumage manipulations also had a significant effect on the size of the second clutch ($\chi^2 = 21.396$, $p=0.002$). Females mated to control males laid significantly fewer eggs than those mated to males with elongated tail streamers ($z_1 = -3.478$, $p=0.0005$), reduced tail streamers ($z_1 = -3.170$, $p=0.002$), control males with enhanced ventral plumage colouration ($z_1 = 3.337$, $p=0.0008$) or unmanipulated males ($z_1 = 3.938$, $p<0.0001$). Likewise, females mated to males with reduced tail streamer length and enhanced ventral plumage colouration laid significantly fewer eggs than those mated to males with elongated tail streamers ($z_1 = -2.034$, $p=0.042$), control males with enhanced ventral plumage colouration ($z_1 = 2.051$, $p=0.040$) or unmanipulated males ($z_1 = 1.975$, $p=0.048$; Table 1; Figure 6).
There was a significant 2-way interaction between throat brightness before manipulation and the abundance of large insects on the hatching success for the second clutch ($\chi^2 = 15.054$, $p<0.001$; Table 1). Hatching success was higher for couples whose members had brighter throat plumage patches, and this difference was much more pronounced for swallows nesting in sites associated with foraging areas with greater
large insect abundance. The highest hatching success was recorded for brighter individuals foraging in sites richer in large insects (Figure 7). Also, the 2-way interaction between ventral plumage colouration before manipulation and large insect abundance had a significant effect on hatching success ($\chi^2 = 8.971$, $p=0.003$; Table 1). Amongst swallows nesting in sites associated with foraging areas with lower abundance of large insects, those with brighter ventral plumage had clutches that hatched much more successfully than the ones with darker ventral colouration. This relationship reversed for individuals foraging in areas with higher large insect abundance, with darker swallows getting a higher hatching success. The highest hatching success was recorded for brighter individuals nesting in sites with lower large insect abundance, and to a lesser extent, for darker ones nesting next to areas with higher large insect abundance (Figure 8). Furthermore, there was a significant 2-way interaction between throat plumage colouration and tail length (both before manipulation) on hatching success ($\chi^2 = 15.332$, $p<0.001$; Table 1). Barn swallows with darker throat plumage patches had a much higher hatching success for their second clutches than brighter-throated individuals. Amongst birds with darker throat patches, those with longer tail streamers had a higher proportion of eggs successfully hatched, whereas amongst individuals with brighter throat colouration, short tail streamered ones had a higher hatching success (Figure 9). Again, plumage manipulations had a significant effect on hatching success ($\chi^2 = 28.293$, $p<0.001$). Clutches from pairs whose males’ tail streamers were artificially elongated had significantly higher (and less variable) hatching success than clutches from couples with males’ tail streamers reduced ($z_1 = -4.009$, $p<0.0001$), streamers reduced and ventral colouration enhanced ($z_1 = -2.535$, $p=0.011$), control males ($z_1 = -4.997$, $p<0.0001$), unmanipulated males ($z_1 = -6.465$, $p<0.0001$) or control males with enhanced ventral plumage colouration ($z_1 = -3.166$,
p=0.002). Males with artificially shortened tail streamers were part of couples with a significantly higher hatching success and less variance than control ($z_1=-2.574$, p=0.010) or unmanipulated males ($z_1=-2.603$, p=0.009). In contrast, control males with enhanced ventral plumage colouration were in couples with significantly lower hatching success than control males ($z_1=-2.509$, p=0.012), or unmanipulated males ($z_1=-2.210$, p=0.027; Table 1, Figure 10).
When we analysed the physical condition of the chicks in the second clutch, there was a significant 2-way interaction between sex and physical condition of the adults before manipulation on the response variable ($\chi^2_1=8.154$, p=0.004; Table 1). Chicks raised by couples in which males were in better condition were also in much better condition. We did not detect a similar relationship with females’ condition (Figure 11). The 2-way interaction between throat plumage brightness before manipulation and sex also had an effect on the physical condition of the chicks ($\chi^2_1=11.486$, p<0.001; Table 1). Chicks raised in nests with darker-throated males were in significantly better condition than those raised by males with brighter throat patches. We observed the opposite trend in females, but it was not significant (Figure 12). Our analyses showed a significant 2-way interaction between tail streamer length before manipulation and sex on chicks’ physical condition ($\chi^2_1=4.140$, p=0.042; Table 1). Males with originally longer tail streamers were part of couples that raised chicks in significantly better physical condition. We didn’t see any comparable relationship for females’ tail streamer length (Figure 13). The 2-way interaction between ventral plumage colouration (before manipulation) and large insect abundance around breeding sites also had an influence on physical condition of chicks ($\chi^2_1=18.391$, p<0.001; Table 1). Physical condition was much better for chicks raised by brighter individuals. Amongst darker barn swallows, those nesting in sites next to foraging areas with a lower large insect abundance had chicks in a better
condition (Figure 14). Further to that, there was a significant 2-way interaction between tail streamer length before manipulation and large insect abundance around breeding sites on physical condition of chicks ($\chi^2 = 11.142$, $p < 0.001$; Table 1). Chicks raised in nests related to foraging areas with a lower large insect abundance were generally in better condition. Both for individuals next to areas with lower or higher large insect abundance, couples whose male’s tail streamers were longer had chicks in better condition (Figure 15). A significant 2-way interaction between ventral plumage colouration and tail length, both before manipulation, on chick condition was found in our study population ($\chi^2 = 38.107$, $p < 0.001$; Table 1). Darker individuals raised chicks in better physical condition than those with brighter ventral plumage patches. In the case of brighter barn swallows, those couples whose males had shorter tail streamers raised chicks in slightly better condition (Figure 16). Finally, plumage manipulation had a significant influence on physical condition of chicks ($\chi^2 = 49.483$, $p < 0.001$). Couples in which males’ tail streamers were artificially elongated raised chicks in significantly poorer physical condition than couples with control males ($z_1 = 8.644$, $p < 0.0001$), control males with enhanced ventral plumage colouration ($z_1 = -3.646$, $p = 0.0003$) or unmanipulated males ($z_1 = 12.710$, $p < 0.0001$). Furthermore, chicks born in nests where males’ tail streamer lengths were shortened were also in worse physical condition than those born in nests with control males ($z_1 = 9.879$, $p < 0.0001$), control males with enhanced ventral colouration ($z_1 = -4.153$, $p < 0.0001$) or unmanipulated males ($z_1 = 22.588$, $p < 0.0001$). Couples whose males were assigned to the control group and their ventral plumage colouration enhanced raised chicks in better condition than couples with control ($z_1 = -11.469$, $p < 0.0001$) or unmanipulated ($z_1 = 19.893$, $p < 0.0001$) males. Finally, chicks raised in nests with control males were in worse physical
condition than those raised in nests with unmanipulated males ($z_1 = -3.244$, $p=0.001$; Table 1, Figure 17).
**Discussion**
The results of our study show that throat and ventral colouration-related ornaments, further to tail streamer length, are part of the European barn swallow’s multi-component signalling system, and influence reproductive success. Darker-throated males were in pairs with significantly higher hatching success in the first clutch, and raised chicks in significantly better physical condition in the second clutch. Furthermore, male tail streamer length before manipulation along with male physical condition, were positively related to chick condition in the second clutch. Thus, darker-throated or longer-tailed males appeared to raise chicks in inherently better condition. This suggests that both traits, elongated tail streamers and throat plumage colouration, qualify as potential honest quality-indicating sexual ornaments (Andersson, 1994). While several previous studies have pointed at the quality-indicating function of tail streamers and their relationship with annual reproductive success (Möller, 1994, 1998b), our results are the first, to the best of our knowledge, to demonstrate this for throat plumage colouration (but see Ninni *et al.*, 2004, for indirect evidence through arrival date).
Throat plumage colouration, together with ventral plumage colouration, along with tail streamer length, also explained variation in reproductive success in interaction with other variables. The interaction between throat plumage colouration and tail length influenced hatching success in both clutches. In both cases, individuals with darker throat patches and longer tail streamers had the highest proportion of eggs in the clutch successfully hatched, with darker-throated, shorter-tailed ones also achieving notably enhanced hatching success. This observation confirms our hypothesised qualityindicating value of plumage colouration, either by itself or together with tail streamer exaggeration. The fact that we did not observe significant sex differences for this interaction might suggest that these ornaments are also subject to sexual selection in females to a certain extent, rather than being just nonadaptive, correlated effects of selection on males (Amundsen, 2000). Although the studies demonstrating functional female ornaments are rare for birds, some previous work has shown enhanced fecundity in the most exaggeratedly ornamented females (Møller, 1993; Safran & McGraw, 2004). Likewise, throat plumage darkness in males alone positively influencing first clutch hatching success might be indicative of differential maternal allocation in relation to male sexiness (Burley, 1986; Osorno et al., 2006; Loyau & Lacroix, 2010). Ventral plumage colouration, interacting with tail length, also influenced physical condition of chicks during the second clutch. Again, individuals with darker plumage patches achieved the higher values of reproductive success by raising chicks in much better condition. For individuals with darker ventral plumage, tail streamer length hardly had any effect on the physical condition on chicks. Amongst brighter-coloured ones, though, shorter-tailed barn swallows raised chicks in better condition than longer-tailed ones. This may suggest a higher importance of “cheaper” (colour-related) sexual signals rather than more “expensive” ones (elongated tail streamers) as quality-indicators in our study population, possibly due to geographic variation in environmental conditions (Safran & McGraw, 2004): if individual quality is mainly signalled by plumage colouration, we would expect brighter individuals with longer tail streamers to find it more difficult to capture appropriate prey items due to aerodynamic constraints (Møller & de Lope, 1994). This would ultimately have repercussions on chick physical condition.
For first clutches, the interaction between throat and ventral plumage colouration on hatching success showed this to be higher for pairs whose members had brighter coloured plumage patches (both throat and ventral). Evidence for a “compensation mechanism” has been found in this European barn swallow population, which would help explain the persistence of relatively high variance in the phenotypic expression of secondary sexual characters that are part of potential multi-component signalling systems (Vaquero-Alba *et al.*, unpublished data; 3). According to this evidence, individuals with lower investment in one or more ornaments involved in sexual signalling should be selected to seek and defend breeding sites associated with foraging sites richer in large insects (the most profitable prey for barn swallows; Turner, 2006). If hatching success increases with large insect abundance, this mechanism might help explain, at least partly, why the highest hatching success was observed for individuals with brighter plumage.
Actually, large insect abundance around breeding sites, in interaction with other variables, explained some of the reproductive success indicators we measured. During the first clutch, chicks in the best physical condition were raised by brighter parents nesting in sites surrounded by areas richer in large insects. Likewise, (originally) brighter parents also raised chicks in better condition in the second clutch, but this time no difference was detected between sites associated with foraging areas with lower or higher large insect abundance. Lower-quality individuals, in terms of ventral plumage colouration, seemed to compensate by feeding their chicks at a higher rate in the first clutch, but only in sites where a greater abundance of large insects permitted it. Paradoxically, in the second clutch, amongst darker individuals, those birds foraging in areas poorer in large insects were the ones that had chicks in better condition. These results, contrary to our initial prediction, suggest that parents with a higher investment
in ventral colouration provide less parental care than those with lower investment, as it has been reported in previous studies for males in relation to tail streamer length (de Lope & Møller, 1993; Møller et al., 1998a). It remains to be tested whether plumage darkness, even as a less costly signal (at least to maintain) than elongated tail streamers, is imposing any kind of limit on foraging efficiency, which would help explain our observations. The condition of chicks in second clutches was also influenced by tail streamer length in interaction with large insect abundance. Counterintuitively, chicks in better condition were raised by individuals nesting next to areas with fewer large insects, especially those with longer tail streamers. Whether tail streamer length exaggeration is mainly due to sexual selection (Møller, 1994) or only partly (Evans, 1998), higher quality individuals in terms of tail streamer length should raise chicks in better physical condition due to their heritable quality (Zahavi & Zahavi, 1997). Furthermore, the “compensation mechanism” mentioned above would help explain the presence of higher-indicator/investing swallows in sites associated to lower large insect abundance (Vaquero-Alba et al., unpublished data; 3).
Also, during the second clutch, when analysing the interaction between ventral plumage brightness and large insect abundance on clutch size, we observed that larger clutches were recorded for brighter individuals, especially those nesting in sites associated with areas richer in large insects. Thus, females with lower investment in ventral plumage colouration and/or mated to lower-investing males might be selected to allocate more resources to laying a larger amount of eggs, perhaps to compensate for their lack of fitness compared to darker individuals. This higher investment in producing more eggs, however, was not reflected in hatching success, which was higher for brighter individuals nesting in sites close to areas with lower large insect abundance, and for darker individuals nesting in sites associated with areas richer in large insects.
Hunt (1973) reported decreased hatching success, as well as an eggshell-thinning, for eggs in unusually large clutches in western gulls. Females of a lower quality and/or mated to lower quality males for the character considered (i.e. with brighter ventral plumage) that invested more in egg production, may have experienced a cost in terms of lower hatching success. This, however, would not explain the increased hatching success for brighter individuals in areas less abundant in large insects. As we are studying a multi-component signalling system, it would not be surprising if these individuals were actually of better quality than is apparent from this character alone, due to more investment in alternative ornaments. When considering the interaction between throat plumage colouration and large insect abundance on hatching success for the second clutch, higher hatching success was found for brighter-throated individuals nesting in sites associated with areas richer in large insects, but also, to a lesser extent, brighter ones nesting next to areas poor in large insect abundance. These results might be due, again, to these individuals being of better quality than the analysis of this single interaction reveals, as other interactions analysed clearly suggest a positive influence of exaggerated sexual ornaments on reproductive success.
In accordance with previous studies, we found, in most cases, that pairs in which male tail streamers were artificially lengthened had higher and less variable hatching success than pairs in which males were assigned to any other experimental group. Evidence suggests that females can affect the quality of eggs by supplying them with carotenoids and other substances that can improve the development of the embryonic immune system (Turner, 2006), which would lead to an enhanced hatching success. Saino *et al.* (2002b) reported a higher transfer of antibodies to first-laid eggs from vaccinated barn swallow females when mated to artificially long-tail, i.e. more attractive-looking, males. In this study, males with experimentally reduced tail streamers were in pairs with higher
and less variable hatching success than control or unmanipulated males. Females barn swallows mated to experimentally short-tailed males transfer more lutein to the eggs than those mated to experimentally long-tailed ones, to compensate for the poor parasite resistance of the chicks of poor quality (shorter-tailed) males (Saino et al., 2002a). Due to luteins’ antioxidant properties, we would expect this transfer to increase hatching success.
Also in accordance with existing evidence, the results we observed for the effect of plumage manipulation on physical condition of chicks can be explained in terms of differential aerodynamic costs for males subject to tail streamer elongation or shortening and differential female reproductive effort according to the degree of ornamentation of their males. Males with experimentally elongated tails undergo an increased flight cost during aerial feeding. Thus, they capture a higher proportion of smaller prey items, which is energetically inefficient (Møller et al., 1995; Turner, 2006). Although it has been reported that females mated to experimentally longer-tailed males adjust their feeding behaviour in order to compensate for impaired male foraging ability (de Lope & Møller, 1993), our study suggests that this compensation may not be enough: chicks raised by such pairs were in significantly worse physical condition than those raised by control males, with or without plumage colour enhancement, or unmanipulated males, and their mates. Females mated to experimentally short-tailed males may also have adjusted their feeding behaviour to being with an apparently lower-quality (but better foraging) mate, by reducing their foraging effort. This would help explain why, despite males within these couples being able to capture more profitable prey items (Møller et al., 1995) their chicks are in poorer condition than those raised by control males, with or without plumage colour enhancement, or unmanipulated males, and their mates.
Fitze & Richner (2002) found a significant decrease in the size of the breast stripe of great tits experimentally infested with parasites compared to uninfested ones. They suggested that this trait serves as an honest signal of quality, as predicted by models of sexual selection. On the other hand, Møller (1991) found smaller second clutches for female barn swallows with mite-infested first clutch nests. So, it seems that female barn swallows can adjust their investment in egg production during the second clutch in order to maximise their reproductive success. Specifically, they should adjust it to the ability of both parents to feed their broods (Turner, 2006). Our results show significantly smaller clutch sizes for females mated to males with experimentally short tail streamers and enhanced ventral plumage colouration. If melanin-based ventral plumage colouration is an honest quality signal, we would predict males with experimentally enhanced ventral darkness to have a higher amount of parasites than expected from their external appearance. If females can assess the quality of their mates throughout the season (de Lope & Møller, 1993) and use this information to adjust their investment in clutch size, we would expect them to decrease the production of eggs in response to a “dishonest” male and/or one made to look unattractive. This is exactly what we observed. It is important to point out that only females mated to males in which with both of these characteristics decreased their egg production. We did not observe the same response in females mated to males with only one of these “weaknesses”. Thus, this observation highlights not only the honesty of ventral plumage colouration as a sexual signal, but also the multi-component nature of the sexual signalling system in the barn swallow. Clutch size was also significantly lower for females mated to control males than for females mated to males assigned to other experimental groups. As clutch size is not significantly lower for females mated to unmanipulated males, we could argue that the manipulation of the tail itself exerted some negative effect on their
bearers and prompted a decrease in investment by the females mated to them. But in that case, we should expect to see a similar response in other manipulation groups, and we did not. An alternative explanation might be that, despite the random assignment of males to experimental groups, those assigned to the control group may be, by chance, of a significantly lower quality than the ones assigned to other groups. Couples with control and ventral colouration-enhanced males had significantly lower hatching success than control or unmanipulated groups, perhaps due to the negative adjustment of female investment to a dishonest quality signal. They also had chicks in significantly better condition.
To sum up, throat and ventral plumage colouration, together with tail streamer length, seem to function as notable quality indicators in the multi-component signalling system of the European barn swallow. We provide evidence, for the first time in this subspecies, that both throat and vent plumage colour patches, in combination with tail length, influence reproductive success, in a similar way to that reported for the North American subspecies. Generally speaking, swallows with higher investment in darker plumage patches, alone or in interaction with tail streamer length or physical condition, seemed to exhibit higher reproductive success. Interactions with habitat quality (i.e. large insect abundance in foraging areas), however, seemed to somehow “reverse” these effects, suggesting the existence of a “compensation” mechanism through habitat selection by individuals with poorer phenotypes, as has been proposed recently. Finally, males with artificially lengthened tail streamers seemed to stimulate higher reproductive investment in the females mated to them, as expected, but plumage colour manipulations did not seem to have any notable effect in this system.
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Table 1 Summary of minimal adequate linear mixed-effect models
| Response variable | N | ΔAIC | Explanatory variable | $X^2(df)$ | p | z ph(df) | p ph |
|-------------------|-----|------|-------------------------------|-----------|------|----------|------|
| (a) First clutch | | | | | | | |
| Clutch size$^a\ddagger$ | 83 | 28.99| Sex | 0.04 (1) | 0.842| | |
| | | | Condition | 0.062 (1) | 0.803| | |
| | | | Tail length | 0.129 (1) | 0.72 | | |
| | | | Ventral bright | 0.211 (1) | 0.646| | |
| | | | Large insects | 0.46 (1) | 0.498| | |
| | | | Throat bright | 0.48 (1) | 0.489| | |
| Hatching success$^a$ | 83 | 10 | Sex | 0.139 (1) | 0.709| | |
| | | | Throat bright | 0.182 (1) | 0.670| | |
| | | | Ventral bright | 2.735(1) | 0.098| | |
| | | | Tail length | 0.389 (1) | 0.533| | |
| | | | Large insects | 32.91 (1) | <0.0001| | |
| | | | Sex (male):throat bright | 4.911 (1) | 0.027| | |
| | | | Ventral bright:throat bright | 9.450 (1) | 0.002| | |
| | | | Tail length:throat bright | 5.158 (1) | 0.023| | |
| Chick condition$^b$ | 86 | 18.5 | Large insects | 2.070 (1) | 0.150| | |
| | | | Ventral bright | 7.004 (1) | 0.008| | |
| | | | Ventral bright:large insects | 4.882 (1) | 0.027| | |
| (b) Second clutch (manipulations) | | | | | | | |
| Clutch size$^c\ddagger$ | 85 | 23.6 | Plumage manip | 21.396 (6)| 0.002| | |
| | | | RC-E | -2.034 (1)| 0.042| | |
| | | | RC-SC | 2.051 (1) | 0.040| | |
| | | | RC-U | 1.975 (1) | 0.048| | |
| | | | S-E | -3.478 (1)| 0.0005| | |
| | | | S-R | -3.170 (1)| 0.002| | |
| | | | S-SC | 3.337 (1) | 0.0008| | |
| | | | S-U | 3.938 (1) | <0.0001| | |
| | | | Ventral bright | 5.584 (1) | 0.018| | |
| | | | Large insects | 0.964 (1) | 0.326| | |
| | | | Ventral bright:large insects | 4.019 (1) | 0.045| | |
| Hatching success$^c$ | 65 | 10 | Plumage manip | 28.293 (6)| <0.0001| | |
| | | | E-R | -4.009 (1)| <0.0001| | |
| | | | E-RC | -2.535 (1)| 0.011| | |
| | | | E-S | -4.997 (1)| <0.0001| | |
| | | | E-SC | -3.166 (1)| 0.002| | |
| | | | E-U | -6.465 (1)| <0.0001| | |
| | | | R-S | -2.574 (1)| 0.010| | |
| | | | R-U | -2.603 (1)| 0.009| | |
| | | | SC-S | 2.509 (1) | 0.012| | |
| | | | SC-U | -2.210 (1)| 0.027| | |
| | | | Throat bright | 4.654 (1) | 0.031| | |
| | | | Ventral bright | 18.204 (1)| <0.0001| | |
| | | | Tail length | 2.052 (1) | 0.152| | |
| | | | Large insects | 22.176 (1)| <0.0001| | |
| | | | Throat bright:large insects | 15.054 (1)| <0.0001| | |
| | | | Ventral bright:large insects | 8.971 (1) | 0.003| | |
| | | | Throat bright:tail length | 15.332 (1)| <0.0001| | |
| Chick condition | 50 | 7.7 | Plumage manip | 49,483 (5) | <0.0001 |
|-----------------|----|-----|---------------|------------|---------|
| E-S | | | | 8.644 (1) | <0.0001 |
| E-SC | | | | -3.646 (1) | 0.0003 |
| E-U | | | | 12.710(1) | <0.0001 |
| R-S | | | | 9.879 (1) | <0.0001 |
| R-SC | | | | -4.153 (1) | <0.0001 |
| R-U | | | | 22.588 (1) | <0.0001 |
| SC-S | | | | -11.469 (1)| <0.0001 |
| SC-U | | | | 19.893 (1) | <0.0001 |
| S-U | | | | -3.244 (1) | 0.001 |
| Sex | | | | 0.0004 (1) | 0.992 |
| Condition | | | | 0.173 (1) | 0.678 |
| Tail length | | | | 0.170 (1) | 0.68 |
| Throat bright | | | | 1.773 (1) | 0.183 |
| Ventral bright | | | | 0.655 (1) | 0.418 |
| Large insects | | | | 0.090 (1) | 0.764 |
| Sex (male):condition | | | | 8.1535 (1) | 0.004 |
| Sex (male):throat bright | | | | 11.486 (1) | 0.0007 |
| Sex (male):tail length | | | | 4.1402 (1) | 0.042 |
| Ventral bright:large insects | | | | 18.391 (1) | <0.0001 |
| Tail length:large insects | | | | 11.142 (1) | 0.0008 |
| Ventral bright:tail length | | | | 38.107 (1) | <0.0001 |
ΔAIC difference between AIC of maximal and minimal model, *condition* physical condition, *covariate*, *large insects* large insect abundance, *covariate*, *measureday* day of measurement, factor with 5 levels, *nest:site* nest location within each site, factor with 37 levels, *plumage manip* plumage manipulation, factor with 7 levels, *E* tail elongation, *EC* tail elongation+colour enhancement, *R* tail reduction, *RC* tail reduction+colour enhancement, *S* control, *SC* control+colour enhancement, *U* unmanipulated, sex factor with 2 levels, *site* geographic location of nesting site, factor with 12 levels, *tail length* tail length, *covariate*, *throat bright* throat brightness, *covariate*, *ventral bright* ventral brightness, *covariate*, *year* factor with 2 levels, *z ph z score* for each pairwise comparison after post-hoc analyses.
---
a Fixed effects and covariates included in maximal models: *sex*, *large insects*, *tail length*, *throat bright*, *ventral bright*, *condition*, *sex:tail length*, *sex:throat bright*, *sex:ventral bright*, *sex:condition*, *large insects:tail length*, *large insects:throat bright*, *large insects:ventral bright*, *large insects:condition*, *tail length:condition*, *throat bright:condition*, *ventral bright:condition*, *tail length:throat bright*, *tail length:ventral bright*, *ventral bright:throat bright*; random effects: *site/nest*, *year*.
b Fixed effects and covariates included in maximal models: *sex*, *large insects*, *tail length*, *throat bright*, *ventral bright*, *condition*, *sex:tail length*, *sex:throat bright*, *sex:ventral bright*, *sex:condition*, *large insects:tail length*, *large insects:throat bright*, *large insects:ventral bright*, *large insects:condition*, *tail length:condition*, *throat bright:condition*, *ventral bright:condition*, *tail length:throat bright*, *tail length:ventral bright*, *ventral bright:throat bright*; random effects: *site/pair*, *year*, *measureday*.
c Fixed effects and covariates included in maximal models: *sex*, *plumage manip*, *large insects*, *tail length*, *throat bright*, *ventral bright*, *condition*, *sex:tail length*, *sex:throat bright*, *sex:ventral bright*, *sex:condition*, *large insects:tail length*, *large insects:throat bright*, *large insects:ventral bright*, *large insects:condition*, *tail length:condition*, *throat bright:condition*, *ventral bright:condition*, *tail length:throat bright*, *tail length:ventral bright*, *ventral bright:throat bright*; random effects: *site/nest*, *year*.
d Fixed effects and covariates included in maximal models: *sex*, *plumage manip*, *large insects*, *tail length*, *throat bright*, *ventral bright*, *condition*, *sex:tail length*, *sex:throat bright*, *sex:ventral bright*, *sex:condition*, *large insects:tail length*, *large insects:throat bright*, *large insects:ventral bright*, *large insects:condition*, *tail length:condition*, *throat bright:condition*, *ventral bright:condition*, *tail length:throat bright*, *tail length:ventral bright*, *ventral bright:throat bright*; random effects: *site/nest*, *year*, *measureday*.
† Explanatory variables were standardized: stand(x)=x-mean(x))/sd(x)
‡ Response variable was gamma transformed
| Type of manipulation | Description |
|----------------------|--------------------------------------------------|
| E | Tail streamer elongation |
| EC | Tail streamer elongation + colour enhancement |
| R | Tail streamer reduction |
| RC | Tail streamer reduction + colour enhancement |
| S | Control |
| SC | Control + colour enhancement |
| U | Unmanipulated |
**Table 2:** Description of the levels in the “plumage manipulation” factor
**Figure 1:** Effect of throat brightness on hatching success
Figure 2: Effect of throat and ventral brightness on hatching success - 1st clutch (Model fit)
Figure 3: The effect of throat brightness and tail length on hatching success - 1st clutch (Model fit)
Figure 4: The effect of ventral brightness and large insect abundance on hatching success - $1^{st}$ clutch (Model fit)
Figure 5: The effect of ventral brightness and large insect abundance on clutch size - 2\textsuperscript{nd} clutch (Model fit)
Figure 6: Effect of plumage manipulation on clutch size (2nd clutch).
(For a description of levels of “plumage modification”, see Table 2)
Figure 7: The effect of throat brightness and large insect abundance on hatching success - $2^{nd}$ clutch (Model fit)
Figure 8: The effect of ventral brightness and large insect abundance on hatching success - 2\textsuperscript{nd} clutch (Model fit)
Figure 9: The effect of throat brightness and tail length on hatching success - $2^{\text{nd}}$ clutch (Model fit)
Figure 10: Effect of plumage manipulation on hatching success (2nd clutch).
(For a description of levels of “plumage modification”, see Table 2)
Figure 11: Effect of adult condition on chick physical condition (2\textsuperscript{nd} clutch)
Figure 12: Effect of throat brightness on chick physical condition (2nd clutch)
Figure 13: Effect of tail streamer length on chick physical condition (2nd clutch)
Figure 14: The effect of ventral brightness and large insect abundance on chick physical condition - 2nd clutch (Model fit)
Figure 15: The effect of tail length and large insect abundance on chick physical condition - 2nd clutch (Model fit)
Figure 16: The effect of ventral brightness and tail length on chicks’ physical condition - $2^{nd}$ clutch (Model fit)
Figure 17: Effect of plumage manipulation on chicks’ physical condition (2\textsuperscript{nd} clutch)
(For a description of levels of “plumage modification”, see Table 2)
Chapter Five
Predator inspection activity in Trinidadian guppies (Poecilia reticulata): drab males are bolder than conspicuous males in the presence of females
Iker Vaquero-Alba*, Claire Canning*, Alessandro Macario†, Darren P. Croft†, Matthew R. Evans* & Sasha R.X. Dall*
*Centre for Ecology and Conservation, University of Exeter, Cornwall Campus
Penryn, Cornwall, TR10 9EZ, UK
†Washington Singer Laboratories, School of Psychology, University of Exeter
Exeter, EX4 4QG, UK
Abstract
Although females prefer to mate with conspicuously coloured males in numerous species, mate choice experiments in Trinidadian guppies (*Poecilia reticulata*) have shown that females prefer bolder males irrespective of their colouration when given the opportunity to observe their behaviour toward a potential fish predator. Given that coevolutionary systems are often subject to non-equilibrium dynamics, traits involved in the multi-component signalling system of guppies may not be expressed at the maximum possible level, which could lead to unreliable intersexual communication through the phenotypically plastic predator inspection behaviour. Here, we test experimentally the hypothesis that visually less conspicuous male guppies may compensate their lower quality-indicating value by being bolder than more conspicuous males in front of females through increased predator inspection activity, i.e. willingness to risk approaching predators during inspection without being killed. In the process we show that less visually conspicuous males, which invest less in one or more ornamental colouration traits, were often bolder than more conspicuous males for those traits. We go on to discuss the implications of this for the evolution of sexual signalling systems.
Introduction
According to communication theory, animal signals, and more specifically body colouration, are used by “signallers” to increase their own fitness by providing information to affect “receiver” behaviour (Johnstone, 1997; Endler, 2000; Dale, 2006). Choosing elaborately ornamented individuals as sexual partners can provide animals with direct and indirect benefits, information about which is transmitted by inter-individual differences in the degree of exaggeration of the ornaments (Pomiankowski, 1987; Andersson, 1994; Garamsziegi et al., 2006; Bro-Jorgensen et al., 2007). Models of sexual selection predict that these ornamental characters will be costly at evolutionary stability (Fisher, 1930; Zahavi, 1975; Iwasa et al., 1991). This cost may be expressed during production and/or maintenance, and also through increased risk of predation or parasitism (Magnhagen, 1991; Folstad & Karter, 1992, Houde, 1997). Costly signals are efficient quality-indicators, as the costs they inflict to their bearers make them likely to be general viability indicators, in accordance with the handicap principle (Zahavi, 1975).
Despite the costs associated with production and reception of signals, animals often engage in multiple signalling systems. For example, the male wolf spider (*Schizocosa stridulans*) produces different components of its complex sexual display by using its abdomen and pedipalp simultaneously (Elias, 2006). Female crickets *Gryllus campestris* consider male call frequency as well as chirp rate to assess a mate, as both features signal different aspects of male quality (Scheuber et al., 2004). And in the European barn swallow *Hirundo rustica rustica*, several ornaments related to reproductive success, such as tail streamer length, white spots in the tail and throat or ventral colouration, have been found to transmit information about quality of males and, perhaps, females (Møller, 1993, 1994; Kose et al., 1999; Amundsen, 2000; Ninni et al.,
Bro-Jørgensen (2009) argues that variation in selection pressures derived from fluctuations in ecological conditions and/or social context might lead to the emergence and maintenance of such multicomponent signalling systems.
Trinidadian guppies (*Poecilia reticulata*) provide an excellent example of multidimensional variation in sexually selected traits (Blows et al., 2003). They show complex colour patches based on carotenoids, pteridines and melanins (Houde, 1997) as well as several behavioural characters are involved in sexual selection. Thus, males exhibit a courtship display called the “sigmoid” display (Baerends et al, 1955; Liley, 1966; Houde, 1997), named that way because of the S-shape of the male’s body when performing the display in front of the female. Another behavioural quality-indicating signal is “boldness”, which is the willingness to risk approaching predators during inspection without being killed (Dugatkin & Godin, 1992; Wilson *et al.*, 1994; Godin & Davies, 1995).
Several experimental studies on guppy populations have found evidence of a mating advantage for more conspicuous colour patterns in males (Haskins *et al.*, 1961, Endler 1980, 1983). To avoid the confounding effects of predation, which may favour more cryptic patterns, Endler (1980) conducted a long term study in artificial streamers free from predators. He observed that males evolved increased conspicuousness in their colour patterns through a mismatch with the gravel background colouration. Carotenoid-related colours (red-orange-yellow) seemed to be the ones for which females showed a higher and more consistent preference. Female guppies preferred either males with bigger total body areas occupied by these pigments (Kodric-Brown, 1985; Houde, 1987; Brooks & Caithness, 1995a) or those with brighter orange spots (Kodric-Brown, 1989; Houde & Torio, 1992). Other studies have also tested other
colour pattern characters, such as the area of structural-iridescent or black spots, but the results have not shown much consistency (see Houde, 1997) or vary among populations (Endler & Houde, 1995). Melanin-based black colouration in male guppies has been suggested to interact with orange colouration to determine mate choice, as a redundant signal (Brooks and Caithness, 1995a), or as an amplifier trait to improve detectability of orange spots (Brooks & Caithness, 1995b; Brooks, 1996). Another kind of quality-indicating secondary sexual character of males that could have an effect on female choice is the courtship display itself. Nicoletto (1993) found a positive correlation between the frequency of male displays and the frequency of female sexual responses. Stoner and Breden (1988) found that female guppies preferentially associated with the more frequently courting males, and Farr (1980) observed that males with higher rates of display obtained a disproportionately higher number of matings than males with lower rates of display.
Predation greatly influences different aspects of guppy biology. Expression of male colour patterns and behavioural characters such as sexual displays, as well as mate choice by females, seem to be determined to a great extent by the costs imposed by predation (Houde, 1997). Even sperm storage in females has been suggested to vary according to predation regime (Magurran, 2005). When they detect a predator, guppies exhibit inspection behaviour, which is thought to function as a way of recognizing and assessing the dangerousness of the predator (Magurran & Seghers, 1990; Dugatkin and Godin, 1992). Bolder males, i.e. those more willing to risk approaching predators during inspection without being killed, are better informed about predators near them, so they may have better chances of surviving encounters with them (Godin & Dugatkin, 1996). In a mate choice experiment in which male guppies were exposed to a cichlid fish predator, either live or a model of it, in the presence or absence of females, Godin and
Dugatkin (1996) demonstrated a positive correlation between the visual conspicuousness of the colour pattern of male guppies and their boldness toward the predator. Additionally, they found that although females prefer the most colourful males as mates, they show a stronger preference for bolder males irrespective of their colouration when they are given the opportunity to observe their inspection behaviour. Cichlid fish predators preferentially and consistently attack brightly coloured males rather than drabber ones (Godin & McDonough, 2003). Furthermore, being bolder toward a predator implies a willingness to incur greater costs in terms of predation risk and lost mating opportunities (Godin & Dugatkin, 1996). Thus, both characters have viability costs associated with them, which reinforces their reliability as indicator traits of male quality.
Predation regimes and other environmental and social factors are highly variable among different streams inhabited by guppies, which leads to an extreme degree of geographic variation in several characters (Endler, 1995; Houde, 1997). These differences result in dynamic selection pressures, which can explain the coexistence of multiple signals in guppy populations (Bro-Jørgensen, 2009). As coevolutionary systems are subject to non-equilibrium dynamics, traits in such systems may not be expressed at their maximum possible levels (see Saino et al., 2003), leaving room for phenotypic plasticity in response to fluctuations in ecological and social environments. We hypothesize that in locations with lower risks of predation, less visually conspicuous males may be selected to increase their boldness at a low cost, in order to balance out their mating disadvantage due to lack of quality. This “compensation mechanism”, which has previously been proposed for barn swallows (Vaquero-Alba et al., unpublished data; 3, 4), would help explain, at least in part, the high phenotypic variability observed in natural guppy populations. It would also contribute to
understanding the persistence, in these populations, of males with lower colouration expression intensities, despite suffering a selective disadvantage due to their poorer performance during mate choice.
In the present study, we investigate the relationship between boldness and visual conspicuousness of male colour patterns of the Trinidadian guppy *Poecilia reticulata* during exposure to three model predators of varying dangerousness and in absence of predators. We predict higher boldness by less visually conspicuous males, as a way of compensating their lower intrinsic attractiveness to females, especially when exposed to models mimicking less dangerous predators when this compensation can be carried out at a lower cost.
**Materials and methods**
Trinidadian guppies are small fish belonging to the cyprinodont family Poeciliidae. They are native to streams and rivers of Trinidad and Tobago and adjacent parts of South America, they have internal fertilization and give birth to live young (Houde, 1997). Male and female guppies used in our experiment were first generation, laboratory-born descendants of wild guppies collected from the Aripo River, Trinidad, West Indies. All females were raised separately from males and were thus virgins when tested, as virgin female guppies are highly receptive to male courtship displays (Liley, 1968). Experimental males were allowed to court and mate with other females (not used in this study) in their home aquaria prior to being used in the current study. Fish holding aquaria for male guppies consisted of individual plastic tanks (20cm x 10cm, with a water level of 12 cm), each housing a single individual taken from a larger stock tank. Tanks contained a plastic plant and gravel substratum and were maintained at 23-25°C under a 12 hour light / 12 hour dark illumination cycle. Females were kept in a large
stock tank (100cm X 36cm, with a water level of 33cm) and under the same lighting and temperature conditions as males. Guppies were fed flake food in the morning, at least one hour before experiments began, and were given live brine shrimp nauplii in the evening. No food was available to the fish during experimental trials.
**Male visual conspicuousness assessment**
Fifteen bright and fifteen drab male guppies were selected based on the conspicuousness of their colouration. Each male was photographed on its left side using a Canon EOS digital SLR camera. Photographs were uploaded and analysed using UTHSCSA ImageTool software to calculate the areas of different colouration patches: orange, yellow, black, white-silver, blue-violet and bronze-green, on the body of each male. The total area occupied by all the colouration patches on the left side was calculated as a proportion of the total left-side surface area. This proportion was then used to categorize males into “conspicuous” and “drab”. Males with a proportion of less than 20% of their total left-side surface area occupied by colouration patches were allocated as drab, and males of 20% and above were considered as conspicuous. The highest recorded colouration cover was 34.79% and the lowest was 10%. Once being categorized as either visually “conspicuous” or visually “drab”, males were assigned into pairs with the most conspicuous males being paired with the least drab males, in order to keep the percentage difference as constant as possible. The average difference in conspicuousness was 14.01%.
**Experimental trials**
Experimental trials were carried out in a tank 120 cm long and 36 cm wide, with a water level of 15 cm and gravel substratum. It was divided into 3 compartments: a small
compartment (15cm in length) that housed the predator model, separated from the rest of the tank by a permanent clear Perspex partition, allowing the model predator to be visible to both male and female guppies throughout experiments. Another small compartment (30 cm in length) at the opposite end of the tank contained the two male guppies during the acclimation period; throughout this time an opaque partition blocked their view from the predator compartment. Two virgin female guppies were present in each experiment and were separated from males in a clear plastic cylinder (10.5 cm in diameter, 32.97 cm in circumference). The cylinder was perforated, so both males and females were able to respond to visual and chemical cues. Females were restricted from free-swimming in the test tank to minimise distraction of the males and to prevent sneak mating attempts. The large section between the predator and acclimatisation compartments was further divided into the risky zone, and safe zones 1 and 2. The risky zone was measured as 15cm from the edge of the predator compartment (approximately 6 guppy body lengths); safe zone 1 was the area between the end of the risk zone and the female guppy cylinder (40cm); safe zone 2 was the area between the female guppy cylinder and the opaque partition (20cm), the farthest from the predator compartment and therefore the least “risky” zone. The risky zone was further divided into 6 smaller zones, according to their risk level due to their proximity to the predator chamber. Three model predators of varying perceived dangerousness were used in our experiment: the high-intensity predator was represented by a model of the pike cichlid fish *Crenicichla alta*, the medium-intensity predator by a model of the blue acara fish *Aequidens pulcher*, and the low-intensity one by a model of the rivulus fish *Rivulus hartii*. All these species are all known natural guppy predators in the Aripo River region (Reznick & Endler, 1982; Reznick *et al.*, 2001). Before each trial, a pair of males was put in the compartment at the opposite end of the predator compartment, and males were allowed
to acclimatise for a period of 30 minutes. Following this period, the opaque partition was raised slowly, allowing the male guppies to view the females in the cylinder and the predator compartment that contained the model predator. Trials began as soon as both males left the acclimatisation zone and lasted for 30 minutes. Once in the main test chamber, each individual male guppy had the choice of either initiating an inspection visit (the riskiest option), following the other male who initiated the approach, or not inspecting at all. Fifteen pairs of males were similarly tested for each of the three aforementioned predator treatments separately. Different females were used for each trial. Therefore, individual fish were only used once in each experiment. Each trial was recorded using a small video camera attached to a tripod and visual observations were made from behind a white blind through a small opening, to minimize any behavioural disruption. Males were randomly allocated to different predator treatments. The order in which males were allocated to different model predators, as well as the time at which each trial was carried out were recorded.
*Shyness score* (named that way because its value increased with increasing distance from the predator) was calculated by allocating each male a score every 30 seconds depending on his position in the test chamber: they were allocated a score of 15 in safe zone 2, a score of 10 in safe zone 1, and a score of 1 (closer to the predator) to 6 (farther from the predator) in risky zone. Therefore, bolder males acquired lower shyness scores. *Time in risky zone* was calculated as the amount of time each male spent in the risky zone in 30 minutes. *Number of first inspections* was calculated as the total number of predators inspections (swimming from safe zones to the risky zone) initiated and led by each male during each trial.
Statistical analyses
Multicollinearity is an interdependency condition that can be observed in a multiple regression in which several linear relationships hold between variables. It can exist independent from any relationships between the response variable and the explanatory variables, and may make the coefficient estimates change erratically in response to small changes in the model or the data (Frisch, 1934; Farrar & Grauber, 1967). As several of our explanatory variables and interactions were related to areas of different body colouration patches of male guppies, we checked them for multicollinearity, using the vif.mer function of mer-utils.R online repository (Frank, 2011). Parameters with values of vif.mer under 5 are considered to suffer from acceptably levels of multicollinearity.
We analysed the variables influencing several indicators of boldness in male guppies, using generalized linear mixed effects models (GLMM; details explained below) implemented with the lmer function of lme4 package (Bates & Sarkar, 2007) of R 2.13.0 (Crawley, 2007; R Development Core Team, 2010). Using GLMMs, it is possible to analyse the influences of both random and fixed effects on response variables (Calabuig et al., 2010).
Factors affecting shyness score and time in risky zone were analysed using a Gaussian error structure and an identity link function. Factors affecting the number of first inspections were analysed using a Poisson error structure and a log link function. For all the analyses, body length and areas of carotenoid-based (orange+yellow), black, and structural (white-silver+blue-violet+bronze-green) colouration patches, were included as covariates, and predator type was included as a fixed factor. “Pair identity” was included as a random term in our models. In doing so, we controlled for the nonindependence of the data points that occurred because individuals within a pair
could have influenced each others’ behaviour. Also, time when each trial took place and order of allocation of pairs to different model predator treatments were included as random effects, to control for the potential confounding effects of these variables.
GLMMs were initially fitted with all explanatory terms included in them, including first-order interactions in which body length and/or colouration ornaments were involved. A summary of the maximal models fitted is given in Table 1. Minimal adequate models were chosen after a process of stepwise deletion of the least significant effects (p>0.05), starting with the highest-order interactions, providing the simplification did not significantly reduce the explanatory power of the model (Crawley, 2002). Consecutive models were compared using chi-square ($\chi^2$) statistics. The residuals from the models were checked for normality and homocedasticity; where appropriate, transformations of the response variables or standardizations of the explanatory variables were done following R Development Core Team (2010).
**Results**
After standardizing the explanatory variables of our models, carotenoid colouration area and structural colouration area yielded *vif* values slightly higher than 5, although still several orders of magnitude lower than the same variables before standardization. The rest of the single variables and 2-way interactions included in the analyses had *vif* values under 5, and most of the times under 4.
Our analyses showed a significant 2-way interaction between body length and carotenoid-related colouration area on shyness score (n=90, $\chi^2_1=5.549$, p=0.018; Table 1). Male guppies with smaller areas of carotenoid-based colouration showed lower shyness scores than those with bigger areas. Amongst males with bigger carotenoid colouration areas, smaller males had lower shyness scores than those with a longer body
(Figure 1). There was also a significant 2-way interaction between area of carotenoid-based colouration and black coloured area on shyness score (n=90, $\chi^2_1=4.855$, p=0.030; Table 1). Males with both bigger carotenoid-based colouration areas and bigger black colouration spots showed lower shyness scores than the rest. Amongst males with smaller black colouration patches, those with bigger carotenoid-based colouration areas had slightly lower shyness scores than the ones with smaller carotenoid colouration areas (Figure 2).
When analysing the number of first inspections by males, we saw a significant effect of male black colouration area on the response variable (n=90, $\chi^2_1=8.655$, p=0.003; Table 1). Male guppies with smaller black colouration areas initiated significantly more predator inspections than those with bigger areas (Figure 3). There was a significant effect of male body length on the number of predator inspections initiated and led by males (n=90, $\chi^2_1=19.562$, p<0.0001; Table 1). Bigger males initiated slightly more predator inspections than smaller sized males, being this effect significant (Figure 4). We observed a significant covariation between the size of carotenoid-based colouration area and the number of first predator inspections (n=90, $\chi^2_1=34.233$, p<0.0001; Table 1). Males with smaller carotenoid colouration areas initiated significantly more predator inspections than those with bigger areas. (Figure 5).
Our analyses also revealed a significant effect of melanin-related (black) colouration area size on time spent in the risky zone (n=90, $\chi^2_1=5.328$, p=0.021; Table 1). Male guppies with smaller areas of black colouration spent significantly more time in the risky area than those with bigger black areas (Figure 6).
We did not detect any effect of predator type on either shyness score (n=90, $\chi^2_3=0.101$, p=0.951; Table 1), number of first inspections (n=90, $\chi^2_3=1.006$, p=0.605; Table 1) or time spent in risky zone (n=90, $\chi^2_3=0.790$, p=0.674; Table 1).
Discussion
The results of our study suggest the existence, at least to a certain extent, of a compensatory strategy through predator inspection in males with poorer indicator traits, as we predicted. Male guppies with smaller areas of black colouration spent more time in the risky zone, closer to the predator chamber, and males with smaller body size or smaller areas of carotenoid-related colouration got lower shyness scores, than bigger and/or more coloured males. Lower shyness scores and longer times in risky zone reflect bolder males. Also, males with either smaller areas of carotenoid-based colouration, black colouration, or both, initiated and led a much larger number of predator inspections than those with bigger areas. All this is indicative of a higher boldness, and a willingness to incur greater costs, in terms of predation risk and lost mating opportunities (Godin & Dugatkin, 1996). Male guppies with more carotenoid colouration, as well as more of other types of colour, are preferred by females as sexual partners (Kodric-Brown, 1985; Houde, 1997). Likewise, females prefer bigger males in certain populations (Reynolds & Gross, 1992). Thus, male guppies with a lower intensity of expression for one or more of these characters should be selected to increase their chances of being chosen by females by increasing their predator inspection activity. This trend somehow reversed when analysing specific interactions. Males with both bigger areas of carotenoid-related and black colouration showed lower shyness scores, whereas bigger-sized males initiated more predator inspections. According to these results, males with a higher total investment in simultaneously exaggerating certain, expensive sexual traits, may still afford to show a bolder behaviour in front of females, suggesting a higher individual quality. When considering these sexual signals separately or in different interactions, however, there seems to be a trade-off between the resources allocated to exaggerating the expression of ornamental traits and those left
for engaging in a bolder behaviour. Alternatively, and more plausibly, the costs of the alternative ways of signalling may vary, allowing only males with less costly combinations of simultaneously exaggerated traits to engage also in predator inspection. A greater conspicuousness means greater conspicuousness to predators (Godin & McDonough, 2003), and good physical condition is required to successfully escape from predator attacks. Thus, only lower-quality males that did not make a heavy investment in one or more colouration-related traits may be selected to maximise the phenotypically plastic predator inspection behaviour, in an attempt to increase their chances of being chosen by females and maximise their reproductive success.
Our study complements that of Godin & Dugatkin (1996), and reinforces the importance of predator inspector behaviour as a sexual signal. They concluded that visual conspicuousness of the colour pattern of males correlates positively with boldness toward a cichlid fish predator. Our results, however, show that this is not necessarily always the case, and boldness can actually serve as a compensatory strategy for less visually attractive males, as well as a redundant sexual signal for more attractive ones. They also highlight the geographic variability in the expression of different traits used by guppies for signalling purposes (Endler, 1995; Houde, 1997), as the fish used by Godin & Dugatkin came from wild guppies collected from Quará River, rather than Aripo River, from which the parental generation of the ones we used came.
Several authors have reported that more conspicuously coloured males are higher quality (Endler, 1980; Nicoletto, 1991, 1993). If this was always the case, we would expect more conspicuous males to signal through predator inspection at a higher rate than less conspicuous ones. However, in our study, this trend was only found for specific interactions between colouration traits, whereas most of the results suggest higher predator inspection activity by less conspicuously coloured males. Female choice
seems to be based on multiple criteria, in guppies (Kodric-Brown, 1993) and many other species (e.g. Kodric-Brown, 1990; Möller, 1994; Elias et al., 2006; Vaquero-Alba et al., unpublished data; 3, 4). The relative importance of each of the traits involved in sexual signalling changes with social and environmental factors, and is determined by the behavioural context (Kodric-Brown, 1993). Contrary to the static point of view of earlier research, the latest studies on multiple-signalling species are helping us understand how multi-component signalling systems, including both redundant or non-redundant signals, can be explained by environmental fluctuations (including both ecological conditions and social contexts; Bro-Jørgensen, 2009). Non-equilibrium dynamics to which signaller-receiver systems are often subject can lead to unreliable communication, at least when considering the quality-indicating value of certain ornamental colouration signals alone, and highlight the importance of more condition-dependent, phenotypically plastic traits (like carotenoid-related colouration and predator inspection behaviour) for coping with dynamic selection pressures. Thus, only higher quality males may be able to maximise both visual conspicuousness and boldness, and counter the predation costs associated with their greater attractiveness to predators, provided they are better at escaping from predators due to their better physical condition (Godin & Dugatkin, 1996). Considering ornamental traits alone or in particular trait combinations, however, only males with lower investment levels in one or more of those traits may show an enhanced boldness. This is actually what we saw in our study.
Contrary to our initial prediction, predator type did not have any effect on predator inspection behaviour, and males were not bolder or more timid in front of predators with varying levels of dangerousness. As the males we used for our experiment had never experienced a real predator before, the only behavioural differences we should expect are those with a genetic basis, shaped by natural selection, rather than those due
to learning and experience. Furthermore, as we used model predators instead of real ones, male guppies did not have the possibility of experiencing different behaviours from the different predators themselves, and react accordingly. Further experimentation with live predators is required, to test whether there is actually a different response of male guppies, in terms of boldness, towards predators with different levels of dangerousness.
To sum up, only male guppies with a high degree of exaggeration in the expression of certain traits or combinations of traits (like body size or carotenoid-based and melanin-based colouration areas) could afford to show also a high degree of boldness through an increased predator inspection activity, presumably due to their higher quality and behavioural vigour. However, when analysing quality-indicating traits alone or in other trait combinations only less visually conspicuous males with lower investments in one or more colouration-related, ornamental traits, seemed to be able and/or willing to compensate their lower attractiveness to females by increasing their levels of predator inspection.
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Table 1 Summary of minimal adequate models
| Response variable | N | ΔAIC | Explanatory variable | $X^2(df)$ | p |
|-------------------|-----|------|-------------------------------|-----------|------|
| Shyness score† | 90 | 22 | Body length | 2.483 (1) | 0.115|
| | | | Carotenoid | 0.1367 (1)| 0.712|
| | | | Black | 1.917 (1) | 0.166|
| | | | Predator | 0.101 (2) | 0.9507|
| | | | Body length:carotenoid | 5.549 (1) | 0.018|
| | | | Carotenoid:black | 4.855 (1) | 0.030|
| Number of first inspections‡ | 90 | 14.8 | Body length | 19.562 (1)| <0.0001|
| | | | Carotenoid | 34.233 (1)| <0.0001|
| | | | Black | 8.655 (1) | 0.003|
| | | | Predator | 1.006 (2) | 0.605|
| Time in risky zone†‡ | 90 | 18.7 | Black | 5.328 (1) | 0.021|
| | | | Predator | 0.790 (2) | 0.674|
ΔAIC difference between AIC of maximal and minimal model, black area of melanin colouration (black), covariate, body length, covariate, carotenoid area of carotenoid colouration (orange+yellow), covariate, pair id pair identity, factor with 15 levels, predator predator treatment, factor with 3 levels, structural area of structural colouration (white-silver+blue-violet+bronze-green), covariate, timeday time of day, factor with 58 levels, trial order order of allocation of pairs to predator treatments, factor with 6 levels.
Fixed effects and covariates included in maximal models: body length, carotenoid, black, structural, predator, body length:carotenoid, body length:black, body length:structural, body length:predator, carotenoid:predator, black:predator, structural:predator, carotenoid:black, carotenoid:struct, black:struct, random effects: pair id, timeday, trial order.
† Explanatory variables were standardized: stand(x)=x-mean(x))/sd(x)
‡ Response variable was tangent transformed
Figure 1: The effect of body length and carotenoid-based colouration area on shyness score (model fit)
Figure 2: The effect of black colouration area and carotenoid colouration area on shyness score (model fit)
Figure 3: Effect of male black colouration area on number of first inspections
Figure 4: Effect of male body length on number of first inspections
Figure 5: Effect of male carotenoid colouration area on number of first inspections
Figure 6: Effect of male black colouration area on time spent in risky zone
Chapter Six
General discussion
In the introduction (I), the conditions that might lead to the emergence and maintenance of multicomponent signalling systems in animals were reviewed, and the evolutionary consequences of dynamic variation in selection pressures and/or non-equilibrium dynamics in sexually antagonistic coevolution were discussed. Through the testing of the so-called “compensation mechanism” hypothesis, the experimental work presented in this thesis explores the possibility of multicomponent sexual signalling systems used by animals being selected to optimise rather than maximise the amount of information they transmit in relation to the costs they incur.
The objective colour assessment conducted in the field and in the lab and the repeatability calculations for each technique separately and between the techniques indicated a higher reliability for the lab option, consisting in collecting feather samples from live birds and assessing their colouration in the lab, after arranging them in a way that mimics the original plumage pattern. As this technique considerably reduces the handling time of the birds, minimising the stress levels inflicted on them, as well as being easier on equipment, it should be the common way of assessing colour objectively and doing repeatability calculations.
We demonstrated the value of the novel GLMM-based method for repeatability calculation (Nakagawa & Schielzeth, 2010). Thanks to the possibility of accounting for random factors, it allows to calculate adjusted repeatabilities, more accurate than those calculated with other methods, e.g. ANOVA-based, increasing the power of the tests.
Pigment-based plumage markings as quality indicators
Using European barn swallows as field models of multicomponent signalling species, we investigated the effect that several ornamental traits potentially used with sexual signalling purposes exert on a number of well-known reproductive success indicators (Vaquero-Alba et al., unpublished data; 3, 4). The ornamental traits studied included ventral plumage colouration, a character largely overlooked in previous sexual selection studies for this subspecies, and a specific consideration was given to interactions between the variables studied. The results of our study suggest that throat and ventral colouration patches, as well as tail streamer length, may play an important role in the sexual signalling system of the European barn swallow. Swallows with higher investment in darker plumage patches, either alone or in interaction with tail streamer length or physical condition, performed generally better according to the values of direct indicators of reproductive success (Vaquero-Alba et al., unpublished data; 4), and females mated to darker-throated males laid significantly sooner during the breeding season, an indirect indicator of fitness (Vaquero-Alba et al., unpublished data; 3). Remarkably enough, elongated tail streamers seemed to have quite a weak effect in predicting reproductive success in our population compared to pigment-based plumage markings, and contrary to previous similar studies in the European barn swallow (reviewed in Møller, 1994; Turner, 2006). Actually, tail streamer length did not predict patterns of assortative mating nor laying date, although it determined chick condition in second clutch to a great extent. A possible explanation could be that the barn swallows in the population we studied have suffered an evolutionary process different from the other European populations studied, as a consequence of the adaptation to local ecological and/or social conditions. For example, intensification of farming practices along the continent, with livestock numbers decreasing and hedgerows and waterbodies
being scarcer than in the past, may be associated with reduced invertebrate numbers (Evans, 2001; Møller, 2001, Vickery et al., 2001). Such changes in ecological conditions may differentially affect barn swallow populations, according to the extent of the habitat manipulation. However, whether this is having an influence in the sexually selected signals used by this species, it may be too soon to make inferences about it, given that the biggest changes in farming activity have been happening essentially for the last few decades. In any case, the findings of our study indicate that dynamic variation in selection pressures (Bro-Jorgensen, 2009) may be shaping the evolution of sexually selected multiple signals in the European barn swallows, perhaps leading to speciation.
It may be difficult to understand how female choice can evolve in species with no apparent direct benefits of choice to females, as it seems to happen in the species we studied. The most extreme case is lekking species, where in spite of males giving no resources to females or parental care to offspring, female preference has evolved and maintained. A possible explanation could be that females receive “good genes” for their offspring from preferred males. But under this assumption, we would expect genetic variance to be depleted by directional selection induced by female choice, as the lek paradox states (Taylor & Williams, 1982; Andersson, 1994; Rowe & Houle, 1996), and multicomponent signalling systems would hardly ever evolve. Rowe and Houle (1996) argue that sexually selected traits may favour genetic variance, given two premises: expression in sexually selected traits being condition dependent, and condition having high genetic variance. Both of which are well supported (see Houle, 1991; Andersson, 1994). This would help resolve the lek paradox. Alternatively, Pomiankowski & Møller (1995), suggest that under directional selection, greater additive genetic variance can be maintained if exaggeration of sexually selected traits increases fitness at a greater than
linear rate. Through their model, they invoke what they call “modifier loci”, arguing that directional selection increases the number of loci that affect the sexually selected trait or increases each locus’ contribution to it. Under certain circumstances, our “compensation mechanism”, largely based on Bro-Jørgensen’s “fluctuating environments” hypothesis (2009) and van Dorn’s “sexually antagonistic coevolution” one (2006) could also explain how multiple signals could emerge. Our hypothesis works especially well, we think, for the case of monogamous species, where the number of available mates decreases with time, and obviously, the quality of the remaining, unchosen ones. Lower-quality individuals could be selected to develop a compensation strategy through phenotypically plastic traits as, at a certain point, that could make them become the most exaggeratedly-signalling ones (when only lower-quality individuals remain available).
Tail streamer manipulations affected reproductive success indicators quite much in the predicted direction. Ventral colour manipulation, however, did not have a remarkable effect on hatching success or chick condition (Vaquero-Alba et al., unpublished data; 4). Nevertheless, females mated to males with simultaneously artificially shortened tails and enhanced ventral colouration seemed to “penalize” their partners’ lack of “honesty” by laying smaller clutches. If this behaviour was due to these males having more parasites than expected from their external appearance, it could be argued that ventral colouration functions as an honest signal of quality through susceptibility to parasite infestation (see Fitze & Richner, 2002), but more consistent results are needed. Tail streamer manipulation has been extensively applied to the European barn swallow during the last couple of decades (e.g. Møller et al., 1995, Evans, 1998) and the technique is well developed and tested. It’s the first time to our
knowledge, however, that ventral colour manipulation technique is applied to this subspecies, although it has been used in the North American barn swallow, a different subspecies, before (e.g. Safran et al., 2005). Thus, the method may still need to be “finely tuned” to the particular idiosyncrasy of the European subspecies, so that more consistence evidence is gathered for quality-indicating value and female preference for darker plumage colouration, as other evidences gathered during our investigation suggest (Vaquero-Alba et al., unpublished data; 4).
**Compensating through habitat quality and predator inspection**
The findings of our investigations support the hypothesised “compensation mechanism”, at least to a considerable extent: barn swallows investing more heavily in “cheaper” ornaments (pigment-based plumage markings), or, for certain combinations of traits, showing some kind of “weakness” either in their sexual displaying intensity or physical condition, seem to be selected to defend breeding sites associated with higher-quality foraging areas (Vaquero-Alba *et al.*, unpublished data; 3). Likewise, brighter individuals breeding in sites next to higher quality areas achieved higher values for several reproductive success indicators (Vaquero-Alba *et al.*, unpublished data; 4). The fact that other equally bright individuals bred in sites surrounded by poorer quality areas and achieved a lower reproductive success may be indicative of differences in individual quality amongst brighter-coloured swallows. If animals are selected to optimise rather than maximise the information content of sexually transmitted signals (1), we may expect some swallows not to express their plumage colouration patches at the maximum possible level, perhaps because they are saving themselves for future breeding attempts. These individuals might be selected to put a higher premium on
foraging and territorial aggression, compensating to a certain extent their lack of signalled quality at a lower cost than using morphological characters, and keeping themselves in good condition.
Using Trinidadian guppies as lab models of multicomponent signalling species, we investigated the effect that several ornamental traits potentially used with sexual signalling purposes exert on various indicators of “boldness”, i.e. the willingness to risk approaching predators during inspection without being killed (Dugatkin & Godin, 1992; Wilson et al., 1994; Godin & Davies, 1995). Although previous studies had shown a bolder behaviour for more conspicuously ornamented males (Godin & Dugatkin, 1996), we present evidence that this is not necessarily always the case, and boldness can serve as a compensatory strategy for less visually attractive males, as well as a redundant sexual signal for more attractive ones (Vaquero-Alba et al., unpublished data; 5). Male guppies with a lower intensity of expression for one or more quality-indicating traits, or with exaggerated combinations of traits which are not very costly to express, should be selected to increase their chances of being chosen by females at a lower cost, by increasing their predator inspection activity. As for the case of the barn swallows, lower intensity in the display of certain ornamental traits in guppies could be due to those traits being costly to express, or to certain individuals decreasing their investment in signal exaggeration in order to save themselves for future mating attempts. These individuals might be selected to engage in a bolder behaviour, which would be a “cheap” way of compensating for the lack of displayed quality while keeping themselves in good condition for the future.
The study of multicomponent signalling systems: future prospects
Future repeatability calculations need to be performed using the GLMM-based method developed by Nakagawa & Schielzeth (2010), in order to improve their accuracy thanks to increased test power. Comparability between field and lab-based objective colour assessment techniques can be carried out for other bird species, especially those with other types of colouration, to consistently assess the reliability of the more convenient lab technique for objective colour assessment (Vaquero-Alba et al., unpublished data; 2).
Further work is needed to gather more empirical evidence for the “fluctuating environments” hypothesis (Bro-Jørgensen, 2009), the “sexually antagonistic coevolution” hypothesis (van Doorn & Weissing, 2006), and the “compensation mechanism” hypothesis(1; Vaquero-Alba et al., unpublished data; 3,4,5). Likewise, hypotheses that have tried to unravel the lek paradox (Rowe & Houle, 1991; Pomiankowski & Møller, 1995) need to be theoretically and empirically tested to increase the amount of evidence supporting them, and see how our predictions can fit into the new ideas about multiple signalling. Using the powerful computing capacity of the new GLMM based statistical tools, new analyses on sexually selected multicomponent signalling systems can be performed with a specific focus on the interactions between the traits studied (e.g. Garamszegi, 2006, Vaquero-Alba et al., unpublished data; 3,4,5), including multiple component systems which have been usually regarded as single traits (Scheuber, 2004; Elias et al., 2006). Additionally, existing work can be critically reviewed to find evidence for multiple cues providing additional information or serving as back-up signals, and sexual conflict favouring the evolution of multiple signalling (Candolin, 2003; Grether et al., 2004). Non-equilibrium
scenarios, triggered by sexual conflicts and favouring the coexistence of multiple sexual signals, need to be investigated in more depth (Holland & Rice, 1998; Arnqvist & Rowe, 2005), so that we can improve our understanding of the complex processes leading to the emergence and permanence of multiple signalling systems, as well as the intercorrelated selective pressures to which traits being part of these systems are subject and the interactions which arise between them.
We hope our work adds some interesting ideas and predictions to a topic which is currently under intense controversy, and suggests some interesting directions which future investigation could take, in order to promote a productive debate in an area of knowledge that we find fascinating and intellectually challenging.
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Virtual Chinese Literature: A Comparative Case Study of Online Poetry Communities*
Michel Hockx
ABSTRACT This article looks at the practices of communities that employ internet technologies in order to produce, distribute, consume and value Chinese poetry. The article is in three parts. The first part provides a brief general overview of the current state of research about the Chinese internet. I take issue with the dominant tendency of English-language research to focus almost exclusively on questions of censorship. The second part looks at the development of “web literature” (wangluo wenxue) in China, briefly outlining the meaning of the term and the content of a protracted debate about web literature that took place in 2001. The debate illustrates the limited extent to which web literature is able to distinguish itself from conventionally published literature. Paradoxically, this has led to “web literature” becoming a recognized genre within print culture. In the final part, I compare a PRC online poetry community with a similar community based in the USA. I conclude by arguing that previous scholarship’s biased focus on the transformative aspects of cyber culture has made it difficult to gain a clear insight into the many positive and culture-specific features of Chinese web literature.
Discussing the distinction between web literature and literature is as boring as discussing the distinction between web love and traditional love. ----“wwwmm”
According to statistics from the China Internet Network Information Center (CNNIC) there were by the end of 2004 approximately 668,900 websites on the Chinese world-wide web. It is reasonable to assume that a good proportion of these contain content that can be described as literary. The intention of my research, and of this article, is to study the practices of communities that employ internet technologies in order to produce, distribute, consume and value literary products. In this article, my emphasis is on poetry communities. As is shown in Maghiel van Crevel’s article in this volume, the genre of poetry, often seen as culturally marginal, continues to occupy a significant niche in PRC elite culture, with some of the most avant-garde groups making good use of
* I am grateful to Charles Laughlin for his perceptive comments as the discussant for the first draft of this paper during the IIAS workshop in October 2004.
1. Quoted from an article entitled “Wangluo wenxue” (“Web literature”), originally published (possibly under a different title) in Zhonghua dushu bao (China Reading Journal), 10 October 2001, reproduced online, URL: http://www.china.org.cn/chinese/RS/65142.htm. The reproduction does not mention the name of the article’s author. “wwwmm” is the screen name of one of the participants in the 2001 debate about web literature, discussed below.
2. Information provided on the CNNIC website, URL: http://www.cnnic.net.cn/download/2005/2005012701.pdf. Most general sources on the Chinese internet referred to in this article were traced using the resources available to members of the Chinese Internet Research Group. For more information about this group, see URL: http://groups.yahoo.com/group/chineseinternetresearch/.
© The China Quarterly, 2005
websites to disseminate and discuss their work and ideas. However, poetry (both modern and classical) also continues to have a more popular appeal among young educated urbanites who gather in virtual communities as part of their lifestyle, without displaying any obvious interest in being avant-garde. It is the latter type of community that I intend to discuss in this article.
The article begins by providing a brief general overview of the current state of research about the Chinese internet. I take issue with the dominant tendency of English-language research to focus almost exclusively on questions of censorship. By foregrounding censorship and by highlighting what does not appear on the Chinese internet, attention is taken away from what does appear. For research on contemporary Chinese web literature this exclusive focus on censorship issues is unnecessary as censorship is a fact of life for Chinese writers and it does not make their work less valuable or interesting.
The article then looks at the development of “web literature” (wangluo wenxue) in China, briefly outlining the meaning of the term and the content of a protracted debate about it that took place in 2001. The debate illustrates the limited extent to which web literature is able to distinguish itself from conventionally published literature. Paradoxically, this has led to “web literature” becoming a recognized genre within print culture.
In the remainder of the article, I compare a PRC online poetry community with a similar community based in the United States. The comparison further emphasizes the limited distinctions between online and printed literature. It also confirms that, as many have begun to suspect, the supposed “globalness” of internet technology does not remove clear cultural differences.
Research on the PRC Internet
The CNNIC statistics mentioned above estimated the number of Chinese internet users at 94,000,000. As is well-known (and as is the practice in some other countries) these users do not have access to the world-wide web in its entirety, as the content of non-PRC sites is continuously monitored, filtered and, if deemed necessary, blocked.\(^3\) Perhaps because of the world-wide web’s reputation of being an exceptionally permissive and uncontrollable space, most researchers of the Chinese internet so far seem to be predominantly interested in methods and technologies of control and censorship, that is, in what does *not* appear on the Chinese web, rather than what does. If initially China watchers were optimistic that the advent of the internet in China would promote freedom of speech and create niches for free thinkers
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3. For an ongoing analysis of web content filtering in China and other countries see Jonathan Zittrain and Benjamin Edelman, “Empirical analysis of internet filtering in China,” URL: http://cyber.law.harvard.edu/filtering/china/.
to express their opinions, recent research seems to deny these expectations, demonstrating instead the effectiveness of state control of the internet in China.\textsuperscript{4} Among the 138 items on Randy Kluver’s “Bibliography on the internet in China,”\textsuperscript{5} which lists English-language studies of the Chinese internet, the vast majority deal with issues of state control versus civil liberties, with e-commerce and economic aspects the second most popular topic. References to research on cultural production on the Chinese internet are almost absent, nor can they easily be found elsewhere. A notable (and very recent) exception is Michael Day’s annotated archive of avant-garde poetry websites that is part of the “Digital archive for Chinese studies (Leiden Division).”\textsuperscript{6} That project, however, also appears to be largely motivated by the fear that government repression will cause websites to disappear without warning: it is mainly an archive of dissident voices. That such disappearances can be immediate and extensive became clear to me on 20 September 2004 when I tried to access some of the tens of thousands of personal web pages (including 2,372 pages classed as “literature” and 25,699 pages classed as “personal manifestos” \textit{(geren xuanyan)} hosted by the Chinese commercial ISP 533.net. They had become inaccessible. An announcement by the ISP explained that it had ceased its free web hosting service with immediate effect because of “problems with the contents of a small minority of the pages” and that it would henceforth only provide web space for paying customers providing full proof of identity.\textsuperscript{7}
Following this, similar announcements began to appear on the websites of many Chinese ISPs, discussion forums and chatrooms. The government policy of making service providers, forum moderators and chatroom operators legally responsible for any “inappropriate content” appearing on their sites has obviously led to extensive self-regulation. The demand for users to provide full proof of identity acts as a deterrent to those possibly planning to post or host (politically) subversive content. Service providers, moderators and operators appear willing to co-operate with the authorities in this way in order to keep their sites up and running. In the context of this volume, this is one example of how the state in contemporary China continues to exercise control over culture, especially popular culture, by appealing to cultural
\textsuperscript{4} For examples of this approach, see for instance Jeroen de Kloet, “Digitisation and its Asian discontent: the internet, politics and hacking in China and Indonesia,” \textit{First Monday}, Vol. 7, No. 9 (2002), URL: http://firstmonday.org/issues/issue7_9/kloet/index.html and Lokman Tsui, “Big Mama is watching you: internet control and the Chinese government” (Leiden: Unpublished MA thesis, 2001; downloadable from URL: http://www.lokman.nu/thesis/).
\textsuperscript{5} This list is available to members of the Chinese Internet Research Group mentioned above. My comments here are based on a visit to the (continuously expanding) list in September 2004.
\textsuperscript{6} URL: http://www.sino.uni-heidelberg.de/dachs/leiden/poetry/index.html.
\textsuperscript{7} The URL of the announcement, being the page that would appear regardless which personal web page one was trying to access, was (and perhaps still is) http://ads.533.net/gonggao/.
brokers’ interest in maintaining a stable marketplace for their enterprises.\(^8\)
**Web Literature: Production-Oriented Analysis**
Although the preference for censorship-oriented research is understandable, there are good reasons to argue for the viability of a more production-oriented approach, that is, an approach that looks at what *does* appear in Chinese web space. Foremost among those reasons is the fact that all Chinese literature of all dynasties and periods has been produced under conditions of state censorship and this has never prevented scholars and critics from taking it serious as literary work, nor has it prevented Chinese writers from writing and readers from reading. Censorship of the internet does not necessarily confront Chinese writers and readers with an unfamiliar situation. Censorship is the norm, rather than the exception.
A second (related) reason is that censors and their practices can and should be studied as part of literary practice as a whole, whether in print culture or in cyber culture. There is no reason to ignore censorship but there is similarly no reason to overemphasize it or isolate it from the practices of other agents within the literary field.
Thirdly, the particular type of censorship practised in China is, for better or for worse, one of the things that make contemporary Chinese web literature different from that of other countries (cultures, language communities). This would support the suggestion that there is such a thing as “PRC web culture” despite the fact that the world-wide web is supposed to work against such nation-based distinctions.\(^9\)
Finally, content analysis of literary websites is important because it aids our understanding of how internet technologies, as distinct from print culture technologies, are challenging familiar concepts of literature, not just in China but everywhere in the world. It is this final point that reverberated most strongly during a lively debate about web literature on the Chinese internet in 2001.
**The PRC Web Literature Debate**
In as far as I can claim to have an overview of the large amount of web space devoted to the debate about web literature, it began on one of the discussion forums (*luntan*), sometimes referred to as “bulletin boards”
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8. It might be mentioned in passing that the techniques used by the Chinese government here are not new. Similar policies, based on mutual interest of government and ISPs, were arrived at in Western countries in order to suppress illegal content such as child pornography or terrorist manuals. The difference is of course that the Chinese government’s definition of what constitutes “inappropriate content” is much wider.
9. Here the formulation of my argument is indebted to Jeroen de Kloet’s research cited above.
or BBS\(^{10}\) of the popular Shanghai-based website “Rongshu xia” (“Under the banyan tree”) (URL: http://www.rongshu.com or http://www.rongshuxia.com).\(^{11}\) The debate was sparked by a short post (\textit{tiezi}) to the forum by the author Chen Cun, known to some as the “father of Chinese web literature,” presumably because he was the first well-known contemporary author to switch to the new medium.\(^{12}\) I have been unable to retrace the original post, but its content has been copied in many places. It reads as follows:
I go online and visit Under the Banyan Tree because I want to see what web literature is really like. I have high hopes for it. But web literature these days is starting to make me reconsider. If the highest achievement of web literature is to publish traditional books offline, if that is what qualifies you as a writer and allows you to brag, then is there still a web literature? Its freedom, its randomness and its non-utilitarian nature have already been polluted. Although I understand these changes, it is still not what I hope to see. Web literature is already past its prime. What Laozi called the period of utter innocence [\textit{chizi zhi xin}] has vanished too quickly.\(^{13}\)
Chen Cun, who later withdrew from the management of “Under the banyan tree,” a site which he had founded and funded, provided with this post an (in my view) accurate assessment of the position of web literature within the Chinese book market. In 2001, and probably still nowadays, it was the case that the main measure of success for a web literature author was to have his or her works appear in print. “Under the banyan tree” itself played a major part in developing these practices, as it published regular book collections of its best online works and secured generous funding from the German Bertelsmann Book Club who used the site to enter the Chinese (printed) book market. It is understandable that someone like Chen Cun, who probably saw himself as a representative of a literary avant-garde, was attracted by the possibilities that HTML writing offers for literary experimentation, ideally leading to novel ways of writing making full use of the characteristics of hypertext and hypermedia. Such writing did indeed not or hardly develop on the Chinese internet. The bulk of web literature, including much online writing that presents itself as new or shocking, is plain (or “linear”) text rather than (“non-linear”) hypertext.\(^{14}\)
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10. In Western cyber-circles, the term “BBS” (bulletin board system) is considered somewhat outdated, referring as it does to the text-only interactive systems of the 1980s and 1990s. In East Asia, however, the acronym is still popular and used to refer to both text-only and multimedia online forums. Cf. the article “Bulletin board systems” on Wikipedia, URL: http://en.wikipedia.org/wiki/Bulletin_board_system.
11. For a partial content analysis of this website, see Michel Hockx, “Links with the past: mainland China’s online literary communities and their antecedents,” \textit{Journal of Contemporary China}, Vol. 13, No. 38 (2004), pp. 105–127.
12. Chen Cun’s status as an important contemporary author is confirmed by the inclusion of one of his stories in Howard Goldblatt (ed.), \textit{Chairman Mao Would Not Be Amused} (New York; Grove Press, 1995).
13. Quoted from the article entitled “Web literature,” see n. 1.
14. This is true, for instance, of the “happening” avant-garde website called “Shi jianghu” (“Poetry vagabonds”), which consists entirely of poems that adhere to the print culture format, despite their often highly unconventional content. (Indeed, the group also publishes
The crossing-over of web literature authors into print culture led, however, to an unusual phenomenon which continues until the present day. Within print culture, “web literature” has become a genre of its own. When I visited Beijing in 2002, I was struck by the fact that the literature sections of all major bookshops had shelves devoted to “web literature” just as they had shelves devoted to, say, “Chinese poetry” or “foreign fiction.” The category also appears as a genre category on some online Chinese bookshops. In short, for a web author (xieshou) to become successful in print culture there is an additional hurdle to overcome: even if one does manage to publish one’s work in print it might still carry a generic label that distinguishes it from “real” literature. On the other hand, for web authors to have this kind of entry point into print culture at all is convenient and valuable. As Howard Becker points out in an article about English-language hypertext fiction, the challenges faced by American web authors are much greater.\footnote{Howard S. Becker, “A new art form: hypertext fiction,” URL: http://www.soc.ucsb.edu/faculty/hbecker/lisbon.html.} I return to this comparison below.
Many of the thousands who responded to Chen Cun’s post after it was first published disagreed with his pessimism. However the most frequently seen response, documented in online articles and in newspapers into which the debate spilled over by October 2001, was that the internet was after all only a medium (zaii) for literature. Those who held this view argued that literature was literature no matter how it was distributed and that web literature, rather than being on the way out, was to have a glorious future. Although the enthusiasm for web literature does seem to have diminished since 2001, the sheer number of literary works produced for the various online forums on a daily basis is still staggering.\footnote{For an overview of related articles see “2001: Qingsuan wangluo wenxue” (“2001: liquidating web literature”), URL: http://www.booker.com.cn/gb/paper253/1/.}
Below, I take a closer look at the practices of a typical Chinese literary website, making a straightforward comparison with a similar American website. Despite the continued adherence to the print culture format, the actual practices of the Chinese website occasionally diverge from the print culture paradigm, especially in the realm of criticism and valuation.
\textit{Online Poetry Communities}
The following sections briefly describe the practices of two online literary communities, one hosted on a server located in the United States
\footnote{In September 2004, the site was no longer accessible at the old URL (http://www.wenxue2000.com – this instead linked, ironically, to the text of a poem by Li Bai). Alternative URLs were available, leading to a collection of old work (http://sh.netsh.com/bbs/3307/) and to a new discussion forum (http://my.clubhi.com/bbs/661502/). In December 2004 the old site was accessible again. At the time of writing (March 2005), it has once more disappeared, but the other two URLs mentioned above remain valid. The translation “poetry vagabonds” for \textit{shi jianghu} was suggested to me by Maghiel van Crevel.}
and using the English language (http://www.everypoet.com) and the other hosted on a server in the People’s Republic of China and using the Chinese language (http://www.chinapoet.net). Both communities boast a large membership, have well-designed websites and apparently a solid financial basis, gained from advertising, donations or the sale of web hosting services. They are independent domains and therefore different from the smaller communities that one finds on portals such as Yahoo. Both sites are dedicated to the genre of poetry, usually a marginal and elitist literary genre, which however seems to enjoy a remarkable popularity online.
The description below is in the form of a straightforward comparison between the US site and the PRC site. It points out where practices are identical or similar, and where they diverge as a result of cultural factors. I look at practices surrounding the production and distribution of poems as well as practices of valuation (“symbolic production”). Following the example set in Howard Becker’s book on “art worlds,” I try to take into account all the skills and tools that are needed to bring an online poem into being. I also refer to a more recent essay by Becker about “hypertext fiction,” already mentioned above. In that article, Becker argues that hypertext fiction, being non-linear writing requiring special software to create, special distributors to sell and special reading strategies to enjoy, is a truly new art form in the sociological sense. The world of printed literature has no way of accommodating it within the existing forms of organization and co-operation between its agents. As mentioned above, such innovative forms of online literature have not yet emerged in the PRC. Similarly, the practices described below are only partially innovative in comparison to those of offline publishing.
**Hardware and Software**
The basic material condition for the authors of online poetry is to have a computer with access to the internet. For most people in the United States and for increasing numbers of people in the urban areas of China,
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17. When I visited the sites in May 2004, over 15,000 members were claimed to subscribe to the forums on the Chinese site; over 18,000 members were claimed to subscribe to the main forum (the “Poetry free for all”) of the US site. When I visited both sites again on 20 September 2004 both were claiming a membership of over 19,000. At the time of writing (March 2005) the American site has “over 21,000 members.” The Chinese site boasts 23,267 subscribers. It has, however, temporarily stopped accepting new subscriptions. Although no reason for this is given, the appearance of a two-line slogan under the announcement (“Let us jointly establish a healthy, civilized, law-abiding poetry community. Actively suppress and eliminate bad information and lend your strength to the cleaning up of the web environment”) suggests that in this case, too, state intervention has brought about stricter self-regulation.
18. I borrow my usage of this term from C.J. van Rees’s model for the description of literary communities. See for instance C.J. van Rees and Jeroen Vermunt, “Event history analysis of authors’ reputation: effects of critics’ attention on debutantes’ careers,” *Poetics*, Vol. 23, No. 5 (1996), pp. 317–333.
19. Howard S. Becker, *Art Worlds* (Berkeley, Los Angeles, London: University of California Press, 1982).
computers are affordable and internet access is cheap and convenient. Membership of the two poetry websites discussed here is free, as is publication of one’s work on the site. This means that most of the cost of making this kind of literature available is incurred by those who run the website. Their position combines that of publisher and book seller in the print culture system. In the PRC case, this means that the website is also subject to censorship regulations. The front page displays, at the bottom, a state registration number and a link to the government website listing the domain registration details of the site.
The basic material conditions for hosting a website like this are server space and software. Large interactive sites that generate much server traffic, like the two sites under discussion, are generally not hosted free by internet service providers. The software needed to operate the interactive forums is also not freely available. However, compared to any kind of print culture venture, the direct costs of running sites like this is small. The American site (Figure 1) seems to derive at least part of its income from advertising in banners and pop-up windows. It also offers members the opportunity to make donations. Initially, the PRC site had very few advertisement banners. In Figure 2, a screenshot of the front page of the PRC site taken in April 2004, the only advertisement present is the one on the right-hand side in the middle, advertising a printed anthology of best works from the site itself. During later visits to the site in September and December 2004 and March 2005 I noticed an increasing number of
Figure 1: Front Page of everypoet.com
advertisements on the front page, presumably an indication of increased popularity. The PRC site also functions as a company offering paid web hosting services. It is unclear in the case of both sites to what extent the enterprises are profit-making or rather based on generous investment of time and personal funds by enthusiastic individuals.
Both sites provide copious information about famous poems and poets, this being an indication of their relative closeness to the print culture tradition. The main attractions of the sites, however, are the “poetry forums” (shige luntan). These are interactive message boards dedicated to different poetic genres, styles and themes. In China at the moment such message boards are hugely popular and literary forums such as the ones discussed here can be found on almost every portal.\(^{20}\) The software used by both sites appears to be identical, with the first point of entry being a page listing the various available boards and inviting the user to choose which one to read or contribute to (Figures 3 and 4). These listings also provide some statistical information about the forums, such as the number of posts and threads they contain. In both cases, the lists also provide the screen names of the moderators of each of the forums.
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20. To mention just one fairly unexpected example, the website of the All China Lawyers Association (ACLA) runs a lively literary forum where lawyers can publish and discuss their own literary writing. URL: http://www.acla.org.cn/forum/postlist.php?Cat = &Board = 2.
Figure 3: (Partial) List of Discussion Boards on everypoet.com
| Forum | Posts | Threads | Last Post | Moderator |
|--------------------------------------------|-------|---------|--------------------|----------------------------|
| Amorphous All-Purpose Anarchy | | | | |
| General Poetry | 11905 | 2280 | 09-29-2004 09:00 AM| Artemis1 |
| General CRC | 26262 | 4435 | 09-29-2004 09:00 AM| Citybrawl |
| Another General CRC | 12875 | 1847 | 09-29-2004 09:00 AM| Minter Micahue |
| Scammy Musings | 4136 | 466 | 09-29-2004 09:00 PM| Stephen1 |
| Charon's Leaky Schooner | 1932 | 229 | 09-29-2004 09:00 PM| Luke_Poe |
| High Critique | 10841 | 1592 | 09-29-2004 09:00 AM| hakai |
| Merciless & Possibly Painful Critique | 8708 | 1160 | 09-29-2004 09:00 PM| Luke_Poe |
Figure 4: (Partial) List of Discussion Boards on www.chinapoet.net
| 网络论坛 | 李可可 | 今日： 05 主题： 经验与力 | 回复： 14940 2004年05月25日 09:49pm |
|----------|--------|---------------------------|-----------------------------------|
| 论坛办公室 | 李可可 | 今日： 7 主题： 最后发表： 剑 | 回复： 4532 2004年05月25日 10:29pm |
| 论坛神精榜区 | 管理者 | 今日： 0 主题： 最后发表： 剑 | 回复： 7825 2004年05月25日 10:31am |
| 网络论坛 | 刘永 | 今日： 12 主题： [原创]谁是造物主 | 回复： 7108 2004年05月25日 08:29pm |
|----------|------|----------------------------------|-----------------------------------|
| 古风古韵的意境 | 田村 | 今日： 142 主题： 拍照 | 回复： 13964 2004年05月25日 10:11pm |
| 中国诗人一百单八将 | 黄秋颖 | 今日： 108 主题： 老者与年轻人 | 回复： 13983 2004年05月25日 10:29pm |
| 先锋—现代诗 | 评论者 | 今日： 72 主题： 小说的写法 | 回复： 7229 2004年05月25日 10:29pm |
| 青春诗语 | 天地马后 | 今日： 230 主题： 老者与年轻人 | 回复： 101202 2004年05月25日 10:29pm |
Agents
The moderators are the key agents involved in the running of this kind of online poetry forum. Combining the roles of editors and censors in print culture, they decide which posts are and which are not included but they do so (at least in the forums discussed here) after the original post has been submitted.\footnote{For a more detailed discussion of the various control mechanisms of online discussion forums in China, see Wenzhao Tao, “Censorship and protest: the regulation of BBS in China people daily,” \textit{First Monday}, Vol. 6, No. 1 (2001), URL: http://www.firstmonday.dk/issues/issue6_1/tao/index.html.} In other words the moderators’ main task is to screen submissions and to ensure that their content is suitable and appropriate for the forum to which they have been sent. The content of this task might vary from removing obscene or abusive messages to moving a poem to another forum where it more appropriately belongs. In the case of the Chinese site, the moderators are also responsible for ensuring that submissions do not violate government censorship regulations.\footnote{It is likely that especially the Chinese site also screens submissions with automatic filters to remove posts containing certain words or terms that are deemed unacceptable. It has recently been suggested that all Chinese messenger services, chatrooms and message boards are forced to operate filters based on a single list of “forbidden” words put together by the authorities. This list was retrieved by Chinese hackers and reproduced in full on various websites. (See Xiao Qiang, “The words you never see in Chinese cyberspace,” \textit{China Digital News}, 30 August 2004, URL: http://journalism.berkeley.edu/projects/chinadn/en/archives/002885.html.) However, I have certainly seen some of the “forbidden” words on message boards; following the publication of the list, others have reported successfully using them in Chinese cyberspace. It is more likely that the list (with entries ranging from “democracy” to “masturbation”) only applies to the messaging software (QQ) in which it was found, or that the report was a hoax.} As is the case in most internet communities, the moderators are themselves regular contributors or visitors to the site. It is unlikely that they receive more than token remuneration for their efforts. This is consistent with Becker’s model of art worlds: if one wants to do things within an art world that are unconventional (such as publish online rather than in print) one must be prepared to do a lot of the work oneself, since other agents within the community might not be willing (or be trained) to provide the assistance you need.
The key agents in keeping these sites alive are of course the members contributing to the forums, either by submitting their own work or by commenting on other work posted. As mentioned before, contributions to the forums are represented as threads – as a series of individual posts on one topic – normally a poem submitted by one of the members, who, by doing so, starts a new thread. A typical forum on the US site (Figure 5) shows the title of the post/poem, the screen name of the author, the number of replies to the original post, the number of times the thread has been visited, the screen name of the last person contributing to the thread and the date and time when that last contribution was made. Various symbols on the left-hand side indicate various aspects of the status of the thread. For instance, the yellow folder symbol turns into a symbol of a flaming folder if the thread is “hot,” meaning it has been responded to or visited more than a certain number of times.
Figure 5: List of Threads on a Discussion Board on everypoet.com
| Thread | Thread Starter | Replies | Views | Rating | Last Post |
|---------------------------------------------|----------------|---------|-------|--------|-----------|
| Sticky On posting to the higher critical forums | Scarvella1 | 0 | 3634 | | AM 2008/09/20 10:10 by Scarvella1 |
| Sympathy in Paradise | lekala | 2 | 112 | | AM 2008/09/20 10:10 by lekala |
| When a Boy Becomes a Man | RainKings | 2 | 87 | | AM 2008/09/20 10:10 by StevenSchroeder |
| World without Glasses | StevenSchroeder| 4 | 238 | | PM 2008/09/20 10:10 by StevenSchroeder |
| Of Half-castes and Rabbits | Melanie | 5 | 327 | | AM 2008/09/20 10:10 by Melanie |
| Dining alone | Searcher | 12 | 406 | | PM 2008/09/20 10:10 by Searcher |
| In Alexandria: A Dialogue (Revised) | Jae Leang | 3 | 199 | | PM 2008/09/20 10:10 by Jae Leang |
| The Old Man and the Sea | Saluda | 8 | 419 | | PM 2008/09/20 10:10 by Saluda |
| Happis Supper | romac | 0 | 522 | | PM 2008/09/20 10:10 by romac |
| Mordedchai Vanunu is Free | Marc Adler | 0 | 343 | | PM 2008/09/20 10:10 by Marc Adler |
| Talking to Lord Newborough | David Anthony | 14 | 477 | | PM 2008/09/20 10:10 by David Anthony |
| (Revised from CBC) The Dualist | XOX | 0 | 231 | | PM 2008/09/20 10:10 by XOX |
| Of Thai Lyric | Jae Leang | 7 | 530 | | PM 2008/09/20 10:10 by Jae Leang |
Figure 6: List of Threads on a Discussion Board on www.chinapoet.net
A typical forum on the PRC site (Figure 6) looks much the same. Apart from some additional symbols (which I am still looking to understand completely), the same information is provided about the various threads. As the word “thread” itself indicates, the poems submitted and the responses by other users are organized in a linear fashion, in chronological order. In other words, the threads themselves are not hypertexts. Nevertheless, the possibility of direct interaction between poet and reader/critic is unusual when compared to print culture. This is discussed in more detail below.
**Symbolic Production**
The main attraction for poets of publishing their poems on a website like this, rather than in a smaller writers’ discussion group or on their own personal website, is the opportunity to expose their work to, obtain feedback from and get in touch with a community of poetically minded individuals. Since “being published” is the easiest thing in the world on the internet, at least part of the attraction of these websites has to be the idea of belonging to a community and obtaining some sort of recognition. Interestingly, though, a poll asking members to indicate their main motivation for writing poetry, carried out by the US site (Figure 7), shows that “emotional catharsis” was considered by a majority to be their main drive. It should be noted, though, that the answers “to get published” or
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**Figure 7: “Why do you write poetry?” (from everypoet.com)**
“to belong to a community” were not given as possible options to respondents.
Unlike most print culture communities, symbolic production in these online communities is not carried out by specialized critics but by other authors (that is, members who themselves are contributing poetry to the site), presumably because it is difficult to find specialist critics willing (or able) to take on the task. Moreover, it is normal for authors to respond directly to comments on their work, which is a new function not available in print culture. The moderators of both sites (themselves also authors and contributors) play a crucial role in attributing recognition to members’ works as they decide which posts or threads are selected for inclusion in regularly published anthologies (the US site\textsuperscript{23}) or on a special board for the best of Chinapoet. This special board in turn provides material for the editors of the two web journals (\textit{wangkan}) which Chinapoet strives to publish each month, one for modern poetry and one for classical poetry.\textsuperscript{24}
The web journals are edited by a small group of moderators. The contents of the journals represent only a very small part of what is contributed to the site every month. The works (poems and essays about poetry) are presented on nicely designed web pages that do not have any interactive functions, that is, they can only be read and not commented on. The web journals are not unlike printed literary magazines of the kind that appeared in China in the 1920s when new printing technologies (and New Literature) were spreading. Those magazines, too, were low-cost ventures produced by small editorial collectives participating in a literary economy that valued swift and frequent production. Furthermore, the fact that editors select a tiny proportion of work submitted to them for inclusion into a journal is also not dissimilar from the editing process of a print culture journal. The main difference, however, is that in this case all contributions get published on the site first. To have one’s work included in the site’s web journal is probably the highest possible form of symbolic recognition that a contributor to Chinapoet can obtain within the site itself. Other literary websites in China have web journals as well and some of them are important mouthpieces for groups that have little access to, or interest in, official print culture. On the other hand, the format of the web journals is so devoid of interactive characteristics that they can also be seen as possible stepping stones into print culture. They could simply be (and presumably sometimes are) printed off and enter the offline literary world. Unlike the world of hypertext fiction discussed by Howard Becker, the world of online Chinese poetry on the whole displays
\textsuperscript{23} Information based on my April 2004 visit to the US site. During a visit in December 2004, I failed to detect references to such anthologies. During that visit the US site did have special boards in which some of its best contributions were preserved. Members were not allowed to add work to those boards, but were allowed to submit comments as usual.
\textsuperscript{24} Recent issues of the site’s web journals (starting from the August 2004 issue) are accessible through links on the page listing the discussion forums: http://www.chinapoet.net/cgi-bin/leobbs.cgi. I have been unable to find an index page of all issues published so far. It is possible that publication did not start until August 2004.
no intention to break away from print culture paradigms, making the boundaries between the two much more fluid.
The web journal phenomenon is worthy of further separate investigation. A useful question to ask would be whether or not the editors’ selection of works for inclusion in the web journals is influenced by the valuation of those same works by contributors to the discussion forums. It is likely that a certain amount of recognition derives from the statistics indicating how often a poem has been read and commented on. I noticed that, as I was browsing the forums, I was generally inclined to click on threads with high statistics, assuming that they would be more interesting or controversial than others. Naturally these numbers can be manipulated by the author, if he or she simply keeps going back to the thread to add new posts. The US site has a rule against such behaviour and moderators are authorized to delete any posts from authors other than those directly responding to critical suggestions from other posters. Moreover, it also asks of each member not to post more than one poem per forum per day, and to post three responses to other people’s work for each original poem posted. Clearly, the US site values the “mutual aid” aspect of the site and is wary of becoming merely a showcase of aspiring poets’ work or (the other extreme) a glorified chat room. No such rules can be found on the PRC site, which does, however, as mentioned above, have strict rules for the content of posts, based on government censorship regulations.
Censorship
Figure 8 shows the rules for submission to Chinapoet (as downloaded in April 2004). There are two main parts. One part lists all the content that is not allowed on the site, including “writings violating the PRC constitution, the policy of reform and opening up and the four cardinal principles,” “writings attacking the PRC government, the Chinese Communist Party and its leaders,” “writings propagating violence, superstition and licentiousness,” “writings exposing state secrets” and (lastly) “all other content forbidden by law.” The second main part lists all types of screen names that members are not allowed to use, including “names, stage names and pseudonyms of Party and government leaders or other celebrities” and “names of state institutions and other institutions.” Most importantly, however, and mentioned three times in bright red font, the site is closed to any and all content alluding to the outlawed *falun gong* movement. The last line states unequivocally: “This site does not welcome *falun gong*. If we see one we delete one” (*jian yi shan yi*).
This is not just paying lip service to government campaigns. When doing research on another paper on web literature three years ago, I found that *falun gong* members and sympathizers were indeed using freely accessible bulletin boards to spread information about their movement.
25. During my last visit to the PRC site, in March 2005, a notice had appeared informing users that no more than three posts per member per day would be allowed. There was however no encouragement for users to post more responses to other poems.
and to denounce government oppression. Failure to remove such contributions in time might lead to a website being closed down. The alternative to outspoken warnings like the one cited above would be to renounce the open character of the forums and screen every contribution before publication, a step which Chinapoet obviously is not willing or able to take, since it would place a much heavier burden on forum moderators. As shown above, there is now a tendency among Chinese ISPs and forum hosts to add membership registration to its strategies of keeping out unwanted content and avoiding legal responsibility.\footnote{As I was making revisions for the final version of this article (in March 2005), there was an uproar among China’s “netizens” about the restrictions imposed on popular discussion forums at Qinghua, Fudan and Nanjing Universities. In this case, the restrictions (suspending off-campus access and making full registration of members compulsory) appeared to have been imposed directly by the authorities, rather than being the result of self-regulation.}
Cultural Differences
A comparison of the content of some of the threads on the two sites reveals some more cultural differences. At the moment I can only characterize these as differences between the cultures of the actual sites, although the Chinese site confirms ideas I have about the cultural specificity of modern Chinese literature and criticism as a whole. Empirical support for those ideas awaits, however, a much more comprehensive reading of these and other websites.
\begin{figure}[h]
\centering
\includegraphics[width=\textwidth]{image.png}
\caption{Rules for Contributors (from www.chinapoet.net)}
\end{figure}
Substantial differences are noticeable in the area of symbolic production. Figure 9 shows a part of a typical thread on the US site. The discussion focuses on whether or not the poem is “good enough” to be included in this particular forum, which is meant for “more than merely moderately experienced poets” (Figure 10). Responses to poems are very specific as to which words in which lines might be changed or omitted. In general, the US site strongly propounds the notion of poetry as a craft or skill which requires considerable training and investment of time and which needs to be subjected to harsh criticism in order to be able to improve. In fact, the US forums are partly subdivided on the basis of the harshness of criticism allowed to be submitted to threads, with the most fiercely critical forum presented as the one to which only the best poets/critics should contribute.
On the PRC site, the issue of skill and discussions about the right word in the right place are much less prominent, although they do appear in the forum dedicated to those writing in the classical style, which of course has very strict prosodic rules. The feedback on poems in the modern poetry forums is much less normative, and often consists of one-liners of the type “I like this poem” or “I don’t like this poem,” without going into much detail. Questions of content and personality are also often debated. A typical example is shown in Figure 11, which shows two responses to a simple poem (not shown) expressing love for a woman. The first response basically dismisses the poem as romantic rubbish. The second response points out that this would be the case if the poet had been a man,
Figure 10: Posting Guidelines, “High Critique” Forum, everypoet.com
A place for critical commentary, mainly for the more than merely moderately experienced. Park your ego outside, embrace critical commentary, and, as always, comment on at least three poems for each one you post. Good luck!
Before you consider posting, please read these guidelines in full:
Welcome to the High Critique forum of the Poetry Free For All. This is where things start to get pithy, and subsequently, where the bar is set higher. As with all the forums, the Posting Guidelines are very much applicable here. So too are the High guidelines below:
1. "More than merely moderately experienced" means that, as a writer, it is assumed you know the basics of poetic knowledge. You should know common poetic terms, common forms, and what the basic distinctions/abstractions are. There should be evidence from the quality of the poetry you post that you have put thought into that some revision has already been done, and that you are ready for and will understand the forthright, honest, and potentially harsh critique you will receive in High.
1a. If the moderators do not feel that your work indicates a fundamental knowledge of poetic basics, and that you have not, in fact, put some work and some thought into it, the moderators reserve the right to delete it, lock it, or move it to a more appropriate forum. We have seen this happen to you lurked in High and figured it out for yourself. If you're not sure you're up to posting in High, post in General C&C instead. Better safe than slapped by mods. Trust me.
2. "More than merely moderately experienced" also means that, as a critic, it is assumed you know the basics of poetic knowledge. With your comments, it should be clear that you are generally able to grasp a writer's intent and evaluate how successful the poem communicates that intent, that you have an appreciation of how sound works in poetry, and that you have a familiarity with figurative language and meter, forms, and the use of rhyme. Lastly, you should be able to tell when a reviewer is actively regressing rather than progressing. If you can't do that, you should hang out in General C&C a while to get the hang of it.
Figure 11: Comments on a Poem (from www.chinapoet.net)
Figure 12: List of Threads, Poetry Translation Forum, www.chinapoet.net
| Thread | Poster | Posts | Date | Time |
|-----------------------------------------------------------------------|--------|-------|------------|--------|
| [原创]Testtry | 老呛 | 1 | 53 | 2004/04/05 00:58 |
| [译诗] zengpush 如花 | 老呛 | 8 | 127 | 2004/03/31 16:33 |
| [原创]《原创新手第一——＜梦＞的日文版》此诗的题目：《诗的翻译困境》 | 春子 | 10 | 164 | 2004/03/30 10:29 |
| [译诗]月牙儿 Sein. | 碧粼水 | 7 | 134 | 2004/02/25 04:10 |
| [译诗]月牙儿《蝴蝶结》（节选） | 碧粼水 | 1 | 110 | 2004/02/24 23:45 |
| [译诗]鸟飞翔 ——Total Silence | 老呛 | 6 | 119 | 2004/02/22 23:48 |
| [译诗]老兄请进，放过墨公造么？ | 水鸟飞翔 | 5 | 95 | 2004/02/21 14:06 |
| [原创]In This Violent Fire | 老呛 | 4 | 80 | 2004/02/21 10:50 |
| [译诗]爱的翅膀——爱若 | 紫荆主人 | 11 | 190 | 2004/02/18 21:10 |
| [翻译]污客不 诗两首 | 浩浩唐歌 | 24 | 328 | 2004/02/15 14:12 |
| [原创]My Lunch Today | 老呛 | 5 | 102 | 2004/02/15 05:38 |
| [原创]Cartoon Ecard | 老呛 | 5 | 111 | 2004/02/14 11:03 |
| [原创]lll | 老呛 | 5 | 108 | 2004/02/11 22:15 |
| [原译诗 Bilingual Pastry 'Mg.' | 碧粼水 | 6 | 152 | 2004/02/11 00:16 |
| [原创]Flying Above Clouds | 老呛 | 2 | 119 | 2004/02/08 19:00 |
| [译诗]家兔白运 To A Fasstery | 老呛 | 3 | 101 | 2004/02/02 15:36 |
| [原创]译 真小邪 (史)乞讨者 | 金舟 | 2 | 76 | 2004/02/02 15:34 |
Figure 13: User Statistics for www.chinapoet.net
| Country of origin | Posts | Percentage |
|-------------------|-------|------------|
| China | 676416| 95.9% |
| Taiwan | 3435 | 8.5% |
| Hong Kong S.A.R. | 2298 | 8.3% |
| Japan | 2218 | 8.3% |
| United States | 1981 | 8.3% |
| Singapore | 1111 | 8.2% |
| Canada | 935 | 8.1% |
| Korea | 639 | 8.1% |
| Malaysia | 534 | 8.1% |
| United Kingdom | 467 | 8.1% |
| Unknown | 13846 | 18.4% |
| The rest | 2253 | 8.3% |
| Total | 787324| 100.0% |
but since the poet is a woman writing about love for a woman, it is actually much more interesting and gives the reader “food for thought” because the poet creates a “contradiction” and a “role reversal.”
Unlike the US site, the PRC site has a lively forum devoted to translation. Although the forum does not limit the languages used, all translations I have seen were either from or into English (Figure 12). The forum has separate “sub-forums” for translation of famous English poems into Chinese and translation of members’ poems into English. These forums naturally attract much normative discussion. The inclusion of translation and the focus on English places the Chinese site firmly in the margins of the system of world literature, whereas the total neglect of translation places the American site squarely in the centre. This mirrors the situation in print culture. The fact that, according to the site statistics, 95.9 per cent of its visitors are from China, and another 1 per cent or so from Sinophone areas such as Taiwan, Hong Kong and Singapore, shows the nature of the predicament.
**Conclusion**
Online literature communities are, on the one hand, part of the print culture community, as are writing clubs, school newspapers or other communities in which aspiring writers practise, discuss and publish in the hope of finding recognition and perhaps one day becoming an acclaimed print culture author. At the same time, however, these communities foster direct interaction across vast geographical distances, coupled with publication for a potentially huge audience, a combination that print culture practices would find difficult to accommodate. The use of similar software and protocols in different cultural settings ensures that the practices of online communities all over the world have certain elements in common, such as their tendency to rely in part on statistics for recognition and the blurring of boundaries between specialized roles such as author, critic and reader, which are so crucial to the practices of print culture. Moreover, the nature of the online medium seems to favour, at least for the moment, the shorter genres such as poetry or the serialized story (or ongoing role-play), creating a more central position for genres that tend to be marginal in print culture. At the same time, cultural differences are observable and demonstrate that cyberspace is not the locus of any kind of transnational cultural expression.
In that respect, a final comment must be made about the relative status of both the US website and the PRC website in the context of internet studies and literary studies. In a provocative newspaper article entitled “Internet studies: what went wrong?” David Gauntlett uses the publication of the first edition of his edited collection *Web.Studies* to criticize
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27. David Gauntlett (ed.), *Web.Studies: Rewiring Media Studies for the Digital Age* (London: Arnold, 2000). (A revised edition, edited by Gauntlett and Ross Horsley, came out with the same publisher in 2004.)
other scholars of the internet for not keeping up with recent developments and for not taking their research beyond the level of description. Gauntlett writes:
The rise of the internet in the past three or four years means that its users know far more about sex, politics, hobbies, and shopping than ever before.
You would expect that internet scholars would be lapping all this up. It’s a transformation of modern society, affecting many spheres of everyday life as well as broader social processes…
But no. Publishers are still churning out books called “Virtual something” and “Cyber something else.” They might as well be called “Wow! Virtual communities!” and “Holy cow! In cyberspace, no-one knows who you are!” Even the journals are still publishing those articles which people were pulling out of the drawer in 1996. Has no-one changed the record? The internet might change politics. It might not. It’s a global phenomenon. It’s not really a global phenomenon. Something funny happened to a bunch of people in a chatroom. Give me a break.\(^{28}\)
As someone interested in the relationship between internet and literature, I disagree with Gauntlett’s statement. I found that what little Western scholarship on web literature exists is almost exclusively focused on the most innovative forms, especially hypertext and hypermedia.\(^{29}\) It is very much up-to-date with current developments, but the majority of literary production by internet users is not considered. Some scholars even present their exclusive focus on hypertext as methodological correctness, as in the following excerpt from an article discussing questions of method in analysing internet texts:
Based on the earlier discussion of the characteristics of hypertext, it is clear that the greater the opportunities of surfing provided by the text, the greater the likelihood that readers are empowered to “write” their own texts. WWW sites that remain “close-ended” do not provide empowerment to readers and could thus be far less suitable as the starting point of the analysis…. These texts are limited in scope and fail to use the full potential of the WWW.\(^{30}\)
At the same time, I found scholarship on the Chinese internet overly concerned with the issue of censorship, while rarely addressing the enthusiasm and creativity of the majority of Chinese internet users, which is so readily observable to anyone who visits Chinese websites. There is, in other words, a tendency among Western scholars to focus on the potential of the internet to bring about a transformation of society (or literature, or culture, or politics). And there is a simultaneous tendency among Western scholars of the Chinese internet to foreground censorship issues, as if to demonstrate that lack of personal freedom will always
\(^{28}\) David Gauntlett, “Internet studies: what went wrong?” originally published in *Times Higher Education Supplement* in 2000, URL: http://www.newmediastudies.com/thes.htm.
\(^{29}\) For instance, the authoritative website for humanities research “Voice of the shuttle” (URL: http://vos.ucsb.edu/) does not have an entry for cyber literature, neither under “cyber culture” nor under “literary theory.” Both categories do have entries about hypertext.
\(^{30}\) Ananda Mitra and Elísia Cohen, “Analyzing the Web: directions and challenges,” in Steve Jones (ed.), *Doing Internet Research: Critical Issues and Methods for Examining the Net* (Thousand Oaks, London, New Delhi: Sage Publications, 1999), p. 193.
cause Chinese society (or literature, or culture, or politics) to lag behind the West when it comes to being transformative, innovative or empowering.
This article has shown that a different perspective is possible. Sure enough, there is no indication that Chinese cyber writing will radically transform Chinese society or even Chinese literature. Partly this is because the state has developed effective mechanisms to ensure that it cannot do so; partly it is because many of its practitioners lack interest in being either transformative or avant-garde or both; partly it is because literature fulfils a different function in Chinese culture. Yet although Chinese web literature is only mildly innovative, it has been the topic of intensive debate among Chinese critics and scholars of literature and it has literally made a name for itself among critics, publishers and book sellers in the larger print culture world, something that Western web literature has conspicuously failed to do.
|
MARY KAY SEMINAR 2024
This is the final month to register. Plus, skin care is on sale!
NOW'S THE TIME FOR SKY-HIGH SUCCESS!
Sherell McDonald Love, an Independent Sales Director and former military gunnery sergeant, shares how trusting herself and learning the importance of work-life balance helped her achieve her goals.
Listen Now
Miss an episode? Check out the archive, and catch up today through the Mary Kay® Digital Showcase App.
TRENDING Now
New!
June Skin Care Product Promotion
June 1–30
Have you heard? Some skin care favorites are on sale all month, and you’ve got options! We’ve bundled the TimeWise® Miracle Set® and the TimeWise Repair® Volu-Firm® Set with some of their skin care besties. You can save on these bundles AND receive the Mary Kay® Travel Roll-Up Bag FREE while supplies last! Limit 13 per bundle per independent sales force member.
Get details!
CELEBRATE PINK!
National Pink Day Is June 23.
Let’s turn social media pink for National Pink Day! You can create excitement with products such as Mary Kay® Skin Care, TimeWise® Moisture Renewing Gel Mask and Mary Kay® Hydrogel Eye Patches.
Get inspired!
June Is Acne Awareness Month.
Help your customers get their confidence back, and they’ll spread the word to their friends. The Clear Proof® Acne System can help clear the way to beautiful skin. And of course, Clear Proof® Deep-Cleansing Charcoal Mask is always a customer-favorite!
GET READY TO POWER UP IN DALLAS!
FINAL MONTH TO REGISTER!
The deadline to register is July 1, 2024, 11:59 p.m. CT.
Make way for THE main event of the year: Seminar 2024!
- **Power UP** your dreams, and unleash boundless belief, gravity-free thinking and skyward goal-setting!
- **Soak UP** all the latest, greatest education to help you reach your new goals, from full-circle selling strategies to team-building tips to the latest digital marketing resources and technology.
- **Team UP** with your fellow goal-getters, and share all the must-know nuggets you’ve learned in your business.
- **Show UP** and **Glow UP** at the Seminar Awards show, celebrating achievers as they **Light UP** the stage.
All of this is waiting for you at Seminar 2024!
MARY KAY SEMINAR 2024
Product Perk!
SEMINAR DATES:
- **Emerald**: July 25–28
- **Diamond**: July 29 – Aug. 1
- **Ruby**: Aug. 2–5
- **Sapphire**: Aug. 6–9
Location:
Kay Bailey Hutchison Convention Center
650 S. Griffin St.
Dallas, Texas 75202
Deadlines:
- Recognition Qualification: June 30, 2024, by 11:59 p.m. CT.
- Registration, Hotel Reservation, Cancellation, Special Needs Requests: July 1, 2024, by 11:59 p.m. CT.
Registration Fees:
- $250 for those who register by the registration deadline, except for new Independent Beauty Consultants whose Agreements are accepted by the Company, July 1, 2023, through July 1, 2024; their registration fee will be $225.
- $275 on-site, space-permitting, except for new Independent Beauty Consultants whose Agreements are accepted by the Company beginning July 1, 2024; their registration fee will be $225.
- $70 On-Site Transfer Fee.
There will be no General Session Viewing option.
Learn more about the Mary Kay National Area Party in Pink Reception and Celebration, and find Seminar Recognition FAQs.
Register today! Hurry! The registration deadline is July 1.
GET READY TO POWER UP IN DALLAS!
FINAL MONTH TO REGISTER!
The deadline to register is July 1, 2024, 11:59 p.m. CT.
Make way for THE main event of the year; Seminar 2024!
- **Power UP** your dreams, and unleash boundless belief, gravity-free thinking and skyward goal-setting!
- **Soak UP** all the latest, greatest education to help you reach your new goals, from full-circle selling strategies to team-building tips to the latest digital marketing resources and technology.
- **Team UP** with your fellow goal-getters, and share all the must-know nuggets you’ve learned in your business.
- **Show UP** and **Glow UP** at the Seminar Awards show, celebrating achievers as they **Light UP** the stage.
All of this is waiting for you at Seminar 2024!
MARY KAY SEMINAR 2024
Product Perk!
If you register for and attend Seminar 2024, you will receive a special bundle of products on-site for you to try! Be sure to save a little space in your checked luggage.
| Product | Dates |
|---------|-------------|
| Diamond | July 29 – Aug. 1 |
| Ruby | Aug. 2–5 |
| Sapphire| Aug. 6–9 |
Location:
Kay Bailey Hutchison Convention Center
650 S. Griffin St.
Dallas, Texas 75202
Deadlines:
- Recognition Qualification: June 30, 2024, by 11:59 p.m. CT.
- Registration, Hotel Reservation, Cancellation, Special Needs Requests: July 1, 2024, by 11:59 p.m. CT.
Registration Fees:
- $250 for those who register by the registration deadline, except for new Independent Beauty Consultants whose Agreements are accepted by the Company, July 1, 2023, through July 1, 2024; their registration fee will be $225.
- $275 on-site, space-permitting, except for new Independent Beauty Consultants whose Agreements are accepted by the Company beginning July 1, 2024; their registration fee will be $225.
- $70 On-Site Transfer Fee.
There will be no General Session Viewing option.
Learn more about the Mary Kay National Area Party in Pink Reception and Celebration, and find Seminar Recognition FAQs.
Register today! Hurry! The registration deadline is July 1.
Close out the Seminar year with these wins! Final month to achieve these challenges! All end June 30, 2024!
**SAMANTHA HILL**
INDEPENDENT SALES DIRECTOR, SENOIA, GA
Five tips to help you finish your Seminar year on top!
“IT’S GO TIME!”
1. **DECIDE** where you will finish. It doesn’t matter where you started, where you’ve been or what your Mary Kay career patch status is. It’s time for you to go today! Find your win, and decide what you want to accomplish. Write it down. Make your goal visible everywhere you are.
2. Make a **PLAN**. Mary Kay Ash said, “Create a plan and work it.” Know what you’re going for. Have a game plan and do the work to make it happen. There are many distractions out there. Put on your blinders and focus on faces. There’s no need to reinvent the wheel – everything you need is right here. Concentrate on the products and on finding faces at your parties. That’s where sales, recruits and new bookings come from.
3. **FOCUS**. Don’t overcomplicate. K.I.S.S. = Keep It Simple, Sweetie.
4. Embrace your Mary Kay **ENTHUSIASM**. Get excited about where you’re going in your Mary Kay business. Your enthusiasm is contagious. Soon you’ll notice others getting behind you in your goals and dreams. Get your goals out there for others to see and be inspired by.
5. Get to **WORK**! You’ve got everything in place now. It’s up to you! You **CAN** do it!
---
**FINAL MONTH!**
**Independent Sales Director 6 Most Important Things Game**
Jan. 1 – June 30, 2024
Are you reaching your goals in the 6 Most Important Things Game? Complete as many game squares as possible within the challenge time frame.
**Rewards**
- **WHEN YOU ACHIEVE ANY 3**
- Custom Mary Kay Cosmetic Bag
- **WHEN YOU ACHIEVE ANY 4**
- Custom Mary Kay® Intro Kit (one sponsor)
- Custom Mary Kay® Cosmetic Bag
- Orange Recognition of Seminar 2024
- **WHEN YOU ACHIEVE ANY 5**
- Tony Bachar Hair Brush
- Custom Mary Kay® Intro Kit (two sponsors)
- Custom Mary Kay® Cosmetic Bag
- Orange Recognition of Seminar 2024
- **WHEN YOU ACHIEVE ALL 6**
- Tony Bachar Hair Brush (2)
- Tony Bachar Hair Brush
- Custom Mary Kay® Intro Kit (three sponsors)
- Custom Mary Kay® Cosmetic Bag
- Custom Mary Kay® Cosmetic Bag
- Orange Recognition of Seminar 2024
Get all the details and FAQs!
---
**FINAL MONTH!**
**Independent Beauty Consultant 6 Most Important Things Game**
April 3 – June 30, 2024
Here’s a fun game designed to help you develop your team and boost your sales as you work toward your goals!
Independent Beauty Consultants can earn a red jacket that they can pick up at Seminar 2024 and can earn onstage recognition when they complete all six game squares on the gameboard within the challenge time frame!
Qualifications: Independent Beauty Consultants must have personal retail sales of $600 or more in wholesale Section 1 products in each of the following months: April, May and June, AND have three or more Great Start™ qualified* new team members during the challenge time frame.
Get all the details and FAQs!
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**FINAL MONTH!**
**Seminar 2024 Power of One More Challenge**
March 1 – June 30, 2024
Embrace the power of one more! Take the challenge to enhance your Seminar 2024 experience, and get access to express lines! Finish out the Seminar year by achieving the new Seminar 2024 Power of One More Challenge!
**Power Pass Qualifications:** Independent sales force members who add three or more Great Start™ qualified* new personal team members and register to attend Seminar 2024 will be entered into a random drawing to earn a Power Pass.
Two hundred independent sales force members will earn a Power Pass at each Seminar.
**Power Pack Qualifications:** The top 5 independent sales force members with the highest number of Great Start™ qualified* new personal team members at each Seminar can earn a Power Pack, which includes:
- Power Pass
- $300 gift card
- NEW! Kurt Geiger tote bag and Jimmy Choo sunglasses.
- Onstage recognition.
Get all the details!
---
*To be successful in the Seminar 2024 Power of One More Challenge, a Great Start™ qualified team member is one who has per serial retail sales of $600 or more in wholesale Section 1 products within the Great Start™ time frame. The wholesale order(s) to support this retail sales amount must be single item or cumulative order. The Great Start™ time frame is the month/ten-day Agreement is received and accepted by the Company. Orders that are placed three or more months’ New Initiative Agreement Contracts and/or Agreements are received March 2024 through June 2024 must meet qualification requirements and must place the qualifying single retail order or cumulative order by June 30, 2024.
Limit of 52 entries into the drawing.
Must be registered to attend Seminar 2024 by July 1, 2024, to qualify.
All brand names and trademarks mentioned herein are the property of their respective owners.
To receive an award, an Independent Beauty Consultant must be in good standing with the Company at the time the award is presented.
As the Seminar year wraps up, you undoubtedly are clear on your plan. Now is the time for that last concerted effort of persistence and work! You’re so close to the finish line – can’t you feel it? Finish the race with extra endurance and hard work, and see how successful you can be!
This gorgeous wing-inspired silver-tone bracelet can be yours when you have personal retail sales of $600* or more in wholesale Section 1 products in June.
FINAL MONTH! SOAR ON SILVER WINGS YEARLONG CONSISTENCY CHALLENGE
When you achieve the Soar Like Never Before Challenge each month July 1, 2023 – June 30, 2024, you can earn this custom Mary Kay® watch.
GET ALL THE SOAR LIKE NEVER BEFORE DETAILS!
*The order(s) to support the personal retail sales of $600 or more in wholesale Section 1 products requirement per month can be placed in one single order or placed in cumulative orders, as long as the orders are placed in the same calendar month. Customer Delivery Service, Guest Checkout and EZ Ship order amounts count toward your personal retail sales of $600 or more in wholesale Section 1 products. You’ll receive your monthly reward inside your qualifying order. One reward per achiever each month.
BE INTENTIONAL WITH YOUR BUSINESS.
These resources can help you glow up your business and reach the next generation of skin care-lovers!
WHAT’S NEW ON MKCONFIDENT™
NEW Product Education modules are now available! These new modules are perfect for revamping your product strategy and brushing up on your product knowledge.
- NEW! Skin Care Module including the new Mary Kay® Skin Care line.
- NEW! Color Module with updated Brow education.
Ready to grow? You can get more information on how to earn a red jacket or to qualify for the Sales Director-in-Qualification Program in these new MKConfident™ modules:
- Ready in RED: Sales Director-in-Qualification
GET INFORMED, GET CONFIDENT! GO TO MKCONFIDENT™.
NEW SOCIAL MEDIA CONTENT ADDED TO THE GREAT START™ MODULE!
Encourage your new unit members to check out the Intro to Social Media and Start Selling lessons within the MKConfident™ “Great Start” module to learn about the importance of following up with new customers. These lessons provide tips for utilizing digital resources, such as paid social media ads, to help you identify Golden Rule customer service and building or expanding your connections via social media. Check them out!
PAID SOCIAL MEDIA ADVERTISING PILOT PROGRAM
As a Mary Kay Social Creator, you know the incredible impact and opportunity that social media marketing can bring. What if, by extending your reach further than ever through social media advertising, you could unleash your true sales potential? Amazing news – you can!
Visit the Paid Social Media Advertising page on Mary Kay In Touch™ to get an introduction to this pilot program. First, set yourself up for success by reviewing and accepting the Terms and Conditions. Then check out the Raise Your Reach With Paid Social Ads video to learn about the importance of this initiative from corporate leaders and a Meta representative.
We also outline the steps to help you get started in paid social media advertising using various educational resources including:
- Step-by-step instructions on how to boost on Instagram.
- Visual examples of what paid social media ads look like and what kind of messaging you can use.
- A video demonstrating how to set up Meta ad campaigns.
WIN WITH TIKTOK!
CHEYENNE HUSAK, INDEPENDENT SALES DIRECTOR, GRIFFIN, GA
“I love using TikTok! It helps me reach people I would never meet in person, and it also helps me reach the Gen Z audience. My biggest tip is to not post only Mary Kay product information, but to post videos of family, friends, DIYs, and, of course, fun trends going viral, even if they don’t have anything to do with your business. TikTok allows you to share – and for new people to discover – your authentic self, which in turn can inspire them to purchase from you!
On TikTok, I enjoy discovering makeup trends and I love replicating these trends using Mary Kay® products. Gen Z is all about skin care, which makes creating a TikTok easier because I can post my skin care routine or even Mary Kay® products that have a specific skin care ingredient that is going viral.
Promoting your business on social media is one step of a layering process. I never post directly about the opportunity due to TikTok’s guidelines, but I do love posting about Mary Kay, sisterhood and even fun Mary Kay events.
Showing people the pink lifestyle through social media makes them want to be part of it. Not everyone has time to come to my customer events, but I guarantee they have time every day to scroll on a social media platform, so why not let them get a glimpse into my business and my life? This can lead to a sale, a facial or even a new team member.
My biggest tip for promoting on social media is to reach people who have never heard of Mary Kay. We want the next generation to learn about Mary Kay now! They could be the future product-lovers and independent sales force leaders who can carry on the Mary Kay legacy for generations to come!”
JOIN US ON TIKTOK!
Follow and share some love on the @MaryKayUS TikTok account! TikTok is an amazing platform Mary Kay is utilizing to expand our reach to the next generation of Mary Kay customers! Join us by showing a new audience what we are all about: beauty inside and out, proven skin care and fabulous makeup!
With the exciting launch of the NEW Mary Kay® Skin Care line, consider creating more skin care content in short, fun and trendy videos! Posting skin care content is a great step into the TikTok world. Not only is it quick and easy to film your regular skin care routine, but skin well always be in a steadfast routine and healthy skin outlook any trend. So if you want to grow your business, turn swaps into sales and start trending toward skin care success on TikTok. Learn more about it on MKConfident™ or from the Social Media Master Class.
READY TO CELEBRATE AMAZING ACHIEVEMENTS?
RECOGNITION CENTRAL IS HERE!
Mary Kay Ash said, “The Go-Give® Award is perhaps the greatest honor a Mary Kay Independent Sales Director can earn. Those who possess the Go-Give spirit are the heart of this Company and our shining hope for the future.” These award recipients best exemplify the Golden Rule – helping others selflessly and supporting adoptees as much as unit members.
Sonia Argueta
Lyndsey Lavallee
Bridget Penberthy
Vickie Harris
Nominate a well-deserving Independent Sales Director who displays the Go-Give spirit!
DIAMOND
Sonia Argueta
Independent Future Executive
Senior Sales Director
Began Mary Kay Business: November 1995
Sales Director Debut: June 1997
Mary Kay National Area
Personal: Lives in Daly City, Calif. Son, David; daughter, Kayla.
“I am motivated to help others because I don’t feel like it’s help. We are called to serve, and it is a way to thank God for all the blessings He gives me day by day. I am passionate about seeing women achieve their goals.”
Testimonial: Independent Senior Sales Director Rosalina Ramirez of Sun Valley, Nev., says, “Since I met her, I realized she has Mary Kay in her veins and deep inside her heart. She is always willing to help others without expecting anything in return. I am very grateful for everything I have learned from her. Thank you very much from the bottom of my heart!”
Nominate a well-deserving Independent Sales Director who displays the Go-Give spirit!
EMERALD
Lyndsey Lavallee
Independent Sales Director
Began Mary Kay Business: May 2015
Sales Director Debut: March 2019
Mary Kay National Area
Personal: Lives in Manchester, N.H.
Husband, Nate.
“I am motivated to help others because many women have poured into me throughout the course of my business and have believed in me. They’ve given of themselves selflessly and instilled the true spirit of Mary Kay within me, which has allowed me to build a life I love in all areas. I could never keep that to myself. I will always pass that on. When I help others grow into who God meant for them to be, there is no greater reward.”
Testimonial: Independent Beauty Consultant Diane Clancy of Manchester, N.H., says, “Lyndsey is not only an amazing Independent Sales Director, but also an amazing person. She is kind and giving, and she helps anyone she meets. Lyndsey helps Beauty Consultants walk through the process and reach their goals. She’s amazing!”
Bridget Penberthy
Independent Senior Sales Director
Began Mary Kay Business: January 2011
Sales Director Debut: July 2012
National Sales Director: Lara McKeever
Personal: Lives in Leesburg, Va. Husband, Bill; sons: Will, John; daughters: Hannah, Charlotte.
“I am motivated to help others because it brings me such joy to see women growing in confidence and believing in their abilities. I know I have done my job when I help a woman see how amazing she is and give her the love that she deserves. I feel so blessed to have the opportunity to do that on a daily basis.”
Testimonial: Independent Beauty Consultant Sharon Schlatter of Severna Park, Md., says, “Bridget is a top Sales Director in my eyes; she really is always learning and passing it on to us. No dust on Bridget. She’s a champion of women in her community and church and of the Mary Kay Ash Foundation™. She loves the Lord, and she represents Him beautifully.”
SAPPHIRE
Vickie Harris
Independent Sales Director
Began Mary Kay Business: January 1995
Sales Director Debut: September 2002
Mary Kay National Area
Personal: Lives in Lawton, Okla. Husband, Bruce; son, Bruce; nieces: Duana, Rellanda.
“I am motivated to help others because of the opportunities with Mary Kay and the community: to be a light, to shine His light and to enrich a life!”
Testimonial: Independent Beauty Consultant Annie Adams of Lawton, Okla., says, “Vickie Harris is my personal recruiter. She has a genuine servant’s heart and a love for all people. She is one of the most humble people I know. Vickie is truly a woman of faith. She is an encourager, a cheerleader and, most of all, a prayer partner. These are just a few of the many ways she gives of herself without complaining.”
Nominate a well-deserving Independent Sales Director who displays the Go-Give spirit!
It was 2018, and Independent Sales Director Sherrell McDonald Love was looking for new ways to meet other women.
As a wife, mother and now retired Marine Corps gunnery sergeant, she missed interaction and connection. “I longed for adult conversation. In my position as logistics specialist, I worked tirelessly with others to plan, execute and put complicated operations in place. Suddenly, I found myself at home with a teenager, a four-year-old and a two-year-old.”
A CHANCE MEETING
It was while volunteering at her daughter’s school that Sherrell met Independent Beauty Consultant Tasha Boyette, who was delivering product to several customers at the school. We started talking, and while I already had some knowledge about Mary Kay, it was just what was needed to get back into the pink bubble, so I joined her team.
PINK CADILLAC DREAMS
Mary Kay was not new to Sherrell. She had been an Independent Beauty Consultant years prior when her then-late husband encouraged her to start her own Mary Kay business. “I remember being super stoked about the Mary Kay business plan, and I always dreamed of earning the use of a pink Cadillac to drive my family to my military retirement ceremony,” she recalls. She was enjoying the Mary Kay franchise life, but then life happened, and she was deployed to Iraq. Sherrell made the decision to stop working her Mary Kay business.
“Women’s lives change every four to six months, and I am a perfect example,” says Sherrell. “Now, more than a decade later, I believe that this time around was the right time for my Mary Kay business. I knew the business well, and I wanted to re-familiarize myself with the products. Once again, I started with inventory as a Star Consultant. That plan worked well for me originally, and I didn’t want to waiver from that.”
Sherrell started reaching out, meeting new faces and holding parties.
AIM HIGH.
This time, Sherrell saw the bigger picture and rationale behind the Mary Kay business plan. “Before, I simply followed what others coached me to do, which was fine and worked, but this time, I knew my journey could be even greater. I was hungry for more education. I set goals, tracked my goals, learning more about myself and my Mary Kay business each day.”
But even with her added focus and attention, Sherrell says she struggled with team-building. “Selling was my forte for sure, and I strived to be a Star Consultant every quarter,” she says.
“At first, I would get discouraged and then become complacent about moving along the Mary Kay career path. I attended Career Conference and Seminar where I was exposed to other sales force leaders and learned so much each time.
“In 2019, I attended Career Conference outside Washington, D.C. My adopted unit was hosting the volunteers who helped with registration. At the event, the volunteers were invited to a luncheon with several Independent National Sales Directors.”
LIGHT BULB MOMENT
“I remember my conversation with Independent National Sales Director Lara McKeever. She listened as I shared my story and my team-building struggles. That conversation was raw and emotional but motivational. She was so encouraging in my own way of success and growth in my business. She shared her own story – the highs and the lows. It was like the lightbulb went off. The saying, ‘it’s not what is taught, it’s what is caught, truly applied’”
Fast-forward five years and Sherrell’s recruiter, Independent Beauty Consultant Tasha Boyette, began talking to her about enrolling in Sales Director Qualification (DIQ) Program together. “Tasha encouraged me to think big, to break down my goals in order to better achieve them. To not sweat the small stuff, and to keep on going despite any setbacks along the way. I was all in.”
STEPPING UP
Sherrell says she began to see the vision of becoming an Independent Sales Director after attending an event hosted by several Independent National Sales Directors. She was ready to take the next step on the Mary Kay journey and entered the DIQ Program in April 2023.
“The floodgates opened,” she says. “I was working full circle to book, sell and now team-build. I completed the DIQ Program and debuted as an Independent Sales Director on May 1, 2023. But in my focus to complete the program, I lost sight of My Why. My sister pulled me aside one day and said, ‘What’s wrong? Are you okay? You look exhausted and don’t seem to be having fun?’ Who else but a sister could be so brutally honest? I needed that reminder!”
READY FOR THE JOURNEY
Today, Sherrell says she’s grateful for the lessons she’s learned on her Mary Kay journey, and knows there are more to come.
“I’m beginning my leadership role, and I am fortunate to have magnificent people here to support me. I’ve learned the importance of balance and of not getting ahead of myself. My military experience definitely helps me prioritize and delegate. I’m humble and I encourage women with others so they can achieve their own goals and dreams. I want to lead with integrity, openness and joy. Mary Kay Ash built her business with ease and grace. That didn’t change the work that had to go into it, but she worked with intention, and that’s what I want to embody – to be authentically me.”
Her best advice for new Independent Beauty Consultants? “Just trust the process. You are exactly where you are supposed to be. Find the joy in working your business. Be patient because what you dream you can achieve. The race is yours, so run your own race.”
It’s here to help you reach the next generation of product-lovers who are new to a skin care regimen and are not yet showing any signs of aging!
**REGIMENS**
Each regimen includes a cleanser and moisturizer, plus a universal scrub and toner.
- NEW! Mary Kay® Hydrating Regimen, $80
- NEW! Mary Kay® Mattifying Regimen, $80
**MIX AND MATCH:** Choose from hydrating or mattifying cleansers and moisturizers based on individual skin care needs, then add the universal Exfoliating Scrub and Balancing Toner.
**HYDRATE**
Designed with normal to dry skin in mind, but is suitable for all skin types. Use morning and night.
- NEW! Mary Kay® Hydrating Cleanser, $20
- NEW! Mary Kay® Hydrating Moisturizer, $20
**MATTIFY**
Designed with combination to oily skin in mind, but is suitable for all skin types. Use morning and night.
- NEW! Mary Kay® Mattifying Cleanser, $20
- NEW! Mary Kay® Mattifying Moisturizer, $20
**UNIVERSAL**
Designed with all skin types in mind.
- NEW! Mary Kay® Exfoliating Scrub, $20
Use 2 or 3 times per week
- NEW! Mary Kay® Balancing Toner, $20
Use morning and night
**GOOD THINGS COME IN SMALL PACKAGES!**
Samples and Go Sets are great to let your customers try before they buy. They’re also great for travel!
- NEW! Mary Kay® Hydrating Go Set, $22
- NEW! Mary Kay® Hydrating Samples, $3, pk./3 on Section 2
- NEW! Mary Kay® Mattifying Go Set, $22
- NEW! Mary Kay® Mattifying Samples, $3, pk./3 on Section 2
Balancing Toner not included in the Samples due to its liquid form.
Visit [Mary Kay In Touch](#) to learn more about the new skin care line, and discover resources to help you sell.
**CONNECT WITH GEN Z!**
TRACEE WILKERSON,
INDEPENDENT SALES DIRECTOR, SAN ANTONIO, TEXAS
“Social media plays an important role when it comes to reaching our Gen Z customers, especially Gen Z. For them, it’s all about keeping up with the latest trends and then asking questions on social media to find out what they’re interested in. For example:
- If they’re into the ‘glass look,’ I let them know they can achieve that same look with Mary Kay Naturally™ Nourishing Oil and that great skin can be achieved with consistent use of Mary Kay® skin care.
- If they like the natural, no-makeup makeup look, I share with them a quick natural look featuring our fabulous mascaras and include a picture of my 18-year-old daughter who loves Lash Intensity® Mascara and Lash Love Eyelash Conditioner.
- Since Mary Kay® Mineral Facial Sunscreen Broad Spectrum SPF 30* is all the rage, I ask if my Gen Z customers and potential new customers are using 100% mineral sunscreen. This is a great opportunity to get the opinions of women aged 45–62 and introduce them to the new Mary Kay® Skin Care line and other products I think they’ll love!”
Reach Gen Z Where They Are.
Learn about the [Paid Social Media Advertising Pilot Program](#) as a resource to help you reach a new audience on Instagram. You’ll find step-by-step instructions and creative examples to help you get started.
All prices are suggested retail.
*One-of-the-counter drug product
All third-party trademarks, registered trademarks and service marks are the property of their respective owners.
BROW HERO
NEW! MARY KAY® CLEAR BROW STYLING GEL, $14
Meet your new brow hero! This is the ultimate bestie for brows that wow. Here are three ways to build your brows:
PREP: Use prior to brow liner to prep.
SET: Use after brow liner to set.
SOLO: Wear alone for a naturally sculpted look.
Complete the look! Recommend a favorite Mary Kay® brow liner and eyeliner!
ULTIMATELY YOU
MARY KAY® ULTIMATE MASCARA®, $16 EACH
The packaging has a new look, but this popular mascara features the same amazing formula and brush inside. Available in Black and Black Brown.
Effortless Application Tips
1. Pull the wand out of the tube without swirling or pumping.
2. Lightly brush mascara onto lashes working back and forth, starting at the base of the lashline and sweeping outward.
3. Using the tip of the wand, brush mascara onto lower lashes in a light, sweeping motion.
SHOW YOUR LIPS SOME LOVE!
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+ Empower
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PINK CHANGING LIVES®
In the United States, from April 26 through Sept. 15, 2024, Mary Kay Inc. will donate $1 from each sale of the limited-edition* Mary Kay® Sparkle Cream Lipstick to the Mary Kay Ash Foundation.*
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Back by popular demand, this Matte Liquid Lipstick has returned in three new shades:
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Benefits:
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ORDER TODAY!
GET SOCIAL!
Check out the Social Media Playbook for creative ideas for social media posts, photos and prompts for effective captions. There is also a paid social media ad tip included for every post to help you amplify your content to new audiences.
All prices are suggested retail.
*Available while supplies last.
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Current TimeWise® Targeted-Action™ Toning Lotion is now available in the while supplies last section of Online Ordering.
*Based on an independent third-party consumer study in which 125 women used the product twice a day.
**Sell-ebrate Summer!**
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Mary Kay’s first 100% mineral sunscreen that protects against both UVA and UVB rays. Lightweight and low shine, it’s perfect for wearing under makeup or for applying to bare skin for daily sun defense. It’s suitable for all skin types, including sensitive skin.
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Apply Mary Kay® Mineral Facial Sunscreen Broad Spectrum SPF 30+ evenly to face, neck, chest and backs of hands—anywhere skin is likely to receive sun exposure.
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**Tip:** Spending time outdoors? It’s recommended to reapply every two hours (more frequently if swimming or sweating).
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All prices are suggested retail.
*Over-the-counter drug product
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NEED SOME SOCIAL MEDIA GUIDE-ANCE?
Check out the Guides section in the My Mary Kay Facebook group for access to the Paid Social Media Advertising Pilot Program and TikTok guides! These guides are curated just for you with helpful information, including insights in the comments from your fellow independent sales force members!
Rise Up and FINISH STRONG
FACEBOOK LIVE
June 12 in the My Mary Kay Facebook Group
English: 11 a.m. CT Featuring Samantha Hill, Independent Sales Director, and Mike Strong, Managing Director, U.S. Sales Force Development.
Spanish: 11:45 a.m. CT Featuring Pricila Martinez, Independent Executive Senior Sales Director, and LuzAngelica Monohan, Managing Director, U.S. Sales Force Development.
Tune in to this exclusive Facebook Live that will provide tips for some last-push power moves as you finish out the last few weeks of the Seminar year.
Not a member? Join the My Mary Kay Facebook group!
|
NOW, MORE THAN EVER,
ALL RADIO DEPENDS ON
THE RADIO SERVICEMAN!
"Keep 'em listening!"
Despite restricted production and material shortages, as a radio service engineer, we urge you to "keep 'em listening!"
We know how essential a home front job you perform. And, you know you can depend on Mallory for practical help in meeting the brain-racking radio service problems imposed by current shortages and restricted production.
Although we are up to our ears in wartime production, we haven't forgotten your needs for one instant.
We are making every effort to provide adequate supplies of Mallory Approved Precision Parts. Our research laboratories are working night and day, coping with the present, anticipating the future. Problems induced by material shortages have, in many cases, resulted in engineering adaptations that mean real progress in good servicing.
And we are getting a fine demonstration of how beautifully the Mallory policies of standardizing parts and simplifying lines have operated to aid servicemen.
You can depend on Mallory and Mallory Distributors for real help, while you work to "keep 'em listening."
P. R. MALLORY & CO., Inc.
INDIANAPOLIS INDIANA
Cable "Pelmallo"
WE’VE GONE OUT FOR VICTORY!
On April 7, the last Camden-built RCA Victrola rolled off the lines, to make way for war work
RCA Victor’s last instrument—number 17,199,547—rolled off the Camden production lines on April 7. This set was presented to the Georgia Warm Springs Foundation by the RCA Victor Family—to uplift the spirits and morale of the Foundation’s young patients.
Actually, the chassis of this last set had been completed on March 5—50 days ahead of the deadline set by the War Production Board!
For as early as September, 1940, we said in a published advertisement, “With RCA Victor, Defense comes first. By comparison, we hold nothing else important.” That policy of preparedness has made it possible for us to “Beat the Promise” to America not once, but many times—hastening the day when RCA Victor Dealers can again sell RCA Victor radios and phonograph-radios to an America at peace.
We are grateful to the many RCA Victor Dealers, whose cooperation helped and is helping us to participate in the war effort on a fuller war footing. By selling RCA Victor radios and RCA Victrolas in the past, you helped us to grow and expand—made possible the facilities now serving America-at-war. By foregoing civilian instruments today, you are helping us to devote our attention to war production.
You, the Dealer—we, the Manufacturer—are now all in the same business. That business is Victory!
BUY WAR BONDS EVERY WEEK!
All out Performance with METAL TUBES
Metal Radio Tubes can be depended upon for all-out performance. That’s why 80,000,000 metal tubes are in use. That’s why we make and recommend metal tubes for best results.
Handle Ken-Rad Metal Tubes and Be Sure of Satisfied Customers.
KEN-RAD
Metal Radio Tubes
KEN-RAD TUBE & LAMP CORPORATION, Owensboro, Kentucky
Keep 'em Playing
Use Meissner Replacement Parts
Now, more than ever before, it is vitally necessary to keep radio receivers in good condition... Meissner replacement parts have proven their ability to satisfy even the most discriminating serviceman.
See your Meissner distributor for replacement Coils of all types... I.F. Transformers... R.F. Coils... Antenna Coils... Coil Assemblies... FM components... R.F. Chokes... Switches... Dials... Variable Condensers... Trimmers and Padders... Relays... Wave Traps... Chokes... Interference Filters... Crystal Calibrator... Signal Splicer... Signal Shifter... Uni-signal Selector.
14-1027 Adjustable R.F. Coil
14-1028 Adjustable Oscillator Coil
14-1026 Adjustable Antenna Coil
Why try to repair defective coils or wait to obtain exact duplicates, or wait to have coils re-wound. Meissner Universal adjustable series will replace defective antenna, RF and oscillator coils in over 85% of present day receivers. Adjustable Ferrocart iron cores used in the Universal replacement coils give additional gain and selectivity to the receiver. Try them on your next coil replacement job. In stock at all Meissner Jobbers.
Meissner Quality Never Varies
Write Dept. RT-5 for Complete Catalog
Meissner
MOUNT CARMEL, ILLINOIS
"PRECISION-BUILT PRODUCTS"
RADIO RETAILING Combined with RADIO TODAY, May, 1942
"Lifers" in a Good Cause
THE radio tubes you see here are good radio tubes—every bit as good as Sylvania’s best. Yet not one of them will ever live to see the inside of a receiving set.
Each has been sentenced to a life term in the testing racks—where, under conditions akin to actual service, it is subjected to the most thorough checks and trials our engineers can devise.
It’s pretty tough on these tubes—but knowledge gained from their ordeal is one reason for the matchless performance and long-lived efficiency of the whole Sylvania line.
Another important reason is the fact that radio tube-making is Sylvania’s one job in radio. That means all of our time and skill are devoted to improving and refining our product, to keeping it first in quality.
So it’s easy to understand why we can offer you such a clean franchise—with no ifs, buts or provisos.
With radio sets now in use slated to stay for the duration, fine tubes are more essential than ever before. Isn’t it wise to deal with an organization in which experience focuses on making only the highest quality you can buy?
IT PAYS TO SELL SYLVANIA
SYLVANIA LOCK-IN RECEIVING TUBES
RECEIVING TUBES REGULAR STYLE
SYLVANIA
RADIO TUBE DIVISION
HYGRADE SYLVANIA CORPORATION
New York City
EMPORIUM, PA.
Salem, Mass.
Also makers of HYGRADE Incandescent Lamps, Fluorescent Lamps and Fixtures
RADIO RETAILING Combined with RADIO TODAY, May, 1942
On their RECORD—
ELECTRICAL instruments have been called upon to do a big job—and that is the kind of job that Simpson Instruments are built to handle.
This fact stands out in the Simpson record. Try to find a Simpson model that has not been a complete success. Try to find a Simpson Instrument that has failed to do its job properly. Can you think of any product for which a better record could be cited?
If your requirements are vital enough to give you the right to buy instruments, they are vital enough to deserve the best.
On their record — past and present — that means Simpson!
SIMPSON ELECTRIC CO., 5208-18 Kinzie St., Chicago, Ill.
MODEL 260
High Sensitivity Tester
Here is a typical example of Simpson leadership. At 20,000 ohms per volt this tester is far more sensitive than any other instrument in its price range—covers a wide range of unusual conditions which cannot be checked by ordinary servicing instruments. Six voltage ranges—0.25, 10, 50, 250, 1000 and 5000 V.—both AC and DC. Current readings from ½ ohm to 10 megohms. Five decibel ranges, -10 to +52 DB.
The SIMPSON Full Bridge Type Movement, with Soft Iron Pole Pieces
• This is the fighting heart of every Simpson Instrument. Soft iron pole pieces distribute magnetic flux evenly, assuring perfect accuracy. Bridge type construction holds the moving assembly always in perfect alignment.
Always Specify Centralab
CONTROLS
Featuring the famous WALL TYPE resistor element which hugs the inner circumference of the black moulded bakelite case. Exclusive non-rubbing contact assures quiet smooth rotation and long life. Available in STANDARD, MIDGET AND ELF with or without switch cover.
RESISTORS
Available in two types: RADIAL LEAD and AXIAL LEAD. Both feature a center core of resistance material, surrounded by a dense shock-proof ceramic providing strength and protection against humidity. Core and jacket are fired together at 2500 degrees F: into a single solid unit, hard and durable as stone.
SWITCHES
Both selector and transmitter switches are available in an infinite number of combinations... ideal for high frequency circuits with minimum losses.
Most switches are supplied with an "adjustable stop" index... permitting the selection of from two to twenty-three positions.
CENTRALAB through every emergency continues to adhere to its policy of "performance plus" at all times.
The slogan "ALWAYS SPECIFY CENTRALAB" which has appeared in our advertising from the very inception of this name is as timely today as it was fifteen years ago.
Today as then... there is no substitute for QUALITY.
Write for Catalogue No. 23
CENTRALAB: Div. of Globe-Union Inc.
Milwaukee • Wisconsin
HIGH FREQUENCIES
Ultra HIGH FREQUENCIES
30,000 KC.
HERE ARE THE INSTRUMENTS FOR YOUR MEASUREMENT NEEDS!
50 kc to 33 mc fundamental frequencies...
WESTON Model 776 OSCILLATOR
For testing broadcast and intermediate frequencies... in vacuum tube circuits. Accurate within 1/2 of 1% on broadcast bands—1% on short wave bands. Has large 330° hand calibrated dial. WESTON AAC (Automatic Amplitude Control) circuit assures uniform output level regardless of frequency (no trimmers or padders used.) Triple shielding eliminates leakage.
22 mc to 150 mc fundamental frequencies...
WESTON Model 787 UHF OSCILLATOR
For testing transmitters and receivers—stationary and mobile—AM and FM types operating in high frequency bands. Inductive tuning system provides fundamental frequencies with higher resonant circuit voltages and broad tuning range. High order of stability and reset-ability over entire frequency band. Reads 40 kc per division at 40 mc. Modulation frequencies 400-1300 and 3000 cycles. Compact, Portable, battery operated.
For potential measurements over broad frequency ranges...
WESTON Model 669 Vacuum Tube Voltmeter
For measuring voltages in vacuum tube circuits where frequency is a factor. Has low input capacity of 5 microfarads with 5" leads. All ranges operate direct to measuring tube grid, providing a maximum input resistance and impedance device. Loading effect on circuit under test is only that of the tube itself, as no grid resistors are used on 0-1-2-3-6-8-12-16 volts ranges. Phone jacks provided for making audible tests. Ranges 0-12-30 -60-80-120-160 volts full scale with multipliers.
for your panel and built-in needs—
WESTON Thermo-Ammeters
WESTON improved design of heating elements provides extreme accuracy on high frequencies (frequency error less than 2% up to 65 mc). Available as ammeters and milliammeters in ranges for all transmitters including portables. Also a complete line of ultra-sensitive portable instruments for high frequency laboratory measurements.
...for complete information on these and other WESTON instruments see your local WESTON representative, or write Weston Electrical Instrument Corporation, 581 Frelinghuysen Avenue, Newark, New Jersey.
Laboratory Standards... Precision DC and AC Portables... Instrument Transformers... Sensitive Relays... DC, AC, and Thermo Switchboard and Panel Instruments.
Specialized Test Equipment... Light Measurement and Control Devices... Exposure Meters... Aircraft Instruments... Electric Tachometers... Dial Thermometers.
FOR OVER 54 YEARS LEADERS IN ELECTRICAL MEASURING INSTRUMENTS
RADIO RETAILING Combined with RADIO TODAY, May, 1942
Next to the Stars and Stripes . . .
AS PROUD A FLAG AS INDUSTRY CAN FLY
Signifying 90 Percent or More Employee Participation in the Pay-Roll Savings Plan
It doesn't go into the smoke of battle, but wherever you see this flag you know that it spells Victory for our boys on the fighting fronts. To everyone, it means that the firm which flies it has attained 90 percent or more employee participation in the Pay-Roll Savings Plan . . . that their employees are turning a part of their earnings into tanks and planes and guns regularly, every pay day, through the systematic purchase of U.S. War Bonds.
You don't need to be engaged in war production activity to fly this flag. Any patriotic firm can qualify and make a vital contribution to Victory by making the Pay-Roll Savings Plan available to its employees, and by securing 90 percent or more employee participation. Then notify your State Defense Savings Staff Administrator that you have reached the goal. He will tell you how you may obtain your flag.
If your firm has already installed the Pay-Roll Savings Plan, now is the time to increase your efforts: (1) To secure wider participation and reach the 90-percent goal; (2) to encourage employees to increase their allotments until 10 percent or more of your gross pay roll is subscribed for Bonds. "Token" allotments will not win this war any more than "token" resistance will keep our enemies from our shores, our homes. If your firm has yet to install the Plan, remember, TIME IS SHORT.
Write or wire for full facts and literature on installing your Pay-Roll Savings Plan now. Address Treasury Department, Section D, 709 12th St., N.W., Washington, D.C.
Make Every Pay Day "Bond Day"
U.S. WAR BONDS • STAMPS
This Space is a Contribution to Victory by RADIO RETAILING-TODAY
RADIO RETAILING Combined with RADIO TODAY, May, 1942
Where lives hang in the balance
They use RAYTHEON tubes!
Submarine duty is exacting work... mistakes cost lives... radio tube performance must be of the highest quality especially when used in submarine signalling work... they must be on the job!
RAYTHEON TUBES are daily performing this difficult task. RAYTHEON quality is meeting all these exacting requirements for dependable performance.
This unusual service is typical of the plus advantages you gain with RAYTHEON tubes... servicemen and dealers are making more money on RAYTHEON replacements... your RAYTHEON distributor can tell you why... see him today!
Raytheon Production Corporation—Newton, Mass.
NOTICE! If you have not obtained RAYTHEON'S interchangeable Tube Chart, it is important to get one of these cards in advance from your RAYTHEON Jobber. Speeds up radio repair service and simplifies your tube stock by elimination of a large number of types.
Los Angeles
New York
Chicago
Atlanta
RAYTHEON RADIO TUBES
DEVOTED TO RESEARCH AND THE MANUFACTURE OF TUBES FOR THE NEW ERA OF ELECTRONICS
"I've found a way to make radio service business pay!"
—BOB KIPLE
If you want to build repair and service business, take a tip from the experience of the Broadway-North Side Radio Co., Chicago.
"One reason I'm not too worried by the shortage of new radios," says Robert R. Kiple, owner and manager of the Broadway-North Side Radio Co., Chicago, Ill., "is that we started out as a service business 10 years ago. And right from the start we hit on the one way to make a service business pay—do the job right the first time!
"When we find a resistor or bypass bad in a set, we do not let it go by simply replacing the part. We bring the set in and go over every circuit carefully. Maybe it has nothing more than that wrong, but often we find little troubles that would be worse in a couple of months if neglected. By telling the customer the truth, we fix the set right the first time, so we're not constantly having to bring it back.
"You'd be surprised how much people talk about this kind of service. It's given us a reputation for fair-dealing and good workmanship. And they don't mind paying an inspection charge, because they know we're giving them something for their money.
"Looking ahead to a boom in service business, we have built up our stock of tubes and parts about 250% over normal. Of course, the Stromberg-Carlson 24-hour service on factory parts is a big help in emphasizing speed on our repairs.
"We are holding our trade in other ways, too. We're continuing our Red Book advertising, which we were the first in this vicinity to adopt. And we are paying extra close attention to our record business, studying the music, composers, and artists for clues to the potential popularity of new records as a guide for our customers and for us in our buying.
"Between our active service business and our healthy record department, we are confident we can hold our customers and carry on successfully until new radios are again available."
Commenting on the customer attitude toward a minimum service charge, Shop Foreman R. F. Graham (above) says: "Most people realize that a thorough test costs money, and agree that a minimum of 75c for this service is sensible."
At the Broadway-North Side Radio Co., records are a large and profitable department. With 12 listening positions (including both head-phone spots and booths), and a diversified assortment, sales of 400 records have been chalked up in a single day!
"We have six working positions," declares Mr. Kiple, "plus a large general bench; all with excellent fluorescent lighting. We have plenty of room for speedy service. And our testing equipment is geared to handle almost any type of trouble."
A "Shot-by-Shot" Report, Via PA
On the day Lieutenant O'Hare shot down five Jap planes, sound amplifiers also did a job, the story of which seems not to have gotten into the news.
Ordinarily the men below decks on an airplane carrier or other ship in action, know little about what is going on, above and around the ship. They have to attend to their jobs, shut away from the stirring events that may be taking place nearby.
But the day of Lt. O'Hare's exploit all that was changed! When the doughty flier went into action, an officer in an observation post on the plane-carrier grabbed a microphone feeding loudspeakers all down through the great ship. And as the American ace dived in, destroying one Jap plane after another, the entire crew of the big carrier, working at their own battle positions, heard a "blow-by-blow" report of the battle, over the ship's loudspeaker system!
War Work for Distributors
Many automobile dealers have turned their salesrooms and repair departments into light assembly plants for war work. Other local business men have become sub-contractors for the manufacture of munitions and war supplies.
Isn't there an idea here for radio men also to think about? It has been suggested that radio manufacturers who have war work, could sub-let some of the simpler assembly jobs to their distributors—who soon may have little merchandise to distribute. Such jobber organizations could thus put their workers and workrooms on the job of war assembly and munitions making.
It would be a way to hold the jobbers employees together. Also it would tie manufacturer and distributor together on a new basis, for the duration—and against the day when distribution will again be needed and in a big way!
The Search for New Lines
Whatever else is gone, the cautious buying habits of radio dealers and jobbers remain. They need new lines to replace their thinning stocks of sets, but they aren't taking on the first fancy merchandise that comes along. They want something suitable for a warring nation, related to radio if possible, and not "chicken feed."
Our inquiries on this subject have ranged across the U.S.A. and some dealer reports are eye-openers. Many are going in for service, and are developing repair promotion and receiver check-up to a high degree. There's also an extra stir in the sound business—public-address installations for war plants. Some outfits have actually turned to light assembly and manufacture, converting their service floors to machine shops, and turning out small radio parts for the Army, Navy or Signal Corps.
But as for merchandise itself, the talk swung to phonograph records for many weeks, and retailers looked with relish at the flourishing disc business. When WPB cracked down on the supply of shellac, however, another good bet went out the window. Now they're talking re-conditioned radios, furniture (particularly if related to radio and records), blackout devices, stirrup-pumps, games and toys, picture frames, books, wallpaper and paint, record cabinets and indexes, and small musical instruments.
By next month, a number of substantial trends should emerge from this "search for substitutes" and our plans are to publish them under the general head of industry conversion. Or maybe the report should appear under several heads, such as resourcefulness, skill, ingenuity, and discretion, and do proper honor to the merchandising judgment of the men in our business.
"Service Women"—or Skirts at the Service Bench!
For years much of the assembly work and soldering in the making of home radio sets, has been done by women. Women are painstaking and attentive to details; they handle tedious, repetitive jobs more easily than men do.
So now, with radio men being called to the colors, it is proposed that this method of using women—so successful in radio manufacturing—be applied to the repair and servicing of home radios. The plan proposed is that receiver troubles would first be diagnosed by a technically-qualified man supervisor, and the necessary work indicated for each set. Actual repairs and replacements would then be completed by women workers.
The weakness of such a plan lies, we think, in the fact that trouble in a radio set usually takes more time to find than to repair. The actual soldering is a small part of the job. Diagnosing the trouble will still be the chief problem.
So the proposed "woman technique" doesn't promise much saving, until Miss Service Gal can be trained to use her noodle and her technical knowledge, as well as her skillful fingers!
Radio WORKS to WIN
Big rally of workers in the war-converted plant of Stromberg-Carlson as Lt. Comm. A. E. Rice, USNR, commends war production drive there.
No more refrigs. from Nash-Kelvinator, Detroit—they're all celebrating start of more war work.
Right, U.S. war birds bristle with antennas and get signal to "peel off" for attack. (U.S. Navy Photo)
Map and speaker unit above illustrate Cleveland's own "radio network" to warn schools in air raids.
At left, NYC television studios produce a bomb-spraying scene as part of official instruction shows for 50,000 air raid wardens.
Above is Mary Small, a Blue Network star singer on "Daughters of Uncle Sam," another show in the Victory spirit.
Upper left, a 6-lb. portable radio developed by the Forest Service, cooperates with the Army in protecting vital U.S. areas.
Directly at left, the CBS actress Ann Eden of the "They Live Forever" program, a powerful dramatization of war heroes' efforts.
Below, an officer aboard a Navy patrol bomber stands guard over the Atlantic seaboard, depending on throat microphone.
Special Radio Devices and Apparatus Needed in Wartime Civilian and Plant Defense Are "Down Radiomen's Alley"
- Present war conditions in every community have opened up new sales and installation opportunities for local radio men.
There are air-raid alarms and switches to be sold and installed, and anti-sabotage devices to be provided. Sound equipment finds many new uses for both civilian and industrial purposes. And for the radio man who makes his place of business local headquarters for war-emergency equipment (most of which is radio-tube apparatus) there are other supplementary products which the public also wants, like luminous paints, blackout lamps and lamp shades, and window black-out material.
The radio man has an inside track to this special wartime business because most of the major devices used in blackout alarms and anti-sabotage devices employ tubes, amplifiers, relays and sound equipment. Because of the radio man's special knowledge he is better equipped than everyone else in town to supply and install this emergency apparatus.
For industrial-plant installations, of course, he can secure equipment under the plant's usual priority when the plant is engaged in war work. And for miscellaneous alarms and sound systems, demanded by his local civilian defense group, the radio man can if necessary adapt existing radio and sound equipment on hand or in stock, to serve special war-time emergency services.
Blackout Switches
Extinguishing lights on business premises and in show-windows at the time of an air-raid alarm, is one service for which there has been the greatest demand. Since the local street lights are invariably extinguished in such a blackout, a photo cell relay tying up the store lighting with the street illumination, will provide automatic extinguishing of the store lights. The photo cell merely has to be focussed on a nearby street light. When the street lights go out, off goes the store lighting, too.
Another method of extinguishing lights utilizes the carrier-wave of some broadcast station received well locally. If the station shuts down, as is expected to happen during an air-raid alarm, the absence of carrier causes a relay to drop back, opening the lighting switch. Even should the broadcast carrier come back on, the lights preferably remain off until manually restored by an authorized person; this precaution is provided to prevent saboteurs from turning on lights during an air raid. Such radio-controlled lighting switches have been developed by a number of different inventors and have been applied to controlling street lights, signs, store lights, show-windows and residence lighting.
Audible Alarms
Such a relay mechanism, actuated by interruption of the carrier, can also be used to sound an alarm. But a simpler air-raid alarm method employs the oscillation of the radio set itself, which sets up a loud howl if the station carrier is interrupted.
Meanwhile the recently publicized "Alert" receiver, which embodies tuned relays sensitive to sub-audible frequencies such as 24 cycles and 36 cycles for "on" and "off" respectively enables local alarms to be registered or turned off, by putting the corresponding sub-audible frequency on the station's carrier. This can be done without the actuating impulses being noticeable on the station's regular broadcast program.
Public-address systems and intercommunicators have been increasingly installed and used in war plants for spreading alarms, delivering messages, providing music, and disseminating inspirational talks to groups of workers.
Siren amplifiers fed by oscillators, also make a valuable form of air-raid alarm for industrial plant and small-town use. The siren sounds can be fed over plant loud speakers, and in towns, through steeple and tower reproducers, to cover wide areas.
Recordings are now available to serve in the same way. By playing one of the "air raid alarm" records, a variable-pitch tone lasting two minutes, goes out over the plant or town sound system. An "all clear" signal is also supplied on the same record. This "all clear" consists of a single continuous, pleasing note, lasting two minutes.
**Anti-Sabotage Protection**
Anti-sabotage protection for industrial plants and key transportation centers, can be provided by photo-cell alarms. These preferably are invisible infra-red or ultra-violet beams, and sound an alarm if any trespasser intercepts the beam. Another new development is the "acoustic fence" which, as described on a following page, employs sound pick-ups at intervals along the fence, so that the entire factory enclosure serves as a listening post if anyone attempts to climb the fence, cut an opening in it, or tunnel under it.
All of these air-raid alarms, blackout switches, and anti-sabotage devices employ tubes, amplifiers, transformers, condensers, relays and other parts familiar to radio men. For war-plant installations carrying the necessary priority, the usual method of priority purchases applies. To meet demands of civilian-defense uses, radio men can often adapt parts and equipment from their own radio stocks, to these new war-time applications.
**Tube Devices Radio Men Can Sell**
*Photo-electric Switches—Turn off house lights or ring alarm if street lights go off.*
*Radio Switches and Alarms—Turn off lights or ring alarm if broadcast station goes off the air.*
*"Radio-alert"—Special receiver which picks up inaudible impulses carried by broadcast station operates alarm.*
*Public Address Systems—Special provision for receiving and spreading air-raid alarms, sabotage alarms, etc. in industrial plants.*
*Siren Amplifiers—Air-raid warnings for plants, towers, steeples.*
*Photo-cell Anti-sabotage Beams — To form invisible fence around plant (infra-red or ultra-violet) for detecting trespassers.*
*"Acoustic Fence"—Phonograph pickups linked by wires, enable faint sounds to be detected and recorded.*
*Intercommunicators—For factory intercommunication and alarms.*
*Portable PA Systems—For alarms, department speeches, etc.*
*Luminous Paints—For marking signs or danger spots in event of blackouts.*
*Blackout lamps—Incandescent bulbs and special shades which reduce visibility of light source.*
Portable sound equipment such as this unit is finding wide use in the entertainment of employees, making special announcements at group meetings, etc.
Confronted by war conditions, every man in the radio business must make new and drastic business plans.
This means facing the situation squarely, and applying the remedies promptly.
Every radio-dealer must control and reduce his expense. Here are some ways to go about cutting business outgo:
1. **Prepare a Budget**
Obviously, before the dealer can do an intelligent job of expense reduction, he must know all he can about his expenses.
This means that, as a business man, you must study your expenses, and challenge every item.
Then, based upon your keen knowledge of your own expenses, prepare a budget, an exacting budget. And resolve that the only way you will depart from that budget will be to reduce it.
2. **Check Systems, Methods, Practices**
Then you must check your system, your methods, what you do and how you do it, in relation to your expenses.
Everything you do—and plenty that you don’t do—costs you money. Anything you do which does not pay for itself, you should ruthlessly discard. Root out every expense which is not absolutely necessary and stop it.
In every business you will find many such expense-producing ideas:
- Records which take time and money to keep, yet are seldom if ever used.
- Needless telephone calls.
- Electricity which is consumed far beyond actual needs, radios left turned on, unnecessary lights burning.
- Stationery wasted, small parts in the service department broken, lost, wasted.
- Cabinets scratched, deliveries poorly timed and routed.
Go on down the list and you will be surprised how much money you waste, how much you do that is not really necessary. And a dollar saved is a dollar earned.
3. **Reduce Sales and Office Force**
You must be prepared to work harder yourself and expect your co-workers to do the same. Because your very future existence is at stake, you should reduce your office, sales and other staff as much as you can—as soon as you can—now!
Only such employees as you know give more to your business than they take from it, can you afford to keep. Sentiment has no place in war.
4. **Consolidate Your Delivery Service**
Your delivery truck, or car costs a lot of money. And much of this outlay can easily be saved by a little planning. Arrange your deliveries so that your truck goes to only one section a day, and makes only one trip a day.
Some customers will have to wait a little, but the saving of tires, gas and oil, is at least as important as saving money.
Since there are lots of other retailers who also have delivery trucks going over the same areas, it would be an economical and patriotic idea for local retailers to consolidate their deliveries and share the expense.
5. **Sub-let Part of Your Store**
Sooner or later, as sales fall off, dealers will have no practical use for the expensive space once needed as a sales floor. In this space, for which you pay rent, but no longer require, is another source of expense reduction. There are other small business men who themselves either have too much space or less well-located space, who would be glad to sub-lease a part of your store space. This is being done in large cities and small towns, by many different lines of retail business.
6. **Get Together With Others**
In almost every town there are many small businesses which would be much more healthy if they were to combine. And now there is a pressing need to save expense, too. It is entirely appropriate to consolidate your business—merge it, with another, save money for both and have a better business, too.
Look around you, at the music dealer, hardware dealer, electrician, gift shop, stationer, furniture dealer, appliance dealer. How many of them, do you think, do not have the same problems you do.
Why not talk it over with them and among them is apt to be one who also sees how he will be better off by sharing his troubles and his business with you.
7. **Take a less Expensive Store**
If merging your business with another seems too drastic and your pride refuses you permission to sub-let part of your store, then you should give serious consideration to taking a smaller, less expensive store as soon as you can, and cut your needless rent expense in that way.
Bear in mind that rent is usually based upon the traffic density of the location. Radio dealers who battle this problem through to the end, no longer require a high traffic location, or as much space as heretofore. You can neither justify the location if it does not provide the business, or afford the rent when it becomes disproportionate to sales.
8. **Renew Lease "on Sales" Basis**
Most dealers will prefer to sub-let a part of their store now and have their good location ready for the days of peace and good business which must come. If your lease expires soon, negotiate a renewal on a "percentage-of-sales" basis. That is, the amount of rent you will agree to pay will depend upon the volume of goods sold.
It is common practice, sound economics, and good business for both landlord and tenant, in the long run.
9. **Turn Off Your Window Lights**
Show windows in a high traffic location are the "eye-ways" to your store. But in war time they are a menace to a blackout—and can hardly help you sell merchandise you do not have for sale. So turn off your display window lights and save the "juice."
10. **Put 2-to-1 Flasher on Electric Sign**
You can also cut down the cost of your large electric signs by 60 per cent—with a 2-to-1 flasher, which you can buy for little, or make for less. Such a flasher should turn your signs on and off—being off twice as long as on. An "on 10, off 20" or "on 15, off 30" second cycle is practical, and even more effective than a steady sign.
11. **Go on a Cash Basis**
Your "bad debts" may only amount to 1 per cent but if you have to borrow money to operate, while you loan your own money to your customers, it costs you more than you think, to give credit. You can save money, headaches and heartaches, if you go on a cash basis.
(Continued on page 22)
Records Limited
WPB order limits shellac and starts new trends
- Outwardly the record business continues on its brisk way with national promotions still in evidence and wartime demand on the upswing. But inwardly there was considerable disturbance over the order from the War Production Board, limiting the amount of shellac available for disc manufacture.
The upshot of the order was that "during the period from the effective date hereof (April 14) to June 30, 1942, and during each calendar quarter thereafter, any person may consume shellac in the manufacture of recording and transcription materials in an amount not to exceed 30 per cent of the amount of shellac consumed in such manufacture by such person during the corresponding period of 1941." Inventories of 10,000 lbs. or over were frozen to the extent of 50 per cent.
Immediate speculation began as to what effect this would have on such matters as summer deliveries, new emphasis on certain types of records, development of substitutes, reclaiming old records, etc. Efforts of the editors of this magazine to get new-policy statements from the manufacturers revealed only that most of them were still studying the WPB order, and had not outlined new steps.
However, discussion in the trade indicated that, in general, the industry would accent higher priced discs; that duplication of popular tunes would be eliminated and some minor artists dropped; that substitutes could be developed of sufficient quality to interest the public; that everybody in the business would begin to emphasize the value of old records of the non-laminated type.
New Trends
Details on some new plans for gathering up old records will probably be announced shortly. This is of special interest to dealers because their stores are naturally the depots for large-scale turn-ins. There were nearly 315,000,000 records sold in the last five years, not counting any of the terrific sales of 1942, and what the resourceful manufacturers can do with the reclaimable part of this mass of shellac, will considerably improve the picture for the retailer.
RCA Victor had already undertaken a nationwide reclaiming of old records before the WPB order was issued. The millions of people who have non-active records lying around in their attics, basements, and storage areas, have already had some indication that they should drag them out for the emergency.
Just the Hits
One record manufacturer, the Classic Record Co., 2 W. 46th St., New York City, has announced that it will continue its 35c Elite label, and will emphasize its new "Hit" disc at 50c. Eli E. Oberstein, a Classic executive, says that "since we concentrated on the hit (every side a hit) merchandise, we will continue this policy and put out few releases and make every record a salable one." He estimates that deliveries will settle down to around 50 to 75 per cent of last year. Classic also offers a 10 in. classical Concertone record at 50c, and a 12 in. Concertone classical at 75c.
Another executive of the Classic firm, A. E. Middleman of the Pittsburgh, Pa., offices at 524 Penn Ave., adds that "we are augmenting our list of recording artists, but we do not intend to drop any of our present orchestras." He reveals also that Classic is announcing several campaigns on scrap records.
Immediate Jobs
Meanwhile, there are several steps that a record retailer can take, to get set for the new situation. In the first place, he should keep up his record promotion within reasonable limits, and cut out all of those bargain sales. This is no time for an exhibition of distress merchandise.
Certainly this is an excellent time for a dealer to merchandise all those accessories which contribute to the long life of records. Folks will be interested in this, sure enough, if there are to be fewer records on the market.
A retailer should also keep in closer touch with his local jobber. Things are moving so fast that a retailer must keep on his toes and get all the news from his immediate source of supply.
In his contacts with record buyers, the dealer should immediately establish himself as headquarters for old records. His customers should have the idea that he will be offering the best values available, and that he stands right in the thick of the effort to help his country and his industry by being headquarters for useful materials.
In peacetime it was WILCOX-GAY RECORDS for the pursuit of happiness.
In wartime it is WILCOX-GAY INSTRUMENTS for the pursuit of the AXIS.
Here Are the Blackout Rules
WILCOX-GAY CORPORATION
CHARLOTTE, MICHIGAN
"Producing for war... planning for peace"
Japan's New East
Send Friends Toward in Move to China
By the NEW DAILY Japanese major offensive of opening a line to isolate China and India at a time Chinese allies operate rearlay the communists' oil wells.
The official Satoween is now
Where does the manufacturer's agent stand in the war picture? Has he revamped his business methods? How does he fit into the new war production plans?
For the answers to these questions, on another important branch of radio parts distribution, Radio Retailing Today has gone to a veteran "rep" who finds himself in the thick of wartime demands. He's David Sonkin, who has for many years been the national secretary of "The Representatives," radio's nationwide organization of agents.
Work Faster
Fundamentally, a rep's job is to sell radio parts to clients who buy in large quantities. But in a sense, the lessons now being learned by these men apply to anyone in the business of dispensing parts, in large quantities or small. Many of the agents handle other lines besides radio parts, of course, but their combined activities may still be regarded as an illustration of the new tempo in distribution.
What the representative must do, these days, is to smooth out the route that runs from his source of supply to his customers. Conditions demand that things go more swiftly along that route. The key word in 1942 is speed.
A rep should now adopt the practice of taking the order first, and working from that. He gets better results if he confronts his supplier with a definite order, rather than a conditional agreement from his client to the effect that "I'll order the parts if you can get me the '10' series by June 1st."
With the definite order in hand, it will often be found that all the production factors will work more efficiently to fill it. The rep's contact with the supplier should be so close and so sensitive these days that he should know exactly how these factors will function under various circumstances.
Adapt if Possible
It often happens, however, that standard stocks can be adapted to meet rush requirements, even if the "specs" seem to forbid it, to start with. Everybody realizes that manufacturers' molds are, as one of them put it, "not made of cream cheese." New and special products cannot be turned out over night. Reasonable concessions are being made.
In a situation like this, it will be seen that a rep should be specially resourceful. It will help if he is a well-grounded technician who understands the conditions operating around his product as well as in it.
Need to Circulate
This brand of activity indicates a lot more personal running around on the part of the rep. More of the "on the lot" activity and less of the sitting at a desk. The take-it easy days of leisurely correspondence are gone, along with a lot of the other pre-war practices.
As far as office work is concerned, it should make an extra attempt to keep up-to-the-minute files of factory prices and delivery conditions. The office should keep in mind that the supplier is up to his neck in production problems, and that the traveling rep needs the latest bulletins in his pocket.
If he takes all these tips, the rep should begin to notice that he is being given more responsibility, both by the suppliers and by those who use his services. Each of his customers will regard him as a technical advisor, almost as though he were a part-time member of the staff. They will have more confidence in the rep, at a time when such a feeling is important to radio's war effort.
COMMUNIQUE TO REPS
1. On each job, take the order first.
2. Learn how to adapt and utilize stocks already on hand.
3. Keep up to the minute on what designs are to be frozen.
4. Give more careful study to specifications on every job.
5. Maintain closer contact with manufacturers.
6. Spend more time out of the office.
7. Expand the technical services.
8. Recognize the vital element of time in all of today's biz.
9. Keep complete delivery and price information at hand.
10. Build up customer confidence in quick service.
11. Review all past experience and errors; correct them for emergency times.
CAP SAYS:—
LOOK THE SITUATION IN THE EYE—AND LICK IT!
• In recent months, many people—including all too many radio dealers—have let their hopes guide their actions.
• Even with war fast closing in on them from all sides, these people had hoped to do “business-as-usual.” And have tried to realize their hopes.
• But this war we are in is a “total war.”
• A democratic war, in which soldier and civilian, man, woman and child, rich and poor, must serve, and suffer together.
• Radio, too, has been called to the colors!
• And radio will measure up to the need of time.
• While the contribution of the manufacturer will be direct and measurable, the role of the radio dealer will be less direct.
• While the manufacturer is striving for new and greater production records, the radio dealer must fight for his business existence.
• Many radio dealers will inevitably fail to measure up and will fall by the wayside, for these are times which try the souls of men, and prove their abilities.
• But determined, alert dealers can and will measure up to current demands.
• They will tighten their belts, look the situation squarely in the eye, and lick it!
You’ll Have to Amputate, Too
(Continued from page 17)
12. Advertise “Service”
Cutting expense can’t save your business if you don’t have sales. And advertising produces sales. But advertising which does not produce sales is an expense, a reducible expense. And so you should subject your advertising to a searching investigation. Any advertising which does not pay its own way by creating good will, or in profits of the sales it creates, should certainly be discontinued.
Since much of your future depends upon your Service Department, you can justify some Service Department advertising for its cumulative and future value, but even this should be carefully checked.
13. Eliminate Night Hours
Night operations of your store are expensive in operating costs—for lights, heat, personnel. Unless your night sales are very good, and cannot be transferred to day sales, night operation should be discontinued.
Even in good times, stores with good night sales, still found that very, very little of this night business was lost when they closed at nights. It could be, and was, transferred to daytime business.
14. Buy Only Equipment That Saves Money
While you are closely inspecting expense, you cannot well afford to overlook similar scrutiny of capital expenditures.
Improvements should be discontinued. And only such machines or equipment as can and will actually save money should even be given a second thought.
15. Rebuild Trade-ins
Every employee must work all the time and particularly the Service Department, which must now carry a larger share of the sales load than usual, must make every hour productive.
This you can do by careful planning. In the Service Department an excellent idea now widely used is to have trade-in and other used sets always awaiting renewal in the Service Department to be worked on whenever other work slackens.
16. “Beat the Budget”
There are other ways, too, to reduce expense, many of which you will think of, and practice.
An excellent idea is to make a game of “beating the budget.”
Let every co-worker play the game. Keep score. Pay some small token prize for every usable suggestion. Each month, tally your “scores” and give some recognition for those who win. During the summer, score on a baseball basis.
The spirit of friendly competition will do much to keep all your coworkers’ minds “expense conscious.”
The “scoreboard” will keep it constantly before them. And results may be as surprising to you as they have been to many others.
RADIO RETAILING TODAY is anxious to help all dealers help themselves.
You are invited to write in any expense reduction ideas or methods you have found effective. Such letters will be printed for the benefit of all dealers.
How New Price Ceilings Affect Dealers
As the General Price Maximum Regulation swung into action, the OPA revoked the temporary regulation on radios which was issued earlier, and released new instructions for retailers. Three important “do’s” came from OPA, indicating immediate steps in getting ready for the May 18 effective date of the General Regulation. These were as follows:
(1) Assemble and preserve immediately all your records regarding all prices charged for goods in March. In addition, begin preparing your statement of highest base period prices for each item sold, so that it will be completed by July 1, 1942.
(2) Check prices of all goods in your store to be sure that they are no higher than the highest prices charged in March, 1942. This job must be completed by May 18, after which time you cannot exceed these maximum prices.
(3) Arrange to post or mark and identify as “ceiling price” or “our ceiling,” your maximum prices on all “cost-of-living” commodities specified in Appendix A of the Regulation. This must be finished by May 18. In adWashington Expert
James H. Simon, prominent jobber of Washington, D.C., who has been serving as radio's key man in the Office of Price Administration, has announced that he will now leave that post. Mr. Simon is credited with much expert work on civilian radio rulings when OPA first faced the huge problems of price regulation.
dition, a list of these items and their ceiling prices must be filed with the War Price and Rationing Board in your area by June 1, 1942.
OPA advised retailers not to bring their problems to Washington personally, not to telephone the Washington administrators, and not to write in before the Regulation has been carefully read and re-read. "Troubleshooters" from OPA's Retail Trade and Services Division are preparing a bulletin of explanations on matters most frequently asked about.
As for the retail licensing provision of the General Regulation, OPA points out that the purpose of the license is to give OPA a basis for action against stores which refuse to conform. OPA may ask a court to suspend the license as long as 12 months. Every retail and wholesale outfit is automatically licensed to sell under the Regulation at the date the ceiling applies to it.
Price ceilings established by the Regulation apply to radio repair charges, as well as to the sale of new or reconditioned sets.
Sylvania Opens Another Plant
Hygrade Sylvania Corp. has purchased a new plant at Mill Hall, Pa., and is now grooming it for the manufacture of radio tubes. Actual operation will start within a few months.
GLASS MASTER DISCS NOW READY FOR DELIVERY
Presto 17¼" Glass Base Master discs are now in stock awaiting your order. Transcription manufacturers have been processing samples of the Presto Glass Master for several months and report it perfect in every respect, easily adapted to their plating equipment, thick enough (.135") for safe handling and having the exceptionally quiet surface characteristic of all Presto discs.
The Presto 17¼" Master is made in two styles. Type 917-D has a removable metal center insert to allow its use with overhead cutting mechanisms driven from the center of the turntable. The 917-E has a solid insert for tables having independent cutting head drive (Presto 8-C).
Priced only slightly higher than previous aluminum master discs. Sold by Graybar Electric Company and leading radio parts distributors throughout the United States and Canada.
PRESTO RECORDING CORP.
242 WEST 55th ST. N.Y.
World's Largest Manufacturers of Instantaneous Sound Recording Equipment and Discs
KEEP A BALANCED TUBE STOCK ALWAYS ON HAND
Win Customer Goodwill With Quick Service
KEEP 'EM PLAYING WITH Electronic RADIO TUBES
FAMOUS FOR LONG LIFE AND FINE RECEPTION
GENERAL ELECTRIC
DEVELOPMENTS IN SOUND ENGINEERING BY utah
A typical result of Utah's ingenuity and ability to meet changing requirements is the new Utah A.C. Field Excited Speaker. In anticipation of the critical shortages of raw materials needed for the fabrication of permanent speakers, it was necessary to develop a new line of substitute speakers. Utah's solution to the problem has all the dependability and high satisfaction of the line of which it becomes a part.
These new Utah speakers have standard Utah weather resistance. They are humless in operation and equivalent in performance to the famous Utah Permo-Dynamic line. A speaker has been designed for every public address and sound requirement. They require only the addition of the A.C. Field Supply shown at the left—to substitute for any Permo-Dynamic application. The Field Supply is properly designed for humless operation of any of the new Field Excited Speakers. The supply may be mounted directly in the speaker baffle.
Look for the Utah trademark. Utah Radio Products Company, Address: 810 Orleans Street, Chicago, Illinois. Canadian Offices: 560 King Street, West, Toronto. In Argentina: Ucoa Radio Products Company, SRL, Buenos Aires. Cable Address: Utaradio, Chicago.
New Utah A.C. Field Excitation Supply. At 117 volts, 60 cycle input, the maximum output is 12 watts at 105 mills.
At 400 miles per hour
You'd Appreciate Metal Tubes!
A POWER DIVE in a pursuit plane is something to write home about! Your engines roar...the wind shrieks and screams...the airspeed indicator climbs past 300, 350, 400, toward terminal velocity. As you ease back on the stick to pull her out, enormous stresses wrench at the vitals of the plane—and at every piece of equipment inside!
Is it any wonder that both the Army and the Navy specify metal tubes for military radio equipment—? Or that RCA is now turning out metal tubes at the fastest pace in our history—? For metal tubes combine extreme ruggedness with positive socket-contact (at the pins, where contact should be!) along with lower interelectrode-capacitances, greater compactness, and efficient self-shielding. Only metal tubes can give the advantages of metal tubes!
With enormous quantities of RCA Metal Tubes going into equipment for the Democracies, you may experience difficulty or delay in the shipment of your orders for non-war purposes. Please be patient. For military equipment comes first...and military equipment needs metal tubes!
METAL TUBES
RADIO RETAILING Combined with RADIO TODAY, May, 1942
Home Service vs. Shop Service
Servicemen some days ago found themselves shoved into the midst of the age-old battle about repairing the set in the home vs. "pulling the chassis" for a shop job. Just when this subject was resting peacefully in each serviceman's policy of handling his jobs, along come some words of advice from Washington for consumption by the general public.
In a statement for consumers from the Office of Price Administration, radio-set owners were advised to do certain things to insure the operation of their sets for the duration. Due to the widespread publicity given in newspapers to this statement by the OPA, servicemen should be prepared to cope with it.
Most Suggestions Good
Most of the suggestions were good. Owners were advised to allow for air circulation around their sets, to check the line cord for good contacts, to see that ground and antenna leads were not broken or loose, to see that tubes were firmly in their sockets, to clean the dust from the set with a small hand vacuum-cleaner. It was further advised that a radio repairman be called if the set were still unsatisfactory.
These suggestions are commendable. They can do much to save time and tires in "fixing" Mrs. Jones' set because the plug was pulled out during a flurry of housecleaning!
However, in the same breath the OPA further states... "Insist that he (radio serviceman) fix the set at your home. Most service firms have portable testing and repair equipment for home calls."
Of course if Mrs. Smith insists you can always use her new double boiler and kitchen stove to melt the pitch out of the can which contains the power transformer, filter coke, three RF coils, and the tuning condenser—the exact replacement for which is no longer being made, in her Super XYZ model.
In spite of the laughability of the situations which could arise if householders insisted on having every type of repair job done in their own homes, servicemen should not be too hasty in "pulling the set" when it is possible for them to do the job in the home conveniently and gracefully.
Plan Your Service Calls
In these days of tire and gasoline shortages don't rush across town to pick up a set, bring it back, put in a tube, and haul it back to the customer. There is one unnecessary round trip in that method. Where to draw the line on doing jobs in the home and in the shop depends upon the business set-up of each individual serviceman. When it is a toss-up, explain what is involved in making the repair and let the customer be the judge as to whether the set shall be fixed in the home or not.
On those jobs which must be done in the shop, you're the doctor. Few set owners will fail to see your point if you tell them that it is unfair and unpatriotic for you to gamble your ability to keep the radios of your other customers working—and if they ever needed them, they need them now—through risking damage to hundreds of dollars worth of delicate and now unreplaceable test equipment, just to fix the set in their home.
Even when you have to bring the set to the shop you can help yourself and your country by routing your calls and deliveries. If you get a call from "across town" early in the morning, plan to make the visit late in the day. The chances are you will get other calls in the general vicinity and save unnecessary long trips.
Further comment by the radio trade on this OPA statement on "home repairs" will be found on following pages.
Auto Radio Service
Basic facts on vibrator power supplies, installation, noise elimination and features of new circuits.
With more than 10 million auto radios in use today, and over 20 million cars and trucks still without sets, the service and installation market is very great. While it is true that in some instances the volume of motoring has been reduced, most of this traffic has been of the pleasure variety. The majority of the business uses of automobiles continues.
The servicing of auto sets calls for extra knowledge and experience in two classifications: (1) vibrator power supplies, and (2) installation and noise elimination. These two subjects are not often encountered in ordinary home set service work. They are factors on nearly every auto radio job.
Vibrator power supplies are often skipped over by some servicemen because of a lack of understanding as to what makes 'em work.
The purpose of the vibrator type power supply is to convert the 6-volt (usually) DC to high voltage DC for application to the plates of the tubes. To do this it is necessary to change the direct battery current into a pulsating or alternating current after which the step-up properties of a transformer can be used. The stepped-up pulsating voltage can then be rectified and filtered to the required DC.
The rest of the power supply consists of a power transformer, buffer condensers, a rectifier (if synchronous vibrator is not used) and the filtering system. The power transformer has a double primary of two 6-volt sections connected in series. The battery current is sent through each primary section in turn by the vibrator. The secondary winding of the transformer is the usual high voltage winding common to other types of radio power units.
Transforming and Rectifying
If the self-rectifying or synchronous type of vibrator is not used, either a gas filled no-filament type rectifier (0Z4) or a regular thermionic tube is used.
Since the vibrator serves only to switch the direct battery current through each section of the primary winding in turn, the current that flows through the winding during the time the vibrator contacts are in one position is nearly a constant or direct current value. Because the contacts cannot be in two places at the same time, there is a period while the vibrating reed is moving from one set of contacts to the other that the current through the primary is zero. The ratio of the time that the contacts remain in the "make" position to the total time for one cycle is the time efficiency of the vibrator and for modern units this efficiency is between 80 and 90 per cent. The frequency of vibrator is usually in the vicinity of 115 cycles per second.
Induced Peaks
If some shunting or damping action were not used across the windings of the power transformer, the sudden breaking of the contacts of the vibrator would cause a high voltage to be developed in the windings since the rapidly decaying flux would induce that voltage. The voltage across the secondary of the transformer could rise to a value several times its normal magnitude and thus damage the rectifier and filter system. To prevent this abnormal voltage from developing during the breaking of the primary vibrator contacts, a buffer or "timing capacity" is placed across either the primary or the secondary, usually the latter. The size of this capacity is determined by the characteristics of the vibrator, transformer and load system. Its purpose is to prevent the excessive voltage peaks by opposing any change in voltage when the primary contacts are broken.
The value of the timing or buffer
Fig. 1.—Interrupter and synchronous vibrator circuits are shown in A and B respectively. An electrically equivalent switch circuit is shown below each type vibrator.
capacity is very important to the proper functioning of the vibrator power supply. The value recommended for the set should be used unless it becomes necessary to change the vibrator type or power transformer. If this is done, the buffer capacity should be adjusted to give a voltage waveform, as viewed on an oscilloscope, as symmetrical and free from sharp peaks as possible. The typical waveform should have sloping sides and a flat top. Both the positive and negative half-cycles should be as nearly alike as possible.
**Buffer Sizes**
If the buffer capacity is too small it will not do the best job of keeping excessive voltage peaks out of the system. If the capacity is too large, the input current from the battery will be higher than necessary and therefore detract from the normal life of the vibrator contacts.
One of the best tests for vibrators is its starting voltage. That is the voltage at which the vibrator goes into operation by itself. A "good" vibrator will start at 5.2 volts or less. If 5.2 to 5.6 volts are required to start the vibrator, it is probably close to the end of its life. If more than 5.6 volts are required to start the unit, it should be replaced. A second test for the vibrator should be the smoothness of the output voltage from a conventional rectifier and filter system under normal load. A volt-meter across the output load should register the normal voltage with very little fluctuation if the vibrator is good. Low or unsteady voltage is a sign of a bad vibrator. With all of these vibrator tests it is assumed that the other parts of the circuit are in good condition.
**Primary Resistors**
Two typical power supply diagrams are shown in Fig. 2. Fig. 2A is the synchronous vibrator type supply used in Truetone model D4255. In addition to the buffer capacity across the secondary of the transformer, the primary is further protected against voltage peaks when the vibrator contacts are opened and closed each half-cycle, by two 100-ohm resistors connected across the two primary sections and grounded at the center. Since the center tap of the primary winding is connected to the "hot" side of the battery and the two 100-ohm resistors return their center point to the other side of the battery circuit, a small direct current will flow through the two sections of the primary at all times. The current flows through each section in opposite directions so that the net result on the flux in the core of the transformer is zero. When the vibrator contacts close, full current flows through the section of the primary connected to the contacts. When the contacts are broken, the resistors (one for each section of the primary) prevent the current from falling to zero and at the same time acts as a protective shunt for the high voltage which would develop from the rapidly falling current.
**Input Filtering**
In Fig. 2B is the power supply used in Motorola model 37D-I. Here an interrupter or non-synchronous type of vibrator is used in conjunction with a gas filled OZ4 rectifier. Low ohmage resistors are used across the primary for the same reasons as in the circuit of Fig 2A.
With a conventional rectifier, the center tap of the secondary is the negative lead, while in most synchronous vibrator systems the center tap is positive. The center tapped capacities across the secondary serve to by-pass high frequency hash and other noise voltages.
The input lead from the "hot" side of the battery is well filtered with inductance and capacity combinations. This is done to block the entrance of ignition noise. Since the receiver is thoroughly shielded, the "hot" A lead and the antenna are the only means of entrance, unless the set is equipped with an external speaker, tuning head or other exposed leads. Because the A lead is a part of the electrical circuit of the automobile, it carries rather high noise voltages developed mainly by the ignition system. To filter this noise out of the A lead to the receiver, a very low impedance path to ground must be provided.
**Antenna Coupling**
Because the lead inductance of ordinary capacitors is high enough to prevent the high frequency ignition noise from being by-passed effectively, a special type of by-pass capacitor known as a spark plate is now widely used to filter out ignition noise. In general, it consists of a metal plate fastened to the chassis and insulated electrically. The inductance of this unit is therefore very low, since the chassis and the plate serve as their own leads.
The use of chokes prevent current surges in the vibrator circuit from seriously affecting the filaments, and they also isolate power supply noise voltages from the tubes.
In Fig. 3 is the antenna coupling system used on many auto sets. Due to the relative high capacity to ground
(Continued on page 38)
A long needed step in the tube industry was taken by the War Production Board when, with the cooperation of the tube makers, it dropped 349 types from the ranks of the tubes being made. These 349 types represented about half of the total types. The large percentage of them were either obsolete, slow moving, or duplicates of other construction types. About 280 of them are familiar to U. S. radio men. The balance of the types were either proposed types never released, or foreign tubes made for export service.
Although the 349 types represented a total of less than 800,000 units per year or less than 1 per cent, the elimination of these types from the production lines will represent some saving in needed materials and plant capacity.
While these tubes will no longer be manufactured, it does not mean that they are not available. In many cases, sufficient stocks are available in manufacturers', jobbers', and dealers' hands. They should, by all means be used as replacements whenever they are available.
The accompanying list are those types which will no longer be made less the foreign and "never released" types. The code letters following each tube refer to remarks in the explanatory table.
### Key Letters Explaining Reasons for Tube Eliminations
- **A**—Replaced by 1F7G.
- **B**—Replaced by 6AB5/6N5.
- **C**—Ceramic type base. Small demand.
- **D**—Replaced by 6U5/6G5.
- **E**—Replaced by 6TTG.
- **F**—Replaced by 1V.
- **G**—Replaced by 84/6Z4.
- **H**—Replaced by same type with plain octal base.
- **I**—Replaced by 7G7/1232.
- **J**—Replaced by 14A7/12B7.
- **K**—Replaced by 1B3/23S.
- **L**—Used in limited number of special sets.
- **M**—Special tube for some Majestic models. Small demand.
- **N**—Replaced by 117L7/M7/GT.
- **O**—Old style; now obsolete.
- **P**—Replaced by 6AC7/1852.
- **Q**—Replaced by 6AB7/1853.
- **R**—Replaced by GT or GT/G construction, or by equivalent metal type.
- **S**—Small demand. Total of these types less than 1 per cent of total yearly sales.
"I've Learned a Lot and Saved a Lot with my MYE"
That's what a young radio serviceman who's just "getting his feet wet" in the business told us recently. He's found his Fourth Edition "MYE", the Mallory Radio Service Encyclopedia, a valuable instruction manual and an invaluable time-and-money saver.
For instance, both veteran and "rookie" radio servicemen have learned that it pays to keep a copy of the "MYE" on counter or workbench, right near the telephone. When a hurry call comes in for service on any model of any make of receiver, it's easy to look up circuits, original part numbers and recommended replacements for volume controls, condensers and vibrators. All this data for any set can usually be found in one compact listing, on one page of the "MYE"!
Also, if a detailed inspection of the entire schematic is necessary, you can quickly refer to your "MYE" for the tube complement, the I.F. peak and the Rider's Reference.
Ask any radio serviceman who has bought one. He'll tell you how he uses his Fourth Edition "MYE" every day to save time and money. This "MYE" is worth many times its nominal price. Get your copy today from your Mallory Distributor!
P. R. MALLORY & CO., Inc.
INDIANAPOLIS INDIANA
Cable Address—PELMALLO
Philco C-1908
Auto Radio Circuit
Permeability tuning is used in the auto radio model C-1908 made by Philco for Chrysler, Desoto, Dodge, and Plymouth cars. In addition to the moving cores used for manual tuning, the five push-buttons are also permeability tuned. Circuit for this set is shown.
The coupling between the first RF tube and the converter is fixed tuned. A series trap in the converter grid circuit is tuned to reject interference at the intermediate frequency. The manual and push-button oscillator grid circuit coils are shunted in parallel with a fixed tuned oscillator coil which has the feedback winding.
The set has a conventional AVC system and also a sensitivity control in the cathode circuit of the RF and converter tubes. A feedback tone control circuit is employed between the secondary of the output transformer and the grid circuit of the 7A4 phase inverter. The inverter circuit uses the divided plate load circuit with half the load resistance in the cathode circuit.
Circuit Alignment
To align the IF transformers, connect the signal generator through a 0.1 mfd. capacitor to the antenna receptacle and with the tuning dial turned to the complete clockwise position, align the trimmers 40, 38, 32, and 30 to 455 KC. Repeat for best results.
With the signal generator connected as before, adjust the trap trimmer for minimum response to 455 kc. This trimmer is located in the top of the RF coil can.
The RF end of the set is adjusted at 1500 kc. and at 580 kc. The signal generator should be connected to the antenna receptacle through a 25 mmfd. capacitor and dummy antenna such as Philco part No. 95-0111. At 1500 kc., adjust the RF trimmer 17 alongside the antenna receptacle. At 580 kc. adjust the iron core in the fixed oscillator coil while rocking the tuning condenser.
When the set is installed in the car, the RF end should be realigned at 1500 kc. and 580 kc. as mentioned before but with the signal radiated from a piece of wire and the car antenna mounted and connected to the set.
Shop Service vs.
Home Service Opinions
From the Trade
The reaction to the suggestions made by the OPA for the repairing of radios has brought forth considerable criticism. (See general story on page 25.)
The following letter is a copy of the one sent by the Radio Technicians' Association of Long Beach, California, to a local newspaper which printed the OPA advice to radio set owners.
Editor
Long Beach Independent
Dear Sir:
In regard to your recent article on radio and radio service published April 10, it seems to me that an outstanding mistake was made. The Independent would investigate existing laws and general conditions before publishing an article of this nature. You have caused the radio dealers and servicemen, through this article, no end of trouble. I would like very much for a retraction or correction to be published to offset such outlandish statements.
In the first place, we are working under a terrific handicap. We cannot take our radio test equipment, which in most stores is valued up to $1,000.00, to any home to check a radio. One instrument used, a channelyst, checks to a micro-volt one-millionth part of a volt. If this instrument is damaged or broken, replacements are not allowed for the duration.
We have been given orders that all radio parts removed from radio chassis must be returned in order for us to procure a new supply, which is the reverse of your published statement.
It seems to me if you were to do
(Continued on page 33)
WHAT ABOUT RESISTORS AND CONTROLS FOR THE HOME FRONT?
A Statement Regarding the IRC War Effort and Its Effect on Normal Production
As a natural consequence of unquestioned leadership in its field, IRC has been called upon to provide fixed and variable resistors for war requirements to an extent which has absorbed a large part of its production capacity. This war demand has steadily increased and has exceeded all expectations. Naturally, this is a tremendous responsibility that we cannot neglect.
Nevertheless, we have by no means forgotten the important requirements of service men and jobbers. We have wanted for some time to make a definite statement concerning our ability to supply replacement parts. This has been impossible since there was no means of knowing the extent of the war demands, and no definite provision has been made to insure a supply of critical materials for radio servicing requirements.
The national authorities in charge of this most difficult task of allotting critical materials for the greatest good of our Nation fully recognize the importance of radio repair and maintenance but, up to now, urgent war production has rightfully had priority over all else. As soon as these authorities decide upon definite plans for the allotment of materials for replacement parts, we shall relay these plans to you.
Meanwhile, we state definitely:
That IRC realizes fully the importance of keeping the Nation's old radios in perfect working order during the emergency.
That IRC thoroughly recognizes its obligation to keep jobbers and service men supplied with dependable replacement parts.
That IRC pledges itself to fulfill its obligation to the utmost, within the limits permitted by authorization of critical materials.
If deliveries have at times been slow, or if you have found an occasional unit missing from jobbers' stocks, we know that you will understand. You will realize that any failure to deliver goods on the home front is only because we have recognized that there must be no failure to deliver them on the war front.
Obviously, all replacement parts production will be faced with many difficulties arising from the war. Through participation in the "Production Requirements Plan" now being expanded to cover replacement parts, plus full utilization of our greatly enlarged manufacturing facilities, IRC hopes to be in a position to handle all legitimate requirements.
The home front has not been forgotten. We'll do our level best to meet its needs!
INTERNATIONAL RESISTANCE COMPANY 401 NORTH BROAD ST. PHILADELPHIA, PA.
New Equipment
SPRAGUE TYPE WR tubular cardboard dry electrolytics replace wet capacitors and also various aluminum can dry types. Have high voltage formation to insure their standing up under the high peak voltages impressed on wet electrolytics. Diameter is same as standard wets. Available in 3 sizes—WR-10 to replace capacities from 4 to 8 mfd.s, WR-16, capacities from 12 to 18 mfd.s, and WR-25 from 20 to 40 mfd.s. Sprague Prods. Co., North Adams, Mass.—RRT.
SQUARE D AIRCRAFT THUMB SWITCH, Class 9390. Contact is made by pressing and holding metal button. Switch body is made of bakelite. Enclosed electrical contacts are fine silver, single pole, double break; hexagon flange, lockwasher and locknut on threaded throat. Designed for flush mounting. Conductive rating is 15 amps at 24 Volts DC. Square D Company, 6060 Rivard St., Detroit, Mich.—RRT.
DURAKOOL BLACKOUT RELAYS—BF and CF mercury relays available for defense applications. Circuit is made and broken in hermetically sealed chambers under hydrogen pressure. Not affected by dust, dirt, moisture or corrosion. Low contact resistance and explosion proof. When coil energized plunger is pulled down and displaces the mercury, causing it to rise in the relay chamber until contact is made. BF relay has capacity up to 30 amps and CF relays up to 65 amps. Durakool, Inc., 1010 N. Main St., Elkhart, Ind.—RRT.
AEROVOX OIL CAPACITORS, type 20 units, cover voltage ratings from 6,000 to 50,000 B.C. Including dual-section units for voltage-doubling circuits of 12,500, 12,500 v. in 0.25-0.25 and 0.5-0.5 mfd. Multi-laminated kraft tissue and aluminum sections are uniformly and accurately wound, and vacuum-treated, oil-impregnated and oil-filled. Capacitors maintain full rated capacity even at freezing temperatures. Hermetically sealed in sturdy welded steel containers. Aerovox Corp., New Bedford, Mass.—RRT.
LEE DE FOREST "BLACKOUTER" is a radio controlled relay device which shuts off lights which may be connected thereto when a local station calls off the air in case of a blackout. A buzzer or bell may also be attached to it to sound an alarm. Lee De Forest Labs., 5106 Wilshire Blvd., Los Angeles, Calif.—RRT.
FEDERAL AIR RAID SIREN, No. 7, 7½ h.p., available for 220 v., 60 cycle, A.C. Single phase. Fuse size 100 amp. Height, 58 in., diameter, 25 in. base. Estimated sound range under average weather conditions, 1 mile, with no obstructions and high noise levels, in 2 miles or better. Possible methods of control are operated by push button only, or with timer. Federal Elec. Co., Inc., 8700 S. State St., Chicago, Ill.—RRT.
NATIONAL UNION AIR RAID ALARM, AR-101 is designed to work on any AVC type of radio set, and is set off by a local "alert" broadcasting station which is on the air 24 hours a day. When the station goes off the air the alarm automatically goes on, creating a loud, penetrating signal in the radio set. Complete in compact metal case, $5. National Union Radio Corp., 57 State St., Newark, N. J.—RRT.
SONORA 5-TUBE AC-DC SUPERHET tunes 535-1720 kc. (incl. 1720 kc. police channel). Table model, with built-in Sonoroscope loop, slide rule dial, dynamic speaker, A.V.C. Plastic knobs. Hand-rubbed walnut cabinets. Sonora Radio & Telev. Corp., 325 N. Hayne Ave., Chicago, Ill.—RRT.
WABASH BLACKOUT BULB UNIT has been changed from the original unit in color of light—a deep orange color. Current consumption has been reduced to 15 w. Improved type of heavy black silicon coating. Will fit almost any household socket and lists at 45c. Wabash Appl. Corp., 335 Carroll St., Brooklyn, N. Y.—RRT.
(Continued from page 30)
the right thing you would contact our Association or any legitimate store before making such an outlandish statement.
The following quotation, made by the Columbia Broadcasting System, was published in the March issue of RADIO RETAILING-TODAY:
"The success of radio's attempts to serve through this crisis will depend on the skill and loyalty of the men who supply the nation with sets and keep those sets in working order. I am sure that the American radio retailer and repair man will rise to his responsibility."
You will find that our Radio Technicians' Association men are above the average technically and have a sincere desire to serve the public at the lowest possible cost. We have always endeavored to cooperate with the dealers.
A copy of your newspaper is being forwarded to the Radio Board, Washington, along with this letter to see if pressure can be made nationally to prevent future articles of this nature from appearing in any publication. Such articles will only undermine the good feeling that has been built up between dealer and customer. It not only leaves a sense of doubt but violates instructions given us by various Boards.
We hope this presents a new light at this time, when the radio serviceman is working under such a handicap.
I am sorry the article was published and hope you see fit to correct or retract the statements made.
In behalf of the Radio Technicians' Association I thank you for this cooperation.
Very respectfully yours,
Harry E. Ward, Jr.,
Public Relations
Hygrade Sylvania experts, Walter E. Jones, Director of Commercial Engineering Dept., and Ralph S. Merkle, Technical Editor of Sylvania News, counter with these statements.
Considerable interest has been evidenced in the advice to consumers issued recently by a Washington Bureau on care and repair of radio sets. While the majority of the points which caution the user on care in cleaning, tuning and proper placing of the set are excellent instructions, it is felt sure that advice to have sets repaired in the home is satisfactory only for minor troubles which can be corrected by tube replacement, possibly cord and plug repair and similar easily changed items. However, where major repairs are concerned, serious complications enter to make home servicing unfeasible and in some cases impossible.
An outline of radio repair procedure to be followed is: 1. Diagnosis of difficulty; 2. Repairing or replacing the defective part or parts; and 3. Testing the receiver to make certain that it is in a satisfactory condition.
Adequate diagnosis takes time and involves the use of cathode-ray oscil-
(Continued on page 35)
Sound Guards War Plants
"Acoustic Fence" Keeps Vigil Around Important War Industries Through All Kinds of Weather
Man has always been forced to bend his brains as much in protecting his possessions as he did in acquiring them.
In wartime the problem of protection takes on far grimmer aspects. Mere thievery is child's play to combat in comparison to the daring modern saboteur intent on much higher stakes.
Several types of protective devices for fences were available to war industry when the likelihood of sabotage grew to menacing proportions—photoelectric, make-and-break circuits, sonic pick-ups, burglar alarm and death-dealing systems—but all had loopholes. Consequently, there was a desperate need for a dependable way of improving the efficiency of a plant's first line of defense—which is the fence.
A Fence With Ears
The engineers of E. I. du Pont de Nemours & Company were accordingly asked to "Invent a saboteur-proof fence." After developing the idea for the protective fence, the Du Pont engineers contacted the Astatic Corp. of Youngstown, Ohio who in turn formed the Automatic Alarms Co. to build the equipment.
Now, a "fence with ears" has passed its test under actual conditions of use. The acoustic fence, so-called, filled protection requirements so completely, it is being said that it cannot be defeated. Even the inventor's efforts are thwarted in trying to climb or jump over, dig under, saw or cut through a fence that has been lent these super-sensitive detector ears.
Picks Up Vibrations
In fact, the sound of approaching footsteps, even the wind's sigh or a bird's chirruping is heard. The acoustic fence passes on greatly amplified sounds to guards who are on the alert at a central control panel for the alarm signal and the flash of the red light which tells just where along miles of fence an intruder is attempting illegal entry.
The device takes advantage of the fact that most plant fences are all-metal or of substantial wood construction. If a continuous wire fence (or substantial wood fence) is disturbed by even an exceedingly small impact, mechanical vibrations will pass from the point of the disturbance and travel along the fence for an indefinite distance. Impact upon the ground will start vibrations in the ground which pass to the fence, and thence along the fence for considerable distances. It is, therefore, possible to place a vibration pick-up against a member of the fence and with suitable amplifying equipment get audible registration of vibrations set up in the fence at remote points.
In actual practice, an especially developed pick-up, similar to phonograph types, of weather-proof construction is attached to the fence at intervals of 1,000 feet. This results in an operating range of 500 feet on each side of each pick-up, which is about one-third the maximum possible range.
Blinker Lights
Where guard towers are placed along the fence at intervals of 800 feet to 3,000 feet, it is only necessary to have pick-ups between guard towers. A loud speaker enables the guard to hear and usually identify the nature of the disturbance to the fence. Also a relay closes and rings a bell in the guard tower and if desired transmits a signal to a central headquarters over the telephone system; in addition, a pilot light indicates that the system has been disturbed and must be reset by hand.
The system enables the guard to hear any disturbance along the fence, also gives a visual indication that a disturbance has occurred and the alarm bell is provided to wake the guard up if he is asleep or drowsy. It also serves as an alarm in the event that the guard is away from the post when the disturbance occurs.
The fence is extremely sensitive, protected from tampering by guards themselves, cannot be put out of action by external tampering and offers the necessary flexibility to meet the requirements of particular installations. The equipment is simple in operation, involves no exceedingly fine adjustments, is independent of terrain, is easily maintained, and is economical to install.
(Continued from page 33)
lographs, tube tester, volt-meters and much other equipment. It is hardly to be expected that any housewife would countenance an operation on her living room set which required five or six large pieces of equipment in addition to a tool kit and spare parts.
A soldering iron must then be hooked up to make the replacement of parts. On completion of the repair work, a signal generator, an output meter and similar equipment may be needed for final checking. To avoid ear-splitting noises that could drive a housewife into frenzy, the loud speaker must be disconnected while aligning the receiver. It is obvious that such procedure takes unnecessary extra time.
Inefficiency and wasted efforts that are a part of servicing sets in the home include extra trips to the customer's home and the parts jobber's store, wear and tear on delicate and important testing equipment. All of this, at a time when greatest conservation of physical and material assets, energy, tires, and electrical equipment is of paramount importance, shows that advice to service all sets in the home does not follow the facts in every case.
Phono and Changer Service Notes
Where the rumble is objectionable, see that the changer or player floats freely in its mountings and does not make solid contact with the cabinet or chassis. See that connecting wires between the chassis and changer or player are not tight, but have plenty of "play." Warped turntables, bent center pins, and records with worn center holes will all cause "wow." The cause can usually be spotted by watching the pickup while the record is playing. If the pickup oscillates slightly back and forth due to some off-center condition, the "wow" will be timed with the oscillations when they are causing the trouble.
Hum, and "Wow" Pickup
AC hum may be due to excessively long leads from the pickup to the amplifier as in the case of separate players. Shorter lines, or high to low impedance transformers at each end, or a pickup with more output voltage will solve this. With the volume control at the player end, the impedance of the line is pretty well established by the setting of the control. With a high output pickup, the control can be set at a correspondingly lower value hence the impedance of the line is less and less hum will result. Examples of this can be observed when playing a "loud" record and a "soft" record at the same room volume. The "soft" record will appear to have more hum on it because the volume control setting is higher and for the reasons just mentioned.
WARTIME restrictions make it difficult to supply wet electrolytic condensers because of their aluminum thread-neck cans—but, thanks to Sprague engineers, you can keep right on making wet electrolytic replacements, and do it with the same assurance as though you were using the finest wet electrolytic condensers now out of production.
The answer is the new Sprague Type WR Replacement Capacitor—a tubular cardboard dry electrolytic of very high voltage formation. Not only will WR's stand the peak voltages often impressed on wet electrolytics, but they'll handle the AC ripples that might cause standard 450-volt dry electrolytics to overheat to the point where they break down. The diameter of WR's is the same as that of standard wets so that they will fit the screw-type can mounting holes. Their metal feet can then be soldered to the chassis for firm mounting.
Sprague Type WR's are now available in three sizes—WR-8 which replaces wets from 4 to 8 mfd.; WR-16 to replace capacities from 12 to 18 mfd., and WR-25 to replace capacities from 20 to 40 mfd. Ask your Sprague jobber today!
WARNING! Don't be fooled! Although standard dry electrolytic condensers can sometimes be used as wet replacements, your safety margin is apt to be mighty thin. High surge voltages and AC ripples may cause trouble. That's why it pays to play safe by using the new Sprague WR Types. They're not substitutes. They're actually built to do a wet electrolytic job. They're the real thing as far as performance and durability are concerned.
SPRAGUE WET REPLACEMENTS (SPECIAL WR TUBULAR DRY'S)
SPRAGUE PRODUCTS COMPANY, North Adams, Mass.
Expediter . . . BOGEN!
BOGEN SOUND SYSTEMS are expediting production in key defense plants throughout the country.
INTERCOMMUNICATION between vital points is achieved instantly. Important messages to the workers are made while work continues. Alarms are sent automatically—in an instant—to every corner of the plant.
BOGEN SOUND SYSTEMS step up production—multiply efficiency—expedite the flow of work—Write for details!
DAVID BOGEN CO., Inc.
663 Broadway, New York City
Success in Servicing
Business plans that have proved "winners" for this New York radio serviceman.
Ray Mattraw has been in the service business, for upwards of sixteen years, ten of which were spent in field service work for a large manufacturer. For the past six-year stretch, he has been engaged in building up a successful service shop at Watertown, N. Y. The suggestions and hints passed on in this article are based on actual trial and error methods which Mattraw himself has tried. His shop probably holds the record for most service customers in the Watertown area.
Calls Monthly
To begin with, Mattraw maintains "you must integrate yourself with your community." He has made personal calls, for example, on all hotel managers, restaurant operatives, business executives, and merchants of all sorts, from the independent shoe retailer to the furniture credit-shop. With hotel managers, he leaves sets of cards, so that when portables or car radios fail to work, he gets the calls. The same holds true for restaurants. With merchants he discusses idea of having a radio receiver in the store, to hear latest news flashes, and allow customers to do likewise. Even with set production stopped entirely, supply of receivers on hand in average dealer's shop is adequate to stock such merchants with second-hand sets. Receivers which servicemen long ago took in, on one trade or another, can be liquidated to merchants, in many instances. Business executives are also prospects for inter-communication systems for their offices and for departments. They also have their own home and car radios which need servicing.
Mattraw leaves cards or blotters with name, imprint and telephone number at every business contact. And he calls on EVERY business contact, at least once monthly. He leaves his cards at all factories, with plant superintendents. Workers are too tired when they come home at night after a long shift, to think of having their radios serviced, so Mattraw has cards inserted with paychecks or handed out at some other time of day. He doesn't neglect seeing the schools, for nowadays, with the war situation acute, history, economics and foreign language classes in junior high and high schools, utilize radio in their instruction periods. This gives him acquaintance with music instructor and with the principal, who may be interested in loudspeaker outlets for classrooms. Many schools now have their own miniature broadcasting studio, with actual studio equipment, which can be operated in cooperation with a local broadcast studio. This school equipment needs constant checking over. Mattraw keeps in with local broadcasters and theatre owners, checking their needs from time to time, when station engineers are tied up or motion-picture apparatus goes haywire and no time is available to contact out-of-town sound experts.
Advertising Pays
So much for personal contacts. In addition, Mattraw keeps constant insertions of advertising in local free-distribution newspaper or "shoppers guide." Many communities have such sheets and Mattraw favors it over insertions in regular newspapers. He finds, he says, that people "do read circular shopping issues, continually and do notice such advertising." Mattraw also is a firm believer in display ads in both phone and city directories. Strangers who need sets serviced, and other forms of transient trade almost invariably look up "Servicemen" in phone or city listings.
Charges Scheduled
Mattraw has some interesting suggestions on price. He charges flat rate of one fifty to go to any home and make ordinary routine inspection. Under no circumstance will he undertake a radio-set repair job, unless trivial, on the family floor. Tube checking at the shop is no longer free, Mattraw charges fifty cents to test tubes, even when customer lugs them in personally. Reason is that under this setup, if tubes are all O.K., Mattraw doesn't have to perjure himself and find one bad, to make operating expenses. Mattraw boasts of being "the highest-priced serviceman in the community," yet has less than 5% kickbacks on price. With war news important, everyone wants sets periodically checked.
Mattraw also advocates that when set is repaired in shop, Western Union messenger return set from shop to customer, to save wear and tear on shop truck. The Western Union messenger boy will pick up payment, and won't leave radio without it, if such are his instructions.
Mattraw advises every serviceman who may be drafted to arrange to have his shop continued under its original name, even if some other individual conducts the business. People are fickle and much goodwill and business worth can be dropped during wartime, if shop goes under new name or is cancelled out entirely. Mattraw also warns that after war, influx of servicemen, trained by Uncle Sam in Signal Corps, is to be expected, "making servicemen a dime a dozen," — at least that's his belief.
As a final suggestion, Mattraw says: "Be a joiner." Join all clubs and organizations possible and put in a personal appearance now and then. Make a speech at Rotary on Radio's Part in War, write a gratis radio column for local newspaper. Publicity is meat and drink for radio servicemen and don't forget it for a moment. Cooperate with Red Cross, Salvation Army, USO, by giving window and poster space in your shop. In short, cultivate the social and community spirit end, and you'll find business.
To industries converting to war production where the use of magnet wire and coils is important...
Anaconda Can Help!
Three Anaconda mills have unfilled capacity on magnet wire and coils...for war work
WRITE US IMMEDIATELY FOR COMPLETE INFORMATION
ANACONDA WIRE & CABLE COMPANY, GENERAL OFFICES:
25 Broadway, New York, CHICAGO OFFICE: 104 N. Wacker Drive, Subsidiary of Anaconda Copper Mining Company, Sales Offices in Principal Cities, 45881A
Magnet wire and coils
ANACONDA WIRE & CABLE COMPANY
Every Radio Service Man Needs This VACO Amberyl Electrolytic Condenser Nut Wrench
It's Great
For easy removal or tightening of electrolytic wet or dry condenser Pal Nuts without damage. Two sizes, deep drawn steel sockets. No interference with leads. Sockets 1½" deep, copper finish. Break proof and shock proof handles.
Write for information regarding our big line of VACO screw drivers.
VACO Products Co. 1603 S. Michigan Ave. CHICAGO, ILL.
HOW MUCH SHOULD A TESTER COST?
First, let's ask what You require. The various RCP Test Instruments are designed to supply maximum sensitivity, flexibility and utility specific features within range. Why buy a Lincoln limousine if a Ford roadster suits your purpose? Compare RCP Test Instruments point-by-point with competing testers, analyzers and signal generators, and you'll convince yourself that RCP offers the finest values.
Send for Catalog No. 125. Glance at RCP's Universal DeLuxe Multitester, Model 414. It embodies advanced design features far outclassing other line instruments, multi-purpose meters. Direct reading, accurate, rugged. Only $28.95. Forty-seven other fine instruments, rock-bottom prices! Available to radio men who are servicing priority plants and public institutions. See your distributor for details.
RADIO CITY PRODUCTS CO., INC.
88 PARK PL. NEW YORK, N.Y.
RAID-O-LARM Safeguards offguard hours
... radio-remote controlled raid warning for homes, air raid wardens, institutions, industrial plants.
A simple adapter, attaching to any set, RAID-O-LARM is the answer to an emergency need.
Born of necessity, but capably engineered, honestly made and properly sold.
Fair profits, fast turnover, for dealer and distributor ... a calling card for new set sales and the sale of repair service and parts.
V Model at $5.00 List
BLACKOUT CONTROL, INC.
545 FIFTH AVENUE NEW YORK
... BLACKOUTERS, Remote Control for Lights, previously announced, manufactured to order only. For information, write above.
AUTO RADIO SERVICE
(Continued from page 27)
of the ordinary auto antenna and its shielded lead-in a special type of coupling circuit must be used if the signal strength is not seriously reduced by this shunt capacity. In the circuit shown the shunt capacity of the antenna is utilized as part of the capacity of the tuned grid circuit by isolating the lower end of the grid coil from ground by a high value resistance. The antenna signal current is introduced into the tuned circuit through the 0.006 mfd. capacitor. The adjustable trimmer is used to balance up the tracking of the RF end of the set at the low frequency end of the dial for the antenna being used, just as the oscillator trimmer is adjusted for tracking. The antenna voltage is thus introduced in series with the tuned circuit.
Fig. 4 is the phase inverter used to drive the push pull audio stage of Motorola model 37D-1. The load resistance is divided into two equal halves with the center grounded for audio frequencies. One of the 6V6GT grids is driven from the plate of the 6J5GT, while the other 6V6GT is driven from the cathode of the 6J5GT. Cathode bias is used on the 6V6GT tubes and zero bias on the 6J5GT.
Interference elimination in automobile set installation generally involves the noises developed by two separate sources. The first source is the car electric system, and the second is the mechanical parts of the car which develop static charges when in motion.
The electric system noises originate in general from the ignition system, and the generator. The sparking of the distributor rotor and the accompanying current leakage from the high tension wiring and plugs produces a broad band type of signal which interferes with most of the radio spectrum. Plug and distributor suppressors help to isolate this high voltage steep wave front noise. Spark-plug suppressors are not usually employed unless absolutely necessary since they rob the engine of some of its "pep."
Noise Sources
As mentioned before, the electrical system interference can enter the set only through the exposed leads, that is, the antenna and battery lead and any other external wires. While the interference can be filtered out of the battery lead to the set, the same technique cannot be applied to the antenna circuit since this would also reduce the desired signal. It is therefore necessary to reduce to a minimum the radiation of the noise energy from the high tension parts of the ignition system and from those parts of the car which are poorly grounded and therefore act as re-radiators of the noise.
Preventing Re-radiation
The prevention of radiation from various parts of the car involves the grounding of the offending part or parts to the chassis. Many parts of cars are quite well insulated from the chassis through rubber shock absorber mountings. Entire engines in the "floating power" cars of a few years ago are rubber mounted. These parts must be connected to "ground" with heavy copper braided straps. If the bonding lead is too small, its inductance will be high enough to defeat its purpose. Ordinary copper wire and narrow braid less than 3/8" are generally too small to do a job.
In many cases, the problem of leaking interference is one of hunting for the offending part. In all cases the instructions from the manufacturer of the radio and the car should be followed completely where they are available.
Static noise voltages are generated mainly by the tires moving over certain types of pavement. It is worse in dry weather, since the insulation resistance of the rubber tires to earth is highest. Front wheels give the most trouble since the grease film in the bearings is an efficient insulator. Spring type connectors are available for grounding these front wheels to the axle shaft. Various conducting powders are available for internal application to tires.
Other electrical system noises include the whine from the generator commutator, fan motors, clicks from gauges, etc. These can usually be reduced or eliminated with a by-pass capacitor to ground at the offending unit.
Tuning Eye Failure on Zenith Sets
All sets employing tuning eye on dial scale. If eye remains stationary, replace 6C5G tube on rear chassis. Failure of this tube also responsible for fading and poor reception.
Wait! WE'RE HAVING OUR OWN TRADE SHOW
In the June issue of
RADIO RETAILING
COMBINED WITH RADIO TODAY
To show the rapid conversion of Distribution from merchandising to servicing ... To feature products available for the critical summer months ... To stress maintenance as a wartime necessity ... To make vital contacts between manufacturers and the trade, now urgently needed as jobbers and manufacturers trim their sales staffs.
CLOSING DATE OF TRADE SHOW ISSUE
JUNE 8
CALDWELL-CLEMENTS, Inc., • 480 Lexington Avenue, New York
What Happens When "Betty Goes to War"
Harry J. Deines, who has developed effective morale campaigns for General Electric Radio-electronics division, now concentrating on war production, is here shown with starlet of his latest production, "Betty Goes to War." Picture sequence tells story of young married couple in GE plant, and how, when Herb is called to Army service, Betty serves her country by building military radios.
Book Reviews
1942 Radio Diagrams and Servicing Information
by M. N. Beitman
Supreme Publications
328 S. Jefferson St.
Chicago, Ill.
The "most often needed" radio diagrams for 1942 radios as compiled by M. N. Beitman has just been published. Like previous editions for sets of other years, this book contains the circuit diagrams and service data on the more popular models. This edition, the fifth, contains 192 pages of diagrams and data, including a complete receiving tube characteristic table.
Many models which were released just before the production shut-down are included. The price is $2.
The Mathematics of Wireless
by Ralph Stronger
Chemical Publishing Co., Inc.
234 King St.
Brooklyn, N. Y.
This is the first American edition of this English book which is aimed at the "wireless experimenter" or the serviceman, and amateur who wishes to improve his mathematical background.
The book is not a complete text on the subject of mathematics but more in the form of an outline of the various branches of that science. The branches, arithmetic, algebra, geometry, trigonometry, differential calculus, integral calculus, and logarithms are explained in a simplified form. The operation and use of the slide rule is illustrated with enlarged pull-out photos. The application of these subjects to radio is illustrated in special chapters and problems. The price of this book is $3.
Acoustic Design Charts
by Frank Massa
The Blakiston Co.,
Philadelphia, Pa.
This unusual book will prove of value to engineers and designers requiring authoritative data for acoustic and related problems. This book is a compilation of acoustic engineering data into graphical form. Each pair of pages consists of a graph representing the relationship of the variables while the opposite page of the pair explains the graph and shows by example how it is used.
Ten Sections
The book is divided into ten sections, dealing with fundamentals, attenuation in various media, mechanical vibrating systems, acoustical elements, radiations from pistons, directional characteristics, reverberation and reproduction, exponential horn speakers, electro-magnetic design data, and a section of miscellaneous data. Most of the charts involving dimensions have both English and Metric curves. The scales cover a range of values which are common in actual designs.
The author is in charge of the Acoustic Division, Brush Development Co. The book contains 228 pages and is priced at $4.
Radio Leaders in Chicago Meetings
The week of June 7th in Chicago will mark several radio gatherings, although the regular Parts Show has been cancelled.
The 18th annual and "all out" war convention of the RMA is set for June 9th, complete with a membership luncheon session and various division and committee meetings later. Featured speaker is William L. Batt, a key official of the War Production Board, director of the WPB Materials Division and a chief aide to Donald M. Nelson.
RMA president Paul V. Galvin will preside at the luncheon meeting and there will be a report by treasurer Leslie F. Muter. The annual convention golf tournament will be held June 10th at the Calumet Country Club.
The annual meeting of the National Radio Parts Distributors Association will be held June 8th. Jobbers throughout the U. S. were asked whether they wanted the meeting; the majority said yes. NRPDA directors will meet June 7th, and the full membership huddle will be the following day.
Program plans will depend upon what happens before June 8th, in the fast-moving war production picture, but tentative plans are to invite representatives from both the WPB and OPA. Jobbers who wish to suggest topics of national importance for the meeting, are invited to send them to NRPDA temporary headquarters at P. O. Box 2, Reading, Pa.
Invited to the meeting on June 8th are "The Reps," the Sales Managers, and any other interested radio men.
The Sales Managers Club, Western Group, has named a Victory Program committee, including J. J. Kahn of Standard Transformer (chairman), W. A. Kuehl of Drake Electric, Ed Singer of Alliance Mfg. Co., and Jerome Prince of Carron Mfg. Co., to plan a joint meet with NRPDA and The Reps. The meeting is set for June 8, Stevens Hotel; dinner at 6:30 p.m.. Manufacturers, jobbers and reps are invited and all are urged to send reservations to Mr. Kahn at 1500 N. Halsted St., Chicago, with checks at rate of $2.50 per plate. Officials from WPB and OPA will be invited to speak, and answer questions on radio parts industry topics, according to Kenneth C. Prince.
"The Representatives" of Radio Parts Manufacturers will get together June 8th at the Bismark Hotel, for the annual meeting and banquet, under the auspices of the Chicagoland Chapter of the organization. On the program are S. K. MacDonald, the Reps' president; representatives from Sales Managers Clubs and from the National Radio Parts Distributors Association; and a representative from the War Production board. The chairman of arrangements is Royal A. Stemm, 21 E. Van Buren St., Chicago. This meeting is for both members and non-members.
Replacing DISCONTINUED WETS...
- Despite shortages of this, that and the other material, Aerovox continues to meet the essential requirements of the radio serviceman. Through engineering ingenuity closely geared to field conditions, plus production foresight and expanded facilities, Aerovox still has available a suitable replacement type for almost every need. To replace discontinued wet electrolytics, for example...
- Type GL, the always popular metal-can dry electrolytic. High filtering efficiency. Necessary choice of capacities, voltages, combinations.
- Type F prong-base dry electrolytic in compact metal can, packing a lot of dependable capacity into minimized bulk. Already a popular replacement in sets initially equipped with this type, it is now being used in general servicing.
- Type PRU, the handy AC-DC universal replacement dry electrolytic unit in tubular cardboard casing, with spade lugs. Also the PRS Dandees and the PBS cardboard-case types.
Ask Our Jobber... He'll gladly show you how Aerovox can help you keep those radio sets going by means of satisfactory replacement condensers. Ask for new 1942 catalog—or write us direct.
NEW BEDFORD, MASS.,
U. S. A.
Sales Offices in All Principal Cities
AEROVOX CORPORATION
I Forgot to Bring Home a Fidelitone PHONOGRAPH NEEDLE
The greatest improvement in 25 years. Up to 5,000 grooves from one needle. The perfect tip prolongs record life. Unique Floating Point construction filters record scratch. Only one dollar...ask your record dealer for a demonstration. Permo Products Corp., Chicago, Ill.
Fleeting Point permits smooth groove contact. Filters out record Scratch. Smooth construction cuts on needle point.
As Advertised in...
the Ladies Home Journal and 10 other magazines
Blackout & Emergency Equipment
Complete Directory of Manufacturers
Compiled by Radio Retailing Today
Air raid alarms, sirens . . . . A
Air raid alarms, radio . . . . AR
Amplifiers . . . . AM
Blackout light bulbs . . . . B
Blackout switches, photo tube . . . BP
Blackout switches, radio . . . BR
Intercommunicators . . . . I
Luminous paint . . . . LP
Photo electric apparatus . . . EE
Photo-tubes . . . . P
Relays . . . . R
Speakers . . . . S
ADVANCE ELECTRIC CO., 1200 W. 2nd St., Los Angeles, Calif.—AM
AMERICAN CO. OF AMERICA, 17 W. 20th St., New York, N. Y.—AM
ATLAS SOUND CORP., 1451 39th St., Brooklyn, N. Y.—AM
AUTOMATIC ELECTRIC MFG. CO., Mankato, Minn.—R, T, AM
BELL SOUND SYSTEMS, INC., 1183 Essex Ave., Columbus, Ohio—L, AM
BENJAMIN ELECTRIC MFG. CO., Des Plaines, Ill.
BLACKOUT CONTROL, INC., 545 5th Ave., New York, N. Y.—AR
DAVID BOOTH CO., INC., 663 Broadway, New York, N. Y.—AM
BREWER ELECTRIC MFG. CO., 5112 N. Ravenswood Ave., Chicago, Ill.—S
CINADAGUARD SPEAKERS, INC., 3911 S. Michigan Blvd., Chicago, Ill.—S
CONTINENTAL ELECTRIC CO., Geneva, Ill. "Cetron" Control, Inc.—S
CONTINENTAL LITHOGRAPH CORP., 953 E. 72nd St., Cleveland, Ohio—S
CRESCENT INDUSTRIES, INC., 4140 Belmont Ave., Chicago, Ill.—S
LEE DEFOREST LABORATORIES, 5106 Wilshire Blvd., Los Angeles, Calif.—BR
DURAKOOL, INC., 1010 N. Main St., Elkurt, Ind.
HUGH H. EBY, INC., 4700 Stanton Ave., Philadelphia, Pa.—R, P, EE
ELECTRONIC PRODS. CO., INC., St. Charles, Ill.—S
ERWOOD SOUND EQUIP. CO., 223 W. Erie St., Chicago, Ill.—AM
FEDERAL ELECTRIC CO., INC., 8700 S. State St., Chicago, Ill.—A
FLORIDIAN COMPONENTS CO., 445 W. 41st St., New York, N. Y.—LP
GENERAL ELECTRIC CO., Schenectady, N. Y.—BP, BR
GENERAL LUMINESCENT CORP., 730-34 Federal St., Chicago, Ill.—LP
GENERAL OUTDOOR ADVERTISING CO., 122 E. 23rd St., New York, N. Y.—BR
G-M LABORATORIES, INC., 4510 N. Knox Ave., Chicago, Ill.
GUARDIAN ELECTRIC MFG. CO., 1621 W. Walnut St., Chicago, Ill.—R
INDUSTRIAL TIMER CO., 117 Edison Ave., Newark, N. J.—BR
JENSEN RADIO MFG. CO., 6601 S. Laramie Ave., Chicago, Ill.—S
KIMBAXCOV CO., 2131 Butler Rd., Ft. Wayne, Ind.—S
JOHN MEAD INDUSTRIES, 1313 W. Randolph St., Chicago, Ill.—AR, AM
MEISSNER MFG. CO., Belmont and 7th Sts., Mt. Carmel, Ill.—R
MICRO SWITCH CORP., Spring St., Freeport, Ill.—R
NATIONAL UNION RADIO CORP., 57 State St., New York, N. Y.—AB
OPERADIO MFG. CO., 13th & Indiana Sts., St. Charles, Ill.—L, AM, S
OXFORD TARTAN RADIO CORP., 915 W. Van Buren St., Chicago, Ill.—S
PERMFLUX CORP., 4010 W. Grand Ave., Chicago, Ill.—S
PHILCO RADIO & TELEV. CORP., Tioga & C Sts., Philadelphia, Pa.—AM
PHOTO BELL CORP., 123 Liberty St., New York, N. Y.—AM
POTTER & BRUMFIELD MFG. CO., Princeton, Ind.—S
PRESTO RECORDING CORP., 242 W. 55th St., New York, N. Y.—AM
QUAM-NICHOLS CO., 33rd Place & Cottage Grove Ave., Chicago, Ill.—S
RACON ELEC. CO., INC., 52 E. 19th St., New York, N. Y.—S
RADIO SPEAKERS, 221 Culbertson St., Chicago, Ill.—S
RADDLEK CO., 601 W. Randolph St., Chicago, Ill.—S
THE RAULAND CORP., 4245 Knox Ave., Chicago, Ill.—S
RBM MFG. CO., Logansport, Ind.—R
RCA MFG. CO., Front & Cooper Sts., Camden, N. J.—R, P, EE, I, AM, S
REGAL AMPLIFIER MFG. CORP., 14 W. 17th St., New York, N. Y.—AM
REHTYRON CORP., 2150 Magnolia Ave., Chicago, Ill.—S
REYNOLDS ELECTRIC CO., 2620 W. Congress St., Chicago, Ill.—Control units for electric sirens.
SPARKS-WITHINGTON CO., 42 North St., Jackson, Mich.—S
STRUTHERS DUNN, INC., 1819 Cherry St., Philadelphia, Pa.—S
TALK-A-PHONE MFG. CO., 1210 W. Van Buren St., Chicago, Ill.—I, AM
TECHNOCRAFT CO., 119-23 E. 24th St., New York, N. Y.—AM
THORDARSON ELECTRIC MFG. CO., 600 W. Huron St., Chicago, Ill.—AM
TRANSFORMER CORP. OF AMERICA, 69 Wooster St., New York, N. Y.—I, AM
UNITED CINEPHONE CORP., Torrington, Conn.—R, S
UNIVERSITY LABORATORIES, 195 Chrystie St., New York, N. Y.—S
UTAH RADIO PRODUCTS CO., 820 Orleans St., Chicago, Ill.—S
VITA-VAR MFG. CO., 55 Albert Ave., Newark, N. J.—LP
WARD EXCHANGE CORP., 335 Carroll St., Brooklyn, N. Y.—B
WARD LEONARD INSTR. CO., 34 South St., Mount Vernon, N. Y.—AM
WEBSTER CHICAGO CORP., 5622 Bloomingdale Ave., Chicago, Ill.—I
WEBSTER ELECTRIC CO., Racine, Wis.—I, AM
WELFORD MFG. CORP., 3094 E. Outer Dr., Detroit, Mich.—EE
WESTERN SOUND & ELEC. LABS., INC., 311 W. Kilbourn Ave., Milwaukee, Wis.—S
WESTERN ELECTRICAL INSTRUMENT CORP., 614 Frelighyser Ave., Newark, N. J. "Photronic"—R, P, EE
WORNER PRODS., 1010 W. Lake St., Chicago, Ill.—RP, EE
Stromberg’s Victory Production Drive
The war production program at the Stromberg Carlson plants went into high speed last month when the firm staged a huge rally of employees to announce winners of the “work for Victory” slogan contest. Army and Navy officials were on hand to stress the importance of the war work being done at the factory. In the SC production drive, employees had submitted more than 3,340 slogans.
The last civilian radio chassis to be built by Stromberg for the duration came off the production lines April 21, and the company is now completing deliveries of all manufactured products.
Mr. Petrie Dead
Robert I. Petrie, vice president and general sales manager of the Crosley Corp.’s manufacturing division, died in Cincinnati on May 8th after a short illness. Mr. Petrie was one of the best known executives in the appliance industry, and was previously president and general sales manager of Barlow & Seelig Mfg. Co., Ripon, Wis. Before that, the popular Crosley official had been sales manager for Nash-Kelvinator Corp.
Jobber Sales Head
Sylvan A. Wolln has been appointed jobber sales manager for Solar Mfg. Co., Bayonne, N. J., makers of capacitors. The well known exec was previously Solar's sales promotion manager and asst. to Wickham Harter, general sales manager.
WPB Shifts Radio Officials
The radio authorities of the War Production Board have been reorganized and new experts are now in charge of industry problems. The shift follows the shut-down last month of civilian radio production, which left few remaining civilian problems except those concerning replacement parts and tubes.
The WPB Radio Section, heretofore directly under Chief Robert C. Berner, and the Consumers Durable Goods Branch, under Chief Louis C. Upton and Assistant Chief Jesse L. Maury, is transferred to the WPB Communications Branch, whose chief is Leighton H. Peebles. Under Mr. Peebles, the new Radio Section Chief is Frank H. McIntosh, an experienced radio engineer, who has just assumed his office. Mr. Maury has also resigned as chairman of the WPB Radio Industry Advisory Committee.
All present and also future radio problems and jurisdiction, including the present limitation "L" orders, and the questions of replacement tubes and parts, are being taken over by the Communications Branch in the WPB reorganization. Some of the Radio Section personnel under Messrs. Maury and Berner are being transferred to the Communications Branch, while others of the present Radio Section staff will remain with the Consumers Durable Goods Branch, with others transferred to the Communications Production Branch under Ray Ellis.
Immediate action-and further orders under the "L" regulations are in a state of transition. The question of future orders and action on appeals under the "L" orders, officials stated, are being handled during the temporary transition period jointly by the retiring Radio Section Chief, Mr. Berner, and the new Communications Branch Radio Section Chief, Mr. McIntosh. Immediate questions affecting set manufacturers and also future supplies of replacement tubes and parts have been discussed by RMA officials in a preliminary way with the new Radio Section Chief, Mr. McIntosh.
Both the Communications Branch under Mr. Peebles and the former Radio Section of the Consumers Durable Goods Branch are in the organization of the Chief of the Bureau of Industry Branches, under Philip D. Reed, in the Division of Industry Operations, whose director is J. S. Knowlson.
GE to Set Up Servicing Centers
The authorization of General Electric dealers (those who offer adequate repair facilities in accordance with minimum standards) as local GE appliance service centers in their communities, is one of the features of a streamlined servicing program announced by the firm's appliance and merchandise department. The plan includes all appliances but radio, furnaces, and air conditioning. A separate plan is being prepared for radio, television and electronics.
A new speed-up on parts supply is also provided in the plan, as GE announces "The establishment of GE owned regional appliance service centers in various sections of the country, each regional service center serving as a pool of replacement parts and repair facilities for several distributorships."
Jobbers Must Keep Complete Records
Parts jobbers are being earnestly advised by NRPDA to keep complete records on their business, as these are emphasized by new priority rulings. Also, it is suggested that jobbers give more details to their suppliers, on priority orders, since "in many cases the addition of the 'end use' and name of the user to the regular priority data will enable the producer to show preference between several A-1-A ratings, especially if one of them covers aircraft radio.
NRPDA is busy with interpretations of forms PD-1X, L-63, and PD-336, in a continual effort to help jobbers get parts. It is expected that many vital aspects of these rulings will be discussed at the annual Chicago meeting of the organization, June 8th.
R.C.P. Expands
Radio City Products Co., Inc., makers of RCP test instruments, have moved their plant and offices to new and larger quarters at 127-133 West 26th St., New York City. The shift doubles RCP's floor space.
NRPDA Chapter Meets in Philly
The latest meeting of the Philadelphia District chapter of the National Radio Parts Distributors Association was held at the Benjamin Franklin Hotel in Philly with some 20 jobbers on hand, including the national NRPDA president, George Barbey.
Discussion centered around priorities and price freezing, disposition of surplus merchandise, plans for national meeting at Chicago, tube resale prices, and cash discounts.
Guests included: Myron E. Wolf, Eshelman Supply Co., Lancaster, Pa.; J. Wellington Kratz and Sam Kratz of Kratz Bros., Norristown, Pa.; Jacob Sherk and R. A. Sylvester, R. A. Sylvester Co., Hazleton, Pa.; Mr. and Mrs. G. O. Zimmerman of Zimmerman Wholesalers, Hagerstown, Md.; R. H. Wile, Eugene Wile Co., Philadelphia; M. P. Roskowitz and M. M. Oberst, M&H Sporting Goods Co., Philadelphia; John Stern and M. Green of Radio Electric, Philadelphia; Aaron Lippman, Aaron Lippman Co., Newark, N. J.; James D. Strauss, J. R. S. Distributors, York, Pa.; Dahl Mack, Scranton Radio Supply, Scranton, Pa.; and R. M. Peffer, Radio Distributing Co., Harrisburg, Pa.
Radio Reps Hold Meetings
The Regional chapters of "The Representatives" continue to hold regular meetings, in spite of the rush of wartime activities.
News from the Wolverine chapter reveals the addition of Joseph P. Davenport, 642 Beaubien, Detroit, as a new member, and the election of R. A. Adams as the new secretary of the chapter. William S. Lee recently resigned that post.
At the Chicagoland chapter, a new member is Harry Hallinton, 4215 N. Newhall, Milwaukee. Another member, Robert Ford Taylor, has received a commission as a Lieutenant in the Army and is now stationed at Aberdeen Proving Grounds, Maryland.
Reps' national records also show that three members have changed their addresses. These are T. C. Ruhling, now at Box 537, Dallas, Tex.; Harrison Reynolds, now at 34 Bay State Road, Belmont, Mass.; and F. E. Harding, 4925 Penn Ave., S., Minneapolis, Minn.
Boston Jobber Cited for A.R.P. Work
A radio man can contribute a great deal to the success of the air raid wardens in his area, if he'll get into the spirit of community enterprise and all-out helpfulness.
A report comes from Boston concerning the well known jobber, Joe DeMambro, who's doing some notable work with the wardens of his city. Recently he donated complete sound equipment for use at Civilian Defense lectures, held at a local high school. On this occasion, one of the Boston police captains wrote Mr. DeMambro a letter of special thanks, which also complimented him on being one of the most efficient wardens in the area.
Here's one example of alertness and initiative on the part of a radio expert whose specialized knowledge is important to local welfare.
Hundreds of Needles!
Illustrating the new emphasis on record accessories is this red-white-and-blue window at Lyon & Henly, Chicago, featuring the complete line of 15 different phono needles made by Duetone, Inc.
Farnsworth Uses Field Force for Procurement
Announcement from the Farnsworth Television & Radio Corp., Ft. Wayne, Ind., is that the personnel of the Farnsworth and Capehart sales staff have been appointed to posts in the firm's new "Materials Procurement Division." The new division is headed by E. J. Hendrickson, who will in turn report to E. H. Vogel, vice president, and to E. S. Needler, general purchasing agent.
Scattered companies which supply war material now number more than 400, and the procurement men will therefore work as near as possible to their respective sales areas and offer whatever support is possible to their distributors and dealers.
GE Starts Big Radio Promotion
Two new ad drives in national magazines have been launched by General Electric; both of them educational programs. One deals with radio and tubes, and helps the public with all its reception problems during wartime. The other is designed to enlighten the public—business men and engineers in particular, about amazing new strides in development of electronics.
The radio ads run in the Sat Eve Post, Life, Esquire, and The New Yorker. GE will also continue sponsorship of its coast-to-coast news program on CBS, thrice weekly, featuring the commentator, Frazier Hunt.
Emerson Announces Full Line of Parts
Just announced by Emerson Radio & Phonograph Corp., 111 Eighth Ave., New York City, is a complete line of replacement parts for servicing all makes of sets.
The line includes receiving tubes, ballast tubes, pilot lights, flashlight bulbs, condensers of all types, resistors, resistance line cords, shielded I.F. transformers, varied phonograph equipment, speakers, transformers, volume controls, drive belts, etc. Emerson now has various kits, such as a volume control kit, drive belt kit, resistor kit, condenser kit, etc. to help in merchandising these parts. Each kit contains an assortment of popular values most generally used in servicing.
Polymet Condensers Get a Play
New announcements of the aggressive service offered on replacement condensers by Polymet Condenser Co., 699 E. 135th St., New York, are now current in the parts business. Polymet, which is one of the oldest names in the condenser business, is urging jobbers and service men to guard their wartime business by using more care in the selection of their by-pass and electrolytic condensers. The firm points to its "21 years of constant research and development" as an indication of its product quality.
To conserve the vital defense materials, Polymet is limiting sizes to those most generally used. The company will send a complete catalog and price sheet on request.
Cornell Dubilier Names Two Vice Presidents
Two new vice-presidents have been named at the Cornell-Dubilier Electric Corp., South Plainfield, N. J., according to news from Octave Blake, president of the firm. They are William M. Bailey, chief engineer in charge of the industrial and transmitter capacitor divisions, and Paul McKnight Deeley, head of the chemical laboratories, the electrolytic capacitor division and the export section.
Patrick for Sylvania
D. C. Patrick, has been named High-grade Sylvania representative in the Denver, Colo., area for all of the company's products including Sylvania Radio Tubes, fluorescent lamps, Miralume fluorescent fixtures, fluorescent accessories and incandescent lamps.
Diversification Improves Radio Volume Outlook
In recent months, the radio man has found himself looking more favorably on the idea of wider diversification of lines.
It means more departments to divide the overhead, more complete service to customers, more traffic and more sources of profit.
Take the example of a store which now sells radio sets, parts, sound equipment, electrical appliances, records and recording accessories, amateur equipment, electronic devices, test instruments, batteries, FM and television. This is the case at Sun Radio Co., 227 Fulton St., New York City and the store has been able to attract a lot more than the usual group of householders.
Sun is now filling orders for government agencies, broadcast stations, industrial organizations, schools and colleges, research labs, amateurs, manufacturers, engineers, mining firms, dealers, servicemen, purchasing commissions and exporters.
Regular Expansion
The "spread-out" policy has worked well for this outfit. This year the firm celebrates its 20th year in the radio parts business.
A wide variety of lines must be properly supported by appropriate personnel. Samuel Schwartz, sole owner of the Sun firm, believes that radio sales success depends almost entirely upon the calibre of salesmen on the floors. Sun has adopted the policy of "specialists for each department" and sees to it that the activities in any one branch of the business are handled by a selected man well qualified in that particular field. Personal service to customers is considered of utmost importance.
Wartime Work
Although diversification of lines is a great help during this wartime period of merchandise shortages, it also involves a mass of bookkeeping in order to keep abreast of priority restrictions and rulings on all this merchandise.
To meet this situation, Sun Radio has created a special division of pri-
A store front with some style and glitter is important where street traffic is heavy, as it often is at this downtown Manhattan locale. The chief material used is black glass; big letters are wooden, with gilt paint. Note how diversified products are given attractive emphasis.
orities, to learn all about the details and to make an enlightened and organized effort to keep stock and deliveries up. Present stocks are "the greatest ever in its history."
Special Jobs
Also to be counted in the wartime activities of the company are its big installations of public address systems in industrial plants, schools, factories and offices for the dissemination of music, general instruction and air raid warnings. It is reported by Harry Adelman, advertising manager for the company, that "industrial research and development laboratories faced by the necessity of exerting speed in completing schedules, appreciate what a many-sided radio man can do to help!"
Thus Sun Radio illustrates again the theory that the more lines a dealer carries, the more likely he is to fit into any situation that arises.
RADIO RETAILING Combined with RADIO TODAY, May, 1942
Captain John Rider to the Colors
John F. Rider, publisher of Rider's Manual and other radio-service books, has been appointed a captain in the United States Army Signal Corps, and is temporarily located at Fort Monmouth in New Jersey. It is understood that Captain Rider will give special attention to organizing methods for the maintenance and repair of Army radio equipment, in connection with the two-billion-dollar radio production program recently undertaken by the Government.
Captain Rider was guest of honor at a dinner at the New Yorker Hotel, New York City, April 29, when a group of his metropolitan friends gathered to wish him godspeed on leaving civilian life to undertake his new Signal Corps duties.
NEW BOOKLETS
Bogen's new Commun-phone catalog features the new deluxe system and also other industrial paging systems together with suggested installations and accessories to the system. Another catalog just released by Bogen is "The Blue Book of Sound Equipment" which gives specifications and illustrations of their line of sound systems, amplifiers, record players, baffles, speakers, mikes, and other sound accessories. David Bogen Co., Inc., 663 Broadway, New York, N. Y.
A new battery replacement chart for 1250 models of portable radios and a cross tabulated equivalent battery table is printed on one side of large heavy sheet suitable for wall mounting. Copies may be obtained from Radio & Technical Publishing Co., 45 Astor Pl., New York, N. Y. for 10c to cover mailing and handling.
The 1942 edition of the Sprague Manual of Radio Interference Elimination covers the problems of identifying, finding and curing many types of interference. Booklet is available from Sprague Products Co., N. Adams, Mass., or through jobbers for 25c.
Now available from distributors of Emerson Radio & Phonograph Corp., 111 Eighth Ave., New York City, is a new parts catalog, describing and illustrating the firm's new line of replacement parts, with prices.
A 20-page booklet on phonograph needles, the care of records, and home recording is available from Duotone, Inc., 799 Broadway, New York City, for distribution to the consumer by dealers and distributors. Initial printing of 100,000 booklets is exhausted; a second printing is ready.
RADIO MAN'S CAR
OWNER ABSENT
PLEASE INFORM POLICE IF YOU SEE ANYONE TAMPERING WITH TIRES OR CAR
Sign used by radio men to prevent tire thefts. Sign is carried inside car and is placed in window facing sidewalk, before locking and leaving car.
A number of radio men have written in to the editors, pointing out that great difficulties face radio servicemen in the near future, if they are not to be given a priority position in the purchase of tires for the cars in which they make their service calls.
We here at Radio Retailing Today recognize the serious situation which will soon confront the service man, and are taking steps to bring the radio man's claims to the attention of the authorities in charge of tire rationing.
In the meantime, radio men should do everything possible to conserve the tires they now have, by careful use and by protecting them against theft. As one means to safeguard tires and car, we bring to your attention the form of anti-tampering sign which several western radio servicemen have been using since the tire situation became acute. In many cities organized thieves have been going about stealing spare tires and actually jacking up cars and stealing tires, wheels and all. A card like the above in the parked car, puts passersby on notice that the owner is absent and that the car is being molested by unauthorized persons. The presence of such a sign in the car will in many cases scare off thieves from attempting tire robberies.
Display to Hike Book Sales
A brand new counter display, available to dealers without charge is the attention-getting unit released by Radio & Technical Publishing Co., 45 Astor Place, New York City, featuring Ghirardi's 972-page "Radio Physics Course." The display is one of the string of sales aids centering around the "Silent Salesman" counter book display supplied to jobbers several months ago.
The new unit appears at a time when the interest in radio training is at an all-time peak, and also when dealers are looking for appropriate books to sell. It's a 4-color job in heavy cardboard, 20 in. by 14 in., richly varnished. The book price of $5 is prominently shown.
Ghirardi's "Radio Troubleshooter's Handbook," the second addition which appeared last Fall, now has a second printing on the presses. The new book aims to help servicemen save more time, and besides its original contents, "will include data on equipment produced right up to the time when receiver production was stopped."
Clarke for Dunco
The new sales and engineering representatives in the Chicago area for Dunco relays and timing devices, is the John W. Clarke Co., 327 S. LaSalle St., Chicago. The appointment was announced by Charles A. Packard, sales manager for Struthers Dunn, Inc., Philadelphia, Pa.
TOO TOUGH A JOB FOR A MAN!
IN MAKING RADIO TUBES, vital to the war effort, it is not a question of training women to do men's work. Assembly of the delicate, lace-like grids and tiny filaments needs the feminine touch! It's something like threading a needle a thousand times a day—and a mere man just can't compete!
Slim, swift fingers are what it takes to fashion the sensitive mechanisms that detect feeble radio waves and amplify them into sharp, clear signals. So go ahead, men, with your rivet-hammers and punch-presses. But when radio, with split-second precision, delivers an urgent message or a broadcast program—or safely guides ship and plane—give thanks to a woman's skillful fingers!
Of the many thousands of workers in the RCA services, nearly two-thirds are women. Most of them do work essential to the war effort, and more efficiently than a man could do it.
Radio Corporation of America
PIONEER IN RADIO • ELECTRONICS • TELEVISION
Radio City, New York
The Services of RCA: RCA Manufacturing Company, Inc. • Radiomarine Corporation of America • National Broadcasting Company, Inc. • RCA Institutes, Inc. • R. C. A. Communications, Inc. • RCA Laboratories • Blue Network, Inc.
SOON IT WILL BE TOO LATE
you can still GET Motorola AUTO RADIOS
IF YOU ACT NOW YOU CAN PROTECT YOURSELF FOR THE DURATION
Hundreds of items are now on the "can't get" list. So quit fretting about your inability to get them...and concentrate on selling the merchandise you CAN GET! Motorola Auto Radios are now at the head of the list of items you can still sell and get. But protect yourself NOW! See your Motorola Distributor for immediate delivery of America's Finest Auto Radio.
MILLIONS OF CARS ARE STILL ROLLING . . . AND WILL CONTINUE TO ROLL
6 out of every 10 cars have no radio. Their owners will buy radios if you ask them to buy! There is a Motorola to FIT and MATCH every car, OLD or NEW. Promote car radio sales and PROFIT NOW!
GET BUSY NOW—TODAY! • Write or Phone Your Distributor
GALVIN MFG. CORPORATION • CHICAGO
|
As Big Country enters its 18th year and presents its awards for the 16th time, RPM reflects on the history of the event and its role in helping develop the Canadian country music industry.
**HIT PICK**
- CAN'T GET ENOUGH OF YOUR LOVE - Taylor Dayne
Arista
**ALBUM PICK**
- ROD STEWART
Unplugged And Seated
Warner Bros - CDW 45289-P
**ALBUM ADDS**
- JANET JACKSON
Janet
KISS
Alive III
**No. 1 ALBUM**
- SPIN DOCTORS
Pocket Full Of Kryptonite
Epic Associated - CK-47461-H
**BIG HITS**
- COAT OF SHAME
Mae Moore
- FIELDS OF GOLD
Sting
- DON'T TAKE AWAY MY HEAVEN
Aaron Neville
- IN THE MEANTIME
The Waltons
- THE TRUTH
Banned In The U.K.
- NO APOLOGIES
Alanis
- BY THE TIME THIS NIGHT IS OVER
Kenny G & Peabo Bryson
- BREAK IT DOWN
Tears For Fears
- ONE TONGUE
Hothouse Flowers
- DOWN WITH THE KING
Run DMC
**COUNTRY ADDS**
- FORTUNE SMILED ON ME
Cassandra Vasik w/ Russell deCarle
- RENO
Doug Supernaw
- HOLDIN' HEAVY
Tracy Byrd
- THANK VIRGINIA
Gibson/Miller Band
**BIG ALBUMS**
- JIMI HENDRIX
The Ultimate Experience
- SWV
It's About Time
- CHRIS ISAAK
San Francisco Days
- WYNONNA JUDD
Tell Me Why
- DIRE STRAITS
On The Night
- JOEY LAWRENCE
Joey Lawrence
- TASMIN ARCHER
Great Expectations
- DANCE POOL VOLUME ONE
Various Artists
**HIT ADDS**
- GIRL I'VE BEEN HURT BEFORE
Snow
- CAPTAIN NEMO
Sarah Brightman
- HERE'S LOOKING AT YOU KID
April Wine
- UNSTOPPABLE
The Kings
- WALKING IN MY SHOES
Depeche Mode
- MOMENTS OF LOVE
Cathy Dennis
- WHAT TIME IS IT
Spin Doctors
Quality Dino reports decreased net revenues
Quality Dino Entertainment Ltd. has released its financial figures for its third fiscal quarter ended March 31.
The company's net revenues from the third quarter decreased to $18-million from $30.6-million for the same quarter the previous year. However, the net loss for the quarter decreased to $143,000 from $733,000 for the same period.
Net revenues for the nine months ended March 31 decreased to $56.3-million from $97.2-million for the same period in the prior fiscal year. The company reported a net loss of $1.2-million compared to net income of $1.8-million for the same period in the prior fiscal year.
For the nine months ended March 31, Quality Dino Entertainment operated within North America, the United Kingdom, Australia and New Zealand. The comparative results for the nine months ended March 31, 1992 include additional operations in nine countries within continental Europe. The company entered into a plan to withdraw from its continental European operations effective June 30, 1992.
Quality Dino Entertainment Ltd. is primarily engaged in the acquisition, production, marketing and distribution of music. The company distributes these products under the Dino and Quality labels. It also selectively markets home videos and small consumer products.
Vancouver and Barrie to host Lollapalooza dates
Vancouver's Thunderbird Stadium will have the distinction of opening Lollapalooza '93 on June 18.
The travelling road show will feature a main stage with Rage Against The Machine, Babes In Toyland, Front 242, Arrested Development, Fishbone, Dinosaur Jr., Alice In Chains and Primus. A secondary stage will feature Tool, Mercury Rev, Mutabaruka and local acts.
In addition to the music, The Village will feature 200 Canadian vendors with a panorama of arts, crafts, hair styles, alternative clothing, jewellery, leather, exotic food and other surprises. The Forum will add to the festival's socio-political slant with lively discussion in a debate arena.
Lollapalooza's other Canadian stop will be at Molson Park in Barrie, Ont. on July 10. The Barrie show will have the same main stage lineup but will substitute A Lighter Shade Of Brown and Unrest for Mercury Rev and Mutabaruka on the secondary stage. Local acts will also be invited to take part.
Any local acts without major label record deals interested in being considered for the secondary stage lineup at either the Vancouver or Barrie shows should send tapes, bios, photos, etc. to: Lollapalooza, P.O. Box 36674, Los Angeles, CA 90036. Please indicate what city you wish to play in.
Sony Music Canada does internal restructuring
Sony Music Canada has made some internal restructuring moves in recent weeks.
Michael Roth has been named creative director of music publishing and artist development.
Vito Luprano has assumed additional responsibility for English and French language A&R in his position as director of A&R for Sony Musique. A&R vice-president Richard Zuckerman will continue to be part of the expanded department and, along with Roth and Luprano, will report to Sony president Rick Camilleri.
Laurie McRae has been appointed vice-president of human resources.
Jeff Shannon has been appointed to the new position of director of business development and will report to Camilleri.
Tom Hay has been given the new position of senior vice-president of operations and information services.
Alan Dyer, vice-president of finance, has taken charge of the finance department. The purchasing and security departments are also reporting to Dyer, who will answer to Camilleri.
Sandra Power, manager of music marketing, will report to Camilleri along with Roth as the two of them continue to expand Sony's music publishing company.
Ian MacKay is the new director of business affairs and will be responsible for all general legal and business affairs while reporting to Camilleri.
Don Oates, senior vice-president of sales, and Chris Black, vice-president of marketing, continue to report to Camilleri.
Toronto Beaches filled with jazz at festival
The fifth annual Beaches International Jazz Festival will fill Toronto's east end with music from July 21-25.
While jazz is highlighted, calypso, Latin, fusion and steel drum music will also be a part of this summer's festival. Each evening from July 21-23, a number of musicians will perform on Queen Street East corners between Woodbine and Beech Avenues. The Tony Carlucci Quintet, the Durham Big Band, J.R. Connection, Steve Lacey, Walle Larsson, Pan Man Pat, Tim Postgate and Todo Mondo Loco will be among those taking part.
The Kew Beach Bandshell will host shows Saturday and Sunday. Another Life, Muthadi & Friends, Francois Bourassa Trio, the Brian Hughes Group, the Pucho Lopez Group from Cuba, Steps Ahead and Robben Ford & The Blue Line are scheduled for the first day. The Bob Brough Quartet, the Lorraine Desmarais Quartet, the Bill King Quartet with Liberty Silver, Archie Alleyne and The Evolution of Jazz, an eight-piece big band featuring Miki Howard, Alex Bugnon, Jon Lucien and Warren Hill, and A.Y. Croce will round out Sunday's lineup.
Proceeds from the festival's sale of T-shirts and other memorabilia will go to Senior Link, a non-profit organization that provides year-round assistance to seniors in the Beaches area. The festival has raised in excess of $60,000 for Senior Link to date.
Warner Music Canada presented INXS with gold records for their Welcome To Wherever You Are album after their Toronto appearance. Warner's Roger Desjardins and Ken Green, INXS' Tim Farriss, Andrew Farriss and Kirk Pengilly, Warner's Randy Sharrard, INXS' Michael Hutchence and Warner's Garry Newman and Steve Waxman stand behind INXS' John Farriss and Garry Gary Beers.
The Miss Saigon extravaganza!!! Never before in the history of live theatre has there ever been such a display of one-upmanship. Larger and smoother-running sets, whirling, make-believe rivers, explosives and more. Not to be outdone, Ed Mirvish and his son David are far and above their closest rival. They actually built a theatre in order to land a helicopter on stage to bring realism to its peak in their production of Miss Saigon. The Princess of Wales Theatre, an awesome, almost technologically-perfect house, and the only one of its kind in North America, is comfortable and as much a spectacle as the on-stage production. There is, however, a problem with the sound. From where I was sitting in the dress circle, I found it difficult to pick up much of the vocal drama -- although the four letter words came through loud and clear. The Evita-esque marching soldiers, the low-flying helicopters, the mob scenes and the highlight -- the helicopter landing -- was the stuff to keep the adrenalin flowing. But getting to the exciting parts was somewhat dull, not to mention confusing. Despite this little annoyance, Miss Saigon is spectacular theatre and it should run well past its projected two-year break-even run. Winnipeg's Ma-Anne Dionisio as Kim, the Vietnamese Madame Butterfly, is superb and emerges as a glittering new jewel in the world of theatre. Her vocal capacity and range is artistically pure. Kim's pimp, the engineer (Kevin Gray), who has the vocal humour and movements of a purveyor of flesh down perfect, is the glue that holds the production together. The U.S. soldier Chris (H.E. Greer), Kim's lover, has difficulty in the vocal credibility field. The support cast rates a perfect 10. Miss Saigon is a must-see happening and, thanks to the Mirvishes, it has firmly established Toronto as a leading production centre for live theatre. (EC: Tell us about the gala . . . !)
Ed and David Mirvish as a team are extraordinary showbusiness people. No matter what the opening, the care and concern for detail in catering to their first-nighters goes without question. After Miss Saigon completed several curtain calls, the patrons, now looking to party, spilled out onto the street. They were ushered to a never-ending lineup of cabs for the trip to an unbelievably Saigon-ish tailored-to-turn-heads warehouse on Toronto's waterfront. Some opted to bump along in the offered foot and/or pedal powered rickshaws, which was very apropos. With the exquisite touch of Daniel Daniel doing the catering, more than 2,000 of Toronto's well-healed glitterati, not to mention the obvious gate-crashers, feasted on what must have been more than a couple of tonnes of deliciously-prepared gourmet essentials and sweets, which they gleefully and noisily washed down with Mums champagne and/or whatever hard liquor or beer they preferred. It was a night designed for tucking away in the memory node. (EC: But the glitterati have such a short memory . . . !)
Where there's a way . . . ! That tamper-free system to monitor retail sales in the U.S. is being tampered with. No one is saying too much about it yet, but there's an undercurrent of suspicion and resentment building. (EC: Oh well! Back to the drawing board . . . !)
The power of radio . . . ! Isn't it amazing how one radio station can cause such a flap in the industry. CISS-FM, Toronto's new country/AC station, has created a hysteria over country music which has never happened before. And now the Johnny-come-lately's are moving in. Newer and bigger country clubs have been opened and more are on the way. Even the dailies are finding country artists newsworthy. Retailers have finally been dragged, kicking and screaming, into believing that country has potential. Those opportunists who hope to make a bundle on this newly-discovered music sensation that's been around longer than a dog's age (certainly much longer than rock 'n roll) better not take country fans for granted. They're very loyal and they know when they're being screwed around. They'll turn off a radio station and avoid a club at the least little hint of someone trying to take advantage of their love for country. Be aware of new country/AC, but don't count the veterans of country out. The most important age group at radio is still 26-plus, and that's the REAL country listener and buyer, and don't forget it. (EC: Right on!!!)
Another west coast grizzly??? They grow them big and dirty on the west coast and one not-so-new character has pissed on the four corners of the province and claims it as his own. He's effectively built an empire, but watch for the self-anointed emperor to lose his crown and toga. (EC: Crown and toga? Sounds like a board game . . . !)
Frack's hit the road . . . ! Hey! The jury's in on the new weaver of corporate shakcousts. He gets a 9 out of 10, even from his competitors. He's constantly on the road asking questions about his company's performance. Some of the answers are hurtful, some patronizing, but most of them are honest and constructive. He has literally hit the road and, armed with a box full of comments and complaints, it's pretty obvious he's out to restore the respect the company once had and that's been eroding for more than a few years. (EC: Ooh! That's a tall order . . . !)
VISITORS
Kim Zayac - Klm Zayac Enterprises
Nat Merenda - Sony Music Canada
Ken Berry - Warner Music Canada
Rick Wharton - MCA Records Canada
Roger Bartel - EMI Music Canada
Pat Bachynski - Columbia/Sony
Linda Nash - Quality
David Lindores - A&M/Island
PROMOPERSONS' PICKS
RICK WHARTON - MCA Records Canada
Hit: Blood Of Eden - Peter Gabriel
Album: The Waterboys - Dream Harder
ROGER BARTEL - EMI Music Canada
Hit: The River - Tea Party
Album: John McDermott - Danny Boy
PAT BACHYNSKI - Columbia/Sony
Hit: Big Gun - AC/DC
Album: Judas Priest - 73-93
LINDA NASH - Quality
Hit: Healing Power Of Love - Dan Hill
Album: Various Artists - Country North
DAVID LINDORES - A&M/Island
Hit: S.O.L. - Kon Kan
Album: Sarah Brightman - Dive
Canadian Music Industry Hall of Fame for his "outstanding contribution to Canadian country music."
The steering committee met the next spring and an interim board of directors was elected from those members of the original CACMA who were present. A board was required so that the developing trade association, from that day officially known as the Academy of Country Music Entertainment, could become a chartered, non-profit organization.
The first board was comprised of: Hank Smith, president; Charlie Russell, vice-president; Gary Buck, secretary-treasurer; and directors, Larry Kunkel, Lanny Salazar, Sammy Jo, and Klees. Their appointment was only an interim measure as they were not elected by a general meeting.
Big Country moved west to the Edmonton Plaza Hotel for the next convention and awards ceremony, Sept. 25-26. A mixture of optimism and doubt was in evidence as ACME's 350 members gathered to try and bring country music to a wider audience by forming a number of specialized committees which could focus on improving various parts of the industry. After some bickering, Dave Charles, the program director of CFGM Radio in Richmond Hill, Ont. (and now president of the Canadian Academy of Recording Arts and Sciences), invited his rival, Barry Nesbitt, general manager of CKFH Radio in Toronto, to work with him in heading up the radio committee.
Charles urged all segments of the industry to "talk to each other, (and) break down the mental walls and barriers in your mind."
Within 45 minutes, eight other committees were also formed. They were: record companies, headed by Barry Haugen; retail, headed by John Davies; artists, headed by Joe Brown; agents and managers, headed by Don Grashay; fundraising, headed by Blake Emmons; publishers and writers, headed by Bev Munro; club owners, headed by Jerry Brown; and public relations, headed by Patti McDonnell and Marghi Cocks. The committees were to report to the board of directors. It was decided that organizational membership would be $100 minimum.
At the meeting, Grealis presented ACME with a cheque for $3,040. The money was the total of the memberships received by CACMA to help launch the new official academy.
This atmosphere of co-operation pleased ACME president Smith, who said, "I finally have a feeling of confidence in ACME. I haven't had it since I became president in March."
ACME also honoured four members who had worked in country music for a quarter-century. Orval Prophet, Joe Brown, Dick Damron and Harold Moon all received commemorative plaques.
Grealis emceed the awards banquet and, between presentations, seven of Alberta's finest country artists performed: Joyce Smith, Tony White, Donna Adams, R. Harlan Smith, Merv Smith, Paul Lahn and Roxanne Goldade.
Comic relief was supplied by Ronnie Hawkins, who had a hard time opening the envelope which held the name of the winner for best country single. The Hawk said he never had that trouble at home because the RCMP opens all his mail before he gets to it.
Soon after the Edmonton meeting, things began to unravel once again as a lack of communication between executive members themselves, and with the membership at large, started causing problems. The geographic distance between executive members was a difficult obstacle to overcome. As a result, Russell eventually tendered his resignation from the executive.
Ottawa hosted Country Music Week from Sept. 19-25 in 1975. The Big Country Awards closed the week-long celebration of country activities at the Skyline Hotel. The show was once again emceed by Grealis. Damron, Dallas Harms, the Family Brown, Prophet, Bob Webb and Baker all performed.
Country Music Week was celebrated for the second consecutive year, this time in Regina, from Sept. 25-29. During the week, the Big Country album was released as a means of raising funds for ACME. The Big Country meeting and awards were held Sept. 30 and Oct. 1 at the Regina Inn.
" . . . making them honorary ACME members in recognition of their founding of both the Big Country Awards and ACME."
On Saturday, expert panel discussions were held on such topics as the country artist in Canada, programming Canadian country music, racking and retailing Canadian country music, and the Canadian country record company. That evening a special showcase featuring Baker, Terry Carisse & Tenderfoot, David Thompson and Jerry Palmer entertained the assembly.
ACME held its annual general meeting the next morning and Charles, who had by that time become president, presented Grealis and Klees with plaques making them honorary ACME members in recognition of their founding of both the Big Country Awards and ACME. Paid memberships to the academy had grown from 50 to 630 in 12 months, with the bulk being individual memberships. ACME was also granted a federal charter enabling it to apply to the federal government for funds for the betterment of the country industry in Canada.
The awards show that evening was emceed by Ronnie Prophet and featured performances by Glory-Anne Carriere, Jerry & Jo'Anne, Rondini, Terry Carisse & Tenderfoot, Orval Prophet, Sheila Ann, Baker, and the host himself. Crews from Global Television, the local CKCK-TV news team, and people from CTV's W5 were on hand busily shooting footage and doing several interviews with many of the artists. Global edited their tape and presented it as a Global Big Country Special on Oct. 14.
Big Country 1979 was held at the Skyline Hotel in Mississauga the weekend of Sept. 22-23. Three panel discussions were held on Saturday covering the subjects of retailing and marketing country music, Canadian productions and the radio programmer, and the role of the record company in marketing and promoting country product.
The awards were once again emceed by Hunter, a veteran of some 20 years as host of a major Canadian country television show. Performing during the evening were Ralph Carlson & Country Mile, Laura Vinson, Gary Fjellgaard, Nancy Ryan, Artie MacLaren, Eddie Eastman and the Family Brown.
Baker and her manager, Grashey, presented a plaque to Grealis and Klees, honouring their ongoing support of the Canadian country music industry.
Carroll Baker, overcome with emotion, accepts the 1975 Big Country Award as Top Country Female Singer from the late Wm. Harold Moon of BMI Music Canada.
The Winnipeg Inn was the scene of the first Big Country event of the 1980s, ending another successful Canadian Country Music Week from Sept. 20-21. By this time, two camps had formed within the industry and, in a keynote speech, Grealis pointed out the definite need for understanding in order to avoid harming Canadian country through interference from either side.
"Since the awards are free from political influences of the industry, ACME control could be detrimental to the awards. RPM is free from these problems. I am not a manager, a recording artist or company, nor am I a songwriter. With ACME control there could be a horrendous clash of personalities. I've been called a dictator. However, if it's prefixed by benevolent, I don't mind. Everyone has the right to suggest changes, and our new voting system came from such suggestions. We are all here for the benefit of the industry, and the award shows are tailored to promote the industry. Others, like ACME, are welcome to have another show. They could make their own significant contribution, especially if their contributions went on for twelve months of the year. Their opportunities are limitless."
After Grealis called Klees to the stage to answer delegates' questions about the new nominating system for the Big Country Awards and for the system of tabulating record movement on the RPM Country 75, the day's panel sessions began. The first panel dealt with retailing and merchandising and was made up of representatives from various record labels and distribution companies. The second was comprised of club owners from across Canada who dealt with questions concerning the importance of clubs, presentation and
BIG COUNTRY
BIG STARS
IAN TYSON
AND STUDIO THREE ABASED
STANLEY FOX
MUSTANG HEART
WARNER MUSIC CANADA
A Time Warner Company
Inn On The Park and Don Harron, who was at his best as the colourful Charlie Farquarson, was the emcee. J.K. Gulley, Anita Perras, Mike Francis and Blue Rodeo provided additional entertainment and also spent a lot of time with a group of Variety’s Kids.
The Big Country Awards were given out on May 28 and WEA’s George Fox pulled off an unprecedented first by becoming the first artist in the history of the awards to win in four categories in one year. The other highlight of the evening was the induction of Weird Harold Kendall into the Canadian Music Industry Hall of Fame. As music director of CKWX Vancouver for 15 years, he had developed a warm and lasting friendship with Canadian country artists.
The May 25, 1990 Variety Club luncheon at the Inn On The Park was hosted by Grealis and featured performances by Michelle Wright and Silver & Degazio.
Baker hosted the Big Country Awards on May 27 at the Inn On The Park and K.D. Lang matched Fox’s feat from the year before of taking home four trophies. The inductee into the Canadian Music Industry Hall of Fame this time was Balmur president Leonard Rambeau. In making the presentation, Klees was effusive in his praise of the man who helped make Anne Murray an international star.
“The industry honours artists with a Grammy, a Juno, a gold record or a Big Country Award, but the people behind the scenes are often forgotten. This award is given very seldom and no one deserves it more than the man who is best in his field . . . Leonard Rambeau.”
The Variety Club luncheon moved to Toronto’s Westin Harbour Castle on May 24, 1991. CHAM Hamilton morning personalities Vickie Van Dyke and Cliff Dumas emceed the show and took turns introducing the performers. Baker, Wright, Fjellgaard, Larry Mercey, Larry Good, Laura and Larry Mattson, Kimberly Richards, Morris P. Rainville, David Hutchins, Mike Peters, Dyanne Halliday, Ron Victors and Terry Hill all sang Variety Club’s There’s No Business Like Show Business theme. Many of these performers, along with Hawkins, also performed numbers of their own.
The awards dinner on May 26 was also held at the Westin Harbour Castle. For the first time, a sponsor carried some of the financial load for the awards. Maclean Hunter Country Radio sponsored the awards dinner, which was attended by 80 per cent of the nominees.
Last year’s Variety Club luncheon attracted almost 400 people to the Frontenac Room of the Westin Harbour Castle on June 5. Sylvia Tyson was the host for the afternoon and she performed along with Larry Mercey, the J.K. Gulley Band, Joel Feeney, The Good Brothers and Lindsay Thomas Morgan.
Two nights later, more than 200 industry people gathered at the same hotel for the awards dinner. BMG Music Canada and its artists dominated the evening as Prairie Oyster swept an unprecedented five awards, Michelle Wright was named top female vocalist and
Historical high points of the
Sept. 22-23, 1973 RPM stages first Big Country Conference at the Holiday Inn in Don Mills, Ont. where 86 paid registrants attend.
Nov. 23-24, 1974 RPM stages second Big Country Conference at the Inn On The Park in Toronto.
June 21, 1975 Country Week is launched as a regular section of RPM Weekly to promote Canadian country music.
Aug. 1, 1975 RPM announces the formation of CACMA (Canadian Academy of Country Music Advancement) to raise funds to start an academy.
Sept. 27-28, 1975 The third Big Country Conference is held at Toronto’s Inn On The Park.
Sept. 28, 1975 The first Big Country Awards Banquet attracts 250 people to the Café de L’Auberge of the Inn On The Park.
Sept. 25-26, 1976 The second Big Country Awards Banquet is part of the fourth conference held at Edmonton’s Plaza Hotel.
Sept. 19-23, 1977 Big Country approves Ottawa Host Committee to stage Canadian Country Music Week, Ottawa 1977, as a consumer event prior to Big Country.
Sept. 24-25, 1977 The fifth conference and third awards presentation is stage at Ottawa’s Skyline Hotel.
Sept. 30-Oct. 1, 1978 Regina hosts Big Country at the Regina Inn. The sixth conference features the fourth year of the Big Country Awards. Global Television films documentary of the Big Country events.
Sept. 17-21, 1979 ACME applies to Big Country to stage Canadian Country Music Week at the Skyline Hotel in Mississauga, Ont. as a consumer event prior to the annual conference and awards show.
Sept. 22-23, 1979 The fifth annual Big Country Awards are held at Mississauga’s Skyline Hotel as part of the seventh Big Country Conference.
Sept. 20-21, 1980 The Winnipeg Inn is the setting for the sixth awards and the eighth annual Big Country Conference.
Sept. 19-20, 1981 The seventh annual Big Country Awards are staged as the finale to Big Country Weekend at Ottawa’s Talisman Motor Hotel.
Jan. 25, 1982 ACME distributes ballot to members asking if they want separate awards from Big Country. Eight-four per cent of respondents say yes.
April 28, 1985 The Big Country Awards are back with a banquet at Toronto’s Inn On The Park. The tradition of having an awards ceremony without musical performances begins. The event is carried nationally on radio via Broadcast News and locally on television by CITY-TV.
May 4, 1986 Toronto’s Inn On The Park hosts the ninth Big Country Awards.
May 24, 1987 The 10th Big Country Awards are held at Toronto’s Inn On The Park.
May 6, 1988 The first Variety Club Special Luncheon Salute to the Big Country Awards is held at Toronto’s Inn On The Park. Tommy Hunter emcees and performs along with Carroll Baker, Ronnie Hawkins and The Good Brothers.
May 8, 1988 Jack Feeney is inducted into the Canadian Music Industry Hall of Fame at the Big Country Awards presentation at Toronto’s Inn On The Park.
May 26, 1989 The Variety Club luncheon becomes an annual event as the second one is emceed by Don Harron (Charlie Farquarson) at Toronto’s Inn On The Park. J.K. Gulley, Anita Perras, Mike Francis and Blue Rodeo perform.
May 28, 1989 The 12th Big Country Awards take place at Toronto’s Inn On The Park.
May 25, 1990 Walt Grealis hosts the Variety Club luncheon at Toronto’s Inn On The Park. Michelle Wright and Silver & Degazio perform.
May 27, 1990 Carroll Baker hosts the Big Country Awards at Toronto’s Inn On The Park. Leonard Rambeau is inducted into the Canadian Music Industry Hall of Fame.
May 24, 1991 The Variety Club luncheon moves to Toronto’s Westin Harbour Castle. CHAM Hamilton’s Cliff Dumas and Vickie Van Dyke emcee the show, which features performances by Carroll Baker, Michelle Wright, Gary Fjellgaard, Larry Mercey, Larry Good, Laura and Larry Mattson, Kimberly Richards, Morris P. Rainville, David Hutchins, Mike Peters, Dyanne Halliday, Ron Victors, Terry Hill and Ronnie Hawkins.
May 26, 1991 The Big Country Awards dinner also moves to Toronto’s Westin Harbour Castle Hotel.
June 5, 1992 The Variety Club luncheon is held at the Westin Harbour Castle and features performances by Larry Mercey, The Good Brothers, J.K. Gulley and his band, Joel Feeney, Lindsay Thomas Morgan and host Sylvia Tyson.
June 7, 1992 Toronto’s Westin Harbour Castle hosts the 15th Big Country Awards.
June 8, 1992 Almost 400,000 people across Canada watch the Big Country Awards on television as the CBC televises the show nationally for the first time.
May 28, 1993 The Variety Club luncheon moves to the Concert Hall of Toronto’s Royal York Hotel Cliff Dumas and Jane Brown of Toronto’s CISS-FM emcee and Cassandra Vasik, Don Neilson, Anita Perras, and Mark LaForme and his band perform.
May 30, 1993 The Big Country Awards are held on the 33rd floor of Toronto’s Sutton Place Hotel.
May 31, 1993 CBC nationally televises the Big Country Awards for the second consecutive time.
The following is a list of the most common types of software that can be used to create and edit digital images:
1. Adobe Photoshop: A professional-grade image editing software that offers a wide range of tools for creating, editing, and manipulating digital images.
2. GIMP: A free and open-source image editing software that offers many of the same features as Photoshop, making it a popular choice for those on a budget.
3. Paint.NET: A lightweight image editing software that is easy to use and offers a variety of tools for creating and editing digital images.
4. CorelDRAW Graphics Suite: A comprehensive design software that includes image editing capabilities, making it a popular choice for graphic designers.
5. Microsoft Paint: A basic image editing software that comes pre-installed on Windows operating systems and offers a limited set of tools for creating and editing digital images.
6. Canva: A user-friendly online tool that allows users to create and edit digital images without any prior experience or technical knowledge.
7. Pixlr: A web-based image editing software that offers a range of tools for creating, editing, and manipulating digital images.
8. Sketchbook Pro: A professional-grade image editing software that is designed specifically for artists and offers a wide range of tools for creating and editing digital images.
9. Affinity Photo: A professional-grade image editing software that offers a range of tools for creating, editing, and manipulating digital images.
10. Lightroom: A photo management and editing software that is designed specifically for photographers and offers a range of tools for organizing, editing, and sharing digital images.
Congratulations to all of Canada's Great Country Artists
92.5 CiSS FM
TORONTO'S NEW COUNTRY FM!
Big Country Awards
Congratulations To Our Nominees
THE RANKIN FAMILY
- Best Group
- Best country Album - 'Fare Thee Well Love'
- Best Country Single - 'Orangedale Whistle'
- Best Country Composer - Jimmy Rankin
- Best Country Producer - Chad Irschuck
STOMPIN' TOM CONNORS
- Best Country Album - 'Believe In Your Country'
LISA BROKOP
- Best Female Vocalist
Try a New Brand of Bourbon
Bourbon Gautier
I can't complain
Featuring the First Single
I can't complain
100 Proof Country
COUNTRY NORTH
A collection of 40 original Country Hits spanning over 3 decades of Canadian Country Music.
Quality Music and Video is pleased to be a part of this fundraising project in support of CIRPA and CARAS, vital elements of the Canadian Music Industry.
QRSPC 1188 1/2 • 2 Cassettes
QRSPD 1188 1/2 • 2 Compact Discs
Coming to retail soon.
Supported by a national advertising campaign.
The following is a list of the most common types of data that can be collected and analyzed using the methods described in this paper:
- **Demographic Data**: Information about the age, gender, race, ethnicity, education level, income, employment status, and other demographic characteristics of individuals or groups.
- **Behavioral Data**: Information about the behaviors, attitudes, and preferences of individuals or groups, such as their shopping habits, social media usage, and political affiliations.
- **Health Data**: Information about the health status, medical history, and treatment of individuals, including data on diseases, injuries, and other health-related issues.
- **Financial Data**: Information about the financial transactions, assets, and liabilities of individuals or organizations, including data on income, expenses, investments, and loans.
- **Environmental Data**: Information about the natural environment, including data on weather, air quality, water quality, and other environmental factors.
- **Educational Data**: Information about the educational attainment, skills, and knowledge of individuals, including data on academic performance, test scores, and graduation rates.
- **Legal Data**: Information about legal proceedings, court cases, and other legal matters, including data on laws, regulations, and court decisions.
- **Political Data**: Information about political parties, candidates, and elections, including data on voting patterns, campaign finance, and public opinion polls.
- **Social Media Data**: Information about the content, interactions, and engagement of individuals on social media platforms, including data on likes, shares, comments, and other social media metrics.
- **Sports Data**: Information about sports teams, athletes, and competitions, including data on scores, statistics, and rankings.
- **Weather Data**: Information about the weather conditions, including data on temperature, humidity, precipitation, wind speed, and other meteorological variables.
- **Traffic Data**: Information about traffic flow, congestion, and accidents, including data on road conditions, vehicle counts, and travel times.
- **Transportation Data**: Information about transportation systems, including data on public transit, car ownership, and travel patterns.
- **Retail Data**: Information about retail sales, inventory, and customer behavior, including data on product demand, pricing, and marketing strategies.
- **Tourism Data**: Information about tourism destinations, attractions, and visitors, including data on visitor numbers, spending, and satisfaction levels.
- **Travel Data**: Information about travel plans, bookings, and experiences, including data on flight schedules, hotel reservations, and travel itineraries.
- **Workplace Data**: Information about workplace environments, policies, and practices, including data on employee satisfaction, productivity, and safety measures.
These are just a few examples of the many types of data that can be collected and analyzed using the methods described in this paper. The specific types of data that are relevant to a particular research question will depend on the context and goals of the study.
CONGRATULATIONS
BIG COUNTRY AWARD
NOMINEES
When you've got the Munchies
Hostess MUNCHIES
Hostess MUNCHIES
Hostess MUNCHIES
Nothing else will do.
*Hostess Potato Chips.
YOU'RE ALL WINNERS
| TW | LW | WO | JUNE 5, 1993 |
|----|----|----|--------------|
| 1 | 1 | I LOVE THE WAY YOU ... (2 weeks at No. 1) | John Michael Montgomery/Lyle A Dance/Warner comp 166-P (V.Shaw/Cannon) D.Johnson (Atlantic) |
| 2 | 11 | AIN'T THAT LOVELY YET | Weight Young/This Time Warner comp 167-P (K.House)/J.Anderson (Reprise) |
| 3 | 9 | TELL ME WHY | Wynonna/Tell Me Why/MCA comp 3-J (K.Brown/R.McCormick) |
| 4 | 16 | ALRIGHT ALREADY | Larry Stewart/Larry Stewart/MGM comp 17-N (B.Hill/B.Rudd) S.Hendricks/L.Stewart (RCA) |
| 5 | 14 | ALBUM | Pat Benatar/Lawrence/Nibbs Warner comp 162-P (P.Bouchard) J.Stroud (Atlantic) |
| 6 | 12 | EVERYTHING AND MORE | Jimi Wittenberg/Wittenberg CD single-F (J.Wittenberg/D.Douglas) (PRE) |
| 7 | 10 | TALK TO MY HEART | Joan Kennedy/Higher Ground/MCA comp 2-J (M.Sanders/J.Allen) M.Francis (MCA) |
| 8 | 24 | BLAME IT ON YOUR HEART | Jimi Wittenberg/What's Real/Promo CD single-H (H.Ward/H.Kostka) J.Gordy Jr. (Epic) |
| 9 | 18 | MUSTANG HEART | George Strait/Mustang Heart/Warner comp 162-P (G.Ford/M.Dickens) B.Dickens (WEA) |
| 10 | 9 | TENDER MOMENT | Lee Roy Parnell/Love Without Mercy/BMG comp 17-N (P.Farrell)/B.McCormick/M.Dickens/B.Beckett (Arista) |
| 11 | 14 | IT MIGHT AS WELL BE ME | Joe Diffie/It Might Be Beyond The Law/Warner comp 162-P (P.Title/G.Nicholson) M.Francis (Savannah) |
| 12 | 5 | HEARTS ARE GONNA ROLL | Hal Ketchum/Hearts Are Gonna Roll/Promo CD single-F (K.Ketchum/M.Stallings) A.Reynolds/C.Rodney (Curb) |
| 13 | 7 | SADLY MISTAKEN | Cassandra Vaski/Feels Like Home/Promo CD single-H (T.Thomery/E.Henn) T.Thomery/E.Henn (Epic) |
| 14 | 11 | YOU'RE MY HOME TOWN | Don Henley/The Circle Side /Promo CD single-H (T.Thomery/E.Henn) M.Francis/T.Thomery (Epic) |
| 15 | 13 | MADE FOR LOVIN' YOU | Don Stone/From The Heart/Album track-H (C.Rodney/M.Dickens/M.Sanderson) |
| 16 | 17 | THE CHANGE | Michelle Wright/How & Then/BMG comp 16-N (S.Bogard/R.Giles) S.Bogard/R.Giles (Arista) |
| 17 | 19 | IF ONLY YOU KNEW | Joe Diffie/If Only You Knew/Promo CD single-H (B.Brown/R.Pressman) (Columbia) |
| 18 | 12 | HIGH ROLLIN' | Gibson/Aller Band/Where There's Smoke/Sony comp 319-H (C.Brown/R.Pressman) |
| 19 | 6 | HOMETOWN HONEYMOON | Alabama/American Pride/BMG comp 17-N (J.Lee/J.Photoch) J.Lee/M.Lee/Alabama (RCA) |
| 20 | 25 | I WANNA TAKE CARE OF YOU | Bill Deane/In The Dark/Capitol comp 6-F (B.Deane/K.King) J.Collins (Liberty) |
| 21 | 23 | HONKY TONK ATTITUDE | Joe Diffie/Honky Tonk Attitude/Album track-H (C.Brown/R.Pressman) C.Brown (Epic) |
| 22 | 28 | ROUBLE ON THE LINE | Sawyer Brown/Cafe On The Corner/CD single-F (M.A.Miller/B.Shore) R.Scraggs/M.Miller (Curb) |
| 23 | 21 | TELL ME ABOUT IT | Travis Tucker/Well I'll Do It/Atlantic comp 16-P (T.Laflin/J.Laflin/M.Laflin) J.Christleff (Liberty) |
| 24 | 9 | T-R-O-U-B-L-E | Travis Tritt/T-R-O-U-B-L-E/Atlantic track-P (T.Laflin/J.Laflin/M.Laflin) |
| 25 | 26 | OH ME, OH MY, SWEET BABY | Diamond Rio/Closet To The Edge/Promo CD single-N (M.Garvin/T.Shapiro) M.Powell/T.Dubois (Arista) |
| 26 | 12 | SHE DON'T KNOW SHE'S BEAUTIFUL | Shania Kershaw/Heartbreak Hotel/PolyGram comp 304-Q (S.McCormick/B.McCormick/B.Dickens) Wilson (Mercury) |
| 27 | 17 | BORN TO LOVE YOU | Mark Collie/Mark Colle/Atlantic track-J (N.Collie/M.Collie) D.Cook (MCA) |
| 28 | 34 | GILLIS MOUNTAIN | The Rankin Family/Fare Thee Well Love/CD single-F (R.Grarkin) R.Irschick (Capitol) |
| 29 | 31 | JUST AS I AM | Randy Van Warmer/Greatest Hits Plus/Sony comp 311-H (L.Brown/P.Nelson) S.Buckingham (Columbia) |
| 30 | 20 | HARD WORKIN' MAN | Brooks & Dunn/Hard Workin'/Warner BMG comp 16-N (D.Brooks/D.Douglas) S.Hendricks (Arista) |
| 31 | 8 | HEARTACHE | Suzzy Boggus/Voices In The Wind/Capitol comp 6-F (L.George) U.J.Bowen/S.Boggus (Liberty) |
| 32 | 52 | AN OLD PAIR OF SHOES | Reba McEntire/Travels With You Vol. 2/Atlantic track-P (J.Lee/J.Photoch) L.Brown (Warner Bros) |
| 33 | 21 | I'D RATHER MISS YOU | Little Texas/I'd Miss You For Everything/Warner comp 157-P (T.Laflin/J.Laflin/M.Laflin) J.Christleff (Liberty) |
| 34 | 22 | TONIGHT I CLIMBED THE WALL | Alan Jackson/A Lot About Lovin'/BMG comp 16-N (A.Jackson) K.Steggall/S.Hendricks (Arista) |
| 35 | 61 | THAT SUMMER | Garth Brooks/The Chase/EMI comp 9-F (P.Algeris/M.Mahf/G.Brooks) A.Reynolds (Liberty) |
| 36 | 59 | WHEN DID YOU STOP LOVING ME | George Strait/Structure Of A Song/Strait/EMI comp 3-J (M.McCormick/D.McCormick) B.Brown (MCA) |
| 37 | 43 | A LITTLE BIT OF HER LOVE | Robert Ellis Orrall/Finding Colors/BMG comp 17-A (R.E.Orrall/M.Orrall) M.Marcan/McCarr (RCA) |
| 38 | 45 | THE HARD WAY | Mary-Chapin Carpenter/Come On ...Sunny comp 312-H (M.Carpenter) J.Jennings/M.C.Carpenter (Columbia) |
| 39 | 41 | NO FUTURE IN THE PAST | Vince Gill/Still Believing In You/Promo comp 2-J (V.Gill/J.James) T.Brown (MCA) |
| 40 | 47 | LET'S NOT CALL IT LOVE, YET | Reba McEntire/No Promio cd single-C (R.McEntire/Hill) A.Donald (RDR) |
| 41 | 42 | DESTINATION YOU | Michael Terry/Nine album/Polo Note E-run-niate (P.Hochstetler/R.Cousins/M.Terry) Same (Roto Noto) |
| 42 | 50 | LOVE ON THE LOW LOW HEART OF THE RUN | Garth Brooks/She's A Rebel & The Ride/Promo comp 2-J (Kostas/A.Graham) S.Gibson/T.Brown (MCA) |
| 43 | 51 | I GUESS YOU HAD TO BE THERE | Lorne Mork/Magic Watch/MGM comp 17-N (L.Mork/M.Collie) C.Lundes (BNA) |
| 44 | 27 | MY BLUE ANGEL | Aaron Tippin/Read Between The Lines/BMG comp 15-N (A.Tippin/R.K.Williams/P.Doug) E.Gordy Jr. (RCA) |
| 45 | 62 | IT'S YOUR CALL | Aaron Tippin/Your Call/MCA comp 3-J (L.Henager/S.H.Burkhardt/Bursh) T.Brown/R.McEntire (MCA) |
| 46 | 57 | MONEY IN THE BANK | John Anderson/So Good/Promo CD single-N (J.Lee/J.Photoch/M.Sanders) J.Stroud/J.Anderson (BNA) |
| 47 | 58 | SOMEBODY ELSE'S MOON | Colin Rayven This Life/Sony comp 311-H (P.Nelson)/R.Shapiro) C.Funding/O.Hobbs (Epic) |
| 48 | 55 | ESSENTIAL THINGS TO SAY | Mark Lamarr/No Album/Polo Note E-run-niate comp (M.LaFerr) R.Cousins/M.Oliveira (Roto Noto) |
| 49 | 65 | BLANK PAGES | Patricia Conroy/Last Day For Trains/Warner comp 170-P (P.Conroy) R.McEntire (RDR) |
| 50 | 30 | JAQUIMA TO FRENO | Jan Tyson/And Stood There ...Warner comp 164-P (T.Tyson/B.McIntyre) L.Sedrak (Slony Pain) |
| 51 | 32 | NEVER SAY GOODBYE | The Bellwethers/On October/Warner comp 164-P (G.Fjeldgaard) K.Vickers (Savannah) |
| 52 | 56 | HEY BABY | Marty Stuart/This One's Gonna Hurt ...MCA comp 3-J (M.Stuart/K.King) M.Francis/T.Brown (MCA) |
| 53 | 54 | IF I HAD A CHEATIN' HEART | Ricky Lynn Gregg/Ricky Lynn Gregg/Capitol comp 6-F (W.Holstadya/T.Turney) C.Howard (Liberty) |
| 54 | 38 | LEARNING TO LIVE AGAIN | Garth Brooks/The Chase/Promo CD single-F (G.Brooks/M.Collie) A.Reynolds (Liberty) |
| 55 | 39 | MENDING FENCES | Restless Heart/Big Iron Horse/Promo CD single-N (A.Brown) R.McEntire (J.Leo)/Restless Heart (RCA) |
| 56 | 37 | WALKIN' TALL | P.J.Jackson/P.J.Jackson/Warner comp 160-P (P.J.Jackson) T.Laflin (Stony Plain) |
| 57 | 40 | NOBODY WINS | The Everly Brothers/Four On The Floor/1993 Promo CD single-N (R.Foster/R.Richard) S.Fielbel (Arista) |
| 58 | 49 | MY BROTHER AND ME | Johnny Brook/My Brother And Me/Atlantic track-P (B.Brown/R.Pressman) |
| 59 | 44 | THE HEART WON'T LIE | Reba McEntire/Win Win Gillis/Your Call/CD single-J (K.Cammett/D.T.Weiss) T.Brown/R.McEntire (MCA) |
| 60 | 46 | I MIGHT BE DOWN (But I Ain't Out Yet) | Lani Michael/No album/Promo CD single-T (L.Michael) L.Michael (MBS) |
| 61 | 53 | SETTIN' MYSELF UP FOR A FALL | The Wiseman Brothers/Secret Places/Album track-P (T.Wiseman) T.Wiseman/Wiseman Wiseman Bros (NHB) |
| 62 | 67 | IT MUST BE THE RAIN | Marty Brown/Wild Kentucky Skies/MCA comp 3-J (M.Brown) R.Bennett (MCA) |
| 63 | 73 | WE GOT THE LOVE | Reba McEntire/Greatest Hits/Promo CD single-N (S.Bogard/R.Giles) J.Jago/Restless Heart (RCA) |
| 64 | 78 | HAUNTED HEART | Sammy Kershaw/Heartbreak Hotel/Sony comp 315-Q (S.Brown) R.McEntire/S.Wilson (Mercury) |
| 65 | 66 | THERE OF ME (Less Of Lonely) | Cody The West/No album/Hillcrest comp 8 (W.Ferguson) W.Ferguson (Hillcrest) |
| 66 | 58 | SOMEONE TO GIVE MY LOVE TO | Trace Byrd/No album/Promo CD single-J (L.Foster/R.Rice) T.Brown (MCA) |
| 67 | 69 | THE SOUND OF ONE HEART BREAKING | Sylvia Tyson/Gypsy Cadillac/Sony comp 311-H (S.Tyson)/T.Russell (Silver City) |
| 68 | 70 | WHEN I PRAY FOR RAIN | Neal McCoy/Where Forever Begins/Warner comp 163-P (L.Satterfield/G.Teran) J.Stroud (Atlantic) |
| 69 | 71 | LET'S PUT PONIES RUN | Pam Tillis/Don't Let Loving Angel/BMG comp 15-N (G.Peters) P.Worley/E.Seay (Arista) |
| 70 | 75 | LIKE A RIVER TO THE SEA | Steve Wariner/Am Ready/BMG comp 16-N (S.Wariner) S.Hernandez/T.Dubois (Arista) |
COUNTRY continued from page 21
recently include Fiddle Man by Bobby Lalonde and Take Her To Heart by Belleville, Ontario's Ken Harnden. "It still mystifies me," says Matheson, "why Ken hasn't been picked up by one of the majors. He is one of the most consistent artists vocally and in the music he chooses to release." Harnden's new release, included on BMG Songwriter Sessions 2, moves up the RPM Country 100 this week to No. 91.
Stony Plain's P.J. Jackson, now descending the chart with Walkin' Tall (No. 56), could be moving back up shortly. The follow-up single is titled Step Back, which was featured on the Northern Exposure TV series. The song is a Jackson original. The track was taken from his self-titled album produced by Tom Lavin and recorded at Lavin's Vancouver studio. By the way that's Patricia Conroy and Billy Cowsill providing background vocals. Mike Douchette supplies the harmonica inserts with Hal Rugg on pedal steel and Rob Hajacos on fiddle.
Kushla has released Here's Hopin', the third single from her self-titled album. The album is released on the Ketari label and is available through IDAC at 1-800-JOE-RADIO. One dollar from each CD or cassette sold, along with 25 per cent of all Canadian performance royalties, is being donated to Child Find Canada.
COUNTRY PICKERS
GUY BROOKS
CFMK 98.5FM - Kingston
Shame, Shame, Shame - Mark Collie
GARRY MACINTOSH
The MIX 610 CKYL - Peace River
We Gotta Get Away, Restless Heart
JULIE LEPERRE
CKDM Radio 730 - Dauphin
Down On My Knees - Trisha Yearwood
AL CAMPAGNOLA
Country 59 - Toronto
CINEO CHANNEL
New Country CHAT - Medicine Hat
Breakfast Alone - George Fox
GREG MACK
Country 630 - Winnipeg
It Sure Is Monday - Mark Chesnutt
BOB LINN
CKEG Country - Nanaimo
It Sure Is Monday - Mark Chesnutt
CHUCK REYNOLDS
CFHY 96.7FM - Leamington
Fortune . . . - Cassandra Vasik w/Russell deCarle
JOEL CHRISTIE
820 CHAM Country - Hamilton
I Can't Complain - Bourbon Gautier
DAN HALE
CKBY-FM - Ottawa
Fortune . . . - Cassandra Vasik w/Russell deCarle
TOM BLIZZARD
KHJ - Fredericton
Money In The Bank - John Anderson
MODE 104.1
CKRM AM 980 - Regina
Fortune . . . - Cassandra Vasik w/Russell deCarle
IAN MCCALLUM
BX-93 - London
Fortune . . . - Cassandra Vasik w/Russell deCarle
TREVOR BATTAMS
CJBQ 800 AM Stereo - Belleville
Hey Baby - Marty Stuart
DEBRAN KAUFENHOFEN
CKMW Country - Calgary Winkler/Morden
Tears Falling Down - Shania Twain
RANDY OWEN and DANN TRAVIS
570 CKGL - Kitchener
Deeper Water - Christian Peterson
KEVIN MATTHEWS
CKCM-FM 104 - Moncton
It Sure Is Monday - Mark Chesnutt
Chris LeDoux backstage at Hamilton Place with EMI Music Canada staffers: Valerie Helmer, Jeff Manderson, Liz McElheran, Al Andrichow, Ann Forbes, LeDoux, Paul Church, Dave Street and a kneeling Roger Bartel.
The Music City News country awards show is ready to roll from the Opry House in Nashville on June 7. Vince Gill, nominated in five categories, and Reba McEntire, in four, will perform along with Alan Jackson, Lorrie Morgan, the Statler Brothers, Ray Stevens, Brooks & Dunn, and co-hosts George Jones, Ricky Van Shelton and Suzy Bogguss. In addition, Sawyer Brown and Shenandoah, both nominated in the vocal band of the year category, will team up for a medley. Winners will be announced in 14 fan-voted categories with Vince Gill, Garth Brooks and George Strait garnering the most nominations with five each.
Mercury recording artist Shania Twain returns home to Timmins, Ontario where she was introduced to a packed Timmins Square by 750 CKGB Radio news director Dale Tonelli.
BMG's Songwriter Sessions Volume 3 has finally been sent out to the print media. Some stations who got the mailing three weeks ago have been giving playlist action to a couple of the tracks, notably: Ken Harnden's Take Her To Heart, produced by Mike Francis; Terry Sheridan's One More Quarter, produced by Ron Demmans; and Cactus Club's I Don't Know, produced by Bob Funk. Also included on the compilation are Ron Irving's self-produced Tell It To My Heart, Terry Tufts' What Am I Supposed To Do With These, and Billy Charles' Loose Caboose, both produced by Randall Prescott.
ALBUMS
PIGFARM - Rock
Plug
Emmusic - PIG-002
Pigfarm's 1989 debut, Hold Your Nose, was the #1 album at campus radio across Canada before the group took a two-year break. During that time, guitarist/vocalist songwriter Adam Fries (formerly of Lost Dakotas and led The Urban Decay Group, while bassist John DesLauriers saw the world with The Doughboys. The two have gotten back together and added noted producer Michael Phillips (Wolverine (Grazed Ladies), Rheostatics, Change Of Heart), who has previously drummed with Plasterscene Replicas and countless others. The old Pigfarm was a fairly punk-oriented band but brought a lot of country overtones to the mix. The current, more varied, sound inflicts decreased aural damage, but is just as powerful in its own way. Campus stations should still gravitate to it but alternative and CAR programmers will probably be the ones. "Ruin Queen" Green was written during the band's first incarnation and the title acts as a metaphor for money, ecology and politics. Mixed Up Day offers a change of pace with its regular ball line, traditional country feel, the organ and the vocals of a little girl towards the end of the song. Paperbag, Midas It Be' and Lipsync are three other strong tracks. Plug was produced by Robi Baneri and the band and its management. Pigfarm has a worldwide publishing deal with Peacock and don't be too surprised if you see their next release on a major label.
BIG COUNTRY - Rock
The Buffalo Skinners
Compulsion - F2 0946 0 21958 2 2-F
Big Country has sold successively fewer records since its ground-breaking 1983 debut, The Crossing. And despite this album's strengths, it's debatable whether it will reverse the trend. On The Buffalo Skinners, the distinctive "say, aren't those blueprints?" vocal delivery, which was a measure, been moved out in favour of loud rock out chords. The heavier sound is also reflected in vocalist/guitarist Stuart Adamson's lyrics, in which he deals with his disgust with the modern scene, that gull-baiting rich and poor, evangelism, war, injustice, rioting, arms races, animal and biological testing, the disappearing ozone layer, logging, pollution, nuclear power, etc. I'm not having arguments. The first single, "Chips," will appeal at CAR and, perhaps, other formats. The song is somewhat folky and introduces some organ to make it less imposing than some of the harder rocker. "Aries" is also gentle, some would say too gentle. Almost all of the 12 songs are recommended, although it will probably take a concerted push at retail to get the album the attention it warrants. -SM
THE WHITELEY BROTHERS - Blues
Bluesology
Pyramid-007 (Trend)
Here's a unique package of soul-driven Chicago blues that treats the ear with an exuberance and bounce rarely heard north of the border. Brothers Chris and Ken Whiteley, Americans who have lived in Toronto since 1956, have tenderly honed their blues craft, obviously with several of the blues masters in mind. The Bluesology album is a journey through the blues, a musically-documented bit of history beginning with the pre-blues sounds of the Georgia Sea Island Singers (Elsie Mae Jones). Toronto is far removed from Memphis or Chicago blues, but the Whiteley Brothers provide a comfortable zone for their own brand of northern blues that should capture audiences on both sides of the border. The respect for the blues is obvious - it's captured with perfection. The Whiteleys display their talents on more than 20 different instruments as well as harnessing the lead guitar work of Chris' son Daniel, who covers B.B. King's Sneaking Around The Garden, and, much when it comes to the blues, he heard acoustic guitar on M&O Blues and Custard Pie, and electric guitar on the finale jam, Wee Wee Baby. A history of the blues wouldn't be complete without a performance by Jackie Richardson, whose smooth vocal may be heard on the Whiteleys' version of We Don't Talk. The keyboard work has that Gene Taylorsignature, with a key being Sugar Sweet. Produced by Chris & Ken Whiteley and recorded at Toronto's Casa Wroxton.
LARI WHITE - New Country
RCA-65117-N
Lari White is one of the bright new lights in country. She is already established with her first single, What A Woman Wants, a sweetly melodic pop-country chugger. The title track, a White original, is the follow-up and it too looks like a chart happener. White's vocals are sometimes cutesy but never pretentious. Her delivery has an edge that draws you right into the emotional content that extra punch. The crossover potential, which new country is all about, is fairly obvious. Other single consideration should be given to Where The Lights Are Low, which she co-wrote with Chris Waters (of the Eagles) and is a blend of Good Love, and her solo writing of Just Thinking. Attractive cover artwork. Should be a front-rack item. Produced by White, Rodney Crowell and Stuart Smith. -WG
TERENCE TRENT D'ARBY - Pop/Rock
Symphony Or Damn
Columbia - CK 53616-H
A lot of comparisons can be drawn between D'Arby and Lenny Kravitz and Prince, who is given special thanks in the album's liner notes. They mix elements of soul, R&B, pop and rock to make for some sultry, and often danceable, music. They enjoy taking complete control of the studio by writing all their own material, singing, playing a wide variety of instruments and producing. They've all got reputations as lady killers, and now they've released albums in the last eight months. Kravitz's Are You Gonna Go My Way? has been a big hit, while Prince, while sales of Prince's Symbol have, by his standards, been somewhat disappointing. D'Arby's 1988 debut album made him an overnight sensation, while he lost some of his audience following. Therefore, Symphony Or Damn is a crucial album for TTD. But the innuendo-drenched lead single, She Kissed Me, should get lots of momentum rolling. The rocker is the high point of the album and should capture people's attention. Penelope Pleasure is a poppy dance tune that drops Chrissie Hynde's name as a hook, while Turn The Page is the most lyrical track on the album. Let Me Down Easy is the closest to the slower paced rock. The inclusion of fiddle harmonics on I Still Love You could test "new country" radio stations to see just how open their formats are to guys with dreads instead of stetsons. -SM
DUBAY - Pop/Rock
Emoceans
Limozine Records - L.R.002
The second album from this band led by Claude Dubé was recorded with engineer Yves Delivigne (who recently worked in Hamilton, Ont.). Dubé once lived in Los Angeles where he sang in a band that later spawned WASP's Blackie Lawless and Motley Crue's Nikki Sixx. But you'll find no heavy metal here. Instead there are various styles and tempos that could appeal to a few radio formats. Ocean Of Tears is the first emphasis track. World's On Fire, a rock song with a catchy pop hook, carries an environmental message. Ghost In Studio '87 is a slow, funky groove. An acoustic-country-oriented piece with some subtly accented accordion work can be found on Into My Life. And How Many Words makes for a roots-rock romp. Perhaps DuBay drummer Kevin Camilleri might attract Sony's interest. -SM
SUEDE - Rock/Pop
Suede
Nude/Columbia - CK 53782-H
Suede had been the darling of massive music press hype in the U.K., which has propelled the group close to stratospheric heights on their charts. I can already smell three modern rock hits from Suede if consultants and programmers give them a chance. The first glam-pop single, Metal Mud, has completed the circle of (valid) comparisons with early '70s David Bowie. Similar things can also be said for the marvellous Animal Nitrate and the sweetly whining The Drowners, I supposea good singer-filled ballad. The album is probably also in order. Bernard Butler's sonically impressive guitar lines weave masterfully through such other tracks as So Young, Moving and Animal Lover. Sleeping Pills and The Next Life are another and another of the same kind of immediate gratification. Brett Anderson's vocals are as thin as his rake-like torso, but they wrap their way around his disconcertingly obtuse lyrics so provocatively that they work. Well. -SM
RICHARD THOMPSON - Folk/Rock
Watching The Dark
Hannibal/Denon - HNCD 5303
If Thompson sold many records as he did, received critical raves, he probably could have retired years ago. Thankfully, he hasn't. This 215-minute three-CD set chronicles his career from the pioneering late '60s folk-rock sounds of Fairport Convention through the '70s and early '80s albums with then-wife Linda and on through his solo work which continues as strong as ever today. Though he's a gifted guitarist and a piercingly talented songwriter, Thompson is best known for releasing the best-selling album in Warner history. Admittedly he's not a great singer, but it's one of life's great mysteries why the public has ignored him for so long. Rock, folk, pop, Celtic, instrumental, Thompson has covered all the bases. And his collection is grand slam for those who are looking for a primer. For the converted, it might not be quite as valuable, but of the 47 tracks, more than half are previously unreleased rare songs, and many performances are limited versions. I could recommend at least 20 tracks, but you're better off investigating on your own. The package also contains rare photos and a 24-page booklet with liner notes written by Greil Marcus and Leslie Berman. -SM
6 AM - Rock
God's A Girl
Traffic/MCA - TFD 8992-J
The accompanying release sheet to God's A Girl calls it a "straight forward hard rock" album. I can add much more than that one short sentence. A lot more facts about 6 AM. The songwriting core of this Montreal group is vocalist/guitarist Sylvain Michel and drummer/background vocalist Yves Leblanc. The lineup is filled out by bassist George Simard and guitarist Richard Charles. God's A Girl was produced by the Felix award-winning Toby Gendron. It was recorded in Montreal and mixed in Paris. The band earlier released an independent mini-album that did well in Quebec; the first single is a power ballad called I'm Coming Home. -SM
DONALD FAGEN
KAMAKIRIAD
The first album in eleven years from the Steely Dan vocalist/keyboardist and the award-winning artist who brought you Nightfly.
Featuring
TOMORROW'S GIRLS
Produced by
WALTER BECKER
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DISPOSITION: STIPULATIONS ADOPTED
In this order, we adopt two stipulations filed by the parties in this case. Adopting the terms of the stipulations will reduce the size of the Oregon Universal Service Fund (OUSF), provide clarity for the next several years, and strengthen reporting and information requirements for non-rural carriers. We intend to revisit the core questions regarding the OUSF that gave rise to this Phase III when more information is available.
I. PROCEDURAL HISTORY
This Phase III was initiated by Order No. 13-162 to address the following three issues:
(a) Accountability for non-rural companies. Identify methods for accurately estimating how OUSF funds are directed to operating expenses in claimed high-cost areas.
(b) Consideration of a methodology to allocate Incumbent Local Exchange Carriers (ILEC) network costs between basic telephone and other services.
(c) Consideration of a methodology, applicable to all current OUSF recipients, for identifying areas of unsubsidized competition and whether OUSF support should continue to be provided there.
The parties to this case have twice attempted to resolve these issues through stipulation. In Order No. 15-005, we declined to adopt the first proposed settlement based on our decision on the need for more review of the issues relating to the need for and administration of the OUSF. Following a Commission workshop, the parties then refiled the stipulation with a revised explanatory brief. In Order No. 15-365, we again declined to adopt the stipulation. We encouraged the parties to develop a new proposed resolution and instructed any proposal should continue further reductions to the overall size of the OUSF and be consistent with the parameters outlined in Order No. 15-365.
In response to our directive, the parties have now submitted two new stipulations for our consideration. On January 29, 2016, Qwest Corporation, dba CenturyLink QC and Frontier Communications Northwest Inc. filed a “Phase IIIa Stipulation” proposing to resolve issue (a) above. On the same day, CenturyLink,\(^1\) Frontier,\(^2\) the Oregon Cable Telecommunications Association (OCTA),\(^3\) the Oregon Telecommunications Association (OTA), Verizon,\(^4\) and Warm Springs Telecommunications Company filed a “Revised Phase III Stipulation” proposing to resolve issues (b) and (c) (Revised Stipulation). The stipulations are attached to this order as appendices.
**II. STIPULATIONS**
The “Phase IIIa Stipulation” modifies the reporting and filing requirements for CenturyLink and Frontier Northwest for their expenses in areas that are eligible for OUSF support. The purpose is to address accountability concerns raised by Staff and the Commission.
The “Revised Phase III Stipulation” adopts an eight and one-half percent cap on the contribution surcharge for the five-year term of the stipulation. It does not extend the plan detailed in the Revised Stipulation beyond 2021. The stipulated phase-down of the OUSF receipts will result in a 27.5 percent reduction in OUSF support for non-rural companies and a 15.2 percent reduction for rural companies according to a capped disbursement schedule. Attachments to the stipulation outline the methodology for the pro rata support reduction process and rural ILEC support reallocation process. In their explanatory brief, the parties conclude that these reductions accomplish the goal of issues (b) and (c) by controlling the size of the OUSF.
The Revised Stipulation also recommends the Commission initiate a new proceeding in 2019 to review the OUSF and issue a final order prior to the end of the term of the Revised Stipulation.
The parties urge that by approving the Revised Stipulation, the Commission will continue to provide support for universal service, provide carriers with clarity and certainty about funding levels for a five-year period, and stabilize the financial burden on Oregon consumers that contribute to the program.
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\(^1\) The CenturyLink companies that are parties to this docket are Qwest Corporation d/b/a CenturyLink QC; United Telephone Company of the Northwest, d/b/a CenturyLink; CenturyTel of Oregon, Inc.; and CenturyTel of Eastern Oregon, Inc.
\(^2\) Frontier Communications Northwest Inc. and Citizen’s Telecommunications Company of Oregon.
\(^3\) OCTA signed the stipulation on behalf of its members other than Charter and BendBroadband.
\(^4\) The Verizon affiliates that are parties to this proceeding are MCImetro Access Transmission Services LLC, d/b/a Verizon Access Transmission Services; MCI Communication Services, Inc., d/b/a Verizon Business Services LLC; TTI National, Inc.; Verizon Long Distance LLC; and Verizon Select Services, Inc.
III. RESOLUTION
The Phase IIIa Stipulation and the Revised Stipulation generally address our immediate concerns. We acknowledge it is important for the public and the ILECs to have reasonable certainty as to the contributions that will be required to meet the goals of the OUSF. Consequently, we approve the stipulations, recognizing there is a need for certitude and these stipulations satisfy that need on an ongoing, temporary basis and will allow the companies and the public to plan appropriately. Most importantly, the parties have responded to our concern about maintaining the existing surcharge rate. While some of the core questions giving rise to this docket and raised in our prior orders are not fully resolved, we will address those issues in this proceeding at a future date.
We find that adopting the stipulations will directly reduce the amounts distributed to carriers by the OUSF, while maintaining customer surcharges at current levels. We also find that the terms in the stipulations will provide the Commission with the flexibility to address previously-raised issues in the near future.
We therefore conclude that the terms of the stipulations will result in just and reasonable rates and adopting the stipulations is in the public interest.
IV. ORDER
IT IS ORDERED that:
1. The Revised Phase III Stipulation between CenturyLink QC, et al; Frontier Communications Northwest, Inc., and the Citizens Telecommunications Company of Oregon; the Oregon Cable Telecommunications Association; the Oregon Telecommunications Association\(^5\); Verizon, et al; and Warm Springs Telecommunications Company, filed on January 29, 2016, attached hereto as Appendix A, is adopted.
2. The Phase IIIa Stipulation between Qwest Corporation, dba CenturyLink QC and Frontier Communications Northwest Inc., filed on January 29, 2016, attached hereto as Appendix B, is adopted.
\(^5\) On behalf of its member companies other than Charter and BendBroadband.
3. The Staff of the Public Utility Commission of Oregon and the Administrator of the Oregon Universal Service Fund shall take such steps as necessary to effectuate changes to the collection and distribution of funds between January 1, 2017 and December 31, 2021, as set forth in the Revised Phase III Stipulation.
Made, entered, and effective MAR 04 2016.
Susan K. Ackerman
Chair
John Savage
Commissioner
Stephen M. Bloom
Commissioner
A party may request rehearing or reconsideration of this order under ORS 756.561. A request for rehearing or reconsideration must be filed with the Commission within 60 days of the date of service of this order. The request must comply with the requirements in OAR 860-001-0720. A copy of the request must also be served on each party to the proceedings as provided in OAR 860-001-0180(2). A party may appeal this order by filing a petition for review with the Court of Appeals in compliance with ORS 183.480 through 183.484.
1) Except to the extent expressly provided herein, this Stipulation has a five year term which will begin on January 1, 2017 and end on December 31, 2021. As a result, for rural companies, see footnote 2, the term of the UM 1481 Phase II Stipulation adopted in Order No. 13-162 is modified accordingly and OUSF support for said companies in 2016 shall be as set out in Paragraph 5, below.
2) Consistent with the discussion in Commission Order No. 15-365, the Parties agree that the OUSF surcharge will not exceed eight and one half percent (8.5%) during the term of the Stipulation and that, to the extent required, the OUSF support amounts agreed upon for the Non-Rural Companies\(^1\) set forth in paragraphs 3 and 4, below, for the Rural Companies\(^2\) set forth below in Paragraphs 5 and 6, below, and for Warm Springs Telecommunications Company, set forth in paragraph 13, below, shall be reduced on a pro rata basis in order to maintain a surcharge of no greater than eight and one half (8.5%).\(^3\) Any pro rata reductions shall be determined for the Rural Companies in aggregate and for the Non-Rural Companies and Warm Springs Telecommunications Company individually. The adjusted aggregate support amount for the Rural Companies will be allocated to them based on their relative shares of support received to the total adjusted support amount.
3) Subject to any additional reductions required pursuant to Paragraph 2, above, beginning with the 2016 OUSF funding level of $17.5 million of annual OUSF support for Non-Rural Companies, consisting of $10.5 million for Qwest Corporation and $7.0 million for Frontier Northwest, funding for the Non-Rural Companies will be reduced in five equal annual steps. Over the life of the stipulation this will result in a reduction of no less than twenty-seven and one-half percent (27.5%). Non-Rural Companies’ OUSF receipts will not be affected by line counts.
4) Subject to any additional reductions required pursuant to Paragraph 2, above, the phase-down of OUSF support for the Non-Rural Companies will occur each January according to the following schedule\(^4\):
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\(^1\) The Non Rural Companies are Qwest Corporation d/b/a CenturyLink QC and Frontier Communications Northwest Inc. (“Frontier Northwest”).
\(^2\) Asotin Telephone Company d/b/a TDS Telecom, Beaver Creek Cooperative Telephone Company, Canby Telephone Association d/b/a Canby Telecom, Cascade Utilities, Inc., d/b/a Reliance Connects, CenturyTel of Oregon, Inc., d/b/a CenturyLink, CenturyTel of Eastern Oregon, Inc., d/b/a CenturyLink, Citizens Telecommunications Company of Oregon, Clear Creek Mutual Telephone Company, Colton Telephone Company d/b/a ColtonTel, Eagle Telephone System, Inc., Gervais Telephone Company, Helix’ Telephone Company, Home Telephone Company, Molalla Telephone Company d/b/a Molalla Communications Company, Monitor Cooperative Telephone Company, Monroe Telephone Company, Mt. Angel Telephone Company, Nehalem Telecommunications, Inc., d/b/a RTI Nehalem Telecom, North-State Telephone Co., Oregon-Idaho Utilities, Inc., Oregon Telephone Corporation, People’s Telephone Co., Pine Telephone Systems, Inc., Pioneer Telephone Cooperative, Roome Telecommunications Inc., St. Paul Cooperative Telephone Association, Scio Mutual Telephone Association, Stayton Cooperative Telephone Company, Trans-Cascades Telephone Company d/b/a Reliance Connects, and United Telephone Company of the Northwest d/b/a CenturyLink.
\(^3\) There is an open legal question of whether the Commission can mandate a cap on the OUSF surcharge. However, that question is avoided by the Parties voluntarily agreeing to a cap and the subsequent effects of that cap.
\(^4\) Payments will be 1/12\(^{th}\) the annual amount specified herein for a given year and will begin with the January OUSF support payment.
5) Subject to any additional reductions required pursuant to Paragraph 2, above, beginning with the 2016 calendar year OUSF funding level of $14,431,170 of annual OUSF support for Rural Companies, the funding will be reduced in five equal steps, taking effect annually. Over the life of the Stipulation this will result in a reduction of no less than fifteen and two-tenths percent (15.2%). Rural Companies’ OUSF receipts will not be affected by line counts, per Order No. 13-162 in Docket UM 1481 Phase II dated May 2, 2013, page 4.
6) Subject to any additional reductions required pursuant to Paragraph 2, above, the phase-down of OUSF support for the Rural Companies will occur each January according to the following schedule:
| Annual OUSF Support | Phase II Stipulation | Phase III Stipulation |
|---------------------|----------------------|-----------------------|
| | 2016 | 2017 | 2018 | 2019 | 2020 | 2021 |
| Rural Companies | $14,431,170 | $13,991,643 | $13,552,115 | $13,112,587 | $12,673,059 | $12,233,531 |
7) By way of illustration only, and based upon available projections\(^5\) of the retail telecommunications service revenue base subject to the OUSF surcharge, the potential impact of the agreed upon eight and one half percent (8.5%) OUSF surcharge cap imposed by agreement of the Parties pursuant to Paragraph 2, above, on the support amounts of the Non-Rural Companies and the Rural Companies would be:
| Capped Disbursements Based on Phase III Shares | 2017 | 2018 | 2019 | 2020 | 2021 |
|-----------------------------------------------|------------|------------|------------|------------|------------|
| Contributions (8.5%) | $30,000,000| $27,600,000| $25,500,000| $23,500,000| $21,600,000|
| Qwest | $9,177,404 | $8,322,819 | $7,567,104 | $6,850,508 | $6,172,341 |
| Frontier | $6,118,269 | $5,548,546 | $5,044,736 | $4,567,005 | $4,114,894 |
| Total Non-Rurals | $15,295,673| $13,871,365| $12,611,840| $11,417,513| $10,287,235|
| Total Rurals | $12,940,989| $12,069,749| $11,317,287| $10,600,353| $9,919,149 |
| Total CLECs | $1,763,338 | $1,658,886 | $1,570,873 | $1,482,135 | $1,393,616 |
| Total | $30,000,000| $27,600,000| $25,500,000| $23,500,000| $21,600,000|
\(^5\) The projections utilized are the projections provided by Commission Staff to the OUSF Advisory Committee. Obviously, projections are only the current estimates based on recent history and are subject to change.
These projected reductions reflect a reduction in overall OUSF support that is approximately ten and one half million dollars ($10.5M) greater than the reductions agreed upon by the Parties in the Stipulation filed with and rejected by the Commission in Order Nos. 15-005 and 15-365.
8) In light of the five-year term of this Stipulation, the Parties request that the Commission cancel any directions to perform a triennial review that may be contained in its prior orders. *See, e.g.*, Order No. 03-082 in Docket UM 1017.
9) Although not a party to the Stipulation, Commission Staff was given the opportunity to review the Stipulation in draft stage. Commission Staff made two requests. The first was that the Stipulation include a description of how pro-rata reductions would occur. The Parties agreed to include the description, which is set out in Attachment 1, and is incorporated into this Stipulation by this reference.
10) The second request made by Commission Staff was to include provisions in the Stipulation that would result in biennial reviews by Commission Staff for the purpose of possible re-allocation of OUSF support. The rural companies initially objected to this position. However, after strenuous negotiations, it was agreed to include a re-allocation process as part of the Stipulation in a manner that was acceptable to Commission Staff. That re-allocation process is described in Attachment 2, and is incorporated into this Stipulation by this reference.
11) This Stipulation resolves the following issues:
(a) Consideration of a methodology for allocation of ILEC network costs between basic telephone service and other services.
(b) Consideration of a methodology for identifying areas in which there is unsubsidized competition and whether OUSF support should be provided in such areas.
(c) Any other issues reasonably related to issues (a) through (B), above.\(^6\)
12) For the period January 1, 2017 through December 31, 2021, a qualified CLEC that is designated as an ETC for purposes of OUSF will draw at the ILEC per-line amount for the area it serves. The ILEC per line support amount will begin with the base per-line support value contained in the “Base Per Line Support Amounts,” which is found on the PUC website. The per-line amount will be adjusted to reflect the percent reduction that has occurred for the specific Non-Rural Company that owns the wire center between 2016 and the date the support amount is being calculated. This reduction method does not apply to the Warm Springs wire center.
13) It is recognized that the Warm Springs Telecommunications Company is in a unique situation as it builds a new network to serve the Confederated Tribes of Warm Springs Reservation and surrounding area. Pursuant to Order No. 13-162, Warm Springs Telecommunications Company will be capped at $1,500,000 per year from the OUSF. Beginning with the calendar year 2017 and
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\(^6\) A separate stipulation between Staff, CenturyLink QC and Frontier Communications resolves the issue of accountability for the non-rural ILECs.
each year thereafter for a period of five (5) years, Warm Springs Telecommunications Company’s adjusted cap will be calculated by reducing the $1,500,000 cap by three percent (3%) per year, subject to any additional reductions required pursuant to Paragraph 2, above.
14) The companies represented by the signing parties and those companies’ subsidiaries or parents, currently collecting the OUSF surcharge, as applicable, will collect the OUSF surcharge based on the company’s intrastate retail revenues (including but not limited to revenues from Voice over Internet Protocol) and pay that amount to the OUSF.
15) The Parties recommend that the Commission commence no later than 2019 a proceeding to review the OUSF in order to issue a final ruling prior to the end of the 5 year term of the Stipulation regarding any and all aspects of the OUSF, without excluding any options available under Oregon law.
16) The parties recommend that the Commission continue to have its Staff perform the following functions: 1) monitor company quarterly OUSF filings of intrastate retail telecommunications service revenues to ensure the reporting of such information is correct and that the companies are applying the surcharge appropriately; and 2) continue to investigate company filings where there appear to be discrepancies.
17) In Order 00-312 in Docket UM 731, Non-Rural Companies were directed to use OUSF support to ensure that basic telephone service is available at a reasonable and affordable rate. In Order 03-082 in Docket UM 1017, Rural Companies were ordered to use OUSF support for the same purpose. The Parties agree to recommend to the Commission that the Commission modify the purpose for which OUSF support is to be used, pursuant to the powers granted to it by ORS 759.425. Beginning January 1, 2017, the use of the OUSF funds will be for the following purpose: investment, construction, operation, maintenance, and repair to ensure that basic telephone service is available at reasonable and affordable rates. The use of the funds will be restricted geographically to the non-rural high-cost areas established in Order No. 12-065 for reporting purposes and to the areas served by the Rural Companies.
18) The Parties agree that any Party may file a petition to request Commission review of this Stipulation if there is a substantive change in Oregon law that materially affects the terms of this Stipulation or there is a substantive change in federal law or Federal Communications Commission precedent that materially affects the terms of the Stipulation. The Parties further agree that the Stipulation will not automatically terminate merely because a Party has filed a petition as described above, but will continue until the Commission issues a final order that grants, denies or takes other appropriate final action upon the petition. Finally, each Party reserves the right to make whatever arguments it deems appropriate in any docket resulting from the filing of the aforementioned petition.
GENERAL PROVISIONS
19) The Parties understand that this Stipulation is not binding upon the Commission unless and until it is approved by the Commission.
20) This Stipulation does not preclude a Party from explaining, as a factual matter, what the Parties agreed to in this Stipulation.
21) The Parties agree that this Stipulation represents the entire agreement of the Parties and that it supersedes any and all prior oral or written understanding, agreements or representation related to this Stipulation, if any, and no such prior understanding, agreement or representations shall be relied upon by any Party.
22) The Parties shall cooperate in submitting this Stipulation promptly to the Commission for acceptance, and cooperate in supporting this Stipulation throughout the Commission’s consideration of the Stipulation.
23) The Parties enter into this Stipulation to avoid further expense, inconvenience, uncertainty and delay. By executing this Stipulation, no Party shall be deemed to have approved, admitted, or consented to the facts, principles, methods, or theories employed in arriving at the terms of this Stipulation. Nor shall any Party be deemed to have agreed that any provision of this Stipulation is appropriate for resolving issues in any other proceeding, except to the extent expressly set forth in this Stipulation.
24) This Stipulation may be executed in counterparts and each signed counterpart shall constitute an original document. A signed signature page that is faxed or emailed is acceptable as an original signature page signed by that Party.
This Stipulation is entered into by each Party as follows:
Qwest Corporation d/b/a CenturyLink QC,
CenturyTel of Oregon, Inc. d/b/a
CenturyLink,
CenturyTel of Eastern Oregon, Inc.
d/b/a CenturyLink,
United Telephone Company of the
Northwest d/b/a CenturyLink
By: _____________________________
Stacey Goff
Executive Vice President &
Corporate Administration Officer
Legal & Corporate Administration
Date: 1/11/16
Frontier Communications
Northwest
Inc.,
Citizens Telecommunications
Company of Oregon
By: _____________________________
George Baker Thomson, Jr.
Associate General Counsel
Date: 1/11/2016
This Stipulation is entered into by each Party as follows:
**Oregon Telecommunications Association**
By: ____________________________
Richard A. Finnigan
Attorney for OTA
Date: 1/11/16
**Oregon Cable Telecommunications Association***
By: ____________________________
Mark Trinchero
Of Attorneys for OCTA
Date: 1/11/16
*OCTA is signing on behalf of its member companies other than Charter and BendBroadband
**Warm Springs Telecommunications Company**
By: ____________________________
Marsha Spellman, JD
Regulatory Director for Warm Springs Telecommunications Company
Date: January 11, 2016
This Stipulation is entered into by each Party as follows:
MCI Communications Services, Inc. d/b/a Verizon Business Services,
MCImetro Access Transmission Services LLC d/b/a Verizon Access Transmission Services,
TTI National, Inc.,
Verizon Select Services Inc.,
Verizon Long Distance LLC
By: Jesús G. Román
Date: January 11, 2016
UM 1481 PHASE III
REVISED STIPULATION
ATTACHMENT I
Pro Rata Support Reduction Process
• **Increasing the Minimum Balance:**
To avoid potentially frequent changes in support to the companies, Staff has proposed and the Parties agree to raise the minimum balance that it uses as a target from 1 month to 1.5 months. Doing this allows for some error in forecasting that arise as a result of quarter-to-quarter fluctuations in the contribution base erosion rate. Raising the minimum balance will result in reductions being taken earlier, but will also provide a buffer so there are fewer reductions.
• **Adopting the following Process:**
1. **Quarterly fund status report be disseminated to a wider audience:**
Currently a rolling forecast is produced on a monthly basis and the fund status is reported to the OUSF board at its quarterly meeting. This fund status report includes an estimate of the timing of any fund deficit as well as a detailed look at the percentage decline in the contributions. Staff plans to distribute this status report to all companies receiving money from the fund and all signatories to the Revised Stipulation.
2. **Disbursement reductions:**
If the monthly forecast for six months out indicates that the fund balance will go below 1.5 times the projected monthly disbursement, a reduction notice will be sent to all companies receiving disbursements. The notice will cover the current as well as all remaining years of the stipulation; however, the reductions will only be applied to the current year. Subsequent years will be addressed on a year-by-year basis.
3. **Basis for the proposed reduction:**
The proposed reduction will be based on the forecasted contributions, which will take into account any historic rate of decline. The rate of decline will also be projected forward.
4. **Implementation date:**
The reduced disbursement amounts will go into effect the quarter before the revenue shortfall is expected to occur.
**Process initiation date:**
The process of reducing the disbursements will begin 3 months prior to when the shortfall is expected to occur. Three months’ notice will give ample time for notification and agreement on the reduced amounts and will provide time for any internal PUC procedures, such as Public Meeting memos, to be completed. Notification will include a calculation of the reduction as a total and on a company level. As per the stipulation, the disbursement amounts will be reduced on a pro rata basis.
ILLUSTRATIVE EXAMPLE:
In this example we are showing how the process would work if the contributions continue to decline by 2% per quarter from 2016 onwards and the stipulated disbursement amounts are adopted. As contributions are lower than disbursements, the fund balance will decline until the fund is no longer to make payments. Staff would intervene 6 months prior to this occurrence and would issue a reduced disbursement schedule for agreement. Because of the increased minimum balance, unless the rate of decline in contributions was substantially greater than expected, further reductions would not be expected within the duration of the stipulation.
- Contributions decrease by 2% per quarter from 2016 onwards:
- The fund is forecast to have a cash balance which will be below 1.5 X the required monthly disbursement in January 2018.
- The process to reduce the disbursements will begin in April 2017 when Staff will issue a revised disbursement schedule. Once agreed the disbursement schedule will be implemented in October 2017.
Commission Staff has requested that a re-allocation process be included in the Stipulation as part of Commission Staff’s oversight responsibilities during the life of the Stipulation. Commission Staff has described the re-allocation process as occurring on a biennial basis during the five-year life of the Stipulation. The first re-allocation process review would occur in 2016 to be effective January 1, 2017. The second re-allocation process review would occur in 2018 to be effective January 1, 2019. The third re-allocation process review will occur in 2020 to be effective January 1, 2021.
Commission Staff has described the re-allocation review process to include the following elements: (1) The total support that the rural companies are receiving at the start of the re-allocation process; (2) The company specific support each company is receiving at the start of the re-allocation process; and (3) The projected amount of support that each rural company would receive based on a current run of the UM 1017 embedded cost model previously adopted by the Commission. Commission Staff describes the re-allocation process as a means to ensure efficient use of the limited resources of the state universal service fund and to be sure that support is being provided where it is most needed.
This re-allocation process is a multi-step transition process that takes companies from what they are currently receiving from the fund towards what the UM 1017 model defines as their need (which can shift over time). Commission Staff believes that this process will provide relative funding stability in the interim period.
If there is a surplus of monies provided by the fund, it will be used to reduce the size of the surcharge rather than providing further support to individual companies.
The process for the re-allocation would be that the Commission Staff would provide its analysis of the appropriate re-allocation for each company by the end of the second quarter of the year in question (2016, 2018 and 2020). Companies would then have until the end of the third quarter of the year in question to bring any objections that they have about the process or the results to the Commission’s attention if the matter cannot be resolved through discussions and negotiations between and among the rural companies and Commission Staff. The Commission would resolve the dispute, if any, and the re-allocation would proceed as authorized by the Commission. In addition, if there are questions about or proposed modifications to the UM 1017 embedded cost model, they will be brought before the Commission for resolution.
Parties recognize that potential re-allocation may affect individual company planning, budgeting and the recovery of investments already made to provide service to customers. As a result of these considerations the re-allocation process has the constraint that no company will have its support reduced by more than twenty percent over the life of the Stipulation as a result of the re-allocation process and no more than seven percent per allocation through the re-allocation process. The methodology that will be used in the re-allocation process is the methodology prepared by Commission Staff as the model based allocation derived through the waterfall process, which is described below.
Waterfall Approach
Overview: This method starts with the unconstrained support that each company would receive under the UM 1017 embedded cost model which is used to determine the percentage share of the rural OUSF support a company may receive. If any company has support less than what is allowed by the reduction constraint described above, that company’s support is adjusted to the minimum allowed by the constraint. New percentage shares are calculated for the remaining companies which are then applied against the remaining rural OUSF support. This process of calculating new percentage shares and new remaining support amount is repeated until no support amount needs to be reset based on the UM 1017 model results. No company shall receive more support than produced by the UM 1017 embedded cost model as adopted by the Commission or, if modified by the Commission, as so modified.
Work Steps:
Step 1) Calculate an adjusted OUSF distribution ratio as described above.
Step 2) Determine the amount of Rural ILEC OUSF support by multiplying the OUSF distribution ratio by the total amount of Rural ILEC OUSF support available.
Step 3) Compare the amount obtained in step (2) above with the current OUSF support for each company identifying any company that realizes a reduction in OUSF support that is greater than any applicable reduction constraint imposed.
Step 4) Companies identified in Step (3) adjust the OUSF support within the applicable reduction constraint.
Step 5) Recalculate the OUSF distribution ratio for each company not identified in Step (3) by dividing the UM 1017 Model based support for each company by the total sum of model based support calculated for each remaining ILEC that was not identified in Step (3) above.
Step 6) Deduct the total sum of the adjusted OUSF support determined in Step (4) above from the total amount of Rural ILEC support available.
Step 7) Repeat Step (2-6) above until no company realizes a reduction in OUSF support greater than the applicable reduction constraint.
Step 8) Determine the support for all companies remaining by multiplying the OUSF distribution ratio by the remaining total sum of available OUSF support for rural ILECs.
UM 1487 Phase IIIa Stipulation
1. The Parties to this Stipulation are Qwest Corporation d/b/a CenturyLink QC and Frontier Communications Northwest, Inc..
2. The purpose of the reporting that the Parties agree to under this Stipulation is to modify current reporting of information regarding CenturyLink QC and Frontier Northwest Inc. ("Reporting Parties") expenses in areas that are eligible for Oregon Universal Service Fund (OUSF) support. Both Reporting Parties currently provide their investment activities and expenses by wire center in the areas eligible for support. The Stipulation modifies the reporting of expenses.
3. **Reporting.** The reporting under this Stipulation for the Reporting Parties will be as depicted in the attached Oregon Universal Service Fund Accountability Report. The report data will be retained for a period of three years and it will be made available for Staff to audit upon request.
4. **Filing.** The Parties agree that the Reporting Parties and Staff, at the time any of the report data is submitted to the Commission, may submit to the Commission an explanation regarding which set of data from the report reflect the appropriate method to estimate how OUSF funding is being directed to operating expenses in high cost areas. The Oregon Universal Service Fund Accountability Report, being dependent upon Form O data, will be submitted annually no later than June 1st or approximately 60 days following the submission of the Form O.
5. **Applicability.** The Parties agree that the data provided in the report cannot be used to change the non-rural OUSF support amounts agreed to in the stipulation in Phase II of this proceeding, approved by the Commission in Order 13-162, or the non-rural OUSF support amounts agreed to in the stipulation in Phase III of this proceeding.
6. **Term.** This Stipulation will have the same termination date as the stipulation settling Parts IIIb and IIIc of this proceeding.
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This is a repository copy of *A critical analysis of calcium carbonate mesocrystals*.
White Rose Research Online URL for this paper:
http://eprints.whiterose.ac.uk/83533/
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**Article:**
Kim, YY orcid.org/0000-0002-8503-4554, Schenk, AS, Ihli, J et al. (7 more authors) (2014) A critical analysis of calcium carbonate mesocrystals. *Nature Communications*, 5. 4341.
https://doi.org/10.1038/ncomms5341
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A critical analysis of calcium carbonate mesocrystals
Yi-Yeoun Kim¹, Anna S. Schenk¹, Johannes Ihli¹, Alex N. Kulak¹, Nicola B.J. Hetherington¹, Chiu C. Tang², Wolfgang W. Schmahl³, Erika Griesshaber³, Geoffrey Hyett⁴ & Fiona C. Meldrum¹
The term mesocrystal has been widely used to describe crystals that form by oriented assembly, and that exhibit nanoparticle substructures. Using calcite crystals co-precipitated with polymers as a suitable test case, this article looks critically at the concept of mesocrystals. Here we demonstrate that the data commonly used to assign mesocrystal structure may be frequently misinterpreted, and that these calcite/polymer crystals do not have nanoparticle substructures. Although morphologies suggest the presence of nanoparticles, these are only present on the crystal surface. High surface areas are only recorded for crystals freshly removed from solution and are again attributed to a thin shell of nanoparticles on a solid calcite core. Line broadening in powder X-ray diffraction spectra is due to lattice strain only, precluding the existence of a nanoparticle sub-structure. Finally, study of the formation mechanism provides no evidence for crystalline precursor particles. A re-evaluation of existing literature on some mesocrystals may therefore be required.
¹School of Chemistry, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK. ²Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, UK. ³Ludwig-Maximilians-Universität München, Department für Geo- und Umweltwissenschaften, Sektion Kristallographie, Theresienstrasse 41, 80333 München, Germany. ⁴Department of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, UK. Correspondence and requests for materials should be addressed to Y.-Y.K. (email: firstname.lastname@example.org) or to F.C.M. (email: email@example.com).
The last decade has seen enormous leaps in our understanding of solution-based crystallization processes. Together with non-classical mechanisms of nucleation\textsuperscript{1–3} and the selective entrapment\textsuperscript{4} or inclusions within single crystals\textsuperscript{5–6}, it is now recognized that crystal growth can often occur by the aggregation of precursor units rather than by ion-by-ion growth\textsuperscript{7–8}. Pioneering work from Banfield \textit{et al.}\textsuperscript{9,10}, in which it was shown that single crystal TiO$_2$ nanowires can form via the oriented attachment of crystalline nanoparticles, inspired much of the current research into aggregation-based crystallization. Indeed, it is now known that single crystals of many materials including ZnO/SiO$_2$ (ref. 11), goethite\textsuperscript{12}, PbSe\textsuperscript{13} and iron oxyhydroxide\textsuperscript{14} can form by oriented attachment, where this process operates at the nanoscale to give one-dimensional wires or irregular nanostructures\textsuperscript{15,16}.
Following early demonstrations of crystal growth by oriented assembly, this concept was extended to the formation of larger, three-dimensional crystals, where these were designated as ‘mesocrystals’. The term mesocrystal was first applied to calcite and vaterite crystals (polymorphs of CaCO$_3$) that were precipitated in the presence of polymer additives\textsuperscript{17–20}. Mesocrystals were proposed to form via the oriented assembly of polymer-stabilized crystalline nanoparticles (Fig. 1), where evidence for this mechanism was derived from structural analysis of the product crystals. These articles created an enormous amount of interest, and >400 examples of mesocrystals have now been proposed in the literature\textsuperscript{21–26}. Looking critically at this mechanism of crystallization, however, the ability to achieve perfect crystallographic register of subunits over large length scales is clearly highly challenging. The term mesocrystal has therefore recently been refined to define these particles based on their structures, rather than their formation mechanism, such that ‘a mesocrystal ideally comprises a three-dimensional array of iso-oriented single crystal particles of size 1–1,000 nm’ (ref. 27). This definition is potentially far more general and could encompass crystals that form by the assembly and subsequent crystallization of amorphous precursor particles, provided that a memory of the precursor particles is retained in the product crystal.
This evolution of ideas about mesocrystal structure and formation has resulted in a marked lack of consensus in the literature, where this problem is further exacerbated by particles sometimes being designated as mesocrystals on the basis of rather superficial structural analyses. In this article, we look critically at the subject of mesocrystals, and use calcite crystals, precipitated in the presence of polymer additives, as a test case to investigate the validity of the characterization methods commonly used. Indeed, although it is now commonplace to assign mesocrystal structures based on nanoparticulate surface structures and analyses of X-ray diffraction (XRD) data using the Scherrer equation, we demonstrate that both of these approaches may be insufficient and unsafe. We then examine the mechanism of formation of these calcite mesocrystals, and show that modifications in the crystal morphology only occur at later stages of growth. An amorphous calcium carbonate (ACC) precursor phase is also not a pre-requisite to the development of classic mesocrystal morphologies. Finally, electron backscatter diffraction (EBSD) is used to investigate the microstructures of synthetic mesocrystals, revealing a sector-like mosaic structure that contrasts markedly with the uniform nanostructure seen for a biogenic mesocrystal—a sea urchin spine\textsuperscript{27}.
**Results**
**Precipitation and characterization of calcite crystals.** A number of calcite particles that have previously been described as mesocrystals were analysed. CaCO$_3$ was precipitated in the presence of the polymeric additives poly(4-styrene sulphonate-alt-maleic acid) (PSS-MA) and poly(styrene-alt-maleic acid) (PS-MA; Supplementary Fig. 1), where they exhibited the anticipated morphologies of platonic/dodecahedral for PSS-MA and rod-shaped for PS-MA (Fig. 2a,b). These crystals, which are designated as calcite/polymer throughout, were then characterized using powder XRD (PXRD), small-angle X-ray scattering (SAXS), surface area analysis (based on the method by Brunauer, Emmett and Teller (BET)), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and electron backscatter diffraction (EBSD), and comparison was made with control samples. These include calcite precipitated in the presence of Co$^{2+}$ ions, which exhibits a rod-shaped form comparable to calcite/PSS-MA and yet is widely accepted to be a single crystal (Fig. 2c)\textsuperscript{28,29}, and also calcite crystals produced by calcite overgrowth of $\approx 3\,\mu$m rhombohedral calcite seeds in the presence of the selected polymers. Finally, the mechanism of formation of these crystals was investigated and was considered in light of proposed routes to calcite mesocrystals. The overgrowth sample formed part of this study and was used to investigate whether classic mesocrystal morphologies could develop on overgrowth of a rhombohedral calcite core.
**Figure 1 | Schematic of calcite crystal growth mechanisms.** (a) The original mechanism proposed to lead to calcite mesocrystal formation, based on the oriented assembly of crystalline nanoparticles; and (b) the mechanism demonstrated in this paper, where a calcite rhombohedron initially forms, and subsequent growth in the presence of polymer results in a modified morphology and a rough, particulate surface.
taking the peak full-width half-maximum (FWHM), removing the instrumental broadening and then assuming that all remaining broadening is caused by particle size effects alone. This has the virtue of being very straightforward and is suitable for samples where domain size (coherence length) is the major contributor to peak broadening (for example, nanoparticles). However, in most cases, ignoring strain effects is far too simplistic a model, and application of the Scherrer equation when lattice strain is present will return an underestimate of the domain size. Full analysis of PXRD patterns using either Rietveld refinement or Williamson–Hall plots overcomes this problem and enables the individual contributions of domain size and lattice strain to the peak broadening to be determined. These approaches differ in that Rietveld refinement applies a sophisticated rigorous model and generates parameters that are refined to fit the whole pattern, while Williamson–Hall makes use of adjusted FWHM or integral breath values, separating out size and strain effects based on how they vary with the diffraction angle $\theta$, and assuming that these effects are simply additive on the FWHM and integral breath. Notably, in the analyses presented here, both methods validate each other by showing the same trends (Supplementary Note 1).
A detailed synchrotron PXRD analysis of the structures of control samples (pure calcite and Co$^{2+}$-doped calcite) and calcite/PSS-MA, seeded calcite/PSS-MA and calcite/PS-MA crystals was carried out, where the recorded patterns were modelled using a Rietveld refinement, Williamson–Hall plots and the Scherrer equation. The results are presented in Tables 1 and 2 and Supplementary Fig. 2, together with the errors associated with the values given, and analyses that assume size-only and strain-only broadening effects are shown for comparison. Rietveld analysis of control calcite crystals, which were 20–30 $\mu$m in size and were precipitated using comparable reaction conditions to those used for the calcite/polymer samples, gave a best fit with a domain size of 870 nm and strain of 0.004%, whereas a Williamson–Hall plot yielded a size and strain of 678 nm and 0.006%. These crystals can be considered virtually strain-free, and thus the Scherrer equation predicts comparable domain sizes of 798–825 nm (depending on the diffraction peak used for analysis). In the case of 40 $\mu$m Co$^{2+}$ calcite crystals, which are widely described as single crystals in which Co$^{2+}$ is substituted for Ca$^{2+}$ in the lattice\textsuperscript{28}, Rietveld analysis yielded a domain size of 300 nm and strain of 0.128%, whereas Williamson–Hall gave an (unphysical) $-1,812$ nm for the size and a strain of 0.21%. This negative-domain size arises because intrinsic errors in the data can cause the Williamson–Hall plot of crystals with large domain sizes to intercept the $y$ axis at a small negative value. As the intercept is inversely proportional to the domain size, a large negative-domain size is returned. Application of the Scherrer equation, in contrast, yielded domain sizes of 92 nm as broadening arising from lattice strain is attributed to reduced domain sizes. These diffraction data are therefore fully consistent with the description of a single crystal exhibiting considerable lattice strain.
Looking in turn at calcite crystals precipitated in the presence of the polymers, the calculated domain sizes and strain determined using the Rietveld and Williamson–Hall methods, respectively, were 553 nm/0.024% and 1,014 nm/0.049% for calcite/PSS-MA, 612 nm/0.035% and 3,228 nm/0.072% for seeded calcite/PSS-MA, and 622 nm/0.016% and 647 nm/0.021% for calcite/PS-MA crystals (Table 1). As these domain sizes are comparable to pure calcite, this shows that the line broadening observed for the calcite/polymer samples is almost entirely due to lattice strain. This is also seen in the underestimate of the domain sizes determined for these samples using the Scherrer equation, which were 321/228 nm (calcite/PSS-MA), 278/185 nm (seeded...
Table 1 | Strain parameters and coherence lengths of calcite crystals.
| | Calcite control, 10 mM | Co-calcite | PSS-MA | PSS-MA seeded | PS-MA |
|------------------------|-------------------------|------------|--------|---------------|-------|
| Rietveld (pseudo–Voigt)| | | | | |
| Size only (nm) | 817 (32) | 86 (7) | 380 (34)| 319 (24) | 485 (15) |
| Strain only (%) | 0.001 (2) | 0.130 (3) | 0.030 (4)| 0.039 (3) | 0.022 (4) |
| Size (nm) and strain (%)| 870 (22)/0.004 (1) | 300 (14)/0.128 (6) | 553 (23)/0.024 (3) | 612 (19)/0.035 (2) | 622 (12)/0.016 (1) |
| Williamson–Hall plot | | | | | |
| Size only (nm) | 446.4 (27) | 54 (5) | 141 (7) | 109 (5) | 243 (8) |
| Strain only (%) | 0.017 (1) | 0.200 (2) | 0.057 (2)| 0.074 (2) | 0.0225 (7) |
| Size (nm) and strain (%)| 678 (19.3)/0.006 (5) | −1,099 (2532)/0.210 (1) | 1,014 (465)/0.049 (8) | 3,228 (395)/0.072 (8) | 647 (112)/0.021 (2) |
| Scherrer equation (104)| | | | | |
| Size only (nm) | 825 (89) | 92 (9) | 321 (32)| 278 (48) | 368 (43) |
| Scherrer equation (O06)| | | | | |
| Size only (nm) | 798 (56) | NA | 228 (25)| 185 (21) | 435 (56) |
NA, not applicable; PS-MA, poly(styrene-alt-maleic acid); PSS-MA, poly(4-styrene sulphonate-co-maleic acid); XRD, X-ray diffraction.
Strain parameters and coherence lengths derived from line profile analysis of powder synchrotron XRD spectra of calcite crystals. The numbers in brackets are the s.d. of the measurements, and the goodness of fitness of the Rietveld analysis and the $\chi^2$-distributions of the Williamson–Hall plots are listed in Supplementary Table 4.
Table 2 | Influence of annealing and ageing on strain parameters and coherence lengths of calcite/polymer crystals.
| | Sample 1 | Sample 2 | Sample 3 |
|------------------------|----------|----------|----------|
| | Fresh | In situ heating 300 °C | Fresh | Ex situ heating 400 °C | Fresh | Aged in air 24 h |
| Rietveld (pseudo–Voigt)| | | | | | |
| Size only (nm) | 224 (19) | 223 (9) | 388 (32) | 295 (54) | 300 (11) | 322 (31) |
| Strain only (%) | 0.046 (5)| 0.050 (2)| 0.029 (3)| 0.040 (3)| 0.034 (2)| 0.032 (1)|
| Size (nm) and strain (%)| 446 (23)/0.041 (5) | 519 (48)/0.046 (1) | 708 (55)/0.025 (2) | 1,493 (23)/0.039 (7) | 643 (35)/0.030 (2) | 666 (43)/0.029 (2) |
| Williamson–Hall plot | | | | | | |
| Size only (nm) | 83 (5) | 74 (4) | 141 (7) | 102 (5) | 112 (5) | 114 (6) |
| Strain only (%) | 0.099 (3)| 0.109 (3)| 0.057 (1)| 0.079 (1)| 0.072 (1)| 0.070 (2)|
| Size (nm) and strain (%)| 2,344 (1159)/0.10 (1) | −1,812 (3087)/0.11 (1) | 1,335 (609)/0.051 (5) | −1,601 (9513)/0.084 (4) | 1,011 (418)/0.064 (5) | 1,036 (371)/0.063 (6) |
| Scherrer equation (104)| | | | | | |
| Size only (nm) | 225 (12) | 223 (19) | 368 (45) | 299 (31) | 319 (27) | 319 (24) |
| Scherrer equation (O06)| | | | | | |
| Size only (nm) | 192 (21) | 189 (13) | 319 (32) | 258 (18) | 266 (14) | 266 (19) |
NA, not applicable; PS-MA, poly(styrene-alt-maleic acid); PSS-MA, poly(4-styrene sulphonate-co-maleic acid); XRD, X-ray diffraction.
Strain parameters and coherence lengths derived from line profile analysis of powder synchrotron XRD spectra of three different batches of calcite/PSS-MA crystals precipitated from [Ca$^{2+}$] = 5 mM and PSS-MA = 125 µg ml$^{-1}$. 1 solution after in situ heating to 300 °C, ex situ heating to 400 °C and ageing in air. The numbers on brackets are the s.d. of the measurements, and the goodness of fitness of the Rietveld analysis and the $\chi^2$-distributions of the Williamson–Hall plots are listed in Supplementary Table 4.
calcite/PSS-MA) and 368/435 nm (calcite/PS-MA) for the {104} and {006} reflections, respectively. That the sizes estimated for the {006} reflections are generally smaller than those for the {104} is consistent with the greater strain present in the ⟨001⟩ direction, as demonstrated in the Williamson–Hall plots (Supplementary Fig. 2). This can be attributed to preferential adsorption of the polymer additives on {001} planes. These data conclusively demonstrate that all of the mesocrystals examined show comparable domain sizes ($\approx 500$ nm), and that the primary source of line broadening is microstrain, where this can arise from organic occlusions within the crystal$^{31–33}$. Notably, the seeded calcite/PSS-MA crystals, which are 10–20 µm in size and contain a 3-µm pure calcite core, gave comparable data to the calcite/PSS-MA crystals, suggesting that they have similar structures.
It has been proposed that the structures of mesocrystals can change with ageing due to fusion of the nanoparticulate subunits or the crystallization of residual ACC within the structure$^{24,26,27}$. These possibilities were studied here by analysing annealed and aged calcite/polymer samples. Freshly prepared calcite/PSS-MA crystals were isochronously heated for 30 min at temperatures of 100, 200 and 300 °C, and synchrotron PXRD patterns were recorded after cooling. A sample was also analysed after ageing in air for 24 h. No changes in the intensities of the diffraction peaks, the domain sizes (from 446 to 519 nm) or microstrains (from 0.041 to 0.046%) were observed after any of these protocols (Table 2 and Supplementary Fig. 3). These analyses were further confirmed using Raman microscopy, where no change in the peak shapes or broadening were recorded (Supplementary Fig. 4). Even though there was no ACC phase observed at all in the pattern initially, as ACC sometimes required temperatures $> 300$ °C to crystallize$^{24,35}$, additional ex situ annealing experiments were performed over the temperature range ambient to 400 °C. Only $> 400$ °C were any changes in the diffractograms observed, where
these were apparent in increases in domain size (from 708 to 1,493 nm) and strain (from 0.025 to 0.039%; Table 2 and Supplementary Fig. 3c). This can be attributed to decomposition of the polymers at 350 °C (Supplementary Fig. 5), and similar behaviour has been observed for biogenic and synthetic calcite crystals occluding organic molecules\textsuperscript{33,34}. Therefore, these studies provide no evidence for ACC in the samples, or for epitaxial fusion of nanoparticle subunits.
Additional information on the internal structures of the calcite samples was obtained using SAXS. The Co-calcite gave rise to a scattering profile consistent with little structural complexity, as shown by its similarity to that of a control calcite sample (Fig. 3a). The minor deviation from the pure calcite scattering curve is most likely because of increased surface roughness\textsuperscript{36}. Looking in turn to the calcite/PS-MA and seeded calcite/PS-MA samples, their radially averaged intensity profiles were similar to those of calcite/PSS particles previously studied using SAXS\textsuperscript{36} (Fig. 3b). The curves can be divided into three regimes, where scattering at low $Q$ (regime 1) is dominated by a steep linear decay, owing to the large external facets of the powder grains, whereas smaller nanostructural features within the mineral particles give rise to a bent curve shape in regime 2. More detailed analysis of the scattering profile obtained from the calcite/PS-MA crystals based on a modified Guinier law shows that the nanostructural heterogeneities within these particles can be described as dilute platelets with average thicknesses of $\approx 2.9$ nm (Fig. 3c). This would be consistent either with a classic mesocrystal in which individual mineral units are separated by organic layers\textsuperscript{27} or with a single crystal containing organic occlusions; SAXS cannot distinguish between the two. Finally, in the limit of high $Q$ values (regime 3), the profiles show a linear decay that corresponds to smooth interfaces. A full evaluation of the SAXS data is provided in the Supplementary Note 2.
**TEM analysis of the internal structures of crystals.** The internal structure of calcite/PS-MA crystals were investigated using TEM of thin sections cut using focussed ion beam (FIB), where samples were prepared immediately after removal of the crystals from solution. A large thin section cut through a crystal grown under conditions $[\text{Ca}^{2+}] = 5$ mM and $[\text{PSS-MA}] = 125 \mu \text{g ml}^{-1}$ is shown in Fig. 4a. This crystal exhibits a classic mosaic structure comprising 1–2 μm domains (Fig. 4b), and selected area diffraction patterns recorded over the entire area of the section (patterns 1–8) confirmed its single crystal structure. The size of these mosaic blocks is consistent with the >500 nm domain sizes identified by PXRD. Calcite/PS-MA crystals grown at lower initial supersaturations ($[\text{Ca}^{2+}] = 1.25$ mM and $[\text{PSS-MA}] = 125 \mu \text{g ml}^{-1}$) were also single crystals but displayed some differences in structure and comprised two distinct regions, namely a central core that appeared smooth and a rougher outer layer (Fig. 4c).
TEM has often been used to provide evidence for a nanoparticulate or porous sub-structure in mesocrystals. However, such structures can arise as artefacts, where these can be readily introduced into specimens during preparation of thin sections or during imaging itself. Roughness in the surfaces of the section can cause contrast variation in TEM images, which can be falsely interpreted as nanoparticle subunits (Fig. 4d,e)\textsuperscript{37}. CaCO$_3$ occluding organic additives is also particularly susceptible to beam damage\textsuperscript{38,39}, and pores were generated in thin sections of synthetic calcite containing organic inclusions if they were not imaged with extreme care (Fig. 4f,g and Supplementary Fig. 6). Geological calcite is much less susceptible to beam damage (Supplementary Fig. 6). It is emphasized that no evidence for porosity and no discontinuity in lattice fringes were observed between the mosaic blocks (Fig. 4h).
(a) Orientation-averaged plot of the scattering intensity versus the modulus of the scattering vector $Q$ (log–log representation) recorded for powdered samples of control calcite (grey circles) and calcite crystals precipitated in the presence of Co$^{2+}$ ions (blue circles, [Co$^{2+}$]/[Ca$^{2+}$] = 50:1). The control sample shows an intensity decay of $I(Q) \propto Q^{-4}$ over the entire $Q$-range covered by the experiment, thus indicating the absence of structural complexity on the nanometre level. In the profile recorded for Co-doped calcite, there is a deviation from Porod-like behaviour in the low $Q$ regime, which might be attributable to the rough-textured topography of the particle surfaces. (b) Orientation-averaged plots of the scattering intensity versus the modulus of the scattering vector $Q$ (log–log representation) recorded for powdered samples of seeded calcite/PS-MA crystals (light green circles) or calcite/PS-MA crystals (dark green circles). The profiles of both specimens provide indications for structural complexity at the length scale of nanometres. (c) Guinier plot $\ln(I(Q)/Q^2)$ versus $Q^2$ valid for dilute platelet-shaped scattering objects applied to the scattering curve of calcite/PS-MA crystals (dark green circles). The linear relationship in the $Q$-range $0.25 \text{nm}^{-1} < Q < 1.3 \text{nm}^{-1}$ (orange circles) points to a platelet shape of the nanostructural heterogeneities. From the slope of the regression line (black) a mean thickness of the platelets of $D = 2.9$ nm can be calculated. The error bars in **a** and **b** represent s.d. calculated according to Gaussian error propagation on the basis of the uncertainty of the intensity in each data point.
Figure 4 | TEM images of thin sections of calcite/PSS-MA crystals. (a) Large FIB-section of a crystal grown at $[\text{Ca}^{2+}] = 5\,\text{mM}$ and $[\text{PSS-MA}] = 125\,\mu\text{g ml}^{-1}$ (the blue outline indicates the boundary between the crystal and the Pt coating). The selected area electron diffraction patterns (from 1 to 8) were recorded across the whole crystal and show the single crystal structure. (b) A higher magnification image of the crystal shown in a, showing large mosaic blocks. (c) A crystal grown at $[\text{Ca}^{2+}] = 1.25\,\text{mM}$ and $[\text{PSS-MA}] = 125\,\mu\text{g ml}^{-1}$ showing a central core and overgrowth layer, where the inset shows the same crystal at lower magnification. (d–h) High-magnification images of the area shown in the green circle in b. (d) An image taken at focus, which shows a continuous structure. (e) The same area imaged significantly under focus, which shows an apparent nanoparticulate structure. (f) A higher magnification image of the area shown in the red box in d before beam damage, and (g) the same area after it has been beam damaged, resulting in porosity. (h) A HRTEM image of the area shown in the blue box in g, showing perfect lattice continuity, even after being beam damaged. Scale bars, 2\,\mu\text{m} (a), 500\,\text{nm} (b,c), 50\,\text{nm} (d,e) and 5\,\text{nm} (f–h).
Analysis of surface areas and structures. High surface areas provide a further feature that is considered characteristic of mesocrystals, although an enormous range of values (varying from 48 to 540\,\text{m}^2\,\text{g}^{-1}) has been quoted in the literature for calcite mesocrystals\textsuperscript{7,9,10}. It is also noted that an assessment of mesocrystal structure is very often made without measurement of
the surface area, possibly owing to the relatively large quantities of sample required for accurate analysis. The surface areas and structures of calcite/PSS-MA and calcite/PS-MA crystals were determined, and the calcite/PSS-MA crystals were studied in detail (Supplementary Table 1). Comparison was also made with the surface areas of a number of reference samples including 20–30 μm synthetic calcite rhombohedra (0.1 m² g⁻¹), ground sea urchin skeletal platelets (1–2 m² g⁻¹), 50–100 nm calcite nanoparticles (22 m² g⁻¹) and calcite precipitated in the presence of Ca²⁺ ions (0.5 m² g⁻¹). The very low surface area of the latter is consistent with their description as single crystals. The calcite/polymer particles, by contrast, showed large variations in specific areas (2–57 m² g⁻¹) depending on the initial solution supersaturation, the polymer concentration and the ageing conditions. Crystals with higher surface areas were obtained at higher initial supersaturations (S₀,calcite = 3.43–4.17 gave surface areas of 1–4 m² g⁻¹ and S₀,calcite = 5.76–6.47 gave surface areas of 25–60 m² g⁻¹), whereas PSS-MA concentrations of 150 and 300 μg ml⁻¹ gave calcite crystals with surface areas of 40 and 56 m² g⁻¹, respectively. Crystals generated by overgrowth of a rhombohedral calcite seed at lower supersaturations also exhibited high surface areas of 28 m² g⁻¹.
Ageing of dry calcite/PSS-MA samples in air had a marked effect on their surface areas, with reductions from 50–60 m² g⁻¹ to 5 m² g⁻¹ being observed after just 2 days under ambient humidity. In contrast, a much more gradual reduction in surface area was observed when samples were aged in the mother solution in the presence of ammonium carbonate, with a comparable reduction in surface area being observed over ≈2 weeks. That significant changes in the surface area occur on ageing the crystals suggests that the surface must recrystallize, changing the roughness. That this occurs more rapidly in humid air than in the original crystallization solution suggests that the residual polymer in the solution inhibits recrystallization. Indeed, calcite surfaces are well known to undergo reconstruction in water, where this process will be modified in the presence of charged polymers. The surfaces of calcite/PSS-MA crystals that had been freshly removed from the crystallization solution were therefore compared with crystals from the same batch after they had been aged in air for 2 days. The results are striking. The surfaces of the fresh crystals are covered with extremely small (<5 nm) particles (Fig. 5a), whereas the aged crystals exhibit much larger (30–40 nm) features (Fig. 5b). Crystals from the same batch were also examined after they had been incubated in the mother solution in the absence of ammonium carbonate for 2 days. Significant recrystallization was again apparent from the 80–90 nm geometric features viewed (Fig. 5c). This recrystallization process was also supported by thermogravimetric analysis of calcite/PSS-MA crystals. While crystals isolated after 1 day contained 4.1 wt% polymer, 10-day-old crystals occluded just 2.9 wt% polymer (Supplementary Fig. 5).
The nanoparticles on the calcite/polymer crystal surfaces are therefore the origin of the high surface areas sometimes measured. Using back-of-the envelope calculations to illustrate, a 10-μm calcite crystal with a 50-nm outer shell comprising 5 nm particles would exhibit a specific surface area of 63 m² g⁻¹. The fact that freshly prepared calcite/polymer crystals typically showed surface areas of 50–60 m² g⁻¹, which reduced to 5–20 m² g⁻¹ on ageing in air for longer than 2 days, is thus fully consistent with this model. It should also be noted that BET measurements are unreliable on small quantities of sample (Supplementary Table 2), which may also contribute to some of the variability observed in the literature.
**Electron backscatter diffraction analysis.** Further information on the microstructures of the calcite/PSS-MA crystals was obtained using EBSD. The spine of the sea urchin *Paracentrotus lividus* (which has been described as a mesocrystal) and pure synthetic calcite were also analysed for comparison. Figure 6a,b shows the EBSD maps of these samples, together with histograms that show the misorientation for each EBSD-measured pixel (width 280 nm) relative to the mean orientation of each crystal. The EBSD maps of the calcite/PSS-MA crystals clearly show that they typically display primary mosaic structures that comprise sectors that radiate from the centre of a crystal (highlighted 1, 2...
and 3 in Fig. 6b). This leads to a distribution of misorientation in the order of 5–7° (Fig. 6c). The growth sectors radiate from what appears to be a common substrate in the centre of the crystal, where the degree of mutual crystallographic misalignment is consistent with imperfect homoepitaxial overgrowth on a smaller calcite crystal. Interestingly, the orientational changes between the sectors are gradual rather than sharp and the distribution of orientations is diffuse in many places, which indicates orientation changes on a length scale of ≈500 nm. This is consistent with the size of the coherently scattering domain as determined by PXRD. Each mosaic sector also has an internal secondary mosaic distribution, which is larger than that of the pure calcite single crystal (the latter corresponds to our experimental resolution); Fig. 6d. The sea urchin spine (Fig. 6e), in contrast, exhibits a misorientation spread of about 4° (Fig. 6f) and shows a gradual change of orientation over the mapped area rather than sectoring. Occluded non-crystalline material in the form of pores and/or organic matrix in the sea urchin spine appears as dark areas in the map.
These data demonstrate that the calcite/PSS-MA crystals do not grow by a continuous process, and that growth was interrupted and re-started several times. Further, as the misalignments present in these crystals are absent in the pure calcite control, they must be ascribed to the effect of the polymer, which disturbs growth. These data also provide a valuable opportunity to compare the microstructures of the calcite/polymer crystals with that of a calcite biomineral. As structure informs the mechanical properties of biominerals, organisms exert strict control over crystallization processes and classic sector-zoning and small-angle boundaries between large mosaic blocks are rare. Instead, the sea urchin spine exhibits a uniform nanoparticulate structure with ubiquitous small-angle-misorientation fluctuations on the 100–200 nm scale, where these are associated with intracrystalline organic matrix\textsuperscript{13–15}. This structure then imparts considerable fracture resistance. Similar nanotextures have been observed in a range of calcite biominerals, where they are used to produce continuous orientation gradients and structures comprising interdigitated crystals\textsuperscript{43–45}.
**Analysis of growth mechanisms.** While ACC has often been observed as a precursor phase of calcite ‘mesocrystals’ precipitated in the presence of anionic polymers\textsuperscript{17,46,47}, their full developmental pathway has remained unclear because of the
problems associated with isolating rapidly growing crystals at precise points in their development. To address this challenge, we investigated the morphological evolution of the calcite/PSS-MA crystals by performing the crystallization reaction within picolitre droplets formed on patterned self-assembled monolayers\textsuperscript{46}. Crystal growth terminates when the limited quantities of reagents within the droplets are depleted, revealing intermediate growth morphologies. Calcite/PSS-MA crystals were studied, and ACC was identified at early times (Fig. 7). Rather surprisingly, however, the first crystalline particles formed were 200–500 nm calcite rhombohedra. Only when the crystals had grown to sizes of 0.5–1 μm did modifications in morphology become apparent, and further growth then gave the characteristic pseudo-dodecahedral morphology of the calcite/PSS-MA crystals. These later growth stages are consistent with those reported for the growth of calcite/PSS-MA crystals at low polymer concentrations by Song \textit{et al.}\textsuperscript{47}, where a morphological transition from pseudo-dodecahedral morphologies to curved, concave surfaces was observed at particle sizes of ≈2–5 μm.
Further confirmation that the characteristic mesocrystal morphologies are not determined at early growth stages was demonstrated through crystal overgrowth experiments in which calcite/PSS-MA was precipitated on 3–5 μm rhombohedral calcite seeds (Fig. 8). The final morphologies of the overgrown crystals and their rough surfaces—which appear to comprise nanoparticle units—were identical to those of crystals precipitated without seeds (Fig. 8a–c). These crystals also exhibited high surface areas (Supplementary Table 1), and mechanical polishing of overgrown crystals embedded in epoxy resin confirmed the overgrowth structure (Fig. 8d). The dimensions of the seed crystal itself also had some influence on its further morphological development, such that less change in morphology was observed when seeds > 10 μm in size were used (Supplementary Fig. 7). This is readily explained as the larger the seed, the more material that is required to produce the same increase in thickness.
Finally, the influence of ACC as a precursor phase on the development of ‘classic’ mesocrystal morphologies was investigated by precipitating calcite/PSS-MA crystals from solutions that were very undersaturated with respect to ACC\textsuperscript{49} (Supplementary Table 3). While very few crystals were precipitated at initial supersaturations of $s_{\text{calcite}} \approx -3.66$ to $-2.24$ and $S_{L,\text{calcite}} \approx 0.11$ to 2.57, particles with characteristic pseudo-dodecahedral morphologies began to emerge at initial supersaturation levels of $-1.76 < S_{\text{calcite}} < -0.45$ and $3.05 < S_{\text{calcite}} < 4.31$, although they did not show the typical ‘scales’ on the surface which appear at higher supersaturations (Fig. 8e and Supplementary Fig. 8). The surface areas (1–4 m$^2$ g$^{-1}$) and polymer contents (1.3 wt%) of crystals grown under these conditions were also significantly lower than for those precipitated at higher supersaturations (Supplementary Table 1 and Supplementary Fig. 5). That these crystals—which were produced in the absence of ACC—exhibited the curved surfaces often associated with this phase is particularly interesting, where this demonstrates that care must be taken in deducing crystal growth mechanisms on the basis of final morphologies alone. With further increase in the supersaturation (but keeping solutions undersaturated with respect to ACC), the calcite/PSS-MA crystals developed more defined pseudo-dodecahedral morphologies and roughened surfaces (Fig. 8f and Supplementary Fig. 9). These experiments therefore provide strong evidence that development of these characteristic morphologies and rough surfaces does not depend on the assembly of crystalline nanoparticles.
**Discussion**
In light of the data presented here, it is valuable to return to the early published work on calcite/polymer mesocrystals. On the basis of structural data, a suggestion was made that these crystals formed by the assembly of crystalline nanoparticles as mediated by adsorbed polymers. Although this mechanism has never been proven experimentally, and the definition of a mesocrystal has now been relaxed such that it is made purely on the basis of structure and not formation mechanism\textsuperscript{27}, it rather caught the
Figure 8 | Crystal morphologies are not defined at early stages of growth. Images of calcite crystals produced by overgrowth of 3–5 μm calcite seeds at [Ca^{2+}] = 2.5 mM in the presence of [PSS-MA] = 125 μg ml^{-1}. (a) Optical micrograph; and (b) SEM image of a crystal produced by overgrowth; and (c) high-magnification SEM image of the surface of a crystal produced by overgrowth, showing a nanoparticulate structure. (d) Optical micrograph of mechanically polished crystals that had been embedded in resin, showing the overgrowth of calcite (labelled O) on rhombohedral cores (labelled S). Calcite crystals grown in the presence of PSS-MA from solutions that are undersaturated with respect to ACC. (e) [Ca^{2+}] = 0.5 mM, [CO_3^{2-}] = 10 mM and [PSS-MA] = 50 μg ml^{-1}, $S_{calcite} = 3.49$ and $S_{ACC} = -1.32$; and (f) [Ca^{2+}] = 0.5 mM, [CO_3^{2-}] = 200 mM and [PSS-MA] = 50 μg ml^{-1}, $S_{calcite} = 4.70$ and $S_{ACC} = -0.19$. Scale bars, 20 μm (a), 5 μm (b-d-f), and 200 nm (e).
imagination. As a result, the current literature is still dominated by a belief that large single crystals can form by the assembly of crystalline nanoparticles, where the external morphology reflects the shape of the basic building block. The extensive and rigorous analysis of the structure and formation of calcite/polymer crystals described in this article contradicts this picture.
Let us first consider the structural data. While it is tempting to view a single crystal as one in which every atom lies in its perfect position, the reality is that almost all crystals are imperfect. Imperfect crystals have been discussed since the early 1900s, when it was recognized that measured diffraction intensities depend on crystal perfection\textsuperscript{50}. While the diffraction intensities in a perfect crystal are proportional to the structure factor F and have angular spreads in the order of seconds of arc, most crystals give intensities orders of magnitude higher with angular spreads of minutes of arc. Attempts to rationalize these erroneous diffraction intensities were therefore made using a range of models to describe imperfect crystals. One of the earliest was that of a ‘mosaic crystal’\textsuperscript{51}, where this envisaged a crystal as a mosaic of perfect crystalline blocks that are slightly misaligned with respect to each other. These misorientations can destroy the coherence between radiation reflected from different depths of the crystal, resulting in an enhancement of the reflected intensity. Although originating as a mathematically tractable model, the concept of a mosaic crystal appears to apply quite well to many natural crystals that comprise micron-sized blocks\textsuperscript{52}.
A significant advance in the understanding of imperfect crystals was then made with the recognition that crystals contain dislocations—atomic-scale defects—and that simple crystal boundaries can be described in terms of arrays of dislocations\textsuperscript{53}. A complete description of a crystal and its diffraction behaviour thus depends on knowledge of the type and positioning of the imperfections present. Common defects/sources of strain in crystals include dislocations, stacking faults, twinning, grain boundaries, chemical heterogeneities and inclusions, where these can manifest themselves in characteristic changes in peak positions, broadening and shape\textsuperscript{54}. Full-pattern analysis, considering the position, width and shape of the peaks can provide some insight into the nature of imperfections in crystals, where dislocations, for example, can contribute to line broadening owing to their mean separation (which is inversely proportional to their density) and microstrains arising from internal stress fields\textsuperscript{55}. That line broadening due to structural errors can also vary with $hkl$ according to the type of fault present (for example, stacking faults or twins) also provides a further source of information.
A further model of crystal imperfection is that of paracrystallinity, where this has been used to describe structures that are intermediate between crystalline and amorphous\textsuperscript{56}. Indeed, this model is often used to interpret the structure of polymers\textsuperscript{57}, and bone has been described as a paracrystalline material\textsuperscript{58}. Analysis of our diffraction data from the calcite/PSS-MA crystals provides no evidence for paracrystallinity, where this is entirely expected given that calcite is well recognized to form exceptionally large, perfect crystals\textsuperscript{53}. The detailed analyses of the high-resolution synchrotron XRD data instead conclusively show that the observed line broadening arises from microstrains within the crystal lattice rather than small domain sizes. We attribute these to the incorporation of polymer within the crystal lattice, as is consistent with the SAXS data. This causes a distortion of the lattice in their vicinity, resulting in a distribution of tensile and compressive forces. Indeed, this effect has also been observed for calcite biominerals\textsuperscript{31-33}, and for calcite single crystals occluding amino acids\textsuperscript{5} and 20 nm block copolymer micelles\textsuperscript{4}. Previous analysis of calcite/PSS crystals also yielded comparable data, which like the calcite/PSS-MA and calcite/PS-MA crystals, demonstrated preferential adsorption of the polymer on [001] planes\textsuperscript{36}. The absence of shifts in the peak positions demonstrates the absence of uniform macrostrains.
Considering then the mechanism of formation of the calcite/polymer crystals, our data again provides no evidence for the assembly of crystalline precursor particles. In showing that
calcite rhombohedra form before the characteristic ‘mesocrystal’ morphologies, and that these morphologies can also develop on overgrowth of calcite seed crystals, we demonstrate that the shape is not defined at nucleation. Instead, a morphological transition does not occur until the rhombohedra reach sizes of 0.5–1 μm. This can be explained by the change in the solution conditions with time. The morphologies of calcite crystals precipitated in the presence of soluble additives are determined by both kinetic and thermodynamic factors, where changes in the shape and separations of the atomic terraces reflect the macroscopic changes in the crystal morphology\textsuperscript{36,60}. Further, it has also been observed that polymeric additives can modify the crystallization pathway of calcite from the typical step flow at dislocations that dominates at supersaturations of SI = 0.8 (ref. 61) to two-dimensional nucleation and growth on the terrace\textsuperscript{62}. Two-dimensional nucleation is expected at the supersaturation levels used here, and is consistent with the crystal morphologies and mosaic blocks observed. As the polymer/Ca\textsuperscript{2+} ratio in the growth solution will increase with time because of the much higher rate of depletion of the Ca\textsuperscript{2+} ions than the polymer—while a near-constant supersaturation level is maintained using the ammonia diffusion method\textsuperscript{35}—the effect of the polymer will become more significant. The nanoparticulate surfaces observed towards the termination of crystal growth are likely to arise owing to the rapid decrease in supersaturation that occurs at this stage of the ammonia diffusion method. With high polymer/[Ca\textsuperscript{2+}] ratios and a low supersaturation, surface poisoning by the polymer additive will become more significant\textsuperscript{35}.
ACC has also been frequently observed at early stages of formation of calcite/polymer mesocrystals, suggesting that ACC may be an essential precursor phase. Indeed, growth of crystals by transformation of an amorphous precursor phase has marked parallels with the formation of biogenic mesocrystals such as sea urchin spines\textsuperscript{27}. Running counter to this argument, our data show that calcite crystals that morphologically resemble ‘classic’ mesocrystals can be precipitated in the presence of polymer, in solutions that are well below the saturation level of ACC. Further, these crystals can exhibit asymmetric morphologies and curved surfaces, demonstrating that such morphologies can actually occur by classical growth mechanisms. This is fully consistent with alternative studies that have demonstrated that CaCO\textsubscript{3} particles with morphologies identical to reported vaterite mesocrystals can be formed below the ACC supersaturation level and in the absence of additives through control of the supersaturation alone\textsuperscript{63}. These studies also demonstrated that supersaturation values orders of magnitude higher would be required to generate sufficient numbers of nanoparticles to support a growth mechanism based on the aggregation of crystalline nanoparticles\textsuperscript{64}. However, given that the majority of calcite/polymer crystals studied here were precipitated at an initial supersaturation level where ACC formation is expected, it is likely that a range of crystal growth mechanisms operate during their transition from nuclei to crystals of over 10 μm in size.
In conclusion, the detailed analysis of calcite crystals presented here provides a clear demonstration that great care needs to be taken when classifying particles as mesocrystals. Indeed, while observations of nanoparticulate surface structures, high surface areas, line broadening of PXRD spectra and characteristic morphologies are all routinely used to assign mesocrystal structure, none of these provide stand alone evidence for the nanoparticulate sub-structure that is now considered to define a mesocrystal\textsuperscript{27}. The misinterpretation of PXRD is particularly widespread, and we re-emphasize that the Scherrer equation cannot be used to estimate particle sizes when lattice strain is also present (as is often the case when crystals are co-precipitated with additives). We also reiterate that our work provides no evidence for the formation of these calcite/polymer crystals by the assembly of crystalline precursor particles, and that morphologies that are considered signatures for mesocrystals can also be generated in the absence of an ACC precursor phase. In moving on, it is therefore essential that researchers are rigorous in their analyses of crystals, and that the many articles describing not only CaCO\textsubscript{3} but also other types of mesocrystals are re-examined, especially where assembly-based formation mechanisms have been proposed.
**Methods**
**Materials.** CaCl\textsubscript{2}·2H\textsubscript{2}O, (NH\textsubscript{4})\textsubscript{2}CO\textsubscript{3} and CaCl\textsubscript{2}·6H\textsubscript{2}O were purchased from Sigma-Aldrich and were used without further purification. PSS-MA and PS-MA were purchased from Sigma-Aldrich and were used after purification by dialysis against copious amounts of water for 2 days to remove impurities, followed by freeze-drying.
**CaCO\textsubscript{3} precipitation.** Calcium carbonate was precipitated in the presence of the soluble anionic block copolymers PSS, PSS-MA and PS-MA. Two precipitation methods were used, such that CaCO\textsubscript{3} was precipitated using either the ammonium carbonate diffusion method or by a double decomposition method (precipitation from a metastable solution) where equal volumes of [(NH\textsubscript{4})\textsubscript{2}CO\textsubscript{3}] = 1–100 mM solution and [CaCl\textsubscript{2}·2H\textsubscript{2}O] = 0.2–5 mM solution were added in a Petri dish and then incubated at room temperature for 24 h. The ammonium carbonate method was principally used, and was referred to unless stated otherwise.
CaCO\textsubscript{3} was precipitated in the presence of the polymers in a polystyrene dish containing 10 ml of solution. Glass substrates were cleaned by soaking in Piranha solution (70:30 H\textsubscript{2}SO\textsubscript{4}:H\textsubscript{2}O\textsubscript{2}) before use, and were placed at the base of the dish. Block copolymers in polymer solutions of [PSS] = [PS-MA] = 0.5–3 mM were first mixed with polymer concentrations of 50–300 mg ml\textsuperscript{-1}. Precipitation of CaCO\textsubscript{3} was then initiated by placing the dish in a sealed desiccator containing a glass Petri dish of (NH\textsubscript{4})\textsubscript{2}CO\textsubscript{3} (5 g), which was covered with a piece of parafilm pierced four times with a needle. Crystallization was typically allowed to proceed for 1–2 days (depending on the otherwise). Following this period, the glass slides with the CaCO\textsubscript{3} crystals were removed from the solutions, washed with Millipore water and dried in air.
For comparison with the experiments generated in the presence of copolymers, CaCO\textsubscript{3} was also co-precipitated with cobalt (II) ions using the Kitano method\textsuperscript{65}. In brief, the solution was bubbled through CO\textsubscript{2} gas for 3–4 h before filtering (200 nm, Millipore filter) to remove any undissolved CaCO\textsubscript{3} particles. CO\textsubscript{2} gas was then bubbled through the solution for a further 10–20 min. CoCl\textsubscript{2}·6H\textsubscript{2}O was then added to the prepared Kitano solution to give a final Co(II) concentration of 0.01–2 mM, and precipitation was induced by opening the prepared solution to air to allow escape of CO\textsubscript{2} gas\textsuperscript{65}.
**Overgrowth on seed calcite crystals.** Overgrowth experiments were performed on seed calcite crystals either 3–5 μm or 10–30 μm in size. Rhombohedral calcite seed crystals were precipitated on mica or glass substrates using the ammonia diffusion method as described above. After brief washing and drying, the substrates supporting the crystals were transferred to a solution of 2.5 mM CaCl\textsubscript{2}·2H\textsubscript{2}O and the desired soluble polymer and precipitation was carried out via ammonium carbonate for 1 day, before isolating the crystals. The grown crystals were then scraped off the substrate using a razor blade dispersed in epoxy resin and subsequently cured on a silicon wafer. The specimen was then mechanically polished and was examined using optical microscopy and SEM.
**Precipitation of calcite crystals in droplet microgravity.** Experiments were performed using established methods\textsuperscript{66}. In brief, 20 nm Au films were deposited onto freshly cleaned glass substrates after pre-deposition of 2–5 nm of Cr to promote adhesion between the glass and the Au. The Au surfaces were then functionalized with hydrophobic self-assembled monolayers of 1H,1H,2H,2H perfluorodecane thiol (Sigma-Aldrich, 99%) by immersing the substrate in perfluorodecane thiolane of 0.1 mM perfluorodecane thiol at 1 atm for 24 h. The substrates were then immersed in deionized water and subjected to UV light source (λ ≤ 255 nm; hν = 4.8 eV) through a circular quartz photomask at a constant distance of 2 cm for 1 h. The mask was patterned with an array of 100–200 μm diameter circles with centre-to-centre spacings of 100 μm. Photoactivated alkylthiols were then displaced by back-filling it with methanol solutions of 0.1 mM mercaptoundecanoic acid (Sigma-Aldrich, 99%) for 1 h at 4°C before rinsing thoroughly in deionized water (18.2 MΩ cm; Milli-Q) and drying under a nitrogen gas stream.
CaCO\textsubscript{3} precipitation was then carried out within a sealed chamber in which the humidity was controlled at 10%. A solution of [CaCl\textsubscript{2}·2H\textsubscript{2}O] = 1–5 mM and [PSS-MA] = 125–500 mg l\textsuperscript{-1} was placed on a freshly cleaned substrate, resulting in the formation of particle-volume domains across the hydrophilic domain. The substrate was then placed in the equilibrated humidity chamber along with 0.1 g solid ammonium carbonate, and precipitation was allowed to proceed for up
to 30 min, before removing the substrate, washing with ethanol and air-drying. Different growth stages were assessed by analysing particles present within different droplets at the same time, and over a range of times.
**Determination of supersaturations.** The supersaturations of the crystallization solutions were calculated using visual Minitc software, where the supersaturation index is defined as:
\[
SI = \ln \frac{IAP}{K_{sp}}
\]
(1)
where IAP = ion activity product, \( K_{sp} \) = solubility constant, \( K_{sp} \) (calcite) \(= 10^{-8.48} \) and \( K_{sp} \) (ACC) \(= 10^{-6.39} \).
The actual Ca\(^{2+}\) concentrations in the reaction solutions containing PSS-MA polymers were measured with a Ca\(^{2+}\)-selective electrode (Metrohm). All solutions were freshly prepared before each experiment and kept fresh. Milli-Q water (resistivity 18.2 MΩ cm at 20 °C) and the electrode were calibrated with the standard CaCl\(_2\)·2H\(_2\)O solutions with concentrations in the range 0.5–10 mM. The desired concentrations of pure CaCl\(_2\)·2H\(_2\)O solutions were first measured, then CaCl\(_2\)·2H\(_2\)O solutions containing PSS-MA polymer were measured after stirring for 30 min. The (Davies-extended) Debye–Hückel equation was used to convert the measured Ca\(^{2+}\) activities into concentration for determination of the activity coefficients.
**Characterization of CaCO\(_3\) particles.** The CaCO\(_3\) particles were analysed using SEM, high-resolution TEM (HRTEM), optical microscopy, Raman microscopy, infrared spectroscopy, thermogravimetric analysis, surface area analysis (BET), atomic absorption spectroscopy and EBSD. Selected samples were also analysed using synchrotron PXRD and SAXS. For SEM, particles were typically transferred onto glass coverslips that had a sufficient amount of carbon coating applied using carbon stick pads. Samples were then coated with 5 nm Pt/Pd using an Agar High Resolution Sputter Coater and were examined using a LEO 1350 Gemini FEG-SEM operating at 3 kV using an in-lens detector mode. For TEM analysis, an FEI Tecnai TF20 FEG-TEM fitted with Oxford Instruments INCA 350 EDX system/80 mm X-Max energy dispersive spectrometer (EDS) operated at 200 kV and 100 kV was used. Raman microscopy and infrared spectroscopy were used to further confirm the polymorph and internal structure change of crystal particles with Raman being carried out using a Renishaw 2000 Raman microscope operating with a 785-nm diode laser, and infrared being performed with a PerkinElmer ATR-IR. The Co content of samples was analysed using a PerkinElmer Atomic Absorption Spectrometer AAnalyst 400 with an air-acetylene flame after dissolving samples in dilute HNO\(_3\).
**Surface area analysis (BET).** Surface area analysis was conducted using N\(_2\) adsorption with a Micromeritics-Tristar 3000 after degassing for 2 h and/or heating up to 80 or 300 °C. The surface area was calculated from the linear part of the BET plot while the pore-size distribution was determined using the Barret–Joyner–Halenda model. For multi point (5 point) BET measurements, the data were treated according to the absorption isotherm given in equation (1):
\[
\frac{1}{V'[(P_0/P) - 1]} = \frac{1}{V_m C} + \frac{C - 1}{V_m C} \left( \frac{P}{P_0} \right)
\]
(2)
where \( P \) = partial vapour pressure of adsorbate gas in equilibrium with the surface at 77.4 K (boiling point of liquid nitrogen), in pascals, \( P_0 \) = saturated pressure of adsorbate gas, in pascals, \( V' \) = volume of gas adsorbed at standard temperature and pressure (STP), 15 K and atmospheric pressure (1.013 × 10\(^5\) Pa), in millilitres, \( V_m \) = volume of gas adsorbed at standard temperature and pressure to produce an apparent monolayer on the sample surface, in millilitres, \( C \) = dimensionless constant that is related to the enthalpy of adsorption of the adsorbate gas on the powder sample. A value of \( V \) was measured at each of 5 values of P/Po. Then, the BET value:
\[
\frac{1}{V'[(P_0/P) - 1]}
\]
(3)
was plotted against \( P/P_0 \), according to equation (2). This plot should yield a straight line in the pressure range \( \approx 0.05–0.3 \). The data are considered acceptable if the correlation coefficient of the line is not \( < 0.9975 \); that is, \( r^2 \) is not \( < 0.995 \) from the resulting linear plot, where the slope and intercepts are obtained as:
\[
\text{slope } s = \frac{C - 1}{V_m C}
\]
(4)
\[
\text{Intercept } i = \frac{1}{V_m C}
\]
(5)
From this value, \( V_m \) is calculated as:
\[
V_m = \frac{1}{s + i}
\]
(6)
From the value of \( V_m \) determined, the total surface area \( S_t \) is calculated by the equation:
\[
S_t = \frac{V_m N_{av} A_w}{M_c}
\]
(7)
\( N_{av} = 6.022 \times 10^{23}, A_w = 0.162 \text{ nm}^2, M_c = 22.414 \text{ ml} \).
Finally, the specific surface area \( S \) is obtained as:
\[
S = \frac{S_t}{m}
\]
(8)
where \( m \) = the mass of sample used.
**FIB and HRTEM analysis.** Images of the lattice structure of the calcite particles were obtained using HRTEM imaging of thin sections prepared by FIB Milling\(^8\). FIB to electron transparency was performed using an FEI Dual Beam system equipped with a 30 kV Ga beam and a field emission gun operated at 5 kV. The samples were then analysed with a FEI Tecnai TF20 FEG-TEM fitted with an Oxford Instruments INCA 350 EDX system/80 mm X-Max SDD detector and a Gatan Orius SC600/1 CCD camera operating at 100 kV. In order to minimize electron beam related damage during the recording of images and diffraction patterns recording, a 10-μm condenser lens and the smallest spot size were used.
**Synchrotron PXRD studies.** The high-resolution PXRD measurements were carried out at the dedicated high-resolution powder diffraction beamline (111) at the Diamond Synchrotron Radiation Facility (Diamond Light Source Ltd, Didcot, UK). The beamline is equipped with a crystal monochromator (a liquid nitrogen-cooled double-crystal silicon monochromator) and a crystal analyser at the incident and diffracted beams, respectively. The optics of the diffracted beam consists of nine (111) Si crystal analysers and the use of the advanced analysing optics yielded diffraction spectra of superior quality that contained intense and extremely narrow diffraction peaks with an insignificant contribution to the peak width, not exceeding 0.004°\(^2\) (ref. 66). Instrument calibration and wavelength refinement were performed with silicon standard samples from the National Bureau of Standards and Technology (NIST; Gaithersburg, MD, USA). Powders for analysis were loaded into 0.7 mm borosilicate glass capillaries, and void intensities arising from internal capillary walls in the capillaries prevented diffraction measurements at a rate of 0.9 r.p.s. using high-resolution multi-analyser crystal diffraction scans, with scan times of 1,800 s. Spectra were recorded both at room temperature and after *in situ* heating of specimens to temperatures of 100, 200 and 300 °C for 30 min using an internal heater.
**XRD analysis.** The structural parameters were refined by Rietveld analysis both using GSAS and using PANalytical XPert HighScore Plus software. There was no evidence of amorphous material, as indicated by a flat baseline in the entire range \( 2\theta = 3–150^\circ \). Strain and size analysis was performed using both Rietveld analysis and line profile analysis. In order to quantify the broadening, Williamson–Hall plots were also prepared. Using this technique, B cos\(\theta\) was plotted against sin\(\theta\) for the [104], [001] and [100] families of planes. The average microstrains and size effects (or coherence lengths) of each sample were determined from the gradient and intercept of the plot, respectively.
**Rietveld size/strain analysis.** At least 20 reflection peaks were refined using a pseudo-Voigt function and the size and strain analysis was plotted using the Caglioti equation\(^{67}\):
\[
\text{FWHM} = \sqrt{(U \tan \theta + V \tan \theta + W)}
\]
(9)
The \( U \) parameter contains the information about the strain broadening, whereas the \( W \) parameter corrects for a possible size broadening. Microstrain and size are then obtained using the following algorithm:
Microstrain is described by:
\[
\varepsilon (\%) = \frac{\sqrt{(U - W)}}{(19)} \frac{1}{4\sqrt{2}} \ln 2 \times 100
\]
(10)
Crystallite size is described by:
\[
D(A) = \left( \frac{180}{\pi} \right) \frac{\lambda}{\sqrt{W}}
\]
(11)
**Williamson-Hall analysis.** The XRD data was also analysed by combining a Williamson–Hall plot with line profile analysis. This method uses the integral breadth as a measure of the peak width and the universal shape factor \( \Phi \) to describe the peak shape variation. These numbers can be determined for single peaks as well as for a whole range of peaks. The algorithm then empirically deconvolutes the profile into a Gaussian and a Lorentzian integral breadth: This is defined as net peak area/peak height, and is equal to a rectangle with the same height and the same area as the peak. The integral FWHM (2\(\theta\)) is defined as the full width at half-maximum intensity of the peak. The shape factor parameter is defined as FWHM/integral breadth. The sample contribution to the total broadening is calculated using an empirical convolution of the Gaussian and the Lorentzian parts,
where the Lorentzian broadening \((B_L)\) is given as:
\[
B_{\text{Lorentzian}} = (2.0207 - 0.4803\Phi - 1.7756\Phi^2) \times \text{integral breadth}
\]
(12)
The Gaussian broadening \((B_G)\):
\[
B_{\text{Gaussian}} = \left(0.6420 + 1.4187\sqrt{\Phi} - 2/\pi - 2.22043\Phi + 1.8706\Phi^2\right) \times \text{integral breadth}
\]
(13)
In order to construct the W–H plot, both Gaussian and Lorentzian broadenings are combined using the empirical convolution, where \(B\) cost\(\theta\) is plotted against sin\(\theta\).
\[
B \cos\theta = \frac{K\lambda}{\text{Crystallite size}} + \text{Strain} \times 4 \sin\theta
\]
(14)
The strain(%) is then obtained from the slope \((= 4 \times \text{strain})\).
The crystallite size (\(\AA\)) is obtained from:
\[
\text{Intercept} = \frac{K\lambda}{\text{Crystallite size}}
\]
(15)
A shape factor of \(K = 1\) was used.
**Scherrer size analysis.** The crystal size was also estimated using the Scherrer equation:
\[
D = \frac{K\lambda}{B \cos\theta}
\]
(16)
\(B = \text{FWHM or integral breadth and a shape factor (or Scherrer constant) of } K = 1\) was applied.
The integral breadth and FWHM as a measure of the peak width of selected peaks were used in the Scherrer equation. The individual peaks were fitted by line profile fitting as described above.
**Small-angle X-ray scattering.** SAXS profiles of powdered samples (control calcite: Co\(^{2+}\)-doped calcite, seeded calcite/PS-MA crystals and calcite/PS-MA crystals) were recorded using a Nanostar Instrument (Bruker AXS, Karlsruhe, Germany) equipped with a single-photon counting area detector (H\(\bar{I}\)STAR, Bruker AXS). The X-ray generator had an anode radius with a wavelength of 1.54 Å (Cu-K\(\alpha\)), and operated at 40 kV and 15 mA, while a parabolic bent grided multilayer (Golm) mirror was used to ensure a parallel and monochromatic X-ray beam. All specimens were measured in borosilicate glass capillaries at a sample-detector distance of 105 cm. Scattering data for the polymer-containing particles were additionally recorded at a smaller sample-detector distance of 26 cm in order to cover the range of scattering vectors for the polymers. The normalized integrated profiles of the scattering intensity versus the modulus of the scattering vector \(Q (Q = 4\pi \sin(\theta)/\lambda)\), where \(2\theta\) is the scattering angle and \(\lambda\) represents the wavelength of the incident beam) were corrected for instrument-related background and sample transmission. \(Q\) was set to scattering attributable to the sample containing the Laue backscattered \((L_{\text{back}})\) origin from a Poisson fit of the profiles \((I(Q) = I_0 + L_{\text{back}} + P\), being the Poisson fit of the profile). For the crystal/polymer samples a \(T\)-parameter analysis was performed according to the method described by Fratzl et al.\(^{49}\) However, due to the strong scattering contribution of the large external surfaces of the powder grains in the low \(Q\) limit \((Q < 0.3 \text{ nm}^{-1}\) for calcite/PS-MA crystals and \(Q < 0.25 \text{ nm}^{-1}\) for calcite/PS-MA crystals), data points in this \(Q\)-range were not included in the experimental integration of the Kratky curve \((I(Q), Q^2 \text{ versus } Q)\), but were approximated by a rectangle curve.
**Electron backscatter diffraction.** EBSD requires that samples have highly smooth surfaces, where this was achieved here by sectioning and polishing the crystals with an ultramicrotome (Leica). The surface of the sample was then coated by 4–6 nm of carbon. EBSD maps were obtained at 15 and 20 kV on a FEI-SEM (JEOL JSM 6400) equipped with an Oxford Instruments NordlysNano EBSD detector and interpreted by CHANNEL 5 EBSD software.
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**Acknowledgements**
This work was supported by an Engineering and Physical Sciences Research Council (EPSRC) Leadership Fellowship (to F.C.M., Y.Y.K. and H.L., EP/H00574/1), and EPSRC grant EP/K008304/1 (to F.C.M. and A.S.S.). F.C.M. and A.S.S. are also supported by an EPSRC Programme Grant (grant EP/I01014/1) that funds the Materials Interface with Biology consortium. E.G. is supported by DFG grant GR1235/9-1. The PXRD work was carried out with the support of the Diamond Light Source. We would like to thank Dr Tim Comyn (Institute for Materials Research, University of Sheffield) for helpful advice and discussion regarding the XRD experiments, and Dr Andrew D’Arcy and Dr Jeff Krause (Micromeritics Instrumentation Corp. Norcross, GA USA) for their helpful discussion on BET analysis and offering additional measurements of samples.
We are also grateful to Professor Peter Fratzl and Dr Barbara Achhammer (MPI, Colloids and Interfaces, Golm) for providing access to SAXS equipment and for fruitful discussions about the SAXS data, and Ingrid Zenker for running some of the SAXS analyses. We would like to thank Dr Andrew D’Arcy (Ganil Facility for Electron Microscopy, University of Ulm) for his help with obtaining crystals for EBSD with a micrometre. We would also like to thank other previous and current members of the Meldrum group for their valuable input and discussion over the many, many years occupied by this study.
**Author contributions**
Y.-Y.K. led the experimental work, preparing samples and carrying out TEM, BET and XRD analyses. A.S.S. performed the SAXS experiments, analysed the SAXS data and participated in sample preparation for EBSD. J.L. participated in the BET study of surface areas, whereas A.N.K. carried out the FIB preparation of samples and assisted with the BET analysis. C.C.T. provided access to Beelime 111 at Diamond and assisted with the XRD experiments. G.H. assisted with analysis and discussion of the XRD data. E.G. and W.W.S. performed the EBSD studies and analyses; N.B.J.H. performed early studies that inspired this work; F.C.M. originated and supervised the project. All authors contributed to the preparation of the manuscript.
**Additional information**
Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications
**Competing financial interests:** The authors declare no competing financial interests.
**Reprints and permission** information is available online at http://npg.nature.com/reprintsandpermissions/
**How to cite this article:** Kim, Y.-Y. *et al.* A critical analysis of calcium carbonate mesocrystals. *Nat. Commun.* 5:341 doi: 10.1038/ncomms3541 (2014).
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b) in returning to his normal and regular place of residence following the conclusion of each Hockey Season; and
c) during the Christmas holiday season of each Hockey Season for one return trip from the city where the Club is located to the Player's normal and regular place of residence.
2.5 The Club will, during the Term of this Agreement, pay or reimburse or cause to be paid, as the case may be, all reasonable expenses associated with the Player's enrollment in a high school or a post-secondary educational institution during the fall and winter term, in the city where the Club is located, including, but not limited to, the reasonable expenses related to tuition fees, compulsory student fees (excluding premiums for health services, which may include medical and dental insurance fees) and textbooks directly related to the Player's course of study (including any applicable sales taxes and goods and services taxes). The obligation of the Club under this paragraph 2.5 to pay or reimburse or cause to be paid, as the case may be, the Player's reasonable expenses related to tuition fees, compulsory student fees (excluding premiums for health services, which may include medical and dental insurance fees) and textbooks shall be limited to an amount which reflects, as a benchmark, the reasonable expenses of a Player attending a mainstream general program of study (mainstream general program of study to be defined as an undergraduate arts, science or general studies program). The Club will, during the Term of this Agreement, also pay or reimburse or cause to be paid, as the case may be, the reasonable expenses
associated with retaining qualified tutors and educational advisors, as deemed reasonably necessary, to assist the Player in his academic studies.
ARTICLE 3 – WHL SCHOLARSHIP PROGRAM – GRADUATE PLAYER
3.1 a) i) Subject to the provisions of paragraphs 3.1(d) and 3.2, but in addition to any benefits the Player may receive pursuant to the provisions of paragraph 2.5 or otherwise under this Agreement, the Player will receive, the benefit of the WHL Scholarship Program pursuant to which the Club will provide financial assistance to the Player in respect of the Player's educational costs to enroll in and attend a post-secondary educational institution as a full time student following completion of the Player's WHL playing career. The Player will have qualified for and the Club shall provide financial educational assistance to the Player, in accordance with this paragraph 3.1, for one half of an academic year if the Player is on the Club's roster at any time on or after October 10 of any Hockey Season; the Player will have qualified for and the Club will provide financial educational assistance to the Player, in accordance with this paragraph 3.1, for one academic year if the Player is on the Club's roster at any time on or after January 10 of any Hockey Season; provided however, the obligation of the Club to provide the Player with financial assistance in accordance with this paragraph 3.1 will be limited to a maximum of one academic year for each Hockey Season or portion thereof played in the WHL to a maximum of five (5) years, regardless of the number of Hockey Seasons or portions thereof that the Player has played in the WHL.
ii) Educational costs shall, subject to paragraph 3.2, include the costs and expenses of tuition fees, compulsory student fees and textbooks directly related to the Player's course of study, including any applicable sales taxes and goods and services taxes to attend, as a full time student, the publicly funded post-secondary educational institution designated by the Player (in accordance with the WHL Standard Player Agreement) in a province of Canada or a state of the United States of America where the Player normally and regularly resides (the "designated post-secondary educational institution"). In the event the Player enrolls in and attends a post-secondary educational institution other than the designated publicly funded post-secondary educational institution, then the obligation of the Club under this paragraph 3.1 to provide financial education assistance shall, subject to paragraph 3.2, be limited to an amount which does not exceed expenses of a similar program of study at the publicly funded post-secondary educational institution designated by the player.
b) Amounts payable by the Club for tuition fees and compulsory student fees shall be limited to the amount published in the official school handbook, calendar or other relevant publication of the designated post-secondary educational institution for the academic year in which the Player is enrolled in and attends a post-secondary educational institution, subject to the provisions of paragraph 3.2. Upon receipt by the Club of evidence of the Player's enrollment in a post-secondary educational institution together with an invoice from that institution for tuition and compulsory student fees, the Club will, subject to the provisions of paragraphs 3.1(a), 3.1(b) and 3.2, pay or reimburse or cause to be paid, as the case may be, such tuition and compulsory student fees directly to the post-secondary educational institution by the date set forth in the invoice. Refunds or reimbursements, if any, to the Player applicable to tuition or compulsory student fees which were paid by the Club, resulting from the Player's withdrawal from the post-secondary educational institution or from classes or failure of the student to maintain an acceptable passing grade in the course of his studies, will be made by the post-secondary educational institution directly to the Club, and if made by the post-secondary educational institution to the Player, will be repaid by the Player to the Club within thirty (30) days of the Player's receipt of same from the post-secondary educational institution.
c) Subject to the provisions of paragraph 3.2, the Club will pay or reimburse or cause to be paid, as the case may be, the Player for school textbook expenses directly related to the Player's course of study, together with any applicable sales taxes and goods and services taxes, within thirty (30) days of the Player providing appropriate receipts therefore to the Club. The maximum reimbursement for the expenses of school textbooks will be based on the estimate for the Player's program of study as outlined in the official school handbook, calendar or other relevant publication of the designated post-secondary educational institution for the academic year in which the Player is enrolled in and attends a post-secondary educational institution, subject to the provisions of paragraph 3.2; if such an estimate is not available in the official school handbook, calendar or other relevant publication of the designated post-secondary educational institution then the maximum reimbursement of such expenses will be based on information obtained by the Club from the designated post-secondary educational institution.
d) The Player will be permitted to play an unlimited number of hockey games in certain hockey leagues which the WHL has, in its sole discretion acting reasonably, designated as professional development hockey leagues without affecting the Player's eligibility to participate in the WHL Scholarship Program pursuant to this paragraph 3.1. The WHL will identify the hockey leagues which will be designated as professional development hockey leagues for the purpose of this Agreement. The Player shall not, however, be eligible for the benefits contained in this paragraph 3.1 if:
i) the Player has executed a professional hockey playing contract with a team in the National Hockey League, a team in the American Hockey League (but excluding an American Hockey League tryout contract under which the Player does not play more than twenty-five (25) games in the American Hockey League) or a professional hockey team in Europe; or
ii) in the hockey season immediately following completion of the Player's eligibility to play in the WHL as a 20 year old, the Player has played more than twenty-five (25) hockey games in the American Hockey League or has played any games in any other professional hockey league which has not been designated by the WHL as a professional development hockey league or
iii) the Player fails by September, after one full academic year or hockey season following completion of his eligibility to play in the WHL as a 20 year old, to enroll in and attend a post-secondary educational institution as a full time student; or
iv) the Player fails by September after two full academic years or hockey seasons following completion of his eligibility to play in the WHL as a 19 year old, to enroll in and attend a post-secondary educational institution as a full time student; or
v) the Player fails at any time to enroll in, attend and maintain the status of a full time student during the fall and winter academic semesters at a post-secondary educational institution in consecutive academic years following the academic year the Player first utilizes the benefits of the WHL Scholarship Program, provided however, the Club may, in consultation with the WHL, upon the written request of the Player, permit the Player to extend the benefit period under paragraph 3.1(a)(i) by permitting the Player to attend a post-secondary institution on a part time basis or in non-consecutive academic years. The Club shall not, however, be liable to the Player for any increase in educational costs due to such extension notwithstanding the consent of the Club to extend the benefit period under paragraph 3.1(a)(i).
e) If the Player, while on the Club's active roster, suffers a career ending injury while participating either in:
i) any hockey game under paragraph 4.1, or
ii) in any sanctioned event or activity,
then notwithstanding the provisions of paragraphs 3.1(a)(i), 3.1(d)(iii) and 3.1(d)(iv), the Club shall pay or reimburse or cause to be paid, as the case may be, the Player's educational costs as provided for in paragraph 3.1(a)(ii) for a maximum of four (4) academic years (unless the Player has qualified for five (5) years of financial educational assistance in accordance with the provisions of paragraph
3.1(a)(i)) in accordance with and subject to the provisions of paragraphs 3.1(a), 3.1(b), 3.1(c) and 3.2 to enroll in and attend a post-secondary educational institution, whether as a part time or full time student.
3.2 It is acknowledged that, for the purposes of paragraph 3.1, there are certain post-secondary educational institution programs of study that are outside of the mainstream of general study with the result that such programs have higher academic costs and expenses associated with them; if the Player enrolls in such a program, the obligation of the Club under paragraph 3.1 to pay or reimburse or cause to be paid, as the case may be, the Player's reasonable expenses associated with such a program shall be limited to an amount which reflects, as a benchmark, the reasonable expenses of a Player attending a mainstream general program of study (mainstream general program of study to be defined as an undergraduate arts, science or general studies program), at a publicly funded post-secondary educational institution designated by the Player in accordance with the WHL Standard Player Agreement in the province or state where the Player normally and regularly resides. It is also understood that compulsory student fees may vary depending on the post-secondary educational institution at which the Player may enrol. For the purpose of this agreement, the obligation of the Club under paragraph 3.1, to pay or reimburse or cause to be paid, as the case may be, all compulsory student fees, excludes premiums for health services, which may include medical and dental insurance fees.
ARTICLE 4 - OBLIGATIONS OF THE PLAYER
4.1 The Player will, during the Term of this Agreement, provide his services as a hockey player except as a result of an injury or with the prior consent of the Club and the WHL, in accordance with the following:
a) except as hereinafter specifically provided in this paragraph 4.1, exclusively for the Club and shall play for the Club in all the Club's exhibition, preseason, regular season and playoff games and all tournament games for the Memorial Cup,
b) in the WHL and Canadian Hockey League (the "CHL") all-star game(s), the CHL Top Prospects Game, or other sanctioned events or activities scheduled or organized by the WHL or CHL,
c) if selected by the Hockey Canada or any other like national governing hockey association in Europe or the United States of America, in the annual World Junior Hockey Championships and all tryouts and team practices leading to such championship, in accordance with release dates and conditions agreed to by the WHL,
d) if selected by any provincial governing hockey association or any like governing hockey association in Europe or the United States of America, in the Under-17 World Hockey Challenge Tournament and the Under-18 World Hockey Championship Tournament and all tryouts and team practices leading to these tournaments, in accordance with release dates and conditions agreed to by the WHL, and
e) at the request of the WHL or the CHL, in other hockey games or tournaments as may be sanctioned by the WHL or the CHL including, without limitation, the Canada Winter Games.
4.2 The Player covenants and agrees:
a) to report, in good physical condition, to the Club's training camp prior to the commencement of the Hockey Season at the time and place designated by the Club;
b) to keep and maintain himself in good physical condition at all times throughout the Hockey Season;
c) at the request and direction of the Club, to cooperate and participate in reasonable promotional activities sponsored by the Club, the WHL or the CHL;
d) to conduct himself at all times, both on and off the ice, in a manner consistent with good standards of honesty, decency, morality, and fair play, and not to conduct himself at any time in any manner that would be detrimental to the well-being of the Club, the WHL, the CHL, Hockey Canada, USA Hockey, or any like provincial, state or federal governing hockey association in Canada, Europe or the United States of America, or hockey in general;
e) to abide by the rules and regulations, directions and instructions governing conduct and behaviour reasonably established by the Club from time to time and applicable to all its players including, without limitation, rules, regulations, directions, and instructions governing the use of tobacco, drugs and alcohol, attendance at school, conduct on and off the ice, curfew, community service and training;
f) to abide by the rules and regulations, directions and instructions governing conduct and behaviour reasonably established by any provincial governing hockey association in Canada, Hockey Canada, USA Hockey, or any like governing hockey association in Europe or the United States of America applicable to all its players participating in the World Junior Hockey Championship, the Under-17 World Hockey Challenge Tournament, the Under-18 World Hockey Championship Tournament and the Canada Winter Games and related events or other like events, including, without limitation, all tryout camps and team practices leading to such events;
g) to participate, at the request of the Club and the WHL, in events or activities organized, scheduled or sanctioned by the WHL or the CHL;
h) to maintain a valid passport for purposes of facilitating customs and immigration processes during the course of the Hockey Season;
i) to execute a Hockey Canada major junior hockey playing card and any other registration card as may be required by the WHL, including, if applicable, a USA Hockey playing card;
j) to provide his services faithfully, diligently and to the best of his abilities as a hockey player;
k) not to engage in hazardous activities or avocations including, without limitation, racing (automobile, go-kart, motorcycle, boat, snowmobile or ski), diving (scuba or sky), parachuting, snow skiing, snow boarding or aviation other than as a passenger, during the Term of this Agreement without the prior consent of the Club; and
l) without the prior written consent of the Club, not to participate in hockey games that are not sanctioned by the WHL.
4.3 The Player irrevocably transfers, conveys and assigns to the Club and the WHL for the Term of this Agreement all rights to the Player’s image. The Player agrees that the Club and/or the WHL may authorize or otherwise license any individual, firm or corporation to take and produce photographs, pictures, films, video or other images of the Player. The Player recognizes that all rights to his image during the Term of this Agreement shall be the sole and exclusive property of the Club and the WHL. The Club and the WHL may use or distribute such photographs, pictures, films, video or other images of the Player for the promotion of the Club, the WHL and the CHL in any manner as the Club or the WHL may reasonably see fit and that such use by the Club, the WHL and/or the CHL may take place during the Term of this Agreement and any time thereafter.
4.4 a) The Player consents and agrees to the use by or on behalf of the Club, the WHL and the CHL and their respective agents, licensees, contractors, successors and assigns, of the name, image, photograph, likeness, statistical record and biographical information of the Player including, without limitation, the use of same by the WHL and the CHL in connection with the manufacture, sale, distribution, marketing and advertising of WHL and/or CHL hockey cards and/or other souvenir material relating to the Club, the WHL or the CHL; in connection therewith, the Player agrees, during the Term of this Agreement, to attend at photograph and film sessions and to pose from time to time in his hockey equipment for pictures and films as may reasonably be required by or on behalf of the Club, the WHL and/or the CHL and their respective agents, licensees, contractors, successors and assigns. The Club, the WHL and the CHL, as applicable, shall pay or reimburse or cause to be paid, as the case may be, the reasonable costs and expenses incurred by the Player to attend such photograph or film sessions.
b) The right to use the Player’s name, image, photograph, likeness, statistical record and biographical information in connection with the WHL and/or CHL hockey cards and/or other souvenir material relating to the Club, the WHL and/or the CHL shall, during the Term of this Agreement and any time thereafter, be the sole and exclusive property of the Club, the WHL and the CHL.
4.5 The Player agrees not to use his own name, image, photograph, likeness, statistical record and biographical information in conjunction with logos, trademarks or copyrights of the Club, the WHL or the CHL, without the prior written consent of the Club, the WHL or the CHL, as applicable.
4.6 Except as provided in this Agreement, the Player will not, during the Term of this Agreement, be restricted from otherwise utilizing his own name, image, photograph, likeness, statistical record or biographical information provided such use does not conflict with the business affairs of the Club and the WHL. Except as provided in paragraphs 4.4 and 4.5, at the expiration of the Term of this Agreement, the Player shall not be restricted from utilizing his own name, image, photograph, statistical record or biographical information in any marketing or advertising materials.
4.7 Except as provided in this Article 4, the Club, the WHL and the CHL shall not utilize the name, image, photograph, likeness, statistical record or biographical information of the Player in connection with any commercial endorsements of particular products, services, firms or corporations, without the prior written consent of the Player.
ARTICLE 5 - OBLIGATIONS OF THE CLUB
5.1 The Club covenants and agrees:
a) to provide the Player in each Hockey Season with professional coaching and training in the fundamentals of hockey together with supervised training periods and other assistance the Club deems necessary, acting reasonably, to enable the Player to develop his hockey playing skills and abilities;
b) to provide the Player with room and board accommodation, during the Hockey Season, in the city where the Club is located; such accommodation will be subject to approval of the Player’s parents or guardian, as applicable;
c) to sign the Player to a Hockey Canada or USA Hockey major junior hockey playing card, as applicable;
d) to provide the Player during the Hockey Season with full WHL officially licensed hockey-playing equipment, including sticks, skates, and other miscellaneous hockey equipment reasonably necessary for playing the game of hockey and for the safety of the Player;
e) to provide the Player with travel, accommodation and meals when traveling with the Club for away games during the Hockey Season;
f) to provide the Player with regular medical attention, as required, for the assessment and rehabilitation of injuries which the Player may sustain during the Hockey Season;
g) to provide the Player with out of country medical coverage;
h) to provide the Player with medical and dental insurance coverage through the Hockey Canada national insurance program, for hockey related injuries;
i) to provide the Player, upon request, with a copy of the Hockey Canada national insurance manual outlining coverage applicable to the Player;
j) to retain, as required by paragraph 2.5, qualified tutors and educational advisors, as deemed reasonably necessary, to assist the Player in his academic studies;
k) to cause the Club’s representatives to conduct themselves, at all times, both on and off the ice, in a manner consistent with good standards of honesty, decency, morality and fair play; and
l) to make available to all professional hockey organizations and others, during and at the completion of the Player’s eligibility to play in the WHL, all relevant data, information and statistics reasonably required to enable the Player to pursue or initiate a professional or other hockey career.
5.2 The Club shall not provide nor does it undertake to provide the Player with any disability insurance coverage; any disability insurance coverage of the Player shall be obtained by the Player and shall be the sole and exclusive responsibility and obligation of the Player and shall be at the Player’s own cost and expense should the Player desire such coverage.
ARTICLE 6 - MEDICAL EXAMINATIONS
6.1 Prior to the commencement of each Hockey Season and from time to time, at the request of the Club, acting reasonably, during the Hockey Season, the Player will submit to and undergo a thorough medical examination with a qualified physician approved by the Club. If, as a result of such medical examination, it is the opinion of the physician acting reasonably, that the Player is not medically fit to play the game of hockey other than as a result of an injury sustained by the Player to which the provisions of paragraphs 11.2 and 11.3 are applicable, then the Club will have the option of terminating this Agreement on written notice to the Player personally delivered by the Club to the Player within seven (7) days following such examination; the Club will set out in the notice the reasons why the Club has terminated this Agreement and will include with the notice a copy of the physician's report and the physician's professional qualifications. Upon such termination of this Agreement by the Club, the provisions of paragraph 10.2 will apply.
6.2 The Player may at any time, acting reasonably, obtain, at his cost, any medical assessments of an injury that the Player deems necessary; the Club will assist the Player in obtaining any such medical assessments.
ARTICLE 7 – CLUB RULES AND SANCTIONS
7.1 The Club may, from time to time, acting reasonably in accordance with guidelines approved by the WHL, establish rules applicable to all the Club’s players, governing the conduct, behavior and physical condition of the Club’s players generally. Such rules will be provided by the Club to the Player and will
form part of this Agreement. The Club may, for any material violation by the Player of such rules, with the prior written approval of the WHL, either:
a) impose a suspension, in accordance with guidelines approved by the WHL, whereby the Player will be suspended, from further play with the Club. During the period of such suspension the Player will not be entitled to receive the Allowance provided for under the provisions of this Agreement, or
b) impose further sanctions, in accordance with guidelines approved by the WHL, as deemed necessary by the Club.
In imposing any suspension or sanction, the Club and the WHL shall at all times act reasonably having regard to the degree of severity of the violation by the Player, the suspensions and sanctions historically imposed by the Club and the WHL on its players for similar violations and the guidelines approved by the WHL.
7.2 The Player acknowledges that the Club has the authority to carry out any order or directive of suspension or expulsion rendered against the Player by the WHL, the CHL, Hockey Canada, USA Hockey, or any like provincial, state or federal governing hockey association in Canada, Europe or the United States of America. In the case of a suspension, at the discretion of the Club, the Allowance of the Player will cease to be paid during the period of the suspension. In the case of an expulsion from the WHL, this Agreement may, at the option of the Club, be terminated on written notice to the Player personally delivered by the Club to the Player within seven (7) days following such expulsion; the notice will set out in reasonable detail the reasons why the Club has terminated this Agreement. Upon such termination of this Agreement by the Club, the provisions of paragraph 10.2 will apply.
ARTICLE 8 - ASSIGNMENT
8.1 a) Except as provided in paragraph 8.2 and subject to the regulations of the WHL in place from time to time, the Club will have the right to assign, trade or otherwise transfer this Agreement to any other member franchise of the WHL (the "Assignee").
b) Upon any assignment, trade or other transfer of this Agreement to the Assignee, each of the Player and the Assignee will be bound by and will dutifully fulfill their respective obligations under this Agreement as if the Player and the Assignee were original parties to this Agreement; further,
i) the Club will be and remain liable to the Player under paragraph 3.1 for financial assistance which the Player is qualified to receive pursuant to paragraph 3.1(a)(i) prior to the date of such assignment, trade or other transfer of this Agreement, and
ii) the Assignee will be and remain liable to the Player under paragraph 3.1 for financial assistance which the Player is qualified to receive pursuant to paragraph 3.1(a)(i) subsequent to the date of such assignment, trade or other transfer of this Agreement.
For greater certainty, if the Player is on the Assignee's roster at any time on or after January 10 of any year, then the Assignee will be and remain liable to the Player in accordance with paragraph 3.1 for one academic year, notwithstanding that the Player was not on the Assignee's roster at any time prior to January 10 of that year. In the event of such assignment, trade or other transfer of this Agreement, the Player will receive the benefits under paragraph 3.1(a)(i) firstly from the Club and at such time when those benefits for which the Club is liable to the Player have been fully utilized by the Player, the Player will then receive the benefits under paragraph 3.1(a)(i) from the Assignee for which the Assignee is liable to the Player, in accordance with WHL Regulations.
8.2 The Club will not, during the term of this Agreement, assign, trade or otherwise transfer this Agreement during the Christmas period of any Hockey Season with the dates to be determined each year by the WHL.
8.3 This Agreement shall be binding and remain in full force and effect for the Term of this Agreement, even if the WHL should change its name, or withdraw from membership in the CHL or Hockey Canada. In the event the Club ceases to operate or to be a member franchise of the WHL, then the WHL may, notwithstanding the provisions of paragraph 8.2, at any time, assign, trade or otherwise transfer this
Agreement to any other member franchise of the WHL (the “Assignee”) in which case the provisions of paragraph 8.1 shall apply to the Player and the Assignee and the WHL will be and remain liable to the Player under paragraph 3.1 for financial assistance which the Player is qualified to receive pursuant to paragraph 3.1(a)(i) prior to the date of such assignment, trade or other transfer of this Agreement. In the event of such assignment, trade or other transfer of this Agreement, the Player will receive the benefits under paragraph 3.1(a)(i) firstly from the Assignee and when those benefits for which the Assignee is liable to the Player have been fully utilized by the Player, the Player will receive the benefits under paragraph 3.1(a)(i) for which the WHL, in accordance with this paragraph 8.3, is liable to the Player.
ARTICLE 9 - REMEDIES OF THE PLAYER
9.1 a) In the event of any breach by the Club of any of its obligations under this Agreement, the Player may give written notice of the nature of the breach to the Club and to the WHL. If the breach is not remedied by the Club within ten (10) days of receipt by the Club of such written notice, then on further written notice by the Player to the Club specifying that the breach has not been remedied by the Club, this Agreement will be null and void and of no further force or effect, except for the obligations of the Club to the Player under Articles 2 and 3 which will remain in full force and effect. Upon such termination, the Player shall forthwith be released by the Club in accordance with the WHL and CHL regulations.
b) Should the Player be entitled to any benefits under the WHL Scholarship Program, the Club will, at the time of termination of this Agreement, outline in writing to the Player, any benefits through the WHL Scholarship Program which the Player is entitled to under the terms of this Agreement.
c) In any dispute between the Club and the Player, either the Club or the Player may at any time refer the matter in dispute to the WHL for determination.
ARTICLE 10 - REMEDIES OF THE CLUB
10.1 a) In the event of any breach by the Player of any of his obligations under this Agreement, the Club may give written notice to the Player of the nature of the breach. If the breach is not remedied by the Player within ten (10) days of receipt by the Player of such written notice, then on further written notice by the Club to the Player specifying that the breach has not been remedied by the Player, this Agreement will be null and void and of no further force or effect, subject to the provisions of paragraph 10.2.
b) Notwithstanding the provisions of paragraph 10.1(a), the Club may terminate this Agreement on written notice to the Player, upon the occurrence of any one of the following events, subject to the provisions of paragraph 10.2:
i) if the Player defaults, refuses, or neglects to provide his services as a hockey player in accordance with paragraph 4.1;
ii) if the Player defaults, refuses or neglects to obey the rules and regulations, directions and instructions reasonably established by the Club, in accordance with guidelines approved by the WHL, governing training, conduct and behaviour of all players on the Club and such default, refusal or neglect reasonably constitutes a material violation of the rules, regulations, directions and instructions of the Club, in accordance with guidelines approved by the WHL, applicable to all the Club's players; or
iii) if the Player fails, in the opinion of the Club, acting reasonably, to demonstrate sufficient skill, competence and ability as a hockey player at the time of termination to retain a position as a hockey player on the Club's roster.
c) Should the Player be entitled to any benefits under the WHL Scholarship Program, the Club will, at the time of termination of this Agreement, outline in writing to the Player, any benefits through the WHL Scholarship Program which the Player is entitled to under the terms of this Agreement.
d) In any dispute between the Club and the Player, either the Club or the Player may at any time refer the matter in dispute to the WHL for determination.
10.2 Upon termination of this Agreement by the Club for any reason:
a) the Allowance in paragraph 2.1 shall forthwith cease to be payable by the Club to the Player. The Player shall be entitled to retain any portion of the Allowance which, prior to termination, had been paid to the Player;
b) the Club shall remain liable for its obligations to the Player for travel expenses pursuant to paragraph 2.4;
c) subject to the provisions of paragraph 8.1(b), the Club shall remain liable for its obligations to the Player for financial assistance pursuant to paragraph 3.1; and
d) provided the Player has not been retained by the Club on the Club's WHL 50 Player Protection List, the Player shall forthwith be released by the Club in accordance with the WHL and Hockey Canada regulations.
ARTICLE 11 - PHYSICAL CONDITION AND INJURIES
11.1 If, in the opinion of the Club acting reasonably, the Player is not in sufficient physical condition, other than as a result of an injury sustained by the Player while performing his obligations under this Agreement, to enable him to play hockey for the Club in an acceptable manner, the Club may, at its option and with the consent of the WHL, either suspend the Player for the period of such incapacity or terminate this Agreement upon written notice to the Player which notice will set forth the Club's reasons for termination. During the period of any such suspension, the Club will not be obligated to pay or reimburse or cause to be paid, as the case may be, the Player the Allowance provided for in paragraph 2.1. If the Club elects to terminate this Agreement, then the provisions of paragraph 10.2 shall apply.
11.2 If the Player is injured in an activity, other than in the performance of his obligations under this Agreement or an activity the Player is involved in as part of his training, that is not scheduled, organized or sanctioned by the Club and if as a result the Player is unable to play hockey for the Club in an acceptable manner for any part of the Hockey Season, the Club may, at its option and with the consent of the WHL, either suspend the Player for the period of such incapacity or terminate this Agreement upon written notice to the Player which notice will set forth the Club's reasons for termination. During the period of any such suspension, the Club will not be obligated to pay or reimburse or cause to be paid, as the case may be, the Player the Allowance provided for in paragraph 2.1. If the Club elects to terminate this Agreement, then the provisions of paragraph 10.2 shall apply. Except as provided herein,
the Player hereby discharges the Club from any and all obligations, responsibilities or monetary compensation of whatever nature that the Player might claim by virtue of this Agreement.
11.3 If the Player is injured in the performance of his obligations under this Agreement or an activity that is scheduled, organized or sanctioned by the Club, the Club shall pay or reimburse or cause to be paid, as the case may be, all reasonable medical and dental expenses the Player incurs in the treatment of his injury together with the expenses of all prescription drugs and medical equipment reasonably required in relation thereto. During the period of such injury, the Player shall be entitled to all the benefits of this Agreement as if the Player had not been injured and was playing. The Player may at any time, acting reasonably, obtain, at his cost, any further medical assessments of the injury he deems necessary; the Club will assist the Player in obtaining such further medical assessments.
ARTICLE 12 - COMPENSATION FOR DEVELOPMENT OF THE PLAYER
12.1 If the Player, has not completed his eligibility to play in the WHL, the Player shall not, during the Term of this Agreement, enter into a contract to play hockey for a professional hockey team unless;
a) the Player has obtained a written release from the WHL, and
b) the Club has been paid the sum of $200,000 in the currency where the Club is located, either by the Player or the professional hockey team with whom the Player has entered into such a contract.
The foregoing provisions of this paragraph 12.1 do proviso does not apply in circumstances where the Player is released by the Club and, in accordance with the WHL regulations, enters into a contract to play for a professional hockey team that is a member of a league that has a written agreement with the WHL covering compensation for player development.
ARTICLE 13 – GENERAL
13.1 If the whole or any portion of this Agreement or the application to any circumstance is held invalid, illegal or unenforceable to any extent that does not affect the operation of this Agreement in a fundamental way, the remainder of the provision in question, or its application to any circumstance other than to which it had been held invalid, illegal or unenforceable and the remainder of this Agreement shall not be affected thereby and shall be valid, legal and enforceable to the fullest extent permitted by law.
13.2 The parties agree that for any litigation arising from this Agreement, the courts of the province of Canada or the state of the United States of America where the Club is located shall have exclusive jurisdiction to determine the issue, according to the laws of such province and country or such state and country, regardless of where the Player or the Club may have executed this Agreement.
13.3 Any notice required, permitted or contemplated in this Agreement shall be in writing. Any notice required to be given by the Player to the Club and the WHL will be personally delivered to the address of or sent by fax to the Club and the WHL, respectively, particulars of which are set forth in the WHL Standard Player Agreement. Any notice required to be given by the Club to the Player shall, during the Hockey Season, be personally delivered to the Player, otherwise any such notice shall be personally delivered to the Player at the address of the Player set forth in the WHL Standard Player Agreement Execution Schedule or sent by fax to the Player at the fax number set forth in the WHL Standard Player Agreement Execution Schedule. Any notice to a parent or guardian of the Player shall be delivered to the address of the parent or guardian or sent by fax, particulars of which are set forth in the WHL Standard Player Agreement Execution Schedule. Any party to this Agreement may change its address for service by providing written notice to the other parties.
13.4 Except for an assignment, trade or other transfer of this Agreement in accordance with the provisions of Article 8, this Agreement is not assignable by either the Player (or, if applicable, the Player's parent or guardian who is a signatory to this Agreement) or the Club.
13.5 The contents of this Agreement are strictly confidential to the parties hereto. Any reproduction or distribution of this Agreement by the Player or the Player's parents or guardian, without the prior written consent of the WHL, is strictly prohibited.
Western Hockey League
Father David Bauer Arena
2424 University Drive NW, Calgary, Alberta, Canada T2N 3Y9
Phone: (403)693-3030 Fax: (403)693-3031
firstname.lastname@example.org www.whl.ca
Agreement dated effective September 15, 2011
Between the Tri-City Americans, hereinafter referred to as the "Club", a member franchise of the Western Hockey League, hereinafter referred to as the "WHL", and LUKAS WALTER, hereinafter referred to as the "Player".
The parties hereto mutually covenant and agree to the following:
1. **Interpretation:**
(a) "Agreement" or "this Agreement" means this agreement between the Club and the Player and is inclusive of the WHL Standard Player Agreement Terms and Conditions Schedule.
(b) The WHL Standard Player Agreement Terms and Conditions Schedule (hereinafter referred to as the "Terms and Conditions Schedule") is hereby incorporated by reference and forms part of this Agreement.
(c) Words and phrases not defined in this WHL Standard Player Agreement have the meaning ascribed to them in the Terms and Conditions Schedule.
(d) The "WHL Scholarship Program" has the meaning ascribed to that term in the Terms and Conditions Schedule.
2. **Term:** Subject to the terms and conditions of this Agreement, the Club hereby retains the services of the Player for a period of 3 years commencing with the 2011 to 2012 Hockey Season and ending with the 2013 to 2014 Hockey Season (the "Term"). For the purposes of this paragraph and this Agreement, the "Hockey Season" means that period of time commencing with the start of the WHL's regular season schedule in the month of September in any given year through to and ending in the following calendar year at the end of the later of: (a) the WHL's regular season schedule in the month of March, or (b) the Club's participation in the WHL playoff season should the Club qualify for the WHL playoffs, including the Club's participation, if any, in the Memorial Cup Championship.
3. **Remuneration:** In consideration of the Player providing his services as a hockey player and otherwise to the Club, and in further consideration of the Player playing hockey exclusively for the Club during the Term of this Agreement, the Club agrees, subject to the limitations, restrictions, provisions and exceptions contained in this Agreement:
(a) to pay or reimburse or cause to be paid, as the case may be, the Player an allowance (the "Allowance") as follows, in the currency of the country where the Club is located, in accordance with the regulations of the WHL in place from time to time:
| HOCKEY SEASON | ALLOWANCE (dollars/month) |
|---------------------|---------------------------|
| 2011 to 2012 | $200.00 |
| 2012 to 2013 | $240.00 |
| 2013 to 2014 | $600.00 |
| 20 to 20 | $ |
| 20 to 20 | $ |
(b) to pay or reimburse or cause to be paid, as the case may be, the reasonable expenses in accordance with the provisions of Article 2 of the Terms and Conditions Schedule associated with the following:
(i) the Player's room and board;
(ii) the Player's travel expenses incurred:
1. for moving from his normal and regular place of residence to the city where the Club is located for the purpose of reporting to the Club at the commencement of each Hockey Season;
2. in returning to his normal and regular place of residence following the conclusion of each Hockey Season; and
3. for one return trip during the Christmas holiday season of each Hockey Season from the city where the Club is located to his normal and regular place of residence;
(iii) the Player's enrollment in a high school or post-secondary educational institution, for the fall and winter term, including tuition fees, compulsory student fees and textbooks directly related to the Player's course of study; and
(iv) tutors and educational advisors as deemed reasonably necessary to assist the Player in his academic studies during the fall and winter term.
4. **WHL Scholarship Program:**
(a) In accordance with the provisions of Article 3 of the Terms and Conditions Schedule, the Club agrees to pay or reimburse or cause to be paid, as the case may be, the Player's educational expenses to enroll in and attend a designated publicly funded post secondary educational institution based on the assessment for a full-time student, following completion of the Player's WHL playing career, including tuition fees, compulsory student fees, and textbooks directly related to the Player's course of study of which payment will be made in the currency of the country where the designated publicly funded post-secondary institution is located in accordance with tuition and fees published in the official calendar of that year.
(b) The Player designates the following publicly funded post-secondary educational institution in a province of Canada or a state of the United States of America where the Player normally and regularly resides, as the "designated post-secondary educational institution" for the purposes of paragraph 3.1 in the Terms and Conditions Schedule;
UNIVERSITY OF BRITISH COLUMBIA (Name of Post Secondary Institution)
5. **Binding Commitment:** The parties hereto hereby accept and agree to the terms, conditions, covenants, agreements and obligations of each other set forth and contained in this Agreement.
6. **Time:** Time shall be of the essence of this Agreement.
7. **Entire Agreement:** This Agreement, inclusive of the Terms and Conditions Schedule which is incorporated herein by reference and forms part hereof, constitutes the whole and entire agreement between the parties hereto and cancels and supersedes any oral and prior agreements, undertakings, declarations, representations and warranties, written or verbal, between the parties hereto.
8. **Governing Law:** This Agreement shall be governed by and construed in accordance with the laws of the province of Canada or the state of the United States of America, as applicable, where the Club is located.
9. **Acknowledgement:** Each of the Player and, if applicable, the Player's parent or guardian who is a signatory to this Agreement, acknowledges that he has read and understands the contents of this Agreement.
In Witness Whereof, the parties have executed this Agreement effective as of the date set forth above and are in agreement with all terms and conditions contained herein:
**WHL MEMBER CLUB:**
Tri-City Americans
WHL Member Club
September 15, 2011
Date of Execution
Bob Tory
Authorized Signing Officer -
WHL Member Club
Signature - Authorized Signing Officer -
WHL Member Club
Address of WHL Member Club: 7100 W. Quinault Ave, Kennewick, WA, USA, 99336
Phone No: (509) 736-0606
Fax No: (509) 783-4591
**PLAYER:**
LUKAS WALTER
Print Name - Player
September 15, 2011
Date of Execution
Signature - Player
Witness as to Signature of Player
Home Address:
25645-82 Ave Langley BC
Phone No: 604 882-3814
Fax No: 604 882-3815
Notes: In the Provinces of Manitoba, Saskatchewan and Alberta and the States of Washington and Oregon, a parent or guardian must execute this Agreement if, at the time of execution of this Agreement by the Player, the Player is under the age of eighteen (18) years. In the Province of British Columbia, a parent or guardian must execute this Agreement if, at the time of execution of this Agreement by the Player, the Player is under the age of nineteen (19) years.
**PARENT OR GUARDIAN OF PLAYER:**
Print Name - Player's Parent or Guardian
September 15, 2011
Date of Execution
Signature - Player's Parent or Guardian
Witness as to Signature of Player's Parent or Guardian
Address of Player's Parent or Guardian:
25645-82 Ave Langley BC, NMAM8
Phone No: 604 882-3814
Fax No: 604 882-3815
**APPROVED BY WESTERN HOCKEY LEAGUE:**
Ron Robison
WHL Commissioner
September 16, 2011
Date of Execution
Signature - WHL Commissioner
WHL Office Address: #1 - 3030 Sunridge Way NE, Calgary, Alberta, Canada T1Y 7K4
Phone No: (403)693-3030
Fax No: (403)693-303
Amending Agreement dated effective September 15, 2011
Between the Tri-City Americans, hereinafter referred to as the "Club",
a member franchise of the Western Hockey League, hereinafter referred to as the "WHL",
And LUKAS WALTER, hereinafter referred to as the "Player".
The parties hereto mutually covenant and agree to the following:
1. **Statement of Principle:** Notwithstanding the provisions of paragraph 7 of the WHL Standard Player Agreement, this Amending Agreement is supplemental to and amends the agreement dated September 15, 2011 (the "Agreement") between the Club and the Player. The provisions of the Agreement are conclusively deemed to have been amended, modified and supplemented by this Amending Agreement.
2. **Construction:** This Amending Agreement and the Agreement shall have effect as far as practicable as though the provisions hereof and thereof were contained in one instrument.
3. **Amendments:** The Agreement shall be and is hereby amended, modified and supplemented as follows:
LUKAS will receive his first year of WHL educational assistance upon playing one exhibition game in the 2011-12 season for the Tri-City Americans.
In Witness Whereof, the parties have executed this Amending Agreement effective as of the date set forth above and are in agreement with all terms and conditions contained herein:
| WHL MEMBER CLUB | Date of Execution: September 15, 2011 |
|-----------------|--------------------------------------|
| Tri-City Americans | Signature - Authorized Signing Officer |
| WHL Member Club | |
| PLAYER | Date of Execution: September 15, 2011 |
|--------|--------------------------------------|
| | Signature - Player |
| | Witness as to Signature of Player |
| PARENT OR GUARDIAN OF PLAYER | Date of Execution: September 15, 2011 |
|------------------------------|--------------------------------------|
| | Signature - Player's Parent or Guardian |
| | Witness as to Signature |
APPROVED BY WESTERN HOCKEY LEAGUE
SEPT 16, 2011
|
Effects of disparity on visual discomfort caused by short-term stereoscopic viewing based on electroencephalograph analysis
Xiao Wang, Liuye Yao, Yuemei Zhao, Lidong Xing, Zhiyu Qian*ID, Weitao Li and Yamin Yang
*Correspondence:
email@example.com
College of Automation Engineering, Nanjing University of Aeronautics and Astronautics, No. 29 Jiangjun Avenue, Jiangning District, Nanjing 211106, China
Abstract
Background: Discomfort evoked by stereoscopic depth has been widely concerned. Previous studies have proposed a comfortable disparity range and considered that disparities exceed this range would cause visual discomfort. Brain activity recordings including Electroencephalograph (EEG) monitoring enable better understanding of perceptual and cognitive processes related to stereo depth-induced visual comfort.
Methods: EEG data was collected using a stereo-visual evoked potential (VEP) test system by providing visual stimulus to subjects aged from 21 to 25 with normal stereoscopic vision. For each type of visual stimulus, data were processed using directed transfer function (DTF) and adaptive directed transfer function (ADTF) in combination with subjective feedbacks (comfort or discomfort). The topographies of information flow were constructed to compare responses stimulated by different stereoscopic depth, and to determine the difference in comfort and discomfort situations upon stimulation with same stereoscopic depth.
Results: Based on EEG analysis results, we found that the occipital P270 was moderately related to the disparity. Moreover, the ADTF of P270 showed that the information flows at frontal lobe and central-parietal lobe changed when stimulation with different stereoscopic depth applied. As to the stereo images with same stereoscopic depth, the DTF outflows at the temporal and temporal-parietal lobes in δ band, central and central-parietal lobes in α and θ bands, and the comparison of inflows in these three bands could be considered as discriminated indexes for matching the stereoscopic effect with viewers’ comfort or discomfort state impacted by disparity. The subjective feedbacks indicated that the comfort judgments remained as a result of cumulative effect.
Conclusions: This study proposed a short-term stereo-VEP experiment that shortened the duration of each stimulus in the experimental scheme to minimize the interference from other factors except the disparity. The occipital P270 had a mid-relevance to the disparity and its ADTF showed the affected areas when viewers are receiving stimulations with different disparities. DTF could be considered as discriminated indexes for matching the stereoscopic effect with viewers’ comfort or discomfort state induced by disparity. This study proposed a preferable experiment to observe the single effect of disparity and provided an intuitive and easy-to-read result in a more convenient manner.
**Keywords:** Stereoscopic depth, Visual discomfort, Visual evoked potential, Directed transfer function/adaptive directed transfer function
**Background**
Visual discomfort in stereo viewing is noted as a result of inappropriate binocular disparity. Depth features of stereoscopic images have thus been widely investigated. Lambboij et al. [1] claimed that disparity beyond one degree could cause noticeable visual discomfort. Cho et al. [2] found that the increase of binocular disparity gave rise to human fatigue level. To obtain more in-depth understanding of stereoscopic depth-induced visual discomfort, the relevance between stereo imagery and potential adverse effects have been studied under a wide variety of situations. Shibata et al. [3] reported that large crossed disparity and small uncrossed disparity led to a marked drop in comfort ratings during stereo viewing based on subjective questionnaires. Although such questionnaire-based survey presents a simple and practical method, individual differences in uncertainty tolerance may influence the results. Functional magnetic resonance imaging (fMRI) was also employed to explore the changes in human cortex during visual stereo stimulation study. Based on fMRI, Liu et al. [4] indicated that stereoscopic depth perception was correlated with several regions (hV3A, LG, hMT/V5, LOS and VIPS), which stands for the precise positions on human cortex in stereoscopic vision processing. Jung et al. [5] also used fMRI to locate the activated areas on the cortex when people felt uncomfortable during 3D viewing. fMRI remains a precise yet expensive method which also requires readers to have professional knowledge of the brain structure. For the detection of stereo visual discomfort in daily life and taking the cost-effectiveness into consideration, a relatively simple, easy-operating, labor- and money-saving method needs to be developed. Recently, electroencephalograph (EEG) has been widely used as an effective way to assess stereoscopic visual fatigue. Li et al. [6] found the power of the high frequency band in EEG became stronger in 3D viewing and the peak difference in P700 at 3D oddball paradigm might be an effective indicator for revealing 3D visual fatigue. Mun et al. [7] found significantly reduced P600 amplitudes and delayed P600 latencies appeared in accordance with 3D visual fatigue, and significant fatigue effects were also observed at P4 and O2 sites during the 8.57 Hz attended task. Zou et al. [8] evaluated visual fatigue level via random dot stereogram (RDS) with six different disparities and found that EEG could be employed as a useful tool to predict visual fatigue caused by vergence-accommodation conflict. Malik et al. [9] compared the EEG absolute power differences, coherence and complexity, then concluded that 3D viewing is more attractive than 2D and may cause high attention and involvement of working memory manipulations. Other researches also demonstrated the relevance between visual fatigue and disparity in stereo viewing based on EEG analysis [10–13]. Kang et al. [14] developed a platform to facilitate comfortable stereo video viewing using EEG-based visual discomfort evaluation technology. Frey et al. used EEG and event related potential (ERP) to assess the visual discomfort in stereoscopic displaying and established a prediction model to assess visual discomfort based on their ERP results and reached an accuracy rate more than 62% on average [15, 16]. Although the above work demonstrated the capability of distinguishing uncomfortable stereoscopic viewing conditions from comfortable ones based on EEG analysis, each stimulus in their experimental trial lasted
for seconds. In the majority of previous studies, overall level of visual discomfort was recorded according to the subjective questionnaires and was determined after certain experimental session. However, it might be expected that the time course of viewing could also cause possible cumulative effects and thus affect discomfort judgments.
Visual evoked potential (VEP), which refers to the evoked potential caused by a visual stimulus, is commonly used for vision check in clinics. VEP measures the functional integrity of the visual pathways from retina via the optic nerves to the visual cortex when retinas receive stimuli, and can be induced by a stimulus repeated at a higher rate [17]. Therefore, in order to capture the fast responses upon short-term visual stimuli (less than 1 s) with various stereoscopic depth, the present pilot study described an experimental protocol which acquires both EEG signals and subjective feedbacks without interrupting the viewing.
Thanks to precise stereoscopic parameters and real-time measures of our stereo-VEP setup, the effects of disparity and to which degree the disparity would evoke visual discomfort immediately over short viewing sequences were systematically investigated in this study. The effects of disparity over short viewing sequences were systematically investigated by directed transfer function (DTF) and adaptive directed transfer function (ADTF) methods. The study will provide more intuitive and easy-to-read results to the public to help them understand their physical status in a more convenient manner and pave the way for better estimating stereoscopic discomfort and optimization of stereoscopic display parameters in the future.
**Methods**
**Experimental protocol**
**Stereo-VEP experimental system**
Figure 1 shows the schematic setup of our experimental system which composed of a stimulation part, a recording part and a feedback part. The simulation part is consisted of an active shutter 3D-TV (LED46XT39G3D, Hisense) and a laptop installed with E-Prime 2.0. The visual stimuli were provided by a visual paradigm written based on E-Prime 2.0. During experiment, the paradigm running on the laptop would be synchronously displayed on the 3D-TV through a high definition multimedia interface (HDMI) cable. Participants watched the stereo stimuli series through a pair of shutter glasses (FPS3D02, Hisense). The detailed information of the paradigm will be introduced in the following section.
EEGs were acquired in real-time with NuAmps electroencephalograph (Australia, Neuroscan) during experiment. Signal recording was performed according to the expanded international 10–20 montage system. Thirty of total thirty-seven electrodes were placed along the scalp for EEG recording. As alternative references, two mastoid electrodes (M1 and M2) at bilateral were used during recording. Four electrodes were used for real-time horizontal and vertical electrooculogram (EOG) recording and the ground was set at FPz. The sampling rate was 1 kHz. The contact resistance of each electrode was less than 5 kΩ. The acquisition process was monitored by the other laptop installed with Curry 7. For each trial, participants stimulated upon corresponding visual cues were asked to click the left button of the mouse as soon as they felt uncomfortable.
The entire experiment was conducted in a quiet room and the temperature was kept at 24 °C. Ten right-handed subjects (male: 9, female: 1, aged from 21 to 25) with normal stereoscopic vision participated in our experiment. All subjects were informed about the general experiment information and signed the consent form before the experiment. All experiments were carried out in accordance with institutional guidelines of Nanjing University of Aeronautics and Astronautics (NUAA). All experimental protocols were approved by the Ethics Committee of NUAA.
**The paradigm**
The pattern onset/offset visual evoked potential (POVEP), including both flash visual evoked potential (F-VEP) and pattern visual evoked potential (P-VEP), was applied to explore the relevance between stereoscopic depth perception and human discomfort in this study. The stereo pattern onset/offset visual evoked potential (Stereo-POVEP) paradigm was shown in the dashed box in Fig. 1. After the subject read through the instructions for experiment description, he or she would tap the space key to start the experiment. A cross would appear at the center of the screen for 5 s to draw the participant’s attention to the screen center. One of four images with different disparities would randomly appear and displayed for 500 ms for each, followed by a black background for 500 ms. A complete set of stimulation series presented 240 trials (60 trials for each image stimulus) and it costed less than 5 min in total. Each subject performed two sets of above stimulation trials.
The disparity parameters of the stereo images are illustrated in Fig. 2a. The image stimulus with zero disparity includes neither uncrossed disparities nor crossed disparities, and in other words, it is a 2D image stimulus (abbreviated as ‘2D’). The “±” sign in the
“disparity” column (abbreviated as ‘3D uncr+cr’) means that, besides the 2D (zero disparity) part of the image, the image stimulus includes uncrossed (orchid bud appeared in front of the screen) and crossed disparities (orchid bud appeared behind the screen) (Fig. 2b). The “+” sign means the uncrossed disparity (abbreviated as ‘3D uncr’), and the “−” sign means the crossed disparity (abbreviated as ‘3D cr’), respectively. For convenience, we numbered these four types of stimuli ‘2D’, ‘3D uncr+cr’, ‘3D cr’ and ‘3D uncr’ as ‘S1’, ‘S2’, ‘S3’ and ‘S4’. ‘S2’ has both negative and positive disparities, and the absolute value of its positive disparity or negative disparity are both in the suggested comfortable range (| positive (or negative) disparity of S2| = 0.5 < 1). The size of the image in left–right format was 1920 × 1080 and the object in the middle of the image was 1280 × 898 (Fig. 2c). The visual angle was approximately 4.08° × 6.67°. All images were provided by Prof. Qiu and his group from the School of Art in Peking University.
**Data processing**
The reference was switched to Cz during offline processing. After baseline correction, 50 Hz notch filtering, 0.01–30 Hz band-pass filtering, eye movement artifacts removal and bad blocks removal, VEPs were averaged by the time-locked and phase-locked EEGs. Valid data used for each averaging was over 90 trials. The processed VEPs were saved for following computations.
**Brain connectivity estimators**
**DTF and ADTF**
Granger causality is used to compute the causal relationship between two time series. Kaminski and Blinowska successfully applied DTF into neurobiological system computations, which expanded the application of Granger causality based on multivariate autoregressive (MVAR) model [18]. A short description of this algorithm is as follows.
\[ X(t) = [X_1(t), X_2(t), X_3(t), \ldots, X_k(t)]^T \] is defined as a k-channel signal at time point t. The superscript T represents the matrix transposition. Then the MVAR model can be described as:
\[ X(t) = \sum_{n=0}^{p} A(n)X(t-n) + E(t) \]
(1)
with
\[ A(0) = I \]
(2)
where \( E(t) \) is the white noise and \( A(1), A(2), \ldots, A(p) \) are \( k \times k \) coefficient matrices. The order \( p \) could be derived from Schwarz Bayesian Criterion (SBC) [19]. Then it is transformed into the frequency domain by Fourier transforms:
\[ A(f)X(f) = E(f) \]
(3)
where
\[ A(f) = \sum_{n=0}^{p} A(n)e^{-2\pi ftn} \]
(4)
So \( X(f) \) could be rewritten as:
\[ X(f) = A^{-1}(f)E(f) \]
(5)
Define \( H(f) = A^{-1}(f) \), and then
\[ X(f) = H(f)E(f) \]
(6)
\( H(f) \) is the transfer matrix of the system, which equals to the inverse of frequency-transformed coefficient matrix. The casual information from channel \( j \) to channel \( i \) is:
\[ \theta_{ij}^2(f) = |H_{ij}|^2 \]
(7)
The DTF measurement \( \gamma_{ij}^2(f) \) is the normalization of \( \theta_{ij}^2(f) \), which describes the directional causal information from channel \( j \) to channel \( i \). The value of \( \gamma_{ij}^2(f) \) is between 0 and 1.
\[ \gamma_{ij}^2(f) = \frac{|H_{ij}(f)|^2}{\sum_{m=1}^{k}|H_{im}(f)|^2} \]
(8)
ADTF was proposed by Wilke in 2008, which was a time-varying multivariate method aiming to estimate rapidly changing connectivity relationship of the brain [20]. Some studies have confirmed that ADTF is suitable for the analysis of short duration signals [19, 21]. Similarly, the signal \( X(t) \) could be described as:
\[ X(t) = \sum_{n=1}^{p} A(n,t)X(t-n) + E(t) \]
(9)
where \( X(t) \) is a vector that changes with time, \( A(n, t) \) is the time-varying coefficient matrices. Other parameters are the same as their definitions in DTF. ADTF measure \( \gamma_{ij}^2(f) \) could be similarly defined as:
\[
\gamma_{ij}^2(f) = \frac{|H_{ij}(f, t)|^2}{\sum_{m=1}^{k} |H_{im}(f, t)|^2}
\]
(10)
Matlab toolbox eConnectome (Biomedical Functional Imaging and Neuroengineering Laboratory, University of Minnesota, Minneapolis, http://econnectome.umn.edu/) was used to compute the DTF and ADTF out-to-in of these signals. The out-to-in information indicates the causal information flows from one channel to another [22]. From out-to-in information we could compute the outflow information (or outflows) and inflow information (or inflows) of each flow. The outflow information of an electrode is the sum of the information from this electrode to all the other electrodes while its inflow information is the sum of all the other electrodes’ information flow into this electrode [22]. The outflow or inflow information value represents the ability of the electrode to affect other electrodes or to be affected from other electrodes. In this paper, the electrode with strong outflows was defined as a key cause node and the electrode with sensitive inflow information was considered as a key result node.
**Surrogate data**
Surrogate data, a time series which fits well with the linear-dynamics null hypothesis, could assess the significance of DTF and ADTF connectivity measures [19, 23]. It demonstrated that this method is suited for DTF and ADTF analysis that are the measurement of frequency-specific causal interactions [19]. The significance setting in this study was \( P < 0.05 \).
**Results**
**Visual comfort comparison among different disparities measured by Stereo-VEPs**
**Waveforms and time–frequency analysis**
According to the subjective feedback, EEG signals in the cases of visual comfort were firstly averaged among subjects. Figure 3 depicts a representative planform of Stereo-VEP from one subject and averaged Stereo-VEP over all subject evoked by four types of stimuli at O1, Oz, O2 electrodes, respectively. In the planform, the magnitude of each amplitude was represented by colors. Colors from blue to red depict the amplitude of EEG signal from low to high. Obviously, the most distinct peak presented in the occipital lobe. In the grand average waves of Stereo-VEP, the P3 component was extremely obvious at nearly 270 ms after the onset of the stimulus (in the following contents, we use P270 for convenience). Table 1 lists the means and standard deviations of P270 from 10 participants. Compared with VEP evoked by ‘S1’, P270 evoked by other stimuli had a delay no less than 10 ms. As shown in Table 1, ‘S2’ caused nearly the same latencies at both the left and the right occipital lobe. In the case of ‘S3’, the latency of P270 at the left occipital lobe (O1) is slightly greater than that at the right part (O2), while ‘S4’ led to the contrary result. It is also quite clear that ‘S3’ evoked the most significant peak in the amplitude of P270.
The Pearson correlation coefficient showed that peak amplitudes of P270 had a mid-relevance with the disparity (O1: Pearson correlation coefficient = −0.474, $P=0.006<0.01$; Oz: Pearson correlation coefficient = −0.480, $P=0.005<0.01$; O2: Pearson correlation coefficient = −0.459, $P=0.008<0.01$). The Paired $T$ test between the occipital P270 from any two different types of visual stimuli was shown in Table 2. It is shown that the difference can be significantly observed by P270 component from O1, Oz, and O2 electrodes at the occipital lobe. Although between-group differences cannot be illustrated using latency and amplitude information from one single electrode, P270 based on the integrated results from three electrodes located at occipital lobe could be used as an effective indicator for differentiating different type of stimuli.
In addition, when the stimulation disappeared, an off-response at about 660 ms in the grand average showed that ‘S3’ caused the most significant changes in amplitude (Fig. 3). Previous study [24] suggested that off-responses had close relationship with visual persistence. It may infer that large crossed disparities would contribute to more off-responses. Another negative potential showed at around 700 ms, which could be due to the open issue of visual N700 [25, 26].
**Table 2 The Paired T-test of P270**
| Pairs | Latency (ms) | Amplitude (μV) |
|-------|--------------|----------------|
| | O1 | Oz | O2 | O1 | Oz | O2 |
| S1–S2 | T = −4.862 | T = −7.735 | T = −4.235 | T = 3.110 | T = 3.233 | T = 3.233 |
| | P = 0.001 | P = 0.000 | P = 0.002 | P = 0.013 | P = 0.01 | |
| S1–S3 | T = −2.648 | T = −2.776 | | | | |
| | P = 0.027 | P = 0.022 | | | | |
| S1–S4 | T = −2.454 | T = −3.995 | T = −2.669 | T = 4.125 | T = 4.302 | |
| | P = 0.037 | P = 0.003 | P = 0.026 | P = 0.003 | P = 0.002 | |
| S2–S3 | | | | T = −3.363 | T = 3.423 | |
| | | | | P = 0.008 | P = 0.008 | |
| S2–S4 | | | | T = 2.504 | | |
| | | | | P = 0.034 | | |
| S3–S4 | | | | T = 3.324 | T = 4.703 | T = 3.887 |
| | | | | P = 0.009 | P = 0.001 | P = 0.004 |
**Fig. 4** **a** Time–frequency map of averaged Stereo-VEP at Oz electrode upon visual stimuli with various disparities. Upper row in from left to right: TF-Analysis images upon stimuli with ‘S1’ (2D image) and ‘S2’ (3D image with both uncrossed and crossed disparities); Bottom row from left to right: TF-Analysis image upon stimuli with ‘S3’ (3D image with large crossed disparity) and ‘S4’ (3D image with large uncrossed disparity); **b** dominant frequency distributions of VEPs at P270 under stimulation with four different types of disparities
Figure 4a describes the time–frequency analysis (TF-Analysis) of averaged Stereo-VEP at Oz electrode within 800 ms after the onset of various visual stimuli. These images showed that P270 component evoked by 3D stereo stimuli includes wider frequency bandwidth (δ, θ and a few α bands) at occipital lobe as compared with the 2D stimulus. Figure 4b represents the dominant frequency distribution of P270, indicating that although the frequency band of Stereo-VEPs became wider upon 3D stereo stimuli, most of leading frequency were still at δ band (about 2.6 Hz). An exception was that the dominant frequency of Stereo-VEP evoked by ‘S3’ was 5.4 Hz, which was in θ band.
**ADTF information flows**
δ, θ, α bands are related to signal matching and decision-making process, attentive processing and sensory processing, respectively [27]. Figure 5 shows the ADTF information flow topographies of P270 at δ, θ, α and δ~α (δ to α) bands (from bottom to top). In Fig. 5a, 2D stimulus evoked strong outflows at F3 electrode at δ~α bands, while 3D stimuli evoked that at F7, CP4, CP3, P4, Pz, T3 and Oz electrodes. It was easy to observe
that 2D stimulus caused more powerful outflows in the left frontal lobe in the decision-making process (δ band), while 3D stimuli caused significant influence in the central lobe and the central-parietal lobe (CP4, CP3, Pz electrodes). In addition, ‘S3’ caused strong outflows at FP1 electrode as well. Although the θ band topographies were almost similar to the δ band topographies, Oz electrode at θ band became the key cause node instead of FP1 electrode in the δ band under ‘S3’ stimulation. In α band, P4 became one of the key cause nodes under ‘S1’ and ‘S3’ stimulation, while the CP3 electrode was never the key cause node under ‘S2’ or ‘S3’ stimulation. F7 and T3 were the key cause nodes under ‘S4’ stimulation. All the nodes mentioned above were marked as bright color in Fig. 5a. Generally, the ADTF outflows of P270 evoked by 2D stimulus mainly centered on the frontal lobe, while upon 3D stimuli the outflows of P270 centered on the posterior brain areas.
The ADTF inflow topographies of P270 were shown in Fig. 5b. The number of disparity types included in one stimulus was defined as depth complexity (D-complex). The D-complex of ‘S2’ was higher than that of ‘S3’ or ‘S4’ for the reason that it includes two types (uncrossed and crossed) of disparities. The result showed that the stimulus with high D-complex (‘S2’) caused stronger inflows at θ and δ bands (especially in the pre-frontal area). Large crossed disparity evoked slightly stronger inflows when comparing the inflows between the two stimuli with low D-complex (‘S3’, ‘S4’). ‘S1’ and ‘S4’ caused relatively strong inflows in α band from the central area to the occipital lobe. The difference was that ‘S1’ acted on the left part while ‘S4’ acted on the right. ‘S3’ also slightly influenced the right frontal and central-parietal lobe at α band. The δ~α band topography for each stimulus showed that ‘S1’ owns the strongest inflows.
**Comparison between visual discomfort and comfort by DTF**
**Subjective feedbacks**
The subjective feedbacks (Table 3) showed that no discomfort was reported during stimulation by ‘S1’, while ‘S3’ caused 93 feedbacks reporting discomfort. That is to say,
Table 3 Subjective feedbacks
| Image | Abbreviation | Feedback | Total number of stimuli |
|-------|----------------|----------|-------------------------|
| S1 | 2D | 0 | 1200 |
| S2 | 3D uncr+cr | 39 | 1200 |
| S3 | 3D cr | 93 | 1200 |
| S4 | 3D uncr | 27 | 1200 |
Fig. 6 The contrast of the feedbacks during a session of the experiment. **a** At the beginning, **b** the point over half of the experimental time
observers are more sensitive to crossed disparity than to uncrossed ones, especially the large ones. This result is in good accordance with our previous study about stereoscopic depth in watching 3D films [28]. Furthermore, we found that most participants did not report feedbacks at the beginning of the experiment. As the experiment continues, discomfort feedbacks were frequently marked. Typically, as shown in Fig. 6a, there was no discomfort feedbacks (marked as ‘1’) recorded at the very beginning of the experimental session upon stimulation by ‘S3’ (marked as ‘30’), while discomfort feedbacks firstly appeared after half of the experimental process (Fig. 6b). Other labels ‘10’, ‘20’, ‘40’ in Fig. 6 represented ‘S1’, ‘S2’, and ‘S4’ respectively. The electrodes named A1 and A2 represented the references M1 and M2. It implied that, although disparity might easily lead to visual discomfort, there could still be an accumulative process.
Comparison between visual discomfort and comfort by DTF
DTF information flows
Based on the subjective feedbacks, Stereo-VEPs were further divided into seven groups, dependent on the types of stimuli and subjective feelings (comfort and discomfort). Because in some cases uncomfortable feedbacks upon certain stimulation were rarely received, differences between comfort and discomfort at a specific time point could not be accurately represented. Therefore, the DTF method rather than the ADTF method was chosen to distinguish the difference between visual comfort and discomfort. Figure 7 depicted the DTF information flow topographies at α, θ, δ bands (from top to bottom). Data from 0 to 500 ms after the onset of the stimulus were analyzed. It was obvious that 3D stereo stimuli activated more and stronger outflows than the 2D stimulus did in posterior brain areas. In Fig. 7a, as compared to that in 2D condition, outflows became weaker in the right frontal lobe (F8 electrode)
when participants received 3D stimuli no matter whether they reported visual discomfort or not. The comparison of comfort in this finding was inconsistent with what has been illustrated in Fig. 5a. In ‘S2’, ‘S3’ and ‘S4’ cases where no discomfort was reported, the outflows in the left temporal lobe (T3 electrode) or in the left temporal-parietal lobe (TP7 electrode) became stronger, while the outflows in the same lobes became weaker if discomfort was marked in the feedback. The central lobe and the central-parietal lobe showed that the outflows at $\alpha$ and $\theta$ bands became relatively strong when participants received ‘S2’ and ‘S4’ stimuli and felt uncomfortable. However, the phenomenon was opposite under ‘S3’ stimulation.
As shown in Fig. 7b, the DTF inflows throughout the whole brain were obviously stronger under 3D stimulations than that under 2D stimulation. Consistent with Fig. 5b, the stimulus with high D-complex (‘S2’) caused stronger DTF inflows when participants felt comfortable. In terms of DTF inflows caused by low D-complex, compared ‘S3’ with ‘S4’, the large crossed disparity rather than uncrossed disparity evoked stronger DTF inflows in comfortable situation. It was also noticeable that the DTF inflows became weaker when subjects received ‘S2’ and ‘S3’ stimulation and felt uncomfortable, but the result was opposite in the ‘S4’ case.
**Discussion**
Considering the safety issue, the largest disparity value used in this experiment was within the comfortable range proposed by Lambooij [1]. However, it is very close to the superior limit of the range and the effect was distinct when comparing with other stimuli in this paradigm. The uncomfortable feelings were frequently reported by participants when they received the stimulus with large crossed disparity.
P270 and other Stereo-VEP components in stereo viewing
As VEP suggests the electrical activity of occipital lobe when the retina receives stimuli [17], VEPs in occipital region indicate that the change of disparity could influence the brain. In the overall averaged Stereo-VEPs, five onset response components C1, C2, C3, N2 and P3 were found. Early components of N75 component (or N1), P100 component (or P1) and N135 component (or N2) are often regarded as a set called the NPN component [29, 30]. Shawkat et al. verified the relevance between pattern reversal visual evoked potential (PRVEP) and POVEP, that is, P100 corresponds to C1, N135 corresponds to C2, and the following positive component may correspond to C3 [31, 32]. Sometimes, there is a small negative wave called C0, which corresponds to N75 [32]. In our study, it is shown in Fig. 3 that ‘S2’ and ‘S3’ caused distinct NPN component.
P1 (C1 in present study), N2 and P3 are regarded as neural correlate of visual consciousness and are usually used to study attention and visual processes [33]. Our results are consistent with previous studies which confirmed that C1 increases due to the depth perception [34, 35]. Compared to the 2D stimulus, a significant increase in the amplitude of C1 component in the 3D conditions was noticed. The difference in C1 amplitude between different stimuli can be explained by the difference between conditions, as the amplitude of C1 is known to be modulated by depth perception of 3D stimulus. As shown in Fig. 3, although ‘S3’ and ‘S2’ both evoked a clear C1 component at around 100 ms, more significant increases in C1 amplitude were found in ‘S3’ condition, which explains that the larger crossed disparity (‘S3’) may result in more distraction of visual attention and lead to more discomfort feedback. Although the result shown in Fig. 3 was the grand averaged amplitude of VEPs, there is no any statistically significance between different individuals participated in our experiment.
In order to attract participants’ attention and to avoid the decrease of attention intensity during stimulation, we used POVEP paradigm and forced participants to give feedbacks whenever there is any visual discomfort. P270 in this paper was P3b which appears in voluntary attention [36]. The TF-Analysis inferred that the P270 component at occipital lobe correlated closely to the decision-making process and attentive processing. As shown in Table 1, the latency of P270 appears to be longer in the case of ‘S2’, indicating that the visual cortex may need longer period of time for fusion and stereopsis upon processing images with increased disparity complexity. Similar to C1, the amplitude of P270 also increases with the depth perception by reflecting enhanced neural activity upon stimuli with larger disparity. Therefore, the occipital P270 could be chosen as an index to distinguish the effect of stereoscopic depth under comfortable feelings in stereo viewing.
The ADTF analysis showed that outflows of P270 caused by 2D stimulus were mainly on the frontal lobe, while 3D stereo stimuli evoked outflows of P270 on the posterior brain areas. It suggests that ADTF could help to judge whether the visual stimuli include any stereoscopic depth information under the comfortable state. Furthermore, the key cause nodes showed no significant change in ‘S1’ and ‘S2’ condition at δ, θ and α bands. However, in the large disparity cases, they changed obviously. According to the topographies of ADTF inflows, the result could be helpful to distinguish which kind of depth the stimulus may contain.
Comparison between comfort and discomfort caused by stereoscopic depth
Researchers have demonstrated that the posterior parietal cortex participated actively in processing depth based on fMRI and functional near infrared spectroscopy (fNIRS) methods [37, 38]. In our study, changes of DTF and ADTF outflows were also found at the central-parietal lobe when stimulated by 3D images with different stereoscopic depth (Figs. 5 and 7). The key cause nodes appeared in posterior brain areas under 3D condition, while the nodes were in the frontal lobe under 2D condition. The subjective feedbacks indicated that ‘S3’ caused the most significant visual discomfort. DTF outflows in Fig. 7 shows that visual discomfort is accompanying with the decrease of the importance of the cause node in left temporal lobe. Based on literatures, temporal lobe relates to visual memory, language comprehension, and emotion association [39]. The ventral part of the temporal cortices also participates in high-level visual processing of complex stimuli. Anterior parts of the ventral stream are involved in object perception and recognition during visual processing [40]. Thus, it implies that discomfort from depth perception may diminish the connection strength between temporal lobe and other parts of the brain. On the other hand, the parietal lobe relates to attention and the central area is correlated with information processing. It was shown that the outflows at θ bands in the central lobe and the central-parietal lobe became relatively strong in participants under ‘S2’ and ‘S4’ stimulation accompanied with visual discomfort. However, the phenomenon evoked by ‘S3’ was opposite. It infers that the discomfort caused by the uncrossed disparity may incline to catch participants’ attention but that caused by the crossed disparity may reduce the attention. Figure 7b shows high depth complexity and large crossed disparity made DTF inflows weaker when participants felt uncomfortable, but it was contrary in the case with large uncrossed disparity. That is to say, the high depth complexity and the large crossed disparity would reduce the connectivity strength but the large uncrossed disparity would increase the connectivity strength when participants felt uncomfortable. This result indicated that DTF information flows from EEG signals could be considered as an index to distinguish the comfort level in stereo viewing.
Generally, this study proposed a Stereo-VEP experiment that can capture the fast responses upon short-term visual stimuli with various stereoscopic depth to minimize the interference from other factors. Compared to previous experiments, it focused more on the comfort level caused by disparity itself. ADTF and DTF results in this study illustrated that the information flows of EEG electrodes could be as effective as other methods to show the effect of disparities and comfort level. For the next step, based on the experimental and EEG analysis results in this paper, we intend to further explore the disparity-induced visual discomfort in stereo viewing in the cerebral cortex. Besides the findings reported herein, we have also found some strongly activated gyri at frontal and temporal lobes when viewers receiving stimuli with visual discomfort, which correlated well with previous studied using fMRI analysis.
Conclusions
In this paper, we established a short-term experimental system which combined viewer-interactive subjective feedback towards better understanding of visual comfort or discomfort impacted by different stereoscopic depth. The results showed that the occipital P270 had a mid-relevance to the disparity of the stimuli and its ADTF showed the
strongly activated areas when viewers are receiving stimulations with different disparities. DTF of P270 during the presence of the stimuli helped understanding the difference between comfort and discomfort stimulated by the same disparity. The change impacted along with the presence of discomfort could be found from the DTF outflows at the temporal and temporal-parietal lobes in $\delta$ band, central and central-parietal lobes in $\alpha$ and $\theta$ bands, and the inflows in these three bands. The subjective feedbacks showed the discomfort situations remained as a result of cumulative effect. Overall, the study provided a preferable experiment to observe the effects of disparity based on the Stereo-VEP experiment. Reconstruction of the information flow following a visual stimulus could be helpful to match the stereoscopic effect with viewers’ state (comfort or discomfort status) induced by disparity and provided an intuitive and easy-to-read result in a more convenient manner.
**Abbreviations**
ADTF: adaptive directed transfer function; D-complex: the depth complexity; DTF: directed transfer function; ECG: electrocardiograph; EEG: electroencephalograph; EOG: electro-oculogram; fMRI: functional magnetic resonance imaging; fNIRS: functional near infrared spectroscopy; F-VEP: flash visual evoked potential; MVAR: multivariate autoregressive; NIRS: near infrared reflectance spectroscopy; POVEP: pattern onset/offset visual evoked potential; PRVEP: pattern reversal visual evoked potential; P-VEP: pattern visual evoked potential; RDS: random dot stereogram; SBC: Schwarz Bayesian Criterion; Stereo-POVEP: stereo pattern onset/offset visual evoked potential; Stereo-VEP: stereo visual evoked potential; TF-Analysis: time-frequency analysis; VEP: visual evoked potential; 2D or S1: zero disparity, i.e. the image stimulus includes neither uncrossed disparities nor crossed disparities; 3D uncr+cr or S2: the image stimulus includes both uncrossed and crossed disparities; 3D cr or S3: the image stimulus only includes the crossed disparity; 3D uncr or S4: the image stimulus only includes the uncrossed disparity.
**Authors’ contributions**
XW was involved in performing experiments, data analyzing and writing the first draft of the manuscript. LY and YZ did the data analysis. LX and ZQ was responsible for the guidance and providing suggestions during the research. WL and YY wrote and modified this paper. All authors read and approved the final manuscript.
**Acknowledgements**
We are thankful to those helped with the experiment and gave suggestions during the research. We also thank Prof. Qiu and his team from the School of Art in Peking University for providing the image stimuli.
**Competing interests**
The authors declare that they have no competing interests.
**Availability of data and materials**
The datasets analyzed in this study are available from the corresponding author on reasonable request.
**Consent for publication**
The manuscript is approved by all authors for publication.
**Ethics approval and consent to participate**
All experimental methods were carried out in accordance with institutional guidelines of Nanjing University of Aeronautics and Astronautics (NUAA). All experimental protocols were approved by the Ethics Committee of NUAA. Participants signed the informed consent before the experiment started in this study.
**Funding**
The research work was supported by Funding of Jiangsu Innovation Program for Graduate Education (KYLX_0248) and the Fundamental Research Funds for the Central Universities; National Major Scientific Instruments and Equipments Development Project Funded by National Natural Science Foundation of China (81277804, 81827803), National Natural Science Foundation of China (61875085, 81601532), Natural Science Foundation of Jiangsu Province (BK20160814), Jiangsu Science and Technology Support Plan (Social Development) (BE2016759).
**Publisher’s Note**
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Received: 17 March 2018 Accepted: 25 October 2018
Published online: 03 November 2018
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Commencement Program, December (2013)
Minnesota State University Moorhead
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Steven Rosenstone
Chancellor of Minnesota State Colleges and Universities
Steven Rosenstone began his term as chancellor of Minnesota State Colleges and Universities in August, 2011. He is responsible for leading the seven state universities and 24 community and technical colleges that serve more than 430,000 students in 57 communities across the state, and for strengthening the contributions MnSCU colleges and universities deliver in growing Minnesota’s workforce and economy.
Previously, Rosenstone served as dean of the College of Liberal Arts and Vice President for Scholarly and Cultural Affairs at the University of Minnesota, where he was awarded the McKnight Presidential Leadership Chair. He previously served as professor of political science at Yale University and professor of political science and program director in the Center for Political Studies at the University of Michigan. He is the author of four books and numerous scholarly articles on elections and political participation, and he is a nationally recognized expert on higher education issues.
Keith Langseth
Former State Senator
Former Senator Keith Langseth's career included 36 years of outstanding service as a member of the Minnesota Legislature. His lasting impact on public policy spanned a range of issues including agriculture and natural resources, transportation, education, tax policy, bonding and small business.
Senator Langseth is responsible for the "Border City" legislation, which gave property tax breaks to local business and apartment owners to help them compete with neighboring states that have lower tax rates. He was the chief author of numerous bills directly benefitting Moorhead and the surrounding communities; including legislation to improve flood control, increase investment in transportation and infrastructure, and provide funding to ensure access to quality education.
Senator Langseth's efforts as a member of the Senate Capital Investment Committee, including 10 years as committee chair, contributed to the success of all higher education institutions and improved higher education throughout the state.
Fall 2013 Commencement
WELCOME ................................................................. Mr. Lawrence Schwartz
Master of Ceremonies
PROCESSIONAL ............................................. “TRUMPET TUNES AND VOLUNTARIES” BY CLARK, MOURTET AND STANLEY
“Pomp and Circumstance” by Elgar
Dr. Thomas J. Strait, Professor of Music; Trumpet
Mr. Michael Olson, Music Director, First Lutheran Church of Fargo, Organ
STAFF BEARER .................................................. Dr. Theodore Graczyk
Faculty Association President
PRESENTATION OF THE COLORS ................................... Tri-College Army ROTC
NATIONAL ANTHEM .............................................. Led by Ms. Maasi Pedersen
Instructor of Music
INTRODUCTION .................................................. Dr. Edna Mora Szymanski
President of Minnesota State University Moorhead
GREETING .................................................. Dr. Steven Rosenstone
Chancellor of Minnesota State Colleges and Universities
CONFERRING OF HONORARY DOCTORATE .................. Dr. Edna Mora Szymanski
President of Minnesota State University Moorhead
COLLEGE OF ARTS, MEDIA AND COMMUNICATION
Bachelor of Arts; Bachelor of Fine Arts;
Bachelor of Music, Bachelor of Science ........................................ Dr. Timothy A. Borchers
Dean of the College of Arts, Media and Communication
COLLEGE OF SCIENCE, HEALTH AND THE ENVIRONMENT
Bachelor of Arts; Bachelor of Science;
Bachelor of Science in Nursing ........................................ Dr. Michelle L. Malott
Dean of the College of Science, Health and the Environment
MUSICAL SELECTION ............................................ “May The Road Rise To Meet You”
Traditional Irish Blessing, Music by Lori True
Performed by Ms. Holly Kaderlik
BS Music Education, Fall 2013
Mr. Michael Olson, Accompanist, Piano
COLLEGE OF BUSINESS AND INNOVATION
Bachelor of Science .................................................. Dr. Marsha L. Weber
Dean of the College of Business and Innovation
COLLEGE OF HUMANITIES AND SOCIAL SCIENCES
Associate in Arts; Bachelor of Arts;
Bachelor of Science .................................................. Dr. Randy L. Cagle
Interim Dean of the College of Humanities and Social Sciences
GRADUATE STUDIES
College of Education & Human Services
Dr. Boyd L. Bradbury, Interim Dean
College Marshal: Bruce Hanson, Associate Professor of Speech/Language/Hearing Sciences
Bachelor of Science
Barker, Kaylee Christine, Bismarck, ND, Elementary Inclusive Education, *Summa Cum Laude*
Bothun, Christopher, Beulah, ND, Speech/Language/Hearing Science, *Cum Laude*
Braaten, Katelyn Marie, St. Paul, MN, Elementary Inclusive Education
Brakefeld, Laurel Lee, Audubon, MN, University Studies
Carmeau, Jennifer Ann, Brownsville, TX, Early Childhood Teacher Education; Special Education, *Cum Laude*
Carry, Ashley Elizabeth, Rochester, MN, Early Childhood Teacher Education; Special Education, *Cum Laude*
Chaisnard, Breanna Leigh, Barnesville, MN, Early Childhood Teacher Education, *Cum Laude*
DeBlieck, Jennifer Marie, Granite Falls, MN, Speech/Language/Hearing Science; Special Education, *Summa Cum Laude*
Egeland, Meredith Ann, Fisher, MN, Speech/Language/Hearing Science; Biology
Ellefson, Victoria Sue, Ada, MN, Elementary Inclusive Education, *Magna Cum Laude*
Feigum, Brenda Joyce, Pine City, MN, Special Education, *Cum Laude*
Floura, Annette Catherine, Blackduck, MN, Speech/Language/Hearing Science
Folden, Alexandra Lee, Wahpeton, ND, Elementary Inclusive Education
Furth, Abigail Marie, Lamberton, MN, Elementary Inclusive Education; Mathematics Education, *Summa Cum Laude*
Goedert, Emily Jo, Britton, SD, Elementary Inclusive Education, *Cum Laude*
Goodburn, Jena Rachel, Madelia, MN, Elementary Inclusive Education, *Magna Cum Laude*
Lahlum, Angela Michelle, Castle Rock, CO, Elementary Inclusive Education, *Summa Cum Laude*
LeDuc, Lindsay Ann, Thief River Falls, MN, Early Childhood Teacher Education
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Marquart, Ashley Lynn, Dilworth, MN, Early Childhood Teacher Education; Special Education
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Ordahl, Samantha Jo, Milnor, ND, Elementary Inclusive Education, Early Childhood Teacher Education
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Qualey, Nanci Elizabeth, Detroit Lakes, MN, Elementary Inclusive Education
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Bachelor of Social Work
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Kretschmar, Sara Lynn, Bernsdijj, MN, Social Work; Psychology
Marshall, Charlotte Deanne, Eagan, MN, Social Work, Summa Cum Laude
Maxwell, Jens Ellen, Fergus Falls, MN, Social Work; Sociology
Mayfield, Lisa Marie, Fraze, MN, Social Work, Magna Cum Laude
McMillan, Andrea Ruth, Wimbledon, ND, Social Work
Moen, Mackenzie Lee, Odessa, MN, Social Work; Spanish, Cum Laude
Sauer, Arica, Hawley, MN, Social Work
Schaefer, Leona Mae, Zimmerman, MN, Social Work, Magna Cum Laude
Sirjord, Kristin Nicole, Gary, MN, Social Work
Spanswick, Jerica Leigh, Perham, MN, Social Work
Stompro, Sara Marie, Fargo, ND, Social Work
Strand, Tyler Leslie, Fargo, ND, Social Work
Sundberg, Arielle Jordan, Red Lake Falls, MN, Social Work
College of Arts, Media & Communication
Dr. Timothy A. Borchers, Dean
College Marshal: Dr. Michael Ruth, Professor of Cinema Arts & Digital Technologies
Bachelor of Arts
Brink, Katharine Ashley, Rosemount, MN, Art
Gregory, Brenna E., Belle Plaine, MN, Communication Studies
Jones, Robert Thomas, Moorhead, MN, Communication Studies
King, Allison, Fargo, ND, Art
Lahlum, Ian Benjamin, Moorhead, MN, Music, Theatre Arts
Low, Emmanuel, Singapore, Singapore, Communication Studies
McDonald, Kayla, Andover, MN, Art; Sociology
Notch, Christopher John, Freeport, MN, Theatre Arts; Communication Studies
Peterson, Mark John, Long Prairie, MN, Theatre Arts
Petter, Stephani Lauren, Plymouth, MN, Music Industry and Entertainment Studies
Pontius, Kelly Ruth, Fergus Falls, MN, Art, Magna Cum Laude
Provost, Rebecca Lynn, Fargo, ND, Theatre Arts
Raymo, Nikko, Bismarck, ND, Theatre Arts
Sebring, Baylee Alexandra, Red Wing, MN, Art
Swanson, Solveig Lynn, West Fargo, ND, Theatre Arts; Theatre Arts Dance, Cum Laude
Tryba, Shannon Kayle, Anoka, MN, Art, Cum Laude
Veno, Abigail Ann, Chanhassen, MN, Communication Studies; Leadership Studies
Viau, Troy Kenneth, Baxter, MN, Music, Philosophy
Bachelor of Music
Barta, Landon James, Lisbon, ND, Music Industry and Entertainment Studies, Cum Laude
Stermer, Andrew John, Montevideo, MN, Music Performance; Spanish, Summa Cum Laude
Bachelor of Science
Boethin, Krista Kay, Bismarck, ND, Mass Communications; Communication Studies, Cum Laude
Christenson, Margo Annette, Britton, SD, Advertising and Public Relations; Marketing, Summa Cum Laude
Christianson, Daniel Jordan, Grand Forks, ND, Music Teacher Education, Cum Laude
Egeland, Macy Rebecca, Bismarck, ND, Mass Communications, Cum Laude
Eisenlohr, Julie Marie, New York Mills, MN, Mass Communications, Summa Cum Laude
Fleming, Jessica Marie, Minnetonka, MN, Mass Communications
Gleeson, Mathew John, Bismarck, ND, Mass Communications, Cum Laude
Goither, Kasey Pamela, Pelican Rapids, MN, Mass Communications
Haugen, Bryce Vincent, Moorhead, MN, Mass Communications, Summa Cum Laude
Heisler, Robert James, Moorhead, MN, Mass Communications
Hendricks, Andrew John, Watertown, SD, Graphic Communications, Cum Laude
Hughes, Logan Michael, Fargo, ND, Graphic Communications, Cum Laude
Jackson, Branton Q., New Brighton, MN, Graphic Communications
Kaderlik, Holly Lynn, Lonsdale, MN, Music Teacher Education
Knutson, Anna Elizabeth, Velva, ND, Advertising
Korsten, Allison Ann, Zumbrota, MN, Graphic Communications; Marketing, Summa Cum Laude
College of Science, Health & the Environment
Dr. Michelle L. Malott, Dean
College Marshal: Dr. Wayne Chen, Professor of Mathematics
Bachelor of Arts
Awosika, Ibiakun Afolami, Plymouth, MN, Psychology; English Writing, Summa Cum Laude
Ballard, Nicole Danielle, Brandon, MN, Biology; Mass Communications, Chemistry, Honors Program
Berg, Sarah Ann, Twin Valley, MN, Biology; Psychology, Magna Cum Laude
Dell, Christina Lynn, Mapleton, ND, Anthropology
Dickman, Brianna Marie, Fargo, ND, Psychology
Engeseth, Eva Maree, Lake Park, MN, Biology
Franklin, Tyler Bryan, Fargo, ND, Biology, Magna Cum Laude
Gapinski, Alyssa Marie, Foley, MN, Psychology; Sociology
Gareis, Molly Erin, New Ulm, MN, Biology; Spanish, Summa Cum Laude
Gonzales, Felicia Marie, Savage, MN, Biology
Grausam, Jenna Rose, Springfield, MN, Psychology, Health and Medical Sciences
Halvorson, Bjorn Davin, New Folden, MN, Biology
Hanson, Eric John, Bismarck, ND, Biology
Hanson, Michael Davis, Hawley, MN, Biology
Heinen, Travis Mark, St. Cloud, MN, Anthropology; Geosciences
Henrich, McKenzie Ashlyn, Bellingham, MN, Biology
Hersch, Brittnee Marie, Perham, MN, Psychology; Women's and Gender Studies, Magna Cum Laude
Schmitz, Amelia Marie, Red Lake Falls, MN, Psychology
Serbus, Ashley Elizabeth, Belgrade, MN, Psychology, Criminal Justice
Smith, Sarah Marie, Monticello, MN, Anthropology
Spanier, Rebecca Judith, Pipestone, MN, Psychology, Spanish, Summa Cum Laude
Tripathi, Jayanti, Kathmandu, Nepal, Biology
Vohs, Jenna Nicole, Chaska, MN, Psychology; Sociology, Magna Cum Laude
Bachelor of Science
Beckel, Annie Marie, Andover, MN, Biochemistry and Biotechnology
Beers, Brittany, Sioux Falls, SD, Life Science Teacher Education, Cum Laude
Bresly, Derek Gordon, Hoffman, MN, Exercise Science: Strength and Conditioning, Coaching
Capra, Cory Thomas, Forest Lake, MN, Exercise Science; Strength and Conditioning, Coaching
Collins, Max Jeffry, North Pole, AK, Health Services Administration
Condon, Benjamin John, Maple Grove, MN, Physical Education
Conejo Cervantes, Cruz Alonso, San Diego, Costa Rica, Physical Education, Cum Laude
Dodds, Walter Donald, Frederic, WI, Physical Education; Coaching
Ekanayake, Rajinda Buwaneka, Kandy, Sri Lanka, Mathematics
Ferdig, Bridgett Jo, Blackduck, MN, Exercise Science
Gareis, Molly Erin, New Ulm, MN, Life Science Teacher Education; Spanish, Summa Cum Laude
Haertter, Johanna Elizabeth, West Fargo, ND, Physical Education, Health Education
Halvorson, Bjorn Davin, New Folden, MN, Life Science Teacher Education
Swanson, Ashley Marie, Lino Lakes, MN, Exercise Science; Health and Medical Sciences
Thotagamuwu, Gayan Arjuna, Auisawella, Sri Lanka, Chemistry; Mathematics
Toepke, Megan, New Salem, ND, Mathematics Teacher Education, Cum Laude
Wagers, Kyle Vincent, Hamilton, OH, Health Education, Physical Education; Coaching
Wallin, Carl Eric, Kensington, MN, Exercise Science
Washenberger, LeAnn Katherine, Mounds View, MN, Mathematics Teacher Education; Physics
Weber, Andrew Scott, Hutchinson, MN, Physical Education, Health Education
Williams, Barik, Milwaukee, WI, Sustainability
Wittner, Abbi Nicole, Cass Lake, MN, Chemistry Teacher Education
Wixo, Alyssa Marie, Fargo, ND, Exercise Science: Strength and Conditioning, Coaching, Summa Cum Laude
Yang, Shoua, Brooklyn Center, MN, Biochemistry and Biotechnology
Zamora Jr., Fernando, Glenwood, MN, Mathematics Teacher Education
Bachelor of Science in Nursing
Allen, Renee Joy, Duluth, MN, Nursing
Anderson, Kristen Lee, Buffalo, MN, Nursing
Blair, Laisha Renae, Browerville, MN, Nursing
Hawkinson, Megan Joy, Cambridge, MN, Nursing
Hemmesch, Lacy Marie, Eden Valley, MN, Nursing
Johnson, Michelle Lynn, Argusville, ND, Nursing
Jones, Mackenzie Ann, Fargo, ND, Nursing
Kiffmeyer, Elizabeth Ellen, Sauk Rapids, MN, Nursing
Larson, Jessica Marie, Minneapolis, MN, Nursing
Lindstrom, Emily Jean, St. Cloud, MN, Nursing
Mack, Amanda Marie, Minneapolis, MN, Nursing
McDonald, Lauren Marie, Minneapolis, MN, Nursing
Meyer, Jessica Marie, Minneapolis, MN, Nursing
Nelson, Jessica Marie, Minneapolis, MN, Nursing
Olsen, Kristin Marie, Minneapolis, MN, Nursing
Peters, Jessica Marie, Minneapolis, MN, Nursing
Rasmussen, Jessica Marie, Minneapolis, MN, Nursing
Reed, Jessica Marie, Minneapolis, MN, Nursing
Rice, Jessica Marie, Minneapolis, MN, Nursing
Rogers, Jessica Marie, Minneapolis, MN, Nursing
Santos, Jessica Marie, Minneapolis, MN, Nursing
Schmidt, Jessica Marie, Minneapolis, MN, Nursing
Shaw, Jessica Marie, Minneapolis, MN, Nursing
Simpson, Jessica Marie, Minneapolis, MN, Nursing
Stevenson, Jessica Marie, Minneapolis, MN, Nursing
Talbot, Jessica Marie, Minneapolis, MN, Nursing
Tolman, Jessica Marie, Minneapolis, MN, Nursing
Tran, Jessica Marie, Minneapolis, MN, Nursing
Vanderlinde, Jessica Marie, Minneapolis, MN, Nursing
Vogt, Jessica Marie, Minneapolis, MN, Nursing
Wagner, Jessica Marie, Minneapolis, MN, Nursing
Wang, Jessica Marie, Minneapolis, MN, Nursing
Ward, Jessica Marie, Minneapolis, MN, Nursing
Watts, Jessica Marie, Minneapolis, MN, Nursing
Wells, Jessica Marie, Minneapolis, MN, Nursing
Wilson, Jessica Marie, Minneapolis, MN, Nursing
Wright, Jessica Marie, Minneapolis, MN, Nursing
Xu, Jessica Marie, Minneapolis, MN, Nursing
Yao, Jessica Marie, Minneapolis, MN, Nursing
Young, Jessica Marie, Minneapolis, MN, Nursing
Zimmerman, Jessica Marie, Minneapolis, MN, Nursing
**Graduate Studies**
Dr. Boyd L. Bradbury, Interim Dean
College Marshal: Dr. Melanie Schicker, Program Coordinator of Healthcare Administration
**Master of Fine Arts**
Bakkum, Karl D., Moorhead, MN, Creative Writing
“In Search of Minh Tran: A Biographical Memoir”
Dr. Thomas Tammaro, Advisor
Bartholomay, Megan J., Fargo, ND, Creative Writing
“Nine Lives”
Dr. Alan Davis, Advisor
Binkard, David P., Barnesville, MN, Creative Writing
“The Wreck”
Dr. Alan Davis, Advisor
Bjorlie, Selina, Fargo, ND, Creative Writing
“Sunlight Playing Over a Mountain: A Novel”
Lin Enger, Advisor
Cole, Ian M., Blackduck, MN, Creative Writing
“Three Years Gone”
Dr. Richard Zinboer, Advisor
Gustafson, Anastasia, Fargo, ND, Creative Writing
“The Only Way Out is Through”
Dr. Richard Zinboer, Advisor
Gustafson, Andreana, Fargo, ND, Creative Writing
“D’sonnance”
Dr. Richard Zinboer, Advisor
Beckstead, Kyle, Grand Forks, ND, Educational Leadership
“Educational Leadership Portfolio”
Dr. Julie Swaggert, Advisor
Boles, Adam J., St. Cloud, MN, School Psychology
“Early Literacy Intervention with Two ELL Students and Reflections on ELL Service Delivery”
Dr. Olivia Melroe, Advisor
Burfeind, Jennifer Ann, Zumbrota, MN, Educational Leadership
“Educational Leadership Portfolio”
Dr. Julie Swaggert, Advisor
Byer, Gretchen, Fargo, ND, Curriculum and Instruction
“Engagement in Music Classes”
Dr. Leah Pigman, Advisor
Coia, Sherry M., Willmar, MN, Nursing
“Dental Examination Experience of Adults Age 65 and Older Living in a Senior Residence in Western Minnesota”
Dr. Barbara Matthees, Advisor
Cowles, Victoria Grace, Alexandria, MN, School Psychology
“Reducing Work Refusals Using the Mystery Motivator Intervention”
Dr. Olivia Melroe, Advisor
Ehlert, Ethan J., Wahpeton, ND, Educational Leadership
“Educational Leadership Portfolio”
Dr. Julie Swaggert, Advisor
Goetz, Sarah M., New London, MN, Special Education
“Attrition Rate Among Special Education Teachers”
Shannon Dahlberg, Advisor
Hansen, Kelsey, Alexandria, MN, School Psychology
Korcuska, Hannah Rae, West Fargo, ND, School Psychology
“Self-Monitoring: Increasing Eye Gaze Through a Behavioral Intervention”
Dr. Lisa Stewart, Advisor
Labrensz, Marilyn, Glyndon, MN, Curriculum and Instruction
“Re-Establishing a Collaborative Relationship”
Dr. David Tack, Advisor
Lardinois, Joseph G., Green Bay, WI, Educational Leadership
“Educational Leadership Portfolio”
Dr. Michael Couqyt, Advisor
Lissick, Alyssa R., Bernidij, MN, Special Education
“Using Games in Special Education to Increase Engagement, Independence and Academic Skills”
Shannon Dahlberg, Advisor
Lynch, Lyndsy, Breckenridge, MN, Educational Leadership
“Educational Leadership Portfolio”
Dr. Michael Couqyt, Advisor
Mayclin, Timothy, Bernidij, MN, Educational Leadership
“Educational Leadership Portfolio”
Dr. Julie Swaggert, Advisor
McArthur, Katharine E., Fargo, ND, Curriculum and Instruction
“Improving Student Vocabulary Through Differentiated Instruction”
Dr. Lynn Mahlum, Advisor
McLean, Margaret L., Hillsboro, ND, School Psychology
“The Use of a Mindful Movement Program to Improve Focus in Second Grade Students”
Dr. Gloria Joyce, Advisor
O’Brien, Lisa K., Bristol, ND, Nursing
“Incivility in Online Nursing Education: Does Incivility Exist in a Virtual World?”
Dr. Leah Pigatti, Advisor
Sellden, Brandy, West Fargo, ND, Curriculum and Instruction
“A Flipped Classroom: Reaching Every Learner at the Middle Level”
Dr. Leah Pigatti, Advisor
Svor, Jami, Fargo, ND, Special Education
“The Impact of the Picture Exchange System on Reducing Challenging Behaviors During Transition Periods”
Shannon Dahlberg, Advisor
Thielke, Lisa, Montevideo, MN, Nursing
“Describing the Relationship Between Stress Levels and Working Demographics in Associate Degree Nursing Students”
Dr. Jane Berglund, Advisor
Underwood, Christopher, Fargo, ND, Curriculum and Instruction
“Finding Our Mistakes: Student Self-Assessment on Pitch Matching and Steady Beat”
Dr. Lynn Mahlum, Advisor
Vaughan, Joshua, Moorhead, MN, Educational Leadership
“Educational Leadership Portfolio”
Dr. Julie Swaggert, Advisor
Yahna, Heather Jane, Eden Prairie, MN, Curriculum and Instruction
“Fluency Development for English Language Learners (ELL’s)”
Dr. Leah Pigatti, Advisor
Specialist
Kjonas, Rachel R., Fargo, ND, Educational Leadership
“Educational Leadership Portfolio”
Dr. Julie Swaggert, Advisor
Majors and Minors
The Commencement Program lists all majors and minors which have been completed. If a student has completed two majors, the majors are separated by a comma (English, History); if a student has completed a major and a minor, the major and minor are separated by a semi-colon (English; History). If a student has earned a minor via the Tri-College University, the home institution is listed after the minor and placed in parentheses, i.e. French (Concordia).
Graduate Candidates
Candidates receiving graduate degrees are listed in the program. The listing also contains the title of their thesis or project and their faculty advisor. Each candidate that participates in the ceremony is wearing the appropriate master’s or specialist hood. The color of the velvet edging on the hood represents the academic discipline and the silk lining (red and white) represents Minnesota State University Moorhead.
Academic Honors
Students who complete at least 60 credits at MSU Moorhead as candidates for baccalaureate degrees may graduate with the following honors:
CUM LAUDE........................................... representing a cumulative grade point average of 3.40 or higher
MAGNA CUM LAUDE.............................. representing a cumulative grade point average of 3.60 or higher
SUMMA CUM LAUDE............................... representing a cumulative grade point average of 3.80 or higher
Such honors are computed from the cumulative grade point average earned in all courses at MSU Moorhead. The graduates who have earned these honors will have the achievement listed on their academic transcripts. The CUM LAUDE graduates wear a gold cord, MAGNA CUM LAUDE graduates wear a silver cord, and SUMMA CUM LAUDE graduates wear a gold cord and a silver cord.
Honor Societies
Alpha Kappa Delta seeks to acknowledge and promote excellence in the study of sociology, the research of social problems, and such other social and intellectual activities to help improve the human condition. Sociology students are nominated by MSUM professors for membership if they are an officially declared sociology major; at least a junior; (b) elected by a standing rank in the top 55% of their class in general scholarship or have at least a 3.3 overall GPA; have at least a 3.0 GPA in sociology courses prior to initiation; and (d) complete at least four regular sociology courses. Students are inducted into the society each spring.
Alpha Phi Sigma is a National Criminal Justice Honor Society. APS recognizes academic excellence of undergraduate criminal justice majors who maintain a GPA of 3.5 or above, nominated by faculty, and in the top 35% of their class. The goals of Alpha Phi Sigma are to honor and promote academic excellence, community service, educational leadership and unity.
Alpha Upsilon Alpha, the honor society of the International Reading Association, has as its purpose the recognition and encouragement of scholarship, the development of personal and professional leadership qualities, and service to the field of reading—with special emphasis at the undergraduate and graduate levels. Established in 1985, the honor society derives its name from the Greek words Anagnosis (reading), Upotropia (scholarship), and Archon (leadership). The requirements for Alpha Tau chapter of Alpha Upsilon Alpha are: (a) completion of at least two semesters of graduate coursework, (a) overall GPA of 3.0, (c) complete at least one course in reading and language arts and earn a 3.0 in that course, (d) recommended by a literacy faculty member at MSUM. All members must be members of the International Reading Association. The motto of the Honor Society is: Legi sapere audie-dare to be wise. The
Beta Gamma Sigma honor society is an academic society for students who major in Business or Accounting. Students ranking in the top 10 percent of the baccalaureate program at schools accredited by AACSB International – The Association to Advance Collegiate Schools of Business – are eligible for this invitation. AACSB Accreditation is known, worldwide, as the longest standing, most recognized form of accreditation that a business program can earn. Since its founding as an honor society in 1933, more than 625,000 outstanding business and accounting graduates at all academic levels have earned recognition through lifetime membership in Beta Gamma Sigma. The blue and gold honor cord worn by Beta Gamma Sigma members feature a gold (metal) key charm attached to the knot above the tassel on one end of the blue cord signifying the wearer as a member of Beta Gamma Sigma.
Lambda Epsilon Chi “LEX” is a national honor society recognizing academic excellence for Paralegal students. Invitations for membership are extended to those students who are declared majors in MSUM’s Paralegal Department and who have met the following criteria: (1) Completion of at least two-thirds of the course requirements for the major, and (2) Cumulative Grade Point Average of 3.5 in Paralegal courses.
Lambda Pi Eta is the official honor society for communication studies students. It is sponsored by the National Communication Association (NCA). Students must have a 3.0 cumulative GPA and a 3.25 GPA for communication studies courses. The pin features the Greek words lambda, pi, and eta which stand for Aristotle’s three forms of proof: logos, pathos, and ethos.
Phi Alpha is a national social work honor society which was established in 1960. The purpose of Phi Alpha is to promote humanitarian goals and ideals and to provide a closer bond among students of social work. The membership requireSigma Phi Omega is the national honor society for students who major in gerontology. The purpose of SPO is to recognize excellence of those who study gerontology and aging. Local chapters serve as links within their respective communities to promote interaction between gerontology educators, students, alumni, and local professionals. Membership is open to undergraduates who are majoring in gerontology and who are in at least their second term of enrollment with a cumulative GPA of at least 3.3. Members of Sigma Phi Omega wear a blue and gold honor cord and a pin that bears the SPO emblem of the Greek letters Sigma, Phi, and Omega.
Sigma Pi Sigma is the physics honor society and was founded in 1921. Election to SPS is earned by a minimum GPA of 3.0 and demonstrated excellence in at least two of four categories: research work, outreach participation, Society of Physics Students involvement or outstanding academic achievement in physics. SPS exists to honor and encourage physics scholarship, to be of service to the various communities it may influence, and to be a fellowship of persons who have shared in the traditions and culture of physics. The name SPS comes from the first letters that compose the society motto Skeiyiz Prostatith Sunesewz which translated from Greek is “investigation, the forerunner of knowledge.” The official insignia and key consists of a voltmeter, lamp and dynamo. The voltmeter symbolizes high accuracy which is the hallmark of careful experimentation; the lamp is the symbol of knowledge (Sophia in Greek) and the dynamo represents the creative energy that is so necessary to productive research. The MSUM Sigma Pi Sigma chapter was founded in 1974.
Sigma Theta Tau International is an honorary society for nursing majors, and was founded in 1922. MSUM belongs to the Xi Kappa chapter. The founders chose the name from the Greek words Storga, Tharsis, and Tima meaning “love,” “courage” and “honor.” The founder’s vision for the society helped bring recognition to nursing as a science. Members of Sigma Theta Tau wear a white cap with a black band and a white, long closed sleeve, square at the end. The doctors gown is characterized by a voluminous bell-shaped sleeve with three velvet bars.
The hoods for all degrees are lined with silk in the official academic color or colors of the institution conferring the degree and have an edging or trim of velvet, the color of which indicates the academic discipline or faculty to which the degree pertains. The bachelors hood is not worn by candidates for the baccalaureate degree but only by those upon whom this degree has been previously conferred. The masters hood is considerably longer, has a wider velvet edging, and exposes more of the lining. The doctors hood has wide panels at the side. It has the widest velvet edging, greatest length, and fullest exposure of the lining.
The black mortarboard cap with tassel is worn with each type of gown. Those holding a doctor’s degree are privileged to wear a gold tassel. Masters and bachelors wear tassels that are either black or of the color appropriate to the academic discipline in which they are graduating.
Some of the colors which may be observed at this commencement and which symbolize various faculties are as follows:
- Arts and Humanities..............................................White
- Business Administration.................................Light Brown
- Education .........................................................Light Blue
- Fine Arts .........................................................Dark Brown
- Law .................................................................Purple
The University Mace
As the official symbol of the university’s power and authority, this staff is carried in academic processions by the University President or her designee. Lyle Laske, Associate Professor Emeritus of Art at Minnesota State University Moorhead, crafted the staff for the university in recognition of his colleague and friend Dr. Roy Royer who served as an Associate Professor of Art from 1967 until his death in 1984. The staff is sculpted from Brazilian rosewood, rubbed and polished to a deep luster. Its tips are cast in bronze and gold plated. The top piece on the staff, also in rosewood, is shaped in a universal form which symbolizes the medieval northern world. The staff rests in a base that is a solid, circular piece of rosewood.
The staff was presented to President Edna Mora Szymanski in 2008. This year, the staff bearer is Dr. Theodore Graczyk, Faculty Association President.
The Presidential Medallion
The Presidential Medallion serves as a sign of the educational mission of MSUM. The President’s commitment to the university’s purposes is renewed in each solemn academic function, when it is appropriate to wear the medallion and be recharged by its symbols.
The medallion presented to Edna Mora Szymanski upon her inauguration was designed and cast in bronze by P. Richard Szetze, a faculty member in the MSUM art and design department from 1966 to 1994.
Minnesota State University Moorhead Alumni Foundation Board Members
Corey Elmer, President
Edna Mora Szymanski (Ex-Officio)
Bob Bowlsby
David Daugherty
Lisa Erickson
Jim Fay
Rose Fortier
Tod Garje
Joe Gehlen
Sue Geyer
Lindsay Hample
Rick Kasper
Sandra Korbel
Lynne Kovash
DeWayne Kurpius
Frank Leidenfrost
Deb Magnuson
Frank Mosier
Scott Nelson
Mary Jo Richard
Tim Roche
Toni Sawyer
Terry Sonke
Mona Tedford
John Thorvilson
Minnesota State Colleges and University System Board of Trustees
Clarence Hightower, Chair
Ann Anaya
Brett Anderson
Dennis Bollig
Alfredo Oliveira
David Paskach
Maria Peluso
Timothy A. Borchers, PhD
Marsha L. Weber, PhD
Michelle L. Malott, PhD
Minnesota State University Moorhead Officers
Edna Mora Szymanski, PhD........... President
Anne Blackhurst, PhD ................. Provost and Sr. Vice President for Academic Affairs
Janet Mahoney, MS..................... Vice President for Finance & Administration
Yvette Underdue Murph, DPA...... Vice President for Enrollment Management &
Student Affairs
Laura Huth, BS .......................... Vice President of Alumni Foundation
Donna Brown, EdD..................... Associate Vice President for Diversity, Inclusion &
Affirmative Action
David Wahlberg, PhD .................. Executive Director for Communications & Marketing
Ginny Bair, MS .......................... Associate Vice President for Academic Affairs
Jean R. Hollaar, MBA .................. Associate Vice President for Finance &
Administration
Denise M. Gorsline, MA .............. Associate Vice President for Academic Planning
Karen Mehnert-Meland, MS ......... Interim Associate Vice President for Student Affairs
Daniel A. Heckaman, MBA .......... Chief Information Officer
Timothy A. Borchers, PhD............ Dean of the College of Arts, Media & Communication
Marsha L. Weber, PhD............... Dean of the College of Business & Innovation
Michelle L. Malott, PhD ............. Dean of the College of Science, Health &
the Environment
|
Lanthorn, vol. 49, no. 45, February 19, 2015
Grand Valley State University
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Recommended Citation
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This Issue is brought to you for free and open access by the Lanthorn, 1968-2017 at ScholarWorks@GVSU. It has been accepted for inclusion in Volume 49, July 7, 2014 - June 1, 2015 by an authorized administrator of ScholarWorks@GVSU. For more information, please contact firstname.lastname@example.org.
Plans to expand rec center, build new housing are approved
BY STEPHANIE BREIZINIKS
email@example.com
The Grand Valley State University Board of Trustees approved plans to expand the Recreation Center and build a new housing and academic building on the Allendale Campus during its Feb. 17 meeting.
The new housing building will be constructed near the freshmen dorms, where Robinson Field is currently located. This project costs $37 million, which will come from university-issued bonds, the University General Fund and the Housing Capital Reserve Fund. Construction will begin in March, with the goal of completion for August 2016.
The facility will include 498 beds, three classrooms, a multi-room and three faculty offices, Einstein's Bagel food service and a multi-purpose room. It will also include study space, laundry facilities and a game room. The university will tear down Icie Macy Hoobler Living Center, a 50-bed residence hall built in 1987, to make room for the new building.
GVSU can currently accommodate 5,675 students on campus, but with the expansion, there will be room for about 6,300 students by fall 2016. With this project, more beds for first-year students – who have the biggest demand for on-campus housing – will be available, which will also free up more beds for upperclassmen.
SEE EXPAND ON A2
GV ranked second for faculty Fulbright Scholars
BY HANNAH LENTZ
firstname.lastname@example.org
For the first time, Grand Valley State University was ranked second in the nation for the number of faculty members regarded as Fulbright Scholars and 15th for the number of Fulbright student recipients.
The U.S. Student Fulbright Scholarship competition is conducted at GVSU through the Frederik Meijer Office of Fellowships. The program offers an individual merit institutional grant based on merit that allow individuals to study, teach, lecture and conduct research in other countries. Faculty Fulbright Scholars are administered through the Padrón International Center.
According to The Chronicle of Higher Education, this ranking was released on Feb. 1 highlighting the high involvement of GVSU in the Fulbright Scholarship program. On top of its high rankings in numbers, GVSU is also well-regarded regarding master’s degree institutions for faculty Fulbright Scholars.
For the 2014-2015 school year, Charles Baker-Clark, associate professor of political science and management, Montenegro; Russ Rhoads, associate professor of anthropology; Sierra Leone; and Jerry Scripps, assistant professor of computing and information systems, Austria received awards to teach and conduct research in their study abroad areas.
“I am happy to represent Grand Valley State University and the people of Montenegro,” Baker-Clark said. “I plan to include my experiences in my teaching after I return.”
Washington Center offers internships
BY ALEX SINN
email@example.com
An adviser from the Washington Center spoke at Grand Valley State University’s Allendale Campus on Tuesday, Feb. 17 to spread the word of opportunities available during the 2015 summer and fall semesters.
With the opportunity presented, students can earn up to 12 credits as they develop professional skills through seminars and pursue internships with a wide range of organizations during their stay in Washington, D.C.
“You can jump into professional development while you’re still working on your degree,” said Washington Center adviser Jaclyn Sheridan.
Students enrolled in the program will participate in a leadership forum and academic courses, in addition to internships.
“You can customize it any way you want,” Sheridan said. “Everybody’s experience is totally different.”
The program is best suited for political science and international affairs majors, but all majors can benefit from the experience, Sheridan said.
The Washington Center’s internship mentorship program finds substantive internships that are tailored to suit students’ needs in a variety of fields in the government, business and nonprofit worlds.
“No matter what you’re interested in, we can find an internship to match,” Sheridan said.
Students will also have the opportunity to explore the capital and live in the city, said having hands on experiences to the students who choose to take part in this educational opportunity.
“The city itself can be such a teacher,” she said. “It’s also a really wonderful cultural center.”
The Washington Center provides apartment-style housing for students, which gives them an opportunity to build connections with other students from around the world, Sheridan said.
“It’s one of the best parts of the whole experience,” she said.
Mark Richards, GVSU Political Science department chair, said GVSU students have had successful experiences through The Washington Center.
“From our students’ perspective, all of the students have been really happy with the time they’ve put in,” he said.
Students who apply must be of sophomore standing or higher, 18 years or older and have a minimum 2.75 GPA. Applications must be approved by campus liaison Donald Zinn, chair of the Political Science department.
The deadline for the summer application is March 18.
There are many scholarship opportunities available to help defray the costs of the program, including a new GVSU scholarship that will be announced this winter.
GO TO:
www.twc.edu
FOR MORE INFORMATION
SEE SCHOLARS ON A2
EXPAND
CONTINUED FROM A1
For the Recreation Center, construction of the $7.8 million project will begin in March 2015 and the new facility is expected to open in the fall of 2016. The current Recreation Center will receive almost 17,000 square feet of additional space, with funding by the Campus Development Fund.
Campus Recreation Director Kate Harmon sat on a university committee that reviewed data from the last five years. The committee looked at the participation rate and demands on the recreation facilities.
"I was co-chair of this committee, as my previous role on campus was to oversee the Recreation Center," Harmon said. "We all agreed that it was getting increasingly harder and harder for our students to access recreation spaces."
The new addition will provide more space for cardio equipment, such as treadmills and elliptical trainers, and strength training equipment, such as free weights and weight machines. In addition, it will have more space for storing personal belongings and a new spin room.
Harmon said the need for more space came from the students.
"The largest growth trend in university recreation is in the area of fitness and wellness-related programs and services," she said. "GVSU students are very involved in recreation. Participation in recreation programming has exploded."
The Rec Center first opened its doors to a student population of about 15,000 in 1995. In 2002, the building received an addition of 18,000 square feet. However, with 25,000 students currently enrolled at GVSU and 360,000 visits last year, Harmon and others decided it is time for another update.
Occupancy is not the only reason to expand the Recreation Center.
"It is in the best interest of our students, as my previous role on campus was to oversee the Recreation Center," Harmon said. "In order to benefit our students positively, students need to have ready access to our recreational facilities and equipment."
"It is our job, as a student-centered institution, to reduce barriers that prevent our students from experiencing the benefits of regular exercise. This addition, especially will provide less wait time for students who wish to use particular equipment."
Harmon said many studies have shown that college students have performed better academically when they exercise regularly. In addition, exercise prevents illness and diseases as well as improves overall mental and physical health.
She added that a 2013 Campus Recreation survey also highlights student, faculty and staff interest in recreation. Seventy-nine percent of students said recreational facilities were important to their decision to attend GVSU.
Andy Beachnau, director of housing and health services, agreed with Harmon that expansion is necessary. Beachnau was part of the planning team that recommended the decision to university leaders, who then made the final decision based on GVSU's mission statement and goals.
"Student success is the primary outcome for program and facility decisions," he said. "Creating opportunities for student wellness is critical for student success, both academically and socially."
Beachnau added that the addition is important because it helps students start to maintain a healthy lifestyle that can continue after graduation.
Student Senate President Andrew Plague said that, although they were not involved in the decision-making process, the expansion is a good idea.
"Student Senate has been voicing its desire to see the Rec Center expanded for a couple of years now, as have many other players on campus," Plague said.
GO TO:
http://bit.ly/1Fuw6ub
FOR MORE INFORMATION
AWARD: Grand Valley State University professors Russ Rhoads and Jerry Scrippes have received Fulbright awards to teach and conduct research. Rhoads went to Sierra Leone and Scrippes went to Austria for the 2014-2015 school year.
SCHOLARS
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Student recipients of the program, who helped GVSU tie for 12th place among master universities for student study abroad, include Lydia Bennett, research grant; Giana Anne Giordano, English Teaching Assistantship, Ukraine; Erin Lutenski, English Teaching Assistantship, Germany; Hayley Pangle, English Teaching Assistantship, Azerbaijan.
Mark Schaub, the international officer for GVSU, said the program is not only important for the campus community, but also for the United States as a whole.
"It has fostered citizen ambassadors for nearly 70 years, with faculty serving as citizen-to-citizen diplomats who bring a perspective on the USA to students and communities around the world," Schaub said. "Plus, they're cheap for the taxpayer – 300,000 Fulbrighters over 70 years cost less than the next three days of the U.S. Defense budget."
With the increase in participants – and faculty and students having opportunities to bring their opportunities to a world level – information learned and experienced throughout the world can be brought back to and shared with the GVSU community, as well as promoting the idea of studying abroad and taking risks.
"The Fulbright program is important for GVSU students in that only 14 percent of GV students study abroad, but their faculty have great international and cultural experiences to bring back to their classrooms," Schaub said. "Thousands of GVSU students benefit (directly and indirectly) for many years to come through the enhanced global understandings that the faculty member has gained."
TOUR OF CAMPUS:
U.S. Senator Gary Peters visited the Grand Valley State University Pew Campus on Feb. 17 as part of his Michigan Economic Listening Tour. He met with Michigan business leaders, educators and workers as part of this tour.
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TO READ THE ARTICLE
KEVIN SIELAFF
GVL
Over 200 employers to attend Career Fair
BY CONSTANCE TURNBULL
firstname.lastname@example.org
The Grand Valley State University Career Center will host the first Career Fair of 2015 on Feb. 24 at the DeVos Place Convention Center. All GVSU students are encouraged to attend the free event, which will take place from 12:30 p.m. to 1 p.m.
Organizers of the event have arranged over 200 employers offering over 8,000 diverse opportunities for internship and employment for GVSU students.
GVSU career fairs are organized mainly by Susan Proctor, Career Center employer development manager. Proctor works with a team of staff to make sure everything runs smoothly. This team also works closely with the employers who will attend the event to plan aspects of parking, lunch, the set-up and layout of booths.
Megan Riksen, assistant director of the Career Center, said a lot of the work that goes into planning the Career Fair is the promotion of the event to students and alumni.
“I conduct classroom presentations and pass out fliers in prime student traffic areas throughout multiple buildings and campuses,” she said. “Our entire staff pitches in at all aspects of the fair.”
The event is not necessarily just for students who are currently job seeking. It is also a place for students to explore types of careers and companies they may want to work for.
“The event will offer thousands of immediate internship and job openings,” Riksen said. “It offers a great opportunity for networking face-to-face with great companies and organizations.”
The Career Fair is set up so students can easily locate potential employers. Organizations will have separate booths, and maps will help students locate companies they may be interested in visiting.
After the event, students will walk away from the event after meeting future employers with whom they can continue a relationship. Those who attend the event will gain information about the companies attending and the opportunities offered, and will potentially get offered a job or internship, she said.
GVSU student Nick Westrate obtained an internship at Aflac Insurance after speaking to his future manager at a previous Career Fair.
“I got called and interviewed by many employers who were interested in offering an internship,” Westrate said. “I loved the management at Aflac and the internship program they had. I learned a lot at the fair.”
The internship taught Westrate how to prospect, create leads and set appointments with HR departments. He learned how to prepare employer presentations and give group presentations for employees, he said.
Westrate, who will graduate in April, said his internship with Aflac gave him an advantage when searching for job opportunities after college.
“I currently have two full time offers, one in sales in the financial advice and insurance industry,” he said. “If you want to land your dream job, an internship is definitely a must to give you that experience and edge on your competition.”
However, in order to have an advantage over other candidates when searching for job and internship opportunities at the Career Fair, students should come to the event prepared.
“I was professionally dressed and had over 40 copies of my resume on hand,” Westrate said. “I also had my 30 second elevator pitch ready because I believe that that’s the amount of time you have to impress a potential employer. First impressions are a big deal; you have to set yourself apart from your colleagues.”
The event, which is sponsored by Anti-Discrimination, is not a required registration. However, students should be professionally dressed, have their student ID card available and expect to sign in upon arrival.
EATING WELL: Laura Burkett, owner of Real Food Wellness, spoke to GVSU about mindful eating, which is being aware of what you eat, to kick off Love Your Body Week. She said that students should appreciate the process of eating.
Speaker defines mindful eating as part of Love Your Body Week
BY LUCAS ESCALDAS
email@example.com
A healthy lifestyle comes from understanding one's body image and mastering mindful eating, nutrition counselor and eating psychology coach told Grand Valley State University students on Monday, Feb. 16.
Laura Burkett, owner of Real Food Wellness, kicked off Love Your Body Week by educating students on the idea and practice of mindful eating.
Mindful eating is the awareness of what is happening when one eats. Understanding mindful eating helps people understand what kind of food one needs, or how and why one is hungry.
"Many times we're so distracted or disconnected from our bodies we don't only register starving or stuffed," Burkett said. "That's still any thing wrong, it's just lost our ability to feel into what happens in between."
However, mindful eating does not necessarily have to deal with the food one eats, but rather with the situation and process involving the food.
Burkett gave simple steps for students to become mindful eaters. Elements such as deciding what to eat and asking oneself what the body is asking for are key. On the other hand, she also recommends taking deep breaths before eating and putting the fork down in between bites.
In addition, she addressed the idea that students need to think about their food and appreciate the process of eating. She criticized certain dieting strategies that do not help because they mislead eaters.
"A person can spend a lifetime always trying to err on the side of eating less, or using smaller plates, or eating with their mouth closed, and yet never be satisfied and still feel like they are fighting themselves."
There are many problems getting in the way of mindful eating. Burkett said a diet-obsessed society is causing more problems than it solves.
However, the largest challenge is the body. Many specifically confusing individuals see their body, or how they think others see their bodies.
"We have to change our life," Burkett said. "Once I change my body, then I'll take better care of myself," or "Once I change my body, then I'll be more mindful of my eating," Burkett said.
It is because of this that Burkett said too many people have negative views of their bodies. Common body image problems include visually or physically checking one's body and weighting frequently. On the other hand, many have body image problems because they ignore their body. Burkett said this is another side of the same coin - ignoring the body because it seems superficial also causes problems.
She told the story of a client who wanted to lose some weight to be healthier. The client had no health problems. What the client wanted was to be smaller because he or she was not happy with their size.
The belief that less body weight equals better health is one of the greater challenges people face, Burkett said.
"Believing weight loss equals better health is actually a problematic belief that many many people into trying all sort of dietetic foods to lose weight all in the name of health," Burkett said.
It is also a possibility that many individuals could become ironic in their appearance and health because they only change a few pounds, not an understanding of their body, Burkett said. Health is not about losing weight, it is about understanding what the body needs, she added.
Understanding and appreciating how you eat and what means can help one become healthier. Burkett said this is about understanding what part of your body is in charge of the decisions you make.
Love Your Body Week continues with different events focused on the idea of a healthy body.
Career Fair advice
BY ALLISON RIBICK
firstname.lastname@example.org
Going to the Career Fair to talk with possible employers takes guts and confidence, but it also takes a little bit of preparation. What do students need to know before going to the Grand Valley State University Career Fair on Feb. 24? Here are some tips:
1. Between 12:30 p.m. and 1 p.m. on the day of the fair, students can attend a career exploration session to get an overview of the room, but students do not need to be there to immediately get an internship or job. For more information, Career Fair can help them learn about what employers are looking for.
"It's really good to get that idea so that, over your next couple years at Grand Valley, you can start to build your resume by joining organizations and getting part-time jobs or internships," said Elizabeth Clark, a graduate assistant at the GVSU Career Center.
The main goal of the Career Fair is networking, Clark said.
2. To prepare for the interactions with employers, individuals should pay attention to their wardrobe and decide what to bring. Individuals should dress in business attire, which includes suits, slacks, blazers, blouses and button-downs.
Clark advised students to wear dark colors and avoid distracting colors and patterns. Depending on what field the student is interested in, however, there may be more flexibility, like in the arts or marketing.
Revealing clothing, excessive jewelry and makeup and strong perfume or cologne should be avoided. Facial piercings and tattoos can be distracting too, so students should be mindful about them.
3. Clark recommended bringing a portfolio or a folder with notepaper in it. Notes can include the names of the employers someone has spoken to, pressing questions and/or notes about what they have heard. Students should bring multiple copies of their resume with them as well.
"If an employer doesn't accept your resume, don't be offended," Clark said. "A lot of them are either going to throw every resume or they're not going to accept any resumes. That doesn't mean that they're not interested in you; it's just a formality that their company does."
The resume should be one page with the most important and important items, the top, like college courses, internships, jobs or organization involvement, Clark said.
4. A list of the companies that will be at the Career Fair is available at the Career Center's website. Individuals can take the opportunity to learn more about the organizations they want to visit and prepare questions for specific organizations.
Clark advised students to follow up with employers within the week by sending them an email, letter or phone call to thank the employer and indicate their interest again.
5. Things to avoid include walking in large groups of friends, having a cell phone or iPod out, bringing extra baggage, asking recruiters obvious questions or simply taking all of the free stuff companies have on their booths.
"Don't just walk around the Career Fair and see who has the coolest stuff and grab that, because it's not about what you can get, it's about getting a job or an internship," Clark said.
GO TO:
www.gvsu.edu/careerfair
FOR MORE INFORMATION AND A FULL LIST OF COMPANIES ATTENDING THE CAREER FAIR
Every newspaper is printed on recycled paper. Every issue left on the stands is then re-recycled.
We call it:
"The cycle of life."
Recently in a session for my sociology class, the students were assigned to watch a documentary on race in the United States.
The professor was not there and expected all students to do the assignment without him having to be there.
About five minutes into the documentary, students began to leave. I was confused because it seems that there is less student participation for assignments either when the professor is not present or when the assignment is not mandatory. In this case, the professor’s absence gave the students the OK to not participate and leave class.
In most cases, I am not offended when students choose not to participate in assignments that are not mandatory. There have been times that I chose not to participate. However, this time was different.
As a black student at a predominantly white institution, I feel that I am being forced to learn things about my people because I am surrounded by them. These students leaving the classroom upset me because they chose not to take an hour out of their day to learn something about me.
Quite often, I hear students making ignorant remarks in the classes I attend about the black community. Now these are students who have not experienced what it is like to live in a black community. They don’t know what it is like to walk around a store and be stared at while leaving.
Watching this movie would have given them an opportunity to try and understand the struggles of being a person of color in America, moreover at a predominantly white institution.
There were so many things that the documentary covered that I didn’t even know, which I am grateful because it gave me a chance to learn more about my ancestors. However, since a lot of the students wouldn’t relate to the black community, they didn’t care to learn about it.
There may be other reasons why the students chose not to view the movie. This documentary talked a lot about white privilege and gave evidence that it exists. I have learned that this is a truth that a lot of people don’t want to accept. This means that racism will have to be acknowledged. This also means that people will have to realize that, because of white privilege, other races are treated as inferior.
There has always been a disregard for the truth in America. People have society’s blind beliefs and self-centered. There are a lot of things going on around the world and people choose not to learn about it.
I’m tired of being in class when the teacher starts to discuss stories in the news about problems and events in other countries and the students in the class don’t know nothing about them. Yet they know what Kim Kardashian and Kanye West wore at the Golden Globe Awards.
This is definitely a huge problem that people have. Everyone should be willing to learn a little bit about another community. Although I’ve never lived nor belong to the same community or belong to the same race, everyone is still connected because of living together as a part of society.
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**QUESTION OF THE ISSUE**
*Does GVSU provide students with enough resources and opportunities to eat healthy on campus?*
**DARJAH TODD**
“I think so. I know you used to be able to request ingredients; there’s a salad bar and Subway too.”
**YEAR:** Sophomore
**MAJOR:** Film & Video
**HOMETOWN:** Detroit, Mich.
**AMBER MARSHALL**
“Yes, there is access to fruits and vegetables if you want to look for them.”
**YEAR:** Junior
**MAJOR:** Anthropology
**HOMETOWN:** Chicago, Ill.
**BRITTANY BOWEN**
“Absolutely not. You often have to pay extra for fruit and vegetable options on campus, and I think that the produce offered needs to be fresher.”
**YEAR:** Freshman
**MAJOR:** Biomedical Science
**HOMETOWN:** Whitelake, Mich.
**ELLIOIT LEE**
“No. There are two places for salad and lots of unhealthy foods all around.”
**YEAR:** Freshman
**MAJOR:** Advertising
**HOMETOWN:** Bloomfield Hills, Mich.
**NICK RANGER**
“No. There’s a lack of fruit and vegetables and lots of fried food on campus.”
**YEAR:** Freshman
**MAJOR:** International Business
**HOMETOWN:** Elk Rapids, Mich.
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**GVL EDITORIAL BOARD**
- Sarah Millenbrand
Editor-in-chief
- Stephanie Brzezinski
Associate editor
- Hannah Lentz
News editor
- Jay Bushen
Sports editor
- Shelby Pendowski
ABE editor
- Colleen Schonfeld
Laker Life editor
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**GVL OPINION POLICY**
The goal of the Grand Valley Lanthorn’s opinion page is to act as a forum for public discussion and comment among students in the Grand Valley State University community. Student opinions published here do not necessarily reflect those of the paper as an entity.
The Grand Valley Lanthorn aims to be a safe space for community discussion. The Lanthorn will not publish or entertain any forms of hate speech, but will not discriminate against any other views, opinions or beliefs. The content, information and views expressed are not approved by nor necessarily endorsed by the university, its Board of Trustees, officers, faculty or staff.
Reader submissions on the opinion page appear as space permits, and are reserved for letters to the editor only, all other reader-generated content can be submitted to the Grand Valley Lanthorn’s YourSpace page by emailing email@example.com.
Letters to the editor should include the author’s full name, address, daytime phone number, a valid email and phone number for confirming the identity of the author. Letters should be approximately 500-800 words in length, and are not edited by the staff of the Grand Valley Lanthorn outside of technical errors for clarity.
To make a submission, email at: firstname.lastname@example.org or by dropping off your submission in person at:
**ODSI KIRKHOFF CENTER**
**GRAND VALLEY STATE UNIVERSITY**
**ALLENDALE, MI 49401**
**616-822-0278**
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**QUESTION OF THE ISSUE**
*Does GVSU provide students with enough resources and opportunities to eat healthy on campus?*
**YES** 56%
**NO** 44%
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**VALLEY VOTE**
Do you feel prepared for midterms?
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**THIS ISSUE’S QUESTION:**
*Does GVSU provide students with enough resources and opportunities to eat healthy on campus?*
**LOG ON & VOTE**
LANTHORN.COM
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**BLOG**
Negotiations across cultures
By Anush Yepremyan
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**EDITORIAL**
Balanced diet
Students who want to eat healthy on campus and establish positive habits for their future have a variety of options at GV
In conjunction with National Eating Disorders Awareness Week, Grand Valley State University is celebrating Love Your Body Week until Feb. 20. Many events and activities have been organized throughout the week to bring attention to topics, such as health eating and overall wellness, eating disorders and body image.
The week began with Laura Burkett, owner of Real Food Wellness, who introduced students to the idea and practice of mindful eating. She explained that, in order to have a healthy lifestyle, one must start with mindful eating – or being aware of what is happening when we eat. Body image is important this because many people think poorly of themselves and believe, incorrectly, that losing weight is the key to a healthy self.
As college students, many of us often make excuses that eating healthy food is time consuming or too expensive for our meager budgets. However, there are many campus resources that students can and should take advantage of during their time at GVSU.
First, the Recreation Center on the Allendale Campus offers many workout classes and special programs, in addition to the standard exercise facilities it provides for members of the Laker community. There are even personal fitness trainers who will help students create a more structured program for optimal health. Students, faculty and staff can get also discounts on these services.
Second, for anyone who need assistance with food options, the Women’s Center provides a food pantry. Anyone can donate items, and anyone can obtain the items they need just by walking in and asking. Don’t be afraid to use these services. They are here for students.
To make a meal plan, campus dining can help maintain a healthy diet. Instead of free meals, students can choose healthier choices like salads or fruit. There has also been a push for more vegetarian and vegan options for those who do without meat or other animal products. While it can be more difficult to find and stick with these options, they are available.
Finally, students can also take another route and make their own food. The bus goes to Meijer, so those without a car still have a way to get to the grocery store. Additionally, cooking for yourself is helping the students prepare for life after college, and it allows them to choose exactly what they want for every meal. Students can also choose to alter recipes when making their own meals to make them healthier. Many campus housing facilities have a kitchenette or full size kitchen, allowing students to choose the route of making their own food if it they feel it is their best option.
GVSU works to provide services for students, so make sure to use these resources when it comes to healthy living. While it may be easier to buy junk food, with all the options readily available, choosing something healthy for a meal on campus is also an option.
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**Purpose of general education courses**
It’s quite common that students complain about having to take general education courses. With each added requirement, all it takes is more time and more money. Those with a declared major just want to graduate already. They want to get hired – not to be stuck in a class that is clearly irrelevant to their future.
That’s completely understandable. College is not cheap. It’s hard. We have a lot of work to do ourselves already and we’re really just trying to get by. But, with that said, general education requirements are a building block of a liberal education.
Yes, at times it’s awful. Especially when those extraneous classes start to drag your GPA. But that’s part of why we are here. To learn diverse perspectives. We look through different lenses – science, social sciences, mathematics, literature, visual, etc.
A lot of the time we discover it’s not a loss we intend to look through again. In that, we are starting to sort out our likes and dislikes. No one is going to like every course we take here at Grand Valley, even if it is for our major. It’s part of the learning experience.
Even if the only thing you can take from a class is that you realize you’re not interested in the subject, something’s helping you figure out your future – especially for the students that are undecided.
It’s important to have these general education courses because most people weren’t exposed to many of the subjects offered here at GVSU. I was fortunate enough to have classes like biology, chemistry, anthropology in high school, but many other schools didn’t. If they were able to learn about the same experience perspective, it was probably through psychology. Sometimes that push is necessary.
We just really don’t know until we try. Last semester I came into a class with a declared major. I was set on the pre-physics therapy track and was super excited. I felt secure, prepared. I had a four-year curriculum planned out.
Part of the general education process is taking a science class with a lab. After taking both biology and chemistry, I started to realize it wasn’t for me. I was thinking more about the actual job’s rather than the actual job. I was so dead set that I didn’t really consider other options, especially with the STEM focus in education.
Being exposed to from different fields introduced me to new interests and other career opportunities. It doesn’t have to be something new with science or math. I also realized how easy it was to change my major, especially as a freshman while still in these general education courses. This is another good thing about them – they truly go leg rooms.
It does feel useful sometimes, but they truly are in the curriculum for a reason.
Lakers create survey for Kentwood residents
BY DREW HOWARD
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The City of Kentwood is teaming up with students at Grand Valley State University to create a survey that will benefit the ways in which city officials communicate with residents.
GVSU students are helping to create the survey in two marketing research classes, both of which are taught by Dr. Kelly Cowart, Assistant Professor of Marketing.
"The two marketing research classes will work together to develop a single questionnaire to help the fire department, police department and the mayor's office better communicate with city residents," Cowart said. "In addition, students will work in teams to conduct other forms of marketing research such as observations and marketing for the city."
The project was initiated after the Kentwood Fire Department contacted the GVSU Marketing Department for help in developing a marketing plan in 2014.
"After speaking with city officials, we determined that additional information was needed to assist with the development of the marketing plan," Cowart said. "We recommend marketing research to gather that information."
One of the reasons Kentwood is enlisting the help of GVSU students is for their expertise on all things social media, Cowart said.
"The City of Kentwood is interested in incorporating social media in its marketing plan," Cowart said. "College students, among the heaviest users of social media, are well-suited to help identify how social media can be best used to meet the city's objectives."
Additionally, many cities are just now realizing the importance of younger generations as direct and indirect influencers of popular culture, Cowart said.
"The City of Kentwood is quite progressive in its realization that the students of today are potential residents," Cowart said. "It's wise to identify this population's preferences as a way of attracting new residents and better serving current residents."
If all goes as planned, the results of the questionnaire will influence how the city communicates with the public, its service offerings and its operations in the future, Cowart said.
The survey process began after Cowart's marketing research class met with the Kentwood deputy police chief, the deputy fire chief and the mayor on Jan. 27.
James Scott, a student in one of Cowart's classes, is among a smaller group of students in the class who are working specifically with the Kentwood Fire Department.
"We did some calls with the fire department to talk about more specifics, like how they will work with us to help distribute surveys, making focus groups and what their target market is," Scott said. "The police chief, fire chief and the mayor all had the same kind of focus as far as reaching out to the community."
The final survey will consist of somewhere from 30 to 40 questions, Scott said.
"In my smaller group, we have 15 questions," Scott said. "What we're doing is generating all the questions and putting them it into one survey for the whole class."
While students in the class will be focusing on different subject matter, the survey is still the same.
"What we're hoping for is that we will find a way to efficiently reach out to the community and raise awareness of all the services they offer," Scott said.
Van Andel Global Trade Center celebrates 15th anniversary
BY ALYSSA RETTELLE
firstname.lastname@example.org
Students, faculty, staff and members of the West Michigan community gathered at the Van Andel Global Trade Center inside the L. William Seidman Center on Feb. 6 to celebrate its 15th anniversary.
According to the Van Andel Global Trade Center's GVSU homepage, the center was founded in 1998 as an outreach arm of Grand Valley State University at the time when the Richard M. DeVos Center in downtown Grand Rapids was opened.
Amway Corporation Co-founder Jay Van Andel recognized that DeVos Center would be a focal point for international business education and activity and saw the opportunity to provide local businesses with access to valuable international resources that he himself required during the early years of developing his business.
"Some Johnson, the current director of the Van Andel Global Trade Center, said Arend Lubbers, who was president of GVSU at the time, had a vision of really connecting with community.
"One of their plans to connect with the community was to build a trade center," Johnson said. "So they implemented a research committee here at the university. They spoke with members of the business community to find out what was needed. Then ended up deciding businesses needed specialized students and assistants to be successful."
"So the goal and objective was to assist businesses in Michigan to be successful internationally. The more successful the businesses are globally, the more they'll need to grow and hopefully hire more Grand Valley graduates."
Beginning in the year 2000, the downtown building was opened and a formal dedication ceremony was held for the Van Andel Global Trade Center. Since then, it has supported more than 6,800 students and 2,000 people through consulting projects.
It has also provided more than 30 GVSU students the opportunity to receive hands-on experience conducting international market research and assisting with global projects.
"We hire a lot of Grand Valley students to work on contracts, and the students get office experience and also event planning," Johnson said. "They do marketing flyers, registrations and they set up materials. They also do a lot of research for us so they get a lot of experience.
"Many of our students aren't only from the college of business -- they span all different majors and minors. We learn a lot from the students, too, because they have fresh minds and innovative thinking. Some students get an internship credit for their time here, and all of our students get paid."
One thing Johnson said she'd like to see continue is the amount of students.
"We would love to see more Grand Valley students come in to take advantage of our resources and to get interviewing skills," Johnson said. "There are different programs we offer and a lot of networking opportunities. A lot of people just stop by and the office is always fun and different. We really, really would like to see more students get engaged and get involved in networking."
Additionally, the Van Andel Global Trade Center has taken area business representatives and GVSU faculty on trade missions to China, Canada, India and Brazil.
ARTS AT A GLANCE
KPOP UNLEASHED
Stop by the Grand Valley State University Grand River room in the Kirkhof Center tonight to catch a special performance of the Kpop Group Evolution’s annual dance showcase. The performances represent Korean pop culture through different genres and dance moves shown in Korean music. Attendees will have the opportunity to watch and learn about Korean culture, and the show will also offer the chance to eat Asian cuisine. The free event occurs from 7 p.m. to 11:45 p.m. tonight.
UNIVERSITY ARTS CHORALE
The Grand Valley State University music department is notorious for its musical excellence. The choral ensemble prides itself on creating new approaches to the choral arts. The University Arts Chorale and Cantate Chamber The Ensemble performance highlights creative aspects of choral music. Associate Professor of Music and Director of Choral Activities Ellen Pool will conduct the concert in the Cook-DeWitt Center auditorium. The event is free to the public and starts at 7:30 p.m. tonight.
LOVE YOUR BODY
GVSU Women, Gender & Sexuality Studies and the Women’s Center are sponsoring the Love Your Body: Express Yourself event Friday night in the Thomas J. Roach Jr. Kirkhof Theater as part of Love Your Body Week, which focuses on positive body image and the role social media and pop culture play in body image satisfaction. Students, faculty and staff members who are open to sharing stories, songs and poems on body image are encouraged to attend.
DR. GRINS PRESENTS CHRIS PORTER
Chris Porter, American comedian and finalist on season four of Last Comic Standing, will grace the stage of Dr. Grins inside The B.O.B. this weekend. He’s been a comic for nearly a decade and has made appearances on Comedy Central’s Live Tour, “The Bob and Tom Show” and Comedy Central’s “Live at Gotham” tour. Porter is known for his quick wit, no puppets and no catch phrases, but brings high energy and unique perspective to help distinguish himself from other comedians. Porter will take the stage tonight through Saturday, with ticket prices ranging from $10 to $20. For show times, visit http://bit.ly/LeZimi.
NEW WORLD SYMPHONY
The Grand Rapids Symphony will present “New World Symphony” to showcase Russian-American violinist Philippe Quint. The concert features Bernstein’s Serenade led by Carnegie Hall veteran artist Marco Pierangeli. Quint will perform “Serenade” and Plato’s Symposium. Tickets are available at the DeVos Place Convention Center and at the Van Andel Arena box offices starting at 9:16. The show will begin at 8 p.m. on Friday and Saturday at the DeVos Performance Hall.
INDOOR/OUTDOOR BY KENNY FINKLE
The Dog Story Theater will present Indoor/Outdoor by Kenny Finkle on Saturday. The play follows the “homeward bound” style adventures of a kitten who leaves home to find her true self. The play features four actors and is directed by Kiara Pipino as a part of the Lake Effect Fringe Festival. The festival celebrates new works and new interpretations of the classics and utilizes the motto “changing the format for live theater.” The first showing is Saturday at 3 p.m. with a second showing on Sunday at 10 p.m. General admission is $14 and $8 for students and seniors.
MOTIONLESS IN WHITE: BEYOND THE BARRICADE TOUR
The Intersection will present Motionless In White: Beyond the Barricade Tour with For Today, New Years Day and Ica Nine Kills All Ages. The tour starts on Friday in Pennsylvania and stops in Grand Rapids on Sunday. The group is known for its Gothic-inspired metalcore act and aggressive music styles. Doors open at 6 p.m. with ticket prices running $16 in advance and $18 the day of. The show is open to all ages.
Walking for a cause
GV fraternity raises awareness for multiple sclerosis
BY RACHEL HUCK
email@example.com
Despite frigid wind chills, 20s of Grand Valley State University’s Alpha Tau Omega brothers will walk from GVSU’s Allendale Campus to Traverse City for their third annual Walk Hard fundraiser next week.
Tyler Dunham, president of ATO, said that Walk Hard is held in an effort to raise both money and awareness for The National Multiple Sclerosis Society. The 160-mile trek takes eight days to complete.
“We saw a need to have a stronger impact on the community around us and made it a goal to see ourselves make a positive change on members of our community,” Dunham said.
In order to ensure that all of the money donated through the walk goes directly to the National Multiple Sclerosis Society, ATO makes it a priority to provide their own meals and sleep in churches along the way.
“We’ve pledged ourselves to ending MS,” Dunham said. “Every day, we see the effects of MS. It’s one of the few things that tell us they have MS, and it helps us realize how great our impact on friends, family and strangers around us truly is.”
Introduced in 2008, ATO was the first GVSU fraternity to emphasize Christian values rather than Greek ones, Dunham said. As a result, ATO focuses on serving as a positive influence for the campus community.
“We walk for those who can’t,” Dunham said. “We’re willing to go to great lengths to do whatever we can to join the fight.”
As ATO reaches out to get more individuals involved in their fundraiser, donations increased from $1,000 the first year to $31,000 the second year.
This year, the brother’s goal is to raise $35,000. Dunham said ATO currently has about 75 percent of its goal.
He hopes that once people are aware of the effects that the disease can have and why it needs to be stopped, we can reach state level work to prevent and combat the disease,” said Jonathan Erickson, chair of public relations for Walk Hard.
Last year, ATO started MS Awareness Week, and they say they are eager to keep it going during this year’s fundraising. Erickson said that the awareness week has become a key part of the fundraiser.
“Greek life and other student organizations have been some of our biggest supporters by helping us raise money and support during awareness awareness week, which occurs the week before the actual walk kick-off,” Erickson said.
In addition, ATO has begun to consider how to incorporate others into the walks this coming year.
“We’ve had many individuals express interest in walking for part of the trip, and that’s definitely something we’re working toward in future walks,” Erickson said. “They support two or three people world to us and those afflicted by MS, and they really do make a difference when it comes to ending this disease.”
Jacob Devine, ATO philanthropy chairman, said that Walk Hard hopes to get more people involved in the cause this year by allowing anyone to join ATO for the first portion of their walk.
“(This) can start the walk with us and walk through campus to take a picture under the arch at the Grand Valley entrance,” Devine said.
ATO will depart from the Kirkhof Center on Feb. 26 at 10 a.m.
Leadership series highlights nonprofit culture
BY MADDIE FORSHEE
firstname.lastname@example.org
Leadership is one of the key values that Grand Valley State University seeks to instill in, and students can find many opportunities to participate in developing or exercising their leadership skills throughout the year.
Through the College of Communication and Public Service, the Johnson Center for Philanthropy has created the Leadership Series to bring national philanthropic leaders from West Michigan to speak about a range of topics relating to the field.
The third installment of SLS took place on Wednesday evening with a lecture titled “Culture is the Board Room.” The four-part series is primarily geared toward students and professionals in the nonprofit or public service field, but it remains open to anyone interested in attending.
“Nonprofit boards face many challenges and opportunities,” said Annie Osburn, nonprofit services program manager for the center. “Boards with essential tools and knowledge...help their organizations run well.”
David Renz served as the speaker for the event, which the Johnson Center is offering different sectors and strategies to event attendees in an effort to bring different cultures into the boards of nonprofits. Renz incorporated his own research into the lecture’s themes.
“Culture is vital to organizational success,” Osburn said. “A healthy culture in the board room is a key piece of (that) success.”
Renz currently holds a chair in the Department of Public Administration at the University of Missouri, Kansas City. In the past, he served as a government executive in Minnesota where he received much of his education.
His research focuses on the leadership and management side of nonprofit and public service business, broadening the scope of the event to more than just professionals and nonprofit students.
Renz was chosen for the event due to the work he’s performed nationally. He founded the National Academic Centers Council and the Forum of Regional Associations of Grantmakers.
The Strategic Leadership Series aims to challenge local nonprofit organizations to increase their impacts and sustainability by bettering their organizational culture. The Johnson Center developed such a series in an effort to bring fresh thinking and the potential to do good to the region by going beyond the typical nonprofit topics and challenging the usual ways that organizations bring about change.
“The goal of the SLS focused on how to build and incorporate philanthropy culture into sustainable organizations.”
(The Johnson Center) believes learning curves shouldn’t get in the way of impact,” Osburn said.
The SLS events are free for students and $10 for professionals. Registration is required and available through The Johnson Center’s website.
GO TO:
www.johnsoncenter.org
FOR MORE INFORMATION
PHILANTHROPY: David Renz speaks at the leadership series. He used his research to highlight nonprofit culture.
Housing Trivia
1. Living Center that is attached to art building of the same name.
2. Housing center attached to the mail room.
3. Only freshman traditional style dorm named after a person.
4. The Nienayev Living Center is traditionally for those that live off campus.
5. Number of new housing facilities that opened in 1987.
6. International Housing
7. First dorm built on campus
**QUICK HITS**
**TBF TEAMS JUMP TO NO. 1, NO. 3 IN NATIONAL POLL**
The GLIAC dominated Monday’s USTFCCCA National Team Computer Rankings as the Grand Valley State men and Ashland women captured the No. 1 spots in the NCAA Division II ranks.
GVSU’s men hurdled conference foe Findlay to snag the top spot. The Lakers sit atop the rankings with 159.29 points. Ashland and Findlay follow closely behind at 148.40 and 145.99 points, respectively.
On the women’s side, the No. 3 Lakers trail No. 1 Ashland by just one point. No. 2 Central Missouri by fewer than 15 team points.
The Lakers wrapped up the GVSU Big Meet on Saturday, netting 80 provisional athletes.
Two athletes who came up big in the event, throwers Chris Sajakis and Laura Schroeder, swept the field portion of the GLIAC Track & Field Athlete of the Week laurels on Tuesday.
**GV, MSU SET FOR FIRST ROUND CLASH IN FLINT**
The Grand Valley State women’s club hockey team starts the 2015 Central Collegiate Women’s Hockey Association (CCWHA) Playoffs pitted against a well-known opponent: Michigan State.
Fourth-seeded GVSU has played fifth-seeded MSU four times this season. The Lakers tied the Spartans, 1-1, on Oct. 26, but rebounded a 5-1 decision at home in November, but found a way to ignite some third-period fireworks to beat the Spartans in back-to-back games earlier this month.
The rivals face off at Flint Iceland Arena on Thursday at 7:30 p.m.
**LAKER WOMEN TABBED AS NO. 8 TEAM IN MIDWEST**
The Grand Valley State women’s basketball team was one of five GLIAC teams listed in the Initial NCAA Midwest Regional Rankings, which were released Wednesday.
GVSU (18-7, 13-6 GLIAC) currently owns second place in the North Division.
**VARSITY SCHEDULE**
**WOMEN’S HOOPS**
- **Tonight @ SVSU 8pm**
- **Saturday vs Hillsdale 8pm**
**MEN’S HOOPS**
- **Tonight @ SVSU 8pm**
- **Saturday vs Hillsdale 8pm**
**TRACK & FIELD**
- **Friday vs GVSU Tune-Up**
**SOFTBALL**
- **Friday - UWP 10am***
- **Friday - UIS 12pm***
- **Saturday - UMSL 7pm***
- **Saturday - Upper Iowa 10pm***
- **Sunday - KWC 10am***
**BASEBALL**
- **Saturday @ USI 3pm**
- **Saturday @ USI 2pm**
- **Sunday @ USI 1pm**
**MEN’S TENNIS**
- **Sunday vs UNOH 8am**
- **Wednesday vs FSU 8pm**
**WOMEN’S TENNIS**
- **Sunday vs UNOH 8am**
*Games played at Lewis Dome Tournament (Rosemont, Ill.)*
---
**COLUMN: THE MOUNT RUSHMORE OF GV**
**BY JAY BUSHEN**
@PETEBARBOWS
SPORTSBLANTHORN.COM
**PB:** Happy Presidents Day, Jay! I say so because its one of my favorite federal holidays – a holiday that can rival Columbus Day at least – but it seems to me that when it comes to holidays, its as criminally underrated as James Polk. How do you celebrate a Spin “Dead President” on your head and your head? Wear a powdered-wig to class? Debate which presidents should comprise Mt. Rushmore?
**JB:** You know I’m living in the Presidents’ Day bubble on this end, brought to me courtesy of the Red, White and Blue and Toby Keith, but what does the rest of you know about Grand Valley State Athletics?
**PB:** I don’t know much about anything, but I am talented at shoveling snow. Just last week I started a fire that was a dead ringer for Louie the Laker – you know if you cock your head and squint. Presidents, Lakers, heads in the wall – it’s too bad there’s not a way to tell all those apart.
**JB:** A sculpture of Louie? Your talents transcend the literary form. But I suppose you should transition from snow to Star and then to the great and snubs of GVSU’s Mt. Rushmore. Contextually, we can take this in a number of directions. Which era shall we cover? Since 2000? 2010? This academic year? Last month? Last week?
**PB:** If we’re keeping it to last week, I want context. When I went down to rec, moved around and got a double-triple. That’s three perfectly set picks and three bricked shots when I got the ball back on the roll.
**JB:** I’m not necessarily a ring guy, but Finney – a guy who means so much to the program – won three of them on his way to becoming college football’s first quarterback with an astounding 51-4 mark under Brian Kelly and Chuck Martin from 2003-06. A winner. Tough decision, but I’ll take Finney.
**PB:** I think our next nomination is a no-brainer and, yes, it’s another gridiron great. Tabbed as GVSU’s most decorated athlete, this Laker legend won two football state titles, also a four-time All-American wrestler. And a golfer. And a baseball player. And a track and field athlete. That man, however, is Jamie Hostos (class of 1977).
**PB:** Anes was GVSU’s Danny Wuerffel. He took home the Harlon Hill – the Division II Heisman – in 2002, and became the first Laker QB to get fitted with a ring that same year by his coach, what is it? Co? But if Anes is Wuerffel, that must make Finney GVSU’s Tim Tebow, and if I’ve learned anything from the Godfather, besides what to ask for at the restaurant, is that the original isn’t always the best. I’m partial to Part II, myself, and no Gator in their right mind is taking Wuerffel over Anes. And they must know something since they’re still living through Michigan winters.
Now that it’s agreed we’ve found our George Washington, it looks like we’re on to Teddy Roosevelt. Names like Kayla Addison and Jamie Potts could be considered for the part on a contemporary list, but there’s really only one better than Anes – GVSU’s Jim Thorpe – to play that role on our wall. The original isn’t always best, but sometimes it is.
That’s not looking at, and at someone’s best. Who’d a thank we could manage?
**JB:** Rolling. Now it’s just you, me, two Rushmore faces and about three readers remaining. At least we haven’t missed the main course.
Hard to overlook multi-sport athletes. You mentioned a pair of solid chaps, Potts has been an All-America performer in football and baseball. Also made a position switch, as you know, from QB to TE – and then TE to WR last fall. Versatile. In 2014, dude hit a GLIAC-best .412 and leads the conference in both receptions in 54 receptions and leading 10 touchdowns. Hard enough to be an All-American, but in two sports? Impressive. No rings, though, at least not yet.
**PB:** I was there when softball slugger Katie Martin smoked out the shot heard around GVSU and with one swing, simultaneously passed Karen Kuhlman’s record for most hits (259), runs (164) and home runs (47). I witnessed Dani Carroll gut out games on the hardwood after being knocked out for turn a game behind the dynamic sport duo of Brianna and Brittany Taylor, the reigning women’s soccer NCAA Division II Player of the Year Marti Cory had been the heart and soul of my team as she helped to guide GVSU to a national title in Georgia in 2013, pole vault extraordinaire Kristen Hixson piece together one of the most prolific seasons in Division II history and fielder ever seen and current Minnesota Vikings receiver Charles Johnson make 23 different NFL scouts gawk at their stop watches, all within a few days. If I tasked to sculpt a Laker Rushmore with only faces of athletes, I’ve covered, I’m sure I could, but there would be difficult decisions to make.
---
**SEE RUSHMORE ON AB**
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**A new era**
Softball players look to new leader, Callihan, in ’15 after longtime coach’s retirement
**BY BRODIE ORENT**
email@example.com
The Grand Valley State softball team will turn the page on a new chapter in 2015 with its best second-full-time softball coach in school history.
Dana Callihan was named head coach of the Grand Valley State softball team in May and her first season as head coach is already looking promising.
GVSU’s season will kick off on Friday, Feb. 20 at 10 a.m. against Wisconsin-Parkside in The Dome at the Ballpark in Rosemont, Ill. The Lakers have a total of five games this weekend scheduled in the Lewis University Flyers 2015 Dome Invitational.
Callihan said he is very proud to have the opportunity to take control after being the top assistant to coaching legend Doug ‘Doc’ Woods for 13 seasons.
“I’m proud to have been handed the reins,” Callihan said. “I am working very hard to continue the established tradition.”
Callihan said that Woods taught him many things, but the most significant was recruiting.
“He had a great eye for talent and a tremendous network; both are crucial to being successful in the future,” she said. “We are quite young with seven freshmen and several sophomores. We have new players at just about every position and we will see who will step up.”
The Lakers lost five starters from last year’s team, leaving big holes to fill. Callihan said it may take a few games to replace them.
One player the Lakers are looking to depend on is junior pitcher Sara Andrusik.
Last year, Andrusik was named to the CoSIDA All-American Division II team and was named the GLIAC Freshman of the Year in 2013. Andrusik finished last season with a 12-7 record and a 4.00 ERA in 124.1 innings pitched last season.
Andrusik said this year’s focus is on strengthening...
Valentine’s day ballad
Column: Mascots, security hoops spark good times at Fieldhouse
by JAY BUSHEN
firstname.lastname@example.org
I should preface this by confessing to a childlike enthusiasm for all types of mascot-related shenanigans. That brand of humor just does it for me.
This story begins with a 14-mile commute from Grand Rapids to Allendale. It’s Saturday night, Valentine’s Day, and my romantic endeavors are placed aside on account of my love for the sports journalism. Go team.
While instructing a faithful way into the Fieldhouse Arena parking lot, I begin my routine: The number of cars suggests a poor turnout. Grand Valley State students are elsewhere, perhaps searching for love. In my head, it’s warmer where they are.
Whatever.
Not as many fans as usual in the house tonight, but I managed to park row safely. The GVSU and Northwood teams are warming up. I do the same, which involves less stretching, more sitting and numbers.
The big one tonight is four, the number of games left. The Lakers are 6-12 in conference play; Northwood is 9-9. The GLIAC Tournament place is becoming clearer by the point: Lake State, Saginaw, Michigan Tech and Ferris are running the North Division show. They’re in. The South gets some spots.
One spot is up for grabs. Does GVSU still have a chance at keeping the 10-year streak of tournament appearances alive? The check schedule doesn’t look good, but I sure hope it’s Lakers and this six-game home slide tonight. Some hope.
Then more hope. I spot the Griffins mascot, Griff, from across the gym. Then I see a basketball-headband head too. I’m told it’s Buckets from the Grand Rapids Drive. Where’s Kris? Crash from the White Caps? And Louie’s here?
Hold up. Four mascots in the same place, at the same time?
I spend what feels like five (but is more like 25) minutes laughing. Griff’s clownin’, turning my stat sheets into paper airplanes and pretending to eat my head. I lose focus completely, but I’m feeling better.
Things are looking good for GVSU, too. Ryan Sabin knocks down a jumper at the 8:41 mark, putting the Lakers up 20-16, and he’s the fifth Laker to score. Meanwhile, Ricky Carbalaj keeps making shots from the low block.
At the 8:24 mark, assistant and Aaron Hayes check in for GVSU. Had a chance to write about Hayes earlier this year. Things haven’t gone as planned thus far: He started late with a hamstring injury, came back, hurt his other hamstring and came back again, and finally came back.
It’s 32-25 GVSU at halftime. I find out why the mascots are here – a dance-off against the Laker Dance Team. I watch as the Lakers compete with the best.
Four points from Sabin make it 35-28, then Luke Ryksamp nets a fast-break layup. Sabin scores the next five, followed by a bucket from Slick Rick down low through the post. Ryan Hayes completes a three-point play -- then a triple, from Carbalaj, and in the blink of an eye it’s 52-32.
GVSU cruised to an 80-63 victory, stayed in the hunt for the title, and it couldn’t be looser and played to its potential. These guys were feeling it. Confident. I saw swagger I haven’t seen since last season.
Keep in mind, this is a Laker team that had just eight of its 15 players healthy enough to practice at all once this year. It’s been tough, but there are reasons to be optimistic with three games left. More than I thought.
All five teams competing for the GLIAC 8 spot (GVSU tied 0-2), Hillsdale (9-10), Malone (8-11), GVSU (7-12) and Northern (7-12); have tough schedules down the stretch, not just GVSU.
The teams are playing at home this Saturday, which could potentially be a big game.
The Lakers will probably have to win at SVSU and at FSU, but they went 4-0 against those teams last year with help from leading point guard Rob Woodson, who led the Lakers to a 0-2 record on those teams, but Woodson’s replacement is back. They’re healthier than they’ve been in weeks, and they’ve finally got some momentum.
I’m in Allendale last night in a good mood. Was it the mascots? Probably, but it was refreshing to see the Lakers stand tall and band together with their season on the line.
Win or lose, this is a team that deserves its home fans on Saturday night. Might not be four mascots there, but still worth traversing through subzero temperatures.
Griff’s clownin’, turning my stat sheets into paper airplanes and pretending to eat me. I lose focus completely, but I’m feeling better.
RUSHMORE
CONTINUED FROM A7
That long, winding list above was just a few of the names I could mention, and that’s just without accomplishments in six-year spans. It’s an embarrassment of record-breaking, going-pro, doing generally-impressive stuff riches.
I think there’s room on the wall for one relatively contemporary name, however – the question is whose? Head track and field and cross country coach Tim Bales. His barn dragon layer/office is overstuffed with hardware, current Iowa State and soccer coach Dave Dillman was accomplished as a coach in Division II and Doc Woods was not only a stellar softball skipper, but also an amiable man that helped build a strong athletic training powerhouse. Kelly and Martin, as you mentioned, have both found success as head coaches in major Division I programs and could be easily argued for, too.
JB: I’ve covered my share of greats, as when I covered baseball while with Grandval Bragroni broke the school’s home-run record. I watched Kaitlyn Wolters rise from “nameless” from the bench to GLIAC Volleyball Player of the Year as she led GVSU to a ridiculous Final Four run. I was a few rows when Kendra Foley earned the championship and confided her perfect game schedules in complete and utter futility. He was all business, but when he turned to speak, he met my eyes. You could tell he didn’t really want to be wasting any more time than he had to on something that wasn’t helping to get his team better, but he was honest, respectful and thoughtful. The exchange took less time than the wait, but it was a quality interview.
The other guy that catches intensely every practice and every meet, but he also is a smart man that seems to have a fatherly rapport with his athletes. And despite all his successes, he’s not too big for the job. Not even to do an interview with a rookie reporter. That’s what makes him great, and I have no problem giving him the nod.
JB: Three down, one to go, and I look like a moron. We’ve covered about 11 percent of the candidates we had originally planned on, but we’ve got to beat the buzzer and make a fourth decision. One assist shy of the triple-double I’ll pass this one off.
PB: And then there was one. There’s a lot of different ways we could finish, but it seems to me there are still a few elements essential to GVSU’s athletic prowess not represented on our list.
Arend D. Lubbers – one of the most highly-touted tenured university presidents in the history of universities – is a shoe-in as head on GVSU’s all-around Rushmore, and should be included on this list, too. Without him, there’s no football at GVSU and Lubbers Stadium – consistently an attendance leader among Division II venues – is a major landmark here in Allendale, Mich.
Tim Selge – also deserving of a spot, and you’d have trouble finding another athletic directors on any level of sport more accomplished than he. If you don’t believe me, stroll past his office and take a look around yourself in the rec. You’ll see more GLIAC Presidents’ Trophies – an award given annually to the best overall athletic program in the conference – in cases than you will see students on the wall. GVSU has won 16 straight, according Selge’s watch.
With those nominations in mind, we’d be crazy not to include Joan Boudin. Plenty of made athletes and teams have had enormous success at GVSU, but the mainstay of this program – even more than football – is its continued excellence in women’s athletics. GVSU has won four national championships, all but four have been won by women’s teams. The list starts with Joan.
Not only did she pioneer women’s athletics at burgeoning GVSU, she held coaching positions in basketball, volleyball, softball and track & field from 1970 until 1994. During that span, she garnered more than 700 total wins, 10 GLIAC championships, two GLIAC coach of the year awards and countless more, as she has for the past 30 years, to serve as an advocate for women’s sports in committees across the country.
The average daily cost to heat GVSU in December was $24,055 (natural gas). Costs for January & February are expected to exceeds that number.
Perfect season rolls along as dodgeball club wins another MDC
BY MARK WASHBURN
email@example.com
If you look into the corner of the gym, you will see a pile of 27 wins from the current season. Add that to the 12 wins to finish out last season, and Grand Valley State has 39 consecutive.
The Lakers (27-0) won all three games on Valentine’s Day in the Michigan Dodgeball Cup by defeating Central Michigan, Michigan State and Saginaw Valley State in East Lansing. Monday was the seventh time that Grand Valley State has claimed the Michigan Dodgeball Cup title.
The Lakers got the run started in a game against CMU. GVSU dominated the Chippewas in a game that featured power and physicality – from the Lakers.
“They had a hard time handling our pace because we were running faster and throwing harder,” said senior assistant captain Dylan Fettig.
Central Michigan also had a hard time handling freshman Kurtis DeYoung.
“(DeYoung) was not on their radar, and he was throwing well so we kept giving him the ball and he kept taking them off,” Fettig said.
The Lakers used a fast-paced play threw the Chippewas off their game, but it was exactly the start the Lakers were looking for in their quest for the cup.
“The game against CMU set the tone for us today,” said day, said senior captain Kevin Bailey. “The last few times (against CMU) have been close, but this really helped us moving forward.”
In the GVSU/MSU matchup, the teams played evenly in the first half. The Lakers were able to get off to a good start scoring a point 4:15 into the stanza, but the Spartans slowed down the pace and were able to secure a point that led to a 1-1 halftime deadlock.
“We’ve played them seven times (heading into the game), so they knew what to expect and they slowed the pace down and made smarter throws,” said senior Austin Morley.
Coming out of the half, the Lakers were on the same page after Bailey gave the team a halftime talk. After the break, GVSU dominated scoring points with their disciplined dodgeball to defeat the Spartans 3-1. MSU’s one point was the only point GVSU gave up all day.
“Being on the same page in the second half really helped us (beat MSU) because we didn’t give them a ball advantage, and we didn’t have any wasted throws,” Fettig said.
Senior Trevor Nordberg has been vital for the Lakers success recently. He stepped up in the national tournament at DePaul, and stepped up again in East Lansing.
“He really excels in a fast-paced game, is an accurate thrower and really helps us get an edge,” said Bailey.
The Lakers finished the day against rival Saginaw Valley State. SVSU was no match for GVSU, as the Lakers topped the Cardinals 4-0.
GVSU won the first point relatively quickly and didn’t look back. The team’s energy and focus was present throughout the match, and fatigue caught up to the Cardinals.
With GVSU up 2-0 at the half, they played younger players to give them more experience. This allowed them to enjoy the big-game atmosphere and develop big-game situations.
Of course, the Lakers feel good about being able to bring home the Michigan Dodgeball Cup after falling to SVSU in last year’s championship.
“It feels good to have the cup back,” said Morley. “Overall we had a pretty good day, and based on how we performed, it gives us confidence going forward.”
The 39 wins in a row is two shy of the National Collegiate Dodgeball Association (NCDA) record. GVSU won 41 straight between 2006-10.
They will have a chance to break their record at the CMU Showdown on March 21. GVSU will play four games that day and tangle with James Madison University, who is also undefeated, in a brawl for No. 1 seed in the national tournament.
|
BEFORE THE PUBLIC UTILITY COMMISSION
OF OREGON
UG 360
In the Matter of
AVISTA CORPORATION, dba AVISTA UTILITIES,
Purchased Gas Cost Adjustment, Schedule 461 and 462.
ORDER
DISPOSITION: STAFF’S RECOMMENDATION ADOPTED
At its Special Public Meeting on October 11, 2018, the Public Utility Commission of Oregon adopted Staff’s recommendation in this matter. The Staff Report with the recommendation is attached as Appendix A.
BY THE COMMISSION:
[Signature]
Michael Grant
Chief Administrative Law Judge
A party may request rehearing or reconsideration of this order under ORS 756.561. A request for rehearing or reconsideration must be filed with the Commission within 60 days of the date of service of this order. The request must comply with the requirements in OAR 860-001-0720. A copy of the request must also be served on each party to the proceedings as provided in OAR 860-001-0180(2). A party may appeal this order by filing a petition for review with the Circuit Court for Marion County in compliance with ORS 183.484.
STAFF RECOMMENDATION:
Staff recommends approval of Avista Corporation dba Avista Utilities’ (Avista or Company) Advice No. 18-01-G, which is the Company’s 2018 annual Purchase Gas Adjustment (PGA) tariff sheet updates, for service rendered on and after November 1, 2018.
DISCUSSION:
Issue
Whether the Commission should approve Avista’s 2018 annual PGA as reflected in its Advice No. 18-01-G.
Applicable Rule or Law
ORS 757.205 requires public utilities to file all rates, rules and charges with the Commission. ORS 757.210 provides that the Commission may approve tariff changes if they are fair, just and reasonable. Filings that make any change in rates, tolls, charges, rules, or regulations must be filed with the Commission at least 30 days before the effective date of the changes.
ORS 757.259(5) states that unless subject to an automatic adjustment clause, amounts deferred under ORS 757.259 shall be allowed in rates only to the extent authorized by the Commission in a proceeding under ORS 757.210 to change rates and upon review of the utility’s earnings at the time of application to amortize the deferral. The
Commission may require that amortization of deferred amounts be subject to refund. The Commission's final determination on the amount of deferrals allowable in the rates of the utility is subject to a finding by the Commission that the amount was prudently incurred by the utility.
ORS 757.259(6) states that the overall average rate impact of the amortizations authorized under this section in any one year may not exceed three percent of the utility's gross revenues for the preceding calendar year. ORS 757.259(7) allows the Commission to consider an overall average rate impact greater than that specified in subsection (6) for natural gas commodity and pipeline transportation costs incurred by a natural gas utility, if the Commission finds that allowing a higher amortization rate is reasonable under the circumstances.
OAR 860-022-0025 requires that revised tariff filings include statements showing the change in rates, the number of customers affected and resulting change in annual revenue, and the reasons for the tariff revision.
OAR 860-022-0030 requires that tariff filings which result in increased rates include statements showing the number of customers affected, the annual revenue under existing schedules, the annual revenue under proposed schedules, the average monthly bills under existing and proposed schedules, and the reasons supporting the proposed tariff.
The PGA mechanism was originally established by Order No. 89-1046 to minimize the frequency of gas cost-related rate changes and the fluctuation of rate levels pursuant to ORS 757.259(2)(e). Since the mechanism's creation in 1989, the Commission has issued a series of orders concerning PGA filings through open-docket UM 1286.\(^1\) Order No. 14-238 is the most recent of these orders, which set out the Commission's procedures and requirements concerning the processing of PGA filings.
**Analysis**
On July 27 and September 9, 2018, Avista submitted Advice 18-01-G, which constitutes its annual PGA filing (Initial 2018 PGA Filing). Avista subsequently filed a supplement to its initial filing (Supplemental 2018 PGA Filing). In aggregate, the filings are commonly referred to as the 2018 PGA filing (2018 PGA Filing). The 2018 PGA Filing is comprised of two parts: a forward-looking part (Projected Purchased Gas Cost for the
---
\(^1\) PGA Guidelines were acknowledged by the Commission in Docket No. UM 1286, Order No. 09-248, on June 23, 2009. The Guidelines in Docket No. UM 1286 have been modified three different times since they were first acknowledged by the Commission, in Order No. 10-197, in Order No. 11-196, and in Order No. 14-238.
2018-2019 Gas Year) and a backward-looking part (True-Up of the 2017-2018 Gas Year).
The Projected Purchased Gas Cost for the 2018-2019 Gas Year projects the costs of natural gas for the upcoming gas year (i.e., 2018-2019 Gas Year)\(^2\) and results in the new rates set forth in Schedule 461.\(^3\) The True-Up of the 2017-2018 Gas Year trues up the costs of natural gas in the previous gas year (2017-2018 Gas Year) by comparing the amount collected from customers in that year with the actual costs incurred by the Company in the same year.\(^4\) Any over- or under-collection from customers in the 2017-2018 Gas Year, together with any over- or under-collection from previous years,\(^5\) is either given back (in the case of over-collection) or surcharged (in the case of under-collection) to customers in the upcoming gas year. The True-Up of the 2017-2018 Gas Year results in the new rates set forth in Schedule 462.\(^6\)
**Projected Purchased Gas Cost for the 2018-2019 Gas Year (Rate Schedule 461)**
The Projected Purchased Gas Cost for the 2018-2019 Gas Year comprises two rate components: 1) the commodity component rate and 2) the capacity or demand component rate. The rates for these components are represented in Table 1 in $ per therm.
---
\(^2\) The 2018-2019 Gas Year begins on November 1, 2018, and ends on October 31, 2019.
\(^3\) Schedule 461 is titled “Purchased Gas Cost Adjustment Provision - Oregon.”
\(^4\) The 2017-2018 Gas Year covers the period beginning on November 1, 2017, and ending on October 31, 2018. However, per page 10 of Appendix A to Order No. 14-238 in Docket No. UM1286 (See: [http://apps.puc.state.or.us/orders/2014ords/14-238.pdf](http://apps.puc.state.or.us/orders/2014ords/14-238.pdf)), all deferrals to be amortized into rates will be based on June deferral balances plus interest for July–October, and the deferrals that occur after June will be carried forward to the next PGA period.
\(^5\) Any over-collection or under-collection from previous years is the result of the fact that actual volumetric sales of natural gas will always be different from forecasted volumetric sales. Since amortizations are intended to be recovered in volumetric forecasted sales, a remaining balance will always be present.
\(^6\) Schedule 462 is titled “Gas Cost Rate Adjustment - Oregon.”
Table 1: Projected Purchased Gas Cost for 2018-2019
(in $/Therm or as noted otherwise)
| Item | Current Rate 2017-2018 Gas Year | Proposed Rate 2018-2019 Gas Year | Change |
|----------|---------------------------------|----------------------------------|--------|
| Commodity| (A) | 0.20072 | 0.20323| 0.00251 |
| Demand | (B) | 0.18539 | 0.16723| -0.01816|
| Total Gas Cost | (C = A+B) | 0.38611<sup>8</sup> | 0.37046<sup>9</sup> | -0.01565 |
The commodity component of the weighted average cost of gas proposed for the 2018-2019 is increasing slightly by $0.00251 per therm. Prices remain generally stable due to continued high natural gas production levels and an abundance of natural gas in storage, as shown above in Table 1. The proposed demand component reflects a decrease of approximately $0.01816 per therm.<sup>10,11</sup> This decrease is largely due to new transportation rates on Williams Northwest Pipeline.<sup>12</sup>
Avista’s 2018 PGA Filing meets the PGA Filing Guidelines and the Natural Gas Portfolio Guidelines. Avista has demonstrated its adherence to these Guidelines with regard to natural gas supplies and financial hedges.<sup>13</sup> Staff’s conclusions are supported by the Company’s comprehensive work papers and by review and discussion as part of the quarterly meetings.
---
<sup>7</sup> See the Supplemental Twelfth Revision Sheet 461 issued on September 15, 2017, “Rate” section approved by the Public Utility Commission of Oregon in Order No. 17-416 of Docket No. UG 339.
<sup>8</sup> The Company requested an out-of-cycle update to its amortization rate for cost of gas purchased in the 2017 PGA. The Commission approved updated Tariff Sheets in Docket UG 345/Advice No. 17-10-G, Order 18-041.
<sup>9</sup> Supplemental 2018 PGA Filing at Schedule 461.
<sup>10</sup> Demand costs reflect the cost of pipeline transportation to the Company’s system, as well as fixed costs associated with natural gas storage.
<sup>11</sup> This rate does not apply to Schedule 440 Interruptible Customers This rate schedule is applicable to commercial and industrial customers who are subject to interruptions in capacity and supply. For interruptible customers, the Company does not secure firm interstate pipeline capacity. As a result, customers served on this rate schedule do not pay transportation or demand charges.
<sup>12</sup> The Williams Northwest Pipeline (WNP) Settlement agreement was approved by the Federal Energy Regulatory Commission (FERC) Settlement Order dated August 18, 2017. The new rates became effective on January 1, 2018, with additional rates to phase in beginning October 1, 2018.
<sup>13</sup> Accepted “best practices” for the purchase of natural gas supply by local distribution companies (LDCs) is portfolio construction that balances the objectives of reliability, cost, and price volatility using the tools of diversity, flexibility, and balance. The “Natural Gas Portfolio Development Guidelines” (Portfolio Guidelines) implement these “best practices” for Oregon LDCs. The Portfolio Guidelines require gas utilities to include certain information related to their gas supply portfolio with their annual PGA filing. This information allows the Commission to determine the prudence of the utility’s costs. Staff’s analysis of and conclusions regarding Avista’s natural gas supply portfolio and related purchasing strategies and actions are based on the Portfolio Guidelines in Docket No. UM 1286.
Staff reviewed Avista’s forecasted commodity and demand costs to determine whether Avista complied with the Commission’s Natural Gas Portfolio Development Guidelines (Portfolio Guidelines).
Accepted “best practices” for the purchase of natural gas supply by a local distribution company (LDC) is through a portfolio that balances the objectives of reliability, cost, and price volatility using the tools of diversity, flexibility, and balance. The Portfolio Guidelines implement these “best practices” for Oregon local distribution companies (LDCs). The Portfolio Guidelines also require each gas utility to include certain information related to its gas supply portfolio with its annual PGA filing. This information assists the Commission in determining the prudence of the LDC’s costs.
Avista’s portfolio preparation and planning process meets the standards in Section III of the Portfolio Guidelines related to portfolio planning, as do Avista’s physical gas contracts and financial transactions relating to natural gas pricing. Avista has also demonstrated its adherence to the Portfolio Guidelines with regard to natural gas supplies and financial hedges. In addition, Avista has provided all the information called for in Section IV (Information and Workpapers), and Section V (Supporting Data and Analysis) of the Portfolio Guidelines.
True-Up of the 2017-2018 Gas Year (Schedule 462)
Table 2: True-Up of the 2017-2018 Gas Year \(^{14}\)
(in $/Therm or as noted otherwise)
| Item | Current Rate | Proposed Rate | Change |
|-----------------------|--------------|---------------|----------|
| Commodity Amortization| -0.05278 | -0.08021 | -0.02743 |
| Demand Amortization | -0.02580 | -0.01383 | 0.01197 |
| Total Amortization | -0.07858\(^{15}\) | -0.09404\(^{16}\) | -0.01546 |
As for the commodity amortization, the new rebate rate of $0.08021 per therm reflects a refund of approximately $6.9 million, which comprises approximately $7.2 million\(^{17}\) of over-collections in the period from November 2017 to June 2018 and approximately $348,857\(^{18}\) of under-collections from prior years. The $6.9 million figure is after
---
\(^{14}\) Positive numbers represent surcharges; negative numbers or numbers in parentheses represent refunds.
\(^{15}\) Supplemental Seventh Revision Sheet 462 issued on July 28, 2017, “Rate” section, approved by the Public Utility Commission of Oregon in Order No. 17-416 of Docket No. UG 339.
\(^{16}\) Supplemental Eighth Revision Sheet 462 attached to Avista’s 2018 PGA Filing.
\(^{17}\) See page 27 of 27 in Avista’s workpapers filed with its Supplemental 2018 PGA Filing (i.e., row “61”, Column “L”.
\(^{18}\) See page 27 of 27 in Avista’s workpapers filed with its Supplemental 2018 PGA Filing (i.e., row “60” Column “L”.
accounting for the commodity cost variance sharing between the Company and customers as required by Order No. 08-504 in Docket No. UM 1286.\textsuperscript{19} For the reasons mentioned previously in this memo, actual wholesale natural gas prices were lower than the level approved in the Company's 2017 PGA.
As for the Demand Amortization portion, the Company is currently rebating $0.02580 per therm to customers (except Rate Schedule 440).\textsuperscript{20} The Company proposes to reduce the amount to be charged to customers to $0.01383 per therm. The new rate reduces the customer rebate by approximately $1.0 million to approximately $1.1 million, which comprises approximately $766,051\textsuperscript{21} of an over-collection in the period from November 2017 to June 2018 and an over-collection of $376,408\textsuperscript{22} from previous years.
\textit{Three Percent Test}
Pursuant to ORS 757.259(6), ORS 757.259(7) and OAR 860-027-0300, the annual average rate impact of the amortizations authorized under the statutes may not exceed three percent of the natural gas utility's gross revenues for the preceding calendar year unless the Commission finds that allowing a higher amortization rate is reasonable under the circumstances. As shown on Attachment C of this public meeting memo, the resulting annual average rate impact from the PGA amortization and four other filings\textsuperscript{23} is negative 7.0 percent when compared to the Company's 2017 total gross revenues and falls within the requirements of the statute. The percentage changes in Table 3 below reflect the change in revenues related to the gas commodity portion of the Company's gross revenues for the 2018-19 gas year based on projected customer usage and differs from the three percent calculation.
\textsuperscript{19} For the 2018-2019 Gas Year, the Company elected a 90/10 variance sharing on July 26, 2018.
\textsuperscript{20} Schedule 440 refers to Interruptible Service Customer. This rate schedule is applicable to commercial and industrial customers who are subject to interruptions in capacity and supply. By choosing to be interruptible, the Company does not secure firm interstate pipeline capacity for these customers. As a result, customers served on this rate schedule do not pay transportation or demand charges.
\textsuperscript{21} See page 27 of 27 in Avista's work papers filed with its Supplemental 2018 PGA Filing (i.e., row "65" Column "L").
\textsuperscript{22} See page 27 of 27 in Avista's work papers filed with its Supplemental 2018 PGA Filing (i.e., row "64" Column "L").
\textsuperscript{23} Schedule 476, Intervenor Funding, Docket No. UG 363/Advice No. 18-04-G; and Schedule 478 DSM, Docket No. UG 362/Advice No. 18-03-G; and Schedule 475, Decoupling Mechanism, Docket No. UG 361/Advice No. 18-02-G; and Schedule 484, Bank Fee Free Program, Docket No. UG 364/Advice No.18-05-G.
Table 3: Overall Revenue and Rate Impact
(in $ or as noted otherwise)
| Schedule | Description | Total Revenues at Current Rates$^{24, 25} | Revenue Increase / (Decrease)$^{26} | Change (%)$^{27} |
|----------|----------------------|-------------------------------------------|-------------------------------------|------------------|
| 410 | Residential | 59,705,765 | -4,952,491 | -8.3 |
| 420 | General | 27,419,349 | -2,214,601 | -8.1 |
| 424 | Large General | 2,118,852 | -341,345 | -16.1 |
| 440 | Interruptible | 1,314,232 | -322,613 | -24.6 |
| 444 | Seasonal | 130,784 | -20,221 | -15.5 |
| 456 | Int. Transportation | 3,149,855 | 22,479 | 0.7 |
| Overall | | 94,051,113$^{28}$ | -7,828,792 | -8.3 |
Conclusion
Avista’s 2018 PGA Filing reflects a revenue decrease of $2.7 million,$^{29} or approximately 2.9 percent$^{30}$ effective November 1, 2018, due to gas costs (Purchased Gas Cost Adjustment Provision; Schedule 461) and amortization of previous deferrals (Gas Cost Rate Adjustment; Schedule 462). Combining the impact of this filing with the four other
---
$^{24}$ See column “Present Annual Revenue” of page 20 of 27 in Avista’s work papers filed with its Supplemental 2018 PGA Filing.
$^{25}$ “Total Revenues” include the revenues from: 1) base rates, 2) gas revenues (Purchased Gas Cost Adjustment Provision; Schedule 461), 3) amortizations (Gas Cost Rate Adjustment; Schedule 462), 4) intervenor funding (Intervenor Funding Grants – Oregon; Rate Schedule 476), and 5) low income assistance (Residential Low Income Rate Assistance Program – Oregon; Schedule 493).
$^{26}$ See column “Total Incremental Change in Revenue” of Attachment A of this public meeting memo.
$^{27}$ See column “Total Change – Proposed Annual Change” of page 20 of 27 in Avista’s work papers filed with its Supplemental 2018 PGA Filing.
$^{28}$ The total of $94,051,113 does not equal the sum of the figures above this number, because it includes revenues of $212,276 from Special Contracts. See page 20 of 27 in Avista’s work papers filed with its Supplemental 2018 PGA Filing (i.e., row “Special Contracts” Column “Present Annual Revenue”).
$^{29}$ $2.7 million is the difference between the revenues at proposed rates and the revenues at current rates only for gas revenues (Purchased Gas Cost Adjustment Provision; Schedule 461) and amortizations (Gas Cost Rate Adjustment; Schedule 462). See Attachment B of this public meeting memo.
$^{30}$ See page 20 of 27 in Avista’s work papers filed with its Supplemental 2018PGA Filing (i.e., row “Total Annual Revenues.” Column “PGA Only – Proposed Annual Change.”)
regulatory filings filed on and after July 27, 2018, results in a revenue decrease of $7.829 million,\textsuperscript{31} or negative 8.3 percent,\textsuperscript{32} as represented in Table 3 above.\textsuperscript{33}
With these changes, effective November 1, 2018, the monthly bill of a residential customer using an average of 47 therms per month will decrease by $4.56, or 8.3 percent, from $54.89 to $50.33.\textsuperscript{34}
**PROPOSED COMMISSION MOTION:**
Approve Avista's Advice No. 18-01-G, which is the Company's 2018 annual Purchase Gas Adjustment (PGA) tariff sheet updates, for service rendered on and after November 1, 2018.
\textsuperscript{31} See Attachment A of the Company's Supplemental 2018 PGA Filing, row "Total," column "Total Incremental Change in revenue."
\textsuperscript{32} See page 20 of 27 in Avista's work papers filed with its Supplemental 2018 PGA Filing (i.e., row "Total Annual Revenues." Column "Proposed Annual Change.")
\textsuperscript{33} The amounts shown in Table 3 include base rates, while the amounts shown in Attachments A and B to this memo are incremental changes without base rates.
\textsuperscript{34} See Attachment D of this public meeting memo.
|
Creep crack initiation and growth in 2.25% chromium - 1% molybdenum alloy steel
COLLINGTON, Rachel A.
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CREEP CRACK INITIATION AND GROWTH IN 2.25% CHROMIUM - 1% MOLYBDENUM ALLOY STEEL
Rachel A. Collington
A thesis submitted in partial fulfilment of the requirements of Sheffield Hallam University for the degree of Doctor of Philosophy
April 2001
Collaborating Establishment: Alstom Energy, UK, Ltd.
CHESTERFIELD HALLAM UNIVERSITY
ADSETTS CENTRE
This thesis is submitted in partial fulfilment of the requirements of Sheffield Hallam University for the degree of Doctor of Philosophy. It contains an account of research carried out, on a part-time basis, between May 1988 and December 2000 in the School of Engineering and Materials Research Institute at Sheffield Hallam University. Supervision of the project was by Dr J. Cawley and Dr R. Stratton of Sheffield Hallam University and Dr S. Holdsworth of Alstom Energy, UK, Ltd. Except where acknowledged and referenced, this work is, to the best of my knowledge, original and has been carried out independently. No part of this thesis has been, or is currently being, submitted for any degree or diploma at this, or any other, university.
Rachel A. Collington
March 2001
Acknowledgements
I wish to acknowledge Dr Jess Cawley for his continuing support and supervision of this project and for not giving up on me during the difficult times.
I would like to thank Dr Rod Stratton and Dr Stuart Holdsworth for their advice and guidance, particularly in the early stages of this project.
For his encouragement and guidance during the arduous writing up stage I would like to express my sincere gratitude to Dr Robin Acheson.
Many thanks go to all my colleagues in both the School of Engineering and The Materials Research Institute who have provided help and given their support throughout.
And last, but definitely not least, I would like to acknowledge the love and support given to me by my partner Lawrence and my children Elliot and Amelia. This is for you!
Abstract
2.25%Cr-1%Mo Steel has been utilised extensively for the manufacture of power plant components.
This study has highlighted the implication of the presence of pre-existing defects in such components and, in particular, the influence of the microstructure on the creep deformation response ahead of a defect during the crack initiation, or incubation, period.
The investigation was conducted by carrying out creep tests on compact tension specimens in three microstructural variations of the alloy and, subsequently, using optical, scanning and transmission electron microscopy techniques to evaluate the creep damage in the "defect" tip region up to a point where ~1mm of crack extension was detected.
Details of two image analysis routines devised specifically for this project have been presented and their suitability for purpose discussed. The first of these routines allowed the distribution and density of creep voids occurring ahead of the simulated defect to be quantified using back scattered electron imaging in the scanning electron microscope. The second allowed carbides, extracted from the alloy, to be classified in the STEM on the basis of their key element ratios.
The microstructural constraint imposed by the material ahead of the defect tip was shown to critically affect the crack initiation and growth process; the order of constraint observed being that of 100% bainite > mixed ferrite / bainite > mixed ferrite / pearlite.
A correlation between the carbide type, distribution and shape and the creep deformation and crack initiation process has been discussed and the implications of this to remanent life assessment suggested. Evolution of $M_{23}C_6$ grain boundary carbides to the equilibrium carbide, $M_6C$, and the affect of the grain boundary sliding on the shape of the $M_6C$, were proposed to be the most important microstructural phenomena contributing to the crack initiation process.
A detailed review of published literature relating to the topic studied and recommendations for further work have also been included in this thesis.
## Contents
| Section | Page |
|------------------------------------------------------------------------|------|
| Preface | ii |
| Acknowledgements | iii |
| Abstract | iv |
| Contents | v |
| Nomenclature and Abbreviations | ix |
| **Chapter 1.0 Introduction** | 1 |
| **Chapter 2.0 Literature Review** | 8 |
| 2.1 Introduction | |
| 2.2 The Chromium – Molybdenum Creep Resisting Steels | 9 |
| 2.2.1 The Transformation Structure | 10 |
| 2.2.2 The Carbide Structure | 13 |
| 2.3 Creep Deformation | 19 |
| 2.3.1 Creep Deformation Mechanisms and Maps | 21 |
| 2.3.2 Creep Deformation of Chromium – Molybdenum, Steels | 25 |
| 2.4 Creep Fracture | 30 |
| 2.4.1 Creep Fracture Mechanisms | 31 |
| 2.4.2 Creep Cavity Initiation and Growth | 33 |
| 2.4.3 Creep in the Presence of a Pre-existing Defect | 41 |
| 2.5 Creep Failure Prediction and Remanent Life Assessment | 47 |
| 2.5.1 Creep Life Assessment of Initially ‘Defect Free’ Structures | 47 |
| 2.5.2 Creep Life Assessment of Structures Containing Pre-existing Defects | 52 |
| 2.5.3 Microstructural Investigation Techniques for Creep Life Prediction Methods | 59 |
| **Chapter 3.0 Experimental Procedures** | 61 |
| 3.1 Introduction | 62 |
| 3.2 Raw Materials | 65 |
## Contents cont.
### 3.3 Heat Treatment
| Section | Page |
|------------------------------------------------------------------------|------|
| 3.3.1 Production of 100% Bainite CT Creep Test Specimens | 67 |
| 3.3.2 Bainite Tempering Investigation | 68 |
### 3.4 Mechanical Testing
| Section | Page |
|------------------------------------------------------------------------|------|
| 3.4.1 Hardness Tests | 69 |
| 3.4.2 Tensile Tests | 70 |
| 3.4.3 Creep Deformation Tests | 70 |
| 3.4.3 (a) Creep Rig Design | 70 |
| 3.4.3 (b) Creep Test Specimens and Loading Regime | 73 |
| 3.4.3 (c) Crack Opening Displacement Measurement | 75 |
### 3.5 Optical Microscopy Techniques
| Section | Page |
|------------------------------------------------------------------------|------|
| 3.5.1 Specimen Preparation and Microstructural Examination | 77 |
| 3.5.2 Grain Size Determination | 77 |
| 3.5.3 Determination of the Volume Fraction of Transformation Products | 78 |
### 3.6 Electron Microscopy Techniques
| Section | Page |
|------------------------------------------------------------------------|------|
| 3.6.1 Scanning Electron Microscopy | 79 |
| 3.6.1(a) SEM Specimen Preparation | 80 |
| 3.6.1(b) General SEM Examination | 81 |
| 3.6.1(c) Creep Damage Quantification using SEM Image Analysis | 81 |
| 3.6.2 Transmission and Scanning Transmission Electron Microscopy | 84 |
| 3.6.2(a) Preparation of Carbon Extraction Replicas | 85 |
| 3.6.2(b) Preparation of Thin Foils | 86 |
| 3.6.2(c) Microstructural Examination in the TEM | 87 |
| 3.6.2(d) Carbide Characterisation by Image Analysis in the STEM | 88 |
### Chapter 4.0 Experimental Results
| Section | Page |
|------------------------------------------------------------------------|------|
| 4.1 Introduction | 93 |
| 4.2 Characterisation of the Starting Materials | 93 |
| Section | Page |
|------------------------------------------------------------------------|------|
| 4.2.1 Mechanical Properties | 94 |
| 4.2.2 Optical Microscopy | 95 |
| 4.2.3 Scanning Electron Microscopy | 96 |
| 4.2.4 Transmission Electron Microscopy | 98 |
| 4.3 The Tempering Behaviour of Fully Bainitic 2.25%Cr-1%Mo Alloy Steel | 110 |
| 4.3.1 Hardness Test Results | 111 |
| 4.3.2 Microstructural Changes During Tempering | 113 |
| 4.3.2(a) Examination by Scanning Electron Microscopy | 113 |
| 4.3.2(b) Examination by Scanning Transmission Electron Microscopy | 114 |
| 4.4 Creep Testing and Deformation Assessment | 129 |
| 4.4.1 Crack Opening Displacement Measurement | 129 |
| 4.4.2 Creep Damage Assessment in the Scanning Electron Microscope | 135 |
| 4.4.2(a) 100% Bainite Specimens | 135 |
| 4.4.2(b) Mixed Pro-eutectoid Ferrite / Bainite Specimens | 147 |
| 4.4.2(c) Mixed Pro-eutectoid Ferrite / Pearlite Specimens | 159 |
| 4.4.3 Correlation and Summary Of The Creep Test Investigation Data | 168 |
| 4.4.4 Carbide Analysis of Creep Tested Materials in the TEM | 174 |
| 4.4.4(a) Carbide Analysis of the 100% Bainite Specimens | 174 |
| 4.4.4(b) Carbide Analysis of the Mixed Ferrite / Bainite Specimens | 176 |
| Chapter 5.0 Discussion | 187 |
| 5.1 Starting Structures and Basic Mechanical Properties of 2.25%Cr-1%Mo | 188 |
| 5.1.1 Transformation Structures | 188 |
| 5.1.2 Carbide Structure | 190 |
| 5.1.3 Basic Mechanical Properties | 194 |
| 5.2 Tempering Characteristics And The Carbide Evolution Process In 2.25%Cr-1%Mo Steel. | 195 |
| 5.3 Creep Deformation in the Presence of a Pre-existing Defect | 200 |
| Section | Page |
|------------------------------------------------------------------------|------|
| 5.3.1 Creep Damage Response | 200 |
| 5.3.2 The Affect of Microstructural Variables on the Creep Crack Initiation and Growth Process | 207 |
| 5.3.3 The Contribution of Grain Boundary Carbides to the Creep Crack Initiation Process | 213 |
| 5.4 Contribution to Life Assessment Strategy | 216 |
| 5.4.1 The Effect of Notch Tip Constraint and Life-Time Prediction | 216 |
| 5.4.2 The Mechanisms of Creep Deformation Occurring Ahead of a Pre-existing Defect and Life-Time Prediction | 219 |
| 5.4.3 Carbide Evolution and Life-Time Prediction | 220 |
| 5.5 Experimental Considerations and Suggestions for Further Work | 221 |
| Chapter 6.0 Conclusions | 223 |
| References | 228 |
| Appendices | 242 |
| Appendix 1 Creep Data For The Derivation Of $\sigma_{ref}$ | 243 |
| Appendix 2 EDX Spot Analysis For The Determination Of Carbide Classification Schemes | 245 |
| Appendix 3 Elemental Factors Determined for Use in the STEM Image Analysis Routine | 247 |
| Appendix 4 Published Paper | 248 |
| Index | 256 |
| Index to Table Nos. | 257 |
| Index to Figure Nos. | 259 |
| Index to Plate Nos. | 263 |
| Symbol | Description |
|--------|-------------|
| $a$ | Crack length |
| $\dot{a}$ | Crack growth rate |
| $A_{C_1}$ | The eutectoid reaction temperature on cooling |
| $A_{C^3}$ | The austenite – ferrite transformation temperature on cooling |
| $b$ | Burgers vector |
| $B$ | Specimen thickness |
| BCC | Body-centred cubic |
| $B_n$ | Net specimen thickness |
| $C$ | Constant in the Larson-Miller equation |
| $C_w$ | Solute concentration in a solvent when a particle has an infinite radius of curvature |
| $C^*$ | Creep crack parameter |
| CCT | Continuous cooling transformation |
| CDM's | Continuum damage mechanics |
| COD | Crack opening displacement |
| CT | Compact tension |
| CTOD | Crack tip opening displacement |
| $d$ | Grain diameter as measured by the mean linear intercept method |
| $D$ | Damage parameter |
| $D_0$ | Material constant in equation for determining creep crack growth rate |
| $E$ | Young’s modulus |
| EDXA | Energy dispersive X-ray analysis |
| $f_0$ | Reaction force of a precipitate against dislocation movement during creep |
| FCC | Face-centred cubic |
| $G$ | Elastic strain energy release rate |
| $H$ | Hardening parameter |
| $J$ | J-contour integral |
| $K$ | Linear elastic stress intensity factor |
| $K_{\text{nom}}$ | Nominal value of stress intensity |
| LEFM | Linear elastic fracture mechanics |
| LMP | Larson-Miller parameter |
| LPD | Load point displacement |
| $m$ | General yield ratio |
| $M$ | Metal atoms in alloy carbides |
| MTDATA | Metallurgical and thermodynamic data |
| $n$ | Creep stress exponent |
| n.a. | Not applicable |
| n.d. | Not determined |
| Symbol | Description |
|--------|-------------|
| p | Diameter of a particle subjected to a stress concentration imparted by grain boundary sliding |
| $P'$ | Applied load |
| Q | Activation energy |
| r | Mean particle radius |
| $r'$ | Radius of the damage zone |
| R | Gas constant |
| SEM | Scanning electron microscope |
| STEM | Scanning transmission electron microscope |
| t | Time |
| $t_f$ | Time to failure |
| $t_g$ | Time to grow a creep crack |
| $t_i$ | Time to initiate a creep crack |
| $t_r$ | Time to rupture |
| $t_x$ | Time at stress $\sigma_x$ and temperature $T_x$ |
| $t_{xf}$ | Failure time at stress $\sigma_x$ and temperature $T_x$ |
| $T_x$ | Temperature at the $x^{th}$ fraction of creep life |
| T | Temperature |
| TEM | Transmission electron microscope |
| Tm | Homologous temperature |
| $V_m$ | Molar volume of a dispersed phase |
| W | Specimen width |
| $x_0$ | Crack initiation criterion |
| Y | Specimen compliance function |
| $\alpha$ | Ferrite |
| $\Delta$ | Specimen load point displacement |
| $\varepsilon$ | Epsilon carbide |
| $\dot{\varepsilon}_0$ | Strain rate in uniaxial creep |
| $\varepsilon_{f0}$ | Creep ductility in uniaxial tension |
| $\varepsilon_t^*$ | Local rupture strain |
| $\dot{\varepsilon}_s$ | Secondary creep rate |
| $\varepsilon_x$ | Strain at stress $\sigma_x$ and time $t_f$ |
| $\varepsilon_{xf}$ | Failure strain at stress $\sigma_x$ and $t_{xf}$ |
| $\phi$ | Material constant in equation for determining creep crack growth rate |
| $\gamma$ | Austenite |
| $\sigma_0$ | Stress in uniaxial creep |
| $\sigma_p$ | Average stress on a particle due to grain boundary sliding |
| $\sigma_{\text{ref}}$ | Reference stress |
| Symbol | Description |
|--------|-------------|
| $\sigma_x$ | Stress at the $x^{th}$ fraction of creep life |
| $\sigma_y$ | Yield strength |
| $\sum_x$ | Sum of the creep life fractions |
| $\tau$ | Shear stress |
| $\nu$ | Poisson’s ratio |
The use of steam power for the generation of electrical power has been utilised in Europe since the late 1800's and since its introduction there has been a continuing drive to improve the efficiency of the process. The 'efficiency' of the thermodynamic process of coal fired power describes how much of the energy fed into the cycle is converted into electrical energy. The greater the output of electrical energy for a given amount of energy input, the higher the efficiency.
It is well understood, by an examination of the Carnot cycle\(^{[1]}\), that the efficiency of a steam turbine is proportional to temperature and pressure and that relatively small increases in these bring a considerable efficiency benefit. If, therefore, the energy input is kept constant the output can be increased by selecting elevated pressures and temperatures for the water-steam cycle\(^{[2]}\), for example a temperature rise from 560°C – 600°C can provide a relative efficiency increase of 2\(^{[3]}\).
Figure 1.1 shows the trend for higher steam conditions utilised between 1988 and the present.
**Figure 1.1: Trend to higher steam conditions for steam power plant – after Scarlin\(^{[3]}\)**
To enable power plant to operate at improved efficiencies there has been, since the process began, a requirement for materials with improved resistance to the high temperature deformation process of creep. The development of these creep resistant alloys is based on fundamental theories of creep deformation and fracture which are well understood and have been extensively reported [4] - [6].
Methods for predicting the lifetime of components destined for operation in high temperature environments have traditionally been based on the steady state period of creep deformation determined by testing specimens subjected to uniaxial loading conditions. It became evident, however, that there was also a need for studies to be carried out to predict the behaviour of material in non-uniaxial situations, for example material ahead of pre-existing defects in large castings or in welds. Research in this area has since been reported by many authors [7] - [14] with the prediction of material behaviour ahead of defects being predominantly based on fracture mechanics theory and on crack propagation rates.
One of the alloys that has been used extensively for high temperature power plant components in the last five decades is 2.25%Cr-1% Mo steel. 2.25%Cr-1%Mo steel confers good creep strength and corrosion resistance up to service temperatures of ~565°C [15] and although the current materials requirements for new plant have superseded that of the relatively low alloy Cr-Mo steels there are still a substantial number of components of this type in operation necessitating means of remanent life prediction.
The long term creep resistance of 2.25%Cr-1%Mo steel is afforded by a combination of interaction solid solution hardening and precipitation hardening effects [16] the combination of which is dependent upon the initial alloy heat treatment and in service conditions. Over a period of time, at elevated temperatures in excess of ~ 400°C, the microstructure of 2.25%Cr-1%Mo steel, and in particular the carbide morphology, has been shown to age [17]. This microstructural ageing process has a direct effect on the creep strength of the
steel and, consequently, any design codes or remaining lifetime prediction models must take into consideration this continuously varying factor.
Many attempts have been made to predict the lifetime of a component containing defects using mathematical models based on fracture mechanics theory [14] and, as a result, standards based on the use of the $C^*$ parameter and critical COD have now been established [18]. Although lifetime prediction models were initially based on crack propagation times alone it has become clear that a significant proportion of component life may also be accounted for by an incubation period prior to crack initiation and that this should also be included [11] [18]. Consequently the most recent life assessment models are based on the equation:
$$t_i + t_g = t_f \quad -\text{Equation 1.1} \quad [11]$$
Where:
- $t_i$ = time to initiate a creep crack
- $t_g$ = time to grow the creep crack to rupture of the remaining ligament
- $t_f$ = component lifetime
To estimate the period of crack initiation, or incubation, accurately it is necessary to understand the various contributory factors that may affect it and the most critical of these is the material microstructure.
During the incubation period, it has been established that, a creep damage zone develops ahead of the pre-existing defect and that there is a redistribution of the stress intensity at the defect tip. The structural degradation that occurs in this period is dependent upon the operating stress and temperature regime and includes changes in chemistry, size, distribution and spacing of second phase particles and nucleation and growth of cavities and cracks.
The gradual accumulation of damage ahead of the defect leads to strain in the component and this can be monitored in terms of crack opening displacement,
(COD). Critical CODs may then be defined for a given crack initiation criterion, where crack initiation may be defined as the formation of the first creep cavity occurring ahead of the crack tip or the formation of an engineering sized crack, 0.5mm to 1mm long.
There is a fundamental requirement to understand the physical mechanisms of crack initiation from its earliest stages and the role of the ageing microstructure on this process. To this end, establishing a crack initiation criterion of 1mm, this research project intended to correlate information regarding COD data and corresponding microstructural evidence from various structures of 2.25%Cr-1%Mo alloy steel.
In large cast components microstructural variations occur due to differences in cooling rate from the core of the castings to the surface and thin sections. The core material in large castings of normalised and tempered 2.25%Cr-1%Mo steel is typically mixed pro-eutectoid ferrite and pearlite and/or bainite and the surface material, and that in central regions of thin sections, are fully bainitic. As each of these microstructures imparts a unique resistance to the creep deformation mechanisms occurring in the alloy, components possessing these structures exhibit a range of crack opening displacement rates and creep crack initiation times. Consequently, there is a need to establish the influence of the microstructure on these parameters so that they may be accounted for in life-time and remanent life-time prediction models.
The main aims of this study then were to:
i) establish a relationship between critical crack opening displacement and damage accumulation in three microstructures of 2.25%Cr-1%Mo steel with a view to improving creep life prediction models and
ii) to make a contribution to knowledge by proposing a mechanism for the creep cavitation and coalescence process occurring in a material ahead of a pre-existing defect.
To achieve the aims of this study a detailed review of the relevant literature was carried out as detailed in chapter 2.
The creep deformation response of material ahead of a pre-existing defect was studied by creep testing compact tension specimens, in three microstructures of 2.25%Cr-1%Mo steel, and performing physical measurement techniques to establish their critical COD values. An extensive microstructural investigation was carried out to quantify the amount of creep damage occurring in the specimens and to establish the mechanisms by which that damage occurred. The various experimental procedures for the techniques established and used in this study are detailed in chapter 3 and are summarised below in Figure 1.2. The experimental results derived are presented and described in detail in chapter 4 and discussed in context with the current knowledge in chapter 5. Finally, chapter 6 defines the major conclusions from the investigation and highlights the contribution to knowledge.
CHARACTERISATION AND GENERATION OF THREE MICROSTRUCTURAL VARIABLES OF 2.25%Cr-1%Mo STEEL
25% BAINITE / 75% PRO-EUTECTOID FERRITE STRUCTURE
90% PRO-EUTECTOID FERRITE / 10% PEARLITE STRUCTURE
100% BAINITE STRUCTURE
CREEP TESTS ON CT SPECIMENS CONTAINING SIMULATED DEFECTS
TEMPERING TRIAL TO INVESTIGATE CARBIDE EVOLUTION SEQUENCE
CRACK LENGTH ($\Delta a$) AND DETERMINATION OF CTOD
CREEP DAMAGE QUANTIFICATION USING IMAGE ANALYSIS IN THE SEM
SEM HIGH RESOLUTION ELECTRON MICROSCOPY OF ALLOY MICROSTRUCTURE
TEM IMAGE ANALYSIS OF CARBIDE MORPHOLOGY
Figure 1.2: Flow diagram summarising the experimental process.
2.1 Introduction
The ideal material for components destined for power plant applications requires a combination of both high creep strength and creep ductility to resist deformation and fracture at high operating temperatures and stress levels. To develop new materials for such applications, and to predict the remaining lifetime of those already in service, it is necessary to fully understand the mechanisms of creep deformation and fracture and to appreciate the effect of these on the inherent and operational variables: composition, microstructure, temperature, stress, stress state, strain rate. These variables are not, of course, mutually exclusive but affect each other leading to complex inter-relations which are difficult to interpret. However, due to the vast amount of research that has been conducted in the last 50 years in this area, methods of predicting the behaviour of components over long periods at elevated temperatures are now in operation and are, on the whole, being utilised successfully. The aim of this section is to review the existing knowledge regarding creep deformation and fracture in chromium - molybdenum power plant steels, and specifically 2.25%Cr-1%Mo steel, with a view to providing a basis to interpret and place the data derived in this study into context. This section necessarily begins with a consideration of the microstructure of chromium – molybdenum steels and their stability at high temperature and finishes with a review of creep remanent life assessment techniques.
2.2 The Chromium – Molybdenum Creep Resisting Steels
The chromium - molybdenum, (Cr-Mo), series of steels are used widely in the power and process industries because they offer high strength at ambient and elevated temperatures combined with adequate toughness [20]. The properties of the steels are a direct consequence of the material microstructure which in turn is controlled by a relatively simple heat treatment process. Microstructure and mechanical properties can, therefore, be optimised for a particular
application by controlling the heat treatment parameters; austenitising temperature, cooling rate and tempering temperature and duration.
2.2.1 The Transformation Structure
The chromium – molybdenum steels belong to a group commonly referred to as ferritic creep resisting steels; the term ferritic being derived from the fact that on cooling from austenite, (γ), the microstructure consists of one, or a combination, of three essentially body centred cubic structures, namely ferrite, bainite or martensite. For a given chemical composition the transformation product that results is dependent upon the cooling rate from the austenitising temperature.
The grain size of the transformed structure can be considered as a direct function of the austenitising temperature but is regarded, generally, as only having a small influence on the long term creep resistance of the alloys [21]. Cane and Fidler [22], however, showed that the grain size effect on creep properties varies from steel to steel and reported a greater dependency of creep properties on grain-size for a fully bainitic tempered 2.25%Cr-1%Mo steel than for a tempered martensitic 9%Cr-1%Mo steel. The fracture ductility of Cr-Mo steels, in particular, is considered to be adversely affected by a large grain size and consequently the austenitising temperature used for these steels does not rise significantly above the $A_{C_3}$ [20].
In commercial applications, Cr-Mo steels are put into service in a microstructurally meta-stable condition. The structure that exists is a result of both continuous cooling from above the $A_{C_3}$ to give the initial transformation product, and any subsequent stress relief / tempering treatment to develop the fine precipitate structure. The transformation product is dependent on not only the alloy composition and cooling environment but also on the section size, as indicated by reference to the continuous cooling diagrams for typical Cr - Mo steels as shown in Figure 2.1. In this figure the transformation structure can be seen to vary from ferrite and pearlite in slow cooled material, (thick sections), to
bainitic or even martensitic structures in rapidly cooled material, (thin sections). In the alloys containing higher chromium contents the transformation time required to produce ferrite, pearlite and bainite is significantly retarded and austenite decomposes directly to martensite. The likelihood of partitioning of elements and their effect on the kinetics of the austenite to ferrite transformation is well documented [23] and the mechanisms of diffusion controlled and diffusionless transformation to ferrite / bainite and martensite respectively have been comprehensively reviewed [24]. Both of the aforementioned papers drew attention to the effect on the creep resistance of the Cr - Mo alloys of the non-equilibrium existence of elements in either precipitate form or in solid solution in the as cooled structure and the move to equilibrium during further exposure to elevated temperatures.
Figure 2.1: Continuous cooling diagrams for some Cr-Mo Steels – after Orr [20].
The remainder of this section will concentrate on the development of the microstructure, for elevated temperature service, of Cr-Mo steels in the annealed or normalised conditions.
During slow cooling through the $A_{C_3} - A_{C_1}$ region, Figure 2.1, transformation to ferrite occurs progressively, enriching the remaining austenite in carbon. Eventually any remaining austenite transforms to pearlite\textsuperscript{[23]} in which the carbon precipitates as cementite, $(Fe_3C)$. In the case where strong carbide formers are present in the alloy, the cementite may be replaced by alloy carbides\textsuperscript{[25]}. Up to 20% of iron atoms in cementite can be replaced by chromium, 10% by vanadium and only 4% by molybdenum without affecting the crystal structure of the carbide. The pearlite interlamellar spacing, detailed morphology and tendency to spheroidise may, however, be more significantly affected.
When cooling of the alloy is sufficiently rapid to avoid the pearlite transformation curve, but not sufficient to produce diffusionless transformation to martensite, the remaining structure will consist of a mixture of ferrite and bainite or may even be fully bainitic. In the austenite to bainite reaction, the austenite may transform to bainite in two ways\textsuperscript{[28]}, i) to upper bainite where cementite forms between the bainitic ferrite laths only, or ii) to lower bainite where the cementite additionally forms within the platelets of ferrite.
During the displacive transformation to bainite\textsuperscript{[26]} there is no diffusion of substitutional iron atoms across the transformation interface. The excess carbon trapped in the bainitic ferrite is removed by a combination of diffusion into the residual austenite and by precipitation of carbides between the ferrite plates.
In the situation where pro-eutectoid ferrite forms prior to the transformation to bainite, the enhanced concentration of carbon in the remaining austenite, due to its rejection from the ferrite, leads to an increased density of cementite.
particles in the bainite phase when compared to that in fully bainitic structure [24].
In all cases, the carbides in the as cooled condition do not exist in their equilibrium state and subsequent heat treatment and service at elevated temperatures leads to a change in their composition and morphology that consequently affects their long term creep properties. This process will be reviewed in the following section.
2.2.2 The Carbide Structure
In developing alloys for service at elevated temperatures the aim is to produce a material which not only withstands the initial service conditions but is capable of withstanding the service conditions throughout the whole of the expected design life, ~20 years for power plant components. The coarsening and evolution of carbides is of great importance in terms of the long term creep resistance of alloys and is controlled by the process of Ostwald ripening. Ostwald ripening involves the growth of large particles at the expense of small particles, which are taken into solution. It is controlled by the transfer of vacancies through the ferrite lattice to the growing particle and by the diffusion of the solute through the matrix. An increase in temperature brings about an increase in the diffusion rate, so that Ostwald ripening occurs more rapidly at high temperatures.
According to Nutting [27] it is possible to describe the particle coarsening process by the following rate equation:
\[
r_t^3 - r_0^3 = \frac{K V_m C_\infty t}{RT}
\]
- Equation 2.1
Where:
\( r_t \) = mean particle size after time \( t \)
\( r_0 \) = mean particle size at the start of the observations
\( K \) = constant related to the rate controlling diffusing species
\( V_m \) = molar volume of the dispersed phase
For optimum performance low alloy Cr-Mo steels usually enter service in either the normalised and tempered or annealed condition [28], thus the evolution process in terms of carbide ripening is already under way. Such steels are in an active metastable condition and further carbide precipitation, accompanied by changes in composition and coarsening, occurs throughout high temperature exposure.
In the normalised condition the microstructure is predominantly bainitic with varying amounts of pro-eutectoid ferrite, dependent upon component section size. The tempering temperature and duration used are selected to optimise the steels properties for a particular application, the knowledge to do so being drawn from works by authors such as Baker and Nutting, [17]. Baker and Nutting determined the sequence of carbide precipitation events which occurred upon tempering a normalised 2.25%Cr-1%Mo steel at temperatures ranging from 400°C to 750°C and times up to 1000 hours, as summarised below in Figure 2.2.
**In bainite –**
\[ \varepsilon \text{ carbide} + M_3C \rightarrow M_6C \rightarrow M_3C + M_2C \rightarrow M_{23}C_6 \rightarrow M_7C_3 \rightarrow M_6C \]
**and in ferrite –**
\[ M_2C \rightarrow M_6C \]
**Figure 2.2:** Carbide evolution sequence for 2.25%Cr-1%Mo steel, where M indicates a mixture of metal atoms - after Baker and Nutting [17].
In the annealed condition the microstructural constituents are basically the same as for the normalised and tempered material but due to the slower cooling rate there is now a significant amount of pro-eutectoid ferrite present and some pearlite.
The nature of the carbides in low alloy creep resisting steels has been extensively investigated [17] [28] [27] and those most likely to be encountered in 2.25%Cr -1%Mo steel are summarised below in, Table 2.1.
| Carbide Type | Crystal Structure | Composition (metal-atom ratios) | Description and Location |
|--------------|-------------------|---------------------------------|--------------------------|
| $\varepsilon$ Carbide | Hexagonal | Fe rich. Cr /Mn - high solubility Mo - low solubility | In the bainite between the ferrite laths. Metastable precursor to $M_3C$ |
| $M_3C$ | Orthorhombic structure of cementite, $Fe_3C$ | Fe rich. Cr - high solubility (0.2) Mn - high solubility Mo - low solubility (0.04) | Replaces $\varepsilon$ carbide on ageing. Predominantly $Fe_3C$. |
| $M_6C_3$ | Pseudo hexagonal structure of $Cr_7C_3$ | Chromium rich. Fe - high solubility (up to 0.6) Mn - high solubility Mo - low solubility (up to 0.055) | Stick like precipitate, bainite side of $\alpha$/bainite grain boundaries. Nucleates in vicinity of $Fe_3C$. |
| $M_{23}C_6$ | FCC structure | Cr rich. Fe - high solubility (up to 0.4) Mn - high solubility Mo - high solubility can replace Cr. | Precipitates in bainite at the expense of $Mo_2C$ and $Fe_3C$. Not observed in immediate vicinity of $Cr_7C_3$, therefore considered to be based on Fe and Mo predominantly. |
| $M_2C$ | Hexagonal | Mo rich Cr - high solubility (up to at least 0.3) Fe - solubility upto 0.2 | Fine matrix precipitate - stable up to long times in $\alpha$. May also form fringe like precipitates at $\alpha$ side of $\alpha$/bainite grain boundaries. |
| $M_6C$ | Complex cubic | Ternary carbide of Fe and Mo from $Fe_4Mo_2C$ -> $Fe_2Mo_2C$ Cr - solubility up to 0.3 | Equilibrium carbide. Forms from $Mo_2C$ in $\alpha$ and eventually from $M_6C_3$ and $M_{23}C_6$ in bainite. |
Table 2.1: Nature of carbides in normalised 2.25%Cr-1%Mo steel [17] [28] [27].
The carbides in the bainite structure of 2.25%Cr-1%Mo steel precipitate from a matrix saturated in carbon, chromium and molybdenum on tempering. The $M_2C$ in the pro-eutectoid ferrite, however, precipitates directly on cooling from above the $Ac_3$ by a process known as interphase precipitation [23].
$M_6C$ has been shown by many workers\textsuperscript{[17]} \textsuperscript{[29]} \textsuperscript{[30]} to be the equilibrium carbide in 2.25%Cr -1%Mo steel. More recent studies\textsuperscript{[31]}, however, suggested that in silicon containing steels, 0.61% in the alloy studied, $M_6C$ precipitates at earlier times than in the steels studied by Baker and Nutting\textsuperscript{[17]} which contained only 0.18% Si, and the equilibrium carbide is $M_{23}C_6$. In the same study, manganese was shown to have a similar effect. Decreasing the carbon content of the alloy has been shown,\textsuperscript{[30]} \textsuperscript{[32]}, to accelerate the precipitation sequence described in Figure 2.2. This has commercial implications when Cr-Mo steels are welded to mild steels as carbon migration will affect weld properties,\textsuperscript{[33]}.
Detailed studies of the enrichment of carbides in chromium during the ageing of 2.25%Cr-1%Mo steel has been carried out by Thomson and Bhadeshia\textsuperscript{[24]} \textsuperscript{[34]} \textsuperscript{[35]}. These studies indicated that small cementite particles become enriched in chromium more quickly than larger ones and that the enrichment rate of cementite in bainite is slower in mixed ferrite / bainite structures than in those which are fully bainitic. MTDATA\textsuperscript{[36]}, a computerised databank that allows the computation of chemical equilibrium as a function of composition and conditions, was used in conjunction with energy dispersive X-ray analysis (EDXA) to investigate the chromium levels of $M_7C_3$ and $M_{23}C_6$ in equilibrium with ferrite. It was concluded that the amount of chromium each carbide can support increases with decreasing tempering temperature. The concentration of chromium in $M_7C_3$ tempered at 565°C, however, was found to increase with time at temperature and the mass percent at extended times exceeded that predicted from the equilibrium constant obtained from MTDATA. This anomaly was considered to be due to the fact that some of the molybdenum predicted to be in the $M_7C_3$, was actually in another carbide, $M_6C$, which was not accounted for in the analysis. Tempering at 750°C for 48 hours allowed the $M_{23}C_6$ and $M_7C_3$ to mature and subsequently the equilibrium compositions of these carbides were determined by energy dispersive X-ray analysis, (EDXA), as described in Table 2.2:
| Carbide Type | Element Mass % |
|-------------|----------------|
| | Cr | Mn | Mo | Fe |
| $M_{23}C_6$ | 31 | 2 | 9 | 58 |
| $M_7C_3$ | 51 | 2.5 | 7 | 39.5|
Table 2.2: Equilibrium compositions of $M_{23}C_6$ and $M_7C_3$ carbide - after Thomson [24].
Data derived from MTDATA was used in conjunction with a model developed by Robson and Bhadeshia [37] to predict precipitation sequences in power plant steels. Figure 2.3 shows the outcome of their prediction for 2.25%Cr-1%Mo steel at 600°C.
Figure 2.3: Predicted evolution of precipitate volume fractions at 600°C in 2.25%Cr-1%Mo steel -after Robson and Bhadeshia [37].
The microstructural changes occurring in 1%Cr-0.5%Mo steel have been investigated by Varin and Haftek [38] and compared with the ageing studies on 2.25%Cr-1%Mo steel by Baker and Nutting [17] and those of Abdel-Latif et al [39]. Discrepancies between the times for the formation of the equilibrium carbide, $M_6C$, to form in the ferrite were attributed to the lower chromium and molybdenum content of the 1%Cr-0.5%Mo steel. In the study carried out by
Abdel-Latif et al [39] the transformation structure of the annealed 2.25%Cr-1%Mo steel was undefined but appeared from representative microstructures to consist of a mixture of pro-eutectoid ferrite, pearlite and possibly some bainite. The authors did not attempt to attribute specific carbides to their location in the microstructure but considered only the general trend for a change from predominantly $M_{23}C_6$ type carbides in the structure at relatively short service times to a mixture of $M_{23}C_6$ and $M_8C$ at greatly extended times. It was noted that the chromium rich carbide, $M_7C_3$, was not detected in this study and that the carbide evolutionary process was, hence, described as:
$$M_2C \rightarrow M_{23}C_6 \rightarrow M_8C.$$
In the study by Varin and Haftek [38] $M_7C_3$ carbides were detected in the pearlite after $1.22 \times 10^5$ hours having precipitated at the expense of Fe$_3$C. In the ferrite grains it was suggested that $M_2C$ gave way to $M_{23}C_6$, which is in contradiction to that described by Baker and Nutting [17] for precipitation in ferrite where $M_2C \rightarrow M_8C$. This variation may be due to the difference in the partitioning of elements between the ferrite / pearlite phases in the former study compared to that between the ferrite / bainite phases in the latter or simply due to the difficulty in uniquely identifying the carbides by the techniques used. $M_8C$, the equilibrium carbide was found in this study only at extended times at the ferrite grain boundaries.
Most of the studies outlined above have only considered the effect of time and temperature on the carbide evolution process, but in fact it has been shown that the action of an applied creep stress plays a role. Gope et al [15] carried out microstructural studies on gauge and shoulder sections of long term creep tested specimens in normalised and tempered, fully bainitic, 2.25%Cr-1%Mo steel. It was found that the applied stress had the effect of accelerating the dissolution of $M_2C$ in the structure, promoting the precipitation of the equilibrium carbide $M_8C$ and aiding the recovery and recrystallization of the ferrite matrix. In both the case of the stressed and unstressed regions the $M_2C$ particles were observed to grow in length initially with time at temperature and then decrease just prior to the precipitation of $M_8C$.
In reviewing the foregoing work it was clear that many anomalies existed in the precipitation sequences defined for the low alloy Cr-Mo steels and, to summarise, these can be attributed to:
i) Variations in the starting microstructures studied; annealed structures of ferrite/pearlite\(^{[38][39]}\), normalised structures of ferrite/bainite\(^{[17]}\) or 100% bainite\(^{[15][17]}\).
ii) Compositional variations; comparing alloys containing different carbon, chromium and molybdenum mass percents.
iii) Uncertainty in the methods used for identifying the precipitate types; X-ray diffraction, energy dispersive X-ray analysis, carbide morphology and electron diffraction.
If estimations of component remaining life are to incorporate changing material microstructural maturity it is obvious that the carbide evolution process must be uniquely understood for a particular composition, initial microstructure and phase constitution and that the techniques used for this identification of the carbides involved must be reproducible and consistent.
### 2.3 Creep Deformation
The creep behaviour of metals and alloys are commonly portrayed in terms of the well known ‘creep curve’ exhibiting the classical three stage deformation process\(^{[6][40]}\). Conventionally the design lifetime of a metallic component has been based upon the secondary creep rate portion of the creep curve, where the deformation rate is governed by the combined action of strain hardening and thermally activated recovery of the dislocation structure\(^{[6]}\). Equation 2.2 describes the generally accepted inverse relationship between secondary creep rate of metals and alloys, \(\dot{\varepsilon}_s\) and their rupture time, \(t_r\)\(^{[41]}\). Both parameters vary similarly as a power function of the applied stress\(^{[42]}\) and Arrhenius function of temperature.
Where:
\[ n = \text{stress exponent (varies with creep mechanism acting)} \]
\[ Q = \text{activation energy} \]
\[ R = \text{gas constant (8.315J/mol.K)} \]
To correlate the vast bank of data for creep tests derived from specific materials under various conditions, several empirical formulae have been derived which describe the dependence of the rupture time on stress and on temperature [41] [43] [44]. One of the most commonly used of these is the Larson – Miller parameter [44], (LMP), which proposes the following relationship between time, stress and temperature:
\[
\text{LMP} = T \left( C + \log t_r \right) / 1000
\]
Where:
\[ T = \text{absolute temperature in Kelvin} \]
\[ C = \text{constant generally accepted to have a value of 20} \]
\[ t_r = \text{time to rupture} \]
The practical application of this parameter is to allow long time rupture to be predicted from short term test as described by Dieter [45].
Lifetime predictions based on such relationships and data extrapolated from relatively short term tests can lead to misinterpretation due to microstructural changes which occur with time. Hence, to prevent under-estimation, lifetime predictions are often conservative estimates incorporating generous safety factors to cover deficiencies in knowledge. Detailed knowledge about a particular material and how its creep behaviour is affected by changes in composition, stress state, temperature and microstructure provides the basis for more accurate prediction of component lifetime and consequently prevents unnecessary, and expensive, premature removal of components from service. The techniques developed for the estimation of remanent life will be reviewed further in section 2.4.
Creep is known to involve a number of diffusion rate controlled processes. The process of atom movement by diffusion, leading to creep deformation, is in turn governed by the temperature and stress regime in which the material operates and can be conveniently summarised for a specific material, by a deformation mechanism map as first proposed by Frost and Ashby [5]. Figure 2.4 shows a schematic representation of a map for the general case.
![Figure 2.4: Typical deformation map – plot of normalised stress against homologous temperature - after Reidel [40].](image)
At low temperatures and high stresses plastic deformation occurs mainly by dislocation glide. At high temperatures, however, above 0.3Tm creep deformation occurs at much lower stresses than those associated with dislocation glide by the processes of either dislocation creep or diffusion creep [5]. As indicated in Figure 2.4 diffusion creep may operate in two ways by either
diffusion of atoms through the bulk of the grain, Nabarro – Herring creep, or by diffusion predominantly along grain boundaries, Coble creep.
The stress required to deform a crystalline material plastically is that needed to move dislocations within its structure. Dislocation movement is resisted by:
i) the intrinsic lattice resistance, (Peierls force), and
ii) the obstructing effect of obstacles, for example, dissolved solute atoms, precipitates or other dislocations.
Diffusion of atoms can unlock dislocations from obstacles and the diffusion of these unlocked dislocations under an applied stress leads to dislocation creep, also known as power law creep.
In this process the glide force, $\tau b$, per unit length is balanced by the reaction force, $f_0$, from the precipitate and unless the dislocation intercepts the precipitate in mid-plane there is a force, $\tau b \tan \theta$, that attempts to deflect the dislocation out of the slip plane, as illustrated in Figure 2.5. Edge dislocations cannot move out of the slip plane by glide upwards by shearing of atom planes, however, they can climb perpendicular to the slip plane if atoms at the bottom of the extra half plane are able to diffuse away, as shown in Figure 2.6. Thus the dislocations appear to climb until they overcome the obstacle to glide.
Where: $\tau =$shear stress and $b=$Burgers vector.
Figure 2.6: Diffusion of atoms leading to dislocation climb - after Ashby and Jones [46].
As indicated in Figure 2.4 the region of dislocation creep can be further divided into sub-zones behaviour dependent on the temperature and stress operating. The approximately horizontal broken line separates the low-stress regime, where grain boundary sliding contributes markedly to the total strain [47] [48] [49], from the high stress regime, where the grain boundaries behave as if they were rigid. Various mechanisms for grain boundary sliding have been proposed [50] [51] [52] [53] and summarised by Pickering [6] to involve:
i) Sliding along the grain interface, in which there may be ledge type obstacles, with accommodation being provided by diffusion of atoms and vacancies.
ii) Sliding in regions adjacent to the boundaries by dislocation climb and glide resulting in the sub-grain formation adjacent to the boundary.
iii) Sliding by the motion of grain boundary dislocations and grain boundary migration.
In each case, grain boundary sliding may be accommodated by the development of local cavities, the formation of which will be reviewed in section 2.4.2.
The vertical dashed line in the low stress regime of dislocation creep in Figure 2.4 separates the zones where low temperature, vacancy diffusion along dislocation lines, known as core diffusion, and illustrated in Figure 2.7, dominates over that where diffusion is mainly through the lattice.
Figure 2.7: Diffusion along dislocation lines, core diffusion, at low T/Tm – after Ashby and Jones [46].
As the externally applied stress decreases the rate of dislocation creep falls quickly but creep does not stop. At high T/Tm ratio, diffusion may occur from one set of grain faces to another and dislocations are not involved. This process is known as the Nabarro-Herring mechanism [54], as shown schematically in Figure 2.8.
Figure 2.8: The process of creep deformation by diffusion at high T/Tm – after Ashby and Jones [46].
The rate of creep by this process is proportional to the diffusion coefficient, $D$, and to the stress, $\sigma$, which provides the driving force for diffusion. Creep rate in this regime varies inversely with (grain-size)$^2$, that is $1/d^2$. Effectively as the grain size increases the atoms have to diffuse further and thus the rate of creep deformation diminishes.
At lower values of $T/T_m$, bulk diffusion becomes more difficult and the mechanism which predominates is that of Coble creep [4]. In this case, atoms diffuse along grain boundaries rather than through the grains and grain boundary sliding takes place in order that holes do not open up between grains.
The accuracy of creep deformation maps derived for specific alloys are, as Ashby [55] points out, "only as good as the constitutive equations used to construct them", but, they do provide an excellent qualitative basis on which to choose material for a particular application, to predict operative mechanisms and to select appropriate strengthening mechanisms [6].
### 2.3.2 Creep Deformation of Chromium-Molybdenum Steels
It is generally recognised [20] [56] that all creep resistant alloys depend primarily on finely dispersed particles for their strength and that inter-particle spacing is an important factor controlling the movement of dislocations. In ferrite areas the dominant carbide to form in molybdenum steels is Mo$_2$C, whereas, in chromium steels it is M$_7$C$_3$ and in vanadium steels V$_4$C$_3$. A fine dispersion of these carbides gives rise to secondary hardening peaks during tempering and initially high creep resistance. The reduction in creep strength with time at elevated temperatures is widely considered to be due to microstructural degradation associated with the coarsening of these precipitates and it is this which is responsible for the initiation of tertiary creep in low alloy steels. Consequently it was recognised that there is a relationship between the critical particle spacing and the onset of rapid creep deformation [57] and this has been
extended to creep life prediction from microstructural assessment [56] [58] [59] [60]. Eventually, with time at temperature the material microstructure matures to its equilibrium state, as outlined in section 2.2.2, and the carbides resulting will be stable. In creep deformation terms this usually occurs towards the end of the component life as the equilibrium structure offers little resistance to the mechanisms of deformation and failure.
It has been shown by Abe and Yagi [61] [62] that although the initial microstructural differences between the chromium – molybdenum steels of various compositions imparts significant differences in creep rupture strength over extended periods, corresponding to Larson Miller Parameter >19x10^3, the properties converge, as illustrated in Figure 2.9. The fundamental creep strength is an inherent property of the Cr-Mo steels at long times and is referred to as “inherent creep strength” [62]. The inherent creep strength is dependent on the chemical composition of the ferrite matrix and not on the initial microstructure as a whole.
Figure 2.9: Creep rupture strength of carbon steels and Cr-Mo steels - after Abe and Yagi [62].
The optimum combination of transformation and precipitate structure for creep resistance in 2.25%Cr-1%Mo steel has been discussed by many authors [17] [29] [22]. It is generally accepted that creep strength is conferred by the presence of fine M$_2$C precipitates in both ferrite and bainite phases, the M$_2$C causing precipitation hardening due to interactions with moving dislocations. The growth and dissolution of M$_2$C is much more rapid in bainite than in ferrite and in the former is followed by recovery and recrystallization of the matrix. Consequently, although bainite is beneficial for short term low temperature strength, this advantage is lost after longer term service or service at high temperatures [29].
In addition to precipitation strengthening, solid solution strengthening also plays an important role in the creep deformation behaviour of 2.25%Cr-1%Mo alloy. Solid solution strengthening involves the inhibition of movement of dislocations decorated by atmospheres of carbon and molybdenum atom clusters [16] [64] [65].
Kleuh [16] showed that creep curves for 2.25% Cr-1%Mo alloy may differ from the classical three stage curve due to a combination of precipitation and solid solution strengthening mechanisms being operative, Figure 2.10. At intermediate stresses non classical curves, exhibiting 2 steady state stages, were observed. It was considered that the first steady state stage was controlled by interaction solid solution strengthening and that the second steady state stage was controlled by precipitation strengthening. In the low stress regime classical curves were produced and deformation was considered to be being controlled by dislocation movement only. At high stresses, classical curves occurred once more, this time due to the combined effect of deformation controlled by both solid solution and precipitation strengthening.
The effect of stress regime on the creep behaviour of 2.25%Cr-1%Mo steel has also been studied by Yamauchi et al [68], who assessed the relevance of the Larson-Miller Parameter constant, commonly taken to be 20, on creep life prediction times. From extrapolation of long term data from short term tests it
was concluded that in the low stress regime, $< 60 \text{MPa}$, the LMP constant should be 15.6 and in the high stress regime, $> 60 \text{MPa}$, 18.8. It was proposed that the difference in the constant for the two stress regimes was due to the different mechanisms controlling the deformation processes and that in the high stress regime deformation was controlled by Orowan bowing between particles and in the lower stress regime by dislocation climb or grain boundary sliding, Figure 2.11.
Creep deformation mechanisms operating in various stress regimes for 2.25%Cr-1%Mo alloy have also been proposed by Maruyama et al [67], their study being portrayed in the form of a deformation map, Figure 2.12, in the manner of Frost and Ashby [5]. The range of conditions depicted in Figure 2.12 were considered to reflect those typical of the service conditions for a 2.25%Cr-1%Mo steel component in operation in a power plant.
Maruyama et al [67] suggested that below the athermal yield stress, $\sigma_A$, creep deformation took place by dislocation creep after initial elastic deformation upon loading. In this regime the dislocation creep rate is controlled by lattice diffusion and the main obstacle to dislocation motion is by fine precipitates in the microstructure. Creep by the process of bulk diffusion did not appear to be a contributing mechanism under the conditions used to obtain the test data from which this map was derived.
Above $\sigma_A$, it was proposed that athermal plastic deformation (dislocation glide) took place during loading and then creep deformation occurred by the same dislocation creep mechanism as at lower stress level. However, in this high stress regime the main obstacle to dislocation movement changes to dislocation substructure. Dislocation creep controlled by core diffusion, (low temperature power law creep), was not observed in this study.
Figure 2.10: Creep curves for various stress regimes at $565^\circ C$ - after Kleuh \textsuperscript{[16]}
Figure 2.11: Creep rupture mechanism diagram for 2.25%Cr-1%Mo steel - after Yamauchi et al \textsuperscript{[66]}.
Figure 2.12: Proposed creep deformation mechanism map of 2.25%Cr-1%Mo steel – after Maruyama et al [67].
2.4 Creep Fracture
In the latter stages of the creep process, the so called tertiary stage, the stressed material undergoes a period of rapid deformation followed by failure. With materials exhibiting low rupture ductility, the advent of tertiary creep can be quite abrupt, the acceleration in the creep rate occurring over a very short period of time immediately prior to fracture.
Many mechanisms have been postulated to explain tertiary creep behaviour. These have been reviewed by Pickering [6] and also by Ashby and Dyson [68] and are summarised as follows:
i) necking leading to an increase in stress by decreasing the load bearing area
ii) the development of grain boundary cracking and cavitation of sufficient intensity to affect the deformation rate, or which can decrease the load bearing area and thus increase the stress
iii) microstructural changes such as precipitate particle coarsening, recovery, recrystallization and grain growth
iv) environmental changes
v) increase in specimen volume due to cracking which makes a contribution to strain.
In terms of industrial significance, the creep ductility of a material is deemed to be of paramount importance as it reflects the ability of the material to resist cavitation and cracking during the period of rapid strain.
The following section aims to review the most industrially significant mechanisms proposed for creep fracture and, in particular, examines the mechanisms for the process of cavity nucleation and growth. The phenomena of notch sensitivity will also be covered with reference to creep damage mechanisms in the presence of a pre-existing defect.
2.4.1 Creep Fracture Mechanisms
Crystalline solids can fracture by one or more of several mechanisms which can be displayed simplistically on a fracture mechanism map [69] [70], analogous to the deformation maps discussed in section 2.2.1. A schematic of such a diagram is shown in Figure 2.13.
The location of the boundaries between the different mechanism fields depends on the material, its heat treatment, the chemical environment and any
additional stress components. Actual fracture mechanisms maps for iron and various steels have been constructed by Fields et al.\textsuperscript{[71]}.
In the stress and temperature regimes experienced by Cr-Mo steel components in power plant applications, failure is most likely to occur by the process of intergranular creep fracture, with ductile transgranular failure being of much lesser significance.
Transgranular creep fracture occurs by a mechanism not unlike that of normal ductile fracture at low temperature, in that voids nucleate and grow and eventually coalesce to give fracture\textsuperscript{[6]}. Nucleation occurs at non-metallic inclusions or other second phase particles, with the void nucleation mechanism depending on the nature of the particle / matrix interface and its degree of coherency and surface energy. Sulphides, for example, usually decohese whilst carbides may either decohese or crack. As most engineering components fail by transgranular ductile failure at room temperature, extensive literature describing this mechanism is available and has been reviewed by Goods and Brown\textsuperscript{[72]} and Knott\textsuperscript{[73]}.
At lower stresses and elevated temperatures, hole growth by plastic straining or creep flow becomes so slow that fracture by grain boundary cavitation intervenes, the strain to fracture is then relatively low and fracture is intergranular\textsuperscript{[40]}.
2.3.2 Creep Cavity Initiation And Growth
Many of the steels operating in power plant applications develop creep cavities under their normal conditions of service, eventually leading to failure. There is, therefore, a need to understand the mechanisms by which the cavities form and grow with a view to improving future generations of power plant steels for increased creep resistance.
Voids in polycrystalline materials nucleate by either wedge type cracking at grain boundary triple points or by more diffuse cavitation at grain boundaries. The mechanisms of grain boundary cavity nucleation and growth have been reviewed by Pickering [6] and more comprehensively covered by Reidel [40]. Wedge cracks of the type shown in Figure 2.14 occur due to grain boundary sliding, building up stress concentrations at triple points and initiating cracks on boundaries normal to the applied stress axis. These types of cracks are found predominantly in samples tested at high stresses or in coarse grained material where grain boundary segregation of impurities decreases the surface energy.
The more widespread type of cavity formation involves the development of "spherical cap" or lenticular voids, mainly on grain boundaries which have a high grain boundary sliding component. The main mechanisms which have been proposed to explain their formation are illustrated in Figure 2.15.
Figure 2.14: Wedge cracking at grain boundary triple point due to sliding – after Lagnebourg [74].
Figure 2.15: Various mechanisms for grain boundary cavity nucleation due to grain boundary sliding – after Evans and Wilshire [60].
In commercial alloys the most common sites for cavity nucleation are second phase grain boundary particles. Numerous investigations have shown that in low alloy steels, cavities are often associated with carbide particles [75] [76]. Although such particles at the boundaries in creep resisting steels are initially beneficial, that is they transmit the shear stresses which the grain boundaries would otherwise not be able to support, eventually stress concentrations may build up at the particle / matrix interface leading to cavity initiation. Once initiated the cavities grow and coalesce forming microcracks, which subsequently, upon reaching a critical size, lead to failure of the remaining ligament.
The role of particles in creating stress concentrations on sliding grain boundaries has been analysed by Reidel [40] and reviewed by Perry [77]. Sliding of grain boundaries, is believed to occur in bursts, causing stress concentrations to be set up at irregularities in the boundaries. These local stresses cause boundary separation to occur and cavities initiate if the process of atom / vacancy diffusion is insufficient to prevent it. Two general mechanisms for the nucleation of cavities have been cited [77]:
i) Steps or ledges in grain boundaries have been supposed to result from the intersection of slip bands with grain boundaries.
ii) Local break down of adhesion associated with precipitates or impurities.
In both cases local plastic strain is a pre-requisite. Evidence for both the mechanisms proposed has been observed during metallographic examination of creep tested material and it is believed that one or other may dominate at any particular instance.
Figure 2.16 summarises Reidel's [40] analysis of the stress concentrations which occur in the steady state after elastic stress concentrations have been relaxed; $\sigma_p$ is the average stress focused on a particle by grain boundary sliding or by inhibited Coble creep and $p$ and $\lambda_p$ are the particle diameter and spacing respectively.
Cavity nucleation has been shown to occur over a substantial fraction of creep life and as such may be used in some materials to estimate remaining life [78].
Once voids have been nucleated, they will continue to grow as creep deformation proceeds. It has been demonstrated that whilst clearly continued grain boundary sliding can result in the progressive growth of both wedge and the rounded type of cavities, another more important growth mechanism is that due to vacancy diffusion to the cavities [79] [80]. Whereas grain boundary sliding would tend to give cavities which are elongated along the boundaries, growth by vacancy diffusion gives the more rounded cavities frequently observed. Additionally, vacancy diffusion would be expected to give more pronounced cavities on boundaries normal to the stress axis, whereas with grain boundary sliding they would be more apparent on boundaries at 45° to the stress axis. In a study by Tipler et al [81] on void formation in chromium – molybdenum steels, it was reported that although the lines of cavities were frequently normal to the applied stress, few of the voids were so oriented. This suggests that the dominant mechanism for void nucleation may differ from that for void growth.
Another factor involved in the growth mechanism of voids was discovered by Taplin [82] who noted that in fine grained materials sliding of grain boundaries tended to encourage void growth on those boundaries which were oriented in the region of 45° to the applied stress, whilst in coarse grained material, voids elongated on boundaries normal to the applied stress. Again there is strong evidence to support cavity growth by both sliding and diffusional mechanisms and it has been suggested [77] that the dominant mechanism may change within a single test.
Under the stress and temperature regimes associated with most power plant applications Hull and Rimmer [83] suggested that the cavity growth mechanism is most likely to be stress-directed diffusion of atoms away from cavities into grain boundaries where they can be deposited. Figure 2.17 attempts to illustrate the mechanism proposed.
**Figure 2.17:** Model for grain boundary cavitation by surface and grain boundary diffusion – after Hull and Rimmer [83]. Adjoining grains are assumed to act as rigid bodies.
Dyson [84] [85] pointed out that the initial models proposed for this mechanism did not take into account that the material surrounding the grain boundary facet may impose a restriction on cavity growth rates. If the material surrounding a cavitating facet was rigid, the excess volume of cavities could not be accommodated and cavity growth would come to a stand still. Thus, the rate of cavity growth may be controlled by the deformation rate of the surrounding material leading to the phenomena known as constrained cavity growth.
Hancock [86] has proposed an alternative growth mechanism which occurs in situations where stresses are high and plastic dislocation creep is sufficiently rapid that, in the limiting case, the transport of matter along the grain boundary can be neglected. This is illustrated below in Figure 2.18. Co-operative diffusion of atoms and vacancies in the vicinity of the void wall tend to make the void increase in volume and decrease in radius. If surface diffusion is rapid, local matter transport along the void surface always serves to retain the quasi-equilibrium spherical cap shape so that in this case the net effect is to increase void radius.
![Figure 2.18: Schematic illustration of cavity growth by the combination of plastic creep flow and rapid surface diffusion – after Hancock [86].](image)
If both the above processes of creep flow and grain boundary diffusion are operative at the same time, as discussed in detail by Needleman and Rice [87], active cavity growth rates can be many times greater than would be the case if either mechanism acted in isolation and the path length over which matter must diffuse is effectively shorter. In turn this means that less stress is required for growth to occur. Conversely, for a given stress level, the growth rate will be more rapid when local stress accommodation of the diffused matter is possible by creep [87].
Figure 2.19 shows rupture life-times of 2.25%Cr-1%Mo steel, as measured by Cane [88], compared to estimated rupture lifetimes based on models proposed for constrained and unconstrained cavity growth involving either instantaneous or continuous nucleation derived from Reidel [40]. The study showed that the
constrained model with continuous nucleation gave the most accurate prediction for time to failure for 2.25%Cr-1%Mo steel.
Figure 2.19: Comparison of creep curves from uniaxial creep data for 2.25%Cr-1%Mo steel compared with predictions for constrained and unconstrained cavity nucleation —after Reidel [40].
As the mechanisms of creep deformation and failure are affected by both the transformation products and precipitate structure of steels it is essential when discussing creep deformation that the condition of the material is accurately described. An example of this effect was apparent in a study by Cane [89] on the effect of transformation structure on creep rupture behaviour of 2.25%Cr-1%Mo steel. In this study an increase in the ferrite / bainite ratio in tempered coarse grained 2.25%Cr-1%Mo alloy was found to modify the rupture properties by improving ductility, increasing rupture life up to intermediate ferrite contents and causing a subsequent decrease thereafter. Additionally the final fracture mode was observed to change from brittle intergranular to ductile transgranular failure. These effects were considered to be due largely to a
reduction in susceptibility to cavitation damage caused by the progressive elimination of prior austenite grain boundaries with increase in ferrite content. Minimum creep rate, $\dot{\varepsilon}$, was shown to be relatively independent of transformation structure at intermediate and high bainite contents but increased markedly at low bainite contents.
At high bainite contents, cavitation damage was considered to have made a significant contribution to the overall creep rate, while at intermediate bainite contents, the respective age hardening and softening in the ferritic and bainitic constituents lead to a corresponding structural insensitivity to creep rate. At low bainite contents, however, coarse precipitation occurring during transformation resulted in a consequent loss in super-saturation and, therefore, any ferrite strengthening potential.
In summary, fracture of power plant steels operating at temperatures of 0.3-0.4 times the melting temperature under creep conditions is governed predominantly by intergranular cracking. The underlying physical mechanisms are nucleation and subsequent growth of microscopic cavities on the boundaries of the grains. When sufficient cavities have reached a critical size, they will coalesce to form a microcrack along a grain boundary. Subsequent linking-up of microcracks leads to the growth of macroscopic cracks and eventually failure of the component remaining ligament. Cavity growth occurs mostly by grain boundary and surface diffusion, other important mechanisms being creep of the grain material and grain boundary sliding. All of these have different time scales and different dependencies on stress state, and it is the competition between them that controls damage evolution and the final lifetime of the component [90].
2.4.3 Creep in the Presence of a Pre-Existing Defect
Prior to the 1970's, the majority of work carried out to estimate the design life of a component in a creep environment was determined from data extrapolated from uniaxial creep tests. It soon became apparent, however, after the occurrence of a few premature failures in electricity power plants, that uniaxial data did not give an accurate estimation of design life if the component contained a pre-existing defect. Consequently many researchers have since carried out investigations relating to this phenomena [14]. The most recent studies include not only a means of estimating and appreciating failure due to crack growth, but also consider the period of initiation, or incubation, prior to crack extension [9] [11] and these will be reviewed in more detail in section 2.5.2.
To accurately predict the safe operating life of a component, which may include a period of damage tolerance, many factors have to be taken into consideration and these can be broadly categorised into two areas:
i) The continuum-mechanics deformation fields around the crack tip, characterized in terms of stress intensity factor, $K_I$ [91], the J-contour integral [92], and creep fracture mechanics parameter, $C^*$ [93] [95].
iii) The micro-mechanisms occurring at the crack tip, such as stress relaxation, microstructural evolution and cavity formation.
The aim of this section is to review the studies which have been carried out on creep crack initiation and growth from pre-existing defects with a view to understanding how the state of stress at the defect tip affects the microstructure and, hence, the creep crack initiation and growth mechanisms in this region.
When a body is loaded under creep conditions in the presence of a pre-existing defect, there may be a period of time before that defect begins to grow, the so-called initiation time. During this period a creep process zone develops at the defect tip, the size and rate of development of which is governed predominantly by the elastic-plastic characteristics of the material [94]. The
damage zone that develops, undergoes microstructural changes due to the influence of the operative creep mechanisms, as described in section 2.3.1, and the effect of the triaxial state of stress at the defect tip. Hence, creep brittleness rather than creep strength of a service component is often the vital factor in failure by creep or thermal fatigue [95] under such conditions.
Relaxation of stress by heat treatment, due to atom / vacancy diffusion, represents displacement – controlled situations where stress can decrease as local creep strain accumulates. The probability of crack initiation and growth depends on the magnitude of the displacement relative to the properties of the local microstructure. Ductile crack growth occurs with a large displacement and is accompanied by widespread microstructural damage, whereas brittle cracking is accompanied by small displacements and little damage visible away from the main crack. It therefore follows that, the greater the notch opening / creep crack length ratio the more ductile the situation [96].
The factors affecting the mode of crack growth and failure has been described schematically by Gooch and King [95], as shown in Figure 2.20.
Figure 2.20: The factors that affect ductile-brittle creep behaviour - after Gooch and King [95].
The three regions of this diagram can be described as follows:
i) The top right hand corner of the diagram portrays the creep brittle situation, where crack growth rates are commonly described by the use of linear elastic fracture mechanics (LEFM)\textsuperscript{[97]} in the form:
\[
\frac{da}{dt} = AK^n \quad \text{-Equation 2.4}
\]
Where,
K = the linear elastic stress intensity factor
a = crack length,
t = time and
A and n = constants
The LEFM approach assumes that stress relaxation is slow in relation to crack growth rate and, hence, the stress and the displacement distribution are represented by K to a reasonable degree of accuracy.
ii) The bottom left hand corner of the diagram represents the case where reference stress, $\sigma_{ref}$, can be used to assess the lifetime of a component by comparison with uniaxial creep rupture tests\textsuperscript{[98]}. This approach assumes that stress relaxation by creep at the crack tip is rapid compared with crack growth in the same region and, hence, there is negligible stress concentration remaining ahead of the crack.
iii) The intermediate region, represents all other cases where crack growth rates need to be calculated from time dependent fracture mechanics methods using C*, J or COD (crack opening displacement) parameters. These methods, the so called post- yield fracture mechanics methods\textsuperscript{[95]} involve complex calculations to account for microstructural changes taking place with time.
The geometrical factors which affect the situation at the notch tip of the pre-existing defect have been defined by Haigh\textsuperscript{[99]} by the equation:
Where:
$K_{\text{nom}} = \text{nominal value of stress intensity}$
$\sigma_{\text{ref}} = \text{reference stress (equivalent stress level in a uniaxial test which causes failure in the same time)}$
$W = \text{specimen width}$
$m = \text{general yield ratio} = \left( \frac{\text{load to yield a cracked specimen}}{\text{load to yield an uncracked specimen}} \right)^{[100]}$
$Y = \text{compliance function} = \frac{KBW^{1/2}}{P'}$
and
$B = \text{specimen thickness}$
$P' = \text{applied load}$
Typical values of $\frac{K_{\text{nom}}}{\sigma_{\text{ref}} \sqrt{W}}$, shown for common specimen geometry's and plotted as a function of $a/W$ (crack length / specimen width) in Figure 2.21, indicate that compact tension (CT) specimens exhibit the most brittle situation at the notch tip.
Figure 2.21: The effect of specimen geometry on brittleness in plane strain—after Gooch and King [85].
Materials operating in power plant predominantly fall into the intermediate regime of Figure 2.20, where the ductile microstructure allows the initially high crack tip stress to relax at the same time as the integrity of the structure is reduced. Haigh [7] has shown that in the situation where constraint is high, such as in the case of CT specimens, and the material is ductile then the C* characterising parameter is valid and the reference stress can be re-written as shown in equation 2.6 for the plane stress situation:
\[
\sigma_{ref} = \left( \frac{P}{m \cdot B \cdot W} \right)
\]
-Equation 2.6
For the plane strain situation the equation is adapted to give equation 2.7:
\[
\sigma_{ref} = \left( \frac{P}{m_{PL\epsilon} \cdot B_n \cdot W} \right)
\]
-Equation 2.7
Where:
\(B_n = \text{net specimen thickness}\)
\(M_{PL\epsilon} = -(1+1.702a/w) + (2.702+4.599(a/w)^2)^{1/2}\) [19]
Whether plane stress or plane strain conditions are operative at the crack tip will depend upon the degree of constraint; this constraint, in turn, will be dependent upon the test piece dimensions, notch geometry and creep deformation which accompanies fracture. The effect of specimen geometry on defect tip constraint has been studied by many authors and some of the factors observed are outlined below.
i) Constraint increases with increased specimen thickness [94]
ii) Constraint increases with the presence of side grooves [101]
iii) Constraint increases as depth of side grooves increase [101]
iv) Constraint varies with specimen geometry and is greatest for CT specimens [102] [103].
v) Constraint varies with notch geometry and is higher for sharp notches than blunt notches [14].
In general, the greater the degree of constraint at the notch tip the more creep brittle the situation.
The microstructure can also have a powerful effect on creep brittleness / constraint as demonstrated by Gooch and King [95] who showed that an increase in the prior austenite grain-size of bainite increased the tendency to brittleness of a Cr-Mo-V steel as did an increase in the bainite content of a mixed ferrite / bainite structure.
The effect of triaxiality, defined as the ratio of the average of the radial, axial and tangential stresses to the Von Mises stress [104], on the deformation process of coupled creep and grain boundary diffusion was shown in a study by Sham and Needleman [105]. They reported that an increase in triaxiality from 0.33 – 4.0 resulted in an increase in the flow of matter from the cavity to the grain boundary but did not significantly alter the effective diffusion path lengths. This hypothesis was later discussed and developed by Van Der Giessen et al [106] who found that for higher triaxialities, >4.0, and in the presence of large voids, the diffusion path essentially vanishes since dislocation creep completely dominates. In a complex study by Maille [107] on the effect of multiaxiality on the creep deformation of 1%Cr-1%Mo steel, it was found that increasing the multiaxiality quotient resulted in a decrease in rupture strain and an increase in the severity of creep damage.
A recent study conducted by Frères and El-Magd [104] showed the influence of a multi-axial stress state on the behaviour of inter-crystalline creep microcracks. Tests carried out on circumferentially notched creep specimens showed that deep, sharp notches enhanced multiaxiality and stresses in the tangential and radial directions, leading to notch constraint and reduced cavitation density. Shallow, sharp notches enhanced the stress in the axial direction. A similar effect was noted by Molinie [108] who not only showed that cavity density decreased with increased triaxiality but also that the maximum damage occurred in the location of the principal stress axis.
The purpose of obtaining creep rupture data is to assess a particular material for service at elevated temperatures. The design code for components is usually based upon uniaxial creep rupture data and defines a maximum allowable stress which can exist in the structure for the required life at the operating temperature. Design codes give a conservative estimate of useful life and the actual life may not be expended in service, therefore, it is economically advantageous to be able to assess components in service and, where possible, extend their life. In service component assessment also avoids costly plant shutdown time due to unexpected failures.
A review of life assessment methods by Cane and Williams [86] details two main approaches to this problem. The first method is based upon data taken during the operation of plant, for example temperature and pressure measurements and the second method is based upon periodic examination during plant shutdown, for example by crack detection, microstructural examination and strain analysis. For the purpose of the review, damage was considered to have occurred if the material life had been reduced by service. Damage characterisation was by assessment of the presence, size and distribution of cavities or second phase particles, the local softening of the material caused by exposure, or any other effect which reduced life under subsequent creep loading.
Additionally, life assessment techniques must be able to account for the situation where components are put into service containing pre-existing defects, such as in large castings, and in the main this is addressed by fracture mechanics concepts.
2.5.1 Creep Life Assessment of Initially ‘Defect Free’ Structures
Traditionally most creep rupture data for design purposes is obtained from uniaxial stress rupture tests which are carried out at higher stresses or
temperatures than those used in service. Long term rupture behaviour is then predicted by extrapolation of the short term data of test stress against a time temperature parameter, as described previously in section 2.3.
Parametric equations have also been used in conjunction with life fraction rules in attempts to predict the creep behaviour of materials under variable stresses and temperatures [88].
The original life fraction rule put forward in 1952 by Robinson [109] suggested that for any series of stress and temperature conditions the life fractions could be linearly summed with failure occurring at unity.
\[
\sum_x \frac{t_x}{t_{xf}} = 1 \quad \text{-Equation 2.8}^{[88]}
\]
Where
\( t_x = \) time at stress \( \sigma_x \) and temperature \( T_x \)
\( t_{xf} = \) failure time at \( \sigma_x, T_x \)
This model assumed that material behaviour was independent of history, which was soon shown to be incorrect [110] when smaller specimens machined from a failed uniaxial creep specimen were found to have a reduced life on re-testing. Many variations of the rules have since been refined to account for the effects of material variability, multiaxial stresses and environmental effects and these have been rationalised by Grounes [111]. The most successful improvement to the life fraction time rules incorporates strain fractions [112].
\[
\sum_x \frac{\varepsilon_x}{\varepsilon_{xf}} = 1 \quad \text{-Equation 2.9}^{[88]}
\]
Where
\( \varepsilon_x = \) strain at stress \( \sigma_x \) and time \( t_x \)
\( \varepsilon_{xf} = \) failure strain at stress \( \sigma_x \) and \( t_{df} \)
These methods are used in conjunction with creep models which simulate time dependent strain accumulation of materials under load at temperature and models have been developed to characterise the growth of creep cavities, wedge cracks and the formation of macroscopic cracks to ultimate failure. Such strain based methods require disciplined monitoring and ensuring that the measurements taken are representative of the component as a whole is difficult. Microstructural examination can provide useful supporting information and studies have been carried out to correlate creep strain with quantitative microstructural damage.
Creep strain data generated under accelerated test conditions can provide residual life estimates if a model can be developed relating creep strain, $\varepsilon$, or strain rate, $\dot{\varepsilon}$, to creep life, $t_r$. To determine remaining lifetime by microstructural examination predictive models are required that relate the relevant damage feature to the life fraction.
The important mechanisms of microstructural damage that must be considered for such a model, and their relationship to strain rate, have been reviewed and defined quantitatively by Dyson and McLean [113]. The so-called, physically based ‘continuum damage mechanics’ (CDM’s) equations, as defined in Table 2.3, are all related to strain rate as a function of applied stress, $\sigma$, temperature, $T$, hardening parameter, $H$ and a damage parameter, $D$.
The use of advanced computer systems allows the various contributions to creep deformation, in the form of CDM parameters, to be assessed both individually and concurrently resulting in a very powerful tool for life assessment. Of particular relevance to the current study is the use of data based on the microstructural aspects of cavity nucleation and growth together with the carbide evolution process.
Several methods have been used to quantify creep damage by the process of cavitation. These methods include the use of parameters, such as A1, A2 and $A^*$ [114], that quantify the number of cavitating grain boundaries on, or in the vicinity of, a reference line, and those due to observations based on a visual classification scheme, such as that derived by Neubauer and Wedel [115]. In a
study by Sandstrom [116] the creep damage classes of Neubauer and Wedel were related to a measure of physical damage in order to allow a direct estimation of remaining life and a suggested time scale for the next plant inspection.
The observation of the changing microstructure in power plant steels has often been proposed as a useful mechanism for estimating the remaining life of a component [117] [118] [119] [120]. In particular the composition of the evolving carbides has been used to predict the onset of failure by tertiary creep. An example of such a study is that of Sugita [120] who found that in the heat affected zone of a 2.25%Cr-1%Mo steel sample, creep voids formed when the equilibrium carbide M₆C was present in volume fractions >60%.
Pioneering work by Bhadeshia et al [34] [37] [121] [122] has shown that the microstructural evolution process in power plant steels can be predicted using a computer programme called MTDATA [38], as mentioned previously in section 2.2.2, from a knowledge of chemical composition and relevant transformation kinetic equations. Knowing the time to reach a particular microstructural maturity, allows components to be assessed by periodic microstructural assessment for suitability for extended service. For example an accurate means of determining data by the predictions of Bhadeshia et al could negate the need for long term creep rupture testing as used in the studies on Cr-Mo steels by Dobrzanski and Hernas [103]. In this latter study the influence of phase constitution and damage process was linked to residual life time, as described in Figure 2.22 and Table 2.4.
Table 2.3: Creep damage categories, mechanisms and incorporation into continuum damage mechanics equations – after Dyson and McLean [113].
| CREEP DAMAGE CATEGORY | DAMAGE MECHANISM | DAMAGE PARAMETER $D$ | DAMAGE RATE $\dot{D}$ | STRAIN RATE $\dot{\varepsilon}$ |
|-----------------------|------------------|----------------------|------------------------|-------------------------------|
| Strain-Induced | Cavity Coarsening Controlled by Growth Constraint | $D_c = \frac{\pi d^2 N}{4} w_{\text{eff}}$ | $\dot{\omega} = \frac{k_1}{k_0} \dot{\varepsilon}$ | $\dot{\varepsilon} = k_{\text{sh}} \left[ \frac{\sigma (1 - H)}{\sigma_c (1 - \omega)} \right]$ |
| | Cavity Growth Controlled by Creep Constraint | $D_c = \left( \frac{r}{l} \right)^2$ | $\dot{\omega} = 0$ | $\dot{\varepsilon} = k_{\text{sh}} \left[ \frac{\sigma (1 - H)}{\sigma_c (1 - \omega)} \right]$ |
| | Dynamic Grain Coarsening | $D_g = 1 - \left( \frac{w_{\text{eff}}}{t_g} \right)$ | $D_g = (1 - D_g) \frac{t_g}{t_g}$ | $\dot{\varepsilon}$ |
| Thermally-Induced | Multiplication of Mobile Dislocations | $D_f = 1 - \frac{p}{p}$ | $D_f = C(1 - D_f) \dot{\varepsilon}$ | $\dot{\varepsilon} = \frac{k_2}{(1 - D_f)} \left[ \frac{\sigma (1 - H)}{\sigma_c} \right]$ |
| | Particle Coarsening | $D_p = 1 - \frac{p}{p}$ | $D_p = \frac{K_p}{2}(1 - D_p)^2$ | $\dot{\varepsilon} = k_{\text{sh}} \left[ \frac{\sigma (1 - H)}{\sigma_c (1 - D_p)} \right]$ |
| | Depletion of solid-solution elements | $D_s = p \phi_s$ | $D_s = K_s D_s^{1/2} (1 - D_s)^2$ | $\dot{\varepsilon} = \frac{k_3}{(1 - p D_s)^{1/2}} \left[ \frac{\sigma (1 - H)}{\sigma_c} \right]$ |
| Environmentally-Induced | Fracture of Surface Oxidation Product | $D_m = \frac{2x}{R}$ | $D_m = \frac{1}{R} \left( \frac{K_x}{2\pi} \right)^{1/2}$ | $\dot{\varepsilon} = k_{\text{sh}} \left[ \frac{\sigma (1 - H)}{\sigma_c (1 - D_m)} \right]$ |
| | Internal Oxidation | $D_m = \frac{2x}{R}$ | $D_m = \frac{K_m}{2\pi T_m}$ | $\dot{\varepsilon} = k_{\text{sh}} \left[ \frac{\sigma (1 - H)}{\sigma_c (1 - D_m)} \right]$ |
Figure 2.22: Influence of phase constitution and damage process on safe residual lifetime of 1Cr-0.5Mo steel - after Dobrzanski and Hernas [103].
Table 2.4: Classification of damage process and exhausted creep life - after Dobrzanski and Hernas [103].
2.5.2 Creep Life Assessment Of Structures Containing Pre-existing Defects
If a component has been put into service containing a pre-existing defect, as described in section 2.4.3, then it is likely that failure will occur before that anticipated by the routine examination of microstructure or even strain. To ensure safe operation, therefore, it is necessary to detect any pre-existing defects and assess the likelihood of them growing to an unacceptable size. Effective procedures for defect assessment and remanent life prediction of power plant components have been developed in recent years [10] [18] [94] [123] based on fracture mechanics concepts and an understanding of material creep ductility.
It has been recognised that when a component containing a defect is loaded in creep, there will be a period of stress redistribution at the defect tip prior to crack growth and that this process could occupy a considerable period of the component lifetime, that is, there will be a period of crack incubation [124] - [126]. The life of a structure operating under creep conditions, therefore, may be regarded as comprising two components [11] as reflected in equation 2.10:
Where:
$t_i =$ time to initiate a crack
$t_g =$ time to grow the crack to failure
Immediately after loading, in the absence of plastic deformation, the stress distribution ahead of the crack tip will be defined by the elastic stress intensity factor, $K$. With time creep will cause stress redistribution until a steady state condition is reached which can be described by the fracture mechanics parameter, $C^*$. The creep strain rate, $\dot{\varepsilon}$, and rupture life, $t_r$, properties of a material can be expressed in terms of stress, $\sigma$ as:
$$\dot{\varepsilon} = \dot{\varepsilon}_0 \left( \frac{\sigma}{\sigma_0} \right)^n$$ \hspace{1cm} -Equation 2.11
and
$$t_r = \frac{\varepsilon_{f0}}{\dot{\varepsilon}_0} \left( \frac{\sigma_0}{\sigma} \right)^\nu$$ \hspace{1cm} -Equation 2.12
Where
$\dot{\varepsilon}_0 =$ strain rate in uniaxial creep
$\varepsilon_{f0} =$ uniaxial creep failure strain at a stress of $\sigma$
$n =$ creep exponent
$\nu =$ Poisson’s ratio
The time taken for this stress distribution to be complete, $t_T$, is given by [40]:
$$t_T = \frac{G}{(n+1)C^*}$$ \hspace{1cm} -Equation 2.13
Where
$G =$ the elastic strain energy release rate.
$C^* =$ steady state crack tip parameter.
According to Webster et al [10], this time is usually a small fraction of the component lifetime and it is found that creep crack growth rate, $\dot{a}$, can be correlated satisfactorily in terms of $C^*$ by the relationship:
$$\dot{a} = D_0 C^{*\phi} \quad \text{-Equation 2.14}$$
Where, $D_0$ and $\phi$ are material constants which can be determined by equations 2.15 and 2.16 respectively:
$$D_0 = \left( \frac{n+1}{n+1-\nu} \right) \frac{\dot{\varepsilon}_0}{\varepsilon_{f0}} \left( \frac{1}{I_n \sigma_0 \dot{\varepsilon}_0} \right)^{\frac{\nu}{n+1}} r_c^{\frac{n+1-\nu}{n+1}} \quad \text{-Equation 2.15}$$
and
$$\phi = \frac{\nu}{n+1} \quad \text{-Equation 2.16}$$
Several broadly compatible models relating creep crack growth to $C^*$ have been postulated [94] [127] [128] [129]. When $C^*$ has been shown to predict the stress situation ahead of a crack tip the following general equation can be used to relate crack growth rate to $C^*$:
$$\dot{a} \propto C^{*\phi} \quad \text{-Equation 2.17}$$
Where
$\dot{a}$ = crack growth rate
$\phi$ = fraction close to unity
Nikbin et al [84] derived an equation of this form for a ductile 2.25%Cr-1%Mo steel CT specimen at 538°C as follows:
$$\dot{a} = \frac{3C^{*0.85}}{\varepsilon_f^*} \quad \text{-Equation 2.18}$$
Where
$\varepsilon^*_f$ = creep ductility at the crack tip as a fraction
(for plane stress $\varepsilon^*_f = \varepsilon_{00}$ and for plane strain $\varepsilon^*_f = \varepsilon_{00}/50$ where $\varepsilon_{00}$ is the uniaxial creep ductility).
A subsequent study by Wang and Wu [101] based on the $\dot{a} - C^*$ relationship of Nikbin et al, showed that the relationship is temperature dependent for 2.25%Cr-1%Mo steel as indicated below:
$$\dot{a} = \frac{4C^{*0.85}}{\varepsilon^*_f} \quad \text{at } 550^\circ\text{C} \quad \text{-Equation 2.19}$$
and
$$\dot{a} = \frac{4.93C^{*0.85}}{\varepsilon^*_f} \quad \text{at } 565^\circ\text{C} \quad \text{-Equation 2.20}$$
In this study the Kubo [130] form of the $C^*$ equation was used for CT specimens where:
$$C^* = \frac{2n}{n+1} \cdot \frac{P'\Delta}{B(W-a)} \quad \text{-Equation 2.21}$$
and
$n =$ creep exponent
$P'$ = applied load
$\Delta =$ load point displacement
In a review of aspects relating to the initiation and early growth of creep cracks from pre-existing defects by Holdsworth [11] it was pointed out that equation 2.10 depends to a large extent on the increment of crack extension, $\Delta a_i$, adopted as the definition of crack initiation ($x_c$). For example, as shown in Figure 2.23, it was shown that the time to generate the critical displacement necessary to initiate a micro-crack, for example $x_{c[1\mu m]}$, was significantly less than the time to develop a macro crack, for example $x_{c[1mm]}$. The advantages and disadvantages of the crack initiation criteria adopted were discussed and overall it was considered that a macro-crack criterion of >0.5mm, had the most benefits.
If the minimum crack extension that can be resolved is $\Delta a$ then the incubation period, $t_i$, is given by \textsuperscript{[126]}.
$$t_i = \int_0^{\Delta a} \frac{dr'}{\dot{a}_i}$$
- Equation 2.22
Where
$t_i =$ incubation period
$r' =$ radius of damage zone
$\dot{a}_i =$ crack growth rate
An upper bound $t_i$ can be obtained by replacing $\dot{a}_i$ in this equation with $\dot{a}_0$ and a lower bound by replacing it with the steady state crack growth rate \textsuperscript{[110]} giving:
$$t_{iL} = \frac{\Delta a}{D_0 C^{*\phi}}$$
- Equation 2.23
Alternatively, when the approximate growth relation is used, equation 2.23, it becomes:
$$t_{iL} = \frac{\Delta a \varepsilon_f^*}{3C^{*0.85}}$$
- Equation 2.24
The critical crack tip opening displacement, $\delta_{ix}$, is an important parameter for characterising the creep crack initiation resistance of a material. Conventionally, in the laboratory tests of CT specimens, it is only the crack opening displacement at the load line which is measured \textsuperscript{[131]} and this does not always convey accurately the situation at the notch tip. An alternative method has been proposed by Holdsworth and Cunnane \textsuperscript{[19]} that accurately assesses the actual notch tip displacement and is based on the displacement measurement of hardness indentations placed either side of the notch and pre-crack in the CT specimen as shown in Figure 2.24.
As reported by Holdsworth [132], deformation and crack development are strongly influenced by the creep ductility in the alloy. Crack initiation occurs in creep brittle materials on the attainment of a relatively low crack tip opening displacement, $\delta_{\text{cr}}$, Figure 2.25a, whereas in creep ductile materials relatively large $\delta_{\text{cr}}$ values, Figure 2.25b, are required to initiate cracking with the onset of cracking occurring later in life.
Based on the above knowledge and relationships developed for crack initiation and growth, defect assessment procedures have now been developed [10] [18] and are in use to monitor in service operation of power plant components.
Figure 2.23: Variation of crack opening displacement and crack length in a fracture mechanics specimen indicating the importance of specifying crack initiation criteria – after Holdsworth [11].
Figure 2.24: Derivation of $\delta_{ix}$ using hardness indentations – after Holdsworth $^{[132]}$
Figure 2.25 a & b: Influence of creep ductility on crack opening displacement and growth behaviour in a fracture mechanics test piece subject to steady load at high temperature, a) brittle 0.5%Cr-0.5%Mo-0.25%V HAZ, b) ductile 2.25%Cr-1%Mo weld metal – after Holdsworth $^{[132]}$.
2.5.3 Microstructural Investigation Techniques For Creep Life Prediction Methods
To ensure accuracy of information from quantitative microstructural analysis for remanent life assessment it is essential that the techniques used are standardised and reproducible. In particular this relates to methods based on damage parameters such as quantification of creep cavitation and microstructural maturity.
Cavitation damage may be assessed from laboratory and in service components by three main techniques:
i) Direct replication \textsuperscript{[133]}
ii) Extraction replication \textsuperscript{[134]}
iii) Bulk sample examination
For each of the techniques the quality of preparation of the material surface is of paramount importance to ensure faithful results. In a study by Silveira and May \textsuperscript{[135]}, based on the Neubauer and Wedel \textsuperscript{[145]} cavitation classification scheme, the sensitivity of the final classification to sample surface preparation was analysed. Prolonged polishing or repeated polish – etch cycles were shown to exaggerate the degree of creep cavitation by the pull out of matrix or precipitates at damage centres along grain boundaries. It was concluded that the extent of creep damage and hence the prediction of remaining life should not be based purely upon the quantitative measurements of cavities and microcracks observed but also on an appreciation of the distribution of the damage centres disclosed. A surface preparation procedure using a minimum of three final stage polish – etch cycles was proposed to define, unequivocally, the presence or absence of creep damage.
In a related study by Samuels et al \textsuperscript{[136]} extended etching was found to widen cavities that had opened up at grain boundaries perpendicular to the applied stress in uniaxial creep specimens. It was noted, however, that etching did not in itself generate any new cavities.
As discussed in section 2.5.1 the evaluation of carbide morphology may be used to assess the fraction of lifetime remaining in a component operating in a creep environment. To avoid removal of bulk samples from components, necessitating costly weld repair, a technique for generating extraction replicas has been devised by Corti et al [134]. In a study by the same authors on extraction replicas taken from steam headers in 2.25%Cr-1%Mo steel, EDX analysis was used, in the mode of Titchmarsh [137] [138] and Li [139], to identify the morphological carbide types and relative Cr/Fe ratios. A linear correlation between the Cr/Fe ratio in M$_{23}$C$_6$ carbide and the cube root of exposure time was obtained for steam pipes service exposed at 540°C for up to 160,000 hours, that is:
$$\text{Cr/Fe} = 0.33362 + 0.01516t^{1/3} \quad \text{-Equation 2.25}$$
In the original work by Titchmarsh [137] it was emphasised that to uniquely identify carbide on the basis of EDX analysis, the carbides types must first be established, for the specific composition and heat treatment, by electron diffraction.
Chapter 3.0
Experimental Procedures
2.25%Cr-1%Mo alloy steel is a microstructurally complex alloy, that is generally put into service in a meta-stable condition. At elevated temperatures, the carbides present evolve with time, and in so doing change the response of the alloy to the processes of creep. The inter-relationship between microstructure and creep behaviour is complex. In the presence of a pre-existing defect, the carbide ripening process and hence the creep response may, for example, be affected by the stress intensity developed at the defect tip. To improve the lifetime of a component by controlling its initial microstructure, or to predict the remanent life, it is therefore essential to fully understand the role of the carbides in the process of creep and the effect that carbide ripening has on this process during service.
The microstructure of 2.25%Cr-1%Mo alloy steel may vary according to the prior heat treatment to which it has been subjected. In large heat-treated components, the microstructure may also vary through the cross-section, due to the differential response to heating and cooling. In general, commercial alloys tend to exhibit three different types of microstructure, namely:
i) mixed pro-eutectoid ferrite + pearlite
ii) mixed pro-eutectoid ferrite + bainite and
iii) a tempered, fully bainite structure.
The purpose of this investigation has been to obtain a detailed understanding of the effect of each of these microstructural profiles, and their subsequent evolution at elevated temperature, on the creep initiation process ahead of a defect, in samples of 2.25%Cr-1%Mo alloy steel. The experimental work progressed in three stages, as explained in outline below:
(a) Characterisation of the starting material:
The first requirement of the investigation was to obtain a full understanding of the microstructural profile of the as received starting material, prior to creep deformation.
Two commercial sources of 2.25%Cr-1%Mo steel alloy, with different thermo-mechanical histories were obtained for this work. These are described in detail in section 3.2. The microstructure of one consisted of a mixture of pro-eutectoid ferrite and pearlite, while that of the other contained pro-eutectoid ferrite and bainite, thus providing representative examples of microstructure (i) and (ii) above. Material with a 100% bainite structure was obtained by heat treatment of the as-received material and subsequently tempered to produce microstructural type (iii) - see section 3.3.
Samples of all three microstructural variables were subjected to microstructural analyses and investigation of their basic mechanical properties. These materials eventually provided the samples on which detailed studies of creep behaviour were carried out.
(b) The tempering characteristics of fully bainitic 2.25%Cr-1%Mo steel:
Exposure of the bainite phase to elevated temperatures results in a dispersion of carbides, which progressively coarsen and evolve with time. To facilitate a comparison with microstructural changes during subsequent creep tests, when there is the added complication of both external and localised internal stresses, a tempering trial on samples initially with the fully bainitic microstructure, was carried out.
The tempered samples were investigated using scanning electron and scanning transmission electron microscopy techniques - see section 3.6. A major part of this work involved the development of an image analysis procedure to aid the interpretation of the carbide evolution process. A thorough understanding of the complex processes involved in carbide precipitate development in this alloy, was thus obtained.
(c) Creep testing and investigation:
A mechanical testing rig, described in section 3.4, was designed and built to accommodate suitable test specimens, in order to generate samples of the steel exhibiting creep damage initiation features, ahead of a pre-existing
defect. The test rig design allowed a number of compact tension (CT) type specimens, each containing a fine spark machined notch, to be creep tested at the same time, under controlled test conditions. After pre-designated time intervals, a specimen was removed from test and the microstructure developing ahead of the notch, assessed for evidence of creep damage.
Scanning electron microscopy was used to assess the extent of creep damage in terms of void formation and flaw/crack extension. Crack extension was measured using a linear measuring device in the microscope and the area percentage of voids and void orientation, was determined with the aid of an image analysis routine, specially devised for the purpose of this project. These techniques are described in section 3.6.
To assess the affect of the pre-existing defect and subsequent crack propagation, on the carbide ripening and creep damage initiation mechanisms, an in depth analysis of the carbide morphology was necessary. A study of the carbide precipitates in the defect tip region was made possible by devising a technique for selectively extracting the carbides from the area of interest and analysing them in a transmission electron microscope using energy dispersive X-ray analysis as detailed in section 3.6.
Samples of 2.25%Cr-1%Mo steel, exhibiting the three different microstructural profiles identified below, were required for the investigation:
i) mixed pro-eutectoid ferrite + pearlite,
ii) tempered mixed pro-eutectoid ferrite + bainite and,
iii) tempered 100% bainite.
The raw material for this work was supplied by courtesy of the collaborating establishment Alstom Energy, UK. Ltd. The material came from two sources, namely:
a) cast and heat treated round bar, and
b) wrought and then heat treated steel pipe.
The composition of each is presented in Table 3.1.
The bulk of the material supplied came from source (a). This consisted of 87mm diameter x 465mm long round bars, extracted from trepanned out bolt holes in the flange of a high quality, cast and heat-treated, steam turbine generator casing. The heat treatment schedule to which it had been subjected is summarised in Table 3.2, producing a microstructure consisting of pro-eutectoid ferrite and tempered bainite. This, therefore, provided the source of all sample material with this microstructural profile. To avoid any end effects due to the casting / heat treatment processes, 50mm was discarded from the end of each of the trepanned bars supplied.
Source (b) provided a lesser amount of material, supplied in the form of microspecimens extracted from creep tested compact tension, (CT), specimens. These had previously been machined from 74mm wall thickness x 419mm outside diameter, wrought and then heat-treated steam pipe. This material, during manufacture, had finally been subjected to a full annealing treatment at 960°C, followed by furnace cooling to room temperature. This produced a microstructure consisting of pro-eutectoid ferrite and pearlite and thus provided the source of all sample material with this microstructural profile.
| Material Source | Element (mass %) |
|-----------------|------------------|
| | C | Si | Mn | P | S | Cr | Mo |
| (a) Casting | 0.11 | 0.35 | 0.59 | 0.006 | 0.005 | 2.34 | 1.00 |
| (b) Steam Pipe | 0.13 | 0.29 | 0.54 | 0.026 | 0.028 | 2.31 | 0.96 |
Table 3.1: Composition of as-received material.
| Material Source | Heat-treatment |
|-----------------|----------------|
| (a) Casting | Normalised at $960^\circ$C and then air cooled + Temper for 12 hours at $705^\circ$C and then furnace cooled + Stress relieve at $700^\circ$C for 12 hours and furnace cooled. |
| (b) Steam Pipe | Annealed at $960^\circ$C and furnace cooled. |
Table 3.2: Final commercial heat-treatment of as-received material.
3.3 Heat Treatment
Heat treatment procedures were necessary for two areas of investigation in the research programme:
a) To produce material with a microstructure consisting of a 100% bainite from which creep test specimens could be machined and,
b) To produce samples with a 100% bainite structure and subsequently study the affect of tempering temperature and time on the carbide evolution process.
In all cases, the heat-treatment procedures were carried out in a Carbolite muffle furnace, controlled by an in-situ chromel-alumel thermocouple and Eurotherm 808 temperature controller to a nominal accuracy of 0.25% x temperature of reading. The temperature distribution profile of the furnace chamber was established using a chromel-alumel type thermocouple, in conjunction with a cold junction corrected Fluke digital temperature recorder.
3.3.1 Production of 100% Bainite CT Creep Test Specimens
The cast and trepanned bar, from source (a) identified in section 3.2, was used as the starting material for manufacturing CT specimen blanks with dimensions of 48mm x 45mm x 19mm. In order to protect them against oxidation during heat treatment, all specimens were coated with Birkatec paint.
Consideration of the relevant continuous cooling transformation diagram, presented in Figure 3.1, for the CT blank dimensions, indicated the necessary cooling conditions, following austenitising, to achieve a 100% bainitic structure. Austenitisation was achieved by soaking each blank at $960^\circ\text{C}$ for one hour, followed by air cooling to room temperature to produce a fully bainite structure. The blanks were subsequently tempered at $700^\circ\text{C}$ for 24 hours, followed by air cooling to room temperature.
Figure 3.1: The continuous cooling transformation diagram for a 2.25%Cr-1.0%Mo steel – after M. Atkins [140].
3.3.2 Bainite Tempering Investigation
Specimens for the tempering studies were obtained from CT test piece blanks, following austenitising at $960^\circ\text{C}$ for one hour and air cooling to room temperature. The fully bainite blanks were then cut into $29\text{mm} \times 16\text{mm} \times 19\text{mm}$ samples, illustrated in Figure 3.2, for the tempering investigation.
All samples were again coated in protective Birkatec paint. Tempering was carried out at temperatures of $650^\circ\text{C}$, $700^\circ\text{C}$ and $750^\circ\text{C}$ for periods of 0.5, 1.0, 5.0, 10, 25, 50 and 100 hours, followed by air-cooling to room temperature. In addition, two specimens were heat treated at $700^\circ\text{C}$ for 1000 and 5000 hours.
Figure 3.2: Plan for sectioning of bainite tempering samples from CT blank (48 x 45 x 20mm).
3.4 Mechanical Testing
3.4.1 Hardness Tests
Hardness measurements were made using a Vickers hardness testing machine. A pyramidal diamond indenter of angularity $136^\circ$, was employed with a 20kg applied load, on specimens previously prepared to a 600 SiC grit surface finish.
The hardness value was determined by measuring the diagonals of the indentation using a calibrated ocular micrometer scale and then converting the average of the two orthogonally opposite diagonals, using a conversion chart, to obtain a Vickers hardness number.
To ensure accuracy, a Vickers certified hardness machine was used and a calibration test block was checked immediately prior to testing, in accordance with BS427 (1990).
3.4.2 Tensile Tests
To gain an appreciation of the general properties of the materials under consideration, tensile specimens, 10mm$^2$ in cross-sectional area, were machined and submitted to a NAMAS approved test house for the determination of their tensile properties at both room temperature and at 550°C. The properties determined were the Young's modulus, 0.2% proof stress, ultimate tensile strength and percentage elongation.
3.4.3 Creep Deformation Tests
The main purpose of this programme of work was to study the mechanism of creep initiation in the presence of a pre-existing defect, in the nominated microstructures of interest, and to correlate this information with critical crack tip opening displacement (CTOD) data.
To facilitate these aims, it was decided to subject a series of $\frac{3}{4}$ size CT specimens, each with a spark eroded notch <0.2mm in width, to a predetermined creep load and test temperature of 550°C, and to interrupt the tests to microstructurally examine the crack tip region after various time intervals.
3.4.3.(a) Creep Test Rig Design
In order to reduce the experimental error, due to temperature fluctuations, and maximise the number of specimens available for examination, a test rig and
furnace were designed to accommodate four CT specimens in series to fit an existing ESH 50kN capacity creep testing machine.
A schematic diagram of the test rig designed for this project is shown in Figure 3.3.
The test rig incorporated a three zone split furnace of sufficient size to accommodate four \( \frac{3}{4} \) size CT specimens. The furnace was supported on a wrought aluminium alloy frame. The key dimensions of the furnace were as follows:
- Internal bore diameter = 90mm
- Centre hot zone length = 350mm
- Top and bottom heating zones = 100mm
The furnace was controlled by three Eurotherm 808 temperature controllers to a claimed accuracy of 0.25% x temperature of reading, i.e. \( \pm 1.37^\circ C \) for a furnace setting of 550°C. In addition to the furnace control thermocouples, three external chromel-alumel, type k, thermocouples were also positioned inside the furnace tube to monitor the actual temperature of the specimens during the tests.
The specimens were linked together and to the loading bars of the creep machine by shackles, pins and bars heat treated and machined, specifically for this project, from the following nickel based Superalloys:
**Loading Bars:**
- Nimonic 80A - heat treated at 1080°C for 8 hours, air cooled, machined and then heat treated at 705°C for 16 hours and finally air cooled.
**Loading Pins:**
- Nimonic 115 - heat treated at 1190°C for 1.5 hours, air cooled, machined and then heat treated at 1100°C for 6 hours and finally air cooled.
**Loading Shackles:**
- PK 33 - heat treated at 1100°C for 2 hours, air cooled, machined and then heat treated at 850°C for 4 hours and finally air cooled.
Figure 3.3: Creep test rig design.
3.4.3(b) Creep Test Specimens and Loading Regime.
Three quarter scale compact tension specimens were selected for the CTOD/creep testing programme. The specimens were machined to the dimensions given in BS5477:1977 and are depicted schematically in Figure 3.4.
Width, $W = 38\text{mm}$
Total thickness, $B = 19\text{mm}$
Depth of side grooves = 1.9mm on each side
Nominal thickness, $B_n = 15.2\text{mm}$
Effective crack length, $a = 11.4\text{mm}$
Figure 3.4: Schematic diagram of CT specimen and dimensions.
To simulate the presence of a defect in the specimen, the final 1.9mm of the effective crack length was machined by spark erosion, using a copper wire electrode 0.2mm in diameter.
The applied stress for the tests was based upon the plane strain reference stress criteria, $\sigma_{\text{ref}}$, as described in section 2.4.3 of the literature review, and applied to the CT specimens by the use of equation 3.1.
\[
\sigma_{ref} = \frac{P}{m_{ple} B_n W}
\]
- Equation 3.1
Where
\( P \) = applied load
\( m_{ple} \) = yield ratio in plane strain
\( B_n \) = net specimen thickness after side grooving
\( W \) = specimen width
and,
\( m_{ple} = - (1 + 1.702 \frac{a}{W}) + (2.702 + 4.599 (\frac{a}{W})^2)^{1/2} \)
In the initial absence of any unidirectional creep testing data specific to the three microstructural profiles identified for 2.25%Cr-1%Mo steel in this project, the loading conditions for the test specimens were initially based upon \( \sigma_{ple} \) values reported by The Creep of Steels Working Party Of Institution Of Mechanical Engineers [141] for this alloy.
The ferrite / pearlite specimens, tested at Alstom Energy, UK. Ltd., were tested at a stress level of 85MPa, predicted to give failure in ~10,000 hours, whereas the stress values for the two bainite structures, tested in house, were selected to give failure in ~2,500 – 5,000 hours and give creep crack initiation, i.e. crack growth up to 1.0mm, in approximately 1,000 hours. After an initial testing programme based on the above, and information gained from newly acquired uniaxial creep testing data obtained from the collaborating establishment, it soon became apparent that the loading regimes did not give an accurate assessment of time to rupture and, hence, time to initiate a creep crack. Subsequently a reference value based on newly acquired uniaxial creep data for the heat-treated as cast material, detailed in Appendix 1, was subsequently used to determine the test stress.
The uniaxial creep test specimens in the as cast and heat treated ferrite / bainite structure, tested at a stress of 110MPa, were found to fail in a rupture.
time of 1589 hours. From literature on similar structures it had been shown by Holdsworth and Cunnane\textsuperscript{[19]} and Ainsworth\textsuperscript{[9]} that the time to initiate a crack in a notched specimen was approximately 0.3-0.4 of the uniaxial rupture time and, therefore, the CT test specimens in this study were subsequently tested at 110 MPa to give crack initiation in a predicted 500-700 hours. Although this prediction proved to be reasonably accurate for the ferrite / bainite structures it was not satisfactory for the bainite structure. The fully bainitic structure when tested at this stress was found to initiate a crack 1mm long after only 300 hours and consequently the stress was further reduced to 100MPa. The final test regime utilised is detailed in Table 3.3 below.
| Microstructure | Estimated time to initiate a crack at 550°C (h) | Test stress / reference stress, $\sigma_{plc}$ (MPa) | Test load – based on equation 3.1 (KN) | Load on creep machine (Lever ratio 25:1) (N) |
|--------------------|-----------------------------------------------|-----------------------------------------------------|--------------------------------------|---------------------------------------------|
| Ferrite / Pearlite | ~8000h | 85 | Tested at Alstom Energy | Tested at Alstom Energy |
| Ferrite / Bainite | ~600 | 110 | 16.125 | 645 |
| Bainite | ~600 | 100 | 14.706 | 588 |
Table 3.3: Creep test loading conditions.
3.4.3(c) Crack Opening Displacement Measurement
Crack opening displacement, (COD), measurement by conventional electronic methods is difficult at elevated temperatures due to specimen oxidation effects, strain gauge degradation and electronic instability. The method utilised in this project was based upon that described in section 2.5.2 after Holdsworth and Cunnane\textsuperscript{[19]} and involved monitoring of the crack opening displacement, $dy$, of Vickers hardness indentations made parallel to the specimen notch at a known distance, $x$, from the front face of the specimen, as illustrated in Figure 3.5a.
Prior to the commencement of a test, diamond indentations were made at intervals of 2.54mm (0.1"), to a distance of about 20.32mm (0.8") from the front face of the specimen, using a Vickers hardness testing machine, with the specimens clamped in a sliding vice arrangement with micrometer stage movement. The displacement values, $dy$, were accurately measured before and after the creep test using an optical microscope and calibrated ocular graticule. A graph of $dy$ against $x$ was subsequently plotted and the relative positions of the load point and notch tip points indicated on the charts, as illustrated in Figure 3.5(b). Values of crack tip opening displacement, (CTOD), could then be read directly from the graph. Extrapolating the displaced indentation lines to $dy = 0$ gives the point of rotation for the crack opening, or the "hinge point", which varies with test duration.
Figure 3.5: Crack opening displacement measurement.
a) Schematic diagram of CT specimen notch and hardness indentations.
b) Schematic diagram of crack opening displacement against indentation position.
3.5 Optical Microscopy Techniques
3.5.1 Specimen Preparation and Microstructural Examination
Microstructural examination of the material under investigation was carried out using both Zeiss and Vannox optical microscopes with 35mm camera attachments.
Examination was carried out on micro-specimens mounted in conducting bakelite, ground and polished to a $1\mu m$ diamond finish and subsequently etched in 2% nitric acid in methanol solution, (2 % Nital). Uniformly etched and stain free surfaces were achieved by using freshly polished specimens and by applying a layer of industrial methanol to the surface of the specimens immediately prior to immersion.
3.5.2 Grain Size Determination
The grain size of an alloy may have an effect on its creep resistance since it is known that:
- grain boundaries can act as easy pathways for the diffusion of elements at high temperatures, i.e. Coble creep
- grain boundaries may act as barriers to dislocation movement, and
- precipitates can pin grain boundaries and thus prevent grain deformation.
A knowledge of the grain-size of the starting material was, therefore, considered important to this study.
The average grain-size of the microstructures of interest was determined by the mean linear intercept (MLI) method, using a Vickers 55 projection microscope. This involved counting the number of grain boundaries, $N$, which crossed a line of known length, $L$, and thus:
\[ \text{"MLI Grain Size"} = \frac{L}{N} \quad - \text{Equation 3.2} \]
The intercept line was provided by superimposing the microstructural image on a calibrated 1mm graticule, at the magnification chosen, prior to the counting procedure.
It is accepted that the true mean diameter of the grains in a sample is greater than the measured MLI, due to grain shape and improbability of measurement at the true grain diameter, \textsuperscript{[142]}. The following generally accepted relationships have been established:
i) \( D \approx 1.75 \text{ MLI} \), - Equation 3.3
where,
\( D = \) mean grain diameter taking into consideration improbability of measuring true grain diameter.
ii) \( D_m \approx 1.86 \text{ MLI} \), - Equation 3.4
where,
\( D_m = \) mean grain diameter taking into consideration true grain shape.
In this study the mean grain diameter quoted was that in accordance with equation 3.3. For each specimen, ~300 intercepts were counted.
\subsection*{3.5.3 Determination of the Volume Fraction of Transformation Products}
The volume fraction of micro-structural phases present in an alloy has a significant affect on its mechanical properties. The starting materials for this project were, therefore, assessed in terms of the volume fraction of their transformation products by 2-D manual point counting, using the technique as described by Pickering \textsuperscript{[142]}.
The procedure was carried out on a Vickers 55 projection microscope using a suitable magnification and point counting grid to ensure that no one phase region intercepted two consecutive grid points. For each sample determination ~3000 points were counted, i.e. ~20 fields per specimen.
3.6 Electron Microscopy Techniques
Electron microscopes allow small objects or features present in a material's microstructure to be magnified with a level of image resolution and depth of field which is far superior to that of the optical microscope.
The scanning electron microscope (SEM) is used for imaging surface morphological features, whereas, the transmission electron microscope (TEM) probes the internal structure of solids and gives access to both microstructural and ultra-structural details. In both techniques, high velocity electrons impinging on the material in a vacuum, produce X-ray spectra characteristic of the elements present. Energy dispersive X-ray (EDX) and wavelength dispersive X-ray (WDX) analysis of the spectra produced, thus provide a means of assessing the chemical composition of micro-structural features observed in electron microscopes.
In this project, both SEM and TEM techniques were employed, in conjunction with energy dispersive X-ray (EDX) analysis systems.
3.6.1 Scanning Electron Microscopy
Examination by SEM was primarily employed in this investigation to aid the identification of the microstructural phases present in the alloy, to study the evolution of carbide morphology with tempering time and temperature and to examine and quantify the creep damage in the tested compact tension specimens.
Scanning electron microscopy was carried out using both a JEOL 840A Scanning Electron Microprobe and a Philips XL 40 Scanning Electron Microscope. Both instruments were equipped with Oxford Instruments EDX analysis systems. The Philips XL 40 microscope was also equipped with
Oxford Instruments analysis software, Feature Scan, which was usefully employed in the quantification of creep damage.
3.6.1(a) SEM Specimen Preparation
Specimens of the as-received and heat-treated steel samples prepared for optical examination, were also suitable for examination by SEM. Apart from ensuring cleanness, no additional preparation was necessary.
To examine the microstructure and creep damage around the notch tip in tested CT specimens, a section as shown in Figure 3.6, was extracted using a Discotom cut-off machine and then mounted in conducting bakelite.
**Figure 3.6:** Micro-specimen extraction from notch tip region.
The mounted sections were ground and subsequently polished to a $1\mu m$ diamond paste finish and then etched for $\sim 20$ seconds by immersion in a solution of 2% nitric acid in methanol. To ensure that the surface was free from preparation damage, the latter two stages, i.e. the $1\mu m$ polish and etch, were repeated three times. Samples were stored in an air tight desiccator to prevent deterioration of the polished and etched surfaces.
To prevent charging of the oxide layer around the notch sample in the SEM, the prepared micro-specimens were coated with a thin layer of carbon using a carbon evaporation process. The coating was achieved by placing the specimens in an Edwards vacuum coating unit at a pressure $\sim 1 \times 10^{-5}$ bar,
beneath a pair of carbon electrodes through which a current passed, thus evaporating carbon onto the specimen surface.
To improve the electrical conductivity of the specimens in the SEM, and hence limit image noise, a thin trail of silver dag conducting paint was used to link the specimen surface to the conducting, aluminium, support stub.
3.6.1(b) General SEM Examination
General micro-structural examination of the starting materials, tempered bainite specimens and creep tested samples was carried out on an SEM using an accelerating voltage of 25keV.
Secondary electron imaging was used to resolve the fine microstructural details in samples of interest whereas back-scattered electron imaging was used to locate creep cavitation and measure crack extension in the tested specimens.
Micro-structural evidence was recorded using either a 35mm camera attachment or digital image capturing device.
3.6.1(c) Creep Damage Quantification using SEM Image Analysis
Microstructural examination and damage accumulation of the creep tested CT specimens was performed utilising a Philips XL 40 SEM and Oxford Instruments image analysis software. The micro-specimens prepared were examined using both back-scattered electron (BSE) and secondary electron (SE) imaging modes. BSE imaging allowed the creep voids and micro-cracks to be clearly delineated from the general micro-structural features for quantification purposes whilst SE imaging enabled the operator to determine whether the damage was present at inter or intra-granular sites.
The accuracy of the analytical process relied on imaging conditions remaining consistent between consecutive fields of view and between samples and, therefore, a systematic routine was devised to ensure reproducible image contrast and brightness and feature detection.
The following microscope conditions were maintained during the analytical procedure:
- Working Distance: 15mm
- Accelerating Voltage: 20Kev
- Beam Current: ~85μA
- Probe Size: Spot size 6 (large)
- Image Type: Back scattered electron
- Magnification: x1000
- Scan Rate: 200ms/line
As the stability of the beam current cannot be guaranteed over long periods of time, or from sample to sample, it was necessary to establish a datum for the contrast and brightness levels to ensure consistency. This was achieved by using the wave-form meter on the SEM control panel to ensure that the average contrast and brightness signal for the general microstructure was at a known position with respect to the “white” level and that the signal from the voids and micro-cracks fell into the “black” level, Figure 3.7.
Figure 3.7: Use of the SEM wave-form meter to ensure image consistency.
The operational procedure for the SEM and Feature Scan image analysis software was as follows:
i) Allow SEM chamber to de-evacuate
ii) Home stage
iii) Place specimen on the microscope stage with the notch facing west.
iv) Evacuate chamber
v) Optimise image
a) check filament saturation
b) Set gun tilt
v) Image specimen at x1000 (screen mag.), focus (slow scan) and adjust stigmators.
vi) Place notch tip in screen centre using cross hairs.
vii) Use “measurement” function to determine distance from notch to top of field of view e.g. ~87µm (Feature Scan software gives image dimensions)
viii) Using “position” move image so that notch tip is just out of view, e.g. ~87µm.
ix) Note the stage x,y co-ordinates.
x) Set up the BSE image as detailed in microscope conditions above.
xi) Go to external x,y, signal controlled by image analysis software.
xii) Select Feature Scan programme in digital imaging programme.
xiii) Set up detection conditions or call up conditions file, e.g. TB samples.
a) features smaller than 10 pixels excluded
b) noise reduction = 20 samples
c) thresholding - collect digital image in slow Kalman (5 frames); levels set to distinguish between the grey levels of features of interest and background.
E.g. Typical threshold band for detection of voids = 0-140 grey levels.
xiv) Set up measurement conditions
a) no. of feret projections for measurements = 36
b) measurement derived - feature area as % of field, centre of gravity positions (x,y), feature length and breadth, orientation (radians with respect to horizontal axis\(^1\)), perimeter and aspect ratio.
\(^1\) This value was subsequently recalculated to give orientation with respect to the notch tip.
xv) Set up feature filter conditions - used to differentiate between low atomic number inclusions and voids (chemical classification bins set up for elements of interest e.g. sulphides in ferrite / pearlite samples).
xvi) Set up image calibration.
E.g. Typical calibration
Field width = 187.778μm, field height = 185.082μm,
Field area = 34754.2μm².
Image resolution = 1024 x 1024 (i.e. 1 pixel = 0.18338μm x 0.18074μm)
xvii) Analyse live image.
3.6.2 Transmission and Scanning Transmission Electron Microscopy
Transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) were performed using a Jeol 100CX microscope with a tungsten filament electron gun and a Philips CM 20 microscope with an LaB₆ electron gun. Both microscopes were fitted with energy dispersive X-ray (EDX), analysis facilities.
TEM was generally employed to examine and identify the micro-structural features of interest in the starting materials, temper trial samples and creep tested specimens. The Jeol 100CX in STEM mode and associated EDX feature detection software, was specifically employed to carry out an assessment of the evolution of carbide morphology in the bainite tempering investigation.
Image contrast in the TEM is due to density and thickness variations in the specimen or diffraction effects due to the interaction of features in the microstructure with the primary electron beam, i.e. kinematic and dynamic conditions.
Specimens for TEM examination generally take the form of either a 3mm diameter solid disc partially thinned to a few nm thick for electron penetration,
thin foils, or extraction replicas in which particles from the bulk material are extracted onto a carbon film and supported on 3mm diameter copper grids.
The majority of the work carried out in this study necessitated the preparation and examination of extraction replicas with only the occasional requirement for preparation and examination of thin foil type specimens.
3.6.2(a) Preparation of Carbon Extraction Replicas
Extraction replicas consist of particles extracted from the specimen of interest, supported on 3mm squares of carbon film. TEM examination of the extracted particles can provide information about their type, morphology, crystallography and chemical analysis.
Specimens for examination were prepared in the conventional metallurgical manner, by grinding and polishing to a $1\mu m$ diamond finish. After polishing the specimens were immediately subjected to a light etch in 2% Nital. The area of interest was then isolated using masking tape, as illustrated in Figure 3.8 for selective extraction of carbides in the notch tip and reference regions. The sample was then placed in an Edwards carbon evaporation coating unit for at least 12 hours to achieve a high vacuum of $\sim 1 \times 10^{-8}$ bar and, on reaching this, carbon was evaporated over the specimen from a height of 100mm in 0.5 second blasts, allowing the vacuum to recover between each layer. Coating at high vacuum is essential to maintain a fine grained carbon structure which will remain intact during subsequent operations.
Figure 3.8: Selective preparation of notch tip and reference region extraction replicas.
After carbon coating, the masking tape was removed from the specimen and a sharp scalpel was used to score a 3mm x 3mm area on the coated surface of interest. The specimen was then immersed in 2% Nital reagent until the surface carbon layer began to blister. At this point, the specimen was transferred via a dish containing analar methanol, to a dish containing distilled water. Separation of the carbon from bulk specimen, was achieved by angling the specimen surface at $45^\circ$ to the water surface during immersion and allowing the surface tension of the water to provide the force for separation.
After removing the bulk specimen from the water 3mm diameter copper grids were used to "catch" the floating carbon squares which were then dried carefully and stored in a clean dry environment until required for TEM examination.
3.6.2(b) Preparation of Thin Foils
TEM examination was necessary to characterise the structure of the 100% bainite starting material produced by heat-treatment. The specimens for this work were produced by conventional jet electro-polishing, using chilled 2% perchloric acid in methanol solution.
3.6.2(c) Microstructural Examination in the TEM
General microstructural assessment of the as received material, tempered bainite material and CT tested specimens was performed on extraction replicas using a Philips CM20 TEM/STEM with EDAX analysis. An accelerating voltage of 200keV was used for the investigation, with the specimen positioned at eucentric height and tilted at an angle of $25^\circ$ towards the detector for chemical analysis.
The carbide classification scheme for this part of the investigation was based upon the ratios of $\text{Fe}_{\text{K} \alpha}/\text{Cr}_{\text{K} \alpha}$, $\text{Mo}/\text{Cr}_{\text{K} \alpha}$ and $\text{Fe}_{\text{K} \alpha}/\text{Mo}_{\text{L} \alpha}$ detected in each of the carbide types, that is $\text{M}_3\text{C}$, $\text{M}_2\text{C}$, $\text{M}_7\text{C}_3$, $\text{M}_{23}\text{C}_6$, $\text{M}_6\text{C}$. The ratios used, as detailed in Table 3.4, were determined by carrying out a series of EDX spot analysis trials on carbides extracted from both starting material and tempered bainite specimens. The procedure for the analysis involved focusing the electron beam, of spot size 6, onto the carbide of interest and collecting the emanating signal utilising the EDAX software. The result of the analysis was displayed in the form of an X-ray spectra that allowed the elements of interest to be identified and X-ray windows in the relevant KeV ranges to be created. The net number of X-ray counts contributing to each of the elemental windows specified in the spectra, as described in section 3.6.2(d), was then displayed and the key elemental ratios determined. The raw data for this determination is shown in Appendix 2.
| Carbide Type | Fe / Cr | Mo / Cr | Mo / Fe |
|--------------|---------|---------|---------|
| $\text{M}_3\text{C}$ | 2.00 – 6.00 | 0.20 – 0.60 | 0.05 – 0.25 |
| $\text{M}_2\text{C}$ | 0.02 – 0.60 | 1.50 – 4.50 | 7.50 – 30.00 |
| $\text{M}_7\text{C}_3$ | 0.50 – 0.90 | 0.05 – 0.50 | 0.05 – 0.35 |
| $\text{M}_{23}\text{C}_6$ | 1.10 – 1.50 | 0.15 – 0.60 | 0.10 – 0.35 |
| $\text{M}_6\text{C}$ | 5.00 – 15.00 | 5.00 – 15.00 | 0.75 – 1.50 |
Table 3.4: Elemental ratios determined by EDX spot analyses and used for carbide identification.
3.6.2(d) Carbide Characterisation by Image Analysis in the STEM
To study the carbide evolution process in the alloy, an image analysis procedure was developed for use with the Jeol 100CX STEM allowing the size, chemical composition and type of carbide particles to be assessed. For this part of the work the STEM unit was used with a LINK Instruments EDX software package called Digiscan, a feature detection and analysis programme that converts an electron microscope image into a digitised image displayed as a range of grey levels. The microscope conditions were set up so that the features of interest, i.e. the carbides, fell within an acceptable grey level band within the STEM image, as illustrated in Figure 3.9(a). The corresponding digital image, see Figure 3.9(b), was then thresholded, i.e. a range of grey levels was selected, to identify more precisely which features were to be considered in the analysis.
Once the thresholds had been set, the microscope beam was controlled by the Digiscan programme and made to scan a grid of points superimposed on the thresholded image. The X-Y co-ordinates of all the features detected were stored, subsequently allowing the beam to return and perform a sizing and analysis routine. All relevant size and compositional information was stored in a file to which data from subsequent image fields was added.
Sizing of the features was based on mean diameter measurements derived from 20 feret projections. Calibration for sizing was achieved by entering the screen width corresponding to the Digiscan rastered area and the working magnification. The analysed field width was equivalent to $5.6 \mu m$ and 1024 pixels each pixel measured approximately $0.005 \mu m \times 0.005 \mu m$. Only features having an area >10 grid points, i.e (pixels) were considered for the analysis.
The determination of the composition of microstructural features using Digiscan involved energy dispersive X-ray analysis in conjunction with user determined window files and chemical classification bins. EDX analysis data, displayed in
the form of a spectra, as illustrated in Figure 3.10 was used to define the X-ray energy limits of elements of interest and regions of background radiation. Areas defined by these limits are known as "windows".
Each element window contains X-ray counts for both the element of interest and background radiation and therefore, background windows are necessary to calculate the net contribution of specified element. For this purpose the background fraction of X-ray counts contributing to a specific element window was determined as follows:
\[
\text{Background fraction in element window} = \left( \frac{X-\text{ray counts in element window on carbon film}}{X-\text{ray counts in background window on carbon film}} \right)
\]
In the case where two elements in the alloy have X-ray energies that overlap, and therefore contribute to the same EDX window, a factor to calculate the contribution of each element has to be made. This was achieved by calculating the contribution of pure element standards to the specified window using bulk samples in the SEM. For example, in the case of 2.25%Cr-1%Mo steel, Figure 3.10, the $K_\alpha$ peak for chromium contributes to the window specified for manganese and the overlap fraction is calculated as follows:
\[
\text{Overlap frac" for Mn}_{K\alpha} = \frac{\text{Net } X-\text{ray counts from Mn std to } W_1}{(\text{Net } X-\text{ray counts from Mn std to } W_1) + (\text{Net } X-\text{ray counts from Cr std to } W_1)}
\]
Where:
$W1 =$ specified window for the element manganese
The elemental factors in each case are detailed in Appendix 3.
Chemical classification by Digiscan image analysis was achieved by setting up chemical classification bins, each of which defined the range of element mass
percentages for a particular class of feature; in this case the mass % of elements in each carbide type.
It has been reported that the types of carbides expected in this alloy are $M_3C$, $M_2C$, $M_7C_3$, $M_{23}C_6$ and $M_6C$ \cite{17} and that the composition of these carbides is known to vary \cite{28}. Based on this knowledge and the EDX spot analyses trials, as described in section 3.6.2c, the classification bins for the image analysis process were derived, as detailed in Table 3.5.
Figure 3.9: STEM images of a) carbide precipitates and b) the corresponding digitised image.
Figure 3.10: EDX spectra of 2.25%Cr-1%Mo steel showing identification of elements of interest by formation of “windows”.
| Carbide Type | Chemical Composition Classification Bins (element mass %) |
|--------------|----------------------------------------------------------|
| | Fe | Cr | Mo |
| M₅C | 56 - 85 | 10 - 20 | 0 - 10 |
| M₂C | 0 - 10 | 15 - 35 | 55 - 75 |
| M₇C₃ | 25 - 45 | 50 - 65 | 5 - 15 |
| M₂₃C₆ | 45 - 60 | 30 - 50 | 5 - 15 |
| M₆C | 35 - 50 | 0 - 15 | 40 - 55 |
Table 3.5: The compositional range of various metal carbides encountered in 2.25%Cr-1%Mo steel, as used in conjunction with EDX-Digiscan image analysis.
4.1 Introduction
The experimental work involved in this investigation progressed in three stages, as explained in detail in Section 3.1, namely:
a) the characterisation of the starting materials
b) a study of the tempering characteristics of the steel with a fully bainitic structure
c) creep testing and microstructural investigation
The results obtained from these three phases of the work are described in detail in the subsequent sections.
4.2 Characterisation of the Starting Materials
Samples of 2.25%Cr-1%Mo steel exhibiting the following microstructural profiles provided the raw material for this investigation:
i) Pro-eutectoid ferrite + pearlite
ii) Pro-eutectoid ferrite + bainite
iii) 100% bainite.
The source, detailed compositions and prior heat-treatment to obtain these structures have been described in detail in sections 3.2 and 3.3.
In order to determine the affect of such microstructural variations on the creep deformation behaviour of material ahead of a defect in 2.25%Cr-1%Mo steel it was necessary to first obtain an understanding of the initial microstructures, and their subsequent evolution at elevated temperatures, and establish the basic mechanical properties of the starting materials.
Hardness and tensile tests were carried out on the starting materials and a combination of optical and electron microscope techniques were used to examine the microstructures.
4.2.1 Mechanical Properties
The room temperature Vickers hardness and tensile test behaviour at room temperature and 550°C were determined as described in section 3.4.1 and 3.4.2 respectively. The results obtained are summarised in Table 4.1.
| Sample Identity | Average $H_{V_{20kg}}$ | 0.2% Proof Stress (MPa) | Ultimate Tensile Strength (MPa) | Elongation % | Young’s Modulus (GPa) |
|-----------------|------------------------|-------------------------|---------------------------------|--------------|-----------------------|
| | RT | RT | 550°C | RT | 550°C | RT | 550°C |
| Ferrite + Pearlite | 142 | 278 | 190 | 488 | 400 | 33 | 29 | 197 | 124 |
| Ferrite + Bainite | 136 | 256 | 176 | 492 | 296 | 30 | 28 | 208 | 131 |
| 100% Bainite | 170 | 413 | 298 | 551 | 380 | 30 | 24 | 208 | 112 |
Table 4.1: Hardness and tensile properties.
The sample exhibiting the fully bainitic structure was found to possess the highest hardness value followed by the sample consisting of the mixed ferrite / pearlite structure and finally the ferrite / bainite sample. The determination of these values was considered relevant to the project in terms of providing a possible link between room temperature hardness and creep brittle / ductile behaviour.
The room temperature tensile properties, for all three structures, were superior to those determined at 550°C. The bainitic structure resulted in the highest room temperature properties but exhibited the most significant reduction in properties on raising the test temperature to 550°C. The decrease in tensile strength and Young’s modulus at the elevated temperature was expected but it was notable that the high temperature ductility of all three structures was lower than that at room temperature. It was anticipated that the bainite constituent of
the mixed ferrite / bainite material would render this structure harder and stronger than that of the ferrite / pearlite structure. Unexpectedly, with the exception of the Young's modulus values, the hardness and tensile properties of the ferrite / pearlite structure were similar or better to those of the ferrite / bainite structure. The reason for this discrepancy may be due to the relative maturity of the carbides in the predominant ferrite phase of these structures. This will be discussed later in section 5.2 in conjunction with a description of the carbide morphology results.
4.2.2 Optical Microscopy
Optical microscopy was carried out on samples extracted and metallurgically prepared from the starting materials as detailed in section 3.5 to determine the microstructural phase constituents, grain-size and phase proportions in each of the materials.
Plates 4.1 - 4.3 are photomicrographs representative of the microstructures of each of the starting materials.
A qualitative metallurgical assessment of the materials, using magnifications up to x800, indicated that the microstructures required for this project had been achieved. With the resolution capability of the optical microscope it was not possible to determine the morphological type of bainite present in the latter sample and, consequently, this was assessed further in the scanning electron and transmission electron microscopes as reported in sections 4.2.3 and 4.2.4 respectively.
The grain size and phase proportions of the micro-structures observed in Plates 4.1 - 4.3 were assessed on a Vickers 55 optical microscope using mean linear intercept and 2D point counting methods as described in sections 3.5.2 and 3.5.3 respectively.
The grain-size and phase volume fraction results are summarised in Table 4.2.
| Micro-structure | Average Grain-Size (mm) | Average Phase Proportions |
|----------------------------------|-------------------------|------------------------------------|
| Pre-eutectoid ferrite / pearlite | 0.035 | 90% ferrite + 10% pearlite |
| Tempered, pro-eutectoid ferrite / bainite | 0.064 | 75% ferrite + 25% bainite |
| Tempered 100% bainite | 0.066 | 100% bainite |
**Table 4.2: Grain size and phase proportions of starting materials.**
The average grain size determined for the tempered bainite and mixed ferrite/bainite structures were found to be similar. The ferrite/pearlite microstructure, however, which was derived from a different source, was found to possess a significantly finer grain structure. This may be significant later in relation to the mechanisms of creep cavity formation and crack initiation.
The phase proportions present in each of the structures is indicative of the relative number of grain boundary types. In the mixed ferrite/pearlite structure the pearlite occurs as discrete islands in the ferrite matrix and the majority of grain boundaries are ferrite/ferrite in nature. In the ferrite/bainite structure the ferrite structure is broken by an almost continuous network of bainite grains and there is a mixture of ferrite/ferrite and ferrite/bainite boundaries with occasional boundaries of bainite/bainite. In the structure classified as fully bainitic no other major phases were observed.
These structures represent the range of microstructures that may occur in a large casting of quality heat-treated 2.25%Cr-1%Mo steel and were, therefore, considered satisfactory for the purpose of this study.
### 4.2.3 Scanning Electron Microscopy
A major part of the creep damage assessment procedure in this programme of work was to be carried out using scanning electron microscopy. It was
necessary, therefore, to establish the appearance of the starting material structures prior to the application of the creep stress and elevated temperature. SEM examination of the starting materials was carried out using a Philips XL40 microscope as described in section 3.6.1. Examples of the structures observed are shown in Plates 4.4 - 4.9.
In the ferrite / pearlite structure, as shown in Plates 4.4 and 4.5, a network of relatively coarse carbides can be seen clearly delineating the position of the prior austenite grain boundaries. Regions of pearlite were found to be homogeneously distributed throughout the structure and were identifiable by the presence of a dense mass of lamella carbide. Growth of the pearlitic lamellae could be seen to occur either into one or both of the adjacent ferrite grains. The ferritic regions could be identified by the presence of a fine, less dense, distribution of carbides which were more globular in nature than those observed in the pearlite.
Plates 4.6 and 4.7 show SEM images of the mixed pro-eutectoid ferrite / bainite microstructure. The regions of ferrite and bainite were, again, easily distinguished by the difference in their carbide size and population. The bainite regions possessed a structure of relatively large carbides which decorated the boundaries of the prior, upper bainite laths. The ferrite, however, exhibited a fine distribution of very small carbides that were uniformly dispersed. The boundaries between the two regions were outlined by a network of globular carbides.
The carbide morphology in the fully bainitic structure, as shown in Plates 4.8 and 4.9, was similar to that described above for the bainite in mixed ferrite / bainite structure. In the case of the fully bainitic structure, however, the carbides in the intra-granular regions appeared to be slightly larger in size and less densely populated.
4.2.4 Transmission Electron Microscopy
Transmission electron microscopy was carried out on all three starting materials using the techniques described in section 3.6.2.
Bainite can exist in two forms, upper bainite and lower bainite. It was necessary to establish the type of bainite formed by the heat treatment regimes employed in this work, as described in section 3.3.1, to produce the 100% bainite starting material. Examination of a thin foil specimen produced from the bainite structure in the normalised condition, Plate 4.10, showed that carbides existed at the boundaries of the bainite laths. This inter-lath precipitation is indicative of the structure being upper bainite.
One of the primary objectives of this investigation was to assess the role of carbides and their evolution process on the mechanism of creep crack initiation and growth in the presence of a pre-existing defect. It was, therefore, necessary to assess the carbide compositional morphology of the three starting micro-structures. This assessment was achieved by examining extraction replicas, taken from each of the three materials, in the TEM and analysing, where possible, the carbides in terms of their chemical composition and type. Chemical analysis of the carbides was by semi-quantitative EDX analysis based on elemental ratios as described in section 3.6.2c of the experimental procedures. Plates 4.11 - 4.14 illustrate the typical microstructures observed and Tables 4.3 - 4.5 detail the carbide compositions found in each case.
Plate 4.11 shows a typical ferrite / pearlite boundary in the mixed pro-eutectoid / ferrite microstructure. The lenticular shaped precipitates in the ferrite regions, designated 'A' in Plate 4.11, were analysed and found to be rich in chromium and molybdenum. Comparison of the elemental ratios for this carbide with those detailed in Table 3.4 of the experimental procedures suggested that their composition was similar to that of M$_2$C but with a greater chromium
concentration. The lamellar carbides observed in the pearlitic regions, designated 'B' in Plate 4.11, were found to have compositions typical of $M_{23}C_6$, being richer in iron and chromium than would be expected for $M_3C$. Carbides existing at the prior austenite grain boundaries were also found to be predominantly $M_{23}C_6$ type, for example carbide 'C', in Plate 4.11.
Plates 4.12 and 4.13 show typical images of the carbide morphology observed in the mixed ferrite / bainite structure. The precipitates in the ferritic regions of were predominantly of one morphology, that is, small, lenticular-shaped carbides, rich in molybdenum. Comparison of the elemental ratios determined by EDX analyses for these carbides with the data in Table 3.4 suggested that these carbides were $M_2C$. Occasional fringes of carbide of a similar composition, but larger in size, were also found adjacent to the ferrite / bainite grain boundaries.
The carbides present in the bainitic regions of the ferrite / bainite structures had a range of morphologies and chemistries including the lenticular-shaped, $M_2C$, carbides observed in the ferrite regions. In addition small and large globular carbides plus stick-like carbides were observed as indicated in Plate 4.13. The stick-like and globular precipitates exhibited a range of overlapping compositions based on iron, chromium and molybdenum which meant that it was impossible to identify the type of carbide on shape alone. Based on the EDX analyses ranges in Table 3.4 the small globular precipitates were found to be predominantly $M_7C_3$, the stick-like precipitates either $M_7C_3$ or $M_{23}C_6$ and the large grain boundary precipitates $M_{23}C_6$ or $M_6C$. The carbides at the ferrite / ferrite boundaries were found to be chromium rich and of the type $M_7C_3$. This was in contrast to the carbides at the ferrite / ferrite boundaries in the ferrite / pearlite structure which were iron rich and typical of $M_{23}C_6$.
Plate 4.14 shows the carbide morphology observed in the fully bainitic structure. As described previously for the SEM images in section 4.2.3, the fully bainitic structure was found to be less densely populated in terms of the carbide distribution than was that in the mixed ferrite / bainite structure. The
types of carbides present were consistent with those detected in the bainite phase region in the ferrite / bainite samples described above, however, it was noted that the relative proportions of $M_7C_3$ and $M_{23}C_6$ carbides in the intra-bainitic regions differed. The majority of carbides in the intra-bainitic regions of the fully bainitic structure were of the $M_{23}C_6$ type with only occasional $M_7C_3$ type carbides being identified, whereas, in the mixed structure there was almost an equal amount of the two carbides. Typical EDX analyses spectra for the range of carbides detected are shown in Figure 4.1.
Plate 4.1: Optical micrograph of the mixed pro-eutectoid ferrite / pearlite structure derived from pipe section fully annealed at 960°C.
Plate 4.2: Optical micrograph of the mixed pro-eutectoid ferrite / bainite structure derived from as-received bar normalised at 960°C, tempered at 705°C and stress relieved at 700°C.
Plate 4.3: Optical micrograph of the 100% bainite structure produced by normalising at 960°C and tempering at 700°C for 24h.
Plate 4.4: Low magnification SEM image of the ferrite / pearlite starting microstructure.
Plate 4.5: High magnification SEM image of the ferrite / pearlite starting microstructure.
Plate 4.6: Low magnification SEM image of the ferrite / bainite starting microstructure.
Plate 4.7: High magnification SEM image of the ferrite / bainite starting microstructure.
Plate 4.8: Low magnification SEM image of the fully bainitic starting microstructure.
Plate 4.9: High SEM magnification image of the fully bainitic starting microstructure.
Plate 4.10: TEM bright field Image of a thin foil specimen extracted from the as normalised material. Carbide precipitation observed between the ferrite laths identified the structure as upper bainite.
Plate 4.11: TEM micrograph of the carbide morphology in the ferrite / pearlite starting material.
| Carbide Designation | Typical Elemental Ratios | Carbide Type |
|---------------------|--------------------------|--------------|
| | Fe:Cr | Mo:Cr | Mo:Fe | |
| A | 0.11 | 0.68 | 6.07 | M$_2$C |
| B | 0.95 | 0.34 | 0.36 | M$_{23}$C$_6$|
| C | 1.41 | 0.32 | 0.22 | M$_{23}$C$_6$|
Table 4.3: Elemental ratios for the carbides observed in the ferrite / pearlite structure as indicated in plate 4.11.
Plate 4.12: TEM micrograph of the carbide morphology in the tempered mixed pro-eutectoid ferrite / bainite starting material.
Plate 4.13: TEM micrograph of the carbide morphology in the tempered, mixed pro-eutectoid ferrite / bainite starting material.
| Carbide Designation | Typical Elemental Ratios | Carbide Type |
|---------------------|--------------------------|--------------|
| | Fe:Cr | Mo:Cr | Mo:Fe | |
| A | 0.21 | 2.03 | 9.92 | $M_6C$ |
| B | 0.62 | 0.11 | 0.18 | $M_7C_3$ |
| C | 1.34 | 0.17 | 0.13 | $M_{23}C_6$ |
| D | 6.89 | 5.60 | 0.81 | $M_6C$ |
Table 4.4: Elemental ratios for the carbides observed in the ferrite / bainite structure as indicated in plate 4.13.
Plate 4.14: TEM micrograph of the carbide morphology in the tempered 100% bainite starting material.
| Carbide designation | Typical Elemental Ratios | Carbide Type |
|---------------------|--------------------------|--------------|
| | Fe:Cr | Mo:Cr | Mo:Fe | |
| A | 0.59 | 0.05 | 0.09 | M$_7$C$_3$ |
| B | 1.22 | 0.10 | 0.09 | M$_{23}$C$_6$|
| C | 1.13 | 0.12 | 0.10 | M$_{23}$C$_6$|
| D | 5.25 | 3.74 | 0.71 | M$_6$C |
Table 4.5: Elemental ratios for the carbides observed in the ferrite / bainite structure as indicated in plate 4.14.
Figure 4.1: Typical EDX spectra for the carbides in 2.25%Cr-1%Mo alloy.
Exposure of the bainite phase to elevated temperatures results in a dispersion of carbides, which progressively coarsen with time. To facilitate a comparison with microstructural changes during subsequent creep tests, when there is the added complication of both external and localised internal stresses, a tempering trial on samples initially with the fully bainitic microstructure, was carried out. Details of the experimental procedures used for this trial are given in section 3.3.2.
Samples of the 100% bainite starting material were tempered at temperatures of 650°C, 700°C and 750°C for times up to 5000 hours as shown below in Table 4.6.
| Tempering Duration (h) | Tempering Temperatures (°C) |
|------------------------|-----------------------------|
| | 650 | 700 | 750 |
| 0.5 | √ | √ | √ |
| 1.0 | √ | √ | √ |
| 5 | √ | √ | √ |
| 10 | √ | √ | √ |
| 25 | √ | √ | √ |
| 50 | √ | √ | √ |
| 100 | √ | √ | √ |
| 1000 | | √ | |
| 5000 | | √ | |
Table 4.6: Temper trial sample conditions.
After heat treatment the Vickers hardness of each sample was determined and specimens prepared for optical microscopy, SEM and TEM examination.
4.3.1 Hardness Test Results
Hardness tests were performed on each of the tempered, fully bainitic samples using a Vickers hardness testing machine and a load of 20Kg. The results are detailed below in Table 4.7 and presented graphically in Figure 4.2.
As can be seen from Figure 4.2 for each tempering temperature the hardness of the material was found to decrease with time at temperature. The rate of the reduction in hardness was highest in the initial period of leading to a period of almost constant hardness in the intermediate period and softening slightly at extended times. As might be expected, the degree of softening after any particular interval was increased with increasing tempering temperature.
No secondary hardening peaks due to the precipitation of M$_2$C, as described by Baker and Nutting [17], were observed in these trials. The hardness test data in Table 4.7 may be represented in the form of a Holloman – Jaffe plot, as described by Honeycombe [23], in order to assess the combined effect of time and temperature on the carbide evolution process as shown in Figure 4.3. Although the general trend observed, after the first 0.5 hour, is for a gradual reduction in hardness with increasing temperature the Holloman - Jaffe plot suggests that at extended times the samples tempered do not follow a linear relationship when a constant of 20 is used.
| Temp. °C | Tempering Time (Hours) / Vickers Hardness No. (HV$_{20kg}$) |
|----------|-------------------------------------------------------------|
| | 0 | 0.5 | 1.0 | 5.0 | 10 | 25 | 50 | 100 | 1000 | 5000 |
| 650 | 233 ±2.55 | 219 ±6.39 | 212 ±4.79 | 206 ±5.99 | 192 ±1.70 | 186 ±1.21 | 183 ±2.69 | 168 ±2.57 | n.a | n.a |
| 700 | 233 ±2.55 | 194 ±4.11 | 194 ±2.07 | 192 ±1.81 | 192 ±1.95 | 173 ±3.81 | 169 ±2.56 | 161 ±2.40 | 159 ±2.76 | 136 ±3.77 |
| 750 | 233 ±2.55 | 185 ±4.24 | 184 ±2.94 | 175 ±4.37 | 176 ±4.93 | 164 ±4.22 | 163 ±3.33 | 148 ±3.26 | n.a | n.a |
Table 4.7: Hardness results for the tempered fully bainitic samples (±2σ).
Note* - n.a = not applicable.
Figure 4.2: Graph showing Vickers hardness values against tempering temperature and time for the fully bainitic specimens.
Figure 4.3: Graph showing Vickers hardness values against the Holloman - Jaffe parameter, $P = T \times (k + \log t)$, for the fully bainitic specimens.
Where: $T =$ temp. (K), $k =$ a constant taken to be 20 and $t =$ time (secs).
4.3.2 Microstructural Changes During Tempering
Specimens were prepared from each of the heat treat samples for examination using both the scanning electron microscope and transmission / scanning transmission electron microscope.
4.3.2(a) Examination by Scanning Electron Microscopy
Scanning electron microscopy was utilised to study the general micro-structural changes taking place in the bainitic microstructure at all three tempering trial temperatures.
Plates 4.15 - 4.21 show micrographs of the samples tempered at 700°C for 0.5, 5, 50, 100 and 5000 hours. General microstructural assessment of the structures depicted in these micrographs indicated that for a constant tempering temperature the microstructure changes with time.
In the sample tempered for 0.5 hours, Plate 4.15, the intra-granular carbides were very fine and observed, generally, to decorate the original bainite lath boundaries. The carbides at the grain boundaries in the 0.5 hour were slightly larger and more globular than those at the intra-granular sites.
With increasing time from 0.5 hours to 5, 50 and 100 hours, Plates 4.15 - 4.18, the carbides at both intra and inter-granular sites were observed to spherodise, grow and reduce in number rendering the original bainite lath boundaries indistinguishable. In addition the samples tempered at 50 and 100 hours clearly showed evidence of sub-boundary formation and the onset of recrystallization.
After 1000 hours the large grain boundary carbides were observed to be growing in an irregular shaped manner and at the expense of carbides in the
neighbouring regions. This is evident from the images shown in Plates 4.19 and 4.20.
After 5000 hours at $700^\circ$C, Plate 4.21, the original bainite morphology was no longer visible and the structure consisted of recrystallized ferrite grains delineated by large spheroidal carbides.
To consider the effect of varying the tempering temperature for a constant tempering time Plates 4.22 and 4.23, depicting the micrographs of the samples tempered for 50 hours at $650^\circ$C and $750^\circ$C respectively, were included for comparison with Plate 4.17. For a constant duration with increasing temperature the microstructure was observed to change in the same manner as that described above for a constant temperature and increasing time. The micrograph of the sample tempered at $750^\circ$C for 50 hours, Plate 4.23, showed a well developed sub-structure similar in nature to that observed in Plate 4.18 for the sample tempered at $700^\circ$C for 100 hours.
A general observation was that a degree of re-crystallisation was found to be occurring in samples having a Holloman - Jaffe parameter value of greater than about 25.
### 4.3.2.b Examination by Scanning Transmission Electron Microscopy
Scanning transmission electron microscopy was used to assess the changes in carbide precipitate morphology, size, number and density in the samples tempered at $700^\circ$C utilising the image analysis routine specifically devised for this project as described in section 3.6.2(d).
The samples tempered at $700^\circ$C for 0.5, 5, 50 and 5000 hours were selected for the analysis routine to show the progression of the carbide evolution process, described qualitatively in section 4.3.2.a.
Tables 4.8 - 4.11 and Figures 4.4 - 4.7 show the size distribution data and charts for the carbides classified in the analysis, Table 4.12 and Figure 4.8 show the change in carbide type with time at 700°C and Figures 4.9 - 4.13 show the combined results of percent carbide type against size distribution.
Figure 4.4 shows that after 0.5 hours at a tempering temperature of 700°C the carbides classified as $M_7C_3$ are the most prevalent and largest of the carbides existing at this stage. $M_{23}C_6$ carbides existed in smaller size ranges and in lesser amounts than $M_7C_3$ in this sample as did $M_3C$ and $M_2C$. $M_6C$ type carbides were not detected at this early stage of tempering.
$M_3C$ carbides were absent in all other samples tempered for duration's longer than 0.5 hours.
With increasing time to 5 hours and subsequently 50 hours, Figures 4.5 and 4.6, the $M_{23}C_6$ carbides were observed to be dominant. The majority of carbides after tempering for 5 hours existed in the 0.1 - 0.3 micron size range with a lesser distribution in the 0.3 - 0.6 micron range. The occurrence of $M_7C_3$ and $M_{23}C_6$ carbides in this larger size range decreased with increasing time to 50 hours as the $M_6C$ carbides began to evolve. Fine $M_2C$ carbides were most abundant in the sample tempered for 5 hours and disappeared after 50 hours. After 5000 hours, as indicated in Figure 4.7, $M_6C$ carbides were the major carbides remaining in the material tempered at 700°C, only a few $M_{23}C_6$ carbides coexisted at this stage. The evolution of the $M_6C$ carbide at the expense of all other carbides is clearly represented in Figure 4.8 and appears to begin at a tempering duration of ~50 hours. The size distribution of each carbide type and their predominance at each stage of the tempering process up to 5000 hours is indicated in Figures 4.9 – 4.13.
Plate 4.15: SEM micrograph of the fully bainitic sample tempered at 700°C for 0.5h.
Plate 4.16: SEM micrograph of the fully bainitic sample tempered at 700°C for 5h.
Plate 4.17: SEM micrograph of the fully bainitic sample tempered at 700°C for 50h.
Plate 4.18: SEM micrograph of the fully bainitic sample tempered at 700°C for 100h.
Plate 4.19: SEM micrograph of the fully bainitic sample tempered at 700°C for 1000h.
Plate 4.20: High magnification SEM image of the area outlined in plate 4.19 showing irregular growth of grain boundary carbides and associated precipitate free zones.
Plate 4.21: SEM micrograph of the fully bainitic sample tempered at 700°C for 5000h.
Plate 4.22: SEM micrograph of the fully bainitic sample tempered at 650°C for 50h.
Plate 4.23: SEM micrograph of fully bainitic sample tempered at 750°C for 50h.
| Particle Mean Diameter (microns) | Percentage Of Total No. Of Carbides |
|---------------------------------|-----------------------------------|
| | M₃C | M₂C | M₇C₃ | M₂₃C₆ | M₆C |
| 0.0-0.1 | 1.8 | 2.1 | 4.8 | 7.4 | 0.3 |
| 0.1-0.2 | 2.7 | 0.0 | 17.2 | 11.6 | 0.0 |
| 0.2-0.3 | 1.8 | 0.0 | 18.2 | 7.4 | 0.0 |
| 0.3-0.4 | 0.0 | 0.0 | 12.8 | 1.8 | 0.0 |
| 0.4-0.5 | 0.0 | 0.0 | 6.0 | 0.0 | 0.0 |
| 0.5-0.6 | 0.3 | 0.0 | 2.1 | 0.0 | 0.0 |
| 0.6-0.7 | 0.0 | 0.0 | 1.2 | 0.0 | 0.0 |
| 0.7-0.8 | 0.0 | 0.0 | 0.6 | 0.0 | 0.0 |
| 0.8-0.9 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
| 0.9-1.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
| Total % | 6.6 | 2.1 | 62.9 | 28.2 | 0.3 |
Table 4.8: Carbide size distribution data for the fully bainitic sample tempered at 700°C for 0.5 hours.
Figure 4.4: Size distribution curves for the various carbides identified in the fully bainitic sample tempered at 700°C for 0.5 hours.
| Particle Mean Diameter (microns) | Percentage Of Total No. Of Carbides |
|---------------------------------|-----------------------------------|
| | M₃C | M₂C | M₇C₃ | M₂₃C₆ | M₆C |
| 0.0-0.1 | 0.0 | 12.0 | 8.0 | 4.0 | 0.0 |
| 0.1-0.2 | 0.0 | 4.0 | 12.0 | 24.0 | 0.0 |
| 0.2-0.3 | 0.0 | 0.0 | 12.0 | 14.0 | 0.0 |
| 0.3-0.4 | 0.0 | 0.0 | 0.0 | 6.0 | 0.0 |
| 0.4-0.5 | 0.0 | 0.0 | 2.0 | 2.0 | 0.0 |
| 0.5-0.6 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
| 0.6-0.7 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
| 0.7-0.8 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
| 0.8-0.9 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
| 0.9-1.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
| Total % | 0.0 | 16.0 | 34.0 | 50.0 | 0.0 |
Table 4.9: Carbide size distribution data for the fully bainitic sample tempered at 700°C for 5 hours.
Figure 4.5: Size distribution curves for the various carbides identified in the fully bainitic sample tempered at 700°C for 5 hours.
| Particle Mean Diameter (microns) | Percentage Of Total No. Of Carbides |
|---------------------------------|-----------------------------------|
| | M₃C | M₂C | M₇C₃ | M₂₃C₆ | M₆C |
| 0.0-0.1 | 0.0 | 0.5 | 13.8 | 19.9 | 0.0 |
| 0.1-0.2 | 0.0 | 0.0 | 11.0 | 22.1 | 0.5 |
| 0.2-0.3 | 0.0 | 0.0 | 4.9 | 12.1 | 1.1 |
| 0.3-0.4 | 0.0 | 0.0 | 2.2 | 2.2 | 1.1 |
| 0.4-0.5 | 0.0 | 0.0 | 1.1 | 1.1 | 1.6 |
| 0.5-0.6 | 0.0 | 0.0 | 0.0 | 0.5 | 0.5 |
| 0.6-0.7 | 0.0 | 0.0 | 0.0 | 0.0 | 1.6 |
| 0.7-0.8 | 0.0 | 0.0 | 0.0 | 1.1 | 0.5 |
| 0.8-0.9 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
| 0.9-1.0 | 0.0 | 0.0 | 0.0 | 0.5 | 0.0 |
| Total % | 0.0 | 0.5 | 33.0 | 59.5 | 6.9 |
Table 4.10: Carbide size distribution data for the fully bainitic sample tempered at 700°C for 50 hours.
Figure 4.6: Size distribution curves for the various carbides identified in the fully bainitic sample tempered at 700°C for 50 hours.
| Particle Mean Diameter (microns) | M₃C | M₂C | M₇C₃ | M₂₃C₆ | M₆C |
|---------------------------------|-----|-----|------|-------|-----|
| 0.0-0.1 | 0.0 | 0.0 | 0.0 | 1.3 | 5.3 |
| 0.1-0.2 | 0.0 | 0.0 | 0.0 | 0.0 | 12.7|
| 0.2-0.3 | 0.0 | 0.0 | 0.0 | 0.0 | 23.9|
| 0.3-0.4 | 0.0 | 0.0 | 0.0 | 0.7 | 21.3|
| 0.4-0.5 | 0.0 | 0.0 | 0.0 | 0.0 | 14.7|
| 0.5-0.6 | 0.0 | 0.0 | 0.0 | 0.0 | 7.4 |
| 0.6-0.7 | 0.0 | 0.0 | 0.0 | 0.0 | 2.0 |
| 0.7-0.8 | 0.0 | 0.0 | 0.0 | 0.7 | 4.0 |
| 0.8-0.9 | 0.0 | 0.0 | 0.0 | 0.0 | 4.0 |
| 0.9-1.0 | 0.0 | 0.0 | 0.0 | 0.0 | 2.0 |
| Total % | 0.0 | 0.0 | 0.0 | 2.7 | 97.3|
Table 4.11: Carbide size distribution data for the fully bainitic sample tempered at 700°C for 5000 hours.
Figure 4.7: Size distribution curves for the various carbides identified in the fully bainitic sample tempered at 700°C for 5000 hours.
| Time (Hours) | M₃C | M₂C | M₇C₃ | M₂₃C₆ | M₆C |
|-------------|------|------|------|-------|------|
| 0.5 | 6.6 | 2.1 | 62.9 | 28.2 | 0.3 |
| 5 | 0.0 | 16.0 | 34.0 | 50.0 | 0.0 |
| 50 | 0.0 | 0.5 | 33.0 | 59.5 | 6.9 |
| 5000 | 0.0 | 0.0 | 0.0 | 2.7 | 97.3 |
Table 4.12: Change in carbide type with tempering time at 700°C for the fully bainitic samples.
Figure 4.8: Change in carbide type with tempering time at 700°C for the fully bainitic samples.
Figure 4.9: Graph showing $M_3C$ type carbides as a percent of the total detected for each tempering duration plotted against mean diameter.
Figure 4.10: Graph showing $M_2C$ type carbides as a percent of the total detected for each tempering duration plotted against mean diameter.
Figure 4.11: Graph showing $M_7C_3$ type carbides as a percent of the total detected for each tempering duration at 700°C plotted against mean diameter.
Figure 4.12: Graph showing $M_{23}C_6$ type carbides as a percent of the total detected for each tempering duration at 700°C plotted against mean diameter.
Figure 4.13: Graph showing $M_6C$ type carbides as a percent of the total detected for each tempering duration at 700°C plotted against mean diameter.
4.4 Creep Testing and Deformation Assessment
4.4.1 Crack Opening Displacement Measurement
The experimental techniques adopted to assess the creep deformation behaviour of the steel being investigated have been described in section 3.4.3. Compact tension specimens of 2.25%Cr-1%Mo steel with the previously specified starting microstructures were prepared, each with a spark eroded notch tip to simulate the presence of a pre-existing defect. These were then creep tested to assess the affect of the presence of the pre-existing defect on the creep crack initiation and growth process in samples exhibiting each of the three microstructural profiles.
Crack opening displacement values for each test specimen were determined by measuring the Y-axis displacement between two parallel lines of hardness indentations which had been made along the specimen width prior to the test as described in section 3.4.3(c).
Tables 4.13 and 4.14 present the results for the x/y displacement values versus time for creep tests carried out at 550°C on the 100% bainitic and ferrite / bainite specimens. The displacement values for the ferrite / pearlite test specimens, tested at the collaborating establishment, were not available for analysis and are, therefore, absent from this section of the results.
The y-axis displacement values were plotted against the x-axis position along the specimen width to produce charts as shown Figures 4.14 - 4.17. The positions of the CT specimen load point, (LP), and spark eroded notch tip, (NT), have been indicated on the charts to show the difference in the degrees of deformation at these two critical points. The y-displacement at the notch tip position also allowed an assessment of the relationship between notch tip opening displacement, creep cavity density and crack extension to be made.
For the crack opening displacement values of the 100% bainite and mixed ferrite / bainite samples two charts were plotted in each case. This was necessary to compare the deformation characteristics of all the test specimens in each microstructural condition concurrently and to assess the crack tip opening displacement values of the specimens tested for the shortest times. The specimens tested for the shorter duration’s, in particular, gave information regarding the critical stages of creep crack initiation and early growth.
Comparison of the crack opening displacement values for the tempered bainite and mixed ferrite / bainite structures show a marked difference in the deformation behaviour of the two materials. Assessment of the deformation lines between the load point and notch tip points for the two materials showed that the 100% bainite structure was less ductile than the mixed ferrite / bainite structure. This is indicated by the straight line nature of the graph in Figure 4.15 as opposed to the curved line in Figure 4.17.
The crack, or notch, tip opening displacement values (CTOD’s), as detailed in Table 4.15, were determined by extrapolating the deformation lines plotted on Figures 4.14 – 4.17 to the notch tip points. Although an absolute like for like comparison of the CTOD results was not possible due to the difference in loading stresses and test duration’s it was noticeable that the CTOD values for the ferrite / bainite material, tested at a stress of 110MPa, were less than those observed for the fully bainitic structure, tested for similar test duration’s and a stress of 100MPa.
Table 4.13: Y-displacement values of hardness indentations for 100% tempered bainite starting material tested at a stress of 100MPa for times between 300 and 1000 hours.
| No. Of Hours at 550°C | X/Y-Displacement (mm) |
|-----------------------|------------------------|
| | 2.54 | 5.08 | 7.62 | 10.16 | 12.7 | 15.24 | 17.78 | 20.32 |
| 300 | 1 | 1 | 0.8 | 0.6 | 0.5 | 0.3 | 0.2 | 0.1 |
| 350 | 1.2 | 1.2 | 1 | 0.8 | 0.6 | 0.6 | 0.4 | 0.3 |
| 400 | 1.3 | 1.2 | 1 | 0.8 | 0.6 | 0.5 | 0.4 | 0.2 |
| 500 | 1.8 | 1.6 | 1.3 | 1.1 | 0.9 | 0.8 | 0.6 | 0.4 |
| 600 | 2 | 1.9 | 1.5 | 1 | 1 | 0.8 | 0.6 | 0.4 |
| 800 | 3.6 | 3.3 | 2.9 | 2.5 | 2 | 1.9 | 1.6 | 0.9 |
| 1000 | 4.9 | 4.5 | 3.8 | 3.8 | 2.9 | 2.6 | 2.1 | 1.6 |
Table 4.14: Y-displacement values of hardness indentations for mixed ferrite / bainite starting material tested at a stress of 110MPa for times between 300 and 1500 hours.
| No. Of Hours at 550°C | X/Y Axis Displacement (mm) |
|-----------------------|-----------------------------|
| | 2.54 | 5.08 | 7.62 | 10.16 | 12.7 | 15.24 | 17.78 | 20.32 |
| 300 | 1 | 1 | 0.7 | 0.6 | 0.5 | 0.3 | 0.2 | 0.1 |
| 700 | 2 | 1.7 | 1.4 | 1.2 | 0.9 | 0.6 | 0.3 | 0.2 |
| 850 | 2.3 | 1.9 | 1.5 | 1.3 | 1 | 0.7 | 0.5 | 0.3 |
| 1000 | 3.3 | 3 | 2.6 | 2 | 1.3 | 0.8 | 0.5 | 4 |
| 1500 | 10.2 | 9.4 | 8.2 | 6.9 | 5.7 | 4.6 | 3.8 | 2.9 |
Figure 4.14: Crack opening displacement values for the fully bainitic sample tested at a stress of 100MPa and 550°C for times between 300 – 1000h.
Figure 4.15: Crack opening displacement values for the fully bainitic sample tested at a stress of 100MPa and 550°C for times between 300 – 600h.
Figure 4.16: Crack opening displacement values for the ferrite / bainite sample tested at a stress of 110MPa and 550°C for times between 300 – 1500h.
Figure 4.17: Crack opening displacement values for the ferrite / bainite sample tested at a stress of 110MPa and 550°C for times between 300 – 1000h.
| Test Duration (h) | Ferrite / Bainite Tested at 110MPa | Bainite Tested at 100MPa | Pearlite Tested at 85MPa |
|------------------|-----------------------------------|--------------------------|-------------------------|
| | 0 | 300 | 700 | 850 | 1000 | 1500 | 0 | 300 | 350 | 400 | 500 | 600 | 800 | 1000 | 0 | 3690 | 6850 |
| Crack Tip Opening Displacement (mm) | 0.00 | 0.08 | 0.16 | 0.27 | 0.27 | 2.75 | 0.00 | 0.07 | 0.18 | 0.28 | 0.35 | 0.35 | 0.88 | 1.52 | n.d | n.d | n.d |
Table 4.15: Crack tip opening displacement results derived from CT specimens manufactured from the starting materials.
Note* - n.d = not determined
4.4.2 Creep Damage Assessment in the Scanning Electron Microscope
Scanning electron microscopy was used to assess the microstructure ahead of the CT notch in each of the tested specimens in order to assess the degree of creep damage and, hence, attempt to determine the mechanisms by which that damage had occurred. Samples were prepared from the test specimens and examined in the SEM, in the polished and etched condition, using the techniques as described in section 3.6.1.
In particular back-scattered electron imaging was used in conjunction with the image analysis routine outlined in section 3.6.1(c) to examine the extent of creep cavitation and its orientation with respect to the "pre-existing defect" created by the machined notch. The percentage of cavitation occurring in an area $1\text{mm}^2$ around the notch tip was determined and graphically correlated against crack extension to ascertain the degree of creep ductility for the purpose of this project, creep crack initiation was considered to be taking place up to a crack extension of 1mm and therefore only specimens exhibiting $\leq 1\text{mm}$ of crack extension were considered for damage accumulation assessment.
Crack extension, measured using a linear measuring device incorporated in the SEM imaging software, was considered to have occurred if creep cavities had coalesced to give a micro-crack traversing more than one grain. To help gain a better understanding of the mechanism of creep crack initiation a number of selected samples were examined in detail using high resolution secondary electron imaging.
4.4.2(a) 100% Bainite Specimens
Compact tension specimens of the steel initially with 100% bainite structure were creep tested at a constant applied stress of 100MPa and a temperature of 550°C for 300, 350, 400, 500, 600, 800 and 1000 hours.
Plate 4.24 shows a low magnification image of the microstructure in the specimen tested for 300 hours in the region close to the spark eroded notch tip. Although cracking had not yet occurred in this sample there was evidence of grain boundary voiding. Table 4.16 and Figure 4.18 show the analysis of void % in the vicinity of the notch and indicate that the densest region of void formation was in an area, $200\mu m \times 200\mu m$, immediately adjacent to the notch tip.
The microstructure of the sample tested for 350 hours, Plate 4.25, revealed a micro-crack emanating from a point approximately $50\mu m$ below the notch tip and slightly to the right of the notch tip centre. The crack extended up to a length of 0.49mm and appeared to follow an intergranular path. The corresponding void % data for the notch tip region for this sample is shown in Table 4.17 and Figure 4.19. Again the densest region of void formation is shown to be in the area closest to the notch tip with the distribution of voids in the surrounding area being slightly unevenly biased toward the right hand side.
After testing for 400 hours the degree of crack extension was found to have increased to a length of 0.62mm, the crack path being similar to that observed in the 350 hour specimen. In addition to the dominant 0.62mm crack a further crack was observed to have initiated, emanating from the left hand side of the notch and propagating towards the main crack path. Overall, as indicated in the void analysis results, Table 4.18 and Figure 4.20, the crack front and region of highest void density remained constrained to the material immediately ahead of the notch tip.
The microstructures examined for the specimens tested for 500 hours and above showed crack extensions exceeding 1mm. These samples were not, therefore, analysed further in terms of void area % and distribution.
A summary of the crack extension values measured for all the 100% tempered bainite CT specimens and the area percent of voids detected in a region extending $1mm^2$ around the notch tip centre is given in Table 4.19.
To assess the area around the notch tip in terms of its evolving microstructure and creep void orientation with respect to the stress axis, a detailed microstructural analysis was carried out on the specimen tested for 400 hours. Plate 4.27 shows a montage of back scattered electron images depicting the areas assessed for void formation immediately ahead and to the right of the notch tip in the specimen tested for 400 hours, i.e., 0 μm to ~800 μm in the y-direction and ~100 μm to 300 μm in the x-direction. These images revealed that the main crack was extending in an intergranular manner at approximately 45° to the notch tip and, hence, 45° to the principal stress axis. Similarly, voiding observed in the area surrounding the crack appeared to be taking place preferentially on grain boundaries oriented at ~45° to the notch tip.
The incidence of void formation occurring in a particular orientation for the areas shown in Plate 4.27 was assessed in the SEM image analysis routine as described in section 3.6.1(c). The orientation of the voids, i.e., the orientation of the longest feret diameter of the void with respect to the notch tip, was subsequently plotted on a frequency chart as shown in Figure 4.21. The chart showed that the voids occurred most frequently at an orientation of ± 50 - 60° with respect to the notch tip.
Plates 4.28 – 4.32 show high magnification secondary electron images resulting from the examination of selected grains exhibiting the initial stages of cavitation in the specimen tested for 400 hours. This examination was necessary to find evidence for the mechanisms of creep crack initiation ahead of the defect. In the majority of cases, as suggested by the low magnification image in Plate 4.27, the voiding occurred along grain boundaries having an angle of 45° to the notch tip. In Plate 4.28, the voids appear to have been initiated in the vicinity of large grain boundary carbides which are elongated in the direction of the boundary. Adjacent to these elongated, and quite often irregular shaped, carbides was evidence of an intra-granular substructure, as indicated by the red arrows in this plate, and a grain boundary area denuded zone. Further evidence of denudation and the presence of sub-structure boundaries adjacent to cavitating grain boundaries is shown in Plates 4.30 and
4.31 respectively. Plate 4.31 also shows evidence of void formation at a triple point. For comparison, Plate 4.32 shows the microstructure from a reference region in the CT specimen remote from the stress field. No evidence of cavitation was detected in this region.
Plate 4.24: Low magnification SEM image of the notch tip region in the fully bainitic sample tested at 550°C and 100MPa for 300 hours.
| Y-Direction (microns) | -400 | -200 | 0 | 200 | 400 |
|-----------------------|------|------|-----|-----|-----|
| 0 | 0.014| 0.075| 0.097| 0.07| 0.005|
| -200 | 0.001| 0.01 | 0.117| 0.023| 0.061|
| -400 | 0.055| 0.012| 0.084| 0.018| 0.018|
| -600 | 0.019| 0.026| 0.039| 0.03 | 0.017|
| -800 | 0.04 | 0.047| 0.001| 0.01 | 0.01 |
| -1000 | 0.041| 0.016| 0.001| 0.009| 0.003|
Table 4.16: Void % analysis results for the 100% tempered bainite sample tested at 550°C and 100MPa For 300h.
Figure 4.18: Void % analysis distribution chart for the 100% tempered bainite sample tested at 550°C and 100MPa for 300h.
Plate 4.25: Low magnification SEM image of the notch tip region in the fully bainitic sample tested at 550°C and 100MPa for 350h.
| Y- Direction (microns) | X-Direction (microns) / Void % |
|------------------------|--------------------------------|
| | -600 | -400 | -200 | 0 | 200 | 400 | 600 |
| 0 | 0.107 | 0.092 | 0.283 | 1.126 | 0.063 | 0.097 | 0.055 |
| -200 | 0.078 | 0.115 | 0.117 | 1.279 | 0.091 | 0.081 | 0.069 |
| -400 | 0.093 | 0.143 | 0.326 | 0.857 | 0.058 | 0.082 | 0.048 |
| -600 | 0.044 | 0.107 | 0.078 | 0.087 | 0.075 | 0.103 | 0.069 |
| -800 | 0.039 | 0.067 | 0.084 | 0.080 | 0.063 | 0.100 | 0.087 |
| -1000 | 0.042 | 0.151 | 0.098 | 0.105 | 0.049 | 0.046 | 0.083 |
Table 4.17: Void % analysis results for the 100% tempered bainite sample tested at 550°C and 100MPa for 350h.
Figure 4.19: Void % analysis distribution chart for the 100% tempered bainite sample tested at 550°C and 100MPa For 350h.
Plate 4.26: Low magnification SEM image of the notch tip region in the fully bainitic sample tested at 550°C and 100MPa for 400h.
| Y Direction (microns) | -1000 | -800 | -600 | -400 | -200 | 0 | 200 | 400 | 600 | 800 | 1000 |
|-----------------------|-------|------|------|------|------|------|-----|-----|-----|-----|------|
| 0 | 0.081 | 0.075| 0.147| 0.259| 0.419| 8.86 | 0.648| 0.248| 0.255| 0.11 | 0.079|
| -200 | 0.092 | 0.083| 0.166| 0.202| 0.429| 11.251| 0.463| 0.357| 0.187| 0.094| 0.096|
| -400 | 0.121 | 0.505| 0.173| 0.285| 0.249| 7.779| 0.513| 0.324| 0.177| 0.131| 0.221|
| -600 | 0.09 | 0.129| 0.142| 0.239| 0.363| 0.828| 0.356| 0.2 | 0.171| 0.17 | 0.119|
| -800 | 0.085 | 0.649| 0.177| 0.195| 0.171| 0.339| 0.261| 0.318| 0.098| 0.22 | 0.054|
| -1000 | 0.087 | 0.077| 0.618| 0.176| 0.112| 0.215| 0.159| 0.217| 0.122| 0.144 | 0.054|
Table 4.18: Void % analysis results for the 100% tempered bainite sample tested at 550°C and 100MPa for 400h.
Figure 4.20: Void % analysis distribution chart for the 100% tempered bainite sample tested at 550°C and 100MPa for 400h.
Centre of notch tip
| Test Duration (h) | 0 | 300 | 350 | 400 | 500 | 600 | 800 | 1000 |
|------------------|-----|-----|-----|-----|-----|-----|-----|------|
| Crack Ext. (mm) | 0.00| 0.00| 0.49| 0.62| 2.50| 4.00| 6.17| 6.81 |
| Void Area % | 0.00| 0.04| 0.22| 1.42| n.d | n.d | n.d | n.d |
Table 4.19: Crack extension and void area \( \%^2 \) values for the tempered, fully bainitic, CT specimens.
Note* -n.d = values not determined
Figure 4.21: Frequency distribution analysis for void orientation in the fully bainitic sample tested at 550°C, 100MPa for 400h.
\(^2\) Void area \% values were taken from data determined in a region 1mm\(^2\) ahead of the notch tip.
Plate 4.28: High magnification SEM image of grain boundary voiding evident in the tempered, fully bainitic, material tested at $550^\circ\text{C}$ and $100\text{MPa}$ for $400\text{h}$. The red arrows indicate evidence of sub-structure boundaries adjacent to the cavitating regions.
Plate 4.29: High magnification SEM image of voiding at a grain boundary in the tempered, fully bainitic, material tested at $550^\circ\text{C}$ and $100\text{MPa}$ for $400\text{h}$. Elongated carbides, adjacent to PFZ's, are evident in the vicinity of the voids.
Plate 4.30: SEM micrograph showing a denuded zone adjacent to grain boundary voiding in the tempered, fully bainitic, material tested at $550^\circ C$ and $100 \text{MPa}$ for $400 \text{h}$.
Plate 4.31: SEM image showing voids opening up at triple points and evidence of atom migration down sub-structure boundaries in the tempered, fully bainitic, material tested at $550^\circ C$ and $100 \text{MPa}$ for $400 \text{h}$.
Plate 4.32: SEM micrograph showing the carbide morphology in a reference region remote from notch tip stress field in the tempered, fully bainitic, material tested at $550^\circ\text{C}$ and $100\text{MPa}$ for $400\text{h}$.
Compact tension specimens of the steel initially with a mixed ferrite / bainite structure were creep tested at a constant stress of 110MPa and a temperature of 550°C for 300, 700, 850, 1000 and 1500 hours. The specimen tested for 300 hours showed no evidence of crack extension and a minimal amount of cavitation, consequently this sample was not analysed further in terms of void analyses.
Plate 4.33 shows a low magnification image of the microstructure ahead of the notch in the specimen tested for 700 hours. In this specimen isolated cavities were visible uniformly distributed in an area approximately $400\mu m^2$ around the notch tip area, as indicated in Table 4.20 and Figure 4.22. The cavities in this specimen appeared to be less constrained by the stress concentration at the notch tip than that observed for the fully bainitic specimen tested for 300 hours which showed a similar density of voiding immediately ahead of the notch.
After 850 hours crack extension had occurred to a length of $\sim 0.39mm$, as shown in Plate 4.34. When compared to the initial crack observed in the tempered bainite specimen after 350 hours, as shown previously in Plate 4.25, this crack, although still propagating in an intergranular manner, appeared to be less acute in its directionality. A fairly large cavity was observed to have opened up approximately $150\mu m$ ahead of the notch tip in the crack path, possibly due to the presence of an inclusion, and in addition an oxidised crack could be seen adjacent to the notch tip at the left hand side. The void % data and corresponding distribution chart for this specimen are given in Table 4.21 and Figure 4.23 respectively.
Plate 4.35 shows the microstructure of the specimen tested for 1000 hours. In this specimen crack extension had occurred up to a length of 0.9mm. Again, as in the specimen tested for 850 hours, the crack emanated from a position close to the left hand side of the notch tip, it then propagated for approximately $300\mu m$ at an angle close to $45^\circ$ and then changed direction through an angle of
The region surrounding the main crack exhibited widespread grain boundary cavitation as indicated in Table 4.22 and the void distribution chart, Figure 4.24.
A summary of the crack extension values measured for all the mixed ferrite / bainite specimens and the area percent of voids detected in a region extending $1\text{mm}^2$ around the notch tip centre is given in Table 4.23.
For the mixed ferrite / bainite structure the CT specimen selected for detailed microstructural assessment was that tested for 1000 hours. Plate 4.36 shows a montage of back scattered electron images depicting the areas assessed for void formation immediately ahead and to the right of the notch tip in this specimen, i.e. an area extending from $0_\mu\text{m}$ to $-800_\mu\text{m}$ in the y-direction and 0 to $300_\mu\text{m}$ in the x-direction. As suggested previously for this specimen the angle of propagation of the dominant crack appears less constrained to the immediate notch tip centre than in the case of the 100% bainite specimens. Cavitation, once again, was observed to occur predominantly at grain boundary sites and in particular, in this mixed structure, at the boundaries between the ferrite and bainite grains. Occasional regions of bainitic intra-granular voiding were also observed.
The incidence of void formation, occurring at a specific orientation with respect to the notch tip for the areas depicted in Plate 4.36, was assessed in the manner described for the 100% tempered bainite specimens. Figure 4.25, showing the frequency of voids occurring at particular orientations, again suggests that the maximum feret diameter of the voids initiated occurred at an angle of $\pm 50 - 60^\circ$ with respect to the notch tip centre.
Plates 4.37 – 4.42 show high magnification, secondary electron, images taken from regions of voiding in the specimen tested for 1000 hours. Plate 4.37 shows an image of both a ferrite / ferrite and ferrite / bainite grain boundary oriented at approximately $45^\circ$ to the notch tip. The carbide at the ferrite / ferrite boundary, whilst delineated by elongated carbides, does not show any
evidence of cavitation. In contrast, as shown in detail in Plate 4.38 the ferrite / bainite boundary shows cavitation at a number of carbide sites. As described previously for the 100% tempered bainite structure, the large grain boundary carbides associated with the void formation appear to be in the vicinity of sub-structure boundaries formed within the bainite grain.
Plate 4.39 shows a large irregular carbide evident on a ferrite / ferrite grain boundary oriented at $90^\circ$ to the notch tip. This carbide appears to be growing into both adjacent ferrite grains, perhaps with some preference towards the grain featured below the carbide. The material surrounding the carbide shows no signs of cavitation unlike the adjacent bainite grain where extensive denudation and cavitation have occurred. A high magnification image of this latter cavitating boundary is shown in Plate 4.40. From the shape of the cavity in the centre of this image it seems likely that it formed at the site of a carbide which grew away from the bainite grain into the ferrite, eventually losing coherency. Further evidence for the likely mechanism of void formation is also visible in Plate 4.41. Cavities can be seen to initiate at the cusp of grain boundary carbides, probably due to the grain boundary sliding, leading to decohesion between the carbides and matrix to one side. Plate 4.42 shows evidence for the fact that cavitation also occurred, very occasionally, at intra-granular sites along sub-boundaries in the bainite.
For comparison Plates 4.43 and 4.44 show images of the microstructure in a reference region of the specimen well away from the notch tip. No evidence of cavitation was found in this area. It was evident from Plate 4.44 that the carbides at the ferrite / bainite boundary were larger than those at the ferrite / ferrite boundary.
Plate 4.33: Low magnification SEM image of the notch tip region in the mixed ferrite / bainite sample tested at 550°C and 110MPa for 700h.
| Y-Direction (microns) | X-Direction (microns) / Void % |
|-----------------------|--------------------------------|
| | -400 | -200 | 0 | 200 | 400 |
| 0 | 0.271 | 0.114 | 0.095 | 0.069 | 0.065 |
| -200 | 0.006 | 0.036 | 0.055 | 0.068 | 0.041 |
| -400 | 0.012 | 0.094 | 0.062 | 0.050 | 0.078 |
| -600 | 0.019 | 0.037 | 0.056 | 0.065 | 0.061 |
| -800 | 0.060 | 0.012 | 0.017 | 0.028 | 0.012 |
| -1000 | 0.127 | 0.041 | 0.094 | 0.039 | 0.100 |
Table 4.20: Void % analysis results for the mixed ferrite / bainite sample tested at 550°C and 110MPa for 700h.
Figure 4.22: Void % analysis distribution chart for the ferrite / bainite sample tested at 550°C and 110MPa for 700h.
Plate 4.34: Low magnification SEM image of notch tip region in the ferrite / bainite sample tested at 550°C and 110MPa for 850h.
| Y Direction (microns) | -1000 | -800 | -600 | -400 | -200 | 0 | 200 | 400 | 600 | 800 | 1000 |
|-----------------------|-------|------|------|------|------|------|-----|-----|-----|-----|------|
| 0 | 0.281 | 0.173| 0.280| 0.350| 22.283| 9.579| 0.714| 0.048| 0.023| 0.104| 0.018|
| -200 | 0.330 | 0.190| 0.379| 0.248| 0.286| 0.582| 6.589| 0.044| 0.054| 0.050| 0.062|
| -400 | 0.303 | 0.206| 0.266| 0.244| 0.205| 0.146| 0.113| 0.047| 0.322| 0.092| 0.082|
| -600 | 0.114 | 0.224| 0.266| 0.244| 0.205| 0.146| 0.113| 0.054| 0.128| 0.104| 0.044| 0.078|
| -800 | 0.189 | 0.128| 0.483| 0.167| 0.235| 0.143| 0.054| 0.086| 0.075| 0.018| 0.058|
| -1000 | 0.145 | 0.159| 0.113| 0.141| 0.118| 0.116| 0.038| 0.086| 0.075| 0.018| 0.058|
Table 4.21: Void % analysis results for the mixed ferrite / bainite sample tested at 550°C and 110MPa for 850h.
Figure 4.23: Void % analysis distribution chart for the mixed ferrite / bainite sample tested at 550°C and 110MPa for 850h.
Plate 4.35: Low magnification SEM image of notch tip region in the mixed ferrite / bainite sample tested at 550°C and 110MPa for 1000h.
| Y Direction (microns) | X Direction (microns) / Void % |
|-----------------------|--------------------------------|
| | -1000 | -800 | -600 | -400 | -200 | 0 | 200 | 400 | 600 | 800 | 1000 |
| 0 | 0.14 | 0.223 | 0.235 | 1.637 | 29.279| 1.129 | 7.028 | 1.122 | 0.503 | 0.356 | 0.206 |
| -200 | 0.302 | 0.209 | 0.292 | 0.443 | 6.299 | 16.559| 0.83 | 1.396 | 0.587 | 0.32 | 0.287 |
| -400 | 0.368 | 0.587 | 0.158 | 0.334 | 32.706| 1.981 | 1.226 | 0.714 | 0.394 | 0.336 | 0.288 |
| -600 | 0.345 | 0.461 | 0.264 | 2.235 | 10.707| 0.379 | 3.233 | 0.589 | 0.772 | 0.451 | 0.472 |
| -800 | 0.522 | 0.344 | 0.46 | 3.265 | 4.188 | 0.522 | 2.005 | 0.495 | 0.291 | 0.165 | 0.378 |
| -1000 | 0.372 | 0.438 | 0.253 | 2.437 | 6.998 | 0.678 | 0.938 | 0.504 | 0.243 | 0.281 | 0.269 |
Table 4.22: Void % analysis results for the mixed ferrite / bainite sample tested at 550°C and 110MPa for 850h.
Figure 4.24: Void % analysis distribution chart for the mixed ferrite / bainite sample tested at 550°C and 110MPa for 1000h.
Centre of notch tip
Figure 10. Micrograph showing the fracture surface of the specimen tested at 20°C.
| Test Duration (h) | Mixed Ferrite/ Bainite (110MPa) |
|------------------|---------------------------------|
| | 0 | 300 | 700 | 850 | 1000 | 1500 |
| Crack Ext. (mm) | 0.00 | 0.00 | 0.00 | 0.39 | 0.90 | 8.22 |
| Void Area % | 0.00 | 0.01 | 0.06 | 1.73 | 5.14 | n.d |
Table 4.23: Crack extension and void area \( \frac{3}{3} \) values for the mixed ferrite / bainite CT specimens.
Note* -n.d = values not determined
Figure 4.25: Frequency distribution analysis for void orientation in the ferrite / bainite sample tested at 550°C and 110MPa for 850h.
\(^3\) Void area % values were taken from data determined in a region 1mm\(^2\) ahead of the notch tip.
Plate 4.37: High magnification SEM image of voiding at a ferrite / bainite grain boundary.
Plate 4.38: High magnification SEM image of an area indicated in plate 4.37 above showing voiding at a grain boundaries and evidence of substructure boundaries in the bainite phase.
Plate 4.39: SEM micrograph showing large, irregular shaped, grain boundary carbide perpendicular to principal stress axis between two ferrite grains and adjacent to a denuded bainite grain.
Plate 4.40: SEM micrograph showing cavitation on a ferrite / bainite grain boundary and associated denuded region.
Plate 4.41: SEM image depicting cavitation on a ferrite / bainite grain boundary and growth of associated grain boundary carbides. PFZ evident in the ferrite grain adjacent to the grain boundary.
Plate 4.42: SEM image showing voiding in an intra-granular bainitic region of the mixed ferrite / bainite specimen tested for 1000 hours.
Plate 4.43: SEM micrograph showing carbide morphology in a reference region, remote from the notch tip stress field, of the ferrite / bainite specimen tested for 1000h.
Plate 4.44: High magnification SEM micrograph showing the carbide morphology in a reference region, remote from notch tip stress field, of the ferrite / bainite specimen tested for 1000h.
Compact tension specimens of the steel initially with mixed ferrite / pearlite structure were creep tested at a constant stress of 85MPa and a temperature 550°C for 1007, 2006, 3530, 3680h and 6850 hours. The first two of these specimens showed no evidence of creep damage and were not, therefore, considered for further analyses.
The samples tested for 3530, 3680 and 6850 hours all showed evidence of damage accumulation and <1mm of crack extension, however, due to the similarity, in terms of test duration, of the first two of these samples only the 3680h and 6850 h specimens were investigated.
Plate 4.45 shows a low magnification image of the notch tip region in the CT specimen tested for 3680 hours. Although cavity coalescence did not appear to have occurred to a length extending more than one grain, extensive voiding was observed, particularly in a region up to 300μm either side and 1mm ahead of the notch tip. The corresponding void % data and distribution chart for this specimen is given in Table 4.24 and Figure 4.26 respectively.
After testing for 6850 hours a small crack extending approximately 80μm, i.e. about 2-3 grain diameters, was observed to have initiated in a position approximately 300μm to the left of centre and below the notch tip, as visible in Plate 4.46. As denoted in the void % data table, Table 4.25, the area initiating the crack was found to be associated with the presence of a large MnS type inclusion as was the area indicated at the x,y co-ordinates (-200μm,-200μm). The area percent of voids detected in the surrounding regions was less than that observed in the previous specimen which had been tested for half the test duration.
A summary of the crack extension values measured for the ferrite / pearlite CT specimens and the area percent of voids detected in a region extending 1mm² around the notch tip is shown in Table 4.26.
For the mixed ferrite / pearlite structure the CT specimen selected for detailed microstructural assessment was that tested for 3680 hours. Plate 4.47 shows a
montage of back scattered electron images depicting the area assessed for void formation, as described previously in parts b) and c) of this section for the 100% bainite and ferrite / bainite structures. The montage of images show that cavity formation was widely distributed throughout the area examined. Although multiple cavities were frequently found to occur on adjacent grain boundaries, evidence of coalescence encompassing more than one grain was not observed. Sulphide inclusions were found to exist in most areas of the sample examined, for example the large inclusion evident in the right hand bottom quadrant of Plate 4.47. In the majority of cases the voids observed appeared to form on grain boundaries occurring between regions of ferrite and pearlite. In contrast to the 100% bainite and ferrite / bainite material, the orientation of the grains upon which cavitation was seen to initiate was not restricted to boundaries oriented at $\sim 45^\circ$. Many grains boundaries exhibiting cavitation appeared to be oriented at much lower angles of incidence. This was confirmed by the void orientation results, Figure 4.28, which showed that many of the voids in the ferrite / pearlite structure initiated at angles between $30^\circ - 50^\circ$ with respect to the notch tip.
Plates 4.48 - 4.51 show high magnification secondary electron images resulting from the examination of selected grains exhibiting the initial stages grain boundary voiding and coalescence in the specimen tested for 3680 hours. In Plate 4.48 cavities were observed to be forming on grain boundaries between a ferrite and pearlite region in the microstructure. The cavities did not, however, appear to be uniquely linked to specific grain boundary carbides and in fact in some areas, as shown in Plates 4.49 and 4.50, the carbides appeared to be pinning the boundaries. Plate 4.52 shows the microstructure taken from a reference region of the 3680 hour CT specimen for comparison.
In contrast to the carbides observed in the ferrite and bainite regions of the ferrite / bainite CT specimens, the carbides present in the two phase regions in the ferrite / pearlite structure are similar in size. Additionally, the boundaries between the ferrite / pearlite regions of the structure were not always clearly delineated by globular grain boundary carbides.
Plate 4.45: Low magnification SEM image of the notch tip region in the ferrite / pearlite sample tested at 550°C and 85MPa for 3680h.
| Y Direction (microns) | -600 | -400 | -200 | 0 | 200 | 400 | 600 |
|-----------------------|------|------|------|-----|-----|-----|-----|
| 0 | 0.062| 0.041| 0.215| 0.582| 0.483| 0.083| 0.218|
| -200 | 0.046| 0.069| 0.208| 0.243| 0.319| 0.150| 0.055|
| -400 | 0.094| 0.095| 0.119| 0.642| 0.292| 0.207| 0.089|
| -600 | 0.085| 0.076| 0.168| 0.169| 0.230| 0.184| 0.042|
| -800 | 0.083| 0.090| 0.148| 0.225| 0.253| 0.141| 0.076|
| -1000 | 0.047| 0.097| 0.099| 0.223| 0.097| 0.216| 0.056|
Table 4.24: Void % analysis results for the mixed ferrite / pearlite sample tested at 550°C and 85MPa for 3680h.
Figure 4.26: Void % analysis distribution chart for the ferrite / pearlite sample tested at 550°C and 85MPa for 3680h.
Plate 4.46: Low magnification SEM image of the notch tip region in the ferrite / pearlite sample tested at 550°C and 85MPa for 6850h.
| Y Direction (microns) | -600 | -400 | -200 | 0 | 200 | 400 | 600 |
|-----------------------|------|------|------|-----|-----|-----|-----|
| 0 | 0.009| 0.033| 0.089| 0.224| 0.056| 0.020| 0.030|
| -200 | 0.022| 0.019| 1.480| 0.095| 0.035| 0.024| 0.028|
| -400 | 0.053| 0.048| 0.079| 0.149| 0.031| 0.022| 0.026|
| -600 | 0.029| 0.017| 0.035| 0.044| 0.291| 0.028| 0.028|
| -800 | 0.012| 0.021| 0.031| 0.020| 0.023| 0.021| 0.034|
| -1000 | 0.021| 0.017| 0.034| 0.109| 0.031| 0.021| 0.025|
Table 4.25: Void % analysis results for the mixed ferrite / pearlite sample tested at 550°C and 85MPa for 6850h.
(Note- Shaded area denote areas containing large cavities due to the presence of sulphide inclusions.)
Figure 4.27: Void % analysis distribution chart for the ferrite / pearlite sample tested at 550°C and 85MPa for 6850h.
Centre of notch tip
| Test Duration (h) | Mixed Ferrite / Pearlite (85MPa) |
|------------------|---------------------------------|
| | 0 | 3680 | 6850 |
| Crack Ext. (mm) | 0 | 0 | 0.08 |
| Void Area % | 0 | 0.22 | 0.05 |
Table 4.26: Crack extension and void area \% values for the mixed ferrite/pearlite CT specimens.
Figure 4.28: Frequency distribution analysis for void orientation in the ferrite/pearlite sample tested @ 550°C, 85MPa and 3680h.
\(^4\) Void area \% values were taken from data determined in a region 1mm\(^2\) ahead of the notch tip.
Plate 4.48: SEM image showing voiding at a grain boundary between the ferrite and pearlite phase.
Plate 4.49: Voiding at a grain boundary between the ferrite and pearlite phase.
Plate 4.50: Voiding at a grain boundary between the ferrite and pearlite phase.
Plate 4.51: Voiding at a grain boundary between the ferrite and pearlite phase.
Plate 4.52: SEM micrograph showing the carbide morphology in a reference region, remote from notch tip stress field, of the ferrite / pearlite specimen tested for 3680h.
4.4.3 Correlation and Summary of the Creep Test Investigation Data
The crack tip opening displacement, crack extension and void analysis results, for all three microstructural variables, as described in sections 4.4.1 and 4.4.2, are summarised in Table 4.27.
The crack opening displacement data for the mixed ferrite / pearlite specimens, tested at the collaborating establishment, were not available for further analysis and are, therefore, absent from this table and subsequent charts. The only data comparison that could be made for all three microstructural variables was that for the crack extension and void area %.
The ability of the microstructure to sustain creep cavities in the structure, prior to the onset of crack initiation, was observed to occur in the following order: ferrite / pearlite > ferrite / bainite > 100% bainite.
Figure 4.29 shows the crack tip opening displacement, crack extension and void area % results plotted against test duration for the 100% tempered bainite structure and the mixed ferrite / bainite structure. As described in section 3.4.3, to achieve a crack extension of about 1mm in 1000 hours, the ferrite / bainite CT specimens were tested at a temperature of 550°C and a creep stress of 110 MPa and the 100% bainite specimens at 550°C and a stress of 100MPa. The results show that even though the ferrite / bainite specimens were tested at a higher stress than the fully bainitic specimens the occurrence of a specific value of CTOD, crack extension or void area % was delayed with respect to the bainitic structure.
In Figure 4.29a it can be seen that the time to achieve a critical CTOD of 0.3mm was ~ 1000 hours in the ferrite / bainite specimens and only ~400 hours in the fully bainitic specimens. Similarly, from Figure 4.29b, a crack extension of a 1mm occurred in the ferrite / bainite specimens after ~1000 hours but after only ~400 hours in the fully bainitic structure. Once the cracks had extended to a length of ~1mm the void area % was observed to increase rapidly as indicated in Figure 4.29.
Overall the results described above suggest that the mixed ferrite / bainite structure was more resistant to ductility exhaustion, i.e. was more creep ductile, than the fully bainitic structure. This was substantiated by further analysis of the CT test results as presented in Figures 4.30 and 4.31.
Figure 4.30a shows that crack extension, i.e. coalescence of grain boundary voids extending more than one grain boundary, occurred upon the attainment of only a very small percent of voiding in the area $1\text{mm}^2$ around the notch tip in the fully bainitic structure. In the mixed structure a greater % of voids were found to exist prior to the onset of crack extension.
Figures 4.30b indicates that, for CTOD’s less than 0.2mm, a greater CTOD was required to generate the same creep damage, in the form of voids and microcracks, for the ferrite / bainite specimen than for the fully bainitic structure.
Correlation of the data derived for CTOD and crack extension, as shown in Figures 4.31a and b, suggested that although CTOD initiation was delayed in the ferrite / bainite sample an almost linear relationship existed for the two structures once a critical CTOD had been reached, i.e. upon the attainment of a CTOD value of $\sim 0.3\text{mm}$ and a crack extension of $\sim 1\text{mm}$.
| Test Duration (h) | 0 | 300 | 700 | 850 | 1000 | 1500 | 0 | 300 | 350 | 400 | 500 | 600 | 800 | 1000 | 0 |
|------------------|-----|-----|-----|-----|------|------|-----|-----|-----|-----|-----|-----|-----|------|-----|
| CTOE (mm) | 0.00| 0.08| 0.16| 0.27| 0.27 | 0.27 | 0.00| 0.07| 0.18| 0.28| 0.35| 0.35| 0.98| 1.52 | n.a |
| Crack Ext. (mm) | 0.00| 0.00| 0.00| 0.39| 0.90 | 8.22 | 0.00| 0.00| 0.00| 0.49| 0.62| 2.50| 4.00| 6.17 | 6.81|
| Void Area % | 0.00| 0.01| 0.05| 1.73| 5.14 | n.d | 0.00| 0.04| 0.22| 1.42| n.d | n.d | n.d | n.d | 0 |
Table 4.27: Summary of creep testing investigation results.
Note* -
n.a = data not available,
n.d = data not determined
Figure 4.29: Variation of a) CTOD, b) Crack extension and c) Void area %, with test duration for 100% tempered bainite and mixed ferrite / bainite specimens.
Figure 4.30: Variation of a) CTOD and b) Crack extension with void area % for 100% tempered bainite and mixed ferrite / bainite specimens.
Figure 4.31: CTOD against crack extension for a) all samples, b) expanded scale delineating crack initiation region, i.e. <1mm of crack extension.
Cavitation due to the presence of a creep stress acting at the notch tip in the CT specimens was found, in the SEM study, to be associated with the grain boundary carbides. To study these carbides in more depth it was necessary to produce extraction replicas from the specimens of interest and analyse them in the TEM as described in section 3.6.2. To establish the effect of the stress on the grain boundary carbide evolution process replicas were taken from an area immediately ahead of the notch tip and from a reference region remote from the stress concentration. The assessment of the replica samples included a study of the distribution, morphology and type of carbides occurring in the two regions. The classification of the carbides detected was based on the ratios of Fe, Cr and Mo found in the carbides, as described in section 3.6.2(c) and detailed in Table 3.4, page 87.
This study was carried out on the specimens tested in the ferrite / bainite and fully bainitic structures only.
4.4.4(a) Carbide Analysis of the 100% Bainite Specimens
The fully bainitic CT specimen analysed in this study was that tested for a duration of 350 hours.
Plates 4.53 and 4.54 show typical grain boundary regions in the reference and notch tip areas of the fully bainitic CT specimen. The intra-granular carbide distribution was similar in both areas, each showing regions consisting of the following:
i) densely populated directional carbides, predominantly $M_{23}C_6$ in type
ii) occasional globular carbides, $M_{23}C_6 / M_6C$ in type
iii) denuded areas adjacent to large carbides situated at grain boundaries.
In the notch tip and reference regions large carbides observed at the grain boundaries were found to be either $M_{23}C_6$ or $M_6C$ carbides or of a composition somewhere between the two. Analysis of a number of carbides existing along a typical grain boundary in each region showed no significant difference in the number of carbides of a particular type. This suggests that the rate of the $M_{23}C_6 \rightarrow M_6C$ evolution process was not affected by the effective stress acting at the notch tip.
Plate 4.55 shows a typical grain boundary, in the notch tip region of the specimen, oriented at an angle with respect to the principal stress axis. To evaluate the evolutionary state of the carbides in this region a number of the carbides were analysed in terms of their key element ratios, i.e. Fe:Cr, Mo:Cr and Mo:Fe using EDX analysis. High magnification images of the carbides analysed, identified by the letters A-C in Plate 4.55 are shown, along with their elemental ratios, in Plates 4.56 – 4.58.
Carbide A, as shown in Plate 4.56, was an 'L' shaped carbide which appeared to have evolved from the diffusion of atoms across the interface between a grain boundary and sub-grain boundary carbide. The dark regions in the carbide had a composition which was typical of $M_{23}C_6$ whereas the lighter, diffuse, regions were typical of $M_6C$, the equilibrium carbide. Carbide B, a large irregular shaped carbide positioned along a sub-grain boundary, was mainly found to be of a chemistry which was consistent with that of $M_6C$. In this case, however, it appeared to have evolved from an $M_7C_3$ type carbide, the remnants of which could be observed in dark field imaging mode as shown in Plate 4.57. Carbide C, which lies directly upon the grain boundary, was identified as an $M_6C$ type carbide and, as shown in Plate 4.58, could be seen to be growing along the bainite / bainite interface. In contrast to the latter carbide, the carbide shown in Plate 4.59 was found to lie on a grain boundary which was parallel to the notch tip / principal stress axis. Although the chemical analysis of this carbide was similar to that of carbide C in Plate 4.58 its growth appeared to be occurring in finger like
protrusions away from the original $M_{23}C_6$ carbide and into the adjacent grain. Plate 4.60 shows the effect of a triple point on the growth characteristics of $M_6C$.
The main difference observed between the notch tip and reference regions was that in the notch tip area there was evidence of carbides elongated along grain boundaries which were oriented at an angle with respect to the notch tip. These carbides were considered to be the ones that were associated with the initiation of grain boundary cavitation as observed in the SEM study section 4.4.2 and as such were investigated further. Plate 4.61 shows a montage of images and elemental ratios derived from an elongated carbide which appeared to be associated with cavitation to one side. The elemental ratios determined for Fe, Cr and Mo along the length of this carbide suggests that, as for carbide C in Plate 4.58, the evolution and growth of $M_6C$ from $M_{23}C_6$ occurs along the grain boundaries. The denuded regions surrounding these large carbides suggest that the atoms necessary for this evolutionary process are provided by the dissolution of carbides in the adjacent grains.
In all the examples outlined above it apparent that the equilibrium carbide, $M_6C$, evolves from existing carbides by the dissolution of carbides in adjacent regions and that growth occurs in the direction of easiest diffusion.
4.4.4(b) Carbide Analysis of the Mixed Ferrite / Bainite Specimens
The ferrite/bainite CT specimen analysed in this study was that tested for a duration 850 hours.
As for the fully bainitic specimen a number of grain boundary carbides in both a reference and notch tip region were analysed to determine any differences between the carbide compositions and morphologies. No significant difference between the number of carbides of a particular type was detected.
For reference Plate 4.62 shows a typical ferrite / bainite grain boundary from a region remote from the notch tip. As described in the TEM study of the starting material, section 4.2.4, the ferrite regions were densely populated with fine $M_2C$ type carbide and were separated from the coarse carbide structure of the bainite regions by large globular grain boundary carbides, predominantly $M_{23}C_6$ or $M_6C$ in nature. The carbides in the bainite were again predominantly $M_{23}C_6$ type carbides with occasional $M_7C_3$ and $M_6C$. Carbides at the ferrite / ferrite grain boundaries were found to be predominantly $M_7C_3$ in nature.
Once again the main difference observed between the notch tip and reference region was the morphology of the evolving carbides on grain boundaries oriented at an angle with respect to the notch tip. Elongated carbides apparently associated with cavitation were observed on ferrite / bainite boundaries, as shown in Plates 4.63 and 4.64. Cavitation associated with grain boundary carbides was not observed on ferrite / ferrite boundaries. The carbide depicted in the montage of Plate 4.64 is similar to that observed for the fully bainitic specimen in Plate 4.61. Growth of the evolving $M_6C$ carbide occurs along the grain boundary between the existing $M_{23}C_6$ carbide and by the corresponding dissolution of surrounding carbides.
Plate 4.65 shows the growth process occurring on a carbide which was not oriented at a critical angle with respect to the notch tip. Carbides adjacent to the grain boundary $M_{23}C_6$ can be seen to be absorbed by the evolving carbide. The growth of an $M_7C_3$ type carbide at a ferrite / ferrite grain boundary is shown in Plate 4.67.
A significant difference between the ferrite / bainite structure and the fully bainitic structure was the presence of ferrite / ferrite boundaries in the former and the type of carbides precipitated there. The carbides at the ferrite / ferrite boundaries were found to be predominantly $M_7C_3$ in nature, as depicted in Plate 4.66, and as such appeared to be more resistant to change. This fact was considered to be due to the difference in the concentration of elements in the immediate vicinity of the grain boundary carbides.
Plate 4.53: TEM micrograph showing the typical carbide morphology observed in a reference region of the tempered bainite CT specimen tested at a temperature $550^\circ\text{C}$ and a stress of $100\text{MPa}$ for 350 hours.
Plate 4.54: TEM micrograph showing the typical carbide morphology observed in the notch tip region of the tempered bainite CT specimen tested at a temperature $550^\circ\text{C}$ and a stress of $100\text{MPa}$ for 350 hours.
Plate 4.55: Typical boundary oriented at an angle with respect to the notch tip in the fully bainitic CT specimen tested at a temperature $550^\circ C$ and a stress of $100 \text{ MPa}$ for 350 hours. Evolving carbides identified for EDX analysis in Plates 4.56 – 4.58.
Plate 4.56: Brightfield image of feature A in micrograph depicted in plate 4.55 and the corresponding elemental ratios for Fe:Cr, Mo:Cr and Mo:Fe determined by EDX analysis.
Plate 4.57: Dark field image of feature B in micrograph depicted in plate 4.55 and the corresponding elemental ratios for Fe:Cr, Mo:Cr and Mo:Fe from EDX analysis.
Plate 4.58: Bright field image of feature C in micrograph depicted in plate 4.55 and the corresponding elemental ratios for Fe:Cr, Mo:Cr and Mo:Fe from EDX analysis.
Plate 4.59: Growth of $M_6C$ from $M_{23}C_6$ on a grain boundary almost parallel to the maximum principal stress in the notch tip region of the fully bainitic CT specimen tested at a temperature of 550°C and a stress of 100MPa for 350 hours and corresponding elemental ratios for Fe:Cr, Mo:Cr and Mo:Fe determined by EDX analysis.
Plate 4.60: $M_6C$ type carbides at grain boundary triple points in the fully bainitic CT specimen tested at a temperature of 550°C and a stress of 100MPa for 350 hours.
Plate 4.61: High magnification TEM image of an elongated grain boundary carbide in the notch tip region of the fully bainitic CT specimen. The elemental ratios of Fe:Cr, Mo:Cr and Mo:Fe, determined by EDX analysis, are indicated in this micrograph.
Plate 4.62: TEM micrograph showing the typical carbide morphology observed in a reference region of the ferrite / bainite CT specimen tested at a temperature $550^\circ\text{C}$ and a stress of $110\text{MPa}$ for 850 hours.
Plate 4.63: TEM micrograph showing elongated carbides along a ferrite / bainite grain boundary oriented at an angle with respect to the notch tip and maximum principal stress.
Plate 4.64: High magnification TEM image of the elongated grain boundary carbide observed in plate 4.63 and corresponding elemental ratios for Fe:Cr, Mo:Cr and Mo:Fe determined by EDX analysis.
Plate 4.65: Evolution of $M_{23}C_6$ to $M_6C$ by the gradual absorption of elements from adjacent carbides on a non-critically oriented ferrite / bainite grain boundary.
Plate 4.66: $M_7C_3$ type carbide at a typical ferrite / ferrite grain boundary in the ferrite / bainite CT specimen and corresponding elemental ratios for Fe:Cr, Mo:Cr and Mo:Fe.
Plate 4.67: Growth of $M_7C_3$ type carbide at a ferrite / ferrite grain boundary by absorption of adjacent $M_2C$ carbides in the ferrite / bainite CT specimen.
5.1 Starting Structures and Basic Mechanical Properties of 2.25%Cr-1%Mo Steel
In large, ferritic alloy steel, cast components a range of microstructures may exist ranging from pro-eutectoid ferrite + pearlite structures, in thick sections, to fully bainitic structures, in thin sections.
In this study, three microstructural variants of 2.25%Cr-1%Mo steel, a material utilised for power plant applications, were selected to assess the effect of the presence of a pre-existing defect on the creep resistance of the alloy. To make this assessment it was first necessary to characterise the starting structures in terms of their microstructural constituents and basic mechanical properties and to consider the principles behind their formation.
5.1.1 Transformation Structures
The three transformation structures generated for this study, i.e. ferrite / pearlite, ferrite / bainite and 100% bainite, were a result of varying the cooling rate of the alloy from the austenitising phase field, above the $Ac_3$. The grain size of the resulting structures was a direct consequence of the austenitising temperature and duration and was determined in this study using the mean linear intercept method.
As anticipated the grain-size of the ferrite / bainite and fully bainitic structures were similar having an average grain diameter of $\sim 0.065\text{mm}$. The ferrite / pearlite structure, however, being from an alternative material source, was found to possess a significantly finer grain structure of average grain diameter $\sim 0.03\text{mm}$. Although the grain size is only regarded as having a small influence on the creep resistance of ferritic alloys \cite{24} \cite{75}, it may have had a significant effect on the tensile properties, as discussed later.
In accordance with the CCT diagram for the alloy [140], cooling to room temperature from above the $A_{C_3}$ results in the transformation of FCC austenite to BCC ferritic phases, the elemental additions in the alloy either remaining in solid solution or precipitating out as discrete second phase particles. The propensity of the elements to be situated in one or the other of these sites depends upon the solubility of the alloying elements in the BCC crystal lattice and the cooling rate from the $A_{C_3}$ to the $A_{C_1}$.
In ferritic, 2.25%Cr-1%Mo, alloy steel the elements that are of particular interest, with respect to the creep resistance of the alloy, are C, Mo and Cr. When precipitation of these elements is suppressed on cooling from above the $A_{C_3}$, C is held interstitially in the Fe lattice, while Cr and Mo substitute for Fe. In both interstitial and substitutional solid solutions the elements create a strain field in the BCC iron lattice leading to solid solution hardening effects.
On slow cooling from above the $A_{C_3}$ to form the ferrite / pearlite transformation structure the pro-eutectoid ferrite formed first, nucleating at the austenite grain boundaries and rejecting carbon into the remaining austenite. On reaching the $A_{C_1}$ any remaining austenite underwent the eutectoid reaction to pearlite, precipitating the majority of carbon as Fe$_3$C or alloy carbide. The relative amounts of pro-eutectoid ferrite and pearlite in the final structure were dependent upon the cooling rate between the $A_{C_1}$ and $A_{C_3}$. In this study only ~10% pearlite phase was found to exist, the dominant phase in the pearlite / ferrite structure being that of newly formed pro-eutectoid ferrite grains.
During the transformation process to pro-eutectoid ferrite + bainite, by faster cooling than that experienced for the ferrite / pearlite structure, the pro-eutectoid ferrite was again formed first. The pearlite reaction was prevented by the faster cooling rate and the remaining austenite, enriched in carbon, transformed to bainite. In the case of upper bainite, as was confirmed for the material in this study, the carbon was held in both interstitial solid solution sites and precipitated out as cementite between the upper bainitic ferrite laths. The Mo and Cr in the as-cooled structure existed in solid solution sites uniformly
dispersed between the pro-eutectoid ferrite and the bainite, in line with the theory of non-partioning as described by Honeycombe [23]. The volume fraction of the pro-eutectoid ferrite and bainite formed was again dependent upon the cooling rate from above the $A_{C_3}$, and in this investigation a structure consisting of 75% ferrite and 25% bainite was observed.
In the case of the fully bainitic structure, where the cooling rate was sufficiently high to bypass the pro-eutectoid ferrite and pearlite transformation curves, the austenite transformed directly to bainite and the carbon, for upper bainite, was distributed as described above for the bainite in the mixed structure. In this case, however, due to the absence of the pro-eutectoid ferrite phase there was less interstitial saturation of carbon in the bainite. Cr and Mo were, again uniformly dispersed in the solid solution structure.
### 5.1.2 Carbide Structure
In the case of the ferrite / bainite and fully bainitic material, the as cooled structures were, subsequently, subjected to a stress relief and tempering treatment. This treatment allowed some of the elements saturated in the solid solution to precipitate out as alloy carbides. As the carbide type, size and distribution in these structures has a significant effect on its mechanical properties the starting structures were also characterised in these terms.
In the mode of Titchmarsh [138], Pilling and Ridley [30] and Abdel-Latif et al [39] semi-quantitative EDX analysis of carbides extracted from the temper trial samples and starting structures enabled a carbide classification scheme, based on the ratios of Fe, Cr and Mo, to be derived. These classification ratios, as detailed in Table 3.4, p87, were then used to identify the carbides in the alloy for the remainder of the study.
From the SEM and TEM studies of the starting structures it became clear that although the various carbide types detected in each structure were similar their distribution and, in some cases, maturity varied.
The carbides in the pro-eutectoid ferrite phase of both the mixed ferrite/pearlite and ferrite/bainite structures were identified as $M_2C$ by their lenticular shape and composition rich in molybdenum and chromium. It was noted, however, that the $M_2C$ in the ferrite phase of the ferrite/pearlite structure was richer in chromium than that detected in the ferrite/bainite structure. This was considered to be due to the increased chromium concentration of the ferrite in the slow cooled structure, that is the slower cooling may have allowed partitioning of the ferrite former.
The pearlitic regions in the ferrite/pearlite specimens were found to exhibit a lamellar structure of eutectoid ferrite and alloy carbide with large, globular or elongated, $M_{23}C_6$ carbides at the prior austenite grain boundaries. The boundaries between the pearlite and pro-eutectoid ferrite regions that did not coincide with those of the prior austenitic structure were not delineated by large carbides. At the ferrite/ferrite boundaries carbides typical of $M_{23}C_6$ were found. No evidence of $M_6C$ carbides were detected in this structure.
From the analysis of the carbides in the bainitic regions of both the mixed ferrite/bainite and fully bainitic structures it became clear that, not only was there a higher density of carbides in the bainite of the mixed structure but that the proportion of carbides of a particular type varied.
The bainitic regions in both structures exhibited a mixture of $M_2C$, $M_7C_3$ and $M_{23}C_6$ carbides, the occurrence of $M_2C$ being minimal compared to that observed in the ferrite grains and also to that of the $M_7C_3$ and $M_{23}C_6$. $M_{23}C_6$ was widely detected in the bainitic regions of both structures but $M_7C_3$ was more prevalent in the mixed structure.
As determined in the tempering trials of this investigation, and described by previous authors [17] [30], $M_7C_3$ in this alloy nucleates in the early stages of tempering in the vicinity of $M_3C$ by absorption of Cr from the surrounding matrix. At extended times and elevated temperatures the $M_7C_3$ carbide begins to dissolve giving way to the independent nucleation and growth of $M_{23}C_6$, having a lower C:metal atom ratio. With this evidence in mind it can be surmised that the increased carbide density and persistence of $M_7C_3$ to longer times observed in the bainite region of the mixed structure was due to an increased C: metal atom ratio compared to that in the fully bainitic structure.
It is proposed that, during the austenite to pro-eutectoid ferrite transformation the remaining austenite was enriched in carbon. On further cooling, when the remaining austenite transformed to upper bainite, the carbon in solution precipitated out between the bainite laths as cementite and, to a lesser extent, remain trapped interstitially in the bainitic ferrite. The increased concentration of carbon in the remaining austenite led to a corresponding increase in the density of carbides in the bainite of the mixed structure.
Evidence for the stabilising effect of carbon on the $M_7C_3 - M_{23}C_6$ evolution process was reported previously by Beech and Warrington [32]. They proposed a relationship between the alloy composition and the time to achieve 50% transformation of the $M_7C_3$ carbides to $M_{23}C_6$, such that:
$$t_{50} = \exp(13.7(1 + C - 0.12Cr))$$
- Equation 5.1
Carbides existing at the bainite / bainite boundaries and ferrite / bainite boundaries in both bainite containing structures were found to be predominantly $M_{23}C_6$ in nature with occasional $M_6C$. Analysis of the carbides at the ferrite / ferrite boundaries in the mixed ferrite / bainite structure, however, were found to be chromium rich and of the type $M_7C_3$.
The explanation for the retention of $M_7C_3$ at the ferrite / ferrite boundaries in the mixed structure may again be linked to the relative carbon: metal atom ratio in this region and the evidence for this comes from a comparison of the current work with that of Thomson [35]. In contrast to the current work Thomson reported that $M_{23}C_6$ was present at the ferrite / ferrite grain boundaries during the early stages of tempering a mixed ferrite / bainite, 2.25%Cr-1%Mo steel. The mixed structure in the Thomson study, however, was that of 50% ferrite / 50% bainite and not 75% ferrite / 25% bainite as in this case.
During the formation of the pro-eutectoid ferrite in the mixed structure of the current study less remaining austenite was available to absorb the carbon displaced from the ferrite, i.e. only 25% austenite remained compared to 50% in the study by Thomson [35]. The outcome of this was that carbon became trapped at the ferrite / ferrite boundaries, increasing the carbon : metal atom ratio of the carbides that formed there, allowing them to persist in the structure to longer times.
Table 5.1 summarises the types and locations of carbides identified in the various starting structures.
| Locations | Transformation Structures |
|------------------------------------------------|---------------------------|
| | Ferrite / Pearlite | Ferrite / Bainite | 100% Bainite |
| Ferrite Intra-granular Regions | $M_2C$ | $M_2C$ | n.a. |
| Pearlite Intra-granular Regions | Lamellar alloy carbides | n.a. | n.a. |
| Bainite Intra-granular Regions | n.a. | $M_7C_3$, $M_{23}C_6$ | $M_{23}C_6$ |
| Ferrite / Ferrite Grain Boundaries | $M_{23}C_6$ | $M_7C_3$ | n.a. |
| Ferrite / Pearlite Grain Boundaries (prior $\gamma$) | $M_{23}C_6$ | n.a. | n.a. |
| Ferrite / Pearlite Boundaries (not prior $\gamma$) | Not delineated by carbides | n.a. | n.a. |
| Ferrite / Bainite Grain Boundaries | n.a. | $M_{23}C_6 + M_6C$ | $M_{23}C_6 + M_6C$ |
| Bainite / Bainite Grain Boundaries | n.a. | n.a. | $M_{23}C_6 + M_6C$ |
**Table 5.1:** Type and location of carbides detected in the three starting structures investigated in the current study.
5.1.3 Basic Mechanical Properties
In light of the evidence given above for the microstructural variables in the three structures studied, the variation in room temperature and elevated temperature properties can now be discussed.
The room temperature hardness and strength of the fully bainitic structure were superior to those of the ferrite containing structures as anticipated. Bainite, and in this particular case upper bainite, is rich in carbon some of which is held interstitially in solid solution and some precipitated out as alloy carbide. The strengthening in such structures results from the strain fields around the interstitial C sites, and precipitate structures, reacting with mobile dislocations, [23]. The effectiveness of precipitates acting as barriers to dislocation movement is dependent on their size which is in turn controlled by Ostwald ripening. In the fully bainitic starting structure the carbides were only in the early stages of evolution and the structure was, therefore, relatively hard and strong.
The room temperature hardness and strength of the ferrite / pearlite and ferrite / bainite structures were similar. This result could not be anticipated from the explanation for strengthening mechanisms given above for bainite as it would be expected that the faster cooled bainite transformation product in the ferrite / bainite structures would be harder and stronger than that of the pearlite in the ferrite / pearlite structure. The grain size of the two structures, however, must also be taken into consideration as this has a direct effect on these properties.
The well known Hall – Petch relationship, as described by Honeycombe [23], shows that the yield strength, $\sigma_y$, increases linearly for a decrease in grain diameter and it is this factor that was considered to be dominating the tensile behaviour in this case. The smaller grain-size of the ferrite / pearlite structure led to an increase in the number of barriers to dislocation movement per unit length of material. The carbide size and distribution at the grain boundaries may also have had an effect on the fracture stress but this was regarded as being of only minor significance here.
The elongation to breaking point was found to be similar in each structure indicating the relative insensitivity of the test to any variation in grain boundary carbide morphology present at this stage.
At 550°C the results of the tensile results were quite different to those of the room temperature tests. The ultimate tensile strength of the bainitic containing structures reduced significantly compared to that of the ferrite / pearlite structure, that became the stronger material at this temperature. This effect was considered, predominantly, to be due to the loss of carbon from the interstitial solid solution sites in the bainite and, secondly, to the growth of existing carbides at the elevated temperature. The ferrite / pearlite structure, being closer to the equilibrium state for the alloy, did not suffer a similar loss in strength, the advantage of the fine grain structure being dominant.
The elongation to breaking point at the elevated temperature was surprising in that the values obtained for all three structures were lower than those obtained at room temperature. This result was also found in tests carried out on this alloy by other authors [143] [144] but was not explained. It may be possible that an increase in the size of the carbides at the grain boundaries, due to the elevated temperature, had an effect on the fracture initiation and growth process but there is insufficient evidence to substantiate this at this time.
It became apparent that even when studying the basic mechanical properties of 2.25%Cr-1%Mo steel, variations in grain-size, transformation products and carbide type and distribution make trends in behaviour very difficult to establish.
5.2 Tempering Characteristics and the Carbide Evolution Process in 2.25%Cr-1%Mo Steel.
To gain an understanding of the carbide evolution process in this alloy a tempering trial was carried out on the fully bainitic structure generated for this
study. It was necessary to achieve this understanding in order to assess the effect of a tri-axial state of stress, as exists ahead of a pre-existing defect, on the microstructural evolutionary process in a power plant component.
Although it was appreciated that the evolutionary process may vary from structure to structure, the fully bainitic structure was selected for the tempering trial on the basis that it possessed, at some stage during tempering, all of the carbides types reported to exist in this alloy. The trial consisted of tempering normalised 2.25%Cr-1%Mo steel for various times and temperatures and examining the resultant microstructures and hardness values.
Vickers hardness values were determined for all the specimens tempered in the trial and plotted concurrently on charts showing the change in hardness with time at temperature and against the Holloman-Jaffe parameter, as described in section 4.3.1
The similar trends in softening apparent for the specimens tempered at all three tempering temperatures suggested that the same softening mechanisms were occurring but at different rates dependent upon the temperature. The largest reduction in hardness was observed in the first 0.5 hours of tempering for all specimens and was the greatest for the specimens tempered at 750°C, as indicated in Figure 4.2, p112. There was no evidence of a peak in the hardness values due the secondary hardening as described for the study on this alloy by Baker and Nutting [17]. As the softening mechanisms were considered to be consistent for all specimens tempered at the various temperatures the remainder of the discussion concentrates on the specimens tempered at 700°C only.
A qualitative assessment of the tempered specimens in terms of their general microstructure, carbide size and distribution was made by SEM examination. The carbide evolution process, however, was assessed by setting up an image analysis programme in the STEM and characterising the carbides in terms of
their size and type. The carbide classification scheme in this trial was based on the key elemental constituents in the carbides.
The microstructure of the bainitic starting structure for the tempering trials was that of the as normalised 2.25%Cr-1%Mo alloy. The carbides present between the upper bainitic, ferrite, laths were Fe and Cr rich and, therefore, believed to be either $\varepsilon$ - carbide or Fe$_3$C as described in the study of Baker and Nutting [17]. The intra-lath regions were free of any precipitation.
After tempering the bainite structure for only 0.5 hours at 700°C a distribution of both small, uniformly dispersed, carbides and larger, grain boundary and lath boundary, carbides were evident throughout the structure. The majority of the small carbides were found to be Fe rich with some contribution from Cr that identified them as M$_3$C type carbides. The remainder of the small carbides were identified as M$_2$C type by their lenticular shape and Mo / Cr rich composition. The large carbides were observed to be either rod-like, trapezoidal or globular in shape and analysed to contain a range of Fe, Cr, and Mo concentrations. This evidence was consistent with these larger carbides being either M$_7$C$_3$ or M$_{23}$C$_6$ type.
The results of the image analysis routine on the carbides extracted from the 0.5 hour sample, as shown in Figure 4.4, p121, indicated that the most abundant carbides at this very early stage of tempering were M$_7$C$_3$ in nature with a significant presence of M$_{23}$C$_6$ carbides. The fine dispersion of M$_3$C and M$_2$C carbides represented only a small percentage of the total of carbides detected in this sample. M$_6$C carbide, the equilibrium carbide in this alloy, was not evident at this stage of tempering.
After tempering for 5 hours at 700°C the image analysis results, as shown in Figure 4.5, p122, indicated that the small M$_3$C carbides had already dissolved at the expense of the other carbides and that M$_{23}$C$_6$ was now the most abundant carbide. A slight increase in the amount of M$_2$C was detected at this
stage of tempering but the quantity of $M_7C_3$ had decreased. Again no evidence for the evolution of the $M_6C$ carbide was found in this sample.
The simultaneous dissolution of $M_7C_3$ and precipitation $M_{23}C_6$ supports the theory that $M_{23}C_6$ grows at the expense of $M_7C_3$. Although the mechanism for the $M_7C_3 - M_{23}C_6$ process, in this study and from literary evidence, is unclear the most support appears to be for the theory that $M_{23}C_6$ precipitates independent of the $M_7C_3$ carbides but may only do so when sufficient Cr has been released back into solution during the dissolution of the $M_7C_3$ [32].
After 50 hours the quantity of $M_7C_3$ carbides remained approximately the same but their size was diminishing as indicated by a comparison of the graphs depicted in Figures 4.6, p123, and Figure 4.8, p125. In contrast the incidence of $M_{23}C_6$ type carbides was on the increase with many more small carbides of this type being detected. The number of large $M_{23}C_6$ carbides, greater than 0.3 $\mu m$ in diameter, however, appeared to be decreasing at the expense of the evolving equilibrium carbide, $M_6C$.
As confirmed at a later stage of the study when analysing the creep tested specimens, and as reported by previous authors [17] [29] [30] [138], $M_6C$ is found to evolve from other carbides, predominantly $M_2C$ in the pro-eutectoid ferrite and $M_{23}C_6$ in bainite. The evolutionary process takes place by the gradual absorption of Mo from the surrounding regions and at the expense of the smaller non-equilibrium carbides. Hence, the significant reduction in the number of $M_2C$ type carbides detected at this stage is likely to be a result of its dissolution at the expense of the larger $M_{23}C_6$ and $M_6C$ carbides.
The growth of the larger $M_{23}C_6$ and $M_6C$ carbides at grain boundaries and sub-boundaries was particularly evident in the tempered microstructures observed after extended times and at higher temperatures, for example; after 1000h at 700°C, as shown in Plate 4.19, p118, and after 50 hours at 750°C, as shown in Plate 4.23, p120. The carbides at the grain boundaries were observed to grow in an irregular manner and at the expense of carbides in the adjacent
grains. Their growth rate appeared to be faster than those of like-carbides in intra-granular regions of the structure due to the increased rate of atom diffusion at the grain boundary interface.
After 5000h at 700°C only a small percentage of M\textsubscript{23}C\textsubscript{6} carbides remained in the structure in addition to the equilibrium carbide M\textsubscript{6}C, that had taken on a more rounded morphology as shown in Plate 4.21, p119. These remaining carbides existed almost exclusively at the prior austenite and recrystallized grain boundaries.
The reduction in hardness trend described previously for the tempered specimens can now be related to the change in microstructure. The initial rapid decrease in hardness coincided with the rapid dissolution of the ε-carbide / M\textsubscript{3}C carbide and the corresponding loss of carbon and other alloying elements from solution by the precipitation of M\textsubscript{2}C, M\textsubscript{7}C\textsubscript{3} and M\textsubscript{23}C\textsubscript{6} type carbides. Subsequent softening, considered to be due to a further loss in solid solution hardening elements from the matrix and growth of the alloy carbides, occurred at a much slower rate but continued gradually up to extended times when M\textsubscript{6}C, the equilibrium carbide, was fully formed.
In the tempering trials on 2.25%Cr-1%Mo steel carried out by Baker and Nutting [17] the bainite region in the normalised steel was shown to soften rapidly and then slow down immediately prior to the precipitation the M\textsubscript{7}C\textsubscript{3}. Once precipitation of the M\textsubscript{7}C\textsubscript{3} had taken place the softening rate increased again. This may have been due to an effect of the carbon from the M\textsubscript{3}C going back into solid solution and for a short period of time forming solute pairs with Cr prior to the precipitation of the Cr rich carbide.
In the well publicised carbide precipitation sequence for normalised 2.25%Cr-1%Mo steel, by Baker and Nutting [17], M\textsubscript{2}C and M\textsubscript{7}C\textsubscript{3} were shown to persist for significantly longer times than those reported here. This anomaly is due to the fact that the normalised structure in the Baker and Nutting study was that of 55% ferrite and 45% bainite, and not 100% bainite as in this case. M\textsubscript{2}C is
known to persist in ferrite for extended times and eventually to dissolve only at the expense of the equilibrium carbide, $M_6C$. The existence of pro-eutectoid ferrite in the normalised structure also meant that the resulting bainite was enriched in carbon and that this, in turn led, to the stabilisation of $M_7C_3$ to longer times.\textsuperscript{[30]} \textsuperscript{[32]}.
5.3 Creep Deformation in the Presence of a Pre-existing Defect
To compare the creep deformation response of the three structures selected for this investigation, CT specimens were tested under creep conditions and the material ahead of the spark machined fine notch subsequently assessed in terms of ‘damage’ accumulation.
The deformation of the CT specimen was monitored by measuring the physical displacement of hardness indentations made parallel to the notch, in the mode of Holdsworth and Cunnane\textsuperscript{[19]}, and the mechanisms of damage accumulation were assessed by qualitative and quantitative microstructural examination of the notch tip material after removing the specimens, sequentially, from test.
Testing of the ferrite / pearlite specimens was carried out at the collaborating establishment and, as a consequence, only micro-samples prepared from the tested specimens were available for further investigation. The ferrite / bainite and fully bainitic specimens were tested in house utilising a creep rig specifically designed for this project, as described in section 3.4.3(a). These specimens were, therefore, fully assessed by all the techniques described in section 3.6.
5.3.1 Creep Damage Response
When a creep load is applied to a body containing a defect, the stress intensity at the defect tip is dependent upon a number of factors\textsuperscript{[14]} \textsuperscript{[95]}.
i) the load transmitted to the defect tip
ii) the defect tip profile
iii) the constraint exerted at the defect tip by the surrounding material.
In the creep tests carried out in this study the load was applied to the specimens by two diametrically opposite loading pins either side of a machined notch, as shown in Figure 3.4, p73, and the load was transmitted along the load line to the notch tip simulating the defect. The ability of the material surrounding the defect tip to resist creep crack initiation and growth describes the materials creep ductility where the greater the notch opening to creep crack length ratio the more ductile the situation [86].
Evidence from the COD evaluation for the 100% bainite and mixed ferrite / bainite CT specimens gave an early indication that the ferrite / bainite structure was more creep ductile than the fully bainitic structure. Examination of the COD curves, depicted in Figure 4.15, p132, and Figure 4.17, p133, showed an almost linear relationship for the displacement of the fully bainitic specimens and a non-linear load line / displacement relationship for the ferrite / bainite specimens. This difference in deformation behaviour highlighted the greater ductility of the mixed ferrite / bainite structure. It also confirmed the observation of Holdsworth et al [19] that, values of CTOD determined from load point displacement measurements, as opposed to physical displacement measurements at the crack tip, are inaccurate for materials exhibiting a high creep ductility.
As described in the literature, section 2.4.2, the application of a steady load to a pre-cracked creep ductile structure at high temperature leads to the progressive build up of a damage zone ahead of the crack tip, subsequent crack propagation and eventually failure of the remaining ligament. The spread of the damage zone, prior to crack initiation, is dependent upon the ability of the material ahead of the defect to redistribute the stress intensity [124] [125] [126] that, in turn, is dependent upon the elastic – plastic characteristics of the material and the degree of notch constraint.
As the geometry of the CT test specimens was the same for all the three microstructural variables studied the constraint due to this factor could be considered equivalent. The test temperature in each case was 550°C and, therefore, the only inconsistency between the specimens, that was non-microstructural, was that of the applied stress used to induce crack initiation in less than 1000 hours, as detailed in section 3.4.3b.
Although it was recognised that the difference in applied stress, and hence the creep deformation rate, may have had an effect on the stress redistribution at the notch tip, it was considered that the observations recorded from the creep tested material were still relevant and significant to the determination of the mechanisms of creep crack initiation and growth ahead of a pre-existing defect in the alloy.
As described by Reidel [40], a crack may be perceived to have initiated at the absolute minimum when only two adjacent cavities coalesce or, on the other hand, as described by Holdsworth [11], when the crack is detectable by non-destructive testing techniques, that is approximately 0.2mm for the electrical potential drop method. In this study a crack was considered to have initiated if grain boundary cavity coalescence spanned more than one grain. Initiation was then considered to be in progress up to 1mm of detectable crack extension, after which the crack was considered to be propagating.
The creep damage analysis results for the 100% bainite, ferrite / bainite and ferrite / pearlite CT specimens were detailed in sections 4.4.2(a)–(c) respectively.
Comparison of the SEM micrographs and void analysis data for the three microstructures showed that the propensity for cavitation in the notch tip region varied from structure to structure.
In the notch tip region of the fully bainitic structure cavitation, and subsequent crack initiation, was constrained predominantly to an area immediately ahead of the notch tip and into the remaining ligament as shown in the SEM image.
and corresponding void analysis chart, Figure 4.20 and Plate 4.26, p141. In the ferrite / bainite structure less constraint was apparent and the damage zone was more widely dispersed, Plate 4.35 and Figure 4.24, p152.
In both the fully bainitic and ferrite / bainite structures cavities were found to have initiated on grain boundaries that were oriented at approximately $45^\circ$ to the notch tip, or principal stress axis, and the area % of cavities was observed to increase with increasing test duration. High resolution, SEM, secondary electron imaging of the cavitating boundaries indicated that cavitation was nearly always associated with the presence of large, and quite often elongated, carbides. Once initiated, the cavities were observed to extend along the interface between the initiating grain boundary carbide and one of the adjacent grains, suggesting a weakness or loss of coherency of the carbide with matrix.
When carbides nucleate and grow at the interface between two adjacent grains they do so by maintaining a degree of coherency with one of the grains, and in the absence of an applied stress, grow preferentially into the other with which the coherency is low. In the two bainite containing structures, the carbides on the boundaries oriented at $\sim 45^\circ$ to the principal stress axis were observed to grow preferentially along the boundaries and the explanation for this phenomenon lies in the consideration of paths of easy diffusion.
As indicated in Plate 4.28, p144, for the fully bainitic structure, growth of the grain boundary carbides appeared to be occurring at the expense of other carbides in the adjacent grain by atom diffusion down the original, bainitic, lath boundaries. However, rather than the grain boundary carbide growing into an adjacent grain, growth appeared to occur along the line of least resistance, i.e. along the grain boundary. The grain boundaries oriented at $45^\circ$ to the principal stress axis in the CT specimen were subjected to the maximum shear stresses in the test specimens and, as such, were attempting to slide, thereby acting as a source of dislocations. The dislocation structure, thus created, then acted as an ideal path for atom redistribution, or vacancy migration.
In the mixed ferrite / bainite structure cavitation was rarely observed on the ferrite / ferrite boundaries, even though elongated carbides were found to occur on the boundaries oriented at $45^\circ$ to the principal stress axis. This phenomenon was considered to be due to the increased stability of the $M_7C_3$, detected at these boundaries compared with that of $M_{23}C_6$, $M_{23}C_8$ showing a greater propensity to evolve to the equilibrium carbide, $M_6C$.
Analysis of the void orientation results for the two bainite containing structures showed that although the creep voids occurred on boundaries oriented at $45^\circ$ to the principal stress axis the highest frequency of voids detected was for those oriented at angles between $\pm 50^\circ$ to $60^\circ$. It was considered that this angle was that related to the longest, feret, diameter of the newly initiated voids, i.e. the angle of the void at the cusp of the carbide, as described by Evans and Wilshire [50].
In the case of the ferrite / pearlite structure an increase in the area % of cavities with test duration was not found for the limited number of ferrite / pearlite specimens examined. The ferrite / pearlite specimen tested at 85MPa for 3689 hours showed extensive cavitation in the notch tip region, as indicated in Plate 4.45 and Table 4.24, p161, but the structure resisted crack initiation by cavity coalescence. In contrast, the ferrite / pearlite sample tested at 85MPa for 6850 hours showed little cavitation in the area ahead of the notch, however, a crack measuring $\sim 0.08\text{mm}$ was found to have initiated, as shown in Plate 4.46, p162. Examination of this crack, using high resolution secondary electron imaging in the SEM and complementary EDX analysis, revealed that it was associated with the presence of a large sulphide inclusion that appeared to have caused its initiation. In a microstructure such as this, that allows gross cavitation to take place without extensive coalescence, it was considered that the incoherent interface between the sulphide and the matrix may have acted as a sink for stress induced dislocations rendering the surrounding material relatively stress and cavity free. This evidence also highlights the importance of fine microstructure on the creep properties of a material containing a defect.
in that a large inclusion, under the action of a triaxial state of stress, may nucleate a crack prematurely.
In contrast to the ferrite / bainite and fully bainite specimens, that exhibited cavitation on boundaries that were approximately 45° to the principal stress axis, cavitation in the ferrite / pearlite specimens took place on grain boundaries which were oriented at much lower angles, the angle of the first formed voids being ~ 30°-40° with respect to the notch tip. This difference in orientation could not be explained mechanistically but it was considered that it may have been due to a preferred orientation effect resulting from the manufacture of the pipe material from which it was derived.
A further difference in the creep deformation behaviour between the two bainite containing structures and the ferrite / pearlite structure was the apparent mechanism by which the initiated grain boundary cavities appeared to be "growing". Whilst cavity growth and crack extension in the aforementioned structures appeared to be a result of concurrent decohesion of elongated carbide / matrices and subsequent coalescence; in the ferrite / pearlite structure cavity growth was considered to be a result of ductile tearing along the carbide / matrices interface.
Due to the lack of data available for ferrite / pearlite specimens the final correlation between the void area %, CTOD and crack extension results was made only for the ferrite / bainite and 100% bainitic microstructures.
A comparison of the behaviour of the two bainite containing structures was made by plotting the creep deformation results on the same axes as shown, and described, in section 4.4.3. The results plotted for the CTOD against crack extension data showed that a CTOD value of ~0.3mm gave crack initiation of 1mm in both bainite containing structures, Figure 4.31, p173. The time to generate this degree of crack extension, however, was found to be only ~400 hours in the fully bainitic structure tested at the lower stress of 100MPa, compared to ~1000 hours in the mixed structure tested at 110MPa.
In the preliminary stages of initiation, i.e. up to 0.5mm of crack extension, cracking was found to proceed for a lesser degree of voiding and CTOD in the bainitic structure than in the ferrite / bainite structure as shown in Figure 4.30a, p172, and Figure 4.31b, p173, respectively. The values of $\Delta a/\Delta$CTOD and $\Delta a$/void area % in this period give an indication of the constraint and, hence, creep ductility of the microstructure in the vicinity of the defect tip and have, therefore, been determined and displayed below in Table 5.2 for the two bainite containing structures. The larger the ratio value the greater the microstructural constraint and the lower creep ductility.
| Transformation Structure | Ratio Values Determined Upon the Attainment of 0.5mm Crack Extension |
|--------------------------|-----------------------------------------------------------------------|
| | $\Delta a/\Delta$CTOD | $\Delta a$/void area % |
| 75% Ferrite / 25% Bainite | 1.85 | 0.21 |
| 100% Bainite | 2.78 | 2.0 |
Table 5.2: Values of $\Delta a/\Delta$CTOD and $\Delta a$/void area %, determined from the ferrite / bainite and 100% bainite CT specimens at a crack extension of 0.5mm.
All the above factors confirm the superior creep ductility of the mixed structure up to a critical CTOD of ~0.3mm. Once this critical value has been obtained crack extension in both structures appeared to progress at a similar rate, Figure 4.29b, p171.
The difference in the deformation behaviour of a material exhibiting either a ferrite / bainite or a fully bainitic microstructure in the presence of a triaxial state of stress was also studied by Gooch and King [96]. In this study on a 0.5%Cr-0.5%Mo-0.25%V steel a microstructure consisting of 100% tempered bainite was found to exhibit a crack extension to COD ratio, ($\Delta a/\Delta$COD), three times the value of that determined for a 15% bainite + pro-eutectoid ferrite. It was considered that, although this observation supports the findings of the
current study, the deformation effects in the work of Gooch and King were exaggerated by the method used for evaluating the COD, i.e. displacement at the loading pins and not physical displacement measurements at the crack tip as in this work.
5.3.2 The Effect of Microstructural Variables on the Creep Crack Initiation and Growth Process
In this section the relationship between the microstructural evolution and the onset of creep crack initiation and growth will be considered for the three microstructural variants investigated.
The difference in the early deformation response of the microstructural variables was considered to be the key to understanding the mechanisms of creep damage in the presence of a pre-existing defect in this alloy.
When a defect is present in a large component operating under creep conditions the stress intensity at the defect tip is dependent upon the ease with which that stress can be redistributed into the surrounding material, i.e. dependent upon the constraint of the material. With reference to the deformation map of Maruyama [67] it was considered that, in the temperature and stress regime relevant to this study, creep stress redistribution was related to the ease that dislocations could glide through the structure and bypass inherent obstacles.
In the microstructures studied, dislocation mobility was restricted by interaction with solid solution atoms, precipitates, other dislocations and grain boundaries and was released by diffusion of atoms, by dislocation climb, leading to power law creep. At elevated temperatures a further degree of freedom to deform was activated by the ability of the grain boundaries to slide past one another.
In the presence of grain boundary carbides, sliding can only occur if there is sufficient driving force for atoms in the vicinity to circumnavigate the obstructing carbide. The driving force for this to occur is provided by the heat in the system which is, generally, less for movement of atoms down dislocation cores, (Coble creep), than diffusion through the bulk, (Nabarro-Herring creep).
From a consideration of the above factors, the following microstructural aspects were regarded to be of importance to evaluate the creep behaviour of the material under investigation in this study:
- The elements in solid solution in the pro-eutectoid ferrite, pearlite and bainitic regions of the structures.
- The chemistry, morphology and distribution of the carbides in the intra-granular regions of the pro-eutectoid ferrite, pearlite and bainite.
- The chemistry, morphology and distribution of the carbides at the ferrite / ferrite, ferrite / pearlite, ferrite / bainite and bainite / bainite grain boundaries.
- The presence, or absence, of precipitates in the zones immediately adjacent to the grain boundaries in the structures.
The resistance to dislocation movement in the intra-granular regions of the structures will be considered first.
By comparing the microstructural constituents of the ferrite / pearlite structure with that of the ferrite / bainite structure and, in turn, the ferrite / bainite structure with that of the 100% bainite structure, the contribution of each phase region to the creep resistance of the alloy could be assessed.
The main difference in the creep response of the ferrite / pearlite and ferrite / bainite structures was attributed to the relative maturity of the carbides in the pearlite and bainite phase regions. The slower cooling regime that produced the ferrite / pearlite structure allowed the majority of the alloying elements in
that structure to precipitate out as alloy carbides, reducing the solid solution strengthening capability and leaving it susceptible to creep deformation. The presence of the faster cooled bainite phase regions in the ferrite / bainite structure were comparatively creep resistant, the carbides existing there being in a less mature state.
Similarly, the significant difference between the two bainite containing structures studied was the presence of pro-eutectoid ferrite grains in the mixed structure.
In terms of the passage of dislocations through the bainite containing structures the presence of the ferrite phase may have had two distinct effects:
i) To impart a restriction to dislocation movement in the ferrite by the precipitation of a fine distribution of $M_2C$.
ii) To cause, during its formation, an increase in the saturation of carbon in the remaining austenite resulting in additional solid solution and/or precipitation hardening in the, finally formed, bainite.
In the starting condition these structures were in a tempered state and the carbides had already undergone a degree of evolution with respect to original transformation structures.
$M_2C$ was the only carbide found to exist in the ferrite of the mixed structure but it had almost completely disappeared at the expense of $M_7C_3$ and $M_{23}C_6$ type carbides in the bainitic regions. If restriction of dislocation movement by the $M_2C$ precipitates was the dominant strengthening mechanism, as has been described for many long term tests, the ferrite / bainite specimens would have been more resistant to creep deformation than the fully bainitic specimens. This was not the case, the ferrite / bainite specimens exhibiting the greater creep ductility.
In addition to the presence of the pro-eutectoid ferrite another difference between the two bainite containing structures was identified. The bainite in
the mixed structure was found to consist of a more dense dispersion of carbides and, as mentioned previously, a higher $M_7C_3 / M_{23}C_6$ carbide ratio than that in the fully bainitic structure. This was evident from the TEM images for the ferrite / bainite and fully bainitic structures, as shown in Plate 4.12, p107, and Plate 4.14, p108, respectively. The variation in the amount of each carbide type present and the increased density of carbides was attributed to the fact that the bainite in the mixed structure was enriched in carbon with respect to that in the fully bainitic condition. The fact that the additional carbon in the bainite of the mixed structure appeared to have precipitated out, as indicated by the greater density of carbides in the bainite of this structure, suggests that its subsequent effect on interstitial solid solution hardening would be minimal, however, an effect of precipitation strengthening may have occurred.
The principle difference in the response to a creep stress of the two bainite containing structures, then, appears to be the percentage of bainite present.
Two strengthening mechanisms may be operable in the bainite, that is:
i) precipitation strengthening from the alloy carbides and
ii) solid solution strengthening from the C, Cr and Mo in solution.
It has been reported by Kleuh [16] and Jones [64] that Cr and Mo may form solute pairs with C leading to dislocation drag and, hence, creep strengthening. In the relatively short term tests of the current study, i.e. where the applied stress was relatively high, this was considered to be a likely mechanism for the strengthening as a substantial amount of C, Cr and Mo still existed in the solid solution. The argument against this theory may be that if a substantial amount of Mo was still present in the solid solution then $M_2C$ would naturally precipitate next as a consequence of this. This was not the case. In this study $M_2C$ was shown to be virtually absent in the bainite after the tempering treatment and prior to creep testing. It was considered unlikely, from existing knowledge, that Cr alone would have this strengthening effect and, therefore, it was believed that in the bainite $M_2C$ dissolved early in the
tempering process, re-enriching the solid solution in Mo, from where it was gradually absorbed by the pre-existing $M_{23}C_6$. Finally when the Cr in the matrix was depleted the continuing enrichment of Mo led to the evolution of the $M_6C$ equilibrium carbide. This enrichment behaviour was observed in the final stages of evolution in this study during the investigation of the grain boundary carbides of the creep tested specimens.
Comparison of the Fe:Cr, Mo:Cr and Mo:Fe elemental ratios for the evolving carbides depicted in Plate 4.59, p181, Plate 4.61, p182, and Plate 4.64, p184, indicated how the carbide could exist in a transition state between the equilibrium compositions of the $M_{23}C_6$ and $M_6C$ carbides. The Fe:Cr and Mo:Cr elemental ratios in the evolving carbides were shown to gradually increase indicating the gradual enrichment of the evolving carbide in molybdenum.
In addition to the solid solution strengthening in the bainite, it was considered that strengthening may also have been afforded by the $M_7C_3$ and $M_{23}C_6$ precipitates. Compared to the $M_2C$ in the ferrite these carbides were relatively coarse, however, it is known that optimum creep strengthening is not always afforded by a fine dispersion of precipitates but, “rather by a somewhat over-aged structure”.\textsuperscript{[6]}.
In summary, dislocation mobility in the intra-granular regions of the specimens tested was considered to be limited by:
i) Mo, Cr and C atoms in solid solution, possibly at atom clusters \textsuperscript{[5]}\textsuperscript{[16]}\textsuperscript{[65]}.
ii) $M_2C$ precipitates in the pro-eutectoid ferrite.
iii) $M_3C$, $M_2C$, $M_7C_3$, $M_{23}C_6$ precipitates in the pearlitic and bainitic regions.
Once freed from barriers, dislocations were able to glide through the structure until they impinged upon a grain boundary. At boundaries the dislocations then piled up creating a stress that may have been sufficient to cause grain boundary sliding and, or, initiation of mobile dislocations in adjacent grains; and so the plastic zone propagated.
Resistance to grain boundary sliding during creep was dependent on two main factors:
i) grain boundary carbide pinning
ii) stress accommodation by precipitate free zones
As described previously in section 5.1.2, large carbides existed, in most cases, at the grain boundary sites of the three starting microstructures investigated in this study. The exception to this was at the ferrite / pearlite boundaries that did not coincide with those of the prior austenitic structure. The effect of these 'pinning' carbides was to resist the sliding motion exerted by the shear stress acting ahead of the notch in the CT specimens. Once the carbides, or the interface between the carbides and the matrix, could no longer support the stress, sliding occurred eventually leading to the onset of cavitation and creep crack initiation. This process has been reported to occur in bursts [40] due to the time taken for a stress concentration to build up at the interface between the grain boundary carbide and slip band within the grain.
In a creep study by Cane and Fidler [22] on uniaxial test specimens in 2.25%Cr-1%Mo alloy steel it was noted that a significant amount of shear took place at grain boundaries during creep. This deformation mechanism was observed to appear, occasionally, as pure grain boundary sliding but also, more generally, as deformation in precipitate free zones (PFZ's) adjacent to the boundaries. Cavity density was shown to decrease with increasing mean PFZ width suggesting local stress accommodation by these zones.
Evidence of PFZ's were observed in this study in the microstructures of the fully bainitic and ferrite / bainite CT tested specimens, as shown in Plate 4.29, p144 and Plate 4.41, p157, respectively. The width of these zones was found to be narrow, $\sim 1 \mu m$, compared to those affording low cavitation densities in the study by Cane and Fidler. It is believed, therefore, that although the stress at the grain boundaries may have been briefly accommodated by the PFZ's most of the stress induced will have been acting directly on the grain boundary itself, and, in particular on the carbide / matrix interface. The resistance to grain
boundary sliding, then, was effected by the presence of the alloy carbides $M_7C_3$, $M_{23}C_6$ and to a lesser extent, $M_6C$.
The types of carbide present at the boundaries between the various microstructural regions of the specimens were dependent upon the local concentration of elements either side of the boundary and the temperature and duration of the elevated temperature exposure. No literary evidence was found to suggest that the carbide type evolving during the creep process is affected by the mode or size of the stress field acting on the grain boundary.
The carbides present at the grain boundaries of the starting structures were, as described above in section 5.1.2, predominantly $M_{23}C_6$ at ferrite / pearlite, ferrite / bainite and bainite / bainite boundaries and $M_7C_3$ or $M_{23}C_6$ at ferrite / ferrite boundaries. Both these carbides are believed to be effective at preventing grain boundary sliding provided that they remain semi-coherent with the adjacent grains. At extended times at elevated temperatures, as demonstrated in the tempering trial, these carbides tend to evolve to the equilibrium carbide $M_6C$, at which time, it has been reported [28], that coherency with the matrix is lost.
5.3.3 The Contribution of Grain Boundary Carbides to the Creep Crack Initiation Process
To ascertain the reason why some carbides, on critically oriented boundaries, initiated creep cavities a TEM investigation was instigated. Carbide replicas, extracted from the 100% bainitic specimen tested for 350 hours and the mixed ferrite / bainite specimen tested for 850 hours, were examined to identify any differences in the composition, morphology or distribution of carbides on the grain boundaries in both notch tip and reference regions of the specimens. Particular attention was paid to those carbides on the grain boundaries orientated at $\sim45^\circ$ to the principal stress axis in the notch tip specimens.
As indicated in the SEM study on these structures, the majority of voids detected were found to have initiated at carbides present at either bainite / bainite or ferrite / bainite grain boundaries. It was also evident from this study that, the carbides associated with cavitation were often elongated in the direction associated with grain boundary sliding. It was with this knowledge that the carbides of interest were located for examination in the TEM.
EDX analyses taken from several positions along representative elongated carbides, as indicated in Plate 4.61, p182 and Plate 4.64, p184, showed that the carbides were composed of a number of $M_{23}C_6$ carbides that had grown along the boundary and evolved, at their extremes, to the equilibrium carbide $M_6C$. The additional elements required for the evolutionary process were made available from the dissolution of carbides in the adjacent grains, as indicated by the presence of PFZ’s described previously.
Grain boundary carbides not oriented at a critical angle with respect to the notch tip, and those analysed in the reference regions, were also found to be evolving to $M_6C$ but with less propensity to grow in the direction of grain boundary. For example, the carbides depicted in Plates 4.59, p181 and Plate 4.65, p185 show growth and evolution by dissolution and absorption of the adjacent carbides but with markedly less directionality. In fact, the carbides growing on these non-critical boundaries were observed to follow the growth mechanism expected, i.e. that of protrusions into the adjacent grains as described by Honeycombe [23] and Benevenuti et al [145].
This evidence shows that it is the grain boundary sliding action that encourages growth of the grain boundary carbides along the boundary, probably due to the increased driving force for diffusion imparted by the atomic disorder created there. By implication then it can be stated that, the effect of the triaxial state of stress in the creeping material was to increase the grain boundary sliding element, thereby, encouraging growth of the grain boundary carbides in the direction of the sliding.
No evidence was found to suggest an increase in the frequency of the M\textsubscript{23}C\textsubscript{6} - M\textsubscript{6}C transformation due to grain boundary sliding, the numbers of both carbides being similar in the notch tip and reference regions.
Although elongated carbides were found to exist at critically oriented ferrite / ferrite boundaries no evidence of the equilibrium carbide was found. These carbides were determined to be M\textsubscript{7}C\textsubscript{3} and, as described for the starting structure, appeared to be more resistant to the evolutionary process if formed between two ferrite grains.
From the evidence presented, the onset of grain boundary cavitation, ahead of a pre-existing defect, appears to be related to both the M\textsubscript{23}C\textsubscript{6} – M\textsubscript{6}C evolutionary process and the growth of the carbide along the sliding grain boundary.
From theory it is known that, normally, precipitates grow in the direction of the interface with which there is least coherency. In the absence of grain boundary sliding then, the grain boundary carbides grew into one of the adjacent grains. Under the influence of sliding, however, due to the increased disorder, growth occurred along the boundary. As the majority of the structure was still resisting deformation by grain boundary sliding there was an increased stress concentration at the extremes of the elongated carbides and, when this coincided with the evolution to M\textsubscript{6}C, cavitation began. Once initiated, with increasing stress, the cavities associated with the evolving carbides were observed to coalesce and grow by dissociation of the carbide with one of the adjacent grains.
Evidence of large voids in the shape of pre-existing carbides, as shown in Plate 4.40, p156, were considered to be indicative of the poor coherency of the grain boundary carbides that caused cavitation.
5.4 Contribution to Life Assessment Strategy
The accurate prediction of component life-time and remaining lifetime necessitates knowledge gained from both accurate microstructural evaluation and mathematical manipulations based on fracture / continuum damage concepts. With this in mind it was considered that the information gained in this current study will aid lifetime assessment strategies in several ways;
i) To add to the knowledge base an appreciation of how certain microstructural variables can affect the degree of constraint at the notch tip of a pre-existing defect loaded in creep and to consider whether these variables have been accounted for, to date, in existing life-time prediction models.
ii) To propose the mechanisms of creep deformation occurring ahead of a pre-existing defect in 2.25%Cr-1%Mo steel so that the microstructural characteristics affecting these can be quantified to aid life-time predictions.
iii) To consider that the observation of the evolving microstructure in a power plant material may give an early indication for the onset of creep crack initiation and growth from a pre-existing defect.
Each of these contributions will now be considered in turn with a view to assessing the time for creep crack initiation and growth in components containing a pre-existing defect.
5.4.1 The Effect of Notch Tip Constraint and Life-Time Prediction
The assessment of creep deformation in CT specimens containing a simulated "pre-existing defect" has shown that the constraint at the defect tip varies with microstructure as described in section 5.3 above. The constraint imparted has
been shown to have a dramatic effect on the initial response of the material ahead of the defect tip in terms of its resistance to creep crack initiation.
The concept of local creep constraint has previously been addressed by Wang and Wu\textsuperscript{[101]} who defined a creep constraint factor $\xi$ where:
$$\xi = \left( 1 - \frac{\varepsilon_f^*}{\varepsilon_f} \right)^{(n-1)/n}$$
- Equation 5.1\textsuperscript{[101]}
And
$\varepsilon_f^*$ = local rupture strain with creep constraint
$\varepsilon_f$ = creep ductility in uniaxial tension
$n$ = material constant of the creep law
The authors demonstrated that if the value of $\xi$ can be determined for a particular material, based on the creep constant, 'n', and a particular specimen geometry, then, knowing the creep ductility value for uniaxial creep failure, the value of $\varepsilon_f^*$ can be determined and used in an equation for crack growth rate, $\dot{a}$.
Where:
$$\dot{a} = \frac{4C^{*0.85}}{\varepsilon_f^*}$$
- Equation 5.2\textsuperscript{[130]}
And
$$C^* = \frac{2n}{n+1} \frac{P\dot{\Delta}}{B(W-a)}$$
- Equation 5.3\textsuperscript{[130]}
Comparing calculated and measured values for $\varepsilon_f^*$ and, hence, crack growth rate, Wang and Wu \cite{101} showed that the equations postulated gave reasonably accurate predictions.
In the current study it is not the crack propagation rate that is being considered but the time to initiate a crack from a pre-existing defect. The mathematical criteria for estimating the constraint factor during the crack initiation, or incubation phase, therefore, may be different to that proposed by Wang and Wu and a further study is required to quantify this.
Due to the possibility of non-linear displacement between the load point and the crack tip, particularly in creep ductile materials, $C^*$ should be calculated from CTOD data as described in this work and by Holdsworth \cite{11} \cite{132}. In equation 5.3 the load point opening displacement rate, $\dot{\Delta}$, is replaced by the crack tip opening displacement rate, $\dot{\delta}$, up to the point of crack initiation, $\delta_{ix}$. If the crack length at crack initiation, defined as $x_c$, is then put equal to the minimum crack extension, $\Delta a$, the incubation period, $t_i$, becomes:
$$t_{i[x_c]} = \frac{\Delta a \varepsilon_f^*}{4C^{*0.85}}$$
To test the validity of this, and any mathematical models postulated for life-time prediction of components containing defects, it is necessary to carry out both uniaxial and notched specimen creep tests. From the evidence portrayed in this study it has been shown that creep performance is critically dependent upon the microstructure of the tested material and, therefore, to ensure accuracy when comparing test data, all test specimens must be extracted from micro-structurally equivalent material.
5.4.2 The Mechanisms of Creep Deformation Occurring Ahead of a Pre-existing Defect and Life-Time Prediction
In this study the mechanisms of creep deformation, up to the point of crack initiation, have been shown to be similar to those experienced in uniaxial creep specimens.
It has been shown that the variation in creep deformation behaviour of the three microstructural variables examined was due, predominantly, to the difference in:
a) the solid solution strengthening effects and
b) the grain boundary carbide stability.
In both cases the success of the resistance to deformation was related to the ability of the molybdenum in the alloy to stay in solid solution. Once the dislocations were freed from the strain field surrounding the solid solution elements they were free to migrate to the grain boundaries. At the boundaries sliding was initially restricted by grain boundary carbide pinning until such a time that the equilibrium carbide, $M_6C$, evolved. At this time it is believed that the effect of the triaxial state of stress acting on the $M_6C$ grain boundary carbide had a significant effect on the subsequent stage of deformation leading to earlier creep crack initiation times that would have been expected in uniaxial specimens.
For the important evolving microstructural variables to be built into mathematical models for life prediction it is necessary to link them to a suitable mathematical variable. As described by Dyson and McLean [113] this can be achieved by using continuum damage mechanics equations involving the use of $\dot{\varepsilon}$. Each parameter affecting $\dot{\varepsilon}$ can be summed to give the total life expectancy in the mode of Grounes and Liberman [111] [112]. In the case of 2.25%Cr-1%Mo steel, then, strain rate ahead of a pre-existing defect should be based initially on damage accumulation in the form of multiplication of mobile dislocations and/or particle coarsening and evolution to $M_6C$ at the grain boundaries. Using these parameters, the final total strain could be estimated.
for the material creeping in uniaxial tension and then this value could be used, subsequently, in equations for components containing a pre-existing defect, such as that proposed by Wang and Wu\textsuperscript{[101]}. Finally, once the effect of notch tip constraint had been determined, the time to initiate a creep crack, $t_{i_{XC}}$, could be estimated.
5.4.3 Carbide Evolution and Life-Time Prediction
A detailed microstructural analysis was carried out in this study to relate the change in carbide morphology in three microstructural variables of 2.2%Cr-1%Mo steel to the creep deformation and creep crack initiation process. It was found that in each case the mechanism of deformation and crack initiation was related to the evolutionary process of the carbides present at each stage. In particular it was related to the depletion of Mo from the solid solution in the bainite phase and, to a lesser extent, the precipitation strengthening of the fine M$_2$C precipitates in the ferrite.
With respect to the two bainite containing structures the dominant creep weakening mechanism was considered to be signalled by the growth of the inter and intragranular M$_{23}$C$_6$ carbides and their subsequent evolution to the equilibrium carbide, M$_6$C. Cavitation was nearly always associated with the decohesion of grain boundary M$_6$C carbides at their interface with an adjacent grain. Consequently any method which could predict the time to evolve M$_6$C at a particular temperature could aid in the prediction of creep life / remanent life.
Such methods have already been proposed and described by Bhadeshia et al\textsuperscript{[12]}\textsuperscript{[119]} based on thermodynamic calculations using programmes such as MTDATA.
From the experimental and literary evidence described in this study, a number of important factors affecting the validity of experimental studies in this area have been noted.
It has become apparent that the fine microstructure critically affects the properties of creep resistant alloys. As such any comparisons between results derived from various sources must recognise these differences. For example, the proportion of phases in the alloy can have a significant bearing on the distribution of carbon in the microstructure and, hence, the relative maturity of the precipitate structures within those phases. Reference stress values must be taken from uniaxial specimens having equivalent chemistries and thermal histories to those to be tested under complex loading conditions.
The effect of varying the volume fraction of transformation structures, and hence microstructural constituents, on the microstructural constraint imposed at the tip of a pre-existing defect could be assessed by carrying out a detailed creep testing and microstructural analysis programme.
It is suggested that for further work, CT and uniaxial creep specimens should be manufactured from material consisting of a range of pro-eutectoid ferrite/bainite contents. Reference stress, creep index and creep ductility's could then be determined from uniaxial test data and used/correlated to creep deformation data obtained from creep testing CT specimens containing spark eroded defects in exactly the same microstructural condition. From this study, the contribution of the various microstructural constituents could be assessed and quantified, subsequently, to be included in models for predicting creep or remanent lifetime.
In the assessment of void area percent, or cavitation damage, it is essential that all micro-specimens are prepared via the same routine. As described by
Silveira and May\textsuperscript{[135]} and Samuels and Le Mann\textsuperscript{[136]} variations in polishing or etching time can alter the size of the cavities detected and as such the value of the damage parameter determined.
In ductile materials local deformation at the crack tip of a notched specimen should be determined from the physical measurement of indentation marks made parallel to the crack opening front, i.e. CTOD measurements should be made by the similar triangles method described by Holdsworth\textsuperscript{[132]}. If the displacement between the loading pins is utilised for the determination of CTOD non-linear deformation results in values of CTOD which are artificially large.
As discussed by Abdel-Latif\textsuperscript{[39]} care must be taken when quoting absolute values for the elemental compositions of carbides analysed by EDX methods in the TEM. Due to the complexity of determining the necessary constants to quote absolute values characterisation of carbides in this study was made only by comparison of the elemental ratios present in the carbides with those derived from a series of experimental trials and by comparison with literary evidence\textsuperscript{[30]}\textsuperscript{[138]}. Accuracy was ensured by consistency in sample preparation and TEM operating conditions. In most cases the carbides were considered to be sufficiently thin, i.e. less than 5000Å, to avoid significant absorption and fluorescence effects.
The aim of this study was to assess the effect of a pre-existing defect on the creep deformation response of three microstructural variables of 2.25%Cr-1%Mo alloy steel. It is believed that the findings of this work will make a significant contribution to the understanding of how the presence, or absence, of key microstructural constituents can affect the deformation response of a component operating under creep conditions and, specifically, a component containing a pre-existing defect.
The conclusions of this study are as follows:
- The local creep constraint at the tip of a pre-existing defect in 2.25%Cr-1%Mo steel has been shown to vary with the microstructural condition of the alloy. The greatest constraint of the three microstructures examined was that exerted by the fully bainitic structure. This was followed by the mixed proeutectoid ferrite + bainite structure and, finally, the annealed ferrite + pearlite.
- The magnitude of the constraint imposed at the defect tip was related to the ease with which the stress concentration was redistributed into the surrounding structure. This, in turn, was related to the microstructural constituents in the alloy preventing the processes of dislocation glide and climb. SEM and TEM analysis of the creep damaged structures indicated that the microstructural constituent most effective in restricting dislocation mobility was that of Mo in solid solution in the bainitic ferrite.
- During the early stages of creep crack initiation from a pre-existing defect, the creep ductility of the alloy was inversely related to the constraint exerted by the material in the vicinity of the defect tip. A high creep ductility, or low constraint, was indicated by a small value of $\Delta a/\Delta CTOD$ or a small value of $\Delta a$/void area %.
In the ferrite / pearlite structure, that imparted only a small degree of constraint, cavitation was observed to occur over a large area surrounding the 'defect' tip. Gross plastic deformation was observed and yet crack initiation did not occur.
In the ferrite / bainite structure more constraint was evident, cavitation was confined to smaller area and a crack was observed to initiate after exceeding a CTOD of ~0.16mm. For a crack extension of 0.5mm the ratios of $\Delta a/\Delta CTOD$ and $\Delta a$/void area % were determined to be 1.85 and 0.21 respectively.
In the fully bainitic structure, where constraint was the highest, cavitation occurred in a confined area close to the 'defect' tip and crack initiation occurred after the CTOD exceeded ~0.07mm. For a crack extension of 0.5mm the ratios of $\Delta a/\Delta CTOD$ and $\Delta a$/void area % were determined to be 2.78 and 2.0 respectively.
- Crack extension occurred at a slower rate in the ferrite / bainite structure than in the fully bainitic structure up to a critical CTOD value of ~0.3mm, and a crack extension of ~1mm. Once these values had been attained crack extension occurred at a similar rate in both structures.
- The presence of an incoherent sulphide inclusion in the vicinity of the defect in the ferrite / pearlite structure resulted in poor stress redistribution and premature creep crack initiation. This occurrence was considered to be due to the inclusion acting as a sink for the dislocations induced at the notch tip.
- Creep cavitation ahead of the pre-existing defects occurred, predominantly, at the interface between grain boundary carbides and their adjacent grains. High resolution SEM and TEM examination, in conjunction with energy dispersive X-ray analysis, showed that the carbides most frequently linked to the cavitation process were large, elongated carbides exhibiting signs of evolution to the equilibrium carbide $M_6C$.
• A triaxial state of stress, created by the presence of a pre-existing defect in a component subject to creep conditions, imparted a significant grain boundary sliding element to the creep deformation process. This was indicated by the large number of cavitating grain boundaries that were detected oriented at $45^\circ$ to the principal stress axis in the fully bainitic and ferrite / bainite creep damaged specimens.
• Grain boundary sliding affected the growth direction of the carbides on critically oriented boundaries, i.e. those at $45^\circ$ to principal stress axis. This reduced the effectiveness of the carbides pinning the grain boundaries allowing further sliding and, as a consequence, grain boundary cavity initiation and cavity growth.
• Crack initiation occurred when adjacent grain boundary cavities coalesced. High resolution SEM imaging of the cavitating structures indicated that the likely mechanisms for cavity coalescence were:
➢ In the ferrite / bainite and fully bainitic structures: vacancy diffusion along paths of least resistance, i.e. along the interface formed between the elongated carbides and the adjacent grains.
➢ In the ferrite / pearlite structure: plastic tearing of the matrix material adjacent to the grain boundary carbides
• The volume fraction of transformation products in the alloy affects the distribution and maturity of the alloy carbides at inter and intra-granular sites in the microstructure.
This was inferred by comparing the types of carbides detected in the microstructures of the current study with those reported by other workers. In particular, with reference to the mixed ferrite / bainite microstructure, the greater the volume fraction of pro-eutectoid ferrite the more stable the carbides in the intra-granular regions of bainite and at the ferrite / ferrite grain boundaries. The main significance of this is to the comparison of
creep rupture data derived from a number of sources. It is evident from these studies that, an appreciation of the fine micro-structure of a alloy is crucial to predict the operative mechanisms of creep deformation and, hence, component lifetime.
- It was possible to identify numerous carbides, based on their elemental ratios, quickly and accurately utilising the STEM / EDX image analysis routine developed in this study. This routine may also be suitable to determine the carbides existing in power plant components at stages of component life by the examination of extraction replicas.
- Image analysis in the SEM was used, successfully, to determine the area % of voids occurring ahead of a pre-existing defect in creep damaged material. The method developed necessitated the analysis of bulk material extracted from the creep damaged specimens. It may, however, be possible to adapt the procedure to assess surface replica specimens extracted from power plant to aid lifetime prediction.
- The lifetime of a large cast component operating in a creep environment can be significantly reduced by the presence of a pre-existing defect in the microstructure. Lifetime, or remanent lifetime, prediction methods should, therefore, include parameters to account for this effect. Models should include not only a parameter to account for the triaxial state of stress at the defect tip but also a parameter to account for the constraint imposed by the evolving component microstructure.
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## Appendix 1 – Creep Data For The Derivation Of $\sigma_{\text{ref}}$
| Time | Strain % |
|------|----------|
| 0 | 0 |
| 18 | 0.3343 |
| 42 | 0.6496 |
| 66 | 0.9033 |
| 90 | 1.0811 |
| 114 | 1.2428 |
| 138 | 1.3905 |
| 162 | 1.5319 |
| 186 | 1.6656 |
| 210 | 1.796 |
| 234 | 1.9187 |
| 258 | 2.2234 |
| 282 | 2.5038 |
| 306 | 2.7631 |
| 330 | 2.9691 |
| 354 | 3.1291 |
| 378 | 3.2798 |
| 402 | 3.4326 |
| 426 | 3.5637 |
| 450 | 3.6937 |
| 474 | 3.8193 |
| 498 | 4.0671 |
| 522 | 4.42 |
| 546 | 4.7213 |
| 570 | 4.9568 |
| 594 | 5.1392 |
| 618 | 5.3084 |
| 642 | 5.4701 |
| 666 | 5.6243 |
| 690 | 5.7739 |
| 714 | 5.9172 |
| 738 | 6.3862 |
| 762 | 6.7582 |
| 786 | 7.0267 |
| 810 | 7.2371 |
| 834 | 7.436 |
| 858 | 7.6251 |
| 882 | 7.8068 |
| 906 | 8.0832 |
| 930 | 8.5974 |
| 954 | 8.9899 |
| 978 | 9.2557 |
| 1002 | 9.5001 |
| 1026 | 9.7252 |
| Time | Strain % |
|------|----------|
| 1050 | 9.9373 |
| 1074 | 10.4839 |
| 1098 | 11.0095 |
| 1122 | 11.341 |
| 1146 | 11.6479 |
| 1170 | 11.9092 |
| 1194 | 12.4496 |
| 1218 | 13.1079 |
| 1242 | 13.5045 |
| 1266 | 13.8575 |
| 1290 | 14.3852 |
| 1314 | 15.227 |
| 1338 | 15.7139 |
| 1362 | 16.1491 |
| 1386 | 17.179 |
| 1410 | 17.4793 |
| 1434 | 18.9457 |
| 1458 | 19.6635 |
| 1482 | 20.9761 |
| 1507 | 21.9371 |
| 1530 | 23.6802 |
| 1554 | 25.8442 |
| 1578 | 30.0659 |
| 1580 | 30.4803 |
| 1589 | Rupture |
**Table A1.1:** Creep data for as received 2.25%Cr-1%Mo steel tested at 550°C and 110MPa by courtesy of Alstom Energy, UK. Ltd.
Figure A1.1: Creep curve plotted from data detailed in Table A1 - 2.25%Cr-1%Mo steel tested at 550°C and 110MPa, by courtesy of Alstom Energy, UK. Ltd.
### Table A2.1: Fe₃C Type Carbides (Determined from an intra-granular region of the fully bainitic specimen tempered @ 650°C for 1h)
| Fe% | Cr% | Mo% | Fe/Cr | Mo/Cr | Mo/Fe |
|-------|-------|-------|-------|-------|-------|
| 73.98 | 18.48 | 7.55 | 4.00 | 0.41 | 0.10 |
| 76.81 | 16.57 | 6.62 | 4.64 | 0.40 | 0.09 |
| 79.17 | 14.79 | 6.04 | 5.35 | 0.41 | 0.08 |
| 76.04 | 17.22 | 6.74 | 4.42 | 0.39 | 0.09 |
| 75.81 | 17.29 | 6.90 | 4.38 | 0.40 | 0.09 |
| 75.73 | 17.43 | 6.84 | 4.35 | 0.39 | 0.09 |
| 75.85 | 16.85 | 7.30 | 4.50 | 0.43 | 0.10 |
| 75.73 | 17.43 | 6.84 | 4.35 | 0.39 | 0.09 |
| 75.85 | 16.85 | 7.30 | 4.50 | 0.43 | 0.10 |
| 76.70 | 16.39 | 6.91 | 4.68 | 0.42 | 0.09 |
| 76.23 | 16.85 | 6.93 | 4.52 | 0.41 | 0.09 |
| 74.14 | 18.02 | 7.85 | 4.12 | 0.44 | 0.11 |
| 64.22 | 24.81 | 10.97 | 2.59 | 0.44 | 0.17 |
| 64.18 | 29.76 | 6.06 | 2.16 | 0.20 | 0.09 |
| 66.46 | 24.31 | 9.24 | 2.73 | 0.38 | 0.14 |
| 69.68 | 22.02 | 8.30 | 3.16 | 0.38 | 0.12 |
| 66.00 | 24.02 | 9.98 | 2.75 | 0.42 | 0.15 |
| 69.99 | 21.25 | 8.76 | 3.29 | 0.41 | 0.13 |
| 59.78 | 26.08 | 14.14 | 2.29 | 0.54 | 0.24 |
| 65.63 | 19.87 | 14.50 | 3.30 | 0.73 | 0.22 |
| 71.34 | 20.29 | 8.37 | 3.52 | 0.41 | 0.12 |
| 71.46 | 20.60 | 7.94 | 3.47 | 0.39 | 0.11 |
| 72.23 | 19.75 | 8.02 | 3.66 | 0.41 | 0.11 |
### Table A2.2: M₂C Type Carbides (Determined from the α region of the as-received mixed ferrite / bainite structure).
| Fe% | Cr% | Mo% | Fe/Cr | Mo/Cr | Mo/Fe |
|-------|-------|-------|-------|-------|-------|
| 0.57 | 25.51 | 73.91 | 0.02 | 2.90 | 129.67|
| 5.19 | 27.24 | 67.57 | 0.19 | 2.48 | 13.02 |
| 4.12 | 25.43 | 70.45 | 0.16 | 2.77 | 17.10 |
| 3.86 | 37.93 | 58.21 | 0.10 | 1.53 | 15.08 |
| 8.03 | 27.58 | 64.39 | 0.29 | 2.33 | 8.02 |
| 3.91 | 31.56 | 64.54 | 0.12 | 2.04 | 16.51 |
| 5 | 26.27 | 68.73 | 0.19 | 2.62 | 13.75 |
| 9.45 | 16.62 | 73.93 | 0.57 | 4.45 | 7.82 |
| 2.43 | 26.89 | 70.68 | 0.09 | 2.63 | 29.09 |
| 6.33 | 30.87 | 62.8 | 0.21 | 2.03 | 9.92 |
| 5.34 | 27.24 | 67.42 | 0.20 | 2.48 | 12.63 |
| 4.92 | 23.81 | 71.27 | 0.21 | 2.99 | 14.49 |
| 3.94 | 30.74 | 65.32 | 0.13 | 2.12 | 16.58 |
| 7.39 | 23.35 | 69.35 | 0.32 | 2.97 | 9.38 |
Table A2.3: $M_7C_3$ Type Carbides (Determined from the bainite region of the as-received mixed ferrite / bainite structure)
| Fe% | Cr% | Mo% | Fe/Cr | Mo/Cr | Mo/Fe |
|------|------|------|-------|-------|-------|
| 34.15| 60.31| 5.54 | 0.57 | 0.09 | 0.16 |
| 31.04| 60.68| 8.3 | 0.51 | 0.14 | 0.27 |
| 43.11| 51.91| 4.98 | 0.83 | 0.10 | 0.12 |
| 35.75| 57.69| 6.56 | 0.62 | 0.11 | 0.18 |
| 37.97| 55.83| 6.2 | 0.68 | 0.11 | 0.16 |
| 35.97| 60.95| 3.08 | 0.59 | 0.05 | 0.09 |
| 33.57| 57.93| 8.49 | 0.58 | 0.15 | 0.25 |
| 34.15| 55.1 | 10.75| 0.62 | 0.20 | 0.31 |
Table A2.4: $M_{23}C_6$ Type Carbides (Determined from the bainite region of the as-received mixed ferrite / bainite structure)
| Fe% | Cr% | Mo% | Fe/Cr | Mo/Cr | Mo/Fe |
|------|------|------|-------|-------|-------|
| 49.6 | 42.1 | 8.3 | 1.18 | 0.20 | 0.17 |
| 55.77| 37.57| 6.65 | 1.48 | 0.18 | 0.12 |
| 53.66| 35.99| 10.36| 1.49 | 0.29 | 0.19 |
| 49.59| 41.24| 9.17 | 1.20 | 0.22 | 0.18 |
| 54.48| 38.76| 6.75 | 1.41 | 0.17 | 0.12 |
| 53.16| 36.33| 10.51| 1.46 | 0.29 | 0.20 |
| 53.33| 39.88| 6.79 | 1.34 | 0.17 | 0.13 |
| 52.82| 39.9 | 7.28 | 1.32 | 0.18 | 0.14 |
| 53.82| 40.33| 5.85 | 1.33 | 0.15 | 0.11 |
| 53.31| 29.49| 17.2 | 1.81 | 0.58 | 0.32 |
| 51.86| 39.68| 8.47 | 1.31 | 0.21 | 0.16 |
Table A2.5: $M_6C$ Type Carbides (Determined from an intra-granular region of the fully bainitic specimen tempered @ 700°C for 10,000h)
### Appendix 3 – Elemental Factors Determined for Use in the STEM Image Analysis Routine.
| Element / Background Window | Window Identification | Window span (ev) | Background Window | Background Fraction | Overlap window | Overlap Fraction |
|-----------------------------|-----------------------|------------------|-------------------|--------------------|----------------|-----------------|
| Si | 1 | 1580 - 1900 | 3 | 1.57 | n.a | 0 |
| Mo | 2 | 2120 - 2520 | 3 | 2.14 | n.a | 0 |
| BG1 | 3 | 2760 - 3000 | 3 | 1.00 | n.a | 0 |
| BG2 | 4 | 4840 - 5140 | 4 | 1.00 | n.a | 0 |
| Cr | 5 | 5200 - 5620 | 4 | 1.18 | n.a | 0 |
| Mn | 6 | 5880 - 6120 | 4 | 0.96 | 5 | 0.13 |
| Fe | 7 | 6160 - 6840 | 4 | 1.18 | n.a | 0 |
Table A3.1: Data For Calculation Of Factors Required For STEM Image Analysis Routine.
---
\(^1\) Overlap contribution from Mn\(_{0.9}\) to Fe\(_{0.6}\) considered to be negligible and therefore not calculated for the purposes of this analysis.
Creep damage quantification of 2·25Cr–1Mo steel using scanning electron microscopy
R. A. Collington, J. Cawley, and S. R. Holdsworth
The material 2·25Cr–1Mo alloy steel has been used extensively for high temperature applications in power generation plant for over five decades owing to its long term creep resistance. It has been recognised that the lifetime of a high temperature component containing pre-existing defects is dependent not only upon the material's crack propagation resistance but also upon an incubation period before crack growth during which a damage zone ahead of the defect tip develops. The extent of the damage occurring during this incubation period, before crack propagation, is dependent upon the stress intensity at the defect tip, the ductility of the material, and the microstructure in the damage zone. The present paper details a technique for quantifying the early stages of creep damage using image analysis in the scanning electron microscope and compares the degree, distribution, and orientation of creep damage occurring in two microstructural variables of 2·25Cr–1Mo alloy steel. The paper describes the procedures necessary for generating consistent and reproducible quantitative analysis results, including specimen preparation, defect detection, and measurement criteria. The image analysis process, its accuracy, and application to the study of creep damage mechanisms occurring ahead of defects are discussed.
Ms Collington and Dr Cawley are at the Materials Research Institute, Sheffield Hallam University, Sheffield, UK and Dr Holdsworth is with Alstom Power, Rugby, Warwickshire, UK. Based on a presentation at the conference on 'Quantitative microscopy of high temperature materials' held at Sheffield Hallam University on 22–24 November 1999; this meeting was one of the Microstructure of High Temperature Materials series sponsored by The Institute of Materials. The full proceedings will be published as an IoM book.
© 2000 IoM Communications Ltd.
Introduction
Assessment of the remaining lifetime of components in a creep environment has been the subject of much research during the past few decades, two approaches to prediction being the use of crack growth models based on time dependent fracture mechanics concepts and microstructural evidence. To predict accurately the lifetime of a component, it has become clear that these two approaches should not be studied in isolation, and that detailed microstructural evidence is required to model creep damage and failure mechanisms of materials of interest.
A number of methods are available to predict the lifetime of a component containing defects using mathematical models based on the theory of fracture mechanics, and assessment procedures based on the use of the $C^*$ loading parameter and critical crack opening displacement (COD) have been published. The importance of estimating the time to initiate cracking, as well as the time for crack propagation $t_p$ has now been recognised, and, therefore, to estimate this period more accurately more detailed microstructural evidence is required.
During the incubation period before crack propagation, a creep damage zone develops at the crack tip and there is a redistribution of the stress intensity. The structural degradation that occurs in the incubation period is dependent upon the operating stress and temperature regime, and includes changes in chemistry, size distribution, and spacing of cavitation phase particles and nucleation and growth of cavities and cracks.
The present paper details experimental procedures designed to assess the progress of cavitation damage in two microstructures of 2·25Cr–1Mo steel at 550°C in the vicinity of a pre-existing defect, and correlates the results from the study with corresponding crack tip opening displacement and crack initiation data.
Experimental procedure
MATERIAL INVESTIGATED
The material for the present study was extracted from the flange of a fully heat treated cast steam turbine casing in 2·25Cr–1Mo steel. Composition of the steel was Fe–0·11C–0·35Si–0·59Mn–0·006P–0·005S–2·34Cr–1·00Mo (wt.%), heat treated as given in Table 1.
The microstructure of quality heat treated, complex shaped casings, such as those used for turbine generator casings in the power industry, may range from fully bainitic structures in thin sections to ferrite–pearlite structures in the core of the casting. This microstructural variation results from differences in the cooling rate experienced by these regions when cooling from the normalising temperature, relative to the position of the ferrite nose in the continuous cooling transformation (CCT) diagram for 2·25Cr–1Mo steel. For the purpose of the present study, two possible microstructural variables were assessed:
(i) the as received microstructure of tempered 25% bainite+75% proeutectoid ferrite (Fig. 1a)
(ii) a fully bainitic structure derived from normalising at 960°C and tempering at 700°C (Fig. 1b)
CREEP DAMAGE TEST PROGRAMME
The aim of the creep test programme was to simulate the stress situation ahead of a pre-existing defect in 2·25Cr–1Mo steel and generate creep damage up to the point of 1 mm crack extension $t_{cr}$. Analysis of the tested specimens would then allow the degree, distribution, and orientation of creep damage to be determined and related to the strain at the defect tip (crack tip opening displacement) and the onset of cracking (crack initiation).
To simulate the stress situation occurring ahead of such a defect, compact tension specimens, containing fine spark eroded notches <0·2 mm dia., were subjected to
creep loads at a temperature of 550°C. Crack tip opening displacement (CTOD)\textsuperscript{6,7} was assessed by measuring the displacement of hardness indentations placed at each side of the compact tension specimen notch before and after the creep test\textsuperscript{8} (Fig. 2).
Creep loads applied were based on plain strain reference stress criteria\textsuperscript{9} $\sigma_{p1e}$, equation (1), and then finally adjusted to give a crack extension of $\sim$1 mm in 1000 h
$$\sigma_{p1e} = \frac{P}{m_{p1e} B_n W}$$ \hspace{1cm} (1)
where $P$ is the applied load, $m_{p1e}$ is the yield ratio in plane strain, $B_n$ is the net section thickness, and $W$ is the specimen width, and
$$m_{p1e} = [1 + 1.702(a/W)] + [2.702 + 4.599(a/W)^2]^{1/2}$$
where $a$ is crack length.
To assess the progress of damage accumulation, CTOD, and crack initiation, up to four compact tension specimens were tested in series and then removed sequentially at various life fractions for evaluation. Damage accumulation was assessed using image analysis in the scanning electron microscope, and CTOD and crack initiation measurements were made using calibrated scalar devices in the optical and scanning electron microscopes.
**CREEP DAMAGE ACCUMULATION BY IMAGE ANALYSIS IN SEM**
**Specimen preparation**
To obtain an accurate appraisal of the microstructural damage induced during the creep process, it was essential to prepare the specimens for examination with the minimum amount of deformation. Sections from around the damaged defect tip were removed from the test specimens using a fine alumina abrasive wheel, mounted in conducting Bakelite, and ground to a 600 grit finish using conventional silicon carbide abrasive papers. After thorough cleansing, the specimens were polished to a 1 $\mu$m finish on diamond impregnated cloths and etched in 2% nitric acid in methanol. The specimens were then subjected to two further polish–etch sequences to ensure that the burred surface had been removed, but without artificially enlarging the voids and microcracks present.\textsuperscript{10} After the final etching process, the specimens were coated with a thin evaporated layer of carbon to prevent charging of the oxide layer in the notch tip and ensure uniformity of image contrast in the scanning electron microscope (SEM).
**Image analysis routine**
Microstructural examination and damage accumulation of the creep tested compact tension specimens was carried out using a Philips XL40 SEM and Oxford Instruments image analysis software. The microspecimens prepared were examined using both backscattered electron (BSE) and secondary electron (SE) imaging modes. The BSE imaging allowed creep voids and microcracks to be clearly delineated from general microstructural features for quantification purposes, while the SE imaging enabled the operator to determine whether damage was present at inter- or intragranular sites.
The accuracy of the analytical process relied on imaging conditions remaining consistent between consecutive fields of view and between specimens, and, therefore, a systematic routine was devised to ensure reproducible image contrast and brightness and feature detection.
The following microscope conditions were maintained during the analytical procedure:
- working distance: 15 mm
- accelerating voltage: 20 keV
- beam current: $\sim$85 $\mu$A
- probe size: spot size 6 (large)
- image type: backscattered electron
- magnification: $\times 1000$
- scan rate: 200 ms/line
As the stability of the beam current cannot be guaranteed over long periods of time, or from specimen to specimen, it was necessary to establish a datum for the contrast and brightness levels to ensure consistency. This was achieved by using the waveform meter on the SEM control panel to ensure that the average contrast and brightness signal for...
3 Use of SEM waveform meter to ensure image consistency
The general microstructure was at a known position with respect to the 'white' level and that the signal from the voids and microcracks fell into the 'black' level (Fig. 3).
The analytical programme was set up to detect the voids and microcracks using a function which isolates the features of interest from the remaining microstructure, known as grey level thresholding. Once set, the threshold levels remained valid, providing that the contrast and brightness levels in the BSE image were maintained.
To assess quantitatively the creep damage, the analysis routine was used to carry out morphological measurements based on an area of 26 field of view locations. The use of a large number of measurements allows the features of interest to be accurately assessed in terms of their size and shape and, specifically for the present study, the area percentage of the voids and microcracks and their orientation with respect to the stress axis. During the image analysis routine, a BSE signal was collected from the field of interest and displayed as a digital image having an image resolution of $1024 \times 1024$ pixels and an area $\sim 185 \times 185 \mu m$, i.e. 1 pixel $\approx 0.18 \times 0.18 \mu m$.
Figure 4 shows typical BSE, SE, and digital images of cavitation and microcracking in a specimen of 2-25Cr–1Mo alloy steel.
For this particular exercise, up to 36 fields of view were studied on each specimen, extending to 1 mm each side of the notch tip and 1 mm into the remaining ligament.
Results
At predetermined intervals, the compact tension specimens were removed from the creep test and assessed for crack-opening displacement. Microspecimens were then extracted and prepared from the 'defect' tip region and analysed in terms of their microstructure, void area percentage, and void orientation in the SEM.
CTOD, CRACK EXTENSION, AND VOID AREA PERCENTAGE
The results determined for CTOD, crack extension, and void area percentage are given in Table 2.
From the results (Fig. 5) it was evident that the onset of CTOD, crack extension, and void nucleation in the ferrite–bainite material was delayed with respect to that in the fully bainitic condition, suggesting that the duplex structure is more resistant to ductility exhaustion (more creep ductile) than the single phase structure. This was substantiated by examination of the results plotted in Fig. 6a for CTOD against void area percentage. This showed that, for CTODs less than 0.2 mm, a greater CTOD was required to generate the same creep damage, in the form of voids and microcracks, for the ferrite–bainite specimen than for the fully bainitic structure.
In the fully bainitic structure, crack extension advanced for a minimal amount of voiding and then appeared to slow down, while in the case of the ferrite–bainite structure there
Table 2 Test data* derived from compact tension specimens creep tested at 550°C
| Test duration, h | CTOD, mm | Crack extension, mm | Cavity area, % |
|------------------|----------|---------------------|---------------|
| **Ferrite–bainite (110 MPa)** | | | |
| 0 | 0.00 | 0.00 | 0.00 |
| 300 | 0.07 | 0.00 | 0.01 |
| 700 | 0.16 | 0.00 | 0.06 |
| 850 | 0.27 | 0.39 | 1.73 |
| 1000 | 0.27 | 0.90 | 5.14 |
| 1500 | 2.75 | 8.22 | ND |
| **Bainite (100 MPa)** | | | |
| 0 | 0.00 | 0.00 | 0.00 |
| 300 | 0.07 | 0.00 | 0.04 |
| 350 | 0.18 | 0.49 | 0.22 |
| 400 | 0.28 | 0.62 | 1.42 |
| 500 | 0.35 | 2.50 | ND |
| 600 | 0.39 | 4.00 | ND |
| 800 | 0.98 | 6.17 | ND |
| 1000 | 1.52 | 6.81 | ND |
*CTOD is crack tip opening displacement, ND is data not determined.
5 Variation of given parameters with test duration
was an almost linear relationship between crack extension and void percentage (Fig. 6a).
Correlation of the data derived for CTOD and crack extension (Fig. 7) suggested that, although CTOD initiation was delayed in the ferrite–bainite specimen, an almost linear relationship existed for the two structures once a critical CTOD had been reached.
MICROSTRUCTURAL EXAMINATION AND VOID ORIENTATION
Low magnification imaging of the notch tip region in each of the specimens analysed showed that voiding and microcrack extension in the fully bainitic structure was constrained to a narrow field width even in the ferrite–bainite region (Fig. 8). The degree of constraint was also evident on plotting the void area percentage data against position relative to the notch tip, as shown in Fig. 9.
Frequency distribution charts showing void orientation with respect to notch tip position (Fig. 10) suggested that void elongation occurred predominantly at angles of 50–60° with respect to the notch tip in both microstructural variables. Microstructural evidence, however, suggested that the orientation of the grain boundaries, along which voiding occurred, was close to the 45° shear axis as might have been predicted in these short term tests (Fig. 11). The occurrence of voids in the microstructures in all cases was predominantly intergranular, being detected mainly at the proeutectoid ferrite/bainite boundaries in the mixed structure (Fig. 11a) and at the prior austenite grain boundaries in the fully bainitic structure (Fig. 11b). Examination of the precipitate morphology depicted in Fig. 11 showed that the carbides in the ferrite region of the mixed structure were much finer than those present in the bainite phase of either material condition. This was also evident from extraction replicas produced from the original starting microstructures of the two materials (Fig. 12).
Discussion
The aim of the present study was to compare the mechanisms of creep damage accumulation ahead of a pre-existing defect in two microstructures of 2-25Cr–1Mo steel, with a view to appreciating how microstructural variables can affect creep life predictions.
Quantitative image analysis in the SEM enabled microstructures from creep tested, compact tension specimens to be studied in terms of creep damage accumulation ahead of a simulated defect tip in conjunction with microstructural examination.
8 Low magnification images of voiding and microcracking around notch tip
In general, the results showed that during the initiation period $t_{\text{init}}$, the ferrite–bainite material required a greater CTOD to extend the damage zone than did the fully bainitic structure. It was evident from Figs. 6b and 7b that a greater degree of strain, in the form of CTOD, could be accommodated at the notch tip of the ferrite–bainite specimen before the onset of damage by void accumulation and crack extension. This additional strain accommodation was considered partly to result from the ferrite phase regions acting as ‘soft zones’, reducing the number of prior austenite grain boundaries, but also because the bainite in the mixed structure existed in a more ‘mature’ evolutionary state (Fig. 12).
Creep crack initiation has been shown previously$^{12,13}$ to occur on the attainment of a critical local crack tip strain, and this was also evident in the present study. Figure 7 demonstrated that, although the CTOD to generate a given $\Delta \varepsilon$ (damage) for the ferrite–bainite material during the incubation period was greater than that for the fully bainitic material, the critical value of CTOD $\delta_{\text{crit}}$ was similar for both microstructural variables and that once the critical value had been exceeded, crack extension occurred at a similar rate in both materials with respect to CTOD.
The quantity and distribution of creep damage, in the form of grain boundary voids and microcracks, is depicted in Figs. 8 and 9 and shows the effect of variation in microstructure on the degree of constraint at the notch tip. In the incubation period it was evident that the higher ductility exhibited by the ferrite–bainite structure resulted in lower notch constraint and, hence, more extensive cavitation and microcracking before the onset of crack growth (crack extension > 1 mm).
To determine the mechanism of creep cavity nucleation in the two microstructural variables, the orientation of the voids and microcracks was determined and studied in conjunction with microstructural evidence detected in the SEM. Void orientation analysis showed that the cavities nucleated most frequently at angles of $\pm 60^\circ$ with respect to the notch tip (Fig. 10). Microstructural evidence, however,
10 Frequency distribution charts for void orientation data
a ferrite–bainite structure, 1000 h; b bainitic structure, 400 h
11 High magnification images of damage at grain boundaries
showed that the boundaries, upon which the cavities nucleated and extended, were generally oriented at angles of ±45° with respect to the notch tip (Fig. 8). This combined evidence suggests that the mechanism of cavity nucleation and growth, in both the ferrite–bainite and fully bainitic structures, was not by grain boundary sliding alone, but by a combination of sliding and atom diffusion. High magnification imaging of the microstructure using secondary electrons in the SEM supported this theory (Fig. 11) in that diffusion of atoms appeared to be occurring away from the bainite phase regions, down substructure boundaries, towards the main grain boundaries. The large grain boundary carbides, predominantly M$_{23}$C$_6$ type, then appeared to grow and elaborate along the boundary until they lost coherency with the matrix resulting in cavity nucleation.
Previous studies of the effect of transformation structure on the creep and rupture behaviour of 2-25Cr–1Mo steel carried out on uniaxial creep test specimens showed that failure in high bainite content material occurred, as in the present study, by intergranular failure. However, in low bainite content material (~20% bainite), failure was found to occur by intragranular cavitation and transgranular ductile fracture. The difference observed in the present study may be caused by the triaxial state of stress ahead of the defect tip, encouraging diffusion and sliding on grain boundaries oriented at 45° to the principal stress axes.
Conclusions
1. Image analysis in the SEM can be used effectively to study creep damage accumulation in steel, providing that image stability can be maintained.
12 Precipitate morphology of materials before testing
2. During the incubation period for creep crack extension ahead of a pre-existing defect in two structures of 2-25Cr–1Mo steel at 550°C:
(i) the material, in both microstructural conditions, undergoes strain accumulation in the form of elastic–plastic deformation, grain boundary voiding, and microcracking
(ii) voiding and microcracking occur predominantly at intergranular sites between the ferrite–bainite grains in the mixed structure and at the prior austenite boundaries in the fully bainitic material
(iii) the ferrite–bainite structure is more creep ductile than the fully bainite structure due to the effective dispersal of applied stress in the ferrite phase and mature bainite phase, leading to enhanced strain-accommodation and reduced notch tip constraint
(iv) grain boundary voiding appears to occur in both microstructural conditions because of the combined effects of atom diffusion down the bainite substructure and grain boundary diffusion, precipitate growth, and sliding.
3. Upon the attainment of a critical notch tip strain, i.e. $\varepsilon$ to give 1 mm of crack extension, the ferrite–bainite and fully bainitic structures appear to deform at a similar rate.
References
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2. A. D. POWELL: ‘Microstructures and mechanical properties of aging materials’, 1–17; 1993. Warrendale, PA: TMS.
3. E. G. ELLISON and M. P. HARPER: J. Strain Anal. (Eng. Des.), 1978, 13, 35–51.
4. R. A. ANSWORTH and P. J. BUDDEN: Mater. High Temp., 1998, 15, 291–297.
5. S. K. HOLDSWORTH: Mater. High Temp., 1992, 10, 127–137.
6. S. M. Beech, J. W. Selway, and A. D. Batte: in 'Creep and fatigue of engineering materials and structures', (ed. B. Wilshire and D. R. J. Owen), Vol. 2, 925–936; 1984, Swansea, Pineridge Press.
7. K. H. Kloos, J. Granacher, and R. Tschuschner: Z. Werkstofftech., 1987, 18, 390.
8. S. R. Holdsworth and D. J. Cunnane: 'Lifetime prediction of components containing defects', COST 501 WP5C Final Report, GEC Alstom Turbine Generators, Rugby, UK, 1993.
9. A. G. Miller: Pressure Vessels Piping, 1988, 32, 197.
10. T. L. Da Silveira and I. Le May: Mater. Charact., 1992, 28, 75–83.
11. B. J. Cane: The effect of transformation structure on the creep and rupture behaviour of 2.25%Cr–1%Mo steel, CEGB Report no. RD/LN/236/75, Leatherhead, UK, March 1976.
12. R. A. Ainsworth: Int. J. Solid Struct., 1982, 18, 873.
13. R. A. Ainsworth: Proc. Conf. Euromech 239: 'Mechanics of creep brittle materials', Leicester, UK, 13; 1988, Oxford, Elsevier.
| Table No. | Title | Page |
|----------|----------------------------------------------------------------------|------|
| 2.1 | Nature Of carbides in normalised 2.25%Cr-1%Mo steel [17][28] [27]. | 15 |
| 2.2 | Equilibrium compositions of M$_{23}$C$_6$ and M$_7$C$_3$ carbide - after Thomson [24]. | 17 |
| 2.3 | Creep damage categories, mechanisms and incorporation into continuum damage mechanics equations – after Dyson and McLean [113]. | 51 |
| 2.4 | Classification of damage process and exhausted creep life - after Dobrzanski and Hernas [103]. | 52 |
| 3.1 | Composition of as-received material. | 65 |
| 3.2 | Final commercial heat-treatment of as-received material. | 65 |
| 3.3 | Creep test loading conditions. | 75 |
| 3.4 | Elemental ratios determined by EDX spot analyses and used for carbide identification. | 87 |
| 3.5 | The compositional range of various metal carbides encountered in 2.25%Cr-1%Mo steel, as used in conjunction with EDX-Digiscan image analysis. | 91 |
| 4.1 | Hardness and tensile properties. | 94 |
| 4.2 | Grain size and phase proportions of starting materials. | 96 |
| 4.3 | Elemental ratios for the carbides observed in the ferrite / pearlite structure as indicated in plate 4.11. | 106 |
| 4.4 | Elemental ratios for the carbides observed in the ferrite / bainite structure as indicated in plate 4.13. | 107 |
| 4.5 | Elemental ratios for the carbides observed in the ferrite / bainite structure as indicated in plate 4.14. | 108 |
| 4.6 | Temper trial sample conditions | 110 |
| 4.7 | Hardness results for the tempered fully bainitic samples (±2σ). | 111 |
| 4.8 | Carbide size distribution data for the fully bainitic sample tempered at 700°C for 0.5 hours. | 121 |
| 4.9 | Carbide size distribution data for the fully bainitic sample tempered at 700°C for 5 hours. | 122 |
| 4.10 | Carbide size distribution data for the fully bainitic sample tempered at 700°C for 50 hours. | 123 |
| 4.11 | Carbide size distribution data for the fully bainitic sample tempered at 700°C for 5000 hours. | 124 |
| Index to Table Nos. cont. | Description | Page |
|---------------------------|-----------------------------------------------------------------------------|------|
| 4.12 | Change in carbide type with tempering time at 700°C for the fully bainitic samples. | 125 |
| 4.13 | Y-displacement values of hardness indentations for 100% tempered bainite starting material tested at a stress of 100MPa for times between 300 and 1000 hours. | 131 |
| 4.14 | Y-displacement values of hardness indentations for mixed ferrite / bainite starting material tested at a stress of 110MPa for times between 300 and 1500 hours. | 131 |
| 4.15 | Crack tip opening displacement results derived from CT specimens manufactured from the starting materials. | 134 |
| 4.16 | Void % analysis results for the 100% tempered bainite sample tested at 550°C and 100MPa for 300h. | 139 |
| 4.17 | Void % analysis results for the 100% tempered bainite sample tested at 550°C and 100MPa for 350h. | 140 |
| 4.18 | Void % analysis results for the 100% tempered bainite sample tested at 550°C and 100MPa for 400h. | 141 |
| 4.19 | Crack extension and void area % values for the tempered, fully bainitic, CT specimens. | 143 |
| 4.20 | Void % analysis results for the mixed ferrite / bainite sample tested at 550°C and 110MPa for 700h. | 150 |
| 4.21 | Void % analysis results for the mixed ferrite / bainite sample tested at 550°C and 110MPa for 850h. | 151 |
| 4.22 | Void % analysis results for the mixed ferrite / bainite sample tested at 550°C and 110MPa for 850h. | 152 |
| 4.23 | Crack extension and void area % values for the mixed ferrite / bainite CT specimens. | 154 |
| 4.24 | Void % analysis results for the mixed ferrite / pearlite sample tested at 550°C and 85MPa for 3680h. | 161 |
| 4.25 | Void % analysis results for the mixed ferrite / pearlite sample tested at 550°C and 85MPa for 6850h. | 162 |
| 4.26 | Crack extension and void area % values for the mixed ferrite / pearlite CT specimens. | 164 |
| 4.27 | Summary of creep testing investigation results. | 170 |
| 5.1 | Type and location of carbides detected in the three starting structures investigated in the current study. | 193 |
| 5.2 | Values of Δa/ΔCTOD and Δa/void area %, determined from the ferrite / bainite and 100% bainite CT specimens at a crack extension of 0.5mm. | 206 |
| Figure No. | Title | Page |
|-----------|----------------------------------------------------------------------|------|
| 1.1 | Trend to higher steam conditions for steam power plant – after Scarlin [9]. | 2 |
| 1.2 | Flow diagram summarising the experimental process. | 7 |
| 2.1 | Continuous cooling diagrams for some Cr-Mo Steels – after Orr [20]. | 11 |
| 2.2 | Carbide evolution sequence for 2.25%Cr-1%Mo steel, where M indicates a mixture of metal atoms- after Baker and Nutting [17]. | 14 |
| 2.3 | Predicted evolution of precipitate volume fractions at 600°C in 2.25%Cr-1%Mo steel -after Robson and Bhadeshia [37]. | 17 |
| 2.4 | Typical deformation map – plot of normalised stress against homologous temperature - after Reidel [40]. | 21 |
| 2.5 | The climb force on a dislocation - after Ashby and Jones [48]. Where: τ = shear stress and b=Burgers vector. | 22 |
| 2.6 | Diffusion of atoms leading to dislocation climb - after Ashby and Jones [48]. | 23 |
| 2.7 | Diffusion along dislocation lines, core diffusion, at low T/Tm – after Ashby and Jones [48]. | 24 |
| 2.8 | The process of creep deformation by diffusion at high T/Tm – after Ashby and Jones [48]. | 24 |
| 2.9 | Creep rupture strength of carbon steels and Cr-Mo steels - after Abe and Yagi [62]. | 26 |
| 2.10 | Creep curves for various stress regimes at 565°C – after Kleuh [16] | 29 |
| 2.11 | Creep rupture mechanism diagram for 2.25%Cr-1%Mo steel - after Yamauchi et al [66]. | 29 |
| 2.12 | Proposed creep deformation mechanism map of 2.25%Cr-1Mo steel – after Maruyama et al [67]. | 30 |
| 2.13 | Fracture Map – after Reidel [40]. | 33 |
| 2.14 | Wedge cracking at grain boundary triple point due to sliding – after Lagnebourg [74]. | 34 |
| 2.15 | Various mechanisms for grain boundary cavity nucleation due to grain boundary sliding – after Evans and Wiltshire [53]. | 34 |
| 2.16 | Stress concentration factor, $\sigma_p/\sigma_m$ at particles versus applied stress – after Reidel [40]. | 36 |
| Figure No. | Description | Page |
|-----------|------------------------------------------------------------------------------|------|
| 2.17 | Model for grain boundary cavitation by surface and grain boundary diffusion – after Hull and Rimmer [85]. Adjoining grains are assumed to act as rigid bodies. | 37 |
| 2.18 | Schematic illustration of cavity growth by the combination of plastic creep flow and rapid surface diffusion – after Hancock [86]. | 38 |
| 2.19 | Comparison of creep curves from uniaxial creep data for 2.25%Cr-1%Mo steel compared with predictions for constrained and unconstrained cavity nucleation – after Reidel [40]. | 39 |
| 2.20 | The factors that affect ductile-brittle creep behaviour – after Gooch and King [86]. | 42 |
| 2.21 | The effect of specimen geometry on brittleness in plane strain – after Gooch and King [86]. | 44 |
| 2.22 | Influence of phase constitution and damage process on safe residual lifetime of 1Cr-0.5Mo steel – after Dobrzanski and Hernas [103]. | 51 |
| 2.23 | Variation of crack opening displacement and crack length in a fracture mechanics specimen indicating the importance of specifying crack initiation criteria – after Holdsworth [11]. | 57 |
| 2.24 | Derivation of $\delta_{\text{cr}}$ using hardness indentations – after Holdsworth [132]. | 58 |
| 2.25 | Influence of creep ductility on crack opening displacement and growth behaviour in a fracture mechanics test piece subject to steady load at high temperature, a) brittle 0.5%Cr-0.5%Mo-0.25%V HAZ, b) ductile 2.25%Cr-1%Mo weld metal – after Holdsworth [132]. | 58 |
| 3.1 | The continuous cooling transformation diagram for a 2.25%Cr-1.0%Mo steel – after M. Atkins [140]. | 68 |
| 3.2 | Plan for sectioning of bainite tempering samples. | 69 |
| 3.3 | Creep test rig design. | 72 |
| 3.4 | Schematic diagram of CT specimen and dimensions. | 73 |
| 3.5 | Crack opening displacement measurement.
a) Schematic diagram of CT specimen notch and hardness indentations.
b) Schematic diagram of crack opening displacement against indentation position. | 76 |
| 3.6 | Micro-specimen extraction from notch tip region. | 80 |
| 3.7 | Use of the SEM wave-form meter to ensure image consistency. | 82 |
| 3.8 | Selective preparation of notch tip and reference region extraction replicas. | 86 |
| Figure No. | Description | Page |
|-----------|------------------------------------------------------------------------------|------|
| 3.9 | STEM images of a) carbide precipitates and b) the corresponding digitised image. | 90 |
| 3.10 | EDX spectra of 2.25%Cr-1%Mo steel showing identification of elements of interest by formation of "windows". | 91 |
| 4.1 | Typical EDX spectra for the carbides in 2.25%Cr-1%Mo alloy. | 109 |
| 4.2 | Graph showing Vickers hardness values against tempering temperature and time. for the fully bainitic specimens. | 112 |
| 4.3 | Graph showing Vickers hardness values against the Larson-Miller parameter, $P = T \times (C + \log t)$, for the fully bainitic specimens. Where: $T =$ temp. (K), $C =$ a constant taken to be 20 and $t =$ time (secs). | 112 |
| 4.4 | Size distribution curves for the various carbides identified in the fully bainitic sample tempered at 700°C for 0.5 hours. | 121 |
| 4.5 | Size distribution curves for the various carbides identified in the fully bainitic sample tempered at 700°C for 5 hours. | 122 |
| 4.6 | Size distribution curves for the various carbides identified in the fully bainitic sample tempered at 700°C for 50 hours. | 123 |
| 4.7 | Size distribution curves for the various carbides identified in the fully bainitic sample tempered at 700°C for 5000 hours. | 124 |
| 4.8 | Change in carbide type with tempering time at 700°C for the fully bainitic samples. | 125 |
| 4.9 | Graph showing M$_{23}$C type carbides as a percent of the total detected for each tempering duration plotted against mean diameter. | 126 |
| 4.10 | Graph showing M$_{23}$C type carbides as a percent of the total detected for each tempering duration plotted against mean diameter. | 126 |
| 4.11 | Graph showing M$_7$C$_3$ type carbides as a percent of the total detected for each tempering duration at 700°C plotted against mean diameter. | 127 |
| 4.12 | Graph showing M$_{23}$C$_6$ type carbides as a percent of the total detected for each tempering duration at 700°C plotted against mean diameter. | 127 |
| 4.13 | Graph showing M$_6$C type carbides as a percent of the total detected for each tempering duration at 700°C plotted against mean diameter. | 128 |
| 4.14 | Y-displacement values of hardness indentations for mixed ferrite / bainite starting material tested at a stress of 110MPa for times between 300 and 1500 hours. | 132 |
| Figure No. | Description | Page |
|-----------|------------------------------------------------------------------------------|------|
| 4.15 | Crack opening displacement values for the fully bainitic sample tested at a stress of 100MPa and 550°C for times between 300 – 600h. | 132 |
| 4.16 | Crack opening displacement values for the ferrite / bainite sample tested at a stress of 110MPa and 550°C for times between 300 – 1500h. | 133 |
| 4.17 | Crack opening displacement values for the ferrite / bainite sample tested at a stress of 110MPa and 550°C for times between 300 – 1000h. | 133 |
| 4.18 | Void % analysis distribution chart for the 100% tempered bainite sample tested at 550°C and 100MPa for 300h. | 139 |
| 4.19 | Void % analysis distribution chart for the 100% tempered bainite sample tested at 550°C and 100MPa for 350h. | 140 |
| 4.20 | Void % analysis distribution chart for the 100% tempered bainite sample tested at 550°C and 100MPa for 400h. | 141 |
| 4.21 | Frequency distribution analysis for void orientation in the fully bainitic sample tested at 550°C, 100MPa for 400h. | 143 |
| 4.22 | Void % analysis distribution chart for the ferrite / bainite sample tested at 550°C and 110MPa for 700h. | 150 |
| 4.23 | Void % analysis distribution chart for the mixed ferrite / bainite sample tested at 550°C and 110MPa for 850h. | 151 |
| 4.24 | Void % analysis distribution chart for the mixed ferrite / bainite sample tested at 550°C and 110MPa for 1000h. | 152 |
| 4.25 | Frequency distribution analysis for void orientation in the ferrite / bainite sample tested at 550°C and 110MPa for 850h. | 154 |
| 4.26 | Void % analysis distribution chart for the ferrite / pearlite sample tested at 550°C and 85MPa for 3680h. | 161 |
| 4.27 | Void % analysis distribution chart for the ferrite / pearlite sample tested at 550°C and 85MPa for 6850h. | 162 |
| 4.28 | Frequency distribution analysis for void orientation in the ferrite / pearlite sample tested @ 550°C, 85MPa and 3680h. | 164 |
| 4.29 | Variation of a) CTOD, b) Crack extension and c) Void area %, with test duration for 100% tempered bainite and mixed ferrite / bainite specimens. | 171 |
| 4.30 | Variation of a) CTOD and b) Crack extension with void area % for 100% tempered bainite and mixed ferrite / bainite specimens. | 172 |
| 4.31 | CTOD against crack extension for a) all samples, b) expanded scale delineating crack initiation region, i.e. <1mm of crack extension. | 173 |
| Plate No. | Title | Page |
|----------|----------------------------------------------------------------------|------|
| 4.1 | Optical micrograph of the mixed pro-eutectoid ferrite / pearlite | 101 |
| | structure derived from pipe section fully annealed at 960°C. | |
| 4.2 | Optical micrograph of the mixed pro-eutectoid ferrite / bainite | 101 |
| | structure derived from as-received bar normalised at 960°C, tempered | |
| | at 705°C and stress relieved at 700°C. | |
| 4.3 | Optical micrograph of the 100% bainite structure produced by | 102 |
| | normalising at 560°C and tempering at 700°C for 24h. | |
| 4.4 | Low magnification SEM image of the ferrite / pearlite original | 102 |
| | microstructure. | |
| 4.5 | High magnification SEM image of the ferrite / pearlite original | 103 |
| | microstructure. | |
| 4.6 | Low magnification SEM image of the ferrite / bainite original | 103 |
| | microstructure. | |
| 4.7 | High magnification SEM image of the ferrite / bainite original | 104 |
| | microstructure. | |
| 4.8 | Low magnification SEM image of the fully bainitic original | 104 |
| | microstructure. | |
| 4.9 | High SEM magnification image of the fully bainitic original | 105 |
| | microstructure. | |
| 4.10 | TEM bright field Image of a thin foil specimen extracted from | 105 |
| | the as normalised material. Carbide precipitation observed between | |
| | the ferrite laths identified the structure as upper bainite. | |
| 4.11 | TEM micrograph of the carbide morphology in the ferrite / | 106 |
| | pearlite, as received, material. | |
| 4.12 | TEM micrograph of the carbide morphology in the tempered mixed | 107 |
| | pro-eutectoid ferrite / bainite. Starting material. | |
| 4.13 | TEM micrograph of the carbide morphology in the tempered, mixed | 107 |
| | pro-eutectoid ferrite / bainite starting material. | |
| 4.14 | TEM micrograph of the carbide morphology in the tempered 100% | 108 |
| | bainite starting material. | |
| 4.15 | SEM micrograph of the fully bainitic sample tempered at | 116 |
| | 700°C for 0.5h. | |
| 4.16 | SEM micrograph of the fully bainitic sample tempered at | 116 |
| | 700°C for 5h. | |
| 4.17 | SEM micrograph of the fully bainitic sample tempered at | 117 |
| | 700°C for 5h. | |
| Plate No. | Description | Page |
|----------|-----------------------------------------------------------------------------|------|
| 4.18 | SEM micrograph of the fully bainitic sample tempered at 700°C For 100h. | 117 |
| 4.19 | SEM micrograph of the fully bainitic sample tempered at 700°C For 1000h. | 118 |
| 4.20 | High magnification SEM image of the area outlined in plate 4.19 showing irregular growth of grain boundary carbides and associated precipitate free zones. | 118 |
| 4.21 | SEM micrograph of the fully bainitic sample tempered at 700°C For 5000h. | 119 |
| 4.22 | SEM micrograph of the fully bainitic sample tempered at 650°C for 50h. | 119 |
| 4.23 | SEM micrograph of fully bainitic sample tempered at 750°C for 50h. | 120 |
| 4.24 | Low magnification SEM image of the notch tip region in the fully bainitic sample tested at 550°C and 100MPa for 300 hours. | 139 |
| 4.25 | Low magnification SEM image of the notch tip region in the fully bainitic sample tested at 550°C and 100MPa for 350h. | 140 |
| 4.26 | Low magnification SEM image of the notch tip region in the fully bainitic sample tested at 550°C and 100MPa for 400h. | 141 |
| 4.27 | Montage of SEM images showing the microstructure in the area immediately adjacent to the notch tip in the 100% tempered bainite sample tested at 550°C and 100MPa For 400h. | 142 |
| 4.28 | High magnification SEM image of grain boundary voiding evident in the tempered, fully bainitic, material tested at 550°C and 100MPa for 400h. The red arrows indicate evidence of sub-structure boundaries adjacent to the cavitating regions. | 144 |
| 4.29 | High magnification SEM image of voiding at a grain boundary in the tempered, fully bainitic, material tested at 550°C and 100MPa for 400h. Elongated carbides, adjacent to PFZ's, are evident in the vicinity of the voids. | 144 |
| 4.30 | SEM micrograph showing a denuded zone adjacent to grain boundary voiding in the tempered, fully bainitic, material tested at 550°C and 100MPa for 400h. | 145 |
| 4.31 | SEM image showing voids opening up at triple points and evidence of atom migration down sub-structure boundaries in the tempered, fully bainitic, material tested at 550°C and 100MPa for 400h. | 145 |
| 4.32 | SEM micrograph showing the carbide morphology in a reference region remote from notch tip stress field in the tempered, fully bainitic, material tested at 550°C and 100MPa for 400h. | 146 |
| Plate No. | Description | Page |
|----------|-----------------------------------------------------------------------------|------|
| 4.33 | Low magnification SEM image of the notch tip region in the mixed ferrite / bainite sample tested at 550°C and 110MPa for 700h. | 150 |
| 4.34 | Low magnification SEM image of notch tip region in the ferrite / bainite sample tested at 550°C and 110MPa for 850h. | 151 |
| 4.35 | Low magnification SEM image of notch tip region in the mixed ferrite / bainite sample tested at 550°C and 110MPa for 1000h. | 152 |
| 4.36 | Montage of SEM images showing the microstructure in an area immediately adjacent to the notch tip in the ferrite / bainite sample tested @550°C and 110MPa for 1000h. | 153 |
| 4.37 | High magnification SEM image of voiding at a ferrite / bainite grain boundary. | 155 |
| 4.38 | High magnification SEM image of an area indicated in plate 4.37 above showing voiding at a grain boundaries and evidence of sub-structure boundaries in the bainite phase. | 155 |
| 4.39 | SEM micrograph showing large, irregular shaped, grain boundary carbide perpendicular to principal stress axis between two ferrite grains and adjacent to a denuded bainite grain. | 156 |
| 4.40 | SEM micrograph showing cavitation on a ferrite / bainite grain boundary and associated denuded region. | 156 |
| 4.41 | SEM image depicting cavitation on a ferrite / bainite grain boundary and growth of associated grain boundary carbides. PFZ evident in the ferrite grain adjacent to the grain boundary. | 157 |
| 4.42 | SEM image showing voiding in an intra-granular bainitic region of the mixed ferrite / bainite specimen tested for 1000 hours. | 157 |
| 4.43 | SEM micrograph showing carbide morphology in a reference region, remote from the notch tip stress field, of the ferrite / bainite specimen tested for 1000h. | 158 |
| 4.44 | High magnification SEM micrograph showing the carbide morphology in a reference region, remote from notch tip stress field, of the ferrite / bainite specimen tested for 1000h. | 158 |
| 4.45 | Low magnification SEM image of the notch tip region in the ferrite / pearlite sample tested at 550°C and 85MPa for 3680h. | 161 |
| 4.46 | Low magnification SEM image of the notch tip region in the ferrite / pearlite sample tested at 550°C and 85MPa for 6850h. | 162 |
| 4.47 | Montage of SEM micrographs showing the area adjacent to the notch tip in the ferrite / pearlite sample tested @550°C and 85MPa For 3680h. | 163 |
| 4.48 | SEM image showing voiding at a grain boundary between the ferrite and pearlite phase. | 165 |
| Plate No. | Description | Page |
|----------|-----------------------------------------------------------------------------|------|
| 4.49 | Voiding at a grain boundary between the ferrite and pearlite phase. | 165 |
| 4.50 | Voiding at a grain boundary between the ferrite and pearlite phase. | 166 |
| 4.51 | Voiding at a grain boundary between the ferrite and pearlite phase. | 166 |
| 4.52 | SEM micrograph showing the carbide morphology in a reference region, remote from notch tip stress field, of the ferrite / pearlite specimen tested for 3680h. | 167 |
| 4.53 | TEM micrograph showing the typical carbide morphology observed in a reference region of the tempered bainite CT specimen tested at a temperature 550°C and a stress of 100MPa for 350 hours. | 178 |
| 4.54 | TEM micrograph showing the typical carbide morphology observed in the notch tip region of the tempered bainite CT specimen tested at a temperature 550°C and a stress of 100MPa for 350 hours. | 178 |
| 4.55 | Typical boundary oriented at an angle with respect to the notch tip in the fully bainitic CT specimen tested at a temperature 550°C and a stress of 100MPa for 350 hours. Evolving carbides identified for EDX analysis in Plates 4.56 – 4.58. | 179 |
| 4.56 | Brightfield image of feature A in micrograph depicted in plate 4.55 and the corresponding elemental ratios for Fe:Cr, Mo:Cr and Mo:Fe determined by EDX analysis. | 179 |
| 4.57 | Dark field image of feature B in micrograph depicted in plate 4.55 and the corresponding elemental ratios for Fe:Cr, Mo:Cr and Mo:Fe from EDX analysis. | 180 |
| 4.58 | Bright field image of feature C in micrograph depicted in plate 4.55 and the corresponding elemental ratios for Fe:Cr, Mo:Cr and Mo:Fe from EDX analysis. | 180 |
| 4.59 | Growth of M₂C from M₂₃C₆ on a grain boundary almost parallel to the maximum principal stress in the notch tip region of the fully bainitic CT specimen tested at a temperature of 550°C and a stress of 100MPa for 350 hours and corresponding elemental ratios for Fe:Cr, Mo:Cr and Mo:Fe determined by EDX analysis. | 181 |
| 4.60 | M₆C type carbides at grain boundary triple points in the fully bainitic CT specimen tested at a temperature of 550°C and a stress of 100MPa for 350 hours. | 181 |
| 4.61 | High magnification TEM image of an elongated grain boundary carbide in the notch tip region of the fully bainitic CT specimen The elemental ratios of Fe:Cr, Mo:Cr and Mo:Fe, determined by EDX analysis, are indicated in this micrograph. | 182 |
| Plate No. | Description | Page |
|----------|-----------------------------------------------------------------------------|------|
| 4.62 | TEM micrograph showing the typical carbide morphology observed in a reference region of the ferrite / bainite CT specimen tested at a temperature 550°C and a stress of 110MPa for 850 hours. | 183 |
| 4.63 | TEM micrograph showing elongated carbides along a ferrite / bainite grain boundary oriented at an angle with respect to the notch tip and maximum principal stress. | 183 |
| 4.64 | High magnification TEM image of the elongated grain boundary carbide observed in plate 4.63 and corresponding elemental ratios for Fe:Cr, Mo:Cr and Mo:Fe determined by EDX analysis. | 184 |
| 4.65 | Evolution of M$_{23}$C$_6$ to M$_6$C by the gradual absorption of elements from adjacent carbides on a non-critically oriented ferrite / bainite grain boundary. | 185 |
| 4.66 | M$_7$C$_3$ type carbide at a typical ferrite / ferrite grain boundary in the ferrite / bainite CT specimen and corresponding elemental ratios for Fe:Cr, Mo:Cr and Mo:Fe. | 185 |
| 4.67 | Growth of M$_7$C$_3$ type carbide at a ferrite / ferrite grain boundary by absorption of adjacent M$_6$C carbides in the ferrite / bainite CT specimen. | 186 |
|
APRIL 1999
APRIL CHAIRMAN: DICK GOLZE
2 Speaker: Charles Forbes, Forbes Management, Inc.
Subject: “Moving the Gem Theatre”
Applicants invited S - Z
9 Speaker: Thomas G. Cutler, Brigadier General, Michigan Air National Guard
Subject: “127th Wing Role in National Defense”
Applicants invited A- E
16 Speaker: Honorable Candice Miller, Michigan Secretary of State
Subject: “Update and Innovations”
Applicants by Reservation
23 Speaker: Sheila R. Ronis, Ph.D., President, The University Group
Subject: “Issues of National Security in the 21st Century”
Applicants invited F - L
30 Speaker: John King, Partner, Foresight Research Group
Subject: “Market Research...It’s much more than Political Polling”
Applicants invited M - R
MAY 1999
7 Speaker: Andrew Aljian, Consultant with Arthur Gallagher & Co.
Subject: “Life Insurance & Estate Planning”
Applicants invited A - E
Discussion Groups
APRIL CHAIRMAN: LARRY PRICE
Apr. 2 Speaker: Beverly Hannett-Price, High School English Teacher
Topic: “Literature for Leisure”
Apr. 9 Speaker: Dick Harper, Senior Men’s Club
Topic: “Member Survey Results and Recommendations”
Apr. 16 Speaker: Bret Tillender, Director of Communications
American Heart Association of Michigan
Topic: “Operation: Heartbeat!”
Apr. 23 Speaker: Glen Shilling, Asst. Headmaster, Detroit Country Day
Topic: “Private Education in Our Community”
Apr. 30 Speaker: Stewart Keeny, Senior Men’s Club
Topic: “Hospitals and Nursing Homes – Patient’s Point of View”
Two Outstanding Special Events
Bus Trip of Detroit
A look at Detroit - past and present – is planned for club members May 5. Conducted by Frank Angelo, the tour by bus will depart from the First Presbyterian of Birmingham Church parking lot (on Maple) at 8:45 a.m. and return about 4:30 p.m.
There will be stops for lunch at the Scarab Club and dessert at the Hellas Cafe in Greektown. Also, there will be time for shopping.
Cost is only $23. Previous Detroit Tour trips have been “sell-outs” so sign up promptly. Tickets will be on sale starting April 2.
DSO Does Jazz
A celebration of musical Americana featuring New Orleans and Dixieland style jazz – with a special appearance of the Preservation Hall Jazz Band – is a Detroit Symphony Orchestra concert offered to Senior Men’s Club members at 10:45 a.m. on June 10 in Detroit’s Orchestra Hall.
A block of 55 main floor tickets has been secured at a reduced rate for the two-hour program in the DSO’s “Pops Series.”
Cost for ticket and transportation is $29 a person. Tickets will be on sale at Club luncheons standing April 16.
Transportation (by bus) will depart from the First United Methodist Church of Birmingham (located on Maple Road and Pleasant) at 9:45 a.m. and will return there about 1:30 p.m. Cars can be left in the church parking lot.
Investment Study
David Sowerby, senior portfolio manager and economist, Loomis Sayles & Associates will speak to the Investment Study Group at its April 14 meeting starting at 2:45 p.m.
Sowerby will discuss “When Will Sanity Return for Value Style Investing?”
Sowerby is a frequent media spokesman on local and national financial issues. He addressed the Birmingham-Bloomfield Chamber of Commerce in February for the seventh time. “He will help us find good value / low risk investments,” says George Miller, group chairman.
Computer Group
Not wanting to be left out, the Computer Group has distributed a questionnaire to find out what kinds of programs group members want.
“The most popular request was to have John Wigman, the owner of Birmingham Computer, back again, says Don Cox.”
“John has talked to us a couple of times previously and he is very capable of diagnosing and fixing computer problems. He will even go to your home to work on your computer.
John has agreed to discuss how we can check our PC’s to make sure they are Y2K ready. He will bring along some floppy disks which we can use to make our PC’s work properly on Jan. 1, 2000.”
The meeting will be on April 21 at 2:30 p.m.
Camera Group
Harold Brown will present “Motoring through Bavaria and Switzerland” to the group on April 13 at 2 p.m. Harold has already established his expertise with tales and pictures of some of his many other trips, so we are looking forward to this presentation too. Members, Applicants, their families and friends are welcome.
New Club Interest Groups Considered
The recent member research study resulted in several suggestions as to possible formation of additional special interest groups within the club. Principal suggestions were:
- Choral Group
- Additional Card Games, especially Pinochle
- Classic Car Owners
- Musical Group (Instruments)
- Book Discussion Group
The Executive Board has approved determination of potential member interest in each of these groups. Any member or applicant who might be interested should contact Corresponding Secretary Ben Ewing either at a Friday meeting or at (248)334-6872.
Phil Martz Top Bowler
The leading team for the second half with 30 1/2 points includes Frank Garland, Dennis Kozak, John Wargelin and Norris Wetters.
Individual Leaders
- High Average Phil Martz 175
- High Game Dick Harper 247
- High Series Phil Martz 640
Team High Scores
- High Team Game 742
(Ray Latovick, Don Doty, Bob Klein)
- High Team Series 1993
(Ed Neill, Harry Mitchell, Vince Caputo, Bob Jordan.)
Competition continues hot for first place. The season ends April 12 when the winners of each half meet for the League championship.
MEMBERSHIP NEWS
Special Birthday Greetings: Charles Latham, will be 91 on April 2.
Associate Member Birthdays: April 5, Richard Jeffries.
Became Life Member: John Frailey, who joined in March, 1989.
Deaths: John Hoffman, Life Member, who joined in Feb., 1972.
Resigned: Irwin Peterson, was on leave..
New Members:
- Auld, Frank, 5436 Fairway Ct., West Bloomfield, 48323, 737-0239. Sponsor: Joe Maertens.
- Horiszny, John, 1058 Arden Lane, Birmingham, 48009, 646-3470. Sponsor: Stan Skaisitis.
- Salrin, James C., (Myra), 4611 Ranch Ln., Bloomfield Hills, 48302, 642-0299. Sponsor: Harold Jackson.
- Sheren, David, (Patricia), 279 S. Cranbrook Cross, Bloomfield Hills, 48301, 540-3792. Sponsor: Cliff McClew.
- Slocum, John D., (Mary), 319 Hillboro Dr., Bloomfield Hills, MI 48301, 646-7230. Sponsor: Don Upward.
- Snapp, Leon A., (Alice) 16360 Beechwood, Beverly Hills, 48025, 642-7896. Sponsor: Fran DeGrande.
New Applicants:
- Cameron, Scott R., 1721 Stanley, Birmingham, 48009, 645-2537. Sponsor: Jim Cameron.
- Catchpole, CLive E., (Gillian), 31050 Stafford, Beverly Hills, 48025, 647-4794. Sponsor: Phil Werner.
- Compton, William A., 1050-A Trailwood Path, Bloomfield Twnshp. 48301, 855-8697. Sponsor: Bill MacLachlan.
- Harder, Arthur S., (Olive), 859 Ridgedale, Birmingham, 48009, 647-0929. Sponsor: Herm Peters.
- Reddy, John W. (Joan), 650 Wattles Road, Bloomfield Hills, 48304, 645-1874. Sponsor: Bob Hagemeyer.
- Saylor, John M., (Annette), 2833 Arlund Way, Troy, 48098, 267-8444. Sponsor: Kirby Callam.
– Walt Meyers & Bob Babcock
Sure Signs of Growing Old
There are a few chuckles about the senior generation.
Everything hurts and what doesn’t hurt doesn’t work.
Your little black book contains only names ending in M.D.
You get winded playing cards.
Your children are beginning to look middle-aged.
You join a health club and don’t go.
A dripping faucet causes an uncontrollable bladder urge.
You know all the answers but nobody asks you the questions.
You look forward to a dull evening.
You need glasses to find your glasses.
You turn out the lights for economic rather than romantic reasons.
You sit in a rocking chair and can’t get it going.
You sink your teeth into a steak and they stay there.
Your knees buckle but your belt won’t.
Your back goes out more than you do.
You have too much room in the house and not in the medicine chest.
—Author Unknown
Golf Begins April 8
Think Spring. Think Swing (golf that is)
**ACTIVE GOLF**
The golf season begins for regular club members at 9 a.m. at Springdale, April 8, weather permitting. Ray Swartz says that foursomes and tee times have been established and are posted on the club bulletin board each Friday. Walk-ons are always needed and encouraged.
Ray indicates that Springdale will have a new experienced manager, and that several course improvements are expected. Fee for 9 holes will be $6.50 – the same as last year. Call Ray at 646-3129 if you have any questions.
**APPLICANT GOLF**
Applicant golf starts April 19 at 8:30 a.m. at White Lake Oaks Golf Club. Tee times and foursomes will be posted on the bulletin board. Fran DeGrande indicates that a few players have yet to pay the season fee of $162. Call him at 489-1064. Walk-ons are always welcome. Players should plan on arriving 20-30 minutes prior to their starting time.
**DOME GOLF**
All Club golfers get one more chance to practice at the Golf Dome on Thursday, April 1.
Lezy and Kern Lead Bridge Players
Mark Lezy took honors in duplicate bridge play with Joe Kern leading the contract players during Feb-March, per Don McNair.
**DUPLICATE**
| Player | Percentage |
|------------|------------|
| Mark Lezy | 70% |
| Omer Petti | 68% |
| Bob Elliott| 64% |
**CONTRACT**
| Player | Score |
|------------|------------|
| Joe Kern | 2400 |
| Carl Schorn| 2098 |
| Russ McCarty| 2073 |
| Jane Piggott| 1845 |
| Henry Dawkins| 1730 |
Club Dates To Remember
**Apr 13:** Executive Board, 9:30 a.m.
**Apr 16:** Reminder Deadline
**Golf:** Active members start April 8 at Springdale (weather permitting) at 9 a.m.
Applicants start April 19 at White Lake Oaks at 8:30 a.m.
**Bridge:** Tuesdays at 9 a.m. and Fridays after lunch.
**Bowling:** At Thunderbird Lanes
Mondays, 9:15 a.m. through April 12.
New Life Member
Applicant Activity
Applicants who plan to attend the Friday, April 16 meeting will require advance reservations to hear speaker Candice Miller, Michigan Secretary of State. Only 35 places available.
For reservations call George Falls at (248) 645-0698 or Tony Zar (248) 647-2274.
Some Observations to bring a few smiles
Have the courage to live. Everyone can die. – Robert Cody.
If Patrick Henry thought that taxation without representation was bad, he should see how bad it is with representation. – Old Farmer’s Almanac.
A magazine once asked J. Paul Getty to submit a short article explaining the secret to success. He returned the following: “Some people find oil. Others don’t.”
By the time you’re 80 years old, you’ve learned everything. You only have to remember it. – George Burns.
“Young men want to be faithful and are not;
Old men want to be faithless but can not.”
“LIVE AND LEARN AND PASS IT ON”
By Harrison Brown, Jr.
“I’ve learned that the prayer I say most is: ‘Lord, please keep your arm around my shoulder and your hand over my mouth.’”
“I’ve learned that an adult is someone who stopped growing except around the middle.”
“I’ve learned that you should hope and work, but never hope more than you work.”
“A peacock today may be a feather duster tomorrow.”
“You should never jump off a diving board when you are wearing a bikini.”
“I’ve learned that after being on a diet for two weeks, all I lose is 14 days.”
“I’ve learned that all people have both good and bad traits. The secret of a happy marriage is to concentrate on your spouse’s good traits.”
“Old age is not a defeat but a victory, not a punishment but a privilege.”
“The only way to grow old gracefully is to keep active.”
“You’re never too old to try something new.”
“All grandchildren are beautiful, brilliant and take after their grandparents.”
“What we’ve done for ourselves dies with us. What we have done for others and the world remains and is immortal.”
“If you die broke, your timing was absolutely right.”
“I’ve learned that home is the place where we grumble the most and are loved the best.”
“I’ve learned that first graders are the only ones who think it’s neat when their teeth fall out.”
“If you finish the toilet paper roll without replacing it, you’re the first person who needs it.”
“Absent-minded people get lots of exercise looking for things they can’t find.”
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Berlin cop serves town and country
By Josh Davis
Associate Editor
(Oct. 20, 2016) Cpl. Merle Bragg was less than 24 hours away from another lengthy deployment with the U.S. Army National Guard. It would have been a familiar story for the 50-year-old veteran, before the state granted a hardship approval memorandum to keep him on duty with the Berlin Police Department.
A native of northern Maine, Bragg said the service “kicked [him] around” between different units until he ended up in Maryland 26 years ago. He has been a member of the Berlin Police force for two decades.
For Bragg, entry into the service had been a foregone conclusion since middle school.
“I joined the delayed entry program with the Army between my junior and senior year of high school. I told my dad when I was 12, I wanted to be a soldier,” he said. “I was a farmer [and] didn’t want to be a farmer. I wanted to do something else.
“In the small towns, you always have the patriotic, loyalty-type duty thing going on, and that was instilled with me,” Bragg continued. “I’ve have members of my family – uncles and grandfathers – that were involved in it. I just decided that was going to be my career path.”
After graduating from high school, Bragg served four years active duty, including Army Airborne school at Fort Benning, Georgia and a tour with the 11th Armored Cavalry.
He also served with the 101st Airborne at Fort Campbell in Kentucky, and then left active service to attend college. Always a soldier, Bragg joined the National Guard in Georgia immediately after his active-duty discharge.
From there, he was transferred to the Maryland Army National Guard, where he remains to this day.
At last count, Bragg said he’s been “well up into the higher teens” in the number of countries in which he has been stationed. He has not had any say in where he goes, he said.
“Uncle Sam is usually pretty good at coordinating my travel itinerary. It’s never been a question – it’s part of the uniform,” he said. “They say this is where you’re going to go and when you’re going to go, and this is how long you’re going to be there. And you stand up, give them a salute, ‘Yes sir,’ and off
See LAST-MINUTE Page 6
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Pocomoke hears grim report from fiscal year 2016
By Brian Gilliland
Associate Editor
(Oct. 20, 2016) Partner Mike Kleger and Manager Leslie Michalik of Pigg, Krahl and Stern accountants delivered Pocomoke’s financial statements and audit report for fiscal 2016, which ended June 30, and noted a deficit in net position of $639,000 from last year.
Net position is the term, adopted in 2011, that replaces net assets. The shorthand in describing the term is if 100 widgets were purchased for one dollar each, but are now trading for $9, the net position is worth $900, or the total current value minus the cost of the initial investment.
“The City’s combined net position, after depreciation, at June 30, 2016 was $10,705,000. Of this total, $13,400,000 is invested in capital assets or restricted for special revenue leaving an unrestricted deficit in net position of $2,696,000,” the report reads.
Meaning, the town’s liabilities outweigh its assets by almost $2.7 million.
The auditors recommended eliminating the “special revenue” fund in a separate report, also delivered during Monday’s regular council meeting, advising that in 2016, only funds from Community Development Block Grants were tracked there. Previously, the fund was used to account for CDBG revolving loans and Section 8 programs.
Revenues increased slightly between 2015 and 2016 according to the report — by about $28,000 and expenses grew at a slower rate year over year during fiscal 2015 and 2016.
In fiscal 2015, the total deficiency in net position, encompassing both governmental and business-type activities is about $374,000. In 2016, the number grew to about $639,000, a difference of about $293,000 between the years.
On the governmental side of the equation, the town was set to realize a deficit of $154,000, but transferred $242,000 to the water/sewer fund to offset a nearly $500,000 loss on the
See INCREASES Page 8
DDC to celebrate decoy culture
By Brian Gilliland
Associate Editor
(Oct. 20, 2016) For the first time in its history, the Delmarva Discovery Center in Pocomoke City will explore the rich history and folklore surrounding Lower Shore hunting traditions and culture this weekend with the inaugural Delmarva Heritage Days event.
Members enjoy free admission to the event that begins on Saturday at 10 a.m. and runs until 4 p.m. and continues Sunday starting at noon and ending at 4 p.m. Non-members can expect single day charges of $10 per adult, and $5 per youth. Children 4 years old and younger are free. Two-day passes are also available at $15 each per adult and $8 per youth.
“We’re trying to promote storytelling — we want to hear the stories,” Barbara Tull, founder and board member of the center, said. “We’re having seminars both days — we want to capture the stories.”
To that end, invited carvers include Don Briddell, of Crisfield whose work is part of the Smithsonian Institution’s permanent collection, recipient of the Ward Museum of Wildfowl Art’s Living Legend award Rich Smoker, from Marion Station, Maryland, Oliver “Toots” Lawson, also of Crisfield who produces highly sought after decoys and Cameron McIntyre, who lives on the Eastern Shore of Virginia and carves decoys exclusively through the use of hand tools.
Also, on the decoy side, will be appraisals of personal pieces or collections, as well as one-on-one access to some of the best carvers the area has to offer and carving demonstrations.
There will also be stories from the old Assateague Island and Barrier Island Hunt Clubs, and more exploration into the market and outlaw gunners of days past.
“The city folk decided they liked wild ducks, so the market hunters filled that need by killing ducks and shipping them to market. The outlaw hunters did the same thing, but they broke the law to do so,” Tull explained.
These types of hunters regularly employed punt guns, or large, boat-mounted shotguns capable of downing a large number of birds should the hunter be able to maneuver close enough. One boat could be equipped with several punt guns at once.
“We’ll also be giving out three heritage awards for perpetuating and sustaining carving traditions,” Tull said. “Each awardee will get a bronze statue from Turner Sculpture.”
Duck blinds, shooting rigs, duck callers and roundtable discussions will also be offered.
Children’s’ activities will be offered, including painting duck silhouettes.
Shucked oysters, oyster sandwiches, hamburgers and hot dogs will be available for purchase.
OPA directors appear to lean toward renovating golf club
By Josh Davis
Associate Editor
(Oct. 20, 2016) While a decision on whether to renovate or replace the ailing country club was not made during a special board of directors meeting Monday in Ocean Pines, the message was clear – the community is not likely to support another costly referendum to spend millions on a new building.
Interim OPA General Manager Brett Hill said mold remediation on the building had been successful, and that bids to replace the roof of the country club would be on the agenda during a regular board meeting on Saturday. He estimated that cost at about $93,000.
While Director Pat Supik said it was obvious most of the directors would favor a rehab of the building, she worried about spending too much money “without knowing the endgame.”
“We’re kind of making decisions on individual pieces in a blind manner,” she said. “Maybe just a little bit longer range or a bigger-picture idea with the country club would be helpful.”
Director Cheryl Jacobs agreed.
“I have a concern that we’re, as Pat said, we’re going little by little with a big number, which maybe we shouldn’t be doing if, in fact, the ultimate decision is tear down and consolidate and make it smaller,” she said.
Hill said that staff was working on a report on different options for the building and that it would be ready for a board meeting in December.
Part of that discussion, he said, would be “what do we want the building to be.” Golf management company Landscapes Unlimited had said the building was too big for their needs.
If OPA opted to replace the building, Hill said trailers would likely need to be rented to accommodate continuing golf operations for up to three years, perhaps costing several hundred thousand dollars.
“That’s why we approached just stabilizing,” he said. “We do have a report that showed that building is absolutely solid. There’s no structural issues. The building could stand for another 100 years.”
Director Slobodan Trendic appeared to question the entire golf amenity, saying losses had totaled almost $5 million dating back to 2010, including depreciation. He said he was not in favor of replacing the building.
“The question we need to ask is about the responsibilities we have to the homeowners,” he said. “Can we continue to carry and subsidize what appears to be a very costly amenity to the community?
“Historically speaking, the numbers are not very attractive when it comes to golf course operations,” Trendic continued. “They’re just a burden, a drain of our financial resources and we need to have a long-term solution to address this problem.”
Director Doug Parks noted that a recent engineering report estimated a new, smaller 8,000-square-foot building had an estimated cost of $1.7 million.
“Looking at that number as kind of a benchmark … might give us a better idea whether or not it’s feasible,” he said, adding that number should be weighed against estimated costs for an extensive renovation. “I want to really investigate those numbers and say, from a repair perspective after the initial required investment, what’s a number that gets us to operational efficiency?”
Vice President Dave Stevens said it would be his choice to renovate the building because that would likely be more cost-effective overall, but admitted that would likely not be the most “straightforward path.”
“If we’re going to replace it, it’s not going to be our choice – it will not be our choice,” he said, hammering on the desk. “We can go out to referendum … but it’s going to be everybody’s choice.
“Maybe we make a really good case for it, we say, ‘Look, we’re saving your money in the long run,’ [but] we have to sell that idea,” Stevens added. “I don’t like my chances at all, therefore I do believe that, at a minimum, we ought to start by focusing on what we have to do to renovate that building, initially to make it operationally useful, and then, not long afterwards, to make it functional – and in a way that will serve the benefit of the entire community.”
When board President Tom Herrick asked for a consensus from the board, Trendic said he did not want to spend money on a new building. Supik and Jacobs both said they needed more information, and Jacobs added she was not as pessimistic about a referendum as some board members were.
Others pointed to the millions recently spent on a new yacht club, opened in 2014, which many in the community were not happy with after the fact.
“We have not had good success at pricing things out,” Stevens said, adding that estimates to replace the yacht club started at about $2.7 million. Final costs were closer to $5 million.
“And the yacht club’s still not done,” Hill said, drawing laughter from those at the meeting.
Three public comments also favored renovation.
“The odds of passing a referendum now to spend $2-to-$3-to-$4 million on a new golf club, I think, are slim-and-none,” resident Joe Reynolds said. “If you want to be generous, five percent, maybe.”
Last-minute hardship order keeps police cpl. in Berlin
■ CPL BRAGG continued you go.”
He toured Iraq during the “early part of the war,” and was part of the military police unit from Salisbury that responded to the Pentagon immediately following the events of Sept. 11, 2001.
“We went to the Pentagon for the initial securing of the scene,” he said. “We were the first unit to go down to Guantanamo to open that facility down there. And when we went into Iraq, we maintained the high-value detainee facility. Part of that was because we had been the first ones to do the Guantanamo mission – we knew we could handle the high-value individuals.”
What will stick with Bragg the most about the events of the 9/11 terrorist attack was how quickly the country rallied.
“From what I observed, initially after 9/11, was the camaraderie where everyone pulled together,” he said. “There was a huge outpouring of patriotism, of loyalty, of ‘U.S.A.’ – all of that. When I went back, three years later, the sense of security had obviously been tightened. People were obviously paying a lot more attention to what was going on.
“I wouldn’t say it made for a bad work environment, but it’s something that everybody should pay attention to, even your everyday life” Bragg added. “Pay attention to what’s going on around you – don’t become an automatic victim. And that goes right in line with the police department. We try to tell the folks constantly – if it doesn’t look right, if it doesn’t smell right, it probably isn’t right. At least have somebody understand and come by and check it out. The military is no different.”
Most recently, in 2012, the Salisbury outfit was deployed to Afghanistan, where Bragg said he was “borrowed” from his current unit to serve as the operations sergeant major.
“That was an interesting tour,” he said. “The Guard is being asked to do a lot that active-duty units used to do, and for the most part it’s showing that we can definitely do that job.”
“I’ve spent more time deployed with the military at the end of my career than I ever did at the beginning of my career,” Bragg continued. “Ever since 9/11, the Berlin Police Department has done really well in supporting our military. We have one other officer here in the department who is still actively engaged with the Maryland Guard, and for a small agency, sometimes that makes for some interesting logistical issues.
“The town was really good back in ’04,” he said. “After I came back from Iraq, I went up and I worked in D.C. for a number of years. The town maintained my job and I came back, obviously out of loyalty to the department.”
All of those experiences have helped Bragg be much more aware of his surroundings in his job with the Berlin Police Department.
“It’s something that almost becomes second nature, because you know you’re in an environment where you never know what’s going to happen, and when you come back you become more lax, but you begin to notice a lot of things that you really didn’t pay attention to before,” he said.
Bragg was due to leave last Friday morning, after finishing a police shift on Thursday that ended at midnight. He was ordered to go to Camp Frederd in Reisterstown for records review with the 29th Infantry. He would have reported at 6:30 a.m. that morning.
At about 3 p.m. last Thursday, he learned the trip – at least his part in it – had been canceled. He would have been deployed in the Middle East, where he said he’s been in and out “a couple dozen times.”
Most orders in the military are written for 400 days, so it was likely that Bragg would have been gone for up to a year.
Asked about how he thought people’s perceptions of current military activity differed from reality, Bragg said, “People just need to remember that the culture is completely different [overseas].”
“It’s not something that isn’t understandable, it’s just that we, here, have had it so good for so long,” he said. “If I want to walk down to the local grocery store and get an apple, I can walk down to the store and get an apple. You don’t always have that same capability over there. It’s dependent upon where they live, how they’re going to get there, what’s available to them, what type of travel means, mode and security are they going to have to do today to get there.
“Sometimes when you sit and compare life here in the United States to somewhere out in the middle of a freakin’ desert somewhere, there’s just no way to make people understand just how good they really do have it,” he said.
Bragg said he has always been a proponent of mandatory service for all U.S. citizens and that everyone should serve a minimum of two years.
“One, it gives them their bearing, because when you’re 17, 18 years old do you really know what you want to do? Two, it gives you a little bit of discipline, and three, you actually get to see something other than what your little bubble has been during your childhood years,” he said. “My momma won’t let me have the newest iPhone. In the big scheme of things, that really doesn’t count for anything.
“It’s like, you wake up the next morning, ‘Momma, am I going to have a roof over my head, are the animals going to die today, and am I going to have anything to eat tonight?’ That makes a big difference,” Bragg added. “The power goes out for 10 minutes, and — oh my God — you don’t know how to read a book anymore.”
“I’m a soldier … I do as I’m ordered”
One thing Bragg won’t be spared because of the cancellation is what he termed the “proverbial honey do-list” that typically greets him upon his return.
“No matter how well you prep before you go, s..t will break,” he said. “Usually [his wife] is really good about being able to take care of the day-to-day stuff, but it’s the oddball stuff. She’s pretty self-sufficient.”
“The first time, when we were doing the initial deployment, I got down to Cuba and she called me and said that something happened to one of the rotors on the front brakes on the pickup truck. She was all upset that she ruined the truck. First off, you haven’t ruined it, and please take it to a mechanic because I’m in Cuba.”
What he misses the most when he’s deployed overseas for an extended period, Bragg said, is “good television.”
“Overseas, if you’re lucky enough to get a signal and have the equipment to be able to watch it, the only thing you’ve got is AFM – the Armed Forces Network,” he said. “Usually things are three or four months behind, except for the news, of course. I miss the ability to sit back in a recliner in your own living room and turn on the TV and watch one of the stupid, brain cell-killing sitcoms that’s on.”
When he returned from his first permanent station, in Germany, Bragg remembers sitting and watching the traffic, just “to watch American cars go by.”
“And believe it or not, American commercials,” he said. “It’s just weird things. Jump on a motorcycle, go a couple hundred miles and get off and have an ice cream, then ride back on the back roads. It’s the sense of being free enough to do what you want to do.”
Bragg admitted he was “coming to end of [his] career” after serving his country for more than three decades.
“It’s a young kids’ game,” he said. “But there’s still gotta be a few of us old dinosaurs to stick around to make sure some of the old traditions are still maintained. There’s been quite the change from when I first came in to what it is now. Some of it’s OK, some of it’s not bad, and some of it sucks.
“That’s another idiom of the military: ‘Joe’s got the right to bitch and complain about something,’” Bragg added. “He’s going to do it no matter what you do.”
As for his most recent order, this time to not deploy, Bragg said he would continue to what he has done for more than three decades – serve his country to the best of his abilities, however it sees fit.
“I’m a soldier,” he said. “I do as I’m ordered. If the military has deemed I am needed more to stay and work emergency services at home, then I stay at home.”
DNR cautions drivers during annual whitetail deer rut
Breeding spree to end next month; most injuries occur as drivers attempt to avoid
By Brian Gilliland
Associate Editor
(Oct. 20, 2016) Most injuries, and a healthy percentage of the fatalities suffered during car accidents involving whitetail deer come from drivers avoiding, rather than striking the animals as they pursue each other during mating season.
That’s, according to Brian Eyler, the deer project leader from Maryland’s Department of Natural Resources.
“It’s just getting started now — bucks are chasing does, the does are chasing bucks and love is in the air,” he said.
In the rare cases there are human fatalities from a crash involving an automobile and an animal. There also are times that motorists suffer severe injuries when the animal ricochets off one car and onto another.
More frequently, however, injuries occur for drivers when they swerve to avoid striking an animal and end up hitting a tree, another vehicle or running off the road instead.
“You’re better off bumping a deer than driving into traffic,” he said.
And forget the deer whistles on cars, Eyler said.
“We’ve got pretty good research that shows none of the deer-repelling devices work,” he said. “They might chase off a deer, but it’s just as possible they’ll run into the road. The best defense is to be aware, first and foremost.”
Deer are typically most active at night, at dusk and at dawn, he said.
Typically, and somewhat apocryphally, the whitetails’ rut begins after the first full moon in October — called a Hunter’s Moon.
“The moon doesn’t really have anything to do with it. The moon is just going through its regular cycle and the mating season begins,” Eyler said.
The Hunter’s Moon was last weekend, but Eyler said, activity will increase until about mid-November until it begins to fall off again. The annual rut is when does are most fertile.
During this time, both sexes engage in the thrill of the chase, even across roads and highways. This year could be particularly dangerous especially in the southern end of Worcester County, where recent flooding has pushed motorists off the more popular routes and onto side streets and back ways to their destinations.
“There is no gender or age discrepancy in which animals are more likely to be struck,” he said. “They’re struck in proportion to their prevalence in the existing population.
Eyler said State Farm Insurance releases annual projections of deer populations by state. According to those figures, he said, there are nearly 30,000 whitetail deer in Maryland.
“The population is stable, which means we still have a lot of deer. We’re looking at ways to bring that number down,” he said.
Increases in salary, wages drive net position downward
■ POCOMOKE BUDGET continued business activity side.
The losses on the government side are explained in the report by increased expenses in general government, public safety and urban development/housing. In addition, the PCPD is adding a 17th officer this month, in addition to promoting several officers and issuing a blanket raise to all PCPD officers, the report states. Another expense related to police is the recent switch to the State Law Enforcement Officers Pension System, which has a cost of $55,000 — but that is expected to be offset by a reduction in overtime.
On the business side, Pocomoke City’s position slid another $243,000 due to increases in salaries and wages; materials and supplies; and utilities.
Finally, as of June 2016 the city’s fund balance was about $170,000 according to the report, which is a decrease from $229,000 last year. Klegler said neither number was particularly healthy and suggested the town attempt to build the balance up to about $750,000.
Board appears to favor hiring search firm to replace GM
■ MANAGEMENT SEARCH continued
agers, but they have HOA experience – they haven’t dealt with a police department,” he said, adding that Ocean Pines was more “county-like” than traditional homeowners’ associations.
“We have a population here that’s larger than two of the counties here on Delmarva – that’s a huge number,” he said. “I think we’re beyond [being] able to recruit and we’re beyond what a management company could do for us. We need the right person and we need a better means to find that person.
“While I know we have many talented residents that live in the neighborhood, I think a working group of our residents is not going to do us the best service and define the correct executive recruiter that could place [an] almost municipal-level position and conduct a search that reaches beyond our geographic footprint here to find all of the candidates so we have a good pool to choose from is the direction we need to go. That’s my personal recommendation,” Hill added.
Director Cheryl Jacobs said that was “exactly what [she] wanted to hear,” adding that the initial job description might have been too narrow.
Trendic maintained he still saw a benefit of having the work group, and asked if the board had considered how it would vet potential general manager candidates. He said he not seen any applications, “except for maybe one.”
He suggested the board examine what similar associations, specifically Columbia, Montgomery Village and Monticello in Charlottesville, Virginia, had done.
“We have the opportunity to really look at and learn from other associations, because we don’t have other unique challenges – there are other associations that have the same kind of operations that we do,” Trendic said.
He added his disappointment that the association could not easily fill the role internally, calling that a failure.
“Whether this was a planned situation or not, I think as an association we have a lack of senior staff,” Trendic said. “It’s one of the major challenges that really, in a way, should not have happened had we had the right, perhaps, approach from a board perspective in terms of setting the priorities, how we govern the association, how we mentor the internal talent. Just the fact that we really have not even said that there is one single person inside this association that could fill a void of one of those two positions, to me, is a failure.”
“I would challenge that, because I believe we’re fulfilling those positions just fine – there is not failure to fulfill anything here,” Hill said.
Trendic clarified that he believed Hill was doing a “phenomenal job” in the interim role, but said “for us to rely on our own opinions ... might be an oversight.”
Hill suggested the board get formal proposals from three different companies “so we can understand the costs, the services they offer, their areas of expertise, and then we as a board can use our sound judgment to use the best authority to search for that position.”
When a final list of candidates came in, he said the entire board should run the interview process together.
“If we have an industry expert recruiting the position, then we have everything we need to bring that person to the table,” Hill said.
Jacobs agreed and Director Pat Supik, added, “I would support that.”
Vice President Dave Stevens said the board must remain involved and kept aware of the process “every step of the way.”
“Hopefully the process is as reasonable and visible as it can be,” Stevens said.
Pines Chamber of Commerce awards recognize members
By Greg Ellison
Staff Writer
(Oct. 20, 2016) The 17th annual Ocean Pines Chamber of Commerce awards ceremony was held Oct. 13 in conjunction with the Ocean Pines Yacht Club’s Harvest Ball.
The honorees this year were: Nonprofit of the year, the Worcester County Veterans Memorial at Ocean Pines Foundation; Business of the Year, 5 Star Plumbing, Heating & Cooling; and Aaron Eckman, sales manager with the Ocean Pines Independent newspaper.
After cocktails and dinner, each recipient was honored with proclamations from Sen. Jim Mathias, Maryland Delegate Mary Beth Carozza and Worcester County Commissioner Chip Bertino.
Accepting the honor for the Veterans Memorial Foundation, which was sponsored by the Community Foundation of the Eastern Shore, was President Marie Gilmore, who deflected praise to her foundation board, after summoning them to stand behind her during her remarks.
“These are the people that truly get things done, they give me more credit than I deserve,” she said. “We promised Ocean Pines we would take care of it into perpetuity and that’s exactly what we intend to do.”
Funded with seed money from the Ocean Pines 35th Anniversary Celebration in 2003, the Veterans Memorial, located on the South Gate Pond in Ocean Pines, was dedicated on Memorial Day in 2005. Donations have also been raised through the sale of bricks and pavers honoring the memory of veterans or current members of the U.S. military.
Mathias praised the work of Gilmore and fellow board members including: Mary Adair, Don Clagett, Carolyn Dryzga, Sarge Garlitz, Ken Ingram, John Lemon, Lou Etta McClaflin, Sharyn O’Hare, Carol Rose, Linda Shanahan, Doug Slingerland, James Spicknall, Steven Zangwill and Sherri Lassahn.
“They say never forget, (and) because of you we won’t,” he said.
In addition to keeping the memorial in good condition, the foundation is committed to teaching Worcester County students about the service of U.S. military members through its Education and Community Outreach Committee.
Carozza opened her remarks by asking veterans to stand for a round of applause before relating her experiences with the foundation’s educational efforts.
“When I watch our veterans with our students, you just see these young people starting to understand the sacrifices involved for their own freedom,” she said. “When I watch children respond to that, it is really uplifting.”
Youthful enthusiasm is sometimes contagious, Carozza said.
“They’re so excited after seeing this veterans memorial they’ll come back and say ‘Ok I’m going to come back and bring my family,’” she said.
Bertino said he was pleased that the Veterans Memorial Foundation was selected as nonprofit of the year.
“For more than a decade, the memorial’s mission has resonated within our community,” he said.
Bertino said the foundation has risen to the task of building and maintaining a suitable memorial to honor and preserve those who risked their own safety to serve in the military.
See WORCESTER Page 13
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Birds of a feather from Scales and Tales flocked together last Saturday during Berlin’s Oktoberfest on Main Street.
A couple put on their dancing shoes for the Alte Kumpel band during Oktoberfest, Saturday in downtown Berlin.
Brooklyn Baking Baron Tony Lanuza brings a special German chocolate cake to the downtown Oktoberfest celebration in Berlin on Saturday.
Berlin Chamber of Commerce Executive Director Larnet St. Amant, right, and a group of volunteers pour Burley Oak beer during Oktoberfest, Saturday.
Artist Ramon Matheu sells original post-Pop Art prints in downtown Berlin during Saturday’s Oktoberfest.
Thousands turn out during Berlin’s annual Oktoberfest in the downtown area, last Saturday.
Worcester County Veteran’s Memorial nonprofit of year
Continued from Page 10
“I think everyone in this room would agree the memorial does that and so much more,” he said.
He also noted the foundation sponsors ceremonies on Memorial Day and Veterans Day.
Bertino, a former board member and foundation president, said the memorial was brought to life through the efforts of numerous parties.
“It started with co-founders Sharyn O’Hare and Roseanne Bridgman, who had a vision and they brought that vision to reality with the help of many people.”
Receiving Business of the Year honors was 5 Star Plumbing, Heating and Cooling, which opened shop in Berlin in 2013. Ginger Fleming, Ocean Pines Chamber of Commerce executive director, said owners Joe and Vicky Magnolia have been a staple in the community since launching their business.
“Joe and Vicky have been to every single one of my events at the chamber,” she said.
Unfortunately the couple broke the streak that evening, as they were unable to attend as they were vacationing in Aruba. Since they were otherwise occupied, their son, Joey, accepted the award.
“We actually stated to coming to Ocean City in 2008 and we met a lot of successful business owners in this area,” he said. “I don’t think we would be this successful without the loyalty and support you’ve shown us over the last three years.”
Taking home the Citizen of the Year award was Aaron Eckman, sales manager with the Ocean Pines Independent newspaper.
Fleming praised Eckman for his community involvement.
“He stands 100 percent behind the chamber,” she said.
The evening concluded with the installation of the Ocean Pines Chamber of Commerce 2016-2017 Board members and officers.
Sworn in were Incoming President, Heather Shamer of M & T Bank; Outgoing President, Amy Unger of Inesse Consulting Inc.; Vice President, Michael Mathers of Michael B. Mathers, P.A.; Treasurer, Celeste Miller of Farmers Bank of Willards; Secretary and new board member Teresa Berger of Atlantic General Hospital.
Serving on the board of directors are Teresa Travatello of the Ocean Pines Association and Patty Dundore of American Granite and Tile, along with two additional new board members, Steven Sweigert of PKS Investment Advisors, and Suzy Taylor of Ayers Creek Adventures.
Also honored were outgoing board members Anna Giles of Merrill Lynch, attorney Will Cathell and Lee Ann Gunning of Diamond Contractors.
Ocean Pines to present Halloween activities
(Oct. 20, 2016) Area residents will find a few tricks and many treats during Halloween activities sponsored by the Ocean Pines Recreation and Parks Department.
Pumpkin artists are invited to Family Fun Night Pumpkin Painting on Friday, Oct. 21 from 6-8 p.m. at the Ocean Pines Community Center. Attendees may bring their own pumpkins or purchase one at the event for $6 (limited quantity available). All decorations and paint will be provided. The cost is $5 for Ocean Pines residents and $6 for non-residents.
Then on Saturday, Oct. 29 from 1-4 p.m. Ocean Pines will celebrate the holiday in spooky style with its annual Halloween Fall Festival in White Horse Park. Admission is free and open to the public.
Costume contests, carnival games, face painting, pony rides, candy, a haunted hay ride, crafts, refreshments for sale and more will be part of the fun. Admission and games are free; there will be a fee for some attractions.
The event will also include a chance to vote for the contestants in the “Pup of the Pines” dog and puppy photo contest. The winner of the contest will be the 2017 face of the Ocean Pines Dog Park and will receive a free dog park registration. The winner will be announced during the Hometown Christmas Tree Lighting on Nov. 26.
Volunteers and candy donations are needed for the Halloween Fall Festival. Contact the Ocean Pines Recreation and Parks Department at 410-641-7052 for more information.
Paddling weekend washed out; event moved to Saturday
By Brian Gilliland
Associate Editor
(Oct. 20, 2016) In an attempt to get back a little of what was lost when torrential rains washed out roads, bridges and the Delmarva Paddling Weekend among other events, the organizers of that event pieced together two paddles leaving from Snow Hill on Saturday.
The first, and earliest paddle begins at 8:30 a.m. from Porter’s Crossing back to Snow Hill, and should be back in town by noon.
Starting at the Pocomoke River Canoe Company in Snow Hill, paddlers will be delivered to the forests of Porter’s Crossing, just north of Snow Hill. From there the boats will be placed in the water and the journey back home begins.
Jim Rapp, half of Conservation Community Consultants and event organizer, said this leg of the paddle will be limited to about 20 participants, but a couple of people give or take isn’t going to ruin anything.
The cost is $40 for a kayak, $50 for a tandem kayak or canoe or $20 if a boat rental is unnecessary. Tandem boaters must provide their own partners for the trip.
The afternoon paddle, scheduled between 1:30 p.m. and 4:30 p.m., and again starting and finishing at the canoe company, travels from Snow Hill Road to Nassawango Creek, ending at Red House Road and the Nas-sawango Creek Preserve. After arrival, the canoe company will ferry the boaters back to Snow Hill.
The pricing structure remains the same, except the afternoon paddle for those bringing their own boats is slightly cheaper at $15.
The total distance for both trips is about 10 miles, Rapp estimated.
“I really just want to go kayaking,” Rapp said. “It’s an attempt to get back a little of what we lost in Delmarva Paddling Weekend, and this will probably be it for 2016.”
Rapp said he wants to get a head count if possible, so pre-registration is encouraged, but not wholly necessary.
“The Pocomoke River Canoe and Kayak Company has plenty of boats available, so there’s no problem there, and it’s an opportunity to be social and be on the water before it gets too cold,” he said.
Rapp will be participating in both excursions, and offering a little bit of interpretation to the explorers.
“We’ll be talking a little bit about the history of the river and the nature we encounter along the way,” Rapp said.
He said the sojourn would be used as a stress test of the system he and partner Dave Wilson devised to manage the Delmarva Paddling Weekend.
“This is a smaller version of that,” Rapp said. “We’re learning all the time, and getting some nice testimonials to help carry it forward to next year, when we hope to have our first paddling weekend.”
Planning commission to tackle standards
Berlin group will again look to craft own design rules; previous effort stalled out
By Josh Davis
Associate Editor
(Oct. 20, 2016) Berlin Planning Director Dave Engelhart called a lack of uniform design standards “the biggest hole we have in our armor” during a planning commission meeting last Wednesday.
The commission had previously attempted to craft such a document, but the effort stalled. Afterwards, the town budgeted to outsource the job and appeared to nail down a firm, when the low bidder dropped out and a satisfactory alternative could not be found.
Last week, Engelhart asked the commission if they would like to try again.
“We’ve discussed it many times and we know we need to do something,” he said. “We can approach it a couple different ways. I’ve talked about this with [Town Administrator] Laura Allen recently again. It doesn’t ever get far from our minds.”
He said members of the commission could form a subcommittee, look at other design standards from similar municipalities, and “come up with something that we can work with and tailor.”
“If not, we still have the ability to put it out to bid and try again,” Engelhart said, adding that standards would help “protect and guide the growth that we’re probably headed for in the future,” especially along the Route 50 corridor.
“That’s good as far as our growth area and our comp plan are concerned because that’s where we said we want it, but we still want to be able to control it,” he said.
Engelhart said he already had a list of links to professionally written, online examples of design standards from towns like Stevensville, Grasonville and Easton.
Committee member Ron Casio asked about the previous effort, “why did we fail?”
“The first bidder that we awarded the contract ended up backing out. They had one of the principals leave the firm, so that was one reason,” Engelhart said.
He said $35,000 had been budgeted for that, and the first bid came just under that. The second bid was more than $50,000.
“Why are we different than any other of these towns and we don’t need any professionals?” Casio asked.
“If somebody has written it and we could adopt it and save the taxpayers $30,000-$50,000, I think that’s a very good direction,” committee member John Barrett said. “I think three of us could probably go through and find something that we could present to the board that we could vote on and possibly adopt.”
“That was my thought from the beginning,” Engelhart said.
Casio said he had his doubts, and worried specifically about the board becoming “citizen planners.”
“I’m concerned that we don’t have the professional knowledge,” he said. “I wouldn’t fix my own automatic transmission. I’m not going to give myself an appendectomy. I don’t know that we have the ability to come up with a really good plan for the town.”
Commission member Pete Cosby said he did not think it was “rocket science,” while Chairman Chris Denny added, “the town burned down in 1894 and they rebuilt it in 1895.”
“It’s not as easy as you portray it to be,” Casio said.
Cosby said he was satisfied with his own effort on a previous attempt at crafting standards, and did not see why a subcommittee could not try to replicate at least part of that.
“Let’s finish up the spec examples and finish this thing,” he said. “We’ve got an ordinance, but we don’t have any guidelines – that’s all. Let’s pass some guidelines, get it done and if there’re not good we’ll amend them later. It’s not a big deal.”
Engelhart said he could recirculate the previous work down towards the standards to the commission members, with the understanding that the discussion would continue during the next regular meeting, in November.
“I think that’s a good approach,” Barrett said. “I don’t think we’re that far away from it.”
Council Briefs
By Josh Davis
Associate Editor
(Oct. 20, 2016) The Berlin Mayor and Council discussed the following items during an Oct. 11 public meeting at Town Hall:
Enterprise zone
The council voted unanimously to redesignate an enterprise zone in Berlin. Managing Director Jeff Fleetwood said the designation would be good for 10 years, and that the most recent businesses added to the enterprise zone were SonRise Church and Twisters.
“Sometimes it’s the smallest things that make the biggest difference,” Mayor Gee Williams said. “Like all of us here, I’m hopeful we’ll bring some new businesses within these preset limits.”
Cannery update
Planning Director Dave Engelhart said Cannery Village had a “punch list walk through” on Oct. 4 of items that needed to be addressed in order to pass a final inspection. Those items are also apparently holding up repaving of Flower Street, where work trucks have damaged the roadway.
Engelhart said he was pressing the developer, Osprey Properties, to speed up the process because he feared Berlin would “run out of asphalt season.”
“We don’t want to have to wait until spring,” he said. “We were trying to get an answer [from the developer] this week or next so we could arrange for that fix of Flower Street.”
Rain report
Public Works and Water Resources Director Jane Kreiter said Hurricane Matthew spilled about 1.5 million gallons into Berlin’s wastewater treatment plant. The average daily load is about 400,000.
During the prior weekend, Kreiter said rains “showed up virtually out of nowhere” and led to a small overflow at the plant, about 200 gallons.
“It was completely treated wastewater – it was in our effluent tank that we use to hold water while we’re pumping it out to our spray irrigation facility, so it was completely treated and disinfected,” she said.
Minor flooding at the power plant on Williams Street during that same weekend led to damage to the oldest generator there. The council unanimously approved a $60,500 requisition order for repairs.
Fleetwood said the town had insurance on the generator with a $10,000 deductible, meaning the majority of the cost would be recouped.
Planning Commission Briefs
By Josh Davis
Associate Editor
(Oct. 20, 2016) The Berlin Planning Commission discussed the following items during an Oct. 12 meeting at Town Hall:
AGH Cancer Center
A new site plan for the Atlantic General Hospital Cancer Center on Old Ocean City Boulevard was unanimously approved by the commission. Slight modifications were made after the State Highway Administration imposed minor changes, moving the building farther north and reducing the parking lot by about 20 spaces. One of the two entrances to the center, from Healthway Drive, was also eliminated.
Other items
Planning Director Dave Engelhart said upcoming items the planning committee would have to tackle included a utility plan for the new SonRise Church complex, and that discussions had been held regarding new hotels and motels in Berlin, but there was “nothing concrete.”
He said work on the Twisters gymnastics and recreation complex on Old Ocean City Boulevard was coming along after several delays, and that the commission would likely soon be discussing approval for items at the new police department near Flower Street and the Oceans East apartment complex, near Stephen Decatur High School.
Permitting could start soon for the new Berlin branch of the Worcester County Public Library, and the new mixed use building on Gay Street could see construction starting before the end of the year, Engelhart said. That building would contain shops on the first floor and apartments overhead.
Engelhart said the town would pursue grants to help remediate the ponds at Berlin Falls park, and that a grant application regarding the design of a bike path that’s tied into the park and runs along existing railroad lines was due back soon.
As for the proposed excursion train, he said, “Someone’s gotta figure out how to make [the train] profitable. I don’t know what other recent movement there’s been – I don’t think there has been much.”
Ocean Pines offers special membership to BJ’s Wholesale
(Oct. 20, 2016) BJ’s Wholesale Club is once again partnering with the Ocean Pines Association to bring a special membership offer, effective Oct. 14-28, to benefit the Worcester County Veterans Memorial at Ocean Pines Foundation.
In addition to offering special membership benefits, BJ’s will donate $5 of each membership fee to the foundation. The offer, which is available to new and renewing members, is not available for purchase online or at any BJ’s location.
Other benefits include receiving $15 off the BJ’s $50 Inner Circle membership fee or $25 off the $100 BJ’s Perks Rewards membership fee, one additional free month of membership and a free second membership card for a household member. With this offer, 13 months will be added to the expiration date of a current BJ’s membership, regardless of when the membership was last renewed.
This special promotion is only offered through Ocean Pines two times a year. The next promotion will be in April of 2017. Current BJ’s members whose memberships will expire before then are encouraged to renew during the fall offer.
BJ’s operates over 200 clubs in 15 states from Maine to Florida, including a location in Millsboro, Delaware.
BJ’s membership applications are available at the Ocean Pines Administration Building at 239 Ocean Parkway and online at OceanPines.org.
Applications must be returned with payment by Friday, Oct. 28. They may be dropped off at the administration building or mailed to Ocean Pines Association, Attn: Teresa Travatello, 239 Ocean Parkway, Ocean Pines, Maryland 21811.
For more information, contact Travatello, director of marketing and public relations for the Ocean Pines Association, at 410-641-7717 ext. 3006.
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Anti-opiate group ‘Warriors’ move in new Berlin office
By Josh Davis
Associate Editor
(Oct. 20, 2016) With their new office now open in Berlin, Heidi McNeeley and the Worcester County Warriors Against Opiate Addiction are hoping to offer a friendly face – and some vital information about treatment – for those who have friends and family suffering from addiction to heroin and other opiates.
McNeeley, her husband Jamie and son Sean were busy setting up the new office, inside the Berlin Visitor’s Center on 14 South Main Street, last Thursday.
For now, the plan is to fill the office with pamphlets, brochures and flyers for local treatment centers and addictions programs, and to meet with people one-on-one as needed.
“I think this space is going to slowly evolve,” McNeeley said. “I think we see it as being, if a mom calls and says, ‘hey I just found out my kid’s in trouble or I need some treatment options,’ we can say, well meet us at the office because we’re going to have all the information here.
“It’s not really a space where people can come in and vent to us, but they can come in and get information and let us guide you,” she added. “Right now my desk at home is overflowing with all the Warriors stuff, so I think coming in here to do some planning and paperwork will be helpful.”
The office will not be staffed, but McNeeley said she and other volunteers would be readily accessible by appointment. If the office is closed, information will still be available outside the doors after hours.
“We’ll kind of see how it evolves,” McNeeley said. “This makes us feel legit, and if nothing else it gets our name out there and gives us a presence. It’s pretty cool.”
“And everybody in town has been great,” Jamie said. “More welcoming than you would think.”
The next meeting of the Worcester County Warriors Against Opiate Addiction is Thursday, Oct. 27 at Stephen Decatur High School starting at 6:30 p.m.
For more information, visit www.facebook.com/WorcesterCountyWarriors or wocowarriors.org. To make an appointment, call McNeeley at 302-381-0569.
Pines directors continue to work toward CPI revisions
Regular meeting this Sat. to include first reading of Resolution M-01, violations
By Josh Davis
Associate Editor
(Oct. 20, 2016) After nearly two hours of debate on Monday, the Ocean Pines Association Board of Directors agreed that nine new compliance, permit and inspection (CPI) violations would be listed on the agenda of a regular board meeting this Saturday, and that the agenda also would include a first reading of a revised resolution governing how those violations would be addressed.
Part of the reason for the prolonged discussion was that several directors questioned whether the updated version of Resolution M-01 is an improvement and streamlined the process as intended.
Director Cheryl Jacobs, who helped write the proposed changes, said the revised document would bring violations to the Architectural Review Committee (ARC) much earlier. That, theoretically, would speed up board or legal action. She said the update included new provisions for foreclosure properties and repeat offenders as well.
Jacobs said attorney Joe Moore vetted the document.
Board President Tom Herrick, however, argued that the revised language did not include the strict timeline that was present in the earlier draft. Language that allowed for an appeal at ARC was also apparently taken out, although Jacobs argued that it was implied by requiring committee hearings that invited the homeowner found in violation to attend.
“How many days do you have to correct the violation? I don’t see that and I saw that in the old one, and I’m a guy that likes black and white,” Herrick said.
Interim General Manager Brett Hill said the current process was clearly defined, but that staff was struggling with how aggressively to pursue enforcement.
“I’m really concerned if we don’t have the dates and timelines clearly spelled out, how I can give direction to staff?” Hill said. “I think the legal action further defined in it is good, [but] quite honestly I’d like to see it go a step further.”
Jacobs said the exact timeline was difficult to determine because homeowners were entitled to certain due process protections and coordinating with regularly scheduled ARC meetings, which were pushed to the front of the process, logistically could not be uniform. Still, she said she would try to develop a timeline before the regular meeting.
“The strength of the old document was the timeline,” Hill said. “The issue is not with M-01 – it’s with the staff.
“With the way we’re written today … I can move something to ARC within 30 days,” Hill said. “I have no problem taking that action today, I just need to know that that’s what the board wants me to do.”
The directors agreed to place the first reading on the agenda with the understanding that further changes could be made before a final vote was held.
State Highway repairs begin on Route 12 in Snow Hill
By Brian Gilliland
Associate Editor
(Oct. 20, 2016) The State Highway Administration announced work has begun to repair Snow Hill Road in the area just south of Sermon Road that was washed out during the heavy rainfall that affected the region on Oct. 1.
Detours will remain in effect until the work is completed.
Charlie Gischlar, spokesman for the SHA, said the repairs would take approximately three to four weeks to complete.
The repair is not only of a simple washout, as several 42-inch drain pipes that date back to when the road was first constructed need to be replaced to conform to current design guidelines before the road can be reopened.
Hurricane Matthew, which threatened but did not strike the shore directly, also affected the effort in two ways. First, repairs were delayed while the storm’s forecasted track directly targeted the shore, and again when the remnants of the hurricane dumped additional water onto the already waterlogged area.
Owner calls new Snow Hill salon dream come true
By Brian Gilliland
Associate Editor
(Oct. 20, 2016) If Betsy Brittingham were asked in April if she thought she’d be running her own salon in Snow Hill by October, the answer would have been “no.”
While it was always her dream to have her own salon, at that point in April, she was just starting two new adventures: starting a job at the town’s Courtney Brooke Salon, and returning to work following the birth of her son, J.J.
After Courtney Brooke announced its closure effective Sept. 24, Brittingham’s timeline accelerated. She found a spot across the street from where Courtney Brooke was located and got to work.
It helped that this wasn’t her first foray into the world of haircuts and color. Brittingham was the former manager of the Great Clips franchise in Salisbury, but was also looking for something a little closer to home when she accepted the job at Courtney Brooke.
“I’m originally from Girdletree and I graduated from Pocomoke High School, and I wanted something a little closer to home than Salisbury,” she said. “I’m taking all the knowledge of the business side I got from Great Clips and putting it with the feeling of home that Courtney Brooke did so well, and I’m making my own thing.”
For example, like Great Clips, Mirror Salon will send reminders the day before an appointment is scheduled, and it will also keep track of how many times a customer has visited, and if they’ve not been in for a bit of freshening up in a while.
“Some of the business processes are a little more detailed than what I was expecting. A franchise operation like Great Clips gives you all the little details. Right now I’m focusing on decorating the shop to match Snow Hill,” Brittingham said.
On the other end of the spectrum, Mirror will also service walk-in customers, and while not offering bargain basement haircuts, her goal is to keep prices comfortable.
“We’re doing hair, we’re doing color and we’re doing men’s cuts too. I want to keep it affordable, but it’ll cost more than $10,” she said.
See GRAND OPENING Page 21
Grand opening of Mirror Salon will run with First Fri.
Continued from Page 20
The soft opening of the shop was on Tuesday, with the grand opening coming in about two weeks at the next First Friday. Mirror Salon will be open for regular business from Tuesday until Saturday, but will be closed to haircut traffic during the grand opening.
“We’ll be dressing up and doing a meet and greet. I’m going to be talking with the chamber of commerce to see if we can work something out for that. I think we might be doing a giveaway, or have some kind of prize,” she said.
Besides Brittingham herself, she will have another full-time staff member, Emily LeMay, from Pocomoke City and another part-time employee, Lashette Dennis.
“I definitely didn’t think I’d be doing this now — not with a six month-old. I had planned to stay on at Courtney Brooke for a few years at least before working more directly towards my dream,” she said. “When Courtney decided to leave, I thought that I should be there to pick up the pieces. The town knows me, and the people know me.”
Improvements to close Berlin center, lobby remains open
(Oct. 20, 2016) The Town of Berlin’s Visitor Center will be closed to the public beginning Wednesday, Oct. 19 for renovations and improvements to the front of the building.
Economic and Community Development Director Ivy Wells applied for and received funds for a façade grant for improvements to the front of the building.
The small lobby inside the door will still be open for visitors to pick up information. The improvements will likely take anywhere from three to four weeks.
Tried formula still truest
The search for a general manager in Ocean Pines is as much about finding the right person for the position as it is determining what kind of operation the Ocean Pines Association wants to be.
All in all, the approach the community has followed over the years — a general manager and staff overseen by a board of directors — has worked out just fine, give or take the occasional political kerfuffle along the way.
It’s also the simplest form of management there is, as compared to those in place in larger planned communities such as Columbia, Maryland and in the south and west, where some homeowners associations are so large that community subdivisions have their own management companies.
Columbia also has multi-tiered management, with the Columbia Association responsible for certain operations and facilities, village associations enforcing covenants and creating master plans, and Howard County government handling essential services such as police, fire and trash collection.
Obviously, something like that wouldn’t work here, which leaves the board and association members two choices: keep doing what has been done or turn over much of the community’s operational and financial responsibility to a professional management company.
As to the latter option, the big question is what would be gained by doing that? Would it save money? Probably not. Would it improve the quality of life for residents? No. Would it do a better job of providing services than the current system and staff? That’s unlikely, since most everyone agrees that a highly capable staff is already in place.
All Ocean Pines needs, as the board has agreed, is the right general manager, someone who has been vetted by a professional executive search company and the directors.
Finding that best manager isn’t the easiest thing to do, as the Town of Ocean City discovered a few years ago with its managerial misstep, but it is still the best course to take, especially considering that no strong case has been made how residents would benefit by doing something different.
Letters
Town support still exemplary
Editor,
The following letter was sent to Berlin Mayor Gee Williams in appreciation for the support the town provided for the recent Cruisers event held on Sept. 8.
Mayor Williams:
Once again we received excellent support from town employees and the town itself during the fall Cruisers event. Despite the weather, a good time was had by all.
We have worked and organized most of these shows, and always are very pleased with the way things click together. This year special thanks to Wendell Purnell and Dave Wheaton for assistance with traffic control devices, Rick Dennis who came very early Saturday to empty and check on the trash cans, later that day to make sure none were overflowing, then planned to come some time afterwards in case any were overfilled over the holiday three-day weekend.
We also received a call from Augie Wienhold on Friday to confirm that we did not need any special electrical needs. We are sure there may be others missed, and as stated above this service is the norm.
Aspecial thanks to Jeff Fleetwood, who volunteered his time on Saturday to be a judge. Town support always seems to extend from the newest employee to the most senior managers.
Tanja Giles, Mike Wiley, Bill Hoshal
Committee Members
Community Foundation awards
(Oct. 20, 2016) The Community Foundation of the Eastern Shore (CFES) awarded a record $452,000 in scholarships to 320 students in the region this past year. The Community Foundation administers 157 scholarship funds that provide financial assistance for college students throughout the Lower Eastern Shore of Maryland.
“During 2016 the Community Foundation reached new records in the amount of scholarships awarded, the total amount of financial assistance for scholarships and the number of scholarship funds administered,” said Eric Joseph, CFES president. “Meeting the rising need for college scholarship assistance and the rising tuition costs is an essential part of the Community Foundation’s mission.”
“Helping young people here on the Lower Shore fulfill their dreams and realize their potential is among the highest goals of the Community Foundation,” added BJ Summers, the Foundation’s director of Development and Donor Services.
Some of the Foundation’s scholarships are available for students in particular high schools, while others target specific college majors and professional careers.
While most Foundation scholarships do not limit students to a particular course of study, there are scholarships available for students wishing to pursue careers in teaching, architecture, engineering or healthcare. A complete listing of Community Foundation of the Eastern Shore scholarships is available by visiting www.cfes.org.
As leaders, grant makers and stewards of philanthropy, The Community Foundation of the Eastern Shore serves the common good of the Lower Eastern Shore. A 501(c)(3) nonprofit, CFES inspires history of fostering charitable endeavors, and has provided more than $60 million in grants and scholarships to the local community since 1984.
CFES collaborates with individuals, families and businesses to match their charitable interests with community needs and strengthen local nonprofits through grants and resources. CFES is devoted to improving the regional community and believes in people helping one another to provide positive impact. Visit CFES.org or call 410-742-9911.
Ocean Pines Association Briefs
By Josh Davis
Associate Editor
(Oct. 20, 2016) The Ocean Pines Association Board of Directors discussed the following items during an Oct. 17 work session in the community center:
Comprehensive plan
In discussing recommendations from the comprehensive planning committee related to a community-wide survey, the directors agreed to set a joint meeting with the committee.
Tentatively, that was set for Nov. 17 at 7 p.m. in the community center.
Golf committee
A second reading for Resolution C-11, reestablishing the dormant golf advisory committee, will be held during a regular board meeting on Saturday.
Board President Tom Herrick asked the other directors to bring appointments to the meeting, and said he had also identified several homeowners who were willing to serve.
Budget guidance
Director Pat Supik, the liaison to the budget and finance committee as well as former chair of that group, went over a series of budget guidance recommendations from the committee.
The board had discussed those recommendations during a joint meeting with the committee on Sept. 21.
Interim General Manager Brett Hill said several of those recommendations were already being acted upon.
There was some confusion among the directors, who initially did not realize Supik had intended to use the committee guidance to work toward a board-approved budget guidance document for the general manager. Still, the directors agreed to let Supik create a draft document that could be discussed and potentially voted on during a regular board meeting, Saturday.
If the board waited until the next regular meeting, in December, Supik said the budget would be “about finished” before the board had a chance to weigh in.
Delinquent assessments
Two final items on the agenda submitted by Director Slobodan Trendic will not appear on the regular session agenda Saturday.
The first, reviewing delinquent assessments, was deemed unnecessary. Although the association apparently has $1.2 million in unpaid dues, the collection process could likely not be improved. Herrick said, Ocean Pines collected assessments from all but 2.86 percent of homeowners last year.
“We’re dealing with a very small pool of homeowners that have gone unpaid for years,” Hill said. “We just go through our process. It’s an unfortunate situation, but it’s a reality of the housing market and the area we live in with the effects of that housing market.”
Hill said outstanding debt was down from last year.
Another item, discussing board priorities, was deferred to a future work session because the meeting had already run for more than five hours.
Wor. Arts Council to judge ‘small wonders’
(Oct. 20, 2016) The Worcester County Arts Council invites all interested artists to participate in a juried art show with the theme, “Small Wonders,” to be exhibited during the months of November and December at the WCAC Gallery located at 6 Jefferson Street in Berlin.
Work must be delivered to the Arts Council’s Gallery in downtown Berlin between Thursday, Oct. 27 and Friday, Oct. 28 to be accepted.
The exhibition is open to all artists, professional or amateur with work in all media.
The 2D entries cannot exceed 11 inches in any one direction (including the frame). All 3D work cannot exceed 7 inches in any one direction. Work must be original and completed within the last three years.
Maximum of two entries. The cost is $10 per entry for WCAC members; $15 for nonmembers.
Artwork will be juried in and judged for the competition by talented and widely recognized artist, Angela Herbert-Hodges.
Detailed entry guidelines are available on the Arts Council’s website, www.worcestercountyartscouncil.org.
Monetary prizes will be awarded for first, second, and third place during the Second Friday Arts Stroll reception on Nov. 11 from 5-8 p.m. For additional information, call 410-641-0809 or email email@example.com.
Elangwe sends 490 pounds of books to Africa
(Oct. 20, 2016) Worcester Prep 10th grader Henry Elangwe, of Salisbury, has not forgotten where he’s from or how fortunate he is, thanks to his family.
Born in Douala, Cameroon, Elangwe moved to the states when he was in third grade and enrolled at WPS in ninth grade. This summer, he packed up over 550 books and hundreds of magazines and shipped them to Little Angels Elementary School in Bamako, Mali, as part of his aunt’s “Ignite Africa” project.
The Little Angels Elementary School opened in 2014, and is now in the process of building a middle school. The books Elangwe sent will help stock the future school library.
“I feel proud and happy going to a school like Worcester Prep that has so many educational tools to help students learn,” he said. “I felt since I had the chance, I was obligated to give other children a chance to feel the same pride and happiness.”
Last year, his aunt, Wele Elangwe, developed the project called “Ignite Africa” to help schools in Africa with limited access to books and other educational items. He got involved by collecting new and used books from his fellow WPS students at the end of last school year, with the help of Head of Upper School Mike Grosso. His WPS bus driver was instrumental in helping transport the books to Elangwe’s home while his cousins, Nde Labah and Xavier Muapi, helped him pack the books to be shipped.
Elangwe is very thankful for everyone, especially his aunt, who helped inspire him along the way.
“I was proud of myself for indirectly making a difference by helping educate the next generation of children,” he said. “I am especially proud of my fellow students, teachers and my Worcester Prep family who greatly supported me on my journey to help others in need from my country. My goal is to continue collecting books every year.”
‘Flames at the Furnace’ to raise funds for heritage
(Oct. 20, 2016) The third annual Flames at the Furnace barbecue fundraiser will take place at historic Furnace Town Living Heritage Village on Saturday, Oct. 22 from 5-8 p.m.
Tickets cost $30 per person. The event is for guests 21 and older. Dress is casual, and participants will enjoy a southern barbecue with craft and domestic beer and wine served. Auctions, both silent and live, will be held along with a raffle and door prizes. Live music will be provided by Apple & Brit.
Tickets can be purchased at Furnace Town by calling 410-632-2032 or online at www.eventbrite.com.
This annual fundraiser helps Furnace Town preserve the cultural heritage of the region, share rich history for the benefit of future generations, and makes history come to life.
Funding for this event is in part provided by the Worcester County Arts Council, Maryland State Arts Council and the National Endowment for the Arts, organizations dedicated to cultivating a vibrant cultural community where the arts thrive.
Furnace Town is dedicated to preserving the historic Nassawango Iron Furnace and engaging the public in the culture of shared past. Throughout the year, the museum features artisans in period dress demonstrating crafts and trades that were practiced in Furnace Town and the Lower Eastern Shore during the 19th century. Every year, Furnace Town, located in Snow Hill, hosts about 10,000 visitors, more than 3,000 of which are school children.
Pocomoke Rotary to raise ‘Heroes’ flags
(Oct. 20, 2016) The Pocomoke City Rotary Club is sponsoring the Flags for Heroes program again this year, on Saturday, Nov. 5, just in time for Veterans Day, Nov.11.
Anyone can nominate a hero (living or deceased) for recognition. It may be a soldier, a fireman, emergency personnel, teacher or anyone who is held in high esteem by that individual.
“Last year on Memorial Day many flags adorned the area downtown beside the big flag, and a very nice program honored all the recipients,” Willie Jackson, Pocomoke Rotary president, said. “The club was so proud to be a part of honoring these special people.”
A flag will be flown, supplied by the club, with a $50 donation. Each participant will receive a certificate in honor of his or her honoree.
The Pocomoke High School ROTC will present colors, and Mayor Bruce Morrison will give the keynote speech, beginning at 11 a.m. Applications may be obtained from any Rotary member.
For more information, contact Carole Krueger at 410-430-2243 or email at firstname.lastname@example.org.
Snapshots
BELL OF BALL
Isabella the Chihuahua greets guests at Salt Water Media in Berlin during the 2nd Friday art stroll, Oct. 14.
SDHS FLASH MOB
Stephen Decatur High School seniors Lexie VanKirk, Claire Porter, Savannah Schultz and Makensie Froman perform during the flashmob dance at the fifth annual Character Kickoff assembly on Sept. 16.
AGH DONATION
The Auxiliary of Atlantic General Hospital and Health System recently presented President and CEO, Michael Franklin, FACHE, with a check in the amount of $50,000 toward the Campaign for the Future for their fiscal year 2016 fundraising efforts. Along with this initial donation, the Auxiliary has made an additional pledge to raise $50,000 each year for the next five years totaling $300,000. Pictured, from left, are Barbara Lischak, Kathleen Loetz, Franklin, Ann Hamilton, Margie DiNenna and Barbara Loffler.
STUDENT ARTIST
The Maryland Association of Realtors awarded second place to Rachel Eure of Snow Hill, a student at Salisbury University, in its “Maryland Door Painting Contest.” The contest was held in conjunction with the association’s annual Conference at the Roland E. Powell Convention Center in Ocean City. Eure, a 2016 Art League of Ocean City scholarship winner, is pictured with Rina Thaler, executive director of the Ocean City Center for the Arts.
CASA HIGHLIGHTED
CASA (Court Appointed Special Advocate) is one of the programs under the umbrella of Worcester Youth & Family Counseling Services, a nonprofit organization serving the community since 1975. CASA depends on volunteers who advocate for abused and neglected children in the court system. Kiwanis is about “Serving the Children of the World,” so hearing about a program that protects children is most important. CASA Director Brigitte Southworth and Kiwanis Club of Greater Ocean Pines - Ocean City President Mark Joseph gather for a photo during a recent club meeting.
FIRE PREVENTION WEEK
The Worcester County Commissioners on Oct. 4 join with Fire Marshal Jeff McMahon, front, center, and Deputy Fire Marshal Matt Owens, front, left, to recognize National Fire Prevention Week, Oct. 9-15, and to support the initiatives of the National Fire Prevention Month theme “Don’t Wait - Check the Date! Replace Smoke Alarms Every 10 Years.”
Lauer named SHHS ‘Teacher Champion,’ foundation liaison
(Oct. 20, 2016) The Worcester County Education Foundation recognizes Pamela Lauer, Snow Hill High School’s Teacher Champion.
Lauer grew up in Philadelphia and graduated from Central High School. She earned a Bachelor in Science from Salisbury University, a Masters in Science (Microbiology Concentration) from Virginia Tech and a Masters of Arts in Teaching (Biology) from Salisbury University.
While attending Salisbury University she substituted in Worcester County. Upon graduation, she taught for three years at Washington High School in Somerset County, and another three years at Parkside High School in Wicomico County.
Lauer is especially pleased to be teaching at Snow Hill High School, because her oldest child just started pre-K at nearby Snow Hill Elementary School.
“Worcester County (has) such a great school system,” Lauer said, adding that she has a strong personal interest in maintaining that excellence now that her own child has entered the system.
As a Teacher Champion, Lauer will act as a liaison between Snow Hill High School and the Worcester County Education Foundation (WCEF). The mission of the WCEF is to create equal access to a world-class education. By bridging community resources with the needs of the schools, foundation members believe that every child in Worcester County can have access to the learning tools necessary in order to succeed.
Working on the front lines, teachers like Lauer have a unique perspective of current and evolving needs of students, teachers and staff, which allow the WCEF to constantly evaluate, define and quickly respond with financial or other support where it’s most needed. To learn more about the WCEF, visit www.wcef.foundation or call 410-632-5076.
Coastal Hospice adds two to provider relations team
Frankie Knight and Caroline Hutchison will join outreach group serving Lower Shore
(Oct. 20, 2016) Coastal Hospice welcomes two new provider relations associates as members of its outreach team.
Provider relations associates go out into the four Lower Shore counties that Coastal Hospice serves to provide physicians and health care facilities with the proper tools to give them a full understanding of hospice and the services the nonprofit provides.
Frankie Knight grew up in Ocean City and attended Wor-Wic Community College and Towson University. She has volunteered for the National Alliance of Mental Illness, Susan G. Komen and CASA. After a cancer diagnosis, she started a women’s peer support group at TU.
Knight is dedicated to working in the nonprofit world.
Caroline Hutchison grew up in Salisbury and attended Bridgewater College in Virginia. Previously, she interned at Coastal Hospice in Provider Relations during the fall and spring of 2014/2015. Hutchison’s goal is to extend a helping hand to her neighbors and have a positive impact on her community.
Founded in 1980, Coastal Hospice is a non-profit health care organization that cares for individuals facing life-limiting conditions but who want to remain as active and engaged as possible. Coastal Hospice cares for patients in their home, nursing home, assisted living facility or at Coastal Hospice at the Lake.
The organization serves Wicomico, Worcester, Dorchester and Somerset counties. Information is available at 410-742-8732 or at CoastalHospice.org.
Spare goose, but don’t spoil appetite
This column originally ran in an Oct. 16, 2014 edition of the Bayside Gazette.
It was a scene out of “Rambo IV,” or maybe I’m thinking “Terminator.” I rolled up to Route 589 just as those geese and ducks – those dreaded monsters – started waddling their way into incoming traffic. What ensued was nothing short of sheer terror. Buildings were ablaze, cars stacked in ditches while innocent bystanders were forced to cover the eyes of their offspring to prevent them from seeing the carnage forged by these fiery-eyed, feathered foes.
They made their way across the road, nary a scratch on any of them, all the while laughing (In hindsight, I imagine they were honking and quacking) at the ensuing destruction. As I think about this, I shiver as I recall the traffic light falling on a new litter of puppies enjoying an innocent frolic around the pond.
And then I woke up. I realized that I can’t imagine the geese being such a problem that we would even consider resorting to killing them to improve traffic conditions. Did I really read that correctly in the paper? The geese are causing New York-style congestion? Are we talking about the same geese and the same intersection? I need to go back to sleep.
I reentered dreamland only to be confronted by a goose dressed as Marlon Brando in “The Godfather,” and I’m tied to a chair. I won’t delve further into that dream, but suffice it to say, I awoke trembling and in a cold sweat. I wonder if I can sue waterfowl for mental trauma and duress.
Every time those beasts get in front of my car, it costs me at least 45 seconds of my life and I can’t get those back. I’m a busy man, and I absolutely refuse to stop for a minute and allow nature to stride in front of my car, taking a brief moment to stop and smell the roses. I won’t stand for it. Wait, now I’m dressed as a lawyer?
I wake up again, and I realize that I am not angry at the geese and I never have been. I wonder why I would agree with anyone who wants to kill the geese or spread chemicals around our natural resources to get rid of an animal that has inhabited our wetlands long before any of us were born. Well, there may be a few residents left who predate the Branta Canadensis, also known as the Canada goose (not “Canadian,” so just stop it), but they are probably few and far between at this point.
We live in their house, not the other way around. The waterfowl at the South Gate was something that truly attracted my wife and me to the Pines 15 years ago, and I was happy to hear many friends respond in kind when I posted on this issue in social media this weekend.
Our kids have always enjoyed waddling along with our feathered friends (and not feeding them bread) and I don’t know that I’ve ever lost control of my vehicle based on the shenanigans of the Crazy Canadas or the Malicious Mallards; but I now know that I must be ever so cautious as I navigate my way through the mire that has apparently become 589.
As I write this, I am inspired. I now have my “why:” I must create The Canada Goose Liberation Front for a Waddle-Free America, if for no other reason than to have the chubby-cheeked goose in my dreams stop telling me to “leave the gun and take the cannoli.”
Duck Confit, Cherry Compote
Makes approximately 12 hors d’oeuvres
2 Duck breast lobes, skin on
EV Olive oil OR rendered duck fat, as needed
1 in-sprig fresh rosemary
3 cloves garlic
Zest from 1 lemon (Microplaned)
1 shallot, halved
1 tsp. Black peppercorns
Salt and Pepper, as needed to season
Remove the skin from the duck breast and score with a knife.
Place in a small pan and put on a low-medium heat to begin to render the fat from the skin.
When the skin has rendered as much fat as possible, remove the skin and cut into small lardons or strips.
Bring the oil to medium-high and fry the skin until it becomes duck cracklings. Set aside until service at room temperature.
Put all other ingredients except for the salt and pepper for seasoning in an ovenproof pan just big enough to hold it.
If there is not enough fat (which there won’t be) just cover with rendered duck fat or olive oil, or both.
Cover with foil.
Cook at 225 if using a conventional oven and 200 if using a convection oven for 12-14 hours. Your house will smell divine all through the evening.
When the chicken easily shreds, it is done. Remove from the oven and cool in the refrigerator, at least 12 hours.
When ready to serve, remove from the oil and drain, and try to strain the juices from the bottom of the pan – a gravy separator will work splendidly here.
Serve on individual spoons with fruit compote and greens. These are rich, satisfying and delightful hors d’oeuvres for the fall and winter months, however please don’t let them take you over so that you aim for the waterfowl on our precious roads.
— Paul G. Suplee is an Assistant Professor of Culinary Arts at Wor-Wic Community College. Find his ePortfolio at www.heartofakitchen.com.
DAR recognizes Barnes
The General Levin Winder Chapter of the Daughters of the American Revolution (DAR) recently recognized Ellen Duncan Barnes, left, for 50 years of DAR membership. Barnes was accepted into the DAR on Oct. 15, 1966 and is a charter member of the chapter which was organized in August 1966. Her Patriot is James Smith of Virginia. Chapter Regent Patricia Ayers presented the certificate to Barnes at her home “Shore Acres” in Pocomoke City. The DAR is a women’s service organization dedicated to promoting patriotism, preserving American history, and securing America’s future through better education. For more information, visit www.dar.org.
Gems’ tea honors county women
(Oct. 20, 2016) The Worcester County Commission for Women, in conjunction with the Friends of the Worcester County Commission for Women, will be celebrating the lives of four extraordinary Worcester County women during a special tea on Sunday, Oct. 30 at the Dunes Manor Hotel on 28th Street in Ocean City.
The event will take place from 2-4 p.m. The Gems’ Tea honors local women whose exemplary lives have influenced others to contribute to Worcester County’s history in various ways.
This year, Vicki Nock of Berlin, Barbara Purnell of Berlin, Ann Coates of Snow Hill and Annette Wallace of Pocomoke will be honored. These women will share their life experiences with interviewer Jack Barnes, resulting in a DVD that captures their history.
The Oral History DVD will be presented to all branches within the Worcester County Library system and may be viewed in the future to serve as an inspiration to other women in the county.
For reservations, checks should be made payable to: FWCCW and mailed to Lou Etta McClaffin, 11108 Dale Road, Whaleystyle, Maryland 21872 by Monday, Oct. 24. The cost is $25 per person.
For more information, call Event Chair Carol Rose at 410-430-7540.
**Sudoku**
HARD – 34
Fill in the blank spaces in the grid so that every vertical column, every horizontal row and every 3 by 3 box contains the numbers 1 through 9, without repeating any. There is really only one solution to each puzzle.
| | | | | | | | | |
|---|---|---|---|---|---|---|---|---|
| 6 | 5 | 2 | 7 | 5 | | | | |
| 3 | | | | | | | | |
| 2 | | | | | | | | |
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**Answers to last week’s puzzles**
| | | | | | | | | |
|---|---|---|---|---|---|---|---|---|
| 2 | 4 | 6 | 9 | 5 | 3 | 1 | 7 | 8 |
| 8 | 7 | 3 | 6 | 4 | 1 | 2 | 5 | 9 |
| 5 | 9 | 1 | 7 | 2 | 8 | 6 | 3 | 4 |
| 3 | 8 | 7 | 2 | 9 | 6 | 4 | 1 | 5 |
| 1 | 6 | 2 | 4 | 8 | 5 | 3 | 9 | 7 |
| 4 | 5 | 9 | 3 | 1 | 7 | 8 | 2 | 6 |
| 6 | 3 | 4 | 1 | 7 | 9 | 5 | 8 | 2 |
| 9 | 2 | 8 | 5 | 3 | 4 | 7 | 6 | 1 |
| 7 | 1 | 5 | 8 | 6 | 2 | 9 | 4 | 3 |
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**Emotion**
BY TOM MCCOY / EDITED BY WILL SHORTZ
**ACROSS**
1 Full of sound and fury
8 Shampooing, e.g.
16 Hemsworth of “The Hunger Games”
20 Mine craft?
21 Endanger
22 ___-European
23 Goodyear’s carefully guarded secrets?
25 Neutral tone
26 Title for Palpatine or Amidala in “Star Wars”: Abbr.
27 Lose it
28 Novelist Pierre
29 Polynesian inn locale, maybe
31 Unnerve
33 Revision that satisfies both author and publisher?
35 Offspring
38 Jog the memory of
40 Amer. money
41 U.F.O. pilots
42 “Would you like me to?”
44 Be inclined (to)
45 Increases
48 What a pianist uses for triple-time pieces?
50 “Principia Discordia” figure
51 ___ donna
53 Ready
56 Japanese honorific
**DOWN**
57 Country with the smallest national capital in the European Union (about 9,000 people)
59 Fall times: Abbr.
60 Scalawag
63 Result of a Morton’s factory explosion?
67 Eng. or hist.
68 Strange and unsettling
69 “Stay” singer Lisa
70 Skater boys?
74 Absorbs
76 Count in Lemony Snicket books
77 Director’s circle?
78 Reaction to a puppy video, say
79 First in command?
83 ___ Kringle
84 Dating site?
88 Dependent (on)
90 Unleavened cornbread
91 Makeup of many a tribal council
92 “Eww!”
95 El ___
96 Ones making lots of bucks?
112 Magna ___
113 Get room service, say
114 Meme
115 Like the coda of “Hey Jude,” seemingly
116 Feudal superior
117 12 9 11 5 10 20 8 9 19 3 12 21 5
118 Adept
119 Gun supporters
120 Team V.I.P.
---
**Online subscriptions:**
Today’s paper and more than 100 other newspapers
nytimes.com/crosswords ($39.95 a year).
---
**Puzzles**
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CAR announces board of directors during ceremony
(Oct. 20, 2016) The Coastal Association of Realtors (CAR) installed its 2016-2017 Board of Directors during a special ceremony recently at Lighthouse Sound in Bishopsville.
Donald Bailey, a Realtor for Coldwell Banker Residential in Salisbury, was installed as president of the association’s board.
“It is an honor to serve as president of the Coastal Association of Realtors for the upcoming year,” Bailey said. “This year, we will focus heavily on continuing education for our membership, while also working to raise money for my charity of choice – Habitat for Humanity.”
Bailey was installed by Dean Cottrill, president of Coldwell Banker Residential Brokerage, Mid-Atlantic.
The following members were also installed during the ceremony by Shelly Murray, 2016-17 president of the Maryland Association of Realtors:
President Elect: Joel Maher, Coldwell Banker Residential, Salisbury
Vice President: Terrence McGowan, Berkshire Hathaway HomeServices PenFed Realty, Ocean City
Secretary: Bernie Flax, EXIT Realty at the Beach, Ocean Pines
Treasurer: Joni Martin-Williamson, ERA Martin Associates, Salisbury
Immediate Past President: Linda Moran, Berkshire Hathaway HomeServices, PenFed Realty, West Ocean City
Directors: Joseph Wilson, Condominium Realty, Ocean City; Courtney Wright, Condominium Realty, Ocean City; Jeff Powell, Powell Real Estate, Salisbury; Tony Prochazka, Vantage Resort Realty, Ocean City; Brandon Johnson, ERA Martin Associates, Salisbury; and Grace Masten, Sea Grace at North Beach Realtors, Ocean City.
“We truly feel we have an exceptional Board of Directors this year and we are grateful to them for their service,” said Page Browning, executive vice President of CAR. “We’re looking for them to lead our association into an exciting and successful year at CAR.”
## NOW PLAYING
### BIG EASY ON 60
5909 Coastal Highway
Ocean City
410-524-2305
www.thebigeasyon60.com
Oct. 21: Judy Sings the Blues, 7 p.m.
Oct. 22: Tear the Roof Off, 7 p.m.
### BJ’S ON THE WATER
75th Street and the bay
Ocean City
410-524-7575
www.bjsonthewater.com
Oct. 21: Monkee Paw, 9 p.m.
Oct. 22: Dust N Bones, 9 p.m.
Oct. 26: 2 Guys & A Mama, 5 p.m.
Oct. 27: Betteneroo, 8 p.m.
### BOURBON STREET ON THE BEACH
116th Street, behind Fountain Head Towers Condominium
Ocean City
443-664-2896
www.bourbonstreetonthebeach.com
Oct. 21: Ricky & Lennon LaRicci, 7-10 p.m.
Oct. 22: Reform School, 7-10 p.m.
Oct. 23: Just Jay, 6-9 p.m.
Oct. 26: Open Mic 7-11 p.m.
Every Thursday: Dave Sherman & Chris Button, 7-10 p.m.
### CAPTAIN’S TABLE
15th St. & Baltimore Ave.
Ocean City
410-289-7192
www.captainstableoc.com
Every Thursday-Tuesday: Phil Perdue, 5:30 p.m.
### CASINO AT OCEAN DOWNS
10218 Racetrack Road
Berlin
410-641-0600
www.oceandowns.com
Oct. 21: Tear the Roof Off, 5:30-9:30 p.m.
Oct. 22: Everett Spells, 4:30-8:30 p.m.; Monkee Paw Duo, 9:30 p.m. to 1 a.m.
### DUFFY’S TAVERN
130th Street in the Montego Bay Shopping Center
410-250-1449
www.duffysoc.com
Every Friday: Bob Hughes, 5-9 p.m.
### FAGER’S ISLAND
60th Street and the bay
Ocean City
410-524-5500
www.fagers.com
Oct. 21: DJ Hook, 9 p.m.; Side Arm, 9:30 p.m.
Oct. 22: DJ Groove, 9 p.m.; Animal House, 9:30 p.m.
Oct. 23: Everett Spells, brunch
Oct. 24: Bryan Clark, 6 p.m.
### HARBORSIDE BAR & GRILL
12841 S. Harbor Road
West Ocean City
410-213-1846
www.ocharbarside.com
Oct. 21: DJ Billy T, 4 p.m.
Oct. 22: Simple Truth/Side Project, 2-6 p.m.; DJ Jeremy, 9 p.m.
Oct. 23: Opposite Directions, 2-6 p.m.; DJ Billy T, 6:30 p.m.
Oct. 26: Karaoke w/DJ Jeremy
Oct. 27: Opposite Directions, 9 p.m. to 1 a.m.
### HARPOON HANNA’S
Route 54 and the bay
Fenwick Island, Del.
800-227-0525
302-539-3095
www.harpoonhannahasrestaurant.com
Oct. 21: Dave Hawkins, 5-10 p.m.
Oct. 22: Ray Holiday, 6-10 p.m.
Oct. 27: Kevin Poole, 6-10 p.m.
### M.R. DUCKS
Talbot Street and the bay
Ocean City
410-289-9125
www.mrducks.com
Oct. 21: DJ Batman, 5 p.m.
Oct. 22: Kevin Poole, 3 p.m.
### OCEAN CLUB NIGHTCLUB
In the Horizons Restaurant
In the Clarion Fontainebleau Hotel
101st Street and the ocean
Ocean City
410-524-3535
www.clarionoc.com
Every Wednesday-Sunday: DJ Dusty, 9 p.m. to 1 a.m.
Oct. 21-22: Power Play, 9:30 p.m. to 2 a.m.
### PURPLE MOOSE
Boardwalk, between Talbot and Caroline streets
Ocean City
410-289-6953
www.purplemoosesaloon.com
Oct. 21: Fuzzbox Piranha, 9 p.m.
Oct. 22: CK the DJ/VJ, 2 p.m.; Fuzzbox Piranha, 9 p.m.
### SEACRETS
49th Street and the bay
Ocean City
410-524-4900
www.seacrets.com
Oct. 21: DJ Tuff, 9 p.m. to 1 a.m.; The Benderz, 10 p.m. to 1:50 a.m.
Oct. 22: Darcy Dawn & Company, 5-9 p.m.; Full Circle Duo, 5-9 p.m. DJ Cruz, 9 p.m. to 1 a.m.; The Lunatics, 9:30 p.m. to 1:30 a.m.; Steal The Sky, 10 p.m. to 1:50 a.m.
Oct. 27: Name That Drink Contest & Ocean 98’s “Live Lixx” Concert, Shelby Blondell, 6-7 p.m.; Bryan Russo & The Tragic Figures & Ocean 98’s “Live Lixx” Concert, 7-8:30 p.m.
### SKYE RAW BAR & GRILLE
66th Street, bayside
Ocean City
410-723-6762
www.skyebaroc.com
Oct. 21: Angeline Leech, 4-8 p.m.
Oct. 22: Elwood Bishop Trio, 4-8 p.m.
### TOUCH OF ITALY
67th Street and Coastal Highway, in the Holiday Inn Oceanfront
Ocean City
302-703-3090
Every Tuesday: Piano Bar w/Bryan Russo, 9 p.m.
### WHISKER’S BAR & GRILLE
11070 Cathell Road, Suite 17
Pines Plaza, Ocean Pines
443-365-2576
www.whiskersbar.com
Oct. 21: Karaoke w/Donnie Berkey, 10 p.m. to 2 a.m.
---
**BRYAN CLARK**
Fager’s Island: Monday, October 24, 6 p.m.
**OPPOSITE DIRECTIONS**
Harborside Bar & Grill: Sunday, October 23, 2-6 p.m.
BIG CRAFT SPIRITS SALE!
CRAFT BOURBON AND WHISKEY TASTINGS
4PM - 7PM FRIDAY
Route 50 and Pocomoke Locations
4PM - 7PM SATURDAY
16th Street and Gold Coast Mall Locations
Local OC DISTILLING CO.
All Flavored Vodkas On Sale $10
| Product | Price | Regular Retail Price |
|--------------------------------|---------|----------------------|
| Ciroc Vodka (L) | $33 | $47.99 |
| Vincent Van Gogh Double Espresso Vodka (L) | $18 | $35.99 |
| All Pinnacle Vodka Flavors (L) | $10 | $14.99 |
| Conch Republic Lite Rum (L) | $8 | $11.99 |
| Calico Jack Coconut Rum (L) | $10 | $14.99 |
| Three Olives Cherry & Grape (L)| $8 | $24.99 |
| White Marlin Gold Tequila (L) | $8 | $11.99 |
| Boston Crème DeCafe (L) | $5 | |
GREAT PRICE....PLUS A GREAT FIGURE!!
Smirnoff Sorbet Light Vodkas (L)
Raspberry Pomegranate • Lemon • Mango
Passion Fruit • Pineapple Coconut
$10 Reg. Retail Price - $15.99
ONLY WHILE SUPPLIES LAST!
**THU, OCT. 20**
**FALL RESTAURANT WEEK**
All Day Participating restaurants will offer special, fixed-price menus. No passes, tickets or coupons needed. Go to the website for list of participating restaurants.
http://www.oceancityrestaurantweek.com
**PINE’ER CRAFT CLUB MEETING**
Ocean Pines Community Center, 235 Ocean Parkway, Ocean Pines, MD, 9:45 a.m. Refreshments served at 9:45 a.m., business meeting at 10 a.m. Following the meeting there will be a glass painting demonstration with Claudia Ford Clancy of Art-C. Sharon Puser, 410-208-3032
**FLU SHOT CLINIC**
Health Fair at Holy Savior Catholic Church, 1705 Philadelphia Ave., Ocean City, MD, 10 a.m. to 1 p.m. Must be 13 years old. If you are allergic to eggs, consult with your health care provider. Donations made during the Flu Clinics will help offset the cost of future Flu Clinics. 410-641-9-FLU (9358)
**HOLY SAVIOR HEALTH FAIR**
Holy Savior, 1705 Philadelphia Ave., Ocean City, MD, 10 a.m. to 1 p.m. Featuring carotid screenings, bone density screening, dreamscape, blood pressure and flu shots as well as other health information. Free and open to the public. Dawn, email@example.com, 410-641-9268, http://www.atlanticgeneral.org/calendar
**COASTAL HOSPICE GRIEF SUPPORT GROUP**
Ocean Pines library, 11107 Cathell Road, Ocean Pines, MD, 11 a.m. The group meets every Thursday, Free and open to anyone who has lost a loved one, not just Coastal Hospice families. 410-251-8163
**BEACH SINGLES**
Harpoon Hanna’s, 39064 Harpoon Road, Fenwick Island, DE, 4 to 7 p.m. Every Thursday, Beach Singles 45-Plus meets for happy hour. Athene or Kate, 302-436-9577 or 410-524-0649
**FREE FINANCIAL SEMINAR**
Ocean Pines Community Center, 235 Ocean Parkway, Ocean Pines, MD, 5:30 to 6:30 p.m. Learn how to create a personal financial plan. William G. Ryon, III and Daniel W. Rowles from Compass Investment Advisors will explain the five-step process. Advance registration required. Ocean Pines Recreation and Parks Department, 410-641-7052
**BINGO**
American Legion Post 166, 2308 Philadelphia Ave., Ocean City, MD, 6:30 p.m. Doors open at 4:30 p.m., games start at 6:30 p.m. Food and non-alcoholic drinks available at 5:15 p.m. Open to the public. 410-289-3166, http://www.alpost166.org
**OC NEIGHBORHOOD PARKS MEETING**
Northside Park Recreation Complex’s Community Room, 200 125th St., Ocean City, MD, 7 p.m. Ocean City Recreation and Parks is holding a public meeting to discuss Northside Park located on 125th Street, bayside, 410-250-0125
**FRI, OCT. 21**
**FALL RESTAURANT WEEK**
Participating restaurants will offer special, fixed-price menus. No passes, tickets or coupons needed. http://www.oceancityrestaurantweek.com
**FREE INTRODUCTORY PICKLEBALL CLINICS**
Ocean Pines Community Center, 235 Ocean Parkway, Ocean Pines, MD, Ocean Pines, MD, 2:30 to 3:30 p.m. Every Friday through Dec. 16. Free clinic for brand new players. Registration is required by calling 410-641-7052.
**FAMILY FALL FESTIVAL**
Most Blessed Sacrament Catholic School, 11242 Racetrack Road, Berlin, MD, 3:30 to 7 p.m. Featuring carnival-style kids’ games, face painting, hay rides, pumpkin painting and more. There will also be a trunk-or-treat contest. For a $10 fee, decorate your trunk or truck bed with family friendly themes and hand out candy. Prizes awarded. A variety of food and refreshments available for sale. Pam Houck, firstname.lastname@example.org
**3RD ANNUAL ZOMBIE 5K/1 MILE WALK**
John Walter Smith Park, 6030 Public Landing Road, Snow Hill, MD, 5 p.m. Open registration begins at 5 p.m., race begins at 6 p.m. Registration fee is $25 for the 5K and $20 for the 1-mile walk. Families of a can register for the 5K course for $90 and the 1-mile course for $70. An additional $5 for registering day of the event. Prizes awarded to top runners. Participants are invited to dress in costume. Benefiting WCRP youth scholarship fund. Lea Catoggio, email@example.com, 410-632-2144, Ext. 2509, http://www.WorcesterRecandParks.org
**FREE FAMILY NIGHT PUMPKIN PAINTING**
Ocean Pines Community Center, 235 Ocean Parkway, Ocean Pines, MD, 6 to 8 p.m. Attendees may bring their own pumpkins or purchase one for $6 (limited quantity). All decorations and paint will be provided. Cost is $5 for Ocean Pines residents and $6 for non-residents. Ocean Pines Recreation and Parks Department, 410-641-7052
**KNIGHTS OF COLUMBUS BINGO**
Columbus Hall (behind St. Luke’s Church), 9901 Coastal Highway, Ocean City, MD, 6:30 p.m. Doors open at 5 p.m. and games begin at 6:30 p.m. Refreshments for sale. 410-524-7994
**SAT, OCT. 22**
**FALL RESTAURANT WEEK**
Participating restaurants will offer special, fixed-price menus. No passes, tickets or coupons needed. http://www.oceancityrestaurantweek.com
**MAKING STRIDES AGAINST BREAST CANCER 5K WALK / RUN**
Ocean City Inlet Parking Lot, 806 Atlantic Ave., Ocean City, MD, 7:30 a.m. Check-in at 7:30 a.m. The 5K run begins at 9 a.m. and walk starts approximately 9:15 a.m. Pre-register online at www.makingstidewalk.org/oceanctydmt. Advance registration is $35 for the 5K; the walk is free. Participants will receive a T-shirt. Top finishers and fundraisers receive awards. Jamie Barrett, firstname.lastname@example.org, 410-726-3230
**DELMARVA HERITAGE DAYS**
Delmarva Discovery Center & Museum, 2 Market St., Pocomoke City, MD, 10 a.m. to 4 p.m. Learn about the art of decoy carving, watch a live carving demonstration and get feedback from an expert on your own decoy. Children can participate in a “Paint a Wooden Duck Silhouette” instruction. Assateague and Barrier Island Hunt Clubs, as well as Market & Outlaw Gunners will be sharing their hunting stories. Cost is $10 for adults and $5 for children. Two-day tickets are $15 for adults and $8 for children. Members are free. email@example.com, 410-957-9933, http://www.DelmarvaDiscoveryCenter.org
**FARMERS MARKET**
White Horse Park, 239 Ocean Parkway, Ocean Pines, MD, 8 a.m. to 1 p.m. Held every Saturday. Locally grown vegetables and fruits, eggs, honey, kettle corn, flowers, artisan breads, seafood, meats and more. 410-641-7717, Ext. 3006
**AIRPORT DRIVE-IN BREAKFAST**
Ocean City Airport, Terminal Building, 12724 Airport Rd, Berlin, MD, 9 a.m. to 12 p.m. Coffee, eggs, bacon, sausage, scrapple and potatoes. Suggested donation is $7. All donations directed to the Huey Veterans Memorial Display and Park. Coleman Bunting, 410-726-7207
**FREE FINANCIAL SEMINAR**
Ocean Pines Community Center, 235 Ocean Parkway, Ocean Pines, MD, 10 to 11 a.m. Identity theft will be the topic of this seminar. Robert Mullen will share how thieves obtain personal information and how to reduce your risk. Advance registration is required. Ocean Pines Recreation and Parks Department, 410-641-7052
**O.C.TOBERFEST BEACH MAZE**
Ocean City beach at N. Division Street, Atlantic Avenue at N. Division Street, Ocean City, MD, 10 a.m. to 5 p.m. Family fun beach maze featuring wicked witches, pirates of the sand, scary scarecrows, ghouls in the graveyard, creepy clowns, zombies and more. Admission is free. http://specialeventpro.com/oc-toberfest
**O.C.TOBERFEST BOUNCING ON THE BEACH**
Ocean City Beach on the north side of the Amusement Pier, 10 a.m. to 5 p.m. Two terrific inflatable bounce houses and slides. A 5-in-1 combo and Tropical Wave Slide will be available. Admission is free. http://www.oceocean.com
**O.C.TOBERFEST BIG TOYS ON THE BOARDWALK**
Ocean City Boardwalk Amusement Pier, 401 S Atlantic Ave., Ocean City, MD, 11 a.m. to 1 p.m. The walkway around the amusement pier will be lined with vehicles to touch, climb in, take photos with and enjoy. Including vehicles such as an Ocean City beach tractor, wheel loader, articulating bus, Beach Patrol ATV, Ocean City Police Cruiser, Boat and more. http://www.oceocean.com
**AMERICAN APPLE FESTIVAL**
Berlin Nursing & Rehabilitation Center, 9715 Healthway Drive, Berlin, MD, 12 to 3 p.m. Featuring live music by Randy Lee Ashcraft, face painting, kids games, raffles, hot dogs, hamburgers, French fries, apple pies, popcorn, son-cones, Magee Farms Farmer’s market and a classic car show with trophies. All proceeds benefit the Resident’s Activity Fund and the Alzheimer’s Association. Take lawn chairs. Angie Latham or Heather Cormack, 410-629-6116 or 410-629-6123
**O.C.TOBERFEST HOWL-O-WEEN PET PARADE**
Ocean City Boardwalk at N. Division Street, Atlantic Avenue and N. Division Street, Ocean City, MD, 12 p.m. Event offers lots of prizes and surprises for best-dressed pets, family and friends. While the event is free to participate, donations of pet supplies and monetary donations will be collected to benefit the Worcester County Humane Society. Registration begins at noon, parade starts at 1 p.m. https://specialeventpro.com/oc-toberfest
**TOWN CATS KITTEN CARNIVAL AND ADOPTION**
OC Pet Spa, 9808 Stephen Decatur Highway #1, West Ocean City, MD, 12 to 4 p.m. Bounce house, games, food, prizes, vendors and lots of kittens with special low adoption fees. Take your leashed costumed pets. Rain date is Sunday, same times.
**2016 HOMETOWN HEROES MILITARY BANNER PRESENTATION CEREMONY**
Ocean City Elks Lodge #2645, 13708 Sinepuxent Road, Ocean City, MD, 2 p.m. Featuring 24 local, living, World War Veterans and 16 Active Duty members of the various branches of our Armed Forces. The public is welcome. Pat Riordan, 443-623-6162, firstname.lastname@example.org
**6TH ANNUAL DRIVE IN DISGUISE BOARDWALK PARADE**
Ocean City Boardwalk at 27th Street, Atlantic Avenue and 27th Street, Ocean City, MD, 3 p.m. Residents and visitors with a vintage Ocean City license plate are invited to ride down the Boardwalk. Anyone wishing to decorate their car will also be invited to participate. Decorated bicycles and riders also welcome. The parade will proceed along the Boardwalk from 27th Street to the inlet parking lot. There is no fee to participate for those with an Ocean City “vintage” plate, a $25 donation fee for all others. Proceeds benefit the Ocean City Downtown Association. To obtain a city vintage plate go to www.oceancitymd.gov. Nancy Howard, email@example.com, 443-235-4405, http://www.downtownassociation.net
**TOWNCENTER BOO-ARDWALK PET & PAL COSTUME PARADE**
67th St. TownCenter, 6701 Coastal Highway, Ocean City, MD, 3:30 to 5 p.m. Little Goblins are encouraged to dress their pets in costume to ‘parade’ around the TownCenter Boardwalk and collect a “treat” from each merchant. SweetFrog will feature their mascot “Cookie” and a prize wheel. Stay and enjoy Mione’s and Longboard Café’s children’s menus. Held rain or shine. Patti, 443-880-7795
O.C. TOBERFEST TRUNK OR TREAT
Ocean City Boardwalk north of the Amusement Pier, 401 S Atlantic Ave., Ocean City, MD, 3:45 p.m. Drive in Disguise participants have the option of participating in this candy and treat give-away. The event will last as long as the participants have treats to give. http://www.ococean.com
ROCKTOBERFEST FISHING TOURNAMENT
Bahia Marina, 2107 Herring Way, Ocean City, MD, 4 p.m. A 24-hour rockfish tournament. Prizes awarded for heaviest rockfish, flounder, trout, tautog and an open category. Concludes with the awards banquet around an “Eastern Shore” bonfire and pig roast on the beach at Fish Tales. http://www.ocrocktoberfest.com
3RD ANNUAL ‘FLAMES AT THE FURNACE’ BARBECUE FUNDRAISER
Furnace Town Living Heritage Village, 3816 Old Furnace Road, Snow Hill, MD, 5 to 8 p.m. Dress is casual for this southern barbecue with craft and domestic beer and wine served. Silent and live auctions, raffle and door prizes. Live music by Apple & Brit. Tickets cost $30 and can be purchased at Furnace Town by calling 410-632-2032 or online at www.eventbrite.com.
SUN, OCT. 23
FALL RESTAURANT WEEK
Participating restaurants will offer special, fixed-price menus. No passes, tickets or coupons needed. Go to the website for list of participating restaurants. http://www.oceancityrestaurantweek.com
ROCKTOBERFEST FISHING TOURNAMENT
Bahia Marina, 2107 Herring Way, Ocean City, MD, 12 a.m. to 4 p.m. A 24-hour rockfish tournament. Prizes awarded for heaviest rockfish, flounder, trout, tautog and an open category. Concludes with the awards banquet around an “Eastern Shore” bonfire and pig roast on the beach at Fish Tales. http://www.ocrocktoberfest.com
AIRPORT DRIVE-IN BREAKFAST
Ocean City Airport, Terminal Building, 12724 Airport Rd, Berlin, MD, 9 a.m. to 12 p.m. Coffee, eggs, bacon, sausage, scrapple and potatoes. Suggested donation is $7. All donations directed to the Huey Veterans Memorial Display Park. Coleman Bunting, 410-726-7207
O.C. TOBERFEST BEACH MAZE
Ocean City Beach at N. Division Street, Atlantic Avenue at N. Division Street, Ocean City, MD, 10 a.m. to 5 p.m. Family fun beach maze featuring wicked witches, pirates of the sand, scary scarecrows, ghouls in the graveyard, creepy clowns, zombies and more. Admission is free. http://specialeventpro.com/oc-toberfest
O.C. TOBERFEST BOUNCING ON THE BEACH
Ocean City Beach on the north side of the Amusement Pier, 10 a.m. to 3 p.m. Two terrific inflatable bounce houses and slides. A 5-in-1 combo and Tropical Wave Slide will be available. Admission is free. http://www.ococean.com
DELMARVA HERITAGE DAYS
Delmarva Discovery Center & Museum, 2 Market St., Pocomoke City, MD, 12 to 4 p.m. Learn about the art of decoy carving, watch a live demonstration and get feedback from an expert on your own decoy. Children can participate in a “Paint a Wooden Duck Silhouette” instruction. Assateague and Barrier Island Hunt Clubs, as well as Market & Outlaw Gunners will be sharing hunting stories. Cost is $10 for adults and $5 for children. Two-day tickets are $15 for adults and $8 for children, Members are free. firstname.lastname@example.org, 410-957-9933; http://www.DelmarvaDiscoveryCenter.org
ALCOHOLICS ANONYMOUS
Atlantic General Hospital, Conference Room 2, 9733 Healthway Drive, Berlin, MD, 12 to 1 p.m. Group shares experience, strength and hope to help others. Open to the community and to AGH patients. Rob, 443-783-3529
WORCESTER COUNTY HISTORICAL SOCIETY’S ANNUAL FALL DINNER
Dunes Manor Hotel, 2800 Baltimore Ave., Ocean City, MD, 1 p.m. Hunter “Bunk” Mann’s talk will focus on the history of Ocean City from the 1940’s to the late 1950’s — problems faced locally during World War II and the growth that followed. Open to the public. Tickets cost $25 and can be purchased by sending a check to Robert Fisher, 230 S. Washington St., Snow Hill, MD 21863.
OVEREATERS ANONYMOUS
Atlantic General Hospital, Conference Room 1, 9733 Healthway Drive, Berlin, MD, 2:30 to 3:30 p.m. Group is a 12-step program for anyone struggling with a compulsive eating problem. No initial meeting charge. Meeting contribution is $1 weekly. Bett, 410-202-9078
MON, OCT. 24
OVEREATERS ANONYMOUS
Atlantic General Hospital, Conference Room 1, 9733 Healthway Drive, Berlin, MD, 6:30 a.m. to 7:30 p.m. Group is a 12-step program for anyone struggling with a compulsive eating problem. No initial meeting charge. Meeting contribution is $1 weekly. Bett, 410-202-9078
FREE ATHLETIC INJURIES SEMINAR
Ocean Pines Community Center, 235 Ocean Parkway, Ocean Pines, MD, 9 to 10 a.m. Atlanic General Hospital’s Dr. Dan Pascucci will share how to prevent common athletic injuries. Learn how to protect joints while remaining active and healthy. Ocean Pines Recreation and Parks Department, 410-641-7052, http://www.OceanPines.org
7TH ANNUAL LOCAL’S APPRECIATION WEEK
Ocean City Life-Saving Station Museum, 813 S Atlantic Ave., Ocean City, MD, 10 a.m. to 5 p.m. The Ocean City Life-Saving Station Museum is offering free admission and 10 percent off gift shop purchases to any visitor who considers themselves a “local” (anyone who loves Ocean City and considers it their home for a day, month, year). http://www.ocmuseum.org
TUE, OCT. 25
SNOW HILL ROTARY CLUB MEETING
All Hallows Church Parish House, 109 W. Market Street, Snow Hill, MD, 7:30 a.m. Contact email@example.com or 410-546-1978 for more information.
FREE WOMEN’S HEART PROGRAM
Pocomoke Public Library, 301 Market St., Pocomoke City, MD, 8 a.m. to 12 p.m. Screening assessments will be performed on board the Wagner Wellness Van. Appointments are required. Women choosing to participate must not currently be under the care of a cardiologist or have a known history of heart disease. 410-543-7026
WED, OCT. 26
KIWANIS CLUB OF GREATER OCEAN PINES/OCEAN CITY
Ocean Pines Community Center, 235 Ocean Parkway, Ocean Pines, MD, 8 a.m. Meets every Wednesday. Doors open at 7 a.m., meeting begins at 8 a.m. 410-641-7330, http://www.kiwanisofopoc.org
DELMARVA SWEET ADELONE CHURCH
Ocean Pines Community Center, 239 Ocean Parkway, Ocean Pines, MD, 7 to 9 p.m. The group meets each Monday. Women interested in learning the craft of a cappella singing welcome. 410-641-6876
DELMARVA WOMEN’S A CAPELLA CHORUS GUEST NIGHT
Ocean Pines Community Center, 235 Ocean Parkway, Ocean Pines, MD, 7 p.m. Music, laughter, fellowship and lots of singing. Jean Beatty, 410-208-4149
FLU SHOT CLINIC
Atlantic General Women’s Health, 38394 Dupont Blvd., Unit H, Selbyville, DE, 1 to 4 p.m. Vaccines should be repeated each year. Must be 13 years old. If you are allergic to eggs, consult with your health care provider. Donations made during the Flu Clinics will help offset the cost of future Flu Clinics. 410-641-91FLU (93558)
KNIGHTS OF COLUMBUS SIMPLE SUPPER
Columbus Hall (behind St. Luke’s Church), 9901 Coastal Highway, Ocean City, MD, 5 to 7 p.m. This month is Italian Sausage subs and a make your own salad bar. Takes place the last Wednesday of each month. Full meal for only $7. Cash bar. 410-524-7994
DELMARVA HAND DANCE CLUB
Elks Lodge, 13708 Sinopexent Ave., Ocean City, MD, 5:30 to 9 p.m. Jitterbug, swing, cha-cha and Carolina Beach music. Meets every Wednesday. firstname.lastname@example.org, 302-200-3262, http://delmarvahanddancing.com
OCEAN CITY/BERLIN ROTARY CLUB MEETING
Captain’s Table Restaurant in the Courtyard by Marriott, 2 15th St, Ocean City, MD, 6 p.m. The group meets every Wednesday. email@example.com, 410-641-1700
BEREAVEMENT SUPPORT GROUP
Atlantic General Hospital, conference room 1, 9733 Healthway Drive, Berlin, MD, 7 to 8 p.m. The group gathers the fourth Wednesday of each month. Pre-registration is not necessary. Pastoral Care Services, firstname.lastname@example.org, 410-641-9725
**HELP WANTED**
**99 SEA LEVEL**
1001 Ocean City Blvd.
Located on the boardwalk in the heart of Bethany Beach. We are a full service, the dining restaurant and bar serving lunch and dinner 7 days a week.
**CASH BONUS**
after completion of training and 60 days of positive employment!
Seeking professional, highly motivated, friendly, and experienced Servers & Banquet Servers
Email your resume to: email@example.com
---
**Now Hiring for Year Round Full Time Delivery Drivers w/own car**
Come in for Interview on Thursdays @ 11:00 am
5601 Coastal Hwy. (Bayside)
---
**Join Team Dunes! Now Hiring:**
- **BARTENDER**
- **DISHWASHER**
- **SERVERS**
- **BANQUET MANAGER**
28th & Oceanfront - "For Shore ... The Best Place to Work"
Please apply online at www.realhospitalitygroup.com
---
**Work At The BEACH... Work With The BEST!!!**
Top wages, excellent benefits package and free employee meal available to successful candidates.
**Employment Opportunities:**
Year Round, Full/Part Time: Banquet Servers, Banquet Houstestaff, Purchasing Agent, Room Attendant (van will pick up in Salisbury), Wash Room (Laundry), Front Desk Agent, Housekeeping Houstestaff, Reservations, Food Runners, Server, Bartender
**Excellent Benefits and Free Employee Meal.**
Clarion Resort Fontainebleau Hotel
Attn: Human Resources Dept.
10100 Coastal Highway, Ocean City, MD 21842
Phone: 410-524-3535 Fax: 410-723-9109
EOE/M/F/D/V
---
**HELP WANTED**
**NOW HIRING!!**
**Production Supervisor**
for our WCC kitchen facility Up to $17/hour
Apply online at: www.delmarvadd.com
---
**HELP WANTED**
**Experienced Servers & Line Cook Needed** for Year Round position. Apply in Person @ Alex’s Italian Restaurant, Rt. 50, West Ocean City.
A busy contractor company in Ocean Pines, MD is currently hiring: HVAC Maintenance Technician, Plumber and Plumber’s Helpers. START IMMEDIATELY. To apply call Marc at 302-682-1777.
**FT/YR Guest Service Rep**
Excellent Benefits & Pay. Must have great customer service skills. Apply in person @ 105 120th St., Ocean City.
**FT Housekeeper Wanted,**
No experience necessary. Apply in person @ SeaTime Condominiums, 6 135th St., Ocean City, Md.
---
**HELP WANTED**
*Alban Service Advisor*
Alban CAT, the Caterpillar dealer for the mid-Atlantic Region is accepting applications for a Service Advisor for our Ocean City Branch. The Service Advisor is responsible for communicating with the customer, gathering information and providing recommendations. Open and closes work orders, orders parts and outside service in an efficient and timely manner. Minimum three years service administrative experience required. Must have excellent customer service and communication skills, problem solving and parts ordering experience in the automotive or related industry preferred. Working knowledge of preventative maintenance and repair of marine, industrial or heavy construction equipment preferred. For more information and to apply, please visit our hiring site: www.albanhring.com
**Employment Authorized**
Alban CAT is an Equal Opportunity/Affirmative Action Employer. All qualified applicants will receive consideration for employment without regards to race, color, religion, sex, national origin, disability, protected veteran status, sexual orientation and gender identity. If you need reasonable accommodation for any part of the application and hiring process, please notify Alban CAT by calling 410.686.7777 and asking to speak with the Resources Department. Alban CAT is a federal contractor. Alban CAT is a drug free workplace.
---
**HELP WANTED**
**Chairside**
DENTAL ASS’T.
Experience Preferred
Ocean View, DE
Email Resume:
firstname.lastname@example.org
---
**HELP WANTED**
**OCEAN BLOCK CONDO** Y/R 2BR/1BA. W/D, DW, AC, furnished, 125th Street. Great views of the bay. Convenient to shopping, bank & Northside Park. 1100/month + utilities. 443-386-5944
**Ocean City**
Winter or Year round rental near canal. NO PETS. Call 443-235-2556.
**YEAR ROUND RENTAL**
Emerson House, 48th and Boardwalk Hy, 1 bed, 1 bath. 1 off-street parking space ocean block. Call 443-365-6169 after 5 pm.
**1BR Apartment For Rent in Old Town OC. 1 person only. No pets. $500/month deposit. NO PETS. Non-smoking. Call 410-289-2344.**
**2BR/2BA Unfurnished Condo, 141st St. Available Nov. 1, can show now. $900/month. Call Ron Steen 302-448-0272.**
**WINTER WEEKLY RENTALS**
Pool Front Rooms $175. Efficiencies $195. 2BR Apartments $280. Burgundy Inn 1201 Philadelphia Ave. 410-285-8851
---
**HELP WANTED**
**NOW HIRING!!**
**Production Crew**
for our WCC kitchen facility Starting at $10.50/hr.
Apply online at: www.delmarvadd.com
---
**SELL REAL ESTATE AT THE BEACH**
**Interested in a career in Real Estate?**
Coldwell Banker School of Real Estate is offering Monthly Licensing Classes
Classroom or Online Available
Contact Lynn Mauk at 410-524-6111
CBRBSchool.com
Free Career Night every Wednesday
---
**Hiring Nurses**
GNA’S, RN’s & LPN’s
All shifts. Please stop by to fill out an application, fax your resume to 410-641-0228 or email it to us at www.marchhealth.com. Come and find out why we are the area's only CMS 5-Star rated skilled nursing facility. EOE
Classifieds
410-723-6397
www.baysideoc.com
www.oceancitytoday.net
---
**RAMBLER MOTEL**
9942 Elm Street, WOC (Behind Starbucks)
Sleeps 4, $250 per week
Manager onsite
410-213-1764
---
**Holiday**
Real Estate Inc.
Yearly & Seasonal Rentals
We Welcome Pets
7700 Coastal Hwy
410-289-8888
www.holidayoc.com
---
**Get the most widely read newspaper classifieds on the coast and get online classifieds free of charge**
Now you can order your classifieds online
Convenient, quick, no waiting, no long distance calls, days, nights and weekends.
CALL BY MONDAY AT 5 P.M.
DIRECTORY
AUTOMOTIVE REPAIR
RACETRACK
Auto & Marine
AUTO & MARINE TIRE CENTER
COMPLETE BODY SHOP
Auto Sales & Service • Complete Computerized Diagnostic Specialists
TRAILER PARTS, SALES & SERVICE
ROUTE 589, RACE TRACK ROAD
410-641-5262
CARPET CLEANING
CENTURY CARPET CLEANING
LIVING ROOM, 2 BEDROOMS AND HALLWAY
All for $70
410-723-2300
Some restrictions apply
CLEANING SERVICES
$100 OFF
New Customer
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|
NEW YORK STATE COUNCIL ON THE ARTS 1962
NEW YORK STATE
Library of Congress Catalogue Card Number 62-62582
| Section | Page |
|----------------------------------------------|------|
| FOREWORD | 3 |
| Nelson A. Rockefeller, Governor | |
| INTRODUCTION | 4 |
| William Schuman | |
| THE CHAIRMAN’S STATEMENT | 6 |
| Seymour H. Knox | |
| REPORT ON CURRENT PROGRAM | 9 |
| THE FUTURE | 42 |
| John H. MacFadyen, Executive Director | |
| THE 1964 NEW YORK WORLD’S FAIR | 45 |
| MEMBERS OF THE COUNCIL | 47 |
| ADVISORS TO THE COUNCIL | 48 |
| CONSULTANTS TO THE COUNCIL | 51 |
| COUNCIL STAFF | 52 |
| FINANCIAL STATEMENT | 52 |
COUNCIL ON THE ARTS
The new stadium will be built on the site of the old one, which was demolished in 1960. The new stadium will have a capacity of 50,000 and will be equipped with all the latest technology. The construction of the stadium is expected to be completed by the end of 2023.
Once again it is my privilege to express my gratitude and the appreciation of the people of our state to the New York State Council on the Arts and to the Legislature, which created it. Under the enabling Act, the Council’s purpose is “to insure that the role of the arts in the life of our communities will continue to grow and will play an ever more significant part in the welfare and educational experience of our citizens and in maintaining the paramount position of this state in the nation and in the world as a cultural center.” A detailed account of the progress and plans pertinent to the role of the arts in the life of our communities is set forth in this report.
In spite of the signal success of its present operation, the Council continues to evaluate and reappraise, to search for its most effective place in fostering and encouraging the Arts. First by its existence, and now through its careful work, the Council is making a significant contribution toward maintaining the paramount position of our state’s leadership in the Arts. In recognizing the national cultural resurgence to which I referred a year ago, and in taking positive legislative action to acknowledge a relationship between government and this resurgence New York continues to lead the Nation. The Council on the Arts is providing a pattern for emulation both for the Federal government and for many of our states.
As the Council progresses, it does so with a keen sense of responsibility, not only to our own citizens but to our sister states. While enjoying our many unique cultural advantages, we recognize that one of the most practicable plans for government encouragement of the arts can emerge from the gradual establishment across the country of State programs similar to ours, with a resulting exchange of ideas and projects. What we do will affect the growth of this movement; it is our deepest concern that as we lead, we will do so with courage and vision.
NELSON A. ROCKEFELLER
Governor
Governor Rockefeller and Lt. Governor Malcolm Wilson, Chairman of the New York State Commission on the World’s Fair, discuss the Council’s plans for the fair with Chairman Seymour H. Knox and Executive Director John H. MacFadyen before a model of the state pavilion.
A few days after the opening of Lincoln Center, I received a letter from a man in Lincoln, Illinois (pop. 17,000). A former New Yorker, he remains a New Yorkophile. He said our community is a cultural oasis, and his is a cultural wasteland. Lincoln Center, he said, is only for New Yorkers and visitors to New York. “I place Lincoln Center in the same category as my government in this respect. Week after week glory will come to Lincoln Center and we’ll be in Lincoln, Illinois. Month after month my government sends artists to all corners of the world, but not to Lincoln, Illinois. I love and respect you both, but do you understand my feelings?”
I understand completely, and I sympathize. But I disagree that Lincoln Center is for New Yorkers alone. Its influence will be national. Indeed it is already having an effect: it has heightened a hunger for the arts in one man in Lincoln, Illinois. And he, I am sure, is but one among millions.
A new hunger for the arts and the enrichments they give to life is a phenomenon of our contemporary American scene. A. E. Housman, the British poet and essayist, once remarked that the effects of physical hunger and thirst are all too apparent. But we cannot see the ravages when man’s innate craving for knowledge is denied satisfaction. Nor can we when he hungers for the arts. In Housman’s words, “though the man does not die altogether, part of him starves to death: as Plato says, he never attains completeness of health, but walks lame to the end of his life.”
Millions of Americans, like my correspondent from Lincoln, Illinois, suffer a lack of cultural opportunity. America has thousands of communities that are artistically underprivileged. They are artistically underdeveloped areas with wholly inadequate access to the arts. For the artistically undernourished who live in these communities, there is but one cure. And the cure is nourishment.
Fortunately, the problem is coming to be recognized. I believe America has come to a historic turning point in its attitude toward the arts. The well-being of the creators, performers, and institutions of the arts is coming to be accepted as a responsibility of the whole community. We are beginning to recognize that the arts — like schools, hospitals, and welfare agencies — need and deserve the continued help of every segment of our society.
Art, from the days of Maecenas, has always had its patrons. For centuries the patron was the individual. Then, in our own day, foundations and corporations have likewise become patrons. And now — the most significant breakthrough of all — government is beginning to do its share. Now, as never before in our country’s history, the people — the body politic — are patron of the arts.
All who are concerned with the cause of the arts cannot fail to recognize a new attitude of the institutions of the arts toward government and of government toward the arts. We are developing a new solution to an old problem; our society is evolving a Twentieth Century American solution toward the support of the arts. The solution is emerging from a dynamic partnership of traditional private philanthropy and enlightened government assistance which, though its forms are various and sometimes subtle, is growing across America and on every level of government.
The artist is learning that the bugaboo of “government interference” is just that — a bugaboo, a fear without foundation in our American society.
And government is changing its attitude towards the arts. Dozens of instances can be cited, but none is more significant than the short but brilliant history of the New York State Council on the Arts. Here is government giving help so desperately needed and the people in communities throughout the State receiving the artistic nourishment they crave.
The Council is a credit to all who have conceived it, and who work in its cause. This report of its service to the people of our State is dramatic evidence of America’s new Twentieth Century solution at work — the partnership of private and public support.
It is working in New York, and from it, my friend in Lincoln, Illinois, should take heart.
William Schuman
President, Lincoln Center for the Performing Arts
The Chairman’s Statement
In the intervening year since the report, *New York State Council on the Arts, 1961* was published, the Council has continued its efforts with a variety of programs and projects. Reviewing the “Statement of Objectives” from that report it would seem appropriate to repeat those broad principles that serve to guide us in our endeavor. They include the recognition of standards as fundamental to the arts, the continued intent to raise these standards a prime obligation of the Council. One prime function of the Council is to bring quality performances and exhibitions to new and expanded audiences.
To help meet the prime obligation, the Council has stepped up its program of Technical Assistance. This has included the assignment of specific advisors to community arts projects requesting this service. And on a regional basis, a number of highly successful workshops have been conducted, bringing together representatives of organizations with problems in common to meet with experts in the handling of these problems.
To help perform the prime function, the Council has continued its program of support for touring performances and exhibitions.
This report, on the year 1962, seeks to demonstrate the diversity and geographical distribution of our work. There follows a chronological listing of Council-supported activities as they have occurred in six broad regions of the State. While the definition of these regions has been somewhat arbitrary, it may serve to suggest the degree of interest that exists or is developing. Of course, this calendar does not reflect the tremendous interest and support for the arts that already exist, without direct aid from the Council, in many communities throughout the State. One frequently reads statistics supporting the national trend towards reawakening this interest. The *New York Times* recently quoted the contention that “there are twice as many people listening to concerts and recitals as at Major League ball games.”
I can only add our factual experience — in Buffalo, during little more than a month, 247,000 people visited the Andrew Wyeth exhibition at the Albright-Knox Art Gallery. This growing local enthusiasm, familiar to residents of every region, is our cultural vitality. Without it, the work of the Council could have no permanent effect.
An architectural treasure of each region is illustrated by a photograph in the geographical sections. At a time when so many of these great buildings are being threatened by the forces of population, highway construction and urban development we must pause to consider the depth of this part of our cultural heritage and rededicate ourselves to its preservation. New York is a state of natural and man-made beauty, it is incumbent on each of us to remain conscious of this beauty and to protect it.
Again, we must express our profound debt to the many organizations, institutions and individuals whose devoted cooperation makes our work possible. Their mere listing in a chronological way is bound to be incomplete and can by no means indicate the dimension of their contribution to our mutual objectives.
The Council acknowledges its deep sense of responsibility to Governor Rockefeller and our Legislators who continue to demonstrate their concern for the enrichment of the lives of our citizens. The 1962 Legislature, beyond approving an increased appropriation to permit the expanding of our activities, passed supplementary legislation extending the life of the Council to March 31, 1967, and investing it with further specific powers to help implement its diverse programs. It is our constant hope that in the evolution of this unique project we may continue to deserve their confidence.
Seymour H. Knox
Chairman
Western Area
Niagara
Orleans
Genesee
Erie
Wyoming
Chautauqua
Cattaraugus
Allegany
Report on Current Program, Western Area
January 3-4, Buffalo, Technical Assistance to the Albright-Knox Art Gallery, Identification of oriental collection by Fong Chow.
January 7-28, Jamestown, AFA Exhibition at Jamestown Community College, Fifteen Years of Award Winning Prints.
January 21, Buffalo, Meeting of the New York State Council on the Arts.
February 11, Buffalo, New York City Ballet, Full company performance.
February 12, Batavia, New York City Ballet, Lecture demonstration.
May 22-23, Wyoming, Technical Assistance to the Middlebury Historical Society, Revision of facilities by Keith Martin.
August 15, Cattaraugus County, Survey of cultural institutions by Assistant Director of the Council.
August 16, Chautauqua County, Survey of cultural institutions by Assistant Director of the Council.
September 26, Medina, Rochester Philharmonic Orchestra, Concert.
The Old State House, built in 1836, is one of the oldest state capitols still in use in the United States. It was designed by architect Thomas U. Walter and is located in Augusta, Maine. The building has served as the seat of government for Maine since its statehood in 1820. It features a distinctive cupola on top and is constructed from granite quarried nearby. The interior includes ornate woodwork and murals depicting scenes from Maine's history. Today, the Old State House houses various offices and departments related to state government.
| Date | Event |
|------------|----------------------------------------------------------------------|
| October 2 | Warsaw, Rochester Philharmonic Orchestra, Concert. |
| October 5 | Buffalo, American Ballet Theatre, Full company performance. |
| October 20 | Alfred, Phoenix Theatre, The Matchmaker. |
| October 23 | Olean, Phoenix Theatre, The Matchmaker. |
| November 7 | Buffalo, Phoenix Theatre, The Matchmaker. |
| November 20| Jamestown, Technical Assistance to the city, Architectural evaluation of the Jamestown City Hall by Harley J. McKee. |
| November 25| Buffalo, New York City Center Opera Company, Rigoletto. |
| November 26| Jamestown, Buffalo Philharmonic Orchestra, Concert. |
| November 27| Buffalo, New York City Center Opera Company, Marriage of Figaro. |
With support from the Council within its budget for the current fiscal year, performances are planned for the winter and spring by the New York Shakespeare Festival in Batavia; Ars Antiqua in Alfred; Jose Limon in Lackawanna; the Metropolitan Opera Studio in Olean, Wellsville and Jamestown; and other programs in music, the visual arts and technical assistance.
*The Genesee County Courthouse, a building in the late Federal style built in Batavia in 1841.*
Finger Lakes Area
January 1-16, Corning, AFA Exhibition at Corning Community College, Masterpieces of Photography.
February 10, Rochester, New York City Ballet, Full company performance.
Rochester, Meeting with the Executive Committee of the York State Craftsmen by the Assistant Director of the Council.
February 11-March 4, Rochester, AFA Exhibition at Rochester Public Library, Indian Art of the United States.
Rochester, AFA Exhibition at University of Rochester, The Hudson River School.
February 12, Geneva, Technical Assistance to the Geneva Historical Society, Cataloguing and display techniques by Francis Cunningham.
Geneseo, New York City Ballet, Lecture demonstration.
Ithaca, New York City Ballet, Lecture demonstration.
Auburn, New York City Ballet, Lecture demonstration.
March 3, Geneseo, Technical Assistance to the Livingston County Historical Society, Display techniques by Beaumont Newhall.
March 4-25, Ithaca, AFA Exhibition at Andrew Dickson White Museum of Art. Masterpieces of Photography.
March 15-16, Aurora, Technical Assistance to the Elbert Hubbard Library, Cataloguing, accessioning and display techniques by Francis Cunningham.
March 20, Rochester, Address before the County Officers Association by the Executive Director of the Council.
The house was built in 1835 by John C. Halsey, a prominent lawyer and judge who served as a member of the New York State Assembly. The design is attributed to architect Alexander Jackson Davis, known for his neoclassical style. The house features a grand portico with six Ionic columns supporting a triangular pediment, which is characteristic of Greek Revival architecture. The interior includes a large ballroom and several reception rooms, all designed to impress guests with their elegance and grandeur. The Halsey House is now part of the National Register of Historic Places and is open to the public for tours, offering visitors a glimpse into the lifestyle of wealthy New Yorkers in the 19th century.
April 1-18,
**Auburn**, AFA Exhibition at Auburn Community College, *How To Look At A Painting*.
April 4,
**Ithaca**, Address before the Cornell University Festival of Contemporary Arts by the Executive Director of the Council.
April 8-29,
**Corning**, AFA Exhibition at Corning Community College, *Three Centuries of Architecture in New York*.
April 16,
**Rochester**, Technical Assistance to Rochester Memorial Art Gallery, Jurying for regional art exhibition by Leroy Flint, Morris Kantor and George Rickey.
April 22-May 13,
**Corning**, AFA Exhibition at Corning Community College, *Fifteen Years of Award Winning Prints*.
May 14-15,
**Geneseo**, Technical Assistance to the Livingston County Historical Society, Display techniques by Per Guldbeck.
May 28,
**Geneva**, Address before the Geneva Historical Society by the Executive Director of the Council.
June 17-July 8,
**Hornell**, AFA Exhibition at Hornell Public Library, *Masterpieces of Photography*.
July 3, 13, 16,
**Brockport**, Technical Assistance to the Brockport Summer Festival, *Performance direction by Richard Stuart Flusser*.
July 17,
**Watkins Glen**, Conference with Arts representatives of Schuyler County regarding formation of Arts Council by Assistant Director of the Council.
July 18,
**Elmira**, Conference with representatives of Chemung County Arts Groups and Institutions by Assistant Director of the Council.
"Rose Hill", a Greek revival house built near Geneva about 1839.
The cast of "The Merry Wives of Windsor" on stage, with the curtain in the background.
July 19, Yates County, Survey of cultural institutions by Assistant Director of the Council.
July 20, Seneca County, Survey of cultural institutions by Assistant Director of the Council.
July 24, Lyons, Conference with President and Curator of Wayne County Historical Society by Assistant Director of the Council.
July 25, Rochester, Survey of cultural institutions by Assistant Director of the Council.
August 13, Watkins Glen, Address to Schuyler County Arts Council by Assistant Director of the Council.
August 13-14, Lyons, Technical Assistance to the Wayne County Historical Society, Display techniques by Jane des Grange.
August 14, Steuben County, Survey of cultural institutions by Assistant Director of the Council.
August 17, Brockport, Conference with Arts representatives regarding formation of Arts Council by Assistant Director of the Council.
September 9-30, Hornell, AFA Exhibition at Hornell Public Library, Indian Art of the United States.
September 10, Seneca Falls, Conference with Director of Seneca Falls Historical Society regarding Workshop for Finger Lakes Historical Societies by Assistant Director of the Council.
September 23-October 14, Rochester, AFA Exhibition at Nazareth College, Masterpieces of Photography.
Scene from "Rigoletto" in Rochester, from the Council-supported performance by the New York City Center Opera Company.
September 27, Wolcott, Rochester Philharmonic Orchestra, Concert.
October 1-18, Corning, AFA Exhibition at Corning Community College, How To Look At A Painting.
October 1, Watkins Glen, Rochester Philharmonic Orchestra, Concert.
October 1-21, Rochester, AFA Exhibition at Rochester Public Library, Japanese Prints.
October 3, Bath, Rochester Philharmonic Orchestra, Concert.
October 4, Corning, American Ballet Theatre, Full company performance.
October 5, Seneca Falls, Rochester Philharmonic Orchestra, Concert.
October 7, Ithaca, American Ballet Theatre, Full company performance.
October 19, Geneva, Phoenix Theatre, The Matchmaker.
October 22, Auburn, Phoenix Theatre, The Matchmaker.
October 26, Corning, Phoenix Theatre, The Matchmaker.
October 29, Ithaca, Phoenix Theatre, The Matchmaker.
October 31, Brockport, Meeting with representatives of local Arts groups by Executive Director of the Council.
November 1-2, Geneseo, Phoenix Theatre, The Matchmaker.
November 5-25. **Geneseo**, AFA Exhibition at State University, *Contemporary Landscapes and Their Antecedents*.
November 12. **Seneca Falls**, Technical Assistance to Finger Lakes Region Historical Societies, *Workshop in administration, conservation and display techniques by Alice Beer, Jane des Grange, Janet MacFarlane and Per Guldbeck*.
November 18-December 18. **Rochester**, AFA Exhibition at University of Rochester, *Masters of American Watercolor*.
November 24. **Rochester**, New York City Center Opera Company, *Rigoletto*.
November 26. **Corning**, New York City Center Opera Company, *Marriage of Figaro*.
November 30. **Ithaca**, New York City Center Opera Company, *Marriage of Figaro*.
December 1. **Ithaca**, New York City Center Opera Company, *Rigoletto*.
December 2-31. **Rochester**, Whitney Museum Exhibition at Rochester Memorial Art Gallery, *Forty Artists Under Forty*.
December 13. **Corning**, Ars Antiqua, *Concert*.
With support from the Council within its budget for the current fiscal year, performances are planned for the winter and spring by the Merry-Go-Rounders in Lyons, Geneva, Burdette, Elmira and Seneca Falls; the New York Shakespeare Festival in Rochester; Ars Antiqua in Yates and Geneva; Jose Limon in Geneseo; the Metropolitan Opera Studio in Hornell and Brockport; Demonstration concerts by Young Audiences, Inc. will be offered in the region with Council support; and other programs in music, the visual arts and technical assistance.
Central Area
Oswego
Oneida
Onondaga
Madison
Cortland
Otsego
Chenango
Delaware
Broome
January 4, Utica, Meeting with representatives of local Arts groups by Executive Director of Council.
January 7-28, Binghamton, AFA Exhibition at Roberson Memorial Center, Masters of American Watercolor.
January 29- February 18, Utica, AFA Exhibition at Munson-Williams-Proctor Institute, Masterpieces of Photography.
February 12, Oswego, New York City Ballet, Lecture demonstration.
Oswego, Meeting with representatives of local Arts groups regarding formation of Arts Council by Assistant Director of the Council.
Syracuse, New York City Ballet, Lecture demonstration.
Binghamton, New York City Ballet, Lecture demonstration.
March 6, Syracuse, Conference with Dean Kenneth Sargent and Harley J. McKee of Syracuse University regarding "Architecture Worth Saving" Project by Assistant Director of the Council.
March 18- April 8, Binghamton, AFA Exhibition at Roberson Memorial Center, The Hudson River School.
April 22- May 13, Syracuse, AFA Exhibition at Everson Museum of Art, Masters of American Watercolor.
Syracuse, AFA Exhibition at Everson Museum of Art, The Hudson River School.
Akron, AFA Exhibition at Newstead Historical Society, Indian Art of the United States.
April 22- June 17, Oswego, Conference with Oswego Arts Council regarding proposed building of new Art Center by Assistant Director of the Council.
April 23, Utica, AFA Exhibition at Munson-Williams-Proctor Institute, How To Look At A Painting.
May 1-18, Syracuse, Technical Assistance to Canal Museum, Administration and general policies for new museum by Francis Cunningham, Fred Rath and Robert Wheeler.
May 10, Oswego, Technical Assistance to Oswego Art Center, Planning on theatre design, architecture and remodeling by Omar K. Lerman and Peter Wingate.
The University of Oregon, founded in 1876, is a public research university located in Eugene, Oregon. It offers over 200 undergraduate and graduate programs across various fields including arts, sciences, engineering, business, and law. The university is known for its strong academic programs, research opportunities, and commitment to sustainability. It has a diverse student body and is recognized for its beautiful campus, which includes the iconic Camas Bell Tower.
May 25,
**Hillcrest**, Eisenberg String Quartet, (Young Audiences, Inc.), *Demonstration concert*.
**Binghamton**, Tri-Cities Opera Workshop, *Premiere Performance of Jeremiah*.
May 27-June 17,
**Utica**, AFA Exhibition at Munson-Williams-Proctor Institute, *Masters of American Watercolor*.
May 28,
**Cortland**, Conference with representatives of local Arts groups by Executive Director of the Council.
June 17-July 8,
**Utica**, AFA Exhibition at Oneida Historical Society, *Three Centuries of Architecture in New York*.
July 1-22,
**Utica**, AFA Exhibition at Munson-Williams-Proctor Institute, *Fifteen Years of Award Winning Prints*.
July 18-August 31,
**Syracuse**, AFA Exhibition at Witter Agricultural Museum, *Three Centuries of Architecture in New York*.
July 24, 25, 26,
**Binghamton**, Technical Assistance to Roberson Memorial Center, *Design for installation of Metropolitan Museum loan by Peter Wingate*.
August 9,
**Syracuse**, Technical Assistance to Canal Museum, *Display techniques by George Montgomery*.
August 25,
**Syracuse**, Technical Assistance to Civil Service Employees Association, *Jurying, awarding of prizes for art display at New York State Exposition by Gertrude Moore*.
August 26-September 9,
**Syracuse**, AFA Exhibition at Women’s Division of New York State Exposition, *Masterpieces of Photography*.
September 1-18,
**Binghamton**, AFA Exhibition at Roberson Memorial Center, *How To Look At A Painting*.
September 7,
**Oneonta**, Technical Assistance to Upper Susquehanna Historical Society, *Administration, cataloguing and display techniques by Paul Perrot*.
September 9-30,
**Oneonta**, AFA Exhibition at State University, *The Hudson River School*.
**Syracuse**, AFA Exhibition at Witter Agricultural Museum, *Three Centuries of Architecture in New York*.
*The Hamilton College Chapel in Clinton, designed by Philip Hooker in 1827.*
The Museum of Art, which opened in 1962, is one of the most important art museums in the country. It has a permanent collection of more than 30,000 works of art, including paintings, sculptures, and decorative arts from around the world. The museum also hosts temporary exhibitions featuring works from renowned artists and galleries.
October 2, Binghamton, American Ballet Theatre, Full company performance.
October 14-November 4, Oneonta, AFA Exhibition at State University, Masters of American Watercolor.
Oneonta, AFA Exhibition at Hartwick College, Three Centuries of Architecture in New York.
Utica, Whitney Museum Exhibition at Munson-Williams-Proctor Institute, Forty Artists Under Forty.
Utica, Phoenix Theatre, The Matchmaker.
Oswego, Technical Assistance to the Gerritt Smith Public Library, Architectural evaluation by Harley J. McKee.
Oneonta, Phoenix Theatre, The Matchmaker.
Binghamton, Phoenix Theatre, The Matchmaker.
Oswego, Phoenix Theatre, The Matchmaker.
Binghamton, Ars Antiqua, Concert.
Rome, Buffalo Philharmonic Orchestra, Concert.
Binghamton, Roberson Memorial Center, Opening of the extended loan exhibition from the Metropolitan Museum of Art.
Delhi, Phoenix Theatre, The Matchmaker.
Binghamton, New York City Center Opera, Marriage of Figaro.
Syracuse, AFA Exhibition at Everson Museum of Art, Masterpieces of Photography.
Syracuse, New York City Center Opera, Rigoletto.
With support from the Council within its budget for the current fiscal year, performances are planned for the winter and spring by the Merry-Go-Rounders in Utica and Delhi; the New York Shakespeare Festival in Syracuse; Jose Limon in Binghamton, Cortland and Utica; the Metropolitan Opera Studio in Oneonta, Norwich, Homer, Owego, Cazenovia and Pulaski; demonstration concerts by Young Audiences, Inc. will be offered in the region with Council support; and other programs in music, the visual arts and technical assistance.
On extended loan from the Metropolitan Museum of Art, a part of the installation of Greek, Roman, Egyptian and Medieval arts at the Roberson Memorial Center in Binghamton.
Eastern Area
Schenectady, Rensselaer, Albany, Schoharie, Greene, Columbia, Ulster, Dutchess, Sullivan, Orange, Putnam
January 4, Albany, Technical Assistance to the Albany Institute of History and Art, Design consultancy for the Dutch Room by Gerald Watland.
January 10, Monroe, Technical Assistance to the Old Museum Village of Smith’s Cove, Display techniques, community relations and sales desk operation by Ralph Miller.
February 13, Albany, New York City Ballet, Full company performance.
February 14, Poughkeepsie, New York City Ballet, Lecture demonstration.
February 15, Middletown, New York City Ballet, Lecture demonstration.
February 17, Troy, New York Philharmonic Orchestra—Leonard Bernstein conducting, Children’s Concert.
February 27, Albany, Meeting of the New York State Council on the Arts.
March 1-18, Schenectady, Technical Assistance to the Schenectady Arts Council, Procedures and policy for Arts Council by Keith Martin.
March 10, Hudson, Address to Columbia County Arts and Crafts Guild by Assistant Director of the Council.
March 18-April 8, Troy, AFA Exhibition at Rensselaer Polytechnic Institute, Indian Art of the United States.
March 23-24, Coxsackie, Technical Assistance to the Greene County Historical Society, Administration and display techniques by Robert Wheeler.
April 2, Albany, Address before the Albany Torch Club by the Executive Director of the Council.
April 30, Albany, Technical Assistance to the Albany Institute of History and Art, Archive evaluation by Herbert Cahoon.
May 4, 5, 6, Schenectady, Northeast Ballet Festival Association, Orchestral Assistance for performances.
May 13-June 3, Troy, AFA Exhibition at Rensselaer Polytechnic Institute, Three Centuries of Architecture in New York.
The house at 103 East 2nd Street, built in 1795, is one of the oldest houses in the city. It was owned by John and Elizabeth Van Rensselaer, who lived there until their deaths in 1846. The house has been restored to its original appearance and is now a museum.
May 21, Albany, Technical Assistance to Albany Institute of History and Art, Jurying for regional art exhibition by Gordon Smith, John Hultberg, Fletcher Martin and William C. Palmer.
June 28, Schenectady, Technical Assistance to Capitol District Music Teachers, Address on Trends in Music Education by Samuel Spurbeck.
July 1-September 30, Albany, AFA Exhibition at Albany Institute of History and Art, Masters of American Watercolor.
September 27, Albany, Meeting of advisors with Council members and staff on problems relating to community arts.
October 1, Middletown, American Ballet Theatre, Full company performance.
October 3, Albany, American Ballet Theatre, Full company performance.
October 6, Albany, Address before the New York State Community Theatre Association by the Executive Director of the Council.
Albany, Technical Assistance to the New York State Community Theatre Association, Workshop and conference lectures by Marc Connelly and others.
October 8, 9, 10, Poughkeepsie, Phoenix Theatre, The Matchmaker.
October 9, Mahopac, Meeting with representatives of local Arts groups regarding formation of Arts Council by Assistant Director of the Council.
October 11, Troy, Phoenix Theatre, The Matchmaker.
October 12, Schenectady, Albany Symphony Orchestra, Concert.
October 14-November 4, Kinderhook, AFA Exhibition at Kinderhook Memorial Library, Indian Art of the United States.
Albany, AFA Exhibition at Albany Institute of History and Art, The Hudson River School.
October 19, Schenectady, Albany Symphony Orchestra, Concert.
October 22, Troy, Eisenberg String Quartet (Young Audiences, Inc.), Demonstration concert.
The "Yates House", an early 18th Century Dutch Colonial Building in Schenectady.
October 23. Schenectady, Buffalo Philharmonic Orchestra, Concert.
October 28. Pawling, Hudson Valley Philharmonic Orchestra, Afternoon concert.
Amenia, Hudson Valley Philharmonic Orchestra, Evening concert.
October 28- November 19. Schenectady, AFA Exhibition at Schenectady Museum Association, Masterpieces of Photography.
November 5, Albany, Phoenix Theatre, The Matchmaker.
November 9, Middletown, Phoenix Theatre, The Matchmaker.
November 10. Cornwall, Phoenix Theatre, The Matchmaker.
November 18. Hudson, Hudson Valley Philharmonic Orchestra, Afternoon concert.
Monroe, Hudson Valley Philharmonic Orchestra, Evening concert.
November 18- December 18. Poughkeepsie, AFA Exhibition at Vassar College, The Hudson River School.
November 18- December 23, New Paltz, AFA Exhibition at State University, Three Centuries of Architecture in New York.
November 29, Troy, Albany Symphony Orchestra, Concert.
December 5, Middletown, New York City Center Opera, Rigoletto.
December 10. East Greenbush, Albany Symphony Orchestra, Concert.
With support from the Council within its budget for the current fiscal year, performances are planned for the winter and spring by the Merry-Go-Rounders in Nyack and Troy; the New York Shakespeare Festival in Albany, Poughkeepsie, Woodstock and Middletown; Jose Limon in Albany; the Metropolitan Opera Studio in Middletown, Kingston and Albany; Demonstration concerts by Young Audiences, Inc., will be offered in the region with Council support; and other programs in music, the visual arts and technical assistance.
A workshop in Museum techniques held in the Rensselaer County Junior Museum at Troy.
Adirondack Area
Report on Current Program, Adirondack Area
February 14, Glens Falls, New York City Ballet, Lecture demonstration.
February 15, Plattsburgh, New York City Ballet, Lecture demonstration.
Massena, New York City Ballet, Lecture demonstration.
May 13-June 3, Massena, AFA Exhibition at Massena High School, Masterpieces of Photography.
July 1-22, Plattsburgh, AFA Exhibition at State University, Indian Art of the United States.
July 22-August 12, Plattsburgh, AFA Exhibition at State University, Masterpieces of Photography.
July 31, Saratoga County, Survey of cultural institutions by Assistant Director of the Council.
October 9, Canton, American Ballet Theatre, Full company performance.
October 10, Plattsburgh, American Ballet Theatre, Full company performance.
October 16, Ogdensburg, Phoenix Theatre, The Matchmaker.
The Old Stone House, built in 1830, is one of the oldest buildings on campus and was used as a dormitory for many years. It now houses the Office of the President.
October 22, Plattsburgh, Eisenberg String Quartet (Young Audiences, Inc.), Demonstration concert.
October 31, Plattsburgh, Buffalo Philharmonic Orchestra, Concert.
November 18-December 16, Canton, AFA Exhibition at St. Lawrence University, Fifteen Years of Award Winning Prints.
November 29, Canton, New York City Center Opera, Marriage of Figaro.
December 3, Glens Falls, New York City Center Opera, Marriage of Figaro.
With support from the Council within its budget for the current fiscal year, performances are planned for the winter and spring by the Merry-Go-Rounders in Amsterdam; the New York Shakespeare Festival in Massena, Ogdensburg, Plattsburgh, Gloversville, and Glens Falls; the Metropolitan Opera Studio in Saratoga, Potsdam, Canton and Watertown; Demonstration concerts by Young Audiences, Inc. will be offered in the region with Council support; and other programs in music, the visual arts and technical assistance.
Guy Park Manor, an English Colonial house built in Amsterdam in 1766.
Metropolitan Area
Rockland
Westchester
New York
Bronx
Kings
Queens
Nassau
Suffolk
Richmond
January 7-28,
**Hempstead**, AFA Exhibition at Hofstra College, *The Hudson River School*.
**Rye**, AFA Exhibition at Milton Elementary School, *Indian Art of the United States*.
January 25,
**New York City**, Release of New York State Council on the Arts Report for 1961, *Press Conference — Governor Rockefeller, Council Members, Staff, Representatives of the Press and Participating Organizations*.
February 1-19,
**Brooklyn**, AFA Exhibition at Long Island University, *Three Centuries of Architecture in New York*.
February 7,
**Huntington**, Technical Assistance to the Heckscher Museum, *Display techniques by Lothar Witteborg*.
February 11-March 4,
**New York City**, AFA Exhibition at Whitney Museum, *Masters of American Watercolor*.
**Richmond**, AFA Exhibition at Staten Island Institute of Science and Art, *Fifteen Years of Award Winning Prints*.
February 28,
**New York City**, Address before the Women’s City Club by the Executive Director of the Council.
March 5-25,
**New York City**, AFA Exhibition at City College, *Three Centuries of Architecture in New York*.
March 16,
**Riverhead**, Address before the George Spelvin Awards Dinner by the Executive Director of the Council.
March 18-April 8,
**Brooklyn**, AFA Exhibition at Long Island University, *Fifteen Years of Award Winning Prints*.
**Richmond**, AFA Exhibition at Staten Island Institute of Science and Art, *Masters of American Watercolor*.
March 19,
**New York City**, Meeting of the New York State Council on the Arts.
March 28,
**Huntington**, Technical Assistance to the Huntington Historical Society, *Display techniques by Jane des Grange*.
April 8-29,
**New York City**, AFA Exhibition at Parsons School of Design, *Masterpieces of Photography*.
April 9-10,
**Huntington**, Technical Assistance to Huntington Historical Society, *Administration and community relations by Keith and Laura Martin*.
The New York Public Library, Central Branch, designed by Carrère and Hastings, was built in 1897-1904. The building is constructed of brick and stone with terra cotta ornamentation. The tower is 235 feet high. The library contains over 5 million books and 100,000 periodicals. It is one of the largest libraries in the world.
April 13,
**Brooklyn**, Technical Assistance to Long Island Historical Society, *Administration and display techniques* by Thomas Buechner.
April 16,
**White Plains**, Address before the Center for the Fine Arts in Westchester by the Executive Director of the Council.
May 8,
**Brooklyn**, Technical Assistance to the Long Island Historical Society, *Administration and display techniques* by Thomas Buechner.
May 14-15,
**Bridgehampton**, Technical Assistance to the Bridgehampton Historical Society, *Display Techniques* by Jane des Grange.
June 1-18,
**Armonk**, AFA Exhibition at Harold Crittendon School, *How To Look At A Painting*.
June 16,
**Brooklyn**, Technical Assistance to Wykoff House Foundation, *Administration and Fund Raising* by Ralph Miller.
June 23,
**Scarsdale**, Technical Assistance to Scarsdale Art Center, *Administration* by Gordon Smith.
June 25,
**New York City**, Commencement Address to the High School of Art and Design by the Executive Director of the Council.
July 1-August 19,
**Brooklyn**, AFA Exhibition at the Long Island University, *How To Look At A Painting*.
July 23-September 16,
**New York City**, Premiere showing at the Whitney Museum of Forty Artists Under Forty, organized by the Whitney Museum of American Art for AFA distribution throughout the State.
August 5,
**Port Washington**, AFA Exhibition at Port Washington Library, *Fifteen Years of Award Winning Prints*.
September 9-30,
**Brooklyn**, AFA Exhibition at Packer Collegiate Institute, *Fifteen Years of Award Winning Prints*.
September 17, 18, 19, 20,
**New York City**, Meeting of the Council Subcommittees with Advisors on Ballet, Opera, Concert, Visual Arts and Theatre.
October 10,
**Port Chester**, Conference with officers of Rotary Club regarding Arts projects by Assistant Director of the Council.
October 11-12,
**Brooklyn**, Equity Library Theatre, *Mr. Roberts*.
*The Jefferson Market Courthouse in lower Manhattan, New York City, designed by Frederick C. Withers and Calvert Vaux in 1876.*
TO LOOK AT A PAINTING
DESIGNED BY THE
ADDISON GALLERY OF AMERICAN ART
PHILLIPS ACADEMY ANDOVER MASSACHUSETTS
IN THE FEDERATION OF ARTS
STATE COUNCIL ON THE ARTS
October 12-13, Huntington, American Ballet Theatre, Full company performance.
October 14, New York City, AFA Exhibition at Parsons School of Design, Fifteen Years of Award Winning Prints.
October 19, Queens, Equity Library Theatre, Mr. Roberts.
October 20-21, Bronx, Equity Library Theatre, Mr. Roberts.
October 21, New York City, Composers Forum, Workshop-concert of contemporary compositions.
October 26, Larchmont, Buffalo Philharmonic Orchestra, Concert.
Brooklyn, Conservation workshop at the Brooklyn Museum for upstate Museum Board Members and Directors, Council Members, and Staff.
New York City, Meeting of the New York State Council on the Arts.
October 28, Riverhead, Buffalo Philharmonic Orchestra, Concert.
November 1-18, Setauket, AFA Exhibition at Setauket School, How To Look At A Painting.
November 18-December 16, Albertson, AFA Exhibition at Meadow Drive Elementary School, Indian Art of the United States.
November 29-30, Brooklyn, Equity Library Theatre, Elizabeth the Queen.
Brooklyn, Technical Assistance to the Long Island Historical Society, Archives and Library by James J. Heslin.
November 30, Hempstead, AFA Exhibition at the Hofstra College, How To Look At A Painting.
December 1-18, Bronx, Technical Assistance to the Bronx Council on the Arts, Administration and public relations by Keith Martin.
December 6, December 7, 8, 9, Farmingdale, New York City Center Opera, Performances of Rigoletto, La Boheme, Marriage of Figaro and Madame Butterfly.
December 7, Queens, Equity Library Theatre, Elizabeth the Queen.
With support from the Council within its budget for the current fiscal year, performances are planned for the winter and spring by Jose Limon in White Plains, Jamaica and New York Public Schools; and other programs in music and the visual arts.
Students viewing a Council-sponsored American Federation of Arts Exhibition at the Setauket School.
Summary of Future Plans
It is reasonable, when seeking solutions to a number of loosely related problems, to attempt to define a fundamental order which will direct one's thought and imagination. For the Arts, such an order is uncomfortably obscure; there is an elusiveness of dimension that is disturbing in an age of measurement. Finding ourselves at home with infinity, where terms like "megaton" and "light years" at least imply our grasp by the commonness of their usage, we too frequently suggest a purpose in means while ends remain unquestioned.
If the Arts have a function in the human experience, what is the nature of that function? If part of it is educational, what is the purpose of education? If part of it is communication, how is the message recognized; if part of it is pleasure, how is the pleasure derived? When asked to define the purpose of education, to which he had devoted his life, an enlightened man equated it to those three concerns of the Declaration of Independence, Life, Liberty and the Pursuit of Happiness. We educate to understand the nature and preservation of physical life, to understand and protect our personal and collective liberties, and to prepare us for the reward, the pursuit of happiness. In the sense that happiness is the highest development of human intuition, the Arts are part of happiness. They communicate in a way that must be unique to each receptor and pleasure lies in sensing the message.
Like all other elements of our Life, Liberty and the Pursuit of Happiness, the Arts depend to some extent on economic factors for their perpetuation and development. Because they are part of the experience of happiness, they have found common patronage from diverse sources. That Government should take its place among these sources is not inconsistent with what we now refer to as our "National Purpose". The question is, where?
The New York State Council on the Arts continues to search for the answer to this question, guided by the broad principles repeated by Mr. Knox earlier in this report.
Since the opportunity to take pleasure from the Arts must be a first concern, the Council will continue to support touring performances. Statewide reception to these events has been enthusiastic indeed, and this enthusiasm reflects the imagination and vigor with which the participating organizations have attacked their assignments. But the ambition of the Council is to reach more audiences in more communities, and towards this end we now propose to support touring in a more flexible manner. Under the new procedures proposed, the Council will designate a wide variety of performances and exhibitions for support on a formula recognizing the relationship between the size of the available community facility and the cost of the performance itself. The Council's control will be limited to assuring the professional quality of the available performances and exhibitions and protecting a proper geographical and categorical distribution of events within its budget.
In reaching out to new audiences it becomes increasingly evident that there is a need, particularly on the part of young audiences, for more basic interpretation, particularly of the form and structure underlying each artistic expression. This is regarded as educational not in the didactic sense, for "appreciation" can not be inculcated, but with the conviction that to understand these values is fundamental to true receptivity. Hence, it seems appropriate for the Council to expand this aspect of its program, seeking new and imaginative ways of developing this audience background.
A year ago in these pages Mr. Knox noted the complex problems involved in any selective aid to the individual creative artist, and he expressed the hope of the Council that its overall program would provide him benefits incidental to our ambitions for increased over-all stimulation in the arts. While this continues to be the long-range goal of the Council, we are forced to recognize through the guidance of our advisors that the lack of opportunity to be seen and heard for these artists is an obstacle so overpowering as to potentially deprive us of the growth of the essential resource. There are a number of organizations dedicated to improving this
climate, and the Council now feels that it is the continuity of creativity that most clearly expresses the culture of an era and renders its image for posterity.
These programs in education and creative aid will include the continued use of technical assistance to community arts enterprises, offering expert advice, within the limits of our budget, to any situation where we find the opportunity to be of assistance in raising standards.
There is a danger in the over-diversification of any limited source of patronage; the Council is conscious of this danger. But there is an even greater danger in our becoming static so early in our development. It is possible that we may move in areas which are more appropriately the responsibility of others. The definition of these areas is still unclear and must await not only the experimental growth of other State Councils across the nation but the emergence of new Federal support. While there may be no fundamental order to the creative forces playing over all the Arts there is very probably an order to the sources of their support. Where it contradicts this order, the Council’s program will be redirected, and its experience may help to show others the way.
As we progress we welcome that objective criticism indispensable to our sound development. At a moment in history where signs point to a new resurgence of support for and pleasure in the Arts, the dedications of many life-times will anticipate just reward. There is a need for a note of caution here, for the best-intentioned and most selfless dedication can never take the place of creative imagination. Some of the problems faced by the Arts grow out of the natural shift of sources of patronage and increased costs, but some of them also grow out of confusing dedication and imagination. Patronage must always be selective; for the Council to attempt an attitude of “something for everyone” would be to set an artistically unsound precedent for emerging government support.
JOHN H. MACFADYEN
Executive Director
The scheduled construction of the New York State Pavilion for the 1964 New York World’s Fair coinciding with the completion and opening of the New York State Theatre at Lincoln Center for the Performing Arts promises a unique opportunity to reaffirm the position of leadership in the Arts throughout the world enjoyed by our State. To take full advantage of this opportunity, the Council plans to work in cooperation with the New York World’s Fair Commission, headed by Lieutenant Governor Malcolm Wilson, and Lincoln Center for the Performing Arts to present exhibitions and performances demonstrating the quality of our artistic heritage and continuing vitality.
A major exhibition of the history of New York State painting is being prepared to include the finest examples, selected from collections throughout the State, from each important period. Complementing this are plans to integrate contemporary arts and crafts in the display and public areas of the Pavilion. Together with catalogues of these shows, the Council intends to publish guides to the Museums and Architecture of New York State and New York City, and it is hoped that Council headquarters in the State Pavilion will function as a cultural clearing house and information center for arts events occurring in the City and State at the time of the Fair.
In the New York State Theatre at Lincoln Center, performances by organizations from throughout the State will be coordinated with international programs of ballet, theatre, opera, dance and concert. A smaller facility as part of the public area of the State Pavilion is envisioned as a show-case for performances of more modest dimension.
In selecting events and works of art for inclusion in this program the only permissible yard-stick, within the realities of our budget, will be quality. But within this dimension the Council will exercise its discretion to present a true and comprehensive image of the Arts in our State.
The play was performed at the National Theatre in 1952, directed by John Gielgud and starring Peggy Ashcroft as Mrs. Bradman, Laurence Olivier as Mr. Bradman, and John Gielgud as the Narrator. The set design was by Oliver Messel, and the costumes were by Cecil Beaton. The play was a critical success and received positive reviews from critics.
Members of the Council
Seymour H. Knox, Chairman
Henry Allen Moe, Vice-Chairman
Reginald Allen
Cass Canfield
Angus Duncan
Theodore M. Hancock
Mrs. W. Averell Harriman
Wallace K. Harrison
Miss Helen Hayes
Louis Clark Jones
David M. Keiser
Richard B. K. McLanathan
Alfred J. Manuti
Richard Rodgers
Lewis A. Swyer
Scene from "Jeremiah" in Binghamton, from the Council-supported premiere performance by the Tri-Cities Opera Company.
Since its formation in 1960, the Council has had the benefit of thoughtful counsel from advisors in all walks of professional and lay life of the Arts. Those listed below have responded, individually and collectively, to our needs; their guidance has been invaluable. To them the Council extends its deepest gratitude. We hope, in the years ahead, to continue to turn to persons outside our membership for help in determining our direction.
| H. Harvard Arnason | Michael Dewell |
|---------------------|---------------|
| Mrs. Hedy Backlin | Donald Engle |
| George Balanchine | Carl Engelhart |
| Richard Barr | Alexander Ewing |
| Miss Genevieve Bazinet | Mrs. Marion Feman |
| Ralph Bellamy | Frank Forest |
| James M. Brown | Wilbur H. Glover |
| Thomas S. Buechner | Lloyd Goodrich |
| Miss Lucia Chase | Charles Graney |
| Langdon Clay | John Gutman |
| Howard Conant | T. Edward Hambleton |
| Marc Connelly | Howard Hanson |
| Francis W. Cunningham | Philip Hart |
| Edgar Curtis | Peyton Hibbitt |
| Stanley Czurles | Mrs. Ada Louise Huxtable |
| Mrs. Edith Dappert | Robert Bruce Inverarity |
| Mrs. John E. Davis | Louis Ismay |
| Miss Agnes DeMille | Mrs. Ninita Johns |
| Mrs. Jane des Grange | Rev. William Kalaidjian |
Melvin Kaplan
Sheldon Keck
Lincoln Kirstein
Arnold Kohn
William Kolodny
Omar K. Lerman
Jose Limon
Howard Lindsay
Harley J. McKee
Maxwell McKnight
Gertrude Macy
Robert Mann
Keith Martin
William Martin
Burt Martinson
Gian Carlo Menotti
Ralph R. Miller
John D. Mitchell
Claude Monteux
Douglas Moore
Miss Carol Morse
Carlos Moseley
Beaumont Newhall
Donald Oenslager
Jerome Patterson
Paul Bruce Pettit
Harris Prior
Miss Janet Reed
Mrs. Robert A. Riester
Jerome Robbins
James Rorimer
George Ruby
Julius Rudel
Allen Sapp
William Schuman
Dean Jeanette Scudder
William Seitz
George Seuffert
Jack Shana
Joseph Shoenfelt
Herman Shumlin
Robert Sinclair
C. Duryea Smith, III
Donald Smith
Gordon M. Smith
Benson Snyder
Samuel Spurbeck
Franz Stone
Carl Streuver
Martin Tahse
Mrs. Mildred Taylor
Richard Turner
Gordon B. Washburn
Harold Weston
The house was built in 1806 by John Brown, a wealthy merchant and landowner. It is a two-story, five-bay, Federal-style house with a gable roof and a central entrance flanked by pilasters. The front facade features a portico with four Doric columns supporting a pediment. The interior includes a central hall with a staircase leading to the second floor. The house has been well-preserved and is now owned by the National Park Service.
Through the Technical Assistance Program, the Council has been able to make significant contributions to the progress of community arts endeavors throughout the State. Professional consultants, assigned temporarily to the Council payroll, have undertaken diverse assignments with imagination and devotion. They are listed below in recognition of their unique contribution.
Miss Dorothy C. Barck
Miss Alice Beer
James M. Brown
Thomas S. Buechner
Herbert Cahoon
Fong Chow
Marc Connelly
Francis W. Cunningham
Mrs. Edith Dappert
Carl C. Dauterman
Mrs. Jane des Grange
Leroy Flint
Richard Stuart Flusser
Wilbur H. Glover
Per Guldbeck
Miss Fran Harris
James J. Heslin
John Hultberg
Morris Kantor
Sheldon Keck
Louis J. Koster
William Lassiter
Omar K. Lerman
Harley J. McKee
Miss Janet MacFarlane
Fletcher Martin
Keith Martin
Mrs. Laura Martin
Ralph R. Miller
George Montgomery
Mrs. Gertrude H. Moore
Beaumont Newhall
Donald E. Nichols
William Palmer
Albert E. Parr
Paul N. Perrot
Fred Rath
Miss Frances Raynolds
George Rickey
Marvin D. Schwartz
Mrs. Rose Senehi
Donald Smith
Gordon M. Smith
Samuel Spurbeck
Philip H. Stevens
Stuart Vaughan
Mrs. Helen Vaughan
Gerald Watland
Robert G. Wheeler
Peter Wingate
Lothar P. Witteborg
“Hyde Hall”, a late Federal style house designed by Philip Hooker and built in 1833. It is the preservation of buildings of this quality in New York State that the Council hopes to insure through its “Architecture Worth Saving” program.
Staff
John H. MacFadyen, Executive Director
William Hull, Assistant Director
Mrs. Herman W. Hertweck, Office Manager, resigned June 1962
Miss Sally Wasylik, Office Manager
Mrs. Sylvia Coleman, Secretary
The Executive Director has outlined the Council’s program in addresses before meetings of the National Music Council, the American Educational Theatre Association, the Women’s Association of the Minneapolis Symphony Orchestra, the United States Institute for Theatre Technology, Community Arts Councils, Incorporated, the Michigan Cultural Commission, the American Association of State and Local History, the National Guild of Community Music Schools, and the International Concert Managers Association. Mr. MacFadyen also testified before the Select Subcommittee on Education of the Committee on Education and Labor of the House of Representatives, and the Assistant Director, Mr. Hull, before a Special Subcommittee of the Committee on Labor and Public Welfare of the United States Senate, both in Washington, D. C.
Financial Statement
Personal Service including
Technical Assistance .......................................................... $ 65,700
Maintenance and Operation
Administrative Expenses
including Workshops and Special Projects ......................... 44,550
Special Expenses including Touring
Programs in the Performing and Visual Arts ....................... 450,000
$560,250
Photographs
Inside cover Gene Baxter, Troy
Facing page 3, Marvin P. Lazarus, White Plains
Facing page 11, NYSPIX, Department of Commerce, Albany
Facing page 15, Clifford E. Orr, Geneva, Courtesy of Carl K. Hersey, Rochester
Facing page 17, Louis Ouzer, Rochester
Facing page 23, Bureau of Public Relations, Hamilton College, Clinton
Facing page 25, John Bolas, Binghamton
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Searching for Worlds beyond the Canon: Narrative, Rhetoric, and Legal Change
Susan Bandes
DePaul College of Law
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Susan Bandes, Searching for Worlds beyond the Canon: Narrative, Rhetoric, and Legal Change, 28 Law & Soc. Inquiry 271 (2003).
Bandes, S. (2003). Searching for worlds beyond the canon: Narrative, rhetoric, and legal change. Law Social Inquiry, 28(1), 271-294.
Bandes, S. (2003). Searching for worlds beyond the canon: Narrative, rhetoric, and legal change. Law Social Inquiry, 28(1), 271-294.
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Searching for Worlds beyond the Canon: Narrative, Rhetoric, and Legal Change
Susan Bandes
Anthony Amsterdam and Jerome Bruner. *Minding the Law*. Cambridge, Mass.: Harvard University Press, 2000. Pp. 453. $35.00 cloth.
Anthony Amsterdam and Jerome Bruner set out, in *Minding the Law*, to “make the already familiar strange again” (p. 1). This phrase captures one central goal of interdisciplinary inquiry: to allow those steeped in a field to step outside its closed, often self-referential world and view it from another vantage point. For legal scholars, studying a field that styles itself as autonomous, and that is rife with “canonical ways of proceeding” (p. 1), the turn to other disciplines has not been easy. The rewards of crossing disciplinary boundaries are palpable: the infusion of information, the jolt to our complacency, the enlarged frame of reference. Yet for the study of law, grounded in practice as well as theory, in real world consequences as well as the love of knowledge, it is worth asking: What exactly should we seek from the interdisciplinary endeavor? Is it a way to see the legal world afresh, or more than that: a source of principles for remaking it?
Amsterdam and Bruner share a conviction, which grew in part from a highly rewarding seminar they have co-taught over the past decade, that it is possible to “[e]xperience fresh ways of looking at . . . [the works that lawyers do] as the ceaseless works-in-progress of a culture that, while binding its members in a common canon, leaves them free to some extent to visualize and even realize possible worlds beyond the canon” (p. 5).
They found that the students’ most vivid insights into the workings of
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Susan Bandes is professor of law, DePaul University College of Law, Chicago. I am grateful to Anne Dailey, Carol Sanger, and Austin Sarat for reading and commenting on earlier drafts.
© 2003 American Bar Foundation. 0897-6546/03/2801-271$10.00
law came from nonlegal sources, that often literary readings helped them connect to texts in disciplines like anthropology or linguistics, and that, taken together, these sources helped liberate the students “from the deadly habit of taking the law for granted” (p. 6). The book undertakes, with great enthusiasm, to provide a similar experience for its readers.
*Minding the Law* is animated by the belief that literary, cultural, and psychological theory can illuminate legal texts, help expose the underlying psychological and cultural dynamics of lawmaking and, ultimately, make a difference in the real world of legal actors and legal consequences. Its authors are ideally suited for making the best case for this claim. Amsterdam is a renowned civil rights litigator, clinical educator, and public law scholar\(^1\) and Bruner is a distinguished cognitive psychologist and a pioneer in the field of cultural psychology.\(^2\) Both are committed to interdisciplinary inquiry in the best sense of the term—they approach each discipline with respect for its complexities but without undue reverence for its formal boundaries.
Amsterdam and Bruner describe their mission as to help us see afresh how legal meaning is created by demystifying several standard (and indeed, inevitable) processes of legal thought—categorization, narrative, and rhetoric—which often serve to mask the choices legal actors make and the consequences that flow from these choices. They also have a more ambitious mission: to help us not just visualize but actually “realize possible worlds beyond the canon” (p. 5). The authors have produced a book that is insightful, readable, and filled with delights. It succeeds at many of its ambitious goals—in particular its goal of demystification. As to whether the expanded understanding it offers brings us closer to achieving justice, this is a question worth focusing on as legal scholars consider the future direction of the interdisciplinary endeavor as a whole.
**STORYTELLING AND PERSUASION IN THE LAW LAID DOWN**
*Minding the Law* explores “the ways in which human beings, including judges and lawyers, must inevitably rely upon culturally shaped processes of categorizing, storytelling, and persuasion in going about their business” (p. 7). The authors’ particular interest is in the influences that shape legal narratives and make them persuasive. This question contains the seeds of their other focus as well: since legal narratives are the product of deeply ingrained cognitive processes and must draw upon culturally accepted conventions in order to persuade, how much room do they contain for the possibility of change?
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1. His pathbreaking article *Perspectives on the Fourth Amendment* (1974) remains a standard in the field.
2. See, e.g., his *Acts of Meaning* and *Actual Minds, Possible Worlds*.
Given the sweep of the authors’ interdisciplinary ambitions and the amorphous boundaries of the fields of narrative and cultural theory, it may be useful to locate the book within these fields. Much of the early narrative analysis offered by the legal academy focused on the insights to be gleaned from the content of literary works (law in literature) (Posner 2000; West 1985) or on storytelling as a means of including the voices of the marginalized in legal discourse (Abrams 1991; Delgado 1989). The pioneering work of James Boyd White and others concerned with the intersection among law, rhetoric, and literature is perhaps the closest early precursor to Amsterdam and Bruner’s approach (White 1973, 1985; Fish 1982; Weisberg 1987b).³ Amsterdam and Bruner’s primary concern in this field is with law as literature; that is, with how narratives pervade and shape the law.⁴ The authors bring formidable strengths to this approach, notably an impressive command of a broad range of related theoretical fields, including narrative theory, cognitive theory, and semiotics. The result is a major contribution to the literature: a rigorous and learned excavation of the narrative, cognitive, and rhetorical structure of legal opinions.
The contribution is further enhanced by the authors’ approach to cultural theory. When Amsterdam and Bruner argue that narratives draw on cultural expectations for their coherence and persuasive power, they avoid the vague notions of culture that can plague such efforts. Instead they offer a sophisticated, fluid, and historically grounded notion of the cultural milieu and its intersection with law, again informed by insights from a range of fields, including anthropology, sociology, and, of course, the law, history, and practice of the struggle for racial equality.
The authors are not, in this book, concerned with the constitutive aspects of law. Their interests vis-à-vis law and culture run mostly one way: toward the beliefs justifying law.⁵ They are concerned with the conceptions of those who produce case law and with the often unacknowledged assumptions that give meaning and coherence to the law laid down. That is, they tend to focus solely on the law as embodied in legal opinions and on the judges who author those opinions. They do not focus on the creation of law by other bodies such as legislatures or administrative agencies, or the legal narratives of ordinary people (see, e.g., Ewick and Silbey 1998), or the ways
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3. See also Weisberg (1987a), calling White the “foremost rhetorician of law in our academic culture.”
4. They are not concerned with law in literature, which Robert Weisberg and Gyora Binder define as “a species of conventional literary criticism and history that treats works of imaginative literature that contain legal themes or depict legal practice” (2000, 3). A prominent example of this type of scholarship is Posner 1988, in which Judge Posner considers literature on legal themes, including Hamlet, Billy Budd, and various works of Kafka. See also Morawetz (1993, 497–99), summarizing the various aspect of the law and literature movement.
5. Paul Kahn is prominently associated with this approach (see, e.g., Kahn 1999). See also Sarat and Simon (2001), and Sarat (2000), locating Kahn in the broader field of cultural studies.
in which law operates by influencing modes of thought or social practice, or even on legal narratives in other contexts, such as the courtroom. Although it may seem greedy to ask for more in a book of such sweeping scope, the authors’ top-down approach does raise a question: would a broader focus on other sources of legal meaning have led to a richer understanding of the cultural milieu within which legal understandings both give and take shape?
Questions about disciplinary scope are inevitable when dealing with such elastic and interlocking fields. They meld, in *Minding the Law*, into other difficult questions about emphasis, viewpoint, and content. The most basic question is, why study categories, rhetoric, and narratives? What is gained by approaching legal opinions as a species of storytelling and by revealing their narrative structure?
One claim is that exploring the usually invisible influences on judicial opinions will help us see the legal world afresh. Why is this a worthy goal? Does its value lie in the ability to demonstrate the existence of multiple perspectives and multiple narrative choices and thus to dispel the illusion of inexorability? If so, is there a value simply in the awareness of multiplicity? Or does awareness of multiplicity imply the possibility of choice?
If the claim is that once we make these underground influences visible and conscious we will be able to use tools like categories and rhetoric more responsibly, difficult questions about agency and choice follow. To the extent the authors consider cognitive processes in which we all engage, are these processes inevitable and deeply imbedded or amenable to change? If change is possible, under what conditions?
The question of change and its requisite conditions is the most difficult of all. The authors ultimately suggest the possibility of something more than just heightened awareness. They suggest that this awareness can be used to imagine and help attain better worlds. If so, ought we turn our attention decisively to distinguishing narratives, categories, and rhetorical strategies that serve justice from those that impede it? What is the relationship between narrative and social change? Does the very act of viewing law stories through the lens of narrative theory provide some normative framework for making value distinctions—is there something inherent in narrative structure that “takes directly into account the normative element upon which law is based” (p. 141)? Are particular genres inherently hegemonic or subversive, or can they become so under certain conditions?
Alternatively, when the authors invoke novels, myths, and other narratives to help illuminate case law, do these narratives function as an alternative version of social, political, or cultural reality to which the judicial
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6. See Sarat and Simon (2000, 18–20), discussing the pioneering contributions of Clifford Geertz and the legal academy’s increasing engagement with constitutive approaches to law and culture.
7. See, e.g., Bennett and Feldman 1981 for a prominent example of this sort of cultural study.
opinion’s version can be compared? If not, how can we best use narrative theory and our understanding of cognitive processes to achieve more just results? From where should our standards for a “better” world derive, and what role should these authors play in helping suggest such standards?
In light of the book’s admirably broad sweep, the questions I raise are not meant to suggest that an even broader focus was in order. Their purpose, instead, is twofold. First, to consider whether the authors succeeded in telling the important story they meant to tell, to the audience they wanted to reach. And second, to convey the excitement, the sense of new possibilities, generated by this unique collaboration, and to suggest some directions for further exploration.
USING UNEXPECTED SOURCES TO OPEN THE MIND
*Minding the Law* seeks to weave together insights from multiple disciplines for an audience consisting of lawyers, generalists, and academics in disciplines ranging from law to the human sciences. What began as a lawyering theory colloquium for “second and third year law students of exceptional insight and perceptiveness” (p. 5) must now make the leap into a wholly different genre: a story about legal storytelling, aimed toward a far wider, more loosely defined audience. The task of defining an audience\(^8\) and “tailoring a story to suit an audience” (p. 136) is daunting in a work that seeks to introduce readers of varying backgrounds to such a wide range of disciplines. It is perhaps inevitable that those schooled in law will experience much of the ground as well trod. Conversely, lay readers may find it rough going at times. Nevertheless, the authors keep these pitfalls firmly in mind. They are true to their promise to avoid undue jargon without oversimplifying complex topics (p. 17). For even the most sophisticated reader, Amsterdam and Bruner’s lively, collaborative approach to law through the lens of anthropology, linguistics, and narrative theory should offer rich rewards.
What sorts of rewards does such an approach offer? Do we gain understanding solely by studying a text, for example, a legal opinion, through the lens of narrative theory? What is gained by thinking of a judicial opinion as a story? Amsterdam and Bruner find narrative theory a useful path toward their goals for several reasons. As they observe, “the law is awash in storytelling” (p. 110). Thus narrative theory is not an optional overlay one might employ to parse legal opinions and other law stories, but a valuable means of making sense of the stories that are inevitably imbedded in the law. The turn to narrative theory is useful because it reminds us that law is not a self-
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8. See, e.g., Martin 1986, 158: “Though the writer may wish to make a narrative accessible to any potential reader, some conception of the audience is necessary to lead individuals to feel that they are the ones to whom it is addressed.”
contained system, but one that can be illuminated by the knowledge of other disciplines. If law is rife with stories, surely narrative theorists, for all their internecine disagreements, can teach us much about how stories work.
How does narrative theory enrich our understanding of law? The authors make two main claims here. Their weaker claim is that it helps strip away the veneer of verisimilitude that, in “well-wrought narratives,” including legal narratives, “virtually blind[s] us to the subtle architecture of their construction” (p. 113). A judicial opinion, for example, whose reasoning and construction might at first glance seem inexorable, leading to the only possible outcome, can be exposed as partial, one narrative choice among many, a product of “perspective, circumstances, interpretive frameworks” (p. 141). The second, stronger claim the authors make for the value of narrative study is that “narrative’s inherent structure fits it for [the] task . . . of justifying legal judgment” (p. 141). I will consider the first of these here and the second in a subsequent part.
The first goal, then, is to strip away the veneer of ordinariness that insulates legal processes from scrutiny. To accomplish this goal, the book begins by introducing the reader to, respectively, categories, narratives, and rhetoric, which the authors characterize as “three commonplace processes of legal thought and practice without which lawyers, judges, and students of the law could not possibly make do” (p. 2). It then illustrates the application of the process to particular legal cases.
This material provides an accessible, well-written, and richly sourced introduction to the processes covered. It does run, perhaps inevitably, into the problem endemic to its loosely defined audience. For the motivated reader unfamiliar with the terrain, these introductory chapters both offer a complex and useful introduction and alert the reader to the multiple disciplinary strands she might explore to gain a richer understanding of the processes of categorization. For the legal reader, some of it—particularly the material on categories—is pedestrian. The message that categories serve certain functions, that these functions are variable and manipulable, and that undue reliance on categories without recognition of these functions can lead us to error is a message with which the educated legal reader even lightly schooled in legal realism will be familiar. Nevertheless, Amsterdam and Bruner provide a well-organized summary of the chaotic world of narrative theory without flattening it of nuance.\(^9\) They approach the minefield with sensitivity, providing an overview of the right breadth, depth, and complexity for the purposes of the book.
Legal scholars have drawn on increasingly sophisticated and diverse sources as they mine narrative structure to help reveal the assumptions and cultural norms undergirding legal thought. Such work is most successful
\(^9\) Though their overview is not much focused on incorporating the work of legal scholars (many of whom are simply string cited in a single footnote, see pp. 355–56, n. 1).
when coupled with extensive knowledge of narrative theory, and when it is capable of giving substance to the notion of cultural norms. Amsterdam and Bruner's unique combination of gifts has made their work on this subject a notably serious and useful look at the structural underpinnings of literary meaning—how narrative frame, plot, character, time line, moral, and other essential aspects of narrative structure are implicated, through the lens of culture, in the production of meaning.
Once the veneer of verisimilitude is stripped away, it is revealed that narrative choices, like where the story should begin and end, what characters should populate it, what motivates them, what fate they meet, and what moral the story conveys, are deeply influenced by cultural and political assumptions. These assumptions, which help shape the raw legal materials, albeit invisibly, into a narrative that will be received as coherent and convincing, can be more easily unearthed when the narrative's structural underpinnings are exposed (Bandes 1999, 1310). This claim is no longer particularly controversial in the abstract. Nevertheless, legal scholars have rarely focused on illustrating it, and this book does so masterfully. The authors' vast collective knowledge of anthropology, rhetoric, cognitive psychology, and of course both law and literature, engender a rich, far-ranging, and often delightful exploration of the ways in which culture influences legal thought. Their thoroughly interdisciplinary approach goes beyond the summary observation that culture influences narrative and gives us a thick description of how it might do so.
For example, even the sophisticated reader should be fascinated by the discussion of *Michael H. v. Gerald D.* (491 U.S. 110 [1989]), a case about the efforts of a child's biological father to gain recognition of his status, despite the child's out-of-wedlock birth. The discussion of *Michael H.* uses both linguistic analysis and an exegesis of the opinion's underlying mythic structure to illustrate how categorization "is performed against a ground of notions and narratives about the nature of the world" (p. 78). The authors' claim here is that the opinion's portrayal of the participants in the lawsuit and what they have at stake is illuminated when viewed as a choice between two traditional mythic structures dealing with a wife and her adulterous lover. Michael H., who seeks the right to visit his biological daughter, Victoria, is portrayed as a predator-invader seeking to despoil the sacred and unsailable family unit constituted by Victoria's mother, Carole, and Carole's husband, Gerald. Justice Scalia's version, they assert, is a story of villainy ("adultery as combat myth") rather than weakness (or "lack of moral or physical capacity") (p. 83). Their linguistic and narrative analysis of how the opinion achieves its effects and masks its legal and cultural choices is nuanced and entirely helpful in making sense of the opinion.
Likewise, the fine discussions of *Prigg v. Pennsylvania* and *Freeman v. Pitts* are particularly insightful about the effect of the American dialectic on changing narratives of racial "trouble" (p. 159) and why the trouble
at hand might at different times require an integrative (increasing federal power) or disintegrative (dispersing federal power to states) fix (pp. 159–160). And the two penultimate chapters, “On the Dialectic of Culture” and “Race, the Court, and America’s Dialectic,” provide a complex and hopeful view of the possibility of cultural change.
One problematic omission from this otherwise fine treatment of narrative is its failure to make use of the work of narrative theorists at the intersection of law and sociology, such as Patricia Ewick and Susan Silbey or Francesca Polleta, who focus on questions of narrative and social change central to this book’s focus. This omission is part of the larger neglect of “the law on the ground”: legal narratives from sources other than judges and how law, thus broadly defined, constitutes culture. This choice of focus creates unfortunate strictures for a book that is centrally concerned with the relationship between law and social change, as arguably little such change occurs through the agency of judges.
MAKING SENSE OF CULTURAL CONTEXT
The concept of culture is crucial to the book’s ambitions. There are many ways to engage in a cultural analysis of law. In Paul Berman’s useful summary, these include (1) helping us go beyond the stated rationale for legal norms by considering their symbolic content; (2) providing us with data about how people actually understand or respond to legal norms; (3) studying narratives used in official legal settings for insight into how “law’s stories both structure and reflect our experience of the world,” and (4) broadening our notions of legal discourse to understand how “the ‘law talk’ that is diffused throughout everyday life and popular culture can become a source of alternative conceptions about law” (Berman 2002, 1134). With a few exceptions, such as their treatment of the iconic stature of *Brown v. Board* and their discussion of legal institutions in two towns in Italy and Ireland, Amsterdam and Bruner are wholly concerned with the third of these possibilities, the study of legal opinions.
In the first several chapters, the authors seek to help readers step back from the law’s “canonical ways of proceeding” (p. 1)\(^{10}\) and see law afresh as the “ceaseless works-in-progress of a culture” (p. 5) that imbues the categories, narratives, and rhetoric through which lawyers ply their trade. In these chapters, Amsterdam and Bruner take on cultural theory and its implications for legal change. This is a daunting task, admirably executed. In a lesser treatment of legal canonicity, the term *culture* might serve simply as an empty rhetorical flourish to elevate the description of the legal canon.
\(^{10}\)E.g., “the proper form for a brief, the right motion to file, the obvious line of precedent to cite, the correct way to advise a client . . . the substantive rules of the corpus juris” (p. 1).
Naomi Mezey recently observed in a pair of insightful articles about cultural theory, it is often a “quirky, ill-defined hash of insights from different disciplines” (2001b, 151) that is “everywhere invoked and virtually nowhere explained” (2001a, 35). But, as she continued, “[w]hen we make the effort to clarify what we mean by the term and are cautious about the sort of work we ask it to do, the concept may still prove to have teeth” (2001a, 39).
A discussion of legal culture divorced from the many overlapping cultural frameworks in which any particular legal culture exists would be profoundly unsatisfying. Happily, the authors’ collaborative method and rich mix of disciplinary interests come to full flower in this section, as they consider of what, precisely, the relevant culture consists.
The authors begin with a heroic attempt to map the contours of the debate about the nature of culture as an anthropological construct. As in their discussions of categories, narrative, and rhetoric, they manage to present a highly contested and complex terrain accessibly. One overriding lesson they take from their exploration of anthropology is the importance of avoiding monolithic, time-bound, exaggeratedly coherent notions of what constitutes a culture. Their method is not to attempt a tidy summary of legal culture or legal culture in America. It is instead to provide some “snapshots of the Court in action” (p. 248) and suggest a possible explanatory theory for the road from *Plessy* through *Brown* to *Pitts* and *Jenkins*, albeit one “full of clashing texts and subtexts” (p. 279). Their method of revisiting these four cases, introduced earlier, allows them to flesh out the concept of culture first explicated in the earlier chapters on categories, narratives, and rhetoric.
The authors posit a deeply ingrained dialectic between what they call “the American Creed and the American Caution,” or, in simplified terms, “the need to be inclusive and the need to be exclusive” (pp. 261–65). They deftly chart the ebb and flow of these warring influences on the historical and cultural forces that led to the landmark racial desegregation opinions they examine. Although they might have made a more explicit link between what they call the “cardinal features” (p. 250) of the opinions (their rhetorical and narrative framework) and the influence of these cultural forces, nevertheless their analysis is beautifully done. Their demonstration of the effect of the American Caution on the discourse of affirmative action, leading to what the authors aptly call “a new kind of racism,” is chillingly acute. Particularly arresting is their discussion of the dissonance between the article of faith that *Brown* remains not only good law but also an icon of our national commitment to equality\(^{11}\) and the bitter reality embodied in cases like *Pitts* and *Jenkins*, which virtually gut *Brown* of legal vitality and, they argue, hew more closely to the de facto legal world of *Plessy* (pp. 248–57).
In light of these achievements, perhaps one should not ask for more. Yet the analysis of the law in the courts, as influenced by culture, would
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\(^{11}\) Or as Reva Siegel has called it, our “civic religion” (1997, 1135).
have been enhanced by discussion of the law on the ground—reflections on how culture experiences legal change. Some sense of the complex interaction between legal and cultural change is necessary for a fuller depiction of the dynamics of law reform, which—as Tony Amsterdam knows better than most of us—does not occur only from the top down.\(^{12}\) If law is a “distinctive manner of imagining the real” (Geertz 1983, 184), it would be a shame for the reader to be trapped solely in the imagination of judges.
**NORMATIVE—AND WHERE TO GO? NARRATIVE AND JUSTICE**
I now turn to the second, stronger claim the authors make for the value of narrative study, which is that “narrative’s inherent structure fits it for [the] task . . . of justifying legal judgment” (p. 141). Or as the authors elaborate in the following paragraph:
[N]arrative is a mode of discourse that takes directly into account the normative element upon which law is based—the existence of a legitimate, canonical state of things that has been complicated by some human action in some particular context or setting. Narrative takes for granted—indeed, takes it as a necessary condition—that in human affairs, the actors are agents whose acts are in some significant way under their own responsible control. Narratives require and provide an accounting of the justifiability of those acts that are alleged to have violated the legitimate expectations of another party or of the community. And in its very nature, narrative requires resolution. (P. 141)
In addition, the authors claim that, because the possibility of alternate narratives always exists, narrative “leaves room for the possibility that things have changed” (p. 141).
The notion of “narrative’s inherent structure” (p. 141), what it is suited for, and whether it has normative content, is a fascinating one. The requisites of particular genres and their amenability to progressive aims offer a ripe topic for further exploration.\(^{13}\) It is also a topic that seems essential to the authors’ thesis. Therefore, it would have been helpful had the authors developed it more fully. In what sense does narrative’s inherent structure suit it for justifying legal judgment? In what sense does the study of legal opinions as narratives help us evaluate legal judgment? And finally, does
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\(^{12}\) For a fascinating treatment of the complex interaction between judicial decisions and cultural and social change in the civil rights era, see Greene 1992.
\(^{13}\) See, e.g., Scheurer 1999 for a fertile exploration of this topic. See also Bandes 2000.
engaging in such study bring us closer to achieving justice, and are the authors making that claim?
Perhaps, as I suggested earlier, the authors’ claim is simply that narrative structure introduces the possibility of conflicting narratives, and therefore brings the reader face to face with the notion of choice and indeterminacy or allows entry into what they later refer to as “noetic space—an imaginative space, teeming with alternatives to the actual” (p. 237). But this claim is more true for literature than it is for law and in both fields is complicated by the narrative drive toward coherence and closure. Literary narratives have a slight edge over legal narratives, because the former do not require resolution, while the latter do. Admittedly, as literary critic Gary Saul Morson put it, a novelist seeking to convey “freedom and contingency” must struggle to escape “the prejudice of structure and closure” (1994, 8). It is possible, though risky, to flout narrative convention and offer novelistic verisimilitude without conventional closure. Mikhail Bakhtin famously praised the novels of Dostoevksy for achieving this feat, which he described thusly: “Nothing conclusive has yet taken place in the world, the ultimate word of the world and about the world has not yet been spoken, the world is open and free, everything in the future is still in the future and will always be in the future” (Bakhtin 1984, 93).
In law, however, resolution, not nuance or complexity, is the reason for the whole exercise. Under no circumstances is it optional. Indeed, the authors cite Robert Cover’s well-known caution about confusing law with literature: that law is played out upon “a field of pain and death” (p. 226). And as the book’s discussion of rhetoric explains, the opinion writer seeking to impose and justify a resolution will make every effort not to acknowledge the possibility of alternative narratives, perhaps not even to himself (p. 176). He will adopt what Robert Ferguson called “the rhetoric of inevitability” (Ferguson 1990, 213) and his doing so cannot be viewed simply as a choice of narrative strategies. Although the authors recognize rhetoric’s inherently conservative function of “concealing contestability” (p. 177), they nevertheless maintain a rather rosy view of rhetoric as having the ability to “open possibilities of interpretation instead of shutting them down” (p. 193). It is the sort of view that Judge Posner attributed to literary critic Wayne Booth and to James Boyd White: the view of rhetoric as “edifying discourse, discourse that preserves and enhances culture and decency” (Posner 2000, 202).
I have argued that judicial narratives are under tremendous pressure to be hegemonic, that “[j]udges feel the powerful pull of the expectations of the genre”; they are “expected to create a story that portrays the evolution of doctrine as the linear result of doctrinal logic” (Bandes 2001, 856, 868). The aura of authority and inevitability is achieved largely by the very refusal to acknowledge alternate viewpoints (Ferguson 1990, 213–16; Cover 1975,
Ferguson describes the adoption of the rhetoric of inevitability, the tendency toward formalist expression, as “an innate psychological impulse” (Ferguson 1990, 208). I do not mean to suggest that judges lack choice in the matter (and, as I will discuss shortly, the extent of judicial choice is a troubling topic in this book). However, the difficulty of breaking the mold of the genre (a genre that bounds and domesticates alternative viewpoints within the structured confines of majority, concurring, and dissenting opinions) and the deeply ingrained forces pulling for the status quo present formidable hurdles (Bandes 2001, 856). The language of activism is not necessarily antithetical to judicial narratives (Polleta 1998, 154–55), but it will take some getting used to.
The difficulty of breaking the mold of the genre brings us back to the original question. What are the requisites of the genre, and do they “take directly into account the normative element upon which law is based” (p. 141)? Perhaps the authors simply mean to say that since a lawsuit involves a disturbance to “the existence of a legitimate, canonical state of things” (p. 141), it must lead to consideration of whether that state of things is indeed legitimate or ought to be disturbed. But does the structure of narrative encourage such consideration?
Amsterdam and Bruner seem to make this argument, calling noetic space “profoundly narrative in its ordering” (p. 238). But as the authors note, a judicial opinion must convey a story that is “culturally comprehensible” (p. 141) and even convincing (p. 238) to its audience. The demand for narrative coherence may be, if anything, profoundly conservative. The canonical state of things is, by definition, the state that seems most familiar, and narratives seeking the reassuring verisimilitude that makes a convincing story must tread carefully when challenging settled expectations (Bandes 2000, 577; Chatman 1978, 18). In this way, narrative “risks merely recapitulating existing community norms” (Kingwell 1994, 331) and “may actually be complicit in constructing and sustaining . . . patterns of silencing and oppression” (Ewick and Silbey 1995, 205). The examples of *Prigg*, *Pitts*, *Michael H.*, and *Jenkins*, and particularly the powerful example of *McCleskey v. Kemp*, seem to demonstrate that it is quite possible to construct an opinion that is internally coherent, that achieves verisimilitude, that draws upon culturally resonant imagery, and yet still reaches a result that both the authors and I would agree is unfortunate or even repugnant, and not legally required.
In other ways, as well, the inherent structure of narrative might insulate the status quo from challenge, thus avoiding normative judgment. The authors note that “narrative takes for granted—indeed takes it as a necessary condition—that in human affairs, the actors are agents whose acts are in some significant way under their own responsible control” (p. 141). They imply that the ascription of agency encourages normative judgment, but, in fact, ascribing human agency to governmental entities may have the opposite effect. The authors explicitly recognize this problem in their discussion of the Whorf-Sapir hypothesis (p. 37). They note narrative’s possible Whorfian “tendency to overemphasize the role of agency rather than circumstances, fault rather than unindictable conditions, intent rather than cultural conscription” (p. 142), because these characteristics endow the narrative with dramatic shape and verisimilitude.
The narrative drive toward character and agency, toward simple causal links, toward good and evil motives is not conducive to convincing portrayals of systemic or structural harms caused by the acts, or failures to act, of multiple governmental actors, often proceeding without malevolence (p. 39; see also Bandes 1990, 2323; Bandes 1999, 1335–38). Surely this is one lesson of *McCleskey v. Kemp* (481 U.S. 279 [1987]), which ignored a long historic pattern of racial discrimination in the administration of capital punishment in North Carolina, ostensibly because the plaintiffs could not demonstrate a simple and intentional causal link between the wrongful acts of particular defendants and the sentence of the particular plaintiff. Conversely, as the authors vividly illustrate in their appendix material comparing the frequency of references to people and institutions in *Prigg*, *Pitts*, and *Brown* (pp. 293–307), an opinion can all but erase humans and their travails from its cast of characters when doing so serves a purpose.
Another possible claim for narrative’s transformative potential is that literary narratives are “intrinsically more authentic or sincere than other social practices” (Binder and Weisberg 2000, 208). This sentimental assumption seems an attractive trap for lawyers—Amsterdam and Bruner included—in the field of law and literature. Sophisticated legal scholars, who are well aware of the political influences that shape legal opinions, at times treat literary works as if they exist free from such influences. For example, in their discussion of culture, the authors seek to support their claim of a dialectical clash between honor and terror at work in racial relations in the changing South as follows: “We know of this inner dialectical clash not from the canonical social- or political-history accounts, but from Lillian Hellman’s *Little Foxes* and from William Faulkner, Tennessee Williams, Robert Penn Warren, and more recently, Harper Lee and Michael Malone”
---
14. The Whorf-Sapir hypothesis holds that “one makes distinctions in experience more easily if the distinction is present in [one’s] language, [and that] we entrench in the lexicon only those categorical distinctions that are useful in a linguistic community” (p. 37).
15. Alan Freeman’s classic article on law’s maintenance of the status quo, although written well before the decision in *McCleskey*, provides a superb explanation for the status-quo-enforcing nature of what he calls “the perpetrator perspective,” the insistence on focusing on a single intentional perpetrator (1978, 1052–57). More recently, Reva Siegel’s elegant piece (1997), in demonstrating the mechanics by which law preserves the prerogatives of power even while ostensibly transforming them, emphasizes the status-enforcing nature of the requirement for intentional discrimination, generally and in the context of *McCleskey*.
(p. 228.) Similarly, in the following chapter on race and culture they claim that:
the racialization of African-Americans has taken many convoluted turns that economic competition alone cannot explain, as we know from [citing several historians] and know still better from the pages and plays of William Faulkner, Langston Hughes, Lillian Hellman, Richard Wright, Robert Penn Warren, and Tennessee Williams. (P. 265)
The references to Lillian Hellman should underscore the problem. Many would agree she was a superb playwright, memoirist, and essayist whose writing about the South was richly evocative, but her reputation is decidedly not based on her factual accuracy (see Goodman 1983; McPherson 1984; Melnick 1997; Bernstein 1998) or on her work’s insulation from political ideology (see, e.g., Rollyson 1988). Literature can provide valuable perspectives on a time and place like the Jim Crow South, but epistemological certainty (p. 184) is as out of place in describing works of literature, even those by less controversial authors, as in works of law.
ARE JUDGES (OR AUTHORS) FREE AGENTS? CHOICE AND CULTURAL CONTEXT
The search for epistemological certainty is not a trap into which these authors are likely to fall very often. On the contrary, the importance of acknowledging perspective is a central tenet of this book. But it exists in uneasy tension with other principles animating the work, specifically the concepts of agency, choice, and subjectivity, both in respect to judicial decision making and in respect to Amsterdam and Bruner’s own authorial role.
Amsterdam and Bruner claim, quite plausibly, that the processes they discuss—categorization, storytelling, the use of rhetoric—are universal (see, e.g., p. 287).\(^{16}\) Yet there is a tension throughout the book between the claim that the constructs around which legal concepts are organized are culturally shaped and deeply imbedded and the implication that they operate on a conscious level. As Naomi Mezey explains, the concept of culture is itself susceptible to this tension. She says: “Culture can be conceived as the almost unconscious meaning-systems that people inhabit and enact without choice” or as a more self-conscious and temporary deployment of symbols (Mezey 2001a, 42). The authors tend to portray judges as choosing certain frameworks, certain narrative structures, certain modes of categorization, certain types of rhetoric. Although this portrayal sends the important message that judges are responsible for the choices they make and the conse-
\(^{16}\) The authors do not claim the processes work identically in various cultures, only that the processes exist cross-culturally and are deeply imbedded in the structure of our thought.
quences these choices engender, it is to some extent at odds with the book’s portrayal of the deeply imbedded nature of the various thought processes it reviews. This is not to suggest that choice and cultural influence are mutually exclusive. Rather, the ambiguity about the nature of their relationship would have benefited from closer inspection.
Moreover, the authors’ claim of the universality of these processes sits uneasily with the cases they choose and with the unarticulated specter of their own judgment of the results in these cases. The failure to articulate their own judgment, in turn, has the effect of obscuring some of their insistent message about the pervasiveness of choice and viewpoint in narrative.
As Barbara Herrnstein Smith observed, “there are always multiple basic stories that can be constructed” (1980, 217). “No narrative version can be independent of a particular teller and occasion of telling, and, therefore . . . we may assume that every narrative version has been constructed in accord with some set of purposes or interests” (Herrnstein Smith 1980, 215). As a general matter, this notion of narrative choice is at the heart of Amsterdam and Bruner’s message. The authors frequently exhort their readers to use the tools they provide to scrutinize the authors’ own ideological leanings and how they influence their interpretations (see, e.g., pp. 8, 248). Such an exhortation could have been underscored had the authors modeled it through more self-reflection about how they shaped their story and the likely effects of their choices on the audience.
Can this ambitious work, which seeks to convey the importance of the concept of noetic space, the “imaginative space where mind can envision other possible worlds” (p. 237) succeed without taking a position on what sorts of worlds we should envision and why the status quo is not sufficient? That is, can their story succeed without a more transparent, self-reflective account of its organizing aesthetic, its prescriptive point, its driving force? It is obvious the authors have struggled with this question, and the reader does, as well.
“Stories go somewhere. They have an end, a telos,” as Amsterdam and Bruner explain (p. 127). The notion of narrative coherence must encompass more than simply the mechanics of plot, character, motivation, and causality (Ricouer 1984, 174). It requires some measure of significance, some standard for shaping these elements into a satisfying whole. A satisfying story is one “in which there is some appropriate match between a story’s telos and the obstacle to its attainment” (p. 127). Amsterdam and Bruner’s book, for all its richness, is at times frustrating, perhaps because of some contradictory signals about its telos.
Essayist Vivian Gornick, in a recent work contemplating the role of the narrator, usefully observed: “Every work of literature has both a situation and a story. The situation is the context or circumstance, sometimes the plot; the story is the emotional experience that preoccupies the writer: the insight, the wisdom, the thing one has come to say” (2001, 13).
Amsterdam and Bruner illustrate their forays into narrative, rhetoric, and categorization with cases whose results strike them as unjust, cases that matter to them (p. 7), but they disclaim any intention to sell their own ideological point of view, desiring instead “to increase vigilance and to sharpen scrutiny” (p. 8). The authors are aware of their conundrum. They understand the impossibility of achieving the “view from nowhere” (p. 6, quoting Thomas Nagel), admit to having “a definite ideological point of view,” but assert that, although they cannot “stand above the battle,” neither will they “seek to fight the battle here” (p. 8). Although they are largely successful in helping the reader see the legal world afresh, perhaps they set themselves an impossible task when they try to avoid taking sides in the battle over how that world should change.
It is not entirely convincing that the need for increased vigilance is really the thing they have come to say, the reason they undertook this important project. The problem that appears to preoccupy the authors, to make the need for vigilance and scrutiny matter, is their deep concern for the injustices perpetrated under cover of these standard processes. The tension between situation and story, or between the authors’ stated aim and what seems to be the unstated telos of their tale, leads ultimately to a less than satisfying match between problem and solution. If the problem is insufficient scrutiny, across the ideological spectrum, of the inevitable tools of legal thought, then heightened scrutiny is a plausible solution. If the problem is injustice—for example deeply etched racial inequality that leads to consequences that include substandard, segregated schools and systematic bias in capital sentencing—the call for heightened scrutiny seems oddly out of sync with the problem.
The reader, whatever the authors’ stated intentions (which, as they are the first to admit, cannot be taken at face value [p. 17]) must grapple with the book’s unstated intentions as well. Its success must be measured both by how well it uses unexpected sources to open the reader’s mind to “worlds beyond the canon” and also by how well it helps the reader understand why these other worlds matter and whether it provides better tools for achieving them.
Although the authors disclaim intent to advance a particular viewpoint, they are candid about choosing examples in which the results seem wrong to them. For example, the authors choose two cases with whose outcome they obviously disagree, Michael H. and Missouri v. Jenkins, to illustrate their treatment of categorization. Categories, Amsterdam and Bruner tell us, “imply a world that contains them” (p. 29). That world is shaped by a variety of cultural influences, and these influences are in turn deeply imbedded in our linguistic, narrative, and other cultural conventions (Lakoff and Johnson 1980, 3–6; Winter 1989, 2225, 2228; White 1985, 168–74). We surely cannot think and reason without the help of categories, but to what extent are we free to stand above and outside their content or to choose
different categorical constructs when those familiar to us lead us into error? This is an important question and indeed an essential one for both purposes of this book—its ambition to open our minds to alternative possibilities and its ambition to help us realize them. Thus it is an odd choice to focus solely on cases in which categorization has led its author into error. In a later treatment of narrative and mythic structure (chapter 5), the authors contrast two cases of this same ilk (that is, with outcomes they consider unjust), *Prigg v. Pennsylvania* (16 Pet. 539 [1842]) and *Freeman v. Pitts* (503 U.S. 467 [1992]) with *Brown v. Board of Education* (347 U.S. 483 [1954]), a case whose approach and outcome they clearly find more desirable. But the 90 pages introducing us to categorization provide no such counterexamples that might illustrate the pervasiveness of the processes they seek to illustrate. The authors thereby seem to flirt with the possibility that Justice Scalia, as well as Chief Justice Rehnquist in *Jenkins*, have used categorization as a convenient tool to mask conscious decisions to choose one path over another. This possibility is particularly plausible as to *Michael H.*, which tends to paint Justice Scalia as actively in charge of the choices he makes, the opinion he signs, and the consequences it engenders.
Another possible interpretation is that the very act of drawing on categories and narratives is itself normatively undesirable. Neither seems to be the intended message. Instead the authors want to encourage more awareness that choices exist, more sensitivity to the influences and assumptions that lead to particular choices, and more individual responsibility for the path eventually chosen. Cultural influence and choice coexist: we are shaped by cultural context, yet we make choices within that context. The question is how much room to move exists and at what level of awareness. It is possible that the tension that seems to surround this topic reflects a similar tension in the field of psychology itself between “scientific” approaches concerned with innate cognitive processes and “humanistic” approaches more concerned with choice and transformation. If so, some clarification of this point would have been useful in elucidating the authors’ own view of the psychology of choice and responsibility in legal decision making.\(^{17}\)
To the extent the authors’ position on these questions is obscured, their failure to examine their own narrative choices contributes to the problem. We are left uncertain both about the pervasiveness and precise contours of the problem they suggest exists and about how it might be addressed. Although their decision not to conduct such an examination was evidently the result of much soul-searching, it nevertheless detracts from the insistent (and important) theme of the book, that transformation is possible and that the failure to examine one’s choices leads to self-deception, deception of
\(^{17}\) Thanks to Anne Dailey for her insights on this topic.
others, failure of responsibility, and, perhaps, unjust results (see, e.g., pp. 8, 176, 188, 216, 285).
The book’s generally enlightening and useful treatment of rhetoric contains a similar tension. The authors suggest that the persuasive legal rhetorician will mask the contestability of his premises, assertions, and arguments, and that, in doing so, he will delude not only others but also himself. Their typology of common rhetorical tricks used for concealment, titled “rhetorical devices for managing contestability” (pp. 177–92) is particularly helpful. It nicely demonstrates not only how contestability may be concealed but also some of the mechanics of how the language of opinions ratifies accepted cultural and legal norms.
What is not entirely clear is on what level these rhetorical strategies operate and, therefore, how best to pierce them. Categories, the authors suggested earlier, are a necessary part of thought (p. 19). Certain types of categorization are, if not innate, at least endemic (p. 39). Recent studies suggest that certain category mistakes are also common, for judges as well as other humans (Guthrie, Rachlinski, and Wistrich 2001). Further, differences in individual use of categorization may go quite deep: it may be that, to use the authors’ terms, some judges are “relatively category-centered” and others “relatively situation-centered” (p. 108).
This uncertainty about the innateness of categories is replicated in the discussion of rhetorical strategies. When judges are in the process of “structuring what’s possible and imaginable” (pp. 181–84), no doubt they, like other people, will be deeply influenced by their notions of “the way the world works” (p. 183). Studies reveal that it is quite common to believe in what psychologists call “just world thinking,” that what is, should be (Bandes 1999, 1319; Staub 1989, 79). I have suggested elsewhere that judges are likely to be particularly susceptible to such thinking (Bandes 1999, 1324–26). Or an appeal to the way the world works might be the product of a particular interpretive framework. Perhaps, for example, a category-centered judge, one who prefers certainty and order, maintains a strong belief in the status quo as an essential part of his worldview. The emerging field of the cultural theory of risk, associated most prominently with the work of Mary Douglas and Aaron Wildavsky (1982), is generating interesting data suggesting, for example, that an individual’s cultural orientation exerts tremendous influence not only over which norms he subscribes to but also over what information he finds relevant and what weight he assigns it. As Dan Kahan and Donald Braman (2002) argue, such findings have important implications for the ways in which our arguments are constructed, if they are to be persuasive.
These interlocking tensions—between universally imbedded ways of thought and the possibility of choice and between the authors’ rejection of objectivity and their own refusal to make value judgments about particular judicial choices—become most pronounced in the treatment of McCleskey
The authors explain their decision to focus on *McCleskey* as based on the hope that the “opinion will offer an instructive study of the means by which rhetorics achieve a self-justification that is strong enough to kill with, although not strong enough to endure much beyond the killing” (p. 194). This statement refers to the fact that Justice Powell, who cast the deciding vote for execution in the 5–4 case, found only four years later, after his retirement, that he could no longer “accept his own grounds for rejecting *McCleskey’s* argument” (p. 194).
The most salient characteristic of the chapter is its pervasive, entirely justifiable, yet never explicitly expressed anger at the result and the reasoning in *McCleskey*. The palpable anger and the failure to acknowledge it or make it manifest send confusing signals. At times the discussion reads as if Justice Powell’s use of rhetoric caused him to fall into self-deception (p. 215), at other times as if it simply provided him a convenient tool for masking choices he made quite consciously (see, e.g., p. 216). The possibility that Justice Powell’s reasoning was shaped by his historical and cultural context is raised in a brief coda to the chapter (p. 216). But the unarticulated message or at least subtext of the discussion seems to be that Justice Powell well knew what choices he was making and well knew that he was using rhetoric to paper them over. This may be a misreading, but had the authors been more direct and self-reflective about their own moral and emotional reaction to the opinion, it might have been easier to disentangle it from their viewpoint about Justice Powell’s role in it.
The emotional undercurrent of the chapter also raises questions about why the authors chose *McCleskey* as the sole means of illustrating a neutral principle about rhetoric rather than coupling it with cases in which rhetoric does not lead its user so far astray. The authors wish us to understand rhetoric as a pervasive feature of the legal system, one which they do not regard as itself problematic. They argue for “more examination, not less rhetoric” (p. 192). This message is not well served by the *McCleskey* chapter.
The specter the chapter raises of “a self-justification that is strong enough to kill with” (p. 194) is powerfully disturbing. But it is not evident that the cure for *McCleskey* is more self-examination or even more dialectic. The *McCleskey* opinion is a testament to the proposition that mere facts, or mere evidence, no matter how strong, cannot persuade judges of a moral proposition they are not inclined to accept. As the authors observe, “the Baldus study—the centerpiece of *McCleskey’s* case—never enters the realm of factuality” (p. 208). Randall Kennedy noted that the opinion was “haunted by anxiety over the consequences of acknowledging candidly the large influence of racial sentiment in the administration of capital punishment in Georgia” (Kennedy 1998, 336–37; see also Bandes 1999, 1320). What set of circumstances could have enabled the plaintiffs to change this central, and fatal, disconnect between their case and the majority’s reaction to it? Alternative viewpoints were well expressed by both the Eleventh Circuit and the four dissenters in the Supreme Court. Yet the majority opinion, rather than engage in a dialectic on the strength of the plaintiff’s case, seems to exist in a separate interpretive universe in which the strength of the plaintiff’s case is beside the point—or perhaps more accurately, is the very problem. The intractable question remains: how can such gaps be bridged?
DISMANTLING THE MASTER’S HOUSE: THE PROBLEM OF CHANGING THE WORLD THROUGH NARRATIVE
Amsterdam and Bruner observe ruefully that their “theoretical journey yields no clue” (p. 120) about why sometimes transformative and sometimes restorative narratives triumph. They sell themselves short; their book is brimming with information about how legal opinions entrench and safeguard existing power relationships. The difficult question, with which they struggle valiantly, is how to mount a successful challenge to the status quo. The soul of the book is its claim that it is possible, not just to see law afresh, but to “visualize and even realize possible worlds beyond the canon” (p. 5). To the extent the struggle to maintain legal power is a struggle to wall off the ability to imagine other worlds (see, e.g., White 1973, 757–61; Gordon 1984, 108), the tools Amsterdam and Bruner provide should be helpful in opening up the noetic space of which they speak. For judges and other legal actors open to increased self-reflection, and indeed for all of us engaged in the national dialogue about what sort of community law ought to strive to establish (p. 193, quoting James Boyd White), this book may make us more alive to the possibilities of interpretation. Yet the question stubbornly remains: What is it that sometimes, but only sometimes, allows us, and particularly those with the power to effectuate change, to “[s]ee the unseeable, speak the unspeakable, and aspire to change what our more usual mythology declares to be natural and unchangeable” (p. 265)? When Amsterdam and Bruner give two examples of the dialectic of culture, examples designed to illustrate the possibility of a productive and stable marriage between canonicity and change, they choose a small Irish village and the city of Reggio Emilia (pp. 239–45). Both examples, while delightful, seem to rest on a historically entrenched (though fluid) equilibrium achieved in a geographically small and fairly homogeneous community. The question, as the authors recognize, is whether such a dialectic can be achieved in a broad, heterogeneous, culturally splintered community with no common core of consensus.
The more basic question is whether an equilibrium that rests on the unequal distribution of power and privilege can be upset through the provision of more or better interpretive tools. As the work of Patricia Ewick and Susan Silbey suggests, sometimes the very existence of a dialectic is the means by which power perpetuates itself. Under their theory, the grand narrative of the law as majestic and impartial could not long exist unless mediated by more cynical and pragmatic counternarratives (Ewick and Silbey 1995, 1998). That is, the existence of the counternarratives does not deal a fatal blow to the rigid and inaccurate grand narrative. Instead, the counternarratives permit it to flourish by absorbing and assimilating evidence of its fallibility (Bandes 2000, 575–76). McCleskey and Brown, unfortunately, illustrate this phenomenon all too well. We pay lip service to the majestic ideals of Brown, but McCleskey illustrates the infinite ability to iconize the ideal of Brown while interpreting away Brown’s guarantee of equality in order to avoid destabilizing an entrenched power regime.
If the goal is not just illumination but reform, it is also worth questioning the intensity of the focus, both in this book and by the legal academy generally, on the work of judges. Should the turn toward other disciplines bring with it a broader focus on the range of actors, legal and nonlegal, who constitute the legal landscape and who, ultimately, create and sustain the cultural milieu in which judges operate?
But no doubt it is too much to expect of a book that it help us “realize possible worlds beyond the canon” (p. 5). The lack of a blueprint for unsettling existing power relations is not a shortcoming one can, in fairness, complain about. To see the legal world afresh, and sometimes even visualize better worlds, through the eyes of two passionately committed, humanistic professors, writing in the best tradition of interdisciplinary work, is a rare delight, a journey well worth taking.
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|
HPC-GAP: engineering a 21st-century high-performance computer algebra system
BEHREND, Reimer, HAMMOND, Kevin, JANJIC, Vladimir, KONOVALOV, Alexander, LINTON, Steve, LOIDL, Hans-Wolfgang, MAIER, Patrick <http://orcid.org/0000-0002-7051-8169> and TRINDER, Phil
Available from Sheffield Hallam University Research Archive (SHURA) at:
https://shura.shu.ac.uk/18621/
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Citation:
BEHREND, Reimer, HAMMOND, Kevin, JANJIC, Vladimir, KONOVALOV, Alexander, LINTON, Steve, LOIDL, Hans-Wolfgang, MAIER, Patrick and TRINDER, Phil (2016). HPC-GAP: engineering a 21st-century high-performance computer algebra system. Concurrency and Computation: Practice and Experience, 28 (13), 3606-3636. [Article]
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HPC-GAP: engineering a 21st-century high-performance computer algebra system
Reimer Behrends\textsuperscript{1}, Kevin Hammond\textsuperscript{2}, Vladimir Janjic\textsuperscript{2}, Alexander Konovalov\textsuperscript{2}, Steve Linton\textsuperscript{2}, Hans-Wolfgang Loidl\textsuperscript{3}, Patrick Maier\textsuperscript{4,*}, Phil Trinder\textsuperscript{4,*,\dagger}
\textsuperscript{1}Department of Mathematics, University of Kaiserslautern, Kaiserslautern, Germany
\textsuperscript{2}School of Computer Science, University of St Andrews, Fife, UK
\textsuperscript{3}School of Mathematical and Computer Sciences, Heriot-Watt University, Edinburgh, UK
\textsuperscript{4}School of Computing Science, University of Glasgow, Glasgow, UK
SUMMARY
Symbolic computation has underpinned a number of key advances in Mathematics and Computer Science. Applications are typically large and potentially highly parallel, making them good candidates for parallel execution at a variety of scales from multi-core to high-performance computing systems. However, much existing work on parallel computing is based around numeric rather than symbolic computations. In particular, symbolic computing presents particular problems in terms of varying granularity and irregular task sizes that do not match conventional approaches to parallelisation. It also presents problems in terms of the structure of the algorithms and data. This paper describes a new implementation of the free open-source GAP computational algebra system that places parallelism at the heart of the design, dealing with the key scalability and cross-platform portability problems. We provide three system layers that deal with the three most important classes of hardware: individual shared memory multi-core nodes, mid-scale distributed clusters of (multi-core) nodes and full-blown high-performance computing systems, comprising large-scale tightly connected networks of multi-core nodes. This requires us to develop new cross-layer programming abstractions in the form of new domain-specific skeletons that allow us to seamlessly target different hardware levels. Our results show that using our approach we can achieve good scalability and speed-ups for two realistic examples on high performance systems comprising up to 32,000 cores, as well as on ultra-high-end multi-core systems and distributed clusters. The work reported here paves the way towards full-scale exploitation of symbolic computation by high-performance computing systems, and we demonstrate the potential with two major case studies. © 2016 The Authors. Concurrency and Computation: Practice and Experience Published by John Wiley & Sons Ltd.
Received 5 May 2015; Revised 27 October 2015; Accepted 2 November 2015
KEY WORDS: parallelism; multicore; high-performance computing; computational algebra
1. INTRODUCTION
This paper considers how parallelism can be provided in a production symbolic computation system, GAP (Groups, Algorithms, Programming [1]), to meet the demands of a variety of users. Symbolic computation has underpinned several key advances in Mathematics and Computer Science, for example, in number theory, cryptography and coding theory. Computational algebra is an important class of symbolic computation, where applications are typically characterised by complex and expensive computations.
*Correspondence to: Phil Trinder, School of Computing Science, University of Glasgow, Glasgow G12 8QQ, UK.
†E-mail: email@example.com
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
‡The copyright line for this article was changed on 28 June 2016, after original online publication.
Many symbolic problems are extremely large, and the algorithms often exhibit a high degree of potential parallelism. This makes them good candidates for execution on both large and small-scale parallel systems, including high-performance computing (HPC) systems. With the widespread availability of multi-core systems, there is an increasing need to provide effective support for symbolic computation on small shared-memory computers. The size and complexity of the problems that are being tackled means, however, that there is also a significant demand for symbolic computation on clusters of distributed-memory systems and potentially for full-blown HPC systems. The HPC-GAP systems described in this paper aim to meet those diverse needs in a way that is idiomatic for the GAP programmer and easy for the domain specialist to exploit.
There are, however, numerous practical challenges that must be overcome if the parallelism potential is to be exploited. Firstly, symbolic algorithms tend to employ complex data and control structures that are not generally studied by the parallel programming community. Secondly, the parallelism that is present is often both dynamically generated and highly irregular, for example, the number and sizes of subtasks may vary by several orders of magnitude. These properties present significant challenges to conventional parallelisation techniques. Finally, the developers of symbolic computing applications are not parallelism experts: they are typically mathematicians or other domain experts, who usually do not possess a background in computer science and who have limited time and inclination to learn complex system interfaces. What is needed is a simple, but highly scalable, way to deal with massive parallelism in symbolic computing systems, which is consistent with existing symbolic programming approaches.
This paper studies how these challenges may be overcome in the context of the widely used GAP computational algebra system. Our work establishes symbolic computation as a new and exciting application domain for HPC. It also provides a *vade mecum* for the process of producing effective high-performance versions of large legacy systems. The primary research contributions of this paper are as follows:
1. The systematic description of HPC-GAP as an integrated suite of new language extensions and libraries for parallel symbolic computation. These are a thread-safe multi-core implementation, GAP5 (Section 2); an MPI binding to exploit clusters (Section 3) and the SymGridPar2 framework that provides symbolic computation at HPC scale (Section 4). Collectively, these allow us to address scalability at multiple levels of abstraction up to large-scale HPC systems (Section 5).
2. We provide two substantial case studies to demonstrate the effectiveness of the language mechanisms for irregular symbolic computations. Specifically, we consider Orbit calculations and a Hecke Algebra representation theory calculation on the HECToR HPC system. The datasets for these, and other experiments in the paper, are available at [2] (Section 6).
3. SymGridPar2 re-establishes symbolic computation as an application domain for HPC for the first time in 20 years (Sections 4, 5.3 and 6).
**Novelty:** We provide the first complete and systematic description of HPC-GAP. Many of the individual components of HPC-GAP and associated case studies have been previously reported piecemeal: with descriptions of GAP5 [3], SymGridPar2 [4], the MPI-GAP case study [5] and the SymGridPar2 case study [6]. A key part of the systematic presentation of the HPC-GAP components is to provide a common SumEuler benchmark to facilitate comparison of the programming models and performance at different scales. Moreover, some key aspects of the components are presented for the first time, for example, Section 4.2, describing the interface between GAP and SymGridPar2. Section 5 that uses SumEuler as a basis for a performance evaluation of the HPC-GAP components, and discusses their interworking, is almost entirely new.
### 1.1. Computational algebra and the GAP system
GAP ('Groups, Algorithms and Programming' [1]) is the leading open source system for computational discrete algebra. GAP supports very efficient linear algebra over small finite fields, multiple representations of groups, subgroups, cosets and different types of group elements, and backtrack search algorithms for permutation groups. The GAP system and extension packages now comprise 360K lines of C and 900K lines of GAP code that is distributed under the GNU Public License. To
date, GAP has been installed at over 2000 sites and is cited in more than 1000 publications. GAP has been used in such landmark computations as the ‘Millennium Project’ to classify all finite groups of order up to 2000 [7]. From its inception, GAP was designed to be natural to use for mathematicians, powerful and flexible for expert users and freely extensible so that it can encompass new areas of mathematics as needed. All three objectives have been met comprehensively. Our achievements were recently recognised by the award of the ACM/SIGSAM Richard D. Jenks Memorial Prize for Excellence in Software Engineering applied to Computer Algebra.
GAP fills an important need in the computer algebra community. Unlike GAP, general ‘computer algebra systems’ such as Maple [8], Mathematica [9] or MATLAB [10] simply do not address computation in abstract algebra in any serious way. Maple, for example, can compute with some finite permutation groups, but is many orders of magnitude slower than GAP and can handle only ‘toy’ examples. Only MAGMA [11] and Sage [12] have any significant overlap with GAP in its core areas of computational algebra and discrete mathematics. MAGMA has a strong focus on number theory and cryptography, and unlike GAP, is closed source, monolithic and difficult to extend. Sage [12] incorporates and derives much of its algebraic functionality from GAP, as permitted by the GNU Public License. Neither of these systems supports parallelism well; Sage has limited support for client-server style distributed computing, but does not support large-scale parallel computing; and MAGMA currently has no support for parallelism.
1.2. Parallelism and high-performance computing
With the widespread adoption of multi-core processors, parallelism has become mainstream. While most laptops, desktops and even mobile platforms comprise single multi-core systems, higher-performance systems will cluster several multi-cores into a single coherent system, connected either by a high-speed network or by a shared memory bus. HPC systems provide a further level of hierarchy, connecting multiple clusters into a coherent, highly parallel supercomputing platform. While the general problems of identifying and controlling parallelism are similar in each level, the increasing scale and complexity of larger systems means that more powerful programming models are needed to deal with them effectively. These models should abstract over low-level architectures, in order to provide scalable parallelism up to HPC-size architectures.
While low-level programming models are still dominant for HPC applications, this low level of abstraction is in stark contrast to the high level of abstraction sought for in the domain of symbolic computation. A typical user of a computational algebra system will naturally aim to develop theories and programs in a style close to mathematical notations and therefore refrain from (prematurely) constraining how the theories are executed. In fact, the bigger gap between model and implementation found in symbolic computation makes parallel realisation of these problems relatively rare, and some exceptions are discussed in [13].
While there are instances of explicit coordination infrastructures in this domain, and we discuss them in the following sections, the most natural combination of technology and domain is a mostly declarative programming style, and the main part of our survey here focuses on such systems.
1.2.1. Threading and message-passing approaches. The most commonly used technologies on today’s HPC systems start from established, if not dated, sequential host languages, such as Fortran or C, and provide added functionality for parallelism in the form of libraries or compiler annotations. Different technologies are often combined, reflecting the underlying architecture, for example, using an explicit message passing library (e.g. MPI) between nodes combined with a dedicated shared memory model (e.g. OpenMP in the form of compiler annotations) within a multi-core node. This combination aims to use the most efficient technology on each level, but necessarily complicates software design. This development is aggravated by the heavy use of Graphic Processing Units (GPUs) and many-core co-processors, such as Xeon Phis, on the latest generation of supercomputers.
In contrast to the mixed paradigm approach common in HPC codes, we advocate a unified, high-level approach. Such an approach abstracts over low-level machine details, reducing the complexity of the parallel programming task, while still enabling the tuning of key parallel execution characteristics, such as the size of the parallel tasks and the distribution of the application data. The latter
is becoming increasingly important with modern applications often being extremely data intensive and the memory-per-core ratio falling. In this aspect, symbolic computation software technology is ahead of mainstream HPC, because symbolic applications often require enormous and complex intermediate data structures. Hence, bespoke libraries have been developed to deal with huge data structures efficiently have been developed, for example, the Roomy library discussed in Section 1.3. Hence, control of data, in addition to control of tasks, features prominently in the design of HPC-GAP.
1.2.2. Parallel patterns and skeletons. Parallel pattern that abstracts and re-uses common parallelism idioms is an increasingly important technology [14]. Algorithmic skeletons [15] provide specific implementations of patterns in the form of functions that have effective parallel implementations and that are instantiated with concrete parameters to specify functionality and behaviour. The practical relevance of such approaches is underlined by recent practitioner oriented textbooks on parallel patterns [16, 17] and by high levels of activity in projects such as Google’s MapReduce [18] and Apache’s Hadoop [19].
Well-known skeleton systems include $P^3L$ [20], Skil [21] and the SCL coordination language [22], which are all based on a functional coordination language. Moreover, numerous skeleton libraries provide skeletons that can be used from a mainstream sequential host language, for example, SkePU [23], Müsli [24] and eSkel [25], which are all embedded in C/C++.
While general purpose skeleton libraries like those mentioned previously can be used across domains, some packages encode domain-specific patterns. One such example is the LinBox [26] exact linear algebra library, which provides a wide selection of linear algebra operations operating over arbitrary precision numbers. Another example is our work on the Orbit pattern [5], a commonly occurring pattern in symbolic computation applications used as a case study in Section 6.1.
An important development in the area of programming languages for HPC systems is the emergence of partitioned global address space (PGAS) languages as a new model of high-level parallel programming. The basic idea in these languages is to provide language support for distributing large data structures, typically flat arrays, over a collection of distributed memory nodes. Parallelism is then expressed in the form of data parallelism, with constructs to place a computation on the machine that holds the relevant segment of the data structure, and synchronisation and communication become implicit. While the focus is on data parallel constructs, general fork-and-join parallelism is also available, although less frequently used. First generation PGAS languages that defined these concepts have been developed by major HPC vendors: Chapel [27] by Cray, X10 [28] by IBM and Fortress [29] by Sun/Oracle. More recently, PGAS constructs have been integrated into mainstream languages: Unified Parallel C [30] and Coarray Fortran [31].
1.3. Parallel computational algebra
Several computer algebra systems offer dedicated support for parallelism and/or distribution ([32, Sec 2.18] and [33]). Early systems focused on shared-memory architectures, and some prominent examples are Aldor [34], PARSAC2 [35] and PACLIB [36]. Because our focus is on distributed memory systems, we do not discuss these further.
Distributed Maple [37] provides a portable Java-based communication layer to permit the interaction of Maple instances over a network. It uses future-based language constructs for synchronisation and communication and has been used to parallelise several computational geometry algorithms. Unlike our system, it does not provide specific multi-core or HPC support, however. The Sugarbush [38] system is another distributed memory extension of Maple, which uses Linda as a coordination language. The GAPMPI [39] package is a distributed-memory parallel extension to GAP, which provides access to the MPI interface from within GAP. Unlike our work, no higher-level abstractions are provided to the programmer.
The TOP-C system provides task-oriented parallelism on top of a distributed shared memory system [40], implementing several symbolic applications, including parallel computations over Hecke algebras [41] on networks of workstations.
The Roomy system [42] takes a data-centric view of parallel symbolic computation, recognising that many applications in the domain manipulate enormous data structures. Roomy provides a library for the distribution of data structures and transparent access to its components, implemented
on top of the MPI message passing library. Example applications implemented on top of Roomy include a determinisation algorithm for non-deterministic finite state automata.
Two decades ago, there were several projects that parallelised algebraic computations, but there has been little work in this area since. Sibert et al. [43] describe the implementation of basic arithmetic over finite fields on a Connection Machine. Roch et al. [44] discuss the implementation of the parallel computer algebra system PAC on the Floating Point System hypercube Tesseract 20 and study the performance of a parallel Gröbner Basis algorithm on up to 16 nodes. Another parallel Gröbner Basis algorithm is implemented on a Cray Y-MP by Neun and Melenek [45] and later on a Connection Machine by Loutstaunau and Wang [46]. We are not aware of any other work within the last 20 years that targets HPC for computational algebra.
More recently mainstream computer algebra systems have developed interfaces for large-scale distribution, aiming to exploit Grid infrastructures [47]. The community effort of defining the Symbolic Computation Software Composability Protocol (SCSCP) for symbolic data exchange on such infrastructures supports distributed execution and interchange between different computer algebra systems [48]. In contrast to these Grid-based infrastructures, our SymGridPar2 framework targets massively parallel supercomputers.
2. PARALLELISM SUPPORT IN GAP5
The shared-memory component of HPC-GAP, which we refer to for this paper as GAP5 reimplements the latest version of GAP (GAP4) to include support for parallelism at a number of levels [3]. This has a number of implications for the language design and implementation. Distributed memory implementations of memory hungry symbolic computations can duplicate large data structures and may even lead to memory exhaustion. For example, the standard GAP library already requires several 100 MB of memory per process, much of which is data stored in read-only tables; a multi-threaded implementation can provide access to that data to multiple threads concurrently without replicating it for each thread. This is why it is crucial to use shared memory to achieve efficient fine-grained parallelisation of GAP applications. Secondly, we need to maintain consistency with the previous version of the GAP system, refactoring it to make it thread-safe. Achieving this has involved rewriting significant amounts of legacy code to eliminate common, but unsafe, practices such as using global variables to store parameters, having mutable data structures that appear to be immutable to the programmer, and so on.
The overall GAP5 system architecture aims to expose an interface to the programmer that is as close to the existing GAP4 implementation as possible in order to facilitate the porting of algorithms and libraries. The two primary new concepts that a GAP5 programmer may need to understand is that of tasks (an implementation of futures [49]) and regions, a GAP-level abstraction for areas of shared memory. Existing sequential code will run largely unchanged, and sequential code can also use parallelised libraries transparently. Only code that exploits GAP5 concurrency or kernel functionality needs to be adapted to the new programming model.
2.1. Task introduction and management
GAP5 adopts a future-based task model [49], identifying tasks using the RunTask primitive:
\[ \text{td} := \text{RunTask}(f, x_1, \ldots, x_n); \]
Here, \( f \) is a GAP function, and the \( x_i \) are the arguments to be passed to that function. A call to RunTask introduces a future into the runtime system, which will eventually yield a parallel task executing \( f(x_1, \ldots, x_n) \). RunTask returns a task descriptor, td, which can be used to further control or observe the task behaviour. In particular, the result of a task can be obtained using a blocking call to the TaskResult primitive; WaitAnyTask can be used to block until one of a number of tasks has completed; and ScheduleTask is a variant of RunTask that allows the creation of a task at some future point when the specified condition becomes true.
\[
\begin{align*}
\text{result} &:= \text{TaskResult(td)}; \\
\ldots \\
\text{td1} &:= \text{ScheduleTask(cond, f, x_1, \ldots, x_n)}; \\
\ldots \\
\text{tdx} &:= \text{WaitAnyTask(tdl, td2, td3)};
\end{align*}
\]
The return value of `TaskResult` is the value returned by the task. The return value of `ScheduleTask` is a task descriptor. `WaitAnyTask` returns a positive integer that denotes which of the tasks passed as arguments (via their descriptors) completed first, starting with 1 for the first argument. In the aforementioned example, `tdx` would be 1 if `td1` completes first, 2 if `td2` completes first or 3 if `td3` completes first.
### 2.2. SumEuler in GAP5
We use the SumEuler computation as a common example to demonstrate the GAP5, MPI-GAP and SymGridPar2 components of HPC-GAP. SumEuler is a symbolic computation that computes the sum of Euler’s totient function over an integer interval. The computation is a fold (the sum) of a map (of the totient function). The computation is irregular as the time required to compute the totient of a large integer is greater than for a small integer. We use chunked data parallel versions here. That is, the time to compute the totient of a single integer is small, so tasks typically compute the totients for a ‘chunk’ or interval of integers.
Figure 1 illustrates how to express SumEuler in GAP5, the source code for this and all other examples is available at [2]. `SumEulerSeq` is a simple sequential implementation, using the standard GAP implementation of the totient function, `Phi`. The `GAPList` function implements a functional `map` operation: taking a list as its first argument and a function or closure as its second, applying that function to each element in the list and returning the resulting list. `SumEulerPar` is a very simple parallelisation of the same code. It starts a task for each number in the range `n1` through `n2`, then collects the results of those tasks. While simple, this is also inefficient because most computations of `Phi(x)` will be very short and the overhead will therefore be high. `SumEulerPar2` is a more efficient implementation that reduces the overhead by aggregating the data into intervals of size 100.
### 2.3. Shared regions in GAP5
Safe mutable state in GAP5 is handled through the concept of *shared regions*. A region is a set of GAP objects; each object in GAP5 belongs to exactly one region, and each region has exactly one associated read-write lock. The GAP5 runtime ensures that an object is only accessed by a thread that has either a read or write lock on the region containing the object. Each thread has an
```gap
SumEulerSeq := function(n1, n2)
return Sum(List([n1..n2], Phi));
end;
SumEulerPar := function(n1, n2)
local tasks;
tasks := List([n1..n2], x -> RunTask(Phi, x));
return Sum(List(tasks, TaskResult));
end;
SumEulerParAux := function(n1, n2, step)
local l, tasks;
tasks := [];
i := n1;
while i <= n2 do
Add(tasks, RunTask(SumEulerSeq, l, Minimum(i + step - 1, n2)));
i := i + step;
od;
return Sum(List(tasks, TaskResult));
end;
SumEulerPar2 := function(n1, n2)
return SumEulerParAux(n1, n2, 100);
end;
```
**Figure 1.** SumEuler in GAP5.
associated *thread-local region*, on which it has a permanent write lock. By default, newly created GAP objects are created in the thread-local region of the current thread. The goal of thread-local regions (in particular, the thread-local region of the main thread) is to present programmers with a quasi-sequential environment. This environment is protected against unexpected interference from other threads, because only the thread associated with the thread-local region can ever access it, thus allowing programmers controlled use of concurrency features.
A programmer can create any number of *shared regions*. The `ShareObj(ob)` primitive takes a GAP object as an argument, creates a new shared region and *migrates* the object and all its sub-objects to the shared region. The `ShareSingleObj(ob)` variant migrates only `ob` and not any sub-objects. Objects in a shared region must be accessed using the GAP5 `atomic` statement, as shown as follows.
```gap
1 SharedList := ShareObj([]);
2 AddShared := function(x)
3 atomic readwrite SharedList do
4 Add(SharedList, x);
5 od;
6 end;
7
9 LastShared := function()
10 atomic readonly SharedList do
11 return SharedList[Length(SharedList)];
12 od;
13 end;
```
Note that we never operate on a region directly. The `atomic` statement operates on GAP objects, and the corresponding region is inferred by the runtime system. The `atomic` statement also allows the simultaneous locking of multiple regions by providing multiple arguments. These regions are then guaranteed to be acquired atomically and without causing deadlock. The following code fragment illustrates this usage of `atomic`.
```gap
1 atomic ob1, ob2 do
2 DoSomethingWith(ob1, ob2);
3 od;
```
A special case of a shared region is the *read-only region*. Every thread holds a permanent read-lock for that region, and objects can be migrated to it using the `MakeReadOnly` primitive. This is primarily intended for the convenient storage of large, constant tables in order to access them without the overhead and inconvenience of an `atomic` statement.
Objects can be *migrated* between shared regions or between the current thread-local region and a shared region. Unlike copying, migration is generally a cheap operation, which only changes the region descriptor of the underlying GAP object (a single word). As already mentioned, `ShareObj(ob)` implicitly migrates its argument to the new region. More generally, `MigrateObj(ob1, ob2)` migrates `ob1` and any sub-objects to the region containing `ob2`. For this to succeed, the current thread must have a write lock on both regions. The `AdoptObj(ob)` primitive is a special case of `MigrateObj` that migrates `ob` and any sub-objects to the current thread-local region.
Figure 2 shows how this works in practice for the parallel multiplication of two square thread-local matrices. `ParMatrixMultiplyRow` is the usual GAP code to multiply the $i$-*th* row vector of `m1` with the matrix `m2`. It is identical to the sequential version except that it is declared to be `atomic`, and `m1` and `m2` are declared to be `readonly`. `ParMatrixMultiply` contains the actual parallel matrix multiplication code. This extends the sequential version slightly.
ParMatrixMultiplyRow :=
atomic function (readonly m1, readonly m2, i)
local result, j, k, n, s;
result := [];
n := Length(m1);
for j in [1..n] do
s := 0;
for k in [1..n] do
s := s + m1[i][k] * m2[k][j];
od;
result[j] := s;
od;
return result;
ParMatrixMultiply := function (m1, m2)
local tasks, result;
ShareObj(m1);
ShareObj(m2);
atomic readonly m1, readonly m2 do
tasks :=
List(1..Length(m1)),
i -> RunTask(ParMatrixMultiplyRow, m1, m2, i));
result := List(tasks, TaskResult);
od;
atomic m1, m2 do
AdoptObj(m1);
AdoptObj(m2);
od;
return result;
end;
Figure 2. Parallel matrix multiplication in GAP5.
Firstly, in lines 18–19, m1 and m2 are placed in shared regions. Then, in lines 21–23, each row vector of m1 is multiplied with m2 using its own GAP task. Line 24 collects the results. Finally, in lines 26–30, m1 and m2 are migrated to the current thread-local region using atomic calls to AdoptObj.
2.4. Comparison with other parallel computational algebra systems
Symbolic Computation Software Composability Protocol. The computational algebra community has defined a common protocol for combining computer algebra systems, the SCSCP [48]. Essentially, it is possible to make a remote procedure call from one system to another, and both protocol messages and data are encoded in the OpenMath format. SCSCP compliant components may be combined to solve scientific problems that cannot be solved within a single CAS, or may be organised into a system for distributed parallel computations. The SCSCP has become a de facto standard for mathematical software with implementations available for seven CAS and libraries for C/C++, Java Haskell, and so on. On a single multicore, GAP5 provides higher performance by allowing direct functions manipulating GAP objects, rather than an SCSCP RPC where the function and arguments are encoded as OpenMath XML objects. Moreover, while SCSCP’s explicit RPC mechanism is suitable for small-scale parallel execution, SymGridPar2 scales onto HPCs.
GAP4. The current stable release of GAP4 provides two ways to run distributed memory parallel computations: (i) the ParGAP package that uses MPI and (ii) an SCSCP package. Both provide basic implementations of the ParList skeleton plus low-level tools to develop new parallel GAP code. Both approaches are prone to limitations of the distributed memory approach concerning serialisation, and so on. In particular, it may not be straightforward or may even be impossible to transmit complex GAP objects from one node to another, limiting performance [50].
Sage has some rudimentary facilities for distributed memory parallel calculations, which are still under development. The current design focuses on task parallelism and uses Python’s
decorator concept to add parallel execution functionality to existing methods. While this adds concurrency to the programming model, it does not provide means of explicit synchronisation or communication. Another parallelism primitive is a parallel iterator. The implementation of parallelism is also seriously hampered by the central scheduler lock in the Python runtime-system.
**Maple** supports multithreading on shared memory architectures through a task programming model. While its kernel is thread-safe, work on a thread-safe library is still in progress. On distributed memory architectures parallelism is supported by the Maple Grid Toolbox [47]. It is implemented on top of MPI and can be configured in either MPI (cluster) or HPC mode. The latter allows for integration with batch job scheduling middleware, whereas the former is targeted at dynamic, self-assembling Grid infrastructures. In terms of language support for parallelism, the Maple Grid Toolbox mainly provides MPI-level primitives for synchronisation and communication. Support for higher-level primitives is rudimentary and at the moment restricted to a parallel map operation. The older Distributed Maple [37] system, with its higher-level coordination primitives, has been discussed in Section 1.3.
**MAGMA** is a single-threaded application that does not support parallelism [11]. Distributed memory computations could be emulated by, for example, interfacing multiple copies to Sage, or by using an SCSCP wrapper for MAGMA [48].
**TRIP** is dedicated to celestial mechanics, specialising in operations with polynomials [51]. Its single-core implementation is multitreaded, reporting good parallel performance on sparse polynomial operations [52], and SCSCP can be used for small-scale distributed memory parallelism.
**MATLAB.** The MATLAB parallel computing toolbox supports multicores, GPUs and clusters using a number of simple primitives to expose data parallelism and loop-parallelism, based on parallel for-loops [10]. Support for heterogeneous devices is provided through a Cuda interface.
Using the MATLAB Distributed Computing Server, parallelism can be orchestrated on large-scale, distributed memory configurations. The preferred programming mode is data parallelism over the pre-defined data structure of distributed arrays. Additionally, a general map-reduce structure is available. On a lower level, primitives for single-program multiple-data parallelism and explicit message passing are also available.
The current version of MATLAB’s symbolic computation toolbox, MuPAD, does not support parallel computing. However, earlier versions of MuPAD provided master-work parallelism on distributed systems in the form of ‘macro parallelism’ [53].
**Mathematica.** Starting with Mathematica 7, the internal Wolfram Language also supports parallel computation. The focus of the language support is on data-parallelism, with pre-defined parallel map and combine operations. Parallel iterators are provided for common data structures. Speculative parallelism is also supported, enabling the programmer to try several approaches to solve a problem and pick up the first successful computation. More generally, fork-and-join parallelism can be defined, using lower-level thread creation and synchronisation primitives. However, the language design favours the usage of the higher-level primitives, in particular for defining map-reduce-style computations. Support for heterogeneous devices is provided through separate interfaces to Cuda and to OpenCL. In a distributed memory setting, the parallelism generated by the primitives is managed by the gridMathematica system, originally developed for large-scale Grid architectures [54]. This system automatically handles marshalling and communication of data structures, as well as retrieving the results from a remote execution. Through the usage of the higher-level parallelism primitives, the details of the architecture are mostly hidden, although aspects such as communication latency need to be taken into account for parallel performance tuning.
**Reflection.** Thus, we see that the majority of existing computational algebra system only provide support for (independent) distributed parallel calculations, or else have limited computational algebra functionality. The work described in this paper considers a wider class of parallel computation, including shared-memory multicores, clusters and HPC system, as well as distributed systems. Through the free GAP system, it makes the benefits of parallelism available to a much wider, open source community. Achieving this, while obtaining significant real speedups, represents
a major technical challenge: GAP is a large dynamically typed, interpreted system, with a complex implementation that manipulates large and complex data structures.
3. MPI-GAP DESIGN AND IMPLEMENTATION
MPI-GAP is an extension of GAP5, which targets distributed-memory systems by building on the *de facto* standard MPI message passing library. MPI-GAP provides both a high-level programming model that supports the same set of high-level parallel task constructs as the shared-memory version of GAP5, `CreateTask`, `RunTask`, `TaskResult`, and so on, and a low-level programming model that offers more control of distributed task and data placement when required. MPI-GAP is implemented in a layered manner (Figure 3), where each layer builds on the constructs provided by the layer below to offer a higher level of abstraction.
**Object marshalling layer.** This layer consists of operations that transform GAP objects into an equivalent binary representation that can be transmitted using MPI primitives. MPI-GAP currently supports two classes of marshalling operations: *native serialisation*, implemented as kernel operations, and *IO pickling*, implemented as GAP-level operations. Native serialisation is much faster, but it is architecture dependent and currently only allows certain GAP objects to be marshalled. IO pickling is significantly slower but is architecture independent and more customisable – more data structures are supported, and programmers can provide functions to marshal custom data structures. In general, if an application needs to transfer large amounts of ‘simple’ data (e.g. lists of simple objects and matrices) and the underlying data processing is sufficiently fine-grained, native serialisation is preferred. Conversely, when transferring smaller objects or when the underlying data processing is coarse-grained, IO pickling should be used.
**MPI binding layer.** This layer consists of GAP bindings to raw MPI functions to send and receive messages, based largely on ParGAP [55]. Currently, only a small subset of MPI functions are supported on the GAP side, including blocking and non-blocking MPI send and receive functions, probing, and so on. Object marshalling primitives must be used to marshal GAP objects so that they can be transmitted using these operations.
**Global object pointer layer.** This layer supports operations on GAP objects (handles) that represent pointers to other GAP objects that may live on remote computing nodes. Handles can be copied
| Layer | Supported primitives/operations |
|------------------------|--------------------------------------------------------------------------------------------------|
| Distributed Tasks | `CreateTask`, `RunTask`, `TaskResult`, `SendTask`... |
| Shared Objects | `RemoteCopyObj`, `RemotePutObj`, `RemoteCloneObj`, `RemotePullObj` |
| Global Object Pointers | `CreateHandleFromObj`, `SendHandle`, `SetHandleObj`, `GetHandleObj` |
| MPI Bindings | `MPI_Send`, `MPI_Isend`, `MPI_Recv`, `MPI_Probe`, `MPI_Iprobe`... |
| Object Marshalling | `SerializeToNativeString`, `DeserializeNativeString`, `IO_Pickle`, `IO_Unpickle` |
Figure 3. MPI-GAP layers and the associated primitives/operations.
between different GAP processes and can then be used to access the same physical GAP object from different nodes. Handles, and the underlying GAP objects, are managed in a semi-automatic way – handles are created, opened, closed and destroyed manually, but a reference-counting mechanism prevents unsafe operations, for example, destroying a handle that exists on multiple distributed nodes and, so, possibly garbage-collecting the underlying object.
Handles are created using the `CreateHandleFromObj` operation that takes a GAP object and returns a handle to it. Depending on the required level of automatic control, handles can be read-only, read-write or volatile. Objects referred to by read-only handles can be freely copied between nodes, but cannot be modified using `SendHandle`, `GetHandleObj` or `SetHandleObj`. Objects referred to by read-write handles can be freely modified, but the underlying objects cannot be copied between nodes. They can, however, be migrated. Finally, volatile handles are unrestricted, that is, a programmer has full control and is responsible for handling consistency of cached copies, and so on. Handles can be transferred between nodes using the `SendHandle` operation. They can be read and modified using the `GetHandleObj` and `SetHandleObj` operations.
**Shared object layer.** This layer supports operations on objects that are shared between multiple distributed nodes and accessed via handles from the global object pointer layer. Objects can be copied using the `RemoteCopyObj` or `RemoteCloneObj` operations and migrated using the `RemotePushObj` and `RemotePullObj` operations, where, in each case, the first version is used by the sender and the second by the recipient. Depending on the handle type, certain operations might be prohibited for safety reasons, for example, `RemoteCopyObj` is prohibited on read-write handles.
```gap
DeclareGlobalFunction("Euler");
InstallGlobalFunction(Euler,
function(handle)
Open(handle);
inputList := GetHandleObj(handle);
resList := List ( EulerPhi, inputList);
res := Sum(resList);
Close(handle);
return resList;
end
);
SumEuler := function (n)
if processId = 0 then
chunkSize := n / commSize;
ts := [];
# send work to other nodes
for nodeid in [1..commSize] do
h := CreateHandleFromObj ([n*chunkSize+1..(n+1)*chunkSize])
SendHandle (h, nodeid);
RemoteBeginJob(h);
t :=nodeid; CreateTask ("Euler", h);
SendTask (t, nodeid);
od;
# do local calculation
result = Sum(Euler([1..chunkSize]));
# fetch the results
for nodeid in [1..commSize] do
remoteResult := TaskResult(ts[nodeid]);
result := result + remoteResult;
od;
return result;
else
return -1; # only master process should call this function
fi;
end;
```
Figure 4. SumEuler in MPI-GAP.
**Distributed task layer.** This layer supports the same task creation and management operations as shared-memory GAP5, for example, `CreateTask`, `RunTask` and `TaskResult`. In addition, it also supports explicit task placement using the `SendTask` operation. Task distribution between nodes in MPI-GAP uses a *random work stealing* work stealing approach, which has been shown to perform well in low-latency settings. Tasks that are not yet executed are kept in node-local *task pools*. Each node apart from the master starts in the *idle* mode and requests work from another randomly chosen node. If a node that has more than one task receives a message requesting work, it offloads one of its tasks. This distributes tasks among the nodes on an as-needed basis.
### 3.1. SumEuler in MPI-GAP
Figure 4 shows the implementation of SumEuler in MPI-GAP. Line 1 declares a global function to map the Euler totient function over a list of values and sum the results. In lines 2–11, we implement this function, passing it a handle to a list of positive integers. We first open the handle (line 4), in order to inform the reference-counting mechanism that the handle is in use by the current thread. We then read the list that the handle points to (line 5). After applying the EulerPhi function to all of the elements of the list, to produce a list of results `resList` (line 6), we close the handle (line 8) to inform the reference-counting mechanism that the current thread has finished using the handle.
The main function, `SumEuler` (lines 12–35), is only called by a master GAP process (i.e. the GAP instance that controls the execution). We first determine the sizes of the chunks of work that will be sent to each worker GAP process (line 15) and initialise a list of tasks (line 16). For each worker process, we create a handle to a list of positive integers (line 19), send the handle to the worker process (line 20), migrate the actual list (line 21), create a task that calls the Euler function on the handle (line 22) and finally send the task.
The program implements *eager* work distribution by explicitly assigning tasks to the processes. After sending work to each worker process, the master process does its own chunk of work (line 26), and then fetches and sums the results of the tasks (lines 28–31).
## 4. THE DESIGN AND IMPLEMENTATION OF SYMGRIDPAR2
SymGridPar2 (SGP2) is middleware for coordinating large-scale task-parallel computations distributed over many networked GAP instances. SymGridPar2 inherits its architecture, and specifically, its skeleton-based programming model, from SymGridPar [56]. The key new feature is the use of HdpH (Haskell distributed parallel Haskell), a novel *domain-specific language* (DSL) for task-parallel programming on large-scale networks, including HPC platforms. The SGP2 middleware comprises three layers:
1. the HdpH DSL for parallel coordination, embedded into Haskell (Section 4.1);
2. a Haskell binding for GAP (Section 4.2); and
3. a set of algorithmic skeletons, written in HdpH and calling into GAP (Section 4.3).
Figure 5 illustrates the SGP2 architecture and its interaction with GAP. In the figure, the outer dotted rectangles represent a host, possibly multi-core, and while two instances are shown, there may be an arbitrary number. The solid rectangles represent operating system processes, and the arrows represent communication.
The intention is that a GAP program invokes appropriate skeletons, essentially GAP functions with parallel semantics, for example, `parList([40..60], fibonacci)` may create 21 tasks to compute the Fibonacci function of the integers between 40 and 60. The GAP skeleton corresponds to an HdpH skeleton that distributes the subproblems to multiple hosts where they are passed to local GAP instances to be solved, and the solutions are returned to the caller.
As the GAP skeleton library has not yet been constructed, SGP2 applications are HdpH programs. While this section presents some key Haskell code, it is intended to be comprehensible without knowledge of Haskell. The skeletons generate tasks, which are distributed and executed by the HdpH runtime; HdpH provides sophisticated distributed work stealing built on top of an MPI...
communication layer. Tasks will typically call GAP functions through the GAP binding. Each SGP2 instance can transparently coordinate a pool of several GAP4 or GAP5 servers running on the same host. Communication between SGP2 and these GAP servers is via Unix IPC. SGP2 requires one dedicated core per host, the remaining cores can be used by GAP servers. On an $n$-core host, one SGP2 instance will typically coordinate $n - 1$ GAP4 instances or a single GAP5 instance running $n - 1$ threads. To reduce memory bus contention on large Non-Uniform Memory Access (NUMA) servers, it is also possible to coordinate several multi-threaded GAP5 instances, each bound to its own NUMA region. Section 5.4 contains some example deployments.
### 4.1. Coordination DSL
SGP2 exposes parallel patterns to the programmer as *algorithmic skeletons* [15], implemented in *HdpH*. HdpH is described in detail elsewhere [57, 58], and here, we discuss only the small part of the API which users of the SGP2 skeleton library are exposed to, as outlined in Figure 6. As is customary in functional programming languages, the HdpH DSL is embedded into the host language, Haskell, by wrapping DSL *computations* in a special computation type. In HdpH, this is performed by the `Par` type constructor, which forms a *monad*, the standard Haskell construct for effectful computations. Almost all HdpH and SGP2 primitives create or transform `Par` computations; ultimately, an SGP2 program is one large `Par` computation. The primitive `runParIO` takes such a computation and executes it on the HdpH runtime system.
In HdpH, parallel computations return results asynchronously through *futures*. These are represented by the type constructor `IVar`. Futures are created by a number of skeletons as well as by the GAP binding. A user may read the value stored in a future by calling the primitive `get`, which blocks the caller until the value is available.
---
**Figure 5.** SymGridPar2 architecture.
---
**Figure 6.** Type signatures of a minimal HdpH API.
As a distributed DSL, HdpH exposes *locations* (i.e. hosts) as values of type `Node`. SGP2 programs can query the current node and obtain a list of all nodes by calling the primitives `myNode` and `allNodes`, respectively. Crucially, the API in Figure 6 does not list any primitives for creating or distributing parallel *tasks*. This functionality is hidden in the skeletons discussed in Section 4.3.
### 4.2. GAP binding
HdpH is a DSL for coordinating task-parallel computations. Ordinarily, HdpH tasks evaluate function calls in Haskell. To coordinate computations in GAP, SGP2 has to provide a way to call from HdpH into GAP. This is performed by providing a *GAP binding*, that is, a library of Haskell functions to call GAP functions, including marshalling arguments from Haskell to GAP and results from GAP to Haskell.
Additionally, the GAP binding also provides functions for starting and stopping GAP servers. Each HdpH node can launch one or more GAP servers, running as independent operating system processes on the same host as the HdpH node and communicating via pipes. The API distinguishes between *stateful* GAP servers, each with a distinct identity, and *stateless* GAP servers, which are anonymous and interchangeable.
#### 4.2.1. Controlling stateful GAP servers
Figure 7 shows the HdpH API for controlling GAP servers; primitives ending in `ST` are concerned with stateful servers. HdpH can start a GAP server by calling `startST`, which expects information on how to call GAP and a list of GAP function calls used to initialise the server. The primitive `startST` immediately returns a future that will eventually provide a handle to the GAP server, once the server is up and running. The primitive
---
**Figure 7.** HdpH Primitives for controlling GAP servers.
barrierST takes a server handle and blocks until the server is idle, that is, no longer busy executing a previous function call. Similarly, stopST blocks until the server is idle and then terminates the server (after which the handle is void). Finally, the primitive callST posts a function call to the given server. It blocks until the server is idle and then immediately returns a future that will eventually yield either an error or the result of the call. GAP function calls (type GAPCall) are simply strings. For convenience, they can be constructed by applying a GAP function name (type GAPFunction) to a list of encoded GAP objects using the function mkGAPCallFuncList; the encoding is discussed in Section 4.2.3.
Note that all primitives operate on stateful GAP servers running on the same host as the calling HdpH node; calls to distributed stateful GAP servers are provided by the higher-level skeletons discussed in Section 4.3. More precisely, any stateful GAP server can only be controlled by the HdpH node that started it, as identified by the primitive atST, and a GAP handle is only valid on the node indicated by atST.
### 4.2.2. Controlling stateless GAP servers
Many SGP2 applications interact with GAP in a stateless fashion, that is, GAP calls are proper function calls that do not manipulate hidden state on the server. For this case, HdpH offers primitives to coordinate a pool of stateless GAP servers.
The stateless primitives mirror the stateful ones (Figure 7). The start primitive takes the same information about calling and initialising GAP. Of course, the GAP calls used to initialise a stateless GAP server, like startST, change the system state, that is, are themselves stateful. The start function takes an additional pool size argument and returns nothing. The choice of pool size is critical for performance, for example, performance suffers if the pool size exceeds the number of physical cores. Because stateless servers are interchangeable, they can be anonymous, so there is no need to return a handle. The primitives barrier and stop behave like their stateful counterparts, except that they block until the whole pool of servers is idle, and terminate the whole pool.
The primitive call posts a function call to any stateless server. It blocks until a server is idle and then immediately returns an IVar that will eventually yield either an error or the result of the call. Note that the GAP function call is expected to be stateless in the sense that any side effects on the GAP server do not affect subsequent calls. Violating this restriction may result in unpredictable behaviour.
### 4.2.3. Marshalling data to and from GAP
Marshalling data between GAP and HdpH requires an encoding and a Haskell representation of encoded GAP objects; the latter is provided by the data type GAPObj. A GAPObj can represent the GAP constant Fail, GAP Booleans, GAP integers, GAP rationals, lists of GAP objects, the empty GAP list [], GAP strings or opaque GAP objects; opaque objects are all those that are not explicitly listed in the aforementioned enumeration. The encoding assumes that GAP can print the object to be marshalled into a string that can be parsed back later; objects that do not support this need to be explicitly serialised to a GAP string prior to marshalling.
The bottom of Figure 7 displays the API for accessing GAPObjs. There are a number of functions testing which GAP type the encoded GAPObj represents. And there are two overloaded functions toGAP and fromGAP, defined on types instantiating class GAPEnc, for encoding to and decoding from GAPObj. The Haskell types, which are instances of GAPEnc, are the standard base types (), Bool, Integer and Rational, as well as ByteString (which encodes GAP strings). Standard list, pair and Maybe data types can also be encoded, provided their components are encodable.
Opaque GAP objects cannot be decoded and remain opaque as there is no instance of class GAPEnc which they can be decoded to. This is justified because, as a coordination layer, SGP2 mainly needs to decode containers (lists or tuples) and numbers; other GAP objects are simply passed as is from one GAP server to another.
4.3. The SymGridPar2 programming model
SGP2 exposes parallel patterns to the programmer as *algorithmic skeletons* [15] where the computations coordinated are GAP functions. Figure 8 lists some basic SGP2 skeletons for starting, stopping and calling stateful and stateless GAP servers. These skeletons use the coordination features of HdpH to extend the scope of the GAP binding in that they transparently control GAP servers beyond the current node.
The `startGAP` skeleton initiates starting a stateful GAP server on a remote node (using the primitive `startST`); it returns a future that will eventually, when the server is up and running, yield a handle. The `startGAPEverywhere` skeleton starts pools of stateless GAP servers on all nodes (using the primitive `start`). However, unlike `startGAP`, this skeleton blocks until all GAP servers are up and running, and then returns nothing. The started GAP servers can be terminated by `stopGAP` and `stopGAPEverywhere`; both skeletons block until the servers in question have actually quit.
The `pushGAPCall` skeleton is a generalisation of the primitive `callST` for calling a stateful GAP server on any node, transparently marshalling call and result across the network. The skeleton returns a future, which will eventually hold the result of the call. In contrast, the `parGAPCalls` skeleton is a generalisation of the `call` primitive for calling stateless GAP servers, handling both lists of calls as well as balancing load between all GAP servers in the system (relying on HdpH random work stealing). Unlike `pushGAPCall`, the `parGAPCalls` skeleton blocks and only returns the list of results after the last result has come in; the skeleton will return the empty list if any of the GAP calls failed.
4.3.1. SumEuler in SymGridPar2. Figure 9 shows the implementation of the SumEuler example in SGP2. The `parSumEuler` starts a number of GAP servers before computing the sum of Euler’s totients in parallel. The computation divides the input interval into smaller chunks and then proceeds by using the skeleton `parGAPCalls`, converting its results (GAPObjs encoding integers) into Haskell integers, and summing up. Incidentally starting GAP servers may take several seconds and hence is usually performed at the start of an application rather than immediately before `parGAPCalls` as here.
The list of GAP calls passed to `parGAPCalls` consists of calls to the GAP function `SumEuler`, the definition of which is passed to every GAP server during the start up phase coordinated by `startGAPEverywhere`. In Haskell, this function definition is a literal string embedded into the Haskell code. Alternatively, the definition can be read from a file.
```haskell
-- Start a stateful GAP server on given (remote) node; returns a future which
-- will yield a server handle once the server is up and running.
startGAP :: GAP -> [GAPCall] -> Node -> Par (IVar GAPHandleST)
-- Start pools of stateless GAP servers on all nodes;
-- blocks until all servers are up and running.
startGAPEverywhere :: GAP -> [GAPCall] -> Int -> Par ()
-- Stop given (remote) stateful GAP server; blocks until server has died.
stopGAP :: GAPHandleST -> Par ()
-- Terminate all stateless GAP servers; blocks until all servers have died.
stopGAPEverywhere :: Par ()
-- Post GAP call to given (remote) stateful GAP server; returns a future
-- which will eventually yield the results.
pushGAPCall :: GAPHandleST -> GAPCall -> Par (Either GAPError GAPObj)
-- Process list of stateless GAP calls in parallel using work stealing;
-- returns [] if any GAP call caused an error.
parGAPCalls :: [GAPCall] -> Par [GAPObj]
```
Figure 8. Example SGP2 skeletons and GAP interface.
-- Define SymEuler function in GAP,
defSumEuler :: GAPCall
defSumEuler = pack $ unlines [
"SumEuler_ := function([lower,upper])",
" return Sum_([lower,upper],X -> Phi(x));",
"end;"
]
funSumEuler :: GAPFunction
funSumEuler = pack "SumEuler"
mkIntervals :: Integer -> Integer -> Integer -> [(Integer, Integer)]
mkIntervals lower upper chunksize = go lower
where
go lb | lb > upper = []
| otherwise = (lb, ub) : go lb'
where
lb' = lb + chunksize
ub = min upper (lb' - 1)
-- Start 'n' stateless GAP servers everywhere, compute the sum of Euler's
-- torsion function on the integral intervals ('lower','upper'), evaluating
-- sub-interval of length 'chunksize' in parallel.
parSumEuler :: Int -> GAP -> Integer -> Integer -> Integer -> Par Integer
parSumEuler n gap lower upper chunksize = do
startGAPsEverywhere gap [defSumEuler] n
let calls = mkGAPCallFuncList funSumEuler [toGAP l, toGAP u] |
(l,u) <- mkIntervals lower upper chunksize]
gapObjs <- parGAPCalls calls
let result = sum $ map fromGAP gapObjs
stopGAPsEverywhere
return result
Figure 9. SumEuler in SGP2.
4.4. Advanced features
The capabilities of SymGridPar2 described so far match largely those of SymGridPar. SGP2 goes beyond SGP in its support for large architectures. Where SGP assumes a uniform communication latency between any two cores, SGP2’s scheduling algorithm can take into account a non-uniform hierarchy of latencies; such latency hierarchies are common in large compute clusters.
Abstract model of hierarchical distances. The coordination DSL HdpH exposes node-to-node latencies abstractly to the programmer as distances in a metric space. More precisely, the distance between nodes is a real-valued binary function satisfying the axioms of ultrametric spaces. As a result, the programmer need not be concerned with actual latencies and can instead express task scheduling constraints in terms of relative distances like here, close by or far away. We refer the reader to [58] for more detail about ultrametric distances in HdpH.
Task scheduling HdpH offers two modes of work distribution: on-demand implicit task placement and eager explicit task placement.
On-demand implicit task placement is realised by a distributed work stealing algorithm. Upon creation, a task is stored in the creating node’s local task pool. Stored with the task is its radius, as chosen by the programmer. The task radius is the maximum distance the task may travel away from its originating node. HdpH’s work stealing algorithm is based on random stealing and has two properties relating to radii: the task radius is a strict bound on the distance between victim and thief; thieves prefer to steal tasks within small radii. The radius can be viewed as expressing a size constraint on a task, where larger radii mean bigger tasks. Adopting this view, HdpH’s work stealing algorithm prefers to keep small tasks near their originating nodes, similar to [59], in the hope of minimising communication overheads related to work stealing.
Many basic SGP2 skeletons, for instance parGAPCalls, rely on HdpH work stealing, typically without imposing constraints on work stealing distances; we refer to [4] for examples of SGP2 skeletons that make use of radii for bounding work stealing.
On-demand random task placement performs well with irregular parallelism. However, it tends to under-utilise large scale architectures at the beginning of the computation. To combat this drawback, HdpH offers *eager explicit task placement* as a complementary placement mode. Explicit placement ships tasks to selected nodes, where they are executed immediately, taking priority over any implicitly placed tasks. Eager execution implies that these tasks are meant to perform coordination, e.g. create further tasks, rather than actual computation.
The downside of explicit placement is that the programmer has to select target nodes, which may jeopardise portability. Fortunately, HdpH’s abstract ultrametric distances offer some help for the purpose of flooding large architectures with work. More precisely, the HdpH runtime identifies an *equidistant basis*, that is, a maximal set of nodes such that any two basis nodes are maximally far apart. At the beginning of a parallel computation, the programmer can explicitly place large tasks at the basis nodes, where each will generate smaller tasks that can be stolen quickly by nearby nodes; stealing may or may not be bounded, depending on the irregularity of the small tasks.
SGP2 offers a number of skeletons implementing such a *two-level task distribution*, for example, the `parMap2Level` and `parMap2LevelRelaxed` skeletons used in Section 5.3, and detailed in [4].
**Reliability.** The coordination DSL HdpH is designed for *transparent fault tolerance* and [60] presents an alternative work stealing scheduler that monitors nodes and pessimistically replicates tasks that may have been lost to node failure. The fault tolerant work stealing is shown to have low runtime overheads. The fault tolerance properties of HdpH are, however, not yet inherited by SGP2 because the current GAP binding is not fault tolerant.
## 5. PERFORMANCE EVALUATION
This section reports a performance and interworking evaluation of the HPC-GAP components. The primary comparators are the SumEuler benchmark implementations from the preceding sections, although we investigate some GAP5 concurrency overheads using microbenchmarks. We measure strong scaling, that is, speedups and runtimes, for GAP5 on a multicore (Section 5.1), and for MPI-GAP and SymGridPar2 (Sections 5.2 and 5.4) on Beowulf clusters. Only weak scaling is appropriate for large architectures, and we measure SymGridPar2 on up to 32K cores of the HECToR HPC (Section 5.3).
Section 5.2 demonstrates the interworking MPI-GAP and GAP4, and Section 5.4 demonstrates the interworking SymGridPar2 with both GAP4 and GAP5.
### 5.1. GAP5 evaluation
We consider both the overheads on sequential programs introduced by managing concurrency in GAP5 and demonstrate the performance gains that can be obtained through parallelisation.
#### 5.1.1. Sources of concurrency overheads
GAP5 exposes essentially the same programming language and libraries as GAP4, augmented with the task and region features described in Section 2. However, supporting multithreading requires many, and often significant, changes even where the functionality is unchanged. While these changes should be transparent to GAP programmers, they affect the performance of purely sequential code. These performance differences can be attributed to one or more of the following factors.
1. Whereas GAP4 has a sequential, generational, compacting garbage collector, GAP5 has a concurrent, non-generational, non-compacting garbage collector. Depending on the workload, either GAP4 or GAP5 can perform better; GAP4 where the generational collection wins out, GAP5 where concurrent collection (with a sufficient number of processors) makes it faster. Generational collection generally wins out when a program allocates a large number of short-lived objects.
2. Memory allocation in GAP4 occurs through a very fast bump allocator made possible by the compacting garbage collector. Memory allocation in GAP5 is more complicated in that it has to support concurrent allocation, which incurs a small, but non-zero, overhead. Most allocations occur through the thread-local allocator, which is nearly as fast as the GAP4 bump allocator, but larger chunks of memory cannot be handled this way and require that their allocation be serialised.
3. To ensure memory safety, the GAP5 kernel code had to be instrumented with checks that no GAP object is being accessed without the corresponding lock being held. Like bound checks for arrays, this incurs an unavoidable overhead. While this instrumentation can be disabled if speed is essential, it is generally assumed that users want these checks to be enabled when running parallel code. The instrumentation is currently performed (almost) fully automatically by a separate tool as part of the build process that performs dataflow analysis and attempts to optimise the instrumentation (such as hoisting checks out of loops). This automated instrumentation can be manually overridden where the optimiser fails to correctly identify such possibilities, though so far, we make very little use of that.
4. The current GAP5 implementation does not yet fully support GAP-to-C compilation for all new features. As a result, some core GAP modules that are compiled in GAP4 are still being interpreted in GAP5.
5. A few internal data structures of the GAP kernel had to be redesigned in order to make them thread-safe. One example is the implementation of global variables in GAP, which required non-trivial changes to ensure that multiple threads can access them concurrently, even where they, and their contents, are immutable.
The overhead varies from program to program, and the combined effect of the concurrency overheads is that sequential code typically executes between 10% and 40% more slowly in GAP5 than in GAP4, based on our observations with various synthetic benchmarks and existing GAP code. Sections 5.1.3 and 5.3 report the overheads for specific programs, and it is reassuring to see that the overheads fall as the parallel system scales. We also expect to be able to eliminate the remaining avoidable overheads in the medium term. Nevertheless, our plans for GAP5 include a purely sequential build option for code that cannot effectively exploit parallelism.
5.1.2. Quantifying concurrency overheads. The first GAP 5 concurrency overhead we investigate is that of instrumenting the GAP5 kernel with guards against race conditions. We execute the
```plaintext
benchmark_base := function()
local i;
for i in [1..10^9] do
cd;
end;
end;
benchmark_inc := function()
local i, s;
s := 0;
for i in [1..10^9] do
s := s + 1;
cd;
end;
end;
benchmark_index := function()
local i, s;
s := [0];
for i in [1..10^9] do
s[1] := s[1] + 1;
cd;
end;
end;
```
Figure 10. Microbenchmarks to measure GAP5 instrumentation overheads.
Table I. GAP5 instrumentation runtime overheads.
| Benchmark | GAP5 (no checks) | GAP5 (checks) | Overhead |
|--------------------|------------------|---------------|----------|
| benchmark_base | 3.4 s | 3.4 s | 0% |
| benchmark_inc | 12.8 s | 13.7 s | 7% |
| benchmark_index | 50.7 s | 66.5 s | 31% |
Figure 11. GAP5 SumEuler runtimes and speedups (multicore).
micro-benchmarks in Figure 10 with and without instrumentation, taking the median value over five runs. All of the micro-benchmarks allocate only a small and fixed amount of memory and do not access global variables, so neither garbage collection nor global variable access has a measurable effect on their performance. Measurements in this section are performed on a 64-core machine with AMD Opteron 6376 processors.
The results in Table I show that for benchmark_base there is almost no overhead for a basic for loop. The benchmark_inc result shows that there is a small amount of overhead for performing local variable increments. The benchmark_index result shows that there is around a 33% overhead (after subtracting the time for the for loop) for performing increments on an array element. In fact, there are no checks in the C code that performs local variable increments, so the performance differences for benchmark_inc are probably due to other effects, such as changes in memory locality as a side effect of instrumentation.
The overhead for benchmark_index is not surprising, because for each iteration, three checks are performed. This is suboptimal as our tool inserts two checks for the assignment (one, before checking that the length of the list is at least 1, and another, before performing the assignment) where one would suffice. So the overhead could be reduced by a third.
Garbage collection overhead can vary greatly by workload. Allocating many short-lived objects can result in an overhead of 30% or more; more typical for GAP applications is an overhead in the range of 10%–20%. In cases where allocations of long-lived objects dominate, the GAP5 garbage collector can also be faster than the GAP4 garbage collector, as it can parallelise its work, while the GAP4 garbage collector gains little from its generational features.
5.1.3. SumEuler performance. We investigate the performance of the GAP5 version of SumEuler outlined in Section 2.2 (page 3611). GAP’s standard implementation of Euler’s totient function relies heavily on cache-based implementation of factorisation routines. This can have unpredictable effects on performance, depending on how number ranges are being broken into chunks and how these chunks are assigned to threads. In order to obtain reproducible results, we therefore use a cache-less implementation of the totient function for this benchmark.
Figure 11 shows the runtime and relative speedup of the GAP5 SumEuler up to 16 cores, taking the median value of three successive runs. The sequential SumEuler runs in 16.5 s on GAP4, so GAP5 (22.0 s) is 33% slower.
5.2. MPI-GAP evaluation
We investigate the performance of the MPI-GAP version of SumEuler outlined in Section 3.1 (page 3617). The evaluation was conducted on a 32-node Beowulf cluster, with each node having 8-core Intel Xeon E5504 2 GHz processor with 16 GB RAM. One node was designated as a master node, and it distributes chunks of work to the slave nodes, who then execute them on a single-threaded GAP4 instance. To exercise a cluster, the input is larger than that for a single GAP5 instance in Section 5.1.3.
The runtime for a single-threaded GAP4 SumEuler computation is 211.7 s, and for the MPI-GAP version on one node is 217.3 s. So the overhead incurred by the MPI-GAP on a single node is approximately 3% for this benchmark. The MPI-GAP runtime on 32-nodes is 9.9 s. These runtimes are reflected in the speedup graph in Figure 12, which shows near-linear speedups up to a maximum of 22 on 32 cores.
5.3. SGP2 evaluation
We investigate the performance of the SymGridPar2 version of SumEuler outlined in Section 4.3.1 (page 16) on the HECToR supercomputer. At the time (2013), HECToR was the UK’s largest public HPC with approximately 90,000 cores [61]. Figure 13 studies weak scaling of two variants of the SumEuler benchmark from 1 to 1024 HECToR nodes (i.e. 32 to 32K cores). The two variants differ only in their use of the underlying skeleton: One uses parMap2Level, and the other uses parMap2LevelRelaxed both outlined in Section 4.4.
The benchmarks start with an input interval of 6.25 million integers on 32 cores, doubling the size of the interval as the number of cores doubles, so that on 32K cores, for example, the interval is 6.4 billion integers long. Doubling the size of the input interval increases the amount of work by more than a factor of 2; by sampling the sequential runtime of small subintervals, we estimate a runtime curve for ideal scaling.
The runtime graphs in Figure 13 show that the two benchmarks do not scale perfectly. However, even on 32K cores, their runtimes are still within 50% of the ideal. Efficiency is calculated with respect to the estimated ideal scaling, and the curves show that efficiency declines steadily, yet remains above 70% even on 32K cores. These graphs also show that the parMap2LevelRelaxed skeleton offers a small efficiency advantage (of 3–4%) over parMap2Level1. This is probably because parMap2LevelRelaxed copes better with the irregularity of the benchmark in the final stages of the computation, because unlike parMap2Level1, it can utilise nodes that have run out of work early.
5.4. HPC-GAP interworking
Section 5.2 demonstrated MPI-GAP coordinating single-threaded GAP4 servers, and this section focuses on the performance implications of SymGridPar2 coordinating GAP4 (version 4.7.6) and multi-threaded GAP5 (version alpha_2015_01_24) servers. We investigate the speedup of SumEuler on the interval \([1, 10^8]\) on a Beowulf cluster with 256 cores, distributed over 16 nodes (Intel Xeon CPUs, 2 GHz, 64 GB RAM per node). The input interval is subdivided evenly into subintervals, and the SGP2 instances distribute the resulting SGP2 tasks by work stealing. When co-ordinating GAP4, each SGP2 instance further subdivides the current task interval and distributes calls to function SumEulerSeq (Figure 1) to 15 single-threaded GAP4 servers. When co-ordinating GAP5, each SGP2 instance calls a single GAP5 server (running on 15 cores) which in turn subdivides the interval and uses the GAP5 RunTask primitive to evaluate calls to SumEulerSeq on subintervals in parallel.
Table II records optimal values for the numbers of SGP2 tasks, GAP calls per SGP2 task and GAP tasks per GAP call. These optima are determined by exhaustive experiment. The table also reports sequential runtimes for GAP4 and GAP5, from which the average runtime of a GAP task, that is, mean time to compute SumEulerSeq, is derived. Given the discussion of GAP5 overheads in Section 5.1, it is unsurprising that the sequential GAP5 runtime is 24% greater than for GAP4.
| | Seq. runtime | SGP2 tasks | GAP calls per SGP2 task | GAP tasks per GAP call | Mean GAP task runtime |
|----------------|--------------|------------|-------------------------|------------------------|-----------------------|
| SGP2/GAP4 | 2160.0 s | 320 | 15 | 1 | 450 ms |
| SGP2/GAP5 | 2836.1 s | 400 | 1 | 300 | 24 ms |
Figures 14 and 15 show log/log plots of runtime and absolute speedup for SGP2/GAP4 and SGP2/GAP5. The numbers of SGP2 tasks, GAP calls and GAP tasks are fixed to the optimal values from Table II, and the runtime data is the median over 11 runs. We find that variance, measured as standard error, is consistently between 1% and 2%.
Figure 14 shows that while SGP2/GAP4 has consistently lower runtime, the advantage falls from about 24% on one core to 6% on one node to only about 2% on 16 nodes. The absolute speedups in Figure 15 are computed with respect to the sequential runtime of the respective GAP version. The curves show that both systems scale similarly, yet the speedup of SGP2/GAP5 is consistently about 25% greater than for SGP2/GAP4. On 1 node, SGP2/GAP5 achieves an absolute speedup of 11.5, corresponding to 76% efficiency: ideal speedup is 15 because only 15 cores are available to GAP while the 16th core is dedicated to SGP2. On 16 nodes, SGP2/GAP5 efficiency drops to 71%, corresponding to a speedup of 170.
Symbolic computations are memory hungry, and on large shared-memory architectures, SGP2/GAP5 can use a small number of GAP processes (possibly just one), where SGP2/GAP4 must use as many GAP processes as there are cores. Sampling memory residency for SumEuler (using Unix tools like `ps`) consistently shows a total per-node residency of SGP2/GAP4 (i.e. 15 GAP4 processes plus 1 SGP2 process) of about 2 GB. In contrast, the residency of SGP2/GAP5 (one GAP5 process plus one SGP2 process) is only 240 MB, or approximately 1/8th of the residency of SGP2/GAP4.
In conclusion, SGP2/GAP5 is able to almost entirely eliminate the GAP5 overheads by achieving better speedups even on mid-scale parallel architectures. While we have not been able to demonstrate SGP2/GAP5 outperforming SGP2/GAP4, we anticipate this may happen on larger architectures. A second advantage of SGP2/GAP5 over SGP2/GAP4 is greatly reduced memory residency on large shared-memory architectures.
6. CASE STUDIES
We present two case studies to demonstrate the capability of the HPC-GAP components to deliver good parallel performance for typically irregular algebraic computations on both NUMA and HPC architectures.
6.1. Orbits in GAP5
Enumerating an orbit is a typical algebraic computation and can be described as follows.
**Definition 1 (Orbit of an element)**
Let $X$ be a set of points, $G$ a set of generators, $f : X \times G \rightarrow X$ a function (where $f(x, g)$ will be denoted by $x \cdot g$) and $x_0 \in X$ a point in $X$. The *orbit* of $x_0$, denoted by $O_G(x_0)$, is the smallest set $T \subseteq X$ such that
1. \( x_0 \in T \)
2. for all \( y \in T \) and all \( g \in G \) we have \( y \cdot g \in T \).
Often the set \( X \) can be very large and is not given \textit{a priori}.
The sequential orbit algorithm in Figure 16 maintains a hash table \( T \) of orbit points discovered so far and a list \( U \) of unprocessed points. The hash table facilitates a fast duplicate test (line 6). Initially both \( T \) and \( U \) contain only \( x_0 \), and thereafter, we iterate, in each step removing the first point \( x \) from \( U \), applying all generators \( g \in G \) to \( x \) and adding any new points to both \( T \) and \( U \).
The computation terminates when there are no unprocessed points (line 2).
As an example of how the algorithm works, let \( X = \{1, 2, 3, 4, 5\}, x_0 = 2, G = \{g_1, g_2\} \) and \( f \) be given by the following table:
| | 1 | 2 | 3 | 4 | 5 |
|---|---|---|---|---|---|
| \( x \cdot g_1 \) | 1 | 3 | 2 | 4 | 5 |
| \( x \cdot g_2 \) | 5 | 2 | 4 | 3 | 1 |
The following table shows the steps in evaluating the orbit \( T \) of \( x_0 \):
| Step | 1 | 2 | 3 | 4 |
|------|---|---|---|---|
| \( T \) | \{2\} | \{2, 3\} | \{2, 3, 4\} | \{2, 3, 4\} |
| \( U \) | \{2\} | \{3\} | \{4\} | \emptyset |
### 6.1.1. The parallel orbit skeleton
At first glance, it may seem that the sequential Orbit algorithm can be parallelised by carrying out step 5 for all generators and all points in \( U \) independently. However, synchronisation is required to avoid duplicates (steps 6–8), and this requires communication among the tasks that apply generators to different elements of \( U \).
Our Parallel Orbit skeleton uses two different kinds of threads:
- **Hash server** threads, which use hash tables to determine which points are duplicates, and
- **Worker** threads that obtain chunks of points from hash servers and apply generators to them, so producing new points.
This means that we have explicit two-way communication between each hash server and each worker, but no communication between different hash servers or different workers. Where there are multiple hash servers, we use a distribution hash function to decide which hash server is responsible for storing each point. Figures 17 and 18 show the pseudocode for a worker thread and a hash server thread, respectively. Initially, all hash servers and input queues are empty, and all workers are idle. We first feed a single point into one of the hash servers, which immediately creates some work. This hash server then sends this work to a worker which, in turn, produces more points that are then sent to the corresponding hash servers, bootstrapping the computation. The number of points in the system increases and, eventually, all input queues become completely full, and the system as a whole becomes busy. Towards the end of the computation, fewer new points are discovered. Eventually, all generators will have been applied to all points and the computation terminates. Now, all workers are idle again and the hash servers collectively know all the points in the orbit. In our GAP implementation, we maintain one channel for the input queue of each hash server. Any worker
Data: the set $G$, the action function $X \times G \to X$, the number $h$ of hash servers and a uniform distribution hash function $f : X \to \{1, \ldots, h\}$
while true do
get a chunk $C$ of points;
$R :=$ a list of length $h$ of empty lists;
for $x \in C$ do
for $y \in G$ do
$y := x \cdot y$;
append $y$ to $R[f(y)]$;
for $j \in \{1, \ldots, h\}$ do
schedule sending $R[j]$ to hash server $j$;
Figure 17. Parallel Orbit worker pseudocode.
Data: A chunk size $s$
initialize a hash table $T$;
while true do
get a chunk $C$ of points from our input queue;
for $x \in C$ do
if $x \notin T$ then
add $x$ to $T$;
if at least $s$ points in $T$ are unscheduled then
schedule a chunk of size $s$ points for some worker;
if there are unscheduled points in $T$ then
schedule a chunk of size $< s$ points for some worker;
Figure 18. Parallel Orbit hash server pseudocode.
can write to any of these channels. We also maintain a single shared channel for the work queue. Because of the chunking mechanism, this is not a bottleneck.
6.1.2. Performance results. This section presents a preliminary evaluation of our Parallel Orbit skeleton on a shared-memory machine consisting of four AMD Opteron 6376 processors, each comprising 16 cores (giving us a 64-core system). Each core runs at 1.4 GHz and has 16 Kb of private L1 data cache. In addition, each processor has 8×64 Kb of L1 instruction caches and 8×2 Mb of L2 caches (shared between two cores) and 2×8 Mb of L3 caches (shared between eight cores). As an instance of an Orbit enumeration, we consider the sporadic finite simple Harada-Norton group in its 760-dimensional representation over the field with two elements, acting on vectors. The set $X$ of all possible points consists of about $10^{228}$ elements. The size of the orbit is 1.4 million points, and we are using ten random generators. On each figure, we consider mean speedups over five runs of the same instance. We have also added error bars in the cases where error margin was notable.
The sequential runtime of this orbit calculation on our test machine was 250 s. Additional experiments with larger instances and a sequential runtime of 1514 s produced similar runtimes and speedups. Figure 19 shows the absolute speedups over the sequential Orbit algorithm that we have obtained with 1 to 4 hash server threads, and 1 to 32 worker threads. We can observe good speedups, up to 23 with four hash servers and 28 worker threads (so, using 32 cores in total). We can also observe that about seven workers produce enough points to fully load one hash server. Also, for a fixed number of hash servers $h$, the speedups grow almost linearly with the increase in the number of workers. Once the number of workers reaches $7h$, we do not observe further increase in speedups. Note further that, on our testbed, it is not possible to obtain linear speedups, because of frequency scaling of cores when multiple cores are used at the same time. Therefore, the figure also shows the ideal speedups that could be obtained. The ideal speedup on $n$ cores was estimated by taking the runtime of the sequential Orbit algorithm in one thread\footnote{With $n - 1$ parallel threads executing a dummy spin locking function that does not use any memory, to scale down the frequency.} and then dividing it by $n$. We obtain similar results on other shared-memory architectures [5].
To test the scalability further, Figure 20 shows the speedups obtained on the same machine with seven and eight hash servers, and up to 57 workers, therefore using all 64 cores in the system. The speedups steadily increase in both cases from 18 to 35 (with seven hash servers) and 36 (with eight hash servers). The fact that the speedup obtained is suboptimal can be attributed both to the problem itself, because in these cases, the bootstrapping and finishing phases (where not enough points are available to utilise all workers) become dominant, and to the hardware, because there are only 16 memory channels, which limits the memory access when more hash servers and workers are used.
6.2. Hecke algebras in SGP2
SGP2 has been used to compute matrices of invariant bilinear forms in the representation theory of Hecke algebras. In the following, we will only sketch this case study and present the main performance results; the reader is referred to [6] for a detailed report and to [62] for background on the problem.
6.2.1. Problem: finding invariant bilinear forms. We consider Hecke algebras $\mathcal{H}$ of exceptional type $E_m$ ($m = 6, 7, 8$), generated by $m$ generators $T_1, \ldots, T_m$.
An $n$-dimensional representation $\rho$ of $\mathcal{H}$ is an $R$-algebra homomorphism from $\mathcal{H}$ to $M_n(R)$, the $R$-algebra of $n \times n$ matrices over $R = \mathbb{Z}[x, x^{-1}]$, the ring of Laurent polynomials in indeterminate $x$. Being a homomorphism, $\rho$ is uniquely determined by the images of the generators $T_1, \ldots, T_m$. Thus, each $n$-dimensional representation $\rho$ can be succinctly expressed by $n \times n$ matrices $\rho(T_1), \ldots, \rho(T_m)$ of polynomials; in a slight abuse of terminology, we call the $\rho(T_i)$ generators.
For any given $\rho$, there exists a non-trivial symmetric matrix $Q \in M_n(R)$ such that
$$Q \cdot \rho(T_i) = \rho(T_i)^T \cdot Q$$
for all generators $\rho(T_i)$. We call $Q$ the matrix of an invariant bilinear form. It suffices to compute $Q$ for so-called cell representations $\rho$, of which there are only finitely many. Depending on $\rho$, finding the invariant bilinear form $Q$ may require substantial computation. The table below lists the number of cell representations $\rho$, the range of dimensions of $\rho$ and the range of degree bounds of Laurent polynomials in $Q$. These numbers, and hence, the difficulty of the problem, vary by several orders of magnitude reflecting the extreme irregularity of symbolic computations.
| Hecke algebra type | $E_6$ | $E_7$ | $E_8$ |
|-------------------|-------|-------|-------|
| Cell representations | 25 | 60 | 112 |
| Dimension of reps | 6–90 | 7–512 | 8–7168|
| Degree of Laurent polys | 29–54 | 45–95 | 65–185|
6.2.2. Sequential algorithm. In principle, $Q$ can be computed by viewing Equation (1) as a system of linear equations and solving for the entries of $Q$. However, solving linear systems over $\mathbb{Z}[x, x^{-1}]$ is too expensive to obtain solutions for high dimensional representations. Instead, we solve the problem by interpolation. We view each entry of $Q$ as a Laurent polynomial with $u - l + 1$ unknown coefficients, where $l$ and $u$ are the lower and upper degree bounds of polynomials in $Q$. Solving Equation (1) at $u - l + 1$ data points will provide enough information to compute the unknown coefficients by solving linear systems over the rationals instead of $\mathbb{Z}[x, x^{-1}]$. To avoid computing with very large rational numbers (because of polynomials of high degree), we solve homomorphic images of Equation (1) modulo small primes and use the Chinese Remainder Theorem to recover the rational values. The algorithm takes as input $m$ generators $\rho(T_i)$ of dimension $n$, lower and upper degree bounds $l$ and $u$ and a finite set of small primes $P$. From the degree bounds, we construct a set $V_{lu}$ of $u - l + 1$ small integers to be used as data points for interpolation.
The algorithm runs in three phases:
1. For all $p \in P$ and $v \in V_{lu}$, GENERATE a modular interpolated solution $Q_{vp}$ of (1) by instantiating the unknown $x$ with $v$ and solving the resulting system modulo $p$.
2. REDUCE the modular matrices $Q_{vp}$ to a matrix of Laurent polynomials $Q$ as follows. First, for all $v \in V_{lu}$, construct a rational interpolated solution $Q_v$ of (1) by Chinese remaindering. Second, construct each Laurent polynomial $q_{ij}$ in $Q$ by gathering the $(i, j)$-entries of all $Q_v$ and solving a rational linear system for the coefficients $q_{ij}$. Because $Q$ is symmetric, there are $(n + 1)n/2$ such systems, each of dimension $u - l + 1$.
3. For all generators $\rho(T_i)$, CHECK that the resulting $Q$ satisfies (1) over $\mathbb{Z}[x, x^{-1}]$.
The theory of Hecke algebras admits a particularly efficient way to GENERATE $Q_{vp}$. Instead of solving a linear system, the rows of $Q_{vp}$ can be computed by spinning a basis (i.e. multiplying a basis vector with certain matrix products).
6.2.3. Parallel algorithm. Each of the three phases of the sequential algorithm to compute $Q$ contains significant amounts of parallelism. Figure 21 shows the parallel structure, where $n$ is the dimension of $Q$ and the $m$ generators $\rho(T_i)$, $l$ and $u$ are lower and upper degree bounds of the
Laurent polynomials and $P$ is the set of primes used in the GENERATE phase. Note that there is a sequential duplicate filtering phase at the beginning of the REDUCE phase.
6.2.4. Evaluation. A systematic evaluation of the SymGridPar2 implementation on all cell representations of E$_6$, E$_7$ and the smallest 16 representations of E$_8$ is reported in [6]. Here, we present performance results for E$_8$ on two different architectures: a 48 core NUMA server (Cantor), and 1024 cores (distributed over 32 nodes) of the HECToR supercomputer [61].
Runtime. Figure 22 plots the runtime of SGP2 with 40 GAP workers on Cantor. It also plots the total work, that is, the cumulative runtime of all tasks, and the time spent in the sequential part of the REDUCE phase. The reported times reflect single experiments as a statistically significant number of repetitions would be prohibitively expensive. We observe that the amount of (total and sequential) work and the parallel runtime appear to be correlated, yet oscillate noisily between representations. This is due to the high degree of irregularity in the number of REDUCE tasks.
Speedup. Figure 23 plots the estimated speedup, computed as total work divided by parallel runtime, on Cantor and on two configurations of HECToR (for representations 11 to 15). On Cantor, speedups stabilise just below 40, which is the maximum expected with 40 GAP workers. With
Figure 21. Structure of parallel algorithm for computing $Q$.
Figure 22. SymGridPar2 computing Hecke algebra $Q$ runtimes (NUMA and HPC).
Figure 23. SymGridPar2 computing Hecke algebra $Q$ speedups (NUMA and HPC).
HECToR’s 4 node configuration (128 cores, running 115 GAP workers). SGP2 achieves speedups of around 80; yet with the 32 node configuration (1024 cores, 992 GAP workers), speedup hovers around 160; representation 11 is the exception achieving a speedup of 548.
7. CONCLUSION
We have provided a systematic description of HPC-GAP, a thorough re-engineering of the GAP computational algebra system for the 21st Century, which incorporates mechanisms to deal with parallelism at the multicore, distributed cluster and HPC levels. At the GAP5 base level, this has involved major work to remove single-threaded assumptions in the runtime system and libraries. We have developed MPIGAP to exploit ubiquitous small clusters and designed and implemented a highly sophisticated coordination system for HPC systems, SymGridPar2, which uses parallel Haskell to coordinate large-scale GAP computations. The result is the most advanced, free and open source, computational algebra system currently in existence.
We evaluate the scalability and performance of the HPC-GAP components using a common SumEuler benchmark on a range of systems from small-scale multicores to HPC platforms. Key results include showing that while the GAP5 concurrency overheads are typically between 10% and 40% (Section 5.1); these overheads are almost completely ameliorated on medium-scale architectures (Section 5.4); we demonstrate good weak scaling to 32K cores on the HECToR HPC (Section 5.3). The Orbit and Hecke Algebra case studies convincingly demonstrate the capability of HPC-GAP to manage large and highly irregular algebraic computations, for example, achieving a maximum speedup of 548 using 992 GAP instances on 1024 cores of the HECToR HPC (Section 6). The datasets for these experiments are available in [2].
There is enormous potential for further exploitation of this work. GAP5 is gaining widespread adoption in the algebraic computation community. We are exploring alternate parallelism options for GAP, like a PGAS model with UPC-GAP. Moreover, reliability is increasingly an issue at HPC scale, and here, the statelessness of many algebraic computations means failed computations can be safely recomputed. The HdP-H-RS extension tracks the location of computations and reinstates any that may have failed [60, 63].
ACKNOWLEDGEMENTS
This work has been partially supported by EPSRC grants HPC-GAP (EP/G05553X), AJITPar (EP/L000687/1) and by EU grants ICT-288570 (ParaPhrase), ICT 287510 (RELEASE), ICT-644235 (RePhrase), by the EU COST Action IC1202: Timing Analysis On Code-Level (TACLe).
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Community in Postmodern Philosophy with an emphasis on the work of Jean-Luc Nancy
M.Phil. thesis
Gender Studies
Candidate:
Oleg Domanov
Supervisors:
Professor Linda Fisher
Professor Gabriele Griffin
Budapest, Novosibirsk 2004
Submission date: 1 December 2004
Award date: 9 March 2005
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How else can one write but of those things which one does not know, or knows badly? It is precisely there that we imagine having something to say.
Gilles Deleuze, *Difference and Repetition*, p. xxi
We write before knowing what to say and how to say it, and in order to find out, if possible.
Jean-François Lyotard, *The Postmodern Explained for Children*, p. 119
# TABLE OF CONTENTS
**INTRODUCTION..................................................................................................................5**
**PRELIMINARY EXPOSITION: BEING, TIME, LANGUAGE...........................................10**
- 1.1 *Being* ..........................................................................................................................10
- 1.2 *Time* ............................................................................................................................15
- 1.3 *Language* .....................................................................................................................20
**DIFFÉRANCE, SUBJECTIVITY, AND LANGUAGE COMMUNITY........................................26**
- 2.1 *Différance and the critique of presence* ......................................................................26
- 2.2 *Two gestures of differentiation in différance* ...............................................................30
- 2.3 *Joining two times of diachrony in différance* ..............................................................34
- 2.4 *Subjectivity: Derrida’s approach* ................................................................................39
- 2.5 *Subjects of involvement and engagement* ....................................................................43
- 2.6 *Engagement and the place of the other* ......................................................................47
**JEAN-LUC NANCY: COMMUNITY OF THE ORIGIN OF COMMUNITY .........................55**
- 3.1 *Community as distribution of singularities. Spatial metaphor* .................................55
- Community interrupted or lost ..........................................................................................59
- Singularity .........................................................................................................................62
- “There is” and the opening of the world and sense .........................................................67
- Sense and truth ..................................................................................................................68
- Citizen and subject. Establishing communal ties .............................................................70
- The distributive model of community .............................................................................75
- 3.2 *The ambiguity of the distributive model* ....................................................................76
- Multiplicity and temporality ............................................................................................76
- Separation versus distribution ..........................................................................................79
- Saying and Nancy’s ontology ..........................................................................................82
- Saying and establishing communal ties ..........................................................................90
- 3.3 *Literature/writing* .......................................................................................................96
- The ontological model of writing ....................................................................................96
- Writing as exposition and addressing the other .............................................................100
CONCLUSIONS ........................................................................................................... 105
REFERENCES ............................................................................................................ 112
Introduction
In the postmodern condition, when values, norms, meanings grow uncertain and lose their ground, when morality, politics, family, and many other social institutions inherited from the so-called modernity cease to serve us one by one, community itself becomes problematic. On what basis can it be erected? God, humanity, nature—everything has become suspect. Perhaps only one possibility remains at our disposal: to take the very lack of consistency as a basis for community. Postmodern philosophy attempts this. It seeks a third path between myth and nihilism, that is, between traditional unitary community and the absence of community. This way is claimed to be found in the community undoing itself, or, better, in the very undoing of community. The absence of myth (as well as a community based on it) is thought, contrary to nihilism, not as lack and nothing. The very rejection of myth is a work—if I can use this word here—of community. The impossibility of immanence and closed up identity is supposed to be a basis for an understanding of community that obviates the abysses of both totalitarianism and nihilism. To put it simply, the impossibility of the One, of the Absolute leads immediately to multiplicity and thus to irreducible community.
Therefore, instead of the alternative between eternal sense and non-sense, this philosophy relies on the undoing of sense and seeks therein all sense of community. Ontology comes to be crucial here, since Being—at least from Heidegger on—is conceived as having a similar structure of undoing: Being presents beings¹ (exists them in the transitive sense of the verb) but does so in such a way that lays no firm ground for their continuous existence. Thus the very Being of community justifies its undoing. The sense of
¹ Terms “Being” and “being” here correspond to German *Sein* and *Seiende*. For a more detailed discussion see the note on p. 10.
community is sought, therefore, in the ontology or in the very fact that "there is something," that Being "is," or existence "exists." The structures of *there is* and the undoing of community are strictly parallel.
The conviction behind this is that the undoing—which is the undoing of the founding myth of community—might provide a sufficient ground for a community to come. A deeper still conviction is that ontology or understanding of "how things truly are" is sufficient for the justification of community. But what does "sufficient" mean? Obviously, it has ethical connotations\(^2\). Undoing or interruption are associated with anti-totalitarianism, multiplicity of voices or cultures, recognition of the other, genuinely ethical attitude toward the other, and so on. The whole project is a response to challenges of the XX century. However, is undoing sufficient? Is such recognition of the other's freedom sufficient for community? This is the question of the present work. I shall argue that there is never merely undoing but always undoing endowed with a meaning. To put it another way, the interruption of sense does make sense by itself but this sense may be different. There is never one sense of *there is* (and any philosophy trying to find out this one sense tends to conceive of it as a pure negativity, death), but different senses. I shall argue as well that the latter have the structure of "for the sake of..." which means that not the undoing itself is important for community but that for the sake of which it is performed.
This opens, I believe, a sphere of meanings different from that of signification\(^3\). Although this sphere is to large extent obscure to me, I can discern two meanings possibly
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\(^2\) Such terms as "sufficient" or "proper" are used here with all their vagueness. They cannot be defined in this work because of their belonging to its ethical project. They point obviously to what I, the author, consider being sufficient and proper (or rather what I consider insufficient and improper). No objective meaning is possible in this case. In this respect the following discussion is prescriptive rather than descriptive.
\(^3\) Perhaps Emmanuel Levinas was the first who discovered this sphere and distinguished it from a
situated in it. The first has to do with the multiplicity of meanings supposedly caused by the impossibility of immanence. I wrote “supposedly” because strictly speaking multiplicity is not an effect of undoing, it is rather its reason: undoing is performed for the sake of multiplicity of meanings. In a sense, this is a life for the future, novelty, always-new experience. This is the openness toward the coming. It is this openness that is frequently interpreted as the openness toward the other able to provide a basis for proper ethical relationships (see, for example, Bauman 1993). This picture is in no way totalitarian nor purely nihilistic (in the Nietzschean sense of overcoming nihilism by saying “Yes” to the world). And subjectivity of this sort—if we can still speak of subjectivity—is not the autonomous self-sufficient subjectivity of the classical philosophy. However, is this subject really concerned with others? Are this acceptance and this “Yes” not rather at bottom just “taking circumstances into account?” Is it not the utilising of others (without however imposing anything on them) with the aim to sustain oneself in the ceaseless movement of becoming? This makes others free, for sure. Moreover, this implies a will to accept them as free (ironically, first of all free of me). But this freeing is rather abandonment and breakup. Perhaps proper social relationships need another structure.
The other meaning in the sphere of *for the sake of* is the offering of community: on the one hand, doing-undoing seems to be the only proper tie with a free being; on the other hand, it has to be performed not for the sake of itself but for others. Doing is necessary because it is the establishing of ties, but undoing is also necessary because these are ties with a free being, free to the point where the other can decline the offer of community. In still another register, undoing presupposes doing—including the opening of doing and undoing themselves. The latter in the following text is interpreted as laying down foundation, or inaugurating language, or a will to have some language and foundation pure negativity.
(which all are undone in the very event of their laying down). The two meanings I have just spoken of are the meanings of the play of doing and undoing. (Is it thus a play at all?) Is it as such purposeless? In this work the two meanings of the doing-undoing are considered. It might be for the sake of 1) maintaining a flowing identity, “Being-there,” or even “Being human”, or 2) establishing ties with others by offering them a common Being (and thus sacrificing one’s own “Being-there” or even “Being human” for the common Being).
How can we reveal these two meanings? In the philosophy I am concerned with the second one appears under the name of writing or literature. It has, however, a contradictory structure comprising both above-mentioned meanings. To clarify this structure I take as an example two of Jean-Luc Nancy’s works devoted entirely to the concepts of community and sense (which is always common, according to Nancy), namely *The Inoperative Community* (Nancy 1991) and *The Sense of the World* (Nancy 1997)\(^4\). In these books writing is two-dimensional. One dimension is the production of meaning and as such is not actually discernible from reading in its hermeneutical understanding. It is responsible for the multiplicity of meanings and plurality of voices in any particular text. The other
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\(^4\) This work was originally planned as a critique of community and ethics in Nancy’s writing. However, this critique demanded a significant preparatory work. Consequently, what was supposed to be an introduction, orientation, and provision of preliminary definitions turned into the work in itself, and Nancy could no longer hold the main place within it. Although his concept still remains a privileged example, I do not insist that it exhausts the theme or may be counted as *the* concept of postmodern community. It is worth noting, nevertheless, that in a recent *Encyclopaedia of Postmodernism* (2001) Nancy is mentioned among three authors (the other being Giorgio Agamben and Maurice Blanchot) who contributed most significantly into the postmodern understanding of community. Without denying the necessity of a more attentive analysis, I claim that the conflict discussed throughout the present work is one of the most important in the postmodern understanding of community, ethics, and—I would say—humanity. It haunts this understanding at least as a problem to be solved.
dimension is what Nancy calls sharing, that is, offering and establishing communal ties performed by any act of writing. In general terms, they correspond to two functions of language: 1) cognitive or creative, consisting in the constituting of the symbolic world to live in, and 2) phatic, consisting in addressing others and opening for their addressing. However, Nancy tends to identify the two and conceive of the world’s multiplicity as already offering and establishing ties. But in fact they are not merely incommensurable. They imply different ethical attitudes. At least this is what I hope to demonstrate.
Therefore, the following text is divided on three chapters. Chapter 1 provides a preliminary view on Being, time, language, and their relationships as they are used throughout the work. Chapter 2 applies this vision to Derrida’s understanding of différence and subjectivity and makes conclusions concerning possible theory of community that it entails. Chapter 3, final and largest, is devoted to a detailed analysis of Nancy’s theory of community as it is presented in his two mentioned books.
Chapter 1
Preliminary Exposition: Being, Time, Language
This introductory chapter is devoted to the background of the postmodern understanding of community. It describes the context wherein this understanding comes up and develops. The discussion of some topics in this chapter might sometimes look redundant or even irrelevant. However, it is in fact the introducing of concepts and structures that I shall apply later. Only at that time will the purpose of their discussion be fully recognised. In addition, the reader has to keep in mind that these concepts and structures themselves are rather preliminary. These are, so to say, starting conditions destined to be transformed, sometimes considerably. Here, in this general exposition, I shall start with ontology, the role of temporality in it and proceed to their interpretation from the viewpoint of language. This latter interpretation will be a leading one throughout the rest of the work.
1.1 Being
I shall call the ontology I am about to describe the ontology of event. Along with Nancy, Levinas and others, I rely on Heidegger’s distinction between *Sein* and *Seiende*\(^5\), so
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\(^5\) Translating these terms into English is not easy because separately both of them are usually rendered as “being”. Translators have offered different solutions such as writing the former with a capital “B” or replacing them with the pair existing-existent but all of these seem barely appropriate. Throughout the following text I translate them as Being (always capitalised) and entity(ies) (or being(s) in phrases such as “human beings”). This is the variant that John Macquarrie and Edward Robinson settled on in their
called ontological difference. I would present Heidegger’s argument as follows. In *Being and Time* Heidegger seeks the meaning of Being. He asks, “What does it mean to be?” and goes on to the analysis of a specific entity which has in a way direct access to Being because “in its Being the Being itself is an issue.” The last formula is the definition of entity that Heidegger calls Dasein. Being is always intentional, or directional, or “Being-towards.” It is toward entity and this difference between Being and entity is irreducible. It would be wrong to speak of Being in terms of entities. The question “What is the Being of Being itself?” cannot be answered in this manner. Being does not have another “Being-toward-Being,” with this process recursively going to infinity. Thus, there must be an entity whose Being is not intentional and which, consequently, bears its meaning in itself. This “entity” is Dasein grasped as event, act, process, all these terms being not fully appropriate. Late Heidegger speaks of event (*Ereignis*) viewed as the coming of an entity to Being, i.e. as a sort of act that makes what it is—the entity—of an entity. Thus the event of Being is the entity’s becoming what it is. However, this sentence sounds ambiguous. Does the entity mentioned become entity as such (“entitiness”), entity of such and such a type, or a concrete singular entity? But in fact Heidegger’s concern goes deeper. He asks about the happening of all these distinctions among which the principal one is between Being and entities. In this sense, as Giorgio Agamben remarks in *Language and Death* (Agamben 1991), the term Dasein should be better translated not as Being-there, but as Being-the-there, i.e. Being the very place where Being itself happens (Heidegger himself points out in *Zollikoner Seminars* (Heidegger 1992, 87) that the French equivalent for Dasein should be *être-le-là* instead of the routinely accepted *être-là*).
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translation of *Being and Time* (see their notes 1 on pages 19 and 22 in Heidegger (1962)). In cases of obscurity I use the most direct solution—German spelling. The only exceptions are quotations, which I left unchanged.
Heidegger proceeds to the description of event in terms of ability-to-be (Seinkönnen)—Dasein’s ability to realise, to perform, to participate in the event of Being. By doing so, Dasein gains, so to say, immediate access to Being, which Heidegger calls understanding (Verstehen). In the final analysis, every entity’s meaning refers, through a series of relations, to Dasein’s abilities-to-be. Dasein therefore is the very Being of meanings discovered in the world. According to Heidegger in the act of understanding (which is to say, in Dasein’s Being, since this Being is essentially understanding) the totality of signification, which he calls significance or meaningfulness (Bedeutsamkeit), is disclosed to Dasein. By its own Being Dasein makes meanings as references to its abilities-to-be possible. Understanding is Dasein’s familiarity with relations that allow these references:
Dasein, in its familiarity with significance, is the ontical condition for the possibility of discovering entities which are encountered in a world with involvement (readiness-to-hand) as their kind of Being, and which can thus make themselves known as they are in themselves [in seinem An-sich] (Heidegger 1962, 120).
However, meanings in understanding are not explicit. In order for that to happen, the interpretation (Auslegung) is necessary. It implies a fore-structure (Vor-Struktur comprising Vorhaben, Vorsicht, and Vorgriff—fore-having, fore-seeing, and fore-concept), which Dasein uses to grasp reality, and only through which meanings become articulated. Therefore, entity’s meaning—its Being-something—becomes possible due to the horizon of fore-structures thrown ahead by understanding. And eventually ontology is possible only as interpretation or hermeneutics. This tough tie between Being and meaning is not accidental. It stems from Heidegger’s project as such: to inquire into the meaning of Being.
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6 Macquarrie and Robinson’s translation is “potentiality-for-Being”.
Being therefore is always meaningful (which the word ontology—discourse on Being—already indicates). Each time when there is something, there is a meaning, and there is understanding.
Here we find two dimensions of Being important for the following. On the one hand, the event of Being consists in laying down a horizon(s) allowing any meaning whatsoever to appear at all. On the other hand, the event opens up the very structure of meaningfulness and in this sense consists in the very fact that there is meaning. That Dasein is Being-the-there means precisely that it is able to be in such a way that there are meanings. This allows Heidegger to say that there is a voice of Being and that it is a response to this voice that opens up the possibility of Being at all. Therefore, event “contains” the fact of meaningfulness, its concrete (namely, horizontal) structure and a set of particular horizons. Logically, meaningfulness does not necessarily implies horizons. Moreover, as we shall see, the postmodern theory of community is claimed to rest exclusively on the fact of Being without any further qualifications. It is not easy, however, to keep the fact of meaningfulness apart from the structure of meaningfulness. For instance, Nancy sometimes fails to do so, as I hope to demonstrate.
Another important trait of Being according to Heidegger is its mineness (Jemeinigkeit—in each case belonging to me\(^7\)). Being thus always implies someone (since it must be performed by someone). It is a human being (in the form of consciousness, Dasein, etc.) who performs the event of Being. One can even say that human being is no more than this performing. Performing designates the fact of Being’s always happening to me. This is not to claim that Being is in any way “created” by subjectivity, however one understands the latter. Rather the event is a “happening” wherein everything—subject, object, their relation, and the “happening” itself—happens. There is so far no activity or
\(^7\)I do not know how to avoid personal pronouns in the contexts such as this one. To distinguish
passivity of the subject. The event precedes both, or, as Levinas put it, we are dealing with the “passivity, more passive than any passivity.” Accordingly, performing does not denote any deed whatsoever that lies within the sphere of the subject’s power. The subject, if any, is constituted precisely in the same event, and performing is its very constitution and definition.
To sum up, three points of Heidegger’s approach to Being are essential for the following exposition. (1) The relational character of Being: it is a relational part of the structure, the other part of which is entity. It is a relation (rather direction, “towardness”) toward entity\(^8\). (2) The specific character of the “Being of Being”. When there is something, there is at the same time the \textit{there is} itself, that is, the very fact of Being or, more exactly, the event of Being. This indicates the singularity of event and we shall see, for example, that this is the main meaning of singularity in Nancy’s works. As I have already pointed out, \textit{there is} has the double structure of the happening of meanings and meaningfulness (or of the signified and significance). (3) The mineness of Being, its in each case happening to \textit{me}.
Heidegger’s fundamental ontology found in \textit{Being and Time} is extremely elaborate and rich in detail. For my purposes, a more schematic variant will suffice—the one that I shall call the ontology of event. This ontology deals with the Being of meaningful entities (which means all entities whatsoever). It conceives of Being transitively as Being towards entities. The latter, in turn, is thought as the event of entities’ (and Being’s itself) coming to Being. The event is always \textit{mine}, always happens to \textit{me}. Beside that, meanings in the ontology of event have the horizontal structure, which means that they are grounded in some embracing system of meaningfulness or significance. What exactly the structure of meaningfulness is—Dasein’s abilities-to-be, founding actions, referential system of signs,
\footnote{them from the pronouns referring to myself, the author, I shall write them in italics.}
narratives, or whatever else—is not specified. But the dependence of meanings on horizons makes them potentially plural in the sense that the same entity may be engaged in various contexts and thereby has a number of meanings at the same time. Although meaningfulness in a way precedes meanings, its structure comes up in the same event. The event is the absolute beginning in the sense that everything it comprises is brought forth in the event itself. This, however, does not prevent the event from having internal temporal structure.
Such is the ontology of event, and I have to show in the following text that it is the implicit (and at times explicit) ontology of the postmodern theory of community. Comprehended as event, Being is inseparable from time. This connection is more complicated than a simple succession of events in the course of time, and we need to discuss it carefully.
1.2 Time
Time belongs perhaps to the deepest structures of human experience and Being. It is not by chance that Husserl saw in temporality the basic structure of consciousness. Heidegger claimed that Being is in the final analysis time. Levinas spoke of diachrony and time as fundamental ties with the other. Psychoanalysis keeps talking about repetition when discussing the most fundamental structures of the human psyche. As for the ontology of event, temporality cannot but play a significant role in it. Indeed, event is the coming to Being, movement from “there was not” to “there is” (and to “there will not be”). I shall rely on the logic of this movement as it appears in Husserl’s *On the Phenomenology of the Consciousness of Internal Time* (1991). Although postmodern philosophy cannot join the whole Husserlian project of the science of consciousness, it is to a large extent indebted to his analysis of temporality. Temporal structures discovered by Husserl—or, more
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8 Perhaps “intentionality” would be more precise.
precisely, the structure of diachrony which Levinas recognised in *Zeitbewußtsein*—often provide a conceptual scheme for grasping signification, significance, and the relational network of the signified. This is why I devote a relatively large space to the depiction of these structures.
In *The Phenomenology of the Consciousness of Internal Time* Husserl examines intentional structures in the experience of time as such. He starts with considering enduring objects and takes a tone or melody as an example. According to Husserl, an enduring object is experienced as a unity of primary impression (*Ur'impression*) in the now (*Jetzt*)-point and primal memory—retention (and also primal anticipation—protention; but it does not play essential role in Husserl’s book). Retention holds the now that has just been. This does not mean that retention modifies the previous now through, for instance, transposing the actual now into memory:
Retention is not a modification in which impressional data are really preserved, only in modified form: on the contrary, it is an intentionality—indeed, an intentionality with a specific character of its own. When a primal datum, a new phase, emerges, the preceding phase does not vanish but is ‘kept in grip’ (that is to say, precisely ‘retained’); and thanks to this retention, a looking-back at what has elapsed is possible. The retention itself is not a looking-back that makes the elapsed phase into an object: while I have the elapsed phase in my grip, I live through the present phase, take it—thanks to retention—‘in addition’ to the elapsed phase; and I am directed towards what is coming (in a protention) (Husserl 1991, 122).
There is therefore the irreducible temporal gap between the now and its retention. The retained now is by no means “the now that was actual in the past.” Nor is it this now’s modification. In a certain way, the retention retains its own now that has been, different
from the now that has been “in reality.” This duplication of nows leads to diachrony that I shall discuss a little later. Furthermore, retention holds not only the now that has been but also all previous retentions:
...each later retention is not only continual modification that has arisen from primal impression; each is also continual modification of all earlier continuous modifications of that same initial point (Husserl 1991, 31).
As Husserl put it, we have a tail of retentions each holding in itself modifications of all previous retentions. The head of this tail is the primal impression temporally separated from the tail itself. In fact, according to Husserl, any constitution of objectivity—as well as self-consciousness—becomes possible thanks to this feature of retention. Any meaning in consciousness is derived from the relationships between temporal phases kept by retention. The latter thus is a referential system that determines all meanings within consciousness. Retention contains everything presented to consciousness, whereas primal impression appears as an indefinite and purely negative source-point separated from the “field of meanings” but guaranteeing somehow its unity and consistency (indeed, it seems to be consistent only because all retentions have a common source in the primal impression). Levinas reveals here two structurally different temporalities (Levinas 1996). One is the succession of temporal phases that retention holds in itself. Although temporally separated, the phases are found in retention simultaneously, “in a flash.” This precisely points to another present not belonging to the line of succession. It is the presence of the succession itself. More exactly, it points to the time of the temporal gap between retention and primal impression. This time has perhaps only two dimensions—“what is” (with the whole differential structure of the temporally separated phases) and “what has been” (with the purely negative source-point of *Ur*impression). It is this structure that Levinas calls diachrony (Levinas 1998).
The status of primal impression in Husserl is ambiguous. On the one hand, each now consists of primal impression and primal retention. On the other hand, retention retains the now that has been (along with past retentions). But the primal impression of this past now, as we have seen, is not a modification of actual impression (having actually been in the past). While the latter does not have any determinations except its having-been, the former is divided in itself and is, in fact, the differentiated experience. The actual impression therefore plays the role of the origin (source-point) but its indefiniteness allows one to consider it as a pure negation of the retentional tail—something whence all retentions originate but which is not accessible to consciousness and has thus no further determinations. One can infer that the only function of primal impression in the Husserlian analysis is to designate the coming-to-Being, the originating. And this is the point of ambiguity. Should we understand the primal impression positively as production or negatively as a pure negation of “what is”? In a certain way, it is this ambiguity that is in the focus of the present work. The philosophy that I discuss transposes the Husserlian structure into the field of ontology (in doing so it relies on Heidegger) and then of community (not always, though, it is a transformation of the Husserlian problematic; one can find a similar structure elsewhere, for instance, in Saussure’s linguistics or in psychoanalysis). But the ambiguity remains and turns into the contradiction between community as pure negation of founding myth and as the negation “for the sake of something”.
The shift from temporality toward ontological problematic is made through the notion of *there is*. It signifies the fact of existence, the fact that there is something (*Daßsein* in its difference from *Wassein*). This fact, however, is grasped precisely as happening. It points therefore to the ontology of event, since event is coming-to-Being. What is the temporality of *there is*? In the ontology of event “there is something” amounts to “there is something meaningful”—simply because any “something” is meaningful. This implies the
disclosedness of meaningfulness or the relational network that makes meanings possible. In this case we face two kinds of precedence. On the one hand, horizons of fore-structures precede meanings, as long as the latter are acquired only through the reference to horizons. On the other hand, the very horizontal structure of meaningfulness must be disclosed beforehand. This latter disclosedness is irreducibly temporal, since it is the very event of bringing forth the former, i.e. the event of the emerging of meanings along with founding horizons. By contrast, horizons and meanings can be treated as in a certain sense simultaneous. Even when meaning is thought of as a result of a movement along a referential chain of signifiers, this chain is "kept in grip" altogether as a whole. In this last case we have the structure of diachrony. Indeed, there is a time of succession of the referential chain whose links, however, are presented simultaneously (as if held by retention or memory), and there is a time of the succession itself (as if from a primal source-point). I have to note, nevertheless, that the temporality of event is not necessarily diachronical. The horizontal structure might equally well be non-temporal, as, for example, in cases of the model of founding actions or that of figure-background. In this sense the ontology of event is supposed to differentiate the logic of meaning and of its appearing. The first might be temporal or non-temporal but it is always structured as something referring to something else (cf. Heidegger's definition of meaning as "something as something" [etwas als etwas] (Heidegger 1962, 32)). That which is referred to may reside in the past, present, or future but in any event temporality seems redundant here. This is to such an extent so, that this logic of meaning can be reduced to the "eternal presence." At the same time, the ontology of event aims to preserve precisely the irreducible time of happening, the time wherein the event of Being happens. Postmodern community is claimed to belong entirely to the second time and in this sense we might have kept to the discussion of it alone. I do not do so because it is postmodernism that often thinks meanings temporarily and, moreover, tends to identify the
two times of diachrony\(^9\) (I shall trace the impact of this identification on the theory of community). Discovering this conjunction is not always easy. For this reason I considered it necessary to give the elaborate description of diachrony. Two distinguished times correspond to two preconditions of meanings. While the former determines them on the side of the content, the latter conditions meanings by letting them be. I shall try to clarify what this means through the interpretation of Being and temporality in terms of language.
1.3 Language
Language, especially under the names of writing and literature, plays a significant, if not the decisive, role in the philosophy in question. It may be safely assumed that it is Being and time that are patterned after language, not the other way around. How then is language conceived of? In *Language and Death* (1991) Giorgio Agamben suggests a fruitful analysis of relationships between language, Being, and temporality. To a large extent, it makes clear the importance of language and literature in the philosophy that I consider and in the theory of community in particular. Agamben argues that the very problem of Being in Western philosophy became possible thanks to language’s ability to signify its own Being. He relies on the understanding of demonstrative and personal pronouns by Émile Benveniste and Roman Jakobson. They comprehended these pronouns as the indication of an utterance. Such words (shifters in Jakobson’s terminology) as *this*, *here*, and also *I*, *you* become endowed with a meaning only in the act of utterance and point to this act itself\(^{10}\). Utterance, therefore, not only says or means something, but also
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\(^9\) Cf. the discussion of Derrida’s *différence* below, p. 34.
\(^{10}\) In this sense the utterance itself is the event of differing between *here* and *there*, *I* and *you*, etc. Besides, from this point of view the Heideggerian interpretation of humanity as Da-sein or Being-the-there becomes possible thanks to the fact that *I* and *there/here* refer to the same reality, namely, to the instance of
shows; and shows precisely its own singular Being. This is the very Being of language, which cannot be named by language itself\(^{11}\). For Agamben the connection of Being and language is not a mere analogy. The fact of Being is the fact of language, and ontology stems from the self-indication of speech. Agamben finds in both Being and language the intention to signify which he calls the Voice (with the capital V to distinguish it from a voice as sound). Language emerges from mere sounds (or voices) thanks to the intention to signify. Without it language simply does not take place. Intention, however, can be defined only negatively: no longer a sound but not yet a signification—a pure bearer of an unknown meaning. It goes without saying that, although Agamben speaks of sounds and voices, his analysis is applicable to any system of meanings as well. Indeed, it has to do with the existence of meaningfulness or significance as such. The intention to signify is a will to have meanings or to search for meaningfulness. This is why (since in ontology we deal precisely with meaningful entities) this structure is the structure of ontology. Moreover, it is the structure of the ontology of event. Indeed, the distinction Being-entity is comparable with that between uttering and the uttered. The first component in both pairs is the production of the second\(^{12}\). In both cases we find a “having-been” as
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\(^{11}\) Agamben does not mention this, but for Benveniste this also points to the place of the subject in discourse. Benveniste defines utterance as the “utilisation of discourse (langue)” and writes: “In some way language puts forth ‘empty’ forms which each speaker, in the exercise of discourse, appropriate to himself and which he relates to his ‘person,’ at the same time defining himself as / and a partner as you. The instance of discourse is thus constitutive of all the coordinates that define the subject...” (Benveniste 1998, 50). This idea will contribute to the analysis below.
\(^{12}\) Production is used here and throughout the text not in the sense of creating by the subject but in the sense of poesis, opposed to praxis; see (Agamben 1999, 68), where Agamben differentiates “poesis (poiein, ‘to pro-duce’ in the sense of bringing into Being) and praxis (prattein, ‘to do’ in the sense of acting).” The same goes for the term “act” and its derivatives: whenever I use them I mean meaningful
necessary but inaccessible condition of meaningfulness. And finally, in both cases there is or the fact of Being-meaningful coincides with the intention to signify of the silent Voice.
Agamben develops Heidegger's examination of Being and language. Dasein can be interpreted (as Agamben actually does) in terms of letting-language-be (that is to say, of the Voice). However, Heidegger's concept of significance has another dimension. Significance which I called horizon in the context of the ontology of event is also unspeakable, that is, can only be shown. Its unspeakability, however, does not amount to that of the instance of uttering. Horizons belong entirely to the structure of language and are conditions of possibility for its meanings. Thus utterance shows (but does not speak out) not only the fact of language but along with it the structure of language (its logic, as Ludwig Wittgenstein would say). Agamben in his book is focused exclusively on the former. He asks what meaningfulness regardless of any particular structure of meaning is. This immediately refers to the Voice, and my conviction is that it is in the interpretation of the Voice as a will to found a community with others that we have to look for primal communal ties. Moreover, as far as I can see, the postmodern theory of community tends to move in this direction. Nevertheless, the author whom I mostly discuss—Jean-Luc Nancy—in many cases does not differentiate the fact of meaningfulness and its structure, although claims to build his theory entirely on the former. This is why I shall carefully distinguish them. Other writers, of course, demand additional analysis, which I have not carried out. At the same time, the distinction is important for me, because the two types of language usage which I am primarily concerned with—using it for self-sustaining and for establishing communal ties—belong to the level of meaningfulness or letting-language-be.
actions as opposed to a mere doing (i.e. producing only itself).
Grasping their difference (which lies in that for the sake of what one lets language be) demands careful separating one from the other.
It will be more convenient for me to speak of saying instead of utterance (that is, following Levinas, to differentiate saying (*le dire*) and the said (*le dit*) (Levinas 1974, 6-9)). However, one needs to keep in mind that saying is not limited to spoken or even written words. It can equally well make use of gestures or another code. Saying is rather *poesis* in the sense of production that I have already referred to (see p.21). Moreover, saying/writing in this sense is not distinguishable from hearing/reading. Indeed, what counts here is the emerging of meaning or, better, meaningfulness out of meaningless material. It matters little whether this material “came out” of the writer or someone else. The event of understanding happens to a writer exactly as to a reader. This is why one can even speak of the absence of the author as such. The theory provided is simply incapable of grasping the reader-writer distinction\(^{13}\).
Therefore, the model of language that I shall use rests on the explication of saying and is as follows. In saying I shall discern:
(1) Signs, which I understand not in Saussure’s sense of the unity of signifier and signified but in the sense that Saussure would rather call material. That is, signs are sounds, gestures, figures on paper, and so on—all material, mnemonic, or other traces that the saying leaves.
(2) Gestures of saying or leaving traces in the first sense. They, or more exactly, their differences from one another, are not independent from the language as a whole (as the same Saussure among others taught us). Even the very existence of some of them may be the consequence of certain grammatical rules (such as writing a semicolon, for example). What is essential about gestures is their repeatability, which implies, of course,
\(^{13}\) In order to grasp it we shall need other means.
the ability of a listener/reader to recognise the same sign in differently pronounced sounds or differently drawn letters. Language’s gestures provide, as it were, a common stock principally accessible for everyone’s repetition.
(3) Significance or differential system of gestures. A gesture never appears alone, but always in a referential network of other gestures. Although these gestures are not performed, they are always present as something able to be done. This constitutes the horizontal structure of meanings. It belongs to this structure that the same saying can utilise different horizons and therefore simultaneously express several, at times contradictory, meanings. On the other hand, different signs can express the same meaning. Furthermore, another important characteristic of differential system consists in that it is precisely that of gestures, not of signs.
(4) The event of saying. As we have seen, it in no way amounts to a mere manipulation with signs or repetition. The event of saying includes the letting-language-be. In this case it implies the acceptance of significance. It dwells, in fact, between signs and meanings and is the very movement from the first to the second. The movement happens to someone, who is always I. And it is me who lets language be, even if “only” by my readiness to accept its meanings.
I am not in a position to judge the identification of Being and language (although it seems convincing to me), but it plays a crucial role in the postmodern theory of community. Moreover, I myself chose to adopt this viewpoint and to interpret this theory fully in terms of language. Obviously, this implies my agreement with the authors in point: the mystery of community is to be found, if at all, only in language conceived of as a milieu (or production) of meanings, but meanings produced for others. It may appear that the suggested theory is too simple to cover all nuances in the understanding of community in postmodernism. And it is simple, in fact. Nonetheless, it does meet the needs of the
present analysis. In order to see how it works let us proceed to the interpretation of Derrida’s view on subjectivity and writing.
Chapter 2
Différance, Subjectivity, and Language Community
The subject is but an effect of language; there is no consciousness before and beyond signs; the subject is not present in its own signs. These statements by Derrida are immediate consequences of the structure of différance which excludes any presence and for the first order the presence of the subject. It is against the self-affection and self-presence (presence à soi) of the subject that this “neither word nor concept” is first of all set. However, if subjects are questioned, how can we think of their community? Any community in this case seems destined to be as anonymous as any system of language. Here, it is rather people who are assigned to no one’s community, not the other way around. My task in this chapter will be to demonstrate that différance does not exclude subjectivity. To be more precise, it excludes one sort of subjectivity whereas there are others that différance does not contest but instead presupposes. Thus the chapter proceeds in two steps. First, I shall describe other patterns of subjectivity that evade Derrida’s critique of presence. Second, I shall consider possible consequences of différance for the understanding of community. The next chapter in its turn will be devoted to the way that Nancy chooses to extend the idea of différance onto community.
2.1 Différance and the critique of presence
In the article entirely devoted to différance (Derrida 1999)\(^{14}\) Derrida starts with a
\(^{14}\) Unfortunately, in the final stages of my work I did not have at hand English translations of some necessary texts. Therefore, I was compelled to translate them from Russian. All those cases will be explicitly
reference to Saussure for whom
the signified is never present in a sign by itself, in that form of presence when it relates only to itself. Any concept is of necessity described in some chain or system in the framework of which it relates to other concepts through a regulate play of differences. (Derrida 1999, 136) (RT)
It is this play that Derrida calls différence. It is not a concept but a condition of conceptuality. It creates the very network of differences wherein concepts are only able to appear:
we shall call différence the movement through which language or any code as well as any referential system as a whole becomes “historically” constituted as a system, as a fabric of differences. (Derrida 1999, 137) (RT)
Derrida provides the following example: phonemes are never heard by themselves but always thanks to their mutual differences. At the same time their differences themselves cannot possibly be heard. This structure is opposed to the ontology of presence. The play of différence (that is, meanings that it gives birth to) is not determined by any presence in thought or intuition (the presence “in reality” is not the case in point at all), that is to say, by any presence beyond signs. On the contrary,
Presence is a determination or/and result existing in the framework of the system that is no longer the system of presence but that of différence.
(Derrida 1999, 143) (RT)
One can ask, however, how is this derivative presence present within the framework of différence? What is its place in this system? We would think—and Derrida marked by the abbreviation RT.
sometimes gives occasion for this\textsuperscript{15}—that presence appears when the results of differentiation—not only signifiers, but also signified, i.e. concepts—are considered self-sufficient and present on their own. As if something behind the differences is present, affects the differences, and determines them. However, the second aspect of \textit{différence} resists this understanding. References to other signs turn out to be temporal:
\textit{Différence} is what makes the movement of signification possible only in the event that each element considered as “existing,” appearing on the scene of presence, correlates with something other, different from it, but retains at the same time the sign of already past and simultaneously remains open to the sign of its relations to the coming. (Derrida 1999, 138) (RT)
In this respect presence is similar to phonemes from the example above. It is never present on its own without reference to another presence. However, what immediately attracts our attention is the similarity between this structure and temporality as described by Husserl. Like there, past and future in this passage are not reducible to the modified present. This, in particular, does not allow this structure to be directly transferred onto the Saussurian model. Indeed, what else are other signs in his model if not a “modified present”? The Saussurian differential system is synchronic; all its differences are given simultaneously. Derrida goes further and applies Saussure’s idea to the temporal structure of the movement of signification. He puts temporal differences peculiar to the constituting movement in one row with differences within the constituted system. In what way can the constituting be transferred into the sphere of the constituted?\textsuperscript{16} As is known, in order to
\textsuperscript{15} For instance, the examples of differences on page 145 (intelligible-sensual, concept-intuition, culture-nature) speak rather for such an interpretation.
\textsuperscript{16} This question is in fact analogous to the one that Levinas posits when he speaks of making-present
demonstrate how this happens Derrida employs the concept of trace. The latter "retains in itself the experience of time" and thereby allows the constitution to appear in the sphere of the constituted. Derrida elaborates two major themes in order to clarify how the past is retained in trace: Freud's *Nachträglichkeit* and the concept of supplement. Freud’s notion of deferred action (*Nachträglichkeit*) points to the re-considering of the psychic experience in late stages of human development\(^{17}\). It is this dependence of the past on the movement of constituting that is essential for Derrida. This phenomenon of the *appearing* of a new past points to what Levinas calls diachrony. Indeed, we have two ‘nows’. The first is constituted as different from past nows, whereas the second is the now within which all of them are constituted. To put it in other words, the first now is opposed to other nows, whereas the second is opposed to ‘non-now’ or non-Being. However, Derrida does not distinguish these temporal orders. Moreover, *différence* itself is essentially based on their identification as we shall see later (see p.34).
Another approach to the trace is connected with the concept of supplement or substitution. Presence is hidden and reveals itself only through a supplement. It emits, as it were, something that substitutes it (Derrida 1999, 148) because in itself it remains not self-sufficient. The substituted—that is, presence—stays in the past which, however, is produced by the movement of *différence*. We see that the trace not so much retains the experience of time as carries it out. The trace—along with *différence*—is a movement in
\(^{17}\) J. Laplanche and J.-B. Pontalis raise an objection against rendering this Freud’s term as “deferred action” (as the English *Standard Edition* does). They write: “Freud’s conception of *Nachträglichkeit* cannot be reduced to the notion of ‘deferred action,’ if we understand it as a temporal gap between excitation and response determined by the gradual accumulation of experience” (Laplanche and Pontalis 1996, 345) (RT). It is rather a post-action that inscribes the experience that could not earlier be endowed with a meaning into a new context. The prototype of such experience is trauma.
which the very difference between the substitution and substituted—that is to say, the signifier and signified—appears. This, of course, is in tune with Derrida’s assertion of the irreducibility of temporality, but we should accentuate things differently. It is not the point that the articulated speech by itself is a trace or that the articulation by itself gives birth to the sign as a trace. *Différance* rather constitutes sign as a sign. In *Of Grammatology* Derrida formulates the question as follows: “What condition must discourse fulfil in order to ‘signify’ something?” (Derrida 1999, 169-70) (RT). He searches for an answer in the temporality of the trace. The temporal gap between saying and the said turns into the gap between the signifier and signified. At the same time, however, Derrida follows Saussure and understands the significance of signifiers through their mutual references within a referential system. According to this scheme, the differences between the signifying, signifier and signified are in fact equal to the intra-system differences (in other words, differences constituting for the fact of the system are identical with intra-system ones). *Différance* then is responsible for generating all language’s differences. We could agree with this but in this case the question might be raised: Does Derrida not identify in *différance* two gestures of differentiation?
2.2 Two gestures of differentiation in *différance*
*Différance* as a movement of differing produces two sorts of differences. Those of the first one are between signifiers. They constitute the network of mutual references of signifiers. Unlike them, the differences of the second sort belong to the movement itself. They are between “parts” of the movement: signifier, signified, and the act of signifying. Therefore, the movement of differentiation can be viewed as constituting either a repeatable differential system of language or the very difference between repeating and repeated.
The distinction drawn helps us to give more details about Derrida’s notions of trace and effacement. Their general definitions (the trace is a signifier that refers to the absent,
and the effacement is a retention of the experience of time) remain the same but specific meanings become different according to two meanings of differentiation. When the latter is considered as constituting the differential system of language, the trace appears as the reference of the signifier to other signifiers: any sign, symbol, text, etc. holds traces of other signs, symbols, texts, etc. These traces may be situated in the past and/or future (we have already seen the corresponding quotation on the page 28). The latter however belong to the constituted time rather than to the time of constituting. Furthermore, effacement in this case should be grasped as forgetting these references, that is to say, as forgetting the referential system wherein the signifier was established. This makes it possible for the gesture of repetition to be repeated independently of the established system. The phenomenon of Nachträglichkeit as well as the appearing of the signifier in different contexts becomes also possible thanks to such a forgetting. Moreover, this can explain one more meaning of trace found in Derrida’s introduction to Husserl’s *The Origin of Geometry*. This is the trace as remnants or residues of the experience that happened, was not understood, but nevertheless remains efficient in the repetition. This is a gesture that appears in the by now forgotten (or even repressed by means of metaphor and metonymy) differential system.
Other meanings of trace and effacement stem from the second meaning of differentiation. The trace now refers to the event of repetition. In this sense the whole differential system (insofar as signifiers are never established outside of such a system) becomes the trace of the event of its establishing. There is no other “signified” except this event. The logic of effacement works here, although it needs no signatum beyond repetition. Moreover, the event itself does not play the role of the inaccessible absent signified or presence ‘behind the scene’. Its presence is of a specific sort—it must be performed in order to be (presented) at all. No non-presentable past is necessary here. Movement and reference to it are simultaneous. The event of establishing is the absolute
beginning (for this reason the logic of desire which I shall describe below does not work here as well). Effacement now denotes the releasing of the said in the act of saying (what Nancy often calls withdrawal). This independence of the said leads to the necessity to repeat the said if one wants to make it exist at all.
We see that repetition lies in the fundament of *différance*. We can isolate a “basic” level of *différance*, that is, the gesture that differentiates what is repeated and the act of repeating. This gesture consists in using a signifier (doing a gesture, uttering a word, drawing a hieroglyph). It is possible in turn only if the signifier is different from other signifiers, that is, other gestures, and therefore implies a differential system of gestures and the ability to discern between them. The possibility of automatic or meaningless repetition (for instance in the case of neurosis) demonstrates that repetition does not necessarily implies signification. At the same time, we are able to recognise here signifiers and signified and to track the logic of effacement and trace. Indeed, the gesture, even when it is produced for the first time, presupposes repetition. In this sense, it leaves a trace—that which allows one to consider the gesture repeatable or the same. Even if it is not fixed in speech or writing, it remains a signifier and settles apart from the repeated gesture. Fixed, it becomes a part of the written language, simultaneously poison and a “remedy for memory and wisdom” (Plato, *Phaedrus*, 274e-5b). However, what it signifies not only appears in the same gesture but is identical to it. The gesture of repeating a particular signifier is this very signifier. We can speak here of archi-writing as the gesture that (archi)writes by the very fact of its repeatability or distinguishability from other gestures. On the other hand, the gesture is not fully present in what is left. It is “effaced” from its own result. But precisely this effacement—that is to say, the possibility of repetition or the difference between repeating and repeated—forms a sign in which the repeated serves as a signifier and the act of repeating as a signified. This structure of repetition is present in *différance* as the movement of differentiation.
Although this description is similar to Derrida’s, it does not follow it in all details. First of all, for Derrida the concept of trace connotes something that happened but was not understood. Like traces precipitated in the unconsciousness, they gain their interpretation in post-action: something happened, remained obscure, and could not even be remembered but suddenly shows up and becomes meaningful. However this repetition, it seems, does not contain what Derrida calls “the past that has never been present.” The latter is not very clear in Derrida. Borrowed from Levinas, for whom it designates the past of *Urimpression* in Husserl’s description of time-consciousness, in Derrida it points sometimes to the past of the differing act and sometimes to the signified preceding this act. This ambiguity extends over the concepts of trace and signification. By separating the level of repetition, I try to clarify this moment (by means of some sort of “deconstruction” of *différance*). Repetition, as it is described here, presupposes only the difference between repeating and repeated which implies the differential system of the repeated referring to one another, leaving a trace, and a gesture of differentiation generating the system of the repeated. This repetition demands no “non-presentable past,” since the act of repetition (which is the act of using a signifier) comes to awareness in the very moment of its performing (speaking, reading, writing…). It is non-presentable, but it is not in the past. This does not contradict its non-determination and dependence on the game of signifiers, Derrida’s main assumption. There are no acts that precede signs and are signified by them backwards (as a “supplement”). What is indeed presupposed by the gesture is a set of other gestures. However, they are not situated in the past or future. They are possible gestures. What is brought about in any gesture’s performing is a system of possible gestures which compose in the final analysis the referential system of significance. Generally speaking, possibilities belong to the present, and assigning them to the past or future is rather contingent. To be more precise, they belong to the constituted time. If they are assigned to some past or future, they are the past or future of the constituted time. Therefore,
differences of the referential system have nothing to do with the temporal gap between repeating and trace. Nevertheless, Derrida explicitly identifies them. This means that he blends in one the two considered meanings of trace and effacement.
2.3 Joining two times of diachrony in différence
As we have already seen, Derrida starts with Saussure’s idea of a differential system but includes into this system the temporal differences of the movement of differentiation. One could even say that this is the core idea of différence as such. It is this interpretation that allows Derrida to qualify différence as differing-deferring. Différence comprises temporal and spatial dimensions and is even, as Derrida puts it, “time becoming-spatial and space becoming-temporal”. However, this becomes possible only because he identifies two times of diachrony—the constituted time and the time of the event of constituting. In order to demonstrate this I shall start with Derrida’s analysis of the Husserlian phenomenology. Husserl’s description of time-consciousness (Zeitbewußtsein translatable as both “consciousness of time” and “time as consciousness”) remains the basis of Derrida’s contemplation on différence. This determines its possibilities and problems.
Derrida’s approach to phenomenology is guided by Saussure’s linguistics. Derrida utilises the latter’s theory of sign systems in order to demonstrate phenomenology’s insufficiency. His whole intention is to undermine the idea of objective experience. Instead, language is supposed to be the only experience and basis of knowledge. For this reason Derrida starts directly with the analysis of language within the foundation of phenomenology. As is well known, according to Husserl phenomenologists need to perform époche, that is a special act of excluding everything not belonging to the sphere of experience that Husserl called phenomenal. To delimit this sphere he starts with the notion of expression. The latter implies and bears a meaning because it “means something to say”. Therefore, such experience as pain or pleasure is excluded from the scope of
phenomenology. By contrast, expression expresses an implicit meaning preceding it, even though appearing only through it. Being in a sense hidden, this meaning is nonetheless fully accessible to the subject in its inner life. It is this claim that becomes the target of Derrida's criticism. In the course of his analysis he reveals a weak point in phenomenology and proposes a solution to deal with it. However, not unaware of this weakness, Husserl himself in his later writings provides his own solution, different from Derrida's. To me, Husserl's own approach seems more productive, but, as we will see, Derrida's elaboration is especially interesting in the context of the present work.
Thus, the matter in question is the usage of signs. Derrida shows that the implicit pattern of phenomenological meaning is "living speech," which he opposes to writing. Their difference, in the final analysis, lies in the relation of the subject to the expressed meaning. Living speech, as well as life itself, becomes living only insofar as the subject possesses fully the meaning that it means to express. In contrast, according to Derrida, written language carries within itself a network of meanings which the subject not only does not possess before the expressive act but also cannot possibly appropriate by means of this act (and by any other means as well). Constituted as a referential network of signs, language holds in itself meanings prior to any possible usage of them and moreover dictates to a large extent, if not totally, the rules of any such usage. This is why, according to Derrida, phenomenology has no right to speak of the intentionally constituted meaning. The latter is but a fiction. Reasonable, as it is, the argument, however, does not cover the whole problematic.
Indeed, Husserl discerns three elements in an intentional act: the object of intention, the intentional act (*noesis*), and its intentional content (*noema*). In expression, which is certainly a language act, we have in addition signs or words. Husserl considers this fact as an unimportant complication because he presumes a strict parallelism between intentional acts and words used for their description. After all, it is this parallelism that makes Husserl
believe that phenomenology can be developed as a pure descriptive science, a pure description of the subject’s intentional life. The essential feature of the intentional act is that it can be repeated and therefore named and idealised. Moreover, in many cases it can be repeated voluntarily, making it possible to mean anything at all (cf. Derrida 1973, 34-5). ‘Voluntarily’ here designates nothing but the act’s independence from the object (more exactly, from its Being or truth): the meaning of expression exists even when the object is absent, and this is the first step in the emancipation of language (the second one being its emancipation from the subject). It is important to note that according to Husserl noema and noesa are experienced differently. Intentional acts are accessible through a special sort of intuition, which a phenomenologist gains as a result of *epoche*. This transcendental intuition has no object, it functions according to the pattern of vision: we see something and at the same time perceive the act of vision itself. Accordingly, objects are perceived through intentional acts directed toward them, whereas the acts themselves do not demand any further intentional acts to be perceived. They are immediately given as performed actions. Later Husserl abandoned this conception and worked out its more complicated version. According to this, any actual meaning depends on the horizon of potential experience. Its limiting case is the life-world (*Lebenswelt*)—the ultimate horizon of all human meanings, the place where people always already dwell. However, the primary horizon appears as temporality. It is described in *The Phenomenology of the Consciousness of Internal Time* (see p.16 et seq. for the discussion). Thus any meaning appears like in Derrida only in the context of a differential system which is fundamentally temporal. In this sense Derrida detects the equivalence between Husserl’s time-consciousness and Saussure’s structure of signs and then identifies them. What happens to diachrony?
Derrida’s discussion of Husserlian phenomenology shows very clearly that he does not distinguish sufficiently between two times of diachrony. Indeed, on the one hand,
spacing of *différance* is described as obviously belonging to the time of event.
It becomes even more obvious with his reference to Heidegger whose *Anwesen* is precisely a movement of inner temporality. Nevertheless, at times Derrida takes one as the other. We can find many examples of this. In a footnote we find the following passage:
...temporalization taken as the openness of the present upon an outside of itself, upon another absolute present. This being outside itself proper to time is its spacing: it is a proto-stage [archi-scène]. This stage, as the relation of one present to another present *as such*, that is, as a nonderived re-presentation (*Vergegenwärtigung* or *Repräsentation*), produces the structure of signs in general as “reference,” as being-for-something (*für etwas sein*) and radically precludes their reduction. (Derrida 1973, 84-5)
Two points are worth mentioning here. The first is the description of the sign’s temporality as being-for-another-sign. Actually this is what Saussure meant when he spoke of the referential nature of signs. The second, however, is a notable identification of spacing—*différance*’s temporal component—with the relation of one present to another. Moreover, later on in the text Derrida clearly identifies this relation with phenomenological retention:
The process by which the living now, produced by spontaneous generation, must, in order to be a now and to be retained in another now, affect itself without recourse to anything empirical but with a new primordial actuality in which it would become a non-now, a past now...
The living present springs forth out of its nonidentity with itself and from the possibility of a retentional trace. . . . the self of the living present is primordially a trace. (Derrida 1973, 85)
Thus, Derrida understands retention as retaining the past living present, “past now”. But this is not exactly so (although it seems at times to be Husserl’s own standpoint). What is retained in retention is not the “past now” but rather “the past in the now”, that is, the past as it is presented in the now. Retention comprises all the chain of such “pasts” along with all past retentions. Retention essentially and completely belongs to the living now. Levinas calls this feature of phenomenological temporality *Vergegenwärtigung*, making-present or turning-into-present\(^{18}\). But the connection of ‘nows’ is not a retention or representation. It is rather a looking for sense or establishing of ties. Derrida is right in that there is an essential gap between the actual present and its representation. It is experienced in the present as a trace, a reference retained in the present itself. But this trace is in no way retention, as Derrida wishes to indicate. Temporalisation is not a reference to another present. It points rather to the non-present, non-now.
Derrida applies the Saussurian theory of sign systems to the Husserlian temporal organisation of experience, in order to undermine the presence of experience. Thus he introduces the idea of writing. He starts by denying the presence of signs (Derrida 1982, 26-8). Each element has traces of other elements in the chain or system. Spacing is this relation of elements. It is temporal because each element refers to other elements in the past or future. Thus, Derrida claims, we have a production of intervals, “generative moment in the play of differences.” *Différance* is a transformation, he concludes. This temporal production of intervals might be viewed in two ways. It can be the play of retentions and protentions in the internal time, but it might also be the production of these retentional relations in the external one. Derrida does not distinguish between these two situations. Perhaps he is right but this certainly demands a more detailed explanation.
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\(^{18}\) The word is borrowed from Husserl himself. Derrida applies it as well, as we have just seen. The usual rendering of *Vergegenwärtigung* is ‘representation’. For example, the German version of Levinas’ work *Diachrony and Representation* is called *Diachronia und Vergegenwärtigung* (Levinas 1991).
Anyway, Derrida’s thesis about time turning into space appears to be ungrounded, at least as far as his understanding of spacing is concerned.
Therefore, Derrida mixes up two times of diachrony and two gestures of differentiation in *différance*. In spite of this I shall explicitly distinguish them with the only goal to delimit the gesture of producing “extra-system” differences between signifier, signified, and signifying. It is in this sphere that I shall look for the subjectivity escaping Derrida’s criticism of presence. But before that let us look closer at his vision of subjectivity.
2.4 Subjectivity: Derrida’s approach.
From Derrida’s standpoint the subject—moreover, the subject in the first place—is nothing but an effect of *différance*. With reference to Saussure, to his idea of the subject as a function of language, Derrida writes:
It is presupposed thereby that the subject (identifying itself or even conscious of its own personality, that is, possessing self-consciousness) is described by language, that it is a “function” of language. It becomes a speaking subject only thanks to subjecting its speech . . . to a system of linguistic rules constructed as a system of differences or the universal law of *différance*. (Derrida 1999, 141) (RT)
Derrida’s answer to the question of the existence of self-consciousness before and beyond signs—that is to say, “the self-consciousness of the subject’s presence in the silent intuitive consciousness” (Derrida 1999, 142) (RT)—is negative. Nevertheless things are not that certain.
Emile Benveniste while discussing the place of subjectivity in language relates it not to the sense of oneself but to the reality of the instance of discourse (*instance de discours*), that is, to the very fact of speech (Benveniste 1974, 292-301) (RT). Personal
pronouns (of which the most important is *I*) as well as deictic words (*now, here*, etc.) refer not to the corresponding concepts but to the moment of their uttering. Developing this theme, Giorgio Agamben asks what is said in every speech act by the very fact of saying. For all that, saying does not amount to a mere repeating of words. Language appears in the transition from indicating to signifying, from the articulated sound to the meaningful word. As we have already seen, Agamben calls this double negation (no longer a sound but not yet a word) the Voice (capitalised in order to discern it from the voice as a mere sound) and interprets it as the intention to signify (that very *vouloir dire* which Derrida subjects to criticism) or will to have language (66). This Voice therefore is accomplished as the effacement of sound, forgetting the origin, that is to say, as trace.
Returning to Derrida, does he take into account this type of subjectivity?
Let us start with a long quotation from *Speech and Phenomena*:
Let us consider the extreme case of a “statement about perception.” Let us suppose that it is produced at the very moment of the perceptual intuition: I say, ‘I see a particular person by the window’ while I really do see him. It is structurally implied in my performance that the content of this expression is ideal and that its unity is not impaired by the absence of perception here and now. Whoever hears this proposition, whether he is next to me or infinitely removed in space and time, should, by right, understand what I mean to say. Since this possibility is constitutive of the possibility of speech, it should structure the very act of him who speaks while perceiving. My nonperception, my nonintuition, my *hic et nunc* absence are expressed by that very thing that I say, by *that* which I say and *because* I say it. This structure will never form an ‘intimately blended unity’ with intuition. The absence of intuition—and therefore of the subject of the intuition—is not only tolerated by speech; it is required by
the general structure of signification, when considered in itself. It is radically requisite: the total absence of the subject and object of a statement—the death of the writer and/or the disappearance of the object he was able to describe—does not prevent a text from ‘meaning’ something. On the contrary, this possibility gives birth to meaning as such, gives it out to be heard and read. (Derrida 1973, 92-3)
One can see how in this passage Derrida repeatedly slides from the “meaning for me” to the “meaning for another” and to “other’s meaning for me”. But these are completely different meanings and Husserl devoted many pages to explaining how the second one is constituted as the third, i.e. as the first at bottom: ultimately every meaning is a “meaning for me,” because only in the inner life of consciousness may any unity of meaning whatsoever appear\(^{19}\). Derrida, on the contrary, argues that there is meaning in the written text itself. But it is also true that any meaning comes into Being only through its “animation” by a speaking subject (Does it not consist in providing a spectre of potentialities for any actual meaning?). Dead languages have no meanings until someone starts looking for them. Which is not to say, of course, that the subject produces this meaning or possesses it. However, Derrida’s point is that in fact meaning is constituted by language (by its play of differences), whereas the subject’s “animation” consists merely in complying with its rules, and the subject itself is but an effect of the play. We need, therefore, to examine more closely the relationship between the subject and language.
The examination of the word ‘I’ seems to clarify these relationships, and Derrida undertakes it. He quotes Husserl: “In solitary speech the meaning of ‘I’ is essentially realised in the immediate idea of one’s own personality” and continues, “Even supposing that such an immediate representation is possible and actually given, . . .” (Derrida 1973,
\(^{19}\) Thus, the ‘meaning of the text’ can serve as all of them depending on who repeats the text.
The phrase clearly shows his misunderstanding, because Husserl does not mean any “representation” at all. This idea of ‘I’ is a—truly immediate—understanding of one’s Being, one’s using of the word ‘I’, if one wishes. *I* is never the object of representation, and Husserl is but Kant’s disciple in this point. This is why Derrida’s phrase “…in the absence of intuitive presence—here, in the absence of myself” misses the point: “myself” is never “intuitive presence,” i.e. intentional content. It is noesis and never noema, and being such, it is perceived without objectifying. Moreover, Derrida changes the issue slightly. Instead of discussing the presence of living speech, he speaks of the intentional object. To put it differently, instead of the presence of intuition, he discusses the presence of the object of intuition, that is to say, instead of saying he discusses the said.
Derrida’s reference to the subject’s death also appears misleading. He claims: “Whether or not I am alive, ‘I am’ ‘means something’”, “there is no need to know who is speaking in order to understand or even utter it [the word “I”—OD]” (Derrida 1973, 95). It is true, but what *is* this “understanding or even uttering?” (And why does Derrida put them in the same row?) Unlike intention, the intuition of ‘I’ cannot be “without an object” because it is its own “object.” Undoubtedly, “I am” means something regardless of anyone’s death and can, moreover, have many different meanings. But all these meanings can be only for *me*—this is what Husserl describes as “the immediate idea of one’s own personality.” The intuition of “I” designates the presence of meaning itself or the presence of significance, but not the presence of “what is meant,” and it is precisely this presence that Derrida aims to contest. This is hardly possible with the argument considered, and, actually, Derrida has more powerful instruments, which I shall discuss later, but they also fail for the same reason: presence in phenomenology has nothing to do with objectification and representation. In “uttering ‘I’” one should distinguish between “what is uttered” and the uttering itself. The former belongs to the differential system of language, whereas the latter does not. The situation here is analogous, if not identical, with Noam Chomsky’s
distinction between competence and performance, used by Judith Butler in her concept of gender as performance (Butler 1990).
Derrida’s point is clear and reasonable. We can use language even without knowing its meanings. Signifiers can be repeated regardless of any presence supposed to guarantee their meanings. But even if we accept Derrida’s criticism of presence, there remain different models of “significance without presence”. Two of them will be important for the following elaboration. As we shall see, they correspond to two types of subjectivity which I shall call that of involvement and engagement.
2.5 Subjects of involvement and engagement
The event is in every case mine. It belongs to me in the sense of happening, not appropriation. I am irreducibly involved into the happening of the event. This subjectivity of involvement differs from that produced by discourse\(^{20}\). Derrida’s criticism of the presence of subjectivity aims at the latter and neglects the former. Subjectivity comes to Being as involvement and the place of repetition. Being so it is always my subjectivity. Furthermore, involvement implies the possibility of decision. Although I always already find myself involved, I can decide on further involvement, at least negatively. This power of rejection is granted to me by the event itself, more exactly by its repetitive character. Having been created by the event, I remain free for another event or for the absence of events. We encounter here precisely the structure of trace (or withdrawal, as Nancy prefers to name it). But unlike a mere sign, the subject is an “active” trace of the event. This
\(^{20}\) Paul Ricoeur in this respect distinguishes between the subjectivity of idem and ipse (Ricoeur 1992). The first is constructed by narratives, whereas the second stems from the very fact of using narratives. As a result, although I cannot know who I am, I know that I am, that something is happening here, where I am (“What I am is as problematic as that I am is apodycitic” (Ricoeur 1995, 371 (RT)). I am the very place of this “happenstance”.
“activity” consists first of all in the agreement to become involved or in letting a discourse be. There is a point whence involvement is impossible without *my* offering the body for that. This is the point where the repetition of discourse ceases merely to happen. Although *I* still in no way determine what happens, it needs *my* participation in order to exist, and thus *I* do determine the fact of its happening. Agreement therefore contains a negative aspect—refusing a mere repetition, transiting from a sound to signification, from *Es* to *Ich*. This is the gesture of refusing meaningless repetition (which is therefore acknowledged as such) in favour of meaning (or nothing). This refusing may be accompanied by the refusing of the current discourse. At least, this possibility to deny its current conditions points primarily to the subject of involvement and its activity. But even without this denial the agreement is marked by negativity, since it refuses to merely repeat. But what then prompts *me* to let language be? What prompts *me* to speak? With this question we come to the subjectivity of engagement.
In Derrida’s *Of Grammatology* (Derrida 2000) a mere constituting of the differential system turns out to be insufficient for presence. One more condition is necessary. Derrida calls it the desire directed to presence (Derrida 2000, 171). This desire is insatiable and structured as a lack, but the lack of what has never taken place (Derrida 2000, 255). *Différence* generates what it immediately prohibits and makes impossible (Derrida 2000, 294). This logic as well as the very choice of the term “desire” refers, of course, to Lacan. Ellie Ragliang in her book on death in psychoanalysis describes the structure of desire in Lacan’s works in the following way:
One of Lacan’s discoveries reveals the ego as itself a defence, or means of resisting the truth of unconscious desire. Indeed, the ego is constructed *for the purpose* of minimizing the anxiety produced by losses that constitute a void place within being and body… [T]he ego is driven to annul the anxiety produced by the unbearable pain of lack and loss. In this
context, it makes sense to argue that the infant’s first corporal satisfaction or pleasurable sensations give rise to desire—not to static fantasy hallucinations—as a dialectical call to the other (such as an infant’s cry). This “demand” is the desire for connection that Freud called Eros, that Lacan called libido or jouissance. Thus seen, drives are themselves constituted on a paradoxical basis, both initiating the cut and attempting to annul the effects of primordial losses that make the primary human goal that of retaining illusions of consistency. (Ragland 1995, 27)
Therefore, the inaccessibility of jouissance is not so much in conflict with desire but rather itself at its service. Desire appears as the desire to be or, as Lacan put it, “not to be nothing.” In this logic—coming back to Derrida—the hidden and inaccessible presence proves to be a condition of language’s existence. This means not only that language stands on the negative fundament but also that this negativity is not reducible to that of originating in the sense of transition from non-Being to Being. To put it differently, the repetition of the articulated system is not sufficient for turning it into the system of significance. The ability to repeat and discern words is not yet sufficient for endowing them with meanings.
To move further we need to recall the Freudian distinction between satisfaction and identification (Freud 1920). Freud associates the latter with the death drive (“something demonic”) and repetition compulsion. It can operate independently of and even contrary to any search for satisfaction. Although this concept is disputable and has not been fully adopted by the post-Freudian psychoanalysis, Lacan makes use of it in order to develop his understanding of desire. Thus desire, as the desire to repeat is not necessarily connected with satisfactory experience. It is the repetition for the sake of repetition, desire to be, regardless of any possible dissatisfaction. It is language that allows this desire to be played out. Signs, fantasies, mis-en-scènes are capable of being repeated without a reference to
any reality whatsoever except themselves. This is a completely artificial world in the sense that it has nothing to do with any "natural" signified, whether it be need, experience of satisfaction, material objects, etc. *Différance* refers precisely to this world. But here we need more subtle investigation.
Let me note that the rejection of presence, as Derrida describes it, does not necessarily exclude any reference to "nature." The thing in point is the illusory character of any certain signified supposedly preceding language. However, the Freudian model of drive depicts the unstructured and undetermined force or pressure that looks for a satisfaction but does not offer in advance any particular way or method for it. The drive pushes out here rather than toward there (in Lacan's wording, "the drive kicks us to the arse"). This does not contradict Derrida's critique of presence. However, it is difficult to understand from Derrida's texts what precisely he thinks of this matter because it is not clear whether he speaks of the presence before the movement of *différance* or in its very moment. At first glance the moment of the act is itself problematic for Derrida. Nevertheless, he grasps the gesture of differentiation as something simultaneous—what is present on the scene of presence refers no doubt to the past and future but this very reference is present "instantly" (see above about the evidence of *différance* and its temporality). Anyway, we can distinguish between two types of repetition depending on its relation to the "extra-discursive reality." Both conform Derrida's refusal of determination or motivation by a pre-sign signified. However, one of them remains bound, as it were, with the "pre-sign implying no signified" that is with satisfaction, sense of conformity, etc., as if something finally obtains satisfaction in the found expressive form (even if in a post-action). Unlike this, the other repetition, to which Derrida is close, does not have even this support. It is completely artificial. This is a part of language that has no signified except the very acts of repeating or using signifiers. It is connected to "reality" through the
ability to perform one or another gesture\textsuperscript{21}, rather than through the correspondence with some “real content.” Nonetheless, both repetitions are in a sense artificial and this is what \textit{différence} calls our attention to. The ability to repeat, not a connection with the signified lies at their basis\textsuperscript{22}.
In a broader sense repetition may be performed for itself or for the sake of something else, but in both cases it is engaged, so to say, in some project. I shall call the corresponding subjectivity that of engagement. Thus this subjectivity does not merely repeat discourse or uses language. It does so for the sake of something. How precisely the logic of this “something” looks, is a specific problem with which I am not concerned here. Instead, I shall take two examples of engagement important for the exposition to follow. They have a direct bearing on the presence of the other and consequently community.
\textbf{2.6 Engagement and the place of the other}
As we have seen, \textit{différence} generates a system of signifiers. There are different types of them. Some comprise a “static” (in the sense of belonging to the internal time) system which can be easily substantivated and turned into a “structure of the world”—objects and state of affairs. Others are the signifiers, such as personal pronouns, that are unthinkable without a reference to the movement of repeating (or saying). Derrida, as we have seen, while analysing the pronoun “I” does not take this feature into consideration. However, this sort of signifier denotes the structural elements of repetition, not the repeated. In other words, these gestures bear in themselves their own signification. For this reason, it is to them most of all that Derrida’s statements about the movement of \textit{différence} belong. Above all that, a special group of signifiers consists of ones referring to the
\textsuperscript{21} We can recall here Heidegger’s \textit{Seinkönnen} or possibly Wittgenstein’s \textit{Sachverhalten}.
\textsuperscript{22} It is possible to repeat mistakes despite facts even in empirical sciences. The phenomenon of repetition compulsion (\textit{Wiederholungszwang}) also consists in repeating regardless of immediate pleasure.
situation of communication. On the one hand, they refer to the gesture of saying. But on the other hand, they seemingly presuppose a mis-en-scène with personages and roles. In what sense can we speak of the presence of these personages?
The issue then is repetition of a special sort, when language (or signifiers) is used for addressing and appealing. It breaks up into two asymmetrical situations corresponding to my appeal to others and their appeal to me (consequently, the appeal is always mine, or in Heidegger’s words, assigned for my taking over). The first is my opening to others, that which Levinas denoted with the phrase *me voici* (“I am here”). It is a self-revealing, exposing, and indicating of one’s own presence. The expression “I am here” contains exclusively the signifiers referring to the moment of saying (since Being also refers to this moment). Moreover, for me, it is not reducible to a mere attestation of the fact or even to the event of existence\(^{23}\). As exposition, it gives birth to the world “there” in relation to which I am “here.” However, what is this “there,” if “here” denotes the very event of saying? Probably, another event. It can be mine, and its structure of repeating (with its feature to be in each case mine) directly presupposes this. However, as soon as it can be repeated, not only I can repeat it. In that case it will become somebody else’s and turns into his or her self-indication. This is not yet the appeal directed towards me but already the openness of the other in her/his ability to speak, not necessarily meaningfully (with signification) but already responsibly. This apparition of the other, which Levinas calls the epiphany of the Face, demands speech in a wide sense, which is close to Being. The face by itself or merely eyes or even the human body as a whole prove to be a gesture of self-revealing. It cannot be reduced, however, to the self-revealing of phenomenon. Therefore, signs, which initially were my gestures (writing or reading, saying or listening), become, thanks to their exteriorisation in repetition, the gestures and speech of the other. The place
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\(^{23}\) Although it is reducible perhaps to the performative “I am saying” in the sense of “I am [a subject
of language, which initially was the place of my saying, becomes a public place. Undoubtedly, this has nothing to do with the mere fact of the precedence of language in relation to the subject as well as with the fact that language is always a "collective product." Rather the point is in distinguishing two functions of language, two ways of its using regardless of how it originated or how speaking subjects learned to use it. The other is the one who speaks, which means that the other does not merely repeat signifiers but says. To recognize the other as a subject of speech means to transpose my own structure of the speaking being to her or him. This, in turn, means two things. First, the other uses language to address me. Second, language itself turns into something that not only me but also others can repeat. Language becomes a common field where ties of community are established.
Now let us come back to repetition, to what I called the completely artificial sphere. If repetition is not automatic\(^{24}\), what prompts me to speak in this sphere? What prompts me to search for another language in the artificial world? Or what makes me, on the contrary, consent to repetition? There may be different answers. Perhaps it is the desire of consistency, as in Lacan's psychoanalysis; or sophisticated criteria for empirical verification, as in the natural sciences; or correspondence with experience; or bodily satisfaction; or fidelity. All these contain my letting the coming to come (even if this letting is but a readiness of my "receiving apparatus"). Its concentrated expression is Nietzsche's eternal return, "the highest form of approval which can be reached at all" (Nietzsche 1990, v.2, 743) (RT). However, the recognition of the other as a subject of speech goes further. Opening myself through language I expose myself to the other's interpretation and judgement (either blaming or praising). Entering a common world means that my letting
\(^{24}\) To note it in passing, as soon as I, like Derrida, understand that différence lies in the basis of signification, repetition ceases to be automatic.
(as Heidegger’s *lassen* can be translated) does not suffice. Language that provides a satisfaction to *me* can be unjust to the other and this prompts *me* to search for a new discourse. The other here is not merely the one who comes unexpectedly, destroys *my* world, and thereby forces *me* to look for a new language. This invasion happens, if at all, on the level of abilities (*Sein*- or *Redenkönnen*), whereas the other as the subject of speech does not change the structure of *my* world at all. He or she rather makes this world be not only *mine*. Neither new experience (like trauma), nor a movement for the sake of the movement, but striving for a common world prompts me to speak. What becomes important is not the event of speaking but the one who speaks. Responsibility for oneself (care or *Sorge*) turns into that for the other, and this does not require from the other to take over the form of presence.
Here the sphere of obligation appears distinct from that of Being. In this sphere such signifiers as good, evil, justice are always open to question—not because of their contingency but insofar as they are liable to agreement. The latter is not a consensus. Nor is it a universal remedy for all imaginable ethical problems. It is closer to Heidegger’s *Verlaffenheit* and happens literally every minute, as long as people live in a world that they do not contest. On the other hand, it is neither unconscious nor automatic. It should be made explicit. The agreement or letting be is what distinguishes saying from mere repetition. In Heidegger’s terms it is what distinguishes *Man* from the authentic (*eigentlich*) mode of Being. Letting be is a taking over (which is of course far from being always possible). However, I do not advocate here self-control as an ethical ideal to be acquired. At the same time, the recognition of the other’s speech does not amount to the recognition of the unconscious (which Lacan calls the Other’s speech). The point is not merely that the other speaks in *my* speech and desires in *my* desire. Rather the other is the one who addresses *me*. A deeper reason for not identifying the other’s speech with unconsciousness is that the interpretation of the unconscious (which essentially implies
letting it speak) aims at the self-clarification and appropriation of the unconscious by means of language. This is why *I myself* am supposed to decide when to finish it, whereas recognising the other’s ability to speak passes to her or him the right to cease the interpretation. Others become every bit as decisive an instance as *me*. It is precisely this that does not allow *me* to accept happily the world “as it is” and be satisfied with *my* interpretation.
Above all, agreement is not equal to argumentative dialogue. It may equally well be the consent to a play, being seduced, and so on. In this sense it does not at all prevent pain or disappointment. Its intention is to build a common world no matter what it is going to be and to take over the responsibility for this world.
Thus we can separate two sorts of the subjectivity of engagement in accordance with that for the sake of which the repetition is performed. In the first case it is performed for self-sustaining, that is to say, for gaining consistency in the movement of repetition. It is a flowing identity concerned with “not-being-nothing”. “Nothing” here designates not death but rather mere repetition or repetition without decision. The subject speaks in order to sustain itself as a subject of speech and because there is no other way for that. Being so, its speech does not need or presupposes others. It is scientific in the broad sense of cognition. By contrast, in the second case the “aim” of the repetition is to establish ties with others. The subject says in order to address the other or to become open to his or her addressing. This does not exclude the constituting of the world but now the world becomes offered to others. From the very beginning it is a common world. Therefore, the logic of repetition is in both cases the same: repetition brings forth a differential system of significance (the world) at the same time leaving it unfounded. Both subjects may perform the same gestures and say the same words. But their goals are different. The first aims at the ceaseless opening of the world, its reinterpretation and saying “yes” to the coming future. The second seeks a community with others and offers a world, always being ready
to reject it for the sake of a new one.
How from this viewpoint does subjectivity look like in the case of Derrida's *différance*? We can recognise there at least four pretenders to the name of subject. The first is constructed by the narrative and is not actually different from any other constructed object in the world. The second appears already in the repetition as its very place (*Dasein*). This is the subject of mineness, and pronouns and deictic words point to it. The third one is the subject of involvement who refuses mere repetition and decides on its own involvement. It is the subject of the desire to signify or to have a system of significance. It does not merely repeat but lets the repetition happen. The last, forth, subject appears as that of engagement. This subject is engaged in a project broader than letting significance be. It does desire language but not for itself. It uses language for the sake of something else. Of these broader projects, as I have already said, two will be the focus of the following study. The one is self-sustaining which uses language for creating an artificial environment, for leading, as it were, an artificial life no matter what it is. The other project uses language for establishing ties with others. It does not exclude creating an artificial world but considers it as offered to others. The corresponding subject addresses others and—ready to be judged—exposes itself toward them. We have come here to two basic functions of language. On the one hand, it can be used for constituting the world, knowledge, phantasm, or whatever—in brief, the world to live in. Generally speaking, others here play the roles of initiators into language or obstacles but not necessarily beings to live with. On the other hand, language is used for speaking toward someone, that is, appealing, asking, answering, proposing help, blaming, or praising. These functions do not necessarily exclude each other. Constituting the world may become addressing others when this world is offered to them.
Now, the question is which of these types of subjectivity does Derrida's thesis about the subject as an effect of the signifiers' play imply? Primarily, no doubt, it is the
first one. But what about the rest? It is necessary to say from the outset that the description of *différance* hardly allows one to answer the question unambiguously. Indeed, the subject is criticised as present, but presence has two meanings. It is something either preceding language, or simultaneous with it (although at the same time independent of it). But what can we say about the gesture of repeating a signifier? After all, is the gesture of *différance* itself present? Or we should speak of presence as a sort of gesture’s substantivation, its “petrification” in trace? To what—the trace as a result or as a movement—does the “non-presentable past” precede? Derrida’s texts are too uncertain to provide the answers. The situation remains vague and I am compelled to give my own interpretation.
Let us start with the note that Derrida does not pay attention to the subjectivity of repetition. *Différance* appears as an anonymous event in which everything, including the subject, emerges (and immediately disappears). But even if we agree with this and accept that the mineness of event points to the subject brought about by the event itself, the status of two other subjects (that of involvement and engagement) in *différance* remains uncertain. Derrida’s critique of presence aims at the signified behind the play of signifiers. Well grounded, as it is in the case of essence-like items, this critique misses the point when it speaks of the event itself. Indeed, it does not say whether the event or signifiers are present. Even if we agree that there is nothing except traces that archi-writing leaves, what can we say about the acts of leaving traces themselves? Furthermore, the analysis of the signifying gestures (see section 1.3 in this thesis) shows that the traces that archi-writing implies are not exactly letters, written words, recorded sounds, and other “material” parts of saying. The trace is rather the whole system of significance: the gesture demonstrates the possibility of this system and in this sense leaves a trace. This reflects the fact that archi-writing is situated on the level of gesture (i.e. saying), not traces (i.e. the said). On this level there is no precedence and Derrida’s logic of supplement and inaccessible past
ceases to work. This, in turn, changes the character of desire. Movement toward the inaccessible presence situated in the radically forgotten past is superseded by the desire to repeat that does not need any presence beyond the repeating itself. This desire is without a lack. What it wants to achieve is perfectly realisable. This may be illusory but it is precisely this illusion that this desire wants. To make a little pun, it has no illusions about the illusory character of its own doing. This fidelity to an illusion is a peculiar feature of the subjectivity of engagement. Hence, the subjectivity of engagement is not corrupted by Derrida’s critique of presence. It dwells on the same level as différence, that of event. Repetition is necessary not because—in the supplementary manner—it provides an indirect access to presence. It is necessary because it is by itself the subject’s Being or the tie with others. It is desired as such.
From the standpoint of the theory of saying Derrida grasps the temporality of saying, the distinction between saying and the said, and their independence. At the same time, he neglects the subjectivity of the one who not only speaks but, more importantly, decides on speaking, on its fact and goals. One can say that Derrida neglects the function of language different from the cognitive ones, its usage for the addressing and self-exposing. Hence, Derrida’s différence is not able to account for both my and the other’s subjectivity. Nevertheless, Nancy’s theory of community can be to a large degree considered as an extension of différence to intersubjective relations. How in this case is he able to grasp the other? Having indicated the problem let us proceed to Nancy’s presentation of community.
Chapter 3
Jean-Luc Nancy: Community of the Origin of Community
The most important characteristic of community, as seen by Jean-Luc Nancy, is its interruption or suspension or undoing or unworking. He derives this trait from the ontology of event: the very Being of a community makes immanence or totality impossible and the community itself inevitably plural. However, at the same time he provides another line of argumentation based on writing or literature understood as establishing communal ties. Although Nancy traces literature/writing back to ontology, it has, I believe, a value of its own, independent of Being and its derivatives. Therefore, I shall start this chapter with the concept of interrupted community and Nancy’s ontological argumentation and end up with the communal meaning of writing and its independence from ontology. My aim is to demonstrate that what I call Nancy’s distributive model of community is based not on the ontology of event, as he claims, but rather on a sort of interpersonal relation exceeding this ontology.
3.1 Community as distribution of singularities. Spatial metaphor
By a genuinely postmodern gesture Nancy denies community immanence. He finds a justification for this in ontology or, more precisely, in the irreducible distinction between Being and entities. I shall discuss this later in the chapter. Now, in this and the following section, I would like to demonstrate that one of Nancy’s presentations of community (he in fact provides several), namely, its presentation as sharing and distribution, demands and rests on more than the impossibility of immanence. It presupposes the impossibility of
fusion with others, that is to say, the impossibility of the immanence of the social body or, even more exactly, the separation of *me* from the other.
Nancy begins by rejecting the notion of community as a number of individuals bound into community with external ties. He insists instead on the notion of singularity\(^{25}\). The latter does not precede community. Singularities rather appear simultaneously and form a community in the very event of this appearance. They are not merely bound by aids of communal ties, they themselves belong to the order of ties, rather than what is tied: “Singularity never takes place at the level of atoms, those identifiable, if not identical identities; rather, it takes place at the level of the *clinamen*” (Nancy 1991, 6). The individual as a closed up identity is impossible. But in what sense? At the beginning of *The Inoperative Community* (Nancy 1991, 4-6) Nancy applies Hegel’s argument in order to show the impossibility of immanence or the impossibility of the Absolute (see Hegel 1977, 158-9). The argument states that the Absolute being, should it exist, must be absolutely separated from everything, that is, must be without relations. But Being without relations is itself a relation, and this makes the individual as such impossible and, according to Nancy, makes a being, of necessity, the being in community: “The relation (community) is, if it *is*, nothing else than what undoes, in its very principle—and at its closure or on its limit—the autarchy of absolute immanence” (Nancy 1991, 4). Roots of community are seen in the impossibility of immanentisation, which is to say, in the impossibility of the One. We can easily recognise here the irreducibility of Being to entity, and Nancy in fact explains his argument adopting Heidegger’s distinction between ontological and ontical (*Sein* and *Seiende*, see sec.1.1). Thus the irreducible ontological relation proves to be the relation of the entity to its own Being: “a relation [of the Absolute] *to* its own being instead of making this being immanent to the absolute totality of beings” (Nancy 1991, 6). “And so, being
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\(^{25}\) On the notion of singularity, see also page 62 et seq. in this thesis.
‘itself’ comes to be defined as relational, as non-absoluteness, and, if you will—in any case this is what I am trying to argue—as community” (Nancy 1991, 6; emphasis added). Therefore, what Nancy calls community is this relational character of Being, Being-ecstatic of Being itself. In fact, the preoccupation of the whole book is revealed here. Let us follow, however, the actual picture of community that Nancy offers to readers.
The key word for this picture is partage translated in The Inoperative Community as sharing. Nancy defines it in spatial terms as distribution, partition, or dis-location. The community of sharing is that of singularities touching one another, but with these singularities constituted by sharing itself. Nancy sees his task in
thinking the sharing [partage] of community and the sovereignty in the sharing or shared sovereignty, shared between Daseins, between singular existences that are not subjects and whose relation—the sharing itself—is not a communion, nor the appropriation of an object, nor a self-recognition, nor even a communication as this is understood to exist between subjects. But these singular beings are themselves constituted by sharing, they are distributed and placed, or rather spaced, by the sharing that makes them others: other for one another, and other, infinitely other for the Subject of their fusion, which is engulfed in the sharing:
“communicating” by not “communing.” These “places of communication” are no longer places of fusion, even though in them one passes from one to the other; they are defined and exposed by their dislocation. Thus, the communication of sharing would be this very dislocation (Nancy 1991, 25).
What is presented here is the picture of a divided space inhabited by touching one another’s singularities. Besides, the passage contains what could be considered as the
definition of sharing: “communicating” by not “communing.” Community thereby stands opposed to communion, which is a society of immanence and “fuses the egos into an Ego or a higher We” (Nancy 1991, 15). The very definition of community and sharing refers to the impossibility of—or rather the resistance to—communion or fusion. What makes the resistance possible is the non-static character of distribution. Sharing is essentially the movement of coming into Being and fusion does not take place thanks to singularities’ co-appearing or, as Nancy terms it, compearing. This means for him that any singularity co-appears with other singularities and can appear only together with them. Whenever the singularity appears, others appear as well. Singularity exists only by and through exposing to outside (Nancy 1991, 29). Nancy says that singularities therefore share identities. This means, in its turn, that the actual event is the appearance not so much of singularities but rather of their very difference. Comppearance
consists in the appearance of the between as such: you and I (between us)—a formula in which the and does not imply juxtaposition, but exposition. What is exposed in comppearance is the following, and we must learn to read it in all its possible combinations: “you (are/and/is) (entirely other than) I” (“toi [e(s)t] [tout autre que] moi”). Or again, more simply: you shares me (“toi partage moi”) (Nancy 1991, 29).
How are we to understand the appearance of the between or exposition? In tune with Nancy’s previous discussion (the community of singularities touching each other), we should probably think of the distribution or dislocation of singularities, of the event of differing and drawing borders (although without any pre-existing space supposed to be divided). As we shall see, this understanding of comppearance is not incorrect. However, it is not complete either. Nancy develops another vision of the “between you and I” essentially based on his reading of writing. This vision will become our theme later. At this point I would like to focus on the distributive model of community. Its further specification
is the interruption of myth.
**Community interrupted or lost**
Another name for the impossibility of communion (opposed to the sharing of community) is the interruption of community\(^{26}\). Its exemplary figure is the interruption of myth. Nancy grasps myth as a founding narrative. Its principal function lies in founding a community. Myth does so through organising the community around itself: “myth necessarily contains a pact, namely, the pact of its own recognition: in a single gesture, in a single sentence, in sum, myth says what is and says that we agree to say that this is (it also says, therefore, what saying is)” (Nancy 1991, 50). Myth does not refer to any reality beyond itself, be it natural or social. It rather creates this reality, the reality of community: “myth says that it says, and says that this is what it says, and in this way organises and distributes the world of humanity with its speech” (Nancy 1991, 48). Saying here is akin to showing—myth shows the foundation, while at the same time inventing it. As origin, myth itself originates from nothing. For this reason it might be regarded as a fiction, but it is, as Nancy put it, “a founding fiction, or a foundation by fiction” (Nancy 1991, 53). Myth conceals no reality, it is itself a condition and foundation of any reality whatsoever. Nancy compares the so comprehended myth with Kant’s schematism—a condition of possibility for any human experience (Nancy 1991, 54). Myth is also language showing itself: “Fundamentally, *mythos* is the *act of language* par excellence, the performing of the paradigm” (Nancy 1991, 54). And, which is more important in our context, Nancy directly equates the founding functions of myth and Being and speaks of “a mythic essence of
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\(^{26}\) Although Nancy distinguishes between community and communion, with the term interruption being more applicable to the latter, he usually speaks of the interruption of community. Probably he means to emphasise that community is itself the movement of interruption.
Being” (Nancy 1991, 54). Thus, myth is “an absolutely foundational, symbolizing, or distributive speech” (Nancy 1991, 52).
Now, this myth is interrupted. This does not mean that it is absent. Although Nancy at times speaks of myth’s absence, he immediately corrects himself: it is the absence of a very special sort, better to be named interruption. What is absent is rather not the myth but its power to found a community, its pact and recognition. This is why the interruption of a myth is always accompanied by the interruption of a community. The loss of community, however, may be a resource for the community of another kind, “community of the absence of community,” so to say. The latter is opposed to fusion:
The absence of community appears with the recognition of the fact that no community, in the fusion that it is essentially seeking, for example in “the ancient festival,” can fail “to create a new individual, that one might call the collective individual” (Nancy 1991, 60). (Nancy quotes Bataille here)
This is another formulation of the impossibility of immanence or totality. Here, it is associated with the impossibility of the collective individual. Nevertheless, for Nancy myth is not merely communal, that is, belonging to communion. Its logic shows a community that does not disappear even after myth’s interruption. Nancy once again exploits the ontology of event. Despite its tendency to totalisation, myth is at bottom comppearance because it gives Being-in-common\textsuperscript{27}. Comppearance thus lurks within myth in spite of its
\textsuperscript{27}“Being \textit{in} common means that singular beings are, present themselves, and appear only to the extent that they comppear (\textit{comparaissent}), to the extent that they are exposed, presented, or offered to one another. This comppearance (\textit{comparution}) is not something added on to their being; rather, their being comes into being in it” (Nancy 1991, 58).
tendency to fusion. Moreover, it resists fusion. Nancy describes this function of myth in a manner of the ontology of event that brings forth everything that it contains.
The very idea of inventing a myth... is a contradiction in terms. Neither the community nor, consequently, the individual (the poet, the priest, or one of their listeners) invents the myth: to the contrary, it is they who are invented or who invent themselves in the myth (Nancy 1991, 58-9).
Hence, myth, like Being, is a distribution and dislocation. This is not to say that compearance is mythic. Myth remains totalitarian for Nancy. But he seems to argue that compearance takes a priority over myth and undermines its pretension to totality\(^{28}\). Myth still founds a community but tends to make of it a collective individual, which it is not. Accordingly, Nancy conceives of the absence of myth not as the absence of the founding but as the movement of “unfounding” or unworking (Nancy 1991, 60). Borrowing the term from Bataille, he calls this movement passion and explains: “the passion of and for community propagates itself, unworked, appealing, demanding to pass beyond every limit and every fulfilment enclosed in the form of an individual” (Nancy 1991, 60). This is a genuine communication because “this interruption once again exposes singularity to its limit, which is to say, to other singularities” (Nancy 1991, 60). It is in this way that Nancy derives the multiplicity of singularities from interruption. Indeed, passing beyond an individual leads apparently to the confronting of other singularities, and hence the distributive character of myth, compearance, and Being acquires the meaning of building a differentiated social body. This step is principal for Nancy’s theory of community and one of my major tasks in this work is to demonstrate its doubtfulness. But before we examine it more carefully, we need to expand on Nancy’s distributive model of community.
\(^{28}\) I would say that myth is parasitic on compearance.
Singularity
It would not be an exaggeration to say that ‘singularity’ and ‘singular’ are the most important words in Nancy’s vocabulary (at least in the two books considered). Not only is community composed of singularities or singular beings, but also all principal Nancy’s notions—compearance, Being, sense, event, etc.—carry the mark of singularity. It is their singular character that first of all stands opposed to totality, fusion, essence, myth, etc. Singularity designates the absence of any pre-given totality. The corresponding community is “formed by an articulation of ‘particularities,’ and not founded in any autonomous essence that would subsist by itself and that would reabsorb or assume singular beings into itself” (Nancy 1991, 75). Nancy recalls Marx as a thinker who was working out the concept of such a community, “a division of tasks that does not divide up a pre-existing generality” (Nancy 1991, 75). Nevertheless, singular beings do form a totality: “they relate, in some respect or in some way,… to a totality that marks their common end…” (Nancy 1991, 75). This sort of totality differs drastically from fusion. It is made up by the articulation of each member:
…articulation is only a juncture, or more exactly the play of the juncture: what takes place where different pieces touch each other without fusing together, where they slide, pivot, or tumble over one another, one at the limit of the other—exactly at its limit… without this mutual play—which always remains, at the same time, a play between them—ever forming into the substance or the higher power of a Whole. Here, the totality is itself the play of the articulations (Nancy 1991, 76).
Nancy goes further and, unlike Marx, conceives of not only totality but also singular beings themselves as formed by the same articulation. This view is first presented in *The Inoperative Community* and then further developed in *The Sense of the World* where the chapter ‘Someone’ (68-75) is entirely devoted to the theme of singularity. It starts with
the following passage:
There are some things, there are *some ones*, there are numerous *ones, singularities*. Sense is the singularity of all the singular ones, in all senses simultaneously. It is singularity first of all in the distributive or disseminative sense of the nonsubstitutable unicity of each singular one (be it, for example, a rock or a man named Peter [*pierre ou Pierre*]. But sense is also the singularity of the singular ones in the transitive or transitional sense of what shares them out and what they all share among themselves (their finitude, common to all, proper to none, as a common impropriety, communicating yet neither communicated nor communion).
And finally, sense is singularity in the collective or worldly sense of what makes of the totality of the existent the singular absolute of being (its infinite spacing). (Nancy 1997, 68)
As we can see, Nancy identifies “some one” with singularity. The latter is therefore the name for the member of community. Nevertheless, this passage does not directly deal with human community and does not even differentiate things (“a rock”) and people (“a man named Peter”). So far, therefore, singularity has nothing to do with community. What we do learn from the passage is that singularity covers three clusters of meaning: distribution, finitude, and “collectivity” of Being. They are closely connected and opposed to the metaphysical subjectivity or the presupposition of presence. The latter is something that precedes its own presentation, although appears only through it. “The *sub-* or *subjecthood* represents in a way the inverted form of the *prae-* of *pres-ence*: the present that precedes itself and thus also remains behind itself” (Nancy 1997, 69). But, according to the logic of event which Nancy applies here, such presence “resolves itself into an *absence* in the very instant of its—supposed—presentation” (Nancy 1997, 69). The foundation thus disappears and we can sink into nihilism but precisely at that point, according to Nancy,
we come to the singular existence of the founding itself, “the simple and absolute position that cuts short all supposition, all sub- or pre- . . . no longer privation of all predicates, but predicates without support, holding each other together mutually, singularly” (Nancy 1997, 69-70). Thus, we shift from the pre-existing presence that presents itself to the logic of presenting by itself.
The next six pages of Nancy’s text are devoted to a more detailed examination of singularity. He offers three traits of the singular (someone): it is “unique”, “whatever”, and “exposed” (Nancy 1997, 71-5).
**Unique.** “The unicity of the singular consists quite exactly in its multiplicity” states Nancy. How are we to understand this statement? Nancy’s explanation shows that its primary meaning points to the simultaneity of singularities and their relations. No one exists alone: “‘One’ means: some ones *and* some other ones, or some ones *with* other ones” (Nancy 1997, 71). The singular does not possess its own singularity: “singular unicity is what shares it out and what it shares with the totality of singular multiplicity” (Nancy 1997, 72). And furthermore: “it does not constitute its singularity on the basis of its own resources—on the contrary, it does so on the basis of the most common resource, the one that comes (down) to each and every one as to none” (Nancy 1997, 72). What is this common resource Nancy refers to? In fact, the unicity of every one is a consequence of the world’s unicity, or more exactly still, the event’s unicity. The singular “is being-as-act or being-in-action,” and the whole analysis of event (see chapter 1) becomes relevant here. Therefore, the unicity of the singular amounts to the unicity of the event or happening. On the other hand, the multiplicity of the singular is situated on the level of what happened, not happening. But it is the latter—as Nancy put it, the signature of existence—that assures the unicity of all singularities and appears thereby as the primary singularity. Given that singularity is conceived in the context of distribution, one might be tempted to attribute unicity to the ‘some ones’, that is, to the members of the community. But in fact unicity is
that of the temporal instance. It cannot structurally be applied to the distributed items. In other words, this unicity points to the temporal rather than spatial multiplicity. Let us note this ambiguity.
Whatever. Nancy stresses a simple fact: “every one is just as singular as every other one”, “always capable of coming in the place of the other”. This substitutability may be understood—and Nancy definitely means that—as a similarity in the structure of happening, that is, as a similarity insofar as the singularity is the event, happening, singular going-coming. This similarity is independent of the (possible) dissimilarity or even incommensurability of what happens: “Birth/death, each as the other, a singular going-coming, represents this intersection of substitution and nonsubstitution, of the replaceable and the irreplaceable, the whatever, and the unique” (Nancy 1997, 73). It is in this sense that “what is commensurable in them [singularities—OD] is their incommensurability” (Nancy 1997, 72). In this way, I believe, one should understand Nancy’s notion of example:
... every one, being born, dying, being-there, exemplifies singularity.
Each proposes itself as an example, if you like, but it exposes this example, every time, as exemplary, in the sense of a remarkable model.
That which is exemplary each time, that which sets an example, is singularity itself, insofar as it is never anything but this or that singularity, inimitable at the very heart of its being-whatever. (Nancy 1997, 73)
Examples are singular in their ontological structure, which essentially implies the uniqueness of the act of existence. Accordingly, that the similarity of examples does not refer to any generality or universality of some “ideal existence” simply means its belonging to the level different from where any “ideal existence” may occur. If it is “generality,” it is that of repetition, not of substance.
The very term ‘substitutability’ and the idea of example as a remarkable model indicate rather that the ‘what’ of happening is repeatable. I shall later rewrite Nancy’s presentation of singularity in terms of saying and repeating but already at this point we can see the affinities of singularity with the act of saying, of the substitutability of “whatever” with the accessibility of the said (note Nancy’s usage of deictic words—“this or that singularity”—in the quotations above), and of the uniqueness of the singular with that of the instance of saying. In terms of repetition, what is repeated and the structure of repeating are commensurable, whereas the event or the fact of repeating is incommensurable. The former belongs to the system of language (including its ‘languageness’), whereas the latter marks the place of the subjectivity of involvement. Thus, the reduction of the event (in fact, saying) to the distributive structure of language amounts to the elimination of the speaker from the event.
Exposed. In a comment on the Russian translation of his paper “Répondre de l’existence” (Nancy 1998, 317) Nancy points out that the notion of exposition is especially important for him. It denotes ex-position as both positioning outside and Being-subjected-(to), risking. It points to the existence as openness towards others in the sense of making oneself accessible. This is the meaning Nancy keeps in mind while describing singularity (1997). However, it is embedded into the context of the ontology of event and thus bears no explicit reference to this idea. Later on, in the consideration of community, exposition acquires precisely the meaning of becoming accessible (this conceptual shift has yet to be dealt with). But here, the main meaning of exposition is the attestation of existence: “what is exposed is exposition itself. What is presented is coming-into-presence, and thus, the différence of its being-present” (Nancy 1997, 74). And further: “What is exposed in this way is thus a singular transitivity of being, and what every one engages in is an attestation of existence” (Nancy 1997, 74). And finally: “Here attestation replaces foundation. In its way, this formula contains all sense” (Nancy 1997, 74-5). This radical expression of the
primary status of the event is simultaneously the expression of the absence of the pre-given “without the least connotation or intonation of an appeal to a hidden sense or revelation” (Nancy 1997, 75). Therefore, this meaning of exposition falls entirely into the pattern of the ontology of event. Happening, the event attests the existence, which needs henceforth no other “foundation” whether it is substance, essence, or pre-given sense. Singularity is the absolute beginning. It is a response with no preceding question, “it is a given guarantee, a promise, an engagement” (Nancy 1997, 71). Is there anyone who gives the guarantee and makes the promise? No, it is rather the event by itself that guaranties and attests. According to the logic of event, nothing precedes it, and anyone capable of promising is to be born in the event itself.
“There is” and the opening of the world and sense
In *The Sense of the World*, as the title shows, the leading theme and problem is the sense of the world. Nancy starts with the absence of sense and comes to the equivalence of world and sense. Since sense is absent, the world no longer refers to it. None the less, there remains the fact of the world’s Being, which precedes any signification. As a consequence,
If we are toward the world, if there is being-toward-the-world in general, that is, if there is world, there is sense. The *there is* makes sense by itself and as such. We no longer have to do with the question, “why is there something in general?” but with the answer, “there is something, and that alone makes sense.” (Nancy 1997, 7)
“The sense of the world” becomes a tautological expression. The world does not *have* sense any longer, but *is* sense. The world comes to be the starting point, the first and the last reference of meanings. Its presence insists absolutely: “‘The world is the origin’ means that the origin is *there* where it opens itself” (Nancy 1997, 160). Or in a short formula: “*There is*: sense is there” (Nancy 1997, 160). At this point Nancy evokes the
notion of significance (significance which makes signification possible). He explains in a note the meaning of significance and cites Jean-Christophe Bailly: “The unlimited, but in all the limits that give access to it despite everything, and that coincide with the arrival of sense, with the fact that there is sense” (Nancy 1997, 173). Sense that comes before any signification and the event of sense/world/Being is in fact the origin of significance. We have arrived at the further specification of the distributive model. Sharing here becomes the origin of the signifying order, which is to say, of the order in which alone signification can appear as such. For this reason I shall also call the distributive model that of the origin of significance.
What precisely, however, is there? Is it a particular sense or the fact of its Being? What is attested in this way, a particular order of significance or the fact that there is whichever significance at all? In his explanations Nancy definitely conceives of sense as purely the fact of sense. In this way, what is opened in the opening of the world/sense is this opening itself. What seems to be important for Nancy’s theory of community is the fact of attestation, no matter what is attested. This is only apparently strange, since, as we shall see soon, what Nancy wants to describe is not such or such a community but rather the very event of its establishing.
**Sense and truth**
Further elaboration of the distributive model can be found in Nancy’s distinction between sense and truth. They are distinguished ontologically:
That one speaks of sense does not mean that one abandons or disdains the category of truth. But one does shift registers. Truth is being-such [l’être-tel], or more exactly it is the quality of the presentation of being such as such. Sense, for its part, is the movement of being-toward [l’être-à], or
being as *coming* into presence or again as transitivity, as passage to presence—and therewith as passage *of* presence. (Nancy 1997, 12)
If we recall Heidegger’s definition of meaning as “something *as* something [etwas als etwas]”\(^{29}\), we shall immediately notice the parallel between the sense-truth and Being-entity distinctions. This is the distinction between Being as *such* and Being as *Being* (Nancy 1997, 12). The former points to the essence (Nancy’s example: the essence of the phenomenon is to appear, *φαίνομαι*), whereas the latter is the action of the verb ‘to be.’ This allows Nancy to speak of the transitive character of Being: Being *is* the entity, it is Being *toward* the entity, “the structure, property, and sense-event of the entity in general” (Nancy 1997, 13). Just as Being is the presentation of entity, so is sense that of truth: “the distinction I am drawing between truth and sense is the distinction between the presentation of a present or the scene of presence and its division within itself” (Nancy 1997, 175). Sense is a presentation, truth is what it presents. At the same time, this is not the only function of sense. Besides presenting, sense provides a connection of truths that Nancy calls enchainment.
Truth punctuates, sense enchains. . . . [Punctuation] is always without spatial or temporal dimension. Enchaining, on the contrary, opens up the dimensional, spaces out punctuations. There is thus an originary spatiality of sense that is spatiality or spaciousness before any distinction between space and time: and this archi-spatiality is the matricial or transcendental form of a *world*. In turn, truth is in principle instantaneous (. . . one could
---
\(^{29}\)“Meaning is the ‘upon-which’ of a projection in terms of which something becomes intelligible as something; it gets its structure from a fore-having, a fore-sight, and a fore-conception” (Heidegger 1962, 193) (emph. by Heidegger).
say that it is the a priori form of a universe, in the literal sense of the gathering-into-one). (Nancy 1997, 14-5)
The instantaneousness of truth does not prevent it from being divided within itself. Although what we have read until now may leave an impression that truth is a temporal instant whereas sense should be associated with temporal interval, this is not exactly so. For example, when explaining the analogy between sense-truth and syntactic-semantic distinctions (Nancy 1997, 15), Nancy conceives of punctuations as rather instantaneously co-existing. In fact, he does not distinguish between ‘inter-’ and ‘intra-instantaneous’ division. This is why the spatiality of sense precedes any distinction between space and time. Here he follows Derrida, and we shall see that he repeats the structure of différence in which every element is endowed with a meaning only thanks to temporal references to other elements (the temporal—retention-protection—division of presence in itself). But, as we have seen, this feature of différence is problematic, since Derrida does not differentiate two times of diachrony and in fact reduces the external time to the internal one (to be more precise, he does differentiate them but only in order to equate them immediately). Consequently, we find the same problem in Nancy’s ontology. This will show itself in the concept of community that Nancy derives from the outlined theory of sense.
Citizen and subject. Establishing communal ties
However important the ontological understanding of sense set forth in the previous section is, its principal model for Nancy is political. It is above all political intuitions that Nancy seeks to explicate in terms of Being, comppearance, and so forth. This is why the image of sense given in the chapter Politics II (Nancy 1997, 103-17) rules, in fact, the whole book.
Politics there is distinguished from love or hate in that it does not presuppose any ties already tied or given (Nancy 1997, 103). This absence of ties is the cardinal feature of
the community Nancy speaks of. Furthermore, he separates the politics of citizenship and subjecthood. “The citizen is, first of all, one, someone, everyone, while the subject is, first of all, self, that is, the circling back through which a one raises its unicity to the power of unity” (Nancy 1997, 104). Citizenship points to a common field of alienated identities, and is nothing else than their sharing:
...the citizen is a mobile complex of rights, obligations, dignities, and virtues. These do not relate to the realisation of any foundation or end other than the mere institution of the city. In a sense, the citizen does nothing other than share with his/her fellow citizens the functions and signs of citizenship, and in this ‘sharing’ his/her being is entirely expressed. (Nancy 1997, 104)
The in-common of the city has no identity other than the space in which the citizens cross each other’s paths, and it has no unity other than the exteriority of their relations. In a certain sense, citizenship in accordance with its pure concept is always virtually citizenship of the ‘world.’
(Nancy 1997, 104)
Therefore, unicity or oneness is the principal characteristic of the citizen. However, Nancy’s description is uncertain. If the citizen is “a mobile complex of rights, etc,” what can we say about the unicity of items (rights, etc) themselves? Do they possess any oneness different from that of the citizen? Later on in the chapter Nancy seems to answer this question negatively, and any unicity appears to emerge in the same event/move of tying. Here, by contrast, Nancy differentiates them, and this reveals a problem. Namely, Nancy’s general intention is to deny any sort of pre-given presence. A citizen, if any, is no more than an effect of the institution of the city, mobile complex of items (rights, etc)
constituted by the institution. But it remains unclear whether one can say the same about the items themselves.
Unlike the politics of citizenship, that of subjecthood implies “the demand for a subjective appropriation of sense” (Nancy 1997, 105). The model of this appropriation is the Hegelian subjectivity which “retains within itself its own negativity” (Nancy 1997, 106). Accordingly, the political subject appropriates the constitutive exteriority of the city (Nancy 1997, 106). The subject, therefore, has no interiority different from the appropriated exteriority. This means, among other things, that the subject presupposes the division of the city as something which the subject is to appropriate. In fact, there is a full reciprocity between citizen and subject. Both “mirror” each other.
The citizen becomes subject at the point where the community gives itself (as) an interiority, and at the point where sovereignty no longer contents itself with residing in the formal autoteleology of a “contract,” or in its autojurisdiction, but expresses also an essence... Reciprocally, the subject makes itself into a citizen at the point where the expressed essence tends to express itself in and as a civic space and, if one can put it like this, to “display” subjective essentiality. (Nancy 1997, 106)
Therefore, as the passage demonstrates, conceptually citizen and subject share the same element. This is the form of the city, which Nancy understands as a network of connected items. The difference between citizenship and subjecthood lies then in the first being, in fact, the contingent institution (appearing) of the network, whereas the second is its interiorisation. More exactly, subjecthood begins with presupposing (instituting, opening up) the sphere of interiority into which the exteriority of the city can be projected. This common element leads, according to Nancy, to the intrinsic defect in both politics — that of citizenship and subjecthood. They take sense as something given or self-sufficient. Social ties in the case of citizenship or the items that they tie in the case of subjecthood are
considered as self-sufficient (Nancy 1997, 111), whereas the problem is precisely these ties themselves. Nancy formulates the task of inventing a politics with “no longer any subject, but no citizen either” (Nancy 1997, 109). He finds this politics in the very activity of establishing ties:
It is therefore a matter of going toward a thought (that means indiscernibly toward a praxis) of the (k)not as such. It is the tying of the (k)not that must come to the crucial point, the place of democracy’s empty truth and subjectivity’s excessive sense. (Nancy 1997, 111)
The tied here is understood in two senses of (k)nots and their connection. Tying thus establishes both (k)nots and the network of their connections. It is a gesture, “the very gesture of tying and enchainment of each to each, tying each time unicities (individuals, groups, nations, or peoples) that have no unity other than the unity of the (k)not, unity enchainied to the other, the enchainment always world-wide and the world having no unity other than that of its enchainment” (Nancy 1997, 112).
Thus, we can recognise in tying a further development of the concept of comppearance from *The Inoperative Community*. Community is again understood within the distributive model of co-appearing singularities. Here, however, it acquires an additional aspect—it is interpreted as a search for sense. From the very outset of *The Sense of the World* Nancy acknowledges the essential absence of sense. The task of the book is stated as follows: “I will suggest that the only chance for sense and its only possible sense reside either this side of or beyond the appropriation of signifieds and the presentation of signifiers, in the very opening of the abandonment of sense, as the opening of the world” (Nancy 1997, 3). The absence of sense is considered as its crucial trait. At the same time, the politics of tying can be summarised in the phrase: neither myth, nor nihilism. This formula stands opposed to two regimes of sense: its absoluteness (myth) or arbitrariness and dissolution (nihilism). Both, Nancy claims, are caught up in the regime of signifying
sense, regime presupposing either presence or absence of sense. Here, sense is "the signifier of a proper and present signified, the signifier of the proper and the present as such" (Nancy 1997, 3). It matters little whether this signified is posed as stable and perennial (myth) or as absent and illusory (nihilism). Rejecting both possibilities, Nancy nonetheless renders sense as in some way present, namely present precisely as something to be established. The search for sense is not a disadvantage or misfortune—"not deprivation but a condition of existence" (Nancy 1997, 15): "Nothing is lacking in our being: the lack of given sense is, rather, precisely what completes our being" (Nancy 1997, 152). Community exists precisely as not given. To make a community—inoperative community—means to move towards the community, with this movement itself being precisely what makes the community. This specific temporal structure constitutes the "coming-and-going" character of community. This marks the task of re-establishing community, re-establishing its ties—the coming community is a task. Nancy compares community/coming with breaking a path (frayage) or creating a melody in a jazz improvisation. In any case it is the establishing of ties that in no sense pre-exist. "Everything remains to be invented" (Nancy 1997, 121) is a formula to have fundamental significance for the understanding of sense. It is the very meaning of coming. It points not to obligation but to assignment, setting a problem. Ethics exists not as rules to comply with, or obligations/commands to execute, but as an ethical problem to be solved: "the absolute injunction of having to establish ties" (Nancy 1997, 121). Its absoluteness consists in the impossibility of avoiding it.
Nancy associates this towardness of sense with the transitivity of Being. The latter designates something which has not yet finished: "that which is not in its end (deferring itself in its end, deferring its end in itself), is toward itself" (Nancy 1997, 28). Nancy grasps this "not being in the end" as breaking path (frayage), in which every new instant is the opening of a new space. The act of Being is not produced: "It 'is produced' in the
remarkable sense of ‘taking place,’ ‘happening’” (Nancy 1997, 28). The absence of sense, therefore, indicates rather the birth of a new sense and impossibility of holding for oneself what was born (this is what Nancy calls the withdrawal of Being in the very event of Being). But here a question can be raised. The happening of Being seems to be anonymous. It merely happens, whereas the search for sense should probably imply some one who searches. It has not yet become clear how Nancy copes with this anonymity of Being when he enters the field of community. In fact, it is in this passage that Nancy’s exposition becomes perhaps most ambiguous. Thus, we are coming to the discussion of this ambiguity. But before we proceed, let me outline the main characteristics of the distributive model that was the theme of the current section.
**The distributive model of community**
- The simplest scheme is that of attestation. According to it, there is a movement that sets up or attests an existence. The latter needs no further elaboration, as the scheme stresses its independence from any pre-given essence.
- A more concrete scheme is that of punctuation and enchainment according to which point-like items with neither temporal nor spatial dimensions are enchaigned due to a movement of towardness directed from one item to another. This is a general scheme of the community’s resistance to totality: the accent shifts from the unity to its establishing. The latter is opposed to the veiling/unveiling of *aletheia* or appearing of phenomena, insofar as they still reside “on the level of punctuation, not on the level of enchainment” (Nancy 1997, 175). They still privilege truth whereas “the *world* invites us to think no longer on the level of the phenomenon, however it might be understood (as surging forth, appearing, becoming visible, brilliance, occurrence, event), but on the level... of the dis-position (spacing, touching, contact, crossing)” (Nancy 1997, 176).
- In the section about suffering and unhappiness Nancy brings forth three formal structures of sense.
There are perhaps only three formal structures of sense: (1) the observance of an order or ritual of the world, where all unhappiness is a tragic omission opening onto truth (Oedipus); (2) salvation, where unhappiness is an illness, a worldly alienation that calls forth the tragedy of its infinite healing/expiation (Parsifal); (3) existence, as the exposition of being-toward-the-world or being-world—where evil seems coextensive with good, the “worst” with the “best,” and where therefore the exposition has to be decided each time. In other words, sense as given, sense as mediated, sense as surprise. Or again, in another register, sense as set of signs, sense as signification, sense as origin of significance.
(Nancy 1997, 147)
This links the distributive model with the concept of signification. As a result, dislocating becomes the birth of the signifying order (which is divided in itself). This can be understood as comppearance but as that of meanings rather than members of community.
3.2 The ambiguity of the distributive model
Multiplicity and temporality
Speaking of community is of course a speaking of the multiplicity of people. Indeed, individuals, groups, unions, and other diverse relationships are always implied in all of Nancy’s discussions. What he argues against is the model of closed up individuals (including collective ones) bound into a pre-established community. Thus multiplicity precedes unity. However, not being fully satisfied with this claim, Nancy tries to derive multiplicity from the structure of Being. Arguments here are various. The simplest one recalls Hegel’s reasoning for the impossibility of pure autonomy (see p. 56). However, this seems rather misleading. Indeed, Hegel’s issue is the irreducibility of entities’ relation to
Being or of the difference between Being and entities. He speaks of the duality of existence, so to say, not its multiplicity. In this argument, we rather deal with the alterity of Being (moreover, it is not clear how this anonymous alterity may be converted into the personal Other). However, Nancy suggests two other lines of argument. One is based on temporality, the other on the concept of voice. Let me start with temporality.
“There is not merely one thing in the world, and only thus can there be something (or rather some things)” (Nancy 1997, 67). In the section entitled Spanne Nancy draws this conclusion from the temporal separation. It is time that is “intrinsically spanned or stretched” (Nancy 1997, 64). In order for the passage of time to take place, the present, the now must be extended. This extended taking place of the present is more original than a succession of punctual presents: “There can be no passage from one present to another, if neither the one nor the other takes place” (Nancy 1997, 64). Nancy starts with arguing against time understood after the model of variations of the ever-present substance. By contrast, Nancy’s theme is not a modification but, if one can say so, the birth and death of substance: “It is the spacing of the present that takes place, as present, when a ‘substance’ or a ‘subject’ is coming along or going away” (Nancy 1997, 64-5). Thus the present features the complex temporality of coming-and-going. Furthermore, the main sense of coming-and-going is the interruption of temporal continuities. Sense as presentation without presentness [présentité] “comes and goes, as an interruption of symbolic enchainment and of substantial continuities. Or more precisely, it comes and goes, as an interruption of symbolic enchainments qua concatenations that ensure, by means of signification, a communication of substances (substances that are thereby signified, supposed, and subjectified)” (Nancy 1997, 134). This all means indeed that temporality is inherent in the present, but in what sense can we say that the present precedes itself? It rather springs up as always-having-already-been. But again, in what sense? Heidegger and Husserl are more precise on this point when they say—each in his own manner—that the
presence is directed or intended towards the past and future (with the past, future, and present each having different structures). As we have seen in the introductory chapter, these intentions belong to the internal time of event, whereas coming-and-going (the gesture of presenting, as Nancy put it (Nancy 1997, 66)) belong to the external time. The latter is structured “in accordance with a not”\(^{30}\), which means that temporal span implies present and non-present. It is a passage but not “the effective step from one existence to another.” Consequently, the multiplicity of presents found in the internal time does not take place in the external one. Thus, Nancy’s inference of the multiplicity of presents from the external time of coming-and-going seems doubtful. But even less can we draw the multiplicity of things from temporal separation. Nonetheless, this is precisely what Nancy does:
Separation as tension—time stretched like a bow of which it would also be itself the arrow. But for that, precisely, in order that there should be separation of the stretching or being-stretched out, no punctuality, no instantaneous instant, but the instant as spacing out, and spacing out as the simul of the several things that make up a world. (Nancy 1997, 66-7)
Obviously, spacing here has a temporal meaning. It is the creation of time itself, the time of event. The following is one more passage that shows that plurality for Nancy definitely carries in itself a temporal meaning: it is, in fact, the plurality of instants.
There is not merely one thing in the world, and only thus can there be something (or rather some things). If there were only one, there would be only pure time, immobile duration [durée]. But there is more than one
\(^{30}\)“Nothing . . . is except in accordance with a not: the crossing from nonbeing to being, that is, from being itself insofar as it is nothing to being itself again insofar as it is (entrances) the existent—and the crossing from being to nonbeing.” (Nancy 1997, 65)
thing, and this means not so much that there are several times one but, rather, more than one—the plural of a singular that is itself always and from the very first plural (*singuli*, for “*singulus*” does not exist)—*more than one* is the more than oneness in the very present of the one, its excess that separates it from itself within itself. (Nancy 1997, 67)
The passages clearly bring out that the multiplicity of things is, in fact, that of instances. Hence, the temporal (interruption) turns into the spatial (dislocation, dividing). This happens in two steps. The first is the identification of the two times of diachrony. The second is the interpretation of the temporal division of the internal time in terms of dislocation and spacing as such (this logic is a Husserlian heritage present also in Derrida’s *différance*: the division of entities is temporal in the first order, since the entity exists only thanks to its reference to other entities in the past and future). The next step that Nancy makes after his identification of the temporal and spatial is the interpretation of multiplicity as social. The goal of these transformations is to suggest a new type of social relation instead of substantial unity (myth) and contingent union (nihilism). However, we can see that his attempt to found this relation on ontology is rather doubtful. Does this mean that the whole theory of community also becomes so? No. As a matter of fact, Nancy proposes another vision of these relations and thus another logic of the inoperative community. It is rather implicit, unlike the distributive model. He relies on the event that is in many respects similar to the event of Being. This is the event of speaking toward the other. It brings about separation rather than distribution.
**Separation versus distribution**
Compearance
consists in the appearance of the *between* as such: you *and* I (between us)—a formula in which the *and* does not imply juxtaposition, but
exposition. What is exposed in compearance is the following, and we must learn to read it in all its possible combinations: “you (are/and/is) (entirely other than) I” (“toi [e(s)t] [tout autre que] moi”). Or again, more simply: *you shares me* (“toi partage moi”) (Nancy 1991, 29).
I have already quoted these lines while discussing the distributive model (see p. 58). They were supposed to demonstrate that not so much items as their differences compear in compearance. Now let us note that the passage also suggests that compearance is not merely a co-appearing of items or differences. It has to do with “you and I,” that is, with very specific singularities, one of which (*I*) is perhaps the most specific of all. For Nancy the difference between “you and I” and between any other singularities are of the same order. This permits him to apply the logic we have examined in the previous section: Nancy starts with the ontological distinction (*Sein-Seiende*) which he interprets as the impossibility of immanence. Then he infers from it the unavoidable co-presence of the other (*you* co-appearing with *I*); after that he assumes others’ presence and applies to them the same structure of the impossible immanence; at last, this all culminates in the picture of singularities compearing in the event of dislocating. However, this is not the only logic of community in Nancy. He also provides another vision of the same situation, in which he accentuates relations between the two rather than multiplicity.
On the one hand, it is a dialogue (“Here, *the totality is itself the play* of the articulation. . . . This totality is the totality of a dialogue” (Nancy 1991, 76)). This dialogue has a specific meaning:
I no longer (no longer essentially) hear in it [dialogue—OD] what the other *wants to say* (to me), but I hear in it that the other, or some other (*de l’autre*) speaks and that there is an essential articulation of the voice and of voices, which constitute the being *in* common itself (Nancy 1991, 76).
This is more than a simple compearance of entities. It accentuates the other’s
saying in its distinction from the said. On the other hand, community is based on the death of others. Nancy quotes Blanchot:
The basis of communication is not necessarily speech, nor even the silence that is its foundation and punctuation, but exposure to death, and no longer my death, but someone else’s, whose living and closest presence is already an eternal and unbearable absence (Nancy 1991, 61).
Paradoxically, a proper communication is that with the absent other. Communication with the other starts with separation. “To be in common” proves to mean “to be separated.” This points to the face-to-face relationships with each particular other rather than to the compearance of multiple singularities. This also gives sense to the words I have already quoted: “…the between as such: you and I (between us). . .”
At the same time, communication in Nancy is linked with my death (and birth):
…each birth exposes another singularity, a supplementary limit, and therefore another communication. This is not the opposite of death, for the death of this singular being who has just been born is also inscribed and communicated by its limit. It is already exposed to its death, and it exposes us to it as well. Which means, essentially, that this death as well as this birth are removed from us, are neither our work nor the work of the collectivity (Nancy 1991, 60).
The inaccessibility of death for our work marks the impossibility of immanence, which is similar to what we have seen in the ontology of event. Although, here we also deal with the relation between the two, these two are rather the singularity and its otherness or alterity. Nancy identifies this otherness with that of the other in dialogue. What is the logic of this identification? Do they rather have different incommensurable structures? In order to answer these questions I shall put into play the concept of saying.
Saying and Nancy’s ontology
The next two sections are devoted to a systematic interpretation of Nancy’s ontology and concept of tying in terms of the saying/said distinction. Let us start with the observation that the structure of event is parallel to that of language-in-act. This is to say that one can discern saying, said, and signs within it. The transitivity of Being appears as language’s feature to produce the said in the act of saying. Being-entities distinction becomes that of saying-said. Repetition peculiar to saying becomes that of example. Nancy’s distinction between signs, signification, and the origin of significance (see Nancy 1997, 147 and page 76 of the present text) can be interpreted in terms of gestures. In the case of signs (myth) gestures are self-consistent rituals and the repetition is nothing but obedience. In the case of signification they start meaning something thanks to their reference to the invisible beyond the gestures. Although the logic of this reference is arguable, in any case it implies a system of significance (which is perhaps by definition the system of references as such). Significance gives birth to the distributive multiplicity of meanings. The same gestures are able henceforth to possess different meanings when they are inscribed into different orders of significance.
What is important is that in any case repetition is an appropriation, and the system of language logically precedes the fact of its usage. Language is pre-given, pre-established. To deny this pre-givenness is to turn to what Nancy calls the origin of significance. This denial destroys the temporal unity of the signifying order (therefore, two steps from signs to signification and then to the origin of significance may be treated as the denial of the distributive and temporal unity respectively). Nancy speaks of the fact of significance and grasps language as its ceaseless originating of significance. The repetitious character of saying is opposed here to the perpetual character of substance. In order for that to be, saying must exclude anything pre-given. This means that Nancy deals with a specific aspect of language—its making meanings possible or its establishing the order of
significance. This is a proper meaning of the absence of the pre-given. The latter stands opposed not to the structure or logic as such but rather to the unchangeable structure of the said—in a sense the logic of saying (i.e. language) is pre-given but its pre-givenness is not like that of essence. Nancy’s ontology has to do with the specific structure of the fact of Being (namely, the structure of the origin of significance) rather than with merely this fact itself.
The absence of any pre-given unity of sense or Being may be safely called the guiding idea of Nancy’s ontology. It is developed against different kinds of totalities. The first of them is temporal. In opposition to the constancy of substance, Nancy relies on the consistency of repetition. Thus he speaks of Being-as-act or Being-in-act. It essentially holds in itself the withdrawal, which is in fact a synonym for the absence of the pre-given. However, along with the negativity of the absence, the act or event points to the positivity of attestation. The gesture of presenting and the fact of Being are undeniable. Sense is there regardless of any foundation it might have or not. The task of philosophy is for Nancy to think the world and sense as such without referring to any pre-established structure whatsoever\(^{31}\). Sense therefore begins with each singularity (and singularity is therefore that of sense). This repetition (rhythm) is the temporal structure of event. We do not have to understand the absence of sense and the “nothing” of “All” as the absence of any structure at all. Nancy himself points out that this would be incorrect. Rhythm is this basic structure without being substantiality (Nancy 1997, 141-2). Repetitious saying possesses this rhythmic structure with its withdrawal at any instant of performing.
The second sort of totality is the “instantaneous” unity of existence. The interpretation from the standpoint of saying allows us to understand resistance to it as the internal multiplicity of saying. This can be done in several ways. First, significance as a
\(^{31}\)In this particular way, Nancy is a phenomenologist.
differential system of gestures implies their multiplicity. A gesture never appears but always co-appears. Second, saying allows the multiplicity of orders of significance. Nancy’s early text (1990) shows how important the hermeneutical notion of horizon is for him. In this text he considers the sharing of voices as the ability for the said to be inscribed into different horizons and thereby to acquire different meanings\(^{32}\). This makes any said inevitably multiple. Third, saying is multiple in the sense of compearing voices, which does not amount to the co-presence of horizons. Of these three types of multiplicity the first two can be understood on the basis of the notion of significance. They reflect different kinds of its internal multiplicity. By contrast, the third one demands others who are not merely other orders of significance. We encounter here the third type of totality different from the temporal or instantaneous totality of meanings. Nancy evokes it when he discusses the passage beyond the individual. This totality, thus, is that of the individual and to exceed it means to touch other individuals (who thereby cease to be individuals and turn into singularities). In the distributive model community is derived from the fragmentation of the world in general. However, does Nancy’s criticism of
\(^{32}\)In this text Nancy espouses the Gadamerian view of the two preconditions of understanding: 1) anticipating or announcing a meaning having to be interpreted and 2) regulating this anticipation on account of prejudices. Having applied it to the experience of the other, Nancy concludes: “the other can neither be signified, nor presented, but only announced. The announcement is, in this way, the mode of the proper presence of the other” (Nancy 1990, 247). On the one hand, this safeguards the other against placing him/her in one row with presence or signified. But on the other hand, this still grasps the other in the framework of meaning, this time not present but announced. To be more precise, this text reveals Nancy’s ambiguity on this point. In his description he persistently moves amongst the announced meaning, the act of announcing, and the one who announces, tending nevertheless to remain within the context of meaning and meaningfulness. In contrast, the description of the other demands rather to account for the addressing which does not necessarily imply any transmitted, coming, or announced meaning.
totality truly lead to community? To answer the question let us turn to the concept of compearance.
Compearance also has more than one meaning in Nancy. However, they all have a common core based on the primary function of compearance: to overcome totality. Event, Being, sense are not total because at bottom they are a compearance (or sharing). However, it is not easy to understand what precisely this means because Nancy persistently moves from the level of saying to the level of the said and vice versa. As a result he tends to understand separation (which has to do with others’ sayings) in terms of the said, that is, of the distribution of meanings or horizons. Indeed, we find three aspects of compearance in accordance with three kinds of the overcoming of totalities. Compearance refers to the instantaneousness of the event, ungroundedness of the appeared, or withdrawal. This all refers in turn to death, but my death, not others’, death as a movement toward a completely unpredictable future (so unpredictable that even its happening is problematic). It has therefore to do with otherness rather than with the other, let alone multiple others\(^{33}\).
Compearance then is interpreted as the co-appearing of things or voices. Since voices here are understood after the model of horizons, this aspect of compearance can be traced, as we have seen, to the multiplicity inherent in significance and saying. Finally, compearance is thought of as “the appearance of the between as such,” that is, as separation rather than distribution. In spite of Nancy’s attempts to identify the three, I would like to insist on their difference. It becomes clear in terms of saying, since the three aspects of compearance correspond to the repetitious character of saying, the internal multiplicity of significance, and the experience of the other in addressing and self-exposing. However, in Nancy’s text
\(^{33}\) Levinas considers death—in opposition to Heidegger—as a primary relation to the other. However, he does not provide strong evidence for this. His argumentation is rather analogical (as to large extent is all his thinking). Moreover, in late writings he tends to speak of the other’s death (for example, Levinas 1996).
these meanings are so closely interwoven that their difference is sometimes very difficult to discern. For example, on few pages (Nancy 1991, 72-6) one can find all three meanings of compearance that I have just discussed: temporality (offering as an act of incompleteness), multiplicity (the instant totality of touching/touched singularities), and the other’s saying (it is here that Nancy writes the phrase I have already quoted: “I no longer… hear in [dialogue] what the other wants to say (to me) but… that the other… speaks…” (Nancy 1991, 76))\textsuperscript{34}. The last one is certainly the most important for Nancy. Community as a dialogue or sharing of voices is an ultimate theme of his elaboration. But he tries to think of the voice of the other as the (silent) Voice of Being. After all, the first two meanings of compearance stem from the Voice of Being and represent its two aspects: the fact of Being and Being as the event of laying foundation. But the other’s voice is reducible to none of them. We see the indefiniteness of Nancy’s position. His basic intuition is undoubtedly the society of subjects completely independent in their production of sense. Language, since it does not limit this independence, is perhaps the only possible connection of such subjects. Hence, Nancy takes language as a basic model of their relations. However, trying to grasp this plurality he departs from the ontology of event. Since the multiplicity of speakers cannot be understood in such a way, he, while in all other respects keeping to the level of saying, slips into the said when speaking of multiplicity (the notions of substitutability and example may serve as good instances of
\textsuperscript{34} One can even observe how the first converts into the second. For example: “There is no singular \textit{being}: there is, and this is different, an essential singularity \textit{of being} itself…. In a sense, every being is absolutely singular: a stone never occupies the space of another stone” (Nancy 1991, 77). Here, the temporal or instantaneous (singularity of Being) turns into the spatial (the multiplicity of the occupied spaces). This identification could be traced perhaps to the temporal interpretation of Dasein’s spatiality (that is, Being-there) in Heidegger’s \textit{Being and Time}.
this). The reason is that Nancy refuses to accept any theory of the subject that is different from the distributive one. The concept of subjectivity is substituted with that of singularity.
Singularity can also be interpreted in terms of saying. To begin with, one can easily notice the distinction between saying and the said in this concept. Three characteristic traits of singularity appear then as follows. The “unicity” of the singular is that of the act of saying, with saying here implying the said and the order of significance making it possible (whether it be structured as the supposing-supported or horizon-figure or differences-sign). The “whatever” then refers to the similarity of acts of saying: all such acts consist in using a language system, including possible generating the whole system itself. The interpretation of the third trait, “exposition”, presents certain difficulties, again because of its ambiguity. The main meaning of exposition in *The Inoperative Community* is the attestation of Being. Now it converts into the attestation of the fact of language (let me remind you that by language act I mean any meaningful act, i.e. the act performed in view of meaning). The attestation is anonymous. However, as I have already pointed out, exposition for Nancy has another meaning (not in *The Inoperative Community*, though). It is understood as exposing toward others and thus requires that we take into account subjectivities of involvement and engagement. Saying in this case is not reducible to the event of sense. It is of another kind, that of responsibility. The difference lies in what I would call a rejective choice, sometimes or perhaps always negative. The event and the world are chosen even though there were no options at all. The power of rejection—even though it does not lead to any positive possibility—is a feature of subjectivity. This means that we cannot avoid subjectivity in our elaboration. In this sense the attestation Nancy speaks of is that of choice—someone has chosen to have the world and to have precisely this particular world. In this sense this is the attestation of someone, namely of someone who is saying. Saying therefore retains in its structure not only what is said and the fact of
saying but also who says. This “who” is precisely the place of the world, the *Da* of *Dasein*. However, it is also something more, insofar as it implies the agreement to be such a place, that is to say, taking responsibility for what happens at this place. We have to distinguish it from the responsibility of promise, although they may seem identical. Promise implies durability and is in this sense always the promise to keep on with what has started. By contrast, the responsibility of *Being* in a way precedes it and is the responsibility of beginning. It is the responsibility of bringing the world into reality, making the world exist (and keeping on with bringing).
Nancy’s representation of singularity as articulation (Nancy 1991, 76; see the discussion on p. 62 in this thesis) provides additional arguments for such reading. This text may serve as an example of Nancy’s ambiguity in the understanding of singularity. On the one hand, the picture there reminds one to a large extent of the coexistence of horizons, which do form a totality (of the event) but entail no fusion. On the other hand, we shall see the full analogy between articulation and saying, if we conceive of saying as in a certain sense multiple, as if the result of saying (that is, the said) is produced by the simultaneous saying of many singularities. Nancy does not distinguish the co-appearance of horizons and voices. Moreover, he seems to conceive of the coexistence of sayings after the model of horizons (despite the fact that he names them voices). For example, juncture in this passage (Nancy 1991, 76) is understood *simultaneously* as separation and as distribution. But if separation is the difference between what the other says and the other him/herself and if what the other says belongs in the final analysis to *my* structure, that is, to the structure of the event that happens to *me* (since it is *me*, who interprets what is said), the separation is the difference between *me* and the other (*you*). In fact, this is the difference between the event of *Being* and something else that is neither an entity nor *Being*, between what “happens to *me*” and what in some way “does not happen to *me*” or “happens not to *me*.” It is not by chance that the other’s death is so fundamental for Nancy’s meditation;
perhaps, the difference I am trying to describe is that between *my* death and the other’s. And here lies the source of—in fact, ethical—dissymmetry between *me* and the other: to accept *my* death is not the same as to accept the other’s. It is not clear how this difference can be described in terms of distribution belonging entirely to the sphere of mineness. Separation does not imply others’ separation from one another. In this sense *I* touch others and they touch *me*, but only through a sort of unlawful approximation can we say that they touch one another. It is when Nancy tries to think the mutual touch of singularities that he moves to the level of the said. He undertakes, therefore, this unlawful approximation. He has to do so because otherwise the mutual touch is unthinkable. But situating the other within the sphere of the said puts it at risk of being objectified. It tends to make of the other a thing, or role, or personage—in any case something determined by the constitution of event. This positions the other within the sphere of mineness. In this sense it is *I* who is/am multiple. The multiplicity by itself, even non-totalisable, does not yet constitute a community\(^{35}\).
Therefore, the examination of Nancy’s concepts of totality, comppearance, and singularity reveals three structurally different strategies of the escape from immanence. In terms of saying they are based on the temporal discontinuing of saying, internal multiplicity of significance, and saying toward the other. The first two of these fit naturally into the distributive model whereas the last one seems to surpass its limits. Let us inspect more closely how Nancy transfers ontology into the sphere of community.
\(^{35}\) The same goes for difference. A diversified society composed of irreducible to one another entities is not yet a community. For this to happen, differences must be “recognised,” or “accepted.” In other words, not differences as such but how we deal with them, how we respond to the plurality, constitutes a community. Or again, not comppearance by itself but a special mode of it is essential for community.
Saying and establishing communal ties
Let us consider now Nancy’s theory of citizenship, subjecthood, and establishing communal ties (Nancy 1997, 103-17 and p.70 in this thesis) from the viewpoint of the saying/said distinction. Nancy changes the language of his description as he elaborates this theory. Instead of division and spacing he speaks of (k)nots and enchainment. This transforms the networks of items which is the common element of citizenship and subjecthood. To begin with, tying (k)nots introduces temporality into the analysis. Indeed, although the institution of the city and its further appropriation seem to be temporal, this time has nothing to do with the time of event. From the standpoint of diachrony, institution and appropriation both belong to the inner time of distribution. Moreover, the self-sufficiency of citizenship and subjecthood denotes precisely this fact, although each in its own way. Citizenship means the self-sufficiency of any instant, its independence from all other instants. It is in this sense that city rests on nothing. Subjecthood means the independence of essence from temporality, its lasting forever through the course of time. For both of these the external time of event is simply redundant. Tying introduces this time first of all in the form of undoing. The mistake of self-sufficiency then consists in viewing ties as already established. Unlike this, the politics of ties takes them as having to be tied. The structure here is the diachronical scheme of event.
Another transformation that Nancy makes on his way toward the politics of tying is the shift from spacing to enchainment. This entails a redefinition of ties. Whereas the city’s ties bind by distributing, in the politics of tying we deal with relationships directed from one unicity to another. Nancy states this clearly speaking of the “tying up of sense from one to the one” (Nancy 1997, 113). How can we think of this sort of tying? It is not accidental that Nancy here speaks of ties between individuals, nations, groups, etc. Now the notion of ties does not possess the ambiguity I discussed before (p. 71). Ties of rights, etc. become unthinkable. Tying becomes a gesture that presupposes the intention to tie up
and enchain. Although Nancy does not speak explicitly about this intention, it is nevertheless present in his analysis under the name of the seizure of speech. Characterising the politics of tying (k)nots, Nancy writes:
Its event could be called the *seizure of speech* [prise de parole]: the emergence or passage of some *one* and every *one* into the enchainment of sense effects, statement and offering in phrase or outline, including the cry, the call, and the complaint as much as the theoretical discourse, the poem, and the song, along with the gesture and even silence. (Nancy 1997, 115)
We have arrived at the very centre of the political model that Nancy proposes in this chapter. His intention becomes clear. Community is constituted by the incessant reformulation or reworking of the sense in common. Nancy, with a reference to Jacques Rancière, calls this the “wandering labour of sense” (Nancy 1997, 115). The condition of its possibility is everyone’s access to this labour. Everyone must have the right and resources allowing him or her to rise to speak and thereby to contribute to the sense in common. In this case the sense is never complete and always exposed to anyone’s rethinking and even rejecting/demolishing. To name it shortly, this is the politics of the recognition of any possible voice. To designate it Nancy also invokes the term compearance from *The Inoperative Community* (Nancy 1997, 113).
Enchainment and tying are supposed to grasp this sort of community. One of their most important features is eventality. Ties exist properly only in the event of tying. Otherwise they come undone by fusion (subjecthood) or atomisation (citizenship) (Nancy 1997, 111). This conviction appears to contradict the intuition that fusion and atomisation also constitute community and are in a certain way ties. For Nancy, however, ties deserve their name only when we consider them in the act of tying, not as already given. But why should we do so? Nancy’s answer is: because Being, including the Being of community,
implies nothing pre-given at all. But why should we then be dissatisfied with ties established by subjecthood or citizenship? Why not to repeat them? Obviously, because the repetition concerns not only *me* but also others in community. But the question is whether this recognition of others can be adequately grasped with the evental character of tying alone; in other words, whether the resources of ontology of event and the distributive model are sufficient for that. I believe that they are not, and this leads to the uncertainty in Nancy’s presentation of the act of tying.
How are ties actually established? We can draw two models from the text. The first one is that of the distribution through tying (k)nots. In fact, this is the already familiar ontological model: tying spaces out and differentiates thereby, constituting both unicities and ties between them:
What would happen if, in the Platonic comparison of the art of politics with the art of the weaver, one no longer considered weaving to be the second and as arriving after a given material, but as primary and as itself comprising the *res*? (Nancy 1997, 113)
The second model of tying is enchainment. The act of tying here is “the act of the enchainment of singular sense to every other singular sense” (Nancy 1997, 115) whereas making sense is identified with tying (k)nots (Nancy 1997, 115). Even more, Nancy seems to conceive of (k)nots as events of making sense itself:
the *(k)n*ot itself is neither a sense nor a goal nor a subject, even and above all if one wants to call it “the law.” It is thus quite the contrary of what one calls today, in the magazines, the “search for meaning” with which our time is driving itself crazy. It is the “wandering labour of sense.”
(Nancy 1997, 115-6)
According to Nancy, tying (k)nots and enactment from the second model are ties from the first one but viewed “in the process,” i.e. as act. However, these ties are structurally different. In this sense Nancy’s characteristic of the politics that he is seeking as that “of the tie as such, rather than of its untying into a space or substance” (Nancy 1997, 114) is rather misleading. Indeed, at the same time enchainment is enacted as a seizure of speech, that is, as a gesture that did not merely happen but was performed by someone. What is this presupposed subjectivity?
Let me try to clarify the situation by means of interpreting tying as saying. To begin with, the seizure of speech should be obviously interpreted as saying. Moreover, it presupposes the subjectivity of involvement, since the seizure of speech points to mineness and the act of taking responsibility for one’s speech. It is not yet the responsibility of engagement but already of involvement and letting-be. But tying is more than that. Enchainment also requires the recognition of others’ speech, that is, others who are also involved in the “labour of sense”. Thus the links of the chain are not merely the results of an anonymous distribution. They address *me* and await *my* response. This is not to say that the said should be taken as meaningful, but rather as appeal aiming at communicating, establishing ties no matter of what kind. The other does not say anything in particular, she or he seeks a contact with *me*, a language community, where language is not given but is to be created in the appeal itself. The other seeks to be *with me*. The other’s saying does not simply amount to the coming of meaning, which equally well may be anonymous. There is a difference between the other’s appeal and a mere coming of the future. Both are enacted as *my* hearing or understanding, but the first bears something more than the unpredictability of the coming. It contains, as it were, the unpredictability of the other’s judgement, which deprives *me* of the power to decide whether *I* am satisfied with the coming or not.
In its turn, *my* saying becomes addressing others, offering them *my* vision of community (including the vision of the chain). The sense of the world thus is not merely produced or accepted but offered to the community. This means that we encounter here the subject of engagement and the structure of the “for the sake of”. However, as we have seen, it cannot be derived from the distributive model or the ontology of event in general. Consequently Nancy’s concepts of enchainment and tying also exceed their limits. Enchainment is a saying toward the other. However, we have to be aware that the self of the enchainment is not a (k)not. Not (k)nots say toward the other, but we, people who are not constructed by saying (insofar as we have no construction at all) but who use saying for our self-constitution. Nancy’s discourse is about people’s ties. His description of the seizure of speech, rights to speak, etc. has in view people (or groups). See, for instance, *The Sense of the World* where the tied unicities are obviously individuals, groups, etc. (112). The whole discourse here is about people, and it is not clear how it can be applied to “mobile complexes of rights, etc.” Since Nancy does not know—or, better, does not want to take into account—identities other than dwelling on the level of the said, he is not capable of conceiving of the ties of the seizure of speech in its full peculiarity. He interprets the identity of the speaker through distribution. Only this sort of self does he know, in spite of the fact of his provisioning another one. Dissatisfied in this concept of identity (along with a number of contemporary philosophers), he tries to find a remedy in the very act of the self’s birth. However, one needs to make the next step in this direction and admit that this birth points to the self that is in some sense inner (*mine*) and irreducible to the singularity of event. Nancy is halfway here. He has departed from subjecthood and distribution but has not arrived at the selfhood of saying.
Let me be more precise. Identity is constituted in the triple act of (1) producing a narrative structure with places for speakers and objects, (2) taking on a place in this structure, and (3) offering the narrative to others. The first of these acts can be described in
Nancy's manner as spacing and distribution. The second is enacted as a seizure of speech. The third appears when a narrative ceases to be only *my* business, when someone else acquires the right to decide about the world *I* live in. Without this dimension tying ceases to be a proper tying and serves for self-sustaining, even if this "self" is a non-substantive self of flow, movement, or joy.
All these three aspects exclude the pre-givenness of communal ties. However, Nancy, in order to escape the ties' having been already established, focuses on the act of tying (which is, in fact, the act of emergence of a communal myth or narrative). He seems to be convinced that this movement by itself makes up communal ties escaping both nihilism and totalitarianism. In a sense it is true, but Nancy's basic intuition of community—that of language, writing, or writers co-participating in the production of meaning—cannot be conceptually grasped in terms of distributing or spacing. The self of the speaker is more than the unicity of distributed items.
In the theory of (k)nots Nancy puts to work the subjectivity of engagement. However, for him the decisive property of this subjectivity is its temporal character. It is precisely this point that I would like to dispute. It is true that the temporal character of saying is a primary source for the resistance to the totality of immanence. But this resistance is not sufficient for community. The relation to the other demands a passing beyond not only the individual but also the ontology of event as such. The relation to the other demands a separation more radical than that of meanings, horizons, or events. Nancy's accent on dislocation and singularity seeks, in fact, to grasp this separation. But due to his tendency to the spatial quasi-geometrical interpretation he fails to do so. This happens as well in the central concept of the theory of inoperative community—that of literature or writing.
3.3 Literature/writing
The ontological model of writing
Nancy gives his view of community the name of literary communism ("clumsy expression," in his own words (Nancy 1991, 26)). This makes the meaning of compearance and Being in common more concrete. This is the heart of Nancy's understanding of community in *The Inoperative Community*. It should be noted that Nancy's "literature" does not mean what it usually does. Sometimes he calls it writing and adds that it can also be speaking, music, painting, dance, or the exercise of thought (Nancy 1991, 64). It can also be reading, provided it complies with the main condition for Being literature: to interrupt myth. By this interruption literature enacts community. Literature is Being-in-common itself, or even stronger: "it is being *in* common that *is* literary (or scriptuary)" (Nancy 1991, 64). Writing/literature is not just some community's activity however important or sacred. It is the very Being of community. Literature, Nancy writes, "designates that singular ontological quality that *gives* being *in* common, that does not hold it in reserve, before or after community, as an essence of man, of God, or of the State achieving its fulfilment in communion, but that rather makes for a being that *is* only when shared *in* common, or rather whose quality of being, whose nature and structure are shared (or exposed)" (Nancy 1991, 64). To be in common, thus, is to write. What is then the writing that interrupts myth? As is usual in Nancy, interruption acquires several meanings: temporality of an origin, sharing of a work, sharing as offering to others, and distributive limit of communication. The primary meaning of interruption is temporal: "community without community [i.e. the interrupted community—OD] is *to come*, in the sense that it is always *coming*, endlessly, at the heart of every collectivity (because it never stops coming. It ceaselessly resists collectivity itself as much as it resists the individual)" (Nancy 1991, 71). However, literature is not only the interruption of myth, it must establish a continuity beyond this interruption (Nancy 1991, 72). That is, the interruption must not cease. In a
way, literature interrupts itself, because myth is the invention of literature: "it inaugurates itself with one stroke, one incision, and it names 'myth' that which it represents to itself as having been present before this stroke" (Nancy 1991, 72). Myth thus is rather a possibility that literature prevents and is supposed to prevent ceaselessly. This feature is the very definition of literature:
Literature interrupts itself: this is, essentially, what makes it literature (writing) and not myth. Or, better, what interrupts itself—discourse or song, gesture or voice, narrative or proof—that is literature (or writing). Precisely what interrupts or suspends its own mythos (that is to say, its logos) (Nancy 1991, 72).
What is prevented or interrupted is a completion or totality. This happens in a 'literary' work but for that "the work... must be offered up for communication" (Nancy 1991, 73). At times Nancy seems to offer singularity itself but more often he speaks of the work, text, myth, foundation. The meaning of the work in the book is quite broad. In effect, Nancy denotes with this word a "result" of literature, the result supposed to be unworked. Work can be complete, and is perhaps always complete, but it becomes incomplete (or, I would say, it becomes engaged in the movement of incompleteness) when it is offered. In general, the offered is capable of—and always tends to—totalisation, which is to say that it is accessible to anyone and can be shared by everyone (a myth, work, foundation can be adopted by all society). What prevents totalisation is the act of sharing itself. Having been offered, myth ceases to be a myth: "The text interrupts itself at the point where it shares itself out—at every moment, to you, from him or her to you, to me, to them. In a sense, it is the sharing of myth. It is community exchanging and distributing its myth. Nothing could resemble more closely our myth of the foundation and communion of a tribe, or a people, indeed of humanity. And yet, this is not what it is" (Nancy 1991, 65). In the offering myth is disjoined or withdrawn from itself (Nancy 1991, 61). Myth holds in itself its own
difference from itself. Nancy obviously thinks of this difference ontologically: writing the myth is equivalent to Being the myth, with Being understood transitively. The logic of limit borrowed from Hegel and Plato and developed on the starting pages of *The Inoperative Community* works here perfectly: myth cannot be absolute inasmuch as it exists.
Therefore, we have to search for the meaning of interruption not in the work itself but in the mode of its appearing. It can appear in the mode of myth or in the mode of literature/writing (offering). Myth turns into a literary work when the mythic hero tells the truth: that he is not a hero, not even, or especially not, the hero of writing or literature, and that there is no hero, there is no figure who alone assumes and presents the heroism of the life and death of commonly singular beings. He tells the truth of the interruption of his myth, the truth of the interruption of all founding speeches, of all creative and poietic speech, of speech that schematizes a world and that fictions an origin and an end. He says, therefore, that foundation, poiesis, and scheme are always offered, endlessly (Nancy 1991, 79).
This passage contains two possible meanings of interruption. It is whether a passing beyond the Absolute or the One (“there is no figure who alone assumes…”) or a temporal movement that dissolves any mythic consistency (“foundation, poiesis, and scheme are always offered, endlessly”). Nancy draws no difference between them. But he is able to do so only thanks to the fact that any passing beyond the One is for him first of all temporal (which is to say, ontological). We have seen that this is Nancy’s main thesis: community is based on the relational character of Being. Hence the ontology of event is set to work. Interruption denotes the unstable ontological character of myth. It is not however merely the need for the act of saying in order to be. Interruption stops the order of signs by introducing the order of significance. Writing is “the breaking of the path [frayage] of
significance” (Nancy 1997, 118), it makes signification possible. Thus, the temporality of interruption is that of the origin of significance or of an inaugural act:
The communism of being-in-common and of writing (of the writing of being-in-common) is neither an idea nor an image, neither a message nor a fable, neither a foundation nor a fiction. It consists, in its entirety—it is total in this respect, not totalitarian—in the inaugural act that each work takes up and that each text retraces: in coming to the limit, in letting the limit appear as such, in interrupting the myth. (Nancy 1991, 68)
Thus, the interruption is thought as “coming to the limit, letting the limit appear as such.” At the same time, the limit appear as a “dividing line” between me and the other:
On this limit, the one who exposes himself and to whom—if we listen, if we read, if our ethical and political condition is one of listening or reading—we expose ourselves, does not deliver a founding speech. On the contrary, he suspends this speech, he interrupts it and says that he is interrupting it. (Nancy 1991, 68)
From here on Nancy comprehends the limit in terms of distributing and dislocating—dividing, to be more exact. Although he warns against seeing the limit as a mere boundary between singularities (Nancy 1991, 32), he means thereby to undermine the picture of singularities as pre-existing entities limited by one another—the limit does not limit but constitutes singularity.
In general, Nancy tends to present the limit in the distributive framework. Thereby the temporality of interruption and the separation of communication both acquire the spatial sense of differential distribution or ontological multiplicity. However, as in cases of singularity, compearance, tying and others, his own description transcends the distributive model.
Writing as exposition and addressing the other
In *The Sense of the World* writing is “the breaking of the path [frayage] of significance through which it becomes possible not only for significations to be signified but for them to make sense in being passed on and shared among individuals” (Nancy 1997, 118). In this fashion writing possesses two dimensions. First, it is différence or the very act of distribution, spacing out, and frayage. Second, it is the exposition of a writer, her/his opening toward others and offering oneself. The sharing out of one’s work (the core meaning of sharing in *The Inoperative Community*) has something to do with communication and “the passage from one to another” (Nancy 1991, 65). It is this passage that, according to Nancy, interrupts myth:
Myth is interrupted by literature precisely to the extent that literature does not come to an end. . . It does not come to an end at the place where the work passes from an author to a reader, and from this reader to another reader or to another author. It does not come to an end at the place where the work passes on to another work by the same author or at the place where it passes into other works of other authors. It does not come to and end where its narrative passes into other narratives, its poem into other poems, its thought into other thoughts, or into the inevitable suspension of the thought or the poem (Nancy 1991, 64-5).
This long list of different entities indicates, in fact, that literature is a passing from one singularity to another (and leaves the impression, by the way, that it is readers, writers, etc. who are singularities in the primordial sense). Nancy states this explicitly: “the text, or the writing, stems only from the singular relationship between singular beings” (Nancy 1991, 66); “A singular being (‘you’, or ‘me’) has the precise structure and nature of a being of writing, of a ‘literary’ being: it resides only in the communication—which does not commune—of its advance and its retreat. It offers itself, it holds itself in suspense” (Nancy
This passing from one singularity to another should not, of course, be understood as a sort of access, or even less as a sending of a message. To pass to another means to arrive at “the limit where being itself, where being-in-common conceals us one from the other” (Nancy 1991, 66). We came across the same paradox again: to be in common is to be separated, to touch the non-traversable limit concealing us one from another. We encounter the notion of limit again but this time it is conceived as separation rather than distribution.
Thus, writing is offering, simultaneous putting into social space and creating the space itself (or, which is the same, creating the space by putting one’s work into it). In this way “it is ‘literature’ that does the sharing” (Nancy 1991, 64). The gesture of offering is dual. On the one hand, it follows the logic of sedimentation: having been said, the myth becomes a form accessible for everyone’s usage, a foundation capable of being taken over by anyone. Offering in this sense is rather detaching, not so much giving as taking away. It does presuppose others but rather negatively as ones who share my myth even if I try to prevent this. On the other hand, offering addresses others. It does not merely leave traces; it offers a foundation and invites others to share it. By definition, “any one who writes for the same, for himself, or for the anonymity of the crowd is not a writer” (Nancy 1991, 66). Nancy calls this sort of offering exposition. Literature is a multiplicity of voices; each of them is a voice as long as it shares itself, i.e. writes for others. And again, Nancy presents the multiplicity of voices in different ways. Some of them fit into the distributive model but others go beyond it. This is visible more than anywhere in the understanding of the voice of the other. This voice has a specific ontological status. Its experience is not passive and depends on one’s efforts: “Everything is a matter of one’s practical, ethical, political—and why not add spiritual?—positioning around this singular eruption of voice. You can always make a myth out of it again. But this voice, or another, will always begin interrupting the myth again—sending us back to the limit” (Nancy 1991, 67-8). Voice is
the experience of the other, limits, and outside. Being so it is essentially ethical. Voice plays a decisive role in the constitution of singularity. Although Nancy insists that singularity is that of Being itself, the consideration of voice in *The Inoperative Community* on page 76 shows that he relies, in fact, on its specific experience. It is here that Nancy writes:
I no longer (no longer essentially) hear in it [dialogue—OD] what the other *wants to say* (to me), but I hear in it that the other, or some other (*de l’autre*) speaks and that there is an essential articulation of the voice and of voices, which constitute the being *in* common itself (Nancy 1991, 76).
*I* hear thus not what is said but the other’s saying as such. What happens here is the acknowledging of the other’s voice and applying to it the structure of *my* voice and its articulation. The mineness of saying becomes multiple. In this sense indeed the hearing of the other’s voice amounts to the Being in common. Nevertheless, Nancy is ambiguous here. This reasoning appears in the context of discussing articulation (Nancy 1991, 75-8; see p. 62 in this thesis) where it remains uncertain whether or not voices are understood as horizons. The sociality of articulation is first of all opposed to that of fusion or immanence. Nancy conceives of totality as the play of articulations. But for this he has no need to go beyond the sphere of mineness. The multiplicity of horizons—and accordingly of the voices each articulated in its own horizon—is already such a totality. By contrast, the acknowledging of the other’s voice demands a completely different gesture irreducible to the ontological event.
Later on Nancy conceives of the “voiceness” of the voice (the fact of voice, that is, the fact of its appearing) as Being-voice, that is, in the ontological framework. This, in turn, allows him to conceive of community ontologically, that is, eventually in terms of revelation (Being reveals beings and itself). Even though Nancy rejects the ontology of
revelation\textsuperscript{36}, his interpretation seems to comply with its logic. But contrary to this, community seems incapable of revelation, be it even that of event. In this sense we are always in the position of extreme uncertainty about community: we cannot know not only whether it will be, but also whether it has been or is at the moment. Perhaps even the coming community is too an optimistic image and this precisely opens up the ethical dimension. We cannot even know if ethics (and community) is possible in the world, although it already demands our decision. This demand does not yet constitute a community but does constitute an ethics. Community is rather a response to this demand, taking on the responsibility for a common world. This view is present in Nancy’s book, explicitly or implicitly, but his ontological assumptions make it difficult to be observed.
We find the already familiar logic in the theory of writing/literature. On the one hand, Nancy proceeds in accordance with the double internal articulation of the voice which is, first, insufficient like any entity and thus implies some sort of otherness and, second, may be inscribed into different incommensurable horizons and is thus inevitably multiple. This double principal non-oneness of the voice is for Nancy a sign of the presence of other voices, i.e. of a community. This is, of course, the logic of the distributive model. On the other hand, Nancy refers to a more immediate experience of the other—that of addressing—although, as happened before, he tries to reduce it to the ontological one. However, the community of the other’s saying is that of neither transcendental Being nor the multiplicity of horizons or orders of significance.
Therefore, the term ‘literary communism’ designates a society of voices each open toward others and existing only through and in this opening. The model of this society is literature where a writer “is not the author, nor is he the hero, and perhaps he is no longer
\textsuperscript{36} He does so explicitly in \textit{The Sense of the World} where the ontology of revelation is named phenomenology (177, note 19). Here, I believe, Nancy is close to this ontology and still relies upon it. His exposition still has visual connotations, although it is already thought of as touch.
what has been called the poet or what has been called the thinker; rather, he is a singular voice” (Nancy 1991, 70). Each voice does produce a myth, that is, a total and closed in itself foundation of the Whole. But the act of offering (which constitutes literature) interrupts this myth and stops its totalisation. In this sense the sharing of myths, texts, works is the Being-in-common itself. Nancy states this explicitly: “being-in-common is literary, … it has its very being in ‘literature’ (in writing, in a certain voice, in a singular music, but also in a painting, in a dance, and in the exercise of thought)” (Nancy 1991, 64).
Beings communicating through writing are called singular beings. Writing is not a revealing of the boundary between pre-existing singularities. They come to being only through writing itself.
However, this appearing singularities—or the ‘between’ as such—in the act of writing can be understood at least in two ways and Nancy actually does so. One way stems, in fact, from Derrida’s idea of différence as a movement of differing-deferring. As I tried to demonstrate in the course of this work, this way does not allow one to surpass the sphere of mineness. The other way, in contrast, is a movement toward the other from the very outset. The incommensurability of the two ways become especially visible when one realises that they correspond to two functions of writing/literature—or, broader, language—the cognitive and communicative (or even phatic) ones. It become immediately clear then that community following from addressing and exposing has nothing in common with that based on the fact of meaning or significance, even when it is conceived as frayage or the origin of significance.
Conclusions
In my conclusion I would like to outline the main themes and turning points of my thesis. Nancy formulates his task as the elaboration of a theory of community that will be based on its own interruption. It should be set against both myth (a pre-given and determined community) and nihilism (an arbitrary and non-obligatory community). To accomplish this end, he must invent a concept of community that is neither unable to be interrupted nor without any ties at all. Nancy seeks a solution in basing community on the very event of interruption understood not merely as disappearing community but rather as re-establishing its ties. This community is not mythic but neither is it without a myth. It is the incessant movement of inventing myths. Hence it is the origin of myth (or sense or significance or community’s ties) that is brought to the focus of Nancy’s work.
At the same time, myth for Nancy invents not only ties but along with them the tied items, that is, not only social relations but also members of society (individuals or groups). This allows him to liken the event of community to that of Being. He relies on the Heideggerian tradition and understands Being transitively as the presentation of entities or attestation of the world. Hence Nancy works out the theme of ‘there is’ which he understands as the attestation or presentation of sense or the world. However, to derive a social theory from Heidegger’s fundamental ontology one needs something more than the movement, gesture, or event of Being. For this reason Nancy undertakes a further elaboration. He borrows the concept of *différance* from Derrida and that of horizon from hermeneutics and applies them to community. The event of Being appears thus as a distribution, dislocation, or *frayage*. But instead of the differing-deferring of meaning or signification he speaks of singularities and social ties. The Being of community consists in
the ceaseless spacing out and differentiation in which not only ties but also the items to be tied are brought about. One of the best examples of this strategy is Nancy’s theory of tying (k)nots.
However, this identification of communal ties with signifying references has a weak point. Neither Heidegger’s ontology nor Derrida’s *différance* goes beyond the sphere of mineness\(^{37}\). This makes their usefulness for a theory of community highly problematical. Indeed, Being as well as *différance* always happen to *me* and it is not clear how *my* sense can be understood as others’ or how *my* view on community can be extended to others’. It would be wrong to interpret this mineness in terms of self-sufficient and self-reflexive subjectivity. It implies, of course, unconsciousness and acquired social practices, which are in a certain way external. Nevertheless, it still has to do with internalised roles, models, gestures, etc.\(^{38}\) In this sense one deals here with the Other within oneself (and thus we can speak of anonymity), whereas the case in point should be the other that is more radically outside than unconsciousness. As we have seen, Nancy turns to the experience of this other when he speaks of writing/literature, offering a work, and exposing toward others.
At the same time, as we have also observed, Nancy reduces writing to Being and offering to presentation. The very term that designates the movement of writing—*partage*—signifies simultaneously dividing or distributing and offering (one’s part). However, Nancy’s interpretation of writing is not reducible to the ontology of event. It is more than the fact of Being, even taking into account that this fact is simultaneously the
---
\(^{37}\) We cannot say the same about hermeneutics, at least in its Gadamerian version. He perfectly realised the importance of addressing in his elaboration of anticipatory and question-answer structure of understanding (see Gadamer 1993, for example).
\(^{38}\) I use the term ‘internalisation’ in the sense of the subjectivity of involvement: to internalise means to learn how to repeat, that is to say, how to be involved in a differential system of gestures.
fact of the *Sein-Seiende* difference and the fact of time. Writing, as Nancy conceives of it, is never anonymous. Not in the sense that it has an author who “expresses his/her thought in words,” but in the sense of someone’s responsibility for giving/offering itself. In the ontology of event subjectivity, if it exists as such, is grasped as a place of the “happening” of Being (for instance, Dasein in *Being and Time*), in this sense irresponsible because it precedes any possible act of taking over responsibility. But writing, inasmuch as it is the offering *for* others, is itself praxis, which can be chosen, or not. More exactly still, writing is rather a response to the event of Being, not this event itself. Ontological difference (actually, *différance*) is not enough to comprehend writing.
On the other hand, writing is not a mode of Being understood hermeneutically or phenomenologically as the meaning of a being’s Being (that is, what means to be this being, this entity). If it were so, to be a work/myth would mean to be written for others. But in this case others would be reduced to the mode of Being: to be the other would mean to be the addressee of a written work. The other would be thus immanentised, which cannot be true for writing. The latter is not merely the inauguration of the world where the other (as well as the writer) has this or that place. The other is rather the one who crashes the world but does so in another way than death does. This difference between the ways of the world’s interruption is precisely what exceeds ontology. Writing is more than a mode of Being because it is Being-for-someone. It is the existence devoted to someone, but devoted without making a work or project out of it. Writing is precisely the suspension of any work or project. It is for the sake of community.
There are, therefore, different ethical attitudes associated with distribution and separation. They correspond to the two types of subjectivity that I have described throughout this thesis. The first one can be characterised as “for the sake of itself or self-sustaining”. This is a subject of flow, incessant withdrawal of itself, radical ungroundedness of any stage, and so forth. This is the courage of the openness toward the
future, heroic acceptance of the coming destiny and opening this destiny with one’s own deeds (Is such a courage not the primary meaning and a hidden ground of Heidegger’s explication of Dasein as resoluteness (*Entschlossenheit*) in *Being and Time*? I believe so.). Community, undoubtedly, demands such courage but it also demands something more. This subjectivity is totalisable in the sense of reducing to the sphere of mineness, whereas community requires rather that one exceeds this sphere. The ethics of this subject is based on the acceptance of internal plurality and the rejection of any attempts to unify oneself. It is indeed the resistance to totality but is not yet a community.
This subject uses a language for self-sustaining. Performing gestures, it aims solely at the performance itself, or, better, at the prolongation of itself as a performing instance. In this sense it opens toward nothing and is ready to accept everything. It does resist the immanence of essence but at the same time forms the immanence of mineness or that of a flow, the very movement of Being revealing itself. This is a strategy of radical scepticism depriving Being of any essence. The best strategy for it is following the flow of the event (be worthy of the event, as Deleuze puts it) because there is nothing beyond, above, or beneath it.
Doubtless, this is not Nancy’s choice. He explicitly argues against this standpoint and devotes the whole of *The Sense of the World* to the demonstration and elaboration of the irreducible sense of Being resisting any sceptical denial. However, this irreducible sense amounts, in fact, to the subjectivity of involvement or, more precisely, to the fact of involvement. “The sense or world is there” means that the structure of ‘senseness’ or ‘worldness’ is there. In terms of language this means that the structure of significance is there, or language itself is there. The passage to community thus appears as a passage from unconscious repetition to the active will to language or will to bring forth the structure of significance, to be involved into originating significance. So structured, this passage is equivalent to that from *Man* to *Entschlossenheit* as described in Heidegger’s *Being and*
Time (see especially p. 60). But the subjectivity of self-sustaining fits into this picture. Hence, if we want to go beyond it we need to focus not merely on the will to language but on that for the sake of which the language is desired.
Thus, we arrive at the second type of subjectivity, which is “for the sake of community”. In what sense? As far as I can see, there are two such senses: 1) this subject uses language not for self-sustaining but for addressing others or making oneself accessible to them, 2) this subject recognises the other’s right to speak. In both cases the fundamental structure of saying remains intact and this subjectivity wills language and world thereby securing the structure of ‘senseness/worldness’. But unlike the subjectivity of self-sustaining, this one implies some aims that surpass the event of sense (or the fact of the origin of sense). This is that for the sake of what the event is performed. Furthermore, the recognition of the other’s right to speak amounts to the recognition of the other’s saying and allows one to escape the logic of event with its tendency to reduce everything to me. Without it compearance fails to ground a proper community. It does reveal the possibility of others by opening up the place they might dwell in. But this place is empty—or perhaps does not even exist—until I recognise the other’s right to speak. This recognition is truly groundless and stems from neither the structure of the world nor its Being. Perhaps its only ground is the ethical decision—to be with others, to keep building a common world with them. No doubt, this is the underlying principle of Nancy’s theory of community, although his “ontological reductionism” makes it obscure.
Therefore, in the course of this thesis we discovered the subjectivity of involvement, which can be described in terms of distribution, spacing, or origin of significance. But above this we found two subjectivities for which this terminology turned out to be insufficient. They demand the invention of something for the sake of which the spacing is performed. Nancy keeps to the concept of distribution and this reveals, I believe, a weak point in the postmodern project in general. The latter can be formulated as follows.
The key theme of postmodern philosophy is the impossibility of universality and its dependence on the play of differences, which is the play of language. In this context, not only universality but also subjectivity appears as a mere effect of the intersection and overlapping of heterogeneous forces. *Différance* or the incessant originating of differential structures lies behind any individual or collective universality and stability, thereby making them no more than simulacra. The same goes for the subject becoming capable of appearing and sustaining only in the context of anonymous discourse. This leads to radical relativism and nihilism. Nancy’s concept of writing surpasses this approach (and in particular Derrida’s concept of writing). He opposes to it the undeniable fact of Being which after a closer consideration proves to be the fact of language understood in the active sense of saying. However, in another sense he still resides within the limits of the so understood postmodernism. For he still relies on the movement of differentiation and seems to be convinced that the ‘active’ distributive model solves the problem of relativism. He aims to demonstrate that the saying always opens a common world, and hence the fact of language is equivalent to the fact of community. However, in so doing he argues from the multiplicity of saying, that is to say, again from the movement of *différance*. And this is so in spite of his own description of writing as a speaking of one to another. It is difficult to say why he does so, but perhaps this is because of a typically postmodern presumption that subjectivity can be nothing but an effect of *différance*.
However, community and ethics, by contrast, call for the deciding subject, the one who is not merely involved in the anonymous movement of differentiation but devotes her-or himself to this or that movement without ever being certain that “that for the sake of which” is approachable at all. This subject of fidelity without illusions does not perhaps precede the movement of saying and does not exist otherwise than in a gesture of saying but once appeared cannot simply repeat the gesture and is liable to take responsibility for it. Thus, postmodernism is right in saying that *I* was brought into the world by external
forces but, since this happened and since I realise that this has happened, it becomes impossible for me to avoid the responsibility for the world arisen well before my birth and for the community in creation of which I am supposed to take part from now on. This is what postmodernism seems not to realise.
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|
RADIO AT WAR
UNITED STATES
GREAT BRITAIN
CHINA
RUSSIA
AUSTRALIA
CANADA
WLAC
50,000 WATTS - NASHVILLE, TENNESSEE
A MESSAGE TO OUR LISTENERS
Dear Friends:
On September 1st, 1942, the power of WLAC was increased to 50,000 watts, making this station as powerful as any standard broadcast station in the United States.
When we look back over the history of our station and note its great expansion, it is only natural that we want to give credit to those agencies which contributed so much to the attainment of our goals.
First must go our thanks to the thousands of radio listeners whose loyalty to WLAC enabled us to "show cause" why we should continue to operate as an essential public service medium.
Then, we always have been blessed with a splendid staff of employees whose sincere interest in their work resulted in consistently good programming of a type that made "listening to WLAC" a highly profitable form of recreation.
Many of these people are now in uniform, and those remaining are (as the pages of this book will reveal) going all-out in their efforts to give unwavering support to our nation's armed forces. During 1942 they sold more than $1/4 of a million dollars worth of War Bonds and Stamps, received the U. S. Treasury Department's "minute Men Award" for adoption of the "Payroll Savings Plan", and broadcast war programs and announcements of a value approximating $1,000 per week throughout the year. You may be sure we shall remain on the job 'til victory is won.
Cordially yours,
F. C. SOWELL
General Manager
WLAC BROADCASTING SERVICE
50,000 Watts..."Power Where Power Counts Most"
F. C. SOWELL
Manager
www.americanradiohistory.com
Serving Uncle Sam
Public Service
... Behind our war effort is a vast organization known as "Our Government". This government is composed of many federal agencies and officials reflecting the policies of the President and Congress. These various agencies and individuals have important missions to accomplish which require widespread understanding and cooperation.
... In a Democracy - even at war - there is a limit to the effectiveness of regulations. In most instances, public acceptance must be secured. To reach our large population of 130,000,000, no medium is more effective than radio.
... And radio, alert to its vital role in this part of the war effort, is generously contributing its facilities, its time, and its trained personnel to serve the government and our people.
How Radio Helps
The Record:
U. S. Army
U. S. Navy
U. S. Civil Service
Maritime Commission
U. S. Employment Service
American Red Cross
War Production Board
Office of Price Administration
U. S. Treasury
U. S. O.
Department of Agriculture
Office of Price Administration
War Production Board
Federal Security Agency
Office of Civilian Defense
Department of Labor
Office of Coordinator of Inter-American Affairs
War Production Board
Department of Agriculture
Department of Agriculture
Department of the Interior
Department of Agriculture
Department of Interior
Department of Agriculture
National Park Service
Department of Interior
NATIONAL
Recruiting for Armed Forces
Recruiting for Navy, Marines, Merchant Marine and Coast Guard
Recruiting for War Production Workers
Recruiting for Shipyards Workers
Recruiting for War Service Specialists
Recruiting for Nurses, Nurses Aids, etc.
Production Drive Information
Price Control Information
Sale of War Bonds & Stamps
Campaigns for Funds
Food Conservation, Rationing
Gas Rationing
Rubber and Scrap Salvage
National Nutrition Drive
Air Raid Precautions
Child Welfare in Wartime
Information on other American Republics
Conservation of Electric Power
Conservation of Household Equipment
REGIONAL
Grain Storage
Reclamation Campaign
Relief for Farm Labor Shortage
Promotion of Power Programs
Promotion of supply of farm products vital to war
Forest Fire Prevention
Mine Service
Each local area can add scores of items to this imposing list.
Orders from headquarters by radio as troops leave bivouac area.
Marine uses portable radio in landing operation.
Portable Army radio outfit operates on maneuvers. Note hand generator.
Reporting by radio from concealed command car. Note transmitter key on radio operator's thigh.
Sergeant in foreground is tank crew member plotting attack on basis of information radioed from outpost.
Report on enemy aircraft is radioed to concealed artillery at rear.
OUR fighting forces throughout the world are linked to Command Headquarters in Washington by a vast network of military communication. Messages are necessarily sent in code -- for in them are the secrets of our future military operations. Our system of radio stations in the United States has been a reservoir which provided our Army and Navy with thousands of skilled specialists who now maintain our important lines of military radio communication. Meanwhile, the services are training thousands of additional men for radio duty on land, on the sea, and in the air. Today, radio is the nerve system of our military might. Crackling messages over the airways will carry the signal of the last great offensive and the first news of the final defeat of our enemies.
ON THE SEA
Some of the delicate radio equipment in a Navy radio room.
Radioman receiving message on U.S. Battleship.
Navy radio operators help to guard the sea lanes.
In the operational radio control of a Naval Air Station.
The Watch Below! Firemen report burner control readings.
Every one a radio operator.
Radio operator on Navy patrol blimp on anti-submarine duty.
Duty officer checking flight board after flight.
Radio operator on Navy bomber.
Coast patrol radio man keeps tabs on weather and directs surface ships to scene of disasters; keeps wary eye for enemy aircraft signals.
"Blind flying" by radio in ground school trainer.
School dismissed. The "desks" in a radio classroom.
Aviation cadets and student officers attend "buzzer" class.
Another class explores intricacies of radio code.
Recruits receive instructions at Signal Corps Training school.
Diagram on wall aids Army Radio instruction.
Flight instructor corrects students' errors after formation flying.
Here's the famous walkie-talkie... "talk as you walk".
Above: In landing operation, soldier reports back to ship.
Left: Lone sailor communicates from beach.
Lower left: The Marines have landed! 'Nuf said.
Walkie Talkie on skis going up hill.
Two-way hook-up at message center.
1. General View Field Transmitter, Power Unit and Antenna.
2. Battery of code keys at message center.
3. Radio-equipped Army Command Car.
4. Close-up of Army Field transmitter.
5. Motorcycle and side-car equipped with Radio.
A MILITARY MISSION
On April 5, 1942, the United States Army started a new kind of military operation. For the first time in history, the War Department was directly sponsoring and producing a radio program with a definite military objective -- "The Army Hour". Since then, the official "Army Hour" has established itself as an integral part of the global fight of the United Nations against the Axis.
Through the "Army Hour", which is broadcast each Sunday, America and the whole world is getting a weekly view of the progress of the war and how it is being fought. The program reaches to all parts of the globe to tell the story of the United Nations fight, with buck privates telling their important role as prominently as the top-ranking military chiefs.
When listeners heard the chatter of machine guns, they were hearing live bullets fired by the gun crew in this photo. Radio microphones enabled the listener to hear also, the bullets striking the target.
From the West Point air training field, the Army Hour introduced J. H. Weikert, Captain Donald Thurmar and Cadet Vincente Lim.
Bill Stern, famous sports commentator, describes how it feels to look through a bomb sight and pull the release that will send bombs from U.S. planes to blast the enemy.
Behind the scenes in any Army Hour broadcast is Art Feldman, the man who gives the signals and makes the check-ups on as high as 25 "switches" on a single program. He is in touch with each remote point, foreign or domestic, until each is off the air.
A message by Secretary of War Henry Stimson inaugurated the first official War Department radio program.
Lt. Gen. Hugh A. Drum, commander of the First Army, addressed an Army Hour audience. On the same program were: Lt. Generals McNair, Lear, Krueger, and DeWitt.
Instrumental in planning and arranging each War Department program is Lt. Col. E. M. Kirby, Col. Ernest R. DuPuy, Major General A. D. Surles and Col. R. B. Lovett.
Private Joe Louis, heavyweight champion, addressed Army Hour listeners, with Col. Ned J. O'Brien, Art Flynn and James Braddock.
WAR INFORMATION
With news, roundtables, speeches, forums, special events and dramatic programs, radio is keeping Americans the most informed people in the world. Today, more than ever before, Americans demand all the facts except those which will give aid and comfort to the enemy. From these truths come American unity and decision. Radio's task is to bring this information to our people as quickly and as clearly as possible.
The first Filipino Battalion in the United States Army staged a demonstration for Army Hour listeners.
ON THE AIR
When they are not too busy learning the methods of warfare, the Army, Navy and Marine Corps can present radio programs with a professional flavor, designed to entertain and inform the folks back home. Hundreds of radio entertainers, musicians, writers, announcers, production men and specialists are in the service, and they welcome the opportunity to resume association with their former civilian pursuits. Meanwhile, radio listeners, they make it possible for friends and relatives to visit camps and training stations, without moving away from the loud-speaker. Through this medium, radio can claim special distinction for building and maintaining our strong morale.
A portable organ, makeshift stage and soldiers entertain during maneuver rest periods.
Hawaiian soldiers find time for broadcast.
Trained Army Public Relations officers proved they could operate a radio station they "captured" during a 1943 maneuver.
Sailors compete in a quiz broadcast while buddies listen in audience.
Microphone catches formal guard mount at West Point.
Tyrone Power, of screen and radio, is sworn in as a private in the Marine Corps by Maj. Wm. Howard, U.S.M.C.
Wayne Morris, called to active duty with the Naval Aviation Cadet Selection Board, interviews flying cadet applicants.
Becoming an army officer doesn't keep Glenn Miller from being a favorite with autograph seekers.
Rudy Vallee, of the Coast Guard, reporting to Lieut. M. A. Sturges.
Clark Gable is now serving with the United States Army Air Forces.
After his driving chores, Robert Young joins the chow lines and loads up his plate.
This Army Air Corps looks on as Sgt. (honorary) McCarthy greets his friend James Stewart (right). Behind Charley is Edgar Bergen.
HOME OF STATION WLAC...
UPPER
Interior view at the 50,000-watt transmitter. WLAC's technical equipment is one hundred per cent Western Electric; every piece brand new and of the finest quality obtainable.
LOWER
Typical executive office at WLAC. These offices were constructed with an eye to comfort, affording an inviting atmosphere for visitors.
UPPER RIGHT
There are few radio stations with more "room space" than that used by WLAC. An entire floor, 12,000 square feet atop the Third National Bank Building, is utilized for this modern radio station.
LOWER RIGHT
WLAC's $175,000 transmitter is ideally located for the inspection of thousands of motorists travelling the Nashville-Chicago highway.
WLAC employs a full-time staff of six experienced announcers. Between them they have put in a total of forty years behind the microphone. Seated, left to right: Wayne Howell, Paul Oliphant and (Your Esso Reporter) Dow Mooney; Standing, left to right: Herman Grizzard, John Richbourg and Charlie Roberts.
Virginia Mansell came down from Pennsylvania to be with her father who is USO chief at Camp Forrest. Virginia is station's traffic manager and handles women's programs.
WLAC houses the Tennessee office of United Press, where three teletype machines bring in world news in an unending stream. Pictured is the Esso Reporter looking over a late bulletin.
WLAC
SERVES THE PEOPLE!
WLAC's increased power enables it to reach a far greater listening audience than ever before, bringing news, information of war interest and entertainment closer to the people of the Great Tennessee Valley.
Above: Wendell Willkie, Republican candidate for President in 1940, was featured speaker at the dedication of the great Willeke Building on the campus of Vanderbilt University. As usual, WLAC was on the spot to bring his message to Nashville.
Below: Station employees at the State Guard. Left to right: Miss C.A. Director Charles H. Smith, WLAC News Director Robert Maddox, Announcer Herman Grizzard, and Operator Oscar Griffin.
One of Nashville's most successful Utilities, WLAC-Lonergan, Bond Salesmen, have been selling bonds and donating to WLAC. Fine musical talent has been discovered among the flyers.
Above: Camp Forrest was the first Tennessee induction center. WLAC was there with its microphone. Announcer Tim Sanders is now a sergeant in the U.S. Marine Corps.
Below: First honors in originating programs from the State Capitol went to WLAC. Some of the best talent go to WLAC. Fine musical talent has been discovered among the flyers.
Top of page: Mayor Thomas L. Cummings of Nashville, accepts "Spuds" from the Air Force Auxiliary, with members of the "Spuds" Club, windshields studded with stars issued by the station in WLAC's name. Frances Rich (Trent's daughter) tells listeners about the advantages of using V-mail.
Left: When a sailor and a SPAR girl get together, they talk about Uncle Sam's Armed Forces provided WLAC provides them with a good quarter-hour of listening.
Right: WLAC employees were among the first in Nashville to adopt the "Payroll Savings Plan." Here, Nurse and Junior Secretary, two employees of the front office, hold the coveted U.S. Treasury Department's "Minute Man" flag.
BUY WAR BONDS!
WLAC STUDIOS...
Left: All studios were designed to acoustical perfection by CBS Engineers. Visitors to Studio "A" are entranced by the sight and sound of the great $20,000 Kilgen Pipe Organ.
Right: Charlie Roberts announces the 'Night Owl Club' program, and pulls stacks of mail from most Everywhere.
Left: From Studio "C" come 'Songs by Frances Hill', a popular feature on the WLAC schedule. Announcer Paul Oliphant at the mike.
Right: The station's ultra-modern control room was the result of expert planning by Chief Engineer David Binns (Standing).
WLAC SERVES THE NATION!
Since "Pearl Harbor", WLAC has employed its facilities in every possible way toward aiding in the country's war cause. Time valued at more than $1,000 per week has consistently been given for this purpose.
Above: Wendell Willkie, Republican candidate for President in 1940, is shown here as featured speaker at the dedication of the great Vultee Aircraft plant near Nashville. As usual, WLAC was on the spot to bring his message to its listeners.
Below: Station employees now serving in the Tennessee State Guard. Left to right: Musical Director Charles Nagy, Merchandise Manager Robert Maddux, Announcer Herman Grizzard, and Operator Oscar Griffin.
One of WLAC's most highly entertaining programs originates at Nashville's Air Corps Classification Center every week. A quiz-type show, it is presented under the title "Stump The Cadet", with talent recruited from the far corners of the nation.
Top of page: Mayor Thomas L. Cummings of Nashville, accepts from Manager F.C. Sowell the first "40-Mile Club" windshield sticker issued by the station in WLAC's novel campaign to reduce speed and save tires, before gas was rationed.
Right: WLAC employees were among the first in Nashville to adopt the "Payroll Savings Plan." Here Adine Fite and Josephine Lister two fair employees of the front office, hold the coveted U.S. Treasury Department's "Minute Man" flag.
BUY WAR BONDS!
WLAC
SERVES THE PEOPLE!
WLAC's increased power enables it to reach a far greater listening audience than ever before; bringing news, information of vital importance and fine entertainment to the people of the Great Tennessee Valley.
Above: Camp Forrest was the first Tennessee induction center and WLAC was first there with a microphone. Announcer Tim Sanders is now a captain in the U.S. Marine Corps.
Below: First honors in originating programs from the Smyrna Bomber Base, also go to WLAC. Fine musical talent has been discovered among the flyers.
One of Nashville's most successful Bond Sale Campaigns was conducted at the WLAC-Loveman 'Bond Site.' Utilizing a prominent downtown show-window and the cooperation of the city's leading civic clubs, the station sold $767,000 worth of bonds in just four weeks.
Above: Representatives of the women's auxiliary units of our armed forces always find a warm welcome at WLAC. Lt. Frances Rich (Irene's daughter) tells listeners about the advantages of using "V" mail.
Left: When a sailor and a SPAR get together, there's plenty to talk about. These two members of Uncle Sam's armed forces provided WLAC's audience a good quarter-hour of listening.
BUY WAR BONDS!
PERSONALITY PARADE
UPPER LEFT
The "Old Dirt Dobber" has received 650,000 letters from listeners to his weekly CBS program, 8:30 to 9:00 CWT, Saturday mornings. His broadcast is now official radio outlet for the U.S. Department of Agriculture.
LOWER LEFT
A "Natural-born" gardener himself, the Old Dirt Dobber knows whereof he speaks. He has been a WLAC feature for nine years.
UPPER RIGHT
Frances Hill guest-starred on the Camel Cigarette program in Hollywood. She is now singing on 9 sponsored programs per week on WLAC.
LOWER RIGHT
Little Texas Daisy, famous yodeling cowgirl, is a great favorite with WLAC listeners. Monthly fan mail for the diminutive singer comes from thirty-five states.
AT STATION WLAC
This station endeavors to inject a wide variety of entertainment into its programs, so as to have an appeal for all types of listeners. The talent portrayed on these pages will illustrate just how well this policy is being followed.
Studio ensemble heard daily over WLAC. This fine musical organization is composed of Mary Elizabeth Hicks at the novachord; Fred Murff, accordian; and Brooks Kirk, guitar.
War Information has an important place on the WLAC schedule. Here Virginia Mansell interviews James R. Aswell, local OWI director, on "What's New?"
Above: The "Chuck Wagon Gang" delivers the "Best there is" in current Western tunes and hill-billy music. This talented group enjoys a wide following throughout the South.
Left: In the deep south, nothing is more pleasing than songs by a good quartet. Especially when they're delivered in the smooth, rhythmic style of the "Fairfield Four". This quartet drew 10,000 letters in a single month.
CBS STARS ON WLAC
Gladys Swarthout Kate Smith
Nan Grey
Frazier Hunt Harry James Al Jolson Burns & Allen Fred Allen Major Bowes Bob Hawk
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Robert Montgomery, U.S. Naval attache in London, salutes 8 year old bomb victim, Alan Locke.
Gene Autry (second from left) finds time to entertain his buddies while training for the Air Corps.
Wayne King now directs army activities instead of "The Waltz You Saved For Me".
Robert Allen (left) gives Drew Pearson the real inside information on army life.
Dave Breger (Left), creator of the "Private Breger" cartoons, cuts a piece of birthday cake for his buddy, Ezra Stone ("Henry Aldrich").
Charles (Buddy) Rogers (right) reports to Lt. Comdr. C.C. McCauley prior to taking up his flying duties.
Jimmy Fidler looks on as Joan Davis (heard with Rudy Vallee) bids goodbye to Jimmy Henaghan, Fidler's ace reporter.
Walter Winchell of the United States Naval Reserve is "back in a flash with a flash".
Naval cadets and sailors sing and play for radio audience.
A corner of the barracks serves as rehearsal room for this "jive" group.
All sergeants are not "hard-boiled". This one burlesques a "home-makers' hour, discussing a topic of child apparel that doesn't seem to impress the young admirers.
Soldiers fresh from field duty accompany Service Club worker in broadcast.
Radio network correspondents, wearing prescribed uniforms, report from maneuver areas.
Aviation cadets at Randolph Field have organized this Glee club for radio appearances.
Soldiers on duty in Washington, D.C. boast this Glee club.
Robert Montgomery, U.S. Naval attache in London, salutes 8 year old bomb victim, Alan Locke.
Gene Autry (second from left) finds time to entertain his buddies while training for the Air Corps.
Wayne King now directs army activities instead of "The Waltz You Saved For Me".
Robert Allen (left) gives Drew Pearson the real inside information on army life.
Dave Breger (Left), creator of the "Private Breger" cartoons, cuts a piece of birthday cake for his buddy, Ezra Stone ("Henry Aldrich").
Charles (Buddy) Rogers (right) reports to Lt. Comdr. C.C. McCauley prior to taking up his flying duties.
Jimmy Fidler looks on as Joan Davis (heard with Rudy Vallee) bids goodbye to Jimmy Henaghan, Fidler's ace reporter.
Walter Winchell of the United States Naval Reserve is "back in a flash with a flash".
Naval cadets and sailors sing and play for radio audience.
A corner of the barracks serves as rehearsal room for this "jive" group.
All sergeants are not "hard-boiled". This one burlesques a "home-makers'" hour, discussing a topic of child apparel that doesn't seem to impress the young admirers.
Soldiers fresh from field duty accompany Service Club worker in broadcast.
Radio network correspondents, wearing prescribed uniforms, report from maneuver areas.
Aviation cadets at Randolph Field have organized this Glee club for radio appearances.
Soldiers on duty in Washington, D.C., boast this Glee club.
Buddies gather 'round to enjoy some boogie woogie on a Service Club piano.
Maj. General Hugh Drum faces a battery of microphones.
No spot is too tough for radio special events men. Here's one following the Army engineers during a river crossing.
Entertainment aboard ship enroute to Australia.
Sailors at Pensacola rehearse before broadcast.
A soldier audience at an open air broadcast as seen by performing artists.
The Famous U.S. Marine Corps Band heard on many broadcasts.
KEEPING IN TOUCH
Wherever they are, in training or in action, U.S. fighting forces look to radio to maintain their association with "home" -- it may be the voice of a friend, word from the home town or news from the good, old U.S.A. It all serves the same purpose for the service men who have no intentions of losing contact with things that were familiar before the war interrupted their lives. Radio does this job, too.
Winter-clad soldiers anchor cable for antenna in far northern base.
Radio and games in the barracks at a Naval Air Station.
Concentrated listening by Army men at a railroad siding.
Short-wave listening at an outpost in Puerto Rico.
U.S. NAVY INSIGNIA OF RANK AND SERVICE DIVISION
OFFICERS' SHOULDER AND SLEEVE INSIGNIA
ADMIRAL VICE-ADMIRAL REAR ADMIRAL CAPTAIN COMMANDER LIEUT COMMANDER LIEUT LIEUT (JG) ENSIGN CHIEF WARRANT WARRANT OFFICER
OFFICERS' CORPS DEVICES
LINE MEDICAL DENTAL SUPPLY CHAPLAIN CONSTRUCTION (Christian) CIVIL ENGINEERING BOATSWAIN MACHINIST PAY CLERK ELECTRICIAN GUNNER CARPENTER RADIO ELECTRICIAN PHARMACIST SERVICE STRIPES
PETTY OFFICERS' RATING MARKS
CHIEF FIRST CLASS SECOND CLASS THIRD CLASS CHIEF
Each service stripe, worn on the left sleeve below the elbow, indicates one year's service in that rating. After 12 years with good conduct, gold stripes are worn. Enlisted men's rating insignia become gold after completing three enlistments with good conduct. Red rating marks are worn on blue uniforms, blue marks are worn on white.
ENLISTED MEN'S SPECIALTY MARKS
BOATSWAINS GUNNER'S TURRET MATE CAPTAIN QUARTER-MASTER PAINTER PHOTOGRAPHER AVIATION ORDNANCE TORPEDOMAN CARPENTER'S MATE AVIATION METALSMITH COOK FIRE CONTROL MAN YEOMAN STORE MUSICIAN KEEPER PHARMACIST'S MATE AEROGRAPHER GUN CAPTAIN SEAMAN GUNNER MASTER HORIZONTAL BOMBER PARACHUTIST
CHIEF PETTY OFFICERS AND PETTY OFFICERS
The rating badges are worn on the sleeve between shoulder and elbow. Petty officers, seaman branch, wear rating badges on the right arm, other petty officers wear them on the left arm.
POCKET OR BREAST INSIGNIA
NAVAL AVIATOR AVIATION OBSERVER SUBMARINE MERCHANT MARINE PARACHUTIST
U.S. ARMY INSIGNIA OF RANK AND SERVICE BRANCH
OFFICERS' INSIGNIA OF RANK
- GENERAL
- LIEUT. GENERAL
- MAJOR GENERAL
- BRIG. GEN., COLONEL
- LIEUT. COLONEL
- MAJOR
- CAPTAIN
- FIRST LIEUT.
- SECOND LIEUT.
- CHIEF WARRANT OFFICER
- WARRANT OFFICER
CAP DEVICES
- OFFICERS
- WARRANT OFFICERS
- ENLISTED MAN
- AIR CADETS
- U. S. MILITARY ACADEMY
- WAAC
OFFICERS' LAPEL OR COLLAR INSIGNIA
- US OFFICERS
- WAAC
- ADJUTANT GENERAL
- AIDE TO GENERAL
- AIR FORCES
- CAVALRY
- CHAPLAINS (CHRISTIAN)
- CHAPLAINS (JEWISH)
- GENERAL STAFF
- INFANTRY
- INSPECTOR GEN.
- JUDGE ADVOCATE
- COAST ARTILLERY
- ENGINEERS
- FIELD ARTILLERY
- FINANCE DEPARTMENT
- CHEMICAL WARFARE SERVICE
- MEDICAL CORPS
- MEDICAL ADM.
- NURSE CORPS
- MILITARY POLICE
- NAT'L GUARD BUREAU
- ORDNANCE
- QUARTERMASTER CORPS
- ARMORED FORCE
- SIGNAL CORPS
- OFFICERS NOT MEMBERS OF A BRANCH
- WARRANT OFFICERS
- U. S. MILITARY ACADEMY
- ARMY TRANSP. CORPS
- ARMY BAND
- ARMY SPECIALIST CORPS
NON-COMMISSIONED OFFICERS' INSIGNIA
- MASTER SERGEANT
- FIRST SERGEANT
- TECHNICAL SERGEANT
- STAFF SERGEANT
- TECHNICIAN 3RD GRADE
- SERGEANT
- TECHNICIAN 4TH GRADE
- CORPORAL
- TECHNICIAN 5TH GRADE
- PRIVATE FIRST CLASS
BREAST INSIGNIA
- COMMAND PILOT
- SENIOR PILOT
- PILOT
- SERVICE PILOT
- GLIDER PILOT
- LIAISON PILOT
- NAVIGATOR
- BOMBARDIER
- AIR CREW MEMBER
- COMBAT OBSERVER
- FLIGHT SURGEON
- PARATROOPS
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U.S. ARMY INSIGNIA OF CORPS AND DIVISION
THE GENERAL COMMANDS
HEADQUARTERS
AIR FORCES GROUND FORCES SERVICE OF SUPPLY
SERVICE COMMANDS
FIRST SECOND THIRD FOURTH FIFTH SIXTH SEVENTH EIGHTH NINTH
ARMY CORPS
FIRST SECOND THIRD FOURTH FIFTH SIXTH SEVENTH EIGHTH NINTH
DIVISIONS
ELEVENTH TWELFTH THIRTEENTH FOURTEENTH 1st 2nd 3rd 4th 5th
6th 7th 8th 9th 26th 27th 28th 29th 30th
31st 32nd 33rd 34th 35th 36th 37th 38th 40th 41st
42nd 43rd 44th 45th 76th 77th 78th 79th 80th 81st
82nd 83rd 84th 85th 88th 89th 90th 91st 92nd 93rd
94th 96th 98th 99th 100th 102nd 103rd 104th HAWAIIAN DIV. FIRST CAVALRY DIVISION
ARMORED FORCE
HQ & HQ COMPANY 1st CORPS 4th DIV. NEW ENGLAND NEW YORK-PHILADELPHIA CHESAPEAKE BAY SOUTHERN COASTAL PACIFIC COASTAL PANAMA CANAL DEPT. HAWAIIAN DEPT.
AVIATION CADET
U.S. MARINES
INSIGNIA OF RANK AND SERVICE
OFFICERS' INSIGNIA OF RANK
LIEUTENANT GENERAL MAJOR GENERAL BRIGADIER GENERAL COLONEL LIEUTENANT COLONEL MAJOR CAPTAIN FIRST LIEUTENANT SECOND LIEUTENANT WARRANT OFFICER
CAP DEVICES
OFFICER SERGEANT MAJOR FIRST SERGEANT PLATOON SERGEANT MASTER TECHNICAL SERGEANT TECHNICAL SERGEANT STAFF SERGEANT SERGEANT CORPORAL PRIVATE FIRST CLASS
ENLISTED MAN
NON-COMMISSIONED OFFICERS' INSIGNIA
DEPARTMENTAL INSIGNIA
ADJUTANT & INSPECTOR'S DEPT. QUARTERMASTER'S DEPT. PAYMASTER'S DEPT. BRIG. GENERAL'S AIDE AVIATION CADET CHIEF GUNNER BAND LEADER
COAST GUARD
RANK AND SERVICE
OFFICERS SHOULDER INSIGNIA
CADET OFFICER WARRANT OFFICER USCG OFFICER'S STEWARD
REAR ADMIRAL CAPTAIN COMMANDER LIEUT. COMMANDER LIEUT. COMMANDER LIEUT. (JG) ENSIGN CHIEF WARRANT CHIEF WARRANT WARRANT
OFFICERS' SLEEVE INSIGNIA
CHIEF PETTY OFFICER REAR ADMIRAL CAPTAIN COMMANDER LIEUT. COMMANDER LIEUTENANT LIEUTENANT (JG) ENSIGN CHIEF WARRANT WARRANT FIRST CLASS CADET SECOND CLASS CADET
ENLISTED MEN'S SPECIALTY MARKS
AVIATION MACHINIST'S MATES AVIATION PILOTS AVIATION METALSMITHS BUGLERS RADIO MEN PHOTOGRAPHERS PRINTERS COOKS, SHIP'S STEWARDS BAND ELECTRICIAN'S MATES FIRST CLASS SECOND CLASS
QUARTER-MASTERS SIGNAL MEN GUNNER'S MATES BOATSWAIN'S MATES, COXSWAINS COMMISSARY STEWARDS PHARMACIST'S MATES YEOMEN MACHINIST'S MATES, WATER TENDERS MASTERS, MUSICIANS FIRST CLASS SECOND CLASS
CADETS' SHOULDER INSIGNIA
The figures presented on this page show American women in the uniforms authorized for their various types of war work. Never before in the history of the country have women played such important parts on the war front and the home front and enlisted in such numbers as today. This wholesale volunteering for war work releases large numbers of men for the actual business of fighting.
Member of the WAAC--Women's Army Auxiliary Corps.
Left: Member of Women's Auxiliary Ferrying Squadron (WAFS)
Right: Member of the WAVES--Women Appointed for Volunteer Emergency Service (Women's Reserve of the Naval Reserve)
Member of American Women's Voluntary Services.
Red Cross Worker
Right: Army Nurse
Left: Navy Nurse
Civilian defense worker.
Member of Red Cross Production Corps.
Member of Red Cross Motor Corps.
Red Cross Nurse
Member of Red Cross Nurse's Aid Corps.
Red Cross Canteen Worker.
Every able-bodied citizen has a part in the national defense of the United States. Any attack upon this country must find each citizen assigned to his or her place, trained in the duties involved, and resolute to carry out those duties, regardless of the danger to be faced.
Thousands of United States communities have organized and trained efficient Civilian Defense units and have conducted tests, drills and exhibitions to determine that each cog in the vital machinery of wardens, police, firemen, nurses, etc., will be capable of meeting any emergency.
Only with the complete cooperation and support of those whom Civilian Defense is designed to serve and protect, can it operate smoothly and efficiently. You will recognize the Civilian Defense Workers by these insignia.
1. DECONTAMINATION CORPS
2. FIRE WATCHER
3. AUXILIARY POLICE
4. RESCUE SQUAD
5. NURSES’ AIDE CORPS
6. DEMOLITION AND CLEARANCE CREW
7. AIR RAID WARDEN
8. MEDICAL CORPS
9. BOMB SQUAD
10. DRIVERS CORPS
11. AUXILIARY FIREMAN
12. ROAD REPAIR CREW
13. EMERGENCY FOOD AND HOUSING CORPS
14. MESSENGER
15. STAFF CORPS
1--When flags of states or cities or pennants of societies are flown on the same halyard with the flag of the United States of America, the latter should always be at the peak. When flown from adjacent staffs the flag of the United States should be hoisted first and lowered last.
2--When displayed with another flag against a wall from crossed staffs, the Flag of the United States should be on the right (the flag's own right), and its staff should be in front of the staff of the other flag.
3--When used on a speaker’s platform, whether indoors or out, the flag should never be reduced to the role of a mere decoration by being tied into knots or draped over the stand. For this purpose bunting should be used. The flag, if displayed, should be either on a staff or secured to the wall or back curtain behind the speaker with the union to the flag’s right.
4--When flags of two or more nations are displayed together they should be flown from separate staffs of the same height and the flags should be of approximately equal size.
5--When the flag is displayed in the body of the church, it should be from a staff placed in the position of honor at the congregation’s right as they face the clergyman. The service flag, the state flag or other flags should be at the left of the congregation. If in the chancel or on the platform, the flag of the United States should be placed on the clergyman’s right as he faces the congregation and the other flags at his left.
6--When the flag is displayed in a manner other than by being flown from a staff, it should be displayed flat, whether indoors or out. When displayed either horizontally or vertically against a wall, the union should be uppermost and to the flag’s own right, that is, to the observer’s left.
7--Whenever a number of flags of states or cities or pennants of societies are to be arranged in a group and displayed from staffs with the flag of the United States, the latter should be placed at the center of that group and on a staff slightly higher than any of the others.
8--When the flag is displayed from a staff projecting horizontally or at an angle from the window sill, balcony or front of a building, the union of the flag should go to the peak of the staff (unless the flag is to be displayed at half-staff).
9--Whenever the flag of the United States is carried in a procession in company with other flags, it should occupy a position in front of the center of the line of flags or on the right of the marching line.
RIGHT!
WRITE RIGHT!
Don’t be discouraged by long delays in receiving replies to your letters to men in Uncle Sam’s fighting forces. This is a World Wide War. The seas are wide and rough sailing. Regular mail travels in convoys and there are many unpredictable factors that may unavoidably delay the delivery of mail to men overseas. Be patient.
Don’t be discouraged by necessary military restrictions. Write often to your servicemen; write long letters, but, remember, your letter may fall into enemy hands. Don’t make it valuable reading for them.
The government considers your mail important—every ship that leaves this country carries mail.
The marines receive an assigned unit number and designation which he sends to the postmaster, either at New York or San Francisco, upon safe arrival overseas. The cards are then mailed to designated friends and relatives, who address mail according to the instructions on the cards.
POSTAGE FEES:
REGULAR MAIL: Three cents on letters addressed through an Army Post Office number.
AIR MAIL: Six cents per half ounce, outside United States.
PARCEL POST: Postage charged only from city of mailing to port of despatch in the United States. (Get exact cost from your local post office).
THE WAR AND NAVY DEPARTMENTS ALWAYS NOTIFY THE NEXT OF KIN IN THE EVENT OF ANY SERIOUS CASUALTIES. IN THE ABSENCE OF ANY SUCH REPORTS, IT IS SAFE TO ASSUME THAT “No News Is Good News”.
OUR HONOR ROLL
Name ____________________________
Entered Service _____________________
First Station _______________________
Promotions _________________________
Service Record _____________________
Decorations _______________________
Discharged _______________________
Name ____________________________
Entered Service _____________________
First Station _______________________
Promotions _________________________
Service Record _____________________
Decorations _______________________
Discharged _______________________
Name ____________________________
Entered Service _____________________
First Station _______________________
Promotions _________________________
Service Record _____________________
Decorations _______________________
Discharged _______________________
Name ____________________________
Entered Service _____________________
First Station _______________________
Promotions _________________________
Service Record _____________________
Decorations _______________________
Discharged _______________________
"Army, Navy and Marine Photos in this book were released for publication by the War and Navy Departments."
U. S. Army Signal Corps Photo
Official U. S. Navy Photograph
Official Photograph, U. S. Army Air Forces
Official U. S. Marine Corps Photograph
Blue Network Photo
NBC Photo
Fort Bragg Photo
INP International News Photos
Compiled and edited by Brooks Watson. Published by National Radio Personalities, Peoria, Illinois.
Additional copies of this book may be obtained by sending 25¢ to the publishers, Peoria, Illinois.
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RADIO STATION
WLAC
"Radio At War"
Album
Nashville Tennessee
FROM
WLAC
THIRD NATIONAL BANK BUILDING
NASHVILLE, TENNESSEE
TO
Mrs. Louella Rainey
1418 Highland Ave.
Nashville (6) Tenn
50,000 WATTS
COLUMBIA PROGRAMS
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|
Climate forcings in Goddard Institute for Space Studies SI2000 simulations
Citation
Hansen, J., M. Sato, L. Nazarenko, R. Ruedy, A. Lacis, D. Koch, I. Tegen, T. Hall, D. Shindell, B. Santer, P. Stone, T. Novakov, L. Thomason, R. Wang, Y. Wang, D. Jacob, S. Hollandsworth, L. Bishop, J. Logan, A. Thompson, R. Stolarski, J. Lean, R. Willson, S. Levitus, J. Antonov, N. Rayner, D. Parkerm and J. Christy. 2002. “Climate Forcings in Goddard Institute for Space Studies SI2000 Simulations.” Journal of Geophysical Research 107, issue D18: ACL 2-1-ACL 2-37.
Published version
https://doi.org/10.1029/2001JD001143
Link
http://nrs.harvard.edu/urn-3:HUL.InstRepos:14117821
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Climate forcings in Goddard Institute for Space Studies SI2000 simulations
J. Hansen,1,2 L. Nazarenko,1,2 R. Ruedy,1,3 A. Lacis,1 D. Koch,1,4 I. Tegen,5 T. Hall,1,6 D. Shindell,1 B. Santer,7 P. Stone,8 T. Novakov,9 L. Thomason,10 R. Wang,11 Y. Wang,12 D. Jacob,13 S. Hollandsworth,14 L. Bishop,15 J. Logan,13 A. Thompson,14 R. Stolarski,14 J. Lean,16 R. Willson,2 S. Levitus,17 J. Antonov,17 N. Rayner,18 D. Parker,18 and J. Christy19
Received 24 July 2001; revised 14 November 2001; accepted 27 November 2001; published 20 September 2002.
[1] We define the radiative forcings used in climate simulations with the SI2000 version of the Goddard Institute for Space Studies (GISS) global climate model. These include temporal variations of well-mixed greenhouse gases, stratospheric aerosols, solar irradiance, ozone, stratospheric water vapor, and tropospheric aerosols. Our illustrations focus on the period 1951–2050, but we make the full data sets available for those forcings for which we have earlier data. We illustrate the global response to these forcings for the SI2000 model with specified sea surface temperature and with a simple $Q$-flux ocean, thus helping to characterize the efficacy of each forcing. The model yields good agreement with observed global temperature change and heat storage in the ocean. This agreement does not yield an improved assessment of climate sensitivity or a confirmation of the net climate forcing because of possible compensations with opposite changes of these quantities. Nevertheless, the results imply that observed global temperature change during the past 50 years is primarily a response to radiative forcings. It is also inferred that the planet is now out of radiation balance by 0.5 to 1 W/m$^2$ and that additional global warming of about 0.5°C is already “in the pipeline.”
INDEX TERMS: 1620 Global Change: Climate dynamics (3309); 1635 Global Change: Oceans (4203); 1650 Global Change: Solar variability;
KEYWORDS: climate forcings, climate models, greenhouse gases, aerosols, solar irradiance, ozone
Citation: Hansen, J., et al., Climate forcings in Goddard Institute for Space Studies SI2000 simulations, J. Geophys. Res., 107(D18), 4347, doi:10.1029/2001JD001143, 2002.
1. Introduction
[2] A fundamental challenge regarding climate is to determine how much of observed climate change is a response to climate forcings, as opposed to chaotic (unforced) variability. A climate forcing is an imposed perturbation of the Earth’s energy balance with space. Forcings arise naturally, as with aerosols injected by volcanic eruptions, and from human activities, as with increasing greenhouse gases.
[3] Climate models provide a tool for investigating the effect of climate forcings. One obstacle to achieving the full potential of the models is the imperfect data for actual forcings. However, knowledge of forcings is improving as modern observations accumulate. Also, some estimated forcings for prior times are tending to become more
Figure 1. Measured greenhouse gas amounts and an extension to 2050 based on the “alternative scenario” of Hansen et al. [2000b]. The sum of the CFC and “other trace gas” forcings is constant after 2000. For comparison, we illustrate IS92a scenarios for CO$_2$, CH$_4$, and N$_2$O [Intergovernmental Panel on Climate Change, IPCC, 1992]. After 2000, “other trace gases” are assumed to increase so as to exactly compensate for predicted declines of CFC-11 and CFC-12.
quantitative and reliable, e.g., based on polar ice core data and improved chemical transport models.
[4] One consequence of continued improvement of forcing data is that current scenarios are sure to be replaced by more realistic ones. Nevertheless, there is reason to document our present scenarios. This is needed for interpretation of our climate simulations, and it will also allow other researchers to use the same forcings or at least make accurate comparisons.
[5] We use the radiative flux change at the tropopause as a primary measure of climate forcings. However, in some cases, especially for absorbing aerosols and ozone changes, this flux change can be a poor predictor of even the global mean climate response [Hansen et al., 1997c] (hereinafter referred to as RF-CR). Therefore we also illustrate the simulated equilibrium climate response to each forcing using a climate model with a mixed layer ocean. This helps to characterize and compare the different forcings.
[6] We define in section 2 the scenarios for well-mixed greenhouse gases, stratospheric aerosols, solar irradiance, ozone, stratospheric water vapor, and tropospheric aerosols, and we compare the climate forcings for each of these mechanisms. In section 3 we discuss the SI2000 model and summarize the experiments that are being carried out with it. In section 4 we illustrate the equilibrium response of the climate model to each of these forcings. The transient model response to these forcings for the period 1951–2050 is presented in section 5 for simple representations of the ocean. Implications of the simulations are discussed in section 6.
2. Climate Forcings
[7] We consider a sequence of six climate forcings in order of how accurately we believe they can be defined: well-mixed greenhouse gases, stratospheric aerosols, solar irradiance, ozone, stratospheric water vapor, and tropospheric aerosols. This sequence, for the first five forcings, is also approximately the order of their importance for timescales of 1–100 years. The sixth forcing, tropospheric aerosols, is probably large, but it is complex with negative and positive components, and its history is uncertain, especially the critical black carbon component. A seventh forcing, the indirect effect of tropospheric aerosols on clouds, could be substantial, but it is even more uncertain. The indirect aerosol effect is being tested in the SI2000 model by S. Menon and A. Del Genio, as will be reported elsewhere. The forcing due to anthropogenic land surface alterations [Sagan et al., 1979; Henderson-Sellers and Gornitz, 1984; Hansen et al., 1998; Govindasamy et al.,
Figure 2. Adjusted climate forcings due to six mechanisms, (a) Global mean forcings and (b) their sum as a function of time.
[8] We calculate both the instantaneous and adjusted forcings for most of the climate change mechanisms that we consider. The instantaneous forcing, $F_i$, is the flux change at the tropopause that occurs when the radiative constituent is changed, but the temperature is kept fixed throughout the atmosphere. The adjusted forcing, $F_a$, is the flux change after the stratospheric temperature has been allowed to adjust to a new radiative equilibrium profile. It has been shown that the adjusted forcing in general provides a better measure to judge the expected climate response [RF-CR], so we usually illustrate the adjusted forcing. We show only the global mean forcing here; global maps are shown in section 4, where they can be compared with global maps of the climate response. We consider only the period since 1950. The historical evolution of forcings since 1750 is discussed by Myhre et al. [2001].
[9] We calculate the instantaneous and adjusted forcings using a full annual cycle of the model control run for which the three-dimensional climate fields were saved at every time step. We define the tropopause the same as RF-CR have, varying from 100 hPa in the tropics to 300 hPa at the poles. We calculate the fluxes at the model levels just above and just below this tropopause level, averaging these results to obtain the flux at the tropopause.
2.1. Well-Mixed Greenhouse Gases
[10] The primary gases that we include (Figure 1) are CO$_2$, CH$_4$, N$_2$O, and the chlorofluorocarbons (CFCs). Data for recent decades are based on in situ observations available from the NOAA Climate Monitoring and Diagnostics Laboratory (CMDL) [1998]. The earlier data for CO$_2$ and CH$_4$ are based on ice core measurements [Etheridge et al., 1996, 1998], as are the earlier N$_2$O data [Machida et al., 1995]. The deviation of the estimated global mean CH$_4$ from the amount measured at the South Pole (Figure 1) is due to the pole-to-pole gradient of CH$_4$. CFC amounts prior to in situ measurements are estimated from industry production data, assuming atmospheric lifetimes of 50 and 100 years for CFC-11 and CFC-12, respectively [Hansen et al., 1998]. We include an estimate for the other well-mixed trace gases, primarily halons [Intergovernmental Panel on Climate Change (IPCC), 1996, 2001; Myhre et al., 1998; Hansen et al., 1998; Highwood and Shine, 2000], which we specify as an additional amount of CFC-12. Annual amounts of the well-mixed greenhouse gases at 5-year intervals are given in Table 1. All annual data are available at www.giss.nasa.gov/data/si2000/ghgases.
[11] We calculate the climate forcing using the SI2000 version of the Goddard Institute for Space Studies three-dimensional climate model [Hansen et al., 2000a], which uses the Lacis and Oinas [1991] correlated $k$ distribution radiative transfer method. The updated absorption coefficients are based on fits to line-by-line radiative transfer calculations with current HITRAN [Rothman et al., 1998] absorption line data. We estimate the possible error in the forcing for the sum of the well-mixed greenhouse gases as about 10%, because of uncertainties in gas amounts and absorption coefficients.
[12] The forcing by the well-mixed greenhouse gases (GHGs) has increased steadily over the past 50 years (Figure 2a). The increase of the GHG forcing in the period 1951–2000, more than 1.6 W/m$^2$, is about 70% of the increase during the Industrial Era, i.e., since 1850 [Hansen et al., 1998]. Although it is barely perceptible in a cumulative graph such as Figure 2, the growth rate of the GHG forcing slowed significantly in the 1990s. This slowdown was mainly in the CFCs and CH$_4$, as illustrated for the individual gases by Hansen et al. [2000b] and Hansen and Sato [2001].
[13] We include for comparison two scenarios for future greenhouse gases in Figure 1: scenario IS92a of IPCC [1992, 1996] and the “alternative” scenario of Hansen et al. [2000b]. In the “alternative” scenario the CO$_2$ growth
Table 1. GHGs With “Alternative” Scenario for 2000–2050a
| Year | CO₂, ppm | CH₄, ppb | N₂O, ppb | CFC-11, ppt | CFC-12, ppt | Others, ppt |
|------|----------|---------|----------|-------------|-------------|------------|
| 1850 | 285.2 | 791 | 275.4 | 0 | 0 | 0 |
| 1900 | 295.6 | 879 | 279.8 | 0 | 0 | 0 |
| 1950 | 310.7 | 1147 | 289.0 | 0.7 | 9.3 | 0 |
| 1955 | 313.1 | 1192 | 290.1 | 4.1 | 19.7 | 0 |
| 1960 | 316.5 | 1247 | 291.6 | 10.7 | 38.1 | 2.9 |
| 1965 | 319.6 | 1312 | 293.8 | 27.8 | 72.0 | 6.4 |
| 1970 | 324.7 | 1386 | 296.2 | 61.7 | 133.0 | 20.5 |
| 1975 | 330.2 | 1465 | 298.8 | 118.0 | 220.9 | 43.1 |
| 1980 | 337.9 | 1547 | 301.2 | 166.6 | 300.3 | 79.9 |
| 1985 | 344.9 | 1618 | 305.0 | 213.3 | 387.4 | 141.5 |
| 1990 | 353.0 | 1676 | 308.7 | 262.7 | 478.7 | 253.4 |
| 1995 | 359.5 | 1709 | 311.6 | 271.4 | 522.8 | 322.8 |
| 2000 | 368.4 | 1740 | 315.4 | 267 | 535 | 333 |
| 2005 | 376.8 | 1765 | 318.8 | 258 | 535 | 340 |
| 2010 | 385.0 | 1779 | 322.1 | 246 | 527 | 358 |
| 2015 | 393.0 | 1783 | 325.4 | 231 | 508 | 389 |
| 2020 | 400.8 | 1776 | 328.5 | 214 | 486 | 425 |
| 2025 | 408.4 | 1759 | 331.5 | 197 | 463 | 462 |
| 2030 | 415.8 | 1731 | 334.5 | 180 | 441 | 498 |
| 2035 | 423.0 | 1693 | 337.3 | 164 | 420 | 532 |
| 2040 | 430.0 | 1644 | 340.0 | 149 | 400 | 564 |
| 2045 | 436.8 | 1585 | 342.7 | 136 | 380 | 595 |
| 2050 | 443.4 | 1515 | 345.2 | 123 | 362 | 624 |
a“Other” trace gases are radiatively equivalent CFC-12 amounts.
rate initially rises slightly but begins to decline after 2020. The CH₄ growth rate continues to decline slowly such that the CH₄ amount peaks in about 2015 at about 1785 ppm and declines to 1515 ppm in 2050 (13% less CH₄ than at present). The CH₄ forcing in 2050 is thus −0.1 W/m² relative to 2000. N₂O continues to increase through the period at a slowly declining rate, such that the additional forcing in 2050 is +0.1 W/m². The “other” well-mixed trace gases are assumed to increase so as to provide a forcing that balances expected decreases of CFC-11 and CFC-12. For computational purposes this is handled by keeping the CFCs and “other” well-mixed gases constant after 2000.
2.2. Stratospheric Aerosols
2.2.1. Aerosol properties
[14] Climate forcing by stratospheric aerosols depends mainly on the aerosol optical depth across the solar spectrum and in the thermal infrared [Lacis et al., 1992; RF-CR]. The optical depth is thus required over almost 2 orders of magnitude of wavelength, which implies that the aerosol size distribution must be known well. This means that the effective radius of the size distribution, i.e., the area weighted mean radius [Hansen and Travis, 1974], must be known accurately. In addition, the effective variance of the size distribution is needed with reasonable accuracy for the sake of calculating infrared heating of the stratosphere.
[15] Accurate aerosol information can be derived for the period with multispectral measurements of atmospheric extinction obtained by the SAGE (Stratospheric Aerosol and Gas Experiment) instrument [McCormick et al., 1995]. The four wavelengths of SAGE, from 0.385 μm to 1.02 μm, cover a range sufficient to define the effective radius of the size distribution. When combined with satellite data for aerosol extinction in the thermal infrared region, the effective variance of the size distribution is also constrained.
[16] One of us (AL) has used the SAGE data at all wavelengths to calculate the effective particle radius that fits the multispectral SAGE data most accurately, with the variance of the size distribution constrained by infrared occultation data [Lambert et al., 1993], as summarized by Hansen et al. [1996]. The retrieval method is defined by Lacis et al. [2000]. The satellite data required for this analysis are available for the Pinatubo era, i.e., the 1990s, but not immediately after the El Chichon eruption of 1982. Thus for El Chichon we employed information extracted from aircraft, balloon, and ground-based observations by P. Russell and colleagues at Ames Research Center [Russell et al., 1996; Hansen et al., 1997d (hereinafter referred to as F-C)]. For still earlier times we used aerosol optical depths inferred from ground-based observations [Sato et al., 1993]. Aerosols in the Mount Agung period, which had an optical depth similar to that of the El Chichon aerosols, were assumed to have the same size distribution as those after El Chichon, but the latitudinal distribution of sizes was reflected about the equator. Lesser volcanoes were assigned the size distribution of the background stratospheric aerosols.
[17] The optical depth at wavelength 550 μm and the effective radius of stratospheric aerosols for our resulting scenario are shown in Figures 3a–3d for the period 1951–2000. The vertical distribution of the aerosol optical depth is based on the SAGE data for Pinatubo, which showed an early injection or lifting of aerosols toward higher levels followed by subsidence. Aerosols from earlier major volcanoes were assumed to follow a similar altitude scenario (Figure 3e). Tabular data for our stratospheric aerosol parameters are available from www.giss.nasa.gov/data/strataer, including updates and minor improvements to the data file of Sato et al. [1993].
2.2.2. Radiative forcing
[18] Even with the aerosol properties known, there is uncertainty in their climate forcing. Using our SI2000 climate model to calculate the adjusted forcing for a
Figure 3. (a) Global and hemispheric mean stratospheric aerosol optical depth at wavelength 550 nm. (b) Variation of this optical depth with latitude. (c and d) Effective radius of the aerosol size distribution. (e) Altitude distribution of stratospheric aerosols employed in simulations with SI2000 model.
globally uniform stratospheric aerosol layer with optical depth $\tau = 0.1$ at wavelength $\lambda = 0.55 \, \mu m$ yields a forcing of $2.1 \, W/m^2$, and thus we infer that for small optical depths
$$F_a \left( W/m^2 \right) \sim -21 \, \tau.$$
This is substantially smaller than values we estimated earlier ($-30 \, \tau$ by Lacis et al. [1992] and $-26.8 \, \tau$ by F-C [1997]). The smaller forcing in our current model may be due in part to increased vertical resolution in the stratosphere, greater warming of the stratosphere by the volcanic aerosols, and thus greater downward thermal radiation. In our earlier 9-layer model stratospheric warming after El Chichon and Pinatubo was about half of observed values (Figure 5 of F-C), while the stratospheric warming in our current model exceeds observations, as shown below. Our calculated forcing is smaller than the $-25.4 \, \tau$ of Andronova et al. [1999], but it is larger than the $\sim -15 \, \tau$ of Ramachandran et al. [2000]. This forcing is sensitive to the assumed aerosol size distribution.
[19] The adjusted forcing resulting from the stratospheric aerosol properties defined above is shown in Figure 2a. The peak global mean forcing was about $-3 \, W/m^2$ after Pinatubo and about $-2 \, W/m^2$ after both Agung and El Chichon. The geographical distribution of aerosols (and the aerosol forcing, which we illustrate in section 4) in the 12 months after Pinatubo peaks at low latitudes and is reasonably symmetric about the equator. The aerosols after Agung and El Chichon were greater in one hemisphere than in the other by a factor of 2–3, as shown in Figure 3a. On the basis of studies and comparisons in our several previous papers on stratospheric aerosols we subjectively estimate the uncertainty in the stratospheric aerosol forcing as 15% for the Pinatubo era, 20% for El Chichon, 30% for Mount Agung, and 50% for the large volcanoes in the period 1880–1915.
[20] Climate forcing by stratospheric aerosols is substantial after large volcanoes, even temporarily exceeding in magnitude the forcing by greenhouse gases, as shown in Figure 2. It is apparent that a clustering of volcanoes could be a significant contributor to long-term climate change. It has been suggested, for example, that volcanic aerosols were the principal cause of the “Little Ice Age” [Lamb, 1970; Pollack et al., 1976]. Free and Robock [1999] carry out simulations of that period with both solar and aerosol forcings, and they argue that volcanoes were at least as important as solar irradiance in climate change of recent centuries.
[21] We note that the stratospheric aerosol optical depth inferred by Andronova et al. [1999] for the period after the Pinatubo eruption is about 50% larger than our value [Andronova et al., 1999, Figure 14]. Their larger aerosol optical depth is the primary reason that they obtain a Pinatubo forcing of about $-5 \, W/m^2$, compared with our maximum forcing of about $-3 \, W/m^2$. (A secondary reason is that their calculated maximum instantaneous forcing of...
Figure 5. Data sources and assumptions used to construct the ozone change scenario $O_3^A$ for different regions in the atmosphere and different time intervals. $O_3^B$ is the same except that the 1979–1996 trends in the stratosphere are based on the analysis of Randel and Wu [1999].
−4.7 W/m² increases to −5.4 W/m² after thermal adjustment of the stratosphere. Our calculated forcing decreases with thermal adjustment (section 4.1), as the aerosols heat the stratosphere thus increasing downward longwave radiation.) The main reason for their larger optical depth may be their assumption of a fixed aerosol size distribution (log-normal with mean radius 0.2 μm). As discussed above, we obtain the aerosol size by finding the effective particle radius that fits the multispectral SAGE data most accurately. The estimated uncertainty of the optical depth in our multiple wavelength retrievals [Lacis et al., 2000] using SAGE observations is typically several percent. There were situations after Pinatubo in which the SAGE observations were saturated and required aide from ground-based lidar observations [McCormick et al., 1995] and other data sources [Russell et al., 1996]. However, this saturation occurred only a small fraction of the time and affected mainly low altitudes. The large differences with Andronova et al. [1999] persist at times and places where there was no saturation. As part of our previous modeling study [F-C] we found that our Pinatubo aerosol properties, derived from SAGE, are in good agreement with analyses of multiple data sources by Russell et al. [1996]. We believe that our stratospheric aerosol climate forcing is accurate within about 15% during the Pinatubo era.
[22] We show in sections 4 and 5 that satellite measurements of the planetary radiation balance and ocean measurements of heat storage are consistent with a Pinatubo forcing of −3 W/m² but not with a forcing of −5 W/m². However, neither of these measures of aerosol forcing can provide an accuracy approaching that obtainable from the precise multispectral occultation measurements of SAGE. The SAGE extinction measurements are simple and precise, including calibration observations of the unocculted Sun. These data can be converted accurately to multispectral aerosol optical depths, and thus they should yield the most accurate available information on climate forcing by stratospheric aerosols.
[23] We assume in our “alternative scenario” that stratospheric aerosol amount in the period 2001–2050 will be comparable to that of the previous 50 years. Specifically, we duplicate the aerosol properties for 1951–2000. Thus a Mount Agung eruption occurs in 2013, an El Chichon eruption occurs in 2032, and a Pinatubo eruption occurs in 2041.
2.3. Solar Irradiance
[24] The total solar irradiance has been measured to a useful accuracy since 1979 [Willson and Hudson, 1991]. For earlier times we must rely on solar irradiance reconstructed from proxy measures of solar variability. Analysis of historical change of the total solar irradiance and its spectrum are described by Lean et al. [1995, 1997]. We use the solar spectral radiance reconstruction provided by J. Lean in 1999 (private communication). The data that we employ in our present total and spectral irradiance scenarios are available from our web site www.giss.nasa.gov/data/si2000/solar.irradiance) or from J. Lean.
[25] Lean’s scenario for total solar irradiance is shown in Figure 4a. We also illustrate the mean spectral irradiance (Figure 4b), the spectral irradiance change, i.e., the difference between solar maximum and solar minimum (Figure 4c), and the corresponding fractional change at each wavelength (Figure 4d). The solar variability is largest in the ultraviolet, in the wavelength range where the solar energy is absorbed in the stratosphere. However, because the irradiance is small in the ultraviolet, only about 15% of the solar variability occurs at wavelengths that are absorbed in the stratosphere [RF-CR]. The other 85% of the solar variability is deposited below the tropopause, mainly at the Earth’s surface.
[26] There are other, indirect, climate forcings caused by solar variability, in addition to the direct forcing from absorbed solar radiation. One indirect solar forcing that has been investigated is the change of ozone induced by solar irradiance variability [Haigh, 1994, 1999; RF-CR; Shindell et al., 1999, 2001]. However, there is uncertainty about
the magnitude of this indirect forcing, and we caution that the ozone forcing provides a very poor measure of the surface temperature response, even regarding its sign (see section 4.2). The ozone change associated with the solar cycle almost certainly provides a positive amplification of the direct solar forcing, but it is complex and may depend significantly on the background state of the stratosphere [Shindell et al., 2001]. Haigh [1994] points out that increased ultraviolet radiation should increase ozone throughout the stratosphere for today’s atmospheric composition. The instantaneous forcing due to ozone increase at any altitude in the stratosphere is negative, but the adjusted forcing is positive for ozone increases at altitudes below about 30 km (Table 3 of RF-CR). Shindell et al. [2001] conclude that in the drier preindustrial stratosphere the temperature dependence of ozone chemistry would cause the indirect ozone forcing to be positive for both upper and lower stratosphere, and the effect on surface climate would be magnified by its influence on the phase of the Arctic Oscillation.
[27] Ozone change in the lower stratosphere (below 30 km) is much more effective in causing radiative forcing than changes at higher altitudes [RF-CR]. If the changes in the lower stratosphere are systematic, they are likely to be the dominant indirect forcing. In our previous [RF-CR] and present calculations the solar cycle ozone forcing are 0.05 and 0.02 W/m$^2$, respectively, both of these in phase with the direct solar forcing. The solar cycle ozone change, inferred in these two cases by S. Hollandsworth and R. Wang, respectively, are difficult to extract from a 2-decade record because of other causes of ozone change during that period and measurement errors.
[28] Study of these solar cycle ozone changes, even though the radiative forcing is small, should be pursued in more detail with a model that resolves the stratosphere well, because of possible dynamical indirect effects. Stuber et al. [2001] suggest that ozone perturbations may in turn amplify stratospheric water vapor changes, which raises the interesting possibility of a second-generation indirect forcing that might significantly amplify the solar forcing.
[29] Other possible amplifications of the solar forcing have long been discussed, usually involving mechanisms that alter cloud properties, for example, solar modulation of cosmic ray flux and thus atmospheric ionization [Svensmark and Friis-Christensen, 1997]. Marginal detection of a change in earthshine during the current solar cycle [Goode et al., 2001] are not inconsistent with a larger cloud reflectivity during solar minimum, but the suggestion remains, at most, a hypothesis.
[30] The direct climate forcing due to solar irradiance variability has a peak-to-peak amplitude of about 0.2 W/m$^2$, over each of the recent solar cycles, as illustrated in Figure 2a. The geographical distribution of the solar irradiance forcing, which we illustrate in section 4, is of course largest at low latitudes. The trend in solar irradiance is near zero for the period 1951–2000. However, there is a long-term change of about 0.25% in the Lean et al. [1995] solar irradiance between 1700 and 1950, corresponding to a forcing of about 0.6 W/m$^2$. It has long been argued that the Sun is the likely cause of the “little ice age” [Eddy, 1976]. The simulations of Shindell et al. [2001] provide a plausible quantitative explanation of how a moderate global forcing may provide a substantial regional climate effect. It appears that volcanic aerosols and solar irradiance could be of comparable importance as climate forcings on century timescales.
[31] We include solar variability in our “alternative scenario” for 2000–2050 with 10-year periodicity, cyclically repeating the data for January 1989 to December 1998. Thus there is no long-term solar trend in our simulations. We argue elsewhere [Hansen, 2000] that solar irradiance could be a significant climate forcing in the next 50 years, but as yet we have no reliable way of predicting future solar changes.
2.4. Ozone
[32] We construct an ozone change scenario from several data sources, models and assumptions. For the presatellite era (until 1979) we use a model calculation to specify the tropospheric ozone change, and we include only cyclic (solar cycle and QBO) stratospheric ozone variations. After 1979 satellite data are the primary basis for both stratospheric and tropospheric ozone change. Because of uncertainties in the data, climate simulations using this scenario should be viewed as a sensitivity study. We hope that this straw man scenario may stimulate construction of a more precise ozone change data set. Indeed, as discussed below, we already include one optional change to the ozone trends in the polar stratospheric regions for the period after 1979, based on the analysis of Randel and Wu [1999].
[33] Figure 5 summarizes data sources employed in the first version of our O$_3$ data set, O$_3^A$. A key component is the “model” of ozone change constructed by R. Wang from SAGE measurements [McCormick et al., 1992] for the altitudes (20–51 km), latitudes (60S-60N), and period (1979–1996) sampled by SAGE. The model includes seasonal, long-term trend, solar cycle, and quasi-biennial ozone changes. Solar cycle and QBO periodicities are based on proxy indicators (10.7 cm solar flux series for the solar cycle and Singapore zonal winds for the QBO) with O$_3$ amplitudes and phases inferred from the SAGE data. Our extrapolation back to 1951 is based on the assumption that the long-term trend of stratospheric ozone was zero in 1951–1970, and in 1970–1979 it was half as large as in 1979–1996. The assumption that there was some stratospheric ozone depletion in 1970–1979 is consistent with limited surface (Dobson) measurements [World Meteorological Organization (WMO), 1999, Tables 4–7] and ozone sonde data [WMO, 1998], as well as with the interpretation that ozone depletion of recent decades is caused by halocarbons. In scenario O$_3^A$ ozone change at the altitude of SAGE data was extrapolated to the poles with reference to latitudinal variations in SBUV [Hollandsworth et al., 1995] and TOMS [WMO, 1999] data. In scenario O$_3^B$ this extrapolation is replaced by the analysis of Randel and Wu [1999] for the ozone trend during 1979–1996. O$_3^B$ is presumed to be more realistic, but O$_3^A$ is also defined here since it was used in some of our climate simulations reported below.
[34] At lower altitudes the ozone change for 1979–1996 is based on the difference between the column ozone change measured by TOMS, as analyzed by S. Hollandsworth, and the SAGE ozone change. The resulting ozone change is distributed with height according to a “SPARC-like” vertical profile [WMO, 1998]. Specifically, we assumed that the maximum ozone depletion occurred about 3 km above the tropopause, with a trend toward less negative or positive ozone...
change closer to the ground. This procedure yielded ozone depletion in the upper troposphere at all latitudes, but ozone increase in the lower troposphere at latitudes 90N to 30S.
[35] For the period prior to 1979 our ozone change is based mainly on the model calculations of Wang and Jacob [1998]. They calculate the tropospheric ozone distributions for preindustrial and 1980 conditions, accounting for fuel combustion, industry, and biomass burning. We assume that the change between 1890 and 1980 occurs exponentially, as suggested by observations in Europe [Marenco et al., 1994], at the rate (which varies from grid box to grid box) required to yield the Wang and Jacob [1998] change.
[36] Our present ozone scenario differs markedly from that employed by F-C. The main change is the omission of the large ozone depletion at 17–20 km in the tropics that earlier SAGE analyses had suggested, but which is omitted from WMO [1998] assessments. This has a large impact on the simulated change of vertical temperature profile, as discussed in sections 5 and 6. Also F-C used SBUV data above the 32 hPa level, but we now use SAGE because it is more consistent with ozone sonde data [WMO, 1998].
[37] We take stratospheric ozone change as being independent of longitude, because of the absence of adequate measurements. It is likely that there are correlations between decadal longitudinal temperature changes and ozone changes, so it is desirable to remove this limitation in the future. We include longitudinal dependence of tropospheric ozone change during the time prior to 1979, when the tropospheric ozone change was taken from the model of Wang and Jacob [1998].
[38] Figure 6 summarizes the resulting ozone history. Total ozone (Figure 6a) shows little trend prior to 1980, as stratospheric ozone depletion during 1970–1980 competes with the longer-term tropospheric ozone increase. Figures 6b–6d shows the ozone change in more detail for the periods 1970–1979 and 1979–1997, with the two alternatives for the latter period, O$_3^A$ and O$_3^B$, both illustrated. Total ozone decreases in spring at high latitudes in both periods, but for 1970–1979 this result is in part an assumption justified by only limited data. Ozone in the lower troposphere increases at tropical and northern latitudes during 1979–1997 but decreases at high southern latitudes. This tropospheric ozone change for 1979–1997 is based on our combination of two satellite data sets, rather than upon any assumption about ozone sources or ozone change.
[39] The global mean ozone forcing is barely noticeable in Figure 2. The tropospheric ozone change by itself yields a positive forcing, but, on the global average, this tends to be balanced by the negative forcing due to recent stratospheric ozone depletion. This does not mean that the climate effect of ozone change is negligible. In fact, we show below that stratospheric ozone loss seems to be the largest cause of polar stratospheric cooling in recent decades, which in turn strengthens the polar vortex in zonal winds and affects the Antarctic and Arctic Oscillations [Thompson and Wallace, 1998; Sexton, 2001]. Over the Industrial Era, 1850–2000, the forcing that we estimate for tropospheric ozone change, $0.4 \pm 0.15$ W/m$^2$ [Hansen et al., 1998], is third in magnitude to CO$_2$ and CH$_4$ among the greenhouse gases. Our tropospheric O$_3$ forcing is consistent with that estimated by several other groups [IPCC, 2001]; however, we note that Mickley et al. [2001] and D. T. Shindell (private communication, 2001) argue that the tropospheric O$_3$ forcing over the Industrial Era could be as large as 0.7–0.8 W/m$^2$. The negative forcing that we calculate due to stratospheric ozone depletion of recent decades, about $-0.1$ W/m$^2$, is less in magnitude than the forcing calculated by RF-CR, because the current reconstructions for ozone change do not include a large depletion near the tropical tropopause.
[40] We do not provide a detailed breakdown of the ozone climate forcing into contributions from the troposphere and stratosphere for different periods, because the forcing is relatively small. However, we note that our results seem to be consistent with other recent calculations. Myhre et al. [2000] find a global forcing of +0.05 W/m$^2$ for tropospheric ozone change over the period 1980–1996. Shine and Forster [1999] obtain $-0.15 \pm 0.12$ W/m$^2$ for the forcing due to stratospheric ozone change in the same period. Our small positive forcing for ozone change over the period 1951–1997 occurs because the positive forcing from tropospheric ozone increase over the full period exceeds the negative forcing due to stratospheric ozone depletion in 1979–1997.
[41] We have zero long-term trend for future ozone in our “alternative scenario.” (The zero trend begins with 1998 in all of our simulations; the rate of ozone change derived from 1979–1996 data was extended through 1997). We include QBO and solar cycle ozone variations with 26 month and 10-year periodicities for the sake of retaining such variability. In reality, a small positive climate forcing is expected because of stratospheric ozone recovery, but conceivably the global mean forcing could be countered by a decrease of tropospheric ozone pollution (or enhanced by an increase of pollution). In any case, our scenario with zero future ozone trend provides climate change results that can be compared with those for more detailed and realistic assumptions about future ozone change.
2.5. Stratospheric Water Vapor
[42] We include the climate forcing due to stratospheric H$_2$O produced by oxidation of increasing CH$_4$. The forcing is small, only about 0.1 W/m$^2$ for the CH$_4$ increase between 1850 and 2000. However, changing H$_2$O, along with changing O$_3$ and CO$_2$, has a significant effect on stratospheric temperature.
[43] Measurements of stratospheric H$_2$O change [Rosenlof et al., 2001] reveal a larger trend, close to 1%/year, than CH$_4$ oxidation alone can account for. If this water vapor change were interpreted as a climate forcing it would yield a value greater than 0.1 W/m$^2$ [de Forster and Shine, 1999; Oinas et al., 2001; Shindell, 2001; Smith et al., 2001]. Although part of the excess stratospheric H$_2$O change could be a consequence of some climate forcing other than CH$_4$ oxidation, most of it is probably a climate feedback, i.e., a climatic response to forcings. Indeed, we find the profile of simulated H$_2$O change to be reasonably consistent with observations. The maximum increases are in the upper stratosphere and near the tropopause, with a minimum increase at 50–100 hPa. In our model, and we believe in the real world, the maximum in the upper stratosphere is from CH$_4$ oxidation and the maximum in the lower stratosphere is a positive climate feedback in response to tropoFigure 6. Ozone change in our scenario for 1951–1997: (a) total ozone versus time and (b–d) ozone trends in two time intervals as a function of month, latitude, and altitude. The third column in Figures 6b–6d shows the recommended alternative scenario $O_3^B$ for 1979–1997 that incorporates stratospheric ozone change according to Randel and Wu [1999]. Dashes in Figure 6d delineate the 12 layers below 10 hPa and the three layers above 10 hPa employed in our model calculations.
spheric temperature increase. The simulated water vapor increase in the lower stratosphere occurs without any warming of the tropopause. (This increase may be caused by an increased flux from the troposphere, where the water vapor amount increases, but we defer quantitative examination until we include a more realistic representation of the stratosphere.)
[44] In our terminology the portion of the H$_2$O change that is a consequence of CH$_4$ oxidation is an indirect climate forcing; specifically, we include it is an indirect forcing
Figure 7. (a) Annual stratospheric H$_2$O production rate for a tropospheric CH$_4$ abundance of 1675 ppmv based on the two-dimensional model of I. Plumb. (b) Equilibrium increase of stratospheric H$_2$O in our 12-layer SI2000 model with fixed SST given the production rate in Figure 7a.
associated with anthropogenic CH$_4$ increase [Hansen et al., 2000b]. The distinction between an indirect climate forcing and a climate feedback is that the indirect forcing occurs in response to a specific imposed change of atmospheric composition, rather than as a hydrologic response of the climate system to temperature change.
[45] We determine the CH$_4$-derived H$_2$O amount at levels below 10 hPa (i.e., in the part of the model with interactive dynamics, water vapor and radiation) by using the source strength for CH$_4$ oxidation as a function of latitude and height (Figure 7a) from a two-dimensional model [Randeniya et al., 1997]. In our transient simulations the H$_2$O production is proportional to surface CH$_4$ at time $t - 5$ years. We include the 5-year delay because it requires several months for a surface CH$_4$ increase to be mixed through the tropopause, a few years for it to reach the 1–10 hPa level where most oxidation occurs, and some additional time for the H$_2$O to mix down into the top climate model layer (10–30 hPa). With H$_2$O added continuously at this rate and with the SST fixed, the 12-layer version of the SI2000 model yields the equilibrium increase of H$_2$O shown in Figure 7b. The mass of the equilibrium increase of stratospheric H$_2$O is 1.6 times the annual column-integrated production, implying an average lifetime in the lower stratosphere (below the 10 hPa level) of 1.6 years for the H$_2$O in our model. The H$_2$O in the model exits the stratosphere mainly via saturation in the winter hemisphere in the polar and midlatitude regions. Although this removal mechanism is perhaps realistic, the stratospheric lifetime of the injected H$_2$O is shorter than estimates based on tracers [Hall and Waugh, 2000]. The too rapid removal is not surprising, given the crude vertical resolution of the model and the low model top.
[46] In the three model layers above 10 hPa, which have only a radiative influence in our 12-layer model, we specify a temporal variation of H$_2$O proportional to surface CH$_4$ at time $t - 5$ years. The distribution of CH$_4$-derived H$_2$O for a specific amount of surface CH$_4$ was calculated by one of us (D.S.) using a 23-layer version of the GISS model [Rind et al., 1988]. The H$_2$O above the 10 hPa level causes only a small forcing of the order of 0.01 W/m$^2$.
[47] Without any CH$_4$ source of H$_2$O, the lower stratosphere in the 12-layer SI2000 model is very dry, with about 1.9 ppm H$_2$O (average for layers 10–12, which corresponds to pressures 100–10 hPa or heights about 16–30 km) in the control run with 1951 boundary conditions. With the CH$_4$ source the stratospheric H$_2$O amount increases to about 2.4 ppm (in 1951), and in our transient simulations (section 5) the stratospheric H$_2$O increases to 2.7 ppm by 1998. However, this is still drier than recent observed values of about 4 ppm [Nedoluha et al., 1998]. Part of this underestimate of stratospheric H$_2$O is likely to be caused by the too brief (1.6 years) lower stratospheric residence time of H$_2$O oxidized from CH$_4$. Another reason for the deficiency of stratospheric H$_2$O is likely to be the fact that the model troposphere is 1°–2°C cooler than recent observations. The cool troposphere is due in part to imprecision in the treatment of longwave absorption by H$_2$O in the SI2000 model, as discussed below.
[48] The lifetime of H$_2$O injected into the lower stratosphere by the CH$_4$ source increased about 20% in a test with the 23-layer version of the GISS model [Rind et al., 1988] with top at 0.05 hPa. However, the 23-layer model also has a coarse vertical resolution, and we suspect that it still removes water vapor from the stratosphere too rapidly. We estimate that the climate forcing by CH$_4$-derived H$_2$O could be underestimated by 20–50% in our 12-layer model.
[49] The global mean climate forcing that we obtain for CH$_4$-derived H$_2$O is small, amounting to only 0.1 W/m$^2$ for the period 1850–2000 (790 ppb → 1700 ppb) and being hardly noticeable in Figure 2. Nevertheless, together with the indirect effect of CH$_4$ on tropospheric O$_3$, it is sufficient to make the climate forcing by CH$_4$ in the Industrial Era (0.7 W/m$^2$) half as large as the climate forcing by CO$_2$. As
illustrated in section 4, the forcing by CH$_4$-derived H$_2$O is relatively more effective than most forcings because it is concentrated at high latitudes.
### 2.6. Tropospheric Aerosols
Tropospheric aerosols cause great uncertainty about anthropogenic climate forcing. The uncertainty is related to the variability and heterogeneity of aerosols, which makes it difficult to accurately characterize and monitor aerosols on a global basis. Improved understanding of the aerosol forcing may be brought about by a combination of global aerosol transport models, global composition-specific aerosol monitoring from satellites, ground-based and in situ observations that constrain the transport models and satellite retrievals, and compilations and analyses of historical emissions. In the mean time, we carry out a sensitivity study using an aerosol scenario derived from an aerosol transport model that employs simple assumptions about time-dependent aerosol emissions.
Aerosols cause climate forcing in several ways: (1) the direct effect of aerosols on solar and infrared radiation, which has been studied extensively, (2) the semidirect-direct effect on clouds, as absorbing aerosols heat the atmosphere and thus tend to reduce large-scale cloud cover [RF-CR; Ackerman et al., 2000], (3) indirect effects on clouds, as increased condensation nuclei lead to smaller cloud drops, which can increase cloud brightness [Twomey, 1974] and cloud lifetime and cloud cover [Albrecht, 1989], (4) cloud absorption of sunlight caused by black carbon nuclei in cloud drops, and (5) snow and ice albedo reduction due to black carbon deposition. The direct and semidirect-direct effects are included in our present climate simulations. The indirect (Twomey and Albrecht) effects are not included here, but they are being investigated in the SI2000 model by S. Menon and A. Del Genio (private communication, 2001). The dirty cloud and dirty snow forcings, which are perhaps smaller than the other aerosol forcings, are not included in our present transient climate simulations.
There are a number of aerosol compositions that contribute significantly to the total atmospheric aerosol load [Andreae, 1995; Penner et al., 1998; Haywood and Boucher, 2000; IPCC, 2001]. Moreover, these aerosols are often internally mixed and their characteristics change with humidity, aerosol age, and other factors. As a first approximation we treat the different aerosols individually, i.e., as an “external mixture” of distinct aerosol compositions; a discussion of the differences between these mixtures is given in Box 7.1 of Harvey [2000]. We can ameliorate the impact of the external mixture approximation when we assign aerosol optical properties and when we assess the results. For example, we can estimate the increased absorption by black carbon when it is internally mixed [Fuller et al., 1999; Jacobson, 2000, 2001a, 2001b]. However, it is desirable to eventually employ explicit realistic representation of multiple composition aerosols.
The aerosols that are included in the SI2000 model are listed in Table 2. The only tropospheric aerosols that are time-dependent in our present experiments, and thus the only ones providing a climate forcing, are sulfates (S), black carbon (BC), and organic carbon (OC) aerosols. The primary sources of these aerosols are associated with fossil fuel use. We do not include a long-term change of aerosols from biomass burning, of soil or desert dust, or of nitrates. There has probably been some increase of biomass burning over the past 50 years, but we do not have data that quantify the temporal variation. Soil dust has probably changed because of human activities, especially land use practices, and there is also a natural variability of airborne soil dust with drought cycles, but it is difficult to quantify these. Calculations by Adams et al. [2001] of climate forcing by nitrates suggest a forcing change of the order of $-0.1 \text{ W/m}^2$ in the past 50 years. This is less than the uncertainties in some of the other aerosol forcings, so the omission may not be too important for our present simulations, but Adams et al. [2001] suggest that nitrates could become increasingly important in the future.
The sources of the aerosol distributions in our model that do not have a secular trend are as follows. The natural sulfate distribution was computed by one of us (D.K.) using the sources specified by Koch et al. [1999] in the transport model described below. The sulfates, black carbon and organic carbon aerosols from biomass burning are based on the transport modeling of Koch et al. [1999], with the carbonaceous aerosols employing the source distribution of Cooke and Wilson [1996]. Soil dust is based on Tegen and Fung [1995] and Tegen and Lacis [1996]. The sea salt distribution is based on Tegen et al. [1997], but it has been increased by a factor of four as suggested by Quinn and Coffman [1999] and Haywood et al. [1999].
Our time-dependent aerosol distributions are based on aerosol transport model calculations carried out by one of us (D.K.) with a 9-layer version of the GISS GCM. We interpolate the results to the 12 layers of the SI2000 model. Koch et al. [1999] and Koch [2001] describe the transport calculations for S, BC, and OC distributions for current emissions. Koch used the same model to calculate the distributions in 1950, 1960, 1970, 1980, and 1990 that we employ in our present climate simulations. The global S emission scenario is from Leffohn et al. [1999]. The method of Cooke and Wilson [1996] was used to derive BC emissions from United Nations energy statistics [Tegen et al., 2000; Koch, 2001]. The UN statistics distribute fuel use among countries, and emissions within countries are distributed in proportion to the population. The fuel data account for temporal changes in sulfur content, but not for changes of combustion technology.
| Aerosol | Optical Depth 1950/1990 | Single Scattering Albedo 1950/1990 |
|--------------------------|-------------------------|------------------------------------|
| Tropospheric sulfate | 0.004 | 1.00 |
| Natural | | |
| Biomass burning | 0.0006 | 1.00 |
| Anthropogenic | 0.0072/0.0184 | 1.00 |
| Black carbon | | |
| Biomass burning | 0.0013 | 0.48 |
| Industrial | 0.0004/0.0016 | 0.31 |
| Organic carbon | | |
| Natural | 0.0009 | 0.98 |
| Biomass burning | 0.0096 | 0.93 |
| Industrial | 0.0016/0.0056 | 0.96 |
| Soil dust | 0.0324 | 0.89 |
| Sea salt | 0.0267 | 1 |
| Stratospheric sulfate | 0.0065/0.0110 | 1 |
| Total | 0.0912/0.1121 | 0.942/0.944 |
Figure 8. (a) Optical thickness at wavelength 0.55 μm and (b) climate forcing for the three time-dependent tropospheric aerosols in the GISS SI2000 model. Optical depth at wavelength 0.55 μm (c) for all aerosols in the SI2000 model in 1990 and (d) the change between 1950 and 1990.
that may affect carbon or sulfur emissions. BC global annual emissions are 6.4 Mt/yr in 1984, based on consumption of hard coal, brown coal and diesel [Cooke et al., 1999]. OC fossil fuel emissions were assumed to be a factor of four larger than the BC emissions, following Lioussse et al. [1996]. The aerosol distributions and global amounts after 1990 are kept fixed at the 1990 values.
2.6.1. Results
The calculated changes of BC, OC, and S aerosol global-mean optical depths are shown in Figure 8a, and the climate forcings are shown in Figure 8b. The net change of the global aerosol forcing is about −0.3 W/m² over the period 1950–1990. Maps of the optical depth in 1990 and the change between 1950 and 1990 are given in Figures 8c and 8d.
Table 2 summarizes the aerosol optical depths and single scattering albedos in the SI2000 model. The small absorption by OC, which is assumed to occur at ultraviolet wavelengths, is based on absorption measurements by one of us (T.N.). The variation of the spectrally integrated single scatter albedo for natural, industrial and biomass burning OC is due to their assumed effective radii of 0.3, 0.5, and 1.0 μm, respectively. The global mean aerosol optical depth (at wavelength 0.55 μm) for all aerosols in the SI2000 model is about 0.09 in 1950 and 0.11 in 1990. The global mean single scattering albedo is about 0.94. This shortwave albedo includes a full spectral integration for desert dust [Tegen and Lacis, 1996] and the mean effect of the small absorption by OC in the ultraviolet region.
2.6.2. Assessment
Koch [2001] makes extensive comparisons of the simulated BC and OC distributions with observations. Although the observations are highly variable, if the measurements near urban locations are excluded the modeled BC and OC are in good agreement with observations on the average [Koch, 2001, Figures 3 and 4]. An exception is remote ocean regions, especially over the Pacific, where the model aerosol amount tends to be too small. One potentially serious problem for climate applications is that the BC does not fall off with height as rapidly as observed. The BC amount in the upper troposphere is about an order of magnitude greater than observed amounts [Koch, 2001, Figure 5], although the amounts there are quite small.
Sulfates are nonabsorbing, so, as far as their direct climate effect is concerned, their vertical distribution is not crucial. The geographical distribution of our sulfate aerosols is similar to that of other investigators, and we believe that it is realistic. The total anthropogenic sulfate forcing (present
minus preindustrial) for our present model [Koch, 2001] is $-0.65$ W/m$^2$, in the middle of the range for other investigators [Adams et al., 2001, Figure 1]. However, Adams et al. [2001] make a strong case that the anthropogenic sulfate forcing may be even larger (more negative), because the swelling of sulfate aerosols at high humidity is probably underestimated.
[60] The total aerosol amount in our model, with a current optical depth of 0.11, is probably less than in the real world. Likely reasons include too little S over the oceans because of a deficient dimethylsulfide (DMS) and insufficient hydration effects on S as mentioned above, the absence of nitrates, and the absence of a trend in biomass burning. For example, Figure 8 has very little aerosol over Indonesia, where recent satellite data [Nakajima et al., 1999] show large aerosol amounts.
[61] The important factor for climate change is the temporal aerosol change. We consider our present aerosol scenario as providing only an initial aerosol sensitivity study. We believe that the time dependence of nonabsorbing and absorbing aerosols is qualitatively realistic, but we have few data to check against. We suspect that this scenario underestimates both negative and positive aerosol forcings, and the transport model may loft the aerosols too efficiently, as discussed above. It may be useful to have a sensitivity study in which the vertical profile of the BC is modified in accord with observations and the BC absorption is increased. One reason to increase absorbing aerosols is their present treatment as an external mixture, which underestimates their absorption [Haywood and Shine, 1995; Fuller et al., 1999; Jacobson, 2001b]. Additional reasons are the evidence for deficient aerosol amount in remote regions and an impression that our single scattering albedos tend to fall toward the high side of typical field observations. Additional aerosol sensitivity studies might include enhancement of the nonabsorbing-absorbing aerosol opacities including a stronger amplification at high humidities, as well as a more complete representation of BC absorption effects (addition of dirty cloud and dirty snow). We suggest elsewhere [Hansen and Sato, 2001; Hansen, 2002] that the total BC forcing, including the albedo effects on clouds and on ice and snow surfaces may be as large as $0.8–1$ W/m$^2$.
3. GISS SI2000 Model
[62] The GISS SI2000 atmospheric model is similar to predecessor versions that are documented in the literature for the $4^\circ \times 5^\circ$ resolution that we employ here. We summarize here recent model changes and model shortcomings that seem particularly relevant to the present applications. We also discuss the climate sensitivity of the model. Finally, we summarize the array of simulations that is underway with the SI2000 model.
3.1. Atmospheric Model
[63] The SI2000 atmospheric model is similar to the SI95 model described by F-C. The primary change is an increase of vertical resolution from 9 to 12 layers in the principal version of the model, with the added resolution in the upper troposphere and stratosphere (Figure 9). This vertical resolution allows stratospheric radiative forcings (e.g., volcanic aerosols and ozone change) to be defined more accurately. We find that the stratospheric thermal response to stratospheric aerosols and ozone changes is typically 50% larger in the 12-layer model than in the 9-layer version. We show in section 5 that the results with 12 layers are in closer agreement with observations. However, the 12-layer resolution is still inadequate for reliable simulation of stratospheric dynamics or stratosphere-troposphere interactions, as discussed below.
[64] The discrepancies in absorbed solar radiation in SI95 compared with ERBE data in regions of sea ice and near the South Pole, illustrated in Figure 1 of F-C, are reduced considerably in SI2000. The excessive absorption of solar radiation by sea ice puddling was found to be a result of a programming error that caused the albedo reduction to be in effect at all times, rather than when the surface of the sea ice reached the melting point. The excessive absorption in Antarctica near the South Pole in SI95 was found to occur when sublimation exceeded snowfall thus exposing dark bare ice. The resulting unrealistic reduction of surface albedo is avoided in SI2000 by fixing the albedo of interior Antarctica (and Greenland) at 0.80. Additional changes were made to the radiation routines in SI2000, as discussed below in conjunction with the model’s climate sensitivity.
[65] The GISS atmospheric model is representative of state-of-the-art GCMs in a number of ways. In a comparison of GCM radiation results with line-by-line calculations [Cess et al., 1993] the GISS model was among the more
accurate. This was also true in comparisons of cloudiness and its variability [Weare et al., 1995] and in comparisons of seasonal changes in cloud radiative forcing [Cess et al., 1996]. A comparison of hydrologic processes in 29 GCMs with observations ranked the GISS model in the upper quartile [Lau et al., 1996]. Comparison among 30 GCMs of the amplitude and seasonality of precipitation over the United States showed the GISS model to be one of the most realistic, with a fidelity similar to that of the finer resolution 19-layer T42 model of the Max Planck Institute [Boyle, 1998]. Model shortcomings are summarized next.
### 3.2. Atmospheric Model Deficiencies
[66] The 12-layer version of SI2000 retains the simple top-layer drag formulation from GISS model II [Hansen et al., 1983] designed to assure numerical stability. The model does not yield a well-defined polar night jet or realistic interannual variability of stratospheric zonal wind, and it is not anticipated that this version of the model can provide realistic dynamical interactions between the stratosphere and troposphere. An immediate strategy, summarized below, is to make trial simulations with a version of the GISS middle atmosphere climate model [Rind et al., 1988], which has higher vertical resolution, a model top in the mesosphere, and a parameterized gravity wave drag representation. The computing time is several times greater with this middle atmosphere model, so in our climate model development we will also seek a model with the top at an intermediate level that can still yield a realistic representation of the lower stratosphere and its variability.
[67] Another continuing problem with the GISS model is inaccurate radiation balance in regions of stratus clouds over the ocean, specifically off the coasts of California, Peru, and southern Africa. There is a deficiency of cloud cover and an excess of absorbed solar radiation of the order of 50–75 W/m$^2$ in the summer in those regions. Although this problem is common among GCMs, the GISS model is among the models that have the largest discrepancy. Tests with higher vertical resolution in the PBL and an improved turbulence representation (Y. Cheng, private communication, 2001) yield some improvement. However, this inaccuracy in the fluxes delivered to the ocean surface by the SI2000 model, as it stands, presents a substantial problem for ocean models.
[68] The GISS model is generally too cool. The surface of SI2000 is about 1°C cooler than observed and the troposphere is typically 1–2°C too cool. In a perhaps related problem the troposphere tends to be too dry, especially at upper levels. Cirrus cloud cover is deficient.
[69] The radiation in the SI2000 model uses the correlated $k$ distribution method of Lacis and Oinas [1991]. This allows explicit realistic representation of gaseous absorption and atmospheric scattering. The SI2000 version of the radiation has 33 $k$ intervals in the thermal spectrum, which allows the climate forcing by the principal greenhouse gases to be calculated accurately. However, recent line-by-line tests [Oinas et al., 2001] show that some adjustments are needed to the $k$ distributions in regions of CH$_4$-N$_2$O-H$_2$O overlap. Radiation for the SI2001 model has not been completed, but tests with an improved parameterization yield a surface and tropospheric warming of about 1°C. The improved parameterization does not alter the model’s sensitivity to CH$_4$-N$_2$O changes, which was already pegged to line-by-line results.
[70] The horizontal resolution and finite difference calculations in the GISS GCM are another concern. The “tracers” in the model, including heat and water vapor, use a quadratic upstream scheme [Prather, 1986], which is both accurate and stable. The momentum equation is more difficult. The SI95 model uses a fourth-order finite differencing scheme that moves storms realistically in the troposphere but is computationally slow and very noisy in the stratosphere. In the SI2000 model we allow the option of either second or fourth-order differencing for the momentum equation. As a test, we are also making simulations with 2° × 2.5° atmospheric resolution, which reduces the importance of the differencing scheme. It should be noted that because of the higher-order differencing schemes in the GISS model and the preservation of concentration gradients within grid boxes, the effective resolution of the GISS model is generally higher than that of other models with the same grid size.
### 3.3. Model Sensitivity
[71] The climate sensitivity of GISS model II [Hansen et al., 1984] was 4.2°C for 2 × CO$_2$. The sensitivity of the SI95 model [F-C] was 3.6°C for 2 × CO$_2$. The sensitivity of the SI2000 model is about 3°C for 2 × CO$_2$. Specifically, it is 3.2°C for the version of SI2000 that uses second-order finite differencing and 2.9°C for the version that uses fourth-order differencing.
[72] A precise quantitative analysis of all the causes for changes in model sensitivity requires a new control run and a 2 × CO$_2$ experiment to be carried out for every model alteration. Although that is impractical, we can provide an indication of the main causes of the lesser sensitivity of the SI2000 model.
[73] Part of the reduced sensitivity to 2 × CO$_2$ is due to a smaller radiative forcing for 2 × CO$_2$, rather than to a change in the model’s sensitivity to a forcing. The forcing for 2 × CO$_2$ was about 4.2 W/m$^2$ in both GISS model II and the SI95 model. The 2 × CO$_2$ forcing in SI2000 (for 311 ppm → 622 ppm) is 3.95 W/m$^2$. This 6% reduction of the forcing accounts for about 0.2°C of the reduced model response to 2 × CO$_2$. The smaller forcing results from higher spectral resolution in the SI2000 radiation, which is calibrated against line-by-line calculations. We estimate that the uncertainty in our present radiative forcing for 2 × CO$_2$ is ≤ 10%. The forcing in SI2000 falls within the range of results for 2 × CO$_2$ reported by IPCC [2001], which is 3.5 to 4.1 W/m$^2$.
[74] Changes of the model sensitivity, as opposed to changes of the forcing, are associated with climate feedbacks. The principal feedbacks involve changes of water vapor, clouds, and sea ice [Hansen et al., 1984; Held and Soden, 2000]. Changes of the temperature lapse rate are also a factor, but lapse rate changes are usually associated with changes of the water vapor profile. The positive feedbacks feed off of each other, in the sense that they cause higher temperature thus increasing the other feedbacks [Hansen et al., 1984]. We note that 2 × CO$_2$ experiments with the earlier GISS model tended to have a larger increase of water vapor, larger decrease of sea ice cover, larger increase of cirrus clouds, and larger decrease of low clouds. However,
this comparison does not identify the model changes that instigated the lesser sensitivity in SI2000.
Among the changes between the SI95 and SI2000 models, prime candidates for affecting the climate sensitivity are changes in the sea ice parameterization and changes of the radiation that affected the clouds. The “aging” of snow on sea ice, which reduces the albedo, occurred in SI95 only when the surface temperature was above the freezing point. Snow on sea ice continuously ages in SI2000, regardless of the temperature. Thus the sea ice in SI2000 tends to be darker, and the reduced contrast in albedo between sea ice and ocean tends to lessen the sea ice feedback effect. It is also possible that several changes in the radiation could have affected the model sensitivity. One of these is the introduction of a parameterization for the effect of finite cloud size on shortwave albedo and on infrared emissivity [Cairns et al., 2000]. However, we have not quantified the effect of the individual changes on model sensitivity.
The difference in sensitivity between the two versions of the SI2000 model (second- or fourth-order differencing in the momentum equation), to at least a large degree, is related to the different amounts of sea ice in their control runs. The sea ice in the fourth-order model covers 4% of the globe, which is realistic. This compares with more than 6% sea ice cover in the second-order model. In retrospect, we should have made adjustments in the sea ice parameterizations in the second-order model control run in an attempt to achieve a realistic sea ice cover. However, that would not necessarily have yielded a model with the same sensitivity as the fourth-order model. Because the atmospheric energy transports are different in the two atmospheric models, the implied ocean heat transports are different and this may affect the climate sensitivity.
The bottom line is that, although there has been some narrowing of the range of climate sensitivities that emerge from realistic models [Del Genio and Wolf, 2000], models still can be made to yield a wide range of sensitivities by altering model parameterizations. We suggest that the best constraint on actual climate sensitivity is provided by paleoclimate data that imply a sensitivity $3 \pm 1^\circ$C for $2 \times CO_2$ [Hansen et al., 1984, 1993, 1997b; Hoffer and Covey, 1992]. It is satisfying that the a priori sensitivities of the SI2000 model comes out near the middle of the empirical range of $2–4^\circ$C for $2 \times CO_2$. However, for the sake of interpreting observed climate change and predicting future change it is appropriate to consider climate sensitivity as an uncertain parameter that may, in fact, be anywhere within that range.
Therefore we include the possibility of altering the model’s climate sensitivity. We do this by adjusting an arbitrary cloud feedback as defined in the appendix of Hansen et al. [1997a]. Specifically, the cloud cover is multiplied by the factor $1 + c\Delta T$, where $\Delta T$, computed every time step, is the deviation of the global mean surface air temperature from the long-term mean in the model control run at the same point in the seasonal cycle and $c$ is an empirical constant. For the SI2000 second-order model we take $c = 0.04$ and $-0.01$ to obtain climate sensitivities of $2^\circ$C and $4^\circ$C for $2 \times CO_2$.
### 3.4. Ocean Representations
Ocean A (observed SST) uses the SSTs and sea ice of HadISST1 (N. A. Rayner et al., Globally complete analyses of SST, sea ice, and night marine air temperature, 1871–2000, manuscript in preparation, 2002) (hereinafter referred to as Rayner et al., manuscript in preparation, 2002), which uses reduced-space optimum interpolation [Kaplan et al., 1997, 1998] to fill in data sparse regions. SSTs at high latitudes have substantial uncertainties. The sea ice records in HadISST1 have been “homogenized” in an attempt to make the different components consistent, but considerable uncertainty remains in the sea ice record, especially in the presatellite era. Ocean A has severe limitations for climate studies, as discussed in sections 3.5 and 5.2.2.
Ocean B ($Q$-flux ocean) includes a deep ocean with diffusive penetration of heat anomalies. The diffusion coefficient varies geographically as described by Hansen et al. [1984]. Ocean B, based on observed rates of ocean mixing of tracers, should provide a useful approximation of global heat uptake by the ocean for climate forcings that do not fundamentally alter the deep ocean circulation.
Oceans C, D, and E are distinctly different dynamical ocean models. Ocean C, developed initially by Russell et al. [1995], uses a vertical coordinate related to pressure. Ocean D, the GFDL Modular Ocean Model (MOM), uses depth (z-level) [Pacanowski and Griffies, 1999]. Ocean E, the Hybrid Coordinate Ocean Model (HYCOM) of Bleck [1998], uses an isopycnic vertical coordinate in the deep ocean with a $z$ level in the less stratified upper ocean.
All of the ocean models are being attached to the same (B grid) version of the SI2000 atmospheric model. This should help to isolate the role of the ocean in the simulated climate response. The wide range of ocean models will provide an indication of the model dependence of any inferences about climate change.
We report in this paper only results of simulations for ocean A (observed SST) and ocean B ($Q$-flux ocean). These simple ocean “models” allow us to characterize the efficacy of each radiative forcing when only limited dynamical interactions are permitted. (By “efficacy” we mean the effectiveness of a forcing for producing a climate response in a general sense. One specific measure of efficacy is the global climate sensitivity, the ratio of the global mean temperature response to the magnitude of the forcing, $\Delta T_s/F_a$ (see section 4.1 and Table 4).)
### 3.5. Model Experiments
Table 3 summarizes model runs (climate simulations) carried out or planned. Our rationale is that we may be able to learn more from a systematic array of experiments in which we vary key factors one by one. The variable factors include the climate forcings, the ocean model, and the atmosphere model.
Each atmosphere-ocean model has one long control run with the atmospheric composition fixed at 1951 conditions. The control runs each cover at least a few hundred years. A few of them are continuing to run and may be extended for thousands of years, if that appears to be useful.
There are also two “long” (or “equilibrium”) runs for each radiative forcing. The first equilibrium run is a 250-year run made with the $Q$-flux mixed layer ocean, without any attached deep ocean. The objective is to help characterize each radiative forcing. Weak forcings, e.g., stratospheric H$_2$O change, are exaggerated by a specified
Table 3. SI2000 Experiments
| Radiative Forcing | Ocean A (Varying SST) | Ocean A (Mean SST) | Ocean A (Stratospheric Model) | Ocean A ($2^\circ \times 2.5^\circ$ Atmosphere) | Ocean B ($Q$-Flux, $3^\circ$C Sensitivity) | Ocean B ($2^\circ$ and $4^\circ$C Sensitivity) | Ocean C (GISS Ocean) | Ocean D (GFDL MOM) | Ocean E (Bleck/Sun Isopycnal) |
|-------------------|-----------------------|--------------------|-------------------------------|-----------------------------------------------|---------------------------------------------|---------------------------------------------|---------------------|------------------|------------------------|
| None | five runs$^b$ | long run | long run | long run | long run | long run | long run | long run | long run |
| GHGs | | long run | | | long run | | | | |
| Stratospheric aerosols | | long run | | | long run | | | | |
| Solar | | long run | | | long run | | | | |
| O$_3$ | | long run | | | long run | | | | |
| Stratospheric H$_2$O | | long run | | | long run | | | | |
| Five forcings | five runs$^b$ | long run | | | five runs | | | | |
| Tropospheric aerosols | | long run | | | long run | | | | |
| Six forcings | five runs$^b$ | five runs | five runs | | five runs | five runs | five runs | five runs | five runs |
| Aerosol indirect | | | | | long run | | | | |
$^a$ The “long run” is a control or a run with time-independent forcing, while “five runs” is an ensemble of runs with transient forcings. Italicized runs are not included in present paper.
$^b$ Runs are made for both second- and fourth-order differencing of atmospheric momentum equation.
factor in the equilibrium runs, so that a reasonable signal-to-noise ratio can be obtained. The second equilibrium run is a 21-year run with ocean A using the mean (seasonally varying) SST for 1946–1955. A good signal-to-noise ratio is obtained with a 21-year run because of the fixed SST. These runs are used to define a fixed SST forcing (section 4).
[87] Ensembles of transient simulations, i.e., with time-dependent forcings, are carried out for the period 1951–2000 or 1951–2050. Ensembles presently completed all contain five runs. The ensembles are run with six forcings, and in some cases with five forcings (fixed 1951 aerosols).
[88] At least two different atmospheric models are also being coupled to certain of the ocean models, as summarized in Table 3. The middle atmosphere model of Rind et al. [1988] has been attached to ocean A and also may be attached to a dynamical ocean model. A $2^\circ \times 2.5^\circ$ version of the SI2000 atmospheric model is being run with observed SSTs (ocean A).
[89] Simulations for ocean A (observed SST) and ocean B ($Q$-flux ocean) are presented in this paper. The inherent constraints in these models limit the degree of realism in the climate response. However, for just this reason the results provide a useful comparison and complement to simulations with more fully interactive coupled models.
[90] Ocean A, for example, has “correct” SSTs, but obviously will not yield realistic ocean-atmosphere fluxes in some cases of interest. A prime example is the North Atlantic Oscillation, a dynamical fluctuation in which the North Atlantic Ocean cools and in the process discharges energy that warms Siberia. Ocean A, by specifying a cool North Atlantic, tends to cool Eurasia. This characteristic limits the merits of ocean A, even though it is a popular model for atmospheric data assimilation and reanalysis.
[91] Ocean B is useful for studying the global mean thermal response to forcings. It is most relevant to forcings of moderate size, for which heat anomalies are likely to penetrate the ocean like passive tracers. Sokolov and Stone [1998] showed that the transient global surface temperature response in a wide variety of ocean models can be matched with the $Q$-flux ocean by choosing the diffusion coefficient appropriately.
[92] Geographical patterns of climate change depend upon realistic representation of climate dynamics. Analysis will require use of a dynamically interactive ocean-atmosphere, including a realistic stratosphere. It has been suggested that even the global mean temperature is altered by dynamical fluctuations [Wallace et al., 1995]. This is the sort of issue that can be examined with the full array of experiments in Table 3.
4. Equilibrium Simulations
4.1. Global Means
[93] We carry out here long (“equilibrium”) GCM runs for each of the six climate forcings used later in our transient climate simulations (section 5). These long runs are made with both ocean A (fixed SST) and ocean B ($Q$-flux ocean). Control runs for oceans A (21 years) and B (250 years) have 1951 atmospheric composition. The fixed SST in ocean A is the mean of years 1945–1955 of the HadISST climatology (Rayner et al., manuscript in preparation, 2002). Individual runs are made for each forcing. The mean of years 2–21 for ocean A and years 51–250 for ocean B are taken as the equilibrium responses.
[94] Most of the six forcing mechanisms were included in our earlier study with the Wonderland model [RF-CR], but the new results are useful for several reasons. First, the forcings used here, including their global distributions, are more specific to our current 1951–2000 simulations and in some cases are more realistic. Second, the present $4^\circ \times 5^\circ$ 12-layer SI2000 model is more realistic than the $8^\circ \times 10^\circ$ 9-layer Wonderland (sector) model that employed the physics of our 1983 climate model. For example, the present model has state-of-the-art cloud modeling [Del Genio and Yao, 1993; Del Genio et al., 1996], which may effect the “semidirect–direct” climate forcing found for absorbing aerosols [RF-CR].
[95] We compute several measures of each climate forcing, as summarized by the cartoon in Figure 10. $F_i$, the instantaneous forcing, is the flux change at the tropopause prior to any thermal response. $F_o$, the adjusted forcing, is the flux change at the tropopause (or any higher level) after the stratospheric temperature reaches radiative equilibrium with the tropospheric temperature held fixed. $F_s$, the fixed SST forcing, is the equilibrium energy flux change at the Earth’s surface (and at all higher levels) with the sea surface temperature (SST) held fixed.
In a previous study [RF-CR] we concluded that for most radiative forcings the adjusted forcing, $F_a$, provides a reasonably good predictor of the equilibrium global-mean temperature response. However, there are notable relevant exceptions, specifically absorbing aerosols and ozone change. In one sense, $F_s$ might be expected to provide a better measure than $F_a$ of the flux perturbation that would beset the real world because the tropospheric lapse rate, as well as stratospheric temperature, can adjust rather quickly to a forcing. So can atmospheric temperature over a large continent, even though the magnitude of the response is limited by exchange of continental and marine air masses. The rationale for $F_s$ is that the ocean’s thermal inertia is the reason for sustained flux imbalance after the first transient atmospheric response to an imposed forcing. We find here, however, that $F_s$ is no better than $F_a$ as a predictor of global temperature change, as the latitude distribution of the forcing is apparently more critical than the vertical profile of temperature change. Therefore it seems better to use the conventional quantity, $F_a$, as our standard measure of climate forcing. However, $F_s$ is the most relevant flux to estimate the initial rate of ocean heat storage and the response time of the climate system.
We take $F_s$ as the mean for years 2–21 of a 21-year GCM run with fixed SST. Although the need for a GCM to calculate $F_s$ may seem to be a disadvantage, most climate studies are now made with GCMs and the run to obtain $F_s$ only needs to be a few years if a precision of 0.1 W/m$^2$ for the global mean forcing is sufficient. $F_s$ is simpler to compute in a consistent fashion than are the other forcings. For example, it removes the uncertainty associated with defining the tropopause level [Harvey, 2000, chapter 7.2]. Thus it is easy to compute $F_s$ in conjunction with GCM studies. It would be informative if $F_s$ were routinely reported as a quantification and verification of forcings actually employed by all models.
Table 4 summarizes the forcings and equilibrium climate response for a large number of climate forcing mechanisms. $\Delta T_s$ is the equilibrium (years 51–250) global mean surface air temperature response. Several of the results in Table 4 warrant comment.
We first compare the responses to the CO$_2$ and solar irradiance ($S_O$) forcings. Manabe and Wetherald [1975] and Wetherald and Manabe [1975] found that the responses of a swamp ocean model to similar forcings, specifically $2 \times$ CO$_2$ and +2% $S_O$, were very similar. Our simulated responses to CO$_2$ and $S_O$ forcings are consistent with their results.
We obtain a climate sensitivity, $\Delta T_s/F_a$, between $\frac{3}{4}^\circ$C and 1°C for each Watt of forcing for most of the forcing mechanisms in Table 4. The two apparent exceptions, O$_3$ and stratospheric H$_2$O, are explained by the geographical distribution of the forcings, as discussed in section 4.2. The change of tropospheric aerosols between 1950 and 1990 (or 2000), for the specific combination of S, BC and OC aerosols that we employ, yields a sensitivity of 1°C per W/m$^2$. However, this sensitivity is a strong function of the assumed amount of aerosol absorption [RF-CR], and it thus depends on the BC history and vertical distribution, which are very uncertain.
We calculate the forcing by stratospheric aerosols for the specific aerosol distribution that existed for the 12-month period July 1991 through June 1992 and for a uniform distribution of aerosols with optical depth 0.1 at wavelength 0.55 µm. Note that although $F_i = -3$ W/m$^2$ for the first year after Pinatubo, $F_a = -2.6$ W/m$^2$ and $F_s = -2.4$ W/m$^2$. Thus, other things being equal, if the SST were truly fixed one would expect the ocean to pump heat into space after Pinatubo at a global rate of only 2.4 W/m$^2$. It is still less when the SST is allowed to respond partially to the forcing (section 5), as the SST would in the real world. One reason that the planet cools at a rate less than 3 W/m$^2$ even when the SST is fixed, i.e., one reason that $F_s < F_i$, is inefficiency of the ocean and atmosphere in pumping heat to the continents for radiation to space. In other words, despite exchange of continental and marine air masses the continents cool in response to the
Table 4. Radiative Forcings (W/m$^2$) and Equilibrium Responses ($^\circ$C)$^a$
| Forcing Mechanism | $F_i$ | $F_a$ | $F_s$ | $\Delta T_s$ | $\Delta T_s/F_a$ |
|--------------------------------------------------------|---------|---------|---------|--------------|------------------|
| Doubled CO$_2$ (311.1 – 622.2 ppm) | 4.46 | 3.95 | 3.94 | 3.2 | 0.81 |
| Greenhouse gases (1951 – 1998) | 1.75 | 1.62 | 1.65 | 1.55/1.21$^b$| 0.96/0.75$^b$ |
| Stratospheric aerosols | | | | | |
| Pinatubo (July 1991 to June 1992) | | | | | |
| $\tau = 0.1$ uniform | | | | | |
| Solar irradiance | | | | | |
| $10 \times^b$ (1951 – 1981) | 1.39 | 1.31 | 1.33 | 1.10 | 0.84 |
| Ozone (1951 – 1998) | | | | | |
| O$_3^A$ data | | | | | |
| stratosphere | 0.08 | −0.085 | | | |
| troposphere | 0.16 | 0.135 | | | |
| entire atmosphere | 0.23 | 0.05 | | −0.20 | −4.00 |
| stratosphere | 0.06 | −0.095 | | | |
| troposphere | 0.15 | 0.125 | | | |
| entire atmosphere | 0.21 | 0.03 | 0.05 | −0.34 | −11.00 |
| Stratospheric water vapor (1951 – 1998) | | | | | |
| 0 – 10 hPa | 0.005 | 0.007 | | | |
| 10 – 150 hPa | 0.037 | 0.029 | | | |
| all altitudes | 0.041 | 0.036 | | 0.00 | |
| $10 \times^b$ all altitudes | 0.37 | 0.31 | 0.36 | 0.48 | 1.55 |
| Tropospheric aerosols (1951 – 1990) | | | | | |
| | −0.29 | −0.28 | −0.13 | −0.28 | 1.00 |
$^a F_i, F_a,$ and $F_s,$ are the instantaneous, adjusted and fixed SST forcings, and $\Delta T_s$ is the equilibrium (years 51 – 250) GCM response. Stratospheric aerosol forcing is computed for Pinatubo aerosols (July 1991 through June 1992) and for a constant uniformly distributed aerosol with optical depth 0.1 at wavelength 0.55 µm. Solar irradiance and stratospheric water vapor changes are multiplied by a factor of 10 to magnify the response. Troposphere aerosol forcing is the change of sulfate, black carbon and organic aerosols between 1950 and 1990 in the model of Koch [2001].
$^b T_s$ is computed by the GCM with second-order differencing, except for the 1951 – 1998 GHG forcing, which is also computed with fourth-order differencing.
aerosols, and thus their rate of heat loss to space is less than 3 W/m$^2$.
4.2. Global Maps
[102] The global distribution of the forcings is shown in the first column of Figure 11. The greenhouse gas, stratospheric aerosol, and solar irradiance forcings are largest at low latitudes, decreasing by a factor of two or so toward the poles. The ozone forcing is positive at low latitudes but becomes negative in the polar regions because of ozone depletion there. The stratospheric water vapor forcing is largest in the polar regions. The tropospheric aerosol forcing is primarily in the Northern Hemisphere.
[103] The equilibrium surface air temperature change, Figure 11b, is computed with the mixed layer $Q$-flux ocean with second-order differencing. The global mean response is consistent with the global mean forcing, with a sensitivity of $\frac{3}{4} – 1^\circ$C per W/m$^2$, for all of the forcings except O$_3$ and stratospheric H$_2$O. As discussed above, the sensitivity of the second-order differencing model, which has excessive sea ice in the Southern Hemisphere, is higher than our best estimate. The sensitivity of the model with fourth-order differencing, which has realistic sea ice cover, is closer to the sensitivity of 0.75°C per W/m$^2$ that we estimate from empirical (paleoclimate) data.
[104] The unusual responses to O$_3$ and stratospheric H$_2$O are explained by the latitude variation of the forcing. The response of our models, and presumably of the real world, is larger for forcings at high latitudes than for forcings at low latitudes, typically by a factor of two [RFCR]. Thus, although the negative forcing from polar ozone depletion and the positive forcing from tropospheric O$_3$ approximately balance on global average, the polar forcing is more effective. The greater sensitivity to high-latitude forcings is due to the ice/snow positive feedback and the relatively stable atmospheric lapse rate at high latitudes. This effect may be exaggerated somewhat in the present model by the excessive sea ice in the control run.
[105] Note that there is some similarity in the geographical pattern of the equilibrium response to different forcings. Even the aerosol forcing, which is mainly in the Northern Hemisphere, evokes a global response. There is only a limited tendency toward a universal response, however. The Southern Hemisphere response to the aerosol forcing is not as intense as the Southern Hemisphere response to a globally uniform forcing. Also, the ozone forcing, which is concentrated near the South Pole, evokes a response that is restricted mainly to the Southern Hemisphere.
[106] The surface air temperature change with fixed SST is small on global average. However, several of the forcings yield a significant warming in Eurasia, which is largest in the winter. The winter warming is usually associated with an increased magnitude of the zonal wind (not illustrated).
4.3. Zonal Means and Altitude Profiles
[107] The equilibrium zonal-mean temperature response to the six forcings is given in Figure 11c for ocean A (fixed SST) and ocean B ($Q$-flux model). Figure 11c (right) gives the altitude profiles of the global mean responses.
[108] The GHGs (specifically CO$_2$) cause substantial stratospheric cooling that increases with altitude. When the SST is allowed to respond (ocean B), there is a relative maximum in the equilibrium warming at about the 300 hPa level in the tropics that is about 30% greater than the surface warming. The maximum in the equilibrium near-surface warming near the South Pole is probably exaggerated by the excessive sea ice in the control run.
[109] The volcanic aerosols cause warming in the lower stratosphere, with the warming concentrated at low latitudes even though the aerosols are uniformly distributed with latitude. The tropospheric thermal response is similar to that for greenhouse gases but with the opposite sign. The tropospheric response exceeds the surface response, conFigure 11. (a) Adjusted forcing for six climate forcing mechanisms. (b) Annual-mean equilibrium surface air temperature response for the $Q$-flux ocean, with the global means given on the upper right corner. (c) Annual-mean $\Delta T$ ($^\circ$C) for both ocean A (fixed SST) and ocean B ($Q$-flux ocean). The SST is fixed in ocean A, so only the atmosphere and land surface can respond to the forcing.
consistent with empirical evidence after El Chichon and Pinatubo [Santer et al., 2001].
[110] The solar irradiance forcing has been magnified by taking 10 times the difference between the solar irradiance near a solar maximum (1981) and near a solar minimum (1951), as estimated by J. Lean (section 2.3). No indirect forcings associated with solar irradiance changes are included. The solar irradiance increase warms the stratosphere, because of absorption of ultraviolet radiation, by an amount that increases with altitude. The spatial structure of the tropospheric temperature change is similar to that for other forcings.
[111] The ozone forcing includes the larger polar depletion estimated by Randel and Wu [1999], i.e., the O$_3^B$ data set (section 2.4). The ozone change causes substantial cooling in the lower stratosphere, particularly near the South
Pole. The tropospheric cooling that occurs when the SST is allowed to respond is associated with increased sea ice in the Southern Hemisphere.
[112] The stratospheric H$_2$O forcing is that for 10 times the 1951–1998 CH$_4$-derived H$_2$O increase in our 12-layer model. However, as discussed in section 2.5, the 12-layer model removes this H$_2$O from the stratosphere too rapidly, and thus we estimate that the forcing used here is only 6–8 times larger than the actual CH$_4$-derived H$_2$O increase in that 47-year period. This specified (magnified) H$_2$O source causes an equilibrium increase of 2.4 ppmv of H$_2$O in the 10–100 hPa layer. Reducing this by the factor 6–8 suggests that methane oxidation should have increased H$_2$O by 0.3–0.4 ppmv during 1951–1998. Indeed, in our transient simulations (section 5) we find an increase of stratospheric H$_2$O from 2.4 ppmv to 2.7 ppmv. Observations suggest that stratospheric H$_2$O may have increased in the past half century by of the order of 1 ppmv [Rosenlof et al., 2001]. Thus either there are additional mechanisms causing H$_2$O to increase in the stratosphere or our simulation underestimates the CH$_4$ impact on H$_2$O, for example via climate feedbacks that are inadequately represented in our 12-layer climate model (transport of water vapor from the troposphere is probably underestimated in both the control run and perturbations because of the excessively dry upper troposphere in the SI2000 model). In either event, the H$_2$O increase employed in Figure 11 is only 2.4 times larger than the observed H$_2$O change during 1951–1998.
[113] The tropospheric aerosols cause a cooling that is rather uniform with height, as the sulfate aerosols are dominant in our current scenario. We mentioned in section 2.6 that BC aerosols are probably underrepresented in our present model, and they may be mixed too uniformly with height. The changes of aerosols that cause cooling are also probably understated. Although our global mean aerosol forcing probably has the right sign, and is perhaps even of the right order of magnitude, we do not have confidence in the vertical and temporal distributions of aerosol changes. Thus the nature of the aerosol forcing and simulated response may change with better aerosol information.
[114] Finally, we note the tendency for different forcings to produce similar spatial responses. This has obvious implications for studies aimed at identifying the causes of climate change based on comparison of modeled and observed spatial-temporal patterns of climate change, although it does not rule out identification and quantification of climate signatures, if the spatial-temporal distributions of the forcings are well known.
5. Transient Simulations
[115] We describe transient simulations for 1951–2000 using the simplest ocean representations, ocean A (observed SST) and ocean B (Q-flux ocean). The simulations are made for “five forcings” (GHGs, stratospheric aerosols, solar irradiance, O$_3$, and stratospheric H$_2$O) and for “six forcings,” which adds the direct forcings by three tropospheric aerosols (sulfates, BC, and OC), all as defined in section 2. We extend the ocean B “six forcing” runs to 2050 for two distinct alternative scenarios of future climate forcings. The “business as usual” (BAU) scenario has a 1%/year CO$_2$ growth rate, which yields an added forcing of almost 3 W/m$^2$ in 2000–2050. The “alternative scenario,” defined in section 2.1, has an added forcing of 1.1 W/m$^2$ in 2000–2050.
[116] We focus on the global mean response, as our aim is to investigate the global efficacy of the forcings. Study of the geographical distribution of climate change requires interactive ocean dynamics and a realistic representation of the stratosphere. Our present simple models are a useful prelude to such dynamical studies, especially if the latter employ the same forcings and atmospheric physics. Furthermore, for a crucial issue such as global ocean heat storage, the specified empirical mixing rates in the Q-flux ocean may provide as realistic an estimate as is possible at this time and, in any case, a standard for comparison. Sokolov and Stone [1998] have shown that heat uptake by the Q-flux ocean provides a good approximation to that by ocean general circulation models, providing that there is no change in the mode of deep circulation. This condition should be satisfied for timescales less than a century with the moderate forcings that we employ.
5.1. Global Mean Response
5.1.1. Period 1951–2000
[117] The global mean response to five forcings and six forcings is shown in Figures 12 and 13. The stratosphere (top row) cools as a result of O$_3$ depletion and CO$_2$ and H$_2$O increase, but it warms temporarily after large volcanoes as a result of thermal infrared and near-infrared heating by the aerosols. Such qualitative results have been reported in the literature many times. The quantitative agreement with MSU (microwave sounding unit) observations [Christy et al., 2000] is better than that found by F-C or Bengtsson et al. [1999]. The increased stratospheric response in our model occurred when we increased the vertical resolution from 9 to 12 layers.
[118] We note that some of our models use a second-order finite differencing scheme for the momentum equation, while others use a fourth-order scheme. The second-order scheme is more diffusive, while the fourth-order scheme is much noisier in the stratosphere, as is apparent in the different levels of variability among the five-member ensembles. To ameliorate uncertainty about the effect of the numerical differencing scheme, we carried out some ocean A and ocean B calculations with both second-order and fourth-order differencing. The results were similar, as summarized in Table 5. We use the fourth-order results as our standard case, because that model has a more realistic sea ice area in its control run.
[119] The tropospheric temperature changes, weighted by the MSU channel 2 (MSU2) weighting function, are shown in the second row of Figures 12 and 13. The agreement with MSU2 data is good for ocean B, but the simulated warming trend with ocean A is larger than the observed warming (Figure 12 and Table 5). Discrepancies with the observed 1979–1998 temperature trend are greater for MSU2LT, which has a weighting function that peaks closer to the surface [Christy et al., 1998]. Specifically, the lower troposphere with ocean B warms slightly (about 0.1°C) more than observed, and ocean A warms significantly (about 0.3°C) more than observed (MSU2LT is included in Table 5 but not in Figures 12 and 13.) The uncertainty in the MSU tropospheric temperature change for the 20-year interval 1979–1998 is probably at least 0.1°C; indeed, an alternative
Figure 12. Transient response of SI2000 model with observed SST to (a) five forcings and (b) six forcings.
Figure 13. Transient response of SI2000 model with $Q$-flux ocean to (a) five forcings and (b) six forcings.
Table 5. Observed and Simulated Global Temperature Change at Several Levels Based on Linear Trends
| Atmospheric Levels | Observations MSU/GISS$^a$ | Radiosondes | Ocean A (Second-Order) | Ocean B (Second-Order) | Ocean B (Fourth-Order) |
|--------------------|---------------------------|-------------|------------------------|------------------------|------------------------|
| | | | Five Forcings | Six Forcings | Five Forcings |
| | | | | | |
| MSU channel 4 | HadRT2.0/2.1 | | | | |
| 1951–1998 | | −0.77 | −0.74 | −0.76 | −0.94 |
| 1958–1998 | | −1.58/−1.31 | −0.83 | −0.83 | −0.82 |
| 1979–1998 | | −0.98 | −1.46/−1.16 | −0.57 | −0.62 |
| MSU channel 2 | | | | | |
| 1951–1998 | | 0.39 | 0.39 | 0.37 | 0.53 |
| 1958–1998 | | 0.38/0.33 | 0.34 | 0.34 | 0.36 |
| 1979–1998 | | 0.08 | 0.00/−0.09 | 0.32 | 0.31 |
| MSU channel 2LT | | | | | |
| 1951–1998 | | 0.52 | 0.51 | 0.50 | 0.70 |
| 1958–1998 | | 0.51/0.46 | 0.49 | 0.48 | 0.52 |
| 1979–1998 | | 0.14 | 0.07/−0.02 | 0.42 | 0.41 |
| Surface | | | | | |
| 1951–1998 | | 0.43 | 0.47 | 0.44 | 0.46 |
| 1958–1998 | | 0.45 | 0.45 | 0.43 | 0.47 |
| 1979–1998 | | 0.29 | 0.32 | 0.31 | 0.18 |
$^a$ Microwave Sounding Unit data for channels 4 (stratosphere), 2 (troposphere), and 2LT (lower troposphere). GISS surface analysis uses SST data of Reynolds and Smith [1994] over the ocean.
analysis by Wentz et al. [2001] suggests that the warming is 0.1°C/decade greater than reported by Christy et al. [1998]. We examine the height dependence of the temperature trends in more detail and discuss the differences between ocean A and ocean B in section 5.3. Note in Table 5 that the model results with six forcings agree well with the observed warming over the longer period, i.e., from the 1950s to the present.
The surface temperature change, the third row in Figures 12 and 13, is in reasonable agreement with the observations. This is an expected result with ocean A, but it is a more meaningful test of the forcings with ocean B. The observed surface warming over the period 1951–1998 is about 0.4°C based on the linear trend. Ocean B yields a warming of about 0.6°C for five forcings and about 0.45°C for six forcings. Thus six forcings, i.e., the inclusion of aerosols, yields better agreement than five forcings. This conclusion becomes more solid on the basis of observations of ocean heat storage (section 5.2).
The net heating at the Earth’s surface is given in the fourth row of Figures 12 and 13. The units are W/m$^2$ averaged over the Earth’s entire surface. Because heat storage in the atmosphere and land is small, the rate of heat storage through the ocean surface is obtained by multiplying our number by 1/0.7. The ocean A model, with fixed SST, would not be expected to be in precise energy balance with space in 1951, unlike the ocean B model, in which the SST of the control run is allowed to adjust until equilibrium (energy balance) is achieved. We evaluated the initial ocean A imbalance by running the model for 20 years with 1949–1953 mean SSTs, obtaining a global mean flux $-0.18$ W/m$^2$. We thus adjusted the zero point of the net heating by this amount. By 1999 both the ocean A and ocean B models are soaking up heat at a rate between +0.5 and +1.0 W/m$^2$. We integrate this time series of the net heat at the ocean surface over time and compare the result with observed ocean heat storage in section 5.2.
Ocean ice cover is specified in the ocean A model, based on HadISST1 (Rayner et al., manuscript in preparation, 2002). Ocean ice cover decreases in the ocean B simulations. Although the magnitude of the simulated sea ice decrease is consistent with observations, the simulated decrease is primarily in the Southern Hemisphere while the observed decrease is mainly in the Northern Hemisphere. Realistic results for the spatial distribution of sea ice change are likely to require realistic representation of both ocean surface circulation and the full ocean dynamics and conceivably even a realistic stratosphere because of its influence on the Arctic Oscillation. We note also that it is possible that Southern Hemisphere sea ice has actually decreased since 1950, as some researchers have suggested [de la Mare, 1997]. The “homogenization” process included in the construction of the HadISST1 (Rayner et al., manuscript in preparation, 2002) data set removed a substantial (and probably unrealistically large) long-term trend that existed in prior versions of the data set, but there is considerable uncertainty in this adjustment process.
5.1.2. Period 2000–2050
Simulations for ocean B were extended to 2050 for two greenhouse gas scenarios: CO$_2$ increasing at 1%/year (yielding a forcing of 2.95 W/m$^2$ in 50 years) and the “alternative” scenario for greenhouse gases illustrated in Figure 1 (yielding a forcing of 1.1 W/m$^2$ by 2050). The 1% CO$_2$ scenario had no other forcings for the sake of simplicity and to allow ready comparison with other climate models. The alternative scenario included stratospheric aerosols with the same sequence of optical depth in 2001–2050 as in 1951–2000.
The rate of stratospheric cooling declines markedly after 2000 in the alternative scenario (Figure 14a). In fact, if anticipated recovery of stratospheric ozone is included, stratospheric temperature would tend to level out as the effects of increasing CO$_2$ and increasing O$_3$ approximately offset each other.
Global warming over 2001–2050 is about 1.5°C and 0.75°C in the 1% CO$_2$ and alternative scenarios (Figure 14b). The response is only twice as large in the 1% CO$_2$ scenario as in the alternative scenario, despite the forcing being almost 3 times as large, because of the disequilibrium of about 0.75 W/m$^2$ in 2000. This planetary energy imbalance is the portion of the forcing to which the atmosphere has not yet responded; that is, this portion of the forcing has not yet
Figure 14. (a) Stratospheric temperature, (b) surface temperature, and (c) heat flux into the Earth’s surface for SI2000 model with six forcings. “Business-as-usual” and “alternative” scenarios have added forcings of 2.9 and 1.07 W/m$^2$ in 2001–2050.
impacted the temperature. Thus the relevant forcing in the 1% CO$_2$ case is $2.95 + 0.75 = 3.7$ W/m$^2$, while it is $1.1 + 0.75 = 1.85$ W/m$^2$ in the alternative scenario.
[126] The planetary energy imbalance approximately doubles to 1.5 W/m$^2$ in the 1% CO$_2$ scenario. On the other hand, the imbalance is rather flat in the alternative scenario, perhaps increasing by 0.1–0.2 W/m$^2$. Thus the unrealized warming, i.e., the amount of warming “in the pipeline,” hardly increases in the alternative scenario. In other words, if climate sensitivity is $\frac{3}{4}$°C per W/m$^2$, the 1% CO$_2$ scenario not only yields a warming of 1.5°C in the next 50 years, but it also leaves more than another 1°C warming in the pipeline. The alternative scenario produces a warming of about $\frac{3}{4}$°C and leaves about 0.5°C warming in the pipeline.
[127] These results depend not only on the climate forcing scenario but also on climate sensitivity. Our results are obtained from a model with sensitivity 3°C for doubled CO$_2$ ($\frac{3}{4}$°C per W/m$^2$). Wetherald et al. [2001] estimate that the present unrealized warming is 1°C, rather than $\frac{3}{4}$°C, at least in part because their model has a higher climate sensitivity (about 4.5°C for doubled CO$_2$). The measured rate of heat storage in the ocean should eventually help distinguish among models. However, unique interpretation will require quantitative information on climate forcings as well as accurate long-term data for heat storage.
5.2. Ocean Heat Storage
[128] A climate forcing, by definition, causes a planetary energy imbalance. An extended planetary energy imbalance must show up as a change of ocean heat content, because of the negligible heat conductivity of the continents and the small heat capacity of other heat reservoirs such as the atmosphere. We inferred previously [F-C] that the Earth had attained a positive rate of heat storage of 0.5–1 W/m$^2$ by the middle 1990s, and we argued that the best confirmation of this planetary disequilibrium would be measurements of ocean temperature adequate to define heat storage. Recent analysis of global ocean data [Levitus et al., 2000] permits comparison of observations with the transient energy imbalance in climate scenarios. Model results for ocean B refer only to the upper 1000 m of the ocean, as the $Q$-flux model in our present simulations only extended to that depth.
[129] Figure 15a shows the observed heat content in the upper 500 m (top graph) and the upper 3000 m (middle and bottom graphs) of the ocean. The heat content is defined as anomalies relative to the mean for the period having both model results and observational data (1951–1994 in Figure 15a (top and middle graphs) and 1979–1994 in Figure 15a (bottom graph)). As done by F-C, we use the units W year/m$^2$ averaged over the entire Earth to allow ready comparison with global climate forcings (1 W year/m$^2$ = $1.61 \times 10^{22}$ Joules). Note that the Levitus et al. [2000] data set has annual data through 1998 for the upper 500 m of the ocean. Because of sparse observations at greater depths, only a 5-year mean (through 1994) is provided for 500–3000 m, and no data are provided for greater depths. Thus in Figure 15a (middle) the heat content for years 1995–1998 includes annual heat gain at 0–500 m, but heat content at 500–3000 m is fixed at the mean value for 1992–1996.
[130] It is possible that the observed heat storage in the Levitus et al. [2000] analysis is an underestimate of the true value. In cases where no observations were available the procedure was to assign climatological values. Also no analysis is incorporated below 3000 m, where in at least some locations significant anomalies do occur [Bindoff and Church, 1992]. However, the vertical profile of the global ocean temperature trend (Figure 15b) suggests that the ocean beneath 3000 m would not contribute much to the full ocean value.
[131] The depth profile of ocean temperature change in Figure 15b is based on the linear trend for two alternative periods, which differ in their beginning date (1951 or 1955). The year 1951 corresponds to the beginning date of our climate model runs. However, the ocean heat content record since 1955 is considered to be more reliable because of a substantial expansion of the data sources that occurred in conjunction with global observing programs initiated in the mid 1950s.
[132] We discuss here possible relations between climate forcings and changes in the ocean heat content. We order the discussion according to timescale, from the brief perturbations (volcanoes) to the trend for the full period.
5.2.1. Volcanoes
[133] We mark in Figure 15a, with the symbol V + 2, the dates (2 years after large volcanoes) at which minima in the ocean heat content would be expected to occur because of large volcanic eruptions. By the third year after the eruption the aerosol forcing is small and tends to be overshadowed by trends in other forcings such as greenhouse gases. The observed ocean heat content shows evidence of cooling after all three large eruptions (Agung, El Chichon, and Pinatubo) since 1950, although the date of the minimum differs from that predicted by −1 year for El Chichon and +1 year for Pinatubo. It is unlikely that there are other short-term competing climate forcings comparable in magnitude to the volcanic aerosol forcings, so the discrepancies in timing are probably an indication of the level of “dynamic” variability of ocean heat uptake and/or the level of measurement uncertainty. By dynamic variability we refer to internal climate system mechanisms rather than global radiative forcings; these could involve, for example, fluctuations in heat transport by the ocean or atmospheric fluctuations including changes of cloud cover. The dynamic variability and measurement uncertainty issues may be related; for example, we have not subsampled the model with the time-varying coverage of the Levitus data, which may account for some of the higher frequency variability in the observations.
[134] The magnitude of the observed negative heat storage anomalies after the volcanoes is reasonably similar to the expected values. The climate simulations for both ocean A and ocean B yield decreases of 1–2 W years/m$^2$ in ocean heat content (ocean heat content is calculated in ocean A by integrating over time the heat flux at the ocean surface; see section 5.1.1). We discussed in section 4.1, in connection with Table 4, reasons that the ocean heat loss is less than would be estimated from instantaneous or even adjusted stratospheric aerosol forcing. In addition, the ocean heat uptake after a volcano is the net effect of the (negative) volcanic aerosol forcing and the (positive) portion of the greenhouse gas forcing that the climate system has not yet responded to. This disequilibrium greenhouse gas forcing was rather large by the time of the Pinatubo eruption.
Figure 15. (a) Ocean heat content anomaly in units of W year/m$^2$ averaged over the entire surface of the Earth (1 W year/m$^2 = 1.61 \times 10^{-22}$ Joules). The anomalies are relative to the common periods of data and simulations: 1951–1994 in top and middle graphs and 1979–1994 in bottom graph. Observed data are annual at 0–500 m (top graph), and the combination of this with 5-year mean data for depths 500–3000 m is shown (middle and bottom graphs). The combined data are repeated in the bottom graph to allow comparison with the SI95 simulations of F-C, which employed the $Q$-flux ocean model (ocean B) as well as two dynamic ocean models. The $Q$-flux model extends only to a depth of 1000 m. (b) Ocean temperature change versus depth based on the linear trend. Observations are annual and extend through 1998 in the upper 500 m. Below 500 m the data are 5-year mean and extend through 1994, which is the reason for the discontinuity in the model results. Note the scale change that occurs at 500 m. The full period is shown in the top graph, and the period of more reliable data, since 1955, is shown in the bottom graph.
(The planetary radiation imbalance probably was small at the time of El Chichon because of the 1976–1981 jump of global temperature, regardless of the (presumably dynamical, in part) cause of that jump.) Our prior modeling (Plate 5 of F-C, which included two dynamical ocean models) and our current results for oceans A and B (Figures 12 and 13) together suggest that the planetary radiative imbalance at the time of the Pinatubo eruption was $0.75 \pm 0.25$ W/m$^2$. This imbalance would reduce the 2-year (negative) heat storage after Pinatubo by 1–2 W year/m$^2$. Indeed, the fact that the ocean did not cool as much after Pinatubo as would have been expected if it were the only forcing, we suggest, could be because of an existing positive planetary energy imbalance at the time of the Pinatubo eruption.
[135] Verification of the negative planetary radiation imbalance that occurred after Pinatubo is provided by Earth Radiation Budget Experiment (ERBE) satellite measurements [Minnis et al., 1993] as illustrated in Figure 12 of Hansen et al. [1996]. The ERBE data yield a radiation balance anomaly of $-2.1$ W year/m$^2$ in the two years after Pinatubo. The magnitude of our simulated ocean heat content anomaly (Figure 15) is reasonably consistent with, but somewhat larger than, the ERBE measured imbalance. The ERBE imbalance shifts back to positive in 1993, consistent with the climate model, but not with the observed ocean heat storage minimum in 1994. Finally, we note that both the ocean heat content anomaly and the ERBE data are inconsistent with a Pinatubo peak forcing as large as the $-5$ W/m$^2$ suggested by Andronova et al. [1999].
5.2.2. Decadal variations
[136] The observed ocean heat content (Figure 15a) has significant decadal variability. The warming from 1968 to 1977 and the cooling from 1977 to 1983, for example, are much larger than the year-to-year variability or the estimated uncertainty in observed year-to-year and decade-to-decade changes of heat content [Levitus et al., 2000].
[137] These heat content changes do not appear to be caused by climate forcings. The simulations with a diffusive ocean model employing all of the known forcings, with and without the uncertain tropospheric aerosols, cannot produce the sharp increase of heat content in the early 1970s or a realistic representation of the cooling in the early 1980s. It is difficult to concoct a plausible underestimated forcing that might account for the observed variation. For example, if one suggested that solar cycle effects were underestimated by neglect of an indirect forcing (such as a forced cloud cover change), one would be faced with the contradiction that the time of minimum heat content in the mid 1980s and maximum heat content in the mid 1970s both occurred at the same phase of the solar cycle.
[138] It is more likely that the fluctuations are dynamical. However, they do not come about simply as a consequence of changing SST patterns that then alter fluxes to the atmosphere. This is shown by the simulation with ocean A (green line in Figure 10a (middle graph)), which used observed (HadISST1) SSTs for the period 1951–1999. On the basis of the energy fluxes at the ocean surface in this run the ocean heat storage is similar to that for the $\mathcal{Q}$-flux ocean model. The failure of observed SSTs to produce the observed change of ocean heat content is not surprising. As discussed in section 3.4 in cases such as the North Atlantic Oscillation, specified SST calculations do not capture correctly the heat exchange between ocean and atmosphere associated with vertical motions in the ocean, and indeed that model (ocean A) can yield the wrong sign for the heat flux anomaly [Bretherton and Battisti, 2000]. That might also happen at low latitudes. For example, the west Indian Ocean warmed substantially over the past half century. It is possible that in reality the ocean warming in that region was associated with increased heat flux into the ocean surface; however, in ocean A the increasingly positive SST anomalies in that region yield an increased heat flux out of the ocean.
[139] Exploration of the decadal variations in ocean heat content will require use of dynamical ocean models, which are outside the scope of our present paper. We note that in previous simulations [F-C] with dynamical ocean models for the period beginning in 1979 (see Figure 15a (bottom)), one model had a variation in heat content in the 1990s that was unrelated to the climate forcings. However, that fluctuation was associated with unrealistic deep water formation in the North Pacific Ocean. Recent coupled model simulations by two different groups [Levitus et al., 2001; Barnett et al., 2001] do not capture the specific observed decadal variations, but Barnett et al. [2001] note that their model does produce decadal fluctuations of the magnitude and timescale of those observed.
5.2.3. Long-Term change
[140] The change in the ocean heat content over the past half century is in good agreement with the climate model driven by known climate forcings. The dominant forcing and the cause of the long-term increase in ocean heat content is the GHG forcing, as shown by Figure 2. The positive ocean heat storage, because it is so directly connected to the planetary energy balance, is probably the best confirmation of the sign of the net climate forcing that has been operating on the planet during the past half century.
[141] Observed temporal change of ocean heat content also has the potential to yield a good, perhaps the best, quantitative measure of the net global climate forcing. However, the rate of heat uptake by the ocean depends upon climate sensitivity and ocean mixing, as well as upon the net climate forcing [Hansen et al., 1984, 1985]. If it were accepted that the mixing in ocean models is reasonably realistic, at least as it affects the global penetration of heat anomalies, and if it were accepted that climate sensitivity is about 3°C for doubled CO$_2$, then the observed ocean heat storage provides an indication that the net climate forcing is positive and of approximately the magnitude that we have assumed. In particular, under these assumptions, we find, as illustrated in Figure 15, that better agreement is obtained with a net climate forcing that includes the climate forcing by aerosols (six forcings) rather than the case without this negative aerosol forcing.
[142] Alternatively, if we knew the net global climate forcing, the rate of heat storage would provide an empirical measure of climate sensitivity. It is only if climate sensitivity is high that there is substantial “unrealized warming” due to the slow increase of greenhouse gases as the dominant climate forcing. Indeed, the recent positive trend of ocean heat storage and the fact that the ocean heat content dropped only slightly after Pinatubo are consistent with high climate sensitivity. However, there is such a large uncertainty in the indirect aerosol forcing that the ocean heat
storage does not provide a very helpful measure of climate sensitivity. Furthermore, all of these inferences are limited by poorly quantified but substantial uncertainty in the observed ocean heat storage, which potentially could be measured with high accuracy.
Barnett et al. [2001] and Levitus et al. [2001] previously reported global climate model results for ocean heat storage, which they found to be reasonably consistent with the Levitus et al. [2000] data. Barnett et al. [2001] used the National Center for Atmospheric Research (NCAR) Parallel Climate Model (PCM) [Dai et al., 2001], which has a sensitivity of $2.1^\circ$C for doubled CO$_2$, and forcing by greenhouse gases and sulfate aerosols, with a net forcing of 2 W/m$^2$ in 2000 relative to 1850. Levitus et al. [2001] used the Geophysical Fluid Dynamics Laboratory (GFDL) model [Delworth et al., 2001], which has a sensitivity $3.7^\circ$C for doubled CO$_2$, and a forcing similar to that of Barnett et al. [2001]. Barnett et al. [2001] found an ocean heat storage of $12 \times 10^{22}$ J in the period 1955–1995, while Levitus et al. [2001] obtained $33 \times 10^{22}$ J. The observed heat storage [Levitus et al., 2000] is about $18 \times 10^{22}$ J (this is reduced to $13–14 \times 10^{22}$ J if the data are first averaged over decades; Barnett et al. [2001] only report their model result after such averaging). When Levitus et al. [2001] added solar and volcanic aerosol forcings, the heat storage was reduced to $20 \times 10^{22}$ J. The reduction in heat storage probably was due mainly to the volcanic aerosols. Their solar forcing was +0.18 W/m$^2$ over the interval 1865–2000. Their volcanic aerosol forcing averaged $-0.54$ W/m$^2$ over 1960–1999; it is based on the data of Andronova et al. [1999] and is thus larger than that which we employ, as discussed in section 2.2. The dependence of the simulated heat storage on the model sensitivity and the climate forcing in these studies is consistent with the discussion above. Their results are also consistent with the heat storage in our model over the same interval, which was $18 \times 10^{22}$ J for five forcings and $14 \times 10^{22}$ J for six forcings; our results refer just to the upper 1000 m of the ocean, because our present Q-flux ocean extended only to that depth.
5.3. Atmospheric Temperature Profile
Climate forcings have a strong effect on the atmospheric temperature profile, as illustrated explicitly in Figure 6 of F-C and by Ramaswamy et al. [2001]. We do not attempt a comprehensive study here, which would require more realistic representations of the stratosphere and ocean as well as better information on the vertical profile of absorbing aerosols. However, our present simulations cover a longer period than those of F-C. This allows us to compare modeled and observed temperature profiles for both the era of satellite data and the longer period with radiosonde data.
The satellite era begins in 1979 with the first MSU data, for which we use version d of Christy et al. [2000]. Radiosonde coverage was extensive by 1958, the International Geophysical Year, although the coverage is only considered to be reasonably global after 1964 [IPCC, 2001]. We use radiosonde data analysis of Parker et al. [1997]. The two radiosonde data sets, HadRT2.0 and HadRT2.1, differ in that the latter has been adjusted with the help of MSU data (version c) in an attempt to correct for bad radiosonde records. We illustrate both data sets, thus providing one indication of data uncertainty.
We present two views of the temperature profile. Figure 16 compares line graphs of observed and modeled temperature profiles for the global mean and for northern latitudes, low latitudes, and southern latitudes. We define low latitudes as 40N–40S, which is the latitude range at which the tropopause extends to about the 100 hPa level. Figure 17, the zonal mean temperature change versus latitude, provides a more pictorial view of the nature of the zonal temperature change.
Qualitatively, there is reasonable agreement between the simulated and observed temperature changes, particularly for the longer time period. However, Figure 16 reveals that the model warms more than observed in the upper troposphere, and the model cools less than observed in the stratosphere. The discrepancies occur primarily in the latter period, 1979–1998, the period with more complete observations of climate forcings. Agreement is best at southern latitudes. The simulated upper tropospheric warming is especially excessive at low latitudes. Simulated stratospheric cooling is too little at northern latitudes and low latitudes. The discrepancies are significant based on the standard deviation among the ensemble members, as illustrated at three pressure levels in Figure 16.
Among the climate forcings, ozone, stratospheric water vapor, and aerosols probably are the best candidates for contributing to the discrepancies in simulated temperature profiles. Ozone should be considered first because it changed dramatically during 1979–1998, yet it was poorly measured in the lower stratosphere and troposphere. Indeed, much closer agreement with the observed change in temperature profile would have been obtained if we had employed the ozone change that was used by F-C. The ozone change of F-C had large ozone depletion near the 100 hPa level at all latitudes, including the tropics, based on the then available analysis of SAGE observations. The SPARC ozone trend assessment [WMO, 1998] excluded SAGE data below the 20-km level because of its uncertainties, but they did not replace it with any other estimate. As a result our current estimate for ozone change, as discussed in section 2.4, has a maximum ozone depletion rate near the tropical tropopause of only about 2% per decade. However, recent analyses of SAGE II data for October 1984 to April 2000 (J. Zawodny, private communication, 2000) yield an ozone depletion of more than 5% per decade with maximum depletion near 20 km altitude. This is less depletion than assumed by F-C, but it is substantial. Furthermore, the SAGE II period of data, beginning in late 1984, misses the period of rapid depletion of column-integrated ozone that occurred in 1980–1985 (Figure 6). Although column-integrated ozone amount does not show much depletion at low latitudes (Figure 6), this could be a result of increases in tropospheric ozone as suggested in the recent SAGE II analyses of J. Zawodny (private communication, 2000). Therefore it seems possible that the ozone depletion rate in the tropopause region for the full period 1979–1998 was larger than that in our current scenario. As shown in Figure 6 of F-C, ozone depletion near the tropopause could cause significant cooling in the upper troposphere and lower stratosphere.
Stratospheric water vapor probably contributes to the discrepancy between the observed and modeled temperature profile change. The positive trend of stratospheric water vapor in the model is less than the observed trend, as
Figure 16. Change of annual-mean temperature profile for 1958–1998 and 1979–1998 based on linear trends. Model results are for oceans A and B, with five and six forcings. Surface observations are the land-ocean data of Hansen et al. [1999], with SSTs of Reynolds and Smith [1994] for ocean areas. The bars on the MSU satellite data [Christy et al., 2000] are twice the standard statistical error adjusted for autocorrelation [Santer et al., 2000]. Radiosonde profiles become unreliable above about the 100-hPa level. Twice the ensemble standard deviation is shown at three pressure levels for ocean B with six forcings.
discussed in section 2.5. Water vapor change at the rate reported by Rosenlof et al. [2001] would increase stratospheric cooling slightly, of the order of 0.1°C in 20 years [Oinas et al., 2001]. However, this can account for only a small fraction of the discrepancy.
Tropospheric aerosols cool the surface but have only modest impact on the temperature profile in our present simulations. However, suspected inaccuracies in the aerosol vertical distribution and temporal change may cause the upper troposphere to warm relative to the near surface layers in the simulations. As discussed in section 2.6, the black carbon aerosols are mixed too high in the troposphere compared with limited available observations, with the amount of black carbon in the upper troposphere
Figure 17. Change of zonal-mean annual-mean temperature for 1958–1998 and 1979–1998 based on linear trends. Radiosonde data in the top row are for versions 2.0 and 2.1 of the HadRT analysis [Parker et al., 1997]. Model results are for oceans A and B, with five and six forcings. Note that ocean A with five forcings employs the ozone $O_3^A$ data set, while the others use $O_3^B$ (the latter has greater ozone depletion in the South Pole region).
perhaps as much as a factor of 10 too large. The temporal issue arises because black carbon (and sulfates and organic carbon) aerosols are taken as proportional to fossil fuel use. However, T. Novakov (private communication, 2001) argues that the proportion of black carbon aerosols has decreased in recent decades in developed countries because of a decrease in inefficient coal burning in domestic and commercial sectors as well as improved efficiency of diesel engines, at least in the United States. Thus it is plausible that more realistic vertical and temporal distributions of black carbon would cause less warming of the troposphere relative to the surface. Quantitative analysis requires better knowledge of aerosol distributions and their temporal change. This topic is discussed further in section 6.
[151] In the period 1979–1998 the discrepancy between model and observations is primarily at low latitudes, and it is larger in ocean A than ocean B. This is most apparent in Figure 17, as the bulls-eye warming in the tropical upper troposphere. Excessive warming at this level did not occur in our previous simulations [F-C], because of greater ozone depletion near the tropopause, as discussed above. Ocean B has slightly less warming at the surface than ocean A at low latitudes during 1979–1998, and this difference increases in the middle to upper troposphere in the way temperature anomalies are observed to change with height in the tropics [Hurrell and Trenberth, 1998; Wentz and Schabel, 2000; Santer et al., 2001]. As shown in Table 5, the discrepancy between MSU lower tropospheric temperature change and the ocean B model results for six forcings is small, although the discrepancy is substantial for ocean A. The discrepancy with radiosonde temperature change for 1979–1998 is larger (Figure 17 and Table 5), but the radiosondes suffer from poor spatial sampling and temporal discontinuities [Gaffen et al., 2000], suggesting that in this case MSU may provide the
more reliable result. However, there are also significant sources of potential error in the MSU temperature trends [Santer et al., 1999; Hurrell et al., 2000; Wentz et al., 2001].
[152] There are several possible interpretations of these results. Perhaps there is an error in the observed trend of low latitude SSTs, as only a small error (about 0.2°C over 2 decades) in the SST is required to explain the tropospheric temperature change. Such an error would be consistent with the smaller warming of tropical nighttime marine air temperature (NMAT) found by Christy et al. [2001] for the period after 1979; that is, it would remove the difference between the NMAT and SST trends. Alternatively, the observed SST temperature trends may be accurate, and the difference between the NMAT and SST trends may be real, but the heat flux anomalies from the ocean to the troposphere may be inaccurately simulated. Given a positive SST anomaly, the model faithfully delivers a larger anomaly to the midtroposphere, but the model could be flawed in its simulated dynamical or thermodynamical energy fluxes or in the forcings that influence those fluxes.
[153] There are also real differences between the model and radiosonde observations for the longer period 1958–1998 at the tropical and northern latitudes, and on the global average, as summarized in Figure 16. We believe that these discrepancies are meaningful and probably related, at least in part, to inaccurate or incomplete representations of the three climate forcings’ ozone, water vapor, and aerosols, as discussed above. Ozone depletion near the tropopause is probably understated in our scenario. We know that the measured stratospheric water vapor increase was larger than in our model. Suspected flaws in the BC aerosol scenario are in the sense to partially account for the discrepancies.
6. Discussion
6.1. Climate Forcings
[154] We have defined six radiative forcings, nominally from first principles, for the period 1951–2000. These forcings, when inserted in a climate model having sensitivity 3°C for doubled CO₂ and an ocean mixing rate for heat based on empirical data from ocean tracers, yield remarkably good agreement with observed global mean surface temperature change over that period. It may seem tempting to take this close agreement as evidence that these forcings accurately reflect the true forcings that influenced climate during this period. We suggest, however, that the close agreement could be, to a substantial degree, fortuitous, with omissions and errors in some forcings countering errors of the opposite sign in other forcings. Nevertheless, we argue that the net forcing has sufficient significance that, together with observed ocean heat storage, we can draw some conclusions about the present state of planetary energy imbalance, as discussed in section 6.2.
[155] We discuss the forcings with the aid of Figure 18. Figure 18a is the estimate of Hansen and Sato [2001] for the
change of climate forcings between 1850 and 2000. The greenhouse gas forcings are known with reasonably good accuracy. CO$_2$ (1.4 W/m$^2$) has the largest forcing, but the CH$_4$ forcing is half as large when its indirect effects on stratospheric H$_2$O and tropospheric O$_3$ are included, and the sum of non-CO$_2$ greenhouse gas forcings exceeds the CO$_2$ forcing. Recent studies [Mickley et al., 2001; D. T. Shindell, private communication, 2001] suggest that the tropospheric O$_3$ forcing may be as large as 0.7–0.8 W/m$^2$. We take 0.1 W/m$^2$ of the O$_3$ forcing as an indirect effect of CH$_4$ for a total O$_3$ forcing of 0.6 W/m$^2$, with a large uncertainty.
[156] Aerosol forcings are still more uncertain. Adams et al. [2001] suggest that the sulfate forcing is $-0.95$ W/m$^2$ when the swelling of sulfate aerosols at high humidities is fully accounted for, and they suggest that the forcing due to anthropogenic nitrate aerosols is about $-0.2$ W/m$^2$. IPCC has a smaller estimate for sulfates and does not consider nitrates. We estimate the sum of the sulfate and nitrate forcings as $-1 \pm 0.25$ W/m$^2$. Forcing by black carbon is estimated by IPCC [1996] as $+0.1$ W/m$^2$ and by IPCC [2001] as $+0.25$ W/m$^2$, but Jacobson [2000a, 2000b] estimates the black carbon forcing at about 0.5 W/m$^2$, and Hansen et al. [2000b] suggest that the total black carbon forcing might be closer to $+1$ W/m$^2$ when indirect effects are included. It should be noted, however, that the surface thermal response to forcing by absorbing aerosols is a strong function of the vertical distribution of the aerosols [RF-CR]. The greatest uncertainty, however, is the indirect effects of aerosols on clouds. We estimate these forced cloud changes to cause a forcing of the order of $-1$ W/m$^2$ with an uncertainty of at least a factor of two.
[157] Figure 18b is the change of forcings between 1951 and 2000 that were used for the simulations in this paper. The net increase in the forcing over this half century was about 1.6 W/m$^2$. Perhaps this net forcing, fortuitously, is reasonably realistic even though the assumed scenario probably underestimates several negative forcings, with the omission of forced cloud changes, nitrates, biomass burning, soil dust, and land cover alterations. These omissions could be at least partially compensated by underestimates of the tropospheric O$_3$ and black carbon forcings. Also, the vertical distribution of black carbon that we employed, being weighted too much toward high altitudes, probably reduced the efficacy of that positive forcing. There may also have been a positive initial disequilibrium forcing in 1951, perhaps as much as 0.1 to 0.2 W/m$^2$, representing the unrealized warming due to the history of forcings in the period 1850–1950. Figures 15 and 16 suggest that a slightly smaller net forcing for 1951–2000 would have yielded closer agreement with observed temperature change.
[158] This unsatisfactory knowledge of the true forcings could be improved in years to come. Measurements of current aerosol distributions, their relation to sources, and their influence on clouds may permit an improved understanding that can be used with chemical transport models to construct improved histories of many of these forcings. Success in this endeavor would require global satellite monitoring of aerosol and cloud particle microphysical properties, global surface monitoring stations, field campaigns, aerosol and cloud modeling, directed laboratory measurements, and the integration of information from all of these into global chemical transport and climate models.
6.2. Heat Storage: Planetary Disequilibrium
[159] The good fit that we obtain between simulated and observed atmospheric and surface temperature changes and the consistency with measured ocean heat storage suggest that the observed climate change in the past 50 years is primarily a response to the radiative forcings. However, it would be possible to obtain a comparably good fit to the observed temperatures with a larger (smaller) positive trend of the net forcing by employing a model with smaller (larger) climate sensitivity. Even the limited range for climate sensitivity that we infer from paleoclimate data, $3 \pm 1^\circ$C for doubled CO$_2$, allows a substantial range of forcings. We are examining this topic further using versions of the Q-flux model with sensitivities 2°C and 4°C for doubled CO$_2$. More realistic ocean circulation models will also be needed for a full investigation.
[160] Using our present reconstruction of climate forcings for 1951–2000, and assuming that climate sensitivity is near 3°C for doubled CO$_2$, leads to a planetary disequilibrium in 2000, with the planet soaking up energy at a rate of about $\frac{3}{4}$ W/m$^2$. The same assumptions imply additional future global warming of about $\frac{1}{2}$°C, even without any additional forcing. The amount of warming “in the pipeline,” however, is a function of climate sensitivity, and, in principle, it could vary by almost a factor of two in either direction for the range of climate sensitivities from 2 to 4°C for doubled CO$_2$ [Hansen et al., 1985]. The fact that the model with sensitivity 3°C for doubled CO$_2$ yields ocean heat storage comparable to observations tends to favor that sensitivity, but the examples given in section 5 illustrate that there is some latitude in the forcings and climate models that yield reasonable agreement with observed ocean heat storage. However, if the trend of ocean heat storage is established more accurately with a longer record, and if the forcings are defined more precisely, it should be possible to narrow the uncertainty in the unrealized warming.
6.3. Temperature Profile
[161] The simulated global-mean surface temperature change is in good agreement with observations. However, there seem to be significant discrepancies between the modeled and observed changes in the temperature profile. There are uncertainties in the observed temperature profile change, and we have not sampled the model at radiosonde locations, but these factors cannot fully account for the discrepancies. Model inadequacies, especially in our current representations of the stratosphere and ocean, may be partly responsible. However, we suggest that the discrepancies between the observed and modeled profiles are due at least partly to inaccuracies in the histories of some of the forcings, specifically, BC aerosols, O$_3$, and stratospheric H$_2$O.
[162] Perhaps the most important observational need is for composition-specific aerosol monitoring. The vertical profile of absorbing aerosols is especially important, but measurements are needed for all aerosols that can influence cloud cover and cloud properties. Troposphere O$_3$ needs to be monitored, especially near the tropopause level, where even small changes have a noticeable impact on the temperature profile. Stratospheric H$_2$O is in part a modeling problem that can probably be solved with higher vertical resolution, but continued monitoring of stratospheric H$_2$O is also important.
6.4. Strategy for Further Experiments
[163] Our strategy of investigation, described in section 2 of F-C, tries to balance the need for systematic experiments using fixed models and well-defined forcings with the need to improve the realism of both the climate model and the forcings that drive it. We want a structured set of simulations to allow comparisons but without inhibiting alternative ideas and initiative. We need to optimize use of limited human and computer resources.
[164] The simulations of F-C, carried out on desktop IBM RISC 6000 workstations, were 17-year runs. The present simulations, carried out on a Silicon Graphics 2000 system, focus on the past 50-year period and include additional forcings and models. Some of the experiments now being carried out for 1951 to present (see Table 3) are using a version of the model reprogrammed, documented, and optimized for parallel computations but nominally with the same physics as in SI2000. The aim is to find a practical path leading to a prompt new round of experiments for a longer period, 1850–2000, including improvements in the realism of both forcings and models.
[165] Acknowledgments. We thank Ian Plumb for providing model results on methane production of water vapor, David Randel for stratospheric ozone change data, Urs Baltensperger for aerosol data prior to publication, David Rind for comparative studies of water vapor in his middle atmosphere model, Tony Broccoli for comparative data from the GFDL model, Joe Zawodny for SAGE ozone data, Mark Chandler, Ralph Cicerone, Jos Leelieveld, Michael Oppenheimer, and Jonathan Overpeck for discussions, and two anonymous referees for helpful reviews. This research was supported by the NASA Earth Science Research Division.
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Analyzing the Diverse Impacts of Conventional Distributed Energy Resources on Distribution System
Muhammad Aamir Aman\textsuperscript{1}, Sanaullah Ahmad\textsuperscript{1}, Azzam ul Asar\textsuperscript{2}, Babar Noor\textsuperscript{1}
\textsuperscript{1}Electrical Engineering Department, IQRA National University, Peshawar, Pakistan
\textsuperscript{2}Electrical Engineering Department, CECOS University, Peshawar, Pakistan
Abstract—In recent years, the rapid boost in energy demand around the globe has put power system in stress. To fulfill the energy demands and confine technical losses, researchers are eager to investigate the diverse impacts of Distributed Generation (DG) on the parameters of distribution network. DG is becoming even more attractive to power producing companies, utilities and consumers due to production of energy near to load centers. Reduction in power losses, better voltage profile and less environmental impact are the benefits of DG. Besides renewable energy resources, conventional energy resources are also a viable option for DG. This research aims to analyze the impact of localized synchronous and induction generators on distributions network. The main objectives are to find optimal type, size and location of DG in distribution network to have better impact on voltage profile and reduction in power losses. Using worldwide recognized software tool ETAP and Kohat road electricity distribution network as a test case. Results depicted that at certain buses, positive impacts on voltage profile were recorded while almost 20% of power losses were decreased when synchronous generator as DG unit was injected in distribution networks. Injecting induction generator as DG unit, the results showed increase in power losses due to absorption of reactive power, while improving voltage profile by injecting active power.
Keywords—Electric power system; distributed generation; voltage profile; power losses; synchronous generator; induction generator
I. INTRODUCTION
In electrical power plants the power is generated which fulfills the demand of energy required. The capacity of generation depends on type and size of the generating unit. Capacities of these traditional power plants vary from hundreds of Megawatt to few Geggawatt [1]. These generation power plants of such a large scale are placed far away from load centers. For transmission of power from the generation station to the customer’s premises transmission lines and distribution feeders are used [2], [3]. As shown in Fig. 1, it is necessary for the utilities to provide standard voltage profile/level to its customers.
To balance demand and supply gap, DG became the feasible option. The term DG can be defined as any power generation unit that is integrated within the distributed system [4]. DG may be a conventional or non conventional energy source which consists of wide range of technologies such as internal combustion engines which are prime movers for synchronous and induction generators, wind turbines, photo voltaic systems, fuel-cells, etc. For generation of power if DG uses non-conventional or renewable energy sources then it is known as renewable distributed generation as shown in Fig. 2 [4].
DG typically ranges from few Kilowatts (KW) to several Megawatts (MW) as they are not centralized [1]. Both conventional (non-renewable) and non-conventional (renewable) resources can be used to generate power for DG [3]. Combustion engines and fuel cell are used as conventional energy resources, while geothermal system, solar energy and wind energy are used as non-conventional energy resources [5]. DG sources accompanied with energy storage technologies is called distributed source of energy as shown in Fig. 2.
As DG is on site generation of power feeding to the distribution network, to acquire a complete and reliable DG system it is more important to have the knowledge of injecting DG at proper optimal location and to determine the source of DG either renewable or non-renewable and also number of DG units. Connection and process of power generation elements linked straightly to distribution system or associated to the network on consumer’s location of the meter defines the location of distributed generation [6], [7].
DG is the power generation from the resources which is not centralized and is injected in the distribution system near the load centers. While the traditional power system is linked to the main grid, contains the generation station, transmission network and distribution system. In distribution system voltage and reactive power control equipment, in a current grid system are mostly operated on unidirectional power flow only, that is from the transmission system to high voltage and medium voltage substations and then to the low voltage distribution system. Thus there is decrease in voltage along the feeder, from the substation to the feeder-end. By installing DG makes this statement no longer valid. When DG is injected it will affect voltage profile. Also, when the output power of DG is high then the power may flow from the distribution system to the transmission system which may start false triggering of protection system [8].
DG unit to be effectively is desirable to operate at full capacity. But if they are injected improperly causes restriction and limits its maximum capacity. So, to have undesirable effects on the power system DG unit must be placed at optimal location to operate to its maximum limit [9]. Researchers are eager to develop methods and algorithms to achieve adequate voltage profile and reduce power losses by optimization of DG.
To manage the increasing demand of power and to reduce line losses and voltage profile, DG is considered to be a power paradigm for the new millennium. This depends on optimal location of DG injection and also type of DG to be injected in the power system. If DG is injected in a power system in un-deterministic way a vital impact of DG occurs, causing variation in voltage profile and power losses. Utility companies are searching for a technique to provide standard voltage to the customer’s premises. Injection of DG at optimum location is one solution to overcome this problem.
In this paper Electrical Transient Analyzer Program (ETAP) software tool is used for simulation and analysis. This paper focuses on the impact of DG on voltage profile and power losses in a radial test system in which three cases are considered: firstly results are evaluated when no DG unit is injected, secondly and foremost impact of synchronous generator and induction generator as a DG units are evaluated and lastly comparison for optimum type and location of DG is carried out.
II. DISTRIBUTED GENERATION AND ITS DIVERSE IMPACT ON DISTRIBUTION SYSTEM
Universally DG is accepted as an effective and economical solution to reduce the increasing demand in power system. In current years, DG achieved much consideration due to its positive impacts on the electrical distribution systems. Some of these are improving transmission and distribution congestion, voltage and power quality, line losses reduction, reliability, power consumption demand, security, and reaching the goal to utilize green and renewable energy resources [10], [11]. Also DG has lesser principal cost because of utilizing renewable energy resources and has nil pollutant radiations. DG can work in the situation of peak shaving during increase in requirement and also as a backup in case of interruption. However, some negative impacts of DG including voltage rise, poor power quality, harmonics, etc. Absence of synchronization between DGs and distribution system results in voltage regulation problems [12].
A. Impact of DG on Voltage Profile and Power Losses
For positive impacts, DG must be appropriately synchronized with the operational system and feeder scheme. This means addressing concerns allied to voltage regulation, flicker, grounding compatibility, overcurrent safety, harmonic variations, consistency, islanding, capability limits and further aspects. Integration of DG and its impact on system is relative to feeder demand and capacity therefore the coordination of DG with system must be optimized accurately to avoid these issues [13].
It is of prodigious significance to clients having a better voltage profile because its an elementary request for electrical equipment running near the valued voltage. Voltage drop may occur across the distribution transformer due to line impedance, line losses, etc. Thus, rated voltage at customer service would change.
DG supports the voltage to rise at the termination of the feeder. Though, where the DG is introduced may raise the voltage above the standard limits. Voltage may be greater at the consumer amenities than on the primary side of the distribution transformer and may even go beyond the voltage above the higher limits. It means that both high and low service voltage can take place due to the mismatch of DG with the radial power stream.
III. POWER FLOW ANALYSIS AND MATHEMATICAL MODELING
The electrical network is signified by non-linear algebraic equations under steady state. Power flow solution uses these
equations. For determining the simultaneous equations iterative methods are used. Different methods are used for solving Gauss-Seidel approach is discussed below.
A. Power Flow Solution by Gauss-Seidel Method
Gauss-Seidel is one of the iterative methods used for solving non-linear algebraic equations. Complex voltage at every node successively sweeps and updates its neighbor bus voltage in this arrangement.
\[ I_{Bus} = Y_{Bus} x V_{Bus} \]
And for any particular Bus “a”
\[ I_a = \sum_{n=1}^{N} Y_{an} V_n \]
The complex power
\[ S_a = V_a I_a^* \]
\[ P_a + jQ_a = V_a [\sum_{n=1}^{N} Y_{an} V_n]^* \]
Where \( a = 1, 2, 3 ... N \)
For complex power
\[ I_a = \frac{P_a + jQ_a}{V_a^*} \]
Also, \( I_a = \sum_{n=1}^{N} Y_{an} V_n \)
Or,
\[ I_a = Y_{a1} V_1 + Y_{a2} V_2 + \ldots + Y_{aa} V_a + \ldots + Y_{aN} V_N \quad (1) \]
From the above equation
\[ V_a = \frac{1}{Y_{aa} [I_a - (\sum_{n=1}^{a-1} Y_{an} V_n + \sum_{a+1}^{N} Y_{an} V_n)]} \]
or,
\[ V_a = \frac{1}{Y_{aa} [\frac{P_a + jQ_a}{V_a^*} - (\sum_{n=1}^{a-1} Y_{an} V_n + \sum_{a+1}^{N} Y_{an} V_n)]} \]
Where, \( a = 1, 2, 3... N \)
However, suitable determination is desired at per iteration, with proliferation in number of iterations the tolerance reduces because the convergence of this technique is linear. This postures important restriction for large system because of the solution time and estimation cost increases.
B. Gauss-Seidel Iterative Procedure
1) Make an initial guess \( |V_i| = 1.0 \) and \( \delta_{i(0)} = 0.00 \).
2) Use this solution in power flow equation to obtain the better first solution and this solution is called improved estimated of \( V_a \).
3) First solution is used to obtain a better second solution and so on, in general equation i.e.
\[ V_a^{i+1} = \frac{1}{Y_{aa} [\frac{P_a + jQ_a}{V_a^*} - (\sum_{n=1}^{a-1} Y_{an} V_n^{i+1} + \sum_{a+1}^{N} Y_{an} V_n^i)]}. \]
IV. Research Methodology
For this research work a residential radial distribution feeder of 11KV located at Kohat Road Peshawar, Khyber Pakhtunkhwa, Pakistan is selected. The selected feeder is modeled and analyzed in Electrical Transient Analyzer Program (ETAP). ETAP software tool is used worldwide for power system planning, designing, operation and analysis.
V. Modeling of Distributed Generation Units
For this research work synchronous and induction generator are selected as a DG units. They will operate in different conditions.
A. Without Distributed Generation Unit
The analysis of the modeled feeder in ETAP will be done when no DG is injected. The results obtained from this case are taken as a reference. The results evaluated from other cases will be compared in order to analyze the impact of different type, size and location of DG on voltage profile and power losses.
B. Synchronous Generator as Distributed Generation Unit
Synchronous generator as DG unit of 2.5 MW will be modeled and will be placed at different buses in radial distribution system as shown in Fig. 3. The DG unit will inject only real power into the system with unity power factor. Two different places bus-3 and bus-6 are selected for connecting DG units randomly.
C. Induction Generator as Distributed Generation Unit
An optimum size of 2.5 MW DG will be modeled and injected in the radial distribution system at bus-3 and bus-6 randomly as shown in Fig. 4. DG will inject active power and will absorb reactive power, working as an induction generator mode with an operating power factor of 0.85 lagging.
VI. TEST CASES
In this research work the following different cases have been conducted:
A. Case-I
Load flow analysis of the radial distribution network in case-I is shown in Fig. 5. The requirement of load is fulfilled by the power grid as in this case no DG unit is injected. This case is taken as a reference for other cases. Real time condition and voltage levels at different buses are shown. As the red color shows critical condition of buses, bus-5 and bus-6 are in critical condition in this case.
The power losses at different locations in distribution network are shown in Table II. These losses are decreased from the previous case when no DG was connected as the total active power losses are 321.3 KW while the total reactive power losses are 481.8 KVAR.
B. Case-II
Load flow analysis of Case-II while injecting DG at bus-3 are shown in Fig. 6. DG unit of 2.5 MW connected with the system at Bus-3 operating at power factor of unity. Requirement of load is fulfilled by the power grid and DG source mutually in this case. By comparing voltages with previous case voltage level is improved at different buses as shown in Fig. 6.
C. Case-III
The load flow analysis of Case-III are shown in Fig. 7. A DG unit of 2.5 MW is connected at Bus-6 operating at power factor of 0.85 lagging. Requirements of load is fulfilled by the power grid and DG source mutually. While real power is injected in the system and reactive power is absorbed from the system by the DG unit as connected DG unit is induction generator.
The power losses at different locations in distribution network are shown in Table III. When an induction generator unit is connected at bus-6 the total power losses i.e. active power losses and the total reactive power losses are 522.8 KW and 660.9 KVAR, respectively, as comparing to DG unit placement at Busbar-3 these losses are increased as shown in Table III.
VII. RESULTS AND DISCUSSIONS
This section discusses the results calculated after the implementation of above cases. Table IV shows a detail report about the real and reactive power injected by DG unit and by grid in each case. The DG unit acts as a negative load when injected in the system.
A. Voltage Profile Analysis
Different impacts on voltage profile occurs when different types of DG unit are connected with the radial distribution system. Table V shows the overall results.
1) Case-I: In this case no DG unit is injected with the system. Fig. 8 shows the radial decrease in voltage profile from source to load. This reduction in voltage level is due to the line impedance.
2) Case-II: 2.5 MW DG unit is installed in this case at Bus-3. Operating power factor of this DG unit is unity. Injection of DG unit is considerable effects on voltage profile. Voltage profile is improved in this case. This case shows the best results as comparing it to the other cases. As depicted in Fig. 9.
3) Case-III: In this case 2.5 MW capacity of DG unit is injected to the system delivering active power while absorbing reactive power. The result obtained from injection of DG unit at Busbar-6 is shown in the Fig. 10.
The results obtained from injecting synchronous generator at bus-3 are far better than injecting induction generator at bus-6.
B. Power Loss Analysis
1) Case-I: No DG unit is injected with the radial distribution network in this case. The total power losses of the system are shown in Fig. 11. These losses occur due to line impedances. These losses also consist of some fixed losses which are constant and cannot be changed e.g. transformer losses. There is no DG unit installed in this case and the total power is fed from the power grid. Current flow from the power grid to the consumer localities through each cable results in huge amount of power losses.
2) **Case-II**: 2.5 MW DG unit injecting active power and power grid mutually fed the load and hence reduces the power losses as shown in Fig. 12. It is clearly seen that losses are reduced by the installation of DG unit.
3) **Case-III**: 2.5 MW induction generator is injected as a DG unit in this case. This DG setup is delivering active power while absorbing reactive power from the system. The losses in this case are increased from the base case as shown in Fig. 13. The logic behind this increase in losses is that the induction generator absorbs reactive power from the system. It is noted that by injecting synchronous generator at busbar-3 shows the outperform results than by injecting induction generator as a DG in busbar-6. Therefore, injecting DG unit at optimum location i.e. busbar-3 and DG type i.e. synchronous generator will give greater reduction in power losses.
**TABLE III.** Case-III Power Losses due to Injection of DG Unit at Bus-6
| Branch/ Circuit ID | Lines Total | Transformers Total | Cables Total | Total |
|--------------------|-------------|--------------------|--------------|-------|
| KW | 360 | 73.9 | 88 | 522.8 |
| KVAR | 80.3 | 567.5 | 12.9 | 660.9 |
**TABLE IV.** Injection of Real and Reactive Power from DG Units and Grid
| Case | DG Injected Power | Grid Injected Power |
|------|-------------------|---------------------|
| | MW | MVAR | MW | MVAR |
| 1 | 0 | 0 | 6.805 | 4.449 |
| 2 | 2.5 | 0 | 6.747 | 4.454 |
| 3 | 2.5 | -1.55 | 4.468 | 6.194 |
**TABLE V.** Different Buses Voltage Levels
| No. of Bus | Case-I | Case-II | Case-III |
|------------|--------|---------|----------|
| 1 | 11 | 11 | 11 |
| 2 | 10.805 | 10.899 | 10.884 |
| 3 | 10.643 | 10.793 | 10.769 |
| 4 | 10.535 | 10.596 | 10.675 |
| 5 | 10.4 | 10.462 | 10.646 |
| 6 | 10.363 | 10.424 | 10.714 |
Fig. 10. Induction generator impacts on voltage profile.
Fig. 11. Total active power losses of the radial distribution system.
Fig. 12. Power losses of the system in case of active power injected by DG unit.
**VIII. Conclusion and Future Work**
Analyzing the assorted impacts of DG units on distributed system is of most significance. Some vital factors such as type, size and location of DG units are important for proper utilization of DG units. The detailed analysis in this research work showed diverse impacts of DG on voltage profile and power losses. It is investigated that connecting synchronous generator as a DG unit near to load centers will improve voltage profile and reduce power losses. While connecting induction generator...
as DG unit near to load centers have positive impact on voltage profile but it caused increase in power losses due to absorption of reactive power by induction generator. In future several DG technologies either conventional or non-conventional, can be evaluated for their various impacts on distribution system.
REFERENCES
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Computers can make it easier, quicker, and cheaper to print formal publications; individuals can do what was once practical only for organizations. ... As the computer increases the freedom of writers, so does it increase the responsibility of readers.
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*The Sachertorte Algorithm and other antidotes to computer anxiety*, Penguin Books, 1986, pp. 18, 20
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**Patrick D. Ion**
Mathematical Reviews
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313-763-6829
**Calvin W. Jackson, Jr.**
1749 Michelorena St
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firstname.lastname@example.org
**Helmut Jürgensen**
Dept of Computer Science
Univ of Western Ontario
London N6A 5B7, Ontario, Canada
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Bitnet: A505@UWOCC1
UUCP: helmut@deepthot
**Arthur Keller**
University of Texas at Austin
Department of Computer Science
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**Bill Kelly**
Academic Computing Center
University of Wisconsin, Madison
1210 W. Dayton Street
Madison, WI 53706
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**John Kennedy**
Mathematical Physics
University College Dublin
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Karl Kleine
Forschungszentrum Informatik
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Donald E. Knuth
Department of Computer Science
Stanford University
Stanford, CA 94305
Arpanet: DEKnuth-AI
Pierre A. MacKay
University of Washington
Department of Computer Science,
FR-35
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Rick Mallett
Computing Services
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Richard S. Palais
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Brandeis University
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Mitch Pfeffer
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Arnold Pizer
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University of Rochester
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Craig Platt
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Tom Rokicki
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Barry Smith
Kellerman & Smith
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Alan Spragens
SLAC Computing Services
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Ralph Stromquist
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Rilla Thedford
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General Delivery
Comments on the Format of This Issue
Barbara Beeton
The last issue of TUGboat was noteworthy for its new design, specified by the designer Martha Gannet, implemented by David Kellerman and Barry Smith, and bearing on its cover the image of our worthy vessel drawn by Duane Bibby.
This issue returns (temporarily, it is hoped) to the old "scrapbook" format. There are several reasons, chief among them the desire to get it into members' hands before the summer TUG meeting. A second reason, interacting strongly with the first, is the fact that we at the Math Society have not yet succeeded in obtaining the new fonts, and the time required to install them on the Alphatype typesetter (once the METAFONT files are in hand) is greater than the time available before copy is due to the printer. There should be enough lead time before the next issue goes to press to allow the new style to be accommodated in a leisurely fashion.
In the meantime, please contemplate your reactions to the new design (and the old one too), and let us know what they are. (Cal Jackson has already sent in his comments; see page 98.) At the summer meeting, David Kellerman will give a session describing what was involved in implementing the new design. I hope this will be a catalyst for starting a lively discussion.
Introducing Donald Knuth
and Computers & Typesetting
Peter Gordon
Addison-Wesley Publishing Co.
at the Computer Museum
Boston, Massachusetts
May 21, 1986
I am especially delighted to be celebrating here, at the Computer Museum, both the TeX system for computer typesetting and the completed work of its author, Don Knuth. It seems fitting that TeX, which is leading a computer-based revolution in the way books and documents of all sorts are produced, should be presented in a thoughtful, historical context. It seems equally appropriate that Don Knuth should himself be introduced in this setting, given his well-known understanding of
the history of computing and his own important contributions to that history.
In promoting TeX, with the publication of both software and books related to it, and in our use of the TeX system in the production of our own books, we at Addison-Wesley hope to make some contribution to the field as well—a contribution to the future of publishing and communications, and, with that, to the future of science, technology, and education.
Before I go further, I should explain what TeX is, and why people are so excited about it. Simply put, TeX is a computer program. It is a sophisticated piece of software developed by Don at Stanford University, through years of design, testing, and refinement, originally to solve problems he perceived in technical book production, and ultimately to show how the best computer science theory can effectively be translated into practice.
TeX is a tool for typesetting written works with the aid of a computer—articles, reports, proposals, books, you name it—a tool with which writers themselves can specify, through a rich language of commands, exactly what each page will look like when finally printed. Another program developed by Don as a companion to TeX, called METAFONT, even allows users to design the type that will appear on these pages.
The TeX system is particularly useful where the document being written contains mathematical expressions, or where book-like quality in appearance is desirable. These two features—math and beauty, if you will—would alone distinguish TeX from other available computer typesetting systems, and lead to comparison, instead, with more expensive typesetting systems used by professional compositors.
But there is more.
TeX for example, is a portable system, running on a wide range of computers, from micros to mainframes, each implementation fully compatible with the others. This is especially important in mixed computing environments, where a variety of machines is in use—which is almost everywhere in the scientific and technical community.
Related to this portability is TeX's printing device independence. Printed output can be obtained from the same TeX-processed file on everything from a CRT screen or dot matrix printer to a laser printer or even a phototypesetter. A writer, in other words, can proofread his or her text in screen display or local printer output before sending the very same file from which it was produced to a phototypesetter.
I won't go into all the features here. You should really see TeX demonstrated, and ask questions until you learn as much about it as you want.
I do want to point out, though, that TeX is rapidly becoming a standard text processing system in many academic departments and research laboratories throughout the world. It is also gaining increasing recognition for its potential in corporate and industrial, in-house publishing, as well as our own more traditional publishing environments. Addison-Wesley editors, for example, are working with more and more manuscripts prepared with TeX. Among my own authors, Fred Brooks, of Mythical Man-Month fame, and Carver Mead, co-author (with Lynn Conway) of the seminal Introduction to VLSI Systems, have both converted to TeX.
As early as seven years ago, Gordon Bell perceived the importance of TeX. He wrote then that "Don Knuth's [TeX] is potentially the most significant invention in typesetting in this century. It introduces a standard language for computer typography and in terms of importance could rank near the introduction of the Gutenberg press."
Addison-Wesley first became interested in TeX as an extension of our book publishing relationship with Don dating back many years. As a publisher of many scientific and technical books, however, we soon recognized ourselves the significance of his system for our own business. TeX offered the opportunity to produce such books more quickly and more cheaply than ever before possible, and to provide our authors with increased convenience and facility in developing their works.
We are now deeply involved with TeX in two distinct ways.
First, we are the publishers of TeX. We publish a variety of books related to TeX, including Don's own Computers & Typesetting series, about which I'll say more in a minute, and books by other authors. One such book I should mention is LATEX: A Document Preparation System, by Leslie Lamport, which describes the "front-end" system he built for TeX, and which he, like Don, placed in the public domain.
We also publish TeX software, as well as other software products related to it. MicroTeX, for example, is a complete implementation of TeX for microcomputers developed by David Fuchs at Stanford. MicroTeX currently runs on the IBM PC family of machines, plus compatibles.
The second way Addison-Wesley is involved with TeX is in the production of our books. Our aim is to learn this new technology as deeply as possible through first-hand experience with it, and thereby
to maintain our position among publishers at the forefront of modern book production technologies.
The publication of Don Knuth's five-volume *Computers & Typesetting* series represents the culmination of his work on TeX and METAFONT. And this, specifically, is what we are celebrating today. You can see the books spread around the room. They were all, of course, typeset, by the author, with TeX.
Volume A is the definitive user's guide and complete reference manual for TeX. This book first appeared in softcover form, and many thousands of copies have already been sold around the world.
Volume B contains the complete source code listings for TeX, and incidentally provides an excellent example of how to write and document a very large program.
Volume C is the user's guide and reference manual for METAFONT, the companion to TeX for font design.
Volume D contains the complete source code listings for METAFONT.
Volume E might become the first coffee-table book in computer typesetting. It graphically depicts over 500 examples of METAFONT programming, the programs that generated all the letters used in the five volumes.
I would like now to introduce Don Knuth. In computer science circles, there would be no need to say anything more. His contribution to the field includes the classic series of books on the *Art of Computer Programming*, about which one reviewer has said, it is as important a work for computer science as Euclid's was for geometry. Don is the recipient of the prestigious Turing Award and National Medal of Science. He is Fletcher Jones Professor of Computer Science at Stanford University.
Don is on sabbatical this year working on a book in theology. If he has been compared to Euclid for his work on the *Art of Computer Programming*, and to Gutenberg for his work on TeX, we can only wonder what the next comparison will be.
With Don today are his wife, Jill Knuth, herself the author of a recently-published book, *Banners without Words*, and daughter Jenny, a student at Brown University. Son John, a student at Stanford, is back home in California minding the computers.
Now, needing no further introduction, here is Don Knuth.
---
Remarks to Celebrate the Publication of *Computers & Typesetting*
Donald Knuth
at the Computer Museum
Boston, Massachusetts
May 21, 1986
The title of the books we're celebrating today is *Computers & Typesetting*, and since we're meeting here in the Computer Museum I think it's appropriate to point out that computers have been intimately associated with typesetting ever since the very beginning. Anybody who reads about the history of computers will soon learn that many of the key ideas go back to 19th century England, where Charles Babbage designed a so-called Difference Engine and went on from there to plan his Analytical Engine.
Babbage's own machines were never completed, but a Swedish author and publisher named Georg Scheutz read about them and was so fascinated that he and his son Edvard actually built a working difference engine. Thus it was that the first sophisticated computing device came to be built in Sweden. And the most interesting thing, to me at least, was that the output of the Scheutz machine was not punched cards or anything like that; their machine actually produced lead stereotype plates from which books could be printed! Several books were, indeed, printed from the output of this early machine. It was demonstrated in 1856 at the Universal Exposition in Paris, and the souvenir album of that exposition contains the following glowing tribute: "This nearly intelligent machine not only effects in seconds calculations that would demand an hour; it prints the results that it obtains, adding the merit of neat calligraphy to the merit of calculation without possible error."¹ I have copied a page from the first computer-produced book—printed in 1857—so that you can see how far we've come since then. As far as I know, this page is the first extant output of an automatic calculator.²
---
¹ Leon Brisse, *Album de l'exposition universelle*, Paris, 1857, p. 194. [Cited in Uta C. Merzbach, *Georg Scheutz and the first printing calculator* (Washington: Smithsonian Institution Press, 1977).]
² (Editor's note.) The page, which was displayed among images of METAFONT letterforms, contains columns of figures, neatly aligned and separated by rules, and clearly displays the traces of ink that indicate the edges of pieces of metal type impressed on moistened paper.
I'd also like to say a few words about the history of my own work on computers and typesetting. Last week I went back to my diary of 1977 and found an entry from Thursday, May 5, where it says 'Design of TeX started'. My diary says that I worked intensely on the design all day Thursday, Friday and Saturday; then I went to see Airport 77 and Earthquake to relax! The entry for the following Thursday says: 'Wrote draft report on TeX, stayed up till 5 a.m. typing it into machine'. That weekend I went with my wife on a tour of the Sacramento area with Stanford's Library Associates. We saw many examples of fine printing during that trip, and this encouraged me to read a lot of books about font design during the following week. My diary entry for Saturday, May 21, 1977—exactly nine years ago today—says that by 5 a.m. that day I had made 'rough drafts of lower case and upper case Roman and italics and digits 0-9'. After a few hours of sleep, I spent the rest of that Saturday writing computer programs to plot curves on a raster. Oh, how little I knew in those days about how difficult it would be to complete this work, which I had sketched out in about two weeks!
Why did I start working on TeX in 1977? The whole thing actually began long before, in connection with my books on The Art of Computer Programming. I had prepared a second edition of volume 2, but when I received galley proofs they looked awful—because printing technology had changed drastically since the first edition had been published. The books were now done with phototypesetting, instead of hot lead Monotype machines; and (alas!) they were being done with the help of computers instead of by hand. The result was poor spacing, especially in the math, and the fonts of type were terrible by comparison with the originals. I was quite discouraged by this, and didn't know what to do. Addison-Wesley offered to reset everything by the old Monotype method, but I knew that the old way was dying out fast; surely by the time I had finished Volume 4 the same problem would arise again, and I didn't want to write a book that would come out looking like the recent galleys I had seen.
Then a nice thing happened. I was on a committee to revise Stanford's reading list for our department's comprehensive exam, and one of the things we had to do was evaluate a book that Pat Winston had just written about Artificial Intelligence. We received galley proofs of that book, and the story we were told was that these galleys had been made on a new machine in Southern California, all based on a discrete high-resolution raster. Apparently one of Winston's students at MIT had flown to Los Angeles with that book on magnetic tape, and the galley proofs we saw were the result. Well, I had had lots of experience with rasterized printing, but only at low resolution, so I thought of it as simply an amusing toy. When I saw these galleys of Winston's book, I was astounded, because the resolution was so good I couldn't tell that the type was actually digital. In fact the digital type looked a lot better than what I had been getting in my own galley proofs.
Digital typesetting means patterns of 0s and 1s, and computer science can be thought of as the study of patterns of 0s and 1s. Therefore, it dawned on me for the first time that I, as a computer scientist, would be able to help solve the printing problem that was worrying me so much. I didn't need to know about metallurgy or optics or chemistry or anything scary like that; all I had to do was construct the right pattern of 0s and 1s and send it to a high-resolution digital typesetter like that machine in Southern California; then I'd have my books the way I wanted them. In other words, the problem of quality printing had been reduced to a problem about 0s and 1s. Therefore it was almost an obligation for a computer scientist like myself to study the problem carefully.
Within a week after seeing the galleys of Winston's book, I decided to drop everything else and to work on digital typography. Although Winston unfortunately couldn't be present here today—Pat, I can't thank you enough for having written that book!
Ever since these beginnings in 1977, the TeX research project that I embarked on was driven by two major goals. The first goal was quality: we wanted to produce documents that were not just nice, but actually the best. Once upon a time, computers could deal only with numbers; then several years passed when they had numbers and uppercase letters; then they became able to deal with both uppercase and lowercase; then they became capable of working with letters of variable width; and by 1977 there were several systems that could produce very attractive documents. My goal was to take the last step and go all the way, to the finest quality that had ever been achieved in printed documents.
It turned out that it was not hard to achieve this level of quality with respect to the formatting of text, after about two years of work. For example, we did experiments with TIME magazine to prove that TIME would look much better if it had been done with TeX. But it turned out that the design
of typefaces was much more difficult than I had anticipated; seven years went by before I was able to generate letterforms that I began to like.
The second major design goal was to be archival: to create systems that would be independent of changes in printing technology as much as possible. When the next generations of printing devices came along, I wanted to be able to retain the same quality already achieved, instead of having to solve all the problems anew. I wanted to design something that would still be usable in 100 years. In other words, my goal was to arrange things so that, if book specifications are saved now, our descendants should be able to produce an equivalent book in the year 2086. Although I expect that there will be a continual development of "front ends" to TeX and Metafont, as well as a continual development of "back ends" or device drivers that operate on the output of the systems, I designed TeX and Metafont themselves so that they will not have to change at all: They should be able to serve as useful fixed points in the middle, solid enough to build on and to rely on.
Today I'd like to brag a little, and say that I think that these goals of top quality and technology independence seem to be achieved; and volumes A, B, C, D, E tell everything about how it was done. Today I'm seeing these books for the first time, and I'm happy that all of you can be here to help me celebrate this event. These books are somewhat unusual because they describe themselves: They describe exactly how they were typeset. All of the formatting was done by the TeX system described in volumes A and B. Also every letter and every symbol that appears in all five volumes, as well as on the covers and book jackets, was done by the Metafont system described in volumes C and D. Volume E tells how I dotted all the i's and crossed all the t's, literally. If copies of these books were sent to Mars, the Martians would be able to use them to recreate the patterns of 0s and 1s that were used in the typesetting. Essentially everything I learned during the past nine years is in here.
All of the methods described in these books are in the public domain; thus anybody can freely use any of the ideas. The only thing I'm retaining control of is the names, TeX and Metafont: products that go by this name are are obliged to conform to the standard. If any changes are made, I won't complain, as long as the changed systems are not called TeX or Metafont.
Volumes A and C are user manuals; I tried to write manuals that would suit users at all levels as they grow with the systems. And I also strove for a high standard of excellence in the choice of the quotations from other works that are included at the end of every chapter.
Volumes B and D contain the complete program listings of TeX and Metafont. These books are specifically for computer scientists, not for casual users, but I'm especially pleased with how they came out because they represent an unexpected payoff of my research. This is something that I had no idea would be possible when I began nine years ago. As I wrote the programs for TeX and Metafont, I wanted to produce systems that would represent the state of the art in computer programming, and this led to the so-called WEB system of structured documentation. I think that WEB might turn out to be the most important thing about all this research—more important in the long run than TeX and Metafont themselves—because WEB represents a new way to write software that I think is really better than any other way. Using WEB, it was possible to write programs that are so readable that I think there already are more people who understand the inner workings of TeX than now understand any other system that is as large. Furthermore I think it's fair to claim that WEB has made TeX and Metafont as portable, as maintainable, and as reliable as any other pieces of software in existence. The programs are now running and producing essentially identical results on almost all large computers; there are thousands of users, yet no bugs have been reported for more than half a year. I think there is at most one more bug in TeX, and I'm willing to pay $20.48 to the first person who finds it. (Next year the reward will double, to $40.96, etc.)\footnote{3}
Volumes B and D also contain another innovation that improves on the basic WEB system previously available: Every pair of facing pages has a mini-index on the right-hand page, for quick cross reference to anything that's referred to on either page; this saves a lot of time thumbing through the master index at the end.
In recent years I've been making a pitch for programs as works of literature. Although there still is no Pulitzer Prize for the best-written computer programs of the year, I tried to write volumes B
\footnote{3}{(Editor's note.) A listener asked, how much had it cost to pay off the finders of bugs in the programs and errors in the books? Depending on how many checks were actually cashed, Don estimated the total to be between $2,000 and $5,000. It is doubtful that the checkbook in question is easily balanced.}
and D in such a way that I would be a candidate for such a prize if it were actually given! More seriously, I intended these books to be useful to computer scientists for self study as well as for study in college seminars. Volume D, in particular, should make a good text for a group of advanced students.
The fifth volume, volume E, is the most fun of all. I hope you will all open a copy and riffle through the pages, so that you can see what I mean. METAFONT is a computer language that is not very much like any other, so my goal in this book was to provide lots of examples of how METAFONT can be used to produce fonts of reasonably good quality. Over 500 examples appear here; they cover every letter, digit, punctuation mark, and other symbol that was used in printing these books.
The fonts you get from these programs have the general name ‘Computer Modern’. My colleague Charles Bigelow has contributed an introduction that talks about Modern fonts in general. The book explains how you can make your own personal variations of the fonts, which are designed with many parameters so that they can be generated in almost limitless variety. At the end of the book there are sample pages that show specimens of 75 standard Computer Modern typefaces; and thousands of additional varieties could be generated with ease.
Even if you don’t read the METAFONT programs in this book, I think it’s appealing just to look at the pictures of these constructed alphabets,\(^4\) and to ‘know’ that the program on the page facing each letter was what ‘drew’ that letter; it’s all there. Somehow this gives a satisfying sense of completeness and order.
The most important thing I want to talk about this morning is HELP. I had lots of help — literally hundreds of people who volunteered to assist this project in significant ways — beginning with Hans Wolf of Addison-Wesley, who taught me the details of the Monotype systems that had been used to typeset *The Art of Computer Programming* in the 60s. I was especially fortunate in my work on font design to have had extensive help from world leaders like Hermann Zapf and Matthew Carter.
Another stroke of luck was to have outstanding research associates like David Fuchs and John Hobby. Furthermore my research project at Stanford had generous financial support, most notably from the National Science Foundation and the System Development Foundation. With so much help, it would have been very hard for my research to fail. And my wife Jill gave the most help of all. (Next month we will celebrate 25 years of marriage!)
One final note: People often ask me why TeX and METAFONT are symbolized in these books by a lion and a lioness. When Duane Bibby first came up with the lion idea, I instinctively felt that it was right, but I never understood exactly why this was, until about a month ago when I was in the Boston Public Library. I passed by the magnificent stone lions on the library’s grand staircase, and I thought: “That’s it! TeX and METAFONT try to be like these lions, fixtures that support a great library.”\(^5\) I love books, and lions represent books!” No wonder I’m so happy when I realize that TeX and METAFONT have already contributed to the making of several dozen books of fine quality; it makes me extremely pleased to think that this research will probably contribute to the making of many more fine books in years to come.
---
\(^4\) (Editor’s note.) The pictures, it was pointed out, were generated separately from the text of the examples, and pasted in. If both the raster images and the text had been incorporated at the same time, it would have exceeded the capacity of the machines used to produce the book.
\(^5\) (Editor’s note.) One is also reminded of the lions that grandly guard the entrance to the New York Public Library, which celebrated its 75\(^{th}\) anniversary during this same week.
of themselves. Their effort, like Knuth's, is one of people that learn from others rather than invent without regard to prior work. It's a fine piece of work and I hope they can find the time to share the nitty-gritty of the experience with TUG members.
I suggest that all TUG members can learn from this experience. How? Have the issue critiqued by several professional designers, typographers, compositors. Publish their critiques. That modality appears to be the predominant one for training people in the graphic arts. Work is not considered right or wrong, or a consensus sought; it is the exposure to critical review that develops the worker.
I hope that there will be other guest editors. I once suggested to Pierre MacKay that there be a competition among \TeX users where a work would be judged by professionals. My objective—provide input to users that would improve their visual literacy of typographic material. I cannot think of a better alternative to that than guest editors and subsequent critique by professionals.
Editor's note: Professional criticism has always been welcome, as have well-thought-out comments and suggestions for improvement in the appearance and utility of TUGboat. There have been presentations at several TUG meetings dealing with design issues, and there is a session scheduled for the Tufts meeting on the creation and implementation of the format used for TUGboat 7, No. 1. If any readers know any designers, typographers or compositors who might be interested in critiquing \TeX documents for publication in TUGboat, please forward their names and other relevant information to the Editor.
Software
VAX Language Sensitive Editor Templates and Guide for Use with \LaTeX
Kathy Hornbach
Lear Siegler/Instrument Division
A Quick Reference Guide and VAX/VMS Language Sensitive Editor (LSEDIT) templates have been made available for distribution by TUG. The package includes both the printed Guide and the software, which consists of an LSEDIT language definition for \LaTeX and several new styles, described below. [The software will be provided on magnetic media; for details, see the current TUG publications list.]
Using LSEDIT and the \LaTeX language definition, a user, regardless of his/her level of experience, can quickly and easily learn to format complex documents using \LaTeX. Use of LSEDIT reduces the amount of typing necessary by automatically supplying the user with a set of templates that define the basic structure of a given \LaTeX style. These templates can be selected and filled in or deleted as appropriate. The novice user will use the templates extensively, while the more experienced user will use the templates as an aid in remembering infrequently used commands or formats.
The default \LaTeX styles supported by the LSEDIT language definition are: article, report, letter and slides (SLiTeX). Also included are three new styles for \LaTeX: memo, MIL-STD-490 documents, and book form documents. These new styles are supported by the LSEDIT language definition.
VAX/VMS format HELP library entries are included for most of the features in version 2.09 of \LaTeX and SLiTeX.
Use of this package requires that LSEDIT be installed.
It would be easy enough for me to natter on about the wonders of the new and improved version of METAFONT: The code for the font you are now reading was only a gleam in my eye in mid-December of 1985; The code for the masthead on this column took less than eight hours for me to write; and so on and so on. I will not belabor the point that METAFONT's way of precisely describing how a character is drawn (rather than simply drawing it) allows the designer literally to go from alpha to omega:
At present I wish to focus on the product, not the process, of METAFONT. Most users of TeX believe (rightly or wrongly) that the process of METAFONT is something wholly beyond their requirements; its product, on the contrary, is fundamental to the effective use of TeX.
Let me digress briefly to enumerate some of the details that must be included in a good font. The first consideration that comes to mind is the wealth of detail in the images of the characters that make up the font. Curves must be smooth and pleasing to the eye. The ratio of descender to mean-height must 'look right' for the character's representation at a given point size, and must be harmonious from one point size to another; in many of the classic typefaces, this visual harmony is much too complex to be adequately captured by simple scaling from a single template.
On the left, we have letters from a 48 point Roman font and the 12 point version produced by simply scaling down from it; the ratios of ascender and descender to point size are the same for both sizes. On the right, we have the same 48 point, but a 12 point version that has, in essence, been completely redrawn by METAFONT to preserve the slight but significant change in ascenders and descenders which typically occurs in traditional typefaces to enhance the legibility of text faces. Completely redrawn, but done so from the same code; thus, METAFONT minimizes effort while maximizing effectiveness.
Widths and heights of component strokes and of the images themselves show subtle changes of their own. METAFONT, with its potential for attention to such necessary niceties, handles all these imaging considerations with far more finesse than is possible with any other digital design system.
All of these imaging adjustments are well and good; but as the reader knows, digital image information is not used by TeX at all. The details that TeX requires are all contained in separate files, the .fm files. For example, to set text, we need to have an idea of the amount of space between words; ideally, such space will grow or shrink within acceptable limits depending on the exigencies of the text being set; an error here can make a font the ultimate inconvenience rather than the ultimate in convenience.
It is imperative that the information contained herein be as precise as possible in order to make a good font, a font capable of rising to the highest level of digital typesetting quality, that is, a font suitable for TeX. If the amount of space at the side of each letter's image is ill-advised, typesetting quality deteriorates and legibility is sacrificed. (Notice how dreadful the seven words preceding this parenthetical remark look.)
Moreover, the ability to let these spaces shrink or grow ever so slightly according to the letter's environment, called kerning, as in
Toffee not Toffee
or to substitute a slightly different image for a character pair or triple, called ligatures, as in
Toffee for Toffee
is a hallmark of the best typesetting. METAFONT has the ability to handle both kerns and ligatures and thus can produce fonts that rise above the level of the merely adequate.
In some cases, such information can be derived 'after the fact' (from existing images) and appended to a digital font; but it is not difficult to imagine that such a shotgun marriage of image information and .tfm information will tend not to result in a happy and harmonious union of the two. Quite simply, the simultaneous creation of image and .tfm information as done with METAFONT produces the best results.
More critically, there are some other subtler bits of information in font metric files that are much more nearly impossible to imagine creating with any tool other than METAFONT. Those are the various tidbits in the math/science and symbols fonts that are crucial to the fine setting of equations and formulae that is one of \TeX's strengths. These fonts must have a wealth of information that controls positioning and even the composition of certain characters (built up curly brackets, square brackets, integrals, radicals).
The modern typographer must now understand that his art has become an interdisciplinary pursuit and involves mathematics and programming skills as well as the traditional design concerns. While most current typographers will fail to adjust to this radically different method of type design, there will be many newcomers who will use METAFONT to contribute the beautiful digital typefaces that \TeX needs for unprecedentedly superb typesetting.
Powell, Ohio
24 May 1986
Every character in this column was created using METAFONT version 0.81. Fonts used include a prototype sans serif in book and slant styles, a proto–prototype Century Schoolbook text style, and a chiseled-look headline font.
The original of this document was printed on a Canon LBP-CX with a resolution of 300 dpi.
The \TeX Logo in Various Fonts
Donald E. Knuth
According to the plain \TeX macro package described in *The \TeXbook*,
\[
\def\TeX{T\kern-.1667em\lower.5ex
\hbox{E}\kern-.125emX}
\]
is the "official" definition of \TeX's logo. But the plain \TeX macros are specifically oriented to the Computer Modern fonts. Other typefaces call for variations in the backspacing, in order to preserve the logo's general flavor.
The definition above seems to work satisfactorily with the main seriffed fonts of Computer Modern (i.e., with all sizes of cmr and cmsl and cmti and cmbx); but sans-serif types are a different story. Indeed, *The \TeXbook* itself gives alternative definitions of '\TeX' on pages 418 and 419, one for the font cmssdc10 at 40pt used in chapter titles (cf. page 36) and one for the cmssq fonts used in quotations at the ends of chapters (cf. page 337).
My purpose in this note is to record the various versions of '\TeX' that were actually used to typeset the books in the *Computers & Typesetting* series, so that it will be easy to make forgeries of the particular style used there.
In every case the 'E' has been lowered by .5ex (half of the x-height); the only variation is in the amount of backspacing represented by the two \kern instructions. Let us therefore consider a "generic" \TeX logo to be defined by
\[
\def\TeX{T\kern\alpha em\lower.5ex
\hbox{E}\kern\beta emX}
\]
for some $\alpha$ and $\beta$. The following values of $(\alpha, \beta)$ were actually used in the published volumes:
| font family | $\alpha$ | $\beta$ |
|-------------|----------|---------|
| cmr | -.1667 | -.125 |
| cmsl | -.1667 | -.125 |
| cmti | -.1667 | -.125 |
| cmbx | -.1667 | -.125 |
| cmssdc | -.2 | -.06 |
| cmssq | -.2 | 0 |
| cmssqi | -.2 | 0 |
| cmss | -.15 | 0 |
| cmssi | -.2 | 0 |
| cmssbx | -.1 | 0 |
(The last three were used only to typeset the jacket copy, not the "real" texts inside. It took a bit of fiddling to get the spacing right.)
I've had little experience with other fonts, but they seem to respond to a similar treatment. For example, my paper on "Literate Programming" in *The Computer Journal* 27 (1984), 97–111, was typeset in a variant of Times Roman, and the standard \TeX macro worked fine. The captions and references in that article were set in Univers; for that sans-serif font we used $(\alpha, \beta) = (-.2, 0)$ as in cmssq.
Meta-METAFONT:
An Exhibit at The Cooper Union, NYC
Alan Hoenig
The Herb Lubalin Study Center of Design and Typography of The Cooper Union (41 Cooper Square, New York, NY 10003) featured an exhibit on METAFONT from March 3 to April 4 of this year. I discovered this fact well after the April 4 termination date, so this is only a second-hand report. The curator of the Lubalin Center, Ms. Ellen Lupton, would like this to be a travelling show, and one reason for this report is to whet the appetites of any institutions who might wish to host this show.
The show concentrated on the humanistic nature of METAFONT, rather than upon its mathematical underpinnings. The exhibit consists of text drawn from a series of articles by Don Knuth, Douglas Hofstadter, and Geoffrey Sampson published in the journal Visible Language between winter 1982 and winter 1985, and is illustrated with work done by Neenie Billawala of Stanford and Georgia K.M. Tobin of The Metafoundry.
This exhibit will appear in an abbreviated form from July 12 through July 19 at the Stevens Institute of Technology in Hoboken, NJ, and will also be in Edinburgh, Scotland, later this summer; dates and exact location are unavailable. For further information, contact Ellen Lupton directly at the above address or by phoning her at (212) 254-6300, ext 211. She also solicits institutions who would be interested in guest-hosting the exhibit.
Editor’s note: The following quote, and the illustrations on this page, are from the “poster” describing the exhibition; the illustrations were created by Neennie Billawalla, Stanford University, and are typical of her experiments with METAFONT.
Metafont, as Knuth describes is, has a humanistic mission; its value is not so much in the variety it may offer consumers, but in the discipline it offers designers. The Lubalin Center is not so interested in the final ‘look’ of Metafont. We are interested in Metafont as an open, unfinished method for making and thinking about letters. The specific language METAFONT is under continuous revision; the concept of a meta-font provides a frame for questioning the history and spirit of letters.
Output Devices
Addenda to the Output Device Charts
The charts giving information on interfaces between computers and output devices are being omitted from this issue, since almost no changes have been received since their publication in the last issue of TUGboat. The following new information can be added to the existing charts.
MPAE Max-Planck-Institut für Aeronomie
HP 9000 Series 500 with HP LaserJet Plus
Helmut Kopka, [49] 5556-41451
Bitnet: M1040L@DGGWDO1
TA&M3 Texas A&M
Commodore Amiga with previewer,
QMS Kiss laser printer
Norman Naugle, 409-845-3104
Terminal serifs with varying heel softness and terminal angle.
Serifs with varying mid-bracket pull and stem slant
Site Reports
TeX on the Amiga
Tomas Rokicki
I am pleased to announce that TeX works on the Commodore Amiga. It first produced a document on March 29, and passed trip shortly thereafter. On a 512K machine, TeX runs with a memmax of 22000; on a 1024K or larger machine, a full sized TeX capable of running LaTeX or any other macro package runs. A hard disk is certainly nice, but not at all necessary; I've been running TeX on a two drive system with no disk swapping at all. A screen previewer is in the works (it works, the user interface is being improved) and should be available with TeX by the end of June. The screen previewer will be full-featured with dynamically changing magnifications (and zoom capability), access to random pages, and page motion through scroll bars. Various drivers are coming into operation; currently we have a driver for the QMS Kiss laser printer. Because the Amiga is a multitasking machine, you can run your editor, previewer, TeX, and printer driver all concurrently, yielding an impressive TeX environment.
The virtex executable comes in at an incredibly small 154,100 bytes; this is smaller than on any other machine I have seen. The final version will be even smaller due to certain optimizations still in progress. It is currently about as fast as a six megahertz PC AT, requiring less than eight seconds per page for WEB documents and between four and six seconds per page for normal text. The optimizations we are performing should make it even faster.
TeX, the screen previewer, and various printer drivers are also being ported to the Atari 1040ST. These should be ready shortly. We are also planning a port to the Macintosh. For details on any of these products, contact Norman Naugle at (409) 845-3104 or Tomas Rokicki at (415) 723-1646.
CDC Cyber Site Report
Jim Fox
University of Washington
My last site report indicated that I did not have fonts to distribute. The problem has been corrected. I now have a set of AM style fonts, in PXL format, and some utilities to work with them. I expect to investigate the PK format and the new CM fonts in the near future.
Prime 50 Series Site report
John Crawford
Things have been going well on the Prime front. They should be even better, as this issue hits the newstands, as my Primos TeX distribution tape will reflect the latest from Stanford. TeX 2.0, the new Computer Modern fonts, and METAFONT 1.0 should all be available.
A technical point of information for Primos users is in order. With Primos revision 19.4.7 and above, my TeX 1.3 should be invoked with the global variable ".tex_typeahead" set to TRUE. Otherwise, you will likely lose screen output during error displays. I had based my code on the results of a faulty, and later fixed, Primos subroutine. Get the latest port, TeX 2.0, and upgrade out of this workaround.
Typesetting on Personal Computers
The Sperry IT: An IBM AT Compatible
M. Pfeffer and A. Hoenig
The high speed of the Sperry IT makes TeXing and previewing comfortable—far more comfortable than on a PC-class machine. A complete system, using the Sperry system unit (with its fast 40 megabyte hard disk, and 1 Mb of RAM), in conjunction with non-Sperry keyboard, monitor, and Hercules-compatible video card, costs about $3,400.
The System unit: Because TeX is a computation-intensive program, the processing speed of your system is important. Processing speed is determined by two factors: the clock frequency, and the number of wait states required by the system.
The higher the frequency of the system clock, the faster the system. The clock in the original IBM AT ran at 6 MHz; the version released in April 1986 runs at 8 MHz. The Sperry's clock can be switched to 6, 7.16, or 8 MHz.
The second consideration is the number of wait states used: in some machines, the computer's memory can't cough up the information requested by the processor fast enough, forcing the processor to wait. To indicate the delay to the processor, the system introduces one or more wait states. Each wait state degrades the system's performance by 25%.
Both versions of the IBM AT use one wait state. The Sperry uses one wait state in the 6 and 8 MHz settings, but runs with no wait states at the 7.16 MHz speed. This means that the 7.16 MHz speed is the fastest of the Sperry's three speeds, and at this setting, it will out-perform the 8 MHz IBM AT.
For comparison, a 6 MHz AT is two to three times faster than a 4.77 MHz PC; an 8 MHz AT is 33% faster than a 6 MHz AT, while the Sperry, at 7.16 MHz, is 45% faster than a 6 MHz AT.
When using the Textset Preview program, the bottle-neck is disk access. Fortunately, the Sperry is equipped with a fast hard disk (30 ms access time). With this disk, the performance of the preview program becomes acceptable—on slower systems, I find that the performance of the preview program (version 2.1) is uncomfortably slow.
The system unit also includes: two serial ports, one printer port, a 1.2 Mb floppy-disk drive, MS-DOS 3.1, GW-Basic, diagnostic diskette, six available slots (after installing the video card): one 8-bit only slot, and five 8- or 16-bit slots, reset switch, one-year warranty, and manuals (including a setup guide). Technical reference manuals are available.
Important: Sperry currently ships the IT with either a 44 Mb drive made by Miniscribe, or a 40 Mb drive made by CDC. Because I've heard of reliability problems with the Miniscribe 44 Mb drive, specify that the drive in your system not be from Miniscribe. Also specify that the serial number be greater than 414001, ensuring that you receive the latest version of the system: unlike the older version, you can now defeat the serial ports and printer port (necessary if you can't defeat the ports that may be present on other cards you install in the system). The BIOS in the current systems is version 1.48, and corrects problems relating to access of the floppy-disk drives (you can ascertain the BIOS version by running the romver program). Also in the current system, the optional math co-processor can run at 8 MHz. (The serial number and drive manufacturer are marked on the outside of the box. A leading digit of '4' in the serial number indicates that the hard drive was installed by Sperry.)
Configuring the System: A limitation in DOS is its inability to access more than 32 Mb on a hard disk. To circumvent this, Sperry allows you to treat the 40 Mb drive as if it were two (or more) drives, each with a separate drive letter. These pseudo-drives will total 40 Mb, and you can specify a size of up to 32 Mb for a pseudo-drive.
The 1 Mb of RAM on the motherboard can be configured in two ways: it can be split into two 512 K segments, with one segment for running programs under MS-DOS, the other segment for use as a ram-disk; or, 640 K can be used for program memory, but then the remaining memory becomes unavailable, and is wasted.
The 512 K/512 K split is handy for users of Personal TeX's Cordata (formally Corona) laser printer driver, as the driver requires a 512 K ram-disk. Squeezing TeX into the other 512 K segment may require some care: you may need to forgo use of RAM-resident software, reduce the number of buffers specified in the config.sys file (I normally use 64 buffers on the Sperry), and eliminate device drivers from the config.sys file.
©1986 M. Pfeffer.
Date submitted: May 21, 1986.
Eliminating device drivers has its drawbacks: a device driver is needed by the Sperry to access the part of the hard disk beyond the 32K DOS limit; also, the ansi.sys driver is needed by the Textset Preview program—without it, the screen goes blank when you exit the program, until you type the cls command.
To avoid these difficulties, you'll want to increase the system's memory—this requires the installation of a RAM card. AMI manufactures RAM cards that do work properly in the Sperry, and cost about $300—be sure to request the PAL chip for using the card in a Sperry. These cards do not insert wait states. When ordering RAM to populate the card, specify an access time of 150 ns. (The Mitsuba RAM card, designed for the IBM AT, was rejected by the Sperry's power-on diagnostics. I've also heard that the AST RAM card will not work.)
Compatibility: No problems with TeX-related software or editors. Many games will not run. Jet, a flight simulator, works in Hercules mode. Symphony requires a math co-processor. The current Hayes internal modems will work, but the older versions will not.
Support: The Sperry support center, 800-328-1015, is staffed by knowledgeable, helpful representatives.
My dealings with one service center showed that location's staff to be eager to correct a problem in one unit; unfortunately, they released the unit before resolving the problem.
Sperry's corporate personnel proved to be conscientious and responsive.
Peripherals
The following recommendations on peripherals apply to other AT- and PC-class computers, including the ACS computer I discussed in volume 6, number 3.
Display: The most comfortable display I've used to date is the Panasonic TR-122MYP. It uses a long-persistence, lime-colored (QD) phosphor, which I find more restful than the IBM green display, or any of the amber displays I've used. (The current versions of the Taxan 122 display discussed in vol. 6, no. 3 no longer seems to have the long-persistence I originally admired.)
Display Adapter: Low-cost Hercules-compatible video cards use slow chips, and may have trouble working in fast machines. However, I did test one such card, made by Fortron, which did work in the Sperry. On special request, you can purchase a version of this card with a defeatable printer port—a necessity if you have a card in your system with a printer port that can't be disabled.
(In my system, I have a video card with a custom PAL chip that produces a non-blinking, inverse-video cursor. I find blinking cursors distracting—they make me feel that the "meter's running" as I plod along. I've seen ads for a program that claims to produce a non-blinking cursor.)
Keyboard: The good news is I've found a keyboard with an excellent, solid feel, in the AT layout, with a separate cursor pad as an option. (This keyboard can also be used on an IBM PC or compatibles.) Most keyboards (including those made by Key Tronic, and those sold by Sperry and Compaq) have a mushy feel—as though you're typing on a sponge (this isn't far from the truth: the capacitive keys used in most keyboards have a foam pad beneath each key). IBM's clacky keyboard is the opposite extreme: it feels as if you're breaking through egg shells as you type. One die-hard fan of IBM's keyboard expressed his preference for this new keyboard after a short period of use.
The bad news is that this keyboard isn't available through normal mail-order channels. But you can seek out other brands with the same touch: the keys use a hard-contact switching technology.
While the legends on some cheap keyboards consist of surface markings, which will rub off with use, the keycaps on this keyboard are made by two-shot molding: this means that the plastic that makes up the legend goes through the entire thickness of the top of the keycap. You can spot this type of keycap by gently removing a keycap and looking at its underside; if you can see an inner layer of plastic of the same color as the legend, then the keycap was made by two-shot molding.
Anti-Glare Screen: A new model of the SuperScreen (vol. 6, no. 3) incorporating a grounding mesh is now available. One potential advantage is that by discharging the static build-up, less dust should be drawn to the screen—dust that would gradually degrade the sharpness of the image, unless the mesh is frequently cleaned.
Surge Suppressors: See the results of comparative testing in PC Magazine, vol. 5, no. 10, pages 107–146, May 27, 1986.
Diskettes: Elek-Tek, 800-621-1269, sells a box of ten 1.2 Mb Dysan diskettes (a top-rated brand) for $33.
Table Making with INRSTeX
Michael J. Ferguson
INRS-Télécommunications
Montréal
The table commands described here were developed as an integral part of INRSTeX. They have also been implemented as a standalone macro package, TABLES.TeX, to run with the standard Plain macro package that comes with TeX. This paper was produced using TABLES.TeX with the standard TUGboat macros (TUGBOT.STY).
The major intent of the table commands is to make the input for tables appear on the editor screen in a reasonable manner. Although this package appears to avoid the basic \align commands and forms of \cr, &, and #, their insertion in tables is not precluded by the structure of the table commands. To use these basic commands will result in some loss of readability but a gain in flexibility.
Below is shown a now classic table (also used in the The TeXbook) for demonstrating the table-making skills of a set of macros. The input which produced the table appears in Figure 1. This table is not identical to the one in the The TeXbook. It is, however, very close.
\centerline{
\begintable
\begintableformat
\center " \center " \center
\endtableformat
\br{} \sa{Dividend} "\sa{Dividend} "\sa{Dividend} \er{} %sample line
\-
\br{\:|} \use{3} AT\&T Common Stock \er{|}
\-
\br{\:|} Year | Price | Dividend \er{|}
\-
\br{\:|} 1971 | 41--54 | \$2.60 \er{|}
\-
\br{\:|} --2 | 41--54 | ~2.70 \er{|}
\-
\br{\:|} --3 | 46--55 | ~2.87 \er{|}
\-
\br{\:|} --4 | 40--53 | ~3.24 \er{|}
\-
\br{\:|} --5 | 45--52 | ~3.40 \er{|}
\-
\br{\:|} --6 | 51--59 | ~.95\rlap* \er{|}
\-
\br{\:}\use{3} \left{* (first quarter only)}
\endtable}
Figure 1. Input for table: AT&T Common Stock
nicer and more readable on the input screen. Other details about this table will be apparent later.
The INRSTeX table commands really use two different types of columns: *rule columns* for insertion (or omission) of vertical rules and *data columns* for the insertion of normal data entries. Generally it is assumed that every pair of data columns is separated by a rule column. In addition, a template for rule columns is provided automatically at the left and right hand sides. The table commands make the insertion or omission of a vertical rule or column as simple as placing a " or a | in the text.
The INRSTeX table commands have been designed to be most useful when the following conventions are obeyed:
- A rule column is specified in the table format using a ". Successive data columns are separated by a rule column. The actual rule is inserted in the text with a | or omitted with a " rather than being put in the format.
- A rule column is appended to each end of the table format statement. In general this means that there is a rule column on both sides of the table. The actual insertion or not of the rule is very easy.
- Struts for maintaining row height are inserted in each row rather than in the table format.
The commands make it very easy to include struts, and insert or omit rules as needed. In fact it is no more difficult than inserting an & in the appropriate place. The advantage of following this discipline is that insertion of partial horizontal lines (rules) is quite easy, as is the exact vertical placement of items in different data columns.
The basic format for building a table is
```
\begin{table}
<special table forms>
\begin{tableformat}
<table format>
\end{tableformat}
<first row>
...
<last row>
\end{table}
```
Each row has the form
```
\br{<..>} <item> | <item> " <item>
... | <item> \er{<..>}
```
where | means to put in a vertical rule and " means to leave it out of the corresponding rule column. \br{..} and \er{..} signify the beginning and end of the row respectively; the optional arguments are intended for rules and/or struts if needed. For instance, \br{\:|} indicates that this line has a standard strut \: and a standard vrule. \br{\:height 2pt} indicates that this row has no strut but that the initial vrule has a height of 2pt. The strut could just as easily have been put in the \er{\:} instead. The form \br{} or \br{"} indicates a row with no initial strut or vrule.
The table commands supply a second flavour of vrule that is user definable. This is specified by the command \l, which may be used in exactly the same way as |. The *insides* of the \l are changed by redefining a command \sprule, a special rule. The default definition is \def\sprule{\tvrule {2.5}\tr} where \tvrule{<dimen>} produces a special centered vrule with width <dimen>, and \tr is TeX default rule thickness, .4pt.
An INRSTeX table format is a template for the table, and corresponds to the normal preamble in an \halign. In fact any valid \halign preamble may be used in the table format, as long as the # are replaced by ##. However, since a rule column is added to the beginning and end of the format list for beginning and terminating rules, a repeating field specification, which is indicated by an (extra) & in the format, should **not** start at a vrule indicator or the result will be two adjacent rules.
Simple column formats are indicated by the use of the commands \left, \center, \right, \math, and \displaymath. The two \..math forms are **never** used alone but rather in conjunction with the first three. These forms are **always** separated† by ", which allows for, but does not put in, a vertical rule. The simplest table format, and probably the most useful, is
```
\begin{tableformat}
&\center
\end{tableformat}
```
This is a repeating format, as indicated by the &. and thus allows for any number of columns. The data in each column is centered.
Another simple table format is
```
\begin{tableformat}
\left " \right " \math\center
\end{tableformat}
```
This table would have three data columns: The first data column is ordinary text, left justified; the second is right justified text; and the last is centered mathematics. Each pair of data columns is separated by a rule column indicated by ".
---
† In fact, any field of the form
```
&<optional stuff>##<optional stuff>&
```
is acceptable.
columns will be added automatically to the left and right hand ends of the specification. The commands \right, \left, and \center insert the appropriate glues and spacing at both sides of the column. The width of this spacing \tcs, the table column separation, is a <dimen>. This may be modified if desired for all tables by putting it in an \everytable token list, or for a specific table by placing it in the special table forms.
The following is a repeating column specification:
\begin{tableformat}
\left " \right " &\math\center
\end{tableformat}
This is identical to the previous example except that the data column format and the implicit rule column added by the table commands, \math\center ", is repeated indefinitely. For almost all tables, the only & that should appear in the table format is the one indicating the start of a repeating field.
Horizontal rules are specified by \- for the standard \tr width rule. An \hrule the full width of the table should appear all by itself on a line between an \vr and the following \br, or immediately after the table format. Other \noalign commands may be inserted as desired. A partial horizontal rule is specified with \use, a special version of multispan, and the \-
The table command place some restrictions on the automatic insertion of tabskip glue. Hopefully these will be minor. The initial and final tabskip glues are set to 0pt. This should create no problems. A parameter \midtabglue may be modified. This is set just after the first rule column and is turned off at the last rule column. Finally, \tablespread = {to <dimen>} will force the table to be <dimen> wide, assuming there is enough tabskip glue available.
The instructions in Figure 2 produce the following rather contrived table.
| XYZ | ABC |
|-----|-----|
| X | Y | Z | A | B | C |
| 372.466 | 493.7 | 45 | 124 | 489 | 280 |
| 372.40 | 493.7 | 45 | 124 | 489 | 280 |
| 372. | 493.7 | 45 | 124 | 489 | 280 |
| XY/A | | | | | |
|------|-----|-----|-----|-----|-----|
| | 832 | abc | 774 | | |
| | qrr | aaa | 799 | | |
This example illustrates the power and flexibility of the table commands. Row i shows a modified
\centerline{
\begin{table}
\def\sprule{\tvrule{5\tr}}
\begin{tableformat}
&\center
\end{tableformat}
\-
\br{} \use{6} XYZABC \er{|\mst{.}{3pt}\rlap{ \it ** i}}
\-
\br{} \use{3} XYZ | \use{3} ABC \er{|\mst{.}{2pt}}
\-
\br{} X | Y | Z | A | B | C \er{}
\-
\br{} 372.466 | 493.7 " 45 | 124 | 489 | 280 \er{}
\-
\br{} 372.40" | 493.7 | 45 | 124 | 489 | 280 \er{}
\br{} " " | \use{3}\- \er{}
\br{} 372.--- | 493.7 | 45 | 124 | 489 | 280 \er{\rlap{ \it ** ii}}
\-
\br{} \use{2} | 832 | abc | 774 |\int$ \er{|\mst{$\int$}{0pt}{3pt}}
\br{} \use{2}\zb{XY/A} \use{4}\- \er{}
\br{} \use{2} | qrr | aaa | 799 | \er{\rlap{ \it ** iii}}
\-
\end{table}
}
Figure 2.
strut, \mst, with changed height and depth (the standard strut is 2.5ex high and .9ex deep). \mst is used again in row iii where some extra space is added at the bottom of the integral sign, but not at the top. \mst takes three arguments or parameters: The first is the character from which the strut is derived, the second is the additional space on the top of the character and the third is on the bottom. The first two rows of this table had additional space at both the top and bottom while the "integral" row was changed only at the bottom. Fine tuning tables in this way can improve their visual form immensely. Row iii demonstrates the use of a \zb or zero-centered box for putting items in the center of larger boxes. This row with the \zb has no struts. In addition, \zb boxes are centered and have zero height and depth. This means that they take up no vertical space, so that a partial horizontal line or rule may be inserted. Here the \use{4}\- tells the table commands to use 4 (data) columns for the horizontal rule.
Many other variations are possible.
Editor's note: The INRSTeX table commands will be made available through the TeX Users Group in both printed form and on magnetic media. Details can be obtained from the TUG office.
Editor's note: This is a gentle request to readers who find they can answer any of the queries published in this column. Please send a copy of your answer to the TUGboat editor as well as to the author; all answers will be published in the next issue following their receipt.
WEB System Extensions
I'm working on D. E. Knuth's "WEB System for Structured Documentation". If you have any experiences with or suggestions for improving this software development and documentation tool, please let me know about them. I want to extend the WEB system so that it is possible to use other programming languages than Pascal. If anybody out there uses the WEB system with another text formatting system than TeX or if you have made extensions to the system, I would also like to hear about that. I'm very interested in all your experiences, in what you use WEB for, and in your suggestions for improvement.
Helmut Becker
Rittershausstr. 4
D-5300 Bonn, West Germany
+49 228 211850
email@example.com
Editor's note: The last issue of TUGboat, 7#1, contained two articles on this subject: R. M. Damerell, "Error detecting changes to Tangle", pages 22–24, and Wolfgang Appelt and Karin Horn, "Multiple changefiles in WEB", pages 20–21. At least one attempt to build a WEB for C is known to Helmut Jürgensen, who would also be interested in hearing of work in this area, for possible publication in future issues.
This query was originally disseminated via TeXhax and UNIX-TeX, and the following responses have been posted.
WEB (TeX, LaTeX etc.) should all conform to ISO-646 (i.e. ASCII) in the following way: In ISO-646 the codes 64, 91–96 and 123–126 are reserved for "national or application oriented use". Many languages use more letters than a–z, these letters are present on keyboards, and the codes above are used for their representation. Every application (e.g. WEB) should be designed so these codes can be used according to the national standards and the documentation should show how this is done.
Staffan Romberger
"Staffan_Romberger_NADA%QZCOM.MAILNET"
@MIT-MULTICS.ARPA
Drama Scripts
Can anyone tell me where I can find macros for formatting a play complete with line numbers and different fonts for characters, text and stage directions?
John Kennedy
Mathematical Physics
University College Dublin
Dublin 4, Ireland
firstname.lastname@example.org
Marking Changes in Revised Documents
I would like to know if any of you have solved the following problem for \TeX generated documents:
When revising a document there is a convention where changed lines are identified by a vertical rule in the right margin.
I would like to be able to define macros that can be invoked at the start and end points of a contiguous sequence of changed text that cause the vertical rule to be automatically inserted. The changed text can span paragraphs and pages.
While it is reasonably straightforward to identify the start and end points of the vertical rule, I have not been able to come up with a scheme to accurately determine the height (or depth) of the rule.
If you have looked at this problem and found a solution, I would very much appreciate hearing from you.
Sylvester Fernandez
Sperry Corporation
Defense Products Group
Sperry Park, P.O. Box 64525
St. Paul, Minnesota 55164-0525
(612) 456-2222
Can it be done? One possibility would be to put the texstuff in an auxiliary file and read the file twice. Can it be done without an auxiliary file?
(2) On page ix of *Joy of \TeX*, Spivak has a paragraph where the first line is in caps-small caps, the remainder in Roman. Can this be done automatically?
James Alexander
University of Maryland
Electrical Engineering Dept
College Park, MD 20742
email@example.com
Editor’s note: The auxiliary file technique was used to set the tables, input and output, in the article by Michael Ferguson on page 106. The verbatim macros are based on those used by Knuth to set *The \TeXbook* (*The \TeXbook*, pp. 420–421) and go by the names \begintt and \endtt. It was found to be expedient to put the \ldots tt commands into the auxiliary file, and to \let them \relax when the content was to be acted on rather than displayed verbatim; this may not be necessary, but after several ineffectual attempts to input the auxiliary file after entering verbatim mode (yielding at most one blank line and no verbatim text), it seemed most productive to take the easy way out.
This technique might be built into a \showoff macro, with one argument giving the name of an auxiliary file, as follows:
\def\showoff#1{\begingroup % process the file
\let\begintt=\relax \let\endtt=\relax
\input #1 \endgroup
\bigskip % separate output from input
\input #1 % print verbatim listing
}
The first and last lines (except for comment lines) of the auxiliary file should be \begintt and \endtt respectively.
In order to preserve line breaks, the verbatim text must be read in while ‘m (the carriage-return character) is active (as it is when \obeylines, set on by \begintt, is in effect). This may or may not be a suitable convention while actually processing the text, and analysis of this problem should determine the answer to the question “can it be done without an auxiliary file?”
The question about fancy first-line processing has been asked before, in TUGboat 4, no. 2: 80–81, but if any answer has surfaced, we have not seen it.
Customized Editors for TeX
Now that there are TeX and LaTeX, what about editors for handling input for these? I would like to compile a list of customized editors. What I expect—well, hope for—is a list of packages for the EMACS family (Gosling, CCA, GNU, Epsilon, . . .), but maybe other people have customized their editors also, or even written special editors (VAX/VMS TPU anybody?). Of particular interest are Unix systems and PC's.
When answering, please quote base editor (if any) and version. Also let me know, please, if and how you're willing to make it available. I will compile a list for publication in TUGboat and redistribution via the TeX bulletin boards. If there is sufficient interest, we might even start a TUGboat column.
Karl Kleine
Forschungszentrum Informatik
Haid-und-Neu-Str. 10-14
D-7500 Karlsruhe, West Germany
firstname.lastname@example.org
email@example.com
...!mcvax!unido!uka!kleine
Editor's note: A set of templates for LaTeX, to be used with DEC's Language-Sensitive Editor (LSEDIT), which runs on top of TPU on VMS systems, has been developed at Lear Siegler, Inc. These templates, and a "Quick Reference Guide" for their use, are available through TUG; see the TUG order form and publications list for details.
Post-LaTeX Index Formatting
Is there a LaTeX addition which formats nice indexes? The amount of manual work remaining after LaTeX leaves off is discouraging (after using Digital standard Runoff).
Jim Ludden
Weyerhaeuser Company
Tacoma, Washington 98477
(206) 924-2345
Letters
To the members of TUG:
Now that *The Joy of TeX* has appeared, old friends (and other acquaintances of old) may be wondering why I've stopped using my first name, inserted a new initial, and joined the ranks of those who waggle their academic degrees after them like little pennants, so I'd like briefly to explain.
The idea was for the cover and title page to list the author as "Spivak, M.D., Ph.D.", and thus exude that air of multi-degreed authority so popular in the books fondly being parodied. The clue to the spurious degree would be the Library of Congress Cataloging-in-Publication Data, listing the author as "Spivak, Michael David". (When I say "idea" I really mean something rather more concrete, since all this appeared on the camera copy supplied to the AMS.) My mother, to whom the book is dedicated, was looking forward to this bit of academic buffoonery, since my initials were chosen so that the M.D. "would always be in front of your name, instead of after it".
Unfortunately, the Society's doyennes of academic stodginess, impervious to the promptings of humor, clumsily altered this to its present obnoxious form. Other petty changes, sneakily effected behind my back, are too numerous to mention here. (I don't know who wrote the copy for the back of the book, so can't identify the source of the hypocritical remarks about my "lively style" that "makes this an entertaining manual.")
It is customary for the author of a book to accept responsibility for its defects, and endeavor to make corrections and improvements in future editions. In this case, I expressly disavow any such responsibilities. If you find things wrong with the book, please don't tell me, tell the AMS; they own it, and apparently feel that they are better qualified than I to decide what should appear in it.
Mike Spivak, né Michael David Spivak
## Calendar
### 1986
**Tufts University, Medford, Massachusetts**
- **July 14–15**: Intermediate TeX
- **July 16–18**: Advanced TeX
- **July 21–23**: TeX Users Group Annual Meeting
- **July 24–25**: TeX Output routines
---
**College of St. Thomas, St. Paul, Minnesota**
- **Aug 18–20**: Beginning TeX
- **Aug 21–22**: Intermediate TeX
---
**Illinois Institute of Technology, Chicago, Illinois**
- **July 28–30**: Beginning TeX
- **July 31–Aug 1**: Intermediate TeX
---
**University of Illinois, Chicago, Illinois**
- **Sept 15–17**: Beginning TeX
- **Sept 18–19**: Intermediate TeX
---
### 1987
**Vanderbilt University, Nashville, Tennessee**
- **Aug 4–6**: Advanced TeX
- **Aug 7–8**: Macro writing
---
**Stanford University, Stanford, California**
- **Aug 12–14**: Beginning TeX
- **Aug 15–16**: Intermediate TeX
- **Aug 18–20**: Advanced TeX
- **Aug 21–22**: Macro writing
---
**TUGboat Volume 8, No. 1:**
Deadline for submission of manuscripts (tentative)
For additional information on items listed above, contact the TUG office (401-272-9500, ext. 232) unless otherwise noted.
### TUG Chapters
Members wishing to form TUG Chapters, either in-house or regional, are invited to contact Ray Goucher at the TUG Headquarters for additional information.
Institutional Members
Aarhus Universitet, Det Regionale EDB-Center (RECAU), Aarhus, Denmark
Addison-Wesley Publishing Company, Reading, Massachusetts
American Mathematical Society, Providence, Rhode Island
ASCII Corporation, Tokyo, Japan
Baumeister College, Witten, West Germany
Bell Northern Research, Inc., Mountain View, California
California Institute of Technology, Pasadena, California
CALMA, Sunnyvale, California
Calvin College, Grand Rapids, Michigan
Canon, Inc., Office System Center, Tokyo, Japan
Carleton University, Ottawa, Ontario, Canada
CDS/WordWorks, Davenport, Iowa
Centre Inter-Régional de Calcul Électronique, CNRS, Orsay, France
Centro Internacional De Mejoramiento De Maiz Y Trigo (CIMMYT), México, D.F., Mexico
City University of New York, New York, New York
College of St. Thomas, St. Paul, Minnesota
Columbia University, Center for Computing Activities, New York, New York
Corbel & Co., Jacksonville, Florida
COS Information, Montreal, P.Q., Canada
Digital Equipment Corporation, Nashua, New Hampshire
Dowell Schlumberger Inc., Tulsa, Oklahoma
Edinboro University of Pennsylvania, Edinboro, Pennsylvania
Educational Testing Service, Princeton, New Jersey
Electricité de France, Clamart, France
European Southern Observatory, Garching bei München, West Germany
FTL Systems Incorporated, Toronto, Ontario, Canada
Geophysical Company of Norway A/S, Stavanger, Norway
Grumman Corporation, Bethpage, New York
GTE Laboratories, Waltham, Massachusetts
Harvard University, Cambridge, Massachusetts
Hewlett-Packard Co., Boise, Idaho
Humboldt State University, Arcata, California
IBM Corporation, Scientific Center, Palo Alto, California
Illinois Institute of Technology, Chicago, Illinois
Imagen, Santa Clara, California
Institute for Advanced Study, Princeton, New Jersey
Institute for Defense Analyses, Communications Research Division, Princeton, New Jersey
Intergraph Corporation, Huntsville, Alabama
Intevep S. A., Caracas, Venezuela
Istituto di Cibernetica, Università degli Studi, Milan, Italy
Kuwait Institute for Scientific Research, Safat, Kuwait
Los Alamos National Laboratory, University of California, Los Alamos, New Mexico
Marquette University, Milwaukee, Wisconsin
Massachusetts Institute of Technology, Artificial Intelligence Laboratory, Cambridge, Massachusetts
Mathematical Reviews, American Mathematical Society, Ann Arbor, Michigan
McGill University, Montreal, Quebec, Canada
McGraw-Hill, Inc., Englewood, Colorado
National Center for Atmospheric Research, Boulder, Colorado
National Institutes of Health, Bethesda, Maryland
National Research Council Canada, Ottawa, Ontario, Canada
Online Computer Library Center, Inc. (OCLC), Dublin, Ohio
QMS, Inc, Mobile, Alabama
Queens College, Flushing, New York
Reed College, Portland, Oregon
RE/SPEC, Inc., Rapid City, South Dakota
Ruhr Universität Bochum, Bochum, West Germany
Sandia National Laboratories, Albuquerque, New Mexico
SAS Institute, Cary, North Carolina
Schlumberger Offshore Services, New Orleans, Louisiana
Schlumberger Well Services, Houston, Texas
Science Applications International Corp., Oak Ridge, Tennessee
I. P. Sharp Associates, Palo Alto, California
Smithsonian Astrophysical Observatory, Computation Facility, Cambridge, Massachusetts
Software Research Associates, Tokyo, Japan
Sony Corporation, Atsugi, Japan
Springer-Verlag, Heidelberg, West Germany
Stanford Linear Accelerator Center (SLAC), *Stanford, California*
University of Chicago, Graduate School of Business, *Chicago, Illinois*
Stanford University, Computer Science Department, *Stanford, California*
University of Delaware, *Newark, Delaware*
Stanford University, ITS Graphics & Computer Systems, *Stanford, California*
University of Glasgow, *Glasgow, Scotland*
State University of New York, *Stony Brook, New York*
University of Groningen, *Groningen, The Netherlands*
Syracuse University, *Syracuse, New York*
University of Maryland, *College Park, Maryland*
Talaris Systems, Inc., *San Diego, California*
University of Massachusetts, *Amherst, Massachusetts*
Texas A&M University, Department of Computer Science, *College Station, Texas*
University of North Carolina, School of Public Health, *Chapel Hill, North Carolina*
Texas A&M University, Department of Mathematics, *College Station, Texas*
University of Oslo, Institute of Informatics, *Blindern, Oslo, Norway*
Texas Accelerator Center, *The Woodlands, Texas*
University of Southern California, Information Sciences Institute, *Marina del Rey, California*
Textset, Inc., *Ann Arbor, Michigan*
University of Texas at Austin, Physics Department, *Austin, Texas*
TRW, Inc., *Redondo Beach, California*
University of Texas at Dallas, Center for Space Science, *Dallas, Texas*
Tufts University, *Medford, Massachusetts*
University of Washington, Department of Computer Science, *Seattle, Washington*
TYX Corporation, *Reston, Virginia*
University of British Columbia, *Vancouver, British Columbia, Canada*
University of Calgary, *Calgary, Alberta, Canada*
University of British Columbia, *Vancouver, British Columbia, Canada*
University of California, Berkeley, Computer Science Division, *Berkeley, California*
University of California, Berkeley, Computing Services, *Berkeley, California*
University of California, San Francisco, *San Francisco, California*
University of California, Computation Center, *Chicago, Illinois*
University of Chicago, Computer Science Department, *Chicago, Illinois*
University of Chicago, Graduate School of Business, *Chicago, Illinois*
University of Delaware, *Newark, Delaware*
University of Glasgow, *Glasgow, Scotland*
University of Groningen, *Groningen, The Netherlands*
University of Maryland, *College Park, Maryland*
University of Massachusetts, *Amherst, Massachusetts*
University of North Carolina, School of Public Health, *Chapel Hill, North Carolina*
University of Oslo, Institute of Informatics, *Blindern, Oslo, Norway*
University of Southern California, Information Sciences Institute, *Marina del Rey, California*
University of Texas at Austin, Physics Department, *Austin, Texas*
University of Texas at Dallas, Center for Space Science, *Dallas, Texas*
University of Washington, Department of Computer Science, *Seattle, Washington*
University of Western Australia, Regional Computing Centre, *Nedlands, Australia*
University of Wisconsin, Academic Computing Center, *Madison, Wisconsin*
University of York, *Heslington, York, England*
Vanderbilt University, *Nashville, Tennessee*
Washington State University, *Pullman, Washington*
Request for Information
The TeX Users Group maintains a database and publishes a membership list containing information about the equipment on which members' organizations plan to or have installed TeX, and about the applications for which TeX would be used. This list is updated periodically and distributed to members with TUGboat, to permit them to identify others with similar interests. Thus, it is important that the information be complete and up-to-date.
Please answer the questions below, in particular those regarding the status of TeX and the hardware on which it runs or is being installed. (Operating system information is particularly important in the case of IBM mainframes and VAX.) This hardware information is used to group members in the listings by computer and output device.
If accurate information has already been provided by another TUG member at your site, you may indicate that member's name, and the information will be repeated.
If your current listing is correct, you need not answer these questions again. Your cooperation is appreciated.
- Send completed form with remittance (checks, money orders, UNESCO coupons) to:
TeX Users Group
P.O. Box 594
Providence, Rhode Island 02901, U.S.A.
- For foreign bank transfers
direct payment to the TeX Users Group,
account #002-031375, at:
Rhode Island Hospital Trust National Bank
One Hospital Trust Plaza
Providence, Rhode Island 02903-2449, U.S.A.
- General correspondence
about TUG should be addressed to:
TeX Users Group
P.O. Box 9506
Providence, Rhode Island 02940-9506, U.S.A.
| QTY | ITEM | AMOUNT |
|-----|----------------------------------------------------------------------|--------|
| | 1986 TUGboat Subscription/TUG Membership (Jan.-Dec.) – North America | |
| | New (first-time): [ ] $25.00 each | |
| | Renewal: [ ] $35.00; [ ] $25.00 – reduced rate if renewed before January 31, 1986 | |
| | 1986 TUGboat Subscription/TUG Membership (Jan.-Dec.) – Outside North America | |
| | New (first-time): [ ] $30.00 each | |
| | Renewal: [ ] $40.00; [ ] $30.00 – reduced rate if renewed before January 31, 1986 | |
| | TUGboat back issues, $15.00 per issue, circle issue(s) desired: | |
| | 1980 (v.1) 1981 (v.2) 1982 (v.3) 1983 (v.4) 1984 (v.5) 1985 (v.6) | |
Air mail postage is included in the rates for all subscriptions and memberships outside North America.
Quantity discounts available on request.
TOTAL ENCLOSED:
(Prepayment in U.S. dollars required)
* * * *
Membership List Information
Institution (if not part of address):
Title:
Phone:
Network address: [ ] Arpanet [ ] BITnet
[ ] CSnet [ ] uucp
Specific applications or reason for interest in TeX:
My installation can offer the following software or technical support to TUG:
Please list high-level TeX users at your site who would not mind being contacted for information; give name, address, and telephone.
Date:
Status of TeX: [ ] Under consideration
[ ] Being installed
[ ] Up and running since
Approximate number of users:
Version of TeX: [ ] SAIL
Pascal: [ ] TeX82 [ ] TeX80
[ ] Other (describe)
From whom obtained:
Hardware on which TeX is to be used:
Operating system(s)
Computer(s) Output device(s)
Please answer the following questions regarding output devices used with TeX
if this form has never been filled out for your site, or if you have new information.
Use a separate form for each output device.
Name ________________________________________ Institution __________________________
A. Output device information
Device name
Model
1. Knowledgeable contact at your site
Name
Telephone
2. Device resolution (dots/inch)
3. Print speed (average feet/minute in graphics mode)
4. Physical size of device (height, width, depth)
5. Purchase price
6. Device type
[ ] photographic [ ] electrostatic
[ ] impact [ ] other (describe)
7. Paper feed [ ] tractor feed
[ ] friction, continuous form
[ ] friction, sheet feed [ ] other (describe)
8. Paper characteristics
a. Paper type required by device
[ ] plain [ ] electrostatic
[ ] photographic [ ] other (describe)
b. Special forms that can be used [ ] none
[ ] preprinted one-part [ ] multi-part
[ ] card stock [ ] other (describe)
c. Paper dimensions (width, length)
maximum
usable
9. Print mode
[ ] Character: ( ) Ascii ( ) Other
[ ] Graphics [ ] Both char/graphics
10. Reliability of device
[ ] Good [ ] Fair [ ] Poor
11. Maintenance required
[ ] Heavy [ ] Medium [ ] Light
12. Recommended usage level
[ ] Heavy [ ] Medium [ ] Light
13. Manufacturer information
a. Manufacturer name
Contact person
Address
Telephone
b. Delivery time
c. Service [ ] Reliable [ ] Unreliable
B. Computer to which this device is interfaced
1. Computer name
2. Model
3. Type of architecture*
4. Operating system
C. Output device driver software
[ ] Obtained from Stanford
[ ] Written in-house
[ ] Other (explain)
D. Separate interface hardware (if any) between host computer and output device (e.g. Z80)
1. Separate interface hardware not needed because:
[ ] Output device is run off-line
[ ] O/D contains user-programmable micro
[ ] Decided to drive O/D direct from host
2. Name of interface device (if more than one, specify for each)
3. Manufacturer information
a. Manufacturer name
Contact person
Address
Telephone
b. Delivery time
c. Purchase price
4. Modifications
[ ] Specified by Stanford
[ ] Designed/built in-house
[ ] Other (explain)
5. Software for interface device
[ ] Obtained from Stanford
[ ] Written in-house
[ ] Other (explain)
E. Fonts being used
[ ] Computer Modern
[ ] Fonts supplied by manufacturer
[ ] Other (explain)
F. What are the strong points of your output device?
G. What are its drawbacks and how have you dealt with them?
H. Comments – overview of output device
TEX82 Order Form
The latest official versions of TEX software and documents are available from Maria Code by special arrangement with the Computer Science Department of Stanford University.
Ten different tapes are available. The generic distribution tape contains the source of TEX82, WEB, and the latest (prototype) version of WEB METAFONT, standard test programs for TEX and METAFONT, a few "change" files, the collection of fonts in TFM format, and other miscellaneous materials; a PASCAL compiler will be required to install programs from a generic tape. The TEX distribution tapes include the AMS-TEX, IATEX and HP TEX macro packages; other macro packages will be added as they become available. The special distribution tapes are for the indicated systems only, and should be ordered for these systems instead of a generic tape. Two tapes are PXL font collections covering various magnifications at 200/240 dots/inch and 300 dots/inch respectively.
Each tape will be a separate 1200 foot reel which you may send in advance or purchase (for the tape media) at $10.00 each. Should you send a tape, you will receive back a different tape. Tapes may be ordered in ASCII or EBCDIC characters. All tapes are 1600 bpi.
The tape price of $82.00 for the first tape and $62.00 for each additional tape (ordered at the same time) covers the cost of duplication, order processing, domestic postage and some of the costs at Stanford University. Extra postage is required for first class or export.
Manuals are available at the approximate cost of duplication and mailing. Prices for manuals are subject to change as revisions and additions are made.
Please send a money order or check (drawn on a US bank) along with your order if possible. Your purchase order will be accepted, as long as you are able to make payment within 30 days of shipment. Make checks payable to Maria Code.
The order form contains a place to record the name and address of the person who will actually use the TEX tapes. This should not be someone in the purchasing department.
Your order will be filled with the most recent versions of software and manuals available from Stanford at the time your order is received. If you are waiting for a specific release, please indicate this. Orders are normally filled within a few days. There may be periods (like short vacations) when it will take longer. You will be notified of any serious delays. If you want to inquire about your order you may call Maria Code at (408) 735-8006 between 9:30 a.m. and 2:30 p.m. West Coast time.
If you have questions regarding the implementation of TEX or the like, you must take these to Stanford University or some other friendly TEX user.
Now, please complete the order form on the reverse side.
TEX82 Order Form
TAPES:
TEX generic distribution tapes (PASCAL compiler required):
ASCII format EBCDIC format
TEX distribution tapes in special formats:
† VAX/VMS Backup format IBM VM/CMS format
† DEC 20/Tops-20 Dumper format * IBM MVS format
† Includes WEB METAFONT * Not yet available; call before ordering
Font tapes:
Font library (200/240 dots/inch) Font library for IBM 4250 printer
Font library (300 dots/inch) Font library for IBM 6670 printer
Total number of tapes.
Tape costs: $82.00 for first tape; $62.00 for each additional.
Media costs: $10.00 for each tape required.
MANUALS:
TEX: The Program – $28.00 A Torture Test for TEX – $8.00
WEB – $10.00 TeXware – $8.00
TeXbook – $20.00 LATEX – $20.00
BibTeX – $10.00
Manuals cost = $ ____________
California orders only: add sales tax = $ ____________
Domestic book rate: no charge.
Domestic first class: $2.50 for each tape and each manual.
Export surface mail: $2.50 for each tape and each manual.
Export air mail to Canada & Mexico: $4.00 each.
Export air mail to Europe: $7.00 each.
Export air mail to other areas: $10.00 each.
(Make checks payable to Maria Code)
Name and address for shipment:
Person to contact (if different):
Telephone
Send to: Maria Code, DP Services, 1371 Sydney Dr., Sunnyvale, CA 94087
IT'S BEEN CALLED A WORK OF GENIUS.
IT'S ALSO A WORK OF ART.
Donald Knuth's Computers and Typesetting series.
Addison-Wesley is proud to present a five-volume collection on two revolutionary typesetting ideas: TeX and METAFONT.
VOLUME A
THE TeXbook.
The definitive guide to TeX, the text-processing system that lets you produce high-quality, technical typeset documents like no other typesetting program. Hardcover version of the popular paperback manual.
VOLUME B
TeX: The Program.
The complete source code for TeX. Never before has a program of this size been documented so clearly and completely.
VOLUME C
The METAFONTbook.
The user's guide and reference manual for METAFONT, the language that helps you design clear, precise letters and symbols more quickly than ever before. Hardcover version of the popular paperback manual.
VOLUME D
METAFONT: The Program.
The complete source code for METAFONT. An excellent lesson by example on how to put programming theory into practice.
VOLUME E
Computer Modern Typefaces.
The ultimate reference for type designers interested in how Knuth created these beautiful letters and symbols. Includes more than 750 illustrations.
Look for the complete Computers and Typesetting series in your bookstore this spring!
☐ Please put me on your computers and typesetting mailing list, and tell me how I can order the series.
Name_____________________________________________________
Address___________________________________________________
City________________________________________ State________ Zip________
Phone______________________________________________
Mail to:
Addison-Wesley Publishing Company
Attn: P. Wiley
Reading, Massachusetts 01867 (617) 944-3700
We publish the leaders.
Personal TEX Inc
Your Complete Supplier of TEX for the PC!
...now offers a list of software, fonts and hardware so that we can be your complete TEX outfitter for PC and AT workstations. We have joined forces with Textset, $n^2$ Computer Consultants, and the Metafoundry to bring you these products:
**SOFTWARE:**
**PCTEX®** A full TeX82, version 2.0, including INITEX, LATEX 2.09, LATEX User's Guide, AMS-TEX, and Mike Spivak's PCTEX Manual and VANILLA macro package.
33% faster than version 1.0! Runs LATEX in 512K RAM! **$249.**
**PCDOT** Device drivers for the following dot-matrix printers: Epson FX, RX and LQ printers, IBM Graphics Printer, and the Toshiba 1340, 1350, P351 printers. Each driver includes over 230 TeX and LATEX fonts. **$95. each.**
**DVILASER/HP** Textset's new device driver for the HP LaserJet Plus. Operates with standard serial or parallel ports - no additional hardware needed. Includes complete TeX and LATEX fonts. **$249.**
**PCLaser** Device drivers for several popular laser printers: Apple LaserWriter (Postscript) (Textset), QMS Lasergrafix 800, 1200 ($n^2$ Computer Consultants, Textset), Imagen 8,12,24/300 (Textset), Corona LP300 and JLASER (HP LaserJet). Includes complete TeX and LATEX fonts. From **$249. each.**
**Preview** Textset's popular Preview is now available for the PC. Requires the Hercules Graphics Card, IBM Enhanced Graphics Adapter, or Tecmar Graphics Master. **$225.**
**FONTS:**
**MF Medley** 44 Popular fonts from the Metafoundry library. Includes printer and screen preview resolutions of Chel fonts (Computer Helvetica, shown here), and Copperplate, Black Letter and Schoolbook headline fonts. Specify printer resolution (180, 240 or 300 DPI) **$100.**
**HARDWARE:**
**Corona Laser Printer** This device will print a full page of TeX output, and employs the same Canon CX print engine found in popular laser printers. Comes with PCTEX and the Corona device driver. Complete software and hardware: only **$2995.**
**JLASER** An interface card for your PC with 1 MB RAM and a direct connection to any Canon CX print engine (HP LaserJet, QMS KISS and others). Allows high resolution full page printing. From **$699.**
Join hundreds of satisfied PCTEX users. Write or call us today for complete product information. Inquire about educational and corporate discounts and site licensing.
System requirements: DOS 2.0 or later, 512K RAM, 10M hard disk. Preview requires appropriate graphics adapter. Corona Laser Printer requires additional 512K RAM disk. Include $8. shipping and handling for each U.S. order. Orders outside the U.S. must be placed through distributors listed below. California orders, add 6% sales tax. MasterCard, Visa accepted.
---
**Personal TEX Inc**
20 Sunnyside, Suite H
Mill Valley, CA 94941
(415) 388-8853 Telex 510-601-0672
Distributors: Canada: Ahearn & Soper, Rexdale, Ontario, (416) 675-3999; UK: UniTeX Systems, Sheffield, (0742) 351 489; Germany: Kettler EDV-Consulting, Lenggries, (08042) 8081; France: Probe Informatique, Trappes, (3062) 2603; Ireland: UniTeX, Dublin, 772941 x1983; Australia: The Wordworks, ACT, (062) 572893.
Trademarks: PCTEX, Personal TeX, Inc.; TeX, American Mathematical Society; IBM PC and AT, IBM Corp. Manufacturer's product names are trademarks of individual manufacturers.
This ad was generated using PCTEX, and printed on a Cordata LP-300.
THE TALARIS 810:
THE MISSING PIECE
IN YOUR PERSONAL TYPESETTING SYSTEM
Now that you have \TeX™ on your IBM PC or compatible, you need a printer that lives up to \TeX's formatting standards—a high-resolution printer with fonts and flexibility. What you don't need is a printer that costs far more than your PC.
Introducing the Talaris 810
The *Talaris 810* is that printer—an 8-page-per-minute, 300 dots-per-inch laser printer that costs just $3,850. Ideal for \TeXnicians on a budget.
And Talaris completes your personal typesetting system with fonts and software: \TeX PROM font cartridges and a DVI file post-processor.
Talaris's \TeX font cartridges
The *Talaris 810* comes with 12 standard word processing fonts. Add one of Talaris's \TeX font cartridges and start \TeXing. Each font cartridge contains from 16 to 30 \TeX fonts. Choose from Plain, Plain + (30 fonts), Dutch Times Roman-like, or Swiss Helvetica-like \TeX font cartridges.
Talaris's Lo\TeX DVI post-processor
Lo\TeX outputs ANSI 3.64 commands to support absolute character positioning as required by \TeX DVI files. Lo\TeX also provides three options for positioning the page on the paper—centered, uncentered, and \TeX standard—as well as options for landscape orientation and printing more than one copy of output.
The Talaris 810 and graphics
Store digitized artwork, forms, and signatures as overlays. Then use \TeX's \special command to call the overlays.
Talaris, the personal typesetting software specialists
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Brown & Root/NORTHROP
The image shows an aerial view of a large industrial or commercial facility, likely from the mid-20th century based on the style of vehicles and architecture. The building is rectangular with a flat roof and appears to be surrounded by open land. There are several parking lots filled with cars, indicating that this is a place where people come to work or visit. The area around the building includes roads and some greenery, suggesting a suburban setting. In the background, there are more buildings and what looks like a power plant or similar infrastructure. The sky is partly cloudy, adding to the overall impression of a typical day at this location.
HOME-AWAY-FROM-HOME FOR APOLLO 11 ASTRONAUTS -- NASA's Lunar Receiving Laboratory, staffed by scientists of Brown and Root/Northrop, will be used to quarantine the world's first men to set foot on the moon. The astronauts, and the samples of lunar material they bring back, will be isolated at the Houston, Texas, facility for preliminary examination. Personnel of Brown and Root/Northrop, a joint-venture company, provide scientific and technical support to this laboratory as well as four other facilities at the Manned Spacecraft Center.
FROM: Public Relations
Northrop Corporation
Palos Verdes, California
06069/dg
Public Relations
Brown & Root/Northrop
Beta Building
Houston, Texas
LRL FUNCTIONAL AREAS
- BIO-BARRIERS
- CREW RECEPTION AREA
- OPERATIONS AREA
- SAMPLE
- RADIATION COUNTING LABORATORY (PARTIALLY UNDERGROUND)
- ADMINISTRATION AND SUPPORT AREA
QUARANTINE BUILDING FOR APOLLO 11 CREW -- The first men to return from the moon will undergo quarantine in NASA's Lunar Receiving Laboratory at the Manned Spacecraft Center. In this view the four key laboratory areas are shown. Personnel of Brown & Root/Northrop, a joint venture firm, provide scientific and technical support to the laboratory located in Houston, Texas.
FROM: Public Relations
Northrop Corporation
Palos Verdes, California
06069/dg
Public Relations
Brown & Root/Northrop
Beta Building
Houston, Texas
Two individuals in white lab coats and caps are seated at control panels, operating large industrial equipment with numerous dials and gauges. The setting appears to be a laboratory or factory environment, with complex machinery and piping visible in the background.
EXTRATERRESTRIAL SAMPLES gathered by the crew of Apollo 11 will be processed and distributed to scientists around the world from the Vacuum Laboratory at NASA's $15-million Lunar Receiving Laboratory, Houston, Texas. The laboratory is manned by scientists and technicians of Brown & Root/Northrop, a joint-venture company. Sample containers will enter the sterile airlock, left, where they undergo decontamination by ultraviolet light before moving to the center cabinet for an acid bath. Hot sterile nitrogen is used at the last station to dry the boxes before they are opened.
FROM: Public Relations
Northrop Corporation
Palos Verdes, California
06239/dg
Public Relations
Brown & Root/Northrop
Beta Building
Houston, Texas
BROWN, ROOT & NORTHROP
This image depicts a laboratory setting with individuals in lab coats engaged in various activities. The room is well-lit with large windows covered by blinds, and there are multiple tables and chairs arranged throughout the space. Some individuals appear to be working at their stations, while others seem to be observing or preparing materials. The overall atmosphere suggests a professional and focused environment typical of a research or development facility.
HOME FOR APOLLO ASTRONAUTS -- NASA's $15-million Lunar Receiving Laboratory, staffed by scientists of Brown & Root/Northrop, will be used to quarantine the world's first men to set foot on the moon. The astronauts, and the samples of lunar material they bring back, will be isolated for approximately 21 days while undergoing preliminary examinations. Shown here is the combination kitchen, dining room and recreation area within the 83,000 square foot biological barrier. Personnel of Brown & Root/Northrop, a joint-venture company, provide scientific and technical support to this laboratory as well as four other facilities at the Manned Spacecraft Center, Houston.
FROM: Public Relations
Northrop Corporation
Palos Verdes, California
06239/dg
Public Relations
Brown & Root/Northrop
Beta Building
Houston, Texas
TRANSPORTATION TO AND FROM LRL
AIRCRAFT CARRIER IN RECOVERY ZONE
DIRECT AIR TRANSPORT
LUNAR SAMPLE
DATA FILM & TAPE
ASTRONAUT BIO SPECIMENS
NEAREST LAND BASE WITH RUNWAYS
LUNAR RECEIVING LAB
MORE THAN 50 SPECIMENS (LATER) FOR INVESTIGATION
AIR TRANSPORT ON ISOLATION VAN
3 ASTRONAUTS
1 MEDIC
1 TECHNICIAN
SURFACE OR AIR TRANSPORT SEALED SPACECRAFT
MORE THAN 50 UNIVERSITIES AND LABS ALL OVER WORLD
SPECIMENS RETURNED FOR STORAGE AND/OR REDISTRIBUTION
(STILL LATER)
MOON-CONQUERING ASTRONAUTS TO USE SPECIAL FACILITY -- Astronauts of the greatest space adventure in history, Apollo 11, and the samples of lunar material will undergo quarantine in a special NASA facility at Houston, Texas.
Called the Lunar Receiving Laboratory, the building is the last stop for men and materials after their 8-day voyage to the moon and back. Lunar samples will be distributed to universities and laboratories all over the world from NASA's Manned Spacecraft Center.
FROM: Public Relations
Northrop Corporation
Palos Verdes, California
06069/dg
Public Relations
Brown & Root/Northrop
Beta Building
Houston, Texas
The isolation or quarantine period is scheduled to last 21 days after liftoff from the lunar surface.
Contact: Roy Gregory Northrop Corporation 1 Research Park Palos Verdes, Calif. (213) 377-4811
Bill Averyt Brown & Root/Northrop 16915 El Camino Real Houston, Texas (713) 488-2500
The $13-million laboratory in Lunar Receiving Laboratory will be the home of the astronauts after they return from their eight day voyage into space. The samples of moon surface material they will gather from the western edge of the Sea of Tranquility also will be brought here for analysis.
Brown & Root/Northrop is a joint venture of Brown & Root, Inc., Houston, Texas, and the Northrop Corporation of Southern California. The organization provides technical support services for a number of Manned Spacecraft Center facilities.
Under supervision of the two directorates, Science and Applications and Medical Research and Operations, at the Manned Spacecraft Center, the astronauts will stay in quarantine to prevent possible contamination of the earth's atmosphere with potentially dangerous particles from the lunar surface.
The isolation or quarantine period is scheduled to last 21 days after liftoff from the lunar surface.
The $15-million laboratory will be the focal point where astronauts Armstrong, Collins, and Aldrin tell their story to the rest of the world. Here, also, the international scientific community will begin an intense study of the contents of the two boxes holding material from the moon's surface programmed to be collected by Armstrong and Aldrin.
While the three men are being carefully examined, the samples will undergo preliminary physical and chemical tests within a biological barrier much like the one surrounding the crew's area.
Heading the 180 man Brown & Root/Northrop effort in the Lunar Receiving Laboratory is Tom Slack. Reporting to him and scheduled to direct Brown & Root/Northrop efforts in the quarantined crew reception area, is Mel Graham, logistics operations officer and senior research analyst.
Quarantined with Graham and the three astronauts will be Laboratory Technologists John Potter, Jerry Terrell, Hank Knippa and Charles Gill, who are specialists in clinical chemistry, hematology, enzyme analysis and the immunology program. Each technologist is cross-trained in all biomedical requirements.
Also reporting to Graham is Chief Steward William Woods and Assistant Stewards Jessie Stewart and Bobby Lartigue. Rounding out the team is Harry Cantu, x-ray technologist.
Dr. Charles Truby, clinical biologist, will head the Brown & Root/Northrop scientists working on biological investigations of the lunar samples. This team will assist in conducting experiments which will provide a basis for decision upon any possible threat to earth life systems.
In the Physical Sciences Area, Dr. Leonard Jones will supervise the Brown & Root/Northrop group that will operate the equipment and do the initial handling of the returned moon samples as well as data collection.
Frank Fato is responsible for all Brown & Root/Northrop engineering and operations within the laboratories.
NASA personnel, including a flight surgeon, a clinical pathologist, a photographer, a public affairs officer, and a landing and recovery technician, will be quarantined with the astronauts.
Even though the possibility of harmful germs or bacteria existing on the moon is considered remote, science--and these experts--are taking no chances. It is equally important that the returned lunar samples not be contaminated by earth organisms which, if such contamination occurred, might lead to false conclusions about the existence of living organisms on the moon.
Under supervision of the two directorates, Science and Applications and Medical Research and Operations, at the Manned Spacecraft Center, the astronauts will stay in quarantine to prevent possible contamination of the earth's atmosphere with potentially dangerous particles from the lunar surface.
Repackaging and distribution of lunar samples will be handled by Brown & Root/Northrop for NASA's Office of Space Sciences. The company will provide the necessary equipment and facilities to perform more than 120 experiments of varying scope and magnitude on the samples.
OPERATIONS AT NASA'S LUNAR RECEIVING LABORATORY
Three years ago ground was broken for the Lunar Receiving Laboratory (LRL) where, within the month, American scientists will have their first look at samples of our nearest neighbor, the moon. The LRL will be the focal point where the Apollo 11 crew will tell the story of their lunar visit and the national and international scientific community will begin an intense investigation of moon material.
The Lunar Receiving Laboratory will be the final stop for Apollo crewmen following their lunar journey. For the samples of the moon brought back, it will be the first stop in a series of scientific investigations.
Staffed by personnel of Brown & Root/Northrop, a joint venture, through a special airtight plastic transfer tunnel, after being brought aboard the recovery ship, the laboratory is under the supervision of the Science and Applications Directorate at NASA's Manned Spacecraft Center.
Main purposes of the multi-level research structure are: remain in the mobile facility during the trip to Houston. In Honolulu, the unit will be transferred to deckside, then to an airport where it will be placed aboard a C-141 transport for the flight to Houston. It is anticipated that the time from recovery to the time the mobile facility reaches the LRL will not exceed two days within the quarantine period.
Repackaging and distribution of the lunar samples to scientists throughout the world for detailed investigation after the quarantine period.
NASA's Office of Space Science and Applications has selected 149 scientists to perform more than 120 experiments of varying scope and magnitude on the initial samples. Investigators from the United States were selected from 21 universities, two industrial firms, three private institutions and 10 government laboratories. Thirty-three experiments were awarded to 27 foreign scientists representing England, Germany, Canada, Japan, Finland, and Switzerland.
The Crew Reception Area will serve as quarters for the flight crew. Astronauts returning from the moon will be transferred to the LRL and attendant technicians for the period during which the three astronauts following their recovery in the Pacific. The lunar samples--photographic film, tapes, and other flight items--will be flown by jet directly to the laboratory where they will undergo a battery of analytical processes.
The crewmen will return to the LRL in a specially designed quarantine facility unit which resembles a mobile home. They will enter it through a special airtight plastic transfer tunnel, after being brought aboard the recovery ship. Like the Crew Reception Area, the Sample Operations Area is contained within a biological barrier system. This barrier is unique in that it will protect the gathered lunar surface samples from earth contamination remain in the mobile facility during the trip to Houston. In Honolulu, the unit as well as protect the outside world from possible contamination by lunar materials.
The astronauts, an MSC physician and a recovery technician, will be transferred to dockside, then to an airport where it will be placed aboard a C-141 transport for the flight to Houston. It is anticipated the time from recovery to the time the mobile facility reaches the LRL will not exceed two days.
After the crewmen leave the Apollo spacecraft and enter the mobile unit, the command module will be resealed. It will be moved to Houston where it will be placed in separate quarantine, adjacent to the crew quarters. Access to the spacecraft interior will be permitted only after the 21-day isolation period is ended. It will, however, be available within this controlled area for inspection.
The Lunar Receiving Laboratory building consists of three adjacent and related functional areas--Crew Reception, Sample Operations, and Support and Administrative.
The Crew Reception Area will serve as quarters for the flight crew and attendant technicians for the period during which the three astronauts will be debriefed and examined. Among the 17 attendant employees (including 10 from Brown & Root/Northrop) will be medical doctors and technicians, housekeepers, and stewards. These rooms also have the capability to serve as a contingency quarantine area in the event it is necessary to place people from the Sample Operations Area in quarantine.
Like the Crew Reception Area, the Sample Operations Area is contained within a biological barrier system. This barrier is unique in that it will protect the gathered lunar surface samples from earth contamination as well as protect the outside world from possible contamination by lunar materials.
Laboratory personnel also will perform studies of the crew microbiology, virology and clinical chemistry.
Studies of the returned material will be performed in the Sample Operations Area, which includes vacuum, magnetics, gas analysis, biological test and radiation counting laboratories in addition to the physical-chemical test area. The entire Sample Area, with the exception of the radiation counting rooms, is within a second biological barrier much like the one surrounding the crew's area.
Containers holding lunar samples first will be brought to the LRL vacuum laboratory. There they will be placed in the ultraclean vacuum system and opened by Brown & Root/Northrop technicians. After preliminary examination of the material, it will be divided, repackaged and transferred to the Biological Preparation and Radiation Counting Laboratories.
A Gas Analysis Laboratory is available to monitor amounts and types of gases given off by the lunar material and such gases that may be contained either within the sample or within the sample containers.
The major responsibility of the Biological Test Laboratory will be to determine if there is life in the material that may multiply on earth. This will be accomplished by the introduction of lunar surface samples into small, germ-free animals and plants.
This laboratory is equipped to do preparation and analysis of exposures to the lunar samples of germ-free and normal animals including mammals, fowls, and invertebrates, plus plants and tissue cultures, through histological, anaerobic, microbiological and biochemical techniques. Laboratory personnel also will perform studies of the crew microbiology, virology and clinical chemistry.
A Radiation Counting Laboratory is located 60 feet underground. This lab will assay low background radioactivity of the samples and is located underground to shield samples from the effects of earth's natural radiation.
After the samples have been through the Vacuum System for examination and identification, they pass to the Physical-Chemical Test Laboratory. It is here that geochemists will test the lunar surface samples for reactions with atmospheric gases and water vapor. In addition, detailed studies of the mineralogic, petrologic and physical properties of the sample will be made.
The Lunar Receiving Laboratory will be the final stop for Apollo crewman following their lunar journey. For the samples of the moon brought back, it will be the first stop in a series of scientific investigations.
Staffed by personnel of Brown & Root/Northrop, a joint venture, the laboratory is under the supervision of the Science and Applications Directorate at NASA's Manned Spacecraft Center.
Main purposes of the multi-level research structure are:
- Quarantine and testing for possible harmful organisms in the lunar samples, spacecraft, and crew.
- Scientific sample investigations that must be accomplished within the quarantine period.
A closed circuit television and standard communications system connects the astronaut debriefing room with the press briefing room.
Contact: Roy Gregory
Northrop Corporation
1 Research Park
Palos Verdes, Calif.
(213) 377-4811
Bill Averyt
Brown & Root/Northrop
16915 El Camino Real
Houston, Texas
(713) 488-2500
The kitchen contains a microwave oven and facilities. The dining room and library; and a room containing standard gym and exercise equipment allows the quarantined personnel to pursue a physical exercise program.
FACT SHEET
LUNAR RECEIVING LABORATORY
Complete medical examination rooms are enclosed within the biological barrier.
The $15-million Lunar Receiving Laboratory--a three-story, 83,000 square feet research facility located at NASA's Manned Spacecraft Center, Houston, Texas--is designed to provide biological containment of specimens, spacecraft, and personnel during the 21-day Apollo 11 quarantine period. The Biological Test Laboratories are housed in this area. Their key purpose is to test the effectiveness of the biological barrier.
The laboratory is divided into three primary functional groups: Crew Reception, Sample Operations, and Administrative and Support.
CREW RECEPTION
This area is enclosed within its own biological barrier, consisting of sealed building construction, filtration of supply and exhaust air, and sterilization of all solid and liquid effluents. A slightly lower air pressure inside the room prevents outflow of any possible contamination.
Designed primarily as the quarantine section for the astronauts and spacecraft, the Crew Reception Area (CRA) is the actual living quarters for quarantined personnel. It includes bedrooms and offices for the astronauts and astronaut medical team, and a dormitory, lockers, showers and toilet facilities.
A closed circuit television and standard communications system connects the astronaut debriefing room with the press briefing room, allowing interviews of crew members by non-quarantined personnel.
The kitchen contains the latest in microwave and infrared cooking facilities. The dining room and lounge area has a television set and small library; and a room containing standard gym and exercise equipment allows the quarantined personnel to pursue a physical exercise program.
Complete medical examination rooms are enclosed within the biological barrier of the CRA, and include an audio booth, minor surgery room, microbiology and biomedical laboratories, x-ray facilities, and a computer room.
**SAMPLE OPERATIONS**
The Biological Test Laboratories are housed in this area. Their key purpose is to test returned lunar samples, spacecraft and crews for the possible existence of harmful moon organisms. All exhaust air passes through two absolute biological filters and is incinerated at 550° F. before it is released. The laboratory houses sterile atmosphere cabinetry with a leakage rate of less than .05 ounces of gas per year.
Steam ethylene oxide sterilizers, a phase contrast microscope and stereo microscope, and special grinding, pulverizing, and centrifugation equipment for preparation of samples are also included. This area also contains the vacuum pumps capable of producing pressures of $10^{-12}$ torr (approximately 1.5-million feet altitude), vacuum and magnetics lab, gas analysis chamber, and the radiation counting laboratory shielded by five-foot thick concrete walls and 26 tons of lead.
ADMINISTRATIVE AND SUPPORT
Biological Support Laboratories are located adjacent to the Sample Operations Area to facilitate control, operation, and circulation of both personnel and materials.
The plant environment laboratory within this section contains four controlled environmental chambers utilized to obtain growth and development data on 21 plant species. It also houses an electron microscope capable of magnification to 80,000 x.
Brown & Root/Northrop, a joint venture of Brown & Root, Inc., and the Northrop Corporation, provides scientific services to NASA in the biological and physical sciences areas, as well as the crew area, of the Lunar Receiving Laboratory.
Mel G. Graham, senior research analyst at the LRL, will head a Brown & Root/Northrop team of x-ray and laboratory technologists, as well as three stewards, who will undergo quarantine with Apollo 11 astronauts Armstrong, Collins and Aldrin.
The firm also provides 180 technical personnel for the operation of the vacuum containment and lunar sample transfer system and the monitoring and verification of the biological containment barrier.
Designed primarily as the quarantine section for the astronauts and spacecraft, the Crew Reception Area (CRA) is the actual living quarters for quarantined personnel. It includes bedrooms and offices for the astronauts and astronaut medical team, and a dormitory, lockers, showers and toilet facilities.
The following background information on Brown & Root, Inc., and the Northrop Corporation is supplied for possible editorial use. Further information may be obtained by contacting the companies concerned.
Brown & Root, Inc., a subsidiary of the Halliburton Company, is one of the largest engineering and construction firms in the world. Employing some 20,000 people in Houston, Texas, has an annual sales volume that reaches several hundred million dollars.
FACT SHEET
BROWN & ROOT/NORTHROP Division, virtually every discipline is represented.
The Division is, in effect, a great pool of experts supported by draftsmen and other technicians from which specialists are drawn, providing the flexibility needed to meet the vast scope of projects for which the company is retained.
Brown & Root/Northrop, with its more than 800 employees, provides scientific and engineering support for NASA's Manned Spacecraft Center, Houston, Texas.
Services include feasibility studies, design, construction engineering supervision, project management, and all the steps between.
Brown & Root/Northrop, a joint venture of Brown & Root, Houston, and the Northrop Corporation of Southern California, was formed in 1964 for the purpose of joining the capabilities of the two companies to provide the kind of industrial facilities on earth including highways, dams, bridges, tunnels, power plants, pulp and paper mills, laboratories and test facilities, chemical process plants, offshore platforms, pipelines, and manufacturing plants.
Northrop Corporation is a diversified, advanced technology company operating on a worldwide basis principally in aerospace, communications, and electronics. Brown & Root, Inc., a subsidiary of the Halliburton Company, for the company activities in working for those who explore for, produce and transmit oil and gas. The company was a pioneer in designing and building offshore platforms for the production of hydrocarbons and has built more than $15-million Lunar Receiving Laboratory as well as the Space Environmental, 6,000 miles of underwater pipeline. The oilfield department provides a variety of services including the building of canals and waterways for oilfield locations access and the development of mars.
NOTE TO EDITORS: The following background material on Brown & Root, Inc., and the Northrop Corporation is supplied for possible editorial use. Further information may be obtained by contacting the companies concerned.
BROWN & ROOT, INC.
Brown & Root, Inc., a subsidiary of the Halliburton Company, is one of the largest engineering and construction firms in the world.
Employing some 20,000 persons, the Houston, Texas-based company has an annual sales volume that reaches several hundred million dollars. Its operations range throughout the free world.
In its Engineering Division, virtually every discipline is represented. The Division is, in effect, a great pool of experts supported by draftsmen and other technicians from which specialists are drawn, providing the flexibility needed to meet the vast scope of projects for which the company is retained. Services include feasibility studies, design, construction engineering supervision, project management.....and all the steps between.
Various projects engineered by the company include practically every kind of industrial facility on earth, including highways, dams, bridges, tunnels, power plants, pulp and paper mills, laboratories and test facilities, chemical process plants, offshore platforms, pipelines, and manufacturing plants.
Brown & Root's Marine, Oilfield and Pipeline Division is responsible for the company activities in working for those who explore for, produce and transmit oil and gas. The company was a pioneer in designing and building offshore platforms for the production of hydrocarbons and has built more than 6,000 miles of underwater pipeline. The oilfield department provides a variety of services including the building of canals and waterways for oilfield locations access and the development of marshland for commercial use.
(MORE)
Brown & Root has five construction divisions with worldwide activities. Ranging in scope from municipal paving to the building of multi-million dollar industrial and defense locations, Brown & Root is involved in almost every phase of today's industrial construction.
Included in the portfolio of Brown-built installations are projects in the following categories:
- Aerospace
- Chemical Plants
- Bridges
- Sulphur Plants
- Airfields
- Dams
- Tunnels
- Mining
- Public Works
- Highways
- Streets
- Steam and Electric Power Generation
- Gas Compressor Stations
- Gasoline and Gas Treating Plants
- Pulp and Paper Plants
- Townsites, Community Projects
- Research and Development Centers
- Oil Field Construction
- Marine Operations
- Steel Mills
- Metal Forming Facilities
Contact: Hal Hazelrigg...Brown & Root, Inc...P.O. Box 3...Houston, Texas
NORTHROP CORPORATION
Northrop Corporation today is a leading contributor to world technology in the areas of aircraft, electronics, communications, advanced weaponry, and space.
Founded more than 30 years ago, the company now has major engineering and manufacturing facilities in six states and the District of Columbia. The company is active in more than 50 countries. Nearly 24,000 men and women are presently employed in operations which are administered from Beverly Hills, California, and include four divisions and five wholly-owned subsidiaries, and a number of joint-venture companies.
Sales for the fiscal year ended July 31, 1968, were a record $486-million. Net earnings were $15.7-million, also a record.
Northrop expects sales to exceed $700-million by 1970. The company has set a goal of $1-billion in annual sales by 1973, evenly divided between defense and commercial programs. Greatest expansion is expected to come from growth in world transportation and communications.
Current major programs at Northrop include: construction of the main fuselage section for Boeing's 747 superjet, production of the F-5 Freedom Fighter, Doppler-Inertial Navigation System for the USAF/Lockheed C-5 cargo-transport, Omega navigation receivers for the U.S. Navy, and multi-national communications projects.
Northrop Corporation's Electro-Mechanical Division, headquartered at Anaheim, California, is the focal point for Support Operations Department activity which includes administrative interface with the Brown & Root/Northrop venture. More than 800 engineers and scientific personnel of Brown & Root/Northrop assist NASA in five laboratories at the Manned Spacecraft Center in Houston, Texas.
Contact: Roy Gregory...Northrop Corporation...1 Research Park...Palos Verdes, California
###
|
CYTOLOGICAL AND GENETICAL STUDIES ON
PUCCINIA STRIIFORMIS WESTEND.
ROBERT GEOFFREY WRIGHT, B.Sc. (Wales)
Doctor of Philosophy
Edinburgh University
1977
DECLARATION
This thesis has been composed by myself and describes experimental work which I carried out between August 1974 and August 1977.
| CONTENTS | Page |
|------------------------------------------------------------------------|------|
| Title page | i |
| Declaration | ii |
| Contents | iii |
| Acknowledgements | v |
| Summary | vi |
| Section 1 General Introduction | 1 |
| Section 2 Cytological Studies | 4 |
| 2.1 Introduction | 4 |
| 2.2 Experimental materials and methods | 11 |
| 2.2.1 Introduction | 11 |
| 2.2.2 Basidiospore studies | 12 |
| 2.2.3 Studies on leaf hyphae | 13 |
| 2.2.4 Teleutospore studies | 14 |
| 2.2.5 Studies on germinating teleutospores | 15 |
| 2.2.6 Studies on hyphal fusion | 16 |
| 2.3 Results | 17 |
| 2.3.1 Basidiospore studies | 17 |
| 2.3.2 Studies on hyphae from infected leaves | 18 |
| 2.3.3 Teleutospore studies | 22 |
| 2.3.4 Studies on germinating teleutospores | 23 |
| 2.3.5 Studies on hyphal fusion | 24 |
| 2.4 Discussion | 37 |
| Section 3 Studies on Induced Variation | 47 |
| 3.1 Introduction | 47 |
| 3.2 Experimental materials and methods | 58 |
| Section | Title | Page |
|---------|----------------------------------------------------------------------|------|
| 3.2 | Maintenance of fungal cultures | 58 |
| | Procedures for genetical studies | 59 |
| | Screening for new genotypes of yellow rust | 65 |
| | Tests on the characteristics of new genetic lines | 66 |
| | Mutation studies | 67 |
| 3.3 | Results | 71 |
| | Mixed inoculation studies | 71 |
| | The characteristics of recombinant and parental lines | 73 |
| | Mutation studies | 75 |
| 3.4 | Discussion | 79 |
| Section 4 | Studies on Quantitative Variation | 87 |
| 4.1 | Introduction | 87 |
| 4.2 | Experimental materials, methods and results | 95 |
| | General experimental procedures | 95 |
| | Quantitative variation between isolates of the pathogen | 98 |
| | Components of quantitative variation | 117 |
| 4.3 | Discussion | 123 |
| Section 5 | Concluding Discussion | 133 |
| | References | 138 |
| | Appendices | 155 |
ACKNOWLEDGEMENTS
I wish to express my sincere thanks to Dr J.H. Lennard for his advice, guidance and criticism throughout the duration of this work and in the preparation of the manuscript.
I offer my thanks to Professor N.F. Robertson for the provision of research facilities and to Dr R.C.F. Macer for proposing the research project.
I also wish to thank Dr E.G. Duncan, Dr D. Harder, Dr R. Johnson, Dr R.H. Priestley, Mr B.D. Smith and Mr G. Finney without whose help the study could not have been completed. My thanks to Mrs C.Y. Carey for typing the manuscript and also to my wife for her patience and encouragement throughout the study.
Finally I wish to acknowledge the financial assistance provided by the Ministry of Agriculture, Fisheries and Food.
SUMMARY
Cytological and genetical studies were carried out on the possible mechanism(s) involved in the production of new physiologic races of the yellow rust pathogen, *Puccinia striiformis*.
The size and structure of dividing and non-dividing nuclei in hyphae and basidiospores was ascertained using light and electron microscopy. The parallel observations of the dividing somatic nucleus by the two techniques permitted a relatively complete sequence of events to be established for mitosis. Chromosome studies revealed that *P. striiformis* has a basic chromosome number of three, although some nuclei from basidiospores were seen to possess four chromosomes. Preparations in which hyphae were possibly fusing were observed using the light and electron microscope. The results of the cytological work are discussed with reference to the physical basis of genetic variation in the yellow rust fungus.
Genetical studies included tests on the progeny of a mixture of two races, following the inoculation of susceptible and differentially resistant varieties. A new race, designated 105E137, was identified following the mixed inoculation of races 104E137 and 33E32. Induced mutation experiments established that the rate of mutation in the pathogen for specific virulence factors was less than one in $2 \times 10^7$. The results of the study are discussed and genetic models are presented to explain how race 105E137 may have evolved.
In a series of experiments on quantitative variation in *P. striiformis* differences between isolates in spore production and colony growth rate were established. An isolate of race 40E8 was the most prolific genotype in experimental studies on a range of host varieties. Isolates tended to rank similarly on the test varieties although there was some evidence of isolate-host interactions. Detailed studies on the various stages of infection revealed that the prolificacy of the race 40E8 isolate could be detected at all stages of pathogen development.
1. GENERAL INTRODUCTION
The development of the space industry has been accompanied by an increasing demand for space technology. The United States has been at the forefront of this development, with its space program being one of the most successful in the world.
In 1961, the Soviet Union launched the first human into space, Yuri Gagarin, and since then, the United States has continued to make significant contributions to the field of space exploration. The Apollo program, which culminated in the landing of astronauts on the moon in 1969, was a major milestone in the history of space exploration.
Today, space technology is used in a variety of applications, including satellite communications, weather forecasting, and navigation systems. The development of new technologies and materials continues to drive the growth of the space industry, and as we look to the future, it is clear that space exploration will continue to play a crucial role in shaping our world.
SECTION 1
General Introduction
*Puccinia striiformis* Westend. was initially described in 1818 by Schmidt and given the name *Uredo glumarum* (Grove, 1913). In 1841 J.S. Henslow recorded the presence of the fungus at Hitcham in Suffolk but at the time considered it to be a stage of *P. graminis* (Wilson and Henderson, 1966). The teleutospore stage was observed by G.D. Westendorp in 1854 and as a result of this finding the pathogen was placed in the genus *Puccinia* (Hassebrauk, 1965). The first detailed study was undertaken in 1896 by Eriksson and Henning who separated *P. rubigo-vera* DC. into two forms, *P. dispersa* (brown rust) and *P. glumarum* (yellow rust) (Grove, 1913). Until 1956 the fungus was referred to as *P. glumarum* but it was renamed *P. striiformis* by Cummings and Stevenson (1956) in their check list of North American rust fungi.
There are a maximum of five spore types in the development of a long cycled rust: spermatia; aecidiospores; uredospores; teleutospores and basidiospores. Each spore type is formed in orderly succession with the alternation of a haplophase and a diplophase. This alternation of generations is often associated with taxonomically diverse host plants. In *P. graminis*, for example, spermatia andaecidia develop on members of the Berberidaceae, while uredosori and teleutosori occur on the Gramineae. Rust species exhibiting this host alternation are described as heteroecious, while those in which the complete life cycle occurs on one host are autoecious (Wilson and Henderson, 1966).
As far as is known, *P. striiformis* has a hemi-form life cycle in which only the uredial and telial stages occur. The teleutospores germinate when mature in the autumn and following spring, to produce basidiospores. Eriksson and Henning inoculated various host genera with basidiospores but were unable to demonstrate the existence of an alternate host (Wilson and Henderson, 1966).
Yellow rust affects wheat, barley, rye and a large number of grass species. It assumes its greatest economic significance on wheat and is generally considered to be the most important rust of wheat in the United Kingdom and N.W. Europe. The fungus has a temperature optimum of 10-15°C but can tolerate extremely low temperatures. Manners (1969) has suggested there is abundant evidence to show that it can overwinter in the uredial state on wheat in parts of western Europe. The following spring the pathogen spreads from such overwintering foci. It has also been suggested that long-distance dispersal by air currents could be responsible for the incidence of yellow rust in many areas (Manners, 1969). Recently, it has been proposed that *P. striiformis* is extending its range to hotter areas of the U.S.A. (Macer, 1972) and other arid regions (Spehar, 1966; Bamadadian, 1972).
Estimates indicate that approximately 10% of world cereal production is lost due to disease caused by rust fungi (Stakman and Harrar, 1957). In the case of yellow rust of wheat in the United Kingdom, Batts and Elliott (1952) estimated that a severe attack could reduce yields by 20%. When a very susceptible variety was attacked, however, losses could be of the order of 50% (Batts, 1957) and results from controlled glasshouse experiments confirm this figure (Doodson, Manners and Myers, 1964). Doling and Doodson (1968) estimated that the maximum loss from foliage infection was approximately 30%. They suggested greater yield losses arose as a result of subsequent ear infection. Labelled C\textsuperscript{14} studies indicated that severe rust infection affected yield primarily by reducing the carbohydrate supply to the developing part of the plant and in particular to the ear (Doodson, Manners and Myers, 1965). Indeed, work by several research groups suggests that most of the assimilates synthesised by the plant following infection are incorporated into developing rust tissue (Manners, 1969).
Control of yellow rust has been principally based on the use of resistant varieties, although the number of commercial fungicides now available for use against the pathogen is increasing rapidly and several have given good results (Line, 1976). Although fungicides do not reduce the level of disease sufficiently to make the source of inoculum unimportant (Gilmour, 1976), they can be extremely successful where the host simultaneously possesses moderate levels of disease resistance (Line, 1976). Zadoks (1972) has described a simulated model for the prediction of yellow rust epidemics and such a model could enable chemical control to be a safety measure in seasons where host resistance is overcome.
Varieties bred for resistance, although providing an attractive method of minimising losses in practice, are liable to become susceptible with the emergence of new races of the fungus. The ephemeral nature of this type of resistance, i.e. race-specific resistance, is well known (Van der Plank, 1968) and highlights the problem of potential variability in pathogens. Despite the absence of a known sexual phase in *P. striiformis*, genetical changes in populations of this fungus arise and have proved of considerable practical significance. Knowledge of the nature and sources of genetical variation are of practical importance in providing a basis for a rational approach to the use of host resistance.
The present work was established in order to gain more information on the mechanism(s) responsible for the production of new races of *P. striiformis* and on the nature and extent of quantitative variation in the fungal population. It involves:
(a) A cytological study of nuclear behaviour in the pathogen.
(b) An attempt to induce variation in virulence characters.
(c) An assessment of quantitative differences between a number of isolates of the pathogen.
2. CYTOLOGICAL STUDIES
SECTION 2
Cytological Studies
2.1 INTRODUCTION
Early cytological studies on rust fungi were concerned mainly with nuclear events in teleutospore cells, involving the fusion of dikaryotic nuclei and the subsequent meiotic division of the diploid nucleus (Sappin-Trouffy, 1896; Holden and Harper, 1903). Allen (1928) investigated the development of the teleutospore stage from its dikaryotic initials in *P. striiformis* and later in *F. malvacearum* (Allen, 1933; 1935) and Savile (1939) described details of the structure and behaviour of the teleutospore nucleus during its reduction division after diploidisation in *P. sorghi* and *Uromyces fabae*. Karyogamy (nuclear fusion) and reduction division in rust fungi normally occurs in the teleutospore stage of the life cycle.
The identification of nuclear fusion outside the sexual cycle was reported to occur in *Aspergillus nidulans* (Pontecorvo and Roper, 1952). This system, referred to as the parasexual cycle, has been shown to determine variation in pathogenicity in a number of plant pathogens (Buxton, 1956) and such a discovery has stimulated great interest in nuclear behaviour in somatic cells with respect to possible scope for combining variation. Although evidence for the parasexual cycle in rust species is mainly of genetic origin (Ellingboe, 1961; Vakili and Caldwell, 1957; Sharma and Prasada, 1970) cytological studies on axenically cultured *P. graminis* (Williams and Hartley, 1971; Williams and Mendgen, 1975) have established that this fungus can exist as a somatic diploid, a condition which is essential for the operation of the parasexual cycle.
A second area of investigation, relating to the establishment of the dikaryophase, was initiated by Blackman (1904) who suggested that
the dikaryophase in *Phragmidium violaceum* was brought about by the migration of the nucleus of one cell into an adjacent one through a narrow pore. However, an alternative process, also leading to the production of a dikaryotic phase was suggested to take place in *P. speciosum*, when two adjacent cells fused to produce a single large binucleate cell (Christman, 1905). Following these reports considerable interest centred around the formation of the dikaryophase and over the next two decades the aecidial stages of some forty species were examined (Savile, 1939). All the theories advanced, however, were founded upon the assumption that the pycniospores were non-functional.
The discovery by Craigie (1927) that the pycniospores of *P. helianthi* and *P. graminis* acted as spermatia, directed the attention of cytologists into a new area of study. It was found that the dikaryotic phase arose from the fusion of spermatia and receptive hyphae, when pycnial nectar from different pustules was mixed. However, if no transfer of nectar occurred, the pustules remained sterile. Craigie's discovery initiated a considerable number of investigations on heterothallism (Allen, 1930; 1932; 1934; Craigie, 1942). Ashworth (1931) suggested that rust fungi which had rudimentary or no pycnia were mainly homothallic, while Lamb (1935) postulated that the production of well developed pycnia was indicative of a species being heterothallic. More recently, it has been suggested that most cereal rust fungi, including short cycled rusts, are heterothallic (Buller, 1950).
Plasmogamy, leading to the establishment of the dikaryotic condition, is normally associated with the spermogonium and aecidial primordia stages. However, fusion of cells may also occur during other phases of the pathogen's life cycle. Experimental studies have shown that the association of genetically dissimilar isolates during the dikaryotic (uredial) stage may give rise to new genetic combinations and it has been
suggested that such combinations arise by hyphal anastomosis followed by nuclear transfer. Such events have been suggested to take place in several rust species: *P. recondita* (Barr, Caldwell and Amacher, 1964); *P. striiformis* (Little and Manners, 1969(a); Goddard, 1974; Taylor, 1976); *Melampsora lini* (Flor, 1964) and *P. graminis* (Nelson, Wilcoxson and Christensen, 1955). Cytological evidence, however, is rather less substantial (Rodenhiser and Hurd-Karrer, 1947; Bampton and Manners, 1957; Little and Manners, 1969(b); Goddard, 1974). Fusion of germ tubes from uredospores was observed frequently in these investigations, although no conclusive evidence was obtained for nuclear transfer between isolates. Fusion bodies arising as a result of anastomosis between uredospore germ tubes were seen to contain large numbers of nuclei (Wilcoxson, Tuite and Tucker, 1958; Goddard, 1974) which suggested the possibility of nuclear transfer.
For a large proportion of the life cycle, cells of most rust fungi are dikaryotic. Nelson, Wilcoxson and Christensen (1955), however, reported an unstable trikaryon of *P. graminis tritici* from a mixture of two races. The trikaryon was virulent upon the previously resistant wheat variety Khapli. Two previously unrecorded genotypes of *P. striiformis* isolated from a mixture of two other races were found on average to contain more multinuclear uredospores than either of the parental genotypes (Little and Manners, 1969(a)). Although instability in nuclear number was one of the salient features of these new races described in *P. striiformis*, no identifiable trikaryon comparable to that described by Nelson et al. (1955) was found. Konovalova and Schekotkova (1970) isolated a new genotype of *P. striiformis*, which they considered to be a forced heterokaryon, after mixing inocula of two races. After three generations the heterokaryon reverted to its
original form. A recent investigation by Goddard (1976(a)) revealed that a new genotype evolved from a mixture of races 104E137 and 41E136 of *P. striiformis* had mainly binucleate uredospores. The new race, designated 105E137, did contain, however, a small proportion of trinucleate uredospores.
Several studies have been concerned with the nuclear size of rust fungi and these indicate dimensions of the order of about 4 µm in diameter for nuclei during the dikaryotic phase (Goddard, 1976(a); Harder, 1976(a)). Earlier studies using light microscopy reported nuclei of *P. striiformis* to be on average 0.7 µm in diameter (Little and Manners, 1969(b)). However, recent investigations have shown uredospore nuclei to be much larger, with dimensions of 4.6 x 3.4 µm, while nucleoli averaged 1.7 x 1.4 µm (Goddard, 1976(a)). Teleutospore nuclei measured in the same study were on average 6.1 µm in diameter. Maclean, Tommerup and Scott (1974) measured teleutospore nuclei of *P. graminis* using phase contrast microscopy and reported average dimensions of 5.0 µm. An ultrastructural study of hyphal nuclei of *P. graminis*, *P. recondita* and *P. coronata* (Harder, 1976(a)) revealed that fungal nuclei from infected wheat leaves were approximately 4.0 x 2.5 µm, while nucleoli measured 2.1 x 1.6 µm.
The small size of rust nuclei has made observations with the light microscope somewhat difficult to interpret and because of this little published information is available on the process of mitosis in rust fungi (Savile, 1939; McGinnis, 1953; 1954; Maclean et al., 1974; Valkoun and Bartos, 1974; Goddard, 1974). However, in recent years more interest has been shown in the mitotic process, not only in rust species but in all groups of fungi and with the aid of phase contrast and electron microscopy there is a growing body of information on this subject. It has
become apparent that certain categories of behaviour exist amongst fungi during the mitotic cycle. Mitosis has been found to occur within an intact nuclear membrane in some species of fungi (Aist, 1969; McKeen, 1972; Huang and Tinline, 1974; Harder, 1976(b)), whereas in other fungi, including a number of basidiomycetes, the nuclear membrane frequently breaks down during mitosis (Robinow, 1963; Motta, 1969). At the metaphase stage the chromosomes may be organised as a metaphase plate (Robinow, 1963; Hartmann, 1964; Ward and Ciurysek, 1961) or in a "two track" configuration (Duncan and Macdonald, 1965; Heale, Gafoor and Rajasingham, 1968; Day, 1972). Ultrastructural observations have indicated that the nucleolus may be persistent (Tanaka, 1970), semi-persistent (Huang, Tinline and Fowke, 1975) or be extruded from the nucleus during mitosis (Harder, 1976(b)).
A recent ultrastructural study of the nuclei in leaf hyphae of a number of rust fungi (Harder, 1976(a); 1976(b)) revealed that mitosis occurred within the confines of the nuclear membrane. During mitosis the spindle became orientated between two (centriolar) bodies referred to as spindle-pole-bodies (SPB) (Fulton, 1971). Following extrusion of the nucleolus, the chromosomes became organised on the mitotic spindle and following separation were seen to migrate to the polar regions of the cell. Daughter nuclei produced as a result of mitosis were then observed to move in pairs into newly formed hyphal branches.
Goddard (1976(a)) has recently reported that *P. striiformis* has a basic chromosome number n = 6. From observations on other *Puccinia* species a basic chromosome number has been reported for the following: n = 6 for *P. graminis* (McGinnis, 1953; Maclean et al., 1974); n = 6 for *P. recondita* (Valkoun and Bartos, 1974); and n = 3 for *P. coronata* (McGinnis, 1954).
It has been suggested by McGinnis (1956) that a polyploid series may exist in rust fungi, since the somatic chromosome number of some species appears to be multiples of others. In some species, such as *P. graminis*, there was some evidence at mitosis of secondary association due to residual terminal attractions. In earlier stages of division an end-to-end arrangement was seen and this was interpreted as reflecting residual homology (McGinnis, 1953). Burnett (1976) has suggested that the evidence presented by McGinnis (1953; 1954; 1956) provides a strong case for autopolyploidy in *Puccinia* species.
Knox-Davies (1967) studied nuclear behaviour in the vegetative hyphae of *Macrophomina phaseoli* and reported that mitotic irregularities were common. Although the commonest chromosome number observed was six, higher counts suggested a continual reassortment of chromosomes, with haploid, diploid, aneuploid and possibly polyploid nuclei occurring in the same mycelium. Aneuploids occur whenever diploid nuclei undergo haploidisation, the condition usually being a transient one. Kafer (1961) isolated and maintained 0.6 to 2.5% aneuploids from strains of *Aspergillus nidulans*. Aneuploids of constitution n + 1 (disomics) have arisen spontaneously in both *Neurospora* and yeast (Pittenger, 1954; Cox and Bevan, 1962) and here too they are transient conditions. Hartley and Williams (1971) have suggested that a non-reciprocal exchange of chromosomes between dividing nuclei in *Puccinia* species could result in aneuploidy. The total genetic constitution of the cell would not be altered, hence the genotype would change but not the phenotype. However, Hartley and Williams (1971) have also reported that an axenic diploid culture of *P. graminis tritici* did not dissociate to the haploid state by sequential chromosome loss but by separating into two dikaryotic daughter nuclei.
They have also suggested that the somatic hybridisation that has been observed among rust fungi may be explained by anastomosis and nuclear exchange because by its very nature a dikaryon may contain multiple genotypes within one phenotype. A dikaryotic cell has the genetic constitution of a diploid cell but each set of homologous chromosomes is enclosed by a separate nuclear membrane. If a reciprocal transfer of whole homologous chromosomes were to occur between the two nuclei of a dikaryon, the overall genetic constitution of the cell would not be altered but the make-up of the individual nuclei would be changed. Such a transfer of chromosomes has been suggested to occur at metaphase of mitosis, when the dikaryotic nuclei are in an adjacent transverse position across the cell and the division of both nuclei is progressing in a synchronised manner. However, as Hartley and Williams (1971) have pointed out, this mechanism ultimately depends on the breakdown of the nuclear membrane during mitosis.
The present cytological investigation was established in order to obtain more information on nuclear behaviour in *P. striiformis*. The primary objectives were to study mitosis in the fungus, to obtain an accurate chromosome count and to assess the involvement of various mechanisms in the evolution of new races of the pathogen.
2.2 EXPERIMENTAL MATERIALS AND METHODS
2.2.1 Introduction
A number of staining techniques have been used successfully for fungal cytology. Singleton (1953) studied chromosome morphology and behaviour in the asci of *Neurospora crassa* with the aceto-orcein stain. Nuclear division in fructifications of certain basidiomycetes was followed through meiosis and the first post-meiotic division with a modified technique, using the Giemsa stain on sectioned tissue (Duncan, 1970; Duncan and Galbraith, 1973). Aceto-orcein, Feulgen and haematoxylin were utilised to study nuclear phenomena in *Marasmius* species (Duncan and Macdonald, 1965).
In the case of rust fungi, satisfactory results were obtained using the Feulgen stain (Savile, 1939; Williams and Mendgen, 1975); Giemsa (Maclean et al., 1974; Goddard, 1974); aceto-orcein (McGinnis, 1953; 1954); acetocarmine (Kapooria, 1971) and propionic iron haematoxylin (Valkoun and Bartos, 1974). Little and Manners (1969(b)) used Heidenhain's haematoxylin to follow migrating dikaryotic nuclei and Flemming's triple stain to observe nuclei in the uredial stage of *P. striiformis*.
By studying the behaviour of living tissue with the aid of phase contrast techniques and complementing the observations with results from fixed and stained material, a greater understanding has developed of the processes involved in fungal nuclear division. Such a method of investigation has been reported on a number of fungi including *Fusarium oxysporum* (Aist and Wilson, 1966), *Ceratobasidium praticolum* (Saksena, 1961) and *Cochliobolus sativus* (Huang and Tinline, 1974).
A further technique used in fungal cytology has been interference contrast microscopy. Using this method it was postulated that *Erysiphe*
had a basic chromosome number of \( n = 2 \) (Kimber and Wolfe, 1966).
The use of the electron microscope for studies on fungal ultrastructure has added a new dimension to mycology and plant pathology (Bracker, 1967). The fine structure of rust fungi has been reported in several papers (Dunkle, Wergin and Allen, 1970; Coffey, Palevitz and Allen, 1972) and more recently electron microscopy has been utilised to investigate mitosis and cell division in a number of cereal rust fungi (Harder, 1976(a); 1976(b)).
In the current investigation observations were based on light microscopy using bright field illumination and some of the staining techniques outlined above. Transmission electron microscopy was also used to obtain more information on the detailed structure and behaviour of the somatic nucleus.
### 2.2.2 Basidiospore studies
Teleutospores of *P. striiformis* were collected from infected barley plants during the summer of 1974. Diseased straws were stored at \( 4^\circ C \), prior to being used during the following winter for cytological investigations. Straw segments bearing teleutosori were surface sterilised for 3 minutes in 1% hypochlorite solution, transferred to sterile deionised water and washed in three changes of distilled water. A high speed blender (MSE Atomix) was used to macerate the straws. After filtration, the teleutosori on the filter paper were placed on the surface of 1% water agar on a glass slide. The slide was then inverted over a clean, grease-free slide supported at each end by pith segments. All slides were placed in a plastic vinyl box and incubated at \( 10^\circ C \) in continuous light (4,000 lux) and a humid atmosphere. After 2-3 days basidiospores were discharged onto the lower slides. Approximately 12-15 hours after the initial signs of discharge, the lower slides were removed and fixed in a solution of absolute ethanol, lactic acid and glacial acetic acid (6:1:1 by volume). The slides were stored
in this fixative for up to 14 days at $4^\circ C$. After hydration in a graded ethanol series, the tissue was hydrolysed for 2 minutes in 1N HCl at $20^\circ C$ and stained in a 2% solution of acet-o-orcein in 60% glacial acetic acid (Singleton, 1953). Observations were made on a Vickers M 15C light microscope using a green filter. Photomicrographs were prepared using a semi-automatic camera loaded with Kodak microfile film.
2.2.3 Studies on leaf hyphae
(1) Light microscopy
In 1975 and 1976 seedlings of the wheat variety Sappo were inoculated with uredospores of race 104E137 of *P. striiformis* by brushing freshly collected uredospores onto the upper surface of seedling leaves. When the leaves were at the 'flecking' stage, tissue was removed for microscopical studies. Hand-cut, transverse sections were made and then immediately immersed in BAC fixative consisting of N-butyl alcohol, glacial acetic acid and 10% aqueous chromic acid (9:6:2 by volume). After storage at $4^\circ C$ for 1-3 days the sections were hydrolysed in a solution of 10.5 N HCl and absolute ethanol (1:1 by volume) for 30 minutes at $20^\circ C$. After washing in 1N HCl for 5 minutes, the sections were placed in Carnoy's solution (absolute ethanol and glacial acetic acid, 3:1 by volume) for 2-3 minutes and then stained with propionic iron haematoxylin (Lu and Raju, 1970). Slides were sealed with Depex mounting medium (Gurr), stored at $4^\circ C$ and observed on the second day after preparation.
(2) Electron microscopy
During 1976, 8-day old seedlings of the wheat variety Sappo were inoculated with race 104E137 of *P. striiformis* as previously described, placed in the infection chamber overnight and returned to the growth
chamber the following morning. Seven days after inoculation, infected areas of leaf tissue (1 cm$^2$) were removed under a solution of 2% glutaraldehyde in 0.025 M phosphate buffer at pH 6.9. After transferring the material to a vacuum chamber, it was fixed until the leaf tissue sank to the bottom of the vial. The tissue was then placed in a solution of 6% glutaraldehyde and stored overnight at 4°C. The next morning the material was washed six times, at hourly intervals, in 0.025 M phosphate buffer. After the final wash the tissue was placed in a 2% solution of osmic acid (0.025 M phosphate buffer) for 2 hours. Following this the material was washed four times at 30 minute intervals in buffer and dehydrated in a graded ethanol series. After two changes in pure ethanol the material was washed three times in propylene oxide and embedded in araldite resin. Silver and gold sections were cut on prepared glass knives using an LKB ultratome. Sections were floated onto uncoated 200 mesh grids, stained in saturated aqueous uranyl acetate (1 hour) followed by saturated aqueous lead citrate (5 minutes) and coated in carbon. Sections were examined and photographed on an AEI E.M.6 operated at 100 KV.
2.2.4 Teleutospore studies
(1) Light microscopy
Teleutosori were prepared as previously described (Section 2.2.2). At the onset of germination the teleutosori were immersed in one of a number of fixatives including Carnoy's solution, BAC fixative, Chrom-acetic solution, Bouin's fixative, Newcome's fixative, Lactic-alcohol-acetic acid solution, FAA, modified Navashin's fluid, Shaudinn's fluid and Zenker's fixative (see appendix for details). After fixation and dehydration, teleutosori were embedded in paraffin wax or water soluble aquax and sections 10 μm thick were cut using a Hearson rotary microtome.
Sections were placed on clean grease-free slides that had been smeared with Haupt's adhesive.
A number of solutions were used to stain the teleutospore nuclei. These included iron alum haematoxylin, Harris' haematoxylin, Giemsa stain, Feulgen stain, propionic iron haematoxylin, aceto-orcein and aceto-carmine. Fast green and orange G were used as counterstains with the iron alum haematoxylin staining technique. Teleutosori were also used for squash preparations, following a technique using Feulgen stain (Tommerup, personal communication) and propionic iron haematoxylin (Lu and Raju, 1970).
(2) Electron microscopy
Teleutosori were prepared and germinated as described in Section 2.2.2. At the first signs of promycelial extension, the teleutosori were immersed in a fixative consisting of 3% glutaraldehyde in 0.05 M cacodylate buffer (pH 6.9) overnight. The following morning the material was washed, post fixed and embedded as described in Section 2.2.3. Sections were cut on prepared glass knives, stained, coated in carbon and viewed on an AEI E.M.6 transmission electron microscope operating at 100 KV.
2.2.5 Studies on germinating teleutospores
Teleutosori were prepared and germinated as previously described (Section 2.2.2). With the aid of a stero-microscope, teleutosori were teased apart on a clean grease-free slide. A drop of sterile deionised water was used to suspend the teleutospores during the dissection of the teleutosorus. Each slide was then placed in a moist plastic vinyl box as a hanging drop culture. Slides were incubated at $10^\circ$C in continuous light (4000 lux). Frequent microscopic observation allowed the tissue to be fixed at a suitable stage, after approximately 4 hours
of incubation. Each slide was then dried on a hot plate at 60°C for 1-2 minutes and fixed in Singleton's fluid (Absolute ethanol, lactic acid, glacial acetic acid, 6:1:1 by volume, Singleton, 1953). The tissue was fixed for 4-6 hours at 20°C, prior to being hydrated in a graded ethanol series and hydrolysed in 1N HCl for 5 minutes (20°C). After washing in 60% glacial acetic acid the slides were stained with 2% aceto-orcein. Preparations were sealed with Depex (Gurr), stored at 4°C and observed the following day.
2.2.6 Studies on hyphal fusion
(1) Light microscopy
Seedlings of the wheat variety Sappo were inoculated with a mixture of isolates 104E137 (m) and 33E32 (1:1 by weight) and incubated as described in Section 2.2.3. When heavy flecking was visible in the leaf, tissue was removed for microscopical studies. With the aid of a sharp double-edged razor blade and fine artery forceps, the upper epidermis was removed from the seedling leaf. The remaining tissue was then cut into 2 to 3 mm squares and fixed immediately in the BAC fixative, prior to staining with the propionic iron haematoxylin stain. Observations were made on the second day after preparation using a Vickers M15C microscope and a semi-automatic camera loaded with Kodak microfile film.
(2) Electron microscopy
Seedlings of the wheat variety Sappo were inoculated with a mixture of isolates 104E137 (m) and 33E32 (1:1 by weight) and the tissue prepared as described earlier for electron microscopy on leaf hyphae (Section 2.2.3). Lateral sections were cut using glass knives, stained in aqueous uranyl acetate (1 hour) followed by saturated aqueous lead citrate (5 minutes) and coated in carbon. Sections were examined and viewed on an AEI E.M.6 operated at 100 KV.
2.3 RESULTS
2.3.1 Basidiospore studies
Basidiospores of *F. striiformis* were approximately 12 x 8 μm at maturity. Initially each basidiospore possessed a single, oval nucleus, but soon became binucleate due to a mitotic division. The non-dividing nuclei measured 3.0-3.5 μm in diameter when stained with 2% aceto-orcein. Non-dividing nuclei contained weakly stained chromatin which showed no obviously organised structure. At the periphery of the nucleus a small, oval, densely stained structure (0.8-1.0 μm in diameter) could be seen (Fig. 1). This structure appeared to be associated with the nuclear membrane and was considered to be the spindle-pole-body, SPB (Fulton, 1971).
The start of mitosis was indicated by the duplication of the SPB. Two daughter bodies (approximately 0.5 μm in diameter) were produced and migrated to opposite ends of the nucleus (Fig. 2). At what seemed to be a later stage, strands of deeply stained material become visible (Fig. 3). This stage of chromatin condensation was considered to represent prophase. The configuration of chromatin was complex: in some instances it was spirally arranged (Figs. 4 and 5). In some cells ring-shaped structures were observed (Fig. 6). These rings were composed of deeply stained granules interconnected by fine, orcein-positive threads. Individual chromosomes could not be seen at this stage, but in some rings a break in the chromatin was noted (Fig. 6).
With further condensation of the chromatin, distinct chromosomal bodies were evident but no metaphase plate was seen (Fig. 7). The chromosomal bodies appeared to be joined by very fine threads of material. The spindle pole bodies could not be seen at this stage. Twenty-one cells were counted at metaphase, when the chromatin bodies seemed most condensed,
and 16 were found to contain three chromosomal bodies (Fig. 7), each being between 0.7 and 0.9 μm in diameter. Five other cells appeared to possess four chromosomal bodies (Fig. 8). Evidence of chromosome disjunction was seen in ten cells with the formation of six chromatids (Fig. 9). The size of each chromatid varied between 0.4 and 0.6 μm in diameter, this being approximately half that of the parental metaphase chromosomes.
Each set of daughter chromatids was joined into a continuous chain interconnected by fine threads of stained material (Figs. 10 and 11). As the chromatids moved apart individual chromosomes lost their identity (Fig. 12). The final product of mitosis was two deeply stained daughter nuclei situated at each end of the basidiospore (Figs. 13 and 14). These bodies became more diffuse and reassumed a typical non-dividing nuclear state. The behaviour of the nucleolus was not clearly followed when cells were stained with aceto-orcein. However, in some dividing cells a faintly staining body could be seen in the cytoplasm and was probably the nucleolus as observed in studies on hyphae from infected leaves.
2.3.2 Studies on hyphae from infected leaves
The hyphae of *P. striiformis* contained many non-dividing nuclei that were irregularly oval in shape with average dimensions of 3.2 x 2.1 μm (Figs. 15 and 16). Mitosis was frequently observed in the two apically situated dikaryotic nuclei. These two nuclei were seen to undergo division side by side or, occasionally, in tandem. The division progressed in a synchronised manner. The first indication of nuclear division was judged to be the ejection of the nucleolus from within the nucleoplasm (Fig. 17). The nucleolus at this time measured about 0.8 x 0.6 μm. After nucleolar extrusion the SPB on the periphery of the nucleus replicated and each nucleus had two darkly stained granular bodies present (Fig. 18). Fig. 18 illustrates an early divisional pair.
of dikaryotic nuclei: the chromatin is situated between two daughter bodies but individual chromosomes are not clear.
The behaviour of the chromatin, however, could not be clearly followed in hand sectioned material stained with propionic iron haematoxylin. Although nuclei in the middle stages of mitosis were seen, accurate chromosome counts could not be made due to poor resolution. Following nuclear division the four daughter nuclei could be clearly seen in the apical cell (Fig. 19). These appeared to measure from 0.9-1.5 μm in diameter, approximately half the size of the initial dikaryotic nuclei.
From electron microscope studies leaf hyphae were seen to contain a number of nuclei that were oval in shape with average dimensions of 3.8 x 2.8 μm (Figs. 20 and 21). Each non-dividing nucleus was surrounded by a double membrane which was interrupted by scattered pores (Fig. 22). A disc-like SPB was situated in a shallow pocket in the nuclear envelope and appeared to be associated with a less densely staining amorphous layer lying within the nucleoplasm (Figs. 23 and 24). Contact was maintained between the SPB and the amorphous region by pores situated in the nuclear envelope (Figs. 25 and 26).
In the nucleoplasm heterochromatic regions were present (Fig. 22). An electron dense nucleolus could also be seen either in the centre or to one side of the nucleus (Figs. 20, 21 and 22). The dimensions of the nucleolus were on average 1.5 x 1.3 μm but showed a wide variation (0.8 x 0.6 to 2.0 x 1.5 μm). In some cells the nucleolus occupied over 50% of the total visible area of the nucleus, whereas in others it was no more than 10%. Part of this variation could be attributed to irregularities in nucleolar shape in relation to the plane of sectioning. However, it was felt that sufficient observations were made on individual nuclei to suggest that different cells varied considerably in their
nucleolar size. The nucleolus was composed of distinctly different components, with a number of centrally positioned lighter staining regions (Figs. 21 and 22). These regions have been termed lacunae (Chouinard, 1974; Harder, 1976(a)), and exhibit similar staining properties to those of the nucleoplasm. Granular regions of material were distributed throughout the nucleolus along with less distinct fibrillar regions (Fig. 27).
Nuclear deformation followed by nucleolar extrusion were the first indications of the onset of mitosis (Fig. 28). Frequently the nucleolar material was seen in a projecting region of the nuclear envelope and in some instances practically the whole of the nucleolus could be seen in this projecting zone (Fig. 29). Eventually, the portion containing the nucleolar material was pinched free of the main nuclear body (Fig. 30). This extruded portion, however, did not always carry with it the whole of the nucleolus. After detachment from the main nuclear body the extruded portion moved into the cytoplasmic matrix and dispersed nucleolar material was observed.
Division of the SPB appeared to occur after nucleolar extrusion. The two daughter bodies that were formed came to lie at opposite ends of the nucleus and an intranuclear spindle was established between these two bodies (Figs. 31 and 32). In Fig. 31 the microfibrils can be seen to radiate from the SPB situated in the lower region of the cell. Serial sections through a number of nuclei indicated that some spindle microfibrils ran from pole to pole (Fig. 32). In Fig. 33 shorter microfibrils are seen to run from the pole to the chromosomal structures. The two types of spindle material were observed in mitotic nuclei. No conclusive evidence was obtained for the presence of kinetochores within the chromosomal material, although the cell shown in Fig. 33 may provide an
example. Cytoplasmic microtubules were observed, but no connection was found between these and the SPB.
Condensed chromatin was not observed in nuclei during early prophase. The dikaryotic nuclei during this stage became squarer with an out-pocketing of the nuclear envelope at the poles (Fig. 32). Metaphase was considered to be represented by the arrangement of chromosomal bodies in a band across the width of the nucleus (Fig. 34). The nuclei at this stage commonly had a spur-like projection in one corner and were surrounded by numerous mitochondria, multi-vesicular bodies and large amounts of endoplasmic reticulum. The endoplasmic reticulum was commonly seen to be in close association with the nuclear membrane (Figs. 31, 32 and 33). A later stage in division, just after the daughter chromosomes have separated, may be seen in Fig. 35. The chromatids are widely distributed within the nucleus and there is some evidence of the presence of spindle material. The SPBs do not appear as prominent as they were during early division.
Towards the end of mitosis the nuclear envelope became constricted and dumbell-shaped nuclei were formed (Fig. 36). The nucleus appeared to cleave apart with a breakdown of the nuclear envelope in the central region. Few cells were observed at this stage and it is likely that daughter nuclei separate rapidly. As the daughter nuclei moved apart the products of mitosis could be seen as four nuclei with average dimensions of approximately 1.8 x 1.4 μm (Fig. 37). Nucleoli were formed soon after the separation of these daughter nuclei, apparently by de novo synthesis (Fig. 38). A diagrammatic representation of mitosis is given in Fig. 39.
2.3.3 Teleutospore studies
The two-celled teleutospores of *P. striiformis* measured approximately 70 x 18 µm with the light microscope. Mesospores were also frequently observed and were approximately half the size of typical two-celled teleutospores. Both types of spore were dark, yellowish-brown in colour and were retained within a compound sorus.
Non-dividing teleutospore nuclei measured 5 µm in diameter, when fixed in FAA and stained with Heidenhain's iron-alum haematoxylin. Most teleutospore nuclei observed were more regular in shape than leaf hyphae nuclei (Fig. 40). The squash technique using propionic iron haematoxylin (Lu and Raju, 1970) on freshly hydrated teleutospores was successful, whereas the squash technique using Feulgen stain did not differentiate any nuclei. Results obtained on sectioned tissue using the Giemsa and Feulgen stains were disappointing. The cytoplasmic material of the teleutospore contained a substance that had an affinity for these two stains and no nuclear differentiation was achieved. The aceto-orcein and aceto-carmine stains were also unsuccessful on sectioned teleutospore material.
Although reasonable results were obtained when staining non-dividing nuclei, no success was achieved when staining germinating teleutospores. A range of fixatives and stains were used on this material but no dividing meiotic nuclei were seen during the investigation.
Sectioned material observed on the electron microscope revealed an electron dense structure, centrally positioned within the teleutospore. This structure had dimensions of 6 x 5 µm and was considered to be the diploid teleutospore nucleus. Little detail was apparent however and no nucleolus, SPB or nuclear membrane could be identified (Fig. 41). The centrally positioned electron dense structure was surrounded by aggregates of material of a similar nature and large amounts of lipid material were present.
2.3.4 Studies on germinating teleutospores
Teleutospores of *P. striiformis* germinated readily in hanging drop cultures, exhibiting no dormancy period. Each cell produced a prominent promycelium which frequently grew to approximately 100 μm in length. The promycelium emerged from near the apex of the upper teleutospore cell and from close to the dividing septum in the lower, basal cell. Growth of each promycelium continued unrestricted when humid conditions were provided.
The diploid nucleus underwent meiosis within the teleutospore cell. When the promycelium was 40-60 μm in length, the haploid meiotic nuclei could be observed migrating along the promycelium towards the apical region (Fig. 42). Septa were produced in an acropetal arrangement within the promycelium and separated the daughter nuclei. The result was a promycelium with four uninucleate cells and a fifth cell at the base of the promycelium, which was anucleate.
In fixed and stained material nuclei could be seen situated along the length of the promycelium. The chromatinic material of each nucleus frequently adopted a ring-like or dumbbell-shaped configuration (Figs. 42 and 43). Some nuclei appeared to be in the process of mitotic division as they migrated along the promycelium. An example of this can be seen in Fig. 44. The micrograph shows a four-celled promycelium. In one cell three deeply stained structures are visible and may represent chromosomes of a nucleus during the first post-meiotic division.
Each of the four cells forming the promycelium produced a papillar sterigmata approximately 6-10 μm in length. The four sterigmata frequently arose from one side of the promycelium (Fig. 45). Basidiospores were produced initially on the sterigmata of the apical promycelial cell; the later formed basal cells being somewhat slower
in initiating basidiospores (Fig. 46). The haploid nuclei became extremely elongate and were seen to migrate through the sterigmata and into the basidiospore. Young basidiospores were mainly uninucleate (Fig. 46) but soon became binucleate by an early mitotic division (Fig. 47).
2.3.5 Studies on hyphal fusion
Seedling leaves approximately 5-7 days after inoculation provided the best material for studying the possibilities of fusion between leaf hyphae. Fungal tissue at this stage did not occupy excessively large areas of the leaf and allowed distinction to be made between independent hyphal cells. Also at this time large runner hyphae were produced and frequently grew 300-400 μm longitudinally through the leaf. These runner hyphae were approximately 10 μm in diameter and on numerous occasions made contact with other cells in the intercellular spaces beneath the stomata. Infected tissue observed 8-10 days after inoculation had the intercellular spaces packed with ramifying hyphal cells and identification of hyphal connections was difficult.
In Figs. 48 and 49 numerous fungal nuclei can be seen distributed along the runner hyphae. The two hyphal cells were followed back to their initial source and ascertained to originate from separate infections. Contact had occurred between the two hyphal cells and differential focusing indicated that erosion of the hyphal walls had taken place on the lower side of the vertically running hyphal cell. However, no further evidence for hyphal fusion was observed. Forty-six slides were prepared and screened but difficulties in resolving the regions of contact made the possibility of identifying fusion bodies difficult.
A number of instances were observed with the electron microscope in which fungal hyphae had made contact and were possibly at some stage
of the fusion process. In Fig. 50 the apical regions of two hyphae are in contact. An enlarged micrograph (Fig. 51) reveals that the zone of contact between the two cells has undergone some modification or dissolution, although no continuity exists between them. Fig. 52 represents what may be a more advanced stage in the process of hyphal fusion. Observation of several serial sections indicated that the hyphae present may have represented two independent cells which had fused. A large number of mitochondria are present in the lower apical cell and may be indicative of increased metabolic activity which would be associated with fusion.
| Abbreviation | Description |
|--------------|------------------------------|
| am | amorphous region |
| ch | chromosome |
| dch | daughter chromosome |
| er | endoplasmic reticulum |
| fn | fungal nucleus |
| ha | haustorial cell |
| hn | host nucleus |
| hy | intercellular fungal hyphae |
| k | kinetochore |
| la | lacuna |
| m | mitochondria |
| mt | spindle microfibrils |
| mvb | multivesicular bodies |
| ne | nuclear envelope |
| np | nuclear pore |
| nu | fungal nucleolus |
| se | septum |
| spb | spindle-pole-body |
| v | vacuole |
Fig. 1 A non-dividing nucleus of *P. striiformis* x 5000.
Fig. 2 Daughter SPBs soon after SPB division has occurred x 4000.
Fig. 3 An early prophase nucleus x 2500.
Fig. 4 A later prophase stage x 5500.
Fig. 5 A later prophase stage x 5500.
Fig. 6 A prophase ring structure containing densely stained granular bodies. A prominent break in the chromatin is visible x 5000.
spb
1 2 3 4 5 6
Fig. 7 Metaphase stage showing three chromosomes x 4000.
Fig. 8 Metaphase stage showing four chromosomes x 2000.
Fig. 9 Post metaphase stage. One set of daughter chromosomes is visible and fibril-like connections are present x 3500.
Fig. 10 Anaphase separation. Chromosomal connections are again present x 5000.
Fig. 11 Late anaphase. Chromosomal connections are present and daughter chromatids are part way to the polar regions x 2000.
Fig. 12 Late separation stage x 3000.
Fig. 13 Daughter nuclei after mitosis in basidiospore x 3000.
Fig. 14 Daughter nuclei after 2nd mitotic division in basidiospore x 3000.
7. ch
8. ch
9. dch
10. dch
11.
12.
13.
14.
| Fig. 15 | Hyphae of *P. striiformis* showing hyphal tissue, host nucleus and fungal nuclei x 1500. |
|---------|-------------------------------------------------------------------------------------|
| Fig. 16 | Apical regions of hyphae are visible where mitosis is occurring x 1500. |
| Fig. 17 | Apical cell of hypha. A nucleolus can be seen lying outside the daughter nucleus x 3000. |
| Fig. 18 | Early dividing nuclei in an apical cell. SPBs can be clearly seen x 3000. |
| Fig. 19 | Daughter nuclei formed as a result of mitosis x 3000. |
Figures 15-19
Figure 15: Hyphae (hy) and hyphal nuclei (hn) with fungal nuclei (fn).
Figure 16: Hyphae with fungal nuclei (fn) and hyphal nuclei (hn).
Figure 17: Nuclei (nu) in hyphae.
Figure 18: Sporangia (sp) and sporangial bases (spb).
Figure 19: Sporangia (sp).
Fig. 20 Longitudinal section through a hypha of *P. striiformis* x 8000.
Fig. 21 Section through several fungal nuclei. The lighter staining lacunar regions within the fungal nucleolus are visible x 12,000.
Fig. 22 A non-dividing nucleus of *P. striiformis* x 16,000.
Fig. 23 Micrograph showing detail of the SPB x 32,000.
Fig. 24 Longitudinal section through a SPB x 64,000.
Fig. 20. A section through the anterior end of a mature oocyte. The nucleus (n) is located in the posterior half of the cell. The cytoplasm contains numerous mitochondria (m), a large amount of rough endoplasmic reticulum (r), and a few lipid droplets (ld). The Golgi apparatus (g) is located near the nucleus. The cytoplasmic membrane (cm) is clearly visible.
Fig. 21. A section through the anterior end of an oocyte. The nucleus (n) is located in the posterior half of the cell. The cytoplasm contains numerous mitochondria (m), a large amount of rough endoplasmic reticulum (r), and a few lipid droplets (ld). The Golgi apparatus (g) is located near the nucleus. The cytoplasmic membrane (cm) is clearly visible.
Fig. 22. A section through the anterior end of an oocyte. The nucleus (n) is located in the posterior half of the cell. The cytoplasm contains numerous mitochondria (m), a large amount of rough endoplasmic reticulum (r), and a few lipid droplets (ld). The Golgi apparatus (g) is located near the nucleus. The cytoplasmic membrane (cm) is clearly visible.
Fig. 23. A section through the anterior end of an oocyte. The nucleus (n) is located in the posterior half of the cell. The cytoplasm contains numerous mitochondria (m), a large amount of rough endoplasmic reticulum (r), and a few lipid droplets (ld). The Golgi apparatus (g) is located near the nucleus. The cytoplasmic membrane (cm) is clearly visible.
Fig. 24. A section through the anterior end of an oocyte. The nucleus (n) is located in the posterior half of the cell. The cytoplasm contains numerous mitochondria (m), a large amount of rough endoplasmic reticulum (r), and a few lipid droplets (ld). The Golgi apparatus (g) is located near the nucleus. The cytoplasmic membrane (cm) is clearly visible.
| Fig. 25 | Nucleus of *P. striiformis*, showing a large pore in the nuclear envelope directly beneath the SPB x 16,000. |
|---------|--------------------------------------------------------------------------------------------------|
| Fig. 26 | Enlarged micrograph of section seen in Fig. 25. Note the multivesicular body near the SPB x 32,000. |
| Fig. 27 | Nucleolar material of *P. striiformis* x 32,000. |
| Fig. 28 | Early stage in extrusion of nucleolar material x 13,000. |
| Fig. 29 | Nucleolus in a projecting zone of the fungal nucleus x 13,000. |
| Fig. 30 | Portion of nucleus containing nucleolar material is extruded from the main nuclear body x 20,000. |
| Fig. 31 | Mitotic nucleus, with SPB and spindle microfibrils visible. Notice the multivesicular bodies and endoplasmic reticulum in close contact with the nucleus x 32,000. |
25-31
25: np
26: np, mvb
27: mvb
28: mvb
29: nu
30: fn, nu
31: mt, spb, mvb, fp
Fig. 32 Mitotic nucleus, showing spindle material running the entire length of the nucleus. Chromatin is centrally positioned but showing little organisation x 32,000.
Fig. 33 Mitotic nucleus showing a possible kinetochore x 60,000.
mt
ne
er
mt
k
Fig. 34 Mitotic nucleus in which discrete chromosomes are visible x 32,000.
Fig. 35 Section of a mitotic nucleus. The daughter chromosomes have moved towards the polar regions x 60,000.
Fig. 36 Mitotic nucleus just after the daughter nuclei have separated x 8000.
Fig. 37 Section through hyphae showing the products of mitosis x 13,000.
Fig. 38 Three daughter nuclei are visible. A nucleolus has formed in one of the cells x 13,000.
ne
er
mt
ch
ch
ch
mvb
fn
fn
fn
fur
Fig. 39. A diagrammatic representation of the process of mitosis in *P. striiformis*
1. Non-dividing nucleus, showing the nucleolus (nu) and spindle pole body (spb).
2. The nucleolus passes into a projection of the nuclear envelope and it is either partly or completely extruded.
3. Spindle formation is taking place in one corner of the nucleus, following replication of the spb.
4. The spindle consists of continuous and discontinuous microfibrils (mt), and frequently chromosomal structures can be seen within the cell.
5. The two daughter nuclei are forming, following chromosomal dis-junction and spindle extension.
6. Two daughter nuclei (fn) are formed and nucleoli soon become visible.
Fig. 40 Teleutospore nuclei x 3000.
Fig. 41 Electron micrograph of teleutospore nucleus x 6000.
Fig. 42 A promycelial cell showing migrating nuclei x 2000.
Fig. 43 A promycelial cell containing ring-like and dumbbell-shaped nuclei x 1500.
Fig. 44 A promycelial cell containing a nucleus, possibly in the process of mitosis x 2000.
Fig. 45 A mature promycelium. The sterigmata are frequently seen to arise from one side x 2000.
Fig. 46 A mature basidiospore of *P. striiformis*. A single nucleus is visible x 2000.
40. 41. 42. 43. 44. 45. 46.
| Fig. 47 | Basidiospores just prior to release from the promycelium. Some are already binucleate x 2000. |
|---------|------------------------------------------------------------------------------------------------------------------|
| Fig. 48 | Fungal hyphae of *P. striiformis* in contact in the intercellular leaf spaces x 1500. |
| Fig. 49 | Enlarged micrograph of Fig. 48. Hyphae may be in the early stages of fusion x 3000. |
| Fig. 50 | Electron micrograph showing two leaf hyphae colliding in the intercellular spaces x 8000. |
| Fig. 51 | Enlarged micrograph of Fig. 50 x 15,000. |
| Fig. 52 | Electron micrograph showing two hyphae that may have fused x 10,000. |
47
hn
hy
fn
50
51
52
2.4 DISCUSSION
Harder (1976(a)) recorded dimensions in the region of 4.0 x 2.5 µm for non-dividing nuclei and 2.1 x 1.6 µm for nucleoli observed in the fungal hyphae of *P. graminis*, *P. recondita* and *P. coronata*. In *P. striiformis*, the average sizes of uredospore nuclei and nucleoli were reported to be 4.6 x 3.4 µm and 1.7 x 1.4 µm respectively (Goddard, 1976(a)). Earlier studies (Little and Manners, 1969(b)) suggested that the uredospore nuclei of *P. striiformis* were less than 1.0 µm in diameter but as suggested by Goddard (1974) such dimensions are probably representative of the nucleolus and not the fungal nucleus. In the present study non-dividing nuclei in basidiospores had dimensions of 3.5 µm in diameter and those in hyphae from infected leaves 3.8 x 2.8 µm. Harder (1976(a)) found that the size of nuclei in the leaf hyphae of several rust species was relatively constant. However, he noted (Harder, 1976(c)) that the size of fully developed nuclei in pedicels differed from those in sporogenous cells and leaf hyphae. The slight difference in the average size of nuclei recorded in this investigation from those reported by Goddard (1976(a)) may be related to the different types of cells examined in the two studies: in keeping with this view the results of observations reported in Section 3.3.2 show that non-dividing uredospore nuclei had an average diameter of 4.5 µm.
Considerable variation in nucleolar size was reported by Harder (1976(a)). According to Harder, nucleolar size is influenced by the physiological condition of the cell and the cell type. Nucleoli in the leaf hyphae of *P. striiformis* were on average 1.5 x 1.3 µm, while Goddard (1976(a)) reported average dimensions for nucleoli in uredospores as being 1.7 x 1.4 µm.
Teleutospore nuclei had average dimensions of 6.0 x 5.0 µm. According to Savile (1939) this larger size relates to their diploid
condition and is in agreement with the dimensions reported by Goddard (1976(a)) for *P. striiformis* and those reported for *P. graminis* by Maclean et al. (1974).
The nucleus of *P. striiformis* was surrounded by a double membrane frequently interrupted by scattered pores, typical of all eucaryotic species (Bracker, 1967). The SPB, although situated outside the nucleus was contiguous with an amorphous region in the nucleoplasm by means of pores in the nuclear envelope. A similar structural arrangement has also been observed in *Fusarium oxysporum* (Aist and Williams, 1972), *Erysiphe graminis* (McKeen, 1972) and several *Puccinia* species (Harder, 1976(a)). It has been suggested that the amorphous region associated with the SPB is a zone of heterochromatin in *P. graminis* (Dunkle, Wergin and Allen, 1970). However, Huang, Tinline and Fowke (1975) have proposed that such amorphous material may be involved in organising microtubules during mitotic division in *Cochliobolus sativus*.
The nucleoli observed in *P. striiformis* appeared similar to those seen in higher plants (Chouinard, 1974) and other rust fungi (Harder, 1976(a)), with light-staining lacunar regions, granular regions and fibrillar regions. The lacuna have been described as meandering channels and are connected with the nucleoplasm, which they resemble in appearance. Harder indicates that ribonucleoprotein synthesis, the primary function of the nucleolus, involves the conversion of fibrillar material into granular material.
Due to the small size of rust nuclei, attempts to follow events during mitosis using only light microscopy are difficult. However, when observations with the light microscope are combined with results obtained from electron microscopy, a clearer picture is obtained of the sequence of mitosis.
The extrusion of the nucleolus as a preliminary to mitosis has been observed in a number of other investigations on rust fungi (Savile, 1939;
Various forms of nucleolar behaviour have however been recognised in other fungi: in some species, the nucleolus may persist during division and pass to daughter nuclei (Tanaka, 1970; Heath, 1974); in others it may be semi-persistent and be dispersed at the end of nuclear division (Huang et al., 1975); in a third group it may be lost during prophase, as in higher plants (Ichida and Fuller, 1968). The early extrusion of nucleolar material during mitosis appears to occur only in the rust fungi (Harder, 1976(b)). Harder has suggested that a variable number of nuclear fragments containing nucleolar material may be extruded early in the mitotic cycle and that this could account for the significant reductions he observed in nuclear size during mitosis, a reduction also observed in the present study.
At an early stage in division the SPB replicated and formed two daughter bodies which became situated at opposite ends of the mitotic nucleus. The presence of a SPB has been recorded in other investigations (Robinow and Caten, 1969; Brushaber and Jenkins, 1971; Valkoun and Bartos, 1974). The daughter SPBs observed in the mitotic nuclei of *P. striiformis* (0.4-0.5 μm) were larger than similar structures (less than 0.3 μm) reported for *P. recondita* (Valkoun and Bartos, 1974). As division progressed in *P. striiformis* it became more difficult to recognise the SPBs and frequently at metaphase and anaphase stages they could not be clearly resolved. In *P. striiformis* and a number of other fungi (McKeen, 1972; Harder, 1976(a)) spindle microfibrils passed into the internally situated amorphous region and then on to the disc-like SPB by means of pores in the nuclear envelope. However, Huang et al. (1975) reported that in *C. sativus* the spindle microfibrils appeared to terminate at the nuclear envelope. The intranuclear spindle observed in the nuclei of *P. striiformis* was situated between two daughter SPBs and consisted of pole to pole and pole to chromosome fibres. Similar fibres have been
described in other fungi (Ichida and Fuller, 1968; McKeen, 1972). Harder (1976(b)) has suggested that the pole to chromatid fibres are involved in chromatid disjunction and the pole to pole fibres involved in daughter nuclei separation.
Day (1972) considered that metaphase plates, as observed in higher eucaryotes, are not a regular feature of dividing fungal nuclei. In some fungi a "two track" or "double bar" stage splits transversely to produce two daughter nuclei (Duncan and Macdonald, 1965; Heale, Gafoor and Rajasingham, 1968; Robinow and Caten, 1969). In other fungi a "metaphase bar" configuration has been observed (Bakerspigel, 1959). There are, however, reports of a discrete metaphase stage in several fungi: *C. sativus* (Huang and Tinline, 1974); *E. graminis* (McKeen, 1972); *Uromyces fabae* (Kapooria, 1971) and several *Puccinia* species (McGinnis, 1953; 1954; Valkoun and Bartos, 1974). The present study indicates that in *P. striiformis* metaphase could be identified and distinct chromosomal bodies were visible.
It was concluded from the present investigation that *P. striiformis* had a basic chromosome number $n = 3$, as seen in haploid basidiospore nuclei. This was the number most frequently recorded in a metaphase equivalent stage and the observations are very similar to those illustrated for *P. coronata* by McGinnis (1954). The higher count ($n = 6$) recorded by Goddard (1976(a)) for *P. striiformis* may have been due to condensed areas of chromatin in early divisional cells being considered as distinct chromosomes. Robinow and Caten (1969) have concluded that the number of condensed regions of chromatin seen during early division cannot be equated to the number of chromosomal bodies present at metaphase of mitosis.
The presence of four chromosomes in a number of basidiospores of *P. striiformis* suggests that aneuploid nuclei may occur in this species. Knox-Davies (1967) reported the presence of aneuploid nuclei in the fungus *Macrophomina phaseoli* and suggested that a continual reassortment
of chromosomes was taking place. Aneuploid nuclei occur when diploid nuclei undergo haploidisation or whenever non-disjunction takes place during chromatid separation (Burnett, 1975). Such an increase in chromosome number could result in an increase in the virulence spectrum of *P. striiformis*. However, aneuploid cells are not normally as vigorous as euploids (Burnett, 1975). The result of aneuploid nuclei in *P. striiformis* could therefore reduce the vigour of such a genotype, thereby imposing a strong selection pressure against its successful establishment in a population. However, it should be noted that the basidiospores of *P. striiformis* are non-functional, as there is no known secondary host. The additional chromosome recorded in basidiospore nuclei may have arisen from non-disjunction during the preceding meiotic division in the germinating teleutospore. Aneuploid cells have not been recorded in previous cytological investigations on rust fungi (McGinnis, 1953; 1954; Valkoun and Bartos, 1974; Maclean *et al.*, 1974; Goddard, 1976(a)).
The chromosomes of *P. striiformis* were similar in size, being 0.7 to 0.9 μm in diameter, while the daughter chromatids varied between 0.4 and 0.6 μm in diameter. These dimensions are similar to those reported for *P. graminis* (McGinnis, 1953), *P. coronata* (McGinnis, 1954) and *P. recondita* (Valkoun and Bartos, 1974).
In order to obtain a confirmatory chromosome count, germinating teleutospores were prepared, stained and examined under the light microscope. However, difficulties experienced in staining the dividing meiotic nucleus prevented such a count being made. Similar problems in differentiating meiotic nuclei were expressed by Savile (1939) and other investigators (Maclean *et al.*, 1974). The cytoplasmic matrix of the teleutospore contained a substance that prevented differentiation of the nuclei when using the Feulgen or Giemsa stain. These two stains are used
widely and are noted for their specificity and excellent differentiation of genetic material. Their use with fungi would seem to be limited, however, especially when cytoplasmic material similar to that in *P. striiformis* is present.
In some rust fungi meiosis takes place within the promycelium (Kapooria, 1971; Savile, 1939). After failing to stain dividing teleutospore nuclei, it seemed that a study of the promycelial cell could offer a suitable alternative, especially if meiotic division could be delayed and studied while taking place in the promycelium. However, teleutospore germination, although not difficult to induce, only occurred under carefully controlled environmental conditions and in these investigations meiosis itself was always completed before any nuclei migrated into the promycelium.
The promycelium was five-celled, the lower cell being anucleate. This was similar to promycelial formation reported in *P. penniseti* (Kapooria, 1968). The condition of the haploid nuclei varied from cell to cell, but in a number of cells the nuclei appeared to be undergoing mitosis in the promycelium. Some nuclei revealed a number of deeply staining bodies, that could have been chromosomal structures. Rust basidiospores are extremely delicate structures and the preparation of these nuclei for division may be a survival mechanism, leading to rapid germination of the basidiospore and infection of the alternate host.
During cytological studies on basidiospore nuclei it was observed that a proportion of spores were bi-nucleate, while others were uni-nucleate, shortly after their release from the promycelium. Depending upon environmental conditions, some nuclei may undergo part of the first mitotic division prior to migration into the basidiospore cell. This non-synchronisation of nuclear division would be advantageous and assist in survival and infection of a secondary host.
Investigations with the light microscope on *P. striiformis* revealed the presence of interconnecting filaments between the chromosomal bodies during mitosis. A number of studies have reported similar structures in other basidiomycetes, e.g. *Puccinia* species (McGinnis, 1953; 1954); *Marasmius* species (Duncan and Macdonald, 1965); *Ustilago violacea* (Day and Jones, 1968) and other fungi such as *Neurospora* (Dowding and Weijer, 1960) and *Verticillium* species (Heale, Gafoor and Rajasingham, 1968).
Weijer and MacDonald (1965) concluded that the interconnecting filament did not contain D.N.A. because it did not stain with Feulgen. Differentiation of the interconnecting fibrils using Feulgen was not possible in this study due to the excessive period of hydrolysis that was necessary and, therefore, no conclusions could be drawn about the properties of these structures. McGinnis (1956) has suggested that a polyploid series may exist in *Puccinia* species. The presence of interchromosomal attachments could therefore be indicative of residual homology and of a species being a functional autopolyploid. However, this explanation is not in keeping with the chromosome number of three recorded for *P. striiformis* in this study. Wagenaar (1969) has proposed that the interchromosomal attachments he observed in *Allium* and *Crepis* species were responsible for the orderly pairing of homologous chromosomes during meiosis. Other reports have suggested that these structures are artefacts of the staining process (Day, 1972) or are otherwise due to chromosomes being closely aligned on a narrow spindle (Aist, 1969). A considerable number of studies, using a wide variety of techniques and stains, have identified the presence of such structures and this fact lends strong support for their existence.
A number of theories have been put forward regarding the mechanism(s) responsible for the origin of new physiologic races of rust fungi. These include the reassortment of whole dikaryotic nuclei (Nelson et al., 1955;
Little and Manners, 1969(a)), the parasexual cycle (Vakili and Caldwell, 1957; Bartos et al., 1969), chromosome exchange (Hartley and Williams, 1971), extra-nuclear inheritance (Johnson, Newton and Brown, 1934) and mutation (Watson, 1957; Griffiths and Carr, 1961). The first three of these mechanisms were considered with respect to the observations made during the current cytological investigation.
Several studies have emphasised the importance of hyphal and germ tube fusion (Rodenhiser and Hurd-Karrer, 1947; Bampton and Manners, 1957; Little and Manners, 1969(b)). Little and Manners (1969(a)) have suggested that the reassortment of whole dikaryotic nuclei may occur as a result of hyphal fusion and be the mechanism primarily responsible for the origin of new races of *P. striiformis*. Such a suggestion has been supported by more recent studies (Goddard, 1974; Taylor, 1976). A spore colour mutant of *P. graminis tritici* was used to demonstrate that different isolates fused with each other (Nelson et al., 1955), while evidence from a later investigation has shown that fusion can occur between *P. graminis tritici* and isolates of other *formae speciales* (Wilcoxson, Tuite and Tucker, 1958).
In a field situation, however, two similar mechanisms may be responsible for the reassortment of dikaryotic nuclei between different isolates of the pathogen. Where a host variety showed some measure of susceptibility to a number of races, anastomosis and the subsequent transfer of cellular contents could take place between leaf hyphae of different genetic origins. Such theories have been propounded by Little and Manners (1969(b)) and Goddard (1974). The vast numbers of nuclei recorded in leaf hyphae and the intimate association of fungal material would, from the evidence of the present study, offer ample opportunity for anastomosis and nuclear transfer.
In the situation where a host variety was resistant to common races of the pathogen, providing a sufficient concentration of uredospores was
Little and Manners, 1969(a)), the parasexual cycle (Vakili and Caldwell, 1957; Bartos et al., 1969), chromosome exchange (Hartley and Williams, 1971), extra-nuclear inheritance (Johnson, Newton and Brown, 1934) and mutation (Watson, 1957; Griffiths and Carr, 1961). The first three of these mechanisms were considered with respect to the observations made during the current cytological investigation.
Several studies have emphasised the importance of hyphal and germ tube fusion (Bodenhiser and Hurd-Karrer, 1947; Bampton and Manners, 1957; Little and Manners, 1969(b)). Little and Manners (1969(a)) have suggested that the reassortment of whole dikaryotic nuclei may occur as a result of hyphal fusion and be the mechanism primarily responsible for the origin of new races of *P. striiformis*. Such a suggestion has been supported by more recent studies (Goddard, 1974; Taylor, 1976). A spore colour mutant of *P. graminis tritici* was used to demonstrate that different isolates fused with each other (Nelson et al., 1955), while evidence from a later investigation has shown that fusion can occur between *P. graminis tritici* and isolates of other *forma speciales* (Wilcoxson, Tuite and Tucker, 1958).
In a field situation, however, two similar mechanisms may be responsible for the reassortment of dikaryotic nuclei between different isolates of the pathogen. Where a host variety showed some measure of susceptibility to a number of races, anastomosis and the subsequent transfer of cellular contents could take place between leaf hyphae of different genetic origins. Such theories have been propounded by Little and Manners (1969(b)) and Goddard (1974). The vast numbers of nuclei recorded in leaf hyphae and the intimate association of fungal material would, from the evidence of the present study, offer ample opportunity for anastomosis and nuclear transfer.
In the situation where a host variety was resistant to common races of the pathogen, providing a sufficient concentration of uredospores was
present to permit contact, fusion of uredospore germ tubes on the leaf surface could offer an alternative means of increasing the host range of *P. striiformis*. Laboratory experiments have shown that on nutrient agar fusion bodies can be formed between different isolates of yellow rust (Little and Manners, 1969(b); Goddard, 1974).
Virtually nothing is known about the genetics of hyphal and germ tube fusion (Burnett, 1975). In the current work, leaf hyphae were seen to be in intimate contact on many occasions but no fusion occurred. The reasons for this are not clear but it appears that a restriction exists with respect to the fusion of hyphal cells. Bartos et al. (1969) mixed races 228 and 393 of *P. coronata avenae* and identified eleven new races. Simultaneously, they mixed six races of *P. recondita* in pairs, and obtained no new races, suggesting that some restriction existed on hyphal fusion between the selected races.
The transfer of dikaryotic nuclei and establishment of a diploid somatic phase is an essential pre-requisite of the parasexual cycle (Bridgmon, 1959). However, in rust fungi a somatic diploid line has been identified only in axenically cultured material of *P. graminis tritici* (Maclean et al., 1974). Such a phase during the vegetative part of the life cycle would be in keeping with the polyploid hypothesis expounded by McGinnis (1956) for *Puccinia* species. Diploid teleutospore nuclei observed in this study were obviously larger than haploid dikaryotic nuclei. It is unlikely that the presence of such large nuclei during the vegetative stage of the pathogen's life cycle would have gone unnoticed. However, there was no evidence of a somatic diploid phase in this investigation.
A reciprocal exchange of whole chromosomes between dividing dikaryotic nuclei has been suggested as a possible source of variation in rust fungi (Hartley and Williams, 1971). In order to determine if such a mechanism
could be responsible for the evolution of new races of *P. striiformis*, an ultrastructural study examined the fine details of nuclear behaviour during mitosis. During the divisional stage when discrete chromosomal bodies were present (metaphase) the nuclear membrane was found to remain intact. This observation would indicate that the reciprocal exchange of chromosomes could not normally take place at this stage. This mechanism, therefore, would seem unlikely to play an important part as a means of genetic recombination and production of new races of yellow rust.
SECTION 3
Studies on Induced Variation
3.1 INTRODUCTION
Physiologic specialisation in *P. striiformis* was first recognised in 1930 by Allison and Isenbeck, and Gassner and Straib (Johnson, Stubbs, Fuchs and Chamberlain, 1972). Later, Gassner and Straib (1932(a)) introduced a system of race identification and nomenclature based on ten differential host varieties. Some of these differential varieties were labile in their reaction and, in 1969, Manners indicated the need to extend the original series of differentials. More recently, a new set of differential host varieties and a more efficient system of nomenclature has been adopted (Johnson et al., 1972). With this system, a set of differential varieties is selected, placed in a fixed linear order, and each variety assigned a value in a decanary notation. For example, variety A = 1, B = 2, C = 4. When an isolate of the pathogen is tested on these varieties, susceptible varieties are given their respective decanary values and resistant varieties are allocated the value 0. The summed value is then used to designate that race and is unique, in that it can only be derived by summation of the values assigned to the varieties on which the race is virulent. In order to make the system as internationally acceptable as possible, and to permit regional variations, two sets of differential varieties exist: a world set and a regional set, which in the case of the United Kingdom is the European set. Thus race 41E136 would be virulent upon Strubes Dickkopf (decanary value = 32), Vilmorin 23 (8) and Chinese 166 (7) in the world set and Heine VII (128) and Nord Desprez (8) in the European set. This system has been used since 1972 and is preferable to earlier methods, whereby races were known by an accession number, or by a name derived from the variety on which they were commonly found (Ubels, Stubbs and S'Jacob, 1965; Chamberlain et al., 1971).
Manners (1950) did the first extensive study on physiologic races of *P. striiformis* present in the United Kingdom. He identified eight races of wheat yellow rust, one race of barley yellow rust, and four races of yellow rust from various grass species. Since this work, new races of *P. striiformis* have been recorded quite frequently and by a number of investigators. In Table 1 a list of these races with their original and up-dated numbers is given.
**TABLE 1.** Some important races of *P. striiformis* identified in the United Kingdom during the last 25 years
| Race | Year first identified | Author/year |
|-----------------------|-----------------------|----------------------|
| 2B (40E72) | 1952 | Batts (1957) |
| 8B (32E128) | 1951 | Batts (1957) |
| 60 (37E132) | 1966 | Macer & Doling (1966)|
| 58C (41E136) | 1967 | Chamberlain et al. (1970) |
| 1B (108E9) | 1969 | Chamberlain et al. (1971) |
| 3/55D (104E137) | 1969 | Chamberlain et al. (1971) |
| 43E138 | 1971 | Chamberlain et al. (1975) |
| 108E173 | 1972 | Priestley & Byford (1975) |
| 45E140 | 1973 | Priestley & Byford (1975) |
| 1, 2, 3, 4, 6 | | |
| 1, 2, 3, 6, 7 | | |
| 1, 2, 3, 4, 6, 10 | | |
Batts (1957) described two races of yellow rust which were widely distributed in the United Kingdom during the early 1950's. One race gave rise to heavy infection of the previously resistant variety Nord Desprez. This race, designated 2B, was similar to race 2 (biotype A) described by Manners (1950) but possessed added virulence on Cappelle
Desprez and Heines Kolben. Race 2B was first identified in 1952 and by 1953 had seemingly replaced the less virulent race 2. Batts also described the characteristics of a second race, designated 8B, which had first been recorded as early as 1951. Manners (1950) had previously described a race which he referred to as 8A. Race 8B, while being similar to 8A, had added virulence on Heine VII and a number of other varieties, previously resistant to race 8A.
The severe outbreak of yellow rust in 1966 was a result of the rapid extension of race 60 (Macer and Doling, 1966). Macer and Doling suggested that race 60 could have evolved from one or more races common at the time, race 27/53, race 8B, or race 54. Since race 8B was then the most widespread race in the United Kingdom, it was postulated that this was the most likely progenitor of race 60. The devastation caused by race 60 during the 1966 epidemic led to the establishment of the Physiologic Race Survey Committee (Cereal Pathogens) in 1967 and the centralisation of investigations into the virulence of *P. striiformis* to the National Institute of Agricultural Botany (NIAB) at Cambridge.
The establishment of a Physiologic Race Survey was quickly followed by the identification of race 58C in 1967 (Chamberlain et al., 1970) and races 1B and 3/55D in 1969 (Chamberlain et al., 1971). Race 58C possessed virulence necessary to infect Chinese 166, Heine VII, Cappelle Desprez and Minister, which contain the resistance genes *Yr1*, *Yr2*, *Yr3a* + *Yr4a* and *Yr3c* respectively (Lupton and Macer, 1962). Combined virulence on Cappelle Desprez and Chinese 166 had only been previously recorded in a glasshouse race, designated race 58 and described by Macer (1967). Tests on supplementary differentials revealed that race 58C differed from race 58 in being capable of infecting the varieties Heine VII, Minister, Professeur Marchal, Rothwell Perdix and Cama. Race 58C may have arisen from race 3/55 Cleo by a single step mutation to virulence on Chinese 166. Alternatively, it may have been produced by heterokaryotic recombination between race 60 (virulent on Chinese 166
and Heine VII) and race 3/55 Cleo (virulent on Vilmorin 23 and Minister).
Race 1B possessed virulence on the varieties Cappelle Desprez, Hybrid 46, Heines Peko and could also overcome the resistance of the commercial variety Maris Ranger. Race 3/55D could overcome the resistance of varieties with genes derived from Heine VII, Cappelle Desprez, Minister and Hybrid 46, as well as causing extensive disease on the commercial variety Maris Beacon. Race 1B differed from a previously widespread race 3/55 Opal, only on the variety Heines Kolben, while 3/55D differed only on the variety Heine VII. It was therefore suggested that races 1B and 3/55D may have arisen by single step mutation (Chamberlain et al., 1971).
The identification of new races has continued in recent years with the occurrence of races 43E138 (Chamberlain et al., 1973), 45E140 and 108E173 (Priestley and Byford, 1975). Race 43E138 possessed virulence on the differential varieties Reichsberg 42 and Lee 1. Combined virulence on these two varieties had not previously been detected in races of yellow rust in the United Kingdom. Race 45E140 had virulence necessary to overcome the resistance of seven of the wheat differential varieties, including Chinese 166, Heine VII, Vilmorin 23 and Heines Peko.
Race 108E173, although being avirulent on Chinese 166, possessed virulence on the varieties Suwon 92 x Omar, Strubes Dickkopf, Vilmorin 23, Heines Kolben, Heine VII, Carstens V, Nord Desprez, Heines Peko and Hybrid 46. Increases in the complexity of the pathogen have continued during 1975 and 1976 and races of the pathogen now exist that can attack varieties with resistance derived from Chinese 166, Heine VII, Vilmorin 23, Hybrid 46 and Heines Kolben. Virulence on the differential variety Riebesel 47/51 was detected for the first time in 1974 and virulence on the variety Compair for the first time in 1976 (Priestley and Byford, 1975; 1976).
Resistance to yellow rust in the host plant is expressed in various forms and at different stages of growth. Manners (1969) recognised these
as seedling resistance, adult plant resistance, environmentally determined resistance and tolerance. The main type of resistance used by plant breeders has been seedling resistance, and it is with the specificity of this type of resistance that this section is primarily concerned.
Seedling resistance is controlled in most cases by Mendelian (major) genes (Biffen, 1907) and resistance of this type (race-specific resistance) has been readily overcome by a simple genetic change in the pathogen, followed by a population change induced as a result of selection pressure imposed on the pathogen (Macer, 1972). With the introduction of race-specific resistance genes in host varieties, complementary changes in the pathogen can be predicted, due to the operation of a co-evolutionary gene for gene system (Flor, 1942). Zadoks (1961) has suggested that such a system may occur with respect to race-specific resistance in the wheat:yellow rust interrelationship.
The resistance genes Yr1, Yr2, Yr3 and Yr4 were initially described by Lupton and Macer (1962). The loci Yr3 and Yr4 were found to be complex and contain more than one resistance allele. Genetic tests revealed that Yr3 could be represented by three alleles, Yr3a, Yr3b and Yr3c, while Yr4 could be represented by two alleles, Yr4a and Yr4b. The resistance genes Yr5, Yr6, Yr7 and Yr8 were described in the varieties Triticum Spelta Album, Peko, Thatcher and Compair (Macer, 1966; Riley, Chapman and Johnson, 1968) but insufficient evidence was produced to discount the possibility of allelism with other resistance genes. The resistance genes associated with Riebesel 47/51 and Moro have been designated as Yr9 and Yr10 respectively (Priestley, Smith and Doodson, 1975; Macer, 1975), but there is no evidence to discount the possibility of these genes being allelic with resistance genes present in other differential host varieties (Johnson, personal communication). Such designations might therefore be viewed as premature.
A number of the original differential varieties on which genetic analysis was undertaken are no longer included in the differential series, but have been replaced by varieties that exhibit similar reactions to races of the pathogen. For example, Vilmorin 23 has replaced Cappelle Desprez, Heines Kolben replaced Peko and Lee 1 has replaced Thatcher. However, there is no genetical evidence to support the presence of identical resistance genes in these varieties. On the contrary, Little and Manners (1969(a)) indicated that possible differences may exist in the resistance genes present in the varieties Cappelle Desprez and Vilmorin 23.
Recently, the resistance genes present in a number of host varieties have been referred to as resistance factors and resistance factors R1 to R14 have been assigned to a number of differential and commercial varieties (Priestley and Byford, 1975). Resistance factors, however, do not always have an established relationship with the Yr genes, the latter usually being designated only after detailed genetic studies.
The word "mutation" has been used in a number of different senses but here it is taken to mean a discontinuous change in the genetic make-up of the pathogen. Mutation occurs in all organisms and rust fungi are no exception. Mutation for pathogenicity has occurred spontaneously in *P. graminis tritici* (Watson, 1957), *P. coronata avenae* (Zimmer, Schafer and Patterson, 1963), *P. anomala* (D'Oliveira, 1939) and *P. striiformis* (Gassner and Straib (1932(b)). Although the rate of mutation in rust fungi appears to be low (Stakman and Harrar, 1957; Little and Manners, 1969(a)), it has been suggested that a single step mutation could have been responsible for the origin of races 41E136 (58C) (Chamberlain et al., 1970), 108E9 (1B) and 104E137 (3/55D) (Chamberlain et al., 1971). However, in the absence of a sexual stage, it is difficult to demonstrate that a particular race is the result of gene mutation. Nevertheless Day (1974) suggests that with
populations of plant pathogens that are sufficiently dense to cause economic loss, natural mutation rates are probably sufficient to generate mutants virulent on any nearby cereal variety protected by a single resistance gene. The useful life of a major resistance gene is thereby governed by the frequency with which the allele mutates from avirulence to virulence and the reproductive capacity of that mutant in the environment, in the presence of other races of the pathogen.
A number of artificially induced mutation studies have indicated that changes from avirulence to virulence can occur and at much higher frequencies than spontaneous events (Flor, 1956; Griffiths and Carr, 1961; Stubbs, 1969). Day (1974) has summarised such investigations and the rates at which mutational events occur. These varied from 0.02% in *P. graminis tritici* using ethyl methane sulphonate to 2.0% in *Melampsori lini* using fast neutrons.
Manners (1969) has suggested that *P. striiformis* is sensitive to prolonged exposure to ultra-violet (UV) light, such as that experienced on a clear summer's day. However, since UV light has been used successfully to induce mutants experimentally (Griffiths and Carr, 1961), it would seem plausible that under field conditions UV light could be important in the production of mutant genotypes. The induction of mutants using UV light arises due to the fact that this wavelength of light causes dimers to form between any two adjacent pyrimidine bases in the same D.N.A. chain. The sensitivity of an organism to UV light depends upon its enzymic capability to excise the dimer and replace it by a normal base complement (Witkin, 1969).
In those fungi with a sexual cycle, e.g. *P. graminis*, meiosis promotes genetic recombination through random assortment of chromosomes into haploid sets and through crossing over (Day, 1974). However, as far as is known rust species with no alternate host, such as *P. striiformis*, have no
functional sexual stage and therefore genetic variation produced as a result of mutation must be recombined by asexual mechanisms.
Heterokaryosis may be defined as the existence of two or more genetically dissimilar nuclei in the same cytoplasm. Rust fungi are in such a condition for most of their life cycle. With species such as *P. striiformis*, it is only during the dikaryotic phase that variants can come together to form new genotypes. Heterokaryosis can also act as a store of genetic variability which might otherwise disappear from the population. In many asexual fungi the dissociation of naturally-occurring heterokaryons offers an explanation for the duality and saltation that has been found to occur (Buxton, 1960). Different nuclei may divide at different rates, so a heterokaryon can vary the genetic balance and thereby adjust to meet the demands of a changing environment. This somatic flexibility could be important to fungi in the wild (Jinks, 1952).
Cytological evidence for the occurrence of hyphal anastomosis and the possible transfer of dikaryotic nuclei in rust fungi has been reviewed in Section 2. Genetic studies on *P. graminis tritici* (Nelson et al., 1955) revealed that an isolate of the fungus that was virulent on the resistant variety Khapli was a trikaryon. The trikaryon was unstable however, and dissociated giving rise to two new genotypes. The presence of such a trinucleate culture provides strong evidence for the transfer of heterokaryotic nuclei.
Little and Manners (1969(a)) obtained two new genotypes from a mixture of races 2B and 8B of *P. striiformis* and suggested that the recombinants arose due to the reassortment of existing factors, rather than the creation of new ones. A similar explanation was proposed by Konovalova and Schekotkova (1970) for a genotype of *P. striiformis* produced by inoculating mixtures of races 7M, 14 and 25 onto the resistant varieties.
Cappelle Desprez and Heine VII. A virulent genotype was obtained when Cappelle Desprez was inoculated with a mixture of 7M and 14. The recombinant, found to be avirulent on all the test varieties except Cappelle Desprez, was unstable and after three generations reverted to its original form.
Goddard (1974) obtained a new genotype from mixing races 104E137 and 41E136 of *P. striiformis*. The new genotype designed 105E137 was isolated on three separate occasions and had the accumulated virulence of the two parental races. Each of the three isolates appeared to be stable for virulence and the uredospores contained two nuclei of normal size. Two new genotypes were produced when races 37E132 and 104E137 were mixed and inoculated onto a range of resistant varieties (Taylor, 1976). Two isolates of race 36E132 were obtained and 19 isolates of race 105E137. It was suggested that the reassortment of whole nuclei may have been responsible for the production of these new genotypes, although the possibility of other mechanisms being involved could not be excluded.
It has been suggested by Buller (1950) that many cereal rust fungi, including *P. striiformis*, are heterothallic (Little and Manners, 1969(a)). Therefore, the exchange of nuclei between different heterokaryons would result in the production of only two new genotypes and this appears to fit in with the majority of evidence at present available on *P. striiformis*.
Heterokaryosis has been thought to be of more significance as the first step in the parasexual cycle (Pontecorvo and Roper, 1952). The parasexual cycle involves the fusion of compatible nuclei, followed by mitotic crossing over and haploidisation. Pontecorvo and Kafer (1958) found that in *Aspergillus nidulans* the frequency with which each event occurred was very low: 1 in $10^6$ divisions for nuclear fusion; 1 in 500 divisions for mitotic crossing over and 1 in 1000 divisions for haploidisation of the diploid nucleus. Although parasexuality is of
little consequence to fungi with a sexual phase, it is of considerable importance in imperfect fungi and heterothallic fungi (Tinline and MacNeil, 1969).
Vakili and Caldwell (1957) described 33 different physiologic races of *P. recondita* f. sp. *tritici* which were derived by mixing uredospores of a red pigmented race 2, with a yellow pigmented race 122. Seventeen of the races had not been previously identified and although the production of some of the races could be accounted for by nuclear reassortment, the number and types obtained suggested that mitotic recombination may have been involved. Bartos, Fleischmann Samborski and Shipton (1969) carried out a similar experiment with *P. recondita*, but obtained no new races, although they were at the same time able to show apparent parasexual recombination in *P. coronata* f. sp. *avenae*. However, a recent study of *P. recondita* produced seven new races from a mixture of races 107CM and 17 and six new races from a mixture of races 107CM and 63 (Sharma and Prasada, 1970).
One of the anomalies of the parasexual cycle is the fact that somatic recombination never appears to occur within a single race (Ellingboe, 1961). Assuming the dikaryotic nuclei were heterozygous for virulence, then new races could arise from the progeny of a single uredospore (Burnett, 1975). However, experimental evidence for the occurrence of the parasexual cycle in rust fungi has been detected only after different races have been mixed. Furthermore, a diploid phase is an essential prerequisite of the parasexual cycle (Bridgmon, 1959) and this has not been observed cytologically in rust fungi, except in axenic cultures of *P. graminis tritici* (Williams and Hartley, 1971) or from rust strains originally derived from axenic culturing (Williams and Mendgen, 1975).
While most emphasis on studies involving the physical basis of heritable factors has been placed on chromosome behaviour and structure,
there is now considerable evidence to demonstrate that particles outside the nucleus are, in some instances, responsible for the variation observed in certain fungi (Jinks, 1966). Jinks (1958) has shown that certain characters in *Aspergillus* species, such as germination, growth rate and pigmentation, are under cytoplasmic control.
Johnson, Newton and Brown (1934) suggested that cytoplasmic particles were responsible for the production of new physiologic races of *P. graminis tritici*. Reciprocal crosses in *P. graminis avenae* and *P. graminis tritici* (Green and McKenzie, 1967; Johnson, 1946) revealed that virulence on some host varieties was inherited cytoplasmically. Since no sexual phase occurs in *P. striiformis*, the possible role of cytoplasmic particles cannot at present be determined. However, with technical advancement, the cytoplasmic control of virulence in asexual rust fungi may be demonstrated by the controlled transfer of cytoplasmic material between compatible dikaryons.
The work undertaken in the present study was intended to complement the cytological investigation presented in Section 2. It was felt that a genetical study might assist in establishing the mechanism(s) by which fungi, such as *P. striiformis*, increase their virulence characteristics.
3.2 EXPERIMENTAL MATERIALS AND METHODS
3.2.1 Maintenance of fungal cultures
Cultures of the pathogen were maintained on susceptible wheat seedlings in Sherer 255 CEL growth cabinets, programmed to give a 16 hour photoperiod (10,000 lux) at $18 \pm 2^\circ C$ and a dark period of 8 hours at $12 \pm 2^\circ C$. One cabinet was used solely for the production of differential host seedlings and another for the multiplication of single spore lines. This routine procedure reduced the risk of contamination of disease free seedlings.
To prevent cross contamination between single spore lines, each pot of seedlings was covered by a plastic vinyl dome (Fig. 53). When seedlings were taken for examination or treatment, pots were placed individually in the Slee 4 Vertical Lamina Flow Cabinet before the domes were removed. A clean, dry dome was placed on each pot before it was returned to the growth cabinet. Using these simple precautions up to 15 isolates of the pathogen were multiplied in the growth cabinet at any one time, without contamination of the white spore mutant (control).
Seedlings were inoculated when the first seedling leaf was fully expanded. Pots (12 cm) of ten seedlings were used, each pot being placed in the flow cabinet and the adaxial leaf surface inoculated with a uredospore culture by means of a sterile cotton wool swab.
After inoculation each pot was covered with a dome (21 cm), the inner surface of which had been sprayed with sterile deionised water. All inoculated pots were then removed to the infection chamber, operating at $8 \pm 2^\circ C$ (darkness). The following morning the pots were returned to the growth cabinets and watered every 2 days until the commencement of sporulation. A sterile glass tube (155 x 10 mm) was used for collecting uredospores. Each infected seedling leaf was carefully inserted into the tube and shaken to release the uredospores. After collection of the
inoculum, each pot was watered and returned to the growth cabinet. Daily collections over a period of 4 or 5 days provided sufficient inoculum for test purposes.
Following collection, each glass tube was sealed with a plug of cotton wool and labelled before storage. For short term storage of up to several weeks, the tubes containing yellow rust isolates were placed in a desiccator containing 50% sulphuric acid (by weight) and stored at $4^\circ C$ (Stevens, 1916). For long term storage, isolates were vacuum dried using the Edwards Speedivac vacuum drier for 2 hours (Hughes and Macer, 1964). Ampoules produced by this process were kept at $4^\circ C$ until required (Figs. 54 and 55).
3.2.2 Procedures for genetical studies
Two races of the fungus, with contrasting virulence, were selected for genetic studies. Race 104E137 was an albino (white-spore) mutant, collected from a wild type culture of race 104E137 (69/10) at the P.B.I., Cambridge. Race 33E32 was a yellow-spore isolate, also obtained from stocks at the P.B.I. (Fig. 56).
Race 104E137 was inoculated onto the wheat variety Gamin and race 33E32 onto the differential variety Chinese 166. Use of these two varieties aided in reducing the level of cross contamination between isolates, since race 104E137 was avirulent on Chinese 166 and race 33E32 avirulent on Gamin. Single spore isolates were established from cultures of the two races and found to be of a consistent genotype, by tests on the differential host series.
For single spore isolation plants were grown in glass tubes (150 x 15 mm) filled with potting compost (Levington). Two seeds of the susceptible variety Sappo were sown in each tube and 12 tubes were retained in a large beaker. Each beaker was then placed in a pot (12 cm) and covered with a dome before removal to the growth cabinet. When the first
Fig. 53 Plastic vinyl domes used during experimental studies
Fig. 54 The Edwards 'speedivac' vacuum drier
leaves were 6 to 8 cm in length, the tubes were removed for inoculation. An infected leaf of the fungal isolate was examined under the binocular microscope and a suitable area, approximately 2 to 4 cm long, removed and placed on the surface of 1% water agar in a sterilised petri dish. Under the binocular microscope (x 75) single uredospores were selected and placed on the surface of a seedling leaf by means of a fine glass needle. After inoculation, each seedling was placed individually in a conical flask inside a 12 cm pot and covered by a moist dome. Each single spore culture was placed in the infection chamber for 48 hours and then returned to the growth cabinet.
Between inoculations the glass needle was washed in 70% ethanol and then rinsed in sterile, deionised water. The infection rate with this technique varied with different batches of seedlings, initially being between 2 and 5%, but rising in later batches to around 15%.
The standard differential varieties of wheat (Johnson et al., 1972) were used to classify isolates of the fungus. Several of the differentials contained major resistance genes, which permitted virulence changes in the pathogen to be detected. Supplementary varieties used for screening isolates were Maris Beacon, Maris Templar, Gamin, Sappo and an F3 hybrid of Heine 110 x Hybrid 46 (Goddard, 1974), supplied by the P.B.I. Reactions of the differential and supplementary varieties to races 104E137 and 33E32 are given in Table 2. Combined virulence of the two races was necessary to overcome the resistance of several of the differential host varieties, including Maris Templar and the F3 hybrid.
TABLE 2. Differential and supplementary varieties, their decanary value and reaction to races $10^4E137$ and $35E32$ of *Puccinia striiformis*
| Decanary value | $10^4E137$ Reaction class | $35E32$ Reaction class |
|----------------|---------------------------|------------------------|
| **World differential series:** | | |
| Triticum Spelta Album | (256) | R | R |
| Riebesel 47/51 | (128) | R | R |
| Suwon 92 x Omar | (64) | S | R |
| Strubes Dickkopf | (32) | S | S |
| Moro | (16) | R | R |
| Vilmorin 23 | (8) | S | R |
| Heines Kolben | (4) | R | R |
| Lee 1 | (2) | R | R |
| Chinese 166 | (1) | R | S |
| **European differential series:** | | |
| Heine VII | (128) | S | R |
| Spaldings Prolific | (64) | R | R |
| Carstens V | (32) | R | S |
| Compair | (16) | R | R |
| Nord Desprez | (8) | S | R |
| Heines Peko | (4) | R | R |
| Reichsberg 42 | (2) | R | R |
| Hybrid 46 | (1) | S | R |
| **Supplementary differentials:** | | |
| Maris Beacon | | S | R |
| Maris Templar | | R | R |
| Gamin | | S | R |
| Heine 110 x Hybrid 46 (F3) | | R | R |
| Sappo | | S | S |
Complete sets of the differential varieties were sown, inoculated and then incubated as previously described in a Sherer 255 CEL growth cabinet. For inoculation, the uredospore sample was diluted with talc (1:20 by weight) and using a sterile cotton wool swab brushed onto the expanded first seedling leaf. After overnight incubation, the inoculated differentials were returned to the growth cabinet. The domes covering the seedlings were removed after a few hours and not replaced until just prior to the onset of sporulation. Each pot was watered daily and all plant growth except the first leaf cut back every 3 to 4 days. When reaction types were fully developed (16 days after inoculation) each set of differential varieties was scored on the scale given in Table 3 and illustrated in Fig. 57.
**TABLE 3.** The reaction type assessment key for scoring differential wheat varieties following inoculation with *P. striiformis*
| Reaction Type | Description |
|---------------|-------------|
| i | No visible reaction |
| oo | Minute chlorotic flecks |
| O | Chlorotic areas varying in extent with or without slight necrosis |
| On | Chlorotic and necrotic areas deforming the shape of the leaf |
| I | Very few small scattered pustules of low spore bearing capacity and with extensive chlorosis and/or necrosis |
| II | Few to many small scattered pustules of low vigour and with extensive chlorosis |
| III | Normal pustule formation of high spore bearing capacity and with chlorosis |
| IV | Normal pustule formation of high spore bearing capacity and without any chlorosis |
Reaction types classified in the immune (i) to II category were considered as resistant responses, while those in the 3-4 range were considered as susceptible reaction types. When an intermediate reaction type \((2^+/3)\) occurred, the isolate was re-inoculated onto the differential variety concerned and the host response reassessed.
Fig. 55 Ampoule storage
Fig. 56 Races 104E137 (white) and 33E32 (yellow)
Fig. 57 Differential variety reaction types following inoculation with yellow rust
3.2.3 Screening for new genotypes of yellow rust
Forty-eight single spore lines were produced from mixed inoculations with races 104E137 and 33E32. Infected seedling leaves of the susceptible variety Sappo were screened with a binocular microscope for uredia that contained both white and yellow uredospores, since it was felt that these uredia would provide the best opportunity of isolating a new race. Yellow and white spores were removed from such pustules and established as single spore cultures. Following multiplication, each single spore line was tested and classified on the differential host series.
In a further investigation single spore lines were produced from the mixed inoculation of differentially resistant varieties. For this procedure a mixture of isolates 104E137 and 33E32 was multiplied on the susceptible variety Sappo. The uredospores collected from Sappo were then used to inoculate a number of varieties that were resistant to one of the parental isolates but susceptible to the other (Table 4). At the onset of sporulation yellow pustules were selected from varieties that were resistant to isolate 33E32 and white pustules from varieties resistant to isolate 104E137. These pustules were used for the production of single spore lines, which were subsequently multiplied and tested on the differential host series.
Some varieties selected for the screening work were resistant to one of the fungal isolates, but still produced low or moderate levels of sporulation following inoculation. Single spore isolates were not established from these varieties since it was not possible to discriminate between parental and recombinant lines (Table 4).
TABLE 4. Varieties used for screening recombinant genotypes of *P. striiformis* from a mixture of races 104E137 and 33E32
| Differential variety | 104E137 Reaction type | 33E32 Reaction type | Number of seedlings inoculated | Number of single spore isolates |
|----------------------|-----------------------|---------------------|-------------------------------|---------------------------------|
| Chinese 166 | R | S | 70 | - |
| Gamin | S | (R) | 90 | GM 1-3 |
| Hybrid 46 | S | R | 120 | HY 1-9 |
| Joss Cambier | S | R | 60 | JC 1-2 |
| Maris Beacon | S | R | 170 | MB 1-8 |
| Maris Huntsman | S | R | 90 | MH 1-7 |
| Maris Templar | R | (R) | 120 | MT 1-4 |
| Nord Desprez | S | (R) | 30 | - |
| Sappo | S | S | Control | - |
| Selpek | S | (R) | 60 | - |
| Suwon 92 x Omar | S | R | 100 | SW 1-4 |
| Tadorna | R | (R) | 60 | - |
*(R) indicates moderate levels of sporulation from a resistant reaction*
### 3.2.4 Tests on the characteristics of new genetic lines
In studying recombinant lines, observations were made on uredospore size, nuclear number and nuclear size. Uredospores of parental and recombinant lines were collected from sporulating host tissue. Inoculum from each of the lines was brushed gently onto each of two 1% water agar plates and incubated at room temperature for 12 hours. Each plate was examined microscopically and the dimensions of five randomly selected uredospores determined. The volume of each isolate was then calculated using the formulae \( v = \frac{4}{3} \times ab^2 \) (Goddard, 1974),
where \( v \) = volume of uredospore
\( a \) = half uredospore length
\( b \) = half uredospore width.
Uredospores of a recombinant isolate (MB6) and the two parental lines, 104E137 and 33E32, were multiplied separately on the variety Sappo. Two to three days after the onset of sporulation tissue was selected from each line, hand-cut transverse sections were prepared, immediately immersed in the BAC fixative and stained in propionic iron haematoxylin (Section 2.2.3). After storage for two to three days at 4°C, the preparations were squashed gently and the number and dimensions of uredospore nuclei ascertained.
3.2.5 Mutation studies
For studies on the effects of ultra-violet (UV) light on uredospore viability, the white spore mutant of isolate 104E137 (69/10) of *P. striiformis*, its wild type (yellow) progenitor and an isolate of *P. recondita* (WBR 73/3) were multiplied on the variety Sappo. Freshly collected uredospores of each isolate were used to inoculate 1% water agar plates, using a spore settling tower (Bell, Schmitt, Miller and Kingsolver, 1952). Three plates of each of the isolates were then irradiated with UV light for 1, 2, 4, 8, 16 and 32 minutes. Three unirradiated plates were used as controls. The plates were fully randomised in the infection chamber and incubated overnight at 8 ± 2°C. The following morning, 500 uredospores were selected at random on each plate and the germination level determined. A uredospore was considered to have germinated when it produced a germ tube at least as long as the diameter of the spore.
Irradiation of the plated uredospores was achieved using two 15 watt UV tubes (Philips). Each of the plates was placed approximately 60 cm from the tube and irradiated for the prescribed period of time. The UV tubes were allowed to operate for 15 minutes before the first plates were treated.
TABLE 5. Varieties inoculated with UV irradiated uredospores of isolate 104E137
| Variety | Reaction type | Ultra-violet treatment | Unirradiated control |
|------------------|---------------|------------------------|----------------------|
| | | Number of seedlings inoculated | |
| Chinese 166 | (O) | 172 | 41 |
| Riebesel 47/51 | (O) | 147 | 35 |
| Heines Peko | (O-1) | 58 | 16 |
| Heines Kolben | (O-1) | 39 | 10 |
| Lee 1 | (O-1) | 30 | 10 |
| Carstens V | (O-1) | 36 | 10 |
| Spaldings Prolific | (O-1) | 36 | 10 |
| Moro | (OO) | 124 | 30 |
| Reichsberg 42 | (O-1) | 83 | 20 |
| T. Spelta Album | (O-O_N) | 63 | 20 |
| Sappo (control) | (3/3^+) | 50 | 41 |
A number of selected varieties which were resistant to race 104E137 were used to screen irradiated uredospores for virulence changes (Table 5). For irradiation treatment, uredospores were spread thinly on a piece of white card, placed in the flow cabinet and irradiated for 15 minutes. This was sufficient to reduce the viability of the inoculum to approximately 5% of the untreated control. Seedlings of the selected varieties were inoculated with a uredospore:talc mixture (1:20 by weight). Ten mg of uredospores were then used to inoculate each of four experiments.
Following inoculation, each pot was placed in the infection chamber for 48 hours and then returned to the growth cabinet. Under these environmental conditions, some selected differentials produced low levels of sporulation following inoculation with race 104E137. Such varieties were excluded from later experiments.
An investigation was established to assess the effects of a second mutagenic substance on uredospore viability. Ten mg batches of freshly collected uredospores of the white spore mutant of race 104E137 were treated with varying concentrations of MNNG (N-methyl-N'Nitro-N'-Nitrosoguadinine) in order to determine the optimum concentration required for virulence studies. MNNG stock solution (2 mg per ml of phosphate buffer, pH 7.0) was diluted with phosphate buffer (0.067 m) to give concentrations of the mutagen of 10, 20, 40 and 100 μg per ml of phosphate buffer. Uredospores were placed in a microcentrifuge tube and covered slowly with the MNNG solution. After gently agitating each tube for 30 minutes, the uredospores were centrifuged down, washed twice in phosphate buffer and inoculated with a soft camel hair brush onto plates of 1% water agar. After overnight incubation 500 randomly selected spores were scored on each of three plates and the germination level calculated for each chemical concentration.
The results of the viability tests were noted and two concentrations of MNNG were used in an attempt to induce changes in the virulence characteristics of the pathogen. Two 10 mg batches of uredospores were treated with 10 μg per ml of MNNG for 30 minutes and two batches with 20 μg per ml. Chemically treated uredospores were inoculated onto the susceptible variety Sappo and multiplied before being inoculated onto the test varieties.(Table 6).
TABLE 6. Varieties inoculated with MNNG treated uredospores of isolate 104E137
| Variety | Reaction type | MNNG treated | Untreated control |
|------------------|---------------|--------------|-------------------|
| Maris Templar | (o) | 74 | 20 |
| Compair | (o) | 62 | 18 |
| Moro | (oo) | 57 | 18 |
| Chinese 166 | (o) | 83 | 20 |
| T. Spelta Album | (o-o_N) | 60 | 20 |
| Riebesel 47/51 | (o) | 58 | 20 |
| Sappo (control) | (3/3^+) | 33 | 21 |
A camel hair brush was used to spread uredospores evenly onto the adaxial surface of each seedling leaf. Each pot was then incubated for 48 hours before being returned to the growth cabinet.
A number of single spore isolates were established from pustules developing on resistant varieties and after multiplication these isolates were tested on the differential host series.
3.3 RESULTS
3.3.1 Mixed inoculation studies
Single spore lines isolated from fully susceptible varieties
Forty-eight single spore lines, isolated from mixed pustules on the susceptible variety Sappo produced only parental races during tests on the differential series. Forty-three yellow isolates, conformed to race 32E32 and five white isolates conformed to race 104E137. Each of the single spore lines behaved identically to the respective parental race during differential testing.
Single spore lines isolated from differential host varieties
TABLE 7. Single spore isolates produced from a number of differential host varieties
| Variety of origin | % yellow pustules | Number of yellow single spore lines | Parental types | Recombinant types |
|-------------------|------------------|------------------------------------|----------------|------------------|
| Gamin | 3-4 | GM 1-3 | 3 | - |
| Hybrid 46 | 0.1 | HY 1-9 | 7 | 2 |
| Joss Cambier | 0.5 | JC 1-2 | 2 | - |
| Maris Beacon | 0.5 | MB 1-8 | 6 | 2 |
| Maris Huntsman | 0.5 | MH 1-7 | 7 | - |
| Maris Templar | 5.0 | MT 1-4 | 4 | - |
| Suwon 92 x Omar | 0.5 | SW 1-4 | 3 | 1 |
Thirty-seven single spore isolates were taken from seven varieties giving differential reactions to the two parental races (Table 7). Five yellow, single spore lines had virulence characteristics in excess of either of the parental races and these five lines were identified as race 105E137 when tested on the differential host series (Table 8). Each of the five isolates was virulent on Chinese 166 but avirulent on Carstens V. Seedlings
of the F3 hybrid variety and Maris Templar were susceptible to this genotype, suggesting that a combination of virulence of the two parental genotypes had taken place (Fig. 58).
**TABLE 8. Reaction of parental and recombinant lines to differential and supplementary varieties**
| Decanary value | 104E137 | 32E32 | 105E177 |
|----------------|---------|-------|---------|
| **World differential series:** | | | |
| Triticum Spelta Album | (256) | R | R | R |
| Riebesel 47/51 | (128) | R | R | R |
| Suwon 92 x Omar | (64) | S | R | S |
| Strubes Dickkopf | (32) | S | S | S |
| Moro | (16) | R | R | R |
| Vilmorin 23 | (8) | S | R | S |
| Heines Kolben | (4) | R | R | R |
| Lee 1 | (2) | R | R | R |
| Chinese 166 | (1) | R | S | S |
| **European differential series:** | | | |
| Heines VII | (128) | S | R | S |
| Spaldings Prolific | (64) | R | R | R |
| Carstens V | (32) | R | S | R |
| Compair | (16) | R | R | R |
| Nord Desprez | (8) | S | R | S |
| Heines Peko | (4) | R | R | R |
| Reichsberg 42 | (2) | R | R | R |
| Hybrid 46 | (1) | S | R | S |
| **Supplementary differentials:** | | | |
| Maris Beacon | | S | R | S |
| Maris Templar | | R | R | S |
| Gamin | | S | R | S |
| Heine 110 x Hybrid 46 (F3) | | R | R | S |
| Sappo | | S | S | S |
In some tests with the five isolates, the differential variety Vilmorin 23 did not always give a fully susceptible reaction. However, when the tests were repeated, sufficient sporulation occurred on Vilmorin 23 to class this as being susceptible to the test race. The differential varieties Chinese 166 and Tadema produced only yellow pustules when inoculated with a
mixture of the two parental races. Therefore, no white single spore lines were produced by differential screening and only yellow uredospores were tested for new virulence combinations.
3.3.2 The characteristics of recombinant and parental lines
Uredospore size
TABLE 9. Uredospore volume of seven yellow rust isolates
| Isolate: | 33E32 | 104E137 | MB6 | MB8 | HY2 | HY9 | SW2 |
|----------|-------|---------|-----|-----|-----|-----|-----|
| Uredospore volume (x 10^{-2} \mu m) | 6.79 | 6.84 | 6.89 | 7.20 | 6.83 | 6.88 | 6.78 |
After rehydration for 12 hours, the volume of the parental and recombinant uredospores was not significantly different. Isolate MB8 had the largest uredospore volume (7.2 x 10^{-3} \mu m) whereas other isolates were on average 6.8 x 10^{-3} \mu m (Table 9).
Nuclear number and nuclear size of isolate MB6 and the parental lines
TABLE 10. Nuclear number and nuclear size of three test isolates of yellow rust
| | 104E137 | 33E32 | MB6 |
|----------------------|---------|-------|-----|
| Number of uredospores in test | 102 | 99 | 106 |
| Uredospores with 2 nuclei | 102 | 99 | 104 |
| Uredospores with 3 nuclei | 0 | 0 | 2 |
| Size of uredospore nuclei | 4.5 \mu m | 4.4 \mu m | 4.5 \mu m |
Uredospore nuclei were clearly differentiated using the propionic iron haematoxylin stain (Fig. 59) and, therefore, it was possible for them to be scored and measured. Only a small sample of uredospores was used in the test, but observations made on other preparations showed the results obtained from
Fig. 58 Reaction types of the F3 hybrid Heine 110 x Hybrid 46 following inoculation with races 104E137, 33E32 and 105E137 (right)
Fig. 59 Uredospore squashes (propionic iron haematoxylin)
the small sample to be representative of the remainder of the fixed material (Table 10). In the two parental lines, no uredospores were observed with greater than two nuclei, whereas in isolate MB6, a recombinant line, trikaryotic uredospores were occasionally seen. Nuclear size in all three lines was very similar, being of the order of 4.5 μm diameter.
3.3.3 Mutation studies
The effect of ultra-violet irradiation on uredospore viability
The two isolates of *P. striiformis* (race 104E137) demonstrated similar responses when irradiated with UV light, the white spore mutant being marginally lower in viability than the yellow spore isolate (Fig. 60). The isolate of brown rust had a lower germination level than either of the yellow rust isolates, when each was exposed to low levels of UV light. At higher irradiation levels, the brown rust isolate gave a higher frequency of germination than the two isolates of yellow rust.
TABLE 11. The effect of UV light on uredospore germ tube development
| UV irradiation (min.) | Average length of germ tube in μm |
|-----------------------|----------------------------------|
| | *P. recondita* (73/3) | *P. striiformis* (104E137) |
| 8 | 600 | 200 |
| 16 | 250 | 100 |
From microscopic observations during germination assessment, it appeared that the average length of the germ tubes formed by the brown rust isolate (WBR 73/3) was considerably greater than those formed by the yellow rust isolate. A comparison of the two rust species confirmed this impression. After 8 minutes' irradiation, germ tubes produced by the brown rust isolate were three times the length of those formed by the yellow rust isolate, and after 16 minutes two and a half times as long (Table 11).
Fig. 60 The effect of UV irradiation on the germination of rust uredospores
- - - P. striiformis - white
- - - P. recondita
- - - P. striiformis - yellow
GERMINATION %
UV IRRADIATION IN MIN
Screening for virulence changes induced by UV irradiation
The differential varieties Chinese 166, Riebesel 47/51, Moro and *Triticum Spelta Album* were resistant to all inocula and no sporulation occurred. Heines Kolben, Heines Peko, Lee 1, Spaldings Prolific, Carstens V and Reichsberg 42 produced low levels of sporulation when inoculated with irradiated uredospores of the white spore line. Uredospores collected from these varieties were avirulent when reinoculated onto their variety of origin and no isolates were found that had virulence characteristics different from the parental genotype. It was apparent, however, that the uredospores irradiated with UV light were 3-4 days later in commencing sporulation on susceptible varieties than the unirradiated uredospores.
As a result of the failure of UV treatment to induce observable virulence changes, a more potent mutagenic compound was used during later experiments.
**The effect of MNNG on uredospore viability**
Treatment of viable uredospores with concentrations of 10, 20 and 40 μg per ml of MNNG produced a relatively small decrease in uredospore viability (Fig. 61). Concentrations of the order of 100 μg per ml of MNNG totally inhibited germination. For preliminary experiments uredospores were treated with a solution of 40 μg per ml of MNNG.
**Screening uredospores for virulence changes after MNNG treatment**
The initial concentration of 40 μg per ml of MNNG produced hypersensitive flecks on the control variety Sappo and therefore lower concentrations of 10 and 20 μg per ml were used for experimental studies. Some sporulation occurred on individual seedlings of the varieties Compair and Moro. However, when reinoculated onto its respective variety of origin, all the inoculum was found to be avirulent. Two single spore lines (WM1 and WM2) were established from a sporulating lesion on the variety Moro. When tested on the differential varieties, the two single spore lines were of the parental genotype with respect to virulence characters. Reaction types were scored somewhat later with these lines, normally around 20 days after inoculation.
Fig. 61 The effect of MNNG on uredospore viability
GERMINATION %
CONC MNNG (ug/ml)
DISCUSSION
The production of race 105E137 following inoculation with a mixture of races 104E137 and 33E32 provides additional evidence that *P. striiformis* can increase its virulence spectrum when two races are grown in close proximity. Race 105E137, although not recorded in the field, has been identified in laboratory experiments on two other occasions. Goddard (1974) mixed races 104E137 and 41E136 and isolated three single spore lines of race 105E137. Similarly, from a mixture of races 104E137 and 37E132, two new genotypes of the pathogen were isolated and identified as races 105E137 and 36E132 (Taylor, 1976). However, the production of new physiologic races is not limited to laboratory experiments and it has been suggested such genotypes may arise in the field by a mechanism(s) similar to the one responsible for the production of race 105E137 (Goddard, 1976(b)).
The rate of emergence of recombinants varies between experimental studies. Little and Manners (1969(a)) obtained 10% recombinants from mixing inocula of races 2B and 8B of *P. striiformis*, while Goddard (1974) recorded a 6% recombination rate with races 104E137 and 41E136. Little and Manners produced single spore lines from a variety equally susceptible to both parental races, while Goddard produced cultures from a number of varieties, most of which were resistant to one of the two parental lines. In the current investigation no recombinants were detected when races 104E137 and 33E32 were mixed and inoculated onto the susceptible variety Sappo. If, however, the two races were inoculated simultaneously onto a susceptible variety, multiplied and re-inoculated onto a differential host variety such as Hybrid 46, then out of 37 lines, five were genetically different from the two parental races. The use of differentially resistant varieties would thus appear to increase the chances of screening out new virulent races of the pathogen. The methods used by Goddard and Little and Manners are not therefore comparable in terms of the frequency of recombination, although their results are in a similar range.
The various mechanisms by which the five isolates of race 105E137, identified in the present study, could have arisen include extra-nuclear inheritance, nuclear reassortment, parasexuality and mutation.
Johnson (1946) discovered that virulence of the stem rust fungus to the wheat variety Marquis was inherited cytoplasmically. Similarly, reciprocal crosses in *P. graminis avenae* revealed virulence on certain differentials to be under cytoplasmic control (Green and McKenzie, 1967). Cytoplasmic determinants may combine by hyphal fusion to produce variation in vegetative progeny and although some elements have been identified, such as yeast mitochondria, which carry several linked markers, other elements are as yet only hypothetical carriers (Day, 1974). The methods most widely used to demonstrate cytoplasmic inheritance in fungi include maternal inheritance and the heterokaryon test (Jinks, 1966). Such tests introduce technical problems that are made increasingly more complex by the absence of a sexual phase in *P. striiformis*. However, the frequency of trinucleate uredospores in isolate MB6 (2%) recorded in the present study reduces the attraction of extra-nuclear inheritance as a suggested mechanism for the production of race 105E137.
Nelson et al. (1955) postulated that the transfer of whole nuclei had occurred, when they obtained a trinucleate culture of *P. graminis tritici* which was virulent on the variety Khapli. This biotype was unstable however and dissociated to give rise to two new biotypes. Little and Manners (1969(a)) isolated two new races from a mixture of races 2B and 8B and suggested that, since *P. striiformis* was probably heterothallic (Buller, 1950), the production of two new races was most easily explained by nuclear reassortment. Similarly, Konovalova and Schekotkova (1970) and Goddard (1974) favoured nuclear reassortment as the mechanism responsible for the new race of *P. striiformis* that each isolated from mixing differentially virulent races. Two new races produced in controlled
experiments by mixing races 104E137 and 37E132 were considered to have arisen by the exchange of dikaryotic nuclei (Taylor, 1976). Only a single recombinant genotype was produced in this investigation and nuclear reassortment as described by Little and Manners (1969(a)) could also account for the formation of such a race.
If it is accepted that most rust fungi are heterothallic (Buller, 1950), then the isolates of race 105E137 may have arisen as follows, where X and Y represent individual dikaryotic nuclei and + or - refer to the compatibility factor in each nucleus.
Race 104E137
\[ X^+X^- \]
Race 33E32
\[ Y^+Y^- \]
\[ (X^+X^-Y^+Y^-) \] hyphal or germ tube fusion
\[ X^+X^- \quad X^+Y^- \quad Y^+X^- \quad Y^+Y^- \]
104E137 Recombinants 33E32
If the assumption is made that nuclei are reassorting to produce new races of *P. striiformis*, a number of possible genetical models may be suggested, taking to account virulence or avirulence on differential varieties as being represented by the following symbols:
\[ V_1 = \text{virulence on Chinese 166} \quad (A_1 \text{ being the allelic factor for avirulence}) \]
\[ V_2 = \text{virulence on the variety Heine VII or Vilmorin 23 or Suwon 92 x Omar or Nord Desprez} \quad (A_2 \text{ being the allelic factor for avirulence}) \]
\[ V_3 = \text{virulence on Carstens V} \quad (A_3 \text{ being the allelic factor for avirulence}) \]
\[ V_4 = \text{virulence on Hybrid 46} \quad (A_4 \text{ being the allelic factor for avirulence}). \]
Race 104E137 must possess \( A_1V_2A_2V_4 \), 33E32 possess \( V_1A_2V_3A_4 \) and 105E137 \( V_1V_2A_3V_4 \), in such a form that they are expressed in the phenotype, i.e. be homozygous if recessive and either homozygous or heterozygous if dominant. Day (1974) suggests that virulence is expressed in many pathogenic fungi when in the recessive condition. Assuming this to be the case, the situation in the present study can be represented as follows:
On some occasions virulence in rust fungi has been found to be dominant (Green and McKenzie, 1967; Kao and Knott, 1969). If this was so in the present case, the situation could be represented as follows:
Race 104E137
\[ X^+ \quad v_1v_2A_3v_4 \]
\[ X^- \quad A_1v_2A_3v_4 \]
Race 33E32
\[ Y^+ \quad v_1A_2v_3A_4 \]
\[ Y^- \quad v_1v_2v_3v_4 \]
Race 105E137
\[ X^+ \quad v_1v_2A_3v_4 \]
\[ Y^- \quad v_1v_2v_3v_4 \]
Race ?
\[ X^- \quad A_1v_2A_3v_4 \]
\[ Y^+ \quad v_1A_2v_3A_4 \]
A number of other genetical interpretations are plausible depending on whether particular alleles are dominant or recessive and until more information is available on this subject, each of the models can only be considered hypothetical.
Only one recombinant genotype was isolated in the present work. This may have been due to the fact that other isolates had a reduced number of virulence factors and were therefore avirulent on the differential varieties. Another possible reason is that further isolates were white and not yellow spore types. Only five white-spore lines were established and tested in the present study and all these conformed to race 104E137. Such a theory cannot be discounted, although when the white mutant line of race 104E137 and a yellow spore line of race 37E132 were mixed, the recombinant isolates were all yellow spore types (Taylor, 1976). A recent investigation at the PEI, however, recovered a white spore recombinant from a mixture of yellow and white parental lines (Johnson, personal communication).
On a number of differential varieties race 105E137 clearly had an advantage over other isolates due to its wider virulence spectrum. Any other recombinant(s) was probably reduced in virulence compared to the parental races and may have been avirulent on all varieties except Strubes Dickkopf or Carstens V. Working on the nuclear reassortment theory a second recombinant may therefore have been designated as race 32E0 or 32E32.
The parasexual cycle (Pontecorvo and Roper, 1952) could account for the production of new genotypes of rust fungi, following the mixed inoculation of two parental races possessing contrasting virulence characteristics. However, in this study and other investigations, no somatic diploid phase has been identified (Little and Manners, 1969(b); Goddard, 1974) and, as Ellingboe (1961) suggests, a diploid phase must occur in the host in order for the parasexual cycle to operate. Little and Manners (1969(b)) concluded that the absence of a diploid stage, the instability in nuclear number and the identification of only two new races suggested nuclear reassortment and not parasexuality as the mechanism.
most likely to be responsible for the production of new physiologic races of *P. striiformis*. In the current work the five isolates of race 105E137 had uredospores similar in size to those of the two parental races and no large spore isolate was recorded, as was the case in other investigations (Goddard, 1974). Each of the isolates of race 105E137 was found to have nuclei comparable in size to those of the parental races and no evidence was obtained to suggest that parasexuality was involved in the production of such a recombinant genotype.
A fourth mechanism that may have been responsible for the production of race 105E137 is mutation. Chamberlain et al. (1970) suggested that a single step mutation may have been involved with the production of races 41E136 from race 40E136 (3/55 Cleo) and for the production of races 104E137 and 108E9 from race 104E9 (Chamberlain et al., 1971).
Spontaneous mutation for pathogenicity has been shown to occur occasionally in a number of rust fungi (Watson, 1957; Zimmer, Schafer and Patterson, 1963), while induced mutation studies have indicated that rates of mutation are much higher, being of the order of 0.02-2.0% (Day, 1974). In the present investigation five independent isolates of race 105E137 were identified and this fact alone casts considerable doubt as to whether direct mutational events could have been responsible for the origin of such a race. Furthermore the isolate of race 104E137 used in the present work was a white spore mutant, so any single step change at the site controlling virulence on Chinese 166 would have to be accompanied simultaneously by a change in the factor(s) controlling the uredospore spore colour, since spores of all five isolates of race 105E137 were yellow.
All forms of life depend ultimately on mutation for the introduction of new variation into a population. Mutation rates do however vary between species and the evidence from this work suggests a low incidence of
mutation in *P. striiformis*, being less than 1 in $2 \times 10^7$. This observation agrees with those of Little and Manners (1969(a)). The low mutation frequency to the white spore type may also be indicative of the rate of mutation in this fungus.
A number of reasons could be responsible for the failure to detect mutant genotypes in this study. Avirulent genotypes may have interfered with the establishment of induced mutants and this was why the uredospores inoculated onto the differential varieties following UV and MNNG treatment were initially of low viability. When the inoculum was of approximately 5% viability, interference between germinating uredospores was minimal. A second possibility is that a virulence allele, produced as a result of mutagenic treatment, may not have been expressed sufficiently rapidly and therefore have been eliminated (Day, 1974). Successful mutation experiments on virulence in the pathogen were conducted on dikaryotic lines of flax rust that were heterozygous for avirulence (Flor, 1956). Stubbs (1969) irradiated races of *P. striiformis* with X-rays and obtained new genotypes, each with an additional gene for virulence. Similarly, a new race of *P. coronata avenae* was obtained after UV irradiation (Griffiths and Carr, 1961). The new race was identical in virulence to race 277, reported independently in Argentina and Israel.
It was observed that isolates treated with either UV or MNNG were several days longer to sporulation than the untreated control. Single spore isolates WM1 and WM2 isolated from the variety Moro commenced sporulation 4-5 days after the control isolates. It appeared that such isolates were of reduced vigour as a result of mutagenic treatment, possibly as a result of damage to cytoplasmic or genetic factors. Griffiths and Carr (1961) noted that uredospores of *P. coronata avenae* treated with UV light were 9 days later in commencing sporulation on inoculated seedlings than unirradiated controls.
The small difference in the effects of UV irradiation on the viability between the yellow and white genotypes of race 104E137 (69/10) was surprising. It was somewhat more surprising to observe that the isolate of *P. recondita* was lower in viability than either of the *P. striiformis* isolates following UV treatment. It has been suggested that *P. recondita* is not as susceptible as *P. striiformis* to certain wavelengths of UV light (Manners, 1969). However, the actual wavelengths of UV used in the present study are not known.
After 16 minutes' irradiation the isolate of *P. recondita* showed a smaller reduction in viability than the white spore line of *P. striiformis* or its wild type progenitor. Furthermore, even after a relatively short period of irradiation, the isolate of *P. recondita* produced longer, more vigorous germ tubes than did the isolates of yellow rust. Schwinghamer (1958) has suggested that assessing the viability of irradiated spores on agar gives results comparable to host infection tests. Evidence from the current work indicates that this may not always be the case, since the more vigorous germ tubes produced by *P. recondita* would probably be more effective in establishing infection than those produced under the same conditions by *P. striiformis*.
SECTION 4
Studies on Quantitative Variation
4.1 INTRODUCTION
Since the recognition of physiologic specialisation in *P. striiformis* (Allison and Isenbeck, 1930) races of the pathogen have been identified by their virulence characteristics on a series of differential host varieties (Gassner and Straib, 1932(a); Johnson et al., 1972). The physiologic race however is an artificial unit, with the number and nature of the races identified depending upon the resistance genes present in the test varieties.
Considerable variation exists within the pathogen and this cannot be defined solely in terms of the infection types produced on a differential host series. Variation is also expressed in quantitative terms relating to such characters as spore production and fungal growth rate. Races and isolates of the pathogen that differ in these quantitative characteristics have been said to vary in aggressiveness (Van der Plank, 1968) or competitive ability or survival ability (Katsuya and Green, 1967).
Johnson and Taylor (1972) found quantitative differences, in levels of spore production, between field isolates of race 104E137 of yellow rust collected from the commercial varieties Maris Beacon and Joss Cambier. They suggested that two genetically distinct biotypes of this race were responsible for field epidemics and that these differed in minor genes for virulence. Further field isolates of this race were subsequently examined (Priestley and Doling, 1974). Four biotypes were distinguished by quantitative tests and it was suggested that these differences were due to varying levels of aggressiveness in the pathogen.
Johnson and Bowyer (1974) found quantitative differences between field isolates of race 41E136, and as a result suggested that distinct genetic differences existed between the various isolates within this race.
A number of experimental techniques have recently been developed that allow quantitative differences between isolates of the pathogen to be evaluated. Such techniques involve harvesting inoculum from sporulating lesions or measuring the growth rate of the pathogen. These methods are of a direct nature. Other quantitative methods measure differences between various host:pathogen combinations by indirect procedures, which entail the simultaneous inoculation of a number of isolates onto a susceptible variety.
Direct quantitative methods include the collection and measurement of propagules, in terms of either the number or weight of spores produced over a period of time. A number of studies have evaluated propagule number by the use of a coulter counter or haemacytometer (Guzman, 1964; Clifford, 1972; Priestley and Doling, 1972; Dickinson and Crute, 1974). The propagules produced in such tests may be collected by washing sporulating host tissue with a stream of water (Lapwood, 1961) or by the use of a solution containing a wetting agent (Clifford, 1972; Ward and Manners, 1974). Hilu and Hooker (1963) transferred a disc of sporulating material direct to a depression slide, before scoring. Discharged spores of *Helminthosporium maydis* were collected onto a piece of filter paper and resuspended in a copper sulphate solution, before evaluation (Nelson and Tung, 1973).
Differences between isolates of the pathogen can be assessed by weighing the spores produced by particular host:pathogen combinations on an electrical microbalance. Johnson and Bowyer (1974) found that the weight of uredospores collected from sporulating material was comparable to results obtained using other experimental techniques. Mehta and Zadoks (1970) collected uredospores of *P. recondita* from the leaves of 50 wheat seedlings using a cyclone spore collector in order to obtain sufficient for weighing. Sufficient uredospores of *P. recondita* were
obtained for the detection of quantitative differences by scraping three seedling leaves (Eyal and Peterson, 1967). A technique developed by Johnson and Bowyer (1974) where discharging uredospores of *P. striiformis* were collected from single seedlings gave sufficient precision to establish differential variation with a number of host:pathogen combinations.
Priestley and Doling (1974) found quantitative differences between isolates by measuring pustule development, following the point inoculation of seedling and adult plant leaves. A similar technique developed by Heitfuss and Dehne (1976) measured the growth rate of the pathogen towards the culm after inoculating the tip of the leaf and the results were used to express differing levels of host resistance to the pathogen.
Quantitative differences between different host:pathogen combinations can be determined indirectly by studying the relative survival ability of a number of isolates inoculated simultaneously onto a susceptible host variety. Isolates included in such experiments are usually distinguished when scoring by the use of differential varieties, or in some instances by spore colour differences. When isolates of a similar spore colour are inoculated together, samples of inoculum are transferred to differential host varieties at the end of each generation and the frequency of the various isolates estimated (Martens, 1973). With colour mutants it is unnecessary to test samples on differential varieties, since direct counts can be made of the number of pustules of each isolate on infected leaves (Brown and Sharp, 1970; Rastegar, 1976). Mixed infection experiments allow the reproductive potential of each isolate to be determined and the relative susceptibility of a variety to each of the test isolates to be assessed.
Small differences between various host:pathogen combinations cannot always be attributed to quantitative variation in the fungus or race.
non-specific resistance in the host. Van der Plank (1963) implies that race non-specific resistance is partial in its effect, whereas race-specific resistance is complete and totally inhibits sporulation of the pathogen. However, closer examination of the host:pathogen system has revealed that slight differences can be produced by the effects of race-specific resistance in the host and by the effects of virulence in the pathogen (Johnson and Taylor, 1976). Johnson and Taylor have used spore production as a test for the detection of specificity in the host:pathogen system. Specificity in this instance is usually expressed by a differential interaction between varieties of the host and races of the pathogen.
Van der Plank (1968) maintains that where no differential interaction occurs, isolates of the fungus differ only in aggressiveness, which is expressed by the uniform ranking of isolates on different host varieties. He cites the work of Wellman and Blaisdell (1940) on Fusarium wilt of tomatoes. Five culture types of the fungus were ranked in the same sequence on a susceptible variety (Bonny Best) and a variety possessing good levels of race non-specific resistance (Marglobe). Such a uniformity in ranking was said to indicate quantitative differences between isolates of the pathogen.
A number of studies have indicated that a race of the pathogen with many virulence genes competes poorly on a susceptible variety with races that are relatively simple and possess few virulence genes (Watson, 1954; Van der Plank, 1963; 1968; Martens, 1973). This has been reported to occur not only in the rusts (Watson and Singh, 1952; Leonard, 1969; Sebesta, 1972) but also in *Rhynchosporium secalis* (Williams and Owen, 1975), *Phytophthora infestans* and *Cladosporium fulvum* (Day, 1968). Black (1952) found that a race of potato blight which possessed three known virulence genes was eliminated in four generations, when in competition with a race carrying only two virulence genes. Contrary to such reports, some
investigations have established that races with the greatest number of identified virulence genes became predominant after a number of generations on a susceptible variety (Ogle and Brown, 1970; Allen, 1975). Brown and Sharp (1970) found that a yellow-spored race of *P. striiformis*, possessing several genes for virulence, predominated in competition with a white-spored mutant of a race possessing fewer genes for virulence.
Changes in the incidence of various races of cereal rust fungi are well known in the field (Stakman and Christensen, 1960). Martens (1973) found that, under field conditions, races of oat stem rust with a wide virulence spectrum performed consistently better than races with a narrow virulence spectrum. However, he concluded that the number of genes for virulence, other than those required for successful parasitism, was probably not the key determinant affecting the success of a race in the field. Similarly, the predominant races of oat stem rust recorded in Australia during the 1950's were those having the minimum number of virulence factors necessary for survival (Watson and Singh, 1952; Watson, 1954).
Races and isolates of *P. striiformis* are known to differ in their reproductive rate when grown as a pure culture (Johnson and Taylor, 1972; Johnson and Bowyer, 1974; Priestley and Doling, 1974) or as mixed infections (Brown and Sharp, 1970) on varieties of wheat apparently equally susceptible to the test isolates. A number of investigations have been carried out in an attempt to reveal causes for the predominance of certain races or isolates in surveys and experimental studies, especially when the predominance could not be explained by selection due to the widespread use of varieties with race-specific resistance (Johnson and Taylor, 1976).
Broyles (1955) investigated the percentage germination, the ability to infect and the ability to produce uredospores of a number of races of *P. graminis tritici* and found that although the races differed widely in
many of these respects, survival ability depended upon the cumulative effects of these factors and perhaps, also, on others. Ogle and Brown (1970) reported that strain 21 Anz-2,3,7 had a higher reproductive potential when used in mixed infections with strain 21 Anz-2,7. Later studies (Ogle and Brown, 1971) revealed such a difference was due to the higher infection levels that were produced by strain 21 Anz-2,3,7. Uredia of strain 21 Anz-2,3,7 also increased more rapidly in size and were ultimately larger in area than those of strain 21 Anz-2,7. Similarly, more uredospores per uredium were produced by strain 21 Anz-2,3,7. It was suggested such differences between strains could account, at least in part, for differences in relative survival ability.
Clifford and Clothier (1974) found that races E and F of *P. hordei*, isolated from the variety Vada, produced higher levels of disease on Vada than did isolates of the same races from other barley varieties. It was suggested that the increased aggressiveness of the Vada isolates may have been due to prepenetration or postpenetration processes. Rastegar (1976) investigated the competitive ability of races F and A1 of *P. hordei* in mixed infections. It was found that race A1 germinated more rapidly than race F and this was a contributing factor to the predominance of race A1 at heavy infection levels. However, race F produced more viable uredospores than race A1 and tended therefore to become prevalent at low infection levels.
Environmental factors, particularly light and temperature, have been found to have profound effects on the host:pathogen interaction. Katsuya and Green (1976) found that race 56 of *P. graminis tritici* predominated in competition with race 15B at 20°C and 25°C but that race 15B predominated at 15°C. Laboratory experiments were used to explain the rise in frequency of race 15B in Canada during 1953 and 1954, when temperatures were lower than average. The subsequent increase of race 56 was considered to be due to the higher than average temperatures which followed. Browder (1965)
found that race 56 sporulated more profusely than race 15B at temperatures of 16-21°C. Races 1, 5, 15, 53 and 104B of *P. recondita* exhibited lower levels of spore production when light and temperature levels were increased from 1600 ft candles and 62°F to 2600 ft candles and 75°F (Eyal and Peterson, 1967).
Yellow rust is particularly sensitive to environmental conditions. Sharp (1965) reported that the pre-penetration and post-penetration environmental conditions in which the host plants were grown were critical to the successful proliferation of the pathogen. He observed the effect of environmental conditions on infection and the subsequent phases of development and found that the dark period temperature was of importance to all development stages. McGregor and Manners (1976) investigated the effects of light on the growth and sporulation of yellow rust on susceptible wheat varieties. The overall spore production was significantly reduced by lowering the level of light falling on the infected plants.
It seems probable that isolates of rust fungi will be adapted to the environment from which they are collected. Therefore, if isolates are collected from different regions, it is possible that they possess different temperature optima (Johnson and Taylor, 1976).
As the host plant matures its interaction with a race or isolate of the pathogen can change, presumably as a result of changes during the process of development in the host. Previously this has been more evident with respect to race-specific resistance. Manners (1950) suggested that a variety that was susceptible as a seedling to an isolate of the fungus, could be susceptible or resistant at the adult plant stage. This adult or mature plant type of resistance can be expressed quantitatively against the pathogen, as exhibited by the varieties Little Joss, Yeoman and Holdfast (Russel and Hudson, 1974). Quantitative assessments of the host:pathogen interaction at the seedling stage are not necessarily a good guide to the situation occurring with more mature plants. However, quantitative
resistance in the variety Vada to *P. hordei* was observed at the seedling stage and also operated in adult plants (Clifford, 1972). Priestley and Doling (1974) tested two isolates of *P. striiformis* on seedling and adult plants and found that the most prolific isolate on seedlings was also the most prolific on more mature plants.
Most studies on variation in rust fungi have been concerned with major gene differences, which have the attraction of providing easily assessed criteria in determining genetic differences between isolates of the pathogen or in determining host resistance. The present work aimed to provide more information on quantitative variation in the wheat:yellow rust system.
4.2 EXPERIMENTAL MATERIALS, METHODS AND RESULTS
4.2.1 General experimental procedures
Glasshouse environmental conditions
A glasshouse was used for a number of spore production experiments during the autumn and spring of 1975 and 1976. Use of the glasshouse was restricted to these periods because of the low light levels during the winter and the high temperatures during the summer months. Natural daylight was supplemented by two banks, each of six Crompton "double life" single coil bulbs (200 watts). This gave light intensities during experiments of the order of 10-12,000 lux but, during December and January, the artificial lighting was insufficient to raise the overall light intensity to this level. The glasshouse compartment was fitted with heating elements, thermostatically controlled to maintain temperatures during the day of $15^\circ - 20^\circ$C. During the dark period (8 hours) the temperature in the glasshouse was allowed to fall to approximately $10^\circ$C.
Air filters were not fitted in the glasshouse and therefore plastic vinyl domes were used during the growth and incubation of the test plants to prevent cross contamination between isolates. The domes were removed once the test plants were sporulating, in order to permit easy access to the seedlings for spore collection. Between experiments the glasshouse was sprinkled with water and the heating turned up to maximum, in order to destroy any viable inoculum present.
Growth cabinet environmental conditions
A number of experiments were conducted in the Sherer 255 CEL growth cabinet, which was programmed to give a photoperiod of 16 hours (12,000 lux) at $18^\circ \pm 2^\circ$C and a dark period of 8 hours at $12^\circ \pm 2^\circ$C. Each growth cabinet was thoroughly cleaned with detergent and hot water between experiments.
Quantitative control of inoculum
Two techniques were used for inoculating the test varieties during the quantitative studies:
Cotton wool swabs
A known weight of freshly collected uredospores was thoroughly mixed for 30 seconds with 20 times its weight of sterilised talc in a test tube. The mixture was then inoculated onto the upper leaf surface by using sterile cotton wool swabs (Medical Wire and Equipment Co, Bath, Wilts). Using the Slee 4 Vertical Lamina Flow Bench, each swab was placed in the mixture of talc and uredospores and then gently brushed along the leaf surface, using the deposit of talc as an indicator of uredospore distribution. After inoculation, each pot (12 cm) was covered with a moist dome, placed in the infection chamber, and treated as previously described so as to permit disease development (Section 3.2.1).
The spore settling tower
More precise control of the inoculum was achieved by the construction of a spore settling tower (Bell, Schmidt, Miller and Kingsolver, 1956). The tower with modifications, suggested by Clifford (personal communication) is illustrated in Fig. 62, and closely follows the specifications of Eyal, Clifford and Caldwell (1968). The tower was calibrated and it was found that a uniform distribution of inoculum was produced when 5 mg of uredospores were allowed to settle on test seedlings for 2 min. The inoculum was introduced into the glass inoculator by means of a small glass funnel. A specially constructed glass tube and a modified pro-pipette were used to introduce each uredospore sample into the tower. The pro-pipette had to be reset for each batch of plants, thus preventing spores from being drawn into the inoculating unit. Between inoculating different isolates, the tower was thoroughly cleaned with 50% ethanol in order to prevent any cross contamination.
Fig. 62. Spore settling tower
upper tower
baffle gate
modified pro-pipette
lower tower
For inoculation the test seedlings were taped to a pot back-support and placed on the base of the tower. Following inoculation, plants were placed in the infection chamber overnight and returned to the glasshouse the following day.
**Isolates of the pathogen**
Isolates in vacuum dried ampoules were obtained from the PBI and the NIAB, Cambridge. An additional isolate used in the experimental work was collected from trial plots of the Scottish Plant Breeding Station in East Lothian. A list of the isolates used in the present study, their variety of origin and year of identification is given in Appendix 2.
Each isolate was multiplied on the susceptible variety Sappo and single spore cultures were then established from the respective isolates. Single spore lines were tested after multiplication on the World and European differential varieties and classified using the race nomenclature of Johnson *et al.* (1972).
**Experimental varieties of wheat**
As indicated above, the standard set of World and European differentials was used to classify isolates of the pathogen (Johnson *et al.*, 1972). For quantitative investigations on *P. striiformis*, varieties of wheat were selected that were highly susceptible at the seedling stage to all known races of yellow rust (see Appendix 3). As far as could be ascertained, no evidence was present in the literature to indicate that any of the test varieties possessed race-specific resistance.
4.2.2 **Quantitative variation between isolates of the pathogen**
Quantitative differences between isolates of yellow rust were investigated using two experimental techniques: measuring colony growth rate and weighing spores.
1. **Colony growth studies**
**Preparation and inoculation of test seedlings (Experiments 1, 2 and 3)**
Ten seeds of the variety Sappo were sown in each 12 cm pot containing John Innes No.2 potting compost. After watering, each pot was transferred to the growth cabinet. Cabinets were set to operate at the conditions previously described (Section 4.2.1). Inoculum of each isolate was multiplied on the susceptible variety Red Bobs and collected by means of a sterile glass tube (see Section 3.2.1). All inoculum was stored for 2 days over 50% sulphuric acid (Stevens, 1916) and then used to inoculate the test seedlings. Seedlings were inoculated by means of a modified pair of steel forceps, which enabled a standard amount of inoculum to be applied to a point source, centrally on the adaxial leaf surface (Figs. 63 and 64) (Priestley and Doling, 1974). Three pots, each consisting of six seedlings, were inoculated with uredospores on the tenth day after sowing in experiments 1 and 2 and six pots in experiment 3. Isolates were arranged in a randomised block layout with one pot of each isolate to each block.
**Incubation**
Following inoculation, each pot was covered with a moist dome and placed in the infection chamber overnight, before returning to the growth cabinet the following morning. The domes were removed and not replaced until heavy flecking of the leaves was visible. In experiments 1 and 3, domes were replaced after 8 days and after 5 days in experiment 2. This provided a humid atmosphere for spore production and symptom expression, as well as preventing cross contamination between isolates. Seedlings were watered daily and every 2 to 3 days the foliage cut back to the first leaf.
**Measurement of sporulating lesions**
Twenty days after inoculation each pot of seedlings was removed to the lamina flow bench and the variation between isolates determined by measuring
Fig. 63 Apparatus utilised for colony growth experiments
Fig. 64 Inoculation of test seedlings - colony growth experiments
the diameter of the fungal colony (Fig. 65). The results were then subjected to an analysis of variance.
**Results**
The results of the colony growth rate experiments are summarised in Table 12 (see Appendices 4-6 for analysis of data). All three experiments confirm that the isolate of race 40E8 produced the largest colonies: the average diameter being significantly greater ($P < 0.05$) than those of isolates 104E137 (71/2) and 37E132 but not significantly greater than the dimensions for the other test isolates. The small colony size for isolate 104E9 in experiment 1 is attributed to a low inoculum viability and probably a more representative value was obtained in experiment 2. Greater colony growth of all isolates in experiment 2 was most likely due to the fact that the domes were placed over the pots three days earlier in this experiment than in the others. The coefficient of variation for each of the colony growth experiments was approximately 10%.
2. **Spore production studies - multiple seedling tests**
*Preparation, inoculation and incubation of test seedlings (Experiments 4 and 5)*
Six seeds of the variety Sappo were sown in each 12 cm pot of John Innes No.2 potting compost. Isolates for testing were multiplied on the variety Red Bobs, the inoculum collected into glass tubes, diluted with talc (1:20 by weight) and inoculated onto four test seedlings in each pot using sterile cotton wool swabs. Each pot was placed in the infection chamber before being returned to the glasshouse the following morning. The pots containing different isolates were arranged in a randomised block layout with five replicates. Six days after inoculation, the talc was washed from the infected leaves with a stream of water. Each plant was cut back every 2 days to the first leaf.
Fig. 65 Colony development - 20 days after inoculation
Measuring variation in spore production between test isolates
At the commencement of sporulation, uredospores were collected in sterile 150 x 15 mm glass tubes. Each seedling leaf was inserted into the tube and gently tapped so that the uredospores fell onto the base of the glass tube. Inoculum from each of the four leaves was collected in this way every 48 hours. The area of sporulating tissue on the adaxial leaf surface was also assessed at each collection, to permit spore production to be calculated for each $10 \text{ cm}^2$ of infected leaf tissue.
At the end of the sporulating period, the accumulated inoculum in each tube was weighed in a plastic weighing boat using a Sartorius electrical microbalance. The data was subjected to an analysis of variance.
| Isolate | Reaction class | Colony diameter (mm) | Spore production (mg/10 cm²) |
|-----------------|----------------|----------------------|-----------------------------|
| | | Experiment No. 1 | 2 | 3 | 4 | 5 |
| 32E160 | 3⁺ | 29.4 | | 29.2 | 20.3 | 13.3 |
| 33E32 | 2⁺/3 | 26.9 | 36.7 | | | 10.9 |
| 37E132 | 3⁺ | | 31.3 | | | |
| 40E8 | 3⁺ | 30.0 | 39.3 | 30.0 | 24.3 | 16.5 |
| 41E136(a) | 3⁺ | | 36.3 | | 20.8 | |
| 41E136(b) | 3⁺ | | | | 21.3 | |
| 104E9 | 3⁺/4 | 23.2 | 37.7 | | 18.5 | 14.9 |
| 104E137 (69/10) | 3⁺ | 28.8 | | | 21.8 | 15.5 |
| 104E137 (69/10)m| 3⁺ | | | 28.0 | 22.6 | |
| 104E137 (71/2) | 3⁺ | 25.0 | | | 20.9 | 13.6 |
| 104E137 (72/23) | 3⁺ | | 38.3 | | | |
| 104E137 (71/26) | 3⁺ | | 36.0 | | 21.3 | |
| 108E9 | 3/3⁺ | | | | 20.6 | |
| 108E141 (2) | 3⁺ | 27.5 | | 27.3 | | 14.4 |
s.e.d. 2 mean values in individual experiments = 1.2 1.2 1.1 0.9 1.0
not included in experiment
Results
Ten isolates were compared in experiment 4 and seven isolates in experiment 5 and the results of the spore production tests are summarised in Table 12. Highly significant differences (P < 0.001) between isolates were found both in experiment 4 (Appendix 7) and in experiment 5 (Appendix 8). The isolate of race 40E9 ranked highest in order of spore production in both experiments. It produced a significantly greater weight of uredospores (P < 0.05) than isolates 32E160, 41E136(a), 104E9, 104E137 (71/2) and 108E9 in experiment 4 and 32E160 and 33E32 in experiment 5. Four isolates of race 104E137 were compared in experiment 4 but these were not statistically different from each other when grown on the variety Sappo. This was also true of the two isolates of race 41E136 which were compared in the same experiment. Inoculum of each isolate was visually assessed for viability before inoculating the experimental seedlings. This entailed a brief microscopic scan of an inoculated plate of 1% water agar. A low inoculum viability was recorded for isolate 104E9 in experiment 5 and the results obtained for this isolate in experiment 4 are probably more representative. The results summarised in Table 12 also indicate the reaction type of each isolate on the variety Sappo after 16 days' incubation and from the table it can be seen that most isolates produced a highly susceptible reaction at this stage. The coefficient of variation for the two experiments was comparable to that produced in the colony growth studies, being 6.9% in experiment 4 and 11.2% in experiment 5.
Experiment 6
Results from colony growth and spore production studies (experiments 1-5) indicated the existence of quantitative variation in isolates of *P. striiformis* on the same susceptible variety. A further experiment was carried out to determine how selected isolates of the pathogen performed on a number of susceptible host varieties. Four isolates, each of which produced a similar reaction type on the control variety (Sappo), were inoculated onto the
varieties Atle, Jufy 1 and Sappo. Isolates 32E160 and 40E8 were selected because they exhibited contrasting characteristics, 40E8 being significantly more prolific than 32E160 in spore production tests. Isolate 104E137 (69/10), a white spore line, possessed a phenotypic characteristic which enabled it to be distinguished from other isolates, while isolate 37E132 was originally identified and isolated from the variety Rothwell Perdix during the severe yellow rust epidemic of 1966.
**Germination level of test isolates**
In order to assess the germination of the test isolates used in this spore production experiment, freshly collected uredospores were inoculated onto each of three 1% water agar plates using the spore settling tower. After overnight incubation in the infection chamber, 500 spores on each plate were scored for viability. A spore was considered to have germinated when a germ tube, at least the diameter of the spore in length, had been produced after incubation for 24 hours. The germination ratio is the fraction of the spores that had formed germ tubes within one day.
**Results**
**Uredospore viability**
Uredospores of isolates 40E8 and 32E160 were similar in viability and both were significantly higher in viability ($P < 0.001$) than isolates 37E132 and 104E137 (Table 13 and Appendix 9). Isolate 37E132 showed a significantly higher viability than isolate 104E137, which had the lowest germination frequency.
**Spore production of test isolates**
In comparing the different test isolates a highly significant difference ($P < 0.001$) was found between isolates in mean spore production on the three wheat varieties (Appendix 10). Isolate 40E8 produced the greatest quantity of uredospores on each of the host varieties (Table 13) and was significantly more productive than any other isolate ($P < 0.01$). Isolates 37E132 and 104E137 produced a similar weight of inoculum but significantly more than isolate 32E160.
Spore production from host varieties
The three wheat varieties showed significant differences in the weight of uredospores produced when each was inoculated with the test isolates of *P. striiformis* (Appendix 10). More spores were produced from Jufy 1 ($P < 0.01$) than from either Atle or Sappo (Table 13). There was no significant difference between Atle and Sappo in overall spore production, although smaller weights of uredospores were produced from Sappo with all test isolates.
**TABLE 13.** Germination ratio and spore production of four test isolates on the host varieties Atle, Jufy 1 and Sappo (multiple seedling technique)
| Variety | Isolates | Spore production mg/10 cm² |
|---------|----------|----------------------------|
| | 40E8 | 37E132 | 104E137 | 32E160 |
| | | Germination ratio | | |
| | 0.90 | 0.65 | 0.40 | 0.96 |
| | s.e.d. | 2 ratio means = 0.06 | | |
| | | Spore production mg/10 cm² | Mean |
| Atle | 14.6 | 11.9 | 12.4 | 11.0 | 12.5 |
| Jufy 1 | 17.1 | 14.8 | 14.8 | 11.2 | 14.5 |
| Sappo | 12.7 | 10.9 | 12.3 | 10.8 | 11.7 |
| Mean | 14.8 | 12.6 | 13.2 | 11.0 | |
s.e.d. 2 variety means = 0.4
s.e.d. 2 race means = 0.4
s.e.d. 2 means within table = 0.7
**Isolate:variety interaction**
A significant interaction ($P < 0.01$) occurred between test isolates and host varieties (Appendix 10). Most of the interaction was due to the behaviour
of isolate 32E160 (Table 13). This isolate produced more inoculum, relative to the average response, on Sappo but less on Jufy 1. Smaller deviations from the general pattern were recorded with isolate 40E8, which produced a greater weight of uredospores on Jufy 1 and a lower weight on Sappo compared with the average response of isolates. Isolate 37E132 produced a greater weight of uredospores on Jufy 1 relative to its behaviour on other varieties.
3. Spore production studies - single seedling tests
Preliminary experiments had shown that the single seedling method used for assessing the host:pathogen interaction (Johnson and Bowyer, 1974) produced results comparable to other techniques. The various host:pathogen combinations selected and used in experiment 6 (Table 13) were reproduced in a single seedling experiment in order to assess this method.
The apparatus used for the single seedling study is illustrated in Fig. 66, and shown during operation in Fig. 67. A plastic seed tray 37 x 23 x 8 cm was modified to hold up to twelve of the seedling units. Plastic piping 3 cm in diameter was held in position in the tray by a stainless steel springclip, while open-ended glass tubes 15 cm long and 1.5 cm in diameter were used for collecting the discharging uredospores.
Preparation, inoculation and incubation of test seedlings (Experiment 7)
The base of each plastic seed tray was covered by a layer (3 cm) of John Innes No.2 potting compost and the single seedling tubes were clipped into position. Seeds of the three varieties were sown at a uniform depth in the plastic piping and covered evenly with fine sand. The sowing was arranged so that each host:pathogen combination could be represented in each of five randomised blocks. Large cellophane domes (Fig. 68) were used to cover the seedlings during early growth and thereby prevent any cross contamination in the glasshouse.
Fig. 66. Single seedling apparatus.
Fig. 67 Single seedling apparatus - in operation
Fig. 68 Large isolation domes
Seedlings were inoculated with each of the test isolates, using cotton wool swabs, when the first leaf was fully expanded. Each of the seed trays was then placed in a large plastic dome, and removed to the infection chamber. Trays were returned to the glasshouse the following morning.
**Collection of inoculum**
The talc used in the inoculating procedure was washed from the seedling leaves after approximately 6 days. Just prior to the onset of sporulation, each leaf was carefully measured, inserted into the glass tubes and clipped into position. Uredospores were initially discharged after 10 days' incubation and in order to ensure no inoculum was lost, the glass tubes were frequently adjusted throughout the duration of the experiment. When the leaves of the test varieties were no longer discharging uredospores, the first leaf was detached and shaken in the glass tube using artery forceps. The weight of uredospores produced by each host:pathogen combination was then determined on an electrical microbalance.
**Results**
**Spore production of test isolates**
There was no significant difference between the test isolates in spore production (Appendix 11). Isolate 40E8 was, however, the most prolific isolate, while isolate 32E160 was the least (Table 14). The experiment had a coefficient of variation of 28%.
TABLE 14. The spore production of four test isolates on the host varieties Atle, Jufy 1 and Sappo (single seedling technique)
| Variety | 40E8 | 37E132 | 104E137 | 32E160 | Mean |
|---------|------|--------|---------|--------|------|
| Atle | 13.1 | 11.7 | 10.4 | 9.2 | 11.1 |
| Jufy 1 | 19.4 | 18.1 | 15.6 | 14.3 | 16.9 |
| Sappo | 12.9 | 14.9 | 13.2 | 12.8 | 13.5 |
Mean
s.e.d. 2 variety means = 1.2
s.e.d. 2 race means = 1.4
s.e.d. 2 means within table = 2.5
Spore production from host varieties
There was a highly significant difference ($P < 0.001$) between varieties in the weight of inoculum produced by the test isolates (Appendix 11). From the variety Jufy 1 significantly more spores were produced ($P < 0.05$) than from either Atle or Sappo, which were not significantly different (Table 14). There was no significant interaction between the test isolates and the host varieties.
4. Spore production studies - a comparison of different isolates in relation to host plant age
Previous experiments in this study have been concerned with seedling responses following inoculation with a number of test isolates of *P. striiformis*. As plants mature they undergo developmental changes and these can be identified by differences in the host:pathogen interaction. A series of experiments were conducted in conjunction with the NIAB, Cambridge, on a number of host:pathogen combinations.
Preparation, inoculation and incubation of test varieties (Experiment 8)
The varieties Gamin, Score and Clement were sown ten seeds per 12 cm pot. Twelve pots of each variety were established in three separate experiments, each consisting of six randomised blocks. Each isolate was multiplied on its variety of origin and freshly collected uredospores were inoculated onto the test varieties 10 days after sowing as previously described. Experiments with the same host:pathogen combinations were established on adult plants in large polythene tunnels at the NIAB.
Experimental varieties and yellow rust isolates
In seedling and mature plant experiments the same host:pathogen combinations were tested for quantitative differences. Isolates 74/298 and 74/129 were inoculated onto the variety Gamin, Score was inoculated with isolates 71/368 and 69/163, while Clement was inoculated with isolates 74/62 and 71/493 (Appendix 2). Uredospores from four seedling leaves were collected into glass tubes every 48 hours and weighed on the electrical microbalance at the end of each experiment. In adult plant experiments the number of pustules produced by each host:pathogen combination were assessed.
Results
Variety Gamin
Adult plant experiments revealed that isolate 74/298 produced a significantly greater incidence of yellow rust on the variety Gamin than did isolate 74/129 (R.Priestley, personal communication). Seedling test results showed that isolate 74/298 produced a reaction type $3^+$ and high levels of sporulation, while isolate 74/129 produced no sporulation and a reaction type 0.
Variety Score
Isolate 71/368 produced a significantly greater amount of yellow rust than isolate 69/163 on adult plants of the variety Score. Seedling tests, however, revealed no comparable difference, with both isolates producing a reaction type 3. Isolate 71/368 yielded on average 9.1 mg uredospores on each $10 \text{ cm}^2$ of leaf tissue while isolate 69/163 yielded 8.6 mg.
Variety Clement
Isolates 74/62 and 71/493 both failed to produce any sporulation on the variety Clement in seedling tests. However, in adult plant tests isolate 74/62 produced significantly higher levels of disease than isolate 71/493.
5. Mixed infection studies
Mixed infection studies were established in an attempt to extend the available information on quantitative variation in the fungus. The test isolates used in the experiments were isolates of races 32E160, 40E8, 104E137 (69/10) and 108E141. Races 32E160 and 40E8 possess a similar number of virulence characters, while 108E141 was selected because it possesses a greater number of identified virulence factors. The albino (white spore) mutant of race 104E137 (69/10) was chosen because it enabled rapid scoring of the experimental material.
Establishment of mixed infections (Experiment 9)
The four test isolates were multiplied individually on the susceptible variety Red Bobs. Each of the yellow isolates (32E160, 40E8 and 108E141) was mixed with the white spore isolate (104E137) in three ratios (90:10; 50:50 and 10:90, by weight). After thoroughly mixing, each of the combinations was inoculated onto six seedlings in each of three replicates, using a sterile camel hair brush. This permitted uredospores to be distributed evenly on the adaxial leaf surface. After inoculation each pot was placed in the infection chamber overnight. The following morning the pots were returned to the growth cabinet and replaced in the latin square arrangement. Every 3 or 4 days following inoculation plants were cut back to the first leaf.
Re-inoculation and scoring of mixed infections
Thirteen days after inoculation, infections were sufficiently well developed for the next generation of seedlings to be inoculated. The three pots comprising each combination were removed to the flow bench and with the
use of a camel hair brush, uredospores were brushed from infected seedlings onto the adaxial surface of uninfected seedling leaves. Seedlings were re-inoculated in this way, until each combination had completed five generations.
Following re-inoculation, the sporulating leaves were detached by means of a modified pair of scissors and 3 cm segments were removed 2 cm behind the leaf tip. The number of yellow and white pustules on each segment was then determined using a stereo-binocular microscope, the percentage wild type pustules calculated for each combination and the data statistically analysed.
**Comparing the yellow and white isolates of race 104E137 (69/10)**
As the results of the mixed infection experiments with the white isolate of race 104E137 were inconsistent with the spore productivity results, a further experiment was carried out to determine if the white and yellow isolates of this race were of equal competitive ability.
**Results**
The results obtained from mixing the various yellow isolates with the white spore line are summarised in Table 15. In every case the white spore isolate decreased in frequency over five uredial generations. However, the relative increase of each of the yellow isolates was not always statistically significant. For example, when the yellow and white isolates of race 104E137 (69/10) were mixed in equal parts, the yellow spore line increased in each of the replicates but this was not statistically significant (Table 15 and Appendix 14).
TABLE 15. Relative proportions of test isolates in mixed infection experiments
| Generation | % wild type pustules |
|------------|----------------------|
| | 1 | 2 | 3 | 4 | 5 | P |
| Isolates | | | | | | |
| 40E8 x M | 97.7| 98.5| 99.3| 99.5| 99.4| NS |
| 108E141 x M| 91.0| 96.4| 96.7| 96.3| 99.7| <0.01|
| ratio | | | | | | |
| 32E160 x M | 96.8| 94.6| 95.8| 95.1| 97.8| NS |
| Generation | % wild type pustules |
|------------|----------------------|
| | 1 | 2 | 3 | 4 | 5 | P |
| Isolates | | | | | | |
| 40E8 x M | 71.5| 82.9| 93.4| 90.3| 93.6| NS |
| 108E141 x M| 66.7| 72.2| 79.1| 78.6| 89.9| NS |
| ratio | | | | | | |
| 32E160 x M | 73.4| 74.3| 76.0| 59.1| 76.4| NS |
| Generation | % wild type pustules |
|------------|----------------------|
| | 1 | 2 | 3 | 4 | 5 | P |
| Isolates | | | | | | |
| 40E8 x M | 25.8| 50.4| 63.4| 69.8| 79.4| <0.01|
| 108E141 x M| 26.6| 25.9| 32.3| 46.6| 47.7| NS |
| ratio | | | | | | |
| 32E160 x M | 45.6| 44.1| 46.7| 65.3| 69.9| <0.05|
| Generation | % wild type pustules |
|------------|----------------------|
| | 1 | 2 | 3 | 4 | 5 | P |
| Isolates | | | | | | |
| 104E137W x M| 65.3| | | | 69.4| NS |
| ratio | | | | | | |
M = white spore mutant of race 104E137
P = probability from t test that a difference exists between generation 1 and generation 5
✓ = not assessed
The frequency of the wild type (yellow) pustules increased noticeably in the 90:10 mixture, although only isolate 108E141 increased significantly (P < 0.01) over the 5 generations (Table 15, Appendix 12). This isolate also increased significantly more (P < 0.05) than did the other wild type
lines (Appendix 13). In the 50:50 mixture isolates 40E8 and 108E141 increased substantially but not significantly (Appendices 12 and 13). The relative increase of the yellow spore lines was more noticeable in the 10:90 mixture. Isolates 40E8 ($P < 0.01$) and 32E160 ($P < 0.05$) increased significantly, while isolate 108E141 did not, although this latter isolate had almost doubled in frequency over the 5 generations. A comparison of isolates revealed that in the 10:90 ratio isolate 40E8 increased significantly more ($P < 0.05$) than the other isolates (Appendix 13).
4.2.3 Components of quantitative variation
A series of experiments were carried out to examine the pattern of development of the pathogen and establish the stages of development or growth where quantitative differences between isolates became evident.
Preparation, inoculation and incubation of test seedlings
Seedlings of the variety Sappo were inoculated with four isolates of yellow rust (32E160, 40E8, 104E137 (69/10) and 108E141) using the spore settling tower. After inoculation, the pots, each containing seven seedlings, were removed to the infection chamber for 18 hours and returned to the growth cabinet the following day. The isolates were arranged in a randomised block lay-out replicated six times.
Studies on developmental stages of the fungus
Germination
When the test seedlings were being inoculated, three water agar plates (1%) were placed on the base of the settling tower. The inoculum density and germination frequency of each of the isolates was assessed using these plates, as described in Section 4.2.2.
Infection efficiency
At the end of the initial 16 hour photoperiod following inoculation, a leaf was selected at random from each pot and detached. A 3 cm segment was removed 2 cm from the tip of each leaf using a pair of modified scissors. Each segment was then placed in a deep freeze unit at minus $20^\circ C$ for 30 minutes.
(Parker, 1976). After allowing the tissue to remain at room temperature for 30 minutes, each segment was placed in a solution of absolute ethanol and lactophenol (2:1 by volume), brought to boil slowly and allowed to boil for 1\(\frac{1}{2}\) minutes. After cooling, each segment was placed in a 0.1% solution of trypan blue for 48 hours and mounted in lactophenol. The number of penetrated stomata on the adaxial leaf surface of each segment was then determined.
**Days to sporulation**
The commencement of sporulation was determined by tapping each infected leaf into a clean glass tube and looking visibly for discharged uredospores. As each leaf commenced sporulation, it was tagged and the day when three of the test leaves were releasing spores was taken as the number of days to sporulation.
**Uredospore and pustule number**
Ten days after inoculation, just prior to pustule eruption, a leaf was selected at random and a 1 cm segment detached 2 cm from the leaf tip. This segment was detached and floated on exactly 10 ml of benzimidazole solution (60 ppm) in a plastic compartmentalised box. The four segments from each block were placed in each of six plastic boxes. The six boxes were then randomised in the growth cabinet. Seven days after being placed in the boxes, a drop of Tween 80 was added to each compartment and each segment agitated with forceps to remove the uredospores from the leaf surface. The number of uredospores present on each segment was then assessed and taken as the mean of three haemacytometer slide counts. The area of each segment was carefully measured and the number of uredosori on each segment determined using a binocular microscope.
**Periodicity and total uredospore production**
Prior to sporulation infected seedlings were transferred from the growth cabinet to a glasshouse to allow easy access for uredospore collection.
Uredospores from each of four seedlings were collected in sterile glass tubes every 48 hours and the weight of uredospores determined using the electrical microbalance. Seedlings were measured twice during sporulation in order to assess the area of sporulating material in each pot. Spore collection continued until the seedling leaves senesced.
**Terms used in the developmental study**
A number of terms used in the results of the development work have been previously defined (Mehta and Zadoks, 1970). The germination ratio is the fraction of spores that formed recognisable germ tubes within one day. The colonisation ratio is the fraction of germinated spores that produced pustules. The infection ratio is the fraction of spores (total of germinated and ungerminated) that produced pustules.
**Results**
**Germination, infection and colonisation by the test isolates**
There was a significant difference ($P < 0.001$) between isolates in the frequency of spores that formed germ tubes (Appendix 15). The proportion of spores that formed germ tubes within 24 hours was high in isolates 40E8 and 32E160, intermediate in 108E141 and low in the isolate of race 104E137 (Table 16).
**TABLE 16.** The germination, infection and colonisation ratios of test isolates on the susceptible variety Sappo
| Isolate | Germination ratio | Colonisation ratio | Infection ratio |
|-----------|-------------------|--------------------|-----------------|
| 40E8 | 0.97 | 0.14 | 0.14 |
| 32E160 | 0.94 | 0.08 | 0.08 |
| 108E141 | 0.78 | 0.07 | 0.06 |
| 104E137 | 0.47 | 0.14 | 0.06 |
| s.e.d. | 2 race means 0.02 | 0.01 | 0.01 |
The number of infection structures established by the different test isolates was not itself a true estimate of the infectivity, since the uredospores of each isolate inoculated onto the test variety varied in concentration and viability. The colonisation and infection ratios were corrected for such variables. It may be seen from Table 16 that isolates 40E8 and 104E137 were not significantly different in their colonisation ratio, whereas the other two isolates had a significantly lower colonisation ratio (Appendix 16).
Isolate 40E8 produced a significantly higher infection ratio \((P < 0.01)\) than the other three isolates which were not significantly different from each other (Table 16 and Appendix 17).
**Days to sporulation (latency)**
Pustules of the isolates were erumpent on some leaves approximately 10 days after inoculation. A highly significant difference \((P < 0.001)\) was found between the test isolates in the number of days it took each to commence sporulation (Appendix 18). Isolates 40E8 and 108E141 were sporulating on average 12 days after inoculation. Isolate 104E137 was sporulating on average after 12.5 days, while isolate 32E160 had the longest latent period of 13 days.
**Pustule density and uredospore production of the test isolates**
The test isolates produced a pustule density varying between 205 and 231 uredia per cm\(^2\) (Table 17). Isolates 40E8 and 108E141 produced a similar concentration of uredia and significantly more \((P < 0.05)\) than isolate 32E160 (Appendix 19).
Isolate 40E8 produced the greatest number of uredospores from each uredium and this was reflected by the greater weight of spores produced by this isolate (Table 17). However, no significant difference existed between the four isolates in the number of uredospores each produced (Appendix 20), although isolates 104E137 and 32E160 produced a significantly lower weight \((P < 0.05)\) of uredospores than isolate 40E8 (Appendix 21).
TABLE 17. The pustule density, uredospore production per pustule and total spore production of the test isolates on the susceptible variety Sappo
| Isolate | Pustule density per cm² | Uredospores per pustule | Total spore production mg/10 cm² |
|-----------|-------------------------|-------------------------|----------------------------------|
| 40E8 | 231 | 2960 | 20.2 |
| 108E141 | 231 | 2653 | 19.4 |
| 104E137 | 223 | 2300 | 18.8 |
| 32E160 | 205 | 2408 | 18.0 |
s.e.d. between 2 race means = 8 311 0.5
Periodicity of spore production of the test isolates
Uredospores of each test isolate were initially harvested 14 days after inoculation. Isolates 40E8, 108E141 and 104E137 produced similar amounts of inoculum at the first collection, whereas isolate 32E160 produced considerably less (Table 18). The pattern of sporulation of each isolate was, however, very similar, with a single peak occurring around day 18, approximately 6 days after the onset of sporulation. At this time spore production increased to 4.3 mg per 10 cm² in isolates 40E8 and 32E160 and slightly less in the other two isolates. After this peak, spore production fell progressively and had ceased in isolates 108E141 and 32E160 28 days after inoculation.
TABLE 18. The periodicity of spore production of isolates of *P. striiformis* on the susceptible variety Sappo
| Days after inoculation | 14 | 16 | 18 | 20 | 22 | 24 | 26 | 28 |
|------------------------|----|----|----|----|----|----|----|----|
| **Isolate** | | | | | | | | |
| 40E8 | 2.8| 3.7| 4.3| 3.6| 2.6| 1.9| 1.0| 0.3|
| 108E141 | 2.9| 3.7| 4.2| 3.5| 2.4| 1.7| 0.8| 0 |
| 104E137 | 2.8| 3.7| 4.1| 3.5| 2.5| 1.4| 0.7| 0.1|
| 32E160 | 2.0| 3.2| 4.3| 3.6| 2.6| 1.5| 0.8| 0 |
Small quantities of uredospores were still being produced by isolates 40E8 and 104E137 at this time when the experiment was terminated. Isolate 40E8 was consistently producing more inoculum at each uredospore harvest and this culminated in isolate 40E8 producing the highest weight of uredospores over the test period. Each of the test isolates produced a reaction type $3^+$ on the sixteenth day and the levels of chlorosis were small. However, after day 18 isolates 32E160 and 108E141 showed greater amounts of chlorosis than the other test isolates and this eventually led to the premature senescence of the seedlings inoculated with these race isolates.
4.3 DISCUSSION
Previous investigations involving genetical aspects of the wheat:yellow rust system have been concerned primarily with the virulence level of the pathogen and the way virulence characters are combined to produce new races of the fungus (Little and Manners, 1969(a); 1969(b); Goddard, 1976(b); Taylor, 1976). Other forms of variation which may be concerned with the success of the pathogen in the field have tended to be neglected and little published information is available on quantitative differences in the fungus. It is possible that quantitative variation in the pathogen may assume an increasing importance as the emphasis in plant breeding moves from race-specific to race-non specific resistance.
In the course of a number of investigations, the characteristics of a pathogen have become defined in the first instance by what seems to be major gene effects (Day, 1974) relating to virulence as discussed in Section 3. There is now also recognised a quantitative component of pathogenicity which has been referred to by various investigators as aggressiveness (Van der Plank, 1968), competitive ability (Katsuya and Green, 1967) and horizontal pathogenicity (Robinson, 1969). Quantitative characteristics studied in a number of fungi, mainly saprophytic in nature, have indicated that this type of variation is possibly controlled by polygenes (minor genes) or by extrachromosomal components (Burnett, 1975). With the wheat:yellow rust system the control of pathogen variation is not so clearly resolved and it would therefore seem appropriate to use the term quantitative variation in preference to polygenic variation.
Differences were found between yellow rust isolates measured in the present study in their rate of spore production and colony growth following the inoculation of a susceptible host variety. Quantitative differences between races of several rust fungi have been reported by a number of
investigators: *P. graminis* (Katsuya and Green, 1967); *P. recondita* (Eyal and Peterson, 1967); *P. coronata* (Sebesta, 1972) and *P. hordei* (Clifford and Clothier, 1974). Isolates of race 104E137 of *P. striiformis* were found to differ in quantitative characteristics on a number of host varieties (Johnson and Taylor, 1972; Priestley and Doling, 1974): as a result of such investigations it was found necessary to subdivide race 104E137 into a number of biotypes. In comparisons of isolates of different races used in the present work, the isolate of race 40E8 proved to be the most prolific in terms of spore production and colony growth rate, in all experiments and with all the host varieties under test. In comparisons of different isolates of race 104E137 there was evidence of within race variation in at least colony growth rate. Although differences between isolates of this race were not significant in the weight of spores produced, the isolates were ranked consistently in each of the spore production experiments in which they appeared.
Van der Plank (1968) has suggested that two types of pathogenic race exist: those that do and those do not interact differentially with host varieties. With reference to testing for the second group, there is no evidence for a differential interaction, if any one variety chosen at random is sufficient to sort out the races into their correct order with respect to quantitative characteristics. The general trend in the present study was for the test isolates to show a consistent order of ranking on the host varieties with respect to spore production, as measured by the multiple seedling technique (experiment 6). However, in one instance, the isolates of races 37E132 and 104E137 were ranked equally on the wheat variety Jufy 1. It was also evident that a significant interaction was present in the analysed data between the yellow rust isolates and the host varieties. Aggressiveness, according to Van der Plank (1968) is a characteristic expressed uniformly against all genotypes of the host. This, however, seems
to be an over-simplification, since evidence of a specific compatibility between isolates and their variety of origin has been demonstrated in *P. striiformis* by Johnson and Taylor (1972) and in *P. hordei* by Clifford and Clothier (1974). It is quite conceivable that if more host varieties had been included in the current study the isolate of race 40E8 may not have been ranked consistently high.
The host varieties used in the quantitative studies were specifically chosen because they exhibited low levels of seedling resistance to isolates of yellow rust (Macer, personal communication). However, the results of seedling tests with rust fungi are not necessarily representative of adult plant studies. Manners (1950) found that a variety that was resistant to a race as a seedling was invariably resistant as an adult plant, while a variety susceptible as a seedling was either susceptible or resistant at the adult plant stage. Priestley and Doling (1974) tested two isolates of race 104E137 on seedling, juvenile and adult plants of the variety Joss Cambier. The results showed that isolates always ranked in the same order at all growth stages. The results from seedling and adult plant experiments in the present study were, however, inconsistent. The isolates inoculated onto the variety Gamin were ranked similarly at both growth stages. However, with the varieties Score and Clement, no significant difference between isolates occurred in seedling tests, but did occur in adult plant tests. Furthermore varieties resistant as seedlings were found to be susceptible at the adult plant stage.
It should be noted, however, that these experiments were limited in scope and require to be repeated for adequate assessment.
Spore production in a host:pathogen combination is the culmination of a number of developmental stages and its extent will depend on the efficiency of infection, the latent period, the spore production of each lesion and the length of the sporulation period (Van der Plank, 1963).
Quantitative differences between isolates, such as those observed in the spore production experiments, may arise as a result of variation in uredospore viability. Cassell (1939) found that race 34 of *P. graminis tritici* produced uredospores with a higher frequency of germination than those of races 11, 36, 38 and 56. Uredospores of strain 21 Anz-2,7 germinated more rapidly than those of strain 21 Anz-2,3,7, although final germination levels were not significantly different (Ogle and Brown, 1971). Browder (1965) found that uredospores of race 15B of wheat stem rust underwent a sharp reduction in viability shortly after collection, while those of race 56 did not. Although races differed in their levels of germination, Broyles (1955) could find no fixed relationship between the germination level and survival ability of races of wheat stem rust over a prolonged period of time.
In the current investigation, isolates 40E8 and 32E160 produced more viable uredospores than a number of other test isolates. Such differences were consistently recorded and did not seem to be due to poor batches of inoculum. However, since isolates 40E8 and 32E160 were significantly different in spore production, it would not appear that viability alone could be responsible for producing such a result. The isolate of race 40E8 produced significantly more infection structures (substomatal vesicles) than isolates 32E160, 104E137 and 108E141 and this difference could, in part, be responsible for the greater spore productivity of isolate 40E8. Allen (1926) found that races 9, 21 and 27 of *P. graminis tritici* differed in their ability to penetrate the variety Khapli. Results of a similar nature have been reported by a number of other investigators (Brown and Shipton, 1964; Ogle and Brown, 1971), who suggested that a high infection efficiency could possibly lead to the eventual predominance of a race.
Investigations on a number of rust fungi have shown that isolates may (Browder, 1965; Katsuya and Green, 1967) or may not vary in their latent period (Loegering, 1951; Ogle and Brown, 1971). Slight differences were found
between isolates in the period from inoculation to sporulation in this work, with the isolates of races 40E8 and 108E141 commencing sporulation before the isolates of 104E137 and 32E160. The higher levels of infection illicited by isolate 40E8 did not, however, necessarily lead to a shorter latent period as has been suggested to occur in other experimental studies on rust fungi (Yarwood, 1961; Mehta and Zadoks, 1970). Although fewer infections were established by isolate 108E141, it may have colonised the host tissue more rapidly than isolate 40E8 and thereby commenced sporulation at the same time. Alternatively, the high infection levels may have delayed the proliferation of isolate 40E8 due to the fact that the host assimilates were limiting and preventing the true quantitative expression of this isolate.
Isolates 40E8 and 108E141 produced significantly more uredia per unit area than isolate 32E160 in pure and in mixed infection studies. At lower infection levels, however, the results may have differed somewhat from those obtained at the high levels of infection established in the current investigation. Katsuya and Green (1967) found that in mixed infections race 56 predominated in light infections and race 15B-1 (Can) in heavy infections. It was suggested that race 15B-1 (Can) was the strongest competitor for host nutrients at high infection levels, whereas race 56 was the better competitor when infection levels were low and less stress was on the nutrient supply. Similarly, race A1 of *P. hordei* predominated in heavy infections and race F in light infections, when the two races were mixed and inoculated onto a susceptible variety (Rastegar, 1976). There is no evidence, therefore, that isolate 40E8 would be the most prolific race at all infection levels.
The number of spores per pustule is not an absolute value but represents the total produced at the time of sampling. Factors such as the longevity of primary pustules, the production of secondary pustules and the integrity of the leaf are critical to the total weight or number of spores produced.
Isolates 40E8 and 108E141 produced the greatest number of uredospores per pustule and also the highest number of spores per unit leaf area at the time of assessment. However, the size and longevity of uredosori, both critical components of overall spore production, were not considered in the present study. Ogle and Brown (1971) found a good correlation between uredial size and the number of uredospores produced, but Katsuya and Green (1967) found that the largest uredia did not necessarily produce the most spores.
The sporulation pattern of the test isolates revealed a single peak in spore production occurring approximately 18 days after inoculation. This peak was of a similar level for all four isolates, being approximately 4 mg per 10 cm$^2$ every 2 days. Following this peak, spore production fell progressively in each of the four test isolates, the decline being most noticeable in isolates 32E160 and 108E141: the amount of visible chlorosis increased rapidly in these two isolates around the twentieth day. Broyles (1955) investigated the spore production of a number of races of *P. graminis tritici* and observed three to four peaks of uredospore production. Each successive peak was smaller and lasted for a shorter time than the previous one. Browder (1965) found that the sporulation peak of races 15B and 56 occurred between 16 and 24 days after inoculation. Similarly, results obtained by Leonard (1969) on *P. graminis avenae* suggested that the rust fungus alternated between periods of sporulation and periods of mycelial growth and that several peaks of uredospore production occurred. Ogle and Brown (1971) recorded one major and three minor peaks of uredia production by strains 21-2,7 and 21-2,3,7 of *P. graminis tritici*. However, Yarwood (1961) found a similar pattern of sporulation in *Uromyces appendiculatus* to that recorded in the present study. The level of infection in the current investigation may have been excessively high and prevented the production of significant numbers of secondary uredia. Primary uredia may
therefore have been totally responsible for the levels of sporulation observed in the quantitative analysis. At lower levels of infection the pathogen may have alternated between periods of mycelial growth and sporulation and thus produced minor peaks in spore production.
The virulence spectrum of the test isolates of *P. striiformis* bore no obvious relationship with the performance of the isolate in quantitative studies. This is illustrated not only in spore production and colony growth studies but also in mixed infections. Isolates 37E132 (five virulence factors) and 104E137 (six virulence factors) produced a greater weight of uredospores than isolate 32E160 (three virulence factors) on the susceptible varieties Atle, Jufy 1 and Sappo. Conversely, isolate 40E8 (three virulence factors) produced greater amounts of spores and colony growth than any of the other test isolates. Similarly, in mixed infection studies, isolate 108E141 (eight virulence factors) increased in frequency at the expense of isolate 104E137 (six virulence factors) as also did isolate 40E8 (three virulence factors). There was no indication that any specific virulence factor was detrimental to the success of a fungal isolate. No evidence was obtained from the present study to support the suggestion that virulence and quantitative characters (aggressiveness) are either positively (Ogle and Brown, 1970; Allen, 1975) or negatively related (Van der Plank, 1963; 1968; Day, 1968). Moreover, quantitative variation was found to occur within a race of the pathogen in the present study and has been observed in a number of other investigations (Johnson and Taylor, 1972; Priestley and Doling, 1974). Although an inverse relationship between the number of genes for virulence and a races reproductive potential may exist in some cases, it is not a general rule for host-pathogen systems (Martens, 1973).
The behaviour of the white spore isolate of race 104E137 (69/10) as a pure culture was not reflected in its behaviour in mixed infections. The albino mutant tended to fall in frequency when inoculated simultaneously
with a number of yellow spore isolates. This was inconsistent with the findings of earlier experiments, in which the white spore mutant produced a greater weight of uredospores than most of the yellow spore lines. Such a discrepancy could be due to the techniques used in the different investigations. Spore production encompasses the complete period of sporulation, whereas inoculum from the mixed infections was transferred after only 13 days and was not representative of the whole developmental cycle. This may also be the reason for the success of isolate 32E160 in colony growth studies but its apparent failure in spore production tests. When assessed 18 days after inoculation this isolate was discharging the same weight of uredospores as the other test isolates. However, after this time a noticeable increase in the level of chlorosis occurred in isolate 32E160 and ultimately led to a rapid decline in spore production. The period over which sporulation occurs is taken into consideration by spore production tests, but not by techniques used in the mixed infection and colony growth experiments. The white spore mutant of *F. striiformis* used by Brown and Sharp (1970) also tended to fall in frequency when established in mixed infections with yellow spore isolates. It was suggested by Brown and Sharp that the white spore isolate and its yellow spore progenitor were of equal competitive ability. However, it would appear that the reason for non-significance was, as found in the present study, due to large levels of variation within individual replicates. The rarity of white spore types in the field may indicate that such genotypes have little competitive ability in nature and are unable to increase in frequency when grown in the presence of yellow spore isolates (Johnson and Taylor, 1976). This latter theory would appear to be supported by the evidence of mixed infection experiments in the current investigation. The simultaneous presence of yellow spore isolates may have altered the host in such a way that it was detrimental to the proliferation of the white spore isolate. After treatment with
UV light, uredospores of the white spore isolate were of lower viability than those of the wild type progenitor. However, there seemed such an insignificant fall in uredospore viability, it did not seem conceivable that UV light alone could be responsible for the elimination of white spore types in nature.
Mehta and Zadoks (1970) determined quantitative differences between treatments by weighing uredospores of *P. recondita* collected from 50 seedlings. Eyal and Peterson (1967) scraped uredospores from three seedlings and obtained sufficient inoculum to identify differences between races of *P. recondita* produced by differing environmental treatments. A technique developed by Johnson and Bowyer (1974) collected inoculum from individual seedlings. However, the technique using single seedlings produced a coefficient of variation for spore weight of between 20 and 30% in experimental work. This level of variation was also observed in the present study and is illustrated by a coefficient of variation of 28% in experiment 7. Such variation was not due to inaccuracies in weighing but probably arose during spore production and collection. The result of such a high level of variation was a lack of sensitivity and possibly a failure to detect small differences between isolates.
The multiple seedling test required approximately the same amount of time as the single seedling technique but was far more sensitive, with a coefficient of variation of between 7 and 9%. Sixty pots, each consisting of four seedlings, could be harvested for uredospores in approximately one hour, with relatively little loss of inoculum. However, the multiple seedling method was not suitable for use in draughty conditions, since uredospores were lost from the infected leaves.
Results obtained using spore production and colony growth rate techniques were comparable, with the exception of one or two isolates. This agrees with the results obtained by Priestley and Doling (1974). The crucial stage in
the colony growth studies is at inoculation, since this can frequently damage seedlings and increase the level of experimental variation. Such a method is less time consuming than spore production procedures but appears to lack the sensitivity of the latter methods, probably due to the fact that the sporulation potential of each isolate is not fully expressed.
5. CONCLUDING DISCUSSION
SECTION 5
CONCLUDING DISCUSSION
This study has been concerned with genetic variation in the yellow rust fungus, *P. striiformis*, taking into account both virulence characters and also quantitative differences in pathogen-host relationships.
The occurrence of mutation creates a source of genetic variability whereby new characters may be introduced into the fungal population. Such characters are then exposed to selection pressure and their eventual prevalence depends upon the advantages they confer to a race of the pathogen. Further developments in genetic changes in a population will also depend upon the extent of recombination to give rise to individuals with a new spectrum of characters. Although mutation rates in the field may be very low, the vast number of uredospores produced during an epidemic provides ample opportunity for relatively rapid genetic shifts in the population. Mutations have been implicated in virulence changes giving rise to new races (Watson, 1957; Zimmer et al., 1963); they may also influence quantitative traits although such changes are gradual and less likely to be recorded. Such quantitative adjustments, however, may be responsible for adapting a fungal isolate to the environment or to a particular host variety: changes may increase the aggressiveness of particular isolates and lead to a specific compatibility between host and pathogen as recorded by a number of investigators (Johnson and Taylor, 1972; Clifford and Clothier, 1974).
The small size of rust nuclei has made them difficult to study by light microscopy. However, recently developed staining techniques and transmission electron microscopy have enabled more detail to be obtained from cytological studies on nuclear behaviour and thus provide a better understanding of the physical basis of genetical variation. It was concluded that the exchange of chromosomes between dividing dikaryotic nuclei
(Hartley and Williams, 1971) was unlikely to be responsible for new genetic combinations due to the intranuclear method of mitotic division. The production of aneuploid nuclei was recorded in the current work, but only in basidiospores. The significance of this finding is not altogether clear. Aneuploid nuclei may be produced during the vegetative stage of fungi but tend not to survive (Burnett, 1975) and were never observed in the functional stages of *P. striiformis* in this study.
The study did not reveal the presence of any diploid nucle in somatic cells of the fungus. However, a diploid line of *P. graminis* has been isolated in axenic culture (Maclean et al., 1974) and therefore the possibility of a parasexual phase in *P. striiformis* cannot be excluded.
The possible fusion and exchange of hyphal components between isolates would on the evidence of the present work be an important means of increasing the pathogen's virulence characteristics. The fact that new races have never been detected experimentally from the progeny of a single race is suggestive of the significance of hyphal fusion. In a field situation, however, hyphal fusion within the leaf is more attractive than germ tube fusion, since a heavy inoculum source is required for the latter and germ tubes are also more vulnerable to environmental changes.
In several investigations on *P. striiformis* only one or two new races have been produced following the mixed inoculation of two differentially virulent parental races (Little and Manners, 1969(a); Goddard, 1974; Taylor, 1976). The low frequency of new races in laboratory investigations is difficult to understand, especially since a diverse array of new genotypes can be produced under similar experimental conditions in both *P. graminis* and *P. recondita* (Ellingboe, 1961; Sharma and Prasada, 1970). Axenic culturing of *P. striiformis* has met with little success at present, possibly because no race has been isolated that can be cultured using today's crude techniques (Williams, personal communication). Growth of the fungus on a
suitable media could quite conceivably assist in elucidating the mechanism responsible for the production of new races and also of assessing if any restriction exists on the frequency with which new races are produced. The presence of a diploid phase in *P. graminis* was confirmed only after the successful axenic culturing of the fungus.
The relative ease with which new races of the fungus are evolved has been suggested as the reason for the instability of race-specific resistance. Where resistance sources can be strategically controlled, race-specific resistance may possibly be exploited more effectively (Wolfe, 1977). Wolfe has suggested that varietal mixtures (multilines) can utilise race-specific resistance genes more effectively and provide a greater degree of host stability than where varieties are grown in genetically pure stands. The substitution of different component lines into a varietal mixture would also assist in overcoming the high level of variability in the pathogen, and this would be particularly relevant to the wheat:yellow rust system. Genetically controlled resistance may be successfully exploited where such high levels of variability exist in the fungal population. Johnson and Allen (1975) have suggested that avirulent races of yellow rust incite an induced form of resistance to virulent races and that this effect would be of some significance where multiline varieties were being exposed to inoculum of epidemic proportions in the field.
Considerable variation was found within and between races of the pathogen in quantitative characters, based on experiments with pure and mixed cultures of the fungus. An isolate of race 40E8 was found to be the most prolific genotype and it might be concluded that this isolate was the most aggressive. However, the expression of quantitative variation is probably dependent upon specific environmental conditions. It may have been that under different environmental conditions other isolates would have been more aggressive. Furthermore, laboratory results cannot be
extrapolated to the field and it cannot be concluded therefore that isolate 40E8 would have been the most prolific genotype under such conditions. This is exemplified by the white spore isolate of race 104E137 (69/10): under laboratory conditions this isolate ranked highly in spore production experiments. However, such types are rarely found under field conditions and therefore the laboratory results should be treated with some caution.
The expression of quantitative variation was observable microscopically at all stages of pathogen development. The results indicated that the isolate of race 40E8 was not only more prolific because of its high viability and infection frequency but because it produced more uredia and more uredospores per pustule. A more aggressive isolate appeared, therefore, in the study to be more vigorous at all stages of the pathogen's life cycle.
A number of the quantitative experiments produced inconclusive results and need to be repeated. For example, some of the test isolates produced unexpected results when inoculated onto a range of host varieties. There was a distinct possibility of a host:pathogen interaction, indicating some degree of specificity may have been present. The mixed infection studies also indicated the possibility that one fungal isolate on a host variety may alter that variety in such a way as to be detrimental to the presence of a second fungal isolate.
There was no evidence of any inter-relationship between virulence and quantitative traits (aggressiveness) in the test isolates of *P. striiformis* in this study. Previous investigations have established either a positive or negative relationship between these characters (Day, 1968; Brown and Sharp, 1970; Ogle and Brown, 1970; Allen, 1975). Most of these investigations however have only examined two or three isolates of the fungus and this would seem an insufficient number from which to draw any reliable conclusions. The theories suggested by Van der Plank (1963; 1968), with
respect to virulence and aggressiveness, are in all probability an over-simplification of a complex host:pathogen interaction.
Until comparatively recently most genetic studies on host resistance or fungal pathogenicity have been undertaken on seedling plants. This was for a number of reasons but it has now become clear that experimental results obtained while using seedlings are frequently dissimilar from those obtained when using mature plants. Since a large proportion of the damage to cereal crops occurs at the time of grain filling it would seem justifiable for more research work to be undertaken at the flag leaf and not at the first or second leaf stages.
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APPENDIX 1
Fixatives used in cytological studies
Carnoys solution
3 parts (by volume) - 95% ethanol
1 part - glacial acetic acid
BAC fixative
9 parts (by volume) - N-butyl alcohol
6 parts - glacial acetic acid
2 (10%) - aqueous chromic acid
Chrom-acetic-solution
1 part (by volume) (10%) - aqueous chromic acid
10 parts (10%) - aqueous acetic acid
89 parts - distilled water
Bouins fixative
7.5 parts (vol.) - saturated aqueous picric acid
2.5 parts - formalin
0.5 parts - glacial acetic acid
Lactic-alcohol-acetic acid solution
1.0 part (vol.) - lactic acid
6.0 parts - absolute ethanol
1.0 part - glacial acetic acid
FAA
9.0 parts (vol.) - 50% ethanol
0.5 parts - glacial acetic acid
0.5 parts - formalin
Newcomers fixative
6.0 (vol.) - isopropyl alcohol
3.0 - propionic acid
1.0 - petroleum ether
1.0 - acetone
1.0 - dioxane
Modified Navashins fluid
Solution A: 1 g - chromic acid
7 cc - glacial acetic acid
92 cc - distilled water
Solution B: 30 cc - neutral formalin
70 cc - distilled water
Mix equal proportions of A and B just before use.
Shaudinns fluid (half strength)
100 ml - sat. aqueous mercuric chloride
50 ml - absolute ethanol
150 ml - distilled water
Zenkers fluid
2.5 g - potassium bichromate
5.0 g - mercuric chloride
5 cc - glacial acetic acid
100 cc - distilled water
## APPENDIX 2
### Isolates of *Puccinia striiformis* West.
| Isolate | Variety and source of inoculum | Year first isolated |
|-------------|-------------------------------------------------|---------------------|
| 33E32 | ? Germany ? (PBI, Cambridge) | ? |
| 37E132 | Rothwell Perdix | UK (PBI) | 1966 |
| 32E160 | Heine VII? | UK (PBI) | 1957 |
| 40E8 | Nord Desprez | UK (PBI) | 1952 |
| 41E136(a) | Cama | UK (PBI) | 1968 |
| 41E136(b) | Maris Huntsman | UK (ESCA) | 1975 |
| 104E9 | Opal | UK (PBI) | 1966 |
| 108E9 | Maris Ranger | UK (PBI) | 1970 |
| 104E137 | Maris Beacon (69/10) | UK (PBI) | 1969 |
| 104E137 | Joss Cambier (71/2) | UK (PBI) | 1971 |
| 104E137 | Maris Bilbo (72/23) | UK (PBI) | 1972 |
| 104E137 | Colour mutant isolated from 104E137 (69/10) | | ? |
| 104E137 | Joss Cambier (71/26) | UK (NIAB) | 1971 |
| 108E141(2) | Maris Nimrod | UK (PBI) | 1972 |
| 71/368 | 1,3 (6) (7) | UK (NIAB) | ? |
| 69/163 | 2,3,4 (6) | UK (NIAB) | ? |
| 74/62 | 1,2,3 (6) | UK (NIAB) | ? |
| 71/493 | 1,2,3 (6) (?) | UK (NIAB) | ? |
| 74/298 | (2) 3,4,6 | UK (NIAB) | ? |
| 74/129 | 1, (2), 3 | UK (NIAB) | ? |
APPENDIX 3
Experimental wheat varieties
1. Differential varieties of wheat
Parentage
Cappelle Desprez - Vilmorin 23 x Hybride du Jonquois
Carstens V - (Squarehead Anglais x Crielwener 104) x Ble de Russie
Chinese 166 - Origin China
Clement - (Hope x Timstein) x Heine VII x Riebesel 47/51
Compair - Chinese Spring x Aegilops comosa
Gamin - (Crystal x Villa Glori) x Champlein
Heines Kolben - Selection from Ble Saumur de Mars
Heines Peko - Paragis x Heines Kolben
Heine VII - Hybride a courte paille x Svalofs Kronen
Hybrid 46 - Benoist 40 x other hybrids
Joss Cambier - (Heine VII x Tadepi) x Cappelle Desprez
Lee 1 - Hope x Timstein
Maris Beacon - (Hybrid 46 x (Cappelle Desprez x CI 12633)) x Professeur Marchal
Maris Huntsman - (CI 12633 x Cappelle Desprez) x Hybrid 467 x Professeur Marchal
Maris Templar - (Heine 110 x Cappelle Desprez) x CI 12633 x Cappelle Desprez x (Nord Desprez x Viking)
Moro - PI 178383X ?
Nord Desprez - Vilmorin 23 x Hybride du Jonquois
Reichsberg 42 - Thatcher x Heine IV
Riebesel 47/51 - Crielwener 104 x Petkuser rye
Sappo - W177/62 x W176/62
Score - Floress x GU 48411
Selpek - (Selkirk x Peko) x Koga II
| Variety | Parentage |
|--------------------------|---------------------------------------------------------------------------|
| Spaldings Prolific | ? |
| Strubes Dickkopf | Selection from English Squarehead |
| Suwon 92 x Omar | ? |
| Tadorna | (Chinese 165 x Panzer III) x Heines IV x (Teutonen x Hindukush 516) x Heine VII) x Merlin |
| Triticum Spelta Album | ? |
| Vilmorin 23 | (Melbor x Gross Tete) x (Japhet x Parsel) |
2. **Varieties of wheat used in quantitative studies**
| Variety | Parentage |
|-------------|------------------------------------------------|
| Atle | Extra Kolben x Saxo |
| Jufy 1 | Jubilegem x Fylgia |
| Little Joss | Squareheads Master x Ghirka |
| Red Bobs | ? |
| Sappo | W177/62 x W176/62 |
| Svenno | (Marquis x Hatif Inversable) x Extra Kolben x Swedish Land Race |
| Yeoman | Red Fife x Browick |
APPENDIX 4
Colony growth studies on seven isolates of *P. striiformis* (Experiment 1)
| Analysis of variance | DF | SS | MS | VR | P |
|----------------------|----|------|------|------|------|
| Blocks | 2 | 0.4 | 0.2 | NS | NS |
| Race | 6 | 105.3| 17.6 | 7.9 | < 0.01 |
| Residual | 12 | 26.2 | 2.2 | | |
| GRAND TOTAL | 20 | 131.9| | | |
APPENDIX 5
Colony growth studies on seven isolates of *P. striiformis* (Experiment 2)
| Analysis of variance | DF | SS | MS | VR | P |
|----------------------|----|------|------|------|------|
| Blocks | 2 | 3.6 | 1.8 | NS | NS |
| Race | 6 | 119.0| 19.8 | 9.9 | < 0.01 |
| Residual | 12 | 24.4 | 2.0 | | |
| GRAND TOTAL | 20 | 147.0| | | |
APPENDIX 6
Colony growth studies on four isolates of *P. striiformis* (Experiment 3)
| Analysis of variance | DF | SS | MS | VR | P |
|----------------------|----|------|-----|----|-----|
| Blocks | 5 | 5.6 | 1.1 | 0.3| NS |
| Race | 3 | 25.5 | 8.5 | 2.2| NS |
| Residual | 15 | 57.8 | 3.9 | | |
| GRAND TOTAL | 23 | 88.9 | | | |
APPENDIX 7
Spore production studies on ten isolates of *P. striiformis* (Experiment 4)
| Analysis of variance | DF | SS | MS | VR | P |
|----------------------|----|------|-----|----|-----|
| Blocks | 4 | 16.6 | 4.1 | 1.9| NS |
| Race | 9 | 101.1| 11.2| 5.3| < 0.001 |
| Residual | 36 | 77.1 | 2.1 | | |
| GRAND TOTAL | 49 | 194.8| | | |
APPENDIX 8
Spore production studies on seven isolates of *P. striiformis* (Experiment 5)
| Analysis of variance | DF | SS | MS | VR | P |
|----------------------|----|------|------|-----|-------|
| Blocks | 4 | 4.7 | 1.2 | 0.5 | NS |
| Race | 6 | 96.0 | 16.0 | 6.4 | < 0.001 |
| Residual | 23 | 57.5 | 2.5 | | |
| GRAND TOTAL | 33 | 158.2| | | |
APPENDIX 9
Germination frequency of four yellow rust isolates on 1% water agar
| Analysis of variance | DF | SS | MS | VR | P |
|----------------------|----|-------|-------|-----|-------|
| Races | 3 | 5800.9| 1933.6| 105.1| < 0.001 |
| Residual | 8 | 147.3 | 18.4 | | |
| TOTAL | 11 | 5948.2| | | |
APPENDIX 10
Spore production studies on four isolates of *P. striiformis* (Experiment 6)
(multiple seedling method)
| Analysis of variance | DF | SS | MS | VR | P |
|----------------------|----|------|------|------|-------|
| Blocks | 4 | 11.7 | 2.9 | 2.2 | NS |
| Races | 3 | 111.7| 37.2 | 29.9 | < 0.001 |
| Variety | 2 | 82.6 | 41.3 | 31.0 | < 0.001 |
| Interaction (RxV) | 6 | 27.6 | 4.6 | 3.4 | < 0.01 |
| Residual | 44 | 58.6 | 1.3 | | |
| GRAND TOTAL | 59 | 292.4| | | |
APPENDIX 11
Spore production studies on four isolates of *P. striiformis* (Experiment 7)
(single seedling method)
| Analysis of variance | DF | SS | MS | VR | P |
|----------------------|----|------|------|------|-------|
| Blocks | 4 | 187.8| 47.0 | 3.1 | < 0.05 |
| Races | 3 | 97.6 | 32.5 | 2.2 | NS |
| Variety | 2 | 332.3| 166.1| 11.1 | < 0.001 |
| Interaction (RxV) | 6 | 41.3 | 6.9 | 0.5 | NS |
| Residual | 43 | 643.4| 15.0 | | |
| GRAND TOTAL | 58 | 1302.4| | | |
APPENDIX 12
Analysis of difference in mixed infection studies between Generation 1 and Generation 5
| Isolate combination | 4OE8 x MUT | 32E160 x MUT | 108E141 x MUT | SED |
|---------------------|------------|--------------|---------------|-----|
| **Ratio** | | | | |
| 90:10 | 1.6 (0.2) | 0.8 (0.5) | 8.7 (< 0.01) | ± 1.3 |
| 50:50 | 22.1 (0.1) | 3.1 (0.7) | 23.3 (< 0.2) | ± 9.1 |
| 10:90 | 53.6 (< 0.01) | 24.2 (< 0.05) | 14.6 (0.1) | ± 6.1 |
The relative proportions of isolates in mixed infection experiments
**Analysis of variance**
**Ratio 90:10**
| | DF | SS | MS | VR | P |
|--------|----|------|------|-----|------|
| Rows | 2 | 1.1 | 0.6 | 0.2 | NS |
| Columns| 2 | 5.1 | 2.5 | 1.0 | NS |
| Race | 2 | 113.0| 56.5 | 22.6| < 0.05 |
| Residual| 2 | 5.0 | 2.5 | | |
| TOTAL | 4 | 118.0| 29.5 | | |
| GRAND TOTAL | 8 | 124.2 | | | |
**Ratio 50:50**
| | DF | SS | MS | VR | P |
|--------|----|------|------|-----|------|
| Rows | 2 | 40.5 | 20.3 | 0.2 | NS |
| Columns| 2 | 233.4| 116.7| 0.9 | NS |
| Race | 2 | 770.7| 385.4| 3.1 | < 0.20 |
| Residual| 2 | 246.9| 123.4| | |
| TOTAL | 4 | 1017.6| 254.4| | |
| GRAND TOTAL | 8 | 1291.5 | | | |
**Ratio 10:90**
| | DF | SS | MS | VR | P |
|--------|----|------|------|-----|------|
| Rows | 2 | 321.0| 160.4| 2.9 | NS |
| Columns| 2 | 47.3 | 23.7 | 0.4 | NS |
| Race | 2 | 2482.1| 1241.0| 22.1| < 0.05 |
| Residual| 2 | 112.3| 56.2 | | |
| TOTAL | 4 | 2594.4| 648.4| | |
| GRAND TOTAL | 8 | 2962.6 | | | |
A comparison of the white and yellow lines of race 104E137 (69/10)
Analysis of variance
| Ratio 50:50 | DF | SS | MS | VR | P |
|------------|----|------|-------|-----|-----|
| Generations| 1 | 148.8| 148.8 | 1.67| NS |
| Replicates | 4 | 81.4 | 20.3 | | NS |
| Error | 30 | 2677.8| 89.3 | | |
| TOTAL | 35 | 2908.0| | | |
APPENDIX 15
Germination frequency of four yellow rust isolates on 1% water agar
| Analysis of variance |
|----------------------|
| DF | SS | MS | VR | P |
|----|------|-------|------|-------|
| Races | 3 | 4649.8 | 1549.9 | 267.2 | < 0.001 |
| Residual | 8 | 46.7 | 5.8 | |
| TOTAL | 11 | 4696.5 | | |
APPENDIX 16
The colonisation ratio of four yellow rust isolates on susceptible wheat seedlings
| Analysis of variance |
|----------------------|
| DF | SS | MS | VR | P |
|----|------|-------|------|-------|
| Blocks | 5 | 36.0 | 7.2 | 1.4 | NS |
| Races | 3 | 220.0 | 73.4 | 14.4 | < 0.001 |
| Residual | 15 | 76.0 | 5.1 | |
| TOTAL | 23 | 332.0 | | |
APPENDIX 17
The infection ratio of four yellow rust isolates on susceptible wheat seedlings
| Analysis of variance |
|----------------------|
| DF | SS | MS | VR | P |
|----|------|-----|-----|------|
| Blocks | 5 | 7.0 | 1.4 | 0.4 | NS |
| Races | 3 | 230.9 | 76.9 | 19.2 | < 0.001 |
| Residual | 15 | 60.0 | 4.0 | | |
| TOTAL | 23 | 297.9 | | | |
APPENDIX 18
Days to sporulation of test isolates on the variety Sappo
| Analysis of variance |
|----------------------|
| DF | SS | MS | VR | P |
|----|------|-----|-----|------|
| Blocks | 5 | 1.0 | 0.2 | NS | NS |
| Races | 3 | 8.0 | 2.7 | 54.0 | < 0.001 |
| Residual | 15 | 0.8 | 0.05 | | |
| TOTAL | 23 | 9.8 | | | |
APPENDIX 19
The pustule density of four yellow rust isolates on the variety Sappo
| Analysis of variance |
|----------------------|
| DF | SS | MS | VR | P |
|----|------|-------|-----|-------|
| Blocks | 5 | 7867 | 1573.4 | 8.4 | < 0.01 |
| Races | 3 | 2640 | 880.0 | 4.7 | < 0.05 |
| Residual | 15 | 2825 | 188.4 | | |
| TOTAL | 23 | 13332 | | |
APPENDIX 20
Uredospore production per pustule of four yellow rust isolates
| Analysis of variance |
|----------------------|
| DF | SS | MS | VR | P |
|----|------------|-----------|-----|-------|
| Blocks | 5 | 4,395,622 | 879,125 | 3.02 | < 0.05 |
| Races | 3 | 1,542,826 | 514,275 | 1.77 | NS |
| Residual | 15 | 4,360,089 | 290,673 | | |
| TOTAL | 23 | 10,298,537 | | |
APPENDIX 21
Weight of uredospores produced by four yellow rust isolates on the variety Sappo
| Analysis of variance | DF | SS | MS | VR | P |
|----------------------|----|------|-----|-----|------|
| Blocks | 5 | 21.4 | 4.3 | 7.2 | < 0.01 |
| Races | 3 | 16.0 | 5.3 | 8.8 | < 0.01 |
| Residual | 15 | 9.1 | 0.6 | | |
| TOTAL | 23 | 46.5 | | | |
Proceedings of the 4th European and Mediterranean Cereal Rusts Conference, Interlaken, Switzerland, 1976
Variation in *Puccinia striiformis* Westend.
Wright, R.G.
Edinburgh School of Agriculture, Department of Crop Production, Edinburgh, Scotland, UK
Introduction
New physiologic races of *Puccinia striiformis* Westend. are frequently recorded in the United Kingdom following the introduction of resistant wheat cultivars (Macer, 1967; Chamberlain and Doodson, 1971; Chamberlain, Smith and Doodson, 1973). The mechanism responsible for the evolution of these new pathogenic genotypes has been a subject of speculation for more than 40 years.
A sexual phase has not been recorded in *Puccinia striiformis* and a parasexual mechanism, as found in other rust species (Bridgmon, 1959; Ellingboe, 1961; Sharma and Prasada, 1969), has not at present been shown to occur in this fungus. Mutation has been suggested as a source of genetic variation (Gassner and Straib, 1932; Stubbs, 1969) as well as the heterokaryotic exchange of whole nuclei (Little and Manners, 1969(a), 1969(b)).
A possible further source of new pathogenic types may be from a whole chromosome exchange between dividing dikaryotic nuclei in leaf hyphae (Hartley and Williams, 1971). A study, utilising light and electron microscopy, was undertaken to establish the behaviour of the dikaryotic nuclei during mitotic division.
Materials and Methods
Seedlings of the susceptible wheat cultivar Sappo were inoculated with race 104E157 of *Puccinia striiformis*. Six to seven days after inoculation infected areas of tissue were removed from the leaf for microscopical studies. For light microscopy the staining technique of Lu and Raju (1970) was followed. Studies with the electron microscope were based on a method described by Harder (1976). Electron micrographs were prepared using an AEI E.M.6 operating at 100 KV.
Results
Leaf hyphae of *Puccinia striiformis* were found to contain a great number of interdivisional nuclei which measured from 2-3 microns in diameter. Septation within the leaf hyphae was delayed and dikaryotic cells were not observed until 8-10 days after inoculation.
The interdivisional nucleus was shown to consist of a double walled nuclear envelope, an electron dense nucleolus (approximately 1-1.5 microns in diameter) and a spindle pole body (SPB), which was associated with the nuclear envelope. The SPB was situated in a shallow pocket of the nuclear envelope and consisted of an external, electron dense disk-shaped structure with an amorphous granular region (AGR) situated internally in the nucleoplasm.
Mitosis was observed only in the two nuclei situated in the apical region of the leaf hyphae. The first indications that mitosis was about to take place was when these two nuclei moved from a tandem position into an adjacent transverse position across the cell. Subsequently, the SPB divided to form two daughter bodies. Between these two structures a system of microtubules was established to form an intra-nuclear spindle. At this stage the nuclei measured 3.5-4.0 microns.
During early division, the nucleolus was expelled from within the nucleoplasm. A small segment of the nuclear envelope was seen to break down,
then, immediately the nucleolus was released, the nuclear envelope regained its integrity. Division of the two nuclei continued in a synchronised manner. Discrete chromosomal bodies were formed. The intra-nuclear spindle connected the SPB with these chromosomal structures.
When the chromosomes were fully condensed a wide chromosomal band could be seen to span the entire width of the nucleus. Later in division the daughter chromosomes separated and became more widely distributed as they moved towards the polar regions. The integrity of the nuclear envelope was maintained until the chromatin constituted two discrete packages within the nucleus.
At this stage each nucleus developed a central constriction and divided into two. The result was two pairs of daughter nuclei of approximately equal size. Nucleoli were formed early, after separation of these daughter nuclei, by de novo synthesis.
**Discussion**
A reciprocal exchange of whole chromosomes between nuclei during dikaryotic mitosis has been suggested as a possible source of variation in rust fungi (Hartley and Williams, 1971). For such an event to take place the nuclear envelope would have to break down during mitosis. This has been shown to occur in some fungi, e.g. *Basidiobolus ranarum* (Robinow, 1968) and *Polystictus versicolor* (Girbardt, 1968). However, in *Erysiphe graminis hordei* (McKeen, 1972), *Ceratocystis fagacearum* and *Fusarium oxysporum* (Aist, 1969) the nuclear envelope was reported to remain intact throughout mitotic division. Observations made during mitosis in *Puccinia striiformis* have shown that in this fungus the nuclear envelope remains intact, except for a short period when the nucleolus is expelled and late in division when the central region of the nucleus constricts and the two daughter nuclei separate. During the divisional stages when discrete chromosomal bodies were evident, the nuclear membrane was found to retain its integrity. Hartley and Williams (1971) have
implied that a reciprocal exchange of whole chromosomes would take place during 'metaphase' of mitosis. The observations made during mitosis in *Puccinia striiformis* would indicate that reciprocal exchange of chromosomes could not take place at this stage due to the presence of an intact nuclear envelope.
One may conclude that other mechanisms are responsible for increasing the virulence spectrum of rust species including *Puccinia striiformis*. Possible mechanisms include mutation (Gassner and Straib, 1932; Griffiths and Carr, 1961), whole nuclear reassortment (Little and Manners, 1969(a) (b), Goddard, 1973), the parasexual cycle (Bridgmon, 1959, Ellingboe, 1961) and cytoplasmic inheritance (Johnson, Newton and Brown, 1934). Of these only the first two have so far been specifically associated with *Puccinia striiformis*.
**References**
AIST, J.R. (1969). *J. Cell. Biol.* **40**, 20-135.
BRIDGMON, G.H. (1959). *Phytopathology* **49**, 386-388.
CHAMBERLAIN, N.H. and J.K. DOODSON (1971). *Pl. Path.* **20**, 92-95.
CHAMBERLAIN, N.H., J.C. SMITH and J.K. DOODSON (1973). *Pl. Path.* **22**, 27-29.
ELLINGBOE, A.M. (1961). *Phytopathology* **51**, 13-15.
GASSNER, G. and W. STRAIB (1932). *Z. Indukt.- u. Vereblehre.* **63**, 155-180.
GIRBARDT, M. (1968). *Symp. Soc. Exp. Biol.* **12**, 249-260.
GODDARD, M.V. (1973). Ph.D. Thesis. University of Cambridge, Cambridge, England.
GRIFFITHS, D.J. and A.J.H. CARR (1961). *Trans. Br. mycol. Soc.* **44**, 601-607.
HARDER, D.E. (1976). *Can. J. Bot.* In press.
HARTLEY, M.J. and P.G. WILLIAMS (1971). *Can. J. Bot.* **49**, 1085-1087.
JOHNSON, T., M. NEWTON and A.M. BROWN (1934). *Scient. Agric.* **14**, 360-373.
LITTLE, R. and J.G. MANNERS (1969(a)). *Trans. Br. mycol. Soc.* **53**, 251-258.
LITTLE, R. and J.G. MANNERS (1969(b)). *Trans. Br. mycol. Soc.* **53**, 259-267.
LU, B.O. and N.B. RAJU (1970). *Chromosoma* **29**, 305-316.
MACER, R.C.F. (1967). *Trans. Br. mycol. Soc.* **50**, 305-310.
McKEEN, W.E. (1972). *Can. J. Microbiol.* **18**, 1915-1922.
|
TREVOR HAS A HIT AND A BIG WEEK
Toronto: When Date recording artist, Van Trevor signed into the Edison Hotel, he was already batting a thousand. Columbia's promotion man Charlie Cannilieri had laid a carpet of promotion that only required a fast moving car and 26 hours a day.
It would appear that Van Trevor is off and running with his first country hit for Canada. The dynamic young Date recording artist was given a fast and very effective promotion tour of the Toronto area, which netted him a "pick" on Canada's most successful country outlet, CFGM, in Richmond Hill, plus an interview with Country Gentlemen, Ted Daigle, himself a very successful recording artist, and a television set-up with Canada's most popular country host, Bill Bessey, on the CBC-TV network. All this added up to one heck of a big boost for Trevor's week long engagement at the Edison Hotel. When he stepped onto the stage, he wasn't just another country singer, he was Date recording artist Van Trevor, with a large sized hit titled "You've Been So Good To Me", although I prefer the flip, "Sunday Morning", which only makes Mr. Trevor that much bigger, in my opinion.
When handsome Van appeared on stage in his gold lame suit, guitar strung over his shoulder, he had his female audience in the palm of his hands, and even before he uttered a note. Before he completed his first number "Ring Of Fire", it was evident that he was loaded with talent. He stuck to straight down the centre of the road pop country and folk hits which was apparently just right for the Edison audience, who responded well to his offering. Being very versatile, Mr. Trevor changes the pace of his act by doing novelty numbers such as "You Don't Roller Skate In A Buffalo Herd" and peppered the house up with audience participation numbers like "Ye All Come".
It's interesting to note that the U.S. Congress and Senate chose Van to represent country music in the U.S. government usually declares one month of the year as "National Music Month".
Coming up next for Van Trevor is his first Date LP, "Mr. Van Trevor" which is expected to be released within the next few weeks.
The Edison Hotel which has recently changed their format to country acts exclusively could very easily become the country palace of the strip. Playing there nightly is one of Canada's top country bands, The Rhythm Masters. This group of five fine musicians started with the idea of the drummer who is the comic. The band has developed a loyal following which has led them to the release of their first LP on the Arc label, "Jet Age Of Country Music".
By the way, if you missed Van Trevor this trip, he'll be back to the Toronto area, playing The Brampton Inn, from Oct. 31 through to Nov. 5. Don't miss him, I won't, and before I leave, let me fill you in on one small but big bundle of talent, that of Pam Smith, who shares the bill nightly with The Rhythm Masters. She soloes and accompanies herself on guitar and sings up one beautiful string of country favourites. Try to catch her.
By Lori Bruner
NOCTURNALS AT EXPO OCT.8-14.
One of Canada's best known west coast groups, The Nocturnals will be appearing at Expo's Garden of Stars - October 8-14.
The Nocturnals are perhaps the most popular group of musicians to ever come east from Vancouver to Montreal. As a matter of fact it is because of this popularity and professionalism that they were picked to play Expo's famous Garden of Stars. The group has been together for almost five years and has one of the most active and largest fan clubs in the nation. Monthly news bulletins keep their thousands of fans well informed of where they'll be playing next and when they've been, and what the reaction has been. When they released their first record, "Between You And Me", they had a ready and waiting market. Overnight there were the talk of the recording industry and became the biggest group in British Columbia. They have since released "Devilit" and "Lovin' Blues" and again met with province-wide acceptance, and finally became known throughout the rest of the nation.
On the eve of their Expo appearance they have released what has to be their best disc ever to date, "Uptown", which was written by drummer Bill McBeth, and the Hip "Ain't No Big Thing", written by bass man Wayne Evans features Bill on the vocals. Most of their repertoire is original material and leans to the very commercial rhythm and blues, but being the talented musicians they are, play to their audience and groove as well with rock, folk-rock and soul.
The Nocturnals is made up of Ron Henschel - leader and plays lead guitar, Roger Skinner is the expert on sax; Chad Thorp - organ, Bill McBeth - drums and Wayne Evans on the bass. They are all members vocalize but Bill is featured as the lead singer on all their recordings.
Montreal audiences have an extra treat in store for them. Besides restricting themselves to original compositions, The Nocturnals are probably the first Canadian group to use special lighting effects and have worked it down to almost a science.
The Nocturnals are taking advantage of their trip to Expo to tie in with a promotion of their new release, and are calling on as many stations as possible. Initial reaction from bookers has already been encouraging. They have been placed on the VIG list of several of the important Upper Canadian booking agencies. They are expected to return to the Toronto area, the latter part of October.
Their new single "Uptown" and "Ain't No Big Thing" has been released on the Trans World label.
GROUP NEWS
By Carada Bill
By now there probably isn't a high schooler in Upper Canada who doesn't know who the Rock Show of the Yeomen is, but for those of you who haven't seen this great group, toddle down to the Night Owl in Toronto's Village and prepare yourself to be entertained. This is one of the hardest working groups in Toronto, and while the not-so-knit all television producers expose groups who are carbon copies of what's going on south of the border, they're overlooking the real performers. The Yeomen have been using the light, that makes the beautiful colours around them. All this from a projector as well as numerous spots. Each member, except Terry the organist, has his own set of drums, and now they've just added tom-toms. During one number, Laurie and John do a weird bit with two horn speakers which creates a psychedelic sound that's easy to take. No rough grating sounds here.
The gang that turns Winnipeg on is in Toronto. Reo recording artists, The Eternals are accepting one-niters in and around the Toronto area, and they're getting a fistful of offers to. Their latest recording isn't doing them any harm either. It's called "Sunshine Boy" and "Rainbow In My Heart".
The Beatles will have a new album out on the market soon. It's titled "Magical Mystery Tour" and from all the trips they've been taking lately it should be a gas. They'll also be appearing in a new UK TVE by the same name. Over a five year period it's been estimated that the Beatles grossed over 70 million dollars.
Well the Mamas and Papas was a pleasant way to spend a lunch hour, and I'm sure you will too. If you can't make the Cinegate Theatre on Bay St. where CBC radio tapes the R&B Thompson Show daily from 1 to 2 P.M. It's a variety show where interesting personalities are interviewed and where top performers are given the spotlight, thanks to Lucio Agostini, the musical director.
Caught Kathryn Grayson's act, yes the same star of many MGM musicals a decade and a half ago, at the Imperial Room of the Royal York Hotel. Kathryn looks as young as ever and can still belt out some of the classics from popular operas. The crowd apparently didn't like her but the patrons of the Imperial Room enjoyed her immensely and after all that's what counts isn't it?
Peter Mann, a member of Yorkshire's Sugar Shoppe, has leased his Playhouse on Bayview Ave., after being closed since early last year. The grand reopening is Oct. 13 with the off-Broadway musical "You're A Good Man Charlie Brown".
Spent an enjoyable evening at the Cave-A-Bob with Charles Dolan, who is right off the boat from London, England. He is personal manager to Natalie De Sylve, who is currently held over for one week at the Cave. Natalie is a very stunning and talented little girl with a big big voice. She has the potential of another Judy Garland. I must commend the waiters of the club for their good service and very good manners. A few of the other clubs around town would be a little more interested in their patrons than the likes of Cave management.
Shirley Bassey, the gal who made a large sized hit out of "Climb Every Mountain", has just jetted in from London to tape a CBC-TV special. I hope to peek in on it tomorrow. Shirley has been off the record scene lately and a new release would be timely about now.
Ronnie Hawkins opens his second Hawk's Nest on Oct. 13. The new club will be called The Other Nest, and is located at Jane and Finch. Opening group is The New Riders. This is Ronnie's first step toward opening a string of teen-age clubs across the province.
On stage at the Royal Alexandra Theatre this week is the Marigold Charlesworth and Jean Roberts production of The Fantasticks. Touring with the show are John Cunningham as the narrator (El Gallo), Constance Moffitt as the girl, Ty McConnell as the boy (it's a boy girl story with a twist) and Wayne Martens and Donald Babcock as their respective fathers. The big hit from this musical is "Try To Remember", but the complete score has some of the most beautiful and haunting music written in the last decade.
Gabor Szabo is playing the Colonial Tavern for the next two weeks. Szabo is an unusual combination of jazz-rock and oriental-jazz, if there is such a thing. It's a modern twist to jazz and should bring out those sometimes hard to find jazz buffs.
The Guess Who have taped a few shows already and the first is to be seen on Oct. 5, which presents a bit of a problem. That's a Thursday, and that is the day show originates in Toronto, and that makes it a little light at the head. But the Winnipeg show is on a Tuesday. Anyway, The Guess Who will be touring the West Coast of the U.S. in October and working their way back to the East Coast of the U.S. They'll be appearing with the Jefferson Airplane Oct. 10 in Fargo, North Dakota and later in Winnipeg. Their Quality single "This Time Long Ago" has been programmed on many of the popular American stations, the latest being KRLA. You'll also be hearing The Guess Who as backers for Coke.
Remember The Sceptres? "Rien Cu'Une Larme" and if that's not enough to curl your eyebrows try "Mai Je Pense Toujour A Toi"? No, I haven't gone overboard (apologising to the Frenchmen), that's the French picture sung by the Sceptres. Actually, when the English single "I Never Had A Lover Like That" but reading the back of cokes boxes has given me enough French parlance to know I got the wrong side. But did you know that the Sceptres were all university graduates? They've just moved from Montreal to Ottawa because there are more Frenchmen in Ottawa than in Montreal and being as they are fluent both French and English, they should be able in being in the capital get the new generation. No, I'm not kidding about the French influence in our capital. It's about 40%.
Hey, what do you think about that newspaper type sounding off about the strange and not so original names of Canadian groups? Have you even taken a look at some of the names being hung on groups in Canada. How about Squid, Camel Hung Jury, Onion Rings, Fun and Games Commission, Uncle Sam & War Machine, Definitive Rock Chorale, Tombstones, Wreck-A-Mended. There's a couple of howntown groups who seem to be reaching out for originality. One is Lampy's Hippies, and the other is Swine & Cheese featuring the dirty musician.
The Evil Nine are straight. They're well known as Rainy Fields and have concentrated so much on their vocals that it's like watching another group, and they're in suits yet. That must have been a real chore, getting John into a suit. They've been playing the local circuit very well as well as keeping their high attendance record at the Club 42 in Stratford.
There's an old nursery rhyme that starts off "The time has come, the walnuts said, to think of many things", and the kicker is "Of Cabbages And Kings". Now, how do you think they came up with anything so stupid? Chad Stuart and Jeremy Clyde, that's who, and it all on one of the greatest albums ever put out by Canada. It's The Process Suite, Movements 1 through 5, which all adds up to a symphony of sounds that are the most original creations on the market. Sound like a commercial? It is, but it's a great album.
Around Town with Lori
RUSS THOMPSON-KATHRYN GRAYSON-CHARLIE BROWN-SHIRLEY BASSEY
THE NIGHT OWL
THE UGLY DUCKLINGS
THE STACCATOS
THE MANDALA
THE STITCH IN TYME
THE MAGIC CYCLE
ROCK SHOW OF THE YEOMEN
THE DICKENS
THE RAINY FIELDS
THE FIFTH
THE STAMPEDERS
DAVID CLAYTON THOMAS
ERNIE LYONS
JIMMY DYBOLD
THE MAGIC CIRCUS
THE RAGGED EDGES
THE REEFERS
THE WEE BEASTIES
THE PASSING FANCY
GRAEME & THE WAIFERS
THE BIG TOWN BOYS
have all appeared at the...........famous
NIGHT OWL
Coffee House
102 Avenue Road, Toronto
CITERAMA AT EXPO '67
By John Norris
Expo '67 has proved to be a marvelous visual and aural experience for the millions who have been seized into its various exhibits.
One of the most impressive has been CITERAMA - the environmental experience in the Man In The Community building. Citerama, as described in the storyline, is "a spectacle, with an introduction, multiple developments, elements of suspense, moods which are either complimentary to or clash with, one another. It is a spectacular dramatization of Man In The Community: multifarious story unfolding before your eyes."
The synopsis goes on to say "This is the world of streets, buildings, concrete, coloured bridges, right angles, spots of colour, planned movements, operating charts, advertisements, the thermite city, the glass city, of the city where, in short, SPACE, TIME, MOVEMENT AND DYNAMICS make up a complete landscape."
To dramatize and heighten the unfolding story, Citerama commissioned Canadian composer Norman Symonds to write the music. It was in four dimensions, to be heard and seen through the use of loudspeakers and used in a classical string quartet, jazz, electronic and concrete and, finally, city noises.
The startling nature of the challenge was unusual enough. What Symonds created was equally startling. He combined these musics into a striking and dynamic montage of sounds that caught the changing pace and seeming time of a crashing car, the roar and howl of city life, the piercing wail of a stark fluting of the saxophone (Fred Stone) and the wailing and sad cries of Ada Lee's voice and the thrusting saxophone of Don Thompson - all adding up to a fascinating kaleidoscope of sounds.
The original scenario was conceived by Jacques Langrandt and he worked closely with Symonds during the entire shaping of the music.
The music was recorded on four separate tape tracks and then mastered in such a way that each part fitted into the whole structure, as originally conceived by the composer.
Citerama was but one commission for Symonds recently. He's now busily writing a concerto for the Winnipeg Symphony Orchestra that will combine the jazz quartet of flugelhornist Fred Stone with the orchestra. It's to be premiered in December. He recently completed his music for Jacques Langrandt's new play L'Age de Pierre, which opens in Montreal in November.
Symonds has been a prolific composer for many years - and his work reflects his deep interest in both jazz and classical fields. His best known work is his Concerto Grosso for Jazz Quartet and Symphony Orchestra.
Symond's music for Citerama was released, earlier this summer, by KL Records as one side of a seven inch LP that was sold only at Expo. The flip side of the disc is an ad lib jazz improvisation by Stone and Thompson that is used while people are being led into the auditorium.
The disc is now being distributed nationally through Disques Select, 500 St. Catherine St. E., Montreal, Quebec.
This music has had a strange effect on those who have experienced it regularly. Employees at the Man In The Community Building have been put on sailor ship duty (volume six off) and a lock had to be placed on the volume control of the amplifier to ensure that the correct volume of sound was maintained. (The very loudness of the music was seriously disturbing a number of people).
Now, many more people will be able to experience at least one part of the Citerama story with the release of Symonds' music. It vividly conjures up an image of the environment - the true transition of man from an agricultural era to the technological world of today.
(For enquiries as to record availability please write to Disques Select, 500 St. Catherine St. E., Montreal, Quebec.)
WILLIAM (BILL) BANNON
PROMO REP - CAPITOL
Toronto; Gord Edwards, national advertising manager Capitol (Canada) Ltd., announces the appointment of William Bannon as Ontario promotion representative.
Bill has been with the promotion department of Capitol for the past five years and has assisted with in-store displays and stock taking which has given him a wide and varied knowledge of Capitol product.
Music BIZ
By Guest Columnist Stan Klees
No matter how well your group is doing, no matter how much you are making and no matter how far ahead you are booked you can't let up on your image building. You must continue to make known what you have to offer.
Although big companies have a reputation as a buy-word and everyone has heard in their product, like a plane in flight they can't turn off the engines because they've reached the maximum speed. You must promote and you must ADVERTISE.
A group is in business to entertain. They usually are employed to entertain and logistics, their advertising should be directed at the booker and the employer and not to the consumer. Drugs will keep you alive and going, but you can have all the fans in the world and if you aren't getting the bookings, the finance company will step in.
If bookings are a little slow, ADVERTISE. If you release a record, ADVERTISE. If your advertising is effective it won't cost a cent when you start getting the bookings. It's a vicious circle, once it gets going.
In some way, even the neighbourhood dry cleaner advertises. He realises that a $50.00 ad will bring in hundreds of dollars of business. It does.
There are groups in Toronto who have never had a hit record but who get more for a gig than the groups who get their records right up the charts and did it with a lot less effort and expense.
Where do you advertise? Quite often a small ad in the personal column of your local daily paper will bring a flood of enquiries and your services as a group or singer. Many papers have a weekly teen supplement where a small display ad will not only make your name known, but will bring in gigs. Groups today play garden parties, birthday's, weddings, baptisms, etc. etc. etc. As adults begin to frug, the groups become entertainers above and beyond the teen dances. Fans are one thing, but working Monday to Thursday will make money.
I would like to mention one thing (that I feel is very important). When you are placing advertising with any paper, be sure you can afford it and why not pay it in full. If you can't afford it, don't do it. Don't promise to pay later. Don't make them sorry they trusted you and then had to chase you for the money. It is part of good public relations for a group to keep on top of their debts and CASH payments could be the best friend you ever had. I have always told my groups never to owe money to a publisher.
Why should a group be interested in a trade paper? I have always felt that there is a complete misunderstanding (in Canada) of what a trade paper for the music industry is. A very unprofessional resentment of trade papers because they only cover the news they are aware of. Often groups sit and wait for a trade paper to send out a reporter to cover their events. Although this may be the intention of a newspaper, it is not the function of a trade paper. The trade paper depends on press releases and must be made aware of your activities to cover them. It is up to you to advise them (or your press agent). Finally I want to say something that has always made a great deal of sense to me. I once spoke to a top record man in the United States, and asked him if his advertising did him that much good. He told me it not only did a good deal of good, but more important it made the trade paper publish stories that were the most important ones. Don't ask what a trade paper can do for you. Ask what you can do to make sure the trade paper can continue to do something for you and the business you are in.
In the past three years, music has come a long way in Canada. Possibly some soul searching might just give you some clue as to WHY.
THE DATE IS SET FOR RELEASE OF THE STAMPEDERS FIRST RECORD!
OCT. 23
MORNING MAGIC
B/W
ALL THE TIME
On MWC Records 2001
Distributed by
CARAVAN RECORD SALES LTD.
for SASK., MAN., ONT. - 117 Midwest Road Scarborough Ont.
WHOLESALE APPLIANCES LTD.
for B.C., ALTA. - 8401 Fraser St., Vancouver B.C.
EASTERN RECORD DISTRIBUTORS
MARITIMES-144 Glenwood, Lewissville, New Brunswick
QUEBEC DISTRIBUTOR TO BE ANNOUNCED
120 ALBERTUS AVENUE - TORONTO 12, ONTARIO - Telephone (416) 489-3742
RPM MUSIC WEEKLY Page 3
## New Record Releases
Each week RPM will list new records available in Canada.
### Singles
**CAPITOL**
- 20th Fox **Rex Harrison**
- 6899 When I Lose In Your Eyes
- f/s Talk To The Animals
- Capitol **BUCK OWENS & HIS BUCKAROOS**
- 2011 I'm Sorry I've Been Away Too Long
- f/s It Takes People Like You
- Capitol **TINEE**
- 5993 There Are The Eyes Of Love
- f/s Just A Memory
- Capitol **TENNESSEE ERNIE FORD**
- 5926 Hand-Me-Down Things
- f/s The Same Old Song
- Capitol **ROBBIE LANE & DISCIPLES**
- 72503 It's Happening (1967)
- f/s Just A Memory
- Capitol **CLIFF RICHARD**
- 72504 The Day I Met Marie
- f/s Our Star
- Capitol **RUPERT'S PEOPLE**
- 72505 The Ballad Of Charles Brown
- f/s Love Opus 193
- Capitol **THE HOLLIERS**
- 72508 I'll Be On Your Mind
- f/s Running Through The Night
- CARAVAN
- Stone **THE PRECISIONS**
- SX 717 If You Love Me
- f/s You'll Soon Be Gone
- Stone **DICK MALLEY**
- SX 718 I'm In Love Again
- f/s Love Winds Blow
- Caravan **JACKIE SHANE**
- CX-102 Knock On Wood
- f/s You're So Good
- Bunky **THE ESQUIRES**
- 7750 Get Up
- f/s Listen To Me
- Verve/F. THE HOMBRÉS
- KF-5020 Let's All Hang Out
- f/s Go Girl, Go
- Verve F. **THE PAUPERS**
- KF-5020 The Thank You Package
- f/s Magic People
**COLUMBIA**
- Columbia **MYRNA LORRIE**
- MUJA-1265 Tell Me What To Do
- f/s You're Free To Return To Your Past
**COLUMBIA GEORGE JONES**
- MU-267 If My Heart Had Windows
- f/s The Honky Tonk Downstairs
- Columbia **LEFTY PRIZZELL**
- 57-5805 This Is The Change Of Me
- f/s Habo's Pride
- Columbia **JOHN HARRELL**
- 4-44244 S-renata
- f/s La Guerre De La Ciel
- Columbia **THE HARDEN TRIO**
- 4-44249 Forbidden
- f/s (I'm Gonna) Cry Enough For Me)
- Columbia **THE BUCKINGHAMAS**
- 4-44254 Baby (They're Playing Our Song)
- f/s Ain't Our Love
- Columbia **ANDRE KOSTELANETZ & ORK**
- 4-44255 All Of Those Songs
- f/s You Are There
- Columbia **TONY BENNETT**
- 4-44256 I'd Rather Be With You
- f/s Something In Your Smile
- Columbia **TOMMY COLLINS**
- 4-44257 Do You Know
- f/s What-cha Gonna Do Now?
- Columbia **ARETHA FRANKLIN**
- 4-44258 Wisdom Of A Fool
- f/s Follow Your Heart
- Columbia **THE ROBBINS**
- 5-44271 In The Valley Of The Rio Grande
- f/s The Last Time
- Columbia **BILLY JOE ROYAL**
- 4-44259 Hush
- f/s Forever From The Bandstand
- Columbia **THE BROTHERS FOUR**
- 4-44278 Here Today And Gone Tomorrow
- f/s I'm A Fool
- Epic **RAY LYNN**
- S-10200 The Wisdom Of A Fool
- f/s I'm Gonna Be That I Ain't Got
- Epic **LEROY**
- S-10201 I'm Gonna Be That I Ain't Got
- RCA VICTOR
- RCA **TED DAIGLE**
- 57-5841 I'm Gonna Go To Go Around
- f/s Labrador Retriever
- RCA **WILL CARTER**
- 57-5841 One More Time
- f/s My Old Yellow Sticker
- RCA **LUC**
- 57-5776 L'Amour T'Ouvre Grand Les Bras
- f/s Toi
- RCA **LES JEROLAS**
- 57-5777 Alors Que Nous Dormons
- f/s Le Manoir Nouveau
- RCA **LES SPECTRES**
- 57-5778 Rien Qu'Un Souvenir A Toi
- f/s Mon Joli Souvenir
- RCA **GINETTE RAVEL**
- 57-5779 Fleur De Soleil
- f/s Apres
- RCA **JEN ROGER**
- 57-5780 Pres De Ton Coeur
- f/s Qui L'Importe L'Age
- RCA **BARBARA ET DICK**
- 57-5781 Mon Amour, Mon Ami
- f/s Corazon Y Angel
- RCA **LES SHARKS**
- 57-5782 Monga
- f/s Melle-Louise
- f/s I Can't See It 'n' Pryin'
- RCA **JEAN-CLAUDE DECAMP**
- 57-5783 Ne Dis Pas
- f/s Guido
- f/s La Vie Le Ciel
- RCA **STEVE ALLEN**
- 57-5787 Here Comes Sgt. Pepper
- f/s I'm A Fool
- RCA **BARRY McGuIRE**
- 57-5788 Stop And Dig It While You Can
- f/s Master Of War
- RCA **THE STORYBOOK PEOPLE**
- 57-5789 I'm A Fool
- f/s Do You Believe (1394)
- RCA **THE DYNAMICS**
- 57-5776 I Can't Make You Feel Good
- f/s Lights Out
- RCA **SCOTT MUNN**
- 57-5791 I'm An Unborn Child
- f/s 23rd Floor
- RCA **JOHN YARBROUGH**
- 47-5903 Ain't You Glad You're Livin', Joe
- f/s Honey And Me
- RCA **ROBERT FISHER**
- 47-5931 There's A Whole Full Of Girls
- f/s Jesus Christ Jerusalem
- RCA **BOBBY BARE**
- 47-5931 They Covered Up The Old Swimm'n' Hole
- f/s I'm Pilgrim
- RCA **ED AMES**
- 47-5970 I'm In Love
- f/s When The Snow Is On The Roses
- RCA **HANK LOCKLIN**
- 47-5923 I'm A Million
- f/s The Country Hall Of Fame
- RCA **WILLIE NELSON**
- 47-5924 I'll Make A Long Story Short
- f/s San Antonio
- RCA **BILLY RED BROWN**
- 47-5929 I Don't Want To Be Any Better
- f/s Bottle, Bottle
- RCA **JIMMY SMITH**
- 47-5935 Only For Me
- f/s Burning Hole In My Mind
- RCA **CARL BURNETT**
- 47-5930 Enter Laughing
- f/s I'm A Fool
- RCA **THE METROS**
- 47-5933 The Replacer
- f/s I'm A Lover
- RCA **STU PHILLIPS**
- 47-5934 Castle, A Cabin
- f/s Juvente Monte
- RCA **HENRY MANCINI & ORK**
- 47-5940 New York
- f/s Theme For Three
- RCA **ELVIS PRESLEY**
- 47-5941 I'm Not A Man
- f/s You Don't Know Me
### Quality
- Cameo **ELVIE SANDS**
- C-125 Angel Of The Morning
- f/s Dear John
- Ball **JAMES & BORRY PURDY**
- 685 Don't Want To Have To Wait
- f/s Let Love Leave Between Us
- Snop **SAM & DAVE**
- 237 Soul Man
- f/s May I Baby
- RCA **EDWARD TATE**
- 10247 I Learned It All The Hard Way
- f/s Part Of The Game
- MGM **SAM THE SHAM**
- 1383 Boomer In Boston
- f/s Money, Money, Pham
- Revue **GARLAND GREEN**
- R-1001 Girl In My Dreams
- f/s It Rained Forty Days And Nights
- TRX **GENE & DEBBIE**
- 30244 I'm Gonna Be Me
- f/s The Torch I Carry
- Acta **THE OTHER HALF**
- 801 I'm Gonna Be Me
- f/s No Doubt About It
- Atlantic **BOBBY DARIN**
- 24-514 I'm Gonna Be Me
- f/s Talk To The Animals
- Columbia **THE FIREWORDS**
- CR-1010 Expressway To Your Heart
- f/s Hey Guy
- UMM **PATRICK AND PAUL**
- 55030 Big City Blues
- f/s Love Me
- Rep. **THE SOUL SET**
- 899 Mickey's Funky Monkey
- f/s Flunkin' Around
- Mercury **MANFRED MANN**
- 127224 So Long Dad
- f/s I'mminin' For You
- A&M **THE MERRY-GO-ROUND**
- 804 You're The Loveliest Woman
- f/s Where Have You Been All My Life
- A&M **JIMMIE RODGERS**
- 87-1001 Child Of My Way
- f/s Turnaround
- A&M **TOMMY BOYCE & BOBBY HART**
- 87-1002 Someday She's A Little Girl
- f/s Love Everyday
- Smash **THE LEFT BANK**
- 2119 I've Got Something On My Mind
- f/s Desired
- Roger Miller **ROGER MILLER**
- 2121 Ballad Of Water Hole No. 3
- f/s Rainbow Road
- Atco **CLARENCE CARTER**
- 1010 Tell Daddy
- f/s Stayin' Alive Too Long
- Atco **THE FIREBALLS**
- 649 Battle Of Mine
- f/s I Can't See It 'n' Pryin'
- Atco **KING CURTIS**
- 651 Memphis Soul Stew
- f/s Blue Blooded
- Atco **THE KINGPINS**
- 652 Billy Billie Joe
- f/s In The Pocket
- Atco **THE BUFFALO SPRINGFIELD**
- 653 Rock Me, Baby Man
- f/s A Child's Claim To Fame
- The Bee Gees **THE BEE GEES**
- 653 Holiday
- f/s Every Christian Lion Hearted Man
- Will Show
### ALBUMS
**ARC**
- Pat Riccio **A Man And A Woman**
- A-725
**CANADIAN MUSIC SALES**
- Expo-67 Original Sound Track
- LP-1000
- Stereo Only - LAB-5005
- Columbia **GABRIEL SINGERS & ORK**
- Anne Of Green Gables
- Stereo Only - 1368-5
- Columbia **ALLEN GOULD**
- Bach/Three Keyboard Concertos
- ML-1400-1401-1402-1403
**LONDON**
- Liberty Ventures
- LP-1000
- LP-1001-1002-1003
- Smash **JAY & THE TECHNIQUES**
- Peaches, Pumpkin Pie
- MGS 2700-2701-2702-2703
**QUALITY**
- London **MUSIC EXPLOSION**
- Little Bit O'Soul
- LPL 2048
- Quality **HERMAN'S HERMITS**
- V 1880/V 1808
**RCA VICTOR**
- Elvis Presley
- Double T-SP-2822
- LPM-3787/LSP-3787
- Porter Wagoner
- The Cold Hard Facts Of Life
- LPM-3797/LSP-3797
- Kate Smith
- Here & Now
- LPM-3821/LSP-3821
- Homer & Jethro
- Nashville Cat
- LPM-3822/LSP-3822
- Len Barry
- My Kind Of Soul
- LPM-3823/LSP-3823
- Hank Snow
- Christmas With Hank Snow
- LPM-3824/LSP-3824
- Julie Andrews
- A Christmas Carol
- LPM-3825/LSP-3825
- Bobby Bare
- A Bird Never Sang Yesterday
- LPM-3831/LSP-3831
- Norma Jean
- Jackson And The Big Town
- LPM-3832/LSP-3832
- Jelly Roll Morton
- Mr. Jelly Roll
- LPV-546
- Mamás & Papás
- Deliver
- D-30014/D-30014
- Floyd Cramer
- Night Train
- CAL-2152/CAS-2152
- Dotte West
- Sound Of Country Music
- CAL-2153/CAS-2153
- Wild Carter
- Waitin' For Someone Leaves To Fall
- CAL-2168/CAS-2168
- Guido Basso
- The Fabulous Guido Di
- CTL-1088/CTL-1088
- The Carlton Showband
- The Carlton Showband
- PC-1173/PC-1173
- Art Tatum & Arthur Fiedler
- At Tanglawood
- LM-2925/LSP-2925
- Stich-Randall/Forrester Young/Quilico/Priestman
- Handel: Harpsichord
- LM-6181/LSP-6181
---
**Van Trevor**
ON DATE - No. 2-1965
(Distributed by Columbia Records)
---
**Bebé Gee Recommends**
An accurate tabulation of RPM's Music Director Miss Bebe Gee's personal preference.
**PEOPLE ARE STRANGE**
- The Doors/Metale - 4582-C
- Natural Woman
- Aretha Franklin/Antalite - 2441-M
**EXPRESSWAY TO YOUR HEART**
- Soul Searchers - 1022-H
- Please Love Me Forever
- Bobbie Gentry - 1022-H
- Rain Park & Other Things
- Cowsills/MGM - 13810-M
---
**ALL CHOICES ARE MADE BY MISS GEE AT HER TUESDAY AFTERNOON PRODUCTION MEETINGS.**
**THE RPM 100**
Canada's Only Official 100 Single Survey
| Rank | Title | Artist/Label |
|------|------------------------------|-------------------------------|
| 1 | I HAD A DREAM | Reeves/Raiders-Columbia-44227-H |
| 2 | GROOVIN' | Boogie-Woogie-Rca Max-224-M |
| 3 | FUNKY BROADWAY | Wilson Pickport-Atlantic-2430-M|
| 4 | NEVER LOVE | Lenny Williams-7073-P |
| 5 | THE BOAT THAT I ROW | Little River-1878-T |
| 6 | BALLAD OF PAULINE & POONIEL | Jefferson Airplane-Rca Victor-9-297-N |
| 7 | THE LETTER | Bob Seger-665-M |
| 8 | PUT YOUR MIND AT EASE | Everly Brothers-13788-M |
| 9 | HOW CAN I BE SURE | Young Recals-Atlantic-2438-M |
| 10 | LITTLE OL' MAN | The Beatles-7074-P |
| 11 | DANDELION | Reeves/Raiders-London-903-X |
| 12 | PEACHES PEACHES PUMPKIN PIE | Jay/Techiques-Smash-2080-M |
| 13 | HEY BAB | Kinks/Columbia-44224-H |
| 14 | THINGS I SHOULDN'T HAVE SAID | Gene Pitney-7075-P |
| 15 | DIG ROCK & ROLL MUSIC | Peter Paul & Mary-WB-7076-P |
| 16 | SAN FRANCISCAN NIGHTS | Ex-Pressions-Atlantic-2439-M |
| 17 | HOLE IN MY SHOE | Bob Seger-665-M |
| 18 | GIMME LITTLE SIGN | Brenton Wood-Double Shot-116-J |
| 19 | THERE IS A MOUNTAIN | Donny Osmond-7077-P |
| 20 | SUNNY GOODE STREET | The Beatles-1878-T |
| 21 | COME BACK WHEN YOU GROW UP | Bobby Vee-Liberty-5596-K |
| 22 | YOU KNOW WHAT I MEAN | The Beatles-7074-P |
| 23 | BRING IT DOWN FRONT | Just Group-Sparrow-1677-D |
| 24 | ASHLIGHT | Ducklings-Yorkville-45013-D |
| 25 | MEMPHIS SOUL STEREO | King-Columbia-44225-H |
| 26 | YOU CAN'T DO THAT | Bob Seger-665-M |
| 27 | I MAKE A FOCUS OF MYSELF | Franklin Valli-Philips-40-465-K|
| 28 | TWELVE STEPS | Mama & Paws-Dunhill-4099-N |
| 29 | LET LOVE COME BETWEEN US | The Beatles-7074-P |
| 30 | CATCH THE LOVE PARADE | Steeptoes-Columbia-7249-F |
| 31 | WHAT NOW MY LOVE | The Beatles-7074-P |
| 32 | ANYTHING GOES | Wilson Pickport-Atlantic-7075-P|
| 33 | THIS TIME I'LL GO LONG | Guess Who-Quality-1874-M |
| 34 | SOUL MAN | Sam & Dave-Stax-231-M |
| Rank | Title | Artist/Label |
|------|------------------------------|-------------------------------|
| 35 | YOU KEEP RUNNING AWAY | Four Tops-Motown-1113-L |
| 36 | BUNKY | Exquise-Bunky-7750-G |
| 37 | THE CAT IN THE WINDOW | Petula Clark-WB-7073-P |
| 38 | I'M A STRANGER AT HOME | The Doors-Elektra-5021-C |
| 39 | BANDA | Arista Records-AM-870-M |
| 40 | TAKE A LOOK | Arista Records-Columbia-44270-H|
| 41 | GET TOGETHER | Youngbloods-Rca Victor-9246-N |
| 42 | FUNSHINE GAME | New York Express-Laurie-3400-M |
| 43 | OUR PRECIOUS LOVE | Gary/Terrance-Tom-3415-L |
| 44 | HURRY UP WILLY JOE | Kings-Atco-6516-M |
| 45 | LIGHTNING'S GIRL | Nashville-Record-6020-P |
| 46 | THE LAST WALTZ | Engelbert Humperdinck-Purch-8079-K|
| 47 | TURN THE WORLD AROUND | Arlo And Dic-River-47-5265-N |
| 48 | EVEN THE BAD TIMES ARE GOOD | The Beatles-1878-T |
| 49 | EYES OF EYED GIRL | Wilson-Promotion-Bang-345-C |
| 50 | IT MUST BE SUMMER | Vikki Carr-Liberty-55986-K |
| 51 | FISHERMAN | The Beatles-7074-P |
| 52 | HOLYDAY | Bert Kaempfert-Arto-6521-M |
| 53 | WHAT'S IT'S WORTH | Staple Singers-Epic-10220-H |
| 54 | THE LOOK OF LOVE | Dickie Valentine-Philips-40-465-K|
| 55 | LET IT OUT | Wilson-Promotion-5058-G |
| 56 | NEW DAWN | Stritch In-Tyme-Yorkville-45011-D|
| 57 | YOU'VE MADE ME SO VERY HAPPY | The Beatles-1878-T |
| 58 | I'M STRONG | The Beatles-1878-T |
| 59 | I'M STRONG | The Beatles-1878-T |
| 60 | NATURAL WOMAN | Aretha Franklin-Atlantic-2441-M|
| 61 | BELIEVE IN LOVE | Petula Clark-WB-7076-M |
| 62 | IF THIS IS LOVE | The Beatles-1878-T |
| 63 | EXPRESS YOUR TO THE HEART | Soul Survivors-Crimson-1010-M |
| 64 | MY GIRL | My Girl-Address/Atlantic-546-G |
| 65 | CORNFLEAK & ICE CREAM | The Beatles-7074-P |
| 66 | CHILD OF CHAOS | Jimmie Rodgers-Ad-A&M-871-M |
| 67 | PLEASE LOVE ME FOREVER | Bobby Vinton-Epic-12229-H |
Compiled from Record Companies, Record stores and Disc Jockey reports.
---
**SEMCCHUK SISTERS RELEASE ON GALAXY**
Dauphin, Man.: The Semchuk Sisters, winners of the "Best Ukrainian Vocal Awards" at the CKDM Centennial Parade of Stars, have just released a long playing record on the Galaxy label. The sisters, who are from Gilbert Plains, Manitoba, were picked by the judges to appear in the final concert of the Centennial series and were awarded the trophy for the best performance of that evening. Prior to their picking up the honours they were signed to a recording contract by Galaxy, which was witnessed by CKDM station manager, Mayor Hugh Dunlop of Daughin. A photo of this signing has been reproduced as part of the back of the album cover.
The Semchuk Sisters have been singing together for a number of years and range in age from nine to sixteen. Their album features Liana Semchuk on guitar, with a large violin assist supplied by Bill Prokopchik, of Saltcoats, Saskatchewan.
Mr. Alex Moodrey, manager of Galaxy Records, advises that the album is now available at most record bars throughout the prairie provinces or can be ordered direct.
---
**CANADIAN GOLD FOR LULU**
Toronto: Lulu's Epic outing of "To Sir With Love" is fast approaching the 100,000 sales mark (Columbia's equivalent of a U.S. gold disc) which should put her in line for a gold record. The release with "The Boat That I Row" as the top side caught chart action back on July 1st when it was "picked" by RPM and rose rapidly on the charts. Its stay popular and also the reason it being flipped was the smashing nationwide success of the Columbia flick, "To Sir With Love", which starred Lulu, and has made the soundtrack one of the big sellers for the Fontana people (London Distrib.)
"To Sir With Love", or as RPM prefers, "The Boat That I Row" is beginning to descend the charts after a record 18 weeks of action. (No. 5, RPM 100).
---
**MASSI PRODUCES SESSION**
NYC: Nick Massi, former member of the Four Seasons, has just produced a record session for The Victorians. The disc, "Wasn't The Summer Short", has been released on the Bang label. Initial reports show the material which is considered very different than any other offered from the company, as having good chart potential.
CANADIAN NEWS FROM THE U.S.
Canada's Sparrow sings no more. The group has broken up because of the disagreements which have grown over the music they were to record. Dennis Edmonton, lead guitarist and writer, is on his own and a single is expected from him in the near future on the Epic label. Mike Young, who left The Buffalo Springfield two months ago, is now back with them. He and Steven Stills produced the Springfield's new single "Rock 'N' Roll Woman"/"Zalman (Zal) Yanovsky is a "Big Boy Now". His first single, "As Long As You Have A Buddah, (a division of Kama Sutra) should establish him as a solo hit-maker.
The Carnival Connection is heading for New York City's Electric Circus. They will appear for two weeks at the nightclub.
I understand that The Richmond Organization is interested in the Carnival's material.
David Clayton Thomas must like to see his name in print. He started out as David Clayton Thomas and The Shays, which later became The David Clayton Thomas Quintet. He then called his act The David Clayton Thomas Show. Now he's with a new group known as The David Clayton Thomas Combine, who's first release for MGM, "Mind Painting Time Machine" will be out shortly.
I hear that some Canadian discs are being played on Buffalo's WKWB. Presently "Gaslight" by The Ugly Ducklings is being given extensive play by the station. I'm looking forward to "Confidence And Ice Cream" by The Lords of London which will be released by Decca within the next week. Wayne Cochran, the nice quiet fellow who tore up Toronto's Embassy Club awhile back, has his first Capitol recording on the material entitled "I Want Your Sweet Love". It's not easy to put his live appearances on a record.
Ian and Sylvia are heading for Western Canada this month, playing concerts in Edmonton and Calgary. They'll start their U.S. tour Oct. 20.
The Guess Who and 49th Parallel are slated for Coca Cola commercials. I remember a year back that David Clayton Thomas wasn't known for his hit songs in Vancouver but was for his hair dressing commercials.
I haven't been to Canada for a year and I am looking forward to my trip, taking me to Toronto, Montreal and Ste. Agathe. It'll be interesting to see how things have changed.
ELVIRA CAPRESE
IT'S TIME I SPOT MY PIECE! It's time for me to give my opinion and it won't be open end either. I am going to follow the guide of radio commentators across Canada and give MY opinion, mind thinking, WASH brains and all the other goodies that the BBG have granted to programmers who take advantage of the airwaves to swing elections, and influence public opinion BEYOND belief. As I listen to the syrupy voice of the worldly commentator and his sadistic attack on the calling listener, I feel a moral satisfaction that I am sure other listeners must be experiencing.
I always feel relieved to know that I can turn this Svengali down in the same way that many of these "gods" turn down the listener on their AIR MOUNTAIN comments in Toronto. There is becoming the opinion of the Canadian public, and the licensees of Canada's airwaves must be very happy about the supreme power that has been granted them, and all in the name of free opinion. Yes, there the people of Canada and particularly young Canada is being molded into exactly what the radio stations that possess this power wants its listeners to be.
One typical example of the "general topics" that might be discussed by one of these geniuses is the radio stations policy that the listener might not agree with. While the station might lambast anyone and everyone, there is also a few "good cues" and one of them might just be the political and legal angles that the radio station itself gets caught up in. These you will not hear discussed. You won't hear anything the screening doesn't permit and that is where this form of radio can fall down.
At the end of every call the commentator makes his point and often favourable towards the party the station might lean toward. There is nothing like equal time in this kind of discussion.
The station can't just put any dunce in this worshipped spot. It is easy for a radio station to hire a dummy and have him on one day. A few minorities a month will eventually find the station's ratings sagging and dying. School kids are an example. Teenagers, shoppers, and the many religions that should you question the old faithful, might possibly might find a whole block of the listening public away to a station where the great voice entertains but doesn't question.
Once the reputation of one of these "wise men" is built up, the country's politics, the provincial government or a municipal election might easily be influenced.
Next time you listen to your friendly "open end" "public opinion" show make the decision. The next time you hear it just come out, "Hello, this is BRAINWASHING UNLIMITED, I'm about to share my thinking with you, dear people".
STAPLE SINGERS & McKINNON COME RIVERBOAT DATES
Toronto: Enjoy fast chart rising Staple Singers have been scheduled for a five day engagement (Oct 10-15) at the Riverboat. Their current single, "For What It's Worth" is happening on many of the charts across the nation and is listed at No. 53 on the RPM 100 this week.
The pretty and very talented Catherine McKinnon is set to appear at the Riverboat Oct 17 for one week. Catherine, who has had much success with her Art Album and single, has been off the recording scene for some time and been following an equally successful stage career, namely with Spring Thaw. After her stay at the Riverboat, Catherine will rejoin the government-sponsored Canadian Tour that will take her and the Travelers to many centres across the nation.
Following Catherine into the Riverboat is Penny Lang, well known folk singer from Montreal.
WEINER-VARIETY TIE-IN OPERATIONS
Winnipeg: Frank Weiner, of the Hungry "I" Agency will be working closely with Variety Theater International, of Duluth, Minnesota. Weiner has become one of the top agencies in the west over the past year and is well respected on both sides of the border. His handling of the Gettysburg Address has made this Franklin recording unit one of the most sought after agencies in Western Canada as well as south of the border. His agency also handles, The Mongrels, Shage, Shondels, Footprints, Finders Keepers, The Guess Who and many others.
His association with Variety will now make available to Canada, through him, The New Christy Minstrels, Jack Jones, Johnny Mathis, Jimmie Rodgers, Peter, Paul & Mary, Nancy Sinatra, and many other top U.S. entertainers.
ELVIRA CAPRESE
IT'S TIME I SPOT MY PIECE! It's time for me to give my opinion and it won't be open end either. I am going to follow the guide of radio commentators across Canada and give MY opinion, mind thinking, WASH brains and all the other goodies that the BBG have granted to programmers who take advantage of the airwaves to swing elections, and influence public opinion BEYOND belief. As I listen to the syrupy voice of the worldly commentator and his sadistic attack on the calling listener, I feel a moral satisfaction that I am sure other listeners must be experiencing.
I always feel relieved to know that I can turn this Svengali down in the same way that many of these "gods" turn down the listener on their AIR MOUNTAIN comments in Toronto. There is becoming the opinion of the Canadian public, and the licensees of Canada's airwaves must be very happy about the supreme power that has been granted them, and all in the name of free opinion. Yes, there the people of Canada and particularly young Canada is being molded into exactly what the radio stations that possess this power wants its listeners to be.
One typical example of the "general topics" that might be discussed by one of these geniuses is the radio stations policy that the listener might not agree with. While the station might lambast anyone and everyone, there is also a few "good cues" and one of them might just be the political and legal angles that the radio station itself gets caught up in. These you will not hear discussed. You won't hear anything the screening doesn't permit and that is where this form of radio can fall down.
At the end of every call the commentator makes his point and often favourable towards the party the station might lean toward. There is nothing like equal time in this kind of discussion.
The station can't just put any dunce in this worshipped spot. It is easy for a radio station to hire a dummy and have him on one day. A few minorities a month will eventually find the station's ratings sagging and dying. School kids are an example. Teenagers, shoppers, and the many religions that should you question the old faithful, might possibly might find a whole block of the listening public away to a station where the great voice entertains but doesn't question.
Once the reputation of one of these "wise men" is built up, the country's politics, the provincial government or a municipal election might easily be influenced.
Next time you listen to your friendly "open end" "public opinion" show make the decision. The next time you hear it just come out, "Hello, this is BRAINWASHING UNLIMITED, I'm about to share my thinking with you, dear people".
NORTHWEST COMPANY DRAWS RECORD CROWDS IN DRYDEN
Dryden, Ont.: One of the first Canadian west coast groups to play east of the Rockies, The Northwest Company, drew one of the largest crowds ever witnessed in the Dryden/Kenora area. Writes Peter L. Harding, program director-CKDR, "The group has great possibilities on the Canadian scene and with dedication for this much needed recognition, ie: their extensive travelling, they deserve it."
The group went stateside after their Dryden success and are expected to move into Upper Canada within the next few months.
The Northwest Company have had major hits on the recording scene as well. Their ditties "I Had To Cry" and "Get Away From It All" reached No. 3 on the published Canadian west coast chart.
ASSOCIATION TO APPEAR ON SMOTHERS BROTHERS SHOW
NYC: The Association, the first pop group to appear at Hollywood's famous Cocoanut Grove, have been signed to appear on the Smothers Brothers TVer, Oct. 8. The taping was apparently already taken place, although the group will also be appearing at Philadelphia's Town Hall that same date.
The month of October will see the Association on one of the most extensive radio tour-nites in their career and includes a college circuit of almost every state in the nation.
Their Warner Bros. single, "Never My Love" is currently riding the top side of most charts.
B&O MARCHING BAND HEADS UP CHARTS
NYC: One of the hottest releases in the Jubilee roster has turned out to be "Lapland" by The Baltimore & Ohio Marching Band. Many stations across the U.S. nation have given the disc "picks" and chart listings.
The material is somewhat similar to that of "Winchester Cathedral", and a natural for halftime entertainment by marching squads during the football season.
RPM COUNTRY CHART
1 HUMAN NATURE
Orval Prophet - Caledon
2 THE SOUND THAT MAKES ME BLUE
Diane Leigh - Capitol
3 TED AND THE GOONE
Lucille Starr - Epic
4 TAKE THE BAD WITH THE GOOD
Linda Davis - Capitol
5 7 MODEL "T"
Irvin Presley - Melbourne
6 LEAVE A MEMORY
Johnny Ellis - Columbia
7 BATTLE OF THE LITTLE BIG HORN
Tommy Cash - RCA
8 I LOVE'S GONNA COME BACK
Glen Buck - Capitol
9 ALL ALONG WITH THE BLUES
Billy Stoltz - Melbourne
10 -- THE HOMECOMING
Ralph Corson - Melbourne
WHERE IT'S AT... R&B chart
2 HIGHER & HIGHER
Maurice Williams & The Zodiacs
3 FUNKY BROADWAY
Wilson Pickett-Atlantic
4 MEAN OLD SOUL STEW
Joe Curtis-Atlantic
5 BABY I LOVE
Maurice Williams-Atlantic
6 APPLES PEACHES PUMPKIN PIE
Jay /Techniques-smash
7 IN THE HEART OF THE NIGHT
Roy Ayers-Sparrow
8 SOUL MAN
Davey Starz
9 GET YOUR SPURS
Esquires-Bunky
10 TAKE A LOOK
Maurice Williams-Atlantic
11 COLD SWEAT
James Brown-Delta
12 DON'T KEEP RUNNING AWAY
4 Tops-Tamla Motown
13 KNO' ON WOOD
Billie Joe & The Seven
14 IT'S GOT TO BE MELLOW
The Four Seasons
15 YOU GOT TO PAY THE PRICE
Al Kent-Ric Tic
16 LOVE BUG, LEAVE MY HEART ALONE
Martha & The Vandellas-Tamla Motown
17 LET LOVE CATCH US
James & Bobby Purify-Bill
18 YOUR PRECIOUS LOVE
Martha & The Vandellas-Motown
19 A NATURAL WOMAN
Aretha Franklin-Atlantic
20 REFLECTIONS
Supremes-Tamla Motown
21 CASANOVA
Rudy Ray Moore-Trans World
22 LITTLE OL' MAN
Billie Joe & The Seven Bros
23 I CAN'T STAY AWAY FROM YOU
Impressions-Sparrow
24 BRING IT DOWN FRONT
Joe Curtis-Atlantic
25 FALL IN LOVE
Ben E. King & The Falcons-wick
26 IF THIS IS LOVE
Precisions-Stone
27 ODE TO BILLY JOE
Kirk Douglas
28 FORGET IT
Shirley Bassey-Times World
29 SOME KIND O' WONDERFUL
Soul Brothers-Six-Atlantic
30 BABY I'M LONE'L
Intercord-Mike
31 KARATE BOOG-A-LOO
Jerry O-Shout
LISTEN TO JOHN DONABIE 1 AM to 6 AM
"WHERE IT'S AT..." CKFH 1430 TORONTO
PRODUCED BY SANFORD PRODUCTIONS - 925-0826
GOT A HOT MASTER you want to place in the USA? Contact Timmie Wax for a complete service to help ARTISTS in the USA.
RPM Box 1000 or telephone Toronto 487-5812.
WANTED an original boxed set of Judy At Campbell Hall. Must be mint condition. Must be in mint shape. Write RPM Box 1560.
LEAD SHEETS of your newly written songs. Fair compensation. Send tape or disc to RPM Box 1561.
YOUNG SINGER looking for group of good musicians to work with. For interested parties call John Downey at Trainco Talent Toronto 725-1111.
GO GO GIRLS wanted to work in Winnipeg and area. Call Hungry Horse Agency 947-0079 or write to: 361 Fort St., Suite #12, Winnipeg.
TOP GROUP looking for lead guitarist-vocalist, Toronto area. Must be young with good stage presence and long hair. Write to RPM Box 1563. Must be available.
YOUNG MUSICIAN would like to write to other group members about CMA. Please send me a copy of your resume. All letters. Send picture. Write Ron at RPM Box 1560.
GUITARIST looking for a solid built-in amp. double manual, bass pedals. Walnut finish. Write to RPM Box 1562.
RPM MUSIC WEEKLY
CLASSIFIED DEADLINE
TUESDAYS AT NOON
CLASSIFIED RATES
SEMI-DISPLAY
70c per line - minimum 3 lines
WE WANT YOU!
IF YOU ARE A TALENTED AND NON-UNION GROUP
ECHO RECORDING SERVICES
BOX 216
SCARBOROUGH, ONTARIO
Telephone: 226-2947
CANADIAN CONTENT?
FOR THE RECORD
MELBOURNE RELEASES
NEW BANFF ALBUM RELEASES
Rodeo's Golden Stereo Series
RALPH CARLSON SINGS
SBS - 5286
THE CELTS SING
SBS - 5287
CURLY KENNY SINGS
SBS - 5288
DISTRIBUTION - LONDON RECORDS
CBC O'KEEFE CENTRE PRESENTS
8p.m. Oct. 16
The Rock Scene - Like It Is!
jefferson airplane • the doors
dionne warwick • noel harrison
sergio mendes • eric anderson
|
Multi-Dimensional Regression Host Utilization algorithm (MDRHU) for Host Overload Detection in Cloud Computing
Ali A. El-Moursy\textsuperscript{1}*, Amany Abdelsamea\textsuperscript{2}, Rukshanda Kamran\textsuperscript{2} and Mohamed Saad\textsuperscript{1}
Abstract
The use of cloud computing data centers is growing rapidly to meet the tremendous increase in demand for high-performance computing (HPC), storage and networking resources for business and scientific applications. Virtual machine (VM) consolidation involves the live migration of VMs to run on fewer physical servers, and thus allowing more servers to be switched off or run on low-power mode, as to improve the energy consumption efficiency, operating cost and CO$_2$ emission. A crucial step in VM consolidation is host overload detection, which attempts to predict whether or not a physical server will be oversubscribed with VMs. In contrast to the majority of previous work which use CPU utilization as the sole indicator for host overload, a recent study has proposed a multiple regression host overload detection algorithm, which takes multiple factors into consideration: CPU, memory and network BW utilization. This paper provides further improvement along two directions. First, we provide Multi-Dimensional Regression Host Utilization (MDRHU) algorithms that combine CPU, memory and network BW utilization via Euclidean Distance (MDRHU-ED) and absolute summation (MDRHU-AS), respectively. This leads to improved results in terms of energy consumption and service level agreement violation. Second, the study explicitly takes real-world HPC workloads into consideration. Our extensive simulation study further illustrates the superiority of our proposed algorithms over existing methods. In particular, as compared to the most recently proposed multiple regression algorithm that is based on Geometric Relation (GR), our proposed algorithms provide an improvement of at least 12% in energy consumption, and an improvement of at least 80% in a metric that combines energy consumption, service-level-violation, and number of VM migrations.
Keywords: Cloud Computing, Power management, Data center management, Virtual machine consolidation, Host overload detection, Multiple regression
Introduction
Cloud computing [1] technology is acquiring a great deal of prominence across the computing and networking research communities. Nowadays, cloud data centers are the basic framework for the computing and data storage communities that offer expanded services to the end users. These data centers consume large amounts of electrical energy to process the cloud services bringing a large quantity of CO$_2$ emissions, high operational cost, and influencing the reliability of hardware equipments [2]. The fundamental drain of power consumption in data centers are processing, network, cooling systems, and disk storage. Since conventional ways [3–5] to decrease the power consumption are no longer suitable for modern data centers, new adaptive software-oriented techniques are inevitable. To reduce energy consumption, it is important to address inefficiencies and waste in the manner electricity is conveyed to computing servers, and in the manner these resources are utilized to satisfy the running workloads. This may be possible by enhancing the data centers physical infrastructure, in addition to resource management and allocation algorithms.
One of the important methods to address the energy inefficiency in cloud data centers is to leverage the capabilities of the virtualization technology. Virtualization is...
a key player in cloud computing [6]. In particular, virtualization permits logical resource abstraction in isolation from their corresponding physical resources; the physical resources are transformed into virtual on-demand resources of cloud data centers. Virtualization is provided by the utilization of a hypervisor to logically allocate virtual machines (VMs) on physical server resources. The hypervisor permits the VM guest operating system to operate as if it were solely in control of the hardware, unaware that other guests are sharing it. Virtualization gives a chance to consolidate various VM instances running on under-utilized server machines into fewer nodes, allowing more servers to be switched-off, which leads to considerable energy savings. Virtual machine consolidation [7] involves scheduling/migrating several virtual machines into a few number of physical servers. Virtual machine consolidation has essentially an important tradeoff, namely reduction of energy consumption without scarifying the Quality of Service (QoS) delivered by the system (energy-performance trade off). The improvement of the system QoS may be achieved indirectly by tuning parameters of the assigned host. The tuning is based on the utilization percentage achieved by the physical resources in a computing node. According to this percentage we can decide if a node is underloaded or overloaded. According to the workload information, a VM can be migrated from an overloaded host to an underloaded host. Server overload results in applications performance degradation and resource shortages, as well as an extensive increase in power consumption. Monitoring the overloaded hosts will enhance the host utilization and prevent thrashing of the VMs.
The capability to manage physical machine (PM) overload is a critical element of next-generation, competitive cloud services. If overload is not handled properly, cloud service providers will take the risk of violating their Service Level Agreements (SLAs) [8]. SLAs provide a level of assurance to customers that their requested resources are available when they request them [9]. An overloaded host directly affects the QoS because, if the resource capacity is fully utilized for a significant time window, it is expected that the applications will encounter performance degradation and resource shortage.
Most of the recent studies migrate VMs based on the CPU utilization of the physical server. This may be suitable for CPU-intensive applications, but will not be accurate for network, I/O and memory intensive applications [10]. Motivated by the fact that high-performance-computing (HPC) applications, and server applications usually utilizing cloud data centers, are sensitive to multiple factors (CPU, Memory and Network BW utilization), we propose a family of novel multi-dimensional regression host overload detection algorithms that explicitly take these orthogonal factors into consideration. In particular, the most crucial aspect of multi-dimensional regression is how to combine the orthogonal factors (CPU, Memory and Network BW utilization) into a composite metric that accurately captures whether or not the host is overloaded. Making such a decision accurately is the ultimate goal of the regression algorithm. To this end, we introduce two alternative mathematical formulas for VM utilization, namely Euclidean Distance (ED) and Absolute Summation (AS), that are more general than the existing approach in [11].
More specifically, the contribution of this paper can be summarized as:
1. This paper introduces a Multi-Dimensional Regression Host Utilization algorithm (MDRHU) for host overload detection. In particular, we introduce two alternative mathematical composite metrics to measure the host utilization based on the VM utilization profiling of three independent factors: CPU, memory and network BW. The proposal is tuned and enhanced to meet the specs of the Cloud. The numerical results reveal the superiority of the proposed approach as compared to the existing state-of-the-art.
2. This study explicitly takes real-world HPC workloads into consideration. This ensures the trustworthiness of our numerical study in revealing the suitability of our proposed approach for HPC applications.
This paper is organized as follows: “Related work” section presents the related work, and summarizes the contribution of this paper. “Multi-dimensional regression host utilization for host overload detection algorithms (MDRHU)” section introduces the proposed regression-based, multi-dimensional host overload detection algorithm. “Evaluation methodology” section presents the evaluation methodology. “Simulation results and analysis” section presents the simulation results. Finally, “Conclusion” section concludes the paper.
**Related work**
Previous techniques to energy efficient host overload detection can be broadly divided into three categories: (1) static threshold based heuristics, (2) adaptive utilization based heuristics and (3) regression based heuristics. All categories enjoyed significant attention from the research community, so we focus here on the most relevant and significant work.
Threshold-based techniques rely on setting a static CPU utilization threshold distinguishing the non-overload and overload states of the node. These techniques compare the current CPU utilization of the host against the predefined threshold. If the threshold is exceeded, a host overload is declared. See, e.g., [12] and [13]. Fixed
| Related work | Parameters | Target | Algorithm name | Advantages | Disadvantages |
|--------------|------------|--------|----------------|------------|---------------|
| Tian et al. [16] | CPU, memory and network BW | Resource scheduling | Dynamic and Integrated Resource Scheduling (DIPS) | - It provides a dynamic and integrated resource scheduling which treats CPU, memory and network BW/integrated for both physical machines and virtual machines.
- It provides integrated measurement of the total imbalance level of a Cloud data center, as well as the average imbalance level of each server for performance evaluation. | It did not consider energy-efficiency for Cloud data centers. |
| Tang et al. [17] | CPU, Communication network in a data center | Virtual Machine Placement | Hybrid GA (HGA) | - It considers the energy consumption in both physical machines and the communication network in a data center.
- Compared with existing heuristic algorithms, the HGA can generate much better solutions as the HGA is basically a global search algorithm while heuristic algorithms are local search algorithms, which may be trapped at a local optimum during their search process. | Its computation time is higher than other existing heuristic algorithms. |
| Castro et al. [18] | CPU and RAM | Virtual machine Placement | CPU and RAM Energy Aware (CREW) | - It improves VM placement by employing a power model that considers the energy consumed by both CPU and RAM.
- CREW offers the best trade-off between energy-saving and SLA violation. | It considers CPU and RAM only and it did not consider the communication network in a data center. |
| Li et al. [19] | CPU and disk | Host overload/ underload detection and VM placement | Multi-resource double threshold method and Modified Particle Swarm Optimization (MPSO) VM placement | - It designed a method of double threshold with multi-resource utilization to trigger the migration of VMs.
- The Modified Particle Swarm Optimization method is introduced into the consolidation of VMs to avoid falling into local optima which is a common defect in traditional heuristic algorithms. | It did not consider the memory and communication network in a data center. |
| Farahnakian et al. [20] | CPU and memory utilizations | A virtual machine consolidation | A Utilization Prediction-aware VM Consolidation (UPvMC) approach | - UPvMC goes beyond the existing works which only consider CPU utilization by also considering memory. Combining both memory and CPU utilization, UPvMC can better identify cases of SLA violations and consequently prevent them from happening. | It should take into account network resource utilization and traffic to optimize VM placement. |
| Abdelsamea et al. [11] | CPU and RAM and network BW | Host overload detection | Multiple Regression Host Overload Detection (MRHOD) | - It significantly reduces energy consumption while ensuring a high level of adherence to Service Level Agreements (SLA). | It uses a workload that is insignificant for BW. |
utilization thresholds are not suitable for environments with dynamic and unpredictable workloads, where various types of applications may share a physical resource. The system should be capable of adjusting automatically its behavior based on the workload patterns. Adaptive utilization based algorithms provide auto-adjustment of the utilization thresholds depending on a statistical analysis of historical data calculated within the lifetime of the VMs. See, e.g., [7, 14, 15]. Adaptive utilization based algorithms are efficient for dynamic environments, but provide poor prediction of host overloading. Hence, host overload detection may also benefit from the estimation of future CPU utilization. Regression-based techniques provide better prediction of host overloading because they depend on estimation of future CPU utilization. Although they are complex, their benefits may payoff. Example regression algorithms include the Local Regression (LR) algorithm [14]. The basic idea of local regression is to
**Fig. 2** CPU, RAM, BW, Geometric Relation (GR), Euclidean Distance (ED) and Absolute Summation(AS)
fit a simple model to the localized subsets of the data to produce a curve that approximates the original data.
The main drawback of the above-mentioned studies is that host overload detection is mainly based on the CPU utilization. Studies that combined multiple criteria, e.g., CPU, memory and/or BW utilization, do exist. Table 1 provides an overview of the main existing studies that considered multiple factors for detecting host overload. In a previous effort, Abdelsamea et al. [11] have developed a multiple regression algorithm that uses CPU utilization, memory utilization and BW utilization for host overload detection. They proposed preliminary results for this methodology using random and planetLab workload traces which are significant for CPU and RAM but are insignificant for BW utilization. This follow-up paper proposes a new family of multi-dimensional regression host overload detection algorithms using Euclidean Distance (ED) and Absolute Summation (AS) that are more general than that in [11]. Also, and in contrast to [11], we use real-life HPC workloads that are sensitive to CPU, RAM and network BW.
It is worth noting that host overload detection is considered as the first step in the VM consolidation process. Once the overloaded hosts are detected, VM consolidation entails two other steps: (1) selecting the VMs to be migrated from the overloaded hosts to other hosts (known as VM migration), and (2) replacement of the VMs selected for migration on new hosts (known as VM placement). Some recent studies on energy-efficient VM placement and VM migration can be found, e.g., in [21–23]. In particular, the study in [21] addressed the VM placement problem with the objective of improving energy consumption and SLA violations. The authors proposed a novel VM placement algorithm based on the bin packing heuristic. The study in [23] also addressed the energy-efficient VM placement problem, but proposed a genetic algorithm meta-heuristic. Finally, the study in [22] presented new energy-efficient VM placement and VM migration policies. Our work, however, focuses on host overload detection.
**Multi-dimensional regression host utilization for host overload detection algorithms (MDRHU)**
In what follows, we present a novel family of multiple regression based algorithms for host overload detection. The presented algorithms explore different models for host utilization using multiple factors, namely CPU,
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**Table 2** Planetlab Workload data [14]
| Date | 03-03-2011 | 06-03-2011 | 09-03-2011 | 22-03-2011 | 25-03-2011 | 03-04-2011 | 09-04-2011 | 11-04-2011 | 12-04-2011 | 20-04-2011 |
|------------|------------|------------|------------|------------|------------|------------|------------|------------|------------|------------|
| Number of VMs | 1052 | 898 | 1061 | 1516 | 1078 | 1463 | 1358 | 1233 | 1054 | 1033 |
memory and BW. The flowchart for the proposed Multi-Dimensional Regression Host Utilization (MDRHU) algorithm is presented in Fig. 1. In order to predict future host utilization values, the regressor requires two major components. The first component is the profiled data for the independent factors (dimensions) of the running VMs that contribute to the host utilization evaluation. This data is readily available through the online profiling of the VMs and can be superimposed/aggregated across all VMs per host.
The second and the most critical component needed to perform the multiple regression process is the function to combine the independent factors together to derive a dependent metric representing the overall host utilization. This non-trivial component is required as the host utilization cannot be measured directly. A successful evaluation of the host utilization leads to a correct decision of whether the host is really overloaded or not, which is the ultimate goal of our proposed algorithm. Although the work in [11] incorporated the Geometric Relation (GR) function for the host utilization formulation, we believe it does not closely trace the actual host utilization behaviour. In this paper, two alternative models considering the space distance (the multi-dimensionality of the problem) for the host utilization are proposed as discussed, namely, Euclidean Distance (ED) and Absolute Summation (AS). The following paragraph discusses these two proposed models. In addition, GR is also discussed as a benchmark for comparison purposes.
1. **MRHOD** [11]: Geometric Relation (GR) is a multi-factorization relationship that combines multiple parameters in one metric that summarizes the overall system behavior. The most critical factors to be considered for VMs are CPU, memory and BW. However, the absolute values for those factors scores are not the desired parameters to be used. The utilization of the aforementioned factors relative to the maximum permissible utilization is more meaningful to make the factors dimensionless and representative for the host overload. The maximum utilization is either defined by the cloud service provider or the absolute available host utilization. Hence, the profiled data is to be normalized to represent a fractional utilization per factor.
Geometric Relation (GR) for host utilization evaluation is used in [24] as shown in Eq. 1. However, the GR lacks the consideration of the orthogonality of the multi-dimensional space among the different factors.
\[ \text{HostUtilization} = \frac{\omega_1}{1 - \text{CPU}} \times \frac{\omega_2}{1 - \text{RAM}} \times \frac{\omega_3}{1 - \text{BW}} \]
where \( \omega_i \) The weight of for factor \( i \), where the factors are CPU, memory and BW
\( \text{CPU} \) The relative CPU utilization
\( \text{RAM} \) The relative memory utilization
\( \text{BW} \) The relative network utilization
An alternative method to the GR relation above is to use different space distance methodologies. Several methods can be used [25] such as Euclidean Distance (ED) or Absolute Summation (AS) to calculate the distances of two points in space.
2. **MDRHU-ED**: As mentioned above, the CPU, RAM and BW utilizations have different scales or measures. Therefore, the host utilization cannot be determined by simply summing up these values. To overcome this difficulty, the objective is divided by a normalization constant (normConst) as shown in Eq. 2.
\[ \text{HostUtilization} = \frac{\text{CPU} + \text{RAM} + \text{BW}}{\text{normConstED}} \]
Our proposed algorithm MDRHU-ED uses the Euclidean distance between the current and previous host utilizations as the normalization constant. In particular, the normalization constant used by MDRHU-ED is shown in Eq. 3:
\[ \text{normConstED} = \sqrt{d(\text{CPU})^2 + d(\text{RAM})^2 + d(\text{BW})^2} \]
Note that \( d(\text{CPU}) \), \( d(\text{RAM}) \) and \( d(\text{BW}) \) denote the relative difference between the current and previous CPU utilizations, memory utilizations and BW utilizations, respectively.
3. **MDRHU-AS**: Similarly to MDRHU-ED, our alternative proposed algorithm MDRHU-AS estimates the host utilization using:
\[ \text{HostUtilization} = \frac{\text{CPU} + \text{RAM} + \text{BW}}{\text{normConstAS}} \]
The normalization constant (normConst), however, is calculated as the summation of the absolute values
### Table 3: Relative Energy consumption for different algorithms vs. Number of VMs
| Algorithm | 898 | 1033 | 1233 | 1463 | 1516 |
|---------------|------|------|------|------|------|
| HLRHOD | -0.077 | -0.121 | -0.054 | -0.076 | -0.096 |
| MRHOD | -0.089 | -0.144 | -0.089 | -0.081 | -0.115 |
| MDRHU-ED | -0.211 | -0.243 | -0.176 | -0.245 | -0.291 |
| MDRHU-AS | -0.207 | -0.244 | -0.206 | -0.242 | -0.289 |
### Table 4: Relative ESV metric vs. Number of VMs
| Algorithm | 898 | 1033 | 1233 | 1463 | 1516 |
|---------------|------|------|------|------|------|
| HLRHOD | -0.161 | -0.113 | -0.078 | -0.136 | -0.072 |
| MRHOD | -0.164 | -0.112 | -0.046 | -0.126 | -0.056 |
| MDRHU-ED | -0.245 | -0.233 | -0.248 | -0.269 | -0.206 |
| MDRHU-AS | -0.267 | -0.216 | -0.223 | -0.27 | -0.234 |
of the utilization distances. In particular, the normalization constant used by MDRHU-AS is shown in Eq. 4:
\[
\text{normConstAS} = |d(\text{CPU})| + |d(\text{RAM})| + |d(\text{BW})|
\] (4)
Again, \(d(\text{CPU})\), \(d(\text{RAM})\) and \(d(\text{BW})\) denote the relative difference between the current and previous CPU utilizations, memory utilizations and BW utilizations, respectively. This strategy results in a bigger divisor as compared to the Euclidean distance strategy, and is considered to be preferable for scaling problems that are considered badly incommensurable. On the other hand, it is characterized by a similar robustness to the Euclidean distance method.
It is worth noting that our proposed alternative approaches (MDRHU-ED and MDRHU-AS) are used to estimate the host utilization using profiled data related to CPU, memory and BW utilizations, respectively. As shown in Fig. 1, the next step is to build a general model that maps the independent variables (here CPU, memory and BW) to their corresponding dependent variable (here host utilization). This is accomplished using a multiple
Fig. 5 Algorithms vs. Number of VMs for Gaia. a Energy Consumption. b SLAV metric. c ESV metric. d VM Migrations. e ESM metric
regression algorithm. See, e.g., [26]. Multiple regression is an extension of simple linear regression. Its objective is to predict the value of a dependent variable (here host utilization) based on the values of multiple independent variables (here CPU, memory and BW). The outcome of the multiple regression algorithm is a prediction of the future host utilization. After the predicted Host Utilization is obtained through the regressor, its value is evaluated. The host is considered to be overloaded if the predicted host utilization exceeds some threshold as recommended by [14]. Consequently, a VM is selected to be migrated from the overloaded host.
To examine the correlation between the multi-dimensional approach and the utilization of the single factor parameters, Fig. 2 depicts the VM utilization for each individual factor and the host utilization calculated through GR, as compared to the two proposed multi-dimensional models (MDRHU-ED and MDRHU-AS) at different time slots. We can observe that the GR host utilization is somewhat averaging the individual utilization behaviour. On the other hand, ED and AS trace the overall utilization behaviour better with more pronounced variation in the curves.
**Evaluation methodology**
**Experimental setup**
Since it is hard to conduct repeatable large scale experiments on a real infrastructure [14], simulations are chosen as an approach to highlight the superiority of our proposed algorithms. The Cloudsim toolkit [27] has been utilized as a simulation framework because of the following reasons. Cloudsim supports VM provisioning at two levels: the host level and the VM level. At both levels, Cloudsim performs space-shared and time-shared provisioning techniques. Space-shared techniques distribute certain CPU cores among the VMs. These techniques behave similarly to the First Come First Serve (FCFS) scheduling algorithm. Time-shared techniques variably distribute the capacity of one core among the VMs. These techniques act similarly to the Round-Robin (RR) scheduling technique. Also Cloudsim permits the modeling of virtualized frameworks, sustaining on demand resource provisioning and resource management. Therefore, we choose the Cloudsim 3.0.3 toolkit as our simulation platform. We have extended Cloudsim with energy-aware simulations, which were originally not available in the core framework [27]. For the multi-dimensional regression process, Ordinary Least Square (OLS) Multiple Regression function [26, 28] is used for the regression coefficient calculation, then host utilization prediction.
To assess the performance of the newly proposed algorithms (MDRHU-ED and MDRHU-AS), we use the following host overload detection algorithms from the literature as benchmarks:
- The Local Regression (LR) algorithm from [14].
- The Hybrid Local Regression Host Overload Detection (HLRHOD) algorithm from [11].
- The Multiple Regression Host Overload Detection (MRHOD) algorithm from [11].
It is worth noting that LR is already implemented in the CloudSim simulator. However, we add an implementation of HLRHOD and MRHOD, as well as our proposed algorithms (MDRHU-ED and MDRHU-AS), to CloudSim.
A critical parameter to be adjusted for the host overload detection process is the threshold used to identify whether the host is overloaded or not. This parameter is called safety parameter in Cloudsim. The safety parameter defines how aggressively the system consolidates VMs on physical servers. If the safety parameter is too tight, opportunities for energy savings become too low. On the other hand, if the safety parameter is too relaxed, the levels of service level agreement violations become too high. Therefore, we perform an experimental (via simulation) selection for the safety parameter value, as to achieve an acceptable tradeoff between energy saving and SLA violation, as indicated by the overall performance metric. All results reported in the following sections are using the experimentally adjusted safety parameter.
Note also that, once a host overload has been detected, the next step is to select particular VMs to be migrated from the overloaded host to other hosts. To this end, we use the Minimum Migration Time (MMT) algorithm [14] for VM selection, and the modified Best Fit Decreasing (BFD) algorithm proposed in [14] for VM migration. It is worth noting that MMT and BFD are already implemented in Cloudsim.
**Power model**
The Power consumed by computing resources in cloud data centers is mostly consumed by the CPU, disk storage, memory, power supplies and cooling devices [29]. Establishing exact analytical models for power consumption is a complicated task due to the complexity of the power models of modern multicore CPUs. Consequently, we utilize real data on power consumption offered by the outcomes of the SPECpower benchmark [14] as an alternative to the usage of an analytical power consumption model. The host overload is frequently examined every scheduling interval chosen to be 300 sec. The host types are: HP ProLiant ML110 G4 (Intel Xeon 3040/2 cores/1860 MHz/4 GB), and HP ProLiant ML110G5 (Intel Xeon 3075/2 cores/2660 MHz/4 GB), and their power consumption features are shown in Fig. 3.
**Performance metrics**
The metrics we use to assess the performance of our proposed algorithms are summarized as follows:
• **Total energy consumption (E)** is defined as the sum of energy consumed by the physical resources of a data center, and is computed using the model presented in [14, 30].
• **Number of VM migrations** is an important metric reflecting the time needed to migrate VMs from the overloaded host to other underloaded hosts.
• **SLA Violation (SLAV)** captures the performance degradation due to host overloading and performance degradation due to VM migration. SLA Violation (SLAV) [14] occurs when a VM cannot obtain its promised Quality of Service (QoS) [31, 32].
• **Energy and SLA Violations (ESV)** is a metric that combines energy consumption and SLA Violations. This metric is originally proposed in [14], and is shown in Eq. 5. Note that the energy consumed by physical hosts and SLAV are adversely related, as energy can be frequently minimized on the expense of increasing SLA violations. The lower the ESV metric lower, the better the performance.
\[
ESV = E \times SLAV
\]
(5)
• **Energy-SLA-Migration (ESM)** [33] captures the simultaneous minimization of energy, SLA violation, and number of VMs migrations, and is given by Eq. 6.
\[
ESM = E \times SLAV \times Number \ of \ VM \ migrations
\]
(6)
**Workloads**
We have used two real workloads for our experiments. The first workload data is provided as a part of the CoMon project, a monitoring infrastructure for PlanetLab. We have randomly chosen 10 days of the workload traces collected during March and April 2011 [14]. Each day is characterized by a specific number of VMs as shown in Table 2.
The second workload contains 3 months worth of HPC data from the Gaia cluster at the University of Luxembourg [34]. The workload data includes CPU and memory usage [35]. The log is available directly in standard workload format (SWF). We use UniLu-Gaia-2014-1.swf. It is based on accounting data collected by the scheduler. We build three utilization models that read the CPU and RAM, and calculate the BW, respectively, from the workload and pass it to the cloudlets.
**Simulation results and analysis**
**Sensitivity analysis**
In this subsection, we assess how the performance of our proposed algorithms are affected by varying the number of VMs, and by varying the scheduling interval. Experiments for the sensitivity analysis uses PlanetLab workload traces by default. However, the sensitivity for the number of VMs is also studied for the Gaia workload.
**Number of VMs**
Varying the number of VMs with fixed 800 hosts and 300 sec. scheduling interval is tested and evaluated. The effect on the E and ESV metrics are shown in Tables 3 and 4, respectively. MRHOD is used as the baseline algorithm using Eq. 7. The higher the energy consumption, the more inferior the algorithm. Accordingly, a positive value for this equation indicates worse performance, while a negative value indicates better performance.
\[
Relative \ Improvement = \frac{Algorithm - Reference}{Reference}
\]
(7)
As shown in Table 3, MDRHU-ED is the best since it causes a reduction in energy consumption by about 21% as compared to the LR algorithm. As the number of VMs increases, MDRHU-AS is the best when the number of VMs is 1033 and 1233. HLRHOD is the simplest multiple regression algorithm since it depends on local regression. Its best improvement is when the number of VMs is 1033 where the energy consumption is enhanced by about 12% compared to LR. MRHOD based on multiple regression outperforms HLRHOD in term of energy consumption. MRHOD gives an improvement more than HLRHOD of about 2% for 898 VMs and about 14% for 1033 VMs. Also it is clear from Table 3 that space distance based multiple regression algorithms (MDRHU-ED and MDRHU-AS) give better enhancement in term of energy consumption than Geometric Relation (GR) based multiple regression algorithm (MRHOD). MDRHU-ED is the best when number of VMs are 898, 1463 and 1516 while MDRHU-AS is the best when number of VMs are 1033 and 1233.
In Table 4, when the number of VMs is small (898), MDRHU-AS gives the best results as it causes a reduction of the ESV metric by about 26%. For the largest number of VMs (1516), MDRHU-AS is the best since it decreases the ESV by about 23%. HLRHOD gives better results than MRHOD across all number of VMs except when the number of VMs is 898. MDRHU-ED is the best when number of VMs are 898, 1463 and 1516, respectively, while MDRHU-AS is the best when number of VMs are 1033 and 1233, respectively. The results in Table 4 prove that multi-dimensional regression algorithms do not sacrifice energy consumption with SLA violations.
**Table 5** Relative Energy consumption vs. Scheduling interval (sec.)
| Algorithm | 300 | 500 | 700 | 900 | 1100 |
|-----------|-------|-------|-------|-------|-------|
| HLRHOD | -0.122| -0.08 | -0.085| -0.084| -0.028|
| MRHOD | -0.144| -0.145| -0.097| -0.099| -0.033|
| MDRHU-ED | -0.243| -0.222| -0.162| -0.205| -0.163|
| MDRHU-AS | -0.244| -0.227| -0.156| -0.208| -0.148|
Figure 4 shows the variation of the results for different number of VMs per algorithm as a box plot to indicate the maximum, minimum, first quartile and third quartile using PlanetLab workloads. Each part of the Figure shows one of the evaluation metrics as (a) E, (b) SLAV, (c) ESV, (d) VM migrations and (e) ESM across different algorithms. For the energy consumption, both newly proposed MDRHU-ED and MDRHU-AS algorithms are very close to each other, and better than all other algorithms. MDRHU-AS is very stable and shows the least variation in its results for the VM migrations, followed by MDRHU-ED which comes in the second place. For the SLAV, HLRHOD is the best. However, for the combined metrics (ESV and ESM) MDRHU-ED and MDRHU-AS are the best. Similar behaviour is noticed for the Gaia workload with even better results for MDRHU-ED and MDRHU-AS, as depicted in Fig. 5. However, it is interesting to see HLRHOD and MRHOD are not performing as good as LR for Gaia workloads. This supports the importance of using the multi-dimensional regression approach for the HPC workloads.
**Scheduling Interval**
Relative energy consumptions with respect to LR while varying the scheduling interval with fixed 800 hosts and 1033 VMs are shown in Table 5. Increasing the scheduling
interval indicates a lower frequency of running the VM consolidation and vice versa. HLRHOD provides better results than single factor algorithm since it causes a reduction in energy consumption by about 12% as compared to LR when the scheduling interval is 300 sec. Our new proposed multi-dimensional regression based algorithms outperform single factor algorithm as well as the HLRHOD multiple regression algorithm across all values of scheduling interval. MDRHU-AS gives the best energy consumption. In particular, it reduces the energy consumption by about 24% when the scheduling interval is 300 sec. It is worth noting that the scheduling interval specifies how often the overload detection is performed. So, a small scheduling interval allows a better prediction of host overloading. This leads to better reduction in energy consumption as compared to when the scheduling interval is large. However, this comes at the cost of an increased computational complexity.
Similar to Fig. 4, Fig. 6 shows the variation of the results for different scheduling intervals. For the energy consumption, MDRHU-ED and MDRHU-AS outperform all other algorithms. MDRHU-AS is very robust, and shows no variation in the SLAVs. However, LR has the lowest SLAV and ESV values among all algorithms. However, it has the worst performance in terms of the VM migrations. MDRHU-ED is the best in terms of the ESM metric.
**Fig. 7** Algorithms Comparison for PlanetLab relative to MRHOD. **a** Energy Consumption. **b** SLAV metric. **c** ESV metric. **d** VM Migrations. **e** ESM metric.
**Algorithms comparative analysis using PlanetLab Workload**
The comparison between our proposed multi-dimensional family of algorithms (MDRHU-AS and MDRHU-ED) and the existing multiple factor algorithms (HLRHOD and MRHOD) is shown in Fig. 7. Note that the results are normalized with respect to MRHOD. Note also that the $y$-axis of Fig. 7e is in log-scale. We use a *PlanetLab* workload trace with the number of hosts set to 800 and the number of VMs set to 1033. For the *PlanetLab* workload we consider two factors only (CPU and RAM) in MDRHU and HLRHOD. The reason is that *PlanetLab* workloads are insensitive to the BW utilization because the VMs are not HPC in nature, and thus not communicating among each other. The next subsection, however, is devoted for results using a Gaia HPC workload trace that is sensitive to all three (CPU, RAM and BW) factors.
Across different metrics for power/performance and QoS, our proposed MDRHU-ED and MDRHU-AS algorithms outperform all other algorithms. In particular, the energy consumption is reduced by about 28% as compared to LR. This can be justified by the robustness of the absolute summation, which resists data outliers. Moreover, our newly proposed MDRHU-ED and MDRHU-AS algorithms outperform HLRHOD and MRHOD algorithms by about 12%. The SLAV is lower in LR as compared to other multiple factor algorithms by a negligible percentage (2%) because the energy and SLAV are negatively correlated. However, MDRHU-AS and MDRHU-ED improve the combined ESV metric by about 25% as compared to LR. MDRHU-AS and MDRHU-ED have lower (better) ESV than the recently proposed MRHOD by 12% and 14%, respectively.
Multi-dimensional regression algorithms outperform multi-factor algorithms due to their superiority in predicting host overloading, which leads to a decreased number of VM migrations as well. The latter leads to a decrease in the SLAV metric. The effect of number of VM migrations on energy consumption and ESV is observed when using the ESM metric. MDRHU-AS and MDRHU-ED have lower (better) ESM than the recently proposed MRHOD by 12% and 15%, respectively.
Although multiple regression enhances the energy consumption, its higher complexity adds overhead as compared to the simpler techniques. For the sake of fair comparison, Fig. 8 depicts the execution times of the different algorithms. HLRHOD is the best in terms of execution time. In spite of the higher execution time of MDRHU, the overhead is insignificant for typical job dispatch rates in cloud data centers. The other components of VM consolidation have even a higher overhead.
**Algorithms comparative analysis using Gaia Workload**
In this part of our simulation study we use a Gaia HPC workload trace with the number of hosts set to 800 and the number of VMs set to 1001. Using the Gaia workload is motivated by its HPC nature, and its significance to the BW utilization parameter. To this end, we re-address the *PlanetLab* workload with 800 hosts and 1033 VMs as to illustrate, in contrast to Gaia, its insignificance to the BW utilization. In particular, we test the recently proposed algorithm MRHOD with only two factors (CPU and RAM) taken into consideration in the regression model, then with all three (CPU, RAM and BW) factors taken into consideration in the regression model. The same (two vs. three factors) comparison is done on the *PlanetLab* workload. Figure 9 depicts the parameters relative improvement results for MRHOD considering all three factors as compared to considering only two factors when using the Gaia HPC workload and the *PlanetLab* workload, respectively. In particular, considering three factors in MRHOD improves the energy consumption by more than 10% for the Gaia workloads, while considering three factors improves the energy consumption by only 0.3% for the *PlanetLab* workloads. MRHOD improves the ESV metric by more than 23% for the Gaia workloads, while considering three factors in the proposed MRHOD only makes the ESV metric worse for *PlanetLab* workloads. This proves that the *PlanetLab* workload is insignificant to the BW utilization, i.e., non-HPC in nature. In contrast, the Gaia workload is indeed HPC in nature due to its dependance on the BW utilization of the VMs. This partly illustrates the importance of this work in testing the proposed algorithms for real HPC workloads, in contrast to [11], which used only the non-HPC *PlanetLab*.
Since there is improvement when using three factors as opposed to using two factors, a comparison between different multiple factors algorithms (MRHOD and HLRHOD) and multi-dimensional algorithms (MDRHU-AS & MDRHU-ED) when taking all three factors (CPU, RAM, BW) into consideration is presented in Fig. 10. The results are normalized with respect to MRHOD. In
particular, Fig. 10 shows that the consideration of the BW utilization in HPC workloads causes an improvement in all metrics for multiple factor algorithms. Using MRHOD leads to about 23% better (less) energy consumption and HLRHOD leads to about 14% better (less) energy consumption as compared to LR. However, the new multi-dimensional regression used in MDRHU-AS and MDRHU-ED achieve a slight energy saving of 2% and 4%, respectively. The SLAV metric is reduced in MRHOD by about 18% as compared to that of LR. MRHOD violates the service level agreement more than 60% as compared to the newly proposed MDRHU-AS and MDRHU-ED. Hence, the ESV metric is greatly improved for MDRHU-AS and MDRHU-ED as compared to using MRHOD or LR algorithm. The number of VM migrations is also reduced significantly by the newly proposed algorithms. MDRHU-AS and MDRHU-ED reduce the VM migrations by about 40% as compared to MRHOD. Hence, the ESM metric shows a significant improvement (reduction) for the new proposals. MDRHU-AS and MDRHU-ED reduce the ESM metric by about 78% and 80%, respectively, as compared to MRHOD. The improvement achieved using the new formulas in multi-dimensional regression using the Gaia HPC workload is six times better than the improvement achieved using the PlanetLab workload. However, we can hardly claim a winner when comparing MDRHU-AS and MDRHU-ED. Both of these multidimensional distance measurement concepts are significantly superior than using the GR formula; yet they are very close to each other to discriminate a winner.
**Conclusion**
The use of cloud computing data centers has gained a lot of interest as a viable solution to satisfy the tremendously increased demand for high-performance computing (HPC), storage and networking resources for business and scientific applications. Such large-scale data centers lead to excessive amounts of energy consumption, operating costs and $CO_2$ emissions. Virtual machine (VM) consolidation, which involves the live migration of VMs to run on fewer physical servers, comes as an important solution because it allows more servers to be switched off or run on low-power mode, which helps reduce the energy consumption, operating cost and $CO_2$ emission. A crucial step in VM consolidation is host overload detection, which attempts to predict whether or not a physical server will be oversubscribed with VMs. Unlike most of the previous work, which use the CPU utilization as the sole indicator for host overload, this paper took multiple factors into consideration: CPU, memory and network BW utilization. This is motivated by the fact that HPC applications are not only constrained by the CPU, but also by the memory and BW requirements. Therefore, this paper presented a family of novel multi-dimensional regression host overload detection algorithms, which combine CPU, memory and network BW utilization via Euclidean Distance (ED) and Absolute Summation (AS), respectively. The contribution of this paper is two-fold. First, the presented algorithms are based on multi-dimensional regression, leading to improved results in terms of energy consumption and service level agreement violation. Second, the proposed algorithms were tested using real-world HPC workloads. Our extensive simulation study illustrated the superiority of our proposed algorithms over existing methods. In particular, as compared to the most recently proposed multiple regression based on Geometric Relation (GR), our proposed algorithms provide an improvement of at least 12% in energy consumption, and an improvement of at least 80% in a metric that combines energy consumption, service level agreement violation and number of VM migrations.
Fig. 10 Algorithms Comparison for Gaia HPC workload relative to MRHOD. **a** Energy Consumption. **b** SLAV metric. **c** ESV metric. **d** VM Migrations. **e** ESM metric
**Abbreviations**
AS: Absolute Summation; E: Total energy consumption; ED: Euclidean Distance; ESV: Energy and SLA Violations; GR: Geometric Relation; HLRHOD: Hybrid Local Regression Host Overload Detection algorithm; LR: Local Regression; MDRHU: Multi-Dimensional Regression Host Utilization; MRHOD: Multiple Regression Host Overload Detection algorithm; VM: Virtual Machine
**Acknowledgements**
Not Applicable.
**Funding**
This research was supported in part by the the cloud computing center of excellence grant number 5220, Science and Technology Development Fund (STDF), Egypt, and in part by the Distributed and Networked Systems Research Group Operating Grant number 150410, University of Sharjah, UAE. Through those grants the Cloud system is established to perform the experiments and simulations.
**Availability of data and materials**
The data, through which the experiment is conducted, is available at: - The Gaia HPC workload. [http://www.cs.hju.ac.il/labs/parallel/workload/L_unilu_gaia/index.html](http://www.cs.hju.ac.il/labs/parallel/workload/L_unilu_gaia/index.html) (2014) - Parallel Workloads Archive. [http://www.cs.hju.ac.il/labs/parallel/workload](http://www.cs.hju.ac.il/labs/parallel/workload) (2017) - [https://www.planet-lab.org/db/pub/slices.php](https://www.planet-lab.org/db/pub/slices.php)
However, the full description of the experiment setup is provided in the manuscript in sections Experimental setup and Workloads.
**Authors’ contributions**
The authors equally contributed to this research and the paper initiated by the first author. All authors read and approved the final manuscript.
**Authors’ information**
Ali A. El-Moursy (Senior Member, IEEE) received the Ph.D. in the area of High-performance Computer Architecture from University of Rochester, Rochester, NY, USA, in 2005. Dr. El-Moursy has worked for Software Solution Group, Intel Corp., CA, USA till early 2007. In 2007, he has joint Electronics
Research Institute, Giza, Egypt. His research interest is in high-performance computer architecture, multi-core multi-threaded micro-architecture, power-aware micro-architecture, simulation and modeling of architecture performance and power, workload profiling and characterization, cell programming, high performance computing, parallel computing and Cloud computing. Dr. El-Moursy has also participated with IBM Cairo Technology Development Center, Egypt, as a visitor research scientist for the period from Feb.2007 till Jan.2010. In Sep.2010 Dr. El-Moursy has joint ECE Dep.at University of Sharjah, Sharjah, UAE as an Assistant Prof.. In Jan.2017, Dr. El-Moursy has been promoted to the Associate Prof. Rank.
Amany Abd El Sameea received the BSc degree from Shoubra Faculty of Engineering in 1999, the MSc degree from Faculty of Engineering Cairo University in 2006 and the PhD degree in Computer Engineering from Faculty of Engineering Cairo University in 2017. In 2000, she has joint Electronics Research Institute, Giza, Egypt. She attended the Chain-Reds school on Cloud Computing held at INFN Catania April 2015. Her research interest focus on the fields of parallel computing, distributed computing, grid computing and cloud computing.
Rukshanda Kamran has completed her Masters in Information Technology in 2005 from Preston University, Pakistan and Bachelors in Computer Science in 1999 from Shah Abdul Latif University Karachi, Pakistan with Honors. Rukshanda Kamran has working experience as Faculty of Information Technology in Bedfordshire University, UK (Dubai Campus) Sep 2011 till January 2014, Preston University Dubai for the period from early 2003 till May 2013 and as Research Assistant at University of Wollongong Dubai from July 2014 till Feb 2015 in the area of A Hybrid Cloud Enabled Supply Chain Network. Rukshandakamran has joined SZABIST University Dubai in Sep 2013 and currently working as Assistant Professor SZABIST University and in Feb 2016 Rukshanda Kamran as joint ECE Dep.at University of Sharjah, Sharjah, UAE and working as Research Assistant. Rukshanda Kamran is an enrolled Student for PhD, in the area of High-Performance Cloud Computing at University of Malaysia, Sarawak (UNIMAS) 2014. Her research interest are High performance computing and Cloud Technologies.
Mohamed Saad (Senior Member, IEEE) received Ph.D. degree in electrical and computer engineering from McMaster University, Hamilton, Canada, in 2004. He is currently an Associate Professor at the Department of Electrical and Computer Engineering, University of Sharjah, UAE. His research interests include networking, communications and optimization, with current activity focused on the optimal design of wireless and wired communication networks, and optimal network resource management. He has also held research positions with the Department of Electrical and Computer Engineering, University of Toronto, Toronto, Canada, and the Advanced Optimization Laboratory at the Department of Computing and Software, McMaster University, Hamilton, Canada. Dr. Saad is an editor for the International Journal of Distributed Sensor Networks. He was the recipient of the best paper award in the IEEE Symposium on Computers and Communications, Riccione, Italy, June 2010. He was the recipient of the University of Sharjah “Annual Incentive Award for Distinguished Faculty Members”, for excellence in research, April 2010 (university-wide). He received also two best teaching awards by the IEEE Women in Engineering Society, University of Sharjah (in 2007 and 2009). He was also the recipient of a 2005–2006 Natural Sciences and Engineering Research Council of Canada (NSERC) post-doctoral fellowship. He is a senior member of the IEEE.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Author details
1Electrical and Computer Engineering Department, University of Sharjah, Sharjah, UAE. 2Computers and Systems Department, Electronics Research Institute, Giza, Egypt. 3Senior IT Faculty, SZABIST University, Sharjah, UAE.
Received: 1 January 2019 Accepted: 17 April 2019
Published online: 13 June 2019
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Devices, methods and computer-readable media for providing control of switching of an interactive session in a communications network. Content may be switched between media presentation screens. Collected data is analyzed and a location of a user within the communications network is monitored. A presentation of a first screen the user is watching may be exchanged for a presentation of a second screen selected based upon a change in the location of a monitored user and the analysis of the collected data.
16 Claims, 11 Drawing Sheets
| Patent Number | Date | Inventors | Classification Code |
|---------------|------------|-------------------------|---------------------|
| 2004/0253940 A1 | 12/2004 | Andrews et al. | |
| 2005/0157660 A1 | 7/2005 | Mandato et al. | |
| 2006/0020646 A1 | 1/2006 | Tee et al. | |
| 2006/0085147 A1 | 4/2006 | Karaoguz et al. | |
| 2007/0093337 A1 * | 5/2007 | Koegi et al. | 340/825.22 |
| 2007/0032345 A1 * | 2/2007 | Prasad et alhanan et al.| 482/8 |
| 2007/0050492 A1 | 3/2007 | Jorgensen | |
| 2007/0133428 A1 | 6/2007 | Taylor et al. | |
| 2007/0198682 A1 * | 8/2007 | Pazhyannur et al. | 709/223 |
| 2007/0202923 A1 * | 8/2007 | Jung et al. | 455/566 |
* cited by examiner
Is This An Appropriate Time For A Screen Switch?
Yes → Determine The "From" Screen And The Candidate "To" Screens
Analyze The Feasibility And Ramifications Of Candidate Switching
Does The Feasibility And Ramifications Meet Acceptable Standards?
No → End
Yes → Implement The Determined Screen Switch
End
Default Preferences Are Provided
After Contracting For The Service, A Range Of User Preferences Are Set
Do The Users Want To Modify The Earlier User Settings?
Yes
No
End
User Movement To, Or Away From, A Location Where Another Screen Is Located Is Monitored
The Device/Screen Capabilities Are Analyzed
Based On Location Measurement, Is This An Appropriate Time For Switching Screen?
Yes → End
No → 634
The Relative Current Screen Movement Is Analyzed
Is The Current Screen Moving To A Fixed-Screen Location?
Yes → The "From" Screen Is Determined To Be A Mobile Device Screen
No → Is The Current Screen Moving Away From A Fixed-Screen Location?
Yes → The "From" Screen Is Determined To Be A Fixed Screen At That Location
No → End
The Relative Screen Movement Location Is Analyzed
Is The Current Screen Moving To A Fixed-Screen Location?
Yes → The "To" Screen Is Determined To Be The Fixed Screen At That Location
No → Is The Current Screen Moving Away From A Fixed-Screen Location?
Yes → The "To" Screen Is Determined To A Mobile Device Screen
No → End
The Network Is Analyzed For Available Bandwidth And/Or Resources Corresponding To New Data Path
The Application And/Or Service Is Analyzed
Based Upon The Analysis Of The Network And The Application/Services, The Feasibility Of Candidate Switching Is Made
End
An Identification Of Other Potentially Relevant Users Is Made
An Analysis Of The Impact On The Identified Other Potentially Relevant Users Is Performed
Are The Identified Other Potentially Relevant Users Unaffected Or Does The Impact Meet Predetermined Criteria?
Yes
Ramifications Are Deemed Acceptable
No
The Priority Levels Of The Affected Users Are Compared
The Priority Of The Other Usage Is Also Analyzed Via Identification Of Usage, Where Possible
Based Upon The Analysis Of The Other Potentially Relevant Users, Whether Those Users Are Affected And Whether The Other Usage Is Determined To Be A Priority Usage, The Ramifications Of Candidate Switching Is Ascertained And An Appropriate Switch Is Made
End
Fig. 10
The Preferences And Rules Are Reviewed
When The Limits Defined By The Preferences And Rules So Indicate, The User May Be Queryed To Verify And Allow The Candidate Screen Switch
Does The Feasibility And Ramifications Allow The Candidate Switch?
Yes
The Candidate Screen Switch Is Performed
A Command Is Sent To The Network To Implement The Candidate Switch
Commands Are Sent To Applications And Services To Implement The Candidate Switch
Exception Handling Is Provided To Rollback The Switch Only When Necessary
End
Fig. 11
DEVICES, METHODS AND COMPUTER-READABLE MEDIA FOR PROVIDING CONTROL OF SWITCHING BETWEEN MEDIA PRESENTATION SCREENS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is related to the following co-pending and commonly-assigned patent applications, which are hereby incorporated herein by reference in its respective entirety:
“DEVICES, METHODS, AND COMPUTER-READABLE MEDIA FOR PROVIDING CALENDAR-BASED COMMUNICATION SYSTEM SERVICES” to Jeffrey Aaron, et al., having U.S. patent application Ser. No. 12/253,137, filed Oct. 16, 2008.
“DEVICES, METHODS, AND COMPUTER-READABLE MEDIA FOR PROVIDING SERVICES BASED UPON IDENTIFICATION OF DECISION MAKERS AND OWNERS ASSOCIATED WITH COMMUNICATION SERVICES” to Jeffrey Aaron, et al., having U.S. patent application Ser. No. 12/253,157, filed Oct. 16, 2008.
“DEVICES, METHODS, AND COMPUTER-READABLE MEDIA FOR PROVIDING BROAD QUALITY OF SERVICE OPTIMIZATION USING POLICY-BASED SELECTIVE QUALITY DEGRADATION” to Jeffrey Aaron, et al., having U.S. patent application Ser. No. 12/253,178, filed Oct. 16, 2008.
“DEVICES, METHODS, AND COMPUTER-READABLE MEDIA FOR PROVIDING QUALITY OF SERVICE OPTIMIZATION VIA POLICY-BASED REARRANGEMENTS” to Jeffrey Aaron, et al., having U.S. patent application Ser. No. 12/253,197, filed Oct. 16, 2008.
BACKGROUND
The present invention relates to systems, methods, and computer-readable media for controlling the presentation of media content to a user. More particularly, the present invention relates to devices, methods and computer-readable media for providing control of switching a presentation of content and/or an interactive session between media presentation screens and/or interactive terminals.
Traditionally, televisions signals have been provided via a single wire cable or terrestrial antenna. In contrast, the telephone has traditionally used twisted-pair copper wires to provide telephone service. Access to the Internet has been provided by either twisted-pair copper wires or via a single wire cable. Recently, both cable operators and telecommunication companies have begun to offer all three services, television, telephone and Internet, on one wire. Significant cost savings and/or other advantages are provided by combining all three services on a single wire.
Wireless cell phone technology is a fourth type of service that offers the convenience of keeping in contact with others at any place and at any time. Cell phones may connect wirelessly with a cell phone network to offer data and communication services. Cell phones often combine communication, digital photography, video game functions, video and audio/video viewing functions, etc. To take advantage of this added functionality, hybrid cell phones that combine short-range (Wi-Fi), high-speed Internet access and cellular service have been marketed to allow users to make connections using a local wireless Internet access point and seamlessly switch over to a cell phone network whenever necessary. Moreover, cellular carriers have spent billions of dollars to upgrade their systems for high-speed data. The upgraded networks offer wide coverage that exceeds Wi-Fi’s short range. However, wireless LAN (local area network) technology still provides a higher bandwidth than cellular high-speed data networks. Accordingly, the ability to seamlessly switch between the different technology networks enables users to more efficiently use high-speed networks.
In view of the popularity of the above type of services, communication service providers covet the eyes and ears of consumers, which today are focused on three screens, i.e., the television, the personal computer and wireless devices such as cell phones integrated with the above listed functionality. One aspect that is common to the delivery of content to the three screens is the Internet Protocol (IP). Moreover, an IP-based platform also allows significant opportunities to make the consumer’s experience more interactive and personalized. For example, television viewers may want to retrieve a player’s statistics while watching a sports game, to access photos or music from their PC on their television or use a wireless phone to schedule a recording of their favorite show.
However, a problem presented by the three screens vision involves determining when to switch the presentation of an application or service from one of the three types of devices or screens to another. For example, if a user wants to switch from viewing a movie television program on a smartphone or personal computer to viewing the movie on a home television, the user has to turn on the television, select the proper channel, set the volume, and then turn off the former viewing source. There may also be additional parameters the user must consider, e.g., whether the television in the media room is available. Other scenarios may provide additional or greater difficulties, or make the transition disruptive to the user.
It can be seen then that there is a need for devices, methods, and computer-readable media that provides control of switching between media presentation screens, including any associated interactive communication sessions.
SUMMARY
According to exemplary embodiments, methods, computer-readable media, and devices provide control for the switching a presentation of media content on one device to another device, including likewise switching of any associated sessions used for interactivity, control, communication, and/or other relevant purposes.
According to one embodiment, a controller is provided for analyzing the collected data and monitoring locations of a user within the communications network. The controller exchanges a presentation of a first screen the user is watching for a presentation of a second screen selected based upon a change in the location of a monitored user and the analysis of the collected data.
These and other features and advantages, which characterize the present invention, will be apparent from a reading of the following detailed description and a review of the associated drawings. It is to be understood that both foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a general block diagram of a media and communication network according to an exemplary embodiment;
FIG. 2 illustrates a system for collecting and controlling the user experience of services according to an exemplary embodiment;
FIG. 3 illustrates an example of a screen switch according to an exemplary embodiment;
FIG. 4 is a flow chart illustrating the control of switching a presentation of content between media presentation screens according to an exemplary embodiment;
FIG. 5 is a flow chart illustrating collection of initial data for controlling the switching a presentation of content between media presentation screens according to an exemplary embodiment;
FIG. 6 is a flow chart illustrating the determination of when switching is appropriate according to an exemplary embodiment;
FIG. 7 is a flow chart illustrating the determination of a “from” screen according to an exemplary embodiment;
FIG. 8 is a flow chart illustrating the determination of a “to” screen according to an exemplary embodiment;
FIG. 9 is a flow chart illustrating the determination of the feasibility of candidate switching according to an exemplary embodiment;
FIG. 10 is a flow chart illustrating the analysis of the ramifications of candidate switching according to an exemplary embodiment; and
FIG. 11 is a flow chart showing the implementation of the candidate switching according to an exemplary embodiment.
DETAILED DESCRIPTION
In the following description of the exemplary embodiments reference is made to the accompanying drawings that form a part hereof, and in which is shown by way of illustration the specific embodiments in which the invention may be practiced. It is to be understood that other embodiments may be equally applicable to controlling the switching of a presentation of content between media presentation screens.
FIG. 1 illustrates a general block diagram 100 of a media and communication network according to an exemplary embodiment. A multimedia carrier 110 is configured to provide hardware and software infrastructure for the delivery of content and for providing communication channels for subscribers or other users. Examples of communication channels includes telephone services, Internet access, voice-over-IP, interactive television and gaming, etc. The multimedia carrier 110 acquires programming from content providers and encodes the content. The multimedia carrier 110 may also add local programming and provide additional digital services such as video on demand, and storage for content according to user directions.
The multimedia carrier 110 is coupled to a network 120 for distributing the content, providing Internet access, telephone services, etc. The network 120 provides the intermediate infrastructure between the point-of-delivery 130 and the multimedia carrier 110. The network 120 may be configured to provide one or more or the intermediate infrastructure 140 for television content delivery, plain-old-telephone system, satellite television, Internet services, wireless communications, such as cellular mobile phone services, etc. Accordingly, the network may include hub offices, IP service offices, local loop carrier facilities, satellite carrier facilities, communication satellites, telecom point-of-presence, mobile switching centers, base stations, and distribution nodes.
At the point-of-delivery 130, a router 132 may be used to provide digital services, such as Internet access. A computer or other Internet-capable device 160 may be coupled to the router 132 by a network cable 162 or wirelessly 164. A residential gateway 134 may be used to provide content, e.g., IPTV, to multimedia-capable viewing devices, such as a television, via a cable 172 or wirelessly 174. A mobile device 150 operates within a mobile operating system. Examples of such mobile devices 150 may include cell phones, smart phones, handheld computers, personal digital assistants, etc.
In addition, mobile devices 150 may be configured to use location-based services (LBS) to receive information specific to a location. Some examples of location-based services include requesting the nearest business or service, such as an ATM or restaurant, and finding a friend. A service provider may obtain the location of a mobile device from a global positioning system (GPS) circuit built into the device, or by using radiolocation and trilateration based on the signal-strength of the closest cell-phone towers (for phones without GPS features). Any other suitable means of obtaining or estimating location may also be used.
FIG. 2 illustrates a system 200, according to an exemplary embodiment, for collecting and controlling the user experience of services, e.g., through an infrastructure as described above. The system operator may provide a processor 210 that collects and controls the system based upon the data 230 collected from the user. The data collection and system control processor 210 gathers information from across the network of users 220. The collected data 230 is stored in a database 240 or other suitable means of storage (e.g. flash memory). The data 230 in the database 240 may be analyzed for further determination of the type of control that is to be provided. Alternatively, or in addition to, the data 230 may represent choices or parameters identified by the user and provided as input to the data collection and system control processor 210. Moreover, data for making decisions may be derived from any point in the network 220. For example, data can be collected from applications, services, servers, network routers, etc. The data may also be automatically collected, collected in response to queries made to a user, or collected via un-prompted feedback from a user. The data collection and system control processor 210 uses the collected data 230 to control access and the delivery of content to the network of users 220. Accordingly, the data collection and system control processor 210 may be configured to manage a switching of an interactive session in a communications network. The control processor 210 may also be configured to switch between the presentation of an application or service from one of multiple devices/screens to another, for instance between three screens such as a television, a computer, and a mobile device.
As shown in FIG. 2, the data collection and system control processor 210 includes a policy decision module 250 that makes decisions based on the collected data 230 obtained from the network 220. Based upon the collected data 230, the data collection and system control processor 210 may identify different system states 222, such as the state of the network, various states of network-related equipment such as routers and firewalls, various states of service-related equipment such servers, the state of a service provided to a user, even the state of a customer, or other more fine-grained managed object states. Then, an appropriate action may be taken. Once that decision is made it is communicated to policy enforcement functions 260 that can be distributed anywhere within the network. Accordingly, within a communications network, different screens, e.g., display device, may be used by a user to enjoy content provided over the communications network. For example, mobile devices such as personal digital assistants (PDAs), smart phones, mobile computers, etc. may be used to access content while a user is mobile. In contrast, at the office or at home, a user is more likely to
access content via personal computers, large flat-panel televisions, projection systems, etc. Policy decision module 250 may be configured to cause the different screens to work cooperatively to allow switching between screens based upon parameters such as the user’s locations, preferences, and other parameters. In general, such decisions may be arrived at via a suitable set of rules, algorithms, threshold checks, etc. as for instance may be embodied via software or hardware or some combination thereof.
FIG. 2 shows one exemplary embodiment. However, other embodiments may take the form of an entirely software embodiment or an embodiment containing both hardware and software elements. Exemplary embodiments may be implemented in software that includes, but is not limited to, firmware, resident software, microcode, etc. Furthermore, exemplary embodiments may take the form of a computer program product 290 accessible from a computer-usable or computer-readable medium 268 providing program code for use by or in connection with a computer or any instruction execution system.
For the purposes of this description, a computer-usable or computer readable medium 268 can be any apparatus that can contain, store, communicate, propagate, or transport the program for use by or in connection with the instruction execution system, apparatus, or device. The medium 268 may be an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system (or apparatus or device) or a propagation medium. Examples of a computer-readable medium include a semiconductor or solid-state memory, magnetic tape, a removable computer diskette, a random access memory (RAM), a read-only memory (ROM), a rigid magnetic disk and an optical disk. Current examples of optical disks include compact disk-read only memory (CD-ROM), compact disk-read/write (CD-R/W) and DVD.
A system suitable for storing and/or executing program code for providing data collection and system control according to an exemplary embodiment may include at least one processor 210 coupled directly or indirectly to memory elements 212. The memory elements 212 can include local memory employed during actual execution of the program code, bulk storage, and cache memories which provide temporary storage of at least some program code in order to reduce the number of times code must be retrieved from bulk storage during execution.
Accordingly, the computer program 290 comprise instructions which, when read and executed by the system 200 of FIG. 2, causes the processor 210 to perform the steps necessary to execute the steps or elements of exemplary embodiments.
Moreover, the functionality associated with the processor for providing data collection and system control 210 may be centralized or partially to fully distributed and included in any piece of equipment or any portion of software in the system 200. Accordingly, the data collection and control framework may be distributed across the system 200. Further, the data collection and control framework is configured as an overlay over the system infrastructure to offer any service to any user on any screen, and to be able to switch from one screen to the other. Still further, the control process may be automated, semi-automated, or manual.
FIG. 3 illustrates an example of a screen switch 300 according to an exemplary embodiment. In FIG. 3, a user of a mobile device 310 is shown at a location 350 away from, e.g., home 330. This position 350 is designated to be at time T1 340. The mobile device 310 of the user is showing displaying a first show 312, e.g., as illustrated by the hot-air balloons on the mobile device 310. At home 330, the family may be watching a travel show 332, as represented by a beach scene on the home television 334. At time T2 342, the user has arrived home as represented by the mobile device 310 positioned within the home 330. Using parameters set earlier, queries presented to the user when the user arrives at home, system default parameters or other criteria, various decisions are made by the user’s service provider.
A key parameter for determining whether to make a screen switch may be based on at least the new location of the user and the state of the mobile device 310 and the home television 334. In response to the decisions, the home television 334 is switched to the show 312 the user was watching on the mobile device 310 without the user missing any part of the show 312. The mobile device 310 may then be turned off. Network connections supplying the show 312 are controlled accordingly so as to support the switch from one screen to another, for instance to accomplish the re-routing of pertinent communications including signaling and data connections. Service-related aspects are likewise controlled.
Though a home is described and illustrated in FIG. 3 for illustrative purposes, it should be appreciated that embodiments are not limited to applications associated with a home or residence but may also include applications within other types of environments, e.g., an office.
FIG. 4 is a flow chart 400 illustrating the processor controlled switching of a presentation of content between media presentation screens according to an exemplary embodiment. First, a determination is made whether the timing of a screen switching is appropriate 410. Determining when switching is appropriate can be done at least in part via location measurement, e.g. detecting when the user’s cell phone moves away from or back to the user’s home, office, etc. If the time is not appropriate for a screen switch 412, the process returns to the start. If the time is appropriate for a screen switch 414, the system determines the “from” screen and candidate “to” screens 420. The feasibility and ramifications of candidate switching is analyzed 430. A determination is made whether the feasibility and ramifications meet acceptable standards 440. When the feasibility and ramifications meet acceptable standards 444, the determined screen switch is implemented 450. Otherwise 442, the process returns to the start.
The determination of the steps 420, 430, 440, 450 subsequent to determining when switching is appropriate 410 may be based upon information regarding the current active service, other active services which may be impacted including active services of other users, profiles and usage histories of the switching user and other relevant users, network capacity and utilization, and various policy rules, algorithms, parameters, heuristics, timers, and thresholds. Policy rules can be modified by the user, network provider and/or service provider so as to properly take preferences, needs, limitation, and contingencies regarding all parties into account. The impact that a switch will have on other users may also be considered.
FIG. 5 is a flow chart 500 illustrating collection of initial data for processor controlled switching of a presentation of content between media presentation screens according to an exemplary embodiment. Default preferences are provided and may be used when the user settings do not provide an override of the default preferences 510. Multiple default sets may be used, wherein such default sets may correspond to predetermined user profiles. A range of user preferences may be set when a user contracts for the service 520. Whether the users want to modify the initial settings, e.g., via web site, are determined 530. Preferences may include whether to switch, in what circumstances, at what locations, at what times/days, other users in a group, family or team and their priorities, priorities of various usage types, programs and applications,
etc. If the user wants to modify the settings 532, the process returns to allow such modifications. Otherwise 534, this portion of the process 500 is completed.
FIG. 6 is a flow chart 600 illustrating the processor controlled determination of when switching is appropriate according to an exemplary embodiment. User movement to, or away from, a location is monitored 610, e.g., home, office, lake cabin, hotel room, etc. For a mobile user, change in location may be used as a key indicator of a need to switch screens. The device/screen capabilities, such as features, size, etc. are also analyzed 620. Based on LBS (Location Based Service) location measurement, e.g., and the analyzed capabilities, a determination of when the switching is appropriate is made 630. If a switch is not now appropriated 632, the process returns to the beginning to continue to monitor the user 610. Otherwise 634, this portion of the process 600 is complete.
FIG. 7 is a flow chart 700 illustrating the processor controlled determination of a “from” screen according to an exemplary embodiment. In FIG. 7, the relative current screen movement is analyzed 710. A determination is made whether the current screen is moving to a fixed-screen location 720. When the current screen is moving to a fixed-screen location 722, the “from” screen is determined to be a mobile device screen 730.
When the current screen is not moving to a fixed-screen location 724, a determination is made whether the current screen is to be moving away from a fixed-screen location 740. When the current screen is moving away from a fixed-screen location 742, the “from” screen is determined to be a fixed screen at that location 750. Otherwise 744, the process 700 is complete.
FIG. 8 is a flow chart 800 illustrating the processor controlled determination of a “to” screen according to an exemplary embodiment. In FIG. 8, the relative screen movement location is analyzed 810. A determination is made whether the current screen is moving to a fixed-screen location 820. When the current screen is moving to a fixed-screen location 822, the “to” screen is determined to be the fixed screen at that location 830. When the current screen is not moving to a fixed-screen location 824, a determination is made whether the current screen is moving away from a fixed-screen location 840. When the current screen is moving away from a fixed-screen location 842, the “to” screen is determined to a mobile device screen 850. Otherwise 844, the process 800 is complete.
FIG. 9 is a flow chart 900 illustrating the processor controlled determination of the feasibility of candidate switching according to an exemplary embodiment. In FIG. 9, the network is analyzed for available bandwidth and/or resources corresponding to new data path 910. The application and/or service is analyzed 920, e.g., for capability, authentication, authorization. Based upon the analysis of the network and the application/services, the feasibility of candidate switching is made 930.
FIG. 10 is a flow chart 1000 illustrating the processor controlled analysis of the ramifications of candidate switching according to an exemplary embodiment. In FIG. 10, an identification of other potentially relevant users is made 1010. The identification of other potentially relevant users is made using the database, profile, and preferences that are stored. An analysis of the impact on the identified other potentially relevant users is performed 1012. The impact on other users may depend on what service other users are using, what device the other users are using, how much associated resources are the other users using, e.g., HDTV versus SDTV, and what is their priority.
A determination is made whether the identified other potentially relevant users are unaffected or whether the impact meets predetermined criteria 1020. When the identified other potentially relevant users are deemed unaffected or the impact meets predetermined criteria 1022, the ramifications are deemed acceptable 1030. When the identified other potentially relevant users are affected or the impact does not meet predetermined criteria 1024, the priority levels of the affected users are compared 1040. The characteristics of the other usage also influences the switching decisions, e.g., is there competing use, is the same content already being watched, is the appropriate device already on or does the device need to be turned on. The priority of the other usage is also analyzed 1050 via identification of usage, where possible, e.g., application/service being used, specific usage such as watching a particular television show, news program, or flipping channels, etc. The priority of other usage may also be determined via a database of usage history, e.g., random usage tends to indicate low priority, whereas religiously watching a particular show and never missing that show tends to indicate a high priority usage. Based upon the analysis of the other potentially relevant users, whether those users are affected and whether the other usage is determined to be a priority usage, the ramifications of candidate switching is ascertained 1060.
FIG. 11 is a flow chart 1100 showing the processor controlled implementation of the candidate switching according to an exemplary embodiment. In FIG. 11, the preferences and rules are reviewed 1110. When the limits defined by the preferences and rules so indicate, the user may be queried to verify and allow the candidate screen switch 1120. A determination is made whether the feasibility and ramifications allow the candidate switch 1130. When the feasibility and ramifications allow the candidate switch 1134, the candidate screen switch is performed 1140. If not 1132, the process 1100 is complete. After the candidate screen switch is performed 1140, a command is sent to the network, and/or to associated equipment such as routers, switches, firewalls, gateways border controllers, etc., to implement the candidate switch 1150. Commands are sent to applications and service, and/or to associated equipment such as servers, proxies, databases, etc., to implement the candidate switch 1160. Exception handling is provided to rollback the switch only when necessary 1170. Errors may be detected. Moreover, user exception triggers, undo commands or go back commands may be issued automatically and/or by the user to indicate a desire to revert to the last working situation.
The above specification, examples and data provide a complete description of the manufacture and use of the composition of the invention. Since many embodiments of the invention can be made without departing from the spirit and scope of the invention, the invention resides in the claims hereinafter appended.
We claim:
1. A device comprising:
- a controller; and
- memory for storing collected network data, the memory comprising executable instructions that when executed by the controller cause the controller to effectuate operations comprising:
- analyzing the collected network data;
- monitoring a location of a mobile device, to or from which a presentation of content is to be switched, within a communications network;
- detecting a change in the monitored location of the mobile device;
responsive to detecting the change in the monitored location of the mobile device;
determining a relative direction of motion of the mobile device relative to another device;
based on the determined relative direction of motion of the mobile device, determining an appropriate time for switching the presentation of content;
identifying others that will be impacted by switching the presentation of content;
determining an impact on the others of the switching of the presentation of the content;
determining a ramification on the others of the switching of the presentation of the content;
identifying a change in a system state within the communications network;
determining active services other than the current active service being provided to the user that may be impacted by the determined switch;
determining network capacity and utilization;
based on the determined relative direction of motion, the determined appropriate time, the collected network data, the determined impact, the determined ramification, the identified change in a system state, the determined active services that may be impacted, and the determined network capacity and utilization, determining whether to perform a switch of the presentation of the content from a first media presentation screen on the mobile device to a second media presentation screen on the other device, or from the second media presentation screen on the other device to the first media presentation screen on the mobile device; and
performing the determined switch of the presentation of the content to or from the first media presentation screen on the mobile device.
2. The device of claim 1, wherein the controller includes a policy decision module configured to make decisions based on the identified change in the system state.
3. The device of claim 1, the operations further comprising: analyzing whether the determined switch is feasible; and evaluating ramifications of the determined switch.
4. The device of claim 1, the operations further comprising performing at least one of determining a current active service being provided to a user, determining profiles and usage histories of the user involved with the determined switch, determining the active services of other users, determining profiles and usage histories of the other users, or determining policy rules, algorithms, parameters, and thresholds.
5. The device of claim 1, the operations further comprising preparing the first media presentation screen or the second media presentation screen for viewing by a user based upon analysis of the collected data.
6. The device of claim 1, the operations further comprising identifying a state of the communications network, a state of a service provided to a user, and a state of a customer based upon an analysis of the collected data.
7. The device of claim 1, wherein the collected data includes parameters set by a user.
8. The device of claim 1, the operations further comprising determining that the first media presentation screen or the second media presentation screen is appropriate for the switch based upon the determined change in the monitored location of the mobile device and an analysis of the collected data and communicate control information within the network.
9. The device of claim 1, the operations further comprising identifying the second media presentation screen as a fixed screen at a fixed-screen location when the mobile device is determined to be moving away from the fixed-screen location and identify the first media presentation screen to be a display on the mobile device, and wherein the controller is further configured to identify the first media presentation screen as the display on the mobile device when the mobile device is determined to be moving toward the fixed-screen location and identify the second media presentation screen as the fixed screen.
10. A computer-readable medium, wherein the computer readable medium is not a transient signal, the computer-readable medium having computer-executable instructions that when executed by a processor cause the processor to effectuate operations comprising:
storing collected network data;
analyzing the collected network data;
monitoring a location of a mobile device, to or from which a presentation of content is to be switched, within a communications network;
detecting a change in the monitored location of the mobile device;
responsive to detecting the change in the monitored location of the mobile device;
determining a relative direction of motion of the mobile device relative to another device;
based on the determined relative direction of motion of the mobile device, determining an appropriate time for switching the presentation of content;
identifying others that will be impacted by switching the presentation of content;
determining an impact on the others of the switching of the presentation of the content;
determining a ramification on the others of the switching of the presentation of the content;
identifying a change in a system state within the communications network;
determining active services other than the current active service being provided to the user that may be impacted by the determined switch;
determining network capacity and utilization;
based on the determined relative direction of motion, the determined appropriate time, the collected network data, the determined impact, the determined ramification, the identified change in a system state, the determined active services that may be impacted, and the determined network capacity and utilization, determining whether to perform a switch of the presentation of the content from a first media presentation screen on the mobile device to a second media presentation screen on the other device, or from the second media presentation screen on the other device to the first media presentation screen on the mobile device; and
performing the determined switch of the presentation of the content to or from the first media presentation screen on the mobile device.
11. The computer-readable medium of claim 10, the operations further comprising identifying parameters set by a user by analyzing the collected data.
12. The computer-readable medium of claim 10, the operations further comprising performing at least one of the following: determine a current active service being provided to a user, determine profiles and usage histories of the user associated with the determined switch, determine the active services of other users, determine profiles and usage histories of the other users, or determine policy rules, algorithms, parameters, and thresholds.
13. The computer-readable medium of claim 10, the operations further comprising:
analyzing whether the determined switch is feasible; and
evaluating ramifications of the determined switch.
14. The computer-readable medium of claim 10, the operations further comprising identifying the second media presentation screen as a fixed screen at a fixed-screen location and identify the second media presentation screen as a display on the mobile device when the mobile device is determined to be moving away from the fixed-screen location, and identify the first media presentation screen as the display on the mobile device and identify the second media presentation screen as the fixed screen when the mobile device is determined to be moving toward the fixed-screen location.
15. The computer-readable medium of claim 10, the operations further comprising determining that the second media presentation screen is appropriate for the switch based upon the determined change in the monitored location of the mobile device and the analysis of collected data and communicating control information within the communications network.
16. A method comprising:
storing collected network data;
analyzing the collected network data;
monitoring a location of a mobile device, to or from which a presentation of content is to be switched, within a communications network;
detecting a change in the monitored location of the mobile device; and
responsive to detecting the change in the monitored location of the mobile device:
determining a relative direction of motion of the mobile device relative to another device;
based on the determined relative direction of motion of the mobile device, determining an appropriate time for switching the presentation of content;
identifying others that will be impacted by switching the presentation of content;
determining an impact on the others of the switching of the presentation of the content;
determining a ramification on the others of the switching of the presentation of the content;
identifying a change in a system state within the communications network;
determining active services other than the current active service being provided to the user that may be impacted by the determined switch;
determining network capacity and utilization;
based on the determined relative direction of motion, the determined appropriate time, and the collected network data, the determined impact, the determined ramification, the identified change in a system state, the determined active services that may be impacted, and the determined network capacity and utilization, determining whether to perform a switch of the presentation of the content from a first media presentation screen on the mobile device to a second media presentation screen on the other device, or from the second media presentation screen on the other device to the first media presentation screen on the mobile device; and
performing the determined switch of the presentation of the content to or from the first media presentation screen on the mobile device.
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THE ROLE OF CELL-TO-CELL INTERACTIONS IN THE SUPPRESSION OF THE TRANSFORMED PHENOTYPE
Iain W. Foulkes
This thesis is submitted in part fulfilment of the degree of Doctor of Philosophy in the University of Glasgow
November 1996
Beatson Institute for Cancer Research, Faculty of Medicine,
CRC Beatson Laboratories, University of Glasgow,
Bearsden, Glasgow. Glasgow.
© I. Foulkes 1996
All rights reserved
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ABSTRACT
The growth and aberrant morphology of certain transformed cells can be suppressed when cultured in the presence of excess resting normal cells. This well established phenomenon has been correlated with the presence of heterologous gap junctional intercellular communication, (GJIC) between the two cell types and the inhibition observed may provide an important defence against cancer. This thesis describes a study into this potentially important form of growth inhibition.
For this project a new assay was developed which enabled this form of inhibition to be studied in detail. The transformed cells were transfected with a β-gal lineage marker to allow identification in mixed culture with normal cells. The proliferative status of both the normal and transformed cells was determined using a growth assay based on thymidine incorporation and analysis by autoradiography. A series of normal and transformed cell lines was selected with a range of homologous and heterologous communication phenotypes and these were used to assess the role of junctional communication in the inhibition phenomenon.
Standard focus assays, in which transformed cells are cultured with excess, growth arrested, normal cells, were performed to determine the extent of transformed cell growth inhibition by normal cells. The majority of foci which formed were markedly smaller than respective control colonies and this size suppression showed a positive correlation with heterologous communication. However, further analysis revealed that in many instances focus compression, mediated by the physical presence of the normal cells, significantly contributes to the explanation of focus size inhibition. Furthermore, the level of proliferation in some small foci was very high and would suggest that some transformed cells are lost from the culture dish due to high cell density coupled with a defect in cell-substrate adhesion. The results show that the use of focus size as an index of growth can generate inaccurate measurements of growth suppression.
Transformed cells can be inhibited by resting normal cells by a mechanism consistent with transfer of inhibition via gap junctions. However, the data appears to show that the presence of heterologous communication between normal and transformed cells is not necessarily sufficient for inhibition to occur and other factors may be involved. It was also shown that inhibition occurred in the absence of detectable communication.
The growth of normal cells can be significantly inhibited when they are co-cultured with excess transformed cells and this does not appear to be due to nutrient deprivation. The data suggests that a high proportion of the normal cells have competed with each other and with the transformed cells, which they recognise as an 'edge', for the amount of space they require to grow and divide, and as a consequence, become growth inhibited. It would appear that two different inhibition phenomena exist, one which may involve the transfer of inhibition from resting cells to growing cells and one which is, at least in part, due to contact-inhibition or the ability of cells to respond to the physical presence of other surrounding cells.
A higher proportion of normal cells (relative to controls) surrounding the majority of foci were found to be dividing. The level of growth stimulation decreased as distance from the focus periphery increased and there appeared to be a wave-like pattern to the distribution of stimulated cells. The stimulation may be caused by the disruption of the contact-inhibition mechanism, which
could be responsible for some of the inhibition observed in normal cell colonies surrounded by excess, transformed cells. This disruption may be caused by the foci expanding and the transformed cells competing for space on the culture dish. Normal cells in direct contact with the transformed cells, were less likely to be stimulated than cells 2-3 cells away, a pattern that may result from contact-inhibition by the transformed cells.
The inhibition of transformed cells by normal cells is more complex than is often suggested and more than one mechanism may be involved. A new inhibition phenomenon has been identified which appears to be mediated by contact-inhibition and may be related to the ability of cells to compete for space on the culture dish. Data from this study has highlighted the need to assess the growth of cells directly rather than using indirect growth indices such as focus size. The assay developed for this study provides detailed and accurate information on cell growth and could be used to look for an inhibition phenomenon *in vivo*.
for my family and Lindsey
# TABLE OF CONTENTS
| Section | Page |
|------------------------------------------------------------------------|------|
| Abstract | ii |
| Acknowledgements | xi |
| Abbreviations | xii |
| List of figures | xiv |
| List of tables | xv |
## CHAPTER ONE
### INTRODUCTION
1.1. **General Introduction.** 2
1.2. **Cell-cell interactions and their role in the determination and maintenance of cell phenotypes.** 4
1.2.1. The cell surface: a primary interface of all cell-cell interactions. 4
1.2.2. Secreted factors. 4
1.2.3. Juxtacrine interactions. 5
1.2.4. Extracellular matrix. 6
1.2.5. Cell-cell adhesion. 8
184.108.40.206. Cadherin-mediated cell-cell adhesion. 8
1.2.6. Gap junctional communication. 10
1.2.7. Summary of the cell-cell interactions involved in the determination and maintenance of cell phenotypes. 11
1.3. **Gap junctional intercellular communication.** 12
1.3.1. Gap junction structure. 12
220.127.116.11. Ductin. 12
18.104.22.168. Connexins. 13
1.3.2. The formation of gap junctions. 14
1.3.3. Gap junction permeability. 14
1.3.4. The regulation of GJIC. 15
22.214.171.124. Cyclic AMP. 16
126.96.36.199. Calcium. 17
188.8.131.52. Phorbol esters. 18
184.108.40.206. Retinoids. 18
220.127.116.11. Adhesion-mediated regulation of GJIC. 19
18.104.22.168. Other effectors of GJIC. 20
| Section | Title | Page |
|---------|----------------------------------------------------------------------|------|
| 1.4.5 | The role of signalling via gap junctions in normal growth and development. | 21 |
| 22.214.171.124 | Communication compartments | 23 |
| 126.96.36.199 | Gap junction specificity | 23 |
| 188.8.131.52.a | Connexins and specificity | 24 |
| 184.108.40.206.b | Cadherins and specificity | 25 |
| 1.4.6 | Gap junctional communication and cancer | 25 |
| 220.127.116.11 | GJIC during multistage carcinogenesis | 27 |
| 18.104.22.168 | Homologous and heterologous communication during carcinogenesis | 29 |
| 1.5 | Embryonal carcinoma cells: an *in vivo* example of extracellular, environmental growth control. | 30 |
| 1.5.1 | General overview | 30 |
| 1.5.2 | Summary | 31 |
| 1.6 | Suppression of the transformed phenotype by normal cells | 32 |
| 1.6.1 | Mechanisms of suppression | 33 |
| 1.7 | Summary and aims | 35 |
# Chapter Two
## Materials & Methods.
| Section | Title | Page |
|---------|----------------------------------------------------------------------|------|
| 2.1. | Materials. | 37 |
| 2.1.1. | Chemicals. | 37 |
| 2.1.2. | Kits. | 38 |
| 2.1.3. | Water. | 38 |
| 2.1.4. | Equipment and Plasticware. | 39 |
| 2.1.5. | Antiserum. | 40 |
| 2.1.6. | Plasmids and bacterial host. | 41 |
| 2.1.7. | Cell Culture Materials. | 41 |
| 2.1.8. | Cell lines. | 41 |
| 2.2. | Tissue Culture Techniques. | 42 |
| 2.2.1. | Cell maintenance. | 42 |
| 2.2.2. | Measurement of growth parameters. | 42 |
| 22.214.171.124.| Determination of cell viability. | 42 |
| 126.96.36.199.| Population doubling time. | 42 |
| 188.8.131.52.| Saturation density. | 42 |
| 184.108.40.206.| Growth in low serum. | 43 |
| 2.2.3. | Transfection of cell lines with a β-gal expression vector. | 43 |
| 220.127.116.11.| Ring cloning of resistant colonies. | 43 |
| 2.2.4. | Preparation of conditioned media. | 44 |
| 2.2.5. | Frozen cell stocks. | 44 |
| 2.3. | Gap junctional communication assays. | 44 |
| 2.3.1. | Dye transfer. | 44 |
| 2.3.2. | Nucleotide transfer | 44 |
| 2.4. | Focus formation assay. | 45 |
| 2.5. | Proliferation assay. | 46 |
| 2.5.1. | Staining procedure for cells expressing the β-gal lineage marker. | 46 |
| 2.5.2. | Autoradiography of cells exposed to [³H]-uridine or [³H]-thymidine. | 47 |
| 2.6. | Apoptosis assay. | 47 |
| Section | Title | Page |
|---------|----------------------------------------------------------------------|------|
| 2.7 | Nucleic acid procedures | 48 |
| 2.7.1 | Growth, transformation and storage of competent cells | 48 |
| 18.104.22.168 | Large scale plasmid preparations (Qiagen) | 49 |
| 2.7.2 | Determining nucleic acid concentration | 49 |
| 2.7.3 | Agarose gel electrophoresis | 49 |
| 2.7.4 | Extraction of RNA from mammalian cells | 50 |
| 22.214.171.124 | mRNA purification | 50 |
| 2.7.5 | Northern Analysis | 51 |
| 126.96.36.199 | Radiolabelling of cDNA probes | 51 |
| 188.8.131.52 | Northern blots | 52 |
| 2.8 | Protein procedures | 52 |
| 2.8.1 | Protein preparations from mammalian cells | 52 |
| 2.8.2 | Liver Preparation for connexin 32 & 26 positive controls | 53 |
| 2.8.3 | Polyacrylamide gel analysis of proteins | 53 |
| 2.8.4 | Western blotting analysis | 54 |
| 2.8.5 | Immunofluorescence | 55 |
# Chapter Three
## Results
| Section | Title | Page |
|---------|----------------------------------------------------------------------|------|
| 3.1 | Introduction. | 57 |
| | Experimental system and selection of cell lines for study. | 57 |
| 3.2 | THE GROWTH AND COMMUNICATION PHENOTYPES OF THE CELL LINES. | 60 |
| 3.2.1 | Growth phenotypes of cell lines under study. | 61 |
| 3.2.2 | Communication Phenotypes. | 61 |
| 184.108.40.206 | Homologous communication. | 61 |
| 220.127.116.11 | Heterologous communication between normal and transformed cells. | 63 |
| 3.2.3 | Expression of gap junction associated proteins. | 65 |
| 18.104.22.168 | Connexin43 expression. | 65 |
| 22.214.171.124 | Cadherin expression. | 67 |
| 126.96.36.199 | NCAM expression. | 67 |
| 3.2.4 | Summary. | 68 |
| 188.8.131.52 | Relationship between Cx43, cell-cell adhesion and communication. | 68 |
| 184.108.40.206 | Gap junction specificity. | 69 |
| 3.3 | THE FOCUS FORMING ABILITY OF THE TRANSFORMED CELLS. | 71 |
| 3.3.1 | Introduction. | 71 |
| 3.3.2 | The effect of normal cells on focus number. | 72 |
| 3.3.3 | The effect of normal cells on focus size. | 73 |
| 3.3.4 | The effect of conditioned media on normal and transformed cell growth.| 75 |
| 3.3.5 | The effect of focus size on the focus forming efficiency of the transformed cells. | 76 |
| 220.127.116.11 | The effect of normal cells on focus number after 4, 8 and 12 days when both cell types are plated simultaneously. | 78 |
| 18.104.22.168 | The effect of normal cells on focus size after 4, 8 and 12 days when both cell types are plated simultaneously. | 78 |
| 22.214.171.124 | The effect of normal cells on focus size when the transformed cells are pre-established for 2 and 4 days. | 79 |
| 3.3.6 | Summary. | 82 |
3.4. CELL-CELL INTERACTIONS BETWEEN THE NORMAL CELL MONOLAYER AND TRANSFORMED FOCI.
3.4.1. Introduction. 84
3.4.2. Does focus compaction contribute to the difference in size between foci and control colonies? 85
3.4.3. Inhibition of cell division within the foci. 88
3.4.4. Summary. 90
3.4.5. The effect of the normal cell monolayer on focus morphology. 92
3.4.6. The effect of transformed cells on surrounding normal cells. 94
126.96.36.199. The effect of expanding foci on normal-cell density. 94
188.8.131.52. Apoptosis in control and co-cultures. 95
184.108.40.206. Do transformed foci affect the proliferation rate of the normal cells? 97
220.127.116.11. Is there a pattern to the stimulation of Rat2 cells surrounding foci? 99
3.4.7. The affect of excess transformed cells on colonies of normal cells. 101
18.104.22.168. Summary. 105
CHAPTER FOUR
DISCUSSION
4.1. Introduction. 108
4.1.2. Current hypotheses regarding the role junctional communication in the inhibition phenomenon. 109
4.1.3. The interpretation of focus formation data. 111
4.1.4. Do the data obtained in this study fit the current working hypothesis? 112
4.1.5. Other mechanisms which may mediate growth inhibition. 114
22.214.171.124. Density-dependent regulation of cell growth (contact-inhibition). 114
126.96.36.199. The role of cadherins and connexins in the inhibition phenomenon. 116
188.8.131.52. Inhibition mediated by transmembrane bound glycoproteins. 117
4.1.6. The stimulation of normal cells surrounding transformed foci. 117
184.108.40.206. Possible mechanisms responsible for the stimulation of the normal cells. 118
4.1.7. Summary. 120
REFERENCES 121
Acknowledgments
I would particularly like to thank Dr. John Pitts for all his help and supervision. I would also like to thank all members of R5, past and present, for their help in the lab and Professor F. Tato for the cell lines. I would like to thank my parents, family and friends for their help and encouragement over the course of the project. Special thanks to Lindsey for putting up with me and putting me up. Finally I would like acknowledge the financial support of the Medical Research Council and Cancer Research Campaign.
Declaration: The work described in this thesis was performed personally unless otherwise acknowledged.
| Abbreviation | Description |
|--------------|-------------|
| β-gal | β-galactosidase |
| BSA | Bovine serum albumin |
| Ca++ | Calcium |
| cAMP | 3', 5'-cyclic adenosine mono-phosphate |
| CDK | Cyclin dependent kinase |
| CM | Conditioned medium |
| Cx | Connexin |
| DEPC | Diethyl pyrocarbonate |
| DMEM10% | Dulbecco's Modified Eagle Medium supplemented with 10% FCS |
| DMSO | Dimethylsulfoxide |
| EC-cells | Embryonal carcinoma cells |
| ECL | Enhanced chemilluminescence |
| ECM | Extracellular matrix |
| EDTA | Ethylenediaminetetra-acetic acid |
| EGF | Epidermal growth factor |
| FAK | Focal adhesion kinase |
| FCS | Foetal calf serum |
| FITC | Fluorescein-isothiocyanate |
| G418 | Geneticin |
| GAPDH | Glyceraldehyde-3-phosphate dehydrogenase |
| GJIC | Gap junctional intercellular communication |
| HEPES | N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesulfonic acid] |
| hp | Horseradish peroxidase |
| Ig | Immunoglobulin |
| IP₃ | Inositol triphosphate |
| kDa | Kilodalton |
| LI | Labelling index |
| MAb | Monoclonal antibody |
| MAP kinase | Mitogen activated protein kinase |
| MOPS | 4-morpholinopropanesulfonic acid |
| MW | Molecular weight |
| NCAM | Neural cell adhesion molecule |
| PAG | Polyacrylamide gel |
| PBS | Phosphate buffered saline |
| PDGF | Platelet derived growth factor |
| SatD | Saturation density |
| Sd | Standard deviations |
| TPA | 12-O-tetradecanoylphormol-13-acetate |
| TdT | Terminal deoxynucleotidyl transferase |
| Abbreviation | Description |
|--------------|-------------|
| [3H]-TdR | Tritiated thymidine |
| TUNEL | Terminal deoxynucleotidyl transferase nick end labelling |
| v/v | Volume to volume |
| w/v | Weight to volume |
| X-gal | 5-Bromo4-Chloro3-Indoyl b-D-Galactopyranoside |
| TGF | Transforming growth factor |
| TK | Thymidine kinase |
| Figure | Description | Page |
|--------|-----------------------------------------------------------------------------|------|
| 3.1 | Schematic representation of the proposed gap junction connexon. | 13 |
| 3.2 | Examples of homologous dye and nucleotide transfer. | 62 |
| 3.3 | Analysis of heterologous communication using fluorescent beads as lineage markers. | 63 |
| 3.4 | Examples of heterologous nucleotide transfer spreads. | 64 |
| 3.5 | Western blot and immunofluorescence analysis of Cx43 expression. | 67 |
| 3.6 | Western blot and immunofluorescence analysis of E cadherin expression. | 67 |
| 3.7 | Western blot and immunofluorescence analysis of NCAM expression. | 67 |
| 3.8 | A comparison of two staining techniques, used to identify foci and colonies of transformed cells. | 71 |
| 3.9 | Representative photographs showing the focus forming ability of the transformed cells on a background of either Rat2, R24E or 10T1/2 cells. | 74 |
| 3.10.a | Focus forming ability of transformed cells when plated at the same time or before the normal cells. | 81 |
| 3.10.b | Colony forming ability of transformed cells - control cultures for Figure 3.10.a. | 81 |
| 3.11 | Average colony and focus size. | 87 |
| 3.12 | Cell density within foci and control colonies. | 87 |
| 3.13 | Cell density within foci and control colonies. | 87 |
| 3.14 | Labelling index of transformed cells within colonies and foci. | 91 |
| 3.15 | Levels of proliferation within foci and control colonies. | 91 |
| 3.16 | The effect of normal cells on focus morphology. | 93 |
| 3.17 | Cell density in separate cultures of Rat2 and 10T1/2 cells. | 94 |
| 3.18 | Normal cell density next to and away from the focus periphery. | 94 |
| 3.19 | The effect of transformed cells on normal cell density and morphology. | 94 |
| 3.20 | Rat2 apoptosis levels in focus formation assays. | 96 |
| 3.21 | The level of proliferation within separate cultures of Rat2 and 10T1/2 cells. | 98 |
| 3.22 | Labelling index of normal cells next to and away from focus periphery. | 98 |
| 3.23 | Stimulation of normal cells by transformed foci. | 98 |
| 3.24 | Analysis of normal cell labelling pattern. | 99 |
| 3.25.A | The pattern of Rat2 cell growth stimulation surrounding S180 foci. | 99 |
| 3.25.B | The pattern of Rat2 cell growth stimulation surrounding S180E217 foci. | 99 |
| 3.25.C | The pattern of Rat2 cell growth stimulation surrounding S180NII foci. | 99 |
| 3.25.D | The pattern of Rat2 cell growth stimulation surrounding BICR foci. | 99 |
| 3.26 | Patterns of Rat2 cell division next to transformed foci. | 99 |
| 3.27 | Cell-cell interactions between excess transformed cells and normal cell colonies. | 102 |
| Table | Description | Page |
|-------|-----------------------------------------------------------------------------|------|
| 3.1 | Growth parameters of cell lines | 60 |
| 3.2 | Homologous GJIC | 62 |
| 3.3 | Heterologous GJIC | 64 |
| 3.4 | Summary of endogenous and exogenous expression of gap junction associated proteins | 67 |
| 3.5 | Co-culture combinations for focus formation assays | 71 |
| 3.6.a | The effect of normal cells on focus number | 74 |
| 3.6.b | The effect of normal cells on focus size | 74 |
| 3.7 | Correlation analysis: Level of communication vs inhibition of focus size and focus number | 74 |
| 3.8 | The effect of normal- and transformed-cell conditioned media on cell growth | 75 |
| 3.9 | Time points and nomenclature used in the focus formation assays | 77 |
| 3.10.a| The effect of normal cells on focus number after 4, 8 and 12 days when both cell types are plated simultaneously | 81 |
| 3.10.b| The effect of normal cells on focus size after 4, 8 and 12 days when both cell types are plated simultaneously | 81 |
| 3.11 | The effect of normal cells on focus size, when transformed cells are pre-established for 2 days | 81 |
| 3.12 | The effect of normal cells on focus size, when transformed cells are pre-established for 4 days | 81 |
| 3.13 | The effect of excess S180NII cells on the growth of Rat2 cells | 102 |
| 3.14 | The effect of excess S180E217 cells on the growth of Rat2 cells | 103 |
| 3.15 | The effect of excess BICR cells on the growth of Rat2 cells | 104 |
CHAPTER ONE
INTRODUCTION
1.1. General Introduction.
The evolution of eucaryotic cells is defined by the development of a nucleus and the diversification of cellular organelles. Cells increased in complexity as the size of the nuclear genome increased which, together with genetic recombination, permitted cells to express their hereditary information in many different ways. The onset of multicellularity allowed for cellular diversification and specialisation which ultimately led to the development of distinct tissues and organs. A principle advantage bestowed on a multicelled organism is its ability to exploit resources, in a way that no single cell can. This is ultimately achieved by the co-operation and co-ordination between the cells which form the organism.
Co-ordination between the cells of a multicellular organism is fundamental to its development and success within its environment. This is achieved by various communication mechanisms which are employed to varying degrees at different stages of development. Essentially three modes of communication amongst animal cells have been discovered to date:
1). Signalling by secreted factors; these signals effect cells close to or distant from the signal source and include endocrine, paracrine and synaptic signalling (Alberts et al 1989 for review).
2). Juxtacrine or adhesion-mediated signalling, in which certain cells displaying membrane-bound growth factors or adhesion receptors bind to and influence other cells displaying appropriate ligands on their cell surface (Rosenburg & Massague 1993)
3). Gap junctional intercellular communication (GJIC) in which cells are connected (coupled) by transmembrane channels that allow the movement of small molecules less than 1kDa in size. Thus cells within a tissue or organ are able to give a co-ordinated response to a signal which only a few cells may have initially received.
Cohesion between the communicating cells is critical and is maintained by a series of cell-cell junctions such as tight junctions (which serve as a physical barrier responsible for separating interstitial cells from other cells within a tissue), adherens junctions, desmosomes in vertebrates and septate junctions in invertebrates.
Although the communication mechanisms outlined above vary in terms of distance, type of signalling molecule and mechanics of signal transmission they are all closely linked in terms of achieving cellular co-ordination and in relationship to each other. Effector molecules such as hormones and growth factors induce cascades of cytoplasmic events following initial signal transduction (Alberts et al 1989). The second messengers such as IP$_3$ and Ca$^{++}$ which are activated during growth factor stimulation can rapidly spread through gap junctions resulting in a co-ordinated response to a given stimulus (Saez et al 1989, Stauffer et al 1993). Juxtacrine and GJIC mediated signalling are very localised and require cell to cell adhesion, they therefore provide a means of
integrating responses to more distant signalling systems; although at present poorly understood, such interactions may prove to be important in tissue morphogenesis.
Of particular importance in development are the mechanisms by which signals generated by one group of cells controls or determines the fate of another population of cells. In early embryonic development this process is termed embryonic induction and is one important mechanism for generating differences between cells. The existence of even two cells offers the potential for communication. If those two cells differ, for example, in their cytoplasmic constituents then the potential exists for inductive interactions to take place (Greenwald & Rubin 1995). In embryonic development interactions between mesenchymal and epithelial cells eventually leads to the subsequent development of different cells and tissues (Hay 1981). As the organism develops, interactions on a long range basis become important, these are mediated by the hormonal, neural and vascular systems. Throughout the developmental process the cell surface and associated extracellular matrix (ECM) assume pivotal roles in translating external signals into altered gene activity (Gimond & Aumailley 1992). Adhesive interactions between cells and the ECM are crucial in many stages of embryonic morphogenesis (for review see Juliano 1993a) and ultimately lead to the translation of basic genetic information into complex three-dimensional tissues and organs.
Mediated by the types of cell-cell interactions described above, the extracellular environment can exert a profound effect on cell phenotype. Embryonal carcinoma cells provide a striking, *in vivo* example where environment influences gene expression. These malignant cells can undergo phenotypic reversion when placed in a normal tissue environment of a blastocyst. This phenomenon may provide a suitable analogy to the phenomenon observed by Stoker and others in which the aberrant growth of transformed cells is abrogated when cultured with excess normal cells (Stoker 1967; section 1.6). The potential role of gap junctional communication as a mediator of this growth control is considered below. A detailed review of the role of GJIC in normal growth and developmental systems is provided followed by an examination of the evidence concerning its part in the process of carcinogenesis. Finally, gap junctional communication is considered as a potential mechanism for regulating cellular phenotypes within the context of the Stoker observations (Stoker 1967; see above). The aims of the project are provided in section 1.7.
1.2. Cell-cell interactions and their role in the determination and maintenance of cell phenotypes.
1.2.1. The cell surface: a primary interface of all cell-cell interactions.
The cell surface is the first point of contact for many of the extracellular signals responsible for influencing, to a large extent, the phenotype of a particular cell. Such signals may be hormonal, ionic, the proximity of other cells, cell shape and ECM products. Included in the cell surface are structures which link cells together for example adhesion molecules and gap junctions, their role in influencing cell phenotype is discussed below.
1.2.2. Secreted factors.
There is a wide variety of secreted factors involved in the regulation and development of cells. They include hormones and other diffusible factors (morphogens). Hormones may bind to a receptor on the cell surface and induce a cascade of secondary signalling events. Others pass through the membrane and influence gene expression more directly e.g. steroid hormones. Many diffusible factors are involved in inductive signalling (a signal emanating from one cell which controls gene expression in a responding cell), for example, the TGF, HGF/SF, Hedgehog, FGF and Wnt families of secreted proteins. These are thought to be of particular importance during embryonic induction (Jessel & Melton 1992) and mesodermal differentiation (Slack et al 1987, Asashima et al 1990, Christian et al 1991, Ingam 1994, Joannou et al 1995, Bradley & Brown 1995). Longer-range inductive signals involving the above molecules are thought to be responsible for the control of patterning in several embryos. However, the way in which they achieve such a distant organising activity is unknown. There are currently two schools of thought: 1) They function as gradient morphogens whereby similar cells respond in distinct ways to different concentrations of the same signal (reviewed by Wolpert 1989, Cooke 1995). 2) They act as short range inducers initiating sequential secondary signals or relay systems (Zecca et al 1995). Much of the current data available supports the second hypothesis, however, recent papers by Nellen et al (1996) and Lecuit et al (1996) identified a gene, (decapentaplegic - dpp gene) responsible for secreting a factor which exerts a long range influence on anterior and posterior compartments of the developing drosophila wing. Furthermore, they show that different genes respond to different concentrations of the DPP protein - endorsing the morphogen gradient concept.
Retinoids represent a major class of non-peptide growth factor signals and have been implicated in several developmental and differentiation pathways including neural and mesodermal tissue differentiation (Strickland & Mahdavi 1973, Glass et al 1990, Helms et al 1996). The developing chick limb has been extensively used as an experimental system for studying retinoic acid (RA) action on gene expression. (see
Much work has focused on events that occur at stage 18 and beyond when a distinct limb bud is present (Tabin 1995). However, limb buds are specified as early as stages 8 and 11 in chick embryos and appear in response to signals secreted from the lateral plate mesoderm (Cohn et al 1995 and Mahmood et al 1995). Two regions of the limb primordium are essential for the outgrowth and subsequent patterning of the chick limb bud, the zone of polarising activity (ZPA) and the apical ectodermal ridge (AER). It has recently been discovered that retinoic acid is a primary signal required for the establishment of the ZPA in these early stages of limb development (Helms et al 1996).
The ECM is known to be involved in the control of the diffusion of secreted factors throughout the developing organism. ECM components may reversibly bind soluble molecules and in doing so control their rate of migration through the cell population. Alternatively, ECM components such as proteoglycans may permanently bind secreted proteins as a means of localising high concentrations of specific factors such as FGF and TGFβ (Klagsburn & Baird 1991). The difficulty in detecting secreted Wnt proteins is thought to be due to rapid binding to heparan sulphate proteoglycans, (Hinck et al 1994). Binding to the ECM may also enhance or even activate several of these growth factors, indeed the association of FGFβ with ECM proteoglycans stimulates muscle cell differentiation (Rapraeger et al 1991).
There are a myriad of secreted factors which have been identified, many of which are intimately involved in gene regulatory activity which is manifest in a complex array of growth and differentiation control pathways. Many secreted factors are also involved in the regulation of other cell-cell interaction mechanisms and have been discussed further in the summary of this section 1.2.7.
1.2.3. Juxtacrine interactions.
Interactions between certain membrane bound proteins on apposing cell membranes can give rise to both adhesive and signalling events. Such processes have been termed juxtacrine signalling (Massague 1990). Although in many instances the membrane-bound factors can be proteolytically converted into soluble active forms, the cleavage represents conversion from one active form to another. Several juxtacrine molecules have now been identified and include: TGF-α, EGF, TNF-α, CD-21 (for references see Massague 1993). A well studied example is that of development of the R7 precursor cell to the R7 neuron in the compound eye of *Drosophila* which occurs after an inductive interaction with the photoreceptor R8 (Tomlinson & Ready 1986). Adhesive interactions between the R8 ligand, (Boss) and the R7 receptor, (sevenless) was demonstrated by the heterotypic aggregation of only cells expressing those structures (Kramer et al 1991).
Data from *Drosophila*, *C. elegans* and vertebrates suggest that intercellular signalling by the Notch pathway plays a pivotal role in the differentiation of uncommitted
cells. The Notch / LIN-12 / GIP-1 receptor family have been implicated in a general mechanism of local cell signalling which mediates the specification of numerous cell fates during development (Heitzler & Simpson 1991, and reviewed in Tsakonas et al 1995). Notch of *Drosophila* is a 300kDa transmembrane receptor protein with 36 extracellular EGF-like tandem repeats. The putative ligands for Notch, which are membrane anchored and include *serrate* and *delta* also contain variable numbers of EGF-like repeats. Cell aggregation assays show that *delta* and *serrate* bind to the extracellular EGF regions of Notch, which is subsequently activated upon binding. Genetic and molecular analysis have revealed that Notch signalling events do not involve the transmission of specific developmental signals, but modulate the ability of cells to respond to such signals.
In general, cell adhesion systems are responsible for maintaining tissue integrity and overall tissue architecture. It has also been suggested that classical adhesion molecules, which link the cytoskeleton of apposing cell membranes and their associated proteins, couple physical adhesion to signalling events during morphogenesis. Because of their potential importance in influencing cell phenotype they are discussed in their own right in section 1.2.5.
### 1.2.4. Extracellular matrix.
The ECM is an important component of the extracellular environment. It is composed of proteoglycans, glycoproteins and glycosaminoglycans. ECM is present at the 2-cell stage of mammalian embryos and is a component of the environment of all cell types. *In vivo* cells attach to the basement membrane (a distinct sheath of ECM surrounding many tissue types) or a collagen rich interstitial matrix. The basement membrane is composed of two distinct layers; basal- and reticular-lamina. Several adhesive glycoproteins have been found within the basal lamina including collagen IV, laminin and fibronectin (Mosher et al 1992). Reticular lamina, produced by fibroblasts, is composed primarily of fibrillar collagens. In addition to offering structural support to cells it can form a physical barrier or selective filter to soluble factors and it has been shown to play a role in regulating the differentiated phenotype of cells.
Evidence for the importance of ECM components in the normal development of organisms comes from mutations which affect ECM proteins and receptors and experimental perturbation studies. *Drosophila* development particularly, has provided a rich source of information. For example, mutations in laminin is lethal in late embryogenesis (Hortsch & Goodman 1991) whilst mutations in the *Scabrous* gene (whose gene product has significant homology to vertebrate fibrinogen) results in altered spacing patterns of R8 photoreceptor cells (Baker et al 1990). In vertebrates many genetic and autoimmune diseases are associated with defective ECM interactions (Olsen 1995). For example, blistering diseases of the skin, such as *Bullous pemphigoid*, result from a disruption of hemidesmosomes, (a cell-to-basement membrane junction linked to
cytokeratin intermediate filament network, the main adhesion receptor being integrin $\alpha_6\beta_4$ - see below).
In vertebrates fibronectin is produced when gastrulation begins and is abundant during morphogenetic migratory cell movements such as neural crest migration (Boucaut et al 1984). Gastrulation is inhibited when the blastocoel cavity is injected with anti-fibronectin antibodies. The examples provided so far are consistent with the ECM playing a structural role during development. However, there is now a substantial body of evidence to suggest that cell interactions with the ECM can regulate many aspects of cell behaviour including proliferation and differentiation. Certain ECM adhesion receptors on the surface of cells interact with cellular signal transduction apparatus.
The most prominent and to-date the best studied cell-to-ECM adhesion receptors are the integrins. Integrins are expressed by a wide variety of cell types and cells often express more than one type (Hynes 1992a). All integrins are $\alpha\beta$ heterodimers, each subunit varying in size between 120-180kDa and 90-110kDa respectively. There is increasing evidence that integrins are involved in the transfer of information into and out of cells (Hynes 1992a).
In culture integrin-mediated cell adhesion to the ECM results in the formation of specialised adhesion sites termed focal adhesions. Localised at these sites are both structural proteins such as $\alpha$-actinin, talin and paxilin and signalling proteins including focal adhesion kinase (FAK), cSrc and PKC. For this reason these adhesion sites have been described as "adhesion-dependent signal transduction organelles" (Lo & Chen 1994). Integrins have no known intrinsic enzymatic activity and are thought to mediate signalling events via their ability to assemble cytoskeletal frameworks such as the focal adhesion sites (Turner & Burridge 1991, Reszka et al 1992, LaFlamme et al 1992). Integrin mediated adhesion has also been shown to trigger the activation of lipid second messenger pathways via the activation of tyrosine kinase-dependent phospholipase C-$\gamma$ (Kanner et al 1993).
There are many examples where the ECM has been shown to affect gene expression (Adams & Watt 1993). In monocytes for example the ligation of $\beta_1$ integrins with anti-integrin monoclonal antibodies induces the same pattern of gene expression seen after complex adhesion with other ECM molecules such as fibronectin (Yurochko et al 1992). A further example of integrin-mediated gene regulation can be found in the production of metalloproteinases in synovial fibroblasts. $\alpha_5\beta_1$ integrin binds to a distinct domain on fibronectin, in doing so it activates the transcription of metalloproteinases. However, the signal is inhibited if an opposing $\alpha_4\beta_1$ integrin binds to fibronectin (Huhtala et al 1995).
Many normal cell types in culture require anchorage to underlying ECM in order to proliferate (a mechanism lost in many transformed cells). Integrins are thought to be involved in the regulation of adhesion-dependent cell proliferation. They are capable of activating mitogen-activated protein (MAP) kinases (Chen et al 1994), which in turn are
known to be involved in the regulation of cell proliferation (Davis 1993). Thus, activation of MAP kinase appears to be dependent upon integrin-mediated changes in cell adhesion and cell shape (Zhu & Assiain 1995). Integrin-mediated interactions allow cells to 'sense' their spatial relationship with neighbouring cells during stages of controlled morphogenesis, tissue growth and tissue repair.
The ECM plays an important role in several majors areas of tissue morphogenesis *in vivo*, including cell spreading, cell migration and epithelial compaction (Gumbiner 1995). In addition to regulating the structure and organisation of cellular architecture the ECM can transduce signals relating to growth and gene expression. It contributes to ensuring that cell differentiation takes place in the right place at the right time.
### 1.2.5. Cell-cell adhesion.
Cell-cell adhesion is required for the assembly, maintenance, phenotype and overall architecture of cells and tissues in addition to providing a mechanical framework to co-ordinate cell-cell interactions. Cell-cell adhesion is a complex process, the molecules involved are often multiprotein complexes and can be involved in adhesion and cell-cell signalling. The adhesion molecules and their associated proteins can be grouped in to 3 categories: i. transmembrane proteins, ii. extracellular proteins, many of which are ligands for the transmembrane proteins, iii. cytoplasmic plaque/peripheral membrane protein. Group i, the transmembrane proteins, include the following superfamilies: cadherins, integrins, immunoglobulins, selectins, proteoglycans (e.g. syndecans). Cadherin-based adhesion forms one of the most important cell-cell interactions. Through their homophilic binding properties cadherins mediate important events during morphogenesis including cell sorting, cell polarisation and tissue morphology. More recently it has been suggested that cadherins may be involved in intercellular signalling. This is thought to arise via associations with cytoplasmic plaque and cytoskeletal proteins such as the catenins. β-catenin for example, has been shown to be phosphorylated in response to growth factors and can in turn decrease cell-cell adhesion (Kinch et al 1995).
#### 220.127.116.11. Cadherin-mediated cell-cell adhesion.
Cadherins and their role in the formation of cell-cell adherens junctions represent one of the best studied adhesion systems. The cadherins are a family of cell surface membrane proteins which mediate calcium dependent homophilic cell-cell adhesion, they localise at points of cell-cell contact and are a principle component of adherens junctions or belt desmosomes (Hirano et al 1987) and are also thought to be involved in the formation of cell-matrix interactions (Geiger et al 1992). Two major sub-families have been identified: the classical cadherins which include, among others, E (epithelial), N (neuronal) and P (placental) cadherin (Takeichi 1988 & 1990) and desmosomal cadherins such as desmogleins and desmocollins (Buxton & Magee 1992). The latter
share conserved extracellular sequences with, and an overall structural similarity to the cadherins (Magee & Buxton 1991). Classical cadherins possess similar structural and functional domains, including sites for calcium binding, adhesive recognition, cytoskeletal interactions and post-translation phosphorylation sites. These proteins are believed to be responsible for the sorting phenomenon observed in culture, whereby cells expressing different cadherins segregate into homotypic populations (Nose et al 1988, Miyantani et al 1989). They are also responsible for mediating epithelial cell polarity (McNeill et al 1990, Watabe et al 1994, Marrs et al 1995). This process is fundamental to the development of a variety of tissues and organs (Rodriguez & Nelson 1989) and involves the redistribution of several important membrane proteins such as Na*K*-ATPase, to specific areas of the cell membrane (in this case basal-lateral).
It is generally believed that for functional adhesion to occur the cadherins must form associations with the intracellular cytoskeletal proteins - catenins (Gumbiner 1993), which themselves transduce signals received from the adhesion molecules. α-catenin links cadherins to the actin filament network (Rimm et al 1995). In PC9 lung carcinoma cells E cadherin is expressed but remains non-functional due to a lack of α-catenin expression (Watabe et al 1994). The re-expression of α-catenin induces a polarised cell phenotype and cell-cell association via the formation of cell-cell junctions (tight and adherens). Furthermore, the growth of the PC9 cells is retarded indicating a role for E cadherin-catenin associated growth control. β-catenin is believed to bind α-catenin to cadherin and is known to be involved in signal transduction (Kinch et al 1995). Post-translational disruption of the cadherin/catenin complex (rather than decreased expression levels) has been shown to be important in the process of dedifferentiation during epithelial transformation by the oncogene v-src (Behrens et al 1993); the transformed cells were subsequently rendered highly invasive. The effects of v-src were found to be associated with increased tyrosine phosphorylation of the β-catenin protein.
The broad function of cadherins in addition to their adhesive role was demonstrated by Herminston et al (1995). Using embryonic stem cells stably transfected with a dominant negative N cadherin mutant (NCADΔ), they were able to show that transfected cells poorly adhered to each other and to the ECM, their polarised phenotype was lost and they tended to apoptose with high frequency.
The development of the nervous system, in particular the neural crest, provides a useful system to study the role of adhesion in key morphogenetic events. Neural crest development begins with the dispersion, segregation and migration of cells. Following migration, the cells attach in various locations of the embryo and differentiate into neurons and the peripheral nervous system (Erickson and Perris 1993). When dispersion and migration begins N cadherin and N-CAM surface expression is lost (Thiery et al 1982) and cells show preferential associations with the ECM (Duband et al 1985). Following migration and reassociation into peripheral ganglia, the process is sequentially reversed and adhesion molecules such as N cadherin and a range of 'novel' cadherins are
expressed (Nakagawa & Takeichi 1995). The cadherin junctions which form are finely controlled by the co-ordinated activity of cellular kinases and phosphatases (Gavelle & Duband 1995)
It is clear that adhesion molecules do more than simply hold tissues and cells together; they provide a mechanism for integrating physical aspects of tissue morphogenesis with cell growth and differentiation. This role is reflected by the loss of adhesion proteins in many tumour cells and subsequent anchorage independent growth in culture and invasive potential *in vivo* (Behrens et al 1993, Birchmeier et al 1993). However, the re-expression of cadherin alone in such cells is often not sufficient to alter the tumorigenic phenotype of these cells (Navarro et al 1993) and this highlights the importance of integrated interactions with cytoskeletal proteins.
Several other types of cell-cell adhesion junctions are known to exist. They include desmosomes, found in epithelia and cardiac muscle which provide the mechanical strength required for the maintenance of tissue integrity and occluding junctions, i.e. tight junctions, which serve to regulate the permeability of the paracellular space between adjacent cells (Gumbiner 1987). Cadherins are also capable of regulating the formation of gap junctions and this is discussed in section 18.104.22.168.
### 1.2.6. Gap junctional communication.
Gap junctions occur in metazoan organisms, they are present at all times of development and lost only in some fully differentiated cells. They provide a potential mechanism for the distribution of developmental, positional and growth regulatory signals, though whether they perform such a role is still under investigation. Gap junctions are transmembrane channels which permit the passive transfer of cytoplasmic molecules, less than 1kDa in size throughout a coupled cell population. These molecules include ions and second messengers such as cAMP and Ca$^{++}$. As a result, they engender a cytoplasmic continuity (with regard to molecules less than 1kDa in size) within a coupled population. This continuity results in the establishment of a homeostatic pressure which is thought to be responsible for maintaining a homogenous cellular phenotype. Communication compartments (cells of the same compartment can communicate with each other but not with cells of other compartments) have been observed during embryogenesis and tissue development (Lo & Gilula 1979a, 1979b, Lee et al 1987, Nishi et al 1991 Kam & Hodgins 1992). Separation into sub-compartments is thought to be a pre-requisite for cells to express new phenotypes.
A developing tumour (essentially a differentiating, emerging cell population with an aberrant growth phenotype) must escape the growth control of the surrounding normal cells. Many published reports have recorded a loss of heterologous communication (communication between two different cell types or cell lines) between normal and transformed cells *in vitro* (Mehta & Loewenstein 1991, Kalami et al 1992,
Mikalsen 1993). A review of the evidence for the role of GJIC in growth, development and carcinogenesis is provided in section 1.4.5.
1.2.7. Summary of the cell-cell interactions involved in the determination and maintenance of cell phenotypes.
The various interactions described appear to play pivotal roles in the initiation and maintenance of cell differentiation. Needless to say the various mechanisms of cell-cell interactions outlined above do not operate independently. Cell differentiation and regulation of morphogenesis require, among other things, complex interactions between adhesion receptors, the cytoskeleton and networks of signalling pathways. Signalling pathways generated by integrin-mediated adhesion (and possibly cell-cell adhesion dependent signalling) are themselves influenced by extrinsic signals arising from classical growth factors. Gap junctions provide the apparatus for intercellular internal signalling pathways. These may be important for determining and maintaining the differentiated state either by suppressing individual phenotypes as a result of homeostatic pressure or by direct signalling molecules (as yet unidentified). GJIC is known to be regulated by cell-cell adhesion systems (Musil et al 1990, Hertig 1996), and modulated in response to various soluble factors such as the Wnt's and ECM components including proteoglycans and glycosaminoglycans (see section 1.3.4).
Cell adhesion interactions can be regulated by long-range signalling pathways, for example, β-catenin is thought to be a component of the Wnt signalling pathway in Xenopus embryos and may contribute to embryonic patterning (Peifer 1995). In mammalian cell culture systems Wnt-1 expression results in the accumulation of β-catenin and its associated protein - plakoglobin, leading to widespread and stronger cell-cell adhesion. Such interactions offer a potential mechanism for the specification of cell type boundaries (Hinck et al 1994).
Tissues are dynamic structures, chemical signals and cell-cell interactions result in altered gene expression and structural changes to cell morphology. A unifying theory of cell migration, proliferation and tissue development has been proposed. It is based on cellular changes that result from alterations in the extended cytoskeleton, (a continuous structural support network spanning from a cell's nucleus through its cytoskeleton, across its associated ECM via intercellular junctions and on into neighbouring cells). The end result is a physical integration of all cells and nuclei within tissues (Wang 1993, Ingber 1994). The role of mechanical forces generated by cell-cell interactions in tissue development is a growing field and is termed cellular tensegrity a full review can be found in Ingber et al (1994).
It has been shown in this section 1.3.4 that the cell phenotype is determined not only by the genes which a particular cell expresses but also by the environment in which it resides and the interactions it makes with other cells. In the following section the role of GJIC in growth and development of organisms is explored further.
1.3. Gap Junctional Intercellular Communication.
The evidence which supports a role for GJIC in development and cell growth control is mainly circumstantial - see section 1.4.5. The homeostatic pressure which arises due to communication within a coupled cell population would appear to suppress the expression of individual cell phenotypes. In order for cells to express new phenotypes they must first escape this growth control by forming new sub-compartments. Gap junctions are known to perform other roles, in excitable tissues for example. At electrical synapses GJIC facilitates rapid propagation of ionic current (Jaslove & Brink 1986), thus providing the means to regulate neuronal assemblies and the ionic environment in which neuronal activities occur (DeVries & Schwartz 1989). Within this section gap junction-mediated cell-cell interactions will be discussed and their role in growth control and the maintenance of cellular phenotypes investigated. However, some basic information on gap junction structure, formation and subsequent permeability is required, this is provided below.
1.3.1. Gap junction structure.
The gap junction channel is composed of two hemi-channels termed connexons. Each hemi-channel joins end-to-end with another, provided by the apposing membrane of another cell. Each connexon is composed of 6 sub-units, (each subunit is composed of 4-α helices), arranged symmetrically around an axial water-filled channel with a diameter of 1-2nm (Figure 3.1). There is controversy as to what protein is the principle channel component of the connexon (for review see Finbow & Pitts 1993). The debate revolves around two proteins, ductin and connexin (Cx).
22.214.171.124. Ductin.
Ductin is a 16kDa protein which is also a major component of vacuolar-ATPase proton channel (Mandel et al 1988) and, as such, is present in all cells regardless of whether they form gap junctions. It has been found in gap junction preparations from both vertebrate and invertebrate sources (Finbow et al 1984 and 1993). Ductin is a highly conserved membrane protein. Sequences obtained from plants, fungi, vertebrates and invertebrates share ~80% sequence homology. There is also little difference in the size of ductin obtained from such diverse sources (150-160 residues; Finbow et al 1992). Ductin is composed of 4 hydrophobic segments joined by short hydrophilic segments. In the connexon, each of the 4 hydrophobic segments are thought to traverse the membrane as an α-helix (Holzenburg et al 1993). Antibodies to invertebrate ductin bind to isolated gap junctions (Buultjens et al 1988) and gap junctional regions in tissue sections (Leitch & Finbow 1990). GJIC has also been inhibited by the injection of anti-ductin antibodies into mammalian and invertebrate cells (Finbow et al 1993). These data (for a more extensive review see Finbow & Pitts 1993) show ductin to be involved in gap junctional
communication and together with the structural studies (Holzenburg et al 1993, Girvin & Fillingham 1994), make duc tin a suitable and likely component of the gap junction channel.
126.96.36.199. Connexins.
Connexins are a family of proteins ranging in size from 26kDa-50kDa and are expressed in a cell specific manner in a variety of mammalian cells. Twelve members of the murine connexin gene family have been described (Willecke et al 1991) and they are expressed in one or more cell type and often more than one connexin is expressed (Dermietzel & Spray 1993). Although it is argued that these proteins form the gap junction channel no connexins have been identified in invertebrates despite the successful isolation of gap junctions from such organisms (Finbow et al 1984). In addition, functional analogies appear to exist between gap junctions and plant plasmadesmata, however, despite concerted efforts (Xu et al 1990, Yaholm et al 1991) no connexin homolog has been discovered (Mushegian & Koonin 1993).
There is currently little structural data available for these proteins and that which is, does not satisfactorily relate to the structure of the gap junction connexon as imaged by X-ray diffraction and AFM studies (Makowski 1988, Hoh et al 1991, Yeager & Nicholson 1996). The different connexins show homology over the N-terminal region (~20kDa) but show considerable variation in length and sequence over the C-terminal domain (Kumar & Gilula 1992). It has been proposed that the 4 hydrophobic domains of the N-terminal region span the membrane as $\alpha$-helices. Although this fits in with the proposed connexon structure the large variable C-terminal domains can not be accounted for. It has been suggested that this region is "disordered" and therefore can not be visualised but it seems unlikely that the extra mass which the C-terminal region accounts for (60% of Cx32 and 110% for Cx43) remains undetected even when analysed by various imaging techniques (Tibbets et al 1990, Sosinsky 1992).
Because many studies of connexins are based upon the supposition that they form the gap junction channel there has been little progress into the potential array of alternative functions which this large family of proteins may have. It has been shown that the formation and control of gap junctional communication is highly complex and is likely to be under the control of multiple gene products (Macdonald 1985). A wide variety of cell-cell interactions are known to influence GJIC (see section 1.3.4) and connexins may be the target of such diverse signalling pathways. Furthermore, they may be involved in generating the patterns of communication observed \textit{in vivo} (e.g. Pitts et al 1986), which change spatially and temporally during different stages of development. The putative role of the connexins in the generation of gap junction specificity leading to the establishment of communication compartments \textit{in-vivo} and in tissue culture is discussed in section 1.4.5.
Figure 3.1. Schematic representation of the proposed gap junction connexon
Hemi-channel subunit - ductin or connexin
Cell 1 plasmamembrane
Hemi-channel
Channel pore
Cell 2 plasmamembrane
Based on a diagram by Makowski et al 1977.
1.3.2. The formation of gap junctions.
Until the issue regarding the structure of the channel is settled there can be little progress made on how the gap junction forms. However, it is known that when gap junctions form they do so rapidly (within minutes) when cells are brought into close contact with each other (Rook et al 1988, 1990). The fact that this can occur in the presence of protein synthesis inhibitors (Rook et al 1990) suggests that channel precursors exist within the cell or at its surface membrane. If this is so then interaction of the two hemi-channels may cause the channel to open. Alternatively, the channel may form as a closed structure which is opened by the interaction of connexins or other, as yet unidentified, gap junction associated proteins on apposing cell membranes. Once the two hemi-channels have docked the channel is subject to gating mechanisms which can rapidly switch the channel between open and closed states (section 1.3.4).
It has been shown that the hemi-channels protrude by only 1-2nm into the extracellular environment (Hoh et al 1991), therefore, the membranes of the apposing cells must be brought into very close apposition before functional channels can form. Cell-to cell adhesion molecules are the most likely candidates to bring about initial close apposition followed by other, as yet unidentified molecules, which bring the membranes even closer together. Connexins may also be involved in bringing two hemi-channels into close apposition. However, the absence of these proteins in invertebrates despite the presence of functional gap junctions would suggest that the connexins play a more regulatory role in vertebrates and add further complexity to the gap junction structure not recognised by current models of the connexon.
1.3.3. Gap junction permeability.
Gap junction channels permit the passive diffusion, between cells, of molecules less than ~1kDa. This includes, ions such as Ca\(^{++}\), small cytoplasmic molecules including second messengers and metabolites such as nucleotides, (Pitts & Simms 1977, Neyton & Trautmann 1985, Saez et al 1989,) but excludes macromolecules such as nucleic acid, polysaccharides and large proteins. This metabolic co-operation can support metabolic activity within cells of a coupled population, when some of which, for some reason, lack a full range of such activities (Pitts 1980). Metabolic co-operation has been readily demonstrated in tissue culture systems; for example, cells deficient in hypoxanthine phosphoribosyl transferase (HPRT) are unable to make purine nucleotides from hypoxathine and cells which lack thymidine kinase (TK) are unable to make thymidine nucleotides from thymidine. When these two cell types are co-cultured in the selective medium, HAT, survival and growth is dependent on the mutual exchange of nucleotides and occurs only when the cells form heterologous gap junctions (Pitts 1971, Pitts & Simms 1977). Newly developing cancer gene therapies based on enzyme pro-drug systems exploit metabolic co-operation (in this case the transfer of toxic metabolites) as
a means of spreading the toxicity to cells not modified by the therapeutic gene (Bi et al 1993).
Gap junctions mediate electrical coupling within excitable tissues. In heart for example gap junctions mediate the propagation of the action potential resulting in a coordinated muscle contraction (Fawcett & McNutt 1969). Gap junctions are known to be permeable to several molecules involved in the growth and regulation of cells such as, cAMP, IP₃ and Ca²⁺. These findings have led to the proposal that GJIC is involved in cell differentiation in normal developmental pathways, see section 1.4.5.
There are essentially three methods used to measure gap junctional communication in culture. The first and most widely used is the microinjection of fluorescent dye (usually Lucifer yellow CH, with a MW of 440 and molecular radius of 0.7nm). A single cell within a monolayer is injected with the dye for a given time. If the population of cells is connected by gap junctions the dye diffuses readily into adjacent cells, demonstrating 'dye coupling'. Another type of assay demonstrates coupling by the transfer of labelled metabolites (and subsequent analysis by autoradiography). The assay is termed nucleotide transfer and was first used by Pitts & Simms (1977). This technique has biological relevance because it is concerned with the transfer of metabolites and is particularly amenable to the detection of gap junctional communication between cells of different types - heterologous communication. The third assay commonly used is the measurement of gap junctional conductance, where the extent of electrical coupling between cells is measured. This technique is often used in conjunction with the paired Xenopus oocyte system. This system permits direct measurements of cell-cell communication to be made and related to expression of specific connexins (Swenson et al 1989).
GJIC has been measured in intact tissues using the dye injection assay. After injection the tissue is fixed, sectioned and dye spreads visualised. Analysis of dye spreads in various tissues including skin and in mouse embryos led to the discovery of communication compartments (Pitts et al 1986, Kalimi & Lo 1988, see section 188.8.131.52).
1.3.4. The regulation of GJIC.
If GJIC is involved in the growth regulation and development of multicellular organisms then control of communication levels is likely to be critical. Gap junctional regulation may also be necessary during states of disease, injury or exposure to toxins. In such instances it is thought likely that normal undamaged cells would wish to isolate themselves from damaged ones to prevent the leakage of small cytoplasmic molecules. Gap junctional communication can be modulated at four levels:
1) Gating. This refers to the modification of channels whilst in the junction and involves switching between an open and closed conformational state. This is likely to be a fast means of control.
2). Modification of gap junction proteins during synthesis and transport to the plasmamembrane. This is likely to include phosphorylation and subsequent conformational changes of gap junction proteins.
3). Turnover. Evidence of such a regulatory mechanism is limited and rests on the observations of connexin-antibody-reactive material present in the vesicles proximate to the plasma membrane (Atkinson et al 1995), in addition to the relatively short half-life (5hrs) of connexins Cx26, Cx32 & Cx43 (Traub et al 1989, Laird et al 1991). Such short turnover times are rapid enough to regulate levels of gap junctions by control of synthesis.
4). Gene regulatory mechanisms. Many studies on GJIC regulation have concentrated on the effects of various modulators (see below). Therefore little has been discovered on how gap junction genes are regulated. Further genetic analysis may lead to a better understanding of the tissue specific expression of certain gap junction associated proteins such as the connexins (for further discussion see Kumar & Gilula 1992).
Rapid and often transient modulation of GJIC is thought to occur by junctional gating (Spray et al 1981, Giaume et al 1989, Veenstra 1990). Two models of gating mechanisms have been proposed. Firstly, one in which the cytoplasmic end of the channel is capped by some form of protein structure (Makowski et al 1984) and secondly one in which the connexon subunits twist along the channel axis, to open/close the channel - an action similar to that of the camera lens aperture (Unwin & Ennis 1984).
Junctional conductance and single channel conductance measurements (see Veenstra & Brink 1992 for methodology and discussion of technique) permit the proportion of open gap junctions to be estimated. The measurements vary depending on the sample, for example, ~1% of channels are open in the endings of Mauthner cell synapses (Lin & Faber 1988) whereas in the crayfish septate axon all junctions appear to be open in their native state (Zamphigi et al 1988). It has also been shown that individual channels can switch between open and closed conformations within milliseconds (DeHann 1988). Further structural analysis of the channel protein and connexon complex may provide further insight into how gating mechanisms may work.
Changes in junctional permeability or conductance can be induced by a variety of different agents, those thought to be of particular importance are listed below, however, in many instances the actual mechanisms by which they exert their effect are poorly understood.
184.108.40.206. Cyclic AMP.
The signalling molecule cAMP is known to be involved in the control of gap junctional permeability (Flagg-Newton et al 1981a & b, Saez et al 1986, Mehta et al 1992, Atkinson et al 1995). Cells generally respond to cAMP by increasing their levels of gap junctional communication (Saez et al 1986), there are however exceptions such as
horizontal cells from fish retina, where a decrease is recorded (Teranishi et al 1983). Atkinson et al (1995) showed that an increase in communication occurred within 30 minutes of continuous exposure to cAMP. Levels increased by four fold after a further 24 hours.
The upregulation of GJIC by cAMP appears to operate via more than one mechanism. For example several reports have shown that cAMP - upregulated gap junctional communication is sensitive to protein synthesis inhibitors (Traub et al 1987) and that connexin mRNA levels increase in parallel to the increased communication (Mehta et al 1992, Schiller et al 1992). Atinkinson et al (1995) has confirmed that an increase in the junctional permeability of mouse mammary tumour cells is associated with an increase in the number of gap junctions, as shown by freeze-fracture EM. However, it was found that Cx43 mRNA and Cx43 protein levels did not change. This would suggest that the formation of further channels is at the expense of the pool of Cx43 elsewhere within the cell.
The stimulation of cAMP-dependent protein kinase A (PKA) in response to elevated levels of cAMP (Taylor et al 1990), activates the transcription factor - CREB, (cAMP response element-binding factor), causing increased transcription of cAMP responsive genes. Sequences similar to the cAMP response element (CRE) are located near the transcription start site of Cx32 gene (Miller et al 1988). This may account for the increased expression of this protein observed by Traub et al (1987) when embryonic hepatocytes were stimulated with cAMP.
In several instances artificially increased levels of cAMP has resulted in a reduction in cell proliferation (Cho-Chung 1985). Thus, if gap junctions are involved in the spread of growth regulatory signals then the effect of cAMP may be to increase both the amount of regulatory molecules and the mechanism by which they can spread - a convergence of cellular signalling pathways.
220.127.116.11. Calcium.
Ca\(^{++}\) is an important second messenger. It is released into the cytosol after receptor-mediated extracellular signalling induces the IP\(_3\) signalling pathway. Ca\(^{++}\) is then bound by calmodulin which binds to other proteins thereby altering their activity. Transmission of Ca\(^{++}\) via gap junctions has been implicated in signalling neuronal death during development (Wolszon 1994a - see section 1.4.5).
Ca\(^{++}\) has been reported to downregulate GJIC (Spray et al 1982). However, the concentration required is atypically high for most Ca\(^{++}\) effects on normal cells. Such high Ca\(^{++}\) levels may only be achieved after injury when it may provide an important means of isolating damaged tissue from surrounding healthy cells.
There has been a prolonged argument regarding the role of Ca\(^{++}\) versus that of H\(^+\) in the regulation of GJIC. Turin & Warner (1977) showed that lowering pH in Xenopus embryo cells led to the down-regulation of junctional communication.
However, in 1978 Rose & Rick showed that Ca$^{++}$ levels were elevated in response to either increasing or decreasing pH and junctional communication was subsequently decreased.
18.104.22.168. Phorbol esters.
Tumour promoting phorbol esters such TPA appear to act as variable modulators of GJIC. Many cell lines show a downregulation of GJIC in response to TPA treatment, (for review see Klaunig & Ruch 1990). However, TPA has also been shown to increase dye transfer across the dermal-epidermal boundary when applied to intact mouse skin (Kam & Pitts 1988) and electrical coupling is also increased in some systems including cardiac muscle (Spray & Burt 1990).
TPA is thought to inhibit gap junctional communication through activation of protein kinase C and subsequent hyperphosphorylation of Cx43 (Brissette et al 1991, Gainer et al 1985). There are 23 serine sites within Cx43 which may potentially be phosphorylated. However, the effects of PKC activation are widespread and complex. The cascade of signalling changes which can occur include the activation MAP kinase (Nori et al 1992). Studies by Matesic et al (1994) showed that the rapid reduction in gap junctional communication, (within 5min) in rat liver epithelial cells was due to Cx43 hyperphosphorylation (this was manifest in the occurrence of a higher molecular weight band during Western blotting analysis) and a concomitant reduction in the number of Cx43 immunostained gap junction plaques. However, the correlation between Cx43 phosphorylation and decreased communication is in contrast to that of Musil et al (1990) who correlated connexin phosphorylation with increased gap junctional communication. These differences are possibly due to the extent and sites of phosphorylation within the protein.
However, the fact that chemicals such as TPA are non-specific and cause a cascade of potentially important phosphorylation events in other signalling pathways (Gosh et al 1990, Nori et al 1992) mean that it is not possible to draw causal relationships between changes in GJIC and changes in cell physiology. TPA-mediated changes in GJIC during multistage carcinogenesis are considered in section 1.4.6.
22.214.171.124. Retinoids.
Retinoids are naturally occurring and synthetic compounds derived from retinol (vitamin A$_{1}$). They are effective modulators of proliferation and differentiation of keratinocytes \textit{in-vivo} and \textit{in-vitro} and have profound yet diverse effects on the patterning of skeletal elements in various vertebrates (Maden 1982 & 1983, Tickle 1983). Nuclear receptors for retinoids (RAR), which modulate gene transcription in the regulatory regions of retinoid-responsive genes, have been discovered (Glass et al 1990) and may be responsible for the diverse range of actions on gene expression. Retinoids have also been used as cancer preventative agents in epithelial tissue. For example, the recurrence rate
of patients at high risk from second primary malignancies of the head, neck or lung decreases after systematic retinoid treatment (Hong et al 1990).
The effects of retinoids on GJIC vary depending on cell type, retinoid type and concentration (Pitts et al 1986, Yamasaki & Katoh 1988, Mehta & Loewenstein 1991). For example, gap junctional communication is blocked in animal cells at high concentrations ($10^{-4}$M; Pitts et al 1986) and blocked in cultures of mouse C3H10T1/2 cells at low concentration ($10^{-6}$M; Mehta et al 1989). At physiological concentrations ($10^{-8}$M) the homologous communication of C3H10T1/2 cells is increased, but their heterologous communication with chemically transformed relatives is decreased (Mehta & Loewenstein 1991). The expression of Cx43 was found to be regulated by retinoic acid in intact human skin (Guo et al 1992).
### 126.96.36.199. Adhesion mediated regulation of GJIC.
There is abundant evidence to suggest that cell-cell adhesion can control GJIC (Mege et al 1988, Keane et al 1988, Jongen et al 1991, Meyer et al 1992). Its role may simply be to bring cells into close apposition so that gap junctions can form. In a broader sense it may be involved together with cell-matrix interactions in ensuring that GJIC occurs in the correct cellular environment.
Cells expressing different cadherins sort out in mixed cultures indicating a role in selective cell-cell adhesion (Miyatani et al 1988 and Nose et al 1988). This adhesive specificity is thought to be involved in the specificity of gap junction formation observed in culture and *in vivo* (see section 188.8.131.52.b). Mege et al (1988), have shown that transfection of poorly coupled S180 cells with a cDNA for E-cadherin results in increased adherens and gap junction formation. This was observed by ultrastructural and dye transfer analysis. The frequency of expression of both types of junction was sharply decreased by treatment with anti-E cadherin Fab' antibodies.
Similarly, Meyer et al (1992) showed that the formation of gap junctions was dependent upon the formation of N cadherin-mediated adherens junctions. Using antibody Fab' fragments against either N cadherin or the 1\textsuperscript{st} and 2\textsuperscript{nd} extracellular domains of either Cx32 and Cx43, they were able to disrupt the reaggregation of Novicoff hepatoma cells. This was associated with a loss of dye coupling and EM analysis confirmed the loss of adherens and gap junctions. These experiments demonstrate the importance of close apposition between cells for the formation of gap junctions and suggest a possible role for connexins in cell-cell adhesion. Further experiments are required to determine whether adhesion-mediated signalling events are also important for junction formation.
Studies by Mege et al (1988), Musil et al (1990) and Meyer et al (1992) indicate that cadherin-based adhesion contributes to the establishment of gap junctional communication. It has been suggested that gap junction-mediated signal transduction modulates cell adhesion during compaction of the preimplantation mouse embryo (see
Lee et al 1987 and references there-in). A recent study by Paul et al (1995) utilised a dominant-negative chimeric Cx32/43 protein to block gap junctional communication. Following injection of chimeric mRNA into anterodorsal blastomeres of 8-cell stage mouse embryos, a loss of gap junctional communication and concomitant delamination of subsequent surface ectoderm occurred. Although the results were similar to those observed by Levine et al (1994) and Heasman et al (1994) in which cadherin based adhesion in early Xenopus embryos was perturbed by the expression of a cadherin dominant-negative mutant, they differ to the results of the connexin-antibody perturbation studies by Warner 1984, Lee et al 1987 and Fraser et al 1987, who found that adhesion was not perturbed. However, Paul et al (1995) were unable to rescue embryos expressing the chimeric connexin by co-injection of E-cadherin mRNA. The results are complex and may reflect the interplay between adhesion and GJIC. However, the possibility that connexins may be forming adherent structures was not considered as current dogma suggests that connexins only play a role in gap junction formation.
The post-translational phosphorylation of Cx43 appears to be a general phenomenon in communication efficient cell lines (Musil et al 1990, Crow et al 1990, Lau et al 1991 and Meyer et al 1992). Musil et al (1990) showed that poorly coupled mouse sarcoma cells (S180) express two species of Cx43. The unphosphorylated species, designated Cx43-NP (43kDa), predominates over the serine phosphorylated Cx43-P$_1$/P$_2$ (45-47kDa) species. However, after transfection of the cells with E cadherin cDNA and subsequent expression of the protein, the phosphorylation pattern of the connexins is reversed. The morphology of the cells changes to one of a more epitheloid type and dye coupling significantly increases. It has been postulated that phosphorylation of Cx43 (though not hyperphosphorylation) may be important in the formation and maintenance of functional gap junctions (Musil & Goodenough 1991).
184.108.40.206. Other effectors of GJIC.
Junctional permeability measured electrically can be influenced by a range of other agents including, anaesthetics (Niggli et al 1989), fatty acids (Giaume et al 1989), growth factors e.g. EGF (Kanemitsu et al 1993), TGF-$\beta$ (Maduhkar et al 1989) and secreted molecules e.g. Wnt-1 and activin B (Olson & Moon 1992). In this latter study it was shown that polarity in gap junctional communication existed in 32-cell stage Xenopus embryos, in that dorsal cells are relatively more coupled than ventral cells. The ability of factors, which are known to induce mesoderm induction, to increase GJIC were examined. Expression of Wnt-1, activin B and TGF-$\beta$ increased communication in ventral cells, however, the other mesoderm inducing factor - FGF did not upregulate gap junctional communication. Furthermore, subsequent defects in the dorsoventral axis correlated to increased junctional communication, although this does not provide direct evidence that junctional communication is involved in the establishment and maintenance of this axis. Studies by Olsen et al (1991) showed that misexpression of Wnt-8 led to
axial duplication in *Xenopus* which in turn was correlated to increased ventral GJIC. Developmental defects were also observed when activin B was misexpressed in *Xenopus* embryos (Thomsen et al 1990).
EGF, PDGF and TGF-β have all been shown to decrease levels of GJIC (Maldonado et al 1988, Madhukar et al 1989). EGF induced disruption is associated with the serine phosphorylation of Cx43. The cascade of events leading from EGF receptor activation and connexin phosphorylation are thought to be mediated by MAP-kinase (Kanemitsu 1993).
Interactions between cells and their associated ECM are believed to be important in regulation of GJIC. For example, levels of junctional communication in primary liver cultures were upregulated by the presence of proetoglycans and glycosaminoglycans in the medium (Spray et al 1987). These polysaccharide and protein-polysaccharide complexes are present in abundance in the ECM of liver cells. It is not yet known if integrin-mediated signalling plays any role in GJIC regulation.
Certain oncogenes, including src, ras and SV40 downregulate GJIC (Bignami et al 1988a, 1988b). These observations have contributed to the hypothesis that modulation of GJIC is an important step during multi-stage carcinogenesis and tumour development. This is reviewed in more detail in section 1.4.6.
### 1.4.5. The role of signalling via gap junctions in normal growth and development.
There are several lines of evidence to suggest that gap junctions are involved in the normal growth and development of multicellular organisms.
1). They are present in all complex multicellular organisms, particularly during early embryonic stages of development when cell-cell interactions leading to cell commitment and differentiation occur (Lo & Gilula 1979a).
2). Several growth regulatory and developmental factors can pass through gap junctions or modulate gap junctional communication (Spray et al 1982, Pitts et al 1986, Mehta & Loewenstein 1991, Atkinson et al 1995).
3). There is spatial and temporal alteration of the pathways of GJIC and gap junction associated proteins during growth, differentiation and development (Risek & Gilula 1991, Brissette et al 1994).
4). The establishment of communication compartments *in vivo*, (groups of cells coupled to cells of the same compartment but not with those of other compartments), is closely associated with the appearance of different cell lineage's occurring during development (e.g. Karn & Hodgins 1992).
5). GJIC is altered in different stages of carcinogenesis and the gap junction associated protein - Cx43 has been shown to restore communication and normalise the growth of transformed cells (Yamasaki 1991, Mehta et al 1991).
Gap junctions connect the cytoplasm of one cell to another and allow access to an array of potential signalling and growth control molecules. In theory, signals received by only a few cells that give rise to second messengers, may be relayed throughout a population of cells providing a co-ordinated response, which may result in that cell population following a new lineage pathway. Alternatively, a cell which may receive a potential inductive signal may be prevented from differentiating by the homeostatic pressure which gap junctions mediate.
In normal mouse embryos morphologically identifiable gap junctions first occur at 8 cell compaction stage (Ducibella et al 1975, Magnuson 1977). This correlates with the onset of electrical coupling between all blastomeres of 8-cell embryos (Lo & Gilula 1979). Cells can be taken from the 8 cell stage embryo and replaced on either the inside or outside of the embryo. Those on the outside become trophoblastic cells and those on the inside contribute to the inner cell mass (ICM). Beyond the 8 cell stage cells become determined (although not irreversibly). For example, if cells of the ICM are removed from late stage blastula and maintained *in vivo* some revert to trophoblastic cells, suggesting cellular phenotypes are influenced by environmental factors such as the presence of other determined cells. Several mechanisms may be responsible for the environmental influence which is exerted, including homeostatic pressure mediated by gap junctions.
In addition to these early embryonic observations gap junctional communication is believed to be involved in later stages of development and has recently been shown to be involved in neuronal growth cone development. In leech embryos developing anterior pagoda (AP) neurons transiently overlap and detect each others presence adjusting their branching pattern accordingly. Removal of an AP neuron by death or by mechanical means results in a nearby AP axon occupying the vacated area. Thus, it appears the neurons communicate there presence or death to other AP neurons (Wolszon 1994a & b). Wolszon et al (1994a) have shown, via dye injection and electrophysiology, that gap junctions are present between the transient, contacting axons. Furthermore, they identified calcium waves passing through gap junctions which appeared to signal neuron death (Wolszon et al 1994b). In humans, mutations in various connexin genes have been implicated in several genetically inherited diseases of the nervous system (Patel & Lupski 1994, Spray & Dermietzel 1995).
Connexin knockout mice are now beginning to be produced. Mice deficient in Cx43 (after targeted mutagenesis) survive to term (Reaume et al 1995). However, at birth the mice die as a result of a failure in pulmonary gas exchange caused by a blockage in the right ventricular outflow tract from the heart. Cx43 is clearly an important protein in foetal heart development, despite this its absence would appear to be compensated for.
220.127.116.11. Communication compartments.
The establishment of communication compartments during embryogenesis and early development and the association of such compartments with the emergence of new lineage's is further evidence that gap junctional communication is involved in growth regulatory and developmental processes (Lo & Gilula 1979a & b, Warner & Lawarence 1982, Karn et al 1987, Serras et al 1989, Yuste et al 1992). This has strengthened the hypothesis that communication compartments restrict an individual cells ability to form new phenotypes and permit groups of cells to differentiate and form new lineage's. Communication compartments have been observed throughout several stages of embryonic morphogenesis (Fraser & Bryant 1985, Kalimi & Lo 1988, Serras et al 1989, Dahl et al 1996) and in several tissues, including skin (Pitts et al 1986), hair follicles (Karn & Hodgins 1992), early chick limb (Allen et al 1990) and neuroepithelium (Martinez et al 1992). The putative mechanisms which generate such communication patterns are discussed in section 18.104.22.168.
The communication compartments in skin (Pitts et al 1986, Karn & Hodgins 1992) are, in general, associated with subpopulations of different differentiated cells. In the developing hair follicle, compartments were established between cells following different differentiation pathways. Karn & Hodgins (1992) found that dye spreads were extensive between cells of the hair bulb matrix but cell populations committed to the development of the inner root sheath were in separate compartments.
Gap junctional communication appears to be important in the patterning of chicken skin and feather development, (Serras et al 1993). Dye transfer studies revealed compartments in mesoderm and epidermis with preferential dye spreads along the anterioposterior axis of the feather placode and restricted spread at the feather bud/interbud boundary during early feather bud development.
In culture, restricted heterologous communication is observed between normal cells and many types of transformed cells. The situation is perhaps analogous to the establishment of a new phenotype (transformed in this case) and may offer a useful paradigm with which to study the process of gap junction specificity which may be responsible for the establishment of communication compartments in vivo.
22.214.171.124. Gap junction specificity.
The molecular basis of gap junctional specificity which leads to the establishment of communication compartments is not clearly understood. Expression studies using dominant negative mutants, chimeric proteins, and antibody perturbation experiments have enabled gap junction formation events to be dissected. The connexins and the cell adhesion molecules may mediate junctional specificity.
126.96.36.199.a. Connexins and specificity.
Because connexins are a large family of proteins and they show spatiotemporal expression during different stages of development (Paul 1985, Beyer et al 1989, Girnlich et al 1990, Risek & Gilula 1991, Goliger & Paul 1994) it has been suggested that they are involved in gap junction specificity.
Communication compartments are present in postimplantation mouse embryos (Dahl et al 1996). These compartments were defined by lucifer yellow dye spreads and were revealed in embryonic and extra-embryonic germ layers of ectoderm, mesoderm and endoderm. Expression studies and immunofluoresence analysis locate Cx43 exclusively in embryonic cells whilst Cx31 localises to extraembryonic cells and was maintained when the axial polarity of the mouse embryo was established. The results suggest that differential connexin expression is responsible for the establishment of these compartments.
However, Risek et al (1992) showed that Cx43 is expressed by different cell lineage's within the hair follicle despite the fact that different lineage's form separate communication compartments (Karn & Hodgins 1992). Connexin expression has also been analysed in developing rat skin and new born mouse epidermis (Risek et al 1991, Kamibayashi et al 1993). From the results it was concluded that multiple connexin genes contributed to epidermal and follicular morphogenesis and that communication compartments were established.
Gap junction specificity has also been observed in a number of *in vitro* cell systems (Pitts and Burk 1976, Prowse 1992). Using the *Xenopus* oocyte expression system it has been shown that expression of certain connexins can lead to both homotypic and heterotypic gap junction formation (Dahl et al 1987, Swenson et al 1989, Barrio et al 1991). However, different connexin combinations are restricted in their ability to induce junction formation. White et al (1995) found that functional channels formed only when Cx40 and Cx37 were expressed. Conversely, junctions formed when Cx46 was expressed together with Cx - 26, 32, 37, 43 and 50. This has been supported by the studies of Bruzzone et al (1993) who showed that *Xenopus* oocytes expressing Cx40 were able to form junctions with oocytes expressing Cx37, however no communication was not detected between oocytes expressing Cx40 and Cx43. The lack of gap junction formation after the heterotypic expression Cx40 and Cx43 may be of physiological relevance: within the vascular wall of Rats Cx40 is expressed in endothelial cells, whereas in adjacent smooth muscle cells Cx43 is expressed. The purpose of this differential expression may be to limit communication between the two cell types. This is supported by the finding *in vivo* that no communication is detected between the two cell types (Segal & Beny 1992). Specificity of gap junction formation has also been observed in connexin transfectants in culture (Tomasetto et al 1993).
Using pairs of *Xenopus* oocytes to express chimeric connexin constructs it has been shown that heterotypic expression of connexins leads to the formation of gap
junctions with different gating properties (Bruzzone 1994). In addition to their possible role in gap junction specificity, connexins have been implicated in the regulation of channel size (Steinberg 1994, Elfgang et al 1995). However, the evidence for this is limited and the majority of reports show that channel size is fixed, when open, at ~1-2nm.
188.8.131.52.b. Cadherins and specificity.
It has already been shown in section 184.108.40.206 that GJIC has a close association with cell-cell adhesion and that cell-cell recognition mediated by the cadherins may well be a pre-requisite for the formation of gap junctions (Mege et al 1988, Jungian et al 1991). This is not surprising from a formation perspective, given that gap junction connexons protrude by only ~1nm and therefore the membranes need to be brought into close apposition to allow the formation of functional channels. This is consistent with the finding that antibodies to N-cadherin disrupts gap junction formation between Novikoff cells (Meyer et al 1992). The cadherin family of adhesion proteins which are expressed in a tissue specific manner are known to be involved in cell recognition and cell sorting during development (Miyatani et al 1989, Takechei 1991). They may therefore provide a mechanism for segregating cells into separate compartments and restricting junctional communication between cells of different compartments.
Unpublished results by Prowse (1992) showed rat epithelial cells (BRL cells) and rat fibroblasts (BICR cells) sort out into communication compartments in culture. Expression studies revealed differential expression of cell adhesion molecules (but not connexins) and the transfection of E cadherin cDNA into both cell types increased the levels of heterologous communication.
GJIC is often altered during various stages of carcinogenesis. The changes often lead to the restricted communication between normal and transformed cells. Loss of heterologous communication (possibly via altered GJIC specificity) may be a prerequisite for initiated cells to escape the growth control of normal cells. In several instances changes in GJIC levels are associated with the altered gene expression of cadherin and connexin proteins and in some cases these proteins are post-translationally modified. The situation may be analogous to the formation of communication compartments *in vivo* and the subsequent formation of new cell lineage's within each compartment.
1.4.6. Gap junctional communication and cancer.
Cancer can be described as a disease resulting from altered differentiation and cell growth. Putative roles for GJIC in normal development and growth of organisms have been described in the previous sections. Given the considerable amount of circumstantial evidence for the role of GJIC in growth control it might be supposed that there is a
relationship between cancer and loss of gap junctional communication. There are several lines of evidence to support this:
1). GJIC is often reduced in transformed cells expressing various oncogenes including, ras, src, E5 and raf (Bignami et al 1988a & b, Filson et al 1990, Finbow et al 1991, Kalarni et al 1992).
2). The gap junction associated proteins, Cx43 and ductin are the target of the oncogenes src and E5 respectively - although the effects may not necessarily be primary events leading to transformation (Swenson et al 1990, Finbow et al 1991).
3). The aberrant growth of transformed cells is suppressed when cultured with excess normal cells (Stoker 1967), and the phenomenon has been correlated to the presence of heterologous communication (Mehta & Loewenstein 1986).
4). Expression of gap junction associated proteins can normalise the growth of transformed cells (Mehta et al 1991, Jou et al 1993, Chen et al 1995).
5). In several model systems, gap junctional communication is abolished by tumour promoting phorbol esters (Asamoto et al 1991, Roseng et al 1993) and enhanced by tumour suppressing agents such as retinoic acid (Mehta et al 1989, Rogers et al 1990).
Carcinogenesis is thought to be multistep process leading to the sequential loss or impairment of growth control mechanisms. The current view of carcinogenesis begins with a potential growth promoting genetic defect occurring in a single (initiated) cell (Armitage & Doll 1954). If the defective cell divides, then further mutations in subsequent cells may arise which provide the cell with further growth advantages. This stage may be promoted if the cells are exposed to tumour promoting agents such as TPA. Tumours may then progress to more aggressive and malignant forms. The final stage of carcinogenesis is metastasis, which typically involves cells from the primary tumour breaking away, moving to new sites where they may lodge and proliferate - forming secondary tumours. Adhesive interactions have important roles in the process of metastasis and adhesive molecules can act as positive and negative modulators of the metastatic process. E cadherin for example, promotes cell-cell adhesion within the tumour and therefore restricts the ability of individual cells to break away to form secondary tumours (Vleminckx et al 1991, Takeichi 1993). Cells which do break away are at an advantage if they can then attach at a new site rapidly (to avoid elimination in the circulation). Therefore cell surface expression of proteins such as integrins positively promotes metastasis (see Zetter 1993 for review).
In section 1.2 it was shown that the environment of a cell is important to that cell's regulation and phenotype. In 1967 Stoker showed that the growth of polyoma-transformed cells could be inhibited by the presence of excess resting normal cells. Subsequently, this form of inhibition has been correlated with the presence of heterologous communication between the normal and transformed cells (Mehta 1986)
and would appear to show that adjacent cell cytoplasm's can contribute to the environment of a cell and its subsequent regulation.
220.127.116.11. GJIC during multistage carcinogenesis.
Many cancer cells show defective gap junctional communication, however, it is not clear at what stage or by what mechanism this occurs. Down-regulation of connexins has been observed in human mammary tumour cells (Lee et al 1992) and post-translational modification of Cx43 has been observed in cell lines transformed with v-src (Swenson et al 1990).
Viral-mediated transformation is often associated with decreased levels of GJIC but different viral oncogenes have varying effects on gap junctional communication. Expression of the oncogenes raf, mos, src, ras, neu and PyMT result in the down-regulation of both homologous and heterologous communication, but v-myc and PyLT do not alter communication levels (Atkinson et al 1981, Atkinson & Sheridan 1984, Atkinson et al 1986, Kalimi et al 1992). The effect of v-src on communication has been correlated with the tyrosine phosphorylation of connexins, (Filson et al 1990). In addition, the bovine papilloma virus-1 oncoprotein, E5, binds to the gap junction protein ductin (Goldstein et al 1992) and the related human papilloma virus oncoprotein, E5, inhibits GJIC. In addition the BPV-4 viral oncoprotein, E8, binds to ductin and communication is downregulated in cells expressing this protein (Facchini et al 1996).
During initiation a genetic mutation may arise which renders the cell 'primed' for transformation. In the mouse skin chemical-carcinogenesis model, in which tumours are induced after skin is treated, first with DMBA followed by several applications of TPA, this initiation is marked by the expression of the H-ras oncogene and a subsequent rise in IP$_3$ levels (Wakelam 1988). Given the proposed role of GJIC during development it might be suggested that such a cell could be suppressed by the homeostatic pressure of the surrounding normal cells via the loss of excess IP$_3$.
However, recent findings by Novelli (1996), suggest that some tumours may arise from a polyclonal origin. Novelli found that precancerous cells appeared to be capable of inducing precancerous states in neighbouring cells. These results concerned the apparent polyclonal origin of adenomas and subsequent tumours in the human colon. However, at present there is no evidence to suggest that such a mechanism is involved in the origin other tumours. The molecular and biochemical signals that were involved in the induction have not been characterised, but could be mediated by the transfer of aberrant growth signals via gap junctions.
In culture, normal cells generally cease growing once they have reached high cell density. It has been shown that this inhibitory growth control can suppress the aberrant growth of certain transformed cells, providing there is heterologous communication between the two cell types (Stoker 1967, Mehta et al 1986). The ability of transformed cells to stimulate the growth of resting normal cells is therefore potentially important, i.e.
by altering the local environment to one which promotes growth, the inhibitory effect of the normal cells may be reduced or cancelled out.
Returning to the mouse skin model, the ability of the initiated cell to clonally expand requires several applications of TPA. TPA transiently inhibits junctional communication *in vitro* (Mehta et al 1986 - see section 18.104.22.168 on TPA action) and can release transformed cells from the growth suppression of normal cells (Dotto et al 1988). Thus, the effect on mouse skin is consistent with the idea that the initiated cell must escape the growth control imposed by the normal cells via GJIC (Mensil & Yamasaki 1993), until a large enough population has been established in which cells are less influenced by surrounding normal cells and can grow, acquiring further relevant genetic changes.
Investigations have been carried out to determine whether the changes in GJIC associated with progression correlate to altered levels of gap junction associated proteins. The levels of Cx26, and Cx43 were examined by immunofluoresence in normal mouse skin, papillomas, squamous cell carcinomas (SCC) and associated metastases (Kamibayashi et al 1995). In papillomas Cx26 and Cx43 co-localised in the same gap junction plaques, whereas the two connexins were differentially expressed in normal and surrounding non-tumourous epidermis. In SCC's levels of Cx26 and Cx43 decreased in comparison to normal tissue and papillomas. When SCC's metastasised into lymph nodes, Cx26 was expressed but few cells appeared to express Cx43. In metastatic tumours Cx43 was found to be very low. Although the morphometric connexin expression data do not necessarily represent functional states several studies have shown that the number or size of immunoreactive spots of connexins is closely related to levels of GJIC (Krutovskikh et al 1991, Asamoto et al 1991). Reports by Klann et al (1989) also show that levels of GJIC are reduced in a step-like manner as cells progress towards an increased aberrant phenotype.
The importance of connexin expression and GJIC during tumourigenesis is highlighted further by the studies of Mehta et al (1991), Jou et al (1993), Rose et al (1993) and Chen et al (1995). They have shown that the exogenous expression of connexins in a many different transformed cell lines restores or increases heterologous communication. The growth of these cells is often normalised in culture as indicated by lower saturation densities, longer population doubling times and rounder, flatter cell shape. The molecular mechanisms responsible for these changes are not known. It has been suggested that the increase in junctional communication allows specific growth inhibitory molecules to pass throughout the coupled population.
Exogenous connexin expression has also been implicated in the suppression of transformed cells by normal cells. Here it is thought that the protein is responsible for increasing heterologous communication between the two cell types, and it is the normal cells which then impose growth control on the transformed cells population. The cells also show loss or reduction in their tumourigenic potential *in vivo*.
Further complexity in our understanding of the process of carcinogenesis is added by the ability of adhesion molecules to regulate GJIC (Mege et al 1988, Jungian et al 1991). In multi-stage carcinogenesis, alterations to these adhesion proteins are thought to be involved in the later stages of progression when their loss or modification confers metastatic potential on the tumour. A reduction in junctional communication has been observed during the progression of squamous cell carcinomas to spindle cell carcinomas. This has been correlated to the loss of E-cadherin (Holden et al 1996). Communication in many tumourigenic cell lines is rarely zero and the residual low level may be important for tumour survival and metastasis. It has been shown by Sabban et al (1991) that dye transfer occurs between metastatic tumour cells and vascular endothelium.
22.214.171.124. Homologous and heterologous communication during carcinogenesis.
Most work on GJIC and its role in carcinogenesis has concentrated on the changes in homologous communication in transformed cells. Most transformed cells show some residual levels of homologous communication (Kalimi 1992) and this fact is currently being exploited for cancer pro-drug therapies (section 1.3.3). The retention of homologous communication may be of advantage to the transformed cells in later stages of progression. For example, in the establishment of a homeostatic pressure which may contribute to maintaining the aberrant phenotype. However, if normal cells are able to control the growth of transformed cells via junctional communication, loss of heterologous communication would be of selective growth advantage to initiated cells.
Several studies have indeed shown that cells transformed by various agents (including chemicals and oncogene expression) lose the ability to communicate with normal cells (Yamasaki & Enomoto 1985, Mehta et al 1991, Kalimi et al 1992). In subsequent studies attempts have been made to restore communication in these cell lines by transfection with Cx32 or Cx43 (Eghbali et al 1990, Mehta et al 1991, Zhu et al 1991, Rose et al 1993, Chen et al 1995). Restoration of heterologous communication resulted in the normalisation of the transformed cells. This was manifest by reduced proliferative capacity in culture and a non-tumourigenic phenotype *in vivo*. The molecular mechanisms for this phenomenon have yet to be characterised but possible mechanisms are discussed in section 1.6.
Many studies regarding cell-cell interactions, particularly GJIC, and their role in growth control have been carried out in culture. The possibility exists that such events are merely tissue culture artefacts. However, there are examples where dramatic alterations in cell phenotype occur *in vivo*, in response to altered environments. One such example is the phenotypic conversion of embryonal carcinoma cells (EC cells) from a malignant form to one in which the cells participate in normal growth and development (in blastocysts). The phenomenon may serve as a complex *in vivo* analogy to that seen in culture when the growth of transformed cells is suppressed by the presence of normal cells. A brief review of this striking phenomenon is given next.
1.5. Embryonal Carcinoma Cells: an *in vivo* example of extra-cellular, environmental growth control.
1.5.1. General overview.
Teratocarcinomas have proved to be a useful model for studying the effect of cellular environment in the process of cell commitment and differentiation. Teratocarcinomas represent tumours of pluripotential cells and can arise spontaneously in murine testis or ovaries; such tumours are rare in humans - Pierce & Dixon 1959. Teratocarcinomas (or embryonal carcinomas) are composed of disorganised mixtures of well-differentiated tissue, typically epithelia, notochord, cartilage (Pierce et al 1967) and undifferentiated embryonal carcinoma cells which confer malignancy upon the tumour. The term, teratoma is used for teratocarcinomas whose EC-cells have all differentiated and are no longer malignant. Transplantable teratocarcinomas can be experimentally produced by grafting early embryos, germinal ridges or germ layers into adult mouse testis. Such teratocarcinomas have a normal diploid set of chromosomes and are indistinguishable from spontaneous ovarian and testicular teratocarcinomas.
The nature and location of teratocarcinomas led investigators to believe that they arose from cells derived from pluripotent cells in the germ layers of early embryos. Several lines of evidence exist to support such a hypothesis. Germinal ridges or early germ layers from mice homozygous for the *steel* gene lack primordial germ cells and do not form teratocarcinomas when grafted (Sherman 1975). Furthermore the ability of germ layers, when grafted, to produce teratocarcinomas is lost by the 8th day of gestation - a time at which cell commitment is restricted.
Definitive evidence that EC-cells are pluripotent came from a series of experiments through the mid 1960's and early 1970's. Embryoid bodies are teratocarcinomas that form in the peritoneal cavity and consist of a thin sheet of endoderm which surrounds a core of EC-cells (Stevens 1970). In 1964 Kleinsmith and Pierce took single cells from such tumours and grafted them into mice. The teratocarcinomas which subsequently formed contained EC-cells as well as various well differentiated tissues, (up to 14 types). Although clearly pluripotent it was not clear whether such cells were totipotent as the tumours lacked several tissue types. In 1975 it was shown that EC-cells could participate in normal development. Mintz & Illmenses (1975) took a single EC-cell and injected it into a mouse blastocyst whereupon a chimeric embryo developed. These cells contributed to various somatic tissues including liver, thymus, kidneys and germ cells.
These experiments clearly highlight the role of the environment in effecting the programming of the genome. The ability of EC-cells to form normal adult tissue and germ cells (Stewart & Mintz 1981) demonstrates that the conversion to neoplasia under certain conditions does not involve permanent structural changes to the genome but rather a change in gene expression in response to altered environments. Evidence from
other sources also highlight the importance of such environmental interactions in determining cell phenotype. For example, epithelial cell carcinomas can revert to a non-tumorigenic phenotype after interaction with normal skin (Cooper & Pinkus 1977). In this instance a genetic change is being suppressed.
The interactions which determine cell phenotypes in teratocarcinomas are still not understood, although progress has been made in understanding, at a molecular level, the changes which result in a switch from a neoplastic to a normal cell phenotype. Retinoic acid (RA) has been well studied as a mediator of inductive signals during EC-cell differentiation. The inductive ability of RA has been shown using an EC-cell line, F9 cells, which have lost the ability to differentiate *in vitro* or *in vivo* (Strickland & Mahdave 1973). The RA-associated molecular changes which drive the differentiation of F9 cells to a new phenotype are now being dissected. A variety of transcription factors have been identified which show altered levels of abundance and activity in response to RA treatment. These factors (which include c-jun & AP-1) modulate the activity of genes responsible for cell proliferation and ECM production (Sleigh 1992).
The role of GJIC in differentiation of EC-cells has not been fully explored. Gap junctions certainly provide an apparatus for establishing and maintaining microenvironments which may be important in embryogenesis and the maintenance of the differentiated state. Using F9 cells it was first demonstrated that gap junction associated genes can be modulated during the differentiation of a single cell type in culture (Nishi et al 1991). Upon exposure to RA, the F9 cells rapidly differentiated into visceral endoderm (VE) or parietal endoderm (PE). All cells expressed Cx43. Cx26 was also expressed in all cells although differentially i.e. PE cells had very low expression and VE cells had very high expression levels. Cx32 was only expressed in VE cells. The modulation of gap junction associated genes during differentiation in this system is in agreement with the analysis of fully differentiated tissue *in vivo*. Nicholson et al (1987) found that foetal liver predominantly expressed Cx32 and Cx26, and VE cells in teratocarcinomas are thought to be the equivalent of foetal liver precursor cells (see Nicholson 1987 and references there-in). Causal relationships between gap junctional communication deficiency and loss of EC-cell differentiative ability has also been suggested by Sheardown & Hooper (1992).
**1.5.2. Summary.**
The occurrence of teratocarcinomas would appear to be due to the ability of stem cells to retain their proliferative capacity. This ability appears to be the result of general tissue disorganisation during development. In a normal environment the aberrant growth phenotype of the stem cells can be completely reversed and it has been shown that this is not the result of selecting non-malignant cells (Mintz & Illmensee 1975). The experiments which show that previously malignant EC-cells can undergo normal differentiation and contribute to the normal growth of a mouse demonstrates that their
initial state of malignancy was due to a change in gene expression rather than a permanent structural change to their genomes.
Although EC-cells and teratocarcinomas have proved a useful model for studying cell differentiation and the importance of the environment in altering gene expression (and hence cell phenotype) the model is a particularly complex one. The fact that the phenomenon occurs *in vivo* makes it difficult to dissect the individual mechanisms which may be involved. However, a similar phenomenon, in terms of the importance of environment in altering cell phenotype, has been observed in culture i.e. the suppression of transformed cell growth by normal cells. The phenomenon is particularly amenable to experimentation.
1.6. Suppression of the transformed phenotype by normal cells.
The observation that the aberrant growth of polyoma virus transformed cells could be inhibited when grown in the presence of excess normal fibroblasts (Stoker 1966 & 1967) provides an *in vitro* system to examine the importance of environment in determining cell phenotypes. The inhibition occurred after the normal cells had reached high density and were themselves, growth inhibited.
A great deal of information regarding the inhibition phenomenon was obtained by Stoker in the original papers (Stoker 1966 & 1967) using a technique which permitted the level of growth inhibition to be measured ([3H]-thymidine incorporation and analysis by autoradiography). It was shown that the growth of the transformed cells was reduced in response to a decrease in the growth of the normal cells when they reached high cell density. The effect was not due to nutrient deprivation or factors secreted into the media. Contact between the two cell types was required and it was shown that there was heterologous metabolic co-operation between the two cell types, via gap junctions. From this data it was hypothesised that the inhibitory effect of the normal cells could arise from the transfer of inhibitory growth signals from normal to transformed cells. The level of normal cell proliferation was higher than background in some of the co-cultures, however, this was not quantified.
In 1986 the growth inhibition of various chemically and virally transformed cells was shown to correlate to the level of heterologous gap junctional communication with surrounding normal cells (Mehta et al 1986). In this work the proliferation assay, used by Stoker to quantify inhibition, was neglected in favour of the quicker, though less informative, focus formation assay. Mehta et al reached the same conclusion as Stoker, that is, inhibitory molecules *may* pass from the normal to the transformed cells. However, such molecules have yet to be identified. Subsequent published results have confirmed the inhibitory effect normal cells can have upon the growth phenotype of transformed cells (Bignami et al 1988, Dotto et al 1988, Martin et al 1991, Crescenzi & Tato 1994). La Rocca et al (1989) found that in addition to suppressing the aberrant
growth phenotype of myc-transformed quail muscle cells, contact with excess surrounding normal fibroblasts were capable of reverting the cells back to their myogenic differentiation program.
These studies, based on a variety of cell systems, used the focus formation assay as a means of detecting whether the normal cells are capable of suppressing the transformed cells. In a typical assay a small number of transformed cells are cultured together with an excess of normal cells or seeded on top of a confluent layer of normal cells. After 1-2 weeks in co-culture the number of foci are counted and/or their size measured and inhibition calculated. However, it provides little other information. Individual cell-cell interactions cannot be observed and suppressed cells cannot be studied (because they cannot be identified). Any subtle changes in growth, such as the stimulation of normal cells, which Stoker (1967) suggests may occur, cannot be detected because no proliferative data, other than indirect measurements gained from focus number or size, is obtained. Furthermore, focus assays provide little scope for *in vivo* studies. Tumourigenicity assays can be carried out, but again, only cells which have escaped suppression and formed tumours can be examined. Such assays raise further questions; for example, why is it necessary to inject such a large mass of cells (>10^3 cells in even highly tumourigenic cell lines) before tumours are detected? And furthermore, what happens to the cells which don't go onto to form tumours - are they suppressed? If they are suppressed until they eventually die, this may be an important defence against cancer.
Attempts have been made to study the role of GJIC in the inhibition phenomenon by experimentally varying the level of communication. However, the agents used in such treatments (e.g. forskolin and retinoids - Mehta et al 1986, 1989, Mehta & Loewenstein 1991) are insufficiently specific modulators of junctional communication and specific conclusions are difficult to draw.
**1.6.1. Mechanisms of suppression.**
The ability of a normal cell to crawl and spread out over a substratum to occupy vacant space strongly affects its ability to divide. The frequency with which a cell divides increases as the cells become more spread out, (O'Neill et al 1986; possibly because a cell with a larger surface area can take in more nutrients and growth factors). Some transformed cells will divide even when the adhesion site is small and leaves no room to spread out. Normal anchorage-dependent cells in culture are characterised by their density-dependent regulation of growth (Wieser et al 1990). The inhibition of transformed cells by normal cells occurs once the normal cells have reached high density and have themselves stopped growing, it is possible that the same growth control mechanisms are responsible for both these phenomena. However, the specific molecular mechanisms are not known.
The mechanism responsible for the growth suppression does not appear to involve a secreted factor, based on the failure to transfer inhibition using conditioned media and the requirement for direct cell-cell contact. However, a secreted factor may act in a paracrine fashion i.e. requires high local concentrations for effectiveness due to rapid internalisation by surrounding cells, binding to the surface or ECM receptors of other cells or rapid degradation. The high concentration required may be difficult to achieve using standard conditioned media experiments.
Inhibition models have been proposed which are centred on either the production of specific growth inhibitory molecules by normal cells or the dilution of stimulatory factors produced by the transformed cells, both processes mediated by gap junctional transfer (Stoker et al 1967, Mehta et al 1989, Mehta et al 1986). As yet, however, no specific inhibitory molecules, which could pass through gap junctions, have been found. GJIC may induce suppression by a more general mechanism such as homeostasis. A general equilibration of small molecules and ions between the two cell types may result in a homeostatic pressure which suppresses differences between cells.
The studies in which connexins were transfected into communication deficient transformed cells to restore GJIC and a normal phenotype would appear to provide direct evidence that the inhibition is mediated by GJIC. However, it has not been unequivocally shown that connexin expression only effects GJIC. Furthermore, many investigators believe that connexins exclusively form the channel component of the connexon but this has not been shown and they may serve other functions which in turn regulate GJIC. Cadherins for example are known to effect GJIC but it is not suggested they form part of the channel structure.
Overexpression of Cx43 in transformed cells, which can cause the cells to revert to a normal phenotype, has been shown to alter the expression levels of specific genes involved in cell cycle regulation - namely cyclins and cyclin dependent kinases (Chen et al 1995). The authors were unable to distinguish between the expression of Cx43 versus the restoration of GJIC as being responsible for the effects observed on cell phenotype. The relationship between Cx43 expression and cell cycle gene expression may arise from a direct exchange of growth regulatory factors through gap junctions or via the interaction of Cx43 on adjacent cells which may directly affect cell signalling or cell adhesion pathways responsible altering specific genes.
Density-dependent inhibition of cell growth has been correlated to the expression of a glycoprotein termed contactinhibin which binds via cell-cell contact to its appropriate contactinhibin receptor (CiR) on apposing cell membranes (Weiser et al 1990, Gradl et al 1995). However, it is not known at present whether this form of inhibition is responsible for that seen by Stoker and others.
1.7. Summary and aims.
There is abundant circumstantial evidence to suggest that GJIC is involved in the control of normal cell growth and development. However, the nature of the signal molecules and the control pathways affected remains unclear. The importance of environment in altering gene activity has been stressed throughout and clearly demonstrated by the altered fates of embryonal carcinoma cells in response to different environments. Gap junctional communication is likely to be just one of a series of important cell-cell interactions involved in defining and maintaining various differentiated phenotypes, and may provide a general means of co-ordinating cellular activities.
The observations by Stoker (1967) have demonstrated further the idea of environment controlling cell growth and phenotype. In this instance subsequent studies have implicated GJIC as a likely mediator of the growth control, transferred from the normal cells to the transformed cells *in vitro*. However, because most studies have been concerned with the role of GJIC in carcinogenesis detailed studies of the inhibition phenomenon *in vitro* or *in vivo* have not been carried out.
Given that such a phenomenon may play an important role as a primary means of defence against cancer this thesis aims to provide a detailed examination of the phenomenon *in vitro* and establish an assay which can be subsequently used *in vivo* to assess the role of the inhibition phenomenon during the process of tumourigenesis. The potential of such assays was recognised by Stoker (1967) who used lineage marking techniques and a proliferation assay based on thymidine incorporation to examine the suppression of the transformed phenotype by normal cells. Despite all of the work carried out there has been little advance in the detailed description or understanding of the phenomenon since these earlier investigations.
CHAPTER TWO
MATERIALS & METHODS.
2.1. Materials.
2.1.1. Chemicals.
Chemicals used were of "AnalR" grade and obtained from BDH Chemicals Ltd., Poole, Dorset, England or Sigma Chemicals Co., Ltd., Poole, Dorset, England, except those from the suppliers listed below.
| SUPPLIER | CHEMICAL |
|-----------------------------------------------|--------------------------------------------------------------------------|
| Amersham International Plc., Aylesbury, Bucks, UK. | $\alpha$[32P]-dCTP (3000Ci/mmol)
[Methyl-3H]-thymidine (25 Ci/mmol)
[5-3H]-uridine (29 Ci/mmol)
Hybond-ECL, Hybond-N+ |
| Boots Plc, Argyll St, Glasgow. | Clear nail varnish |
| BRL (UK), Gibco Ltd., Paisley, Scotland. | All restriction enzymes and buffer |
| Biogenesis Ltd., Bournemouth, England. | RNAzol B. |
| Boehringer Mannheim UK Ltd., Lewes, East Sussex, England. | DNA molecular weight markers IV and VI
Proteinase K. |
| J. Burrough (FAD) Ltd., Witham, Essex, England. | Ethanol. |
| Central Services, Beatson Institute. | Sterile distilled water and PBS
L-broth, CT buffer (NaCl 6g, Trisodium Citrate 2.96g, Tricine 1.76g, Phenol red 5mg, Water 700ml, pH7.8), PBS, Glycerol. |
| Difco Labs., Detroit, Michigan, USA. | Bacto-agar. Bacto-tryptone. |
| Gateway Plc, Glasgow Scotland. | Marvel Dried non-fat milk powder. |
| Supplier | Product Description |
|-----------------------------------------------|----------------------------------------------------------|
| Oxoid Ltd., Basingstoke, England. | PBS tablets. |
| Pharmacia Biotech Ltd., Milton Keynes, England.| Micro spin columns s-400 HR |
| Rathburn Chemicals Ltd., Walkerburn, Scotland.| Phenol (water saturated) |
| Sigma Chemical Co., Ltd., Dorset, England. | Pre-stained protein molecular weight markers |
| Vector Labs Inc. Burlington, USA | Vector shield mounting media. |
### 2.1.2. Kits.
| Supplier | Kit |
|-----------------------------------------------|----------------------------------------------------------|
| Amersham International Plc. Bucks, England. | ECL Western Blotting Analysis System. Cell Proliferation Kit |
| Bio 101 Inc. Luton, England | Geneclean Kit |
| Boehringer Mannheim UK Ltd., Lewes, East Sussex, England. | Apoptosis, cell death detection kit |
| Molecular Probes, Cambridge Bioscience, Swanns Rd, Cambridge, England | Imagene lacZ Expression Detection Kit. FluoReporter lacZ Quantitation Kit. Fluorescent microspheres |
| Pharmacia Ltd, Milton Keynes, England | Oligo-labelling Kit. |
### 2.1.3. Water.
Distilled water for solutions was obtained from a Millipore MilliRO 15 system, and for nucleic acid procedures it was further purified on a Millipore MilliQ system.
2.1.4. Equipment and Plasticware.
Main pieces of equipment are referred to in the appropriate sections. The suppliers of the most commonly used items are listed below:
| SUPPLIER | EQUIPMENT |
|--------------------------------------------------------------------------|---------------------------------------------------------------------------|
| Beckton Dickinson Labware, Plymouth, Devon, England. | Tissue culture dishes (35, 60, 90mm) |
| Bibby-sterelin Ltd, Stoney, Staffs., England. | Bacteriological dishes (90 mm) |
| Chance Propper Ltd., Warley, England. | 16-22mm glass coverslips |
| Clarke Electromedical Instruments, Reading, England. | Thin wall glass capillaries. |
| Costar, Cambridge, Massachusetts, USA. | 96 well plates, Disposable cell scrapers. |
| Eastman Kodak Co., Rochester, New York, USA. | X-ray film (XAR-5) |
| Fuji Photo Ltd, Japan. | X-ray film (RX) |
| Gibco Europe, Life Technologies, Paisley, Scotland. | Nunc 1ml cryotubes, Chamber slides (8, 4, 2, 1 well), Tissue culture flasks (25, 80 and 175 cm$^3$) |
| Griener Labortechnik Ltd., Dursley, England. | Eppendorf tubes, Pipette tips (200 and 500ul) |
| Whatman International Ltd., Maidstone, England. | 3mm chromatography paper. |
### 2.1.5. Antiserum.
| SUPPLIER | ANTIBODY |
|-----------------------------------------------|--------------------------------------------------------------------------|
| Takara Shozo Co., Ltd., Seta, Otsu, Japan. | Mouse monoclonal anti-E cadherin (ECCD-1 and ECCD-2) |
| | Mouse monoclonal anti-P cadherin (PCD-1) |
| Sigma Chemical Co., Ltd., Dorset, England. | Mouse monoclonal anti-Ncadherin (GC4) |
| | Mouse monoclonal anti-N CAM (OB11). |
| | Goat anti-mouse IgG FITC conjugate. Goat anti-rabbit IgG FITC conjugate. |
| | Goat anti-rat IgG FITC conjugate. |
| | Rat monoclonal Anti-Uvomorolin (DECMA1). |
| Zymega Research, San Francisco, CA 94080 USA. | Mouse monoclonal anti-connexin 43 |
| | Mouse monoclonal anti-connexin 32 |
| | Mouse monoclonal anti-connexin 26 |
| Amersham International PLC., Bucks., England.| Sheep anti-mouse hp-linked whole antiybody. |
| | Sheep anti-rabbit hp-linked whole antiybody. |
| | Sheep anti-rat hp-linked whole antiybody. |
2.1.6. Plasmids and bacterial host.
Compotent E.coli DH5α were obtained from B.R.L., Gibco Ltd.
pSVL - β-gal expression vector. A gift from S. Frame, Beatson Institute.
pNeo - neo resistance expression vector. B.R.L., Gibco Ltd.
2.1.7. Cell Culture Materials.
| SUPPLIER | MATERIAL |
|-----------------------------------------------|--------------------------------------------------------------------------|
| Life Technologies Ltd (Gibco Europe), Paisley, Scotland. | 2.5% (w/v) Trypsin
Penicillin / streptomycin / gentamycin / Amphotericin
200mM Glutamine
7.5% Sodium Bicarbonate
100mM Sodium pyruvate
Hepes Buffer
DMEM x10 |
| Northumbria Biologicals. | Foetal calf serum |
| Sigma Chemical Co., St Louis, USA. | Trypan blue |
2.1.8. Cell lines.
| CELL LINE | DESCRIPTION |
|-------------|------------------------------------------------------------------------------|
| Rat2 | Normal rat fibroblast, TK deficient* |
| R24E | Rat2 - E cadherin transfectant* |
| 10T1/2 | Normal mouse fibroblast |
| S180 | Mouse sarcoma derived fibroblast* |
| S180E217 | S180 E cadherin transfectant* |
| S180NII | S180 N cadherin transfectant* |
| S180N2A1 | S180 N cadherin transfectant* |
| BICR | Rat mammary carcinoma fibroblast |
| SVT2 | 3T3 - SV40 transformed cell line |
* Cell lines were a gift from Prof. F. Tato, Rome, Italy.
2.2. Tissue Culture Techniques.
2.2.1. Cell maintenance.
Cell lines used throughout this thesis are listed in the materials section 2.1.8. All cell lines were maintained in DMEM, which was supplemented with 10% (v/v) foetal calf serum, (unless stated otherwise) and 2mM L-glutamine. This media is referred to as DMEM10%. The following cell lines were maintained in DMEM10% supplemented with 500µg/ml hygromycin: S180, S180E217, S180NII, S180N2A1, R24E. Cell lines which were transfected with the β-gal expression vector were maintained in media containing 500ug/ml G418.
Cells were routinely sub-cultured every 4 days unless stated otherwise in the text or figure legends. The cells were cultured in 90mm tissue culture plates in a 7% CO₂ atmosphere at 37°C. Maintenance of the cells was carried out in a laminar-flow-hood. All of the cell lines were routinely examined for mycoplasma infection and were consistently negative.
2.2.2. Measurement of growth parameters.
126.96.36.199. Determination of cell viability
Cell viability was determined by mixing 25µl of cell suspension with 75 µl of 0.05% (w/v) trypan blue in PBS. The membranes of dead cells lose their integrity allowing trypan blue to enter the cell. The numbers of live and dead cells were counted using a Neubauer haemocytometer. The total number of cells in 4 x 16 square grids was multiplied by $10^4$ to estimate the number of cells per ml of culture:
\[
\% \text{ viability} = \left( \frac{\text{number of live cells}}{\text{total number of cells}} \right) \times 100
\]
188.8.131.52. Population doubling time.
Cells were seeded at $5 \times 10^5$ cells per 90mm dish. The number of live cells (using trypan blue to determine cell viability) were counted every 12 hours using a haemocytometer. Growth curves were plotted, from which population doubling times were determined.
184.108.40.206. Saturation density.
Cells were seeded at $5 \times 10^5$ and counted every 12 hours. Saturation density was considered to have occurred once the size of the cell population no longer increased. The labelling index (the proportion of cells incorporating [³H]-thymidine) of the cultures was taken every 24 hours (see section for 2.5 procedure).
220.127.116.11. Growth in low serum.
Cells were plated at $5 \times 10^5$ per 90mm dish in normal media and left overnight to attach. The media was removed and the cells washed twice with PBS. Fresh, low serum media, (0.5%FCS) was added and the cell number counted every 12-18hrs.
2.2.3. Transfection of cell lines with a $\beta$-gal expression vector.
Transfections were performed using the GIBCO Lipofectamine™ transfection kit following the procedure below. Cells were co-transfected with the plasmids pSVI ($\beta$-gal expression vector) and pNeo (plasmid containing a G418 resistance gene). Cells expressing the pNeo vector were selected in media containing G418 at the appropriate concentration (500µg/ml for the majority of cell lines with the exception of Rat2 cells: 900µg/ml).
• Exponentially growing cells were seeded at $3 \times 10^5$ cells per 35mm dish in DMEM10% and left for 24hours at 37°C in an atmosphere of 5% CO$_2$.
• 10µl of lipofectamine was mixed with 100µl of serum free media in a 1ml eppendorf. In a separate eppendorf 100µl of serum free media was mixed with 1µg of pNeo and 10µg of pSVI. The contents of the two eppendorfs were mixed and left for 45 minutes or until a precipitate formed. The precipitate was added to the cells and left overnight. Two mock control mock transfections were set up, one in which only pNeo was transfected and one in which plasmids were replaced by an equal volume of PBS.
• The following day the media was removed and the cells washed with PBS. Fresh media was added and the cells left for a further 24 hours. At this point the cells were split at 1:10 into 10, 90mm petri dishes and 10mls of selection media added. This selection regime was applied for 2-3 weeks, and the media replace every 3 days. Resistant colonies appearing over this time were ring cloned, (see below).
18.104.22.168. Ring cloning of resistant colonies.
• The position of G418 resistant colonies were marked on the bottom of the culture dish and the media removed. Cells were washed twice with PBS. Sterile rings were carefully applied to the marked colonies, (rings were prepared by cutting the end of yellow pipette tips with a scalpel, autoclaving and greasing the base with a small amount of sterile Vaseline prior to use).
• 50µl of trypsin was added to each ring and left for 1-3 mins. The cells were resuspended in the trypsin and added to 1ml of normal medium in a well of a 24 well plate.
• Successful ring cloned colonies were maintained in media containing G418 at appropriate concentrations and expanded by subcloning into 90mm dishes.
2.2.4. Preparation of conditioned media.
Conditioned media was prepared by growing cultures to confluence in DMEM10% medium. Conditioned media was removed after 10 days and centrifuged at 3000xg for 10 min to remove debris and used directly or stored at -20°C (for a max. of 7 days).
2.2.5. Frozen cell stocks.
Frozen cell stocks of each cell type were maintained in liquid nitrogen. Cell lines were trypsinised and resuspended at $10^6$ cells per 1ml of appropriate media supplemented with 10%DMSO. The cell suspension was added to 1ml Nunc cryotubes, frozen at -70°C overnight and transferred to liquid nitrogen vats for long term storage.
2.3. Gap junctional communication assays.
2.3.1. Dye transfer.
Levels of homologous communication were measured by micro-injecting the cells with the tracer dye Lucifer Yellow CH. Cells were grown in 60mm tissue culture dishes until 80-100% confluent. Immediately prior to the dye injection the media was removed and replaced with fresh media buffered with 25mM Hepes (in place of Sodium Bicarbonate). The cells were then transferred to a 37°C microscope stage and the lid of the dish removed. An individual cell was selected and iontophoretically injected with dye using micro electrodes made from "kwik-fill" thin-wall glass capillaries which were filled with 10µl of 4% Lucifer Yellow CH as described by Pitts and Kam (1985). The cells were injected with dye for 2 mins using a current of 10nA in 0.5 second pulses at 1Hz and the process monitored using a Leitz Diavert inverted microscope with UV (epi-illumination) or visible (phase contrast) light sources. The extent of dye spread to neighbouring cells was recorded and photographed immediately.
2.3.2. Nucleotide transfer
Nucleotide transfer was used to examine the levels of heterologous and homologous communication amongst the cell lines selected. The technique of nucleotide transfer is based on the transfer of $[^3H]$-nucleotides (derived from $[^3H]$-uridine) through gap junctions. The labelled nucleotides are incorporated into the
RNA of coupled cells and the extent of transfer can be analysed using autoradiography.
- $2.5 \times 10^5$ exponentially growing cells representing the recipients were seeded onto a single well chamber slide. $1.5 \times 10^5$ cells, cells representing the nucleotide donor cells (the same cell line as the recipients for homologous communication and a different cell line if studying heterologous communication) were seeded onto a separate single well chamber slide.
- When the recipients were 70-80% confluent, the donor cells were pulsed with 0.46MBq of $^{3}H$-uridine per ml of media for 4 hours. Cells were then washed 4 times with unlabelled medium and trypsinised. After 2-3 mins the trypsin was removed and the cells resuspended in 2mls of DMEM10%. Approximately 10µl of this suspension was added to the recipient cells and incubated at 37°C for 4 hours.
- The co-cultures were then washed 3 times with PBS and fixed in formal saline (0.9% NaCl, 10% Formaldehyde) for 30 mins.
- Cultures were briefly washed with PBS followed by a 5 minute incubation in cold 5% trichloracetic acid (TCA). Cultures were then submerged into gently running cold water for 30-60 mins, washed once with cold ethanol and allowed to dry prior to autoradiography (see section 2.5.2 for autoradiography procedure).
2.4. Focus formation assay.
In a basic focus formation assay normal and transformed cells were plated at $10^6$ and 500 cells per 90mm dish respectively. Controls were represented by separate cultures of each cell type seeded at the same densities as above. All experiments were set up in at least duplicate. Once experiments were set up, cultures were maintained in a humidified incubator at 37°C. After 12 days cultures were fixed in 1% formaldehyde and 0.1 % gluteraldehyde in PBS they were then stained with x-gal for 8 hours (section 2.5.1) and counterstained with Giemsa (1:10) for 10 mins. The number of colonies or foci per plate were counted and their average size (area) determined. In initial experiments a minimum threshold size of 0.4mm (0.125mm$^2$) was used as a cut-off point below which colonies were neither counted or measured. Deviations from this basic format are described in detail where appropriate in the results text and figure legends.
In some focus assays the average focus cell density was measured. Cell density is given as the number of cells / UA. A UA of 0.175mm$^2$ was chosen as this was the size of the eye piece grid and large enough to obtain sufficient data with statistically confidence. Average focus cell density was obtained from examining 5 foci and 5
respective control colonies per plate. For each focus cell density was derived from 10 UA's (or the maximum possible when focus size was small).
2.5. Proliferation assay.
Focus assays were set up in the same way as the focus formation assay unless stated otherwise. However, 18 hours prior to fixing, the cells were pulsed with $^{[3]}$H-thymidine (0.185MBq/ml). The media was removed and the cells washed 3 times with PBS. Cultures were fixed in 1% formaldehyde and 0.1% gluteraldehyde in PBS and stained in x-gal for 8hrs (section 2.5.1). The cultures were then submerged in a 5% solution of TCA for 5 mins, washed in running water for 45 mins and rinsed with cold ethanol. The cells were then exposed to photographic emulsion following the procedure in section 2.5.2.
2.5.1. Staining procedure for cells expressing the $\beta$-gal lineage marker
Prior to staining the following solutions were prepared:
**Fixative:** 1% formaldehyde and 0.1% gluteraldehyde in PBS.
**Stocks for staining solution:**
a). 40mg/ml of 5-Bromo4-Chloro3-Indolyl$\beta$-D-Galactopyranoside (x-gal) in DMSO. Prepared before use.
b). 0.5M K$_3$Fe (CN)$_6$, (Potassium Ferricyanide). 16.42g in 100 mls dH$_2$O.
c). 0.5M K$_4$Fe (CN)$_6$3H$_2$O, (Potassium Ferrocyanide). 21.12g in 100mls dH$_2$O.
d). 1M MgCl$_2$ 6H$_2$O
**Staining Solution:**
To make 10mls of stain, sufficient for one 90mm plate the following solutions were added to a universal in the order shown.
9.45mls PBS, 250µl of (a) + 100µl of (b) + 100µl of (c) + 20µl of (d).
Cultures were incubated in the fixative for 30 mins and washed once with PBS. The cultures were then incubated for 8hrs at 37°C in the staining solution detailed above. Those cells that expressed $\beta$-gal turned a dark blue, the staining solution was removed, and the cultures washed twice with PBS and the cells either counterstained in a 1:10 solution of Giemsa for 10 mins, washed twice with PBS and mounted in 50% PBS/Glycerol or exposed to photographic emulsion for autoradiographic analysis (section 2.5.2).
2.5.2. Autoradiography of cells exposed to $^{3}H$-uridine or $^{3}H$-thymidine
Cells pulsed with $^{3}H$-thymidine or $^{3}H$-uridine were exposed to photographic emulsion following the procedure detailed below.
• A stock solution of Ilford-K5 photographic emulsion was prepared (in total darkness) by melting 100mls of emulsion at 40°C for 25mins, and diluting with 33mls of warm water, (this solution was stored in complete darkness at 4°C and re-melted prior to use).
• TCA treated cells on dishes or slides were, in total darkness, coated with a thin layer of this stock solution. Approximately 3mls of the molten K5 solution was applied to a 90mm plate, whilst slides were dipped in to a slide mailer containing 15mls of emulsion, the excess poured off and the plates / slides stored in a light proof box with a LARGE sachet of silica gel. After 5-7 days the slides or plates were developed:
• In complete darkness, slides and plates were submerged in D19 Kodak developing solution for 5 mins followed by washing in cold water for 1 minute. Samples were then submerged for a further 5 mins in a solution of 1:10 Ilford Hypam fixer, followed by a final wash in water at which point samples were either stored or counter stained with Giemsa (1:10).
2.6. Apoptosis assay.
An in situ cell death detection kit (supplied by Boehringer Ltd; 2.1.2) was used to determine if cells in culture were undergoing apoptosis. In general, cells entering an apoptotic state undergo a characteristic pattern of structural changes in the nucleus and cytoplasm. A rapid degradation of the plasma membrane is associated with extensive damage to chromatin and DNA cleavage into oligonucleosomal length DNA fragments. The kit utilises immunocytochemical detection of apoptosis at the single cell level based on in-situ labelling of apoptosis induced DNA strand breaks. The procedure below was followed:
• Cultures were prepared in 90mm dishes (see figure legends for details), grown for desired period and fixed in freshly prepared 4% paraformaldehyde in PBS (pH7.4) for 30 mins at room temperature.
• Cells were then permeabilised in a 0.1% Triton X-100 in 0.1% sodium citrate solution for 20 mins. They were then rinsed twice with PBS and 50µl of TUNEL reaction mixture (Terminal deoxynucleotidyl transferase-mediated nick end labelling) was applied for 60 mins. To prevent evaporative loss of the TUNEL solution samples were covered with coverslips and incubated at 37°C in a humidified chamber.
• After rinsing the samples 3 times with PBS they were mounted in 50% glycerol/PBS and viewed under UV light using a Leitz vario orthomat microscope.
• A negative control was included in each experiment - fixed and permeabilised cells were incubated with Label solution (supplied) rather than TUNEL, incubated at 37°C for 60 mins and viewed under UV illumination. As positive controls, cells were treated 100µl of DNase 1 (supplied) for 10 mins prior to rinsing in PBS and application of TUNEL.
2.7. Nucleic acid procedures.
2.7.1. Growth, transformation and storage of competent cells.
The competent *E.coli* strain DH5α, (which is suitable for transformation with plasmid DNA) was purchased from B.R.L. and stored in 20µl aliquot's at -70°C.
• Cells to be used in the transformation were thawed on ice and gently mixed. To each 20µl of competent cells 100ng of DNA (in a final volume of 100µl) was added and incubated on ice for 30 mins. The DH5α cells were then heat shocked at 42°C for 45 seconds followed by incubation for 2 mins on ice, after which 80µl of SOC medium (2% bacto-tryptone, 0.5% bacto-yeast extract, 9mM NaCl, 2.5mM KCl, 10mM MgCl₂, 20mM glucose) was added.
• To allow the expression of the antibiotic resistance marker, (ampicillin) the cells were transferred to an incubator and shaken for 1hr at 37°C. The cells were then spread onto agar plates (1.5% agar in L-broth) containing 100mg/ml of ampicillin and the plates incubated upside down at 37°C overnight to allow for the formation of colonies.
• Sterile pipette tips were used to pick colonies which were then transferred to a universal container containing 3ml of L-broth and 100mg/ml of ampicillin and incubated for 4-6 hours at 37°C in a shaking incubator. From this solution subsequent mini or maxi plasmid preps could be made.
• Glycerol stocks were prepared from DH5α cells containing the plasmid required by mixing 1ml of DH5α culture with 1ml of 40% glycerol and frozen in Eppendorf tubes at -70°C.
22.214.171.124. Large scale plasmid preparations (Qiagen).
• 3ml of the bacterial solution prepared in section 2.7.1. was transferred to a 500ml L-broth culture, containing 50µg/ml ampicillin and left shaking overnight at 37°C.
• The bacteria were pelleted by centrifugation at 5000rpm for 10 mins in a Sorval centrifuge (GS-3 rotor) and the supernatant removed. To obtain a preparation of approximately 1mg plasmid DNA per ml the following procedure was followed:
Solutions and buffers, supplied in the Qiagen kit.
P1- 50mM tris, 10mM EDTA, RNase 100µg/ml, pH 8.0
P2- 0.2M NaOH, 1% SDS
P3- 2.55M KAc, pH8.4
QBT- 750mM NaCl, 50mM MOPS, 15% ethanol, 0.15% Triton X-100
QC- 1M NaCl, 50mM MOPS, 15% ethanol, pH7.0
QF- 1.25M NaCl, 50mM MOPS, 15% ethanol, pH8.2
Procedure:
• The bacterial pellet was resuspended in 10ml of buffer P1, to which 10ml of buffer P2 was added and incubated at room temperature for 5 mins.
• 10ml of P3 buffer was mixed with the above solution and incubated on ice for 20 mins. The precipitated material was pelleted by centrifugation at 4ºC for 30 mins at 10000rpm in a Sorval centrifuge (HB-4 rotor). A Qiagen-tip 500 column was equilibrated with 10ml of buffer QBT and the supernatant promptly applied to it.
• The column was washed twice with QC buffer and the plasmid DNA eluted by passing 15ml of QF buffer through the column.
• The DNA was precipitated with 0.7 volumes of isopropanol and pelleted by centrifugation at 10000rpm at 4ºC for 30 mins in a Sorval centrifuge (HB-4 rotor). 70% ethanol was used to wash the DNA pellet which was then allowed to air dry before being redissolved in 500µl of water and stored at -20ºC.
2.7.2. Determining nucleic acid concentration.
The concentration of nucleic acid in a solution was determined spectrophotometrically, by first calibrating the spectrophotometer (Beckman) using a water blank and then diluting the samples in water and reading the absorbency at wavelengths 260nm and 280nm in a quartz cuvette with a pathway of 1cm. For plasmid and genomic DNA samples an A260 of 1 was taken to correspond to 50mg/ml and for RNA an A260 of 1 was taken to correspond to 40mg/ml.
2.7.3. Agarose gel electrophoresis.
• Gels were prepared by dissolving 1g of agarose in 100ml of 1x TBE buffer (5x TBE is 0.45M Tris, 0.45 M boric acid, 12.5mM EDTA, pH8.3) and boiling until the agarose dissolved. The gel mix was allowed to cool to approximately 50ºC before ethidium bromide was added, to a final concentration of 1mg/ml.
• The gel was cast and allowed to set at room temperature before the comb was removed and the gel placed in the gel tank.
• The DNA samples and DNA standards (Boehringer Mannheim) for electrophoresis were mixed with 1/6 vol of DNA gel loading buffer, loaded and resolved by electrophoresis. Electrophoresis was carried out in 1x TBE buffer and run for 30-60 mins at 100v.
• On completion of electrophoresis the size of the DNA fragments were assessed by illuminating the gel on a 312nm transilluminator and compared to the positions of the DNA standards. A digital and photographic record of the UV illuminated gel was taken on a Appligene imager.
2.7.4. Extraction of RNA from mammalian cells.
RNA was extracted from the cells of interest using the Biogenesis RNA isolation kit RNAzol-B. Samples obtained were used directly for Northern analysis or purified further to obtain mRNA (section 126.96.36.199).
• The cells to be assayed were grown to approximately 80% confluence on 90mm dishes. They were then scraped and homogenised (using a cell scraper) in 0.8ml of RNAzol B.
• The homogenates were transferred to a sterile 1ml eppendorf tube to which 80µl of chloroform was added. The tubes were stored for 5 mins at 4ºC and then centrifuged for 15 mins at 12,000g.
• The aqueous phase was collected and RNA precipitated with 0.4ml of isopropanol for 45mins. The RNA was pelleted by a 15 minute spin at 12,000g and washed once with 70% ethanol. Once the ethanol was removed the RNA was resuspended in 30-100µl of water, quantified and stored at -20ºC.
188.8.131.52. mRNA purification.
RNA extracted from mammalian cells were further purified to obtain mRNA using the DYNAL™ purification kit. Solutions and procedures are listed below:
Solutions and buffers.
2X Binding buffer: 20mM Tris-HCL pH7.5, 1M LiCl, 2mM EDTA in a volume of 2ml.
1X Washing buffer: 10mM Tris-HCL pH7.5, 0.15M LiCl, 1M EDTA in a volume of 2ml.
1X Elution Buffer: 2mM EDTA pH7.5 in a volume of 2ml.
Procedure:
• 100µg of total RNA was diluted into 100µl of water and heated to 65ºC to disrupt the secondary structures. 0.2ml of dynobead suspension was aliquoted to a 1ml eppendorf and placed in the Magnetic Particle Holder (MPH), supplied. After 30 secs the supernatant was removed and discarded leaving behind the beads.
• The eppendorf was removed from the MPH and the beads washed with 100µl of 2x binding buffer. A further 100µl of binding buffer was added and mixed gently with the RNA / bead solution. This suspension was left to stand for 3 mins at room temperature.
• The eppendorf was returned to the MPH and the supernatant removed. The remaining beads were washed twice with 200µl of washing buffer and RNA eluted from the beads by the addition of 20µl 1X Elution buffer. This solution was heated to 65°C and placed back in the MPH where the mRNA solution was removed quantified and stored at -70°C until required.
2.7.5. Northern Analysis.
• Total RNA was loaded at 10-20µg/well and polyA RNA loaded at 3-6µg/well. Before loading, RNA volumes of approximately 5µl were mixed with 3 volumes (15µl) of RNA loading buffer (350µl formaldehyde, 1µl formamide, 150µl DNA loading buffer, 10µl ethidium bromide [stock 10mg/ml] and 200µl 5x MOPS buffer containing 0.1M MOPS, 40mM CH₃COONa and 5mM EDTA, pH7.0) and heated for 15 mins at 65°C.
• The samples were then loaded onto a 150ml flat-bed 1% (w/v) agarose gel containing 30ml of 5x MOPS buffer and 26.3ml of formaldehyde. The electrophoresis buffer was 1x MOPS buffer and this was recirculated during an 12hr run at 30V.
• Prior to blotting the gel was washed in dH₂O for 15 mins to remove the formaldehyde. RNA was transferred to nylon membrane (Hybond N+) by overnight capillary blotting (described by Sambrook et al., 1989) using 20xSSC buffer. The RNA was UV-crosslinked using a UV Stratalinker 1800 and the positions of 18S and 28S ribosomal RNA marked. Hybridisation to specific probes was carried out as described in section 184.108.40.206.
220.127.116.11. Radiolabelling of cDNA probes.
For Northern blot analysis, α[³²P]dCTP labelled DNA fragments were produced using the random priming technique. An Oligo-labelling kit (Pharmacia) was used to synthesise new cDNA strands in the presence of radioactively labelled dCTP.
• For each probe, 50-100ng of appropriate DNA, in a volume of 34µl with dH₂O, was linearised by boiling and cooled on ice.
• 10µl of reaction buffer (containing random primer sequences, dATP, dGTP and dTTP, at the required salt concentrations) were added along with 5µl of α[³²P]dCTP and 1µl of Klenow fragment. The reaction was incubated at 37°C for 1 hour.
• After incubation was complete, the unincorporated nucleotides were removed by centrifugation at 3000 rpm for 4 min through a Pharmacia spin column made of
Sephadex resin. The unincorporated nucleotides remained in the column while the labelled probe was collected in a clean Eppendorf.
• The degree of $\alpha^{[32P]}$dCTP incorporation was determined by analysis of 1µl aliquots in a Beckman LS5000CE counter and was $1-2 \times 10^9$ dpm/µg for each probe. The probe was boiled for 10 mins, placed on ice for 5 min and added to the hybridisation solution.
18.104.22.168. Northern blots.
• The nylon membranes were pre-hybridised at 42°C in Hybaid bottles or in plastic bags for 6-16 hrs in hybridisation buffer (5x SSPE, 10x Denhardt's, 2% SDS, 50% deionised formamide and 1mg/ml yeast RNA).
• Fresh hybridisation buffer was then added and the blot hybridised for 16 hrs with the relevant $^{32}$P-labelled cDNA probe at 42°C.
After hybridisation, Northern blot membranes were washed as follows:-
• 2 x SSC / 0.1% SDS 4 washes of 15 min. each at room temperature
• 1 x SSC / 0.1% SDS 1 wash of 20 min at 65°C
• 0.1 x SSC / 0.1% SDS 1 wash of 15 min at 65°C
The membranes were then exposed to Kodak XAR-5 film for 1 to 7 days at -70°C, with an intensifying screen. After exposure the membranes were stripped by incubating them for 2hrs in a solution of 0.1% SDS at 65°C in a shaking waterbath. The membranes were reprobed with GAPDH cDNA probe, as a loading control.
2.8. Protein procedures.
2.8.1. Protein preparations from mammalian cells.
• Total cell lysates were prepared for Western blot analysis. 1ml of cell lysate (4ml 1M Tris pH6.8, 5ml glycerol, 25ml 10%SDS, 200mg BrdB, 16ml dH$_2$O, 160µl of 2-Mercaptoethano) was added to a confluent 90mm plate of cells which were then homogenised using a cell scraper.
• The homogenate was transferred to a 1ml eppendorf and the DNA broken up by sonication, using a Fisons soniprep 150 MSE, for 30 seconds at 12 amplitude microns followed by a 5 minute spin at 12000g. At this point preparations were either stored at -20°C or boiled for 3 mins and loaded directly onto the gel (unless stated otherwise).
2.8.2. Liver Preparation for connexin 32 & 26 positive controls.
• The liver of one adult Spretus mouse was homogenised in 20mls of buffer (0.01% NaHCO₃). This homogenate was spun at 5000rpm for 15 mins in a Sorval ss34 rotor at 4ºC.
• The pellet fraction was resuspended in 3 mls of dH₂O and added to a small 50 ml beaker containing 20g of Sucrose. A further 40 mls of dH₂O was added to the beaker and the sucrose allowed to dissolve.
• The sucrose solution was divided equally into two Beckman centrifuge tubes and 15mls of dH₂O added carefully to the top of the sucrose solution. This sucrose density gradient was spun for 1.5 hrs at 25000rpm.
• The resulting interface was carefully removed and spundown at 10000rpm for 15 mins in a sorval ss34 rotor. The pellet fraction was divided into 50ml aliquots and either stored at -20ºC or loaded onto an 8% SDS-PAGE gel for Western blot analysis.
2.8.3. Polyacrylamide gel analysis of proteins.
Protein samples were resolved according to their molecular weight using sodium doecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), in all instances 15cm gels were used. Polyacrylamide content varied according to the protein which was being resolved, (connexins 10%, cadherins and CAMs 8%). For a single 15cm gel the following solutions were prepared (two gels were made one to be used for Western analysis and the second to confirm equal loading.
| Component volumes per 25 mls | Component solutions |
|-----------------------------|---------------------|
| **8% gel** | |
| 6.7ml | 8.3ml | 30% acrylamide/ 0.8% bisacrylamide |
| 6.55ml | 6.55ml | Running buffer (1.5M Tris, 4% SDS, pH8.9) |
| 11.5ml | 9.9ml | water |
| 250µl | 250µl | 10% ammonium persulphate (freshly prepared) |
| 15µl | 12µl | TEMED |
This resolving gel mix was poured between a sandwich of two glass plates sealed on three sides with a 0.75mm gasket. The solution was overlaid with isopropanol and left to polymerise at room temperature. Once the gel was set, the isopropanol was removed using Whatman 3MM filter paper and a 5% stacking gel solution added, together with the comb. The stacking gel was prepared as follows:
| Component volumes per 10ml | Component |
|---------------------------|-----------|
| 2ml | 30% acrylamide/ 0.8% bisacrylamide |
| 3ml | Stacking buffer (0.5M Tris, 4%SDS, pH6.7) |
| 7ml | water |
| 100µl | 10% ammonium persulphate (freshly prepared) |
| 10µl | TEMED |
Once the stacking gel had polymerised the whole gel was transferred to an electrophoresis tank. The tank reservoirs were filled with Tris-glycine electrophoresis buffer (50mM Tris, 50mM glycine and 1% SDS in water). After removal of the combs, the wells were flushed out with electrophoresis buffer to remove any excess stacking buffer. Total cell lysates were added direct to the gel. To the liver preparations an equal volume of 2x SDS gel loading buffer, (4% SDS, 0.2% bromophenol blue, 20% glycerol, 100mM Tris, pH6.8 and 4% 2-Mercaptoethanol) was added. Once loaded the samples were resolved by electrophoresis at a constant current 30mA/gel for 2-3 hours. Once the dye front had reached the bottom of the gels, they were removed, one was stained for 3 hours in Coomassie blue (2.5g Coomassie brilliant blue, 100ml glacial acetic acid, 450ml methanol and 450ml dH$_2$O), followed by incubation in destain (100ml glacial acetic acid, 450ml methanol, 450ml dH20) until protein bands were visible, in order to confirm equal loading. The second gel was used for Western analysis.
### 2.8.4. Western blotting analysis.
Protein samples (prepared as described in section 2.8.1) and resolved by SDS-PAGE (section 2.8.3).
- After removal from the tank, the gel was trimmed and measured. Six pieces of Whatman 3MM filter paper and 1 piece of Nitrocellulose membrane (ECL-hybond) were cut to an equal size. Transfer of proteins from the gel to the Nitrocellulose was performed on a semi-dry electroblotter.
- 3 pieces of pre-sized filter paper were soaked in the transfer buffer and placed on the semi-dry blotter.
- The nitrocellulose membrane was soaked in dH$_2$O and placed on top of the gel. The gel and membrane were then placed on top of the filter papers with the gel uppermost.
- 3 more pieces of buffer soaked filter paper were placed on the gel, the lid replaced and electroblotting performed at a current of Amps for 1hr 30 mins.
- Once the transfer was complete, the nitrocellulose membrane was placed in blotto, (blocking solution: 5% Marvel in PBS) and shaken for 1 hour at room temperature.
The membrane was then incubated overnight at 4°C with the primary antibody diluted in blotto to the appropriate concentration (see figure legends).
- This was followed by 3 washes with blotto for a duration of 20, 15 and 10 mins respectively. The horseradish peroxidase secondary antibody was diluted in blotto at 1:1000 and incubated with the membrane for 1 hour at room temperature.
- After a quick rinse with PBS the membrane received two 5 minute washes in blotto followed by a further two 5 minute washes in TBS-Tween (100mMTris pH 7.5, 0.9% NaCl, 0.1% Tween in a final volume of 100mls). Chemiluminescence was used to detect signals.
To develop the blots, an Amersham, *Enhanced chemilluminescence (ECL)* kit was used, according to manufacturers instructions. 10mls of Solutions 1 and 2 were mixed in a small beaker, added to a box containing the membrane and incubated for 1-2 mins. The solution was poured away and excess liquid was removed. The membrane was quickly wrapped in Saran wrap and exposed to Fuji RX film for 5 seconds to 60 mins, (depending on the strength of the signal).
**2.8.5. Immunofluorescence**
- Cells were grown until 80% confluent on single well glass chamber slides. After removal of tissue culture media the cells were washed once with PBS and fixed in ice-cold acetone for 30 mins.
- The acetone was removed and a 1.5% Marvel / PBS blocking solution applied to the cells for 10 mins. After removing the blocking solution 50-100µl of primary antibody was applied at the appropriate concentration (see figure legends) and left in a moist chamber at room temperature for 1-2 hours.
- The cells were then washed 3 times with the blocking solution and incubated with 50-100µl of secondary FITC-conjugated antibody for 1 hour. The cells received a final wash in blocking solution and were then mounted in 50% glycerol in PBS. Cells were analysed under UV illumination using a Leitz vario orthomat microscope.
CHAPTER THREE
RESULTS
3.1. Introduction
The aberrant growth of certain transformed cells can be suppressed when cultured in the presence of excess resting normal cells (Stoker 1967; section 1.6). This phenomenon has been correlated with the presence of heterologous gap junctional communication between the two cell types (Yamasaki and Katoh, 1988). The focus formation assay, which has been the major analytical tool for studying the phenomenon, typically consists of a co-culture of normal and transformed cells seeded at $10^6$ and 500 cells per dish respectively. After a period of 7-14 days the number of foci are counted and measured, compared to the control (separate cultures of 500 transformed cells) and the extent of inhibition calculated. In its present form the focus assay can only be used to study those cells that escape inhibition; single cells or small, possibly slow growing, colonies cannot be identified. The information that can be gained from this assay is limited to determining whether suppression occurs. As a result investigators have concentrated on the role of junctional communication in the inhibition phenomenon and neglected to examine, in any detail, the phenomenon itself. There are several important questions regarding this mode of suppression which remain to be answered:
1. Is there a threshold colony size, above which the transformed cells maintain their aberrant growth phenotypes?
2. In large foci is the inhibition greatest at the focus periphery rather than in the centre, as might be expected if GJIC is the pathway of suppression?
3. Several reports have shown that even where there is no inhibition of focus number there is often inhibition of focus size. Is this due to compression of the foci by the physical presence of the normal cells or to a reduction in growth rate relative to the controls?
4. Do transformed cells respond in different ways when different normal cells are present?
5. Do the transformed cells stimulate normal cells to divide?
6. Is the growth of the transformed cells sometimes stimulated by the presence of the normal cells (possibly by a feeder effect)?
3.1.1. Experimental system and selection of cell lines for study.
For this project a new assay has been developed which will facilitate the study of suppression in greater detail. A lineage marker, $\beta$-galactosidase has been introduced into the transformed cells to enable them to be unambiguously identified and counted in coculture. The proliferative status of normal and transformed cells has been determined using a growth assay based on $[^3H]$-thymidine incorporation and analysis by autoradiography. This has allowed the study of cell-cell interactions which lead to changes in growth, to be assessed by analysis of individual cells. Answers to questions
such as those listed above should provide more information on a phenomenon that may play an important role in primary defence against cancer (Section 1.6).
In order to examine the inhibition in detail, cell lines were required which showed the phenomenon described by Stoker. Ideally, a set should be included that contains a tumour cell type that is poorly coupled, produces foci on a background of normal cells and upon genetic modification to correct the defect in junctional communication show an increase in junctional communication and inhibition of focus formation. In previous studies many investigators have used phorbol-esters (usually TPA) and growth regulators such as cAMP as a means of manipulating the levels of gap junctional communication between cells. However, these chemicals act only transiently and are not specific modulators of GJIC; their mode of action on this pathway is also poorly understood. The genetic manipulation of protein expression to stably regulate GJIC provides a more specific means of analysis. Appropriate cell lines were obtained from Prof. F. Tato (Rome).
The tumour cell line S180 is poorly coupled (Musil et al 1990, Prowse 1992) and forms foci in the presence of excess, growth inhibited normal Rat2 cells (Tato, personal communication). However, upon transfection with a cDNA for E or N cadherin their focus forming ability is markedly reduced (Tato personal communication) and it might be expected, on the basis of work by Musil et al (1990; section 1.4.6) that these cadherin transfected cells (S180E217 - E cadherin transfectant, S180NII & S180N2A1 - N cadherin transfectants) should show increased levels of GJIC. In addition to Rat2, an E cadherin transfected Rat2 clone (R24E) was available that was reported to have an elevated level of GJIC (Tato, personal communication). This will provide an opportunity to examine if changes in the communication phenotype of the normal cell effect the inhibition phenomenon.
The low level of homologous communication observed in cultures of S180 cells is typical of many tumour cell types and it is often suggested that the reduced coupling contributes to the escape from normal growth control (section 1.4.6). However, tumourigenesis is not always associated with loss of coupling (section 1.4.6), so it is unlikely that all well coupled tumourigenic or transformed cells are suppressed. Such cells may have partially, or completely, lost the ability to respond to inhibitory growth signals that use the junctional pathway. Analysis of transformed cell lines that are coupled to normal cell lines, but are not inhibited by them, should provide a useful comparison, as well as a control for focus inhibition caused by other factors such as cell crowding or nutrient deprivation. Therefore, focus forming cell lines which show high levels of communication have been included in this study. With these cell lines even low levels of growth inhibition should be detectable using the more sensitive assays developed for this project. Furthermore, analysis of uninhibited transformed cell lines can be used, together with the S180 cells, to examine whether normal cells are stimulated by proliferating transformed cells.
To obtain cell lines with these characteristics the communication phenotypes of a range of transformed cells was surveyed. Two were selected; BICR, a rat mammary carcinoma cell line that is very well coupled relative to S180, and SVT2, a 3T3-SV40 transformed cell line that shows intermediate levels of communication and high tumourigenicity (tumours are produced within 5-7 days of a subcutaneous injection, Fernandez et al 1992). The results are presented in the following section.
The focus forming ability of the selected cells was then examined in the presence of normal Rat2 and 10T1/2 cells. The 10T1/2 cell line has been used extensively in previous studies that examined the focus forming potential of 10T1/2 transformed cells (Mehta et al 1986 & 1991). Here the effects of 10T1/2 cells on unrelated transformed cell lines are examined. Furthermore, the inclusion of a second normal cell line in this study provides an opportunity to examine whether different normal cells have similar abilities to inhibit the growth of specific transformed cells.
The Rat2 cells used in this study have a low level of thymidine kinase and therefore incorporate $^3$H-thymidine at low levels. This leads to a reduced, but detectable grain count above those cells that are going through S-phase, when analysed by autoradiography. However, this should provide a convenient reporter for GJIC as dividing Rat2 cells in co-culture with dividing transformed cells will have higher grain counts when the two cell types metabolically co-operate.
The following results chapter has been divided into 3 parts. Part 1 deals with the characterisation of the growth and communication phenotypes of the different cell lines. Part 2 examines the focus forming ability of the various transformed cells on different backgrounds of normal cells. Part 3 describes the new assay system, developed for this project, and its application to the study of growth inhibition in the cell lines characterised.
3.2. THE GROWTH AND COMMUNICATION PHENOTYPES OF THE CELL LINES.
3.2.1. Growth phenotypes of cell lines under study.
For this, study growth inhibition of the transformed cells is contingent on the presence of excess, growth inhibited normal cells. Therefore conditions must be established which markedly reduce the growth of normal cells, but not the growth of transformed cells in separate cultures. The two methods commonly used are high cell density or serum deprivation. During preliminary experiments it was found that growth inhibition of Rat2 and 10T1/2 cells could be achieved more reproducibly by growing cultures to saturation density (SatD) rather than by reducing the serum concentration in the media to 0.5%. The saturation density of all of the cell lines was examined using a proliferation assay based on $^{[3}H$]-thymidine incorporation and analysis by autoradiography.
Separate cultures of each of the cell lines were set up at $5 \times 10^5$ cells / 90mm dish, in DMEM10% (DMEM growth medium supplemented with 10% FCS). Cell counts were then taken every 12 hours, using a haemocytometer, to measure total cell number. Trypan blue staining was used to measure cell viability (section 2.2.2). Growth curves were plotted from which the population doubling time (PDT) and terminal density (TD) were determined. TD refers to the stauaration density of the normal cells and the density at which the transformed cells begin to die due, for example, to high pH. The $\beta$-gal transfected cell lines were also analysed to ensure the exogenous gene did not alter their growth characteristics.
The labelling index (proportion of cells incorporating $^{[3}H$]-thymidine) was also measured in order to determine the proliferative activity of the cell population. Cultures were set up at $5 \times 10^5$ cells / 90mm dish in DMEM10% and analysed every 24 hours. Prior to fixation (in 1% formaldehyde and 0.1% gluteraldehyde in PBS for 30 mins) the cells were pulsed for 18 hours with $^{[3}H$]-thymidine (0.185MBq/ml). The cultures were then analysed using autoradiography (section 2.5.2). Growth parameters for each cell line are shown in Table 3.1.
Table 3.1. Growth parameters of cell lines.
| Cell lines | TD x10^6 | PDT (hours) |
|------------|----------|-------------|
| | β-gal - | β-gal + | |
| Rat2 | 4.5 (1.1)| 4.5 (0.8) | 19 (3.3) |
| R24E | 4.9 (0.6)| 4.8 (1.1) | 20 (2.8) |
| 10T1/2 | 4.4 (0.6)| 5.0 (1.0) | 25 (4.8) |
| S180 | 8.2 (1.1)| 7.1 (1.5) | * | 14 (2.0) |
| S180N2AI | 10 (2.4) | 10 (1.8) | * | 16 (1.6) |
| S180NII | 7.6 (0.8)| 7.3 (1.5) | * | 13 (3.5) |
| S180E217 | 6.7 (1.1)| 7.8 (2.1) | * | 14 (2.4) |
| SVT2 | 11 (1.8) | 10 (1.7) | * | 17 (4.2) |
| BICR | 4.5 (0.5)| 5.0 (0.8) | ‡ | 13 (1.5) |
Continued over page.
Table 3.1. Continued.
| Cell lines | LI (%) at 24 hrs | LI (%) at 48 hrs | LI (%) at TD |
|------------|-----------------|-----------------|--------------|
| | β-gal - | β-gal + | β-gal - | β-gal + | β-gal - | β-gal + |
| Rat2 | 100 | 100 | 4.5 (0.7) | 5.2 (2.4) | 2.4 (0.4) | 3.5 (1.5) |
| R24E | 100 | 100 | 4.8 (0.5) | 4.0 (1.8) | 2.7 (0.2) | 2.5 (0.4) |
| 10T1/2 | 100 | 100 | 8.2 (1.0) | 10 (3.5) | 4.8 (0.6) | 4.0 (1.0) |
| S180 | 100 | 100 | 75 (3.5) | 68 (5.4) | 43 (6.5)* | 38 (1.5)* |
| S180N2AI | 100 | 100 | 60 (5.0) | 66 (7.0) | 45 (9.3)* | 52 (5.5)* |
| S180NI | 100 | 100 | 67 (3.0) | 54 (6.0) | 36 (5.2)* | 30 (4.3)* |
| S180E217 | 100 | 100 | 58 (4.8) | 77 (5.4) | 32 (3.3)* | 35 (7.8)* |
| SVT2 | 100 | 100 | 80 (6.0) | 67 (9.0) | 47 (6.8)* | 52 (8.9)* |
| BICR | 100 | 100 | 50 (2.5) | 48 (3.5) | ‡ | ‡ |
Table 3.1. To obtain the population doubling time (PDT) and terminal density (TD) cultures were plated at 5 x 10^5 cells / 90mm dish in DMEM10%. Total cell counts were taken every 12 hours. For the proliferation analysis, cultures of each cell line were seeded at 5 x 10^5 cells / 90mm dish in DMEM10% and analysed every 24 hours. 18 hours prior to fixation cultures pulsed with [^3H]-thymidine (0.185MBq/ml) fixed in 1% formaldehyde and 0.1% gluteraldehyde in PBS for 30 mins. Cultures were then processed for autoradiography according to the method given in section 2.5. LI - Labelling Index. Standard deviations are given in parentheses. Data provided are averages from at least three replicate cultures. PDT values were not significantly different for β-gal+ and β-gal- cell lines (P>5%). (*) Indicates cultures where cells continued to grow at confluence and began to multi-layer and often detach. (‡) Indicates that BICR cells began to dye soon after reaching confluence, attempts were made to obtain the LI at confluence but the majority of cells detached during fixation.
The LI's for the Rat2 and 10T1/2 cells drop from 100% at 24hrs to 4.5% and 10%, respectively, after 48 hours. This drops further, to 2.4% and 4.8% upon reaching their respective terminal densities. The transformed cells maintain higher LI's (between 48% - 80% after 48 hrs and between 30% and 52% at the time when the normal cells reach saturation density). The transformed cell lines did not reach a defined saturation density but continued to grow, multi-layer and detach. Student t-tests carried out on the growth parameters obtained from the β-gal⁺ and β-gal⁻ cell lines indicates that the expression of the transfected β-gal gene in the transformed cells has no significant effect on their growth phenotypes (in all instances P>5%). Transfection of E or N cadherin into the S180 cells does not have a significant effect on the growth parameters measured here (P>5%). On the basis of these results the various cell lines provide the opportunity to examine whether, in co-culture, the normal cells, upon reaching high cell density, are capable of suppressing the growth of the transformed cells.
3.2.2. Communication Phenotypes.
22.214.171.124. Homologous communication.
The selected cell lines were examined for levels of homologous communication. Levels of communication in both β-gal⁺ and β-gal⁻ cell lines were compared to ensure the exogenous gene had no effect on their communication phenotypes. The levels of homologous communication which were recorded (Table 3.2), were obtained using two techniques; nucleotide transfer and dye transfer. The technique of nucleotide transfer is based on the transfer of [³H]-nucleotides (derived from ³H-uridine) through gap junctions. The labelled nucleotides are incorporated into the RNA of coupled cells and the extent of transfer can be analysed using autoradiography. The technique has advantages over dye transfer in that the cells incur no physical damage, as is the case in micro-injection, and the principle behind the technique has a direct relevance to biological events i.e. the exchange of metabolites over a long time course (4 hours rather than the 1-2 mins used for the analysis of dye transfer). Furthermore, it can be easily applied to determine levels of heterologous communication, as the donor cells can be intrinsically distinguished from the recipient cells by the larger grain counts over the donors, due to the preparative labelling period (Pitts & Simms 1977). However, dye transfer is more widely used and has, therefore, been carried out also, to see if the two techniques generate comparable data (for methods see section 2.3). Representative nucleotide and dye spreads are illustrated in Figure 3.2 and the average levels of homologous communication recorded for each cell line, are given in Table 3.2.
The two techniques appear to generate comparable data. For example, S180 and BICR cells are, respectively, the poorest and best coupled as determined by both methods of analysis. In the majority of instances nucleotide transfer is the more sensitive of the two techniques; this is not surprising given that transfer and incorporation of nucleotides occurs over 4 hours rather than the 2 mins allowed for dye transfer (a time chosen that generates large enough differences between coupled and uncoupled cells whilst limiting cell damage). Attempts to determine levels of communication for BICR cells using nucleotide transfer were made difficult by the fact that some of the cells were lost during fixation (see Figure 3.2.b.iii), leading to a decreased count. Fixation did not effect the other cell lines in this way.
Both Rat2 and 10T1/2 cell lines show levels of communication typical of many fibroblasts. The transformed cells, SVT2 and BICR, show comparable levels of communication with the normal cells, suggesting, that in these instances, homologous communication is a poor indicator of cell tumourigenicity.
S180 cells show a low level of communication which is typical of many tumour derived cell lines (Kalimi et al 1992). Transfection of the S180 cells with a cDNA for E or N cadherin increases the level of communication markedly. This is associated with a distinct change in cell shape to a flatter, fibroblast-like morphology, (Figure 3.2.c.ii & iii). In contrast to the S180 E & N cadherin transfectants, R24E cells (Rat2 E cadherin transfected cell line) show a significant reduction (P<1%) in homologous transfer in comparison to the Rat2 parental cell line. Two other clones of Rat2 E cadherin transfected cells were examined, R2013E and R206E, their respective homologous communication levels (as determined by dye transfer) were 8.8 (sd 3.9) and 13 (sd 4.7) respectively. Although the E cadherin expression appears to decrease the level of homologous communication in these cell lines, no apparent difference in morphology was observed between these cells and the parental Rat2 cell line.
Table 3.2. Homologous GJIC.
| CELL LINE | DYE TRANSFER β-gal⁻ | DYE TRANSFER β-gal⁺ | NUCLEOTIDE TRANSFER β-gal⁺ |
|-----------|---------------------|---------------------|---------------------------|
| Rat 2 | 19.4 (6.7) | 16.8 (4.9) | 32.3 (12) |
| R24E | 12.5 (6.4) | 12.0 (2.5) | 19.5 (8.7) |
| 10T1/2 | 10.6 (4.4) | 9.3 (1.2) | 22.4 (8.4) |
| S180 | 1.6 (1.3) | 2.7 (2.0) | 2.2 (1.5) |
| S180E217 | 12.0 (3.1) | 13.3 (4.2) | 20.3 (5.5) |
| S180N2A1 | 13.4 (3.7) | 12.3 (5.0) | 17.7 (6.5) |
| S180NII | 18.6 (7.7) | 20.8 (8.5) | 15.8 (6.6) |
| SVT2 | 25.3 (8.9) | 23.1 (4.5) | 19.6 (3.2) |
| BICR | 41.5 (9.5) | 47.5 (11) | 34.7 (8.6) |
Table 3.2. The proportion of injected or donor cells which showed transfer to at least one surrounding cell (frequency of transfer) was 100% for the majority of cell lines with the exception of S180 where the frequency of transfer was 60%. For dye injection, individual cells were injected ionophoretically with Lucifer Yellow CH for 2 minutes and the number of cells into which dye had spread were counted. The mean dye spreads shown are calculated from a minimum of 30 injections (10-20 injections per dish, 2-3 replicate dishes) and the mean nucleotide spreads calculated from the analysis of 30-50 donor cells, (10 donors per slide, 3-5 slides per cell line). Donor cells were prepared by growing cells to 70% confluence, labelling for 4 hours with [³H]-uridine (0.46 MBq/ml), then washing 4 times with unlabelled medium. After trypsinisation 100-500 donor cells were added to 80-100% confluent monolayers of recipient cells. After 4 hours the co-cultures were washed 3 times in PBS and fixed in formal saline (0.9% NaCl, 10% formaldehyde in PBS) for 30 mins. They were then extracted with TCA and processed for autoradiography according to the method given in section 2.5. Background levels of [³H]-uridine were determined by counting the number of grains over a minimum of 50 cells which were situated away from any donor cells and the average grain count per cell determined. Transfer was recorded when grain counts directly above the cells were greater than twice the background. Standard deviations are given in parentheses. The difference between the values obtained from the β-gal⁺ and β-gal⁻ cell lines is not significant (P>5%) for all of the cell lines.
Figure 3.2. Examples of homologous dye and nucleotide transfer.
ai. Rat 2
bi. BICR
ci. S180
di. S180 NIH
aii.
bii.
cii.
dii.
aiii.
biii.
ciii.
diii.
Figure 3.2. Photomicrographs of dye spreads, after microinjection of Lucifer Yellow CH, and of nucleotide transfer. Blue dot indicates cell injected with tracer dye. See Table 3.2. legend for experimental details. Figures ai-di are fluorescence images of the phase contrast micrographs shown in aii-dii. Nucleotide transfer micrographs are shown in Figures aiii-diii. Cell lines correspond to those shown in ai-di. Green arrows indicate donor cells. Bar 50μm.
126.96.36.199. Heterologous communication between normal and transformed cells.
Attempts were made to use dye transfer as a means of examining heterologous communication. In order to distinguish the two cell types in co-culture the transformed cells were first labelled with fluorescent microspheres (separate cultures of transformed cells were incubated for 5hrs with 4µl of the microsphere suspension supplied by Cambridge Bioscience - section 2.1.2). Cultures were then washed 4 times with PBS, trypsinised and approximately 1000 labelled cells were added to 80% confluent cultures of normal cells and left to attach for 4 hours.
However, during the dye transfer assays it became clear that the fluorescent glow from the microspheres was the same colour and in some instances the same intensity as the Lucifer Yellow CH tracer dye. Ambiguity arose when measuring dye transfer, particularly when the dye spreads were faint. Thus it was not always possible to determine with confidence when the dye had transferred into a labelled cell or whether the glow of the microspheres was responsible for the fluorescence observed - see Figure 3.3. Furthermore in some instances beads which had not been endocytosed by the transformed cells but were loosely attached to the cell surface, often detached when transferred to the co-culture, leaving the possibility that they could become endocytosed by the normal cell line leading to false identification. The technical problems associated with this technique made nucleotide transfer the preferred assay.
The experimental procedure for examining heterologous communication using nucleotide transfer is the same as that used to measure homologous communication (section 188.8.131.52), however, in this instance the donor cells were the transformed cells and the recipient cells were the normal cells.
Due to specificity of gap junction formation (section 184.108.40.206) two cell lines that each form homologous gap junctions may not necessarily form heterologous gap junctions. For example, communication is often restricted between epithelial cells and fibroblasts (section 220.127.116.11). The efficiency with which heterologous gap junctions form between the cell combinations examined here, varies in terms of the frequency and the level of nucleotide transfer. Average nucleotide transfers obtained for the cell combinations examined are provided in Table 3.3 and representative spreads are shown in Figure 3.4
In terms of levels of heterologous communication, R24E cells form the best normal cell partner with the transformed cell lines followed by Rat2 and 10T1/2 cells. However, the order (ranked by level of heterologous communication) in which the transformed cells communicate with the normal cells is generally the same regardless of which normal cell line is used. For example, BICR cells show the highest level of coupling to the normal cells, regardless of which normal cell line is the heterologous partner, and S180 cells show the lowest coupling to the normal cells.
Figure 3.3. Analysis of heterologous communication using fluorescent beads as lineage markers.
Figure 3.3. 80% confluent cultures of SVT2 cells were labelled with 4 µl of microsphere suspension (supplied by Cambridge Bioscience; section 2.1.2) for 5 hrs. These cultures were then washed 4 times with fresh medium, trypsinised and approximately 1000 cells added to 80% confluent cultures of unlabelled Rat2 cells. Co-cultures were left for 4 hours prior to dye injection analysis. In the above macrographs a Rat2 cell has been injected with Lucifer Yellow for 2 mins (injected cell indicated by blue dot). However, the fluorescent halo, which can be seen around the beads in the above figure, could be misinterpreted as faint dye spread, particularly when dye spreads in the surrounding cells were also faint. Furthermore, beads which were attached to the surface of the cells (but not endocytosed) often detached in co-culture and the possibility exists that they could be incorporated by unlabelled cells. Bar 50 µm.
BICR cells are very well coupled to Rat2 & R24E cells whereas S180 & S180E217 cells are poorly coupled to these cells. The remaining transformed cell lines show intermediate levels of communication with Rat2 & R24E cells (relative to BICR and S180 cells). All transformed cells communicate relatively poorly with 10T1/2 cells and no detectable transfer is observed when S180 cells are the heterologous partner. In all instances the S180 E and N cadherin transfected cells show increased heterologous communication relative to the S180 parental cells.
An example of gap junction specificity is illustrated here by the S180E217 cells which are well coupled to each other (Table 3.2) but are poorly coupled to the normal cells (Table 3.3). E cadherin tends to confer an epithelial phenotype on the cells which express it (Musil et al 1991) and the change in morphology of the S180E217 cells would tend to support this. However, these cells were relatively poorly coupled to R24E cells, which have also been transfected with E cadherin. This would suggest that E cadherin expression in both cell types is insufficient to ensure heterologous gap junction formation. However, R24E cells maintained a fibroblast morphology and an apparent decrease in homologous communication, and the possibility exists that although transfected with a E cadherin cDNA they do not express the protein. Protein expression has been examined in section 3.2.3.
It is not clear why the N cadherin transfected S180 cells show high levels of communication with the Rat2 cell lines yet are poorly coupled to the 10T1/2 cells. Studies have been carried out in the following section in order to examine the expression of specific adhesion and connexin proteins which may mediate the phenomenon of gap junction specificity seen here.
Table 3.3. Heterologous communication.
| CELL LINE | Rat 2 | R24E | 10T1/2 |
|-----------|-------|------|--------|
| | level | freq. % | level | freq. % | level | freq. % |
| S180 | 0.47 (0.2) | 23 | 1.07 (0.3) | 40 | 0.00 | 0 |
| S180E217 | 2.0 (1.5) | 37 | 4.80 (1.9) | 77 | 2.0 (1.4) | 53 |
| S180N2A1 | 12.3 (4.3) | 100 | 14.8 (5.0) | 100 | 3.70 (1.6) | 70 |
| S180NII | 6.10 (2.7) | 87 | 14.7 (4.7) | 100 | 0.9 (0.5) | 43 |
| SVT2 | 5.73 (3.8) | 80 | 8.30 (2.6) | 97 | 2.33 (1.3) | 33.3 |
| BICR | 15.9 (2.2) | 100 | 29.9 (9.5) | 100 | 4.57 (3.2) | 90 |
Table 3.3. For experimental details see section 18.104.22.168 & legend for Table 3.2. The mean level of transfer for each cell combination was calculated from the analysis of 30-50 donor cells, (10 donors per slide, 3-5 slides per cell line). The frequency (freq.) of transfer (proportion of donor cells which showed transfer to at least one surrounding cell) is given as a percentage. Standard deviations are given in parentheses.
Figure 3.4. Examples of heterologous nucleotide transfer spreads.
a). Rat2 + S180NH
b). Rat2 + S180N2A1
c). R24E + S180E217
d). Rat2 + SVT2
e). R24E + BICR
f). 10T1/2 + S180
g). 10T1/2 + S180NII
h). 10T1/2 + BICR
Figure 3.4. a-h show examples of nucleotide transfer between various normal and transformed cells in contact (cell combinations are given above each figure). Donor cells (transformed cells) are indicated by green arrows. See Table legend 3.3 & section 2.3.2. for experimental procedure. The above cultures were over-exposed to enhance visualisation. Bar 50 µm.
3.2.3. Expression of gap junction associated proteins.
It has been suggested that communication compartments *in vivo* are a manifestation of specificity of gap junction formation (section 22.214.171.124). It has been observed that the cells of such compartments follow different lineage pathways from cells of different compartments (Kam & Hodgins 1992; section 126.96.36.199). In culture, specificity of gap junction formation has been reported between a variety of transformed and normal cell lines (levels of heterologous communication are often very low or zero; Kalamí et al 1992). This is thought to arise due to a defect in either adhesion-mediated cell-cell recognition or the loss or modification of gap junction associated proteins (section 1.4.6). Restoration of the heterologous junctional pathway can result in the growth inhibition of the transformed cells (Mehta et al 1991; section 1.6).
The molecular basis of gap junction specificity is, however, not clearly understood. In an attempt to explain the observed patterns of heterologous communication among the cell lines in this study, expression levels of the following adhesion molecules have been examined; E cadherin, N cadherin and NCAM, together with the expression of the gap junction associated protein - connexin43 (Cx43).
Western blot analysis was used to detect level of expression and immunofluorescence was employed to localise protein expression within the cell.
188.8.131.52 Connexin43 expression.
Although the evidence is mainly correlative connexins have been widely reported as playing an essential role in the formation and regulation of gap junctions (section 184.108.40.206). Cx43 is the most widespread connexin and is expressed by a wide variety of vertebrate cell lines (Beyer et al 1989, Rogers et al 1990). It has been shown that cells which previously expressed other connexin types *in vivo*, express Cx43 in culture (Stutenkemper et al 1992). Cx43 is thought to be synthesised as an unphosphorylated 43kDa species (Cx43-NP). However, many cell lines which are well coupled, also express a phosphorylated, 47kDa species (Cx43-P$_2$; Saez et al 1986, Musil et al 1990, Musil & Goodenough 1991). Intermediate forms (44-46 kDa; Cx43-P$_1$) have also been observed (Musil et al 1990 & 1991). Figure 3.5.a. shows the results of Western blot analysis using a rabbit monoclonal antibody against Cx43 (section 2.1.5). Equal loading of samples was confirmed by Coomassie blue staining of replicate gels.
Cx43-P$_2$ is observed in all of the cell lines examined here, although the level of protein expression, as indicated by the intensity of the band, varies between the different cell lines (Figure 3.5.a). In the S180 set of cell lines, S180-parental cells predominantly express Cx43-NP and only low levels of Cx43-P$_2$. After these cells are transfected with cDNA for E cadherin, the expression level of Cx43-P$_2$ increases. This correlates with an increase in homologous communication (Table 3.2) and confirms reports published by Musil et al (1990) who showed that E cadherin expression in S180 cells results in
increased conversion of Cx43-NP to Cx43-P\textsubscript{2} and subsequently higher levels of communication. Results from the study carried out here also show that expression of N cadherin in S180 cells results in increased expression of Cx43-P\textsubscript{2} and increased communication. It is not known whether cadherins directly regulate GJIC or do so via the regulation of Cx43-P\textsubscript{2}.
Rat2 parental cells predominantly express Cx43-P\textsubscript{1} and only relatively low levels of Cx43-P\textsubscript{2}. 10T1/2 cells express very little Cx43 (of any form), yet both of these cell lines form gap junctions very efficiently (Table 3.2). Transfection of the Rat2 cells with E cadherin appears to increase the expression of Cx43 (phosphorylated and unphosphorylated). This is consistent with the data from the S180 series and would suggest that cadherins can regulate the expression of Cx43. The increased levels of Cx43-P\textsubscript{2} expression in R24E cells are not, however, associated with an increase in homologous communication within this cell line (Table 3.2).
High levels of Cx43 are expressed in BICR cells which is consistent with the finding of others (Prowse 1992).
It has been suggested that discrete, punctate Cx43 immunostaining at points of cell-cell contact is associated with EM-identifiable gap junction structures (Beyer et al 1989, Dermietzel et al 1989, Musil et al 1990). Immunofluorescence analysis was used to examine whether Cx43 distribution relates to the communication phenotypes off the various cell lines examined.
BICR cells showed the greatest amount of immunostaining predominantly cytoplasmic and perinuclear but also at points of cell-cell contact (Figure 3.5.b) and this is consistent with the high expression levels of this protein as indicated by Western blotting analysis. S180 cells show no detectable Cx43 immunostaining (Figures 3.5.c). However, the detection of Cx43 in these cells by Western blotting analysis would suggest that the immunofluorescence assay is not sufficiently sensitive or that the protein is masked and inaccessible to the antibody. This data does not support that of Musil (1991) who found that S180 cells do show very low levels of immunostaining although only at perinuclear sites. The S180 E and N cadherin transfectants do show discrete, punctate Cx43 immunostaining at points of cell-cell contact (Figure 3.5.d. & e). It is not clear which Cx43 species is responsible for this punctate staining as the antibody does not distinguish between the phosphorylated and unphosphorylated forms. Reports by Musil et al (1992) suggest that it is Cx43-P\textsubscript{2}, as they found that Cx43-NP distribution in S180 cells was limited to perinuclear staining and conversion to Cx43-P\textsubscript{2} correlated to a punctate staining pattern at cell contact points, which in turn would suggest that Cx43 phosphorylation is involved in gap junction formation. R24E cells show greater levels of Cx43 immunostaining than Rat2 or 10T1/2 cells. In all 3 cell lines the protein is localised throughout the cells in general, including points of cell-cell contact.
The expression of Cx26 and Cx32 was examined using appropriate rabbit monoclonal antibodies (section 2.1.5). None of the cell lines examined appear to express Cx32 or Cx26. Data not shown.
220.127.116.11. Cadherin expression.
Western blotting analysis revealed no endogenous E cadherin expression in S180 and Rat2 parental cells, SVT2, BICR or 10T1/2 cells. However, expression is observed in the cell lines transfected with E cadherin cDNA (S180E217 and R24E cells), together with the epidermal positive control cell line - P6 (Figure 3.6.a). S180E217 cells appear to express the highest amount of the 120kDa cell-cell adhesion protein. Immunofluorescence analysis reveals that the protein, in R24E and S180E217 cells is distributed along areas of cell-to-cell contact (see Figures 3.6.b & c). S180E217 cells show higher levels of immunostaining than R24E cells which is consistent with the Western blot data.
The group that originally carried out the transfections confirmed that the cell lines, S180NII and S180N2A1 both expressed N cadherin at the Western level and immunofluorescence analysis localised the protein to areas of cell-cell contact. In addition, S180, Rat2 and R24E cells, do not express N cadherin (Prof. F. Tato - personal communication). 10T1/2 and SVT2 cells do not express N cadherin - data not shown. BICR cells have previously been shown to express N cadherin at areas of cell-cell contact (Prowse 1993).
Using Western blotting analysis it was shown that none of the cell lines examined here expressed P cadherin - data not shown.
18.104.22.168. NCAM expression.
Expression studies using Western analysis, (Figure 3.7.a) reveal that the normal cell lines, Rat2, 10T1/2 and R24E and the transformed cell line SVT2 express NCAM (180kDa). Other published reports have shown (using different cell lines) that the protein can also be expressed as a 140kDa protein (Santoni et al 1989), and although recognised by the antibody used here, this protein species does not appear to be expressed in the cell lines examined. It can be seen in Figures 3.7.b-e. that the 180kDa NCAM species localises to the periphery of the cells, though not necessarily at areas of cell-cell contact.
Although the 16kDa protein, ductin, is believed to form the gap junction channel (section 22.214.171.124), antibodies against mammalian forms of the protein for immunofluorescence studies are not available. Examining expression of this protein at the transcriptional level via Northern analysis is complicated by the fact that it is ubiquitously expressed in mammalian cells, forming part of the vacuolar ATPase. Thus little information can be gained in how this protein is regulated in gap junctions using these types of analysis techniques.
Figure 3.5. Western blotting and immunofluorescence analysis of Cx43 expression.
a).
kDa 47 →
b) BICR
c) S180
d) S180NII
e) S180E217
f) R24E
bii)
cii)
dii)
eii)
fii)
Figure 3.5. a) Western blotting analysis of Cx43 expression. Protein samples were prepared from each cell line (as described in section 2.8) and ~20μg (estimated from Coomassie blue stained gels) loaded into separate wells of a 10% PAG. Samples were not boiled prior to loading. Primary antibody was Zymega mouse monoclonal anti-Cx43 at 1:1000. Arrow indicates Cx43 (47kDa species). Figures b-f Cx43 immunofluorescence analysis. b-i-fi are fluorescence micrographs of the phase contrast images shown in bii-fi. Cultures were prepared as described in section 2.8.4. Primary antibody was Zymega mouse monoclonal anti-Cx43 at 1:100. Bar 50μm.
Figure 3.6. Western and immunofluorescence analysis of E-cadherin expression
a). Western blotting analysis of E-cadherin expression. Protein samples were prepared from each cell line (as described in section 2.8) and ~20μg (estimated from Coomassie blue stained gels) loaded into separate wells of an 8% PAG. Samples were not boiled prior to loading. Primary antibody was Takara mouse monoclonal anti-E cadherin ECCD-2 at 1:5000. Arrow indicates E-cadherin (120kDa species).
Figures b-c. E-cadherin immunofluorescence analysis. b-i-c-i are fluorescence micrographs of the phase contrast images shown in bii-cii. Cultures were prepared as described in section 2.8.4. Primary antibody was Takara mouse monoclonal anti-E cadherin ECCD-2 at 1:100. Figure c-i was taken at a higher magnification than the corresponding phase image in Figure cii for which there was no equivalent objective available). Bar 50 μm.
Figure 3.7 Western and immunofluorescence analysis of N-CAM expression.
a).
180kDa
b) Rat2
c) R24E
d) 10T1/2
e) SVT2
bii) Rat2
cli) R24E
dii) 10T1/2
eli) SVT2
Figure 3.7: a) Western blotting analysis of NCAM expression. Protein samples were prepared from each cell line (as described in section 2.8) and ~20μg (estimated from Coomassie blue stained gels) loaded into separate wells of an 8% PAG. Primary antibody was Sigma mouse monoclonal anti-NCAM at 1:500. Arrow indicates NCAM (180kDa species). Figures b-e NCAM immunofluorescence analysis. b-i are fluorescence micrographs of the phase contrast images shown in bii-eii. Cultures were prepared as described in section 2.8.4. Primary antibody was Sigma mouse monoclonal anti-NCAM at 1:500. Bar 50μm.
Table 3.4. Summary of endogenous and exogenous expression of gap junction associated proteins.
| | Rat2 W IF | R24E W IF | 10T1/2 W IF | S180 W IF | S180E217 W IF | S180N2A1 W IF | S180NII W IF | SVT2 W IF | BICR W IF |
|-------|-----------|-----------|-------------|-----------|---------------|---------------|--------------|-----------|-----------|
| Cx43 | + + | + + | + + | + - | + + | + + | + + | + + | + + |
| E cad' | - - | + + | - - | - - | + + | - - | - - | - - | - - |
| N cad' | - - | - - | - - | - - | - - | + + | + + | - - | + + |
| NCAM | + + | + + | + + | - - | - - | - - | - - | + + | - - |
W - Western analysis, IF - immunofluorescence analysis. (+) indicates positive expression of the protein, (-) indicates no detectable levels of protein expression. Expression of N cadherin in S180NII and S180N2A1 cells was analysed by F. Tato, Rome. Expression of N cadherin in BICR was analysed by D. Prowse. All cells tested negative for Cx26 & Cx32, and P cadherin.
3.2.4. Summary.
A panel of cell lines has been established which meet the requirements outlined in section 3.1. S180 cells, which are poorly coupled, show increased gap junctional communication after transfection with cDNA for E or N cadherin. Well coupled transformed cells are represented by BICR cells and SVT2 cells. The growth phenotypes of the normal cell lines selected are such that they provide suitable background monolayers on which to test the growth of the transformed cells and the role of GJIC in the inhibition pathway.
The results presented here do not support a general hypothesis (Loewenstein 1979, Kalimi et al 1992) that levels of homologous communication are indicative of a cells tumourigenicity. The transformed and tumourigenic cell lines BICR and SVT2 (Brummer 1987, Fernandez et al 1992) have similar levels of communication to those of the normal, non-tumourigenic cell lines. Although the sarcoma derived S180 cell line shows very low levels of communication, the findings show that an aberrant growth phenotype is not always associated with loss of homologous GJIC.
In several studies where heterologous communication has been correlated to the inhibition phenomenon (Mehta et al 1986, Mikalsen 1993) only transfer frequency (the proportion of cells which showed transfer to at least one surrounding cell) has been used to measure communication, however this gives only a partial indication of the extent of communication. Frequency of communication is dynamic, i.e. a frequency of 50% does not necessarily mean that 50% of cells can communicate and 50% can't, rather, that at a point in time 50% are coupled. However, when cells are in contact, for however short a period, the level of communication (number of cells into which transfer occurs) is likely to depend on the number of channels and extent of coupling. The level of communication is likely to be more important for understanding the inhibition phenomenon. It has been suggested that the number of channels and extent of coupling is determined, at least in part, by the level of connexin expression.
126.96.36.199. Relationship between Cx43, cell-cell adhesion and communication.
Connexins are thought to be involved in the formation and/or regulation of vertebrate gap junctions (section 1.3). However, the mechanisms that regulate their function are poorly understood. The results obtained here using the S180 series of cell lines are consistent with reports published by Musil et al (1991). That is, expression of exogenous E cadherin in S180 cells results in increased conversion of Cx43-NP to the phosphorylated form (Cx43-P₂) and increased homologous communication. In addition, it has been shown here that N cadherin can also increase the conversion of Cx43 to Cx43-P₂ in S180 cells and increase their levels of homologous communication. This data would suggest that phosphorylated Cx43 is involved in gap junction formation and that the original defect in GJIC within the S180 cells was not due to a genetic defect in the Cx43
gene but rather a defect in the post-translational modification mechanism. The expression of Cx43-P2 may modulate homologous communication by regulating the formation of the channels or their open and closed states. However, it is not known how E & N cadherin regulate the phosphorylation states of Cx43. The data from the S180 series of cell lines would suggest that Cx43 is required for gap junction formation and that cells which express phosphorylated species of the protein form gap junctions more efficiently. However, gap junctions also form with high efficiency in Rat2 and 10T1/2 cells despite the fact that Cx43 expression (phosphorylated and unphosphorylated) in these cell lines is relatively low. Furthermore, Rat2 cells which have been transfected with E cadherin show increased expression of Cx43 (Cx43-P2 in particular), yet no increase in homologous communication.
BICR cells express high levels of Cx43 as indicated by Western blot analysis and are very well coupled. However, examination of Cx43 immunostaining reveals that the majority of the Cx43 localises at sites away from cell-cell contact points suggesting the most of the expressed protein is not involved in gap junction formation. The antibody used in the immunostaining study here is unable to determine whether the Cx43 that accumulates at cell-cell contact points is phosphorylated or unphosphorylated.
Although the Cx43 expression appears to be necessary for cells to communicate the data obtained would appear to show that other factors are involved in mediating high levels of gap junction formation. A likely candidate is the putative channel protein, ductin. However, expression analysis could not be performed due to the unavailability of appropriate antibodies.
### 188.8.131.52. Gap junction specificity.
The molecular basis of gap junction specificity is poorly understood. It has been suggested that the homotypic binding properties of adhesion molecules such as the cadherins and the ability of connexins to selectively bind to other connexins make them suitable candidates (section 184.108.40.206.b). However, data obtained from expression studies (section 3.4.3) would suggest that these proteins are not involved in the junctional specificity observed here. For example, low levels of heterologous communication are observed when the normal cells (Rat2, R24E and 10T1/2) are paired with S180E217 cells or SVT2 cells (Table 3.3). However, all of these cell lines express Cx43 (phosphorylated & unphosphorylated) and show high levels of homologous communication (Table 3.2).
The adhesion molecules NCAM and E cadherin are also unlikely to be responsible for the specificity observed. For example, S180E217 cells and R24E both express E cadherin and yet show very poor heterologous communication. The possibility exists that the E cadherin expressed in the R24E cells may not be fully functional due to the possible absence of cadherin associated proteins, e.g. the catenins, which are responsible for regulating cadherin function (Hirano et al 1992, Shimoyama et al 1992). The immunostaining in the R24E cells was weak in comparison to that in S180E217 cells. Ecadherin would appear to be functioning in the S180 cells because of their altered morphology and increased homologous communication.
SVT2 cells and the normal cells express NCAM but show relatively poor heterologous communication. This would suggest that other factors, possibly other connexins (but not Cx26 & Cx32), are involved in specificity and that the process of gap junction formation is not as simple as the literature suggests.
The rate of gap junction formation is likely to be related to the frequency with which the apposed cell membranes come close enough together to allow interaction between channels that protrude 1nm. Increased adhesion through the expression of any adhesion molecule and perhaps by expression of any connexin, may increase this frequency and it may not be possible to associate specificity with any one given product.
The possibility exists that transformed cells reduce the levels of homologous communication of the normal cells. Such effects have been reported: Diener et al (1995) observed that malignantly transformed non-parenchyma cells suppressed the homologous communication of co-cultured rat liver parenchyma cells. However, these effects were seen over several days and it is not known by what mechanism this was achieved.
Despite being unable to determine the specific molecules responsible for the different levels of heterologous communication the importance of GJIC in the suppression of transformed cell growth can be assessed using these cell lines. Taken within the context of the current working hypothesis, one would predict the greatest inhibition to occur when R24E or Rat2 cells form the normal cell partner and the least inhibition when S180, S180E217 cells are co-cultured with 10T1/2 cells. Focus formation assays are now required to establish which transformed cells are suppressed by the presence of the normal cells and what the relationship is between gap junctional communication and growth suppression.
3.3. THE FOCUS FORMING ABILITY OF THE TRANSFORMED CELLS.
3.3.1. Introduction.
Focus assays were carried out on the cell combinations shown in Table 3.5 to establish whether the transformed cells have suppressible growth phenotypes when co-cultured with excess resting normal cells. And, if so, whether the inhibition correlates with the presence of heterologous GJIC.
In initial experiments foci were located after staining the co-cultures with Giemsa (a cell stain often used in this type of analysis), however, the colonies have to be of a sufficient size and density to avoid the possibility of interpreting clumps of normal cells or cell debris as small foci. To overcome any possible ambiguity an x-gal stain was used to locate those cells expressing the exogenous β-gal gene within the co-culture, i.e. the transformed cells. A preliminary focus assay was performed in order to determine if the different stains, when applied to replicate cultures, yielded significantly different results (based on focus number per plate & average focus size per plate). An example from this experiment can be seen in Figure 3.8. Although there was no statistically significant difference between the staining methods in terms of the number of foci recorded (P>5%) and the size of foci recorded (P>5%), small foci (0.125mm²) were difficult to see by Giemsa staining alone. Foci stained with x-gal were much clearer and the use of the stain avoided the possibility that clumps of normal cells or cell debris could be misinterpreted as foci. Cultures were frequently counter-stained with Giemsa as it did not interfere with the x-gal stain.
In the focus assays, only foci above a diameter of 0.4mm (focus area of 0.125mm²) were examined. In most of the assays some foci below 0.125mm² were observed, however, these were expected even if the growth of the transformed cells were inhibited since the normal cells continued to divide for 24-36 hours after seeding (section 3.1), allowing time for the transformed cells to pass through 2 or 3 divisions before any inhibitory effect by the normal cells could be imposed.
In focus formation assays normal and transformed cells were plated together at densities of $10^6$ and 500 cells / per 90mm dish respectively. Duplicate (or triplicate when focus numbers were small) co-cultures and control cultures (separate cultures of normal and transformed cells seeded at the same initial densities as above) were set up in each experiment. After 12 days in culture the cells were fixed, and stained with x-gal for 8 hours (section 2.5.1) to locate the transformed cells. The numbers of foci (or colonies in control cultures) on each dish were counted and average focus area determined from 40 foci/colony samples, (or maximum possible when less than 40 formed).
The results of the focus formation assays are presented in Tables 3.6.a & 3.6.b. Photographs of representative examples are shown in Figures 3.9.a & 3.9.b.
Table 3.5. Co-culture combinations for focus formation assays.
| Transformed cells | Normal cells |
|-------------------|--------------|
| | Rat 2 | R24E | 10T1/2 |
| S180 | * | * | * |
| S180E217 | * | * | * |
| S180NII | * | * | * |
| S180N2A1 | * | * | * |
| SVT2 | * | | |
| BICR | * | | |
* Indicates focus assays carried out. Transformed cell lines are β-gal⁺.
Figure 3.8. A comparison of two staining techniques, used to identify foci and colonies of transformed cells
a). 10T1/2 / BICR (x-gal)
b). 10T1/2 / BICR (giemsa)
Figure 3.8. The figures above are examples from an experiment performed to compare two different cell staining methods. 10T1/2 cells and BICR-βgal⁺ cells were seeded at $10^5$ and 500 cells per dish respectively. All cultures were grown for 12 days. They were then fixed in 1% Formaldehyde and 0.1% Gluteraldehyde in PBS for 30 mins, washed with PBS and stained with either x-gal for 8hrs (Figure a), which stains blue those cells expressing an exogenous β-gal gene, or stained for 10 mins in a 1:10 solution of Giemsa (Figure b). See section 2.5.1 for staining procedures. Bar 3mm.
3.3.2. The effect of normal cells on focus number.
The S180 E & N cadherin transfectants form more colonies than S180 parental cells (Table 3.6.a. - controls) which would suggest that these cells can form better attachments to the culture dish or those that do attach grow better under low density conditions. It can be seen in the photographs of Figure 3.9.a. that there is a subtle difference in colony morphology between the S180-cadherin transfectants and the S180 parental cells. The S180E217 and S180NII cell colonies appear more compact, particularly at the colony periphery (a phenomenon most likely attributed to increased cell to cell adhesion in the transfectants). BICR cells have the highest colony forming ability whereas SVT2 cells have a relatively poor colony forming ability (Table 3.6.a; Figure 3.9.b).
The data in Table 3.6.a. shows that the number of S180 foci which form in the presence of the normal cells is similar to or slightly greater than the number of colonies formed in control cultures. In contrast, the S180 E and N cadherin transfectants show marked inhibition of focus number when co-cultured with Rat2 and R24E cells (10T1/2 cells do not inhibit the number of foci formed by these cells). Examples of foci formed by the S180 series of cell lines is illustrated in the photographs in Figure 3.9.a.
The ability of the Rat2 and R24E cells to inhibit the number of foci formed by the S180 cadherin transfectants is consistent with the hypothesis that heterologous communication between the two cell types is required. For example, S180 cells show low levels of communication with, and are not suppressed by, the normal cells (Table 3.3 & 3.6.a.), whereas S180NII cells are well coupled to the Rat2 and R24E cells and their focus forming ability is markedly inhibited. A correlation analysis was performed in which the relationship between the number of foci formed by the S180 series of cell lines and their level of communication with the normal cells was examined. A strong positive correlation was revealed (Table 3.7). However, some of the transformed cells (S180E217 & S180NII) also communicate with 10T1/2 cells (albeit relatively poorly; Table 3.3) but show very low and often no inhibition of focus number when co-cultured with them. This would suggest that heterologous communication, although necessary is not sufficient to inhibit the number of foci formed.
SVT2 cells, which form functional heterologous gap junctions with the normal cells, are not restricted in terms of the number of foci which they form, indeed focus formation is increased. Despite the high levels of heterologous communication recorded between BICR cells and the normal cells the inhibition of focus number is relatively poor (Table 3.6.a). Together these results would suggest that SVT2 cells have lost the ability to be inhibited by normal cells whereas the BICR cells would appear to respond poorly to the inhibition mechanism. These cell lines serve as useful controls, (in conjunction with the conditioned media data - section 3.3.4) for the possibility that inhibition is caused from nutrient deprivation. The increased efficiency with which SVT2 and S180
cells form foci in the presence of the normal cells would suggest some form of feeder effect is occurring. This effect may be mediated by factors secreted from the normal cells into the media. To test this possibility conditioned media experiments were performed with these cell lines. Conditioned media experiments were also performed to confirm that the inhibition observed was not due to factors secreted into the media by normal cells - the results are presented in section 3.3.4.
3.3.3. The effect of normal cells on focus size.
Examination of focus size (Table 3.6.b) reveals a more complex picture than the focus number analysis and suggests that all of the transformed cells are growth inhibited to varying degrees. The S180 foci, formed in the presence of any of the normal cells, are considerably smaller than control colonies. The lowest level of focus size suppression for the S180 series, is recorded when the S180 cells are co-cultured with the 10T1/2 cells. Foci formed by S180E217 or S180NII cells are also smaller than their respective control colonies and smaller than the foci formed by S180 parental cells. Although the inhibition of focus size occurs throughout the S180 set of cell lines, a correlation exists between focus size inhibition and heterologous communication ($r = 0.6$, $P < 0.1\%$; Table 3.7). The differences between focus size and control colony size is illustrated clearly by the representative photographs in Figure 3.9.a & 3.9.b.
In the previous section it was shown that BICR and SVT2 cells show little or no inhibition of focus number, respectively. However, these cell lines show considerable focus size suppression, but, there does not appear to be a relationship between focus size inhibition and heterologous communication for these cell lines. BICR and SVT2 cells show higher levels of communication with Rat2 cells than with 10T1/2 cells and greater focus size inhibition when cultured with Rat2 cells. However, Rat2 cells are coupled better to BICR cells than to SVT2 cells (Table 3.3) but impose greater focus size inhibition on the SVT2 cells (Table 3.6.b). This data would appear to show that suppression is more complicated than is often suggested.
The photographs in Figure 3.9.a & 3.9.b. show examples of the foci formed by the transformed cells. It can be seen that the foci are not only smaller than the control colonies but have a different morphology. In addition, they appear more compact, (indicated by darker staining); possibly as a result of the physical presence of the normal cells. If the normal cells are imposing a restriction on focus expansion then the value of focus size as an index of growth inhibition may be limited.
Some smaller-than-average colonies were observed in the all of the cell combinations examined. These colonies appeared to be too small to be the result of increased cell compression alone and they therefore raise the possibility that cells within these foci are growth suppressed. An examination of the cell number and/or proliferative status of the cells within the foci is required to determine whether this is so. Many
S180E217 and S180NII foci below the 0.4mm threshold were also observed (Figure 3.9.a) and further analysis is required to determine if these foci have grown over the 12 day co-culture or represent colonies of cells established before any inhibitory effect could be imposed by the normal cells.
Table 3.6.a. The effect of normal cells on focus number.
| Transformed cell line | Control | Rat2 % of ctl | R24E % of ctl | 10T1/2 % of ctl |
|-----------------------|---------|---------------|---------------|-----------------|
| S180 | 152 (18) | 155 (16) | 198 (18) | 217 (25) |
| | | | | |
| S180E217 | 164 (8) | 60 (4) | 103 (10) | 215 (28) |
| | | | | |
| S180NII | 173 (8) | 4 (1.4) | 3 (4) | 175 (14) |
| | | | | |
| S180N2A1 | 196 (14) | 24 (19) | 28 (4) | 195 (14) |
| | | | | |
| SVT2 | 75 (33) | 111 (23) | 99 (2) | |
| | | | | |
| BICR | 291 (15) | 250 (50) | 264 (52) | |
Table 3.6.b. The effect of normal cells on focus size.
| Transformed cell line | Control | Rat2 % ctl | R24E % ctl | 10T1/2 % ctl |
|-----------------------|---------|------------|------------|--------------|
| S180 | 16 (2.8)| 4 (1.5) | 4.5 (1.3) | 6.2 (2.3) |
| | | | | |
| S180E217 | 10 (3.4)| 0.5 (0.3) | 0.6 (0.2) | 3 (1.2) |
| | | | | |
| S180NII | 17 (1.5)| 0.3 (0.1) | 0.1 (0.1) | 3 (1.0) |
| | | | | |
| S180N2A1 | 15 (4.6)| 0.2 (0.1) | 0.2 (0.1) | 2 (0.7) |
| | | | | |
| SVT2 | 13 (3.9)| 0.8 (0.4) | 3.8 (0.6) | |
| | | | | |
| BICR | 6 (1.6) | 1.5 (0.9) | 3.8 (1.2) | |
Table 3.6.a & b. For controls, replicate cultures of each transformed cell line were plated at 500 cells / 90mm dish. In co-cultures, transformed and normal cells were plated simultaneously, in replicate, at initial densities of 500 and $10^6$ cells / 90mm dish respectively. After 12 days the control and co-cultures were fixed in 1% Formaldehyde and 0.1% Glutaraldehyde in PBS for 30 mins. Foci were located after staining with x-gal for 8 hrs (see section 2.5.1) and Giemsa (1:10 solution) for 10 mins. The number of foci and colonies, per plate, above 0.4mm diameter (focus area 0.125mm$^2$) were counted (Table 3.6.a) and 40 colony / focus diameters (or max. possible when less than 40 formed) were measured from which average area (mm$^2$) was calculated (Table 3.6.b). Standard deviations in parentheses. Transformed cell lines have been placed in order of increasing heterologous communication (see Table 3.3 for values).
Table 3.7. Correlation analysis: Level of communication vs inhibition of focus size and focus number.
| Coefficients | % inhibition focus No. | % inhibition focus size |
|--------------|------------------------|-------------------------|
| r | 0.8 | 0.6 |
| P | <0.1% | <0.1% |
Table 3.7. A correlation analysis was performed on the focus formation data obtained from experiments with the S180 series of cell lines, examining the relationship between their level of communication with, and inhibition by, the normal cells. The individual growth-inhibition values (derived from Table 3.6.a. & 3.6.b.) were categorised by communication level and subjected to linear regression analysis. The correlation coefficient (r) and confidence level (P) are given for each analysis. The type of calculation performed is described in Wardlaw (1992).
Figure 3.9.a. Representative photographs showing the focus forming ability of the transformed cells on a background of either Rat2, R2E4F or 10T1/2 cells.
Transformed cells in control cultures were plated at 500 cells / 90mm dish. In co-cultures normal and transformed cells were plated simultaneously at 10^4 & 500 cells / 90mm dish respectively. The cultures were grown for 12 days and then fixed in 1% Formaldehyde and 0.1% Glutaraldehyde in PBS for 30 mins. The cultures were then washed twice in PBS and stained in either x-gal (for S180) followed by counter staining in Giemsa (1:16 solution) for 10 mins, or Stained only with Giemsa or x-gal. The type of stain applied to each plate shown is given in parentheses. See section 2.5.1 for staining procedure. Bar 5mm.
Figure 3.9.a,b. Representative photographs showing the focus forming ability of the transformed cells on a background of Rat2 or IOT1/2 cells.
See Figure 3.9.a for experimental details. The cell lines and cell combinations are given above each figure together with the types of stain used. Bar 50mm.
3.3.4. The effect of conditioned media on normal and transformed cell growth.
Experiments were performed in an attempt to answer the following questions:
1). Is focus formation inhibition mediated by factors secreted into the media by the normal cells?
2). Is the inhibition of focus number and focus size due to nutrient starvation?
3). Is transformed-cell conditioned media capable of stimulating the growth of high density Rat2 cultures?
4). Is increased focus formation mediated by factors secreted into the media by normal cells?
Proliferation assays, based on measurement of cell density and $^{[3}H$]-thymidine incorporation and analysis by autoradiography, were used to examine the effects of conditioned media on cell growth and focus formation. Four separate experiments were set up, each in duplicate, to answer the above questions. As a control for experiment B, cultures of 500 S180NII cells / 90 mm dish were grown in DMEM10% media and their proliferative status analysed after 1 and 4 days (experiment A; Table 3.8). To determine whether Rat2-cell conditioned media (conditioned for 10 days - see legend for Table 3.8) had any inhibitory growth effect on transformed cells, 500 S180NII cells/90mm dish (which showed the greatest levels of focus number and focus size inhibition in the previous section) were cultured in conditioned media and their growth analysed after 1 and 4 days (experiment B; Table 3.8).
As an control for experiment D, cultures of 500 Rat2 cells were grown in DMEM10% and their growth analysed after 1 and 4 days (experiment C; Table 3.8). The ability of the Rat2-cell conditioned media to sustain the growth of Rat2 cells (plated at 500 per 90mm dish) was examined (experiment D; Table 3.8); growth of these cells would indicate that their growth inhibition at high density is due to contact inhibition, rather than nutrient deprivation and that media taken from confluent 10 day old cultures (see Table 3.8) is still capable of sustaining normal cell growth. In experiment E (Table 3.8), the ability of S180NII-cell conditioned media, to stimulate the growth of Rat2 cells (500 cells / 90mm dish) was examined. All cultures were pulsed for 18hrs, prior to analysis, with $^{[3}H$]-thymidine. Cultures were then fixed and processed for autoradiography according to the method in section 2.5.
Finally, in an attempt to answer question 4, SVT2 cells were plated at 500 cells / 90mm dish and cultured in 10mls of Rat2 conditioned media for 12 days. They were then fixed, stained in x-gal for 8 hours and the number of foci counted.
Table 3.8. The effect of normal- and transformed-cell conditioned media on cell growth.
| Experiment | Conditioned media | Cell line applied to [initial cell density/dish] | Hours in conditioned media |
|------------|-------------------|--------------------------------------------------|----------------------------|
| | | | 24 | 96 |
| | | | Cells/UA | LI % | Cells/UA | LI % |
| A | - | S180NII [500] | 5 (2) | 100 | 12 (4) | 100 |
| B | Rat2 | S180NII [500] | 2.8 (1) | 100 | 15 (2) | 100 |
| C | - | Rat2 [500] | 3.5 (2) | 100 | 14 (8) | 100 |
| D | Rat2 | Rat2 [500] | 2.5 (1) | 100 | 19 (7) | 100 |
| E | S180NII | Rat2 [10^6] | 32 (7) | 85 | 36 (11) | 5 |
Table 3.8. Rat2 and S180NII conditioned media was prepared by growing separate cultures of each cell line to confluence in DMEM10%. Conditioned media was removed after 10 days and centrifuged at 3000xg for 10 min to remove debris and used directly or stored at -20°C (for a max. of 7 days). In each experiment, (set up in duplicate), cells were plated in DMEM10% and left to attach for 12 hours before any conditioned media was added. Cultures which received conditioned medium were first washed twice with PBS before conditioned media was added at 10mls/90mm dish. Cultures were then analysed after 24 and 96 hours. 18hrs prior to analysis cultures were pulsed with [³H]-thymidine (0.185MBq/ml), washed twice in PBS and fixed in 0.1% Formaldehyde and 0.1% Gluteraldehyde in PBS for 30 mins. Cultures were then processed for autoradiography as described in section 2.5. Cell density (cells/UA) and LI was determined over the colonies (Exp A-D) and the monolayers (Exp E) by counting the total number of cells (labelled and unlabelled) in 10 Unit Areas (or maximum possible when colonies were small; 1 UA = 0.175mm²). The experiments were as follows: A) Fresh DMEM10% on S180NII cells plated at 500 cells/ 90mm dish. B) Rat2 conditioned media on S180NII cells plated at 500 cells/ 90mm dish. C) Fresh DMEM10% on Rat2 cells plated at 500 cells / 90mm dish. D) Rat2 conditioned media on Rat2 cells plated at 500 cells/ 90 mm dish. E) S180NII conditioned media on Rat2 cells plated at 10⁶ cells/ 90mm dish. (-) indicates where fresh DMEM10% was added. Standard deviations in parentheses.
The data in Table 3.8 (Exp. B) shows that Rat2 conditioned media has no detectable inhibitory effect on the high level of cell proliferation in low density cultures of S180NII cells (in comparison to controls - Exp A). Furthermore, the Rat2-cell conditioned media is able to sustain the growth of low density S180NII and Rat2 cultures (Exp. B & D), indicating that the inhibition of focus size and number, observed in the previous section, was unlikely to be due to nutrient starvation.
S180NII-cell conditioned media did not appear to stimulate the growth of high density Rat2 cultures (Exp. E). After 1 day in culture the LI of these Rat cultures is high (~85%), however, after 4 days the cells have reached their saturation density (~4.5\(^6\); section 3.1.1) and their LI is appropriately low (~5%).
An experiment was also performed to determine if Rat2-cell conditioned media was capable of increasing the colony forming efficiency of SVT2 cells. The number of SVT2 colonies after 12 days in culture with Rat2 cell conditioned media was 87 (sd 23) which is not significantly different from the number of colonies recorded in control cultures for this cell line; 75 (sd 33); Table 3.6.a. This would suggest that the increased colony forming efficiency when co-cultured with Rat2 cells is dependent on the presence of the Rat2 cells and/or direct contact with them.
These experiments would suggest that Rat2 conditioned media is unable to inhibit cell growth and S180NII conditioned media is unable to stimulate cell growth. Furthermore, these experiments show that nutrient deprivation is unlikely to contribute to any inhibition of cell growth recorded.
### 3.3.5. The effect of focus size on the focus forming efficiency of the transformed cells.
In sections 3.3.2 & 3.3.3 many small foci below the 0.4mm focus diameter cutoff point were observed in x-gal stained cultures. It was not clear whether these small foci had grown over the 12 day culture period or represented colonies which had grown prior to any inhibition being imposed by the normal cells. In addition, many smaller than average foci were observed in cultures of cells which, on average, showed little focus inhibition. These results suggest that there is considerable variability within a population of transformed cells, in terms of the ability of individual cells to form foci in the presence of resting normal cells. To determine whether these small foci were inhibited or whether they were only partially suppressed and continued growing at a slower rate over the 12 day co-culture, focus assays were set up, and analysed over a 12 day time course. To increase the sensitivity of the analysis the threshold focus size, previously 0.4mm focus diameter (focus area = 0.125mm\(^2\)), was lowered to 0.1mm focus diameter (focus area = 0.008mm\(^2\)). This size is equivalent to ~100 cells i.e. the approximate size of a transformed colony after 4 days growth. This size accounts for transformed cell growth before the normal cells impose any inhibition and is therefore a logical cut off to choose.
All of the transformed cells examined showed some degree of focus size inhibition (section 3.3.3) which would suggest that the not all of the transformed cells totally escape inhibition during the first 4 days in co-culture. Within each co-culture there was a degree of variability in terms of focus size. This size heterogeneity may be caused by the degree to which foci are inhibited and at what stage in the time course they escaped. That is, inhibition may reach a maximum after 3 or 4 days. There may be a high frequency with which foci initially escape and a lower frequency of continuing escape. This would give a burst of larger foci (those which escape at day 3/4), some intermediate sized foci which escaped later and some small foci which did not escape at all. The level of heterologous communication between the normal and transformed cells may affect the frequency with which foci are inhibited. To determine whether a focus size exists, beyond which, the central cells at least escape inhibition and maintain aberrant growth phenotypes, colonies of these cells (and S180 parental cells) were pre-established for 2 and 4 days prior to the addition of Rat2 or 10T1/2 cells.
Focus assays were carried out on the cell combinations shown in Table 3.10.a. They include S180NII cells which appear to be well suppressed by Rat2 cells and S180E217 cells which show intermediate levels of focus size inhibition relative to the poorly suppressed S180 parental cells. To compare the effects of different normal cell lines 10T1/2 cells were co-cultured with S180 and S180E217 cells. These combinations also provide suitable communications phenotypes with which to assess the role of GJIC in the inhibition pathway.
Table 3.9 describes the nomenclature, which has been used here and throughout the remainder of the thesis, to indicate the number of days the transformed cells were grown in separate cultures and co-cultures. For example a time point of d6d4 would indicate that the transformed cells have spent a total of 6 days in culture (this is all the information that is required when examining control cultures) and 4 days in co-culture with the normal cells.
Focus assays were performed in which transformed cells were plated simultaneously with the normal cells or pre-established for 2 and 4 days prior to the addition of the normal cells. In co-cultures, transformed and normal cells were plated at 500 cells and $10^6$ cells per 90mm dish respectively, control cultures were represented by separate cultures of each cell line, plated at the same initial density as above. All cultures were set up in duplicate. Recordings of focus number and size (area) were taken at 4, 8 and 12 days. This allowed any increase in colony or focus size over a 12 day period to be detected and measured. After the specific culture period co-cultures were fixed and stained with x-gal (section 2.5.1).
At time points where the two cell types were plated simultaneously, (d4d4, d8d8 & d12d12) both focus size and number have been used as an index of suppression, (Tables 3.10.a & 3.10.b). However, in time points, where transformed cell colonies were pre-established, the colony and focus forming efficiencies (foci above 0.008mm$^2$) of the
Table 3.9. Time points and nomenclature used in the focus formation assays.
| Co-culture description | Total time transformed cells have spent in culture (& control times) | Time transformed & normal cells have spent in co-culture | Nomenclature Code |
|------------------------|---------------------------------------------------------------|------------------------------------------------------|------------------|
| | d | d | |
| Transformed & normal cells plated simultaneously | 4 | 4 | d4d4 |
| | 8 | 8 | d8d8 |
| | 12 | 12 | d12d12 |
| Transformed cells pre-established for 2 days | 6 | 4 | d6d4 |
| | 10 | 8 | d10d8 |
| | 14 | 12 | d14d12 |
| Transformed cells pre-established for 4 days | 8 | 4 | d8d4 |
| | 12 | 8 | d12d8 |
| | 16 | 12 | d16d12 |
d = days in culture.
transformed cells showed no statistical difference (P>10%) and therefore only focus size was used to measure inhibition (Tables 3.11 and 3.12).
220.127.116.11. The effect of normal cells on focus number after 4, 8 and 12 days when both cell types are plated simultaneously.
The number of transformed cell colonies within all control cultures increases over the 12 day culture period (Table 3.10.a) i.e. some are grow slowly to begin with. After 12 days in culture the number of colonies, formed by all of the cell lines, above the 0.008mm$^2$ threshold area were similar to the number recorded in the previous section (3.3.2) using a higher threshold area of 0.125mm$^2$. This indicates that after 12 days in culture the majority of colonies that have grown are above the 0.125mm$^2$ minimum threshold area.
The number of foci in the co-cultures also increased over the 12 day culture (Table 3.10.a). However, more foci were recorded at day 12 when a threshold size of 0.008mm$^2$ was used (compare with data in Table 3.6.b when a threshold of 0.125mm$^2$ was used). This was particularly evident in cultures of Rat2/S180E217 cells and Rat2/S180NII cells. Although these cells form fewer foci the number of foci increases over the 12 day co-culture and suggests the cells grow more slowly in co-culture or their growth is initially delayed. These results are consistent with observations made in the previous section where many small foci (below 0.125mm$^2$) were observed.
18.104.22.168. The effect of normal cells on focus size after 4, 8 and 12 days, when both cell types are plated simultaneously.
To examine whether the small foci (particularly in the inhibited cultures) had grown over the 12 day culture period, their average size (area) was determined at 4, 8 and 12 days. The data is presented in Table 3.10.b. It is clear from the results that there is variability in terms of focus size within each experiment. Such variability is common in these types of experiments and may be due, for example, to differences in the time taken for individual cells to start dividing or differences in local normal cell density. Despite this variability, trends within each focus assay can be identified.
S180 foci above the threshold area were recorded after 4 days in culture with either Rat2 or 10T1/2 cells. Those S180 foci recorded at day 8 (in co-cultures with Rat2 cells) increased in size considerably over the following 4 days of culture (from an average of 0.1mm$^2$ at d8 to 4mm$^2$ at d12), however, they are significantly smaller at both time points, than the respective control colonies (16mm$^2$ at d8 & 18mm$^2$ at d12).
Foci formed by S180E217 cells in co-culture with Rat2 cells continue to grow over the 12 day culture period (from 0.1mm$^2$ to 0.5mm$^2$) but remain markedly smaller than their respective control colonies which grow from 3.1mm$^2$ to 12.1mm$^2$ and foci formed by S180 parental cells. Thus, S180E217 foci show size suppression over a 12
day culture period when plated simultaneously with the Rat2 cells, however, the suppression isn't total and the foci do expand over time.
There were no S180NII foci above the minimum threshold area of 0.008mm$^2$ observed after 4 days in co-culture with Rat2 cells. After 8 days co-culture, foci with an average area of 0.1mm$^2$ were present and these continued to grow over the next 4 days to an average size of 0.2mm$^2$. These data would suggest that the small foci observed in the previous section are not simply colonies of transformed cells which were established prior to the normal cells imposing any growth inhibition. However, an examination of the proliferative status of the cells within the foci is required in order to confirm this interpretation and to determine the proportion of dividing cells.
In general the level of inhibition (focus number and size) imposed by the normal cells tends to decrease over the 12 day culture period. This would suggest that the larger a focus grows the less effective the inhibition mechanism, imposed by the normal cells, becomes.
The results presented in this section would suggest that the enhanced levels of heterologous communication between the S180 E & N cadherin transfectants and the normal cells (relative to S180-parental cells), leads to increased levels of focus size inhibition. However, this inhibition is not total and foci continue to expand, albeit at a reduced rate relative to control colonies.
22.214.171.124. The effect of normal cells on focus size when the transformed cells are pre-established for 2 and 4 days.
In the previous sections it has been shown that S180 cells transfected with E or N cadherin, show increased focus size suppression relative to the S180 parental cells. To determine whether these cells escape this inhibition upon reaching a critical colony size, colonies of transformed cells were pre-established for 2 and 4 days and co-cultured for 4, 8 and 12 days with normal cells. Focus size was used an index of suppression and the results of the analysis are presented in Tables 3.11 & Table 3.12. The photographs in Figures 3.10.a & 3.10.b provide representative examples of focus/colony size and morphology at different analysis time points.
Control colonies of all the transformed cell lines examined continued to grow over all of the time periods analysed (with the exception of BICR colonies between d12 and d16). It can be seen from the examples in Figure 3.10.b. that as colonies increased in size, cell density, particularly within the centre of the colonies also increased - as indicated by darker staining. In later time points (e.g. d16), cells within the centre of some colonies detached during fixation. It is not clear whether this was because cells were unhealthy or whether they were only weakly attached to the culture dish, due to over crowding.
Within the co-cultures the pre-established colonies of all the transformed cells examined continued to grow in the presence of the normal cells (Table 3.11.& 3.12).
Despite the level of variability in terms of focus size (% of control) within each of the separate focus assays it is possible to identify consistent trends within the experiment as a whole. There is still considerable focus size suppression in the majority of cultures examined. However, the results would appear to indicate that those cell lines which showed the greatest level of focus size suppression when plated simultaneously with the normal cells (i.e. S180E217 & S180NII cells; Table 3.10.b) have a reduced response to inhibition by normal cells when they are pre-established for 2 or 4 days (Table 3.11 & 3.12; Figure 3.10.a).
Over the majority of time points, however, S180NII cells are suppressed to a greater degree than both S180E217 cells and S180 parental cells. In several of the focus assays (e.g. Rat2 / S180E217 or 10T1/2 / S180E217; Table 3.12) the level of inhibition imposed by the normal cells decreases as the foci grow in size.
In general the ability of 10T1/2 cells to suppress the growth of pre-established S180 and S180217E cells is less than that of Rat2 cells. In some instances S180E217 cells (which are coupled to 10T1/2 cells) are inhibited less than the parental S180 cells, which show no detectable levels of heterologous communication with 10T1/2 cells. This suggests that GJIC does not play a dominant role in the inhibition observed here.
The results here would suggest that when inhibition occurs it is at its strongest when the two cell types are plated together simultaneously. Pre-establishing cell lines which, when plated simultaneously with normal cells are well inhibited, reduces their response to the inhibitory mechanism suggesting that the larger the focus of transformed cells the less inhibited the cells are.
In the majority of instances all of the transformed cells showed some size suppression even when pre-established. However, because cell lines which show low or zero levels of communication with the normal cells (and are therefore unlikely to be inhibited by a GJIC-mediated mechanism) also show considerable focus size suppression, the possibility exists that increased focus cell density, as a result of the physical presence of the normal cells, leads to differences in size between foci and colonies.
The detailed study carried out here highlights a major limitation of this type of experimental approach. That is, variability within a plate and between plates means that only general conclusions can be drawn. Foci within a plate vary in size and estimates of focus number depend on the arbitrary choice of a cut-off size, this can significantly alter the apparent result. Although focus size would appear to provide a more detailed picture of the differences in growth inhibition it is not clear if focus size directly reflects the level of growth inhibition. Because of the limitations of the focus assay it is not possible to state whether a focus ceases to be inhibited at a specific size. However, the data would appear to show that when a population of transformed cells (including those which have a suppressible phenotype when plated simultaneously with the normal cells) grows
beyond 100-200 cells a proportion of those cells become refractory to the inhibition mechanism.
Table 3.10.a. The effect of normal cells on focus number after 4, 8 and 12 days when both cell types are plated simultaneously.
| Transformed cell lines | Analysis time points | Control | Rat2 co-culture | 10T1/2 co-culture |
|------------------------|----------------------|---------|-----------------|------------------|
| | | avg | sd | avg | sd | % of ctl | avg | sd | % of ctl |
| S180 | d4d4 | 100 | 15 | 96 | 10 | 96 | 100 | 14 | 100 |
| | d8d8 | 113 | 11 | 131 | 8 | 116 | 95 | 5 | 84 |
| | d12d12 | 159 | 4 | 156 | 13 | 98 | 175 | 15 | 110 |
| S180E217 | d4d4 | 85 | 7 | 72 | 5 | 85 | 100 | 11 | 118 |
| | d8d8 | 163 | 3 | 139 | 20 | 85 | 208 | 8 | 128 |
| | d12d12 | 170 | 0 | 129 | 14 | 92 | 232 | 22 | 166 |
| S180NII | d4d4 | 60 | 14 | - | - | - | | | |
| | d8d8 | 154 | 20 | 30 | 9 | 19 | | | |
| | d12d12 | 180 | 22 | 27 | 4 | 15 | | | |
| BICR | d4d4 | 130 | 6 | 140 | 10 | 108 | | | |
| | d8d8 | 260 | 14 | 260 | 18 | 100 | | | |
| | d12d12 | 300 | 0 | 360 | 25 | 120 | | | |
Table 3.10.b. The effect of normal cells on focus size after 4, 8 and 12 days when both cell types are plated simultaneously.
| Transformed cell lines | Analysis time points | Control | Rat2 co-culture | 10T1/2 co-culture |
|------------------------|----------------------|---------|-----------------|------------------|
| | | avg | sd | avg | sd | % of ctl | avg | sd | % of ctl |
| S180 | d4d4 | 2.0 | 0.9 | 0.1 | 0.01 | 5 | 0.2 | 0.1 | 10 |
| | d8d8 | 18.5 | 2.2 | 0.1 | 0.01 | 0.5 | 2.0 | 0.8 | 11 |
| | d12d12 | 16.0 | 2.5 | 4.0 | 1.2 | 25 | 6.0 | 2.2 | 38 |
| S180E217 | d4d4 | 3.1 | 1.0 | 0.1 | 0.01 | 2 | 0.5 | 0.1 | 16 |
| | d8d8 | 12.1 | 3.8 | 0.4 | 0.1 | 3 | 3.0 | 0.9 | 25 |
| | d12d12 | 10.0 | 4.0 | 0.5 | 0.2 | 3 | 2.8 | 0.2 | 28 |
| S180NII | d4d4 | 0.1 | 0.00 | - | - | - | | | |
| | d8d8 | 8.7 | 2.0 | 0.1 | 0.01 | 1 | | | |
| | d12d12 | 17.0 | 5.0 | 0.3 | 0.15 | 1 | | | |
| BICR | d4d4 | 0.3 | 0.1 | 0.2 | 0.1 | 69 | | | |
| | d8d8 | 4.4 | 2.2 | 0.9 | 0.2 | 21 | | | |
| | d12d12 | 6.0 | 1.9 | 1.3 | 0.4 | 22 | | | |
Table 3.10.a & b. In co-cultures, transformed and normal cells were plated simultaneously at 500 and $10^6$ cells / 90mm dish respectively. Control cultures were represented by separate cultures of each cell line plated at the same densities as above. Cultures were fixed in 1% Formaldehyde and 0.1% Gluteraldehyde in PBS for 30 mins after 4, 8 and 12 days. Foci were located after staining with x-gal for 8hrs (see section 2.5.1) and Giemsa (1:10 solution) for 10 mins. In Table 3.10.a. Total number of foci, above 0.1mm diameter (area 0.008mm$^2$), were counted. Average focus/colony area (Table 3.10.b) was obtained from 40 samples (or the maximum possible when less than 40 formed). Percentage of the control was calculated from the equation: $\frac{C_2}{C_1} \times 100$ where $C_1 =$ control colony area and $C_2 =$ focus area. Results are based on counts from at least two replicates. Cell lines have been placed in order of increasing heterologous communication (see Table 3.3 for values). (-) indicates where no colonies were detected.
Table 3.11. The effect of normal cells on focus size, when transformed cells are pre-established for 2 days.
| Transformed cell lines | Analysis time points | Control avg sd | Rat2 co-culture avg sd % of ctl | 10T1/2 co-culture avg sd % of ctl |
|------------------------|----------------------|----------------|---------------------------------|----------------------------------|
| S180 | d6d4 | 2.5 0.8 | 0.4 0.2 17 | 0.1 0.03 5 |
| | d10d8 | 10.9 1.5 | 2.0 1.0 18.4 | 4.2 1.0 38 |
| | d14d12 | 15.4 1.5 | 3.5 1.5 22.5 | 3.5 1.0 22 |
| S180E217 | d6d4 | 2.0 0.8 | 0.01 0 0.4 | 1.1 0.1 57 |
| | d10d8 | 3.6 1.2 | 3.1 1.8 88 | 4.9 0.3 138 |
| | d14d12 | 10.1 3.5 | 4.9 3.1 48 | 7.1 1.0 70 |
| S180NII | d6d4 | 1.7 1.0 | 0.7 0.2 42 | |
| | d10d8 | 8.0 1.2 | 0.5 0.1 6.25 | |
| | d14d12 | 16.9 2.8 | 2.2 0.13 13 | |
| BICR | d6d4 | 1.3 0.1 | 0.2 0.1 15 | |
| | d10d8 | 4.5 1.5 | 1.8 0.5 40 | |
| | d14d12 | 5.0 1.2 | 1.1 0.8 22 | |
Table 3.11. The effect on focus size was determined when the transformed cells were pre-established for 2 days. Controls were represented by separate cultures of each transformed cell line plated at 500 cells / 90mm dish. In co-cultures, transformed cells were pre-established for 2 days (500 cells/dish), normal cells were added (in < 0.5 mls of media) at $10^6$ cells/dish. Cultures were fixed in 1% Formaldehyde and 0.1% Gluteraldehyde in PBS for 30 mins after 4, 8 and 12 days. Foci were located after staining with x-gal for 8 hrs (section 2.5.1) and Giemsa (1:10 solution) for 10 mins. A hand-held magnifier with an eyepiece graticule was used to locate microscopic colonies. The smallest division on the graticule, 0.1mm, was used as the threshold diameter (area = 0.008mm$^2$), foci smaller than this were ignored. Average colony / focus area was obtained from 40 colonies / foci or the maximum possible when less than 40 formed. (sd) standard deviation, (avg) average. Percentage of the control was calculated from the equation: $\frac{C_2}{C_1} \times 100$ where $C_1 =$ control colony area and $C_2 =$ focus area. Results are based on measurements from at least two replicate cultures. Cell lines have been placed in order of increasing heterologous communication (see Table 3.3 for values).
Table 3.12. The effect of normal cells on focus size, when transformed cells are pre-established for 4 days.
| Transformed cell lines | Analysis time points | Control avg sd | Rat2 co-culture avg sd % of ctl | 10T1/2 co-culture avg sd % of ctl |
|------------------------|----------------------|----------------|---------------------------------|----------------------------------|
| S180 | d8d4 | 18.5 2.2 | 1.8 0.8 10 | 3.8 0.5 20 |
| | d12d8 | 16.5 2.5 | 2.5 1.5 15 | 4.2 2.2 25 |
| | d16d12 | 26.4 4.5 | 4.5 2.0 17 | 9.6 4.0 36 |
| S180E217 | d8d4 | 12.1 3.8 | 3.1 2.0 26 | 3.1 0.1 25 |
| | d12d8 | 10.0 4.0 | 3.1 1.0 31 | 4.9 1.0 24 |
| | d16d12 | 19.2 6.0 | 12.6 6.6 65 | 8.0 0.5 42 |
| S180NII | d8d4 | 8.7 2.0 | 0.7 0.05 8 | |
| | d12d8 | 17.0 5.0 | 0.85 0.08 5 | |
| | d16d12 | 27.5 5.9 | 2.8 0.2 6.0 | |
| BICR | d8d4 | 4.4 2.2 | 0.8 0.5 18 | |
| | d12d8 | 6.0 1.9 | 1.3 0.8 22 | |
| | d16d12 | 4.5 0.4 | 1.3 0.8 29 | |
Table 3.12. The effect on focus size was determined when the transformed cells were pre-established for 4 days. Controls were represented by separate cultures of each transformed cell line plated at 500 cells / 90mm dish. In co-cultures, transformed cells were pre-established for 4 days (500 cells/dish), normal cells were added (in < 0.5 mls of media) at $10^6$ cells/dish. See legend for Table 3.11 for experimental details.
Figure 3.10a. Focus forming ability of transformed cells when plated at the same time or before the normal cells.
All normal and transformed cells were plated at $10^4$ and $500$ cells/dish respectively. Controls were represented by separate cultures of each cell lines at the same densities as above (see Figure 3.10b) for respective control cultures. Cell combinations in each column. Culture times are given at the top of each column. All cultures were fixed in 1% formaldehyde and 0.1% Glutaraldehyde in PBS for 30 mins, washed once in PBS and stained with either Giemsa or Giemsa and 1% or Giemsa stain (1:10 solution) for 10 mins (the type of stain applied to each plate shown is given in parentheses above each figure; see section 2.5.1 for staining procedure). Bar: 5mm.
Figure 3.10A.1. Colony forming ability of transformed cells - control cultures for Figure 3.10A. See Figure 3.10A.1 for experimental details. Cell lines are given in the first column. Culture times are given at the top of each column. Cultures were stained with either X-gal (for 8 hrs, blue stain) or Giemsa Stain (1:10 solution) for 10 mins. (The strain used in the plates, shown are given in parentheses; see section 2.5.1 for staining procedure.) Red arrows point to examples of colonies which are difficult to see at this resolution. Bar 5mm.
3.3.6. Summary.
Preliminary focus assays (using a cut-off size of 0.4mm focus diameter), in which the S180 and S180 E & N cadherin transfectants were plated simultaneously with the normal cells (section 3.3.2) revealed a strong positive correlation between the inhibition of focus number and the level of heterologous communication. However, this correlation did not apply to the well coupled transformed cell lines, BICR and SVT2, which were poorly inhibited in terms of focus number. This would suggest that these tumourigenic cell lines (Fernandez et al 1990 & Brummer 1987) have lost the ability to respond to the inhibition mechanism.
The level of focus number inhibition varies depending on the arbitrary cut-off point which is used. Using a lower cut-off (focus diameter of 0.1mm) revealed that only S180 N cadherin transfectants were significantly inhibited in terms of focus number. However, analysis of focus size reveals that, in the majority of instances, all of the cell lines appear to be inhibited. Focus size inhibition is also seen in cell combinations which show low or undetectable levels of heterologous communication (e.g. 10T1/2 / S180) and in cell combinations where focus number is not suppressed (e.g. Rat2 / SVT2).
Conditioned media experiments would appear to show that the inhibition is not mediated by factors secreted into the media or due nutrient deprivation. This is consistent with the observations of Stoker (1966 & 1967) which suggest that the inhibition is mediated by direct cell-cell interaction between the two cell types. However, the type of conditioned media experiments carried out here do not rule out the possibility that the inhibition may be mediated by paracrine signals which act in a localised manner (due to rapid internalisation or degradation - see section 1.2.4).
A more extensive focus formation analysis, using a lower cut-off size, carried out in section 3.3.5 revealed that the cell lines which appeared to be well suppressed, in terms of focus number and/or size, during the initial focus assays (section 3.3.2; e.g. S180NII & S180E217 cells) appeared to be growing, albeit slowly, over the 12 day coculture period. A greater number of foci were recorded and if the transformed cells were established for 2 or 4 days in the absence of normal cells, there subsequent response to the inhibitory mechanism was reduced. This would suggest that when a population of transformed cells (including those with suppressible phenotypes) grow to a sufficiently large size (>100cells) a proportion of those cells become refractory to the inhibition mechanism. If GJIC does mediate the inhibition then it might be expected that those cells towards the centre of the focus would escape, particularly if the transformed cells are poorly coupled to each other. As the focus grows then the transformed cells may stimulate each other to divide possibly by the production of high levels of stimulatory growth signals. This increase in local growth factor concentration may also lead to the stimulation of normal cells surrounding the foci.
The observation that larger transformed cell populations appear more likely to escape inhibition may be analogous to the observation *in vivo* in which initial masses of $10^3$-$10^6$ transformed cells are required for tumourigenicity assays, any less and generally no tumours occur. Cells which don't go onto form tumours may simply die or be attacked by the immune system. Alternatively they may be suppressed by a gap junction mediated form of growth suppression. However, if a clump of cells is established within the host then their ability to be suppressed depends not only on heterologous communication but also on the transfer of the putative inhibitory signals or loss of putative stimulatory signals, throughout their population, (mediated via homologous communication). A similar pattern of inhibition may be occurring in tissue culture when colonies of transformed cells are allowed to grow prior to the addition of normal cells. If this were the case, in instances where transformed-cell homologous communication is low, one would expect to see more inhibition of cell growth at the periphery of colonies than in the centre. An investigation into patterns of growth inhibition across the foci has been carried out in the following results section.
Secreted factors do not appear to mediate the focus formation inhibition, and there is evidence to suggest that GJIC is involved in the inhibition of focus size where heterologous communication is high (e.g. S180E217 and S180NII cells), however, it is unlikely to be the inhibitory pathway in certain combinations which show focus size inhibition (e.g. 10T1/2 /S180). In these instances it is possible that the physical presence of the normal cells restricts focus expansion and the transformed cells continue to multilayer (rather than their growth being inhibited). Cell compression within the foci may therefore account for why focus size inhibition is observed in instances where there is no heterologous communication and where there is no focus number suppression (and often increased focus formation).
Although the focus assay experiments have shown that an inhibition phenomenon appears to exist within some of the cell lines examined, it can not be accurately measured using focus number or focus size. Until the growth of the individual cell populations are examined it is not possible to determine the role of GJIC in the phenomenon. The proliferative status of the transformed cells when cultured with the normal cell lines has been examined in the following section together with an investigation into the role of cell compression in focus size suppression.
3.4. CELL-CELL INTERACTIONS BETWEEN THE NORMAL CELL MONOLAYER AND TRANSFORMED FOCI.
3.4.1. Introduction.
In the previous section, discrete transformed cell populations above 0.1mm in diameter were regarded as foci and therefore likely to be formed by cells which had escaped the inhibitory growth control of surrounding normal cells. In several focus assays, such as those using S180, S180E217 and SVT2 cells, the number of foci which formed were similar to or greater than the number of control colonies formed. In addition, many such foci continued to increase in size over time suggesting cells were not growth suppressed. The majority of foci which formed, however, were markedly smaller than respective control colonies, which would suggest a high degree of growth inhibition. If focus size suppression is indicative of cell growth suppression, the current gap junction-mediated inhibition hypothesis would not appear to apply to all of the cell combinations examined here (e.g. 10T1/2 / S180). The possibility exists that focus size may not be an accurate reflection of cell growth. It was shown in section 3.3 that the majority of foci appeared more compact than control colonies, as indicated by darker staining. It is possible that increased focus cell density due to the presence of the normal cells may contribute to the explanation of focus size inhibition.
To examine this possibility cell density within the foci has been examined using the β-gal lineage marker to identify the transformed cells. A proliferation assay, based on [3H]-thymidine incorporation and analysis by autoradiography, was used to determine the proportion and distribution of dividing transformed cells. The results of this analysis are presented in section 3.4.3.
In addition to the inhibition phenomenon, Stoker (1967) also observed that a small proportion of normal cells (when co-cultured with the transformed cells) were proliferating above background levels. It was suggested that this was possibly due to the growth media being changed prior to exposing the cells to [3H]-thymidine. However, in Stoker's investigation, the normal cells that were used, maintained LI's of 10-20% even at high cell density, making it difficult to confidently identify small changes in cell growth. The normal cell lines used in this study, maintain very low labelling indices once they have reached their saturation density (section 3.1.1) and this should facilitate the analysis of any possible normal cell growth stimulation surrounding the foci.
To address the above points and the questions raised in the Introduction to the results (section 3.1), and to limit the number of experiments to a manageable size, only selected combinations of those used in section 3.3. have been analysed here. These include cell lines from the S180 series (S180, S180E217 & S180NII) in co-culture with Rat2 cells. In addition to these cell combinations, co-cultures of Rat2 / BICR cells and co-cultures of 10T1/2 / S180 cells will be used to assess the contribution, if any, of
increased cell density (relative to control colonies) contributes to the explanation of focus size suppression.
3.4.2. Does focus compaction contribute to the difference in size between foci and control colonies?
Focus cell density was measured to determine whether increased cell density (relative to the control colonies) could contribute to the explanation of focus size suppression. Focus assays were set up as described in section 3.3.5 using the cell combinations given above. For cell proliferation analysis, all cultures were pulsed with $^{[3}H$]-thymidine (0.185MBq/ml) for 18 hours prior to fixation, once fixed they were stained with x-gal and processed for autoradiography (section 2.5). For each time point 40 foci (or max. possible when less than 40 formed) and 40 control colonies, above a diameter of 0.1mm (area=0.008mm$^2$), were measured and average focus/colony area determined. To provide greater statistical confidence this data was combined with the data obtained from the focus formation experiments, presented in section 3.3.5. The combined averaged results have been presented graphically in Figure 3.11.
Cell density, within foci and control colonies was measured and is given as the number of cells / UA (1UA=0.175mm$^2$; see section 2.4). For each time point 5 foci and 5 colonies were analysed. Foci/colonies selected for analysis were not in contact with other foci/colonies and close to average size for each time point. For each focus/colony examined, cell density was derived from 10 UA's (or max. possible when focus/colony size was small) and the results from the 5 foci/colonies averaged and presented in the graphs of Figure 3.12; relevant points made throughout the text are illustrated in Figure 3.13. In some instances cell density together with the intensity of the $\beta$-gal staining within the focus was too high to distinguish individual cells, for example Figure 3.13.a. Attempts were made to overcome this problem by decreasing the staining time but x-gal staining is difficult to control and tends to be all or none. The intensity of staining varies between different cell lines which is presumably due to different levels of $\beta$-gal expression. For example, in Figure 3.13.b, a focus with high cell density is shown and although blue, individual cells can be distinguished. It was sometimes possible to overcome the problem by increasing the light intensity passing through the sample on the microscope stage.
It can be seen in Figures 3.11 that within each assay there is sometimes significant variability in control colony size. For example, S180 and S180E217 control colonies (Figures 3.11 A & B) at day 8 are larger than respective colonies recorded at day 12. This level of variability is perhaps to be expected in a colony formation assay, due to variation in the time taken for individual cells to attach and divide and colony distribution within the plate i.e. if two cells attach to the dish close to one another there may be local feeder effects. In focus assays the frequency of inhibition may vary and foci may
therefore grow at different rates. Despite the variability it is possible to determine significant trends within each experiment.
Cell density within S180 control colonies initially increases over time (Figure 3.12.A) but begins to level off after 10 days in culture. It was not possible to measure cell density in S180 foci at all time points. However, the fact that individual cells could not be identified in these foci (indicated by the asterisks in Figure 3.12.A) is itself indicative that cell density was high. These observations would suggest that high cell density, mediated by the physical presence of the surrounding Rat2 cells contributes, at least in part, to the difference in size between S180 foci and respective S180 colonies. It is not clear at this stage whether there is any cell growth inhibition within the foci. One method to determine the level of growth inhibition is to determine the total number of cells per focus (area x density) and compare it to the number of cells in respective control colonies. However, the calculation only provides a very rough approximation of total cell number, since the variations associated with focus area and cell density (which could not always be measured) are also multiplied. The proliferation assay enables a more accurate and detailed growth analysis to be carried out and, by examining the general degree of $[^{3}H]$-thymidine labelling above each focus, provides a better indication of cell growth within dense foci.
It can be seen from the data in Figure 3.12.B. that cell density within S180E217 foci, at all time points analysed, is higher than in respective control colonies. This difference in cell density can be seen in the focus and colony examples shown in Figures 3.13.c & d. It would appear that the high cell densities within the foci contribute to the explanation of focus size suppression (Figure 3.11.B). However, the difference in size between the foci and colonies at certain time points is so great (e.g. Figure 3.10.a) that it would suggest some degree of cell growth inhibition is imposed on these cells. The proliferative status of the S180E217 cells has been examined (section 3.4.3).
S180NII cells showed the highest level of focus number inhibition (section 3.3.2) and focus size inhibition (Figure 3.11.C). However, when plated simultaneously with the Rat2 cells they showed a very small increase in average focus size over the 12 day culture period and a marked increase in focus size over time when colonies were pre-established for 2 or 4 days (Figure 3.11.C). Cell density within the S180NII foci was generally less than in respective control colonies (Figure 3.12.C). Over the first 3 time points, when both cell types were plated simultaneously, cell density remains constant (~10 cells / UA). Cell densities within the pre-established foci was higher but also remained relatively constant over time (~20 cells / UA), and below respective colony cell density. Together, these data would suggest that increased cell density does contribute towards the explanation of focus size inhibition with this cell line.
Cell density within S180NII and S180E217 colonies increases over time but generally remains less than the level of cell density in S180 parental colonies. It would perhaps be expected that the cadherin transfectants would show higher colony cell
density than parental S180 cells on the basis that they adhere to each other better, due to the expression of N or E cadherin. However, these cells appear less inclined to multilayer, particularly at the colony periphery, thereby leading to a lower cell density when compared to that in S180 foci (e.g. Figure 3.13. g & h).
BICR cells show very little inhibition of focus number when cultured with the Rat2 cells, however they did show significant suppression in terms of focus size (Figure 3.11.D). Cell density within the foci (Figure 3.12.D) is higher than in control colonies which would suggest that cell compression and increased cell density contributes to the explanation of focus size inhibition (Figure 3.11.D). Increased cell density within the foci can be seen by comparing the focus shown in Figure 13.e with the respective control colony shown in Figure 13.f.
10T1/2 cells do not inhibit the number of foci formed by S180 cells (section 3.2), which would suggest that S180 cells escape growth inhibition. However, those foci which formed were significantly smaller than their respective control colonies (Figure 3.11.E). High cell density within the foci (Figure 3.12.E) indicates that the foci are compressed by the surrounding normal cells and this would appear to contribute significantly to the difference in size between S180 foci and S180 colonies. However, smaller than average foci were observed which would indicate the cells can be growth inhibited. The level of cell proliferation within these small foci and the averaged size foci has been examined in the following section.
The majority of foci examined were smaller than respective control colonies. In many published reports (e.g. Mehta et al 1986, Martin et al 1992) this type of result is frequently interpreted as suppression. However, further analysis of the foci shows that in many instances (with the exception of S180NII foci) cell density within the foci is markedly higher than cell density in control colonies. The data would suggest that the normal cells restrict the transformed cells from gaining access to the culture dish causing them to multilayer and their density within the foci to increase. The level of focus compression observed would appear to contribute to the explanation of focus size suppression and would imply that cell growth inhibition is not as high as the focus size data would suggest. To determine the level of growth inhibition within the compressed foci, a detailed analysis of cell growth within the foci and control colonies has been carried out. The results are presented next.
Figure 3.11. Average colony and focus size.
In co-cultures, normal and transformed cells were plated at $10^6$ and 500 cells /90mm dish. Separate cultures of each cell lines plated at the same density as above were set up as controls. The average colony and focus area at each time point was obtained from measuring 40 colonies and 40 foci (or max. possible when less than 40 formed) above a diameter of 0.1mm. The same S180 colony control data has been presented in Figures A & E.
Figure 3.11. Continued: Average focus and colony size
Figure 3.12. Cell density within foci and control colonies.
In co-cultures normal and transformed cells were plated at $10^6$ and 500 cells / 90mm dish. Controls were represented by separate cultures of each cell lines seeded at the same densities as above. Cell density within foci and control colonies is given as the average number of cells per UA. For each time point 5 foci and 5 colonies were analysed. For each focus, cell density was determined from 10 UA's (or max. possible where focus/colony was small). The results from the 5 foci/colonies were averaged and plotted in the above graphs. Data missing in Rat2/S180 cultures (indicated by *) was due to high cell density, which prevented individual cells from being distinguished. Data missing at d16d12 in Rat2/S180NII and Rat2/S180E217 cultures is due to loss of cells during fixation. The S180 data has been used in Figures A & E.
Figure 3.11. Continued: Average focus and colony size
Figure 3.12. Cell density within foci and control colonies.
In co-cultures normal and transformed cells were plated at $10^6$ and 500 cells / 90mm dish. Controls were represented by separate cultures of each cell lines seeded at the same densities as above. Cell density within foci and control colonies is given as the average number of cells per UA. For each time point 5 foci and 5 colonies were analysed. For each focus, cell density was determined from 10 UA's (or max. possible where focus/colony was small). The results from the 5 foci/colonies were averaged and plotted in the above graphs. Data missing in Rat2/S180 cultures (indicated by *) was due to high cell density, which prevented individual cells from being distinguished. Data missing at d16d12 in Rat2/S180NII and Rat2/S180E217 cultures is due to loss of cells during fixation. The S180 data has been used in Figures A & E.
Figure 3.12. Continued: Cell density within foci and control colonies
Figure 3.13. The above micrographs illustrate cell density within foci and colonies. In co-cultures normal and transformed cells were plated at $10^4$ and $500$ cells / dish respectively. In colony controls, cells were plated at $500$ cells / dish. In Figure a) individual cells are difficult to distinguish due to high cell density and high levels of $\beta$-gal - which leads to a dark blue stain, however, in b), cells are also at high density but the level of staining still enables single cell identification. In Figures c) - f) cell density in transformed foci and respective control colonies is compared. In Figures g) & h) cell density in colonies of related cell lines is compared, for details see text. Bar represents scale.
3.4.3. Inhibition of cell division within the foci.
It is clear from the results in section 3.3 that focus formation assays provide only limited information on the inhibition phenomenon. Cell density analysis has shown that foci can be compressed by surrounding normal cells and this appears to contribute to the explanation of focus size suppression. This would imply that estimates of growth inhibition based only on focus size may be inaccurate. It is not yet clear what the actual level of growth within the foci is and until this is ascertained the role of GJIC can not be properly assessed. Only when these points are addressed can the questions detailed in the Introduction to the results (see section 3.1) be answered. The level of cell proliferation within the foci and control colonies has been measured using a growth assay based on $^{3}H$-thymidine incorporation and analysis by autoradiography.
Section 3.4.2 describes the experimental set up and the cell combinations used. At each time point the percentage of dividing transformed cells in control and co-cultures was obtained after analysing 5 colonies and 5 foci. For each focus/colony the average number of labelled and unlabelled cells/UA was obtained from 10 UA's (or max. possible when focus/colony was small). The results from the 5 foci/colonies were averaged and plotted as percentages in the graphs presented in Figure 3.14. The LI of the normal cells was measured in all co-cultures to confirm they had reached saturation density and were growth inhibited. The LI was obtained from counting the number of labelled and unlabelled cells in 20 UA's (samples were taken as far away as possible from any foci).
The average LI obtained for the normal cells over all time points was 3.7% ± 1.2 for Rat2 cells and 4.2% ± 1.4 for 10T1/2 cells. These figures indicate that the normal cells have reached their saturation density (section 3.1.1) and are significantly growth inhibited. However, growth of Rat2 cells (but not 10T1/2 cells) above the background level was observed close to many foci in all of the co-cultures and this has been analysed further in section 126.96.36.199.
S180 cells (in culture with Rat2 cells):
In control cultures of S180 cells, the LI within the control colonies drops considerably after 12 days in culture (Figure 3.14.A). This is consistent with earlier data (section 3.1) which shows that the growth of the S180 cell population begins to slow down after long periods in culture. However, this does not appear to be due to nutrient deprivation or saturation density. This decrease in cell proliferation corresponds with cell density levelling off over time (Figure 3.12.A). Analysis of cell growth in many of the S180 foci in co-culture with Rat2 cells was hindered by the high cell density and high intensity β-gal staining of the cells (as indicated by asterisks in Figure 3.14.A). Although individual cells within the foci could not be distinguished at all time points it was clear that proliferation was high, as indicated by the high level of labelling above the foci.
However, it can be seen in Figure 3.14.A. that inhibition was recorded at d10d8 and d8d4. There were also several examples of smaller than average foci present in many of the Rat2/S180 co-cultures, (e.g. Figure 3.15.e). The proportion of proliferating cells in such foci was low, in Figure 3.15.e, for example only ~16% of cells are dividing whereas in respective control colonies the proliferation was close to 100% - Figure 3.14.A.
**S180E217 cells (in culture with Rat2 cells):**
S180E217 cells in control colonies follow a similar labelling pattern over time as their parental S180 cells i.e. by day 12 their LI has significantly dropped (Figure 3.14.B). When the S180E217 cells are plated simultaneously with the Rat2 cells and a focus diameter cut-off point of 0.1mm (area: 0.008mm²) is used, there is little inhibition of focus number (section 3.3.5). However, the size difference between the foci and respective colonies is marked (e.g. at d12d12 average colony size is 10mm² & average focus size, formed on a background of Rat2 cells is 0.5mm²; Table 3.10.b). This would suggest that there is a high level of growth inhibition occurring. However, analysis of the foci which do form at these time points, reveals that cell growth is not significantly suppressed (Figure 3.14.B). Similarly, when S180E217 cells are pre-established for 2 or 4 days before the normal cells are added, there is very little, if any, cell growth inhibition in the subsequent foci (e.g. Figure 3.13.c).
Although cell density within these foci is higher than in control colonies it is clearly not sufficient to account for the difference in size between the foci and the control colonies (e.g. Figure 3.10.a). This would suggest that a high proportion of cells within the foci are unable to attach to the culture dish (due to high cell density) and are therefore pushed off the dish.
**S180NII cells (in culture with Rat2 cells):**
The level of cell proliferation in control cultures of S180NII cells remains high in the majority of time points analysed (Figure 3.14.C & for example Figure 3.15.a). The level of proliferation in these colonies is consistent with an increase in colony size and colony density over time (Figure 3.11.C & Figure 3.12.C respectively).
When S180NII cells are co-cultured with Rat2 cells the highest level of inhibition occurs when the two cell types are plated simultaneously (Figure 3.14.C) and examples of inhibited foci can be seen in Figure 3.15. b & c. However, by the 12th day of the co-culture, a small proportion of cells within many of the S180NII foci are beginning to escape the growth suppression (Figure 3.14.C. e.g. Figure 3.15.d). This is consistent with a small increase in their average focus size (Figure 3.11.C). Foci formed from S180NII cells which were pre-established for 2 and 4 days have a higher LI than foci formed when the two cell types were plated simultaneously, suggesting that when a focus has grown to a certain size (>0.1mm²) there response to the inhibitory mechanism is markedly reduced. However, unlike S180E217 cells, S180NII cells are still
significantly suppressed (30-60% inhibition; Figure 3.14 C). There appeared to be no pattern to the distribution of inhibited cells within the S180NII foci, i.e. cells at the edge of the focus appeared to be no more likely to be inhibited than cells at the centre.
**BICR cells (in culture with Rat2 cells):**
BICR cells are well coupled to Rat2 cells but only show a very low level of growth inhibition when co-cultured with them (Figure 3.14.D). This low level of growth inhibition is maintained even when the BICR cells are pre-established. However, the difference in size between foci and respective colonies recorded (see Figure 3.14.D) was large, which would suggest that increased focus cell density significantly contributes to the explanation of focus size inhibition or cells are lost from the culture dish due to overcrowding.
**S180 cells (in culture with 10T1/2 cells):**
The number of foci formed by S180 cells in co-culture with 10T1/2 cells was greater than the number of colonies formed in control cultures and would appear to show that some form of feeder effect was taking place (section 3.3.2). However, the cell proliferation data (Figure 3.14.E) shows that a significant, but generally low level of inhibition is imposed on the S180 cells at most time points, even when the S180 cells are pre-established. It is possible that the feeder effect may occur before the 10T1/2 cells reach their saturation density and therefore before any inhibition is imposed on the S180 cells. Subsequent foci show low levels of growth suppression which together with increased cell density (section 3.4.2) leads to smaller foci in comparison to controls. It is not clear what causes the reduction in the growth of the S180 cells. Earlier results (section 3.1) would suggest that as the age of the culture increases the level of proliferation decreases but these changes are not associated with a saturation density or nutrient deprivation. Many smaller than average foci were observed in the 10T1/2 /S180 co-cultures (e.g. Figure 3.15.f). The level of cell proliferation in these foci is generally low in comparison to control colonies and the average size foci. It is unlikely that the inhibition is mediated by GJIC since the two cell lines do not appear to communicate with each other (Table 3.3).
**3.4.4. Summary.**
The cell proliferation analysis would appear to show that inhibition phenomenon is not as common as the literature tends to suggest (Section 1.6). Focus size measurements are presented in many published reports as indications of growth suppression, however, it has been shown here that focus compression (as indicated by increased focus cell density) can contribute significantly to the difference in size between
the foci and control colonies. Furthermore, detailed examination shows that the proportion of cells dividing within many compressed foci can often be very high.
The results from the cell proliferation analysis have added further complexity to the understanding of the role of gap junctional communication in the inhibition pathway. Transformed cell lines which are well coupled to normal cell lines may not necessarily be suppressed in co-culture with them. In this study for example, BICR cells, which are coupled to the Rat2 cells, are poorly growth inhibited by them. This would suggest that these cells do not respond to the putative inhibition factors transmitted from the normal cells and/or that the growth control mechanisms impaired in this cell line are not associated with loss of GJIC. 10T1/2 and S180 cells do not appear to communicate with each other, yet when they are cultured together a low level of S180 growth inhibition is recorded. And in some instances several small, highly inhibited foci were observed. However, communication between the two cell lines may be very low and therefore go undetected with the type of communication assay used here. Over long periods of time, transfer of putative inhibitory factors may be sufficient to cause low level growth suppression.
It has been shown that transfection of the poorly coupled S180 cells with E or N cadherin results in increased homologous and heterologous communication (section 3.2.2). However, only S180NII cells are growth inhibited by the Rat2 cells. The S180E217 cells appear to remain in cell cycle in the presence of the Rat2 cells although the difference in size between the foci and control colonies is sometimes very large. This would suggest that the physical presence of the surrounding normal cells (which results in high focus cell density) may cause cells within the foci to detach due to overcrowding. It is not clear why N cadherin but not E cadherin expression can confer suppressible phenotypes on the S180 cells. Both cell lines show increased communication relative to parental S180 cells it would appear that GJIC is necessary but not sufficient for inhibition to occur. It is possible that adhesion, rather than GJIC, is important for suppression and the increase in GJIC is brought about by the increase in adhesion. However, it is not clear why N cadherin, but not, it would seem, E cadherin alters the interactions between S180 and Rat2 cells to one which favours suppression.
The size of the initial transformed cell colony appears to be important in terms of the level of growth inhibition which is imposed on the S180NII foci. When the S180NII cells are plated simultaneously with the Rat2 cells they are able to go through 2-3 cell divisions before the Rat2 cells reach saturation density and become growth inhibited. Most of the colonies over the first 12 days of co-culture (when plated simultaneously with the normal cells) remain below the 0.1mm diameter threshold size and are normally ignored. Of those S180NII foci which have reached the threshold size, a high proportion of cells are still inhibited. However, when the S180NII cells are pre-established the foci show increased levels of cell proliferation. These data suggest that the larger the initial colony size the less likely the cells are to be inhibited by surrounding normal cells. The
Figure 3.14. Labelling index of transformed cells within colonies and foci
In co-cultures normal and transformed cells were plated at $10^5$ and 500 cells /90mm dish respectively. Controls were represented by separate cultures of each cell line seeded at the same densities as above. For each time point 5 colonies and 5 foci were analysed. For each colony/focus the average number of labelled & non-labelled cells/UA was determined from 10 UA's (or max. possible when foci/colonies were small). The results from the 5 foci/colonies were averaged and plotted as percentage labelled cells in the above graphs. The asterisk indicates that focus density was too high to be able to distinguish individual cells. No data could be obtained in Rat2/S180NII or Rat2/S180E217 cultures at the time point d16d12 because cultures detached during fixation.
Figure 3.14. Continued: Labelling index of transformed cells within colonies and foci
Figure 3.15. Levels of proliferation within foci and control colonies
a). S180NII d8d8 control
b). Rat2 / S180NII d8d8
c). Rat2/S180NII d8d8
d). Rat2 / S180NII d12d12
e). Rat2 / S180 d8d4
f). 10T1/2 /S180 d8d4
Figure 3.15. The above figures are examples from proliferation assays designed to study the effect of excess normal cells on the growth of transformed cells. Normal and transformed cells were plated at $10^6$ and 500 cells/dish respectively. Controls were represented by separate cultures of each cell line at the density as above. Figure a) shows the typical level of proliferation within a transformed cell colony (S180NII in this example) at the time point shown. Figures b) & c) show S180NII cells cultured with excess Rat2 cells for 8 days. Note the high level of growth inhibition. In d) it can be seen that after 12 days culture with the Rat2 cells, S180NII cells are beginning to escape suppression. In most instances S180 parental cells appeared to escape suppression, however, smaller than average S180 foci were observed in Rat2 and 10T1/2 co-cultures, such as those shown in Figures e) & f). However, not all cells within these foci were growth inhibited. Bar represents scale.
size of colony which can escape growth inhibition may be determined by the level of GJIC between the transformed and surrounding normal cells. For example S180NII cells show higher heterologous communication than S180E217 cells. The rate of inhibition transfer may therefore be important, coupled with the ability of the transformed cells to maintain their aberrant phenotype (possibly by the production of stimulatory growth factors), which increases as the focus grows in size.
3.4.5. The effect of the normal cell monolayer on focus morphology.
It has been shown throughout previous sections that the physical presence of the normal cells appears to restrict focus expansion as indicated by higher cell density in many foci. However, transformed cell proliferation remained high in most of the cultures which led to marked increases in the size of many foci over time. The morphology of these growing foci appears to be influenced by the surrounding normal cell monolayer. Examination of focus and cell morphology may provide information on whether the expanding foci grow over the surrounding monolayer or whether the normal cells act as a barrier to focus expansion and are pushed back as the foci expand.
The morphology of the foci and colonies, whose growth was analysed in section 3.4.3, have been examined. S180 and BICR control colonies tend to be circular in shape with ragged edges. The edges of S180E217 and S180NII colonies tended to be smoother because these cells are less inclined to overlap (section 3.4.2; Figure 3.13. g & h) and this is likely to be caused by better cell-cell adhesion. The majority of foci had uneven edges which is probably generated by the ability of the cells at the periphery of the colony to move into areas clear of other cells. Cell density at the periphery of the colonies is generally less than in the centre where competition for space on the culture dish is likely to be greater and where multi-layering is more common (e.g. Figure 3.16.a & b).
Different transformed cells formed foci with different morphologies, however, all appeared to be influenced by the presence of the normal cells. Focus morphology also varied depending on the length of time the foci spent in co-culture with normal cells and whether the transformed cells were pre-established.
In general, foci formed by BICR cells (when plated simultaneously with the Rat2 cells) tended to be circular (e.g. Figure 3.16.c) whereas foci formed by S180 cells or the S180-cadherin transfectants, when the cells had been plated simultaneously with the Rat2 cells, tended to align with the 'grain' of the normal cell monolayer and were generally longer and thinner (e.g. Figure 3.16.d). In the majority of instances the foci formed by all of the transformed cells tended to have smoother edges than the control colonies.
Foci formed from pre-established transformed cells varied in morphology from foci formed when both cell types were plated simultaneously. When the normal cells
were added to pre-established colonies of BICR, S180 or S180-derived cells, the subsequent foci initially had ragged edges (e.g. Figure 3.16.e). This would suggest that the normal cells were able to move and divide in-between the transformed cells at the focus periphery where cell density was initially lower. However, as the culture period increased and the transformed cells continued to divide, the average focus cell density in the majority of instances also increased and often to a much greater extent than control colonies (Figure 3.12). This would suggest that the normal cells, upon reaching confluence, compete with the transformed cells for space on the culture dish, thereby reducing the rate of focus outgrowth (leading to increased cell compression, multilayering and consequently an increase in focus cell density). In many instances, particularly in later time points, the edges of most foci of all cell types became smoother although, in general, the pre-established foci remained more irregularly shaped (e.g. Figure 3.16.f).
These observations would also suggest that there is little transformed-cell over growth of the normal cell monolayer. That is, within the majority of foci, cell multilayering / overlapping is frequent, yet cells at the focus periphery rarely project over the 'grain' of the monolayer (e.g. Figure 3.16. c). In Figures 3.16. g, h & i, for example, it can be seen that transformed cell processes rarely project across the 'grain' of the monolayer, but do project into it when they are oriented with the 'grain' of the monolayer. This would suggest that the transformed cells can grow more easily in-between the normal cells but not across them. One common feature of normal cells is that they generally do not show nuclear overlap (but can show cytoplasmic overlap). In general the transformed cells also appear to avoid nuclear overlap with the normal cells (though not with themselves), for example, Figures 3.16. g, h & j (red arrows). Clearly, in some instances the transformed cells do grow over the normal cells and this has been highlighted in Figure 3.16. h (green arrows).
It would appear that the transformed cells avoid, or are partially prevented from growing over the monolayer. However, despite the apparent competition for space on the culture dish many foci continue to expand. If, as the focus and cell morphology analysis suggests, there is generally little transformed-cell overgrowth of the surrounding monolayer then an increase in normal cell density would be expected as the foci expand and push back the normal cells. The increase in normal cell density is likely to be exacerbated further by the apparent growth stimulation of normal cells which surround many foci (e.g. Figure 3.16. c & d - yellow arrows; this phenomenon has been examined further in section 188.8.131.52).
The question therefore remains as to what happens to the normal cells which lose space on the culture dish to the dividing transformed cells. This is addressed next.
Figure 3.16. The effect of normal cells on focus morphology.
a). BICR d8d8
b). BICR d8d8
c). Rat2 / BICR d12d12
d). Rat2/S180E217 d8d8
e). Rat2 / BICR d6d4
f). Rat2 / S180E217 d10d8
Figure 3.16. The micrographs above show the effect of normal cells on focus morphology. See Figure 3.14 for experimental details. Cell lines and cell combinations used, together with culture times are given above each figure. A BICR colony is shown in a) which in terms of shape and a ragged periphery is typical of most transformed cell colonies. In b) it can be seen that the cells at the periphery of the colony are at lower density than at the centre. In c) the BICR focus shown has smoother edges than the BICR colony but maintains a circular shape. The cells of the foci formed by S180 or S180-derived cells were generally aligned with the grain of the surrounding monolayer, foci were longer than those formed by BICR cells but also had smooth edges, e.g. Figure d). The effect of pre-establishing the transformed cells on subsequent focus morphology can be seen in Figures e) & f). In general foci begin with ragged edges, eg. Figure e), but over time the edges of the foci become smoother leading to irregularly shaped foci, eg. Figure f). Yellow arrows indicate examples of growth stimulated normal cells - a phenomenon which has been analysed further in section 184.108.40.206. Bar represents scale.
Figure 3.16. Continued: The effect of normal cells on focus morphology
g). Rat2 / S180 d10d8
h). Rat2/S180 d8d8
i). Rat2/S180d14d12
j). Rat2/S180 d12d8
Figure 3.16: Continued. Cell lines and cell combinations used, together with culture times are given above each figure. Red arrows indicate examples where the transformed cells appear to have avoided growing over the nuclei of normal cells. Green arrows indicate examples where the transformed cells appear to overlap the nuclei of normal cells. Bar represents scale.
3.4.6. The effect of transformed cells on surrounding normal cells.
It would appear from the focus morphology analysis that, in general, the transformed cells do not grow over the normal cells. However, there is continued transformed cell growth in many of the co-cultures and marked increases in the size of some foci in some of the cultures. For example after 12 days in co-culture with Rat2 cells the S180NII foci take up ~0.2% of the culture dish and S180E217 foci take up ~12% of the culture dish (these approximate figures are based on the total number of foci x the average area of foci; obtained from data in Tables 3.10.a & b).
Since there appears to be little overgrowth of the two cell types it is not clear what happens to the normal cells as the foci expand. An increase in normal cell density, particularly around the focus periphery where demand for extra space is presumably high, would be expected. This is likely to be exacerbated by the apparent small increase in normal cell proliferation surrounding many of the foci. The possibilities as to how the normal cells are accommodated when the foci expand are listed below:
1. As normal cells lose space on the culture dish to the expanding foci, they may shuffle up. The increase in density may be dissipated over the surface area of the culture dish which, over time, may lead to a general increase in normal cell density over the entire dish.
2. The normal cells may be physically pushed off the plate by the expanding foci or apoptose (possibly as a result of a signal from the transformed cells or as a result of over crowding).
These possibilities have been examined and the results presented in the following section.
220.127.116.11. The effect of expanding foci on normal-cell density.
To determine if normal cell density increases next to expanding foci and over the monolayer in general, normal-cell density (cells per UA) was determined within 0.175mm of the focus periphery and as far away as possible from any foci. Cell density was also measured in control cultures ($10^6$ cells / 90mm dish) at all time points. For each time point the cell density in the control cultures was obtained from 20 sample UA's and the results presented in Figure 3.17. In co-cultures, for each time point, cell density was determined next to 5 foci. Cell density was calculated from 10 Sample UA's around each focus (or max. possible where foci were small). The results from the 5 foci were then averaged and plotted in the graphs presented in Figure 3.18. Cell density as far away as possible from any foci at each time point was obtained from 20 sample UA's and the results plotted with the cell density data obtained next to the foci in Figure 3.18.
Rat2 cell density directly adjacent to either S180, S180E217 or S180NII foci is not significantly different (P>5%) from cell density away from the foci (Figure 3.18, A, B & C; e.g. Figure 3.19.b). However, in general cell density next to the foci is slightly
Figure 3.17. Cell density in separate cultures of Rat2 and 10T1/2 cells.
Separate cultures of Rat2 and 10T1/2 cells were plated at $10^6$ cells per 90mm dish. For each time point the average number of cells per UA was determined from 20 sample UA's.
Figure 3.18. Normal cell density next to and away from the focus periphery.
Normal and transformed cells were plated at $10^6$ and 500 cells / 90mm dish respectively. For each time point normal cell density (average number cells / UA) was determined next to 5 foci. Normal-cell density was calculated from 10 sample UA's taken around the focus. The results from the 5 foci were averaged and plotted in the graphs above. Normal cell density was also taken as far away as possible from any foci. For each time point cell density was calculated from 20 sample UA's. Data was not obtained at d16d12 in Rat2/S180E217 & Rat2/ S180NII co-cultures due to loss of cells during fixation.
Figure 3.18. Continued: Normal cell density next to and away from the focus periphery
Figure 3.19. The effect of transformed cells on normal cell density and morphology.
a). Rat2 d8d8
b). Rat2/S180 d8d8
c). Rat2/S180NII d6d4
d). Rat2/S180 d8d8
Figure 3.19. The examples above show the effect transformed foci have on normal cell density. The transformed cells were plated at 500cells/dish and the normal cells at $10^5$/dish. The period of time spent in culture is given above each micrograph. Cultures were fixed in 1% Formaldehyde and 0.1% Gluteraldehyde in PBS for 30mins. They were then washed with PBS and stained with x-gal for 3hrs and Giemsa (1:10) for 10mins (section 2.5.1). In Figure a) cell density in a control culture of Rat2 cells is shown. In Figure b) normal cell density appears unaffected by the presence of the compact focus. However, Rat2 cells directly adjacent to the focus are generally thinner than cells away from the focus periphery. In Figure c), again, normal cell density appears unaffected by the compact focus, however, general variations in normal cell density throughout the monolayer can be observed to the left of the focus. In Figure d) a local increase in Rat2 cell density can be seen next to the S180NII foci. Bar represents scale.
higher (though not significantly) than cell density away from the periphery of the foci. In the few exceptions where there was an obvious increase in normal cell density around the focus periphery, it was very localised, e.g. Figure 3.19.c. However, such increases in cell density occurred infrequently and not in any particular cell combination. Within most of the co-cultures there were areas of the monolayer in which cell density varied from the average (e.g. to the left of the focus shown in Figure 3.19.d), however, these variations were not always associated with the foci and were also observed in many of the control cultures.
In Rat2 / BICR co-cultures (Figure 3.18.D) the density of the normal cells shows considerable variability in comparison to control cultures over many of the time points. It is not clear why such variability occurred with this particular cell combination, however, there appears to be no specific trends to the changes in density over time.
In 10T1/2 / S180 co-cultures (Figure 3.18.E) the general trend appears to be a reduction in normal cell density over time. This is perhaps due to cell death as a result of the ageing of the media (section 3.3) and unknown factors.
It can be concluded from the data presented in this section that there appears to be an insufficient increase in the normal-cell density around the foci over time, even when foci grow relatively large, to account for space on the dish lost to the expanding foci. Furthermore, expanding foci do not appear to cause a significant increase in the overall density of the culture.
### 18.104.22.168. Apoptosis in control and co-cultures.
Loss of normal cells due to apoptosis may account for the general absence of increased cell density throughout the monolayer. Furthermore, apoptosis may indirectly cause the normal-cell growth stimulation which was observed surrounding the periphery of many foci i.e. as the cells die the subsequent space may signal other normal cells to divide, which must then compete with the transformed cells for the available space on the culture dish.
To examine this possibility immunocytochemistry was used to detect apoptosis-induced DNA strand breaks (section 2.1.2). Focus assays were set up using co-cultures of Rat2 and S180E217 cells (foci formed by this cell lines are not growth suppressed and show marked increases in focus size over time). Rat2 cells were seeded at $10^6$ / 90mm dish and S180E217 cells plated at 500 / 90mm dish either simultaneously with the normal cells or pre-established for 4 days. After 4 days the co-cultures were fixed in fresh 4% paraformaldehyde for 30mins and DNA strand breaks visualised, as described in section 2.6. Controls were represented by separate cultures of Rat2 cells, (plated at $10^6$ / 90mm dish) fixed after the same time periods as the co-cultures. As positive controls, parallel cultures were treated with DNase I for 10mins (the procedure is described in section
As negative controls, fixed cultures of Rat2 cells were only treated with the TUNEL reaction solution (see section 2.6)
Low levels of apoptosis were found throughout the general population of Rat2 control cultures (Figure 3.20.c). The space vacated by the dead cells may signal other cells to divide. And the proportion of cells apoptosing per plate is would appear to account for the residual labelling index of 2-4 %, recorded in these cultures at saturation density (section 3.1.1). A similar level of Rat2 cell apoptosis was observed in co-cultures of Rat2 / S180E217 cells (Figure 3.20.d). There was, however, no specific localised apoptosis of normal cells surrounding the foci (Figure 3.20.e).
The normal cells may be pushed off the culture dish by the expanding foci. However, in general, there did not appear to be a significant increase in the amount of floating debris within the growth media. The exceptions were invariably in older cultures (d16d12), which appeared generally less healthy.
From the results presented in this section it would appear that the normal cells do not significantly increase in density over time, even as the foci expand and take up more space on the culture dish. The transformed cells do not appear to cause an increase in the level of normal-cell apoptosis and the normal cells do not appear to be physically pushed off the plate by the expanding foci. Evidence from the focus morphology analysis would suggest that the transformed cells can, to some extent, grow over the normal cell monolayer, however, the fact that foci are compact in comparison to control colonies, would suggest that they are restricted in doing so. In order to measure the level of overgrowth, attempts were made to transfect the normal cells with the β-gal gene and then repeat the focus experiments with β-gal⁻ transformed cells. However, the normal cells were unreceptive to the transfection protocol and only transient transfectants were obtained. Time constraints meant that alternative transfection regimes could not be explored.
The data would suggest that if the normal cells shuffle up, the increase in density is dissipated over the surface of the culture dish and the increase in space taken up by the foci appears to be insufficient to generate detectable increases in the density of the normal cells.
Figure 3.20. Rat2 apoptosis levels in focus formation assays
a(i). Rat2/S180E217^{-}ve control d8
b(i). Rat 2^{-}ve control d8
c(i). Rat2 d8
d(i) Rat / S180E217 d8d4
e(i). Rat2 / S180E217 d8d4
a(ii).
b(ii).
c(ii).
d(ii)
e(ii).
Figure 3.20. The above micrographs are examples from experiments designed to determine the level of apoptosis within the Rat2 monolayer when co-cultured with S180E217 cells. Rat2 and S180E217 cells were plated at $10^5$ and 500 cells/dish respectively. Cell combinations and culture times are given above each micrograph. Figures a(i) - e(i) are immunofluorescence images of corresponding phase contrast images a(ii) - e(ii). Method of apoptosis detection is described in section 2.6. Positive and negative controls (Figures a & b respectively) were set up as described in section 2.6. The general level of apoptosis observed within a separate culture of Rat2 cells is shown in Figure c). Levels of apoptosis within co-cultures of Rat2/S180E217 cells was similar to controls (Figure d) and it can be seen in Figure e) that levels were no higher directly adjacent to S180E217 foci (Figure e is at higher mag. than the corresponding phase contrast image -e(ii; no equivalent phase objective was available).
22.214.171.124. Do transformed foci affect the proliferation rate of the normal cells?
One advantage of the combined focus and proliferation assay developed for this project is that it provides direct evidence for the proliferation status of all cells in the culture dish. Observations made in earlier parts of the study would suggest that the growth of Rat2 cells close to transformed foci are growth stimulated. When investigating the inhibition phenomenon, Stoker also observed increased normal-cell proliferation. Stoker suggested that this was possibly due to the media being changed prior to analysis - an effect first observed by Todaro (1965). This possibility can be ruled out in the experiments carried out here because the culture media throughout the time course of the focus assays was not changed. Since Stokers initial observations there has been no further investigation into any possible growth stimulation phenomenon.
Growth stimulation of the normal cells adjacent to transformed foci may reduce the efficacy of the proposed inhibition mechanism since it has been shown that growth inhibition occurs only after the normal cells have reached saturation density (section 3.4.3) and are themselves growth inhibited. The proportion and distribution of stimulated normal cells has been analysed in more detail in this section.
At each time point, in the normal-cell control cultures, the average number of labelled and unlabelled cells per UA was determined from 20 UA's. The results are plotted as percentage labelled cells in (Figure 3.21. A & B). At each time point in the cocultures the level of normal-cell growth stimulation was measured within 0.175mm of the focus periphery. 5 foci were analysed and around each focus the number of labelled and unlabelled cells / UA was determined from 10 UA's (or the max. possible when foci were small). The results from around the 5 foci were averaged and plotted as percentage labelled cells in the graphs presented in Figure 3.22. In addition the level of background cell proliferation away from the focus periphery, at all time points, was determined from 20 sample UA's taken as far away as possible from any foci and the results also plotted in the graphs presented in Figure 3.22.
In the control cultures, the labelling index of Rat2 and 10T1/2 cells remains relatively constant over all time points (Figure 3.21. A & B). In contrast, a relatively high level of cell proliferation was observed next to most foci in most focus assays (Figure 3.22; e.g. Figure 3.23.a).
In Rat2/S180 co-cultures the level of Rat2 cell proliferation away from the foci (Figure 3.22.A) remains at similar levels to control cultures (Figures 3.21.A). However, a significantly higher proportion of Rat2 cells within 1 UA of S180 foci, are dividing. The level of proliferation remains relatively constant over time.
The level of Rat2 cell proliferation surrounding S180E217 foci (Figure 3.22.B) is also significantly higher than in control cultures and away from any foci (e.g. Figure
Furthermore, it appears that cells in direct contact with the focus periphery were less likely to be dividing than cells 2-3 cells away (e.g. Figure 3.23.e & f).
Although there was stimulation of Rat2 cells surrounding S180NII foci (Figure 3.22.C), the level of stimulation was generally less than that recorded next to S180 & S180E217 foci. This would appear to correlate with the level of proliferation within the foci (i.e. transformed cells which are not growth inhibited stimulate a greater proportion of normal cells). However, cell proliferation within S180 foci decreases over time (section 3.4.3 Figure 3.14.A) but this does not correspond to a decrease in Rat2 cell stimulation next to these foci. The size of the foci may be of more significance, i.e. the larger the focus the greater the disturbance to the surrounding monolayer, which may disrupt the contact-inhibition mechanism or the more transformed cells there are the greater the stimulatory signal, if the signal is mediated by a concentration-dependent factor.
The proportion of Rat2 cells dividing next to BICR foci is higher than that away from the foci and in control cultures (Figure 3.22.D; e.g. Figure 3.23. a & b). The highest level of stimulation is seen when the two cell types are co-cultured for 4 days. In the majority of instances the pattern of stimulation is similar to that observed next to S180 cells & S180 cadherin transfectants i.e. the majority of dividing cells appear to be situated 2 or 3 cells away from the focus periphery (e.g. Figure 3.23.c). However, it can be seen from the data in Figure 3.22.D that the level of growth stimulation decreases over time. The level of BICR proliferation with the foci remains relatively constant over time (Figure 3.14.D) and the foci continue to grow (Figure 3.11.D). This does not support the hypothesis that focus size in some way affects the level of surrounding stimulation. However, it is possible that the BICR cells deplete local concentrations of specific growth factors required for the normal cells to divide.
The proportion of 10T1/2 cells growing next to the S180 foci is not significantly different from cell growth away from the foci (Figure 3.22.E; e.g. Figure 3.23.d). However, 10T1/2 cell proliferation over the monolayer in general, at d4d4, d6d4 & d8d4, is higher than in control cultures. This level of proliferation is not seen in later time points. Experiments in section 3.3.1 show that 10T1/2 cells, when plated at an initial density of $10^6$, reach saturation density within 4 days. It is not clear why the mechanism responsible for inhibiting the growth of the 10T1/2 cells appears to be less effective at day 4 in the presence of the S180 cells, but would suggest that a concentration-dependent soluble factor is not involved since there are fewer S180 cells at these time points.
In instances where there is increased normal cell growth it is not clear how it is mediated. Experiments carried out in section 3.3.4 showed that transformed cell conditioned media was not capable of stimulating high density cultures of normal cells. The fact that the stimulation is localised around the foci suggests that stimulation may be
Figure 3.21. The level of proliferation within separate cultures of Rat2 and 10T1/2 cells.
Separate cultures of Rat2 and 10T1/2 cells were set up at $10^6$ cells per dish. For each time point the number of dividing and non-dividing cells / UA was determined from 20 sample UA's. The % of dividing cells was calculated and the results presented above.
Figure 3.22. Labelling index of normal cells next to and away from focus periphery.
See Figure 3.15 for experimental details. At each time point 5 foci were examined. Around each focus the average number of dividing cells/UA within 0.175mm of the focus periphery was derived from 10 sample UA's. The results from around the 5 foci were averaged and presented as % labelled cells above. The % of labelled cells away from the focus periphery was determined after counting the number of labelled cells per UA in 20 sample UA's. No data was obtained at d16d12 in Rat2/S1870E217 & Rat2/S180NII co-cultures due to loss of cells during fixation.
Figure 3.22. Continued: Labelling index of normal cells next to and away from focus periphery.
Figure 3.23. Stimulation of normal cells by transformed foci.
a). Rat2 / BICR d6d4
b). Rat2 / BICR d6d4
c). Rat2 / BICR d8d4
d). 10T1/2 / S180 d8d8
e). Rat2/S180E217 d8d4
f). Rat2/S180E217 d12d8
Figure 3.23. a)-f) show the effect of various transformed foci on the proliferative status of surrounding normal cells. The normal and transformed cells were plated at $10^5$/ dish and $500$/ dish respectively. The cell combinations and the culture times are given above each micrograph. Cultures were pulsed with [$^3$H]-thymidine (0.185MBq/ml) for 18hrs, then washed twice with PBS and fixed in 1% Formaldehyde and 0.1% Glutaraldehyde in PBS for 30mins. Cultures were then stained in x-gal for 8hrs, washed with PBS and processed for autoradiography (section 2.5). Figures a) & b) show increased levels of Rat2 cell growth localised around the BICR foci. In Figure c) this stimulation appears to be limited to a narrow band of cells surrounding the focus shown. In the majority of instances 10T1/2 cells were not growth stimulated by S180 foci, e.g. Figure d). In many instances growth stimulation appears to be more common when the Rat2 cells are close to but not in direct contact with the foci e.g. Figures c), e) & f).
caused by factors transmitted from the transformed cells. This may be achieved by a signal transmitted by GJIC or a paracrine factor (hence the localised effect). There is evidence to suggest that gap junctions mediate the stimulatory effect. This is on the basis that 10T1/2 and S180 cells do not appear to communicate with each other and 10T1/2 cells are not growth stimulated by the S180 foci. However, the level of Rat2 growth stimulation does not correlate with the level of communication with the transformed cells. That is, the level of stimulation is comparitively similar when the Rat2 cells are cultured with S180 cells (with which they are very poorly coupled) or with S180E217 cells & S180NII cells (with which they are well coupled).
Many of the Rat2 cells which are stimulated by the transformed foci are metabolically co-operating with the transformed cells via gap junctions. This is evident by the increased levels of $^{3}\text{H}$-thymidine labelling above individual, dividing Rat2 cells. For example, in Figure 3.26.a. a control population of Rat2 cells is shown with a relatively high proportion of cells dividing. Although the density of grains above the dividing cells is relatively low (Rat2 cells incorporate only low levels of $^{3}\text{H}$-thymidine due to a deficiency in thymidine kinase) it is sufficient to distinguish dividing cells from non-dividing cells. Rat2 cells dividing next to foci however, have a significantly higher grain density above their nuclei.
Growth stimulation may be caused by a physical disruption in the density-dependent growth inhibition mechanism of the normal cells and this is discussed further in section 126.96.36.199.
From these experiments it can be concluded that transformed foci appear to cause the growth stimulation of surrounding Rat2 cells but not 10T1/2 cells. However, the observations made during the analysis would suggest that cells in direct contact with the foci are less likely to be dividing than those a few cells away. The pattern of Rat2 cell stimulation surrounding foci was examined in greater detail and the results presented in the following section.
**188.8.131.52. Is there a pattern to the stimulation of Rat2 cells surrounding foci?**
The pattern of Rat2 cell growth stimulation next to the transformed foci has been investigated. No analysis of the 10T1/2 / S180 co-culture was performed since, in the majority of instances, 10T1/2 cells showed no significant levels of growth stimulation next to the S180 foci. To analyse the pattern of stimulation the UA, measuring 0.175mm square, was subdivided into 10 rows, (Figure 3.24). The numbers of labelled cells in each row (designated by the distance from the focus periphery) were counted. 10 UA's were sampled around each focus (or max. possible when foci were small) and 5 foci were analysed for each time point; averaged results are presented in Figure 3.25. In instances where the periphery of the focus was uneven the transformed cells nearest to the normal cells were taken as being the focus edge.
Figure 3.24. Analysis of normal cell labelling pattern.
Rat2 / S180E217 d8d4
Figure 3.24. The above diagram shows a single UA placed at the edge of a focus. The number of labelled cells per row were counted. This was repeated 10 times around each of 5 foci per time point. The results were averaged and plotted in Figures 3.25.
Figure 3.25.A. The pattern of Rat2 cell growth stimulation surrounding S180 foci.
For each time point the proportion and distribution of Rat2 cell stimulation surrounding 5 foci was analysed. 10 Sample UAs were taken around each focus (or the max. possible when foci were small) and the average number of dividing cells at specific distances from the focus periphery determined (see Figure 3.25). The results from these 5 foci were averaged and plotted above. It was not possible to obtain data at D16/D12 due to cell loss during fixation. Culture times are given in the top right corner of each graph.
Figure 3.25.B. The pattern of Rat12 cell growth stimulation surrounding S180/E17 foci.
See Figure 3.26.A. for experimental details. Culture times are given in the top right corner of each graph. No data was obtained at D16D12 due to cell loss during fixation.
Figure 3.25.C. The pattern of Rat2 cell growth stimulation surrounding S180NII food.
See Figure 3.26.A. for experimental details. Culture times are given in top right corner of each graph. No data was obtained at D1GD12 due to cell loss during fixation.
Figure 3.25.D The level and pattern of Rat2 cell growth stimulation surrounding BICR foci.
See Figure 3.26.A for experimental details. Culture times are given in top right corner of each graph. No data was obtained at D16D12 due to cell loss during fixation.
Figure 3.26. Patterns of Rat2 cell division next to transformed foci.
a). Rat2 d2
b). Rat2 / S180 d10d8
c). Rat2 / S180E217 d6d4
d). Rat2 / S180 d12d12
e). Rat2 / S180E217 d6d4
f). Rat2/S180E217 d8d4
Figure 3.26. The above figures are examples of Rat2-cell growth stimulation next to various transformed foci. See Figure 3.24. for experimental details. Figure a) shows a control Rat2 culture ~2 days before reaching saturation density. Although the level of labelling is relatively low over individual cells (due to low thymidine kinase levels) it is sufficient to identify dividing cells. In Figure b) it can be seen that cells 30-50µm from the focus periphery are more likely to be labelled than cells in direct contact with the focus. Note also, the increased labelling over individual dividing Rat2 cells, this is indicative of metabolic co-operation. In Figures c) - f) examples are shown of the range of labelling patterns which were observed.
In all of the focus assays at the majority of time points (Figure 3.25. A - D), the Rat2 cells, near to the foci, are more likely to be dividing if they are situated 1 or 2 cells away (30-50μm) from the focus periphery rather than in direct contact with it. In all of the assays, the level of stimulation generally decreases with distance from the focus periphery. However, at the majority of time points, there appears to be a wave-like pattern of growth stimulation. Although the differences between the peaks and troughs of each wave is often only a few cells the pattern is consistently similar regardless of which transformed cell type forms the foci. Examples of different patterns of stimulation are shown in Figure 3.26.b, c, d, e & f. In many instances the labelled cells appeared to be in either groups or rows surrounding the focus.
Data from the previous section (184.108.40.206) would suggest that stimulation of normal cell growth surrounding the foci is not due to the 'removal' of Rat2 cells, thereby making room for other cells to gain space and divide. There is evidence to suggest that the stimulation of the normal cells is caused by a specific signal(s). This is based on the data which shows that the level of growth stimulation decreases with distance from the focus periphery and would suggest some form of concentration dependence on a factor is involved.
If such a signal is involved it may be transmitted via gap junctions. This is based on the observation that stimulation only occurs between cell combinations which show heterologous communication. However, this hypothesis and that of a single concentration-dependent paracrine factor is weakened by several lines of evidence. Firstly, the level of Rat2 stimulation by the transformed cells does not correlate with the level of communication between the two cells types. Furthermore, the pattern of stimulation recorded is not compatible with the junctional or paracrine hypothesis, i.e. normal cells which are in direct contact with the transformed foci are less likely to be labelled than cells 2-3 cells away. If the signal were to be passed via gap junctions or in a paracrine fashion then cells in direct contact with the transformed cells would receive the signal first when it is at its most concentrated.
The growth stimulation may not necessarily arise from a direct signalling event but may be the result of a physical disturbance in the density-dependent contact inhibition mechanism which is responsible for the autonomous growth control of the Rat2 cells. This growth inhibition occurs when the normal cells reach high cell density (section 1.6) and may be responsible for the phenomenon of transformed cell growth inhibition first observed by Stoker (1967). However, the mechanism of contact inhibition is not known. Several models have been proposed and all require direct cell-cell contact and high cell density.
If the growth stimulation is caused by a concentration-dependent factor, transmitted via gap junctions or by a paracrine mechanism then it might be expected that when the transformed cells out-number the normal cells the proportion of normal-cell
stimulation would be higher. To examine this, experiments have been performed in which the transformed cells are cultured in excess of the normal cells. The results are presented next.
3.4.7. The affect of excess transformed cells on colonies of normal cells.
It has been shown that the presence of various transformed foci stimulates the growth of surrounding Rat2 cells. The level of stimulation decreases as distance from the focus periphery increases and there appears to be a wave-like pattern to the growth stimulation. It is not clear however, if the stimulation is caused by a specific stimulatory signal, transmitted via gap junctions, secreted from the transformed cells in a paracrine fashion or is the result of physical disruption to the contact-inhibition between cells.
To examine this possibility, experiments were set-up in which the ratio of normal to transformed cells (usually $10^6$ and 500 per dish respectively) was reversed. Thus any signalling factors transmitted from the transformed cells would pass into a relatively small pool of Rat2 cells and presumably reach high concentrations leading to a more pronounced effect.
Co-cultures of S180/Rat2, S180E217/Rat2, S180NII/Rat2 and BiCR/Rat2 and were set-up. Transformed cells were seeded at $10^6$ cells/90mm dish and normal cells at 500 cells/90mm dish (in triplicate). The two cell types were either plated simultaneously and analysed after 4 days co-culture or the normal cells were pre-established for 4 days the transformed cells then added and the co-cultures analysed after a further 4 days. As controls, separate cultures of normal and transformed cells seeded at 500 and $10^6$ per dish respectively, were set-up in triplicate and analysed at the same time points as respective co-cultures. Cultures were pulsed with $[^3H]$-thymidine (0.185MBq/ml) for 18 hours prior to fixation in 1% formaldehyde and 0.1% gluteraldehyde in PBS for 30 mins. Cultures were then stained with x-gal and processed for autoradiography according to the method in section 2.5.
At each time point the average normal-cell colony area in control and co-cultures was determined after measuring 40 colonies (or max. possible if less than 40 formed). Average cell density (cells/UA) within 5 colonies was determined. For each colony the number of cells / UA in 10 sample UA's (or max. possible when colonies were small), was counted, this was repeated for each of the 5 colonies and the average calculated. The same procedure as above was followed when determining the number of labelled cells within the colonies. The average number of labelled transformed cells/UA away from the colony periphery was determined from 20 sample UA's. The average number of labelled transformed cells/UA around 5 colonies was calculated from 10 sample UA's (or max. possible when colonies were small) taken within 0.175mm of the colony periphery.
In co-cultures of S180/Rat2 cells, many Rat2 colonies detached during the 4 day co-culture or during the fixation process, which would suggest that cells were unhealthy or dying. Because of these problems no analysis could be performed on this cell combination.
Data from the experiments using co-cultures of S180NII / Rat2 cells is presented in Table 3.13. Control colonies of Rat2 cells increase in size and cell density over the two culture periods. Their LI remains very high at both of the time points analysed (d4d4:100% & d8d4:95%). Rat2 colonies in co-culture with excess S180NII cells are significantly smaller than control colonies and have a slightly higher cell density. The LI of the Rat2 cells within the co-culture colonies is markedly lower than in controls which would indicate that some form of growth inhibition, by the transformed cells, is taking place. However, there does not appear to be a pattern to the distribution of the labelled Rat2 cells within the colonies. The morphology of the colonies and the cells are not significantly different from control colonies and cells (e.g. Figure 3.27.a & b). Although the colony size data shows that colony expansion is restricted in the co-culture their morphology suggests that the transformed cells do not severely encroach on the colonies and compress the cells, unlike the normal cells in focus assays which compress and restrict expansion of the transformed foci (section 3.4.5). This would suggest that the Rat2 cells compete more efficiently for space on the culture dish. The interface between the colonies and the surrounding transformed cell monolayer is distinct and there appears to be little or no overlap of either cell type which would suggest there is no overgrowth (e.g. Figure 3.27.b). Furthermore, the lack of increased density of S180NII cells around the Rat2 colonies, supports the possibility that the cells shuffle up when space on the culture dish is taken up by the expanding foci and would suggest that the S180NII cells have a lower efficiency for the plastic than the Rat2 cells.
The number of S180NII cells per UA in control cultures (d4d4:39, d8d4:46) is similar to that recorded in co-culture away from the colony periphery (d4d4:45, d8d4:48). There is no significant increase (P>5%) in the density of these cells in the presence of growing Rat2 colonies. The density of S180NII cells directly next to the colony periphery is not significantly higher than control or background levels (P>5%), e.g. Figure 3.27.b. and the labelling index of the S180NII cells remains constant regardless of whether the cells are situated next to or away from the colony periphery.
Table 3.13. The effect of excess S180NII cells on the growth of Rat2 cells.
| Rat2 / S180NII | d4d4 Control | Co-culture | d8d4 Control | Co-culture |
|----------------|--------------|------------|--------------|------------|
| Ave. colony area mm² | 0.9 (0.4) | 0.3 (0.1) | 6.5 (0.3) | 1.5 (0.1) |
| Ave. No. Rat2 cells per UA | 6 (1) | 10 (3) | 21 (3) | 17 (5) |
| Labelling index of Rat2 cells | 100 (0) | 20 (1.8) | 95 (4.5) | 11 (5) |
| Ave. No. S180NII cells/UA [away from colony edge] | 39 (11) | 45 (7) | 46 (9) | 48 (10) |
| Labelling index | 45 (7) | 47 (9) | 36 (7) | 51 (5) |
| Ave. No. S180NII cells /UA next to colony edge | 42 (4) | | 38 (4) | |
| Labelling index | 38 (11) | | 40 (3) | |
Table 3.13. Controls were represented by separate cultures of Rat2 and S180NII cells seeded at 500 and $10^6$ cells per 90mm dish respectively. In co-cultures the cells were seeded at the same respective densities as above. Rat2 cells were either seeded simultaneously with the S180NII cells or pre-established for 4 days at which point the transformed cells were added and co-cultured for 4 days. 18 hours prior to fixation (in 1% formaldehyde and 0.1% gluteraldehyde in PBS for 30mins), the cells were pulsed with $[^3]$H-thymidine (0.185MBq/ml). Once fixed, cultures were stained with x-gal and processed for autoradiography according to the method described in section 2.5. Standard deviations in parentheses.
Figure 3.27. Cell - cell interactions between excess transformed cells and normal cell colonies.
a). Rat2 d4d4
b). S180NII / Rat2 d4d4
c). S180E217 / Rat2 d8d4
d). S180E217 / Rat2 d8d4
e). S180E217 / Rat2 d8d4
f). S180E217 / Rat2 d8d4
Figure 3.27. The above figures are examples from experiments performed to examine the effect of excess transformed cells on the growth of normal cells. Transformed and normal cells were seeded at $10^6$ and 500 cells / 90mm dish. Controls were represented by separate cultures of each cell type seeded at the same densities as above. Cultures were pulsed with $^3$H-thymidine (0.185MBq/ml) for 18hrs, then washed twice in PBS and fixed in 1% Formaldehyde and 0.1% Glutaraldehyde in PBS for 30mins. Cultures were then stained in x-gal for 8hrs, washed with PBS and processed for autoradiography (section 2.5). Figure a) shows a Rat2 control colony. In Figure b) a Rat2 colony is surrounded by excess proliferating S180NII cells. Note that the Rat2 cells do not appear to be compressed. The level of S180E217 cell growth away from Rat2 colonies is shown in Figure c). In Figures d) - f) Rat2 colonies are surrounded by excess S180E217 cells. The Rat2 cells in d) do not appear to be compressed but do show relatively higher labelling than was observed in most Rat2 co-culture colonies. In Figures e) & f), S180E217 cell density and the proportion of cells dividing is greater next to the Rat2 colonies than away from them. Although a common observation, however, this effect was not observed around all Rat2 colonies e.g. Figure g) - see over page.
Figure 3.27. Cell-cell interactions between excess transformed cells and normal cell colonies.
g). S180E217 / Rat2 d8d4
h). S180E217 / Rat2 d8d4
i). S180E217 / Rat2 d8d4
j). BICR / Rat2 d8d4
k). BICR / Rat2 d8d4
Figure 3.27: continued. The above micrographs are further examples from experiments designed to examine the effect of excess transformed cells on normal cell colonies. Figure g) illustrates the observation that S180E217 cells surrounding Rat2 colonies often, but not always, showed increased proliferation. In Figure h) it can be seen that the proportion of Rat2 cells dividing within the colony is relatively low and those which are, are generally not in direct contact with the transformed cells. Furthermore, the morphology of the Rat2 cells appears to be unaffected by the presence of the S180E217 cells. In Figure i) it can be seen that the Rat2 colony has completely detached from the culture dish, suggesting loss of cell-substrate adhesion. Many Rat2 colonies in co-culture with BICR cells also detached (e.g. Figure j). Increased density of BICR cells, but not proliferation, was also observed next to many Rat2 colonies (e.g. Figure k).
Data from the experiments using co-cultures of S180E217 / Rat2 cells is presented in Table 3.14. It can be seen from the data that Rat2 co-culture colonies are markedly smaller than control colonies. The cells are at a higher density per UA than in controls but their morphology would suggest that they are not severely compressed by the transformed cells (e.g. Figure 3.27.h) unlike the S180E217 foci formed in the presence of Rat2 cells (section 3.4.2) and would suggest that they compete better for space on the culture dish. In the majority of instances only a small proportion of Rat2 cells within the colonies are dividing (d4d4:24% and d8d4:17% compared to 100% - 95% in respective controls) and would suggest that the S180E217 cells are able to partially inhibit the growth of the Rat2 colonies. Even in the instances where Rat2 cell proliferation was relatively high such as that seen in Figure 3.27.d, it was not as high as controls.
The density and LI of the S180E217 cells away from the periphery of the colonies remains similar over both time points and in comparison to the controls (Table 3.14). However, an increase in S180E217 cell density is recorded around the periphery of Rat2 cell colonies and is particularly obvious at d8d4 (an increase of low significance, P=5% from 37 cells/UA to 48 cells/UA is recorded), for example, Figure 3.27.e. Furthermore, in many instances, the labelling index of S180E217 cells surrounding the Rat2 colonies is higher than the LI away from the colony periphery and in the control cultures (Table 3.14). This localised proliferation can be clearly seen in Figure 3.27.f and can be compared to background levels in Figure 3.27.c. However, this labelling pattern was not observed around all colonies (~50% did not show increased labelling), e.g. Figure 3.27.g.
In several colonies examined at d8d4 it was apparent that the Rat2 cells had detached during the co-culture or fixing process e.g. Figure 3.27.i. It can also be seen in Figure 3.27.i that β-gal expression is lost in a relatively high proportion of the S180E217 cells. This is probably because cells were grown in non-selective growth media during the co-culture period. Once cells were returned to selective media 100% β-gal expression was restored. Despite this problem it was possible to identify the two cell types in co-culture, firstly because Rat2 cells were present in distinct colonies and secondly there were morphological differences between the two cell types.
In many of the colonies the distribution of the labelled Rat2 cells was similar to the pattern observed next to transformed foci in section 220.127.116.11. That is, most labelled cells were situated 1-2 cells from the interface of the 2 cell types (e.g. Figure 3.27.h). However, this pattern was not observed in all colonies, so no statistical analysis was performed.
Table 3.14. The effect of excess S180E217 cells on the growth of Rat2 cells.
| Rat2 / S180E217 | d4d4 Control | Co-culture | d8d4 Control | Co-culture |
|-----------------|--------------|------------|--------------|------------|
| Ave. colony area mm² | 0.9 (0.4) | 0.2 (0.1) | 6.5 (0.3) | 0.8 (0.1) |
| Ave. No. Rat2 cells per UA. | 6 (1) | 11 (4) | 21 (3) | 50 (6.5) |
| Labelling index of Rat2 cells | 100 (0) | 24 (9) | 95 (4.5) | 17.6 (3) |
| Ave. No. S180E217 cells/UA away from colony edge | 42 (9) | 35 (5) | 39 (3) | 37 (2) |
| Labelling index | 36 (7) | 32 (12) | 35 (3) | 28 (4) |
| Ave. No. S180E217 cells next to colony edge | 41 (5) | | 48 (2) | |
| Labelling index | 48 (8) | | 32 (4) | |
Table 3.14. Controls were represented by separate cultures of Rat2 and S180E217 cells seeded at 500 and $10^6$ cells per 90mm dish respectively. In co-cultures the cells were seeded at the same respective densities as above. Rat2 cells were either seeded simultaneously with the S180E217 cells or pre-established for 4 days at which point the transformed cells were added and co-cultured for 4 days. See legend for Table 3.13 for experimental details. Standard deviations in parentheses.
The results obtained for BICR / Rat2 co-cultures are presented in Table 3.15. As with the other cell combinations examined the Rat2 colonies are markedly smaller than the control colonies. The majority of cells within the co-culture colonies are at a similar density to control colonies but have a considerably lower labelling index (Table 3.15). This would suggest that the presence of the BICR cells partially suppresses the growth of the Rat2 colonies. The loss of several colonies was observed in cultures analysed at d8d4 and an example of this can be found in Figure 3.27.j. The fact that all of the cells within these colonies (and in several Rat2 colonies in S180E217 co-cultures) detached as a single sheet of cells during the culture period would suggest that the cells have lost the ability to attach to the plastic rather than, or in addition to each other. The cells within the colonies which did remain (e.g. Figure 3.27.j) appear normal in terms of their general morphology and not markedly compressed by the presence of the surrounding BICR cells. In addition many of these cells are dividing which would suggest the cell detachment is not necessarily due to cell death.
The average density of BICR cells in the co-cultures remained approximately the same throughout the population. Although the average number of BICR cells at the colony periphery was not significantly different from the background and control densities there were occasional exceptions such as that seen in Figure 3.27.k. In most instances the extent of BICR cell division next to the Rat2 colonies was not significantly different to those away from the colony and unlike the Rat2 cells in some S180E217 co-cultures no specific labelling patterns were observed in the colonies themselves.
Table 3.15. The effect of excess BICR cells on the growth of Rat2 cells.
| Rat2 / BICR | d4d4 Control | Co-culture | d8d4 Control | Co-culture |
|-------------|--------------|------------|--------------|------------|
| Ave. colony area mm² | 0.9 (0.4) | 0.1 (0.1) | 6.5 (0.3) | 0.3 (0.1) |
| Ave. No. Rat2 cells per UA. | 6 (1) | 6 (1.5) | 21 (3) | 19 (3) |
| Labelling index of Rat2 cells | 100 (0) | 37 (12) | 95 (4.5) | 33 (4.8) |
| Ave. No. BICR cells/UA [away from colony edge] | 37 (3) | 42 (4) | 40 (2.6) | 40 (6) |
| Labelling index | 24 (8) | 55 (9) | 31 (3.5) | 45 (8) |
| Ave. No. BICR cells next to colony edge | 45 (3) | 43 (11) |
| Labelling index | 43 (6) | 37 (8.8) |
Table 3.15. Controls were represented by separate cultures of Rat2 and BICR cells seeded at 500 and $10^6$ cells per 90mm dish respectively. In co-cultures the cells were seeded at the same respective densities as above. Rat2 cells were either seeded simultaneously with the BICR cells or pre-established for 4 days at which point the transformed cells were added and co-cultured for 4 days. See legend for Table 3.13 for experimental details. Standard deviations in parentheses.
18.104.22.168. Summary.
The results presented in this section would suggest that the mechanism responsible for the stimulation of the Rat2 cells, observed in the focus assays, does not involve a specific concentration-dependent factor transmitted from the transformed cells. This is based on the observation that when a small population of Rat2 cells are surrounded by excess transformed cells, the proportion of Rat2 cells which are proliferating is relatively small. This is consistent with earlier suggestions that the distribution of dividing Rat2 cells surrounding the transformed foci was incompatible with a specific concentration-dependent factor.
In some instances the dividing Rat2 cells (surrounded by excess transformed cells) are situated towards the edge of the colony at the interface with the transformed cells (but not necessarily in direct contact with the transformed cells). This is similar to the distribution of dividing Rat2 cells seen in the focus assay experiments (stimulated Rat2 cells were situated with 2-3 cells from the focus periphery). These data would suggest that the growth stimulation arises through indirect interactions at the focus - monolayer interface. Further discussion of the possible mechanisms can be found in section 4.1.6.
In several instances when S180E217 cells were plated in excess of the Rat2 cells there was higher-than-background levels of S180E217 cell proliferation immediately next to the colony periphery. This stimulation may be caused by a specific signal arising from the Rat2 cells or alternatively, the normal cells may be contributing to their proliferation via some sort of feeder effect. However, the stimulation is unlikely to be mediated directly by gap junctions since the level of heterologous communication between Rat2 cells and S180NII cells (where there is not a significant increase in cell division surrounding the colonies) is higher than between Rat2 and S180E217 cells. The stimulation may be caused indirectly as a consequence of physical interaction between the two cell types; similar to that believed to be responsible for the stimulation Rat2 cells surrounding foci. It is not clear why this pattern occurred specifically with this cell combination and not with other transformed cell lines, particularly other S180 derived cells.
The results in section 3.4.3 show that the highest level of transformed-cell growth inhibition is observed with S180NII cells in co-culture with Rat2 cells. However, there appears to be no detectable inhibition of these cells when they surround the Rat2 colonies. This is consistent with the finding in section 3.4.3 that the larger the population of S180NII cells the lower the level of inhibition was imposed on them by the Rat2 cells. In addition there appears to be no growth stimulation of the S180NII cells surrounding the Rat2 colonies.
Rat2 cells within the colonies surrounded by transformed cells are generally at a higher cell density than control colonies, but show few signs of excess compression. This
is in contrast to the compressed transformed cells within foci surrounded by normal cells (section 3.4.5). This is consistent with the observations made in section 3.4.6. in which the morphology and density of the Rat2 cells surrounding expanding foci was very similar to control monolayers. This would appear to show that transformed cells do not encroach upon the normal cells and shuffle up as the Rat2 colonies expand. Furthermore, the data would appear to show that Rat2 cells can compete better for space on the culture dish.
The data suggests that excess transformed cells are able to inhibit the growth of Rat2 cells. This inhibition phenomenon is not caused by nutrient deprivation and would appear to be caused by contact-inhibition or the ability of cells to respond to the presence of adjacent cells. This new inhibition phenomenon is discussed further in Chapter four in addition to possible mechanisms which may mediate the stimulation of Rat2 cells surrounding transformed foci.
CHAPTER FOUR
GENERAL DISCUSSION
4.1. Introduction.
Various normal and transformed cell lines, with a range of homologous and heterologous communication phenotypes, were selected to study the role of gap junctional communication in the inhibition of transformed cells by resting, excess, normal cells (see section 3.1 for cell lines). In the focus formation assays carried out here the majority of foci, in all of the co-cultures, were markedly smaller than respective control colonies (section 3.3). The difference in size was initially interpreted as growth suppression, however, further analysis revealed that cell density within many of the foci was significantly higher than cell density in respective control colonies. This would suggest that the physical presence of the normal cells restricts the transformed cells from gaining access to the culture dish, leading to increased focus cell density and results in a significant size difference between foci and colonies. In some instances cell density within the foci was too high to distinguish individual cells and it was therefore difficult to accurately identify the level of proliferation in these foci. However, the size of these foci together with the cell density data would suggest that inhibition was low. Two transformed cell lines were clearly shown to be growth inhibited; S180NII cells showed a high level of suppression when co-cultured with Rat2 cells and S180 cells showed a low level of suppression when co-cultured with 10T1/2 cells.
In some instances the difference in size between foci and respective control colonies was very large, even allowing for focus compression. For example, foci formed by S180E217 cells were composed of far fewer cells than respective control colonies. This would imply the cells are suppressed, however, a proliferation analysis showed that a high proportion of the cells were growing. The results suggest that a high proportion of S180E217 cells were detaching from the culture dish due to a lack of space, perhaps coupled with a defect in cell-substrate adhesion. Some foci formed by other transformed cells also showed large differences in size from respective control colonies. However, in most instances the difference was not as great as that seen with S180E217 foci and it is not clear to what extent the loss of cells from the dish contributed to the explanation of focus size suppression.
The data obtained in this study appears to contradict the generally held view that transfer of inhibition from normal to transformed cells, only involves GJIC. For example, transformed cell lines which communicate with the normal cells are not necessarily inhibited by them and the restoration of heterologous communication does not appear to be sufficient for inhibition to occur. Inhibition was also observed when heterologous communication was not detected (i.e. 10T1/2 / S180 co-cultures). Furthermore, the growth of normal cells was inhibited when they were co-cultured with excess transformed cells. This would appear to show that two different inhibition phenomenon exist, one which involves the transfer of inhibition from resting cells to growing cells
(possibly via gap junctions) and one which is, at least in part, due to contact-inhibition or the ability of cells to respond to the physical presence of other surrounding cells.
The results regarding the inhibition phenomena and the ability of the transformed cells to suppress the growth of normal cell colonies will be discussed in this section and the implications for the current working hypotheses considered. The limitations of the various experimental systems that are used to examine the phenomenon will also be discussed.
The advantage of the proliferation assay developed for this project is that it allows the growth of both cell types in the co-culture to be examined. During the proliferation analysis it was clear that Rat2 cells (but not 10T1/2 cells) surrounding the majority transformed foci were growth stimulated. The level of stimulation decreased as distance from the focus periphery increased and there appeared to be a wave-like pattern to the distribution of stimulated cells. It is not known what caused the stimulation but possible mechanisms are discussed.
4.1.2. Current hypotheses regarding the role junctional communication in the inhibition phenomenon.
It has been suggested that growth inhibition is transferred from normal cells to transformed cells via GJIC (Loewenstein 1979), however, the specific mechanism by which inhibition is imposed is not known; two possible mechanisms are frequently discussed. In one hypothesis the inhibition is mediated via a general homeostatic pressure set up within the coupled population of cells. Excess, cytoplasmic, stimulatory growth control molecules (e.g. second messengers), which are produced at higher levels by transformed cells, may be diluted by spreading to the normal cell monolayer. If this is so, a higher concentration of such factors may be found in normal cells surrounding the foci and may cause localised normal-cell growth stimulation. Limited evidence consistent with this has been obtained in this study and is discussed in section 4.1.6. Alternatively, the converse may occur. The contact-inhibition, in the normal cell monolayer, may be mediated by cell surface, membrane bound receptors that switch off the cell cycle. Interaction of the receptors on apposing membranes may trigger intracellular signalling cascades involving the release of second messengers that can diffuse through gap junctions into the transformed cells.
A second hypothesis, which is frequently discussed, suggests that specific growth inhibitory molecules, originating from discrete source cells within the normal cell population, pass through heterologous gap junctions into the transformed cells (Mehta et al 1986). The distribution of the signal is governed by junctional permeability and growth inhibition is therefore ruled by the number of channels and the extent of coupling. The level of inhibition will increase with the number of open channels and with the number of signal source cells. Working on the same principle it has been suggested (Mehta et al
that increased homologous communication within transformed cells can normalise their growth. However, no such inhibitory signalling molecule has been found and it is not clear why the production of such signals should be limited to random source cells.
In an attempt to show a role for gap junctions in the inhibition phenomenon, many investigators have used various chemical agents to modulate the level of communication between cells, in an attempt to show a correlation (section 1.3.4). However, these chemicals do not act exclusively on this pathway and other important cellular functions may be altered. In an attempt to provide direct evidence that GJIC is involved in the inhibition phenomenon connexins have also been used to manipulate the communication phenotype of transformed cells (Mehta et al 1991, Rose et al 1993, Chen et al 1995). The connexin transfectants often have a more normal phenotype in culture (e.g. longer PDT's, higher TD's and a flatter, more epithelioid morphology) and a more suppressible phenotype when co-cultured with normal cells. These changes are generally attributed to increased junctional communication and hence increased transfer of inhibition.
It is usually assumed that connexins are the channel components of gap junctions (which has yet to be proved; section 1.3.1), but evidence is accumulating to support the idea that they may have other functions. For example, a disease of the nervous system (X-linked Charcot-Marie-Tooth disease) which causes demyelination of axons is associated with mutations in the Cx32 gene (Bergoffen et al 1993). However, communication has not been shown to occur in mature Schwann cells and it is not clear what purpose intracellular gap junctions would serve, particularly considering the myelin sheath serves as an insulator. Cx32 may be involved in the formation of the myelin layers or contribute to the maintenance of myelin integrity (which is lost during the disease). This hypothesis is consistent with the idea that connexins are involved in bringing membranes into close contact in order that gap junctions can form (Finbow & Pitts 1993). The unusually close membrane apposition found in gap junctions is similar to that in myelin and such a role would explain the requirement of connexins for gap junction formation. This idea is supported further by the finding that connexins remain associated with dense cytoskeletal material when gap junctions (possibly formed by ductin) are extracted from mouse liver using Triton X-100 (Finbow & Meagher 1992). Connexins have also been implicated as tumour suppressor genes (Lee et al 1991) and regulators of gene expression. A recent paper by Chen et al (1995), showed that Cx43 expression can normalise the growth phenotype of transformed cells and alter their morphology (which is often associated with changes in the adhesion properties of the cells). It was not clear whether the changes in growth phenotype were due to the increased formation of gap junctions or due to changes in cell shape or expression levels of specific cell cycle regulatory genes including cyclin A, D1, D2 and the cyclin-dependent kinases 5 & 6.
4.1.3. The interpretation of focus formation data.
Much of the information regarding the inhibition phenomenon has been obtained from focus formation assays (e.g. Mehta et al 1986 & 1991, Martin et al 1991, Zhu et al 1992). Focus number and/or focus size are generally used as indices of growth. However, it has been shown in this study that values obtained from these measurements can vary considerably within an experiment due to some form of intrinsic variability, which is not understood (see section 3.3.6). Furthermore, the number of foci counted within an individual plate can vary depending on the size cut-off point which is used (section 3.3) and estimates of growth inhibition based only on focus size do not take into account the fact that foci can be compressed by the surrounding normal cells. Even in instances where inhibition is observed (e.g. 10T1/2 / S180 co-cultures) the level of transformed cell proliferation may be greater than the size of foci would suggest. Cell density within these foci can be markedly higher than in respective control colonies (e.g. Figure 3.12.E) and this would appear to contribute to the explanation of focus size suppression.
In some reports attempts have been made to calculate the proportion of transformed cells which have escaped inhibition by measuring the increase in total cell number within the co-culture over time (e.g. Mehta et al 1991). It is assumed that any increase in the number of cells within the co-culture is due to the growth of the transformed cell population (on the basis that separate cultures of normal cells remain static, in terms of total cell number, once they have reached saturation density). However, this does not take into account any increase in the number of normal cells due, for example, to growth stimulation by the transformed cells (see section 22.214.171.124). To overcome this problem the transformed cells could be transfected with the lineage marker β-gal and the cells of the co-culture counted using a FACS analysis (Fiering et al 1991). This technique would also overcome the problem, occasionally incurred in this study (section 3.4.2), of being unable to count the number of cells in each focus due to high focus cell density. However, the data obtained from such an analysis would not provide information on variability in growth within foci or between foci or whether focus size affects the extent of inhibition.
Although data from standard focus formation assays can be misinterpreted, some form of inhibition phenomenon has been shown to exist both here (section 3.4.3) and in several published reports (section 1.6). For example, Mehta and co-workers (1986) were able to show that foci formed from various transformed cells decreased in size as their level of heterologous communication with normal cells was increased by various chemical agents. In many instances the large differences in size between foci and colonies are unlikely to be due to focus compression alone. However, it has been shown in this study that foci which are markedly smaller than controls may not necessarily contain cells which are growth suppressed.
For example, an apparent discrepancy was frequently observed between the size of S180E217 foci and the proliferation status of the S180E217 cells (Figure 3.11.B & Figure 3.14.B). It is possible that S180E217 cells compete poorly for space on the culture dish which, coupled with high cell density within the culture, leads to the loss of cells from the focus to the medium and would therefore account for the reduced rate of focus expansion. The loss of cells is likely to occur whilst the cells are dividing, during which time they round up and the number of attachments to other cells and to the plastic may decrease. As the cells detach, surrounding cells may fill the space and divide. However, S180 parental cells did not appear to be affected in this way and high focus cell density would appear to contribute largely to the explanation of S180 focus size suppression. This would suggest that E cadherin expression in S180 cells decreases their ability to attach to the culture dish. It is not clear if N cadherin has the same effect on S180 cells because the S180NII cells were growth suppressed and did not reach a high cell density.
The data obtained from the combined focus and proliferation assay used in this study have shown the need to measure the growth status of the individual cells directly rather than trying to relate indirect growth indices to the level of proliferation within foci. It is possible that inhibition is greater when a single transformed cell is surrounded by growth arrested normal cells (a situation that would be analogous to the possible inhibition of a single mutated cell from which a tumour might develop). However, it has not been possible in this study to examine the phenotype of a single transformed cell within the monolayer of normal cells. This was because even when plated simultaneously with the normal cells the transformed cells went through 2 or 3 divisions before the normal cells had themselves stopped growing. One approach to overcome this problem would be to might be to seed the transformed cells on top of a confluent monolayer of normal cells which have already stopped dividing.
4.1.4. Do the data obtained in this study fit the current working hypothesis?
Mehta et al (1991) suggested that increased homologous communication can lead to the normalisation of transformed cells on the basis that gap junctions transmit growth-regulatory signals. It was found that MCA-10 cells (chemically transformed 10T1/2 cells) transfected with a cDNA for Cx43, show a 2-4 fold increase in homologous communication and have lower terminal densities and longer PDT's than parental controls. This hypothesis, however, would not appear to apply to the S180 series of cell lines examined here. Homologous communication in the S180 cells is markedly increased after they are transfected with a cDNA for E or N cadherin (Table 3.1). The cells become flatter with a decreased tendency to overlap. However, the PDT's and terminal densities of these cells is not significantly different from parental cells (Table 3.1). This may be because the transformed phenotype of the S180 cells is not directly associated
with the loss of cadherin expression or with the loss of communication, or the S180 cells may not produce the putative growth-regulatory control molecules. It is also possible that the results obtained by Mehta et al (see above) were not due to GJIC but to some other consequence of Cx43 expression, which may affect the growth control of cells via the regulation of specific cell cycle genes (see section 4.1.2). The S180 derived cell lines used here did not show an increase in Cx43 expression (but did show an increase in Cx43 phosphorylation; section 3.2.3).
In the introduction to the results (section 3.1) it was suggested that if GJIC was involved in the transfer of inhibition from the normal to the transformed cells it might be expected to see a greater level of inhibition at the periphery of large foci, where transformed cells receive the putative signal first and at the highest concentration, rather than in the centre. This did not appear to be the case. For example foci formed from pre-established S180NII cells showed a decrease in the level of suppression imposed by the Rat2 cells (relative to foci from cells plated at the same time; section 3.4.3) however, a significant proportion of cells were still suppressed but these cells were distributed, apparently evenly, throughout the focus and not with increased incidence at the interface with the surrounding monolayer.
It is often suggested that inhibition can occur even when the level of heterologous communication is very low. For example, Mehta et al (1991) found that MCA-10 cells are very poorly coupled to normal 10T1/2 cells. In focus formation assays these cells showed significant levels of inhibition (~37%; inhibition values are based on changes in total cell number). An MCA-10 Cx43 clone showed a 3-fold increase in heterologous communication with the 10T1/2 cells and the level of inhibition increased to ~99%. In this study S180 cells show a low level of communication with the Rat2 cells but do not appear to be suppressed (Figure 3.14.A). The S180E217 cells show a ~4-fold increase (relative to S180 cells) in their level of communication with the Rat2 cells but a very high proportion of S180E217 cells remain in cell cycle (Figure 3.14.B). S180NII cells which show a ~12-fold increase in heterologous communication do show a high degree of suppression (Figure 3.14.C).
It is not clear why N cadherin expression but not E cadherin expression can confer suppressible growth phenotypes on the S180 cells. Both cadherin transfectants show increased heterologous communication (relative to S180 parental cells) with the Rat2 cells, but appear to have different abilities to compete for space on the culture dish and this may be an influencing factor. These data would appear to show that GJIC is necessary but not sufficient for inhibition to occur and this is supported by the data which shows that BICR cells, which are well coupled to Rat2 cells, are poorly inhibited by them. There is also evidence from this study which shows that inhibition can occur in the absence of detectable GJIC, suggesting other factors may be involved.
In experiments where small numbers of Rat2 cells were plated with excess transformed cells, an unexpectedly high level of growth inhibition was recorded within 4
days in the Rat2 cell colonies, which would also suggest that other factors besides GJIC are involved in cell growth inhibition. It appears that growing transformed cells are able to suppress the growth of the Rat2 cells. It is unlikely that this inhibition is due to nutrient deprivation since media taken from cultures of an older age (8-10 days) can support normal Rat2 cell growth. These results suggest that the suppression of the transformed phenotype may be more complex than is often suggested. It is possible that other mechanisms may be involved in suppression and that GJIC is only a contributory factor.
4.1.5. Other mechanisms which may mediate growth inhibition.
The presence of junctional communication between the normal and transformed cells does not appear to be sufficient for inhibition to occur and other mechanisms may be involved. These are discussed next.
126.96.36.199. Density-dependent regulation of cell growth (contact-inhibition).
The growth of mammalian cells in tissues is thought to be under the control of various stimulatory and inhibitory factors. Inhibitory factors are presumably responsible for determining organ size and controlling tissue maintenance and may involve a specific inhibitory signal(s) which competes with the stimulatory signals or signals stimulation to be turned off. A considerable amount is now known about stimulatory stimuli such as mitogenic growth factors and their respective receptors. However, less is known about inhibitory factors and in particular the phenomenon known as *in vitro* density-dependent growth inhibition or contact-inhibition (section 1.6). This phenomenon describes the characteristic property of many mammalian cells to cease growing once they have reached high cell density within the culture dish (for normal cells this generally occurs soon after confluence is reached). The mechanism of contact-inhibition is not known but may be an important contributory factor to the Stoker phenomenon.
There are several lines of evidence, based on data obtained in this study, to suggest that the Stoker phenomenon does not only involve the direct transfer (via gap junctions) of inhibition from normal to transformed cells (section 4.1.4). The inhibition may be mediated by the ability of cells (normal or transformed, in separate or co-culture) to cease growing, or grow more slowly, at high cell densities. This may be determined by the ability of the cells to recognise the presence of adjacent cells and may be governed by their ability to compete for space on the culture dish. This in turn may be affected by how well the cells adhere to each other and to the plastic.
Evidence obtained throughout this study would suggest that the normal cells are better at competing for space than the transformed cells. For example, transformed foci show a large degree of compression and high cell density which would indicate that the cells are restricted in their ability to gain access to the plastic and some can survive more
easily on a smaller area. Furthermore, Rat2 cells show few signs of compression when they surround the foci (section 3.4.6) which would suggest they compete better for space and shuffle up in response to focus expansion. When low numbers of Rat2 cells are plated with excess transformed cells (section 3.4.7), the Rat2 colonies expand and do not show marked compression, supporting the suggestion that these cells are more effective at competing for space.
Cell proliferation analysis reveals that the normal cells (in standard focus assays) are contact-inhibited throughout the monolayer (section 188.8.131.52), although less so around transformed foci (this stimulation is addressed in section 4.1.6). When transformed cells are present in excess, the LI of the Rat2 cells within the colonies drops markedly in comparison to control colonies (section 3.4.7; e.g. Table 3.13). This shows that the transformed cells are able to inhibit the growth of the normal cells. This would suggest that a high proportion of Rat2 cells have competed with each other and with the transformed cells, which they recognise as an 'edge', for the amount of space they require to grow and divide, and as a consequence, become growth inhibited. In some instances (e.g. Figure 3.27.h, Table 3.14), Rat2 cells within the colonies which were dividing were found more frequently at the interface with the transformed cells. This would suggest that these cells are competing successfully for space with the transformed cells and can continue to divide.
An explanation of the observed growth inhibition in terms of the ability of the cells to compete for space on the culture dish seems consistent with much of the data, but not all. The inhibition of growth in S180NII foci by Rat2 cells, is the only instance of marked inhibition of transformed cell proliferation by normal cells in the focus assays (section 3.4.3). The cells in the inhibited foci are not significantly compressed and the cell density in the foci is often lower than in the control colonies. This suggests that these cells can effectively compete with the Rat2 cells for space. It would appear that they have sufficient space to divide but some other factor inhibits division (possibly mediated by GJIC). The inhibition is reduced when the foci are larger; this happens when the transformed cells are pre-established and the normal cells are added later (Figure 3.14.C). This suggests that inhibition is limited to the interface of the Rat2 and S180NII cells, but the inhibition is evenly distributed through the foci and not concentrated around the periphery. Conditioned media experiments show that the inhibition is not due to nutrient deprivation and is unlikely to be caused by secreted factors. These observations, unlike many others in this study (see section 4.1.4), are consistent with inhibition by transfer of small growth control molecules via gap junctions (either loss of stimulatory factors from the S180NII cells or invasion by inhibitory factors produced by normal cells). It appears that there are two separate phenomena which can cause growth suppression, one of which may require GJIC between the normal and transformed cells junctions and the other which can occur in the absence of heterologous communication and may depend on the ability of cells to recognise adjacent cells or an 'edge'.
184.108.40.206. The role of cadherins and connexins in the inhibition phenomenon.
Cadherins: Adhesion and communication events often go hand in hand, for example, increased adhesion between cells can lead to increased levels of homologous GJIC (Mege et al 1988, Keane et al 1988, Musil et al 1990, Jongen et al 1991, Meyer et al 1992, Hertig 1996) and it is therefore difficult to determine whether a particular change in cell behaviour is caused by one or the other. It has been suggested that cadherins may be involved in intercellular signalling and growth regulation by a pathway(s) not involving GJIC (see section 220.127.116.11). The effects of cadherins on growth are thought to be mediated by the cadherin-associated proteins, the catenins (Kinch et al 1995). β-catenin for example, has been shown to be phosphorylated in response to growth factors and can in turn decrease cell-cell adhesion. The E cadherin - catenin complex has been shown to retard the growth of a carcinoma cell line (Watabe et al 1994) and cadherin interactions between the normal and transformed cells may be involved in the inhibition observed in this study.
However, in the vast majority of instances, cadherin mediated adhesion requires homotypic interaction of the proteins on the apposing cell membranes; Rat2 cells do not express detectable levels of either N or E cadherin (section 3.2.3). However, adhesion between cells is a complex process and is the sum of contributory events (section 1.2.5). The expression of N cadherin (but, for some reason, not E cadherin) may alter the interactions between S180NII and Rat2 cells such that growth regulatory process are triggered when the two cell lines interact and may also affect the ability of the cells to compete for space.
Connexins. In several studies it has been shown that the expression of exogenous Cx43 in communication deficient transformed cell lines can restore junctional communication and a suppressible phenotype (see section 4.3.2). Chen et al (1995) showed that Cx43 transfectants were suppressed when co-cultured with normal cells. They also had altered expression levels of various proteins including cyclin A, D1, D2 and the cyclin dependent kinases, CDK5 and CDK6. Cx43 mutational analysis may provide the means to define specific connexin functional domains which may be responsible for these effects. At present, however, it is not possible to separate GJIC mediated events from other events which may be mediated by connexins. This may be overcome if specific inhibitors of junctional communication (aimed at ductin for example) become available which could be used to knockout junctional transfer but maintain connexin function.
In this study it was shown that E and N cadherin can increase the proportion of Cx43 which is phosphorylated. However, the increase in the expression of this Cx43 species does not appear to affect the growth parameters of these cell lines in homologous cultures (section 3.1). Furthermore, increased expression of phosphorylated Cx43 does not appear to affect the growth phenotype of the S180E217 cells in co-culture. BICR
and SVT2 cells also express relatively high levels of Cx43 (phosphorylated and unphosphorylated) but maintain aberrant growth phenotypes in homologous and heterologous co-cultures. If Cx43 is involved in the control of growth these data would suggest that the protein expressed in these cells is either defective or some other component is involved in the pathway which is not expressed or is not functioning in these cells.
It has been observed in several studies that connexin transfectants have a different morphology to parental cells; generally flatter with fewer processes (Mehta et al 1991, Rose et al 1993, Chen et al 1995). Changes in morphology are often associated with changes in cell-cell or cell-substrate adhesion (Marrs et al 1995, Gumbiner 1991) and would suggest that connexins are involved in or influence cell adhesion. In this study, the S180 cadherin transfectants had a different morphology to S180 parental cells. But is more likely that it is the cadherins which are causing the changes in cell morphology.
18.104.22.168. Inhibition mediated by transmembrane bound glycoproteins.
The molecular mechanism of contact-inhibition is not known but transmembrane glycoproteins have been implicated (Weiser 1990). It is suggested that these proteins interact in an adhesion-dependent manner with appropriate receptors on apposing cell membranes. A plasma membrane glycoprotein has been identified (contact-inhibin) that has density-dependent growth regulatory activity (Wieser 1990) when it binds to a specific 92kD protein (the contact-inhibin receptor; CiR) on apposing cells. Polyclonal antibodies against CiR release the cells from density-dependent growth control (Gradl et al 1995). SV40-transformed fibroblasts (different from the cells used in this study) were found to have reduced levels of CiR and a reduced capacity to bind to contact-inhibin. However, other transformed cells have been shown to express the protein in an active form but are not growth inhibited suggesting that the defects which led to their aberrant phenotype are located down stream of contact-inhibin. The role played by GJIC in these events was not studied.
It is not known if this form of growth inhibition has any significance for the results obtained here since there was insufficient time to analyse the cells for CiR and contact-inhibin expression. However, increased cell-cell adhesion mediated by cadherins or possibly by connexins may regulate the efficiency with which CiR and contact-inhibin bind to each other.
4.1.6. The stimulation of normal cells surrounding transformed foci.
Whilst investigating the inhibition phenomenon, Stoker (1967) observed that a higher proportion of normal cells (relative to controls) were dividing in the presence of the transformed cells. However, no detailed analysis was performed and it was suggested that the stimulation was due to the addition of fresh medium prior to the analysis. This
explanation could not account for the stimulation observed in this study (section 22.214.171.124) because the growth medium was not changed during the experiments. Since Stoker's original experiments, only the inhibition phenomenon has been examined in any detail and the possibility that transformed cells may alter the phenotype of surrounding normal cells has not been considered.
An increased proportion of Rat2 cells (but not 10T1/2 cells), surrounding the foci in most of the different co-cultures used in this study, was dividing (section 126.96.36.199). The stimulation was localised, generally within 0.175mm of the focus periphery and the proportion of dividing cells decreased as distance from the focus periphery increased. However, cells in direct contact with the focus periphery were less likely to be labelled than those 2-3 cells away and the distribution of dividing cells often followed a wave-like pattern (section 188.8.131.52).
The stimulation observed in this study is of interest in its own right but may also affect the ability of the normal cells to impose inhibition on the growth of the transformed cells. That is, by altering the surrounding environment to one which is not as growth inhibitory to the transformed cells, the level of suppression may be reduced. *In vivo*, at early stages of tumour formation if transformed cells were capable of stimulating the growth of surrounding normal cells they may have a selective growth advantage i.e. by reducing the ability of normal cells to impose growth inhibition the initiated cells could continue to divide, increasing the likelihood of more mutations which may provide the cell with further growth advantages.
**184.108.40.206. Possible mechanisms responsible for the stimulation of the normal cells.**
Several possible mechanisms may be involved in the stimulation of normal cells surrounding the transformed foci. The data from preliminary investigations carried out in this study do not provide conclusive evidence that any one in particular causes the stimulation but some would appear to be more likely candidates than others.
1. Stimulatory growth signals (e.g. paracrine growth factors) may be secreted from the cells and act in a localised and transient manner.
2. Stimulatory growth signals may be transmitted from the transformed cells to the normal cells via gap junctions.
3. The loss of normal cells from the culture dish as a consequence of competition for space with the transformed cells at the focus - monolayer interface may loosen up the monolayer or leave spaces which signal surrounding normal cells to divide.
4. The expanding foci may generate a physical disturbance which may disrupt the cell-cell contact-mediated inhibition which is responsible for the inhibiting the growth of normal cells at saturation density.
Paracrine factors: Conditioned media experiments would appear to show that stimulation is not mediated by a stable soluble factor secreted from the transformed cells. However, the factor may be short lived and require high concentrations which can only be achieved within a short distance from the source cell. In general, stimulation decreases with distance from the focus periphery suggesting a paracrine factor may be involved. However, more detailed analysis shows that the pattern of stimulation is more complex and is difficult to explain in terms of a single stimulatory factor. In experiments where low numbers of Rat2 cells are plated with excess transformed cells it might be expected that the level of stimulation would increase. However, the Rat2 cells show high levels of inhibition relative to controls and the interpretation of these experiments is more complicated. It appears unlikely that the stimulation is mediated by a paracrine factor secreted from the transformed cells.
Gap junction mediated stimulation: Some of the data are consistent with the idea that a growth stimulatory signal(s) is transmitted via gap junctions. In one cell combination (10T1/2 S180) there is a correlation between the absence of GJIC and the absence of stimulation (section 220.127.116.11; Table 3.3). It is also clear that Rat2 cells, which are stimulated near the transformed foci, are metabolically co-operating with the transformed cells. This is because there is an increase in the level of $^{[3H]}$-thymidine labelling above dividing Rat2 cells, relative to dividing Rat2 cells in separate cultures (see section 3.1 on the thymidine kinase deficiency in Rat2 cells). However, the level of heterologous communication does not correlate with the level of proliferation and the unexpectedly complex pattern of dividing Rat2 cells is not consistent with it being mediated by a single stimulatory factor transmitted by GJIC. To test whether gap junctions are involved in the stimulation mechanism, specific GJIC inhibitors would be required to block communication during the focus assay. These are not yet available.
Loss of cells from the dish: The loss of Rat2 cells surrounding the foci may, for a short time at least, leave a space on the dish which may allow other cells to fill the space and divide. However, it has been shown here (section 18.104.22.168) that the presence of transformed foci does not lead to increased normal-cell apoptosis. In experiments where the transformed cells were plated in excess of normal cells (section 3.4.7), the normal cell colonies which formed, continued to expand which would suggest they compete better for space than the transformed cells and are therefore unlikely to detach.
Disruption of the contact-inhibition mechanism: The stimulation may not be due to a specific stimulatory factor, but may be caused by the disruption of the contact-inhibition mechanism which causes the normal cells to cease growing at confluence and which may be responsible for some of the inhibition observed in Rat2 colonies surrounded by transformed cells. This disruption may be caused by expanding foci as the transformed
cells compete for space on the culture dish. As the transformed cells lay down new processes in between the Rat2 cells they may disrupt the surrounding monolayer such that the Rat2 cells are forced to shuffle up. As the foci continue to grow the Rat2 cells continue to shuffle back and it is possible that this may give rise to a wave-like pattern of stimulation. As distance from the focus periphery increases the disruption of the monolayer is dissipated and hence the number of cells dividing decreases.
It was shown in section 22.214.171.124 that Rat2 cells which were in direct contact with the transformed cells, were less likely to be stimulated than cells 2-3 cells away. Inhibition of Rat2 cells was observed when they were plated with excess transformed cells and this may have been caused by contact-inhibition. Therefore, in the focus assays, the Rat2 cells which are in direct contact with the transformed cells may remain contact-inhibited.
It is not clear why the 10T1/2 cells should not be stimulated when they are close to expanding S180 foci. The cell-cell contacts which lead to contact-inhibition may be stronger between the 10T1/2 cells and therefore reduce the disruptive effect of the transformed foci.
4.1.7. Summary
The inhibition of transformed cells by normal cells is more complex than is often suggested and more than one mechanism may be involved. It has been shown that the presence of excess transformed cells can inhibit the growth of normal cells. This is a new inhibition phenomenon which appears to be mediated by contact-inhibition and may be related to the ability of cells to compete for space on the culture dish. The frequency of inhibition reported in the literature may not be accurate. This is because indirect indices of growth, such as focus size, do not account for focus compaction by surrounding normal cells, which can contribute significantly to the explanation of focus size suppression. The assay developed for this study provides detailed and accurate information on cell growth and could be used to look for an inhibition phenomenon *in vivo*.
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|
INTERACT HANDBOOK
A Guide for Interactors
This handbook is for youth interested in forming or participating in an Interact club. You will learn how to:
- Start an Interact club
- Build a dynamic club that offers all members meaningful service and leadership opportunities
- Promote your club’s work to your community and the Rotary world
Throughout the handbook you will find question prompts and spaces with the pencil icon to take notes to organize your thoughts and focus your goals for your Interact club.
Your club adviser and sponsors can help if you have any questions or need support. You can also send questions or comments about Interact to firstname.lastname@example.org.
Policy references are based on the Rotary Code of Policies. Decisions made by the Rotary International Board of Directors take precedence over information in this publication.
# Contents
| Chapter | Title | Page |
|---------|--------------------------------------------|------|
| 1 | What Is Interact? | 4 |
| 2 | Starting an Interact Club | 6 |
| 3 | Safety and Inclusion | 10 |
| 4 | Building a Foundation | 16 |
| 5 | Make Membership Meaningful | 24 |
| 6 | Promoting Your Club | 29 |
| 7 | Support From Rotary | 32 |
| 8 | Beyond Interact | 36 |
| 9 | Rotary Resources | 38 |
| APPENDIX 1 | Sample Club Charter Ceremony | 40 |
| APPENDIX 2 | Guiding Questions for Setting Goals | 41 |
| APPENDIX 3 | Rotary Citation for Interact Clubs | 42 |
| APPENDIX 4 | Specific Goals for Your Club | 45 |
| APPENDIX 5 | Sample Meeting Agenda | 46 |
WHAT IS INTERACT?
Interact is a service club for young people ages 12-18 who are passionate about making a difference while learning about the world and themselves. And having fun! Your Interact club will be expected to organize at least two projects every year: one that helps your school or community and one that promotes international understanding.
WHY PARTICIPATE IN INTERACT?
You’ll find that Interact makes an impact in so many ways: on you personally, on your community, and beyond. Here are just a few reasons to join or start an Interact club:
**Taking the lead.** You and your fellow club members get to shape your own club. You’ll plan and do everything from service projects to leadership development and social activities, all tailored to your members’ interests and your community’s needs. In doing so, you’ll gain lifelong leadership skills.
**Learning the value of service.** Your club’s projects will create real and lasting change. You’ll get to observe the results of your service and understand the enormous impact that Rotary’s members and other volunteers make around the world.
**Becoming a global citizen.** When you join an Interact club, you become part of a global community. You connect with other young leaders from around the world, gain a deeper understanding of international events, and forge new friendships.
BE PART OF SOMETHING BIGGER
When you join Interact, you become part of something bigger: Rotary! More than 1.4 million Rotary and Rotaract club members worldwide are working to create lasting change. Interact has a global reach, too, with more than 300,000 Interactors around the world. And just like you, they are all dedicated to making a difference in their communities and beyond.
Through this network of Interact, Rotaract, and Rotary clubs, you can share ideas with an international group of leaders. To foster this connection to Rotary, your club must be sponsored by a Rotary club. Your sponsor club will tell you about the other programs Rotary offers for young leaders, connect you to other clubs in your district, and help you get involved in districtwide training and service opportunities. Finally, sponsor club members will serve as mentors for your club.
GOOD TO KNOW
You can participate in other Rotary youth programs to enhance your leadership skills and become even more of a global citizen. Learn about [Rotary Youth Leadership Awards](#) and [Rotary Youth Exchange](#).
What interests you about an Interact club: leadership, service, learning about people and other countries, or making friends? What does an Interact club look like to you?
Your club will also receive guidance from an Interact adviser, whose name must be reported to Rotary International every year through My Rotary, the online portal for members. Your adviser provides support, answers questions, and ensures that all of Rotary’s youth protection policies are followed. You will learn more about the role of the Interact adviser throughout this handbook.
HOW IT ALL BEGAN
Rotary International started with the vision of one man, Paul Harris. An attorney in Chicago, Illinois, USA, Harris founded the Rotary Club of Chicago in 1905. Harris realized that in joining together with other professionals from different backgrounds, that he and others could exchange ideas and develop lifelong friendships. Over time, Rotary’s reach gradually extended to humanitarian service. Members have a long record of addressing challenges in their communities and around the world.
Empowering young people has always been an essential part of Rotary’s work. In the late 1950s, Rotary members began seeking ways to strengthen these efforts, through a Rotary-wide program designed to encourage service among young people and offer them opportunities to develop their leadership skills. After a few years of study under the direction of 1959-60 President Harold Thomas, Rotary approved plans for the Interact program.
In November 1962, Rotary members helped students at Melbourne High School in Melbourne, Florida, USA, form the first Interact club. The name “Interact” came from combining the words “international” and “action.”
Starting an Interact Club
What if you’re passionate about service but there’s no Interact club in your area? Follow just seven steps to start an Interact club.
1. FIND A CLUB SPONSOR AND ADVISER
First, you’ll need to find a Rotary club to sponsor your Interact club. A sponsor Rotary club maintains a connection to Rotary’s vast network and resources, mentors your Interact club, and connects you to additional service and leadership opportunities through Rotary. To find a sponsor, contact a local Rotary club. Explain why you want to start an Interact club and what you hope to accomplish. When you have found a sponsor club, its members will guide you through the process of starting an Interact club.
If you need help connecting with a Rotary club, use the Club Finder on My Rotary, Rotary’s online member portal.
You will also need to find an adviser for your Interact club. Your club adviser can be a member of a sponsor Rotary or Rotaract club, a teacher, a parent, or a community volunteer. This person will oversee the day-to-day activities of your club, communicate with your club sponsors and Rotary International, and ensure that Rotary’s youth protection policies are followed. If your club is school-based, a teacher or school administrator may need to serve as your adviser, depending on your school’s policies.
Learn more about advisers and club sponsors in chapter 7.
2. CUSTOMIZE YOUR CLUB
Next, you will need to choose the kind of club that will work best for your club’s members. Consider these factors.
CLUB BASE
You can decide whether your Interact club is school-based or community-based. In a school-based club, all the members attend one school. You’ll need approval from your school, and depending on its policies, you may need a teacher or administrator to serve as your club adviser.
If you organize a community-based club, you’ll be able to recruit members from several schools and from local youth programs.
AGE RANGE
Interact is open to people ages 12-18, but you can choose a narrower age range for your club. Doing so may help your club meet members’ needs and interests, especially if it’s a community-based club, which will have a larger pool of potential members.
For a school-based club, be sure to check your school’s policies on eligibility for activities like Interact. If they require that students of all ages be allowed to join, your club may admit members who are younger than 12 or older than 18. Note also that Interactors can remain active in school-based clubs until they graduate, no matter their age.
MEETING FORMAT
Your club can choose to meet in person, online, or a combination of both. Ask members which format works best for them. If your club chooses to meet in person, try to select a location that’s convenient for everyone, such as a place where members already gather to study, play sports, or socialize.
Where will your club be based?
What age range will you include in your club?
How will you meet?
3 RECRUIT MEMBERS
Create promotional materials that tell potential members about your new club and explain how they can learn more. For a school-based club, hang flyers around the school and work with faculty to encourage students to join. For a community-based club, display your promotional materials at youth centers or other local gathering spots. You can also develop content for social media and other online platforms to reach more of your peers. And don’t forget to invite your friends! Learn more about attracting new members in chapter 6.
Where will you find club members and how will you spread the word?
4 DETERMINE HOW YOUR CLUB WILL BE GOVERNED
Review the Standard Interact Club Constitution and fill in your club information. It includes important policies on membership, sponsorship, meeting structure, and more. Anyone who joins your Interact club will need to comply with the principles outlined in the Standard Interact Club Constitution.
When you’ve recruited a group of members, your club can also adopt the Recommended Interact Club Bylaws. Your club bylaws supplement the Standard Interact Club Constitution and help develop common club practices. Members can work together to customize the bylaws to meet your club’s needs, as long as they don’t conflict with the policies in the constitution and the Rotary Code of Policies.
Once you have read these documents, check them off your list.
- [ ] Standard Interact Club Constitution
- [ ] Recommended Interact Club Bylaws
- [ ] Rotary Code of Policies
What bylaws would you like to add for your club?
5 ELECT A BOARD OF DIRECTORS
Your club can now hold elections for its board of directors using the guidelines you developed in your bylaws. Each Interact club should have a president, vice president, secretary, and treasurer, and its bylaws should outline their roles and responsibilities. Learn more about the responsibilities of club officers in chapter 4.
Your club can establish other officer roles as needed. Just be sure that your sponsor club approves of any added roles.
6 MAKE IT OFFICIAL
You can now officially charter your new Interact club! Working with your club adviser, complete the *Interact Club Certification Form*.
Save or scan and email your completed form to email@example.com. You can also email, fax, or mail a copy of the form to your region’s international office.
There is no fee to charter an Interact club. After Rotary receives your form, it will take four to six weeks to prepare your official Certificate of Organization. Rotary will email your certificate to the sponsor Rotary clubs’ presidents to sign and present to your club.
7 CELEBRATE!
When your certificate arrives, plan an event to commemorate the official start of your new club. There’s no standard ceremony when you start an Interact club, induct members, or recognize club officers, so be creative! Organize a celebration that combines your district’s Rotary traditions and local customs into something new. To get ideas, see the sample ceremony in appendix 1.
As an Interactor, you can have fun, meet new people, and learn lifelong skills. We all make that possible by creating clubs that are safe, welcoming, and inclusive.
This chapter covers what you can expect from your club’s adult advisers, sponsors, and mentors. It also outlines steps you can take to create a safe club.
WHAT YOU CAN EXPECT FROM YOUR ADVISERS, SPONSORS, AND MENTORS
Your Interact club advisers and sponsors are responsible for creating and maintaining a safe environment for all Interact club members. They must safeguard Interactors from physical, sexual, and psychological abuse and harassment.
Rotary provides your advisers, sponsors, and district leaders with resources and information on our youth protection policies so they can confidently implement them. These include:
- Rotary Youth Protection Guide
- Protecting Youth Program Participants (online course)
- Electronic and Online Safety Considerations
- Developing a Crisis Management Plan
- Rotary Code of Policies, section 2.120.
YOUR ROLE IN SAFETY
Your adviser is responsible for ensuring that your club follows Rotary’s youth protection policies, but you can also do your part to create a safe environment for your peers.
A safe environment begins with a culture of accountability, where everyone takes responsibility for their words and actions and holds others to the same standard. In the Rotary community, one of our guiding principles is The Four-Way Test. This refers to four things we consider about our words, thoughts, and behavior:
1. Is it the TRUTH?
2. Is it FAIR to all concerned?
3. Will it build GOODWILL and BETTER FRIENDSHIPS?
4. Will it be BENEFICIAL to all concerned?
You can use The Four-Way Test to help create a club environment that promotes safety, courtesy, dignity, and respect for all.
Here are some more things you can do to promote a safe and welcoming environment:
- Consider developing a code of conduct for your club that reflects the principles of The Four-Way Test. Include clear expectations for acceptable and unacceptable behavior, including physical, verbal, and electronic interactions. Learn more in chapter 4 of the *Rotary Youth Protection Guide*.
- Emphasize to all members that your club will not tolerate disrespectful behavior and that it is everyone’s responsibility to create a respectful environment.
- Empower members to stand up to disrespectful behavior when it occurs and report it to your advisers, sponsors, or district leaders.
**Sample code of conduct**
As an Interactor, I will:
- Follow Rotary’s youth protection policies
- Treat others with respect, regardless of their race, ethnicity, sexual orientation, or background
- Create an environment in which members feel safe from physical, verbal, and sexual harassment or abuse
**GOOD TO KNOW**
Leaders of Rotary districts also work to create safe environments for Interactors. Your district governor and district youth protection officer help clubs develop additional youth protection policies, training, and resources to reflect local and regional laws and practices.
REPORT YOUTH PROTECTION CONCERNS
Even with clear rules, abuse or other misconduct can occur in any organization or program. If something happens that affects your safety or the safety of another Interactor, or if you have a safety concern, here’s how to respond:
- **Tell a trusted adult.** Tell your parent or guardian, adviser, sponsor, mentor, or district leader. Share the details of what happened, when, and who was involved.
- **Report it online.** If you’re not comfortable telling an adult, or if you want to report what happened anonymously, you can use Rotary International’s [online youth protection report form](https://rotary.org/youthprotection). Find a link for reporting a youth protection concern at rotary.org/youthprotection.
Rotary’s policies require any allegation of sexual abuse or harassment that involves youth to be reported to local law enforcement.
The safety and well-being of youth program participants and all young people are Rotary’s highest priority, and we take all youth protection concerns seriously. If you have any general questions or concerns about how to protect yourself or your fellow Interactors, visit our [Youth Protection page](https://rotary.org/youthprotection) or write to firstname.lastname@example.org.
ONLINE SAFETY
As we noted in chapter 2, your club has the option to organize itself online and meet virtually. If you choose to meet online, work with your club adviser to review the safety features of online tools and platforms you use. Ask your adviser to provide guidelines for online communications, not only between club members but also between members and adults.
Social media can also be a great way to connect with others and share important information. When you’re using social media, remember:
- Never post photos or personal information about club members without their permission and the permission of a parent or legal guardian.
- Never tag a club member or share their username or handle on your club’s official social media platforms. This could expose your and their personal information, interests, and favorite locations and make you vulnerable to unwanted communication.
CREATING AN INCLUSIVE CLUB
In Rotary, we believe that demonstrating and embracing diversity, equity, and inclusion (DEI) should be part of everything we do. These principles are key to fostering a welcoming and vibrant club.
At Rotary, we understand that cultivating a diverse, equitable, and inclusive culture is essential to realizing our vision of a world where people unite and take action to create lasting change.
We value diversity and celebrate the contributions of people of all backgrounds, across age, ethnicity, race, color, disability, learning style, religion, faith, socioeconomic status, culture, marital status, languages spoken, sex, sexual orientation, and gender identity as well as differences in ideas, thoughts, values, and beliefs.
Recognizing that individuals from certain groups have historically experienced barriers to membership, participation, and leadership, we commit to advancing equity in all aspects of Rotary, including in our community partnerships, so that each person has the necessary access to resources, opportunities, networks, and support to thrive.
We believe that all people hold visible and invisible qualities that inherently make them unique, and we strive to create an inclusive culture where each person knows they are valued and belong.
In line with our value of integrity, we are committed to being honest and transparent about where we are in our DEI journey as an organization, and to continuing to learn and do better.
WHAT DOES DIVERSITY, EQUITY, AND INCLUSION MEAN?
- Diversity = representation from people with different identities/characteristics within a group
- Equity = people are treated with fairness (not equally)
- Inclusion = everyone has a chance to be seen, heard, and has a seat at the table for decisions that affect them
Your Interact club is encouraged to develop its own DEI statement. Work together to write a statement that all club members agree to follow to make the club inclusive for current and future members.
When you develop a DEI statement, consider taking the following steps.
**Welcome and introduction:**
- Make sure your club uses inclusive introductions. If appropriate, consider inviting participants to mention their pronouns when they introduce themselves if they wish.
- Ask participants what diversity, equity, and inclusion mean to them, and give them an opportunity to contribute to your club’s DEI statement.
**Reflection:**
- Allow time for discussing DEI during club meetings. Ask members to review the club’s DEI statement and consider how the club could better support diversity, equity, and inclusion.
**Feedback:**
- Plan to collect feedback from club members every year. Based on their answers, identify any gaps in your club’s DEI statement, and consider whether the club should add or change any policies for the coming year. Before you make any changes, make sure all club members agree and consult your club constitution and bylaws.
**Recruitment:**
- Develop a recruitment strategy that seeks out participants from underrepresented groups and encourages them to join.
- Create a fair and unbiased process for admitting new members.
**Service projects:**
- Design service projects that are physically accessible to all of your club’s members.
- Make sure all members are comfortable with the topics or issues that your service projects are related to.
**Training:**
- Make DEI the foundation of any training your club offers, including orientation for new members.
Social activities:
- Organize social activities that are inclusive, making sure that everyone can participate equally regardless of their physical, cognitive, or cultural needs.
- Remember that some participants might be uncomfortable with, or unable to participate in, certain social activities. If that’s a concern, consider making social events optional or offering alternative activities.
Venues:
- Be sure to select venues that are accessible to all participants, whether they’re for service projects, training, social activities, or club meetings.
What are three things that your club can do to be more diverse, equitable, and inclusive? What are some ideas for your DEI statement?
Building a Foundation
You will likely want to start some club service projects and other activities right away. But taking some time to get organized will prepare your club for success. In this chapter, you’ll learn more about organizing your club, writing its bylaws, electing members to leadership roles, establishing committees, and recruiting and retaining members.
CONSTITUTION AND BYLAWS
Your club’s constitution and bylaws provide guidance on how to run and structure your club. All Interact clubs automatically adopt the Standard Interact Club Constitution. This universal constitution helps unite all the world’s Interact clubs around the same goals. After your club reviews and adopts the constitution, you can customize your club by writing bylaws that will guide members. Review the Recommended Interact Club Bylaws and adapt them to meet your club’s needs. In the bylaws, you can establish the committees, meeting frequency, fees, and other practices that work for your club.
Refer to the standard Interact constitution and recommended bylaws as you review this chapter. The topics covered here will help you decide what to include in your bylaws.
CLUB LEADERS
In Interact, young people take the lead in designing their club. You and your fellow members shape your club’s agenda, plan projects, and work together to implement real change. You can play an even bigger part in club decisions by taking on a club leadership role. Serving as a club leader will also help you develop critical skills like management, negotiation, and relationship building.
Your club’s board of directors will serve as the leaders of your club. They will hold regular meetings to discuss club business and will be responsible for writing an annual report to share important information about club operations with the adviser and sponsoring club and district interact committee.
The directors are members of your club who are elected to serve one-year terms unless your bylaws specify otherwise. (Read more about club elections later in this chapter.) The board should have a president, vice president, treasurer, and secretary.
Here are their usual responsibilities:
**PRESIDENT**
- Oversees and leads most club meetings
- Plans creative club programming
- Arranges speakers, panel discussions, trips, and other activities
- Appoints all standing and special committees with the approval of the board
- Communicates regularly with sponsor clubs, the club adviser, and the district Interact committee
- Promotes the club’s DEI statement
**VICE PRESIDENT**
- Presides over club and board meetings when the president is absent
- Handles special assignments as directed by the president
- Coordinates with your club committees and stays informed of their work
**TREASURER**
- Oversees all funds
- Chairs the club finance committee
- Manages the distribution of funds, with oversight from the board of directors
**SECRETARY**
- Maintains all club records
- Takes attendance at club meetings and service projects
- Records minutes of all meetings of the club and board of directors. These are notes that record the key points and ideas of the meeting and who says them. It is a way to keep track of tasks, activities, responsibilities, and goals.
While each director has specific responsibilities, the board is meant to collaborate. That’s why it’s important for your club’s directors to feel comfortable working together. Icebreakers and social activities can help directors get to know one another. Activities could include game nights or talent shows, themed dinners, and more.
As a club, what can members do to get to know each other?
CLUB COMMITTEES
Committees play an important role in planning projects and ensuring that your club meets its goals. Interact clubs usually appoint the following committees.
**International understanding:** Plans at least one major activity each year to promote international understanding within the club, school, or community. Most, if not all, club members should be involved in the activity.
**Service:** Organizes at least one service project that benefits the school or community. Most, if not all, club members should be involved in the activity.
**Finance:** Develops a plan to finance all club activities.
**Club:** Tracks attendance, develops membership strategies, communicates club updates, and promotes club activities.
Your club can choose to elect committee chairs, as outlined in your bylaws to lead committee activities. They will report back to the board of directors on their committees’ progress and share updates at club meetings. Additional committees may be appointed as needed. Just remember that your club bylaws will need to define the responsibilities of all committees and that each one should meet regularly to discuss plans and activities.
**GOOD TO KNOW**
Check whether your Rotary district organizes a training event for Interact club leaders. If not, ask about participating in district training events like the presidents-elect training seminar (PETS). At PETS, incoming Rotary club presidents prepare for their year in office while district governors-elect and incoming assistant governors build their motivation and working relationships. Your club adviser can connect you to district training opportunities. (Learn more in chapter 7.) You can also complete some of the courses under Club Leadership in Rotary’s Learning Center.
What leadership roles are you interested in? What skills do you want to contribute or develop through a leadership role?
ELECTIONS
Your club will elect directors and, if applicable, committee chairs every year. You have the flexibility to determine your own election process so it will be compatible with local customs.
When organizing club elections, remember:
- Candidates must accept their nomination in order to be elected.
- To win an election, candidates should receive a simple majority of votes.
- Candidates should be current club members in good standing.
- All voting should be anonymous.
- Election procedures should be recorded in your club bylaws.
Here are a few ways you could organize elections:
- Candidates apply for a position using an online form that asks them about their goals. All active club members vote based on their answers.
- Candidates give speeches about what they plan to achieve in the role. Members who attend vote by secret ballot.
- Candidates record videos of themselves talking about their vision for the club and how they plan to achieve their goals. Members watch each video and vote online.
List your ideas for how to hold elections:
SETTING GOALS
With your club leaders and committees in place, you are ready to begin setting goals. Setting goals annually will give your club direction and help it succeed.
Need help with setting goals? Find questions to guide you in appendix 2, information about the Rotary Citation for Interact Clubs in appendix 3, and space to list your club’s goals in appendix 4.
How will you set goals as a club?
The Rotary Citation for Interact Clubs
Each year, Rotary International recognizes Interact clubs for achieving goals that strengthen both the clubs and Rotary. Working toward the Rotary Citation can help your club set annual goals and track its progress. Learn more in these resources:
- **Citation Goals and Instructions for Interact Clubs (PDF):** Review the citation goals, indicate which ones you plan to achieve, and set a target.
- **Nomination form:** Work with your sponsor Rotary club officers or Interact club adviser to complete the nomination form by 15 August.
FUNDING CLUB ACTIVITIES
Your club may need money to carry out its activities and achieve its goals. Costs could include supplies for service projects and meetings or promotional materials like club T-shirts, for example. Your club treasurer can lead the development of a funding strategy. Here are a few options:
- **Annual membership fees.** Your club can choose to ask members to pay a minimal fee, only to cover administrative costs as defined in your bylaws. Consider whether a fee is necessary, and whether it could prevent some potential members from joining.
- **Support from your sponsors.** Your club may occasionally request minimal financial support from your sponsor Rotary or Rotaract clubs.
Fundraisers. Your club can raise funds from other sources. Consider these suggestions when you plan a fundraiser:
- Be creative. Use your club members’ skills, talents, and contacts.
- Be specific. When you raise funds from individuals, businesses, or organizations, clearly explain what the money will be used for and the impact their donations will make.
- Be open to new ideas. Ask your sponsor club, local nonprofit organizations, and other Interactors in your district for fundraising suggestions.
GOOD TO KNOW
Learn more about the types of fundraisers your club can organize in the online course *Youth Guide to Service-Learning*.
Ask your adviser and sponsor clubs for financial guidance, including on how to set up bank accounts and designate funds based on club activities.
Which strategies will you use to fund your club activities?
RUNNING AN EFFECTIVE MEETING
Club meetings are an opportunity for members to come together, share news and announcements, plan service projects and events, and have fun! They are also a chance to invite experts and local leaders to speak.
Your club will first need to determine how often to meet and where. Be sure to include the meeting frequency in your club bylaws. Your club will also need to decide how important member attendance is. You can specify how many meetings or projects members can miss and how they can make up for their absences.
See appendix 5 for a worksheet to help you plan club meetings.
ADMITTING NEW MEMBERS
New members will help your club thrive and enable it to make a larger impact. As your club develops its process for admitting new members, consider these suggestions:
- Allow any member to propose a candidate for membership.
- Develop a way for prospective members to ask to join the club.
- Allow other Interact clubs to nominate a transferring or former member to join your club.
- Tell prospective members within a reasonable time frame whether they have been approved to join your club. In your club bylaws, specify how many days club members have to review a candidate.
- Develop fair, unbiased, and inclusive criteria for admitting new members. Your club’s DEI statement can inform this policy.
GOOD TO KNOW
At least one member of your sponsor clubs should attend your club and board meetings. Work with your Interact adviser to coordinate with your sponsor clubs, making sure they know when and where your club meets and how often they should attend.
What strategies will you use to appeal to and invite new members?
MEMBER ENGAGEMENT
ENERGIZING NEW MEMBERS
It’s important to engage new members in club activities as soon as they join. It tends to work well when clubs:
- Offer an orientation. Describe your club’s key activities and the various ways new members can get involved, including by serving on club committees.
Get new members’ ideas. Ask them about the types of service projects and social activities that interest them and try to use their suggestions.
Mentor new members. Pair experienced club members with new ones. The experienced members can help make sure new members feel included.
KEEPING MEMBERS ENGAGED
The best way to retain members is by keeping them engaged. Here are some ideas:
- Gather feedback from members regularly and make adjustments to your club as needed.
- Allow all members to contribute ideas for service projects and social activities.
- Make sure all members are informed of upcoming activities.
- Plan social events and icebreakers so members can get to know one another.
- Encourage members to get involved in club leadership roles.
ROTARY’S LEADERSHIP OPPORTUNITIES
Rotary offers young people many ways to get involved other than through Interact. By highlighting these opportunities, you’ll help members stay excited about your club and Rotary. Encourage members to participate in district training events, and connect them to Rotary Youth Leadership Awards, Rotary Youth Exchange, and other Rotary opportunities in your area.
GOOD TO KNOW
Learn more about promoting your club in chapter 6.
How will you engage and communicate with members?
Make Membership Meaningful
Through your Interact club, you can lead service projects, promote international understanding, and help your fellow members develop leadership skills. Interact clubs organize at least two activities every year: one that helps their school or community and one that promotes international understanding. But your club has the potential to accomplish much more. Members can have fun and find purpose in working on causes they care about. This chapter covers the basic steps for conducting club activities and offers ideas for deepening your experience and expanding your impact.
SERVICE PROJECTS
Effective service projects require preparation and planning. To guide your club through a successful project, you are encouraged to use a service-learning approach.
Service-learning has five steps:
- **Investigation.** Through research and inquiry, your club identifies a genuine community need and the factors that cause it.
- **Planning and preparation.** Based on your research, your club plans a realistic and meaningful service project with clear goals, timelines, roles, and follow-up plans.
- **Action.** Your club implements the plan through direct, indirect, or advocacy-based service. Throughout this step, collect data about your project and its impact.
- **Reflection.** Think more deeply about what went well, what went wrong, and what you learned. Reflection is important at every phase of service-learning, not just after the action step.
- **Demonstration.** Show what you learned and the impact you made in your community to others who supported your action. Share recommendations based on findings from your reflection activities. Learn more about showcasing your club’s success in chapter 6.
START YOUR SERVICE-LEARNING JOURNEY
In partnership with the National Youth Leadership Council, Rotary International developed a set of resources to support you and your club advisers through each step of service-learning. Complete the online course Youth Guide to Service-Learning, available in Rotary’s Learning Center, and use the interactive handbook Service-Learning Workbook for Youth as you carry out service projects. The course will teach you how to conduct a community assessment, find partners, and develop a detailed timeline and budget, and it includes tools such as a checklist for a day of service.
Rotary also has resources just for advisers. Encourage your adviser to explore the Service-Learning for Advisers learning plan in for more information.
In the early steps of service-learning, decide which types of service will be most effective for addressing the need you’ve identified. The types of service are:
- **Direct service** — Working directly with the people who benefit from your service. Examples include documenting oral histories from older people, tutoring younger students, and delivering meals to people who have limited mobility.
- **Indirect service** — Working to support other efforts that help people, such as cleaning up a playground, raising funds for a cause, or collecting books for a school or library that needs them.
- **Advocacy** — Raising awareness about an issue. Examples include speaking to a city council to support a policy or launching a public information campaign about an issue like eradicating polio.
Looking for inspiration and service project ideas?
- Support one of [Rotary’s causes](#):
- Promoting peace
- Fighting disease
- Providing clean water and sanitation
- Saving mothers and children
- Supporting education
- Growing local economies
- Protecting the environment
- Review nominees for the [Interact Awards](#) which recognize innovative projects that are featured in videos, photos, and essays by Interact clubs around the world.
- Learn about service projects that Interactors have successfully completed on [Rotary Showcase](#). Search for the keyword “Interact” to read stories and share your ideas.
**GOOD TO KNOW**
To use the [Learning Center](#), you will need to create a [My Rotary](#) account. If you are age 16 or 17, you can create a My Rotary account with the permission of a parent or legal guardian. If you are younger than 16, a club adviser can share the printed resources with you.
What service projects are you passionate about?
PROMOTE INTERNATIONAL UNDERSTANDING
Rotary is a global network that strives to advance international understanding, goodwill, and peace. As an Interactor, you are part of this network, and you can use it to connect with other young people around the world. Here are some ways you can use Rotary’s global network to promote international understanding and learn about other cultures:
- **Find a twin club.** Build a long-term partnership with an Interact club in another country. You can work together on an international understanding or service project and organize virtual opportunities for members to learn about the other country and club. It is helpful to partner with a club whose members speak a language you know. Ask your adviser or sponsor club to connect you with an Interact club in another country.
- **Promote peace.** One of Rotary’s causes, promoting peace, can be a way to build international understanding. Work with your sponsor club to find opportunities for conversations and collaborations that will foster understanding within and across cultures. You can also review the [Top 10+ Ways You Can Be an Everyday Peacebuilder](#) on My Rotary for more ideas.
- **Use Rotary’s global network.** Learn about [Rotary Action Groups](#) and [Rotary Fellowships](#) and find ways to collaborate with one.
- **Introduce members to Rotary’s exchanges.** Through Rotary, club members can participate in informal virtual or in-person exchanges, experience [Rotary Youth Exchange](#) as either a host or an exchange student, and look forward to opportunities after Interact through [New Generations Service Exchange](#) and [Rotary Friendship Exchange](#).
LEADERSHIP DEVELOPMENT
As an Interactor, you have lots of opportunities to develop lifelong leadership skills. Organizing service projects and taking on club leadership roles are great ways to learn how to build relationships, solve problems, and make decisions as a group.
You can also support other members’ leadership development, which will not only enhance your own membership but also further your own skills. Consider covering leadership topics during club meetings. Here are some ideas:
## SKILLS
| Leadership theories | Learn the main concepts of various leadership theories. Use a worksheet, survey, or group activity to identify club members’ leadership styles. |
|---------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Communication skills| Let club members take turns leading group activities. Organize a speech competition in which club members speak about a topic of their choice and get constructive comments from other members. |
| Collaboration | Emphasize the importance of acknowledging and respecting different perspectives and ideas. Design challenges that club members can complete successfully only if they work together. |
| Ethical leadership | Learn about the components of ethical leadership, including respect, service, community, justice, and honesty. Consider what ethical leadership means to you, and have club members create their own code of ethics. |
**GOOD TO KNOW**
As you’re developing meaningful experiences for your club, review the goals of the [Rotary Citation for Interact Clubs](#), which can spark ideas and help keep your club on track.
What skills do you already have as a leader? What leadership skills would you like to develop in Interact?
Promoting Your Club
You and your fellow club members work hard to make a difference. Make sure you tell people about your work, so they’ll see your impact and want to get involved. In this chapter, you’ll learn about Rotary resources and how to promote your club.
PUBLIC IMAGE AND BRANDING
Rotary’s public image is how people outside of the organization think about us. The Rotary brand, built over more than a century, can help bring attention to your Interact club when you use it correctly. Proper branding is critical because not everyone understands what we do and why our clubs matter. When you create promotional materials for your club, make sure they follow our branding guidelines. Find these resources in the Brand Center:
- **Guidelines**: Develop communications that are clear, concise, and effective with our messaging and visual guidelines.
- **Logo templates**: Create a club signature, which adds your club’s name to the universal Interact logo and clearly communicates its connection to Rotary.
- **Promotional materials**: Customize promotional cards, posters, banners, and news releases.
- **Images and videos**: Find high-quality photos and professional videos that can help you share information about Interact and Rotary through your website, social media accounts, and events.
USING THE INTERACT LOGO
A key part of branding your club activities is using the Interact logo correctly with an official Interact club signature. The signature system makes it easier for the public to recognize your club across all your communications. Every club can create its own signature in the Brand Center. Here are some examples:
PUBLICIZING YOUR CLUB’S IMPACT
After you create marketing materials, you’ll be ready to showcase your club’s impact. By sharing its work, your club will enhance its public image and possibly attract new members, along with donors to support your projects. Here are some ideas:
SHOWCASE
| Events | Use events, like a celebratory dinner, cultural exhibition, art show, or musical performance, to showcase your club’s accomplishments. Encourage attendees to collaborate with and donate to your future projects and fundraisers. |
|---|---|
| Portfolios | Report on a project from start to finish. For example, design a webpage that includes updates on the project and clearly tells visitors how they can get involved and contribute. |
| Presentations | Give presentations to reach audiences with similar interests. Consider speaking to Rotary, Rotaract, or Interact clubs, at your district conference, or at other service-related events. |
| Local media | Send a press release to television, radio, and newspaper outlets, an essential step if you want widespread attention for your project. |
| Social media | Post about your project on social media, one of the best ways to tell people about your work. You can also ask your sponsor Rotary or Rotaract club to post your project on Rotary Showcase. |
| Community leaders | Write to local leaders about your project, or hold meetings to discuss the issue, why you care about it, and what they can do to help. |
| Interact Awards | Share your projects in a video, photo, or essay for a chance to earn special recognition from Rotary International. |
RECRUITING NEW MEMBERS
When you promote your club effectively, you’ll be more likely to attract prospective members from diverse backgrounds. Here are some recruitment strategies to try:
| SCHOOL-BASED CLUB | COMMUNITY-BASED CLUB |
|-------------------|----------------------|
| Connect with school leaders and share information about your club. | Contact schools and youth organizations and share information about your club. |
| Post club materials around the school, especially in areas where students gather. | Work with district leaders, including the district Interact chair, to promote your club across the district. |
| Use school events, such as assemblies and activity fairs, to tell people about your club. | Ask past participants of Rotary Youth Leadership Awards and Rotary Youth Exchange if they are interested in joining your club. |
| Share information about your club on social media, working with your adviser to create club accounts. | Share information about your club on social media, working with your adviser to create club accounts. |
How will your club reach and recruit prospective members?
As an Interactor, you are part of the Rotary family. That means you can expect support from the club, district, and international levels of Rotary. Read on to learn about Rotary’s organizational structure, the people and resources that can help you, and opportunities to connect with Interactors across districts.
**INTERACT CLUB ADVISERS**
Your Interact club adviser supervises, guides, and supports your club. Your adviser is appointed by your sponsor club (see below) and may be a member of a sponsor Rotary or Rotaract club, an adult nonmember vetted by the sponsor club, or in the case of school-based Interact clubs, a member of the school’s faculty or administration.
Ideal advisers have:
- Strong familiarity with Interact and your club’s activities
- Experience working with young people
- The ability to be a champion for youth
- A passion for service and a desire to make a difference in your community
All potential advisers will need to be screened, vetted, and trained in how to uphold Rotary’s youth protection policies before they formally become your club adviser.
**INTERACT CLUB SPONSORS**
An effective Interact club depends on the guidance, support, and active participation of sponsors.
There are over 36,000 Rotary clubs and 10,000 Rotaract clubs in nearly 200 countries and geographic areas, working to unite people and create lasting change in their communities, around the world, and in themselves.
Sponsor Rotary and Rotaract clubs should help train Interact club members, officers, and committee chairs. They should also encourage Interactors to participate in district, multidistrict, and international training opportunities. Your sponsor clubs are also expected to appoint mentors for your club, have a member attend your club meetings, and invite you to participate in their meetings, service projects, and events.
It takes effort to develop a strong partnership with your sponsor clubs. But by keeping them involved in your activities and maintaining good relationships, you create new connections and opportunities for your club. It’s also easier to ask for help when your sponsor clubs are familiar with your club members and activities. You can ask your club adviser or club president to serve as a liaison to your sponsor clubs, or you can create a separate role for that. The liaison should share information about your club meetings, social activities, and service projects with your sponsors and encourage them to participate.
Here are some tips for fostering a strong partnership with a sponsor club:
- Exchange event calendars and encourage your club members to participate in your sponsor club’s activities, and vice versa.
- Ask some of your club members to attend sponsor club meetings and give updates on your club’s activities.
- Consider asking a representative from each of your Interact club’s committees to connect with the corresponding committee of your sponsor club. For instance, a member of your service committee can attend your sponsor club’s service committee.
- Start a mentorship program between your Interact club and your sponsor club.
**GOOD TO KNOW**
You can build a collaborative relationship with your sponsor club when you succession plan and prepare for the year ahead. Elect your club president well in advance of their term, and encourage them to meet with the incoming president of your sponsor club to share any important information and documents.
DISTRICT SUPPORT
In Rotary, clubs are grouped into more than 500 districts. District leaders include the governor (see below) and district committees, who work to strengthen and serve clubs and programs in the district.
DISTRICT INTERACT COMMITTEE
The **district Interact committee** oversees all Interact activities in the district. It’s responsible for empowering Interact clubs, growing and promoting Interact across your district, and providing training and support to ensure that Rotary’s youth protection policies are followed. This committee should also connect Interact clubs with one another and strengthen the link between Interact and Rotary. The Interact committee can include Rotary, Rotaract, and Interact members.
The **district Interact chair** oversees the Interact committee. Appointed by the district governor, the chair can be either a Rotary or a Rotaract member. This person manages the district’s Interact program and ensures that all clubs submit their contact information to Rotary International every year.
The district Interact committee may be co-chaired by an Interactor who also serves as the **district Interact representative**. This representative is elected by all the Interactors in the district and offers guidance and inspiration to Interact clubs.
ADDITIONAL DISTRICT ROLES
Your club can expect support from other district leaders, including:
**District governor.** The governor supports all programs and activities in the district. The governor approves new Interact clubs and all Interact club sponsors. They appoint the district Interact chair, as well as other leaders.
**District youth protection officer.** This person advises on Rotary’s youth protection policies and procedures.
**District alumni chair.** The alumni chair helps Interact members transition to Rotaract and connects alumni with other Rotary opportunities.
BEYOND THE DISTRICT
You can find even more support and more opportunities for Interactors at the multidistrict, zone, and international levels of Rotary.
**Multidistrict**
Districts often work together on multidistrict events, training, and service projects. It’s a way to bring large, diverse groups of Interactors together to make new friends and learn from one
another. If this sparks your interest, work with your district Interact chair to find a multidistrict event that you could attend or to develop one. Multidistrict events always require an agreement from the governors of the participating districts.
**Zone**
Rotary districts are organized into more than 30 zones around the world. Zones are overseen by a team of regional leaders and elected directors. Interactors are often invited to participate in zone conferences, training meetings, and other events.
**Rotary International staff**
Rotary members and programs are supported by an international staff of hundreds of professionals in six international offices and its world headquarters in Evanston, Illinois, USA. The Programs for Young Leaders staff is responsible for supporting Interact by chartering new clubs, sending regular updates to clubs and district leaders, designing opportunities like the Interact Awards and World Interact Week, and developing resources for clubs. If you have any questions, write to email@example.com or your region’s international office.
How can your district or zone support your Interact club?
Beyond Interact
Supporting Interact is just one of the ways that Rotary invests in young leaders. Through Rotary, you have access to a variety of programs developed to empower you at every age. Contact your district leaders to learn more about these opportunities for young people.
**Rotary Youth Leadership Awards (RYLA):** RYLA is an intensive leadership program for young people ages 14-30. Organized by clubs, districts, and multidistricts, RYLA events allow participants to learn new skills through civic engagement and personal and professional development. To learn more about RYLA and its impact, read about [past recipients of the Leaders in RYLA Awards](#).
**Rotary Youth Exchange:** Through Rotary Youth Exchange, students ages 15-19 can learn a new language, discover another culture, and serve as ambassadors for peace. Exchanges are sponsored by Rotary clubs and can be either short-term or long-term. To learn more about Rotary Youth Exchange, [read stories from exchange students and alumni](#) on the Rotary Voices blog.
**AFTER INTERACT**
Don’t worry about what’s next after Interact. Rotary has something for you at every stage in life. As your experience with Interact comes to an end, explore these opportunities to stay involved.
**Alumni groups:** As a former Interactor, you are part of Rotary’s alumni network and can connect with fellow leaders engaged in service around the world.
**Rotary Peace Fellowships:** Designed for leaders with work experience in peace and development, these fellowships fund study at our Rotary Peace Centers. Fellows are committed to community and international service and the pursuit of peace.
**New Generations Service Exchange:** New Generations Service Exchange is a short-term, customizable international exchange program with a humanitarian or vocational focus for university students and professionals up to age 30.
**Rotary Action Groups:** Rotary Action Groups are independent, Rotary-affiliated groups of people from around the world who are experts in a particular field, such as economic development, peace, addiction prevention, the environment, or water.
**Rotary Community Corps (RCC):** A Rotary Community Corps is a group of volunteers who aren’t Rotary members but share Rotary’s commitment to changing the world through service projects. Sponsored by a Rotary club, an RCC leverages Rotary’s network, brand, and
mentorship by partnering with clubs to plan and implement service projects. More than 11,000 RCCs are active around the world.
**Rotary Friendship Exchange:** Rotary Friendship Exchange is an international exchange program for Rotary members and friends that allows participants to take turns hosting one another in their homes and clubs. Participants may travel as individuals, couples, families, or groups, and may or may not be Rotary members.
**Rotary or Rotaract clubs:** When the time comes, you might want to join a Rotaract or Rotary club to make new friends, continue serving your community, and hone your leadership skills. Ask your district leaders to help you find a club that meets your needs.
Which other Rotary programs interest you?
Rotary Resources
ONLINE TOOLS
- **Brand Center** — Create your own Interact club logo to use on promotional materials, club shirts, and more.
- **Rotary Showcase** — Get inspired! Read about completed service projects, and have your sponsor post yours for everyone to see.
- **Interact Facebook page** — Connect with fellow Interactors and the Rotary members who support Interact.
- **Rotary’s Learning Center**
- **Service-learning resources** — Find interactive courses and a workbook on designing youth-led service projects.
- **Protecting Youth Program Participants** — Refer your adviser, sponsor, and other leaders to understand, recognize, address, and prevent abuse and harassment.
OTHER RESOURCES
- Interact Club Certification Form (PDF)
- Standard Interact Club Constitution (PDF)
- Recommended Interact Club Bylaws (DOC)
- How to Update Interact Club Information (PDF)
- Interact Identification Card (PDF)
- Young Leaders in Action — Subscribe to this monthly e-newsletter about Rotary’s programs for young leaders, including Interact.
- Rotary’s causes — Learn more about our causes and the work we’re doing to advance them.
- Youth Programs page — Discover Rotary’s other programs for young leaders.
- Rotary Code of Policies — Read policies and procedures established by the RI Board of Directors for the organization, including Interact and other programs for young leaders.
APPENDIX 1: Sample Club Charter Ceremony
Learn more about starting an Interact club and holding a charter ceremony in chapter 2.
When it’s time to celebrate your new club’s charter, be creative! Plan a ceremony that reflects your club and community. Use this outline and the spaces below to plan and create a charter ceremony for your new club.
INVITATIONS
Consider inviting your sponsor club or district leaders to officiate or attend the ceremony:
- District governor
- District Interact committee chair
- District Interact representative
You can also invite club members’ families, school teachers and staff (if applicable), and community leaders.
CEREMONY IDEAS
| Share Rotary’s history. Consider providing a brief history of Rotary and Interact, including the founding of Rotary, the start of the first Interact club, and the missions of Rotary and Interact. |
| --- |
| Highlight the international family of Rotary. Emphasize that when young people join an Interact club, they become part of a global community. Mention the current membership numbers for Rotary and Interact. |
| Introduce members. Announce the name of each new club member, and consider presenting everyone with a small token, such as a pin or membership card. |
| Announce the club’s board of directors. Introduce its members and briefly explain their roles. You might ask the club president and other officers to say a few words about their goals for the year ahead. |
PHOTO OPPORTUNITY
End the ceremony with a group photo!
APPENDIX 2: Guiding Questions for Setting Goals
Learn more about setting goals in chapter 4.
Setting goals every year will help your club plan its activities and events and help it stay active and productive. Working together as a club, consider the questions below, and refer back to your answers throughout the year.
NEW CLUBS
What characteristics do we want our club to have?
What will we consider success to be?
What do we want to do to further DEI?
ESTABLISHED CLUBS
How are we reaching our current goals? What are some examples? What has worked well?
What goals are we not reaching? Why? What isn’t working well?
What are our club’s strengths? How can we use these strengths?
How can we continue to gather members’ feedback and make the club experience better for all?
APPENDIX 3: Rotary Citation for Interact Clubs
Learn more about setting goals in chapter 4.
As you set goals for your club, get inspired by the Rotary Citation for Interact Clubs.
The citation recognizes clubs that increase our impact, expand our reach, enhance participant engagement, and increase our ability to adapt.
**Instructions:**
Select at least 11 of the 20 goals listed below. This flexibility allows you to choose the goals that are the most relevant and attainable. You have the entire Rotary year (1 July to 30 June) to achieve your goals, and if you do, your club will receive the citation. Your sponsor club officers and club adviser can refer to this worksheet when they complete your nomination form.
To achieve the citation:
- Review the details of the goals.
- With guidance from your club adviser, select at least 11 goals to achieve.
- Indicate the goals you plan to achieve and set a target, if appropriate, in the SET YOUR GOAL column.
- When your club meets a goal, indicate that in the GOAL ACHIEVED column.
- Your sponsor club officers and club adviser should refer to this worksheet throughout the year to make sure that your club is on track to meet its goals.
- Work with your sponsor Rotary club officers or Interact club adviser to submit your nomination form by 15 August. Have them refer to this worksheet to complete the form.
| CITATION CATEGORY | GOAL DETAILS | SET YOUR GOAL | GOAL ACHIEVED |
|-------------------|--------------|---------------|---------------|
| Club membership | How many total members does your club want by the end of the Rotary year? | | |
| Service participation | How many members will participate in club service activities during the Rotary year? | | |
| Rotary Action Group engagement | During the Rotary year, will club members have the opportunity to learn more about Rotary Action Groups and get inspiration for service projects? | | |
| Leadership development participation | How many leadership development programs will your club organize to enhance members’ skills? | | |
| CITATION CATEGORY | GOAL DETAILS | SET YOUR GOAL | GOAL ACHIEVED |
|----------------------------|------------------------------------------------------------------------------|---------------|---------------|
| District conference | How many members will attend your district conference? | | |
| attendance | | | |
| Rotary Fellowship | During the Rotary year, will club members learn more about the opportunities available through Rotary Fellowships? | | |
| engagement | | | |
| District training | Will your club members attend district training? | | |
| participation | | | |
| Learning Center | Will your club adviser or a member of your club complete the service-learning training in Rotary’s Learning Center? | | |
| engagement | | | |
| Annual Fund contributions | During the Rotary year, how will your club raise funds for or increase awareness of Rotary’s Annual Fund? | | |
| PolioPlus Fund | During the Rotary year, how many events will your club hold to raise funds for or increase awareness of Rotary’s work to eradicate polio? | | |
| contributions | | | |
| Service projects | Will your club partner with your sponsor club or adviser on a significant local or international service project in one of Rotary’s areas of focus? | | |
| RYLA participation | How many of your club members will participate in Rotary Youth Leadership Awards? | | |
| CITATION CATEGORY | GOAL DETAILS | SET YOUR GOAL | GOAL ACHIEVED |
|---------------------------------|------------------------------------------------------------------------------|---------------|---------------|
| Programs for young leaders | Will your club host a meeting that introduces members to other Rotary programs for young leaders, including RYLA and Rotary Youth Exchange? | | |
| Alumni engagement | Will your club engage with your sponsor club to connect graduating Interactors with university- or community-based Rotaract clubs? | | |
| Strategic plan | Will your club follow its strategic plan or update it if it doesn’t reflect your current priorities? | | |
| Update website and social media | During the Rotary year, how many times per month will your club’s website or social media accounts be updated? | | |
| Media stories about club projects| Will your club promote an activity it holds during World Interact Week on social media? | | |
| Use the Brand Center | Will you use logos and materials that were created using Brand Center templates and that correctly reflect Rotary’s brand? | | |
| Use of official Rotary promotional materials | Will your club use Rotary International’s messaging and public service materials, such as broadcast videos, print ads, and other materials from the Brand Center, to promote Interact in your community? | | |
| Promote your club | Will your club participate in the annual Interact Awards? | | |
APPENDIX 4: Specific Goals for Your Club
After you review the goals of the Rotary Citation, you might decide to set additional goals for your club.
1. List your goals in the column labeled GOAL. Make them specific.
2. Under NOTES, write how your club plans to achieve each goal. Include details such as the committee responsible for overseeing progress, any funding or additional resources needed, and due dates.
3. When your club has met a goal, indicate that in the GOAL ACHIEVED column.
| GOAL | NOTES | GOAL ACHIEVED |
|------|-------|---------------|
| | | |
| | | |
| | | |
| | | |
| | | |
APPENDIX 5: Sample Meeting Agenda
Productive club meetings are well organized, with a clear agenda. Here’s an outline you can use for your meetings.
AGENDA ITEMS
1. **Call to order and agenda.** Write a list of things the group needs to discuss and accomplish in the meeting, so you can present it to club members.
2. **Guest speakers or new members.** Note the names of any guest speakers or new members, along with a few details about them so you can introduce them.
3. **Icebreaker.** Plan a fun activity to start your meeting.
4. **Announcements.** List important announcements and reminders so you won’t forget to mention them at your meeting.
AGENDA ITEMS
5. **Board and committee updates.** Note any reports or news that will be presented by the board of directors or committee members.
6. **Open forum.** Give club members time to ask questions or discuss club business. Note any questions or items that your board of directors will need to follow up on.
7. **Adjournment.** Write any closing words you’d like to say to your club.
One Rotary Center
1560 Sherman Avenue
Evanston, IL 60201-3698 USA
Rotary.org
EN—(1022)
|
Microtubule-associated protein tau in oligodendrocytes following acute brain injury
Elaine Alison Irving BSc (Hons)
A thesis submitted for the Degree of Doctor of Philosophy to the Faculty of Medicine, University of Glasgow.
Wellcome Surgical Institute and Hugh Fraser Neuroscience Laboratories,
Garscube Estate,
Bearsden Road,
Glasgow,
G61 1QH
© Elaine A Irving, December 1996.
All rights reserved
INFORMATION TO ALL USERS
The quality of this reproduction is dependent upon the quality of the copy submitted.
In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion.
Published by ProQuest LLC (2018). Copyright of the Dissertation is held by the Author.
All rights reserved.
This work is protected against unauthorized copying under Title 17, United States Code
Microform Edition © ProQuest LLC.
ProQuest LLC,
789 East Eisenhower Parkway
P.O. Box 1346
Ann Arbor, MI 48106 – 1346
Thesis 10772 Cpg 1
## CONTENTS
| Contents | Pages |
|-----------------------------------------------|-------|
| Contents | i |
| List of tables | viii |
| List of figures | viii |
| List of abbreviations | x |
| Acknowledgements | xii |
| Preface and declaration | xiii |
| Abstract | xiv |
### CHAPTER 1 INTRODUCTION
#### 1.1 General introduction
#### 1.2 Acute brain injury
- **1.2.1 Stroke**
- **1.2.2 Head injury**
- **1.2.3 Mechanisms of acute brain injury**
- *Glutamate toxicity*
- *Free radical mediated toxicity*
- *Interaction of glutamate toxicity and free radical damage*
- **1.2.4 Animal models of acute brain injury**
- *Permanent focal cerebral ischaemia*
- *Glutamate toxicity in vivo*
#### 1.3 Structure and function of the neuronal cytoskeleton
- **1.3.1 Components of the neuronal cytoskeleton**
- *Microfilaments*
- *Intermediate filaments*
- *Microtubules*
- **1.3.2 The microtubule-associated proteins**
- *MAP2*
- *MAP5 (MAP1B)*
- *Tau*
- **1.3.3 Normal distribution of tau in the CNS**
- **1.3.4 Cellular functions of tau**
## 1.4 Cytoskeletal breakdown in brain degeneration
| Subsection | Page |
|-------------------------------------------------|------|
| 1.4.1 Acute brain injury | 21 |
| 1.4.2 Chronic neurodegenerative disease | 24 |
| 1.4.3 Neurofibrillary tangles | 24 |
| 1.4.4 Glial fibrillary tangles | 27 |
## 1.5 The neuroglial cells
| Subsection | Page |
|-------------------------------------------------|------|
| 1.5.1 Introduction | 29 |
| 1.5.2 Astrocytes | 29 |
| 1.5.3 Microglia | 29 |
| 1.5.4 Oligodendrocytes | 30 |
| 1.5.5 Oligodendrocyte function | |
| *Myelination* | 32 |
| *Inhibition of neurite outgrowth* | 32 |
| *Neuronal-glial signalling* | 33 |
| 1.5.6 The oligodendrocyte cytoskeleton | 34 |
| 1.5.7 The effect of ischaemic injury on oligodendrocytes | 35 |
## 1.6 Aims of the thesis
| Subsection | Page |
|-------------------------------------------------|------|
| 1.6 Aims of the thesis | 39 |
## CHAPTER 2 MATERIALS AND METHODS
### PART I: ANALYTICAL END POINTS
#### 2.1 Immunohistochemistry
| Subsection | Page |
|-------------------------------------------------|------|
| 2.1.1 Description of antibodies | 40 |
| 2.1.2 Dephosphorylation of tissue prior to immunostaining | 41 |
| 2.1.3 Single label immunohistochemistry | 41 |
| 2.1.4 Double label immunohistochemistry (chromagen) | 43 |
| 2.1.5 Double label immunohistochemistry (fluorescence) | 43 |
#### 2.2 Western Blot Analysis
| Subsection | Page |
|-------------------------------------------------|------|
| 2.2.1 Preparation of cell cultures | 45 |
| 2.2.2 Preparation of whole brain homogenates | 45 |
| 2.2.3 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) | 45 |
| 2.2.4 Semi-dry electroblotting | 46 |
| 2.2.5 Western blot (Enhanced Chemiluminescence -ECL™) | 46 |
## 2.3 In situ hybridisation
| Section | Page |
|------------------------------------------------------------------------|------|
| 2.3.1 Core materials and solutions | 47 |
| 2.3.2 Preparation of tau DNA for riboprobe production | |
| Amplification of tau cDNA | 47 |
| Linearisation of plasmid DNA | 48 |
| 2.3.3 Preparation of $^{35}$S labelled riboprobes | 49 |
| 2.3.4 In situ hybridisation procedure | |
| Pre-treatment of tissue | 52 |
| Hybridisation | 52 |
| Post-hybridisation treatment | 52 |
| Dehydration | 53 |
## PART II: PREPARATION OF TISSUE
### 2.4 Intracortical perfusion of monosodium glutamate in vivo
| Section | Page |
|------------------------------------------------------------------------|------|
| 2.4.1 Surgical preparation of animals | 53 |
| 2.4.2 Microdialysis probe implantation | 55 |
| 2.4.3 Glutamate and sodium chloride perfusion | 56 |
| 2.4.4 Transcardiac perfusion | 56 |
| 2.4.5 Processing of brain tissue for histology and immunohistochemistry | 56 |
| 2.4.6 Processing of tissue for electron microscopy | 57 |
### 2.5 Human post-mortem brain tissue
| Section | Page |
|------------------------------------------------------------------------|------|
| 2.5.1 Sources of post-mortem brain tissue | |
| Head injury | 57 |
| Stroke | 58 |
| Controls | 58 |
| 2.5.2 Histology and immunohistochemistry | 58 |
| 2.5.3 Antigen retrieval | 59 |
### 2.6 Permanent focal cerebral ischaemia in vivo
| Section | Page |
|------------------------------------------------------------------------|------|
| 2.6.1 Surgical preparation of animals | 61 |
| 2.6.2 Induction of middle cerebral artery occlusion | 61 |
| 2.6.3 Immunohistochemical studies | 65 |
| 2.6.4 Quantification of tau-positive oligodendrocytes following cerebral ischaemia | 65 |
| 2.6.5 In situ hybridisation studies | 68 |
## 2.7 Oligodendrocyte cultures
| Subsection | Page |
|---------------------------------------------------------------------------|------|
| 2.7.1 Preparation of oligodendrocyte cultures | 68 |
| 2.7.2 Exposure of oligodendrocytes to glutamate and sodium chloride | 69 |
| 2.7.3 Immunocytochemistry | 69 |
## CHAPTER 3 RESULTS
### 3.1 Intracortical perfusion of glutamate *in vivo*
| Subsection | Page |
|---------------------------------------------------------------------------|------|
| 3.1.1 Histology - light microscope | 70 |
| 3.1.2 The effect of intracortical perfusion on the distribution of tau, MAP2 and MAP5 immunoreactivity in neurons | |
| Tau | 74 |
| MAP2 and MAP5 | 78 |
| 3.1.3 MAPs in oligodendrocytes following intracortical glutamate perfusion | |
| Tau | 78 |
| MAP2 and MAP5 | 84 |
| 3.1.4 Electron microscopy | 85 |
### 3.2 Human post-mortem brain studies
| Subsection | Page |
|---------------------------------------------------------------------------|------|
| 3.2.1 Tau-positive glial cells following severe head injury | 87 |
| 3.2.2 Tau-positive glia following stroke | 90 |
| 3.2.3 Identification of tau-positive glia | 94 |
### 3.3 Tau-positive oligodendrocytes following permanent focal cerebral ischaemia in the rat
| Subsection | Page |
|---------------------------------------------------------------------------|------|
| 3.3.1 MAP2 as a marker for ischaemic damage | 95 |
| 3.3.2 Time course of increased tau immunoreactivity in oligodendrocytes following permanent MCAO in the rat | 97 |
| 3.3.3 Neuronal distribution of MAPs following permanent focal cerebral ischaemia in the rat | |
| Tau | 102 |
| MAP2 | 106 |
| MAP5 | 106 |
| 3.3.4 Mechanisms underlying increased tau immunoreactivity in oligodendrocytes following focal cerebral ischaemia | |
| Ipsilateral hemisphere | 110 |
| Contralateral hemisphere | 112 |
3.4 The effect of permanent focal cerebral ischaemia on the synthesis of tau in oligodendrocytes 114
3.5 Tau protein in an O-2A oligodendrocyte cell line
3.5.1 Western blot analysis of tau in oligodendrocyte cultures 118
3.5.2 Tau immunoreactivity in oligodendrocyte cultures
Tau immunoreactivity in control cultures 118
Monosodium glutamate dose response curve 120
Effect of 100µM monosodium glutamate and NaCl on tau immunoreactivity in oligodendrocyte cultures 120
CHAPTER 4 DISCUSSION
4.1 Detection of the neuronal protein tau in oligodendrocytes following acute brain injury
4.1.1 Distribution of tau in histologically normal brain tissue 124
4.1.2 Tau immunoreactivity in oligodendrocytes following acute brain injury 125
4.1.3 Tau in oligodendrocyte cell cultures 126
4.2 The induction of tau immunoreactivity in oligodendrocytes is a rapid response to acute brain injury 129
4.3 Mechanisms underlying the induction of tau immunoreactivity in oligodendrocytes
4.3.1 Glutamate toxicity *in vivo* induces tau immunoreactivity in oligodendrocytes 130
4.3.2 Induction of tau immunoreactivity in oligodendrocytes is a direct response of these cells to injury 132
4.3.3 The spin trap agent PBN reduces the number of tau-positive oligodendrocytes following focal cerebral ischaemia in the rat 133
4.4 Functional significance of tau in oligodendrocytes following injury
4.4.1 Protective response? 135
4.4.2 Degenerative response? 138
4.5 The neuronal distribution of MAPs following acute brain injury
4.5.1 Tau 139
4.5.2 MAP2 and MAP5 141
4.6 Oligodendrocyte versus neuronal tau following acute brain injury 142
Appendix 144
References 145
Publications 176
List of tables
| No. | Title | Page |
|-----|----------------------------------------------------------------------|------|
| 1. | Physiological variables - Glutamate toxicity study | 54 |
| 2. | Case details | 60 |
| 3. | Physiological variables - Time course study | 62 |
| 4. | Physiological variables - Drug intervention study | 63 |
| 5. | Physiological variables - In situ hybridisation study | 64 |
List of figures
| No. | Title | Page |
|-----|----------------------------------------------------------------------|------|
| 1. | Ischaemic cell change in neurons | 5 |
| 2. | Extracellular glutamate concentrations following MCAO in the rat | 6 |
| 3. | Schematic representation of the tau isoforms found in human brain | 18 |
| 4. | Morphological characteristics of neurofibrillary tangles and glial inclusions | 28 |
| 5. | Astrocyte and oligodendrocyte morphology *in situ* | 31 |
| 6. | Schematic representation of tau antibody specificity | 40 |
| 7. | Tau 1 immunoreactivity prior to and after dephosphorylation of tissue | 42 |
| 8. | Single and double label immunohistochemistry-negative controls | 44 |
| 9. | Gel showing digests used for tau $^{35}$S riboprobe preparation | 50 |
| 10. | Linearisation of plasmid DNA with SmaI | 51 |
| No. | Title | Page |
|-----|----------------------------------------------------------------------|------|
| 11. | Schematic representation of microdialysis probe placement in the parietal cortex | 55 |
| 12. | AD 2 immunoreactivity in Alzheimer brain | 59 |
| 13. | Schematic representation of the sections in which the number of tau-positive oligodendrocytes/mm² was determined in subcortical white matter. | 66 |
| 14. | Linear regression analysis of repeated measures | 67 |
| 15. | H&E stained coronal sections showing the area of pallor characterising the lesion induced by the intracortical perfusion of 1M monosodium glutamate or 1M NaCl | 71 |
| 16. | H&E stained coronal sections showing the area of damage resulting from the intracortical perfusion of artificial CSF | 72 |
| 17. | H&E stained coronal sections showing the subcortical white matter immediately underlying cortical lesions induced by the perfusion of 1M monosodium glutamate or 1M NaCl | 73 |
| 18. | Distribution of Tau 1 and AT8 immunoreactivity in rat cerebral cortex | 75 |
| 19. | The effect of alkaline phosphatase pre-treatment on the distribution of Tau 1 immunoreactivity in neurons and glia following intracortical perfusion of 1M monosodium glutamate or 1M NaCl | 76 |
| 20. | Distribution of B19, TP70 and TP007 immunoreactivity in rat cerebral cortex | 77 |
| 21. | Distribution of MAP2 and MAP5 immunoreactivity in neurons following intracortical perfusion of 1M monosodium glutamate or 1M NaCl perfusion | 79 |
| 22. | Tau immunoreactivity in glial cells of the subcortical white matter following 1M monosodium glutamate or 1M NaCl perfusion in the rat | 80 |
| 23. | Distribution of Tau 1 and AT8 immunoreactivity in the subcortical white matter following perfusion of 1M monosodium glutamate or 1M NaCl | 81 |
| 24. | Tau immunoreactivity in the subcortical white matter following alkaline phosphatase pre-treatment of histologically normal rat brain tissue | 82 |
| 25. | Confocal images showing Tau 1, GFAP and transferrin immunoreactivity in the subcortical white matter immediately underlying a cortical lesions induced by the perfusion of 1M monosodium glutamate or 1M NaCl | 83 |
| 26. | Distribution of MAP2 and MAP5 immunoreactivity in the subcortical white matter following intracortical perfusion of 1M monosodium glutamate or 1M NaCl | 84 |
| 27. | Electron micrographs showing oligodendrocyte morphology in the contralateral and ipsilateral subcortical white matter following intracortical perfusion of 1M monosodium glutamate | 86 |
| No. | Title | Page |
|-----|----------------------------------------------------------------------|------|
| 28. | Tau 1 and AD2 immunoreactivity within the corpus callosum from control patients and 6 h and 23 h following head injury | 88 |
| 29. | Distribution of TP70 and TP007 immunoreactivity in the corpus callosum of control and head injured patients 6 h and 23 h following head injury | 89 |
| 30. | Corpus callosum of control and head injured patients stained with H&E | 90 |
| 31. | Cingulate cortex from control and stroke patients stained with H&E | 91 |
| 32. | Distribution of TP70 and TP007 immunoreactivity in the cingulate cortex from control and stroke patients | 92 |
| 33. | Distribution of Tau 1 and AD2 immunoreactivity in the cingulate cortex from control and stroke patients | 93 |
| 34. | Identification of tau-positive glia in human post-mortem tissue | 94 |
| 35. | Photomicrograph of a rat brain section stained with MAP2 showing the area of ischaemic damage following 40 min MCAO | 96 |
| 36. | Tau immunostaining of subcortical white matter 40 min following MCAO in the rat | 98 |
| 37. | The number of tau-positive oligodendrocytes increased with time following MCAO | 99 |
| 38. | Identification of tau-positive glia present after MCAO as oligodendrocytes Tau immunoreactivity within neurons 40 min following MCAO in the rat | 100 |
| 39. | MAP2, MAP5 and c-fos immunostaining of subcortical white matter 40 min following the induction of focal cerebral ischaemia in the rat | 101 |
| 40. | Tau 1 immunoreactivity within neurons 40 min following MCAO in the rat | 103 |
| 41. | TP70 immunoreactivity within neurons 40 min following MCAO in the rat | 104 |
| 42. | TP007 immunoreactivity within neurons 40 min following MCAO in the rat | 105 |
| 43. | MAP2 immunoreactivity within neurons 40 min following MCAO in the rat | 107 |
| 44. | MAP5 immunoreactivity within neurons 40 min following MCAO in the rat | 108 |
| 45. | Effect of PBN pre-treatment on the number of tau-positive oligodendrocytes in the ipsilateral subcortical white matter 40 min following MCAO in the rat | 110 |
| 46. | Effect of MK801 and NBQX pre-treatment on the number of tau-positive oligodendrocytes in the ipsilateral subcortical white matter 40 min following MCAO in the rat | 111 |
| 47. | Effect of PBN pre-treatment on the number of tau-positive oligodendrocytes in the contralateral subcortical white matter 40 min following MCAO | 112 |
| 48. | Effect of MK801 and NBQX pre-treatment on the number of tau-positive oligodendrocytes in the contralateral subcortical white matter 40 min following MCAO | 113 |
| 49. | Distribution of tau mRNA in normal rat brain tissue | 115 |
| No. | Title | Page |
|-----|----------------------------------------------------------------------|------|
| 50. | Distribution of tau mRNA in normal rat brain tissue following MCAO in the rat | 116 |
| 51. | Distribution of tau mRNA in neurons following MCAO in the rat | 117 |
| 52. | Western blot analysis of tau in cultured oligodendrocytes, using TP70 and Tau 1 | 119 |
| 53. | Tau 1 immunoreactivity in oligodendrocyte cultures treated with 10µM, 100µM or 1mM monosodium glutamate for 4 h | 121 |
| 54. | Tau 1 immunoreactivity in oligodendrocyte cultures derived from an O-2A cell line | 122 |
| 55. | TP70 and TP007 immunoreactivity in oligodendrocyte cultures derived from an O-2A cell line | 123 |
**List of Abbreviations**
| Abbreviation | Description |
|--------------|-----------------------------------------------------------------------------|
| AD | Alzheimer's disease |
| APES | Amino propyltriethoxy-silane |
| AMPA | 2-amino-3-hydroxy-5-methylisoxazole-4-propionic acid |
| APP | amyloid precursor protein |
| BFGF | basic fibroblast growth factor |
| BSA | bovine serum albumin |
| CNS | central nervous system |
| CSF | cerebral spinal fluid |
| CNP | 2',3'-cyclic nucleotide 3'phosphodiesterase |
| DEPC | di-ethyl pyrocarbonate |
| DTT | dithiothreitol |
| GFAP | glial fibrillary acidic protein |
| GTE | glucose Tris EDTA |
| H & E | haematoxylin and eosin |
| HBSS | Hank's balanced salt solution |
| HRP | horse-radish peroxidase |
| IF | intermediate filament |
| MABP | mean arterial blood pressure |
| MAP | microtubule-associated protein |
| MCA | middle cerebral artery |
| MCAO | middle cerebral artery occlusion |
| MSA | multiple system atrophy |
| Abbreviation | Description |
|--------------|-------------|
| MBP | myelin basic protein |
| MK 801 | dizocilpine |
| NFT | neurofibrillary tangle |
| NMDA | N-methyl-D-aspartate |
| PHF | paired helical filament |
| PD | Parkinson's disease |
| PBN | α-phenyl-tert-butyl-nitrone |
| PBS | phosphate buffered saline |
| PDGF | platelet derived growth factor |
| PSP | progressive supranuclear palsy |
| PLP | proteolipid proteins |
| SDS-PAGE | sodium dodecyl sulphate polyacrylamide gel electrophoresis |
| SDW | sterile distilled water |
| TBI | traumatic brain injury |
| TAE | Tris acetate EDTA |
| TBS-T | Tris buffered saline |
Acknowledgements
I have thoroughly enjoyed the time I have spent working at the Wellcome Surgical Institute and would like to take this opportunity to thank everyone in the department for their support and friendship throughout the duration of my PhD.
I would like to extend my greatest thanks to Dr. Deborah Dewar for her continual support and guidance which gave me a source of constant encouragement and determination - to her I am indebted. My thanks also to Prof. James McCulloch for his regular input and interest in my research. Together they have instilled in me a high standard of experimental rigour that I am sure will stand me in good stead for the future.
I also extend a special thanks to Prof. Ian Griffiths, Department of Applied Neurobiology (SACS), for introducing me to oligodendrocytes and allowing me free access to his laboratory and technical staff! In particular I would like to thank Dr. Peter Dickinson, Mrs Jennifer Barrie and Mr E. Kyriakides for their help, expertise and constant patience. Thanks to all the members of staff for their friendship and generosity which made me feel part of the team - even though I'm not a vet!
Thanks to also to Dr. Susan C. Barnett, Dept. of Neurology, for her continuous supply of oligodendrocyte cultures and for finding me a bench in her busy laboratory. I must also thank Prof. David L Graham and Dr. James Nicoll for their expert histopathological advice and allowing me access to human brain tissue.
The technical expertise of the Wellcome Surgical Institute is gratefully acknowledged. I extend a special thanks to Ms. Marion Steele, Ms. Joan Stewart, Ms. Lindsey Gallagher and Ms. Margaret Stewart who often helped me out of a crisis. And to my colleagues, Ailsa McGregor, Dr. Gail MacDonald, Dr. Karen Horsburgh, Dr. Maihri MacCrae and David Paterson for both their invaluable advice and constant good humour. A special thanks goes to all the PhD students that I shot gunned into proof reading this thesis.
Last but by no means least I would like to thank all my friends and family. In particular my parents for their unconditional encouragement and moral (not to mention financial) support. Thanks also to my boyfriend Nick who after teaching me how to study as an undergraduate has had faith in me and encouraged me through all my University years.
Preface and Declaration
This thesis presents results from investigations conducted within 4 broadly defined areas:
1) To investigate the effect of acute brain injury on the distribution of the microtubule-associated proteins tau, MAP2 and MAP5 in oligodendrocytes.
2) To assess the mechanisms underlying the accumulation of tau immunoreactivity in oligodendrocytes following acute brain injury.
3) To determine if increased tau immunoreactivity within these cells reflected increased synthesis or protein modification.
4) To compare the distribution of tau, MAP2 and MAP5 within oligodendrocytes with that in neurons following acute brain injury.
Results from these studies are presented and discussed separately. The thesis comprises my own original work and has not been presented previously as a thesis in any form. Dr. K. Yatsuhiro performed the permanent middle cerebral artery occlusions. In vitro studies were carried out in collaboration with Dr. S.C. Barnett who kindly supplied the oligodendrocyte cultures. In situ hybridisation studies were carried out in collaboration with Dr. P. Dickinson. Both the electron microscopy and confocal microscopy were performed by Prof. I.R. Griffiths.
Abstract
With the growing evidence supporting glia-neuronal signalling it is becoming increasingly apparent that it is crucial to gain an understanding of the mechanisms underlying the degeneration of both neurons and glia to fully understand the pathogenesis of acute and chronic degenerative diseases of the brain. Breakdown of the cytoskeleton is thought to represent a common pathway mediating irreversible neuronal damage in a variety of both acute and chronic neurodegenerative conditions. One of the most well documented alterations to the cytoskeleton, occurring in various neurodegenerative disorders including Alzheimer's disease, Parkinson's disease and progressive supranuclear palsy, is the formation of neurofibrillary tangles. The formation of neurofibrillary tangles is thought to involve alterations in the microtubule-associated protein tau, the protein being hyperphosphorylated compared to its normal form. Tau is traditionally believed to be a neuron-specific protein where it is predominantly located within the axonal compartment. Supporting this specific localisation of tau, immunoreactivity was found only within axons in histologically normal rat or human brain tissue throughout the investigations carried out in this thesis. However, the novel finding of this thesis was the presence of tau immunoreactivity within oligodendrocytes in both rat and human brain tissue following acute brain injury. The studies described in this thesis were aimed at characterising the increased tau immunoreactivity present in oligodendrocytes in animal models of acute brain injury, in human post-mortem brain tissue from patients who died following a stroke or head injury and in an oligodendrocyte cell line.
In the initial study of the thesis tau-positive oligodendrocytes were present 4 h following glutamate-induced toxicity in the rat. In order to confirm that the induction of tau immunoreactivity in these cells following injury was not a phenomenon characteristic to the rat, human post-mortem brain tissue from acute brain injured patients was examined for the presence of tau-positive oligodendrocytes. Tau-positive oligodendrocytes were detected within areas of tissue, as defined by decreased haematoxylin and eosin staining, affected by brain injury, but not in tissue obtained from control patients who had no previous history of neurological or psychiatric disorders. Tau-positive oligodendrocytes were detected as early as 2 h following head injury, suggesting that this is a rapid response of these cells to acute brain injury. In support of this, the induction of tau immunoreactivity in oligodendrocytes was detected as early as 40 min following the induction of permanent focal cerebral ischaemia in the rat.
Glutamate toxicity has been shown to be involved in neuronal degeneration following ischaemic brain injury, and tau-positive oligodendrocytes were detected 4 h following intracortical perfusion of 1M monosodium glutamate, both within the resulting cortical lesion and in the white matter immediately underlying this area. This result implicated glutamate in the mechanisms underlying the accumulation of tau in oligodendrocytes following injury. However, the ability of NaCl to induce a similar response in oligodendrocytes suggested that mechanisms other than glutamate toxicity may be involved. Using pure oligodendrocyte cultures exposed to monosodium glutamate or NaCl it was shown that alteration of tau in these cells was a direct effect, not mediated through neuronal degeneration. Taken together the results of these studies indicated that some aspect of the brain injury *in vivo* other than glutamate receptor activation may be involved in the alteration of tau in oligodendrocytes. In order to investigate further the mechanisms which may be involved in this oligodendrocyte response, the effects of 3 pharmacological agents on the density of tau-positive oligodendrocytes following focal cerebral ischaemia in the rat were determined. Pre-treatment with the spin trap agent a-phenyl-tert-butyl-nitrone reduced the number of tau-positive oligodendrocytes by 55% in the subcortical white matter of the ischaemic hemisphere compared to untreated animals at 40 min after middle cerebral artery occlusion. In contrast, pre-treatment with glutamate receptor antagonists dizocilpine or 2,3-dihydroxy-6-nitro-7-sulpfamoyl-benzo(F) quinoxaline, failed to reduce the number of tau-positive oligodendrocytes following 40 min of ischaemia. Thus free radical mediated mechanisms were involved in the induction of tau immunoreactivity in oligodendrocytes following ischaemic brain injury.
In both the animal models of brain injury and the human post-mortem brain tissue obtained from patients who died following a stroke or head injury, tau-positive oligodendrocytes were detected with a range of antibodies to different epitopes of the protein. This suggested that there may be increased levels of the full-length protein. In order to investigate whether this increased immunoreactivity was due to increased protein synthesis, *in situ* hybridisation studies were performed in rat brain tissue obtained from the focal cerebral ischaemia model. However, these *in situ* hybridisation studies failed to detect increased tau mRNA within the ipsilateral white matter up to 4 h after the induction of focal cerebral ischaemia. This suggests that increased tau immunoreactivity in oligodendrocytes does not reflect increased *de novo* synthesis of the protein.
In contrast to the accumulation of full-length tau in oligodendrocytes, neuronal tau was dephosphorylated and/or degraded following acute brain injury in the rat. Dephosphorylation of tau in neurons was detected as early as 20 min following the induction of focal cerebral ischaemia in the rat. In addition, degradation of tau in neurons
but not in oligodendrocytes suggests that tau present in neuronal perikarya may be more susceptible to ischaemic brain damage than that in oligodendrocytes. However these results demonstrate that tau present in both neuronal perikarya and oligodendrocytes undergoes rapid alteration following ischaemic challenge. Glutamate excitotoxicity induced changes in tau similar to those occurring following focal cerebral ischaemia in the rat suggesting that while the accumulation of tau in oligodendrocytes is initiated through free radical mediated mechanisms, dephosphorylation and/or degradation of tau in neurons appears to be mediated through glutamate mediated mechanisms.
The results presented in this thesis provide compelling evidence that tau is present in oligodendrocytes and therefore can no longer be considered as a neuron-specific protein. Moreover these results show that tau undergoes rapid alteration within oligodendrocytes in response to acute brain injury which is initiated through free radical mediated mechanisms. Alterations in the distribution of microtubule-associated proteins in neurons following injury are thought to represent altered cytoskeletal integrity which may result in irreversible neuronal damage. It is possible to speculate therefore that alterations to tau in oligodendrocytes, following acute brain injury may also represent early stages of cytoskeletal breakdown within these cells. Oligodendrocytes have been until recently considered to be relatively unresponsive to ischaemic brain injury, and neuroprotective agents have been evaluated solely on their ability to reduce neuronal damage. One of the most successful strategies involves the blockade of glutamate receptor activation which was without effect in the reduction of the induction of tau immunoreactivity in oligodendrocytes following focal cerebral ischaemia. The results of this thesis may therefore have important consequences for the development of new therapeutic strategies, since protection of neuronal elements without the protection of glial cells would lead to delayed neuronal death rather than complete protection of the brain following acute brain injury.
CHAPTER 1 INTRODUCTION
1.1 General introduction
Until recently neurons were believed to be the most important cells in the functioning of the central nervous system (CNS) with neuroglia providing a purely supportive role. With this view much attention has been directed at elucidating the mechanisms underlying neuronal degeneration, in both acute and chronic degenerative diseases of the brain. Assuming that similar mechanisms underlie neuronal degeneration in both acute and chronic disorders, animal models of acute brain injury have been used to gain insight into such mechanisms in order to uncover suitable targets for therapeutic intervention. As a result, neuroprotective agents have been evaluated on their ability to reduce the extent of neuronal death following acute brain injury.
Glutamate is a major neurotransmitter in the CNS and is essential for neuronal signalling under normal conditions (Palmer and Gerhon, 1990; Salt and Herrling, 1991). In acute brain injury, including stroke and head injury, excessive glutamate receptor activation, subsequent calcium influx and free radical formation plays a pivotal role in neuronal degeneration (see section 1.2.3). It is now widely accepted that both astrocytes and oligodendrocytes possess glutamate receptors (for review see Gallo and Russell, 1995), suggesting that receptor activation in these cells may also occur during the presence of elevated extracellular glutamate concentrations. In neurons glutamate toxicity whether via calcium release and/or free radical toxicity, has been shown to be mediated largely through the activation of N-methyl-D-aspartate (NMDA) receptors and therefore many of the most effective neuroprotective agents have been directed towards NMDA receptor blockade. Astrocytes and oligodendrocytes however appear to lack NMDA receptor subtypes and glutamate toxicity has been shown to be mediated through non-NMDA receptors in these cells (Gallo and Russell, 1995; Patneau, et. al. 1994; Puchalski, et. al. 1994; Yoshioka, et. al. 1994). In light of this it is possible to envisage that although neurons are protected successfully by NMDA antagonists, glial cells remain at risk in conditions where there are high concentrations of extracellular glutamate.
The role of astrocytes in the uptake of glutamate and other neurotoxic substances and the proliferation of these glial cells following acute brain injury has been well documented. In contrast however, oligodendrocytes have been considered to be relatively unresponsive to acute brain injury. With the exception of the undisputed role of these cells in the pathology of multiple sclerosis, the elucidation of possible roles for oligodendrocytes in the pathogenesis of both acute and chronic neurodegenerative conditions has therefore on the whole been neglected. However, the discovery that these glial cells possess glutamate
receptors suggest that oligodendrocytes may have a function in neuron-glial signalling and therefore play an important role in disease pathology (see section 1.5.5). A limited number of investigators have provided evidence that in addition to astrocytes and neurons, oligodendrocytes may have the ability to respond rapidly to ischaemic, hypoxic and traumatic brain injury (see section 1.5.7).
Cytoskeletal breakdown is thought to represent a final common pathway of neuronal degeneration in a variety of both acute and chronic degenerative conditions. Accumulation of tau within neuronal perikarya and altered distribution of the microtubule associated proteins tau, MAP2 and MAP5, may illustrate alterations in cytoskeletal integrity within these cells. Abnormal cytoskeletal profiles have recently been detected in both oligodendrocytes and astrocytes in various chronic degenerative diseases such as multiple system atrophy (MSA) and progressive supranuclear palsy (PSP) (see section 1.4.4). The distribution and density of these oligodendrocytes in MSA closely parallels the severity of the disease, in addition these cells can occur in areas where neuronal degeneration is absent. This suggests that the formation of these cytoskeletal inclusions in oligodendrocytes may represent an early and important event in the pathogenesis of this chronic degenerative disease.
Together these findings suggest that oligodendrocytes, once thought to be relatively inactive in disease, may play an important role in the progression of both acute and chronic disease of the CNS. In order to fully understand the pathogenesis of acute and chronic brain injury and to highlight areas for new therapeutic intervention, it is crucial to determine the response of these cells to brain injury, the possible mechanisms underlying such responses and the consequence of these responses to the structural integrity of the neuron. Any similarities between mechanisms underlying neuronal and oligodendrogial degeneration may lead to a target for drug intervention protecting both neuronal and glial elements of the CNS essential for effective and prolonged neuroprotection. The main focus of this thesis therefore was to examine the effect of acute brain injury on one particular cytoskeletal protein; tau in oligodendrocytes and the mechanisms through which such effects are mediated.
1.2 Acute brain injury
1.2.1 Stroke
Stroke results from a reduction of blood flow to a given area of the brain, normally caused by the blockage of a major artery, however, spontaneous intracerebral haemorrhage or decreased cardiac output are also minor causes of stroke. Cerebrovascular disease, accounts for approximately 10% of all deaths in the United Kingdom surpassed only by cancer and
heart disease. The incidence of stroke increases with age, however strokes can occur in all age groups (Bamford, et. al. 1990). For those that survive a stroke lasting for 24 hr, 50% remain permanently disabled with only 10% returning to normal activity.
Structural damage resulting from stroke or hypoxia can be limited to neurons (selective neuronal necrosis) or may also affect glia, nerve fibres and blood vessels (cerebral infarction) (Nedergaard, 1988). It is now widely accepted that neurons followed by astrocytes and oligodendrocytes are most vulnerable to hypoxia and stroke. The degree of infarction is dependant on the reduction of cerebral blood flow and the duration of hypoperfusion. A threshold for infarction of 35 % of normal cerebral blood flow or 17ml/100mg tissue has been found in sustained ischaemia (Jones, et. al. 1981). Normally infarction occurs within the territory of a given arterial supply, the extent of which is dependant on the efficiency of collateral blood supply to the affected area. Through the use of post-mortem brain tissue, the evolution of ischaemic brain damage has been well documented in man (Adams and Graham, 1988). At post-mortem, recent infarcts result in soft very swollen brain tissue and can be anaemic or haemorrhagic depending on whether or not some blood flow has been restored or if blood vessel necrosis has occurred. Histological assessment by haematoxylin and eosin (H&E) staining shows dead neurons present in the grey matter, recognised by intensely eosinophilic cytoplasm and a poorly stained nucleus with haematoxylin 4-6 h after cessation of blood flow. Areas of ischaemic brain damage are extremely difficult to define at survival times of less than 4 h following the onset of stroke. Within 12-25 h the infarct has a sharply delineated boundary due to decreased H&E staining of neuropil caused by a combination of the swelling of astrocyte processes and axons. The next stage of development occurs within 24 h and involves proliferation of microglia and astrocytes this proliferation extends to lipid phagocytes 1-2 weeks later which phagocytose the affected tissue. The grey matter becomes shrunken and granular and if the patient survives for several months following stroke the dead tissue is removed by phagocytosis and the infarct presents as a shrunken cystic lesion.
1.2.2 Head injury
Trauma is the largest cause of death of under 45 yr olds in the United Kingdom with head injury being largely responsible. In non-missile head injury a sudden acceleration or deceleration of the head is usually involved with or without skull fracture. The brain moves within the skull resulting in a number of shearing forces and impact of the brain against the bony protuberances of the skull. The majority of head injured patients make an uneventful recovery, however, others sustain significant irreversible brain damage to the extent that may leave them dependant on family members or institutions for the rest of their lives. In the UK alone 1 in 300 families has a member permanently disabled by head injury.
Brain damage resulting from head injury is classified by describing the damage as focal or diffuse or as primary or secondary. Primary damage involves fracture of the skull, cerebral contusions and diffuse axonal injury while secondary damage includes that due to intracranial haematoma, raised intracranial pressure, brain swelling, hypoxic brain damage or infection. Diffuse axonal injury is now widely accepted to represent the most important factor in the outcome of patients following non-missile head injury (Graham, et. al. 1995) often responsible for concussion up to and including post-traumatic coma (Blumbergs, et. al. 1989; Gennarelli, 1993). In this type of injury there is widespread disruption of axons due to the shearing stresses of acceleration/deceleration injury resulting in discrete lesions in the dorsolateral quadrant of the rostral brain stem and in the corpus callosum as well as diffuse damage to axons throughout the brain. Axonal damage can be detected histologically by H&E staining or silver staining and immunologically with anti-amyloid precursor protein antibodies (Gentleman, et. al. 1993; 1995; McKenzie, et. al. 1995) or anti-neurofilament antibodies (Christman, et. al. 1996; Grady, et. al. 1993). In patients surviving only a short time following head injury axonal swellings and axonal bulbs can be detected, however as survival time increases (several weeks) the most conspicuous feature is the presence of multiple clusters of microglia with a few axonal bulbs still detectable 2 months following injury.
Approximately 90% of fatal head injured patients show ischaemic cell change of varying degrees at post-mortem (Graham, et. al. 1978) suggesting that ischaemic brain damage plays pivotal role in patient outcome after head injury. 40% of these patients spoke following head injury implying that cerebral ischaemia may represent a secondary and therefore treatable event (Bullock and Teasdale, 1990). Ischaemic brain injury may therefore be crucial in the degree of brain damage sustained following both moderate and severe head injury. Cerebral ischaemia may be caused by intracranial haematoma or systemic insults such as hypoxia and hypotension which may result in raised intracranial pressure and therefore reduced cerebral perfusion. Further supporting a role for ischaemic brain damage in outcome after head injury, a body of evidence now exists showing that events at the time of the primary injury may leave the brain susceptible to ischaemic injury. This may occur through altered metabolism and function or by alteration of normal vascular reactivity, such that mild ischaemic insults which would normally be tolerated, may cause major ischaemic brain damage (Jenkins, et. al. 1989; Wei, et. al. 1980).
Figure 1. Photomicrograph of neurons displaying ischaemic cell change. Post-mortem tissue obtained from the cingulate cortex of a patient surviving 5 d following a stroke stained with H&E. Note the triangulation of the nuclei present in neurons and the eosinophilic cytoplasm of these cells. Neuropil staining is reduced, probably through swelling of axons, dendrites and astrocytic processes. This reduction in H&E staining of the neuropil clearly delineates the boundary between areas of tissue affected by ischaemic brain damage and histologically normal tissue. Scale bar = 100μm.
1.2.3 Mechanisms of acute brain injury
Glutamate toxicity
There is a large body of evidence supporting a role for glutamate excitotoxicity in the pathogenesis of cerebral ischaemia. In experimental models of cerebral ischaemia there is a marked, immediate rise in extracellular glutamate concentration, irrespective of the cause of the ischaemic episode (Beneviste, et. al. 1984; Bullock, et. al. 1990; Butcher, et. al. 1990; Faden, et. al. 1989). The largest elevation of 20 fold in the cerebral cortex and 80 fold in the caudate nucleus occurred following focal cerebral ischaemia in the rat (Butcher, et. al. 1990; Graham, et. al. 1990). This elevation peaks within 40 min after middle cerebral artery occlusion (MCAO) in the rat and probably results due to an increase release of glutamate from neurons and reduced uptake into both neurons and astrocytes (Drejer, et. al. 1985). This release appears to be dependent on the severity of blood flow reduction, elevation being triggered by blood flows below 20ml/100g/min (Shimada, et. al. 1989).
Levels of glutamate released within 10-30 min of focal cerebral ischaemia have been shown to be comparable with a glutamate concentration (60-500μM) which is toxic to neurons *in vitro* (Choi, 1987).
**Figure 2.** Extracellular glutamate levels are elevated following permanent MCAO in the rat. Microdialysis probes were placed in the cerebral cortex and dialysates collected every 20 min. Extracellular glutamate concentrations were increased approximately 20-fold 40 min after MCAO. Arrow represents the onset of focal cerebral ischaemia by permanent occlusion of the middle cerebral artery (MCA). O represent sham operated controls; □ represent proximal occlusion of the MCA; ▲ represent distal occlusion of the MCA. Reproduced from Butcher, et. al. (1990).
Since the initial discovery of the toxic potential of glutamate by Lucas and Newhouse in (1957), when they described the degeneration of retinal neurons in neonatal mice injected systemically with glutamate, it is now widely accepted that high extracellular concentrations of glutamate are neurotoxic (Rothman and Olney, 1986; Choi, 1991). Glutamate acts on 4 major types of receptor, the NMDA receptor, the 2-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor, the kainate receptor and metabotropic receptors (Collingridge and Lester, 1989; Monaghan, et. al. 1989; Watkins, et. al. 1990). The NMDA, kainate and AMPA receptors are linked to receptor activated ion channels while metabotropic subtypes are thought to be linked to phosphoinositide metabolism as a second messenger system (Schoepp, et. al. 1990). Under normal conditions, AMPA and kainate receptors which are permeable to Na⁺ and K⁺ (there are also some reports of Ca⁺⁺
permeability, see below) are responsible for fast excitatory synaptic transmission in the CNS since NMDA receptors are blocked through a voltage dependant blockade by Mg\(^{++}\) (MacDermott and Dale, 1987; Mayer and Westbrook, 1987).
Glutamate toxicity has been shown to involve both a sodium chloride and a calcium dependant component (Dessi, et. al. 1994). Cellular swelling following glutamate toxicity is thought to result from the influx of Na\(^+\) through the activated NMDA receptor channel, which subsequently leads to an influx of Cl\(^-\) and water through this channel due to the osmotic properties of these ions. Excessive activation of NMDA receptors leads to toxic levels of intracellular calcium (Choi, 1987; 1991; MacDermott, et. al. 1986; Michaels and Rothman, 1990). Depolarisation frees the Mg\(^{++}\) channel block allowing the passage of Ca\(^{++}\) through the receptor mediated channel, voltage-gated calcium channels and results in the mobilisation of intracellular Ca\(^{++}\) stores. Although, originally believed to be impermeable to Ca\(^{++}\), a large body of evidence has accumulated showing that certain configurations of non-NMDA receptor subunits form ionophores permeable to Ca\(^{++}\) (Hollman, et. al. 1991; Murphy and Miller, 1989; Murphy, et. al. 1992; Ogura, et. al. 1990; Ozawa, et. al. 1991). Activation of non-NMDA receptors therefore may also contribute to calcium influx in pathological circumstances. Although the exact mechanisms involved in glutamate excitotoxicity are not fully understood, most evidence to date suggests that the influx of Ca\(^{++}\) is probably largely responsible (Siesjö, et. al. 1991; Müller, et. al. 1992).
Calcium plays an important role in many normal cell functions including the control of membrane permeability, modulation of neurite extension and synapse formation, triggering neurotransmitter release and the regulation of gene expression such as the induction of immediate early genes. In addition Ca\(^{++}\) modulates the activities of various enzymes including calcium-dependant phosphatases, kinases and proteases and therefore plays a role in the maintenance of neuronal structure, since cytoskeletal proteins are substrates for these enzymes (for review see Siesjö, 1988). Intracellular and extracellular Ca\(^{++}\) concentrations of neurons are around \(10^{-7}\) and \(10^{-3}\)M respectively (Choi, 1988) the intracellular concentration being strictly controlled by an ATP-dependent Ca\(^{++}/\text{Na}^+\) exchange (Mayer and Miller, 1990) for normal cellular function. Under pathological conditions such as ischaemia or head injury however, where excessive glutamate receptor stimulation is thought to occur, the resultant influx of Ca\(^{++}\) results in the loss of calcium homeostasis. Calcium "overload" in the cell results in abnormal activation of proteases, such as calpain, resulting in the breakdown of cytoskeletal proteins, activated nucleases result in DNA fragmentation, while activated lipases result in membrane damage and the production of free radicals. Together these events ultimately result in cell death (Siesjö, 1988; Siesjö and Bengtsson, 1989).
In addition to the increase in glutamate release, the most compelling evidence for a role of glutamate receptor activation in the pathogenesis of cerebral ischaemia is the large number of studies carried out, in various species, which show significant reduction in infarct size following treatment with glutamate receptor antagonists (for review see McCulloch, et. al. 1991). It is now widely accepted that glutamate antagonists reduce the volume of infarcted tissue following cerebral ischaemia independent of the species or model of ischaemia involved. The non-competative NMDA antagonist dizocilpine (MK801) and the 2,3-dihydroxy-6-nitro-7-sufomyl-benzo(F)quinoxaline (NBQX) have shown the largest and most reproducible reduction in infarct volume (Gill, et. al. 1992; Park, et. al. 1988). In the rat, pretreatment with MK801 reduced the volume of ischaemic brain damage by more than 50%, a similar reduction was also seen when administered up to 2 h following the onset of ischaemia (Gill, et. al. 1992; Park, et. al. 1988). Pre-treatment with MK801 (0.5mg/kg) 30 min prior to MCAO in the rat significantly reduced the volume of infarcted tissue (Park, et. al. 1988). Similarly pre- or post- treatment of rats with NBQX reduced the volume of infarcted tissue following MCAO in the rat (Gill, et. al. 1992) however this reduction was not a great as that of MK801. NBQX is selective antagonist at the AMPA and kainate preferring receptors with little or no affinity for the NMDA receptor complex (Sheardown, et. al. 1990).
**Free radical mediated toxicity**
Free radicals are atoms that have an orbital with an unpaired electron and are formed under normal conditions during reduction and oxidation reactions. Some free radicals are highly reactive and are able to extract an electron from neighbouring molecules in order to fill the vacancy in their orbital. Reactions such as these can damage a variety of critical biological molecules including DNA, proteins and lipids (Halliwell & Gutteridge, 1985). Superoxide radicals ($\text{O}_2^-$), hydrogen peroxide ($\text{H}_2\text{O}_2$) and the hydroxyl radical ($\text{OH}^-$) are produced through the reduction of molecular oxygen to water by oxidative phosphorylation. Free oxygen can accept ions from transition metals such as iron that can exist in many different valence states and therefore donate, or accept, electrons in redox reactions resulting in radical formation. Neither the superoxide radical or hydrogen peroxide is very reactive, and it is the hydroxyl radical, formed from hydrogen peroxide in a reaction catalysed by a transition metal such as iron, which is thought to be the mediator of radical toxicity. The hydroxyl radical has the ability to combine with most biological molecules, and therefore results in widespread oxidative damage (for review see Olanow, 1992).
Under normal conditions, a series of naturally occurring antioxidant defense mechanisms, prevent or limit free radical production and tissue damage (Rose and Bode, 1993). In pathological conditions such as ischaemia and trauma, an imbalance of the equilibrium of
production and protection, favouring free radical production is defined as a state of free radical stress. Cellular injury appears to increase the availability of metal ions, therefore once cellular injury has begun, damaging radical reactions tend to be amplified, especially during reperfusion when oxygen is exposed to an excess of metal ions (Choi, 1988; Hall and Braughler, 1989; Siesjö, et. al., 1989). For several biochemical, physiological and anatomical reasons the nervous system may be particularly susceptible to free radical injury compared to other organs of the body. These include a high rate of metabolic activity, high concentrations of readily oxidisable substrate, in particular membrane lipid polyunsaturated fatty acids, and low levels of intrinsic free radical scavengers such as catalase and glutathione peroxidase. Membrane disruption and destabilisation of calcium homeostasis have been proposed as major mechanisms by which free radicals induce neuronal damage (Boobis, et. al. 1989; Braughler, et. al. 1985; Orrenius et. al. 1989; Siesjo, et. al. 1981). However, the mechanisms of free radical release, the mode of action of radicals, including the molecular targets and the sequence of events that lead to cell degeneration remain largely undefined.
Although the concept of free radical mediated brain damage is widely accepted, it has proved difficult to obtain definitive evidence of free radical-mediated damage following ischaemia. Watson et. al. (1984) provided some evidence of diene conjugation in unsaturated fatty acids, which is generally accepted as the first structural manifestation of free radical damage, however, other studies failed to show significant levels of lipid peroxidation. Negative results obtained could be explained by the fact that lipid peroxidation is neither a prominent nor early sign of free radical damage (Halliwell, 1987). Superoxide ions and OH$^-$ have been shown to be generated in trauma and ischaemia (Hall, et. al. 1993; Kontos, 1989; Sen and Phillis, 1993; Zini, et. al. 1992). An important role for free radicals in ischaemic brain damage was suggested by Floyd, et. al. (1990) and by Oliver, et. al. (1990) who described the presence of oxidated proteins in gerbils subjected to transient ischaemia, however no oxidated proteins were detected in the dog (Folbergrová, et. al. 1993). Pretreatment with free radical scavangers such as dimethylthiourea and allopurinol and the enzymes superoxide dismutase or catalase have been shown to moderately reduce the extent of ischaemic brain damage (Liu, et. al. 1989; Martz, et. al. 1989). However the most compelling evidence implicating a role for free radical release in the genesis of ischaemic brain injury comes from studies showing significant reductions in brain damage following both transient (Folbergrová, et. al. 1995; Phalmark and Siesjö, 1996; Phillis and Clough-Helfman, 1990) and permanent focal (Cao and Phillis, 1994) cerebral ischaemia by the spin trap agent α-phenyl-tert-butyl-nitron (PBN).
PBN is one of the most widely used compounds for the detection of reactive free radicals *in vitro*. Spin trap agents are nitronе or nitroso compounds with which all forms of free radicals can react producing long lived nitroxide compounds. PBN is rapidly absorbed when injected intraperitoneally in rats, with the plasma level peaking within 15 min. Brain levels of PBN peak around 30 min after administration and decrease steadily over the next 8 h (Chen, et. al. 1990). The brain concentration of PBN is much higher than that in the blood due to the lipophilicity of the compound (Cheng, et. al. 1993). PBN has been shown to facilitate post-ischaemic reperfusion (Schultz, et. al. 1995) and improve energy status following transient ischaemia in the rat (Folbergrová, et. al. 1995) both of which could be involved in the mechanisms of tissue salvage following ischaemic injury.
**Interaction of glutamate toxicity and free radical damage**
Both glutamate excitotoxicity and free radical release have been implicated in the pathogenesis of cerebral ischaemia as described above and have until recently been independently investigated. However, observations suggest that the mechanisms of free radical damage and glutamate excitotoxicity may be at least partially interdependent. Glutamate toxicity has been shown to be markedly reduced *in vitro* (Bondy and Lee, 1993; Dykens et. al. 1987; Murphy, et. al. 1990; Miyamoto, et. al. 1989; Nakao, et. al. 1996) by various antioxidant agents and *in vivo* using PBN (Nakao, et. al. 1996; Schulz, et. al. 1995) suggesting that oxygen-free radicals contribute to glutamate toxicity. Both the glutamate antagonist MK801 and the antioxidant dimethylthiourea have been shown to reduce ischaemic brain oedema in rats indicating that both excitatory and free radical mediated mechanisms are involved in this process. Combination therapy with these two agents failed to show any significant additive effect in oedema reduction (Oh and Betz, 1991) further suggesting that glutamate toxicity and oxidative stress may be acting through similar mechanistic pathways.
Increased extracellular glutamate has been shown to inhibit the uptake of cystine from the surrounding medium into neurons through the action of the cystine/glutamate transporter (Bannai, 1986). Cystine is metabolized to cysteine within the cell which is a precursor for glutathione synthesis, reduced cystine uptake therefore results in glutathione depletion within these cells. Glutathione is an intrinsic free radical scavenger (for review see Bast, 1991) therefore its depletion renders the cell vulnerable to oxidative stress (Murphy, et. al. 1990; Miyamoto, et. al. 1989). Similar mechanism have been postulated to mediate glutamate excitotoxicity in oligodendrocyte cell cultures (Oka, et. al. 1993; Yonezawa, et. al. 1996) (see below). Free radicals have been shown to inhibit the uptake of glutamate into astrocytes (Volterra, et. al. 1994a; 1994b). Astrocytes are responsible for the uptake of glutamate and other substances that are toxic to the neuron (Hertz, et. al. 1992; Schousboe,
et. al. 1988), therefore inhibition of glutamate uptake will have profound effects on the extracellular glutamate levels in pathological conditions. In addition, free radical generating systems such as xanthine/xanthine oxidase, have been shown to cause increased release of glutamate from rat hippocampal slices *in vitro* (Pellegrini-Giampietero, et. al. 1990). Together these results give evidence for the existence of a "vicious circle" between glutamate toxicity and oxidative stress in the pathological conditions such as cerebral ischaemia.
### 1.2.4 Animal models of acute brain injury
As described above stroke and head injury are multi-factorial insults, it is impossible therefore to encompass all the events in a single animal model. However many models have been devised in an attempt to explore individual events. These models include fluid percussion, cortical impact, optic nerve stretch, subdural haematoma, glutamate toxicity and global and focal ischaemia. For the purpose of this thesis however I will focus on a model of permanent focal cerebral ischaemia and a model of glutamate toxicity, as these models were used in the studies reported here.
**Permanent focal cerebral ischaemia**
Models of focal cerebral ischaemia in the rat have gained increasing recognition due to their relation to events occurring in human stroke. In humans the most common cause of stroke is temporary or permanent occlusion of a major artery, usually the middle cerebral artery (MCA) (Brierly and Graham, 1984). Permanent and transient occlusion of the MCA in the rat can be carried out by a variety of methods (for review see Ginsberg and Busto, 1989). The subtemporal approach of Tamura, et. al. (1981) and electrocoagulation of the MCA described briefly in section 2.6.2, has become the gold standard method of permanent proximal occlusion of the MCA. In the rat this results in reduced blood flow within the territory supplied by the MCA including most of the caudate nucleus, the lateral segment of the globus pallidus, much of the cerebral cortex, the internal capsule and adjacent subcortical white matter. Permanent occlusion results in cerebral infarction within this area. At the periphery of this region there is an area where blood flow is reduced, but is not below critical levels termed the "penumbra" (Nedergaard, 1988; Tamura, et. al. 1981; Siesjö, 1992). The boundary between viable and non-viable tissue in this model is well defined making volumetric analysis possible. This model is therefore favoured for the volumetric quantification of neuroprotection given by drugs and has been used in such investigations in the rat and other animal species (for review see McCulloch, et. al. 1991).
Histological assessment of brain tissue can not accurately be performed less than four hours following MCA occlusion (MCAO), however immunological staining using an antibody directed towards MAP2 can be used to delineate ischaemic damage at shorter survival
periods (Dawson and Hallenbeck, 1996). Following 4 hr MCAO conventional H&E staining of tissue reveals swollen or lysed cells in the core of the MCA territory, they appear light due to their inability to stain with haematoxylin. In surrounding areas where blood flow is reduced shrunken and pyknotic neurons are apparent. Twenty-four hr following MCAO all of this territory may become infarcted (Graham, et. al. 1993). Most of the swollen cells disappear 24-48 hr after MCAO, small eosinophilic cells are found in the penumbra, and the lesion area is clearly delineated from normal tissue.
**Glutamate toxicity in vivo**
Glutamate toxicity is involved in neuronal death following cerebral ischaemia (see section 1.2.3), and many *in vitro* models of excitotoxicity have been employed to decipher the pathological progression of neuronal degeneration. However *in vitro* experiments lack the cellular complexity of the intact adult CNS, therefore various animal models have been devised to examine the effects of glutamate toxicity. These models allow the examination of the progression of neuronal death and the effect of glutamate antagonists on neuronal survival without the complications of reduced energy metabolism and blood flow present in ischaemia (for review see Meldrum, 1990). In such studies, selective excitatory amino acid receptor agonists are used to produce excitotoxic lesions, which avoids significant uptake by intrinsic mechanisms and interference with CNS metabolism. However in this thesis the use of monosodium glutamate was chosen for the induction of a cytotoxic lesion in the rat cortex to examine the effects of glutamate toxicity on cytoskeletal proteins. This model of glutamate toxicity has been described previously (Fujisawa, et. al. 1993a; 1993b; 1996; Landolt, et. al. 1993) and involves retrograde dialysis of glutamate into the cortex resulting in histological alterations consistent with excitotoxic neuronal damage (see section 3.1). The perfusion of 1M monosodium glutamate results in a sizeable lesion which extends in anterior and posterior directions and is characterised by an area of pallor as shown by haematoxylin and eosin staining. The histopathological features of the lesion include loss of H&E stained neurons, triangulation of the nucleus, and shrinkage of neurons and neuropil. A sharp boundary between the area of the lesion and histologically normal tissue can be readily detected in H&E stained sections (Fig. 15). In contrast, intracortical perfusion of the control solution, 1M sodium chloride, results in a smaller area of pallor on H&E stained sections compared to that resulting from glutamate perfusion. The boundary between the area of pallor and histologically normal tissue in the NaCl perfused brains was less well delined than in glutamate infused animals (Fig. 15). Within the core of a sodium chloride induced lesion, the number of H&E stained neurons are reduced compared to histologically normal tissue, while towards the edge of the lesion nuclei are triangulated.
1.3 Structure and function of the neuronal cytoskeleton
1.3.1 Components of the neuronal cytoskeleton
As mentioned above, cytoskeletal proteins are excellent substrates for calcium activated proteases therefore leaving the cytoskeletal vulnerable to ischaemic injury. Cytoskeletal breakdown is thought to represent a final common pathway of irreversible neuronal degeneration in a variety of both chronic and acute degenerative diseases of the brain. Before I go on to discuss the effect of acute brain injury on these structural proteins I will briefly discuss the structure and function of the neuronal cytoskeleton. The cytoskeleton is a highly dynamic structure, involved in many essential cellular processes including cell movement, process elongation and intracellular transport. The cytoskeleton is unique to eukaryotic cells and consists of three major classes of cytoskeletal fibres: microfilaments; neurofilaments and microtubules (for review see Ludin and Matus, 1993).
Microfilaments
Microfilaments consist of actin fibres which are polymers of actin protein with a diameter of 6nm. Actin therefore plays a pivotal role in the shape and growth of axons and dendrites. At the leading edge of the elongating process is the growth cone which, through controlled polymerisation and rearrangement of the actin fibres is responsible for process extension. Microfilaments are not readily detectable in mature differentiated neurons, however actin is present concentrated beneath the axolemma.
Intermediate filaments
Intermediate filaments (7-11nm in diameter) are polymers of several intermediate filament (IF) proteins, which vary according to cell type, but are restricted to six classes of IF protein. Neurons of the central nervous system contain class IV proteins - the neurofilament proteins, NF-H (heavy chain; 115kDa), NF-M (medium chain; 110kDa) and NF-L (light chain, 60kDa). Axonal neurofilaments are connected together by numerous side arms in a rope like manner, in contrast to microtubules and microfilaments which are globular. Neurofilaments are present in axons often in association with microtubules, together they provide strength and rigidity to the axon.
Microtubules
Microtubules, so called as they are made of tubulin, are long cylindrical structures with a diameter of approximately 22nm. They are composed of tubulin heterodimers which consist of an α- and β-monomer, aligned longitudinally to form protofilaments which in turn join laterally to form a tube. In the neuron, microtubules are highly organised and have a defined polarity with respect to the microtubule organising centre, thus defining the polarity
of the cell. This results in a fast growing end called the plus end which is guanosine triphosphate-dependent and a slow growing end called the minus end (Wiesenber, et. al. 1976). They are very stable, but generally dynamic structures, which go through periods of growth at the plus end that are interrupted by episodes of sudden collapse (Mitchison and Kirschner, 1984). In addition to tubulin, microtubules contain minor protein components called the microtubule associated proteins (MAP). These are a heterogeneous group of proteins originally described to co-assemble with microtubules, but have now been shown to be a major determinant of microtubule function. Microtubules are essential for the formation and structural maintenance of neurites. The highly complex morphology of neurons correlates with the abundance of microtubules in this cell type (Bray, et. al. 1978; Matus, et. al. 1986). Axons and dendrites lack the capability for protein synthesis, therefore in addition to pre-synthesised proteins, organelles and ribosomes carrying mRNA essential for protein synthesis have to be transported to the axons and dendrites to allow local protein synthesis. These functions are all mediated through the microtubule framework of the cell in conjunction with motor proteins (Bloom, 1992).
1.3.2 The microtubule associated proteins
The MAPs appear to be the major determinants of microtubule function and all share the ability to bind to polymerised tubulin *in vitro*. In the neuron they can be separated into 2 groups. Firstly, the motor proteins such as kinesins and dyneins which are responsible for intracellular transport of organelles and proteins from the cell body to the axons and dendrites. This is an ATP dependant function essential for neuronal survival since axons and dendrites cannot synthesize proteins (Bloom, 1992). Secondly, the structural MAPs: MAP1A/B; MAP2a/b/c; MAP4 and tau, all of which share the ability to promote tubulin polymerisation and stabilise microtubules *in vitro* (Tucker, 1990; Weingarten, et. al. 1975) suggesting that they may regulate the state of assembly of tubulin in neuronal processes and influence the stability of microtubules. MAP expression is developmentally regulated and persists in areas of the brain in which neurons continue to divide, suggesting that they may play a role in neuronal plasticity (Burgoyne, 1986; Matus, 1988; Viercek, et. al. 1989). The structural MAPs of interest in this thesis are described in more detail below.
**MAP2**
MAP2 consists of several isoforms, produced through alternative splicing of a single gene transcript, referred to as: MAP2a (280kDa); MAP2b (270kDa) and MAP2c(70kDa). MAP2c mRNA encodes a polypeptide lacking amino acids 152-1514 of the MAP2 sequence (Doll, et. al. 1990). MAP2 has 2 domains: a 200kDa N-terminal projecting arm that forms regularly spaced 100nm lateral projections from the microtubule surface and a C-terminal microtubule binding domain. It has been proposed that the projection domains of these molecules are involved in the arrangement of microtubules by acting as mechanical struts maintaining regular spacing between microtubules (Weishaar and Matus, 1993). Alternatively, it is possible that the projection domain may play a role in the interaction of tubulin with MAP2 during microtubule assembly (Fellous, et. al. 1994). The tubulin binding domain of both the high and low molecular weight proteins contains a series of 3 or 4 imperfect 18 amino acid repeats each separated by 13-14 amino acids (Kindler and Garner, 1994), which act in conjunction with adjacent sequences to affect microtubule binding affinity (Lewis, et. al. 1988). MAP2 is a phosphoprotein and the degree of phosphorylation affects the affinity of the protein for microtubules. Extensive phosphorylation reduces its ability to stimulate microtubule assembly and to bind to preformed microtubule (Burns, et. al. 1984; Murthy and Flavin 1983) and this may be a means whereby MAP2 can modulate neuronal plasticity. MAP2a is present at very low levels in embryonic and newborn brain, but becomes the major MAP in adult brain. In contrast, MAP2c is the major MAP in embryonic brain, persisting in adult brain where differentiation continues (Binder, et. al. 1984; Burgoyne and Cumming, 1984; Przyborski and Cambray-Deakin, 1995). In normal brain MAP2a and b are predominantly localised in the cytoplasm of dendrites and cell bodies where they are found associated with microtubules (Bernhardt and Matus, 1984; Caceres, et. al. 1988). In contrast, MAP2c is also found in axons of the developing brain (Riederer and Matus, 1985). This differential pattern of distribution can be explained by the compartmentalisation of MAP2 mRNA through selective transport which allows regulation of MAP2 synthesis (Tucker, et. al. 1989). MAP2a and b mRNA is found localised in dendrites (Garner, et. al. 1988; Landry, et. al. 1994) while MAP2c mRNA is present in cell bodies (Papadrikopoalou, et. al. 1986). The use of antisense oligonucleotides has provided evidence for the involvement of MAP2 in neurite extension and the maintenance of existing neurites (Caceres, et. al. 1992; Sharma, et. al. 1994).
MAP5 (MAP1B)
The earliest structural MAP to appear in the developing brain is MAP5 which is already present in axons at the time of their emergence from the cell body (Tucker and Matus, 1988; Tucker, et. al. 1988). In contrast to MAP2 and tau, MAP5 lacks the 3-4 repeat region at the C-terminus, however microtubule binding is mediated through 2 repeat regions. The first consists of a motif composed of 2 basic amino acids followed by 2 acidic amino acids (Noble, et. al. 1989) near the N-terminus. The second is a set of 12 imperfect repeats of 15 amino acids each near the C-terminus. MAP5 mRNA is detected in perikarya of developing neurons suggesting that synthesis occurs in the perikarya and the protein is subsequently therefore transported into the processes (Tucker, et. al. 1989). The expression of MAP5 is most prominent in extending axons (Calvert and Anderton, 1985; Black, et. al. 1994) however, protein levels decrease following termination of neurite outgrowth (Tucker and Matus, 1988), with the exception of areas of the brain where neuronal growth is continuous (Tucker and Matus, 1988; Viereck, et. al. 1989). Phosphorylation of MAP5 occurs during brain development and appears to correlate with axon growth (Calvert, et. al. 1987; Fischer and Romano-Clarke, 1990) suggesting that the function of MAP5 in axonal growth may be regulated by phosphorylation (Tucker, 1990; Gordon-Weeks, 1991). In PC12 cells stimulated with nerve growth factor, neurite extension was inhibited by the presence of MAP5 anti-sense oligonucleotides providing direct evidence for a role of MAP5 in process extension (Brugg, et. al. 1993).
Tau
The microtubule associated protein tau has gained much interest since the discovery, using immunohistochemical studies, that tau is the major component of neurofibrillary tangles (NFTs) found in Alzheimer's disease (AD) (Delacourte and Defossez, 1986; Grundke-Iqbal, et. al. 1986; Kosik, et. al. 1986) (see section 1.4.3). Tau was the first MAP to be isolated and shown to stimulate microtubule assembly *in vitro* (Weingarten, et. al. 1975) and molecular cloning of tau has now confirmed that there are at least six isoforms arising from alternative splicing of a single transcript encoded by a single gene located on the long arm of chromosome 17 (Druben, et. al. 1984; Himmler, et. al. 1989; Neve, et. al. 1986). Multiple isoforms of tau exist, ranging in size from 352-441 amino acids in the human brain (Goedert, et. al. 1988; 1989; Himmler, 1989; Himmler, et. al. 1989; Kanai, et. al. 1989; Kosik, et. al. 1989a; Lee, et. al. 1988), due to the differential expression of 0, 1 or 2, 29 amino acid inserts in the N-terminal region and the expression of 3 or 4 imperfect tandem repeats, 31 or 32 amino acids in length in the C-terminal region of the protein (Lee, et. al. 1988; Goedert, et. al. 1989a; Himmler, 1989) shown in Fig. 3. Tau expression is developmentally regulated, the shortest form containing three repeats and no N-terminal insert, being present in very low levels in adult brain although is abundant in fetal brain.
(Goedert, et. al., 1989a,b). The longest tau transcript containing 4 repeats and a 58 amino acid insert is abundant in adult but not fetal brain. The switch from the short to longer forms of protein has been shown to occur in neurons *in vitro* when process extension is complete and synapses are forming, suggesting that this pattern of expression is probably involved in the maturation of neuronal processes (Smith, et. al. 1995; Pizzi, et. al. 1995a).
Recently an additional form of tau has been identified named "big tau", because of its higher molecular weight (110-120kDa). This isoform was originally found in the PNS (Geogrieff, et. al. 1991; Oblinger, et. al. 1991) and peripheral neuron-like cell lines (Drubin, et. al. 1988) and has recently been reported to be present in a wide range of peripheral tissues (Gu, et. al. 1996). Cloning of big tau cDNA showed that, compared to tau found in brain tissue, this tau contains additional inserts of 237 and 66 amino acids present in the middle of the protein (Couchie, et. al. 1992) or of 254 amino acids present in the N-terminal portion (Goedert, et. al. 1992), respectively in the mouse and rat. In addition big tau is transcribed from an 8kb mRNA, as opposed to the 6kb mRNA that encodes normal tau, generated through alternative splicing from the same gene, located on chromosome 17, that encodes normal tau. Another larger protein, 83kDa, reacting with tau antibodies has been detected within astrocytes (Couchie, et. al. 1988). Together this data suggests that there may be multiple isoforms of big tau present in neuronal and non-neuronal cells.
Tau is believed to be an elongated molecule with very little α-helix or β-sheet structure (Cleveland, et. al. 1977b; Lee, et. al. 1988) and a highly asymmetrical shape (Hirokawa, et. al. 1988). The most striking feature of the tau sequence is the presence of three or four imperfect tandem repeats of 31 or 32 residues located at the C-terminal which is thought to represent the microtubule binding domain (Brandt and Lee, 1993; Butner and Kischner, 1991; Lee, et. al. 1989). Ionic interactions between the basic residues of the tau protein within this repeat region and the acidic residues at the C-terminal of tubulin is partially responsible for the binding of tau to microtubules (Butner and Kischner, 1991). Tau isoforms containing four repeat regions have been shown to be more effective at binding microtubules than those with three repeats probably due to increased ionic interactions with tubulin (Goedert and Jakes, 1990). Recently however, studies carried out *in vitro* have implicated other regions of the tau protein in microtubule binding. Using fragments of tau, the regions flanking this repeat sequence have been shown to increase the affinity of tau for microtubules (Butner and Kischner, 1991; Gustke, et. al. 1994; Trinczek, et. al. 1995). In addition, Goode and Feinstein (1994) have demonstrated that intermediate regions between the repeat sequences have a higher affinity for microtubules than any of the repeat regions themselves. Together this data suggest that a complex interaction between tau and microtubules exists which cannot be explained purely through ionic interactions.
Figure 3. Schematic representation of the six tau isoforms found in human brain. Isoforms are formed through alternative splicing of a single tau transcript and exist due to the presence or absence of 1 or 2 inserts at the N-terminal (closely hatched shading) and the presence of 3 or 4 repeats in the C-terminal region (hatched shading). The number of amino acids in each isoform is indicated at the right of each diagram and are termed 4R, 58, 4R, 29, 4R, 0, 3R, 58, 3R, 29, 3R, 0, from top to bottom according to the nomenclature of Goedert and Jakes, (1990).
Tau contains many potential phosphorylation sites and exists as a phosphoprotein *in vivo* which contributes to the complex pattern of bands after SDS-PAGE. Phosphorylation of tau is the only post-translational modification of the protein known to date and is thought to be of functional importance, as it leads to a reduced affinity of tau for microtubules (Brandt and Lee, 1994; Lindwall and Cole, 1984; Trinczek, et. al. 1995). Fetal forms of tau which are more phosphorylated than adult forms have a decreased affinity for microtubules. Hyperphosphorylated tau present in NFTs (Gustke, et. al. 1992) lacks the ability to bind to and stabilise microtubules, however this ability can be restored following dephosphorylation of the protein (Iqbal, et. al. 1994). It has also been postulated that phosphorylation may be involved in the compartmentalisation of tau within cells (Kosik, 1990). Tau present in perikarya is phosphorylated which prevents microtubule binding and therefore allows tau to move rapidly by diffusion to the axon. However, once reaching the initial segment of the axon tau is dephosphorylated and becomes associated with microtubules, therefore entering the slow transport system that exists within axons, subsequently leading to its predominant location within the axon.
### 1.3.3 Normal distribution of tau in the CNS
Within the normal CNS the predominant localisation of tau within the axonal compartment of neurons is widely accepted (Binder, et. al. 1985; Brion, et. al. 1988; Kowall and Kosik, 1987; Migheli, et. al. 1988; Papasozomenos and Binder, 1987; Trojanowski, et. al. 1989). However the presence of tau in other cell types remains controversial. Tau has been detected in neuronal perikarya, oligodendrocytes and astrocytes following dephosphorylation of tissue sections or using phosphorylation independent antibodies (Migheli, et. al. 1988; Papasozomenos and Binder, 1987). In support of its presence in oligodendrocytes, tau was detected in primary ovine oligodendrocyte cultures using western blot analysis, immunohistochemistry and *in situ* hybridisation (Lopresti, et. al. 1995). In contrast, Vouyiouklis and Brophy (1995) have failed to show the presence of tau in rat primary oligodendrocytes cultures using an antibody directed towards neuronal tau. Discrepancies between these two studies may reflect differences in the species from which the oligodendrocyte culture were derived. In addition, the absence of tau in oligodendrocytes *in situ* may suggest that cultured oligodendrocytes have different properties to those *in situ*. Electron microscopy studies of the rat brain showed that tau immunostaining is found associated with microtubules in axons and in the cytoplasm of cell bodies (Migheli, et. al. 1988). In agreement with the immunohistochemical studies, *in situ* hybridisation showed that tau mRNAs occurred in neurons and that different neuronal subtypes express different isoforms (Goedert, et. al. 1989a; Kosik, et. al. 1989a). However, tau mRNA was not detected in glial cells supporting the predominant localisation of the protein in neurons. Within the neuron, tau mRNA is localised within the cell body.
and proximal part of the axon, this distribution being thought to be due to the interaction of mRNA with microtubules (Litman, et. al. 1993; 1994). Recently however, tau mRNA has been detected in ovine oligodendrocyte cultures *in vitro* (Lopresti, et. al. 1995) supporting the idea that tau protein may be present in non-neuronal cells within the CNS.
### 1.3.4 Cellular functions of tau
Information about the cellular functions of tau originated from studies employing purified microtubule preparations. These studies showed that tau promotes microtubule assembly (Cleveland, et. al. 1977a; Wiengarten, et. al. 1975) and is able to decrease the rate of transition between microtubule elongation and shortening (Dreschel, et. al. 1992; Pryer, et. al. 1992). Further evidence supporting a role for tau in microtubule binding has evolved from the use of cell lines lacking endogenous tau microinjected or transfected with human tau cDNA. In HeLa, 3T3 and rat fibroblast cells, tau expressed from transfected cDNA associated with microtubules, altered the post-translational modification of tubulin, induced microtubule bundling and protected microtubules against drug-induced depolymerisation (Drubin, et. al. 1984; Drubin and Kirschner, 1986; Lee and Rook, 1992; Lewis, et. al. 1989; Takemura, et. al. 1992). In CHO cells however, although tau expressed from transfected tau cDNA protected against drug-induced depolymerisation, tau had no effect on the monomer-polymer equilibrium (Barlow, et. al. 1994). Transfection of tau cDNA into fibroblasts was shown to change the organisation of microtubules and induced microtubule bundle formation similar to that of microtubule domains in nerve cells (Lewis, et. al. 1989; Kanai, et. al. 1992), although, no morphological changes were reported in these cells. In contrast to transfected fibroblasts, sf9 cells transfected with tau expressed in a baculovirus, produced neurite-like processes in which microtubule bundles were formed and orientated as in axons with their plus end distal to the cell body (Baas, et. al. 1991; Knops, et. al. 1991). Inhibition of tau expression with antisense oligonucleotides inhibited the development of axon-like structures in cultured neurons (Cáceres and Kosik, 1990) and in differentiated neuronal cultures resulted in the loss of axon-like structures (Cáceres, et. al. 1991). Together these results suggest that tau is actively involved in the elongation and maintenance of neuronal processes.
As well as its presence in neuronal perikarya and axons, tau has also been shown to be localised within the nucleus of human neuroblasts (Loomis, et. al. 1990; Wang, et. al. 1993). Nuclear tau has not yet been identified in mature neuronal cells suggesting that this may play a role in neuronal differentiation. The nucleus lacks microtubules therefore these results together with the non-microtubule associated localisation of tau to ribosomes (Papasozomenos and Binder, 1987) suggests that tau may have additional roles rather than purely binding to and stabilising microtubules. It is possible to speculate that tau may target
ribosomes to microtubules for transport in the somatodendritic compartments of neurons to facilitate local protein synthesis within axons and dendrites.
As discussed in the previous section (1.3.3), tau has recently been identified in oligodendrocyte cultures. Localisation of tau at the tips of oligodendrocyte processes suggests that, as in neurons, tau may be involved in process elongation in these cells. Oligodendrocytes have abundant microtubules in their cytoplasm which have been postulated to be key elements in the transportation of myelin basic protein (MBP) mRNA and other myelin components along cellular processes to the site of myelination (Barbarese, 1991; Brophy, et. al. 1993; Colman, et. al. 1982; Trapp, et. al. 1987). Co-localization of tau with MBP within oligodendrocytes (Lopresti, et. al. 1995) suggests that tau may interact directly with MBP possibly cross-linking MBP, its RNA and the microtubule.
1.4 Cytoskeletal breakdown in brain degeneration
1.4.1 Acute brain injury
In cerebral ischaemia and traumatic brain injury (TBI), increased extracellular glutamate concentrations and the subsequent elevation of intracellular Ca$^{++}$ are thought to trigger a cascade of neurochemical events resulting in irreversible neuronal damage. Breakdown of the cytoskeleton due to the activation of calcium activated proteases, such as calpain, is thought to be one of these events. Many elements of the cytoskeleton are substrates for calpain including NF (Banik, et. al. 1987; Schlaeffer and Zimmerman, 1985), MAP2 (Johnson, et. al. 1991) and tau (Kampfl, et. al. 1996). The majority of studies investigating the effects of ischaemia and TBI have concentrated mainly on the distribution of NF, tubulin and MAP2 following injury.
Selective degradation of the neurofilament proteins NF68, NF150 and NF200 has been shown to occur following both permanent and reversible cerebral ischaemia in the rat (Nakamura, et. al. 1992; Ogata, et. al. 1989). Decreased NF 150 and NF200 immunoreactivity could be detected as early as 10 min following the induction of cerebral ischaemia, at which time degradation of other cytoskeletal components such as actin, tubulin and NF68 was not apparent suggesting that these neurofilament proteins are extremely sensitive to ischaemic injury (Ogata, et. al. 1989). The loss of NF68 immunoreactivity within the CA1 region of the hippocampus has been suggested to lead to the selective vulnerability of this area to transient global ischaemia in the rat (Nakamura, et. al. 1992). Similarly decreased NF68 and NF200 immunoreactivity was decreased in the rat 3 h following cortical impact (Postmantur, et. al. 1994). In contrast to these studies however, NF68, NF150, and NF200 immunoreactivity were increased 1-72 h following fluid
percussion induced TBI in the rat (Yaghmait and Povlishock, 1992). It is possible to speculate that the discrepancies between these studies may reflect the type and severity of brain injury used. Decreased NF immunoreactivity was detected following severe cortical impact (Postmantur, et. al. 1994) however fluid percussion resulted in a mild TBI and an increase in NF immunoreactivity (Yaghmait and Povlishock, 1992). Interestingly increased NF68 immunoreactivity has been used to show early reactive changes in axons in human diffuse axonal injury. Axonal swellings can be detected using NF68 early following head injury before significant morphological changes can be detected using routine histological staining methods (Christman, et. al. 1994; Grady, et. al. 1993).
In both transient and permanent models of cerebral ischaemia in the gerbil, MAP2 and tubulin immunoreactivity have been reported to be particularly sensitive to ischaemic damage (Hatakeyama, et. al. 1988; Kitagawa, et. al. 1989; Yanagihara, et. al. 1990). In the rat however, decreased MAP2 immunoreactivity was not detected following transient cerebral ischaemia (Geddes, et. al. 1995; Tomoika, et. al. 1992). Loss of MAP2 immunoreactivity within dendrites and neuronal perikarya was however reported following permanent focal cerebral ischaemia (Dawson and Hallenbeck, 1996). Routine histological stains fail to allow accurate estimation of lesion size less than 4 h following MCAO in the rat, however decreased MAP2 immunoreactivity was clearly delineated 1h following MCAO allowing quantitative analysis of lesion size (Dawson and Hallenbeck, 1996) showing that loss of MAP2 immunostaining can be used as a sensitive marker of early ischaemic brain damage induced by MCAO in the rat. Similarly, MAP2 immunoreactivity has also been reported to be decreased in both neuronal perikarya and dendrites following TBI (Postmantur, et. al. 1996a; 1996b; Taft, et. al. 1992) within 3 h of injury. MAP2 is associated with dendrites and may therefore reflect the sensitivity of this neuronal compartment to brain damage. Antigenic changes in tubulin and MAP2 are consistent with changes in dendrite structure observed at the electron microscope level in the gerbil following transient cerebral ischaemia (Tomioka, et. al. 1992; Yamamoto, et. al. 1986).
In contrast, although tau has gained great interest through its presence in NFTs, few studies have investigated changes in tau following cerebral ischaemia. Tau has been reported to accumulate within neuronal perikarya following transient cerebral ischaemia in the rat (Geddes, et. al. 1994) with a loss of immunoreactivity in the axon, suggesting redistribution of the protein from the axon to the cell body. Two and 6 h following permanent focal cerebral ischaemia in the rat, increased tau immunoreactivity was detected using a phosphorylation dependent antibody but not with TP70 a polyclonal antibody raised against the C-terminal of tau, immunoreactivity of which is independent of phosphorylation state. This study indicated that dephosphorylation of existing tau protein in neuronal perikarya,
rather than accumulation (Dewar and Dawson, 1995). Discrepancies between these two studies may reflect the different models of cerebral ischaemia employed. In contrast to that present in NFTs (see section 1.4.1), tau present in perikarya following both types of cerebral ischaemic injury is dephosphorylated at the Tau 1 epitope (Dewar and Dawson, 1995; Geddes, et. al. 1994). This suggests that different mechanisms may underly changes in tau occurring in acute and chronic degenerative diseases, however the possibility that tau becomes phosphorylated over time cannot be ruled out.
Glutamate excitotoxicity is involved in the pathogenesis of cerebral ischaemia and has also been implicated in Alzheimer's disease, however its involvement in the alterations of microtubule associated proteins which occur in these conditions at present remains unclear. Increased MAP2 immunoreactivity was within perikarya following exposure of neuronal cultures to high concentrations of glutamate (Bigot and Hunt, 1990, Bigot, et. al. 1991), suggesting that mechanisms other than glutamate excitotoxicity may be involved in the decreased MAP2 immunoreactivity seen following cerebral ischaemia or TBI. In support of this, pre-treatment of animals with MK801 failed to prevent decreased MAP2 immunoreactivity following TBI in the rat (Lewén, et. al. 1996). The effects of glutamate toxicity on the neuronal distribution of tau remain controversial with some groups reporting the accumulated protein to have antigenic and biochemical properties similar to tau present in neurofibrillary tangles (Mattson, 1990; Sautiere, et. al. 1992; Sindou, et. al. 1994), while others report the tau protein to be dephosphorylated at the Tau 1 epitope (Bigot and Hunt, 1990; Davis, et. al. 1995; Pizzi, et. al. 1993; 1994; 1995a), which is phosphorylated in Alzheimer's disease. Dephosphorylation of the protein following glutamate toxicity is in accordance with the dephosphorylation of tau seen following cerebral ischaemia, suggesting that glutamate may be involved in the mechanism underlying neuronal alterations to tau seen following cerebral ischaemia but not NFT formation. Discrepancies regarding the nature of accumulated tau within neurons following glutamate exposure *in vitro*, compared to those observed *in vivo* may be partially explained by the maturity of the neuronal cultures. Cells are cultured from immature tissue and immature tau differs from the adult forms of the protein being similar to those present in neurofibrillary tangles (Bramblett, et. al. 1993; Watanabe, et. al. 1993; Yoshida and Ihara, 1993). Variation in the stage of culture maturity may therefore influence the data obtained from such experiments. This highlights the care that should be exercised when extrapolating data obtained *in vitro* to events occurring in the intact adult CNS where the cellular milieu is more complex.
Together these studies show that the neuronal cytoskeleton undergoes marked alteration rapidly following acute brain injury. It is possible to speculate that altered distribution of cytoskeletal proteins following ischaemic or traumatic brain injury may reflect loss of
cytoskeletal integrity which may subsequently result in irreversible neuronal damage. The rapid alteration of cytoskeletal proteins following acute brain injury has important implications for the development of effective neuroprotective strategies, since irreversible brain damage may be triggered extremely early following injury.
1.4.2 Chronic neurodegenerative diseases
Alzheimer's disease, originally described by Alois Alzheimer in 1907, is the most common cause of dementia in the elderly. It is characterised by the progressive loss of memory and other cognitive functions, however diagnosis can only be confirmed by the presence of extracellular and intracellular fibrous deposits in the post-mortem brain. These deposits are amyloid plaques and NFTs respectively. Abnormal processing of the amyloid precursor protein (APP) is thought to lead to amyloid deposition (Price and Sisodia, 1994; Yankner, 1996 for reviews) and much research has concentrated on the role of this protein in the pathogenesis of AD since the discovery of familial forms of AD showing mutations in the APP gene. However recent studies have shown that the distribution and abundance of NFTs, rather than amyloid deposits, closely correlates with the severity of dementia in life (Arriagada, et. al. 1992). The density of NFTs is greatest in the nerve cells of the cerebral cortex, hippocampal formation and some subcortical nuclei, which are the areas most commonly affected in the disease. Neuronal loss results in decreased synapse numbers and therefore disruption of neurotransmission occurs. The nucleus of Meynert is severely affected by NFT formation and its disruption leads to loss of cholinergic terminals within the neocortex, which is thought to be involved in the cognitive impairment displayed by these patients. In addition to their presence in AD, NFTs have also been reported to be present in degenerating neurons of patients suffering from other chronic degenerative conditions such as progressive supranuclear palsy (PSP) (Vermersch, et. al. 1994) and Parkinson's disease (Vermersch, et. al. 1993). In PSP neurofibrillary lesions are present in the absence of amyloid deposits suggesting that NFT formation may represent a common pathway in neurodegeneration present in a variety of chronic disorders of the brain.
1.4.3 Neurofibrillary tangles
Neurofibrillary lesions are found in cell bodies and apical dendrites as NFTs and in distal dendrites as neuropil threads. All the lesions consist of abnormal paired helical filaments (PHF) as their major fibrous component and straight filaments as a minor component. In electron micrographs PHF consist of two strands twisted in a helix with a periodicity of approximately 80nm and a width that varies from 8-20nm (Crowther and Wischni 1985). The straight filaments are found as a minor component in PHF preparations, they are 15nm wide and appear to lack the variation in width of the PHF. In contrast to those present in AD, NFTs in PSP are composed mainly of straight filaments with a diameter of 13-22nm.
(Montpetit, et. al. 1985). Despite the morphological differences, the biochemical properties of these two structures are very similar (Vermersch, et. al. 1994).
Immunohistochemical studies have suggested the presence of MAP2 (Six, et. al. 1992), MAP5 (Geddes, et. al. 1991; Ulloa, et. al. 1994a), ubiquitin (Morishima-Kawashima, et. al. 1993) and APP (Perry, et. al. 1993) in PHF. However the major constituent shown by both immunohistochemical and biochemical procedures, is tau protein (Brion, et. al. 1985; Grundke-Iqbal, et. al. 1986; Goedert, et. al. 1988; Wischik, et. al. 1988). Labelling of PHF with antibodies directed towards both the N- and C-terminal of tau protein suggests the presence of full length tau (Brion, et. al. 1991a; Goedert, et. al. 1992). More recently biochemical analysis showed that PHF tau runs as 3 major bands of 60, 64 and 68 kDa and a minor band of 72 kDa following sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Alkaline phosphatase treatment at high temperature resulted in the presence of 6 bands following SDS-PAGE showing that PHF contains all 6 tau isoforms in an abnormality phosphorylated state (Brion, et. al. 1991b; Greenberg, et. al. 1990). PHF tau is hyperphosphorylated, i.e. it is phosphorylated at more sites than normal tau and for each site a higher percentage than normal of tau molecules are phosphorylated. Sites identified through the use of phosphorylation dependant antibodies include threonine residues 185, 205 and 231 and serine residues 202, 396 and 404 (Biernat, et. al. 1992; Goedert, et. al. 1993; 1994; Otvos, et. al. 1995). Some of these sites are phosphorylated in fetal tau, therefore hyperphosphorylation of tau appears to occur through an exaggeration of normal phosphorylation mechanisms. Much research has therefore been concentrated on determining possible candidates for this abnormal phosphorylation. MAP kinase, GSK3 and cdc-2 kinase and calcium calmodulin dependant kinases have all been implicated in this process (Steiner, et. al. 1990; Goedert, et. al. 1995). The hyperphosphorylation of tau and subsequent lack of affinity for microtubules is thought to contribute to the formation of PHF. However, whether tau accumulation within neuronal perikarya occurs before or after phosphorylation is as yet undetermined. Loss of Tau 1 immunostaining (Tau 1 recognises tau dephosphorylated at ser 202) has been reported to occur prior to NFT formation (Bancher, et. al. 1989) suggesting that phosphorylation occurs before PHF formation. However, tau has also been shown to form PHF without prior phosphorylation (Crowther, et. al. 1994) and fragments of tau containing the repeat region which does not encompass the phosphorylation sites have also been shown to form filaments similar to PHF (Crowther et. al. 1992; Wilie, et. al 1992). In addition, phosphorylated tau transfected into COS-1 cells failed to form PHFs (Baum, et. al. 1995). The PHF core consists of the repeat region of tau leaving the N- and C-terminals in a disorganised pronase sensitive state which becomes pronase resistant following hyperphosphorylation (Crowther, et. al. 1992; Vincent, et. al. 1994). Recently Mena, et. al. (1996) have suggested that PHF formation
occurs in two stages. Firstly, the formation of tau complexes truncated at the C-terminal of the protein. Secondly, these complexes then bind together to form PHFs. It is probable therefore that phosphorylation is not the only factor involved in PHF formation.
The formation of PHF in neurons is thought to be involved in the degeneration of neurons in AD. Accumulation of NFT accompanies a decrease in the number of microtubules, decreased affinity for microtubule binding and altered microtubule morphology resulting in loss of microtubule function (Lu and Wood, 1993). As well as decreased affinity of PHF tau for microtubules, the presence of PHF tau appears to inhibit the binding of normal tau to microtubules possibly through binding to normal tau itself leaving it unavailable for microtubule binding (Alonso, et. al. 1994). Within neurons presenting NFTs there is often an accumulation of degraded mitochondria, lysosomes and multivesicular bodies. These organelles are transported along the axon by interaction with microtubules in an ATP dependant manner. This loss of vital functions is thought to lead to rapid degeneration of the cell.
In addition to genetic factors such as APP mutations leading to abnormal amyloid deposition (Price and Sisodia, 1994; Yankner, 1996 for reviews) factors including head injury (Roberts et. al. 1991), free radical damage (Evans, 1993; Friedlich and Butcher, 1994) and glutamate excitotoxicity (Choi, et. al. 1988; Greenamayre and Young, 1989) have been implicated to play a role in the pathogenesis of AD. Clinical investigations have shown that treatment of patients with antioxidant nutrients such as vitamin E and C, selenium and zinc have indicated a reduction in the progression of the disease (McLachlan, et. al. 1991). Furthermore age related increases in oxidatively-inactivated enzymes in the brain and impaired memory in aged gerbils has been reduced by the spin trap agent PBN (Carney, et. al. 1991). It has been postulated that abnormal energy production in the disease may result in increased susceptibility of cells to excitotoxic injury (Greenamyre, 1991). Thus excitotoxicity would occur at physiological levels of glutamate.
At present however, it is unknown if free radical damage and/or glutamate toxicity are involved in the mechanisms underlying NFT formation. As discussed in section 1.4.1, glutamate toxicity has been shown to increase tau in neuronal perikarya (Bigot and Hunt, 1990; Davis, et. al. 1995; Mattson, 1990; Pizzi, et. al. 1993; 1994; 1995; Sautiere, et. al. 1992; Sindou, et. al. 1994), however the antigenic properties of the accumulated protein remains controversial. Some groups report the presence of tau similar to that of PHF tau (Mattson, 1990; Sautiere, et. al. 1992; Sindou, et. al. 1994) while others report accumulated tau to be dephosphorylated at the Tau 1 epitope (Bigot and Hunt, 1990; Davis, et. al. 1995).
1.4.4 Glial fibrillary tangles
Argyrophilic oligodendroglial inclusions were first described by Papp, et. al. (1989) and since this time they have been used routinely in histological assessment of multiple system atrophy (MSA). These inclusions are most commonly detected within the cytoplasm, but have also been detected in the nucleus of the cell (Papp and Lantos, 1992). MSA is a chronic neurodegenerative condition comprising a combination of striatonigral degeneration (Adams, et. al. 1964), olivopontocerebellar atrophy (Duvoisin, 1984) and autonomic failure (Shy and Drager, 1960). Although glial inclusions have been used as a diagnostic marker for this disease for many years, it is only recently been recognised that the formation of these cytoplasmic inclusions may play a pivotal role in the disease pathogenesis. The density and distribution of oligodendroglial inclusions rather than neuronal degeneration correlates with the areas of affected brain and the severity of the disease (Papp and Lantos, 1994). Inclusion bearing cells are found mainly within regions in which myelinated fibres were affected by MSA lesions (Abe, et. al. 1992; Papp and Lantos, 1994) and also in areas of grey matter where little or no neuronal loss was detected, suggesting that neuronal degeneration is not required to induce inclusion formation within oligodendrocytes. The presence of these inclusion bearing cells in areas of tissue which appear normal, suggests that the formation of oligodendrocyte inclusions may be an early event in the degeneration characteristic of MSA (Abe, et. al. 1992; Papp and Lantos, 1994). Although the presence of oligodendroglial inclusions has been used in the diagnosis of MSA similar inclusions have also been described in post-mortem brain tissue of patients dying as the result of AD, PD and PSP (Iwatsubo, et. al. 1994; Nishimura, et. al. 1995; Yamada and McGeer, 1990). There are relatively few oligodendroglial inclusions compared to NFTs in AD and are therefore unlikely to be involved in the pathogenesis of this disease (Nishimura, et. al. 1995). In PSP, glial inclusions are found mainly within astrocytes, although oligodendroglial inclusions are detected in some cases (Iwatsubo, et. al. 1994; Nishimura, et. al. 1995; Yamada and McGeer, 1990). Cytoplasmic inclusions found within astrocytes in PSP are distinct from those occurring in MSA in that they can not be stained using ubiquitin antibodies (Yamada, et. al. 1992).
Neither the ultrastructure nor the biochemical composition of oligodendroglial inclusions is well defined. Some studies however have reported that these inclusions contain fibrils between 20 and 30nm in diameter, however whether these fibrils are microtubules or another form of cytoskeletal component remains controversial (Abe, et. al. 1992; Kato, et. al. 1991). In support of the presence of microtubules, oligodendroglial inclusions have been reported to stain positive for α and β-tubulin subunits (Abe, et. al. 1992; Aria, et. al. 1992; Kato, et. al. 1991). Independent of cell type or the underlying disease, immunohistochemical studies have shown that all forms of glial inclusion can be stained
with antibodies directed against tau (Murayama, et. al. 1992; Iwatsubo, et. al. 1994; Nishimura, et. al. 1992; Yamada and McGeer, 1990; Yamada, et. al. 1992) and antibodies directed towards PHF tau (Iwatsubo, et. al. 1994). Together this data suggests that tau may undergo similar modification in both neuronal and glial cells in chronic neurodegenerative conditions. Immunohistochemical studies have also shown the presence of MAP5 with oligodendrocyte inclusions present in MSA.
**Figure 4.** Photomicrograph showing NFT and GFT morphology. A) shows NFTs present in the hippocampus of a 85 year old female diagnosed with Alzheimer's disease at post-mortem stained with Bielschowsky modified silver stain. B) shows the morphological appearance of oligodendrocyte cytoplasmic inclusions, stained with an anti-ubiquitin antibody, from the pons of a 52 year old female who died with MSA. Scale bar = 50μm.
1.5 The neuroglial cells
1.5.1 Introduction
Recognition and characterisation of glial cells occurred long after the description of neurons. Neuroglia were first described by Virchow in 1846 as "nerve glue" which following the advent of electron microscopy on tissue from the central nervous system fixed by perfusion was found to consist of perikarya and processes. Knowledge of the morphologies of these cells emerged with the development of metallic impregnation techniques. The "astrocyte" was then identified and separated from other neuroglia and thus became the second element of the central nervous system next to the neuron (Cajal, 1913, 1916). In 1897 and 1899 Robertson was the first to identify the "mesoglia" cell, so named due to its different morphology compared to the commonly accepted neuroglial cell. This cell type was later characterised and named the "oligodendroglia" (del Rio-Hortega, 1919; 1921) comprising the third element of the central nervous system. Since these original characterisations the glial cells have been separated into the macroglia, consisting of the astrocytes and oligodendroglia and the microglia. Unlike neurons, macroglia have only one type of process and have the ability to divide throughout life.
1.5.2 Astrocytes
Of the 3 types of glial cells the astrocyte has been the most extensively studied. At the light microscope level astrocytes are recognised as star shaped cells whose processes extend into the surrounding neuropil. In white matter these cells have many fibrils and are referred to as fibrous astrocytes, however those in the grey matter have fewer fibrils and are thus called protoplasmic astrocytes. The astrocyte contains all normal organelles found in eukaryotic cells, however these are more sparse than in other glia. The most prominent cytoplasmic components of the astrocyte are filaments 8 nm in diameter which occur in the perikarya and extend into the processes. These filaments closely resemble intermediate filaments found in neurons, however their dimensions are smaller and they regularly appear as closely packed bundles. Glial fibrillary acidic protein (GFAP, MW 47kDa) one of the intermediate filament proteins is known to be the major component of these filaments (Eng and Bigbee, 1978). In the CNS, GFAP is unique to astrocytes and therefore antibodies raised against this protein are routinely used as specific cellular markers to distinguish these cells from other glia and neurons (Martin, et. al., 1991). Astrocytes can therefore be identified in the CNS at the light microscope level by both immunological and morphological assessment (see Fig. 5).
1.5.3 Microglia
Although microglia were detected in the early 1900's it was 1932 before del Rio Hortega classified the microglia. These cells are small compared to astrocytes, have an elongated or triangular nucleus and display many fine spine-like branches. Microglia are found in both white and grey matter, but are more abundant in grey matter, where they are often situated near neurons or blood vessels. In the normal adult brain microglial cells appear to be inactive, however become activated by any inflammatory or degenerative process. Once activated they move to the site of injury, where they turn into macrophages and remove the debris. At this time, the cells may assume a rounded or rodlike shape as they fill with lipids and cell constituents. They then transport the debris to the vicinity of blood vessels for clearance from the brain tissue.
1.5.4 Oligodendrocytes
As mentioned above the oligodendrocyte was first described by Robertson (1899) however it was decades later before this cell received its name and definitive description by del Rio Hortega (1921; 1928). The cell was described as having a small soma with sparse cytoplasm and in contrast to the astrocyte which shows an elaborate array of processes these cells have few (between 2-10) processes thus leading to the name oligodendrocyte meaning "cell with few branches". Since its original identification the oligodendrocyte has become well characterised both at the light and electron microscope level. Del Rio Hortega separated oligodendrocytes into two major classes according to the anatomical position: 1) perineuronal - where the cell body is adjacent to a neuron and located in the grey matter; 2) intrafascicular - oligodendrocytes positioned between nerve fibres in white matter. Electron microscopy has confirmed del Rio Hortega's descriptions (Stensaas and Stensaas, 1968), the mature oligodendrocyte is a small cell (10-20µm) in diameter with a dense nucleus and cytoplasm (Peters, et. al. 1976) due to the presence of heterochromatin and an unknown dense material occupying the cytosol. Oligodendrocytes can display different morphological profiles involving their cytoplasmic density and the clumping of nuclear chromatin, described as light, medium and dark (Mori and Leblond, 1970). These authors speculated that in the corpus callosum of young rats the different morphologies may reflect cells in different stages of maturation, with the larger light ones being the most actively dividing cells and the most mature being the small dark ones. There are no unique organelles within the oligodendrocyte cytoplasm, however they contain an abundance of ribosomes both free and associated with membranes. In contrast to astrocytes, oligodendrocytes lack GFAP containing filaments, however have an abundance of microtubules similar to those present in neurons.
At the light microscope level oligodendrocytes are morphologically distinct from astrocytes as they display a small perikarya with little cytoplasm. In contrast to astrocytes oligodendrocyte processes are not readily detected *in situ*. In vitro, oligodendrocytes can be easily identified by surface antigens which change through each stage of maturation. However, successful immunological markers for oligodendrocytes in tissue sections have been more difficult to develop. Oligodendrocytes have an abundance of transferrin which although is not unique to oligodendrocytes allows immunological detection of these cells at levels below the threshold for detection in other cell types. Anti-transferrin antibodies are therefore used routinely in conjunction with anti-GFAP status and morphology to identify oligodendrocytes *in situ* (Conner and Finne, 1986; Martin, et. al. 1991) (see Fig. 5).
**Figure 5.** Rat corpus callosum showing astrocytes immunostained with GFAP (a) and oligodendrocytes stained with transferrin (b). *In situ* astrocytes and oligodendrocytes can be easily distinguished by both immunological and morphological criteria. Astrocytes contain abundant intermediate filaments which consist mainly of GFAP, which in the CNS is unique to astrocytes. Immunohistochemical staining of tissue with antibodies raised against GFAP therefore provides a cellular marker for astrocytes (a). Oligodendrocytes contain an abundance of transferrin, therefore antibodies directed against this protein can serve as a good cellular marker in the CNS (b). Staining with these markers distinguishes these cells immunologically and also displays the cellular morphology of these cells. The oligodendrocyte has a small soma with sparse cytoplasm and processes are not readily detectable *in situ*. In contrast however, astrocytes have a larger soma and display an elaborate array of cellular processes (arrows). Scale bar = 50μm.
1.5.5 Oligodendrocyte function
Myelination
Myelination is the most well documented function of oligodendrocytes and detailed discussion of this process is outwith the scope of this thesis, however see McLaurin and Yong, (1994) for review. During development, axons in the CNS become ensheathed with myelin produced by oligodendrocytes, each cell being capable of myelinating many segments of different axons (Bunge, et. al. 1962; Bunge, 1968; Mathews and Duncan, 1971). Myelin is a highly complex and specialised extension of the oligodendrocyte plasma membrane, with each cell capable of making approximately three times its weight in membrane each day during active myelination (Norton and Poduslo, 1973). Myelin is composed mainly of lipids, none of which are unique to oligodendrocytes, however cerebroside is the most characteristic of oligodendrocytes and is often used as a cellular marker (Raff, et. al. 1978). Lipid accounts for approximately 70% of myelin the rest of which consists of protein, the major ones being proteolipid protein (PLP) and myelin basic protein (MBP) which account for 50% and 30% of total myelin protein respectively (Lees and Brostoff, 1984). More minor protein components include myelin-associated oligodendrocyte basic protein, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and myelin associated glycoprotein.
As the oligodendrocyte process encircles its axon, the cytoplasm is eliminated forming a compacted multilamellar sheath. Each cell generating myelin only provides myelin for a segment of the axon which therefore results in a discontinuous sheath with areas of axon uncovered called the nodes of Ranvier. At the node of Ranvier there is a high concentration of ion channels and this is the only point where the axon comes into contact with the extracellular fluid. As the nerve impulse passes along the axon they "jump" from node to node. This feature permits signal transmission to occur at a greater velocity, frequency and efficiency than unmyelinated fibers. Demyelination during disease such as multiple sclerosis therefore results in impaired signal transmission, leading to devastating neurological symptoms.
Inhibition of neurite outgrowth
Using video time lapse cinematography, Schwab and colleagues have provided evidence for a role of oligodendrocytes and myelin in the inhibition of neurite outgrowth. Astrocytes were shown to be a good substrate for neurite extension while contact with oligodendrocytes or myelin resulted in growth cone collapse (Schwab, et. al. 1988). Biochemical analysis has revealed two groups of proteins of molecular weights 35kDa and 250kDa that are uniquely found tightly bound to membrane proteins of CNS.
oligodendrocytes and have been implicated in the inhibitory action of oligodendrocytes to neurite extension (Schwab, et. al. 1993). These proteins are expressed late in development and may therefore play a role in guiding late developing CNS tracts in order to avoid abnormal sprouting and regulate axon number (Cadelli, et. al. 1992; Chiquet, 1989; Collelo and Schwab, 1994). The continued expression of these factors on oligodendrocytes following brain injury may therefore be a key feature in the failure of axonal regeneration. Biochemical analysis of these proteins has revealed two groups of proteins of molecular weights 35kDa and 250kDa that are uniquely found tightly bound to membrane proteins of CNS oligodendrocytes (Schwab, et. al. 1993).
**Neuronal-glial signalling**
The presence of glutamate receptor subtypes on oligodendrocytes and astrocytes (see section 1.5.7) raises the possibility that neurotransmitters released at the synapse or along axons can not only influence neurons, but also glial cells that are intimately associated with neuronal elements (for review see Finkbeiner, 1993). This raises the suggestion that signalling may occur between neurons and glia, via excitatory amino acids. In support of glutamate mediated signalling between neurons and glia, Dani, et. al (1992) showed that glial cells receive signals during neuronal activity in hippocampal organotypic cultures possibly mediated through glutamate. Excitatory amino acid receptor activation may result in the release of growth factor, neuropeptides, or neurotransmitters from glia (Parpura, et. al. 1994) and the release of such substances from glia may participate in activity-dependant plasticity in the adult brain (Kang and Schuman, 1995). Glutamate receptor activation has also been shown to increase *c-fos* and *c-jun* (immediate early genes) expression and inhibit proliferation of oligodendrocyte progenitors through non-NMDA receptor activation (Liu and Almazan, 1995; Pende, et. al. 1994). Repetitive axonal stimulation or treatment of the axons with glutamate in neonatal rat optic nerve also stimulated this response in oligodendrocytes (Mack, et. al. 1994). Immediate early genes encode transcription factors that regulate the expression of late genes which may result in permanent phenotypic alterations (Sheng and Greenberg, 1990).
Most studies to date have concentrated on the role of astrocytes in glia-neuronal signalling, for example K⁺ buffering. During neuronal activity K⁺ levels increase immediately surrounding the neuron, which may lead to prolonged depolarisation if not removed. Glutamate receptor activation in astrocytes during neuronal activity leads to increased intracellular Ca⁺⁺ within these cells resulting in K⁺ uptake through the activity of the Na⁺/Ca⁺⁺ exchanger and Na⁺/K⁺ pump (for review see Barres, et. al 1991). These Ca⁺⁺ signals can be propagated as waves through astrocyte gap junctions (Giaume, et. al. 1991). Recently Nedergaard (1994) showed that Ca⁺⁺ signals can pass from astrocytes to neurons
through gap junctions suggesting that neurons can signal to glia which in turn can signal back to neurons. Due to the presence of glutamate receptors on oligodendrocytes it is possible to speculate that these cells also have the capacity to respond to neuronal signals such as glutamate release. Glutamate receptor activation on these cells results in increased intracellular calcium suggesting that oligodendrocytes may also be capable of K⁺ buffering.
1.5.6 The oligodendrocyte cytoskeleton
In contrast to astrocytes, mature oligodendrocytes *in vivo* do not contain intermediate filaments, they do however contain large amounts of microtubules. Similarly to those found in neurons (see section 1.3.1) these microtubules are 25 nm in diameter and consist of tubulin dimers. They are arranged randomly throughout the perikaryal cytoplasm although in the processes they are arranged parallel to one another as in axons and dendrites of neurons. Since myelination involves the extension of long processes requiring substantial changes in the shape of the oligodendrocyte and given what is known about neuronal cytoskeletal functions it seems likely that the cytoskeleton of oligodendrocytes may play an important role in process extension. In support of this theory, tubulin has been identified as a constituent of myelin isolated from rat brain (Gozes and Richter-Landsberg, 1978). This protein is synthesized prior to myelination and has therefore been suggested to be involved in the formation and maintenance of myelin shape. In addition, purified MBP can form complexes with tubulin and actin (Modesti and Barra, 1986). Using cell culture both MBP and CNP have been shown to associate with the cytoskeleton of oligodendrocytes. CNP and MBP are synthesized on free ribosomes (Colman, et. al. 1982; Gillespie, et. al. 1989) and have to be transported into the processes for myelin formation. It is possible to speculate therefore that microtubular networks within these cells are essential for the transportation of the machinery required for myelin formation into the processes. In immature cells with many processes, CNP appears to localise with filamentous actin which is more abundant than microtubules at this stage. In more mature oligodendrocytes, where the number of processes is decreased and the diameter thickened, filaments appear to be lost and microtubules predominate to which MBP is found to colocalize (Wilson and Brophy, 1989). It is possible to speculate that actin filaments are responsible for the direction of growth however microtubules are involved in the elongation and thickening of processes.
The microtubule associated proteins as discussed in sections 1.3.2 play an important role in microtubule function in neurons. In neurons these proteins appear to be developmentally regulated and are thought to be involved in the formation and extension of axons and dendrites. Although traditionally believed to be neuron specific, MAP2, MAP4, MAP5 and tau have recently been identified in oligodendrocyte cultures (Diaz-Nido and Avila, 1989;
Fischer, et. al. 1990; Lopresti, et. al. 1995; Ulloa, et. al. 1994b; Vouyiouklis and Brophy, 1995). MAP2c expression in neurons is present during development which regresses and is replaced by the higher molecular weight isoforms. In oligodendrocytes a novel MAP2c isoform containing four repeat sequences is present in progenitor cells. This expression of MAP2c is higher in pre-oligodendrocytes than progenitors but decreased again when reaching the final stages of maturation suggesting that MAP2c is required with the transitional pre-oligodendrogial stage (Vouyiouklis and Brophy, 1995). MAP2c is therefore unlikely to be involved in stabilizing myelin processes but important in initiating the extension of processes. The heterogeneous nature of MAP2c may represent various phosphorylation states of the protein (Vouyiouklis and Brophy, 1995). Similarly to neurons, MAP5 expression is developmentally regulated and its appearance coincides with the development of pre-oligodendrocytes from progenitors (Vouyiouklis and Brophy, 1995), when the cells start to develop a network of extensive processes. MAP5 found in oligodendrocytes colocalizes with tubulin (Fischer, et. al. 1990) and has a phosphorylation pattern distinct from that in neurons (Ulloa, et. al. 1994). In contrast to neurons, however MAP5 levels appear to remain elevated in mature oligodendrocytes. Together these results suggest a role for MAP5 in the stabilization of the cytoskeleton and extension of processes in mature oligodendrocytes. The presence of the microtubule-associated protein tau in oligodendrocytes remains a controversial issue. Many groups have failed to detect the presence of tau in non-neuronal cells using antibodies directed towards neuronal tau (Brion, et. al. 1988; Trojanowski, et. al. 1989; Vouyiouklis and Brophy, 1995). However, tau has been reported in oligodendrocytes of rat brain tissue before (Migheli, et. al. 1988) and after enzymatic dephosphorylation of the tissue (Papasozomenos and Binder, 1987). It should be noted however, that tau was not detected in intrafascicular oligodendrocytes following dephosphorylation of the tissue and was only detected in perineuronal oligodendrocytes (Papasozomenos and Binder, 1987). Lopresti, et. al. (1995) failed to detect tau within oligodendrocytes of rat brain tissue with Tau 1 however did detect tau in these cells using Tau 5, an antibody raised against glial tau. In addition Lopresti et. al. (1995) provided evidence for the presence of tau protein and tau mRNA in oligodendrocyte cultures and that tau appears to colocalize with MBP. This raises the possibility that tau may be involved in the transportation of MBP to the site of myelination.
1.5.7 The effect of ischaemic injury on oligodendrocytes
Research to date has mainly concentrated on the mechanisms underlying neuronal death in both acute and chronic degenerative disorders of the brain. However in light of the growing evidence showing the importance of glia in the maintenance of neuronal integrity it is essential to gain an understanding of events occurring within these cells under pathological conditions. The role of astrocytes in glutamate uptake and their proliferation following
cerebral ischaemia has been well documented (Hertz, et. al. 1992; Petito and Babbiak, 1982; Schousboe, et. al. 1988). In contrast however the reaction of oligodendrocytes to ischaemia is not well known.
Oligodendrocytes are relatively resistant to ischaemia (Brierly and Graham, 1984), but can be selectively damaged following cardiac arrest (Plum, et. al. 1962) or severe hypotension (Ginsberg, et. al. 1976). Using a model of transient cerebral ischaemia in the rat Petito (1986) showed that in addition to neurons and astrocytes, oligodendrocytes can also respond rapidly to ischaemic injury. Medium-light oligodendrocytes increased in size 3, 30 and 180 min following ischaemia. These cells showed increased microtubules and tubovascular profiles and accounted for approximately 50% of all glial cells within 2 h of injury, however the number present decreased to 6.5% by 3 h. In contrast, dark cells thought to represent the most mature oligodendrocytes, showed little change following cerebral ischaemia. Petito suggests that cell loss may represent a transition of medium-light oligodendrocytes to intermediate glia which have some characteristics of both oligodendrocytes and astrocytes. This may represent an "active" oligodendrocyte. Further evidence showing that oligodendrocytes are capable of responding rapidly to injury *in vivo* was provided by Ludwin, (1984) who showed that medium oligodendrocytes proliferate 24 h following TBI in the mouse. The density of responsive cells was greatest in the cortex immediately surrounding the wound and decreased in as the distance from the wound increased. Proliferating oligodendrocytes were also detected in the white matter immediately below the wound and across the midline of the corpus callosum similar to the spread of oedema in this model.
Recently rat neonatal oligodendrocytes and human oligodendroglioma cells have been shown to increase ferritin synthesis following 6 h of hypoxia *in vitro* (Qi and Dawson, 1992; 1994; 1995). This increase in ferritin synthesis was seen in conjunction with a decrease in MBP levels but no change in structural proteins such as actin. The increased synthesis of ferritin occurs at a post-transcriptional level, and has been suggested to be part of a protective response of oligodendrocytes to hypoxic injury (Qi, et. al. 1995). Ferritin is a natural iron chelator (Balla, et. al. 1992) and may therefore play an important role in the protection of oligodendrocytes and myelin from free-radical mediated damage. Iron is a catalyst for the production of OH$^-$ from H$_2$O$_2$ therefore chelation of iron would protect the cells from the damaging effects of OH$^-$. Exposure of the same cultures to hydrogen peroxide induced ferritin synthesis (Qi, et. al. 1995). Together these results show that oligodendrocytes can respond rapidly to hypoxic injury *in vitro* which involves free radical mediated mechanisms.
The studies described above suggest that oligodendrocytes may play a more active role in the pathogenesis of brain damage resulting from cerebral ischaemia than originally believed. The mechanisms underlying the changes described following ischaemic injury are however unknown. As described above glutamate toxicity and free radical mechanisms have been implicated in ischaemic injury, however whether these mechanisms are involved in the responses of oligodendrocytes to ischaemia is not known. Electrophysiological studies have shown that mature oligodendrocytes respond directly to glutamate exposure *in vitro* (Ballanyi and Kettenman, 1990; Butt and Tutton, 1992; Patneau, et. al. 1994; Puchalski, et. al. 1994). Molecular analysis of glutamate receptor subunit expression in progenitors, O1 and O4 positive oligodendrocytes revealed the expression of AMPA and kainate preferring subunit transcripts (Patneau, et. al. 1994). Neither mature oligodendrocytes nor their progenitors appear to express NMDA receptors (Patneau, et. al. 1994; for review Gallo and Russell, 1995). Electrophysiology studies showed that the currents elicited by glutamate receptor activation were coincident with cells expressing both AMPA and kainate preferring receptors. This was shown by their different affinities for these receptor agonists, AMPA and kainate and the sensitivity to the AMPA modulator cyclothiazide (Patneau, et. al. 1994). Cyclothiazide acts to block the rapid desensitisation of AMPA receptors following glutamate activation and produces marked potentiation of the response to kainate (Patneau, et. al. 1993; Yamada and Tang, 1993).
Glutamate induced injury has been reported to be mediated through non-NMDA receptors in oligodendrocytes in a Ca++ dependant manner (Patneau, et. al. 1994; Puchalski, et. al. 1994; Yoshioka, et. al 1995). Contrasting studies however, report that glutamate induced injury in oligodendrocytes is mediated, not through glutamate receptor activation, but through the actions of free radicals. Similarly to the transport system present in neurons increased extracellular glutamate concentrations lead to the depletion of cystine and therefore glutathione within these cells rendering them vulnerable to oxidative stress (Oka, et. al. 1993; Yonezawa, et. al. 1996). In support of this, primary cultures of bovine oligodendrocytes were shown to be susceptible to oxygen radicals derived from glucose/glucose oxidase and hypoxanthine/xanthine oxidase reactions (Kim and Kim, 1991). In this study cytotoxicity was measured by tryptan blue exclusion and lactate dehydrogenase leakage, showing that oxygen radicals induce significant membrane damage to oligodendrocytes within 2 h of exposure.
Calcium influx is involved in neuronal degeneration and in glutamate receptor mediated toxicity described above. It has previously been shown that complement activated injury to oligodendrocytes characteristic of demyelinating diseases such as multiple sclerosis, is accompanied by a significant rise in intracellular calcium (Scolding, et. al. 1989).
Oligodendrocytes possess myelin-specific calcium dependant enzymes, including a basic protein kinase (Petrali and Sulakhe, 1982) and a neutral protease responsible for MBP hydrolysis (Banik, et. al. 1985). These enzymes may render oligodendrocytes more susceptible to changes in intracellular calcium than other glial cells (Scolding, et. al. 1990). Moreover, there is evidence that the effects of anoxia on white matter tracts may reflect calcium-mediated injury to the oligodendrocyte and myelin unit (Stys, et. al. 1990; 1992; Waxman, et. al. 1994). Calcium ionophores have been shown *in vitro* to cause cell body swelling, the appearance of cytoplasmic granules and the gradual disintegration of oligodendrocyte processes, leading to cell lysis (Scolding, et. al. 1992) and *in vivo* to cause demyelination of white matter tracts (Smith and Hall, 1994). In addition, MBP has been shown to be a substrate for the calcium activated protease calpain further implicating a role for calcium in the degeneration of the oligodendrocyte-myelin unit (Banik, et. al. 1994).
Together these studies provide evidence that oligodendrocytes can respond to glutamate, free radicals and Ca$^{++}$, all of which have the potential to cause degeneration of these cells. In light of this it is possible to envisage that during ischaemia and TBI, where the levels of all these substances are elevated, oligodendrocytes may become stimulated and/or undergo degeneration. Due to the importance of these cells in maintaining neuronal integrity oligodendrogial injury or death may have profound consequences for neuronal function and survival. Glutamate excitotoxicity is thought to be predominantly mediated through NMDA receptor activation. Although the use of animal models of acute brain injury have shown significant protection of neuronal elements by NMDA antagonists, compounds such as these will not protect oligodendrocytes since glutamate toxicity is mediated through non-NMDA receptors and/or free radical damage in these cells. It is possible to speculate that protection of neurons without a similar protection of glial cells may result in delayed neuronal degeneration rather than neuron protection since the degeneration of glial elements would ultimately result in neuronal death. An understanding of the mechanisms underlying glial responses to ischaemic brain injury is therefore pertinent to highlighting new areas of therapeutic intervention.
1.6. Aims of the thesis
Animal models of acute brain injury have been utilised to determine the mechanisms underlying neuronal degeneration in pathological circumstances. Glutamate toxicity and free radical mediated mechanisms are two such pathways which have been shown to be involved in neuronal degeneration following ischaemic brain injury. Assuming that similar mechanisms underly neuronal degeneration in both acute and chronic degenerative disorders these factors have also been implicated in the pathogenesis of chronic degenerative conditions. Although traditionally considered to be relatively resistant to ischaemic brain damage, oligodendrocytes have recently been shown to be susceptible to both glutamate and free radicals *in vitro*, suggesting that oligodendrocytes may also be susceptible to ischaemic brain damage. Breakdown of the neuronal cytoskeleton is thought to be one of the neurochemical events triggered by elevated extracellular concentrations of glutamate and free radicals resulting in irreversible neuronal death. Changes in the neuronal cytoskeleton resulting from acute brain injury have been well documented, although the mechanisms underlying these changes for the most part remain unclear. In contrast to the intensive investigations of neuronal responses to acute injury, the response of oligodendrocytes to ischaemia has been largely ignored.
The main aims of this thesis were therefore: 1) to investigate the distribution of the cytoskeletal proteins tau, MAP2 and MAP5 in oligodendrocytes in various rat models of acute brain injury and in human post-mortem brain tissue from patients who died following head injury or a stroke and to compare this to changes occurring in neurons and 2) to determine the mechanisms through which changes in oligodendrocyte and neuronal cytoskeletal proteins occur.
CHAPTER 2 MATERIALS AND METHODS
PART 1: ANALYTICAL END POINTS
A variety of different techniques were employed to assess tau protein in animal models of acute brain injury; human post-mortem brain tissue or oligodendrocyte cultures. These included: immunohistochemistry; western blotting and *in situ* hybridisation.
2.1 Immunohistochemistry
2.1.1 Description of antibodies
Tau was detected using six different antibodies: Tau 1; AT8; B19; TP70, TP007 and AD2 which have been previously characterised by others (Fig. 6). The monoclonal antibody Tau 1 labels a dephosphorylated epitope between residues Ser 199 and 204 (Binder, et. al. 1985; Papasozomenos and Binder, 1987). AT8 is a monoclonal antibody which labels tau phosphorylated at residue 200 (Biernat, et. al. 1992). B19 is a polyclonal antibody raised against full length tau (Brion, et. al. 1991b). TP70 is a polyclonal antibody directed against a synthetic peptide corresponding to residues 428-441 at the C-terminal of the tau protein (Brion, et. al. 1993). TP007 is a polyclonal antibody raised to a synthetic peptide corresponding to the 16 most N-terminal residues of human tau protein (Davis, et. al. 1995). Tau 1, B19, TP70 and TP007 were the kind gift of Professor B.H. Anderton (Institute of Psychiatry, London).
**Figure 6.** Schematic representation of the epitopes to which the tau antibodies used in this study bind. Residue numbers correspond to those of the longest human isoform of tau containing 4 C-terminal repeats (hatched area) and 2 N-terminal inserts (shaded area). Tau 1 and AT8 recognise tau dephosphorylated and dephosphorylated between residues 199-204 respectively. AD2 immunoreactivity is dependant on the phosphorylation of tau at residues 396 and 404. In contrast, detection of tau with B19, TP70 and TP007 is independent of the phosphorylation state of the protein.
AD2 is a phosphorylation dependant antibody raised against tau proteins found in AD. The epitope for this antibody includes Ser-396 (phosphorylated) and the participation of Ser-404 (phosphorylated) (BueeScherrer, et. al. 1996).
MAP2 was labelled using a monoclonal antibody which recognises MAP2a, b and c (clone HM-2; Sigma). MAP5 was labelled using an anti-MAP5 monoclonal antibody (clone AA6; Sigma).
Anti-transferrin was used as a cell marker for oligodendrocytes (Conner and Fine, 1986; Martin, et. al. 1991) and was detected using a polyclonal anti-transferrin (1:300) antibody from DAKO. GFAP was used as a cell marker for astrocytes (Martin, et. al. 1991) and was detected using a polyclonal anti-GFAP antibody (1:750) from DAKO.
2.1.2 Dephosphorylation of tissue prior to immunostaining
In some experiments, dephosphorylation of the tissue was performed prior to tau immunostaining using the method previously described by Papasozomenos & Binder (1987). Tissue sections were incubated with 130mg/ml of type VII-L alkaline phosphatase from bovine intestinal mucosa (Sigma) in 100mM Tris-HCl, pH8 for 2.5 h in a shaking water bath at 32ºC. To inhibit proteolysis, 1mM phenylmethylsulfonyl fluoride, 10µg/ml pepstatin and 10µg/ml leupeptin were included in the incubation fluid. For controls, 100mM sodium pyrophosphate was included in the incubation solution as a competitive inhibitor of alkaline phosphatase. The dephosphorylation reaction was quenched by bathing the sections in ice-cold Tris saline (50mM Tris-HCl, 0.2M sodium chloride, pH7.6), and immunostained immediately as described below. Tau 1 immunoreactivity is localised within the axon in histologically normal tissue, however following dephosphorylation Tau 1 immunoreactivity was also detected within neuronal perikarya indicating that tau present in neuronal perikarya is phosphorylated at the Tau 1 epitope therefore preventing antibody binding (Fig. 7). Dephosphorylation of tissue prior to immunostaining was performed in situations where Tau 1 immunoreactivity was decreased, to determine whether this was due to phosphorylation of existing protein or due to protein loss.
2.1.3 Single label immunohistochemistry
The immunohistochemical procedures described below were performed on free floating rat brain sections, modification of this procedure for immunostaining of human post-mortem brain tissue and cell cultures are described in sections 2.5 & 2.7 respectively. Sections were rinsed in phosphate buffered saline (PBS) and incubated in PBS containing 0.2% Triton X-100 for 30 min. After rinsing with PBS the endogenous peroxidase activity
Figure 7. Photomicrographs showing the pattern of Tau 1 immunoreactivity in the parietal cortex and subcortical white matter of the rat before and after dephosphorylation. Tau 1 recognises tau protein dephosphorylated between residues 199-204 and therefore immunoreactivity is localised to axons in both grey matter and white matter. No neuronal perikarya or glial cells are detected in normal brain tissue. Following dephosphorylation of the tissue, in addition to axonal staining, Tau 1 immunoreactivity became apparent within neuronal perikarya of grey matter. In white matter however, immunoreactivity remained predominantly localised to axons, suggesting the absence of tau in glial cells rather than tau being masked by phosphorylation. Thus tau is compartmentalised within the neuron; phosphorylated at the Tau 1 epitope within neuronal perikarya but dephosphorylated at this epitope in the axonal compartment of both grey and white matter. Scale bar = 50µm.
was blocked by incubating in 3% hydrogen peroxide (Sigma) for 20 min. To block non-specific binding, sections were incubated in PBS containing 10% normal horse serum and 1% bovine serum albumin (BSA; Sigma) for monoclonal antibodies (Tau 1, AT8, MAP 2 and MAP5) or PBS containing 10% normal goat serum and 1% BSA for polyclonal antibodies (B19, TP70 & TP007) for 1 h. Dilution curves were carried out to determine the optimum dilution for each antibody i.e. that which produced the best signal to background ratio. Incubation with primary antibody in appropriate blocking solution (Tau 1, 1:1000; AT8, 1:50; MAP2, 1:750; MAP5, 1:1000; B19 1:750; TP70, 1:2000; TP007, 1:500) was overnight at 4°C. Following two rinses with PBS, monoclonal antibody binding was detected by incubating sections with a biotinylated horse anti-mouse IgG secondary antibody preadsorbed against rat serum (1:100; Vector Labs.) for 1 h, while polyclonal antibody binding was detected using a biotinylated goat anti-rabbit IgG secondary antibody (1:400; DAKO). All sections were then incubated with an ABC complex (Vector Labs.) for one hour and peroxidase visualised with 3'-3'-diaminobenzidine (10mg DAB tablet (Sigma); 0.024% hydrogen peroxide in 50mM Tris HCl pH7.4). Sections were then mounted onto poly-L-lysine coated slides, dehydrated through graded alcohols, cleared and coverslipped ready for microscopic examination. Negative controls were included in each experiment, in which the primary antibody was omitted from the procedure. Non-specific immunoreactivity was not detected in these sections (Fig. 8).
2.1.4 Double label immunohistochemistry (chromagen)
Sections were stained as described above for single label immunohistochemistry using Tau1, however antibody binding was visualised using the purple chromagen VIP (Vector Labs.). Following this procedure the sections were washed in water followed by PBS and then incubated with PBS containing 10% goat serum and 1% BSA for 1h. Incubation with the second primary antibody (GFAP or transferrin) was for 1h at room temperature in blocking solution. Sections were then rinsed in PBS (2x10 min) and incubated with species specific anti-rabbit biotinylated secondary antibody (1:400) for 1 h. All sections were then incubated with an ABC complex for one hour and peroxidase visualised with a grey chromagen SG (Vector Labs.). Negative controls were included in each experiment, where the primary antibody was omitted from the procedure. Non-specific immunoreactivity was not detected in these sections (Fig. 8).
2.1.5 Double label immunohistochemistry (fluorescence)
Double label experiments were also carried out using immunofluorescence techniques. Free floating sections were treated as described above however it is unnecessary to block endogenous peroxidase activity since this is not involved in the visualisation of antibody
binding using this technique. Non-specific binding was prevented by incubating sections in PBS containing 10% normal horse serum / 10% normal goat serum and 1% BSA for 1h. Primary antibodies were added together Tau 1 (1:1000) + anti-GFAP (1:750) or Tau 1 + anti-transferrin (1:750) in blocking solution, overnight at 4ºC. Sections were rinsed in PBS and incubated with a biotinylated horse anti-mouse secondary antibody (1:100 in PBS) for 30min (this binds to the Tau 1 antibody only). Following two rinses in PBS sections were incubated in PBS containing streptavidin-FITC conjugate (1:75, Sigma) and Texas Red labelled goat anti-rabbit IgG antibody (1:100, Vector Labs.). Sections were rinsed in PBS and water and mounted onto poly-L-lysine coated slides and coverslipped using citifluor anti-fading agent (Citifluor, Labs.). The FITC conjugate binds to the biotinylated secondary antibody and therefore detects Tau 1 antibody binding. FITC absorbs light of wavelength 495nm and emits at 525nm and therefore can be visualised as green light using a blue filter. The Texas red labelled goat anti-rabbit binds to the transferrin or GFAP polyclonal antibody (rabbit IgG) and absorbs light at 596nm and emits at 620nm and therefore can be visualised as red light using a green filter.
Figure 8. Typical examples of negative controls for single (A) and double (B) label immunohistochemistry using DAB, VIP or SG as chromagens. Negative controls in which the primary antibody was omitted were carried out for every immunohistochemical experiment: A) shows a rat brain section processed for single label immunohistochemistry using DAB as the chromagen as described in section (2.1.3); B) shows a rat brain section processed for double label immunohistochemistry using Vector VIP and SG as chromagens as described in section (2.1.4). Non-specific immunoreactivity was not detected in these tissue sections. Scale bar = 50µm.
2.2 Western blot analyses
2.2.1 Preparation of cell cultures
Cell cultures were removed from -70°C and thawed on ice prior to use (for details of cultures used see section 2.7). 250µl of sample buffer (2% SDS, 5% mercaptoethanol, 10% glycerol in 62.5mM Tris pH 6.8) was added to each culture flask and a suspension of lysed cells made using a rubber spatula. Once all cells were lysed and dissociated from the flask the suspension was transferred to a sterile eppendorf, heated to 100°C for 5 min, centrifuged at 13000rpm for 5 min and returned to ice. Bromophenol blue was added to each tube and a 30µl aliquot loaded onto the gel. Remaning homogenates were stored at -70°C until further use.
2.2.2 Preparation of whole brain homogenate
An adult rat was killed by an overdose of halothane, decapitated and the brain was rapidly dissected and frozen in liquid nitrogen. Whole brain homogenates were prepared by homogenising the brain sample in 5mls of sample buffer and centrifuged at 13000rpm at 4°C for 30 min. The supernatant was retained and heated at 100°C for 5 min, spun at 13000rpm for 5 min, the supernatant harvested and placed on ice. For protein quantification, brain proteins were precipitated with tri-chloroacetic acids as follows. An appropriate volume of brain homogenate was made up to 1ml with distilled water in an eppendorf tube, and sodium deoxy cholate was added to a concentration of 1%. The samples were vortexed and ice-cold tri-chloracetic acid was added to a final concentration of 10%. The samples were vortexed and placed on ice for 30 min to precipitate the protein which was pelleted by spinning briefly in a microcentrifuge. The protein pellet was resuspended in a small volume of 1M Tris/HCl pH 8 and a sample removed for protein analysis as described by Lowry et. al. (1951), using bovine serum albumin as a standard. The remaining brain samples were then diluted in 5x sample buffer.
2.2.3 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
The denatured proteins were separated by discontinuous slab gel electrophoresis (Laemmli, 1970). The resolving gel, consisted of 12% acrylamide, 0.32% bis acrylamide, 0.38M Tris/HCl pH8.8, 0.1% SDS and was polymerised using 0.1% ammonium persulphate and 0.04% TEMED. The gel was cast between 2 glass plates in an ATTA gel setter to give a gel approximately 13 cm in length. While polymerising the top surface of the gel was covered with isopropanol. Once polymerised the isopropanol was removed and the stacking gel, consisting of 4.8% acrylamide, 0.13% bis acrylamide, 0.1% SDS, 0.14M Tris/HCl pH 6.8, 0.1% ammonium persulphate and 0.04% TEMED, poured above
the resolving gel and a 14 well comb inserted. Once solidified the gel was placed in an electrophoresis rig, the comb removed and the rig filled with electrophoresis buffer (190mM glycine, 25mM Tris and 0.1% SDS). 30µl of each of the cell homogenates and 20µg of brain homogenate were loaded onto the gel alongside pre-stained molecular weight markers (14-220kDa; Amersham). Proteins were electrophoresed at 200mV for 2-3 h until the dye front had reached the bottom of the gel and all molecular weight markers could be seen. Proteins were then transferred to PVDF membrane (Millipore) by means of semi-dry electroblotting.
2.2.4 Semi - dry electroblotting
After electrophoresis, the resolving gel was separated from the stacking gel and twelve sheets of 3mm filter paper (Watmann) and one sheet of PVDF membrane were cut to fit the gel. The filter paper was soaked in semi-dry transfer buffer (60mM Tris, 50mM glycine, 1.6mM SDS, 30% methanol) for 15 min and 6 sheets placed on the electroblotter, ensuring there were no air bubbles. The PVDF membrane was soaked in methanol and placed on top of gel, and the gel sandwiched with the remaining six sheets of filter paper. Transfer was carried out at 200mA for 45 min. Efficient transfer was confirmed by the appearance of the pre-stained molecular weight standards on the PVDF membrane sheet, the positions of these were highlighted with pencil. Following transfer the PVDF membrane was soaked in methanol and cut ready for Western blotting. The gel was placed in Coomassie stain (10% glacial acetic acid; 50% methanol; 0.25% Coomassie Brilliant Blue R) and agitated overnight at 4ºC. The following day the gel was placed in destain (7% acetic acid; 25% methanol) during western blot procedure, to confirm equal protein loading of cell samples.
2.2.5 Western blot (Enhanced chemiluminescence-ECL™)
Non-specific binding was prevented by incubating the PVDF blots in blocking solution (3% non-fat dried milk in Tris buffered saline pH8 containing and Tween-20 (TBS-T: 10mM Tris; 150mM NaCl; 0.05% Tween-20)) for 1h at room temperature. Blots were rinsed in TBS-T (1 x 10 min, 2 x 5 min) and incubated with primary antibody (Tau 1, 1:500 or TP70, 1: 3000) in blocker overnight at 4ºC. Following rinses in TBS-T (1 x 15 min, 2 x 5 min) blots were incubated with HRP labelled sheep anti-mouse IgG (1:5000; Amersham), or donkey anti-rabbit IgG (1:2000; Amersham) in blocker for Tau 1 or TP70 respectively, at room temperature for 1h. Blots were rinsed in TBS-T (6 x 10 min), incubated in ECL reagent (equal volumes of each detection reagent; Amersham) for 1 min, blotted dry with filter paper, wrapped in Saranwrap and exposed to blue light sensitive autoradiography film (Fuji) for 1-10 min depending on the amount of antigen present. Films were developed using an automatic processor. The light produced by ECL peaks
after 5-20 min and has a half life of 60 min therefore exposure to film must be within 20 min of the reaction for maximum signal detection.
2.3 In situ hybridisation
2.3.1 Core materials and solutions
Di-ethyl pyrocarbonate (DEPC) minimises RNase activity, therefore all solutions were made using DEPC treated sterile distilled water (SDW). This was prepared by treating distilled water with 0.01% DEPC overnight and sterilising by autoclaving at 15lb/in$^2$ for 15 min. Glasswear (once cleaned by soaking in 6% sulphuric acid and 6% potassium dichromate overnight) and plastics were soaked in DEPC SDW prior to sterilisation by baking at 180°C or autoclaving at 15lb/in$^2$ for 15 min respectively. 3-Aminopropyltriethoxy-silane (APES) coated slides were prepared using DEPC treated glass slides. Slides were soaked in 5% Decon 90 (Decon Labs. Ltd.) overnight, washed well, wrapped in foil and oven dried. Once dry slides were soaked in 0.25% APES (Sigma) in methylated spirit for 2 min in a fume hood. Slides were then rinsed in DEPC SDW for 2 min and oven dried in foil and stored at room temperature.
2.3.2 Preparation of tau DNA for riboprobe production
Amplification of tau cDNA
cDNA encoding full length adult rat tau (Kosik, et. al. 1989a) was kindly gifted by Dr. K. Kosik. Two copies of this adult rat cDNA were cloned into a pGEM3 vector head to tail at the EcoRI site. Competent cells, kindly prepared by Dr. P. Dickinson, were transformed with plasmid to amplify the tau cDNA. Briefly, transformations were performed in sterile 40ml polypropylene tubes pre-chilled on ice. 25ng of tau cDNA was aliquoted and made up to a final volume of 100µl with Tris EDTA buffer (TE) pH8 (10mM Tris HCl pH8 / 1mM EDTA pH8). An aliquot of competent cells was thawed and a 100µl aliquot added to the tau DNA, swirled gently and incubated on ice for 30 min. The competent culture was heat shocked at 420°C for 45 s and rapidly transferred to ice for 2 min. 800µl of SOC medium (see appendix for preparation) was added to the DNA and incubated at 370°C at 100rpm for 1hr in an orbital incubator. 2x 200µl aliquots were then plated out onto Luria-Bertrani agar indicator plates (see appendix for preparation) using a sterile bent glass rod. Once all liquid had been absorbed, the plates were inverted and incubated at 370°C overnight.
Single white colonies were removed from the indicator plates and the plasmid DNA isolated and purified from bacterial cells using a modified technique based on the alkaline lysis procedure (Birnboim and Doly, 1979). Single colonies of cells were incubated overnight at 37°C in Terrific Broth (see appendix for preparation) in an orbital incubator at 100rpm. 4.5ml aliquots of cells were spun for 1 min, the supernatant removed and the pellet resuspended in 200µl of GTE buffer (50mM glucose, 25mM Tris, 10mM EDTA, pH8). The cell suspension was then mixed with 300ml 0.2M NaOH/1% SDS (SDS denatures bacterial proteins and NaOH denatures DNA) and incubated on ice for 5 min. The solution was then neutralised by mixing with 300µl of 3M potassium acetate pH4.8 on ice. This causes plasmid DNA to reanneal, while the chromosomal DNA and bacterial proteins are precipitated with the SDS and form a complex with the potassium. This is removed by centrifuging at 13000rpm for 10 min at room temperature and removing the supernatant to a clean tube. The solution was then incubated with RNase A (DNase free) at 37°C for 20 min to remove any RNA present. The supernatant was then extracted by adding 400µl of chloroform, mixing for 30 s, centrifuging for 1 min and removing the aqueous phase into a clean tube. This step was then and the two aqueous phase added together. The DNA was then precipitated from the solution by adding an equal volume of 100% isopropanol and centrifuging for 10 min at 13000rpm at room temperature. The supernatant was removed and the pellet washed with 500µl 70% ethanol by spinning at 13000rpm for 15 min, the ethanol was then removed and the pellet vacuum dried. The pellet was dissolved in 32µl SDW and DNA precipitated by adding 8µl of 4M NaCl and then 40µl of filter sterilised 13% PEG$_{8000}$ overnight at -20°C. Plasmid DNA was pelleted by centrifuging at 13000rpm for 15 min at 4°C, the supernatant removed and the pellet washed in 500µl of 70% ethanol by spinning at 13000rpm for 15 min at 4°C. After vacuum drying the pellet was resuspended in 20µl SDW and the quantity of DNA measured by using a RNA/DNA calculator (Pharmacia Biotech).
**Linearisation of plasmid DNA**
Plasmid DNA was incubated with appropriate restriction enzymes to linearise the plasmid. EcoR1 restriction was used to confirm that the tau cDNA was cloned in at this site on the vector. Restriction enzyme Sma 1 was used to confirm the orientation of the tau cDNA in the plasmid and the template for the generation of the anti-sense tau riboprobe. Xho 1 was used to provide the template for the generation of the sense riboprobe (Fig. 9 and 10). Restriction reactions consisted of 5µg plasmid DNA, 20U restriction enzyme and the appropriate reaction buffer, incubated for 2-3 h at 30°C for Sma 1 or 37°C for Xho 1 and EcoR1. 1% agarose gels were prepared using ultra pure electrophoresis grade agarose (Gibco BRL). Agarose was melted in the presence of Tris acetate EDTA (TAE) buffer with a final concentration of 0.04M Tris acetate, 0.001M EDTA and 0.5µg/ml ethidium
bromide. The melted agarose was cooled slightly and poured into a mini gel rig, a comb inserted and allowed to set. The solidified gel was then immersed in TAE buffer. 500ng of linearised product was loaded with 6x TAE gel loading buffer (6x TAE, 30% glycerol (Sigma), 0.25% bromophenol blue (BDH) and 0.25% xylene cyanole FF (Sigma)) and electrophoresed at 50mV for 1.5 h to confirm linearisation of plasmid DNA (see Fig. 9). Gels were viewed using a "Fotoprep I" ultraviolet transilluminator (Fotodyne Inc.) and photographed using a Polaroid MP4 land camera (Polaroid), a T2201 transilluminator (Sigma), a Wratten 22A filter (Kodak) an Polaroid 667 (ASA 3000) film.
Following conformation of linearisation, the digestion product from each reaction was ethanol precipitated by adding 1/10 x volume 3M sodium acetate (pH 5.2) and 3 x volume of ethanol (-20°C) overnight at -20°C. The solution was then centrifuged at 13000 rpm at 4°C for 30min, the supernatant removed using a finely pulled glass pipette and the pellet washed with 500µl of 70% ethanol/SDW by spinning at 13000rpm for 15 min. The supernatant was removed, the pellet air-dried and resuspended in SDW at 500ng/µl and stored at -20°C until use.
2.3.3 Preparation of 35S labelled riboprobes
Sense and anti-sense 35S riboprobes were generated using a SP6/T7 transcription kit (Boehringer Mannheim). Labelling reactions contained 2ng linearised plasmid DNA, 0.5mM ATP, 0.5mM GTP, 0.5mM UTP, 50µCi 35S-CTP (specific activity 37TBq/mM; <1000Ci/mM; Amersham), 20U RNase inhibitor, 20U SP6 or T7 RNA polymerase and transcription buffer and were carried out in a DEPC treated sterile eppendorfs. The mixture was incubated at 37°C for 30 min, a further 20U of polymerase was then added and the incubation repeated. 20U RNase free DNase was added to this mixture and incubated at 37°C for 15 min to remove the DNA template. To deproteinise the sample an equal volume of phenol chloroform was added and the sample, vortexed for 10 s, centrifuged at 13000rpm for 3 min and the aqueous layer (containing nucleic acid) removed into a DEPC treated eppendorf. The solvent was then back extracted by adding an equal volume of DEPC SDW. The sample was centrifuged at 13000rpm for 3 min and the aqueous phase harvested and added to other aqueous phase. The RNA was precipitated using 1/10 volume 7.5M ammonium acetate and 2.5 x volume of ethanol (-20°C) for 3 h at -20°C. A 5µl aliquot was removed prior to centrifugation and placed in 5ml "Ecoscint" in a scintillation vial (total amount of isotope present). Following centrifugation at 13000rpm for 30min at 4°C, 5µl of the supernatant was removed and placed in 5ml "Ecoscint" (amount of isotope unincorporated). The remaining supernatant was discarded and the pellet resuspended in 100µl DEPC.SDW, 5µl of resuspended pellet was removed and placed in "Ecoscint" (amount of incorporated isotope). The vials were
counted on a Beckman LS 1801 scintillation counter and the percentage of incorporated isotope calculated. RNA was then re-precipitated overnight at -20°C with 1/10 x volume 3M sodium acetate (pH5.2) and 3 x volume of ethanol (-20°C). The preparation was centrifuged at 13000rpm at 4°C for 30min the pellet washed with 70% ethanol/DEPC SDW, air dried and resuspended at 1ng/μl/kb. The probes were stored at -20°C and used within 6-8weeks.
**Figure 9.** 1% agarose gel showing plasmid digests used for tau $^{35}$S riboprobe preparation. Lane 1; lambda Hind III fragments (Gibco, BRL). Lane 2; 100bp ladder (Gibco, BRL). Lane 3; XhoI digestion products (4308 - linearised plasmid and 1441bp - tau cDNA), used for antisense riboprobe preparation. Lane 4; SmaI digestion products, bands were detected at approximately 3792 (linearised plasmid), 1441 (tau cDNA) and 516bp (small tau fragment), confirming that the tau inserts were positioned head to tail 5'-35'-3' as expected (see Fig. 10). Sma I was used to linearise cDNA for the preparation of the tau sense riboprobe. Lane 5; undigested plasmid containing tau inserts (5794bp). Lane 6; EcoR1 digestion products show linearised plasmid (3792bp) and tau DNA (1441bp).
Figure 10. Linearisation of plasmid DNA with SmaI. a) A schematic representation of the location of the two tau cDNA fragments within the pGEM3 plasmid and the site of linearisation of the plasmid with the restriction enzyme SmaI. b) Shows the fragment sizes resulting from digestion of the plasmid containing tau cDNA (pGTau) with SmaI depending on the orientation of the tau inserts. Linearisation of pGTau with SmaI resulted in fragments approximately 4000, 1400 and 500bp (see Fig. 9) confirming that tau cDNA was inserted within the plasmid 5'-3'5'-3'.
2.3.4 In situ hybridisation procedure
Pre-treatment of tissue
Pre-treatment of the tissue involved several steps that increased the availability of RNA to the riboprobe, while maintaining tissue morphology. Frozen rat brain sections were mounted onto APES coated slides and allowed to dry. Sections were rinsed in PBS and fixed for 20 min in 4% paraformaldehyde in PBS. After rinsing in PBS, sections were placed in a solution of 0.1M triethanolamine and 0.25% acetic anhydride for 5 min. A further 0.25% acetic anhydride was then added for a further 5 min, this decreases non-specific binding of the probe to the tissue through electrostatic interactions. Sections were then placed in PBS (5 min), 0.85% saline (5 min), methylated spirits (5 min) and dehydrated in absolute alcohol (2 x 5 min).
Hybridisation
Hybridisation buffer (50% formamide, 10% dextran sulphate, 1 x Denhardt's, 20mM Tris HCl pH8, 0.3M NaCl, 5mM EDTA, 10mM NaPO$_4$ pH8 and 0.5mg/ml yeast tRNA) was heated to 50ºC to reduce viscosity. Hybridisation mixture, consisting of hybridisation buffer, 1mM dithiothreitol (DTT) and appropriate amount of probe (1:10 dilution) was heated to 80ºC for 2 min to denature the RNA and then placed on ice. 8µl of diluted probe was placed at the edge of each section and covered with a siliconised coverslips (clean coverslips soaked in Repelcort™ (BDH) for 20 min, rinsed and baked at 130ºC for 90 min). Slides were placed horizontally in a slide box containing tissue paper soaked in 50% formamide and 5 x SSC (0.75M sodium chloride, 0.075M sodium citrate) to prevent dehydration of the sections. The box was sealed with tape and placed in two vacuum sealed bags and incubated at 50ºC overnight in a waterbath.
Post hybridisation treatment
Stringency washes remove unbound or poorly hybridised probe therefore reducing non-specific labelling of tissue. Sections were placed in: 5 x SSC/10mM DTT at 50ºC for 30 min to remove coverslips; 2 x SSC in 50% formamide/10mM DTT at 65ºC for 20 min; 500mM NaCl, 5mM EDTA, 10mM Tris HCl pH7.5 at 37ºC for 10 min (x3); 0.02mg/ml RNase A, 500mM NaCl, 5mM EDTA, 10mM Tris HCl pH7.5 at 37ºC for 30 min to remove single stranded RNA; 500mM NaCl, 5mM EDTA, 10mM Tris HCl pH7.5 at 37ºC for 15 min; 2 x SSC in 50% formamide/10mM DTT at 65ºC for 20 min; 2 x SSC at room temperature for 15 min and finally 0.1 x SSC at room temperature for 15 min.
Dehydration
Sections were dehydrated through graded alcohols - 30%, 60%, 80%, 95% containing 0.3M ammonium acetate, and finally absolute alcohol. The presence of ammonium acetate prevents denaturation of the hybridisation. Sections were then allowed to air dry and exposed to Cronex (DuPont) medical screen film inside a radiographic cassette at room temperature overnight. Sections were then dipped in Ilford K5 emulsion (1:1 dilution with distilled water containing 1% glycerol) at 42°C. Slides were air dried for 4-6 h and stored at 4°C in light tight boxes containing a sachet of silica gel, for the required exposure time (determined by intensity of film image), between 1 and 7 days. This procedure was carried out by Prof. I.R. Griffiths. Sections were washed in water, allowed to air dry, counter-stained with haematoxylin and permanently mounted using DPX mountant for microscopic analysis.
PART II - PREPARATION OF TISSUE
Tau was examined by the methods described in Part I in brain tissue from either rat models of acute brain injury or human post-mortem brain tissue and in oligodendrocyte cultures.
2.4 Intracortical perfusion of monosodium glutamate *in vivo*
A model of glutamate toxicity *in vivo* was developed in this laboratory by Bullock and colleagues (Fujisawa, et. al. 1993a; 1993b; 1996). This model was used to determine the effects of high extracellular glutamate concentrations on tau immunoreactivity *in vivo*.
2.4.1 Surgical preparation of animals
Twenty-three adult male Sprague-Dawley rats, each weighing between 322-424g were used in this study. Animals were initially anaesthetised with a mixture of 5% halothane, 70% nitrous oxide and 30% oxygen. A tracheostomy was performed to allow artificial ventilation and anaesthesia subsequently maintained between 1 and 1.25% halothane. Cannulation of a femoral artery and vein allowed the monitoring of blood pressure and arterial blood gases. Blood gas analyses was performed immediately after arterial cannulation, prior to glutamate perfusion and at 1 h intervals thereafter until the end of the experiment. Animals were maintained normocapnic (pCO$_2$ 38-42mmHg), normoxic (pO$_2$ > 100mmHg) and normothermic (37°C ± 1) throughout the experimental period by adjusting the volume of gases delivered to the animal and controlling rectal temperature using a heat lamp (Table 1).
### TABLE 1 Physiological Variables - Glutamate toxicity study
| PHYSIOLOGICAL PARAMETER | Glutamate (n=8) | NaCl (n=10) | CSF (n=5) |
|-------------------------|-----------------|-------------|-----------|
| **Temperature °C** | | | |
| at probe placement | 36.8 ± 0.1 | 36.8 ± 0.1 | 36.8 ± 0.1|
| 4 h after probe placement | 36.9 ± 0.0 | 37.0 ± 0.1 | 36.8 ± 0.1|
| **Blood Pressure (mmHg)** | | | |
| at probe placement | 91 ± 1 | 90 ± 2 | 93 ± 1 |
| 4 h after probe placement | 92 ± 1 | 92 ± 1 | 93 ± 1 |
| **Arterial pH** | | | |
| at probe placement | 7.44 ± 0.01 | 7.43 ± 0.01 | 7.41 ± 0.02|
| 4 h after probe placement | 7.44 ± 0.01 | 7.42 ± 0.01 | 7.40 ± 0.01|
| **pCO₂ (mmHg)** | | | |
| at probe placement | 40 ± 1 | 40 ± 1 | 40 ± 1 |
| 4 h after probe placement | 39 ± 1 | 39 ± 0 | 39 ± 1 |
| **pO₂ (mmHg)** | | | |
| at probe placement | 182 ± 5 | 177 ± 6 | 179 ± 5 |
| 4 h after probe placement | 188 ± 5 | 180 ± 3 | 183 ± 5 |
Animals were maintained normocapnic (pCO₂ 38-42mmHg), normoxic (pO₂ > 100mmHg) and normothermic (37°C ± 1) throughout the experimental period. Values are mean ± SEM. No differences in these physiological variables were seen between experimental groups.
2.4.2 Microdialysis probe implantation
Animals were turned prone and mounted onto a Kopf stereotaxic frame (Clark Electromedical, London, UK.). Using an operating microscope, the left parietal skull was exposed and a burr hole made with a saline cooled dentist drill (stereotactic co-ordinates anterior -1.0mm, lateral +4.0mm relative to bregma). The microdialysis probe (CMA/12 membrane length 1mm, outside diameter 0.5 mm, Biotech Instruments Ltd.) was angled at 15° to the sagittal plane and lowered 1mm into the cortex through the burr hole after the dura-arachnoid had been carefully incised using a fine dental needle (for schematic representation see Fig. 11). Once in position, the probe was perfused with CSF for 30 min prior to the perfusion of 1M monosodium glutamate or 1M NaCl.
Schematic representation of the area of tissue damage resulting from the perfusion of 1M monosodium glutamate
**Figure 11.** Schematic representation of microdialysis probe placement in the parietal cortex. 1M monosodium glutamate or 1M NaCl was the perfused at 1.5µl/min for 90 min using a microinjection pump. Intracortical perfusion of 1M monosodium glutamate resulted in a sizeable lesion which extended in anterior and posterior directions and was characterised by an area of pallor as shown by H&E staining (see Figure 15).
2.4.3 Glutamate and sodium chloride perfusion
1M monosodium glutamate (pH7.4) was prepared in artificial cerebrospinal fluid (CSF) (NaCl 135mM, KCl 1mM, KH₂PO₄ 2mM, CaCl₂ 1.2mM, MgCl₂ 1mM, pH7.4). 1M glutamate was selected for this study on the basis of a prior investigation of dose dependency and lesion size (Landolt, et. al. 1993). The concentration of glutamate being delivered to the CNS is less than that within the fibre, being approximately 100mM due to the dynamics of the dialysis membrane. The vehicle control solution consisted of 1M sodium chloride in artificial CSF (pH7.4). Mechanical damage induced by probe placement was assessed by the perfusion of artificial CSF (pH7.4) alone. After placement in the cortex, the probe was perfused with the appropriate solution at a flow rate of 1.5µl/min for 90 min using a Carnegie Medicine CMA 100 infusion pump (Biotech Instruments, Luton, UK). After 90 min of perfusion, the probe was removed from the cortex and the scalp wound closely sutured. Two-and-a-half hours after the removal of the probe (i.e. 4 h from the start of perfusion), the animals were killed by transcardiac perfusion as described below.
2.4.4 Transcardiac perfusion
At the end of the experimental procedure anaesthesia was deepened using a mixture of 2% halothane, 70% nitrous oxide and 30% oxygen, the heart exposed and a needle inserted through the left ventricle to the aorta. Animals were exsanguinated by perfusing 0.9% heparinised saline at 120mmHg (approximately 150mls) then fixed by perfusing 150-200mls of ice cold 4% paraformaldehyde until the animal was rigid.
2.4.5 Processing of brain tissue for histology and immunohistochemistry
Following transcardiac perfusion, brains were removed and post-fixed in 4% paraformaldehyde at 4ºC for 48 h and cryoprotected in 30% sucrose in 50mM phosphate buffer at 4ºC for 2-3 days. The brains were then frozen in isopentane chilled on dry-ice to -42ºC for 2 min and 30µm coronal sections cut on a cryostat. Sections were stored in multiwell plates containing cryoprotectant (30% glycerol / 30% ethylene glycol in 50mM sodium phosphate buffer) at -20ºC until use. Sections for H&E staining were washed in 50mM PBS, mounted onto poly-L-lysine coated slides, allowed to dry and stained using standard methods. Free floating sections were rinsed in PBS and immunostained as described in section 2.1.
2.4.6 Processing of tissue for electron microscopy
Four additional animals each weighing 338g & 380g were used to produce tissue for electron microscopy. Two animals received intracortical perfusion of 1M monosodium glutamate and 2 received 1M NaCl as described above. At the end of the experimental procedure, the animals were killed by transcardiac perfusion of 150 ml, 0.9% saline followed by 150-200 ml of strong fix (4% paraformaldehyde; 5% gluteraldehyde; 0.05% calcium chloride in 0.08M sodium cacodylate buffer, pH7.2). Once removed, brains were post-fixed in strong fix for 24 h. A coronal section was cut through the resulting cortical lesion using a scalpel blade and a triangular section cut through the cortical lesion to the underlying corpus callosum. This section was embedded in araldite and processed on a Lynx tissue processor.
In collaboration with Prof. I.R. Griffiths, ultra thin sections were cut at 70nm on a Riechart-Jung Ultracut E ultratome and mounted onto copper grids. Sections were then stained by covering grids with saturated solution of uranyl acetate in 50% ethanol for 5-15 min at room temperature and then rinsed in 50% and 75% ethanol, distilled water (x2) and air-dried. Grids were then stained with Reynold's lead citrate for 5-10 min inside a NaOH moisturised chamber and washed 3 times in 1M NaOH and 5 times with distilled water.
2.5 Human post-mortem brain tissue
2.5.1 Sources of post-mortem brain tissue
Head injury
Formalin-fixed post-mortem brain tissue was obtained from 11 patients dying within <1 and 23 h following severe head injury (Table 2). The age of the patients ranged from 12 to 58 years and the post-mortem delay (i.e. the time between death and tissue fixation) ranged from 24 - 72 h. The corpus callosum was the brain area of choice in this study, since this area of the brain is commonly affected by diffuse axonal injury following head injury (Adams and Graham, 1989). Head injury in these patients occurred during a road traffic accident. The accumulation of APP immunoreactivity within axons was used as a marker for the early stages of axonal bulb formation and all patients used in this study had an APP score of at least 1 (McKenzie, et. al. 1995). In patients surviving for more than 2 h following head injury axonal swellings were detected with antibodies directed towards APP, 23 h following head injury many axonal bulbs were detected. APP immunostaining and classification of axonal injury was carried out by Prof. D.I. Graham in the Dept. of Neuropathology, University of Glasgow.
**Stroke**
Formalin fixed post-mortem brain tissue was obtained from 4 patients dying between 2 days and several weeks following a stroke (Table 2). The age of these patients ranged from 70 - 95 years. Post-mortem delay ranged from <24 - 48 h, with one patient outstanding at 6 days, however, no significant differences were detected within this tissue compared to other cases and was therefore included in this study. The corpus callosum, cingulate cortex and thalamus was selected for this study as these were areas affected by ischaemic brain injury. All strokes were occlusive, with no evidence of haemorrhage.
**Controls**
Formalin fixed post-mortem tissue from 7 patients with no known history of neurological or psychiatric disorders and no significant neuropathological abnormalities served as controls in this study (Table 2). The distribution of MAPs has been shown to be altered by post-mortem delay in the rat (Irving and Dewar, in press). This highlights the importance of using control tissue closely matched for post-mortem delay when extrapolating information from post-mortem tissue of brain injured patients. The control patients in this study were therefore selected according to both age and post-mortem delay. The age of control cases ranged from 12 - 59 years for comparison with head injured patients and 71 - 84 years for comparison to stroke patients. Post-mortem delay in the control cases ranged from 19 - 72 h.
### 2.5.2 Histology and immunohistochemistry
At post-mortem the whole brain was removed and immersed in 4% formalin for 48 h. Dissected blocks were then embedded in paraffin wax, 12µm sections cut on a microtome and mounted onto microscope slides. Prior to histological or immunohistochemical staining paraffin wax was removed by heating the sections at 60ºC in an oven for 60 min and then placing in histoclear for 10 min. Sections for H&E staining were rehydrated and stained with H&E by standard methods. Sections for immunohistochemical staining were immersed in: 100% alcohol (2 x 10 min); methanol containing 0.5% hydrogen peroxide for 30 min to inhibit endogenous peroxidase activity and water for 10 min. At this stage microwave antigen retrieval was carried out on sections to be stained with Tau 1 (see below). Following two rinses in PBS all sections were incubated with PBS containing 0.2% Triton X-100. Sections were then rinsed in PBS (2 x 10 min) and immunostained as described in section 2.1.3. In addition to those antibodies described in section 2.1.3, human brain tissue sections were also stained using AD2, an antibody raised against PHF tau. The concentration curve to determine the optimum concentration of AD2 was therefore carried out using formalin-fixed paraffin embedded post-mortem tissue from a patient diagnosed with AD. The concentration found produce the best signal was 1:2000.
and this dilution was therefore used throughout the investigations carried out in this thesis. As a positive control, an Alzheimer brain section was included in every immunohistochemical experiment (see Fig. 12). Following antigen detection the sections were rinsed and allowed to dry. Once dry, sections were dehydrated using graded alcohols and coverslipped using DPX mountant ready for microscopic analysis.
**Figure 12.** Photomicrograph showing NFTs immunostained with AD2 in the hippocampus of a patient diagnosed with AD. AD2 is a monoclonal antibody raised against PHF tau (see section 2.1.1) and therefore fails to detect normal tau, immunoreactivity is therefore specifically localised to NFTs in AD tissue. Staining of Alzheimer tissue was included in each immunohistochemistry experiment involving AD2, as a positive control. Scale bar = 50µm.
### 2.5.3 Antigen retrieval
Microwave pre-treatment was carried out on tissue being used for Tau 1 immunohistochemistry as this antibody does not react well with formalin fixed paraffin embedded tissue. 10mM citric acid pH6 or distilled water was heated for 5 min in a 65watt microwave at 70% power. The sections were then placed in the warmed solutions and heated for a further 5 min at 70% power. The solution volumes, lowered through heating, were topped up using distilled water and the tissue heated for a further 5 min. The sections were then removed from the microwave and allowed to stand in their solution for 20 min, rinsed in PBS and immunostained as described above. Citric acid gave a better antigen retrieval than water and so experiments were carried out using this method.
Post-mortem brain tissue was obtained from 11 patients dying following severe head injury and 4 patients dying following stroke. Survival time of patients following severe head injury ranged from <1-23 h, while that of stroke patients was 2->21 days. Seven patients with no history of neurological deficit served as controls. Control tissue was matched for age and post-mortem delay. The corpus callosum of head injured patients was examined as this area is commonly affected in head injury. For stroke patients, the cingulate cortex and thalamus was utilised as these were the areas affected by ischaemic brain damage. Both the corpus callosum and cingulate cortex were examined in the control brain tissue. N/A: not appropriate; N/K: not known.
2.6 Permanent focal cerebral ischaemia
The MCAO model of permanent focal cerebral ischaemia in the rat was used (Tamura, et. al. 1981). In this model both glutamate toxicity and free radical mediated mechanisms have been shown to be involved in the mechanisms underlying neuronal degeneration which occurs following decreased cerebral blood flow within the territory of the MCA (see section 1.2.3).
2.6.1 Surgical preparation of animals
Thirty adult male Fischer-344 rats, each weighing between 250-300g were used in this study. Animals were initially prepared as described in section 2.4.1. Arterial blood gas and glucose analyses was performed prior to and immediately following middle cerebral artery occlusion and prior to transcardiac perfusion. Animals were maintained normocapnic, normoxic and normothermic throughout the experimental period by adjusting the volume of gases delivered to the animal and controlling temperature using a heat lamp (Tables 3, 4 & 5).
2.6.2 Induction of cerebral ischaemia
The left middle cerebral artery (MCA) was exposed using a subtemporal approach based on the method originally described by Tamura et. al. (1981). This surgical procedure was performed by Dr. K. Yatsuhiro. The animals were placed on their left side and a 2cm vertical incision made between the left orbit and the external auditory canal. The temporalis muscle was dissected from the cranium and retracted to expose the inferotemporal fossa. The zygomatic arch was left intact throughout the procedure. Under a high magnification operating microscope, a small craniectomy was made using a saline cooled dentist drill, at the junction between the medial wall and the roof of the inferotemporal fossa. The underlying dura was opened by an incision using a fine needle and retracted to reveal the main branch of the MCA. The exposed MCA was occluded by bipolar electrocoagulation from the area where it crossed the inferior cerebral vein to proximal to the origin of the lenticulostriate branch. All side branches including the lenticulostriate were coagulated and the artery transected. The muscle and skin was then sutured and the animals maintained under anaesthesia for appropriate intervals. Separate groups of animals were used for three distinct studies: 1) to determine the time of the induction of tau-positive oligodendrocytes following the onset of focal cerebral ischaemia; 2) to investigate the effect of drug intervention on the induction of tau-positive oligodendrocytes and 3) to determine whether increased tau-immunoreactivity in oligodendrocytes following focal cerebral ischaemia reflects increased de novo synthesis of the protein.
### TABLE 3 Physiological variables - Time course study
| Physiological Parameter | Time after MCAO |
|----------------------------------|-----------------|
| | 20 min | 40 min | 80 min |
| **MABP (mmHg)** | | | |
| Pre | 93 ± 4 | 88 ± 7 | 102 ± 6|
| Post | 88 ± 4 | 90 ± 5 | 85 ± 5 |
| **Rectal Temperature (°C)** | | | |
| Pre | 37.0 ± 0.2 | 37.2 ± 0.1 | 36.8 ± 0.3 |
| Post | 36.8 ± 0.1 | 37.0 ± 0.0 | 37.0 ± 0.3 |
| **Arterial pH** | | | |
| Pre | 7.45 ± 0.01 | 7.42 ± 0.01 | 7.39 ± 0.02 |
| Post | 7.44 ± 0.01 | 7.44 ± 0.03 | 7.40 ± 0.01 |
| **pCO₂ (mmHg)** | | | |
| Pre | 38 ± 1 | 40 ± 1 | 44 ± 1 |
| Post | 36 ± 2 | 37 ± 1 | 40 ± 0 |
| **pO₂ (mmHg)** | | | |
| Pre | 156 ± 8 | 151 ± 10 | 175 ± 12 |
| Post | 155 ± 4 | 179 ± 14 | 160 ± 17 |
| **Plasma Glucose (mM)** | | | |
| Pre | 8.6 ± 0.8 | 9.2 ± 0.6 | 9.4 ± 0.3 |
| Post | 7.3 ± 0.9 | 7.8 ± 0.6 | 9.1 ± 0.3 |
Mean arterial blood pressure (MABP) and physiological parameters were recorded both immediately after femoral vessel cannulation (pre) and after MCAO (post). Groups of rats were killed: 20 min (n=3); 40 min (n=4) and 80 min following MCAO. There were no significant differences in the physiological parameters detected throughout the experimental period between experimental groups.
### TABLE 4 Physiological variables - Drug intervention study
| Physiological Parameter | MK-801 | NBQX | PBN |
|----------------------------------|----------|----------|----------|
| **MABP (mmHg)** | | | |
| Pre | 84 ± 7 | 91 ± 1 | 88 ± 5 |
| Post | 82 ± 1 | 89 ± 5 | 85 ± 4 |
| **Rectal Temperature (°C)** | | | |
| Pre | 37.1 ± 0.2 | 37.1 ± 0.2 | 36.8 ± 0.1 |
| Post | 37.2 ± 0.2 | 37.0 ± 0.1 | 37.4 ± 0.3 |
| **Arterial pH** | | | |
| Pre | 7.45 ± 0.02 | 7.42 ± 0.02 | 7.40 ± 0.02 |
| Post | 7.38 ± 0.02 | 7.39 ± 0.02 | 7.40 ± 0.01 |
| **pCO₂ (mmHg)** | | | |
| Pre | 39 ± 1 | 40 ± 1 | 39 ± 1 |
| Post | 39 ± 1 | 39 ± 2 | 38 ± 1 |
| **pO₂ (mmHg)** | | | |
| Pre | 166 ± 7 | 167 ± 13 | 171 ± 7 |
| Post | 160 ± 9 | 164 ± 12 | 175 ± 5 |
| **Plasma Glucose (mM)** | | | |
| Pre | 8.5 ± 0.4 | 10.0 ± 0.6 | 9.5 ± 0.8 |
| Post | 8.3 ± 0.3 | 11.8 ± 0.9 | 9.1 ± 0.5 |
Mean arterial blood pressure (MABP) and physiological parameters were recorded both immediately after femoral vessel cannulation (pre) and after MCAO (post). Rats received drug pretreatment 30 min prior to MCAO and were killed 40 min after MCAO. The following pre-treatments were examined: MK-801 (0.5 mg/kg, i.v.), n=5; NBQX (30 mg/kg, i.v.; 30 min prior to and 30 min after MCAO), n=5; PBN (100 mg/kg, i.p.), n=5. There were no significant differences detected throughout the experimental period between experimental groups, with the exception of increased plasma glucose levels following NBQX administration.
### TABLE 5 Physiological variables - In situ hybridisation study
| Physiological Parameter | Time after MCAO |
|-------------------------|-----------------|
| | 40 min | 120 min | 240 min |
| **MABP (mmHg)** | | | |
| Pre | 80 ± 0 | 90 ± 5 | 83 ± 3 |
| Post | 75 ± 0 | 87 ± 7 | 75 ± 0 |
| **Rectal Temperature (°C)** | | | |
| Pre | 37.2 ± 0.3 | 36.9 ± 0.3 | 36.8 ± 0.2 |
| Post | 36.9 ± 0.1 | 36.6 ± 0.2 | 36.8 ± 0.1 |
| **Arterial pH** | | | |
| Pre | 7.39 ± 0.04 | 7.37 ± 0.05 | 7.44 ± 0.03 |
| Post | 7.40 ± 0.01 | 7.39 ± 0.03 | 7.36 ± 0.04 |
| **pCO₂ (mmHg)** | | | |
| Pre | 44 ± 1 | 40 ± 1 | 37 ± 1 |
| Post | 38 ± 2 | 42 ± 1 | 37 ± 1 |
| **pO₂ (mmHg)** | | | |
| Pre | 145 ± 14 | 148 ± 11 | 168 ± 5 |
| Post | 145 ± 6 | 144 ± 3 | 156 ± 11 |
Mean arterial blood pressure (MABP) and physiological parameters were recorded both immediately after femoral vessel cannulation (pre) and prior to kill (post). Animals remained normocapnic, normoxic and normothermic during the experimental period. Groups of rats were killed: 40 min (n=2), 120 min (n=2) and 240 min (n=2) after MCAO.
2.6.3 Immunohistochemical studies
To determine the earliest time at which tau-positive oligodendrocytes could be detected following permanent focal cerebral ischaemia, animals were killed by transcardiac perfusion of 4% paraformaldehyde, at 20 min (n=3); 40 min (n=4) or 80 min (n=3) after MCAO. In order to investigate the effect of drug intervention on the induction of tau immunoreactivity in oligodendrocytes 40 min following MCAO rats were treated with: MK801 (0.5mg/kg, i.v.)(n=5); NBQX (30mg/kg, i.v.)(n=4) or PBN (100mg/kg i.p.)(n=5). All drugs were administered 30 min prior to MCA occlusion, NBQX was administered again 30 min after MCA occlusion. MK801 was prepared as a 1mg/ml solution in 0.9% saline and PBN as a 25mg/ml solution in distilled water. NBQX was prepared as a 15mg/ml solution by dissolving 15mg of compound in 20µl 1M NaOH and adding 9.98ml of 5.5% glucose (made in distilled water). The doses for each drug used in this study has previously been shown to reduce infarct volume following focal cerebral ischaemia in the rat (Cao and Phillis, 1994; Gill, et. al. 1992; Park, et. al. 1988). For the drug intervention studies animals were killed, by transcardiac perfusion of 4% paraformaldehyde, 40 min after MCAO.
Following transcardiac perfusion, brains were removed and processed for histology and immunohistochemistry as described in section 2.4.5. Free floating sections were immunostained as described in section 2.1.
2.6.4 Quantification of tau positive oligodendrocytes following cerebral ischaemia
For each animal from both the time course study and the drug intervention study, the total number of tau-positive oligodendrocytes in the entire subcortical white matter, extending from that underlying the perirhinal cortex to that underlying cingulate cortex was counted in one section from each of the two brain levels shown in Fig. 13. Sections were viewed at x40 magnification, blind to the drug treatments of each animal. The area of subcortical white matter in each section counted was measured using an MCID image analyser and the number of tau-positive oligodendrocytes / mm$^2$ calculated for the white matter of each brain level. From this the average number of tau-positive oligodendrocytes / mm$^2$ present in the 2 sections counted was calculated for each individual animal. The mean number of tau-positive oligodendrocytes / mm$^2$ in each treatment group was calculated and data expressed as mean ± SEM. The same quantitative analysis was performed in both hemispheres, ipsilateral and contralateral to the occluded MCA. To determine the reproducibility of this quantitative approach, the number of tau-positive oligodendrocytes was determined and the area of corpus callosum measured in one animal from each treatment group 1 week later (see Fig. 14). The coefficient of variance for repeated measurements 1 week apart was 7%. For comparison of drug treated groups statistical
analyses consisted of an ANOVA followed by Students one-tailed t-test with appropriate Bonferroni correction. The *a priori* hypothesis which was tested was that drug-treatment would reduce the number of tau-positive oligodendrocytes present following 40 min MCAO as compared to untreated animals.
**Figure 13.** Schematic representation of the sections in which the number of tau-positive oligodendrocytes/mm$^2$ was determined in subcortical white matter. The sections corresponded to those approximately 1.2 mm and 0.7 mm to Bregma in the atlas of Paxinos and Watson. The arrow indicates the region of subcortical white matter from which photographs shown in Fig. 36 were taken.
Figure 14 Relationship between the number of oligodendrocytes / mm$^2$ subcortical white matter calculated on day 1 and day 2 (1 week later). The number of oligodendrocytes / mm$^2$ was calculated at two brain levels for each animal in the study as described above. This analysis was repeated for 1 animal from each treatment group 1 week later, the correlation coefficient for repeated measures was 7%. The line of linear regression is shown in the graph and the correlation coefficient ($r^2$) is presented.
2.6.5 In situ hybridisation studies
In situ hybridisation studies were carried out to determine whether increased tau immunoreactivity in oligodendrocytes following permanent focal cerebral ischaemia represents increased de novo synthesis of the protein. Animals were killed by decapitation: 40 min (n=2); 120 min (n=2) or 240 min (n=2) following the induction of cerebral ischaemia. One animal was anaesthetised and decapitated without any surgical intervention. Following decapitation, the brains were rapidly removed and submerged in a small volume of tissue mountant contained within a small cup made with aluminum foil. This was then lowered into isopentane chilled in liquid nitrogen until the tissue mountant solidified. 15µm sections were then cut using a cryostat, mounted on to APES coated slides and processed for tau in situ hybridisation using $^{35}$S riboprobes as described in section 2.3.
2.7. Oligodendrocyte cultures
Pure oligodendrocyte cultures, were derived from an O-2A cell line kindly provided by Dr. S.C. Barnett (Department of Neurology, University of Glasgow). This cell line has recently been described by Barnett and Crouch, (1995) and preparation of cultures for immunohistochemistry and immunoblotting experiments was kindly carried out by Dr. S.C. Barnett.
2.7.1 Preparation of oligodendrocyte cultures
Cell line clones were maintained in 25cm$^2$ flasks (NUNC) at -70°C until use. Cells were detached from the flasks by washing in 2.5mg/ml trypsin (bovine pancreas, Sigma) for a few minutes at room temperature. Cells were then pipetted into a centrifuge tube and trypsin activity inhibited using an equal volume of Soya bean trypsin inhibitor: 0.52mg/ml soya bean trypsin inhibitor; 0.4mg/ml bovine pancreas DNase; 3mg/ml BSA fraction V (Sigma). The suspension was spun and the cells resuspended in modified Sato medium (Bottenstein and Sato, 1990) containing 5ng/ml platelet derived growth factor (PDGF) and 5ng/ml basic fibroblast growth factor (bFGF) - PF medium (Barnett and Crouch, 1995). The presence of these growth factors allows self-renewal of O-2A progenitor cells while preventing their differentiation (Bogler et. al. 1990). Cells were counted using a haemocytometer and approximately 7,000 placed on poly-L-lysine coated coverslips in 24 well plates for immunohistochemical experiments. For immunoblotting experiments an equal number of cells were placed on the surface of individual 25cm$^2$ NUNC flasks. All cells were incubated at 37ºC in a CO$_2$ incubator until they had adhered to the coverslips or flasks respectively. PF medium was added and cells were incubated at 37ºC in a CO$_2$ incubator until the cells had reached 80% confluency. PF medium was then replaced with Sato medium to allow differentiation of the cells into oligodendrocytes. Every second day
half the medium was removed and replaced with fresh Sato's medium. Following 4-5 days in culture oligodendrocyte cultures for immunocytochemical analysis were exposed to cytotoxic injury, as described below, and processed for tau immunocytochemistry. Batch cultures for immunoblotting experiments were washed in cold PBS and frozen on dry ice. Cultures were stored at -70°C until use for western blot analyses.
2.7.2 Exposure of oligodendrocytes to glutamate and sodium chloride
In a laminar flow hood, using sterile techniques, oligodendrocyte cultures prepared on coverslips as described above, were exposed to 10µM, 100µM or 1mM monosodium glutamate or sodium chloride (made up in Sato medium) for 4 h at 37°C in a CO₂ incubator. Controls were maintained in Sato medium during this time. Some cultures were dephosphorylated prior to immunostaining, this was carried out in the multiwell plates as described in section 2.1.3.
2.7.3 Immunocytochemistry
A control culture from each experimental run was stained with 01 antibody to confirm the presence of mature oligodendrocytes within the cultures. On removal from the incubator cells were rinsed with Hanks balanced salt solution (HBSS; 3 x 10 min) and 01 added (1:3 in HBSS) or 30 min at room temperature. Following 2 x 10 min washes with HBSS rhodamine labelled IgG was added (1:3) in HBSS for 30 min. Cells were then fixed and stained for tau as described below.
Following incubation, the cells were removed from the incubator washed in Hanks Hepes, fixed with ice cold 4% paraformaldehyde for 15 min, washed with PBS (2 x 5 min) and permeabilised in 0.2% Triton X-100 for 30 min. Following two washes with PBS (2 x 5 min) endogenous peroxidase activity was blocked with 0.5% hydrogen peroxide in PBS, and the cells immunostained for tau as described in section 2.1.3 using 3 different antibodies: Tau 1, 1:1250; TP70, 1:3000; TP007 1:750. Following immunostaining, cells were dehydrated in 70%, 90% and 100% alcohol (10 min each), immersed in histoclear for 10-15 min and permanently mounted onto poly-L-lysine coated slides using DPX, for microscopic analysis.
CHAPTER 3 RESULTS
3.1 Intracortical perfusion of glutamate in vivo
Glutamate excitotoxicity has been implicated in the pathogenesis of a variety of neurodegenerative conditions in which NFT pathology is observed. However, whether increased extracellular concentrations of glutamate are involved in the mechanisms underlying tangle formation is unknown. This study was designed to investigate the effects of high extracellular concentrations of glutamate on the distribution of tau within neurons to determine if glutamate induces any changes in tau similar to those occurring in NFT formation. In vivo microdialysis was used to perfuse 1M monosodium glutamate or the control solution, 1M NaCl into the rat parietal cortex at 1.5µl/min for 90 min. Four h from the start of perfusion the animals were killed and the brain processed for histology and immunohistochemistry.
3.1.1 Histology - light microscope
Intracortical perfusion of 1M monosodium glutamate resulted in a sizeable lesion which extended in anterior and posterior directions and was characterised by an area of pallor as shown by H&E staining. In agreement with previous studies (Fujisawa, et. al. 1993a; 1993b; 1996; Landolt, et. al. 1993) the histopathological features of the lesion included a loss of H&E stained neurons, triangulation of the nucleus and shrinkage of neurons and neuropil. There was a sharp boundary between the area of the lesion and histologically normal tissue observed in the H&E stained sections (Fig. 15). In contrast, intracortical perfusion of the control solution, 1M NaCl, resulted in a smaller area of pallor on H&E stained sections compared to that resulting from glutamate perfusion. The boundary between the area of pallor and histologically normal tissue in the NaCl perfused brains was less well delineated than in glutamate perfused animals (Fig. 15). Within the core of the NaCl induced lesion, the number of H&E stained neurons appeared to be reduced compared to histologically normal tissue, while towards the edge of the lesion nuclei had undergone triangulation. The perfusion of artificial CSF did not cause significant neuronal degeneration (Fig. 16) and failed to show any significant alteration to the normal distribution of immunostaining obtained for all the antibodies used in this study, therefore only the results obtained with glutamate and NaCl perfusion are presented.
Figure 15. H&E stained coronal sections showing the area of pallor characterising the lesioned area produced by the intracortical perfusion of 1M monosodium glutamate (a) or 1M NaCl (b). Note the sharp boundary between lesioned and histologically normal tissue following 1M monosodium glutamate perfusion (a). This boundary was less well delineated after 1M NaCl perfusion (b). Both photographs are the same magnification; bar = 250μm. A - necrotic core; B - boundary region of the lesion; cort - parietal cortex; wm - subcortical white matter.
Figure 16. H&E stained coronal sections showing the area of damage resulting from the intracortical perfusion of artificial CSF. In comparison to intracortical perfusion of 1M monosodium glutamate or 1M NaCl, the perfusion of artificial CSF did not induce significant neuronal damage. Only mechanical damage caused by microdialysis probe placement was evident. Scale bar = 400µm.
In addition to histological damage present in the cortical grey matter, areas of pallor were detected in the subcortical white matter immediately underlying glutamate induced lesions, as compared to histologically normal tissue (Fig. 17). In contrast the white matter underlying NaCl induced cortical lesions appeared to be histologically normal as compared to that of the contralateral hemisphere. This suggests that the neuropil of the subcortical white matter is damaged by the perfusion of glutamate but not NaCl. Examination of H&E stained sections using light microscopy failed to detect significant histological changes in oligodendrocytes within the subcortical white matter following intracortical perfusion of glutamate or NaCl (Fig. 17), although ultrastructural changes in oligodendrocytes were detected using electron microscopy (see section 3.1.4).
Figure 17. H&E stained coronal sections showing the subcortical white matter immediately underlying cortical lesions induced by the perfusion of 1M monosodium glutamate (b) or 1M NaCl (c). Areas where H&E staining was decreased in the neuropil were detected within the subcortical white matter immediately underlying the lesion induced by intracortical perfusion of 1M monosodium glutamate (a). Reduced staining of the neuropil, probably due to the swelling of axons, dendrites and astrocytic processes, suggests that in addition to the cortex, the subcortical white matter is also affected by the intracortical perfusion of glutamate. In contrast, H&E staining of the subcortical white matter immediately underlying the lesion induced by the intracortical perfusion of 1M NaCl (c) was not significantly different from that of the contralateral subcortical white matter (a). No significant alteration in glial cell morphology was detected in the subcortical white matter immediately underlying 1M monosodium glutamate or 1M NaCl induced cortical lesions. Scale bar = 50μm.
3.1.2 The effect of intracortical glutamate perfusion on the distribution of tau, MAP2 and MAPS immunoreactivity in neurons
Tau
The Tau 1 antibody detects tau dephosphorylated between residues 199-202 and therefore in normal rat brain immunoreactivity was detected predominantly within axons where tau is dephosphorylated at this epitope. In contrast Tau 1 immunoreactivity was not detected in neuronal perikarya where tau is phosphorylated at this epitope. This pattern of Tau 1 immunoreactivity in histologically normal tissue is similar to that described previously (Binder, et. al. 1985; Papasozomenos and Binder, 1987). Following intracortical glutamate perfusion this pattern of Tau 1 immunoreactivity was altered (Fig. 18). At the core of the lesion, Tau 1 immunoreactivity was increased within neuronal perikarya and decreased in the neuropil compared to histologically normal tissue. Furthermore, it should be noted that perikaryal staining induced by intracortical glutamate perfusion was much more intense than that of histologically normal tissue incubated with alkaline phosphatase. In contrast to glutamate, NaCl perfusion did not increase Tau 1 immunoreactivity within neuronal perikarya, however neuropil staining was reduced (Fig.18). Alkaline phosphatase pretreatment failed to alter the pattern of Tau 1 immunostaining present within the neurons following glutamate or NaCl perfusion (Fig. 19), suggesting that decreased Tau 1 staining was not due to phosphorylation of the protein at this epitope. In contrast to Tau 1, AT8 detects tau phosphorylated at residue 200 and therefore predominantly detects tau within neuronal perikarya in histologically normal tissue (Fig. 18). Following intracortical perfusion of either glutamate (Fig. 18) or NaCl (Fig. 18), AT8 immunoreactivity was decreased in neuronal perikarya, confirming that tau present within neuronal perikarya following glutamate perfusion is dephosphorylated at this epitope.
The distribution of B19 (directed towards full-length tau), TP70 (directed towards C-terminal of tau) and TP007 (directed towards N-terminal of tau) in histologically normal tissue is shown in Fig. 20. These antibodies are phosphorylation independent and therefore detect tau present in both neuronal perikarya and axons. In contrast to Tau 1; B19, TP70 and TP007 immunoreactivity was decreased within neuronal perikarya following glutamate perfusion (Fig. 20) compared to histologically normal tissue. Similarly to Tau 1, neuropil staining with B19 and TP007 was reduced following both glutamate and NaCl perfusion, however TP70 remained relatively unchanged. B19, TP70 and TP007 staining of neuronal perikarya was reduced following NaCl perfusion (Fig. 20).
Figure 18. Distribution of Tau 1 and AT8 immunoreactivity in rat cerebral cortex. Tau 1 immunoreactivity, most abundant in axons in histologically normal tissue, was increased in neuronal perikarya at the core of the lesion produced by the intracortical perfusion of 1M monosodium glutamate. In contrast, AT8 immunoreactivity, most abundant within neuronal perikarya in histologically normal tissue, decreased following glutamate perfusion. Tau 1 immunoreactivity was decreased in axons following glutamate or NaCl perfusion compared to histologically normal tissue. AT8 immunoreactivity was not detected following glutamate or NaCl perfusion. In addition to the alterations in neuronal immunoreactivity, increased Tau 1 immunoreactivity was detected in small cells with the morphological appearance of glia within the cortical lesion induced by 1M NaCl perfusion (arrows) (see section 3.1.3). Scale bar = 50µm
Figure 19. The effect of alkaline phosphatase pre-treatment on the distribution of Tau 1 immunoreactivity in neurons and glia following intracortical perfusion of 1M monosodium glutamate or 1M NaCl. Following intracortical perfusion of 1M monosodium glutamate, increased Tau 1 immunoreactivity was detected in neuronal perikarya while staining was decreased in the axons. Intracortical perfusion of 1M NaCl resulted in increased Tau 1 immunoreactivity within small cells with the morphological appearance of glia. This pattern of Tau 1 immunoreactivity described within the cortex following glutamate or NaCl perfusion was not altered by dephosphorylation of the tissue prior to immunostaining. This data confirms that decreased Tau 1 immunoreactivity within the neuropil detected following the perfusion of glutamate or NaCl was not due to phosphorylation of tau at the Tau 1 epitope. Scale bar = 50μm.
Figure 20. Distribution of B19, TP007 and TP70 immunoreactivity in rat cerebral cortex. B19, TP70 and TP007 detect tau in both axons and neuronal perikarya of histologically normal tissue. Following intracortical perfusion of 1M monosodium glutamate or 1M NaCl, B19, TP70 and TP007 immunoreactivity was decreased in neuronal perikarya. Similarly, B19 and TP007 staining of axons was reduced following both glutamate and NaCl perfusion however TP70 immunoreactivity remained relatively unchanged. Increased tau immunoreactivity was detected in small cells with the morphological appearance of glia within the cortical lesion resulting from the perfusion of 1M monosodium glutamate or 1M NaCl (see section 3.1.3). Scale bar = 50µm.
**MAP2 and MAP5**
To compare the changes in tau immunoreactivity with that of other MAPs, adjacent sections to those stained with tau, were labelled using antibodies directed towards MAP2 and MAP5. MAP2 immunoreactivity was detected mainly in dendrites and neuronal perikarya in histologically normal tissue (Fig. 21) as described previously (Cáceres, et. al. 1984; Huber and Matus, 1984). At the core of the lesion produced by the intracortical perfusion of 1M monosodium glutamate neuronal perikaryal staining with the MAP2 antibody was increased while dendritic staining was reduced (Fig. 21). In contrast to glutamate, 1M NaCl perfusion did not increase neuronal perikaryal immunostaining, instead both dendritic and perikaryal MAP2 immunoreactivity was reduced (Fig. 21). MAP5 immunoreactivity was detected in both perikarya and neuropil in histologically normal tissue (Fig. 21). Similarly to MAP2, MAP5 immunoreactivity increased within neuronal perikarya and decreased in neuropil within the core of the lesion produced by glutamate perfusion (Fig. 21). NaCl perfusion resulted in decreased MAP5 immunostaining in both perikarya and neuropil (Fig. 21). Thus the effects of intracortical perfusion of glutamate or NaCl on the immunostaining of MAPs within neurons were not specific for tau, but also included MAP2 and MAP5.
**3.1.3 MAPs in oligodendrocytes following intracortical glutamate perfusion**
In addition to changes in the distribution of tau, MAP2 and MAP5 immunoreactivity detected in neurons, tau immunoreactivity was detected in small cells with the morphological appearance of oligodendrocytes following intracortical glutamate or NaCl perfusion. This was an unexpected finding since tau is thought to be a neuron specific protein. However increased tau immunoreactivity was detected in oligodendrocytes within the cortical lesion and the subcortical white matter immediately underlying this area, both in glutamate and NaCl perfused animals. In light of this novel and unexpected finding of increased tau staining in glial cells, it was decided to pursue this finding. Unravelling this response became the focus of future studies.
**Tau**
Within the subcortical white matter contralateral to the glutamate or NaCl induced cortical lesion, tau, as detected with Tau 1, TP70, TP007 and B19 was localised predominantly within axons (Fig. 22 & 23). Following intracortical perfusion of 1M monosodium glutamate or 1M NaCl, tau immunoreactivity was observed in small cells with the morphological appearance of glia. These tau-positive glial cells were found scattered throughout the cortical lesion (Fig. 18 & 20) but also abundantly within the subcortical white matter immediately underlying this area (Fig. 22 & 23). Following intracortical perfusion of 1M monosodium glutamate or 1M NaCl, tau positive glial cells were detected using B19, TP70 and TP007 suggesting the presence of full-length protein.
Figure 21. Distribution of MAP2 and MAP5 immunoreactivity in neurons following intracortical perfusion of 1M monosodium glutamate or 1M NaCl. MAP2 and MAP5 immunoreactivity was detected within neuronal perikarya and neuropil of histologically normal tissue. Following intracortical glutamate perfusion, both MAP2 and MAP5 immunoreactivity increased in perikarya and decreased in neuropil within the resulting cortical lesion. By contrast, MAP2 and MAP5 immunoreactivity was decreased in both neuronal perikarya and neuropil within the cortical lesion induced by the perfusion of 1M NaCl. Scale bar = 50μm.
Figure 22. Tau immunoreactivity in glial cells of the subcortical white matter following intracortical perfusion 1M monosodium glutamate or 1M NaCl in the rat. B19, TP70 and TP007 immunoreactivity was localised within the axons of the contralateral subcortical white matter. However, following intracortical perfusion of 1M monosodium glutamate or 1M NaCl, increased tau immunoreactivity was detected within cells with the morphological appearance of glia both within the resulting cortical lesion and in the subcortical white matter immediately underlying this area. These glial cells were detected using B19, TP70 and TP007, suggesting the presence of full length tau protein. Scale bar = 50µm.
Tau-positive glia were also detected with the Tau 1 antibody, both within the cortical lesion induced by the perfusion of 1M monosodium glutamate or 1M NaCl and in the subcortical white matter immediately underlying this area. This suggests that tau present in these cells is dephosphorylated at the Tau 1 epitope. In support of this finding, the phosphorylation dependant antibody AT8 failed to detect these glial cells (Fig. 23), confirming that tau present within these cells is dephosphorylated at the Tau 1 epitope. Tau immunoreactivity was not detected within glial cells in histologically normal tissue with any of the tau antibodies used in this study with (Fig. 24) or without alkaline phosphatase pretreatment (Figs. 22 & 23).
**Figure 23.** Distribution of Tau 1 and AT8 immunoreactivity in the subcortical white matter following intracortical perfusion of 1M monosodium glutamate or 1M NaCl. Tau 1 immunoreactivity was predominantly localised to axons within the contralateral subcortical white matter. However following the intracortical perfusion of either 1M monosodium glutamate or 1M NaCl, Tau 1 immunoreactivity was detected in glial cells both within the resulting cortical lesion and in the subcortical white matter immediately underlying this area. AT8 failed to detect glial cells prior to, or following intracortical perfusion of 1M monosodium glutamate or 1M NaCl. Scale bar = 50μm.
Figure 24. Tau immunoreactivity in the subcortical white matter following alkaline phosphatase pre-treatment of histologically normal rat brain tissue using Tau 1, B19, TP70 or TP007. Tau 1, B19, TP70 and TP007 immunoreactivity is located predominantly within axons of the subcortical white matter in histologically normal tissue. Alkaline phosphatase pre-treatment of rat brain tissue prior to immunostaining failed to alter this pattern of immunoreactivity as detected with all 4 tau antibodies. Glial cells were not detected using Tau 1, B19, TP70 or TP007 before, or after dephosphorylation of the tissue. This data suggests that the epitopes of tau recognised by these antibodies are not masked by phosphorylation within glial cells present in control tissue. Furthermore, the presence of tau immunoreactivity in glial cells, as detected with all 4 antibodies, following intracortical perfusion of 1M monosodium glutamate or 1M NaCl cannot be explained simply by the dephosphorylation of existing tau protein within these cells. Scale bar = 50µm.
Tau-positive cells displayed a small perikaryon and lacked visible processes consistent with the morphological appearance of oligodendrocytes in situ. Within the subcortical white matter tau-positive cells were aligned in rows, a feature characteristic of intraspecific oligodendrocytes. Moreover, tau-positive oligodendrocytes, detected following intracortical perfusion of glutamate or NaCl, stained negative for GFAP and positive for transferrin therefore showing that these tau-positive cells were immunologically similar to oligodendrocytes (Fig. 25). Together this data confirms that tau-positive glial cells detected in response to intracortical perfusion of 1M monosodium glutamate or 1M NaCl were oligodendrocytes.
**Figure 25.** Confocal images showing Tau 1, GFAP and transferrin immunoreactivity in the subcortical white matter immediately underlying cortical lesions induced by the perfusion of 1M monosodium glutamate (a,c) or 1M NaCl (b,d). (a&b) GFAP shown in red and Tau 1 shown in green. Tau-positive cells were GFAP negative and morphologically distinct from astrocytes which displayed an elaborate array of processes. (c&d) Transferrin shown in red and Tau 1 shown in green. Yellow staining represents areas of overlapping immunoreactivity, showing that tau-positive cells are also positive for transferrin. Tau-positive cells were therefore immunologically and morphologically similar to oligodendrocytes. Confocal images were kindly provided by Prof. I.R. Griffiths. Scale bar = 50μm.
**MAP 2 and MAP5**
Neither MAP2 nor MAP5 immunoreactivity was detected in glial cells of the hemisphere contralateral to the occluded MCA. In contrast to tau, MAP2 and MAP5 immunoreactivity was not detected in oligodendrocytes following intracortical perfusion of 1M monosodium glutamate or 1M NaCl neither in the resulting cortical lesion nor in the subcortical white matter underlying this area (Fig. 26). In contrast to neurons, where tau, MAP2 and MAP5 underwent marked alteration in response to intracortical perfusion of both glutamate and NaCl, changes in MAPs within oligodendrocytes following injury appears to be specific for tau.
**Figure 26.** Distribution of MAP2 and MAP5 immunoreactivity in the subcortical white matter following intracortical perfusion of 1M monosodium glutamate or 1M NaCl. MAP2 and MAP5 immunoreactivity was localised predominantly within axons and dendrites of the contralateral subcortical white matter. In contrast to tau, neither MAP2 nor MAP5 immunoreactivity was detected in glial cells of the ipsilateral subcortical white matter following either 1M monosodium glutamate or 1M NaCl perfusion. Scale bar = 50μm.
3.1.4 Electron microscopy
Structural changes in glial cells present within the subcortical white matter immediately underlying the glutamate or NaCl induced lesion could not be detected by light microscopy. In order to determine if such changes could be detected at the ultrastructural level, the brains from animals which received intracortical perfusion of either 1M monosodium glutamate (n=2) or 1M NaCl (n=2) were processed for electron microscopy. The white matter of the corpus callosum immediately underlying cortical lesions induced by glutamate or NaCl perfusion, was markedly oedematous. Astrocytic swelling was marked, involving both nuclei and cytoplasm, while oligodendrocytes were involved to a lesser degree. Oligodendrocytes of the contralateral corpus callosum, had an electron dense nucleus and abundant microtubules (Fig. 27). Following the intracortical perfusion of 1M monosodium glutamate, the most obvious ultrastructural changes were that the oligodendrocyte nucleus became swollen and less electron dense as did the cytoplasm, as compared to oligodendrocytes in the contralateral hemisphere (Fig. 27). Oligodendrocyte processes were swollen however all cytoplasmic organelles were recognisable (Fig. 27). The ultrastructural changes in oligodendrocytes reported following intracortical perfusion of 1M monosodium glutamate were also seen following 1M NaCl perfusion, there were no significant differences detected between the two treatment groups. The swelling of oligodendrocytes and their processes confirmed that these cells, present within the subcortical white matter, were affected by the intracortical perfusion of glutamate or NaCl. Electron microscopy was performed by Prof. I.R. Griffiths.
Figure 27. Electron micrograph showing oligodendrocyte morphology in the contralateral (a) and ipsilateral (b) subcortical white matter following intracortical perfusion of 1M monosodium glutamate. Oligodendrocytes present in the contralateral subcortical white matter had an electron dense nucleus and cytoplasm which contained abundant microtubules. Following intracortical perfusion of 1M monosodium glutamate (b) or 1M NaCl, the oligodendrocyte nucleus and cytoplasm became swollen and less electron dense, compared to those oligodendrocytes present in the contralateral hemisphere. Magnification factor = x 20,000.
3.2 Human post-mortem brain studies
Increased tau immunoreactivity was detected in oligodendrocytes following glutamate toxicity in rat brain. Previously, extracellular concentrations of glutamate were shown to be increased immediately following both traumatic brain injury and stroke in the rat (Butcher, et. al. 1990, Bullock, et. al. 1990). Therefore in the next study, human post-mortem brain tissue from patients dying following stroke or head injury was examined for the presence of tau-positive oligodendrocytes, in order to determine whether or not the presence of tau-positive oligodendrocytes following excitotoxic brain injury was a phenomenon peculiar to the rat brain.
3.2.1 Tau positive glial cells following severe head injury
For this study, cases of head injury were selected in which there was evidence of diffuse axonal injury within the corpus callosum. The accumulation of APP immunoreactivity within axons was used as a marker for the early stages of axonal bulb formation and all patients used in this study had an APP score of at least 1 (McKenzie, et. al. 1995). APP immunostaining and classification of axonal injury was carried out by Prof. D.I. Graham in the Dept. of Neuropathology, University of Glasgow. In the corpus callosum of control cases, tau immunoreactivity as detected with TP70, TP007 and Tau 1 was localised predominantly within axons (Fig. 28 & 29). However, in the corpus callosum from patients dying following head injury, in addition to its presence in axons, tau immunoreactivity was also detected within cells with the morphological appearance of glia (Fig. 28 & 29). The alignment of these glial cells in rows within the corpus callosum suggests that these tau-positive cells were oligodendrocytes. These tau-positive glia were detected using Tau 1, TP70 and TP007 suggesting the presence of full-length protein which was dephosphorylated at the Tau 1 epitope in these cells. It should be noted however that the number of oligodendrocytes detected with Tau 1 was less than that detected by TP70 and TP007 in all cases examined. The Tau 1 epitope is normally phosphorylated in NFTs, therefore in order to confirm that tau present in glial cells reported here was dephosphorylated, rather than hyperphosphorylated as seen in NFTs, sections were stained with the AD2 antibody. AD2 is an antibody raised against tau proteins found in Alzheimer's disease which recognises tau phosphorylated at Ser-396 and Ser-404 (BueeScherrer, et. al. 1996). The lack of staining of glial cells in the corpus callosum of head injured patients with AD2 suggests that in contrast to that of PHF tau, tau present in these cells is not phosphorylated at the AD2 epitopes (Fig. 28). The possibility that tau may be phosphorylated at different residues, similar to that in of PHF tau however, cannot be ruled out. Tau was detected within a few glia in the corpus callosum of patients surviving <1 h following head injury and the number of these tau-positive cells appeared to increase with survival time after injury, although quantitative analyses were not undertaken (Fig. 28 &
In patients surviving for 6 h or more, areas of pallor within the corpus callosum were detected in H&E stained sections outlining areas of tissue affected by injury (Fig. 29). In these patients tau-positive glia were localised within and immediately surrounding the area of tissue delineated by reduced H&E staining. In tissue distant from these affected areas where there was no histological evidence of brain injury, tau-positive glia were not detected. Tau-positive glia were detected in all brain injured patients examined in this study however, it should be noted that only a proportion of the glial population became tau-positive within damaged areas especially at early times following head injury.
**Figure 28.** Tau 1 and AD2 immunoreactivity within the corpus callosum of control patients and 6 h and 23 h following head injury. In control tissue, Tau 1 immunoreactivity was localised predominantly within the axonal compartment where tau is dephosphorylated at this epitope. AD2 recognises PHF tau and therefore fails to detect tau in histologically normal tissue. 6 h following head injury Tau 1 immunoreactivity was increased in small cells with the morphological appearance of oligodendroglia, however the number of these cells detected was less than that detected by TP70 and TP007 (Fig. 29). 23 h following head injury Tau 1 positive oligodendrocytes were difficult to detect. AD2 immunoreactivity was not detected in glial cells of histologically normal or head injured tissue. Scale bar = 50 μm.
In one patient surviving for 23 h following severe head injury, TP70 and TP007 immunoreactivity was intense in the cytoplasm of glial cells to one side of the cell (Fig. 29), within the corpus callosum where H&E staining was markedly decreased (Fig. 30). The morphological appearance of these tau-positive glial cells was similar to that reported previously for glial inclusions in neurodegenerative conditions such as MSA (Iwatsubo, et. al. 1994; Kato, et. al. 1991; Yamada, et. al. 1992). In this single case, glial cells were not detected with the Tau 1 antibody (Fig. 28), although they were stained with TP70 and TP007, suggesting that tau may be phosphorylated at the Tau 1 epitope. AD2 failed to detect these tau-positive cells showing that in contrast to PHF tau, tau present in these oligodendrocytes was not phosphorylated at the AD2 epitope (Fig. 28).
**Figure 29.** Distribution of TP70 and TP007 immunoreactivity in the corpus callosum of control and head injured patients 6 and 23 h following injury. In control tissue, immunoreactivity is localised predominantly within the axonal compartment. Following head injury however TP70 and TP007 immunoreactivity was increased in small cells with the morphological appearance of oligodendrogliosis. In the patient surviving for 23 h following head injury, TP70 and TP007, immunoreactivity was intense within the cytoplasm of some oligodendrocytes, showing a morphology similar to that of glial inclusions (arrows). Scale bar = 50μm.
Figure 30. Corpus callosum of control and head injured patients stained with H&E. In patients surviving for 6 h or more after head injury, areas where H&E staining was decreased within the neuropil outlined the regions of the corpus callosum affected by brain injury. 6 and 23 h following head injury the number of glial cells present in the corpus callosum decreased as compared to histologically normal tissue. In addition nuclei appeared eosinophilic. 23 h following head injury H&E staining was markedly reduced within the neuropil as compared to that of control tissue. Scale bar = 100μm.
3.2.2 Tau-positive glia following stroke
Post-mortem brain tissue was obtained from patients who died following occlusive stroke. Brain areas examined encompassed the cingulate cortex and thalamus, depending on the location of the ischaemic injury. In brain tissue obtained from all four patients who died following a stroke, tau-positive glia were detected within and immediately surrounding areas of tissue in which there was histological evidence of ischaemic brain damage (Fig. 31). Tau-positive glia were detected at the core of the infarct, however were most abundant in the grey matter immediately surrounding the infarcted tissue (Fig. 32 & 33) and in the white matter lying in close proximity to the infarcted tissue. The detection of tau-positive glia with TP70 and TP007 suggests the presence of full-length protein in these cells (Fig. 32). Detection of glial cells with the Tau 1 antibody shows that, similarly to that occurring following head injury, tau is dephosphorylated at the Tau 1 epitope (Fig. 33). AD2 failed to
to detect tau present within glial cells showing that in contrast to PHF tau, tau present in these cells is not phosphorylated at the AD2 epitope (Fig. 33).
In the patient surviving for several weeks following stroke, many glia within the infarcted tissue contained intense tau staining within the cell cytoplasm to one side of the cell as detected with TP70 and TP007 (Fig. 32). The morphological appearance of these tau-positive glial cells was similar to that reported previously for glial inclusions in neurodegenerative conditions such as MSA (Iwatsubo, et. al. 1994; Kato, et. al. 1991; Yamada, et. al. 1992). This pattern of tau immunoreactivity was not detected with the Tau 1 antibody (Fig. 33) suggesting that tau present in these cells may be phosphorylated at the Tau 1 epitope. Similarly to oligodendrocytes detected 23 h following head injury, AD2 failed to detect tau accumulations within the oligodendrocyte cytoplasm showing that in contrast to PHF tau, tau present within these inclusions was not phosphorylated at the AD2 epitope (Fig. 33). Tau present in glial inclusions can be detected with antibodies raised against PHF tau which is phosphorylated at many sites including the Tau 1 epitope, the possibility that tau present in these cells is phosphorylated at other residues of the tau protein similar to those occurring in glial inclusions cannot be ruled out.
**Figure 31.** Cingulate cortex from control and stroke patients stained with H&E. In all cases areas where H&E staining was decreased outlined the regions of tissue affected by ischaemic brain injury. 5 days and following stroke, neurons showed triangulation of the nucleus and eosinophilic cytoplasm, in addition neuropil staining was decreased. In the patient surviving for several weeks following stroke, infarcted tissue was characterised by decreased H&E staining, of all cellular elements. In all cases neuropil staining was reduced within areas of tissue affected by ischaemic brain injury. Scale bar = 100μm.
Figure 32. Distribution of TP70 and TP007 immunoreactivity in the cingulate cortex from control and stroke patients. In post-mortem brain tissue obtained from control patients, TP70 and TP007 immunoreactivity was localised predominantly within the axonal compartment. However, following stroke tau immunoreactivity was increased within oligodendrocytes in, and immediately surrounding the region of infarct. Figure shows tau-positive oligodendrocytes in the cingulate cortex 5 days and several weeks following stroke. In the patient surviving several weeks following a stroke, tau-positive glia displaying morphological features similar to that of glial inclusions, were detected with TP70 and TP007 (arrows) within the region of infarct. Scale bar = 50μm.
Figure 33. Distribution of Tau 1 and AD2 immunoreactivity in the cingulate cortex from control and stroke patients. In post-mortem brain tissue obtained from control patients, Tau 1 immunoreactivity was localised predominantly within the axonal compartment. However, following stroke Tau 1 immunoreactivity was increased within oligodendrocytes in, and immediately surrounding the region of infarct. Figure shows tau-positive oligodendrocytes in the cingulate cortex 5 days and several weeks following stroke. AD2 failed to detect tau-positive oligodendrocytes in both control and stroke tissue. Scale bar = 50μm.
3.2.3 Identification of tau-positive glia
Sections double immunostained with GFAP, as a marker for astrocytes, and Tau 1 showed that tau-positive glial cells present in the corpus callosum following both head injury and stroke did not stain with a GFAP antibody and were morphologically distinct from astrocytes (Fig. 34). In contrast to the large cell body and elaborate array of processes displayed by astrocytes, tau-positive glial cells had small perikarya, limited cytoplasm and lacked visible cellular processes. This morphology is consistent to that described for oligodendrocytes. In addition, tau-positive glial cells also stained positive for transferrin, a specific marker for oligodendrocytes (Fig. 34) confirming that tau-positive glial cells were both immunologically and morphologically similar to oligodendrocytes.
**Figure 34.** Identification of tau-positive glia in human post-mortem tissue. Sections of the corpus callosum of a patient dying 10 h following severe head injury were immunostained with A) GFAP as a marker for astrocytes shown in grey and Tau 1 shown in purple. Tau-positive cells were GFAP negative and morphologically distinct from astrocytes which displayed an elaborate array of processes (arrows) or B) transferrin as a marker for oligodendrocytes shown in grey and Tau 1 shown in purple. Tau-positive glial were positive for transferrin and Tau 1, the dark purple colour showing areas of overlapping immunostaining. Tau-positive cells detected in human post-mortem tissue after acute brain injury were therefore immunologically and morphologically similar to oligodendrocytes. Scale bar = 50μm.
3.3 Tau-positive oligodendrocytes following permanent focal cerebral ischaemia in the rat.
The presence of tau-positive oligodendrocytes in human brain following acute brain injury confirmed that the appearance of tau in oligodendrocytes was not a phenomenon unique to rat brain, but a response of these cells to acute brain injury. In order to further characterise the presence of tau in oligodendrocytes, the next study was designed to determine the earliest time following focal cerebral ischaemia in the rat that tau-positive oligodendrocytes could be detected. In addition, in order to determine whether the number of tau-positive oligodendrocytes increased over time, quantitative analysis of cell density was performed. Permanent MCAO in the rat was performed by Dr. K. Yatsuhiro as originally described by Tamura et. al. (1981). Twenty min (n=3), 40 min (n=4) and 80 min (n=3) following the induction of cerebral ischaemia, the animals were killed by transcardiac perfusion and the brains processed for tau, MAP2 and MAP5 immunohistochemistry. The distribution of tau, MAP2 and MAP5 immunoreactivity in neurons was also investigated for comparison.
3.3.1 MAP2 as a marker of ischaemic damage
MAP2 immunostaining has been shown to be a sensitive marker of ischaemic damage in animal models of cerebral ischaemia (Dawson and Hallenbeck, 1996; Kitagawa, et. al. 1989; Yamamoto, et. al. 1986; Yanagihara, et. al. 1990). Although traditional histological stains such as H&E have been employed within 4 h of permanent focal ischaemia at shorter survival times, these stains are difficult to interpret. Therefore, in this study involving very short survival times following MCAO, the anatomical extent of ischaemic damage was detected using MAP2 immunostaining (Fig. 35). Twenty min post-MCAO, an area where MAP2 immunostaining was decreased was detected within the caudate nucleus in the ipsilateral hemisphere. Forty and 80 min post-occlusion, MAP2 immunostaining was decreased within the caudate nucleus within the territory ipsilateral to the occluded MCA. In addition, areas of decreased immunoreactivity were also detected within the ipsilateral cortex, however this was variable and patchy in its distribution. Decreased MAP2 immunostaining was detected in all animals used in this study. For a more detailed description of altered MAP2 distribution in neurons following ischaemic brain injury see section 3.3.3.
Figure 35. Photomicrograph of a rat brain section stained with MAP2 showing the area of ischaemic damage following 40 min MCAO. Immunoreactivity was markedly reduced in perikarya and neuropil of the caudate nucleus ipsilateral to the occluded MCA. The boundary of ischaemic tissue could also be detected in the cortex (arrow). All animals used in this study showed areas of decreased MAP2 immunoreactivity within the caudate nucleus, two animals also showed patches of decreased immunoreactivity in the cortex. Scale bar = 50μm.
3.3.2 Time course of increased tau immunoreactivity in oligodendrocytes following permanent MCAO in the rat.
In the hemisphere contralateral to the occluded MCA of all animals in this study the distribution of Tau 1, TP70 and TP007 immunoreactivity within the cortex and caudate nucleus was predominantly located within neurons. In previous studies no tau-positive oligodendrocytes were detected in rat tissue with any of the tau antibodies before or after dephosphorylation of the tissue (see section 3.1). In this study however, occasional tau-positive oligodendrocytes were detected in the subcortical white matter of the contralateral hemisphere 20 min following MCAO, using Tau 1, TP70 and TP007. This number increased slightly with time, however the number of tau-positive oligodendrocytes / mm$^2$ of subcortical white matter remained low compared to the contralateral hemisphere (Fig. 37). This suggests that the mechanisms underlying increased tau immunoreactivity in oligodendrocytes are induced throughout the entire corpus callosum following MCAO. Twenty min following MCAO, only a few tau-positive oligodendrocytes were detected within the ipsilateral subcortical white matter however this was greater than that in the contralateral hemisphere. In rats subjected to 40 min (Fig. 36 & 37) or 80 min of focal ischaemia, the number of tau-positive oligodendrocytes in the ipsilateral subcortical white matter, detected with all three tau antibodies, was increased compared to the contralateral white matter (Fig. 37). While increased tau immunoreactivity was most prominent within intrafascicular oligodendrocytes located in the subcortical white matter, oligodendrocytes were also present to a lesser extent in the grey matter of the caudate nucleus and the neocortex ipsilateral to the occluded MCA. These oligodendrocytes, detected using all three tau antibodies, were present in grey matter only within the boundary of ischaemic damage as determined by MAP2 immunostaining. Double label immunohistochemistry confirmed that similarly to the previous studies, these tau-positive glial cells with the morphological appearance of oligodendrocytes present in the subcortical white matter ipsilateral to the occluded MCA were morphologically and immunologically similar to oligodendrocytes (Fig. 38).
Figure 36. Tau immunostaining of subcortical white matter 40 min following MCAO in the rat. Tau immunostaining was localised mainly within the axons of the contralateral subcortical white matter 40 min following MCAO as detected with Tau 1, TP70 and TP007. In contrast, ipsilateral to the occluded MCA, numerous tau-positive oligodendrocytes were present in the subcortical white matter 40 min following the induction of cerebral ischaemia. Tau-positive oligodendrocytes in the ipsilateral hemisphere were detected with Tau 1, TP70 and TP007. Scale bar = 50μm.
Figure 37. Quantification (described in section 2.6.4) showed the number of tau-positive oligodendrocytes to increase with time following MCAO. Twenty min following MCAO the number of tau-positive oligodendrocytes / mm$^2$ in the ipsilateral subcortical white matter was increased slightly as compared to contralateral white matter. The number of tau-positive oligodendrocytes / mm$^2$ increased significantly 40 and 80 min following MCAO in the white matter ipsilateral to the occluded MCA. Increased cell density was detected with all three antibodies: Tau 1, TP70 and TP007. Data are presented as Mean ± SEM, 20 min (n=3), 40 min (n=4) and 80 min (n=3).
Figure 38. Identification of tau-positive glia present after MCAO as oligodendrocytes. A: Section labelled with Tau 1 shown in purple and GFAP shown in grey. Tau-positive cells were clearly GFAP-negative and therefore immunologically distinct from astrocytes. In addition tau-positive cells were morphologically distinct from astrocytes, which displayed an elaborate array of processes (arrows), having a compact, rounded appearance consistent with that of oligodendrocytes. B: Section double labelled with Tau 1 shown in purple and transferrin shown in grey. Tau-positive cells stained positive for transferrin, the coincident staining appears black. These cells were therefore immunologically and morphologically similar to oligodendrocytes. Scale bar = 50µm.
In contrast to those directed towards tau, oligodendrocytes were not detected with antibodies directed towards MAP2, MAP5 or c-fos in the subcortical white matter contralateral or ipsilateral to the occluded MCA at any time following the induction of cerebral ischaemia (Fig. 39).
**Figure 39.** MAP2, MAP5 and c-fos immunostaining of subcortical white matter 40 min following the induction of focal cerebral ischaemia in the rat. In contrast to tau, MAP2, MAP5 or c-fos immunoreactivity was not detected in oligodendrocytes of the ipsilateral subcortical white matter 40 min following MCAO. Together this data suggests that the induction of tau immunoreactivity in oligodendrocytes following MCAO in the rat is a specific response of these cells to injury. Scale bar = 50μm.
3.3.3 Neuronal distribution of MAPs following permanent focal cerebral ischaemia in the rat
The purpose of the study described above was to examine tau immunostaining in oligodendrocytes in response to MCAO. However, in addition to changes in tau described within oligodendrocytes in the previous section, changes in the neuronal distribution of tau were also observed. Alteration to the neuronal distribution of tau were distinct from those observed in oligodendrocytes and therefore for comparison the following section describes these changes.
**Tau**
The distribution of Tau 1 immunoreactivity was predominantly located within axons of the hemisphere contralateral to the occluded MCA (Fig. 40). Twenty min following MCAO, Tau 1 immunoreactivity was increased within neuronal perikarya of both the cortex and caudate nucleus ipsilateral to the occluded MCA (Fig. 40). In contrast, there was no significant alteration in Tau 1 staining of the neuropil within these areas. Forty min after MCAO, Tau 1 immunoreactivity was increased within neuronal perikarya of both the cortex and caudate nucleus. In addition, increased immunoreactivity was detected within the neuropil of the caudate nucleus (Fig. 40). Eighty min following MCAO, the number of Tau 1 positive neuronal perikarya detected within the cortex, ipsilateral to the occluded MCA, decreased as compared to that present 40 min after MCAO. Within this area of the cortex, decreased neuropil staining was also detected as compared to the contralateral hemisphere. Tau 1 staining remained increased within the neuropil of the caudate nucleus ipsilateral to the occluded MCA.
TP70 and TP007 detected tau both within neuronal perikarya and axons in the hemisphere contralateral to the occluded MCA (Fig 41 & 42). TP70 and TP007 immunoreactivity remained relatively unchanged within neuronal perikarya and neuropil of the cortex and caudate nucleus ipsilateral to the occluded MCA, 20 min following the induction of cerebral ischaemia. In contrast to Tau 1, TP70 (Fig. 41) and TP007 (Fig. 42) staining of neuronal perikarya was decreased in the cortex and caudate nucleus, ipsilateral to the occluded MCA, 40 and 80 min following the induction of cerebral ischaemia, as compared to the contralateral hemisphere. TP70 immunostaining was decreased slightly within the neuropil of the cortex and caudate nucleus, ipsilateral to the occluded MCA (Fig. 41), in contrast TP007 immunoreactivity remained relatively unaltered (Fig. 42).
Figure 40. Tau 1 immunoreactivity within neurons 40 min following MCAO in the rat. Tau 1 immunoreactivity was increased in neuronal perikarya of the cortex and caudate nucleus in the hemisphere ipsilateral to the occluded MCA, 40 min after the induction of cerebral ischaemia as compared to controls. Axonal staining was not significantly altered within the cortex as compared to controls, however increased axonal staining was detected in the caudate nucleus 40 min after MCAO as compared to controls. Scale bar = 100μm.
Figure 41. TP70 immunoreactivity within neurons 40 min following MCAO in the rat. In contrast to Tau 1, TP70 immunoreactivity was decreased within neuronal perikarya of the cortex and caudate nucleus ipsilateral to the occluded MCA, 40 min following the induction of cerebral ischaemia, as compared to control. Axonal staining was decreased within the cortex and caudate nucleus ipsilateral to the occluded MCA 40 min following MCAO. Note the tau positive glia (arrows) within the caudate nucleus. Scale bar = 100 μm.
Figure 42. TP007 immunoreactivity within neurons 40 min following MCAO in the rat. Similarly to TP70, TP007 immunoreactivity was decreased within neuronal perikarya of the cortex and caudate nucleus ipsilateral to the occluded MCA, 40 min following the induction of cerebral ischaemia as compared to control. Axonal staining was not significantly altered in the cortex or caudate nucleus ipsilateral to the occluded MCA. Scale bar = 100μm.
MAP2
Twenty min following MCAO, MAP2 immunoreactivity was increased in neuronal perikarya of both the cortex and caudate nucleus ipsilateral to the occluded MCA as compared to the contralateral hemisphere (Fig. 43). An area where MAP2 staining was decreased within neuropil outlined the boundary of tissue affected by MCAO within the caudate nucleus and cerebral cortex. Forty or 80 min following MCAO, decreased MAP2 immunoreactivity was detected within neuronal perikarya and neuropil of the caudate nucleus within the hemisphere ipsilateral to the occluded MCA (Fig. 43). MAP2 immunoreactivity was increased in neuronal perikarya and decreased in the neuropil of the cortex, however the number of positive cells was decreased compared to that present following 20 min MCAO (Fig. 43).
MAP5
MAP5 immunoreactivity was detected both within neuronal perikarya and neuropil of the cortex and caudate nucleus contralateral to the occluded MCA (Fig. 44). Twenty min following MCAO, increased MAP5 immunoreactivity was detected in neuronal perikarya of both the cortex and caudate nucleus ipsilateral to the occluded MCA as compared to the contralateral hemisphere. No significant difference in MAP5 immunostaining was detected within the neuropil. Forty and 80 min after MCAO MAP5 immunoreactivity remained increased in perikarya of the cortex and caudate nucleus ipsilateral to the occluded MCA, however the number of cells stained with MAP5 was decreased in the cortex as compared to that following 20 min MCAO (Fig. 44). Decreased staining of the neuropil was detected within the cortex ipsilateral to the occluded MCA, as compared to that of the contralateral hemisphere.
Figure 43. MAP2 immunoreactivity within neurons 40 min following MCAO in the rat. Increased MAP2 immunoreactivity was detected in neuronal perikarya, while neuropil staining decreased within the cortex ipsilateral to the occluded MCA 40 min following the induction of cerebral ischaemia. In the hemisphere ipsilateral to the occluded MCA, MAP2 staining was decreased within both the neuronal perikarya and neuropil of the caudate nucleus, as compared to the contralateral hemisphere. Scale bar = 100μm.
Figure 44. MAP5 immunoreactivity in neurons 40 min following MCAO in the rat. Increased MAP5 immunoreactivity was detected in neuronal perikarya within the cortex and caudate nucleus, in the hemisphere ipsilateral to the occluded MCA 40 min following the induction of cerebral ischaemia as compared to control. In the hemisphere ipsilateral to the occluded MCA, MAP5 immunoreactivity was decreased in neuropil within the cortex and caudate nucleus as compared to the contralateral hemisphere. Scale bar = 100μm.
3.3.4 Mechanisms underlying increased tau immunoreactivity in oligodendrocytes following focal cerebral ischaemia.
Glutamate excitotoxicity is involved in the mechanisms underlying the neurodegeneration which occurs following ischaemic brain injury (for review see McCulloch, et. al. 1991) and intracortical perfusion in the rat induced tau-positive oligodendrocytes. Therefore it might be speculated that the presence of tau immunoreactivity within oligodendrocytes in the focal ischaemia model may be induced by the actions of elevated extracellular concentrations of glutamate (Butcher, et. al. 1990). However the ability of intracortical NaCl perfusion to induce a similar response suggests that additional mechanisms may be involved in the induction of tau immunoreactivity within these cells. In addition to glutamate receptor activation, free radical mediated mechanisms have also been implicated in the pathology of cerebral ischaemia (see section 1.2.3). *In vitro* studies employing pure oligodendrocyte cultures have shown that oligodendrocytes are susceptible to both glutamate toxicity and free radical mediated damage (see section 1.5.7). The next study was therefore designed to determine whether the presence of tau immunoreactivity within oligodendrocytes following focal cerebral ischaemia in the rat, was initiated through glutamate receptor activation or free radical mediated mechanisms. In order to do this, rats were pre-treated with the NMDA receptor antagonist MK-801, the AMPA receptor antagonist NBQX or the spin trap agent PBN. Previous studies using the MCAO rat model of focal ischaemia have shown these agents at the doses used in this study reduced the volume of infarcted tissue following permanent MCAO in the rat (Cao and Phillis, 1994; Gill, et. al. 1992; Park, et. al. 1988).
The time course study described in section 3.3.2 demonstrated that the number of tau-positive oligodendrocytes / mm$^2$ detected in the subcortical white matter ipsilateral to the occluded MCA was markedly increased 40 min after the induction of focal cerebral ischaemia in the rat. This was the earliest time point following MCAO that significant numbers of tau-positive cells were detected within the subcortical white matter ipsilateral to the occluded MCA and was therefore selected for the drug intervention study. Similarly to the previous quantitative study, for each animal the total number of tau-positive oligodendrocytes in the entire subcortical white matter, extending from that underlying the perirhinal cortex to that underlying cingulate cortex was counted at two distinct brain levels, as described in section 2.6.4. The area of subcortical white matter within which tau-positive cells were counted was measured for each animal and the number of tau-positive cells / mm$^2$ subcortical white matter was calculated for both the ipsilateral and contralateral hemispheres. Thirty min prior to MCAO rats received PBN (100mg/kg; n=5), MK801 (0.5mg/kg, n=5) or NBQX (30mg/kg; n=4), NBQX was administered again 30 min following MCAO. Forty min following MCAO animals were killed by transcardiac perfusion of 4%
paraformaldehyde, and the brains processed for tau immunohistochemistry. Animals subjected to 40 min MCAO with no therapeutic intervention served as controls in this study.
**Ipsilateral hemisphere**
Pre-treatment with the spin trap agent PBN reduced the number of tau-positive oligodendrocytes / mm$^2$ by approximately 50% within the subcortical white matter ipsilateral to the occluded MCA of animals subjected to 40 min cerebral ischaemia as compared to untreated animals (Fig. 45). This marked reduction in the number of oligodendrocytes / mm$^2$ subcortical white matter ipsilateral to the occluded MCA was consistently detected using all 3 tau antibodies: Tau 1 (recognises a dephosphorylated epitope between residues 199-202); TP70 (directed towards the C-terminal of tau) and TP007 (directed towards the N-terminal of tau) (Fig 45).
**Figure 45.** Effect of PBN pre-treatment on the number of tau-positive oligodendrocytes present in the ipsilateral succortical white matter 40 min following MCAO. Pre-treatment with the spin trap agent PBN (100mg/kg) significantly reduced the number of tau-positive oligodendrocytes / mm$^2$, as detected with Tau 1, TP70 and TP007, in the ipsilateral subcortical white matter as compared to untreated animals (* p<0.05). Data is presented as Mean ± SEM. Statistical analysis consisted of ANOVA followed by Student's t-test with Bonferroni correction.
By contrast, pre-treatment with the glutamate receptor antagonists MK801 or NBQX failed to decrease the number of tau-positive oligodendrocytes / mm$^2$, as detected with Tau 1, TP70 and TP007, in the subcortical white matter ipsilateral to the occluded MCA compared to untreated animals (Fig. 46). Moreover, pre-treatment with MK801 and NBQX increased the number of tau-positive oligodendrocytes in the ipsilateral subcortical white matter by up to 50% and 25% respectively. The statistical significance of this increase was not tested due to the *a priori* hypothesis being tested i.e. that drug intervention would decrease the response in the ischaemic hemisphere. Together these data suggest that the mechanisms which lead to increased tau immunoreactivity in oligodendrocytes following focal cerebral ischaemia at least in part involve free radicals but not glutamate receptor activation.
**Figure 46.** Effect of MK801 and NBQX pre-treatment on the number of tau-positive oligodendrocytes 40 min following MCAO. Pre-treatment with the NMDA receptor antagonist MK801 (0.5mg/kg) or the AMPA receptor antagonist NBQX (30mg/kg, x2) failed to significantly reduced the number of tau-positive oligodendrocytes / mm$^2$, as detected with Tau 1, TP70 and TP007, in the ipsilateral subcortical white matter compared to untreated animals. Data is presented as Mean ± SEM. Statistical analysis consisted of ANOVA followed by Student’s $t$-test with Bonferroni correction.
**Contralateral hemisphere**
Due to the unilateral nature of the ischaemic brain damage resulting from MCAO, the contralateral hemisphere served as a control for the effect of drug treatment alone on tau immunoreactivity in oligodendrocytes. In animals pre-treated with PBN, the number of tau-positive oligodendrocytes / mm$^2$ in the contralateral hemisphere 40 min following MCAO was not significantly different from that present in untreated animals (Fig. 47). This shows that PBN alone did not have an effect on tau immunoreactivity within oligodendrocytes distant from the area of ischaemic brain injury.
**Figure 47.** The effect of PBN pre-treatment on tau-positive oligodendrocytes within the contralateral subcortical white matter 40 min following MCAO. Pre-treatment with PBN (100mg/kg) did not significantly alter the number of tau-positive oligodendrocytes / mm$^2$, as detected with Tau 1, TP70 or TP007 as compared to untreated animals. This shows that PBN alone at this concentration does not have an effect on tau immunoreactivity in oligodendrocytes. Data is presented as Mean ± SEM.
Pre-treatment with MK801 or NBQX however increased the number of tau-positive oligodendrocytes in the contralateral subcortical white matter compared to untreated animals (Fig. 48). Thus treatment with these glutamate receptor antagonists alone at the concentrations employed in the present study have an effect on tau in oligodendrocytes distant from the area of ischaemia. Again the statistical significance of this increase was not tested due to the *a priori* hypothesis being tested i.e. that drug intervention would decrease the response in the ischaemic hemisphere.
**Figure 48.** The effect of MK801 and NBQX pre-treatment on tau-positive oligodendrocytes within the contralateral subcortical white matter 40 min following MCAO. Following pre-treatment with MK801 (100mg/kg) or NBQX (2 x 30mg/kg), the number of tau-positive oligodendrocytes / mm$^2$ subcortical white matter increased by up to 300% and 100% respectively as compared to untreated animals. This increase was detected with each of the 3 tau antibodies used: Tau 1; TP70 and TP007, showing that MK801 and NBQX alone have an effect on tau immunoreactivity in oligodendrocytes. Data is presented as Mean ± SEM.
3.4 The effect of permanent focal cerebral ischaemia on the synthesis of tau in oligodendrocytes
In all the experimental models used so far and in the human post-mortem brain tissue, increased tau immunoreactivity was detected by antibodies directed towards the N- and C-terminals, as well as the middle portion of the protein. This suggests that full-length tau may be present in oligodendrocytes and that increased staining represents increased protein levels. The next question to be addressed therefore, was whether tau synthesis was increased under the same pathological circumstances as those in which increased immunoreactivity was present. In situ hybridisation studies were performed in order to determine whether tau immunoreactivity detected within oligodendrocytes following permanent MCAO in the rat reflects increased de novo synthesis of the protein. In this study rats were killed 40 min (n=2), 120 min (n=2) and 240 min (n=2) after the induction of permanent cerebral ischaemia and the brains processed for in situ hybridisation.
An anti-sense $^{35}$S labelled riboprobe was synthesized from a 1031bp fragment generated from the coding region of rat tau cDNA (see section 2.3). Dark field microscopy, revealed neurons of the cerebral cortex as the most intensely labelled cells in rat brain sections (Fig. 49). High signal was also detected in neurons of the caudate nucleus and other areas of grey matter, however these were labelled to a lesser extent than cortical neurons. Low levels of signal were detected in the subcortical white matter, however the cellular localisation of this signal was difficult to distinguish. The level of signal detected in the white matter was only slightly higher than that detected in sections labelled with a sense riboprobe, suggesting that this signal may represent non-specific hybridisation (Fig. 49).
Forty, 120 or 240 min following the induction of ischaemia, no significant difference in the distribution of tau mRNA in oligodendrocytes was detected within the white matter ipsilateral to the occluded MCA, as compared to the contralateral hemisphere (Fig. 50). However, 120 or 240 min after MCAO tau mRNA was decreased within the neurons of the cortex within the region of ischaemic tissue (Fig. 51)
Figure 49. Distribution of tau mRNA in normal rat brain tissue. Tau mRNA was detected using a $^{35}$S labelled riboprobe directed towards the coding region of rat tau cDNA. Dark field microscopy revealed that tau mRNA was detected predominantly within neurons of the cerebral cortex in histologically normal tissue. Signal present in the subcortical white matter was, by contrast, much lower than that present in grey matter. In situ hybridisation using a sense riboprobe resulted in similar levels of signal within the subcortical white matter, suggesting that signal detected in the subcortical white matter using the antisense riboprobe represent non-specific hybridisation of the probe. Scale bar = 200μm.
Figure 50. Distribution of tau mRNA in the subcortical white matter following MCAO in the rat. No significant increase in tau mRNA was detected in the subcortical white matter ipsilateral to the occluded MCA, 40 min (b), 120 min (c) or 240 min (d) following MCAO as compared to the contralateral white matter (a). Scale bar = 200 μm.
Figure 51. The distribution of tau mRNA in neurons following MCAO in the rat. Tau mRNA as detected with a $^{35}$S riboprobe directed against the coding region of tau, was decreased in the ipsilateral hemisphere 120 min (c) and 240 min (d) following MCAO as compared to control (a). No significant alteration in the pattern of tau mRNA was seen 40 min following MCAO as compared to controls. Scale bar = 200 $\mu$m.
3.5 Tau protein in an O-2A oligodendrocyte cell line
The previous experiments show that the induction of tau immunoreactivity in oligodendrocytes is a rapid response of these cells to acute brain injury and is initiated through free radical mediated mechanisms. However, whether the presence of tau in oligodendrocytes results through direct injury to these cells or is mediated through neuronal degeneration is not known. Pure oligodendrocyte cultures derived from an O-2A cell line, kindly prepared by Dr S.C. Barnett were therefore utilised to investigate the direct effects of pathological stimuli on tau immunoreactivity.
3.5.1 Western blot analysis of tau in oligodendrocyte cultures
In normal rat brain tau exists as six isoforms. To determine whether tau was present in oligodendrocytes in culture, extracts from these cultures were resolved on SDS-PAGE and blotted with Tau 1 and TP70 antibodies. Both antibodies detected multiple isoforms of tau in oligodendrocyte cultures, however each resulted in a different banding pattern. TP70 detected four major bands between 40kDa and 60kDa (Fig. 52). Tau 1 detected five bands within a similar molecular weight range, however the highest molecular weight band (~60kDa) detected with TP70 was not detected with this antibody, suggesting that this isoform may be phosphorylated at the Tau 1 epitope. These results show that oligodendrocyte cultures used in this study contain tau protein.
3.5.2 Tau immunoreactivity in oligodendrocyte cultures
Tau immunoreactivity in control cultures
In contrast to oligodendrocytes in normal rat brain tissue, oligodendrocyte cultures derived from an O-2A cell line, maintained under normal culture conditions, stained positive for tau as detected with Tau 1, TP70 and TP007 (Fig. 53, 54 & 55). This is in agreement with western blot analysis of these cultures which showed the presence of multiple tau isoforms in control cultures and suggests that full length tau protein is present in these cells. Each antibody showed a distinct pattern of immunoreactivity. TP70 and Tau 1 stained the cellular cytoplasm within the cell body and processes, while TP007 immunoreactivity appeared to be within the nucleus. Nuclear staining often represents non-specific binding, it is possible therefore that TP007 may recognise an unrelated epitope in the nucleus of these cells. TP007 immunoreactivity remained nuclear following exposure to glutamate and NaCl (Fig. 55) and the results were therefore disregarded.
Figure 52. Western blot analysis of tau in cultured oligodendrocytes, using TP70 and Tau 1. Similarly to tau present in whole rat brain homogenates (lanes 1 & 3), tau extracted from oligodendrocyte cultures consisted of multiple isoforms (lanes 2&4). Rat brain homogenates displayed tau isoforms between 52 & 68kDa as described previously (Drubin, et. al. (1984). In oligodendrocyte extracts, TP70 detected 3 major bands ranging from 44-66kDa (lane 2). Tau 1 however detected 5 bands ranging from 44-60kDa (lane 4).
Monosodium glutamate dose response curve
All experiments described below were carried out in duplicate on three occasions using different batches of oligodendrocyte cultures. Pure oligodendrocyte cultures were exposed to 10µM, 100µM or 1mM monosodium glutamate for 4 h at 37°C in a CO₂ incubator. Immediately following incubation, the cells were rinsed and immunostained with Tau 1 (Fig. 53). Tau 1 immunoreactivity remained relatively unchanged following exposure of oligodendrocyte cultures to 10µM monosodium glutamate as compared to control cultures. Following exposure to 100µM monosodium glutamate Tau 1 immunoreactivity was significantly increased both within the cytoplasm of the cell body and processes. Tau 1 immunoreactivity was increased following exposure to 1mM monosodium glutamate as compared to those treated with 100µM monosodium glutamate. However, these tau-positive oligodendrocytes exhibited signs of degeneration, the cells were swollen in appearance and disintegration of processes was detected. From these results 100µM monosodium glutamate was selected to investigate the effect of sublethal glutamate induced injury on tau immunoreactivity in pure oligodendrocyte cultures.
Effect of 100µM monosodium glutamate and NaCl on tau immunoreactivity in oligodendrocyte cultures
As a control for these studies, oligodendrocyte cultures were exposed to 100µM NaCl for 4 h prior to tau immunostaining. Increased Tau 1 immunoreactivity was detected in oligodendrocyte cultures treated with either 100µM monosodium glutamate or 100µM NaCl (Fig. 54). However, the intensity of Tau 1 staining was less in NaCl treated cultures as compared to those treated with glutamate (Fig. 54). Dephosphorylation of control cultures prior to Tau 1 immunostaining resulted in a similar increase in the intensity of Tau 1 staining following treatment with 100µM monosodium glutamate and NaCl (Fig. 54). In contrast to the increased Tau 1 immunoreactivity detected following exposure of cultures to 100µM monosodium glutamate or NaCl, TP70 staining remained largely unchanged as compared to controls (Fig. 55). Together these results suggest that, in contrast to the accumulation of tau in oligodendrocytes detected in vivo following glutamate or NaCl perfusion, tau undergoes dephosphorylation following cytotoxic injury in vitro. All results were obtained in duplicate on three separate occasions.
Figure 53. Tau 1 immunoreactivity in oligodendrocyte cultures treated with 10µM, 100µM or 1mM monosodium glutamate for 4 h. Similarly to that present in control cultures (a), Tau 1 immunoreactivity was detected within the cytoplasm of the cell body and processes of oligodendrocytes following exposure of these cultures to 10µM monosodium glutamate (b). However, following exposure to 100µM monosodium glutamate (c), Tau 1 immunoreactivity was increased both within the cell body and processes. Treatment of cultures with 1mM monosodium glutamate resulted in increased Tau 1 immunoreactivity, however cells showed signs of degeneration, such as the disintegration of cellular processes (arrows) and swelling of the cell body (d). Scale bar = 100µm.
Figure 54. Tau 1 immunoreactivity in oligodendrocyte cultures derived from an O-2A cell line. Tau 1 immunoreactivity was detected within the cytoplasm of the cell body and processes of oligodendrocytes maintained in Sato’s growth medium. Following exposure of these cultures to 100µM monosodium glutamate or 100µM NaCl, Tau 1 immunoreactivity was increased both within the cell body and processes. Dephosphorylation of control cultures prior to immunostaining resulted in a similar pattern of Tau 1 immunoreactivity within these cells as detected following treatment with 100µM monosodium glutamate or NaCl. Scale bar = 100µm.
Figure 55. TP70 and TP007 immunoreactivity in oligodendrocyte cultures derived from an O-2A cell line. TP70 immunoreactivity was detected in oligodendrocyte cultures maintained in Sato’s growth medium. Immunoreactivity was detected within the cytoplasm of the cell body and processes, while TP007 was localised mainly to the cell nucleus. Following treatment of cultures with 100µM monosodium glutamate or 100µM NaCl, the distribution of TP70 and TP007 immunoreactivity was relatively unchanged. The localisation of TP007 immunoreactivity to the nucleus, suggests that this antibody may bind non-specifically to an unrelated protein within these cells. Scale bar = 100µm
CHAPTER 4 DISCUSSION
The original aim of my thesis was to investigate the possible mechanisms underlying cytoskeletal breakdown in neurons following acute brain injury. However, in the first study investigating the possible role of glutamate excitotoxicity in this process, tau immunoreactivity was detected within oligodendrocytes. This intriguing finding indicated that cells other than neurons within the CNS may respond to acute brain injury with changes in tau protein. My studies were therefore directed at further characterising the presence of tau in oligodendrocytes following acute brain injury and determining the mechanisms underlying this response. Until recently oligodendrocytes have been considered to be relatively inactive during cerebral ischaemia, however the work in this thesis shows that oligodendrocytes have the ability to respond rapidly to acute brain injury and may therefore play an important role in the progression of ischaemic brain damage. I will firstly describe changes in the oligodendrocyte cytoskeleton that occur following acute brain injury and then go on and compare these to the alterations of the cytoskeleton which occur in neurons.
4.1 Detection of the "neuronal" protein tau in oligodendrocytes following acute brain injury
4.1.1 Distribution of tau in histologically normal brain tissue
The normal distribution of tau in the CNS has been well documented (see section 1.4.2) and it is generally accepted that tau is predominantly located within neurons where it is most abundant in the axonal compartment (Binder, et. al. 1985; Brion, et. al. 1988; Papasozomenos and Binder, 1987; Trojanowski, et.al. 1989). Moreover, tau mRNA was shown to be located specifically within neurons *in situ* (Goedert, et. al. 1989; Kosik, et. al. 1989a; 1989b). In the studies carried out throughout this thesis, tau immunoreactivity was found located mainly within neurons in histologically normal rat and human brain tissue, supporting a predominantly neuronal localisation of tau. Tau immunoreactivity was not detected within glia in histologically normal tissue using antibodies directed towards various regions of tau: Tau 1 (recognises a dephosphorylated epitope in the middle portion of the protein); TP70 (raised against C-terminal portion of tau); TP007 (raised against the N-terminal portion of tau) and B19 (raised against full-length tau). In addition, dephosphorylation of histologically normal rat or human brain tissue failed to induce Tau 1 immunoreactivity in glial cells, suggesting that the lack of Tau 1 immunoreactivity in these cells is not due to masking of the protein by phosphorylation. In support of this TP70, TP007 and B19 antibodies, which detect tau independent of its phosphorylation state, also failed to detect tau in glial cells in normal rat or human brain tissue. These results suggest that tau may be absent from glial cells in histologically normal human and rat brain tissue, or
may reflect very low levels of tau within mature glia *in vivo* which are undetectable using standard immunohistochemical techniques. This is in contrast to a previous study which reported the presence of tau immunoreactivity in glial cells and neurons (Migheli, et. al. 1988). In addition, Tau 1 immunoreactivity was detected in perineuronal oligodendrocytes and astrocytes but not intrafascicular oligodendrocytes in rat brain following enzymatic dephosphorylation of the tissue (Papazosmenos and Binder, 1987). However, in agreement with the present study, Lopresti, et. al. (1995) failed to detect oligodendrocytes in normal rat brain using Tau 1 however did detect tau in intrafascicular oligodendrocytes using Tau 5, an antibody raised specifically towards glial tau. The possibility that tau present in intrafascicular oligodendrocytes in normal brain may be folded in a particular conformation within the oligodendrocyte thus preventing antibody binding cannot be ruled out. However, it is difficult to envisage that epitopes located in the middle of the protein and at the N- and C- terminals of the protein would all be masked under normal conditions and all uncovered following injury. Furthermore the immunostaining detected using the polyclonal antibodies TP70 and TP007 raised against synthetic fragments of tau is unlikely to be altered by conformational change in the protein.
**4.1.2 Tau immunoreactivity in oligodendrocytes following acute brain injury**
Due to the vast amount of evidence suggesting that tau is a neuron-specific protein and the inability of the tau antibodies used throughout this thesis to detect tau in glial cells in histologically normal brain tissue, the most intriguing and novel finding of this thesis was the induction of tau immunoreactivity in small cells with the morphological appearance of glia following acute brain injury. Double label immunohistochemistry, utilising GFAP as a cellular marker for astrocytes and transferrin as a cellular marker for oligodendrocytes, identified these tau-positive glial cells as oligodendrocytes. Tau-positive oligodendrocytes were detected in all animal models of acute brain injury and in post-mortem brain tissue from patients who died following a stroke or head injury. These tau-positive cells were found located in both white and grey matter depending on the location of the brain injury. In white matter these cells were arranged in rows with the appearance of intrafascicular oligodendrocytes, while those in grey matter were found located around neurons and were therefore assumed to be perineuronal oligodendrocytes. Tau immunostaining was observed with antibodies directed towards full length tau, and the N- and C-terminals of the protein suggesting the accumulation of full-length tau protein within these cells. Tau-positive oligodendrocytes were also detected using the Tau 1 antibody, showing that tau present in these cells following injury was dephosphorylated at the Tau 1 epitope. It is possible that injury to oligodendrocytes may result in a conformational change in tau which results in increased antigenicity, however as discussed above it is unlikely that all the epitopes
examined in this study would be masked and unmasked equally before and after injury. Together these findings do not support the complete absence of tau in intrafascicular or perineuronal oligodendrocytes within the adult rat and human brain. In addition, to the evidence described above showing tau within oligodendrocytes in normal rat brain tissue, glial inclusions characteristic of chronic neurodegenerative conditions such as MSA, PSP and PD (Abe, et. al. 1992; Iwatsubo, et. al. 1994; Nishimura, et. al. 1992; Yamada, et. al. 1992) have been shown to stain positive for tau. Tau present in these cells can be detected with antibodies directed towards PHF tau, suggesting that in addition to neurons, tangle formation may also occur in glial cells during chronic progressive degeneration of the brain.
Although oligodendrocytes contain abundant microtubules and would therefore be expected to contain many MAPs, in contrast to tau, neither MAP2 nor MAP5 immunoreactivity was detected in oligodendrocytes before or after acute brain injury in human or rat brain tissue. This is in contrast to previous studies which report the presence of both MAP2 and MAP5 immunoreactivity in oligodendrocytes (Fischer, et. al. 1990; Ulloa, et. al. 1994b; Vouyiouklis and Brophy, 1995). The possibility that oligodendrocytes *in situ* express levels of these proteins below the detection limits of standard immunohistochemical techniques cannot be ruled out. However, mRNA for MAP2 and MAP5 has been located only within neurons *in vivo* (Garner, et. al. 1988; Landry, et. al. 1994; Papadrikopoalou, et. al. 1986; Tucker, et. al. 1989) suggesting that glial cells *in situ* do not express these proteins. These studies highlight the caution that should be exercised when extrapolating events occurring *in vitro* to those occurring *in vivo* where the cellular milieu is more complex.
### 4.1.3 Tau in oligodendrocyte cell cultures
Further supporting the presence of tau in non-neuronal cells, the results presented in this thesis show the presence of tau in pure oligodendrocyte cultures derived from an O-2A cell line. In the adult rat brain, tau protein comprises six isoforms produced by alternative splicing of a single mRNA transcript. Similarly, tau present in the oligodendrocyte cultures also comprised several isoforms. This finding is in agreement with those of Lopresti, et. al. (1995) who also showed the presence of multiple tau isoforms in primary oligodendrocyte cultures. Immunoblotting with TP70 detected 3 major bands ranging from 40-66kDa. In contrast Tau 1 detected 5 major bands within a similar molecular weight range, however failed to detect the highest molecular weight isoform detected with TP70. In addition to the multiple isoforms reported here, Lopresti, et. al. (1995) also showed the presence of a higher molecular weight band of approximately 80kDa. The distinct pattern of isoform detection with Tau 1 and TP70 suggests that, as with neuronal tau, isoforms present in oligodendrocytes are modified by phosphorylation. Together these results confirm the
presence of tau in oligodendrocytes maintained in normal culture conditions. To ascertain whether these bands represent an oligodendrocyte specific isoform pattern or whether each band represents differentially phosphorylated forms of a single isoform would require isolation and characterisation of the tau protein and characterisation of the tau transcripts present within these cells.
In contrast to oligodendrocytes present in the normal rat CNS, tau as detected with Tau 1 and TP70 was present within oligodendrocytes *in vitro* maintained in normal growth conditions. In addition, the distribution of tau was different in oligodendrocyte cultures as compared to those *in situ* following acute brain injury. In oligodendrocytes resident within adult rat brain, tau immunoreactivity was located only within the cell body, no staining was present in the cellular processes. This is probably not surprising however due to the intricate architecture of the mature CNS; oligodendrocyte processes are not readily detected *in situ* at the light microscope level. *In vitro* however TP70 and Tau 1 immunoreactivity was detected both within the cytoplasm of the oligodendrocyte cell body and the cellular processes. The possibility that discrepancies between *in vivo* and *in vitro* studies is due to the accessibility of the tau epitopes to the antibodies cannot be ruled out.
Detection of tau with TP70 and to a lesser extent Tau 1, suggests the presence of full-length tau protein in these cells under normal culture conditions. It should be noted that although oligodendrocytes were detected with Tau 1, the intensity of this staining was far less than that of TP70, suggesting that a proportion of tau present in these cells is phosphorylated at the Tau 1 epitope. Immunostaining with Tau 1 and TP70 was continuous along the length of the oligodendrocyte processes. This contrasts with the findings of Lopresti, et. al. (1995) who reported discontinuous staining within the cellular processes. In their studies, tau staining was segmental, present at the tips of the processes and in the membrane expansions present at the end of each process. It is possible to speculate that discrepancies between these 2 studies may reflect differential stages of maturity of the cultures used in the 2 studies, since cells used in the investigations carried out in this thesis did not show any membrane expansions present in their cultures. Alternatively this may reflect species differences since cultures used throughout this thesis were derived from rat tissue while those employed by Lopresti, et. al. 1995 were derived from ovine brain. In contrast to these 2 studies Vouyiouklis and Brophy (1995) failed to show the presence of tau in rat primary oligodendrocyte cultures using the monoclonal antibody Tau 2 directed towards full-length tau. This may be partly explained by the poor cross-reactivity of this antibody with rat tissue (Binder, et. al. 1985).
The apparent lack of tau immunoreactivity in oligodendrocytes *in situ* may reflect levels of protein in these cells which are below the limits of detection using standard immunohistochemical techniques, however tau protein may be more abundant in oligodendrocyte cultures than those present *in vivo*. Cell cultures are derived from immature tissue and, although these cells were grown for 5 days and showed immunological characteristics of mature oligodendrocytes, it is possible that they may still be undergoing development. Oligodendrocytes have to extend multiple processes required for the effective myelination of axons and in addition, proteins required for myelin formation have to be transported to the site of myelinogenesis. There is a general consensus that the oligodendrocyte cytoskeleton is involved in oligodendrocyte process extension and myelin assembly (Brophy, et. al. 1993). Tau has been shown to be involved in process extension in neurons (Cáceres and Kosik, 1990; Cáceres, et. al. 1991) and presuming that tau has similar functional properties in both neurons and oligodendrocytes, tau may also play a key role in process extension in oligodendrocytes. Supporting this theory, the tips of growing oligodendrocyte processes, like neuronal growth cones are known to be actin rich (Kachar, et. al. 1986) and previous studies have shown that tau binds to actin *in vitro* (Seldon and Pollard, 1983). Taken together this data supports a role for tau in oligodendrocytes similar to that proposed for neuronal tau in the growth cones of cerebellar neurons. That is, mediating interactions between elements of the growth cone cytoskeleton, therefore maintaining its structural organization (DiTella, et. al. 1994). It is possible therefore that until the extension of processes and myelination is established, oligodendrocytes may express high levels of tau protein. Therefore, oligodendrocytes present in culture, although expressing markers of mature oligodendrocytes, may still be extending processes and thus contain high levels of tau protein. In contrast, fully developed oligodendrocytes present within the adult CNS may lack the necessity for high levels of tau protein.
The differential distribution of tau present in oligodendrocytes *in vitro* and *in vivo* highlight the care that should be exercised when extrapolating findings *in vitro* to events occurring within the adult CNS where the cellular milieu is more complex. To ascertain whether tau present in these cells is similar to that present in neurons requires further characterisation, however together the results presented in this thesis strongly indicate that tau is present in oligodendrocytes and therefore can no longer be viewed as a neuron-specific protein.
4.2 The induction of tau immunoreactivity in oligodendrocytes is a rapid response to acute brain injury
The results presented in this thesis provide compelling evidence that the induction of tau immunoreactivity in oligodendrocytes is a rapid response of these cells to acute brain injury. The first study in this thesis, showed the presence of tau-positive oligodendrocytes 4 h following intracortical perfusion of 1M monosodium glutamate both within the resulting cortical lesion and in the subcortical white matter immediately underlying this area. The presence of tau-positive oligodendrocytes in human post-mortem tissue from patients who died within 2 h following head injury confirmed that the induction of tau immunoreactivity in oligodendrocytes was not a phenomenon characteristic of rat brain, but also occurred rapidly in human brain. Tau-positive cells were detected within and immediately surrounding areas of tissue shown by decreased H&E staining to be damaged by the injury. Following severe head injury in the human, the proportion of tau-positive cells detected increased with survival time and it is possible to speculate that this pattern may reflect the evolution of brain damage over time following injury. The detection of tau-positive oligodendrocytes in post-mortem brain tissue of patients who died 23 h following head injury and several weeks following a stroke suggests that the induction of tau immunoreactivity in these glial cells is maintained over long periods of time. In order to determine just how rapidly the presence of tau-positive oligodendrocytes could be detected, animals were killed 20, 40 or 80 min following MCAO and the brains processed for tau immunohistochemistry. Twenty minutes following MCAO the number of tau-positive oligodendrocytes / mm$^2$ subcortical white matter was not significantly different in the ipsilateral hemisphere compared to the contralateral hemisphere. However, 40 and 80 min following MCAO the number of tau-positive oligodendrocytes / mm$^2$ subcortical white matter in the ischaemic hemisphere, as detected with all three antibodies: Tau 1, TP70 and TP007, was increased compared to the contralateral hemisphere. Tau-positive oligodendrocytes were located predominantly within the subcortical white matter ipsilateral to the occluded MCA where they were found aligned in rows with the appearance of intrafascicular oligodendrocytes. In addition tau-positive perineuronal oligodendrocytes were also found scattered throughout the caudate nucleus and cortex within the area of ischaemic tissue as defined by decreased MAP2 immunostaining. Thus, tau immunostaining in oligodendrocytes increased within 40 min of the ischaemic challenge, indicating that these glial cells, like neurons, have the ability to respond rapidly to injury.
Proteins coded by proto-oncogenes such as $c-fos$, act to carry cytoplasmic signals to the nucleus where they interact with the DNA enhancing the expression of several genes (Morgan and Curran, 1989; 1991). $C-fos$ mRNA expression has been shown to increase
rapidly within neurons following ischaemic brain injury in the rat (Abe and Kogure, 1993; Hsu, et. al. 1993). Increased expression of \( c-fos \) and other immediate early genes following brain injury is thought to be involved in the ability of neurons to survive following injury (Magnusson and Wieloch, 1989), however, the real significance of this response is unknown. Since tau immunoreactivity in oligodendrocytes is detected rapidly following injury, it is possible to speculate that this may be part of a "stress" response of these cells to brain injury. \( C-fos \) has recently been shown to be induced in oligodendrocytes progenitor cells \( in vitro \) following exposure to glutamate (Liu and Almazan, 1995), suggesting that increased \( c-fos \) may occur in response to injury in these cells. The distribution of \( c-fos \) immunoreactivity in oligodendrocytes was therefore examined in the rat brain following MCAO this gene product was also increased in these oligodendrocytes following injury \( in vivo \). In contrast to increased \( c-fos \) levels seen in neurons 20 min after MCAO, and in oligodendrocyte progenitor cells in vitro, \( c-fos \) was undetectable within oligodendrocytes \( in situ \) before or after MCAO. Together these results suggest that acute brain injury in the rat specifically alters tau protein in oligodendrocytes and the alteration is not due to a generalised "stress" response within these cells.
4.3 Mechanisms underlying the induction of tau immunoreactivity in oligodendrocytes
4.3.1 Glutamate toxicity \( in vivo \) induces tau immunoreactivity in oligodendrocytes
The \( in vivo \) model of glutamate toxicity used in this study has been well characterised previously (Landolt, et. al. 1993; Fujisawa, et. al. 1993; 1996) and provides a means of studying the effects of glutamate toxicity without the interference of factors such as reduced blood flow, present in models of cerebral ischaemia. In the \( in vivo \) study described in section 3.1, 1M monosodium glutamate was perfused through the dialysis probe, at a concentration far in excess of those used in previous \( in vitro \) studies (\( \mu M \) range). However, due to the dynamics of the dialysis membrane, only approximately 10% of the solution being perfused reaches the tissue where glutamate uptake into astrocytes (Hertz, et. al. 1992; Schousboe, et. al. 1988) further reduces the concentration of extracellular glutamate remaining in the tissue. The extracellular concentration of glutamate decreases further in relation to the distance from the probe as the glutamate diffuses through the tissue. The extracellular concentration of glutamate reaching the corpus callosum is therefore estimated to be within the \( \mu M \)-\( mM \) range.
Tau immunoreactivity was detected in intrafascicular and peri-neuronal oligodendrocytes within the subcortical white matter and the cortical lesion respectively, following
intracortical perfusion of 1M monosodium glutamate, suggesting that glutamate may be involved in the accumulation of tau in oligodendrocytes following acute brain injury. Monosodium glutamate was chosen to avoid the use of glutamic acid which proves problematic when maintaining a neutral pH and 1M NaCl was therefore the control solution for this study. The ability of NaCl perfusion to induce tau immunoreactivity within oligodendrocytes however, suggests that mechanisms other than glutamate toxicity may be involved in this response. It is possible to speculate that the mechanisms underlying tau accumulation in oligodendrocytes involves the presence of excessive extracellular Na$^+$. Glutamate excitotoxicity to neurons has been shown to consist of both a NaCl component and a Ca$^{++}$ component (Dessi, et. al. 1994). Influx of NaCl results in neuronal swelling and can induce neuronal degeneration without concurrent influx of Ca$^{++}$. In addition, increased extracellular sodium exacerbates the damage produced by glutamate, possibly through the disturbance of the Na$^+/K^+$ glutamate transporter. Cells from the oligodendrocyte lineage have been shown to express many ion channels \textit{in vitro} including two types of voltage dependant Ca$^{++}$ channels (Blankenfield, et. al. 1992; Verkhratsky, et al. 1990), and Na$^+$ channels (for review see Barres, et. al. 1990). In light of this it is possible to envisage that high extracellular concentrations of Na$^+$ would result in Na$^+$ influx through Na$^+/K^+$ channels, resulting in membrane depolarisation. Such depolarisation of the membrane may lead to the initiation of Ca$^{++}$ influx through voltage gated Ca$^{++}$ channels and through the mobilisation of intracellular Ca$^{++}$ stores. Moreover, it is possible that glutamate toxicity in oligodendrocytes may also consist of both a NaCl and a Ca$^{++}$ component, since glutamate and the non-NMDA receptor agonists, kainate and AMPA have been shown to cause an influx of Na$^+$ in oligodendrocyte cultures (Borges and Kettenman, 1995). Furthermore glutamate toxicity in oligodendrocytes \textit{in vitro} has also been shown to be Ca$^{++}$ dependent (Yoshioka, et. al. 1995). Disruption of Ca$^{++}$ homeostasis is thought to be involved in the formation of free radicals (Siesjö, et. al. 1989). Once free radicals are formed these cause further disruption of calcium homeostasis and therefore a vicious circle continues. It is possible to speculate therefore that free radical mediated mechanisms initiated through increased intracellular Ca$^{++}$ levels, whether through glutamate receptor activation or excess Na$^+$ ions, may be involved in the induction of tau immunoreactivity in oligodendrocytes as outlined below.
In support of this theory oligodendrocytes have been shown to be particularly susceptible to free radical damage (Kim and Kim, 1991; Oka, et. al. 1993; Thorburne and Juurlink, 1996). Moreover, in the present studies pre-treatment of animals with the spin trap agent PBN significantly reduced the number of tau-positive oligodendrocytes / mm$^2$ present in the subcortical white matter following focal cerebral ischaemia. This will be discussed more extensively in section 4.3.3.
4.3.2 Increased Tau 1 immunoreactivity in oligodendrocytes is a direct response of these cells to cytotoxic injury
Experiments employing pure oligodendrocyte cultures provided evidence that changes in tau occurring in oligodendrocytes following cytotoxic injury are induced through a direct effect on oligodendrocytes and is not mediated through neuronal degeneration. Electron microscopy showed oligodendrocytes did not show significant signs of degeneration following *in vivo* perfusion of 1M monosodium glutamate or NaCl. The *in vitro* study was therefore designed to treat the cells with a sublethal dose of monosodium glutamate and NaCl. Exposure to 100µM glutamate over 24 h has previously been reported to be sublethal to oligodendrocyte cultures (Oka, et. al. 1993). In the present study oligodendrocyte cultures were exposed to 10µM, 100µM and 1mM monosodium glutamate for 4 h. Exposure of oligodendrocytes to 10µM monosodium glutamate failed to induce changes in tau immunoreactivity within these cells, in contrast tau immunoreactivity was increased following exposure to 100 or 1mM monosodium glutamate. However morphological signs of degeneration were detected oligodendrocyte cultures exposed to 1mM monosodium glutamate, therefore 100µM monosodium glutamate was selected for this study.
Tau 1 immunoreactivity was increased in the cytoplasm of both the oligodendrocyte perikarya and processes following treatment of the cells with 100µM monosodium glutamate or 100µM NaCl for 4 h. In contrast to the *in vivo* study however, TP70 immunoreactivity remained largely unchanged following exposure to 100µM monosodium
glutamate or NaCl, although staining within the cellular processes became slightly more prominent with TP70. Dephosphorylation of cultures maintained in Sato's medium resulted in a similar intensity of Tau 1 immunoreactivity present following treatment with 100μM monosodium glutamate or NaCl. Together these results suggest that rather than accumulation of tau protein within these cells as seen *in situ*, increased Tau 1 immunoreactivity reflects dephosphorylation of existing protein in response to glutamate or NaCl exposure. However the possibility that increased TP70 levels were not detected due to the high level of immunoreactivity present in control cells cannot be ruled out.
These results show that alterations to tau in oligodendrocytes following exposure to monosodium glutamate or NaCl is a direct response of these cells to injury. This suggests that the accumulation of tau in oligodendrocytes *in vivo* following acute brain injury is not a consequence of neuronal degeneration. However they do highlight again the caution that must be exercised when relating events occurring *in vitro* to those occurring *in vivo* where the cellular milieu is more complex.
### 4.3.3 The spin trap agent PBN reduces the number of tau-positive oligodendrocytes following focal cerebral ischaemia in the rat
The presence of tau-positive oligodendrocytes following exposure to both glutamate and NaCl suggests that additional mechanisms other than glutamate receptor activation are involved in the initiation of this response. Both glutamate toxicity and free radical mediated mechanisms have been implicated in the evolution of ischaemic brain damage. Oligodendrocytes have been shown *in vitro* to be susceptible to both glutamate and free radical mediated toxicity therefore permanent MCAO in the rat was used to determine if glutamate receptor activation or free radical mediated events are involved in the mechanisms underlying the accumulation of tau in oligodendrocytes following ischaemic brain injury.
Pre-treatment of animals with the spin trap agent PBN prior to MCAO significantly reduced the number of tau-positive oligodendrocytes / mm$^2$ in the subcortical white matter ipsilateral to the occluded MCA as compared to untreated animals 40 min after the induction of focal cerebral ischaemia. This indicates that the initiating stimulus for the response involves events mediated by free radicals. Oligodendrocytes are thought to be particularly susceptible to free radical damage due to low levels of the free radical scavenger glutathione and high levels of iron which is a catalyst for free radical production (Thorburne and Juurlink, 1996). Primary cultures of bovine oligodendrocytes were shown to be susceptible to oxygen radicals derived from glucose/glucose oxidase and hypoxanthine/xanthine oxidase reactions (Kim and Kim, 1991). In that study, cytotoxicity was measured by tryptan blue exclusion and lactate dehydrogenase leakage, and it showed that oxygen radicals induce significant
membrane damage to oligodendrocytes within 2 h of exposure. In addition, Oka, et. al. (1993) showed in rat primary oligodendrocyte cultures that glutamate induced cell death was mediated, not through glutamate receptor activation, but through free radical mediated mechanisms. Excessive extracellular glutamate concentrations led to the efflux of cystine from these cells through the actions of the glutamate/cystine transporter which in turn resulted in glutathione depletion. Glutathione is an important protective agent against oxidative stress (for review see Bast et. al. 1991) and its depletion therefore renders the cell vulnerable to oxidative stress. PBN is a nitrone compound which reacts with many types of free radicals resulting in the formation of the stable nitroxide radical adducts (Oliver, et. al. 1990). Glutamate toxicity and free radical generation have been shown to be interdependent, many studies demonstrating that glutamate toxicity can be markedly reduced by treatment with free radical scavenger systems both *in vitro* (Bondy and Lee, 1993; Dykens, et. al. 1987; Murphy, et. al. 1990; Miyamoto, et. al. 1989; Nakao, et. al. 1996) and *in vivo* (Nakao, et. al. 1996; Schulz et. al. 1995). Extracellular glutamate concentrations peak at 40 min following MCAO in the rat (Butcher, et. al. 1990) and it is possible to speculate therefore that treatment with PBN protects oligodendrocytes indirectly from glutamate toxicity and directly from free radical mediated damage resulting from ischaemic injury and thus prevents accumulation of tau within these cells.
In contrast to PBN neither the NMDA receptor antagonist MK801 nor the AMPA receptor antagonist NBQX prevented increased tau immunoreactivity in oligodendrocytes following 40 min cerebral ischaemia further indicating that glutamate receptor activation is not involved in the response. However, both primary oligodendrocyte cultures and immortalised cultures derived from rat brain express non-NMDA receptor subtypes (Patneau, et. al. 1994; Puchalski, et. al. 1994; Yoshioka, et. al. 1995). Moreover, damage to these cells was mediated by non-NMDA receptor activation. Thus in our *in vivo* experimental paradigm, while it is possible that activation of AMPA receptors by excessive extracellular glutamate concentrations may damage oligodendrocytes, this signalling pathway does not appear to be involved in the tau protein response. Alternatively, the rapid desensitisation of AMPA receptors in oligodendrocytes in response to glutamate (Yoshioka, et. al. 1995) may have masked any effect of receptor blockade by NBQX. Finally although the selection of the drug doses were based on those previously shown to reduce infarct volume following permanent focal cerebral ischaemia in the rat (Cao and Phillis, 1994; Gill, et. al., 1992; Park, et. al. 1988), the possibility that the concentration of NBQX used in this study may be too low to prevent ischaemia induced accumulation of tau in oligodendrocytes cannot be ruled out. Mature oligodendrocytes have been reported to contain non-NMDA receptors, although mRNA for NMDA receptors was not detected (Patneau, 1994; Puchalski, et. al. 1994; Yoshioka, et. al 1995) and therefore it is perhaps not surprising that MK801 failed to
prevent increased tau immunoreactivity in these cells following ischaemic injury. However, the mean value of tau-positive oligodendrocytes present in both the ipsilateral and contralateral white matter was greater following MK-801 treatment compared to controls. This suggests that MK-801 alone may be able to induce increased tau in oligodendrocytes. In the absence of a direct effect on oligodendrocytes themselves, increased tau immunoreactivity within these cells may arise as an indirect consequence of neuronal changes both in proximity and distant to ischaemia. MK-801 and other NMDA ion channel blockers induce a range of marked neurochemical alterations within neurons which include glucose utilisation and heat shock protein gene expression (Kurumaji et al., 1988; Sharp, et. al. 1992). In light of the growing literature on neuronal-glia communication it may be envisaged that MK-801-induced changes in neuronal activity in turn induce responses in associated oligodendrocytes. Alterations in the cytoskeletal protein tau may represent such a response. Together the results of these studies show that the induction of tau immunoreactivity in oligodendrocytes is initiated through free radical mediated mechanisms.
4.4 Functional significance of tau in oligodendrocytes following injury
4.4.1 Protective response?
At this stage the functional significance of tau in oligodendrocytes following acute brain injury is unknown. If tau is a neuron-specific protein it is intriguing that oligodendrocytes express a neuronal protein following injury. It is possible to speculate the accumulation of tau in oligodendrocytes following injury may be part of a protective response in that dephosphorylated tau is able to bind to and stabilise microtubules (Brandt and Lee, 1994; Kosik, 1993; Trinczek, et. al. 1995). Increased tau immunoreactivity was detected with antibodies directed towards full length protein and both the N- and C-terminals of the protein suggesting the accumulation of full length protein within these cells following acute brain injury both in the rat and the human. *In situ* hybridisation studies were therefore carried out to determine if this increased immunoreactivity in oligodendrocytes following ischaemic brain injury reflects increased *de novo* synthesis of tau.
*In situ* hybridisation using a $^{35}$S-labelled riboprobe synthesized from a 1031bp fragment of the coding region of adult rat tau cDNA revealed that tau mRNA was predominantly located within the neurons of the cerebral cortex in rat brain contralateral to the occluded MCA. A low level of message was detected in white matter tracts, however the cellular localisation of this signal was difficult to interpret. Control experiments using a sense riboprobe generated from the same coding region of tau cDNA revealed a similar level of signal within the subcortical white matter, suggesting that white matter signal reflects non-specific
hybridisation of the riboprobe. This is in agreement with previous studies which detected tau mRNA in neurons, however failed to detect tau mRNA in glial cells (Goedert, et. al. 1989; Kosik, et. al. 1989). Following 40, 120 or 240 min MCAO in the rat, no significant changes in the pattern of tau mRNA distribution was detected ipsilateral to the occluded MCA. This suggests that increased tau immunoreactivity within oligodendrocytes following ischaemic brain injury does not reflect increased *de novo* synthesis of the protein. This is maybe not surprising considering the rapidity of tau accumulation in oligodendrocytes following injury. However the possibility that our detection system was not sensitive enough to detect small increases in mRNA levels within these cells cannot be ruled out.
In addition, it is possible to speculate that mRNA present in oligodendrocytes may differ from that present in neurons. Recently a 2kb and 8kb tau mRNA have been detected in a neuroblastoma cell line and the peripheral nervous system respectively, encoding nuclear tau and big tau, as opposed to the 6kb mRNA originally described for neuronal tau (Couchie, et. al. 1992; Goedert, et. al. 1992; Loomis, et. al. 1990; Wang, et. al. 1993). The riboprobe used in the present study was derived from a 1031bp fragment of the coding region of a tau cDNA encoding the 6kb mRNA of tau which includes exons 2, 3 and 10 (Kosik, et. al. 1989). However, it is unknown what type of tau mRNA is present in oligodendrocytes. Tau cDNA encoding the 8kb mRNA, contains an extra 762bp region encoded by exon 4A situated down stream of exons 2 and 3 which encode the N-terminal inserts (Goedert, et. al. 1992). In addition exon 6 further downstream from exon 4A has also been reported to be expressed in this larger isoform of tau (Couchie, et. al. 1992). The riboprobe generated in the present study encompasses 1031bp of the coding region from the 3' end of the tau cDNA. It is possible to speculate therefore that the expression of exon 4A and / or 6 would reduce the strength of hybridisation of the probe with this area of the mRNA. Signal obtained from weaker hybridisation reactions between the probe and the mRNA therefore may be lost during the stringency washes designed to reduce non-specific hybridisation.
Recently increased ferritin synthesis was reported in oligodendrocytes following 6h hypoxia *in vitro* (Qi and Dawson, 1992, 1994, 1995). In these studies the authors showed that increased protein levels were due to a post-transcriptional modification of protein synthesis. It is possible to speculate therefore that increased tau immunoreactivity within oligodendrocytes following acute brain injury may also represent post-transcriptional modification of protein synthesis, which may account for the rapid appearance of tau in these cells. If increased tau immunoreactivity does reflect a protective response of these
cells to injury, it is possible to envisage that post-transcriptional modification of protein synthesis would result in the rapid increase in tau protein levels required for their protection.
There is growing evidence that the oligodendrocyte cytoskeleton is involved in process extension and the transport of myelin proteins to the site of myelin assembly (Brophy, et al. 1993). The tips of growing oligodendrocyte processes, like neuronal growth cones are known to be actin rich (Kachar, et al. 1986) and previous studies have shown that tau binds to actin *in vitro* (Seldon and Pollard, 1983). Taken together this data supports a role for tau in oligodendrocytes similar to that proposed for neuronal tau in the growth cones of cerebellar neurons (DiTella, et al. 1994). Tau has been shown to be involved in process extension in neurons (Cáceres and Kosik, 1990; Cáceres, et al. 1991) therefore it is possible to speculate that tau also plays a key role in the extension of oligodendrocyte processes. Oligodendrocytes contain abundant microtubules which have been postulated to be key elements in the transportation of myelin proteins to the site of myelin assembly (Barbarese, et al. 1991; Brophy, et al. 1993; Colman, et al. 1982). Purified MBP can form complexes with tubulin and actin (Modesti and Barra, 1986), and using cell culture both MBP and CNP have been shown to associate with the cytoskeleton of oligodendrocytes (Colman, et al. 1982; Gillespie, et al. 1989). Both CNP and MBP are synthesized on free ribosomes and have to be transported into the processes for myelin formation. It is possible to speculate therefore that microtubular networks within these cells are essential for the transportation of the machinery required for myelin formation into the processes. Recently MBP and tau have been shown to co-localize within the cell soma, processes and myelin expansions of oligodendrocyte cultures (Lopresti, et al. 1995) and tau has also been postulated to play a role in the transportation of MBP mRNA and protein to the site of myelinogenesis (Lopresti et al. 1995). It is attractive to hypothesise therefore that increased levels of dephosphorylated tau in these cells following injury may act to stabilise existing processes and increase the transportation of myelin components to sites where myelin sheaths have been damaged.
Petito, et al. (1985) showed that medium-light oligodendrocytes increased in size following permanent focal cerebral ischaemia in the rat (Petito, et al. 1985). These cells showed increased microtubule profiles and accounted for approximately 50% of all glial cells within 2 hr of injury, however the number present decreased to 6.5% by 3 h. Petito, et al. suggest that cell loss may represent a transition of medium-light oligodendrocytes to intermediate glia which have some characteristics of both oligodendrocytes and astrocytes. This may represent an "active" oligodendrocyte. Since tau is postulated to be involved in process expansion and therefore cell morphology, it is possible to speculate that increased tau levels within these cells following injury may be involved in the morphological changes
described following ischaemia and may therefore represent a marker for "active" oligodendrocytes. In addition oligodendrocytes have been shown to proliferate 24 hr following trauma in the mouse (Ludwin, 1984). It could be envisaged that the accumulation of tau following acute brain injury may be involved in the proliferation of oligodendrocytes, which would require the extension of new cellular processes. Tau-positive oligodendrocytes found following acute brain injury may thus represent cells about to undergo or are undergoing proliferation.
4.4.2 Degenerative response?
In contrast to a protective role, the accumulation of tau in oligodendrocytes may represent an early stage of cellular degeneration. Tau has recently been reported to be present in glial inclusions characteristic of chronic neurodegenerative conditions such as MSA, PSP and PD (Abe, et. al. 1992; Iwatsubo, et. al. 1994; Nishimura, et. al. 1992; Yamada, et. al. 1992). Tau present in these glial inclusions can be detected using antibodies directed towards PHF tau, suggesting that tau in these glial inclusions is abnormally phosphorylated. The detection of tau immunoreactivity in oligodendrocytes present in human post-mortem brain tissue from patients who died following a stroke or head injury indicates that the accumulation of tau occurs in both acute and chronic neurodegenerative conditions. In contrast to tau present in glial inclusions (Abe, et. al. 1992; Iwatsubo, et. al. 1994; Kato, et. al. 1991; Nishimura et. al., 1992; Yamada , 1992; Yamada and McGeer, 1990) however, tau present in oligodendrocytes early following acute brain injury is dephosphorylated at the Tau 1 epitope. In one patient who survived 23 h following severe head injury, and one patient surviving several weeks following stroke, TP70 and TPO07 immunoreactivity was found accumulated to one side of oligodendrocyte cytoplasm with the morphological appearance similar to the tau-positive glial inclusions previously described in MSA (Abe, et. al. 1992; Iwatsubo, et. al. 1994; Kato, et. al. 1991). These cells were detected within areas of tissue severely affected by head injury and within infarcted tissue of the stroke patient, as determined by decreased H&E staining. Together these results suggest that accumulation of tau in oligodendrocytes is an acute response of these cells to injury, however the formation of cytoplasmic inclusions may occur over time. However such tau-positive inclusions were not detected in tissue obtained from patients dying up to 12 days following a stroke, suggesting that factors other than survival time following injury may be involved in the formation of these tau positive accumulations. Tau present in the cytoplasm of oligodendrocytes with a morphology similar to glial inclusions, were not detected with Tau 1 and in addition the number of tau-positive oligodendrocytes detected with Tau 1 was decreased 23 h following head injury, as compared to earlier survival times, within these areas of tissue where these inclusion like accumulations were detected, suggesting that tau within these cells may be phosphorylated at this epitope. Failure of AD2, an antibody
directed towards PHF tau, to detect tau in oligodendrocytes showed that tau present within these cells, in contrast to PHF tau, was not phosphorylated at the AD2 epitope. The possibility that tau is phosphorylated at other sites similar to PHF tau cannot be ruled out. It is attractive to speculate therefore that tau accumulation in oligodendrocytes may be a rapid response of these cells to acute injury, however changes in antigenicity similar to those of PHF tau may occur over longer periods of time. NFT formation is thought to lead to neuronal degeneration in a variety of chronic degenerative disorders such as AD, PD and PSP, it is possible to speculate that tangle formation in oligodendrocytes will also lead to the subsequent degeneration of these cells.
4.5 The neuronal distribution of MAPs following acute brain injury
4.5.1 Tau
Following intracortical perfusion of glutamate, axonal Tau 1 staining was reduced while perikaryal staining was increased within the core of the lesion. This *in vivo* result concords with previous *in vitro* studies which showed increased Tau 1 immunoreactivity within cultured neuronal perikarya following glutamate exposure (Bigot and Hunt, 1990; Davis, et. al. 1995; Pizzi, et. al. 1993; 1994; 1995). Studies using antisense oligonucleotides suggested that increased tau immunoreactivity *in vitro* reflected increased synthesis of the protein supported by the increased Tau 2 staining which recognises full length tau protein (Pizzi, et. al. 1993; 1994; 1995). This is in contrast to the results presented in this thesis, which suggests that rather than accumulation of protein, increased Tau 1 immunoreactivity reflects dephosphorylation of existing protein in response to glutamate toxicity *in vivo*. In support of this theory AT8 immunoreactivity was lost from neuronal perikarya within the lesioned area which is consistent with dephosphorylation of tau at this epitope. In addition, accumulation of the protein was not indicated with B19, which labels full length tau, or with tau antibodies directed at the N- and C-terminals of the protein.
In contrast to the findings of the present study, other *in vitro* studies have shown the converse; increased AT8 immunoreactivity following glutamate exposure (Sindou, et. al. 1994). Although increased Tau 2 immunoreactivity was detected by Sindou and colleagues they suggested that the accumulation of phosphorylated tau was not due to increased synthesis of the protein (Sindou, et. al. 1992). Discrepancies between different *in vitro* studies may be related to the maturation stage of the neuronal cultures since immature tau has antigenic properties similar to that present in neurofibrillary tangles. It is interesting to note that in experiments employing neuronal cultures grown for 8 days or less (Mattson, et. al. 1990; Sautiere, et. al. 1992; Sindou, et. al. 1994) glutamate was proposed to induce
changes in tau similar to those found in neurofibrillary tangles. However in cultures grown for more than 8 days tau was dephosphorylated at the Tau 1 epitope in response to glutamate (Bigot and Hunt, 1990; Pizzi, et. al. 1993; 1994; 1995). It is also interesting to note that these investigators showed using antisense oligonucleotides, that increased tau immunoreactivity within these older neuronal cultures did represent increased synthesis of the protein (Pizzi, et. al. 1993; 1994; 1995). Such discrepancies between *in vitro* and *in vivo* again highlight the care that should be taken when relating observations made in cell cultures to pathophysiological events proposed to occur in the intact CNS, where the cellular milieu is more complex.
Monosodium glutamate was used in the present study and it is possible therefore that some of the changes in tau immunoreactivity observed may be due to the presence of excessive sodium ions. NaCl however failed to induce increased Tau 1 immunoreactivity in perikarya seen following glutamate perfusion, indicating that glutamate was responsible for the altered pattern of Tau 1 immunoreactivity. However, NaCl perfusion resulted in decreased tau immunoreactivity within the neuropil, suggesting that this pattern of immunoreactivity also seen following glutamate perfusion may result from excessive levels of sodium ions. In addition, decreased B19, TP007 and TP70 was also detected in perikarya following NaCl perfusion *in vivo* further implicating a role for Na$^+$ ions in the disruption of cytoskeletal proteins.
Increased Tau 1 immunostaining within neuronal perikarya of the cortex and caudate nucleus was detected within 20 min following MCAO in the rat. 40 min following the onset of cerebral ischaemia, TP70 and TP007 staining was decreased within neuronal perikarya of the cortex and caudate nucleus, ipsilateral to the occluded MCA. Together these results suggest that neuronal tau undergoes rapid dephosphorylation, which is closely followed by loss of C- and N-terminal immunoreactivity suggesting degradation of the protein, both of which may be mediated through glutamate excitotoxicity. In agreement with this, the *in situ* hybridisation studies carried out showed that tau mRNA was decreased early following ischaemic brain injury, which implies that tau mRNA is degraded following focal cerebral ischaemia. This is in agreement with a previous study showing increased Tau 1 and decreased TP70 immunoreactivity within neuronal perikarya of the cortex and caudate nucleus 2 h and 6 h following permanent MCAO in the rat (Dewar and Dawson, 1995). These results suggest that tau undergoes rapid alteration following MCAO in the rat and that these alterations are maintained up to 6 h or more following the induction of cerebral ischaemia. However in axons, while Tau 1 immunoreactivity was reduced by both glutamate and sodium chloride perfusion *in vivo*, Tau 1 immunoreactivity was increased in the axons of the cortex and caudate nucleus ipsilateral to the occluded MCA 40 min
following the induction of cerebral ischaemia. A similar increase in Tau 1 staining of axons was reported in permanent MCAO in the rat by Dewar and Dawson, (1995). Together this suggests that mechanisms other than those involving high extracellular glutamate and / or Na$^+$ levels may be involved in the changes in tau occurring in the axonal compartment following ischaemic brain damage.
4.5.2 MAP2 and MAP5
Both MAP2 and MAP5 immunoreactivity was increased in neuronal perikarya and decreased in neuropil within the lesion resulting from the intracortical perfusion of 1M monosodium glutamate. In contrast to tau which undergoes dephosphorylation and / or degradation, MAP2 and MAP5 appear to undergo redistribution from the neuropil to the perikarya. This is in agreement with previous studies showing a similar redistribution of MAP2 within neurons in culture following exposure to glutamate (Bigot and Hunt, 1990). Similarly, MAP2 and MAP5 immunoreactivity was increased in neuronal perikarya ipsilateral to the occluded MCA 20 min following the induction of cerebral ischaemia in the rat. 40 min following the onset of ischaemia both MAP2 and MAP5 immunoreactivity was decreased in the neuropil of the cortex of the ipsilateral hemisphere, implicating glutamate excitotoxicity in the mechanisms underlying the redistribution of these proteins following focal cerebral ischaemia. In addition, MAP2 immunoreactivity was decreased within both neuronal perikarya and neuropil of the caudate nucleus and clearly delineated the region of ischaemic tissue. This is similar to the findings of Dawson and Hallenbeck, (1996) who reported decreased MAP2 immunoreactivity within neuropil and neuronal perikarya of the caudate nucleus and cortex 1 h following focal cerebral ischaemia in the rat. This study further demonstrates the sensitivity of MAP2 to cerebral ischaemia, highlighting its use as a sensitive marker for ischaemic damage as early as 40 min of MCAO. NaCl perfusion also resulted in reduced MAP2 staining in perikarya and dendrites suggesting that excess Na$^+$ ions may be involved in the loss of MAP2 immunoreactivity following 40 min cerebral ischaemia.
In conclusion the present study demonstrates that neuronal tau, MAP2 and MAP5 undergo marked alteration in their cellular distribution as early as 20 min following MCAO in the rat indicating that the neuronal cytoskeleton is extremely vulnerable to ischaemic brain injury. Glutamate perfusion \textit{in vivo} resulted in many of the alterations of tau, MAP2 and MAP5 described following cerebral ischaemia in the rat; it is possible to speculate therefore that in conditions such as cerebral ischaemia and head injury, where excessive extracellular concentrations of glutamate are present, alterations in cytoskeletal proteins may be involved in the evolution of irreversible brain damage. These results have important implications for the development of neuroprotective strategies, since cytoskeletal abnormalities are detected
within neurons as early as 20 min following the induction of cerebral ischaemia which may represent the initial stages of irreversible brain damage.
4.6 Oligodendrocyte versus neuronal tau following acute brain injury
Following acute brain injury in the rat and the human, increased tau immunoreactivity was detected in oligodendrocytes using a variety of tau antibodies, suggesting the accumulation of full-length protein within these cells. In contrast, neuronal tau appears to undergo dephosphorylation and/or degradation in response to acute brain injury. The accumulation of tau in oligodendrocytes was detected as early as 40 min following MCAO in the rat suggesting that the cytoskeleton within these cells undergoes rapid alteration following acute brain injury. As early as 20 min following MCAO in the rat, increased tau immunoreactivity was detected in neuronal perikarya with Tau 1, however this was not detected with antibodies raised against the N- and C-terminals of the protein. Together these results suggest that in contrast to the accumulation of full-length tau in oligodendrocytes, neuronal tau becomes dephosphorylated and/or degraded following acute brain injury in the rat. Dephosphorylation of tau in neurons was detected as early as 20 min following MCAO in the rat, however tau-positive oligodendrocytes were not observed until 40 min after the induction of cerebral ischaemia. In addition degradation of tau in neurons but not in oligodendrocytes suggests that tau present in neuronal perikarya may be more susceptible to ischaemic brain damage than that in oligodendrocytes.
Dephosphorylated tau has the ability to bind to and stabilise microtubules. It is therefore possible to speculate that increased Tau 1 immunoreactivity detected in both these cell types following acute brain injury is an attempt to try and stabilise the cytoskeletal architecture. The observation that tau may have undergone some degree of proteolysis within neurons may reflect a loss or modification of the C-terminal of the protein which encompasses the microtubule binding domain, dephosphorylated tau present in neurons following acute brain injury may therefore lack the ability to bind to and stabilise microtubules. In contrast, the presence of full length tau within oligodendrocytes following injury suggests that successful microtubule stabilisation may occur within these cells therefore helping to maintain their cytoskeletal integrity. In contrast to the accumulation of MAP2 and MAP5 in neurons following acute brain injury, these proteins were not detected in oligodendrocytes. It is possible to speculate that MAP2 and MAP5 remain unaltered in oligodendrocytes following injury, however the possibility that oligodendrocytes do not contain these proteins cannot be ruled out. Breakdown of the neuronal cytoskeleton is thought to represent a common final pathway of irreversible cell damage following ischaemic brain injury. Neurons are generally considered to be the most vulnerable cells of the CNS to cerebral ischaemia, while
oligodendrocytes are thought to be fairly resistant. The differential sensitivities of the cytoskeleton to cellular injury in these two cell types may help explain their differential sensitivity to acute brain injury.
In addition, the mechanisms underlying the altered distribution of tau in neuronal perikarya and oligodendrocytes appear to be different. Glutamate excitotoxicity appears to mediate the changes in tau occurring within neurons, while free radical mediated mechanisms appear to be responsible for those occurring in oligodendrocytes. Activation of the NMDA receptor and subsequent influx of Ca$^{++}$ is thought to be one of the most important mechanism underlying glutamate mediated neurotoxicity following cerebral ischaemia (see McCulloch, et. al. (1991) for review). In contrast to neurons which contain abundant NMDA receptors, oligodendrocytes only contain mRNA encoding non-NMDA glutamate receptors (for review see Gallo and Russell, 1995). Glutamate release occurs immediately following MCAO (Butcher, et. al. 1990) and is thought to lead to the release of free radicals (Bondy and Lee, 1993; Dykens et. al. 1987; Miyamoto, et. al. 1989; Nakao, et. al. 1996; Schulz, et. al. 1995). It is tempting to speculate therefore that neurons are susceptible to the initial release of glutamate following the onset of ischaemia, however oligodendrocytes which lack the presence of NMDA receptors are more susceptible to the secondary release of free radicals. Indeed oligodendrocytes are thought to be particularly susceptible to free radical damage due to their high levels of iron and low levels of the endogenous antioxidant glutathione (Thorburne and Juurlink, 1996). Further supporting this theory glutamate toxicity has been shown to be mediated not through receptor activation, but through free radical mediated mechanisms in oligodendrocyte cultures (Oka, et. al. 1993; Yonezawa, et. al. 1996).
In conclusion, the results of this thesis demonstrate that tau is present in oligodendrocytes, which undergoes rapid alteration in response to acute brain injury. Alterations in the distribution of microtubule-associated proteins in neurons following injury are thought to represent altered cytoskeletal integrity which may result in irreversible neuronal damage. It is possible to speculate therefore that alterations to tau in oligodendrocytes, following acute brain injury may also represent early stages of cytoskeletal breakdown within these cells. Oligodendrocytes have been until recently considered to be relatively unresponsive to ischaemic brain injury, and neuroprotective agents have been evaluated solely on their ability to reduce neuronal damage. One of the most successful strategies involves the blockade of glutamate receptor activation which was without effect in the reduction of the induction of tau immunoreactivity in oligodendrocytes following focal cerebral ischaemia. The protection of neurons without concurrent protection of glial cells following acute brain injury would lead to delayed neuronal death due to the degeneration of glia rather than complete protection of the brain following injury.
Appendix
Growth media
SOC medium
1 litre - 20g tryptone, 5g yeast extract, 0.5g NaCl in distilled water. Add 10ml of 250mM KCl and adjust to pH7 using NaOH and sterilise by autoclaving for 20 min at 15lb/in$^2$. Once cooled to <60°C 20ml of filter sterilised, 1M glucose solution was added.
Luria-Bertrani (LB) agar indicator plates
1 litre - 20g Tryptone, 5g Yeast extract, 10g NaCl; dissolved in distilled water and adjusted to pH7; 12g agar/L added and the solution sterilised by autoclaving for 20 min at 15lb/in$^2$. Agar was allowed to cool to 50°C and the following added prior to pouring the plates: 0.004% X-gal (5-Bromo-4-chloro-3-indoyl-β-L-galactoside), 0.012% IPTG (isopropylthio-β-D-galactoside) and 100μg/ml ampicillin.
Terrific Broth
500ml - 6g Tryptone, 12g Yeast extract, 2ml glycerol, make up to 450ml on distilled water (soln A). 0.17MKH$_2$PO$_4$ and 0.72M K$_2$HPO$_4$ in 100ml distilled water (soln. B). Autoclave both solutions separately for 20 min at 15lb/in$^2$, allow to cool and add 50ml of soln B and 100μg/ml ampicillin to soln A.
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Publications
Intracortical perfusion of glutamate in vivo induces alterations of tau and MAP2 immunoreactivity in the rat. Irving, E.A., McCulloch, J. and Dewar, D. *Acta Neuropathologica*, 92 (1996) 186-196.
Increased tau immunoreactivity in oligodendrocytes following human stroke and head injury. E.A. Irving, J. Nicoll, D.I. Graham and D.Dewar. *Neuroscience Letters*, 213 (1996) 189-192.
The effect of post-mortem delay on the distribution of microtubule-associated proteins tau, MAP2 and MAP5 in the rat. E.A. Irving and D. Dewar. *Molecular and Chemical Neuropathology*, in press
Rapid alteration of tau in oligodendrocytes following focal ischaemic injury in the rat: involvement of free radicals. E.A. Irving, K. Yatsuhiro, J. McCulloch and D.Dewar. *Journal of Cerebral Blood Flow and Metabolism* in submission.
Abstracts
Alterations of tau protein in ischaemic and excitotoxic brain damage in vivo. D.Dewar, D.Dawson and E.A. Irving. *Society for Neuroscience Abstracts* Vol 20 (1994) 257.17
Alterations of tau and amyloid precursor protein in excitotoxic brain damage in vivo. E.A. Irving, D. Dewar, J. McCulloch and D.I Graham. *Neuropathology Society abstract* (1994)
Increased tau immunoreactivity within oligodendrocytes following cytotoxic injury. E.A. Irving, S.C. Barnett, P. Dickinson, J. McCulloch, D. Dewar and I.R. Griffiths. *Society for Neuroscience Abstracts*, vol 21 (1995) No. 134.9
Increased tau immunoreactivity in oligodendrocytes following acute brain injury. E.A. Irving, J. Nicoll, D.I. Graham and D. Dewar. *Journal of Neurotrauma* 12 (1995) No. 153
Alteration of the cytoskeletal protein tau in oligodendrocytes following focal cerebral ischaemia in the rat. E.A. Irving, K. Yatsuhiro, J. McCulloch and D.Dewar. *Journal of Neurotrauma*, 13 (1996) No. 138
Spin trap agent prevents tau accumulation in oligodendrocytes following focal cerebral ischaemia in the rat. E.A. Irving, K. Yatsushiro, J. McCulloch and D.Dewar. British Journal of Pharmacology, (1996) in press
Tau-positive oligodendrocyte density in white matter is increased after focal cerebral ischaemia and reduced by PBN but not MK801 or NBQX. E.A. Irving, K. Yatsushiro, J. McCulloch and D.Dewar. Journal of Cerebral Blood Flow and Metabolism, (1997) in submission.
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SOME NITROSONIUM AND NITRONIUM SALTS
OF STRONG ACIDS
THESIS
submitted to the
UNIVERSITY OF GLASGOW
in fulfilment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
by
John Thorley
Chemistry Department
University of Strathclyde
formerly
The Royal College of Science & Technology,
Glasgow.
October, 1964
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# CONTENTS
| Chapter | Title | Page |
|---------|----------------------------------------------------------------------|------|
| ABSTRACT| | i - iv |
| ACKNOWLEDGMENTS | | |
| INTRODUCTION | | |
| CHAPTER I | Studies on Previously Reported Nitrosonium Fluoro-Chloro-adducts | 37 |
| CHAPTER II | Solvent Properties of Nitrosyl Chloride and Dinitrogen Tetraoxide: the Nitrato ligand. | 93 |
| CHAPTER III| Reactions of Nitrogen Oxides and Nitrosyl Chloride with Lewis Acids of groups III B, IV B and V B | 108 |
| CHAPTER IV | Reactions of Nitrogen Oxides and Nitrosyl halides with compounds of Titonium and Zirconium | 136 |
| CHAPTER V | Nitrosonium and Nitrato- compounds of Vanadium, Niobium and Tantalum | 143 |
| CHAPTER VI | Nitrosonium and Nitrato compounds of Chromium and Molybdenum | 197 |
BIBLIOGRAPHY
ABSTRACT
A series of compounds, reported in the literature, which can be formulated as nitrosonium salts of fluoro-acids, have been re-prepared. Several metal chloride-nitrosyl chloride adducts, also reported in the literature, have been re-prepared, and both these groups of compounds have been shown to contain the nitrosonium ion, NO⁺, by infrared diagnostic methods.
The isomorphism of the nitrosonium salts of the anions fluoroborate, chlorostannate and chloroplatinate with their potassium analogues has been confirmed, and nitrosonium and potassium fluorosulphates have been shown to be isostructural. These observations have been explained structurally, with reference to the relative sizes of nitrosonium and halide ions.
The free (i.e. unco-ordinated) nitrosonium ion was found to absorb in the region 2160 cm⁻¹ - 2400 cm⁻¹. This wide variation was partially explicable in terms of polarisation by the anion but it is apparent that other factors were important. In general, it was found that fluoro-acid salts gave the highest nitrosonium ion stretching frequencies, followed by oxy-acid salts and chloro-acid salts respectively.
The behaviour of group IVB and VB Lewis acid halides was observed with nitrogen oxides and nitrosyl chloride. With dinitrogen tetroxide, all the compounds examined, except arsenic trichloride, gave a mixture of products which it was not possible to separate. Arsenic trichloride gave a nitrosonium nitrato-arsenate salt. In the other cases, there was evidence for the formation of nitrosonium and nitronium salt mixtures, in accord with earlier observations on the behaviour of dinitrogen tetroxide with Lewis acids, and strong acids such as perchloric acid. With nitrosyl chloride, the products were either nitrosonium chloro-acid salts or molecular adducts. Reactions involving nitric oxide were not investigated extensively but in reactions with high valency metal chlorides, the primary reaction step was always a reduction by the nitric oxide.
The behaviour of transition metal chlorides and oxides of the titanium, vanadium and chromium groups was observed in nitrosyl chloride and dinitrogen tetroxide. These reactions could all be explained assuming auto-ionisation of the solvent.
The reactions in nitrosyl chloride yielded mainly nitrosonium salts of chloro-acids, and several of the anions thus formed were bi- and tri-nuclear species.
The previously reported compounds NO.VCl₄ and NO.V₂Cl₇ were obtained and characterised as nitrosonium salts. With niobium and tantalum, the new compounds NO.NbCl₆ and NO.TaCl₆ were prepared, containing the previously unknown NbCl₆⁻ and TaCl₆⁻ anions. The new compounds (NO)₃Cr₂Cl₉ and (NO)₃Mo₃Cl₁₀ were obtained. A surprising feature of several of these ionic compounds was their high volatility.
In the reaction between chromium (VI) trioxide and nitrosyl chloride, a new compound (NO)₂Cr₃O₈Cl was characterised, in which chromium has undergone a partial reduction. Vanadium pentoxide underwent a similar reaction, but in this case it was not reproducible.
The reactions involving dinitrogen tetroxide with transition metal halides generally gave rise to one product only, in contrast to the Lewis acid - dinitrogen tetroxide reactions. The products were usually metal nitrate-dinitrogen tetroxide adducts and these could be formulated as nitrosonium metal nitrato-salts, on the basis of their infrared spectra. Vanadium and chromium chlorides gave the adducts VO₂NO₃·N₂O₄ and Cr(NO₃)₃·2N₂O₄ respectively, but molybdenum trichloride gave Mo(V)O(NO₃)₃, which may be polymeric.
No reaction occurred between chromium (III) chloride and either nitrosyl chloride or dinitrogen tetroxide, but in the presence of chromous chloride, reaction proceeded.
readily in both instances.
No reaction was observed between high valency metal oxides and dinitrogen tetroxide, but this may have been due to insufficiently vigorous conditions.
The reflectance spectra of the majority of the above compounds were observed, and, in most cases it was possible to make satisfactory band assignments.
ACKNOWLEDGMENTS.
The author wishes to express his sincere thanks to Dr. D.W.A. Sharp for his encouragement, direction and correction during the supervision of this work.
Thanks are also extended to Dr. D.R. Russell for assistance in using a Ferranti Sirius Computer for X-ray calculations, and to the other members of the Inorganic Chemistry Research Department for many helpful discussions.
Finally, the author would like to thank the European Research Office of the U.S. Department of Army, whose financial support and sponsorship during the years 1961 - 1964 made this research possible.
INTRODUCTION
Much of the early work on systems involving nitric oxide complexes stemmed from observations on two important reactions, one in the industrial context, the other in analysis:
1. The lead chamber process for the commercial production of sulphuric acid was known to proceed via a blue crystalline intermediate, the familiar 'chamber crystals'; this substance was known to be an essential step to the eventual formation of sulphuric acid, and from it a yellow hygroscopic crystalline solid was obtained, $NO_2HSO_4$, nitrosyl hydrogen sulphate, whose structure aroused considerable interest. The outstanding pioneer in this field was Hantzsch, who, between 1905 and 1930, published a series of papers on the behaviour of nitrosyl hydrogen sulphate in solutions of strong acids, and later extended this to nitric acid/strong acid systems. His work and conclusions were largely concerned with cryoscopic and electrolytic observations, and he was the first person to postulate the existence of the nitrosonium ion $NO^+$.
2. The 'brown ring' test for nitrate ion, involving formation of an unstable complex of ferrous sulphate, has been familiar for many years but only recently has the
constitution of this complex been unambiguously assigned. Manchot was prominent among the early workers in this field, and he observed that the 'brown ring' complex was identical to that formed when ferrous sulphate solution absorbed nitric oxide. Manchot and his co-workers were able to establish the metal: nitric oxide mole ratio in the complex, and in 1924, electrolytic experiments confirmed that the nitric oxide was associated with the metal. In addition, nitric oxide complexes of cupric and palladium salts were studied; from the former, by comparison with cuprous/carbon monoxide systems, a relationship was noted between nitric oxide and carbon monoxide complexes, which was later amplified by Hieber and Anderson.
Both these areas of research began in the early twentieth century. In the nineteen twenties another significant field appeared when Gall & Mengdehl, and Rheinboldt & Wasserfuhr, simultaneously reported the formation of adducts between nitrosyl chloride and various metal and non-metal chlorides. These complexes, like nitrosyl hydrogen sulphate, were hygroscopic solids; they were susceptible to x-ray examination, and by these means Klinkenberg, in 1937, confirmed the existence of the nitrosonium ion in some of the adducts. Magnetic
susceptibility determinations were carried out by Asmussen in 1939 but, structurally, these were much less conclusive. Meanwhile, Angus & Leekie, in 1935, had laid the basis for possibly the most powerful diagnostic tools in this field, Raman and infra-red spectroscopy. By observations on the Raman spectrum of nitrosyl hydrogen sulphate in sulphuric acid/water solutions of varying concentrations, they were able to assign a Raman band to the nitrosonium ion.
After the war, studies were extended to nitrosonium and nitronium metal fluorides, and to metal nitrate: dinitrogen tetroxide complexes, which are analogous to the metal chloride: nitrosyl chloride adducts in some respects; this will be enlarged upon in a later chapter. Addison and his school have published a number of papers on the dinitrogen tetroxide solvent system, and more recently, this has been extended to studies on anhydrous transition metal nitrates, which were, until recently, unknown.
Raman and infrared spectroscopy have been developed much more than the other diagnostic methods. Ingold and his co-workers extended this to cover systems containing nitronium, NO$_2^+$ ions, and Griffith, Lewis & Wilkinson extended infrared spectroscopy to other nitric oxide-metal complexes, and established it as a means of
distinguishing between types of complexes. Cryoscopic and conductimetric experiments have been carried out, but these have much more limited application.
Nomenclature.
The nitrosonium ion is derived from the nitric oxide radical molecule by loss of one electron; by gaining an electron the nitric oxide molecule yields the nitrosyl anion.
Thus:
\[
\text{NO}^+ \xrightarrow{+e^-} \text{NO} \xrightarrow{-e^-} \text{NO}^-
\]
nitrosonium ion nitric oxide nitrosyl anion
The ionisation potential for the reaction
\[
\text{NO} \longrightarrow \text{NO}^+ + e^-
\]
is equal to 9.5 e.v. (Hagstrum & Tate 1941).
This is much lower than the values for nitrogen and oxygen, viz:-
\[
\text{N}_2 \longrightarrow \text{N}_2^+ + e^- \quad I = 15.5 - 17.2 \text{ e.v.}
\]
\[
\text{O}_2 \longrightarrow \text{O}_2^+ + e^- \quad I = 12.2 - 18.2 \text{ e.v. depending on the electron state}
\]
Hence the nitric oxide molecule loses an electron much more readily than the oxygen or nitrogen molecule.
The bond lengths of the species are:
\[
\begin{align*}
\text{Nitric oxide} & \quad - \quad 1.14 \, \text{\AA} \\
\text{Nitrosonium ion} & \quad - \quad 1.06 \, \text{\AA}
\end{align*}
\]
Pauling's values for the double and triple bond lengths of the N - O bond are 1.18 \, \text{\AA} and 1.06 \, \text{\AA} respectively, which gives nitric oxide a bond order intermediate between double and triple. Pauling explained this by postulating that the N - O bond in nitric oxide was composed of a double bond plus a three-electron bond. The latter bond would have about half the strength of an electron-pair bond, and would only be energetically feasible when the atoms involved in the bonding are of very similar electronegativities. Loss of an electron from the nitric oxide molecule would convert the 3-electron bond into a normal electron-pair bond, with consequent increase of bond order and shortening of the N - O bond, as observed in the nitrosonium ion. This treatment is fairly satisfactory but a simple molecular orbital approach explains these observations much more elegantly.
Below is given the molecular orbital scheme for the nitric oxide molecule, and its derivation from the nitrogen and oxygen atoms.
The unstarred molecular orbitals are the bonding orbitals, and the starred are the anti-bonding orbitals. The 1s and 2s bonding and anti-bonding orbitals are completely filled and hence these orbitals play no significant part in the N - O bond. The nitrogen and
oxygen atoms contribute 7 electrons, in all, to the molecular orbitals. The bonding sigma 2p orbital can accommodate two, and the degenerate pi-2p(y) and pi-2p(z) can each accommodate two more. Hence one electron must go into the degenerate anti-bonding pi-2p orbitals. The effect of placing electrons in bonding orbitals is to draw the constituent atoms together, while electrons in anti-bonding orbitals force them apart. Hence the presence of one electron in an anti-bonding orbital in conjunction with a triple (\(\sigma + 2\pi\)) bond will be to increase the bond length somewhat compared to the pure triple bond value. Also, electron loss to yield a cation involves the loss of the anti-bonding electron with consequent shortening of the N-O bond. This is consistent with the data already cited regarding N-O bond lengths in nitric oxide and the nitrosomium ion. Addition of an electron to the nitric oxide molecule to give the nitrosyl anion, NO\(^-\), must involve placement of the extra electron in the anti-bonding pi-2p orbitals; this is evident from consideration of the molecular orbital scheme. This will lengthen the N-O bond still further, and the presence of two electrons in the anti-bonding orbitals will cause considerable bond weakening. This is consistent with the known properties of the nitrosyl anion; compounds in
which this species occurs are much rarer than compounds containing nitrosonium ions.
Being a radical species, the nitric oxide molecule would be expected to dimerise quite readily to give diamagnetic species, that it does not must be largely due to the similar electro-negativities of nitrogen and oxygen, causing the anti-bonding unpaired electron to be well delocalised over the whole molecule. However, in the liquid phase, i.e. between $-163^\circ C$ and $-151^\circ C$, nitric oxide is known to be very largely dimerised; also the reactions between diethylamine and nitric oxide in ether at $-78^\circ C$ can best be explained by the presence of $O = N - N = O$ dimer molecules. (Drago & Paulik 1960).
The nitrosonium ion is isoelectronic with carbon monoxide and the cyanide anion. The relationship between nitric oxide and carbon monoxide was first noted by Manchot (1910), and extended by the preparation of the 'pseudo-nickel' carbonyls or nitrosyl carbonyls of Lieber and Anderson (1932, 1933), viz:-
$$\text{Fe(NO)}_2 \text{(CO)}_2 \quad \text{Co(NO)(CO)}_3 \quad \text{Ni(CO)}_4$$
In this isoelectronic species, a unit decrease in the atomic number of the metal atom is compensated for by the substitution of a nitric oxide molecule for a carbon
monoxide molecule. The compounds have very similar properties and gradations can be explained by the increased polarity of the nitrosyl (or, strictly speaking, nitrosonium) group.
Although nitric oxide is stable towards dimerisation at ordinary temperatures, it is readily oxidised to give another paramagnetic species, nitrogen dioxide.
\[ \text{NO} + \frac{1}{2} \text{O}_2 \rightarrow \text{NO}_2 \]
The nitrogen dioxide radical-molecule, is also electronically amphoteric:
\[
\begin{align*}
\text{NO}_2^+ & \xrightarrow{-e^-} \text{NO}_2 \\
\text{nitronium ion} & \quad \text{nitrogen dioxide} \\
& \quad \text{N}_2\text{O}_4 \\
& \quad \text{dinitrogen tetroxide}
\end{align*}
\]
\[
\begin{align*}
\text{NO}_2^- & \xrightarrow{+e^-} \text{NO}_2^- \\
\text{nitrite anion} &
\end{align*}
\]
The nitrogen dioxide molecule is bent, viz:-
with the bond angle, \( A \), equal to 132°, and the N - O bond lengths being both equal to 1.20Å (Claesson et al. 1948); addition of an electron gives the nitrite anion, which is
also bent. In this case the bond angle is between $120^\circ$ and $130^\circ$, and the N - O bond distances are 1.13Å (Langseth & Walles, 1934). It is more difficult in this case to construct a molecular orbital energy level scheme similar to the nitric oxide scheme; the simplest representation is the valence bond scheme, involving resonance between the canonical forms:
\[
\begin{array}{c}
\text{O} \\
\text{N} \\
\text{O}
\end{array}
\quad \rightleftharpoons \quad
\begin{array}{c}
\text{O} \\
\text{N} \\
\text{O}
\end{array}
\]
This gives the N - O bond a bond order somewhat less than two, which is in agreement with the measured value of 1.20Å cited above. A qualitative molecular orbital picture would place the odd electron in a $\pi$-orbital extending over all three atoms. In a molecular orbital treatment of this molecule the unpaired electron is placed in a slightly anti-bonding orbital (Green & Linnett 1961). The short bond lengths in the nitrite anion suggests some degree of triple bonding between nitrogen and oxygen and this is difficult to explain in terms of canonical forms such as:-
\[
\begin{array}{c}
\text{O} \\
\text{N} \\
\text{O}
\end{array}
\]
which would give rise to a much greater N - O bond length. The nitrite anion is much more stable and much better characterised than its analogue in the nitric oxide system, the nitrosyl anion.
Loss of an electron from the nitrogen dioxide molecule gives the linear intronium cation NO$_2^+$, isoelectronic with carbon dioxide. The bond length N - O, is 1.18Å (Eriks 1950), identical to the calculated value for an N - O double bond, so that this is consistent with the formulation:
$$\text{O} \equiv \text{N} \equiv \text{O}$$
Nitrogen dioxide readily dimerises at ordinary temperatures to dinitrogen tetroxide, N$_2$O$_4$, and the extent of dimerisation increases with decreasing temperature. In the solid state the structure is:
$$\begin{array}{c}
\text{O} \\
\text{N} \\
\text{N} \\
\text{O}
\end{array}$$
A = 126°
N - N = 1.64Å
N - O = 1.17Å
(Broadley et al. 1949)
and the liquid can be considered as an equilibrium:
$$\text{N}_2\text{O}_4 \rightleftharpoons 2\text{NO}_2$$
This will be largely due to the absence of the unpaired electron in the latter case. Since both nitric oxide and nitrogen dioxide are radical molecules there should be a stabilisation in the latter case, due to the greater possible delocalisation of the odd electron. With removal of the unpaired electron this is no longer the case.
Both the nitrosonium and nitronium ions are very susceptible to hydrolysis
\[
\text{NO}^+ + \text{H}_2\text{O} \rightleftharpoons \text{H}^+ + \text{HNO}_2 \rightleftharpoons 2\text{H}^+ + \text{NO}_2^-
\]
\[
\text{NO}_2^+ + \text{H}_2\text{O} \rightleftharpoons \text{H}^+ + \text{HNO}_3 \rightleftharpoons 2\text{H}^+ + \text{NO}_3^-
\]
and Millen (1950b) has shown that the nitronium ion is much less stable to hydrolysis than the nitrosonium ion; the latter is virtually unaffected by concentrations of water which will completely hydrolyse the former. As the above equilibria imply, nitrosonium and nitronium ions are formed in systems of high acidity containing respectively nitrite or nitrate ion.
Systems containing nitrosonium ions, under high pressures of nitric oxide give species with one-electron bonds (Seel et al. 1953; Seel & Sauer 1957).
\[
\text{NO}^+ + \text{NO} \rightleftharpoons \text{N}_2\text{O}_2^+
\]
This cation dissociates readily in the absence of nitric oxide but is stable for appreciable lengths of time at normal temperatures and pressures in solutions of high viscosity. It exists in two isomeric forms, corresponding to different electronic states, one form being blue, the other red. It is probable that this cation is responsible for the blue colour of the 'chamber crystals' of the lead chamber process.
An analogous cation, \( \text{N}_2\text{O}_3^+ \), formed by the equilibrium
\[
\text{NO}^+ + \text{NO}_2 \rightleftharpoons \text{N}_2\text{O}_3^+
\]
seems probable (Goulden & Miller 1950). The cation \( \text{N}_2\text{O}_3^+ \) has two possible canonical forms:
\[
\text{NO} \cdot \text{NO}_2^- \quad (1) \quad \text{and} \quad \text{NO}^- \cdot \text{NO}_2^+ \quad (II)
\]
and Goulden & Millen cite Raman spectroscopic evidence for the predominance of species (1).
The nitrosonium ion also appears to solvate in nitrosyl chloride solution (Burg & MacKenzie 1952) and in acetonitrile solution (Fraser & Dasent 1960) but the species formed probably involve normal electron-pair bonds.
The types of compound involving nitrosonium ions or the nitrosyl group have been reviewed by Addison & Lewis (Quart. Revs. 1955); the main types are
1. Nitrosonium Salts $NO^+ X^-$
These contain the free nitrosonium ion; examples are $NO^+ BF_4^-$, $NO^+ ClO_4^-$.
2. Compounds in which the $NO^+$ group is bonded to a metal atom $M \rightleftharpoons NO$
In this case the nitric oxide molecule formally donates one electron to the metal and then forms a metal-nitrosyl bond; almost invariably bonding to the metal occurs via the nitrogen atom but some instances have been reported in which bonding to the metal is postulated to occur via the oxygen (Yamada et al 1960); also, the compound $(C_3H_7)_2 - Zn - O - N < C_3H_7$ has been postulated to form via a $C_3H_7 - Zn - O = N$ intermediate (Abraham et al 1962). An example of this type of compound is cobalt nitrosyl tricarbonyl, which was mentioned previously in connection with the 'pseudo' nickel carbonyls. In this type of compound two types of electron interchange are believed to occur between the nitrosonium ion and the metal atom:
1. Sigma-donation from the nitrosonium ion to the metal. This will have the effect of placing an excessive negative charge on the metal atom which could be relieved by 11) 'Back-donation' from the metal d-orbitals to the vacant anti-bonding pi-orbitals of the nitrosonium ion, which are of a suitable size and symmetry to participate in this form of bonding.
This multiple bonding is believed to contribute largely to the facility with which nitric oxide behaves as a ligand.
3. Compounds in which the nitrosyl anion bonds to a metal atom.
These are much less common than those of groups 1, and 2; the lower stability of the nitrosyl anion is responsible for this. The unstable iron nitrosyl Fe(NO)$_4$ is believed to have the structure:
\[
\begin{array}{c}
+ \\
(NO)_3 \rightarrow Fe \leftarrow NO^- \\
\end{array}
\]
(Griffith et al. 1958b)
The co-ordinated nitrosyl anion is also believed to occur in the nitrosepentamine cobalt salts [Co(NH$_3$)$_5$(NO)] X$_2$, which can occur in two forms, viz, a black form originally believed to contain dimeric cationic species and co-ordinated nitrosyl anions and nitrosonium ions, and a red series containing the monomeric cation.
with all the NO grouping present as the nitrosyl anion (Griffith et al. 1958a). However, a recent examination of the conductivity of solutions of the red isomer has shown that it is dimeric; the black isomer was too unstable to examine by these means (Feltham 1964).
As will be explained later in this chapter, infrared spectroscopy can distinguish between these three possibilities, usually without any ambiguity.
Other significant types of compound containing the nitrosyl grouping are
4. Compounds containing a bridging nitrosyl group.
Besides behaving as a cationic or anionic donor ligand, nitric oxide can also behave as a bridge in binuclear complexes. Again the nitric oxide molecule can be considered as losing an electron to the metal atom centres, and the effective donor group is the nitrosonium ion. Few compounds of this structure are known, the first to be prepared being \((C_5H_5)_3Mn_2(NO)_3\). (Piper and Wilkinson 1956)
\[
\begin{array}{c}
\pi \cdot C_P \\
\pi \cdot C_P \\
\end{array}
\begin{array}{c}
Mn \\
N \\
O \\
\end{array}
\begin{array}{c}
N \\
O \\
\end{array}
\begin{array}{c}
\pi \cdot C_P \\
NO \\
\end{array}
\]
Again, as in the majority of nitrosyl metal complexes, bonding from the nitrosyl group occurs via the nitrogen atom. Recently, a cyclopentadienyl nitrosyl complex of chromium has been prepared, believed to contain bridging nitrosyl groups; also, in this case the chromium - chromium bond is believed to be short enough for metal-metal bonding to occur:
\[
\begin{array}{c}
\text{ON} \\
\text{Cr} \\
\text{N} \\
\text{NO} \\
\text{Cr} \\
\text{N} \\
\text{NO} \\
\text{Cp} \\
\text{Cp} \\
\end{array}
\]
(King & Bisnette 1964)
Structures involving bridging nitrosyl groups have been assigned in the above cases on the basis of infrared evidence.
5. The free nitrosyl anion has been postulated to occur in 'sodium nitrosyl', a compound first prepared by Zintl & Harder (1933) and postulated as being \(\text{Na}^+ \text{NO}^-\) on the basis of its diamagnetism (Frazer & Long 1938). The method of preparation involved treatment of a sodium/ammonia solution with dry nitric oxide, a white solid.
separating out; when Goubeau & Laitenberger (1963) repeated this they found that this compound was identical with sodium hyponitrite $\text{Na}_2\text{N}_2\text{O}_2$, which they depicted as:
$$\left[\text{Na}^+\right]_2 \quad \left[ \begin{array}{c} \text{O} \\ \text{O} \end{array} \right] \quad \text{N} = \text{N} \quad \left[ \begin{array}{c} \text{O} \\ \text{O} \end{array} \right]$$
This compound would be expected to be diamagnetic; by examination of the molecular orbital scheme for nitric oxide, it can be seen that the NO$^-$ anion would be expected to be paramagnetic, like the oxygen molecule with which it is isoelectronic. The two unpaired electrons in both the nitrosyl anion and the oxygen molecule are in the two degenerate pi-antibonding orbitals; unless the energy of electron pairing is abnormally low, Hund's Rule will operate and the electrons will occupy different orbitals and thus possess parallel spins.
Hence, the existence of the free nitrosyl anion is still in question.
6. It is possible that some compounds exist in which the nitric oxide molecule donates two electrons to a metal atom or ion and retains an unpaired electron. Such a structure would be:
$$\text{M} \leftarrow \text{N} = \text{O} \quad \text{M} = \text{metal atom}$$
If the metal centre was paramagnetic, there would be two paramagnetic centres in the molecule, but no compounds
have been prepared which could be assigned such a structure.
As emphasised before, in practically all cases where metal-nitrosyl bonding occurs, the linkage occurs via the nitrogen atom; also the systems
\[ M - N = O \]
are always linear.
Nitrogen dioxide is a much less versatile group than nitric oxide, and the compounds of main interest in this work are those containing the free nitronium ion,
\[ NO_2^+ \]
This has been well characterised by Ingold and his school in a series of papers in 1950.
Nitrogen dioxide will behave as a ligand, bonding like the nitric oxide molecule, via the nitrogen atom. The co-ordinated nitrogen dioxide molecule is bent, and has the same symmetry properties as the nitrite ion, \( NO_2^- \), (Gatehouse 1958). No instances are known in which the nitronium ion behaves as a ligand.
Formation of nitrosonium ions and nitronium ions.
Systems in which the nitrosonium or nitronium ion occurs, or can be produced, are as follows:
1. Electric Discharge.
Passage of an electric discharge through nitric oxide at low pressure causes ionisation; the ionisation potential for this reaction is 9.5 e.v. The method is of
no importance in the synthesis of nitrosonium salts or nitrosyl complexes.
2. Self ionisation of nitrosylating solvents
Nitrosyl chloride is believed to ionise in the liquid phase, viz
\[ \text{NOCl} \rightleftharpoons \text{NO}^+ + \text{Cl}^- \]
By following the rate of chlorine exchange in the systems nitrosyl chloride/tetra-ethyl or methyl ammonium chloride, using radioactive chlorine, Lewis and Wilkins (1955) concluded that the data could best be explained by transfer via chloride ions and postulated the equilibrium \( \text{NOCl} \rightleftharpoons \text{NO}^+ + \text{Cl}^- \). Nitrosonium ions probably exist to some extent in solid nitrosyl chloride, the extent of ionisation being inversely proportional to temperature. Liquid nitrosyl chloride is deep red, as is solid nitrosyl chloride at \(-78^\circ\text{C}\), but the colour lightens gradually until at \(-196^\circ\text{C}\) it is pale yellow. The nitrosonium ion, itself, is colourless, and any colour in simple nitrosonium systems can be explained by charge transfer reactions of the type:
\[ \text{NO}^+ \quad X^- \quad \xrightarrow{\text{charge}} \quad \text{NO} \quad \dot{X} \]
and it is this mechanism which causes the deep colours of the nitrosyl halides. Hence although nitrosyl chloride
is primarily covalent, some self ionisation may occur. The same applies to nitrosyl bromide, NOBr.
Further evidence that self ionisation may occur comes from the structures of nitrosyl chloride and bromide; these both have the structure:
\[
\begin{array}{ccc}
X & A \\
N = O \\
\end{array}
\]
**Nitrosyl chloride**
1.14Å
1.95Å
116°
**Nitrosyl bromide**
1.15Å
2.14Å
117°
Band Length N - O
" " M - X
Bond angle X - N - O
For nitrogen-halogen single bonds, the radius sum values are 1.73Å (N - Cl) and 1.88 (N - Br); thus in the nitrosyl halides the nitrogen - halogen bonds are weaker than single bonds, and it is quite possible that self ionisation will occur. (Ketelaar & Palmer 1937).
As previously stated, dinitrogen tetroxide dissociates in the liquid state
\[
N_2O_4 \rightleftharpoons 2NO_2
\]
Besides this, there are two self-ionisations theoretically possible also:
though the latter one does not appear to occur, in practice. This is unusual, since the structure of the dinitrogen tetroxide molecule is:
\[
\begin{array}{c}
O \\
N - N - O \\
O \\
\end{array}
\]
(Broadley & Robertson 1949) - solid state.
(Smith & Hedberg 1956) - gas phase.
The specific conductance has been determined, and is very small; \( K = 1.3 \times 10^{-12} \text{ ohm}^{-1} \text{ cm}^{-1} \) compared to water \( 4 \times 10^{-8} \text{ ohm}^{-1} \text{ cm}^{-1} \) at the same temperature (18°C). (Addison et al 1951a).
In anhydrous nitric acid, dinitrogen tetroxide dissociates almost completely into nitrosonium and nitrate ions; no evidence was found for the ionisation
\[
\text{N}_2\text{O}_4 \rightleftharpoons \text{NO}_2^+ + \text{NO}_2^-
\]
and the dissociation into nitrogen dioxide species was extremely small (Millon & Watson 1957).
The equilibria
\[
\text{NO}^+ + \text{OH}^- \rightleftharpoons \text{HNO}_2 \rightleftharpoons \text{H}^+ + \text{NO}_2^-
\]
have been mentioned.
Since dinitrogen trioxide, $N_2O_3$, is formally the anhydride of nitrous acid, it would be expected that in strongly acid solutions, it would give nitrosonium ions. This is the case. The following ionisation has been established:
$$N_2O_3 + 3H_2SO_4 \rightarrow 2NO^+ + H_3O^+ + 3HSO_4^-$$
by cryoscopic measurements (Gillespie et al 1950a) and confirmed by Raman observations (Millen 1950b).
By analogy with the above, dinitrogen tetroxide would be expected under similar conditions, to give a mixture of nitrosonium and nitronium ions, since it is the mixed anhydride of nitrous and nitric acids. Also dinitrogen pentoxide, $N_2O_5$, should yield nitronium ions only. The same workers found this to be the case and the following ionisations were verified:
$$N_2O_4 + 3H_2SO_4 \rightarrow NO^+ + NO_2^+ + H_3O^+ + 3HSO_4^-$$
$$N_2O_5 + 3H_2SO_4 \rightarrow 2NO_2^+ + H_3O^+ + 3HSO_4^-$$ (Gillespie et al 1950b)
In addition to this, dinitrogen pentoxide behaves as a binary electrolyte in absolute nitric acid, ionising thus:
$$N_2O_5 \rightleftharpoons NO_2^+ + NO_3^-$$ (Gillespie et al 1950b).
This was an extension of earlier work on the nitric acid/sulphuric acid system which established the ionisation
\[ \text{HNO}_3 + 2\text{H}_2\text{SO}_4 \rightarrow \text{H}_3\text{O}^+ + \text{NO}_2^+ + 2\text{HSO}_4^- \]
(Gillespie et al 1946),
and also from pioneer work by Hantzsch (1925) and Hantzsch & Berger (1928). Those latter workers studied the nitric acid/sulphuric acid and nitric acid perchloric acid systems and obtained compounds which they formulated as
\[ \left[ \text{H}_2\text{NO}_3^+ \right] \left[ \text{ClO}_4^- \right] \quad \text{and} \quad \left[ \text{H}_2\text{NO}_3^+ \right] \left[ \text{HSO}_4^- \right] \]
Goddard et al (1950) repeated this work with perchloric acid and found that the compound
\[ \text{H}_2\text{NO}_3^+ \cdot \text{ClO}_4^- \]
could be separated into nitronium perchlorate \( \text{NO}_2^+ \cdot \text{ClO}_4^- \) and hydroxonium perchlorate \( \text{H}_3\text{O}^+ \cdot \text{ClO}_4^- \), and no evidence existed for the presence of \( \text{H}_2\text{NO}_3^+ \) species, either from cryoscopic or Raman spectroscopic data. This was confirmed for the system \( \text{HNO}_3/\text{H}_2\text{S}_2\text{O}_7 \) by Millen (1950a) who observed the Raman spectra of such solutions at varying concentrations.
The production of nitronium ions in strong acid systems in the manner described above has been utilised by Kuhn (1962) to prepare nitronium salts of the fluoborate, fluophosphate and fluoarsenate anions, viz:
\[ 2\text{HF} + \text{HNO}_3 \rightleftharpoons \text{NO}_2^+ + \text{H}_3\text{O}^+ + 2\text{F}^- \quad (\text{Gillespie & Millen 1947}). \]
This equilibrium is exactly analogous to the ionisation of nitric acid in sulphuric acid. By addition of boron trifluoride, the reaction
\[ \text{BF}_3 + \text{F}^- \rightarrow \text{BF}_4^- \]
occurs and nitronium fluoborate is obtained. In practice the reaction is carried out in nitromethane solution and the reaction is
\[ \text{HF} + 2\text{BF}_3 + \text{HNO}_3 \rightarrow \text{NO}_2\text{BF}_4 + \text{H}_2\text{O}_2\text{BF}_3 \]
Similarly sodium nitrite has been employed as a source of nitrosonium ions in the formation of certain metal nitrosyl complexes, viz:-
A stoicheimetric excess of sodium nitrite will react with bis (triphenylphosphine) nickel dibromide to give bis (triphenylphosphine) nickel nitrosyl bromide,
\[ \left[(\text{C}_{6}\text{H}_{5})_{3}\text{P}\right]_2\text{NiNOBr}, \]
among other products. (Feltham 1960); infrared evidence assigns an Ni<—NO⁺ linkage to this compound. Although the reaction was not carried out in strongly acid solution, the prescribed conditions for obtaining nitrosonium ion from nitrite ion, the sodium nitrite must have been the source of nitrosonium ion.
Diagnostic Methods for nitrosonium ions.
1. Cryoscopy.
Besides the applications referred to above in the elucidation of the species present in nitric acid/strong acid systems, this method is historically interesting in that Hantzsch, on the basis of cryoscopic studies on nitrosyl hydrogen sulphate solutions in sulphuric acid, first postulated the existence of the nitrosonium ion species. He postulated the ionisation:
\[ \text{NOHSO}_4 \rightarrow \text{NO}^+ + \text{HSO}_4^- \quad (\text{Hantzsch 1909}) \]
In cases such as
\[ \text{N}_2\text{O}_3 + 3\text{H}_2\text{SO}_4 \rightarrow 2\text{NO}^+ + 3\text{HSO}_4^- + \text{H}_3\text{O}^+ \]
the value of the van't Hoff factor i enables the number of ionic species produced per molecule of dinitrogen trioxide to be obtained, and cryoscopy can distinguish between possible reaction paths which yield differing numbers of ionic species. The most notable case of this occurs in the nitric acid/perchloric acid system, in which Hantzsch postulated the ionisation
\[ \text{HClO}_4 + \text{HNO}_3 \rightarrow \text{H}_2\text{NO}_3^+ + \text{ClO}_4^- \]
giving two ions per nitric acid molecule.
Goddard et al. (1950) showed that the following ionisation occurred:
\[ 2\text{HClO}_4 + \text{HNO}_3 \rightarrow \text{H}_3\text{O}^+ + \text{NO}_2^+ + 2\text{ClO}_4^- \]
giving four ions per nitric acid molecule.
2. Conductivity and electrolytic data.
The compounds NO\textsubscript{2}\text{AlCl}_4, NO\textsubscript{2}\text{FeCl}_4 and NO\textsubscript{2}\text{SbCl}_6 dissolve readily in nitrosyl chloride solution to give strongly conducting solutions (Burg & Campbell 1948); this indicates that ionisation occurs in solution though it does not necessarily infer that the solids are ionic.
Equilibrium studies provided evidence for the existence of NO\textsubscript{2}\text{AlCl}_4, NOCl and NO\textsubscript{2}\text{FeCl}_4, NOCl species, and these were interpreted as compounds in which the nitrosonium ion is solvated by nitrosyl chloride (Burg & MacKenzie 1952). The extremely high transport number calculated for the nitrosonium ion in nitrosyl chloride solution implies the existence of a chain transfer mechanism analogous to that occurring in the electrolysis of water.
Dinitrogen tetroxide yields nitrosonium nitrate-complexes, viz:-
\[ \text{Zn(NO}_3)_2 + 2\text{N}_2\text{O}_4 \rightarrow (\text{NO}^+)_2 (\text{Zn(NO}_3)_4^{2-}) \] (Addison et al 1951b). When a solution of nitrosonium tetranitratozincate is dissolved in nitromethane, electrolysis liberates nitric oxide at the cathode. The same salt will undergo acid-base reactions with anhydrous nitrates and these can be followed conductimetrically:
\[(\text{NO}^+)_{2} (\text{Zn(NO}_3)_4^{2-}) + 2(\text{C}_2\text{H}_5\text{NH}_3^+ \text{NO}_3^-) \rightarrow \]
\[(\text{C}_2\text{H}_5^+ \text{NH}_3^+)_{2} (\text{Zn(NO}_3)_4^{2-}) + 2\text{N}_2\text{O}_4 \] (Addison & Lodge 1954a)
A reaction such as this also characterises the anion of the nitrosonium salt.
3. Magnetic susceptibilities
Asmussen (1939) assembled magnetic data on several metal chloride-nitrosyl chloride adducts and classified them according to their molar susceptibilities and the apparent or 'induced' molar susceptibility of the attached nitrosyl chloride. In no instances were definite structure assignments possible. The compounds giving low 'induced' molar susceptibilities for nitrosyl chloride were the adducts ZnCl_2·NOCl, HgCl_2·NOCl and MnCl_2·NOCl. All these are thermally unstable with respect to dissociation and Asmussen formulated them as molecular addition compounds. The adducts SnCl_4·2NOCl and PtCl_4·2NOCl were found to give much higher induced molar susceptibilities for the attached nitrosyl chloride and were formulated as ionic nitrosonium salts; these were also more stable thermally.
4. Chloride Exchange.
Zinc, cadmium and mercuric chlorides react with liquid nitrosyl chloride to give insoluble adducts. The rate of chloride exchange between nitrosyl chloride and the adduct has been followed (Lewis & Sowerby 1956), and they found that rapid exchange occurred between absorbed nitrosyl chloride and the metal chloride, while slow heterogeneous exchange occurred between absorbed and liquid nitrosyl chloride. To account for this, the formation of $MCl_3^-$ species ($M = Zn, Cd, Hg$) was postulated and hence $NO^+ MCl_3^-$ were considered to be produced. The anions of the type $MCl_3^-$ were postulated to be polymeric species; this will be considered later.
The systems AsCl$_3$/NOCl and POCl$_3$/NOCl were studied; a slow exchange in the latter case indicated lack of compound formation, while a rapid rate in the AsCl$_3$/NOCl case, together with equilibrium studies, suggested an adduct AsCl$_3 \cdot 2$NOCl which was postulated as being $(NO^+)_2 (AsCl_5)^{2-}$ or $NO_2 NOCl + AsCl_4^-$. (Lewis & Sowerby 1957). This will be dealt with further, in a later section.
5. Infrared and Raman Spectroscopy.
By studying the Raman spectra of nitrosyl hydrogen sulphate in sulphuric acid/water solutions, Angus & Leckie (1935) assigned a displacement of 2340 cm\(^{-1}\) to the nitrosonium ion. The intensity of the band was highly dependent on the concentration of sulphuric acid and became very strong at a sulphuric acid concentration of over 70%; the position of the band also depended slightly on the sulphuric acid concentration, occurring at a higher frequency as the concentration increased. The observed displacement was found to be similar to that recorded for the isoelectronic nitrogen molecule, and solid nitrosyl hydrogen sulphate was found to give a Raman displacement of 2311 cm\(^{-1}\).
Nitric oxide itself absorbs at 1877 cm\(^{-1}\) (Addison & Lewis 1955) and nitrosyl chloride gives a band due to the -N=O bond at 1799 cm\(^{-1}\) (Burns & Bernstein 1950); these are both infrared bands.
Gerding & Houtgraaf (1953) have shown that the adduct AlCl\(_3\)·NOCl gives a Raman displacement of 2236 cm\(^{-1}\), which is of the same order as Angus & Leckie's value for nitrosyl hydrogen sulphate; in addition, the same compound gives a low frequency Raman band identical to that in AlCl\(_3\)·NaCl. This presumably indicates the presence of the AlCl\(_4^-\) ion in both cases.
Lewis, Irving & Wilkinson (1958) describe a series of complexes which are regarded as involving nitrosonium ion - metal co-ordination and these all give prominent infrared absorptions in the 1580 - 1930 cm\(^{-1}\) region. Complexes of the type in which the nitrosyl anion is the ligand absorb at a much lower frequency, in the region of 1100 cm\(^{-1}\) (Griffith et al 1958, a, b). The compound \((C_{5}H_{5})_{3}Mn_{2}(NO)_{3}\), mentioned before as possibly containing bridging nitrosyl groups gives an infrared absorption at 1510 cm\(^{-1}\) (Piper & Wilkinson 1956).
From the molecular orbital scheme constructed for the nitric oxide molecule, it is evident that the N - O bond in the nitrosonium ion, consisting of a sigma and two pi-bonds, is stronger than the N - O bond in nitric oxide which is weakened by the odd electron having to go into the pi-antibonding orbital. Hence the former is shorter and would be expected to give a higher vibrational frequency.
The nitrosonium ion is isoelectronic with the nitrogen molecule, and the total number of 2s and 2p electrons in both cases is ten. They can both be considered to be built up as follows, on the basis of both atoms being in an sp hybridized state.
The orbitals shaded are those participating in pi-bonding (the $2p_y$ and $2p_z$ orbitals). One of the $sp$ hybrid orbitals on each atom overlaps to give what is equivalent to the sigma-bonding orbital. Two electrons are involved in this. The py and pz orbitals on each atom overlap to give the pi-bonding molecular orbitals. Thus six electrons have been used in the N - O bond and the other four available electrons go into the two vacant sp orbitals, one on each atom. It is this orbital on the nitrogen atom which is responsible for the nitrosonium ions donor properties. These sp orbitals not involved in bonding between the atoms could be equivalent to the antibonding $\pi$ orbitals of the molecular orbital energy level scheme.
When the nitrosonium ion donates these two electrons to a metal ion, the latter is believed to get rid of some of the excess negative charge thus acquired via back-donation from the metal d-orbitals to the vacant pi-antibonding orbitals which are of suitable shape and symmetry for this. The presence of electrons in the antibonding orbitals will lengthen the N-O bond and lower the vibrational frequency. The further lowering of the vibrational frequency in complexes involving nitrosyl anions is just an extension of this. In this case, little or no metal $\rightarrow$ ligand back-bonding can be expected to occur since the acceptor orbitals on the nitrosyl anions are already occupied; and this will lengthen the N-O bond and lower the vibrational frequency of the mode.
The cases of nitrosyl chloride and complexes involving bridging nitrosyl groups are slightly different; whereas the metal-N=O bonding system in the above cases are linear, these other cases involve bent X-N=O systems. On a valence band treatment this requires the nitrogen atom to have considerable sp$^2$ character. This will lessen the pi-bonding character between nitrogen and oxygen, lengthening the bond, and lowering the frequency.
The nitric oxide molecule and its derivatives have only one vibrational frequency, that corresponding to bond stretching and it is both Raman and infrared active.
The data can be summarised:
| Type of compound | Vibrational frequency region |
|-----------------------------------------|-----------------------------|
| Nitrosonium salt (NO⁺ X⁻) | 2200 - 2400 cm⁻¹ |
| Nitrosonium donor complex | 1580 - 1930 cm⁻¹ |
| (M ↔ NO⁺) | |
| Bridging nitrosyl complex | c. 1500 cm⁻¹ |
| Nitrosyl donor complex | 1050 - 1200 cm⁻¹ |
Infrared data can usually be used to assign structures to nitric oxide compounds, with little ambiguity. Difficulty may arise when systems containing nitronium ions are examined.
As mentioned before, the nitronium ion is linear:
\[ \begin{bmatrix} O = N = O \end{bmatrix}^+ \]
It has three vibrational modes:
\[ O \leftrightarrow N \rightarrow O \quad V_1 \text{ Symmetrical stretch} \]
Raman active
infrared inactive.
\[ \begin{array}{ccc}
\uparrow & \downarrow & \uparrow \\
0 = N = 0 & V_2 - \text{deformation} & \text{Raman active} \\
& & \text{infrared active} \\
\overrightarrow{0} = \overrightarrow{N} = \overleftarrow{0} & V_3 - \text{asymmetric stretch} & \text{Raman inactive} \\
& & \text{infrared active}
\end{array} \]
These modes occur at the following frequencies:
- \( V_1V_1 = 1400 \, \text{cm}^{-1} \) (Ingold, Millen & Poole 1950)
- \( V_3 = 2350-2400 \, \text{cm}^{-1} \)
- \( V_2 = 538 \, \text{cm}^{-1} \) Teranishi & Decius 1954
The infrared spectrum of the nitronium ion usually consists of bands in the following regions, with the assignments given.
- c. 540 cm\(^{-1}\) weak - medium (\( V_2 \))
- 2350 - 2400 strong (\( V_3 \))
- 3750 - 3800 weak (\( V_1 \) & \( V_3 \))
Thus, in the presence of bands which could overlap the \( V_2 \) and (\( V_1 + V_3 \)) modes the only prominent band is the \( V_3 \) fundamental, lying in the same region as that for the free nitrosonium ion; hence ambiguity is possible.
Obviously if Raman spectra are available the presence of the 1400 cm\(^{-1}\) band would definitely confirm the presence of nitrosonium ion.
6. X-ray Crystallography.
Several compounds are known which from their stoichiometry would be expected to contain the nitrosonium ion. Klinkenberg (1937, 1938) studied the X-ray powder photographs of the compounds $NO_2BF_4$, $NO_2ClO_4$, $SnCl_4 \cdot 2NOCl$ and $PtCl_4 \cdot 2NOCl$, and compared them with the potassium analogues. In all cases the complexes were isomorphous with the potassium analogues. Since the potassium salts all contain the potassium ion, it must be assumed that the complexes contain the nitrosonium ion. This ion is formally cylindrical but by rotation in the lattice spherical symmetry can be obtained. By comparison of nitrosonium fluoborate and perchlorate with the isomorphous hydronium and ammonium compounds, Klinkenberg also calculated the effective radius of the nitrosonium ion in free rotation to be $1.40 \text{ Å}$; this makes it larger than the ammonium ion and very similar to the hydronium ion.
Obviously this method only verifies the existence of nitrosonium ions in compounds which have known ionic analogues, and is thus limited in application.
CHAPTER I
STUDIES ON PREVIOUSLY REPORTED
NITROSONIUM FLUORO AND CHLORO-ADDUCTS
As mentioned in the introduction, several metal and non-metal chlorides will react with nitrosyl chloride to form adducts. (Gall & Mengdehl 1927; Rheinboldt & Wasserfuhr 1927; Klinkenberg 1938; Partington & Whynnes 1948, 1949). In addition, nitrogen oxides are known to react with sulphur dioxide or sulphur trioxide to yield products. (Hart-Jones 1929; Goddard et al 1950; Lehmann & Kluge 1951; Gerding & Eriks 1952). In addition, several nitrosonium and nitronium fluoro-metal or non-metal compounds have been formed. (Woolf & Emelius 1950; Woolf 1950; Robinson & Westland 1956; Clark & Emelius 1958; Geichmann et al 1962, 1963). All of these compounds can be formulated as containing nitrosonium or nitronium ions and some have been shown to do so, by the methods of x-ray crystallography (Klinkenberg 1937, 1938), Raman spectroscopy (Gerding & Eriks 1952, Gerding & Eriks 1953) and infra-red spectroscopy (Geichmann et al 1962, 1963). It is possible to examine all the adducts by infra-red
spectroscopy, and in the present work, this was used as a means of determining the bonding state of the nitrosyl group in the compound. As stated in the introduction, compounds containing the free nitrosonium ion should absorb in the infra-red region at about 2200-2400 cm\(^{-1}\), those containing this cation bonded to a metal atom at 1580-1930 cm\(^{-1}\) and those containing a metal-nitrosyl anion bond at 1050-1200 cm\(^{-1}\). These ranges of frequency are sufficiently separate to enable the bonding state to be determined unambiguously.
The compounds studied, with their nitrosonium ion stretching frequencies are tabulated on the following page, together with data obtained by other workers. In addition, x-ray data were obtained for the majority of the compounds and compared with x-ray data for the potassium analogues, where possible, to establish whether or not isomorphism occurred.
The infra-red Raman data show that all the compounds listed can be classified as nitrosonium salts, though the absorption range of the free nitrosonium ion is very large, being nearly 250 cm\(^{-1}\).
X-ray data on the nitrosonium and potassium salts which are isomorphous are tabulated elsewhere in this chapter (table 2); and spectroscopic data on the anions studied are tabulated in tables 3 → 5.
The compounds tabulated in table 1 will be discussed separately (regarding their structure, etc.) starting with those giving high frequency absorptions. General statements and conclusions will be made at the end of the chapter.
\[(\text{NO})_2\text{GeF}_6\]
This is not isomorphous with the potassium analogue \[K_2\text{GeF}_6\]; the infra-red spectrum shows two bands in the free nitrosonium ion region, the main band at 2391 cm\(^{-1}\) with a shoulder at 2336 cm\(^{-1}\). Gerding & Eriks (1952) have shown that the compound \((\text{NO})(\text{NO}_2)(S_3O_{10})\) gives two Raman peaks at 2277 cm\(^{-1}\) and 2308 cm\(^{-1}\) and assigned the former peak to the antisymmetrical \((V_3)\) mode of the nitronium ion, rendered Raman active by the ion-site symmetry in the crystal lattice. In view of the analytical data for \((\text{NO})_2\text{GeF}_6\) it is most unlikely that the same explanation holds; also, since the reaction system was nitrosyl chloride/germanium dioxide/bromine trifluoride, and the nitrosyl chloride was spectroscopically free from dinitrogen tetroxide, it is improbable that any mechanism for the formation of nitronium ions existed. It was not possible to check the absence of nitronium ion by observing the infra-red regions where the \((V_1 + V_3)\)
and $V_2$ vibrational modes were expected, due to the rapidity with which $(\text{NO})_2\text{GeF}_6$ attacked mujol. The $(V_1+V_3)$ region was examined in florube, and no evidence was found for nitronium ion. Hence the most probable explanation is that the nitrosonium ion in $(\text{NO})_2\text{GeF}_6$ occupies two different types of lattice site, so that it is in two positions of different local symmetry.
$(\text{NO})_2\text{GeF}_6$ is extremely hygroscopic, decomposing rapidly even in the 'dry-box' with copious nitrogen dioxide evolution leaving a germanium dioxide residue. In vacuo, and in the complete absence of moisture it is quite stable, and is thermally stable.
$(\text{NO})_2\text{VF}_6$
This compound, like $(\text{NO})_2\text{GeF}_6$, is not isomorphous with its potassium analogue K.VF$_6$, and also gives two infra-red bands; one at 2391 cm$^{-1}$, with a shoulder at 2328 cm$^{-1}$. The explanation is likely to be similar to that in the instance of $(\text{NO})_2\text{GeF}_6$, the nitrosonium ion being present in two different types of lattice position.
Like $(\text{NO})_2\text{GeF}_6$ it is extremely hygroscopic and rapidly hydrolyses in air to leave a vanadium pentoxide residue; it is quite stable in vacuo.
The reaction nitrosyl chloride/vanadium pentoxide/bromine trifluoride gives $NO_2VF_6$ without any contamination by $NO_2VOF_4$; if a metal cation, such as sodium, is substituted for the nitrosonium cation, the main product is $Na_2VOF_4$, and even prolonged refluxing in bromine trifluoride will not give pure $Na_2VF_6$. (Sharpe & Woolf 1951). The preparation of the sodium salt of the hexafluorovanadate ion requires that vanadium chloride be used instead of vanadium pentoxide in the preparation; under these conditions pure $Na_2VF_6$ results.
Aynsley et al (1954) reported the following reaction scheme for the vanadium pentoxide/nitryl fluoride systems:
$$\begin{align*}
V_{20}^0S & \xrightarrow{\text{NO}_2F} VOF_3 \\
& \downarrow \\
& NO_2VOF_4
\end{align*}$$
This demonstrates that the nitronium ion system can give both the fluovanadate and the oxyfluovanadate. This type of reaction has not been repeated using nitrosyl fluoride, due to the difficulty of handling this reagent, so that the compound $NO_2VOF_4$ is as yet unknown.
Recently, reaction of nitric oxide or nitrogen dioxide with vanadium pentafluoride suggests that NO$^+$ VF$_5^-$ and NO$_2^+$ VF$_5^-$ species may be formed, though the reactions
$$\text{NO}_x + \text{VF}_5 \rightarrow \text{NOVF}_6 + \text{VF}_4 \quad (x = 1 \text{ or } 2)$$
occur more readily than
$$\text{NO}_x + \text{VF}_5 \rightarrow \text{NO}_x \cdot \text{VF}_5$$
(Ogle et al 1964).
This compound was shown to be isomorphous with potassium fluoborate by Klinkenberg (1937), and this was confirmed in the present work. The x-ray data reported latterly (Sharp & Thorley 1963) were incorrect and the corrected data are given in table 2; the values show a reasonable agreement with those of Klinkenberg.
The stretching frequency for the nitrosonium ion is considerably at variance with that given by Sprague et al (1960) and Evans (1963). Evans reports that the nitronium ion V$_3$ frequency in NO$_2$·BF$_4$ is 2387 cm$^{-1}$, and suggested that the value obtained in the present work was due to this same mode, the nitronium ion being present as an impurity. However, x-ray data submitted by the same worker for the nitrosonium salt, were identical with
data obtained in the present work. It is most unlikely that nitrosonium and nitronium fluoborates have the same unit cell sizes; thus the compounds in both cases must have been pure nitrosonium fluoborate and the explanation must lie elsewhere.
Incidentally, the nitronium ion $V_3$ frequency reported by Evans does disagree somewhat with the value of 2358 cm$^{-1}$ obtained by Cook (1960).
The x-ray data show that substitution of nitrosonium ion for potassium ion in a fluoborate lattice increases the unit cell dimensions slightly. The volume of the unit cell increases, due to the accommodation of the larger nitrosonium ion. The nitrosonium ion is cylindrical, but it can obtain spherical symmetry when it is allowed to rotate freely in a lattice, and under these conditions, the effective radius is 1.40Å (Klinkenberg 1937), which is larger than the value $r = 1.33Å$, for the potassium ion. Hence an increase in unit cell size would be expected.
NO$_2$SbF$_6$
This compound is not isomorphous with its potassium analogue; unlike the vanadate and germanates it gives quite a broad absorption in the
nitrosonium ion region. Again, the existence of a prominent shoulder indicates that two types of nitrosonium lattice site are present in the unit cell.
This reacted much less rapidly with nujol than did \((\text{NO})_2\text{GeF}_6\) and \(\text{NO.VF}_6\), and it was possible to obtain data on the \(\text{SbF}_6^-\) anion; this was found to absorb at 660 cm\(^{-1}\), in good agreement with a value of 660 cm\(^{-1}\) for the potassium salt (Peacock & Sharp 1959). (See table 5).
\[\text{NO.PF}_6\]
This compound gave a single sharp absorption at 2379 cm\(^{-1}\) in the infra-red region; it was not isomorphous with potassium fluophosphate.
Table 5 tabulates data obtained in the present work on the \(\text{PF}_6^-\) anion in the nitrosonium salt, and by other workers, with other cations. In all cases bands appear in the 830-840 cm\(^{-1}\) and 720-740 cm\(^{-1}\) regions.
The undistorted \(\text{PF}_6^-\) ion has \(O_h\) symmetry, and six normal modes of vibration. In a non-cubic lattice, the symmetry will definitely be lower than \(O_h\) due to lack of symmetry elements; in a cubic lattice the symmetry will probably be \(O_h\), but not necessarily so. Slight anion asymmetry may occur which would not affect the crystal lattice. Silver, potassium, rubidium and
caesium fluophosphates have all been shown to possess cubic structures (Kemmitt et al 1963 b), and all these give strong absorptions in the 830-840 cm\(^{-1}\) region, with much weaker absorptions around 720-740 cm\(^{-1}\), in all except potassium (Peacock & Sharp 1959; Sharp & Sharpe 1956). The strong absorption was assigned to the \(V_3\) vibrational mode. Woodward and Anderson (1956) measured the Raman spectrum of potassium fluophosphate, and assigned the observed band at 741 cm\(^{-1}\) to the \(V_1\) mode. This is the totally symmetric stretching mode of the ion, and is infra-red inactive. Thus it is possible that the 720-740 cm\(^{-1}\) bands observed are the \(V_1\) modes in the PF\(_6^-\) anion of the salts studied, rendered slightly infra-red active by lowering of the \(O_h\) symmetry. In the nitrosonium salt, the bands at 834 cm\(^{-1}\) and 740 cm\(^{-1}\) are equally strong, and it could be that the PF\(_6^-\) anion has been rendered sufficiently asymmetric by the nitrosonium ion, that the \(V_1\) mode is now strongly infra-red active.
The radii of the various cations are:
| Ion | Radius (Å) |
|-------|------------|
| K\(^+\) | 1.33 |
| Rb\(^+\) | 1.48 |
| Ca\(^+\) | 1.69 |
| Ag\(^+\) | 1.26 |
| NO\(^+\) | 1.40 (effective) |
It appears that, from the infra-red data, in potassium hexafluophosphate the cation size is such that the $O_h$ symmetry of the anion is undistorted, whereas in the other instances, anion distortion and asymmetry occurs in varying degrees. It might be expected that the extent of asymmetry in the anion, measurable by the relative intensities of the $V_1$ and $V_3$ modes, would be dependent on cation radius. However the nitrosonium salt is the only one in which large anion distortion occurs and its effective radius lies between radius values for the other cations; it may be that the shape of the nitrosonium ion is responsible for the extremely large distortion which occurs.
$\text{NO}_2\text{SO}_3\text{F}$
This salt is isomorphous with potassium fluosulphate, and both these salts are isostructural with the nitrosonium and potassium fluoborates. This is again due to the non close packed structure of these salts, permitting nitrosonium-potassium replacement without structural change. In both these instances the structure of the anion is tetrahedral or distorted tetrahedral, and the lattice interstices in those structures are much larger than in the hexafluo-metal nitrosonium salts.
As would be expected for isomorphous salts, the infra-red spectrum of the fluosulphate ion is the same for the nitrosonium salt, as for the potassium salt (Sharp 1957). The early preparations of nitrosonium fluosulphate gave two bands in the nitrosonium region, but the lower one, in the $2280 \text{ cm}^{-1}$ region was assigned to residual nitrosonium pyrosulphate from the reaction between this, and bromine trifluoride.
$\text{NO}_2\text{SnF}_6$
This salt is not isomorphous with the potassium analogue; the presence of a single band in the nitrosonium ion region, at $2342 \text{ cm}^{-1}$, indicates the presence of only one type of lattice site for the nitrosonium ion.
The solid is very hygroscopic, but reacts sufficiently slowly with rigorously dried nujol to obtain infra-red data on the $\text{SnF}_6^{2-}$ anion. The value of $547 \text{ cm}^{-1}$ is in good agreement with the literature value of $552 \text{ cm}^{-1}$ for potassium fluostannate (Peacock & Sharp 1959).
$\text{NO}_2\text{AsF}_6$
Like all the other hexafluo-metal salts studied, $\text{NO}_2\text{AsF}_6$ was non-isomorphous with its potassium analogue.
It gave a single infra-red absorption assignable to nitrosonium ion, and the absorption at 700 cm\(^{-1}\) assignable to the V\(_3\) mode of the fluoarsenate anion, is the same as that obtained by Peacock & Sharp (1959) for the potassium salt.
**NO.HSO\(_4\)**
The literature data on nitrosyl hydrogen sulphate gives the nitrosonium ion a Raman frequency of 2340 cm\(^{-1}\) (Gerding & Houtgraaf 1953), although Angus & Leckie (1935) reported a value of 2311 cm\(^{-1}\) for the solid salt; in sulphuric acid, they reported values of 2330-2338 cm\(^{-1}\) depending on the concentration of water in the sulphuric acid.
On heating, decomposition occurs:
\[ \text{NO.HSO}_4 \rightarrow (\text{NO})_2\text{S}_2\text{O}_7 \]
and, as explained in a later chapter, attempts to prepare \((\text{NO})_2\text{SO}_4\) have been unsuccessful.
**NO.UF\(_6\) and NO.MoF\(_6\)**
These compounds were obtained as white solids by the gas phase interaction of uranium or molybdenum hexafluorides with nitric oxide (Geichmann et al 1962)
\[ \text{MF}_6 + \text{NO} \rightarrow \text{MNO.MF}_6 \quad (M = U_2\text{Mo}) \]
The same workers found that no corresponding reaction occurred with tungsten hexfluoride, and that
nitrous oxide reacted with none of these metal fluorides. Also, reactions of the type
\[ \text{NOF} + \text{MF}_5 \rightarrow \text{NO.MF}_6 \quad (M = U, Mo) \]
gave identical products, and again no reaction occurred with tungsten. Seel & Birnkraut (1962) used the reagent NOF (HF) to prepare compounds of the formula, \( \text{NO.MoF}_6, \text{NO.UF}_6 \) and \( \text{WF}_6.\text{NOF} \). The first two are almost definitely identical with the compounds of Geichmann et al (1962). A compound of the same formula as the last mentioned was prepared by Geichmann et al (1963) by the reaction
\[ \text{NOF} + \text{WF}_6 \rightarrow \text{NO.WF}_7 \]
The presence of bands at 2330 cm\(^{-1}\) and 620 cm\(^{-1}\) was assigned respectively to the nitrosonium cation and the heptafluotungstate anion, and it would appear that this is identical with Seel & Birnkraut's \( \text{WF}_6.\text{NOF} \).
Geichmann et al (1963) also prepared the compound nitrosonium heptafluotungstate by the reaction
\[ \text{NO}_2\text{F} + \text{WF}_6 \rightarrow \text{NO}_2.\text{WF}_7 \]
and the nitronium and nitrosonium salts of the hepta-fluo-molybdate and uranate were obtained by analogous reactions. These were all identified and characterised on the basis of their infra-red spectra.
The same workers also found that the reaction between nitrosyl chloride and either molybdenum or uranium hexafluorides, gave respectively NO.MoF$_6$ or NO.UF$_6$. They suggested that this reaction proceeded through an unstable (MoF$_6$Cl$^-$) anion which readily dissociated. Again, tungsten hexafluoride did not react.
It is surprising that the attempts to form nitrosonium hexafluotungstate were unsuccessful. The WF$_6^-$ anion is quite well characterised (Sharpe 1960). An explanation of this would probably require a knowledge of the electron affinities MF$_6$/MF$_6^-$ (where M = Mo, W, U); it may be that the value of this couple for tungsten is considerably less than for molybdenum or uranium.
Other nitronium and nitrosonium salts have been prepared by gas phase interaction but they have not yet been characterised (Robinson & Westland 1956). They are the hexafluoiridates (NO)$_2$IrF$_6$ and (NO$_2$)$_2$IrF$_6$, obtained thus:-
$$2\text{NO} + \text{IrF}_6 \rightarrow (\text{NO})_2\text{IrF}_6$$
$$2\text{NO}_2 + \text{IrF}_6 \rightarrow (\text{NO}_2)_2\text{IrF}_6$$
These presumably contain iridium (IV) which is well characterised. On heating \((\text{NO})_2\text{IrF}_6\) and \((\text{NO}_2)_2\text{IrF}_6\) give, respectively, \((\text{NO})_2\text{IrF}_5\) and \((\text{NO}_2)_2\text{IrF}_5\), whose structures are unknown.
Geichmann et al (1962, 1963) make no mention of the thermal stability of the nitrosonium metal hexafluorides, but report that several of the hexafluorides do tend to dissociate, though not to lower complex metal fluorides.
**NO·BiCl₄**
This was one of the first metal chloride : nitrosyl chloride adducts to be prepared, (Rheinboldt & Wasserfuhr 1927) by direct interaction of bismuth trichloride with nitrosyl chloride to give a yellow-orange powder. This is very unstable to heat
\[
\text{NO·BiCl}_4 \rightarrow \text{BiCl}_3 + \text{NOCl}
\]
The orange colour suggests that some anion-cation interaction may occur in a case such as this, since BiCl₃ is colourless. As mentioned in the introduction, in the absence of other chromophoric species colour in nitrosonium ion systems will be due to charge transfer mechanisms of the type
\[
\text{NO}^+ \quad X^- \leftrightarrow \text{NO} \quad X
\]
and the possibility of a reaction such as this being
appreciable is, in this particular instance, supported by the observation that NO₂BiCl₄ dissociates very readily so that considerable anion-cation interaction must occur.
\[(NO)₂PdCl₄\]
This compound was first reported by Partington & Whynnes (1949), who prepared it by reaction of palladous chloride with nitrosyl chloride at 100°C; the reaction time was 5 hours. In the present work it was found that palladous chloride and liquid nitrosyl chloride reacted almost instantaneously to give PdCl₂·2NOCl, a bright red compound. An infra-red band at 2330 cm⁻¹ established the presence of free nitrosonium ion, and in the absence of bands characteristic of metal ←nitrosonium ion bonding, the compounds can be formulated \[\left[NO^+\right]_2\left[PdCl_4^{2-}\right]\], containing palladium (II). Complexes of the general formula M₂PdCl₄ (M = alkali metal ion) have been reported in the literature (Sidgwick 1950), and the colours range from yellow to brown, so in this case it is likely that the colour in (NO)₂PdCl₄ arises mainly from d-d transitions in the palladium ion.
Reaction of palladium metal with nitrosyl chloride gives PdCl₂·2NO (Partington & Whynnes 1949) and this may contain palladium ←—nitrosonium bonds. This type of reaction is unusual, in that the solvolysis reactions involving nitrosyl chloride usually yield nitrosonium salts and not nitrosonium covalent complexes; though gas phase reactions involving nitrosyl chloride do yield the latter (Addison & Johnson 1962).
NO₃ClO₄
This compound has been shown to be isomorphous with potassium perchlorate (Klinkenberg 1937); the presence of the nitrosonium ion has been confirmed by the presence of a Raman band at 2313 cm⁻¹ (Gerding & Houtgraaf). It can be prepared by the solution of dinitrogen trioxide in perchloric acid; as explained in the introduction, the dinitrogen trioxide will yield nitrosonium ion on solution and nitrosonium perchlorate can be precipitated. It has also been prepared by bubbling nitric oxide into an aqueous sulphuric acid/perchloric acid mixture (Cruse et al 1949).
It is very hygroscopic, and thermally unstable, decomposing below 100° without melting:
\[ 2\text{NO}_3\text{ClO}_4 \rightarrow \text{N}_2\text{O}_4 + 3\text{O}_2 + \text{Cl}_2 \]
Like the other nitrosonium salts which are isomorphous with their potassium analogues, nitrosonium perchlorate contains a tetrahedral anion; this, again, gives lattice interstices large enough to allow nitrosonium-potassium substitution without structural change.
\[(\text{NO})_2(\text{NO}_2)_3\text{S}_3\text{O}_{10} \text{ and } (\text{NO})_2\text{S}_2\text{O}_7\]
Both these compounds are products of nitrogen oxide/sulphur oxide reactions. The nature of these reactions will be more fully dealt with in a later chapter. The structure \((\text{NO})_2(\text{NO}_2)_3\text{S}_3\text{O}_{10}\) was assigned on the basis of Raman data, which will be fully covered later.
The compound nitrosonium pyrosulphate appears to contain two nitrosonium ion lattice sites per unit cell, since the infra-red spectrum shows a strong shoulder at 2294 cm\(^{-1}\) as well as the main band at 2278 cm\(^{-1}\). Although the structure of the anion is probably based on linked SO\(_4\) tetrahedra, the salt is not
isomorphous with potassium pyrosulphate.
\[\text{NO}_2\text{MnCl}_3\]
This compound was obtained initially by the passage of nitrosyl chloride over manganous chloride at 260° (Gall & Mengdehl 1927); this seems rather surprising in view of the thermal instability of the compound. Gall & Mengdehl reported a yellow powder; the present work gave, on reaction of manganous chloride and liquid nitrosyl chloride, a golden product. Infra-red examination showed a band at 2271 cm\(^{-1}\) and thus this compound could be formulated \(\text{NO}^+\text{MnCl}_3^-\).
Originally, Asmussen (1939), on the basis of magnetic susceptibility data and thermal stability observations, assigned this compound the structure \([\text{MnNO}]^{3+}\text{Cl}^-_3\). In this case magnetic data are not conclusive since the manganese (II) ion is itself paramagnetic, and the compound was found, also, to be paramagnetic.
The pyridinium salt \([C_5H_5NH]^+\text{[MnCl}_3^-\text{]}\) has been prepared (Taylor 1934) and this recrystallises from water as \([C_5H_5NH]^+\text{[MnCl}_3\text{H}_2\text{O]}\), so it is possible that the anion is monomeric; in the absence of single crystal crystallographic data no definite conclusions can be made about the structure of the anion.
The value obtained from the Mn-Cl stretching frequency of 268 cm\(^{-1}\) in NO.\(MnCl_3\) agrees quite well with the value of 282 cm\(^{-1}\) for this mode in the tetrachloromanganite \(MnCl_4^{2-}\) anion (9). If the \(MnCl_3^-\) anion were octahedral, the manganese-chlorine stretching frequency would be less than if it were tetrahedral; hence the infra-red data suggests an octahedral structure but this cannot be regarded as conclusive.
The visible spectrum of the \(MnCl_4^{2-}\) anion shows bands in the 445 and 430 m\(\mu\) regions, (Gill & Nyholm 1959), and the reflectance spectrum of NO.\(MnCl_3\) shows a weak band centred about 450 m\(\mu\), which may indicate the presence of linked tetrahedral \(MnCl_4^{2-}\) units. Compounds which formally contain the \(MnF_3^-\) anion are known (Sharpe 1960) though these are in fact perovskites; no fluomanganites or manganate nitrosyl complexes have been reported, so NO.\(MnCl_3\) was reacted with bromine trifluoride to see if any nitrosonium fluo-manganese compound could be formed. The only product was manganese trifluoride \(MnF_3\); the nitrosonium chloro-manganite presumably dissociates in bromine trifluoride, manganese trifluoride results and is precipitated out. It has been noted previously that
nitrosonium or nitronium fluo-metal salts are not formed when the metallic 'base' or fluoride is unsoluble in bromine trifluoride. (Woolf & Emeleus 1950).
\[ \text{NO}_2\text{CuCl}_2 \]
Gall & Mengdehl (1927) reacted cuprous chloride and nitrosyl chloride and obtained a product which they formulated \( \text{Cu}_2\text{Cl}_2 \cdot 2\text{NOCl} \); a similar product was obtained from the reaction between nitric oxide and cupric chloride (Manchot 1910). The latter worker observed that ethanolic cupric solutions, on passage of nitric oxide, became very deep blue or violet. The nitric oxide copper mole ratio was established as being less than 1:1, and by electrolysis experiments it was shown that the nitric oxide was associated with the cation (Manchot 1914).
Asmussen (1939) studied the magnetic behaviour of the compound, and, on the basis of its apparent paramagnetism, formulated it as \( [\text{CuNO}^{2+}] [\text{Cl}^-]_2 \), assuming the complex ion \( [\text{CuNO}^{2+}] \) to be paramagnetic due to unpaired electrons on both the copper and nitric oxide groups. However, Burg and MacKenzie (1952)
showed that the freshly prepared compound was diamagnetic. Paramagnetism only came about on standing.
Partington & Whynnes (1948) repeated Gall & Mengdehl's work, and found also that the adduct CuCl.NOCl was obtainable by reacting either copper metal or copper oxide with nitrosyl chloride; no structural observations were made.
Griffith et al (1958 b) studied the infra-red spectra of the compounds Cu(C₂H₅OH)₃NO .Cl₂ and Cu(C₂H₅OH)₃NO .Br₂ in ethanolic solution; strong bands in the 1850 cm⁻¹ region in each instance indicated that a Cu⁺ ← NO⁺ linkage was present and the cation would thus be:
\[
\begin{array}{c}
C_2H_5 \cdot OH \\
\downarrow \\
C_2H_5OH \rightarrow Cu \leftarrow N=O \\
\uparrow \\
C_2H_5OH
\end{array}
\]
The diamagnetism of the solutions confirmed a Cu⁺ ← NO⁺ linkage, since cuprous ion has d¹⁰ configuration and is diamagnetic.
Fraser (1960, 1961) and Fraser & Dasent (1960) have carried out several studies dealing with cupric
halide/nitric oxide equilibria in various solvents. By studying the cupric chloride and bromide systems with nitric oxide in ethanolic solution, Fraser & Dasant concluded that with cupric chloride/ethanol chloride, and that in cupric bromide, both neutral cupric bromide and the ionic species CuBr⁺ absorbed.
A structure for the complex was postulated:
\[
\begin{align*}
\text{Solvent} \\
\downarrow \\
X \rightarrow \text{Cu} \leftarrow X \\
\downarrow \\
X \rightarrow \text{Cu} \leftarrow X \\
\uparrow \\
\text{NO}
\end{align*}
\]
and this was assumed to dissociate, to account for the conductivity of these solutions. However, the previous explanation (Griffith et al 1958) seems more probable, since solvation of the copper ion by ethanol molecules seems a more feasible structure than that in the diagram above.
Fraser (1960) also reported the formation of a postulated cupric fluoride/nitric oxide complex in tert-butanol; the deep violet colour of this complex was suggested as being due to N₂O₂⁺ ion.
From methanol/carbon tetrachloride/cupric halide/nitric oxide systems, the isolation of solids corresponding to the formulas, $\text{CuBr}_2.\text{NO}$ and $\text{CuCl}_2.\text{NO}$, has been reported (Mercer & Fraser 1963). These are black solids and give infra-red bands at $1840 \text{ cm}^{-1}$ and $1854 \text{ cm}^{-1}$ respectively, which definitely classifies them as containing a $\text{Cu} \leftarrow \text{NO}^+$ linkages. These data are not consistent with the present work; in the present work reaction of cuprous chloride with liquid nitrosyl chloride gave, very rapidly, a black solid partially soluble in the excess solvent. On removal of solvent, an ochre-coloured product remained which corresponded to the formula $\text{CuCl}_2.\text{NO}$; the freshly prepared compound was diamagnetic, confirming the observation of Burg & MacKenzie (1952), and the band at $2271 \text{ cm}^{-1}$ in the infra-red spectrum definitely signified the existence of free nitrosonium ion.
It is possible that the black product in nitrosyl chloride solution was a solvate of the compound $\text{NO}_2\text{CuCl}_2$, and that the black $\text{CuBr}_2.\text{NO}$ and $\text{CuCl}_2.\text{NO}$ of Mercer & Fraser (1963) are in fact solvates, possibly containing methanol as the ligand. Alternatively it
is possible that the ionic NO$^+$.CuCl$_2^-$ does exist in a different form in solution, and that equilibria of the type
$$\text{NO}^+.\text{CuCl}_2^- \rightleftharpoons \text{NO}^+ \leftarrow \text{CuCl}_2$$
exist, and the form in which it is isolated, is dependent upon the mode of preparation. The nature of the solid NO$^+ \leftarrow \text{CuCl}_2$ species could then be either entirely covalent or $[\text{Cu} \leftarrow \text{NO}^+]^{2+}.2\text{Cl}^-$.
The value obtained for the Cu-Cl stretching frequency of 245 cm$^{-1}$ is slightly lower than the values reported by Clark & Dunn (1964) for Cu(II)-Cl frequencies in $[\text{CuCl}_4]^{2-}$ salts. This would be expected, since metal-ligand bond frequencies are very sensitive to the oxidation state of the metal.
The structure of the $[\text{CuCl}_2]^-$ anion is not known; since it contains Cu(I) which has a d$^{10}$ configuration, no information can be obtained from reflectant spectroscopic data.
**NO.HgCl$_3$**
Gall & Mengdehl (1927) prepared this by the interaction of mercuric chloride and nitrosyl chloride, to yield a red-brown solid. It dissociates very readily.
Asmussen (1939) obtained magnetic susceptibility data on this compound and from these data, and its known thermal instability, postulated it as being a molecular addition compound. Lewis and Sowerby (1957) studied the rate of chloride ion exchange between the compound and liquid nitrosyl chloride, and found evidence for anionic species of the type $\text{HgCl}_3^-$, which, it was suggested, were polymeric. The present work confirmed the presence of ionic species; the infra-red absorption band at $2268 \text{ cm}^{-1}$ is in the characteristic region for nitrosonium ion.
No evidence was obtained to prove or disprove the presence of polymeric anionic species. The far infra-red spectrum gave a single absorption at $261 \text{ cm}^{-1}$, due to an Hg-Cl stretching mode.
**NO.$\Delta$AlCl$_4$**
Reaction of anhydrous aluminium chloride and liquid nitrosyl chloride gives a white solid, soluble in nitrosyl chloride. This confirmed the observations of Gording & Houtgraaf (1953) who obtained a similar product; Partington & Whynnes (1948) had obtained a lemon-yellow product melting with decomposition. This has been explained as being due to the existence of
two allotropic forms, the metastable one being the latter (Addison & Lewis 1955).
Gerding & Houtgraaf (1953) obtained a Raman displacement of $2236 \text{ cm}^{-1}$ for the compound, and the compound $\text{AlCl}_3\cdot\text{NaCl}$, gave identical low frequency Raman displacements to $\text{NO}\cdot\text{AlCl}_4^-$ confirming the presence of $\text{AlCl}_4^-$ in the latter.
Because the value of $2236 \text{ cm}^{-1}$ obtained for the stretching mode of the nitrosonium ion was much lower than that of the same ion in nitrosyl hydrogen sulphate, Gerding & Houtgraaf postulated that anion-cation polarisation occurred in $\text{NO}\cdot\text{AlCl}_4$ to lengthen the N-O bond in the cation and thus lower the frequency. They postulated an equilibrium:
$$\text{NO}^+\cdot\text{AlCl}_4^- \rightleftharpoons \text{AlCl}_3\ldots\ldots\text{Cl}\ldots\ldots\text{NO}$$
and the value for the nitrosonium ion stretching mode would depend on which form, the ionic or molecular, was predominant. This type of polarisation probably does occur and will be discussed later in this chapter.
Partington & Whynnes (1948) also prepared adducts closely related to $\text{NO}\cdot\text{AlCl}_4$, viz:-
$\text{GaCl}_3\cdot\text{NOCl}$, $\text{InCl}_3\cdot\text{NOCl}$ and $\text{TlCl}_3\cdot\text{NOCl}$. These have not been examined to verify the presence of free
nitrosonium ion, but it is most probable that they are of the same structure as the aluminium adduct. Seel & Sauer (1957) examined their behaviour under nitric oxide pressure in sulphur dioxide solution and observed the purple colouration typical of $N_2O_2^+$ ion; thus it can be assumed that the above adducts are ionic compounds.
$NO)_2PtCl_6$
Klinkenberg (1938) has shown this to be isomorphous with potassium chloroplatinate; the present work confirmed this (table 2), and obtained the nitrosonium ion frequency as being consistent with the formulation $(NO^+)_2(PtCl_6^{2-})$. The compound is red, and fairly stable compared to many other nitrosonium salts.
The data on the $PtCl_6^{2-}$ anion (table 3) show it to be unaffected by the change in the cation; this would be expected for isomorphous salts of different cations.
$NO.FeCl_4$
This was first obtained by Rheinboldt & Wasserfuhr (1927) by direct interaction of ferric chloride and nitrosyl chloride. The behaviour of
the compound in nitrosyl chloride solution was studied by Burg & MacKenzie (1952), who concluded that it ionised in solution into NO$^+$ and FeCl$_4^-$, the former probably solvating to [NOCl.NO]$^+$ species.
The present work confirmed the existence of the compound in the solid state as NO$^+$FeCl$_4^-$. The reflectance spectrum gave a broad absorption centred about 530 mμ; this agrees with other data on chloroferrates by Friedmann (1952), who observed a 532 mμ band in solid potassium chloroferrate and in an FeCl$_3$/12M HCl mixture, and Gill (1961) who studied tetraethylammonium chloroferrate and observed strong bands at 533 mμ and 685 mμ. Neither authors assigned these bands.
The data on the FeCl$_4^-$ anion agrees quite well with that for the tetraethylammonium salt (table 3); the literature data on the chloroferrate and -ferrite shows the marked dependence on the oxidation state of the metal atom.
Seel & Birnkraut (1962) reacted iron with nitrosyl hydrogen fluoride NOF(HF)$_3$ and obtained an adduct NOF.FeF$_3$, which may be NO$^+$FeF$_4^-$; if this is so, it is the first reported example of FeF$_4^-$ ion.
In an attempt to prepare Seal & Birnkraut's compound, NO.FeCl₄ was dissolved in bromine trifluoride, and the excess solvent removed. The only product was ferric fluoride.
\[(NO)_2SnCl_6\]
This has been shown to be isomorphous with potassium chlorostannate (Klinkenberg 1938) and the present work confirmed this. Since the two salts are isomorphous anion-cation polarisation is likely to be small, if present at all.
\[(NO)_2SnCl_6\] is a pale yellow solid; it is much more stable in moist air than its fluo-analogue \[(NO)_2SnF_6\]. Asmussen (1939) observed its thermal stability and from this and magnetic susceptibility data formulated it as an ionic compound \((NO^+)_2(SnCl_6^{2-})\).
The far infra-red data on the SnCl₆²⁻ anion (table 3) shows a good agreement between the potassium and nitrosonium salts as would be expected, for isomorphous compounds. The 310 cm⁻¹ band is presumably the \(V_3\) mode.
\[(NO)_2SbCl_6\]
This compound, and the previous one, both prepared by reaction of nitrosyl chloride with the high valency metal chloride, in carbon tetrachloride
solution (Rheinboldt & Wasserfuhr 1927). NO.SbCl₆ is pale yellow, and stable to heat. Like the chloro-stannate couple, it is more stable to hydrolysis than its fluo analogue NO.SbF₆. The far infra-red shows an absorption at 345 cm⁻¹ (table 3) presumably due to the V₃ mode.
**NO.ZnCl₃**
This compound was prepared by Asmussen (1939); like AgCl₂.NOCl, it was found to be thermally unstable, and the two compounds gave similar magnetic properties which led Asmussen to formulate it as a molecular addition compound. However, the present work shows it to be a nitrosenium salt NO⁺ZnCl₃⁻, although the colour suggests that anion-cation polarisation may occur. Apart from the charge transfer system
\[ \text{NO}^+ \quad \text{Cl}^- \rightleftharpoons \text{NO}^- \quad \text{Cl}^+ \]
no other chromophore is present.
The far infra-red data on the anion agrees quite well with other far infra-red data on Zn(II)-Cl band frequencies (table 4). As in NO.AgCl₃, it has been suggested that the ZnCl₃⁻ anion is polymeric (Lewis & Sowerby 1957) but no evidence for this has been obtained.
Like the chloro-stannate and antimonate, \((\text{NO})_2\text{TiCl}_6\) is obtained by the reaction of nitrosyl chloride and a high valency metal chloride, in this case titanium tetrachloride, in nitrosyl chloride (Rheinboldt & Wasserfuhr 1927). It is very readily hydrolysed, leaving a residue of titanium dioxide, even in moist air. It sublimes very readily, even at room temperature.
It is not isomorphous with its potassium analogue; all this suggests that a high degree of anion-cation polarisation occurs, similar to that postulated by Gerding & Houtgraaf for \(\text{NO}.\text{AlCl}_4\).
The far infra-red band at \(478 \text{ cm}^{-1}\) is due to the \(\text{TiCl}_6^{2-}\) anion but due to the possible distortion of the anion, it is not possible to assign this band.
**Discussion.** Table 1 shows that the range of the \(\text{NO}^+\) absorption extends over nearly \(230 \text{ cm}^{-1}\); Seel (1950) and Gerding & Houtgraaf (1953) explained this in terms of partial covalent character:
\[
\text{NO}^+ \cdot \text{MCl}_n^- \leftrightarrow \text{NO...Cl...MCl}^-_{n-1}
\]
The partial formation of an N-Cl bond like this would
lengthen the N-O bond and so lower the vibrational frequency of the N-O stretching mode. This explanation was adequate to describe the bond frequency differences in compounds such as NO.HSO$_4$, NO.AlCl$_4$ and NO.SbCl$_6$. In the latter two, partial cation polarisation would occur due to partial N-Cl bond formation; this would not occur with nitrosyl hydrogen sulphate to the same extent.
Table 1 shows that this explanation is not adequate. The compounds NO.BF$_4$, NO.SO$_3$F, NO.CLO$_4$, (NO)$_2$PtCl$_6$ and (NO)$_2$SnCl$_6$ are all isomorphous with their potassium analogues. This makes it unlikely that anion-cation polarisation occurs in these instances since such polarisation would be very likely to distort the lattice. Also, since in all cases except the perchlorate, the infra-red spectra of the anions of the nitrosonium salt and the corresponding potassium salt are identical, it seems most unlikely that any polarisation occurs in these compounds, yet their nitrosonium ion stretching frequencies range from 2387 cm$^{-1}$ (NO.BF$_4$) to 2191 cm$^{-1}$ (NO)$_2$SnCl$_6$.
No clear trends in nitrosonium ion frequencies are evident from Table 1; in general, it appears that salts of fluo-acids give higher frequencies than salts of oxy-acids, which in turn, give higher frequencies than salts of chloro-acids. Also, the observations mentioned before about the greater tendency towards hydrolysis of \((\text{NO})_2\text{SnF}_6\) and \(\text{NOSbF}_6\) than their chloro-analogues, suggests that in the latter instances, the nitrosonium ion is less susceptible to nucleophilic attack by water molecules; this could be due to polarisation occurring to lower the electropositive nature of the cation, but this would probably distort the lattice, and with \((\text{NO})_2\text{SnCl}_6\), this has been shown not to be the case.
Obviously the factors influencing the nitrosonium ion stretching frequency are very complex, and two interactions may be responsible:
1. Cation - Halogen interaction.
2. Cation - Central metal atom interaction.
In cases involving hexa-co-ordinate anions, the latter interaction is unlikely to be very important, since the central metal atom is well shielded by halogens; this will probably be the case.
with four co-ordinate anions also. Cation-Metal atom interaction may well be important in some of the lower chloro-metal salts. Shielding by halogens is unlikely to be so marked and, if polymeric anions do occur in any of these instances as has been suggested (Lewis & Sowerby 1957), the compounds could well have a layer lattice structure as has been shown to exist in \((\text{NH}_4)_2\text{HgCl}_3\) (Harmsen 1938), in which both types of interaction are possible.
There is little correlation between the stability of the compound, either towards hydrolysis or towards dissociation, and its nitrosonium ion absorption frequency. The fluoro-salts showed little tendency towards dissociation, and the majority of the chloro-salts tend to dissociate fairly readily, irrespective of nitrosonium ion frequency. Many of the fluoro-salts with high nitrosonium ion frequencies hydrolyse extremely readily, but in several cases hydrolysis may involve the anion, which forms oxide or oxyfluoride species.
All of the chloro-adducts except \((\text{NO})_2\text{PtCl}_6\), \((\text{NO})_2\text{SnCl}_6\) and \(\text{NO}_2\text{SbCl}_6\) gave additional bands at 1800 cm\(^{-1}\), and these were originally assigned to metal—\(\text{NO}^+\) bonding. However, the bands were not present in fresh samples and increased in intensity with time; dissociation, giving nitrosyl chloride, with subsequent infra-red absorption by nitrosyl chloride seems a more probable explanation:
\[
\text{NO}_2\text{MCl}_n \rightarrow \text{M}_2\text{Cl}_{n-1} + \text{NOCl}
\]
though isomeric change may occur.
The x-ray data (table 2) shows that the only nitrosonium salts of hexa co-ordinate anions isomorphous with their potassium analogues, were two of the chloro-salts. None of the fluo-salts exhibited isomorphism. Complex halides generally have structures based on close-packing of halide ions and cations. The radii of the fluoride ion and potassium ion are equal (1.33Å); that of the nitrosonium ion is 1.40 when spherical, hence a fluoride ion lattice is unable to accommodate nitrosonium ions without distortion. The radius of the chloride ion is 1.81Å and this is large enough to accommodate the nitrosonium ion in a chloride-ion lattice, and enable
it to achieve spherical symmetry by rotation.
Nitrosonium fluoborate, fluosulphate and perchlorate, isomorphous with their potassium analogues, do not have close-packed structures.
The far infra-red spectra of the anions studied (tables 3 → 5), do show the presence of metal-halogen bonds, but give no information about the structure of the anions. If an anion $MX_3^-$ is planar it has $D_{3h}$ symmetry; if dimeric, with an $\text{Al}_2\text{Cl}_6$ type structure, the symmetry is $V_h$. In the former case, three of the four vibrational modes would be infra-red active, in the latter case eight of the eighteen modes would be infra-red active. In practice, several of the infra-red active modes of the anion of $V_h$ symmetry are deformation modes of such low frequency that they came below 200 cm$^{-1}$, the lower frequency limit of the investigation. In addition, decomposition in the nujol mulls complicated the spectra, and resolution of the instrument was often poor. Hence only the major infra-red bands could be obtained.
In the chloroplatinates, the bands observed may be the $V_3(300 \text{ cm}^{-1})$ and $V_4(265 \text{ cm}^{-1})$ modes; these are the only infra-red active modes of an anion of
of the $\text{PtCl}_6^{2-}$ type ($O_h$ symmetry group) and the $V_3$ vibration is usually the highest (Nakamoto 1963). Hence the bands observed in the chlorostannates and in nitrosonium chloroantimonate are likely to be the $V_3$ modes also.
The 370 cm$^{-1}$ band in nitrosonium chloroferrate will be the $V_3$ mode of the tetrahedral, or distorted tetrahedral $\text{FeCl}_4^-$ anion.
Since the structures of the anions $\text{CuCl}_2^-$, $\text{ZnCl}_3^-$, $\text{HgCl}_3^-$ and $\text{MnCl}_3^-$ are unknown, it is not possible to assign the observed bands for these compounds.
EXPERIMENTAL
Nitrosyl chloride was prepared as described in the experimental section of the following chapter; bromine trifluoride was prepared by bubbling chlorine trifluoride through bromine at c. 0°C, until the dark colour of the bromine had been replaced by the straw-colour of pure bromine trifluoride. The reaction usually took 1 - 1½ hours for 20 - 30 mls. bromine. It was found that reaction proceeded most smoothly if the initial temperature of reaction was 15° - 20°C, i.e. about room temperature. The reaction was allowed to proceed at this temperature for 10 - 15 minutes, thereafter being cooled to 0°C.
All the nitrosonium fluo-salts were prepared by Woolf's method (Woolf 1950) and the potassium fluo-salts by that of Woolf & Emeleus (1949).
\[(\text{NO})_2\text{GeF}_6\]
Nitrosyl chloride, in about 20% - 50% excess was transferred by vacuum-distillation into a silica reaction flask containing germanium dioxide. The flask was removed from the vacuum line and bromine trifluoride added dropwise through a copper funnel, with the reaction mixture cooled to -184°C (liquid oxygen temperature).
When an excess of bromine trifluoride had been added the reaction was allowed to come up to room temperature. After the reaction had stopped, the excess nitrosyl chloride and bromine trifluoride was removed by distillation in vacuo at $80^\circ - 90^\circ$. A white solid remained.
Analysis: Found N:10.9% $(\text{NO})_2\text{GeF}_6$ requires N:11.4%.
$\text{NO.VF}_6$
This was prepared as above, using vanadium pentoxide instead of germanium dioxide. A white solid was the reaction product.
Analysis: Found N:6.2% $\text{NO.VF}_6$ requires N:7.0%. The low value in this case is probably due to the rapidity with which $\text{NO.VF}_6$ hydrolysed.
$\text{NO.BF}_4$
Bromine trifluoride was reacted with boric oxide and nitrosyl chloride, as described above. The white product remaining was analysed.
Found N:11.6% $\text{NO.BF}_4$ requires N:12.0%.
$\text{NO.SbF}_6$
Bromine trifluoride was reacted with antimony pentoxide and nitrosyl chloride; after removal of excess solvent a white solid remained.
Found N:5.0% $\text{NO.SbF}_6$ requires N:5.2%.
NO\textsubscript{3}PF\textsubscript{6}
Bromine trifluoride was reacted with phosphorous pentoxide and nitrosyl chloride; this reaction was violent, inflaming at times. On removal of excess solvent a white product remained.
Analysis: Found N:7.8% NO\textsubscript{3}PF\textsubscript{6} requires N:8.0%.
NO\textsubscript{2}SO\textsubscript{3}F
Nitrosonium pyrosulphate (NO)\textsubscript{2}S\textsubscript{2}O\textsubscript{7} was dissolved in bromine trifluoride. On evaporation of excess solvent, a white solid remained.
Analysis: Found N:10.5% NO\textsubscript{2}SO\textsubscript{3}F requires N:10.8%.
(NO)\textsubscript{2}SnF\textsubscript{6}
Stannous chloride was dissolved in bromine trifluoride and excess solvent removed in vacuo at 200°C to give stannic fluoride. This was reacted with nitrosyl chloride and bromine trifluoride to give, on evaporation of excess solvent, a white solid.
Analysis: Found N:9.3% (NO)\textsubscript{2}SnF\textsubscript{6} requires N:9.6%.
NO\textsubscript{2}AsF\textsubscript{6}
Bromine trifluoride was reacted with arsenious oxide and nitrosyl chloride. After removal of excess solvent, a white solid remained.
Analysis: Found N:6.1% NO\textsubscript{2}AsF\textsubscript{6} requires N:6.4%.
NO.BiCl₄
This was prepared by the method of Rheinboldt & Wasserfuhr (1927). Nitrosyl chloride was transferred by vacuum distillation into a Carius tube containing dry bismuth trichloride. The tube was sealed in vacuo and the mixture raised to room temperature. On careful removal of solvent, a yellow-orange powder remained. If too high a vacuum was applied, dissociation occurred yielding bismuth trichloride.
Analysis: N: 3.6% Cl: 36.2% NO.BiCl₄ requires N: 3.7%
Cl: 37.0%.
(NO)₂S₂O₇
Nitrogen dioxide, whose preparation is described in the experimental section of the next chapter, and sulphur dioxide, were separately vacuum distilled into Carius tube, at -184°C. After sealing the tube, the reaction was allowed to proceed at room temperature for 24 hours (Hart-Jones et al 1929). A pink-violet solid resulted which became white on evacuation. The colour was presumably due to N₂O₂⁺ ion.
Analysis: Found N: 10.0% (NO)₂S₂O₇ requires N: 10.2%.
NO\textsubscript{2}MnCl\textsubscript{3}
Nitrosyl chloride was transferred by vacuum distillation into a Carius tube containing anhydrous manganous chloride, prepared by heating MnCl\textsubscript{2}.6H\textsubscript{2}O at 150° - 200° for several hours. The tube was sealed and the mixture raised to room temperature; on evaporation of excess solvent, a golden solid remained.
Analysis:
Found N:7.1% Cl:55.0% NO\textsubscript{2}MnCl\textsubscript{3} requires N:7.3%
Cl:55.1%.
NO\textsubscript{2}CuCl\textsubscript{2}
Nitrosyl chloride was transferred by vacuum distillation into a Carius tube containing cuprous chloride, and the tube sealed off. On raising to room temperature a black solid and solution resulted, which on evaporation of excess solvent gave an ochre solid.
Analysis: N:8.3% Cl:42.6% NO\textsubscript{2}CuCl\textsubscript{2} requires N:8.5%
Cl:43.1%.
NO\textsubscript{2}HgCl\textsubscript{2}
Nitrosyl chloride was transferred by vacuum distillation into a Carius tube containing mercuric chloride, and the tube sealed. After reaction at room temperature, excess solvent was carefully removed to
leave a red-brown solid. If too high a vacuum was applied during this stage, all the nitrosyl chloride was removed leaving mercuric chloride.
Analysis:
\[ \text{N: 3.8\%} \quad \text{Cl: 30.1\%} \quad \text{NO.HgCl}_3 \text{ requires N: 4.2\% Cl: 31.6\%.} \]
\[ \text{NO.AlCl}_4 \]
Nitrosyl chloride was condensed on to anhydrous aluminium chloride in a Carius tube, and the tube sealed. After reaction at room temperature the excess solvent was removed under vacuum, leaving a white solid.
Analysis:
\[ \text{N: 6.7\%} \quad \text{Cl: 70.6\%} \quad \text{NO.AlCl}_4 \text{ requires N: 7.0\% Cl: 71.3\%.} \]
\[ (\text{NO})_2\text{PtCl}_6 \]
Nitrosyl chloride was condensed on to chloroplatinic acid \( \text{H}_2\text{PtCl}_6 \cdot \text{H}_2\text{O} \) in a Carius tube, and the tube sealed. After reaction at room temperature the excess solvent was removed and a yellow-brown solid remained.
Analysis:
\[ \text{N: 5.9\%} \quad \text{Cl: 45.6\%} \quad (\text{NO})_2\text{PtCl}_6 \text{ requires N: 6.0\% Cl: 45.5\%.} \]
\[(\text{NO})_2\text{PdCl}_4\]
Nitrosyl chloride was condensed on to palladous chloride in a Carius tube and the tube sealed. After reaction at room temperature, the excess solvent was removed, leaving a red-brown solid.
Analysis:
\[N: 9.0\% \quad Cl: 46.3\% \quad (\text{NO})_2\text{PdCl}_4 \text{ requires } N: 9.1\% \quad Cl: 46.1\%\]
\[(\text{NO})_2\text{FeCl}_4\]
Nitrosyl chloride was condensed on to anhydrous ferric chloride in a Carius tube, and the tube sealed. After reaction at room temperature, removal of excess solvent left a yellow-ochre solid.
Analysis:
\[N: 6.1\% \quad Cl: 62.0\% \quad (\text{NO})_2\text{FeCl}_4 \text{ requires } N: 6.2\% \quad Cl: 62.3\%\]
\[(\text{NO})_2\text{SnCl}_6\]
Nitrosyl chloride, stannic chloride and carbon tetrachloride were separately distilled into a reaction flask (100 ml.) in vacuo. Dry air was admitted and the reaction allowed to come up to room temperature, when a pale yellow solid rapidly separated out. Excess nitrosyl chloride and carbon tetrachloride were removed in vacuo and the pale yellow solid remained.
Analysis:
\[N: 6.8\% \quad Cl: 54.0\% \quad (\text{NO})_2\text{SnCl}_6 \text{ requires } N: 7.2\% \quad Cl: 54.4\%\]
This was prepared in a similar manner to the above, using antimony pentachloride instead of stannic chloride. The yellow solid product obtained was analysed.
Analysis:
N: 3.7% Cl: 58.2% NO₃SbCl₆ requires N: 3.8% Cl: 58.3%.
NO₂ZnCl₃
Nitrosyl chloride was condensed on to zinc chloride in a Carius tube, in vacuo, and the tube sealed. After reaction of room temperature, removal of excess solvent left a pale yellow product which tended to dissociate on application of a vacuum.
Analysis:
N: 6.6% Cl: 52.0% NO₂ZnCl₃ requires N: 6.9% Cl: 52.7%.
(NO)₂TiCl₆
Nitrosyl chloride, titanium tetrachloride and carbon tetrachloride were transferred separately by vacuum distillation into a reaction flask. Dry air was admitted and the reaction mixture raised to room temperature, when a pale yellow solid separated out. Excess carbon tetrachloride and nitrosyl chloride were
removed in vacuo.
Analysis:
N: 8.2% Cl: 65.4% (NO)₂TiCl₆ requires N: 8.7% Cl: 66.3%.
Preparation of potassium salts.
K₂GeF₆
Potassium chloride and germanium dioxide were mixed in a 2:1 mole ratio, and placed in a silica reaction flask. Bromine trifluoride was added dropwise until an excess was present; the reaction took place at room temperature and the excess solvent was removed in vacuo at 200°C. A white solid remained.
Analysis:
Ge: 27.4% K₂GeF₆ requires Ge: 27.6%.
K.VF₆
Potassium chloride and vanadium trichloride in a 1:1 mole ratio (for preparation, see chapter on vanadium group metals) were reacted in the same manner as above. A white solid was obtained.
Analysis: V as V₂O₅: 24.8% K.VF₆ requires V: 25.0%.
K.SbF₆
Potassium chloride and antimony pentoxide in a 2:1 mole ratio were reacted in the same manner as above.
A white solid product was obtained.
Analysis:
Sb: 43.9% K.SbF₆ requires Sb: 44.4%.
K₂SO₃F
Potassium persulphate K₂S₂O₈ was dissolved in bromine trifluoride. On evaporation of excess solvent as above, a white solid remained.
Analysis:
S: 23.2% K₂SO₃F requires S: 23.2%.
KPF₆
Potassium chloride and phosphorous pentoxide were reacted in a 2:1 mole ratio with bromine trifluoride, and the excess solvent removed, as above. A white solid remained.
Analysis:
F = 60.9% KPF₆ requires F = 61.9%.
K₂SnF₆
Stannic fluoride was prepared by reacting stannous chloride with bromine trifluoride and removing the excess solvent at 200°C. This was then mixed with potassium chloride (SnF₄:KCl = 1:2 mole ratio) and treated with bromine trifluoride as above. After
removal of excess solvent at 200°C a white solid remained.
Analysis:
Sn: 38.1% $K_2SnF_6$ requires Sn: 38.3%.
$KAsF_6$
Potassium chloride and arsenous oxide were mixed in a 2:1 ratio and the same procedure followed as with $K_2GeF_6$ etc. A white solid was obtained.
Analysis:
As: 33.0% $KAsF_6$ requires As: 32.9%.
$K_2SnCl_6$
A solution of hydrated stannic chloride was made up in concentrated hydrochloric acid, potassium chloride added, and then chlorine bubbled through with constant stirring. The solution soon turned yellow, and pale yellow crystals started to be precipitated. The reaction was stopped after 40 - 50 minutes and the super-natant liquor allowed to settle, before most of it was decanted. The residue was filtered by suction, washed with glacial acetic acid, then ethanol and finally ether. The bulk of the ether was removed by rapid suction, and the remainder dried in vacuo. The
product was a pale yellow solid.
Analysis:
Cl: 52.0% K₂SnCl₆ requires Cl: 51.9%.
X-ray photographs were taken in Lindemann glass capillaries with Cu-Kα radiation; the photographs were measured visually, but calculation of Sin²θ values and indexing of powder lines were carried out on a Ferranti Sirius Computer, using programmes developed by Russell (1963).
Infra-red spectra were measured as mulls in florube grease using calcium fluoride windows. It was found that even the most reactive nitrosonium salts were fairly stable in florube mulls. The nitrosonium salts tended to decompose very readily in nujol, even when rigorously dried, and also in hexachlorobutadiene.
All preparations of mulls had to be carried out in the dry box. Spectra were measured on a Grubb Parsons DB1 spectrophotometer, using sodium chloride optics. All the values of nitrosonium ion frequencies tabulated (table 1) were calibrated against a polystyrene spectrum, using the 3.42μ and 6.24μ bands as references.
| Compound | $V(NO^+)$ in cm$^{-1}$ |
|---------------------------|-----------------------|
| $(NO)_2GeF_6$ | 2391s. shoulder at 2336. |
| $NO.VF_6$ | 2391s. shoulder at 2328. |
| $NO.BF_4$ | 2387s. 2341 (e,f). |
| $NO.SBF_6$ | 2385b. shoulder at 2342. |
| $NO.PF_6$ | 2379s. |
| $NO.SO_3F$ | 2377b. |
| $(NO)_2SnF_6$ | 2342s. |
| $NO.AsF_6$ | 2340s. |
| $NO.HSO_4$ | 2340 (a): 2311 (b) Both Raman spectra. |
| $NO.UF_6$ | 2333 measured as (c) sublimed films |
| $NO.MoF_6$ | 2331 |
| $NO.BiCl_4$ | 2331b. |
| $(NO)_2PdCl_4$ | 2330s. |
| $NO.ClO_4$ | 2313 (d) Raman spectra. |
| $(NO)_2(NO_2)_2.(S_3O_10)$| 2308, 2277 (d) Raman spectra. |
| $(NO)_2S_2O_7$ | 2278s. shoulder at 2294. |
| $NO.MnCl_3$ | 2271b. |
| $NO.CuCl_2$ | 2271b. |
| $NO.HgCl_3$ | 2268b |
| $NO.AlCl_4$ | 2242s. shoulder at 2370; 2236 (a) Raman spectra. |
| $(NO)_2PtCl_6$ | 2201s. |
| Compound | Wavelength |
|-------------------|------------|
| NO₃SbCl₆ | 2189s |
| NO₂ZnCl₃ | 2187b |
| NO₂TiCl₆ | 2165b |
All data obtained from the present work, except:
(a) Gording & Houtgraaf 1953.
(b) Angus & Leckie 1935.
(c) Geichmann et al 1962.
(d) Gording & Eriks 1952.
(e) Sprague et al 1960.
(f) Evans et al 1964.
The letter i - indicates nitrosonium compounds found to be isomorphous with their potassium analogues.
## Table 2
### X-RAY DATA
#### ORTHORHOMBIC STRUCTURES
| Compound | Unit Cell Dimensions (Å) | Unit Cell Volumes (ų) |
|----------|--------------------------|------------------------|
| | a | b | c | |
| NO₂BF₄ | 8.88 | 5.64 | 7.03 | 352.2 |
| | 8.79 | 5.66 | 7.10 | 1) |
| K₂BF₄ | 7.83 | 5.67 | 7.35 | 326.3 |
| | 7.85 | 5.68 | 7.37 | 2) |
| NO₂SO₃F | 8.71 | 6.20 | 7.31 | 403.9 |
| K₂SO₃F | 8.55 | 5.94 | 7.34 | 372.8 |
| | 8.56 | 5.95 | 7.33 | 3) |
#### CUBIC STRUCTURES
| Compound | a (Å) |
|----------|-------|
| (NO)₂SnCl₆ | 10.24 |
| K₂SnCl₆ | 10.14 |
| | 10.14 |
| (NO)₂PtCl₆ | 11.27 |
| K₂PtCl₆ | 11.20 |
| | 11.18 |
All present work except –
1) Klinkenberg 1937.
2) Wykoff, 'Crystal Structures'.
3) Sharp 1957.
| Compound | $V$ (cm$^{-1}$) |
|---------------------------|-----------------|
| $(\text{NO})_2\text{SnCl}_6$ | 310 |
| $\text{K}_2\text{SnCl}_6$ | 312 |
| $(\text{NO})_2\text{PtCl}_6$ | 267,300 |
| $\text{K}_2\text{PtCl}_6$ | 265,300 |
| $\text{NO}_4\text{SbCl}_6$ | 345 |
| $\text{NO}_4\text{FeCl}_4$ | 370 |
| $(\text{C}_2\text{H}_5)_4\text{N}_4\text{FeCl}_4$ | 378 a) |
| $(\text{C}_2\text{H}_5)_4\text{N}_2\text{FeCl}_4$ | 282 a) |
lower values are probably the $V_4$ modes
| Compound | Frequency (cm⁻¹) |
|---------------------------|------------------|
| NO₂CuCl₂ | 245 |
| (C₂H₅)₄N₂CuCl₄ | 247m; 268s; 289sh. |
| Cs₂CuCl₄ | 256m; 288sh. |
| NO₂ZnCl₃ | 270 |
| (C₂H₅)₄N₂ZnCl₄ | 273s; 281m. |
| Cs₂ZnCl₄ | 285m; 292s. |
| NO₂MnCl₃ | 268 |
| (C₂H₅)₄N₂MnCl₄ | 282 |
| NO₂HgCl₃ | 261 |
| (NO)₂TiCl₆ | 478 |
s - strong, m - medium, sh - shoulder
All present work except -
a) Clark & Dunn 1964.
| Compound | Bands (cm⁻¹) |
|----------------|--------------------------------------------------|
| NO₄BF₄ | 770w, 1061s, 1075s, 1305w, 1790w. |
| K₂BF₄ | 771w, 1032w, 1058s, 1072s, 1302w, 1784w. |
| NO₃SO₃F | 1300s, 1282s, 1076s, 731s. |
| K₂SO₃F | 1299s, 1277s, 1073s, 732s. |
| NO₄PF₆ | 834s, 740s. |
| K₂PF₆ | 845a), 741e) (Raman active) |
| Rb₂PF₆ | 845s, 802w, 740-720w. f) |
| Cs₂PF₆ | 843s, 802w, 740-720w. f) |
| Ag₂PF₆ | 830-840s, 740w. f) |
| (NO)₂SnF₆ | 547 |
| K₂SnF₆ | 552 d) |
| NO₄SbF₆ | 660 |
| K₂SbF₆ | 660 d) |
References
a) Cote & Thompson 1951.
b) Edwards et al 1955.
c) Sharp 1957.
d) Peacock & Sharp 1959.
e) Woodward & Anderson 1956.
f) Sharp & Sharpe 1956.
CHAPTER II
SOLVENT PROPERTIES OF NITROSYL CHLORIDE AND DINITROGEN TETROXIDE
THE NITRATO - LIGAND
The majority of the reactions described in the following chapters are effectively solvolysis reactions of metal and non-metal chlorides, in either nitrosyl chloride or dinitrogen tetroxide solution; in some cases metal bromide - nitrosyl bromide reactions have been attempted.
In the case of a metal chloride/nitrosyl chloride reaction, it is possible to write the following general equation
\[ \text{MCl}_n + \text{NOCl} \rightarrow \text{MCl}_n - \text{NOCl} \]
i.e. an adduct is formed which may react either with another molecule of nitrosyl chloride or the metal chloride; in the former case an adduct of the general formula MCl_n. 2NOCl is formed, in the latter case 2MCl_n.NOCl.
When a variety of products are formed, it is normally possible to manipulate the reaction to give a predominant yield of one product by varying the relative concentrations of the metal chloride and nitrosyl chloride used.
With dinitrogen tetroxide solvolyses, the situation is more complex due to the possibility of more reactions occurring and hence a greater variety of products being formed. The initial reaction step is of the type:
\[ \text{MCl}_n + n\text{N}_2\text{O}_4 \rightarrow \text{M(NO}_3)_n + n\text{NOCl} \]
The nitrosyl chloride may then react with the metal chloride to yield adducts such as \( \text{MCl}_n \text{ NOCl} \) as in reaction 1.
It is possible that the nitrate \( \text{M(NO}_3)_n \) may undergo several types of decomposition or dissociation, viz.:
\[ \text{M(NO}_3)_n \rightarrow \text{MO}_2(\text{NO}_3)_{n-2} + \text{N}_2\text{O}_4 \]
In this case the oxidation number of M increases by two. Also the dissociation
\[ \text{M(NO}_3)_n \rightarrow \text{MO(NO}_3)_{n-2} + \text{N}_2\text{O}_4 + \frac{1}{2}\text{O}_2 \]
can occur, in which the oxidation state of M is unchanged.
Reaction 3 is obviously favoured when the original oxidation state of the metal is not the highest possible; also, the M - O bonds formed are usually multiple bonds and have considerable bond strength. Since no such bonding exists in the pure nitrates, this makes the formation of the oxynitrates in reactions 3 and 4 thermodynamically favourable. Whether a particular metal
nitrate will undergo reaction 3 or 4 will depend on the relative stabilities of the oxidation states formed, the most stable one being favoured. The nitrate and oxynitrates formed in reactions 2, 3, and 4 are all potentially capable of forming adducts with dinitrogen tetroxide, thus the variety of products which can be formed is potentially very large. Particular cases in which this situation appears to occur will be dealt with individually in later chapters.
Reaction 2 illustrates the production of nitrosyl chloride in a dinitrogen tetroxide system. As the concentration of dinitrogen tetroxide falls, the concentration of nitrosyl chloride increases. It has been shown that, with uranium, the solvolytic behaviour of nitrosyl chloride/dinitrogen tetroxide mixtures is markedly dependent on the ratio of the components (Addison & Hodge 1961a). These workers found that dinitrogen tetroxide will not react with uranium except in the presence of nitrosyl chloride, but that up to a nitrosyl chloride percentage concentration of 85%, the only product was $UO_2(NO_3)_2 \cdot N_2O_4$. At nitrosyl chloride concentrations above this $UO_2Cl_2 \cdot NOCl$ was formed.
In metal chloride/dinitrogen tetroxide systems, the amount of nitrosyl chloride formed is dependent on the concentration of the metal chloride; this is evident from equation 1. Hence the composition of the nitrosyl chloride/dinitrogen tetroxide mixture at any stage of such a reaction is dependent on the original metal chloride: dinitrogen tetroxide ratio, and thus the products formed may also be dependent on this ratio.
These are, in all cases, reactions in dinitrogen tetroxide or nitrosyl chloride solution, in which the reactant species invariably appear to be ions, in the former case, NO⁺ and NO₃⁻, in the latter NO⁺ and Cl⁻. In the gas phase reactions are markedly different since the reactive species are either NO₂ radicals or NOCl molecules. To illustrate the difference in gas and liquid phase reactions, the reaction
\[ \text{Ni(CO)}_4 + 2\text{NOCl} \rightarrow \text{NiCl}_2 + 4\text{CO} + 2\text{NO} \]
occurs in the liquid phase, while the same components give dichloronitrosylnickel (I) in the gas phase (Addison & Johnson 1962); nickel carbonyl reacts in the liquid phase with dinitrogen tetroxide to give to Ni(NO₃)₂·2N₂O₄, whereas in the gas phase the product is nickel dinitrite Ni(NO₂)₂. (Addison & Sutton 1963). The subject of nitrogen dioxide free radical reactions in this context has been reviewed by Addison (1962).
When nitrate groups are involved in metal systems they can exist either as free nitrate ions, or as bonded nitrate - ligands. Straub et al (1962) have prepared diethyl sulphoxide complexes of manganese, cobalt and nickel which contain the anions $M(NO_3)_4^{2-}$ where $M = Mn, Co, Ni$; cationic complexes containing the nitrate - ligand have been postulated in $Sb(NO_3)_4^+$ and $As(NO_3)_4^+$ cations respectively (Dehmicke 1964). The first workers to examine the infrared spectra of nitrates were Miller & Wilkins (1952), who were attempting to characterise the band frequencies of the nitrate ion. They amassed a considerable amount of spectroscopic data but did not make assignments of any bands, though they noted that a broad absorption in the $1300 - 1400 \text{ cm}^{-1}$ region seemed to be characteristic of nitrate ion.
The infrared spectra of nitrates have been more fully examined by Gatehouse et al (1957), Addison & Gatehouse (1960) and Katzin (1962). The symmetry properties of the free nitrate ion and the co-ordinated nitrate - group are set out below:
By co-ordination of one oxygen atom, the symmetry of the nitrate - group is lowered, relative to the free ion.
The nitrate ion has 4 fundamental frequencies; these have been assigned and the frequency ranges obtained (Gatehouse et al 1957; Addison & Gatehouse 1960).
**NITRATE ION**
| TYPE | RAMAN | INFRARED | RANGE | ASSIGNMENT |
|------|-------|----------|---------|-----------------------------|
| A | (V₁) | a | 1a | c.1050 cm⁻¹ | N-O symmetric stretch |
| A | (V₂) | ia | a | c.830 cm⁻¹ | NO₂ deformation |
| E | (V₃) | a | a | c.1390 cm⁻¹ | NO₂ asymmetric stretch |
| E | (V₄) | a | a | c.720 cm⁻¹ | Planar Rock |
a - active ia - inactive.
The same workers have carried out a similar analysis for the co-ordinated nitrate-group.
| NITRATE-GROUP | TYPE | RAMAN | INFRARED | RANGE (cm⁻¹) | ASSIGNMENT |
|---------------|------|-------|----------|--------------|------------|
| | A₁ | (ν₃) | | c. 739 | NO₂ banding|
| | A₁ | (ν₁) | all active | 1290-1253 | NO₂ symmetric stretch |
| | A₁ | (ν₂) | | 1034-970 | NO stretch |
| | B₂ | (ν₆) | | 800-781 | non-planar rock |
| | B₁ | (ν₄) | | 1531-1481 | NO₂ asymm. stretch |
| | B₁ | (ν₅) | | c.713 | planar rock |
Effectively, co-ordination of the nitrate ion, and hence conversion into nitrate-ligand, has split the ν₃ vibration of NO₃⁻ ion into the ν₁ and ν₄ modes of the nitrate-group, the symmetric and asymmetric stretching modes of the -NO₂ group; also, the ν₄ vibration of the NO₃⁻ ion has been split into the ν₃ and ν₅ modes of the nitrate-group, which gives a much smaller splitting than that due to (ν₄ - ν₁). The lowering of the symmetry by co-ordination has rendered the symmetric N-O stretching mode, infrared inactive in the nitrate ion, active in the co-ordinated ligand as the ν₂ vibration.
From the preceding tables it is observed that for both free nitrate ion and the co-ordinated nitrato-group, bands occur in the 700-750 cm\(^{-1}\) region. The nitrato-group shows the \(V_6\) band at a quite significantly lower range (781-800 cm\(^{-1}\)) than the \(V_2\) nitrate mode (830 cm\(^{-1}\)). In both instances, bands occur in the 1050 cm\(^{-1}\) region but the \(V_1\) mode of the nitrate ion is only Raman active, hence presence of a band in this region of the infrared spectrum is a good indication of co-ordinated nitrato-groups.
Finally, the \(V_3\) band of the nitrate ion at 1390 cm\(^{-1}\) is split into two widely separate bands in approximately the 1500 cm\(^{-1}\) and 1270 cm\(^{-1}\) regions in the co-ordinated nitrato-group and this is used as the primary evidence for the presence of co-ordinated nitrato-ligands.
The amount of the \((V_4-V_1)\) splitting in the co-ordinated ligand is dependent on the extent of deformation caused by co-ordination. If metal-ligand bonding is weak, ligand deformation will be small and the subsequent splitting will also be small. Thus it is possible to use the magnitude of this splitting as a roughly quantitative guide to the covalency of a nitrato-complex. Katzin (1962) has studied the spectra of several nitrates in tritutylphosphate solution and has found that even with obviously ionic nitrates, some splitting of the \(V_3\) vibration of the nitrate
ion occurs, but this was of a magnitude (less than 65 cm$^{-1}$) that could be explained by electrical asymmetry. When the splitting was greater than 125 cm$^{-1}$, it was stated that significant covalency occurred, and the higher this splitting, the greater the extent of covalency.
Several compounds are known in which a ($V_4 - V_1$) splitting greater than 300 cm$^{-1}$ occurs, which can be taken to indicate high covalency in the compound. Also, cases have been postulated in which the nitrate-group behaves as a bidentate ligand, in trinitratomolybium oxide (Field & Hardy 1963a) and anhydrous chromium trinitrate (Addison & Chapman 1964).
Dinitrogen pentoxide is known to exist in the solid state as nitronium nitrate NO$_2^+$ NO$_3^-$ (Teranishi & Decius 1953, 1954) and in nitric acid dissociates into the same ions. (Millen 1950b). It would thus be expected to be a good reagent for introducing the nitrate or nitrate-group into metallic compounds and it has been employed for this purpose (Schmeisser & Lutzow 1953; Schmeisser & Elischer 1957; Ferraro et al. 1955; Gibson et al. 1960; Field & Hardy 1962, 1963a,b; Addison & Chapman 1964; Bagnall et al 1964). In general, it is a much more useful reagent for introduction of nitrate groups, than is dinitrogen tetroxide, since the side reactions are, much
fewer. The initial reaction stage in the solvolysis of a metal halide by dinitrogen pentoxide is
\[ \text{MCl}_n + n\text{N}_2\text{O}_5 \rightarrow \text{M(NO}_3)_n + n\text{NO}_2\text{Cl} \]
The nitryl chloride thus formed does not tend to form adducts with the metal chloride in the same manner as nitrosyl chloride. Also the tendency to form adducts with the metal nitrate is much smaller with dinitrogen pentoxide than dinitrogen tetroxide, though the adduct \( \text{Th(NO}_3)_4 \cdot 2\text{N}_2\text{O}_5 \) is stable up to over 100°C (Ferraro et al 1955), and in this case is more stable than the dinitrogen tetroxide adduct.
The adducts of the types \( \text{M(NO}_3)_n \cdot \text{N}_2\text{O}_4 \), and MCl\(_n\). NOCl are analogous; the former refers to the dinitrogen tetroxide solvent system, the latter to the nitrosyl chloride system.
However, the two systems are not entirely analogous. For instance, Zinc Chloride forms the adduct ZnCl\(_2\)·NOCl, which is structurally NO\(^+\). ZnCl\(_3^-\). With dinitrogen tetroxide, zinc nitrate forms the adduct Zn(NO\(_3\))\(_2\)·2N\(_2\)O\(_4\); no 1:1 adduct in this system is known. Moreover, although this adduct dissociates in solution, thus
\[ \text{Zn(NO}_3)_2 \cdot 2\text{N}_2\text{O}_4 \rightarrow (\text{NO}^+)_2 + \text{Zn(NO}_3)_4^{2-} \]
(Addison & Lodge, 1954b)
the dinitrogen tetroxide is believed to be present merely as a 'lattice compound' in the solid state (Addison et al 1958). Several other instances occur where analogy is not complete, and it is thus not possible to draw too many structural conclusions on various adducts on the basis of analogy with other solvent systems.
With many of the solid products obtained in the reactions described in later chapters, infrared examination in vigorously dried nujol mulls was not invariably accurate due to the decomposition and in many cases it was necessary to determine bands due to the original compounds by observing the time dependence of the spectroscopic bands. Field & Hardy (1963,b) have also noted this; they reported that nitrate-compounds tended to nitrate nujol and postulated a radical mechanism breakdown:
\[ \text{MO}_2\text{NO}_2 \rightarrow \text{MO} + \text{NO}_2 \]
which Addison (1962) had reported. Florube was a much less reactive mulling agent but its range was rather limited (4000 - 1350 cm\(^{-1}\)).
The preparations of nitrosyl chloride, nitrosyl bromide and dinitrogen tetroxide are described in the experimental section of this chapter. Although nitric oxide does come into a different category than these reagents, it is also described here for convenience.
EXPERIMENTAL.
As mentioned previously in this chapter, it is highly important that nitrosyl chloride used in solvolysis reactions should be free of dinitrogen tetroxide, and vice versa.
The preparations of nitrosyl chloride, nitrosyl bromide and dinitrogen tetroxide are described below.
NITROSYL CHLORIDE.
Nitrosonium hydrogen sulphate was obtained by reaction of sulphur dioxide and nitric acid for 4 - 6 hours at c.0°C.
\[ \text{HNO}_3 + \text{SO}_2 \rightarrow \text{NO.HSO}_4 \]
The yellow solid was rapidly filtered by suction; it was then mixed intimately with dried sodium chloride. During this stage the reaction was frozen down to prevent too much loss of nitrosyl chloride due to the following reaction:
\[ \text{NO.HSO}_4 + \text{NaCl} \rightarrow \text{NOCl} + \text{NaHSO}_4 \]
The nitrosyl chloride evolved was condensed in a trap at -78°C; at this temperature any hydrogen chloride impurities are not condensed out.
The gas phase spectrum of the nitrosyl chloride gave bands as follows:
1818 m, 17998, 1740 m, 738, 746, 758 (s). (cm\(^{-1}\))
The first three of these bands are due to the $-N=O$ bond stretching mode and the split bands centred about 746 cm$^{-1}$ are due to the N-Cl stretching vibration.
**NITROSYL BROMIDE**
The analogous reaction to the above for the preparation of nitrosyl bromide was unsatisfactory; the NO.HSO$_4$ / NaBr had to be heated to 50° - 60° to get steady nitrosyl bromide evolution, and the following reactions occurred.
$$\text{NO.HSO}_4 + \text{NaBr} \rightarrow \text{NO.Br} + \text{NaHSO}_4$$
$$2\text{NO.Br} \rightarrow 2\text{NO} + \text{Br}_2$$
$$2\text{NO} + \text{O}_2 \rightarrow 2\text{NO}_2 \rightleftharpoons \text{N}_2\text{O}_4$$
The nitrosyl bromide dissociates quite easily, and the nitric oxide formed reacted with atmospheric oxygen as shown, to give nitrogen dioxide impurity, which condenses out as dinitrogen tetroxide with nitrosyl bromide. As will be enlarged upon later, the presence of dinitrogen tetroxide impurities had a considerable influence on the vanadium bromides / nitrosyl bromide systems.
The most satisfactory method was the reaction in vacuo of nitrosonium pyrosulphate and sodium bromide; the reaction proceeded quite steadily at 110°-130°. The volatile products were condensed out at 198°.
\[(NO)_2S_2O_7 + 2NaBr \rightarrow Na_2S_2O_7 + 2NOBr\]
Obviously, at \(110^\circ-130^\circ\), nitrosyl bromide dissociates markedly but in this system no atmospheric oxidation can occur, and the product appears to recombine at low temperatures.
\[2NOBr \rightleftharpoons 2NO + Br_2\]
The infrared spectrum gave strong bands at 1768 cm\(^{-1}\) and 1760 cm\(^{-1}\) due to the \(-N=O\) bond stretching frequencies, and a weak band at 1880 cm\(^{-1}\) due to nitric oxide.
**DINITROGEN TETROXIDE,**
Lead nitrate was heated strongly, and the dinitrogen tetroxide condensed out in acetone/CO\(_2\).
\[2Pb(NO_3)_2 \rightarrow 2PbO + 4NO_2 + O_2\]
The infrared spectrum shows strong bands at 1698 cm\(^{-1}\) and 1208 cm\(^{-1}\) due to asymmetric and symmetric stretching modes of the bent nitrogen dioxide; also a weak combinational band at 2896 cm\(^{-1}\) is present.
Hence, if dinitrogen tetroxide is present as an impurity in nitrosyl halides, it will be easily distinguishable from the gas phase infrared spectrum, and for this reason, gas spectra were run on every batch of nitrosyl halides and dinitrogen tetroxide prepared.
NITRIC OXIDE
This was prepared as in Inorganic Syntheses, Volume 2, p.126; the reduction of sodium nitrite by acidified ferrous sulphate solution in the absence of oxygen. Acidified ferrous sulphate was added dropwise to sodium nitrite solution, the system being previously swept out with nitrogen, the gas was evolved steadily, passed through concentrated sodium hydroxide to remove traces of nitrogen dioxide, then passed through phosphorus pentoxide tubes to remove water vapour, and condensed in a trap cooled down in liquid oxygen (at -184°C). The nitric oxide separated out as a very pale blue solid. Any formation of nitrogen dioxide due to leakages in the system, was very easy to follow because of the brown coloration produced.
In all the above cases, the product was redistilled in vacuo over phosphorus pentoxide at least twice, before reactions, to remove residual moisture.
CHAPTER XIII
REACTIONS OF NITROGEN OXIDES AND NITROSYL CHLORIDE WITH LEWIS ACIDS OF GROUPS IIIB, IVB, AND VB.
BORON TRIFLUORIDE/NITROGEN OXIDES
Nitrogen oxides are known to react with Lewis acids such as boron trifluoride (Bachmann et al 1955; Sprague et al 1960; Kuhn & Olah 1961; Olah & Tolgyesi 1961; Evans et al 1964). These products can be formulated as adducts of the reactants, and because of their ability to act as nitrating and nitrosylating agents, they were formulated as salts containing nitronium or nitrosonium ions and complex anions.
Bachmann, from a study of the nitrating properties of the $\text{BF}_3\cdot\text{N}_2\text{O}_4$ complex, formulated it as $\text{NO}_2^+ \cdot \text{BF}_3\cdot\text{NO}_2^-$. The composition of the product was independent of the reaction phase, i.e., gas, liquid or solute phase. Later, Kuhn & Olah (1961) observed that the same complex had nitrosylating properties, and from Raman investigations, suggested that the structure was an equilibrium, viz:-
$$\text{NO}_2^+ \cdot \text{BF}_3\cdot\text{NO}_2^- \rightleftharpoons \text{NO}^+ \cdot \text{BF}_3\cdot\text{NO}_3^-$$
Sprague and his colleagues (1960) suggested, from infrared and x-ray observations, that the reaction product was \( \text{NO}^+ \cdot \text{NO}_2^+ \cdot (\text{BF}_3)_2\text{O}^{2-} \); the anion in the latter case was postulated to have the structure
\[
\begin{array}{c}
F \\
F - B - O - B - F \\
F \\
F
\end{array}
\]
and so for x-ray purposes would be very similar to the tetrafluoborate ion.
Finally, Evans et al (1964) showed that the reaction could be explained without recourse to postulating complex anion formation. They studied the reaction of boron trifluoride with dinitrogen trioxide, tetroxide and pentoxide, and their conclusions can be summarized thus:
\[
\begin{align*}
\text{N}_2\text{O}_3/\text{BF}_3 & \rightarrow \text{NO} \cdot \text{BF}_4 + \text{borates} \\
\text{N}_2\text{O}_4/\text{BF}_3 & \rightarrow \text{NO} \cdot \text{BF}_4 + \text{NO}_2 \cdot \text{BF}_4 + \text{borates} \\
\text{N}_2\text{O}_5/\text{BF}_3 & \rightarrow \text{NO}_2 \cdot \text{BF}_4 + \text{borates}
\end{align*}
\]
In all these cases the main products were nitrosonium or nitronium fluoborate with only small quantities of borate species being formed. Reaction 2 explains why the \( \text{BF}_3 \cdot \text{N}_2\text{O}_4 \) adduct possesses both nitrating and nitrosylating properties.
Other reactions involving boron halides which have been studied are:
1. Boron trichloride/Nitrosyl chloride.
Olah & Tolgyesi (1961) reported the formation of a solid complex when these components react at \(-50^\circ C\); the solid dissociates at room temperature. The infrared spectrum shows a band at \(2123 \text{ cm}^{-1}\) and so the compound can be regarded as \(\text{NO}^+ \cdot \text{BCl}_4^-\).
2. Boron trifluoride/Nitrosyl chloride.
These components react in liquid hydrogen chloride solution to give a product of composition \(\text{BF}_3 \cdot \text{NOCl}\), which is orange, and sublimes with dissociation at \(25^\circ C\) (Waddington & Klanberg 1960). The infrared spectrum shows a band at \(2335 \text{ cm}^{-1}\) which indicates free nitrosonium ion, hence Waddington & Klanberg formulate the adduct as \(\text{NO}^+ \cdot \text{BF}_3\text{Cl}^-\). However, Kemmitt et al (1963a), on the basis of infrared and nuclear resonance studies on haloborates, conclude that there is no definite evidence for the existence of mixed haloborate anions.
Since the nitrosonium ion is colorless, the orange colour of this complex implies that significant anion - cation charge transfer must occur.
Comparing these last two adducts with nitrosonium fluoborate, the order of thermal stability is:
as would be expected. The fall in the nitrosonium ion vibrational frequency suggests that anion-cation polarisation depends on the number of chloride groups present in the anion.
In addition Waddington & Klanberg (1960) report the compound \( \text{NO}^+ \cdot \text{B(C}_2\text{H}_5)_3\text{Cl}^- \), which gives an abnormally high infrared band at 2480 cm\(^{-1}\) which is assigned to the nitrosonium ion, but no mention is made of its stability.
**GROUP IV HALIDES/NITROGEN OXIDES**
The reactions of silicon and germanium halides have not been studied in the present work; however, Devin & Perrot (1958) demonstrated that \( (\text{NO})_2\text{SiCl}_6 \) and \( (\text{NO})_2\text{GeCl}_6 \) could not be formed by direct interaction of silicon or germanium tetrachloride with nitrosyl chloride. Waddington & Klanberg (1960) confirmed this, attempting to react the components in liquid hydrogen chloride.
**Stannic Chloride/Nitric Oxide.**
Nitrosyl chloride reacts with stannous chloride to give nitrosonium hexachlorostannate \( (\text{NO})_2\text{SnCl}_6 \). If any intermediates such \( (\text{NO})_2\text{SnCl}_4 \) are formed, they do not exist long enough in the presence of nitrosyl chloride.
to be studied. Nitric oxide and stannic chloride were reacted in carbon tetrachloride to see if any such intermediates could be isolated. A white solid was rapidly precipitated giving a very weak absorption in the free nitrosonium ion region but repeated analyses were inconsistent. No other bands in the infrared spectrum were observed. It appears that these reactions are best explained thus:
\[
\begin{align*}
\text{SnCl}_4 + 2\text{NO} & \rightarrow \text{SnCl}_2 + 2\text{NOCl} \\
\text{SnCl}_4 + 2\text{NOCl} & \rightarrow (\text{NO})_2\text{SnCl}_6
\end{align*}
\]
Thus the main product is stannous chloride, with a small quantity of nitrosonium chlorostannate, accounting for the weak NO⁺ absorption.
**Stannic Chloride/Dinitrogen tetroxide.**
This reaction gives at least two products. It was carried out as a possible means of obtaining anhydrous stannic nitrate Sn(NO₃)₄. The reaction had already been carried out by Partington & Whynnes (1949), who obtained an adduct which they formulated SnCl₄·2.5NO₂, which was probably a mixture of products.
The present work gave a white solid, insoluble in dinitrogen tetroxide which could not be separated by sublimation. It was highly moisture sensitive, hydrolysing with copious evolution of nitrogen dioxide, indicating
the presence of nitrosonium ion. The infrared spectrum gave a strong band at 2200 cm\(^{-1}\); and bands in the 1250 cm\(^{-1}\) and 1600 cm\(^{-1}\) regions which could be the \(V_1\) and \(V_4\) modes of a covalently bonded nitrato-group. Other bands were present at 1000 cm\(^{-1}\) and 800 cm\(^{-1}\). The product was found to contain chloride so that it could not be a simple nitrato compound. It would appear that the following types of reaction occurred:
\[
\text{SnCl}_4 + 4\text{N}_2\text{O}_4 \rightarrow \text{Sn(NO}_3)_4 + 4\text{NOCl}
\]
\[
\text{SnCl}_4 + 2\text{NOCl} \rightarrow (\text{NO})_2\text{SnCl}_6
\]
\[
\text{Sn(NO}_3)_4 + \text{N}_2\text{O}_4 \rightarrow \text{adducts}.
\]
The infrared spectrum definitely indicates covalently bonded nitrato-groups but cannot be regarded as conclusive.
In addition the reaction
\[
\text{Sn(NO}_3)_4 \rightarrow \text{SnO(NO}_3)_2 + \text{N}_2\text{O}_4 + \frac{1}{2}\text{O}_2
\]
may occur; this type of dissociation is known to occur with titanium tetranitrate (Gutmann 1956). Hence, as pointed out previously, this type of reaction can yield several products. Analysis, together with the above data, suggests primarily a mixture of tetranitratotin and nitrosonium chlorostannate.
GROUP V HALIDES/NITROGEN OXIDES
Previous work in this field has been carried out on adducts of dinitrogen tetroxide with arsenic and antimony pentafluorides (Aynsley et al 1951); phosphorus chlorides and oxychlorides with dinitrogen tetroxide (Klement & Wolf 1955); arsenic and antimony trichlorides with nitrosyl chloride (Lewis & Sowerby 1957; Waddington & Klanberg 1960); and the antimony pentachloride/nitric oxide reaction (Bowen 1954).
The present work involved the partial study of the reactions of phosphorus pentafluoride with nitric oxide and dinitrogen tetroxide.
Phosphorus pentafluoride/nitric oxide.
These react to give a very small yield of a white product which rapidly decomposes with evolution of nitrogen dioxide fumes. No studies were possible on this, but it may be similar to the BF₃·NO and SO₃·NO adducts reported in the literature; these may be further examples of compounds containing one electron bonds (Dues 1952).
Phosphorus pentafluoride/dinitrogen tetroxide.
These react in the liquid phase very readily at room temperature to give immediate separation of a white solid. This gave a very complicated infrared spectrum
due to decomposition taking place in the Nujol mull. Decomposition even occurred in floube mulls. Measuring spectra as a function of time it was possible to assign the following bands to the mixture of complexes believed to be formed:
3700w, 2390s, 2360(Sh), 1780w, 1700w, 1120m, 1030s, 840s, 740m, 660m. (all cm$^{-1}$)
This suggests that a mixture of nitrosonium and nitronium species were formed, and the following assignments can be made on this basis:
| Wavenumber | Assignment |
|------------|-------------------------------------------------|
| 3700 | $(V_1 + V_3)$ NO$_2^+$ ion |
| 2390 | $V_3$ NO$_2^+$ ion |
| 2360 | $V_1$ NO$^+$ ion |
| 1780 | not assignable |
| 1700 | possibly $2V_3$ PF$_6^-$ |
| 1120 | P - O multiple bond |
| 1030 | stretching frequencies |
| 840 | probably $V_3$ PF$_6^-$ |
| 740 | probably $V_1$ PF$_6^-$ (strictly infrared inactive) |
| 660 | $V_2$ NO$_2^+$ ion |
Nitronium fluoborate and fluoantimonate have been shown to give spectra agreeing quite well with these assignments (Cook et al. 1960). Values for $V_3$ of the nitronium ion vary from 2387 cm$^{-1}$ (Cook et al 1960) in the fluoantimonate.
The existence of the nitronium ion is supported by the weak combinational band at 3700 cm\(^{-1}\). Cook et al (1960) also report a band at 855 cm\(^{-1}\) assigned as \(V_3\) of the fluophosphate ion. In addition, Sharp & Sharpe (1956) report that the spectra of the fluophosphates of rubidium, caesium and silver give bands in the 840 cm\(^{-1}\) and 720-740 cm\(^{-1}\) regions. As stated in a previous chapter, the spectrum of nitrosonium fluophosphate shows bands at 834 cm\(^{-1}\) and 740 cm\(^{-1}\). Hence it seems reasonable to assign the two bands at 840 cm\(^{-1}\) and 740 cm\(^{-1}\) to the \(V_3\) and \(V_1\) modes of the fluophosphate ion. The bands at 1120 cm\(^{-1}\) and 1030 cm\(^{-1}\) suggest the formation of phosphate entities involving P = O multiple bonding, possibly leading to polyanion formation. It will be seen that this reaction is analogous to the boron trifluoride/dinitrogen tetroxide reaction in that a mixture of nitrosonium and nitronium species are apparently formed.
Hence the behaviour of nitrogen oxides with Lewis Acids bears a strong resemblance to the behaviour of nitrogen oxides in strong acid systems, which is covered in the introductory chapter.
Phosphorus trichloride and oxytrichloride/dinitrogen tetroxide,
Phosphorus trichloride reacted violently with dinitrogen tetroxide at about 0°C to give a number of
products. One of the volatile products of the reaction was phosphorus oxytrichloride identified by its melting point and boiling point. The solid residue could not be further purified and inconsistent analyses suggested a mixture of products.
Reaction of dinitrogen tetroxide with phosphorus oxychloride yielded one product in the solid residue, analysing as $P_4O_5Cl$.
The infrared spectrum showed the following bands: 3700 vw, 2380 sh, 2250 s, 1600 w, 1050 - 1000 s. (all cm$^{-1}$). Given the formula $\left(P_4O_5Cl\right)_n$, and assuming phosphorus to be in the +5 oxidation state, a reasonable structure for the above compound incorporating nitrosonium ions cannot be formulated unless nitronium ions exist also.
Assuming $n = 2$, the following formula is possible:
$$\left[NO^+\right] \quad \left[NO_2^+\right] \quad \left[P_2O_5Cl_2^{2-}\right]$$
in which the complex anion involved could have the following structure:
$$\begin{array}{c}
O \\
\text{Cl} \\
\text{Cl}
\end{array} \quad P - O - P - O \quad \begin{array}{c}
\text{Cl} \\
\text{Cl}
\end{array} \quad 2^-$$
Any structures containing $-ONO$ or $-ONO_2$ groups linked to the phosphorus atoms would be easily distinguishable from
the infrared spectrum. The gas phase reaction between phosphorus trichloride and nitrogen dioxide has been studied (Klement & Wolf 1955) and as might be expected the reaction course is slightly different, since nitrogen dioxide radicals are now the reactive species. The same may possibly occur in dinitrogen tetroxide solution, but, as has been observed previously, the reactive species in the liquid phase always appear to be ions.
Klement and Wolf postulate the initial formation of a highly reactive radical intermediate which can dissociate in two ways:
\[
\text{PCl}_3 + \text{NO}_2 \rightarrow \text{Cl}_3P - O_2\text{NO}
\]
\[
\text{POCl}_2 + \text{NOCl} \rightarrow \text{O-P-O-NO} \rightarrow \text{O-P-O} + \text{NO} \rightarrow \text{O-P-O-P-O}
\]
Reaction of phosphorus oxytrichloride with nitrogen dioxide yields \( \text{P}_2\text{O}_6\text{NCl}_2 \) (Klement & Wolf 1955), presumably by a similar type of mechanism; no structure is suggested for the species. The above mechanism could operate in dinitrogen tetroxide solution with initial cleavage of \( \text{N}_2\text{O}_4 \) species; or attack could be by nitrate ion,
eliminating chloride ions with nitrogen dioxide eventually cleaving from the reaction species. This would give largely the same reaction pattern.
Other reactions studied involving phosphorus chlorides were:
Phosphorus pentachloride/Dinitrogen tetroxide.
This reaction gave a white solid, stable under pressure, but which rapidly dissociated at room temperature and atmospheric pressure.
Phosphorus trichloride or pentachloride/nitrosyl chloride.
Attempts to form $\text{PCl}_4^+ \text{NO}$ species and to form $\text{NO}^+ \text{PCl}_6^-$ were not successful; the former reaction gave only phosphorus pentachloride, and no sign of reaction occurred in the latter case.
Arsenic trichloride/Nitrosyl chloride.
This reaction was found to give two products, a red liquid, $\text{AsCl}_4 \cdot \text{NO}$, and a white solid stable under pressure of nitrosyl chloride but which dissociated at room temperature and atmospheric pressure.
Lewis & Sowerby (1957) studied the arsenic trichloride/nitrosyl chloride equilibrium and found evidence for the formation of a weak complex $\text{AsCl}_3 \cdot 2\text{NOCl}$, to which the white solid observed in the present work
may correspond. Waddington & Klanberg (1960) carried out the reaction in liquid hydrogen chloride and observed only the species AsCl₄·NO with no other adduct formation. Their compound AsCl₄·NO was a red liquid, with a strong infrared absorption at 1860 cm⁻¹ indicating co-ordinated nitrosonium ion, the present work confirms this.
The gas phase spectrum gave a strong bond at 1852 cm⁻¹ in good agreement with Waddington & Klanberg's value. The volatility of the compound (boiling point 60° - 70°) indicates monomeric structure, implying that arsenic is tripositive and penta-co-ordinate.
The structure of this may be based on a hexaco-ordinate arsenic atom, with a pair of electrons occupying one of the positions, viz:-
As yet, there is no proof that the nitrosyl group is bonded to the arsenic atom via the nitrogen atom, or that the As—NO system is linear. This system could possibly be similar to the nitrosyl halides, which are bent molecules.
ARSENIC TRICHLORIDE/DINITROGEN TETROXIDE.
These react rapidly to give a white solid and a little of the compound AsCl₄•NO mentioned above; the solid analysed as As(NO₃)₃•N₂O₄.
The postulated reaction scheme follows the normal pattern for this type of reaction.
\[
\text{AsCl}_3 + 3\text{N}_2\text{O}_4 \rightarrow \text{As(NO}_3)_3 + 3\text{NOCl}
\]
\[
\text{As(NO}_3)_3 + \text{N}_2\text{O}_4 \rightarrow \text{As(NO}_3)_3\cdot\text{N}_2\text{O}_4
\]
\[
\text{AsCl}_3 + \text{NOCl} \rightarrow \text{AsCl}_4\cdot\text{NO}
\]
As with the reaction products in similar systems, the compound decomposes in Nujol mulls, and time-dependent spectra were used to assign the following bands to the compound As(NO₃)₃•N₂O₄:
2220m; 1850w; 1613m; 1252m; 1012s; 805s; 750w.
Assuming that the adduct is the nitrosonium salt (NO⁺) (As(NO₃)₄⁻), the following assignments can be made:
2220 - \( V_1(\text{NO}^+) \)
1850 - \( (V_2 + V_6) \) nitrato-group
1613 - \( V_4 \) nitrato-group
1252 - \( V_1 \) nitrato-group
1012 - \( V_2 \) nitrato-group
805 - \( V_6 \) nitrato-group
750 - \( V_3 \) or \( V_5 \) nitrato group.
The bands assignments are satisfactorily in agreement with the ionic formulation, and the \((V_4 - V_1)\) splitting, in the nitrato-group, of \(361 \text{ cm}^{-1}\), indicated a high degree of covalency in the \(\text{As(NO}_3)_4^-\) anion, on Katzin's criterion (1962).
Attempts to obtain the anhydrous nitrate \(\text{As(NO}_3)_3\) by heating the adduct were unsuccessful; the only products were arsenic trioxide and nitrogen oxides.
**ANTIMONY PENTACHLORIDE/NITRIC OXIDE**
This reaction followed initially a similar pattern to the analogous stannic chloride reaction; i.e. reduction occurs:
\[
\text{SbCl}_5 + 2\text{NO} \rightarrow \text{SbCl}_3 + 2\text{NOCl}
\]
Unlike stannous chloride, antimony trichloride is soluble in carbon tetrachloride, the reaction medium, and the second reaction is:
\[
\text{SbCl}_3 + \text{NOCl} \rightarrow \text{SbCl}_4 \cdot \text{NO}
\]
This compound was first prepared by Waddington and Klanberg (1960) who reacted antimony trichloride and nitrosyl chloride in liquid hydrogen chloride; in addition, the compound \((\text{NO})_2 \text{SbCl}_5\) was observed in this system, which was not observed in the present work.
In the present work, also it was found that the following reaction interfered when the antimony pentachloride concentration was too high:
\[ \text{SbCl}_5 + \text{NOCl} \rightarrow \text{NO.SbCl}_6 \]
However, when low concentrations of antimony pentachloride were used, it was found that practically complete reduction (reaction (1)) occurred, before reaction 3 became significant.
The infrared spectrum of SbCl\(_4\)NO gave a band at 1900 cm\(^{-1}\), agreeing with Waddington & Klanberg's formulation of this as a covalent compound containing -Sb-N=O bonding, though, as in AsCl\(_4\)NO, bonding to the central atom could occur via the oxygen atom.
The gas phase reaction of antimony pentachloride and nitric oxide has been carried out, and the product reported to be a yellow crystalline polymer \( (\text{SbCl}_5)_2\text{NO}_n \) (Bowen 1954), but no structural data were given. He reported that when the reaction mixture was cooled to -196°C, a purple coloration appeared which vanished above -110°C. This purple coloration is typical of ions involving one-electron bonds, i.e. of the type N\(_2^0_2^+\), formed under pressure of nitric oxide by the reaction
\[ \text{NO}^+ + \text{NO} \rightleftharpoons \text{N}_2^0_2^+ \] (Seel et al 1953; Seel & Sauer 1957).
Thus Bowen's compound is likely to contain nitrosonium ions and may be a mixture of compounds such as NO.SbCl₆ and NO.SbCl₄.
ANTIMONY PENTACHLORIDE/DINITROGEN TETROXIDE
This reaction is difficult to study for the reasons outlined in the previous chapter, i.e., many competing reactions occur and most of the products are solids and difficult to separate.
Infrared examination showed evidence for formation of nitrosonium and nitronium ions, and covalently bonded nitrato-groups, but no products could be satisfactorily isolated. Heating caused degradation to the pentoxide with evolution of nitrogen oxides, and no products could be sublimed out.
ADDUCTS OF ARSENIC AND ANTIMONY PENTAFLUORIDES.
Arsenic and antimony pentafluorides have been shown to react with nitrogen dioxide (Aynsley et al 1951) to form adducts analysing as AsF₅·NO₂ and SbF₅·NO₂ but nothing is known of their structures. These appear to be definitely single compounds as AsF₅·NO₂ sublimes at 57°C without decomposition, and nitrosonium and nitronium fluoarsenate sublime at much higher temperatures than this.
Whereas reactions of the type $MCl_n/N_2O_4$ always seem to involve considerable replacement of chloride by nitrate-groups, this occurs only to a very small extent if at all, with $MF_n/N_2O_4$ reactions. In general $MX_n/N_2O_4$ reactions ($M=$central metal or non-metal atom; $X=$halogen) can occur in the following ways:
1. Salt formation
$$MX_n + N_2O_4 \rightarrow NO_xMX_{n+1} + NO_2MX_{n+1} + MOX \text{ species}$$
2. Adduct formation
$$MX_n + \frac{1}{2}N_2O_4 \rightarrow MX_nNO_2 : \text{these could be ionic}$$
3. Substitution Reactions
$$MX_n + nN_2O_4 \rightarrow M(NO_3)_n + nNO X$$
The first two reactions do not involve M-X bond fission and the second would be favoured by a high concentration of $NO_2$ radicals and thus by gas phase reaction. Fluorides undergo these reactions (1 and 2) preferentially, probably on account of the higher M-F bond strength, compared to the M-Cl bond. Also, it is possible that reactions of type 3 are favoured because of the free energy of formation of nitrosyl chloride compared nitrosyl fluoride.
Considering fluorides, boron trifluoride and phosphorus penta-fluoride came into category 1, with arsenic and antimony pentafluorides in category 2.
Both the reaction types involve the fluoride behaving as acceptors; electrostatically the latter two will be the stronger acceptors because of the lower electronegativity of the arsenic and antimony atom, but it is difficult to see how this favours $-NO_2$ co-ordination, rather than fluoride ion co-ordination. Conclusive statements are not possible without a knowledge of the structures of the AsF$_5$-$NO_2$ and SbF$_5$-$NO_2$ adducts. Also, it is possible that under exactly identical reaction conditions, all these compounds would react similarly.
**NITROGEN OXIDE/SULPHUR OXIDE REACTIONS**
These reactions can be considered as coming into the same category as the ones previously discussed, since sulphur dioxide and trioxide can both behave as Lewis acids; sulphur trioxide, in particular, is highly analogous to boron trifluoride and phosphorus pentafluoride:
$$SO_3 + O^{2-} \rightarrow SO_4^{2-}$$
$$BF_3 + F^- \rightarrow BF_4^-$$
$$PF_5 + F^- \rightarrow PF_6^-$$
Sulphur dioxide also tends to behave similarly but in this case the sulphite species produced are very susceptible to oxidation to give sulphates.
Reactions between sulphur trioxide and nitrogen oxides have been extensively covered by several workers (Goddard et al 1950; Lehmann & Kluge 1951; Gerding & Eriks 1952), and are highly similar to the nitrogen oxide/sulphuric acid systems studied by other workers and discussed in the introductory chapter.
The reaction between sodium nitrite and sulphur trioxide yields on vacuum distillation $N_2O_3 \cdot 3SO_3$; and similar treatment of the potassium nitrate/sulphur trioxide mixture yields $N_2O_5 \cdot 4SO_3$ (Lehmann & Kluge 1951). The latter product dissociates at 60°C to $N_2O_5 \cdot 3SO_3$ and sulphur trioxide; also some $N_2O_4 \cdot 3SO_3$ is formed.
Gerding & Eriks (1952) examined the $N_2O_4 \cdot 3SO_3$ adduct and from the Raman spectrum assigned the structure $(NO^+) (NO_2^+) (S_3O_{10}^{2-})$; bands at 1400 cm$^{-1}$ and 2277 cm$^{-1}$ were assigned to the $V_1$ and $V_3$ modes of the nitronium ion, and an absorption at 2308 cm$^{-1}$ was assigned to the nitrosonium ion.
Similarly, the other complexes were assigned the following structures.
It will be seen that these reactions fall into the same pattern as the nitrogen oxide/strong acid reactions in that dinitrogen trioxide adducts yield only nitrosonium compounds, dinitrogen pentoxide adduct gives only nitronium compounds, and dinitrogen tetroxide gives mixed species.
The reaction between sulphur dioxide and dinitrogen tetroxide (Hart-Jones et al 1929) is slightly different, due to the susceptibility of sulphur dioxide to oxidation; the product is nitrosonium pyrosulphate \((\text{NO})_2\text{S}_2\text{O}_7\), so an oxidation-reduction must have occurred.
It is significant that all the nitrosonium and nitronium sulphates prepared so far, contain polymeric anionic species. No \((\text{NO})_2\text{SO}_4\) is known (Hart-Jones et al 1929), neither has \((\text{NO}_2)_2\text{SO}_4\) been isolated in the solid.
state, though in the former case the hydrogen sulphate
\[\text{NO}_2\text{HSO}_4\] is well known. No explanations have been put forward for this, though possibly the high tendency of \(\text{SO}_4\) tetrahedra to catenate is contributory; the role of the cations is not clear.
Lehmann & Kluge (1951) have put forward tentative reaction mechanisms for the salts formed involving initial adducts formation, followed by heterolytic dissociation; this seems unlikely in the instances where sulphur trioxide is reacted with nitrates or nitrates. Also, taking the formation of nitrosonium pyrosulphate as an example, the reaction proceeds fairly slowly, and a mechanism involving ions might serve equally, since the concentrations of ions in liquid dinitrogen tetroxide and sulphur dioxide are known to be small.
**Selenium Oxide/Nitrogen Oxides.**
One of the earliest nitrosonium salts to be made was \((\text{NO})_2\text{SeO}_4\) (Lenher & Matthews 1906) who prepared it by reaction of excess liquid dinitrogen tetroxide with 83% selenic acid; a blue solid crystallises out, decomposing above -13°C. The deep blue colour suggests that this may not be \((\text{NO}^+)_2 (\text{SeO}_4^{2-})\) but may contain covalently bonded nitrosonium groups also this coloration
could be due to the $N_2O_2^+$ ion, but this would put the selenium atom in unusual oxidation state.
The selenium analogue to nitrosyl hydrogen sulphate was prepared by Meyer & Wagner (1922) by reaction of excess dinitrogen trioxide with pure selenic acid. This is analogous to the dissociation of dinitrogen trioxide in sulphuric acid to give $NO^+$ and $HSO_4^-$ ions. $NO.HSeO_4$ is much more static than $(NO)_2SeO_4$ but less so than the hydrogen sulphate.
The reactions of selenium trioxide with nitrogen oxides are similar to those with sulphur trioxide except that no mixed nitrosonium-nitronium species could be formed. (Kempe et al 1964).
$$\text{SeO}_3/N_2O_5 \rightarrow (NO_2)_2Se_3O_{10}$$
$$\text{SeO}_3/N_2O_3 \rightarrow (NO)_2Se_2O_7$$
The latter product, with a limited amount of water, gave $NO^+.HSe_2O_7^-$. These compounds were all assigned their structures on the basis of Raman spectra.
Attempts to prepare nitronium selenate or the hydrogen selenate were unsuccessful (Goddard et al 1950). The compound $(NO_2^+)(HSe_2O_7^-)$ was formed, possibly because the dissociation:
$$2(NO_2^+)(HSeO_4^-) \rightarrow (NO_2^+)(HSe_2O_7^-) + HNO_3$$
occurs.
EXPERIMENTAL
Stannic Chloride/Nitric Oxide.
Nitric oxide, formed by the reduction of sodium nitrate solution with ferrous sulphate (Inorganic Syntheses Vol 2, p.126), was bubbled into stannic chloride/carbon tetrachloride solution for approximately 30 minutes. The white solid analysed largely as stannous chloride.
Found: - Sn: 33.1% Cl: 65.0%; Stannous chloride requires Sn: 36.8% Cl: 63.2%.
Stannic Chloride/Dinitrogen tetroxide.
Excess dinitrogen tetroxide was prepared as mentioned before, and redistilled twice over phosphorus pentoxide in vacuo, before being condensed into a Carius tube containing distilled stannic chloride, frozen down in vacuo. The tube was sealed off and the reaction mixture allowed to come up to room temperature. The gaseous products were pumped off after freezing down the reaction again and opening the Carius tube, and the solid residue examined. The analysis of freshly prepared samples corresponded roughly to a 1 : 1 (NO)₂SnCl₆·Sn(NO₃)₄ mixture.
Calculated: Sn 31.4% Cl 28.3% total N 12.7% NO₃⁻ 33.0%
Found: Sn 33.1% Cl 24.7% total N 15.2% NO₃⁻ 30.0%
Phosphorus Pentafluoride/Nitric Oxide.
Phosphorus pentafluoride was prepared by heating Phosflorogen (Ozark-Mahoning Co., Ltd.) and condensing out the impurities in acetone/CO₂; it was purified by redistillation at c.-78°C. Nitric oxide was prepared as before and the two reactants condensed into a bomb in vacuo; at -196°C. The reaction was allowed to come up to room temperature, then the bomb was evacuated and the solid product examined.
Phosphorus Pentafluoride/Dinitrogen tetroxide.
The two reactants were prepared as before, redistilled separately in vacuo over phosphorus pentoxide and condensed into a Carius tube at -196°C. After raising to room temperature, at which stage reaction occurred, the system was recooled to 198°C and the gas products pumped off and the solid products examined.
Phosphorus trichloride or Phosphorus oxychloride/dinitrogen tetroxide.
Phosphorus trichloride and dinitrogen tetroxide were condensed in a Carius tube as in other systems and the reaction allowed to come to room temperature. Phosphorus oxychloride was isolated amongst the volatile products by fractionation in vacuo, after removal of
excess dinitrogen tetroxide and nitrosyl chloride at -23°C (carbon tetrachloride slush bath) and characterised by its melting and boiling points (lit 1.25°C; found 0.9°C, (M.P.): lit: 105.1°C found: 105.0°C (B.P.))
The solid product from the phosphorus oxychloride/dinitrogen tetroxide reaction, obtained in the similar manner to the other reactions, and was analysed:
Found (%) P: 21.49 N: 9.86 Cl: 25.50. \((\text{PO}_4\text{NCl})_n\) requires P: 21.38 N: 9.70 Cl: 25.49
Arsenic Trichloride/Nitrosyl Chloride
Nitrosyl chloride, prepared as previously, was dried over phosphorus pentoxide in vacuo and condensed, in excess, into a Carius tube containing distilled arsenic trichloride, in vacuo at -196°C. The reaction mixture was raised to room temperature. On evacuation of the reaction mixture the white solid was soon to dissociate. The red liquid was condensed in a trap at -78°C, and the system opened to the atmosphere. On raising the room temperature, excess nitrosyl chloride boiled off.
Analysis of red liquid: Found(%) As: 29.0 Cl: 56.2 N: 5.8
AsCl₄·NO requires As: 29.8 Cl: 56.4 N: 5.5
Arsenic Trichloride/Dinitrogen Tetroxide.
Arsenic trichloride and dinitrogen tetroxide were separately vacuum distilled into a Carius tube, which was then sealed, and the reaction mixture raised to room temperature. A white solid rapidly precipitated out at room temperature. After evacuation of excess solvent, this was transferred to the dry box and examined.
Analysis Found: As: 21.0 N: 19.5 NO₃: 71.1
As(NO₃)₃·2N₂O₄ required As: 21.3 N: 19.9 NO₃: 71.3
Antimony Pentachloride/Nitric Oxide.
A stream of nitric oxide was bubbled through an antimony pentachloride (5 gms)/carbon tetrachloride (100 mls) solution for 30 - 40 minutes. The yellow solid product was filtered by suction in a dry box, then vigorously removed of all traces of solvent in vacuo. It was then examined by infrared spectroscopy, and analysed.
Analysis found (%) Sb: 39.3 Cl: 47.0 N: 4.6
SbCl₄·NO requires Sb: 39.5 Cl: 46.8 N: 4.6
At concentrations of antimony pentachloride in the region of 10 gms/100 mls carbon tetrachloride, or above, significant quantities of NO·SbCl₆ started to be formed.
Antimony pentachloride/Dinitrogen tetroxide.
These were reacted in a Carius tube in a similar manner to the SnCl₄/N₂O₄ reaction, and the gaseous products removed in vacuo, and the solid products examined. Analyses of the reaction product mixtures were highly inconsistent, but there was considerable residual chlorine in all cases.
Dinitrogen tetroxide/Sulphur dioxide.
These reactants, separately dried in vacuo, were condensed in a Carius tube and reacted at room temperature for about 24 - 30 hours. The solid residue had a purple tinge due probably to N₂O₂⁺ ion formation; on pumping down this vanished, leaving (NO)₂S₂O₇
| Found (%) | N : 11.5 | S : 26.6 |
|-----------|----------|----------|
| Calculated for (NO)₂S₂O₇ | N : 11.7 | S : 26.9 |
CHAPTER IV
REACTIONS OF NITROGEN OXIDES AND NITROSYL HALIDES
WITH COMPOUNDS OF TITANIUM AND ZIRCONIUM
The Group IV Elements Titanium, Zirconium.
The first studies on the behaviour of titanium compounds in nitrosylating systems were by Rheinboldt & Wasserfuhr (1927), who prepared \((\text{NO})_2\text{TiCl}_6\) by reaction of titanium tetrachloride and nitrosyl chloride in carbon tetrachloride solution. This has been shown, in Chapter I, to be \((\text{NO}^+)_2(\text{TiCl}_6^{2-})\).
Partington & Whynnes (1949) demonstrated that the same product \((\text{NO})_2\text{TiCl}_6\) was obtained from reacting nitrosyl chloride with either titanium tetrachloride or trichloride.
In addition they carried out the reaction between dinitrogen tetroxide and titanium tetrachloride and reported that the products were probably a mixture of the adduct \((\text{NO})_2\text{TiCl}_6\) and titanium dinitrate \(\text{Ti(NO}_3)_2\). The existence of the latter compound seems highly questionable since the Titanium (II) oxidation state is a very strong reducing agent, and with a highly oxidising group like the nitrate anion present,
immediate oxidation-reduction of the type
\[ \text{Ti(NO}_3)_2 \rightarrow \text{TiO}_2 + \text{N}_2\text{O}_4 \]
seems very probable.
The formation of \( (\text{NO})_2\text{TiCl}_6 \) would be due to reactions of the type
\[ \text{TiCl}_4 + 4\text{N}_2\text{O}_4 \rightarrow \text{Ti(NO}_3)_4 + 4\text{NOCl} \]
\[ 2\text{NOCl} + \text{TiCl}_4 \rightarrow (\text{NO})_2\text{TiCl}_6 \]
Gutmann & Tannenberger (1956) confirmed the formation of \( (\text{NO})_2\text{TiCl}_6 \) in the titanium tetrachloride/dinitrogen tetroxide reaction system; they attempted to prepare titanium tetranitrate by reaction of the tetraiodide with dinitrogen tetroxide.
\[ \text{TiI}_4 + 4\text{N}_2\text{O}_4 \rightarrow \text{Ti(NO}_3)_4 + 4\text{NO} + 2\text{I}_2. \]
Nitrosyl iodide is unknown, hence no undesirable side-reactions, involving a nitrosyl halide, could occur. They believed that titanium tetranitrate was a product of this reaction but above 10°C it dissociated.
\[ \text{Ti(NO}_3)_4 \rightarrow \text{TiO(NO}_3)_2 + \text{N}_2\text{O}_4 + \frac{1}{2}\text{O}_2. \]
The residue, titanium oxydinitrate, was a white hygroscopic solid. No structural studies have been carried out on this compound. It is interesting to note that it apparently shows no tendency to form adducts of the type \( \text{TiO(NO}_3)_2 \cdot \text{N}_2\text{O}_4 \).
However, the compound Ti(NO₃)₄, tetranitratotitanium, has been prepared by the reaction of hydrated titanium tetranitrates with dinitrogen pentoxide (Field & Hardy 1963 b). Infra-red examination showed the compound to be covalent, containing nitrato-groups. The splitting V₄-V₁ was 401 cm⁻¹, showing the compound to be highly covalent. It has a low melting point, 58°C, as would be expected for a highly covalent compound. Field & Hardy noted that in nujol mulls, the compound decomposed even over the period during which the spectrum was run. This has been noticed with similar compounds in the present work. Reaction with n-dodecane gave alkynitrate and nitroalkane fractions, probably due to the tendency of nitrato-groups to radical dissociation.
\[ \text{M} - \text{O} - \text{N} \quad \rightarrow \quad \text{M} - \text{O} + \text{NO}_2 \]
as has been mentioned by Addison (1962).
(NO)₂TiF₆
The preparation of nitrosonium fluotitanate (NO)₂TiF₆ has been attempted previously by the reaction of titanium metal with bromine trifluoride to yield titanium tetra-fluoride followed by further treatment.
with bromine trifluoride and nitrosyl chloride (Woolf 1950). It was found that the product was a mixture of \((\text{NO})_2\text{TiF}_6\) and \(\text{TiF}_4 \cdot x\text{BrF}_3\). In the present work nitrosonium chlorotitanate was dissolved in bromine trifluoride, and the excess of solvent removed. The product, as with Woolf, was a mixture of \((\text{NO})_2\text{TiF}_6\) and \(\text{TiF}_4 \cdot x\text{BrF}_3\). Infra-red examination showed a band at \(2308 \text{ cm}^{-1}\), which would almost certainly be due to nitrosonium ion in \((\text{NO})_2\text{TiF}_6\); the bromine trifluoride impurity is probably present as \((\text{BrF}_2^+)_2(\text{TiF}_6^{2-})\). The \(\text{BrF}_2^+\) ion has been postulated as occurring in the bromine trifluoride solvent system (Woolf 1950).
**Zirconium**
By direct reaction of zirconium tetrachloride and nitrosyl chloride, Devin & Perrot (1958) obtained \((\text{NO})_2\text{ZrCl}_6\). The present work confirmed this; \((\text{NO})_2\text{ZrCl}_6\) is a pale yellow powder, fairly hygroscopic, and an infra-red band at \(2188 \text{ cm}^{-1}\) confirmed the existence of free nitrosonium ion so that the compound could be formulated \((\text{NO}^+)_2(\text{ZrCl}_6^{2-})\).
Reaction of zirconium tetrachloride and nitrosyl chloride in bromine trifluoride gave \((\text{NO}^+)_2(\text{ZrF}_6^{2-})\); this was confirmed by the presence
of an infra-red band at 2310 cm\(^{-1}\). It was observed that the fluo-salt was much less stable to hydrolysis than the chloro-salt. This has been noted before in the cases of the corresponding stannates and antimonates. Also, in the present case, the nitrosonium ion absorption again occurs at a much higher frequency in the fluo- than the chloro-derivative.
No reactions were carried out between zirconium tetrachloride and dinitrogen tetoxide, but the reaction with dinitrogen pentoxide is known to give tetranitrato-zirconium, Zr(NO\(_3\))\(_4\) (Field & Hardy 1962).
\[ 4N_2O_5 + ZrCl_4 \rightarrow Zr(NO_3)_4 + 4NO_2Cl \]
Like its titanium analogue, the zirconium compound has been shown to contain co-ordinated nitrato-groups. In this case the \(V_4 - V_1\) splitting is approximately 350 cm\(^{-1}\), considerably less than that observed in the titanium compound, but still indicative of strong covalent character.
In all the above examples involving the group IVA metals, the metals are invariably in the +4 oxidation state, which is much the most stable state for these metals.
EXPERIMENTAL
(NO)₂TiCl₆
This preparation is described in the experimental section of Chapter I.
(NO)₂TiF₆
Nitrosonium chlorotitanate was prepared as before, and about 1.5 gm placed in a silica reaction flask; then bromine trifluoride was added, with the mixture cooled down to about -20° (carbon tetrachloride slush-bath). The mixture was allowed to rise to room temperature and the excess bromine trifluoride was then removed in vacuo at 80°-90°C. A white, faintly brown powder remained. Hydrolysis, and addition of silver nitrate solution, showed that some bromine was present.
Analysis:
N:12.9% Ti:23.2% (NO)₂TiF₆ requires N:14.6% Ti:25.0%
(NO)₂ZrCl₆
Nitrosyl chloride, dried by distillation over phosphorus pentoxide in vacuo, was transferred into a Carius tube containing zirconium tetrachloride, by vacuum distillation. The tube was sealed and the mixture allowed to rise to room temperature, when
reaction took place. The mixture was frozen down and the excess nitrosyl chloride removed in vacuo. A yellow solid remained.
Analysis:
\[ \text{N:7.4\% Cl:58.6\%} \quad (\text{NO})_2\text{ZrCl}_6 \text{ requires N:7.7\% Cl:58.5\%} \]
\[ (\text{NO})_2\text{ZrF}_6 \]
Nitrosyl chloride, dried as before, was transferred by vacuum distillation into a silica reaction flask containing zirconium tetrachloride; this was removed from the vacuum system and cooled to -184°C, then bromine trifluoride was added. The mixture was allowed to come up to room temperature, when reaction took place, then the excess volatile products were removed in vacuo at 80°-90°C. A white solid remained.
Analysis:
\[ \text{N:10.3\% F:42.8\%} \quad (\text{NO})_2\text{ZrF}_6 \text{ requires N:10.6\% F:43.2\%} \]
In this case the product showed no trace of bromine.
Infra-red spectra were measured, as previously, on a Grubb Parsons DBI spectrophotometer, and the spectra were measured as mulls in florube grease. The quoted nitrosonium ion frequencies were calibrated against polystyrene.
CHAPTER V
THE GROUP VA METALS. VANADIUM, NIOBIUM, TANTALUM
1. Vanadium.
Vanadium is known to exist in the oxidation states -1, 0, +2, +3, +4, +5. In addition, it has a strong tendency to form vanadium-oxygen multiple bonds of high bond strength in systems containing oxygen-donor groups. The stability of the vanadium-oxygen bond has an important effect in vanadium preparative chemistry. When dealing with a system containing vanadium-oxygen bonding, it is often very difficult to obtain oxygen-free vanadium compounds. Hence, the best characterised state of vanadium (IV) is as the vanadyl, $VO^{2+}$ cation; also, several vanadium (V) compounds containing the $VO$ (III) group are well known, e.g. the oxytrihalides $VOF_3$ and $VOCl_3$, and more recently, compounds containing the $VO_2^+$ group have been characterised. (Pantonin et al 1960).
It has been noted, in chapter I, that the V-O bonds in vanadium pentoxide can be broken by nitryl fluoride (Aynsley et al 1954, & Hetherington and
Robinson 1957) and by the nitrosyl chloride/bromine trifluoride system (Woolf 1950). Probably in the latter case the fluorinating agent is nitrosyl fluoride, since the simple vanadium pentoxide/bromine trifluoride system gives the oxytrifluoride VO\textsubscript{2}F\textsubscript{3}. If this is the case, the fluorination reactions could be of the type:
\[ \text{VOF}_3 + 2\text{NO}_2\text{F} \rightarrow \text{VF}_5 + \text{N}_2\text{O}_5 \quad (\text{Or } 2\text{NO}_2 + \frac{1}{2}\text{O}_2) \]
\[ \text{VOF}_3 + 2\text{NOF} \rightarrow \text{VF}_5 + \text{NO} + \text{NO}_2 \]
The free energies of formation of the nitrogen oxide species NO and NO\textsubscript{2} may be the driving force of the reaction, supplying the energy for V-O bond cleavage. This suggests that pure alkali-metal hexafluorovanadates could be obtained by a reaction system of the type NaVO\textsubscript{3}/NOCl/BrF\textsubscript{3}.
Another method of 'deoxygenation' of vanadyl compounds was discovered in the course of the present work, in an attempt to prepare anhydrous vanadyl chloride VOCl\textsubscript{2}. The use of thionyl chloride as a dehydrating agent to convert transition metal chloride
hydrates into the anhydrous transition metals is well known (Inorganic Syntheses, Vol. 5, p.153), and this method was attempted using vanadyl chloride hydrate and thionyl chloride. The expected reaction
\[ \text{VOCl}_2 \cdot 5\text{H}_2\text{O} + 5\text{SOCl}_2 \rightarrow \text{VOCl}_2 + 5\text{SO}_2 + 5\text{H}_2\text{O}. \]
did not occur.
Instead, the products were vanadium trichloride, a little vanadium tetrachloride or oxytrichloride, and sulphuric acid. It was difficult to ascertain whether vanadium tetrachloride or oxytrichloride was produced. Both are volatile compounds, and in the pure state the latter is yellow and the former red. However, when vanadium oxytrichloride is slightly impure, it readily turns red and so is difficult to distinguish visually between the two.
The reaction may have proceeded either by
a) initial formation of anhydrous vanadyl dichloride, then reaction of this giving vanadium trichloride,
or b) simultaneous removal of the oxo-oxygen with one or more of the water molecules.
The structure of the aquated vanadyl ion is /
with the V=O bond length much shorter than the V-OH$_2$ bond lengths. The visible and ultraviolet spectra of the vanadyl ion have been satisfactorily interpreted on this basis (Ballhausen & Gray, 1961). Hence it is possible that the V-OH$_2$ bonds would be cleaved before the V=O bond is cleaved. The presence of sulphuric acid indicates that the sulphur has been oxidised from +4, in thionyl chloride, to +6. The corresponding reduction is
\[ \text{V(IV)O}^{2+} \rightarrow \text{V}^{3+}(\text{III}) \]
It was not possible to stop the reaction at an intermediate stage to obtain anhydrous vanadyl chloride, which suggests that reaction b, above, may be the one that occurs.
However, for a proper reaction mechanism study, the reaction between anhydrous vanadyl chloride and thionyl chloride would need to be examined; in addition, the other products of reaction would have to be identified more accurately than in the present instance.
Vanadium-nitrosonium complexes are known. The compound $K_5 V(CN)_5 NO$ gives an infra-red band at 1575 cm$^{-1}$ which is attributed to a $V - NO^+$ linkage. (Hieber & Jahn 1958).
Also, the nitrosonium and nitronium fluorovanadates have been mentioned previously (Chapter I). In the literature, the reactions between nitric oxide and vanadium tetrachloride in liquid, solution and gas phases have been reported (Whittaker & Yost 1949); no structural information was given.
The System Vanadium Trichloride/Nitrosyl Chloride.
These compounds react readily to give a mixture of purple solids, identified as $NO.VCl_4$ and $NO.V_2Cl_7$; both these compounds were reported by Whittaker & Yost (1949). They arise from the reactions:
$$NO.Cl + VCl_3 \rightarrow NO.VCl_4$$
$$NO.VCl_4 + VCl_3 \rightarrow NO.V_2Cl_7$$
There was no evidence for compounds of the type $(NO)_2VCl_5$ or $(NO)_3VCl_6$. The proportions of the compounds formed obviously depended on the ratios of the original components. In dilute solutions ($VCl_3:NOCl$ mole ratio = c.1:3) the bulk product was $NO.VCl_4$. When the ratio of the components approached
unity, the formation of NO\textsubscript{2}V\textsubscript{2}Cl\textsubscript{7} was favoured; in all cases, however, both products were formed and an equilibrium
\[ \text{NO}_2\text{VCl}_4 + \text{VCl}_3 \rightleftharpoons \text{NO}_2\text{V}_2\text{Cl}_7 \]
may have existed.
Whittaker & Yost found that bubbling nitric oxide through liquid vanadium tetrachloride gave NO\textsubscript{2}V\textsubscript{2}Cl\textsubscript{7}; when the vanadium tetrachloride was in carbon tetrachloride solution the product was NO\textsubscript{2}VCl\textsubscript{4}. This agrees with the present work, in that the former constitutes a high vanadium tetrachloride concentration giving the dimer, while the lower concentration gives the monomer form.
**NO\textsubscript{2}VCl\textsubscript{4}**
This compound was isolated as a very deep purple crystalline solid; it was found to be exceedingly hygroscopic and hydrolysed appreciably even when being handled in the dry-box.
It sublimed completely at 30°-35°C, in vacuo, and this was taken to imply the existence of covalent species probably containing V4—NO\textsuperscript{+} bonds.
The infra-red spectrum gave a band at 2179 cm\(^{-1}\), and a further band at 1800 cm\(^{-1}\); time-dependence studies showed that the latter band was a decomposition band, probably due to nitrosyl chloride. The N-O bond in nitrosyl chloride absorbs at 1799 cm\(^{-1}\) (Burns & Bornstein 1950). This behaviour has been observed before in the compounds NO.\(AlCl_4\), NO.\(ZnCl_3\), NO.\(HgCl_3\), NO.\(BiCl_4\), NO.\(MnCl_3\) and NO.\(FeCl_4\); in all cases it was attributed to the same behaviour.
The 2179 cm\(^{-1}\) band is at the lower end of the free nitrosonium region, which indicates considerable interaction; this interaction would explain the volatility of the compound, while the predominantly ionic formulation at room temperature can account for the solubility in polar solvents such as nitrosyl chloride and sulphur dioxide, and the insolubility in non-polar solvents, e.g. carbon tetrachloride, as reported by Whittaker & Yost (1949).
The existence of the nitrosonium ion necessitates the species be formulated \((NO^+)(VCl_4^-)\). The \(VCl_4^-\) anion contains vanadium (III); in this oxidation state the outer electronic state of the vanadium ion is \(d^2\), and it should give a characteristic d-d absorption spectrum.
The vanadium (III) ion has been obtained in tetrahedral co-ordination by substitution in a $C_5AlCl_4$ host lattice, in which the aluminium is assumed to be tetrahedrally surrounded by chlorine atoms (Gruen & Gut 1961). In addition, $(C_2H_5)_4N.VCl_4$ has been reported (Scaife, 1959) and its ultraviolet and visible spectrum is stated as being markedly different from octahedrally co-ordinated vanadium (III), but no details of band positions were given.
Calculations for a $d^2$ field in a tetrahedral environment predict two $^3T_2$ levels at 16,000 cm$^{-1}$ and 9,000 cm$^{-1}$ (625 $\mu\mu$ and 1,110 $\mu\mu$) and a $^3T_1$ level at 5,000 cm$^{-1}$ (2,000 $\mu\mu$); this is based on pure crystal field calculations assuming V$^{3+}$ and Cl$^-$ ions (Liehr & Ballhausen 1959).
Table 1 and the diagrams of spectra (Diagrams 1 → 4) indicate that wide discrepancies exist between theoretical data and the experimental data on the compounds cited. This is explained as being due to vanadium-chlorine covalent bonding, which will obviously affect the energy level system of the anion. (Gruen & Gut 1961). This explanation can also be used to account for the further discrepancies observed in the N$_2$.VCl$_4$ compound.
In the spectrum of $VCl_4^-$ in $NO.VCl_4$, the bands at 670 $\mu\mu$ and 960 $\mu\mu$ are very similar to the 667 $\mu\mu$ and 985 $\mu\mu$ bands of the $V^{3+}/CsAlCl_4$ system. In $NO.VCl_4$, the band at 870 $\mu\mu$ could be due to octahedral vanadium (III) species, brought about by hydrolysis.
The other bands, at 580 $\mu\mu$ and 420-430 $\mu\mu$ are probably characteristic of $VCl_4^-$ in $NO.VCl_4$.
Irregularities in the $VCl_4^-$ anion in $NO.VCl_4$ can be brought about in two ways:
1) Nitrosonium ion-chlorine interaction can distort the tetrahedral symmetry of the anion, and thus the energy levels of the anion will be affected.
2) Nitrosonium Ion-Vanadium Interaction.
If this occurred, a structure based partially on 5-co-ordination would be formed, and this would affect the energy levels of $VCl_4^-$ anion very much.
Despite these, it seems highly probable that $NO.VCl_4$ contains the $VCl_4^-$ anion, albeit non-tetrahedral due to interactions of the types 1) and 2) above.
Reaction of $NO.VCl_4$ with tetraethyl ammonium chloride gave blue crystals of $(C_2H_5)_4N.VCl_4$.
$$NO.VCl_4 + (C_2H_5)_4N.Cl \rightarrow (C_2H_5)_4N.VCl_4 + NOCl.$$
This has been prepared by Scaife (1959). From Table 1 and the spectra diagrams, $(C_2H_5)_4N.VCl_4$ bears more
resemblance to the $V^{3+}/CsAlCl_4$ than to $NO.VCl_4$. In particular the 580 $\mu\mu$ and 420-430 $\mu\mu$ are absent, indicating that they are characteristic of $NO.VCl_4$.
**$NO.V_2Cl_7$**
This compound was, like $NO.VCl_4$, isolated as a deep purple crystalline solid; it was extremely hygroscopic, and hydrolysed to give a green solution, vanadium (III).
It sublimed at a higher temperature than $NO.VCl_4$, i.e. at 60°-70°C. This is in accord with the observations of Whittaker & Yost, who reported that $NO.VCl_4$ was more volatile than $NO.V_2Cl_7$.
The infra-red spectrum was indicative of a free nitrosonium ion, with a strong band occurring at 2309 cm$^{-1}$, notably higher than in $NO.VCl_4$. The volatility still suggests considerable anion-cation interaction.
The compound can be formulated $(NO)^+(V_2Cl_7)^-$, with the anion consisting of two $VCl_4$ tetrahedra bridged by one chlorine atom.
$$\begin{array}{c}
Cl \\
Cl \quad V \quad Cl \\
Cl \quad Cl \quad V \quad Cl \\
Cl \quad Cl
\end{array}$$
It is unlikely that the V-Cl-V bond is linear. In fact, this bond angle will probably be much nearer the value for the tetrahedral angle. If the anion be looked upon as two VCl₃ units linked by a chloride ion, this latter will probably be sp³ hybridised, donating one pair of electrons to each of the VCl₃ units, leaving two lone pairs. On this basis, assuming absence of steric interaction between the VCl₃ groups, the V-Cl-V will be slightly less than the tetrahedral angle.
There is no spectroscopic evidence that, in the V₂Cl₇⁻ anion, vanadium is six-co-ordinate.
Some similarities should exist between the VCl₄⁻ and V₂Cl₇⁻ anions but important differences are likely due to the different symmetries. A comparison of the spectra of NO.VCl₄ and NO.V₂Cl₇ shows this.
The most important differences are -
1) The disappearance of the band C.670 mμ present both in NO.VCl₄ and the V³⁺/CSA1Cl₄ system.
2) The disappearance of the 420-430 mμ shoulder present in NO.VCl₄, and the appearance of a shoulder at 490 mμ.
3) The splitting of the 580 mμ band of NO.VCl₄ into two components in NO.V₂Cl₇ at 570 mμ and 620 mμ.
This may be an instance in which interaction between the two VCl₃ groups causes splitting of one of the transitions.
It is not sufficient to account for the differences in the spectra of NO.VCl₄ and NO.V₂Cl₇ in terms of the different symmetries of the two ions. As has been pointed out, the VCl₄⁻ anion in NO.VCl₄ probably undergoes considerable distortion due to anion-cation polarisation; this distortion will effect the transitions in the d-d spectrum. Anion-cation polarisation is probably very important in NO.V₂Cl₇ as its volatility would suggest; it is very unlikely that polarisation by the nitrosonium ion will affect the two anions equally. In particular, the greater size of the V₂Cl₇⁻ anion will alter the charge distribution throughout the lattice. This may be one of the causes of the large difference between the nitrosonium ion stretching frequencies in the two cases. Since the ratios NO⁺:Cl and NO⁺:V are different in both cases, these types of interaction are most unlikely to affect both anions equally.
The reactions between vanadium trichloride and nitrosyl chloride invariably yielded a small quantity of a pale green residue. This residue was involatile,
and hydrolysed with mild effervescence. The infra-red spectrum showed a strong band at 2326 cm\(^{-1}\) and rather broad bands at about 1100 cm\(^{-1}\) and 1000 cm\(^{-1}\). No other bands were present.
It seems reasonable to assign the 2326 cm\(^{-1}\) band to free nitrosenium ion; the bands at 1100 cm\(^{-1}\) and 1000 cm\(^{-1}\) may have been due to vanadium-oxygen double bonds. It has been shown that vanadium-oxygen bonding systems do absorb in this region (Barradough et al 1959).
Analysis suggests that the product is mainly NO.VOCl\(_4\). However, the nitrosyl chloride/vanadium oxytrichloride gives no evidence of compound formation, so that the existence of NO.VOCl\(_4\) is highly doubtful.
The compound is probably formed when the vanadium trichloride/nitrosyl chloride solid reaction product mixture is transferred in the dry-box, from the Carius tube to the sublimor. Although this operation was carried out as rapidly as possible it was inevitable that some contact with slightly moist air occurred and so hydrolysis could take place.
The infra-red spectroscopic data suggests very strongly that the VCl\(_4^-\) and V\(_2\)Cl\(_7^-\) anions are much more susceptible to hydrolysis than is the nitrosenium ion.
Whittaker & Yost (1949) isolated one further compound from the nitric oxide/vanadium tetrachloride reaction system. In the gas phase, the two compounds reacted to give $V_2Cl_8.(NO)_5$. No such compound was isolated in the vanadium trichloride/nitrosyl chloride reaction.
The difference between gas and liquid phase reactions involving nitrosyl chloride nitrogen dioxide has already been noted. In the liquid phase, these behave as ionising solvents
$$\text{NOCl} \rightleftharpoons \text{NO}^+ + \text{Cl}^-$$
$$2\text{NO}_2 \rightleftharpoons \text{N}_2\text{O}_4 \rightleftharpoons \text{NO}^+ + \text{NO}_3^-$$
whereas in the gas phase, they behave as molecular or radical-molecular species. However nitric oxide behaves both in gas and liquid phase reactions as a radical-molecule. Thus with the vanadium tetrachloride/nitric oxide system the species should be independent of the phase, but apparently the mechanism of reaction does depend on the phase; it is possible that the compound $V_2Cl_8(NO)_5$ contains $V \leftarrow NO^+$ linkages.
The extreme volatility of ionic compounds such as $NO.VCl_4$ and $NO.V_2Cl_7$ is obviously interesting, but it has been noticed previously, in the case of $(NO)_2TiCl_6$; this ionic compound sublimes readily at room temperature.
It is not essential that the species existing in gas and solid phases are identical, and it is possible that NO\textsubscript{2}VCl\textsubscript{4} sublimes as a five co-ordinate covalent species. This has been postulated to occur with the adduct Fe(NO\textsubscript{3})\textsubscript{3}·N\textsubscript{2}O\textsubscript{4}, which, it has been suggested, exists in the vapour phase as Fe(NO)(NO\textsubscript{3})\textsubscript{4} involving penta-co-ordinate iron similar to iron pentacarbonyl (Addison et al 1958).
It is unlikely that NO\textsubscript{2}VCl\textsubscript{4} sublimes with dissociation, though this could occur with (NO)\textsubscript{2}TiCl\textsubscript{6}
\[ \text{NO}_2\text{VCl}_4 \rightarrow \text{VCl}_3 + \text{NOCl} \quad A \]
\[ (\text{NO})_2\text{TiCl}_6 \rightarrow \text{TiCl}_4 + 2\text{NOCl} \quad B \]
In reaction \( A \), dissociation would lead to deposition of involatile vanadium trichloride. In \( B \), both titanium tetrachloride and nitrosyl chloride are volatile and dissociation could occur, with recombination on cooling. Hence, it seems probable that NO\textsubscript{2}VCl\textsubscript{4} and NO\textsubscript{2}V\textsubscript{2}Cl\textsubscript{7} do exist in the vapour phase as molecular species, though in the solid state they are definitely ionic, with some covalent character.
It is possible, that in reaction \( A \), NO\textsubscript{2}VCl\textsubscript{4} may dissociate to give VCl\textsubscript{3} monomer units, which might sublime, and recombine with nitrosyl chloride on condensing.
Normally, vanadium trichloride is polymeric at room temperature (Wells, 1962, p.345).
In this context, anhydrous cupric nitrate, $\text{Cu(NO}_3)_2$ sublimes at $150-200^\circ\text{C}$; the solid state infra-red spectrum indicates covalently bonded nitrato-ligands (Addison & Gatehouse 1958) and this was interpreted as suggesting the existence of $\text{Cu(NO}_3)_2$ monomer species in the solid state. However, a crystallographic examination of anhydrous cupric nitrate indicates that it can be formulated as a polymeric structure of the type:-
$$\text{Cu(NO}_3)_2 + \text{NO}_3^- \text{ (Wallwork 1959).}$$
The classic example of a volatile ionic compound is ammonium chloride. This exists in the solid state as $(\text{NH}_4^+)(\text{Cl}^-)$, and sublimes as $\text{NH}_3$ and $\text{HCl}$ molecular species:
$$\text{NH}_4^+ . \text{Cl}^- \rightarrow \text{NH}_3 + \text{HCl}.$$
Thus the volatility of a compound may not be too clear an indication of its structure in the solid state, since the possibility of structure change coincident with phase change has to be taken into account. In particular, high volatility of a solid cannot necessarily be assumed to be due to covalent, probably
monomeric species existing in the solid state.
It may be that an ionic compound will be volatile, if the ions are so situated in the crystal lattice to permit appreciable orbital overlap as the magnitude of thermal vibrations increase. Obviously, only under special circumstances will this kind of orbital overlap give rise to covalent bond formation, and it will require the presence of particularly suitable anions or cations. This is well illustrated by taking ammonium chloride as an example. When the magnitude of thermal excitation is low (i.e. at room temperature) there is little opportunity for H-Cl bond formation. However, as thermal excitation increases, the constituent ammonium and chloride ions will approach more closely, until orbital overlap between hydrogen and chloride species is sufficient to permit H-Cl bond formation.
However, it would be interesting to observe the gas phase spectra of these volatile nitrosonium salts, and, especially, the solid state spectra in the region of the sublimation temperature.
Both NO.VCl₄ and NO.V₂Cl₇ dissolve quite readily in nitrosyl chloride to give rise to solutions which are very deeply coloured reddish-brown. NO.VCl₄
and NO\textsubscript{2}V\textsubscript{2}Cl\textsubscript{7} will almost certainly be ionised in such solutions, and the VCl\textsubscript{4}\textsuperscript{-} and V\textsubscript{2}Cl\textsubscript{7}\textsuperscript{-} ions may give rise to VCl\textsubscript{5}\textsuperscript{2-} or higher species, or possibly more highly polymeric anions.
Vanadium Oxytrichloride/Nitrosyl Chloride.
No reaction appeared to take place between these components. There was no solid residue, and gas phase spectra indicated only unchanged nitrosyl chloride and vanadium oxytrichloride.
Vanadium Tetrachloride/Nitrosyl Chloride.
As with the previous system, no reaction took place between nitrosyl chloride and vanadium tetrachloride. Both starting materials were recovered unchanged.
Thus, it appears that nitrosyl chloride will not attack four-co-ordinate vanadium chloro-species; this may possibly be due to steric reasons though it does seem unlikely.
However, it is quite possible that chloride exchange occurs between the species in the VCl\textsubscript{4}/NOCl and VOCl\textsubscript{3}/NOCl systems, and this could be verified by tracer studies of the type carried out by Lewis & Sowerby (1957).
Vanadium Trichloride/Dinitrogen Tetroxide.
This reaction yielded a yellow-orange solid product $V\text{N}_3\text{O}_9$, which was originally considered to be vanadium trinitrate $V(\text{NO}_3)_3$ formed thus
$$\text{VCl}_3 + 3\text{N}_2\text{O}_4 \rightarrow V(\text{NO}_3)_3 + 3\text{NOCl}.$$
However, chemical evidence and magnetic evidence showed that the compound was definitely vanadium (V); it reacted with water to give an orange solution which could be easily reduced with sulphur dioxide, and it was observed to have zero-magnetic moment, indicating a d⁰ configuration.
The following series of reactions is proposed; to explain the $\text{VCl}_3/\text{N}_2\text{O}_4$ reaction:
1) Initial formation of $V(\text{NO}_3)_3$, as above; this compound could have a covalent structure of the type:
$$\begin{array}{c}
\text{ONO}_2 \\
\big| \\
\text{V} \\
\text{ONO}_2 \\
\text{O}_2\text{NO} \\
\end{array}$$
The nitrate-ligand is a potential oxidising agent, and the oxidation of vanadium (III) to vanadium (V) would be accompanied by V-O double bond formation which would facilitate the reaction:
2) /
2) \( \text{V(NO}_3)_3 \rightarrow \text{VO}_2\cdot\text{NO}_3 + \text{N}_2\text{O}_4 \)
The compound \( \text{VO}_2\cdot\text{NO}_3 \) had been prepared by Pantonin et al (1960) and has been shown to contain V-O double bonding, probably in the form of the \( \text{VO}_2^+ \) ion. Then adduct formation could occur:
\[ \text{VO}_2\text{NO}_3 + \text{N}_2\text{O}_4 \rightarrow \text{VO}_2\cdot\text{NO}_3\cdot\text{N}_2\text{O}_4 \]
This adduct is of the same empirical composition as \( \text{V(NO}_3)_3 \). The compound \( \text{VO}_2\text{NO}_3\cdot\text{N}_2\text{O}_4 \) is fairly hygroscopic, but quite easy to handle in the dry-box. The infra-red spectrum gave bands in the following regions:
| Wavenumber | Intensity |
|------------|-----------|
| 3360 w | 1014 vs |
| 2326 m | 793 s |
| 1601 m | 713 m |
| 1301 m | 660 w |
| 1256 s | |
| 1149 w | |
| 1081 w | |
(all numbers in cm\(^{-1}\))
s - strong, m - medium, w - weak
The 2326 cm\(^{-1}\) band was assigned to the free nitrosonium ion; on this basis, the compound was formulated \( (\text{NO})^+(\text{VO}_2(\text{NO}_3)_2)^- \), and the bands assigned thus:
| Wavenumber | Assignment |
|------------|---------------------|
| 3360 | water band |
| 2326 | \( \nu_1 \) nitrosonium ion |
| 1601 | \( \nu_4 \) nitrato-group |
| 1301 | \( \nu_1 \) nitrato-group |
| 1256 | \( \nu_1 \) nitrato-group |
1149
1081
1014 - V-0 stretching mode
793 - $V_6$ nitrato-group
713 $V_3$, $V_5$ nitrato-group
660
The 1301 cm$^{-1}$ band was originally assigned to nitrogen dioxide, present as a decomposition product. The symmetrical stretching mode of nitrogen dioxide absorbs at 1298 cm$^{-1}$, as stated in Chapter II. However, no asymmetric mode at about 1700 cm$^{-1}$ is present, so it is unlikely that the 1301 cm$^{-1}$ can be assigned to the dinitrogen dioxide molecule. It is more likely that the $V_1$ level is split; both the 1301 cm$^{-1}$ and 1250 cm$^{-1}$ bands were too intense to be overtone or combinational bands, and neither were they due to decomposition products.
The 1149 cm$^{-1}$ band could not be assigned; it was weak, and may have been an overtone or combinational band of low frequency modes. The 1081 cm$^{-1}$ band may have been an abnormally high nitrato-$V_2$ mode; this mode usually absorbs between 970 cm$^{-1}$ and 1034 cm$^{-1}$. However it is more likely that the very strong absorption centred about 1014 cm$^{-1}$, and assigned to
the V-O bond, contains the $V_2$ mode of the nitrato-group.
The 793 cm$^{-1}$ band falls with the region for the $V_6$ vibrational mode of the nitrato-ligand, and the lower bands at 713 cm$^{-1}$ and 660 cm$^{-1}$ may be due to the $V_3$ and $V_5$ modes of the same ligand.
Hence, the infrared spectrum of VO$_2$NO$_3$N$_2$O$_4$ can be interpreted satisfactorily in terms of a (NO)$^+(VO_2(NO_3)_2)^-$ structure, with the anion containing covalent nitrato-groups. The $V_4-V_1$ splitting of 300-350 cm$^{-1}$ means that this anion is highly covalent; there is no tendency for a dissociation of the type
\[(VO_2(NO_3)_2)^- \rightarrow VO_2NO_3 + NO_3^- \rightarrow VO_2^+ + 2NO_3^-\]
There is no infra-red evidence for nitrate ion. The absence of bands in the 1390 cm$^{-1}$ and 830 cm$^{-1}$ regions, corresponding to the $V_3$ and $V_4$ modes respectively, indicates that this ion is not present.
The uranyl nitrate-dinitrogen tetroxide adduct has been studied (Addison et al, 1964), and the infra-red spectrum was observed to give bands in the 2200 cm$^{-1}$ and 2300 cm$^{-1}$ regions. No chemical evidence was found to support a nitrosonium ion structure such as (NO)$^+(UO_2(NO_3)_3)^-$, although the anion $(UO_2(NO_3)_3)^-$ is known to occur in other compounds. The 2200 cm$^{-1}$ and 2300 cm$^{-1}$ bands were assigned as overtones or combinational modes.
on account of their weakness. However, Addison et al also observed that the dinitrogen tetroxide of the adduct $UO_2(NO_3)_2 \cdot N_2O_4$ was easily replaced by other ligands such as pyridine, and that electrolysis experiments demonstrated that the uranium was present in the cation.
It is possible that the $VO_2NO_3 \cdot N_2O_4$ adduct has a similar structure; however, the 2326 cm$^{-1}$ band in this adduct is too strong to be assigned to an overtone or combinational mode, and the only possible fundamental modes falling in this region are the nitrosonium $V_3$ mode and the nitrosonium $V_1$ mode. The former cannot be entirely ruled out, but the absence of a combinational band in the 3700 cm$^{-1}$ region suggests that nitronium ion is not present.
The anion $VO_2(NO_3)_2^-$ may have a tetrahedral structure, and be essentially monomeric
\[
\begin{array}{c}
\text{ONO}_2 \\
\text{O} \quad \text{V} \quad \text{O} \\
\text{ONO}_2
\end{array}
\]
or the vanadium could be octahedrally surrounded by oxygen-atoms, giving rise to a polymeric structure which could involve simple V-O-V bridges and vanadiumnitrato-vanadium bridges also:
If the structure were of this nature, the vanadium-oxygen band frequencies suggest that they are double bands, non-bridging, and so would occupy the axial positions of such a structure, in which case the equatorial positions would be occupied by nitrato-groups, viz:-
As has been mentioned, the compound $VO_2NO_3$ is known (Pantonin et al 1960); it was thought that this compound might be obtained from $VO_2NO_3 \cdot N_2O_4$ by heating:
$$VO_2NO_3 \cdot N_2O_4 \rightarrow VO_2 \cdot NO_3 + N_2O_4$$
The adduct $VO_2NO_3 \cdot N_2O_4$ was heated in vacuo up to $200^\circ C$ for 4-5 hours—the only solid product was vanadium pentoxide, though a mixture of nitrogen oxides, largely nitrogen dioxide, were evolved.
It has been reported that the reaction
\[ \text{UO}_2(\text{NO}_3)_2 \cdot \text{N}_2\text{O}_4 \rightarrow \text{UO}_2(\text{NO}_3)_2 + \text{N}_2\text{O}_4 \]
requires highly specific conditions (Addison et al 1964), and the same probably applies to the \( \text{VO}_2\text{NO}_3 \cdot \text{N}_2\text{O}_4 \) dissociation.
Vanadium Oxychloride/Dinitrogen Tetroxide.
This reaction was carried out in an attempt to prepare vanadyl oxytrinitrate, by the following type of reaction:
\[ \text{VOCl}_3 + 3\text{N}_2\text{O}_4 \rightarrow \text{VO}(\text{NO}_3)_3 + 3\text{NOCl}. \]
\( \text{VO}(\text{NO}_3)_3 \) has been prepared by the reaction of dinitrogen pentoxide with vanadium oxychloride or vanadium pentoxide
\[ \text{VOCl}_3 + 3\text{N}_2\text{O}_5 \rightarrow \text{VO}(\text{NO}_3)_3 + 3\text{NO}_2\text{Cl} \]
\[ \text{V}_2\text{O}_5 + 3\text{N}_2\text{O}_5 \rightarrow 2\text{VO}(\text{NO}_3)_3 \quad (\text{Schmeisser & Lutzow 1954}) \]
and is a pale golden liquid, boiling under reduced pressure at 28°-30°.
Evidence of reaction in the \( \text{VOCl}_3/\text{N}_2\text{O}_4 \) system came from examination of the gas phase spectra during the course of the reaction; the spectra indicated the presence of nitrosyl chloride.
After the reaction was complete, and the nitrosyl chloride and dinitrogen tetroxide removed as much as possible, a yellow-brown liquid remained, which was believed to be \( \text{VO}(\text{NO}_3)_3 \).
However, a gas phase spectrum showed only weak bands in the 1000-1050 cm\(^{-1}\) region, where vanadium-oxygen bands normally absorb. Strong bands in the 1700 cm\(^{-1}\) and 1300 cm\(^{-1}\) regions indicated nitrogen dioxide, and a very strong band was present at 1370 cm\(^{-1}\). These were thought to be due to the following series of reactions:
\[
\text{VOCl}_3 + 3\text{N}_2\text{O}_4 \rightarrow \text{VO(NO}_3)_3 + 3\text{NOCl}
\]
\[
\text{VO(NO}_3)_3 + \text{N}_2\text{O}_4 \rightarrow \text{VO(NO}_3)_3\cdot\text{N}_2\text{O}_4
\]
In the gas phase, the postulated adduct \(\text{VO(NO}_3)_3\cdot\text{N}_2\text{O}_4\) may dissociate to yield the reactants. Thus, bands due to nitrogen dioxide will be present in the gas phase.
Finally, reaction between the sodium chloride cell windows and the \(\text{VO(NO}_3)_3\) may occur:
\[
\text{VO(NO}_3)_3 + 3\text{NaCl} \rightarrow \text{VOCl}_3 + 3\text{NaNO}_3
\]
The strong band at 1370 cm\(^{-1}\) may be due to nitrate ion formed in sodium nitrate by the above reaction; however, if this were the case, gaseous VO(III) species would still be present as vanadium oxytrichloride, and hence infra-red absorptions in the 1000-1050 cm\(^{-1}\) mark would be expected.
The liquid was evaporated to dryness and a red solid remained, which was found to be \(\text{VO}_2\cdot\text{NO}_3\), as prepared by Pantonin et al (1960).
This was postulated to be formed thus:
\[ \text{VO(NO}_3)_3 \rightarrow \text{VO}_2\text{NO}_3 + \text{N}_2\text{O}_5 \]
or, alternatively, the yellow-brown liquid could have been a solution of VO\textsubscript{2}NO\textsubscript{3} in dinitrogen pentoxide, and evaporation to dryness merely removed the solvent. In either instance, this suggests that VO\textsubscript{2}NO\textsubscript{3} does not form a dinitrogen pentoxide adduct, i.e. the nitronium ion analogue to (NO)\textsuperscript{+}(VO\textsubscript{2}(NO\textsubscript{3})\textsubscript{2})\textsuperscript{-} does not exist.
**Vanadium Dibromide/Nitrosyl Bromide.**
These compounds react to give an ochre-yellow solid VBr\textsubscript{3}NO, which is hygroscopic and hydrolyses to give a vanadium (III) solution. It gives a very simple infra-red spectrum with bands at 1923 cm\textsuperscript{-1} and 1768 cm\textsuperscript{-1}; bands occurring lower in the infra-red region are compatible with reaction between Nujol, the mulling agent, and the nitrosyl group on the compound.
These bands at 1923 cm\textsuperscript{-1} and 1768 cm\textsuperscript{-1} are in the region characteristic of metal nitrosonium ion bonding; the presence of two bands in the region implies that the vanadium $\leftarrow$ nitrosonium ion system is in two different environments in the molecule. This immediately suggests a polymeric structure, since monomeric VBr\textsubscript{3}NO would have nitrosonium ion present in one environment only. Both the frequencies are well
above those recorded for bridging nitrosonium ions (Piper & Wilkinson 1956; King & Bisnette 1964) so the nitrosonium ions present must all be terminal.
Early attempts to carry out this reaction gave a yellow-brown product, or mixture of products; the main constituent of this was finally identified as the adduct $VO_2(NO_3)_2 \cdot N_2O_4$.
This product resulted from preparations involving nitrosyl bromide prepared by the $NO_2HSO_4/NaBr$ reaction; as explained in Chapter II, this method yielded appreciable quantities of nitrogen dioxide, giving dinitrogen tetroxide in a liquid phase reaction. Thus the nitrosyl bromide was appreciably contaminated with dinitrogen dioxide, and this was confirmed by a gas phase spectrum of the nitrosyl bromide used.
In this context, the systems uranium/NOCl and uranium/$N_2O_4$ have been reported in the literature (Addison & Hodge 1961 a), together with uranium/NOCl/$N_2O_4$ systems. It was found that this latter system yielded only $UO_2(NO_3)_2$, unless the nitrosyl chloride percentage composition was 85% or over; in addition, $UO_2(NO_3)_2 \cdot N_2O_4$ was still one of the products of reaction until the nitrosyl chloride concentration rose above 98%. Hence, if slightly impure nitrosyl chloride gives
rise to nitrates or dinitrogen tetroxide adducts, it is reasonable to assume that NOBr/N₂O₄ mixtures will behave in the same way. This seems the most valid explanation of the formation of VO₂NO₃·N₂O₄; the reaction VBr₂/NOBr occurs less readily than the reaction VBr₂/N₂O₄, again, probably due to vanadium-oxygen double bond formation supplying the free energy for reaction.
Vanadium Pentoxide/Nitrosyl Chloride.
Under specific conditions, the nature of which were not elucidated, vanadium pentoxide will react with nitrosyl chloride, over a period of 3-5 hours to give a very deep blue solid. This reaction proceeded as stated, on the first attempt, but later attempts, even on heating, gave no reaction at all, although the nitrosyl chloride solution became very faintly green due, presumably, to slight solution of vanadium pentoxide. It is possible that the reaction was brought about catalytically by small quantities of adsorbed species on the inside of the Carius tube.
The blue product was not obtained in sufficient yield for analysis; however, infra-red analysis gave a very simple spectrum:
\[
\begin{align*}
2300-2350 & \quad \text{cm}^{-1} \quad \text{m.} \\
1800 & \quad \text{cm}^{-1} \quad \text{w.} \\
900-920 & \quad \text{cm}^{-1} \quad \text{s.}
\end{align*}
\]
Time dependence spectra showed the 1800 cm$^{-1}$ band to be a decomposition band, presumably due to nitrosyl chloride. The 900-920 cm$^{-1}$ band, a broad absorption, is assigned to vanadium-oxygen banding, though it is markedly lower than the analogous mode in VO$_2$NO$_3$·N$_2$O$_4$; this may be because the vanadium is in a lower oxidation state in the V$_2$O$_5$/NOCl reaction product.
There is no doubt that reduction has occurred; the intense colour could be that associated with the vanadyl ion in compounds such as vanadyl sulphate. Alternatively it could be due to charge transfer occurring between different oxidation states of vanadium in the same ion.
**Niobium.**
Niobium is known in a less wide range of oxidation states than is vanadium. The +3 and +5 are the best characterised with niobium (III) being reducing. Vanadium (III) shows no such tendency. Thus, all the compounds dealt with in this section are compounds of niobium (V).
The compound NO.NbF$_6$ is known; it was prepared by reaction of niobium pentoxide and nitrosyl chloride in bromine trifluoride (Clark & Emeleus 1958).
In the present work, this reaction was repeated but using niobium pentachloride instead of the pentoxide. The compound was the same; it was very hygroscopic, and the presence of an infra-red absorption band at 2390 cm\(^{-1}\) confirms the structure as \((\text{NO})^+(\text{NbF}_6)^-\).
There are no examples of the anion \(\text{NbCl}_6^-\) reported in the literature; hence the reaction between nitrosyl chloride and niobium pentachloride was carried out. A pale yellow solid was isolated which gave an infra-red absorption at 2174 cm\(^{-1}\); the intensity of this band diminished very rapidly with time and it was observed that the reaction product did dissociate very rapidly. Thus the reaction
\[
\text{NOCl} + \text{NbCl}_5 \rightarrow \text{NO}^+.\text{NbCl}_6^-
\]
was proposed, with the high nitrosonium ion - chlorine interaction giving rise to the ease of dissociation, and the low frequency free nitrosonium ion stretching frequency.
The high instability of \(\text{NO}.\text{NbCl}_6\) suggests that the niobium (V) ion is not large enough to accommodate six chlorine atoms distributed hexagonally around it. Niobium pentachloride consists of monomeric \(\text{NbCl}_5\) units,
* This was later found to be incorrect: see Adams, Chatt, Davidson & Garratt, J. Chem. Soc., 1963, 2189. The author thanks Dr. K.W. Bagnall for pointing out this error.
with the five chlorine atoms arranged in a trigonal bipyramid about the niobium atom (Skinner & Sutton 1940).
The reactions involving niobium pentachloride with dinitrogen tetroxide (Dagnall et al 1964) and dinitrogen pentoxide (Field & Hardy 1963 a) are reported in the literature.
The latter reaction proceeds readily to yield the compound, niobium oxytrinitrate $\text{NbO(NO}_3)_3$.
$$\text{NbCl}_5 + 4\text{N}_2\text{O}_5 \rightarrow \text{NbO(NO}_3)_3 + 5\text{NO}_2\text{Cl}$$
No tendency for $\text{NbO(NO}_3)_3\cdot\text{N}_2\text{O}_5$ or $\text{NbCl}_5\cdot\text{NO}_2\text{Cl}$ adduct formation was observed. Infra-red examination of the compound showed that covalent nitrato-ligands are present, and that niobium-oxygen double bonds exist also. The compound $\text{NbO(NO}_3)_3$ starts to decompose when heated in vacuo up to $120^\circ\text{C}$; Field & Hardy postulated it as containing niobium-oxygen double bonds and Nb-O-Nb bridging groups; those bridging groups may be present as bridging nitrato-groups, viz:-
$$\begin{array}{c}
\text{O} \\
\text{N} \\
\text{O} \\
\text{Nb} & \text{Nb}
\end{array}$$
Niobium pentachloride will only react with dinitrogen tetroxide in the presence of ionising solvents, to give solvated $NbO_2NO_3$ species (Bagnall et al 1964). Assuming that the reactions in both cases involve attack by nitrate ion, it appears that the concentration of nitrate ion provided by dinitrogen tetroxide auto-ionisation:
$$N_2O_4 \rightleftharpoons NO^+ + NO_3^-$$
is unsufficient to cause formation of niobium nitrate or nitrato-species. In the presence of ionising solvents such as acetonitrile a species of the empirical formula $NbO_2NO_3\cdot0.67MeCN$ is formed, which in moist air picks up water to give $NbO_2NO_3\cdot0.67MeCN\cdot0.3OH_2O$. For $NbO_2NO_3\cdot0.67MeCN$, the formula:
\[
\begin{array}{c}
O \\
O \quad Nb \quad O \quad Nb \quad O \quad Nb \quad O \\
O \quad O \quad O \quad O \quad O \quad O \\
MeCN \quad MeCN
\end{array}
\]
is suggested (Bagnall et al 1964). In the presence of moist air, a water molecule bonds to the central niobium atom.
Tantalum.
In the present context, the chemistry of tantalum is very similar to niobium. It forms a nitrosonium fluoro-salt NO.TaF6 which was first prepared by Clark & Emeleus (1958); in the present work, the infra-red spectrum of nitrosonium fluorotantalate was shown to give a strong band at 2391 cm\(^{-1}\), confirming the structure as \((NO)^+(TaF_6)^-\).
The reaction between nitrosyl chloride and tantalum pentachloride gives NO.TaCl6.
\[ \text{NOCl} + \text{TaCl}_5 \rightarrow \text{NO.TaCl}_6 \]
Infra-red examination of the complex gave an absorption band at 2165 cm\(^{-1}\), which is at the lower end of the scale for nitrosonium ion frequencies. Unlike the niobium analogue, it is fairly stable to dissociation; although the slightly lower nitrosonium ion frequency implies a greater degree of anion-cation interaction than with NO.NbCl6. The enhanced stability of NO.TaCl6 cannot be explained in terms of ionic radii of the niobium (V) and tantalum (V) species, since the latter would be expected to be rather smaller and so would be less able to accommodate chlorine atoms octahedrally about it. However, tantalum has extra
orbitals available which may be able to accommodate the chlorine atoms.
Metathetical reaction of tetraethyl ammonium chloride and NO\textsubscript{2}TaCl\textsubscript{6} in liquid sulphur dioxide gave beautiful white needle-like crystals of (C\textsubscript{2}H\textsubscript{5})\textsubscript{4}N.TaCl\textsubscript{6}.
(C\textsubscript{2}H\textsubscript{5})\textsubscript{4}N.TaCl\textsubscript{6} + NOCl → (C\textsubscript{2}H\textsubscript{5})\textsubscript{4}N.TaCl\textsubscript{6} + NOCl.
The infra-red spectrum confirmed the presence of (C\textsubscript{2}H\textsubscript{5})\textsubscript{4}N\textsuperscript{+} ion, and hence verified the presence of the previously unknown TaCl\textsubscript{6}\textsuperscript{-} anion.
Bagnall et al (1964) carried out the reactions of tantalum pentachloride with dinitrogen tetroxide and dinitrogen pentoxide; both reactions were analogous to the niobium reactions
TaCl\textsubscript{5} + 4N\textsubscript{2}O\textsubscript{5} → TaO(NO\textsubscript{3})\textsubscript{3} + 5NO\textsubscript{2}Cl.
Tantalum oxytrinitrate, like its niobium analogue, contains covalent nitrato-groups which may be present as bridging groups. It has less thermal stability than niobium oxytrinitrate which is postulated as being due to weaker bridging nitrato-groups.
Reaction of tantalum pentachloride with dinitrogen tetroxide proceeds only in the presence of ionising solvents, and the species produced are solvated TaO\textsubscript{2}NO\textsubscript{3}.
species; these presumably have analogous structures to those proposed for the niobium analogues.
It is notable that in the niobium and tantalum pentachloride reactions with dinitrogen tetroxide do not yield nitrosonium ion species; this may be because in acetonitrile solution, the nitrosonium ion is solvated and is less easy to fit in a crystal lattice.
EXPERIMENTAL
1. Vanadium Trichloride.
Vanadyl dichloride hydrate was prepared by gently boiling vanadium pentoxide in concentrated hydrochloric acid for 40-50 minutes (Sidgwick (1950, p.820)) then evaporating to small bulk. After cooling, methanol was added to the deep blue solution and evaporation to small bulk repeated. On repeating this procedure 2-3 times, vanadyl chloride hydrate eventually separated out on cooling as a very dark blue mass with little evidence of crystallinity.
Calculated for VOCl₂·5H₂O Cl:30.2% Found Cl:28.7%.
The rather low chlorine value indicates that methanol may be co-ordinated on to the vanadyl ion also.
About 5-6 gms. of the hydrate, or mixed methanolate-hydrate, were refluxed with 50 mls. thionyl chloride for 1½-2 hours; the bulk of the thionyl chloride was distilled off, and a further 25-30 mls. fresh thionyl chloride added, and the mixture refluxed for another 40-60 minutes. A purple residue remained, and the thionyl chloride was coloured orange-brown, which may well have been due to dissolved vanadium oxytrichloride. The bulk of the thionyl chloride distilled off readily at
75°-80°, but a gummy residue remained. Heating up to 150°C would not remove this effectively so it was decided that the residual liquid would probably be sulphuric acid since it gave a positive test with barium chloride solution; this could be formed either by hydrolysis of sulphur trioxide or sulphuryl chloride
\[ \text{SO}_3 + \text{H}_2\text{O} \rightarrow \text{H}_2\text{SO}_4 \]
\[ \text{SO}_2\text{Cl}_2 + 2\text{H}_2\text{O} \rightarrow \text{H}_2\text{SO}_4 + 2\text{HCl}. \]
Red-brown vapours at these most elevated temperatures indicated the presence of vanadium oxytrichloride. The solid was then filtered as dry as possible by suction, washed twice with thionyl chloride, dried by suction, and then finally washed 3 times with dry carbon disulphide, and dried by suction again. Removal of all traces of residual acid was ensured by storing the purple product in vacuo over sodium hydroxide for 18-24 hours. (Yield = 3.5-4 gms. vanadium trichloride - about 90% based on a VOCl₂·5H₂O → VCl₃ conversion).
Analysis:
Found V: 32.8% Cl: 67.3% VCl₃ requires V: 32.8% Cl: 67.2%
Vanadium Trichloride/Nitrosyl Chloride.
1-1.5 gms. vanadium trichloride, prepared as above, was transferred to a Carius tube in a dry box to avoid hydrolysis. This was evacuated and about a threefold excess of nitrosyl chloride condensed on to the vanadium trichloride by vacuum distillation. The tube was sealed and the reaction mixture allowed to rise to room temperature. A very deep red-brown solution resulted, after vigorous reaction between the two components. On evaporation of the residual liquid by vacuum distillation a purple solid remained which was observed to sublime very early into the cold trap. To avoid too great a loss of the product, the residual nitrosyl chloride was removed at c.-20°C, cooling the Carius tube in a carbon-chloride slush bath. Still under vacuum, the Carius tube was transferred to a dry-box, and the solid transferred as rapidly as possible to sublimer and re-evacuated once more. The purple solid sublimed readily onto a probe cooled to -78°C with solid carbon dioxide; sublimation of one fraction appeared to be complete at 30°-35°C and a small purple residue still remained. After removal of the most volatile fraction, the residue was heated and found to sublime at 66°-70°. A very small green residue still
remained but this could not be sublimed further.
The purple fraction subliming at 30°-35° analysed as NO.VCl₄.
Found: N:6.0% Cl:62.4% NO.VCl₄ requires N:6.2% Cl:62.6%.
The purple fraction subliming at 66°-70° analysed as NO.V₂Cl₇.
Found: N:3.2% Cl:63.5% NO.V₂Cl₇ requires N:3.6% Cl:63.8%.
The infra-red spectrum of NO.VCl₄ was found to give a strong absorption at 2179 cm⁻¹; that of NO.V₂Cl₇ gave a strong absorption of 2309 cm⁻¹.
In the above reaction the main product was NO.VCl₄ with only a little NO.V₂Cl₇.
When, instead of using a threefold excess of nitrosyl chloride for the reaction, the mole ratio was made approximately unity, the reaction behaved slightly differently. After removal of excess solvent and transfer of the reaction mixture to a sublimer, sublimation up to 35°C gave only a very tiny yield of the product NO.VCl₄. The bulk of the reaction mixture sublimed at 66°-70°, again leaving a small quantity of a green residue. In this instance the analyses were -
Fraction 1) 30°-35°
Found: N:6.0% Cl:62.8% NO.VCl₄ requires N:6.2% Cl:62.6%.
Fraction 2) $66^\circ-70^\circ$
Found N:3.5% Cl:63.6% NO.V$_2$Cl$_7$ requires N:3.6% Cl:63.8%.
Fraction 1) gave a strong infra-red absorption at 2178 cm$^{-1}$; fraction 2) gave a strong infra-red absorption at 2312 cm$^{-1}$. Thus the two products are the same in both instances.
The green residue was observed to dissolve in water with mild effervescence; analyses were
1) N:5.1% Cl:58.2%.
2) N:5.7% Cl:55.9%.
NO.VOCl$_4$ requires N:5.9% Cl:59.4%.
The infra-red spectrum of the green residue gave a sharp band at 2325 cm$^{-1}$, and further broad bands in the 1100 cm$^{-1}$ and 1000 cm$^{-1}$ regions.
Tetraethyl ammonium chlorovanadate.
0.46 gms (0.002 moles) NO.VCl$_4$ and 0.33 gms (0.002 moles) $(C_2H_5)_4N\cdot Cl$ were put in a Carius tube. This operation was carried out in the dry-box as rapidly as possible to avoid hydrolysis of NO.VCl$_4$. It was transferred to the vacuum system and evacuated. Sulphur dioxide, from a cylinder, was dried by vacuum distillation over phosphorus pentoxide, and then transferred similarly into the Carius tube. The tube was sealed, and the
reaction mixture raised to room temperature. The reaction was shaken for 24 hours after which time the sulphur dioxide had turned brown due to the formation of nitrosyl chloride.
\[(C_2H_5)_4N.Cl + NO.VCl_4 \rightarrow (C_2H_5)_4N.VCl_4 + NOCl\]
The sulphur dioxide and nitrosyl chloride were removed in vacuo, leaving blue \((C_2H_5)_4N.VCl_4\). This was washed with liquid sulphur dioxide in a Carius tube to remove any traces of the reactants that may have been left.
**Analysis:**
- \(C: 28.1\% \quad H: 5.5\% \quad Cl: 42.1\% \quad N: 3.8\%\)
- \((C_2H_5)_4N.VCl_4\) requires \(C: 28.9\% \quad H: 6.0\% \quad Cl: 42.8\% \quad N: 4.2\%\).
**Vanadium tetrachloride/Nitrosyl chloride.**
Vanadium tetrachloride was prepared by heating vanadium trichloride in vacuo
\[2VCl_3 \rightarrow VCl_4 + VCl_2\]
The volatile vanadium tetrachloride fraction was condensed in a cold trap \((-184^\circ C)\) as a red solid. By vacuum distillation, twice, over phosphorous pentoxide it was thoroughly dried and then transferred similarly into a Carius tube. Nitrosyl chloride was vacuum-distilled separately into the same Carius tube and the tube sealed. After raising to room temperature, the
reaction mixture was frozen down and the tube opened and evacuated. No solid product was formed.
The gas mixture was analysed by infra-red spectroscopy and the spectrum was identical to that of nitrosyl chloride. A cold trap at c.-20°C condensed out a red liquid which was vanadium tetrachloride; a further cold trap at -75°C condensed out nitrosyl chloride.
There was no evidence for any reaction.
Vanadium Oxytrichloride.
This can be prepared by heating a mixture of vanadium pentoxide and aluminium trichloride at 400°C. (Inorganic Syntheses Vol. 6, p. 119).
\[ \text{V}_2\text{O}_5 + 2\text{AlCl}_3 \rightarrow 2\text{VOCl}_3 + \text{Al}_2\text{O}_3 \]
The volatile vanadium oxytrichloride distils off.
The present method is a very slight modification of this in that the reaction was carried out in vacuo and thus proceeded readily at a much lower temperature (160° - 180°C). Anhydrous aluminium trichloride was sublimed in vacuo to rid it of moisture traces. It was then intimately ground up with an excess of vanadium pentoxide, previously heated to 300°-400° for 2-3 hours. The mixture was then
evacuated, and the temperature raised slowly to 160°. If the temperature increase was made too rapid it was found that instead of reaction occurring to give vanadium oxytrichloride, aluminium trichloride sublimed out of the reaction mixture. Early trials showed that aluminium trichloride always tended to sublime out, to some extent, and collect in the cold trap used for condensing vanadium oxytrichloride out. To eliminate this undesirable effect, the tube between the reaction flask and the cold trap was partially packed with dried glass wool; this measure was successful. After vanadium oxytrichloride stopped distilling over, it was redistilled twice over phosphorus pentoxide in vacuo to dry it and used for reactions. Vanadium oxytrichloride so obtained is a yellow liquid, freezing to a yellow solid, and very hygroscopic. If any traces of moisture are present during the preparation the product has a distinct reddish colouration.
Analysis:
Found V: 29.3% Cl: 61.2% VOCl₃ requires V: 29.4% Cl: 61.4%.
Vanadium Oxytrichloride/Nitrosyl Chloride.
Vanadium oxytrichloride, prepared and dried as above, was transferred by vacuum-distillation into a
Carius tube, and nitrosyl chloride transferred similarly into the same Carius tube. The tube was sealed and raised to room temperature. After cooling and re-opening, the tube was evacuated. No solid residue remained. A gas phase infra-red spectrum of the volatile material showed bands in the 1798-1810 cm\(^{-1}\) region, a band in the 1030 cm\(^{-1}\) region and a triplet centred about 760 cm\(^{-1}\). A cold trap at -20°C condensed out vanadium oxytrichloride, and one at -78°C condensed out nitrosyl chloride. There was no evidence for any reaction.
**Vanadium Trichloride/Dinitrogen Tetroxide.**
Vanadium trichloride was prepared as described earlier in this section. Dinitrogen tetroxide was prepared as in Chapter II. 2-3 gms. of vanadium trichloride were placed in a reaction vessel (50 mls. approximately), which was evacuated. An excess of dinitrogen tetroxide was condensed on to the trichloride in vacuo at -184°C, and the reaction allowed slowly to warm. As soon as the dinitrogen tetroxide melted, reaction was observed between the components. The reaction was allowed to proceed slowly and samples of gas were taken off for infra-red analysis during the reaction. The residual solid product was a pale yellow
brown powder $VN_3O_9$.
Analysis:
Found V:24.5% N:20.9% $VN_3O_9$ requires V:24.6% N:20.3%.
This product was heated in vacuo to 200°C. A mixture of nitrogen oxides condensed in a cold trap and the residue was vanadium pentoxide.
Analysis:
Found V:54.8% $V_2O_5$ requires V:55.5%.
Vanadium Oxytrichloride/Dinitrogen Tetroxide.
Vanadium oxytrichloride and dinitrogen tetroxide were condensed separately into a small reaction flask, by vacuum distillation. The reaction mixture was allowed to warm up slowly and reaction appeared to take place when the components melted. Samples of the gas products were taken off from time to time. Excess of the highly volatile residue were removed in vacuo, keeping the reaction mixture at -20°C to avoid loss of volatile fractions in the reaction mixture. A yellow-brown liquid remained. Gas phase infra-red spectra showed no evidence of V=O linkages so it was evacuated to dryness and a red-brown solid obtained, analysing as $VO_2NO_3$.
Found: $NO_3^-$:35.0% V:42.3% $VO_2NO_3$ requires V:35.2%
$NO_3^-$:42.8%.
Vanadium Bromide VBr₂.
A stream of bromine, diluted with nitrogen, was bubbled through concentrated sulphuric acid to dry it. It was then passed slowly over metallic vanadium heated to 300°-400°. Reaction occurred immediately and a mixture of vanadium bromides VBr₃ and VBr₂ sublimed out of the reaction mixture. Heating was continued for a reasonable length along the reaction tube to ensure complete decomposition of vanadium tribromide to the black dibromide. The latter condensed out on the cooler parts of the reaction tube. On completion of reaction the tube was swept out with dry nitrogen and transferred to the dry-box.
For preparation of the red vanadium tribromide the bromine was heated to about 200° before passing over the vanadium. The product sublimed out as red scaly plates.
Analyses
Black V:28.2 Br:71.9 VBr₂ requires V:28.6% Br:71.4%.
Red V:18.0% Br:82.1% VBr₃ requires V:17.3% Br:82.7%
Vanadium Dibromide VBr₂/Nitrosyl Bromide.
Vanadium dibromide was prepared as above, and nitrosyl bromide as in Chapter II. The latter was dried by
vacuum distillation over phosphorus pentoxide and transferred similarly into a Carius tube containing vanadium dibromide. The tube was sealed and the reaction raised to room temperature. On removal of excess nitrosyl bromide by evacuation, an ochre-yellow solid remained. It analysed fairly well as VBr₃NO.
Found V: 20.0% N: 3.9% Br: 75.0%
VBr₃NO requires V: 20.8% N: 4.4% Br: 74.7%.
It was quite susceptible to moisture, and its infra-red spectrum changed significantly with time.
Vanadium Pentoxide/Nitrosyl Chloride.
Vanadium pentoxide was rigorously dried at 300-400°C and put in a Carius tube. This was evacuated, and a two-three fold excess of nitrosyl chloride was transferred into the tube by vacuum distillation. The tube was sealed and allowed to come up to room temperature. On several occasions when this reaction was attempted, no reaction occurred and the starting materials were recovered intact. On one occasion, however, removal of excess nitrosyl chloride left a small quantity of very deep blue compound. Unfortunately insufficient was available for analysis. Its infra-red spectrum was
observed. It hydrolysed vigorously with evolution of nitrogen dioxide.
The specific conditions which were responsible for the formation of the deep blue solid were not elucidated.
Vanadium Pentoxide/Dinitrogen Tetroxide.
Rigorously dried vanadium pentoxide was put in a Carius tube, evacuated, and dinitrogen tetroxide vacuum distilled into the same tube. The tube was sealed and the reaction mixture raised to room temperature. The dinitrogen tetroxide solution was very pale green, but little vanadium pentoxide had dissolved. When the tube was evacuated, the gaseous products were examined by infra-red spectroscopy.
The vanadium pentoxide was recovered unchanged.
Niobium Pentachloride/Nitrosyl Chloride.
Nitrosyl chloride was transferred by vacuum distillation into a Carius tube containing niobium pentachloride (Stauffer Chemical Coy.), and the tube sealed. The reaction mixture was raised to room temperature. On careful evacuation of the volatile products a pale yellow solid remained. Its infra-red
spectrum was measured.
Analysis:
N: 3.7% Cl: 64.4% NO.NbCl₆ requires N: 4.2% Cl: 63.4%.
The wide discrepancy will be due to the ease with which the compound NO.NbCl₄ dissociates.
Tantalum Pentachloride/Nitrosyl Chloride.
Nitrosyl chloride was transferred by vacuum distillation into a Carius tube containing tantalum pentachloride (Stauffer Chemical Coy.). The tube was sealed and the reaction allowed to rise to room temperature. After cooling, the excess nitrosyl chloride was removed in vacuo. A white solid remained, fairly stable to hydrolysis.
Analysis:
N: 3.1% Cl: 49.7% NOTaCl₆ requires N: 3.3% Cl: 50.1%
Nitrosonium Fluoniobiate.
This was prepared by Woolf's method (1950). Nitrosyl chloride was transferred by vacuum distillation into a Silica reaction flask containing niobium pentachloride. The flask was removed from the vacuum system after the admission of dry air, and then bromine trifluoride was added dropwise to the mixture cooled to -184°C. After an excess of bromine trifluoride had
been added, the reaction mixture was allowed to warm slowly to room temperature. Reaction occurred vigorously, and after completion of reaction, the volatile products were removed in vacuo at 80°-90°. A white solid remained.
Analysis:
N: 5.5% F: 47.2% NO.NbF₆ requires N: 5.9% F: 48.1%.
Nitrosonium Fluotantalate.
Nitrosyl chloride was transferred by vacuum distillation into a silica reaction flask containing tantalum pentachloride. Dry air was admitted into the vacuum system and the flask removed. The reaction mixture was cooled to -184°C and bromine trifluoride added dropwise from a stainless steel capsule. After an excess had been added, the mixture was allowed to warm slowly to room temperature. After the reaction had ceased, the volatile products were removed in vacuo at 80°-90°. A white solid remained.
Analysis:
N: 4.1% F: 34.2% NO.TaF₆ requires N: 4.3% F: 35.1%.
Tetraethylammonium chlorotantalate.
0.88 gm. (0.002 moles) NO.TaCl₆ and 0.33 gm. (0.002 moles)(C₂H₅)₄N.Cl were placed in a Carius tube; this was then evacuated and sulphur dioxide, dried by
distillation over phosphorus pentoxide, was vacuum distilled into the tube, which was then sealed. After coming up to room temperature the reaction mixture was shaken for 24 hours after which time beautiful long white needles crystallised out.
\[(C_2H_5)_4N.TaCl_6 \rightarrow (C_2H_5)_4N.TaCl_6 + NOCl\]
The excess sulphur dioxide and nitrosyl chloride were removed in vacuo; the crystals of \((C_2H_5)_4N.TaCl_6\) were washed with liquid sulphur dioxide in a Carius tube before analysis.
Analysis Found
- \(C: 17.0\% \quad H: 3.4\% \quad Cl: 41.2\%\)
\((C_2H_5)_4N.TaCl_6\) requires
- \(C: 17.3\% \quad H: 3.8\% \quad Cl: 40.6\%\)
Reflectance spectra were taken of \(NO.VCl_4\) and \(NO.V_2Cl_7\). The compounds were diluted with rigorously dried potassium chloride and the mixtures finely ground and the reflectance cell holder packed with the mixture. This operation had to be completed rapidly to avoid excessive hydrolysis. Hydrolysis could be observed by the appearance of a distinct green colouration in the mixture.
Reflectance spectra were run on a Milger-Watts Uvispek Photoelectric Spectrophotometer Model H700 307, between the limits 200 \(\mu m\) and 1000 \(\mu m\).
Infra-red spectra were run on a Grubb Parsons DB1 Spectrophotometer. In the cases of NO.VCl₄, NO.V₂Cl₇, NO.NbCl₆, NO.NbF₆, NO.TaCl₆ and NO.TaF₆ the observed nitrosonium ion bands were calibrated against a polystyrene spectrum.
In instances where reaction with a nujol mull was not rapid, nujol was used as the mulling agent. In all other cases, and invariably for calibrated peaks, florube was used.
Reflectance Spectra of Vanadium (III) Complexes
\[ V^{3+}/CsAlCl_4 \quad (\text{Gruen & Gut, 1961}) \]
\[ A - \text{NO}_2V_2Cl_7 \]
\[ B - \text{NO}_2VCl_4 \]
\[(C_2H_5)_4N.VCl_4\]
| Calculated Values for tetrahedral d² system. (Dq = -500 cm⁻¹) | v³⁺ in CSA1Cl₄ host lattice | NO.VCl₄ |
|-------------------------------------------------------------|---------------------------------|---------|
| 625 m | 3T₁ | 667 m | 420-430 m (sh.) |
| 1110 m | | 985 m | 580 m |
| 2000 m | 3T₂ | 1064 m | 670 m |
| | | 1212 m | 870 m |
| | | | 960 m |
(C₂H₅)₄N VCl₄
| 675 m | 940 m |
|-------|-------|
| 870 m | 865 m |
| 955 m | 750 m |
| | 620 m |
| | 570 m |
| | 490 m (Sh.) |
CHAPTER VI
CHROMIUM AND MOLYBDENUM
Although a number of compounds containing chromium or molybdenum - nitric oxide bonds are known, very little is known of the behaviour of compounds of molybdenum and chromium in nitrating and nitrosylating solvents.
Chromium-nitrosonium ion bonds have been characterised by infrared spectroscopy in a series of covalent compounds of the general formula \((\text{NO})_2\text{Cr}(\pi-\text{C}_5\text{H}_5)_x\) where \(X = -\text{Cl} - \text{Br} - \text{SCN} - \text{CH}_3 - \text{C}_2\text{H}_5 - \text{CH}_2\text{Cl}\) - \text{C}_5\text{H}_5 - \text{C}_6\text{H}_5\) (Lewis et al 1958). The same diagnostic method also characterised the Cr-Mo\(^+\) bond in the complex anion \([\text{Cr(CN)}_5\text{NO}]^{3-}\) (Wieber & Jahn 1958).
The complex cation \([\text{Cr(H}_2\text{O)}_5\text{NO}]^+\) is postulated as containing Cr\(^{2+}\)-NO\(^-\) linkages (Ardon & Herman 1962).
Terminal and bridging nitrosonium ion ligands are believed to be present in the binuclear complex:
\[
\begin{array}{c}
\text{O} \\
\text{N} \\
\text{Cr} \\
\text{N} \\
\text{O} \\
\text{Cr} \\
\text{N} \\
\text{O} \\
\text{NO} \\
\end{array}
\]
(King & Bisnette 1964)
Infrared spectroscopy has been used to characterise the molybdenum - nitrosonium ion bond in the covalent species \((\text{NO})_2\text{Mo(CO)}_2(\pi\text{C}_5\text{H}_5)\), (Lewis et al 1958) and in the anionic species \(\text{Mo(CN)}_6^3\text{NO}\) (Heber & Jahn 1958).
Nitrosonium salts of fluoro-acids of molybdenum, tungsten and uranium have been prepared (Geichmann et al 1962, 1963; Seel & Birnkraut 1962) and have been discussed in chapter I. Seel & Birnkraut prepared \(\text{NO}.\text{MoF}_6\), \(\text{NO}.\text{WF}_7\) and \(\text{NO}.\text{UF}_6\) using the metallic elements and the reagent \(\text{NOF(HF)}_3\); attempts, using the same reagent, to obtain nitrosonium fluorochromates were unsuccessful, the only products being chromium(III)fluoride.
In the present work, the behaviour of chromic and chromous chlorides with nitrosyl chloride and dinitrogen tetroxide was observed, and this was partially extended to chromium oxides and oxychlorides. Molybdenum was covered in an analogous manner.
**REACTIONS OF NITROSYL CHLORIDE WITH CHROMOUS AND CHROMIC CHLORIDE.**
Chromic chloride will not react with nitrosyl chloride, even after shaking for 2 - 3 weeks, or on heating up to \(100^\circ\text{C}\); chromous chloride reacts almost instantaneously with liquid nitrosyl chloride to give a
yellow product \((\text{NO})_3\text{CH}_2\text{Cl}_9\), which contains only Chromium (III).
When a trace (i.e., c. 1%) of chromous chloride is added to chromic chloride the resultant mixture will react very readily with liquid nitrosyl chloride to give the same product \((\text{NO})_3\text{Cr}_2\text{Cl}_9\).
This ability of chromous chloride to activate reactions involving chromic chloride is well known; the commonest example of this being the behaviour of chromic chloride in water. In the absence of chromous chloride, chromic chloride is highly insoluble in water, but in the presence of a trace of the chromous salt (Sidgwick (1950) quotes the ratio 1 part in 40,000), chromic chloride dissolves readily and crystallises out as the hexahydrate. The ability of other reducing agents, such as ferrous and stannans chlorides, to similarly activate the dissolution of chromic chloride was observed by Drucker as early as 1901.
Taube and Myers (1954) studied the activation of chromic chloride and chromic iodide and the chlorochromate (III) \(\text{CrCl}^{2+}\) anion by chromous chloride, and in all of these cases postulated the formation of an activated binuclear complex with a halogen bridge, viz:
Hence chromium (III) passes into solution as the CrCl$^{2+}$ species, and Taube and Myers also cite evidence that the retained chlorine in this cation did not pass through the solution. Hence the most reasonable mechanism is atom-transfer via a bridged complex.
It is very probable that the same type of mechanism is responsible for the behaviour of chromous/chromic mixtures in nitrosyl chloride.
The compound $(\text{NO})_3\text{Cr}_2\text{Cl}_9$ gives one prominent infrared absorption band at 2229 cm$^{-1}$. This is in the free nitrosonium ion absorption region, and the absence of other bands indicates that all the nitric oxide groups are present as nitrosonium ions. Hence the compounds must be formulated $(\text{NO}^+)_3(\text{Cr}_2\text{Cl}_9)^{3-}$.
The $\text{Cr}_2\text{Cl}_9^{3-}$ anion has been characterised in the compound $\text{Cs}_3\text{Cr}_2\text{Cl}_9$ (Wessel & Ijdo 1957).
Complex chlorides of this type are built up from a close-packed halogen lattice with cations, in this instance caesium ions, fitting into lattice holes, and chromium ions fitting into certain pairs of octahedral
holes to maintain the stoichiometry. Hence each chromium ion is octahedrally surrounded by six chloride ions and the structure of the anion is thus one of two CrCl₆ octahedra linked across a face, i.e. there are three bridging chlorine atoms, viz:-
\[
\begin{array}{c}
\text{Cl} \\
\text{Cl} \quad \text{Cr} \quad \text{Cl} \\
\text{Cl} \quad \text{Cr} \quad \text{Cl} \\
\text{Cl} \quad \text{Cl} \quad \text{Cl} \\
\end{array}
\]
The chromium (III) ion has a d³ external configuration, and hence gives rise to a characteristic ultraviolet spectrum, in the presence of a perturbing electrostatic field, i.e. in a ligand field.
The spectrum of chromic chloride, CrCl₃, and the (Cr₂Cl₉)³⁻ anion should be fairly similar, since in both cases the chromium ion is in an octahedral field of six chloride ions. Differences may arise in the two instances due to:
a) In the Cr₂Cl₉³⁻ anion, Cr - Cl - Cr bridging may distort the symmetry of the CrCl₆ units from the perfect octahedral structure. Since this type of bridging will be absent in chromic chloride, the chromium ion in this latter instance will be in a more regular octahedral environment.
b) Distortions of the $\text{Cr}_2\text{Cl}_9^{3-}$ anion enforced by the crystal lattice in which it is set; such distortions could remove the symmetry of the anion and give rise to band splittings.
The ultraviolet and visible spectra of $(\text{NO})_3\text{Cr}_2\text{Cl}_9$ and $[(\text{C}_2\text{H}_5)_4\text{N}]_3\text{Cr}_2\text{Cl}_9$ are given; in addition, the main bands are tabulated (table 1), together with those given in the literature for chromic chloride (Jorgensen 1962).
The tetraethyl ammonium salt was prepared by the following metathetical reaction in liquid sulphur dioxide:
$$3(\text{C}_2\text{H}_5)_4\text{N}\cdot\text{Cl} + (\text{NO})_3\text{Cr}_2\text{Cl}_9 \rightarrow [(C_2H_5)_4N]_3\text{Cr}_2\text{Cl}_9 + 3\text{NOCl}$$
$[(\text{C}_2\text{H}_5)_4\text{N}]_3\text{Cr}_2\text{Cl}_9$ separated out as a red-brown solid.
The visible and ultraviolet spectra of chromium (III) complexes are best understood from a consideration of the energy levels of a d$^3$ ion in a ligand field (see diagram).
In chromic chloride the main bands are assigned as shown in table 1 (Jorgensen, 1962, pp. 290 - 291). By comparison, the $A_{T_2} \rightarrow A_{A_2}$ transition in $[(\text{C}_2\text{H}_5)_4\text{N}]_3\text{Cr}_2\text{Cl}_9$ occurs at 720 nm, and in $(\text{NO})_3\text{Cr}_2\text{Cl}_9$ it seems likely that the triply split bands centred about 695 nm are due to this same transition.
The other allowed transition arising from ground state terms, the $^4T_1 \rightarrow ^4A_2$ occurs at 529 mμ in chromic chloride. It seems likely that the 490 mμ band in $\left[(C_2H_5)_4N\right]_3Cr_2Cl_9$ is due to a $^4T_1 \rightarrow ^4A_2$ transition. The shoulder at 540 mμ in the spectrum of $(NO)_3Cr_2Cl_9$ is likely to be due to a forbidden transition and the strong band at 420 mμ is probably due to the $^4T_1 \rightarrow ^4A_2$ transition. If this is the case, it is notable that this transition is shifted to significantly higher energies in both instances of the $Cr_2Cl_9^{3-}$ anion, than in chromic chloride.
The weaker bands, in $\left[(C_2H_5)_4N\right]_3Cr_2Cl_9$ and $(NO)_3Cr_2Cl_9$, above 900 mμ may be due to forbidden transitions of the $^2T_1$ or $^2E \rightarrow ^4A_2$ type.
Consideration of the energy diagram of a d$^3$ ion enables a tentative assignment of the 540 mμ band in $(NO)_3Cr_2Cl_9$ to be made, to a $^2T_2 \rightarrow ^4A_2$ transition, which is strictly forbidden.
Thus, the spectroscopic evidence indicates that the $Cr_2Cl_9^{3-}$ anion in both the above instances are based on bridged CrCl$_6$ octahedra but the band splittings and band displacements observed, indicate that significant loss of symmetry has almost certainly occurred.
CHROMOUS AND CHROMIC CHLORIDES/DINITROGEN TETROXIDE.
As with nitrosyl chloride, no reaction occurred between chromic chloride and dinitrogen tetroxide, even when the products were shaken together for several days. The reaction was unaffected by the presence of ionising solvents, such as acetonitrile and nitromethane. This demonstrated that an increase in nitrate ion concentration did not affect the reaction.
The auto-ionisation
\[ \text{N}_2\text{O}_4 \rightleftharpoons \text{NO}^+ + \text{NO}_3^- \]
is small, but this would be considerably increased in an ionising solvent, which would probably complex the nitrosonium ion, and thus drive the equilibrium towards the right hand side.
Chromous chloride reacted quite rapidly with liquid dinitrogen tetroxide to give a pale green solid and a green solution. The solid analysed as an adduct of anhydrous chromium nitrate, \( \text{Cr(NO}_3)_3 \cdot 2\text{N}_2\text{O}_4 \). This has also been reported in the literature as being formed in the chromium hexacarbonyl/dinitrogen tetroxide reaction (Addison & Chapman 1964).
Chromic chloride, with about 5 - 6% chromous chloride, reacts with dinitrogen tetroxide, but much more slowly than with nitrosyl chloride. The reaction product
is, again, the same as in the system involving chromous chloride only, but the reaction in this case requires shaking for 3 - 4 hours at room temperature.
This is understandable, remembering the electron transfer reaction by which chromic ion goes into solution:
\[
\text{CrCl}_3 + \text{Cr}^{+2+} \rightarrow \left[ \text{Cl}_2\text{Cr} \right]^{2+}
\]
\[
2\text{Cl}^- + \text{Cr}^{2+} + \text{Cr}^{+2+}\text{Cl}_2^{2+}
\]
i.e. chromic ion goes into solution as the CrCl$^{2+}$ cationic species; this will only marginally affect the reaction rate in the formation of (NO$_3$)$_3$Cr$_2$Cl$_9$ since there is no necessity to cleave the Cr - Cl bond, as the (Cr$_2$Cl$_9$)$^3-$ anion only involves Cr - Cl bonding. However, in the reaction involving dinitrogen tetroxide, the situation is different.
The fast reaction step is probably the 'solubilisation' of chromic ion as the CrCl$^{2+}$ species; the slow reaction step is the substitution reaction of the type:
\[
\text{CrCl}^{2+} + \text{N}_2\text{O}_4 \rightarrow \text{Cr(NO}_3)^{2+} + \text{NOCl}
\]
or \[ \text{CrCl}^{2+} + \text{NO}_3^- \rightarrow \text{Cr(NO}_3)^{2+} + \text{Cl}^- \]
giving chromium nitrate or nitrato species which will eventually give the product $\text{Cr(NO}_3)_3 \cdot 2\text{N}_2\text{O}_4$.
The chromous chloride/dinitrogen tetroxide reaction will be different because in this case there are no chromic species to go into solution; the reactant species will be either $\text{Cr}^{2+}$ ions or $\text{CrCl}^+$ ions. If the latter react with nitrate ion from dinitrogen tetroxide autoionisation, it is very likely that an oxidation-reduction reaction will take place, to give chloride-free chromium (III) species viz.:
$$\text{CrCl}^+ + \text{NO}_3^- \rightarrow \text{Cr} - \text{Cl} \rightarrow \text{Cr} + \text{Cl}^-$$
$$\text{NO}_2 + \text{Cr} = \text{O}^+ + \text{Cl}^-.$$
Naturally this is only a schematic representation, since the chromium is very likely to be more highly coordinated that the above mechanism shows.
The infrared spectrum of the $\text{Cr(NO}_3)_3 \cdot 2\text{N}_2\text{O}_4$ was examined, and its behaviour in Nujol mull found to be highly unusual. In a Florube mull two prominent bands were present at $2336 \text{ cm}^{-1}$ and $2278 \text{ cm}^{-1}$, with a further strong band at $1471 \text{ cm}^{-1}$. When the spectrum was run in Nujol mull, the bands at $2336 \text{ cm}^{-1}$ and $2278 \text{ cm}^{-1}$ disappeared almost completely, even when the spectrum
was run as quickly as possible. A sharp band appeared at 1751 cm\(^{-1}\), and the 1471 cm\(^{-1}\) peak seemed to be largely unaffected, though this was difficult to verify as Nujol itself absorbs very strongly at 1460 cm\(^{-1}\) and 1377 cm\(^{-1}\) approximately, and, in all the spectra of Cr(NO\(_3\))\(_3\)·2N\(_2\)O\(_4\) run in Nujol, a high level of absorption due to the compound was observed in the 1540 - 1330 cm\(^{-1}\) region, but the location of band peaks was difficult to ascertain.
It would have been expected that the remainder of the bands in the infrared spectrum of Cr(NO\(_3\))\(_3\)·2N\(_2\)O\(_4\) would show a time-dependence with regard to their relative intensities, such a dependence enabling decomposition product bands to be eliminated. However, this was not the case.
The infrared spectrum in Nujol showed further bands at:
1297s, 1256m, 1167w, 1012s, 800m, 719s.
The relative intensities of these bands were little affected by time. Hence, either all the bands mentioned above are decomposition bands, or else only the species responsible for the 2336 cm\(^{-1}\) and 2278 cm\(^{-1}\) bands are affected by Nujol. Provided the Nujol was thoroughly dry, no obvious signs of decomposition were
apparent in $\text{Cr(NO}_3)_3 \cdot 2\text{N}_2\text{O}_4$ Nujol mulls, even over a period of time, but obviously this is not conclusive; decomposition products of chromium (III) would be expected to be green.
The reflectance spectrum of $\text{Cr(NO}_3)_3 \cdot 2\text{N}_2\text{O}_4$ was observed the complete time for a run over the 200 $m\mu - 1000 m\mu$ region was 30 - 40 minutes, and practically no decomposition occurred over this period, if all the components, including the cell and the potassium chloride diluent, were rigorously dried beforehand, and the non-entry of air assured, during the course of the run. The bands in the spectrum of $\text{Cr(NO}_3)_3 \cdot 2\text{N}_2\text{O}_4$ are tabulated in table 2. Bands and assignments for the $[\text{Cr(oxalate)}_3]^{3-}$ anion (Jorgensen 1962, pp 290 - 291) are tabulated also. In the latter instance, chromium (III) is known to be in an octahedral environment of oxygen atoms, and the same is possible in the case of $\text{Cr(NO}_3)_3 \cdot 2\text{N}_2\text{O}_4$.
By comparison with the data on the $[\text{Cr(oxalate)}_3]^{3-}$ anion, the following assignments can be made for the compound $\text{Cr(NO}_3)_3 \cdot 2\text{N}_2\text{O}_4$. The strong band at 610 $m\mu$ can be assigned to the $^4T_2 \rightarrow ^4A_2$ transition, and it seems likely that the shoulder at 680 $m\mu$ will be due to the Laporte - forbidden $^2T_1, ^2E \rightarrow ^4A_2$ transition. Unfortunately this means that the broad absorption centred about 900 $m\mu$ cannot be characterised.
The higher energy bands are more complicated to assign; it is very probable that one of the bands at 460 mμ or 400 mμ can be assigned to the $^4T_1 \rightarrow ^4A_2$ transition, in which both terms arise from the $^4F$ ground state. Since both these bands have intensities of the same order of magnitude, they are both likely to be allowed transitions. It is possible that the $^4T_1$ level has been split due to loss of symmetry, and that both these bands arise from the same transition, $^4T_1 \rightarrow ^4A_2$.
The band at 320 mμ may be due to the $^4T_1(^4P) \rightarrow ^4A_2$ transition, but the intensity suggests that a charge-transfer process may be responsible for this band, possibly involving 'adducted' dinitrogen tetroxide.
However, there is sufficient evidence to show that the reflectance spectrum of Cr(NO$_3$)$_3$·2N$_2$O$_4$ can be interpreted in terms of chromium existing in an octahedral environment, probably distorted, and probably of oxygen atoms, though the possibility of some co-ordination via nitrogen cannot be ignored.
If the chromium is six-co-ordinate, and this seems highly probable, the nitrate-groups are most likely to be behaving as bidentate ligands, either bridging or donating to just one chromium ion.
It is not possible to assign a structural formula unambiguously. The infrared data are inconclusive, and
the following possibilities exist:
\[(\text{NO}^+)_{2} \cdot (\text{Cr(NO}_3)_5)^{2-}\]
or
\[(\text{NO}^+) \cdot (\text{Cr(NO}_3)_4)^{-} \cdot \text{N}_2\text{O}_4.\]
If 'adducted' dinitrogen tetroxide is present as in the latter formulation, it may behave as a ligand, and may possibly donate via the nitrogen atom.
It seems unlikely, from thermal decomposition studies, that the latter structure is the correct one. Such a structure would be expected to decompose according to the equation
\[(\text{NO}^+) \cdot (\text{Cr(NO}_3)_4)^{-} \cdot \text{N}_2\text{O}_4 \rightarrow (\text{NO}^+) \cdot (\text{Cr(NO}_3)_4)^{-} + 2\text{NO}_2.\]
No evidence was found for such a dissociation; the products of thermal decomposition could not be characterised, but seemed to consist largely of chromic oxide Cr\(_2\)O\(_3\). No formation of the anhydrous trinitrate, Cr(NO\(_3\))\(_3\), occurred by these means, and this is in accord with the observations of Addison & Chapman (1964).
However, chromic trinitrate has been prepared by the latter workers by the reaction of dinitrogen pentoxide with chromium hexacarbonyl; this demonstrates, again, that in general, reaction of a metal compound with
dinitrogen pentoxide affords a better route to the anhydrous metal nitrate, than does reaction with dinitrogen tetroxide.
Addison & Chapman (1964) have shown that chromium trinitrate is a covalent compound, containing co-ordinated nitrato-ligands, on the basis of the infrared spectrum of the compound.
**CHROMIUM TRIOXIDE, CrO₃ + NITROSYL CHLORIDE.**
Chromium trioxide reacts fairly readily with liquid nitrosyl chloride to give a shiny black crystalline product, analysing as \((\text{NO})_2\text{Cr}_3\text{O}_8\text{Cl}\).
It dissolves in water with effervescence to give a yellow-brown solution which contains both Cr(III) and Cr(VI) species. Redox titrations showed that the ratio of the species was \(\text{Cr(VI)} : \text{Cr(III)} = 2 : 1\), which makes the overall oxidation state of chromium in this compound equal to \(+5\). It is rather unlikely that chromium exists in the \(+5\) oxidation state in the compound \((\text{NO})_2\text{Cr}_3\text{O}_8\text{Cl}\), and a mixture of Cr(III) and Cr(VI) is much more likely. In addition, such a structure would explain the intense colour of the compound as being due to Cr(III) \(\rightarrow\) Cr(VI) charge transfer reactions.
The infrared spectrum of the compound is very simple and the bands are tabulated below, with other
compounds containing chromium-oxygen linkages for comparison.
\[
\begin{array}{cccccc}
K_2CrO_4 & K_2Cr_2O_7 & CrO_3 & CrO_2Cl_2 & (NO)_2Cr_3O_8Cl \\
\text{all values in cm}^{-1} \\
885s & 934s & 965s & 1026s & 2340m \\
850m & 900s & 890m & 909m & 964s \\
878s & & 847s & & 887m \\
746 broad, vs & & 820-794 split, s.
\end{array}
\]
In \((NO)_2Cr_3O_8Cl\), the band at 2340 cm\(^{-1}\) is indicative of free nitrosonium ion, and the absence of bands in the 1600-2000 cm\(^{-1}\) region shows that metal - nitrosonium ion bonding does not occur.
The lower frequency bands in the infrared spectrum of \((NO)_2Cr_3O_8Cl\) show a marked similarity to chromium trioxide, \(CrO_3\), which consists structurally of linked \(CrO_4\) tetrahedra (Systrom & Wilhelmi 1950), viz:-
\[
\begin{array}{ccc}
O & O & O \\
Cr & Cr & Cr \\
O & O & O \\
O & O & O
\end{array}
\]
hence it seems probable that \((NO)_2Cr_3O_8Cl\) incorporates \(Cr(VI)\) in this type of environment, with the \(Cr(VI)O_4\)
units having $C_{2v}$ symmetry.
$K_2CrO_4$ contains discrete $\left[CrO_4\right]^{2-}$ tetrahedra (Zachariasen & Ziegler, 1931) and $K_2Cr_2O_7$ contains the $Cr_2O_7^{2-}$ anion, which consists of two $CrO_4$ tetrahedra linked via one corner (Bystrom & Wilhelmi 1951).
Hence it can be inferred from the infrared data that in $(NO)_2Cr_3O_8Cl$, $CrO_4$ units are doubly linked, either to one another as in chromium trioxide, or to other units.
Reflectance data on $(NO)_2Cr_3O_8Cl$ gave bands in the following regions:
| Wavelength | Description |
|------------|-------------|
| 760 mμ | Sh, w |
| 710 " | w |
| 570 " | Sh, m |
| 350 " | s |
(See also Diagram 4)
The band at 710 mμ is characteristic of Cr(III) in an octahedral environment. This band presumably is weak because Cr(III) is in a 'dilute' state in the compound.
Diagram 4 shows that the intensity of absorption increases with decrease in wavelength (increasing energy), and it is likely that this increase is due to charge-transfer reactions between Cr(III) and Cr(VI). A black compound $KCr_3O_8$ is known. It has been shown to contain tetrahedrally co-ordinated Cr(VI) and octahedrally co-ordinated Cr(III). These Cr(VI)$O_4$ and Cr(III)$O_6$ units link together to form layers of composition $Cr_3O_8$ in which
the ratio Cr(IV) : Cr(III) is equal to 2 : 1. These layers are held by potassium ions (Wilhelmi, 1958).
In KCr$_3$O$_8$, the anion is thus polymeric,
\[ \left[ \text{Cr}_3\text{O}_8 \right]^{n-} \]
and \( n \) probably has a very large value.
The anion Cr$_3$O$_8$Cl $^{2-}$ could consist of Cl$^-$ and Cr$_3$O$_8^{-}$ anions, with the latter having the same structure as in KCr$_3$O$_8$. Alternatively, Cr$_3$O$_8$Cl $^{2-}$ units may exist which would give rise to polymeric structures.
Assuming octahedrally co-ordinate Cr(III) and tetrahedrally co-ordinate Cr(IV) it is not possible to construct a satisfactory monomeric Cr$_3$O$_8$Cl $^{2-}$ anion. Also, the infrared evidence suggests that the symmetry of the Cr(IV)O$_4$ units is similar to that in chromium trioxide, i.e. the units link to two other units via two oxygen atoms:
```
O
|
Cr
|
O
```
Such considerations probably necessitate a structure of the type shown below, involving bridging oxygen and chlorine atoms, viz:-
```
O
|
Cr
|
O
|
O
```
The chlorine atoms bridge between $\text{Cr}_3\text{O}_8$ units.
This compares with the structure of the $\text{Cr}_3\text{O}_8^2^-$ layers in $\text{KCr}_3\text{O}_8$:
In the latter instance, Cr - O - Cr bridging causes polyanion formation along the same axis as the chromium ions; in the former instance, Cr - Cl - Cr bridging causes polyanion formation perpendicular to the plane of the chromium ions, but it is apparent from the diagrams that such structures are closely related.
However, distinction between the $\text{Cr}_3\text{O}_8\text{Cl}^2^-$ and $\text{Cr}_3\text{O}_8^- + \text{Cl}^-$ types of structure would require a full crystallographic study.
Since the compound $KCr_3O_8$ is known, it was thought to be of interest to heat $(NO)_2Cr_3O_8Cl$ to attempt to bring about the reaction:
$$(NO)_2Cr_3O_8Cl \rightarrow NO.Cr_3O_8 + NOCl.$$
i.e. to attempt to obtain the nitrosonium analogue of $KCr_3O_8$. On heating to 200°C, nitrosyl chloride was evolved, together with nitrogen dioxide; a blackish-green residue remained which seemed to be a mixture of $Cr_2O_3$ and $CrO_2$. It appears that a redox reaction probably occurred between nitrosonium ion and chromium (VI). There was no evidence for the formation of $NO.Cr_3O_8$; nitrosyl chloride and nitrogen dioxide were evolved simultaneously.
This reaction is similar in some respects to the Vanadium pentoxide/nitrosyl chloride reaction mentioned in the last chapter. Unfortunately a proper comparison is not possible since the products were not identified in the latter case, but it is apparent that in both instances the nitrosyl chloride has behaved as a reducing agent.
**CHROMIUM TRIOXIDE/DINITROGEN TETROXIDE.**
Chromium trioxide will dissolve very slightly in dinitrogen tetroxide to give a green solution; the extent of solution seems to be greater than with vanadium.
pentoxide and dinitrogen tetroxide.
It was hoped that formation of $\text{CrO}_2(\text{NO}_3)_2$ species might occur, but gas phase spectra indicated only the presence of nitrogen dioxide, and no information about the dissolved species was obtained. No solid product was formed and the solid residue consisted entirely of chromium trioxide.
It is possible that the conditions were not vigorous enough for reaction to occur, Gibson & Katz (1951) have carried out the reaction between uranium trioxide and dinitrogen tetroxide, at $90^\circ\text{C}$ and under a pressure of 14.5 atmospheres of the latter, to obtain $\text{UO}_2(\text{NO}_3)_2 \cdot \text{N}_2\text{O}_4$.
Chromyl nitrate, $\text{Cr}_2(\text{NO}_3)_2$, has been prepared by the reaction of chromyl chloride or chromium trioxide with dinitrogen pentoxide:
$$\text{CrO}_3 + \text{N}_2\text{O}_5 \rightarrow \text{CrO}_2(\text{NO}_3)_2$$
$$\text{CrO}_2\text{Cl}_2 + 2\text{N}_2\text{O}_5 \rightarrow \text{CrO}_2(\text{NO}_3)_2 + 2\text{NO}_2\text{Cl}$$
(Schmeisser & Lutzow, 1954)
It was obtained as a volatile red liquid and thus is most likely to exist as monomeric species at room temperature, with the nitrate groups behaving as unidentate ligands. This contrasts strongly with $\text{UO}_2(\text{NO}_3)_2$, which is a solid.
(Gibson & Katz 1951, Gibson et al 1960, Addison et al 1964), and may contain the $UO_2^{2+}$ cation (Addison et al 1964).
No examples are known of the free chromyl cation $CrO_2^{2+}$.
**CHROMYL CHLORIDE:**
**REACTIONS WITH NITROSYL CHLORIDE AND DINITROGEN TETROXIDE.**
These reactions have not been fully investigated, and merit further study. The reaction $CrO_2Cl_2/N_2O_4$ was hoped to yield chromyl nitrate, thus:
$$CrO_2Cl_2 + 2N_2O_4 \rightarrow CrO_2(NO_3)_2 + 2NOCl \quad 1$$
A very small quantity of a blue-green solid was recovered; gas phase spectra indicated that only a mixture of nitrogen dioxide and nitrosyl chloride was present, thus showing that some reaction had occurred, but there was no evidence for the formation of nitrato-ligands or chromyl groups.
However the presence of nitrosyl chloride does suggest an initial reaction of type 1, above, but if chromyl nitrate was formed, further reaction could occur between
a) $CrO_2(NO_3)_2$ and $N_2O_4$, towards adduct formation.
b) $CrO_2Cl_2$ and the NOCl formed.
The reaction between chromyl chloride and nitrosyl chloride yielded a very small quantity of a bright green solid; as with the $\text{CrO}_2\text{Cl}_2/\text{N}_2\text{O}_4$ reaction product, no analysis was possible. The colour definitely suggests that reduction of chromium (VI) has occurred. In both these instances, the infrared spectra of the reaction products are identical, thus two effects could have occurred:
a) The two reactions could yield largely identical products, due to favourable secondary reactions occurring in one of the instances.
b) Reaction could have occurred in Nujol mull, thus making most of the bands decomposition bands.
The latter possibility does seem rather unlikely, since even in florube, which is highly unreactive, both products gave identical spectra.
It is very probable that reduction of chromium (VI) occurs in the main reaction but the oxidation state which chromium finally attains is not known.
**ATTEMPTED REACTION**
**INVOLVING METALLIC CHROMIUM.**
The reaction between vanadium and dinitrogen tetroxide in acetonitrile solution to give $\text{VO}_2\text{NO}_3$ (Pantonin et al 1960) has been mentioned in the previous chapter.
When the analogous chromium reaction was attempted, no reaction occurred either in pure dinitrogen tetroxide or with ionising solvents, e.g. nitromethane and acetonitrile.
MOLYBDENUM
The trend towards enhanced stability of higher oxidation states with increasing atomic number, as observed in the vanadium-niobium-tantalum series, is repeated in the chromium-molybdenum-tungsten series. The oxidation states +3 and +6 are particularly well characterised in chromium, and this is also the case with molybdenum. However, with the latter the +4 and +5 oxidation states are much better known, both as halides and oxyhalides and in complex anions.
As has been pointed out, systems are known in which molybdenum-nitrosonium covalent bonds exist, both in purely covalent species such as $\text{NO}_2\text{Mo}(\text{CO})_2(\pi-\text{C}_5\text{H}_5)$ and anionic species such as $(\text{Mo}(\text{CN})_5\text{NO})^{3-}$.
The nitrosonium salts $\text{NO}_2\text{MoF}_6$ (Geichmann et al 1963; Seol & Birkkvant 1962) and $\text{NO}_2\text{MoF}_7$ (Geichmann et al 1963) are known. The latter workers have also obtained the nitronium salt $\text{NO}_2\text{MoF}_7$. Apart from this, no other nitrosonium or nitronium halomolybdates have been reported in the literature.
In addition, little work has been reported regarding nitrates and complex nitrates of molybdenum and tungsten, although the data on chemistry of uranyl nitrate are much better covered, and has been reviewed (Comyns 1960). The infrared spectrum of the uranyl nitrate hydrates have been examined (Gatehouse & Comyns 1958).
The present work covers the behaviour of molybdenum chlorides in nitrosyl chloride; to a more limited extent, the behaviour in dinitrogen tetroxide solution is examined.
MOLYBDENUM TRICHLORIDE/NETROSYL CHLORIDE.
Molybdenum trichloride reacts with liquid nitrosyl chloride to give a mixture of products; these were identified as molybdenum tetrachloride MoCl₄, and a deep purple, volatile solid of the formula (NO)₃Mo₃Cl₁₀. They were conveniently separated by vacuum sublimation; the gas phase spectra gave no evidence of volatile molybdena-nitrosonium species.
(No)₃Mo₃Cl₁₀
This compound is very deep purple; it is volatile, subliming in vacuo at 90°-93°C. It bears no resemblance to the compound (No)₃Cr₂Cl₉, except that it is a polymeric species.
The infrared spectrum is very simple; a sharp, strong bond is present at 2175 cm\(^{-1}\), with a weak decomposition band at 1802 cm\(^{-1}\). In the spectrum observed in a Nujol mull, a further band is observed at 1000 cm\(^{-1}\).
The 2175 cm\(^{-1}\) band is definitely due to the free nitrosonium ion, NO\(^+\), and the 1802 cm\(^{-1}\) will be due to formation of nitrosyl chloride; the N-O bond in the latter absorbs at 1799 cm\(^{-1}\) (Burns & Burnstein 1950).
It was possible that the 1000 cm\(^{-1}\) band could be due to a molybdenum-nitrosyl anion structure; the nitrosyl group in such a system is known to absorb in the 1050-1200 cm\(^{-1}\) region. However, it is more likely that this is due to molybdenum - oxygen bond formation by reaction of the complex with Nujol. Chromium-oxygen bond frequencies fall in the range 890 cm\(^{-1}\) - 1020 cm\(^{-1}\), depending on the bond multiplicity, and the antisymmetric stretching frequency of the uranyl UO\(_{2}^{2+}\) cation appears at 930 cm\(^{-1}\) (Gatchouse & Comyns 1958); hence it is likely that molybdenum-oxygen multiple bonds will appear in the 900-1000 cm\(^{-1}\) region.
The compound (NO)\(_{3}\)Mo\(_{3}\)Cl\(_{10}\) is extremely hygroscopic, but can be handled in the dry-box fairly readily. It is like the vanadium compounds NOVCl\(_{4}\) and NOV\(_{2}\)Cl\(_{7}\) in this respect.
The absence of bands in the 1600 - 1900 cm$^{-1}$ infrared region implies that all the nitrosonium ions must be unco-ordinated. Hence the compound becomes $(\text{NO}^+)_3(\text{Mo}_3\text{Cl}_{10}^{3-})$. It does seem surprising that a compound with such a high molecular weight should be volatile, but it may sublime with dissociation and structure change; this possibility has been discussed with respect to the compounds $\text{NO}_4\text{VCl}_4$ and $\text{NO}_2\text{V}_2\text{Cl}_7$ in the previous chapter.
The above formula $(\text{NO}^+)_3(\text{Mo}_3\text{Cl}_{10}^{3-})$ necessitates molybdenum being present in more than one oxidation state; since the three molybdenum atoms in each anion have a total oxidation state of +7, it seems most reasonable that molybdenum +2 and +3 should be present, in the ratio:
$$\text{molybdenum (II)} : \text{molybdenum (III)} = 2 : 1$$
Both molybdenum (II) and molybdenum (III) have incomplete outer electron orbitals, the external configuration of Mo(II) is $4d^4$, that of Mo(III) is $4d^3$; hence they should give rise to characteristic d-d absorption spectra. The available spectroscopic data in the literature cover only molybdenum (III) (Hartmann 1957; Jorgensen 1959,) in anionic species such as the octahedral $\text{MoCl}_6^{3-}$ and $\text{MoCl}_5(\text{H}_2\text{O})^{2-}$. Molybdenum (III) has an external $d^3$ electronic configuration, and hence will give rise to
a similar energy level diagram to chromium (III); however since molybdenum is in the second transition series, it gives rise to a higher $Dq$ value than does chromium (Jorgenson, 1962, p.114), so that for the same ligand the two cations Cr(III) and Mo(III) will yield different $Dq$ values. Hence, although the energy diagrams are similar, the magnitude of transition energies may differ, and this is observed. In chromium (III), the forbidden $^2T_2 \rightarrow ^4A_2$ transition is of similar energy to the $^4T_2 \rightarrow ^4A_2$ transition, while in molybdenum (III), the latter occurs at a much higher energy; this is consistent with molybdenum (III) having a higher $Dq$ value.
The reflectance spectrum of $(\text{NO})_3\text{Mo}_3\text{Cl}_{10}$ was observed; the spectrum is shown (Diagram 5), and the main bands are tabulated in table 3 with the data for the $\text{MoCl}_6^{3-}$ anion for comparison.
It will be seen that the spectrum of $(\text{NO})_3\text{Mo}_3\text{Cl}_{10}$ is fairly consistent with the presence of octahedral $\text{MoCl}_6^{3-}$ units, especially with regard to the bands at 680 nm and 425 nm. By comparison with the data for the $\text{MoCl}_6^{3-}$ anion these can be assigned to the $^4T_1 \rightarrow ^4A_2$ and $^4T_2 \rightarrow ^4A_2$ transitions respectively.
The other bands may be due to the molybdenum (II) ion, for which no literature data could be found; it is unknown in the monomeric state.
When the reaction between molybdenum trichloride and nitrosyl chloride is stopped at an intermediate stage, the product isolated is mainly molybdenum tetrachloride.
This suggests that the primary reaction is of the type:-
\[ \text{MoCl}_3 + \text{NOCl} \rightarrow \text{MoCl}_4 + \text{NO} \]
which may occur via an unstable intermediate \( \text{NO}_2\text{MoCl}_4 \). The nitric oxide thus formed may give species such as \( \text{NO}_2\text{MoCl}_3 \), thus:-
\[ \text{NO} + \text{MoCl}_3 \rightarrow \text{NO}_2\text{MoCl}_3 \]
and reaction of \( \text{NO}_2\text{MoCl}_4 \) and \( \text{NO}_2\text{MoCl}_3 \) species could occur:-
\[ 2 \text{NO}_2\text{MoCl}_3 + \text{NO}_2\text{MoCl}_4 \rightarrow (\text{NO})_3\text{Mo}_3\text{Cl}_{10} \]
If such a mechanism is correct, the formation of the complex species is dependent on initial formation of nitric oxide.
Alternatively, the reaction could proceed from a primary disproportionation of the type:-
\[ 2 \text{MoCl}_3 \rightleftharpoons \text{MoCl}_4 + \text{MoCl}_2 \]
with molybdenum tetrachloride precipitating out, and the monomeric \( \text{MoCl}_2 \) species reacting as above with nitrosyl chloride.
MOLYBDENUM DICHLORIDE/NITROSYL CHLORIDE.
No reaction occurred between these components. This is understandable since the species present in molybdenum dichloride are \([Mo_6Cl_8]^{4+}\) cations and \(Cl^-\) anions.
The structure of the former involves considerable metal-metal bonding, as well as metal-chloride bonding. It is thus a very stable unit and difficult to disrupt.
Similarly, no reaction occurred with dinitrogen tetroxide.
Molybdenum Trichloride/Dinitrogen Tetroxide.
Molybdenum trichloride reacts steadily with dinitrogen tetroxide to yield a very pale green solid \(MoO(NO_3)_3\); this compound is insoluble in water, and fairly stable to heat, decomposition starting at 130° - 140°C in vacuo.
Only the one solid product was observed from this reaction, although gas phase spectra of the volatile residues at the end of the reaction indicated that nitrosyl chloride was present, no molybdenum tetrachloride or molybdenum nitrosyl chlorides were present. It will be seen that the product, unlike the products from many other dinitrogen tetroxide solvolysis reactions, is not an adduct or a nitrosonium salt. Its inactivity towards water
verifies the latter statement.
The infrared spectrum shows only a weak absorption in the 2200-2400 cm\(^{-1}\) region, and it appears that this absorption is due to a combinational or overtone mode. The intensity of this absorption is independent of the mulling agent used, this is contrary to the behaviour of the compound Cr(NO\(_3\))\(_3\)·2N\(_2\)O\(_4\).
The infrared spectrum gave bands as follows, with assignments:
| Wavenumber | Assignment |
|------------|--------------------------------------------------|
| 3378 w. | - |
| 2336 w. | - |
| 2304 w. | (V\(_3\) or V\(_5\) + V\(_4\)) - nitrate group (split). |
| 1597 m. | V\(_4\) - nitrate group |
| 1408 w. | 2V\(_3\) or 2V\(_5\) nitrate group |
| 1299 m. | V\(_3\) nitrate group |
| 1149 w. | - |
| 1017 w. | - |
| 974 s. | V\(_2\) nitrate group |
| 917 vs. | Mo-O bond stretching mode |
| 800 w. | V\(_6\) - nitrate group |
| 719 vs. | V\(_3\), V\(_5\) - nitrate group |
These bands can be assigned fairly satisfactorily on the basis of a structure involving co-ordinated nitrate-groups and molybdenum-oxygen multiple bonding, as shown; the (V\(_4\)-V\(_3\)) splitting is 298 cm\(^{-1}\) indicating a high degree of covalent bonding.
The structure may be monomeric, but it is more likely that it is polymeric as has been suggested for niobium oxytrinitrate (Field & Hardy 1963,a), with a structure that may involve nitrate-groups acting as bridging ligands, viz:-
\[
\begin{array}{c}
O \\
N \\
O \\
Mo \quad Mo \\
\end{array}
\]
or
\[
\begin{array}{c}
O \\
N \\
O \\
Mo \quad O \quad Mo \\
\end{array}
\]
Assuming that in the above systems the nitrate-groups are planar, they will both have the same symmetry \(C_{2v}\) and will thus be indistinguishable from their infrared spectra.
The reflectance spectrum of \(\text{MoO(NO}_3)_3\) was observed and compared with that for ammonium pentachloro-oxymolybdate \((\text{NH}_4)_2 \cdot \text{MoOCl}_5\) (Gray & Hare 1962). There is little similarity between the two sets of data, but this could mean that in \(\text{MoO(NO}_3)_3\), the molybdenum ions are not six co-ordinate, and hence the energy diagrams of the two species will be considerably different.
From a comparison of reflectance data for \(\text{MoO(NO}_3)_3\) with that for molybdenum (III) species (diagrams 5 and 6), the reflectance data suggest that the
compound contains molybdenum (III) species, but the analytical data do not support this.
The reaction steps were probably of the type:
\[ \text{MoCl}_3 + 3\text{N}_2\text{O}_4 \rightarrow \text{Mo(NO}_3)_3 + 3\text{NOCl} \]
The Mo(NO}_3)_3 could then decompose in two ways:
\[ \text{Mo(NO}_3)_3 \rightarrow \text{MoO(NO}_3)_2 + \text{N}_2\text{O}_5 \quad (\text{or N}_2\text{O}_4 + \frac{1}{2}\text{O}_2) \]
\[ \text{Mo(NO}_3)_3 \rightarrow \text{MoO}_2(\text{NO}_3)_2 + \text{N}_2\text{O}_4 \]
The molybdenyl species formed would then have to react with dinitrogen tetroxide, effectively abstracting nitrate ion:
\[ \text{MoO(NO}_3)_2 + 2\text{N}_2\text{O}_4 \rightarrow \text{MoO(NO}_3)_3 + 2\text{NO} \]
or
\[ \text{MoO}_2(\text{NO}_3)_2 + 2\text{N}_2\text{O}_4 \rightarrow \text{MoO(NO}_3)_3 + \text{N}_2\text{O}_3 \quad (\text{or NO + NO}_2) \]
There appeared to be no tendency for adduct formation of the types:
\[ \text{MoO(NO}_3)_3 \cdot \text{N}_2\text{O}_4; \text{MoO}_2(\text{NO}_3)_2 \cdot \text{N}_2\text{O}_4; \text{MoO(NO}_3)_2 \cdot \text{N}_2\text{O}_4 \]
It would appear that if such adducts form, they dissociate very readily. Possibly if MoO(NO}_3)_3 does have a polymeric structure, this will not be conducive to nitrosonium compound formation, and any adducts formed
will be just 'lattice compounds', with the dinitrogen tetroxide easily removed by heat or reduced pressure.
Molybdenum Pentachloride/Nitrosyl Chloride.
These components reacted quite readily to give NO Mo Cl₆; this was a grey powder which hydrolysed very readily in moist air to give dioxy-molybdenum dichloride MoO₂Cl₂; in dry air, dissociation into the original components occurred very readily.
The infrared spectrum showed a band, which weakened rapidly with time, at 2180 cm⁻¹, indicating free nitrosonium ion. Hence the compound can be formulated NO⁺ MoCl₆⁻.
The ease of dissociation is similar to that observed with NO₂NbCl₆; this was discussed in the previous chapter.
The Energy Diagram of a $d^3$ ion in an Octahedral Field:
Chromium (III) has an outer electronic $d^3$ configuration and in an octahedral electrostatic field, the $^4F$ ground state gives rise to the $^4A_2$, $^4T_2$ and $^4T_1$ energy terms.
The allowed transitions are:
$$^4P_1 \ (^4F) \rightarrow ^4A_2$$
$$^4T_2 \ (^4F) \rightarrow ^4A_2$$
$$^4T_1 \ (^4F) \rightarrow ^4A_2$$
and the following forbidden transitions may occur:
$$^2T_1 \text{ or } ^2E \ (^2G) \rightarrow ^4A_2$$
Absorbance Spectra of Chromium and Molybdenum Complexes
1) \((\text{NO}_3)_3\text{Cr}_2\text{Cl}_9\)
2) \(((\text{C}_2\text{H}_5)_4\text{N})_3\text{Cr}_2\text{Cl}_9\)
3) \(\text{Cr}(\text{NO}_3)_3 \cdot 2\text{N}_2\text{O}_4\)
4) \((\text{HO})_2\text{Cr}_3\text{O}_8\text{Cl}\)
5) \((\text{NO})_3\text{Mo}_3\text{O}_7\text{Cl}_{10}\)
6) \(\text{MoO}(\text{NO}_3)_3\)
**TABLE 1**
**REFLECTANCE SPECTRUM OF THE Cr₂Cl₉³⁻ ANION**
\[
\begin{align*}
(\text{NO})_3\text{Cr}_2\text{Cl}_9 & \quad \left[(\text{C}_2\text{H}_5)_4\text{N}\right]_3\text{Cr}_2\text{Cl}_9 & \quad \text{CrCl}_3 + \text{Assignments} \\
(m\mu) & \quad (m\mu) & \quad (m\mu) \\
950 & \quad 930 \text{ m} & \quad 760 \text{ sh} & \quad ^2T_1, ^2E \rightarrow ^4A_2 \\
920 & \quad 720 \text{ s} & \quad 730 & \quad ^4T_2 \rightarrow ^4A_2 \\
885 & \quad 490 \text{ s} & \quad 529 & \quad ^4T_1 \rightarrow ^4A_2 \\
665 & & & \\
695 & \quad s & & \\
730 & & & \\
540 & \quad \text{sh} & & \\
420 & \quad s & & \\
\end{align*}
\]
**TABLE 2**
**REFLECTANCE SPECTRUM OF Cr(NO₃)₃·2N₂O₄**
\[
\begin{align*}
\text{Cr(NO}_3)_3\cdot2\text{N}_2\text{O}_4 & \quad [\text{Cr(oxalate)}_3]^{3-} + \text{ASSIGNMENTS} \quad (\text{Jorgensen 1962}) \\
(m\mu) & \quad (m\mu) \\
900 & \quad \text{broad} & \quad 696. & \quad ^2T_1, ^2E \rightarrow ^4A_2 \\
680 & \quad \text{Sh} & \quad 571. & \quad ^4T_2 \rightarrow ^4A_2 \\
610 & \quad s & \quad 418. & \quad ^4T_1 \rightarrow ^4A_2 \\
540 & \quad \text{m} & & \\
460 & \quad \text{m, broad} & & \\
400 & \quad \text{m.} & & \\
320 & \quad s. & & \\
\end{align*}
\]
**TABLE 3**
REFLECTANCE SPECTRUM OF THE \([Mo_3Cl_{10}]^{3-}\) ANION
\((NO)_3 Mo_3Cl_{10}\)
\((m\mu)\)
| \(870\) m | \(1036\) \(^2T_2, ^2E \rightarrow ^4A_2\) |
| \(750\) m | \(676\) \(^4T_2 \rightarrow ^4A_2\) |
| \(680\) s | \(506\) |
| \(640\) s | \(407\) \(^4T_1 \rightarrow ^4A_2\) |
| \(560\) s | |
| \(425\) s | |
\((MoCl_6)^{3-}\) (Jorgensen, 1957, 1962)
\((m\mu)\)
---
**TABLE 4**
REFLECTANCE SPECTRUM OF \(MoO(NO_3)_3\)
\(MoO(NO_3)_3\)
\((m\mu)\)
| \(930\) m | \(725\) |
| \(685\) m | \(435\) |
| \(590\) m | \(375\) |
| \(520\) w | \(312\) |
| \(400\) sh | \(280\) |
| | \(240\) |
| | \(211\) |
\(NH_4_2(MoOCl_5)\) (Gray & Hare 1962)
EXPERIMENTAL
CHROMIUM
Chromic Chloride/Nitrosyl Chloride.
Nitrosyl chloride, dried by vacuum distillation over phosphorus pentoxide, was vacuum distilled into a Carius tube containing 1 - 1.5 gms anhydrous chromic chloride. The tube was sealed, and the reaction mixture allowed to rise to room temperature. No reaction occurred, even on heating, and the original materials were recovered without change.
Chromous Chloride.
Chromous Chloride, CrCl₂, was prepared by passing hydrogen chloride gas, dried by bubbling through concentrated sulphuric acid, over metallic chromium at 900 - 1000°. The reaction was carried out in a silica tube. The chromous chloride sublimed from the reaction tube and was condensed in a receiver at room temperature. On completion of reaction, the system was flushed with dry nitrogen, and the receiver containing chromous chloride sealed and transferred to the dry-box.
Analysis Cr : 42.3 Cl : 57.6
CrCl₂ required Cr : 42.3 Cl : 57.7
Chromous Chloride/Nitrosyl Chloride.
Nitrosyl chloride was dried by distillation over phosphorus pentoxide and then vacuum distilled into a Carius tube containing chromous chloride. The tube was sealed and the reaction mixture raised to room temperature. Reaction was rapid at this temperature and a yellow-brown solid separated out. When the excess solvent was removed in vacuo, a yellow-brown residue remained. This was transferred to the dry-box for further handling.
Analysis: \( N = 7.9 \) \( Cr : 20.0 \) \( Cl : 61.9 \)
\((NO)_3Cr_2Cl_9\) requires \( N = 8.2 \) \( Cr : 20.2 \) \( Cl : 62.2 \)
Chromous Chloride/Chromic Chloride/Nitrosyl Chloride.
A mixture of chromic chloride (c 1.5 gms) and chromous chloride (c 0.05 gms) was made up in the dry-box and transferred to a Carius tube, and then evacuated. Dry nitrosyl chloride was vacuum distilled onto the mixture, the tube sealed, and the reaction allowed to come up to room temperature. Reaction was very rapid, and a yellow-brown solid precipitated out. On removal of excess solvent, a yellow-brown solid remained.
Analysis: \( N: 8.3 \) \( Cr: 20.0 \) \( Cl : 62.0 \)
\((NO)_3Cr_2Cl_9\) requires \( N: 8.2 \) \( Cr: 20.2 \) \( Cl : 62.2 \)
Chromic Chloride/Dinitrogen Tetroxide.
Dry dinitrogen tetroxide was vacuum distilled into a Carius tube containing anhydrous chromic chloride. The tube was sealed and the mixture allowed to rise to room temperature. No reaction occurred. The mixture was heated up to 100°C, but no reaction occurred.
This procedure was repeated, distilling dry acetonitrile into the Carius tube, as well as dinitrogen tetroxide. No reaction occurred in this instance or when nitromethane was used.
Chromous Chloride/Dinitrogen Tetroxide
Dry dinitrogen tetroxide was vacuum distilled into a Carius tube containing chromous chloride. The tube was sealed, and when the mixture rose to room temperature, rapid reaction occurred, a green solid being precipitated out of solution. The excess solvent was removed by vacuum distillation, and the green solid transferred to the dry-box for further examination.
Analysis.
\[
\text{Cr: 11.8% Total N: 21.2% NO}_3^-:67.8%
\]
\[
\text{Cr(NO}_3)_3 \cdot 2N_2O_4 \text{ requires Cr: 11.7% Total N: 21.7% NO}_3^-:68.7%
\]
Chromic Chloride/Chromous Chloride/Dinitrogen Tetroxide.
A mixture of chromic chloride (c.1.5 gms) and chromous chloride (c.0.1 gms) was made up in the dry-box,
and transferred to a Carius tube, then transferred to the vacuum line. Dry dinitrogen tetroxide was distilled into the Carius tube, and the tube sealed. On raising the mixture to room temperature, slow reaction occurred, while at 40°-50°C the rate increased; at room temperature, complete reaction required 3 - 4 hours in a 'shaker'.
Analysis Cr : 11.3% Total N : 21.9% NO₃⁻: 68.0%
Cr(NO₃)₃·2N₂O₄ requires Cr:11.7% Total N: 21.7% NO₃⁻:68.7%
Chromium Trioxide/Nitrosyl Chloride.
Dry nitrosyl chloride was vacuum distilled into a small reaction flask (c.50 mls) containing 1.5 - 2.0 gms chromium trioxide CrO₃. Dry air was admitted to the reaction mixture, and the flask removed from the vacuum system. It was allowed to warm up under atmospheric pressure with a calcium chloride guard tube. At about 0°C, fairly vigorous reaction took place, and a black solid was formed. Removal of excess solvent and the volatile reaction products left a very shiny black crystalline solid, which was transferred to the dry box for further handling.
Chromate was analysed by precipitation as barium chromate; total chromium was analysed for by oxidation of the chromium (III) to chromium (VI) with sodium peroxide Na₂O₂, and precipitation of this, plus the original chromate,
as barium chromate.
Analysis: Found
\[ N : 7.2\% \quad Cr(VI) : 27.1\% \quad Total \ Cr : 41.1\% \quad Cl : 9.5\% \]
\[ (NO)_2 \cdot Cr_3O_8Cl \text{ requires} \]
\[ N : 7.4\% \quad Cr(VI) : 27.5\% \quad Total \ Cr : 41.2\% \quad Cl : 9.4\% \]
Chromium Trioxide/Dinitrogen Tetroxide.
Dry dinitrogen tetroxide was vacuum distilled into a Carius tube containing chromium trioxide. The tube was sealed, and the reaction raised to room temperature. The dinitrogen tetroxide turned fairly bright green, but the chromium trioxide appeared to be only very slightly soluble.
Gas phase infrared analysis of the dinitrogen tetroxide gave no indication of chromyl linkages being present, and complete removal of the solvent in vacuo left only a residue of chromium trioxide.
Chromyl Chloride.
Chromyl chloride was prepared by a method analogous to the preparation of vanadium oxytrichloride (previous chapter - experimental section).
Anhydrous aluminium trichloride was sublimed to purify it and remove traces of moisture. A solid mixture of anhydrous AlCl₃ and 40 - 50% excess of chromium trioxide,
according to the equation below, was made up.
\[ 3\text{CrO}_3 + 2\text{AlCl}_3 \rightarrow 3\text{CrO}_2\text{Cl}_2 + \text{Al}_2\text{O}_3 \]
This mixture was ground up intimately in the dry-box, transferred to a 100 mls reaction flask, then evacuated. Even at room temperature, chromyl chloride distilled slowly out of this mixture, and the rate became quite rapid at \(70^\circ - 80^\circ\text{C}\). The chromyl chloride was condensed in a trap containing phosphorus pentoxide, and redistilled in vacuo to dry it thoroughly.
Analysis Found Cr (as chromate) : 33.5% Cl: 45.4%
\[ \text{CrO}_2\text{Cl}_2 \text{ requires Cr} : 33.6\% \quad \text{Cl} : 45.8\% \]
**Chromyl Chloride/Nitrosyl Chloride**
Chromyl chloride, prepared as previously explained, was vacuum distilled into a 50 ml. reaction flask; dry nitrosyl chloride was vacuum distilled separately into the same flask. Dry air was admitted, and the reaction allowed to rise to room temperature protected from moisture with a calcium chloride guard tube. No vigorous reaction occurred but around \(0^\circ\text{C}\) a small quantity of solid separated out. Gas phase infrared examination of the volatile residue indicated only excess of \(\text{NOCl}\) present. The volatile residue was removed in vacuo and the green
solid residue examined. There was insufficient for analysis, but infrared data were obtained.
**Chromyl Chloride/Dinitrogen Tetroxide.**
Chromyl chloride and dinitrogen tetroxide were separately distilled in vacuo into the same reaction flask, dry air admitted, and the mixture allowed to rise to room temperature protected from moisture with a calcium chloride guard tube. After completion of reaction, a small solid residue was observed. Infrared examination of the volatile residue indicated that a mixture of dinitrogen tetroxide and nitrosyl chloride was present but no evidence was observed for chromyl groups or co-ordinated nitrato-groups.
After removal of the volatile residue, a blue-green solid remained. Insufficient was recovered for analysis, but infrared data were obtained.
**Chromium/Dinitrogen Tetroxide/Acetonitrile.**
Dinitrogen tetroxide was vacuum distilled into a Carius tube containing metallic chromium, and acetonitrile separately distilled into the same tube. The tube was then sealed, and the mixture raised to room temperature. No reaction was observed. On heating to 80°C for 2 - 3 hours no reaction could be initiated;
neither could shaking at room temperature for 2 - 3 weeks initiate a reaction.
Molybdenum Trichloride/Nitrosyl Chloride.
Nitrosyl chloride was vacuum-distilled in about 3 : 1 stoichiometric excess into a Carius tube containing molybdenum trichloride. The tube was sealed and the reaction allowed to rise to room temperature, when steady reaction occurred. The reaction was left for 24 hours.
The tube was opened and excess solvent removed in vacuo, maintaining the reaction mixture at \(-20^\circ\text{C}\) (carbon tetrachloride slush bath) to avoid loss of the volatile reaction product. When all the excess nitrosyl chloride had been removed, the solid residue was transferred rapidly, in a dry-box, into a sublimer. The volatile fraction sublimed out at \(90^\circ-93^\circ\text{C}\), as a very deep blue-purple solid.
Analysis
\[
\begin{align*}
N & : 5.5\% \\
Mo & : 39.1\% \\
Cl & : 48.7\%
\end{align*}
\]
\((\text{NO})_3\text{Mo}_3\text{Cl}_{10}\) required
\[
\begin{align*}
N & : 5.7\% \\
Mo & : 39.3\% \\
Cl & : 48.4\%
\end{align*}
\]
The pale brown product was involatile up to \(200^\circ\text{C}\).
Analysis
\[
\begin{align*}
Mo & : 40.0\% \\
Cl & : 59.5\%
\end{align*}
\]
\(\text{MoCl}_4\) requires
\[
\begin{align*}
Mo & : 40.3\% \\
Cl & : 59.7\%
\end{align*}
\]
Molybdenum Trichloride/Dinitrogen Tetroxide.
Dinitrogen tetroxide was vacuum distilled into a Carius tube containing molybdenum trichloride, and the tube sealed. The mixture was raised to room temperature.
and a steady reaction was observed to occur, which came to completion in 3 - 4 hours. On removal of excess solvent a very pale green solid remained, which was transferred to the dry-box for further handling.
Analysis
Mo : 31.5% NO₃⁻ : 61.6%
MoO(NO₃)₃ requires Mo : 31.9% NO₃⁻ : 61.9%
Molybdenum Pentachloride.
A stream of chlorine, from a cylinder, was dried by bubbling through concentrated sulphuric acid, then passed over molybdenum metal heated to 200° - 250° in a long reaction tube. The molybdenum pentachloride was formed immediately and sublimed off the reaction surface, condensing on the cooler parts of the tube. On completion of reaction the tube was swept out with nitrogen, then transferred to the dry-box.
Analysis
Mo : 35.15% Cl : 64.82%
MoCl₅ requires Mo : 35.10% Cl : 64.90%
Molybdenum Pentachloride/Nitrosyl Chloride.
Nitrosyl chloride was vacuum distilled into a Carius tube containing molybdenum pentachloride, prepared as above. The tube was sealed and the mixture raised to room temperature, when reaction was observed to occur. The excess solvent was removed in vacuo, and the pale grey
solid residue examined.
Analysis N : 3.8% Mo : 27.8% Cl : 62.1%
NO₃MoCl₆ requires N : 4.1% Mo : 28.3% Cl : 62.8%
Infrared spectra were measured on a Grubb-Parsons DBI spectrophotometer in Nujol mulls. The free nitrosonium ion bonds of (NO₃)₃Cr₂Cl₉ and (NO₃)₃Mo₃Cl₁₀ were calibrated in florube mulls against polystyrene film. Since the structure of Cr(NO₃)₃·2H₂O₄ was not definitely ascertained, the 2336 cm⁻¹ and 2278 cm⁻¹ bonds were not calibrated.
All reflectance spectra were measured, diluted in potassium chloride, on a Hilger Watts Uvispek Spectrophotometer Model H700 307; the dry-box was invariably used for making up the reflectance samples.
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ABSTRACT
A series of compounds, reported in the literature, which can be formulated as nitrosonium salts of fluoro-acids, have been re-prepared. Several metal chloride-nitrosyl chloride adducts, also reported in the literature, have been re-prepared, and both these groups of compounds have been shown to contain the nitrosonium ion, NO⁺, by infrared diagnostic methods.
The isomorphism of the nitrosonium salts of the anions fluoroborate, chlorostannate and chloroplatinate with their potassium analogues has been confirmed, and nitrosonium and potassium fluorosulphates have been shown to be isostructural. These observations have been explained structurally, with reference to the relative sizes of nitrosonium and halide ions.
The free (i.e., unco-ordinated) nitrosonium ion was found to absorb in the region 2160 cm⁻¹ - 2400 cm⁻¹. This wide variation was partially explicable in terms of polarisation by the anion but it is apparent that other factors were important. In general, it was found that fluoro-acid salts gave the highest nitrosonium ion stretching frequencies, followed by oxy-acid salts and chloro-acid salts respectively.
The behaviour of group IVB and VB Lewis acid halides was observed with nitrogen oxides and nitrosyl chloride. With dinitrogen tetroxide, all the compounds examined, except arsenic trichloride, gave a mixture of products which it was not possible to separate. Arsenic trichloride gave a nitrosonium nitrate-arsenate salt. In the other cases, there was evidence for the formation of nitrosonium and nitronium salt mixtures, in accord with earlier observations on the behaviour of dinitrogen tetroxide with Lewis acids, and strong acids such as perchloric acid. With nitrosyl chloride, the products were either nitrosonium chloro-acid salts or molecular adducts. Reactions involving nitric oxide were not investigated extensively but in reactions with high valency metal chlorides, the primary reaction steps was always a reduction by the nitric oxide.
The behaviour of transition metal chlorides and oxides of the titanium, vanadium and chromium groups was observed in nitrosyl chloride and dinitrogen tetroxide. These reactions could all be explained assuming auto-ionisation of the solvent.
The reactions in nitrosyl chloride yielded mainly nitrosonium salts of chloro-acids, and several of the anions thus formed were bi- and tri-nuclear species.
The previously reported compounds NO.VCl₄ and NO.V₂Cl₇ were obtained and characterised as nitrosonium salts. With niobium and tantalum, the new compounds NO.NbCl₆ and NO.TaCl₆ were prepared, containing the previously unknown NbCl₆⁻ and TaCl₆⁻ anions. The new compounds (NO)₃Cr₂Cl₉ and (NO)₃Mo₃Cl₁₀ were obtained. A surprising feature of several of these ionic compounds was their high volatility.
In the reaction between chromium (VI) trioxide and nitrosyl chloride, a new compound (NO)₂Cr₃O₈Cl was characterised, in which chromium has undergone a partial reduction. Vanadium pentoxide underwent a similar reaction, but in this case it was not reproducible.
The reactions involving dinitrogen tetroxide generally gave rise to one product only, in contrast to the Lewis acid - dinitrogen tetroxide reactions. The products were usually metal nitrate-dinitrogen tetroxide adducts and these could be formulated as nitrosonium metal nitrate-salts, on the basis of their infrared spectra. Vanadium and chromium chlorides gave the adducts VO₂NO₃·N₂O₄ and Cr(NO₃)₃·2N₂O₄ respectively, but molybdenum trichloride gave Mo(V)O(NO₃)₃, which may be polymeric.
No reaction occurred between chromium (III) chloride and either nitrosyl chloride or dinitrogen tetroxide, but in the presence of chromous chloride, reaction proceeded
readily in both instances.
No reaction was observed between high valency metal oxides and dinitrogen tetroxide, but this may have been due to insufficiently vigorous conditions.
The reflectance spectra of the majority of the above compounds were observed, and, in most cases it was possible to make satisfactory band assignments.
|
Electric Dockyard Crane
A No.10 Set model built & described by B.N. LOVE
Because of the size and complexity of this outstanding, advanced model we are unable to provide the full building instructions in this one feature. They would take up most of the magazine! We are therefore splitting the instructions into two instalments with PART 1, here, dealing with the travelling bogies and tower. PART 2 in our next issue will conclude with the jib, hoisting gear and wiring, etc. We are indebted to Mr. Bert Love, the builder, both for the accompanying illustrations and the comprehensive building instructions.
BOGIE CONSTRUCTION
All four bogies are of slightly different construction and their general arrangement may be seen from the overall view of the Dockyard Crane in Fig. 1. Two of the bogies are directly chain driven through gearing from a Powerdrive Motor inside the Tower and a third bogie picks up its drive by external Sprocket Chain on the far side of the model. Fig 2 shows the construction for a powered or non-powered bogie, one of each being required at both sides of the Tower but a "mirror-image" or reversal of each construction is needed to complete the set. It should be noted that the non-powered Flanged Wheels in each bogie are free to slide on their Axle Rods and are thus self-aligning in the sunken rails required for dockyard working where vehicular traffic must pass beneath the portals of the crane. The two Bell Cranks in the No. 10 Set secure the trailing Axle Rod in two of the bogies while Collars and the two ½" Pulleys with Boss do the same job in the other pair. It is left to the ingenuity of the builder to place the limited number of Collars (24 of them) in the most appropriate places on the model and to bear in mind that any spare gears or, indeed, any spare wheel with boss can double-up as a Collar if applied sensibly.
Large flanged wheels are built up by bolting Face Plates to Wheel Flanges, but for the Sprocket-driven pair, the boss of the Face Plate is pushed through the Wheel Flange and the ½" Sprocket Wheels employed are positioned at the same time, but stood away from the Face Plate by double Nuts to give clearance for the Chain drive. On the plain bogie, (and the far side bogie picking up its drive by external Sprocket Chain) the built-up flanged wheels protrude between a pair of Formed Slotted Strips, but for the Sprocket mounted internally only one formed Slotted Strip is used on each bogie again to give clearance for the Sprocket Chain. Construction of the bogies is clear from Fig. 2, each one being topped by 5½" or 4½" Angle Girders, reinforced externally on the lower edges by overlaid 5½" Strips and braced internally by a 1½" x ½" Double Angle Strip in the position shown. Ten Flat Trunnions, four 2½" Semi-Circular Plates and two 4½"
Flat Girders provide reinforced journals for the driven and trailing axles in the bogies. As the bogies are free to move on the track to accommodate slight fluctuations in rail level, they are provided with journals beneath the feet of each Tower portal. A pair of 2½" Curved Stepped Strips are mounted on the bogie Girders, placed centrally and stood off with one Washer to allow for the distance between the Trunnions bolted to the base of the portal legs. Any convenient size of Axle Rods (3½" in the illustrations) are used to pivot the bogies to the portal and the sheer weight of the model holds them in place without using Collars. Smaller Axle Rods and Collars may be added as extras if preferred.
BUILDING THE TOWER
There are four sections to the Tower, namely the two portals or side frames, the platform and the turntable. Construction is started by building identical portals and reference should be made to Figs. 1 and 3. Each portal is 12¾" high and 15¾" long at the top edges. Pairs of 18¾" Angle Girders 1 brace the bottom and overlap the portal legs by 1½" at each end. Outer faces of the portal frames are made from five 12¾" x 2½" Strip Plates, vertical pairs overlapping and sandwiching horizontal Plate 2 by two holes at each end, thus providing the 15¾" dimension of the top edge. The inside corners resulting are overlaid externally with Corner Gussets 3 for reinforcing the corners and providing a neat curvature in the design. Verticals below the Gussets 3, 9½" Strips 4 continue the overlay for additional support while outer edges of the portal frames are all reinforced by Girders. A compound girder is made from two 7½" Girders joined by a 2½" x 1½" Flanged Plate, leaving a ½" gap at the join, and this girder runs inside the top edge of the horizontal Strip Plate 2. Outside, a 2½" Strip is overlaid and lengthened by 2½" Strips at each end to make up the 15¾" span. The bottom edge of the horizontal Strip Plate is reinforced internally by a 1½" Strip and is held in place by the Bolts which attach the Corner Gussets at that level. Leading vertical edges of the portal legs are reinforced by 12¾" Angle Girders.
The inner faces of the portal frames are somewhat different in construction. This time, the horizontal Strip Plates at the top are doubled up to sandwich 4½" x 2½" Flat Plates (22), allowing 1½" of Plate to extend at each end. The holes provide attachment points for the Crank Handles across the top of the Tower. The inner faces of all four portal legs are lined with double thicknesses of 5½" x 2½" Flexible Plates 5, reinforced at their leading edges with 12¾" Angle Girders and by vertical 1½" Strips as overlays. The Flexible Plates overlap by one hole. When the internal and external faces of the portal frames are complete, each plate may be completed by joining the faces with 1½" Flat Girders at the bottom of each portal leg and by 1½" Girders 6 at the top. This can be seen in Figs. 1 and 4. One 5½" x 1½" Flexible Plate 7 is bolted into the top of the legs to extend beyond the casters (any extra plate shown, as in Fig. 1, should be ignored). Just inside each portal leg, at the height of the lapover of the 5½" Flexible Plates, a 2½" Angle Girder is bolted on horizontally by its round holes to form ledges as anchoring points for a double layer of 5½" Curved Strips 8 which form bracing struts from the legs to the platform.
Internal bracing of the portal legs is achieved by using four Double Angle Strips with 1½" lugs. The location of the 3" x 1½" D.A.S. 9 is shown in Fig. 5, and can be seen to carry the vertical drive shaft for the gear wheel housing. Angle Brackets fix the three end of the strut to the portal legs as shown. The legs which have no transmission shaft inside are reinforced in a similar manner using a pair of 2½" x 1½" D.A. Strips. Some additional support to the portal feet is provided by a pair of 2½" x 1½" Triangular Flexible Plates 10 on three of the legs and one pair of 3½" x 1½" Plates on the fourth leg carrying the ladder. The shorter Plates carry vertical overlays of 2" Strips and the longer pair are covered by 3" Strips. This gives a neat line of continuity up the corner Angle Girders of the portal.
At this stage the pairs of 18¾" Angle Girders can be bolted, slotted flanges upwards, to the bottom of the portal legs as shown in Fig. 1, being secured in place with Washers under the boltheads. It will be noted as construction progresses that various illustrations show more standard Washers than are available in the No. 10 Set, but most of them, are for 'show' and are not essential to the construction. At each end of 18¾" Girders 11, pads made from double thicknesses of 2½" x 2½" Flexible Plates 11 are bolted on below and fixed in place by Trunnions in the positions shown. It is necessary to have the elongated holes of the Plates available because of the slightly expanded width of the portal legs. A similar double thickness Pad 12 is bolted under the centre point of the 18¾" Girders. Four 9½" Strips complete the portal frames as diagonal struts bolted from the vertical flanges of Angle Girders 1 to the vertical overlaid 1½" Strips on the inner faces of the frames.
Continued →
Fig. 6, above left, shows the cable drum mounting of the No. 10 Set Electric Dockyard Crane. Reinforced Plastic Plates give insulated feed for the flexible cable and note the counterweight giving automatic reel-in. Fig. 7, above right, is a view from beneath the tower platform showing the mounting for travelling and slewing motors. Note transverse reinforcing provided by 12½" Braced Girders.
ELECTRICAL FEED TO TOWER
Construction of the automatic reel-in, reel-out power feed is quite straightforward. A 4" Axle Rod is capped with a Steering Wheel followed by the face of a 3" Sprocket 13, boss inwards. Two 1" Motor Tyres are passed over the boss which are jammed in place, roughly centred, by a second 3" Sprocket secured tightly with one Grab Screw. The boss of this Sprocket also points inwards towards the model. Very flexible multi-stranded plastic covered wire is required and that used in the model is known as 'Radiosensitive Flexible Instrument Lead Wire'. A 2 amp carrying-capacity is adequate, but the cable must be very flexible. Two metres of wire are adequate. A standard Bolt fitted with a Nut and then a Washer is screwed into the second hole of the boss on the inner Sprocket. Two cm's of wire is bared of insulation and passed through an inner hole of the Sprocket face, round the Bolt and trapped in place by the Nut and Washer. Most of the insulated flexible cable is then wrapped round the drum between the Sprocket faces.
It is obvious that the Axle Rod is now 'live' but will be carrying only 12 volts for the Power Drive Motors (N.B. Under NO circumstances must this method of electrical feed ever be used at model stage!) It is now necessary to ensure that this 'live' Rod runs in insulated journals and these are supplied by a pair of 2½" Strips reinforcing the top edges of a pair of 2½" x 1½" PLASTIC Plates 14. These Plates are bolted directly to the vertical flanges of 12½" Angle Girders 1, at their centre, and two ¼" Sscrewed Rods make a rigid cross connection to the top of the plates, as shown in fig. 6. The electrical drum shaft is now passed through the middle holes of the 2½" Strips and a pair of 6-hole Bush Wheels, boss to boss, are slipped on between the Strips to make a simple winding drum for the counterweight. A Collar and Washer secures the drum shaft and the Washer is arranged to rub against a Fishplate or freely-turning solder tag to which the ordinary insulated wiring is carried up the tower to the motors. Two 2" Pulleys 15 revolve freely on Axle Rods passing through the tower frame, shown being arranged to receive a vertical roll of cord from the winding drum and a vertical fall of cord to the counterweight 16, which consists of a pair of Channel Bearings joined by a 2½" x ½" Double Angle Strip and a 2½" x 1½" D.A.S. This makes the latter to be swung out of the way while the counterweight is formed with Meccano Steel Balls. A 2½" x 1½" Plastic Plate, Transparent or coloured is inserted as a 'U' shape to prevent balls from spilling through the gap between the Channel Bearings. One pair of 4½" Angle Brackets are bolted to the centre holes of the lower Channel Bearing, slotted lugs outwards, and these act as guides running on a single loop of Spring Cord secured by Hooks inside the portal frame at the top and passing through the slotted ends of a pair of Fishplates bolted to the bottom 18½" Girders 1, and set to make the Spring Cord run vertically and parallel.
General arrangement of the reel and counterweight is shown in Fig. 6. Sealing the counterweight is done by winding the 2½" x 1" D.A.S. back into place and tightening up the lower Bolt and then the upper one, which is a ½" Bolt (or Threaded Pin) carrying an End Bearing to which the Meccano Cord is attached. This completes the insulated supply line for the crane and the remainder of the wiring may be carried out in any low voltage, single strand insulated wire generally available.
All motors in this model have one terminal wired directly to the nearest point of the model's framework as a 'common' earth return and no trouble should be experienced if the model is made in parts using the zinc finish. With the latest finish on Meccano Strips and Girders, it may be necessary to run a separate earth wire down to a pick-up point on the rails as there can be sufficient insulation from the new enamels to prevent circuit continuity through the model's framework.
MAIN PLATFORM
As this will support the full weight of the boom and its loads, a sturdy platform of deep box-web girder construction is used to give a firm base for the roller bearing. Figs. 7 and 8 should be studied carefully when constructing this part of the model. Assembly is started by fixing the slotted flange of a 9½" Angle Girder over one edge of a 9½" x 2½" Strip Plate 17 and a 9½" Strip on the other edge of the Plate, separating and overlaying them with 2½" Strips at each end. Angle Brackets are fitted internally (the Strips and Angle Girder being fixed to the outside of the model) to their round holes at each end of the 9½" Strip. This operation is repeated to make an identical pair. The top edge of a 12½" x 2½" Strip Plate is now sandwiched between the slotted flange of 12½" Girder 18 and the 9½" x 2½" Strip. The same thing is repeated and then the 9½" assemblies are bolted to the 12½" assemblies by means of the round holes in the Girder flanges to form a 9½" square, the four Angle Brackets being bolted to the bottom edges of the longer Strip Plates. Internal reinforcing is carried out on the bottom edges of all four Strip Plates by 4½" Angle Girders 19 located as shown in Fig. 7 and each pair is braced at their outer ends by 4½" Curved Strips to make rigid corners. The top inside edge of each portal frame is fitted with a 12½" Angle Girder, slotted holes downwards, overlaid inside the frame with a 12½" Strip. Secured to the Angle Brackets to this Strip, at the top centres of each portal frame, are small Flanged Plates 20, the securing Bolts passing through the Girder on the other side at the same time. The top edges of the portals are thus reinforced and may now be bolted on, by the round holes in the Girders, to the centre section of the main platform just described. The bottom corners of the two 12½" Strip Plates across the main platform are now fitted with pairs of ½" Angle Brackets, each pair back-to-back by the round holes, and four of these Brackets are then bolted by their slotted lugs to the portal frames horizontal internal Strip Plates. At this stage, the Tower assembly should already be taking on a rugged appearance.
Additional bracing for the Tower is supplied by front and rear deep web Girders as seen in Fig. 1. This time a 12½" Strip Plate 21 is fitted externally at the top with a 12½" Girder by the round holes and then another a 12½" Strip overlays the bottom row of holes as shown. At each end, vertical 2½" Girders are bolted on by their round holes behind the ends of the Strip Plates and they overlap for reinforcement by 2½" Strips at the front. This allows the front-over-running slotted flange of the short Girders to tuck in neatly and to be bolted to the perforations of the 4½" Flat Plates 22 extending at either end of the insides of the portal frames. Fig. 7, showing the view of the Tower from below, illustrates additional support for the front and rear deep girders by horizontal 12½" Braced Girders 23. The lower edges of Strip Plates 21 are fitted with 7½" Flat Girders 24, placed centrally as shown in Fig. 1, and these are used to secure four 2½" x ½" Double Angle Strips 25, which are bolted at their inside ends to two of the ¾" Angle Girders providing internal bracing. The Braced Girders are bolted, in turn, to the D.A. Strips and further reinforced by ½" Angle Brackets, four of which are already in place at the bottom corners of the central 9½" square assembly. A careful check should be made to ensure that the portal frames are vertical and parallel at this stage. Pairs of 5½" Curved Strips 26 are now fitted with 1" x ½" Angle Brackets, lugs forward, and are fixed to the four horizontal 2½" Girders mounted inside.
the portal legs. The upper ends of the Curved Strips sandwich the frontal 7½" Flat Girders 24 at each end and then, after checking once again that all portals are equally spaced all the way across the Tower, the Curved Strips and the 1" x ½" Angle Brackets are bolted securely in place. All nuts and bolts round the Tower assembly should now be checked.
TRAVELLING AND SLEWING MECHANISM
A simple, but strong, motor platform is made from a pair of Flanged Sector Plates fixed to the 4½" Angle Girders and joined by an 8-hole Bush Wheel 27, bossed outwards in the centre. The Bush Wheel is secured by a 3" Screwed Rod through one hole and a 1½" Bolt through its opposite hole. Lock-nutted to both the Screwed Rod and to the end of the long Bolt is a 1½" Strip and its centre hole forms a second journal, with the boss of the Bush Wheel for a 2½" Rod on which a 1½" Bevel Gear 28 picks up the slewing drive from a first Power Drive Motor. One Collar is used to space the large Bevel from the Bush Wheel and a 19-t Pinion 29 is secured to the top end of the 2½" Rod.
Attached to the top of the main platform is a 6" diameter Circular Plate 30 located by two 3/8" Strips and two 3/8" x 1½" Double Angle Strips, as seen in Fig. 8. No weight is placed on the Sector Plate which simply carries a 6-hole Wheel Disc at its centre to centralise the pivot rod from the boom's roller race. Journalled in this Plate, and in the Sector Plate below, are the second and third shafts for the slewing drive using 3/8" x 1½" Rods. Mounted on the second shaft is a 57-t Gear meshing with 19-t Pinion 29, and a 19-t Pinion which meshes with a 60-t Gear 31 on the third shaft. At the top of the third shaft, spaced by four Washers above the surface of the Circular Plate, is the final drive 19-t Pinion 32. This will engage a 3/8" Roller Wheel attached to the crane boom. All of this gearing needs careful setting up for free running without slop and should then be lightly lubricated. This arrangement of shafts and the general selection of gears in the No. 10 Set permits changes of gear ratios to suit individual tasks for "scale" slewing speed. The Power Drive Motor should be set in its highest ratio (60 : 1).
Mounting for the travelling motor is also shown in Fig. 7 where the leading edge of the base plate is set one hole in from the broad end of the Sector Plate. A 2½" Axle Rod is journalled through the rear edge of the portal leg carrying the ladder, nine holes below the top of the portal frame, and is fitted with a 2" Pulley 33 and a Swivel Bearing. Between the 2" Pulley and the portal leg, a 19-t Pinion is fitted to the Rod, boss inwards. After looping a 10" Heavy Driving Band over the Pulley, an 11½" Axle Rod is also fitted with a 19-t Pinion 34 and then locked in the swivel of the Swivel Bearing. Once the second 19-t Pinion is locked in place as shown in Fig. 1, no Collars are required to hold this compound shaft in place and the Swivel Bearing gives all the flexibility required.
Again, the motor is set to the highest ratio and connected up via a ½" Pulley (supplied with the Motor) by the Driving Band to the 2" Pulley. Just below the 19-t Pinions, 57-t Gears are mounted on 2½" Rods also passing through the portal legs and they carry 7/8" Bevel Gears just inside the legs which also position these shafts without the use of Collars.
Fig. 8 shows how the drive is carried down a second Bevel and Contrate Gears to a ¾" Sprocket Wheel 35 which provides the final drive to the bogies. All of the travelling bogies may now be fitted and Sprocket Chain connected up for a test run. It is important that the Chain is slightly slack; rather than tight otherwise the large driving flanged wheel will actually be lifted off the track, thus losing traction. On the far side of the crane, each bogie is fitted outboard with a 2" Sprocket on the driving shaft and connected up by a length of Sprocket Chain running from one bogie to the next. The fourth bogie remains unpowered.
TURNTABLE AND ROLLER BEARING
Eight 4½" x 2½" Flexible Plates 36 with centre holes are required for the roller bearing drum which is simple to construct, but which must be assembled with care. Each Plate is attached by a ½" or ¾" Bolt through every second hole on the outside rim of a Large Flanged Ring, but the Plates must be spaced internally from the Ring by two Washers and one Spring 37. The centre holes in each Plate are used for the initial attachment, but Nuts are left fingertight. The ends of each Plate are now overlapped in turn, overlaid with vertical 2½" Strips and bolted to the Large Flanged Ring with the same stand-off spacing just mentioned. The assembly should be stood on a flat table with the chosen centre plates aligned at their bottom edges and are standing vertical to the Flanged Ring. All Nuts are tightened in turn and then Bolts and Nuts are added to the bottom end of each 2½" Strip, but not to the upper end. These should be left clear to avoid fouling the internal rollers.
Fig. 9 shows the drum in position on the Tower and it is attached to ¾" Bolts set in Double Bent Strips which can be seen centrally located on the sides of the 9½" sturze at the top of the Tower in Fig. 8. A roller race is made from a 7½" Circular Strip fitted with four Couplings 37, attached by pairs of ½" Bolts and four Collars mounted by standard Bolts. Washers and lock-nuts. The Couplings carry 2½" Rods and the Collars are fitted with 1½" Rods. Two 1½" Collars are fitted to 2½" Flanged Wheels on a fixed radius so that these must run inside the upturned flange of the Large Flanged Ring. When a second Flanged Ring, fixed to the boom above, is located over the rollers, they become self-centring, allowing electrical contact to be passed up the crane through the centre of the rollers bearing to the machinery house. It will be noticed in Fig. 9 that a 2½" x 1½" Plastic or Transparent Plate 38 is bolted to the Flanged Ring and has a ¾" Washer attached. This acts as an insulated electrical wiper to pass current up to an insulated ring in the upper half of the roller bearing and this will be dealt with in PART 2 of this feature in our September issue.
When fixing the roller bearing drum to the Double Bent Strips, lock-nuts must be placed on the bolt shanks to ensure level plane attachment without distorting the Large Flanged Ring.
TOWER LADDERWORK
A pair of 12½" Strips 39 are joined at their centres by a 1½" Screwed Rod, four locknuts and Washers (optional). Intermediate holes of each Strip ¾" Bolts are fixed with lock-nut to form rungs and ½" Reversed Angle Brackets are bolted to the foot of the ladder, attaching these to the front holes of the horizontal 18½" Angle Girders 1 as shown in Fig. 4. At the other portal, Rod and Strip Connectors are attached to hold a pair of Double Axle Rods acting as the first section of handrails. These Rods pass through Right-angled Rod and Strip Connectors and these are attached by a pair of ¾" Bolts with lock-nuts to the ladder, ten holes down from the top. The lower pair of holes in the ladder are left open and the second holes are fitted with Angle Brackets with which the ladder is secured to the vertical 12½" Angle Girders of the Tower portals, one hole down from the top. A pair of 3½" Crank Handles continue the handrails, terminating in Handrail Couplings at the top of the ladder. From here, handrails may be extended to suit any residual Axle Rods or spare Couplings.
TO BE CONCLUDED IN OUR NEXT ISSUE
Electric Dockyard Crane Part 2
A No.10 Set model built & described by B.N. LOVE
In the first part of this feature, published in the July MM, we gave building instructions for the bogies and travelling tower of this highly impressive No. 10 Set model. Now we conclude the building instructions by covering the boom, the upper part of the turntable, the machinery house, the travelling crab and the hoist system.
MAIN BOOM CENTRE PORTION
Figs. 10, 11 and 1.3 show the simple box structure and upper turntable ring forming the centre portion of the boom and of these, the turntable ring should be constructed first. A 'flexible joint' arrangement is used here by attaching a large Flanged Ring to a sandwich of two 6" Circular Plates via four 3½" x 2½" Flexible Plates 40 as shown in Fig. 10. This allows the inevitable flexing of the boom under load without distorting the centralising of the gear drive to the turntable. 2½" Curved Strips 41 are overlaid on these, sloped up over the Flexible Plates for additional anchoring where they are attached to the Flanged Ring. Four Double Arm Cranks 42 are bolted to the Circular Plates, as shown, and they carry 2½" or 3" Rods to form a 'cage' axle adding considerable rigidity to the structure and ensuring that the 3½" Gear Wheel 43 cannot slip. All four rods are locked into their Double Arm Cranks, but only two of them are fixed to the 3½" Gear Wheel by means of Rod Sockets, the other two Rods simply protruding through the Gear by a few millimetres. When securing the latter, bolts into the 8-hole Bush Wheel is inserted through the Circular Plates' centre holes and fitted with a 4" Rod which is also locked into the boss of the 3½" Gear Wheel, allowing about one inch of Rod beyond the boss. This extension will locate the upper turntable ring on the 8-hole Wheel Disc which is already mounted in the centre of the lower portion of the turntable.
At this stage, the electrical pick-up is fitted for power to the hoisting motor. Fig. 11 shows a second 5½" Circular Girder 44 suspended and insulated by a pair of 5½" x 1½" Plastic or Transparent Plates, 1½" below the first Circular Girder. A feed wire is bolted to the insulated Circular Girder and passed up eventually to one terminal of the Powerdrive Motor in the machinery house. Modern Meccano is well enamelled so the second terminal of the motor is fitted with an 'earth' (chassis) return lead fastened by a Bolt and lock-nut into a Collar at the top of the Axle Rod passing through the 3½" Gear Wheel. This ensures a good return path for the power supply to the crane tower.
Fig. 13 shows the central portion of the boom which is of box construction, the base being a pair of 5½" x 2½" Flanged Plates, upside down and secured at the extreme ends of the 9½" Angle Girder 45 shown externally. Standing vertically at the four corners of this 5½" x 9½" base are 5½" Angle Girders 46 sandwiched between the 9½" Girders and the Flanged Plates. Four vertical 5½" x 2½" Flexible Plates reinforce the uprights, overlaid by 5½" Strips and spaced by another pair of similar Strips horizontally to form the windings of the central section. Transparent Plates and 2½" Narrow Strips complete the windows and the construction is clear from Fig. 13.
At this stage, the first of the long girder sections is fitted by placing 18½" Angle Girders...
47 on the top of the centre box, allowing seven holes clear overhang to the rear which gives eleven holes clear overhang to the front. One 5\(\frac{1}{2}\)'' x 2\(\frac{3}{8}\)'' Flanged Plate is placed on the top of the structure in its centre and bolted to the Angle Girders and then the first of the upper deck plating is attached by fitting 5\(\frac{1}{2}\)'' x 3\(\frac{3}{8}\)'' Flat Plates at either side of the Flanged Plate. Anchoring plates for the front end of the boom are provided by four Corner Brackets 48 while 2\(\frac{1}{2}\)'' Strips are bolted on at the back for the rear section and these can be seen in the various illustrations. One 5\(\frac{1}{2}\)'' Braced Girder 49 is used at the front of the centre box section to give lateral support between the 5\(\frac{1}{2}\)'' vertical Girders. The completed section can now be bolted to the lower turntable ring. This is done at two points only through the centre holes of the leading edges of both inverted Flanged Plates which coincide with the holes on opposite sides of the large Flanged Ring (see Fig. 11).
A trial run should be carried out to ensure that the centre section of the boom is running properly on the turntable. Adjustment of the Spring Clips on the roller race will be required to ensure that the small Flanged Wheels run just clear inside the flange of the lower Flanged Rings. Making the final turntable drive Pinion from the tower to the Tower Gear Wheel is best adjusted by setting the 19-t Pinion up or down on Washers to obtain centre line engagement. A check should be made to ensure that the small Flanged Wheels do not scrape against the inside of the turntable drum and that they are all properly located inside the flanges of the turntable rings. Rotation from the Powerdrive Motor below should be almost effortless at this stage. Light lubrication of the rollers and a smear of graphite grease will assist smooth running on the ring flanges.
**FORWARD BOOM;**
Double bracing of channel section girders is employed throughout the forward section of the boom and these must be assembled by judicious use of the Angle Girders and Strips left in the No. 10 Set. Two side frames are made of identical, but mirror image reversal, construction. Fig. 12 should be carefully studied as this gives most of the required visual information. A start is made by bolting a 24\(\frac{1}{2}\)'' Angle Girder by its round holes to the 5\(\frac{1}{2}\)'' x 2\(\frac{3}{8}\)'' Flat Plate 50 which forms the small platform on the top of the leading edge of the boom. This is the upper and outer girder. The inner and upper girder is another 24\(\frac{1}{2}\)'' Angle Girder, but this time it has only one hole lapped on to the 5\(\frac{1}{2}\)'' Flat Plate, the space in front of this Girder being taken up by a 2'' Girder 51 bolted on to the Flat Plate by its round holes. A joint between these two Girders is made by the five-hole edge of a Corner Gusset 52 and this is clearly seen in Fig. 12. Note the arrangement of the slotted flanges of all Girders throughout the construction of the boom.
Twenty-six holes back from the boom front, the upper 24\(\frac{1}{2}\)'' Girders are joined by a reinforcing plate which is a 2\(\frac{1}{2}\)'' Flat Girder 53. The inner Bolts for this Flat Girder also hold a 12\(\frac{1}{2}\)'' Girder, slotted flange downwards, under the upper 24\(\frac{1}{2}\)'' Girder. Location of the 12\(\frac{1}{2}\)'' Girder is fifteen holes back from the front of the boom. A second channel section for the lower edge of the boom is another pair of 24\(\frac{1}{2}\)'' Girders, but this time they are staggered by five holes instead of four. The outer lower 24\(\frac{1}{2}\)'' Girder 54 is bolted to a second 5\(\frac{1}{2}\)'' x 2\(\frac{3}{8}\)'' Flat Plate running across the boom head at the lower level and the other 24\(\frac{1}{2}\)'' Girder has its leading vertical slotted hole lapped on the rear edge of the Flat Plate by a Right-hand Corner Bracket and this can be seen in Fig. 12. Twenty holes back from the boom head, a 2\(\frac{1}{2}\)'' Flat Girder makes an overlay joint under the lower pair of 24\(\frac{1}{2}\)'' Girders.
All of the double bracing struts for the boom are set at an angle as shown, starting with 2\(\frac{1}{2}\)'' Strips five holes in for the top girders, angled to seven holes in for the lower girders. Across the boom head, a 5\(\frac{1}{2}\)'' Braced Girder 55 forms a bracing panel from hole deep and this is reinforced by 2'' Girders vertically at the corners and fronted by Girder Brackets 56 which also includes the machinery house and service gantry. Note the counterweight compartment beneath the machinery house, the author filling this with sand held in a strong quality plastic bag. Fig. 15, below right, shows the rear boom section viewed from above, with the machinery housing removed to show the hoist and trolley winch and driving motor. Also, note the solid deck plating...
carrying 2½" Curved Strips, stood off by Fishplates at the upper ends to form journals for the head wheel of the crab-trolley chain-drive. Note that Cord Anchoring Springs locate this Axle Rod at the boom head and that a 50–t Gear Wheel 67 plays the role of a sprocket wheel. One 4½" Angle Girder runs behind the upper edge of the Braced Girder and is fixed under the top 5½" Flat Plate to give a firm bracing.
The second pair of bracing struts for the boom are almost vertical and are made from 2½" Double Angle Strips 58. Due to the limited numbers of short Strips in the No. 10 Set, Double Angle Strips are pressed into service to brace the boom where possible. A general pattern for the arrangement of bracings is clear from Figs. 12 and 13, provided through 3", 3½", and 4" Strips as available. Where obvious joints are shown in the bracings, combinations of lapped 2½" or 3½" Strips are used. Towards the back, the boom frame opens up to accept 3½" Strips and these are shown in Fig. 13 but these are not yet as the boom frames are near completion when they are married to the 18½" Girders protruding from the centre box section of the boom. Because of the staggers in the 24½" Girders, rear extensions are required. Lower channels are extended rearwards by a 9½" Girder 59 outwards and a 9½" Girder inside. To ensure straight and strong joints here, the underside of the channel is lapped by a 9½" Flat Girder 60, first portions of which can be seen in Fig. 10. The rounded hole flange of the topmost pair of 24½" Angle Girders form the running rails for the crab trolley and the inward ends are bolted on to the leading edges of the 9½" Flat Plate running across the boom just in front of the crab driving gear. Three holes overlap is required on to the Flat Plate and then, starting at the top of the vertical forward 5½" Girders of the central box section first a 3½" and then a 3" Flat Girder are used to overlays on the top girders at the same time. The join of the outer top 24½" Girder to the 18½" Girder is overlayed at the side by a 5½" Strip 61.
A pair of 3½" Angle Girders form journals for the crab driving gear and are joined to the rear end of the crab rails by 2" Flat Girders 62, mounted vertically as shown in Fig. 13, together with 1½" Corner Brackets which act as rear stops for the crab trolley. A slipping clutch mechanism is provided as follows: on the final drive shaft for the crab chain is a 57–t Gear Wheel taken the place of a sprocket and is pressed against a 1" Fixed Pulley with Rubber or Plastic Ring, by a Compression Spring and Collar. No Grub Screw is put into the Gear Wheel which revolves only under the friction drag and will slip when overloaded at either end of the crab chain run. A second gear wheel, this time a 50–t Gear, is also fixed to the shaft and is then driven by a 25–t Pinion and this can be seen in Fig. 13. A Journal for the longitudinal Worm drive shaft 63 is provided by a 2½" Curved Strip bolted to the vertical slotted flange of a 4½" Angle Girder across the boom and shown. The Worm at the end of the 11½" Rod engages with a 19–t Pinion on the reduction shaft.
Lifted, additional ballast in the form of lead weights should be substituted for the sand bag. Additional reinforcing for the lower 12½" Flat Girders on the rear boom is provided by internal 12½" Strips and a cross bracing below is provided by sandwiching 3½" x 2½" Flexible Plates and attaching them across the lower rear of the boom by Angle Brackets. Single strut bracing is added as shown, using appropriate Strips and Double Angle Strips as available.
**HOIST AND CRAB TROLLEY GEAR**
Figs. 15 and 16 show the hoist and trolley mechanism which is mounted in a pair of 3½" x 2½" Flanged Plates 66 supported by 3½" Angle Girders secured to the decking. One Plate has been removed to show the gearing clearly as seen in Fig. 16. The position of the Plates and Powerdrive Motor is also clear from the illustrations and it will be seen that a 19–t Pinion on the motor shaft engages with a small Contrate Wheel to drive the first shaft of the gear box. From here, a 25–t Pinion drives the worm gear shaft via a 50–t Gear Wheel 67 and this shaft drives the drive by 1" Sprockets to a take-off shaft, three holes back from the front of the gearbox and two holes up. This shaft runs continuously when the motor is switched on. Drives to the crab or hoist are taken off by sliding Pinions from a 19–t Pinion 70 fitted in a hole just inside the sideplate. A rear bearing for the 11½" Axle Rod 63 to the Worm drive is provided by a 4½" Double Angle Strip 69 mounted across the front of the gearbox two holes up and the Rod is fitted with a small Contrate Wheel at its far end. A Shaft, operated by a hand sliding shaft, moves the 19–t Pinion into engagement with either side of the small Contrate Wheel to effect reversing of the crab-trolley drive. Two Pawls with Boss 70 are fixed to the gear-shift shaft and the rear Pawl drops between a pair of Collars. In the fully 'off' position the 19–t Pinion for the crab is completely free of the long-face Pinion.
A take-off drive to the hoisting drum is provided by a 19–t Pinion 71 operated by yet another sliding shaft and this can be seen in the centre of the mechanism shown in Fig. 16. A Socket Coupling 71 is locked both to its shaft and to a 19–t Pinion and on the far end of the shaft a ½" face 19–t Pinion 72 passes on the drive to the winding drum and also
**Fig. 17**, a close-up view of the service hoist which, despite its comparatively simple construction, is nicely operational.
provides the 'stop' position when the shaft is disengaged. A simple, but effective, brake is provided by a Set Screw (NOT a standard Bolt!) in the Collar outside the gearbox on the take-off shaft mentioned so that when the hoist is not in use and pulling the control lever inwards, the Set Screw locks against the external Collar on the adjacent shaft. It is important to fit that Collar with a 3mm Grub Screw to prevent 'knocking' every time the Collar rotates, which it will do all the time the motor is running. The control lever for the hoist is a long Bolt in a Collar, threaded on a 4½" Rod with a Coupling in the centre of the Rod, fitted with two 1" Rods as a 'fork' for shifting the hoisting gear into mesh via the Socket Coupling.
Fig. 15 shows the twin hoisting drums in position which are made up as follows: first, a 2" Pulley 72 acts as a spacer, boss outwards, followed by a 2½" Gear Wheel also boss outwards. Preceding to the centre of the gear is a Conical Disc taking the closed end of a Chimney Adaptor against it. Inside the Chimney Adaptor is a Cord Anchoring Spring to which one end of a hank of Cord is attached after first passing the Cord through the side hole of the Chimney Adaptor. The process sounds fiddly, but is quite straightforward if done with a bit of patience and common sense and care is taken to ensure that the spring is rotated in the 'uncolling' sense when locating it centrally inside the Chimney Adaptor. A back-to-back pair of Conical Discs 73 are now put on to the shaft to separate the twin drums and the second drum fitted as described. Finally a second 2" Pulley traps the outer Conical Disc in place and all bosses are securely locked with Grub Screws. With the spacing described, the 2½" Gear Wheel will mesh correctly with the ½" face 19-Pinion below. A control switch rod for the Powerdrive motor is mounted in a 2½" x 1" Double Angle Strip 74, and passes through of the boom for reversing and stopping. Two 2½" Narrow Strips, spaced by Nuts, straddle the motor lever as shown in Fig. 15 and are held in place by a Driving Band through a ½" Angle Bracket bolted to the deck. Cranks, or long Bolts in Collars are used as shift levers on the Rod operating the motor switch.
CRAB TROLLEY
This is shown in Figs. 12 and 18 and the view from below should be studied first. The original trolley shown in Fig. 18 uses two Double Bent Strips 75 to support and space the lower pulley shaft. However, four Double Bent Strips are required to give the proper support for the turntable drum mounting on the crane. These are the Corner Gussets to replace these parts and this will hold the shaft in place. Both Ratchet Wheels are retained to give correct spacing, with the internal Spring Clips, for location of the two 1" fixed Pulleys which are free to turn on the shaft and form part of the drive system. It will also be noticed that our Cord Anchoring Springs are used on the front and rear draw bars of the trolley to locate the Axle Rods in place. These should be replaced with Spring Clips, one lug of which is trapped between the side Flat Girders of the trolley and the flange of the 3½" x 25½" flanged Rod. Double Position Plates are provided to take up slack in the Sprocket Chain drive and thus maintain tension throughout. All four 1" fixed Pulleys used as travelling wheels for the crab are free to turn independently, one being fixed by a Grub Screw on each 4½" Axle Rod while the second Pulley on each Rod is held in place by a Collar.
Upper construction of the crab is shown in Fig. 16, overlaying 5¾" Strips 76 on the Flat Girders forming journals for the travelling wheel shafts. A pair of 2½" Triangular Plates 77 are joined across the top by a 3½" Screwed Rod running into Threaded Bosses and 2" Slotted Strips 78 are separated by a Spring Clip and lock-butteted into the centre of the Slotted Rod. These form internal spacers for a pair of 1" loose Pulleys which themselves are spaced by a Washer or two from 1½" Pulleys 79, boss outwards, these being located by Collars. The 'spring' effect of the Formed Slotted Strips securing the fore and aft 3½" x 25½" Flanged Plates 80 holds the pulley shaft in place. The important fact is that the sides frames of the trolley are spaced from the flanges of the 3½" Flanged Plates by Spring Clips on ¾" Bolts, eight of each being required and located in the positions shown. The slight upwards tilting of the Flanged Plates gives added rigidity to the trolley, apart from improving its appearance.
HOOK BEAM AND PULLEY BLOCK
Details of the block can be seen in Fig. 12 where six 2" Strips 81 form separators for the four 1" loose Pulleys running on a 1½" Axle Rod. This is trapped between the centre 2" Strips by a Spring Clip. Long Bolts are provided with a pair of lock-nuts at the top and bottom of the pulley block to set up critical spacing for free running of each Pulley. When fitting the lower 1 ½" Bolt, a 1" x ½" Double Bracket 82 has to be juggled into position and set to swing freely. The 1½" Bolt with Collar spacing passes through the Double Bracket into the suitable Strip and is secured by lock-nuts, allowing the hook beam to revolve freely. Reversed pairs of 5¼" Curved Strips form the actual beam and the Pivot Bolts employed use internal Washer spacing to maintain the universal pivoting properties of the beam blocks which are attached by Lock-nut Bolts to the outer Double Bent Strips. All bolted elements of the hook beam must be set and spaced to swing freely.
HOIST SYSTEM
The double hoist system employed permits the winding drums to "coil" at slight differences without tilting the pulley block and the first reeving of each separate cord from the twin drums is over the top 1½" Pulleys 79 of the crab, through the lower block, up round the top inner 1" loose Pulleys, down through the block and then up, and over the lower pair of 1" fixed Pulleys (four in all) running in the lower portion of the crab. The twin tails of the cords then go up round the compensating Pulley 83 at the boom head where they are knotted together. Very little movement of the cord will ever be seen at this point, but the compensation for drum 'coiling' differences is quite automatic. Two 3½" Double Angle Strips form supports for the compensating Pulley, a 1" fixed Pulley free to revolve over a Wheel Disc on a 1" Rod fixed in a 2" Pulley bolted to the boom head brackets (Corner Gussets 52).
Fig. 18, an underside view of the gantry crab trolley. Note that one of the two Double Bent Strips 75 should be replaced with a Crank.
MACHINERY HOUSE & SERVICE GANTRY
Fig 14, shows most of the machinery house and service gantry which is of simple and open construction. The housing is 12½' long, using 12½" Strips with 5½" Double Angle Strips (preferred) so that Threaded Pins, either long or short, can be fitted to the bottom lugs of the Double Angle Strips and will thus allow the machinery house to be easily and quickly removed or located in the rear boom. It should be remembered that the structure forms part of the general balance or counterweighting of the whole boom. A gantry is formed from the last remaining pair of 18½" Angle Girders 84 in the No. 10 Set and these are supported by a pair of 9½" Strips 85 and 10" struts, see Fig. 14. Curved Strips (4½") 86 and 1" x 1½" Angle Brackets make vertical supports for the gantry rails and spacing for the rails is supplied by lock-nuts on a 3" Screwed Rod at the rear end and by a double thickness of 3" Curved Strips under the front end of the 18½" Girders. Rail 87 is made from twelve 5½" x 2½" Flexible Plates curved to a radius of combined 3" and 2½" Curved Strips at either end, each side of the roof overhanging the machinery housing by the width of Double Brackets or ½" Reversed Angle Brackets. Four 12½" Strips are used on the roof, two along the top, 2½' apart, and two more along the lower edges each side.
There are enough parts left in the No. 10 Set to make a working service hoist as shown in Fig. 17. One 28½" x 12½" Double Angle Strip and one 2½" x 1" Double Angle Strip 87 are bolted to a pair of 8-hole Wheel Discs and fitted with four 1½" Plastic Pulleys, as shown, by ¾" Bolts. A 1½" Rod runs lengthwise through the bottom holes of the Wheel Discs and is rotated by Sprocket Chain in 10" loop over a 1" Sprocket Wheel. This shaft carries a Worm which meshes with a 19-t Pinion 88 on a 2" Rod across the centre of the hoist. One end of an 18½' length of Sprocket Chain is secured to an end Bolt of the hoist and then passed over a 1½" Sprocket 89 above the centre shaft and fitted with a Hook at the free end of the chain. Setting of the gantry rails should be to allow full run of the hoist right through the machinery house.
Final details are added by last stages of ladder work right up to the machinery house where even the 1½" Rack Strips 90 are pressed into service (as ladder sides! see Fig. 19). Six ½" Double Brackets form the first section of steps and a Handrail Support set in a Threaded Crank at the top of the steps takes a Crank Handle for the last section of safety rail.
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SINGLES TO WATCH
HARD SUN
Indio
HARRY HOUDINI
Kon Kan
WILDFIRE
The Nylons
KEEP EACH OTHER WARM
Barry Manilow
DON'T SAY YOU LOVE ME
Billy Squier
WE CAN LAST FOREVER
Chicago
TOY SOLDIERS
Martika
HOT ALBUMS
MADONNA
Like A Prayer
ROXETTE
Look Sharp
GUNS 'N' ROSES
G'N'R Lies
SIMPLY RED
A New Flame
ROD STEWART
Out Of Order
THE CURE
Disintegration
SIMPLE MINDS
Street Fighting Years
TIN MACHINE
Tin Machine
STEVIE NICKS
The Other Side Of The Mirror
THE DOOBIE BROTHERS
Cycles
CYNDI LAUPER
A Night To Remember
ROADHOUSE
Soundtrack
JEFF HEALEY BAND
See The Light
GREAT WHITE
Twice Shy
K.D. LANG
Absolute Torch & Twang
ANDREW CASH
Boomtown
THE OUTFIELD
Voices Of Babylon
COUNTRY
TO WATCH
ALBUMS
IAN TYSON
I Outgrew The Wagon
SINGLES
MORE THAN A NAME ON THE WALL
Statler Bros.
TIMBER I'M FALLING IN LOVE
Patty Loveless
FULL MOON FULL OF LOVE
K.D. Lang And The Reclines
COULD YOU LOVE A WORKING MAN
Staker Bros.
TALK TO A LONELY MAN
Tim Taylor
CRY
Waterfront
BUFFALO STANCE
Nenah Cherry
THE DOCTOR
The Doobie Bros.
EXPRESS YOURSELF
Madonna
BABY DON'T FORGET MY NUMBER
Milli Vanilli
IF YOU DON'T KNOW ME BY NOW
Simply Red
THIS TIME I KNOW IT'S FOR REAL
Donna Summer
SO ALIVE
Love And Rockets
LAY YOUR HANDS ON ME
Bon Jovi
I'LL BE LOVIN' YOU (Forever)
New Kids On The Block
BOOMTOWN
Andrew Cash
WHAT YOU DON'T KNOW
Explosions In The Sky
THINKING OF YOU
Sa-Fire
DRESSED FOR SUCCESS
Roxette
CRAZY ABOUT HER
Rod Stewart
DO YOU BELIEVE
One 2 One
SINGLE PICK
DONNY OSMOND
SACRED EMOTION
Donny Osmond
Capitol - 7PRO-79608-F
ALBUM PICK
THE B-52's
Cosmic Thing
Reprise - 92-58541-P
ALBUMS TO WATCH
LOVE AND ROCKETS
Love And Rockets
BLUE MURDER
Blue Murder
HERBERT GRONEMEYER
What's All This
Roxette's Per Gessle and Marie Fredriksson discuss their breakout of Sweden. The duo is rapidly closing in on the success of 1970's supergroup, ABBA, with the hope of finding the same magic in North America.
MCAG challenges CAB's Strategic Objective No. 2
In a tersely worded press release, the Music Copyright Action Group (MCAG) challenges Strategic Objective No. 2. This plan was recently presented by the Canadian Association of Broadcasters (CAB) to "promote the development of a Canadian music strategy that effectively mobilizes the resources, interests and capabilities of broadcasters, the music industry and the Federal Government."
The group was annoyed that the CAB "would take it upon themselves to speak on behalf of the music industry and outline plans affecting some of the most vital interests of Canadian songwriters, music publishers, record producers and record companies with no discussion whatsoever, while representing that the strategy is in the 'best' interests of the music industry."
The MCAG further suggests that the CAB strategy "focuses on commandeering a proposed home taping royalty for rights holders, opposing a performing right and a performers right in sound recordings and rejecting the idea of increasing Canadian content regulations on the FM-band to match those in effect on AM radio."
The CAB proposal apparently suggests that monies from a government run home taping levy "should be used as the primary source of funding for a proposed 'Canadian Music Foundation', the major aim of which would be to increase the production of Canadian recordings, making many more 'Canadian content' records available for airplay."
In answer to this, the MCAG points out that both the organization and its individual members have, "over the years, witnessed many actions originating from the Canadian Association of Broadcasters or their individual members which have run contrary to the vital interests of Canadian Copyright owners: the broadcasters strongly oppose the increase of Canadian content levels and generally regard minimum content quotas as a maximum when it comes to programming of Canadian music."
It was further suggested by the music group that in view of the Canadian government seriously considering introducing a home taping royalty, "The CAB would like to see these new payments flow into a yet to be established 'Canadian Music Foundation' having wide ranging goals from support of record production to domestic touring," but would, as the group suggests, "virtually exclude any compensation to copyright owners who should be the primary beneficiaries of the home taping royalty."
The music group then zeroes in on the fact that broadcasters "not only refuse to pay for use of their prime programming tool, the recordings they play, a payment which broadcasters in many countries fully accept, but they also wish to use monies from another piece of the creators' rights to reach their goal of more records to play, while not increasing the level of Canadian content."
In conclusion, the MCAG broadsides the broadcasters with, "The message is very clear, 'let's use your money to solve our problems'. It is extremely regrettable that such an approach should be put forward as a policy option."
For further information contact Brian Chater at 416-593-1665 or Paul Berry at 416-926-1666.
First quarter sales up 12 percent - Cinram
Cinram's Chief Financial Officer, Lewis Ritchie, has revealed that Cinram's sales for the first quarter (ended March 31/89) were $10,153,000 over sales of $9,080,000 for the corresponding period last year. This increase in sales of about 12 percent was the result of increased shipments of cassettes and compact discs.
Earnings, however, were down, $642,000 or $0.08 per share for the first quarter, as opposed to $818,000 or $0.11 per share for the comparable 1988 period. The average number of shares outstanding during the first quarter was 8,309,000 vs. 7,641,000 for the same period last year.
As Ritchie points out, earnings for the first quarter were "adversely affected by a faster than expected decline in demand for vinyl records and by higher labour costs."
Vinyl record production apparently made a "positive contribution" to Cinram's earnings of last year, but created a reduction in earnings for the first quarter of this year. Higher labour costs have resulted from lower productivity in record manufacturing and what Ritchie describes as a "tight Toronto labour market."
Ritchie goes on to stress that "compact disc operations were profitable in the first quarter of 1989 and are expected to add significantly to earnings in the latter part of the year when business is traditionally strongest."
In conclusion, Ritchie is enthusiastic over the strong showing of cassette and compact disc configurations, "and based on anticipated demand for both cassettes and compact discs," he concludes, "we are optimistic about the balance of the year."
I.R.S. Canada to release Moranis comedy album
I.R.S. Records Canada has entered into an "exclusive" arrangement with comedian Rick Moranis for the release of his debut solo comedy album, entitled You, Me, The Music and Me. The album, which mixes music with spoken word, will be released later this month.
A native of Hamilton, Ontario, Moranis began his career as a comedian with the SCTV troupe. He teamed with Dave Thomas as the popular "hoser" duo of Bob and Doug Mackenzie, both of whom became household words in Canada and the U.S. He went on to establish his film career with appearances in Ghostbusters and Little Shop of Horrors, and this year appears in three major motion pictures, Ghostbusters II, Honey I Shrunk The Kids and Parenthood, all set for release this summer.
His I.R.S. debut introduces another SCTV favourite, Gerry Todd, and his own strange world of radio, music and the weather. A music video clip of Ipanema Rap teams Moranis with Eugene Levy and "a cast of thousands" to update the classic Girl From Ipanema.
Laing and Timmins out in Westwood One shuffle
In a recent restructuring, Westwood One Canada has lost two of its highly experienced top management people, one who was let go, the other who left on his own.
Gerald Laing, a very popular and dedicated broadcaster, particularly in the syndication field, was one of the major architects in building a base in Canada for the U.S. company. Laing, who was Vice President and General Manager of the Canadian operation, was advised in mid-March that his services were no longer needed.
Randy Timmins, who has been with the syndication company for almost three years, has tendered his resignation, effective the end of June. Timmins was also a major player in the company's establishing itself as a leader in the syndication field. He will now base himself in London, Ontario and concern himself with consulting work. Prior to joining Westwood One Canada, he worked with J. Robert Wood on his application for a new Toronto radio station. Timmins will be replaced by Don Bradley, Corporate Sales Director of CFMX-FM in Cobourg.
The following is a list of the most important and frequently used terms in the field of computer science:
1. Algorithm: A step-by-step procedure for solving a problem or performing a task.
2. Data Structure: A way of organizing data that allows efficient access, modification, and manipulation.
3. Database: An organized collection of data stored in a computer system.
4. Database Management System (DBMS): Software that manages databases and provides an interface for users to interact with them.
5. Encryption: The process of converting information into a code so that only authorized parties can understand it.
6. Hashing: A process of converting data into a fixed-size string of characters.
7. Interface: A way for two systems to communicate with each other.
8. Network: A collection of computers and devices connected together.
9. Operating System (OS): A software program that controls the hardware and software resources of a computer.
10. Programming Language: A set of instructions that a computer can understand and execute.
11. Query: A request for information from a database.
12. Security: The protection of data and systems from unauthorized access, use, disclosure, disruption, modification, or destruction.
13. Software: A set of instructions that tell a computer what to do.
14. System: A collection of related components that work together to achieve a common goal.
15. User: An individual who uses a computer or other electronic device to perform tasks.
16. Virtualization: The creation of virtual versions of physical resources such as servers, storage, and networks.
17. Web Application: A software application that runs on a web server and is accessed through a web browser.
18. Wireless Network: A network that uses radio waves to transmit data between devices.
19. XML: eXtensible Markup Language, a markup language that defines a set of rules for encoding documents in a format that is both human-readable and machine-readable.
20. YAML: Yet Another Markup Language, a markup language that is used to represent structured data.
Boring press receptions . . . ! Parties, press conferences, showcases . . . and all in the same few places. It gets to be monotonous going to the same few rooms to attend these events. Too bad the industry doesn't instill some excitement into these events. They can't seem to entice the dailies into coming back to industry bashes. It seems that every invitation wants you to come to the same place where you were a couple of nights before. There are some exceptions, but when it comes to record oriented events . . . the same old place, same old bad service and the same old street-wise hangers-on. Sometimes I feel that someone out there is going to come up with a guest list of people who OUGHT to be there. As I said, there are some exceptions! (EC: You should name the clubs that have been used to death!) They know . . . they know!
Out goes the 7" single . . . ! I got it from pretty good authority that a major will, within the next month, discontinue their 7" product to radio. They will be including these tracks on their CD compilation disc. Sounds fairly straight ahead . . . wouldn't you say? Why all the secrecy? I would think they would like to be the first out of the gate with this information. (EC: They have to think it over first . . . !)
A jewel of an idea . . . ! Those CD singles that come in jewel boxes are such a waste. They are cumbersome, heavy and hard to handle. It's good to see a new plastic sleeve that just holds the CD single like the old paper record sleeves. It even makes the product look better. We've already seen a few and I hope the idea catches on. (EC: You read it here first!)
The unsung heroes . . . ! One of the disturbing things about this industry is the general don't give a damn attitude by recording artists. They don't seem to realize, nor care that very concerned people are working on their behalf to ensure there is an industry for them. They seem to think their talent . . . and their talent alone is all that's necessary to launch their careers. Oh! How wrong they are. I think it would do them a lot of good to get involved in some of the organizations that are working for them. They haven't got the time . . . you say? Pity! Read page one this week! (EC: Having talent doesn't mean you can read . . . !)
What a sound . . . ! I thought something new was added at Roy Thomson Hall for the Roger Whittaker concert . . . and I was right.
Roger brought in his own sound equipment, and, for the first time in that supposedly acoustically-perfect hall, I could hear every word, and what a difference it made in the performance. By the way, Roger has never looked nor sounded better . . . from colourful customing to perform Cabaret and another quick costume change for Send In The Clowns . . . and a "right on" Presley bit . . . a delightful concert. I didn't see any of the snoops from the dailies there. (EC: This was a class concert . . . !)
Now . . . that's teamwork! Once again I was invited to attend the annual convention of CBS Records Canada . . . and once again I was taken with the dedication and team spirit shown by the 120 or so staff who were in attendance. CBS may not be on top of the heap when it comes to "hit" product, but they know how to sell catalogue and that's the bread and butter when the going gets tough with mainstream product. But you know what they say . . . the industry works in cycles, and when it's their turn, the CBS crew will be ready to move. (EC: The industry moves in circles . . . ?)
Here's a real troupier . . . ! I have just learned that a well-known industry personality will shortly undergo a very painful operation . . . for the benefit of someone else, which I'm not at liberty to reveal at this time. Anyway, his parting comment was "Tell 'em JB is now hooked on Demerol!" (EC: That's a relief!)
Publicists are hard to find! I was sorry to hear that Rhonda Ross is leaving Capitol, where she was in charge of National Artist and Media Relations. Mary Ann Girrard and Steve McAuley are filling her ever so unique shoes, temporarily. (EC: Ever so unique shoes . . . ?)
Facts about FAX...paper! Boy, the FAX people saw us all coming. At this end we get a lead sheet to tell us a lead sheet is coming and at the end of the day we have a wastepaper basket filled with lead sheets. The FAX paper suppliers must be laughing in their sleep. But watch those FAX messages that come FROM RPM and you'll see a header on the first page that says everything. No funny drawings, no empty sheets with a few words on it. We don't waste your FAX paper or line the pockets of the FAX paper suppliers. (EC: How many trees have been cut down in the name of the record industry . . . in wasted lead sheets alone?)
Winnipeg throws support behind White Heat
Winnipeg's Rock For Harvest was both a fund-raiser and a video shoot, and the major benefactors were local band White Heat.
The event to raise food items for the Winnipeg Harvest Food Bank was co-sponsored by 92 CITI-FM and the Winnipeg Sun.
The game plan was for White Heat to shoot a major production concert video for its debut album release due this month on The Record Label, an independent label distributed nationally by CBS Records. Joining White Heat and helping pack out Winnipeg's 800-seat Rendez-Vous Club was Jeffrey Hatcher & The Big Heat, The Johns and Monuments Galore.
The video for the first single; Rollin' With The Thunder, was shot under the supervision of director Don Allan, who has worked with Glass Tiger, Triumph, Gowan and others. "It was an amazing event," says White Heat's vocalist Jim Galloway, "Winnipeg really got behind this and made it the rock charity event of the year."
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ROACHFORD - Pop
Roachford - Epic - BFE-45097-H
Considered by some members of the British music press to be one of the hottest acts of the year, the debut release by Andrew Roachford has reached the side of the Atlantic and the reception has been great. All of the ten tracks on this LP were written by the artist himself, and they are performed with a rawness and energy reminiscent of the days of Sly and The Family Stone. This guy should achieve mega-success, both in the clubs and at the record level. A recent showcase at Toronto's Diamond Club proved he's no slouch on the stage either. The first single, Cusky Tony (Don't Make Me), has been a strong add on CHR stations and don't be surprised if Family Man is chosen for the follow-up. This is a strong debut from an artist who will certainly be one to watch over the next few months, and by early indications, he is well on his way to making his mark on the pop/dance scene. - DS
ARETHA FRANKLIN - Pop
Through The Storm
Arista - AL-8572-N
On the opening track, Gimme Your Love, Franklin belts out "I'm the Queen of Soul - you mess with me and you're gonna burn", and by the sound of this LP she's not kidding. Over twenty years in the business, she's never sounded better from mellowing out. Highlights here include a duet with Whitney Houston (It's So Hard To Be In Love), a duet with George Benson, and the title track, a duet with Elton John that was a great choice for the first single. Also worth checking out is Think (1986). This is the first time she's recorded this track (1968 and 1980), and this time she has really funk'd it up. One of her best collections in years. - DS
WILLIE P. BENNETT - Country
The Lucky Ones
Duke Street - DSR-31059-J
A couple of decades later this smooth, country crooner finally crashes the barrier of indifference, perhaps some of it belonging to himself, but certainly the media of the day who have too conveniently written him off. When Canadian recordings. He was no slouch when it came to record releases... more than a few with the exception of a solo album, but this time, it's for real: a real record label and a real media audience. Willie dishes up a potpourri of what could be the highlights of his life, and no one could illustrate these life excursions better than Willie himself. He has a simple, clean, smooth vocal delivery, and he has the best session people in the business to cushion his vocals with mouth harping, including a few tracks done on fiddle and mandolin, keyboardist John Sheard and drummer Al Cross. The title track is already charting. Also key are Train Tracks and Andrew's Waltz. Produced by Danny Greenspoon at Toronto's Inception Sound. - WG
HANDSOMENED - Country
The Ballad Of Handsome Ned
Virgin - V-2004-1
Handsome Ned was a cult figure of Toronto's Queen Street, who became somewhat of a legend with music critics even before his early and untimely death in 1987 at the age of 29. Handsome Ned was a clever story teller, more in the folk vein, but with his own unique style and distinctive stylings. These are studio recordings of The Sidewinders and The Handsome Ned's from 1984 to 1985, plus live solo performance of one of Rail Blues. This isn't perfection as production goes, but it's real and it connects with a purity that's somewhat awesome. - WG
BLACK SABBATH - HM
Headless Cross - I.R.S.-IRS-82002-J
Once the leading force in heavy metal, Black Sabbath has resorted to relying on the cliches of the genre to generate they pleased. Dungeons & Dragons lyrics and this type of guitar heroism have been done to death the past two decades. Even the Dark Lord himself must wish these aging rockers would give it a rest. - CM
FIGURES ON A BEACH - Pop
Figures On A Beach - Sire - 92-5044-15
Why is it that many new bands choose to release a cover as their first single? It's usually a sign of no originality on the band's part, but not this time. Figures On A Beach's Ain't Seen Nothin' Yet may be getting the group noticed (especially in this country) but one would only guess that these Detroit natives haven't fade into obscurity once the novelty of the single wears off. There's good material for campus radio and club DJs to choose from, Accidentally 4th St. and (Don't Make Me) Nervous being the best of the bunch. A strong debut that deserves more than just an 'oh no, another cover' reception. - DS
DIK VAN DYKES - Rock
Waste Mor Vinyl - OG-24-E
Fans of this campus cult group will not be disappointed by this, the band's second album. Acoustically, the fourteen tracks vary from barely produced to tastefully under-produced, and form a rich, tongue-in-cheek humor that won them their song audience. Standout songs include Chain Letter Massacre, Leave This City and the title track, Among The Men Of Love. Rightfully so, jacket thank-you go to the Ramones, clearly a tremendous influence on this Hamilton, Ontario outfit. - CM
PATO BANTON - Pop
Visions Of The World
IRS - IRS-82003-J
A disappointing, inconsistent effort from ex-cash former DJ. While Bad Man And Woman, Ready Me Ready and Pressure hearken back to previous dance hall material, the remainder of the album is devoted to lifeless slow funk and reggae-influenced reggae distinctly lacking in balls. - CM
ROBERT HEATH - Country/Pop
Play On - Big Stick - BS-1001-E
This is the debut LP from Edmonton's Robert Heath, who has captured more than a few ears from A/C and country programmers. He is currently charted on the RPM Country 60, BMI / Warner Bros. Radio and RC and CHR programmers are going with another single release, Let's Go Round The World Tonight produced by Heath and Van Willink at Edmonton's Damon/Soundtrax Studios. Good stuff, should have a long chart life. - WG
THE GODFATHERS - Pop
More Sex, More Love & Hate
Epic - FE-45084-H
Danceable poprock with a hint of an edge, this effort sounds remarkably like it was made in the late '70s, which is not to be taken as a criticism. However, the title is apt and unfortunately, there is nothing refreshing or groundbreaking to be found here. - CM
THE WAILERS BAND - Reggae
I.D. - Atlantic - 76-19001-P
Considering what these musicians were produced under the direction of Bob Marley, the lifeless backbeat of The Wailers Band is not entirely disappointing. Victims of technology, the group has adopted synthesizers and sampled drums, spent time in first rate studios and delivered a perfectly clean package sorely lacking in bite. - CM
UIC - Rock
Like Ninety - OG-22-E
Recorded live at Toronto's Lee Palace, this album is full of the kind of raunchy rock and roll that made the pogo such a dance craze. The sound quality, while not on par with the newer UIC major release, is not bad but, more importantly, the performances are given with intensity and guts. Campus and alternative radio should definitely be grateful for such a genuine artifact! - CM
THE STOKER BROTHERS - Country
The Wild One
Northern Star - NS-001
An appropriate title for this tight unit from Brantford, Ontario, who are vocally powerful and whose sound is awesome. Their lyrics have a rock edge that demands attention and their vocal projection goes far beyond the usual country limits. This is best illustrated by their current Comstock single, Could You Love A Working Man, which has an almost gospel impact as a video. Produced by Rick Whitehead and Mickey Sloker at Toronto's Inception Sound. Also key are the title track and Coupe DeVille. - WG
ROCKY SWANSON - Country
Rocky Swanson
Music Canada - ML-0004-E
Although a veteran in British Columbia country music, it was Swanson's debut single with Music Line, Give You Need, that finally put that brought him into national prominence. He enjoyed eleven weeks on the national chart, peaking at No. 10. His follow-up single, When A Heart Explodes, is now being readied for shipping. Swanson has a rare vocal smoothness that lends itself beautifully to the country sound and interpretation of the lyrics. Includes Tip Top, one of the top request tracks in the province. Also, written years ago by his dad and uncle, and released by the Swanson family as a tribute to the terminally ill uncle. Produced by Ralph Murphy in Nashville and Vancouver. Cancon is not indicated on the label copy. - WG
DALE JABOBS - Jazz
Coastal Zone
Narada - NMR-4031-W
This is contemporary jazz that should easily find itself a home on any one of a number of jazz radio programs across the country. Jacobs' bluesy style, the keys, and his smooth arrangements make this one a sure bet with fans of such contemporary artists as David Sanborn or Larry Carlton. All the cuts were written and produced by Jacobs himself, Philly Blues and Tinsel Town being two that stand out. - DS
BARBARA MANDRELL - Gospel
Precious Memories
Quartet Singers Products - RSPC-186
Released as a direct-mail album, this package will be one of the "hottest" inspirational albums on the market. With its gorgeous, soul-stirring, Mandrell following in the country field goes without saying, and with these types of selections, her vocal beauty is put to the ultimate test, in particular with Old Rugged Cross, Just A Little Talk With Jesus, Peace In The Valley, When We All Get To Heaven, Peace In The Blood and Let Me Live, plus more, twenty to be exact. Let Me Live will be released as a single. Produced by Tom Collins. Watch for a high-powered television campaign. - WG
Canyon Creek's Bev Marie and Backstage's Steve Thomson present Top Group award to Randall Prescott, Lawanda and Barry Brown.
CHAM's Cliff Dumas accepts his Country Radio Personality award from Carmen Westfall and Balmur's Leonard Rambeau.
A very emotional Weird Harold (Kendall) of CKWX Vancouver, is inducted into the Canadian Music Industry Hall of Fame.
Outstanding New Artist award presented to Carmen Westfall by CHAM's Keith James and Lawanda Brown of The Family Brown.
FACTOR's Heather Sym and Canyon Creek's Bart (The General) Barton present Randall Prescott with Producer of the Year award.
PROCAN's Nancy Gyokeres and CHOO's Joey Frechette present George Fox with his Best Country Single award.
CHAM's Keith James with Program Directors Gord Eno, Music Director Mark LaPointe and Cliff Dumas, accepting Top Station award.
Composer award to Fox from this year's Country Radio Personality, CHAM's Cliff Dumas (l) and '88 winner, CKGL's Randy Owen.
Savannah's Michelle Wright accepting her Big Country Award as Top Female Vocalist, as voted by subscribers to RPM Magazine.
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## RPM Country Singles
### Singles
| Rank | Artist | Title | Label | Catalog | Date |
|------|--------|-------|-------|---------|------|
| 1 | Steve Wariner | WHERE DID I GO WRONG | MCA | 53856-J | 7-87 |
| 2 | Ricky Van Shelton | BETTER MAN | MCA | 42272-J | 7-87 |
| 3 | Ricky Van Shelton | I DON'T WANT TO SPOIL THE PARTY | Columbia | 38-68959-H | 7-87 |
| 4 | Garth Brooks | THEY RAGE ON | Capitol | P-B-44349-F | 7-87 |
| 5 | Garth Brooks | I GUESS | Warner Bros. | 92-75677-P | 7-87 |
| 6 | Garth Brooks | AFTER ALL THIS TIME | Columbia | 38-68958-H | 7-87 |
| 7 | Garth Brooks | I WISH I WERE ONLY LONELY | Warner Bros. | 92-75676-P | 7-87 |
| 8 | Garth Brooks | LOVE OUT LOUD | RCA | 8824-7-R-N | 7-87 |
| 9 | Garth Brooks | SHE DON'T LOVE NOBODY | Warner Bros. | 92-75675-J | 7-87 |
| 10 | Garth Brooks | CALL ON ME | Capitol | 92-75674-P | 7-87 |
| 11 | Garth Brooks | HOLE IN MY POCKET | Columbia | 38-68948-H | 7-87 |
| 12 | Garth Brooks | I KNOW WHAT I'VE GOT | Warner Bros. | 92-75673-J | 7-87 |
| 13 | Garth Brooks | LOVING ONLY ME | Epic | 34-06893-H | 7-87 |
| 14 | Garth Brooks | BEYOND THOSE YEARS | MCA | 53625-C | 7-87 |
| 15 | Garth Brooks | 5:01 BLUES | MCA | 42205-P | 7-87 |
| 16 | Garth Brooks | WHAT'S GOING ON IN YOUR WORLD | MCA | 52648-J | 7-87 |
| 17 | Garth Brooks | BLUE JEANS BOY | RCA | PB-51027-N | 7-87 |
| 18 | Garth Brooks | UP AND GONE | MCA | 42211-H | 7-87 |
| 19 | Garth Brooks | IF I HAD YOU | MCA | 42210-H | 7-87 |
| 20 | Garth Brooks | THE MOON IS OUT TO GET ME | MCA | 42209-H | 7-87 |
### Albums
| Rank | Artist | Title | Label | Catalog | Date |
|------|--------|-------|-------|---------|------|
| 1 | Ricky Van Shelton | LIVING PROOF | Columbia | PC-44221 | 7-87 |
| 2 | The Judds | THE JUDDS | MCA | 42266-J | 7-87 |
| 3 | Blue Rodeo | DIAMOND MINE | Mercury | 92-56244 | 7-87 |
| 4 | Nitty Gritty Dirt Band | CIRCLE II | Universal | UVLC2-12500 | 7-87 |
| 5 | Randy Travis | RANDY TRAVIS | Warner Bros. | 92-57381 | 7-87 |
| 6 | Alabama | ALABAMA | MCA | 42265-J | 7-87 |
| 7 | K.T. Oslin | K.T. OSLIN | MCA | 42264-J | 7-87 |
| 8 | George Strait | BEYOND THE NEON | MCA | 42266-J | 7-87 |
| 9 | Reba McEntire | REBA MCENTIRE | MCA | 42265-J | 7-87 |
| 10 | Clint Black | KILLING TIME | MCA | 42264-J | 7-87 |
| 11 | Rosanne Cash | HITS 1978 - 1988 | Columbia | CK-45054-H | 7-87 |
| 12 | Shenandoah | SHENANDOAH | Columbia | PC-44468 | 7-87 |
| 13 | Rodney Crowell | DIAMONDS & DIRT | Columbia | PC-44076 | 7-87 |
| 14 | Merle Haggard | STRANGER THINGS HAVE HAPPENED | RCA | FE-40986 | 7-87 |
| 15 | Patty Loveless | THE BEST OF PATTY LOVELESS | MCA | 42223 | 7-87 |
| 16 | Randy Travis | ALWAYS AND FOREVER | Warner Bros. | 92-58347 | 7-87 |
| 17 | Hank Williams Jr. | HANK WILLIAMS JR. | Warner Bros. | 92-58344 | 7-87 |
| 18 | Anne Murray | ANNE MURRAY | MCA | 42224 | 7-87 |
| 19 | Dwight Yoakam | BREAKING FROM A LONELY ROOM | Warner Bros. | 92-57467 | 7-87 |
| 20 | George Fox | GEORGE FOX | MCA | 42223 | 7-87 |
| 21 | Kenny Rogers | SO LONG (REPRISE) | MCA | 42224 | 7-87 |
| 22 | Roy Orbison | ORBISON | MCA | 42225 | 7-87 |
| 23 | The Shooters | SOLD AS A BUNCH OF FLOWERS | MCA | 44326 | 7-87 |
| 24 | Murray McLauchlan | SWINGING ON A STAR | Capitol | C-1-92126 | 7-87 |
| 25 | Tanya Tucker | TANYA TUCKER | MCA | 42224 | 7-87 |
| 26 | Ronnie Milsap | STRANGER THINGS HAVE HAPPENED | RCA | FE-40986 | 7-87 |
| 27 | New York Tycoon | NEW YORK TYCOON | Sire | 92-57764 | 7-87 |
| 28 | Dan Seals | DAN SEALS | MCA | 42225 | 7-87 |
| 29 | The Family Brown | THE FAMILY BROWN | MCA | 42226 | 7-87 |
| 30 | Emmylou Harris | EMMYLOU HARRIS | Warner Bros. | 92-57765 | 7-87 |
| 31 | Billy Joe Royal | BILLY JOE ROYAL | MCA | 42227 | 7-87 |
| 32 | Michelle Wright | MICHELLE WRIGHT | MCA | 42228 | 7-87 |
| 33 | K.D. Lang | K.D. LANG | Sire | 92-57766 | 7-87 |
| 34 | Loretta Lynn | LORRETTA LYNN | MCA | 42229 | 7-87 |
| 35 | J.K. Gulley | J.K. GULLEY | MCA | 42230 | 7-87 |
| 36 | Steve Earle | STEVE EARLE | MCA | 42231 | 7-87 |
### Singles
| Rank | Artist | Title | Label | Catalog | Date |
|------|--------|-------|-------|---------|------|
| 21 | Dolly Parton | WHY'D YOU COME IN HERE LOOKIN'... | Columbia | 38-68766-H | 7-87 |
| 22 | Garth Brooks | NEVER SAY NEVER | Capitol | P-B-44349-F | 7-87 |
| 23 | Garth Brooks | SHE'S GOT A SINGLE THING IN MIND | Capitol | P-B-44349-F | 7-87 |
| 24 | Garth Brooks | MUCH TOO YOUNG (TOO DAMN OLD) | Capitol | 92-44329-F | 7-87 |
| 25 | Garth Brooks | CATHY'S CLOWN | Capitol | 92-44329-F | 7-87 |
| 26 | Garth Brooks | PLANET TEXAS | Capitol | 92-44329-F | 7-87 |
| 27 | Garth Brooks | LOVE WITH A CAPITAL "L" | Capitol | 92-44329-F | 7-87 |
| 28 | Garth Brooks | HOUSTON SOLUTION | Capitol | 92-44329-F | 7-87 |
| 29 | Garth Brooks | YOU AIN'T GOIN' NOWHERE | Capitol | 92-44329-F | 7-87 |
| 30 | Garth Brooks | LOVE HAS NO RIGHT | Capitol | 92-44329-F | 7-87 |
| 31 | Garth Brooks | HEAVEN ONLY KNOWS | Capitol | 92-44329-F | 7-87 |
| 32 | Garth Brooks | COME FROM THE HEART | Capitol | 92-44329-F | 7-87 |
| 33 | Garth Brooks | I THINK THAT I'LL BE NEEDING YOU | Capitol | 92-44329-F | 7-87 |
| 34 | Garth Brooks | I DIDN'T KNOW YOU | Capitol | 92-44329-F | 7-87 |
| 35 | Garth Brooks | SURE LOOKS GOOD | Capitol | 92-44329-F | 7-87 |
| 36 | Garth Brooks | SATURDAY NIGHT | Capitol | 92-44329-F | 7-87 |
| 37 | Garth Brooks | NEW MORÉ THAN A NAME ON THE WALL | Capitol | 92-44329-F | 7-87 |
| 38 | Garth Brooks | IT WON'T BE LONG | Capitol | 92-44329-F | 7-87 |
| 39 | Garth Brooks | IN A LETTER TO YOU | Capitol | 92-44329-F | 7-87 |
| 40 | Garth Brooks | SOWIN' LOVE | Capitol | 92-44329-F | 7-87 |
| 41 | Garth Brooks | TURN OF THE CENTURY | Capitol | 92-44329-F | 7-87 |
| 42 | Garth Brooks | GOLDMINE | Capitol | 92-44329-F | 7-87 |
| 43 | Garth Brooks | ONE GOOD WELL | Capitol | 92-44329-F | 7-87 |
| 44 | Garth Brooks | BROKEN HEART | Capitol | 92-44329-F | 7-87 |
| 45 | Garth Brooks | TIMBER | Capitol | 92-44329-F | 7-87 |
| 46 | Garth Brooks | FULL MOON FULL OF LOVE | Capitol | 92-44329-F | 7-87 |
| 47 | Garth Brooks | AM I THE ONLY ONE | Capitol | 92-44329-F | 7-87 |
| 48 | Garth Brooks | COULD YOU LOVE A WORKING MAN | Capitol | 92-44329-F | 7-87 |
| 49 | Garth Brooks | TALK TO A LONELY MAN | Capitol | 92-44329-F | 7-87 |
| 50 | Garth Brooks | BABY I WANNA DO RIGHT | Capitol | 92-44329-F | 7-87 |
| 51 | Garth Brooks | AM I THE ONLY ONE | Capitol | 92-44329-F | 7-87 |
| 52 | Garth Brooks | COULD YOU LOVE A WORKING MAN | Capitol | 92-44329-F | 7-87 |
| 53 | Garth Brooks | TALK TO A LONELY MAN | Capitol | 92-44329-F | 7-87 |
| 54 | Garth Brooks | BABY I WANNA DO RIGHT | Capitol | 92-44329-F | 7-87 |
| 55 | Garth Brooks | AM I THE ONLY ONE | Capitol | 92-44329-F | 7-87 |
| 56 | Garth Brooks | COULD YOU LOVE A WORKING MAN | Capitol | 92-44329-F | 7-87 |
| 57 | Garth Brooks | TALK TO A LONELY MAN | Capitol | 92-44329-F | 7-87 |
| 58 | Garth Brooks | BABY I WANNA DO RIGHT | Capitol | 92-44329-F | 7-87 |
| 59 | Garth Brooks | AM I THE ONLY ONE | Capitol | 92-44329-F | 7-87 |
| 60 | Garth Brooks | COULD YOU LOVE A WORKING MAN | Capitol | 92-44329-F | 7-87 |
| 61 | Garth Brooks | TALK TO A LONELY MAN | Capitol | 92-44329-F | 7-87 |
## RPM 40 CANCON SINGLES
| # | Artist | Title | Label | Catalog |
|---|--------|-------|-------|---------|
| 1 | Scott Jordan | DOUBLE TROUBLE | AOR 6044-F | Q1-532-E |
| 2 | Alannah Myles | LOVE IS | Atlantic | 78-9187-P |
| 3 | Candi | ANGEL EYES | Arista | AS1-9806-N |
| 4 | Diamond Mine | DIAMOND MINE | PRO-652-P | 25-62681-P |
| 5 | Candi | LOVE MAKES NO PROMISES | IRS | 53622-J |
| 6 | Victory Day | VICTORY DAY | Capitol | B-73085-F |
| 7 | Diamond Mine | (Watching) WORLDS CRUMBLE | PRO-652-P | 25-62681-P |
| 8 | Candi | NO SUCH THING | Capitol | B-73082-F |
| 9 | Collin James | CHICKS 'N CARS | Virgin | VS-1492-W |
| 10 | Real Love | UNLOST HEART | Sire | 92-76274-H |
| 11 | Candi | BEAUTIFUL WHITE | Capitol | B-73081-F |
| 12 | Bruce Cockburn | DON'T FEEL YOUR TOUCH | True North | TN7421-H |
| 13 | Bozomtom | BOZOM | Cash | 97908-J |
| 14 | Kipon Kan | HARRY HOUDINI | Atlantic | 78-95907-P |
| 15 | Spillbound | HARD SUN | Columbia | C-30828-H |
| 16 | Big Marwell | SHOOTING FROM MY HEART | SP-5257-W |
| 17 | Chantel | A LITTLE LOVIN' | Columbia | C-3087-H |
| 18 | The Nytons | DREAM COME TRUE | Atco | AT-988-P |
| 19 | Chantel | BABY DON'T FORGET MY NUMBER | Warner Bros | WB-80133-H |
| 20 | Rockapella | PATIENCE | CBS | X018-24186 |
## RPM 30 RETAIL SINGLES
| # | Artist | Title | Label | Catalog |
|---|--------|-------|-------|---------|
| 1 | Roxette | THE LOOK | Warner Bros | WB-80190-F |
| 2 | With Midas | WIND BENEATH MY WINGS | Atlantic | 78-95727-P |
| 3 | Like A Prayer | LIKE A PRAYER | Mercury | 874-012-Q |
| 4 | The Who | ROCK ON | Epic | 34-88541-P |
| 5 | Paula Abdul | FOREVER YOUR GIRL | Virgin | VS-1481-W |
| 6 | Girl You Know It's True | GIRL YOU KNOW IT'S TRUE | Epic | 34-88541-P |
| 7 | She Drives Me Crazy | SHE DRIVES ME CRAZY | Mercury | 874-012-Q |
| 8 | Lloyd Osmond | SOLDIER OF LOVE | Capitol | 7-PRO-79585-F |
| 9 | Tony Orlando | FUNKY COLD MEDINA | Warner Bros | WB-80124-J |
| 10 | Pat Benatar | PATIENCE | Geffen | SRL-29867-P |
## 20 DANCE SINGLES
| # | Artist | Title | Label | Catalog |
|---|--------|-------|-------|---------|
| 1 | Bobby Brown | EVERY LITTLE STEP | MCA | 23933-J |
| 2 | Milli Vanilli | THIS TIME I KNOW IT'S FOR REAL | Atlantic | 78-90987-P |
| 3 | George Michael | STAND | Warner Bros | 92-76887-P |
| 4 | Mike & The Mechanics | THE LIVING YEARS | Atlantic | 78-65647-P |
| 5 | Baby Don't Forget My Number | BABY DON'T FORGET MY NUMBER | Warner Bros | WB-80133-H |
| 6 | Milli Vanilli | REAL LOVE | Atlantic | 78-90987-P |
| 7 | Buffalo Stance | BUFFALO STANCE | Warner Bros | WB-80133-H |
| 8 | Undercover Lover | UNDERCOVER LOVER | MCA | 23933-J |
| 9 | Everything Counts | EVERYTHING COUNTS | Warner Bros | WB-80133-H |
| 10 | Que Te Pasa | QUE TE PASA | Warner Bros | WB-80133-H |
| 11 | Iko Iko | IKO IKO | Warner Bros | WB-80133-H |
| 12 | That's The Way Love Is | THAT'S THE WAY LOVE IS | Atlantic | 78-90987-P |
| 13 | Workin' Overtime | WORKIN' OVERTIME | Warner Bros | WB-80133-H |
| 14 | Best Friend | BEST FRIEND | Warner Bros | WB-80133-H |
| 15 | This Is Acid | THIS IS ACID | Warner Bros | WB-80133-H |
| 16 | Electric Youth | ELECTRIC YOUTH | Warner Bros | WB-80133-H |
| 17 | Once Around The Block | ONCE AROUND THE BLOCK | Warner Bros | WB-80133-H |
| 18 | What You Don't Know | WHAT YOU DON'T KNOW | Warner Bros | WB-80133-H |
| 19 | Bring Me Edelweiss | BRING ME EDELWEISS | Warner Bros | WB-80133-H |
| 20 | Sleep Talk | SLEEP TALK | Warner Bros | WB-80133-H |
## RPM 30 RETAIL SINGLES
| # | Artist | Title | Label | Catalog |
|---|--------|-------|-------|---------|
| 1 | Cher & Peter Cetera | AFTER ALL | Geffen | 92-75297-P |
| 2 | Pop Singer | POP SINGER | Mercury | 874-012-Q |
| 3 | Boyz II Men | REAL LOVE | MCA | 23933-J |
| 4 | Buffalo Stance | BUFFALO STANCE | Warner Bros | WB-80133-H |
| 5 | Iko Iko | IKO IKO | Warner Bros | WB-80133-H |
| 6 | Angel Eyes | ANGEL EYES | Arista | AS1-9806-N |
| 7 | Living Colour | CULT OF PERSONALITY | Epic | 34-88511-H |
| 8 | Heaven Help Me | HEAVEN HELP ME | Warner Bros | WB-80133-H |
| 9 | Every Little Step | EVERY LITTLE STEP | Warner Bros | WB-80133-H |
| 10 | Good Thing | GOOD THING | Warner Bros | WB-80133-H |
| 11 | Baby Don't Forget My Number | BABY DON'T FORGET MY NUMBER | Warner Bros | WB-80133-H |
| 12 | This Time I Know It's For Real | THIS TIME I KNOW IT'S FOR REAL | Atlantic | 78-90987-P |
| 13 | Stand | STAND | Warner Bros | 92-76887-P |
| 14 | The Living Years | THE LIVING YEARS | Atlantic | 78-65647-P |
| 15 | Satisfied | SATISFIED | Warner Bros | WB-80133-H |
| 16 | Dream Come True | DREAM COME TRUE | Warner Bros | WB-80133-H |
| 17 | The Doctor | THE DOCTOR | Warner Bros | WB-80133-H |
| 18 | Love Is | LOVE IS | Atlantic | 78-90987-P |
| 19 | Dressed For Success | DRESSED FOR SUCCESS | Warner Bros | WB-80133-H |
| 20 | Superwoman | SUPERWOMAN | Warner Bros | WB-80133-H |
1927
A BAND OF DISTINCTION
The Australian Band Whose Debut Album
...ISH
Is Triple Platinum In Australia
Featuring The Single
That's When I Think Of You
CD: CD 59968 • Cass: 25 59684 • LP: 25 59681 • 7" 25 79667
MARKETED BY
WEA
MUSIC OF CANADA LTD.
|
EDITOR'S NOTE:
This issue of the FLY PAPER really is a flying paper! Thru an error, Boss Riddle boarded a northbound Eastern Air Lines ship with the FLY PAPER copy in his brief case. We caught him by telegraph at Jacksonville airport, and he shipped the copy back by the next returning plane. Apologies for being a day late!
***
FRONT PAGE NEWS
So many big stories this week, we don't know where to start, for example, Bob English and Danny Bowen, both sheet metal graduates at the Tech School, have been called to work at Intercontinent Aircraft Corp. in Miami. That Intercontinent is expanding its activities is really good news.
Under Alumni Club News you will find that other Tech School grads have gone to work for Glenn L. Martin up in Baltimore, - more good news.
(Continued on Page 3)
FOR R.A.I. NEWS SEE PAGE 8
Fastest growing industry in the world today is the aviation industry, and yet it's the youngest! An infant in arms, as it were, but we don't know of any other business in the world which offers such tremendous opportunities for young men and women - in every branch - production, sales, flight, management, legal requirements and instructional services.
We have to laugh when we hear a person make such statements as "I've been with this company 43 years. We sold President Lincoln his first coal oil lamp, and the company's been doing business for over 200 years". Swell enough, an established business and reputation is all very well, but to the young man seeking fast success, as who isn't, don't miss aviation!
And what other industry has so many men still actively employed who were among the very first to start that industry? In a trip thru the sheet metal department the other day we say Bob English, Charles Carpenter and Bob Baessler repairing a pontoon under the direction of James Henry Halstead, Instructor, master mechanic, A & E mechanic, inventor and pilot, one of the real "First Men In Aviation", still actively engaged in the industry, 68 years old and going strong. Mr. Halstead began his aviation career back in 1905, just two years after the Wright brothers first flight; in 1906 he built his own plane and taught himself to fly it, since then building 18 planes of his own design, inventing a new wing, a new motor, a synchronized multiple machine gun, among other things, and flying over 12,000 hours. And you know what he says about the aviation industry? He says, "It's just beginning! Watch the next few years, especially after the war is over!"
Bob Iba, primary and secondary flight graduate at Embry-Riddle, came back to Miami for a two weeks vacation after winning his commission as Ensign in the U. S. Naval Air Corps at Pensacola. At the end of the vacation, Bob goes back to become a naval flight instructor. And he brings news of many other Embry-Riddle flight grads, -- TOMMIE COLES, long missing on these pages, is at Pensacola and doing right nicely in the Second Battalion...F. Webster Wiggin is there, too, as a Lieutenant, junior grade, taking his instructor refresher course...Wiggle doesn't like all the traffic and distinctly dislikes the Navy style of steep climbing turns on the take-off...J. Russell Wilson is in the patrol bombers now and will complete his course within a couple of weeks...Bill Moore is flying in Squadron One and George Nasworthy is plugging right along...Irwin Carter and Bob Shelley (Ruth's brother) are both at the Naval Air Station in Jacksonville...Ensign Iba tells us that Embry-Riddle is the best represented of all air schools, and that our graduates are outstanding in the ability as student fliers...
* * *
FLASH! REPORT ON THE DANCE
Hi, kids, an early Saturday morning roundup on the Embry-Riddle dance at the Coral Gables Country Club last night indicates that it was more than a big success, despite the rain storm which threatened to cut down our crowd, and everybody had a super excellent time! A few facts and figures, and stuff...by actual count, we had 290 people at the party...89 tickets sold at the door...the music played from 10 to 2...an extra hour because Boss Riddle thought the gang were having too much fun to go home...the midnight supper hit the spot!
Particularly satisfying was the way all the old graduates, flight and tech school, turned out for the affair...the Navy was there...and Intercontinent Aircraft...and Pan American Airways...and the Civil Aeronautics Authority...an aviation "jam session" went on most of the night...the Public Address System broke down...and squelched our graduation ceremony for the CPTPers...but the "wings" will be sent the kids in the mail...
Ye Olde Editor felt pretty sentimental about all the gang stopping past the table to tell us what a good time they were having...that was nice of them, but all credit for the party goes to those who pitched in on the ticket sales,-- and to the gang themselves for turning out for the dance...after all, it's their party!
And what about the next party??? It'll be held on a Saturday night,
just about a month from now. No definite date set yet, but we are planning to have all the Royal Air Force cadets down from R.A.I. at Arcadia. As soon as everything is set we'll let you know details in the Fly Paper - meanwhile, many thanks to all the gang for making the party last night such a grand success!
* * *
BOWLING TEAMS ARE ROLLING ALONG
Tech School won 3 games Thursday night to maintain its place as leader in the league, while the PILOTS won 1 game and lost 2, still in last place. The payoff, tho, is going to be this Thursday evening, the 26th, when the Pilots bowl against the Tech boys. That should be plenty good, with blood money running knee deep in the gutters. Don't miss it.
New faces in the rooters gallery last week included Mrs. John Paul Riddle, Clyde Pieper, Steve Anderson's room-mate and Mr. and Mrs. Jim McShane, these in addition to all our old gang of pals.
The scores, with no boasting on anyone's part, were:
| TECH | PILOTS |
|------------|------------|
| J. Ordway | 106 130 |
| P. Ordway | 117 152 129|
| S. Anderson| 168 148 139|
| B. Belland | 119 -- 131 |
| L. Hamm | 168 161 177|
| J. Lacinak | -- 145 150 |
| J. Garcia | 103 170 116|
| G. Royce | 148 139 109|
| C. Rexrode | 106 131 86 (?)|
| A. Gibbons | 97 129 147 |
ALTERNATES ON THE CPT. PROGRAM
Arthur Gibbons tells us that in addition to the students accepted for flight training on the summer CPTP program, an additional 20% will be taken in as "alternates". This group will participate in the ground schooling with the regular flight appointees, and will be used to replace any regular appointee who might drop out during the "A" stage. In the event that an alternate does not get an opportunity to take the flight training on this program, he will be first to be accepted on the next training program.
* * *
BETTER LATE THAN NEVER is the news (?) that Jake Lacinak, Mac Lowery, Burrill Hamon and Howard Beazel have returned from Chanute Field, Rantoul, Ill., after taking extensive refresher courses in the U. S.
Army mechanical training procedure. Welcome home, fellows, or have you-all gone again?
* * *
Biggest new student story of the week concerns Dorothy "Tommie" Bailey, first girl to ever take a complete flight AND technical course with Embry-Riddle. Of course, we've had plenty of girl students before, but never one who came in and took EVERYTHING in the book. Look at this course - Spanish, Portuguese, Instrument Technician, 1 and 2 S flight ratings on land and seaplanes, complete Aircraft and Engine mechanics course, yeah, be nice to the gal, she'll be here for more than a year on that one! "Tommie's" idea in taking all this is that she believes that there will be a tremendous demand for trained executives in aviation, particularly in the South American field, and she's determined to be ready for such opportunities. Good goin', gal. We're all for you!
* * *
-5-
TECH SCHOOL STUFF
By Don Watson
What With the War ... and all.
There seems to be an epidemic of car trading. Word has finally seeped into the deep south that there will be less production on new autos in 1942 than heretofore and that those produced will have very severe treatment; no white side wall tires, very little if any chrome and a lot of other extras that in the past we have taken for granted. So maybe it's good business to trade the old bus now while there are still many good used cars to be picked up on the local market.
* * *
Students and graduated students are respectfully requested to refrain from visiting the Intercontinent Plant for employment interviews until directly called or advised by the school to go over. The Intercontinent plant is a capable organization and have classified all the applications students have submitted and the latest report is that the following men have been employed.
Daniel Bowen Sheet Metal Graduate
Robert English Sheet Metal Graduate
James Jenkins Sheet Metal Graduate
Raymond Sullivan Sheet Metal Graduate
Jack Barco Sheet Metal Graduate
Alban Reid Sheet Metal Graduate
E. R. Welch Sheet Metal Graduate
Chances will be given all qualified men to be interviewed for positions and we feel reasonably certain the absorption over a period of time will be fairly good. The best thing about all of this activity is that the boys already in the plant to a man intend to further their capabilities by continuing their schooling with Embry-Riddle and in so doing make themselves practically indispensable to their present and future employers. The logical follow up of the Sheet Metal course is Wood Working and fabric work; further along the road to the Aircraft Ticket is the Welding and Finishing. We are very glad to see the men returning for evening instruction after completing their days' work in the plant.
* * *
From the building maintenance department at Tech, we have dubbed Frank Marshall "Termite" after the way he has been cutting holes thru the walls for ventilation purposes.
And if that guy doesn't stop hammering soon, after all, what's the rap for murder these days??? And another of our carpenter friends, George "Scotty" Hope just told us that he sends the FLY PAPER back to his folks in Scotland each week after he reads it. Whatta circulation manager he'd make!
* * *
A MIGHTY IMPORTANT ANNOUNCEMENT FOR ALL EMPLOYEES!
Comes to our attention that Embry-Riddle will very soon have a cooperative company insurance plan for all employees, whereby employees will receive sick, accident and death benefits. Worked out by Boss Riddle, George Wheeler and Glen Kuhl, this is the same type insurance plan adopted by Pan American Airways and many other big timers in the aviation business, and is decidedly worth your sincere investigation. Complete details of the Embry-Riddle insurance plan are being made into a booklet which will be distributed to the employees this week.
* * *
SOCIETY DEPARTMENT
PARTIES - PARTIES - PARTIES
With the advent of summer, seems like like most everyone has moved the cooking department to the charcoal grill in the backyard. Anyhow, Ye Editor has been getting an awful lot of free meals in back yards these days -
Take Saturday evening, last week, for example, Bonnie and Jake Lacinak had us over for broiled hamburgers and weiners, with plenty of onions, potato salad, home baked beans and stuff, and just about a tub full of excellent lemonade! Oh, boy, what food! Other guests included Mr. and Mrs. Ed Riopel, Mr. and Mrs. Howard Beazel and their 6 year old son Peter, Harriett Erpenbeck and Mac Lowery, Golda Jackson and E. M. Smith, from Canton, Ohio.
Then Sunday evening, to Virginia and Don Watson's for charcoaled steaks, (you bring the steak) and the traditional potato salad, olives, pickles, etc., etc., and a case and a half of Coca Colas. Guests included Bob Bowen, Alice Gordon, Harriett Erpenbeck and Mac Lowery (don't they ever eat at home?), David and Owen Hard, Dave Beatty, and Grace and Bill Roome. Nice party, and swell victrola recordings far into the night.
The British Cadets, who are officially known as 42-A, are now becoming fully acclimated and are getting into the old groove. Flight schedules started on the 11th., and rapid progress is already being made.
We find that there are important people in the group. One of the Cadets, Mr. Trapp, is a golf pro, and was a candidate for the Ryder Cup Team. Also in the group are two members of the press, so we understand. We haven't become acquainted with them yet, but hope to in the very near future. How about it fellows, can we have a little contribution?
* * *
The Cadet contribution this week comes from Flying Cadet Bowers, and it throws some interesting light on the matter. Really, it is our first bit of insight into the way our boys and the British are getting along together. Anyhoo, we give you Cadet Bowers report.
We haven't yet much to report on our new British Cadets. They are settling into U. S. Army life in fine style. There was an inspection last Saturday which caused some consternation - but all in all, Carlstrom sees them as another class - and no different.
That's all for now. By the next issue the British "Right turn" will probably have been changed to "Right face", and a snappy uniform will be the only difference. And, by the way, the Saturday walk on the ramp has not differentiated between nation!
ED. NOTE: How about it, 42-A? Do you have any replies you would care to make? This is an open column, and we certainly will welcome your contributions?
* * *
Two new instructors at the Post are Mr. E. J. Smith and Mr. Hunsiker, who arrived via Luscombe the other day from Santa Maria, California. They were previously employed by an Army school there, and after finishing up their contract at that establishment they came down here to be with their old friend G. Willis Tyson.
The latest instructor refresher class has about 12 new men, with more scheduled to come all the time. This will bring the total instructing force up to about 55. The new men are now taking the course under Clete Huff, with Gordon Mougey and Lloyd Lampman assisting.
It might seem like the instructors were copycats, but if the truth be known, they had planned for some time to adopt Sun helmets for the official uniform during the summer. However, the idea sprang into actuality at almost the precise moment the British lads arrived with the sun helmets that are issued to them. The difference being that the instructors wear an "inexpensive" pressed fibre hat, while the Cadets have the real McCoy, which, I believe, are called "Topoys". (Let us know about that word if we're wrong, fellas.)
* * *
STUFF AND THINGS
Random Chatter (mostly nonsense) ---- One of the flight instructors, while chastising his cadet for not looking around enough, said "Looky here, Son, I don't want you to become 'Jerry bait'". ---- Some of the Army Sgts. having a field day against yours truly for some of our recent writings. The occasion was a forced landing at Punta Gorda due to bad weather. ---- Kay Bramlitt getting all excited about a trip to Sarasota.
THE EDITOR'S MAIL BAG
Just received a letter from Bud Heck in Mobile, Alabama. Bud is still "covering the territory" for Paris, and is still promising us that pair of free garters. He says the food is wonderful in New Orleans.
* * *
AROUND AND ABOUT THE SEAPLANE BASE
Biggest news around the Seaplane Base is the rather sad story that our old pal Instructor Mike Covert has been called north by the death of his brother-in-law, Dr. Stephens of Rochester, N.Y. Mike, having other business in the north, will spend the summer there, returning to Miami next fall. Taking Michael's place as an instructor at the Seaplane Base is Roger Carley, transferred from the Municipal Base.
* * *
On the happier side of the ledger, we offer all congratulations to Ad Thompson, who successfully passed his commercial flight test, after
many an hour of solid plugging. The CAA inspector in the case was Dick Vavrina, and now Ad is going ahead for his instructor's rating. "Stick to it", Ad!
* * *
This and that ... George Hall got his water rating last week ... Dot Bailey has begun on her primary flight instruction ... and Dr. Margaret Williams, who is flying on a private ticket, is vacationing in Indiana and will be back July 15th.
* * *
The successful passing of flight tests by Harry Reeder and Fred Cunningham winds up the spring CPT program at the seaplane base. Of the three girls and seven fellows to start in this group, one girl, was washed out, and one fellow, discontinued, the remaining eight successfully completing the program and obtaining private pilots licenses. Nice!!
* * * * *
VISITOR OF THE WEEK
Flying in via Eastern Air Lines from Birmingham was N. A. "Dick" Brown, AWA editor of the Dixie Air News, who came to Miami to visit his two brothers and sister, as well as renew old friends in the aviation industry. He left Monday evening to return to his home port at Charlotte, North Carolina.
* * *
Among other visitors were Bruz Carpenter's parents, who came to Miami to spend a couple of weeks with Bruz before leaving for the Pocono Mountains up in Pennsylvania. This is the first time Bruz has seen the family for about 18 months.
* * *
And another visitor, Lieut. Van Burgin's little (?) son, Van, Jr., who is spending a week in Miami before leaving for the Athens (Ga.) "Y" camp where he will be instructor in tennis and track. Van, Jr., will return to Miami for a month after camp is out on August 20th.
* * *
VACATION DAYS!
By being the first to complete the boys under his care on the Secondary CPTP program, Flight Instructor George May is getting himself a well deserved vacation. He and Tommie Turner left Miami Monday morning for R.A.I. at Arcadia, where Tommie took his Army instructors check flight, after which they went on to Orlando, taking off Tuesday morning in the "Airknocker" Chief which Tommie and Al Janos just bought, - enroute to
"Yeah, I been here quite a while. My 'chute didn't open."
Reprinted from Squads Riot, 1941
Milwaukee, Wisc. Up yonder, George will visit his family who live near Madison, while Tommie, the old devil, will visit THE GIRL! He met her here last winter while she was vacationing, and the purpose of this flying trip is to meet the family, present the ring and set a date for the wedding. Good luck, fellow, and we'll be seeing you again in about 10 days.
AND ANOTHER WEDDING IN THE OFFING!
Comes Friday, June 27, David Abrams, Jr., will marry Betty Serpas. Swell going, fella! Dave is a Chicago, Ill., laddie, the son of Mr. and Mrs. David Abrams, Sr., 4534 N. Albany Avenue; a graduate of the University of Miami, and taught mathematics at Shenandoah Jr. High School one year before joining the Embry-Riddle Staff.
* * *
MENTIONING MUNICIPAL
By Bill Jaster
Ex-flight instructor and old pal ANDY STINIS came last Tuesday to get his Waco sky writing ship and ferry it back to New York City for a new job there. Couldn't stay long, but Andy said to say hello to all the gang for him.
* * *
Bob Johnston's SAFETY THOUGHT FOR TODAY:
The pilots who fly in Japan
Are indeed a peculiar clan,-
They fly in kimonas-
Zoom all the pagodas-
And keep their nose down when they can!
* * *
Jack McKay, Jr., on a cross country flight, just phoned in from Belle Glade, saying that he was "weathered in" and would return next day. Could be Jack has a girl up there, or did he just want to get in some fishing in Lake Okeechobee. Anyway, it was a good idea, and we hope the next CPTP program doesn't get wise, or they'll all be spending the night "out". Seriously, tho, Jack used good judgment in not hesitating to set 'er down. By taking no chances, he averted the possibility of
a serious disaster, - a terrific storm blew up less than an hour after he called. 'Twere a good example for all students, please note!
* * *
When Ye Olde Editor visits Municipal something exciting always happens, - (Ed. Note,- this is Jaster talking!) usually he brings beautiful models for publicity purposes, but this time he came alone - found a ball bat and 10 minutes later he had a ball game going on. And all this at 7:30 in the morning, too!
* * *
Instructor refresher student Mary Brooks beat her instructor, Joe Garcia, out for the 7:00 A.M. appointment one morning, and has been razzing him ever since. Joe has no comeback now when Mary chooses to be late.
* * *
Newest pilot instructor to join the family at Municipal Base is Clarence O. ("Speed") SNYDER, who was transferred to Miami from the Carlstrom Field base in Arcadia because an appendicitis operation temporarily disqualified him from the advanced acrobatic flying practiced there. Well, welcome in "Speed", and hello to wife, Helen, too. "Speed" was born up in Syracuse, N. Y., where he learned to do his flying act, and went to Carlstrom Field after spending a year at the Peter O. Knight Field in Tampa. He has better than 2,793 hours, about half of which was on 2 S ships.
* * *
The swell comradeship which exists among our students and employees was well demonstrated last week when the "gang" at Municipal Base clubbed together and bought Rocco Famigilietti a super suit case as a "going away" present. Rocco who has been on the line crew, just finished his primary flight training under the CPT program, and is returning to his home in Waterbury, Conn., for a short visit before being assigned to the Army Air Corps. Sorry to see you go, Rocco, but we're glad to have known you, - and all best luck in the future!
Keith Phillips passed his private flight test with Bob Johnston on the 14th, which winds up the Spring Primary Program. Out of the 29 who started this program at Municipal Base, 22 successfully completed the course and graduated with private licenses, - the other 7 having been washed out for one reason or another. Nice going, lads and lassies!
* * *
After Instructor Jim Cousin's name on the Primary Progress Board, there are marked down 5 demerits and we are wondering why - It must be a good story whatever it is.
* * *
TALK OF THE TOWER - WMAO
We borrow Maybell Manning's column name from the Daily News long enough to tell you of our first experience with the Control Tower out at our Municipal Base. Along about 7:30 Wednesday morning at Municipal we got curious, so Quintus Feland drew a pair of head-phones from the stock room and we went out to trainer #2 and plugged in. About the first thing we heard was, "Miami Tower to Embry-Riddle trainer #5, NE of Municipal. If you road tower, dip wing please". We peeked out NE, and sure enough, there was a trainer coming in, and the wing dipped. Immediately the Tower continued, "Circle field to left and use Southeast runway. Surface velocity 8 to 10 miles. All clear".
Ship 5 circled the field and made a perfect landing, and the Tower cut in again, advising future action of the ship, this time, "Ship 5, all clear. Turn right and taxi to hangar area". And we watch as the ship turns and taxies to our hangar. And who climbs out but Jack McKay, who had been out all night on a cross country flight, forced down up-state by bad weather.
Well, kids, all this radio talk just made us real curious, so we went up into the Control Tower, where we met Jimmie Wynne, chief senior control tower operator, who showed us all the equipment, 11 different receivers among other things, and introduced us to his gang, Martin Hansen, Clyde Cohron, Kenneth Schwinger, and Bill Fuss.
'Twas a mighty interesting half hour we spent there. We saw Bob Thompson take off in his Cub, using the light gun in absence of a radio, and also Andy Stinis take off in his Waco for New York. "This is just a temporary tower", Jimmie told us, and, pointing to some new construction near the tracks between Municipal and the new Master Airport, "the permanent tower is being built over there, and will have more complete equipment, - an altimeter and anemometer and all that stuff."
Jimmie asked us to pass the word along to all our fliers that he certainly appreciates the way they've taken hold and are cooperating with the operators in the control tower. "After all", he said, "it's new to all of us, but believe me, when we get used to it, it will certainly be a wonderful safety measure, and after all, those pilots in training should be accustomed to flying off a controlled field. Eventually, all airports will be controlled, and it is good training for them".
And he gave us his final recommendation, "Tell your gang that the secret of good reception is fine tuning and high volume. And don't forget to increase your volume after takeoff!" Well, All Right, Jimmie! We'll do that, and hats off to you and your boys for doing a fine job!
* * *
Joining the stockroom personnel at the Tech School is David Abrams who came to us from a year of teaching math at Shenandoah Jr. High School. Dave is a University of Miami graduate and former member of the football squad.
* * *
Arriving June 17 as newest addition to the Tech faculty is Dr. T. D. Phillips who will teach ground school and act as consultant on technical aviation problems, a position for which he is well suited, having been one of the 9 men who wrote the text books for the Civilian Pilot Training Program under which 50,000 pilots have been trained. "Doc" has a string of those college degrees after his name, and among his other accomplishments he is head of the physics department at Marietta (Ohio) College, was on the research staff of the U. S. Bureau of Standards and wrote the "Digest of Civil Air Regulations" for the government. Some Stuff!
* * *
OLD TIMERS CLUB WILL NOW MEET!
A babe in arms, as it were, it's fun to check back over the records and see who's who and how long they've been with the company, Flight Instructor, Bob Johnston, takes the cake as being the oldest pilot with the company, in length of service. Ad Thompson comes next. He and Bob, we think, were about the first two employees of the Embry-Riddle company when we first began operating the Miami Seaplane Base. Next oldest pilot is Charlie Barnhardt, who came with the company June 15, 1940. And there's Registrar Arthur Gibbons and Comptroller George Wheeler, both of whom came in on June 14, 1940. Way back in history, Ye Editor joined the company on June 7, 1940. Tempus certainly fidgets, doesn't it??
HEY! HEY! AND A BIG WELCOME
Plenty of new students at the Tech School! And do you notice the trend to the South American business? The students are beginning to come up from South America, so let's exercise that "Good Neighbor" policy and welcome them into our family. Enrolling in the flight division was Sr. Eduardo D. Hernandez, Cuban Counsul in Miami, who is going to take a private pilots course at the Municipal base, and from Medellin, Columbia, S. A., comes Luis Emiro Jaramillo, 24-year old son of a prominent real estate dealer there, who is taking a complete Aircraft and Engine course to prepare himself for the growing aircraft industry in his country.
And a great big welcome to all the other students who have enrolled in the following courses:
INSTRUMENTS: Donald Uhle, James Girton, L. M. Bassler, Forrest Frazier, Philip Smith, A. Harrison, Geo. W. Haffner.
AIRCRAFT SHEET METAL: John Tierney, G. R. Lyon
WELDING: Fate A. Crews, Jeffie K. Waldrip, Claud Jones, A. Darnes, M. W. Jahn, Asa F. Pilchard, Harry Mayer
DRAFTING: Dick Momand
AIRLINE MAINTENANCE: Webster Clay Bright, Mark Hall Fredericks, Raymond Bayner, Thos. B. Winkikus, Robert N. Gray, W. L. Bell, C. J. Sedlmayr.
RIVETING: Garland Brown, Frank Brillo, W. P. Stewart, Don B. Gray.
SHEET METAL: Robert McDonald, Robert H. Garrison, L. M. Sperling, Ralph Walters, Sidney Hyman, Ross Lambert, H. V. Bender, Morris Zukorsky, Curtis Teems, Burton Tood, Jos. H. Walker, E. Leatherman, T. Galloway, Bernard M. Heitman, Fred Pasquarello, Phillip Salokar, A. M. Ewald, Aaron Shephard, Robert C. Bartholomew, R. H. Patrick, E. J. B. Huguelet, L. M. Sears, J. A. Tanaw, Frank Magnuson.
A & E: Luis Jaramillo, K. Painter, John J. Hufnagel, Arthur L. Walter, Jr.
ENGINE: Myron G. Kintz, James W. Pierce, Jr., G. A. Johnson, Jr., John H. Uhle, Max C. Keating, John A. Niles, R. L. McCamy, W. T. S. Crichfield.
AIRCRAFT: Vaughn Dekle, Larry Austin, Phil Giberson, A. V. Williams, Leonard Bruce, D. W. Esmond, R. R. Dierdorff, David Beatty.
Here's good news about one of our ex-employees who was probably one of the best liked lads to ever pass thru our memory. 'Tis JOE NEISER, who left the dispatcher's tower at our Municipal Base to enter Northwestern University at Chicago under his Naval Reserve Commission. Our last letter from Joe came in April 2, and told of mighty hard work, and the doubt in his mind that he would be able to pass the stiff requirements, but, latest word on Joe was published in the local papers Monday, and confirmed our belief in him, he did pass the course, and was given his commission as ensign. Congratulations, Joey, boy, and write to us again. Anyone wanting to write to Joe can address mail to him at 801 N. W. 63rd Street, Miami, Florida, with the notation "Please Forward".
POST SCRIPT: JOE NEISER WAS IN MIAMI! After the above was written, Joe dropped into the office to say "Hello" to Ye Editor. He was in Miami for only a couple of days vacation, and left Saturday morning on an undisclosed official Naval assignment. Good luck, fellah!
Comes a letter from Secondary flight graduate Seaborn Chiles, Air Corps Training Detachment, Americus, Ga., who tells of meeting Tom Schepis there. Seaborn says to tell all the gang, "Hello!"
A letter from flight graduate Bud Saeger to Wilbur Sheffield tells of Bud getting a job with Eastern Air Lines. Nice going!
Kenneth Manion, Tech School graduate returned last Sunday after a motor trip around the eastern part of the country looking over the job prospects. Kenny reports that Vultee up in Tennessee will take on 3000 men within a couple of weeks, Piper Aircraft is still wanting welders, and Glenn Martin and Curtis Wright are very receptive to applications from qualified aircraft men. Incidentally, Kenny's brother, Arnold Manion, and Jamos Sackett and Pat Stowart, all Embry-Riddle Tech graduates were accepted for employment by Curtis Wright at Columbus, Ohio, and went to work there last Monday morning.
There've been some changes made since last reporting the whereabouts of some of our Tech graduates, IRVING BLOOMBERG is with Brewster, Lynn Bottom and John W. Patterson are with Bell Aircraft at Buffalo, Ernest H. Cutler is with Sikorsky at Bridgeport, Conn., and Bob Shively is
with Allison Engineering at Indianapolis, Ind. Write in fellows, and tell us all about things and stuff! Flash! A letter just in tells that Dunn, Cooper and Huffman are now with Glen L. Martin, making bombers!
* * *
BART SCHWARZENBEK, who finished his primary CPTP last January at the Seaplane Base has successfully passed his physical exam for the Army Air Corps and is now waiting at his home in Nutley, N. J., waiting to be assigned to a training base. Bart started his primary training with Jimmie Cousins and finished with Wiggin.
* * *
A postal card from 2nd Lieut. Al Bachmann, one of the first U. S. Army Air Corps men assigned to R.A.I. at Carlstrom Field, "I am being transferred to Curtis in Buffalo and want to keep up with the news at E.R. Please send me the FLY PAPER, and say hello to Len Povey for me and have him drop me a line, will you? Many thanks and lots of luck to all the Cadets at Embry-Riddle". Sincerely, 2nd Lieut. Al Bachmann
U. S. Army Air Corps,
Address: 581 Potomac Ave., Buffalo, N. Y.
* * *
STUDENTS! ATTENTION!
FLASH! Due to being late with this issue of the Fly Paper, we're able to get in the last minute news that next Tuesday and Wednesday, July 2 and 3, Arthur Barr will show some very special moving pictures covering every phase of primary and advanced welding. These pictures, thru the courtesy of Mr. Mittenmeyer, of the Linde Air Products Company, are excellent examples of visual education, and are well worth being seen by not only the welding students, but also, all those interested in aircraft construction and maintenance.
* * *
MORE LATE NEWS!
Tuesday morning, June 24, and we learned that the following Air Craft Line Maintenance graduates reported to work today at R.A.I. Carlstrom Field, Arcadia; John Concannon, Elmer Hilbrant, Roger Saxon, James Groves, Homer Taylor, Eldon Farwell, Robert Ohlinger, Clark Batchelder, and Norwood Latimer. This, the first group to go from Tech School to R. A. I.
Best wishes and good luck, Fellahs!
* * *
The boys are wondering who this is???? "Dale Delanty" -- NO!!! Couldn't be!
By special, secret messenger, Kay Bramlitt sent another Odom cartoon down to the FLY PAPER, telling all about Ace Correspondent DALE DELANTY getting lost in a rain squall the other day. Kay says the boys wanted it sent straight down, 'cause they were afraid Dale might censor it. Why, Dale, - you wouldn't duck out of a good laugh, would you? Anyway, fella, it's all in fun!
Speed is vital in America's need for trained men in Aviation. And there will never be a better moment than this present one for you to take advantage of the opportunity! Don't delay -- enroll today for the Embry-Riddle Government-Approved course that will open the door to your place in Aviation - in the air or on the ground.
EMBRY-RIDDLE
SCHOOL OF AVIATION
3240 N.W. 27th AVENUE, MIAMI, FLORIDA
TEL. 3-0711
|
REINFORCED CONCRETE STRUCTURES UNDER IMPACT LOADS
Abdul Jabbar Sangi BE, M.Engg
Submitted for the degree of Doctor of Philosophy
Heriot-Watt University
School of the Built Environment
April 2011
The copyright in this thesis is owned by the author. Any quotation from the thesis or use of any of the information contained in it must acknowledge this thesis as the source of the quotation or information.
The effects of extreme loading conditions such as impact and blast on reinforced concrete structures have been investigated by many researchers in order to develop safe and efficient design procedures. The integrity and safety of sensitive structures, for example nuclear containment structures, against these loads is a matter of great public concern. The majority of studies carried out in the military sector mainly focused on high-velocity impacts on reinforced concrete structures. However, there is a clear need of an investigation into the impact behaviour of reinforced concrete members under low-velocity impacts. The present study focuses on the impact behaviour of reinforced concrete beams and slabs subjected to low-velocity falling-weight impacts. LS-DYNA, a commercial finite element code has been used to perform three-dimensional finite element modelling of the beams using the Lagrange formulation. Concrete has been modelled using eight-noded hexahedrons with single point integration. Reinforcing bars have been explicitly modelled into concrete and a perfect bond was assumed. The results of the analyses have been compared with the beams tested at Heriot-Watt University. Two series of beams 3 m and 1.8 m long were subjected to a falling weight of 98 kg impacting the beams at the centre. The impact forces, accelerations and crack patterns have been compared, and good agreement was found. The analyses highlighted a further need of experiments, therefore, a series of beams was tested under similar conditions and additional test
data including the displacements at four locations along one half of the beam and the reactions at the two supports were recorded. Four beams were tested, two under single impacts and the other two with three impacts on each to investigate the effects of damage conditions. The new data served as additional validation of the finite element models. Good agreement was found for the peak displacements and reactions. The finite element models were extended to include the effects of damage conditions and results were compared with the tests. The analytical methodology developed for the beams was extended to analyse reinforced concrete slabs subjected to impacts. The results were compared with the tests carried out at Heriot-Watt University and reasonable agreement has been obtained.
To my loving parents
for their love and hard work
Acknowledgements
Firstly, I would like to express my gratitude to Prof. Ian M. May for his expert supervision and continued guidance throughout the course of this study. I am particularly grateful to him for his enthusiasm, patience, motivation and invaluable advice during all the phases of the research and completion of the thesis.
I am greatly indebted to NED University of Engineering & Technology, Karachi and Higher Education Commission, Government of Pakistan for providing the financial support to carry out this research. Particularly, I would like to express my deepest appreciation and thanks to Prof. SFA Rafeequi and Prof. SH Lodi, my teachers and mentors at NED for their continued guidance, support and encouragement during many difficult phases of this study.
I would like to thank many friends and colleagues at NED and Heriot-Watt University for their support and useful suggestions. Especially, acknowledgments are due to my close friend and colleague Dr. Rashid A. Khan for his moral support and help in carrying out the experimental tests. Many thanks to Muhammad Aslam Bhutto and Dr. Atif Mustafa for a memorable time in Edinburgh. Special thanks to Mukesh Kumar at Imperial College, London for his help in digging out many useful references.
Finally, I express my gratitude to my family for their support, patience and understanding throughout the period of study. My father spent
all his life working really hard to support my studies, for which I can not thank him enough. The last stages of this journey were really painstaking and would have been impossible to complete without the love and support of my wife and lovely daughters Aleena and Arcesha.
## Contents
Abstract ........................................... ii
Acknowledgements ................................. v
List of Tables .................................... xii
List of Figures .................................. xiii
Notations .......................................... xxii
1 Introduction ..................................... 1
1.1 Background .................................. 1
1.2 Objectives ................................... 3
1.3 Contents of the thesis ....................... 4
2 Literature Review ............................... 7
2.1 Introduction ................................ 7
2.2 Impact behaviour of concrete beams .......... 11
2.3 Impact behaviour of concrete slabs .......... 39
2.4 Significance of the current study ............ 47
3 Finite Element Modelling ....................... 48
3.1 Introduction ................................ 48
3.2 The Finite Element Method ................... 48
3.3 Generalized finite element formulation ...... 50
3.4 Finite element code LS-DYNA .......................... 53
3.4.1 Solution of the governing equations ............... 54
3.4.2 FE modelling of reinforced concrete ............... 57
3.4.3 Contact force determination ....................... 60
3.5 Verification of LS-DYNA dynamic analysis .............. 62
3.5.1 Static loading .................................. 63
3.5.2 Free vibrations response ......................... 64
3.5.3 Impulse forces response ......................... 65
4 Constitutive Modelling .................................. 68
4.1 Introduction ..................................... 68
4.2 Concrete constitutive modelling ..................... 68
4.2.1 Uniaxial behaviour of concrete ................. 69
4.2.2 Multiaxial behaviour of concrete ............... 69
4.2.3 Factors involved in concrete modelling ......... 71
4.3 Strain-rate effects ................................ 74
4.4 Concrete models in LS-DYNA ........................ 79
4.4.1 Pseudo Tensor Geological Model ................. 79
4.4.2 Soil Concrete Model ............................ 81
4.4.3 Concrete Damage Model ......................... 81
4.4.4 Winfrith Concrete Model ....................... 83
4.4.5 Johnson Holmquist Concrete Model ............... 85
4.4.6 Continuous Surface Cap Model (CSCM) .......... 86
4.5 Concrete models adopted ............................ 86
4.6 Reinforcement material model ....................... 87
4.7 Material model for impactor ......................... 89
5 Finite Element Analysis of RC Beams under Impact 90
5.1 Introduction .................................................. 90
5.2 Description of tests ........................................... 91
5.2.1 Details of beams ......................................... 91
5.2.2 Instrumentation and data acquisition .................... 94
5.3 Experimental results .......................................... 95
5.4 Finite element modelling ..................................... 95
5.5 Mesh sensitivity ............................................... 98
5.6 Influence of time step ........................................ 101
5.7 Stability of the solution ..................................... 103
5.8 Comparison of finite element results and tests ............... 103
5.8.1 Transient impact forces .................................. 104
5.8.2 Transient accelerations .................................. 115
5.8.3 Transient reinforcement strains .......................... 116
5.8.4 Crack patterns and damage ................................. 116
5.8.5 Correlation between transient load and crack development 123
5.8.6 Displacement histories ................................... 125
5.9 Conclusions ................................................... 127
6 RC Beams under Multiple Impacts 131
6.1 Introduction .................................................. 131
6.2 Description of tests .......................................... 132
6.2.1 Details of beams ......................................... 132
6.2.2 Instrumentation ........................................... 133
6.3 Experimental results .......................................... 133
6.3.1 Transient impact forces .................................. 134
6.3.2 Transient displacements .................................. 142
6.3.3 Damage and crack patterns ................................. 147
6.4 Finite element modelling ..................................... 151
6.5 Comparison of finite element results and tests .......................... 152
6.5.1 Transient impact forces ........................................... 152
6.5.2 Displacements ..................................................... 156
6.5.3 Crack patterns .................................................... 156
6.6 Conclusions ........................................................................ 158
7 Finite Element Analysis of RC Slabs under Impact ...................... 161
7.1 Introduction .................................................................... 161
7.2 Description of slab tests .................................................. 161
7.3 Experimental results ....................................................... 164
7.3.1 Transient impact forces ............................................. 164
7.3.2 Transient accelerations ............................................. 164
7.3.3 Transient reinforcement strains .................................. 166
7.3.4 Local damage ......................................................... 168
7.4 Finite element modelling ................................................ 168
7.5 Mesh sensitivity ................................................................ 173
7.6 Comparison of finite element results and tests ....................... 175
7.6.1 Transient impact forces ............................................. 176
7.6.2 Crack patterns and damage ....................................... 180
7.6.3 Displacement histories ............................................. 186
7.7 Effect of contact interface ............................................... 188
7.8 Slab-on-slab impact ......................................................... 190
7.8.1 Description of the geometry ...................................... 192
7.8.2 Finite element model ............................................... 192
7.8.3 Simulation results ................................................... 193
7.9 Conclusions ...................................................................... 197
8 Conclusions 199
8.1 Conclusions 199
8.2 Recommendation for Future Work 203
A Analytical response for a SDOF system under impulse loading 204
B Sample LS-DYNA input deck for RC beam 206
References 211
| Table | Description | Page |
|-------|-----------------------------------------------------------------------------|------|
| 5.1 | Details of beam tests by Chen & May (2009) | 93 |
| 5.2 | System specifications | 95 |
| 5.3 | Material properties of beams using Winfrith model | 99 |
| 5.4 | Mesh data for beam A1-2 | 101 |
| 6.1 | Maximum impact force, reactions and peak times of beam tests | 135 |
| 6.2 | Maximum displacements recorded in the beam tests | 142 |
| 7.1 | Details of slab tests of Chen & May (2009) | 162 |
| 7.2 | Material properties of concrete | 173 |
| 7.3 | Material properties of steel reinforcement | 173 |
| 7.4 | Mesh size data for 760 mm slabs | 174 |
| 7.5 | Mesh size data for 2320 mm slabs | 174 |
2.14 Impact tests by Fujikake et al. (2009): Test setup .......................... 28
2.15 Failure modes: (a) S1616 series; (b) S1322 series; and (c) S2222 series (Fujikake et al., 2009) ......................................................... 29
2.16 Impact response for S1616: (a) drop height = 0.15 m; (b) drop height = 0.3 m; (c) drop height = 0.6 m; and (d) drop height = 1.2 m (Fujikake et al., 2009) ......................................................... 30
2.17 Impact responses: (a) maximum impact load; (b) impulse; (c) duration of impact load; (d) maximum midspan deflection; and (e) time taken for maximum midspan deflection (Fujikake et al., 2009) ......................................................... 31
2.18 Impulse and duration of impact load (Fujikake et al., 2009) ............ 31
2.19 Midspan impact of a pin-ended beam (Hughes & Beeby, 1982) ....... 33
2.20 First three symmetrical vibration modes of a pin-ended beam (Hughes & Beeby, 1982) ................................................................. 34
2.21 Measured and theoretical force histories (Hughes & Beeby, 1982) .... 34
2.22 Mass-spring model for impact (CEB, 1988) ..................................... 35
2.23 High-speed photo at mid-span and numerical damage plot after 20 ms (Unosson, 2001) ................................................................. 38
2.24 Comparison of concrete penetration depths calculated by various formulae for the case of a typical missile (Yankelevsky, 1997) .......... 40
2.25 Comparison of concrete perforation thickness calculated by various formulae for the case of a typical missile (Yankelevsky, 1997) .......... 41
2.26 A two-stage perforation model (Yankelevsky, 1997) ....................... 42
2.27 Load-time histories of slabs under 610 mm drop (Zineddin & Krauthammer, 2007) ................................................................. 45
2.28 Penetration of projectile fragment into concrete slab (Agardh & Laine, 1999) ................................................................. 46
2.29 Finite element model of slab and loading (Izzat et al., 2009) ............ 46
3.1 General three-dimensional body (Bathe, 1996) ........................................... 50
3.2 Eight node hexahedron element ........................................................................ 58
3.3 Hourglass modes of an eight node element with one point integration, 12 modes exist (Belytschko et al., 2000) ................................................................. 59
3.4 Representation of reinforcement in concrete model ............................................. 60
3.5 Contact formulation based on Penalty method (taken from Tavrez, 2001) .......... 62
3.6 Example test structure (Saatci, 2007) and finite element model ....................... 63
3.7 Response of example test structure under static loading .................................. 64
3.8 Comparison of analytical and numerical free vibration response ....................... 65
3.9 Impulse loading on the example test structure (Saatci, 2007) .......................... 66
3.10 Comparison of the analytical and numerical response under short impulse ........ 66
3.11 Comparison of the analytical and numerical response under long impulse ........ 67
4.1 Typical uniaxial stress-strain curve for concrete specimen in compression, reported in Kupfer et al. (1969) ................................................................. 70
4.2 Typical multiaxial stress-strain curve for concrete specimen in compression (Palaniswamy & Shah, 1974) ................................................................. 70
4.3 Stress-strain curves for concrete in uniaxial compression (Wischers, 1978) ....... 72
4.4 Stress-strain curves for concrete in uniaxial tension (Peterson, 1981) .............. 73
4.5 Volumetric strain under biaxial compression (Shah & Chandra, 1968) ............ 74
4.6 Cyclic uniaxial stress-strain curve in compression (Sinha et al., 1964) ............ 75
4.7 Log-log scale of strength increase versus strain rate showing that CEB equation fits for available data, reported in Bischoff & Perry (1991). ................................. 77
4.8 Pressure versus volumetric strain curve. ......................................................... 80
4.9 Strength model for concrete: (a) two-curves strength model and the yield surface; (b) typical stress-strain curve .......................... 82
4.10 Three failure surface material model 72(first K & C revision of Model 16 by Malvar & Ross (1998)) ........................................... 83
4.11 Volume compaction curve for concrete ........................................ 84
4.12 The Ottosen failure model implemented in Winfrith concrete model (Ottosen, 1977) ................................................................. 84
4.13 General shape of the concrete model yield surface in two dimensions 86
4.14 Elastic-plastic behaviour with kinematic and isotropic hardening where $l_0$ and $l$ are undeformed and deformed lengths of uniaxial tension specimen. $E_t$ is the slope of the bilinear stress strain curve. 88
5.1 Details of beams tested by Chen & May (2009) ............................ 92
5.2 Pin-ended beam support (Chen & May, 2009) ............................... 92
5.3 Schematic diagram of Series A beam model ..................................... 97
5.4 Finite element model of Series A beam (a) Concrete beam mesh (b) Reinforcement mesh ......................................................... 100
5.5 Mesh sensitivity: influence of mesh discretization on transient displacement histories, beam A1-2 (Winfrith model) ...................... 101
5.6 Influence of time-step scale factor on transient impact force histories, beam A1-2 (Winfrith model) ........................................... 102
5.7 Transient energy-time histories, beam A1-2 (Winfrith model) .......... 103
5.8 Comparison of recorded and simulation impact force histories for beams A1(1-7) using Winfrith model ........................................ 106
5.9 Transient impact force and reaction force histories for beam A1-2 (Winfrith model) ............................................................. 107
5.10 Comparison of recorded and simulation impact force histories for beams A2(8-9) using Winfrith model ...................................... 108
5.11 Comparison of recorded and simulation impact force histories for beams B1(12-13) using Winfrith model .......................... 110
5.12 Comparison of recorded and simulation impact force histories for beams B3(16-17) using Winfrith model .......................... 111
5.13 Recorded and simulation impact force histories for beam A3-10 (Winfrith model) .................................................. 113
5.14 Recorded and simulation impact force histories for beam B2-14 (Winfrith model) .................................................. 113
5.15 Recorded and simulation impact force histories for beam B4-18 (Winfrith model) .................................................. 114
5.16 Influence of contact interface on peak impact force for beam B4-18 (Winfrith model) .................................................. 115
5.17 Comparison of transient acceleration-time histories for beam A2-9 (Winfrith model) .................................................. 116
5.18 Recorded and simulation transient reinforcement histories for beam B3-16 (Winfrith model) ........................................... 117
5.19 Comparison of final crack patterns and damage on beams A1-2 and A1-6 ................................................................. 118
5.20 Comparison of final crack patterns and damage on beams A2(8-9) ................................................................. 119
5.21 Comparison of final crack patterns and damage on beam B1-12 ................................................................. 120
5.22 Comparison of final crack patterns and damage on beams B3-16 and B3*-17 ................................................................. 120
5.23 Comparison of final crack patterns and damage on beam A3-10 ................................................................. 121
5.24 Comparison of final crack patterns and damage on beam B2-14 ................................................................. 122
5.25 Comparison of final crack patterns and damage on beam B4-18 ................................................................. 122
5.26 Correlation between impact load and crack propagation for beam A1-2 from test and analysis using Winfrith model ............ 124
5.27 Transient displacement histories of 2.7 m span beams A1-6 and A2-8 (Winfrith model) .................................................. 126
5.28 Transient displacement histories of 2.7 m span beams B1-12 and B3*-17 (Winfrith model) ........................................... 126
5.29 Transient displacement histories of 1.5 m span beams A3-10, B2-14 and B4-18 (Winfrith model) .................................... 127
6.1 Beam support with load cell .............................................. 133
6.2 Schematic diagram of test set-up ......................................... 134
6.3 Transient impact force and reaction force histories, first impact (NB1-1) ................................................................. 136
6.4 Transient impact force and reaction force histories, second impact (NB1-2) ............................................................... 137
6.5 Transient impact force and reaction force histories, third impact (NB1-3) ................................................................. 137
6.6 Impact and reaction force histories, first impact (NB3-1) ........ 138
6.7 Impact and reaction force histories, second impact (NB3-2) .... 139
6.8 Impact and reaction force histories, third impact (NB3-3) ...... 139
6.9 Transient impact and reaction force histories for single impact, beam NB2 ............................................................... 140
6.10 Transient impact and reaction force histories for single impact, beam NB4 ............................................................... 141
6.11 Transient displacement histories, first impact (NB1-1) .......... 143
6.12 Transient displacement histories, second impact (NB1-2) ...... 144
6.13 Transient displacement histories, third impact (NB1-3) ........ 144
6.14 Transient displacement histories after three impacts on NB3 ... 145
6.15 Transient displacement histories after single impact on NB2 ... 146
6.16 Transient displacement histories after single impact on NB4 ... 146
6.17 Crack patterns and damage after three impact on NB1 .......... 148
7.22 Comparison of simulation damage and cracks with the test for slab 5 (Winfrith model) ........................................... 185
7.23 Comparison of simulation damage and cracks with the test for slab 6 (Winfrith model) ........................................... 186
7.24 Comparison of simulation damage and cracks with the test for slab 6 (Concrete damage model) .................................. 187
7.25 Transient displacement histories obtained from analysis at the centre of slabs 2 to 4 (Winfrith model) ......................... 188
7.26 Transient displacement histories obtained from analysis at the centre of slabs 5 and 6 (Winfrith model) ......................... 189
7.27 Recorded and simulation impact force histories for slab 3 (Winfrith model) .............................................................. 190
7.28 Comparison of simulation damage and cracking for slab 3 (plywood interface) using Winfrith concrete model ............... 191
7.29 Details of the slabs: (a) 3.5 m square slab (impactor); (b) 4.5 m square target slab ..................................................... 192
7.30 Finite element model for the slab-on-slab impact .................. 193
7.31 Simulation damage for slab impacted by a falling slab .......... 194
7.32 Cracking in impacting slab from simulation ....................... 195
7.33 Impact force history for slab-on-slab impact ....................... 195
7.34 Velocity time history of target slab .................................. 196
7.35 Displacement time history of target slab ............................ 196
A.1 (a) SDOF system; (b) Impulse force .................................. 204
| Symbol | Description |
|--------|-------------|
| $a$ | Local deformation at the impact zone |
| $A$ | Cross-sectional area |
| $A$ | Parameter for Ottosen (1977) failure surface |
| $\mathcal{A}$ | Matrix of differential operators |
| $A_e$ | Area of master segment |
| $A_{e(max)}$ | Area of the largest side |
| $B$ | Parameter for Ottosen (1977) failure surface |
| $\mathcal{B}$ | Strain-displacement matrix |
| $c$ | Wave speed |
| $C$ | Cowper and Symonds strain rate parameter (Jones, 1989) |
| $D$ | Diagonal of the interface stiffness matrix |
| $\mathcal{D}$ | Constitutive matrix |
| $e$ | Minimum thickness to prevent perforation |
| $E$ | Modulus of elasticity |
| $E$ | Internal energy per initial volume (Chapter 4) |
| $E_a$ | Absorbed energy |
| $E_k$ | Input kinetic energy |
| $E_t$ | Tangent modulus of steel |
| Symbol | Description |
|--------|-------------|
| $f'_c$ | Unconfined compressive strength of concrete |
| $f'_{cd}$ | Dynamic unconfined compressive strength of concrete |
| $f_{cu}$ | Unconfined compressive strength of concrete cube |
| $f_t$ | Dynamic tensile strength of concrete (Chapter 4) |
| $f_t$ | Tensile strength of concrete |
| $f_{ts}$ | Quasi-static tensile strength of concrete |
| $f^B$ | Body force |
| $\underline{f}^S$ | Surface traction |
| $F$ | Impact force |
| $F_m$ | Peak impact force |
| $F_R$ | Resultant penetration resistance force |
| $F^n_s$ | Interface force |
| $h$ | Height of the structure |
| $h_s$ | Minimum thickness to prevent scabbing |
| $H$ | Drop-height of impactor |
| $I$ | Second moment of area |
| $k$ | Stiffness of the structure |
| $k_s$ | Stiffness of a contact segment in an interface |
| $K$ | Deformation constant for the stiffness of the impact zone |
| $\underline{K}$ | Stiffness matrix of the structure |
| $K_1$ | Parameter for Ottosen (1977) failure surface |
| $K_2$ | Parameter for Ottosen (1977) failure surface |
| $K_B$ | Bulk modulus of the master segment |
| $l_e$ | Characteristic length of a finite element |
| $L$ | Span of the beam |
| Symbol | Description |
|--------|-------------|
| $m$ | Mass of the structure (Saatci, 2007) |
| $m$ | Finite element (Chapter 3) |
| $\bar{m}$ | Mass per unit length |
| $m_1$ | Mass of the beam |
| $m_2$ | Impacting mass |
| $m_b$ | Mass of beam |
| $m_s$ | Mass of striker |
| $\underline{M}$ | Mass matrix of the structure |
| $n$ | Number of finite elements |
| $n$ | Number of time steps (Chapter 3) |
| $N$ | Total number of modes |
| $N$ | Number of elements (Chapter 3) |
| $\underline{N}$ | Displacement interpolation matrix |
| $p$ | Pressure |
| $P$ | Cowper and Symonds strain rate parameter (Jones, 1989) |
| $P_f$ | Penalty factor |
| $P(t)$ | Impact force-time history |
| $R_1$ | Stiffness of the beam |
| $R_2$ | Contact resistance |
| $R_m$ | Peak reaction force |
| $R_{ud}$ | Impact shear capacity |
| $\underline{R}$ | Vector of the externally applied forces |
| $t$ | Time |
| $t_0$ | Current time |
| $t_m$ | Time at peak force |
| $t_r$ | Time at peak reaction |
| Symbol | Description |
|--------|-------------|
| $T_1$ | First natural period of vibration |
| $\underline{u}^m$ | Displacement vector in local coordinate system for element $m$ |
| $u_1$ | Displacement of the beam |
| $\dot{u}$ | Velocity |
| $\ddot{u}$ | Acceleration |
| $\ddot{u}_1$ | Acceleration of the beam |
| $u_2$ | Displacement of the impacting mass |
| $\ddot{u}_2$ | Acceleration of the impacting mass |
| $\underline{U}$ | Displacement of the body |
| $\underline{U}^m$ | Nodal displacement |
| $\ddot{\underline{U}}$ | Acceleration of the body |
| $v_o$ | Impact velocity of striker |
| $v_e$ | Element volume |
| $V_e$ | Element volume |
| $V_{BL}$ | Minimum velocity to perforate the target |
| $V_{usc}$ | Calculated static shear capacity |
| $V_{usd}$ | Required static shear capacity |
| $W_{ext}$ | External virtual work |
| $W_{int}$ | Internal virtual work |
| $x$ | Penetration depth |
| $x_p$ | Relative displacement between a slave node and master surface |
| $y_b$ | Beam midspan displacement |
| $y_s$ | Striker midspan displacement |
| $\alpha$ | Ratio of beam mass to striker mass |
| Symbol | Description |
|--------|-------------|
| $\alpha$ | Time step scale factor (Chapter 3) |
| $\beta$ | Ratio of pulse duration to first natural period of vibration |
| $\delta_r$ | Design residual displacement |
| $\delta \epsilon$ | Virtual strain corresponding to virtual displacement |
| $\delta U$ | Virtual displacement in the body |
| $\Delta t$ | Time step |
| $\Delta t_{cr}$ | Maximum time step for stable solution |
| $\Delta u$ | Differential displacement |
| $\Delta \sigma$ | Stress difference |
| $\epsilon_r$ | Strain rate |
| $\epsilon_v$ | Volumetric strain |
| $\epsilon$ | Strains in the body |
| $\epsilon^m$ | Strain at any point in element $m$ |
| $\dot{\epsilon}_c$ | Dynamic strain rate in concrete in compression |
| $\dot{\epsilon}_{co}$ | Static strain rate in concrete in compression |
| $\dot{\epsilon}_{ct}$ | Dynamic strain rate in concrete in tension |
| $\dot{\epsilon}_{eto}$ | Static strain rate in concrete in tension |
| $\phi(x)$ | Assumed displaced shape normalized by the mid-span deflection |
| $\gamma$ | Ratio of specific heats |
| $\eta$ | Parameter function of modified effective plastic strain |
| $\nu$ | Poisson’s ratio |
| $\rho$ | Material density |
| Symbol | Description |
|--------|-------------|
| $\sigma_D$ | Dynamic yield stress |
| $\sigma_Y$ | Static yield stress |
| $\sigma$ | Stresses in the body |
| $\sigma^m$ | Stress in element $m$ |
| $\tau_\infty$ | Pulse duration |
| $\omega_1$ | First fundamental frequency |
| $\omega_n$ | Natural circular frequency |
Chapter 1
Introduction
1.1 Background
The analysis and design against abnormal loading conditions, such as impacts and blasts, are of paramount importance in conventional structures in general and sensitive structures in particular. The loading conditions may be encountered in a wide variety of situations, for example earthquakes, blasts and resulting fragments, wind and tornado generated missiles, accidental collisions involving vehicles, aircrafts, trains, dropped objects, aircraft missiles, bombs etc. Public concerns are very high over the integrity and safety of sensitive structures, for example nuclear containment structures, especially in the wake of recent earthquake and tsunami in Japan (2011) resulting in a major nuclear disaster at the Fukushima nuclear power plant.
Reinforced concrete because of its versatility, is perhaps the most commonly used construction material. It has a very complex behaviour, involving several factors including nonlinear behaviour of concrete in terms of stress strain response, tension cracking, biaxial stiffening, and strain softening phenomena; the definition of material failure under multiaxial stress state; modelling of post-fracturing behaviour; interaction effects between the concrete and reinforcement, etc. Furthermore if the structure is subjected to transient loads such as impact
of falling weights or explosion, this compounds the complexity in the modelling of reinforced concrete.
A concrete structure subjected to impact loading may deform both locally and globally. For example, the local response of a concrete structure due to a projectile impact may result in a number of failure mechanisms such as crushing, plug formation and scabbing. The global response usually results in a bending failure. There are a number of approaches to investigate the response of a member subjected to impact loading: experimental tests usually leading to empirical formulae; analytical approaches using mass-spring systems and/or vibrations and numerical techniques such as the finite element method. The empirical formulae are usually considered valid for the range of tests and are used to determine the local response of the member independent of its global response. The global deformations using the analytical formulations are determined usually by neglecting the local effects. These approaches, therefore, may result in an inaccurate determination of the response of concrete members under impact loading. The local response of a concrete member may have a significant influence on its global response, for example localized shear failure at the point of impact may adversely affect the integrity of the member.
Numerical methods such as the finite element method provide the necessary tools to study the impact behaviour of reinforced concrete members without separating their local and global responses. The majority of numerical investigations of the behaviour of reinforced concrete members involve two-dimensional idealizations. This simplification provides results, which may be valid for most cases, especially under static loading. However, under the dynamic impact loading, the results may be of questionable accuracy. Three-dimensional modelling has many advantages over the two-dimensional idealizations. The structure is modelled as close to the real structure as possible. The loads applied to structure are also modelled as close as possible to the real case. The modelling of triaxial stress
conditions ensures that all the critical effects e.g. the dilation of concrete and confinement effects are also taken into account, which are difficult to model in two-dimensional cases.
There has been a number of studies on the impact behaviour of reinforced concrete members, which involve extensive use of experimental tests and numerical methods such as the finite element method. The majority of these studies were performed in the military sector, therefore mainly focused on the high-velocity ballistic impacts. The nuclear power industry has also been a major contributor towards this research. However, little work has been reported on the low-velocity impacts on reinforced concrete structures. There have been some attempts on investigating the impact behaviour of reinforced concrete members using experimental and numerical methods. Mostly finite element analysis using two-dimensional idealizations has been employed. Some researchers have carried out three-dimensional modelling as well, mostly using smeared reinforcement.
1.2 Objectives
The need for three-dimensional finite element modelling of reinforced concrete members under low-velocity impacts is clearly acknowledged. The study would provide a valuable tool to determine the impact behaviour of reinforced concrete members. The main objective of this study is to develop such a three-dimensional finite element modelling procedure capable of analyzing reinforced concrete members under low-velocity impacts. In order to achieve this objective, a three-dimensional finite element program LS-DYNA (Hallquist, 2007) has been selected.
As part of the principle objective, the following investigations were carried out.
• study of the impact behaviour of reinforced concrete members subjected to falling-weight loads.
• establish a rational procedure for the dynamic structural analysis of reinforced concrete members under low-velocity regimes.
• use of the finite element method as a tool to simulate the impact response of the reinforced concrete members under falling-weight loads.
• validation of the finite element analyses using suitable material constitutive models by comparing it with the experimental results.
• further additional experimental tests on reinforced concrete beams including multiple impacts.
The modelling procedure adopted in this study simulates the impact event as close as possible to the actual event without any geometrical or material simplifications. Moreover, three-dimensional models for the complete structure have been created, so that effects of non-symmetry, for example due to support conditions are included. Furthermore, the reinforcement has been modelled explicitly to include the effects of reinforcement on the local response of the members.
1.3 Contents of the thesis
A comprehensive review of the literature pertaining to the impact behaviour of reinforced concrete members is presented in Chapter 2. Local and global responses of reinforced concrete members subjected to impact loading are discussed for experimental, analytical and numerical approaches. A critical analysis of each approach is presented for reinforced concrete beams and slabs. A vast body of literature is related to the ballistic impacts, where as the focus of the current study was on low-velocity impacts. Therefore, a selection of research on low-velocity
impacts is described. Following the review, a case is presented for a need of the present study and its significance is highlighted.
A three-dimensional finite element program LS-DYNA was selected for the analysis of reinforced concrete members under falling-weights impacts. Chapter 3 briefly describes the governing equations of the finite element method followed by the salient features of LS-DYNA and its application to reinforced concrete. A linear elastic verification of the code for a set of benchmark problems is carried out.
Chapter 4 presents a brief discussion of concrete constitutive modelling in general followed by a description of the material models available in LS-DYNA. The details of the models selected for the present study are also presented.
A comprehensive test program was conducted at Heriot-Watt University by Chen & May (2009) to investigate the impact behaviour of reinforced concrete beams under falling-weight impacts. In Chapter 5, the finite element modelling of these tests is carried out using LS-DYNA. A detailed description of the finite element models of the beams is presented followed by the analytical results and its comparison with the tests.
The results of the analysis in Chapter 5 highlighted a further need of experimental tests to generate additional data, which was not included in the original tests. Chapter 6, therefore, describes a series of experimental tests on reinforced concrete beams similar to those of Chen & May (2009) with additional data for the validation of finite element models developed in the previous chapter. The tests also included multiple impacts on the beams.
In Chapter 7, the methodology developed for the finite element modelling of reinforced concrete beams described in Chapters 5 and 6 has been extended to reinforced concrete slabs tested by Chen & May (2009). The results obtained from the analysis of different slabs are presented and a detailed comparison with the tests is described.
The conclusions drawn from the investigation into the impact behaviour of reinforced concrete beams and slabs under low-velocity, falling-weight impacts are given in Chapter 8 followed by recommendations for future research.
Chapter 2
Literature Review
2.1 Introduction
The study of impact phenomena covers an extremely wide range of situations and is of interest to researchers and engineers from a number of different fields. For example, vehicle manufacturers use their understanding of the response of structures to impact loading to improve the safety of their products; military engineers need to understand the phenomenon in order to design structures that are more efficient at withstanding projectile impact; and civil engineers have to consider the effects of abnormal loads like blasts, earthquakes, and falling objects onto the structures for the safe and efficient design. The earliest studies dealt with the impact problem as a missile striking and penetrating into a semi-infinite medium like earth and tried to predict the depth of penetration of the missile as a function of the mass, velocity, and resistance characteristics of the semi-infinite medium (Corbett et al., 1996). With the complexities in the design of reinforced concrete structures and the need to consider impact loads, this type of approach has proved to be inadequate.
During the 1940s, a great amount of experimental research was carried out in the military sector for the fortification of reinforced concrete structures against ballistic weapons. However, with the emergence of the nuclear reactor industry,
the research on the subject increased dramatically. In nuclear power structures, concrete containment walls and internal concrete barrier walls are required to withstand the effects of accidental impact loads. These include aircraft crashes, impact of tornado generated missiles (steel pipes, steel rods, wooden poles, automobiles, etc.), and internal accident generated missiles resulting from pipe or turbine breaks. This resulted in a more systematic research on the impact problem. The research done in the nuclear power industry to this date provides a solid foundation for the understanding of the effects of impact loads on reinforced concrete structures.
Missile impacts can be classified as soft or hard depending on the deformation of the missile compared to the target. In a soft impact, the missile itself undergoes significant deformation, while in a hard impact, the missile hardly deforms compared to the impacted structure. When subjected to impact, the target structure may respond in several ways depending on the nature of the impact.
**Local response:** Local damage only, as majority of the impact energy is dissipated around the impact zone.
**Global response:** Bending and deformation of the entire reinforced concrete member.
**Combined response:** Combination of both local and global damage.
Several researchers have conducted reviews of impact problems. Bangash (2009) covers the effects of shock, impact and explosion in various fields. Kennedy (1976) provides a detailed review for the effects of missile impacts on concrete structures. Corbett *et al.* (1996) reviewed the impact effects of plates and shells subjected to free-flying objects. More recently, an extensive review of concrete impact problems for local effects has been provided by Li *et al.* (2005). The phenomena generally associated with missile impact effects on concrete targets are shown in Figure 2.1 and have been defined by Li *et al.* (2005).
Figure 2.1: Missile impact effects on concrete target, (a) Penetration, (b) Cone cracking, (c) Spalling, (d) Cracks on (i) proximal face and (ii) distal face, (e) Scabbing, (f) Perforation, and (g) Overall target response (Li et al., 2005).
Penetration: Crater developed in the target at the zone of impact.
Cone cracking and plugging: Formation of a cone-like crack under the projectile and the possible subsequent punching-shear plug.
Spalling: Ejection of target material from the proximal face of the target.
Radial cracking: Global cracks radiating from the impact point and appearing on either the proximal or distal face of the concrete slab or both, when cracks develop through the target thickness.
Scabbing: Ejection of fragments from the distal face of the target.
Perforation: Complete passage of the projectile through the target with or without a residual velocity.
Overall structural responses and failures: Global bending, shear and membrane responses, and their induced failures throughout the target.
From the above impact effects, penetration, spalling, cone cracking, scabbing and perforation are considered as local impact effects and are generally quantified by the following measurements as defined by Li et al. (2005):
Penetration depth ($x$): The depth to which a projectile penetrates into a massive concrete target without perforation.
Scabbing limit ($h_s$): The minimum thickness of the target required to prevent scabbing.
Perforation limit ($e$): The minimum thickness of the target required to prevent perforation.
Ballistic limit ($V_{BL}$): The minimum initial impact velocity to perforate the target.
The local and global response of reinforced concrete structures subjected to impact loads can be investigated using a number of different approaches.
- Experimental approach
- Analytical approach
- Numerical approach
The majority of the research on global response was carried out on reinforced concrete beams, since local damage becomes much more important in the case of slabs and shells subjected to impact loading. Earlier studies done mostly by military engineers on the design of fortification structures mainly focused on high velocity (150–1000 m/s) hard impacts that cause extensive local damage without any significant global response. These studies were mainly experimental resulting in the development of empirical formulae, which had little theoretical basis. However, for civil applications, the empirical formulae had very limited application as they were only applicable to the range of available test data (Kennedy, 1976). To overcome the limitations of the experimental studies, several theoretical studies have been carried out to-date, but the major breakthrough has been the development of numerical methods to analyze the structures under impact loads, thus eliminating the need of expensive and time consuming experimental investigations. The most popular numerical technique for such analytical simulations is the Finite Element Method. These days, most of the experimental studies are carried out to generate useful data to be used for the validation of numerical investigations.
2.2 Impact behaviour of concrete beams
Figure 2.2 shows the most dominant mechanisms when a reinforced concrete beam is subjected to impact loading. Thabet & Haldane (2000) have described
the following mechanisms.
**Surface crushing**
As the impactor strikes the beam, stress waves are transmitted into the contact area on the beam during the first few microseconds. Concrete in this area is crushed and a crater on the surface of the member is formed, Figure 2.2(b) and (c).
**Concrete plug**
As the stress waves travel into the member, they encounter a large number of internal wave reflectors, such as aggregate particles, voids and cement paste. The momentum is progressively accumulated within the concrete as the stress waves are dissipated. If the momentum deposited under the impacted area is large, a local punching shear failure can occur before the beam has time to respond in flexure. This is commonly referred as a concrete plug and is usually accompanied by the development of cracks, Figure 2.2(e).
**Scabbing**
The reflection of the incident compressive stress waves results in tension failure in the concrete normal to its free surface. This localized detachment of an area of concrete normally along the flexural reinforcing bars at right angles to the direction of the impact load is referred to as scabbing and occurs on the opposite face to the impact area, Figure 2.2(d).
**Global flexural response**
As the momentum is transferred away from the impact area towards the supports region, the whole beam progressively responds in flexure, which occurs over a long period compared to the formation of the concrete plug.
A reinforced beam when subjected to static loading will exhibit a ductile flexural response or brittle shear-critical behaviour depending on the design parameters. The ductile flexural failure is characterized by the initiation and development of vertical flexural cracks at the centre. As the beam continues to deform,
Figure 2.2: Contact-impact problem involving a concrete beam (Thabet, 1994)
the flexural cracks widen and propagate towards the top of the beam.
A shear-critical beam under static loading shows a brittle behaviour characterized by the development of diagonal shear cracks near the supports. However, under impact loading, formation of diagonal cracks, originating at the impact point and propagating downward with an angle of approximately 45 degrees forming shear plug has been reported by many researchers regardless of the static behaviour (Saatci & Vecchio, 2009a). Further diagonal cracks parallel to the major shear-plug cracks may also develop. Flexural cracks at the midspan and at the supports develop and usually propagate vertically. The vertical cracks at the midspan start from the bottom surface, whereas those near the support start from the top surface. The vertical cracks starting from the top in a region of the beam away from the impact zone are associated with the traveling of the stress waves from the impact zone towards the supports in the beam.
The failure modes and crack patterns in a reinforced concrete beam subjected to impact loading at the midspan may be broadly generalized depending on their static behaviour.
Mode 1: Flexural failure with some crushing under the impactor and diagonal cracks in the impact zone forming a shear plug. Vertical flexural cracks form in the centre and near the supports. Flexure-critical beams may exhibit this mode of failure.
Mode 2: Shear failure with some crushing under the impactor and diagonal cracks in the impact zone forming a shear plug. Additional diagonal cracks develop alongside the shear-plug, which start near the supports, propagate at an angle of approximately 45 degrees upward and become horizontal close to the top of the beam. Shear-critical beams may exhibit this mode of failure.
Mode 3: Localized failure at the impact zone with extensive concrete crushing below the impactor and yielding of the tension reinforcement.
As mentioned earlier, the impact behaviour of a reinforced concrete beam
mainly depends upon its global response to impact loading as compared to its local response. A great amount of experimental work was carried out in order to develop analytical models for impact and impulsive loadings on reinforced concrete beams. Fundamental theory of vibration and SDOF systems have also been used (CEB, 1988; Hughes & Beeby, 1982; Hughes & Speirs, 1982). With the development of numerical methods, especially the Finite Element Method, many researchers have carried out extensive numerical studies supported by experiments. In the following sections, research on the impact response of reinforced concrete beams using experimental, analytical and numerical approaches is presented.
The experimental research into the impact behaviour of reinforced concrete beams was initiated by Myleea (1940), who tested 8 feet span beams without shear reinforcement, subjected to falling weights. The beams developed severe diagonal cracking. He concluded that the beams has significant impact resistance based on his failure criteria of the rupture of the longitudinal reinforcement. In the 1950s, Feldman et al. (1956, 1958, 1962) carried out a comprehensive series of impact tests on reinforced concrete beams. A total of 43 beams were tested under mid-span and two-point loading, which was applied using a pneumatic loading system comprising a piston with loading and unloading by pressurized gases. The tests were very well instrumented to record the time-histories of impact load, reactions, deflections, accelerations and strains in the concrete and the reinforcement. Based on their tests, they developed an analytical model assuming a SDOF system, which later became the basis of CEB (1988) formulations.
An extensive program of impact tests on reinforced concrete beams was conducted by Hughes & Speirs (1982). There were 80 impact tests on pin-ended reinforced concrete beams and 12 tests on simply supported beams. Impact force histories and beam displacements were measured. In most of the tests, the beam failed in a flexural mode, with flexural cracks at both the bottom of the beam, concentrated towards mid-span, and at the top close to the supports. There were
shear cracks also at both 1/3 spans. No shear failure was observed although diagonal cracks appeared in many of the beams. It was found that stiffness of the impact zone, which was a function of the impactor, the plywood pad and the local stiffness of the beam, had a more significant influence than the supports on the response of the beams. The simple beam vibration model developed was shown to be applicable over the test range and gave good correlation with the measured impact force - time history, but was shown to be inadequate for impacts with very stiff impact zones due to the likelihood of higher modes of vibration being excited.
In Japan, extensive work has been done on the dynamic behaviour of RC beams and slabs under falling weight impact loading (Ando et al., 1999; Bhatti et al., 2006; Kishi et al., 1997, 2001, 2002). A series of experimental studies for the impact resistance of slabs and beams, and their analytical simulations have been carried out.
The flexural behaviour of RC beams under impact loading predicted analytically has been presented by Ando et al. (1999). Three-dimensional FEM analysis was conducted on simply supported rectangular RC beams using LS-DYNA (Hallquist, 2007). Thirteen RC beams were analyzed of varying cross sectional dimensions and area of main reinforcement. A 200 kg steel weight was used to impact with a predetermined velocity onto the mid span of the RC beam. Material models for concrete and reinforcement were bi-linear elastic plastic model and elasto-plastic model with isotropic hardening, respectively. The computed time histories of impact force, reaction force and displacement at mid span were compared with experimental results and showed good agreement.
To investigate the shear behaviour of RC beams, Kishi et al. (2001) conducted impact tests and carried out FE impact analysis. They tested thirteen simply supported rectangular RC beams, each with dimensions of $200 \times 400 \times 2,400$ mm, Figure 2.3. The impact was applied at the mid-span using a 400 kg steel weight.
The impact velocity (3.7 - 10.2 m/s) and the shear reinforcement ratio were taken as variables. The impact force, reaction forces and mid-span deflections were recorded. These were later used to validate numerical simulations carried out using LS-DYNA. A simple elasto-plastic FE analysis was then used to predict the time histories of impact force, reaction force, mid-span displacement and the crack pattern on the side surface of RC beams and good agreement was found (Bhatti et al., 2009).
Further studies were carried out by Kishi et al. (2002) to establish a rational design procedure for shear-failure-type reinforced concrete beams under impact loads. They tested twenty seven simply supported rectangular RC beams, all without shear reinforcement. The longitudinal reinforcement and shear-span ratios were taken as variables. A free-falling weight of 300 kg was dropped at the mid span and recordings for impact force, reactions and mid-span deflections were made. The experimental results were utilized to propose a simplified impact-resistance design procedure for RC beams without shear reinforcement. They suggested a simplified model for the reaction force vs. displacement loop for shear failure as a triangle, Figure 2.4. For their range of test results, they
assumed the maximum reaction force $R_{ad}$ to be 1.5 times the calculated static shear capacity $V_{asc}$ and the absorbed energy $E_a$ given by the loop-area of the reaction force vs mid-span displacement curve to be 0.6 times the input kinetic energy $E_k$. They calculated the required static shear capacity $V_{asd}$ against the dynamic loading in terms of the maximum reaction force as
$$V_{asd} = R_{ad}/1.5 \quad (2.1)$$
The absorbed energy $E_a$ is calculated using the simplified reaction force vs displacement curve (Figure 2.4) as
$$E_a = \frac{R_{ad}\delta_r}{2} \quad (2.2)$$
The design input kinetic energy $E_{kd}$ and absorbed energy $E_a$ are related as
$$E_a = 0.6E_{kd} \quad (2.3)$$
Substituting Equations (2.1) and (2.2) into (2.3), the required shear capacity $V_{asd}$ can be found as
$$V_{asd} = \frac{1.2E_{kd}}{1.5\delta_r} = 0.8\frac{E_{kd}}{\delta_r} \quad (2.4)$$
They concluded that RC beams without shear reinforcement, failing in shear could be designed for impact loads with a certain margin of safety by assuming a dynamic response ratio of 1.5 and absorbed input energy ratio of 0.6.
The research has been further extended to large scale RC girders under falling-weight impact loads (Bhatti et al., 2006; Kishi & Bhatti, 2010). A RC girder having rectangular cross-section of 500 mm x 850 mm, with a clear span of 8 m was subjected to falling-weight impact of 2000 kg dropped freely from the height of 10 m. This type of girders has been used in the roofs of RC rock-sheds, constructed over the highways to ensure the safety of public and vehicles. Measurements of impact force, reaction forces and mid-span deflections were made,
which were later compared with analytical model developed in LS-DYNA. Figures 2.5 and 2.6 show the finite element model of the girder and material models used for concrete and steel reinforcement. They have extended the concept of simple elasto-plastic modelling of concrete used in their earlier studies on beams by including an equivalent tensile fracture energy concept. The equivalent tensile strength for a given mesh size is computed based on the fracture energy of a control element, thus allowing larger mesh sizes. The introduction of a fictitious tensile strength for a concrete element resulted in similar results for a coarser mesh in span directions to those obtained for a control element size of 35 mm.
In 1999, the investigation of the impact behaviour of beams was carried out in Sweden by Magnusson et al. (2000). They have tested high strength reinforced concrete beams under impact loads. The concrete had a compressive strength of 112 MPa. A total of eight beams were tested, three of which were subjected to quasi-static loading and five beams were subjected to impact loading. For the dynamic tests, the striker with a mass of 718 kg was dropped from a height of 2.68 m, and struck a steel pad placed at the mid-span of the 4000 mm long beam.
Figure 2.5: Finite element model of RC girder (Kishi & Bhatti, 2010)
Figure 2.6: Stress-strain relations of materials (a) concrete (b) reinforcement (Kishi & Bhatti, 2010)
The test set up is shown in Figure 2.7. Various measuring devices were used including accelerometers, strain gauges on the concrete and the reinforcement. The tests were also recorded by a high speed film camera at 1000 frames per second, which was increased to 1540 for the last test. Acceleration time histories obtained were used to calculate velocity and displacement time histories, which were then compared with photos and pulse transducer readings. Strain rates in reinforcement and concrete were also computed.
May *et al.* (2005, 2006) and Chen & May (2009) have described the results of an investigation into high mass-low velocity impact behaviour of reinforced concrete beams. They conducted tests on fifteen 2.7 $m$ or 1.5 $m$ span beams under drop-weight loads. A high-speed video camera was also used which recorded the
images at the rate of up to 4,500 frames per second. Durham strain gauges were also used in some tests to measure reinforcement strain, because of the location of the gauges inside the bar without affecting the bond between the concrete and the reinforcement (Scott & Marchand, 2000). Impact loads, strains, accelerations etc. were recorded to obtain time histories. Figure 2.8 shows the test set up for the impact tests. A software package ELFEN (2004) was tested using the data obtained from the tests. The results of these tests have been used by the author to validate finite element modelling using LS-DYNA and the analysis will be described in Chapters 5 and 6.
To investigate the impact behaviour of reinforced concrete beams in shear, Saatci & Vecchio (2009a) carried out an experimental program at University of Toronto. Eight RC beams, four pairs, were tested under free-falling drop-weights, impacting the specimens at the midspan. Longitudinal reinforcement was identical for all the specimens, but shear reinforcement ratio was varied. A total of 20 impact tests were performed which included multiple tests on some specimens. The specimens were 250 mm wide, 410 mm deep and 4880 mm long. All the beams were simply supported with a shear span of 1500 mm, with a 940 mm overhang at each end, Figure 2.9.
The test data was later used to verify a two-dimensional nonlinear finite element analysis procedure using the Disturbed Stress Field Model (Saatci & Vecchio, 2009b). NLFEA procedure was implemented into a two-dimensional, nonlinear finite element analysis program based on rotating smeared-crack approach. The 2-D model of half of the test beam comprised of a total of 992 rectangular elements to represent concrete and 124 truss bar elements for longitudinal steel, Figure 2.10. Midspan displacements, crack profiles and longitudinal reinforcement strains were compared with the experiments. Figures 2.11 and 2.12 show the comparison of midspan displacements and crack profiles, respectively.
The analyses were also carried out for the multiple impact tests on the same
Figure 2.8: Drop-weight test set up at Heriot-Watt University (Chen & May, 2009)
Figure 2.9: Details of beams tested by Saatci & Vecchio (2009a)
Figure 2.10: Finite element model by Saatci & Vecchio (2009b)
Figure 2.11: Comparison of midspan displacements for first impacts on undamaged specimens by Saatci & Vecchio (2009b)
specimens. The second impacts were analyzed, starting from the results of the analyses performed on the undamaged specimens and the results compared for midspan displacements, crack profiles and reinforcement strains. The program failed to analyze the same specimens for the third impact tests, because of accumulated errors from the previous analyses and numerical problems. Analysis of the second impact tests for some specimens were also omitted because of the specimens suffered extensive damage, which was beyond the analysis capabilities of the software.
An experimental study was performed by Fujikake et al. (2009), using drop-hammer impact tests on reinforced concrete beams to investigate the influence of drop height and the effect of the amount of longitudinal steel reinforcement. A total of twelve beams were tested. Figures 2.13 and 2.14 show the specimen details and test setup, respectively. A drop hammer with a mass of 400 kg was dropped freely onto the top surface of the beam at midspan from four different
heights for three series of beams. The impact force was recorded using a dynamic load cell, which was rigidly connected to the drop hammer. A laser displacement sensor was used to measure the midspan deflections. For series S1616, overall flexural failure was recorded at all the drop heights. For the other two series S1322 and S2222, the overall flexural failure was observed only at a drop height of no more than 0.6 m. Local failure with heavy crushing under the loading point was observed at a drop height of not less than 1.2 m. Figure 2.15 shows the typical failure modes for the impact tests. Measured impact loads and midspan deflections are shown in Figure 2.16 for series S1616 beams.
Figure 2.17 shows the maximum impact load, the impulse, and the duration of the impact load, the maximum midspan deflection, and the time taken for the maximum midspan deflection obtained at each drop height. The impulse and duration of impact load were defined, as shown in Figure 2.18. Based on the results of the impact tests, following conclusions were presented:
- The amount of longitudinal reinforcement significantly affected the failure modes of RC beams under impact loading. The RC beam with comparatively lower amounts of longitudinal steel reinforcement exhibited only
overall flexural failure, while the RC beam with the comparatively higher amounts of longitudinal reinforcement exhibited not only the overall flexural failure but also local failure located near impact loading point.
- The amount of longitudinal compression reinforcement affected the degree of the local failure. Local failure was substantially reduced when heavy longitudinal compression reinforcement was provided.
- The maximum impact load, the impulse, the duration of impact load, the maximum midspan deflection, and the time taken for the maximum midspan deflection increased as the drop height was increased. The duration of impact load, the maximum midspan deflection, and the time taken for the maximum midspan deflection were affected by the flexural rigidity of the RC beams.
Figure 2.15: Failure modes: (a) S1616 series; (b) S1322 series; and (c) S2222 series (Fujikake et al., 2009)
Figure 2.16: Impact response for S1616: (a) drop height = 0.15 m; (b) drop height = 0.3 m; (c) drop height = 0.6 m; and (d) drop height = 1.2 m (Fujikake et al., 2009)
Figure 2.17: Impact responses: (a) maximum impact load; (b) impulse; (c) duration of impact load; (d) maximum midspan deflection; and (e) time taken for maximum midspan deflection (Fujikake et al., 2009)
Figure 2.18: Impulse and duration of impact load (Fujikake et al., 2009)
There have been a number of attempts to develop analytical models for impact and impulsive loadings on reinforced concrete beams. Most of the models are based on fundamental theory of vibration using SDOF systems (Bischoff et al., 1990; CEB, 1988; Eibl, 1987; Feldman et al., 1958; Hughes & Beeby, 1982; Hughes & Speirs, 1982; Krauthammer et al., 1990). In some of the models, two-degree-of-freedom systems have also been employed (Fujikake et al., 2009).
Hughes & Speirs (1982) and Hughes & Beeby (1982) have proposed a ‘simple’ beam vibration model to analyze midspan impact of pin-ended and simply supported beams by a rigid striker. Figure 2.19 shows the impact of a rigid spherical striker of mass $m_s$ with impact velocity $v_o$ on the midspan of a pin-ended beam of mass $m_b$ and first fundamental frequency $w_1$. The local deformation at the impact zone, $a$, is given by net compression at the impact zone as
$$a = y_s - y_b \quad (2.5)$$
where $y_s$ and $y_b$ are the striker and beam midspan displacements, respectively. If each term of the above equation is expressed as a function of the force $F$, it in-fact becomes the impact equation. They used the Hertz contact law, $F = Ka^{3/2}$ to relate the deformation to the corresponding impact force $F$. Therefore
$$a = (F/K)^{2/3} \quad (2.6)$$
The displacement of the rigid striker was given by
$$y_s = v_o t - (1/m_s) \int_0^t \left[ \int_0^\tau F(\tau) d\tau \right] d\tau \quad (2.7)$$
Assuming the overall flexural response of the beam remained elastic, they modelled the beam displacement in terms of its free vibration modes by solving the simple beam equation of free vibration
$$EI \frac{\partial^4 y}{\partial x^4} + \rho A \frac{\partial^2 y}{\partial t^2} = 0 \quad (2.8)$$
They assumed that for a midspan impact, only symmetrical modes were excited (Figure 2.20) and calculated the midspan displacement as
\[
y_b = \left( \frac{2}{m_b} \right) \sum_{i=1,3}^{\infty} \left( \frac{1}{w_i} \right) \int_0^t F(\bar{t}) \sin \{ w_i(t - \bar{t}) \} \, d\bar{t}
\]
with \( w_1 = \frac{\pi^2}{L^2} \sqrt{\frac{EI}{\rho A}} \) and \( w_i = i^2 w_1 \)
Therefore, the impact equation could be expressed as
\[
(F/K)^{2/3} = v_o t - \left( \frac{1}{m_s} \right) \int_0^t \left[ \int_0^\tau F(\tau) d\tau \right] d\tau - \left( \frac{2}{m_b} \right)
\]
\[
\sum_{i=1,3}^{\infty} \left( \frac{1}{w_i} \right) \int_0^t F(\bar{t}) \sin \{ w_i(t - \bar{t}) \} \, d\bar{t}
\]
The above equation is an integral equation of impact force \( F \) and its solution in terms of the known quantities \( m_s, v_o, K, m_b \) and \( w_1 \) defines the impact. They suggested that the accurate solution could be obtained using a finite number of
modes $i = 1, 3 \ldots N$. The solution was also related to a limiting case of a massive beam with movement limited to deformation at the impact zone. They treated this limiting case essentially to be an input pulse, soluble for quantities $m_s$, $v_0$ and $K$, which depended on only two ‘input’ parameters of mass ratio ($\alpha = m_b/m_s$) and pulse ratio ($\beta = \tau_\infty/T_1$), which is the ratio of pulse duration $\tau_\infty$ and first natural period of vibration ($T_1 = 2\pi/\omega_1$). The analysis was compared with the experimental tests, which were described earlier. Figure 2.21 shows measured and theoretical force histories for one of the beams.
Figure 2.20: First three symmetrical vibration modes of a pin-ended beam (Hughes & Beeby, 1982)
Figure 2.21: Measured and theoretical force histories (Hughes & Beeby, 1982)
Idealizing the beam as a single degree of freedom system was also recommended by the Comité Euro-International du Béton (CEB, 1988). They classified the impact problem into soft and hard impacts. A single spring-mass system was proposed for soft impacts, where as for hard impact problems, two-spring mass model was suggested, Figure 2.22. For the soft impact case, the force-time history $P(t)$ is determined from the dynamics of the crashing body with a one mass model for the subsequent SDOF analysis. In the two mass model for hard impact on a beam, the distributed mass of the beam is replaced by an approximate
\begin{figure}[h]
\centering
\begin{subfigure}{0.45\textwidth}
\centering
\includegraphics[width=\textwidth]{soft_impact.png}
\caption{Single mass model for soft impact}
\end{subfigure}
\begin{subfigure}{0.45\textwidth}
\centering
\includegraphics[width=\textwidth]{hard_impact.png}
\caption{Two mass model for hard impact}
\end{subfigure}
\caption{Mass-spring model for impact (CEB, 1988)}
\end{figure}
single mass, $m_1$, which is impacted by mass $m_2$. Springs $R_1$ and $R_2$ represent the stiffness of the beam and the contact resistance, respectively. In this approach, the “participating mass” $m_1$ is determined from an estimated displaced shape resulting from energy considerations, given as
$$m_1 = \int_0^L \bar{m} \phi^2(x) dx$$ \hspace{1cm} (2.11)
where $\bar{m}$ is the distributed mass, $L$ is the span and $\phi(x)$ is the assumed displaced shape normalized with respect to the mid-span deflection. Following equations of equilibrium are required for hard impact case.
$$m_2 \ddot{u}_2 + R_2 = 0$$ \hspace{1cm} (2.12)
$$m_1 \ddot{u}_1 + R_1 - R_2 = 0$$ \hspace{1cm} (2.13)
where $R_1 = R_1(u_1)$; $R_2 = R_2(\Delta u)$; $\Delta u = u_2 - u_1$. Displacement and force time histories can be determined easily using, for example a finite difference scheme, if $R_1(u_1)$ and $R_2(\Delta u)$ are known.
The analytical methods based on simple mass-spring models using SDOF approaches have severe limitations. Determination of the force-deformation relationships for $R_1(u_1)$ and $R_2(\Delta u_2)$ would involve a number of idealizations. For impact loads, the impact force history is required which would have to be either measured or estimated as an impulse force. The force deformation relationship for the contact spring also requires thorough investigation and may be affected by several factors including impacting mass, its shape and the material properties, friction conditions etc. Moreover, SDOF idealizations can be applied only to simple structures with predominant deflection mode that can be justified by SDOF simplification. Loading patterns may also limit the use of SDOF methods. In order to overcome these limitations and analyze more complex structures, use of numerical methods such as the finite element method has been preferred over the analytical methods.
There has been tremendous growth in the development of numerical methods, especially the finite element method. Recently, codes have also been developed combining different numerical methods. For example, ELFEN (2004) combines finite element and discrete element technologies and has been used for the dynamic analysis of reinforced concrete structures (Bere, 2004). Most of these methods can analyze various types of structures under typical loading conditions. There are several commercial packages with the ability to solve complex problems including impact simulations, such as ABAQUS, AUTODYN, ELFEN, LS-DYNA, LUSAS etc. Many of these packages utilize different techniques of mesh descriptions such as Lagrangian, Eulerian, arbitrary Lagrangian and Eulerian, smooth particle hydrodynamics (SPH). The choice of time-integration (explicit or implicit) is also available. Anderson (1987) and Hamouda & Hashmi (1996) provide a detailed discussion of the numerical techniques for the simulation of highly dynamic events.
Several researchers have employed the LS-DYNA (Hallquist, 2007) commercial finite element package for the numerical simulations involving highly transient loading including impact and blast effects. In the present study also, LS-DYNA has been used for its various modelling capabilities, which will be described in further details in Chapter 3. Some of the studies described earlier in the chapter were also carried out using LS-DYNA. In the following sections, a selection of numerical studies involving low-velocity impacts on reinforced concrete beams is described.
The data obtained from the experimental tests by Magnusson et al. (2000) has been utilized by Unosson (2001) for numerical simulation using LS-DYNA together with a mechanical constitutive model for concrete, the K&C concrete model (Malvar et al., 1997). The finite element model comprised of Lagrangian elements. The concrete beam and drop weight were discretized in space with eight-node cube elements. One-point Gauss integration and viscous hourglass
control was used for the beam and the weight. The reinforcement bars were discretized with beam elements and the stirrups with truss elements. The contact surfaces, striker-steel pad and steel pad-beam, were modelled using a surface to surface constraint algorithm. The analysis was carried out up to 20 ms after impact. Figure 2.23 shows the comparison of high-speed photo at mid-span and numerical damage after 20 ms. The results of the simulations differed from the experiments, which was attributed to the material model used. The author concluded that the concrete material model was able to accurately describe the material response for standard tests such as uniaxial extension and compression. However, it did not seem to be able to reproduce the structural response in the tests performed when the available data on material properties was used in the model.
Figure 2.23: High-speed photo at mid-span and numerical damage plot after 20 ms (Unosson, 2001)
One of the main features of an advanced code like LS-DYNA is the ability to implement user-defined material models. Many researchers have formulated their own material models and used LS-DYNA as a base code. For example, Thabet (1994) implemented a new elastic-plastic fracture model incorporating a triaxial
failure criterion. Under tensile loading, an elastic stress-strain relationship was used together with smeared cracking, tension softening and shear retention model for the retained stresses. Failure of the concrete was based on either cracking or crushing which depended on a crushing coefficient determined as a function of the triaxial stress conditions. The reinforcement was considered in a distributed manner over the entire concrete element in the appropriate directions. The model was validated by analyzing a number of structural concrete members, which included standard concrete specimens (Thabet & Haldane, 2001) and reinforced concrete beams (Thabet & Haldane, 2000) and good agreement was found.
### 2.3 Impact behaviour of concrete slabs
As discussed in Section 2.1, reinforced concrete slabs are mostly subjected to local damage due to impact loads. There has been a number of experimental studies on the local effects of hard projectiles on reinforced concrete targets (mainly slabs), which have resulted in a large number of empirical formulae (Kennedy, 1976; Li *et al.*, 2005). These investigations mainly suggested the formulae for the penetration depth of the missile, and minimum thickness to prevent scabbing and perforation under impact loading. These formulae were mostly developed based on curve fitting of the test data, thus are limited to an applicable range. Petry’s formula (Kennedy, 1976; Li *et al.*, 2005) developed in 1910 was most commonly used prior to the second world war, which was later modified to include the effects of concrete strength. Several other formulae were later developed, mostly in the military including the Army Corps of Engineers (ACE) formula, the National Defense Research Committee (NDRC) formula, the UK Atomic Energy Agency (UKAEA) formula and the Ballistic Research Laboratory (BRL) formula. Details of these and many other formulae have been discussed by many researchers (Corbett *et al.*, 1996; Kennedy, 1976; Li *et al.*, 2005), therefore are not included in this review for brevity.
As mentioned earlier, the empirical formulae are based on experimental tests and are valid only for a specific range. Therefore, each formula can result in a different prediction for each local effect. For example, Yankelevsky (1997) presents the comparison between several empirical formulae for prediction of the penetration depth and the perforation thickness in a thick target, Figures 2.24 and 2.25. As seen in the figures, the compared empirical formulae give different results for penetration and perforation predictions. The modified NDRC formula provides the predictions for penetration depth and perforation thickness which are consistent and in the middle of the range. For this reason, it was widely used in the nuclear industry. However, for scabbing thicknesses, the modified NDRC formula is reported to severely underestimate the phenomenon especially for low-velocity impacts (William, 1994).
Figure 2.24: Comparison of concrete penetration depths calculated by various formulae for the case of a typical missile (Yankelevsky, 1997)
There are severe limitations associated with the use of these empirical formulae due to a large scatter in the underlying experimental tests. Moreover, the
formulae predict the local effects independent of the influence of global response of the member, i.e. bending and shear which may affect the local response especially in the low-velocity impacts. Most of these formulae also do not account for the influence of the reinforcement, which may be significant for heavily reinforced concrete members. Only the UKAEA formula (Barr, 1990) and more recently the UMIST formula (Li et al., 2005) include the effect of reinforcement. The formulae are also limited to the case of normal impact only.
There has also been considerable amount of effort spent on developing the analytical models for the prediction of local impact effects of a hard projectile on a concrete target. Li et al. (2005) identified the three main considerations associated with these models, the formulation of penetration resistance, multi-stage models and the limitation of the rigid projectile assumption. The penetration resistance of the target is defined as the resultant penetration resistance force $F_R$, applied on the projectile by the target medium during the penetration process.
It is usually defined by a binomial function of the instantaneous projectile velocity. The dynamic cavity expansion theory provides the theoretical foundation for the penetration resistance function (Forrestal & Tzou, 1997; Li et al., 2005). Further details of the analytical models for penetration resistance are discussed by Li et al. (2005) and Zaidi (2008).
The analytical models for perforation prediction are usually based on multi-stage models. Yankelevsky (1997) suggested a two-stage model to determine the perforation limit of a concrete target, Figure 2.26.
Yankelevsky (1997) describes the perforation model:
In the first stage [Figure 2.26(a)] the missile penetrates a semi-infinite medium where no effect of the rear slab face affects the penetration process. When the plastic shock front ahead of the missile, which carries a considerably larger amount of energy than the elastic wave ahead of it, meets with the rear boundary, curved shear cracks are developed and a bellshaped plug is formed [Figure 2.26(b)]. In the second state [Figure 2.26(c)] the missile pushes the plug, shears it off the surrounding concrete and continues penetrating through it. The perforation thickness is then the thickness required to penetrate and completely shear off the plug, and bring to a complete stop the missile and the plug at the end of this process.
Li & Tong (2003) extended the analytical model of Yankelevsky (1997) by combining the shear plug model with the penetration model to formulate an approach for the perforation of the concrete target. The analytical models for penetration and perforation provide reasonable results, however, the main shortcomings are the violation of the assumption of non-deformable projectile and the application being limited to normal impacts (Li et al., 2005).
With the development of numerical methods, the study of local impact effects on concrete targets using such methods has several advantages over empirical and analytical methods. The use of the finite element method along with other numerical methods, not only determines the local effects, but also predicts the influence of global response. To validate the numerical models, several experimental studies have been carried out. A selection of experimental investigations on reinforced concrete slabs is described followed by the numerical simulations.
Sawan & Abdel-Rohman (1986) carried out low-velocity impact tests on reinforced concrete slabs 75 x 75 x 5 cm in dimensions. The slabs were impacted by a 12 cm diameter steel ball at the centre from heights of up to 120 cm and the
deflection was measured. They investigated the effect of the percentage of steel reinforcement and impact velocity on the deflection response of the slabs.
To investigate the dynamic behaviour of slabs, large scale RC slabs were tested under impact loading by Kishi et al. (1997). Nine rectangular specimens measuring 4 m wide and 5 m long were repeatedly loaded onto the centre by a steel weight falling freely. Masses of 1000, 3000 and 5000 kg were used depending on slab thickness. The collapse was assumed when the accumulated residual displacement reached 1/200th of span length. Variations of the slab thickness (25, 50, 75 cm), reinforcement ratio (0.5, 1.0 %), reinforcement arrangement type (single and double arrangements) and reinforcement material were considered. The impact behaviour was considered by recording maximum impact force, reaction forces, residual displacements and crack patterns. The experiments showed that the maximum impact force was more affected by slab thickness than reinforcement ratio, material strength and the reinforcement arrangement type. The failure under repeated impact loading was initiated by flexural cracks, but final failure was due to punching failure. They estimated the punching shear capacity assuming a conical shape shear failure neglecting the reinforcement.
Zineddin & Krauthammer (2007) tested 90 x 1524 x 3353 mm slabs with three different reinforcement configurations. They used welded steel wire meshes on both faces, No. 3 steel bars located at the middle of the slab and No.3 bars on both faces for three configurations, respectively. A 2608 kg mass was dropped from three different heights of 152, 305 and 610 mm at the centre of the slabs. The tests were instrumented using load cell, accelerometers, deflection guages, reinforcement strains and high-speed videos. Figure 2.27 shows the load-time histories of slabs subjected to 610 mm drop.
Chen & May (2009) carried out a series of experimental studies to investigate the high-mass, low-velocity impact behaviour of reinforced concrete beams and slabs. The tests were conducted to generate high quality input data to validate
Figure 2.27: Load-time histories of slabs under 610 mm drop (Zineddin & Krauthammer, 2007)
numerical modelling. They tested four 760 mm square, 76 mm thick and two 2320 mm square, 150 mm thick slabs. A drop-weight system was used to drop a mass of up to 380 kg with velocities of up to 8.7 m/s. Supports were provided by clamping at the four corners to restrain horizontal and vertical movement. The author has used the results of these tests to perform the three-dimensional finite element analysis, which will be described in Chapter 7.
Agardh & Laine (1999) performed a three-dimensional simulation of projectile perforation in 60 mm thick reinforced concrete slab. They used LS-DYNA with material 78 and incorporated erosion in the model. They compared the penetration and exit velocities as well as the weight of the projectile fragments. The diameters of the exit craters were also compared and were in reasonable agreement. Figure 2.28 shows the penetration of the projectile fragment into the slab after 30 μ s.
Algaard et al. (2005) and Izzat et al. (2009) have used the results of slabs tests carried out at Heriot-Watt University to assess the impact behaviour for
perforation of slabs. They have modelled the numerical simulation using LSDYNA. The FE model used non-linear material properties for both the steel and concrete. They modelled reinforced concrete using the Winfrith concrete and reinforcement models with element erosion. The results have been compared with empirical penetration formulae for concrete structures and experimental tests, and perforation limit agreement has been found.
Figure 2.29: Finite element model of slab and loading (Izzat et al., 2009)
2.4 Significance of the current study
This thesis will describe investigations into the impact behaviour of reinforced concrete beams and slabs subjected to falling weight loads. While a number of experimental and numerical studies have been carried out for ballistic regimes; there is need of a greater effort for low velocity situations, which is of most relevance to civil engineering structures. The main objective of the present study is to establish a rational procedure for the dynamic structural analysis of reinforced concrete structures under low velocity impacts. The finite element analysis has been carried out using LS-DYNA (Hallquist, 2006, 2007), an explicit finite element program used for the non linear transient numerical analysis of structures. The three-dimensional finite element analysis results have been compared with the experimental data for the beams and slabs tested at Heriot-Watt University. This data was produced as a result of a series of well monitored tests conducted on reinforced concrete beams and slabs impacted by falling weights. The test programme was specifically designed to produce high quality input data for the computational solution of the phenomenon and its validation. In addition to the three-dimensional analysis of beams and slabs under impacts, experimental tests on reinforced concrete beams have also been carried out to generate test data, in addition to those reported in Chen & May (2009).
Chapter 3
Finite Element Modelling
3.1 Introduction
The finite element method has emerged as one of the most powerful numerical techniques used for the solution of engineering problems. It has provided the necessary tools to model and analyze virtually any engineering structural system. In particular, finite element methods have been very useful for modeling problems involving reinforced concrete. This Chapter briefly covers the fundamentals of the finite element method in general and its application to the dynamic analysis in a commercial finite element code LS-DYNA (Hallquist, 2006, 2007). A generalized formulation of the governing equations of a continuum is described. A brief description of the salient features of the code and its application to modelling reinforced concrete is also discussed. In the end, a linear elastic verification study is carried out for a set of example problems taken from Saatci (2007).
3.2 The Finite Element Method
The finite element method is a numerical technique for obtaining approximate solutions to the partial differential equations that are encountered in many scientific and engineering problems. In general, solving these equations by classical
analytical methods for arbitrary shapes is almost impossible. The basic concept of FEM is to divide the body into a number of subdivisions, called *finite elements*. A finite number of points or *nodes* are identified on the element where the finite elements are to be connected and the conditions of equilibrium and displacement continuity between the elements are to be satisfied. For each element, simple polynomial or interpolation functions are defined, which relate the geometry and the displacement at each point in the finite element to its nodal values. The accuracy of the approximate solution depends upon the number of elements (*discretization*) as well as the choice of interpolation functions used in the modelling of the continuum (Zienkiewicz & Taylor, 2005).
The origin of the finite element method has often been traced back to Hrenikoff (1941) and McHenry (1943) who decomposed a two-dimensional problem domain into an assembly of one-dimensional discrete elements. Most of the early research and development in the 1950s took place in aerospace industry. Argyris (1954) and Argyris & Kelsey (1955) formulated an efficient technique to analyze complex aircraft structures, which was based on matrix operations. Turner *et al.* (1956) published one of the first papers that established the procedure of element matrix assembly and element formulations. They produced the actual solution to the plane stress problems using triangular elements, but did not use the term ‘*finite elements*’. Clough (1960) appears to have been the first to use this term. This is widely regarded as the beginning of the finite element era. Since then the finite element method has been widely used for the solution of complex problems in the field of engineering.
Since the early 1960s, a large amount of research has been carried out and a very large number of publications on the finite element method are available in the literature (see for example Noor, 1991, for a compilation of references). Several text books provide a detailed discussion of the method and its applications (Cook *et al.*, 1989; Hughes, 1987; Hutton, 2004; Zienkiewicz & Taylor, 2005).
3.3 Generalized finite element formulation
The first practical application of finite element procedures was mainly in the linear analysis of solids and structures (Bathe, 1996), which led to the subsequent development of the method. Various formulations have been presented in the literature. For solids, which is the primary area of application in this study, formulation based on the principle of virtual work is widely used. Therefore, a generalized displacement-based formulation of a solid continuum based on the above principle is developed.
Firstly, the equilibrium equations governing the behaviour of a continuum subjected to an externally applied force are derived. Figure 3.1 shows a general three-dimensional body in space defined by a system of Cartesian coordinates. The body is subjected to external forces, surface traction $\underline{f}^S$ and body forces $\underline{f}^B$. In general, the externally applied forces, which include all the forces including reactions, have three components corresponding to the $X$, $Y$, $Z$ coordinate axes. The displacement of a particle within the body initially at point $P(X,Y,Z)$ is given by $\underline{U}$, having components $U$, $V$ and $W$ in the $X$, $Y$ and $Z$ directions, respectively.
Figure 3.1: General three-dimensional body (Bathe, 1996)
If the externally applied forces and geometry of the body are assumed to be given, the solution is sought for the displacements $\underline{U}$ of the body and the corresponding strains $\underline{\varepsilon}$ and stresses $\underline{\sigma}$. To obtain a solution, the governing equations of equilibrium are required. The displacement-based formulation in the finite element method uses the principle of virtual work to establish the governing equations of equilibrium for the body. The principle states that “the equilibrium of a deformable body requires that for any compatible small virtual displacements imposed on the body in its state of equilibrium, the total internal virtual work is equal to the total external virtual work” (Bathe, 1996).
$$W_{int} = W_{ext} \quad (3.1)$$
$$\int_V \delta \underline{\varepsilon}^T \underline{\sigma} dV = \int_V \delta \underline{U}^T f^B dV + \int_S \delta \underline{U}^T f^S dS \quad (3.2)$$
The internal work given on the left hand side of Equation (3.2) is equal to the actual stress $\underline{\sigma}$ resulting from the virtual strain $\delta \underline{\varepsilon}$ corresponding to the virtual displacement $\delta \underline{U}$. The external work on the right hand side of Equation (3.2) is equal to the actual external forces times the virtual displacement $\delta \underline{U}$. Equation (3.2) is a statement of the static equilibrium of the body. The applied forces may vary with time, in which case the displacements also vary with time and Equation (3.2) is considered for any specific point in time. However, if the loads are applied rapidly, inertial forces need to be considered, which results in a dynamic problem. The principle of virtual work can be extended to dynamic equilibrium using d’Alembert’s principle (Chopra, 2001), which simplifies the problem by the inclusion of inertia forces as part of the body forces. Assuming that the accelerations are approximated in the same ways as the displacements, Equation (3.2) can now be written as:
$$\int_V \delta \underline{\varepsilon}^T \underline{\sigma} dV = \int_V (\delta \underline{U}^T \underline{f}^B - \delta \underline{U}^T \rho \dot{\underline{U}}) dV + \int_S \delta \underline{U}^T f^S dS \quad (3.3)$$
or
\[
\int_V \delta U^T \rho \dot{U} dV + \int_V \delta \varepsilon^T \sigma dV - \int_V \delta U^T f^B dV - \int_S \delta U^T f^S dS = 0
\]
(3.4)
Equation (3.4) states the dynamic equilibrium of the body.
**Finite Element Equations**
The governing finite element equations are now derived from the discretization of the equilibrium equations. In the finite element analysis, the body is approximated by an assemblage of an arbitrary number of finite elements, \( n \), interconnected at nodal points on the element boundaries. Consider a finite element \( m \). The displacement vector measured in a local coordinate system \( x, y, z \) at any point within the element, \( u^m \), is interpolated as
\[
u^m = N U^m
\]
(3.5)
where \( N \) is the displacement interpolation matrix, which relates the displacement at any point within the element, \( u^m \), to the nodal displacement \( U^m \). With the displacements computed at all the points within the element, the corresponding strain at any point \( \varepsilon^m \), is evaluated by differentiating the displacement:
\[
\varepsilon^m = A u^m
\]
(3.6)
where \( A \) is a matrix of differential operators. Substituting Equation (3.5) into Equation (3.6) yields:
\[
\varepsilon^m = B U^m
\]
(3.7)
where \( B = A N \) is the strain-displacement matrix. It gives the values of strain at any point within the element, resulting from the unit values of the nodal displacements. The dynamic equilibrium of the body defined by Equation (3.4)
can be written as the sum of the integrations over volume and areas of all the finite elements:
\[
\sum_{n} = \left\{ \int_{V} \rho N^T N dV \ddot{U}^m + \int_{V} B^T \sigma^m dV - \int_{V} N^T f^B dV - \int_{S} N^T f^S dS \right\} = 0
\]
(3.8)
The stresses in a finite element are related to the strains through the constitutive matrix \( D \) as
\[
\sigma^m = D \varepsilon^m
\]
(3.9)
Therefore, Equation (3.8) can now be written as:
\[
\sum_{n} = \left\{ \int_{V} \rho N^T N dV \ddot{U}^m + \int_{V} B^T D B dV U^m - \int_{V} N^T f^B dV - \int_{S} N^T f^S dS \right\} = 0
\]
(3.10)
The above equation may be re-written as follows:
\[
M \ddot{U} + K U - R = 0
\]
(3.11)
where \( M \) and \( K \) are the mass and stiffness matrices of the structure, respectively, and \( R \) represents the vector of the externally applied forces. Equation (3.10) is a system of equations which represents the displacement-based finite element formulation for a generalized continuum.
### 3.4 Finite element code LS-DYNA
LS-DYNA (Hallquist, 2007) is one of the most popular commercial FEM programmes used for numerical analysis involving impacts, blast and many other transient phenomena. It was formerly known as DYNA3D and originated at the Lawrence Livermore National Laboratory (Hallquist, 1976). LS-DYNA is a three-dimensional FEM package with advanced modelling capabilities for contact-impact problems. It offers many formulations based on Lagrange, Eulerian, ALE
and meshless methods. The programme has a comprehensive material library with the options to include user defined materials. Since the early development of the code took place for military purposes, therefore, it has many advanced capabilities for modelling blasts, explosives and missile penetrations. In the following sections, a brief description of the salient features of the code is provided. The details of the constitutive models are given in Chapter 4.
3.4.1 Solution of the governing equations
In Section 3.3, the equations of equilibrium governing the dynamic response of a system of finite elements were derived. It should be recalled that Equation (3.11) has been derived using considerations of statics at time $t$, therefore, it can be re-written in the following form:
$$M \ddot{U} + K U = R$$ \hspace{1cm} (3.12)
Above equation represents a system of second order differential equations, the solution of which can be obtained by standard procedures for the solution of differential equations with constant coefficients. However, if the order of matrices is very large, the procedures become very expensive. Therefore, numerical time integration techniques, for example explicit or implicit methods are used at discrete time intervals.
In the explicit method, the solution for accelerations is obtained using the equilibrium conditions at time $t_0$. The displacements and velocities are then determined using a central difference procedure at time $t_1 = t_0 + \Delta t$. In an explicit finite element method, the solution is obtained on an element-by-element basis i.e. the internal nodal forces at time $t_0$ can be computed and therefore a global stiffness matrix does not need to be formed. As a result, explicit methods can treat large three-dimensional models having thousands of degrees of freedom with comparatively modest computer storage requirements. However, explicit
methods are conditionally stable and therefore small time steps must be used. For stable computations, the time step is selected such that it is less than the time required for a stress wave to travel through the shortest element, and therefore this could result in excessive run times as the level of discretization increases.
The implicit method obtains the solution of the displacements at time $t_1$ using the equilibrium conditions at time $t_1 = t_0 + \Delta t$. The method is unconditionally stable, but is very expensive for dynamic problems as the stiffness matrix must be fully assembled and inverted because it is the coefficient matrix of the unknowns in the system of equations.
**Explicit time integration**
LS-DYNA employs explicit time integration for dynamic problems based on the central difference scheme. The solutions procedure for Equation (3.12) using central difference method is briefly described here. It is assumed that the displacement $\underline{U}_0$, velocity $\dot{\underline{U}}_0$, and acceleration $\ddot{\underline{U}}_0$ vectors at time $t = 0$ to $t = t_0$ are known. After a short time interval $\Delta t$ at the point $t_1 = t_0 + \Delta t$, a linear forward step in time is taken by expanding the displacement vector $\underline{U}$ with respect to time using Taylor series at time $t = t_0$.
$$\underline{U}_1 = \underline{U}_0 + \Delta t \dot{\underline{U}}_0 + \frac{\Delta t^2}{2} \ddot{\underline{U}}_0 + \ldots \quad (3.13)$$
and
$$\underline{U}_{-1} = \underline{U}_0 - \Delta t \dot{\underline{U}}_0 + \frac{\Delta t^2}{2} \ddot{\underline{U}}_0 - \ldots \quad (3.14)$$
where the subscripts 0, −1 and 1 denote the values at times $t_0$, $t_0 - \Delta t$ and $t_0 + \Delta t$, respectively. The error in the above expansion is of the order of $(\Delta t^2)$. Similarly, at time $t_1$,
$$\underline{U}_0 = \underline{U}_1 - \Delta t \dot{\underline{U}}_1 + \frac{\Delta t^2}{2} \ddot{\underline{U}}_1 \quad (3.15)$$
Now substituting $\dot{U}_1$ from Equation (3.13) into Equation (3.15) yields the following:
$$\dot{U}_1 = \dot{U}_0 + \frac{1}{2}(\ddot{U}_0 + \ddot{U}_1)\Delta t$$ \hspace{1cm} (3.16)
The displacements, velocities and accelerations at time $t_1$ are determined using Equations (3.13) and (3.15), respectively. The process is moved forward to the next time step $t_2 = t_1 + \Delta t$, as $\dot{U}_1$, $\ddot{U}_1$ and $\ddot{U}_1$ are known from the previous step.
**Time-step calculation**
As discussed in the preceding section, explicit time integration is conditionally stable and the time step size used should be less than the natural period of vibration in the model. Determination of the highest natural frequency is often very complicated and computationally expensive, therefore, an approximation in terms of the ratio of the characteristic length of the element to wave speed is used. To ensure a stable solution, the time step used should be less than the critical time step, which is the time needed for a wave to propagate through the length of an element.
$$\Delta t \leq \Delta t_{cr}$$ \hspace{1cm} (3.17)
and
$$\Delta t_{cr} = \frac{l_e}{c}$$ \hspace{1cm} (3.18)
where $\Delta t_{cr}$ is the maximum time step required for a stable solution, $l_e$ is the characteristic length of a finite element and $c$ is the wave speed. Characteristic length $l_e$ depends upon the type of the element used. For one-dimensional beam and truss elements, it is simply the length of the element. The wave speed $c$ is given as:
$$c = \sqrt{\frac{E}{\rho}}$$ \hspace{1cm} (3.19)
where $E$ and $\rho$ are the Young’s modulus and the density of the material, respectively. For 8-noded solid element, the characteristic length $l_e$ is defined as:
$$l_e = \frac{v_e}{A_{e(max)}}$$
(3.20)
where $v_e$ is the element volume and $A_{e(max)}$ is the area of the largest side. For elastic material and constant bulk viscosity, the wave speed $c$ is given by
$$c = \sqrt{\frac{E(1-\nu)}{(1+\nu)(1-2\nu)\rho}}$$
(3.21)
During the solution loops in LS-DYNA, a new time step size is automatically determined for each subsequent loop by taking the minimum value over all the elements.
$$\Delta t^{n+1} = \alpha \cdot \min \left\{ \Delta t_1, \Delta t_2, \Delta t_3, \ldots, \Delta t_N \right\}$$
(3.22)
where $N$ is the number of elements and $\alpha$ is the time step scale factor. The default value of $\alpha$ is taken as 0.9 based on the stability considerations.
### 3.4.2 FE modelling of reinforced concrete
Finite element modelling of reinforced concrete requires a rigorous application of the method because of a number of idealizations and complexities involved in concrete. Two dimensional modelling of reinforced concrete has been widely used and has proved adequate for a number of conditions. However, for problems involving contact-impact of reinforced concrete, only three dimensional modelling can adequately represent the local and global behaviour. This is true specially at the point of impact where the states of stress and strain are in three dimensions. To model a member using three dimensional finite elements, tetrahedral or hexahedral elements can be used.
Eight Node hexahedral element
In LS-DYNA, the most popular choice for modelling concrete in three dimensions is to use an eight noded hexahedral element. Figure 3.2 shows the eight node hexahedron element. Full details of the element formulation are given elsewhere (Hallquist, 2006; Liu & Quek, 2003). The element uses a reduced integration scheme (one-point integration) and has been found to be very efficient (Belytschko et al., 2000). Especially in the problems involving contact-impacts, the computational efficiency is very high compared to the fully integrated element. The biggest disadvantage to the one-point integration is the need to control the zero energy modes or hourglassing, Figure 3.3. LS-DYNA offers a number of formulations to resist undesirable hourglassing for example by applying viscous damping or small elastic stiffness capable of stopping the formation of zero energy modes without having any significant effect on stable global modes. Schwer (2005) has provided a detailed comparative study of hourglass formulations in LS-DYNA.
Beam element for reinforcement
Reinforcing bars in concrete are idealized using one dimensional beam elements. LS-DYNA offers a number of beam element formulations. The details are given elsewhere (Hallquist, 2007). The interactions between the concrete elements and
the reinforcement beam elements can be modelled by a discrete model or an embedded model, Figure 3.4. In the discrete representation, the concrete elements and the bar elements share common nodes, Figure 3.4(a). Full bond is generally assumed between the reinforcement and the concrete. In some cases, linkage elements having a spring stiffness determined by a specific bond-slip relationship may also be defined. The main drawback of this approach is that the mesh discretization is restricted by the location of the reinforcement. On the other hand, the embedded formulation overcomes this problem by allowing the placement of the reinforcement in any layout such that the displacements of the reinforcing bars are compatible with those of the concrete element. In concrete structures where the reinforcement layout is often complex, the embedded representation may be advantageous. The third approach is the distributed or smeared representation, where the reinforcement is assumed to be distributed over the concrete element in the appropriate direction. In this approach, reinforcing bars are not discretely
modelled as in the other two approaches, but the contribution of reinforcement is obtained using a ratio, which is dependent on the size of the concrete element and the quantity of steel present within it. In LS-DYNA, all the three approaches can be modelled. Another approach available is by coupling of the reinforcement with the concrete solid elements using the keyword *Constrained_Lagrange_In_Solid (Hallquist, 2007), which is similar to the embedded reinforcement but with the advantage of no additional nodes required as is the case in embedded approach.
 
(a) Discrete model
(b) Embedded model
Figure 3.4: Representation of reinforcement in concrete model
### 3.4.3 Contact force determination
In an impact analysis, the forces acting between the impactor and the target are not known in advance. These forces depend upon geometric and material properties of the contact interface as well as the velocity of the impactor at the time of contact. There are several techniques available to determine these unknown forces. For example, discrete spring elements between the contacting bodies can be used, which would require a constraint to the global governing equations at the time of impact. LS-DYNA offers several formulations to address this problem. Contacts in LS-DYNA are treated using the penalty stiffness method, which uses the concept of master and slave segments sliding on each other. The method
consists of placing normal interface springs to resist interpenetration between element surfaces. Figure 3.5 shows a contact between an impactor and a target using penalty formulation. As shown in this figure, when a slave node penetrates a master surface in a time step, the penetration is detected automatically and an internal force given by the spring is applied to resist penetration and keep the node outside the surface. The internal forces added to the slave nodes are a function of the penetrated distance and a calculated stiffness for the master surface. The stiffness is computed as a function of the bulk modulus, volume, and face area of the elements in the master surface.
The interface force $F_s^n$ causing relative displacement $x_p$ between a slave node and its master surface at time step $n$ is initially calculated as:
$$F_s^n = Dx_p \quad (3.23)$$
where $D = \text{diag}(k_s, k_s, k_s)$ and $x_p = (u_x, u_y, u_z)^T$, the elastic displacement between a given slave node and associated master surface. At time step $n + 1$ the trial interface force is calculated as
$$F_s^{n+1} = F_s^n + D\Delta x_p \quad (3.24)$$
where the stiffness of a contact segment in an interface, $k_s$, can be calculated from:
$$k_s = \frac{P_f A_e^2 K_B}{V_e} \quad (3.25)$$
where $P_f$ is a penalty factor, $A_e$ is the area of the master segment, $K_B$ is the bulk modulus of the master segment and $V_e$ is the element volume. Use of excessively large penalty stiffnesses can affect the stability of the solution and could result in an unstable oscillating solution (Hunek, 1993).
3.5 Verification of LS-DYNA dynamic analysis
A simple test structure proposed by Saatci (2007) has been selected to perform verification of dynamic analysis in LS-DYNA under linear elastic conditions. The test example was a single-mass vertical structure with fixed base, 1000 mm high and 100 mm wide with 50 mm thickness, Figure 3.6. The finite element model was created in LS-DYNA using 4 noded plane stress shell elements. The mesh consisted of 891 nodes and 800 elements with $10 \times 12.5$ mm in size. The middle node at the top of the structure (node 96) was assigned a lumped mass of 500 kg, whereas the rest of the structure had no mass. A fixed-base condition was simulated by restraining the nodes corresponding to the base of the structure against movements in all directions. In order to facilitate the determination of the analytical response of the structure, it was idealized as a SDOF system.
The example structure was subjected to lateral static loads, initial velocity inducing free vibrations and impulse loads to determine its analytical and numerical response under linear elastic conditions. The analysis was performed in LS-DYNA and the results compared with the analytical solutions. The details of
the comparisons for each type of loading are described in the following sections.
 
Figure 3.6: Example test structure (Saatci, 2007) and finite element model
### 3.5.1 Static loading
As mentioned earlier, the example structure was idealized as a linear elastic SDOF system, with elastic modulus $E$ assumed as 40 $GPa$. For a vertical structure fixed at the base and subjected to lateral displacement at the top, the lateral stiffness $k$ is given as:
$$k = \frac{3EI}{h^3} = \frac{3(40000)\frac{50(100^3)}{12}}{1000^3} = 500 \text{ N/mm}$$ \hspace{1cm} (3.26)
where $I$ and $h$ are the second moment of area and the height of the structure, respectively. In order to verify the calculated stiffness of the structure, a series of lateral static loads was applied at the top central node 96, Figure 3.6(b) resulting in the lateral displacement of the structure. Figure 3.7 shows the load vs displacement curve for the structure. The slope of the curve shows the average stiffness of 500 $N/mm$, which confirms to the stiffness calculations in Equation (3.26). The idealization of the structure as a SDOF system was thus justified.
3.5 Verification of LS-DYNA dynamic analysis
Figure 3.7: Response of example test structure under static loading
### 3.5.2 Free vibrations response
The differential equation governing free vibration of an undamped SDOF system is given as:
\[
m \ddot{u} + k u = 0 \tag{3.27}
\]
Free vibration in the system is initiated by disturbing the static equilibrium by assigning the mass some displacement \( u(0) \) and velocity \( \dot{u}(0) \) at time zero. The initial conditions \( u = u(0) \) and \( \dot{u} = \dot{u}(0) \) lead to the solution of Equation (3.27) (Clough & Penzien, 2003) as given below.
\[
u(t) = u(0) \cos \omega_n t + \frac{\dot{u}(0)}{\omega_n} \sin \omega_n t \tag{3.28}
\]
The analytical dynamic displacement response of the example structure was determined by assigning an initial velocity of 0.5 \( m/s \) and initial displacement of zero to the lumped mass at the top node 96. The natural period of vibration \( T_n \) and the natural circular frequency \( \omega_n \) were calculated based on \( k = 500 \ N/mm \), and were found to be 0.2 s and 31.63 \( rad/s \), respectively. A dynamic analysis using LS-DYNA for the example structure was carried out, which is compared
with the exact analytical response in Figure 3.8. As seen in the Figure, the two solutions are in very good agreement.
### 3.5.3 Impulse forces response
The dynamic response of the example structure was determined for two sets of impulse forces applied at the top node 96. Both sets of the impulse forces had different durations and were considered as short and long duration impulse forces (Saatci, 2007). The short impulse with $0.1 \text{ s}$ duration initiated a free vibration, whereas the long impulse with $0.8 \text{ s}$ duration caused a relatively irregular response. Figure 3.9 shows the force-time histories for the applied impulse forces. The numerical analysis was carried out using the same parameters as those for free vibration response described in the preceding section.
The determination of the analytical response for a SDOF system subjected to impulse loading is given in Appendix A. The analytical displacement response of the example structure was determined using Equations (A.1), (A.2) and (A.3) for the two sets of impulse forces. Figures 3.10 and 3.11 show the comparison
between the numerical and analytical response for the short and long impulse forces, respectively. Both Figures demonstrate a very good agreement between the LS-DYNA numerical solution and the exact analytical solution.
Figure 3.10: Comparison of the analytical and numerical response under short impulse
The comparison of LS-DYNA numerical solutions for the example structure with the exact analytical solutions for different loading conditions in the linear elastic range show very good agreement. Discrepancies between the two solutions were found to be less than 1%. These minimal errors could be attributed to a number of idealizations and discretization errors inherent with the finite element formulations.
Chapter 4
Constitutive Modelling
4.1 Introduction
In this Chapter, a detailed discussion of the material constitutive models used for the finite element analysis is presented. The Chapter starts with the general discussion about the behaviour of concrete under uniaxial and multiaxial loading conditions. The factors influencing it have been discussed briefly, highlighting the complexities involved in the modelling of concrete. The concrete material models available in LS-DYNA are briefly discussed along with the models used for steel reinforcement and the falling-weight impactor.
4.2 Concrete constitutive modelling
Extensive research over the past two decades has resulted in a variety of constitutive models that are capable of representing the various aspects of concrete behaviour. Given the complex nature of concrete, it is not surprising that a large number of constitutive models has been developed. Concrete material formulations can be classified into elasticity-based models, plasticity-based models, plastic-fracturing models, elastic-plastic damage models, and endochronic models. Despite the extensive research effort, there is still a need to develop a reliable
concrete material model capable of representing the complex behaviour of different types of concrete structures, and therefore it is still an area of continuing research.
4.2.1 Uniaxial behaviour of concrete
The physical behaviour of concrete is very complex. It largely depends on the structure of the composite material, which comprises of coarse aggregates and continuous matrix of cement mortar and finer aggregate particles. Concrete is considered to be a brittle material, the stress-strain behaviour of which being affected by the development of micro and macro cracks. Even before the application of any external load, a large number of micro cracks caused by shrinkage, segregation and thermal expansion in the cement paste will have developed. With the application of external loads, these cracks progress into larger cracks mainly contributing to the nonlinear stress-strain behaviour.
A typical stress-strain curve of concrete under uniaxial compression is shown in Figure 4.1 (Kupfer et al., 1969). There are three deformation stages, which can be identified qualitatively: the linear elastic stage, the inelastic stage, and the localized stage.
4.2.2 Multiaxial behaviour of concrete
The multiaxial behaviour of concrete is very complex because of the confinement effects in compression. A typical stress-strain curve of concrete under multiaxial compression is shown in Figure 4.2. From the tests of cylindrical specimens subjected to constant lateral pressures, $\sigma_2 = \sigma_3$, it has been observed that the confining pressure has significant effects on the deformation behaviour of the specimen (Palaniswamy & Shah, 1974). Compared to uniaxial compression, confined concrete specimens undergo much larger strains and concrete exhibits a certain degree of ductility before failure.
Figure 4.1: Typical uniaxial stress-strain curve for concrete specimen in compression, reported in Kupfer et al. (1969)
Figure 4.2: Typical multiaxial stress-strain curve for concrete specimen in compression (Palaniswamy & Shah, 1974)
4.2.3 Factors involved in concrete modelling
As discussed in sections 4.2.1 and 4.2.2, concrete has a very complex nonlinear behaviour involving a number of factors. The three deformation stages: linear elastic stage, inelastic stage, and the localized stage have to be considered in modelling concrete behaviour. The important factors that significantly affect the behaviour of concrete include: nonlinear behaviour in both uniaxial and multiaxial loading; different responses in tension and compression; volume expansion under compressive loading; strain softening; tension stiffening; and stiffness degradation under cyclic loading. Some of these factors are discussed in the following sections.
**Strain softening**
Strain softening is generally defined as the decrease in the strength of the material beyond its peak failure stress. It is characterized by the descending branch of the stress-strain curve and by a degradation of the elastic modulus on unloading. Concrete, like many other engineering materials such as rocks and soils, exhibits this behaviour. Typical uniaxial compressive stress-strain curves obtained from strain-controlled tests are shown in Figure 4.3 (Wischers, 1978). Each of these curves has a sharp descending branch beyond peak failure stress.
There has been a serious debate about the softening behaviour being considered as the response of the structure formed by specimen together with test set up rather than a material property (van Mier, 1984). It has been argued that since the post-peak deformation was localized, the descending branch of the stress-strain curve could not be considered a material property.
**Tensile response**
Concrete has different responses in tension and compression. Its tensile strength is very low, approximately $1/10^{th}$ of the compressive strength. Figure 4.4 shows a
Figure 4.3: Stress-strain curves for concrete in uniaxial compression (Wischers, 1978)
typical uniaxial stress-strain curve in tension (Peterson, 1981). The deformation behaviour of concrete in tension is quite brittle in nature.
Figure 4.4: Stress-strain curves for concrete in uniaxial tension (Peterson, 1981)
**Tension stiffening**
The effect of tensile stresses is usually neglected in a cracked cross section below the neutral axis. This may result in overestimation of the deformation, since at sections between successive cracks, some tensile stresses are retained in concrete around steel bars. This is due to the action of bond and is called as the “tension stiffening” effect. The tension stiffening effect represents the capacity of the intact concrete between neighbouring cracks to carry a limited amount of tensile forces.
**Volume dilatation**
Concrete tends to expand under the compression load. A typical curve showing volumetric strain plotted against the stress in biaxial compression is shown in Figure 4.5. This expansion in volume has been attributed to the composite nature of concrete (Shah & Chandra, 1968). It was observed by Shah & Chandra (1968) that the cement paste itself did not expand under the compression load. Volume dilatation was observed only when the cement paste was mixed with aggregate.
Stiffness degradation
Under cyclic loading, concrete tends to lose its stiffness. The loading and unloading paths are different and the elastic modulus or slope degrades with increasing straining. Figure 4.6 shows a typical uniaxial compressive stress-strain curve of concrete under cyclic loading (Sinha et al., 1964). This stiffness degradation behaviour is often related to the damage caused by microcracks or microvoids. It becomes much more significant in the post-peak range.
4.3 Strain-rate effects
The modeling of the strain rate effect in concrete is not very easy to tackle. A mathematical model is useful in order to describe the observed material behaviour such as strain rate effects only when a well-understood phenomenon is available. From the material point of view, concrete shows an increase in strength as the strain rate increases, a phenomenon called the strain rate effect. Particuparticularly when impact and penetration simulations are considered, importance of the strain rate effect becomes paramount. The effect of strain rate on the concrete compressive and tensile strengths is typically considered in terms of a dynamic increase factor (DIF) or the ratio of the measured dynamic to the quasi-static strength. This is found to be a convenient format for implementation into numerical simulations.
The CEB Model Code (1993) presents a detailed formulation for strain rate effects, which is widely used. It is considered to be the most comprehensive model for strain rate enhancement of concrete, both in compression and tension (Malvar & Ross, 1998).
In compression, the dynamic increase factor (DIF) of the compressive strength recommended by CEB, is given by
\[
\frac{f'_{cd}}{f'_c} = \begin{cases}
(\dot{\varepsilon}_c/\dot{\varepsilon}_{co})^{1.026\alpha} & \text{for } |\dot{\varepsilon}_c| \leq 30 \text{ s}^{-1} \\
\gamma_\delta (\dot{\varepsilon}_c/\dot{\varepsilon}_{co})^{1/3} & \text{for } |\dot{\varepsilon}_c| > 30 \text{ s}^{-1}
\end{cases}
\]
(4.1)
where
\(f'_c\) = quasi-static unconfined compressive strength at \( \dot{\varepsilon}_{co} \)
$f'_{cd} = \text{dynamic unconfined compressive strength at } \dot{\varepsilon}_c$
$\dot{\varepsilon}_c = \text{dynamic strain rate in the range of } 30 \times 10^{-6} \text{ to } 300 \text{ s}^{-1}$
$\dot{\varepsilon}_{co} = 30 \times 10^{-6} \text{ (static strain rate)}$
$\alpha = \frac{1}{5 + 9f'_c/f'_o}$
$\log \gamma_s = 6.156\alpha - 2$
$f'_o = 10 \text{ MPa}$
A significant amount of work was done by Bischoff & Perry (1991) collecting large amounts of data and comparing it with CEB formulations. Although, there was a large scatter in reported strength increases, Bischoff & Perry (1991) showed that the CEB model appears to fit reasonably well the available data as shown in Figure 4.7. The CEB equations represent bi-linear curves. The discontinuity in the slope of their model occurs at a strain rate of $30 \text{ s}^{-1}$.
The dynamic increase factor (DIF) for tensile strength is given by
$$\frac{f_t}{f_{ts}} = \begin{cases}
(\dot{\varepsilon}_{ct}/\dot{\varepsilon}_{cto})^{1.016\delta} & \text{for } \dot{\varepsilon}_{ct} \leq 30 \text{ s}^{-1} \\
\beta_s (\dot{\varepsilon}_{ct}/\dot{\varepsilon}_{cto})^{1/3} & \text{for } \dot{\varepsilon}_{ct} > 30 \text{ s}^{-1}
\end{cases} \quad (4.2)$$
where
$f_{ts} = \text{quasi-static tensile strength at } \dot{\varepsilon}_{cto}$
$f_t = \text{dynamic tensile strength at } \dot{\varepsilon}_{ct}$
$\dot{\varepsilon}_{ct} = \text{dynamic strain rate in the range of } 3 \times 10^{-6} \text{ to } 300 \text{ s}^{-1}$
$\dot{\varepsilon}_{cto} = 3 \times 10^{-6} \text{ s}^{-1} \text{ (static strain rate)}$
$\delta = \frac{1}{10 + 6f'_c/f'_o}$
$\log \beta_s = 7.112\delta - 2.33$
Figure 4.7: Log-log scale of strength increase versus strain rate showing that CEB equation fits for available data, reported in Bischoff & Perry (1991).
When plotted on a log-log scale, the curves obtained are bilinear. As with the compression case, the discontinuity in their slope occurs at a strain rate of 30 s\(^{-1}\). Malvar & Ross (1998) conducted a review of strain rate effects for concrete in tension and proposed an alternate formulation based on experimental data. They suggested that the change in slope of the bilinear curves occurs close to 1 s\(^{-1}\) instead of at 30 s\(^{-1}\) as given by CEB.
Recent studies on the strain-rate effects in concrete have led to a considerable discussion among researchers about including these effects in the finite element modelling. Li & Meng (2003) have attributed the increase in the strength observed in the tests under high strain-rates to the confining stresses due to the lateral inertia of the test specimens. They carried out the numerical simulation of Split Hopkinson pressure bar (SHPB) test on concrete specimen using Drucker Prager model that did not explicitly include strain-rate effects and compared the results with SHPB tests. Based on their numerical investigation, they concluded that the apparent increase in the strength of concrete-like materials in a SHPB test was caused by the lateral confinement. This effect was more significant when the nominal strain-rate was around \(10^2\) s\(^{-1}\) and was termed as a pseudo-strain-rate effect. They concluded that using both strain-rate dependent dynamic increase factors (DIF) and lateral confinement due to inertia may result in overestimating the dynamic compressive strength of concrete. Further experimental and numerical studies (Li et al., 2009; Zhang et al., 2009) have been conducted to support their arguments. The study of strain-rate effects on the ultimate uniaxial compressive strength of concrete is still an area of active research and more studies may be required to fully understand the phenomenon and separate pseudo-strain-rate effects from the genuine effects.
4.4 Concrete models in LS-DYNA
LS-DYNA offers a number of material models that can be used for concrete. These include material models 5, 14, 16, 25, 72, 78, 84, 96, 145 and 159. Most of these models rely on multiple and complex parameters, which can only be determined after extensive material characterization tests. The description of these models is not documented in detail in either the theoretical or keyword manuals of LS-DYNA. Some of these models can generate default parameters for a range of concrete based on the basic parameters, for example unconfined compressive strength ($f'_c$), Poisson’s ratio ($\nu$) and aggregate size. An evaluation of the more commonly used concrete models in LS-DYNA is given by Yonten et al. (2002).
In the following sections, a brief description of some of these models is given. The models used in the finite element analyses described in Chapter 5, 6 and 7 are Winfrith concrete (model 84) and Concrete damage rel.3 (model 72). These are discussed in greater detail.
4.4.1 Pseudo Tensor Geological Model
Material Model 16 (MAT_PSEUDO_TENSOR) is a pseudo tensor concrete/geological model. It has two major modes - a simple pressure-dependent Mohr-Coulomb yield surface with Tresca limit, and two yield versus pressure curves with the capability to migrate from one curve to another. The first mode is more suitable for standard geologic models (Hallquist, 2007).
Mode two of the model is employed to model the concrete. In this model, the volumetric and deviatoric responses are decoupled. Volumetric changes are given by the hydrostatic tensor and shape deformations are covered by the deviatoric stress tensor. Strain rate effects are incorporated by multiplying the yield strength of concrete by a strain rate multiplier.
In hydrostatic condition, the model is a multilinear approximation in internal energy. Pressure is given by
\[ p = C(\epsilon_v) + \gamma T(\epsilon_v)E \]
(4.3)
where \( E \) is the internal energy per initial volume, \( \gamma \) is ratio of specific heats. The volumetric strain, \( \epsilon_v \), is given by the natural logarithm of the relative volume. Figure 4.8 shows the pressure versus volumetric strain curve.
The deviatoric stress tensor is modelled by a two-curve model, as shown in Figure 4.9(a). The upper and lower curves represent the maximum yield strength curve and failed material residual strength curve, respectively. The function \( \Delta\sigma \) that limits the deviatoric stresses is defined as a linear combination of two fixed strength curves which are function of pressure:
\[ \Delta\sigma = \eta \Delta\sigma_m + (1 - \eta) \Delta\sigma_r \]
(4.4)
where
\[ \Delta\sigma = \sqrt{3J_2}, \]
\[ \Delta\sigma_m = a_0 + \frac{p}{a_1 + a_2 p} \quad \text{(maximum strength curve)}, \]
\[ \Delta \sigma_r = a_{0f} + \frac{p}{a_{1f} + a_{2p}} \] (residual strength curve),
and \( p = -\left( \sigma_{xx} + \sigma_{yy} + \sigma_{zz} \right)/3 \) is the pressure (stresses are positive in tension, pressure is positive in compression). \( J_2 \) is the second invariant of the deviatoric stress tensor. The parameter \( \eta \) is a user-defined function of a modified effective plastic strain measure \( \lambda \). The function \( \eta(\lambda) \) is intended to first increase from some initial value up to unity, then decrease to zero representing softening. The yield surface migrates between \( \Delta \sigma_r \) (residual strength) and \( \Delta \sigma_m \) (maximum strength). The initial yield surface is given by
\[
\Delta \sigma_y = \eta_y \Delta \sigma_m + (1 - \eta_y) \Delta \sigma_r
\] (4.5)
where \( \eta_y = \eta(0) \) is the initial value of \( \eta \) before any plasticity has occurred. If \( \Delta \sigma \) is greater than \( \Delta \sigma_y \), plastic flow occurs. Figure 4.9(b) shows a typical stress-strain diagram.
### 4.4.2 Soil Concrete Model
Material Model 78 (MAT_SOIL_CONCRETE) is defined by a deviatoric perfectly plastic, pressure dependent yield function. Cracking can be invoked by setting the residual strength factor, B between 0 and 1. It requires four load curves: pressure-volumetric strain, pressure-yield stress, and pressure-plastic strain at which fracture begins, and lastly, pressure-plastic strain at which residual strength is reached. These sets of required data are not readily accessible in the literature.
### 4.4.3 Concrete Damage Model
Material Model 72 (MAT_CONCRETE_DAMAGE) is an extension of Material Model 16 implemented by Malvar et al. (1997) with additional parameters to include damage features. Version 971 of LS-DYNA also offers Release III of the model (MAT_CONCRETE_DAMAGE_REL3), which is a three-invariant model
Figure 4.9: Strength model for concrete: (a) two-curves strength model and the yield surface; (b) typical stress-strain curve
using three shear failure surfaces and including damage and strain-rate effects, Figure 4.10. The significant change from the original model to Release III is the model parameter generation capability, based solely on the unconfined compression strength of concrete (Schwer & Malvar, 2005).
### 4.4.4 Winfrith Concrete Model
Material Model 84 (MAT_WINFRITH_CONCRETE) was developed over many years in response to the requirements of the UK nuclear industry, and has been validated against experiments (Broadhouse, 1995; Broadhouse & Attwood, 1993; Broadhouse & Neilson, 1987). The concrete is modelled using the smeared fixed crack approach, where as the reinforcement can be modelled explicitly or smeared into the concrete. The model is implemented in the hexahedron element with a single integration point. The stress state is separated into the hydrostatic and deviatoric states. The hydrostatic stress state in the concrete is defined by a non-dimensionalised volume compaction curve, Figure 4.11; or a user defined pressure-volumetric strain curve may be input. The deviatoric stresses are updated by
elastic increments, using a locally rate dependent modulus. The model uses Ottosen yield surface shown in Figure 4.12 to limit the deviatoric stresses (Ottosen, 1977).
Figure 4.11: Volume compaction curve for concrete
 
Figure 4.12: The Ottosen failure model implemented in Winfrith concrete model (Ottosen, 1977)
The failure surface is defined analytically as given below.
\[
A \frac{J_2}{\sigma_c^2} + \lambda \sqrt{\frac{J_2}{\sigma_c}} + B \frac{I_1}{\sigma_c} - 1 = 0 \tag{4.6}
\]
where $\lambda$ is a function of $\cos 3\theta$:
$$
\lambda = \begin{cases}
K_1 \cos \left[ \frac{1}{3} \cos^{-1} (K_2 \cos 3\theta) \right] & \text{for } \cos 3\theta \geq 0 \\
K_1 \cos \left[ \frac{\pi}{3} - \frac{1}{3} \cos^{-1} (-K_2 \cos 3\theta) \right] & \text{for } \cos 3\theta \leq 0
\end{cases}
$$
(4.7)
and $A$, $B$, $K_1$ and $K_2$ are functions of $(\sigma_t / \sigma_c)$.
The yield surface expands as the hydrostatic stresses increase. The radii at the compressive and tensile meridia are determined by the locally rate sensitive compressive and tensile strengths, which are based on CEB (1988) formulations described in Section 4.3. The model also has the option to exclude the rate-effects.
A concrete element is treated as failed if the maximum principal stress at yield is greater than half the value of the current tensile strength. When tensile failure is indicated, a crack is flagged in a plane normal to the maximum tensile principal stress. A post-failure treatment initiates a decay of the crack-normal stress as the crack continues to open. The current crack-normal stress is determined using a non-dimensionalised crack stress decay function given in Wittmann et al. (1988). The rate of stress decay is dependent on the local fracture energy. The model allows up to three orthogonal cracks in any element. A tri-axial compression failure treats the concrete to be crushed by generating three instantaneous (closed) cracks such that the material has no tensile capacity on unloading (Broadhouse, 1995).
### 4.4.5 Johnson Holmquist Concrete Model
This is Material Type 111. This model can be used for concrete subjected to large strains, high strain rates and high pressures. The equivalent strength is expressed as a function of the pressure, strain rate, and damage. The pressure is expressed as a function of the volumetric strain and includes the effect of permanent crushing. The damage is accumulated as a function of the plastic volumetric strain, equivalent plastic strain and pressure. A more detailed description of this model can be found in the paper by Holmquist & Johnson (1993).
4.4.6 Continuous Surface Cap Model (CSCM)
Material Model 159 (MAT_CSCM_CONCRETE) has been implemented in Version 971 of LS-DYNA. The model was mainly developed to evaluate concrete road side safety applications, but it can also be applied to many dynamic problems (Murray, 2004, 2007). The concrete model is commonly referred to as a smooth or continuous surface cap model, Figure 4.13. The model comprises of isotropic constitutive equations, three invariant yield surface with translation for pre-peak hardening, a hardening cap that expands and contracts, damage-based softening with erosion and modulus reduction, and strain rate effects. The model input can be set up in two ways: (MAT_CSCM) requiring all the material parameters, such as moduli, strengths, hardening, softening, and rate effects parameters; and (MAT_CSCM_CONCRETE) with default parameters which are a fit to data for unconfined compression strengths between about 20 and 58 MPa, and aggregate sizes between 8 mm and 32 mm (Murray et al., 2007).
Figure 4.13: General shape of the concrete model yield surface in two dimensions
4.5 Concrete models adopted
The majority of concrete models in LS-DYNA require complex parameters, which are not readily available without performing multiple material tests. However,
recent versions of a few models have incorporated a capability to generate default parameters requiring only basic material properties like unconfined compression strength, Poisson’s ratio and aggregate size. Material Model 16, Material Model 72 (Rel. 3) and Material Model 159, described above, possess such a capability. The input parameters for Material Model 84 can also be easily established. In the present study, concrete damage model (72 Rel. 3) and the Winfrith model (84) have been employed.
### 4.6 Reinforcement material model
Material Model 3 (MAT\_PLASTIC\_KINEMATIC) has been used for modelling of steel reinforcement. The model is capable of isotropic and kinematic hardening plasticity with the option to include strain-rate effects, Figure 4.14. It is a relatively simple bi-linear model as compared to other models for steel, but is very cost effective in computational resources.
The effect of strain rate on yield strength is modelled using the Cowper and Symonds (Jones, 1989) strain rate model, which scales the yield strength by a strain rate dependant factor, Equation 4.8.
\[
\sigma_D = \sigma_Y \left[ 1 + \left( \frac{\epsilon_r}{C} \right)^{1/P} \right]
\]
where:
- \( \sigma_D \) = Dynamic yield stress
- \( \sigma_Y \) = Material static yield stress
- \( \epsilon_r \) = strain rate
- \( C, P \) = Strain rate parameters (material constants)
Figure 4.14: Elastic-plastic behaviour with kinematic and isotropic hardening where $l_o$ and $l$ are undeformed and deformed lengths of uniaxial tension specimen. $E_t$ is the slope of the bilinear stress strain curve.
4.7 Material model for impactor
A rigid material model in LS-DYNA (MAT_RIGID) was used to model the falling weight (impactor) in the finite element model. In LS-DYNA, elements that are rigid are bypassed in the element processing and no storage is allocated for storing history variables. Consequently, the rigid material type is very cost efficient. However, properties such as elastic modulus $E$ and Poisson’s ratio $\nu$ are used for determining sliding interface parameters if the rigid body interacts in a contact definition. Given that this is the case in present study, realistic values for these constants should be defined since unrealistic values may contribute to numerical problems in contact. The elastic modulus for steel, the impactor material, is 200 GPa and Poisson’s ratio of 0.3 for all simulations.
Chapter 5
Finite Element Analysis of RC Beams under Impact
5.1 Introduction
This Chapter describes the high-mass, low-velocity impact analysis of reinforced concrete beams. Finite element analysis has been performed on reinforced concrete beams tested by Chen & May (2009) at Heriot-Watt University, which were part of a series of studies to provide high-quality input data to validate numerical modelling. A detailed description of the finite element models is provided. The FE models are created using Hypermesh (2008), a general purpose pre-processing software widely used in the industry. The input files are then analysed in LSDYNA (Hallquist, 2006, 2007). The processing of the results is performed in LS-PREPOST, a post-processor bundled with LS-DYNA. The results of the analysis are compared with the tests to demonstrate the validation and suitability of the analysis procedure employed. In the following sections, a brief description of the impact tests is presented, followed by details of finite element modelling.
5.2 Description of tests
A series of well monitored drop-weight tests was performed at Heriot Watt University by Chen & May (2009) to study the high-mass, low-velocity impact behaviour of reinforced concrete members. The tests were carried out on fourteen 2.7 m and four 1.5 m span beams, four 0.76 m square 76 mm thick and two 2.3 m square 150 mm thick slabs under falling-weight impact loads. The results of the slab tests have also been used to validate finite element analysis, which will be described in Chapter 7.
5.2.1 Details of beams
In the beam tests, reinforced concrete beams were subjected to impact loading using a drop-weight system, Figure 2.8. The beams were 3 m or 1.8 m long with a $100 \times 200$ mm cross-section and pin-ended or simply supported 150 mm from the ends. The reinforcement consisted of two 12 mm diameter high yield steel bars at the bottom and two 6 mm diameter mild bars at the top, 6 mm diameter mild steel shear links at 200 mm centres with 25 mm concrete cover to the main steel, Figure 5.1. There were two series of tests, classified as type A and type B. Series A beams had a 12 mm thick plywood pad placed between the beam and the striker, similar to the beams of Hughes & Speirs (1982). A number of tests in series A were repeated to check the instrumentation and test procedure. Series B beams had a direct contact of impactor striking the beam. The drop-weight had a mass of 98 kg and the striker impacted the beams at a velocity of 7.3 m/s. There were two types of impactors used in the tests, a 100 mm diameter with a flat contact face and a 90 mm diameter with a spherical nose of 125 mm radius referred to as ‘hemispherical’ from here onwards.
The beams were either pin-ended or simply supported at the two ends. Figure 5.2 shows the ‘pin-ended’ supports, which allowed rotation of the beam and
provided axial and vertical restraints by means of plates that clamped the beam. Simply supported condition was obtained by releasing the clamps to allow horizontal movement and up to 50 mm vertical movement at top of the beam. The details of the beam tests and material properties are given in Table 5.1.
Table 5.1: Details of beam tests by Chen & May (2009)
| Group no | Test no | $L_{sp}$ (m) | $f'_c$ (N/mm²) | $f'_t$ (N/mm²) | Support | Impactor | Impact interface |
|----------|---------|--------------|----------------|----------------|---------|----------|------------------|
| A1 | 1 | 2.7 | 49.2 | 1.71 | pin-ended | hemispherical | 12mm plywood |
| | 2 | 2.7 | 49.2 | 1.71 | pin-ended | hemispherical | 12mm plywood |
| | 3 | 2.7 | 49.2 | 1.71 | pin-ended | hemispherical | 12mm plywood |
| | 4 | 2.7 | 49.2 | 1.71 | pin-ended | hemispherical | 12mm plywood |
| | 5 | 2.7 | 45.8 | 1.86 | pin-ended | hemispherical | 12mm plywood |
| | 6 | 2.7 | 45.8 | 1.86 | pin-ended | hemispherical | 12mm plywood |
| A1* | 7 | 2.7 | 33.6 | 1.53 | pin-ended | hemispherical | 12mm plywood |
| | 8 | 2.7 | 45.8 | 1.86 | simply supported | hemispherical | 12mm plywood |
| | 9 | 2.7 | 42.8 | 1.64 | simply supported | hemispherical | 12mm plywood |
| A2 | 10 | 1.5 | 35.6 | 1.73 | pin-ended | hemispherical | 12mm plywood |
| | 11 | 2.7 | 33.6 | 1.53 | pin-ended | hemispherical | direct contact |
| | 12 | 2.7 | 33.6 | 1.53 | pin-ended | hemispherical | direct contact |
| B1 | 13 | 2.7 | 45.8 | 1.86 | pin-ended | hemispherical | direct contact |
| B1* | 14 | 1.5 | 35.6 | 1.73 | pin-ended | hemispherical | direct contact |
| | 15 | 1.5 | 35.6 | 1.73 | pin-ended | hemispherical | direct contact |
| B2 | 16 | 2.7 | 42.8 | 1.64 | pin-ended | flat | direct contact |
| B3 | 17 | 2.7 | 33.6 | 1.53 | pin-ended | flat | direct contact |
| B3* | 18 | 1.5 | 35.6 | 1.73 | pin-ended | flat | direct contact |
Notes:
1 All tests were conducted under a drop-weight of 98 kg with an impact velocity of 7.3 m/s.
2 $L_{sp}$ is the distance (span) between the centres of 200 mm long supports at two ends.
3 $f'_c$ the tensile strength of concrete, is based on the results of the splitting test of cylinders.
4 * Denotes the repeated test in which all conditions were the same as the test without a *, except concrete with lower strengths.
5 Properties of the mild steel: Young’s modulus – 190 kN/mm², 0.2% proof tensile strength – 510 N/mm², Ultimate tensile strength – 600 N/mm².
6 Tension steel percentage, 0.85%
5.2.2 Instrumentation and data acquisition
The tests were monitored to acquire high-quality reliable data. The following instruments and sensors were used.
- A high speed camera operating at a rate of 4500 frames per second was used to obtain a transient record of the details of local failure, such as crack propagation, spalling or scabbing of concrete, etc.
- The transient impact forces were measured using a load cell placed between the mass and the impactor. Two load cells of 40 and 200 tonne capacity were used. For tests where the impact load was expected to exceed the load capacity of the load cell, the transient force was derived from accelerometers fitted to the striker. However, in all beam tests a load cell was used.
- Accelerometers were also attached at various positions on the beams to record acceleration history. To reduce noise, the results from the accelerometers were filtered with a Butterworth filter (Butterworth, 1930) with a cut-off frequency of 2000 Hz.
- Strain gauges were fitted to steel reinforcement for some tests to record the strains without affecting the bond between the reinforcement and surrounding concrete.
- Electronic triggers spaced at 40 mm were used to measure velocity of the striker. An additional trigger was used to ensure simultaneous recording of the transducers and the high-speed camera.
- An automated data acquisition system was used to record the signals from load cell, accelerometers and strain gauges.
The impactor was unrestrained vertically, which resulted in rebound and subsequent impacts after the first strike. The response of the structure under these
Table 5.2: System specifications
| Computer | Dell Precision 690 |
|-------------------|--------------------|
| Processor | 2.33 GHz Dual Intel Xeon |
| Main memory | 4 GB |
| Operating system | Windows XP Pro-SP2 |
| Pre-processor software | HyperMesh version 8.0 |
| Finite element analysis software | LS-DYNA version 971.7600 |
| Post-processor software | LS-PREPOST version 2.1 |
subsequent impacts was not as significant as under the first impact. Therefore, the response of the beams was investigated under the first impact only, which lasted for about 50 ms.
5.3 Experimental results
The experimental programme provided a large amount of data, which included transient impact forces, accelerations, reinforcement strains and high-speed camera recordings for the local response of the beams in the impact zone throughout the loading. The test results have been used to validate the finite element analysis of the beams under impact loading, which will be described in the following sections. Further details of the tests and results are thoroughly presented in Chen & May (2009).
5.4 Finite element modelling
Three-dimensional finite element models have been created using the Lagrangian formulation in LS-DYNA. A high-powered windows based computer system was used for the simulations, which helped to reduce the solution times to a manageable level. The details of the computer system are given in Table 5.2.
The concrete beam, plywood interface and the drop-weight were discretized in space with eight-node hexahedron elements. The elements for the concrete beam have an aspect ratio of 1. The solid elements employ one-point Gauss integration. To avoid spurious modes in the analysis because of single integration point elements, hourglass control using the viscous hourglass formulation was employed. The reinforcement bars were spatially discretized with beam elements and stirrups with truss elements. The interaction between the concrete solid elements and reinforcement beam elements has been modelled by merging their common nodes. In some of the models, the bond between concrete solid elements and reinforcement beam elements has been achieved by coupling the beam elements with solid elements using the LS-DYNA keyword *CONSTRAINED_LAGRANGE_IN_SOLID. This technique has been successfully used in modelling reinforced concrete safety barriers by Abu-Odeh (2006, 2008).
The supports have been modelled by providing 20 mm thick support plates at the two ends of the beam. Vertical restraints have also been applied to the nodes at the top of the beam at the support position to stop the beam from bouncing off, this was also the case in the tests. The model has been created for the entire beam so that effects of non-symmetry because of support conditions in some cases could be investigated. The falling-weight was modelled as a rigid body and assigned an initial velocity of 7.3 m/s.
For the numerical simulations involving contact, the selection of appropriate contact algorithm is vital to the stability of the solution. LS-DYNA offers various contact interface algorithms, as discussed in Chapter 3, which have been investigated for use in the simulations. For the analysis presented here, an automatic surface to surface contact algorithm based on a penalty formulation is used for the interface between the concrete beam and the impactor. Friction between the two surfaces is neglected. Acceleration due to gravity was included in the analysis for initial runs, however, it did not affect the results, therefore, it was not
included in the subsequent analyses. Figure 5.3 shows the schematic diagram of a Series A beam.
**Figure 5.3:** Schematic diagram of Series A beam model
As discussed in Chapter 4, LS-DYNA offers a number of material models, which can be used to model reinforced concrete. The majority of these models require a large number of complex parameters, which can only be determined by specific material characterization tests (Schwer, 2001). However, some of these models have the option to generate the required parameters based on minimal input, for example unconfined compression test, concrete aggregate size and fracture energy (Abu-Odeh, 2008; Schwer, 2008). For the present study, the Winfrith concrete model (Broadhouse, 1995) has been used, which has been discussed in detail in Chapter 4. Table 5.3 shows the material properties and Ottosen (1977) constants used in the Winfrith model. Steel reinforcement was modelled using material model no. 3 in LS-DYNA, an elastic-plastic model with hardening effects, which has been described in detail in Chapter 4. For plywood, an elastic material model was used.
As discussed in Section 4.3, there has been a considerable debate among the researchers about the inclusion of strain-rate effects in concrete. The increase in compressive strength of concrete is attributed to the lateral confinement due to inertia of the test specimen rather than the higher rate of loading or strain rate. The inclusion of strength increase factors due to high rate of loading and lateral confinement in the model may result in overestimating the dynamic compressive strength of concrete (Li & Meng, 2003). The Winfrith model has the option to include these effects based on CEB (1988) formulation. The preliminary analysis carried out with and without the strength enhancements due to strain-rate did not produce any significant difference in results for the beam tests. This is most likely attributed to the fact that the strain-rates for the range of test in this investigation are low. The maximum strain rate recorded in the tests was 32.5 $s^{-1}$ (Chen & May, 2009). Therefore, based on the findings of the recent research, the subsequent models did not explicitly include the strain-rate effects.
### 5.5 Mesh sensitivity
As discussed in Section 5.2, there were two series of beams, Series A with plywood interface and Series B having direct contact between the impactor and the beam. There were a number of models created for each series using different mesh sizes to investigate the sensitivity of mesh discretization. An initial mesh size of 20 mm for the concrete solid and reinforcement beam elements was refined to two finer mesh sizes of 15 mm and 10 mm. In a typical model of 2.7 m span beam, there are 7800 solid elements and 900 beam elements for a mesh size of 20 mm. For 10 mm mesh, the number of solid elements and beam elements was 61,500 and 1800, respectively. For 1.5 m span beams, a mesh size of 20 mm resulted in 5000 solid and 530 beam elements, whereas the number of solid and beam elements for 10 mm mesh was 38,000 and 1050, respectively. Figure 5.4 shows the finite element model of a series A beam showing meshing for reinforced concrete beam
Table 5.3: Material properties of beams using Winfrith model
| Group no | Test no | $L_0$ (m) | $f_{cv}$ ($N/mm^2$) | $f_t$ ($N/mm^2$) | Ottosen (1977) constants |
|----------|---------|-----------|---------------------|-----------------|------------------------|
| | | | | | $A$ | $B$ | $K_1$ | $K_2$ |
| A1 | 1 | 2.7 | 49.2 | 1.71 | 47.7857 | 91.2568 | 29.9059 | 0.9998 |
| | 2 | 2.7 | 49.2 | 1.71 | 47.7857 | 91.2568 | 29.9059 | 0.9998 |
| | 3 | 2.7 | 49.2 | 1.71 | 47.7857 | 91.2568 | 29.9059 | 0.9998 |
| | 4 | 2.7 | 49.2 | 1.71 | 47.7857 | 91.2568 | 29.9059 | 0.9998 |
| | 5 | 2.7 | 45.8 | 1.86 | 43.8913 | 84.6643 | 27.8825 | 1.0000 |
| | 6 | 2.7 | 45.8 | 1.86 | 43.8913 | 84.6643 | 27.8825 | 1.0000 |
| A1* | 7 | 2.7 | 33.6 | 1.53 | 38.2185 | 75.0616 | 24.9359 | 1.0000 |
| | 8 | 2.7 | 45.8 | 1.86 | 43.8913 | 84.6643 | 27.8825 | 1.0000 |
| A2 | 9 | 2.7 | 42.8 | 1.64 | 45.9921 | 88.2206 | 28.9740 | 0.9999 |
| A3 | 10 | 1.5 | 35.6 | 1.73 | 25.9176 | 54.2389 | 18.5509 | 0.9978 |
| | 11 | 2.7 | 33.6 | 1.53 | 38.2185 | 75.0616 | 24.9359 | 1.0000 |
| B1 | 12 | 2.7 | 33.6 | 1.53 | 38.2185 | 75.0616 | 24.9359 | 1.0000 |
| B1* | 13 | 2.7 | 45.8 | 1.86 | 43.8913 | 84.6643 | 27.8825 | 1.0000 |
| | 14 | 1.5 | 35.6 | 1.73 | 25.9176 | 54.2389 | 18.5509 | 0.9978 |
| B2 | 15 | 1.5 | 35.6 | 1.73 | 25.9176 | 54.2389 | 18.5509 | 0.9978 |
| B3 | 16 | 2.7 | 42.8 | 1.64 | 45.9921 | 88.2206 | 28.9740 | 0.9999 |
| B3* | 17 | 2.7 | 33.6 | 1.53 | 38.2185 | 75.0616 | 24.9359 | 1.0000 |
| B4 | 18 | 1.5 | 35.6 | 1.73 | 25.9176 | 54.2389 | 18.5509 | 0.9978 |
and reinforcement arrangement. The detail of the mesh sizes for 2.7 m span beam A1-2 is given in Tables 5.4.
Figure 5.4: Finite element model of Series A beam (a) Concrete beam mesh (b) Reinforcement mesh
Figure 5.5 shows the transient displacement histories obtained at the centre of the beam A1-2 from the analyses using three different mesh sizes. The model appears to be moderately sensitive to mesh size, as shown by the difference in the peak displacement and periods. The coarse mesh of 20 mm predicted a peak displacement of 33 mm at 13.2 ms, against 38.6 mm at 18.7 ms for 15 mm mesh. The refined mesh of 10 mm resulted in peak displacement of 43.3 mm at 18.5 ms. The results seem to be converging as the mesh is refined as indicated by two finer mesh sizes of 15 mm and 10 mm. The variations in the peak displacements and
Table 5.4: Mesh data for beam A1-2
| Beam A1-2 | Mesh 1 | Mesh 2 | Mesh 3 |
|-----------|--------------|--------------|--------------|
| Nodes | 10406 | 21660 | 71490 |
| Solid elements | 7810 | 16263 | 61577 |
| Beam elements | 900 | 1188 | 1800 |
| Element size | (5 x 10 x 150) | (7 x 13 x 200) | (10 x 20 x 300) |
| | 20 mm for solids | 15 mm for solids | 10 mm for solids |
| | 20 mm for beam | 15 mm for beam | 10 mm for beam |
time periods are smaller between 10 mm mesh and 15 mm mesh as compared to those with 20 mm mesh. Based on the findings of this investigation, a mesh size of 10 mm was considered to be relatively insensitive to mesh discretization.
Figure 5.5: Mesh sensitivity: influence of mesh discretization on transient displacement histories, beam A1-2 (Winfrith model)
5.6 Influence of time step
As described in Chapter 3, LS-DYNA automatically computes time steps based on the critical time step, which is defined as the time taken for an elastic wave
to traverse the smallest element. The critical time step is factored by 0.9, which is a default value. In order to investigate the effect of time step on the analysis, this default value was changed to 0.45 and 0.225. This resulted in even smaller time steps than the default values based on a factor of 0.9. Beam A1-2 with a mesh size of 10 mm (10 x 20 x 300) was used. Figure 5.6 shows the comparison of transient impact force histories for beam A1-2 using the three factors of 0.9, 0.45 and 0.225. There appears to be no significant difference in the peak forces, peak times, as well as overall shape of the three curves. The only influence of lower time-step scale factors was on the computational time, which increased substantially with each reduction in the factor. Based on this investigation, it implies that the default factor of 0.9 on the critical time step would be adequate provided a reasonably finer mesh is used.
Figure 5.6: Influence of time-step scale factor on transient impact force histories, beam A1-2 (Winfirth model)
5.7 Stability of the solution
As mentioned in Chapter 3, LS-DYNA employs explicit time integration for dynamic analysis. In explicit schemes, the stability of the solution is ensured by using a time step that is lower than a critical value based on wave traversal in the smallest element. Another useful check on the stability of the solution is the energy ratio, which has been discussed in detail in Chapter 3. Further to the energy balance, the energy introduced by hourglassing should be minimal, typically not more than $2 - 3\%$ of total energy. Figure 5.7 presents the energy-time histories for beam A1-2. As seen in the Figure, the total energy during the simulation remains constant at 2.6 kJ indicating the conservation of energy in the system. The hourglass energy had a maximum value of 80 J, which was about 3% of the total energy ensuring the stability of the solution.
5.8 Comparison of finite element results and tests
Following the mesh sensitivity studies, the beams were modelled using a mesh size of 10 mm. All the beams were subjected to a single impact of a 98 kg mass
impacting at the centre of the beams with an initial velocity of 7.3 m/s. As discussed in Section 5.2.2, various instruments were used for data acquisition. The analysis was performed for 50 ms and the results were post-processed to compare with the tests. A typical finite element analysis for 2.7 m beam with a mesh size of 10 mm ran for about 90 minutes on the high-powered machine detailed in Table 5.2. Earlier analyses, which were carried out using a standard Pentium 4 machine, took considerably longer times typically in excess of 15 hours for the same problem.
In the following sections, a detailed comparison of the finite element results with the tests is presented for each beam. As typical of any finite element analysis, a large amount of output data were generated. For comparison with the tests, transient impact force histories, accelerations and crack patterns only are used. The transient reaction forces and displacement histories are also presented from the analysis, although those were not recorded in the tests.
### 5.8.1 Transient impact forces
The transient impact force histories in the tests were recorded using a load cell placed between the impactor mass and the impactor head. In the analysis, the impact force histories were obtained using the interface forces generated when the impactor contacted the beam. These forces are dependent on the type of contact and the stiffness of the interface. As described earlier in Section 5.4, a two-way, automatic surface to surface contact algorithm based on a penalty formulation was used. In this type of contact, master and slave surfaces are defined using surface segments. The impactor surface was treated as master surface and the plywood in case of A series beams was considered as slave surface. The results are presented for 2.7 m span beams with plywood interface (Series A) and with direct contact (Series B), followed by those of 1.5 m span beam.
2.7 m span beams
Figure 5.8 shows a comparison of transient impact force histories for beams A1(1-7) using the Winfrith concrete model. There were seven beams tested in this series. All the beams were pin-ended with plywood interface. Beams A1(1-4) had identical properties, therefore, the analysis was performed on one beam A1-2. The transient force history for this beam is given in Figure 5.8(a), which shows a peak impact force of 306 kN at 1.7 ms against 230 kN at 1.8 ms, both average values for the first six A1 beams (Chen & May, 2009) in the tests. Figure 5.8(b) shows the comparison of transient impact force history of beam A1-6 with the test, which is also in reasonable agreement for both the peak forces and the peak times. Beam A1*-7 had a lower strength compared to the first six beam in the A1 series. The impact force history in the test recorded a significantly lower value of peak impact force, 128 kN at 1.6 ms. The analysis, though picked up the reduction in the peak impact force, but the force was significantly larger than that in the test, Figure 5.8(c). The overall shapes of the impact force histories from the analysis are in reasonable agreement with the test. The analysis also seemed to pick up the short separation or loss of contact between the impactor and the beam, which is indicated by zero force at about 8 ms.
Figure 5.9 shows the transient impact force histories for the impact force and reaction force obtained from the analysis of beam A1-2. The reaction was determined on the left support and had a peak value of 70 kN at 7 ms. It can be seen in Figure 5.9 that the reaction force reached its peak value when the impact force had already passed its peak at 1.7 ms resulting in a time lag of 5.3 ms.
Beams A2-8 and A2-9 were similar to the beams in A1 series, except that they were simply-supported. The comparison of the impact force histories for both beams is presented in Figure 5.10. The analysis yielded a peak impact force of 295 kN at 1.1 ms compared to 230 kN at 1.6 ms for beam A2-9 as shown in Figure 5.10(b). The shapes of the impact force histories for the two beams are
Figure 5.8: Comparison of recorded and simulation impact force histories for beams A1(1-7) using Winfrith model
in good agreement with those in the test. The change of boundary conditions from pin-ended to simply supported did not appear to have much influence on the transient impact force histories for beams A2-8 and A2-9.
The beams in Series B had no plywood placed between the impactor and the beam as they were struck directly by the striker. Figure 5.11 shows the comparison of the transient impact force histories for beams B1-12 and B1*-13. Beam B1-12 had similar properties as beam A1*-7 and beam B1*-13 was identical to beams A1-5 and A1-6. The comparison of transient impact force histories for the two beams with the test indicate significant variation for peak forces and post-peak curves. The peak times, though, are in reasonable agreement. The maximum force determined from the analysis for beam B1-12 was 282 $kN$ at 1 $ms$ and that from the test was 161 $kN$ at 1.3 $ms$, Figure 5.11(a). In a similar beam A1*-7, but with plywood interface the analysis predicted a peak force of 222 $kN$ at 1 $ms$, which indicated that for a direct contact between the striker and the beam, the analysis resulted in a slightly higher peak force.
In Figure 5.11(b), the comparison of transient impact force histories for beam
Figure 5.10: Comparison of recorded and simulation impact force histories for beams A2(8-9) using Winfrith model
B1*-13 is shown. The analysis yields a peak force of 377 kN at 1 ms, which is significantly higher than test peak force of 183 kN at 1.4 ms. Beam B1*-13 was identical in material properties to beams A1-5, A1-6 and A2-8. However, when the test results for this beam were compared with those similar beams, they showed significantly lower value of peak force and different post peak curve. Despite the fact that other beams with similar properties had plywood interface, whereas beam B1*-13 was struck directly. The analysis impact force histories for beam B1*-13, when compared to similar A series beams for both tests and analyses, were remarkably similar and consistent except a slightly higher value of peak impact force. This was also consistent with a similar observation for beam B1-12.
The last two 2.7 m beams in this series, B3-16 and B3*-17 were impacted by a flat impactor directly striking the beams. The comparison of impact force histories for the two beams is shown in Figure 5.12. The analysis results for beam B3-16 predicted a peak force of 516 kN at 0.3 ms when compared to the test results of 654 kN at 0.3 ms, Figure 5.12(a). The two curves also have a similar post-peak shape. Figure 5.12(b) shows the comparison of transient impact force histories for beam B3*-17. A peak impact force of 281 kN at 1 ms was predicted by the analysis, whereas the test results showed a peak force of 215 kN at 1 ms. For the two beams struck by a flat impactor, the analysis results for transient impact forces are in good agreement. Beam B3*-17 also had identical properties to beam B1-12, which was impacted by a hemispherical impactor. The transient impact force histories for both the beams from analysis are almost identical indicating that the shape of the impactor did not appear to have significant effect on the impact forces.
Figure 5.11: Comparison of recorded and simulation impact force histories for beams B1(12-13) using Winfrith model
Figure 5.12: Comparison of recorded and simulation impact force histories for beams B3(16-17) using Winfrith model
1.5 m span beams
There were four 1.5 m span beams tested, only beam A3-10 had a plywood interface placed between the beam and the impactor. All the beams had identical material properties. Figure 5.13 presents the comparison of transient impact force histories for beam A3-10. The analysis predicted a peak impact force of 228 kN at 1.6 ms against 194 kN at 2 ms. The overall shapes of the post-peak curves are also very similar.
The comparison of impact force histories for beam B2-14, which was impacted by a hemispherical impactor directly falling at the centre, is shown in Figure 5.14 for the Winfrith concrete model. A peak force of 245 kN at 1.4 ms was predicted by the analysis. The tests showed a maximum impact force of 169 kN at 1.5 ms. There is some variation in the post-peak curves between the test and the analysis.
Figure 5.15 presents the transient impact force histories of beam B4-18 for the test and simulation. The beam was impacted by a flat impactor. The peak impact force predicted by the analysis was 285 kN at 0.85 ms and that recorded in the test was 241 kN at 1 ms. Good agreement was also found in the post-peak curves between the analysis and the test. The comparison of peak forces from analysis for beam B4-18 with a similar beam B2-14 impacted by a hemispherical impactor indicated that the shape of the impactor did not influence the transient impact force histories. This was in agreement with an earlier observation made in case of 2.7m Series B beams.
Influence of contact interface
The impact force histories obtained from the analyses of beams show some discrepancies for peak impact forces. When compared to the tests, the analyses generally yield higher peak forces. As discussed in Section 3.4.3, the contact forces between the impactor and a target depend upon geometric and material properties of the contact interface as well as the velocity of the impactor at the
Figure 5.13: Recorded and simulation impact force histories for beam A3-10 (Winfrith model)
Figure 5.14: Recorded and simulation impact force histories for beam B2-14 (Winfrith model)
time of contact. The stiffness of the contact interface is calculated using penalty formulation (Hallquist, 2007). In this investigation, segment based contact was defined between the impactor and the target beam, which were treated as slave and master, respectively. The impactor was assigned material properties of steel, which was the case in the tests. For numerical contact definitions in LS-DYNA, contact treatment is internally represented by linear springs between the slave nodes and the nearest master segments. The stiffness of these springs determines the force that will be applied to the slave nodes and the master nodes. The penalty method uses the size of the contact segment and its material properties to determine the contact stiffness and works effectively when the material parameters between the contacting surfaces are of the same order. For dissimilar contacting surfaces, the penalty stiffness of the contact surface can be scaled to reduce the numerical problems (Hallquist, 2007).
In order to investigate the influence of contact interface on the impact force, the default penalty scale factor of 1.0 for the contact interface was changed to 0.9. Figure 5.16 shows the comparison of transient impact force histories for
beam B4-18 using scale factors 1.0 and 0.9 with the test. The peak impact force recorded in the test was 241 $kN$ and that predicted by the analysis using default scale factor was 285 $kN$. When the scale factor was changed to 0.9, the peak force of 246 $kN$ was predicted by the analysis, which was very close to that in the test. The peak times and the post peak curves are also in reasonable agreement. The change of penalty scale factor affected the peak forces only.
### 5.8.2 Transient accelerations
Accelerometers were attached to some beams to record transient acceleration-time histories. Beam A2-9 had three accelerometers mounted to the top face at 300 mm, 600 mm and 900 mm off the centre of the beam. Figure 5.17 shows the comparison of transient acceleration histories off 300 mm and 900 mm centre for beam A2-9. The accelerations were obtained by differentiating the nodal velocities at 300 mm and 600 mm off the centre. The peak values of accelerations and peak times are remarkably in good agreement with the test.
5.8 Comparison of finite element results and tests
Figure 5.17: Comparison of transient acceleration-time histories for beam A2-9 (Winfrith model)
5.8.3 Transient reinforcement strains
In beam B3-16, the strains in the steel reinforcement were measured using strain gauges placed inside the reinforcing bars at different locations. Figure 5.18 shows the comparison of transient reinforcement strains at two locations. Strain gauge e5 was attached near the mid-span of the beam at 140 mm from the centre, whereas gauge e3 was mounted at 440 mm from the mid-span of the beam. The strain histories obtained from the analysis are in good agreement with the test. The yield strain of the steel reinforcement was of the order of 2500 με, which indicates that the reinforcement has yielded during the initial impact.
5.8.4 Crack patterns and damage
2.7 m span beams
The numerical simulations presented here have been performed employing the Winfrith model for concrete. It provides a useful assessment to the extent of cracking, crack sizes and their locations. The crack patterns and damage obtained from the experimental tests have been compared with the patterns from
Figure 5.18: Recorded and simulation transient reinforcement histories for beam B3-16 (Winfrith model)
the simulations. Figure 5.19 presents the crack patterns for the beams A1-2 and A1-6 from the tests and the analyses. As mentioned earlier, these beams were impacted by a hemispherical impactor with plywood placed between the beam and the striker. The crack patterns predicted by the analysis show the development of diagonal cracks, originating at the impact point and propagating downwards with an angle of approximately 45 degrees, forming a shear-plug. This was also observed in the tests. Vertical cracks starting from the top of a beam away from the impact zone were also formed in the analysis. A zone of crushing under the impactor was also picked up in the simulations, as was the case in the tests.
The comparison of final crack patterns for beams A2-8 and A2-9 is shown in Figure 5.20. These beams were simply supported, which seems to have a minor influence on crack patterns, specially the vertical cracks along the beam section away from the impact zone as shown in Figure 5.20(b) and (d). Shear cracks in the impact zone and crushing under the impactor were in reasonable agreement with the tests. The analysis also predicted horizontal cracks at the level of the bottom steel reinforcement below the impact zone, which were also found in the
Figure 5.19: Comparison of final crack patterns and damage on beams A1-2 and A1-6
test on beam A2-9 shown in Figure 5.20(c).
(a) Final crack pattern from test, beam A2-8
(b) Crack pattern from analysis, beam A2-8
(c) Final crack pattern from test, beam A2-9
(d) Crack pattern from analysis, beam A2-9
Figure 5.20: Comparison of final crack patterns and damage on beams A2(8-9)
Figure 5.21 and 5.22 show the comparison of crack patterns and damage from the test for Series B beams B1-12, B3-16 and B3*-17 with the analysis. These beams were impacted directly by the striker, therefore, showed more damage due to crushing of concrete at the point of impact in the tests. The analysis predicted overall crack patterns agreeing to the tests. The zone of crushing was similar to those in Series A beams. The loss of concrete cover due to scabbing of concrete at the bottom of the beams could be compared with the large number of horizontal cracks at the level of the bottom reinforcement in the analysis, Figure 5.22.
Figure 5.21: Comparison of final crack patterns and damage on beam B1-12
Figure 5.22: Comparison of final crack patterns and damage on beams B3-16 and B3*-17
1.5 m span beams
Figures 5.23, 5.24 and 5.25 show the crack patterns from the test and analysis for three 1.5m span beams A3-10, B2-14 and B4-18, respectively. The crack patterns obtained from analysis for beam A3-10 show diagonal cracks starting at the point of impact and propagating downwards forming a shear plug, similar to those observed in the test, Figure 5.23(a) and (b). Further to these cracks, more diagonal cracks developed propagating towards the supports, which are also picked up by the analysis. Vertical cracks away from the impact zone at the top of the beam are also shown. Compared to beam A3-10, the analysis predicted severe cracking in the impact zone along with diagonal cracks for beam B2-14. The beam was directly impacted by the hemispherical impactor in the test and had severe crushing at the point of impact and diagonal cracking, Figure 5.24. Beam B4-18, apart from diagonal cracking in the test, also showed a loss of concrete cover to the tension steel due to scabbing of the concrete. This could also be correlated to the presence of horizontal cracks at the level of tension steel in the analysis, Figure 5.25.
(a) Final crack pattern from test
(b) Crack pattern from analysis
Figure 5.23: Comparison of final crack patterns and damage on beam A3-10
Figure 5.24: Comparison of final crack patterns and damage on beam B2-14
Figure 5.25: Comparison of final crack patterns and damage on beam B4-18
5.8.5 Correlation between transient load and crack development
In Figure 5.26 the impact force time histories, together with a series of images at different time intervals are presented from the test and analysis on beam A1-2, to show the correlation between the impact force and crack development in the impact zone. Figure 5.26(a) shows initial diagonal shear cracks forming on beam A1-2 as the load reached its maximum at 2 ms. The analysis shown in Figure 5.26(b) almost exactly reproduces the initial diagonal cracks at the point of maximum impact load at 1.7 ms. Vertical flexural cracks start to develop at about 5 ms followed by a short period of separation between the impactor and the beam at about 10 ms as the beam deformed at a faster rate. The simulation also picks up this phenomenon, the separation indicated by zero impact load is predicted at about 8 ms. Both the beam and the striker continue to move downwards. Some spalling in concrete directly beneath the impactor is observed in the test at this point, which is also captured by the simulation. From 10 ms to 30 ms, the beam and the impactor regained the contact resulting in more cracking and spalling both in the test and simulation. At about 50 ms, the impactor started to move upwards and the beam and impactor separated again. The correlation between the impact load and crack development in the test and simulation are remarkably similar. Formation of initial diagonal shear cracks, followed by vertical flexural cracks and spalling are picked up accurately by the analysis. The short periods of separation followed by regaining of contact are also captured well. However, the analysis also predicts a number of horizontal cracks at the level of the bottom steel reinforcement below the impact zone. These cracks are not that apparent in beam A1-2, although they have been observed in a similar beam B3-16.
Figure 5.26: Correlation between impact load and crack propagation for beam A1-2 from test and analysis using Winfrith model
5.8.6 Displacement histories
The finite element analyses produced large volumes of output, which was processed. The transient records of displacement histories were obtained at the centre of the beams. In the tests, there was no instrumentation to record the displacements. However, the results of the analyses for the displacements are discussed in the following sections for 2.7 m and 1.5 m span beams.
2.7 m span beams
In Figure 5.27, the transient displacement histories are shown for two 2.7 m span beams A1-6 and A2-8. Both the beams had identical material properties. The beams were impacted by the hemispherical impactor with a plywood interface between the beam and the striker. The only difference was in the boundary conditions. Beam A1-6 was pin-ended, whereas beam A2-8 was simply-supported at the ends. The analysis yielded a maximum displacement of 43.2 mm at the centre of beam A1-6 at 18.4 ms. Beam A2-8 had a peak displacement of 61.4 mm at 25 ms. The two beams showed a significant difference in the peak displacements despite similar properties and test conditions, except boundary conditions. The apparent increase in the displacement in case of a simply-supported beam may be attributed to the lack of longitudinal restraint as compared to the pin-ended beam resulting in un-restricted wave-propagation in the longitudinal direction. In the case of a pin-ended beam, an opposing axial force might develop, which could have a pre-stress like effect in the beam.
Figure 5.28 shows the transient displacement histories at the centre of beams B1-12 and B3*-17. The beams shared same material properties and boundary conditions, having different impactor head only. Beam B1-12 was impacted by a 90 mm diameter hemispherical impactor, while a flat striker of 100 mm diameter was used on beam B3*-17. The peak displacements determined by the analysis were 57.3 mm at 23.5 ms and 56.4 mm at 22.6 ms for beams B1-12 and B3*-17,
respectively. It is obvious from Figure 5.28 that there is hardly any effect on the displacement histories owing to this slight variation in impactor head.
Figure 5.27: Transient displacement histories of $2.7 \ m$ span beams A1-6 and A2-8 (Winfrith model)
Figure 5.28: Transient displacement histories of $2.7 \ m$ span beams B1-12 and B3*-17 (Winfrith model)
1.5 m span beams
The transient displacement histories at the centre of the beams were determined from the analysis for the 1.5 m span beams. Figure 5.29 shows the displacement histories for the three beams A3-10, B2-14 and B4-18 using the Winfrith concrete model. As already described in Section 5.2, beam A3-10 had a plywood placed between the striker and the beam, whereas the other two beams were struck directly. Moreover, beam B4-18 was impacted by a flat impactor and the other two beams by hemispherical impactor. The maximum and the residual displacements for the three beams, perhaps not surprisingly, are almost identical. The beams were cast in the same batch, therefore, had identical material properties. There appears to be no apparent effect of plywood interface or impactor type on the transient displacement histories for the three beams.
5.9 Conclusions
Detailed finite element modelling of the reinforced concrete beams tested by Chen & May (2009) has been described. The results of the analyses were compared
with the tests and following conclusions can be drawn.
- The transient impact force histories obtained from the analyses were in reasonable agreement with the tests for peak impact forces, peak times and overall shape for Series A beams, which had a plywood interface between the impactor and the beam.
- The change of supports from pin-ended to simply supported, as in case of beams A2-8 and A2-9 did not appear to have significant effect on the transient impact force histories, when compared to similar pin-ended beams.
- There were some discrepancies, specially in the peak forces for some Series B beams, which were directly impacted by hemispherical impactor. Beam B3*-13, which had identical material properties to beams A1-5, A1-6 and A1-8, recorded much lower peak forces in the test.
- The Series B beams impacted by the flat impactor, B3-16 and B3*-17 were in good agreement for transient impact force histories with the tests.
- The 1.5m span beams A3-10, B2-14 and B4-18 showed reasonable agreement with the tests for transient impact force histories.
- The reaction forces obtained from the analysis showed a time-lag to the transient impact forces, which reached their peak value well before the reactions, Figure 5.9.
- The transient acceleration-time histories obtained by differentiating the nodal velocities, showed remarkably good agreement with the test for beam A2-9, Figure 5.17.
- The transient reinforcement strain histories obtained from the analysis showed very good agreement with the tests.
• The crack patterns obtained from the analysis using the Winfrith concrete model matched reasonably with the cracks and damage patterns in the tests. The analyses showed the development of diagonal cracks, originating at the impact point and propagating downwards with an angle of approximately 45 degrees, forming a shear-plug, which was also found in the tests. Vertical cracks away from the impact zone were also picked up in the analyses. A zone of crushing under the impactor head was also marked.
• The development of horizontal cracks at the level of tension steel reinforcement in the impact zone was predicted by the analyses. This could be correlated to the loss of concrete cover due to scabbing at the bottom in some beams.
• The correlation between the transient load and crack development, shown in Figure 5.26, matched exactly with the test. The development of main diagonal shear cracks and flexural cracks along with crushing of concrete was shown to have taken place after the impact load had passed its peak value.
• The transient displacement histories determined from the analysis at the centre of the beams showed significant difference in the peak displacements due to the change of supports from pin-ended to simply supported beams.
• The change in the striker head from hemispherical to flat did not have any effect on transient displacements histories, Figure 5.28.
• In the $1.5m$ span beams, the transient displacement histories were identical. These beams had same material properties, though slightly different test conditions. Despite this, there appeared to be no apparent effect of plywood interface or impactor type on the transient displacement histories for the three beams, Figure 5.29.
• There appears to be no significant influence of time step on the analyses for the mesh used. The only influence of the lower time-step scale factors was on the computational time, which increased substantially with each reduction in the factor. Based on this investigation, it implies that the default factor of 0.9 on the critical time step would be adequate provided a reasonably fine mesh is used.
• Based on findings of this Chapter, it can be concluded that the Winfrith concrete model provides robust and consistent results for the impact analysis of reinforced concrete beams subjected to falling-weight impacts, despite its simple input requiring very few parameters.
Chapter 6
RC Beams under Multiple Impacts
6.1 Introduction
The behaviour of reinforced concrete beams under falling-weight impacts has been discussed in the previous Chapter. The study identified a further need for experimental studies to generate additional data, which was not included in the beams tested by Chen & May (2009). This Chapter, therefore, describes a series of experimental tests to produce additional data, which would serve as an additional validation of the numerical models developed in Chapter 5. The tests also include multiple impacts on the beams to study the effect of initial damage on their subsequent impact behaviour. The experiments have been conducted on reinforced concrete beams similar to the beams tested by Chen & May (2009) and Hughes & Speirs (1982) under multiple falling loads. Numerical modelling of the tests has also been carried out using the methodology developed in the preceding Chapter. The behaviour of beams under undamaged and damaged conditions is discussed.
6.2 Description of tests
6.2.1 Details of beams
The experiments were conducted on 3 m long beams, which had the same cross-section and reinforcement as those described in Section 5.2. A drop-weight system was used to drop a mass of 98 kg at the mid-span of the beam. The striker comprised a mass, a load cell and an impactor of 100 mm diameter with a flat contact face. The beams were simply supported 150 mm from the ends. The dimensions of the beam and reinforcement details are shown in Figure 5.1.
Four beams were tested in this series, two under single impacts essentially repeating Chen’s tests with additional output. The other two were subjected to three impacts on each. The tests on the beams are essentially the same as those of Chen & May (2009), with additional output parameters, the measurement of reactions at the supports and deflections at various points along the length using LVDTs. Hence, the output from the tests included time histories of impact force, reactions and displacements at various positions along the length. Accelerometers were also used on the impactor to record its acceleration history. The beams tested under single impact were impacted by a 98 kg mass falling from a height of 2.7 m resulting in an impact velocity of 7.3 m/s, similar to the tests by Chen & May. For the multiple impacts, the beams were subjected to three impacts each of a lower energy compared to specimens tested under a single impact until the failure of the beams. The first impact was from a height of 1.5 m using the mass of 98 kg resulting in an impact velocity of 5.2 m/s. The next two impacts on the specimen were each conducted from a height of 1 m with an impact velocity of 4.2 m/s.
6.2.2 Instrumentation
A number of sensors were employed to monitor the tests and record high-quality data. The impact forces were measured using a load cell placed between the impactor-mass and the impactor-head. Reactions at the supports were recorded using load cells, which were sandwiched between the beam and bottom support plates, as shown in Figure 6.1.
Displacements at four locations along one half of the beam were measured using LVDTs. Figure 6.2 shows the schematic diagram of the test set-up. The outputs of the load cells and LVDTs were amplified and fed into a data acquisition system at a sampling rate of 500 kHz. The striker was unrestrained vertically which resulted in rebound and subsequent impacts after the first impact. However, the response of the beams under these impacts was not as significant as the response under the first impact. Therefore, the behaviour of the beams under the first impact only is considered.
6.3 Experimental results
The results for the beams NB1 & NB3, which were subjected to multiple impacts and NB2 & NB4 under single impacts, are presented here. The beams NB1 and NB3 were identical except that the longitudinal reinforcement in NB3 was
instrumented with strain-gauges. The first impact on NB1 (NB1-1) and NB3 (NB3-1) was from a height of 1.5 m. The next two impacts on the same beams (NB1-2 & NB1-3 and NB3-2 & NB3-3) were from a height of 1 m. Beams NB2 and NB4 were tested under single impact of 98 kg mass falling from a height of 2.7 m. Both the beams were tested in similar conditions to the beams tested by Chen & May (2009). Beam NB4 also had instrumented reinforcement like NB3. In the following sections, the transient records of impact forces, reactions and displacements are described. The resulting damage and cracking alongwith modes of failure are also discussed.
6.3.1 Transient impact forces
The details of the maximum impact forces, peak times, maximum reactions and peak reaction times for each beam are given in Table 6.1. In the following sections, the results are described in further detail for each beam.
Table 6.1: Maximum impact force, reactions and peak times of beam tests
| Beam no. | Test no. | $H$ (m) | $F_m$ (kN) | $t_m$ (ms) | $R_m$ (kN) | $t_r$ (ms) |
|----------|----------|---------|------------|------------|------------|------------|
| NB1 | NB1-1 | 1.5 | 250 | 1.6 | 46 | 8.0 |
| | NB1-2 | 1.0 | 203 | 1.3 | 37 | 8.0 |
| | NB1-3 | 1.0 | 145 | 1.4 | 33 | 8.5 |
| NB2 | NB2 | 2.8 | 371 | 1.5 | 42 | 8.4 |
| NB3 | NB3-1 | 1.5 | 151 | 1.2 | 17 | 7.2 |
| | NB3-2 | 1.0 | 120 | 1.0 | 14 | 9.0 |
| | NB3-3 | 1.0 | 99 | 1.2 | 14 | 11 |
| NB4 | NB4 | 2.8 | 342 | 1 | 43 | 8.6 |
**Beam NB1**
The beam was tested under three impacts. The impact force and reaction force histories are presented in Figure 6.3, for the first impact from a height of 1.5 m. The impact force reached a peak value of 250 kN at 1.6 ms. There are two smaller peaks of 60 and 90 kN between 5 and 15 ms after the impact. There was a very short period of separation between the impactor and the beam from 7.5 to 12.5 ms. Then the impactor caught up with the beam and they contacted again.
The reactions were recorded using load cells placed under both the supports. The reactions lag behind the impacts force by about 6 ms. The maximum reaction of 46 kN was recorded on the right support after 8 ms, where as that on the left support was measured 34 kN at about the same time. The shape of the reaction histories for both the supports is very similar. The lag between the impact force and reactions is attributed to the time taken by the stress wave to travel from the point of impact (centre of the beam) to the supports.
Figure 6.4 and 6.5 show the impact force and reaction force histories for the second and third impact on NB1 each from a height of 1 m. For the second
impact (NB1-2) a peak impact force of 203 $kN$ was recorded at 1.25 $ms$ and a maximum reaction of 37 $kN$ was measured at the left support after 8 $ms$. The maximum reaction recorded on the right support was 30 $kN$ at about the same time and both the reaction histories are very similar.
The third impact from 1 $m$ on the same beam resulted in a peak force of 145 $kN$ and a maximum reaction of 33 $kN$ on the left support. A peak reaction of 27 $kN$ was recorded at the right support. The shape of the reaction force histories for both the supports is very similar. Following the second impact the beam was heavily damaged, therefore a reduction in peak impact force was recorded for the third impact. The shapes of the impact force and reaction histories for the three impacts on the same beam are very similar. The main difference is in the reduction in peak forces as the beam gets further damaged after each impact.
Figure 6.4: Transient impact force and reaction force histories, second impact (NB1-2)
Figure 6.5: Transient impact force and reaction force histories, third impact (NB1-3)
Beam NB3
As mentioned earlier, beam NB3 was tested under same loading as beam NB1. Figures 6.6, 6.7 and 6.8 show the impact force and reaction force histories for the three impacts on NB3, respectively. For the first impact (NB3-1), a maximum impact force of $151 \ kN$ was recorded after $1.2 \ ms$, whereas a maximum reaction of $17 \ kN$ was measured at the left support after $7.2 \ ms$. The second and third impacts (NB3-2 and NB3-3) resulted in peak forces of $120 \ kN$ and $99 \ kN$, after $1.0 \ ms$ and $1.2 \ ms$, respectively. The corresponding reaction was $14 \ kN$ on the left support for both the impacts. Comparing the results of transient impact and reaction forces with NB1, beam NB3 appears to have smaller peak loads. Since only two beams were tested under identical setup, the reasons for this apparent variation in peak forces can most likely be attributed to the stiffness of the members and the interface between the impactor and the beams.
 
(a) Transient impact force (b) Transient reaction force
Figure 6.6: Impact and reaction force histories, first impact (NB3-1)
Beam NB2
The transient impact force and reaction force histories for NB2, which was subjected to a single impact are shown in Figure 6.9. The impact force reached a
Figure 6.7: Impact and reaction force histories, second impact (NB3-2)
Figure 6.8: Impact and reaction force histories, third impact (NB3-3)
peak value of 371 $kN$ after 1.5 $ms$. The beam was identical to Chen’s beams and was tested under the same conditions. The overall shape and magnitude of the impact force history are very similar to those obtained for the beams tested by Chen. The maximum reaction of 42 $kN$ was measured on the right support. The left and right support reactions, as shown in Figure 6.9(b) are almost identical.
(a) Transient impact force
(b) Transient reaction force
Figure 6.9: Transient impact and reaction force histories for single impact, beam NB2
Beam NB4
Beam NB4 was a repeat of the test on beam NB2, remarkably similar results were recorded. Figure 6.10 presents the impact force and reaction histories for NB4. The peak impact force of $342 \ kN$ and maximum reaction of $43 \ kN$ are very close to those of NB2. The shapes of the impact force and reaction histories are also almost identical.
(a) Transient impact force
(b) Transient reaction force
Figure 6.10: Transient impact and reaction force histories for single impact, beam NB4
Table 6.2: Maximum displacements recorded in the beam tests
| Beam no. | Test no. | $H$ (m) | $\delta_1$ (mm) | $\delta_2$ (mm) | $\delta_3$ (mm) | $\delta_4$ (mm) |
|----------|----------|---------|-----------------|-----------------|-----------------|-----------------|
| NB1 | NB1-1 | 1.5 | 30.2 | 24.3 | 16.5 | 9.1 |
| | NB1-2 | 1.0 | 27.0 | 21.9 | 15.6 | 8.2 |
| | NB1-3 | 1.0 | 32.5 | 25.5 | 15.7 | 8.6 |
| NB2 | NB2 | 2.8 | 51.9 | 40.9 | 20.6 | 10.4 |
| NB3 | NB3-1 | 1.5 | 33.1 | 27.6 | 18.7 | 10.2 |
| | NB3-2 | 1.0 | 28.3 | 23.7 | 16.4 | 9.3 |
| | NB3-3 | 1.0 | 32.1 | 25.7 | 15.3 | 9.3 |
| NB4 | NB4 | 2.8 | 48.8 | 34.7 | 17.7 | 7.4 |
$\delta_1$: displacement near centre of the beam
$\delta_2$: 300 mm off centre
$\delta_3$: 600 mm off centre
$\delta_4$: near support
### 6.3.2 Transient displacements
The details of the transient displacements are recorded in Table 6.2. The displacements were measured using LVDTs at four locations along one half of the beam to obtain a displacement profile for the beam. The first LVDT was mounted near the guide rail of the impactor at 300 mm from the centre to record the displacement as near to the mid-span as practically possible. The other three transducers were attached at a spacing of 300 mm from each other, Figure 6.2.
**Beam NB1**
The displacement histories for the first impact (NB1-1) are presented in Figure 6.11. A peak displacement of 30 mm was measured near mid-span and a residual displacement of 12 mm was present at the end of the first impact (NB1-1). The presence of the peaks in the displacement histories show the beam vibrating until about 750 ms. The displacement measured at 600 mm from the centre and near the support also show reversed displacement at about 65 ms, which can be related to vertical cracks starting from the top in that region.
Figures 6.12 and 6.13 show the displacement histories for second and third impacts, respectively. As seen in Figure 6.12, the maximum and residual displacements near the centre were $27 \text{ mm}$ and $8 \text{ mm}$, respectively for the second impact. During the third impact (NB1-3), a peak displacement of $33 \text{ mm}$ was measured with a residual displacement of $12 \text{ mm}$. At the end of the test after three impacts on NB1, the total residual displacement measured near the centre was $32 \text{ mm}$.
**Beam NB3**
The displacement histories for the three impacts (NB3-1, NB3-2 and NB3-3) on NB3 are presented in Figure 6.14(a), (b) and (c), respectively. The maximum displacements near the centre of the beam were measured as $33.1 \text{ mm}$, $28.3 \text{ mm}$ and $32.1 \text{ mm}$ for the three impacts. On closer examination of Figure 6.14, it can be observed that there is a slight lag in the transient displacements from centre of the beam to the supports. This lag is attributed to time taken by the stress wave to travel from the centre of the beam to the supports.
Figure 6.12: Transient displacement histories, second impact (NB1-2)
Figure 6.13: Transient displacement histories, third impact (NB1-3)
Figure 6.14: Transient displacement histories after three impacts on NB3
Beam NB2 and NB4
Figures 6.15 and 6.16 show the transient displacement histories recorded for the beams NB2 and NB4, respectively. The peak values of displacements for the two beams are very close near the centre, $51.9 \text{ mm}$ for NB2 and $48.8 \text{ mm}$ for NB4. Both the beams were subjected to single impacts with higher energies than NB1 and NB3, which were impacted by three lower energy impacts. They have a lower frequency because the higher energy has induced more damage.
Figure 6.15: Transient displacement histories after single impact on NB2
Figure 6.16: Transient displacement histories after single impact on NB4
6.3.3 Damage and crack patterns
Beam NB1 and NB3
The first impact on beams NB1 and NB3 from a height of 1.5 m resulted in minor cracks appearing in the centre at the bottom of the beam and vertical cracks near the supports at the top of the beam. There was also a small zone of crushing at the point of impact, which was localized. The crack patterns formed after the first impact (NB1-1) on beam NB1 are shown in Figure 6.17(a). The beam developed diagonal cracks, originating at the impact point and propagating downwards with an angle of approximately 45 degrees, forming a shear-plug. In addition, a diagonal crack virtually parallel to the shear-plug crack also developed. Vertical flexural cracks near the supports also developed, which started from the top of the beam. There was also some damage under the impactor due to the crushing of concrete at the point of contact. Figure 6.17(b) presents the crack patterns and damage after the second impact (NB1-2). The cracks formed in the first impact have grown in size along with the development of new cracks. There was more damage at the point of impact because of crushing of the concrete. The final crack pattern of the beam NB1 after the third impact is shown in Figure 6.17(c). The beam showed a predominantly flexural mode with crushing beneath the impactor and shear cracks in the impact zone. The large diagonal crack virtually parallel to the shear-plug cracks opened further and extended from the top to the bottom of the beam. Vertical cracks starting from the top of the beam extended to the bottom along the beam section near supports. There was severe crushing of concrete underneath the impactor, resulting in broken pieces of concrete leading to exposure of top steel, Figure 6.18.
The final crack patterns at the end of each impact on beam NB3 are presented in Figure 6.19(a), (b) and (c), respectively for the three impacts NB3-1, NB3-2 and NB3-3. Compared to beam NB1, the first impact on beam NB3 (NB3-1), resulted in slightly less cracks and damage, as seen in Figure 6.19(a). The beam
(a) after first impact (NB1-1)
(b) after second impact (NB1-2)
(c) after third impact (NB1-3)
Figure 6.17: Crack patterns and damage after three impact on NB1
Figure 6.18: Severe crushing and exposed top steel after final drop of 1 m (NB1-3)
did develop diagonal cracks, originating at the impact point and propagating downwards with an angle of approximately 45 degrees, but they were smaller than those in NB1-1. The damage under the impactor was also less than NB1-1.
 after first impact (NB3-1)
 after second impact (NB3-2)
 after third impact (NB3-3)
Figure 6.19: Crack patterns and damage after three impact on NB3
The second impact on NB3 produced considerable cracking, similar to those produced in the second impact on beam NB1. The diagonal cracks formed during the first impact, now formed a shear plug. There were also well distributed vertical flexural cracks formed in the region between supports and the beam centre. Diagonal cracks parallel to the shear plug were also formed. The pattern was similar to NB1, however, the cracks were more evenly distributed in NB3. The final impact on NB3 resulted in the cracks opening up and extending further. The diagonal cracks forming the shear plug extended now from the top to the bottom. The vertical cracks near the point of impact also extended and joined the diagonal
cracks. There was also considerable crushing underneath the impactor. After the third impact on NB3, the pattern of cracking and crushing was similar to that in beam NB1.
**Beam NB2 and NB4**
Figures 6.20 and 6.21 show the damage and final crack patterns at the end of the single impact tests on beams NB2 and NB4, respectively. As already described in Section 6.3, both the beams were tested under the same conditions as Chen’s beams. The damage and crack patterns are, therefore, very similar. The beams demonstrated a predominantly flexural mode with some crushing beneath the impactor and shear cracking in the impact zone. Vertical cracks starting from the top in a section between the supports and the centre of the beam were also formed. There was no loss of the concrete cover to the tension steel reinforcement, although it was observed in some of Chen’s beams tested under similar conditions.
6.4 Finite element modelling
The tests presented above have been analyzed using the finite element code LS-DYNA using the methodology developed in Chapter 5. A three-dimensional numerical model has been created using the Lagrangian formulation. The concrete beam and the drop weight were discretized in space with eight-nodes hexahedron elements. The elements for the concrete beam have an aspect ratio of 1. One-point Gauss integration and hourglass control were used. The reinforcement bars were spatially discretized using beam elements. The model has been created for the entire member. The concrete solid elements and reinforcement beam elements share the common nodes. In some of the models, a numerical technique available in LS-DYNA was used to couple the beam elements with solid elements using the keyword CONSTRAINED.LAGRANGE.IN.SOLID. The finite element model for the beam NB1 is shown in Figure 6.22. In a typical beam model, there are about 60,000 solid elements and 1800 beam elements.
There are a number of material models for concrete available in LS-DYNA. Full material characterization is required by the majority of these concrete models in order to determine the required parameters. However, recent versions of
a few models have incorporated a capability to generate default parameters requiring only basic material properties such as unconfined compression strength, Poisson’s ratio and aggregate size. In the present study, the Winfrith concrete model (Broadhouse, 1995) has been used. Steel reinforcement has been modeled using an elastic-plastic material model MAT\_PLASTIC\_KINEMATIC (Hallquist, 2007). The impactor was modelled as rigid. Surface to surface contacts were defined between the impactor and the target beam. Further details of the modelling are given in Chapter 5.
### 6.5 Comparison of finite element results and tests
As mentioned in Section 6.3, the test on beam NB1 involved multiple impacts, resulting in the beam being damaged after the first impact (NB1-1). LS-DYNA uses several techniques to model the initial damage conditions, which can then be used in the input stage for subsequent analyses. For the first impact (NB1-1), the analysis was performed for a normal impact of 98 kg mass impacting the beam with an initial velocity of 5.2 m/s. The final stage of the first analysis was used as the input state for the second impact (NB1-2) with an impactor initial velocity of 4.2 m/s. The third impact (NB1-3) was simulated using the final state from the second impact (NB1-2) as the input. The analysis provided extensive output including concrete and reinforcement stresses, strains, crack and damage profiles, displacements and transient forces. In the following sections, the analysis results for transient impact forces, reactions, displacement histories and crack patterns are compared with the tests.
#### 6.5.1 Transient impact forces
The impact force and reaction force histories are presented in Figures 6.23, 6.24 and 6.25 for the three impacts NB1-1, NB1-2 and NB1-3 on beam NB1. The
impact force histories obtained from the analysis are in good agreement with the tests. The shapes of the curves from analysis for all the three impacts are very similar to the tests. The peak impact forces are also captured with reasonable accuracy, especially for the second and third impact. The comparison of reaction force histories shows reasonable agreement for peak reactions, however the shape of the curves from analysis shows some noise, which might be improved by better modeling of the supports.
Figure 6.23: Comparison of simulation impact force and reaction histories with tests for beam NB1 after first impact (NB1-1)
Figure 6.24: Comparison of simulation impact force and reaction histories with tests for beam NB1 after second impact (NB1-2)
Figure 6.25: Comparison of simulation impact force and reaction histories with tests for beam NB1 after third impact (NB1-3)
6.5.2 Displacements
Figures 6.26, 6.27 and 6.28 show the comparison of displacement histories near mid-span for the three impacts NB1-1, NB1-2 and NB1-3, respectively. For the first impact, the peak displacement near mid-span from the analysis is $27 \text{ mm}$, where as the test recorded $30.2 \text{ mm}$. For the second and third impacts, the analysis showed peak displacements of $25 \text{ mm}$ and $31 \text{ mm}$, which are very close to test displacements of $27 \text{ mm}$ and $32.5 \text{ mm}$, respectively. The shapes of the displacement curves from the analyses of the three impacts are in reasonable agreement with the tests, however, the analysis curves seem to dampen out earlier as compared to the tests.
Figure 6.26: Comparison of displacement near mid-span for beam NB1 after first impact (NB1-1)
6.5.3 Crack patterns
Comparison of the final crack patterns after the three impacts on NB1 are shown in Figure 6.29. The crack patterns predicted by the analysis show the development of diagonal cracks, originating at the impact point and propagating downwards.
Figure 6.27: Comparison of displacement near mid-span for beam NB1 after second impact (NB1-2)
Figure 6.28: Comparison of displacement near mid-span for beam NB1 after third impact (NB1-3)
with an angle of approximately 45 degrees, forming a shear-plug, which was found in the tests. Vertical cracks away from the impact zone are also shown in the analysis. There is a similar zone of concrete crushing under the impactor. Overall, the damage and crack patterns obtained from the analysis at the end of the simulation are in reasonable agreement with the tests.
(a) Final crack pattern from test
(b) Crack pattern from analysis
Figure 6.29: Comparison of final crack patterns and damage after third impact (NB1-3) on damaged specimen
6.6 Conclusions
A series of tests on reinforced concrete beams under falling-weight impacts has been described. Four beams were tested, two under single impacts and the other two subjected to three lower energy impacts. Additional high-quality data including transient reactions and displacements, was recorded, which was later used to validate the finite element analysis. Finite element modeling of the beams under multiple impact loads has been described. Following conclusions are drawn.
• Beams NB1 and NB3 were tested under three impacts. The impact force histories recorded for both beams showed a reduction in the peak forces after each impact as the beams were damaged after each impact.
6.6 Conclusions
- The reaction force histories showed a time lag of about 6 ms to the impact forces. This observation validated the similar findings from the finite element analysis of beams described in Chapter 5.
- Beams NB2 and NB4 were tested under identical conditions to Chen’s beams. The impact force histories recorded during the test are remarkably similar to Chen’s beams, which confirms the validity of the test results.
- Displacement histories recorded along the beams show a time lag in the transient displacements from centre of the beam to the supports. This lag is attributed to the time taken by the stress waves to travel from the centre of the beam to the supports.
- The displacement measured at 600 mm from the centre and near the support also show reversed displacement at about 65 ms, which can be related to the vertical cracks starting from the top in that region.
- The transient displacement histories for beams NB2 and NB4, which were subjected to single impact showed lower frequencies compared to beams NB1 and NB3, which were impacted by lower energy impacts three times such that the total energy was similar to NB2 and NB4.
- The damage and crack patterns for beams NB1 and NB3 were similar to those of Chen & May (2009). The first impact produced diagonal cracks starting at the point of impact. Vertical cracks near the supports were also found. Two subsequent impacts on the same beams resulted in more damage under the impactor, the initial cracks opened further and additional extensive cracking occurred.
- Beams NB2 and NB4 showed similar damage and crack patterns to those of Chen & May (2009). The beams demonstrated a predominantly flexural
mode with some crushing beneath the impactor and shear cracking in the impact zone. There was no loss of the concrete cover to the tension steel reinforcement, although it was observed in some of Chen’s beams tested under similar conditions.
- Finite element modelling of beam NB1, which was subjected to three impacts, was carried out. The impact force histories obtained from the analysis were in good agreement with the tests for the three impacts. The shapes of the curves and peak forces are captured with reasonable accuracy, especially for the second and third impacts.
- The comparison of reactions force histories also shows reasonable agreement for peak reactions and peak times. The time lag between the impact forces and reactions is also confirmed from the analysis.
- The peak displacements from the analysis are in good agreement with those in the tests for the three impacts on beam NB1. However, some discrepancies were found for the periods of vibrations between the test and the simulation. This was more significant following the second and third impact.
- The comparison of the final crack patterns for beam NB1 for the test and the simulation, show a very good agreement. Diagonal shear cracks in the impact zone, vertical cracks near the supports and zone of crushing under the impact were all captured with reasonable accuracy.
It is therefore reasonable to state that LS-DYNA could be used to model impacts on members which have damage from previous loadings.
Chapter 7
Finite Element Analysis of RC Slabs under Impact
7.1 Introduction
This Chapter describes the finite element modelling and analysis of reinforced concrete slabs under drop-weight impacts. The methodology developed for the three-dimensional analysis of reinforced concrete beams, as described in Chapters 5 and 6, has been applied to reinforced concrete slabs. The results obtained from the numerical simulations have been compared with the tests that were carried out at Heriot-Watt University by Chen & May (2009) to generate high quality input data to validate numerical modelling. A brief description of the impact tests on the slabs is provided followed by details of the finite element models. The results obtained from the analysis of different slabs are presented and a detailed comparison with the tests is described.
7.2 Description of slab tests
A series of experimental studies to investigate the high-mass, low-velocity impact behaviour of reinforced concrete members was performed by Chen & May (2009).
The details of the testing procedure, drop-weight system, instrumentation, etc have already been briefly described in Chapters 2 and 5. The results of the tests for reinforced concrete beams were employed to validate the finite element analysis, as described in Chapters 5 and 6. The results for the slabs will be employed to validate the finite element analysis of the slabs.
In the slab tests, four 760 mm square, 76 mm thick and two 2320 mm square, 150 mm thick slabs were tested under drop-weight loads of up to 380 kg with velocities of up to 8.7 m/s. The details of the slabs are given in Table 7.1 and Figure 7.1. Slabs 1 to 4 (small slabs) had identical material properties for the concrete and steel reinforcement. Slab 4 had a higher reinforcement ratio of 1.1% compared to 0.6% for slabs 1, 2 and 3. Slab 1 to 3 were subjected to a falling mass of 98.7 kg impacting with a velocity of 6.5 m/s. The same mass was used on slab 4, but with a higher velocity of 8 m/s.
Table 7.1: Details of slab tests of Chen & May (2009)
| Slab no. | Striker mass (kg) | Impactor head | Impact velocity (m/s) | Steel ratio (%) | $f_{cu}$ (N/mm$^2$) | $f_t$ (N/mm$^2$) |
|----------|-------------------|-------------------|-----------------------|-----------------|---------------------|------------------|
| 1 | 98.7 | Hemispherical | 6.5 | 0.6 | 60 | 4.06 |
| 2 | 98.7 | Hemispherical | 6.5 | 0.6 | 60 | 4.06 |
| 3 | 98.7 | Flat | 6.5 | 0.6 | 60 | 4.06 |
| 4 | 98.7 | Hemispherical | 8 | 1.1 | 60 | 4.06 |
| 5 | 196.7 | Hemispherical | 8.7 | 0.5 | 47.3 | 2.93 |
| 6 | 380 | Hemispherical | 8.3 | 0.5 | 55.7 | 2.93 |
Slab 5 and 6 (large slabs) each had the same reinforcement ratio of 0.5% but different concrete strengths, Table 7.1. Slab 5 was impacted by 196.7 kg mass with an impact velocity of 8.7 m/s. A larger mass of 382 kg with velocity of 8.3 m/s was used on slab 6.
Figure 7.1: Details of the slabs: (a) $0.76 \text{ m}$ square slabs; (b) $2.3 \text{ m}$ square slabs (dimensions in mm) (Chen & May, 2009)
7.3 Experimental results
The output from the tests included time histories of impact force, acceleration, reinforcement strains and video footage. In the following sections, the results of the tests are briefly described.
7.3.1 Transient impact forces
Figure 7.2 shows the impact force-time histories for the smaller slabs 2 to 4. There was no record of the impact force for slab 1, as it was tested without a load cell to check if perforation of the slab occurred, which could have resulted in the damage to the load cell. For the smaller slabs, a peak load of $174 \ kN$ was recorded in slab 3 at $1.1 \ ms$, which fell until it reached a plateau of approximately $55 \ kN$. In slab 2 and 4, peak loads of $136 \ kN$ and $156 \ kN$, respectively were measured at $1.4 \ ms$. The shapes of the transient impact histories for slabs 2 to 4 are very similar.
The impact force histories for larger slabs 5 and 6 are presented in Figure 7.3. Slabs 5 and 6 had peak impact forces of $583 \ kN$ and $1380 \ kN$ at $1.8 \ ms$ and $1 \ ms$, respectively. A lower plateau of about one-eighth of the peak force was present in slab 6; where as in slab 5, the plateau was absent. Local failures accompanied by yielding of the reinforcement, were observed to various degrees during this time in all the slabs, which will be further discussed in Section 7.3.4.
7.3.2 Transient accelerations
Accelerometers were attached to the impactor mass and the slabs. In the test on slab 6, the acceleration history was used to determine the impact force history, because the impact load exceeded the capacity of the load cell. On smaller slabs 2 to 4, the accelerometers were mounted on the top of the slab at $150 \ mm$ and $250 \ mm$ from the centre, and for slabs 5 and 6, they were located at $300 \ mm$
Figure 7.2: Transient impact force of slab 2–4 (Chen & May, 2009)
Figure 7.3: Transient impact force of slabs 5 and 6 (Chen & May, 2009)
and 600 mm off the centre. Figure 7.4 and 7.5 show the transient accelerations measured on the top surfaces of slabs 4 and 6, respectively. The two accelerations measured on slab 4 are reasonably in phase. For slab 6, there is some difference in phases of accelerations for the first 5 ms, which reduces afterwards. This difference on larger slabs 5 and 6 was attributed to the lag in the response of the various parts of the 2.3 m square slab due to wider spacing of the accelerometers.
Figure 7.4: Transient acceleration of slab 4 (Chen & May, 2009)
### 7.3.3 Transient reinforcement strains
The reinforcement in slab 6 had strain gauges placed inside the bars to measure the strains. A total of 10 strain gauges were used on both sides from the centre of the slab. Figure 7.6 shows the strain history for slab 6. The strains indicated that some of the reinforcement yielded during the test.
Figure 7.5: Transient acceleration of slab 6 (Chen & May, 2009)
Figure 7.6: Transient reinforcement strain in slab 6 (Chen & May, 2009)
7.3.4 Local damage
Figure 7.7 shows the damage, after the impact, to the top and bottom faces of slabs 1 to 4. There was a significant amount of penetration of the impactor in the 76 mm slabs. A zone of damage was formed at the bottom face owing to scabbing of the concrete. Figure 7.8 presents the damage on the larger slabs 5 and 6. There was some scabbing on slab 6 but on slab 5 scabbing was not fully developed. There was also a significant amount of penetration of the impactor in slab 6 but very little in slab 5, as it was impacted by approximately half of the impactor mass of slab 6.
7.4 Finite element modelling
The tests have been numerically analyzed using the finite element code LS-DYNA. A similar modelling approach has been adopted to that employed for the beams described in Chapters 5 and 6. A three-dimensional finite element model has been created for the entire slab using the Lagrangian formulation. The concrete slab and drop-weight were discretized in space with eight-noded hexahedron elements. The elements for the concrete slab have an aspect ratio of 1. One-point Gauss integration and a viscous hourglass control formulation were used for the elements in the slab and the impactor. The reinforcement bars were spatially discretized with beam elements. Full bond between the concrete solid elements and the reinforcement beam elements has been achieved by coupling the beam elements with solid elements using LS-DYNA keyword *CONSTRAINED_LAGRANGE_IN_SOLID. The supports at the four corners of the slab have been applied using restraints in the vertical and the horizontal direction. The model has been created for the whole problem. The finite element model for one of the smaller slabs is shown in Figure 7.9. In a typical model of a small slab, there are about 8,000 solid and beam elements for a mesh size of 20 mm.
Figure 7.7: Damage on slabs 1 to 4 after impact
Figure 7.8: Damage on slabs 5 and 6 after impact
Figure 7.9: Finite element model of a typical small slab
Figure 7.10 shows the finite element model of 2.3 $m$ slab. A typical model of large slab comprised of about 58,000 solid and beam elements of 25 $mm$ mesh.
(a) Concrete and impactor elements
(b) Reinforcement mesh
Figure 7.10: Finite element model of a typical large slab
As already discussed in Chapters 4 and 5, LS-DYNA offers a number of material models for modelling concrete and reinforcement. Some of the models have
Table 7.2: Material properties of concrete
| Property | Value |
|---------------------------------|---------|
| Density ($kg/m^3$) | 2400.0 |
| Compression strength, $f'_c$ (MPa) | 48 |
| Tensile strength, $f_t$ (MPa) | 4.06 |
| Poisson’s ratio, $\nu$ | 0.19 |
Table 7.3: Material properties of steel reinforcement
| Property | Value |
|---------------------------------|-------------|
| Density ($kg/m^3$) | 7800.0 |
| Yield strength (MPa) | 510 |
| Elastic modulus (MPa) | 190000.0 |
| Poisson’s ratio, $\nu$ | 0.3 |
| Tangent modulus (MPa) | 19000.0 |
been evaluated and used for modelling of impact on reinforced concrete beams, as described in Chapter 5 and 6. In the present study, the Winfrith concrete (Broadhouse, 1995) and Concrete Damage Rel.3 (Malvar et al., 1997) have been used for the concrete slabs. The material properties for concrete used in the simulations of small slab are given in Table 7.2. The material model used for reinforcement is *MAT\_PLASTIC\_KINEMATIC with the properties as shown in Table 7.3. The impactor was modelled as a rigid body with a density of 7800 $kg/m^3$ with an elastic modulus of 210 $GPa$, and a Poisson’s ratio of 0.30. An automatic surface to surface contact formulation was used to define the interface between the impactor and the target slab.
### 7.5 Mesh sensitivity
In order to study the effect of element mesh sizes on the analysis, three mesh sizes were used to model the slabs. Table 7.4 and 7.5 show the details of the mesh data for smaller slabs 2 to 4 and larger slabs 5 and 6, respectively. The standard mesh size of 20 $mm$ for slabs 2 to 4, was refined to 15 $mm$ and 10 $mm$. The analysis
Table 7.4: Mesh size data for 760 mm slabs
| | Mesh 1 | Mesh 2 | Mesh 3 |
|------------------|-----------------|-----------------|-----------------|
| Nodes | 51564 | 19256 | 10153 |
| Solid elements | 40802 | 13375 | 6146 |
| Beam elements | 3344 | 2244 | 1760 |
| Element size | 10 x 10 x 10 mm for solid elements, 10 mm for beam elements | 15 x 15 x 15 mm for solid elements, 15 mm for beam elements | 20 x 20 x 20 mm for solid elements, 20 mm for beam elements |
Table 7.5: Mesh size data for 2320 mm slabs
| | Mesh 1 | Mesh 2 | Mesh 3 |
|------------------|-----------------|-----------------|-----------------|
| Nodes | 882793 | 177450 | 67801 |
| Solid elements | 807670 | 148278 | 52204 |
| Beam elements | 13680 | 8040 | 5460 |
| Element size | 10 x 10 x 10 mm for solid elements, 10 mm for beam elements | 17 x 17 x 17 mm for solid elements, 17 mm for beam elements | 25 x 25 x 25 mm for solid elements, 25 mm for beam elements |
was carried out using the refined mesh sizes for each of the slabs and the results were compared for any variation in the impact force and displacement histories. Figure 7.11 presents the comparison of transient impact force histories for the three mesh sizes of slab 4 using the Winfrith model.
The three curves are very similar in shape with very small variation in the peak force only. This indicates that the analysis performed using the coarser mesh of 20 mm is in close agreement with that of refined meshes of 15 mm and 10 mm for slab 4.
Figure 7.12 shows the comparison of transient displacement histories from the analysis for three different mesh sizes of slab 4 using the Winfrith model. The peak displacements are 10.1, 10.5 and 10.8 mm for mesh sizes of 20, 15 and 10 mm, respectively. The peak displacements and peak times for the three mesh
sizes are in very close agreement. It can be concluded that the results obtained using the mesh size of 20 mm for slab 4 can be considered to be valid for the series of analysis for small slabs to be shown in the following sections. Similar study was also carried out for large slab 5 and based on the results a mesh size of 25 mm was selected for slabs 5 and 6.
7.6 Comparison of finite element results and tests
A number of models were created with varying mesh sizes for both the 760 mm and 2320 mm slabs. As described in Section 7.2, slabs 1 to 3 had identical material properties and reinforcement, slab 4 had the same material properties but a higher reinforcement ratio. Slabs 1, 2 & 4 were impacted by a 90 mm hemispherical impactor, slab 3 was impacted by a 100 mm flat impactor. Slabs 5 and 6 had different properties for concrete and were impacted by different mass and velocities. In the following sections, the analysis results for the transient impact forces, displacement histories, damage and crack patterns are described and compared with the tests for each slab.
Figure 7.12: Comparison of transient displacement histories obtained from the analysis for three different mesh sizes of slab 4 (Winfrith model)
### 7.6.1 Transient impact forces
**Slab 2**
A comparison of transient impact force history with the test for slab 2 is presented in Figure 7.13 using Winfrith model for concrete. The forces reach the peak values at the same time at $2 \text{ ms}$, however the force obtained from the analysis is larger than the test. After the peak is passed, both the force histories are similar. The slab and the impactor remain in contact for about $11 \text{ ms}$ and $15 \text{ ms}$ in the analysis and the test, respectively. The increase in the peak force obtained in the analysis could be attributed to the stiffness of the interface between the impactor and the slab.
**Slab 3**
Figure 7.14 shows the comparison of impact force history for slabs 3 with the test using the Winfrith model for concrete. A similar increase in the peak force was observed as slab 2. A peak time of about $2 \text{ ms}$ was recorded in both the test and the analysis. As mentioned earlier, slab 3 was impacted by $100 \text{ mm}$ flat
Figure 7.13: Recorded and simulation impact force histories for slab 2 (Winfrith model)
impactor. Therefore, there is a slight difference in the post peak curve from slab 2, which terminated after 4 ms.
Figure 7.14: Recorded and simulation impact force histories for slab 3 (Winfrith model)
**Slab 4**
Slab 4 was impacted by the same mass with higher impact velocity of $8.0 \, m/s$ as compared to slabs 2 and 3. Therefore, the analysis predicts a higher peak in the impact force than slabs 2 and 3, as shown in Figure 7.15. The test, however, did not record any substantial increase over slabs 2 and 3 and the load history was almost identical. The analysis seems to pick up one larger peak and two slightly lower peaks very close to each other. This may be a numerical problem and could be attributed to the contact algorithm used in the simulations. The results could have been filtered to reduce the noise, however it was not attempted.
For the three smaller slabs 2 to 4, the analyses predicted higher peak forces than the test. The peak times obtained from the analysis are in very good agreement with the tests.
**Slab 5**
Figure 7.16 shows the comparison of impact force history for slabs 5 with the test using the Winfrith model for concrete. The analysis predicts a peak force of
700 kN at 1.5 ms, whereas the test recorded a peak force of 600 kN at 2.5 ms. The post peak curves start to decrease similarly, however the analysis shows a smaller peak between 6 and 9 ms. Both the curves show the loss of contact between the impactor and the slab at about 10 ms.
**Slab 6**
Slab 6 was impacted by double the mass used on slab 5. However, the velocities were similar. Figure 7.17 presents the impact force histories obtained from the analysis and the test. The peak forces and the peak times obtained from the analysis are in very good agreement with those of the test. As mentioned earlier, the load history for slabs 6 in the test, was obtained from the acceleration history. Therefore, the curve is not as smooth as that of slab 5. The overall shape and decrease after the peak forces is also similar despite many smaller peaks in the curve obtained from the acceleration history.
For the two large slabs 5 and 6, the analyses predict the peak forces, peak
times and post peak curves, which are in good agreement with those obtained from the tests.
### 7.6.2 Crack patterns and damage
The crack patterns obtained from the analyses using the Winfrith concrete model have been compared with those from the tests on the top and bottom face for each slab. Although the Winfrith model only gives the extent of cracking and directions, it can be used to assess the extent of damage of the structure. The assessment of damage due to penetration and scabbing is also discussed in the following sections.
#### Slab 2
A comparison of the damage and crack patterns for the analysis and the test at the top and bottom faces of slab 2 is shown in Figure 7.18 for the Winfrith model. On the top face of slab 2, a similar zone of damage at the point of impact can be
observed as in the test. The impactor showed a small penetration and bounced back as observed in the test. There is no cracking on the top face, except at the point of impact. The bottom face of the slab shows a zone of cracking in the centre of the slab, which can also be observed in the test. The diameter of the damage zone is in reasonable agreement with the test.
(a) Top face of slab 2
(b) Bottom face of slab 2
Figure 7.18: Comparison of simulation damage and cracks with the test for slab 2 (Winfrith model)
Figure 7.19 presents the plot of axial stresses in the reinforcement mesh for slab 2. The analysis results show that some elements in the centre of the slab yielded, which are highlighted by red colour in Figure 7.19.
Figure 7.19: Plot of axial stresses in steel reinforcement for slab 2 (Winfrith model)
**Slab 3**
Figure 7.20 shows the comparison of the damage and crack patterns to the top and bottom faces of slabs 3 after the test and analysis. The damage to the top face of the slab is very similar to slab 2, although slab 3 was impacted by a slightly larger diameter and flat impactor as compared to that used for slabs 2 & 4. Compared to slab 2, the diameter of damage zone at the top face is slightly bigger. The cracking and extent of damage to the bottom face is in reasonable agreement with the test and would indicate some degree of scabbing as seen in the test.
**Slab 4**
Slab 4 showed much more damage and cracking, since it was impacted by a higher velocity of $8.0 \ m/s$ compared to $6.5 \ m/s$ used for slabs 2 and 3. The top face of the slab shows extensive damage and cracking at the point of impact as shown in Figure 7.21 (a). The impactor did not fully penetrate the slab and was arrested. There was extensive cracking and damage to the bottom face of the slab as observed during the test. The diameter of the zone of cracking is in good agreement with that in the test. The cracks are spaced more closer as compared
Figure 7.20: Comparison of simulation damage and cracks with the test for slab 3 (Winfirth model)
to slabs 2 and 3, showing the extent of scabbing from the bottom of the slab.
(a) Top face of slab 4
(b) Bottom face of slab 4
Figure 7.21: Comparison of simulation damage and cracks with the test for slab 4 (Winfrith model)
**Slab 5**
The comparison of damage and cracking to the top and bottom faces of larger slab 5 is shown in Figure 7.22. The diameter of the zone of damage and cracking to the top and bottom faces is very similar to the test. There was not much damage observed in slab 5, compared to slabs 2 to 4. This could be attributed to
the proportionally more significant amount of energy in the impacts on smaller slabs.
(a) Top face of slab 5
(b) Bottom face of slab 5
Figure 7.22: Comparison of simulation damage and cracks with the test for slab 5 (Winfrith model)
**Slab 6**
For slab 6, the mass of the impactor was approximately double that for slab 5. The analysis using the Winfrith model showed more damage and cracking to the top and bottom faces of the slab compared to slab 5, Figure 7.23. There was a significant amount of penetration of the impactor as observed in the tests. The extent of cracking at the bottom face indicates that some scabbing developed in slab 6.
Figure 7.24 shows the damage on the top and bottom faces of the slab 6 using Concrete damage model (Malvar et al., 1997). Comparison with the test indicates a very good agreement, specially for damage to the bottom face of the slab where the reinforcement is exposed due to scabbing as was observed in the test.
(a) Top face of slab 6
(b) Bottom face of slab 6
Figure 7.23: Comparison of simulation damage and cracks with the test for slab 6 (Winfrith model)
7.6.3 Displacement histories
There was no instrumentation in the tests to record the displacement histories for the slabs. From the analysis, transient displacement histories are obtained at the centre of each slab to demonstrate the efficiency of the models. Figure 7.25 shows the transient displacement histories plotted at the centre of slabs 2 to 4 using the Winfrith model. The analysis predicts a peak displacement of 8.2 mm.
Figure 7.24: Comparison of simulation damage and cracks with the test for slab 6 (Concrete damage model)
after 2.8 ms for slab 2. The peak displacements for slab 3 and 4 are 6.2 mm and 10 mm, respectively. The displacement curves do not show further peaks and reduce to about 0.5 mm to 2 mm residual displacements.
The transient displacement histories for slabs 5 and 6 obtained from the analysis are presented in Figure 7.26. The analysis shows peak displacements of 11.5 mm and 18 mm for slabs 5 and 6, respectively. The difference in the peak displacements is due to the larger amount of impact energy on slab 6 than slab 5. The displacement curves for both slabs also show upward displacements, which indicates that the slabs continued to vibrate after the impact.
### 7.7 Effect of contact interface
The results of the analysis described in Section 7.6.1 showed some variation in the peak impact forces for slabs 2 to 4, the analysis predicting higher impact forces than those in the tests. This increase was attributed to the stiffness of the contact interface between the impactor and the slab. In order to investigate it further,
the analysis was carried by introducing a 12 mm layer of plywood between the impactor and the slab, as in the beam tests described in Chapter 5. The results from the analysis are described for two mesh sizes of 10 mm and 20 mm for slab 3.
In Figure 7.27, the transient impact force histories are compared for two mesh sizes of 10 mm and 20 mm with the test. As shown in Figure 7.27, there is no significant difference in the peak impact forces. The shapes of the curves are also in very good agreement with the test.
Figure 7.28 compares the damage and crack patterns on the top and bottom faces using the Winfrith concrete model for slab 3 with the tests. As shown in Figure 7.28 (a), the local damage on the top face of the slab is in close agreement with the experiment. In Figure 7.28 (b), the damage on the bottom face of the slab is compared. The diameter of the damage zone in the experiment is similar to the simulation. Closer examination of results for the two mesh sizes, shows a small variation in the size of the damage zones. The finer mesh size of 10 mm seems to show a more accurate size and pattern of the damage zone at the top.
and bottom of the slab, compared to that of the coarse mesh of 20 mm.
7.8 Slab-on-slab impact
The methodology developed to carry out numerical simulations of the low-velocity impacts on reinforced concrete members, as described above has been extended to consider a case of slab-on-slab impact. This type of impact could be a result of a floor slab falling, during for example progressive collapse of a structure, on a reinforced concrete floor slab below, resulting in serious damage to the structure. A hypothetical case is considered where a floor slab is allowed to fall freely on a reinforced concrete slab, resulting in a low-velocity impact. The behaviour of target slab is investigated in the numerical simulation using the methodology adopted for low-velocity impacts on reinforced concrete beams and slabs.
Figure 7.28: Comparison of simulation damage and cracking for slab 3 (plywood interface) using Winfrith concrete model
7.8.1 Description of the geometry
A square concrete slab $3.5 \, m \times 3.5 \, m \times 150 \, mm$ is assumed to fall freely from a typical storey height of $4 \, m$ onto a reinforced concrete floor slab. The target slab is considered to be $4.5 \, m$ square slab reinforced with $0.5\%$ steel reinforcement in both directions, as shown in Figure 7.29. These dimensions have been selected to fit typical reinforced concrete floor slab in ordinary buildings.
7.8.2 Finite element model
A three-dimensional finite element model has been created for the problem using a Lagrangian formulation. Both the impactor slab and the target slab were discretized using eight-noded solid elements with an aspect ratio of 1. The steel reinforcement in the target slab and the impacting slab was smeared into concrete elements. One-point Gauss integration and viscous hourglass control were used.
The models have been created for the whole problem. Sides of the target slab were fully restrained. The finite element model for the problem is shown in Figure 7.30. In a typical model of the simulation, there are about 270,000 elements for a mesh size of 25 mm.
### 7.8.3 Simulation results
A number of models were created with varying mesh sizes for slab-on-slab impact simulation. Based on the sensitivity study, a mesh size of 25 mm was used. The results of the simulation using the Winfrith Concrete model are presented. The target slab was impacted by a falling slab with an impact velocity of 8.85 m/s. Figure 7.31 shows the damage for the top and bottom faces of the target slab. The simulation results show considerable damage on the top face of the slab. The bottom face of the slab is fully damaged and indicates complete failure of the slab, as shown in Figure 7.31(b).
The results for impacting slab also reveal continuous lines of well connected cracks, suggesting that the slab is severely damaged and would probably be split into many pieces if it was not reinforced. The cracks and the damage on the impacting slab are shown in Figure 7.32.
Time history plots for contact impact force, velocity and displacement of the target slab are shown in Figure 7.33, 7.34 and 7.35, respectively. The plot of
impact force shows a single impact event resulting in complete failure of the target slab.
The displacement time history of the target slab shows a continuing downward displacement indicating complete failure of the target slab. The energy ratio for the simulation was very close to unity, which demonstrates the stability of the solution. This suggests that there is no spurious loss or gain of energy in the system compromising stability of the solution.
The simulation results show severe cracking and damage on the target slab, resulting in complete failure when impacted by falling slab. The damage and cracking plots indicate full damage at the top and bottom faces of the target slab. The simulation also shows severe well connected cracks in the impacting slab. Time history plot for impact force shows a main impact event resulting in collapse of the target slab. The displacement history shows a continuous downward displacement indicating total collapse of the target slab. The results from numerical simulation provide a significant insight into the behaviour of reinforced
Figure 7.32: Cracking in impacting slab from simulation
Figure 7.33: Impact force history for slab-on-slab impact
Figure 7.34: Velocity time history of target slab
Figure 7.35: Displacement time history of target slab
7.9 Conclusions
Finite element modelling of reinforced concrete slabs subjected to impact loads has been described. A brief description of the impact tests on reinforced concrete slabs carried out at the Heriot-Watt University is presented followed by series of finite element analysis. The results of the analysis have been described and compared with the tests for transient impact force histories, displacements, damage and crack patterns. Following conclusions are drawn.
- The transient impact force histories obtained from analysis of the smaller slabs 2, 3 and 4 showed higher peak forces compared to the tests, although the peak times were in good agreement. This increase in peak force could be attributed to the stiffness of the interface between the impactor and the slab.
- For slab 4, which was impacted with a higher velocity of $8 \text{ m/s}$, the analysis did not record any substantial increase in the impact force when compared to slabs 2 and 3, which had an impact velocity of $6.5 \text{ m/s}$.
- For the larger slabs 5 and 6, the analysis predicted the peak impact forces and peak times and post peak curves, which were in very good agreement with the tests.
- The comparison of crack patterns at the top and the bottom faces of all the slabs was in very good agreement with the tests.
- The zone of damage at the point of impact on the slabs is captured showing some amount of penetration as was observed in the tests. The diameter of this damage zone is in very good agreement with the tests. No perforation
was predicted, similar to the tests. The extent of damage and cracking to the bottom face indicates some degree of scabbing as seen in the tests.
- The higher peak forces from the analysis for smaller slabs were attributed to the stiffness of the contact zone. Upon the introduction in the analysis of a plywood layer between the slab and the impactor, the transient impact forces histories from the analysis were found to be in very good agreement with the tests.
Chapter 8
Conclusions
8.1 Conclusions
An investigation into the impact behaviour of reinforced concrete members has been described in this thesis. The study mainly focused on the high-mass, low-velocity impacts on reinforced concrete beams and slabs to study their local and global response under dynamic loading. The investigations involved numerical modelling using a commercial code LS-DYNA and experimental testing. Three-dimensional finite element modelling was carried out on the beams tested by Chen & May (2009) and has been described in detail in Chapter 5. The results of the analyses were compared with the tests and following conclusions can be drawn.
- The transient impact force histories obtained from the analyses were in reasonable agreement with the tests for peak impact forces, peak times and overall shape for Series A beams, which had a plywood interface between the impactor and the beam. There were some discrepancies, especially in the peak forces for some Series B beams, which were directly impacted by a hemispherical impactor. The change of supports from pin-ended to simply supported did not appear to have a significant effect on the transient impact force histories.
The reaction forces obtained from the analysis showed a time-lag to the transient impact forces, which reached their peak value well before the reactions started.
The transient acceleration-time histories obtained by differentiating the nodal velocities, showed remarkably good agreement with the tests.
The transient reinforcement strain histories obtained from the analysis showed very good agreement with the tests.
The crack patterns obtained from the analysis using the Winfrith concrete model matched reasonably well with the cracks and damage patterns in the tests. The analyses showed the development of diagonal cracks, originating at the impact point and propagating downwards with an angle of approximately 45 degrees, forming a shear-plug, which was also found in the tests. Vertical cracks away from the impact zone were also predicted in the analyses. The zone of crushing observed in the tests under the impactor head was also predicted. The development of horizontal cracks at the level of the tension steel reinforcement in the impact zone was predicted by the analyses. The correlation between the transient load and crack development predicted by the analysis matched exactly with the test. The development of diagonal shear cracks forming a plug and the flexural cracks along with crushing of concrete was shown to have taken place after the impact load had passed its peak value.
The numerical modelling performed on the beams tested by Chen & May (2009) identified a need to carry out further the experimental studies in order to generate additional data for the validation of the finite element models. Therefore, a series of experimental studies was conducted on similar beams with additional instrumentation. The additional measurements were the displacements at four
different locations along one half of the beam, recorded using LVDT’s, and the reactions at the supports. The tests also included multiple impacts on the beams to study the effect of initial damage on their subsequent impact behaviour.
- The reaction force histories recorded in the tests showed a time lag of about 6 ms to the impact forces. This observation validated the similar findings from the finite element analysis of beams described in Chapter 5.
- Displacement histories recorded along the beams show a time lag in the transient displacements from centre of the beam to the supports. This lag is attributed to the time taken by the stress waves to travel from the centre of the beam to the supports.
- The damage and crack patterns for beams under multiple impacts were similar to those of Chen & May (2009). The first impact produced diagonal cracks starting at the point of impact. Vertical cracks near the supports were also found. Two subsequent impacts on the same beams resulted in more damage under the impactor, the initial cracks opened further and additional extensive cracking occurred. Beams tested under single impacts also showed similar behaviour to those of Chen & May (2009).
- Finite element modelling of the beams under multiple impacts was carried out. The results show good agreement for transient impact forces, reactions and displacement histories. The final crack patterns are also accurately predicted by the analysis.
The methodology used for the numerical analysis of reinforced concrete beams was extended to carryout the three-dimensional finite element modelling of slabs tested by Chen & May (2009). The analysis results were compared with the tests for transient impact forces and crack patterns. The impact force histories obtained from the analyses of the 2.3 m slabs were in good agreement with the
tests, however, for the 0.76 m slabs, some discrepancies were found for peak forces. The comparison of crack patterns at the top and the bottom faces of all the slabs was in very good agreement with the tests. The zone of damage at the point of impact on the slabs was captured showing some amount of penetration as was observed in the tests. The diameter of this damage zone is in very good agreement with the tests. No perforation was predicted, similar to the tests. The extent of damage and cracking to the bottom face indicates some degree of scabbing as seen in the tests.
The impact behaviour of reinforced concrete members under high-mass, low-velocity impacts has been described in this investigation. The analytical procedure employed successfully captured the response of the members and mechanisms associated with low-velocity impacts. Reinforced concrete beams subjected to low-velocity impact loads can generally undergo some crushing at the point of impact, followed by the formation of a shear plug and overall flexural response. These mechanisms have been identified in the analyses and the tests carried out in this study. The actual mode of failure such as flexural or shear failure, may depend upon the static behaviour of the beams. A shear failure under impact loading at the centre of a beam is usually due to the formation of a diagonal shear crack parallel to the shear plug, which starts near the support and extends upwards becoming horizontal near the impact zone. The flexural failure is characterized by the formation of the concrete plug in the impact zone and vertical flexural cracks in the centre of the beam. Vertical flexural cracks starting from the top of the beam in a section away from the impact zone are also found. The yielding of tension steel reinforcement in the impact zone is also identified. The loss of concrete cover to the tension steel because of scabbing can be associated with flexural failure.
The proposed analytical procedure does not require the impact force history as input. Only the mass and velocity of the impactor or the impact energy is
required. This can be very useful for the design or assessment of structures under impact loads, as impact energy (combination of mass and velocity) is usually a known design parameter.
Based on findings of this investigation, it can be concluded that LS-DYNA and the Winfrith concrete model provide robust and consistent results for the impact analysis of reinforced concrete beams and slabs subjected to low-velocity, falling-weight impacts, despite its simple input requiring relatively few parameters.
8.2 Recommendation for Future Work
Based on the investigations carried out in this study, further areas of studies can be identified to help understand the impact behaviour of reinforced concrete members. Following recommendations can be made for future studies:
1. An experimental programme to investigate the residual capacity of the members following an impact event. This could be very useful, specially for restrengthening and retrofitting of structures subjected to highly dynamic accidental loading.
2. Detailed instrumentation, especially closely spaced accelerometers to accurately determine the inertia forces.
3. Effect of pre-stressing on the impact behaviour of beams. A large number of reinforced concrete structures involve pre-stressing, therefore, its effects on the impact behaviour of reinforced concrete members can be quite significant.
4. A comprehensive programme to investigate the strain-rate effects in the high-mass, low-velocity impacts on reinforced concrete members.
Appendix A
Analytical response for a SDOF system under impulse loading
Figure A.1 shows a mass-spring model and impulse force acting on a SDOF system. In order to determine the analytical response of the system, there are a number of methods available for the solution of the governing differential equation for such a loading condition. However, the use of Duhamel’s integral (see Chopra, 2001, chap. 4) was preferred because of its simplicity. The analytical solution is summarized below.
 
Figure A.1: (a) SDOF system; (b) Impulse force
For \( t \leq t_a \):
\[
u(t) = \frac{P_a}{k} \left( \frac{t}{t_a} - \frac{\sin(\omega_n t)}{\omega_n t_a} \right)
\]
(A.1)
For \( t_a < t \leq t_b \):
\[
u(t) = u(t_a) \cos(\omega_n (t - t_a)) + \frac{\dot{u}(t_a)}{\omega_n} \sin(\omega_n (t - t_a)) + \frac{P_a}{k} [1 - \cos(\omega_n (t - t_a))] \\
+ \frac{P_b - P_a}{k} \left( \frac{t - t_a}{t_b - t_a} - \frac{\sin(\omega_n (t - t_a))}{\omega_n (t_b - t_a)} \right)
\]
(A.2)
For \( t_b < t \leq t_c \):
\[
u(t) = u(t_b) \cos(\omega_n (t - t_b)) + \frac{\dot{u}(t_b)}{\omega_n} \sin(\omega_n (t - t_b)) + \frac{P_b}{k} [1 - \cos(\omega_n (t - t_b))] \\
+ \frac{-P_b}{k} \left( \frac{t - t_b}{t_c - t_b} - \frac{\sin(\omega_n (t - t_b))}{\omega_n (t_c - t_b)} \right)
\]
(A.3)
For \( t \geq t_c \):
\[
u(t) = u(t_c) \cos(\omega_n (t - t_c)) + \frac{\dot{u}(t_c)}{\omega_n} \sin(\omega_n (t - t_c))
\]
(A.4)
where
\[
\dot{u}(t_a) = \frac{P_a}{k} \left( \frac{1}{t_a} - \frac{\cos(\omega_n t_a)}{t_a} \right)
\]
(A.5)
\[
\dot{u}(t_b) = -u(t_a)\omega_n \sin(\omega_n (t_b - t_a)) + \dot{u}(t_a) \cos(\omega_n (t_b - t_a)) + \frac{P_a}{k} \omega_n \sin(\omega_n (t_b - t_a)} \\
+ \frac{P_b - P_a}{k} \left( \frac{1}{t_b - t_a} - \frac{\cos(\omega_n (t_b - t_a))}{t_b - t_a} \right)
\]
(A.6)
\[
\dot{u}(t_c) = -u(t_b)\omega_n \sin(\omega_n (t_c - t_b)) + \dot{u}(t_b) \cos(\omega_n (t_c - t_b)) + \frac{P_b}{k} \omega_n \sin(\omega_n (t_c - t_b)} \\
+ \frac{-P_b}{k} \left( \frac{1}{t_c - t_b} - \frac{\cos(\omega_n (t_c - t_b))}{t_c - t_b} \right)
\]
(A.7)
Appendix B
Sample LS-DYNA input deck for RC beam
*KEYWORD
*TITLE
$# title
IMPACT TEST ON REINFORCED CONCRETE BEAM
$$$$$$$$$$$$$ UNITS $$$$$$$$$$$$$$$$
S N, mm, Ton, S $
$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$
S Control cards $
$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$
*CONTROL ENERGY
$# lgen rwen slnten rylen
2 2 1 1
*CONTROL_OUTPUT
$# npopt nescho nrrefup iaccop opifs ipnint ikedit iflush
0 3 0 0 0.000 0 0 0
$# iprf ierode tet10 msgmax ipcurv
0 0 2 50 0
*CONTROL_TERMINATION
$# endtlim endycyc dtmin endeng endmas
0.050000 0 0.000 0.000 0.000
*CONTROL_TIMESTEP
$# dtinit tssfac isdo tslimt dt2ms lctm erode ms1st
0.000 0.800000 0 0.000 0.000 0 0 0
$# dt2msf dt2mslc imsc1
0.000 0 0
*DATABASE_ELOUT
$# dt binary lcur ioopt
1.000E-4 1 0 1
*DATABASE_GCEOUT
$# dt binary lcur ioopt
1.000E-4 1 0 1
*DATABASE_GLSTAT
$# dt binary lcur icopt
1.000E-4 1 0 1
*DATABASE_MATSUM
$# dt binary lcur icopt
1.000E-4 1 0 1
*DATABASE_NCFORC
$# dt binary lcur icopt
1.000E-4 1 0 1
*DATABASE_NODEFOR
$# dt binary lcur icopt
1.000E-4 1 0 1
*DATABASE_NODOUT
$# dt binary lcur icopt dthf binhf
1.000E-4 1 0 1 0.000 0
*DATABASE_RCFORC
$# dt binary lcur icopt
1.000E-4 1 0 1
*DATABASE_SLEOUT
$# dt binary lcur icopt
1.000E-4 1 0 1
*DATABASE_SPCFORC
$# dt binary lcur icopt
1.000E-4 1 0 1
*DATABASE_BINARY_D3PLOT
$# dt lcdt beam npltc psetid
1.000E-4 0 0 0 0
$# icopt
0
*DATABASE_BINARY_D3THDT
$# dt lcdt beam npltc psetid
1.000E-4 0 0 0 0
*BOUNDARY_SPC_SET
$# nsid cid dofx dofy dofz dofrx dofry dofrz
1 0 0 1 1 0 0 1
*SET_NODE_LIST_TITLE
Left Support Nodes
$# sid dal da2 da3 da4
1 0.000 0.000 0.000 0.000
$# nid1 nid2 nid3 nid4 nid5 nid6 nid7 nid8
286 587 888 287 285 586 588 889
887 1188 1189 1190 1491 1490 1489 1790
1791 1792 2093 2092 2091 2392 2393 2394
2695 2694 2693 2994 2995 2996 3297 3296
3295
*BOUNDARY_SPC_SET
$# nsid cid dofx dofy dofz dofrx dofry dofrz
2 0 0 1 1 0 0 1
*SET_NODE_LIST_TITLE
Right support nodes
$# sid dal da2 da3 da4
2 0.000 0.000 0.000 0.000
$# nid1 nid2 nid3 nid4 nid5 nid6 nid7 nid8
16 17 15 318 317 316 519 618
617 920 918 1221 1220 1219 1522
1521 1520 1823 1822 1821 2124 2123 2122
2425 2424 2423 2726 2725 2724 3027 3026
3025
*SET_SEGMENT
$# sid dal da2 da3 da4
1 0.000 0.000 0.000 0.000
| n1 | n2 | n3 | n4 | a1 | a2 | a3 | a4 |
|------|------|------|------|------|------|------|------|
| 700396 | 700395 | 700404 | 700407 | | | | |
| 700385 | 700384 | 700395 | 700396 | | | | |
| 700395 | 700394 | 700405 | 700406 | | | | |
| | | | | | | | |
| | | | | | | | |
| 700288 | 700287 | 700298 | 700299 | | | | |
*SET_SEGMENT
| sid | dal | da2 | da3 | da4 |
|-----|-----|-----|-----|-----|
| 2 | 0.000 | 0.000 | 0.000 | 0.000 |
| n1 | n2 | n3 | n4 | a1 | a2 | a3 | a4 |
|------|------|------|------|------|------|------|------|
| 201694 | 200040 | 200789 | 201677 | | | | |
| 200040 | 200541 | 200181 | 200789 | | | | |
| 201711 | 200073 | 200040 | 201694 | | | | |
| | | | | | | | |
| | | | | | | | |
| 201490 | 200145 | 201197 | 201473 | | | | |
*CONTACT_ERODING_SURFACE_TO_SURFACE
| cid | title |
|-----|-------|
| | |
| ssid | msid | sstyp | mstyp | sboxid | mboxid | spr | mpr |
|------|------|-------|-------|--------|--------|-----|-----|
| 1 | 2 | 0 | 0 | 0 | 0 | 1 | 1 |
| fs | fd | ds | va | vdc | penchk | bt | dt |
|-----|-----|-----|-----|------|--------|-----|-----|
| 0.200000 | 0.200000 | 0.000 | 0.000 | 0.000 | 0 | 0.000 | 0.000 |
| sfs | sfm | sst | mst | sfst | sfmt | fsf | vsf |
|-----|-----|-----|-----|------|------|------|------|
| 0.600000 | 0.600000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 |
| isym | erosop | iadj |
|------|--------|------|
| 0 | 1 | 1 |
*PART
*title
CONCRETE BEAM
| pid | secid | mid | eosid | hgid | grav | adpopt | tmid |
|-----|-------|-----|-------|------|------|--------|------|
| 1 | 1 | 1 | 0 | 1 | 0 | 0 | 0 |
*SECTION_SOLID
| secid | elform | aet |
|-------|--------|-----|
| 1 | 1 | 0 |
*MAT_WINFRITH_CONCRETE
| mid | ro | tm | pr | ucs | uts | fe | asize |
|-----|------|-----|-----|------|------|------|-------|
| 1 | 2.4000E-8 | 33950.000 | 0.19000 | 36.4 | 1.8600 | 0.090000 | 8.000000 |
| e | ys | eh | belong | rate | conn | conl | cont |
|-----|-----|-----|--------|------|------|------|------|
| 0.000 | 0.000 | 0.000 | 0.000 | 0.000 -4.000000 | 0.000 | 0.000 |
| eps1 | eps2 | eps3 | eps4 | eps5 | eps6 | eps7 | eps8 |
|------|------|------|------|------|------|------|------|
| 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 |
| p1 | p2 | p3 | p4 | p5 | p6 | p7 | p8 |
|-----|-----|-----|-----|-----|-----|-----|-----|
| 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 |
*HOURGLASS
| hgid | ihq | qm | ibq | ql | q2 | qb/vdc | qw |
|------|-----|-----|-----|-----|-----|--------|-----|
| 1 | 4 | 0.000 | 0 | 0.000 | 0.000 | 0.000 | 0.000 |
*PART
*title
FALLING WEIGHT
| pid | secid | mid | eosid | hgid | grav | adpopt | tmid |
|-----|-------|-----|-------|------|------|--------|------|
| 2 | 2 | 2 | 0 | 0 | 0 | 0 | 0 |
*SECTION_SOLID
| secid | elform | aet |
|-------|--------|-----|
| 2 | 1 | 0 |
*MAT_RIGID
| mid | ro | e | pr | n | couple | m | alias |
|-----|------|-----|-----|-----|--------|-----|-------|
| 2 | 7.8000E-8 | 1.6000E+5 | 0.300000 | 0.000 | 0.000 | 0.000 | |
| cmo | conl | conz |
|-----|------|------|
| | | |
*PART
$# title
TOP BARS
$#
pid secid mid eosid hgid grav adpopt tmid
3 3 3 0 0 0 0 0
*SECTION_BEAM
$#
secid elform shrf qr/irid cst scoor nsm
3 1 0.000 0 1 0.000 0.000
$#
tsl ts2 ttl tt2 nsloc ntloc
6.000000 6.000000 0.000 0.000 0.000 0.000
*MAT_PLASTIC_KINEMATIC
$#
mid ro e pr sigy etan beta
4 7.8500E-9 2.1000E+5 0.300000 560.00000 2100.0000 0.000
$#
src srp fs vp
0.000 0.000 0.200000 0.000
*PART
$# title
BOTTOM BARS
$#
pid secid mid eosid hgid grav adpopt tmid
4 4 3 0 0 0 0 0
*SECTION_BEAM
$#
secid elform shrf qr/irid cst scoor nsm
4 1 0.000 0 1 0.000 0.000
$#
tsl ts2 ttl tt2 nsloc ntloc
12.000000 12.000000 0.000 0.000 0.000 0.000
*PART
$# title
STIRRUPS
$#
pid secid mid eosid hgid grav adpopt tmid
5 5 4 0 0 0 0 0
*SECTION_BEAM
$#
secid elform shrf qr/irid cst scoor nsm
5 3 0.000 0 1 0.000 0.000
$#
a ramp1 str
28.260000 0.000 0.000
*MAT_PLASTIC_KINEMATIC
$#
mid ro e pr sigy etan beta
4 7.8500E-9 2.1000E+5 0.300000 250.00000 2100.0000 0.000
$#
src srp fs vp
0.000 0.000 0.200000 0.000
*PART
$# title
plywood interface
$#
pid secid mid eosid hgid grav adpopt tmid
7 2 5 0 1 0 0 0
*MAT_ELASTIC
$ Material for plywood
$#
mid ro e pr da db not used
5 1.5000E-9 1500.0000 0.300000 0.000 0.000 0
*MAT_ADD_EROSION
$#
mid excl mxpres mneps effeps voleps numfip ncs
1 0.000 0.000 0.000 0.000 0.000 0.000 0.000
$#
mpres sigpl sigvm mxeps epssh sigth impulse failtm
0.000 0.000 0.000 0.100000 0.000 0.000 0.000 0.000
*INITIAL_VELOCITY_RIGID_BODY
$#
pid vx vy vz vxr vyr vzr
2 0.000 0.000 -0.730000 0.000000 0.000000 0.000000
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SOME AETIOLOGICAL FACTORS IN DEPRESSIVE ILLNESS
Thesis presented for the degree of Doctor of Medicine in the University of Glasgow by Alistair Munro, M.B., Ch.B., Glas., M.R.C.P. Ed., D.P.M., Eng.
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It is in vain to speak of cures, or think of remedies, until such time as we have considered of the causes.
Galen (c. 130 - 200).
FOREWORD
The main purpose of this study is to determine whether individuals with depressive illness have suffered from parental loss during childhood to a greater extent than normal individuals.
An important part of the investigation is the demonstration that medical and surgical out-patients attending a general hospital are suitable to act as a control series for comparison with the depressive patients. Since the method of study involves interviewing a considerable number of depressive and control individuals, the opportunity has been taken to enquire into a number of other factors, mainly of a socio-demographic nature, which are said to be of importance in the causation of depressive illness.
## Contents
**Introduction**
Review and Discussion of the Relevant Literature
- Phenomenology of depressive illness. 9
- Diagnosis and classification of depressive illness. 10
- Demographic and genetic factors in depressive illness. 16
- 1. Prevalence and incidence. 16
- 2. Social class. 17
- 3. Cultural factors. 18
- 4. Family environment. 20
- 5. Marriage and fertility. 21
- 6. Genetics of depressive illness. 22
- Parental deprivation and mental illness. 23
- Methods of studying the effects of parental deprivation. 29
- Parental deprivation in relation to psychiatric syndromes:
- A. Psychiatric disorder following parental deprivation. 31
- B. The frequency of parental deprivation in psychiatric conditions. 32
- 1. Psychiatric illness. 32
- 2. Psychosis. 32
- 3. Psychoneurosis. 33
- 4. Suicide and attempted suicide. 34
- 5. Schizophrenia. 36
- 6. Depressive illness. 36
- Parental deprivation: its significance to mental illness. 38
- Use of epidemiological methods in the study of psychiatric illness. 39
General Methods 42
| Section | Page |
|----------------------------------------------|------|
| The Investigation | 46 |
| Object and Aim | 47 |
| Definitions | 48 |
| Auspices of the Study | 48 |
| Time schedule | 49 |
| Preliminary formulation of hypotheses | 49 |
| Geographical area in which the study took place | 51 |
| Hospitals in the Edinburgh area | 51 |
| The type of study | 53 |
| Selection of patients | 56 |
| The interview | 59 |
| The questionnaire | 63 |
| Personnel and equipment used in the study | 68 |
| The Pilot Study | 70 |
| Control Series | 73 |
| The outpatient material | 80 |
| Results | 81 |
| Discussion and conclusion | 86 |
| The control series | 87 |
| Comparability of the depressive and control series | 88 |
| Statistical Procedure Employed in the Present Study | 92 |
| The Hypotheses : Results | 96 |
| Miscellaneous Findings | 114 |
| Discussion of the Results | 124 |
| Aspects of parental deprivation | 128 |
| Demographic characteristics of depressive patients | 131 |
| Parental morbidity and mortality | 133 |
| Miscellaneous results | 135 |
| Conclusions | 137 |
| Indications for future research | 138 |
| Summary of Results | 141 |
| Acknowledgements | 145 |
| Appendix | Description |
|----------|-----------------------------------------------------------------------------|
| 1 | The outpatient material. Fig. 1. Tables 1 to 18. |
| 2 | Comparability of depressives and controls. Tables 19 to 24. |
| 3a | The hypotheses: results. Table 25 to 53. |
| 3b | Miscellaneous results. Tables 54 to 61. |
| 4 | The Questionnaire. |
| 5 | The reject series. |
| 6 | The symptom-sign inventory. |
INTRODUCTION
INTRODUCTION
Psychiatric literature contains numerous references to the important role played by the parents, especially mother, in the normal emotional and intellectual development of the individual and many authors testify to the profound effects on the young of disruption of the relationship with the parent. Particularly in psychoanalytic writings, stress is laid on the child's need for a continuing relationship with the mother-figure during the formative years and distortion or interruption of the mother-child relationship is regarded as a factor of great importance in the genesis of mental disorder. Animal experiments in which the young are reared in isolation appear to provide striking confirmation of this viewpoint. Such animals fail to thrive, not only emotionally and intellectually, but also physically, since they do not gain weight at a normal rate and are unduly prone to intercurrent disease.
Actual loss of the parent during childhood has been held to be of particular moment in the subsequent development of various psychiatric conditions; for example, personality disorder, psychoneurosis, schizophrenia and depression. If such a diverse range of pathology can result from one particular disturbance then it would seem that the disturbance must be a fairly non-specific one. This being so, it could be argued that it is scarcely worth studying since an outstanding need in psychiatry is the identification of specific precipitating factors. However, it must be pointed out that much of the work on parental deprivation can be severely criticized on methodological grounds: authors frequently fail to define their terms, the number of subjects studied is often
very small and the views expressed in conclusion are mostly impressionistic. Although the published results often appear striking, lack of a suitable control series, or indeed of any standard of comparison, renders the work of little value in many cases.
There is fairly good evidence for a significant relationship between parental deprivation and the subsequent development of a personality disorder, but the alleged relationship between parental deprivation and other psychiatric illnesses has not been convincingly demonstrated. In particular, this aspect of depressive illness is poorly documented and since the connection between deprivation, mourning and depression is discussed at length in the literature, it seems an important aspect to consider. This project is designed to ascertain whether a group of patients suffering from depressive illness has in fact suffered parental loss in childhood more often than individuals in a control group.
If individuals suffering from a mental illness such as depression can be shown to have an excess of parental loss, it is tempting thereafter to apply post hoc reasoning and to assert that the loss of the parent is a cause of the illness. Such an assumption is frequently made but is not necessarily justifiable. It could equally well be argued that if depressive patients show an excess of parental deprivation it is because the parents of depressives are somehow unusually prone to die while their children are still immature. Following this line of reasoning, it is suggested that the parents of depressives are older than average when the depressives are born and the project undertakes to investigate this.
The term, 'Parental Deprivation', does not represent a single entity
Deprivation may occur in various ways: by death, desertion, absence due to work or war-service and so on. The deprivation may be primarily physical or emotional. It would seem reasonable to suppose that the death of a parent would be of profounder significance than loss of a parent for a reason other than death and it is conventionally accepted that loss of the mother during childhood is of considerably greater importance than loss of the father. Furthermore, it could be held that a deeply disturbed relationship between the child and the parent, not necessarily involving separation, might predispose the child to subsequent depressive illness. Information has been gathered to examine these hypothetical situations.
It has been suggested that the patient's ordinal position in the sibship of his family of upbringing may influence his predisposition to depression and also that the larger the sibship the more apt is he to develop depression since he is less likely to be able to form a deep and stable relationship with his parents and sibs in the period of childhood. Data on these points have been obtained. Enquiry has been made into the civil state and fertility of the depressive patients as compared with the controls since previous references to these factors give contradictory results.
Both depressives and controls have been questioned about the presence of a family history of mental disorder with the intention of confirming previous studies which show an excess frequency of mental illness in the families of manic-depressive patients. As well as this, it was considered that patients suffering from severe depressive illness might show a higher
prevalence of psychiatric disorder in their families as compared with the families of patients with less severe depression. If this were so, it would suggest that depression 'breeds true' in its degree of severity, since the relatives of severely depressed individuals who themselves develop depression are more likely to be recognized as ill if their attacks of depression are severe.
A thorough examination of the literature on depressive illness has produced few satisfactory answers to the various hypotheses mentioned in these preceding paragraphs. This project attempts to examine these hypotheses as impartially as possible, using simple but objective techniques. Patients admitted to psychiatric hospital suffering from a primary depressive illness have been interviewed and their answers to a number of standardized questions recorded. Identical interviews have been conducted with a similar number of non-depressed individuals in order to obtain a standard of comparison. Epidemiological method is used, not to assess the incidence or prevalence of depressive illness in the community, but in this instance to assess the prevalence, and thus indirectly the importance, of certain factors said to be involved in the aetiology of depression. It must again be stressed that, although such a technique may show a significant relationship between an illness and any number of factors, it cannot demonstrate cause and effect.
In summary, this project is designed to examine the following hypotheses:
1. Parental deprivation, by death or by other cause, predisposes towards depressive illness.
2. The age of the parents is of significance in the etiology of depressive illness in that the older the parent at the time of the individual's birth, the more likely is that individual to develop depressive illness.
3. Loss of a parent in childhood by death is more likely to cause a tendency to depressive illness than loss of a parent by a cause other than death.
4. Loss of mother during childhood is more important in predisposing to depressive illness than loss of father.
5. A disturbed parent-child relationship in early life is important in producing a tendency to depressive illness.
6. Persons suffering from depressive illness belong to a larger-than-average sibship in their family of upbringing.
7. The ordinal position of the depressive individual in his sibship is of importance in the etiology of his depressive illness.
8. Depressive patients tend to remain unmarried more than do normal individuals and the fertility of married depressives differs from that of married non-depressives.
9. Depressive patients more often present with a positive family history of severe mental illness than do normal individuals.
10. Individuals with severe depression more often have a positive family history of severe mental illness than do individuals with less-severe depression.
REVIEW AND DISCUSSION OF THE RELEVANT LITERATURE
This review and discussion of the literature on depressive illness makes no attempt to cover the whole field of depression but concentrates instead on those aspects of the subject which are of relevance to the present study. The review falls into five main sections in the first of which depression is defined for the purpose of this study and a brief outline of the phenomenology of the conditions is given.
In the second section, difficulties which arise in the diagnosis of depressive illness are discussed and methods of minimising such difficulties when they occur in the course of an investigation are suggested. Also in this section, problems of classification and nomenclature are dealt with.
The third section reviews the literature on demographic and genetic factors in depression, while the fourth considers the available evidence on parental deprivation and its relation to mental illness, especially depression. In both these sections stress is necessarily laid on the inconclusiveness of the findings at the present time. As will be shown, poor experimental technique is frequently a basic cause of this lack of reliable data.
The review and discussion of the literature ends with a brief appraisal of the application of epidemiology to psychiatric research.
PHENOMENOLOGY OF DEPRESSIVE ILLNESS
Depressive illness, for the purpose of this study, is defined as a primary disturbance of affect in the direction of sadness. This definition is adapted from that of Meyer-Gross et al (1960).
Recognition of a typical case of depressive illness is not usually difficult and most authorities agree on the cardinal features of the condition. The essential element is the presence of pathological sadness although in some cases this feature is so altered or overlaid as to make its recognition difficult. Other symptoms which may be present in varying degree are guilt feelings, insomnia, retardation, apathy and somatic complaints: in more severe cases, suicidal tendencies and delusions may occur. At times, agitation and anxiety are prominent and may even be the presenting symptoms. Foulds (1960) has criticized the traditional use of delayed insomnia and diurnal variation of symptoms as criteria in the diagnosis of depression and he finds that these symptoms are related to advancing age rather than to the illness itself.
Depression belongs to the group of affective illnesses and an outstanding characteristic of these disorders is their tendency to run a periodic course. However, by no means all the patients who develop affective illness suffer from a recurrence and Stenstedt (1952) reports that 5% of his manic-depressive subjects have had only one attack in their lives. As a rule there is complete recovery from the individual attack with no impairment of mental function. Alternation of depression and mania may occur, or recurrence of depression or mania alone, but manic episodes are much rarer than attacks of depression and among
Stenstedt's (1952) subjects the first illness was depressive in 83% of cases. Affective illness is commoner in women than in men, according to hospital admission statistics (Roth, 1959) and results obtained from a community survey, (Stenstedt, 1952). Roth (1959) states that the highest frequency of first admissions to hospital for depression is in late middle age but its first onset in the individual may occur from adolescence onward.
**DIAGNOSIS AND CLASSIFICATION OF DEPRESSIVE ILLNESS**
If the existence of a nosological entity known as depressive illness is accepted, the above statements are fairly unexceptionable and widely agreed upon, but many practical difficulties may arise in the diagnosis of the individual case of depression. This study is concerned to examine the primary depressive syndrome and not the depressive symptom which may occur as a secondary feature in any psychiatric condition. An attempt has been made to gather a group of cases corresponding as closely as possible to the classical descriptions of depressive illness to ensure that the findings of the study will not be contaminated by extraneous psychopathology. The difficulties which stand in the way of accurate diagnosis are considered in some detail in the following paragraphs to demonstrate that, not only must these difficulties be recognized, but also precautions must be taken to overcome them.
The diagnosis of depression is essentially a clinical one which rests on the empirical recognition of depressed affect. Unfortunately, there has been no very striking advance in diagnostic technique since Mapother and Lewis's (1937) statement that, 'Analysis feature by feature is less informative than a single glance'. There is no physical or psychological test
which can provide unequivocal proof of the presence of affective disorder and ultimately the diagnosis is made on the basis of clinical examination. Nowadays, rating scales of signs and symptoms are popular in the study of depression because they allow the investigator to quantify his clinical impressions to some extent but rating scales can only be applied once the initial diagnosis has been made. (Knowles, 1963; Hamilton, 1960). The accuracy of diagnosis may be increased if the patient is examined by more than one psychiatrist and the results of examination combined. This multiple-examination procedure is considered valuable even where the comparative objectivity of a rating-scale is employed, (Hamilton, 1960).
Depression may present in many ways. Apart from variation in severity and chronicity, the appearance of a case may differ greatly according to the stage in its natural history at which the illness is observed. In some instances the appearances are more suggestive of psychoneurosis or a psychosis other than depression. At times the diagnosis of depression is only confirmed by a natural history typical of affective disorder, by the presence of a family history of affective disorder, or by an appropriate response to antidepressant treatment. On the other hand, an apparently unmistakeable depression may prove in the course of time to be a totally different condition and in this context, Degard (1961) notes that of 263 patients diagnosed as suffering from affective psychosis, 54 were found at follow-up to be schizophrenic. Similarly, Clark and Mallet (1963) describe a follow-up study of 92 patients diagnosed as depressive at the time of discharge from hospital: 20% of these patients required re-admission.
within three years of discharge and a third of these readmissions had to be re-diagnosed as schizophrenic. It seems likely that any study on depression must inevitably contain a small proportion of other illnesses masquerading as affective disorder unless prolonged contact with the patient can be maintained to ensure near-complete accuracy in diagnosis. In the short-term study, only rigid exclusion of doubtful cases can minimize the difficulty although this in itself is open to the criticism that it may cause undue bias in the selection of patients.
The depressive patient may make no complaint of a psychological nature and his presenting symptoms may be purely physical. This is especially true of the more severe type of depressive illness (Jones and Hall, 1965). Brown (1936) has made the suggestion that physical complaints may have a basis in the lax musculature, low blood-pressure, constipation and insomnia typical of the severe depressive. Bradley (1963) notes two types of localized pain associated with depression: chronic pain followed by depression and pain which appears simultaneously with depression. Antidepressant treatment dispels the depression but not the pain in the first group, while in the second group both pain and depression disappear with treatment. The pain in the latter group may be regarded as an 'affective equivalent' and at times it may completely obscure the depressive aspect of the illness. da Fonseca (1963) notes that affective equivalents have the same tendency to periodic recurrence as depression and often may occur as a familial phenomenon. It is of course necessary to perform a physical examination in every case of depression to assess the relative importance of physical and psychological symptoms: in a study such as
the present one it is especially important to exclude depression which is purely reactive to physical ill-being and conversely to include those cases in which physical symptoms are unquestionably an expression of the depressive illness. From this point of view it is an advantage to study cases of depression admitted to hospital since each patient has had a thorough examination at the time of admission.
Many studies of psychiatric illness are carried out in hospital and this is not wholly advantageous. It has to be recognized that patients admitted to hospital with depression are a highly-selected group. The less severe forms of depression may go unrecognized or even uncomplained-of and if they are recognized they may well be treated by the general practitioner or the outpatient psychiatrist. Nowadays, admission to hospital because of depression often occurs only when the patient has failed to respond to outpatient treatment. It is probably justifiable to assume that hospitalized depressives represent the most severe end of the depression-spectrum but there does not appear to be any study which confirms this impression. Stengel (1960) has pointed out that investigations on depression carried out in the psychiatric hospital may produce quite different results from those performed in the outpatient clinic or private consulting-room, mainly because the patient-populations seen in these different surroundings are themselves so different. It is therefore necessary to be explicit about the source of patients under investigation and care must be taken not to generalize rashly from data obtained in rather specialized circumstances.
There is still much controversy over the desirability of attempting to distinguish between endogenous and neurotic types of depression. Some
investigators, for example Lewis (1934), Curran (1937), Curran and Mallinson (1941) and Germany (1958), find no evidence to justify such a distinction but recently the opposite view has been taken by Astrup (1959), Poulds (1960) and Kiloh and Garside (1963). In practice it does seem convenient to accept some form of subdivision, even if it simply implies a difference in degree of severity rather than any connotation of aetiology. The distinction between endogenous and reactive depression, that is, depression which arises spontaneously and that which apparently develops as a reaction to some harmful stimulus, has lost favour recently. It can be shown that many so-called endogenous depressive illnesses appear to have clearly-defined precipitating factors and a study which demonstrates this is that of Morozova and Shumskii (1963). These workers report that 36.9% of the depressive phases in patients with recurrent endogenous depressive illness are related to an extrinsic provoking factor, although the frequency of the association diminishes from first to subsequent phases, a finding which the authors explain by postulating an endogenous predisposition which develops with age and advance of the illness. The concept of reactive depression does not seem, in the light of these findings, to be of any classificatory value.
A number of authors, including Roth (1959), state that in neurotic depression the premorbid personality is often unstable and the response to electroconvulsive therapy is poor, with a high relapse rate. This is not a description of a primary depressive illness but most probably that of a depression secondary to psychoneurosis or personality disorder, and the prognosis, by and large, is that of the underlying condition. A
semantic misapprehension has crept in here. The term 'Neurotic Depression' should imply a depressive illness of neurotic intensity or quality arising from a psychoneurosis. In this study neurotic depression is regarded as a primary condition similar to, but less severe than, endogenous depression.
Following present-day custom (Stengel, 1960) the cases of severe primary depression in this study are included in the manic-depressive category (301.1 in the International Classification of Diseases) and those few cases of depression with a history of manic episodes (304.0) are regarded as falling into the same diagnostic group. Primary neurotic depression corresponds to category 314 of the International Classification but again it is stressed that the distinction between it and the endogenous form seems largely an artificial one.
The concept of involutional depression no longer finds favour and there seems no call for its continuing use. Until quite recently it was regarded by many as a diagnostic entity (Titley, 1936; Malsberg, 1948; Dewabory, 1954) and Kallmann (1953, 1959) put forward evidence for a specific hereditary pattern and pre-psychotic personality-types. Recent work by Slater (1953), Tait et al. (1957) and Stenstedt (1959) provides convincing proof that the condition is endogenous depression of late onset and these workers cannot confirm Kallmann's finding of a genetic relationship with schizophrenics. Stenstedt, for example, finds that 10% of his patients have a parent with endogenous affective illness.
Depression in the elderly is a different matter. This is now recognized to be relatively common (Roth, 1955; Kay, 1959; Roth and Kay, 1962)
and also to be to some extent aetiologically distinct from depression occurring in earlier life. (Roth and Kay, 1956). There is less genetic predisposition in depression of old age and immediate precipitating factors are of relatively greater importance: particularly factors like physical illness, acute physical stress and sensory deprivation. Depression in the elderly is a subject which requires quite separate study and in this project patients over the age of sixty are excluded.
Finally, mention must be made of puerperal depression. Psychotic illness is rare in the gestational period but there seems little doubt that a significant excess of psychosis, especially manic-depressive, occurs in the puerperium. (Pugh et al., 1963). However, it is not known whether puerperal depression is qualitatively different from depression occurring at other times. Hemphill (1952) states that puerperal depression responds poorly to treatment, whereas Meyer-Gross et al. (1960) regard the response to electroconvulsive therapy as good in many cases. A small number of cases of puerperal depression is included in this study: they have been classified as endogenous depression as they are clinically indistinguishable from that condition.
**DEMOGRAPHIC AND GENETIC FACTORS IN DEPRESSIVE ILLNESS**
**Prevalence and Incidence of Depressive Illness**
The number of published studies on the epidemiology of depression is small and the lack of uniformity in methods of study makes comparison difficult. Until quite recently, estimates of prevalence and incidence were usually based on hospital admission statistics with their many
attendant fallacies (Lin and Standley, 1962). Ødegård (1961) provides figures based on such data but he expresses confidence that these are a fair reflection of the community situation in Norway. He states that the life-time risk of developing affective psychosis in Norway is about 1.2% for men and 1.6% for women. Sørensen and Stromgren (1961) report that, on the Danish island of Samso at a given date, 3.0% of the population over 20 years of age suffer from a depressive illness, but 77% of the cases are regarded as neurotic. (A prevalence rate for psychotic depression of approximately 0.3% of the population over 20 years of age). Stenstedt (1952) estimates that the prevalence of manic-depressive psychosis in Sweden is about 1%, a figure he notes as comparable with that found for Denmark. Juel-Nielsen et al. (1961) calculate an incidence rate for depressive states in the County of Aarhus, Denmark, of 0.2 to 0.5% per annum, with a high female preponderance, but only about a quarter of these come to psychiatric attention. The Taiwan (Formosa) study of Lin (1953) provides the much lower prevalence figure of 0.7 per 1,000 for cases of depression.
It may be said that, despite the figures quoted above, the true incidence, prevalence and lifetime morbidity-risk figures for depressive illness are not known, partly because of inability to agree on what constitutes a case of depressive illness and partly because of overwhelming difficulties in comprehensive case-finding.
Social Class Factors in Depressive Illness
There is fairly widespread agreement that schizophrenia tends to be commoner at the lower end of the socio-economic scale but the findings
on depression are somewhat contradictory. Faris and Dunham (1960) report no evidence of a differential distribution of manic-depressive psychoses in the City of Chicago in contrast to schizophrenia which is found much more frequently in areas of marked social disorganization. Clark (1949) and Hare (1955) find that the social class distribution for depressives is similar to that for the general population but Malzberg (1956 a & b) notes a higher prevalence of manic-depressive psychosis in the upper social classes while Tietze et al. (1941) and the Report of the Registrar General (1953, 1955) agree on a slight increase of manic-depressive illness towards the lower end of the social scale. These anomalies can probably be explained by social class bias in the types of population studied, and most authors seem agreed that if the social class distribution differs from that of the general population, the divergence is not a large one.
Cultural Factors Influencing the Prevalence of Depressive Illness
There are many allusions to allegedly wide variations in the prevalence of depression in different cultures and these are used to support arguments for the environmental aetiology of depression. Few of them stand up to scrutiny, but two which seem more than usually authoritative are the works of Eaton and Weil (1955) and Böök (1953). The first is a study of mental illness in the Hutterite communities of the American prairies and the authors report a high prevalence of manic-depressive psychosis with a low prevalence of schizophrenia. They attribute this finding to the Hutterite way of life which is characterised by living in small and extremely cohesive groups in a state of religious communism and where aggression must be turned inward rather than be outwardly expressed.
Böök's study of an isolated North Swedish community produces quite different results. This investigator finds an excess of schizophrenia among the mentally ill and very little manic-depressive disease and he postulates that many generations of isolated existence have selectively favoured the survival and procreation of the schizoid personality.
The findings of these two studies are still regarded as controversial and are against the trend of present-day thought. It is possible that differences in the methods of case-identification may have influenced the results. It is also possible that a genetic factor is at work here, since the Mennonites for example are descended from a very small group of families and in-breeding is common. (Lin and Standley, 1962).
Most present-day workers stress that the prevalence of depressive illness, so far as it can be determined, seems remarkably constant in different populations and cultures. Ødegård (1961) finds little difference in the prevalence of depression in different parts of Norway and Helgason (1961) reports that there is no marked variance between the morbid risk and lifetime prevalence of depression in Denmark and Iceland. Leighton and Dembro (1963) stress that depression, which previous workers had regarded as rare in the African negro, is not only common among the Yoruba of Nigeria but is possibly slightly commoner than in Stirling County, Canada. These authors make the important point that, although cultural differences do not appear to affect the basic prevalence rate of depression, they may alter some of the presenting features of the illness and this may be one reason for some of the divergent results obtained in intercultural studies.
While Norton (1952) reports that individuals with psychoneurosis or personality disorder have a tendency to appear in the higher birth ranks and Goodman (1957) finds that schizophrenics tend to be the youngest in the family, Malzberg (1938) can discern no significant relationship between birth-order and manic-depressive illness. Fromm-Reichmann (1949), whose impressions do not appear to be supported by statistics, states that manic-depressives often come from large sibships or otherwise large families in which several father-figures share responsibility. In her opinion, such a situation prevents the child from relating meaningfully to a specific father-figure and this may predispose him to depression in later life. Munroe (1959), paraphrasing Adler, states that the youngest child is typically spoiled by everyone, but he is also the weakest of the family and he is in a position of general competition. Although no specific tendency to psychiatric illness is hypothesised here, the implication is that the youngest child has more difficulty than the rest in attaining psychological maturity.
Enquiry has failed to reveal any reference dealing with the age of the parents at the time of the depressive individual's birth, although Norton has noted that the proportion of patients with psychoneurosis or personality disorder born to mothers at higher maternal ages is greater than in a group of controls and Goodman (1957) similarly notes that in schizophrenia there is a slightly raised maternal age with a deficit of mothers under 25 and an excess aged 35 years or more at the time of the patient's birth.
Barry and Barry (1961) make the interesting but unexplained observation, which is based on figures obtained from very large numbers of patients, that the births of individuals who subsequently develop manic-depressive illness or schizophrenic tend to occur to significant excess in the first three months of the year.
Marriage and Fertility in Depressive Illness
There is disagreement about both these factors. Ødegård (1946, 1953) finds that the marriage rate in depressives is similar to that in normals but Stenstedt (1952), Kraines (1957) and Kallmann (1953) all report a lower marriage-rate in manic-depressive psychosis. Essen-Möller (1935) finds that the marriage-rate is about normal until the time of the first onset of depression and thereafter it falls to approximately half the original level. The group of patients with involutional depression described by Stenstedt (1959) shows a high rate of both celibacy and divorce but Ødegård (1946) notes no increase of affective illness among the divorced.
As regards the fertility of depressives, Ødegård (1961) states that manic-depressives are only slightly sub-fertile till the time of their first admission to hospital with the illness whereas Kallmann (1953) finds that their fertility is significantly reduced. Stenstedt (1952) and Essen-Möller (1935) both report normal fertility in their married depressive subjects and Lewis (1958) remarks that manic-depressives are normally fertile. Hopkinson (1963a) finds that fertility is significantly higher in those women whose illness begins after the age of 50, as compared with women who develop depression prior to that age. The age of onset of the illness does not seem to be of importance in the fertility of male
The Genetics of Depressive Illness
It is fairly generally accepted that a strong heredo-familial tendency to manic-depressive illness exists, but the exact nature of this tendency is not clear. Rüdin (1930) reports that one-third of the children of a manic-depressive parent themselves develop the illness and the rate increases to 62.5% where both parents are manic-depressive. On the other hand, three-quarters of his manic-depressive subjects are born of apparently normal parents and there is no excess of parental consanguinity in his series. Slater (1936) postulates a dominant inheritance of the condition but suggests that inhibition of manifestation can occur. Stenstedt (1952) agrees with this view and adds that manifestation appears to occur more readily in the female than in the male. Hopkinson (1964) notes a lower frequency of genetic loading in depressive illness whose onset occurs after the age of 50 years, as compared with depression of earlier onset. Roth and Kay (1956) similarly find that in patients who develop depressive illness for the first time over the age of 60 years, physical illness and sensory deprivation are more important precipitating causes than an hereditary predisposition.
The closer the degree of consanguinity to a depressive patient, the greater appears to be the risk of being affected by the illness. (Rüdin, 1930; Slater, 1936). Twin studies appear to show the overwhelming importance of hereditary factors in manic-depressive illness (Kallmann, 1959), but recently there has been growing criticism of twin studies. Jackson (1960) points out that not only are twins in any case a highly-selected group, but there is frequently doubt in the diagnosis of zygosity.
and preferential reporting of concordant identical twins. The genetic evidence supplied by twin studies must therefore be accepted with some caution.
Slater (1936) finds no relationship between manic-depressive illness and mental deficiency but he reports an excess of schizophrenics occurring in the families of his depressive subjects. Other investigators (Hanson, 1938; Stenstedt, 1952; Hopkinson, 1964) have failed to confirm any genetic affinity between depression and schizophrenia and Kallmann's (1959) finding of an excess of schizophrenics among the relatives of individuals with involutional depression has been discounted by Stenstedt (1959).
It therefore seems likely that there is a specific hereditary element in manic-depressive illness but it is not possible to define it accurately at present. It is probable that expression can be evoked or inhibited by certain factors and that the earlier the onset of the illness, the stronger is the genetic influence. Stenstedt (1952) points out that an outstanding difficulty in the way of obtaining an accurate estimate of hereditary factors is the ease with which many mild cases of manic-depressive illness may be overlooked.
PARENTAL DEPRIVATION AND MENTAL ILLNESS
Since in most cultures it is normal practice for children to be reared by their parents there is a general consensus of opinion that it is undesirable to be deprived of one's parents in childhood. This can scarcely be disputed but to assert, as many authorities do, that a variety of severe psychological abnormalities may arise in the child as a result
of such deprivation is to make an assumption of some magnitude. It requires an even larger assumption to postulate a causal connection between some long-past occurrence such as childhood bereavement and a psychiatric illness which develops in adult life. It will be contended here that, save in a small number of instances, the evidence for such a connection is meagre.
Goldfarb (1955) suggests that the family of upbringing performs five main functions for the young child and these are:
1) it provides parents who bestow emotional warmth and love on the child and who minister to its needs;
2) it provides for continuous contact, especially between the child and the mother-figure;
3) the mother is a source of safety and gratification and a means of reducing tension and discomfort by feeding, cleaning, fondling and so on;
4) the parents provide constant stimulation which is necessary for normal intellectual and emotional development;
5) the relationship with the parents allows the child to reciprocate and by a process of reality-testing to master its environment.
This list, which is by no means exhaustive, demonstrates some of the complexity of the mother-child relationship and it would not be surprising to learn that its severance while the child is still immature and very dependent could cause serious effects, both immediate and distant. But does it invariably, or even frequently, do so? Bowlby (1962), whose name keeps recurring in this field of study, describes the short-term effects on the young child of admission to a residential nursery or hospital. He defines three distinct stages in the child's reaction to the loss of its
mother: the stage of Protest, then after a variable time that of Despair and finally, after a very variable period, the stage of Detachment when he appears to forget his mother. The last stage becomes permanent if separation is prolonged enough. This course of events is probably a protective mechanism; in the first place to draw attention to neglect and later to allow the child to form a relationship with a substitute mother-figure. Bowlby regards the process as a form of mourning for the lost love-object and this may partly be true. However, as will subsequently be discussed, the situation is a far more complex one than he makes out.
In his early papers on the subject of parental deprivation, Bowlby (1940, 1944) reports the finding of severe intellectual and emotional disturbances in deprived, institutionalized children and he claims (1944) that these children are predisposed to severe psychiatric disorder in adult life, especially psychopathic and affectionless personality disorders. These claims are supported by a number of roughly contemporaneous studies, for example those of Ribble (1943) and Spitz (1945), but disagreement with such findings is growing stronger. Bender (1945) agrees that institutionally-reared children are psychologically disturbed but she finds no evidence of the concomitant physical ill-being that the earlier workers frequently note. Rheingold (1956) and Rheingold and Bayley (1959) are unable to confirm the presence even of psychological disturbance. Bowlby (1962), although he has not changed his theoretical standpoint on the effects of childhood bereavement, now says that it is probably only a small proportion of deprived children who go on to develop personality disorder. Some authorities, for example Lin and Standley (1962), would
insist that there is little evidence even for such a qualified statement.
The effects of institutionalization are complex, but the earlier workers chose mother-child separation as the cardinal feature because it fitted well with current psychiatric theory. Although the descriptions give hints of other influences these are usually not pursued because mourning for mother is the main preoccupation. For example, when Spitz (1945) describes children being reared in conditions of 'impeccable hygiene with precautions against contagion', he actually implies that these children spend their early months in an environment of almost complete isolation, visited by members of staff as infrequently as possible and handled briefly only when being fed or changed.
Experiments in which animals are raised in isolation are often quoted in support of theories of maternal deprivation. These animals show impaired physical and mental development but there is good evidence, for example that contained in the work of McLelland (1956) and Levine (1958), that it is not maternal deprivation but sensory deprivation that is the important factor here. These and other investigators have demonstrated that animals reared apart from their mothers but frequently handled and petted in early life show more liveliness and curiosity, grow more rapidly and to a greater size, and are more resistant to disease and stress than animals deprived of such stimulation. Similar conclusions have been reached by workers studying the human infant. Montagu (1953) and Fischer (1958) believe that sufficient tactile stimulation of the very young infant is more important for its normal development than the presence of a specific mother-figure. Some confirmation of this is lent by the work of Rabin (1957) who finds that Kibbutz-reared
children in Israel are no less emotionally stable and mature than children raised in private homes.
Casler (1961) who is a severe critic of the traditional views on maternal deprivation does not believe that the infant is capable of distinguishing a specific mother-figure before the age of six months and he claims that, provided the child is well-cared for and adequately stimulated before that age, it will develop normally even in institutional surroundings. Maternal deprivation after this age may be perceived by the child but provided an adequate mother-substitute (or substitutes) is available, the great majority of children will only be transiently affected by their bereavement. Casler holds that the original observations made by Bowlby and others stem largely from bad institutional conditions and not from the effects of maternal deprivation per se. He also points out that institutionalized children are a highly selected group: it is possible that a considerable proportion have been admitted to institutions for reasons which imply an adverse heredity or home-environment which may have affected them even before admission.
The literature on parental deprivation contains a great deal of impressionistic material and there is often great unwillingness to define terms. For example, it is often assumed that parental deprivation is synonymous with maternal deprivation and loss of father is rarely taken into account, although Batchelor and Napier (1953) suggest that it may be a factor of importance in attempted suicide. As can be seen, views on parental deprivation have been coloured by the work done on institutionalized children but parental deprivation must occur frequently in children
who never leave their own homes. Indeed, the question arises; what is parental deprivation? When the phrase comes to be examined, it is realized that it covers a whole constellation of factors.
Parental deprivation is often used to denote loss of the parent by death, but may also include physical absence of the parent due to other causes. It sometimes implies irrevocable loss but may be used to cover all types of parent-loss, permanent or temporary. At times it is even used to describe the emotional deprivation which occurs in a disturbed parent-child relationship with or without actual physical separation. Since many workers do not disclose what meaning they attach to the term 'parental deprivation' comparison between their results is unrewarding. The complications do not end there, because the age of the child when deprivation occurs and the duration of the deprivation-experience if it is temporary must be taken into account. Then again, it is customary to assume that parental deprivation is a serious occurrence but this is not necessarily so. The child whose father dies a hero's death or the child deserted by a brutal parent may not take a tragic view of the circumstance. The adequacy of the parent-substitute in continuing the rearing of the child may well be of paramount importance for the child's future mental well-being, but this figure is frequently ignored. Any study which professes accuracy on the subject of parental deprivation must state its position on all these variables.
A great difficulty in the assessment of the effects of childhood bereavement arises from our ignorance of what constitutes a normal level of parental deprivation in the general population. Few studies are extant which give information on this, but those which do, offer surprisingly high results. The Statistical Bulletin of the Metropolitan Life Insurance
Company (1944) states that 16.7% of the population of the United States have lost one or both parents before the age of 18 years. Norton (1952) calculates that 14.2% of a group of non-psychiatric hospital in-patients have lost one or both parents before the age of 16 years. Hilgard, Newman and Fisk (1960) report that in a general population census of individuals aged 19 to 49 years, 21% are found to have lost one or both parents before the age of 19 years. Stein and Sklaroff (1957), studying an apparently stable population in an Edinburgh suburb, make the surprising discovery that families with schoolchildren have an over-all rate of 16% broken homes.
Gregory (1958) has pointed out that rates of parental deprivation may vary considerably in time and place whereas there is no very convincing evidence that rates for mental disorders fluctuate in sympathy, even allowing for a prolonged time-gap. He remarks that parental deprivation rates are closely related to prevailing rates of mortality, separation and divorce. Almost invariably, children lose their fathers more often than their mothers because men are usually older than their wives, males have a higher death-rate than females of the same age and it is father rather than mother who disappears in circumstances such as war or divorce. Factors such as these may introduce considerable bias into a study if they are not allowed for.
Methods of Studying the Effects of Parental Deprivation
Potentially the most valuable method of studying parental deprivation is a prospective one in which a cohort of individuals deprived of parents in childhood is observed and any characteristic psychological reaction occurring thereafter is noted. Ideally, such a study should continue for
the entire life-time of the subjects but of course this method is enormously laborious and expensive and it is rarely used. In the field of social psychology, the outstanding example of the cohort study is the Terman Gifted Group Study which began in 1921 and is still continuing. In the field of parental deprivation, Bowlby is engaged on a lifelong project using the cohort method but unfortunately his description (1962) of experimental methods suggests that the design of the project leaves something to be desired.
Valuable clues about the importance of parental deprivation may be obtained in retrospective studies. These may concentrate on the frequency of mental illness occurring in individuals who have lost parents in childhood or may enquire into the frequency of childhood parent-loss in the mentally ill. The latter is the more popular method since it is easier to gather a group of individuals suffering from mental illness than one of individuals who have suffered parental loss. Examples of both methods are mentioned in the ensuing paragraphs.
Parental Deprivation and its Relation to Psychiatric Syndromes
It has been suggested at various times that parental deprivation is a causal, or at least a concomitant, factor in the aetiology of a number of psychiatric conditions. It is difficult to accept its specificity as a predisposing element when it is apparently found to excess in a number of disparate illnesses and it is therefore important to justify parental deprivation as a worthwhile area of investigation. To do this it is necessary to discuss the literature on parental deprivation as it pertains to various psychiatric disorders in order to demonstrate how flimsy is the evidence linking it to at least some of these disorders. In this way it
may be possible to show that it is rather less non-specific in its effects than at present seems likely. The occurrence of psychiatric disorder in the parentally deprived is considered first and then a number of psychiatric conditions are examined in turn for evidence of an excess of parental deprivation.
A. Psychiatric Disorder following Parental Deprivation
Studies which begin with the gathering of a group of parentally deprived individuals are sparse because of the difficulties of identifying such a group. Earle and Earle (1961) report on 100 adults in whom separation from the mother had occurred for a period of longer than six months in the first six years of life. The subjects are compared with controls matched for age and sex. It is found that early maternal deprivation is significantly related to the diagnosis of sociopathic personality and perhaps dissociative hysteria, to broken marriage, poor work-record and having been in prison or reformatory, but not to any other psychiatric diagnosis. Imboden et al. (1963) have studied the effects of childhood separation experiences in ostensibly healthy adults. Some 25% of their 500 subjects admit to separation as defined in the study and these individuals show a significantly higher score in the Cornell Medical Index and Morale-Loss Scale than do the non-separation individuals. However, the two groups show no difference in frequency of visiting a dispensary with symptomatic complaints and this leads the authors to suggest that the separation experience influences their reporting characteristics (as measured by the Cornell Medical Index) rather than predisposing them to illness.
B. The Frequency of Parental Deprivation in Psychiatric Conditions
1) Psychiatric Illness
In this category is discussed the small number of studies in which the authors give no details of diagnosis except that the patients are psychiatrically ill. Oswald (1953) reports that in young Serviceman presenting for medical examination the frequency of parental deprivation is significantly higher in psychiatric as opposed to neurological cases. Hilgard et al. (1960), in an examination of 3,579 patients in a state mental hospital, find that 27% of the patients had lost a parent before the age of 19 years, as compared with 21% of a group of normal controls; unfortunately they do not disclose if this difference is a significant one. Pollock (1962) gives details of parental deprivation in a group of patients seen by him in private practice, using the 1959 Statistical Abstract of the U.S. for comparison. The patients are admitted to be predominantly from the higher socio-economic strata. He finds that an excess of his female subjects have lost father in childhood as compared with the males, but a slight excess of males have lost mother. In the females, the highest incidence of parental death occurs in adolescence but in the males the peak is in the first two years of life.
As can be seen, the results in this type of study can only be vague at best. For all one knows, one particular psychiatric condition may be contaminating the results for all the others. The need for investigations involving much narrower categories of illness is obvious.
2) Psychotic Illness
Barry (1939) finds that maternal deaths occur three times more
commonly in the childhood of psychotic patients as compared with normal controls whereas paternal deaths show no such excess.
3) Psychoneurotic Illness
Stengel (1943) reports that all but two of eleven cases of psychogenic fugue have a history of gross disturbance of home life as a child. A positive correlation is found by Madow and Hardy (1947) between war neurosis and broken home while in a comparison of a group of psychoneurotic students with a matched group of non-neurotic students Ingham (1949) notes a relationship between psychoneurosis and intrafamily conflict. Other important concomitants he finds are mental illness in the parents and parental separation.
These studies emphasize the role of the disturbed home rather than of actual parental loss in the etiology of psychoneurosis and this may be an important point. However, an excess of childhood bereavement in neurotics is stressed by Barry and Lindemann (1960). These investigators claim that their results show a significant relationship between psychoneurosis and death of the mother before the subject's fifth birthday, especially for females. Unhappily they base their results on a group of 247 patients with 'psychoneurosis or psychosomatic disorder' and do not differentiate between these categories.
The work on psychosis and neurosis as exemplified by the above studies again stresses the need for greater specificity in diagnosis and the rashness of claiming results which the study is incapable of providing.
4.) Suicide and Attempted Suicide
It is customary to discuss these two topics together although it is probable that those who commit suicide and those who attempt it are two fairly distinct populations. From the available evidence it seems likely that they have at least one common link: both groups suffer to an excessive degree from the effects of parental deprivation and broken home. Two studies which suggest that parental loss is an important factor in the background of consummated suicide are those of Palmer (1941) and Reitman (1942). Although neither uses a control series, the findings certainly indicate an excess of deprivation. In Palmer's study, 11 out of 25 suicides had lost a parent before the age of 14 years and Reitman reports that 15 of his 25 subjects had lost a parent by death, desertion or other cause by the age of 15 years.
Similar findings are reported for attempted suicide. Simon (1950) notes that 29 out of 50 ex-servicemen hospitalized because of suicidal attempts have a broken-home background and Batchelor and Napier (1953) find that 116 of the 200 attempted suicides in their series come from broken homes while all of their teenage subjects are products of such a background. Harrington and Cross (1959) state that 22 out of 102 attempted suicides admitted to a general hospital have had an emotionally disturbed childhood; 11 of the 102 were separated from one or both parents by the age of 14 years. (The findings in this last study do not suggest any marked excess of deprivation but no controls are provided for direct comparison). Robins et al. (1957) arrange their attempted suicides into categories according to diagnosis and they note a differential frequency of broken-home background in the various psychiatric disorders: lowest
in depressives (4.7%) and highest in sociopaths (8%).
Suicide and attempted suicide can be associated with virtually any form of psychiatric illness and in Western societies at least, the majority of suicidal attempts are probably made in the presence of some form of psychological disturbance even if the disturbance is a very transient one. Since this association with psychiatric illness exists, it would be interesting to know if the degree of parental deprivation increases in suicidal as compared with non-suicidal individuals suffering from an otherwise similar psychiatric disorder. An investigation on these lines has been carried out by Walton (1958) who compares non-suicidal depressive patients with depressive patients who had threatened or attempted suicide and he demonstrates a significant excess of childhood bereavement in the suicidal group. Bruhn (1962) similarly finds an excess of broken-home background in psychiatric patients who have attempted suicide as compared with matched psychiatric patients who have not made such an attempt.
This finding of an excess of parental deprivation in the suicidal as compared with the non-suicidal patient is of practical importance. It is more than likely that a number of studies on parental deprivation have contained a proportion of patients who have attempted suicide. Their presence will almost certainly inflate the prevalence-figure for childhood bereavement and the credit for this may well go to the particular illness being studied rather than to the suicide-factor.
5) Schizophrenia
Wahl (1956) uses highly subjective criteria in reporting that 50.3% of 568 male schizophrenics come from homes where there is severe rejection and/or overprotection by one or both parents, but he also finds that 41% of the patients have lost a parent by death, divorce or separation before the age of 15 years, as compared with 11.4% of controls. Lidz and Lidz (1949) note a similar degree of parental loss in a group of 50 schizophrenics whose illness began before their 21st birthday. 20 of these subjects lost a parent by death or separation before the 19th birthday, about half of the parents being lost on account of serious emotional illness. Oltman et al. (1951) remark on a tendency to increased parental deprivation in schizophrenics whose illness begins before the age of 20 years but not in those with a later onset. Hilgard and Newman (1963) report a significant excess of maternal, but not paternal, deprivation in childhood in a group of schizophrenics aged 20 to 30 years at the time of admission to hospital.
An impression which is gained from these authors' findings is that the family background of many schizophrenics is disorganized to an abnormal degree and that parental deprivation may often be a result of this. In addition, such deprivation may influence the severity of the illness, causing a tendency to earlier onset.
6) Depressive Illness
Oltman and McGarry (1951) find little difference in the degree of parental deprivation in individuals with manic-depressive psychosis and in normals but Stenstedt (1952) states that dissolution of the home
before the age of 15 years or serious parental conflict may increase the risk that sibs of manic-depressive patients will themselves develop the illness. In his study of involutional depression Stenstedt (1959) further finds that 33% of his depressive subjects come from an unfavourable childhood environment.
Brown (1964) has conducted a study of 216 outpatients suffering from depression who are compared with controls attending local general practitioners. He also uses the 1921 Census results for comparison purposes, this particular census being employed on the grounds that it gives some indication of the amount of childhood parental loss among individuals who are now approximately of the same age-group as his depressive subjects. The findings include a significantly higher loss of parents in childhood among the depressives and especially loss of father, except for the period 0 to 4 years when father loss is no greater than that of the control group. The author reports that 12% of the depressive patients do not know if one or other parent is dead, which he regards as a possible reflection of family disruption in depression but which may equally raise doubts as to his diagnostic methods since the literature generally emphasizes the tendency to family cohesiveness of depressives. It is not made clear exactly what type of depressions the author is studying and out-patient depressives often include a large admixture of depressions secondary to other conditions. Finally, the proportion of those with suicidal tendencies is not mentioned.
Beck et al. (1963) report on a carefully-performed study which unfortunately has no normal comparison-group. These investigators have
classified 297 depressive subjects, by means of a depression inventory and by clinical evaluation, into a 'high-depressed' group and a 'low-depressed' group. The high-depressed group (the most severely ill) shows a significantly higher prevalence (27%) of orphanhood before the age of 16 years than does the low-depressed group (12%). There is no obvious relationship between the age of the parents, the occurrence of orphanhood and the onset of depression and it is concluded that death of a parent in childhood may be a factor influencing chiefly the degree of severity of a depressive illness.
This last point seems an important one. It is conceivable that loss of a parent in early life is one of the factors which geneticists postulate on theoretical grounds to explain the variable expression of the hereditary tendency to depression.
Parental Deprivation: Its Significance to Mental Illness
From this survey of the literature it can be asserted that there is no convincing proof of a causal relationship between parental deprivation and mental illness. Loss of the parent in childhood may have effects on the developing personality but it is likely that these effects become noticeable only where deprivation occurs in an already-vulnerable personality or where the child is not protected from the consequences of losing the parent.
It is possible that parental deprivation may be an important modifying influence in the natural history of several psychiatric conditions but not perhaps in the rather indiscriminate fashion that a first glance at the evidence would suggest. For example, it may on the one hand
accentuate the severity of a depressive illness but on the other hand, accelerate the onset of schizophrenia. In psychoneurosis, parental disharmony rather than actual parental loss may be of importance but in suicide and attempted suicide, both parental disharmony and parental deprivation appear to be prominent factors.
If, as is suggested, parental deprivation does have such comparatively specific effects it is certainly a subject worth further examination, particularly if it can provide help in delineating populations at risk of psychiatric illness. In the belief that this is so, the present study has been carried out.
**THE USE OF EPIDEMIOLOGICAL METHODS IN THE STUDY OF PSYCHIATRIC ILLNESS**
Lilienfeld (1957) defines epidemiology as the study of the distribution of a disease in space and time within a population, and of the factors that influence this distribution.
Originally applied to the study of communicable disease, the value of epidemiology in studying non-communicable, including psychiatric, illness is now widely accepted. (Roid, 1960). Unfortunately, its contribution to accuracy and impartiality in psychiatric research is still severely hampered by such limiting factors as vagueness of diagnostic criteria, controversy over nomenclature and lack of objective standards of severity of an illness. Even with the application of epidemiological and statistical techniques the results of closely similar studies on psychiatric disorders may not be comparable because of the inability of the investigators to agree on basic theoretical premises. It is a paradox that, in the study of psychiatric research-literature, it is often easier to understand the description of experimental method than it is to understand the
psychiatric content. It is obvious that the use of factual methods in psychiatric research must be extended; if it is possible to agree on techniques it may eventually be possible to agree on principles.
The use of epidemiology in psychiatry is increasing, largely because it offers one method of avoiding the somewhat anecdotal approach which is still very prevalent in research into mental illness. However, its suitability as a research tool is not universal and the four main uses to which epidemiology can be put in psychiatric research are enumerated by Lin and Standley (1962):
1) to assess the prevalence of different types of mental ill-health in a population as a basis for the prevention, treatment and control of these diseases;
2) to uncover associations between population characteristics and disease that may clarify the origins of mental disorder;
3) to test aetiological hypotheses originating from laboratory or clinical studies;
4) to assess rates of spontaneous recovery to evaluate the effectiveness of preventive and therapeutic measures.
The use of epidemiological technique in this study does not imply that an attempt is being made to estimate, for example, the prevalence of depressive illness in the population at large. The study begins with a selected group of depressive patients and methods 2) and 3) (above) are employed to investigate a possible relationship between depressive illness and certain factors in the lives of the individuals concerned. Should these methods reveal any significant relationships it must be
stressed that it is not justifiable thereupon to assume that the factors have caused the illness; it can only be stated that they are somehow related to it. This is no disadvantage, since the demonstration by objective means that such relationships exist allows future research to be carried out to define their nature more exactly.
GENERAL METHOD
GENERAL METHOD
This study consisted of an investigation into a number of aetiological factors in depressive illness and in order to enhance its validity, considerable care was taken with its planning. At each stage of the planning, the design was discussed and criticised at meetings with colleagues expert in psychiatric research. Difficulties or oversights which occurred were thus dealt with in the preparatory period and no modification of method was found necessary when the study itself began.
It was decided to investigate patients admitted to psychiatric hospitals in the Edinburgh area on account of depression and a maximum of two years of full-time research was allotted to the study. A retrospective method of investigation was chosen as being most economic of time and it was arranged that the investigator would obtain data on a total of approximately 150 depressive patients. A questionnaire was devised which enabled the investigator to obtain all the relevant information at a single interview with each patient and without recourse to any outside source of information.
When the form of the study had been finalised, a pilot study was carried out. Ten depressive in-patients were interviewed and a questionnaire was completed in each case. No unforeseen difficulties arose: the interviews were uneventful, the patients co-operative and the data apparently satisfactory. So smoothly did the pilot study run that these patients were able to be included in the definitive series.
It was obvious that the results of an investigation of this type
could be shown to be significant only if they could be compared with some normal standard. The need for a satisfactory control series was self-evident and it was necessary to be sure at an early stage that a source of control-material was at hand. It was suggested that out-patients attending medical and surgical clinics in a general hospital were likely to be fairly representative of the general public. With the co-operation of the physicians of the Western General Hospital, Edinburgh, the investigator interviewed a total of 210 medical and surgical out-patients, completing for each one a questionnaire schedule identical to that used for the depressive patients. The results of this investigation suggested that medical and surgical out-patients as a group were not unrepresentative of the population as a whole. By the completion of a questionnaire in each case, all the data required for comparison with a group of depressives was available and so these patients were used as the control series.
At this point the method of investigation was demonstrably satisfactory and a suitable control series had been obtained. It was now possible to proceed with the collection of data from depressive patients. The investigator arranged to visit each psychiatric hospital in the Edinburgh area at a particular time each week and at each visit patients admitted with depressive illness since the previous week were referred for interview. The diagnosis of depression was checked by means of a clinical interview with the patient and then a questionnaire was completed for each case. A total of 183 depressive patients was interviewed but it was decided that 30 of these were suffering from depression secondary
to another psychiatric condition and they were excluded from the final series.
During the course of the study, information from each depressive patient and each control individual was transferred from the questionnaire schedules to punch-cards. As a result, analysis of the data could be started as soon as the collection of information was complete.
As may be seen from this outline description of the research, the study proceeded in a series of stages and in the following section these stages will be described in detail.
THE INVESTIGATION
THE INVESTIGATION
Object To examine a number of hypotheses on aetiological factors in depressive illness with especial regard to those which seek to demonstrate a causal relationship between certain occurrences in childhood and the subsequent development of depressive illness.
Aim To study a group of patients suffering from depressive illness and to compare them with a matched group of non-depressive individuals in order to test the following hypotheses:
1) Parental deprivation, by death or by other cause, predisposes towards depressive illness.
2) The age of the parents is of significance in the aetiology of depressive illness in that the older the parent at the time of the individual's birth, the more likely is that individual to develop depressive illness.
3) Loss of a parent in childhood by death is more likely to cause a tendency to depressive illness than loss of a parent by a cause other than death.
4) Loss of mother during childhood is more important in predisposing to depressive illness than loss of father.
5) A disturbed parent-child relationship in early life is important in producing a tendency to depressive illness.
6) Persons suffering from depressive illness belong to a larger-than-average sibship in their family of upbringing.
7) The ordinal position of the depressive individual in his sibship is of importance in the aetiology of depressive illness.
8) Depressive patients tend to remain unmarried more than do normal individuals and the fertility of married depressives differs from that of married non-depressives.
9) Depressive patients more often present a positive family history of severe mental illness than do normal individuals.
10) Individuals with severe depression more often have a positive family history of severe mental illness than do individuals with less-severe depression.
Definitions
1) Depressive illness is defined as a primary disturbance of affect in the direction of sadness.
2) A case of depressive illness: a patient will be acceptable for inclusion in the study if he has been diagnosed by a psychiatrist as suffering from depressive illness (as defined above) and the investigator making an independent examination, agrees with this diagnosis.
3) A case of suicidal depression: a case of depressive illness where the patient, as part of this or a previous illness, has deliberately tried to harm himself. A patient with thoughts of suicide who has never attempted to harm himself will not be regarded as a case of suicidal depression.
4) Childhood is taken arbitrarily to mean the time from birth till the 16th birthday.
5) Parental deprivation refers to the complete absence of a parent for any reason during a continuous period of three months or more in the patient's childhood.
Auspices of the Study
This study was carried out during the investigator's tenure of the
post of clinical member of the scientific staff of the Medical Research Council Unit for Research on the Epidemiology of Psychiatric Illness, Edinburgh. Permission to undertake the study was granted by the Honorary Director of the Unit, Professor G. M. Carstairs.
**Time Schedule of the Study**
February - March 1963 : Preliminary formulation of hypotheses and study-method.
March 1963 : Approval of the project.
April - July 1963 : Review of the literature and design of the project.
September 1963 : Pilot study.
October 1963 - January 1964 : Obtaining the control series.
January - July 1964 : Data-collection on depressive patients.
August - October 1964 : Processing of data.
**Preliminary Formulation of Hypotheses**
In an investigation of this type it is essential to know at the outset exactly what information is being sought and how it is to be obtained. If the results of the research are to be acceptable it must be shown that the experimental method was held constant throughout the process of data-collection, so the necessity for careful planning and prior elimination of inconsistencies is vital.
This study began as a surmise arising from an hypothesis, the hypothesis being that individuals suffering from depressive illness tended to be deprived of their parents during childhood to a significantly
greater degree than non-depressives. As has been demonstrated in the preceding review of the literature there is some evidence to support this contention but the evidence is equivocal and there is certainly no proof for the frequent assumption that the parental deprivation is a cause of the depression. If depressives are excessively prone to childhood parent-loss, other explanations than cause-and-effect are possible: at this point the surmise arose. It seemed not unreasonable to suggest that depressives lost their parents in childhood to excess because they had been born of older-than-average parents who would, in the natural course of events, tend to die at an earlier stage in the lives of their children.
A close examination of the literature revealed a small number of references to parental age in mental illnesses other than depression (see page ) but none to this aspect of depressive illness. On consideration, it seemed a worthwhile avenue of enquiry, firstly because the findings which suggested that parental deprivation occurred to excess in the childhood of depressives were not overtly convincing and would need to be confirmed or refuted as a preliminary to research into parental age; and secondly, if it could be shown that depressives had a tendency to be born of older parents, interesting aetiological possibilities might arise. As is well-known, a high proportion of infants suffering from Mongolism are born of older mothers and a genetic basis has now been established in this condition. If a similar situation were found to obtain in depressive illness it would suggest a possible new line of enquiry into the aetiology of affective disorder.
After it had been agreed that the relationship between depressive illness, parental deprivation and parental age should be studied, another
through examination of the literature on depression and parental deprivation was undertaken. As a result of this, certain other aspects of the aetiology of depression were noted which could profitably be examined and which could readily be included in the projected investigation. Ultimately, the ten hypotheses listed above were constructed and it was possible to proceed to the design of the experiment.
The Geographical Area in which the Study took place
The study was conducted in a number of hospitals in the Edinburgh area. Edinburgh, with a population according to the 1961 Census of 468,361, is the Capital of Scotland and the County town of the County of Midlothian. It is largely a commercial and residential city but it possesses a growing number of industrial concerns and Leith, the port of Edinburgh, is an active centre of trade, especially with the Continent. The city's hinterland is largely given over to farming but there are a number of coal-mining areas and several small towns associated with light industries. Thus, the city of Edinburgh tends to be residential and substantially middle-class but in the area as a whole a wide distribution of occupations and social classes obtains.
Hospitals in the Edinburgh Area
Edinburgh is renowned for its University and Medical School. Closely associated with the latter is the Royal Infirmary which, with 1,123 beds, is the largest of the city's general hospitals. There are a number of specialized hospitals in the city and also several district hospitals, the largest of which is the Western General Hospital with 510 beds. The
majority of this hospital's beds are given over to the major specialities and there are active out-patient clinics to which come patients from a wide range of socio-economic levels.
Neither the Royal Infirmary nor the Western General Hospital has in-patient psychiatric beds although the former has a Professorial Department of Psychological Medicine and both have psychiatric out-patient clinics. The main psychiatric in-patient centre is the Royal Edinburgh Hospital which has 1,441 beds. This is really a complex of hospitals all administered by a single board of management and its constituent elements are: Jordanburn Hospital, which is the Professorial in-patient unit, West House Hospital, Craig House Hospital and associated nursing homes. Two other hospitals, Bangour in West Lothian (1,080 beds) and Rosslynlee in Midlothian (402 beds) lie outwith the city boundaries and serve their respective counties but also admit patients from Edinburgh. West House, Bangour and Rosslynlee act as district psychiatric hospitals for particular sectors of the city.
On the whole, patients tend to be admitted to the hospital which is responsible for their home area but this system is by no means rigid and, for example, it is usually considered more advisable to re-admit a patient to the hospital where he was previously treated than to admit him to another hospital because he has moved house to another part of the city. Admission to Jordanburn is more selective because of its teaching and research functions while admission to Craig House and the nursing homes is usually on a paying basis. Each hospital has its own out-patient clinics, usually held in the districts which it supervises.
The psychiatric facilities available to Edinburgh citizens are fairly comprehensive and it is probable that the majority of individuals from the city and its surrounding districts who require in-patient psychiatric treatment receive it at one of the hospitals described above. From the point of view of the present research-project the area was compact enough to enable an investigator to screen all the psychiatric hospitals for cases of depressive illness and thus to obtain a group of patients representing all strata of society.
**The Type of Study**
The study was planned to be carried out in its entirety in the relatively limited maximum time of two years. In an illness such as depression whose natural history may span the greater part of a lifetime, an ongoing study of such short duration would be of small value. It was therefore decided that the study should be an anamnestic one in which a group of depressive subjects would be questioned to obtain the desired information. Since this information was for the most part of an objective nature it seemed reasonable to set about obtaining it in this way.
Before the study began no reliable estimate was available of the number of patients with primary depressive illness who would be likely to require admission to Edinburgh psychiatric hospitals in a given period. It was agreed that a series of some 150 depressive subjects would provide sufficient material for statistical analysis. Since most estimates concurred that the admission rate for female depressives was approximately twice that for male depressives it was arranged that the series would be complete when 50 male subjects had been interviewed. The decision to
interview patients in all the psychiatric hospitals in the Edinburgh area was made partly to facilitate the speed of gathering cases but mainly to ensure that there would be adequate representation of both sexes, all age-groups (within the defined limits) and all socio-economic levels among the subjects. It should be stressed however that no attempt was made to estimate prevalence or incidence rates for depressive illness, this being no part of the study, and despite the use of epidemiological methods, the research was not in any way a community survey.
The design of the study was made as simple and straightforward as possible and in the interests of scientific communication terms were defined closely and criteria were rendered as objective as could be. This conceivably sacrificed a number of subtle nuances but this loss was compensated by the opportunity to obtain some firm data in an area of psychiatric theory notably lacking in the commodity. It was not found possible to set up an experimental design which closely followed previous work because previous work was usually so loosely constructed. It was hoped that the present study, by virtue of its simplicity of design, would be capable of being reproduced with little difficulty.
The study contains a control series which can be criticized on the grounds that it does not necessarily represent the general population, but an attempt has been made to demonstrate that it is not markedly unrepresentative and this does not seem to have been done in previous investigations in this field. In addition, the information about both depressives and controls was obtained by a single investigator who carried out personal interviews on all the patients using a standard interview-technique based
on a questionnaire. The standard of the information obtained should therefore be consistent throughout the depressive and the control series.
Only in-patients were included in the depressive series so as to obtain as little diagnostic variation as possible, and since the patients were drawn from several different hospitals it was hoped that no consistent diagnostic bias would be present. Any patient whose diagnosis did not meet the criteria of the study was rejected by the investigator with the result that all the patients in the final series were regarded as undoubtedly depressed in the opinions of two psychiatrists.
The use of a retrospective method of collecting information is a possible source of criticism since retrospective enquiry may produce unreliable data. The human memory is notoriously fallible and it is desirable to have some independent means of checking the material obtained by this method. Unfortunately, most of the information obtained in this study was not readily confirmable but the consistency of the results suggests that it was not unduly inaccurate. Although it was feared that the reporting characteristics of depressive individuals might be affected by the illness and that they might be more likely to repress psychological material relating to their early lives, this did not appear to happen.
Despite the precautions taken to ensure diagnostic accuracy, the diagnosis of depressive illness was a clinical one because no more reliable method was available. Although not part of the original design of the study, in a proportion of the depressive patients the investigator completed a symptom-sign inventory (see Appendix 6) in an attempt to differentiate objectively between endogenous and neurotic depression, but this
failed because virtually all the patients were receiving antidepressant treatment by the time they came to interview and this invalidated the test. This difficulty in diagnostic precision is unfortunately inherent in most psychiatric studies and must inevitably reduce the confidence with which results can be accepted. Then again, as pointed out in the review of the literature (page 11), in a short-term study it is likely that a number of cases are actually other conditions in masquerade. Only the rigorous exclusion of doubtful cases, as was carried out in this research, can reduce this possibility to a minimum.
It is not suggested that this study is free of errors but it is possible that it has been carried out at least as accurately as any research which is reported in the literature on parental deprivation.
Selection of Patients for the Study
The aim of the study has been to examine certain concomitants of depressive illness and in order to avoid confusion by extraneous psychopathology it was essential to obtain a group of patients who, so far as could be determined, had no history of psychiatric disorder other than affective illness. For this reason, any patient who, in addition to his depressive illness, showed one or more of the following characteristics was excluded from the study:
1) a psychiatric condition other than affective illness;
2) thought disorder, thought-blocking, thought-interference, thought-incongruity;
3) emotional incongruity;
4) passivity of feelings;
5) clouding of consciousness and disorientation;
6) a history of convulsions;
7) a history of severe head-injury;
8) evidence of neurological abnormalities.
It was pointed out in the review of the literature (page 10) that depression in the elderly appeared to differ in several important respects from depression of earlier onset and that it required separate consideration. Consequently, no patient who had celebrated his 61st birthday was admitted to the depressive series. The lower age limit was set at the 16th birthday since this, by definition, marked the end of childhood.
To increase diagnostic accuracy it was decided to study only in-patient depressives although these admittedly form a highly-selected group and many of the reasons for selection are only imperfectly known. A sizeable number are admitted to hospital because antidepressant treatment at home has not improved them; others are admitted because of the severity of the illness or the danger of suicide. It is probable that depressive in-patients represent the more severe end of the depressive spectrum but at present this is assumption. However, in this study it was more important to obtain subjects undoubtedly suffering from depressive illness than to obtain a 'representative' sample of depressives and it was simpler to attain this end under in-patient conditions. In addition, the in-patient situation was more conducive to the conducting of a formal interview than the conditions in a busy out-patient clinic and in-patients were more readily available for re-interview if, on occasion, all the information was not gathered at the first session.
Permission was obtained from the medical superintendents or senior consultants of the psychiatric hospitals in the Edinburgh area (Jordanburn, West House, Craig House, Bangour and Rosslynlee) to visit each hospital once a week and on each occasion to interview those patients admitted since the previous visit because of depressive illness. In this way, it was possible to see virtually all the in-patient cases of depressive illness from Edinburgh and the surrounding districts admitted during the period of the study.
To minimize the possibility of personal bias in diagnostic procedure it was arranged that at each visit the investigator would be given a list of patients between the ages of 16 and 60 admitted in the preceding week whom the medical staff had diagnosed as suffering from depressive illness. Each of these patients was then interviewed and clinically assessed by the investigator and a questionnaire schedule was completed in every case. In all, 183 patients were referred in this way and all were undoubtedly depressed. However, 30 were found to be suffering from a depression secondary to a psychoneurosis or a personality disorder and, although details were recorded in these cases exactly as in the others, they were excluded from the final series, leaving a total of 153 primary depressions.
At the end of every visit to a hospital, a check was made with the member of the secretarial staff dealing with admission formalities to ensure that no case of depression had been overlooked. If any appeared to have been so, a member of the medical staff was informed and if he agreed that the case met the criteria for the study, the patient was interviewed.
It was found to be more satisfactory if the investigator called at the hospital in person each week rather than telephoning to enquire if suitable cases were available since his regular appearance prompted the medical staff to prepare lists of patients beforehand and there was less likelihood of patients being overlooked.
The system worked admirably. The minimum of work was required of hospital staff and this ensured their goodwill in the research. If it was ever inconvenient from the hospital's point of view for the investigator to call at a particular time a different time was mutually arranged and if, for any reason, the investigator was unable to visit at his expected time, the hospital was informed. These and similar courtesies enhanced the willingness of medical, nursing and secretarial staff to co-operate and no instance of obstructiveness was encountered in the course of the study.
**The Interview**
Insofar as it was possible the interview followed a set pattern, this being made comparatively easy by the fact that a single individual was conducting all the interviews. The investigator opened the interview by introducing himself by name and by explaining that he was a doctor on the staff of Edinburgh University. (The use of the name of the University was justifiable because the investigator was an honorary lecturer: it was considered that mention of the University would mean more to a patient than reference to the Medical Research Council about which many people know little). The patient was then told that he was being seen from a research point of view and not as part of his personal treatment regime.
and he was asked if he agreed to be interviewed. Only three depressive patients refused permission on the first occasion of asking, probably because of agitation associated with the depression, but in each case full co-operation was obtained at the next visit.
The first two or three minutes of the interview were given over to this introduction and to informal conversation to put the patient at ease. Thereafter, the conversation was led, without apparent break, into an enquiry about the patient's current illness, its symptoms, severity and duration. The patient was asked about a history of previous psychiatric illness and what form it assumed; about a family history of affective disorder; about possible precipitating causes of the present illness; and about the treatment which he was receiving for the current illness. The cardinal feature sought for in the course of this examination was the presence of depressive affect. In some cases this was already responding to treatment and more reliance had to be put on history than on clinical appearances.
Hamilton and White (1959) list a series of signs and symptoms which they regard as typical of depressive illness and this symptom inventory was used as a guide for questioning the patient about his illness. At first it was intended to complete an inventory-schedule for each patient but this was found to be impracticable. However, the list of signs and symptoms provided a convenient and standard method of checking the patient's complaints. The list was as follows:
1) Depressed mood.
2) Guilt
3) Suicide
4) Insomnia (initial)
5) Insomnia (middle)
6) Insomnia (delayed)
7) Work and Interests
8) Retardation
9) Agitation
10) Anxiety (psychic)
11) Anxiety (somatic)
12) Gastro-intestinal symptoms
13) General somatic symptoms
14) Genital symptoms (loss of libido)
15) Hypochondriasis
16) Loss of insight
17) Loss of weight
Because of the careful prior selection of cases it was rarely difficult to be sure of the diagnosis of depression but it was frequently difficult to differentiate into endogenous and neurotic types. In 60 of the 163 depressive patients in the study, a symptom-sign inventory, adapted from that of Foulds (1962), was completed in an attempt to objectify the differentiation (for details see Appendix 6) but this gave no help, largely because the clinical picture had usually been altered by the effects of treatment by the time the inventory was applied to the patient. Despite this difficulty, each case was clinically assessed as either an endogenous or a neurotic type of depression. In the opinion of the investigator this was mainly a division into severe and less-severe forms.
Endogenous depression was diagnosed in the presence of one or more of the following circumstances:
1) if the depression of mood was severe;
2) if delusions, somatic delusions or severe guilt feelings were present;
3) if the depression was disproportionately severe in relation to the nature of the precipitating factors if these were present, or if it persisted unduly when they were removed. (Otherwise the presence or absence of precipitating factors was not considered as a means of differentiation);
4) if the depression was recurrent in the absence of adequate provoking factors or if there was a previous history of manic illness.
The degree of agitation or anxiety was not regarded as per se an indication of the degree of depression and an attempt to commit suicide was not regarded as a criterion of severity of the illness unless the circumstances of the attempt were obviously psychotic.
The clinical assessment of the patient's condition lasted approximately fifteen minutes and when it was complete the investigator decided whether the patient was indeed suffering from a primary depressive illness and whether this was endogenous or neurotic in its degree of severity. Once these decisions were made they were irrevocable so that they could not be affected by subsequently-obtained information about factors whose association with the illness was under investigation.
The next stage of the interview was the completion of the questionnaire which was carried out in all cases, even if the patient had been diagnosed
as suffering from a secondary type of depression. The questionnaire was completed by the investigator who recorded the answers on the schedule. While an attempt was made to complete it systematically, it was found that information was often obtained without asking and as much as possible the patient was allowed to express himself freely. In this way it was ensured that the investigator's personality intruded itself as little as possible. Garrulous patients at times had to be brought back to the point, but in the great majority of cases it was not difficult to acquire the data by careful listening and the interjection of simple, non-committal questions as required by the questionnaire. Great care was taken that the questions asked by the investigator should remain unvaried in essence throughout the study, even though there was a frequent temptation to attempt to amplify certain points as the research progressed.
Completing the questionnaire lasted 10 to 15 minutes in most cases and it was necessary in only a small number of cases for the investigator to have to arrange a second interview to finish the schedule. A patient's inability to provide all the information required of him at a single session was almost invariably due to his having had electro-convulsive therapy earlier in the day. The following interview was then arranged so as not to fall on a treatment day.
The questionnaire items will now be considered in detail.
**The Questionnaire**
The Questionnaire Schedule is reproduced in this thesis (Appendix 4). In practice it consisted of a sheet of foolscap paper printed
on both sides but for convenience it is shown here on quarto-size paper. Answers to questions were written in the spaces provided or, in some cases alternative answers were provided and the correct choice had to be marked by an encircling line.
The questionnaire was designed to be suitable for application both to depressive patients and to non-depressive control patients. It contained eight sections, each dealing with a particular aspect of the case, and these were as follows:
1) Routine Information: for reference purposes, the date of interview, the name of the hospital and the date of the patient's admission to it, and the name of the psychiatrist who referred the patient for interview were noted.
2) Personal Data: the patient's name and address and the name and address of his general practitioner were recorded. These details were of no significance to the present study but provided a means of tracing patients should there be a desire to follow-up the group of depressives at a later date.
The sex, date of birth, age and civil state of the patient were all recorded as being pertinent to the investigation. Religious persuasion was enquired after because it was likely to be an important variable in any study concerned with family size. The patient's occupation (or husband's occupation in the case of a female) was noted in order to assess his socio-economic position as defined by the Registrar General's Classification of Occupations (1960).
3) Previous History of Psychiatric Illness: since a depressive
illness is frequently only one episode in the natural history of an affective disorder, it was thought necessary to record the details of the first and any intervening episodes. Where there was a positive history of psychiatric illness, the approximate date of the episode, the form it took, its duration and its treatment were noted. The name of the hospital where treatment had been carried out was obtained lest in some future study more precise details of the past history would be required. Taking this fairly comprehensive past history was found to be of great assistance in confirming the current diagnosis.
4.) The Current Psychiatric Illness: this by definition was always depressive illness but the term on the schedule was a general one so that it could apply to any current psychiatric condition presenting in a control-patient. In the depressive patients it was noted whether the illness was endogenous or neurotic and how long the present attack had lasted. In order to decide whether the depression was primary or secondary the presence of the following entities was sought for:
1. Personality disorder
2. Psychosis other than depression
3. Psychoneurosis
4. Alcoholism
5. Mental defect
6. Epilepsy
7. Any other psychiatric illness
Evidence of a cyclothymic personality was not regarded as inconsistent with the diagnosis of primary depression and below-average intelligence of
itself did not exclude a patient from the series, but the presence of one or more of the other conditions mentioned above led to his being rejected. If there was doubt whether the patient was truly a chronic alcoholic or merely over-indulgent in alcohol he was not accepted for the series.
Although the presence of demonstrable precipitating factors was not regarded as of great diagnostic significance, the schedule allowed for them to be recorded if they occurred.
5) Family History: In this section the first purpose was to obtain details of parental age at the time of the patient's birth and of the patient's age at the time of his parents' deaths. The patient was asked if the parent was alive and if so, what age the parent was. Subtraction of the patient's age from the parent's age gave the age of the parent at the time of the patient's birth without having to enquire specifically for it. If the parent was dead, the same result was obtained by finding the age of the patient at the time of the parent's death and by subtracting the result from the age of the parent at the time of death. In this way, all the information was obtained by asking the patient only for easily-remembered events, dates and ages.
Where the parent was dead, details of his name, birthplace, last address and the place where death occurred were noted so that they could be used to obtain more precise information from Register House, Edinburgh, if this were necessary to a future study.
A family history of mental illness was then enquired for and a note was made of its presence or absence, who was affected and the form the
illness took, if this was known.
Next, as an integral part of the investigation into sibship size in the family of upbringing, the number of sibs of the patient was recorded, their sex, the order in which they came and the patient's ordinal position in the family. Note was also taken of parental re-marriage, the presence of step-sibs, the death of sibs during the patient's childhood and whether the patient was an adopted child. To investigate the fertility of depressive patients, the age, sex and number of his own children were noted and he was asked if he had lost any children by death.
6) Personal History: here the patient was asked if he had been a twin at birth and then information was sought about separation from parents during the period of childhood. If separation had occurred the reasons for it, the nature of the separation and the period in childhood in which it had taken place were gone into. If the separation was temporary its duration was obtained and details of the parental surrogate were enquired for.
7) Factors Causing Disturbance in Family Relationships: it seemed important here that the investigator should not attempt to suggest any factors to the patient but should instead allow him to express his own opinion about disturbed relationships in his home of upbringing. The questions asked on the topic were made as neutral as possible, the patient first being asked, "Was there anything in your childhood home which you now regard as having been particularly unhappy or upsetting?" If the answer was in the affirmative, his complaint was recorded and he was asked, "Was there anything else which was unpleasant at home when you were young?"
Any further complaint was recorded.
If the answer to the first question was negative, the second question was altered slightly to, "Was there anything at all which was unpleasant at home when you were young?" Otherwise, no further pressure was put on the patient to recall unhappy childhood events and if the response remained negative this was noted.
8) Physical Illness, Present and Past: The main purpose of this section was to provide a means of recording the presenting physical complaint and the previous history of physical illness of the control patients, but the same details were collected from the depressive patients to ensure uniformity of data.
The questionnaire schedule did not contain any section dealing with attempted suicide but from the beginning of the study details of this if it had occurred were noted at the top of the front page of the schedule.
On completion of the questionnaire the formal interview was at an end. Many patients wished to talk a little more and it was now possible to be perfectly informal, but care was taken not to express any opinion on their condition or on the treatment being received lest the hospital staff be antagonized by apparent interference.
The entire interview took on average approximately half-an-hour.
Personnel and Equipment Used in the Study
The study was carried out almost entirely by the investigator with of course the readily accessible advice of Medical Research Council colleagues and the assistance of these psychiatrists who prepared lists of patients to
be interviewed at each visit to hospital. Access to secretarial assistance was available when required but virtually all that was required of the secretary was to prepare stencils and to print batches of questionnaire schedules and symptom-sign inventories as required.
The investigator completed each schedule during the interview with the patient and as soon as possible thereafter he transferred the information to punch-cards.
The equipment used in the study was of the simplest nature:
1) Approximately 400 sheets of foolscap plain paper for questionnaire schedules.
2) Approximately 100 sheets of quarto plain paper for symptom-sign inventories.
3) Approximately 400 Copeland-Chatterson punch-cards for recording data.
4) Approximately 150 plain cards for recording article-references.
5) A notebook into which was entered the name and place and date of interview of each patient. This was carried by the investigator and was a useful form of reference when visiting a hospital as to which patients had already been seen.
Since visiting each psychiatric hospital in the area once a week involved some 72 miles of travel, the use of a motor-car was of considerable assistance.
THE PILOT STUDY
THE PILOT STUDY
By the beginning of September, 1963, the project's design had reached a stage where it required practical application to test its efficacy. All foreseeable theoretical difficulties had been dealt with, the questionnaire appeared adequate to obtain the required information and, as described previously, the type of patient to be studied had been closely defined.
It was decided that 10 depressive patients should be interviewed to test the research method. Permission was obtained from Professor G. M. Carstairs to interview in-patients in Jordanburn Hospital, the Professorial in-patient unit where there was a high admission-rate and thus a good chance of finding depressive patients with relative ease. The investigator therefore conferred with the psychiatrist in Jordanburn who dealt with the admission of cases, explaining to him the purpose of the investigation and he agreed to refer those cases which appeared to him to agree with the criteria of the study.
Following this, the investigator visited the hospital on three occasions at weekly intervals and 10 depressive patients (5 male, 5 female) were referred for interview. No difficulty was encountered: the patients were co-operative, even enthusiastic, and gave no indication of resentment at being included in a research-project. The interviews followed a smooth course and the required information was obtained without difficulty.
As a result, it was practicable to proceed with the study without changing the experimental method in any way. Of the 10 patients seen,
it was possible to include 8 in the definitive series of depressive patients. The remaining 2 (1 male, 1 female) were regarded by the investigator as suffering from depression secondary to another condition and they were relegated to a separate 'Reject' category. As the investigation proceeded, similar cases of secondary depression were included in this category.
No further details of the pilot-study patients are given here since they are most appropriately discussed in the context of the entire series.
THE CONTROL SERIES
THE CONTROL SERIES
It would be of great assistance in any epidemiological study of psychiatric illness to have a ready supply of control individuals but this is difficult to ensure. In planning this study it was realised at an early stage that suitable controls were necessary to render the results meaningful and in considering available sources, the possibility of using general hospital out-patients was entertained. This had something to commend it since it seemed likely that individuals who were themselves in a hospital setting would be more willing to co-operate in a medical research project. As well as this, out-patients are usually available in large numbers: for example, in a typical week the Royal Infirmary of Edinburgh deals with well over a thousand surgical out-patients and in a typical month with some six hundred medical out-patients. Therefore, in any month, the Infirmary's medical and surgical clinics alone will see approximately five thousand out-patients, about half of whom attend by appointment.
From these figures it can be seen that, during a comparatively brief period of time, the Royal Infirmary of Edinburgh will pass through its out-patient clinics a sizeable proportion of the population of the area it serves, and this is true of any active general hospital. Where such large numbers of people are involved it seems possible that a random sample of medical and surgical out-patients would not differ greatly from the population at large, apart from the illness which caused attendance at the hospital.
Fortunately, for the purposes of this study it was not necessary for the controls to be absolutely typical of the general public. The main characteristic required of them was that they should have no history of depressive illness. However, it was certainly desirable to show that a control group was not unduly biased in any direction. A comparatively straightforward method of demonstrating this in the present instance was to choose a number of socio-demographic factors and to compare the medical and surgical out-patients according to these factors. If it could be shown (as in the event it was) that the two groups of patients closely resembled each other, it would encourage the belief that, taken together, medical and surgical out-patients did not differ too markedly from the general population and that they were suitable to act as controls in this study.
As well as this, it seemed logical to use hospital patients as controls for depressive in-patients. Referral to hospital is the result of a complicated process of selection and it seemed possible that some of the variables involved might be eliminated in this way. It was therefore decided to investigate the suitability of general hospital out-patients as controls by interviewing them in the same way as was used for the depressive patients in the study. Out-patients were chosen rather than in-patients because it is simpler in practice to obtain a comparatively unselected group of out-patients. It seemed wiser to study only out-patients attending by appointment since it would be unfair to subject emergency cases to an apparently irrelevant interview. Patients under the age of 16 years were not interviewed as they were still in the period of childhood defined in the study, but there was no upper age limit.
Aim To study a group of medical and surgical out-patients to ascertain
if they are suitable to act as a control series in the present study.
Method of Investigation
At first it was intended to carry out this part of the study in the Royal Infirmary of Edinburgh but this proved impossible because of the pressure on the out-patient facilities there, a situation which will continue till new premises are available. Permission was therefore obtained to attend the out-patient department at the Western General Hospital, Edinburgh, which is about half the size of the Royal Infirmary. In this department it was easier to conduct an enquiry without taking up much-needed accommodation or interrupting the routine of the clinic.
The investigator arranged to attend six clinics, each held once a week. Three of the clinics were medical, dealing mainly with general cases although one had a slight bias to endocrinology and another a slight bias to rheumatic heart disease. Of the other three clinics, two dealt with general surgical cases and the third was orthopaedic. The consultants in charge agreed that, once they had completed their examination, the patient could be seen by the investigator. As it was not possible to interview every patient attending a busy session a working arrangement was devised with the co-operation of the nursing staff. As the first patient left the consulting-room he was taken to another room to be interviewed. When this was completed and the patient had left the room, the next patient to leave the consulting-room was brought along, and so on till the end of the clinic. Any patient whose examination ended while
an interview was in progress was allowed to leave. In this way, it was ensured that no patients were kept waiting, the rhythm of the clinic was not disturbed and if anything, the randomness of the patient sample was enhanced.
The purpose of the investigation was explained to every patient at the outset of an interview and each was asked if he agreed to take part. Only one patient, a female attending the orthopaedic clinic, refused to take part and she had to be excluded from the series. Otherwise there was complete co-operation from all patients.
The first task in the interview was to enquire whether the patient had a history of affective illness and to note whether he appeared to be suffering from depression at the time of examination. When either of these circumstances occurred, the interview was brought to an end and no details about the patient were recorded, so that the series would be as free as possible of depressive individuals. Of 222 out-patients, 12 were excluded in this way as the accompanying table shows.
Patients excluded from the series because of affective disorder
| Description | No. of cases |
|--------------------------------------------------|--------------|
| Patients depressed at interview | 2 |
| Almost-certain history of affective disorder | 3 |
| Doubtful history of affective disorder | 5 |
| History of depression following drug-treatment | 2 |
| **Total** | **12** |
In the remaining 210 patients, who constitute the final sample
the interview was carried through to completion. A questionnaire was completed in each case, using exactly the same technique of obtaining information as was used for the depressive patients in the pilot study. The entire session lasted about 15 minutes and no difficulty was experienced in obtaining the necessary information.
It had been decided that approximately 200 patients would form the series, so data-collection ceased after the last clinic of the week in which the 200th patient was seen. As information was gathered it was transferred from questionnaire to punch-cards. Thereafter, 14 items from the information obtained by the questionnaire were selected and a statistical comparison by the chi-square method was carried out to see whether medical and surgical out-patients differed significantly in respect of any of these items. Next, the entire sample was divided according to sex and the same comparison was made between male and female patients.
The items chosen for comparison were:
1) The age-distribution of the patients.
2) Civil state.
3) Social class distribution.
4) Religious persuasion.
5) Size of the sibship in the patient's family of upbringing.
6) Number of children in the patient's own family.
7) Loss of a parent by death before the patient's 16th birthday.
8) Loss of a parent for any reason, for at least three months, before the patient's 16th birthday.
9) Complaint by the patient of a disturbed relationship with a
parent during childhood.
10) Family history of mental disorder.
11) Age of mother at the time of the patient's birth.
12) Age of father at the time of the patient's birth.
13) Age of the patient at the time of mother's death.
14) Age of the patient at the time of father's death.
The results of the comparisons will now be discussed in detail.
The Outpatient Material
(All tables and figures referred to in this section are contained in Appendix 1 pp. 57 to 77).
The material consists of 210 out-patients of the Western General hospital, Edinburgh, the majority of whom live in Edinburgh, Midlothian or West Lothian, with only a small proportion dwelling outwith these areas (Table 1). Their ages range from 16 to 79 years with a mean age for the entire group of 49.34 years. (Tables 2a & b). There is a preponderance of middle-aged individuals in the sample and this can best be seen in Figure 1 where the age-distribution of the male and female patients is shown in comparison with the age-distribution (according to the 1961 Census) of the male and female inhabitants of Edinburgh. As will be seen later, this excess of the middle-aged is of positive advantage to the study since depressive illness shows a similar tendency to occur especially in the middle years of life.
Table 3 compares the pattern of celibacy, marriage and separation in the out-patients with that prevailing among the population of Edinburgh (as measured by the 1961 Census). Again, a considerable discrepancy occurs, there being an apparent excess of married individuals in the out-patient group. This is easily explained and is due mainly to the small number of out-patients between the ages of 16 and 30 years. Although the celibacy rate falls steadily during the course of this period, the 16-30 age-group provides the highest proportion of unmarried individuals in the adult population.
Of the 210 out-patients, 117 were attending a medical clinic and
93 a surgical clinic at the time of interview. Females outnumber the males in both the medical and the surgical categories, particularly in the former, and this can be seen in Table 4 where the patients are shown distributed according to sex and clinic status. The mean age of the surgical patients is found to be somewhat higher than that of the medical patients (Table 2a) and the males are on average rather older than the females. (Table 2b).
The out-patient data will now be examined in detail. The whole group will first be divided according to clinic-status and the medical patients will be compared with the surgical patients to see whether they differ significantly in any of the 14 socio-demographic characteristics already enumerated. Thereafter, the group will be divided according to the sex of the patients and the comparison will be repeated, this time between males and females. The chi-square method is used for the calculations.
Results
Age Distribution (Table 5a & b). As has already been mentioned, the surgical patients are on average a somewhat older group than the medical patients and the males are rather older than the females. The differences in mean age between the groups is not large but it would not be surprising to find that the distribution of the patients' ages varied markedly since, for example, it is quite possible that medical and surgical conditions attack different age-groups. However, it is found that the difference which is present is not a significant one, either between the medical and surgical patients \((p > .30)\) or between the males and females. \((p > .10)\)
The next three items to be considered are civil state, social class and religious persuasion. These are fairly basic demographic factors and any striking difference which might occur within any subgroup of the sample in relation to them would certainly give rise to the suspicion that the entire group of out-patients was an atypical section of the general population.
**Civil State** (Table 6a & b). Patients were recorded as being unmarried, married, widowed or divorced. (The last category included all individuals permanently separated from the spouse, even if not by legal arrangement). The number of divorced persons is so small (2 out of 210) that for purposes of calculation, divorcees are included with the widowed. There is no significant difference between medical and surgical patients \((p > .10)\) or between males and females \((p > .20)\) as to the civil state pattern.
**Social Class** (Table 7a & b). The five-class system according to occupation (General Register Office, 1960) is used but since the number of individuals involved is rather small, the figures for classes 1 and 2 are combined as are the figures for classes 4 and 5. No recent figures for the social class distribution of the population of Edinburgh are available but among the out-patients there is a considerable predominance of social class 3 which probably reflects the middle-class nature of the area. When the medical and surgical patients are compared, there is no significant difference in social class distribution \((p > .50)\) and the same is true of the males and females \((p > .80)\).
Religious Persuasion (Table 8a & b). Patients were asked to state their religious persuasion or the religion prevailing in the childhood home if they themselves professed none. The particular religious affiliation was noted in each case but since only one patient was Jewish and all the rest declared either Protestantism or Roman Catholicism, for the purposes of calculation the patients were denoted either Protestant or non-Protestant. There is no significant difference in the proportion of medical or surgical patients professing these religions \((p > .05)\) and this holds true for the males and females. \((p > .20)\).
Lack of any significant difference occurring until now is gratifying, particularly when fairly small numbers are involved, and it suggests that, in a broad way, the group of out-patients is a fairly homogeneous one. However, the suitability of the out-patients as a comparison group for the depressive patients in this study is under examination and the ensuing items are a fairly rigorous test of this. The characteristics now to be scrutinised are among those which will be used as normal standards when the depressives come to be compared with the controls.
Size of the sibship in the patient's family of upbringing (Table 9a & b). The sibship was taken as consisting of one individual if the patient was an only child. If brothers and sisters were present, the size of the sibship was recorded as being 2, 3, 4, and 5 and over. Sibs who died in early life were included in the calculations but not miscarriages or stillbirths. There is found to be a close resemblance as to sibship-size
between medical and surgical patients ($p > .80$) and between males and females ($p > .50$).
**Number of children in the patient's own family** (Table 10a & b). Only married patients were considered in this respect since no unmarried patient admitted to having had children. The size of the married patient's family was recorded thus: no children, 1 child, 2 to 4 children, 5 or more children. There is no significant difference between the medical and surgical patients ($p > .05$) or between the males and females ($p > .10$) with regard to the number of children in their families.
**Loss of a parent by death before the patient's 16th birthday** (Table 11). Loss of a parent by death before the patient's 16th birthday, parent-loss in either medical and surgical patients ($p > .05$) or in males and females ($p > .10$).
**Loss of a parent for any reason, for at least three months, occurring prior to the patient's 16th birthday** (Table 12a & b). Separation of the patient and his parent for any cause during the former's childhood was recorded and a close resemblance in respect of such separation exists between medical and surgical patients ($p > .50$) and between males and females ($p > .50$).
**Complaint by the patient of a disturbed relationship with a parent in childhood** (Table 13a & b). There is close resemblance in the proportion of medical and surgical patients ($p > .30$) and of males and females ($p > .80$) who make such a complaint.
Positive family history of mental disorder (Table 14a & b). Doubt would be raised about the suitability of the out-patients as comparison-material for depressives if it were shown that certain types of out-patient were more prone to familial mental illness than others. In this context, a positive family history refers to the individual's reporting a psychiatric illness occurring in a relative which was severe enough to require formal medical treatment. In fact, neither the medical and surgical patients \((p > .10)\) nor the males and females \((p > .30)\) report any such differential tendency.
Age of the parents at the time of the patient's birth (Tables 15a & b and 16a & b). There is a tendency for fewer mothers of the surgical patients, as compared with those of the medical patients, to be aged 36 years or more at the time of the patient's birth but the difference does not reach a significant level. \((p > .05)\). There is no significant difference in maternal age between the males and females. \((p > .10)\). As regards paternal age at the time of the patient's birth, there is close resemblance between the medical and surgical patients \((p > .50)\) and between the males and females. \((p > .20)\).
Age of the patient at the time of the parent's death (Tables 17a & b and 18a & b). Since the medical patients are younger on average than the surgical patients, rather more parents of the former group were still alive when the patient was interviewed and the same is true of the females as compared with the males. However, the differences are not significant, either for the age at mother's death or father's death.
Discussion
It must be stressed that the fourteen characteristics just described in detail were selected for comparison purposes before any data had been collected on the out-patients. It was expected that some significant differences would be revealed when they came to be examined in relation to the out-patients and it was hoped that they would be reasonably few. The use of a two-way comparison, that between medical and surgical patients and between males and females, greatly increased the chance that inconsistencies within the whole group would be shown up. That no significant difference at all has been demonstrated suggests a considerable uniformity of environmental background and it seems not unreasonable to propose that this indicates a strong resemblance between this group of out-patients and the population of the Edinburgh area. To contradict this assertion, it would be necessary to show that male and female patients attending medical and surgical out-patient clinics all differed similarly from normal human beings in a considerable number of respects. Since a great many members of the general public become hospital out-patients at one time or another in their lives, this seems a most unlikely situation and it will be accepted in this study that the out-patients who have been examined are not unrepresentative of the general population.
Conclusion
A group consisting of medical and surgical out-patients is suitable for use as a control series in this study.
The Control Series
It was originally intended that, if out-patients proved suitable, a further group would be gathered to act as controls. However, when data-collection on the depressive patients was completed it was found that the depressive group and the group of out-patients already collected were closely matched in a number of ways and it was decided to use these out-patients as controls. Thus, the somewhat unusual situation occurred that the control series was completed before the definitive series was gathered.
Since the depressive patient group consists of individuals aged 16 to 60 years and the out-patient group of individuals aged 16 and over, those out-patients who were aged 61 and over were excluded from the control series in order to make the groups comparable for age-distribution. 47 patients were excluded in this way (23 males, 24 females), leaving a control series consisting of 163 out-patients. (69 males, 94 females). Otherwise, no adjustment was made to the out-patient group which will from now on be referred to as the control series.
Comparability of the depressive series and the control series
By the early part of January, 1964, the study had reached a stage where it was practicable to begin the collection of data on the definitive series of depressive patients. The pilot study had demonstrated the aptness of the experimental method and the information gathered from medical and surgical out-patients had confirmed their suitability as a source of control material. Arrangements were therefore made to visit each of the psychiatric hospitals in the Edinburgh area at weekly intervals, beginning in the second half of January, 1964. On the investigator's first call at each hospital, all the cases of primary depressive illness there at that time were referred for interview. Thereafter, at each weekly visit those patients who had been admitted with depressive illness in the intervening week were seen by the investigator.
A detailed account of the process of data-collection has been given already. It suffices to say that no unforeseen obstacles arose and the study continued without interruption until July 1964 when the 50th male patient was admitted to the depressive series. No further patients were interviewed, since the intention was to obtain approximately 150 depressive individuals and the series now consisted of 153 such persons. In addition there were 30 patients who had been found to suffer from depressive illness secondary to another psychiatric condition, but after interview these were consigned to a separate 'Reject' category.
Normally, it would now have been necessary to obtain the control series. At first it had been intended to match each depressive patient
with a control individual comparable in age, sex, social class and religious persuasion. However, Medical Research Council colleagues advised that this type of comparison often resulted in a most atypical group of controls and that it was greatly preferable to compare matched groups rather than matched individuals. Fortunately, when the data obtained from the depressive patients and that from the medical and surgical out-patients was studied it was found that the two groups resembled each other closely in a number of demographic characteristics. When those medical and surgical out-patients over the age of 60 were excluded, the two series proved even more similar. As has already been explained, it was decided that the out-patients aged 16 to 60 years on whom information was already available could act most satisfactorily as a control series and so the need to seek further controls was obviated.
In order to demonstrate that this control series is suitable, statistical comparison by the chi-square method will be carried out between the depressives and the controls with regard to the following characteristics: distribution of age, sex, social class and religious persuasion.
Preliminary comparison of depressive patients and controls (All tables and figures referred to in this section will be found in Appendix 2, pp 178 to 184).
The depressive series consists of 153 patients (50 males, 103 females) aged 16 to 60 years. These patients live mainly in Edinburgh, Midlothian or West Lothian and only a small number come from elsewhere. As can be
seen in Table 19 the proportion of patients living out with the City of Edinburgh is somewhat higher in the depressives than the controls. This is mainly because two of the psychiatric hospitals (Bangour and Rosslynlee) act as area hospitals for West Lothian and Midlothian respectively. Broadly speaking, however, it can be said that the two groups come from very much the same geographical area.
Age distribution Tables 20 and 21 show that the age-distribution of the depressives and the controls is very similar ($p = > .30$). Table 20 also shows the mean ages of the groups: that of the depressive group is 44.83 years, the males being on average rather older than the females (47.00 years and 42.54 years respectively). This compares very closely with the control group's mean age of 44.43 years (males 47.00 years and females 42.54 years). The two series are therefore closely matched for age.
Sex distribution Table 22 shows the sex-distribution of the depressives and the controls. It can be seen that the proportion of females is somewhat higher among the depressives than among the controls but the difference does not reach a level of significance ($p = > .05$). The two series are satisfactorily matched for sex-distribution.
Social class The social class pattern is very similar in the depressives and the controls. Table 23 shows that the depressives and the controls are both predominantly middle-class groups, probably reflecting fairly accurately the social class pattern in the district. The two series are closely matched for social class.
Religious persuasion. Since the patients of both groups are, almost without exception, either Protestant or Roman Catholic, the religious affiliation has been designated Protestant and non-Protestant, the latter category being entirely Roman Catholic except for one patient who did not claim any faith. Once more there is a close resemblance between the depressives and the controls, (p = > .50) and this is shown in table 24. The two series are therefore closely matched for religious persuasion.
Obviously, the depressives and the controls are very similar as groups and it is accepted that the hypotheses in this study can usefully be tested by comparing the two series. It is suggested that, since the groups resemble each other so much in the above characteristics, any differences which may subsequently be found between them may be accepted with considerable confidence as being real and not due to artefact.
STATISTICAL PROCEDURE EMPLOYED IN PRESENT STUDY
The use of epidemiological techniques in psychiatric research, both generally and with particular reference to this study, is discussed on p. 39. In the interpretation of data obtained by these techniques, statistics plays a vital part. It seems important to stress that statistical method in psychiatric research should be as simple as is consistent with accuracy and usefulness: the application of subtle mathematical manoeuvres to as yet relatively unsophisticated and non-objective psychiatric data is incongruous, save in the hands of the expert statistician.
The delineation of the depressive and control series has already been fully described. Having obtained the appropriate information from the subjects it was then possible to test the hypotheses on which the study was based. Each hypothesis deals with a particular factor said to be related to depressive illness and the main function of the statistical procedure is to demonstrate significant differences between depressives and controls in relation to possession of these factors.
**The method employed**
As each factor is considered in turn, the assumption is made that the factor and the depressive illness are unrelated, so that the proportion of individuals in the control and depressive groups demonstrating the factor should be the same. A table is constructed, dividing depressives and controls according to the presence or absence of the factor. On the assumption of no relation, the difference between the expected and observed numbers of individuals is calculated and thereafter, further calculation
is required to discover whether the differences found are of a magnitude likely to be due to chance or not.
The Chi-square ($x^2$) test is used for this purpose in the present study. The basic formula for this test is:
$$x^2 = \sum \frac{(O_i - E_i)^2}{E_i}$$
where $O_i$ represents the observed frequencies, $E_i$ the expected frequencies and $i$ represents one to $n$, the number of cells in the table.
Where the difference between the observed and expected values is nil, $x^2$ is nil. As the differences increase, the value of $x^2$ increases. As the number of cells in the table increases, the value of $x^2$ can also be expected to increase and this introduces the necessity of considering the degree of freedom which is calculated thus:
$$n = (c - 1)(r - 1)$$
where $n$ is the degree of freedom, $c$ the number of columns and $r$ the number of rows in the table.
With the values of $x^2$ and $n$ to hand it is possible to obtain the level of significance from Fisher's Tables of $x^2$. This significance value denotes the degree of probability of a finding occurring by chance. The larger the value of $x^2$, the smaller becomes the level of probability: i.e. the less is the likelihood that the event is due simply to chance. The 5% level of probability ($p = .05$) is conventionally accepted as the critical level of probability and is used as such in this study. A value of probability ($p$) of .05 or less is considered to be significant.
Where the numbers in one or more of the cells in a four-fold table are small, the value of $x^2$ is more accurately obtained from a modification of the basic formula known as Yates' Correction:
$$x^2 = \frac{(ad - bc - \frac{a + b + c + d}{2})^2}{(a + b)(c + d)(a + c)(b + d)}$$
where $ad$ is the larger of the two cross-products.
**Interpretation of the results**
When the $x^2$ test shows a probability level of 5% or less (i.e., an association occurring by chance only once in twenty occasions or less) it is accepted that the particular factor under examination occurs in depressives and controls with a significantly different frequency and the hypothesis is regarded as confirmed. Where the value is greater than 5%, the hypothesis is not confirmed.
It should be noted that the $x^2$ test is one of association but not of direction of association. The demonstration of a significant relationship between two events by this test does not allow the conclusion that the relationship is one of cause-and-effect, even where one event has occurred earlier in time than the other. However, the demonstration of parameters on which depressives differ significantly from normals provides a necessary starting-point for more interpretative forms of research.
THE HYPOTHESES : RESULTS
THE HYPOTHESES: RESULTS
(All tables referred to in this section will be found in Appendix 3, pp. 85 to 217)
It is now possible, in the light of data obtained from the depressive patients and the controls, to examine each of the ten hypotheses with which this study is concerned.
Of the 153 depressive patients, 102 (66.7%) were diagnosed as suffering from endogenous depression and 51 (33.3%) from neurotic depression. Since these two varieties of depression are considered by many to be discrete illnesses, each hypothesis will have to be considered first of all in relation to the entire depressive group and then in relation to endogenous and neurotic depression.
In addition, it should be noted that 31 (20.3%) of the 153 depressives had, at some time in their lives, made a suicide attempt. Since there is evidence (see p. 34) that individuals who attempt suicide may be excessively prone to parental deprivation in childhood it is possible that the presence of 31 such patients may accentuate any apparent tendency the depressives have to an excess of childhood parent-loss. This has to be taken into account in certain of the hypotheses and will be dealt with where appropriate.
It should be noted that, while the proportions of attempted suicides in the endogenous and neurotic depressive categories are not significantly different, the percentage of attempted suicides is actually a good deal higher among the less severely depressed patients. It is, of course, very likely that successful suicide is commoner among the severely depressed, but it does seem that there is no necessary connection between the severity of a depressive illness and the tendency to make a suicide attempt. (Table 25).
Each hypothesis will now be discussed in turn.
1. Parental deprivation, by death or by other cause, predisposes towards depressive illness
a) Deprivation of parents by death
An individual whose parent died before his 16th birthday is considered to have been deprived of that parent by death during childhood.
Loss of a parent. Here, the sex of the parent is not considered but simply whether the patient lost a parent by death during his childhood. A small proportion report the loss of both parents before the 16th birthday but these cases are not differentiated from the ones who have lost a single parent.
It is found that the proportion of depressives who have lost a parent is very similar indeed to that of the controls (Table 26a) but when the depressives are divided into endogenous and neurotic categories the picture changes considerably. It is now found (Table 26a) that almost twice the proportion of endogenous as/ or neurotic depressives have lost a parent in childhood with the controls lying almost exactly halfway between these two groups. As shown in table 3, the difference between the endogenous and neurotic depressives in this respect is not quite significant \((p > .05)\).
It is of interest to note that the percentages of endogenous and neurotic depressives in this series who have lost a parent in childhood (25.5% and 13.7% respectively) are very similar to those in patients with 'high depressive' and 'low depressive'
scores (27% and 12% respectively) reported by Beck et al. (1963) as having suffered parental bereavement before the age of 16 (see p. 37).
Loss of father. Depressives as a whole show little difference from the controls as to the proportion who have suffered paternal deprivation by death during the entire duration of childhood (Table 26b). However, when childhood is divided into the time-periods 0 to 5, 6 to 10 and 11 to 15 years, it is found that the depressives appear to show a trend towards excess paternal death occurring in the 11 to 15 years period. This trend is not quite significant (p > .05) as Table 28 shows.
Endogenous and neurotic depressives do not differ significantly from the controls as to death of father during childhood.
Loss of mother. There is a slight excess of depressives over controls who have lost mother by death before their 16th birthdays, but this excess does not reach anything like a significant level (Table 26c). The difference is much more marked for endogenous depressives (p > .05) but does not quite reach significance; neurotic depressives show a very slight deficiency of maternal death in childhood. Although the numbers involved are too small for useful statistical analysis, Table 28 shows that there does not seem to be a noticeable trend for loss of mother to occur at any particular time in childhood.
Suicidal depressives. Let it be argued that the presence of patients who have attempted suicide materially alters the proportion of depressive individuals who have suffered parent loss, Table 29 shows percentage parent-loss (loss of either parent, mother and father) in various
sub-categories of depression. It can be seen that removal of the attempted suicide group makes little difference to the figures for parental bereavement in endogenous and neurotic depression.
It may therefore be said that depressives as a whole do not appear to be more prone to lose parents by death before their 16th birthdays.
It is possible, although this study does not provide statistical confirmation, that endogenous depressives are excessively prone to parental bereavement and that neurotic depressives are correspondingly less likely to have suffered such bereavement. This is rather against the theory that neurotic depression owes more of its origin to environmental influences than endogenous depression.
It is also possible, but again not proven, that depressives show an excess of paternal deaths during the age-period 11 to 15 years and of maternal deaths throughout childhood.
There is no evidence whatsoever of the importance of parental death occurring early in childhood.
b) Deprivation of parents by causes other than death
In this section are examined patients whose parents have not died during their childhood but who have been separated from one or other parent for any reason other than death for a period of three months or more before their 16th birthday. The reasons for separation are not differentiated and may be pathological (e.g., desertion by a parent), of
neutral significance (e.g., absence of parent due to nature of his work) or fortuitous (e.g., hospitalization of a parent or the patient).
**Absence of a parent** The percentage of individuals separated from their parents during childhood is somewhat lower in the depressives than in the controls, but not to a significant degree (Table 30). The endogenous depressives display rather less separation from the parents than do the neurotic depressives, but again not significantly so (Table 33) and neither group exceeds the degree of separation shown by the controls.
**Absence of father** The neurotic depressives show a rather higher percentage than the endogenous depressives and the controls of paternal absence in childhood but the difference is small (Table 21). This difference is slightly accentuated when the suicidal depressives are extracted from the endogenous and neurotic series but no significant difference can be demonstrated between any of the depressive sub-groups and the controls as to separation from father during childhood (Table 34).
**Absence of mother** The differences in maternal deprivation between depressives, endogenous depressives, neurotic depressives and the controls nowhere approach significance and none of the depressive groups shows a percentage of separation from mother higher than that of the controls (Tables 32, 33, 34 and 35).
Therefore, in this study it can be stated that the depressives and the controls are very similar in respect of separation from the parents in childhood for a cause other than death.
Conclusion
The hypothesis is not proved. This study finds no confirmation that parental deprivation in childhood, by death or by other cause, predisposes toward depressive illness.
There is, however, inconclusive evidence that the presence of parental deprivation by death may be associated with an increased severity of depressive illness and that depressive illness may be associated with a tendency to excess loss of father by death during the period 11 to 15 years of age and of mother throughout the period of childhood.
2. The age of the parents is of significance in the etiology of depressive illness in that the older the parent at the time of the individual's birth, the more likely is that individual to develop depressive illness.
This hypothesis was devised in an attempt to explain the alleged association between parental deprivation and depressive illness on a basis other than cause-and-effect. If, as this study appears to show, there is no evidence that parental deprivation and depression are especially related to each other, it is scarcely necessary to explain a relationship. However, the question of parental age at the time of the patient's birth is one worth examining in its own right.
The mean age of the fathers of the depressive subjects at the time of birth of the subjects is very similar to the mean age of the fathers of the control individuals; 33.19 years and 33.08 years respectively. (Table 2). The mean age at the birth of the subjects is also very similar in depressives' mothers and controls' mothers; 30.15 years and 30.37 years,
as shown in table 36. When the depressives are divided into endogenous and neurotic categories, the mean parental age at the birth of the subjects remains very constant. (Tables 37 and 38).
The parents of the depressives and the controls have been allotted to age-categories according to their age at the birth of the subjects to see if there is any specific trend in parental age at the time a depressive individual is born. However, comparison between depressives and controls shows marked similarity for paternal and maternal age, (Tables 35 and 36) and the same is true for comparison between endogenous depressives and controls, and neurotic depressives and controls. (Tables 37 and 38).
When endogenous and neurotic depressives are compared for paternal and maternal age, no significant difference is found. (Tables 37 and 38).
**Conclusion**
The hypothesis is not confirmed. There is no evidence that depressive individuals are born of older parents than are normal individuals.
3. Loss of a parent in childhood by death is more likely to cause a tendency to depressive illness than loss of a parent by a cause other than death.
It has already been shown that there is no significant difference between depressives and controls as to deprivation by death of a parent during childhood; nor is there a significant difference between depressives and controls as to the proportion who are deprived of parents by a cause other than death. It therefore cannot be said that either type of
deprivation is of importance in the causation of depression.
It is possible (see p. 106) that loss of a parent by death has an effect in increasing the severity of the depressive illness, an effect which loss by a cause other than death does not seem to have.
**Conclusion**
The hypothesis is not confirmed. There is no evidence that loss of a parent by death during childhood is of greater or lesser importance in the causation of depression than loss of a parent by a cause other than death.
4. Loss of mother during childhood is more important in predisposing to depressive illness than loss of father
Again, it must be stressed that this study has provided no proof that depressives and normals differ significantly as to loss of either parent during childhood, by death or by other cause. It is possible (Table 3) that there is a tendency to excessive paternal mortality in the late childhood (11 to 15 years) of depressives but this has not been proved. There is a slight excess of maternal deprivation by death throughout the childhood of depressives but the difference from the controls is not striking (Table 2). There is no evidence whatsoever that loss of either parent in early childhood is of especial importance in the aetiology of depressive illness.
**Conclusion**
The hypothesis is not confirmed. There is no definite evidence that
loss of mother in childhood is of more importance than loss of father; such evidence as there is points to the opposite conclusion.
5. A disturbed parent-child relationship in childhood is important in producing a tendency to depressive illness
The information in this study about disturbed relationship with a parent in childhood has been obtained from the depressive himself. It could well be maintained that depressive patients, because of their gloomy frame of mind, are more likely to complain of an unhappy childhood than the less-introspective non-depressives and this argument is not easy to refute. It seemed that, during the investigation, the depressives and the controls were very similar in the manner in which they provided information: if anything, the very severely depressed patients were inclined to under-report because of difficulty in rousing themselves to recall events but this is merely an impression.
Because of the relatively small numbers involved, scrutiny of the causes of parent-child disharmony does not provide any indication that a particular type of relationship-disturbance can be incriminated. However, parental strictness and paternal alcoholism do seem to stand out as being somewhat commoner in depressives than controls. (Table 42a).
39
a) Disturbed relationship with a parent (Table 43), It is found that there is a highly significant difference between the depressives and the controls \((p < .01)\) as to a complaint of disturbed relationship with a parent during childhood, the depressives being much more apt to
report such an occurrence. When endogenous depressives and suicidal depressives are separately compared with the controls, this significant difference is maintained. On the other hand, the difference between neurotic depressives and controls is small and non-significant.
It would therefore appear that individuals with the severer types of depression, but not those with less-severe depression, are more liable to a disturbed relationship with their parents than are normals.
b) Disturbed relationship with father (Table 40). No significant difference is found between the depressives as a whole and the controls as to disturbance of relationship with father, but endogenous depressives are significantly prone to such a disturbance ($p < .02$). Neurotic depressives and suicidal depressives show no significant difference from controls in this respect.
It can be said that there is a significant association between endogenous depression, but not neurotic depression, and a disturbed relationship with father during childhood.
c) Disturbed relationship with mother (Table 41). There is a very significant excess of depressives over controls ($p < .01$) who complain of disturbed relationship with mother in childhood and this excess is largely accounted for by the endogenous depressives. There is a somewhat smaller but still significant excess in the suicidal depressives, but there is once more a small and non-significant difference between the neurotic depressives and the controls.
It seems that severe depression, but not the less-severe variety, is
significantly related to a disturbed relationship between the patient and his mother-figure.
**Conclusion**
The hypothesis is partially confirmed. Depressives are excessively prone to a disturbed relationship with mother during childhood. Endogenous depressives are significantly prone to a disturbed relationship with both mother and father during childhood. Neurotic depressives do not differ significantly from normals as to disturbed relationship with either parent.
6. Persons suffering from depressive illness belong to a larger-than-average sibship in their family of upbringing.
It has been postulated, on purely theoretical grounds, that depressives tend to come from larger sibships than normal individuals (see p. 20). However, when the depressive group in this study is compared with the controls no such tendency is found. (Table 13). The endogenous depressives also show little difference from the controls. Neurotic depressives do show a slight tendency (Table 14) to belong to larger sibships but the difference does not approach a significant level.
**Conclusion**
The hypothesis is not confirmed. Depressives appear to belong to sibships which are of the same size as those to which normal individuals belong.
7. The ordinal position of the depressive individual in his sibship is of importance in the etiology of his depressive illness.
Attempts have been made at various times to demonstrate that the
ordinal position of the individual in his sibship has an influence on his liability to mental illness. (see p. 20). However, when depressive patients are assigned their ordinate rank and are compared with controls (Table 44) it is found that the difference between the depressives and the controls is almost non-existent ($p > .95$). When the depressives are divided into endogenous and neurotic categories, the similarities with the controls remain high. There is therefore no evidence that ordinal position has any influence on the etiology of depressive illness.
**Conclusion**
The hypothesis is not confirmed.
There is a qualification to this conclusion. On reflection, it seemed that consideration of the ordinal position in the sibship was an unrewarding exercise anyway. To say that an individual is 4th in his sibship is a relatively meaningless statement. He could, for example be the 4th and last child or he could be the 4th of ten children; the differences of family relationship implied in these two situations may be profound. It therefore seemed more profitable to examine the position of the patient in the sibship relative to the other sibs.
Patients were designated as being first or last in the family and if they did not fall into either of these positions they were considered as belonging to the middle of the family. Only children were divided equally between the first children and the last. When the depressives were compared with similarly designated controls, highly significant differences emerged.
Table 45 shows that depressives as a group differ significantly from the controls as to the position of the individual in the sibship \((p < .05)\). This difference is almost entirely due to the neurotic depressives who differ in a highly significant fashion from the controls \((p < .01)\), whereas the endogenous depressives closely resemble the controls \((p > .30)\).
The neurotic depressives are further compared with the controls in Table 46. Here, each sibship position - first, middle and last - is examined separately. There is no significant difference between neurotic depressives and controls so far as the first child in the family is concerned, but amongst the neurotic depressives there is a significant excess \((p < .01)\) of individuals who belong to the middle of the family and a significant deficiency \((p < .01)\) of those who belong to the last position in the family. In other words, neurotic depressives tend not to be the last child in a sibship.
A supplementary hypothesis has therefore been devised as follows: the position of an individual in his sibship relative to the other sibs is of importance in the aetiology of neurotic, but not endogenous, depressive illness.
This supplementary hypothesis is confirmed.
8. Depressive patients tend to remain unmarried more than do normal individuals and the fertility of married depressives differs from that of married non-depressives.
a) Celibacy in depressive patients. The available evidence regarding
the civil state of depressive patients has been inconclusive till now. (see p. 21). When the depressive patients of this study are compared with the controls as to civil state (Table 47) it is found that a significant difference \((p < .02)\) exists between them. A significant difference \((p < .01)\) also occurs between the neurotic depressives and the controls but not between the endogenous depressives and the controls. Inspection of Table 47 suggests that there is a deficiency of married individuals and an excess of the unmarried among the depressives. When the depressives and the controls are compared for the proportion of individuals in each group who are unmarried, it is confirmed that there is a significant \((p < .05)\) excess of the celibate among the depressives. A similar excess, but not quite reaching a level of significance, is found in the endogenous and neurotic depressives. (Table 48).
Table 47 also shows that there is an excess of divorced or separated individuals among the depressives, but the numbers are too small to allow of accurate statistical assessment.
Hopkinson (1963) differentiates between depressives of early and late onset (i.e., by his definition, where the first attack of affective illness has occurred before or after the age of 50 years) but fails to find either a diminished marriage-rate in manic-depressive illness or any effect on the marriage-rate of the age of onset of the condition. (see p. 21). The data in this study allow his conclusions to be tested, but since the upper age limit of the present series of patients is 60 years there is a comparatively small number whose illness began after the age of 50. Therefore, they have been divided according to whether the first
onset of affective disorder appeared by the age of 45 years (early onset group) or after that (late onset group). When this is done it is found that there is an excess of the unmarried in both groups as compared with controls. This excess is highly significant in the late onset group \((p < .01)\) but is not significant in the early-onset group. (Table 25). This finding is the more striking since a younger group would normally contain a higher proportion of unmarried individuals.
It therefore appears that depressives remain unmarried more often than normal people and this is especially true of depressives whose illness begins later in life.
b) Fertility of married depressives. Since the marriage-rate seems to be lower in depressives than in normals it is likely that the overall fertility of depressives will be below average. So far as the fertility of married depressives is concerned, the evidence to date has been controversial.
When the present depressive series is compared with the controls, little difference is noted between them so far as fertility pattern is concerned (Table 24). Endogenous and neurotic depressives also closely resemble the controls in this respect. When the married depressives belonging to the early and late onset groups are compared with the controls there is again no significant difference (Table 25) although there does seem a tendency for fewer of the early onset individuals to have very large families, possibly because the illness itself has intervened to prevent childbearing.
**Conclusion**
The hypothesis is partly confirmed. Depressive patients are more
likely to remain unmarried than normal individuals and this is particularly true where the illness has begun after the age of 45 years: on the other hand, depressive illness seems to have no marked effect on the fertility of married patients.
9. Depressive patients more often present with a positive family history or severe mental illness than normal individuals.
This hypothesis scarcely needs confirmation in the present study since affective disorder is well-known for its familial tendency. However, it is necessary to show the importance of a positive family history before the next hypothesis can be tested.
A positive family history is taken to mean that a close relative of the subject has had a psychiatric illness severe enough to require medical treatment. Reports of relatives who were 'highly-strung', 'nervous', 'eccentric' etc., were not accepted unless medical intervention had occurred. Relatives accepted for inclusion were: grandparents, parents, sibs, children, aunts and uncles, nephews and nieces and cousins.
As expected, there is found to be a highly significant association between depressive illness and a positive family history of severe mental disorder (p < .01) as can be seen in Table 52. This is also true of endogenous and neurotic depressions.
Conclusion
The hypothesis is confirmed.
10. Individuals with severe depression more often have a positive history of severe mental illness than do individuals with less-severe depression.
It is probable that the positive family history obtained from the depressive individuals is mostly that of affective disorder although no reliability can be placed on the diagnosis reported by the patients themselves. Since depressive illness has a strong heredo-familial background it would be interesting to discover whether it bred true not only in its form but also in its degree of severity. If so, the relatives of severely depressed patients should themselves suffer from a severe degree of affective illness and thus more often be recognized as psychiatrically ill.
Table 53 shows the result of comparison of the endogenous and neurotic depressives for the frequency with which they report a positive family history. Although the percentage of such reports is certainly higher in the endogenous group, the difference is not in any degree significant \((p > .20)\).
Therefore, although the depressive illness itself has a strong tendency to familial occurrence it is likely that its degree of severity is ordained by factors other than heredity.
**Conclusion**
The hypothesis is not confirmed.
MISCELLANEOUS FINDINGS
MISCELLANEOUS FINDINGS
(All tables referred to in this section will be found in Appendix 3b, pp 286 to 227).
1 Maternal mortality and depressive illness
It has already been demonstrated (Table 2c and 4b) that there is no evidence that depressives lose their mothers in childhood more than other people. However, in preparing the data for the hypothesis dealing with that subject, the depressives and the controls were categorized according to their age at the time of mother's death and then compared. (Table 54). It was found that they differed significantly \((p < .02)\) and inspection of the data suggested that the difference was due to an excess of maternal mortality occurring before the depressives' 26th birthday. Table 55 confirms that this excess is a significant one and demonstrates that it occurs mainly in the endogenous depressives. The same trend is discernible in the neurotic depressives but not to a level of significance \((p > .20)\).
It was shown in Table 36 that depressives and controls differ little in maternal age at the time of their birth. However, Table 56a shows that those mothers who die before their depressive children have reached the age of 26 years were considerably older on average when the depressive subject was born, than were the mothers of controls dying in the comparable period. It would seem that the older mothers of depressive patients have a tendency to die somewhat prematurely.
This is a puzzling finding, but Table 56b provides a partial explanation. Here, the causes of death of those mothers who died before the individual's
26th birthday are recorded. The numbers are too small for statistical evaluation but it can be seen that much of the difference in maternal mortality between depressives and controls is accounted for by an excess of mothers of depressives who die of cancer.
Following this clue, the causes of maternal death in all depressives and controls have been studied (Table 57) and it is found that a highly significant excess \((p < .01)\) of the depressives' mothers have been reported as dying of cancer. It is therefore suggested that some connection exists between cancer in the mother and depression in the child and that a number of mothers dying in middle age of cancer cause the excess of bereavement observed in the under 26 group of depressive patients. The fact that these women are slightly older to begin with probably means that they have reached the danger-period for cancer at a rather earlier stage in the lives of their children.
This finding can be criticized on the grounds that it is based entirely on the patient's evidence, but it seems likely that a diagnosis of cancer, of all diseases, would register itself on the mind of anyone whose mother died of it. It is interesting to note that depressives and controls show no significant difference in the age at which they lose father by death (Table 58) and paternal death due to cancer occurs to an almost identical degree \((p > .50)\) in both groups. (Table 59). This tends to highlight the significance of the findings for the cause of death in mother.
It is not possible, on available evidence, to speculate whether there is some constitutional link between depression and cancer, or
whether the suffering undergone by the mother who develops cancer has some predisposing effect towards depression in the child. The latter explanation seems a little more likely since the cancer occurring in the mothers does not appear to be of a specific site.
**Conclusion**
Endogenous depressive illness is significantly related to an excessive maternal mortality occurring before the depressive's 26th birthday. This may be connected with a significant relationship which exists between depression and a history of death due to cancer in the mother.
2. **Twins and depressive illness**
It has proved impossible to obtain an accurate estimate from the literature of the proportion of adults in the general population who are twins. Slater (1953) suggests that about 2.2% of British adults are twins but admits that this is only an informed guess. Gates (1952) points out that the incidence of twin births appears to vary widely with race, stature and geographical location, but that in Western Europe and the United States about 1 birth in 30 produces twins. This means that approximately 1 newborn infant in 40 (2.5%) is an individual twin. Since twins are usually smaller at birth than other children and are excessively prone to intercurrent illnesses their mortality rate is somewhat higher than normal. It is probable that between 1 in 40 and 1 in 50 of the adult British population are twins.
In this study, the control group of 163 medical and surgical out-patient
contains 3 individual twins and this proportion (1 in 54.3) agrees quite closely with the estimated figure. The ages of these three twins are 37, 44 and 58 (average 46.33 years), all are male and all had partners of the same sex although two lost their partners in infancy.
The situation is quite strikingly different among the depressives. Here 11 twins have been identified in 153 patients, giving the very high proportion of 1 in 13.9. Of the 11, 5 are same-sex twins: one individual was aware of being an identical twin and a second of being non-identical, but the other same-sex twins were unable to be certain of this. 4 of the twins had lost their partner in infancy.
Only one of the depressives reported that the twin-partner had ever required psychiatric treatment; she is the identical twin and she stated that her sister had required electroconvulsive therapy for a depression similar to her own. Another twin, a male, reported that his twin brother was nervous but had never sought treatment.
The twins seem to occur in fairly even proportion among depressive males and females: 4 out of 50 males and 7 out of 103 females. This also holds for the distribution between endogenous (7 out of 102) and neurotic (4 out of 51) depression. As a group, the depressive twins are considerably younger on average than the rest of the depressives: 37.55 years as opposed to 45.45 years for non-twins.
It is interesting to note that none of the twins, either depressives or controls, has lost a parent by death before the age of 24 years, but 5 (45.5%) of the depressive twins report a disturbed relationship with father
in childhood, 6 depressive twins (54.5%) have a positive family history of severe mental illness; 5 of the 6 suffer from endogenous depression, (i.e., 5 out of the 7 twins with endogenous depression have a positive family history).
None of the depressive twins has a history of affective illness occurring more than four years prior to the present episode but this may be because they are a comparatively youthful group. The average age of first onset of affective illness is 36.48 years as compared with 39.26 years for the depressives as a whole. While it does look as though these twins are liable to develop depression at an earlier age than other people, the numbers are too small to provide statistical confirmation of this.
Since none of the twins has a long history of affective disorder it is not possible to compare them with the other depressives to discover whether they require more frequent admissions to hospital than non-twins. It is certainly possible, if depression begins earlier in twins, that they will be readmitted to hospital more often than non-twins and so cause an apparent increase in the numbers occurring within a given time. However, this seems unlikely to cause such a striking excess as has been obtained in the present series.
So far as can be discovered, there has been no previous suggestion of an excess of twins in a group of patients suffering from depressive illness,
Conclusion
In the present group of depressive patients twins are present greatly in excess of the expected number. These depressive twins are notable for:
1) the early age at which the first onset of effective illness occurs;
2) the high proportion who have a positive family history of severe mental illness;
3) the high proportion who report a disturbed relationship with father in childhood;
4) the complete absence of parental deprivation by death during the individual's childhood and early adult life.
3. Gynaecological operations and depressive illness
When the medical and surgical out-patients who eventually became the control series were interviewed, note was taken of the presenting physical complaint and also of any previous physical illness. To ensure uniformity of data, similar enquiries were subsequently made of the depressive patients.
As a result, it was found that 10 (9.7%) of the 103 female depressive patients had undergone some form of gynaecological operation since the beginning of 1963: that is, in approximately the year prior to interview. At least 6 of these 10 women had had a dilatation and curettage which is often a non-specific form of operation. In the similar period prior to interview only 2 (2.1%) of the 94 female control patients had undergone any form of gynaecological procedure.
The numbers are too small for useful statistical assessment but it is interesting that such a comparatively high proportion of female depressive in-patients should have so recently required gynaecological intervention.
It is possible that an operation could have precipitated a depressive illness but Hopkinson (1963b) has pointed out that a considerable proportion of depressive individuals suffer from a prolonged prodromal phase before
frank symptoms of affective illness appear. It seems not unlikely that a number of these women have attended the gynaecologist while in the early stage of a depressive illness and that their complaints relating to the genital tract were somatic symptoms of depression.
**Conclusion**
It is probable that many women in the prodromal stage of depressive illness undergo unnecessary operative procedures because the psychological basis of their physical complaints is not appreciated.
4. **Readmission of depressive patients to hospital**
Nowadays it is the enlightened policy of most psychiatric hospitals to discharge in-patients as soon as treatment has been effective and this of course applies to patients with affective disorder. However, it must be emphasized that even the individual attack of depression can be very prolonged and this long-term aspect of the illness does not seem to be greatly affected by modern treatment.
It is noteworthy that, of the 153 depressive in-patients in this study, 33 (21.6%) have had a previous period of in-patient treatment for affective illness within the year prior to interview and all but 3 of these suffer from endogenous depression. 60 (39.2%) of the 153 depressives have had at least one previous admission in the five years prior to this present attack and of those, 51 are endogenous depressives.
There is no need to stress the tendency to recurrence of depressive illness but these figures indicate how high the recurrence rate may be.
It is obvious that close supervision of depressive patients, especially those with endogenous depression, is required for a considerable period after discharge from hospital.
**Conclusion**
21.6% of the depressive subjects in this study (29.4% of those with endogenous depression) had a previous period of in-patient psychiatric treatment within the year prior to their present admission. 39.2% of the depressive subjects (50% of the endogenous depressives) had at least one previous admission to hospital for psychiatric treatment in the five years prior to the present admission.
5. Loss of a sib by death during childhood and the subsequent development of depressive illness
The loss of a child is likely to cause considerable grief in a household and it seems possible that the sibs of the dead child would be deeply impressed by the event. On an assumption similar to that which claims parental deprivation as an aetiological factor in depression it was decided to ascertain whether depressives were more often deprived of sibs during their childhood than were normals.
In the course of obtaining information about the size of the individual's sibship each person was asked whether his parents had lost any children by death, so a record of sibling mortality was available for both depressives and controls. Examination of these data shows that a similar number of depressives and controls report the death of a pre-adult sib
in their families ($p > .50$): 35 (22.9%) of depressives and 34 (20.9%) of controls. (Table 60).
When the deaths are divided into those which occurred before the subject's birth and those which occurred during his childhood, a slight excess of the latter is found. (Table 61). This excess is not significant ($p > .30$). It therefore seems that child-mortality is not excessively high in the depressive's family of upbringing and the depressive himself does not suffer bereavement of a sib during childhood to a significantly greater degree than normal.
**Conclusion**
There is no evidence that losing a sib by death during childhood predisposes an individual to depressive illness.
DISCUSSION OF THE RESULTS
DISCUSSION OF THE RESULTS
A leading article in the British Medical Journal (1964) bemoans the prevailing vagueness about the nature of depression and the lack of any generally acceptable classification of the illness. This situation is not unique to depression which is, if anything, one of the more readily-definable of the psychiatric syndromes. It is obviously difficult to investigate mental illness when it is known beforehand that the basic premises, methods and results of the research will fail to find general approval. This sometimes engenders the nihilistic attitude that diagnosis is irrelevant in psychiatry. This study has been conducted in the opposite belief: namely, that the present imprecision in diagnosis must be improved upon.
Partly towards this end, an attempt has been made to diagnose the cases under study as carefully as possible and to accept only those cases which most psychiatrists would agree as suffering from a primary depressive illness. By this means it is hoped that, should the findings be considered worthy of confirmation, it will be comparatively simple for other workers to be certain that they are examining the same condition.
As well as this it is obviously important when studying a particular condition to eliminate as many irrelevant variables as possible. It is more than possible that previous studies on the relationship between parental deprivation and depression have produced false results because the patients were not cases of pure depression. Unfortunately it is
almost impossible to find an uncontaminated case, but the nearer to the ideal one gets in this type of research the greater will be the confidence that any correlations demonstrated will be referable to the illness under examination. The depressives in this study are perhaps not typical of depressives as a whole but it can surely be said that each one is suffering from a typical case of depressive illness and the aim of this research is to investigate such cases. That the patients are hospitalized and probably represent the severer types of depression may be advantageous since it seems not unlikely that the more severe the depressive illness the grosser and more easily recognizable may be the aetiological factors which are being sought.
It is accepted that a retrospective method of study is potentially inaccurate and it is agreed that information obtained from patients alone may sometimes be suspect. For this reason, the information sought was simple and factual and of a type which the layman could fairly be expected to provide. In the event, it seemed that the patients, even the iller ones, were able to respond much more satisfactorily than was at first feared would be the case. In any case, this study is not intended to produce the last word in its field. Rather, it represents an attempt to discover facts about certain aspects of the background of depressive illness. These facts may be valuable in themselves but their main purpose is to provide a basis for the more efficient deployment of future research into the condition.
The importance of careful design of a research-project is often overlooked in psychiatry. Research must be planned so that the results
avoid the charge of being either esoteric or patently unsubstantiated by facts. The planning of the present study was a prolonged affair but this paid handsome dividends because the investigation, once under way, ran perfectly smoothly.
A vital part of the investigation was the obtaining of a suitable control series and it will be noted that this process took almost as long as the collection of data on the depressive patients. Such attention to detail is essential. One of the surprising findings in the study is the high frequency of parental death in childhood among the controls, a group in which there is no reason to believe that childhood bereavement is excessively high. It appears to be quite normal for approximately one-fifth of the population to lose a parent by death before the 16th birthday and lack of appreciation of this has probably led a number of workers into quite erroneous speculation about a relationship between bereavement and mental illness.
As a by-product of the present study, it is possible that other workers may consider medical and surgical out-patients attending a general hospital as potentially suitable to act as controls for some types of psychiatric research.
Wherever necessary, the terms used in this research have been defined in the interests of communication. The objection may be raised that at times the undefinable has been defined with consequent concretization of essentially abstract concepts. It is suggested that the apparent specificity
of some of the results will encourage belief in the meaningfulness of the concepts employed and vindicate the objective outlook adopted in the study.
The results of the study are fairly wide-ranging and a number of them are quite unexpected. They have been placed under four general headings: aspects of parental deprivation; demographic characteristics; parental morbidity and mortality; miscellaneous findings. Each category will be discussed in turn.
Aspects of parental deprivation
In the review of the literature an attempt was made to show that much of present-day thinking on parental deprivation and its relation to mental illness has been derived from studies performed on children raised in bad institutional conditions. The evidence that the profound psychological ill-effects often observed in these children resulted from separation from the parents, especially mother, was never good, but the concept has been widely accepted. Parental deprivation in relation to depressive illness has been the subject of a number of inconclusive investigations (p. 36) but the tendency of psychoanalytic psychiatry to equate mourning for the loved object with depression has led many people to believe that an association between loss of a parent in childhood and the subsequent development of depressive illness has been proved.
Unfortunately for these theories, the first negative finding in this study is that depressives as a whole are no more apt to lose parents by death during childhood than are the controls. It is true that there is a small excess of depressives losing mother before the 16th birthday but
the difference does not approach a significant level and there is certainly no hint of a raised frequency of maternal mortality in the allegedly crucial first five years of life of the depressive patient. Although the literature usually stresses the importance of the mother-child relationship, there is some evidence that loss of the father by death when the individual is aged 11 to 15 years may occur to excess in depressives but the numbers involved are too small to allow statistical certainty.
It may be thought that death of a parent is too unsubtle an occurrence to be of aetiological importance in depression, so separation of the child and parent for causes other than death has been investigated. However, it is found that the depressives are actually very slightly less liable to have been separated from a parent for a period of three months or more during childhood than the controls, the difference not being significant. Neurotic depressives show a small, and once-more non-significant, excess of paternal absence as compared with the controls and this is quite contrary to most of the theory on the effects of childhood parental deprivation.
It therefore appears that neither deprivation of the parent by death nor deprivation by a cause other than death is an important factor in the aetiology of depression itself. However, death of a parent before the individual's 16th birthday may well influence the severity of a
depressive illness when it occurs. This is implied by the finding that endogenous depressives show a considerably higher frequency than the controls, and the neurotic depressives a correspondingly lower frequency than the controls, of parental death during childhood. The differences between endogenous, neurotic and control groups are not significant but almost certainly would be if the numbers involved were larger. It is interesting that the percentages of endogenous and neurotic depressives found to have lost a parent before the 16th birthday (25.5% and 13.7% respectively) are almost identical with the figures obtained for 'high depressed' and 'low depressed' patients (27% and 12% respectively) by Beck et al. (1963) in one of the more painstaking studies of parental deprivation in depression. It is unfortunate that this last-named study contains no control series, but it is suggested that the finding in the present study that neurotic depressives are less liable than controls to suffer from death of a parent during childhood must indicate that parental deprivation is not an aetiological factor in depressive illness.
Although physical separation from the parents does not seem to relate to the aetiology of depression, even if it is capable of increasing the severity of the illness, it is reasonable to enquire whether emotional deprivation in childhood has any important effect. Each patient and control was asked to state if there had been a disturbed relationship with either parent during the period of childhood. The endogenous depressives report such a disturbance with both mother and father to a degree
significantly in excess of that reported by the controls. The neurotic depressives do not differ to any significant extent from the controls. This suggests that there may be some aetiological effect and while the numbers involved do not allow of statistical interpretation it is thought that parental overstrictness and habitual overindulgence in alcohol by father may be important causes of the impaired emotional rapport between the endogenous depressive and his parent.
Following the now-discarded assumption that parental death in childhood might be aetiologically related to depression, it was decided to investigate the death of sibs occurring during the early life of the individual. There is no evidence that depressives are excessively prone to experience the death of a sib.
**Demographic characteristics of depressive patients**
It has been a marked impression when interviewing most of the depressive patients that, apart from their illness, they differ very little from the controls. Without attempting to arrange it so, it was found that the depressive and control series were closely matched for social class and religious persuasion. It is impossible to be dogmatic about these similarities since the depressives in the study are a highly-selected group, but other investigators have had much the same experience (p.17).
The results obtained in the present investigation regarding marriage and fertility in depressive illness must take their place in a
controversial field, but the differences from the controls appear quite striking. It is found that, as an entire group, depressives are significantly more liable than controls to remain unmarried. This is true especially of those patients whose first onset of affective disorder is after the age of 45 years. At first this may appear anomalous since it would seem more likely that an earlier onset of depression would act as a barrier to marriage. Certainly the early onset group has a slightly higher proportion of the unmarried than the controls but the difference is not significant. The explanation of the high rate of celibacy in late-onset depressives is probably that suggested by Šdegard (1946) who finds a higher prevalence of mental illness in the unmarried and who attributes this to an excess of abnormal pre-psychotic personalities which militates against marriage.
In those depressives who are married, the illness appears to have little effect on fertility except that early-onset depressives show a slight tendency to have fewer families of very large size. This effect may well be due to the intervention of the illness itself.
The ordinal position of the patient in his sibship seems to be of no significance in depressive illness but as already explained (p.108) the concept of ordinal position is probably almost meaningless anyway. When the position of the individual in the sibship relative to the other sibs (i.e., whether he is first, middle or last in the family) is observed instead, the results are very interesting indeed. It is found that
neurotic depressives, but not endogenous depressives, have a very marked tendency to be in the middle of the sibship and an equally marked tendency not to be the youngest child. The finding is so positive as to give confidence as to its integrity, but the nature of the data in the study does not allow any interpretation of the phenomenon. It may be that being last in the family protects an individual to some extent from developing depressive illness, despite the view of Munroe to the contrary (p. 20) or it may be that the hurly-burly of life in the middle of the family has a predisposing effect towards depression. The protective effect of being a youngest child seems to be the more likely explanation.
Parental age has proved to be of no significance in the aetiology of depression as there is no tendency for depressives to be born of older parents. Since this possibility was postulated in an attempt to explain an excess of parental deprivation in depression which no longer seems to need explanation, it is not surprising that the finding is negative.
**Parental morbidity and mortality**
A not-expected finding is the highly significant excess of depressives who report a history of severe mental illness in their families. This excess is presumably due in the main to relatives who suffer from affective disorder but the information supplied by the patient-informants is not sufficiently precise to make sure of this. When the severe and less-severe depressives are compared it is found that a slightly higher proportion of the former admit to a positive family history of mental
disorder but the difference is not large. Thus, while the familial tendency of depression is undoubted, it cannot be said that the evidence from this study indicates a similar tendency for the degree of severity of the condition. This may suggest that depression owes its origin to hereditary influences but that the manner in which it manifests itself is determined by environmental factors.
Although depressives have not been found to be unusually liable to suffer from parental mortality in childhood, it has emerged that they exhibit a significant excess over the controls of maternal mortality prior to their 26th birthday. This is mainly due to a relatively large number of mothers of depressives dying when the subjects are aged 16 to 25 years. At first this appears inexplicable but examination of the data indicates that it is associated with a striking tendency for the mothers of depressives to die of cancer. So far as is known, this highly significant association between depression in the child and cancer in the mother has never previously been reported. The finding is emphasized by the fact that no excess of paternal death from cancer is reported by the depressives. Fascinating possibilities are raised here which will require further studies to elucidate. It seems unlikely that the finding indicates a constitutional link between depression and cancer because the cancer appears to be of a variety of types, but the possibility cannot be ruled out. A more reasonable explanation perhaps is that individuals who must perform watch mother die of a prolonged and painful illness when they themselves
are at an impressionable age are somehow predisposed to develop depressive illness in later life. Either way, it would appear that a population at risk of depression has been delineated.
Miscellaneous findings
Another fortuitous discovery which may be of considerable importance is that the depressive series contains much more than the expected number of twins. The previous literature contains no reference, so far as can be seen, to an association between twinning and depression. The depressive twins are in some ways distinct from the other depressives: they tend to develop depression earlier and they show a total lack of parental deprivation by death, but a family history of mental illness and a history of disturbed relationship with father during childhood is commoner. It cannot altogether be an upbringing as a twin which predisposes to depression since a proportion of them have lost their twin-partners in infancy. Is it possible that this is an example of a pre-natal environmental influence which somehow produces a tendency for twins to develop depression with less provocation than other people? Or are depressive families more apt to produce twins?
The rationale of twin studies of hereditary factors in mental illness has been under a good deal of criticism lately. (Tienari, 1964). If it could be shown that concordance-rates for manic-depressive illness are artificially high because twins are in any case more liable to depression than normal this would raise further doubts as to the method's usefulness.
It is somewhat unexpected to discover that attempted suicide is rather more common in neurotic than in endogenous depression. It may be that severely depressed individuals are too retarded to attempt suicide although this seems unlikely since there is a strong association between psychotic depression and successful suicide. Perhaps it is more feasible to suggest that attempted suicide, while often associated with depression, is a separate syndrome with its own precipitating factors and natural history and is fairly independent of the degree of severity of the depression.
Finally there are two findings which are of practical rather than theoretical importance. The first is that a considerable proportion (9.7%) of the females in the depressive series have undergone a gynaecological operation in the year prior to their present admission to hospital, as compared with 2.1% of the control females in the comparable period. 6 of the 10 depressive women involved underwent a dilatation and curettage, a procedure often carried out for want of something more specific to do, and it is a fair likelihood that at least some of these women could have been saved an operation had they been recognized as suffering from a psychiatric disorder. It is obvious that a screening method at the level of the gynaecological clinic is urgently required and that there is great scope for psychiatric education and research in the field of gynaecology.
The second practical finding deals with readmission of depressives to hospital. While it is well known that affective disorders have a phasic tendency and that the readmission rate for depression is necessarily high, it is somewhat alarming to find that 21.6% of all the depressives in the
series (29.4% of the endogenous depressives) have had a previous period of in-patient psychiatric treatment during the year prior to the present admission. Furthermore, it is found that 39.2% of the whole depressive group (exactly half of the endogenous depressives) have had at least one psychiatric admission in the five years before the present admission. It is evident that early readmission is one facet of the policy of early discharge of depressive patients. There is no need to stress that depressives, and especially those admitted with the endogenous form of the illness, require much more prolonged follow-up and supervision than they often receive at present.
**Conclusions**
It is difficult to discuss the rather varied results of this study in overall terms. It seems that the importance of the hereditary aspect of depressive illness has been stressed but there is a strong indication that the expression of the illness -- for example, the age at first onset and the degree of severity -- is influenced to a great extent by environmental factors. These influences may possibly be prenatal, as with twinning; may occur in childhood, as with position within the sibship or disturbed relationship with a parent; or may occur even in adolescence and early adult life, as with death of the mother from cancer. It is as though a number of more or less specific provocative or protective factors exist and as each individual progresses through life he is bound to meet at least some of them. The algebraic sum of their effects, acting on an hereditary predisposition, will apparently determine the manifestation of the depressive illness.
It has not been possible to demonstrate whether the position of an individual in his sibship is a provocative or a protective factor, but if it should be the latter, it raises a topic of great importance. The demonstration of a specific factor protecting against depressive illness would give rise to the hope that other such factors might exist. If these could be discovered, it could ultimately lead to the formulation of more precise therapeutic tools or even to a workable concept of preventive psychiatry.
Obviously, all the findings in this study must be confirmed before they can be finally accepted, but that such apparently specific results can be obtained by the relatively crude methods of present-day psychiatric research is an indication of the wealth of factual material which is to hand. There is certainly no need for a pessimistic attitude in the investigation of psychiatric illness.
**Indications for future research**
All the findings in this study suggest possible lines of enquiry and, for example, each of the factors which appear to predispose towards depression needs to be investigated in greater depth to demonstrate the nature of the relationship between the factor and the illness. As regards the importance of parental deprivation to depressive illness, it is unlikely that further retrospective studies will produce much information of note. It would be more valuable to set up a carefully-designed and controlled ongoing study of a large cohort of infants and actually observe the
immediate and late effects of parental deprivation. This would have to encompass the entire lifetime of the subjects and would be extremely expensive of manpower, time and resources. Whether the results as they relate to parental deprivation would be commensurate with the effort is a matter of some doubt, but certainly there would be great enhancement of knowledge on the development, both normal and abnormal, of the individual.
Of greater immediate interest is the type of research which could produce fairly specific results in the shorter term and perhaps the most fascinating would be the study of depressive twins. If twins are indeed more liable to depression than other people, it might be easier to distinguish in them specific predisposing factors which in turn would provide insight into the aetiology of depression in general.
The association of cancer in the mother and depression in the child suggests two main avenues of research. Obviously there must be psychopathological factors of great interest involved in the relationship and these require exploration in depth. As well as this, it would be most illuminating to ascertain the nature of the relationship: is the cancer a predisposing psychological factor or is there some physical connection between a tendency to cancer and a tendency to depression? If the latter, the prospects for biochemical research are limitless.
Lastly, it is suggested that the gynaecological out-patient clinic would be a most fruitful source of psychiatric material for investigation. Not only must there be a considerable number of almost purely psychiatric
cases attending such a clinic but there must be many patients with an admixture of physical and psychiatric complaints who would provide excellent material for research into psychosomatic aspects of gynaecological illness.
SUMMARY OF RESULTS
SUMMARY OF RESULTS
1. Depressives as a whole show no greater tendency than a group of control individuals to lose a parent by death prior to the 16th birthday.
2. Endogenous depressives are more liable, and neurotic depressives less liable, to lose a parent by death prior to the 16th birthday as compared with the group of control individuals.
3. There is no evidence that death of the mother during childhood is more important in the etiology of depression than death of the father, nor is there evidence that death of the mother in early childhood predisposes the individual to depression.
4. There is no evidence that death of the mother during childhood is more important in the etiology of depression than loss of the mother for a cause other than death.
5. There is no evidence that death of a sib during childhood of an individual predisposes that individual to depressive illness.
6. As compared with controls, endogenous depressives report a highly significant excess of disturbed relationship with both mother and father during childhood whereas neurotic depressives report a frequency of disharmony with the parents very similar to that of the controls.
7. With reference to the disturbed relationship between the endogenous depressive and his parents, parental overstrictness and habitual drunkenness in the father may be factors of importance.
8. The size of the sibship in the individual's family of upbringing appears to have no significant relationship with a tendency to depressive illness.
9. The ordinal position of the individual in his sibship does not predispose to depression but the position of the individual in the sibship relative to the other sibs is significantly related to neurotic depression: neurotic depressives, as compared with controls, show a significant tendency to be middle children and not to be the youngest of the family.
10. There is a significant excess of unmarried individuals in the depressive group, largely due to an excess of the celibate in those depressives whose first onset of affective illness occurred after the age of 45 years.
11. Depressive illness does not have a significant effect in reducing the ability of married individuals to bear children.
12. Depressive patients appear to be very similar to controls with regard to social class distribution and religious persuasion.
13. The age of the parent at the time of the individual's birth is of no significance to the etiology of depressive illness.
14. Depressive patients report a significantly higher frequency of family history of severe mental illness than do the controls.
15. Endogenous depressives do not report a significantly higher frequency of family history of severe mental illness than do neurotic depressives.
16. Endogenous depressives show a significantly greater tendency to lose mother by death before their 26th birthday than do controls.
17. A highly significant excess of depressive patients as compared with the controls, report that mother died of cancer. There is no difference between depressives and controls as to death of father due to cancer.
18. A much higher than expected number of twins is found in the depressive series.
19. Although the difference is not significant, the proportion of individuals who have attempted suicide is somewhat higher among neurotic than among endogenous depressives.
20. 9.7% of the female depressive patients have undergone a gynaecological operation in the year prior to their present psychiatric admission as compared with 2.1% of the female controls.
21. 21.6% of the patients in the depressive series (29.4% of the endogenous depressives) have had a previous psychiatric admission in the year prior to the present admission. 39.2% of all the depressives (50% of the endogenous group) have had at least one previous admission for psychiatric illness in the five years prior to the present admission.
22. 20.2% of the control patients, in whom there is no reason to think that parental deprivation has occurred to excess, have lost at least one parent by death before the 16th birthday.
ACKNOWLEDGEMENTS
ACKNOWLEDGEMENTS
My grateful thanks are due to Professor G.M. Carstairs who gave permission for this study to be carried out and whose help has been freely available throughout its course; to Professor T. Ferguson Rodger for his encouragement and for his advice in planning this thesis; to Dr. W.I.N. Kessel for supplying the initial impetus to the study; and to Dr. G.A. Foulds for his help in the use of the Runwell Symptom-Sign Inventory.
I am very aware of the debt I owe my Medical Research Council colleagues whose suggestions in the stage of planning saved me many pitfalls later on. I am also aware of the privilege it has been to carry out this investigation under the auspices of the Medical Research Council.
I am most pleased to record my thanks to:
the superintendents, consultants and medical staffs of Jordanburn, West House, Craig House, Bangour and Rosslynlee hospitals for the friendliness they showed and the help they gave in allowing me to interview cases;
the consultant physicians and surgeons of the Western General Hospital, Edinburgh, and especially Dr. J.A. Strong, for their enthusiastic co-operation in the gathering of a control series;
the secretarial and nursing staffs in each hospital who helped to ensure that no cases were overlooked;
the secretaries of the Medical Research Council Unit who have helped me during the study; and especially Miss Judith Townroe who has prepared this manuscript and whose amiability has never lessened as she has slowly
disappeared under a mass of typescript;
my wife and children, who can always be relied upon to restore my sense of proportion.
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APPENDIX 1
The outpatient material. Fig. 1, Tables 1 to 18.
FIGURE 1
SEX AND AGE DISTRIBUTION OF THE POPULATION OF THE CITY OF EDINBURGH AND OF THE GENERAL HOSPITAL OUTPATIENTS INTERVIEWED IN THIS STUDY.
EDINBURGH FROM 1961 CENSUS
GENERAL HOSPITAL OUTPATIENTS IN PRESENT STUDY
% MALES % FEMALES
% MALES % FEMALES
Table 1
Place of Residence of the Out-patients
| Place of Residence | No. of Patients |
|-----------------------------|-----------------|
| Edinburgh | 172 |
| Midlothian or West Lothian | 27 |
| Elsewhere | 11 |
| **Total** | **210** |
Table 2a.
Ages of the Out-patients: a) Medical and Surgical
Medical Out-patients
| Age group | Ages of Patients |
|-----------|------------------|
| 16 - 25 | 17 18 20 20 20 |
| | 20 21 22 23 25 |
| 26 - 35 | 26 28 28 28 29 |
| | 29 30 30 33 33 |
| | 34 34 34 34 35 |
| 36 - 45 | 36 36 37 37 37 |
| | 37 39 39 39 39 |
| | 40 41 41 41 |
| | 42 43 43 43 43 |
| | 44 44 45 45 |
| 46 - 55 | 46 46 46 47 48 |
| | 48 49 50 50 50 |
| | 50 50 51 51 51 |
| | 51 52 52 53 53 |
| | 53 54 54 55 55 |
| | 55 55 |
| 56 - 65 | 56 56 56 56 57 |
| | 58 58 58 58 58 |
| | 59 59 59 59 59 |
| | 60 60 60 61 61 |
| | 61 61 61 61 61 |
| | 62 62 62 63 64 |
| | 64 64 |
| 66 and over | 67 68 68 68 72 |
| | 76 77 79 79 |
Surgical Out-patients
| Age group | Ages of Patients |
|-----------|------------------|
| 16 - 25 | 16 21 25 25 |
| 26 - 35 | 27 27 28 30 30 |
| | 32 33 34 34 |
| 36 - 45 | 37 37 37 38 39 |
| | 40 40 40 42 43 |
| | 43 44 44 44 44 |
| | 45 |
| 46 - 55 | 46 47 48 49 49 |
| | 49 49 50 51 51 |
| | 51 51 51 52 53 |
| | 53 54 54 54 55 |
| | 55 |
| 56 - 65 | 56 56 56 57 57 |
| | 57 57 58 58 58 |
| | 58 59 59 59 59 |
| | 59 60 60 60 61 |
| | 61 61 61 63 63 |
| | 64 64 65 65 65 |
| | 65 |
| 66 and over | 66 67 67 67 68 |
| | 68 68 68 72 74 |
| | 75 79 |
Mean age 47.61 years
Mean age 51.52 years
Mean age of the 210 medical and surgical out-patients, 49.34 years.
Table 2b.
Ages of the Out-patients: b) Male and Female
| Age Group | Ages of Patients | Age Group | Ages of Patients |
|-----------|------------------|-----------|------------------|
| 16 - 25 | 16 21 25 25 | 16 - 25 | 17 18 20 20 20 |
| | | | 20 24 22 23 25 |
| | | | 25 |
| 26 - 35 | 27 28 28 33 35 | 26 - 35 | 26 27 28 29 29 |
| | | | 30 30 30 30 32 |
| | | | 33 33 34 34 34 |
| | | | 34 34 34 |
| 36 - 45 | 37 37 37 37 38 | 36 - 45 | 36 36 37 37 37 |
| | 39 40 41 41 43 | | 39 39 39 39 40 |
| | 43 43 44 44 44 | | 40 40 41 42 42 |
| | 44 45 | | 43 43 43 44 44 |
| | | | 45 45 |
| 46 - 55 | 46 46 48 48 49 | 46 - 55 | 46 46 47 47 48 |
| | 49 49 49 50 50 | | 49 50 50 50 50 |
| | 51 51 51 51 52 | | 51 51 51 51 51 |
| | 53 53 53 54 55 | | 52 52 53 53 54 |
| | 55 55 | | 54 54 54 55 55 |
| | | | 55 |
| 56 - 65 | 56 56 56 57 57 | 56 - 65 | 56 56 56 56 57 |
| | 57 57 58 58 58 | | 58 58 58 58 58 |
| | 58 59 59 59 59 | | 59 59 59 59 60 |
| | 59 59 60 60 60 | | 60 60 61 61 61 |
| | 61 61 61 61 61 | | 61 61 61 62 62 |
| | 63 63 64 64 64 | | 62 64 65 65 65 |
| | 64 65 | | |
| 66 and over | 67 67 67 68 68 | 66 and over | 66 67 68 68 68 |
| | 72 75 76 79 79 | | 68 68 72 74 77 |
| | | | 79 |
Mean age 51.92 years
Mean age 47.33 years
Table 3
Civil State of the Out-patients compared with that of the Edinburgh Population
i) Male Out-patients
| Civil State | Out-patients% | Edinburgh Population % |
|-------------|---------------|------------------------|
| Single | 10.9 | 27.1 |
| Married | 79.3 | 67.8 |
| Widowed | 9.8 | 4.5 |
| Divorced | - | 0.6 |
Total: 100% 100%
ii) Female Out-patients
| Civil State | Out-patients% | Edinburgh Population % |
|-------------|---------------|------------------------|
| Single | 11.9 | 28.4 |
| Married | 72.9 | 56.2 |
| Widowed | 13.6 | 14.4 |
| Divorced | 1.7 | 1.1 |
Total: 100% 100%
* Figures obtained from the 1961 Census report.
Table 4.
Distribution of the Out-patients according to Sex and Clinic Status
| Number of Patients | Male | Female | Total |
|--------------------|------|--------|-------|
| Medical | 48 | 69 | 117 |
| Surgical | 44 | 49 | 93 |
| Total | 92 | 118 | 210 |
Table 5
Distribution of the Out-patients according to Age Group
a) Medical-Surgical
| Age Group | No. of Patients |
|-----------|-----------------|
| | Medical | Surgical |
| 16 - 35 | 26 | 13 |
| 36 - 45 | 23 | 16 |
| 46 - 55 | 27 | 24 |
| 56 - 65 | 32 | 31 |
| 66 and over | 9 | 12 |
| Total | 117 | 93 |
\[ \chi^2 = 4.131 \text{ d.f.} \]
\[ p = > .30 \text{ N.S.} \]
b) Male-Female
| Age Group | No. of Patients |
|-----------|-----------------|
| | Male | Female |
| 16 - 35 | 10 | 29 |
| 36 - 45 | 17 | 22 |
| 46 - 55 | 22 | 26 |
| 56 - 65 | 33 | 30 |
| 66 and over | 10 | 11 |
| Total | 92 | 118 |
\[ \chi^2 = 7.340 \text{ d.f.} \]
\[ p = > .10 \text{ N.S.} \]
Table 6
Distribution of the Out-patients according to Civil State
a) Medical-Surgical
| Civil State | Medical | Surgical |
|-------------|---------|----------|
| Single | 16 | 8 |
| Married | 90 | 69 |
| Widowed | 10 | 15 |
| Divorced | 1 | 1 |
Total: 117 93
\[ \chi^2 = 3.592 \quad 2 \text{ d.f.} \]
\[ p = > .10 \quad \text{N.S.} \]
b) Male-Female
| Civil State | Male | Female |
|-------------|------|--------|
| Single | 10 | 14 |
| Married | 73 | 86 |
| Widowed | 9 | 16 |
| Divorced | - | 2 |
Total: 92 118
\[ \chi^2 = 1.677 \quad 2 \text{ d.f.} \]
\[ p = > .20 \quad \text{N.S.} \]
N.B. In the calculation of significances, the figures for divorce were so small that they had to be included with those for the widowed.
Table 7
Distribution of the Out-patients according to Social Class
a) Medical-Surgical
| Social Class | No. of Patients |
|--------------|-----------------|
| | Medical | Surgical |
| 1 and 2 | 29 | 25 |
| 3 | 52 | 46 |
| 4 and 5 | 36 | 22 |
Total: 117 93
\[ \chi^2 = 1.328 \quad 2 \text{ d.f.} \]
\[ p = > .50 \quad N.S. \]
b) Male-Female
| Social Class | No. of Patients |
|--------------|-----------------|
| | Male | Female |
| 1 and 2 | 22 | 32 |
| 3 | 44 | 54 |
| 4 and 5 | 26 | 32 |
Total: 92 118
\[ \chi^2 = 0.292 \quad 2 \text{ d.f.} \]
\[ p = > .80 \quad N.S. \]
Table 8
Distribution of the Out-patients according to Religious Persuasion
a) Medical-Surgical
| Religious Persuasion | Medical | Surgical | \( \chi^2 = 2.755 \) 1 d.f. |
|----------------------|---------|----------|-----------------------------|
| Protestant | 106 | 77 | \( p = > .05 \) N.S. |
| Non-Protestant | 11 | 16 | |
| Total | 117 | 93 | |
b) Male-Female
| Religious Persuasion | Male | Female | \( \chi^2 = 1.354 \) 1 d.f. |
|----------------------|---------|----------|-----------------------------|
| Protestant | 83 | 100 | \( p = > .20 \) N.S. |
| Non-Protestant | 9 | 18 | |
| Total | 92 | 118 | |
Table 9
Size of the Sibship in the Patient's Family of Upbringing
a) Medical-Surgical
| Size of Sibship | Medical | Surgical |
|-----------------|---------|----------|
| 1 | 7 | 8 |
| 2 | 16 | 13 |
| 3 | 21 | 20 |
| 4 | 13 | 10 |
| 5 and over | 60 | 42 |
Total: 117 93
\[ \chi^2 = 1.269 \text{ d.f.} \]
\[ p = > .80 \quad \text{N.S.} \]
b) Male-Female
| Size of Sibship | Male | Female |
|-----------------|------|--------|
| 1 | 4 | 11 |
| 2 | 11 | 18 |
| 3 | 20 | 21 |
| 4 | 11 | 12 |
| 5 and over | 46 | 56 |
Total: 92 118
\[ \chi^2 = 2.640 \text{ d.f.} \]
\[ p = > .50 \quad \text{N.S.} \]
Table 10
Number of Children in the Patient's Own Family
a) Medical-Surgical
| No. of Children | Medical | Surgical |
|-----------------|---------|----------|
| Patient unmarried | 16 | 8 |
| 0 | 20 | 14 |
| 1 | 18 | 19 |
| 2 - 4 | 49 | 49 |
| 5 and over | 14 | 3 |
| Total | 101 | 85 |
\[ x^2 = 6.931 \quad 3d.f. \]
\[ p = > .05 \quad N.S. \]
b) Male-Female
| No. of Children | Male | Female |
|-----------------|------|--------|
| Patient unmarried | 10 | 14 |
| 0 | 10 | 24 |
| 1 | 17 | 20 |
| 2 - 4 | 49 | 49 |
| 5 and over | 6 | 11 |
| Total | 82 | 104 |
\[ x^2 = 4.964 \quad 3d.f. \]
\[ p = > .10 \quad N.S. \]
In both instances, unmarried patients were excluded from these calculations as none admitted to having had children.
Table 11
Loss of a Parent by Death before the Patient's 16th Birthday
a) Medical-Surgical
| No. of Patients | Medical | Surgical |
|-----------------|---------|----------|
| Parent lost | 28 | 13 |
| Parent not lost | 89 | 80 |
| Total | 117 | 93 |
\[ \chi^2 = 3.320 \quad 1 \text{ d.f.} \]
\[ p = > .05 \quad \text{N.S.} \]
b) Male-Female
| No. of Patients | Male | Female |
|-----------------|------|--------|
| Parent lost | 15 | 26 |
| Parent not lost | 77 | 92 |
| Total | 92 | 118 |
\[ \chi^2 = 1.108 \quad 1 \text{ d.f.} \]
\[ p = > .10 \quad \text{N.S.} \]
Table 12
Loss of a Parent due to Any Cause,
for a Period of at least Three Months,
prior to the Patient's 16th Birthday
a) Medical-Surgical
| | No. of Patients |
|------------------|-----------------|
| | Medical | Surgical |
| Parent lost | 52 | 45 |
| Parent not lost | 65 | 48 |
\[ x^2 = 0.310 \quad 1 \text{ d.f.} \]
\[ p = > .50 \quad \text{N.S.} \]
Total: 117 93
b) Male-Female
| | No. of Patients |
|------------------|-----------------|
| | Male | Female |
| Parent lost | 42 | 55 |
| Parent not lost | 50 | 63 |
\[ x^2 = 0.195 \quad 1 \text{ d.f.} \]
\[ p = > .50 \quad \text{N.S.} \]
Total: 92 118
Table 12
Complaint by the Patient of a Disturbed Relationship with a Parent during Childhood
a) Medical-Surgical
| No. of Patients | Medical | Surgical |
|-----------------|---------|----------|
| Disturbed relationship | 18 | 20 |
| No disturbed relationship | 99 | 73 |
\[ \chi^2 = 1.334, 1 \text{ d.f.} \]
\[ p = > .30 \quad \text{N.S.} \]
Total: 117 93
b) Male-Female
| No. of Patients | Male | Female |
|-----------------|------|--------|
| Disturbed relationship | 16 | 22 |
| No disturbed relationship | 76 | 96 |
\[ \chi^2 = 0.048, 1 \text{ d.f.} \]
\[ p = > .80 \quad \text{N.S.} \]
Total: 92 118
Table 14.
Presence of a Family History of Mental Disorder
a) Medical-Surgical
| | No. of Patients |
|----------------------|-----------------|
| | Medical | Surgical |
| Positive family history | 19 | 9 |
| No family history | 98 | 84 |
| Total | 117 | 93 |
\[ x^2 = 1.930 \quad 1 \text{ d.f.} \]
\[ p = > .10 \quad \text{N.S.} \]
b) Male-Female
| | No. of Patients |
|----------------------|-----------------|
| | Male | Female |
| Positive family history | 14 | 14 |
| No family history | 78 | 104 |
| Total | 92 | 118 |
\[ x^2 = 0.485 \quad 1 \text{ d.f.} \]
\[ p = > .30 \quad \text{N.S.} \]
Table 15
Age of Mother at the Time of the Patient's Birth
a) Medical-Surgical
| Mother's age group | Medical | Surgical |
|--------------------|---------|----------|
| 16 - 25 | 35 | 28 |
| 26 - 35 | 53 | 53 |
| 36 and over | 29 | 12 |
Total: 117 93
\[ \chi^2 = 5.220 \quad 2 \text{ d.f.} \]
\[ p = > .05 \quad \text{N.S.} \]
b) Male-Female
| Mother's age group | Male | Female |
|--------------------|------|--------|
| 16 - 25 | 24 | 39 |
| 26 - 35 | 53 | 53 |
| 36 and over | 15 | 26 |
Total: 92 118
\[ \chi^2 = 3.297 \quad 2 \text{ d.f.} \]
\[ p = > .10 \quad \text{N.S.} \]
N.B. In dealing with maternal age at the time of the patient's birth a smaller number of age-categories was utilized than for paternal age (Table 16a & b) as it was surmised that the maternal ages would cluster more closely.
Table 16
Age of Father at the Time of the Patient's Birth
a) Medical-Surgical
| Father's age group | No. of Patients |
|--------------------|-----------------|
| | Medical | Surgical |
| Before 25 | 19 | 16 |
| 26-30 | 35 | 26 |
| 31-35 | 23 | 26 |
| 36-40 | 20 | 11 |
| 41 and over | 20 | 14 |
| Total | 117 | 93 |
\[ \chi^2 = 2.721 \text{ d.f.} \]
\[ p = > .50 \quad \text{N.S.} \]
b) Male-Female
| Father's age group | No. of Patients |
|--------------------|-----------------|
| | Male | Female |
| Before 25 | 12 | 23 |
| 26-30 | 29 | 32 |
| 31-35 | 27 | 22 |
| 36-40 | 11 | 20 |
| 41 and over | 13 | 24 |
| Total | 92 | 118 |
\[ \chi^2 = 5.441 \text{ d.f.} \]
\[ p = > .20 \quad \text{N.S.} \]
Table 17
Age of the Patient at the Time of Mother's Death
a) Medical-Surgical
| Age of the Patient | Medical | Surgical |
|--------------------|---------|----------|
| 0-15 | 12 | 6 |
| 16-25 | 5 | 6 |
| 26-35 | 21 | 19 |
| 36 and over | 38 | 32 |
| Mother still alive | 41 | 30 |
| Total | 117 | 93 |
\[ x^2 = 1.685 \text{ d.f.} \]
\[ p = > .70 \text{ N.S.} \]
b) Male-Female
| Age of the Patient | Male | Female |
|--------------------|------|--------|
| 0-15 | 9 | 9 |
| 16-25 | 5 | 6 |
| 26-35 | 20 | 20 |
| 36 and over | 36 | 34 |
| Mother still alive | 22 | 49 |
| Total | 92 | 118 |
\[ x^2 = 7.291 \text{ d.f.} \]
\[ p = > .10 \text{ N.S.} \]
Table 18
Age of the Patient at the Time of Father's Death
a) Medical-Surgical
| Age of the Patient | Medical | Surgical |
|--------------------|---------|----------|
| 0-15 | 17 | 10 |
| 16-25 | 18 | 11 |
| 26-35 | 25 | 18 |
| 36 and over | 26 | 33 |
| Father still alive | 31 | 21 |
| Total | 117 | 93 |
\[ \chi^2 = 4.731 \text{ d.f.} \]
\[ p = > .30 \quad \text{N.S.} \]
b) Male-Female
| Age of the Patient | Male | Female |
|--------------------|------|--------|
| 0-15 | 8 | 19 |
| 16-25 | 13 | 16 |
| 26-35 | 18 | 25 |
| 36 and over | 35 | 24 |
| Father still alive | 18 | 34 |
| Total | 92 | 118 |
\[ \chi^2 = 9.394 \text{ d.f.} \]
\[ p = > .05 \quad \text{N.S.} \]
APPENDIX 2
Comparability of depressives and controls. Tables19 to 24.
Table 19
Geographical area in which the Depressives and Controls dwell.
| Area | Depressives | Controls |
|-----------------------|-------------|----------|
| | No. of Patients | % | No. of Patients | % |
| City of Edinburgh | 95 | 62.1 | 130 | 79.7 |
| Midlothian/West Lothian | 49 | 32.0 | 22 | 13.5 |
| Elsewhere | 9 | 5.9 | 11 | 6.8 |
| Total | 153 | 100% | 163 | 100% |
Ages of the Depressive Patients
(The ages of the control patients will be found in Appendix I, Tables 2a & b)
Age Group
16 - 25 16, 17, 17, 17, 18, 21, 23, 23, 23, 24, 25, 25.
26 - 35 26, 26, 26, 27, 28, 28, 29, 29, 30, 30, 30, 30,
31, 31, 31, 32, 33, 33, 34, 34, 34.
36 - 45 36, 37, 37, 38, 38, 38, 39, 39, 40, 40, 40,
41, 41, 41, 41, 41, 42, 42, 42, 42, 42,
43, 43, 43, 43, 43, 43, 43, 44, 44, 44,
44, 44, 44, 45, 45, 45, 45, 45, 45.
46 - 55 46, 46, 47, 47, 47, 47, 48, 49, 49, 50, 50,
50, 50, 50, 50, 50, 51, 51, 51, 51, 52, 52,
52, 52, 52, 52, 53, 53, 54, 54, 54, 54,
55, 55, 55, 55.
56 - 60 56, 56, 56, 56, 56, 56, 56, 56, 57, 57, 57,
57, 58, 58, 58, 58, 58, 58, 59, 59, 59,
59, 60, 60, 60, 60, 60, 60, 60, 60, 60.
Mean age of the depressive patients 44.88 years:
Males 47.00 years
Females 42.54 years
Mean age of the control patients 44.43 years:
Males 47.00 years
Females 42.54 years
Table 21
Comparison of depressive patients and controls as to age-distribution
| Age-group | Depressives | Controls |
|-----------|-------------|----------|
| | No. of Patients | % | No. of Patients | % |
| 16 - 25 | 12 | 7.8 | 15 | 9.2 |
| 26 - 35 | 21 | 13.7 | 24 | 14.7 |
| 36 - 45 | 45 | 29.4 | 39 | 23.9 |
| 46 - 55 | 40 | 26.1 | 48 | 29.4 |
| 56 - 60 | 35 | 22.9 | 37 | 22.7 |
Total: 153 100% 163 100%
\[ \chi^2 = 1.425 \quad 4 \text{ d.f.} \quad p = > .80 \quad \text{N.S.} \]
Table 22
Comparison of depressive patients and controls as to sex-distribution
| Sex of the Patient | No. of Patients | % |
|--------------------|-----------------|-----|
| | Depressives | Controls |
| Male | 50 | 69 |
| Female | 103 | 94 |
| Total | 153 | 163 |
$x^2 = 3.117$ 1 d.f. $p > .05$ N.S.
Table 23
Distribution of depressive patients and controls as to social class
| Social Class | Depressives No. of Patients | % | Controls No. of Patients | % |
|--------------|-----------------------------|---|--------------------------|---|
| 1 and 2 | 34 | 22.2 | 42 | 25.8 |
| 3 | 75 | 49.0 | 73 | 44.8 |
| 4 and 5 | 44 | 28.8 | 48 | 29.4 |
Total 153 100% 163 100%
\[ x^2 = 0.719 \quad 2 \text{ d.f.} \quad p = > .50 \quad \text{N.S.} \]
Table 24
Distribution of depressive patients and controls as to religious persuasion
| Religious Persuasion | Depressives No. of Patients % | Controls No. of Patients % |
|----------------------|-------------------------------|----------------------------|
| Protestant | 130 | 85.0 |
| Non-Protestant | 23 | 15.0 |
| Total | 153 | 100% |
| | | 163 |
| | | 100% |
\[ \chi^2 = 0.149 \quad 1 \text{ d.f.} \quad p = > .50 \quad \text{N.S.} \]
APPENDIX 3a
The hypotheses : results. Tables 25 to 53.
Table 25
Comparison of the proportion of endogenous and neurotic depressives who have attempted suicide
Total number of depressive patients: 153
Total number of patients who have attempted suicide: 31
| Patients who have attempted suicide | Endogenous depressives | Neurotic depressives |
|------------------------------------|------------------------|----------------------|
| No. of patients | % | No. of patients | % |
| Non-suicidal patients | 84 | 82.4 | 38 | 74.5 |
| Total | 102 | 100% | 51 | 100% |
\[ \chi^2 = 1.163, \text{ d.f.} = 1, p > .20, \text{ N.S.} \]
Table 26a.
Childhood deprivation due to parental death: comparison between depressives and controls
a) Loss of either parent
| | All depressives | Endogenous | Neurotic | Controls |
|--------------------------|-----------------|------------|----------|----------|
| | No. | % | No. | % | No. | % | No. | % |
| Deprived of parent | 35 | 21.6| 26 | 25.5| 7 | 13.7| 35 | 20.2|
| Not deprived | 120 | 78.4| 76 | 74.5| 44 | 86.3| 130 | 79.8|
| Totals | 155 | 100%| 102 | 100%| 51 | 100%| 163 | 100%|
$\chi^2$ = 0.075 1 d.f. 1.003 1 d.f. 1.061 1 d.f.
Level of significance (p)
> .70 > .30 > .30
N.S. N.S. N.S.
Table 26b.
Childhood deprivation due to parental death: comparison between depressives and controls
b) Loss of father
| | All depressives | Histrionics | Neurotics | Controls |
|--------------------------|-----------------|-------------|-----------|----------|
| | No. | % | No. | % | No. | % | No. | % |
| Deprived of father | 20 | 13.4| 15 | 14.7 | 5 | 9.8 | 22 | 13.5 |
| Not deprived | 133 | 86.9| 87 | 85.3 | 46 | 90.2 | 144 | 86.5 |
| Totals | 153 | 100%| 102 | 100% | 51 | 100% | 163 | 100% |
$\chi^2$ = .010 1 d.f. .084 1 d.f. 0.459 1 d.f.
Level of significance (p)
> .90 > .70 > .30
N.S. N.S. N.S.
Table 26c.
Childhood deprivation due to parental death: comparison between depressives and controls
c) Loss of mother
| | All depressives | Endogenous | Neurotic | Controls |
|--------------------------|-----------------|------------|----------|----------|
| | No. | % | No. | % | No. | % | No. | % |
| Deprived of mother | 17 | 11.1| 14 | 13.7 | 5 | 5.9 | 12 | 7.4 |
| Not deprived | 136 | 88.9| 88 | 86.3 | 48 | 94.1 | 154 | 92.6 |
| Totals | 153 | 100%| 102 | 100% | 51 | 100% | 165 | 100% |
\[ \chi^2 = 1.359, 1 d.f., 2.853, 1 d.f., .002, 1 d.f. \]
Level of significance (p)
- Deprived of mother: > .20
- Not deprived: > .05, > .95
N.S.
| | Loss of Father | Loss of Mother |
|------------------|----------------|----------------|
| **Parent Lost** | | |
| **Not Lost** | | |
| **Father Lost** | | |
| **Not Lost** | | |
| **Mother Lost** | | |
| **Not Lost** | | |
| **Endogenous Depression** | 26 | 87 |
| **Neurotic Depression** | 7 | 46 |
| **Totals** | 33 | 133 |
| **2** | 2,782 | 1,409 |
Level of significance (p)
- > .05
- > .30
- > .20
- N.S.
*2,782 + 1,409 = 4,191*
*746 + 1 = 747*
*1,409 + 1 = 1,410*
Table 28
The period in childhood in which the parent died: comparison between depressives and controls
a) Death of Father
| Period in Childhood | 0-5 years | 6-10 years | 11-15 years | Total |
|---------------------|-----------|------------|-------------|-------|
| Depressives | 5 | 4 | 11 | 20 |
| Controls | 12 | 5 | 5 | 22 |
\[ \chi^2 = 5.203 \quad d.f. = 2 \quad p = > .05 \quad N.S. \]
b) Death of Mother
| Period in Childhood | 0-5 years | 6-10 years | 11-15 years | Total |
|---------------------|-----------|------------|-------------|-------|
| Depressives | 7 | 5 | 5 | 17 |
| Controls | 4 | 4 | 4 | 12 |
Table 29
The percentage of depressives who have been deprived of a parent by death during childhood: comparison between different types of depressive illness in this respect.
| Diagnostic category | Death of either parent | Death of father | Death of mother |
|--------------------------------------|------------------------|-----------------|-----------------|
| All depressives | 21.6 | 13.7 | 41.4 |
| Endogenous depressives | 25.5 | 14.7 | 13.7 |
| Neurotic depressives | 13.7 | 9.8 | 5.9 |
| Non-suicidal endogenous depressives | 23.8 | 13.4 | 14.3 |
| Non-suicidal neurotic depressives | 13.2 | 7.9 | 7.9 |
| Suicidal depressives | 25.8 | 19.4 | 6.5 |
| Controls | 20.2 | 13.5 | 7.4 |
Table 30
Absence of a parent, for a cause other than death, during a period of more than three months in the subject's childhood: comparison between depressives and controls.
a) Absence of either parent
| | All depressives | Endogenous | Neurotic |
|------------------|-----------------|------------|----------|
| Parent absent | 42 | 10 | 5 |
| Total | 111 | 72.5 | 74.5 |
| Totals | 153 | 102 | 51 |
Level of significance (p)
- 54.9 1 d.f. 1.009 1 d.f. .000 1 d.f.
- > .30
- N.S. N.S.
2 193
Table 31
Absence of a parent, for a cause other than death, during a period of more than three months in the subject's childhood: comparison between depressives and controls.
b) Absence of father
| | All depressives | Endogenous | Neurotic | Controls |
|------------------|-----------------|------------|----------|----------|
| Father absent | No. | % | No. | % | No. | % | No. | % |
| | 40 | 26.1| 24 | 23.5| 16 | 31.4| 48 | 29.4|
| Not absent | 113 | 73.9| 78 | 76.5| 35 | 68.6| 115 | 70.6|
| Totals | 153 | 100%| 102 | 100%| 51 | 100%| 163 | 100%|
\[ \chi^2 = 4.26 \text{ 1 d.f.} \quad 1.099 \text{ 1 d.f.} \quad 0.91 \text{ 1 d.f.} \]
Level of significance (p)
> .50 > .20 > .80
N.S. N.S. N.S.
Table 32
Absence of a parent, for a cause other than death, during a period of more than three months in the subject's childhood: comparison between depressives and controls.
a) Absence of mother
| | All depressives | Endogenous | Neurotic | Controls |
|----------------|-----------------|------------|----------|----------|
| No. | % | % | % | % |
| Mother absent | 12 | 7.8 | 9 | 8.8 | 3 | 6.3 | 16 | 10.9 |
| Not absent | 141 | 92.2 | 95 | 91.2 | 48 | 93.7 | 147 | 89.1 |
| Totals | 153 | 100% | 102 | 100% | 51 | 100% | 163 | 100% |
$\chi^2$
- 4.00 1 d.f.
- 0.073 1 d.f.
- 3.36 1 d.f.
Level of significance ($p$)
- > .50
- > .70
- > .50
N.S. N.S. N.S.
Table 32
Comparison of Endogenous and Neurotic Depressives as to absence of parents, for a cause other than death, during a period of more than three months in the subject's childhood.
| Absence of Either Parent | Absence of Father | Absence of Mother |
|--------------------------|------------------|------------------|
| | Father Absent | Not Absent | Father Absent | Not Absent |
| Endogenous depression | 26 | 76 | 24 | 78 | 9 | 93 |
| Neurotic depression | 16 | 35 | 16 | 35 | 3 | 48 |
| Totals | 42 | 111 | 40 | 113 | 12 | 141 |
\[ \chi^2 = 59.1 \text{ d.f.} \]
Level of significance (p)
- \( > .30 \)
- \( > .20 \)
- N.S.
\[ \cdot 110 \text{ 1 d.f.} \]
\[ \cdot 102 \text{ 1 d.f.} \]
\[ > .70 \]
N.S.
Table 74: The percentage of depressives who have lost a parent in childhood for a period of more than three months, due to a cause other than the parent's death: comparison between different types of depressive illness in this respect.
| Diagnostic category | Absence of either parent | Absence of father | Absence of mother |
|--------------------------------------|--------------------------|-------------------|------------------|
| | (Figures percent) | | |
| All depressives | 27.5 | 26.1 | 7.8 |
| Endogenous depressives | 25.5 | 23.5 | 8.8 |
| Neurotic depressives | 31.4 | 34.4 | 6.3 |
| Non-suicidal endogenous depressives | 22.6 | 20.2 | 9.5 |
| Non-suicidal neurotic depressives | 34.2 | 34.2 | 7.9 |
| Suicidal depressives | 32.3 | 32.3 | 3.2 |
| Controls | 34.3 | 29.4 | 10.9 |
Table 35
Age of father at time of the individual's birth: depressives v. controls
Average age of father at time of depressive patient's birth: 35.19 years
Average age of father at time of control individual's birth: 33.08 years
| Age of father at time of birth | Depressives | Controls |
|--------------------------------|-------------|----------|
| - 25 | 24 | 26 |
| 26 - 30 | 36 | 48 |
| 31 - 35 | 43 | 42 |
| 36 - 40 | 29 | 22 |
| 41 - | 23 | 25 |
Totals 152* 163
\[ \chi^2 = 2.536 \quad d.f. = 4 \quad D = > .50 \quad N.S. \]
* 1 patient could provide no information about father's age
Table 36
Age of mother at time of the individual's birth: Depressives vs. Controls
Average age of mother at time of depressive patient's birth: 30.45 years
Average age of mother at time of control individual's birth: 30.37 years
| Age of mother at time of birth | Depressives | Controls |
|--------------------------------|-------------|----------|
| 16 - 20 | 7 | 5 |
| 21 - 25 | 34 | 44 |
| 26 - 30 | 44 | 54 |
| 31 - 35 | 37 | 32 |
| 36 - 40 | 20 | 22 |
| 41 - | 11 | 9 |
| Totals | 153 | 163 |
\[ \chi^2 = 2.335, \text{ d.f.} = 5, p = > .80, \text{ N.S.} \]
Table 37
Age of father at time of the individual's birth: endogenous and neurotic depressives v. controls
Average age of father at time of endogenous depressive's birth: 33.54 years
Average age of father at time of neurotic depressive's birth: 32.49 years
Average age of father at time of control individual's birth: 33.08 years
| Age of father at time of birth | Endogenous | Neurotic | Controls |
|--------------------------------|------------|----------|----------|
| - 25 | 11 | 10 | 26 |
| 26 - 30 | 26 | 10 | 48 |
| 31 - 35 | 26 | 17 | 42 |
| 36 - | 38 | 14 | 47 |
Totals 101* 54 163
Endogenous depressives v. controls $x^2 = 2.980$ 3 d.f. $p = > .50$ N.S.
Neurotic depressives v. controls $x^2 = 2.478$ 3 d.f. $p = > .50$ N.S.
Endogenous depressives v. neurotic depressives $x^2 = 4.161$ 3 d.f. $p = > .20$ N.S.
* 1 patient could provide no information about father's age.
Table 38
Age of mother at time of the individual's birth: endogenous and neurotic depressives v. controls
Average age of mother at time of endogenous depressive's birth: 30.40 years
Average age of mother at time of neurotic depressive's birth: 29.35 years
Average age of mother at time of control individual's birth: 30.37 years
| Age of mother at time of birth | Endogenous | Neurotic | Controls |
|--------------------------------|------------|----------|----------|
| - 25 | 25 | 16 | 46 |
| 26 - 30 | 30 | 14 | 34 |
| 31 - 35 | 27 | 10 | 32 |
| 36 - 40 | 20 | 11 | 34 |
Totals 102 54 163
Endogenous depressives v. controls $\chi^2 = 1.948$ d.f. $P => .50$ N.S.
Neurotic depressives v. controls $\chi^2 = 0.525$ d.f. $P => .90$ N.S.
Endogenous depressives v. neurotic depressives $\chi^2 = 1.346$ d.f. $P => .70$ N.S.
Table 39
Comparison by the individual of a disturbed relationship with a parent during childhood: comparison between depressives and controls
| | Depressives | Controls |
|----------------|-------------|----------|
| | Indogenous | Neurotic | Unioial |
| | No. | % | No. | % | No. | % | No. | % |
| Disturbed relationship | 49 | 32.0 | 37 | 36.3 | 12 | 23.5 | 11 | 35.5 |
| No disturbance | 104 | 68.0 | 65 | 63.7 | 39 | 76.5 | 20 | 64.5 |
| Totals | 153 | 100% | 102 | 100% | 51 | 100% | 31 | 100% |
\[
\chi^2 = 8.548 \quad d.f. = 11.666 + d.f. = 0.823 \quad d.f. = 4.960 \quad d.f.
\]
Level of significance (p) < .01 < .01 > .01 < .05
Sig. N.S. Sig.
Comparison between each category of depression and the controls
Table 40
Complaint by the individual of a disturbed relationship with father during childhood:
Comparison between depressives and controls
| | All depressives | Indigenous | Neurotic | Suicidal | Controls |
|------------------------|-----------------|------------|----------|----------|----------|
| | No. | % | No. | % | No. | % |
| Disturbed relationship | 36 | 23.5 | 28 | 27.4 | 8 | 15.7 | 8 | 25.8 | 25 | 15.2 |
| No disturbance | 117 | 76.5 | 74 | 72.6 | 43 | 81.5 | 23 | 74.2 | 158 | 84.7 |
| Totals | 153 | 100% | 102 | 100% | 51 | 100% | 31 | 100% | 163 | 100% |
\[ \chi^2 = 3.136, 1 \text{ d.f.} \quad 5.754, 1 \text{ d.f.} \quad .005, 1 \text{ d.f.} \quad 1.976, 1 \text{ d.f.} \]
Level of significance (p) > .05 < .02 > .95 > .10
Sig. Sig. N.S.
Comparison between each category of depression and the controls
Table 4.1
Complaint by the individual of a disturbed relationship with mother during childhood: comparison between depressives and controls.
| | Depressives | Controls |
|------------------------|-------------|----------|
| | No. | % | No. | % | No. | % | No. | % |
| Disturbed relationship | | | | | | | | |
| 28 | 16.3| 22 | 21.6| 6 | 14.8| 6 | 19.4| 11 |
| No disturbance | 125 | 84.7| 80 | 78.4| 45 | 88.2| 80.6| 152 |
| Totals | 153 | 100%| 102 | 100%| 51 | 100%| 31 | 100%|
\[ \chi^2 = 9.698 \quad 1 \text{ d.f.} \quad 12.666 \quad 1 \text{ d.f.} \quad 4.261 \quad 1 \text{ d.f.} \quad 5.235 \quad 1 \text{ d.f.} \]
Level of significance (\( p \)) < .01 < .01 > .20 < .05
Sig. Sig. N.S. Sig.
Comparison between each category of depression and the controls.
Table 42a.
Causes of disturbed relationship with a parent during childhood, as related by the patient
a) Disturbed relationship with father or father-figure
| Cause of disturbance | Depressives | Controls |
|---------------------------------------|-------------|----------|
| Habitual drunkenness of father | 9 | 5 |
| Father overstrict | 8 | 4 |
| Father aloof or absent much of the time | 7 | 7 |
| Father's quarrelsomeness | 3 | 3 |
| Physical illness | 3 | 3 |
| Mental illness | 3 | 1 |
| Poor relationship with stepfather | 3 | 2 |
Totals: 36 25
Table 42b.
Causes of disturbed relationship with a parent during childhood, as related by the patient
b) Disturbed relationship with mother or mother-figure
| Cause of disturbance | Depressives | Controls |
|---------------------------------------|-------------|----------|
| Mother overstrict | 7 | 1 |
| Mother alcoholic or absent much of the time | 5 | 4 |
| Mother's quarrelsome ness | 7 | 2 |
| Physical illness | 5 | 1 |
| Poor relationship with stepmother | 2 | 3 |
| Adultery committed by mother | 2 | 0 |
| Totals | 28 | 11 |
Table 43
The size of the sibship in the patient's family at upbringing: comparison between depressives and controls
| Size of Sibship | All depressives | Endogenous | Neurotic | Controls |
|-----------------|-----------------|------------|----------|----------|
| | No. | % | No. | % | No. | % | No. | % |
| 1 (only child) | 13 | 8.5 | 11 | 10.8| 2 | 3.9 | 13 | 8.0 |
| 2 | 19 | 12.4| 12 | 11.8| 7 | 13.7| 28 | 17.2|
| 3 | 27 | 17.6| 21 | 20.6| 6 | 11.8| 36 | 22.1|
| 4 | 24 | 13.7| 15 | 14.7| 6 | 11.8| 17 | 10.4|
| 5 | 22 | 14.4| 15 | 14.7| 7 | 13.7| 18 | 11.0|
| 6 | 15 | 9.8 | 10 | 9.8 | 5 | 9.8 | 15 | 9.2 |
| 7 and over | 36 | 23.5| 18 | 17.6| 18 | 35.2| 36 | 22.4|
| Totals | 153 | 100%| 102 | 100%| 51 | 100%| 163 | 100%|
$\chi^2$ = 3.523 d.f. = 4.024, 6 d.f. = 5.967 5 d.f.
Level of significance ($p$)
> .70 > .50 > .30
N.S. N.S. N.S.
a Categories 1 and 2 combined because of small numbers
Table 14
Original position of the individual in his family of upbringing:
Comparison between depressives and controls
| Original position | All depressives | Endogenous | Neurotic | Controls |
|-------------------|-----------------|------------|----------|----------|
| | No. | % | No. | % | No. | % | No. | % |
| 1 | 45 | 29.4| 32 | 31.4| 13 | 25.4| 53 | 32.5|
| 2 | 36 | 23.5| 25 | 24.5| 11 | 21.5| 40 | 24.5|
| 3 | 28 | 18.3| 19 | 18.6| 9 | 17.6| 23 | 14.4|
| 4 | 12 | 7.8 | 6 | 5.9 | 6 | 11.8| 12 | 7.4 |
| 5 | 8 | 5.2 | 5 | 4.9 | 3 | 5.9 | 11 | 6.7 |
| 6 | 9 | 5.9 | 7 | 6.9 | 2 | 3.9 | 9 | 5.5 |
| 7 and over | 15 | 9.8 | 8 | 7.8 | 7 | 13.7| 15 | 9.2 |
| Totals | 153 | 100%| 102 | 100%| 51 | 100%| 163 | 100%|
χ² = 1.315 d.f. = 6 p = 1.697 d.f. = 2 p = 2.459 d.f. = 4 p = 2.8
Level of significance (p)
> .95 > .90 > .50
N.S. N.S. N.S.
* Categories 5, 6, 7 and over combined in this calculation because of the small numbers involved.
Table 45 Position of the individual in family of upbringing in relation to other side: comparison between depressives and controls
| Position in family | All depressives | Homogeneous | Neurotic | Controls |
|--------------------|-----------------|-------------|----------|----------|
| | No. | % | No. | % | No. | % | No. | % |
| First | 37.5| 24.5| 25.5| 25.0| 12 | 23.5| 46.5| 28.5|
| Middle | 76 | 49.7| 45 | 44.4| 31 | 60.7| 59 | 36.2|
| Last | 39.5| 25.8| 31.5| 30.9| 8 | 15.7| 57.5| 35.3|
| Totals | 153 | 100%| 102 | 100%| 51 | 100%| 163 | 100%|
$^2$
| Level of significance (p) | $x \cdot 05$ | $> \cdot 30$ | N.S. | SIG. | < $\cdot 01$ |
N.B. 1. If the patient is neither first nor last in the sibship he is considered as belonging to the middle of the family.
2. Only children are counted as half-first, half-last, in the family.
Table 46
Position of the neurotic depressive patient in family of upbringing in relation to other sibs: comparison with controls
a) Individuals who are first in the family compared with the rest
| | First in the family | The remainder | Total |
|----------------------|---------------------|---------------|-------|
| Neurotic depressives | 12 | 39 | 51 |
| Controls | 46.5 | 116.5 | 163 |
\[ x^2 = 4.68 \quad 1 \text{ d.f.} \quad p = > .30 \quad \text{N.S.} \]
b) Individuals in the middle of the family compared with the rest
| | Middle of the family | The remainder | Total |
|----------------------|----------------------|---------------|-------|
| Neurotic depressives | 31 | 20 | 51 |
| Controls | 59 | 104 | 163 |
\[ x^2 = 9.740 \quad 1 \text{ d.f.} \quad p = < .01 \quad \text{Sig.} \]
c) Individuals who are last in the family compared with the rest
| | Last in the family | The remainder | Total |
|----------------------|--------------------|---------------|-------|
| Neurotic depressives | 8 | 43 | 51 |
| Controls | 57.5 | 105.5 | 163 |
\[ x^2 = 7.004 \quad 1 \text{ d.f.} \quad p = < .01 \quad \text{Sig.} \]
N.B. Only children are counted as half-first, half-last
Table 4.7
Distribution of depressive patients and controls as to civil state
| Civil State | Depressives | Endogenous | Neurotic |
|----------------------|-------------|------------|----------|
| | No. | % | No. | % | No. | % | No. | % |
| Unmarried | 37 | 24.2 | 24 | 23.5 | 13 | 25.4 | 23 | 14.1 |
| Married | 98 | 64.1 | 69 | 67.6 | 29 | 56.8 | 129 | 79.1 |
| Widowed | 11 | 7.2 | 7 | 6.9 | 4 | 7.8 | 9 | 5.5 |
| Divorced or separated| 7 | 4.5 | 2 | 2.0 | 5 | 9.8 | 2 | 1.2 |
| Totals | 153 | 100% | 102 | 100% | 51 | 100% | 163 | 100% |
$\chi^2$ = 8.872 2 d.f.
Level of significance (p) < .02 > .10 < .01
Sig. N.S. Sig.
As the numbers of divorced and separated individuals is small, for the purposes of this calculation they have been included in the group of widowed individuals.
Table 48
Comparison between depressives and controls as to celibacy
| Civil State | All depressives | Endogenous | Neurotic | Controls |
|-------------|-----------------|------------|----------|----------|
| | No. | % | No. | % | No. | % | No. | % |
| Unmarried | 37 | 24.2| 24 | 23.5 | 15 | 25.4 | 25 | 12.1 |
| Others | 116 | 75.8| 78 | 76.5 | 38 | 74.6 | 140 | 85.9 |
| Totals | 153 | 100%| 102 | 100% | 51 | 100% | 165 | 100%|
$\chi^2$
$5.139$ 1 d.f.
$3.802$ 1 d.f.
$3.558$ 1 d.f.
Level of significance ($\beta$)
< .05
> .05
> .05
Sig.
N.S.
N.S.
Table 42
Celibacy in patients whose depression is of early or late onset: comparison with controls.
| Civil state | Early onset depressives | Late onset depressives | Controls |
|-------------|-------------------------|------------------------|----------|
| | No. | % | No. | % | No. | % |
| Unmarried | 21 | 20.4| 16 | 32 | 23 | 14.1|
| Others | 82 | 79.6| 34 | 68 | 140 | 85.9|
| Totals | 103 | 100%| 50 | 100%| 163 | 100%|
$\chi^2$
1.838 1 d.f. 8.051 1 d.f.
Level of significance (p)
> .10 < .01
N.S. 51%
Table 50
The number of children in the married individual's family:
A comparison between depressives and controls
| No. of children | All depressives | Endogenous | Neurotic | Controls |
|-----------------|----------------|------------|----------|----------|
| | No. | % | No. | % | No. | % | No. | % |
| 0 | 14 | 12.1| 10 | 12.8| 4 | 10.5| 24 | 17.4|
| 1 | 25 | 21.6| 20 | 25.6| 5 | 13.2| 28 | 20.0|
| 2 | 40 | 34.5| 27 | 34.6| 13 | 34.2| 42 | 30.0|
| 3 | 23 | 19.8| 14 | 17.9| 9 | 23.7| 19 | 13.6|
| 4 | 5 | 4.3 | 2 | 2.6 | 3 | 7.9 | 14 | 10.0|
| 5 and over | 9 | 7.6 | 5 | 6.4 | 4 | 10.5| 13 | 9.3 |
Totals | 116 | 100%| 78 | 100%| 38 | 100%| 140 | 100%
$\chi^2$ | 6.022| 5 d.f.*| 5.757| 4 d.f.*| 3.682| 4 d.f.*|
Level of significance (p) > .30 N.S. > .20 N.S. > .30 N.S.
* Categories 4 and 5 and over combined because of small numbers.
Table 51
The number of children in the married individual's family: a comparison between depressives of early and late onset and controls.
| No. of children | Early onset depressives | Late onset depressives | Controls |
|-----------------|-------------------------|------------------------|----------|
| | No. | % | No. | % | No. | % |
| 0 | 11 | 13.4| 3 | 8.8 | 24 | 17.1|
| 1 | 15 | 18.2| 10 | 29.4| 28 | 20.0|
| 2 | 27 | 32.9| 13 | 38.2| 42 | 30.0|
| 3 | 20 | 24.3| 3 | 8.8 | 19 | 13.6|
| 4 | 2 | 2.4 | 3 | 8.8 | 14 | 10.0|
| 5 and over | 7 | 8.5 | 2 | 5.9 | 13 | 9.3 |
Totals | 82 | 100%| 34 | 100%| 140 | 100%
$\chi^2$ | 6.319 4 d.f. | 3.072 4 d.f.
Level of significance (p) | > .10 | > .50
N.S. | N.S.
Categories 4 and 5 and over combined because of small numbers.
Table 52
A positive family history of severe mental disorder: comparison between depressives and controls.
| | All depressives | Indolentous | Neurotic | Controls |
|------------------------|-----------------|-------------|----------|----------|
| | No. | % | No. | % | No. | % | No. | % |
| Positive family history| 55 | 35.9| 40 | 39.2| 15 | 29.4| 21 | 12.9|
| Negative family history| 98 | 64.1| 62 | 60.8| 36 | 70.6| 44.2 | 87.1|
| Totals | 153 | 100%| 102 | 100%| 51 | 100%| 163 | 100%|
| $\chi^2$ | 22.978 | 1 d.f.| 24.432 | 1 d.f.| 7.525 | 1 d.f.|
| Level of significance (p) | < .01 | < .01 | < .01 |
Sig. Sig. Sig.
Table 53
Comparison between endogenous and neurotic depressives as to a positive family history of severe mental disorder.
| Positive family history | Endogenous depressives | Neurotic depressives |
|-------------------------|------------------------|----------------------|
| | 40 | 15 |
| Negative family history | 62 | 36 |
| Totals | 102 | 51 |
\[ \chi^2 = 1.392 \quad d.f. = 1 \quad p = > .20 \quad N.S. \]
APPENDIX 3b
Miscellaneous results. Tables 54 to 61.
Table 54
The age of the depressive at the time of mother's death: a comparison with the controls
| Age of the individual at mother's death | Depressives | Controls |
|----------------------------------------|-------------|----------|
| 0 - 15 | 17 | 12 |
| 16 - 25 | 16 | 7 |
| 26 - 35 | 14 | 30 |
| 36 and over | 50 | 43 |
| Mother still alive | 56 | 71 |
Totals: 153 163
$\chi^2 = 12.305$ d.f. $p = .02$ Sig.
Table 55
The age of the individual at the time of mother's death: comparison between depressives and controls.
| Age of individual at mother's death | Depressives | Endogenous | Neurotic | Controls |
|-------------------------------------|-------------|------------|----------|----------|
| | No. | % | No. | % | No. | % | No. | % |
| 0 - 25 years | 33 | 21.6| 24 | 23.5| 9 | 17.6| 19 | 11.7|
| 26 years and over | 120 | 78.4| 78 | 76.5| 42 | 82.4| 144 | 88.3|
| Totals | 153 | 100%| 102 | 100%| 51 | 100%| 163 | 100%|
χ² = 5.607 1 d.f.* 6.415 1 d.f.* 1.194 1 d.f.*
Level of significance (p)
< .02 < .02 > .20
Sig. Sig. N.S.
Table 56a. Causes of death, as reported by the subjects, in those mothers who died before the subject's 26th birthday.
| Cause of maternal death | No. of maternal deaths occurring when the subjects are aged: |
|-------------------------|-------------------------------------------------------------|
| | 0 - 15 years | 16 - 25 years | Total |
| Cancer | 6 | 4 | 10 |
| Intercurrent infection | 3 | 2 | 5 |
| Heart disease | 0 | 3 | 3 |
| Childbirth | 4 | 0 | 4 |
| Miscellaneous or unknown| 4 | 7 | 11 |
Totals
Mean age of mothers dying when the subject is aged 0 - 25 years: 32.13 years
Mean age of mothers dying when the subject is aged 0 - 15 years: 29.82 years
Mean age of mothers dying when the subject is aged 16-25 years: 34.43 years.
Table 56b.
Causes of death, as reported by the subjects, in those mothers who died before the subject's 26th birthday.
b) Controls
| Cause of maternal death | No. of maternal deaths occurring when the controls are aged: |
|-------------------------|-------------------------------------------------------------|
| | 0 - 15 years | 16 - 25 years | Total |
| Cancer | 0 | 2 | 2 |
| Intercurrent infection | 3 | 0 | 3 |
| Heart disease | 0 | 1 | 1 |
| Childbirth | 1 | 0 | 1 |
| Miscellaneous or unknown| 8 | 4 | 12 |
Totals 12 7 19
Mean age of mothers dying when the control is aged 0 - 25 years: 29.77 years
Mean age of mothers dying when the control is aged 0 - 15 years: 30.33 years
Mean age of mothers dying when the control is aged 16 - 25 years: 28.80 years
Table 57
Mothers who die of cancer: a comparison between depressives and controls.
| | Before individual's 26th birthday | After individual's 26th birthday | Totals |
|------------------------|-----------------------------------|----------------------------------|--------|
| **Depressives** | 10 | 4 | 24 |
| **Controls** | 2 | 3 | 5 |
\[ \chi^2 = 12.204, \text{ 1 d.f., } P < .01, \text{ SIG.} \]
Table 58
The age of the depressive at the time of father's death: a comparison with the controls.
| Age of the individual at father's death | Depressives | Controls |
|----------------------------------------|-------------|----------|
| 0 - 15 | 20 | 22 |
| 16 - 25 | 20 | 19 |
| 26 - 35 | 23 | 29 |
| 36 and over | 43 | 42 |
| Father still alive | 46 | 51 |
Totals 152* 163
\[ \chi^2 = .712 \text{ d.f.} \quad D = > .80 \quad \text{N.S.} \]
* 1 patient had no information regarding father's death
Table 59
Fathers who die of cancer: a comparison between depressives and controls.
| | No. of individuals | Totals |
|----------------|---------------------|--------|
| | Father died of cancer | Father did not die of cancer |
| Depressives | 7 | 145 | 152a |
| Controls | 10 | 153 | 163 |
\[ \chi^2 = .359 \quad d.f. = 2 \quad p > .50 \quad N.S. \]
*1 patient had no information regarding father's death
Table 50
Report by an individual that his parents lost one or more children by death:
Comparison between depressives and controls.
| | Death of a sib | No death of a sib | Totals |
|----------------|----------------|-------------------|--------|
| Depressives | 35 | 118 | 153 |
| Controls | 34 | 129 | 163 |
\[ \chi^2 = .188 \quad 1 \text{ d.f.} \quad 2 \geq .50 \quad \text{N.S.} \]
Table 52
Death of one or more sibs during the individual's childhood:
Comparison between depressives and controls
| Death of a sib: | Before individual's birth | During individual's childhood | No sib-death | Totals |
|-----------------|----------------------------|--------------------------------|---------------|--------|
| Depressives | 11 | 24 | 118 | 153 |
| Controls | 16 | 18 | 129 | 163 |
\[ \chi^2 = 4.999, \text{ d.f.} = 2, p = .230, \text{ N.S.} \]
APPENDIX 4
The Questionnaire.
1. Date of Interview ............... First Interviewed by Dr. .................. | CODE NO.
Type of Patient: (Ring) Med. O.P. Surg.O.P. Psych.t.P.
Hospital ......................... If I.P., Date of Admission ..............
2. SURNAME ........................................ MARITAL STATE: (Ring) S M D Sep W
FIRST NAMES ........................................ OCCUPATION ...........................................
ADDRESS ........................................ HUSBAND'S OCCUPATION ........................................
SOCIAL CLASS: .........................................
AGE .............. SEX .............. RELIGIONS: (Ring) Prot. R.C. Jewish Other None
D.O.B. .....................
GENERAL PRACTITIONER: NAME .......................... ADDRESS ...........................................
3. History of Psychiatric Illness: (Ring) Yes No
Previous Psychiatric Illness
| Approx. Year | Type of Illness | Duration | Treatment | Where Treated | I.P./O.P. |
|--------------|-----------------|----------|-----------|---------------|-----------|
4. PRESENT PSYCHIATRIC ILLNESS
Clinical Diagnosis ......................... If Depression: (Ring) Neurotic Endogenous
Duration ...........................................
Personality Disorder: (Ring) Present Absent Type ...........................................
Associated Factors: (Ring) Other Psychosis Neurosis Mental Defect Alcoholism Epilepsy Other
Precipitating Factors: (Ring) Present Absent
Details of any Above:
5. FAMILY HISTORY
MOTHER'S SURNAME .......................................................... FATHER'S SURNAME ..........................................................
FIRST NAMES ........................................................................ FIRST NAMES ..................................................................
MAIDEN NAME ....................................................................... BIRTHPLACE ..................................................................
BIRTHPLACE ........................................................................... BIRTHPLACE ..................................................................
LAST ADDRESS ...................................................................... LAST ADDRESS ..................................................................
PLACE OF DEATH .................................................................... PLACE OF DEATH ..................................................................
CAUSE OF DEATH ................................................................... CAUSE OF DEATH ..................................................................
AGE AT PATIENT'S BIRTH ..................................................... AGE AT PATIENT'S BIRTH ...................................................
PATIENT'S AGE AT MOTHER'S DEATH ................................. PATIENT'S AGE AT FATHER'S DEATH ..................................
FAMILY HISTORY OF MENTAL ILLNESS: (Ring) Yes No
NO. OF CHILDREN BORN ALIVE TO MOTHER
AGE AND SEX OF PATIENT'S CHILDREN Age
Sex
6. PERSONAL HISTORY
MULTIPLE BIRTH: (Ring) Yes No
MOTHER ABSENT DURING CHILDHOOD: (Ring) Yes No FATHER ABSENT DURING CHILDHOOD: Yes No
REASON ................................................................................ REASON ..................................................................
PERIOD: 0-2 3-5 6-10 11-15 PERIOD: 0-2 3-5 6-10 11-15
DURATION: <3/12 <6/12 <1 year > 1 year DURATION: <3/12 <6/12 <1 year > 1 year
SURROGATE ........................................................................ SURROGATE .................................................................
APPENDIX 5
The reject series.
THE REJECT SERIES
Although it was arranged that the investigator should see only cases of primary depressive illness it was inevitable that a certain number of patients with depression secondary to other psychiatric conditions would be referred for interview. 30 patients were rejected from the depressive series on this account but information was gathered on them exactly as with the other depressives.
All of these 30 individuals suffer unmistakeably from a depressive illness but in their case this is secondary to a personality disorder or some form of neurotic illness. (No case of depression secondary to another psychosis was apparently referred). In 27 (90%) a form of personality disorder (usually long-term manifestations of anxiety or inadequacy) is present and in 10 (33.3%) neurotic symptoms are prominent alongside the depression. It will be remembered that in the depressive series, 102 patients (66.7%) were diagnosed as suffering from endogenous, and 51 (33.3%) as suffering from neurotic, depression: in the rejects the ratio is exactly reversed, being 10 (33.3%) with endogenous, and 20 (66.7) with neurotic, depression. The proportion of women to men is high in the rejects, there being 24 females (80%) and 6 males (20%). There is, incidentally, one twin (a female) in the reject series. A high proportion (33.3%) have attempted suicide at some time.
The rejects tend to fall into a narrower age-range than the patients in the depressive series: 23 (76.7%) are found in the age-group 36-55 years as compared with 56.3% of the depressives. There is a tendency for
a higher proportion of rejects as compared with depressives (40% as opposed to 28.7%) to come from the two lowest social classes. Probably because of this social class bias the rejects tend to have been born into rather larger families than the depressives and they themselves tend to produce a larger number of children.
As compared with the depressives a somewhat higher proportion of rejects (16.7% as against 13.1%) report that they lost father by death in childhood and a higher proportion (46.7% as against 32.0%) complain of a disturbed relationship with a parent during childhood.
Apart from the presence of another psychiatric condition in the reject patients, none of the comparisons between rejects and depressives mentioned till now have been found to reach a level of significance according to the chi-square calculation, but this is probably due to the small number of individuals in the reject series. In fact, the only significant difference which has been demonstrated between the two groups is the presence of an excess of Roman Catholic individuals among the rejects (30% as opposed to 15% of the depressives; p < .05) and this is probably a reflection of the social class differences between the groups.
Patients with other psychiatric conditions often develop depression, sometimes of very severe degree, and the depressive symptoms may be quite indistinguishable from those of primary depression. However, there will be a history of previous or concurrent symptoms of the primary disorder. Some authorities describe such cases as neurotic depressive but this term,
as previously explained (p.14) is used quite differently in the present study. It is considered that there was full justification for excluding such patients from the definitive series of depressives since the presence of extraneous psychological factors could only complicate the investigation into the aetiology of the depression itself.
APPENDIX 6
The symptom-sign inventory.
THE SYMPTOM-SIGN INVENTORY
It is generally agreed that clinical diagnosis of psychiatric conditions is thoroughly unsatisfactory, partly because of vagueness of criteria and partly because psychiatrists often find difficulty in agreeing on diagnosis, not only with other psychiatrists, but even with themselves on subsequent occasions. Foulds (1962) says, "Even if psychiatrists could be shown to be highly reliable under optimal conditions, their diagnosis could only serve as the criterion by which to develop some more objective, public and quantitative means of classification." Recognizing the especial need for objectivity in research work, it seemed desirable to employ some form of psychological test to verify clinical assessment.
The diagnosis of primary depression was made on the basis of two independent opinions: those of the referring psychiatrist and the investigator. This introduced a modicum of objectivity, but it was hoped that it would be possible to demonstrate by means of a test that the investigator's classification into endogenous and neurotic depression was an acceptable one.
Although this was an addition to the original concept of the study, it did not in any way interfere with the method of data-collection. The test which was introduced was a modification of the Runwell Symptom-Sign Inventory (Foulds, 1962). The original Inventory is a broad-focus diagnostic test, containing sub-sections dealing with the various psychiatric conditions and the section on depression is designed to differentiate between
endogenous and neurotic types. With the advice of Dr. Foulds, the questions on depressive illness were extracted and were formed into a questionnaire, a copy of which is reproduced at the end of this appendix.
This questionnaire was used by the investigator on 60 patients. Each patient was asked the twenty questions it contained and their answers were recorded on an all-or-none basis: an affirmative was regarded as 'True' and a negative answer as 'False'.
The application of the Inventory presented no difficulties but unfortunately its value was nullified because most of the patients were receiving antidepressant treatment at the time of testing. In fact, many of the patients had been receiving treatment for a considerable period, even before admission. It was found that their responses were greatly modified and as psychological tests are largely dependent on the patient's current mental state no conclusions could be drawn from the results as to his pre-treatment mental condition. On the other hand, the clinician is able to draw on the previous history in his assessment of the illness. The invalidation of the test in this way is obviously an important point to remember in this era of vigorous therapy of affective illness.
Inspection of the Inventory-data showed that it could not be analyzed, although with considerably larger numbers interpretation might well have been possible. Therefore, it was not possible to make a more objective verification and the differentiation of endogenous from neurotic depression regrettably remains on a clinical basis.
SURNAME
CHRISTIAN NAME
SEX
REFERENCE NUMBER
AGE
OCCUPATION
MARITAL STATUS S M W D Sep.
DATE OF TESTING
Are you afraid that you might be going insane?
Have you a pain, or feeling of tension, in the back of the neck?
Are you afraid of going out alone?
Do you cry rather easily?
Does the future seem pointless?
Do you ever seriously think of doing away with yourself because you are no longer able to cope with your difficulties?
Can people read your thoughts and make you do things against your will by a sort of hypnotism?
Are you unnecessarily careful in carrying out even simple everyday tasks like folding clothes, reading notices etc?
Do you think other people regard you as very odd?
Do you feel you cannot communicate with other people because you don't seem to be on the same "wave-length"?
Is there something unusual about your body - like one side being different from the other and meaning something different?
Do you ever do things in a dream-like state without remembering afterwards what you have been doing?
Are you worried about having said things that have injured others?
Are you an unworthy person in your own eyes?
Are you a condemned person because of your sins?
Are you troubled by waking in the early hours and being unable to get off to sleep again (If you don't have sleeping pills)?
Because of things you have done wrong, are people talking about you and criticising you?
Do you cause harm to people because of what you are?
Are you ever so "worked-up" that you pace about wringing your hands?
Do you ever go to bed feeling you wouldn't care if you never woke up again?
|
October 23, 2009
Via Electronic Filing and U.S. Mail
Public Utility Commission of Oregon
Attention: Filing Center
PO Box 2148
Salem, OR 97308-2148
Re: INTERNATIONAL PAPER COMPANY, Complainant, vs.
PACIFICORP dba PACIFC POWER, Respondent
OPUC Docket No. UM 1449
Attention Filing Center:
Enclosed for filing in the above-captioned docket are an original and one copy of DIRECT TESTIMONY OF JIM SCHROEDER ON BEHALF OF PACIFICORP.
An extra copy of this cover letter is enclosed. Please date stamp the extra copy and return it to me in the envelope provided.
Thank you in advance for your assistance.
Sincerely,
[Signature]
Jeff Lovinger
cc: UM 1449 Service List
Enclosures
BEFORE THE PUBLIC UTILITY COMMISSION OF OREGON
UM 1449
INTERNATIONAL PAPER COMPANY,
Complainant,
vs.
PACIFICORP dba PACIFIC POWER,
Respondent.
DIRECT TESTIMONY OF
JIM SCHROEDER
ON BEHALF OF
PACIFICORP
October 23, 2009
Q. PLEASE STATE YOUR NAME AND BUSINESS ADDRESS.
A. Jim Schroeder. PacifiCorp, 825 NE Multnomah, Suite 600, Portland, Oregon 97232.
Q. WHAT IS YOUR OCCUPATION AND BY WHOM ARE YOU EMPLOYED?
A. I am employed by PacifiCorp full-time in the Commercial & Trading department as an Originator.
Q. PLEASE DESCRIBE YOUR FAMILIARITY WITH INTERNATIONAL PAPER COMPANY’S (“INTERNATIONAL”) NEGOTIATIONS FOR A POWER PURCHASE AGREEMENT (“PPA”) WITH PACIFICORP.
A. I was PacifiCorp’s primary contact with International. I took part in relevant correspondence with International. I was involved in preparing and negotiating terms for a PPA.
Q. PLEASE DESCRIBE EXHIBIT PACIFICORP/101 TO THIS TESTIMONY.
A. Exhibit PacifiCorp/101 contains selected pages from the draft PPA proposed by PacifiCorp on August 10, 2009. The selected pages contain my handwritten notes in red felt pen that I made during a phone call with Greg Comatas, International’s Strategic Sourcing Manager, on August 11, 2009 in which we discussed the August 10 draft PPA. My notes in red are issues that Mr. Comatas expressed concern about during the August 11 call.
Regarding Recital G on page 2 of the draft PPA, we discussed the status of International’s generator interconnection agreement, which had been recently assigned from Weyerhaeuser Company to International.
Regarding Section 3.2.8 on page 8, Mr. Comatas inquired whether Moody’s could be the source for International’s credit rating.
Regarding Section 6.1 on page 11, Mr. Comatas inquired as to whether curtailments under that section during on-peak hours could be eliminated from the availability calculation. Mr. Comatas also inquired as to whether maintenance outages would also be eliminated from the availability calculation.
Regarding Section 9.3 on page 12, Mr. Comatas inquired how often PacifiCorp would test the metering equipment and what would be done with the results of the test.
Regarding Section 12.7.8 on page 16, Mr. Comatas inquired about the option to self-insure.
Mr. Comatas’ comments on these issues were provided orally and were never presented to PacifiCorp in writing.
Q. DID INTERNATIONAL PROVIDE ANY WRITTEN COMMENTS TO THE DRAFT PPA YOU SENT INTERNATIONAL ON AUGUST 10?
A. Mr. Comatas sent me an e-mail on August 11 with an attachment containing updated contact information to Section 22 of the PPA. I have attached the e-mail and attachment as Exhibit PacifiCorp/102 to this testimony and have redacted potentially confidential information.
Q. PLEASE DESCRIBE EXHIBIT PACIFICORP/103 TO THIS TESTIMONY.
A. Exhibit PacifiCorp/103 is pages 1, 12, and 16 from the proposed terms that I emailed as an attachment to Mr. Comatas on August 21, 2009. On page 12, I proposed revisions to credit requirements in Section 8.1. On page 16, I proposed changes to the insurance requirements in Sections 12.7.1 through 12.7.6. The
proposal I e-mailed to Mr. Comatas on August 21, 2009 did not include a recitation of proposed pricing terms.
Q. PLEASE DESCRIBE EXHIBIT PACIFICORP/104 TO THIS TESTIMONY.
A. Exhibit PacifiCorp/104 is the cover e-mail that I sent to Mr. Comatas on August 21 with revised proposed terms attached. In this e-mail, I explained that pricing would be finalized per Oregon Public Utility Commission directions.
Q. PLEASE DESCRIBE EXHIBIT PACIFICORP/105 TO THIS TESTIMONY.
A. Exhibit PacifiCorp/105 is pages 1, 8, and 14 of the proposed terms that I e-mailed as an attachment to Mr. Comatas on August 13, 2009. The first page contains a note at the top designating the draft as a working draft, not a binding offer. I noted on page 8 that Section 3.2.8 is subject to review by PacifiCorp’s internal credit division. I also noted on page 14 that the insurance section is under review by PacifiCorp’s Risk Management Department.
Q. DOES THIS CONCLUDE YOUR TESTIMONY?
A. Yes.
PACIFICORP/101
Excerpts of August 10, 2009 Draft PPA
THIS WORKING DRAFT DOES NOT CONSTITUTE A BINDING OFFER, SHALL NOT FORM THE BASIS FOR AN AGREEMENT BY ESTOPPEL OR OTHERWISE, AND IS CONDITIONED UPON EACH PARTY’S RECEIPT OF ALL REQUIRED MANAGEMENT APPROVALS (INCLUDING FINAL CREDIT AND LEGAL APPROVAL). ANY ACTIONS TAKEN BY A PARTY IN RELIANCE ON THE TERMS SET FORTH IN THIS WORKING DRAFT OR ON STATEMENTS MADE DURING NEGOTIATIONS PURSUANT TO THIS WORKING DRAFT SHALL BE AT THAT PARTY’S OWN RISK. UNTIL THIS AGREEMENT IS NEGOTIATED, APPROVED BY MANAGEMENT, SIGNED AND DELIVERED, NO PARTY SHALL HAVE ANY OTHER LEGAL OBLIGATIONS, EXPRESSED OR IMPLIED, OR ARISING IN ANY OTHER MANNER UNDER THIS WORKING DRAFT OR IN THE COURSE OF NEGOTIATIONS.
POWER PURCHASE AGREEMENT
BETWEEN
INTERNATIONAL PAPER COMPANY
AND
PACIFICORP
THIS POWER PURCHASE AGREEMENT (the “Agreement”), entered into this ____day of August 2009, is by and between International Paper Company, a New York corporation, (“Seller”) and PacifiCorp (d/b/a Pacific Power & Light Company), an Oregon corporation (“PacifiCorp”) individually, a “Party” and collectively, the “Parties.”
RECITALS
A. Seller owns and operates a cogeneration facility comprised of a combustion gas turbine generator with heat recovery steam generator, and a steam turbine generator, located in Albany, Linn County, OR with a combined Nameplate Capacity Rating of 93.02 megawatts (“MW”) (as more fully described in Exhibit A, the "Facility"). Because Seller’s combined Nameplate Capacity Rating exceeds the 45 MW capacity of the PacifiCorp Western Kraft
Substation, Seller will self-limit delivery of Net Delivered Energy to 45 MW ("Maximum Delivery Rate"); and
B. Seller has self-certified the cogeneration facility as a qualifying facility under the Public Utility Regulatory Policies Act of 1978 in Federal Energy Regulatory Commission ("FERC") Docket No. QF95-233. Seller intends to operate the Facility as a Qualifying Facility, as such term is defined in Section 3.2.6 below; and
C. Commission Order No. 07-360 mandated utilities offering non-standard power purchase agreements to qualifying facilities to make specified adjustments to Avoided Costs Prices offered in standard power purchase agreements under PacifiCorp’s Schedule 37. Those changes to pricing and terms have been incorporated into this Agreement; and
D. Seller estimates the Net Delivered Energy during the two-year term of the contract, starting January 1, 2010, is 676,134 megawatt-hours ("MWh") net of anticipated scheduled maintenance (set forth in Exhibit C attached hereto); and
E. PacifiCorp has determined that it will be in a state of resource sufficiency for the term of this Agreement, and has adjusted the Contract Prices accordingly; and
F. Seller desires to sell and PacifiCorp desires to purchase the Net Delivered Energy from the Facility in accordance with the terms and conditions of this Agreement; and
G. Seller applied to PacifiCorp Transmission for a Large Generation Interconnection Agreement on ____________; and
H. Seller elects to purchase its partial requirements for the Albany Paper Mill retail load from PacifiCorp. For its retail energy purchases, Seller will be served by PacifiCorp under Oregon Schedule 47, as more particularly described in a separate Master Electric Service Agreement; and
I. This Agreement is a "New QF Contract" under the PacifiCorp Inter-Jurisdictional Cost Allocation Revised Protocol and, as such, the costs of QF energy under this Agreement shall be allocated as a system resource unless any portion of those costs exceeds the cost PacifiCorp would have otherwise incurred acquiring comparable resources. In that event, the Revised Protocol assigns those excess costs on a situs basis to the State of Oregon. In addition, for the purposes of inter-jurisdictional cost allocation, PacifiCorp represents that the costs of this Agreement do not exceed the costs PacifiCorp would have otherwise incurred acquiring resources in the market that are defined as “Comparable Resources” in Appendix A to the Inter-Jurisdictional Cost Allocation Revised Protocol. For the purposes of inter-jurisdictional cost allocation, PacifiCorp represents that the costs and revenues from the energy and capacity sold to Seller by PacifiCorp will be assigned on a situs basis to the State of Oregon.
NOW, THEREFORE, the Parties mutually agree as follows:
- assigned Aug 5, 2009
- redraft contract to come in mail next week
with the Seller or the Facility, stating that the Facility is a QF and has been operated lawfully as a QF under this Agreement. The attorney’s opinion shall provide sufficient documentation to demonstrate that Seller has maintained and will continue to maintain the Facility as a QF. If such legal opinion states that Seller has maintained the Facility as a QF, then PacifiCorp shall pay for said legal opinion. If such legal opinion states that Seller’s Facility is not a QF, then Seller shall pay for said legal opinion and Seller shall be deemed to have received written notice from PacifiCorp that Seller is in default of this representation for the purposes of section 11.1.1.
3.2.7 Under no circumstances shall the Net Delivered Energy exceed the Net Output of the Facility.
3.2.8 Seller’s credit rating on its senior, unsecured debt currently is rated “BBB” by the Standard and Poor’s Rating Service.
3.2.9 Seller will take retail electric service for the Facility pursuant to an effective Master Electric Service Agreement under PacifiCorp’s Oregon Schedule 47 during the Term of this Agreement; and
3.2.10 Seller, at its option, elects to self-insure. Seller maintains and will maintain insurance applicable to losses in excess of self-insured amount. Seller agrees to provide PacifiCorp all the benefits that would otherwise be available and provided under an insurance policy in accordance with the requirements set forth in Section 12.7, including but not limited to defense of claims.
SECTION 4: DELIVERY OF POWER
4.1 Commencing at 12:01 a.m. on January 1, 2010 (or any subsequent date agreed upon by the Parties in writing) and continuing through all hours of the Term of this Agreement, Seller shall sell and make available to PacifiCorp (and to PacifiCorp only), and PacifiCorp shall purchase, the Net Delivered Energy from the Facility at the Point of Delivery as more particularly described in Section 4.2 hereto.
4.2 **Net Delivered Energy.**
4.2.1 Seller’s “**Net Delivered Energy**” (MWh) shall be equal to:
a) The Generation Meter (MW) if the Load Meter and Generation Meter are both less than or equal to 45 MW; or
b) 45 MW if the Load Meter is less than or equal to 45 MW and the Generation Meter is greater than 45 MW; or
c) the lesser of (i) the Generation Meter minus the Load Meter plus 45 MW, or (ii) 45 MW, if the Load Meter is greater than 45 MW.
hereto, illustrates how Net Delivered Energy, Liquidated Damages, and the resulting Payment are calculated under this Section.
SECTION 6: OPERATION AND CONTROL
6.1 Seller shall operate and maintain the Facility in a safe manner in accordance with the Generation Interconnection Agreement, Prudent Electrical Practices and in accordance with the requirements of all applicable federal, state and local laws and the National Electric Safety Code as such laws and code may be amended from time to time. PacifiCorp shall have no obligation to purchase Net Delivered Energy from the Facility to the extent the interconnection between the Facility and PacifiCorp’s electric system is disconnected, suspended or interrupted, in whole or in part, pursuant to the Generation Interconnection Agreement, or to the extent generation curtailment is required as a result of Seller’s non-compliance with the Generation Interconnection Agreement. PacifiCorp shall have the right to inspect the Facility to confirm that Seller is operating the Facility in accordance with the provisions of this Section 6.3 upon reasonable notice to Seller. Seller is solely responsible for the operation and maintenance of the Facility. PacifiCorp shall not, by reason of its decision to inspect or not to inspect the Facility, or by any action or inaction taken with respect to any such inspection, assume or be held responsible for any liability or occurrence arising from the operation and maintenance by Seller of the Facility.
6.2 To the extent not otherwise provided in the Generation Interconnection Agreements, all reasonable costs incurred that are associated with the modifications to PacifiCorp's interconnection facilities or electric system occasioned by or related to the interconnection of the Facility with PacifiCorp’s system consistent with applicable interconnection standards, or any increase in generating capability of the Facility, or any increase of delivery of Net Delivered Energy from the Facility, shall be borne by Seller.
6.3 Approved Outages. On-Peak hours of Approved Outages shall be deducted from On-Peak Delivery Hours for the month in which the outage occurs.
6.4 Unapproved Outages. If any unit in the Facility ceases operation for any hour other than pursuant to an Approved Outage, Seller shall within fifteen minutes notify PacifiCorp of the necessity of such unapproved outage, the time when the outage has occurred or will occur and the anticipated duration of the outage. Notification shall be to the Real Time Generation Desk, as identified in Section 22. Seller shall make best efforts to meet its delivery obligations during utility system emergencies. When feasible, Seller shall schedule routine outages lasting less than twelve hours during Off-Peak Hours. Hours of unapproved outages shall not be deducted from Net Delivery Hours.
SECTION 7: FUEL/MOTIVE FORCE
Seller shall procure at its cost all inputs necessary to deliver Net Delivered Energy.
SECTION 8: CREDIT
8.1 As of the Effective Date and for the Term, Seller shall satisfy the Credit Requirements. In the event of a Material Adverse Change, or at any time during the term of this agreement when Seller does not meet the Credit Requirements, the Seller will be required to post performance assurances, as reasonably determined by PacifiCorp.
8.2 If Seller’s financial statements are not publicly available, and if requested by PacifiCorp, the Seller shall provide copies of its audited year-end financial statements for the previous two years, prepared in accordance with generally accepted accounting principles.
SECTION 9: METERING
9.1 PacifiCorp shall design, furnish, install, own, inspect, test, maintain and replace all metering equipment as provided in the Generation Interconnection Agreements.
9.2 Metering shall be performed at the location and in a manner consistent with this Agreement and as specified in the Generation Interconnection Agreement. All quantities of energy purchased hereunder shall be adjusted to account for electrical losses, if any, between the point of metering and the Point of Delivery, so that the purchased amount reflects the net amount of power flowing into PacifiCorp’s system at the Point of Delivery.
9.3 PacifiCorp shall periodically inspect, test, repair and replace the metering equipment as provided in the Generation Interconnection Agreement, if applicable. If any of the inspections or tests discloses an error exceeding two percent (2%) either fast or slow, proper correction, based upon the inaccuracy found, shall be made of previous readings for the actual period during which the metering equipment rendered inaccurate measurements if that period can be ascertained. If the actual period cannot be ascertained, the proper correction shall be made to the measurements taken during the time the metering equipment was in service since last tested, but not exceeding three (3) Billing Periods, in the amount the metering equipment shall have been shown to be in error by such test. Any correction in billings or payments resulting from a correction in the meter records shall be made in the next monthly billing or payment rendered following the repair of the meter.
SECTION 10: BILLINGS, COMPUTATIONS AND PAYMENTS
10.1 All bills or payments shall be paid within thirty (30) days after issuance of the bill, but no later than the due date specified by PacifiCorp on each monthly invoice for service under the Master Electric Service Agreement. PacifiCorp shall send to Seller an invoice or payment for Seller's purchases or deliveries, as the case may be, together with computations supporting such invoice or payment. PacifiCorp or Seller may offset any such payment to reflect amounts owing from Seller to PacifiCorp or from PacifiCorp to Seller pursuant to this Agreement or the Master Electric Service Agreement.
including coverage for earth movement, flood, and boiler and machinery. The Risk policy may contain separate sub-limits and deductibles subject to insurance company underwriting guidelines. The Risk Policy will be maintained in accordance with terms available in the insurance market for similar facilities.
12.7.2 **Employers’ Liability** insurance with a minimum limit of $1,000,000.
12.7.3 **Commercial General Liability** insurance, to include contractual liability, with a minimum single limit of $1,000,000 to protect against and from all loss by reason of injury to persons or damage to property based upon and arising out of the activity under this Agreement.
12.7.4 **Business Automobile Liability** insurance with a minimum single limit of $1,000,000 for bodily injury and property damage with respect to vehicles whether owned, hired or non-owned, assigned to or used in connection with this Agreement.
12.7.5 The Commercial General Liability policy required herein shall include i) provisions or endorsements naming PacifiCorp, its Board of Directors, Officers and employees as additional insureds, and ii) cross liability coverage so that the insurance applies separately to each insured against whom claim is made or suit is brought, even in instances where one insured claims against or sues another insured.
12.7.6 All liability policies required by this Agreement shall include provisions that such insurance is primary insurance with respect to the interests of PacifiCorp and that any other insurance maintained by PacifiCorp is excess and not contributory insurance with the insurance required hereunder, and provisions that such policies shall not be canceled or their limits of liability reduced without 1) ten (10) days prior written notice to PacifiCorp if canceled for nonpayment of premium, or 2) thirty (30) days prior written notice to PacifiCorp if canceled for any other reason. A certificate in a form satisfactory to PacifiCorp certifying to the issuance of such insurance, shall be furnished to PacifiCorp. Commercial General Liability coverage written on a "claims-made" basis, if any, shall be specifically identified on the certificate. If requested by PacifiCorp, a copy of each insurance policy, certified as a true copy by an authorized representative of the issuing insurance company, shall be furnished to PacifiCorp.
12.7.7 Insurance coverage provided on a "claims-made" basis shall be maintained by Seller for a minimum period of five (5) years after the completion of this Agreement.
12.7.8 **Option to Self-insure.** Seller, at its option, may self-insure for any or all of the coverage described above. In the event and to the extent Seller so elects, Seller shall advise PacifiCorp in writing. For all such self-insurance maintained by Seller, Seller agrees to provide PacifiCorp all the benefits that would otherwise be available and provided under an insurance policy in accordance with the requirements set forth above in this Section 12.7, including but not limited to defense of claims.
**SECTION 13: FORCE MAJEURE**
PACIFICORP/102
August 11, 2009 E-mail from Greg Comatas
Greg Comatas
Manager - Strategic Sourcing
International Paper
901-419-1654 Office
901-326-2799 Mobile
(See attached file: Updated SECTION 22.docx)
22.1 All notices except as otherwise provided in this Agreement shall be in writing, and all notices and payments shall be directed as follows and shall be considered delivered if delivered in person or when deposited in the U.S. Mail, postage prepaid by certified or registered mail and return receipt requested.
To Seller:
All Notices:
Shipping Address: 3251 OLD SALEM RD NE
ALBANY OR 97321
Mailing Address: PO BOX 339
ALBANY OR 97321
Attn: Michael Culver
Phone: (541) 924-4657
Facsimile: (541) 967-2343
Duns: [redacted]
Federal Tax ID Number: [redacted]
Invoices:
Attn: Karen Crawford
Phone: (541) 924-3686
Facsimile: (541) 967-2343
Scheduling:
Attn: Andrew Cooper
Phone: (541) 924-4638
Facsimile: (541) 924-3697
Payments:
Attn: Karen Crawford
Phone: (541) 924-3686
Facsimile: (541) 967-2343
Wire Transfer:
International Paper
JP Morgan Chase Bank
New York, NY 10081
ABA: [redacted]
Swift ID: [redacted]
ACCT: [redacted]
Bank Contact: Jeanne Muino (212) 552-1481
PACIFICORP/103
Excerpts of August 21, 2009 Proposed Terms
THIS WORKING DRAFT DOES NOT CONSTITUTE A BINDING OFFER, SHALL NOT FORM THE BASIS FOR AN AGREEMENT BY ESTOPPEL OR OTHERWISE, AND IS CONDITIONED UPON EACH PARTY’S RECEIPT OF ALL REQUIRED MANAGEMENT APPROVALS (INCLUDING FINAL CREDIT AND LEGAL APPROVAL). ANY ACTIONS TAKEN BY A PARTY IN RELIANCE ON THE TERMS SET FORTH IN THIS WORKING DRAFT OR ON STATEMENTS MADE DURING NEGOTIATIONS PURSUANT TO THIS WORKING DRAFT SHALL BE AT THAT PARTY’S OWN RISK. UNTIL THIS AGREEMENT IS NEGOTIATED, APPROVED BY MANAGEMENT, SIGNED AND DELIVERED, NO PARTY SHALL HAVE ANY OTHER LEGAL OBLIGATIONS, EXPRESSED OR IMPLIED, OR ARISING IN ANY OTHER MANNER UNDER THIS WORKING DRAFT OR IN THE COURSE OF NEGOTIATIONS.
POWER PURCHASE AGREEMENT
BETWEEN
INTERNATIONAL PAPER COMPANY
AND
PACIFICORP
THIS POWER PURCHASE AGREEMENT (the “Agreement”), entered into this ____day of August 2009, is by and between International Paper Company, a New York corporation, (“Seller”) and PacifiCorp (d/b/a Pacific Power & Light Company), an Oregon corporation (“PacifiCorp”) individually, a “Party” and collectively, the “Parties.”
RECITALS
A. Seller owns and operates a cogeneration facility comprised of a combustion gas turbine generator with heat recovery steam generator, and a steam turbine generator, located in Albany, Linn County, OR with a combined Nameplate Capacity Rating of 93.02 megawatts (“MW”) (as more fully described in Exhibit A, the "Facility"). Because Seller’s combined Nameplate Capacity Rating exceeds the 45 MW capacity of the PacifiCorp Western Kraft
SECTION 7: FUEL/MOTIVE FORCE
Seller shall procure at its cost all inputs necessary to deliver Net Delivered Energy.
SECTION 8: CREDIT
8.1 As of the Effective Date and for the Term, Seller shall satisfy the Credit Requirements. In the event of a Material Adverse Change, or at any time during the term of this agreement when Seller does not meet the Credit Requirements, the Seller will be required to post credit assurances, as reasonably determined by PacifiCorp.
8.2 If Seller’s financial statements are not publicly available, and if requested by PacifiCorp, the Seller shall provide copies of its audited year-end financial statements for the previous two years, prepared in accordance with generally accepted accounting principles.
SECTION 9: METERING
9.1 PacifiCorp shall design, furnish, install, own, inspect, test, maintain and replace all metering equipment as provided in the Generation Interconnection Agreements.
9.2 Metering shall be performed at the location and in a manner consistent with this Agreement and as specified in the Generation Interconnection Agreement. All quantities of energy purchased hereunder shall be adjusted to account for electrical losses, if any, between the point of metering and the Point of Delivery, so that the purchased amount reflects the net amount of power flowing into PacifiCorp’s system at the Point of Delivery.
9.3 PacifiCorp shall periodically inspect, test, repair and replace the metering equipment as provided in the Generation Interconnection Agreement, if applicable. If any of the inspections or tests discloses an error exceeding two percent (2%), either fast or slow, proper correction, based upon the inaccuracy found, shall be made of previous readings for the actual period during which the metering equipment rendered inaccurate measurements if that period can be ascertained. If the actual period cannot be ascertained, the proper correction shall be made to the measurements taken during the time the metering equipment was in service since last tested, but not exceeding three (3) Billing Periods, in the amount the metering equipment shall have been shown to be in error by such test. Any correction in billings or payments resulting from a correction in the meter records shall be made in the next monthly billing or payment rendered following the repair of the meter.
SECTION 10: BILLINGS, COMPUTATIONS AND PAYMENTS
10.1 All bills or payments shall be paid within thirty (30) days after issuance of the bill, but no later than the due date specified by PacifiCorp on each monthly invoice for service under the Master Electric Service Agreement. PacifiCorp shall send to Seller an invoice or payment for Seller’s purchases or deliveries, as the case may be, together with computations supporting such
12.7.1 Special Form Property insurance providing coverage in an amount at least equal to the full replacement value of the Facility against special form property physical loss or damage with normal and customary exclusions, including coverage for earthquake, flood, and boiler and machinery. This property insurance may contain separate sub-limits and deductibles. This property insurance will be maintained in accordance with terms available in the insurance market for similar facilities.
12.7.2 Employers' Liability insurance with minimum limits of $1,000,000 applicable to each accident/disease-each employee/disease-policy limit.
12.7.3 Commercial General Liability insurance, to include contractual liability, premises and operations, and broad form property damage, with a minimum single limit of $1,000,000 each occurrence/$2,000,000 general aggregate to protect against and from loss by reason of injury to persons or damage to property based upon and arising out of the activity under this Agreement.
12.7.4 Business Automobile Liability insurance with a minimum single limit of $1,000,000 each accident for bodily injury and property damage with respect to Seller's vehicles whether owned, hired or non-owned, assigned to or used in connection with this Agreement.
12.7.5 Umbrella Liability insurance with a minimum limit of $5,000,000 each occurrence/aggregate where applicable to be excess of the coverages and limits required in Employers' Liability insurance, Commercial General Liability insurance, and Business Automobile Liability insurance above. Seller shall notify PacifiCorp. if at any time this minimum umbrella limit is not available during the term of this Agreement, and may be required to purchase additional limits of coverage.
12.7.6 The Commercial General Liability policy required herein shall include i) provisions or endorsements naming PacifiCorp, its Directors, Officers, agents and employees as additional insureds, and ii) cross liability coverage so that the insurance applies separately to each insured against whom claim is made or suit is brought, even in instances where one insured claims against or sues another insured.
12.7.7 All liability policies required by this Agreement shall include provisions that such insurance is primary insurance with respect to the interests of PacifiCorp and that any other insurance maintained by PacifiCorp is excess and not contributory insurance with the insurance required hereunder, and provisions that such policies shall not be canceled or their limits of liability reduced without 1) ten (10) days prior written notice to PacifiCorp if canceled for nonpayment of premium, or 2) thirty (30) days prior written notice to PacifiCorp if canceled for any other reason. A certificate in a form satisfactory to PacifiCorp certifying to the issuance of such insurance, shall be furnished to PacifiCorp. Commercial General Liability coverage written on a "claims-made" basis, if any, shall be specifically identified on the certificate. If requested by PacifiCorp, a copy of each insurance policy, certified as a true copy by an authorized representative of the issuing insurance company, shall be furnished to PacifiCorp.
PACIFICORP/104
August 11, 2009 E-mail from Jim Schroeder
Greg
Please find attached a draft QF agreement for your Albany Paper mill 2 year proposed QF sale beginning January 1, 2010. Per our discussion earlier today and with the pending OPUC hearing on Tuesday, August 25, 2009, PacifiCorp will finalize the pricing per OPUC directions as a result of the Tuesday meeting. Once the pricing has been finalized we will prepare a complete final draft for your approval. After your approval, we will route the transaction internally for senior management approval. Please review and provide me any comments.
Jim Schroeder
Origination, Commercial & Trading
PacifiCorp
voice: (503) 813-5380
cell: (503) 701-6688
fax: (503) 813-6260
email@example.com
PACIFICORP/105
Excerpts of August 13, 2009 Draft PPA
THIS WORKING DRAFT DOES NOT CONSTITUTE A BINDING OFFER, SHALL NOT FORM THE BASIS FOR AN AGREEMENT BY ESTOPPEL OR OTHERWISE, AND IS CONDITIONED UPON EACH PARTY’S RECEIPT OF ALL REQUIRED MANAGEMENT APPROVALS (INCLUDING FINAL CREDIT AND LEGAL APPROVAL). ANY ACTIONS TAKEN BY A PARTY IN RELIANCE ON THE TERMS SET FORTH IN THIS WORKING DRAFT OR ON STATEMENTS MADE DURING NEGOTIATIONS PURSUANT TO THIS WORKING DRAFT SHALL BE AT THAT PARTY’S OWN RISK. UNTIL THIS AGREEMENT IS NEGOTIATED, APPROVED BY MANAGEMENT, SIGNED AND DELIVERED, NO PARTY SHALL HAVE ANY OTHER LEGAL OBLIGATIONS, EXPRESSED OR IMPLIED, OR ARISING IN ANY OTHER MANNER UNDER THIS WORKING DRAFT OR IN THE COURSE OF NEGOTIATIONS.
POWER PURCHASE AGREEMENT
BETWEEN
INTERNATIONAL PAPER COMPANY
AND
PACIFICORP
THIS POWER PURCHASE AGREEMENT (the “Agreement”), entered into this ____day of August 2009, is by and between International Paper Company, a New York corporation, (“Seller”) and PacifiCorp (d/b/a Pacific Power & Light Company), an Oregon corporation (“PacifiCorp”) individually, a “Party” and collectively, the “Parties.”
RECITALS
A. Seller owns and operates a cogeneration facility comprised of a combustion gas turbine generator with heat recovery steam generator, and a steam turbine generator, located in Albany, Linn County, OR with a combined Nameplate Capacity Rating of 93.02 megawatts (“MW”) (as more fully described in Exhibit A, the “Facility”). Because Seller’s combined Nameplate Capacity Rating exceeds the 45 MW capacity of the PacifiCorp Western Kraft
with the Seller or the Facility, stating that the Facility is a QF and has been operated lawfully as a QF under this Agreement. The attorney’s opinion shall provide sufficient documentation to demonstrate that Seller has maintained and will continue to maintain the Facility as a QF. If such legal opinion states that Seller has maintained the Facility as a QF, then PacifiCorp shall pay for said legal opinion. If such legal opinion states that Seller’s Facility is not a QF, then Seller shall pay for said legal opinion and Seller shall be deemed to have received written notice from PacifiCorp that Seller is in default of this representation for the purposes of section 11.1.1.
3.2.7 Under no circumstances shall the Net Delivered Energy exceed the Net Output of the Facility.
3.2.8 Seller’s credit rating on its senior, unsecured debt currently is rated “BBB” by the Standard and Poor’s Rating Service, and ________ by Moody’s rating service.
3.2.9 Seller will take retail electric service for the Facility pursuant to an effective Master Electric Service Agreement under PacifiCorp’s Oregon Schedule 47 during the Term of this Agreement; and
3.2.10 Seller, at its option, elects to self-insure. Seller maintains and will maintain insurance applicable to losses in excess of self-insured amount. Seller agrees to provide PacifiCorp all the benefits that would otherwise be available and provided under an insurance policy in accordance with the requirements set forth in Section 12.7, including but not limited to defense of claims.
SECTION 4: DELIVERY OF POWER
4.1 Commencing at 12:01 a.m. on January 1, 2010 (or any subsequent date agreed upon by the Parties in writing) and continuing through all hours of the Term of this Agreement, Seller shall sell and make available to PacifiCorp (and to PacifiCorp only), and PacifiCorp shall purchase, the Net Delivered Energy from the Facility at the Point of Delivery as more particularly described in Section 4.2 hereto.
4.2 Net Delivered Energy.
4.2.1 Seller’s “Net Delivered Energy” (MWh) shall be equal to:
a) The Generation Meter (MW) if the Load Meter and Generation Meter are both less than or equal to 45 MW; or
b) 45 MW if the Load Meter is less than or equal to 45 MW and the Generation Meter is greater than 45 MW; or
c) the lesser of (i) the Generation Meter minus the Load Meter plus 45 MW, or (ii) 45 MW, if the Load Meter is greater than 45 MW.
Parties agree that the Mid-C market price shall be the average of three contemporaneous quotations from energy brokers (namely ICAP Energy, Prebon Energy, and Natsource) for firm, flat delivery of Seller’s minimum delivery obligation from the date of cessation of delivery through the Termination Date. The cost to cover may be less than zero, in which case the termination payment shall be zero. The Parties agree that the amount of termination payment shall be calculated by PacifiCorp within thirty (30) days after termination of the Agreement. Amounts owed pursuant to this Section shall be due within five (5) days after PacifiCorp provides Seller notice of amount due. Prior damages and the termination payment shall be the sole remedy for default; provided however, that Seller shall not intentionally breach this Agreement in order to sell Facility Output to another buyer.
11.4 In the event this Agreement is terminated because of Seller’s Event of Default and Seller or Seller’s successor wishes to again sell Net Output to PacifiCorp following such termination, PacifiCorp in its sole discretion may require that Seller or Seller’s successor shall do so subject to the terms of this Agreement, including but not limited to the contract pricing of Section 5.1, until January 1, 2012. At such time Seller and PacifiCorp agree to execute a written document ratifying the terms of this Agreement. This Section 11.4 shall be deemed a covenant running with the land and shall survive any early termination of this Agreement. Concurrently with the execution and delivery of this Agreement, the Parties shall execute and PacifiCorp shall record, in the appropriate real property records of Linn County, Oregon, a memorandum in the form of Exhibit H to provide record notice to third parties of Seller’s agreements under this Section 11.4.
11.5 Dispute Resolution. Whenever a Party believes that a dispute has arisen in connection with this Agreement, that Party shall promptly provide written notice to the other Party describing the matter in dispute. Unless the dispute is resolved sooner, the Senior Vice President of Commercial and Trading for PacifiCorp and the Vice President, Global Sourcing, for Seller shall meet at least once within a period of thirty days following notice to attempt in good faith to resolve the dispute. If the Parties’ senior management does not resolve the dispute, each Party shall have the right to pursue whatever remedies it may have under the terms of this Agreement or applicable regulations, laws, or principles of equity.
11.6 Upon an event of default or termination event under this Agreement, in addition to and not in limitation of any other right or remedy under contract or applicable law (including any right to set-off, counterclaim, or otherwise withhold payment), the non-defaulting Party may at its option set-off, against any amounts owed to the defaulting Party, any amounts owed by the defaulting Party under any contract or agreement between the Parties. The obligations of the Parties shall be deemed satisfied and discharged to the extent of any such set-off. The non-defaulting Party shall give the defaulting Party written notice of any set-off, but failure to give such notice shall not affect the validity of the set-off.
SECTION 12: INDEMNIFICATION, LIABILITY AND INSURANCE
12.1 Seller agrees to release, indemnify and hold harmless PacifiCorp, its directors, officers, agents, and representatives against and from any and all loss, claims, actions or suits, including costs and attorney’s fees, both at trial and on appeal, resulting from, or arising out of or
CERTIFICATE OF SERVICE
I hereby certify that, on October 23, 2009, I served a true and correct copy of the foregoing DIRECT TESTIMONY OF JIM SCHROEDER ON BEHALF OF PACIFICORP on the following named persons/entities by depositing a true copy thereof in the United States Mail at Portland, Oregon:
| JESSE E COWELL | MELINDA J DAVISON |
|-------------------------|-------------------|
| DAVISON VAN CLEVE | DAVISON VAN CLEVE PC |
| 333 SW TAYLOR ST., SUITE 400 | 333 SW TAYLOR - STE 400 |
| PORTLAND OR 97204 | PORTLAND OR 97204 |
| firstname.lastname@example.org | email@example.com |
I hereby certify that, on October 23, 2009, I served a true and correct copy of the foregoing DIRECT TESTIMONY OF JIM SCHROEDER ON BEHALF OF PACIFICORP on the following named persons/entities by electronic mail:
| JORDAN A WHITE (W) | OREGON DOCKETS (W) |
|-------------------------|--------------------|
| SENIOR COUNSEL | |
| PACIFIC POWER & LIGHT | |
| 825 NE MULTNOMAH STE 1800 | 825 NE MULTNOMAH ST STE 2000 |
| PORTLAND OR 97232 | PORTLAND OR 97232 |
| firstname.lastname@example.org | email@example.com |
Jeffrey Zovinger
Attorney for PacifiCorp
|
NATIONAL ADVISORY COMMITTEE FOR AERONAUTICS
WARTIME REPORT
ORIGINALLY ISSUED
March 1946 as Advance Confidential Report 5K27
THEORY AND APPLICATION OF HOT-WIRE INSTRUMENTS IN THE INVESTIGATION OF TURBULENT BOUNDARY LAYERS
By G. B. Schubauer and P. S. Klebanoff
National Bureau of Standards
WASHINGTON
NACA WARTIME REPORTS are reprints of papers originally issued to provide rapid distribution of advance research results to an authorized group requiring them for the war effort. They were previously held under a security status but are now unclassified. Some of these reports were not technically edited. All have been reproduced without change in order to expedite general distribution.
LANGLEY MEMORIAL AERONAUTICAL LABORATORY
Langley Field, Va.
SUMMARY
An account is given of the recent developments in hot-wire instruments for use in turbulent boundary layers to determine the magnitude of the several components of the turbulent velocity fluctuations together with the correlation between them and turbulent shearing stress. The instruments were developed in order to make possible a study of the turbulent characteristics of the layer as well as the average characteristics in a general investigation of the turbulent boundary layer under conditions producing separation. The experimental setup used to provide a thick turbulent boundary layer is described, and results are included to show the general nature of the boundary layer, as well as examples of the measured fluctuations, correlation coefficient, and turbulent shearing stress. Finally the errors inherent in the hot-wire method are discussed.
I. INTRODUCTION
The object of this investigation of the characteristics of turbulent boundary layers is to provide a sound basis of understanding of the turbulent type of boundary layer through careful study of its development and separation. The investigation involves the measurement of pressure distribution, distribution of mean velocity across the layer, boundary layer thickness, and shape parameter, as well as measurements of the turbulent characteristics of the layer.
While a considerable fund of information exists on the mean flow in turbulent boundary layers, little is known about the turbulence itself. All concerned with the boundary-layer problem agree, however, that turbulent mixing processes are
the fundamental processes in the boundary layer and contain
the explanation of such average characteristics as velocity
distribution, internal friction or shearing stress, and sep-
aration in an adverse pressure gradient. It is therefore
not for lack of importance that turbulence measurements have
been neglected, but rather from a lack of suitable instru-
ments and techniques for making the necessary measurements.
When the present boundary-layer investigation was undertaken,
it was decided to determine as many characteristics of the
turbulence as possible, along with the average characteris-
tics of the layer, to fill in the gap left by former investi-
gators. In order to make the turbulence measurements, it
has been necessary first to develop some special type of hot-
wire anemometer instruments. The present report covers re-
search on development of the various hot-wire instruments
and their adaptation to the turbulent boundary-layer investi-
gation.
In order to see what turbulent characteristics of the
layer should be measured to best further an understanding of
turbulent boundary layers, it is well to look into the usual
boundary-layer concepts associated with turbulence. It is
customary to think in terms of the velocity fluctuations and
to regard these fluctuations as the velocity of migration of
fluid masses relative to the mean flow. The three mutually
perpendicular components of the fluctuations are denoted here
by $u$, $v$, and $w$ and are defined under Symbols in the fol-
lowing section. As expressed by Von Kármán in reference 1,
the main characteristics of turbulent flow at a given point
are the magnitudes of the fluctuations and the correlation
between them. The average product of the fluctuations, as,
for example, $uv$, multiplied by the density are the turbu-
lent shearing stresses. Momentum transfer, which gives rise
to the shearing stresses, involves the concept of a length,
or characteristic size, of the region involved in the turbu-
lent exchange. The descriptive term "mixing length" to de-
note this characteristic length was first used by Prandtl.
The migratory processes are imagined to be somewhat
analogous to those involved in the kinetic theory of gases,
with the turbulent motions corresponding in the analogy to
molecular motions in a gas and the mixing length correspond-
ing to the mean free path. While such concepts have been in
use for many years, little is known about the actual nature
of the processes, and assumptions about them have had to be
made in formulating fundamental laws. In relatively simple
cases, such as turbulent flow in pipes and turbulent boundary
layers on plane surfaces, the assumptions have yielded formulas for mean velocity distribution in fair agreement with experiment; but in cases where a turbulent boundary layer develops under the combined action of friction and large pressure gradients, the usual assumptions lead to erroneous results and are obviously not valid. This is particularly true in large adverse pressure gradients, and it is here that the greatest need arises for an understanding of the actual nature of the turbulent processes.
There is need, therefore, for experimental determinations of the magnitude of the various components of the fluctuations, the correlation between components, the turbulent shearing stresses, and mixing lengths in turbulent boundary layers. These will provide the basic information on which to base laws governing the mean motion and the development of the layer. The object in instrument development has been to obtain the means for making measurements that will permit the determination of as many of these quantities as possible.
This investigation, conducted at the National Bureau of Standards, was sponsored by and conducted with the financial assistance of the National Advisory Committee for Aeronautics.
II. SYMBOLS
$U_0$ free-stream velocity, defined here as the velocity that would exist in the region occupied by the wall with the wall absent
$q_0$ free-stream dynamic pressure corresponding to $U_0$
$U$ local mean velocity at any point
$U_1$ particular local mean velocity just outside of boundary layer
$x$ distance along surface measured from the forward stagnation point
$y$ distance normal to surface, measured from the surface
$z$ coordinate normal to $x$ and $y$ with origin on the center line (In the two-dimensional case considered here, mean flow conditions do not vary in the $z$-direction.)
x, y, z coordinates of a point in boundary layer
xy-plane plane of mean flow
xz-plane plane normal to xy-plane and tangent to local direction of mean flow at all points
u x-component of velocity fluctuation lying in xy- and xz-plane
v y-component of velocity fluctuation lying in xy-plane
w z-component of velocity fluctuation lying in xz-plane
\[ u', v', w' \] root-mean-square values of u, v, and w; that is,
\[ u' = \sqrt{u^2}, v' = \sqrt{v^2}, w' = \sqrt{w^2} \], where the bar denotes mean value. The primed symbols are used to avoid an awkward notation in equations
\[ \bar{uv} \] mean value of product of u and v
φ angle between axis of hot wire and direction of wind at the location of the wire
h rate of heat loss from hot wire, watts per second
T instantaneous temperature of wire
\[ T_e \] equilibrium temperature of wire
\[ \bar{T} \] mean temperature of hot wire, degrees C
\[ T_a \] air temperature, degrees C; also temperature of wire when unheated
R mean resistance of hot wire at temperature \( \bar{T} \), ohms
\[ R_a \] resistance of wire at temperature \( T_a \), ohms
\[ R_0 \] resistance of unheated wire at 0° C
\[ \rho_o \] resistivity of the material of the wire at 0° C
| Symbol | Description |
|--------|-------------|
| α | temperature coefficient of resistance referred to 0° C |
| $R_0\alpha$ | slope of temperature-resistance curve |
| i | current through hot wire |
| $E = iR$ | mean voltage across hot wire (When two wires are used, $E_1$ denotes mean voltage across wire I and $E_2$ denotes mean voltage across wire II.) |
| $E_a = E_1 + E_2$ | sum of mean voltage across pair of wires |
| $E_b = E_1 - E_2$ | difference of mean voltage across pair of wires |
| $\Delta E_b$ | change in $E_b$ per radian |
| e | (with various subscripts) voltage fluctuation compensated for wire lag |
| t | time |
| $M_d$ | lag constant of wire |
| M | time constant, seconds |
| γ | phase angle |
| f | frequency, cycles per second |
| m | mass of the hot wire |
| s | specific heat of material of the wire |
| $\rho_i$ | density of material of the wire |
| r | radius of wire |
| ρ | density of air, slugs per cubic foot |
| μ | viscosity of air, slug ft$^{-1}$ sec$^{-1}$ |
| $v = \mu/\rho$ | kinematic viscosity of air, ft$^2$ sec$^{-1}$ |
| $T = -\rho \overline{uv}$ | shearing stress, pound per square foot |
$T_0$ shearing stress on the surface or skin friction
$C_f = \frac{T}{1/2 \rho U_1^2} = \frac{2\overline{uv}}{U_1^2}$ friction coefficient
$K = \frac{\overline{uv}}{u'v'}$ correlation coefficient
$l$ mixing length
$\delta$ boundary-layer thickness
$\delta^*$ displacement thickness $\left( \int_0^\infty \left( 1 - \frac{U}{U_1} \right) dy \right)$
$\theta$ momentum thickness $\left( \int_0^\infty \frac{U}{U_1} \left( 1 - \frac{U}{U_1} \right) dy \right)$
$H = \frac{\delta^*}{\theta}$ shape parameter
$c$ chord of wall, 27.9 feet
$x_s$ position of separation point
$Re = U_0 c / v$ Reynolds number
III. THE NATURE OF THE PROBLEMS INVOLVED
The so-called "turbulence wire," consisting of a hot-wire anemometer with a single wire normal to the wind, has been successfully used in the past in both laminar and turbulent boundary layers to measure the $u$-component of the fluctuations. An attempt was made about 1937 by H. K. Skramstad to determine $v$- and $w$-components of the fluctuations in a turbulent layer by the method of thermal diffusion. (Results unpublished. Method and theory given in references 2 and 3.) He found that the interpretation of results was uncertain because of effects of velocity gradient. Skramstad found, however, that a hot-wire anemometer with the wire making an angle of about $45^\circ$ to the wind could be used with apparent success to measure turbulent shearing stress (reference 4). In recent years, specially constructed
hot-wire anemometers have taken the place of thermal diffusion apparatus and have found considerable use in the measurement of the cross-stream component of turbulence in wind tunnels. Early attempts to use these instruments in turbulent boundary layers, while not entirely successful, indicated at least that under proper conditions reliable results should be obtainable. The principal condition was that the instruments be small compared to the thickness of the boundary layer. This same condition applies to the measurement of shearing stress.
On the basis of this experience an investigation of the turbulent boundary layer again was undertaken. The requirements of the experimental setup were that the boundary layer should be as thick as possible in order to avoid the necessity of building hot-wire instruments vanishingly small and that the adverse pressure gradient parallel to the surface should be sufficiently large to produce separation and yet be negligible normal to the surface. This required a long surface with small curvature, and accordingly the "wall" shown in figure 1 was constructed in the National Bureau of Standards' largest wind tunnel, namely the 10-foot open-air tunnel. The shape and dimensions of this wall are shown in figure 2. The working side, which is the side with curvature on the downstream end, has a smooth surface 28 feet long. The blister on the side of the tunnel was added to steepen the pressure rise and produce separation at the point indicated. By suitable control of the secondary flow near the floor, the flow along the central section was rendered two-dimensional up to and somewhat beyond the separation point.
During the course of the investigation difficulties were encountered with the hot-wire instruments. While the primary cause of the difficulties was the dirt and flying particles carried by the wind, the difficulties manifested themselves in such a way as to show that the requirements for accurate measurements had not been fully appreciated at the outset of the investigation. For example, flying particles broke and bent the delicate platinum wires used as the sensitive element on all hot-wire instruments. Screening the entrance to the tunnel improved matters somewhat, but collisions with smaller particles still getting through the screen bent the wires and changed the calibration of those instruments the characteristics of which depended on the angle between the wire and the wind. Platinum wires could not be placed under tension, and an impact, however slight, with a solid body changed the shape of the wire. In fact, it has since become
clear that it was only a matter of good fortune that measurements of shearing stress were ever possible without having the wire under tension to insure constancy of angle. Another difficulty equally detrimental to accuracy was the accumulation of dirt on the wires.
While these difficulties were probably at their worst in an open-air wind tunnel, they were of such a nature as to indicate that the hot-wire instruments generally might not be practicable for all the measurements theoretically possible. Having found the requirements to be met by a suitable instrument, new methods of construction were tried using tungsten wire and the performance of these new types was investigated in detail. As the result, satisfactory instruments were found, and methods of reducing observed data were improved on the basis of more complete information on the characteristics of such instruments. Thus the stage has been reached where it is possible to state what measurements are possible in practice as well as in theory and give an estimate of the order of the accuracy obtainable. The purpose of this report is to make this information available and show examples of typical results. So far, no work has been done at velocities in excess of 160 feet per second, but with the new instruments, velocity should not be a limiting factor.
IV. TYPES OF HOT-WIRE INSTRUMENTS AND MEASURABLE CHARACTERISTICS OF THE TURBULENCE
All hot-wire instruments considered in this report depend on the rate of heat loss from an electrically heated wire in the wind stream. While some of these instruments are so designed that the rate of heat loss depends on both magnitude and direction of the wind, they may all be regarded as types of hot-wire anemometers. The electrical apparatus is arranged for operation of the wire with constant heating current. This means that the current through the wire may be set at any desired value, but once set is maintained constant either manually or automatically and the wire temperature is allowed to vary as the rate of cooling varies with changes in speed or direction. The resulting change in resistance gives rise to a change in voltage across the wire. Voltage fluctuations, when treated in the manner explained in the following sections, serve to indicate certain characteristics of the turbulence.
The simplest kind of hot-wire anemometer, and the kind that has been used for many years, consists simply of a single wire placed normal to the wind. This kind of instrument, shown in figure 3a responds only to the magnitude of the velocity. Several schemes have been suggested for producing hot-wire instruments with directional characteristics. One such scheme is to place two wires very close together and so obtain a differential heat loss that depends on the direction of the wind. While some investigators have reported success with this scheme, the instruments appear to be difficult to construct. The writers' experience has shown that it is much easier from the standpoint of technique to take advantage of the directional characteristics of a single wire set at an angle to the wind. Such wires may be used singly as shown in figure 3b or in pairs as shown in figures 3c and 3d. When used in pairs the wires are close together, but lie in separate parallel planes. The characteristics of such instruments are treated in section VII, and the method of construction is described in section V. The development and use of hot-wire instruments has been restricted to the types shown in figure 3.
The single wire shown in figure 3a is sensitive to $u$ and is used to measure $u'$. The single slanting wire shown in figure 3b is sensitive to $u$ and $v$ when lying in the $xy$-plane and to $u$ and $w$ when lying in the $xz$-plane, and is used in the $xy$-plane to measure $\overline{uv}$. It is pointed out that so far all experimental work has been confined to the two-dimensional case in which $\overline{uw}$ is zero. The pair of wires intersecting at about $90^\circ$ shown in figure 3c may also be used to measure $\overline{uv}$, and when properly constructed, an instrument of this type has certain advantages over one with a single slanting wire. The $x$-wire arrangement shown in figure 3d differs from figure 3c only by the smaller angle of intersection and is used so as to be sensitive only to $v$ when the wires lie in the $xy$-plane and to $w$ when the wires lie in the $xz$-plane. This instrument is used in the respective planes to measure $v'$ and $w'$.
The measured quantities are therefore $u'$, $v'$, $w'$, and $\overline{uv}$. It is desirable that these values should pertain to a point in the boundary layer. Actually, they are averages over the space occupied by the wires. The wires are made short and the arrangement is made compact so that the space occupied will be small compared to the thickness of the boundary layer. In this connection the advantage of a thick boundary layer is obvious. The $x$-arrangement with the wires
as close together as practicable is employed in order to place both wires as nearly as possible in a region where the mean velocity will be the same for both.
The turbulent shearing stress \( \tau \) and the correlation coefficient \( K \) are obtained from
\[
\tau = -\rho \bar{u} \bar{v}
\]
(1)
\[
K = \frac{\overline{uv}}{\overline{u'}\overline{v'}}
\]
(2)
So far, no method has been found for the direct measurement of the mixing length \( l \). However, on the assumption of similarity of flow patterns in turbulent exchange, proposed by Von Karman (reference 1), \( \tau \) is everywhere proportional to \( \rho l^2 (\frac{dU}{dy})^2 \); and since \( l \) is only a relative measure of the size of the flow pattern, \( l \) may include the factor of proportionality and be defined by
\[
\tau = \rho l^2 (\frac{dU}{dy})^2
\]
(3)
Relation (3) involves the additional assumption that the viscous shearing stress given by \( \mu \frac{dU}{dy} \) may be neglected in comparison to the turbulent shearing stress. This assumption is valid everywhere outside of the laminar sublayer. When \( \frac{dU}{dy} \) is known from the measured velocity distribution, \( l \) may be calculated by means of equations (1) and (3).
It would, of course, be desirable to measure the scale of the turbulence by means of the correlation between like components at different points, as has been done for isotropic turbulence (reference 5). Certain measurements of this sort appear to be possible, say with separate instruments at different points, but at present such measurements are regarded as problems for future development. Only quantities that have actually been measured are to be considered here. These, it is thought, are among the more important quantities needed for the immediate solution of the turbulent boundary-layer problem.
V. NEW DESIGNS OF HOT-WIRE INSTRUMENTS
AND METHOD OF CONSTRUCTION
(a) General Features of the New Design
The instruments shown in figure 3 are the types recently developed for boundary-layer investigation. The prongs with the wires attached across the tips are shown by a, b, c, and d. Figure 3e shows a complete instrument with the stem consisting of a 1/8-inch brass rod 5\(\frac{1}{2}\) inches long by which the instrument is supported on a traversing apparatus, and the flexible leads attached to each prong by which connection is made to long leads running to the electrical equipment outside of the tunnel.
These instruments employ tungsten wires 0.00031 inch in diameter and about 1/16 inch long. The wires were made as short as sensitivity requirements would permit with wire of this diameter. Smaller diameters would make it possible to use shorter lengths, but a diameter of 0.00031 inch appears to be the smallest commercially available in tungsten wire at present. The prongs are about 1/2 inch in length and are made from 0.013-inch phosphor bronze wire. These prongs are flexible and supply spring tension to keep the tungsten wires straight and at a fixed angle with respect to the stem.
The distinctive feature of these instruments is the use of tungsten wire instead of the platinum wire that was always used on older types. Platinum wire has certain advantages over tungsten, such as availability in smaller diameters and higher allowable operating temperatures, but it has very much lower tensile strength and cannot be supported under tension. Platinum wire about 0.0002 inch in diameter, drawn by the Wollaston process, was usually employed. Without tension it was impossible to keep the wires straight and in a fixed orientation, and as a result the calibration of the older instruments could not be maintained. Tungsten wire permits the use of the necessary tension and, in addition, produces a very much more rugged instrument. As mentioned in the introduction, platinum wires could not be used at all in the open-air wind tunnel where it was impossible to keep the air clean and entirely free from flying particles. The two principal difficulties were breakage from collision with solid particles and the accumulation of dirt on the wire.
Breakage rarely occurs with tungsten wire, but the dirt accumulates just the same. However, tungsten wires are sufficiently rugged to permit the removal of dirt by brushing with a small brush.
(b) Method of Attaching Tungsten Wire
The advantages of the superior strength of tungsten wire for application to hot-wire anemometers has been pointed out by Weske in reference 6. However, there has always been one serious drawback to the use of tungsten - namely the difficulty of attaching it to prongs or holders. Whereas platinum wire can be readily soldered with ordinary tin-lead solder, no known solders are entirely satisfactory for tungsten wire. Welding is a possibility, but it is unlikely that the average experimenter will have the skill and equipment required. Weske first called attention to the fact that electroplating the wire with a metal that could be soldered made it possible to use ordinary soldering methods. However, Weske's scheme of plating a thin layer of platinum over the whole wire has the disadvantage of increasing the diameter of the wire and thereby increasing the lag. A variation of the plating scheme was therefore tried with the object of plating the wire only where contact was to be made with the prongs. For this purpose a special copper-plating bath was devised as shown in figure 4. Copper sulphate solution is contained in two wells separated from each other by an air gap. The wire is threaded through the holes in the wells as shown, and solution is then added until the level is slightly above the holes. If the walls are dry on the outside and the holes are small, surface tension prevents leakage. With this arrangement two copper-plated sections are obtained with an unplated section in the middle. The middle section is used as the hot-wire element, and its length is thus definitely defined by the width of the air gap. A microphotograph of a wire processed in this way is shown in figure 4. Any number of hot-wire elements of equal length may be made up by merely pulling another portion of wire into place and repeating the process.
Wires prepared in this way were then attached to prongs by soldering with ordinary tin-lead solder and a flux of zinc chloride solution to produce the instruments shown in figure 3. While a satisfactory mechanical connection was always obtained, the electrical connection was at first unsatisfactory because of an erratic contact resistance. In certain cases there was no evidence of contact resistance for days or possibly
weeks, and then for no accountable reason a resistance suddenly appeared. After much experimentation a technique was found for making satisfactory and lasting connections, which, in essence, amounted to plating slowly enough to get good adhesion between the copper and the tungsten. The thickness of the deposit did not appear to matter, although it was suspected that the differential expansion between copper and tungsten might have a tendency to break the contact if the copper deposit was thick. A clean tungsten surface was obviously to be desired, but no sure method of cleaning was found. Fortunately for the method, contact troubles disappeared when a sufficiently slow plating rate was adopted. The electrolyte used was the same as that ordinarily used in copper plating: 250 grams of crystallized copper sulphate and 75 grams of sulphuric acid per liter of electrolyte. As a precaution against corrosion from the soldering flux, the wires and prongs were washed with a soda solution.
Regardless of whether the plating is thick or thin, the plated and unplated portions are well defined. The copper-plated portions are readily tinned by the solder, and no solder adheres to the bare tungsten. There is, therefore, no difficulty about placing the tips of the prongs at the edge of the copper and thus definitely defining the length of the wire.
(c) Temperature Limitation
The only undesirable property of tungsten wire found so far is its inability to withstand temperatures as high as those withstood by platinum. Tungsten will, of course, oxidize in air at yellow heat, but fine tungsten wires apparently deteriorate at considerably lower temperatures. It has been found that 0.00031-inch-diameter wire will withstand temperatures in the neighborhood of 300°C for an indefinite length of time without showing any signs of deterioration or weakening. Above 350°C, the resistance gradually increases with time and rupture finally occurs. These temperatures are averages over wires about 1/16 inch long. Since wires of this length are considerably hotter in the middle than near the ends, the deterioration probably occurs in the middle at some temperature above the average. It follows, therefore, that longer wires with proportionately smaller end effects will withstand higher average temperatures. The instruments shown in figure 3 had adequate sensitivity for the amplification available with wire temperatures no higher than 250°C.
While platinum wire may be run at much higher temperatures, even to the point of glowing brightly, the wire usually lasted longer and maintained its calibration better when the temperatures were less than 400° C.
VI. ELECTRICAL EQUIPMENT
The electrical equipment associated with the hot-wire instruments is fundamentally the same as that described in reference 7. The general scheme always has been to heat the wire with a known constant current and to measure the mean voltage and the fluctuating voltage across the wire. This is known as constant-current operation. Since the publication of reference 7, the hot-wire circuits have been modified by Mr. Mock to include circuits for two hot wires; the controls have been modified for greater convenience of operation, and the equipment generally has been made lighter and more portable. The assembled equipment is shown in figure 5, where the three basic units are: the amplifier A, its power supply B, and the control unit C. All units are properly shielded internally and externally to prevent pickup from stray electric fields. The circuit diagrams for these three units are shown in figures 6, 7, and 8. The circuit diagrams are given here mainly to help the reader to understand the functions of the control unit and the amplifier. For those who may wish to construct such equipment, the diagrams will be useful in conjunction with the detailed treatments given in references 7, 8, 9, and 10. Only those features of the circuits that determine the performance of the hot-wire instruments will be discussed here. It should be remarked that a radically different type of hot-wire circuit described in reference 11, permitting constant-temperature operation, is worthy of consideration, especially for highly turbulent flow where the temperature fluctuations with constant-current operation may on occasions exceed the high-temperature limit for tungsten wire.
(a) Control Unit
The control unit contains, in addition to the controls, the potentiometer and Wheatstone bridge for measuring current, mean voltage, and resistance. The various components are found in the circuit diagram shown in figure 6. The heating current is supplied by storage batteries, requiring from 6 to 12 volts for each wire depending on the current.
For 0.00031-inch diameter tungsten wire the current is usually no greater than 180 milliamperes. Separate batteries are used for each wire. The 12-henry chokes $L$ in each battery circuit provide sufficient impedance to afford constant-current operation during velocity variations associated with turbulence. In more precise terms, the reactance $2\pi f L$ together with the ohmic resistance in the circuit is sufficient to reduce current variations to negligible proportions for all frequencies above the cutoff frequency of the amplifier. For different mean velocities the current must be reset manually by means of rheostats. The 4-dial decade voltage divider is placed in the circuit as potentiometer or bridge, depending on the position of circuit selector No. 2. Circuit selector No. 1 permits the operator to measure the current through either wire, the mean voltage across either wire or the sum or the difference of the voltages across the wires, and the resistance of either wire at air temperature or the sum of the resistances. Another position on selector No. 2 impresses the voltages on the amplifier, individually or the sum or the difference as selected by selector No. 1. When measuring resistance, R12 is thrown in the bridge circuit to make the bridge current sufficiently small to prevent heating of the wires. The leads are always in series with the wires, and correction must always be made for the leads to obtain the resistance of the wires only or the mean voltage across the wires only. An alternating voltage is supplied by an oscillator through the control unit for calibrating the amplifier. This voltage is the drop across $R9$ and is measured by the thermocouple and microammeter. The various functions of this rather complex control unit will be made clear by tracing the circuits for various positions of the selectors.
(b) Amplifier
Since the voltages may be combined in the control unit when the response of two wires is to be obtained simultaneously, only one amplifier is necessary. The circuit diagram of the amplifier is shown in figure 7. Electrically, this amplifier may be described as a 7-stage capacitance-compensated amplifier. The purpose of compensation is to obtain an amplification increasing with frequency in the same manner as the fluctuating voltage across a hot wire decreases with frequency. As will be shown in section VII, this characteristic of a hot wire, known as lag, may be expressed in terms of a time constant $M$, which varies with the size of the wire and the operating conditions.
For a harmonic velocity variation of constant amplitude, the ratio of the voltage fluctuation across the wire at frequency \( f \) to the voltage fluctuation at zero frequency is given by
\[
\frac{1}{\sqrt{1 + 4\pi^2 f^2 M^2}}
\]
The input to the amplifier therefore decreases as frequency increases, in accordance with the above-mentioned relation. If the input voltage to one of the stages is made proportional to
\[
\sqrt{1 + 4\pi^2 f^2 M^2}
\]
the final result is an output independent of frequency. Under proper conditions as described in reference 8, this may be done with a load reactance consisting of inductance and resistance or capacitance and resistance. In the present amplifier capacitance and resistance are used as shown under "time constant selector" in the circuit diagram. In this case the time constants are C9 times R12, C8 times R12, and so forth, giving values of \( M \) of 0.001, 0.002, 0.003, 0.004, 0.005, and also 0 second. For proper compensation the time constant should be set to the same values as the time constant for the wire. When the time constant for the wire falls between the settings provided, as it usually does, readings must be taken for two settings and the correct result obtained by interpolation.
The design of the amplifier was dictated largely by the impedance requirements for the capacitance type of compensation (reference 8) and by the over-all frequency characteristics desired. The ideal compensated amplifier is one that works in conjunction with a hot wire to give an output truly representative of all frequencies present in turbulent flow. The present amplifier has an error of less than 2 percent between 10 and 2000 cycles per second with the error increasing to 10 percent at 5000 cycles per second. The error below 10 cycles per second has not been experimentally determined, but the amplification begins to drop with decreasing frequency due to the characteristic low-frequency cut-off of resistance-capacitance coupling. According to the values of \( R_1 \) and \( C_1 \), the output voltage at 1 cycle per second is reduced to about
70 percent of that at frequencies above 10 cycles per second. As shown by Dryden in reference 10, these frequency characteristics are satisfactory for measurements of fluctuations ordinarily encountered in isotropic turbulence. The frequency distribution in turbulent boundary layers is not known and probably varies in different parts of the layer. It is believed, however, that the amplifier is just as satisfactory for measurements in turbulent boundary layers as in fields of isotropic turbulence if the proportion of the turbulent energy lying below 10 cycles per second is the same in the two cases.
Other features of the amplifier are evident in the circuit diagram. The gain control permits the amplification to be varied over wide limits. A phase inverter and balanced output are used to prevent direct current from flowing through the thermoelements. Thus only the alternating current produced by the fluctuating hot-wire voltage is read on the output meter. The amplifier is calibrated by reading the output meter with known input voltages. An unknown mean-square input voltage may then be determined from the output meter reading. The "eye" (6E5) gives a rough indication of the reading to be expected and is used to enable the operator to judge whether he is likely to burn out the fuse, and possibly the thermoelements, when depressing the key.
(c) Power Supply
By means of the dual power supply shown in figure 8, the amplifier is operated completely on 115-volt, 60-cycle current. The power-supply circuit is conventional except for the voltage-regulator circuits lying to the right of the filter circuits. The voltage regulator serves two purposes. First it holds the output voltage practically constant regardless of reasonable changes in load and input voltage to the unit. Secondly, because of its regulating action, it acts as an efficient filter circuit, and also causes the power supply to appear to have a very low internal resistance. Since the requirements to be met by a satisfactory power supply are treated in detail in reference 7, no further discussion is given here.
VII. CHARACTERISTICS OF HOT WIRES AND THEORY
OF HOT-WIRE MEASUREMENTS
The relation between the air velocity normal to a heated wire and the rate of heat loss as given by King (reference 12) is
\[
\frac{h}{T - T_a} = D_1 + F_1 \sqrt{U}
\]
(4)
where the terms \(D_1\) and \(F_1\) depend on the thermal conductivity, density, and specific heat of the air and the dimensions of the wire as shown in reference 12. Owing to the methods of using hot wires in boundary-layer investigations, \(D_1\) and \(F_1\) may be regarded as constants. When the wire is heated by an electric current \(i\), the heat is supplied at the rate \(i^2 R\), where \(R\) is the resistance of the wire at temperature \(T\). The temperature head \(T - T_a\) is
\[
T - T_a = \frac{R - R_a}{R_a \alpha}
\]
\[
\alpha = \frac{L}{K} \cdot \frac{R}{R_a}
\]
Under equilibrium conditions the rate of heat loss must be equal to the rate at which heat is supplied, and equation (4) may be written
\[
\frac{i^2 R (R_a \alpha)}{R - R_a} = D_1 + F_1 \sqrt{U}
\]
(5)
Equation (5) is the usual form of the relation used in hot-wire anemometry. Equation (5) is found to hold true over a wide range of velocities.
In the expressions to follow it will be convenient to disregard the distinction between mean quantities and instantaneous quantities. In the few cases where a distinction is necessary, appropriate symbols will be used. The usual distinction between mean quantities and fluctuations still will be made.
As previously mentioned, the control unit has been designed for constant-current operation, which means that \(i\)
is constant. For convenience, the constant terms in equation (5) are included in the constants on the right, and the equation is written
\[
\frac{R}{R - R_A} = D + F \sqrt{U}
\]
(6)
Equation (6) expresses the relation between \(\frac{R}{R - R_A}\) and \(U\) for a given wire heated with a given constant current. The voltage \(E\) across the wire is simply \(iR\), and is the quantity usually observed rather than \(R\). The observations are usually expressed in terms of \(\frac{R}{R - R_A}\) because of the linear relation between this quantity and \(\sqrt{U}\).
Equation (6) is found to hold true regardless of the angle between the axis of the wire and the wind. The constant \(F\), however, depends on the angle. When the voltage is observed for various angles between the axis of the wire and the wind at fixed values of \(U\), \(E\) is found to vary as shown in figure 9. When \(\frac{R}{R - R_A}\) is calculated from the voltage and the measured \(R_A\) and is plotted against \(\varphi\), the curves shown in figure 10 are obtained. If as seems logical, the heat loss is a function of the component of the velocity normal to the wire, the general form of equation (6) should be
\[
\frac{R}{R - R_A} = D + F \sqrt{U \sin \varphi}
\]
(7)
In order to test this relation, \(\frac{R}{R - R_A}\) is plotted against \(\sqrt{U \sin \varphi}\) as shown in figure 11. If equation (7) is valid, all points should lie on a single straight line. The line is reasonably straight above \(\sqrt{U \sin \varphi} = 4\). The fact that not all the points lie on the same line is attributed to an accumulation of dirt on the wire as the run progressed. Equation (7) therefore may be regarded as a reasonably good approximation over the linear range. Since equation (6) is valid over a wide range of velocities, the linear range in figure 11
depends on the value of $\varphi$. If $\varphi$ lies between $20^\circ$ and $90^\circ$, the curve is linear for all values of $U \sin \varphi$. It should be noted that, when $\varphi$ is constant, the curve is always linear regardless of the value of $\varphi$, for then equation (7) reduces to the form of equation (6) with $F \sim \sin \varphi$ taking the place of the constant $F$ in equation (6). However, separate linear curves are obtained for each value of $\varphi$ below $20^\circ$.
When the velocity varies either in magnitude or direction, the voltage across the wire varies. The instantaneous velocities may be regarded as a superposition of $u$, $v$, and $w$, on the velocity $U$. Since $v$ and $w$ are by definition at right angles to $U$, these two fluctuations, if they are small compared to $U$, produce only a variation in the local direction of the stream. The angle variation, expressed in radians, for a wire lying in the $xy$-plane is $v/U$ and for a wire lying in the $xz$-plane is $w/U$. Thus
\[
(xy\text{-plane}) \quad \Delta \varphi = \frac{v}{U}
\]
\[
(xz\text{-plane}) \quad \Delta \varphi = \frac{w}{U} \tag{8}
\]
It is assumed that $u$, $v$, and $w$ are small compared to $U$, and therefore that the voltage fluctuations associated with $u$, $v$, and $w$ are proportional to $u$, $v$, and $w$ and may be expressed by
\[
e_1 = Au
\]
\[
e_2 = Bv
\]
\[
e_3 = Fw \tag{9}
\]
The corresponding root-mean-square values are written
\[
e_1' = \sqrt{A^2} u'
\]
\[
e_2' = \sqrt{B^2} v'
\]
\[
e_3' = \sqrt{F^2} w' \tag{10}
\]
In equations (9) and (10) the voltages are assumed to be the
voltages properly compensated for the lag of wire; that is, they are determined from the output of a properly compensated amplifier. When proper compensation is used; A and B may be evaluated by the aid of equation (7). In other words, compensation for the lag of the wire makes it possible to apply equilibrium relations even though the equilibrium condition assumed in equations (5), (6), and (7) may not actually exist.
Inasmuch as relations (9) are assumed, it is permissible to regard the fluctuating voltages, angles, and velocities, represented by e, Δφ, u, v, and w, respectively, as differentials and evaluate A and B by differentiating equation (7). If dE is regarded as e, dU as u, and Udφ as v or w in equations (8) and (9),
\[ A = \frac{dE}{dU}, \quad B = \frac{dE}{Ud\varphi} \]
Differentiating (7) with respect to U, regarding φ as constant yields,
\[ \frac{dR}{dU} \left( -\frac{R_a}{(R-R_a)^2} \right) = \frac{F \sqrt{\sin\varphi}}{2 \sqrt{U}} \]
and since \( dE = idR \)
\[ \frac{dE}{dU} \left( -\frac{R_a}{(R-R_a)^2} \right) = \frac{i F \sqrt{\sin\varphi}}{2 \sqrt{U}} \]
and
\[ A = -\frac{i F \sqrt{\sin\varphi}}{2 \sqrt{U}} \frac{(R-R_a)^2}{R_a} \] \hspace{1cm} (11)
Differentiating (7) with respect to φ, regarding U as constant yields
\[ \frac{dR}{d\varphi} \left( -\frac{R_a}{(R-R_a)^2} \right) = \frac{F \sqrt{U \cos\varphi}}{2 \sqrt{\sin\varphi}} \]
\[ \frac{dE}{U d\varphi} \left( -\frac{R_a}{(R-R_a)^2} \right) = \frac{i F \cos\varphi}{2 \sqrt{U \sin\varphi}} \]
\[ B = -\frac{1}{2} \frac{F \cos \varphi}{\sqrt{U \sin \varphi}} \left( \frac{R - R_a}{R_a} \right)^2 \]
The fact that \( A \) and \( B \) are negative means simply that the voltages in equations (9) decrease as \( u, v, \) and \( w \) increase.
The method of evaluating the constants \( A \) and \( B \) and the manner in which they are used will be considered in connection with each of the quantities to be determined. The purpose of the different arrangements of wires shown in figure 3 is to obtain instruments that will measure one component at a time; for example, measure \( u' \) without contamination from \( \bar{u}v, v', \) and \( w', \) or measure \( v' \) while excluding \( u', \bar{u}v, \) and \( w', \) and so forth. This can be done more easily in some cases than in others, and in all cases the ideal can be approached only when \( u, v, \) and \( w \) become small with respect to \( U. \) Regardless of the type of instrument, the separation of components is possible only insofar as variations in \( \varphi \) are independent of \( u \) and variations in \( U \) are independent of \( v \) and \( w. \) This again requires that \( u, v, \) and \( w \) be small compared to \( U. \)
(a) Determination of \( u' \)
For the measurement of \( u' \) the wire is placed normal to the wind and parallel to the surface producing the boundary layer. The angle \( \varphi \) is then 90° and \( A \) and \( B \) as given by equations (11) and (12) become
\[ A = -\frac{1}{2} \frac{F}{\sqrt{U}} \left( \frac{R - R_a}{R_a} \right)^2 \]
\[ B = 0 \]
Thus according to equations (10)
\[ e_1' = \frac{1}{2} \frac{F}{\sqrt{U}} \left( \frac{R - R_a}{R_a} \right)^2 u' \]
and \( e_2' \) and \( e_3' \) are zero. The relations show that this instrument is sensitive to \( u \) and insensitive to \( v \) and \( w. \)
Solving (14) for $u'$ and dividing by $U$ gives
$$\frac{u'}{U} = \frac{2 R_A e_1'}{i F \sqrt{U(R-R_A)}}.$$ \hspace{1cm} (15)
All quantities on the right of equation (15), with the exception of $F$, are determined at each point where $\frac{u'}{U}$ is being measured. The constant $F$ is determined for the particular wire used by placing the instrument in the free stream and measuring the mean voltage $E$ at several values of the velocity $U$ at the location of the wire. The quantity $\frac{E}{R-R_A}$ is then calculated and plotted against $U$ to obtain a calibration curve as shown in figure 12. The slope of this curve is $F$, since $\varphi = 90^\circ$. The current $i$ must not only be constant during a run, but must be the same for the calibration as for the determination of $\frac{u'}{U}$.
It is well to see how, and to what extent, a single wire normal to the wind with its axis in the $xz$-plane responds to $u$ and not to $v$ and $w$. Since the wire has cylindrical symmetry, the only way in which $v$ can change the rate of heat loss is through changes in the resultant velocity by vector addition with $U$. The error in a measurement of $\frac{u'}{U}$ arising from $v$ is small until $v/U$ becomes quite large, and when this is the case, the type of error discussed in section IX is so large by comparison that the error arising from $v$ becomes insignificant. The $w$-fluctuations also change the resultant velocity, but do not change the normal component. Since it has been shown that the heat loss depends only on the normal component, no error arises from $w$ regardless of the size of $w/U$.
(b) Determination of Turbulent Shearing Stress.
According to equation (1) the determination of turbulent shearing stress involves the measurement of $uv$. For this purpose, use is made of either the single slanting wire shown in figure 3b, making an angle $\varphi$ of about $45^\circ$ to the wind, or the x-wires shown in figure 3c, set approximately $90^\circ$ to each other and each making about $45^\circ$ to the wind. The manner in which these wires are used is illustrated in figure 13. Consider first the single wire, first in position
I and then rotated $180^\circ$ about an axis in line with the wind to position II. Figure 9 shows that the voltage decreases as $U$ and $\varphi$ increase. Therefore in position I, $+u$ and $+v$, as defined in figure 13, both decrease the voltage; and the resultant voltage change is the sum of $e_1$ and $e_2$ in accordance with equations (9). In position II $+u$ decreases the voltage while $+v$ increases the voltage, and the resultant voltage is the difference between $e_1$ and $e_2$. As previously explained, the mean-square of the resultant voltage fluctuation may be determined from the output meter reading of the compensated amplifier. If the mean-square resultant for position I is denoted by $a$ and the mean-square resultant for position II is denoted by $b$,
$$a = (-e_1 - e_2)^2, \quad b = (-e_1 + e_2)^2$$
From equations (9)
$$a = (-A_I u - B_I v)^2 = A_I^2 u^2 + 2A_I B_I uv + B_I^2 v^2 \quad (16)$$
$$b = (-A_{II} u + B_{II} v)^2 = A_{II}^2 u^2 - 2A_{II} B_{II} uv + B_{II}^2 v^2 \quad (17)$$
where subscripts I and II refer to the value of the constants for positions I and II, respectively.
In using the wire, $a$ and $b$ are determined in positions I and II, care being taken to make the $180^\circ$ rotation from one position to the other about an axis through the center of the wire and along the average wind direction. When rotated in this way, there is no change in the average value of $\varphi$. If, in addition, the average condition of the boundary layer remains the same, equations (11) and (12) show that $A_I = A_{II} = A$ and $B_I = B_{II} = B$. Under these conditions the sum and difference of equations (16) and (17) become
$$a + b = 2(A^2 u^2 + B^2 v^2) \quad (18)$$
$$a - b = 4AB uv \quad (19)$$
From equations (1) and (19) is found
\[ \tau = -\frac{P(a-b)}{4AB} \] \hspace{1cm} (20)
where, according to equations (11) and (12),
\[ AB = \frac{i^2F^2(R-R_a)^4 \cos \varphi}{4U R_a^2} \] \hspace{1cm} (21)
All the quantities on the right of equations (20) and (21), except \( \varphi \) and \( F \), are determined at each point where \( \tau \) is being determined. According to equation (7), \( F \) is the slope of the straight portion of the curve in figure 11, and \( F \) is determined from calibration curves of this kind. The angle \( \varphi \) may be determined by plotting \( R/R-R_a \) against \( \sqrt{U} \), when the wire is set at the proper angle, and, finding the slope of the curve which is equal to \( F \sqrt{\sin \varphi} \).
As mentioned above, the x-wire arrangement may be used in place of the single wire for the measurement of shearing stress. If the wires are now labeled I and II as shown in figure 13, the same relations apply as for positions I and II. The 180° rotation is unnecessary; but in order for the conditions \( A_I = A_{II} = A \) and \( B_I = B_{II} = B \) to apply, the wires must be identical and each must subtend the same angle to the wind. When the wires are prepared by the electroplating method described in section V, it is not difficult to obtain matched wires. Calibration will show whether the wires are sufficiently well matched to be usable, and equal voltages across the wires will indicate the proper setting for the instrument to place the wires I and II at equal angles to the wind. The angle \( \varphi \) is half the angle subtended by the wires and may be determined by direct measurement.
In the earlier type of instrument using platinum wires it was difficult to obtain two wires sufficiently alike to use the x-wire for shearing stress, and consequently the early measurements were all made with single slanting wires. The 180° rotation of the wire is a troublesome feature of this method, not so much because of mechanical complications,
but because of the difficulty of passing the axis of rotation through the midpoint of the wire. It is seldom possible to avoid some amount of displacement to and from the surface in rotating from one position to the other. This difficulty is avoided with the x-wires, and the instrument support system may be simplified by the elimination of a rotation mechanism. The disadvantages of the x-wire are that the instrument is more difficult to construct even when tungsten wires are used and two wires must hold their calibration instead of one. An accident to either wire puts the instrument out of service.
(c) Determination of Correlation Coefficient
The correlation coefficient $K$ is defined by equation (2). Since $K$ involves $\overline{uv}$, $u'$ and $v'$, its evaluation depends on the measurements of these three quantities. There are in fact three ways to evaluate $K$, all three being interrelated but involving $u'$ and $v'$ in different ways. Because of experimental errors, the three methods, that should yield identical results, will in general produce three somewhat different values of $K$. It is therefore advisable to label them $K_1$, $K_2$, and $K_3$ to correspond to the first, second, and third method.
The first method depends mainly on the results obtained in the measurement of shearing stress. Equation (18) is solved for $v'$ and equation (19) is solved for $\overline{uv}$. Then it follows that
$$v' = \sqrt{\frac{a+b}{2B^2} - \frac{A^2}{B^2} u'}$$ \hspace{1cm} (22)
$$\overline{uv} = \frac{a-b}{4AB}$$ \hspace{1cm} (23)
where by definition $u'$ and $v'$ stand for $\sqrt{u^2}$ and $\sqrt{v^2}$. If (22) is multiplied by $u'$ and (23) divided by the result, there is obtained
$$K_1 = \frac{\overline{uv}}{u'v'} = u' \sqrt{\frac{a-b}{2B^2} - \frac{A^2}{B^2} u'^2}$$ \hspace{1cm} (24)
Thus $K_1$ may be calculated from the data taken in connection with a determination of shearing stress together with an independently measured value of $u'$.
The second expression for the correlation coefficient is obtained by dividing equation (19) by (18) and solving for $\overline{uv}/u'v'$. It is found that
$$K_2 = \left(\frac{a-b}{a+b}\right) \frac{\frac{A^2}{B^2} \frac{u'}{v'} + \frac{v'}{u'}}{2 \frac{A}{B}}$$ \hspace{1cm} (25)
Here the data taken in connection with a determination of shearing stress are used in such a way that only the ratio of the constants $A$ and $B$ and the ratio of $u'$ to $v'$ enter into the expression for the correlation coefficient. In this case both $u'$ and $v'$ must be determined by independent measurement.
Finally, by solving equation (19) for $\overline{uv}$ and dividing by $u'$ and $v'$ it is found that
$$K_3 = \frac{a-b}{4AB u'v'}$$ \hspace{1cm} (26)
or by equation (20)
$$K_3 = -\frac{\tau}{\rho u'v'}$$ \hspace{1cm} (27)
As shown by equation (27), the third method amounts simply to using the measured shearing stress and the independently measured $u'$ and $v'$.
Calculation of the correlation coefficient by all three methods affords a check on the accuracy of the measurements. Complete agreement can scarcely be expected, and there is probably a best value depending on the accuracy with which the various terms are known. As noted, $K_2$ involves only the ratios $u'/v'$, and $A/B$. According to equations (11) and (12)
$$\frac{A}{B} = \tan \phi$$ \hspace{1cm} (28)
and therefore $A/B$ is less subject to error than $A$ and $B$ separately. When $\varphi = 45^\circ$, the percentage error in $\tan \varphi$ is a minimum for an error in $\varphi$. Furthermore, according to equation (25) no error in $K_2$ results from an error in $u'/v'$ when $A/B = v'/u'$. Since $v'/u'$ is not greatly different from unity, the optimum conditions appear to be
$$\frac{A}{B} = \tan \varphi = 1, \quad \varphi = 45^\circ$$
It is concluded therefore that $K_2$ is probably the best value of the correlation coefficient. On the other hand, only $K_3$ will be consistent with the measured values of $T$, $u'$ and $v'$. If the difference between $K_2$ and $K_3$ is significant, the least certain of the quantities $T$, $u'$, and $v'$ should be derived from equation (27) using $K_2$. In this way the best consistent set of results will be obtained.
So far it has been assumed that a slanting wire or an x-wire in the xy-plane is insensitive to $w$. On the average there is no flow across the xy-plane by definition of this plane. At any instant, however, the flow makes an angle with the xy-plane given by $w/U$. In order to see how well the assumption is justified it is necessary to find out how much of a change $w/U$ produces in the angle between the wire and the wind. If the angle $w/U$ is denoted by $\Delta \psi$ and the instantaneous angle between the wire and the wind by $\varphi_1$, it is found from geometrical relations that
$$\sin \varphi_1 = \cos \varphi \sqrt{\sin^2 \Delta \psi + \tan^2 \varphi} \quad (29)$$
It is seen from equation (29), for example when $\varphi = 45^\circ$, that $\Delta \psi$ must exceed 0.175 radian or $10^\circ$ before $\varphi_1 - \varphi (= \Delta \varphi)$ is greater than $1^\circ$. The smallness of the effect on $\varphi$ is the reason for assuming that wires in the xy-plane are insensitive to $w$. Just how large $w/U$ may become before this assumption is no longer justified is difficult to estimate because of the distorted nature of the voltage fluctuation resulting from $w$.
In two-dimensional flow there is no error from $w$ in the measured shearing stress because of the fact that $w$ is not correlated with $u$ or $v$. Whether or not there is an error in the measured correlation coefficient depends on
the effect of $v$ and $w$ in the measurement of $u'$ and of $u$ and $w$ in a measurement of $v'$. It has already been shown that the effect of $v$ and $w$ is negligible in a measurement of $u'$. The effect of $u$ and $w$ in the measurement of $v'$ will be considered presently.
(d) Determination of $v'$ and $w'$
As far as the theory is concerned, there is no difference between the measurement of $v'$ and $w'$. The x-wire instrument is used in both cases — in the xy-plane for $v'$ and in the $xz$-plane for $w'$. Therefore the relations for $v'$ will be derived, and the same relations hold true for $w'$.
Figure 13 illustrates the position of the wires for a measurement of $v'$. Whereas for the measurement of shearing stress, the mean-square voltages, denoted by $a$ and $b$, were determined for the wires separately, now the mean-square resultant voltage is determined for the pair. In other words, the voltage across the pair is impressed on the amplifier and only the resultant is measured. This is illustrated as follows: Referring to figure 13, let $-e_1 - e_2$, as defined by equation (9), be the voltage across wire I and $-e_1 + e_2$ be the voltage across wire II. The mean-square resultant voltage when added is
$$\overline{e_a^2} = \left[(-e_1-e_2)_I + (-e_1+e_2)_II\right]^2$$ \hspace{1cm} (30)
and when subtracted is
$$\overline{e_b^2} = \left[(-e_1-e_2)_I - (-e_1+e_2)_II\right]^2$$ \hspace{1cm} (31)
According to equations (9), using constants $A_I$ and $B_I$ for wire I and $A_{II}$ and $B_{II}$ for wire II, equations (30) and (31) become
\[
\overline{e_a^2} = (-A_I u - B_I v - A_{II} u + B_{II} v)^2
\]
\[
\overline{e_a^2} = A_I^2 \overline{u^2} + B_I^2 \overline{v^2} + A_{II}^2 \overline{u^2} + B_{II}^2 \overline{v^2} + 2A_I B_I \overline{uv} + 2A_I A_{II} \overline{u^2}
\]
\[-2A_I B_{II} \overline{uv} + 2B_I A_{II} \overline{uv} - 2B_I B_{II} \overline{v^2} - 2A_{II} B_{II} \overline{uv}\]
(32)
\[
\overline{e_b^2} = (-A_I u - B_I v + A_{II} u - B_{II} v)^2
\]
\[
\overline{e_b^2} = A_I^2 \overline{u^2} + B_I^2 \overline{v^2} + A_{II}^2 \overline{u^2} + B_{II}^2 \overline{v^2} + 2A_I B_I \overline{uv} - 2A_I A_{II} \overline{u^2}
\]
\[+2A_I B_{II} \overline{uv} - 2B_I A_{II} \overline{uv} + 2B_I B_{II} \overline{v^2} - 2A_{II} B_{II} \overline{uv}\]
(33)
When \(A_I = A_{II} = A\) and \(B_I = B_{II} = B\), (32) and (33) reduce to
\[
\overline{e_a^2} = 4A^2 \overline{u^2}
\]
(34)
\[
\overline{e_b^2} = 4B^2 \overline{v^2}
\]
(35)
Thus, in accordance with equations (11) and (12), when the two wires are identical and each subtends the same angle to the wind, \(\overline{e_a^2}\) is a measure of \(\overline{u^2}\) only and \(\overline{e_b^2}\) is a measure of \(\overline{v^2}\). When the two wires are not identical or do not subtend the same angle to the wind, the result is a mixture of \(\overline{u^2}, \overline{v^2},\) and \(\overline{uv}\). Equation (34) shows that the
X-wire instrument may be used to measure $u'$, but due to the possibility of obtaining a mixture of components, the single wire normal to the wind is preferable. In order to lessen the possibility of a mixture of components in the measurement of $v'$, $B$ is made larger than $A$; that is, the angle $\phi$ is made less than 45°.
While the foregoing relations are useful to illustrate how the characteristics of individual wires can be combined to produce an instrument sensitive to $v$, these relations, as such, are not used to determine $v'$. In practice it is unnecessary, as well as too laborious to determine the constants $B$ for the individual wires. Instead, the sum and the difference of the mean voltage across the pair of wires are determined as a function of velocity and angle, and in this way a calibration curve is obtained for determining $v'/U$ from the root-mean-square voltage fluctuation directly. The basis for this procedure is illustrated by the performance characteristics given in figures 14 and 15.
Figure 14 shows the mean voltages as measured on the potentiometer for wires I and II as a function of angle between the stem of the instrument and the wind at a velocity of 124 feet per second. The difference voltage $E_b$, also measured on the potentiometer, is shown by the broken curve. It is seen that while the curves for the wires individually are far from linear in the neighborhood of zero angle, $E_b$ is practically linear with angle over a range of 30°. The reciprocal of the slope indicates the sensitivity of the pair of wires to $v$. When the slope is expressed in terms of volts per radian and is denoted by $\Delta E_b$, there exists the simple relation
$$\frac{v'}{U} = \sqrt{\frac{e_b^2}{\Delta E_b}}$$ \hspace{1cm} (36)
where $\sqrt{e_b^2}$ is determined from the compensated output reading of the amplifier and $\Delta E_b$ is determined by calibration. The calibration consists of determining $\Delta E_b$ at various velocities. A calibration curve shown in figure 15 is obtained by plotting $\Delta E_b$ against $R/R - R_A$ for the pair of wires. This affords a convenient means for obtaining $\Delta E_b$ without having to know the mean velocity at each point in the boundary layer.
In use, the proper angle for setting the instrument is found by making $M_b$ zero regardless of the angle between the stem of the instrument and the wind. If the stem is out of alignment with the wind, for example, 5° as shown in figure 14, the two wires are not set at the same angle with respect to the stem or the wires are imperfectly matched.
The voltage fluctuation produced by $w$ is removed as a potential source of error in the measurement of $v'$ because the subtraction indicated in equation (31) is made before squaring. In other words, if equation (29) were used to find $\Delta \varphi$, a positive $\Delta \varphi$ would be found for both wires, and there would be no net effect on the difference voltage.
The effect of $u$ in a measurement of $v'$ may be found quite simply by considering that $\varphi$ is assumed to be $v/U$ when actually it is $\frac{v}{U+u}$. Then actually $v/U+u$ is being measured or approximately
$$\frac{v}{U} \left(1 + \frac{u}{U}\right)$$
when $\frac{u}{U}$ is small compared to 1. The root-mean-square value of this expression is
$$\frac{v'}{U} \left(1 + \frac{1}{2} \frac{u^2}{U^2}\right) \quad \text{approximately} \quad (37)$$
From this expression the error in $v'/U$ may be found. If, for example, $u'/U$ is 0.5, the measured $v'/U$ is too high by 4% percent.
(e) Hot-Wire Lag
Because of the heat capacity of the wire, a certain time is required for the wire temperature to reach equilibrium after a change has occurred in the rate of heat loss accompanying a change in magnitude or direction of the wind. If $T_e - T$ is small compared to $T - T_a$, the relation between the instantaneous temperature $T$ and the equilibrium temperature $T_e$ is given by
\[ M \frac{dT}{dt} = T_e - T \]
(38)
where \( M \) is termed "the time constant." The solution of (38) consists of a transient term \( \exp \left( -\frac{t}{M} \right) \) and a periodic term, if \( T_e \) is periodic. Since the transient term soon becomes negligible, only the periodic term will be considered. As shown in reference 9, if the temperature variation is periodic with frequency \( f \), the temperature variations and corresponding voltage variations are reduced in amplitude below that for zero frequency by the factor
\[ \frac{1}{\sqrt{1 + 4 \pi^2 f^2 M^2}} \]
(39)
and lag in phase by the angle
\[ \gamma = \tan^{-1} 2 \pi f M \]
(40)
With regard to the irregular wave form of turbulent fluctuations, which results from a superposition of many frequencies, (39) and (40) mean that the voltage fluctuation from each component frequency is reduced in the ratio of 1 to \( \sqrt{1 + 4 \pi^2 f^2 M^2} \) and lags in phase by the angle \( \gamma \). Consequently the wave form of the voltage across the wire fails to reproduce the wave form of the turbulence.
The expression for \( M \) is derived in reference 9 for the case where the heating current is constant and is given as
\[ M = \frac{4.18 ms (R - R_a)}{i^2 R_a R_o \alpha} \]
(41)
where
\( m \) mass of the wire
\( s \) specific heat of the material of the wire
4.18 mechanical equivalent of heat, joules per calorie
In reference 10 an expression for $M$ is derived for the more general case in which the heating current varies with the resistance of the wire. However, only constant-current operation and therefore equation (41) will be considered here.
As a matter of convenience, the terms on the right of equation (41) are separated into those that depend on the wire and those that depend on the operating conditions. Equation (41) then becomes
$$M = M_C \left( \frac{R - R_a}{I^2 R_a} \right) \quad (42)$$
where $M_C$ depends on the wire and $\frac{R - R_a}{I^2 R_a}$ depends on operating conditions. According to equation (41) $M_C$ is given by
$$M_C = \frac{4.18 \text{ ms}}{R_0 \alpha} = \frac{4.18 \pi^2 r^4 \rho_{1s}}{\sigma_0 \alpha} \quad (43)$$
where
$r$ radius of the wire
$\rho_{1s}$ density of material of the wire
$\sigma_0$ resistivity of the material of the wire at $0^\circ C$
If $M_0$ is known, $M$ may be calculated by equation (42) for any working condition. While it is possible in principle to calculate $M_C$ by equation (43) from the radius of the wire and the properties of the material, there are two reasons why it is not feasible to do so. First, it is difficult to measure $r$ with the accuracy required by the fourth-power relation; and second, equation (41) is not strictly valid for short wires because of end effects. The expression $M_C$ is therefore determined by measuring the lag of samples of the wire under known operating conditions on an apparatus which vibrates the hot wire in a steady air stream at various known frequencies. Several experimental values of $M_C$ for tungsten wire with a nominal diameter of 0.00031 inch are as follows:
| Wire length (mm) | $M_C$ |
|-----------------|-------|
| 1.1 | $504 \times 10^{-7}$ |
| 2.5 | 389 |
| 3 | 366 |
The value of $M_C$ calculated by equation (43) is $263 \times 10^{-7}$.
It will be observed that the experimentally determined $M_C$ increases with decreasing wire length; whereas, according to equation (43), $M_C$ should be independent of wire length. This effect is due to the conduction of heat from the ends of the wire to the prongs, which is not taken into account in the derivation of equation (41). Because of this additional heat loss, a greater current is required to produce a given temperature rise than would be required otherwise. This leads to the conclusion that the principal effect of wire length appears in the quantity $\frac{R - R_a}{i^2 R_a}$, used to calculate $M_C$ in a lag determination; and this conclusion is borne out by the experimental results which show that $\frac{R - R_a}{i^2 R_a}$ changes with wire length rather than $M$ itself.
When the wire is used to measure turbulence, $\frac{R - R_e}{i^2 R_a}$ is found for each determination, and $M$ is calculated by equation (42) with the value of $M_0$ for the wire length used. The calculated time constant is found to be about the same for short wires as for long wires. Unfortunately this does not mean that the procedure is entirely correct, and it is thought that end effects introduce some uncertainty in the evaluation of the time constant. Since the uncertainty increases with decreasing length-diameter ratio, wire of 0.00021-inch diameter is not regarded as satisfactory in lengths less than about 1.5 millimeters.
In most applications $M$ is usually between 0.001 and 0.002 second. This is well within the range of time constants provided in the amplifier.
In cases where the sum or difference of instantaneous voltages is taken, as in the determination of $w'$ and $\overline{uv}$, it is important that both wires have the same time constant in order that there shall be no difference in phase introduced. This is another reason for having the wires as nearly identical as possible and operated at the same mean angle and velocity.
VIII. APPLICATION OF HOT-WIRE INSTRUMENTS AND RESULTS
Following the development and study of new instruments and methods, measurements of $u'$, $v'$, $w'$, and $\overline{uv}$ were made in the turbulent boundary layer along the wall shown in figures 1 and 2. The results obtained so far are far from being sufficiently complete to make a significant contribution to turbulent boundary-layer theory. They are given here mainly as examples of the kind of results obtainable.
Prior to undertaking work with hot-wire instruments, the pressure distribution along the surface was measured and the position of the separation point was determined. A certain amount of preliminary work had to be done to obtain two-dimensional flow over the after portion of the wall and to obtain a straight and well-defined line of separation. The final pressure distribution and separation point at a Reynolds number $R_N$ of 15.3 million is shown in figure 16. Attention is called to the region of adverse pressure gradient near the leading edge and to the occurrence of transition therein. This condition resulted from the high effective angle of attack and the small radius of curvature of the leading edge. The turbulence of the free stream was found to be closely isotropic with an intensity of about 1/2 percent.
Mean velocity distributions through the boundary layer were determined at the same Reynolds number by traversing normal to the surface with a small pitot-static tube mounted on the traversing apparatus and support system shown in figure 1. From such distributions, obtained at many stations along the surface, $\delta$, $\delta^*$, $\theta$, and $H$ were found.
The velocity distributions showed certain anomalies near the separation point that are believed to be due to an effect of turbulence on the pitot tube. Effects of this sort appear
in the results of other investigators (reference 13). For this reason it is planned to redetermine some of the velocity distributions at the same time $u'$ measurements are made, since the same hot-wire instrument serves both purposes. The velocity distributions determined so far by the hot wire are shown in figure 17. The velocity distributions are an important adjunct to turbulence data for a number of purposes, one being to calculate mixing length by equation (3).
The results that demonstrate the application of hot-wire instruments are given in figures 19, 20, 21, and 22. The mean velocity contour and the boundary-layer thickness, shown in figure 18, are included here along with figures 18 and 17 to show the kind of boundary layer in which the turbulence measurements were made. The measurements were made at RN = 15.3 million. All these curves were obtained by taking observations at various distances from the surface with the traversing device shown in figure 1. A description of this device will be omitted here, partly because the manner of traversing is incidental to the investigation and partly because the remote-control feature of the device has not proved to be entirely satisfactory for hot-wire work. However, the support system and traversing device shown in figure 1 satisfied an important condition that must be met by any system—namely, that it should not alter the condition of the boundary layer at the position of the hot wire. It is remarked that complete remote control is attended with considerable difficulty due to the requirement that the instruments must be kept in proper alignment with the mean wind in the measurement of $v'$, $\overline{uv}$, and $w'$. Traversing is therefore a matter that must be worked out to meet particular needs.
Figure 19 shows the distribution of $u'/U_1$, $v'/U_1$, and $w'/U_1$ through the boundary layer at $x = 17\frac{1}{2}$ feet. Figure 20 gives the distribution of friction coefficient $c_f$ for the same position. The friction coefficient is obtained from the shearing stress by the following relation:
$$c_f = \frac{\tau}{1/2 \rho U_1^2} = \frac{2 \overline{uv}}{U_1^2}$$
The local skin friction coefficient $\tau_o/1/2\rho U_1^2$, estimated from the momentum equation (reference 13) is indicated in the figure. The limits indicate the uncertainty in the local skin
friction at this point. Theoretically, the turbulent shearing stress should decrease in the laminar sublayer and actually fall to zero at the surface. Beyond the laminar sublayer—but still close to the surface—the turbulent shearing stress may be expected to agree with $T_0$. This appears to be the case, since the points nearest the surface are well outside of the laminar sublayer. The correlation coefficients calculated by the three different methods (equations (24), (25), and (27)) are given in figure 21. Coefficients $K_1$ and $K_2$ agree, but $K_3$ is about 17 percent above the other two. This difference, which indicates experimental error, has not yet been accounted for. More observations are necessary before it is possible to decide which of the quantities $\overline{uv}$, $u'$, or $v'$ is least certain and thereby make use of the procedure suggested in section VII for obtaining the best consistent set of results.
Figure 22 shows the distribution of $u'/U$ at several stations in the region of adverse pressure gradient. It will be noted that the ordinates here are the ratio of $u'$ to the mean local velocity instead of the mean velocity at the outer edge of the layer. This method of presenting the results is useful when it is desirable to know whether or not the fluctuations are small compared to the mean local velocity. This subject will be considered in the following section. Figure 22 shows that $u'/U$ increases progressively as $x$ increases. No turbulence measurements have yet been made beyond $x = 22$ feet.
IX. ERRORS WHEN FLUCTUATIONS ARE NOT SMALL
It will be recalled that $u$, $v$, $w$ small compared to $U$ was a basic assumption in the development of the equations in section VII. The results in figure 22 may well raise doubts about the smallness of these quantities in a turbulent boundary layer. Beyond a doubt $u'/U$ will continue to increase as the separation point at $x_s = 25.7$ feet is approached, and just how high the value will go is not known. The components $v'/U$ and $w'/U$ and the shearing stress have been determined only at $x = 17\frac{1}{2}$ feet, but it is reasonable to assume that these too will increase with $x$. The questions are then, how large may $u'/U$, $v'/U$, and $w'/U$ become before the fluctuations may no longer be considered small compared to $U$, and how does the error depend on the size of these quantities?
Unfortunately the answers to these questions are not
easily obtained. When the fluctuations are no longer small, errors arise from the interaction of components, improper compensation for lag, and the nonlinearity of the voltage-velocity curve and the voltage-angle curve for a hot wire operated with constant heating current. The latter concerns the errors in $A$ and $B$ resulting from the assumption that $u$, $v$, and $w$ are infinitesimals. When the several kinds of errors are considered, it is found that errors in $A$ and $B$ are probably the largest and therefore deserve first consideration.
Since the wave form of turbulent fluctuations is irregular and jagged, and qualitatively has the appearance of complete randomness, an accurate estimate of the errors in $A$ and $B$ seems to be impossible. A crude method is to assume sinusoidal fluctuations and to find $A$ from experimental voltage-velocity curves and $B$ from experimental voltage-angle curves with the relations for $A$ and $B$ given by
$$A = \frac{\Delta E_{\text{max}}}{\Delta U_{\text{max}}} \quad B = \frac{\Delta E_{\text{max}}}{U \Delta \varphi_{\text{max}}}$$
The ratios $A$ and $B$ are determined by arbitrarily assigning larger and larger increments to $\Delta U_{\text{max}}$ and to $\Delta \varphi_{\text{max}}$ about a point on their respective curves and determining the corresponding $\Delta E_{\text{max}}$. For a sine wave
$$\Delta U_{\text{max}} = \sqrt{2} u', \quad U \Delta \varphi_{\text{max}} = \sqrt{2} v' = \sqrt{3} w'$$
The values of $A$ and $B$ found in this way are the correct ones for sinusoidal variations in $\Delta U$ and $\Delta \varphi$, at least until $\Delta U_{\text{max}}$ and $\Delta \varphi_{\text{max}}$ become so large that the voltage wave is distorted by a significant amount by the nonlinearity of the curves. Obviously, the weakness in this method is the inference that there exist relations like
$$u_{\text{max}} = \sqrt{2} u', \quad v_{\text{max}} = \sqrt{2} v', \quad w_{\text{max}} = \sqrt{2} w'$$
for turbulent fluctuations. For want of a better procedure, $A$ and $B$ were determined by this method, and were found to
increase with increasing $\Delta U_{\text{max}}$ and $\Delta \phi_{\text{max}}$. Denoting the values of $A$ given by equations (11) and (13) by $A_{11}$ and $A_{13}$ and the value of $B$ given by equation (12) by $B_{12}$, the errors as given in table I are found.
### TABLE I
| $\frac{u'}{U}$, $\frac{v'}{U}$, $\frac{w'}{U}$ | Wire normal to wind | Wire 45° to wind |
|---------------------------------|---------------------|------------------|
| | $A-A_{13}$ | $A-A_{11}$ | $B-B_{12}$ |
| | $A_{13}$ | $A_{11}$ | $B_{12}$ |
| | (percent) | (percent) | (percent) |
| 0.05 | Too small to estimate | Too small to estimate | Too small to estimate |
| 0.10 | 0.2 | Too small to estimate | 1.0 |
| 0.15 | 1.5 | 1.0 | 5.0 |
| 0.20 | 7.0 | 5.0 | 9.0 |
| 0.25 | 12.5 | 9.0 | 14.0 |
| 0.30 | 19.0 | 15.0 | 20.0 |
| 0.35 | 27.0 | 20 | - |
The errors were found to depend on the velocity to a slight extent, but not enough to warrant consideration in a table of this sort, which, after all, is intended mainly to indicate the order of magnitude of the errors.
The errors indicated in column 2 apply to a measurement of $u'/U$. They are in such a direction as to make the measured value too high by the percentages given. The errors indicated in columns 3 and 4 apply to the measurement of shearing stress. According to equation (20) the error in the shearing stress is the error in $A_{11}$ plus the error in $B_{12}$ for the appropriate values of $u'/U$ and $v'/U$. Again the error
is in such a direction as to make the measured shearing stress too high.
Before investigating the error in correlation coefficient, it is noted that errors in $v'/U$ and $w'/U$ are not those indicated in column 4 of table I. The controlling factor here is the shape of the broken curve in figure 14. If the amplitude of the fluctuation does not go beyond the linear portion of this curve, the only error in $v'/U$ and $w'/U$ associated with size arises from the assumption that $v/U$ and $w/U$ are given by $\Delta \varphi$ when actually they are given by $\tan \Delta \varphi$. This error will generally be negligible for amplitudes that lie within the linear portion of the curve. According to figure 14, the amplitude of a sinusoidal fluctuation will go beyond the linear portion when $v'/U$ or $w'/U$ exceeds 0.19. It will be assumed that the error arising from $u'/U$ may be corrected by means of equation (37), and $v'/U$ and $w'/U$ will be regarded as being free from error due to size for values up to 0.19. It will be necessary to pass over the question of errors for values greater than 0.19.
If no error in $v'$ is assumed, it will be possible to get some idea of the error in correlation coefficient from table I. It is found, for example, that the error in $K_1$ (equation (24)) is given approximately by the difference between the errors in $A_{13}$ and $A_{11}$ in columns 2 and 3. The error in $K_2$ (equation (25)) reaches about 2 percent for a value of 0.3 in column 1. The error in $K_3$ (equation (27)) is the error in shearing stress minus that in $u'$.
At the 22-foot position the maximum value of $u'/U$ is found from figure 22 to be 0.19. If $u'$ and $v'$ are in the same ratio here as at the 17$\frac{1}{2}$-foot position, the predicted $v'/U$ will be about 0.11. The errors charged to size in measurements at the point nearest the surface in the 22-foot position are estimated to be:
6 percent in $u'/U$
6 percent predicted in $c_f$
Not more than 1 percent predicted in $K$
No error predicted in $v'/U$ and $w'/U$
According to reference 9 there is an error in compensation involved in the use of expression (39), which depends on
the size of a term
\[ 2\pi f \alpha M \]
where \( \alpha \) is the amplitude of the resistance changes divided by \( R - R_0 \). It is shown that the error is less than 2 percent when \( 2\pi f \alpha M \) is no greater than 0.14. Since the distribution of amplitude with frequency in a turbulent boundary layer is not known, no estimate of the actual error can be made. The fact that an error in compensation is likely to enter to a greater and greater degree as the fluctuations increase in size is further argument for using wires of the smallest possible diameter.
X. CONCLUSIONS
An account has been given of the recent developments in hot-wire instruments for use in turbulent boundary layers. From the theory of hot-wire measurements and the characteristics of the various instruments it is concluded that \( u' \), \( v' \), \( w' \), \( uv \), and \( K \) may be measured. These quantities are among the more important characteristics of the turbulence needed to further an understanding of turbulent boundary layers. The results so far obtained in the boundary-layer investigation for which the instruments were developed show that these quantities can be measured in the thick turbulent boundary layer used in the present experiment. The average characteristics of the layer are shown by the pressure distribution, mean velocity distribution, thickness, and separation point.
More observations are necessary before the experimental uncertainties can be properly appraised. In theory at least, the important errors are those that increase with the relative magnitude of the fluctuations and are inherent in the constant-current method of operating hot-wire anemometers. The relations between the voltage and the magnitude and direction of the wind are not linear; and it is for this reason that the proportionality factors \( A \) and \( B \) are not constant, but are rather some involved function of the root-mean-square value of the fluctuations. This fact has long been recognized, but has never been a serious drawback in the measurement of free-stream turbulence where the fluctuations rarely exceed a few percent of the mean velocity. These errors are a matter of concern in boundary-layer applications, but are not sufficient to condemn the method. They do, however, show the need for
further development of other methods of operation that are better adapted to the measurement of large fluctuations, such as the constant-temperature method with linearizing circuits proposed by Weske in reference 11.
National Bureau of Standards
Washington, D. C., June 29, 1945.
REFERENCES
1. von Kármán, Th.: Turbulence and Skin Friction. Jour. Aero. Sci., vol 1, no. 1, Jan. 1934, pp. 1-20.
2. Schubauer, G. B.: A Turbulence Indicator Utilizing the Diffusion of Heat. NACA Rep. No. 524, 1935.
3. Taylor, G. I.: Diffusion by Continuous Movements. Proc. London Math. Soc., vol 20, Aug. 1921, pp. 196-211.
4. Dryden, Hugh L.: Turbulence Investigations at the National Bureau of Standards. Proc. Fifth Int. Cong. Appl. Mech., Cambridge, Mass., 1938, pp. 362-368.
5. Dryden, Hugh L., Schubauer, G. B., Mock, W. C., Jr., and Skramstad, H. K.: Measurements of Intensity and Scale of Wind-Tunnel Turbulence and Their Relation to the Critical Reynolds Number of Spheres. NACA Rep. No. 581, 1937.
6. Weske, John R.: Methods of Measurement of High Air Velocities by the Hot-Wire Method. NACA TN No. 880, 1943.
7. Mock, W. C., Jr.: Alternating-Current Equipment for the Measurement of Fluctuations of Air Speed in Turbulent Flow. NACA Rep. No. 598, 1937.
8. Mock, W. C., Jr., and Dryden, H. L.: Improved Apparatus for the Measurement of Fluctuations of Air Speed in Turbulent Flow. NACA Rep. No. 448, 1932.
9. Dryden, H. L., and Kuethe, A. M.: The Measurement of Fluctuations of Air Speed by the Hot-Wire Anemometer. NACA Rep. No. 320, 1929.
10. Dryden, Hugh L.: Isotropic Turbulence in Theory and Experiment. Appl. Mechanics, Theodore von Karman Anniversary Vol., May 11, 1941, pp. 85-102.
11. Weske, John R.: A Hot-Wire Circuit with Very Small Time Lag. NACA TN No. 881, 1943.
12. King, L. V.: On the Convection of Heat from Small Cylinders in a Stream of Fluid: Determination of the Convection Constants of Small Platinum Wires with Applications to Hot-Wire Anemometry. Phil. Trans. Roy. Soc., London, ser. A, vol. 214, 1914, pp. 373-432.
13. von Doenhoff, Albert M., and Tetervin, Neal: Determination of General Relations for the Behavior of Turbulent Boundary Layers. NACA ACR No. 3G13, 1943.
Figure 1.- Front view of "boundary-layer wall" in 10-foot wind tunnel. Height of wall is 10 feet. Instrument support shown on working side.
Figure 2.- Diagram of "boundary-layer wall" with scale of x on working side.
Figure 3.- Magnified view of hot-wire arrangements. (a), arrangement for measuring $u'$; (b), (c), arrangements for measuring turbulent shearing stress; (d), arrangement for measuring $v'$ and $w'$; (e), one complete instrument. Actual size of (a), (b), (c), (d) shown by larger 1/16-inch scale divisions, actual size of (e) shown by smaller 1/16-inch scale divisions.
METHOD FOR ELECTROPLATING TUNGSTEN WIRE
Figure 4.- Electroplating bath. Insert shows a microphotograph of 0.00031-inch diameter tungsten wire with plated sections.
Figure 5.- Assembled electrical equipment. A, amplifier; B, power supply; C, control unit.
PORTABLE TURBULENCE MEASUREMENT EQUIPMENT
CONTROL UNIT
CONTROL UNIT COMPONENTS
R1, 6 ohms
R2, 200 "
R3, 10 "
R4, 100 "
R5, 1,000 " 4 dial decade
R6, 1,000 "
R7, 100 "
R8, 500 "
R9, Special resistor adjusted to give 0.0025 volt drop when carrying 1/2 scale oscillator meter current.
R10, 1,000 ohms
R11, 250 "
R12, 5,000 "
R13, 10,000 "
R14, 1,000 "
C, 0.5 mf.
L, 12 henries, 105 ohms.
Figure 6.- Diagram of control-unit circuit.
PORTABLE TURBULENCE MEASUREMENT EQUIPMENT
AMPLIFIER
AMPLIFIER COMPONENTS
| Component | Value |
|-----------|----------------|
| R1 | 100,000 ohms |
| R2 | 2,000 " |
| R3 | 2,000,000 " |
| R4 | 500,000 " |
| R5 | 3,000,000 " |
| R6 | 1,000 " |
| R7 | 25,000 " |
| R8 | 150 " |
| R9 | 750 " |
| R10 | 50,000 " |
| R11 | 1,000,000 " |
| R12 | 1,000,000 " |
| R13 | 2,000 " |
| R14 | 2,500 ohms |
| R15 | 3,333 " |
| R16 | 5,000 " |
| R17 | 10,000 " |
| R18 | 3,500 " |
| R19 | 2,000,000 " |
| R20 | 1,000,000 " |
| R21 | 1,500 " |
| R22 | 500 " |
| R23 | 10,000,000 " |
| R24 | 1,000,000 " |
| R25 | 500,000 " |
| RGC | 10,240 ohms total |
SA, 2 ma. full scale meter shunt
SB, 20 " " " " "
SC, 20 " " " " "
SD, 2 " " " " "
SE, 2 " " " " "
SF, 2 " " " " "
SG, 200 " " " " "
SH, 10 " " " " "
C1, 2 mf. C5, 0.005 mf. C9, 0.001 mf. C13, 16 mf.
C2, 4 " C6, 0.004 mf. C10, 0.1 " L, 60 millihenry
C3, 0.02 " C7, 0.003 " C11, 0.01 " "
C4, 0.04 " C8, 0.002 " C12, 0.02 " "
Figure 7.- Diagram of amplifier circuit.
PORTABLE TURBULENCE MEASUREMENT EQUIPMENT
POWER SUPPLIES
POWER SUPPLY COMPONENTS
R1, 15,000 ohms
R2, 10,000 "
R3, 250,000 "
R4, 1,000 "
R5, 10,000 "
R6, 3,500 "
R7, 20 " center tapped
C1, 8 mf.
C2, 32 "
C3, 0.5 "
L, 12 henries, 231 ohms
T1, 750 Vac. center tapped, .18A; 5V., 3A.; 6.3V, 3.3A.;
2.5V., 6A.
T2, 6.3V., 3A.
Figure 8.- Diagram of power-supply circuit.
Figure 9.- Curves showing variation of voltage with angle at three wind velocities. $E =$ voltage across wire, $\varphi =$ angle between axis of wire and wind.
Figure 10.- Curves showing variation of $R/R_a$ with angle at three wind velocities. $R =$ resistance of wire when heated, $R_a =$ resistance of wire at air temperature, $\varphi =$ angle between axis of wire and wind.
Figure 11.- Diagram showing linear relation between $R/R - R_a$ and $\sqrt{U \sin \varphi}$ (equation 7).
Figure 12.- Diagram showing linear relation between $R/R - R_a$ and $\sqrt{U}$ when $\varphi = 90$ degrees (equation 6).
Figure 13.- Diagram illustrating position of wires for a measurement of turbulent shearing stress. Arrows indicate positive direction of x, y, U, u, and v.
Figure 14.- Voltages $E_1$ and $E_2$ across each wire of an x-wire arrangement and difference voltage $E_b$ at various angles. $U = 124$ feet per second.
Figure 15.- Calibration curve used in the determination of $v'$ and $w'$.
Figure 16.- Distribution of dynamic pressure at outer limit of boundary layer derived from static pressure measurements.
Figure 17.- Distribution of mean velocity determined by the hot-wire method.
Figure 18.- Mean-velocity contour lines and outer limit of boundary layer. $\delta$ = boundary layer thickness.
Figure 19.- Distribution of $u'/U_1$, $v'/U_1$, and $w'/U_1$ across boundary layer 17-1/2 feet from forward stagnation point.
Figure 20.- Distribution of friction coefficient across boundary layer 17-1/2 feet from forward stagnation point. $cf = 2 \frac{uv}{U_1^2}$.
Figure 21.- Distribution of correlation coefficient across boundary layer 17-1/2 feet from forward stagnation point. $K = uv/u'v'$. $K_1$, $K_2$, and $K_3$, values obtained from equations (24), (25), and (27) respectively.
Figure 22.— Distribution of $u'/U$ across the boundary layer at several distances $x$ from the forward stagnation point.
TITLE: Theory and Application of Hot-Wire Instruments in the Investigation of Turbulent Boundary Layers
AUTHOR(S): Schubauer, G. B.; Klebanoff, P. S.
ORIGINATING AGENCY: National Advisory Committee for Aeronautics, Washington, D. C.
PUBLISHED BY: (Same)
DATE: March '46
DOC. CLASS.: Unclass.
COUNTRY: U.S.
LANGUAGE: Eng.
PAGES: 61
ILLUSTRATIONS: photos, tables, diagrs, graphs
ABSTRACT:
Investigation involves the measurement of pressure distribution, distribution of mean velocity across the layer, boundary layer thickness, and shape parameter as well as measurements of the turbulent characteristics of the layer. It was found that the important characteristics of the turbulence needed to further an understanding of turbulent layers may be measured. Average characteristics of the layer are shown by the pressure distribution, mean velocity distribution, thickness, and separation point.
DISTRIBUTION: Request copies of this report only from Originating Agency
DIVISION: Aerodynamics (2)
SECTION: Boundary Layer (5)
SUBJECT HEADINGS: Boundary layer, Turbulent - Theory (18470); Boundary layer, Turbulent - Measurement (18402)
|
SINISTER WISDOM
Inside the Archives
Founders and publishers: Harriet Desmoines & Catherine Nicholson
Editors: C. Colette, Harriet Desmoines, Catherine Nicholson, Carolyn Shafer
Poetry Editor: Susan Leigh Star
Review Editor: Julia Penelope
Contributing Editors: Sarah Hoagland, Beth Hodges, Mab Segrest
Cover Design by Beth Hodges
Cover Photograph by JEB
Staff: C. Colette, Lynda Koolish, Cheryl Long, Janis Morariu, Vicki Nogle,
Julia Penelope, Donna Saunders
Special thanks to: Dodie Bellamy, Sandy Boucher, and Donna Travis
Printed by Iowa City Women's Press
This issue was funded in part by a grant from the Coordinating Council of Literary Magazines.
Copyright © Sinister Wisdom, 1979. All rights reserved to individual authors and artists.
Published quarterly
Individual subscriptions: 1 year (4 issues) $7.50
2 years (8 issues) $13.00
(For out-of-the-U.S. subs: $9 for 1 year; $15 for 2 years; U.S. dollars only)
Institutional subscriptions: 4 issues, $12.00
Back issues, while available:
Issues 1, 2, 3 sold out
Issues 4, 5: $2.25 each
Issues 6, 7, 8, 9: $2.50 each
(Add 60 cents postage when ordering 1-2 single copies)
Address: SINISTER WISDOM, Box 30541, Lincoln, Nebraska 68503
CONTENTS
Photographs by JEB 2, 3, 6, 11, 13, 66, 68, 70
Photographs by Lynda Koolish 23, 40
Photograph by Katherine Bouton 62
Pen and inks by Sarah Clark 29, 43
Lesbian Herstory Archives: An Interview by Beth Hodges 3
Why There Are No Ghosts of Wimmin Martyrs Meg Jochild 14
Poems Marilyn Hacker 16
Beginnings a story J. Z. Grover 18
THEORY:
Lesbian Celibacy Susan L. Yarbrough 24
Random Thoughts on Victimization Andrée Collard 30
Stillness and Motion Barbara Hammer 39
Conceiving Women Joyce Trebilcot 43
RESPONSE:
Pat Kuras, Martha Yates, Martha Courtot, Joanna Russ,
Eleanor, Judith Schwarz, JR Roberts, Tracy Moore, Sue-Ellen Case 51
REVIEWS:
Letter to Readers Julia Penelope 67
Review of Eye to Eye Deborah Snow 69
Review of Wanderground Julia Penelope 71
Photographs of Energy and Color a story Katherine Sturtevant 74
POETRY: "Embroidered Breast" Quimetta Perle 83
Cher'rie Moraga Lawrence 84
Jan Hardy 87
Janice Maiman 88
Elizabeth Alexander 90
Teresa Anderson 91
Becky Birtha 92
Julie Blackwomon 93
Terry Wolverton 94
Diane Stein 96
Announcements 100
Contributors' Notes 102
"We also have our own voices":
AN INTERVIEW WITH JOAN AND DEBORAH OF THE LESBIAN HERSTORY ARCHIVES
by Beth Hodges
Photographs by JEB
In 1974 there was a pantry, dark, and empty except for a single file cabinet. I knelt on the floor to examine the contents of the cabinet, a complete set of THE LADDER. This was the embryo of the Lesbian Herstory Archives.
Five years later the Archives has outgrown the pantry, spills into the kitchen, the dining room, the front hall, the bedrooms of the upper West Side apartment that houses it. In the pantry where I first held THE LADDER, shelves of our serial publications line an entire wall. And all over the apartment I see walls of books, of file cabinets, flyers, posters, collages, framed photographs, and a bulletin board whose announcements include notice of lesbian raft trips, a black bibliography, a southeastern lesbian writers' conference, a Jewish lesbian anthology.
Today there are over three thousand volumes in the Archives collection. How many unpublished papers and letters and articles, clippings, taped interviews, radio shows, videotapes, photographs and manuscripts there are, no one knows exactly.
The Archives collective has lost members and gained members, but three have been constant from the beginning. Valerie Itnyre, Deborah Edel and Joan Nestle together have done the day-to-day work of the Archives these five years.
In April I spoke with Deborah and Joan, the two who share their home with the Archives collection and with the hundreds of women who visit each year. Since much of our conversation concerns beginnings, visions and quests, Joan starts out with the story of a woman who chose to begin her journey at the Archives.
Joan: The Archives reaches out in ways that we didn't plan for. One day we were sitting around and there was a knock on the door. I went to answer it and it was a woman, probably in her mid- to late forties, and she had a huge backpack on.
Deborah: A little lady.
J: She said her name, she said she was from Hawaii and she'd tried to call us but the line was busy so she just took the risk of coming. Her story was that she had been married for many years. She had raised, I think, five children—the oldest was in his twenties. She had gotten a divorce, gone to law school, and come out as a lesbian woman. She'd just finished law school, and "before she got married to another institution," is how she put it, she wanted to make a pilgrimage through, or to, the lesbian community as she had understood it to be from Hawaii; and she had certain places on her journey that she was going to stop. She'd made no previous arrangements.
The place she wanted to begin her journey was the Archives. So we welcomed her, and I went to take her backpack from her, and it must have weighed at least sixty pounds. She stayed here for three days. What she would do, she would get up in the morning, have her healthy breakfast, and she would sit—she didn't even sit on a chair, she sat on the floor in a corner of the Archives—and she would just reach out and pull out things.
We would have dinner together and she would tell us her itinerary. She would say she was going to Buffalo and we would say, "but"—I don't want to use her name—"but woman," we'd say, "it's terrible snow storms up there now, you know: you're from Hawaii." "No, no, that's o.k., that's where my spirit tells me I have to go," and then when she left she said she knew she was right to begin here, that it was like sitting under a waterfall, in the Archives room.
We said good-bye to her and we told her to keep in touch with us. And around two months later two women came to the Archives from the Actors' Sorority, a lesbian theater group in Kansas City, and they said Jackie says hello. So she was making her trek. It's this image of an older woman launching herself into the lesbian world, and finding it, that symbolizes the Archives.
D: She did something that I thought was very brave also. She had written a series of coming-out letters to old friends, and if their responses weren't fully real, she wanted to go deal with them directly.
J: Her courage and her spiritual vision and her faith that we'd all be there is symbolic of the Archives. Her courage to journey at this point in her life.
Something we found from the Archives is that many, many of our women are on journeys. The Archives gives them a stopping-off place, a renourishing place. We are a very brave people.
Beth: Will you tell about Mabel, a brave woman who has been important to you on your journey?
J: Mabel was the first lesbian woman I knew. I was around ten years old when I met Mabel. She was sort of a buddy of my mother and also was hired by my mother to take care of me—my mother was a working woman. Mabel used to read old lesbian paperbacks and she would keep them in her raincoat pocket with the book cover turned outward so I couldn't see the covers. And one day I took it out of her pocket—it was an invasion of privacy—and I read, devoured this lesbian novel. And Mabel just watched me grow.
D: How did you link back up with Mabel?
J: Mabel always stayed friends with my mother because they went to the race track together all the time, and they helped each other through hard times. So I knew Mabel, and Lillian, the woman she lived with for forty years who died this June.
Mabel was the witness to my coming-out, and to my mother’s upsetness. Mabel told me something the last time she was here, that when I was coming out, my mother called her in the middle of the night and said, “If my daughter is a lesbian, I’m going to kill myself”; and Mabel said to her, “There’s nothing you can do, she chooses, that’s her life, you have to let her do it.” Mabel was sort of the bridge between me and my mother.
I remember the first woman I was involved with, Susan. Mabel was at the house and my mother came home drunk and was in a very bad way. Susan got very scared and ran out of the apartment. I was holding on to my mother, trying to get her to calm down, and Mabel turned to me and said, “Now you leave your mother alone and go after your woman.” And I ran out the door after Susan. This was the first recognition and support for my relationship.
But how we got together again was, when I was out, I guess around 1960, I was with Carol, and I’d been off and on in touch with Mabel. And Mabel told me there was going to be this big dance up in the South Bronx. It was going to be drag dance, for male and female homosexuals. I wanted Mabel to meet Carol so we went to their home and spent the night there; and we went to this dance at this ballroom. It was just incredible, hundreds of people, women in suits and men in dresses.
D: What did you wear?
J: I wore a dress, Carol wore a suit, and I remember to this day coming down the steps and a woman saying to Carol, “Can I borrow your woman? She’s really saying something.”
D: And you love it, you love it!
J: I love it to this day, being an old femme.
B: You said once that the Archives really began with Mabel. That she was an example for you of strength and self-cherishing.
J: Yes. Mabel was raised in the south by her grandmother and then came to New York when she was about seven. She stayed for a short time with an uncle who molested her. She ran away and worked in white people’s houses from the age of nine. She said she was a lesbian from when she was a little girl playing in Winston-Salem.
Years ago she was doing the things that we as a community are doing now. In her own way she was an archivist. Her whole life she was always looking for lesbian images. She taught herself to read; and she’d save the Wonder Woman comics because they were images of strong women.
D: She saved the old paperbacks.
J: She had a lot of the fifties paperbacks, which she prized.
She was political in her own way. She was cherishing of her own history, and knew there were other women like her. She tried to find the hidden images. And she was also seeking another way of knowledge; she joined the Rosicrucians in a search for a spiritual vision which she preserves today in
the Eastern Star. Because she had a strong sense that things were wrong, that men were keeping women from their full power.
B: What is Mabel's relationship to the Lesbian Herstory Archives today?
J: She has become very important to the Archives. In fact, she uses it more than any other woman. Even though reading for her is very hard because she's had cataract operations, she spends hours in that room, reading through what she's missed. It's like a hunger. She's just finished reading Patience and Sarah. And she just read Ethel Waters...
D: And Ann Shockley.
J: Yes, if Ann Shockley could hear this, Mabel wants so much to thank her for *Loving Her* which she read three times and made sure we gave copies to every one of her friends. She felt it was such an important work for her.
We sent her the newsletter. And Mabel, who is living on social security, sent us a donation—when she’s donated her whole life. And she tells all her friends about the Archives.
D: Also she gave us her thirties, forties, fifties paperback collection. Our collection has grown since then, but it’s what she had saved all these years that started the Archives’ collection.
J: We’ve been doing an oral history with Mabel. And finally around a month ago she said, “All I want to do is be remembered.”
B: I don’t think she needs to worry.
J: It’s beautiful to see the kind of recognition she’s getting now. One day there was a woman working here in the Archives and she just kept seeing we had all these pictures of Mabel, Mabel working at her desk, and the woman said, “Is this woman a writer? Why do you have so many of her pictures?” And we said, “No, she’s Mabel Hampton. She’s a lesbian woman.” And really it’s about her that the Archives is. And all the other women like her.
B: Judith Schwarz has been a member of the collective for about two months now, hasn’t she? How did you get together with her?
J: One of the very exciting things about the Archives is that so many women are beginning things, particularly research in lesbian history. They’re working without support, those that aren’t academically based. It’s exciting that we share beginning moments, and one was when Judith Schwarz, whom we did not know at all, who has now become a member of the Archives and also a member of our family—she wrote us a letter about how she’d been doing grass roots research in the Library of Congress, hours and hours, after her ACLU typist job, and women told her she was crazy, all she was interested in was dirty linen, why did she want to know about women’s private lives, that was gossip.
She wrote us a letter saying, “Am I crazy?” It was a very finely typed and finely worded letter. I read it and I got so excited, I sat down and typed one of my emotional outpourings about how it was just the opposite, that what she was doing was putting the center back into things, that she could not listen to those who questioned the importance of what she was doing because they were the ones who created the emptiness in the first place. And I was so afraid that my letter was going to be too crazy...
D: ...for this proper woman, who typed so neatly.
J: She wrote back and she was so grateful we just exchanged letters almost weekly after that.
D: Also, both of you had discovered the connection of your mothers’ deaths which was very present for both of you at the time, and it was another bond between you.
B: Does this happen often, that you bond with women who write you or who come to use the Archives?
J: That’s another thing that makes the Archives so special. Since the Archives is in our home, when women use it, they do share in what is going on in our lives. One of the principles of the Archives is that it be an integral part of lesbian reality, not an isolated collection. We try not to let what’s
going on here personally get in the way of a woman who has something concrete and special to do. But what we found—we've had hard times here—is an incredible caring on the part of women who we have never met before and may never meet again but who come here and, either through a conversation or through something they overhear us say, become involved.
I have gotten incredible caring and support for my own difficulties. It is a world of caring that grows out of that room, a center that radiates. A caring for the collectiveness of all of us also deepens the caring for us individually. So whenever I hear women saying they don't know where the lesbian community is, it's very hard for me, because we always feel that...
B: ...you live there...
J: ...we live there, that the Archives is at the heart of things.
D: It's also been incredible because it's never stopped; no matter what was going on in our lives, it's always managed to keep on flowing. The week Joan was in the hospital two women were staying here...
J: ...working on their anarchist record of lesbian music...
D: ...taking telephone calls and occasionally letting women in for us.
B: Will you talk about what the Archives has meant to you?
J: It has meant life to me.
It started as a political and philosophical and personal issue, but I never imagined it would be as personal as it has become. It's, I would use the word magic—how can I say it?—it's almost as if it has an understanding of things, almost as a living person. During the time I've been ill, whatever I can do, the Archives has something for me to do. It has never made me feel useless or valueless or completely dependent. There is always something to create with it, even if it's just clipping articles, which is what I do sometimes.
B: You once said that you would remember these early years of the Archives as its golden age. Why?
J: I just see it as very gentle and very personal, the way it is now. There's a glory to it, in its simplicity, in the smallness of the room, and in its coherency. We don't have large amounts of money to worry about now and it's all very manageable. Now every woman who comes, helps create it, and it's still small enough that every woman can see her own impact, can touch everything that's there. Women sit shoulder to shoulder, as if the voices could all hear each other still. As we grow as a culture, or as we accumulate more as a culture, some of that immediacy won't be there. But now there's a quiet strength to it that I wish all lesbian women could share in.
B: Fran Winant is an example of a woman seeing her impact on the Archives.
J: Yes, I had known Fran superficially for years and years but never have I been able to tell her how really important she is. To me she is one of our women who has kept her own voice and her own imagination and is constantly growing with it. There's a wonderful integrity to her. And she finally came the other night to the Archives. Way before we knew she was coming, we had made a blow-up of one of her paintings of her dog who's very dear to her.
B: She wrote a whole volume of dog poems.
D: Yes, and did a whole series of paintings...
J: ...with her special language. And we had posted it. Someday we'd like to have the originals, but we do what we can, so we made a slide and we
blew it up. She walked into the Archives room, this woman who I thought would know for sure how at the center of things she was, and she wept.
What she wrote in the book, we read afterwards, was, "This has brought tears to my eyes. You understand." And for us it was such a gift to be able to say to her, "You have to understand too, you have to know how it's voices like yours who've kept our spirits intact." So it was a beautiful moment of being able to say thank you to someone I wanted to thank for many years.
B: Do you dread the future, that the Archives will change so much and your relationship with it will change?
D: We realize that the Archives has to grow...
J: ...into its own entity. We have a future vision of it, of its having a house with various rooms for all aspects of lesbian culture. So our visual artists would have space, our performing artists would have space, our sculptors would have space, and there would be room for women to sleep in and to eat together in. There would be a living creating of culture at the same time it is being documented. The Archives house would be a living symbol of our cherishing of generations. And it would have its own kind of excitement and its own kind of spirit.
D: I hope that it will always have a sense of caring that so many larger spaces lose in the process of becoming larger and dealing with more money and more objects and more things. There can be a hollowness to a building. I'm sure that won't happen, because in the shaping of the Archives, we will have already created a nurturing space, and so it can't grow into a hollow.
B: How are you shaping the Archives?
J: We thought that the first, say, ten years of our life with the Archives would be spent building an atmosphere as much as building a collection. We would be creating a world of confidence in us who are working with the Archives, an attitude of acceptance, and getting our community used to the idea that there would be an on-going intergenerational place that would be for all lesbian women. Not for a specific school of thought or a specific age group or specific class or specific cultural group, but for all of us.
How the Archives does things is as much a part of the culture it has created as what it collects. For instance, it wouldn't be our Archives—it wouldn't be a lesbian archives—if it ever was some place where lesbian women didn't have access to it. It wouldn't be our Archives if you ever needed a letter of referral to be able to use it or if there wasn't a place for women to rest when they were tired or to eat when they were hungry.
B: Or if there was a fee to use it.
D: Or if we got swallowed up into someone else's collection. Even if it was a feminist library.
J: We drew up some principles that we hope will be picked up by the next generation. One principle we hope will always stay loud and clear is that the word lesbian will never be diluted, will never be lost. And our Archives will never be turned into a woman's archives or a gay archives. But will be the one place that the word, the noun, lesbian will echo through the generations.
Though we know that women each time may choose a different word to call themselves—I mean, when I first came out, dyke was a very hard word
and now it's a wonderful word. Each generation will take the glory of naming itself. That's the spirit of the Archives, that we take what has been abused and turn it into cherishing.
B: Are you training another generation to come along and take over for you?
D: We see that as part of what we have to do.
J: The first thing that we have done—we hope when this interview is published we'll be incorporated—is set up a legal identity that gives us a way to hand down what we've created and keep it safe from the patriarchal society. We have created a foundation, and we hope that the Lesbian Herstory Educational Foundation will become an umbrella group to encourage and provide sustenance for lesbian cultural workers in all different fields, and that out of these women working will come those who take on the Archives as their generational commitment.
But we've realized that in any time there'll be only a small group of women who can say, or will want to say, "Yes, this is my way of being political; this is how I want to live, giving all my time and energies to the Archives." So we're working out other ways. For instance, having what we call Daughters of the Archives, women who undertake a project for a short period of time and don't have to say, "I'm giving all my life, I will become a vestal virgin of the Archives," but instead can say, "I will work," for instance, "on documenting lesbian photography from 1970 to 1980."
Once we build up a sense of our endurance and our integrity and our commitment, and we do as much of the shit work as possible, like getting the incorporation out of the way, getting our cataloging, setting up the procedures so that when women come into it they don't have to do the paperwork but can be more imaginative—once we do this, we think that we really won't have any problems in getting women to commit themselves to working on the Archives.
Also, there's a whole generation now of lesbian archivists. We've got in touch with several who've been trained in patriarchal archival schools who are now saying, "How can I contribute my skills to the Archives?"
And we hope eventually the Archives will be able to pay us a salary, will be able to reimburse women who are giving their skills.
B: Do you have a lifetime commitment to the Archives? Do you see yourself working in the Archives, in the middle of the Archives, until you die?
J: We do have a lifelong commitment. We've also learned that lifelong can be as long as tomorrow or ten years or fifty years.
B: Deb, do you have any reservations about a lifetime commitment to the Archives?
D: As long as I can see myself in a fluid relationship to it, no. But I would, if I thought we would be so involved and so tied to it that we would end up creating an atmosphere that would exclude other women. I have a strong sense of commitment to the Archives, but it doesn't mean that thirty years from now I'm going to be living with the collection, either literally or figuratively, on such a day-to-day basis. That I have no way of knowing. Ten
years ago I had no vision of the Archives, so it would be wrong of me to try to be literal and say, "Yes, in ten years I still will be cataloging." But I have committed myself to the Archives, to helping it grow.
B: Can you talk about how your life has changed, living with this collection?
J: Our days are in layers; we never know what's going to happen. The telephone rings all the time. The other night it began with a woman here using the Archives. She was going to hear Kate Millett speak the same place we were and since she lived in Westchester and didn't want to go all the way back, we said, "OK, why don't you stay for dinner?" Then another friend came, and then a woman from Boston, Monica, called and we ended up having ten for potluck dinner.
B: Are you kidding!
J: No, it just grew. And then we all traipsed over to Gay Women's Alternative. But very much, that's what happens, depending on the women's needs and what's going on in the house. A visit to the Archives seldom stops with using the material. It becomes women sitting down at the table and talking about "why are you interested in this?" or about an issue in the community; it's women sharing food.
B: Do you find there are greater numbers of women coming here?
J: Yes. Sometimes there are so many women working here that nobody knows who lives in the house—just the final statement that we've changed the nature of the apartment. Tee Corinne was one of the women working here and never knew who the women of the Archives collective were until she saw us much later. If women have been here several times, they'll give the tour of the Archives. Whoever is in the house with the most knowledge about things sort of takes over.
B: Do women ever come as groups?
J: Yes. Groups are using the house, which is wonderful. I never went to a private school; it gives me the first feeling of a dormitory. There are field trips to the Archives. And there's a group taking a course in lesbian literature at Barnard, so they come in groups of three and four to work here; and there's a wonderful feeling of all this young energy. I can shut up them with coffee and soup.
B: I remember the first time I stayed here. It was the 1974 Gay Academic Union, and you had women sleeping all over the floor. You were so happy to have a lesbian houseparty.
J: I haven't changed much in five years.
D: She's still trying for pajama parties all the time.
B: And you, Deb, how has your life changed, living with the collection?
D: It's been incredible, the sense of women passing through, and they are always passing through. It's trying to hold onto a certain amount of privacy and space that I need, at the same time that we've opened up our house to women coming into it. It's been incredible because we've met wonderful wonderful women.
But there are some days that I think if I see another piece of paper or an index card I will puke.
B: I find your generosity amazing.
J: The Archives is based on our principles of resource-sharing. We have a whole history of resources that allows the Archives to come into being. If we didn't have the size apartment we had, which means, if we didn't have the jobs that we have...
We say, "What is it that we want to do? What do we have that we can share?" We didn't have money to rent a separate building, so we used our apartment. We didn't have the money or the knowledge—we hadn't gone to archival school—so we went to libraries and we spoke to women who did. We learned about archiving and we found places we could buy things cheaper.
Every time a woman comes to the Archives there's another sharing. Women will say to us, "Oh, you need stationery? OK, I'll rip it off from my office" or "You need xeroxing? I'll do that."
The Archives is an act of empowering. We have taken a power or we have created a power. We must not stop at the limits imposed upon us but must think imaginatively, "How far can we take what we have?" And I think one of the givens of being a lesbian is that we have huge amounts of imagination and strength.
This is a message to the whole community: I see us as a colonized people,
and one thing a colonized people know is that the society who thinks you shouldn't exist in the first place isn't going to make it easy for you to create or to survive. And so, rather than talk about what we don't have, we use what we do have.
B: How did you come to see us as an oppressed people?
J: It was reading a passage in a book. This was around five years ago. I had been teaching a third world studies program for six years. I had been reading the literature of colonized peoples and part of me knew that being a lesbian in this country is to be colonized. But it didn't hit home until I read *The Colonizer and the Colonized* and I started to change the pronouns to *she*. There was one incredible paragraph about how the colonized are ruled out of time, and how they lose their sense of lineage. The last sentence in the paragraph was, "The colonized are condemned to lose their memory."
It was the word *condemned. Condemned*. It's the image of imprisonment to death. Without our memories we are in an endless prison. It became the banner of the Archives, to reverse the condemnation.
B: What is the motto of the Archives?
J: In memory of the voices we have lost.
**END OF PART ONE**
You may reach the LESBIAN HERSTORY ARCHIVES at PO Box 1258, New York, N.Y. 10001, 212-874-7232 or 873-9443.
WHY THERE ARE NO SKELETONS OF WIMMIN MARTYRS
A Story
"Of course, many of my acquaintances from the outside are struck by the oddity of this lady who lives with me. They have said she seems to be, at times, removed from reality, or a visionary. I think I will have to agree with them on the word visionary. She is a visionary; she has been called such for several centuries, though they do not know it.
"As for the lady, she is very happy here, incredibly happy. If she were not, I would return her, or find another place for her. But she wishes to stay by me, in my bed, in my world; and upon her real death, what she has written will fill several more volumes. The dry period of her last decade before I came has vanished.
"I think she is most pleased when I show her the books we were taught from in school, for in them she is held up as the example, the Writer, that she feared she never was. It must be a stretch of the self-concept to know that children are reading words she wrote five hundred years ago, reading and understanding and being swept with an emotion half a millennium old. —No, wait—her greatest pleasure came the time I took her to a play based on her life. She laughed long afterward, and when she could finally speak, she said, 'They were so close, and yet so far from knowing.'
"Here in the collective, of course, she can be herself, and that is who we know and love. Age has not lessened the 'boldness like a wren' of her, nor the chestnut in her hair. And she still wears white, but her hair falls free over her shoulders, and the Amazon just arrived next door is teaching her to ride a horse.
"And when we love, she is a girl again, a wild-hearted girl who loved too greatly for her time but not for mine. I am trying, very hard, to make up for the decades she lived with a broken heart. I think I can do this because I loved her for decades, reading the lines both written and silent that told how like me she was.
"And her oddity to those not part of the collective, part of the secret, is no real threat. We are all considered to be odd, we here on the sprawl of land and mountain we have claimed as ours for a livelihood and a home. And if our numbers seem to grow suddenly, it is explained by the appeal of our freedom, the lure that calls in wimmin needing a sanctuary.
"And this is no lie. From the very moment I made my discovery (or was given the secret by the Mother, as Beata insists), I knew how I would use it. I had waited too long for the woman separated from me by my
birthtime to consider anything else. The rest of it came when I realized that my dream was not alone, that other of us here had room and need for their own heroines. And so now we are a great gathering of lovers, poets old and new, who listen to one another with an intensity that can only grow from having been torn apart.
"Next to the first journey, where I gained my love, the best was whisking the French maid from the flames. She wears trousers and short hair with no fear now, and hears the voices of angels each time she speaks with us. Her eyes are so very brown, and the pain is faded altogether.
"Last week I returned with the Amazon, from the Steppes, who could see the erosion of her nation-tribe coming soon. I am going back often to that place—there are many who wished to come. What? Yes, of course they lied, all the accounts of what happened to our eldermothers were lies; they couldn't very well say that a witch appeared as they neared death and they both chuckled merrily as they vanished, now could they?
"I tell you all this, my friend, because you have joined our clan and you can be trusted with the secret. Also, I sense that you may have your own request to make of me, a need to save someone from the woman-hatred of her time. Aha! I thought so; well, it won't be so difficult. Can you get me her last known coordinates and the date of her disappearance? Good. What did friends call her? Melly? Alright, then, I shall bring Melly to you tomorrow. Only you must promise to give her all the room she needs to adjust—and you must let her return cheerfully if she prefers that to being part of now.
"Yes, I would have returned my lady if she had asked it. But I think I would have gone with her, to ease the loneliness of the huge old house and that cold world. You see, I have always loved her. And I wanted to show my love from the first time I read the plea in 'My life closed twice before its close'—
"Hush, now, here comes the mother of us all. Yes, she is short, and quite dark, but the Greeks were in those days. Wait till you hear the verse she composed yesterday..."
Another story still: a porch with trees—maple and oak, sharpening younger shoots against the screen; privileged solitude with early sunlight pouring in a thin wash on flat leaves like milk on a child’s chin.
Light shifts and dulls. I want to love a woman with my radical skin, reactionary imagination. My body is cored with hunger; my mind is gnarled in oily knots of anger that push back words: inelegant defeat of female aspiration. First we’re taught men’s love is what we cannot do without; obliged to do without precisely that: too fat, too smart, too loud, too shy, too old. Unloved and underpaid, tonight untold women will click our failings off, each bead inflating to a bathysphere, our need encapsulated in a metal skin, which we, subaqueous monsters, cannot infiltrate. The middle of the road is noon. Reactive creature with inconstant moon-tides (no doubt amendable as near-sightedness, but sacred to How Things Are) my blood came down and I swarmed up a tree, intoxicated with maturity.
Woman? Well, maybe—but I was a Grown-Up, entitled to make up my own mind, manners, morals myths—menses small price to pay for midnight and my own advice.
By next September, something was revenged on me. Muffled in sweat-soaked wool. I lunged out of seventh-grade science lab, just quick enough to get to the Girls’ Room and be sick. Blotched cheeks sucked to my teeth, intestines turning themselves out, hunched over a churning womb fisting itself, not quite thirteen, my green age turned me regularly green.
Our Jewish man G.P. to whom I carried myself hinted sex helped; once you were married. Those weren’t days I fancied getting laid. Feet pillowed up, belly on heating pad, head lolled toward Russian novel on the floor, I served my time each hour of the four days of the week of the month for the next ten years, during which I fucked a dozen men,
not therapeutically, and just as well.
Married to boot, each month still hurt like hell.
The sky thickens, seeps rain. I retrospectively add my annals to our tribe's collective Book of Passage Rites, and do not say a woman gave notebook leaves to me today whose argument was what I knew: desire, and all the old excuses ranked, conspired: avoid, misunderstand, procrastinate; say you're monogamous, or celibate, sex is too messy, better to be friends (thirsty for draughts of amity beyond this hesitation, which has less to do with her than my quixotic body's too pertinacious—tua tam pertinax valetudo, neither forward nor backward—malingering, I ask, or healing.) I like her: smart, strong, sane, companionate. I still love a man: true, but irrelevant. Then, unavoidably, why not?
She was gone (of course) by this time; I sat mirrored, eye-to-eye, cornered between two scalp-high windows framing persistent rain.
HOW IT HAPPENS
Really, it's a co-educational boarding-school. The big girls complain about the boys: spotty, spoken-for. They do without those gymnastic recreational pastimes, compensate in the dining-hall with Scrabble and lime fizz. One gawky sweet long-limbed math whiz and one dour Semitic Latin grind are keeping their council on the topic, trudge in the woods in the rain instead, get giddy at intramural events, slope off, slanting, before the bell. Doors wedged shut in a pink-wallpapered room they prime their adolescent epicene genius on specialized curriculum.
—Marilyn Hacker
BEGINNINGS
A Story
Beginnings: where did it start, then? Before consciousness, with Nana braiding my hair (the envy of Rumpelstiltskin, she assured me), breathing love and need into my neck as she combed, telling me I was her golden girl, her pearl beyond price? Or much later, with Maureen seducing my scruples at the Chicago MLA? With her silver flash, winking away a world as she cast it in that silent noon-day forest ten years ago?
It is a tradition in my trade (when I had a trade; when I had something to trade) to begin, like all good narrators, *in media res*. I opt instead to begin with my beginning, that free-flying point I have scooped and caught and taught to be my thirty-three year old perception of the truth and where it started.
In the beginning, then.
In 1960, I was a sophomore in college. Yes, indeed. At Thanksgiving in my sophomore year I drove to San Diego to eat dinner with two of my mother's old friends from World War II working days. Dorothy and Jean had retired as Lieutenant-Commanders out of Point Magu and headed, like true Navy lemmings, for San Diego. They had written my mother imploring (so she said) that dear Virginia's daughter spend the holiday with them.
I drove skeptically down the San Diego Freeway from Immaculate Heart and up into the hills above the harbor. There was a turkey waiting for me in the oven. There were two Scotties and a Siamese waiting for me in the living room. And of course there were Dorie and Jean. Their eyes crinkled at the edges (years of gazing out to sea?), they both chain-smoked and they wore good tailored clothes. Their house was filled with flowers.
We sat out on their patio; the railings and light were blinding white looking down to the sea. Pink and purple petunias ("Imagine that! at Thanksgiving!" Jean enthused) and ice-plant grew in mounds, marred only by brown scars where the male Scottie lifted his habitual leg. We drank daiquiris.
We laughed and guzzled through the afternoon and I was near delight at talking for the first time on a parity with people my mother's age, people who had seen the world, for with a child's casual cruelty, I had expelled my mother from that earthly paradise for the simple crime of answering to my own demands, sure sign of her essential frivolity.
We joked about Jean's tan, acquired through ritual daily sunning on the beach below, and I admired the scarabs she wore on a chain around her brown neck. I remember the necklace, my library-white hand holding the limp gold away from Jean's neck as I marveled at how lean and unlined she was compared to my mother.
The turkey dinner was not like later holidays' meagre Swanson's fare: we had three kinds of stuffing and cranberry sherbet and four pies. We drank wine, fed the Scotties and Siamese immoderate amounts of turkey skin and later we drank Cointreau in the living room as the lights over San Diego harbor glided out of the fog. As coup de grace, Dorie and Jean took the kid for a drive around the harbor, where they pointed out different kinds of navy ships, a friend's ketch, an old clipper-ship, all with proprietary pride. We drove home laughing and singing and I fell asleep surprised and happy that I had taken up my mother's suggestion. Two such likeable ladies...
Next morning, the unsuspecting heart trod out into the living room. Dorie was emptying ash trays, pulling open the drapes overlooking the harbor. "Morning," she said; "How's the beach sound?"
"Fine." A little awkwardness there; in daylight, behind nothing but a hangover and near-sobriety, friendship with two middle-aged women struck me as improbable and futureless. I wondered if they were doing this out of loyalty towards my mother, if she had implored them to take me on as a cause to get me out of the library and into what she sanguinely referred to as "life."
Dorie gave me a clap on the back and looked me straight in the eye. "Come on," she said. "Let's see what kind of a breakfast we can throw together out of that bird before Jean staggers out."
We made turkey egg foo yung. Dorie drank a beer while she made sweet-and-sour sauce and whistled "The Washington Post." Her silence was companionable, accessible, and I fell into ease. Her life seemed plausible, attainable and happy; I wondered what had kept her from marrying.
Jean emerged with a Scottie. Her hair was wrapped in a towel and she wore a terry robe with a monogram on the breast-pocket. From the pocket she drew a pack of Camels. She lit one. "Whew," she said. She sat down and smoked the cigarette in silence.
Dorie smiled at her, one of those direct eye smiles. "Coffee?"
"For sure."
"You eating?"
Jean eyed our meal. "You're going to tell me we're applying ourselves to that damned bird again. I can see it. What is that shit?"
"Turkey egg foo yung."
"I'll pass. Any cranberry sauce left?"
We ate in silence. After finishing her cranberry sauce, Jean lit another cigarette and clapped her hands together. They were tan and lean like the rest of her, like her neck, and they made a dry sound as she rubbed them together. "Well. Anyone for the beach?"
I looked over, watching her rub her dry hands together. Then I saw her neck.
It was covered with fresh little bites.
They hadn't been made by a Scottie.
I stared, I felt I could never drag my guilty eyes from Jean's neck. I don't know how long I looked; time dropped away, we all stopped, the three of us frozen in tableau. I tried to drag my eyes from there, turning to look casually at Dorie, but I only swivelled my stare to her neck.
Clean.
Back to Jean's.
Nobody moved. Nobody said anything.
I felt a horror rising, a dulled recognition, a feeling of having been co-opted, duped, trapped into someone else's game. I began replaying the previous day in my head, trying to find hints in what we'd done or said that would have led ineluctably to this moment: glances, phrases, insinuations... It was like a drug-field: time imploded, outward correspondences dropped away. I do not know how long we sat there while dull Toad Ellen struggled to sort out what had happened and what my relation to it was.
With the vindictiveness of the ambushed, I almost hoped one of them would try to explain it away so that I could return some snappy, vicious rejoinder, proof that I was nobody to fool around with.
Instead Dorie said to me, "Yeah—I'm vicious."
Jean just sat and looked stricken.
Dorie began to clear the table. "Jean. Wake up. You want a beer?"
Jean swam out of her daze. "Theodora," she began to me.
Dorie cut in. "For christssake, babe, it's not the first time the kid's seen a monkey bite. Or do they call them something else these days?"
"No... Monkey bites," I said, trying to be helpful. "Although sometimes I've heard, oh, some people call them hickies, PB's..." Chartless, this was a chartless sea.
"What's a PB?" Dorie asked, all alert for new topics.
"Passion bite."
"Oh." Silence, as all tred in fathomless waters.
Jean surfaced. "Does anyone want a cigarette?" she asked; there was such urgency in her voice that I took one just to distract her.
"I didn't know you smoked," she whispered.
"I don't. I thought maybe I could learn," I said. "It seems as good a time as any."
Jean smiled. "Maybe you're going just a bit too fast, Theodora," she said. Her smile wobbled, but she kept urging it up.
"You don't want to start smoking," Dorie yelled. "Christ! Talk about perverted habits—you'll die an old dyke like me." She grinned, marshalling a sinister theatricality. She added, "You will please notice that I have now broken the ice."
But the graceless Toad Ellen didn't choose to tread on it; after seconds of silence in which the aura of disaster dissipated, I loaded the dishwasher and walked out of the kitchen with stagey promises of taking a long shower and washing my hair.
I figured that would give them time enough to hold a conference and get a plausible front together...
I stood in the shower, washing away my outrage with a half-tube of Prell, working its green jelly through my hair again and again. I cowered under the spout: could I stay here until they both went away, hide years under the tap as they aged and died, were buried in each other's arms, dust and monograms,
terry robes and all, in the house’s other rooms?
Someone tried the bathroom door; I shrank against the body-warm tile. Would they actually pursue me into the shower to present their case, rip open the glass door to plead for understanding and forgiveness?
Perhaps they proselytized for their cause...
I stayed through water hot to warm to cold and blue. When it ran wholly cold and hard, I sighed and stopped, using the black towels with new suspicion (black...?).
Back in the living room, Dorie and Jean were assembled formally along one edge of the couch. I stalked in, my head a malt resisting thought or feeling.
Dorie got up after fixing me with an elliptical stare. “I’m going to put on some Duke Ellington,” she announced. “You mind Duke Ellington, kid?”
“I don’t know,” I said. “I think my parents listen to him.” Dumb, dumb, you are so dumb, Theodora.
Dorie laughed. “Yeah, your mother—she always loved jazz. I remember when we were all stationed together at Magu during the War—”
“Dorie,” Jean said.
“For christssake, Jean, you’re not my moral monitor... Let me finish what I was going to say: your mother, Theodora, your mother used to go to service clubs alone and sit in the corner with a bourbon and water and get so wrapped up in the music that she became invisible. You couldn’t see her. Her whole soul was caught up in it; there wasn’t room for normal human conversation with her at all. She’d sit with her mouth open, transfixed... Servicemen didn’t bother her; no one ever went near her. This little girl in brown with her whole heart pouring out into the band...”
I sat, mute witness to my mother’s past, Dorie shiny-eyed and inward as she told her tale.
“Dorie was very fond of your mother,” Jean translated.
Dorie pulled out of her fix to stare at Jean. “Boy,” she said finally. “That was brilliant. A brilliant conclusion.”
“How fond?” I wondered.
Dorie stared back at me from twenty-five years ago, distantly. “Fond.”
Swept away. First the maiden totems of my childhood unmasked as old Navy dykes, then my mother revealed for a jazz fiend pursued by (at least) one of the naval invertis. I stood picking the lint from my (mother-made) robe while edges of my mind tried to crawl, lichen-like, across the opening sutures.
Jean got up, flapping in her terry, and squirreled through the drawers of their walnut secretary. “Here, Theodora,” she said. “Maybe you should read about us. I’m sure this is a shock for you, but it’s not all that unusual. There are a lot of books we’ve collected over the years,” and she began spilling paperbacks, hardbacks onto the floor, peering nearsightedly at their titles, tossing them over the couch back at me.
“Here, this one’s good; it was written a long time ago,” and The Well of Loneliness flew in my direction.
“What about that study, hon? The one where he talked to every dyke in the land?” Dorie asked, watching Jean, smiling at her, her absorption, her activity.
I turned away; I could not witness her terrible tenderness.
"Oh, yeah, *The Grapevine*, that's a good one," and a paperback sailed across the room.
More books flew, their covers racy or sinister, titles a welter of despair and damnation: women embracing in shiny black slips and curling red hair, whip-brandishers and caressers of net-stockinged legs. Red lips, cutting eyes, *pecador, pecador*.
When it was over, Dorie and Jean stood smiling together, Tweedledum and Tweedledee in their monogrammed robes. "Maybe you'll learn a little about the whole thing, Theodora," they said, and exited, stage left, leaving me with their dreaded wares.
Or merely dreadful. From my afternoon's reading, that dizzy deoxygenated spin through the unfamiliar coils of nicotine and sapphism, I surmised that damnation was certain, if painfully enjoyable; that women who were *that way* came in two shapes (crypto-cock and cunt); and that (let's hear it again) The Tale of the Young Invert is an unhappy one. Much gin-drinking (fatal prophesy), much apartment and bar-hopping, an edema of consciousness.
I smoked, gagged and read, my eyes a fundament of tears. Was this what Dorie and Jean's (my mother's?) lives meant?
When the light cut too low for reading, Dorie materialized on cue from the back of the house. "How's tricks, kid?" she asked, her old snappy self again.
I had long since sunk below depression, realizing my own life-curse in the grim tracts I'd read.
"How can you go on?" I wept.
She stared back humorously. "Go on? Why not? Want a drink?" She patted my shoulder, lingeringly.
"And the drinking," I cried.
"Shit, you probably do more drinking with your little college boys. It's no worse for me to have a drink than it is for a fraternity kid. Drinkers—you know what they have in common? Drinking."
She put her blue terry arm around me. "Hey. Nobody writes books about happy people, right? They wouldn't sell."
I honked and wheezed, uncontrollable before my great recognition. Loneliness and waste: I could feel them sucking me out of that brave pure and white tower at Immaculate Heart where only two days before I had cavorted in happy unconsciousness with Matthew Arnold, Thomas Carlyle, Gerard Manley Hopkins and the boys.
"I don't want to go, I don't want to go," I cried, and threw myself in Dorie's lap like a hatchet.
She bent to my neck and kissed it softly, running her flat dry palm down to the cleft in my back. "My dear, my dear, it's not so very bad in here," she said.
THEORY
photograph by Lynda Koolish
LESBIAN CELIBACY
"Lesbian Celibacy" was a paper given to the Sixth Annual National Conference on Feminist Psychology held in March 1979 in Dallas, Texas.
I do not know exactly why I'm here today, but I am sure that it is not without anxiety. I am not a "woman in psychology," but I am one of your clients—formerly a very frequent one, but now rather infrequent. By professional training, I am a teacher of Women's Studies, and I am also a celibate lesbian.
You will, however, be delighted to know that, in spite of my clienthood and my celibacy (which I define to include masturbation), this is not a "confessional" paper.\(^1\) Rather, it is a brief and preliminary consideration of lesbian celibacy\(^2\) from four loosely related perspectives: (1) the non-treatment it is receiving in readily accessible and putatively feminist books about women's psychology, (2) some social difficulties and political criticisms encountered by celibates, (3) the possible impact of celibacy on the client-therapist relationship, and (4) the nexus between lesbian celibacy, self-nurturance, and radical feminism.
My research methodology (to the extent that I have employed one at all) has been crude: I have not worked with psychological abstracts or journals, nor have I bothered to read any library books about celibacy, for most of them appear to be by and about religious men. Instead, I have looked at those women's psychology books which are both intellectually challenging and economically accessible to many laywomen, and to many lesbians who are puzzled and mystified by what professionals say and do not say about us and the lives of our minds.
Primarily, of course, authors of women's psychology books have chosen to ignore lesbianism entirely, and not since Phyllis Chesler gave us one whole chapter seven years ago has anyone ever again given us one whole chapter, except for Charlotte Wolff, who gave us one whole dreadful book. Indeed, a quick check of several indexes and tables of contents of books long since read and shelved reveals that Jean Baker Miller's and Jean Strouse's anthologies got no further than a man's ideas about bisexuality, that Juliet Mitchell mentioned lesbianism only three times in the course of *Psychoanalysis and Feminism*, that Baker Miller once again ignored lesbians in *Toward a New Psychology of Women*, as did Jungian Irene Claremont de Castillejo in *Knowing Woman*. Most recently, Dorothy Dinnerstein reduced lesbians to a footnote, and all of what Nancy Chodorow has to say about us could be put on one page. Needless to say, none of these books mentions celibacy, either
lesbian or non-lesbian. Interestingly, some earlier women psychologists gave more attention to lesbianism, although none to celibacy. *On Women*, a selection of Clara Thompson's work edited and prefaced and foreworded by men, contains two "pieces" about lesbianism, both of which reflect Thompson's pathological and heterosexist approach to homosexuality—an approach also apparent in the works of Karen Horney in the collection entitled *Feminine Psychology*.
Obviously, this dual underrepresentation of both lesbianism and celibacy in popular books about women's psychology is discouraging, for there is virtually no printed word to help us toward self-understanding. And self-understanding is what we greatly need, for a consciously celibate lesbian is most assuredly embattled on at least three borders—those of her womanness, her lesbianism, and her celibacy—and the fear of being deeply violated by the perpetual thrusting of this oversexed yet undereroticized phallocentric culture is very hard to still, especially without books and words to comfort and encourage.³ Lesbian celibacy, then, asserts itself as a particularly female-identified erotic style, necessarily and acutely counterpoised against all other permutations of active and inactive heterosexualities and homosexualities.
This is a difficult position to maintain, both socially and politically. Even other lesbians sometimes react with incredulity and skepticism tinged, perhaps, with some resentment and reproach toward a woman who is so completely in control of her own life. Still others say nothing directly, but subtly imply that celibacy is symptomatic of extreme emotional shriveled-upness—that the celibate is so "totally into herself" that she is unable to give and receive love. What is not perceived, however, is that lesbian celibacy is, as Mary Daly would say, a "state of Self-possession." In other words, our celibacy is not merely or simply a reaction to a culture which so daily and brutally insults and assaults our lesbian aesthetics and sensibilities. Rather, it is an affirmative action—a woman's moving toward her self and her center—and, as such, it is worthy of strong social and therapeutic support, if not occasional recommendation.
Lesbian celibacy can also be targeted for political criticism, e.g., that it is an "individual solution" and therefore impermissible. Such pejorative labeling however, is based on false consciousness from any feminist political perspective. From a lesbian separatist point of view, celibacy should be endorsed as an act of deeply authentic separatism. From a radical feminist viewpoint, celibacy must be respected as a paradigm of self-empowerment and of the taking back of one's body. And even from a mainstream feminist perspective, a celibate should be seen as a healthy role model for female independence⁴ and for the liberation of personal energy towards political work in the women's movement. As Dana Densmore once wrote, "Erotic energy is just life energy and is quickly worked off if you are doing interesting, absorbing things."⁵
Moreover, lesbians who politically condemn celibates on the basis of such facile slogans as "an army of lovers cannot fail" not only implicitly adopt the saying of a man (Oscar Wilde) and his explicitly naive military mentality, but they also contribute to the replication of those male and heterosexual patterns of promiscuity which dominate present culture. The message is clear: in order not to be a straggling failure, one must enlist in the army of lovers. Lesbians,
however, must guard against cooptation by catchy phrases, cultivate and value our ability to be self-loving women, and know that revolutionary potential is only as strong as the individual woman. To paraphrase Adrienne Rich, each woman must constantly expand the meaning of her love for herself as a woman,\textsuperscript{6} but in the process of doing so, we must also resist the label of narcissism, for the protagonist of that mighty myth was a male.
Which somehow brings me to the topic of therapy. With regard to the potential effects of lesbian celibacy on the client-therapist\textsuperscript{7} relationship, I would suggest that it might lead to some distancing or pulling away by the client, for celibacy often seems like an unspeakably private\textsuperscript{8} affair, never had by many people in this culture, and, consequently, not well enough understood to risk talking about in depth. In addition to withdrawal by the client, however, there has surely been a failure by therapists to treat lesbian celibacy’s radical finding of the female self\textsuperscript{9} as the welcome realization and end of what the best of feminist therapy is all about—bringing a client to love and have high regard for herself as an individual woman\textsuperscript{10} to become compassionate\textsuperscript{11} and affectionate with herself, to be so empowered as a radical female that she is strong, competent, and self-centered in the woman-defined meaning of the term. In other words, lesbian celibacy could be viewed as a healthy breaking away from dependence on therapy, and a therapist of high consciousness and integrity\textsuperscript{12} might want to raise the question of whether or not it is time for the formal therapeutic relationship to end.
If it is not, then the therapist can help the client focus on the special insights being gotten through this rare and valuable closeness to the self. Additionally, celibacy might lead to disentanglement of some of the more troubling aspects of the ongoing transference process, and, freed from many distractions, the client could thus be enabled to work especially productively and effectively in therapy, and to approach the inside of her psychic self more easily.
Finally, I want to speak of the connection between lesbian celibacy, self-nurturance, and radical feminism. For the purpose of this paper, I will adopt Jane Flax’s definition of nurturance, as articulated in her recent article in \textit{Feminist Studies}: “[Nurturance is] the expression of love that conveys a deep concern for the well-being of the person receiving it, without requiring that the person prove herself worthy or fulfill the nurturer’s own needs, fantasies and so forth as the condition of receiving such care. Nurturance also has a sensual aspect as well because the care extends to the recipient’s body as well as her psyche.”\textsuperscript{13} Fortunately, some of our best lesbian word artists—Adrienne Rich, Audre Lorde, Mary Daly, Monique Wittig—have begun to talk to us (as have some women in psychology) about the intricacies and intimacies of all types of relationships between and among women. Rich speaks of our eroticism with each other, and of our shared perception of danger,\textsuperscript{14} while Lorde talks of self-definition and bonding among Black women.\textsuperscript{15} In her new book, \textit{Gyn/Ecology}, Daly describes radical female friends, sisters, and lovers; and in the novel \textit{Les Guérillères}, Wittig conjures up a mythically strong vision of an unconstrained society of females who protect and care for and work with each other as they live close to the earth.
Without naming it celibacy, Mary Daly refers to the ability to be radically alone as a requisite for the ability to be a friend to another woman, and she also states that radical friendship between women means "loving our own freedom, loving/encouraging the freedom of the other, the friend, and therefore loving freely." Furthermore, a woman who is a friend to herself possesses an inherent and highly individualized strength upon which the best of female-identified bonding is based. Because she knows that only she can judge herself, and because she has a strong sense of her own worth, she has the courage to accept her self, and is thus neither self-sacrificing nor demanding of self-sacrifice from her sisters. Wittig has also caught some of the rising exuberance and revolutionary potential of celibacy, as evidenced in the middle stanza of the frontispiece poem for *Les Guerilleres*:
CONSPIRACIES REVOLUTIONS
FERVOUR FOR THE STRUGGLE
INTENSE HEAT DEATH AND HAPPINESS
IN THE BREASTED TORSOS
THE PHOENIXES THE PHOENIXES
FREE CELIBATE GOLDEN
THEIR OUTSPREAD WINGS ARE HEARD
And the connection among all these female things? First, on a very material level, by consciously becoming celibate a lesbian takes back her body in the most fundamental form of physical reclamation possible. She has thus effected a condition of aloneness which is extremely radical within the context of an actively heterosexual male culture. Within her radical aloneness, she learns to nurture herself uncritically—that is, she develops concern for her own well-being, gives to herself without requiring proof of worthiness, cares for her lesbian body and its sexual health and pleasure of expression. In the process of learning and practicing self-nurturance, the celibate lesbian also replaces self-destructive behavior and anti-woman indoctrination with female life-givingness to herself. As she begins to center and sustain herself, her energy escalates, and, rising free of cultural and sexual expectations and entanglements, she is able to love and work with other women in supportive and unencumbered ways.
In conclusion, then, lesbian celibacy is a physical and psychic condition which has not received sufficient therapeutic attention or support. Although it is a unique opportunity for a woman to experience self-strengthening aloneness, it is nevertheless a socially and politically uncomfortable position, and intelligent, sensitive feminist therapy will recognize feelings both of isolation and of strength, and the often difficult emotional dialectic which they produce.
It is exceedingly special to choose one's self as friend, as sister, as lover. Certainly, celibacy is a new space for lesbians to build in, where our outspread wings will be heard.
NOTES
1. See Julia Penelope Stanley and Susan J. Wolfe (Robbins), "Toward a Feminist Aesthetic," *Chrysalis* 6, pp. 57, 58.
2. For the purpose of this paper, "celibacy" is defined as the voluntary and conscious abstinence, unfactored by religious vows, from sexual relations with other women.
3. I write this word this way in respectful imitation of Mary Daly and her "A-mazing" way of enriching words by breaking them down and up.
4. See Barbara Love, "A Case for Lesbians as Role Models for Healthy Adult Women" (paper presented at the meetings of the American Psychological Association, Chicago, 1975).
5. "On Celibacy" (heterosexual context) appears in *Voices from Women's Liberation* (Signet, 1970, ed. by Leslie B. Tanner), pp. 264-65.
6. "The Meaning of Our Love for Women Is What We Have Constantly to Expand" (speech at New York Lesbian Pride Rally, June 26, 1977), Out and Out Books, Pamphlet No. 1, 1977.
7. For the purpose of this paper, the therapist is presumed to be a woman.
8. Janice Raymond has enlightened me about the difference between the personal and the private.
9. Again, Mary Daly's influence in this phrase.
10. Sometime early, Eileen spoke to me of seeing the self as "very precious."
11. Judith has spoken to me recently about this.
12. Janice Raymond has multiplied the meaning of this word for me a thousandfold.
13. "The Conflict between Nurturance and Autonomy in Mother-Daughter Relationships and within Feminism," *Feminist Studies* 4, pp. 171, 187-88 (n. 4).
14. Elly Bulkin, "An Interview with Adrienne Rich," *Conditions: Two* (October 1977), pp. 53, 57.
15. "Scratching the Surface: Some Notes on Barriers to Women and Loving," *The Black Scholar* (April 1978), pp. 31-35.
BIBLIOGRAPHY
Phyllis Chesler, *Women and Madness* (Avon, 1972). Nancy Chodorow, *The Reproduction of Mothering: Psychoanalysis and the Sociology of Gender* (Univ. of California Press, 1978). Mary Daly, *Gyn/Ecology: The Metaethics of Radical Feminism* (Beacon, 1978). Irene Claremont de Castillejo, *Knowing Woman* (Harper and Row, 1974). Dorothy Dinnerstein, *The Mermaid and the Minotaur* (Harper and Row, 1977). Karen Horney, *Feminine Psychology* (Norton, 1967). Jean Baker Miller, *Toward a New Psychology of Women* (Beacon, 1976). Jean Baker Miller, ed. *Psychoanalysis and Women* (Penguin, 1973). Juliet Mitchell, *Psychoanalysis and Feminism* (Vintage, 1974). Edna Rawlings, ed. *Psychotherapy for Women: Treatment toward Equality* (C.C. Thomas, 1977). Jean Strouse, *Women and Analysis* (Dell, 1974). Clara Thompson, *On Women* (Signet, 1971). Monique Wittig, *Les Guerilleres* (Avon, 1969).
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RANDOM THOUGHTS ON VICTIMIZATION
"Victimization" was a speech given at the April 8, 1979 rally held to protest Boston College's harassment of Mary Daly.
You know that scientists are investigating "solutions" to the problems they have created through modern technology. All over the world, their drawing boards are littered with blueprints that further remove the human race from its roots in nature. Men have always shown a profound alienation from nature, but it is more obvious today as both overpopulation and the tools of technology threaten life on this planet with destruction that is as painful as it is complete. In their re-combined world, nature as we know it no longer exists: they plan an environment fit only for the humanoids they are hard at work designing, frighteningly artificial and sterile. This raises a number of questions. When the frame of reference that served to define our humanity no longer exists, is it appropriate to continue to call the species human? Or perhaps human is the right word, as the etymologies I'm going to sketch will suggest. In which case it is an insult, and we need to invent another word for ourselves.
Another question: How did this all happen?
Since the scientists who engineer the human future do all their basic research on animals, I wanted to start with the area of animal experimentation as one of the ways to approach this question. Why animals? What does that say about their attitude to life, to nature, to pain? I think a great deal about these things, and today I will try to relate some of my thoughts to the oppression of women in general, hags in particular.
I said I was going to sketch some etymologies. It is very significant that the words we use to designate the non-human world are closely related to the life principle and the process of growth. *Animal* is formed directly on the Latin *anima*, meaning “soul,” “breath of life.” *Vegetable* derives from a Latin verb (*vegetare*) meaning “to grow, rouse, excite,” which is the same root that gives “to wake,” in the sense of being watchful and aware. *Nature* itself comes from a Latin participle meaning “born” (*natus*). In Greek, *zoion* (“animal”) and *physis* (“nature”) have the same connotations of soul and birth. As for *grain* and *corn*, they both derive from a Greek word meaning “old age,” which refers to the goddess Demeter—who also gives the word *cereal* through her Latin name, Ceres. *Legume* means “that which is gathered” and is related to the Greek *logos*, meaning “speech, word, reason.” And *fruit* is something that simply means “to enjoy.”
By contrast, neither Greek nor Latin have kept the identification with these vital processes in their word for “man.” Men formed both *anthropos* and *homo* (*homo* and its derivatives *human, humanity*, etc.) in opposition to these processes, with the clear intention of differentiating themselves from nature, of becoming non-divine, non-mythical, non-animal, non-mother, non-natural (*natural* being taken to mean “irrational—that is, not subject to man’s reason). Only the word *mother* retains its identification with the creative energy that characterizes non-human life forms. It comes from the Greek *meter*, as in Demeter, and gives many interesting derivatives. *Metropolis*, for instance. I like that. Metropolis: Mother-city. As for *father* all it means is “chief” in Greek. *Patriarchy* is formed on that word meaning chief. The true counterpart of “chief,” the one that corresponds to the social reality that gave birth to these words, is queen, *gyn*.
These etymologies I think explain much about the stress the fathers have placed on the earth, about the classification of life into superior/inferior categories (as in higher and lower animals), and about the fact that nature, animals, and women have a parallel history and suffer similar treatment. From the beginning of “human” life (I wish I had another word), women celebrated animals, nature, and themselves as equal manifestations of power. Then, as man invades the metropolis, he cannot identify with what is going on in them because he is predator (he hunts animals), rapist (he subdues women through rape), and above all he does not create life. In the beginning was the Universal Egg, Plato said. Which divided by itself, he said. So man names himself basically from a position of deficiency. History, in the sense of patriarchal forging of values and ethical systems, is the story of his search to compensate for this lack by enforcing as immutable law the supremacy of that one attribute he claims distinguishes him: rationality, reason. I think it is this perception of himself that conditions the curious ambivalence with which he has treated nature, animals, and women. There is, there has always been, a split between his self-image as benevolent nature-loving man and the
actuality of his behavior as agent of destruction. His need of her has led him abstractly to worship her while his fear and envy of her independent power have led him concretely to degrade, dominate, and violate the core of her being, her integrity. In addition, his envy placed him in a competitive position with respect to Mother/woman/Nature. He will out-do her by “prying open her secrets” (these are his words), by eliminating her “imperfections” and “improving” what has worked best for him. In this frame of mind he is trying to manufacture his idea of what life is about. The sole purpose of the genetic engineers, the thing for which they rationalize the sacrifice of thousands of animals every day, is the re-designing of life and the complete monitoring of all its functions. Since I’m not bound by their scientific code that prevents them from making value judgments, I call this immoral.
When you criticize the immorality of patriarchal values (the damage they cause, the directions they take), when you criticize them from a radical feminist perspective you become very dangerous. It is then that your differentness is most obvious. You are making it difficult for him to pretend that you are his intellectual clone, which is how he understands the notion of equality. “Those who are not with me are against me,” said the Lord. The patriarchal way of dealing with differentness is through punishment, and he has many ways to punish. If he can’t “harness” this differentness—that means exploiting it to his advantage—he can try various tactics to silence it: torture, death, isolation, etc. Radical feminism intrudes upon the false sense of security they achieve and maintain through uniformity of thought patterns and behavior (homo also means sameness); it intrudes upon their consciousness in a way that threatens the survival of their homogeneous humanity. They react as if they had just stepped on an electric eel.
To go to the roots of things, which is what radical means, is dangerous only to those who will not leave the surface. I suppose the word radical evokes images of cutting vital parts, judging from how they use it: radical mastectomies, radical hysterectomies, the eradication of animal species and the uprooting of trees and people. Animals and trees don’t need to go to the root of things, they are already there. Nature and animals and women—if only we could be left alone—don’t need to “harness the keys to Paradise,” we are Paradise. (I’m using the title of a recent Nova program here: “Will we harness the keys to Paradise?”) Anyway—for all intents and human purposes, nature and animals have been made utterly powerless; they are forced to live in Hell and to die in Hell.
To get back to the idea that radical feminists are perceived as dangerous. You know how commonplace it is to say that animals are dangerous to man. In their programs on animal conservation, animal behaviorists and ethnologists are always portrayed as heroes who expose their lives by confronting the so-called dangerous wolves, bears, lions, etc. in their natural habitat. In reality, it is man who is, and has always been, dangerous to animals. Animals are dangerous to man only when man has cornered them and the animals fight back. They talk about dangerous women, too: the femme fatale, the vamp, the witch, the castrating bitch, the vagina dentata . . . Perhaps there lingers in the collective memory of men a vague sense of once having been offered as sacrifice to the Great Goddess. The sacrificial king, as he was called, was consort to the goddess for only one year, at the end of which he was killed ritually. Animal sacrifice is said to have replaced this human sacrifice. It is said to be a cultural advance. Artemis is credited with this change, but we don’t really know at what time in patriarchal history this accreditation was made.
I want to read a paragraph from a book review of something called *By Reason of Insanity*, by Shane Stevens. The review is from the New York Times book review section, by a man called Jack Sullivan, who shows no awareness of the paradox in the title. *By reason of insanity*? (He himself has written a book called *Elegant Nightmares*). The name of the main character in *Insanity* is Thomas Bishop, introduced as “the most resourceful mass murderer of modern times.” He is being pursued by a “power-obsessed reporter who states the novel’s theme when he insists that Bishop is ‘still the child living the horror and doing whatever the terrified beast in his nature can do to survive . . . When pushed for survival we all revert to animality, you know.’ The world is a jungle best described with crude animal metaphors: ‘Where else could he find anything like New York, with all its women and places to hide? . . . For what he was hunting, New York was the biggest national game preserve there was.’” Bishop. A child living whose horror? We revert. Crude animal metaphors. Women/game. The reviewer tells us that “Mr. Stevens wants very much for us to take *By Reason of Insanity* seriously.” The title of the review is “Chopping Up Women.” This reminds me of a bumper sticker I once saw while I was driving. It said: “Do it in the woods with a bow.” It had an inset of a deer’s head in a corner. Mr. Stevens might as well have said: Do it in New York with a bow, since that is what the sticker implies, even though it refers to the deer-hunting season in which bow and arrow are allowed. While I am on this gruesome theme, I will mention one more parallel since the seal-hunting season is open in Canada. I don’t see much difference really between the fantasy-image of cavemen who clobber women and drag them by the hair back to their dens and the clobbering of flawless white-skinned baby seals that end up in people’s houses in the form of coats and trinkets. There is a connection, too, between these images/practices and the dredging of the ocean floor.
I mentioned earlier that man is split between his self-image as benevolent lover of nature/animal/woman and the actuality of his behavior as agent of destruction that so often verges on sadism. A leading British physiologist recommends and licenses the use of living animals in medical research. He himself has performed many experiments, often on dogs. He talks about how he loves dogs. He loves them so much that he and his wife go to dogshows regularly with their own beloved pets. This picture of a scientist is very common. I myself know a she-biophysicist who fits it to a T. They believe that a dog is man’s best friend. But when they go to the laboratory every morning, this knowledge vanishes as if by magic. The lab attendants have done the preliminary work: they strap the dog to the experiment table and remove its vocal cords so the scientist can perform his “experiment” and not be disturbed by the dog’s cries of pain. What happens to a woman who doesn’t play man’s helpmate and insists on being herself is similar to what happens to the dog, although the means they use to silence her, real and painful as they may be, are not that extreme. They invade her privacy, violate her integrity, and use
degrading ways in an attempt to silence her. They want to continue to preach the catholicity of their gospel undisturbed by the woman's voice. To grab a dog and force it to suffer pain within the inner space of its psyche without allowing it to defend itself or seek relief in howling is as immoral as to invade a teacher's classroom and try to silence her through binding statements, isolation, and demeaning harassment. But still, the woman is far better off than the dog. She is not the Total Victim because she is able to fight back.
I'd like to go back a little to the idea that the antithesis of animal is intellectual, logical, rational. According to Aristotle, who lumps women and squid together (tentacles?), non-men are all instinct and intuition and, in addition, women suffer from impaired judgment because of our tendency to get lost in our feelings. Lower animals are like inferior women when rational man uses himself as standard of comparison. The ape didn't come into its own as higher animal until Darwin tried to show that man descends from the ape. More recently, dolphins have been found to have a sophisticated sound system they say "qualifies" as language: like apes, dolphins are "higher" because they can be taught to speak humanese, which is seen as a sign and measure of their intelligence. Personally, I would be far more impressed if a human learned to speak dolphinese. I think the Cretan women knew the ways of dolphins when they drew them—as they did so many animals—far more intimately, far more intelligently, than any of these re-searchers can ever hope to know them.
Since man is, by his own definition, the opposite of woman, animal, and nature yet needs us in order to survive, he cannot consciously and systematically destroy the givers of life, the givers of food. That is, not yet. He can and does, however, breed them in order to insure that his supply will not dwindle and disappear, for he relentlessly pursues his quest to create life by himself and to engineer brain, emotions, and motor responses the better to suit his needs and to fulfill his fantasy of "the good life." The breeding of fruit flies, rodents, cats and dogs (to name but a few of the most common experimental animals) has the same purpose as the breeding of humans through in vitro fertilization, embryo implantation, and the cloning of living tissues. Again, the language un/dis-veils. To breed you know means "to hatch out by warmth," the idea being that warming is breathing upon (breed and breath being etymologically related). What could be further from warming than the cold sterility of artificially manufactured life, of artificially manufactured environment, and the uses to which this "life" is destined to "live"? Whether for human consumption, scientific experimentation or as professional wombs, these breeds can at best be left to brood over the loss of their original shape and inherent characteristics. Does a featherless chicken brood over her lost feathers, her nakedness? I believe she does, in her own silent way, miss the well-being of her feathers that regulate her body temperature and protect her from outside discomforts. With respect to human breeding and/or cloning, the object is eventually to reduce the female population to a desirable twenty per cent, according to a team of scientists working in Chicago. These broods of female bodies will be programmed, much more so than we are, to carry out man's intended use of them. Furthermore, they supposedly won't miss a thing, as techniques to alter memory are well developed—and so are the means to control moods, emotions, and pain.
Talking about pain, Descartes, who flaunts his Latin when it suits him (cogito, ergo sum), must have been struck with selective amnesia when he came to the word animal. (You remember that anima means "soul," or "mind," as in psyche, the Greek equivalent of anima). He states categorically that animals cannot feel pain because they have no mind/soul. You can imagine what a boon this was to the then budding physical sciences. Their practitioners could slit open a live animal's body and believe that the animal did not suffer pain. They didn't use anaesthesia on animals until the last few decades, and even now they claim that, in some cases, anaesthesia would hamper their research. Descartes' decree must have helped the 19th-century French physiologist Claude Bernard, who is called the first biochemist because of his work on the chemistry of the liver. He is described by a visitor as follows: "In a narrow, damp corridor . . . he stood before his animal table . . . his fingers in the abdomen of a large dog which was howling mournfully." When I read this, I flashed back to a conversation I had with a French friend of mine who had an abortion in France. She said that the physician made sure that she would howl, he said, to punish her for aborting life. In her fight to liberate the abortion laws in France, Simone de Beauvoir discusses the sadism of doctors in much the same terms. There are two more interesting facts about Bernard. He is remembered today for having established that the stability of the inner self (milieu intérieur) is the precondition of a free independent life." Whose life was he talking about? The dog's? His wife's? Because the second interesting fact is that his wife deplored his treatment of animals and contributed lavishly to humane societies. After years of distress, she separated from him.
I would like to end these thoughts with two more analogies that bring animals, women, and nature close together. Both are mythic transpositions of patriarchal invasions of women's space through trickery and rape. I am referring of course to Zeus and Hades who erupt on the peaceful, playful scenes of women in the fields and who abduct two of them. Zeus swims away across the waters with Europa on his back, and Hades carries Persephone down to Hell. The other image is that of Penelope and Argus the dog, waiting for Odysseus to come home. She at her loom, weaving and unwrapping to keep her suitors away; he on a dung pile, waiting to see his master before letting himself die. I wanted to connect the image of Zeus in the shape of a bull and the bulldozing of the earth—also, the idea of Hades, whose Latin name is Pluto, with the element plutonium and the recent "accident" at Three Mile Island. But I can only mention them because I'm running out of time.
It is really important to remember these images and not spend our lifetime spinning and unspinning in this sterile way but rather to un-spin their deadly fabric as we spin, in Mary Daly's words, "our own cosmic tapestries." It is important not to keep sitting on their dung pile, but to fight back and re-member flowers and animals even as we re-member ourselves, to set free our creative spark and re-member the squid, the dove, the deer, the dog, the frog, the cat, the rat, the rabbit, the mouse, the horse, the cow, the
goat, the sheep, the hen, the leopard, the tiger, the lion, the sea-urchin, the fruit-fly, the owl, the duck, the dolphin, the ant, the whale, the seal, the snake . . . and the spider.
The spider is a fascinating creature. It is a master of disguise, able to blend in with its surroundings, and it can be found in almost every habitat on Earth. Spiders are known for their intricate webs, which they use to catch prey. They are also known for their venomous bites, which can be dangerous to humans. Despite their fearsome reputation, spiders play an important role in the ecosystem, helping to control insect populations.
Spiders come in all shapes and sizes, from tiny harvestmen to massive tarantulas. Some species are solitary, while others live in colonies. Spiders have eight legs, two pairs of pedipalps (which are used for sensing and grasping), and a pair of chelicerae (which are used for biting and chewing). Their bodies are divided into two parts: the cephalothorax (head and thorax) and the abdomen.
Spiders are arachnids, which means they belong to the same class as scorpions and mites. They are not insects, despite their name. Spiders are predators, feeding on other insects and small animals. They are also known for their silk, which they use to build their webs and to wrap up their prey.
Spiders are fascinating creatures, and there is much to learn about them. Whether you are a spider enthusiast or just curious about these amazing creatures, there is always something new to discover.
STILLNESS AND MOTION
A STUDY OF RELATIONSHIP AND FILM
Stillness. Together. I spent an entire day in complete and total relaxation with my lover in a space where we were together yet separate, she reading hers and me reading mine, our legs and feet touching across the bed. A trust. A commitment. To being there. The picnic, the baseball, the social grouping and cocktail-like party did not disrupt the continuity of our stillness. Across a throng of noisy people we were there in our togetherness and separateness like boulders lying under a passing stream. So there that in an instance of harmony we could go from quiet reading to walking out the front door to see a movie as if we were simultaneously moved to action.
Stillness. Heightened by premenstrual sensitivity, vulnerability, huggability, touchiness; the time, she said, when I clung, the only time of the cycle when I held on. The quality of the time was sameness. Although we might be engaged in different activities—eating, reading, hiking, loving—time hung like an evenly strung necklace of mandalas encircling us. The spaces were even, the balance weighted perfectly. No disruption, no dis-harmony, no ups or downs, but continuity. That must be the serene blessing of a longlasting relationship I think, that stretching on and on of even time.
Motion. Abrupt. I move the luxurious quiet to a precipice, daring motion, so unused to periods of stillness am I, unable to accept my inner peace and so the world's. If you don't jump, I will. I introduce into our tranquility, judgment.
She is too such and such. Instead of accepting the wonderful time we spend. I get scared that this might mean an end to my search for the perfect lover, and I introduce criticism, prompting my withdrawal. Not content with stillness for any length of time, afraid it might mean I am not growing, I disrupt. The motion produced is like a little whirlwind going nowhere. A still disruption. Frenetic activity as I try and succeed at befuddling myself. You aren't good enough for me in this way or that, I challenge, and we go around and around in a little dance discharging and recharging without evolution. Revolution without evolution—so that the motion is meaningless, does not produce change. Negative motion. For naught.
To the adolescent, commitment by choice means the end of possibilities. The closure of a world. If I were to accept the peace and harmony I found here, if I were to accept my lover, I would be putting a stop to
my possibilities. A foreclosure on the adventures of life. Actually, I was closing off to the adventuring with commitment, within a frame. The bounding ego, boundless, shall know no home and always be seeking. The adventure is determined as much by the question asked as any thing. At the moment I was fooled. I thought I had adventure only in the patterned way of new lover after new lover; but this format which seemed like change to me was an illusion, for the habit was as rigid as a nun's. Change and adventure for me now would become experiencing a "longterm relationship" (would that there were some new words to replace these prison-sentence-like terms!).
There was a moment recently when many of my past relationships seemed merged into the present. A sense of deja vu. Time played backwards. When life seemed a rerun and stood still. The sense of repetition, of having been here before, gives a sense of stillness.
The experience of deja vu came from a phone call from my love. She said she had presents for me and would give them to me on Saturday. The day she called was Monday. As I lay on my cushion listening to her, my studio seemed to change from a place where I was engaged in the present, moving from moment to moment, to a frozen tableau. The table and rug were the same, but my experience of them changed. Instead of my taking them for granted, they became solid, unmoving, unchanging objects of my past, and I felt caught and stolen away to this ice-cube scene of the past. Sometime before I had been promised presents on a Saturday, presents that never came.
Time moves slowly when we do similar things and moves rapidly when we are in new situations with unfamiliar stimuli. A new relationship can seem like one of years, and an old established one that varies little in habits can make days seem like weeks. In my case of deja vu, time was still because a similar event/situation from the past reconnected with a present one and gave the sense of an ellipsis of time, as if nothing had happened inbetween.
From a study of physics we know that space and time cannot be separated but are coordinates in a relationship. Time stood still and space was frozen in a recall from the past. In the presence of new love time flies, and space is charged by objects and people who take on a glow of special significance. Space and time exist only in relation to a particularizing consciousness. The stillness of a plateau in a relationship is tantamount to a peaceful rest or stifling boredom, depending on who is experiencing it. Recently my lover and I experience stillness in motion, plateaus in our relationships, in two different ways.
She experienced me cutting her out of my life. This reminded her of early feelings of abandonment, and she went in a deep spiral down into herself where the ultimate emptiness and stillness of loneliness lay like an unending hole. Her therapist recommended she feel this utterly painful sense of nothingness and aloneness, the stillness that is so still and quiet like death that it frightens, rather than find distraction in placebo motions of business, filling her life with events and people to evade this stillness.
I experienced shutting off to motion naturally and willingly, seeing fewer people, going out less, in order to have the stillness and aloneness with myself my work requires. I appreciate and love these quiet, centered times when the world of newspapers, daily events, friends and lovers spin around me in their ongoing cycles yet I remain still and quiet. In fact, I shut off the swirl like turning off a faucet, that my inner images might flow. There is no way I can concentrate on a film, or writing, on a performance when I am moving from one date to the next so the week appears a veritable long weekend in my datebook. I need at this time—and the cycle moves me here without my willing it—a long date with myself.
This plateau of stillness where I exist alone gives me the opportunity to concentrate on Self and the images that appear like rocks and craters on the moon. I have filled my time with motion to avoid the stillness for fear that there will be nothing there, but because I have built my identity, a self-construct, on being an artist, I am fed and nourished by whatever—even nothing should I find that—is in my stillness. This is the stuff from which art is made; and if I don’t go here, then I have nothing to make. The social butterfly flapping her wings living years in a day with fluttery, hopping motions is at the opposite side from the still cocoon where the meditation harbors, the images form and grow. In stillness alone I find the motion for the moving images in film. It’s like the persistence of vision where series of still photographs stopped in the film gate of projected light for a short moment, 1/24th of a second, appear as motion. Owing to memory—our memory of the image that precedes—and the continuous present that changes the moment we see it, the still photographs appear to move.
In the stillness of my center, images come one after the other with the appearance of motion, of fluidity, of continuance. There is this flow, this continuity, this progressing which eventually makes the work, but it is all contained in the stillness and cannot be without it.
My lover who enters her stillness afraid of a terrifying loneliness, even she, claims that she would not do away with these periods, for she always brings away new understanding. After we have an intimate sharing time with another, it is an inner voice that demands we give attention to our private selves. The voice says, “pay attention to me,” “reflect on what I am telling you.” Each of us has an inner voice (or several) which guides us in taking care of ourselves. If we don’t listen and follow that voice, we miss out on a relationship with our very self. In that case we could go for days without knowing how we are really feeling, what it is we want to do, what we need. By practicing a following of the spirit, of the intuition, of inner suggestion, a balance will take place, and a circle of flow without practice will eventually establish itself. At first it will take constant and attentive listening to the voice; later it will be second nature (truly the first and primary nature), and one action or contemplation will naturally follow another.
This morning I awoke from a deep and dreamful sleep wanting to be alone. I had spent hours of the day before in intense and wonderfully intimate relating with my lover; much of the time was exceptional. This morning I needed to reflect, to let the unconscious surface if she would.
photographs by Lynda Koolish
I told my lover and she felt the same way; she needed time to herself as well. A time of stillness so that the intensities of the day before could settle themselves, find an interior home. We took our separate rooms.
Yesterday on the couch I was fallen in her arms, sunk into her body in restful giving as I can find in me when I need to, can find in me because she's there for me to lie upon and be held by. As I lay upon her I needed to reveal myself, reveal the mind/body split that tossed me, restless with insomnia, night after night. I told her I was fearing an impending conflict between my love for her and for another. That my love for her was a physical and emotional one, that my body loved her, and that my love for the other was an intellectual, ambitious head. Neither love was so clearly defined, but to make the distinction and understand the conflict I let the division grow. I told her this and she heard without reaction or moral judgment, and my telling and her hearing released an incredible and first-felt surge of surrender in me. I don't have better words for it now and it deserves better, but it was as if I gave up my ego-strength, my power of being, my holding back, my resistances—and when I gave up those boundaries of the self, I experienced a very deep soul-felt release and acceptance. I had given up my definition of myself and was still loved and accepted. I experienced a sense of relief at being able to let go.
This was not an easy letting go. I felt frightened and warmed simultaneously. Frightened that if I let go of myself, she might dominate me in a way that would not be good for me. At the same time I wanted to be dominated, just a little bit. Dominate is not the right word. I wanted her to be as strong, as powerful as I experienced myself—and that was quite powerful; hence, the strong sense in giving it up. I decided then not to make judgments about myself but to experience my emotion without condemnation.
When we were in bed that evening and she began the delicious massage of my back which led to her lying her full body weight on mine, and after she began a movement with her buttocks and vulva pushing against mine trying to get in over and over in a rhythm I couldn't and didn't want to stop I began to make a judgment again. A judgment that this was heterosexual physical movement in our loveplay. And then I let that go, let the whole left side of the brain go, and felt good because I wanted to. I wanted her apushing and abeating on me like that, and I wanted her fiercely and was beginning to wonder if I would come just from the slight crumple of the sheet under me that would lightly brush my clitoris in our mutual thrusting movements. Her hand slid down my hips, down to my fur, her finger reaching, and she went for me. She went for me with all her intensity and push and I was there but not carried away, not carried away to climax, and when I felt her extended and expended and she pulled back, rolled off and over, belly up to me, wide breasts falling, white globes in the candlelight, I didn't care that I hadn't come and that too was new. The stillness came in with her loving me when we were moving in unison. In our movement there was stillness at the center of the ride.
That evening everytime we kissed we were unable to pull away, and one kiss led to another or lasted itself a very long time. We danced and discoed and I got wet pants and could have come with just a little more of her
right then and there but for the interruptions: the closing of the door, the washing of parts that might be open to love licking later, the undressing, the pulling back of the blankets. Another type of stillness. A cutting off, our habit of stopping where we were to make the setting more perfect. Not the stillness at the center of motion, but the stillness that comes from a knife slice, a scissors cut, a snap. However and besides, we did move beyond that stop to our riding passion movement, and she rolled off now all white and bellyside up like a porpoise in play, a live animal exposed. I couldn't help it, I couldn't consider, I went for her breasts, sucking and pulling and getting a sweet sweat I wanted to drink and bullying and tossing them and rubbing them circular with my hard-pressed mouth all over her chest and attugging I went. She was the animal. I was the animal. We were deer lost in the woods to our fancy, and she'd be lost and I'd be lost and we gave ourselves permission together to be lost and nonjudgmental and to do what we wanted lost and most to find ourselves through our very lostness.
It happened everytime we made love. She, more and more aroused, would give a sign, a signal, a cry of surrender that would heighten my passion, my wanting her, my surrender to my own passions that would become more intense by the surrender until only her cry of come would stop me, and I too could roll belly over and up to her again.
No wonder this morning we needed our separate stills away from motion together, away from relationship, away from the comes and goes between us; we needed the stillness and quiet inside each one of us. To listen to that still lake, that still quiet lake of come of the self.
As the eye retains the image seen, I retain the image of the "other" during periods of absence in the mind's eye or memory to give the sense of continuity. Persistence of vision, the time that the eye retains the image just seen, varies among individuals. I may need to see my lover/other every day or have verbal connection with her to continue the sense of continuity. Someone else can have an ongoing sense of relationship from weekend meetings. Still others carry a still image in wallets or prop them on their desk in their workplace to recall a chain of images, a sense of flow and continuance in the present from this frozen image of the past.
The continuity of flow or motion we bring to a still photograph is not an illusion, since we experience the continuance rather than the short, jagged, cut-off events of the actual time/space spent with the loved one. If we fashion a continuance, a persistence of vision, subjectively then it exists; and as our regulating body organs continue and are the basic sense of life to us, so does the ongoing quality of relationship give us a sense of meaning. I saw her this morning; I hugged her and was stroked; she is gone and I won't see her for two days. But the sense of her is with me in the present from the visual and tactile memory I keep.
CONCEIVING WOMEN: Notes on the Logic of Feminism
by Joyce Trebilcot
Feminism is not just a matter of reordering what exists, of, say, moving women from one "place" to another; feminism involves, rather, changes in the very nature of things. In this paper I want first to explicate one reason why feminism requires ontological change and then to give some examples of ways in which these changes are taking place in our redefinings of women.
If we begin with the assumption that women should not be mutilated, violated, locked up, exploited, restricted-dominated, because we are women, we have the basis for feminism in a narrow sense, a feminism that would concern itself only with the mistreatments of women which arise out of perceptions of us as female. But to be a feminist is to care about women, and it is arbitrary to limit our concern to just those aspects of our sufferings and limitations which arise from a particular cause; so feminism, it seems to me, must be concerned with all of the harm done to women, regardless of its source. This means that because women are oppressed not only because we are women but also because we are Black or Hispanic or Jewish, because we are Lesbian, because we are poor or handicapped or fat or young or old or ignorant or (fill in the blanks), we must, as feminists, seek to identify, understand, and eliminate all these oppressions.
They all have something in common. Their form is that a distinction is made, which is a division into two; and one part or half is held to be superior to the other; and it is claimed that the "superior" is justified in having power over the "inferior." This, of course, is dualism. It is a dualism which is essentially evaluative and which functions primarily as an excuse for power over.
The dualistic pair is the unit of hierarchy—that is, a hierarchy consists of overlapping dualistic pairs. A classical rendering is the pyramid with God at the apex, then angels, then man, then beasts, then the rest of the natural world. On this scheme, God is distinct from and better than man, and entitled to exercise control over him; man is distinct from and better than beasts, and entitled to exercise control over them; etc. The pyramid, of course, represents quantity: one on top, then a few, then more and more. While this particular version of the pyramid is perhaps antiquated, the form itself permeates dominant Western cultures.
It permeates received reality. At the epistemological level, it determines perception: we see—sight rather than, for example, touch, is primary—it is objects that are seen, and they are seen as having sharp boundaries. More obviously, dualism/hierarchy is the form of social organizations—governments, businesses, schools, families. It structures the person—self/other, mind/body, conscious and unconscious. There is even a dualism of right and left—the right hand, the right side, take precedence over the left. And so on.
In feminism, there is movement toward the elimination of all dualism, not just of those manifestations of it which oppress women as women or as members of other groups. One reason for this movement is the belief that only by getting rid of dualism in all of its manifestations can oppressions such as sexism, racism, and classism be permanently and thoroughly eradicated. Another reason is the sense that dualism as a form is discordant with women's values: even if no "inferior" category included women, dualism would block the flowering of women's modes.
The elimination of dualism—or either a specific manifestation of it or all of it—requires change in the nature of entities which are related to one another in dualistic/hierarchical orderings. This is because the nature of the entities is determined at least in part by their participation in the orderings. In academic terms, the relations among them are internal rather than external. An example of an external relation is the distance between two cups on a table; a change in the distance does not alter the nature of the cups. A change in an internal relation, however, does alter the nature of the things related. A nurse, for instance, cares for the sick by assisting and following the orders of doctors; if we eliminate her subordinate relationship to doctors, we change the meaning of the word "nurse." Similarly, what it is to be a worker, an Indian, a wife—or, of course, a woman—is determined in part by relations between these and other concepts in hierarchical orderings. Because the relationships among items in dualistic/hierarchical orderings are internal, then, feminism, insofar as it is committed to the elimination of dualism/hierarchy, is committed to changing the nature of things.
Here I want to give examples of just some of the ways in which internal connections among items in hierarchies are broken by feminist reconceivings. I focus on reconceivings of women because they are starting points and centers of feminist theorizings. These reconceivings are, I take it, political strategies which weave bridges from patriarchy to women's spaces.
I use the distinction from academic philosophy between the descriptive and evaluative meanings of a term. The descriptive meaning is characterized as value neutral; it is supposed to imply only some facts about the thing in question. The evaluative meaning commends or condemns the facts described. To call someone forceful, for example, is to ascribe a certain style of behavior; this is the descriptive meaning of "forceful." But in addition, in most contexts, to say that someone is forceful commends if the subject is male and condemns if she is female; commending or condemning is the evaluative meaning of "forceful."
Now because we engage in feminist theorizings in order to re-experience, to reconceive, that is, to bring forth new meanings, when we use a conventional word as central in our theorizings, the descriptive meaning of that word shifts. Sometimes the evaluative meaning shifts as well. Here I discuss three patterns of shift in evaluative and descriptive meaning. The words used to exemplify these shifts are "strong," "nurturant," and "lesbian." As these words, which are used by feminists to describe women, are given new meanings, the meaning of "woman" changes too—and the conceptual bonds which hold us in our traditional places in dualistic/hierarchical orderings are frayed.
1. **Descriptive meaning changes, evaluative meaning remains the same: Strength.** In patriarchy, a strong man is one who deals with difficulty calmly, quietly, unobtrusively. According to the cliché, he is the strong silent type—paradigmatically, the John Wayne character.
The patriarchal strong woman, like the strong man, handles or endures difficulties quietly, without disturbing others. But in other respects she differs from him. In particular, in accordance with the patriarchal practice of defining women in terms of our relationships to men and of our sexuality, the strong woman is typically one who both lacks a heterosexual partner and is thought to be sexually unattractive to men. She has no husband or mate (or, if she has, he is absent from the situation in which she is called strong) because if an appropriate male is present it is presumed that he takes care of and protects her, not that she does this for herself, and so the term "strong" is reserved for him. But even if she has no man, she can't be strong unless she is heterosexually unattractive—usually she is imaged as old—because if she is attractive she should or might have a man, and again the term "strong" is reserved, this time not for an actual but for a possible man. In patriarchy, then, we generally must be both unattached to a man and unattractive to men in order to qualify for strength.
The feminist concept of the strong woman preserves the notion of strength as dealing well with adversity—this is the common thread by virtue of which the same term, "strength," is used in both contexts. But of course the heterosexist limitations of women's strength drop out. A feminist strong woman may have a man—in many cases her strength is manifested primarily in her
struggle against a husband or lover. And she need not be unattractive to men—we know that there are many young and conventionally beautiful women who are strong indeed.
Further, in feminism the idea of strength as quietly, privately, unobtrusively dealing with difficulty drops out. The feminist strong woman is likely to be noisy, even loud; she is inclined to protest, to complain, to call attention to her difficulty. This difference of course is based on the political difference between patriarchy’s interest in preserving present systems and feminism’s interest in changing them.
Thus, while the evaluative meaning of the expression “a strong woman” is the same in patriarchy and in feminism—it is positive in both contexts—the descriptive meaning shifts: what is valued changes. This change is the basis for a partial redefinition of the concept of woman. Whereas in patriarchy, strength in women is an anomaly, in feminism there is a tendency to understand it as essential, as an element in the definition of woman: all women are potentially strong. But this move breaks one link in the internal connection between the concepts of woman and man. In patriarchy, women are weak as compared to men; therefore women are inferior; therefore women may justifiably be dominated by men. But if women are by nature strong, the syllogism fails.
2. Descriptive meaning changes, evaluative meaning changes: Nurturance.
The term “nurturance,” whose root idea, of course, is that of a mother nursing her child, is being given both new descriptive and new evaluative meaning by feminists. Barbara Love and Elizabeth Shanklin, in their article, “The Answer is Matriarchy,” provide an account of a feminist definition of nurturance which clearly differs from patriarchal ones. To nurture a child, they say, is to support “the unique will of the child to grow into its full potential as a self-regulating individual”; again, to nurture is to strengthen “the unique will of each individual to form open, trusting, creative bonds with others” (184).
This concept of nurturing retains the connection with the mother-child relationships, but otherwise contrasts sharply with patriarchal views of nurturance. In patriarchy, the aim of childrearing is not to strengthen the child’s will, but to direct and control it, to dominate it. In patriarchy, the aim of the “training” of children is not to enable them to think for themselves, but rather to prepare them to take their places in existing institutions. Barbara and Elizabeth note that “in capitalism the child’s will is directed toward serving the interests of corporations; in socialism it is directed toward serving the state” (184). Their concept of nurturing as supporting the unique will of each individual to develop as self-regulating and self-realizing constitutes, then, a clear shift in the descriptive meaning of “nurturance.”
Evaluatively, nurturance is viewed positively in patriarchy, but as second-rate. Nurturing is a good thing for women to do, but not good enough for men. In matriarchal theory, however, the value of nurturing is expanded. The nurturant mother-child relationship is to serve as the model of all relationships, and all social institutions are to be designed so as to support nurturing. Here, nurturing, a women’s value, becomes the primary value.
But from a feminist perspective it is probably a mistake to say that matriarchal theories make nurturing primary, for feminists tend not to be concerned to order values hierarchically. We may say, then, that what matriarchal theory does is to horizontally expand the range of nurturance. Traditionally, only women are supposed to be nurturant, we are expected to nurture men and children but not one another or ourselves, and nurturing is supposed to take place only privately, in the home. But a matriarchal society is one in which nurturing is valued for everyone, in all contexts. Thus, matriarchal theory not only gives nurturing a new descriptive meaning, it also radically expands its sphere as a value. And if nurturing is not second-rate, then women are not in this respect inferior to men, and one of the struts holding up the patriarchal hierarchy is toppled.
3. Descriptive meaning changes, evaluative meaning is reversed: Lesbian. Another way in which patriarchal evaluative meanings are altered in feminism is by simply reversing them. The patriarchal intention to keep us in line by calling us dykes or bitches or hags is blocked when we describe ourselves in these terms with pride and pleasure. We break out of secondary status, out of hierarchy, by flipping their evaluations over, by gladly acknowledging that we are what they condemn.
This reversal of value is based on a reconceptualizing of the descriptive meaning of the term in question. While patriarchal concepts of lesbianism focus on women "having sex" with women—and on men (lesbians are women who can't get men, who need men, who hate men, etc.)—feminist conceivings retain only the emphasis on women, and transform it. Here are some samples. From the Radicalesbians: "A lesbian is the rage of all women condensed to the point of explosion."22 From Ti-Grace Atkinson: Lesbianism is the "commitment, by choice, full-time, of one woman to others of her class."23 From Sally Miller Gearhart: "A lesbian is a woman who seeks her own self-nurturance."24 Such ways of understanding lesbianism depart from the patriarchal concept in description and reverse it in evaluation. And they not only move lesbians out of our patriarchal places as inferior to heterosexuals and to men, they also have implications for the concept of woman: if some women—lesbians—are not dependent on and inferior to men, then no women need be.
III
I have so far talked about ways feminists recreate ourselves by giving new meanings to words which are used in both patriarchy and feminism to describe women. But not everything we need to say about ourselves can be expressed in this way. So feminists also describe women in terms not conventionally used to describe women at all. I have chosen three examples here. One is "together," an adverb in standard English, an adjective in slang, and a feminist adjective in the work of Inez Smith Reid. A different sort of example is a new combination of ordinary words: "woman-identified." The third case is a materially new word, a word formed by combining parts of standard words—"gynergy." These are terms we have appropriated or invented for ourselves. One might say that, unlike the words I discussed earlier, they are not old words given new meanings, but rather new words; they are, anyway, new descriptions of women. So to understand them as contributing to feminist conceivings of ourselves is not to trace shifts in their meanings from patriarchy
to feminism—"gynergy," for example, has no meaning in patriarchy at all; it is, rather, just to understand why we need these new words, what they mean.
"Together" is used by Inez Smith Reid in her book "Together" Black Women to describe politically conscious Black women. Many of these women do not define themselves as feminists, but in their struggle against racism as a hierarchy of power they are clearly sisters of feminists. Inez's account of how she came to use the word "together" in thinking about these women helps to illuminate one sort of politically-based conceptual shift.
Inez began her project with the intention of studying militant Black women and so she selected her subjects on the basis of their "reputation in the community for militancy." One of the questions the women were asked was to define militancy. Among the responses: "To be militant you have to be aware . . ."; "Militancy is when you can actually analyze . . ."; "a militant person is, for Black people, anybody who decides it's time for White folks to stop kicking my ass. It's just anybody who's tired of being messed over" (17, 18).
But most importantly, many of the responses carry a sense of dissatisfaction with the term "militant" itself. This sense is perhaps best expressed by the woman who says: "I really feel it's just a name that the White people gave to the Black people. They call them that name because they speak the right things for the race . . . I don't think there's such a word as that" (20). There is no such word as that. This woman not merely refuses to accept a characterization imposed on her by the dominant group, she denies the very existence of the word.
As Inez listened to the women she was studying and began to appreciate their tendency to reject the standard concept of militancy, she herself moved away from this notion and came to think of these women not as militant, but as "together." She cites the definition of "together" from the Dictionary of Afro-American Slang: "to have one's mind free of confusion; to be positive; functional; to emerge as a whole person." Inez adds that in her work, "together" has "a more collective connotation," it is "characterized by a spiritual closeness in a common endeavor—that of a singular or peculiar commitment to erase oppression." Further, the term "denotes a refusal to take on, uncritically, the total value structure of the White community" (29). So here we have the discovery of the negative evaluative meaning of a term and its replacement with a new word, that is, with a word which in the standard language of the dominant culture is not used to describe persons at all.
The description of some women as "together" has, of course, implications for the concept of woman. Inez Smith Reid's work suggests that all women can be "together"—that is, free of confusion, whole, and sharing commitment; insofar as this conception of women is inconsistent with patriarchal ideas of women as scattered, incomplete, and at odds with one another, it tends toward transforming the concept of woman and so breaking the bonds which hold us in our traditional places in hierarchical orderings.
Another approach to reconceiving ourselves is to make up terms. The expression "woman-identified woman," introduced in the Radicalesbians' 1970 paper, names a woman who creates her identity in relation to women rather than in relation to men and who makes women her primary commitment:
"...we must be available and supportive to one another, give our commitment and our love, give the emotional support necessary to sustain this movement. Our energies must flow toward our sisters, not backward toward our oppressors" (215). The paper provides a new, positive description of the lesbian feminist, along with a new name for her: woman-identified.
The meaning of the expression "woman-identified woman" has shifted somewhat within feminism. The expression has been used widely since its introduction, with the understanding that while not all lesbians are woman-identified, all women-identified women are perceived by patriarchy as lesbians. But then at the 1978 founding convention of the National Lesbian Feminist Organization, a resolution was adopted which specified that membership in the new organization is open to "all lesbians and/or woman-identified women who agree with the purposes of this organization." "Woman-identified woman" was included partly to make space for lesbians who cannot publicly say they are lesbians. But it was understood also that this provision would admit to membership women who in patriarchal terms are not lesbians.
So the concept of the woman-identified woman has taken on a life of its own. Arising out of the rejection of the male practice of defining women in narrowly sexual terms, and specifically out of the rejection of the narrowly sexual patriarchal definition of lesbianism, the new concept at first simply added to the sexual: a woman-identified woman was a lesbian who lived primarily with and for women. But then the term was used in such a way that the sexual criterion dropped out entirely. Thus our reconceivings of women change.
Another new word invented by feminists is "gynergy." Like "woman-identified woman," it has become a regular part of the vocabulary of many feminists. (The women I know pronounce it with a soft "g.") Janice Raymond describes gynergy as "the woman power/spirit/strength that is building up in 'woman identified women'" and as both individual and social: "...it proceeds not only from an individual woman's realization of her own power of being but from a collective consciousness, i.e., a feminist collective consciousness."
Unlike the other concepts discussed here, gynergy is not an attribute of individual women: we do not speak of a "gynergetic woman." Gynergy is something we as individuals feel ourselves participating in. I can think of no patriarchal concept which works in the same way—that is, which expresses something personal (not merely an atmosphere) which is at the same time not an attribute of persons. It may happen, of course, that we will come to say things like "Sybil has great gynergy." Then the term "gynergy" would be rather like "spirit." But for now, it appears that gynergy is not only a new concept, but a new kind of concept. And it too is part of our redefinings of "woman."
IV
These are just some of the ways in which we are reconceiving ourselves: taking on conventionally positive characteristics like strength, and changing them; redescribing and reevaluating aspects of ourselves like nurturance and lesbianism; and using words not conventionally used to describe women to express our becoming. These shifts all break internal definitional connections
which exist in patriarchal systems between concepts of women and other concepts, particularly between concepts of women and of men. By breaking away from definitional connections which determine women's place in patriarchal conceptual systems and so in "the world," we move out of traditional dualistic/hierarchical orderings.
From a patriarchal perspective, all the concepts of women discussed here would be said to carry positive evaluative force, or to establish new ideals for women. But this account misrepresents the spirit in which these ideas are set forth. It is men who have invented the concepts of good and bad and for whom the making of value judgments is a major occupation. Feminists make vigorous value judgments about the patriarchy, but in our own worlds our concern tends to be more one of understanding and making space for processes than of evaluating persons and acts. Feminists are highly sensitive to the needs of women to create ourselves out of our own experiences, to our needs not to be told what we should do or be. The concepts of women I have discussed here then are not ideals or models, but gifts from some women to others, to be modified, transformed, abandoned, as each woman feels.
These conceivings of women are exercises of power: the power of naming ourselves, and the power in our new names. This is not different from "real" power. Although I have written here of words and concepts, the shifts I have sketched are also ontological; they are shifts from being women in patriarchy to becoming women in our own times and spaces. What patriarchy might see as logic, feminism understands as political strategy.
NOTES
1. In Our Right to Love: A Lesbian Resource Book edited by Ginny Vida (Englewood Cliffs, N.J.: Prentice-Hall, 1978).
2. "The Woman Identified Woman" in Radical Feminism edited by Ellen Levine and Anita Rapone (New York: Quadrangle/The New York Times Book Co., 1973), p. 240.
3. Amazon Odyssey (New York: Links Books, 1974), p. 132.
4. "The Spiritual Dimension: Death and Resurrection of a Hallelujah Dyke" in Our Right to Love, p. 187.
5. Together Black Women (New York: Emerson Hall Publishers, 1972).
6. "The Illusion of Androgyny" in Quest: A Feminist Quarterly, Vol. II, No. 1, Summer 1975, pp. 64, 65.
Dear Women:
The following paragraph is taken from an untitled essay written by Martha Yates, which appeared in Sinister Wisdom 8:
Recently in Boston women organized "Women take back the night." Along the way, there were small groups of men, each man with his hands around a thin candle, about seven inches high, lit, which he held slightly below his waist. It seems they think they must still bring us their light (so that we can see in the dark), and women are still letting them in so many different ways: semen, the seed of enlightenment, their word...”
I am outraged by Ms. Yates' paragraph, which I have quoted here, in full. I was present at this "Women Take Back the Night" March and I would like to comment on Ms. Yates' remarks and the men she has mentioned.
Men were urged not to participate in the march itself, which occurred on August 26, 1978. However, they were encouraged to attend the rally that followed afterwards. In my article "The History of Take Back the Night," which appeared in the Gay Community News, Vol. 6, No. 6, August 26, 1978, I briefly commented on the proposed route of the Boston march. Here is a terse quote from my article: "The march will proceed through a section of the Fens, dimly-lit tree-shrouded parks which, ironically enough, have been the site of many attacks on gay men."
Now, Boston is a city known for its violence that stems from racism. What is lesser known is the fact that Boston also has a rampant brand of homophobic violence. In the years that I have been out as a lesbian, I have been bombarded with (true) horror stories of gay men being beaten, stabbed, gassed and strangled. Gay men know all too well the brunt of violence.
Mid-way through the Boston "Take Back the Night" march, my eyes beheld a sight that was so wondrously beautiful, I almost wept. The march was approaching the Symphony subway stop. There, perched and standing near the subway entrance, was a group of men holding single white candles of support. Propped against a wall was a placard that signified them as: "Men Against Violence Against Women." (For documentation of this scene, may I refer you to the actual photograph of these men from this event which appears on the cover of the Boston-produced "Walls to Roses" album.)
These men were faggots from the Gay Community News (GCN)—gay men that I knew personally. (I had no idea they planned to show up during the march.) They stood in silent support, smiling and waving at familiar faces among the thousands of women that marched past them. I was so proud that immediately upon seeing them, I shouted, "Those men are from GCN!" Then I cheered and applauded them. The women marchers around me responded with warmth and appreciation. They understood the solidarity of the faggot presence—no women were offended that these gay men stood on the sidelines.
I am quite angry with Ms. Yates for taking this beautifully memorable incident (omitting some very serious facts) and twisting it to apply to her
vague, poorly-written essay. (Another group of men and women, standing further along the route holding candles, were campaign workers for Rep. Mel King, a black man who is currently running for Mayor of Boston.) Furthermore, I am amused by Ms. Yates' phallic paranoia. The faggots (as well as the campaigners) who held candles did so as a sign of support—if there is any symbolism in this, it is the (genuinely concerned) warmth of the flame, and not an oppressive, male-oriented "light" as Ms. Yates suggests. If Ms. Yates objects to the actual phallic shape of candles, I suggest that she vent her anger at the people who manufacture candles and not the men who hold them.
I am furious that Ms. Yates misconstrued this incident to fit her rambling prose. If Ms. Yates honestly perceived the incident as she reported it (threatening, rather than supportive), then I am very sorry that Ms. Yates is incapable of identifying true allies when she sees them. For all the lesbians who read your periodical, particularly those who have political faggot allies, I hope you will publish this letter in its entirety. I will also deliver a copy of this letter to the editors of the Gay Community News. Thank you for your attention.
—Pat M. Kuras
Martha Yates replies:
In the footnote that Pat Kuras refers to I meant to convey a quick, not particularly solemn impression. I was working on an essay uncovering the ideology of light and sight which runs through Western and some Eastern philosophical and religious traditions. Knowing how the physical and philosophical properties of light/sight can disguise a very patriarchal and oppressive consciousness and, then, at the same time, encountering men holding up their candles at the "Women Take Back the Night" march made for a very ironic and ludicrous sight. I was amused—I still am.
The part of Pat Kuras' letter which interested me was her description of men as allies. This implies shared fears, shared risks, a shared and common cause. What, really, is common between men and women? In the light, in the daytime, there seem to be mutualities. For instance, men and women seem to share a so-called "human body." In the dark, on city streets, women have found out how false this is. The "human body" is an illusion, ideological at its roots.
I also doubt whether women and gay men share the same attackers or their attackers the same motives. My feeling is that those who are trying to move beyond what is given find that they do not genuinely share anything with men, anything, that is, which is critical and at the edge of a radical understanding.
The word alien is more descriptive of men. No one should be tricked into thinking that all of us, so-called "members of the human species," come from the same place. Aliens can be dependent upon one another and can even converse easily with each other, as James Triptree, the science fiction author, has suggested in her short story "The Haploid Heart."
Of course, I didn't write all of this in the footnote. For it was just that—a footnote to a complex and difficult subject, the meeting with an abyss in language, an abyss within thought itself.
LISTEN
i have listened
as a slave listens
leaning my ear into the wind
as if the world could be a seashell
and my ear the only one
to hear the roar of the ocean
and what it insists on telling
i have held lives to my ear
and taken their hollow sounds inside me
and made of myself a fluted hollow sound
and i have sung these songs back to the world
if you saw me with my ear to the rail
hands clawing the dirt
and i told you the train of your life
was coming you would have believed me
packed your bags and left
forgetting to wave good-bye
i am trying to tell you something
can you hear me?
or is my voice like the sound of traffic
sage and steady a murmuring a lullaby
to drift off to sleep by
listen:
i want you now to open
to be open to me as an ear is open
to open like the womb opens
when it wants to surround life
i want you to listen to hear me
as if i too were a crucial note
you must invent
-Martha Courtot
The following letter is in response to two articles in Sinister Wisdom 9: Bertha Harris's "Melancholia, and Why It Feels Good" and Irena Klepfisz's "Lesbian Literature and Criticism."
Dear Sinister Wisdom,
Someone ought to step between Irena Klepfisz and Bertha Harris. Bertha is being outrageous, as usual, and she rather deserves the Response, but Bertha is—in her indirect, dramatized way—right too, especially in one throwaway line: "the onerous inhibitions lesbian-feminist politics seek to place on the writer of genuine talent."
Bertha Harris is the author of a very fine book, the best Lesbian novel I've ever read and possibly the best novel of the last thirty years, Lover. Lover has been mostly ignored in the women's press and when it hasn't, it's been called Politically Incorrect (to my knowledge) though a Feminist Review of Books reviewer recently rediscovered it.
Why?
Most artistic and literary criticism in the women's press is very bad. It reacts to having its P.C. buttons pushed. Much of it is practiced by refugees from the misuse of the high culture tradition in high schools and colleges to bully and stupidify the young—this is largely class warfare, owing most of its virulence to the teachers' own insecure class position and their defensiveness about it, teaching having become (since high schools and colleges lost their elite character some time in this century) a road to upward mobility for children of the lower middle class. There is also the problem of the compensatory Instant Junk Food commercial culture which pretends to be a popular alternative to the poisoned (and often poisonous) high culture, and the consequent false split between "art" and "entertainment." And of course the priggishness of certain revolutionaries who really wish to escape from individual personality, individual voice, idiosyncrasy, and any interpretation of life that demands all three. Women (as Phyllis Chesler once said) have a real terror of difference.
What Bertha is trying to defend, in her exasperated, flamboyantly offensive, Southern Gothic fashion is (I think) her right to her own artistic obsessions and her own sense of fantasy—that is, she's defending in a deliberately nasty way (because attacked and exasperated) what every artist must defend: the absolutely inescapable, crucial fact that expression is logically and chronologically absolutely prior to analysis.
Bertha Harris has given much of her life to feminist publishing, has finally cut her ties to a self-sacrificing job in women's studies which was extremely harassing and energy-draining, and has been either ignored or belittled by the very women's community she's been trying so hard to serve, and has had to watch the stupidest sort of mediocrity praised far above her work. (I'm not talking about Irena Klepfisz.) I'm not surprised that she exploded—and knowing Bertha, that she did so elliptically, cryptically, and as angrily as possible.
Every few weeks someone sends me some incredibly clunky artifact of women's culture which falls helplessly on my desk and instantly expires of
sheer unworthiness to live. In between the lousy poems and the excruciatingly dull fairy tales, some singer who can’t stay on key and whose untrained voice can’t sustain a note for more than two seconds arises and proclaims: *Wooh-mun/Will be far-ree-hee-hee!* wrenching her diction, choosing the wrong vowels for her words, letting air escape uncontrollably past her glottis, and radiantly telling interviewers she is “self-taught” and therefore innocent of sexism. This isn’t a new, woman’s style, it’s not a blues style, it’s not any style. It’s simply incompetent.
Incompetence—praised incompetence, revered incompetence—has (I suspect) driven Bertha Harris mad.
It drives me mad.
Do I want therefore to suppress such efforts? Not at all. They’re absolutely necessary. (Irena Klepfisz is not talking from the point of view of incompetence, of course. She’s talking as someone who’s absorbed everything The Boys teach as “art” and is busy throwing much of it out, in an extremely sophisticated and analytical fashion.)
But The Boys are still, by and large, custodians of the most technically rich and adroit literary culture that exists in English, even though it isn’t the only literary culture (they pretend it is) and it has no monopoly on all the other virtues. (They pretend it does.)
Books aren’t bad because they include long words and subtle literary devices. (Surely a classless world will include education in all literary traditions, even the one now called “high culture.”) They also aren’t—quite obviously—bad because they don’t.
I’m speaking from Harris’s corner and to Klepfisz because the latter is being rationally persuasive, I agree with her, and Bertha, instead of arguing, simply went Stomp Smash Crash Blonk and walked out.
We must try to exercise some sense of where people are coming from and what they really mean instead of fastening on each individual statement and walloping it. The latter leads to nothing but aggravated tempers, hurt feelings, and increased insecurity for everyone. I’m not suggesting a meaningless “toleration”—only that language is a very imprecise medium and that 85% of most statements are in a code that really means *I want, I need, I feel, my situation is*... To consider these as positive, precise statements of a considered political position is male-style linear thinking. And how much more politically incorrect than that can you get?
Different needs aren’t betrayals. Incompletenesses (Mary Daly’s lack of awareness of economics, Ellen Moers’ white solipsism) are not deliberate betrayals, either. Though if we handle them that way long enough, we may produce plenty of real betrayals. And a large lack of women left to demonstrate any kind of solidarity with.
Bertha, come home. We love you. A dozen long-stemmed American Beauty roses await you. The intelligent, rationally persuasive, brilliantly talented Irena Klepfisz is favorably reviewing *Lover*. The entire membership of The Oppressed Lesbian Mothers’ Grim Denim Bikeathlon and Deprivation Society has donned elegant riding habits and mounted chestnut mares and is exquisitely dashing about the bois, calling your romantic name. Come home!
All is forgiven.
Joanna Russ
The two following responses are portions of letters that Ann Allen Shockley received after a short story of hers appeared in SW 8 (Spring 1979). That story, "A Meeting of the Sapphic Daughters," is an account of Lettie and Patrice's reactions to the behavior of white lesbians at a meeting in which they are the only black lesbians present. With the permission of all concerned, we're printing these letters—which were not written for publication—as a first step toward sharing information in SW about how racism manifests itself among lesbians—what it looks like, what it feels like, where it comes from, how to stop it.
1. "Patrice applied lipstick..." My first reaction was that in 1979 you won't find any Lesbian caught dead wearing makeup. Then I remembered a new bar that Cheryl and I went to recently. I thought I had stepped backward in time. It was mainly black and Latina and strictly butch/femme, complete with superfly outfits, sheer disco dresses, and the makeup.
Another thought: in 1970 at my first and only DOB meeting, I deliberately wore a dress to let any prospects know that I wasn't butch. Even then I rejected the role-playing bit; but, forced to make a choice, the "butchy" woman was more attractive to me than the simpering, dependent female. At that time only feminists were in combat boots and lumber jackets, and they were all white and straight.
2. The location of the meeting is perfect! My first contact with DOB was in New York: a dance in a loft, in the warehouse district. Your description is absolutely photographic.
3. "... large husky woman with hostile face..." Yes, I still see that face, distorted with the distaste of a racist. I used to see it in all the bars and the streets of Provincetown. I see it in bars now, and yes at Mountain Moving, though rarely. I'd long ago stopped allowing that face to get to me. In the past, B.C. (Before Cheryl), it was affirmation of my fears that a particular place among Lesbians would not offer me a source of finding a woman—that place would therefore become even more inhospitable than straight places, where my mind was at ease with the acceptance of there being no possibility.
4. Patrice and Lettie were lucky: the DOBers in Boston wouldn't even look at me ("...gazing at them from behind cold masks..."). That's what I remember most—the collective consent not to recognize my presence, not to acknowledge my existence. And I, like Patrice, still stupid enough to think that my non-negro, college-educated accent would make them welcome me. Ann, I never did find out how you became aware of this phenomenon. Did you experience this personally?
5. Oh yes, those dreadful potluck suppers. How I hated them. One or two were plenty for me. Heavy, starchy, tasteless food of questionable quality and so-called Lesbians everywhere but all unapproachable.
6. Trollope's "mannish appearance." This is the image I would have had of the type of Lesbian you describe but in terms I would not let myself use to describe her. What I'm expressing is an ambiguity/conflict within myself:
a refusal to accept the definition of a Lesbian as an imitation man; along with a refusal to adopt the Lesbian costume of the 50s or the once-feminist-now-Lesbian/feminist-and-even-ordinary-population garb for wemyn of the 70s (simply because I reject the idea of a uniform and have always hated to dress to prove a point and have always hated anything that smacks of playacting or pretense); along with an uneasiness about dressing in a “feminine” manner that might indicate a fear of being identified as a Dyke, which I certainly do not have (I really do dress just from what appeals to my eye, then what’s comfortable—only); along with always liking to see Lesbians in “Dyke garb”—it having great sensual appeal to me, probably stemming from it being a mark of identification. This conflict was demonstrated recently on the Donahue show when a Lesbian complained of society’s bigoted stereotyping, and Donahue asked why then did she wear a man’s shirt, tie, and vest. His question silenced her; and a response came from a faggot whose feeble retort was, “When Diane Keaton wears the same outfit, it’s called fashionable.”
I’ve asked myself the same question: If you reject prickness and prickdom, why adopt the clothes/characteristics of pricks? I personally feel that I dress in a way that rejects the male ideal and standards set for woman’s dress by my not adopting the dress of prickdom, which to me is the dress of whores, stripteasers, and men in drag. I wear no heels, but I also wear no combat boots. Anyway, I think I just answered the question recently. The description of the mannish dress—compared to what? Compared to prick definitions of what is male and female, with separate sets of everything according to his standards. Well, why not take a vest and a tie and short hair and call it not mannish but Lesbian? Man has set the standards, created all the definitions, established all the opinions. My answer involved my right (our right as wemyn) to set our own standards, make our own definitions, etc. If we want to wear a tie and say it’s proper to us, then “abracadabra” it’s proper to us. I understand now how artificial and manufactured are definitions of masculinity and femininity and how we are unwillingly and unconsciously caught up in them. At one time I could only call a Lesbian such as Trollope “mannish.” I had no other image or words, but I always felt guilty about it. Now I know there’s no such thing as “mannish” or “effeminate” except for the pricks who use those terms as a means of controlling thought and social development. So, that expression of the “mannish” Dyke is offensive to me because I hate to associate anything of man with woman (including his designation of himself), and I reject the idea that the “Dyke garb” is mannish because there is no such thing as mannish. There’s no such thing as male vs. female characteristics, but only human characteristics. An aggressive woman is not mannish but strong, determined, independent. A Dyke in a tie is not wearing man’s clothes—just clothes. In other words I am choosing to make my own definitions, which have got to be better than his. I become the authority, the “bearer of standards.” I’ll set the tone, and he can accept or go to hell—it’s immaterial. (This too is my reason for rejecting the spellings woman/women.) Thus had I written that description, I would have left out the word “mannish.” Also in that sentence is an indication that her voice was that of a woman despite her clothes being that of a man. Again, I’ve come to see this as meaning that a woman’s voice should be of a certain quality only or else she’s mannish, while the opposite would be true for the “effeminate” man—and I reject these definitions. It’s
also saying that a woman is less a woman to the degree that she's aggressive, bold, determined, strong, assured, etc. I do not see a laughable contradiction in a woman who wears what someone called "Dyke schlep" but may have a high-pitched voice—this is because I do not accept prickdom's definitions of what is proper to man and not to woman.
7. May I say that I find this story as something I certainly could have dictated just as you have written it, at a time B.C. Then I was so hungry and so vulnerable, I rejoiced to find myself among Lesbians only to find myself attacked because I was black. This story expresses all the rage and venom I then felt, and still remember, as I looked at their racism and then in vengenace proceeded to condemn everything about those damned white dykes—their appearance, manner, speech, philosophy, politics, ideas, books, togetherness, emotion—all I saw. Like jesus in the temple, this story rises in an all-consuming anger, sweeping the tables clean, scattering their symbols, rejecting all, ridiculing all. Being kept from having any part of it I would destroy it all. I say this because now, from a secure position, I am sometimes able to look upon such a scene with feelings of sharing and love. Thus at this point in time my first reaction to your story was: "But she is ridiculing and deriding." But an instant later I took a different and maybe unique reaction, a reaction I don't know whether or not you had any intention of drawing. I find validity in the story, including your terms and characterizations, with one purpose in mind—a chance to get some "gitback." A chance for me to use these written, tangible lines of derision and ridicule to fight back, to get even, to take delight in punishing "them" for what I've suffered from them. In other words the story for me is a cussing at them, a chance to rant and rave at all those white dykes, past and present and future who have looked at me with racist eyes. It's saying, "This is what I think of you, and if it hurts your feelings, good! This is what I want to do—hurt you back." I can laugh at racism from the white man; it is negligible, no matter the genocide, compared to what I feel in the face of racism from Latinos, Native Americans, Lesbians—all those who should know better. So I say more power to you, Ann, for giving me, through your words, my chance at a delayed scream, a running amok at a DOB meeting, potluck, bar, etc.—even though I doubt you wrote toward this end. Everything communicated by one means something different to each hearer.
8. I still have problems with "Lesbian community," too. I still feel that the concept is a bunch of bull, just as I feel the concept of black community is a bunch of bull. And I have no life to waste addressing what is to me pie-in-the-sky nonsense. Cheryl and I together, plus whatever wemyn we can from time to time spend some time with, are plenty community for me.
9. Movements of resistance always drop off the edge of the opposite extreme, and those movers always bounce back toward the middle in later years with egg on their faces, looking stupid. I refuse to let myself go so far out on a tangent that I can't get back on the road with my dignity still on straight. I will not jump on anyone's bandwagon.
10. Your depiction of J. L. is all too accurate. The first bar I went to was at the invitation of some wemyn I met at a DOB softball game. It took a phenomenal amount of courage to finally ask one to dance—oh the distaste
and revulsion in her eyes as she said, “Let’s wait for a fast one.” Conversation, or rather her notification, during the “dance” was an unsolicited, “Not too many black lesbians come here. I don’t know how to advise you where to find some.” Translation: get this straight, nigger, none of us wants the likes of you. Some poetic justice a year or two later. I ran into this woman, same bar, on a date with a woman I had gone to bed with and who just days prior to that I had refused to see. In effect she wound up with my cast-offs.
11. Unfortunately, you will undoubtedly pick up a lot of denials from white Lesbians; a lot of “But it’s not like that anymore”; and a lot of zeroing in on criticism of details, like I started with “mannish”; and a lot of “But the time is here for solidarity,” etc. However, I’d be surprised if your reaction from black Lesbians is not one of knowing head shakes and gratitude. There’ll also be criticism (and I can see this point, except that I have no solutions I really want to offer—I mean, Lesbians or no, if a racist, I’d rather see them dead than reformed) saying that the story gave no message in terms of how to approach it or what to do about it: no hope on the part of Patrice and Lettie that things would change, but rather a retreat toward a wish for a black Lesbian community (witness notices of such in the literature, and I just found out about all-black Lesbian bars in D.C. that refuse admittance to white wemyn). But again, for me, it is complete as a simple and accurate statement of what for me personally has been a devastating reality, and I really don’t give a damn as to whether or not anything is done about it. It does not resolve itself in the story because it isn’t resolved now—and won’t be. That’s the way it is, and I offer only condemnation, not solution. Your story is purely an “In case you didn’t know—this is what goes on.” My final reaction to “Sapphic Daughters” is very strong, but that is my nature. This is precisely why I don’t jump on bandwagons—I tend to be dictatorial and fanatic, and I might find myself being just stupid enough to die for something or other. So I keep on a tight rein. I’ve taken note of the pathetic Eldridge Cleaver, the silenced Angela Davis, the pimpin’ Huey Newton, and the mousy Andrew Young—I will not follow them. I’ll just stay on the outside and grin a lot.
—Eleanor
Dear Ann,
...Your short story in Sinister Wisdom was, of course, excellent writing, but more than that, I finished reading it full of anger and annoyance. Memories of 1960’s Daughters of Bilitis meetings in San Francisco came back to haunt me—none of them so hostile or so blatantly open in their racism, but surely uncomfortable for any black, chicana, or Chinese lesbians who came to the meetings or dances. I remember wondering why they never came back to a second meeting, the constant questioning by the group, “How can we make our organization more attractive to third world women?” No one ever thought to ask the women themselves about what we could do to make them want to stay. I wondered how it felt to be isolated not only in terms of sexual preference, but also within a white world when that wasn’t your own skin
shade. We made bad jokes, my one black friend and I, about summer tans and how nice it would be if one day everyone woke up and were shaded like cats, some with stripes, spots, patches of this color and that. Sometimes it all seemed so senseless, and often for long periods the whole subject of racism wouldn't come up—until the next time two or three (seldom one woman by herself) black or brown women came to a meeting. Black lesbians had their own bar, I remember being told. It never occurred to most of us to go there. We "won't feel welcome." The rumor was out that a woman we sort of knew had gone there and been beaten up. Who knew or asked why? Who was excluding who from what, we asked? Several women I worked with in the factory were very open in their fear and hatred of any women not completely like themselves in background, color, religion, etc. We who considered ourselves liberal before we learned to be ashamed of that word simply avoided them as much as possible. I need to examine that time and experience more.
Bells went off in my head while reading your story in terms of similar incidents where I felt people were avoiding me or didn't want me around because I wasn't college educated at the time, or they saw me as "different," "crippled," or whatever went on in their sick little brains. The anger just eats you up whenever that happens. It's a wonder more people don't let it out in violent ways. Even now, I sometimes wonder whether some people are only friendly with me because I'm writing things that they like, not because I'm personally their type at all. Will we ever get past all the garbage in our heads we grew up hearing and unthinkingly incorporating into our beliefs? A very thought-provoking, strong story, Ann.
-Judith Schwarz
conditions: five
the black women's issue
Available August, 1979
Conditions is a magazine of women's writing with an emphasis on writing by lesbians. Conditions: Five is an issue devoted entirely to writing by Black women, guest edited by Lorraine Bethel and Barbara Smith.
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CONDITIONS, P.O. Box 56, Van Brunt Station, Brooklyn, NY 11215.
Notes on the Etymology and Usage of "Dyke"
The following is in addition to my article, "In America They Call Us Dykes: Notes on the Etymology and Usage of 'Dyke'," which appeared in Sinister Wisdom 9. Feedback from my friend and associate, Chris Czernik, made me aware of my tendency to oversimplify complex historical processes. I would like to say more about the changes which transformed women's roles in the late nineteenth century, and may have led to the demise of the nineteenth century female culture identified and described by Smith-Rosenberg, and thereon to the development of strong taboos in the twentieth century against women's relationships with each other. It was during this period of change that the meaning of *dike* also appears to have changed, as did the meanings of *gay* and *Lesbian*—they took on homosexual connotations.
Researchers are only beginning to unravel how and why women's intimate and close relationships with one another, once commonly accepted as a way of life in nineteenth century middle class America, became so taboo in the twentieth century. This complex social change can only be briefly and tentatively stated here with reference to other researchers' work.¹ The nineteenth century female networks, which were based on mother-daughter relationships and extended kinship and friendship systems, seem to have gradually disappeared by the first quarter of the twentieth century. It was within the context of these networks that women's close, intimate and long-lived relationships had existed. Echols speculates that these networks died out due to changes in family structure and function, and in women's sexual, social, and economic roles resulting from industrialization. As the factory displaced the home as the economic center of productivity, the roles of middle-class women became more based on their economically dependent position in the nuclear family as wife, mother, and homemaker. Women as a group became a source of cheap, temporary labor whose work outside the home was not valued. Women could be exploited along with minority groups, children, and the working class generally. Thus, it was increasingly profitable in an industrial capitalist patriarchy for women to be tied to men where they not only served as unpaid domestics but also as underpaid laborers.
Echols also demonstrates the increasing push towards male/female relationships for women, whereas in the nineteenth century there seems to have been a considerable social and emotional gulf between the sexes. The trend away from positive same-sex relationships during the first quarter of the twentieth century was aided and abetted by the medical/psychiatric system, and was likewise reflected in magazine literature for adults as well as children.²
No doubt during the nineteenth century, when women's close relationships with one another were more or less the norm, there probably were some relationships which were overt, carrying over into the sexual. Still, although a certain amount of physical interaction was permissible, there probably was
photograph by Katherine A. Bouton
a point beyond which such overtness would not have been acceptable. However, these relationships wouldn't have been so noticeable because the "norm" acted, in a sense, to camouflage or hide them, somewhat like an immense "closet." But as the female networks disappeared and the taboo against women's intimate relationships with one another developed, these lesbian women would have become more noticeable, with society attempting to define and label those women who did not "fit" for one reason or another: radical feminists, lesbians, women who sought alternatives to traditional family structures, women who refused society's dress codes, those who desired careers, education, and independence from structures such as marriage and motherhood. Such "deviants" were often seen as "masculine" since there was no female role model which they represented. It was feared that women's emancipation would turn women into men. According to Faderman, this was one of the greatest fears of the twentieth century. It was also at this time that medical and psychiatric science were labelling love between women as "unnatural," "degenerate," and "sick." "Masculine" characteristics of both a biological and psychological nature were attributed to lesbian women, as well as to other women who deviated from male-defined norms. Thus, the word dike, which in the nineteenth century had meant a "well-dressed male," or "the full set of male clothing itself," could have evolved into a twentieth century slang term used to describe lesbians and other women who dressed in "male clothing," passed as men, or who acted and appeared "masculine" in other ways, i.e. being assertive, independent, and self-defining. The changing definitions of dike and lesbian probably reflect the changing ideas and conceptions about women's roles and lesbianism.
I am interested in hearing from readers and researchers who might come across the word dike being used to describe a "masculine" woman or lesbian in 1930s and pre-1930s literature. Please send citations c/o Sinister Wisdom.
Endnotes
1 Carroll Smith-Rosenberg, "The Female World of Love and Ritual: Relations Between Women in Nineteenth Century America," Signs 1:1 (Autumn 1975), 1-24; Alice Echols, "The Demise of Female Intimacy in the Nineteenth Century or 'There wasn't a Dyke in the Land'," unpublished paper, n.d.; Janet Cooper, "Female Crushes, Affections, and Friendships in Children's Literature: or, Covert Feminism in the Children's Book Industry," unpublished paper, April 1977; Lillian Faderman, "Lesbian Magazine Fiction in the Early Twentieth Century," Journal of Popular Culture XI:4 (Spring 1978), 800-17.
2 See Cooper and Faderman.
Dear Harriet & Catherine,
Enclosed is a submission for the “dyke research” feature in SW. It is from my Lesbian Things collection, and is of particular interest to me/us because of the original inscription and date. It is probably midwestern, possibly Iowa, in locale, as it was found in an Iowa junk shop.
-Tracy Moore
Dear Sinister Wisdom,
I was impressed and educated by SW 9. I particularly liked the article on the etymology of the word dyke. Last May I wrote and directed a play at the University of California at Berkeley entitled The Roaring Girl. It was an all-woman infiltration of a classical male text. There was a scene about the word dyke I would like to draw to your attention and that of JR Roberts, who has done much good work on the subject already.
ANN: Did you hear the rumors about Gillian and Alexandra?
LOUISE: What?
ANN: That they're Lesbians.
LOUISE: (laughs) Oh, that old word.
ANN: What's wrong with it?
LOUISE: It's not specific enough—it could refer to anyone on the entire island of Lesbos.
ANN: So I'll say "gay," is that better?
LOUISE: No, say Dyke.
ANN: Dyke? I thought that was kind of like "nigger."
LOUISE: No, it comes from Sappho—she had a lover named Mnasidica and she called her Dike. (Quotes Sappho)
Although clumsy
Mnasidica has a more
shapely figure than
our gentle Gyrinno
ANN: It's a little artsy-fartsy, don't you think? I hate it when people defend sex by making it so . . . Greek.
LOUISE: (quoting Sappho again)
Tomorrow you had better
Use your soft hands
Dica, to tear off
dill shoots, to cap
your lovely curls
She who wears flowers
attracts the happy
Graces: they turn
back from a bare head.
The scene continues, but turns away from this subject. Of course, I invented this etymology, but it is possible and worth claiming for our own—it is certainly a use of the word closer to our own than "one who wore men's clothing." So let's add it to our lexicon . . .
I will be in Berlin most of next year and want to show your journal to the Lesbian feminists there (of which there are many).
We will all keep up the good something together.
-Sue-Ellen Case
DYKE
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Dear Readers:
With this issue of *Sinister Wisdom* I have become the Book Review Editor, and wanted to begin my work in this capacity by describing for you my reasons for believing that it is an important task and the purpose I hope our book reviews will serve. First, and most obviously, books are an indispensable medium in our lives at this time. If they weren't important to us, none of us would be writing them and none of us would be reading them. The print medium provides all of us with a means of transmitting our ideas and images to each other across space and time; it enables us to share in the creation of our culture whether or not we've met each other or know each other's name. Because print transcends both spatial and temporal distances, it is also the means by which we will transmit ourselves and the news of our lives to those Lesbians of the future who seek us. A book review is one way of celebrating the appearance of yet another document and telling other wimmin of its existence; it is a way of connecting us and acknowledging the work that wimmin are doing for all of us.
I choose to work with *Sinister Wisdom* because I believe that this magazine is one of the connecting links in a network of vision, and it is that vision that I hope our book reviews will serve. Since I offered to handle the work involved with soliciting and accepting reviews, I have thought about how I conceive the function of the critic with respect to our lives and the culture we are creating together. I believe that all of us are working in our own ways to make real our vision of a Lesbian culture. Some of us work with wood, some with words, some with microscopes, others with metal, electricity, the soil, animals, stone, relationships. All of these activities are integral to our lives because Lesbians are doing them, and each of us pursues her chosen work within the context of our vision. As I see it, it is the function of the critic to articulate the place of a given book in the context of our lives and our vision, to tell us what it brings to us, whether it's new insights, new connections, or explorations. Such a definition assumes that the Lesbian critic brings to her understanding of a book its relationship to the many facets of our experience and our culture, and her judgments of the worth of a book will grow out of the connections it makes possible in our lives. In some way that I haven't yet articulated for myself, a book "fails" to the extent that it fails to enlarge or clarify our lives. If it "succeeds," it is because it is the medium of the author's participation with us in the daily struggle to maintain our vision and deepen our grasp of the bonds we are weaving in these days. I hope that the book reviews that appear in *Sinister Wisdom* will be yet one more link in our bonding together.
--Julia Penelope
photograph by JEB
A review of *Eye to Eye: Portraits of Lesbians* by JEB; includes a herstory of lesbian photography by Judith Schwarz; Foreword by Joan Nestle. Glad Hag Books, P.O. Box 2934, Washington, D.C. 20013. $8.95. Distributed by Persephone Press and Naiad Press.
During the second wave of feminism Lesbians have been emerging from closets, becoming visible not only to an unwilling world, but more importantly to ourselves who have been straining to see. *Eye to Eye: Portraits of Lesbians* is an important and necessary volume. It makes our seeing easier.
With this collection of thirty-nine photographic images, JEB has given us a bold yet tender portrait of our Lesbian community. As a photojournalist, she has looked at our culture from the inside out, capturing our dignity and integrity. We are a diverse group with a collective spirit shining through our individuality, and JEB has visually portrayed this.
As both Lesbian and photographer, I love looking at Lesbians, and am aware of our need for visual images. Through the lens of a Lesbian photographer, we can touch our laughter and tears. We can trace the contours of our bodies and the hollows and wrinkles of our faces. We can feel the serene beauty of a naked body in candle light or be mesmerized by the mutual love of mother and daughter in the soft glow of a morning light. Most importantly, we can be moved to open our eyes and see into the spirit and pulse of a movement.
I wish there were more images and fewer words. There needs to be trust in the strength of visual images, and here is JEB's major weakness. When I look at Chris, Spotts, Connie, Priscella and Regina, Darquita and Denyeta, I do not need words to guide or influence my thoughts. In fact, I often found the corresponding text distracting. Such is the case with the image of Mara and the excerpt from *Mothers and Amazons*. Looking at Mara, I am taken into the flow and tranquility of an internal exploration; that flow is interrupted when I read an academic interpretation of the egg. In other cases, the words may complement the image, but they are still extraneous, as with the image of Priscella and Regina adjacent to Audre Lorde's "Woman." The composition, the understanding and use of light, and the subject work together to make a powerfully poetic statement. I am in awe of the beauty of these two women: the fullness of their lips, the warmth of their skin, the trust with which they lay in each other's arms. This image needs no words just as the poem needs no picture.
*Eye to Eye: Portraits of Lesbians* is a pioneering work. The strength of the cover will draw you in. Let yourself be drawn—the volume is a special treat.
—Deborah Snow
**Editor's Note:** Although the topic is briefly dealt with here, Deborah Snow's review raises the aesthetic question of what relationship there is/can/should be between visual and verbal reflections of our culture. We are aware, as Deborah is, of the complexity of the arguments on both sides of the issue, and hope that our readers will offer us their opinions on the subject in future issues of Sinister Wisdom.
photographs by JEB
IMAGINING OUR FUTURE: VISION AND REVISION
A review of *The Wanderground: Stories of the Hill Women* by Sally Gearhart, Persephone Press, P.O. Box 7222, Watertown, Mass. 02172, 196 pp., $5.00.
Those of us who still find pleasure in reading books, especially those written by and about wimmin, have many reasons for doing so, all of them connected to our needs and determined by a complex of situational variables, including current physical, emotional, and intellectual states and the external context in which our struggle to survive is going on. Our reasons for reading may be labeled “good” or “bad,” but only in some immediately subjective fashion. Thus, some of us read for “pleasure,” an adjective that covers a wide range of experiences, while others take to reading in order to “escape” from a harrowing or humdrum existence. Others read to learn something new (and possibly useful), to pass the time waiting for ____________________________ (fill in the blank with whatever seems to be missing from your life these days), to find out something about one’s self (usually a secondary, purely fortuitous result, but one that earns a book the description “good”), to gain insight into one’s situation in the world, to acquire a better understanding of our struggle, not only to survive in the most basic senses, but to perpetuate ourselves as a movement, as the bearers of a vision. All of the above are possible reasons for reading a book; none of them are mutually exclusive, and there are more besides.
Rarely, however, do we manage to find a book that manages to satisfy all of the expectations that most of us bring to the printed page. Sally Gearhart’s *The Wanderground* is such a book. It has, to be sure, its flaws, its loose ends, discontinuities, inconsistencies, its typos. It is not a “perfect” book. Who cares? It’s the second book in my life that I’ve read slowly, only because it brought such pleasure to me, lingering over each sentence, each story, because I didn’t want it to end. (The first book I didn’t want to end was Isabel Miller’s *Patience and Sarah*, which, in spite of the roles that the two wimmin use to define their relationship, delighted me with the clarity of their mutual honesty and their successful struggle to be together. It was my first fictional experience of a Lesbian relationship that wasn’t sordid, except, perhaps, for *The Price of Salt.*) Let me blurt out the awful cliche immediately and get it out of my system: I couldn’t put *The Wanderground* down! Now I have to tell you why.
*The Wanderground* is unique in that it serves those purposes that justify the publication of a book: its stories give immense pleasure, and its vision of the future is a welcome “escape” from the boring (and often unpleasant) details of our lives; but the tensions that hold the stories together are drawn from the world of struggle that we all know too well, and it was from these tensions, and the confrontation of them, that I learned new things about myself and what I want to live for and how I want to live.
It is a deceptively "easy" book, by which I mean I found myself savoring the experience of windriding, getting in touch with my lonth, enjoying the facility of mind-to-mind communication with its demand for honesty, craving the ability to communicate with other animals, to sing with cats, to talk to trees, and have them talk, too. In a very important sense, the stories of the hill wimmin fed my fantasies, something I needed to have nourished. I find it hard to keep my fantasies alive; sometimes I even forget them, a dangerous loss in my life. But beneath and within the pleasure of playing with the possibilities of our as yet largely unclaimed powers of being, there is the tension of the painful struggle to survive on a world where men still hold some measure of power, and it is the complications of survival that weave these stories together.
Bit by bit, we learn that the several groups of hill wimmin are largely descendants of the wimmin who escaped from the cities when the New Witch Trials began. The story is fragmented; we learn only what the wimmin themselves have been able to re-cover and piece together in the Remember Rooms of the Kochlias, or from the memories of wimmin who survived and are still living. The individual tales of horror and escape are real enough; we hear and read about their contemporary versions every day. Sometime after the New Witch Trials had begun (some states passed laws requiring every woman to be married, polygyny was sanctioned, "freaky-looking" wimmin were rounded up and "rehabilitated," many were killed, or raped and killed), the Earth herself rebelled against her own rape and destruction. Male engines of power, their guns, their penises, their bulldozers, ceased to function outside their cities. Rape, war, the mining of the Earth's resources became impossible. The cities were now clearly the bastions of male arrogance and privilege, and only within the cities were wimmin still the sexual and domestic slaves of men.
Within the city, men still nurture their love of destruction; without, the ensconcements of the hill wimmin endeavor to re-claim the Earth for all beings that cherish life. The lines are more clearly drawn and there is less ambiguity, but it is our world, perhaps only a few years in the future. Linking the two cultures is an espionage network of those wimmin willing (and able to "pass" as men) to rotate in and out of the cities, and a few men, the "gentles," who help the wimmin maintain their cover. But unpleasant things start to happen that indicate that the men may be regaining some of their power beyond the confines of the city: wimmin are raped outside the city; a few miles up the river, some men get a factory to run for a few months; farm machinery is started up again. The "gentles" are disturbed; they contact the hill wimmin, requesting a meeting.
The four quarters (the Quarterfold) are called together for the first Gatherstretch; all of the wimmin will gather their collective energies together for the first time to deliberate the wisdom of meeting with the gentles. Nothing is decided. A few wimmin volunteer to meet with the gentles as their own representatives; it will be clear that they do not speak for all the hill wimmin. The meeting is not an unqualified success. The gentles have noticed that the numbers of wimmin working in the cities have been lower than usual. When the numbers of wimmin in the city are too low, the gentles hypothesize, the men become potent beyond the city's limits. They want the hill
wimmin to insure that there are always sufficient wimmin in the cities to keep the men in check.
Is the story beginning to sound familiar? The first collective gathering of womanpower occurs at the request of the gentles. Some wimmin must be willing to endure the hazards of the city in order to insure that male power cannot extend itself and thereby threaten the rest of the earth. As Evona says at the meeting: "Why does it have to be the women? Always the women!" (Well said, sister!) There is no easy answer to that question, and Sally Gearhart doesn't attempt one. But the meeting with the gentles is not kind; even the so-called "gentles" are still banging fists on tables, roaring, and mocking the autonomy of wimmin. We are reminded that even those men who claim to be our allies do so with an intense ego investment, as Andros (one of the gentles—aptly named) reminds Evona: "We're not just your protectors anymore. ...You have to trust us now, lady." Who hasn't heard that particular combination of scorn, pride, and wheedling from the lips of a man?
Those words must still be echoing somewhere in the mazes of the Kochlias. We should never forget them, or the hatred they betray, for it is that hatred and its consequences with which the hill wimmin, all of us, must live. Like them, we cannot be satisfied with what we have salvaged from the past or maintained into the present; our strategies must insure our survival into the future that *The Wanderground* foretells. Just as there are no easy solutions, there are no simple compromises with the enemy. For those of us who are torn by the compromises and contradictions of our lives, Sally Gearhart's stories of the hill wimmin is a rare gift. Speaking only for myself, she reminded me of my own vision of a world of wimmin, made it real for me, made it possible for me once more, at a time in my life when I had begun to forget why I do the things I do; I didn't even realize how hungry I was for an affirmation of that vision, and I need that vision to sustain me.
---
**FRONTIERS A Journal of Women Studies**
For the past four years FRONTIERS has been a unique journal which has aimed itself at bridging the gap between community and academic women. Each issue features a cluster on one topic plus other articles, including creative work. Two recent issues:
- **Women As Verbal Artists** The ways women communicate in a male-dominated world and how and why female verbal artists have been ignored.
- **Literature of the Women's Movement** What are the new women writers' concerns? How do they express them? And how is the women's movement being ripped off?
- **Future Issues:** Equal Opportunity Addiction Chemical Dependency Among Women and Lesbian History
Subscriptions are $9.00 (Issues) a year; $15.00 for institutions. Single copies are $3.25. Write to FRONTIERS Women Studies Program University of Colorado, Boulder, Colorado 80309.
PHOTOGRAPHS OF ENERGY AND COLOR
A Story
Maline Corliss, twenty-eight year old lesbian and feminist, felt a need to leave San Francisco. First of all, she was tired of typing insurance policies. Second of all, she was tired of arguing about separatism, art, class, the politics of shoplifting and the authenticity of astral travel. Third of all, she had just broken up with her lover, Kathleen, and could not bear to see her anywhere.
Fortunately, there was somewhere to go. Maline's sister Iris and Iris's husband Alan owned a small but sleek second home on the shores of a Northern California lake. Maline had money enough for an extended vacation—say, six months if the food stamp program proved cooperative. It did. Maline turned the bar into a darkroom and settled down to solitary bliss. It was there at the lake that she met Carol Pine, thirty-four, married, no children.
Carol was the most talked about woman in the small winter lakeside community. The men thought that she was a bitch, and half the women agreed unequivocally. The other half of the women agreed but loved her in spite of her outrageous bluntness and quick, catty tongue. Carol knew that she was controversial, and felt both proud and hurt because of it. She knew that she was strong (armored). She also knew—and hid from the community, yet felt injured that her friends did not realize it—that she was hopelessly dependent upon her husband, Kent. The community thought he was hen-pecked. He was hen-pecked. Carol demanded a thousand tiny services from him in order to make up for the fact that she needed him more than he needed her. She needed his constancy, and she needed his money—he was in construction. She drew her strength from the high-beamed house that Kent built, from the blue lake glinting through thermal glass in the living room. Microwave oven, crock-pot, the freedom to read Newsweek and Sunset and Gourmet magazines in peace.
They had decided, six years back, not to have children (Carol had a tubal ligation). She thought children were like kittens, marvelous to watch, but a nuisance after awhile. Cats were different. The Pine household was always full of them; remarkably colored, aloof, almost hostile animals; variations on their tense, dignified mistress. When Carol went into the bedroom and shut the door, they cried until she let them in.
The lakefront was crowded with houses (long panes of glass, multi-layered redwood decks) but when Maline arrived, shortly before the first of the year, she counted only twelve occupied houses in the two-mile stretch between the market and her new home. The other houses stood hollow and elegant, and Maline experienced a rich, satisfying feeling of solitude. New Year's Eve she spent mixing screwdrivers in the darkroom/bar and carrying them over
to the couch, where she sat with her feet on the coffee table, gazing into the fire before her (she was rather proud of it; it glowed bright orange in the midst of dark ash, and, when she poked it, splintered obediently into yellow flame) or at the dark plate glass to her left. There she could see her round white face reflected, and a faint, shadowy movement of needled branches settling. At eleven she wrote her New Year's Resolutions. There were two of them.
1. I resolve to explore the issues of political pressure and political responsibility, and their relationship to each other in my life.
2. I resolve to discover and understand the true reason that Kathleen broke up with me.
"Because you never let me alone!" Kathleen cried in one of their last arguments. "Even now, you keep pushing me!"
"I've been pushing you for two years! That's the way I am, that's what you value about me! Why should it suddenly be so terrible? Because of Paula?"
"Yes, because she's helped me to see that it's not what I need after all. I need space." Kathleen was crying. "Please see that. Loving you has made me less and less myself. It's made me shrink instead of grow, I'm only half a person."
At two in the morning she called Kathleen. She called to say: Are you growing, Kathleen? Are you bigger, taller, fatter? What are your measurements? Have you become an Amazon yet? She called to say: You talk about wanting your own space but what you mean is that you don't want to have to look at yourself, at who you are and how you are in a relationship. That's what I push you to do, and you can't face it.
When the phone rang, Paula answered, sounding sleepy. Pain shot through Maline's body, comprehensive. Propriety and territory. Paula had answered the phone.
"Are you living there now?" she asked.
"What? Who is this?" Paula demanded angrily.
Maline hung up, humiliated, wondering if Paula had been able to recognize her voice.
A week later the landscape was magnificently leveled by a record-breaking snowfall. Maline used snowshoes to reach the top of the driveway where the Volkswagen sat half buried; she swung her legs in large, stiff circles, gathering fuzzy clumps of snow around the ankles of her Levis. For mail and groceries, she hiked to the market, carrying a canvas pack on her back. The ploughs had scraped the snow into a tall, clean crust on either side of the road, and she felt as if she were walking through a tunnel.
The market was owned and managed by Roy and Nancy Dock—Maline had met them previously when vacationing with Iris. When Maline entered the store Nancy was at the counter, looking friendly and efficient, with her grey hair smoothed and sprayed behind her head. They exchanged exclamations about the storm while Nancy got Maline's mail from the postal window (a letter from her friend Jill, nothing from Kathleen). Maline bought stamps.
Then she prowled the aisles of the little store, gathering together vegetables and red winter apples, Cream of Wheat, bread, milk, and liverwurst.
Carol Pine came in, wearing a burnt orange sweater. Maline was attracted at once to the fullness and bluntness of Carol’s body, and to her rich coloring. . .tight dark skin, glistening black hair and brows, shoulders that drooped forward and breasts that slumped comfortably against her chest. Nancy introduced them.
“Carol lives just beyond you, where the road goes up to the golf course. I’m sure she’d be glad to give you a ride home.”
“Oh, did you walk? That’s why your cheeks are so red!” Carol said.
Maline reached automatically to feel her cold nose, hot cheek. “Yes. It’s not so bad a hike, really. Invigorating.” She made her voice put quotes around the word.
Carol drove a copper colored van. “Four-wheel drive,” she explained as Maline climbed in beside her and lifted in her pack full of groceries. “I wouldn’t live up there without a van.”
Maline told Carol that she was a free-lance photographer. She had never even tried to sell or publish a photograph, but she needed an identity and wanted to represent something independent and artistic.
“How interesting,” Carol said. “People have told me that I’d make a good photographer’s model, because of my coloring, but I don’t know. . .what do you think?”
“Yes, it occurred to me right away,” Maline agreed. “Unfortunately, I haven’t got the equipment for developing color photos. But of course, even black and white portraits would reflect the. . .” she faltered, nervous. “Individuality of your face,” she finished at last, glancing sideways at the curve of Carol’s large, soft ear.
“I’d love to have my picture taken. I don’t think I’ve seen a picture of myself since my wedding, and Kent is always saying I should have a studio portrait done. How much do you charge?”
“Actually, I wasn’t meaning to fish—”
“Oh, I know that,” Carol assured her. “But I’m interested, really.”
“Well, why don’t you look at some of my work, and if you like it I’ll try a couple of pictures for you at cost.”
Carol didn’t want to come in with Maline to see the photos. She had frozen foods to put away, and besides, no snowshoes. “Can’t you drop off your groceries and grab some pictures, and we’ll have coffee at my place?” she suggested. “I’ll be glad to take you home afterwards.” She waited in the van with the engine running while Maline, wanting to hurry but unable, labored on snowshoes over the deep reservoir of snow.
Inside the house she ran to her bedroom and began untacking photographs from the walls. Pictures of the ocean and the park, of women hugging each other on Gay Pride Day, of Jill, of Kathleen, and Kathleen’s dog, and Kathleen’s back yard. Carol looked at them one by one as they sat at her glossy wooden dining table. Maline looked from the photos to the white mist over the lake and back again.
"That's my friend Jill," she said. "And that's my friend Kathleen." Kathleen naked, Kathleen in overalls with a paintbrush (the day they re-did the kitchen in yellow), Kathleen crying, her face dark and shrivelled and wet. Maline looked at the photo, remembering the odd, painful argument during which she had taken it, in cold revenge, while Kathleen in vulnerable anguish had faced her through the separating, magnifying lens.
Carol glanced at the picture of two women hugging and put it aside without comment. The tension in Maline's stomach (the lake is not San Francisco, she thought) eased.
They made a date to take photos, and Maline walked home after all, wanting the cold, stiff air as a conductor for her energy and excitement. She liked the strength Carol seemed to infuse into conventionality.
At home she read Jill's letter, detailing the political struggles of a food co-op, arguments about fundraising, Jill's personal economic problems of the moment—and hi from Kathleen. She glanced around at the blue and blond living room, at the bar, the knick-knacks on the mantle, trying to pin down affluence. The true wealth was the lake, of course: the mist darkening over it, shadows sliding across it, bordered by a row of silver icicles and a redwood window-sill.
Carol and Maline became friends. Maline took pictures of Carol in slacks and sweater, or in a hostess gown slit to the knee. Pictures of her holding a cat, stirring a soup, looking out at the lake—stern, blunt-chinned. Maline told her to look contemplative, but she could not. Carol was built of conviction without a gap.
Maline came out.
"I thought you might be," Carol said.
"Did you think I was attracted to you?"
"Well, I certainly hope so. I think you're attractive."
"Yes, but finding someone attractive is a matter of aesthetics, being attracted to someone is a sexual thing."
"Oh, I see what you mean. I hadn't thought about it like that—are you?"
Maline grinned. "Oh, my appreciation is mainly aesthetic," she said. "So far."
"Terrific," Carol said. "From now on I'll think of myself as a work of art."
"What about me?" Maline said, hating herself for asking.
"Oh, you're a work of art too," Carol assured her.
Maline hesitated. "How do you feel about my being a lesbian?" she asked finally.
"Well, all right. I mean, I can understand women sleeping together. You know the way men are at cocktail parties, always leering." She shrugged and got up to let one of the cats in.
But Carol was not a feminist. She had no sympathy for women who, unlike herself, were trapped in unhappy marriages, or stuffy kitchens, or waiting on tables. "I chose my life and it's what I want," she said. "They should have done the same."
"Not everyone has the same choices," Maline insisted.
"That's not my problem. I mean, I believe in equal pay for equal work and all that, but don't expect me to think all women are wonderful because most of them are very silly. You take Violet Jackson—I play bridge with her. She's always fluttering about Kent's marvelous blue eyes and talking about sex like it was the be-all and end-all of existence. I mean, I like sex too, but I try not to be adolescent about it. She thinks I'm a bitch—I know because Nancy Dock told me. But she won't say it to my face. That's one thing no one can accuse me of, everything I'm saying to you I've said to her."
Maline was scandalized and half-envious of Carol's dismissiveness, her refusal to take responsibility at any level for the oppression of other women. She tried to make Carol see, pushed her to see the ways women are oppressed, and to recognize the limits of her own life.
"Frankly," she said finally, "I don't see how you can find this life fulfilling. You're removed from reality, isolated with your toys. Bridge, cooking, television. Don't you want something more?" She expected to shock Carol, to offend her. But Carol was neither yielding nor resistant.
"No," she said, shrugging. "I have everything I want."
And for a moment, Maline could see it. Lakeside beauty, idle fulfillment, and Kent. The three of them had dinner together one evening at the Pine House, and Maline found him likeable. He was mildly witty, easily gratified, affectionate. Maline analyzed rapidly: Kent thrived on Carol's power, Carol on Kent's playfulness.
One morning in March (while the snow was peeling away from the manzanita under the sun's pressure, leaving twiggy little holes) Maline received a letter from Kathleen.
Now that we have had time and distance, and can write without anger, perhaps we can exchange helpful feedback about our relationship and where it failed. I know that I closed myself off to you a lot, and that you pushed me too hard—those two things. But what else? How did we fail to connect so often, why were the missed connections so electrical, so painful?
Maline wanted to write: We never failed, it was you who failed—faltered—could not endure (a suitably dramatic reply). Or to quote: "The unexamined life is not worth living." But she was beginning not to believe in those answers. She thought of writing instead: The guilt that we collectively impose upon ourselves as individuals when we insist upon political responsibility killed our love. But that did not seem true either.
Finally she wrote:
How can I answer you? You've gone on to a new part of your life, and are looking back in peaceful, intellectual consideration. I am still trying to encompass my solitude, to believe in the fact of our separation, to understand the truth that you no longer love me (as you see, I am unchanged—still frankly vulnerable).
Adding: P.S. We failed because you were too yielding and too resistant.
Maline began to want rather badly to become lovers with Carol, so that she could feel herself superior, a conqueror, a seducer, and so that she would not be alone anymore. And so that she could write Kathleen: I have a new lover. Or better yet: I am having an affair with a married woman. She knew all this (continually self-challenging, continually self-critical) and was ashamed, but not closed off to the actualization of her shabby needs if it proved possible. And it was—because Carol was flattered and curious, and perhaps, in spite of what she said, a little bored with her life. And, finally, in this stew of unromantic motives—because they were attractive to each other: Maline’s energy, Carol’s color.
So they became lovers, invariably making love in Carol and Kent’s big bed, throwing aside the deep blue bedspread. Carol did not consider herself unfaithful to Kent because Maline was not a man, but did not tell him about the relationship “because of his prejudices.” Maline gave up trying to pierce the solidity of Carol’s vision. She allowed herself to feel politically superior to Carol and gave up arguing with her. Instead, she lavished all her energy—nervous energy, artistic energy, political energy—upon the appreciation of Carol’s body. She let her camera savor Carol’s brown flesh as well, took pictures of Carol lying on the bed, naked; holding a cat, naked. But Carol was uncomfortable. “My clothes give me confidence,” she said. “Even now I’m being more vulnerable with you than I’ve ever been with anyone—even Kent.”
Maline could hardly believe this. “You don’t seem vulnerable,” she said.
“Maybe you’re insensitive.”
“I don’t think so,” Maline said, angry but controlled. “Maybe you’re just so used to keeping things in, being repressed, that even when you want to you can’t let loose, can’t express the vulnerability you feel inside.”
“Who wants to?” Carol said, shrugging her naked shoulders. Maline watched her breasts roll against her chest.
“Why don’t you ever come to my house?”
“Same reason.”
“Carol—don’t you think you’re being awfully insecure? Maybe you’d find you don’t need the trappings you think you need.”
“Maybe I’d find you and your rhetoric ahead of me at every turn if I dared to set foot outside my—domain.”
“I don’t think I’m as awful as all that,” Maline said, beginning to feel miserable, as though everything were spoiled. “Look, Carol—” knowing it was a ploy “—you seem awfully defensive. Maybe you’d be more comfortable—maybe it would be wiser for both of us, if we just quit now—called it off?” Feeling Carol’s flesh still beside her, and then:
“No. . . let’s not do that.”
And so they continued being lovers into the spring, now that Maline had the evidence, verbal: No, let’s don’t do that.
She wrote to Jill: She is casually competent, lazily creative, shrewdly intent on power, and absolutely without guilt. This last, of course, is the real attraction. Being with her eases me. And at least now I know it’s not just me.
She did not write to Kathleen. It seemed unnecessary; she would hear it from Jill.
The hours that Maline spent with Carol were dictated by Kent's schedule. Maline expected to be infuriated by this but wasn't. She was anxious for her hours alone, they were what she had come for. She photographed landscapes, read volumes of women's autobiographical writings, and began an autobiography of her own, synthesizing and enlarging journals kept for over five years.
When Maline and Carol were together they shared meals, watched TV, played cards, went (after the snow melted) on photographic expeditions, and once rented a boat, taking it out to the middle of the lake where they drifted, swam, ate a picnic lunch, cuddled and caressed, wet then hot—
Carol drove the boat. Maline watched her at the propeller, watched her bare arms on the steering wheel. She admired Carol's competence; remembered Carol in her kitchen or in the copper colored van. At the dock Carol made quick loops and knots with nylon cord, then repeated them slowly, so Maline could watch and learn.
Carol paid for the boat rental, and Maline began to think about money seriously. "Another month is all I have funds for," she told Carol. "What do you think—should I look for a job in town?"
"I don't know," Carol said. "Do you want to?" She was sitting in a lawn chair on the deck, looking dreamily out at the lake. Maline stood behind, playing with the long dark fabric of Carol's hair.
"I don't know," Maline returned. "How do you feel about it?" *Do we love each other, or are we just playing, killing time? Shall I go back and face the community now—untangle the political issues I keep tripping over when I fall in love... shall I learn to be like you, Carol, closed, calm, at peace?* "Ever think of leaving Kent and moving with me to San Francisco?" she inquired of Carol finally.
"I thought of it, yes. I mean, it occurred to me. I would never do it, of course."
"Why not?"
"Oh, I like it here." Carol waved a hand at the flat blue water before them. A boat buzzed and skimmed at its center. "The city's so dirty," she said.
Maline was quiet, hurt by the casualness of Carol's reply. She let go the hair; it fell down the back of the chair.
It was spring. Chains of carpenter ants wound their black, glistening way through both houses, and the cats brought home mice and shrews. There was a bird's nest in Carol's mailbox, so she and Maline walked to the store daily to get their mail. It was a ritual, a way of fabricating a little of the routine of marriage for themselves.
Maline got a job in a restaurant in town that was taking on extra help for the tourist season. She wore a dress and white apron, and flowers in her hair. It was part of the restaurant's motif. She hated it, but smiled politely at jovial men and their irritable wives, scooped the tips into her apron pockets.
One evening Carol and Kent came to the restaurant with Roy and Nancy Dock. Maline saw the hostess seat them, and her stomach sent up ribbons of
protesting acid: anger and humiliation that Carol should bring affluence and convention to mock her. She took their order sullen-faced.
"What's the matter, Maline? You look like you have a toothache!" Kent teased.
"My feet hurt," she said, giving him a slight, obedient smile.
"Ah, that's the working life. What'll it be, Sweetheart?"
Maline looked steadily at Carol, who looked calmly from the menu to her lover/waitress. "The veal scallopini, I think," she said. Maline chatted with the Docks, and promised to send their regards to Iris.
The next day at Carol's, sitting cross-legged before the fireplace while the wind played the evergreens like pipes outdoors, Carol said, "I tried to talk them into another restaurant, but they were determined. I didn't want to be obvious."
"How could you just sit there as though I were nobody?"
"What did you want? Did you want me to throw you tragic, romantic glances?"
"Oh, I suppose so." Maline gave a tired smile. The fire's heat was giving her a headache. Her anger felt forced; months of hurt, desire, and energy seemed artificial.
"Don't look like that," Carol said. "You know I love you." She said it confidently, complacently.
"Do you?"
"Yes," Carol said, rolling across the rug to where Maline sat, reaching her head up like a snake to kiss Maline's forehead, lips, breasts underneath Maline's polo shirt. They made love, Maline passive under Carol's slow hands and tongue.
Two weeks later Maline got a letter from Kathleen. Carol was with her at the store the day she got it, and when they were outside she said, "What does Kathleen have to say?"
Maline, who had read aloud to Carol letters from Jill and Iris, slipped the envelope into her back pants pocket, saying "I think I'll read it later." Carol raised her heavy brows, then tossed her head (the long hair skittered on her back) and began to talk about summer vacation: she and Kent were going to Los Angeles to see Kent's parents.
Maline did not read the letter until she got home from work that night. It said:
Paula and I have broken up. I'll always be glad she came along, because of what I learned from her about what I need in my life. In my next relationship, I think I'll be able to stand firmer, more solidly for what I need, and not let myself be bullied by my lovers...like you, like Paula.
Sometimes I get sad, think of you and me and how we can never get back to what we had—the finest of what we had was the finest I've ever had. I don't think anyone will ever mean as much to either of us as we've meant to each other (correct me if I'm already wrong!). But I know we can never go back.
I hope, though, that when you come back to San Francisco (you haven't settled there permanently, have you?) we'll be able to see each other in new ways, relate to each other with more health and more strength.
She signed it love.
Maline called her up immediately. "Do you have any idea of how manipulative the letter you sent me is?" she asked.
"Yes," Kathleen said. "But there are no lies in it."
"The whole letter is a lie," Maline said.
"No it's not. Are you going to come back?"
"I don't know," Maline said stubbornly, knowing that she would.
She told Carol the next morning, while they sat with coffee at the dining table. "I'm going back to San Francisco," she said gently. "I have to."
Carol stared into her coffee cup, then out over the water, choppy today, blotted and mottled blue and grey. "I've been expecting it," she said steadily, "But I really don't know why you're going."
"Because I need the culture, the support...and total commitment instead of a piece of someone's life." And because I still love Kathleen and she and Paula have broken up.
Carol pushed her chair back but did not get up. She looked down at her knees—blue slacks—and closed her eyes.
"You have Kent," Maline said.
"I hate Kent," Carol said, childish for the first time, and began to make a sound, a high wailing, with an abandonment of which she was incapable, scarring her peace, the glass, the lake...a skinny tortoise-shell came bounding across the room to Carol's feet then stood, round-eyed and tense...Maline put her arms around Carol's broad shoulders and comforted. "And it's too late for me to come with you, isn't it?" Carol sobbed.
A wave of shocked blood rocked through Maline: a chain of disbelief, desire, fear. She thought of Kathleen and her dog, the two of them in Golden Gate Park, and of Kathleen singing around the house. Of Carol's hostess gowns—then felt their breasts together and Carol's tears on her neck, and said, "No, it's not too late."
"Yes it is, yes it is." Her sobs were coming chunky now, softer, final. The cat jumped up to the table, and Carol pushed Maline away to take the cat into her lap. "Hello furry-purry," she crooned. The cat purred as she cuddled it to her chest. "Will you love me forever?" Carol asked it.
They said their last goodbyes, for Maline planned to leave the following morning. Maline presented Carol with a dozen beautiful photographs of herself, and went back to Iris's to pack.
At midnight that night, in a final moment of panic, Carol left Kent drowsing over Johnny Carson and hurried through the blackness to Iris's house. She found it dark, and the Volkswagen gone. Maline had become restless, and by that time was miles down the Feather River Canyon, going home, feeling guilt-burnt, but as strong as she had ever been.
"Embroidered Breast" by Quimetta Perle
embroidery, beading
6" x 6" x 4"
photograph by Vicky Johnson
IT'S THE POVERTY
for Kim
you say to me
"take a drive with me up the coast, babe
and bring your typewriter"
all the way down the coast
you and she stopped at motels
your typewriters tucked under your free arm
dodging the rain fast to the shelter of metal awnings
red and white I imagine them
you two snorting brandy
in those vinyl rooms
propping your each machine onto an endtable
this story becomes you
a fiction I invent with my ears
evoking heroism
in the first description of the weather
I say my typewriter sticks in the wet
I have been using the same ribbon over and over and over again
yes, we both agree I could use a new ribbon
but it's the poverty
the poverty of my imagination we agree
I lack imagination you say
No. I lack language
the language to clarify my resistance to the literate
words are a war to me
they threaten my family
to gain the word
to describe the loss
I risk losing everything
I may create a monster
the word's length and body
swelling up colorful and thrilling
looming over my mother, characterized
her voice in the distance
unintelligible
illiterate
these are the monster's words
understand
my family is poor
poor
I can't afford a new ribbon
the risk of this one
is enough to keep me moving
through it accountable
the repetition like my mother's stories re-told
each time reveals more particulars
gains more familiarity
You can't get me in your car so fast
you tell me how you've learned to write
while you drive
how I can leave my droning machine behind
for all
you care
I say not so fast not so fast
the drone, a chant to my ears
a common blend of histories repeatedly inarticulate
not so fast
I am poorer than you
from my experience
fictions are for hearing
not living
—Cherrie Moraga Lawrence
WHAT DOES IT TAKE?
for Sally Gearhart
upon the death of Harvey Milk
I
the martyrs they give us
have all been men
my friend she traces her life
through them a series of assassinations
but not one not
one making her bleed
this is not the death of my mother
but my father
the kind one/the provider
pressed into newsprint
in honest
good will
If they took you I would take to the streets
scream
bloody murder
but the deaths of our mothers
are never that public
they have happened before
and we were not informed
women do not coagulate into one hero's death
we bleed out of many pores
so constant
that it has come to be seen as the way things are
2
my mother's death
was not eventful
expecting it
I put a hole in my arm
no TNT blast
but a slow excavation
my nails
in silent opposition
digging down to the raw part
inside the elbow
If they took you I would take to the streets
scream
bloody murder
what does it take
to move me?
(your death that I have ignored
in the deaths of other women)
Isn't the possibility of your dying
enough?
—Cher'rie Moraga Lawrence
MOVING OUT
"You are not going to see the promised land, sisters and brothers, you are not. But you better get your ass out into the desert because if you don't start on this journey, nobody'll see the promised land."
—Rita Mae Brown
I can’t imagine dying out here, not seeing trees or tasting water, tongue thick from silence these years Our packs grown to our spines we hobble like hunchbacks, our holy robes as yet only ghostly costumes flapping at thin wrists and ankles. This air like every stinging word thrown at our faces we have lived in this desert even unaware, our love flattened like these dunes, distorted, brushed aside. No footprints, no guide but the sun heat of shame newly turned to anger We protect ourselves like cactus grow sharp and hard, drawing rain from an earth closed against us. Still, reaching across glaring stretches of sand toward some land where rain makes music night comes, not to hide but to bless.
—Jan Hardy
THE HOUSEWIFE
the spoon sweeping
the pot's tin,
the bottom,
in smooth circles
sweeping again and a
gain and
the pudding thickens in
the spoon's swirl
ing, the pudding
hot and heavy
and turning, the spoon's
trail,
the diamond of the
ring fogged and the gold
hot and heavy,
turning on the skin
as the pudding hardens
on the pan's side,
the spoon sweeping arc
after arc af
ter arc,
the hand
sweeping again and a
gain and the radio
crackling
the top ten
ten times each spin
of the hand
turning again and a
gain and the bubbles
beginning
as the spoon spins
scraping the tin
as the hand tells three
and the books fall
and the t.v. turns on,
and channels one after one after
turning while the
pudding burns
and the spoon
strikes the pot's tin again and
again and
again
—Janice Maiman
THE DIRT ON THE SIDES
thighs,
in stirrups,
brown,
my mother's legs,
six feet,
cold,
and bones,
my lover's spine,
in the walls,
mud,
deeper,
winter's reign,
numb space,
deaf,
(in a box,
a pencil,
plunging,
my mother's hole,
the wood,
thighs unsettled,
the legs kick,
my mother's legs,
dirt on the sides,
lumps hard,
and flexing,
head first,
the mud,
my mother's womb,
like a fish,
full moon eyed,
limp,
plunging,
my mother's hole,
the wood,
stones,
and bone,
my grandmother's,
in the walls,
fragments,
my mother's womb,
numb space,
hollow,
and deaf)
my mother's legs,
the muscles move,
flexing,
her vagina,
dark,
rocks,
my grandmother's hip,
fragments,
my mother's womb,
yesterday's rain,
frozen walls,
my mother's womb,
hollow.
—Janice Mainan
Clouds muffle the moon:
only the river carries conviction.
In your woods deer must be moving,
their antlers confounded with branches,
like a reflection.
The river’s arrangements are tangled in ice;
Let such knots be unraveled,
and moonlight break free,
and I’m up and away from that house like a witch.
My bones are as clean and as cold
as the ridges I cross, to where you are
breathing in darkness.
Still under quilts, you are little expecting
My body to slide in beside you,
Unsettling your sleeping,
and clearing the air, like a frost.
PHILADELPHIA, 4 A.M.
She woke up from a dream
of two strict aunts in Colorado;
she was shaken by it, grooved
into the agony of childhood;
I tried to reach her there, but
“back off,” she said, “leave me alone.”
So I got up, in that strange expensive
town apartment, and, standing by the window,
felt my spirit spread away from me,
across the rooftops of the downtown city.
I told myself, oh yes, and, naked,
I made tea.
I watched the jasmine flowers open,
and float slowly on the surface
of the cup; pale and bloodless,
they spread themselves, like me.
Oh yes, I thought, all these grey hairs, these long used muscles, bones, and tissues, are dissolving, and every sad truth is coming home: oh yes, I am a woman taking in her washing at long last, stained by the rain.
—Elizabeth Alexander
SONG OF THE MOLE MOTHER
for Alice
These soft, dark places are mine,
the wet, black heart of earth
here under the porch where moss grows hair roots for mushrooms gliding by pearl-white spiders of tentative plans who wait ominously under their caps.
The tender shoots are not hard to find and we follow them awhile and reach the embryo of snakeroots and onions gleaming like eggs suspended in dark water.
One day the sun will not rise and a blind green fish from the deepest crevice will call us to grasp her tail and follow her to a warm center where we will touch in whispers and give birth to love children with no eyes.
—Teresa Anderson
SMALLNESS, SHARON
smallness scares me
smallness, sharon
yours. all gathered in
arms around your knees,
chin tucked under
compact.
this once
I wanted to reach across
to you
bridge this crazy canyon of sizes.
but I cringe from diminutives
and return to my house tonight
because I fit
because no one there is smaller than I
because
(smallness scares me)
paula was small.
—Becky Birtha
Sun-up, second of November
6:15 a.m. cold
wind ripples
the morning a starched white curtain
You in the
frayed blue coat faded
kerchief black stockings kick
round the corner back
up Camae
street all to yourself about
wide as your wide-stretched arms
clog mark hard on the
cobblestones echo up the
hollow, ring out the high brick walls
Come from your lover
you running your morning home
rose sky in your eyes and her
kisses all over your face.
Becky Birtha
GHOST STORY
II
(for my father)
I feel you
crawling through my rear passageways
returning through doors marked
one way,
this way out, and exit only
your persistent footfalls
echo along my narrow corridors
constricting,
drawing in my center.
I feel you
stumbling through my nightmares
a blind vagrant who returns to me
because your cane remembers the way
and there is no place else to go.
I hear the splinter of wood,
the clatter of falling hinges
as you assault doors that open into yesterday
seeking sunlit rooms
in which I used to call you daddy.
–Julie Blackwomon
CAROL
she is clearly the lioness the golden sun ruby fire goddess, the flame, the spark she is truly new in her learning she is truly free in her giving
in the cage. she paces back forth back forth face creased by the effort to hold onto what she knows she could remember if only the bars would lift, the glass shatter, the doors spring open and fall away. pacing eyes watchful, narrowed at those who observe her: caged symbol of roaring and blazing.
clearly the chariot, pulled by winged lions, angel lions, clearly the bringer of light, the gleaming trail across a darkened universe announcing rebirth delight magic
pulled down. the angels bear incisions where their wings should be/ deep slices. looking into those crevices we can read the history of removal. hope chest opens to reveal the remnants of bloodied down, dripping scarlet feathers. now they are fans, creased by the effort to remember flight.
clearly the free flier the golden space dreamer the power raiser the sunlit dancer the radiant mover the sweeper the spinner the runner the leaper
walls disrupt her. she watches crumbling, decay. walls, ceiling, structure cave in, collapse. she remembers this, loving dissolution. glowing orange against the rubble, the dying framework
she is essence escaping
light/heat
light/heat
light/heat
-Terry Wolverton
for iva
the woman who is cat
whose brown fur sparkles/who crouches and watches slit eyes green and knowing/who hides under the bed when the doorbell rings/who crawls into your lap, snuggles down, stretches, kneads/who grrrrrrrs into your ear, deep down humming moaning whiskers sigh/who curls up, stays home, waits and rubs against your memory
encircles satin pillows velvet skins/who will not settle for less than what she wants and expects nothing/who delights in licking herself clean/who cannot be coaxed into the street/gazes amethyst through windows drinking the flight of birds leaves snow/whose lean muscles ease across the floor but tightly-coiled, springing
the woman who cries for food or the stroke of a finger across her throat/who would sink her teeth in to get away but always comes back for milk or a bite of fish
woman who crinkles her face into mirrors and does not see her reflection.
-Terry Wolverton
"I AM FRIGHTENED BY THE SORROW"
The thing that hit me was her eyes, wild and sharp, ready first to flee and then in terror to lash out. She was afraid to strike or stay. Four months of this in hiding was too long; locked from any men but jailors, those with guns, you grow away from life, turning into lynx or hare but always in a cage. When I stepped in, she spread her claws. She saw me something different and shrank back, then chancing all, she ran into my arms. "I'll get you out of here," I said, "You just hold on. Fight me hard where they can see. I'll get you out." I held her close a moment and then held her off, telling those outside to put her in my car, telling them my orders were to take her north, telling them to do it and to do it fast. They listened, dragging her and kicking at her legs, tying tight her hands and feet, shoving her still wrestling them onto the floor in front.
* * *
I started up the Ford and we moved out. Knelt below the seat she shook with tears. "We'll get away from here," I said, "Are you alright?" She nodded and I watched the road. I tried to stop and bring her from her knees but she said, "no" and I drove on, struggling with anger and within her pain, till finally I pulled aside and lifted her and opened ropes and rubbed the burns with carefulness and love. Her limbs were hurt less badly than her heart. She shivered small against me, her head on my lap, silently and tense. We travelled hours into darkness waiting out the miles, and halting only once for drive-in burgers, and a bathroom, and to fill the tank.
That night I picked a dim motel, and asking single space, we chose the furthest from the path. The diner there was open through till dawn. "I'm so dirty," she said, "and so tired. I look a mess." I washed her gently in the shower, she too beaten now to protest or resist, then half walked, half carried her onto the double bed. "My clothes will fit you," I said, "We'll throw these out. What happened to your own?" She only knew that they were gone. The bruises on her back and ribs were new; I touched them; she started to relax. "Where will you take me?" she said. "Anywhere you want to go. North first, as far from here till they can't find us, then I'll take you home." She slept, her fingers clutched in mine, and I slid in beside. In dream she cried and woke. "Diane," she said, "I have no home." "You have a home," I said, "as long as I'm alive."
In the next hotel at evening we sat together as I soothed a cream into her skin. She was stiff and sore, and thinner than a child. "Di," she said, "What if I don't want to stay with you?" "Then you can go," I said, "I'll help you get to anywhere you want." "Now?" she said. I asked her, "Can you do it now?" "No..." She looked at me. "Di," she said, "I want to stay with you. Do you still want me?" "You know I do," I said, and kissed her hair, and held her as she fell asleep. Later waking her was hard. She startled, lost, and was too weak to rise. "We won't go so far today," I said. "Tonight we'll be in Washington, and we can stay there till you get some rest." "But," she said, they'll find us and they'll take me back." I said, "Over my dead body!" "Yes," she said, and stroked my cheek, "I think you would!" I felt her staring as we rode, felt her try to talk to me and hold it down. I brought her water in a cup I
filled in Maryland where we stopped for gas. "I trust you, Di," she said, "I want to stay with you. We won't be lonely anymore."
*
She was too frightened yet to let me bring her into downtown noise. "I know they're hunting us," she said, "I can't go back." I said, "They haven't found us until this, they'll let us go." "I know they'll find us. Please," she said, and when we reached the cut off lane I took the one away. "This takes us to Bethesda and to Pennsylvania," I said. "You're too fatigued to go much further, and we ought to stop. I'm getting punchy watching guardrails, too." "I don't know how you've gone this far," she said. I slipped an arm around her and we took an exit to a grey old town of cottages to choose a room. "Five more hours driving time will take us home," I said. I brought our suitcase in. She said, "How did you find me?" I said that "I just knew. And I just had to try." She said, "I had no hope. I can't believe its true." "I promise you it is," I said, "and that you're safe."
*
We stayed there four more days, just loafing under blankets at the inn. First I brought her meals in bed from someone's Home Cooked Kitchen up the way. We slept and woke and slept again and talked and went for walks. One day she asked to go alone. I put two twenties in her pocket and a paper with my own address. "In case we separate," I said. She saw my apprehension and she grinned: "I have to see if I can do it, but I won't go far." She came back minutes late with an ice cream cone and flowers from somebody's yard. "It's been four months," she said. I knew I had to show her she could really go, but still she seemed content, and didn't want to leave, and seemed a bit upset if I was gone from her too long.
Sometimes she went herself to bring our food, but mostly she stayed with me, and she grew less nervous and with less of nightmares and of fear. She held her head up higher. And she bloomed.
*
The final lap to Pittsburgh was a happiness we shared. More calm than I had seen her, she was stronger and her timidity was halfly gone. We took the turnpike easily, with often halts to try the shops or just to wait and sit there and enjoy the view. Only among people would she sometimes flinch, and then the ache that I once saw as part of her would draw against her face. She retreated to a shell then, though a word from me would bring her out, and she would need me to be near. We reached the limits of the city in mid-afternoon and stopped again for groceries for bringing home. “Just don’t expect my place to be too much,” I said. She said, “It doesn’t have to be if you’ll be there.” She moved to lean against me when we drove the streets, and at the house I had to keep beside her or she wouldn’t come. Her eyes were watching mine as I unlocked our door. We climbed the stairs together, holding hands.
—Diane Stein
for judy
[Author’s note: The title is a quote by James Wright, *In the Face of Hatred*.]
ANNOUNCEMENTS
SOLICITING MANUSCRIPTS:
Linda McDonnell and Toni McNaron, two lesbian/feminists, are soliciting manuscripts for a collection of women's poems and prose focusing on our experiences with incest. We are interested in all types of incest: overt, covert, same sex, different sex, physical, emotional. We don't want sociological or political tracts; or interviews. Aside from these limitations, we are open to all forms—letters, short stories, poetry, journal entries, prose-poems. As incest victims we have conceived this project out of our own and other women's work. We believe breaking silence around this taboo is both transformational and healing. We have been co-editors of So's Your Old Lady, a lesbian/feminist journal, for the past three years.
Please send manuscripts to: Linda McDonnell, 2533 Lyndale Ave. South, Minneapolis, MN 55405. If you wish your copy returned, enclose a SASE. Because we want to print soon, our deadline is February 1, 1980.
We are planning an anthology of writings on the subject of child molestation. We are seeking first-person writing: stories, poems, journal entries, and excerpts from longer works, by people who have been molested as children. We are also interested in writing by other family members, from their own point of view. And, with the idea that this anthology might be useful for young people in schools, we are open to writing by children as well. All material should be true, although you may change names or use a pen-name. Material may be published or unpublished (let us know about permission to reprint). Please type, if possible, and enclose a SASE if you want your manuscript returned. Mail as soon as possible to Ellen Bass, 240 Day Valley Road, Aptos, CA95003.
FEMINARY
A FEMINIST JOURNAL FOR THE SOUTH
Emphasizing the Lesbian Vision
published 3 times a year
Subs: $5 for individuals
$10 for institutions
P.O.Box 954, Chapel Hill, N.C. 27514
EYE TO EYE:
PORTRAITS OF LESBIANS
PHOTOGRAPHS BY JEB
A rare & powerful book
39 b&w photographs;
a herstory of lesbian photography; Postpaid
$10.50 to Glad Hag,
Box 2934, Wash. D.C.
20013 or bookstores.
FOR HANDICAPPED READERS:
*Sinister Wisdom* in conjunction with FEMINIST TAPING FOR HANDICAPPED READERS is making periodicals and books available to handicapped readers. At present the project consists of ten women volunteers who tape *Sinister Wisdom*, *Off Our Backs*, and *Feminary*. Since we would like to make these tapes available for no more than the cover price of the book or periodical itself, we are asking women to help us by donating some money (any amount) that could be used to subsidize this undertaking. Or to donate the equipment needed to do the tape processing ourselves: a cassette-tocassette high speed duplicator, cassette records, and 90-minute cassette tapes.
If you know anyone who would like access to the journals we are taping, and is a handicapped reader—please tell them about FTHR. Donations should be made out to Susan Wood-Thompson and sent to her at FEMINIST TAPING FOR HANDICAPPED READERS, Box 6516, T Street Station, Washington, DC 20009.
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COMING — — — in SINISTER WISDOM
2 Special Issues
SW 13 — LESBIAN PUBLISHING & WRITING. Guest edited by Beth Hodges. Interviews, Reviews, and Theory. Send submissions with SASE to Beth Hodges
64R Sacramento Street
Cambridge, MA02138
Tel.: 617-547-0704
DEADLINE: March 15, 1980
SW 15 — LESBIANS AND PORNOGRAPHY. Guest edited by Julia Penelope. We are seeking poems, articles, and short stories that deal with any of the issues of pornography and violence and the ways in which these facets of patriarchal culture affect Lesbian lives, the way we define ourselves, our sexuality, our relationships with other wimmin, racism and pornography, classism and pornography, incest as a specific form of sexual violence in the lives of Lesbians, the "compromise" distinction between "pornography" and "erotica," the use of pseudo-Lesbian imagery in advertising (to whom do such ads appeal?). Also of interest, in contrast, would be articles and fiction that explore Lesbian sensuality as it is evolving now.
Address all correspondence and manuscripts (include an SASE) to
Julia Penelope
box 30541
Lincoln, NE68503
Subscribers, please read: If you move, please send us your address change as soon as possible. The post office will not forward your magazine. If you have moved recently and think you may have missed an issue, it was probably mailed to your old address and then returned to us. In that case, we ask that you send $1.00 to partially cover the cost of remailing it.
CONTRIBUTORS' NOTES
Elizabeth Alexander is a poet from Concord, New Hampshire.
Teresa Anderson is a poet from Oklahoma.
Becky Birtha. "Thirty years old, I'm a member of a lesbian writers' workshop here in Philadelphia, and do public readings whenever I have an opportunity. My poetry has appeared in the special Black Women's issue of *Conditions*, and one of my short stories is included in *A Woman's Touch*, an anthology exploring lesbian sensuality and eroticism, available from Amazon Reality."
Julie Blackwoman is a poet living in Philadelphia.
Katherine A. Bouton lives in Santa Cruz, California.
Andrée Collard is writing a book on the patriarchal treatment of nature, animals, and women, to be published by Beacon Press.
J. Z. Grover is a writer living in Sacramento who photographs her friends as an avocation.
Marilyn Hacker is the author of *Separations* (Knopf, 1976) and *Presentation Piece* (Viking, 1974), which received the National Book Award for poetry in 1975. She lives in New York City with her daughter, Iva.
Barbara Hammer. "I am a lesbian/feminist/ poetic filmmaker and writer currently working on a symbolic quest film of an aging heroine who encounters her crone counterparts of seductress, guardian angel of consciousness, and wise woman of pain and suffering in her backpack journey around San Francisco Bay. I self-distribute my films through Goddess Films, P.O. Box 2446, Berkeley, CA 94702."
Jan Hardy. "I'm active in a lesbian-feminist theater/poetry/propaganda group in Pittsburgh and trying to use separatism to stay sane in all this missionary work."
Beth Hodges lives in Cambridge, Mass., where she has taught writing workshops for women and is now studying book design. She has been active in lesbian feminist publishing since 1975.
JEB (Joan E. Biren) is a left-handed glad hag photographer. She's been photographing lesbians for nine years.
Meg Jochild is a Texan dyke who has moved to San Francisco so she can be homesick with more style. This is her first time to be published.
Lynda Koolish is a poet and photographer. She is writing a book on contemporary women poets.
Cherríe Moraga Lawrence is an L.A.-turned-Bay-Area poet who waits tables for a living. She is presently involved in editing an anthology of essays by Radical Third World Feminists along with other Bay Area women.
Janice Maiman is a poet living in Virginia.
Tracy Moore is a dyke trying like many others to live in California and Iowa at the same time. Someday her writing may appear in *Better Homes & Dykes*.
Julia Penelope is dropping her patronymic, Stanley. The name Julia Penelope has been handed down on the maternal side of her family every other generation since her great-great aunt Julia Penelope defied General Sherman by chewing and swallowing the map which showed where her husband was hidden with his Confederate troops. More recently, she has created herself as a buttercup, a dragonfly, a boa constrictor, and a bumblebee. Not all at the same time, though. She also, on occasion, appears as the Winter Solstice Elf, so be sure to leave out granola bars for her on December 21.
Quimetta Perle. "I am an artist living and working in Minneapolis. I think of my work as sewing a silk purse from a sow's ear. It is the process of transforming the raw material from my guts into beautiful, rich and complex images.
Deborah Snow is a co-publisher of Persephone Press.
Diane Stein. "I am 31 years of age, holding a Master's Degree in English Literature, and working at minimum wage as a file clerk. My writing is my life. I have been writing since high school, publishing for the last 10 years."
Katherine Sturtevant studied creative writing at San Francisco State. She is currently living in Portland, Oregon.
Joyce Trebilcot teaches women's studies and feminist philosophy at Washington University in St. Louis.
Terry Wolverton is a Los Angeles writer and a director and producer of feminist theater. She is also the co-director of the Lesbian Art Project, an exploration of lesbian sensibility. Susan L. Yarbrough teaches women's studies and legal studies at the University of Massachusetts in Amherst.
Second Class, Working Class
What it is like to join the union in Mexico . . . take off the veil in Iran . . . strike in Belgium . . . be a prostitute from Thailand . . . have an abortion in Italy.
An anthology of original documents, analyses and interviews on working women. Articles selected from the international women's press and translated for the first time into English.
$3/issue if ordered before Nov 30, 1979
(add $5.00 for postage after Nov 30, 1979)
Inquire for rates on quantity orders
Order from:
Peoples Translation Service
4228 Telegraph Ave.
Oakland, CA 94609
off our backs
an independent women's newsjournal
women in struggle
politics, health, work, prison
news coverage and political analysis on the issues that affect women's lives
contributing sub $12 or more
one year sub $6 sample copy $6
foreign $13 Canada $7
business and institutions $10
oob, 1724 20th st. nw,
wash. dc 20009
Sinister Wisdom issues 6 through 11 are now on cassette tape. The recordings are made in the style of Recordings for the Blind, with the donated labor of Susan Wood-Thompson; she reads the text, describes the graphics, etc., for women who cannot read the print copies. Each taped issue costs $3, and a year's subscription costs $12. Make check payable to Susan Wood-Thompson and mail your order to Susan Wood-Thompson, Box 6516, T Street Station, Washington, DC 20009.
SINISTER WISDOM POSTER STILL AVAILABLE
In the spring of 1977, a Tee Corinne solarized photograph of two women making love appeared on the cover of Sinister Wisdom 3, followed by a deluge of requests for a poster. The poster was printed in the summer of 1977: a duplicate of that cover, black on gray, 17" x 22". You can have your own for a contribution of $3.00 toward the survival of Sinister Wisdom plus 50 cents to cover mailing costs. (They make nice gifts for friends, too; bulk rates available.)
Send $3.50 per poster to: Sinister Wisdom, Box 30541, Lincoln, Ne. 68503.
Also available in feminist bookstores.
Sinister Wisdom
Box 30541
Lincoln, Ne. 68503
I would like to subscribe to Sinister Wisdom.
______ Enclosed is $7.50 for one year (4 issues), beginning with issue 12
______ Enclosed is $13.00 for 8 issues, beginning with issue 12
NAME ____________________________________________________________
ADDRESS _______________________________________________________
_____________________________________________________________ ZIP _________
Enclosed is ___________. Please send SW to my friend.
NAME ____________________________________________________________
ADDRESS _______________________________________________________
_____________________________________________________________ ZIP _________
SUBMISSIONS
*Sinister Wisdom* welcomes unsolicited manuscripts and art work. Please type (double-spaced) all written work. Reviews should be no longer than 3500 words; articles no longer than 5000 words. Enclose self-addressed, stamped envelope and a 2-3 line description of yourself. At this time, we can pay only with copies of the issue in which your work appears. Send all manuscripts except poetry to *SW*, Box 30541, Lincoln, Nebraska 68503.
*Sinister Wisdom* will not be accepting poetry manuscripts again until after March 1, 1980. After that time, send poetry to Susan Leigh Star, poetry editor, 52 Mars Street, San Francisco, California 94114.
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BACK ISSUES, WHILE THEY LAST
**Issue 4** (Fall 1977): stories of mothers and daughters and witches and lovers; Joanna Russ's tale for the girlchild in all of us; Lesbian separatism from the inside; photo-essay; interview; reviews, letters and poetry. 96 pp., $2.25.
**Issue 5** (Winter 1978): Susan Leigh Star, “Lesbian Feminism as an Altered State of Consciousness”; Judith Schwarz, “Researching Lesbian History”; Michelle Cliff on speechlessness; Lesbian Day speech by Barbara Grier; fiction; poetry; photo-essay; reviews; interview. 104 pp., $2.25.
**Issue 6** (Summer 1978): Julia Stanley, Mary Daly, Audre Lorde, Judith McDaniel, Adrienne Rich on language and silence; Marilyn Frye on separatism and power; fiction by Sandy Boucher, Thyme Seagull; poetry; interviews; essays by Sarah Hoagland and Peggy Holland; drawings and photos. 104 pp., $2.50.
**Issue 7** (Fall 1978): myths, monsters, teeth, etc. Jane Caputi on patriarchal fish stories; Judith Schwarz on being physically different; self-portraits in prose and poetry by Alice Bloch and Susan Wood-Thompson; feminist musings by Melanie Kaye; plus a wealth of fiction, poetry, reviews, drawings, and photographs. 104 pp., $2.50
**Issue 8** (Winter 1979): “did you say lu-uv?”: storytelling by Sherry Thomas and Audre Lorde; photographs by Deborah Snow; poetry by Judy Grahn; notes on deafness; essay on “that place where nothing is”; interview; reviews; “Scrambled Eggs”; and much more poetry and fiction. 104 pp. $2.50.
**Issue 9** (Spring 1979): JR Roberts on the history of *dyke*, Michelle Cliff on anonymity, Melanie Kaye on violence, Susan Cavin on Lesbian origins, Bertha Harris, Harriet Desmoines, and Irena Klepfisz on Lesbian literature; fiction by Becky Birtha, Jan Clausen, Linda Marie, Kathryn Missett, Ann Shockley, Pearl Time’sChild; an outpouring of fine poetry; photography; reviews; and a puzzle. 104 pp. $2.50.
**Issue 10** (Summer 1979): Special issue on being old and age, guest-edited by Susan Leigh Star. Includes Barbara Macdonald and Baba Copper on their 65th and 59th year, respectively; Audre Lorde on surviving cancer; interview with Mabel Hampton; essays on nursing home women, the time trip, being under 21; poetry; photo-essay; fiction. 104 pp. $2.50.
(Add 60 cents postage for every 1-2 copies ordered)
no. 11 fall 1979
2.50
|
WIN A VIDEO RECORDER!
MARCH 9, 1985 45p
DOUBLE EXCLUSIVE!
TINA TURNER
tells
PETE BURNS
blabs
GARY NUMAN + TC CURTIS GO WEST
STUBBLE, BUBBLE, toll and trouble: as a million small girls recoil in horror at Georgie Michael's recently acquired beard, RM presents its exclusive guide to pop's flirtation with facial hair. Was there a time when Holly Johnson couldn't afford a Jean Paul Gaultier designer razor blade? Was Bowie's 'Baal' look a close shave with poor taste? What about Marilyn? And has Robin Smith got a chin? Index says: Hirsute y'self.
LOST LOVED ONES is a tragic name for a band — containing a sense of unfulfilled promise. But the same can't be said for the musicians. Leader Terry Adams (vocals and bass), Robert Downes (guitar) and Alan Rowland (drums) are ploughing a fertile furrow in the renaissance of guitar rock.
Already a support slot to the Alarm last year, their live sessions on Radio One have earmarked them as Lost Loved Ones to watch. Check out their new single 'Raise The Flag'.
ANYONE OUT THERE with single vision? Or to put it another way, have a listen to the current 45 'Tears Idle Tears' from Sheffield hotshots Vision. Produced by ace knob twiddler Martin Rushent, it's backed with the indie favourite 'Lucifer's Friend' from the days when they were going under the moniker Spiral Visions.
The line up includes original members Andy Beaumont (keyboards, flute) and Gary Gillott (drums, percussion) with Jeremy Cousans (lead vocals), Gary Steadman (guitar, keyboards, vocals) and Pete Dineley (bass, vocals). Seeing is believing.
EIGHTH WONDER are something entering your life pretty soon. It's inevitable really, big management, skyscraper talk from record companies and a face to shift the product. Eighth Wonder are saccharine pop, Motown and Blondie and youth ... they've got plenty of that. Jamie Kensit (guitar), Jeff Beauchamp (lead guitar), Lawrence Lewis (bass), Jake Walters (drums) and Alex Godsen (keyboards) play the music, Patsy Kensit, 16 year old vocalist, plays the face game.
An acting veteran before acne — 'The Great Gatsby' and Birdseye's Peapod Girl at five, 'The Corsican Brothers' and TV's 'Diana' recently, Crepe Suzette in the soon to come 'Absolute Beginners' — Patsy has all the assurance of the child star. So sure — "I want to be successful, I want people to like our records" — it's gotta come true.
The musicbiz might be cranking up to make Patsy a star, but for now she tells it strictly democratically: "Everybody knew each other socially, my brother (Jamie), Jeff and the bass player used to rehearse on Sundays then we found the keyboards player six months ago...
"I might get an idea for a song, then we'll all battle it out. We feed off each other's ideas really, everybody chips in. We're looking for a long term deal. Every group has a front person, but really I'm just a small part of the group."
You'll be hearing more.
AS THEY anxiously bite their nails hoping Norwich make the Milk Cup Final, the Farmer's Boys pass the time with another nifty five-gear, four-door 45, once again a BazFrogMarkStan original called 'I Built The World'. It's from their forthcoming second EMI album 'With These Hands'. A Mike Channon Wembley winner and the farming fellows high in the hit parade? I've been having these dreams for some time now, doc...
INDEX
COMPILED BY DI CROSS
Cover photography by PAUL COX
THE SMITHS go for Shakespeare, Alison Moyet goes for a Billie Holliday classic and Howie Jones gets a new haircut. Whilst elsewhere in the wacky world of pop, we've spotted a Strawberry Switchblade single, a leek donation to Band Aid, and this week's touch of seasonal madness brought to you by Morons.
TEARS FOR FEARS have added two more dates to their tour. They'll be appearing at Southampton Gaumont May 13 and Manchester Apollo 16. The Chippenham Goldiggers gig has been changed from March 21 to 26 and the Preston Guildhall show is now May 20 instead of 22.
The band have had to cancel their Irish dates due to pressing American commitments.
THE DEP International label skanks back into action with the single 'Knock Knock' from Mikey Dread out on March 11.
ALISON MOYET releases her version of the Billie Holliday classic 'That Ole Devil Called Love' on March 8. This, her fourth solo single, is produced by Pete Wingfield and is backed by 'Don't Burn Down The Bridge'.
It is available in seven and 12 inch formats, with a limited edition double pack with the extra single featuring 'That Ole Devil Called Love' and 'Twisting The Knife' recorded live at the Dominion Theatre for Radio One's 'In Concert'.
THE RELEASE date for Joboxers' single 'Is This Really The First Time (You've Been In Love)' has been put back to March 22.
AMII STEWART has an album and single out this week. The LP 'Try Love' is already a hit in her Italian homeland, and it includes a Dutch version of 'Friends' plus the current single 'That Loving Feeling'.
Watch out for some UK appearances in the spring.
GODLEY AND CREME take time off from the production of epic videos to release their first single for nearly a year. 'Cry' is co-produced by the duo and ace knob twiddler Trevor Horn. The B-side is 'Love Bombs' and the 12 inch will feature an extended version of 'Cry'.
Kev and Loi have an album scheduled for April release, and yes, there is a video to accompany the new 45.
GRANDMASTER MELLE MEL AND THE FURIOUS FIVE
CAMDEN PALACE
CAMDEN HIGH STREET
SUNDAY 10th MARCH 7.30pm
TICKETS £5.00 ADV. AVAILABLE FROM THE BOX OFFICE (TEL: 01-387 0428) AND USUAL AGENTS
BRYAN ADAMS
STOP PRESS...
SPECIAL HEADLINE SHOWS
HAMMERSMITH ODEON
APRIL 19th & 20th
SOMEBODY
THE NEW SINGLE
7" & 3-TRACK 12" VERSIONS RELEASED THIS WEEK
12" INCLUDES FULL COLOUR U.K. TOUR POSTER (LIMITED EDITION)
FROM THE NEW ALBUM "RECKLESS"
SHAKESPEARIAN SMITHS
- MORE FOOD for thought, as those lovable Smiths release a single 'Shakespeare's Sister' on March 18. The previously unreleased Morrissey/Marr masterpiece is flipped with 'What She Said', from the lads' chart topping 'Meat Is Murder' album. The 12 inch has the additional track 'Stretch Out And Wait' — another first timer for you collectors out there.
The sleeve depicts that model of Mancunian womanhood Pat Phoenix.
- CURRENTLY HAVING a rest after wearing the boot leather thin on a jaunt round Britain, King will be back in time to see the release of their single 'Won't You Hold My Hand Now' on March 11.
The first 10,000 copies come in a poster bag, and the B-side is a live version of 'Fish', recorded at the Glasgow gig during their current tour. There will also be a 12 inch three track dance 'Youth Mix'.
- RUNNING MAN Bryan Adams brings out a single 'Somebody', with a limited edition 12 inch featuring a hunky Bry poster.
He will also be playing two headlining dates at London Hammersmith Odeon on April 19 and 20. Tickets are priced £6.50 and £5.50.
MURRAY HEAD recovers from 'One Night In Bangkok' for a special one off concert at London Dominion Theatre on April 2. Tickets priced £6 and £5.50 are available from the box office and usual agents, or on the credit card hotline 01-439 3371.
SHAKATAK'S KEYBOARD maestro Bill Sharpe releases his first solo LP 'Famous People' on March 15. The 10 tracks include his current Numan partnered hit 'Change Your Mind'.
A SEVEN minute remix of the Commodores current top 10 hit 'Nightshift', is being rush released by Motown this week. It will replace current supplies of the 12 inch as they run out.
MORE DOTS before the eyes, as Strawberry Switchblade follow up their 'Since Yesterday' hit with another original track 'Let Her Go'. The seven inch B-side is 'Beautiful End', with the extra track 'Michael Who Walks By Night' on the 12 inch.
TRICKY NICKY HEYWARD follows up his hit 'Warning Sign' with the track 'Move It Up' on March 22. The B-side is 'Don't Go, But Don't Stay Either', and the seven inch is available in a poster bag. There will be a different poster available with the 12 inch.
ACTION MAN
- HAPPY MAN Howard Jones, sporting a new haircut, releases his second proper album on March 15. 'Dream Into Action' features 12 tracks, including his current hit 'Things Can Only Get Better'.
Produced by Rupert Hine, the all original tracks also feature Howie's brother Martin on bass.
Those of you who were unlucky in your quest for tickets for the man's April 16 Wembley Arena bash, cry no more. An extra date has been added on the following night. Tickets priced £7.80 and £6.80 are available from Howard Jones Concerts, PO Box 77, London SW4 9LH. Make cheques and postal orders payable to Howard Jones Concerts and enclose an sae. Tickets are also available by personal application to the Wembley Box Office.
PURPLE
- HIS PURPLE Highness Prince, fresh back from collecting three Grammy awards in Los Angeles, will be painting the small screen purple with the unleashing of his 'Purple Rain' flick on video. It should retail for around £37.50.
HRH recently played a two hour concert for 3,000 physically and mentally handicapped children in Santa Monica. One of the songs featured in the concert was 'Tears In Your Eyes', which he has donated to the Ethiopian appeal. It will appear on a special fund raising LP in the spring.
- WITH POP'S baby boom well under way, Pat Benatar announces the birth of a baby girl and a single 'Love Is A Battlefront' from her album 'Tropico'. Alison Moyet is expected to get sprog handed any moment now. Plenty of hot water, please!
- BIG SOUND AUTHORITY are rescheduling some dates on their tour due to the ill health of two of the members. New dates are Plymouth Academy March 6, Cardiff New Ocean Club 13, Swansea University 14, Birmingham Powerhouse 19, Brighton Top Rank 20, London Hippodrome 21 and Leicester Polytechnic 22.
- KING KURT have added some regional dates to their tour at Manchester Cloud Nine March 12, Edinburgh Coasters 13, Aberdeen Victoria Hotel 14 and Glasgow Queen Margaret Union 15.
SIGHTINGS OF circular black plastic objects coming into the earth's atmosphere have been positively identified by NASA as the current Morons single 'Moons From Outer Space'. Is there no end to this comic commotion?
The charts invaders on this occasion are none other than Griff Rhys Jones, Mel Smith, Jimmy Nail (Oz from 'Auf Weidersehen Pet'), Paul Bown and Joanne Pearce.
ABSOLUTE REALITY
IS A LIMITED EDITION DOUBLE PACK FROM THE ALARM
THE ALARM DOUBLE PACK INCLUDES 'ABSOLUTE REALITY' 'BLAZE OF GLORY' PLUS TWO NEW TRACKS IN SPECIAL GATEFOLD SLEEVE
* ALSO AVAILABLE ON 7" & 12"
THE ALARM ON TOUR
APRIL
21. COLSTON HALL, BRISTOL
22. ST GEORGE'S, CARDIFF
24. GUILD HALL, PORTSMOUTH
25. ARTS CENTRE, POOLE
26. THE DOME, BRIGHTON
27. APOLLO, OXFORD
29. UNIVERSITY OF EAST ANGLIA
30. ROYAL COURT, NOTTINGHAM
MAY
1. ROYAL COURT THEATRE LIVERPOOL
2. CITY HALL, SHEFFIELD
4. BARROWLANDS, GLASGOW
5. CALEY PALAIS, EDINBURGH
6. CITY HALL, LEICESTER
7. MAIN HALL, LEEDS UNIVERSITY
9. APOLLO, MANCHESTER
10. ODEON, BIRMINGHAM
11. ODEON, HAMMERSMITH
13. PALAIS, HAMMERSMITH
STING MADE his first solo appearance in New York last week prior to the commencement of recording for his solo LP. Backed by famous NY jazz musicians — Omar Hakim, drums, Branford Marsalis, trumpet — Sting played three sell-out nights at the Ritz. The set included four new numbers, old Police songs and some blues standards. From NY Sting flew to Eddy Grant's studio in Barbados where he will be recording for five weeks before going to Paris to mix his album. Sting will be taking the set out on the road in the summer.
THE BOOTHILL Foot Tappers follow up their excellent 'Get Your Feet Out Of My Show' with the single 'Jogging' on March 15.
The band will be touring to promote the record. Catch them at Leeds University March 12, Bradford University 13, Hull University 15, London Dingwalls 16, Leatherhead Riviera 17, Liverpool University 21, Salford University 22, Sheffield Pantehouse 23, Leicester Polytechnic 24, Glasgow Tech 29, Aberdeen Victoria Hotel 30 and Edinburgh Hoochie Coochie Club 31.
'COULD IT Be I'm Falling In Love', the old Detroit Spinners classic, has been covered by David Grant and Jaki Graham. Available from March 15, it was produced by Derek Bramble, co-producer of Bowie's 'Tonight' album.
THE POET laureate of pop, Paul Weller, is asking for contributions for another collection of art, poetry and prose planned for his publishing venture Riot Stories. Send artistic and literary efforts to Dave Potter at Riot Stories, 45-53 Sinclair Road, London W14, enclosing an sae for returned work.
THE RAMONES are celebrating their highly successful visit to the UK with a new version of their single. It will have 'Chasing The Night' and 'Howling At The Moon' as suitable A-sides. It will be 5,000 copies with a gatefold sleeve, with a bonus record featuring 'Smash You' and the Stones' 'Street Fighting Man'.
OLD HAND Eric Clapton releases an album "Behind The Sun" on March 15. All but two of the 11 cuts are produced by Phil Collins.
DANCE MAESTROS Second Image release their debut LP 'Ove Mexico' to coincide with a tour. A single 'Starting Again', from the album, will be out on March 10.
The tour kicks off at Colchester Embassy Suite March 10, Bournemouth Academy 11, Harrow Weald Middx and Herts Country Club 12, Birmingham Bobby Brown's 14, Maidstone Greenways Hotel 15, Leicester Mr Keisa's 16, Luton Pink Elephant 20, Yeovil Electric Studio 22, Slough Fulcrum Centre 23, Southend Queens Hotel 24, Sunderland Barbary Coast 25, Wakefield Casanova's 26 and London Dominion Theatre 27.
FRIDAY's 'Tube' (C4, 5.30pm) has Dead Or Alive and the Bluebells live, plus an interview with ol' rock'n'roller Little Richard. 'The Oxford Road Show' (BBC 2, 7.30pm) is presented by that man with the social conscience Billy Bragg and will feature The Alarm plus new group Red Guitars.
SATURDAY'S 'Saturday Superstore' (BBC 1, 9am) has the latest video from Alvin Stardust, then switch channels for 'No 73' (ITV, 10am). 'Punk To Present' (Radio 1, 1pm) looks at some of the old songs and sounds revived today, while 'The Other Side Of The Tracks' (C4, 6pm) has Paul Gambaccini looking at the careers of ex-Eagles Don Henley and Alison Moyet. 'In Concert' (Radio 1, 6.30pm) features Yes Let's and Go West.
SUNDAY has Andy Partridge and Keren from Bananarama among the contestants in 'The Great Rock 'N' Roll Hit-via Quiz' (Radio 1, 4.30pm).
TUESDAY finds controversy on 'Whistle Test' (BBC 2) with the Jesus And Mary Chain.
THE EUROPEAN Economic Community has finally benefited the British music charts. The organisation responsible for the wine lake and the beef and butter mountain has invented a 'Shaft' skyscraper. Isaac Hayes' 14-year-old 'Theme From Shaft' has been given a new lift by two versions — from Belgium and Holland respectively.
The original was composed for the soundtrack of the film 'Shaft', starring Richard Roundtree in the title role as a black detective. The single won an Academy Award for Best Song, a Golden Globe and four Grammies: the film, a couple of sequels — 'Shaft's Big Score' and 'Shaft In Africa' — and a TV series.
The front runner in the race for chart glory is the Dutch entry by Eddy And The Soulband. The Eddy in question is 28-year-old Eddy Conard; a native of Gary, Indiana — the home of the Jacksons and Deneice Williams.
In fact they all attended the same school and Eddy claims to have been big friends with Deneice — the 'Let's Hear It For The Boy' girl.
It was in the early Seventies that Eddy took up residence in Holland while studying sociology at university. At 20 he took up playing percussion and ended up in bands that supported the likes of the Supremes, Stylistics, Natalie Cole and Crown Heights Affair.
As his reputation grew he became a respected session musician and later a producer. His first production became a Dutch number one. It was an inevitable move to become a vocalist.
He took a remake of Van 'Hustle' McCoy's 'Soul Cha Cha' to the Dutch number one slot in 1983. He claims that a chance remark by a Dutch radio DJ gave him the inspiration to re-record Isaac Hayes' homage to the "black private dick that's a sex machine to all the chicks."
The Belgian entry for the Eurovision 'Shaft' Contest is from Van Twist — a three piece group consisting of Mike Delanney, Jean-Marc Vederman and the enigmatically monickered Jenny B.
Bass player Mike is not a man to be messed about with. He can boast the fact that he's a karate champion and can obviously be relied upon to know his chops well enough to supply the punch behind the band. Like Mike, dancer Jenny B is from Africa. She provides the vocals in the band.
But it's Jean-Marc who provides the credibility, having used his keyboard skills with Fad Gadget, Matt Johnson's The The and Gene Loves Jezebel. He was also the founder member of the cult group Kid Montana. Can ya dig it?
MIKE GARDNER
GIRLS LIKE Tina Turner because they all want her like that, a voice like that, sex appeal like that, looks like that, and they wouldn’t mind them now, never mind when they’re 45. Boys like Tina Turner for practically the same reasons, excepting perhaps the legs and adding on a few fantasies about being dominated by an older, aggressive woman.
Most men love being intimidated but also love being imply-coddled. Tina Turner can manage both these things because, let’s face it, she’s a bit of a goddess. Never mind yer Sades or Kinks, Kirsty and Abba, this is the real thing.
She’s larger than life, but very warm and accessible at the same time: see her vamping it on Wogan, then melting him into submission; see her on the BPI Awards, lapping up the applause, custodizing in that dress, but also seeming distant and unattainable. If you don’t like her music, you must admire the energy. Who doesn’t think of saying ‘she’s amazing for her age?
Having whizzed through London for chat shows and awards ceremonies, Tina Turner is now in Norway.
It's snowing in Oslo, Tina's just done a press conference following the first night of her European tour in Helsinki.
She's veritably bubbling this cold and frosty day, talking ten to the dozen. She's still grinning about her Grammy triumph. Three awards: Record Of The Year and Best Pop Vocal Performance (Female) for 'What's Love Got To Do With It' and Best Rock Vocal Performance (Female) for 'Better Be Good To Me'. Ironic, perhaps, that Tina was also nominated for Best R'n'B Vocal for 'Let's Stay Together', but didn't win it.
Tina is professional, but doesn't throw platitudes at you. Still, everything is great: "The reviews in Helsinki were great," she enthuses. "They're translations of course, but they're great."
So now The Big UK Tour is nigh: about time? Absolutely. "I've never done a tour like this before, it's incredible," the lady enthuses even more. "I don't believe it yet... it's unbelievable that I'm finally doing something like this after all this time." Oh, but it's never too late... "Once I see it I want to photograph it. I can't wait. Those people have all supported me so much."
Even in Norway, she's just taken delivery of a gold LP. "I seem to be getting awards everywhere," reveals a genuinely astonished Tina. "People love it," (the album, 'Private Dancer', of course) "they seem to be buying it at least twice over, when they wear the first one out."
But it's her mega success in America that makes her feel all funny inside: "That was a total surprise, it feels wonderful. It felt so good that they finally realised I have talent. It feels good to have success worldwide, but to have success at home, where my roots are, was the best thing."
Momentarily sidestepping her golden wall of discs and awards, we come to the sixth (yes, sixth) single from her album, the Ann Peebles classic 'I Can't Stand The Rain': "I'm not a fan of Ann Peebles, I just liked the song when a demo was given to me with a new arrangement. It's the only song I know of hers, the one that reached me; it really gets to you.
"It'll be the last single taken from the album — it's been like pulling rabbits out of the hat, I know. The new LP will be out in April/May."
Tina's always favoured English musicians, and she'll be using more on the newie — exactly who is under wraps at the moment, though Rupert Hine and Terry Britten will still be around. Are they all responsible for her huge crossover? "I'm not trying to do so many different styles," says Tina, "but I'm determined not to do r'n'b at the moment, it's a change in my life. I get that crossover because of the people I'm using, yes, they're not r'n'b people. It's OK, because it's accepted that I do r'n'b vocals, I have credibility as that kind of performer. At heart, I'm still rhythm and blues, it's my soul."
Tina didn't actually win anything at the BPI Awards recently (surely some mistake) but nevertheless she was conspicuous there, flashing her teeth and her pins at the adoring throng. "I had to attend," says Tina, "because of the energy that Britain has, and because it all started here. Being a part of it was great, I had to be a part of such an extravaganza. I feel at home here; I have a great feeling for London because so many good things have happened here."
Now, we all know that the B-side of 'I Can't Stand The Rain' is 'Let's Pretend We're Married', a outsome ditty penned by now neolegendary perfectly-formed minxie purple hero Prince. There's something of a mutual admiration society between these two bundles of sensuality; a duet would be well in order. But have they actually ever met?
"No, I've never actually met Prince," admits a disappointed Tina, "not even at the BPI Awards. I should try and defend him, though — he's very shy, he doesn't meet anyone.
"He came to see my show in his home town but he didn't come and say hello to me; all his people did, but he couldn't muster it. It's hard for people to understand — people say 'speak up, boy, they don't understand him. He's brilliant... maybe one day I'll meet him."
Which brings us neatly to the other megahunk in her life, the equally perfectly formed and magnificently proportioned Mel Gibson. Yep, Tina's just completed her guest role in the third of the Mad Max series, tantalisingly titled 'Beyond Thunder Dome'. Yum yum. How was it for you, Tina? "It was a pleasure for me to be a guest in Mad Max," she sizzles. "The director, George Miller, was the one who said I could do it, and he got the performance out of me, although it took time for me to settle into it.
"It was an exciting part — very strong, very physical, but it was straight drama. I've never really done that before. Mel Gibson was great — a real rock'n'roll actor, all that leather. He's beautiful, and cute as well, a great actor. He really helped me sink into the part, and he and I are close friends now."
What's the part like? "I play a queen who owns a town after a nuclear war, this warrior woman that drives cars, climbs rocks... it's very complimentary to Tina Turner, a chance to play someone similar to my own character."
But you played the Acid Queen in 'Tommy', wasn't a little bit of that a little bit of you? "Yes, but this is a different sort of queen. I don't like everyone to think I'm always playing a queen. Oh, and yes, there are some great costumes." Bate your breath folks, for the film, to be unleashed in July in America, December over here.
But first things first. What excitement do we have in store when Tina tours? "I'm doing every song on the LP in the show, except '1984'. I've got Tim Capello on sax, Phil Palmer on guitar, and Jamie West-Oram from the Fixx. I can't say how things'll go, I'm using a particular strategy and I want it to be a surprise."
Which leaves just one thing. We've discovered, courtesy Tel Wogan, that there's no truth in the Keith Richards-esque rumour that Tina has her blood changed twice a year. So how does she keep mind and body together in such harmony? Look to the East, butterflies: "I've been a Buddhist for 10 years," she confirms. "My first phase of teaching came then, I was started on chakabudl by a friend. It's made life better for me, it helps me to centre myself. I know where I'm going, it helps me make decisions and there's less stress in my life. I deal with life more calmly. I was asked to explain how it affects me exactly, but it deals with consciousness, cleaning out old habits and giving new life. It's about spiritual renewal."
And with that reassuring note, she's whisked off the line to meet more record company people, more press, and to collect more gold discs and awards. Life is very hectic for TT, but she deals with it all charmingly and without a hint of cynicism. A genuine enthusiasm pervades her every word.
She's had success and years in the wilderness, but has survived it because she's come to terms with her inner self: with a healthy mind, a healthy body always follows (courtesy happy dope oil). Still I bet every girl wishes she'll look as good as Tina will at 50. And a few boys, too.
WHEN DAVID Cassidy smiled, a million hearts smiled with him. When he laughed, we laughed too. Pre-pubescent fans would follow him everywhere — leaving a trail of broken concert halls, satin scarves and puddles on the floor when the excitement got TOO much.
Aah, the swoons, the screams, the spots, the Partridge Family... the good old days of teenybop. Eleanor Levy goes gooey reminiscing with the George Michael of the Seventies, David Cassidy
David is deadly serious about his comeback. He spent much of the last 10 years acting, and it was while on stage on Broadway that he realised how much he missed being in front of an audience. He readily admits he missed the adulation, but more importantly, he says, he feels he now has a chance to make a record in which he isn't "creatively" stifled.
"I have a renewed enthusiasm for making this record," he explains earnestly. "I've made a lot of albums in my life — but it's almost like I never have. This is 'mine'. If you love it or hate it, for me it's the purest record I've ever made. It's revealing who I am and what gets to me.
"I have been somebody in the constant pursuit of romance. It's not just a romantic album — it's not strings and stuff — it's about somebody who seeks passion. And losing one's innocence. A thread through my life has been the pursuit of romance."
Why?
"Because I'm an extremely passionate person. I love it, it's what makes me tick. It makes me feel good and alive." Silly me for asking.
As an old David Cassidy fan I blushed deeply with joy on hearing he was making a new record. But will others react in the same way? David is quietly confident they will:
"People have a knowledge of who I was and I think there is a certain curiosity as to 'what is he doing now, what does he look like, what is he singing?'"
"I like being on the edge, I like risk taking. Every time you close a door another door opens. Every time you really stand on the edge and think you can fly, you can fly.
"It's a great compliment to have had people love you — and it's easy to put people down who are in that position. They did it to Sinatra, they did it to Elvis, they did it to the Beatles and they did it to me. They'll do it to Wham! and Duran Duran and the next one that comes along too. But talent survives.
"It's so stupid when I read something lousy about George Michael because I've been there and I like him — he's a nice fella. And he's a very good writer. This is the music business, let's face it. (Yeah, really deep, Dave — Powerful Insights Ed). This is not a lovely little artistic business any more. Reality is that stars are stars — whether you like it or I like it, how can you deny that?
"As I look back I did some incredible things. I left it, you see, it didn't leave me and that's very important.
"What I remember is standing at the focal point of all this incredible energy with thousands of people screaming and yelling. You can look on that as an hysterical thing, but what it is really is an unbelievable exchange of love. To stand there and have people screaming at you that they love you is unbelievable, you have no idea."
Happens all the time David, happens all the time.
kiss me
stephen tintin duffy
7" & 12"
available now
produced by jj jeczalik & nicholas froome
limited edition
7" double pack
includes both old & new versions of 'kiss me'
out now
SINGLES
Reviewed by Mike Gardner
SINGLE OF THE WEEK
PHILIP BAILEY AND PHIL COLLINS 'Easy Lover' (CBS/Virgin) Now here hangs an interesting tale. This duet from the Earth, Wind And Fire's top falsetto and the slightly talented Genesis sidesman was nearly refused release. Virgin and CBS, who hold the contracts of the pair as individuals, had agreed a November 1984 deadline for releasing this track from Bailey's solo album 'Chinese Wall' so as not to clash with Collins' work. CBS declined the offer and the deadline expired. The disc then proceeded to go crazy worldwide. Protracted negotiations secured the release with both companies sharing in the profits. You may ask, is it worth the hassle? You bet your jellybeans. It's slick, smooth and as addictive as a packet of peanuts, and far less fattening.
GOOD 'UNS
ALISON MOYET 'That Ole Devil Called Love' (CBS) The lady sings the blues, and very good it is too. This lazy band styled song is as relaxing and refreshing as a neck massage from your favourite partner. Anyone who's been to Basildon will understand why its inhabitants sing the blues — but it isn't usually this good. The B-side — 'Don't Burn Down The Bridge' — is an excellent evocation of Aretha Franklin at her devil-damning best. By the way, with Alf. Benatar, McColl and Hynde all producing excellent records and children, shouldn't 'Top Of The Pops' producer Michael Hurll think about installing a creche... or have they got one already for the use of the DJs?
AL GREEN 'Never Met Nobody Like You' (Hi) Anyone who's croaked out a tune in the bath must envy the vocal talents Al Green's been given. If you were deaf for your whole life, except one day, then Al's got to be one of the first things worth cramming into your shell-like. There's not many singers who can make average material like this sound as vital as taking your next breath. Wonderful.
MAXI PRIEST AND CAUTION 'Should I' (10) Reggae has been down so long, with few exceptions, that it has only one way to go — up. Following in the skanking footsteps of Barrington Levy and Smiley Culture comes Maxi Priest's reggae chart topper. It's worthy of anybody seeking aural refreshment.
NIK KERSHAW 'Wide Boy' (MCA) Carp all you like about his lack of passion on vinyl and his kiddie following but there's no denying his deft touch in constructing insidiously catchy melodies. He's aided by a smart line in lyrics that sound interesting, even if they don't mean much, and another slick production. Mark it down as one to guiltily hum on the bus while reading the credits to your newly acquired copy of 'Meat Is Murder'.
THE REST
THE POWER STATION 'Some Like It Hot' (Parlophone) Now here's a turn up for the books. The result of the power group collaboration with Robert Palmer, Duran's John and Andy Taylor and Chic's Tony Thompson and producer Bernard Edwards sounds exactly like... wait for it... Robert Palmer singing a latter period Chic song. It's a cool groove that's livened by a smart chorus and a ripping guitar solo from the 'Beat It' school of finger pyrotechnics. File under — no disgrace but a definite underachiever.
BRYAN ADAMS 'Somebody' (A&M) Coming from the land of the lumberjack and the mountie, Bryan's rawk'n'roll is suitably muscular. Luckily he has a light touch and a likeable drive that prevents it going the sludgy way of most 'Stations' releases. It's not quite real meat but it's more substantial than a hamburger.
CHANGE 'Let's Go Together' (Cool Tempo) Jacques Fred Petrus's spin-off disco machine — so bad because his early records had backing tracks recorded in Bologna, Italy — pushes out a singularly undistinguished piece of slowburn disco. It's a great production and a creamy female vocal lead in search of a strong song.
CHINA CRISIS 'Black Man Ray' (Virgin) Donald Fagen's old Steely Dan partner, Walter Becker, produces this soft rock effort — and it shows. Those soft harmonies and instrumental motifs show strong Steely Dan influences. Unfortunately, like all Dan material, this easy going opus grows with every play.
ROCKWELL 'He's A Cobra' (Mercury/Mary Jane Girls In My House) (Motown) Poor old Rockwell seems, on this form, to be destined to be a one-hit-wonder. The record suffers from terminal averageness. Rick James can't inspire much enthusiasm for the equally one-hit-wonderish Mary Jane Girls, having served them with a nursery rhyme over a reharrfed riff from his 'Street Songs' period.
OLIE AND JERRY 'Electric Boogie' (Polydor) After the sheer sparkle of last year's ' Ain't No Stoppin' ', this disappoints. As flat as a Dutch landscape and so laboured as to be of use only to an arthritic breaker.
SIMON MAY 'EastEnders' (BBC) There are many annoying things in life. There's the clashing schedules of 'Auf Weiderschen Pet' and 'Hill Street Blues' on Saturday; the fact that Spurs haven't played a home league match since December; and the demise of 'Dr Who' and 'Star Trek' to make way for Wogan's drivel and this dismal parade of stereotypes. The tune? Well, it ain't quite 'Dr Kildare' or 'Blockbusters' or the aforementioned 'Hill Street Blues' (which, chided sequence it borrows in places) but it's far better than 'The Price Is Right'.
SHEER HEAVEN 'Touch — I Am Rhythm' (EMI) This is EMI's version of the Weather Girls... I kid you not. The Nottingham pair weigh in with 34 stone and a so hi-NRG stomper that lacks both personality and punch.
URIAH HEEP 'Rockarama' (Portrait) On this form these doyens of rock's second division are doomed to relegation — this being limp heavy metal with the musicality of coffee beans in a grinder.
EVELYN 'CHAMPAGNE' KING 'Give Me One Reason' (RCA) All those expecting the bubbly Ms King to produce another stone cold classic like 'Shame' or 'Love Come Down' shouldn't waste too much time delving into this piece of pop/soul ordinaire.
JOURNEY 'Only The Young' (Geffen) Is this really Journey doing a passable Moody Blues impersonation or is it time for my ear syringe? This is part of the soundtrack for the film called 'Vision Quest'. With all due respect, it'd be better on 'Vision On' where at least the majority of the audience wouldn't have to hear it.
JONATHAN PERKINS' SILVER SPURS 'Believe In Me' (Checkmount) Normally I'll always support anything to do with Spurs but after Perkins' great track record, XTC founder, the fabulous Origin Of Movers and live work with Toyah and Police — this is as disappointing as finding no loo roll in the toilet.
IT'S NEW.
MICK JAGGER
SHE'S THE BOSS
GOT IT?
ON RECORD OR CASSETTE £4.99
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WHSMITH
Price correct at time of going to press. Subject to availability. Available where you see this sign.
WIN A VIDEO RECORDER
Fed up with sitting round Wayne's mum and dad's watching the umpteenth slow motion repeat of Prince at the BPI Awards? Sick as a parrot with having to beg and plead to use Darren's really naff hired VHS that's well out of date? Fret no more, video buffs, help is at hand! Yes, your magnificently magnanimous RM offers you the chance to become the most modular person on your block!
By answering one supremely simple question, you can scoop this nifty package: one Ferguson Videostar Compendium Video Recorder plus five top music videos.
Here's more: The Ferguson Videostar 3V52 is a brand new model retailing at about £650 and is just 70mm high, with a removable tape deck. This means all you budding Godley and Creme's can match the recorder with a video camera to make up a portable home movie unit.
The plodders can simply use the whole unit as a portable recorder; y'know, retire to the black and white in the boudoir when the pets get too involved in 'Thriller'.
And these videos to start your library: Tina Turner's 'Private Dancer' EP, Meat Loaf's 'Hits Out Of Hell', U2's 'Live At Redrock', Queen's 'We Will Rock You' and Ultravox's 'The Collection'.
And they're the subject of our extremely easy competition clincher: Who directed Ultravox's epic 'Vienna' video? Clue: he recently won a BPI Award for his work with Duran Duran. First correctly spelt answer wins.
Your answer, plus name and address, to: RM Video Comp, 40 Long Acre, London WC2.
shake with LEVEL Shake
their second album
TC/V2320
CASHMERE 'Cashmere' (Fourth & Broadway BRLP 503)
SOMETIMES A previously unremarkable soul outfit comes along with a single so slick and self-assured that they suddenly expect the world from them — and when their album turns out to be only so-so, you're maybe unreasonably disappointed. That's the story with Cashmere.
Cash was one of the most stylish dance 'n'b records in ages with a very fine vocal by Dwight Dukes, and if TOTP had given it anything like a fair shake it'd have been a far bigger hit. Dukes comes through the whole LP smiling, of roses and real natural, comfortable and down-tempo.
But his voice is capable of more than it's asked, which is to take the band through a series of rather formalised dancers like 'Someone Like You', and 'Fascinating', and the obligatory weepie 'You're All I Need'.
If there'd never been a 'Can I', I'd probably be applauding this as a competent set, but with that gem, they got big by being ambitious and they never get back to that. Nothing coming five different production teams and backups from Eugene Wilde.
Paul Sexton
THOMAS McClARY 'Thomas McClary' (Motown ZL72349)
UNTIL ABOUT two months ago, everyone thought Thomas McClary had timed his jump off a sinking ship just right when he left the Commodores. Since then things have taken a dramatic turn. The hard facts about McClary's solo debut are that his songs are at least as good as his former band's material of the recent past, so hell, he thought, why sit around having them turned down, like the Commodores' pre-'Nightshift' work was? So it's no recommendation in itself; and that the sudden promotion from team member to player-manager is just too much for him.
Partly because the songs, pleasant and unassuming as they are almost without exception, just don't have that cutting edge they need to really draw blood, and partly because Thomas has an unfortunately thin and anonymous voice that lends very little character to his work. Apart from which he can't get his old writing partner Lionel Richie out of his system — he imitates the 'Sailin' country' sound on 'Marianne' and even copies one of his titles, 'Love Will Find A Way'. Not to be unrelentingly hard on the poor bloke, the single 'Thin Walls' was and is an adventurous experiment that deserved better, and 'Contagious' moves very competently. In his heart of hearts though, I wonder if he'd rather be singing 'Nightshift'.
Paul Sexton
AMII STEWART 'Try Love' (RCA 70042)
BETTER STILL, try getting a few decent songs together. Amii Stewart is the most boring singer I've heard since Kim Wilde. They've tried to market her as a poor girl as some kind of vampy and she even reveals a hint of pert nipple on the cover, but the songs are safe, cosy and as boring as hell.
Amii labours her way through all of them with about as much passion as a kipper. 'Dangerous Rhythm' is the most ludicrous song on the album. She sounds as if she's been prodded awake after having one too many. I would be embarrassed to hear this record being played down the Co-Op.
Robin Smith
UB40 'The UB40 File' (Virgin VGN 35111)
A YEAR in a group's life captured on vinyl, 1980 to be precise, the first 12 months of UB's unplugged grasp on socially aware grit-Brit skanking. The nuts and bolts of it all are: the debut LP 'Signing Off' plus free EP included therein, the single 'My Way Of Thinking' and their final single on Graduate, 'The Earth Dies Screaming'/ 'Dream A Lie'.
Delicious stuff sure, but more importantly, history neatly encapsulated - recording in Bob Lamb's home track studio, the first indie single in the UK, 'King Rock Against Racism'. The UBs have remained, give or take a Jerry Dammers, the most consistent popland mouthpiece of black and white unity. This is their dole wage, black unity beginnings and it speaks volumes for their current stature that they've barely shifted from the social/political environment in which they were formed.
Jim Reid
GO WEST member Richard Drummie has a special reason to give thanks for a recording contract — it saved him from jail.
Richard, who plays keyboards and bass on their successful debut single 'We Close Our Eyes', was closer to having to remember the numbers on a prison uniform than the numbers in the pop charts.
"I was living on the never-never," he says. "I had an overdraft, a loan and I'd run up a huge bill on Barclaycard. I'd even borrowed a lot from my friends so I was heavily in debt.
"I'd given up a good job to play music but I was soon in financial trouble. I had the credit agencies chasing me. They'd pester my friends in an effort to embarrass me into paying them.
"Our recording deal came the day before I was going to get banged up," he says. "I paid everybody as soon as we'd got signed. Luckily all my friends had faith in me and didn't mind helping me out."
Both he and partner Peter Cox (vocals, guitar and keyboards) were scraping a meagre living at the time.
"We got so bad we'd have to put garlic in baked beans to make it more interesting," recalls Richard.
"In fact we were so poor that we could only afford to keep putting a pound's worth of petrol in the car every time we went into the garage," adds Peter.
The irony is that Richard turned his back on a lucrative career in favour of poverty. He was a classified ad manager for a newspaper.
"I had everything," he says. "I had a company car, a flat overlooking the River Thames by Teddington and a good wage.
"I was going to be a solicitor. I could have made a fortune by the time I was 45 with a wife, kids and a house. I'm the argumentative type. I get on the case and get wound up about things. But I knew it wasn't what I wanted to do. I knew I'd be happier playing music."
RICHARD FROM Hammersmith and Peter from Richmond met on the London gig circuit and kept in touch. Peter then took the less precarious musical route onto the Mecca circuit in Sheffield, Coventry and Gloucester in a showband.
The band also included Mike and Shaun Ward, now the fulcrum of Floy Joy.
"It was the worst job I ever had," recalls Peter. "It's soul destroying to play to an audience that's not interested in what you're doing and can't wait for you to get off so they can have their disco back on."
"The company would send out a list of 15 songs from the top 20 each week which we would have to learn. We couldn't play anything that was a bit livelier. I remember once we had to audition before a manager. We played two really hot funk numbers — one by Chaka Khan and the other by the Brecker Brothers. We threw ourselves around the stage and put everything we had into it. The manager stood cool throughout. When we finished he said, 'I don't want to hear those songs again in my club.'"
Far from being the quiet life he found that danger lurked on stage.
"We had a riot in Gloucester," he says. "A rugby club got a bit excited and there were 20 arrests. One guy got a fractured skull. Even I got clobbered by some clever sod!
"The trouble was that the mark of a good band was whether they got people onto the dancefloor or not but they just wanted to hear records. We tried everything. We dressed in pyjamas for a pyjama party. We had Caribbean and Hawaiian nights. We even dressed in black lame — we looked like Kool And The Gang without the cool.
"You have to have some sort of ego to get up on stage when nobody's paying any attention. It's destroying. You've got to have given up any hope of success to really enjoy it. I definitely got into drinking heavily at the time — it was the only consolation."
Even the sleazy image of the resident musician pulling girls by the score is put into perspective by Peter. "I can't remember any of the band getting off with anybody in all the time I was there."
THE PAIR got together as a songwriting team and impressed quickly enough to get a songwriting deal. The publishing deal enabled them to make better demos.
"We had to Quick produce some of them," says Richard. "They were idols of ours at the time. They were a two-man act — now, that's boring but then it was very rare.
"At the time nobody wanted American music, especially from an English band," adds Peter. "The vibe then was to be rough, ready and loud. We were bland in comparison."
The pair also found it hard to get a name.
"We tried lots of names only to find that they'd been used. Every time we walked into our publishing company and proudly announced a name like Partners In Crime or Moving Pictures, they'd pull out a record sleeve with the name on. We were almost afraid to say Go West," admits Richard.
One of their demo tapes was spotted by an American A&R man looking through British publishing houses for songs for Pat Benetar. It was he who rushed them into signing a contract with Chrysalis Records.
Go West lined up in the starting blocks for the annual race as the big record companies launch their new discovery bands like the Roaring Boys, Spelt Like This and Dark City are attempting a media blitz that would embarrass even the Frankies.
Only Go West have managed any success. There's no denying the weight of robust marketing that's taken them into the charts. But there's also no denying the fact that 'We Close Our Eyes' is a good song.
The band have had a £90,000 video made by Godley and Creme — the first time they've ever worked on a debut single and only the third video they made in 1984. Even now they're planning their next video 'Call Me'
"We looked like Kool And The Gang — without the cool"
with top director Russell Mulcahy — the man behind many of Duran Duran's epics.
They've even had some of Frankie's styling team working on them. But the boys are quick to deny any suggestion of record company moulding.
"We haven't got a strong image," admits Richard. "The record company have been very open to ideas. Having read previous interviews with other bands we assumed we'd just raise our arms and they'd slip an image over our heads but that hasn't happened.
Are they not afraid of falling on their faces with all those resources behind them?
"It hadn't crossed my mind," says Richard. "If you'd asked me a week ago I'd have been terrified. Our backing band was got together a week before our first gig — a Radio One 'In Concert'. Our second gig was 'The Tube'. As far as I can see that's the worse it'll ever be. I'm really looking forward to everything now."
Eee, it’s a hard life getting to be a pop star. For Go West it involved dodging jail, playing in showbands and eating garlic-flavoured baked beans. This sad story is related by Mike Gardner.
...and gimme the hits, the limos, the mink coats too.
So says the original post-punk primadonna, Dead Or Alive's Pete Burns. On hand with the tranquillisers: Betty Page. Rose-tinted shots by Paul Cox
THE MAN in black with the wicked twinkle in his eye is probably, at this very moment, having a ball. His single is in the top five and all is well with the world. He is quite conceivably languishing in a silk-sheeted bed, handmade truffles resting in exquisitely manicured hand, blinds drawn, living out the primadonna pop star myth to its logical conclusion.
The man is Pete Burns, the single is Dead Or Alive's 'You Spin Me Round (Like A Record)', and it's been rotating round the charts (Frankie style) since last November — for about 15 weeks already. All this in the face of extreme critical indifference to DOA's transformation from outcast, untrendy Liverpool cult band to trashy, flashy highly energetic chartbound dance machine.
We've had Strawberry Switchblade, King, it was only natural that DOA should crack the nuts. Pete Burns reminisces: "We've been a big cult band, we've been lucky. When I started off in an indie group in Liverpool, which was very arty farty, I was really on the outside — all those groups didn't wanna know.
"In the indie scene you never thought about the mainstream charts, but now it's all geared towards one thing. I hadn't dedicated my life to appearing on TOTP — honest to God, it never mattered. But now it's becoming a major obsession, is chartland.
"I got this letter recently from somebody who bought all my independent singles — mug — and had written this six page character assassination of how I'd once given people who cared something good and now I'd gone along with the record company and sold myself out. But the silly git, if only he knew the fight we had since day one — it was hard to get taken seriously."
DOA signed to Epic Records a couple of years ago, probably as token Scouse weirdos that could be softened up round the edges. The lads didn't soften, there was no sudden desire to be Kajagoogoo, and they made a very saucy album — 'Sophisticated Boom Boom' — which no-one seemed to realise was dead rude.
Pete smiles to himself: "A lot of it's send up. I'm gonna have my laugh out of this if it's the last thing I do. I think the way I'm supposed to come across is really humourless. I'm so flippant, and I'm trying to curb it to present this serious deadpan thing, but I'm not really. I have a laugh, a good sense of humour. When we did our last album, nobody twigged to the humour."
AND THUS, their cutesome cover of KC's 'That's The Way (I Like It)' had to be a hit, despite Pete's wondrous flirtation with rubber leotards on TOTP. But then there was an ominous silence. Pete recalls:
"After Frankie got banned, the BBC went through everything with a toothcomb. When we put out 'What I Want', the reissue, they said it was suitable, they'd clicked. It wasn't outright banned, but they wouldn't play it. They won't even ban you now, they just ignore it — that's what happened to Divine, it mysteriously disappeared."
And so did Dead Or Alive, to America. It was the beginning of the end of an era. "We actually had no intention of thinking about the American market," says Pete, "you don't think like that unless you're capitalist about it. Suddenly we were whisked over there 'cos they thought they had the next big thing. I didn't like touring, it nearly killed me. I was having nervous breakdowns, losing suitcases, I hated it. Giggings's for rock people."
East coast traumas heralded a total management restructure situation. "When I went to New York I had to go to the doctor to get some tranquillisers," remembers sensitive Mr B. "I haven't got a mental weakness, but I was so wound up all the time thinking I'm fighting everyone, a constant 24 hour fight, I was going apeshit. I got nervous asthma and couldn't breath. 25 and taking tranquillisers like a middle-aged housewife — time to change things."
So now the butterfly has new wings, the image has been toned down and spruced up; it seems, eventually, to have worked. This is what Pete said last November: "Everyone's predicting this record to be top five. I don't know if it will be — I'll be disappointed if it isn't. If it gets to 45 it'll be a victory for us, 'cos we didn't go along with a lot of things we were supposed to do, so a lot of doors were shut for us. But I know that probably in a year I'll have disappeared and people will be saying 'what's he doing now?'"
UNCANNY, EH? Maybe it won't be long before Pete engineers himself the ideal pop enigma scenario: no interviews, please. "If I have my way I'll never have to do them again after this," says the fragile one. "We're trying to concentrate on the record, not roping me out for everything except suitable things. I don't think you'll see me in Titbits or Woman's Own, that's not where I wanna be.
"That bastard Prince doesn't even have to do an interview, they just put him on the cover, he's so lucky. It's high camp but I wonder how long it'll wash with people. I think it's quite funny the way he gets all over everything. I think his music sounds a bit dated. I blushed all the way through 'Purple Rain' — that album's banned in our house. I didn't like the film even though I've just bought wardrobes full of purple stuff."
Purple's rather a neurotic colour, didn'tcha know, Pete me old dear? "Neurotic? Hmmm, suitable, maybe that's why I like it. We tried to do our new flat in relaxing colours. The last one was hilarious — leopardskin walls, ceilings, couches — you saw spots. This time we've gone very Conran. Still, purple must reflect something in my personality."
So out go the spots, and out goes the mouth'n'stick and the young man stance.
Pete: "Specially coming from Liverpool, they're used to bands being angry young men, what with the Wylie thing. I know he used to be the angry responsible young man. I'm not. I'm pretty stupid and irresponsible, really. I'm not angry about much.
"People are always trying to make you speak on politics, but I'm just not interested really. I'm doing OK, I'm alright Jack. Frankie made no difference, neither did 'The War Song', it didn't tell anybody anything. I doubt if you'll see us hammering out a protest song."
Out also is Pete Burns as all-round bitch; he'll be reluctant to get involved in public slagging sessions. "I think it's unfair to judge what other musicians have been doing," he says in an uncharacteristically restrained moment. "Like that one Nick Heyward did about us, when we got him with fire extinguishers. He shouldn't have been so flippant. It's so easy to say 'pile of crap', I won't do it. I couldn't go to the toilet with a clear conscience."
THE HIT single hurdle successfully negotiated, we await the supersmash album. How will it sound? Pete: "We actually found a production team that was churning it out — Pete Waterman who did the Divine single, and Hazell Dean. We couldn't mess around experimenting, we wanted it done really quickly. It'll be funny, though not as rude as the last one — you can't keep telling the same joke. 'Spin Me Round' is quite different from that, more of a proper song. We've done this great track called 'Lover Come Back To Me', it's really funny. I hope that's a single."
Aaah, sweet success: a lifestyle beckons into which Mr Burns will definitely fit like a velveteen shoulder-length glove. He won't have to wait for the limousines much longer. "I was pining for the limos when we weren't working. I nearly went potty on them before. It's the best bit of it — anyone, sign a deal, make a record and get a limo sent for you — that's funny. We used to commandeer them for shopping sprees. I mean, how many normal people get to sit in limos? You can be sick in it, shit in it, shag in it and they've all got to put up with it 'cos they go 'this is rock'n'roll'."
This is where we came in: picture the scene — Pete, reclining, cucumber on his eyes, icepack on his brow ... "I can really get carried away," he confirms ... "it's wonderful — blinds drawn full time, in bed with me chocolates saying 'no, no, tell them I won't do it'. Last year I acted out every fantasy about every freaked out flapped out Hollywood queen I ever had.
"When we have hit records I could order a mink, really go potty. I'm a prize prat. I had a great time before and I'll go just the same way now. I bought a diamond nose ring, got me teeth capped with gold ... I think the word's ostentatious ..."
CAPTURED BY the magic lens of Joe Shutter (clockwise from top): Tom Browne, Lonnie Liston Smith, Jean Carn, Roy Ayers
LONNIE LISTON SMITH, TOM BROWNE, ROY AYERS, JEAN CARN, Hammersmith Odeon, London
JUST BEAUTIFULLY paced... and it had to be. Four bill toppers in one package don't always equal good arithmetic, but with the NY Jazz Explosion, boy, you just got one great SHOWBOAT. Four acts perfectly in tandem, complementing each other in the nearest thing to pure jazz-funk cabaret.
As such, everybody stood in a perfect line: the piano tinkled pleasantries of Lonnie Liston Smith moving into the mid-paced trumpet 'n' keyboard of Tom Browne, the outtights vibes of Roy Ayers and finally Jean Carn in glittering finale.
Lonnie Liston Smith is a cagey critter gifted with a deceptively light touch. His tunes smooth effortlessly into a dreamy state where everything is just sooo laid back.
Tom Browne raised that heart breaking just a little higher. Showing a sure grasp of synth-thrown grooves he used his trumpet as a sparing, yet effective exclamation mark midst a whole run of flowing sentences.
'Funkin' For Jamaica' was, of course, the stand out — pulled into an audience participatory jamboree, Browne again jammed those keyboards across his song, paused, picked up the trumpet and just flew. Controlled and concise...
So is Roy Ayers... and a whole lot more. Coming on like a holiday camp Red Coat, but tasteful like, Roy races through a set that is part music hall, part testimony to his 20 odd years in control of the vibes.
Ayers, assisted by a scantily clad chanteuse, is like an older, on the button Kid Creole. The wise guy without the bullshit. Thus on the giggle galloping 'Poo Poo La La' Roy enters with sweaty comic routine, flipping the traumas of courtship and marriage through a smoky soul backdrop. Leering, but never slack, Ayers is the perfect red carpet for Ms Carn's entrance...
Stuck inside a dress that shimmers like a sackful of small fish, Jean announces herself with Jocelyn Brown's 'Somebody Else's Guy' and then proceeds to lay some heavy showbiz on y'all.
Her three co-stars are out there laying down tracks with the backing band (the same for each artist) as Joan schmaltzes every song with a voice filtered through years of experience... out back everybody is hammering on tambourines, pushing on keyboards and melting the whole into one great Vesuvius of sound. Some night.
JIM REID
"YOU HAVE to be careful about the ego thing, and the clash of personalities, sometimes they don't go together. You have to have a keen eye, which I think I have, and check it out thoroughly. But basically I think most entertainers have one thing in common, that is that they like to play music."
ROY AYERS
WE'VE BEEN on the road for five months, we've done about 50 shows. Rehearsals? The first couple of gigs were rehearsals! The majority have gone well. When I started I thought we'd be finishing up around October, November, but they've been going so well..."
TOM BROWNE
## US SINGLES
| Rank | Title | Artist/Group | Label |
|------|------------------------------|---------------------------------------|----------------|
| 1 | CAN'T FIGHT THIS FEELING | REO Speedwagon | Epic |
| 2 | CARELESS WHISPER | George Michael | Columbia/CBS |
| 3 | THE HEAT IS ON | Glenn Frey | MCA |
| 4 | CALIFORNIA GIRLS | David Lee Roth | Warner Brothers|
| 5 | MATERIAL GIRL | Madonna | Sire |
| 6 | TOO LATE FOR GOODBYES | Julian Lennon | Atlantic |
| 7 | NEUTRON DANCE | Pointer Sisters | Planet |
| 8 | I WANT TO KNOW WHAT LOVE IS | Foreigner | Atlantic |
| 9 | SHIVER | Shania Easton | EMI America |
| 10 | MISLED | Kool And The Gang | De-Lite |
| 11 | LOVERBOY | Billy Ocean | Jive/Arista |
| 12 | LOVERGIRL | Teena Marie | Epic |
| 13 | THE OLD MAN DOWN THE ROAD | John Fogerty | Warner Brothers|
## US ALBUMS
| Rank | Title | Artist/Group | Label |
|------|------------------------------|---------------------------------------|----------------|
| 1 | MAKE IT BIG | Wham! | Columbia/CBS |
| 2 | CENTERFIELD | John Fogerty | Warner Brothers|
| 3 | LIKE A VIRGIN | Madonna | Sire |
| 4 | BORN IN THE USA | Bruce Springsteen | Columbia/CBS |
| 5 | DENIM AND LEATHER | Foreigner | Atlantic |
| 6 | BEVERLY HILLS COP | Soundtrack | MCA |
| 7 | NEW EDITION | New Edition | MCA |
| 8 | WHEELS ARE TURNING | REO Speedwagon | Epic |
| 9 | PRIVATE DANCER | Tina Turner | Capitol |
| 10 | RECKLESS | Bryan Adams | A&M |
| 11 | PURPLE RAIN | Prince And The Revolution | Warner Brothers|
| 12 | 17 | Chicago | MCA |
| 13 | SWEET FREEDOM | Billy Ocean | Jive/Arista |
| 14 | BUILDING THE PERFECT BEAST | Don Henley | Geffen |
| 15 | BIG BAM BOOM | Daryl Hall And John Oates | RCA |
| 16 | CRAZY FROM THE HEAT | David Lee Roth | Warner Brothers|
| 17 | BREAK OUT | Pointer Sisters | Planet |
| 18 | A PRIVATE HEAVEN | Sheena Easton | EMI America |
| 19 | THE LONE RANGER | Tom Jones | Atlantic |
| 20 | CAN'T SLOW DOWN | Lionel Richie | Motown |
| 21 | SHE'S SO UNUSUAL | Cyndi Lauper | Portrait |
| 22 | CHINESE WALL | Philip Bailey | Columbia/CBS |
| 23 | PERFECT STRANGERS | Deep Purple | Mercury |
| 24 | NO JACKET REQUIRED | Phil Collins | Atlantic |
| 25 | ICE CREAM CASTLE | the Time | Warner Brothers|
| 26 | THE BEST OF | Stevie Wonder | Columbia/Curb |
| 27 | ARENA | Duran Duran | Capitol |
| 28 | EMERGENCY | Kool And The Gang | De-Lite |
| 29 | SOLID | Ashford And Simpson | Capitol |
| 30 | THE FIRM | the Firm | Atlantic |
| 31 | SPORTS | Huey Lewis And the News | Chrysalis |
| 32 | ALL THE RAGE | General Public | IRS |
| 33 | THE RAIN | Billy Ocean | RCA |
| 34 | TROPICO | Pat Benatar | Chrysalis |
| 35 | THUNDER SEVEN | Triumph | MCA |
| 36 | THE AGE OF CONSENT | Bronski Beat | MCA |
| 37 | STARCHILD | Teena Marie | Epic |
| 38 | WELCOME TO THE PLEASUREDOME| Frankie Goes To Hollywood | Island |
| 39 | VITAL SIGNS | Survivor | Scotti Brothers|
| 40 | DIAMOND LIFE | Sade | Portrait |
| 41 | HEARTBEAT CITY | the Cars | Elektra |
| 42 | VOLUME ONE | the Honeydrippers | Es Paranza |
| 43 | STREET TALK | Steve Perry | Columbia/CBS |
| 44 | PLANETARY INVASION | Midnight Star | Solar |
| 45 | SLOW AWAY | Diana Ross | RCA |
| 46 | ESCAPE | Judas Priest | Mercury |
| 47 | HOW WILL THE WOLF SURVIVE | Los Lobos | Warner Brothers|
| 48 | NIGHTSHIFT | Commodores | Motown |
| 49 | JOHN PARR | John Parr | Atlantic |
| 50 | 40 HOUR WEEK | Alabama | RCA |
Compiled by Billboard
---
## BULLETS
| Rank | Title | Artist/Group | Label |
|------|------------------------------|---------------------------------------|----------------|
| 62 | DON'T YOU (FORGET ABOUT ME) | Simple Minds | A&M |
| 63 | KISS AND TELL | E Isley, C Jasper, M Isley | CBS Associated |
---
NEW EDITION: smooth operators
---
PIA 'N' JERMAINE: don't ya just Zadora?
THE LEAGUE OF GENTLEMEN
as told by JIM REID
Sensible, Kenny Jones, Nick Heyward, Thereza Bazar, Samantha Fox and Angie Best. Amen...
"And if all of that wasn't enough to get me reaching for a Mashie Niblick and a decent corporation golf course, there was an incredibly bacchanalian Ramones party. As Joe Shutter hung out of the window of the Embassy Club and relieved his stomach, Pete Wylie, Ian Mac, Motorhead, Phil Lynott and the old Buzzcocks all swapped stories about the Roxy Club...
"Well enough, onto the Savoy New York Hotel Club, where there were absolutely no pop stars, but the coolest people this side of Leon Trotsky...
"Talking of clubs, my dear manservant Gourmand K Gourmand, currently of the Tube TV prog, has got himself involved with something called 'Hit The Deck'. You can catch the groove and watch his hind quarters grow at Casablanca's in Newcastle on a Wednesday night...
"And while I have a bottle or three in my hand: bottoms up to UB40, who have donated £100,000 to the poor of Zimbabwe, and Mr Prince, who has reputedly raised £500,000 for underprivileged kids in America.
"Make that another drink for Mr Bob Geldof's roasting of Maggie Milk Snatcher at a newspaper awards ceremony last week...
"And more cultural cross fertilisation... Jaz of Killing Joke is considering moving to the jungles of South America... not since the Maharajah Taffye moved to Clapham...
"Though the horrors of Clapham are but sixpenn'th of chips compared with the daredevil stunt Gazza Numan is planning to put himself through. Gazza is to play a kamikaze pilot in an air display at Biggin Hill, Kent, where he will pretend to have been shot down...
"And finally latest news from the Culture Club rehearsal studio is that the boys all... wow... have their own film director style chairs with their names on 'em... and wait for this, Boy George takes his chair home with him every night. Now you know."
Indeed we did, but at the moment our most pressing concern was the correct application of HP Sauce to one's special fry baconburger. It was a life of decisions in the League Of Gentlemen.
YELLO
NEW SINGLE
'VICIOUS GAMES'
SPECIAL 6.50 VERSION ON 12''
RM DISCO
Compiled by Record Mirror's James Hamilton and Alan Jones on Monday on the streets on Wednesday.
| | | |
|---|---|---|
| 1 | HANGING ON A STRING/SILENT TALKING, Loose Ends, Virgin 12in |
| 2 | YOU SHOULD HAVE KNOWN BETTER, T.C. Curtis, Virgin/Hot Melt 12in |
| 3 | BAD HABITS/LET'S GET BACK TO LOVE, Jenny Burton, Atlantic 12in |
| 4 | WHEN IT COMES TO BOOGIE, Little Benny & The Masters, Bluebird/10 12in |
| 5 | THEME FROM 'SHAFT' (HOT PURSUIT MIX), Eddy and the Soulband, Club 12in |
| 6 | BUMPIN' STRIDE, Maze featuring Frankie Beverly, Capitol 12in |
| 7 | SOLID, Aswad, Virgin/Hot Melt 12in/LP Mix promo |
| 8 | PERSONALITY (COMPLEX)/LET HER FEEL IT (RETROUCHED), Eugene Wilde, Fourth & Broadway 12in |
| 9 | BUMP LOOSE/IT'S A CHUCK BROWN & THE SOUL SEARCHERS, Source 12in |
| 10 | I'M SO HAPPY, Julia & Co., London 12in |
| 11 | OPERATOR, Midnight Sun, Solar 12in |
| 12 | GIRLS ON MY MIND, Fatback, US Collition 12in |
| 13 | CAN'T STOP THE LOVE/TOO MANY GAMES/I WANT TO FEEL I'M WANTED, Maze featuring Frankie Beverly, Capitol LP |
| 14 | ANYTHING?, Direct Drive, Polydor 12in |
| 15 | NIGHTSHIFT, Commodores, Motown 12in |
| 16 | AFTER THE DANCE IS THROUGH, Krystal, US Epic 12in |
| 17 | STEP BY STEP (REMIX), Jeff Lorber featuring Audrey Wheeler, US Arista 12in |
| 18 | NIGHTSHIFT (REMIX), Commodores, Motown 12in |
| 19 | CAN'T CASHMORE, Fourth & Broadway 12in |
| 20 | 'TIL WE MEET AGAIN (HELLO KITTY REMIX), James Ingram, Qwest 12in |
| 21 | CURIOUS/PLANETARY INVASION/SCIENTIFIC LOVE/LET'S CELEBRATE, Midnight Star, Solar LP |
| 22 | LET'S GO TOGETHER/PART OF ME, Change, Cooltempo 12in |
| 23 | MY HOPE, The O'Jays, CBS 12in |
| 24 | MOVE CLOSER (NEW MIX), Phyllis Nelson, Carrere 12in |
| 25 | FEEL SO REAL, Steve Arrington, US Atlantic 12in |
| 26 | NOW THAT WE'VE FOUND LOVE/INSTRUMENTAL (PAUL HARDCASTLE REMIXED), Thin Lizzy, Islam 12in |
| 27 | I CAN FEEL YOUR LOVE SLIPPIN' AWAY, Samson & Delilah, US Saturn 12in |
| 28 | I'VE GOT YOUR NUMBER/WORK FOR LOVE/CAUGHT IN THE ACT/WE BELONG TOGETHER/YOU FINALLY FOUND THE ONE/STAY WITH ME, Rockie Robbins, US MCA LP |
| 29 | LOVE TONIGHT, David Simmons, US Atlantic 12in |
| 30 | I'M MAKING HEADWAY (I GET) I'LL STILL BE LOOKIN' UP TO YOU, Wilton Faidley, MCA 12in |
| 31 | EVERY WOMAN NEEDS IT/STEP BY STEP/GRVOACIOUS/ON THE WILD SIDE/PACIFIC COAST HIGHWAY, Jeff Lorber, US Arista LP |
| 32 | WISH YOU WERE HERE/PIE/FASCINATION/KEEP ME UP, Cashmere, Fourth & Broadway LP |
| 33 | MOVIN' AND GROOVIN', Redds And The Boys, Fourth & Broadway 12in/US TTED 12in mix |
| 34 | (GUESS) I DON'T BE LOVE, Thelma Houston, MCA 12in |
| 35 | I DIDN'T MEAN IT AT ALL/CITY LIFE, Sassa, 10 Records 12in |
| 36 | DO YOU REALLY (WANT MY LOVE), Junior, London 12in |
| 37 | FOREST FIRE/RAIN FOREST, Paul Hardcastle, Bluebird/10 12in |
| 38 | HEARTBEAT, Barrington Levy, Island 12in |
| 39 | MR TELEPHONE/MARL, New Edition, MCA 12in |
| 40 | ANYTHING? (MARK KING/P. PAUL HARDCASTLE REMIXES), Direct Drive, Polydor 12in promo |
| 41 | MISTER DANNE MAN!, Kool & The Gang, De-Lite 12in |
| 42 | WE NEED SOME MONEY, Chuck Brown & The Soul Searchers, Master Mix 12in |
| 43 | DO WHAT YOU DO (REMIX)/TELL ME I'M NOT DREAMIN', Jermaine Jackson, Ariola 12in |
| 44 | THINGS ARE NOT THE SAME (WITHOUT YOU), First Love, 10 Records 12in |
| 45 | 'TIL MY BABY COMES HOME, Luther Vandross, US Epic 12in |
| 46 | CHANGE YOUR WICKED WAYS (REMIX), Pennye Ford, US Total Experience 12in |
| 47 | OHH BABY/YOU, Spank, German Metrovyn LP |
| 48 | RHYTHM OF THE NIGHT, DeBarge, US Gordy 7in |
| 49 | OUT OF CONTROL (REMIX), Evelyn 'Champagne' King, RCA 12in |
| | | |
|---|---|---|
| 50 | IN THE SAND, Level, Virgin 12in |
| 51 | PARTY TIME, Kurtis Blow, Mercury 12in/US LP remix |
| 52 | BEYOND THE SEA/20/20, George Benson, Warner Bros LP |
| 53 | BIG ROSE/MORE THAN I CAN BEAR/MATT'S MOOD (REMIXES), Matt Bianco, WEA 12in |
| 54 | DONT STOP ME NOW/PUMP ME UP, Trouble Funk, Sugarhill 12in |
| 55 | LOOKING FOR THE REAL THING/BFFRIEND/I DON'T PLAY THAT, Shirley Brown, US Soundtown LP |
| 56 | FLY GIRLS/Iniquity, World Trade Records Inc 12in |
| 57 | STANDING AGAIN/OUYO, Second Image, MCA 12in |
| 58 | SECRET FANTASY (EXTENDED VERSION), Tom Browne, US Arista 12in |
| 59 | WAR, Beverley Skeete, Elite 12in |
| 60 | THIS IS MY NIGGA (DANCE REMIX), Chaika Khan, Warner Bros 12in |
| 61 | MYSTIC MOON, Redd Foxx, Twisted 22, US Vanguard 12in |
| 62 | LOVERIDE, Nuance featuring Vikki Love, Fourth & Broadway 12in |
| 63 | 1999, Prince, Warner Bros 12in |
| 64 | COME AND TAKE SOME OF THIS, Mass Production, US Paran 12in |
| 65 | TIE ME UP/TIE ME DOWN, US Epic 12in |
| 66 | CONTAGIOUS, The Whispers, Solar 12in |
| 67 | PAPAIA, Alex Maitheiros, US Milestone LP |
| 68 | GET UP I LIKE BEING A SEX MACHINE/GET UP OFFA THAT THING, James Brown, Peacock 12in |
| 69 | SPEND THE NIGHT, The Cool Notes, Abstract Dance 12in |
| 70 | SHAFT, Van Twist, Polydor 12in |
| 71 | PEOPLE OF THE SUNSHI, Clair Hicks, And Love Exchange, US KN 12in |
| 72 | POO POO / LA VIDA ES UN JUEGO, Roy Ayers, CBS 12in |
| 73 | AMAZING MIND, Canari, EMI 12in |
| 74 | YOU SHOULD HAVE KNOWN BETTER (MELT DOWN MIX), T.C. Curtis, Virgin/Hot Melt 12in |
| 75 | SHOULD I (PUT MY TRUST IN YOU), Caution/Maxi Priest, 10 Records 12in |
| 76 | ROXANNE, ROXANNE/ THE REAL ROXANNE/ROXANNE'S BACK SIDE (SCRATCHED BY UFTO), Roxanne (with UFTO), Streetwave LP |
| 77 | BUSTING LOOSE, D.C. Masters, Streetwave 12in |
| 78 | SIGN OF THE TIMES/LARRY'S DANCE THEME, Grandmaster Flash, Elektra 12in |
| 79 | FREESTYLE/MEANS COME OUT AT NIGHT, Whodini, Jive 12in EP |
| 80 | CALIFORNIA BAY, The Jacksons, Epic 12in |
| 81 | YOU TURN ME ON, Bruni Pagani, US Motown 12in |
| 82 | COOL OUT, Magnum Force, US Paula Records 12in |
Hi-NRG
| | | |
|---|---|---|
| 1 | CRUISING, Sinitta, Fanfare 12in |
| 2 | SINDERELLA, Betty Wright, US Jamaica 12in |
| 3 | KNOCKING ON HEAVEN'S DOOR, Barbara Tucker, IRS Profile 12in |
| 4 | R.S.V.P., James & Susan Walker, Fanfare 12in |
| 5 | LET ME FEEL IT, DISCONET REMIX, Samantha Gilles, Record Shack 12in |
| 6 | SET OVER THE PHONE (REMAKE), Village People, Record Shack 12in |
| 7 | NEW YORK CITY, Village People, French Scorpio LP |
| 8 | YOU'RE MY HEART YOU'RE MY SOUL, Modern Talking, German Hansa 12in |
| 9 | BELIEVE IN THE BEAT, Carol Lynn Touress, Polydor 12in |
| 10 | I'M NO ANGEL/ECSTASY, Madleen Kane, US TSR 12in |
| 11 | STARGAZING, Earlene Bentley featuring Sylvester, Record Shack 12in |
| 12 | CHINESE EYES/COME INSIDE (REMAKE), Fanny, US Personal 12in |
| 13 | CRUSHING, The Cramps, Italian Crushin' 12in |
| 14 | DON'T PLAY WITH FIRE, Paul Parker, Record Shack 12in |
| 15 | TOUCH ME IN THE MORNING, Lydie Steiman, Long Island Sound 12in promo/remix |
| 16 | TAKE ME TO HEAVEN/SEX (REMAKE), Sylvester, Cooltempo 12in |
| 17 | TOTAL ECLIPSE OF THE HEART, Koffi, US Paradisco 12in |
| 18 | CRASHIN' DOWN (REMAKE), Legear, Proto 12in |
| 19 | JUNGLE BEMWARE, June Brown, Bolts Records 12in |
| 20 | NEW WORLD, De La La Belle, US MCA 12in |
| 21 | DANCING IN THE RAIN/DON'T LEAVE ME THIS WAY, Carol Jiani, Streetwave LP |
| 22 | NIGHT OF THE FULL MOON, Gaby Lang, Dutch Casablanca 12in |
| 23 | WAITING THE NIGHT AWAY, Boyz Canadien, Sun 12in |
| 24 | HEARTS ON FIRE (DANCE MIX), Sara Harris, Music 12in |
| 25 | YOU SPIN ME ROUND (LIKE A RECORD), Dead Or Alive, Epic 12in |
| 26 | AMERICAN, Vivien Vee, Dutch Bread 12in |
| 27 | NO FOOD FOR YOU, Josephine, US Profile 12in |
| 28 | DATE WITH THE RAIN, Anita's Love, UK Profile 12in |
| 29 | NO REGRETS, Martinique, German Teldec 12in |
| 30 | GIRLS IT AIN'T EASY, Peggy Blu, Dutch Injection 12in |
A NEW SINGLE FROM WORKING WEEK
INNER CITY BLUES
AN EXHILARATING SLICE OF SOUL - JAZZ
7" VERSION and 12" URBANE GUERRILLA MIXES
VS 745 (-12)
World Radio History
help!
Problems? Need some ideas or information fast? Or would it help to talk things over? Write to Susanne Garrett, 'Help', Record Mirror, 40 Long Acre, London WC2. Please enclose a stamped addressed envelope for a personal reply. Alternatively, ring our Helpline, office hours, Monday to Friday on 01-836 1147.
I HAD to tell someone my problem, as it's driving me crazy and has totally ruined my life. When I was 11 years old, I was beaten up and tortured several times by a load of thugs, and, to cut a long story short, after recovering from the pain and mental torture of all that, I discovered that my right testicle was badly damaged and not in working order.
Since it happened, my testicles haven't grown much, and there is something wrong with the size of my genitals. Could the fact that I had mumps as a child have anything to do with this lack of development? I realise, as I'm 22 years old, it may be too late to do anything for me.
J. Peterborough
You don't say whether you've had any kind of medical check up since having mumps or being beaten up, or whether any diagnosis has been made in the past. Whatever happened then, it is important to seek medical advice now. See your GP, or, if you have had hospital treatment in the past, you can just go back to the hospital itself.
It is possible, although extremely rare, for someone who catches mumps after the age of puberty to suffer permanent damage to the testicles and sterility, resulting from severe gland inflammation. A doctor will set your mind at rest on this score.
Meanwhile, it will help to talk about that bad experience with the bullies and the rest. Why not contact your nearest young people's counselling and advisory service? CHAT, City Centre, 439 Lincoln Road, Peterborough. (Tel: Peterborough 310704 — any weekday evening, Monday to Friday, 7.30-9.30pm). Calls from anyone aged between 16 and 25 are welcomed.
WHERE CAN I buy a Prince songbook? I've been hunting for one for ages, with no luck.
Mandy, Lincs
Publishers Music Sales will certainly be issuing a special Prince songbook anthology later this summer. For full fax, write to Music Sales, 78 Newman Street, London W1.
I'M A male heterosexual transvestite. Is there anywhere I can write for help, support and information?
Rick, Dagenham
Yes. Drop a line, including a large stamped addressed envelope, to Beaumont Society, BM 3084, London WC1N 3XX. They'll supply useful information, and a reading list as well as contact with others if this is what you want.
PLEASE END my search for a Dead Or Alive fan club. Other music mags can't help.
Willy, Windsor
No problem. For membership information, simply send along a stamped addressed envelope to Dead Or Alive, PO Box 65, Liverpool L69 4LG.
SOMETIMES BETWEEN March and October this year I'm thinking of trying a paid working holiday abroad. Any ideas on how to find out what's on offer, and do I need a work permit or visa just a working holiday? I'm 19 and a UK citizen.
Barry, Lancs
Check out dozens of detailed ideas for an unusual working holiday abroad, ranging from work on farms, in conservation, nature reserves, summer camps and schools; and much more in 'Working Holidays 1985', by Hilary Sewell, available, price £4 from bookshops or direct from Central Bureau For Educational Visits And Exchanges, Seymour Mews House, Seymour Mews, London W1H 9PE. Postage is included.
There's coverage of just about every country you can think of and chapters detail info on cheap travel, accommodation, passports, health requirements, and job contacts.
ABOUT A year ago I contacted you about ways of meeting other people and was considering trying computer dating agencies and contact magazines. I phoned you and you told me about the National Federation Of 18 Plus Groups which is a social organisation for young single people.
That was last February, and since then, my life has changed. I'm now liaison officer for Coventry 18 Plus and responsible for recruiting new members. But, more than that, I'm engaged to a girl I met at 18 Plus too, and we plan to marry in a couple of years.
Pete, Coventry
It's good to publish a success story from time to time, isn't it? It isn't everyone's glass of milk, but the National Federation of 18 Plus Groups, a country-wide network of some 230 local clubs welcomes anyone aged between 18 and 30 and into meeting new people.
For details of your nearest group, ring 0533 821210, or write, with a stamped addressed envelope, for info on your group contact to National Federation Of 18 Plus Groups, Nicholson House, Old Court Road, Nwent, Glos.
CHANGE
LET'S GO TOGETHER
NEW 7" AND 12" SINGLE
TAKEN FROM THE FORTHCOMING ALBUM
"TURN ON THE RADIO"
7" COOL 107
12" COOLX107
If you've got to Go-Go, go-go with JAMES HAMILTON
ODDS 'N' BODS
SUNDAY'S CONVENTION at London's Hippodrome sees mixing finalists Jeff Cree, Mapin, Marky, Mike Johnson, Lee Reynolds, Ricky Santini and seven European winners compete plus panels on radio and the biz — with Alan, Stuart Grun, Wiltshire's Johnnie Walker, Luxembourg's Richard Swainson, a Home Office official (pirates can put their case from the Isle of Sussex) and Chris Hill, Plymouth Academy's Kelly, Bathus's Tony Savill, Phonogram's Jeff Young, MCA's Hughie, MCA Music's Charlie Crane and much more. Steve 'Touch The Speaker' Dennis will take last-minute bookings on 021-351 3217. DMC Fee Mixes remain in late but not all barry, Alan Coulthard's medleys of Madonna and Boystown oldies, Sanny's medley of Little Miss and remix of '85 Les Adams' medley of oldies — incidentally Sanny has remixed Ray Parker Jr's next compo, the single of Lorraine Jackson's current hit, Alan doing a megamix medley for the latter's flip and remixing Whitney Houston's debut single. Kool's Misted Dance Mix and Mark Barry's remix but a close copy by the group themselves.
Disco 85 breakers include Amii Stewart & Manhattans' In, Robert White's Way, The Muddies, Prince, David Roach, Pink Rhythm, Levers, Jazzy Jay — all of which a few weeks ago with the small support would have hit easily. DJs please always post your charts early enough to reach us by Wednesday morning, but just in case next week you get it to us by this coming Tuesday — or give it to me in person on Sunday? ...
Alan James Jewell is again after extending the showdown for high class Hong Kong work: contact Lee Taylor (01-472 5355) — who himself sent me a brilliant fast-cued flow of his megamixes — but what jocks not bothered with the contest can do too ... Dr Who freak Ian Levine is not only calling on all fans to let him know if he can get BBC controller Michael Grade into not suspending the series for 18 months but also, rather than make a good HNR, he's going to do it himself! He's about to produce a Band Aid-style group containing Frankie Goes To Hollywood, Madness, Imagination, Blancmange, Kim Wilde, Joe Jackson, Dean, Village People, Alexi Sayle, Slade, Capt Sensible, Limahl and many more to actually raise the money to finance the series — there's no precedent for any pressure group now: do any superstars fancy bringing back
to my mind by then pervasive unsubtle strings and synthesizers, the forerunner of modern compromises. My subsequent was my prejudice then that it was ages before I could appreciate much of Sam Cooke's songings-badly work, which has just entered better than most — but will its modern equivalent? ... I only ever saw Sam Cooke performing at the Norwich Arts Centre in October '62 when he supported Little Richard's first UK tour (and my first rock 'n' roll show!), but then in 1964 while working in New York I met him in Sam Cooke Jr's dressing room at the Copacabana, following which on separate occasions we really got talking when watching a film demonstration of Jamaica Ska dancing at Trude Heller's in Greenwich Village, and even more memorably drove around his Warwick Hotel suite together with Floyd Price after we'd been to see the latter, in his 'Misty' phase backed by Slide Hampton's big band and young Erma Franklin at the Cop Cab — where I also met Miles Davis ... Record Mirror, where the name dropping was shameless, and Little Richard's documentary on The South Bank Show was essential viewing (find a video): I met him, with Angel Lee, backstage at the Fillmore, Apollo where his (briefly glimpsed on Sunday) comeback engagement was 10 rather than the usual 7 days, spent mainly rearranging his appearance with quotes like "New York is the world's capital for sissies — you live here, I don't" and "Makeup can't fix up!" — he insisted on staying at the Park Sheraton Hotel. Luckily not on orgy night, where with his tour managing brother and a whole mixed crowd limbering up, he was so incensed ("I'm so glad you faggots came over tonight!") we talked till dawn ... Esquire's also called SQ (Eskew) Reed of 'Greatest Hits' fame, used to hang out in a Broadway burlesque joint I haunted (along with Donald Height and so many more of my 'sof' names) — he was in a miniskirt too, better overcoats and camper than Richard ... 1984, and 'Prince is me in this generation' as the more talkative Little Richard's quote ...
Jennifer Holliday will play gospel singer Mahalia Jackson — a role originally offered to Martha Graham — in the Broadway-bound 'Sister Malaia Sings' Radio London's Soul Night Out this Thursday (7) is at Hammarsteins Ballroom, the first of its own suffix, Friday (8) finds the Cool Notes at Bolton Dance Factory, soulful Pete Tong & jazzy Bob Jones at London Bridge, and Oak Room's Second Set start tonight Sun (10) Colchester Embassy Suite, Mon (11) Bournemouth Academy, Tues (12) Harrow Weald Middlesex & Herts continues over
LORRAINE & HEATHER look and sound cool as they lead THE COOL NOTES 'Spend The Night' (Abstract Dance ADT 3, via EMI) through their by now usual 105½/bpm hot tempo fusion of lovers rock and soul, the true Britfunk, this insistent little jittery tripper really nagging into the brain even if maybe it does seem less strong than their last two on first hearing — and in fact there's an emptier 108bpm remix of 'I Forgot' as flip, with the short 124½/0bpm jazz-funk instrumental 'Halui (Spring)'.
'Juke Box Jury'? The miners' strike (It's over in case you hadn't heard!)? Hanging? ... EMI are bringing back Banbara 'Shack Up' on 12in for the first time and David Grant & Jaki Graham are reissuing Detroit Spinners' 12 years old 106½/bpm 'Could It Be I'm Falling In Love' next week and the Pagans' 'Don't Turn Me On' was withdrawn for some reason in the States, and David Roach snuck out here in limited numbers by mistake ahead of its late March release ... 'Better Than The Rest' is George Benson's US follow-up, on 7in ... Diana Ross 'Missing You' topped US Black Singles, Chaka Khan 'This Is My Night' replaced Sheena Easton 'Sugar Walls' atop Dance Disco ... America's Grammy Awards went to homegrown rock star Tina Turner (3), Prince (3), Lionel Richie (2), Pointer Sisters and even Michael Jackson for his video — hopefully the pop crossovers compromises were worth it to further reawaken white radio (and MTV) to The Sound Of Young America? ... 25 or so of the major current chart-aimed black pop records were ruined
from previous page
Country Club Thur (14) Birmingham
Bobbie Brooks' '70s reggae plays
Edinburgh Fire Island - Canzani
Luton Bolts and Phyllis Nelson
Bournemouth Bolts Sun (10), Carol
Jillian Lomax's Hippogriff Man (11)
... Sasha Vitek, which 'Wednesdays
of fashion, makeup, video and live
music are an arty alternative at
Rosa's' 25-30 South Kensington
Harrington Gardens - 'Something
different every week ... Rotherhithe's
new club is Bloomers, not Bootles
(thanks Graham)' Roy Ayers' Running
Away will be on Sun 15/11/85-
116/bpm ... Streetwave/
StreetSounds have moved from their
crazy "Hollywood bungalow" in West
Acton to nearby 1 Harrow Road in
Ealing, W5 2UJ ... DEE ON DA DA!
HOT VINYL
KURTIS BLOW: 'Party Time (The
Go-Go Edition)' (Club JABX 12)
Spliced from the LP by Jeff Young into
a more frenetic 102'/103'bpm version,
this classic percussive party
jitterer was go go before we realized it
in '83 (listen to the lyrics!), and has
filled floors ever since. As well as its
103'bpm original flip, it has the
influential 1980 rap 'The Breaks' —
actually 112%-113-113'bpm
percussive go go too in all but city of
origin (flip).
RAH BAND: 'Clouds Across The
Moon' (RCA PT 40026) The one
we've been waiting for, a gorgeous
dreamily drifting 102'bpm one-sided
telephone conversation in which Mrs
Johnson implores her Flight
Commander husband to come home
... from his star fighter base on Mars,
the gentle melancholy and the subtle
banality of her domestic preoccupations,
will really intrigue
everyone about the procedure and
problem of placing a telephone
phonecall complying with operators
and lost connections! Tremendously
clever (freshly Super Nova dub and the
pirate's chopped original old 102'bpm
rough mix on flip).
T.C. CURTIS: 'You Should Have
Known Better (Melt Down Mix)'
(Virgin/Mot Mot VG 754-13)
Stately 102'bpm original, 113'bpm remix
adding extra clomp and stuttering
trickery to a track that without being
damaged isn't improved either as it
was so good to start with — it's now
marginally different (as is the newly
stringing dub).
TWILIGHT 22: 'Mysterious (Remix)'
(WEA YZ367) Gordon Bahary's the
very Jimmy Jam-ish sneaky slow burning
hot tempo wriggling stark 102bpm
tapper, stunningly sung in spurts
between allusive instrumental leaps and
infectious smurfs, finally appeared here
flipped by its dub plus the original LP
version.
SUN: 'Dancel' (AIR City Records
12AIR 3701, via PRU) A solidly
burning 111'bpm whooping
infectious funk groove from early last
year, which more sensibly should have
been flipped by the now Fatback-bish
lascivious 'I Get' ... faster and with
indeterminate 125bpm 'Reggae Man'.
LOOSE ENDS: 'Silent Talking'
(Virgin VS748-13) Striking
martini-sipping limited edition of
'Hangin' On A String' now has the
added bonus track of this Tom Browne
trumpeted very complex 108%-110-
111'bpm jazz instrumental, strictly
speaking.
WORKING WEEK: 'Inner City Blues'
(Virgin VS745-12) Julie Roberts now
fronts a commendable brassy 98bpm
jazz reworking of Marvin Gaye's
classic 'Inner City Blues' on the
floor but worth keeping, while the
flip's classy latin instrumental 0-129-
0-131-132-129%-0bpm 'No Cure, No
Pay' has a Stax-Santa Fe feel with
cinematic free form blowing by Harry
Beckett) really will raise educated
sweet. It cooks!
PROCESS AND THE DOG RAGS:
'Process Is The Answer' (US Columbia
VG 40-1873) Rick James-grooved/penned
falsetto-led bubbling rather than
blasting 123'-0bpm brassy chantier
with a nice 'rockin' first flip, by a five
studiedly dated sputterer which
the '50s/60s Little Richard/Temptations-
type tank straightened and pomaded
handily ... with a run-off duty
moments was kept precisely firmly in
place by a tightly turbaned kerchief,
known as a doo rag.
THE TEMPTATIONS: 'My Love Is
True (True To Your Own)' (Motown
TMGT 1873) Hollywood, not too over
tenuous, dreamy sweet falsetto 85-
0bpm smoother ending in romantic
rap, on 12in with the sharper jogging
groove of 'I'm Gonna Be There' Light In
My Window' and — sho 'nuff! — the
jiggle 113bpm M&M Remix of 'Treat
Her Like A Lady.
WHITNEY HOUSTON: 'Take Good
Care Of My Heart' (LP Whitney
Houston: US Arista ALB-8212)
Cissy's stunning daughter, long limbed
and leonine, is flying high for Sunday's
DJ contest, and this debut
solo set put together by several
producers, this lightly tripping 112bpm
swayler being a duet with Jermaine
Jackson (they're due to tour soon on
the 30%-61/62'bpm 'Nobody Loves Me
Like You Do'), Michael Masser's
deceptively innocent 66/33bpm 'Saving
All My Love For You', a slow
then searing builder with Tony Scott's
sax (although he's truer to form with
the Teddy Pendergrass duetted 0-29/
68bpm 'Hold Me', 0-33/66bpm 'All At
Once', All's theme 0-32/55-0bpm
'Greatest Love Of All'), tightest of the
other slower beings Karla's 37/-
73bpm 'Give Me Love' and here
his lurching wiggly 120bpm 'Thinking
About You', Jermaine's leaping (0-
124)bpm 'Someone For Me' and
Nancy Wilson's Walter's 108'bpm
strutting 118'bpm 'How Will I Know'
are the only fast 'uns. Another auntie
Dionne?
PHILIP BAILEY & PHIL COLLINS:
'Easy Lover' (CBS TA 8515) Finally
released after inter-label hawking,
over Phil's billing were sorted out, this
US smash 12in remixed choppily
jolted guitar-driven 127/bpm breezy
rock stroller will obviously be here
too here, like Prince (modifier semi-
slow 107/12bpm 'Woman' flip, pop
started like much of his Collins-
produced LP).
RUN-D.M.C.: 'King Of Rock'
(Fourth & Broadway 12BRW 21)
Too raucously raw for many, I expect,
this shouted 100'bpm rapper has
angular, angular, rocking energy which
may make it a big pop hit, and as well as
the flip's 100bpm inst guitarist Eddie
Martinez also yowls through the older
more monotonous 101'bpm 'Rock
Box'.
FIONA FRANKLYN: 'Busted Up
On Love (The Don Was Remix)' (Virgin
VS731-13) The flip's distinguished
dreamy pop vocal bedecked with
immediately into an ultra tough
tempoed 111%-112-111%bpm
electronically tugged and tapped
dubwise instrumental, hot on soul
radio.
RICHARD JON SMITH: 'ABC Of
Kissing' (Jive JIVE T 85) RJ's latest
adopted style is Phil Fearon's on this
typical 125bpm 'Galaxy Skipper' (inst
flip), and calypso-125'bpm 'Jessica'.
BELIOUS SOME: 'I'm Going
(Parlophone 12R 6092) Bowie vocal
and Chic guitar combined on a
H-I-N-R-G RELEASES
SAMANTHA GILLES: 'Let Me Feel It'
(Record Shack SOHOT 35) Good
excitingly pumping 130/3/(intro)-131bpm
leaper in the classic tradition with
many rhythm breaks on its rare Discomix
X-side (132bpm original & inst
flip).
VILLAGE PEOPLE: 'Sex Over The
Phone (Remix)' (Record Shack
SOHOT 34) Remade rather than
remixed, this now is H-NRG and 7bpm
faster at 125bpm, a galloping bounder less
distinctive maybe than the original
but much looser (ditto inst).
LEEGAR: 'Crashin' Down (Remix)' (Proto
ENAT 124) Sylvester-style
squeaker meets the (oddly edited) 126%-126'bpm
Hazell Dean/Dead Or Alive
rattle rhythm.
DENNY DWYER: 'Be My Baby' (DAT
DATT-001) Richard Jones-produced
beautifully dislocated 134bpm Hi-NRG/pop
update of the Ronettes, now remixed,
good for general radio.
KIM CARNES: 'Invitation To Dance'
(EMI America 12EA 1911) Nile
Rodgers-produced 122'bpm techno
chugger from a film that's otherwise
mainly of old musical movie clips.
52nd STREET: 'Can't Afford' (Factory
FAC 118) UK-recorded
undisciplined frantic bass skittered
by a pulsating chick-swing racer
influenced by 'Slick' by David and Goliath,
now a US dance hit on Priority.
MUSIC ACADEMY: 'Ring My Bell' (Record
Shack SOHOT 36) Chaps
charted 'organ' chorded (0-)125bpm
spurring skitterer.
ASTAIRE: 'In The Name Of Love'
(Passion PASH 1241) Chick sung
0-139bpm looncoating filler, 135bpm
'Shout It Out' flip, both with probably
more hi-hat than balls.
KERRY DELIUS: 'They Say It's Gonna Rain'
(Arrival 12PIK 16) Commercial
12in remix of a hisping chick's 113bpm
Europop-type throbbing
from last year. Yup, she's related.
N I G H T C L U B
POP JOX are playing: 1 (1) Ashford & Simpson, 2 (6) Eugene Wilde A/B, 3 (8)
Commodores, 4 (3) James Ingram, 5 (5) Little Benny, 6 (2) King, 7 (4) Prince
'1999', 8 (7) Cashmere 12in, 9 (3) Dead Or Alive, 10 (17) Eddy & The
Souls, 11 (1) The Jacksons, 12 (11) Billy Ocean, 13 (1) The O'Jays, 14 (23)
R&B, 15 (16) George Benson, 16 (14) Madonna 'LAV', 17 (15) Direct Drive,
18 (36) Junior, 19 (32) TC Curtis, 20 (19) Van Twist, 21 (18)
Art Of Noise, 22 (21) Bruce Springsteen, 23 (35) Jermaine Jackson, 24 (22)
Bobby Jean, 25 (20) George Brown, 26 (13) Chaka Khan, 27 (46)
Nightline 'O', 28 (12) Philip Bailey, 29 (14) Billy Ocean, 30 (1) Pet Shop Boys
/EC/LG/TMWU 31 (-) West End (mailing list promo), 32 (29) KoKo-Pop, 33 (-)
Third World, 34 (41) Victoria Pink, 35 (45) Jenny Burton, 36 (46) Sharpe &
Nelson, 37 (1) The Staple Singers 12in, 38 (re) (42) Bar-Kays, 39 (42)
Bar-Kays, 40 (51) Bowie T/MWU, 41 (22) Sinitta, 42 (34) Dazz Band A/B, 44 (30)
Phyllis Nelson, 45 (26) Julia & Co, 46 (-) Madonna 'MG', 47 (25) Kool & The
Gang 'F', 48 (re) Temptations (old), 49 (-) Belouis Some (mailing list promo),
50 (-) TinTin (original). And jolly useful I hope you find it!
Take a bow, Wilton Felder, star and starmaker. This is the man who's been blowing a mean sax with the Crusaders for a quarter of a century — and also the man who helped to bring Randy Crawford to centre stage, recharge the careers of Bill Withers and Joe Cocker, and maybe even save the life of his soul partner of the moment, Bobby Womack.
Wilton it was who gave Womack his first even break in years when he invited him to do the vocal on 'Inherit The Wind' in 1980, and now they're back in business, and really booming, with '(No Matter How High I Get) I'll Still Be Lookin' Up To You' and another Bobby dazzler on Wilts' 'Secrets' album, 'The Truth Song'. In the meantime, of course, Bobby's career has exploded back into life and no one's smiling wider about that than Felder himself.
"The first thing we did together was the 'Inherit The Wind' album," says Wilton. "Things weren't going too well for him back then." The master of the understatement, is the Crusader. Womack had been drowning in a sea of drugs. "He had stopped touring and stopped recording for a while. And I thought about him, I knew he wanted to do something, and it went well. Now of course I'm very happy to see him getting the recognition he deserves."
Bobby, for his part, told me last year he was happy to repay Wilton's recognition by adding his name to the 'Secrets' album, so there's beaming faces all round. The reason we were discussing the "new" Felder/Womack collaboration so long ago is that the LP's been finished for yonks. "The album was completed in March last year," Felder says. "But because of the situation with Bobby and the record company it took a long time before it could come out." (BW's old label Beverly Glen stopped him recording new material until his old contract was up, so his new association with MCA hasn't seen the light of day until now). "The idea was to come out with it right away, but complications set in. That causes you to rethink a few things, it makes a difference to the way that you're feeling. It would have been really nice to have it out then, when you still have the same emotions that went onto the record.
"But it makes you appreciate it even more. It still sounds good, so we must have been doing something right."
'Still Be Lookin'' also gives some vinyl acclaim to new soul siren Alltrina Grayson. "I think she's signed to Bobby," says Wilton. "She's a singer he discovered in Cleveland. She's very good, she's going to go quite a few places." Another discovery, another feather for the cap. "With the Crusaders, we're always looking for that new discovery, but not ruling out the established artist. They have to fit in as far as possible with the sound of the Crusaders. Like Randy Crawford, she was an extension of the Crusaders, we weren't backup to her."
The band's last album 'Ghetto Blasters' was uneasily received by a lot of fans and Wilton doesn't shy away from that. "It was a period of transition, because of the players changing" — drummer Stix Hooper had just left after a lifetime in the group, replaced by Leon Ndugu Chancler — "it represented a phase we were going through at the time.
"When you've worked with someone for that many years it's really like a divorce, and it takes its toll in the way of musical content." Was there any ill-feeling when Stix finally broke the ties? "No, just a feeling of disappointment that the differences could have been worked out. But we just saw him recently, we have offices and we see each other, and our families see each other."
Solo albums by Wilton, Joe Sample and co always seem to use the same musicians as Crusaders albums. All the Cru's are on 'Secrets' for instance — so what is this keeping it in the family routine? How about bringing in some new blood for a change? "I've thought about doing that, and Joe has too, but we've realised that the way we play and feel, is alien to a lot of musicians today."
Anyway even if the occasional album gets the thumbs-down, there's not much danger of Wilton having to blow his horn on street corners to scratch a living — so just what is there left for the Crusaders to achieve? "If we can, before our death, leave our mark like some of the greats like Basie and Ellington."
'Gary's image is cool and almost unapproachable but he's actually a really nice guy' — BILL SHARPE
BILL SHARPE'S voice comes excitedly over a crackly telephone line. "It's great, I'm really chuffed actually," Bill's in Germany touring with Shakatak — the group he is both keyboard wizard and chief songwriter for. Yes, it's all his fault.
Solo for the first time, Bill has teamed up with Gary Numan for 'Change Your Mind' — an electro disco pop song currently nestled in the upper reaches of the charts. It's the first single from his debut solo album 'Famous People' due out on the Ides of March.
For a man who generally plays the sort of music only people in the latter stages of brain damage could enjoy, Bill's a surprisingly lively and coherent man. And, as he said before, "chuffed" at his solo success.
"It helped a lot having Gary on the track," he explains. "His is a well known name — mine isn't. Obviously people know Shakatak but they don't know who I am."
Sharpe and Numan are an odd alliance which, like so many of the great partnerships of history — Romeo and Juliet, Marks and Spencer — came about purely by fate. Bill was
recording in Gary's Rock City studio in Shep-
perton and was without a singer for a song.
His engineer knew Gary. Gary had nothing to
do that day — and that was it. Fate.
"If the song had been recorded by someone
else it wouldn't have sounded so good," Bill
adds generously. "I had a go myself actually
— and that was pretty awful, so I knocked
that idea on the head. I wasn't sure if Gary
would do it because I didn't know him at the
time. If you only know a person through his
image — and Gary's is cool and almost un-
approachable — you think they'll be like that
in real life. Gary's not at all like his image —
he's actually a real nice guy."
Having worked together, Bill obviously sees
nothing strange about mixing the two camps
he and Numan represent. How did the fans
react, though? Have the loyal legions of the
Numan army taken to wearing medallions?
Have Shakatak fans dyed their hair blue?
"Shakatak fans seem to like it," Bill replies.
"The song still retains a little bit of that dance
thing. I think there's been mixed feelings ab-
out Gary's vocal, though. Some of them aren't
quite sure about that."
With the success of 'Change Your Mind', Bill
seems to have taken a fancy to the idea of
working with Gary again.
"I had a really good time doing this and if
there was time I'd love to work with him
again. I've got a little track that would work
quite well. I'll have to give Gary a ring — see
if he's interested!"
AS THE other, more visible, half of the
duo, Gary Numan provides both vocals
and 'oddity value' to the team. As he
puts it simply: "It's an unusual combination. It
gives DJs something to talk about and gives
them a reason to play the record."
Gary doesn't waste pretty words on the
business he's been in for the past six years.
Disillusioned would be one description,
bitter perhaps more apt. He tells you that the
best thing that ever happened in his life was
becoming "famous" but adds, quietly but
pointedly:
"It was tarnished a little bit by your side —
the press. I'll never forget about it — never
forget what they said. Not when it's all gone
... finished. Ever."
The success of 'Change Your Mind' has
been a welcome bit of happiness for Gary at
a time when he seems at a very low ebb.
"It's been a very pleasant surprise," he tells
you smiling. "It's done me a lot of good.
"I've never got on well working with other
people. I worked with Robert Palmer and that
was quite good, but this was easy because all
I had to do was sing somebody else's song so
there wasn't a clash of ideas. That's where I
run into problems because I'm, not stubborn,
but I always have a very clear idea of what I
want. I'd like to work with some people. I
think Dolby's very clever, I liked Howard
Jones ... not many really I suppose.
"I wouldn't mind doing something for fun. I
think the problem is that my images are al-
ways a little bit ... strong, so you get the
impression that I'd be serious about it. But
I'm not, it's just a f***ing job for Chrissakes."
GARY'S A strange one. At one moment
he'll be joking about the problems of
his blue hair colour coming off on his
pillow, the embarrassment of blue dandruff
or, with rare infectious enthusiasm, his plane.
Then he'll suddenly fall back into thinking ab-
out the differences between him and Bill.
"We're on different sides really," he begins
quite happily. "There's my bit which is all the
image and the hoo-ha that goes with it and
then there's them. They're doing Bailey's
(nightclub in sunny Watford) and that kind of
tour — and I'm not!"
Why does he continue to work in the busi-
ness?
"Because it's a nice life — it's very exciting
and you get to meet people. Not that I'm that
keen on meeting people but it keeps ideas
coming because people are a never ending
source of ideas. What wonderful people you
meet. The world's full of shits.
"It's like, nobody writes about my plane un-
less I crash," he continues sadly. "I got two
awards last year for it — best new display or
something and the other one was just for
handling that particular aeroplane because the
one I have is quite hard. And nobody said
anything about that.
"There are certain people who, if they flew
around the world, would be heroes or a
wonderful example of British manhood. If I do
it I'm a hero or I've got more money than
sense — I did it for the public. There's al-
ways somebody who's gonna pull it apart.
"It's like this Bob Geldof thing with Band
Aid. They said he did it to bolster his career —
but he might have, he might not. He still got
f**k knows how many millions for them, it
doesn't matter what his purposes were. Who
got hurt? No-one. Yet they're still gonna
knock the poor f***er for it.
"But there's people for you — a never end-
ing source of ideas. I don't like people much
— as a rule.
"We're more to some people individually,
but as a force we're nothing. Most of us
aren't really well educated, and then people
come and ask us our political views.
"I mean, I don't really know what left and
right means — no idea. Why ask me?"
One likes aeroplanes and
funny make-up. The other
prefers gold chains and
tinkly pianos. Eleanor Levy
investigates the strange
partnership that is Bill
Sharpe and Gary Numan
MOST FRIENDS get to call him TC, only trouble is, even Mr Curtis isn't quite sure what it stands for. The 'You Should Have Known Better' hitman says: "Yeah, I've been trying to come up with something for it myself. I made it up, you see. I used to be called Terry Curtis before, but TC sounded better. Now everybody's asking me what it stands for. They all think it's like 'Top Cat'."
Well, Tel boy, that would give you the perfect excuse to put [Little] Benny on your records, wouldn't it? Not to mention some nifty guitar work from Offices Dibble.
Anyway, while TC's rounding up all the alleycats in town, he's already managed to get some star guests into the studio with him, because 'You Should Have Known Better' features backing vocals by not just the Galaxy girls but Phil Farnes too.
"I know Phil very well, he's been a very good friend of mine for years, since he was in Kandidate," says TC. "I just met him in a studio, we started talking and we've been friends ever since.
Now unless you're an absolute disco swot and you remember 'Bump And Slide' and 'Dance To The Beat', the first two releases on his own Hot Melt Records label, then TC's going to be a fairly new name to you, but we're dealing with a superstar who's played the Albert Hall here.
OK, it was 10 years ago, and back then his music sounded a little different, but in 1976 Curtis' own gospel group, The Date Religion, played at a religious convention in the arena of the Albert. TC was born in Jamaica and came to England when he was 14. After his gospel youth, Curtis took on soul leanings. Deals were slow in coming, but it wasn't until, and by '83 our man was well fed up with record companies big and small. "There were too many complications," he says. "So I decided to go it alone."
After those two earlier dance stabs he came up with the current jackpot number and all the grabbing hands of the majors became just too demanding to refuse. "The record's been around for a while, but probably all the time you heard it wasn't actually released, it was just a promo copy for some of the soul stations. I only decided to put it out in February, and I decided to get a major to pick it up.
"There were so many companies after it, I mean you name it, it was practically every company. And in America, they're going mad, every minute they're phoning up for this record. I can't rest, I mean right up to nine o'clock at night. Virgin won the battle of the big 'uns and they haven't messed about with piddling one-off deals, either. "There's an option for six albums," says TC. "So they really have to have faith in me. It seems like a lifetime. But all I have to do is get the stuff together.
Which he's doing right now, preparing the first of the six which he says should have a good four or five singles material numbers on it. "I've got three or four albums together. In America they're crazy about albums. But I've been writing for years, so material's not a problem."
Top Cat's pains to point out that he wants his music to stand up against, and not just merge with, the current American dance output. "I've always tried to put my own style across, from disco right down to pop. It can stand up against the Americans. They're saying it's the best thing that's ever come out of Britain, it can stand up against Prince or any one of those people.
"At the moment, I'm under pressure as well to do the next single, I've got two in mind. But I never do covers, I do all my own songs. I'm not one for listening to a lot of stuff by other people because I'm always busy, I'm writing and I've always got something to do so I haven't got time. I listen to the pirate stations, quite a lot, though." Live work, perhaps? "I have to stay away from live for the moment, because I've got to get to work on the album. And I've been doing so many PAs, as well."
So, Tel boy gets down to making more records to outdo Prince and conjure a picture of huge grizzly bear TC Curtis bodyguards surrounding him at next year's music industry awards. As long as he remembers to thank Offices Dibble in his speech he'll be OK. For the time being though, things are slightly more down to earth. Especially when you're running your own record label and supplying the shops yourself.
"One old lady, she walked into this record shop seven times asking for the record. The seventh time, I just happened to be walking in to the shop, so I gave her one myself. I don't think she could quite believe it."
PAUL SEXTON
HE might be Terry Curtis to his Mom and Dad but to any self respecting cartoon fan, he is, indisputably, leader of the gang...
TOP CAT
CROSSWORD
ACROSS
1 David's discovery (4,2,3,7)
6 September Song singer (3,9)
9 Siouxsie's house (5)
12 When out of town take Bow Wow Wow's advice (2,4,2,3,7)
14 Bowie's lover (5,4)
16 Virgin singer (7)
19 U2 song heard on January 1st (3,5,3)
21 Village People No 1 (1,1,1,1)
22 Good you may hear first thing in the morning (5)
23 Feeling like the wolf (6)
24 Spandau's medal (4)
29 Eurovision song contest winners from 1974 (4)
30 The Style Council like it when you're like a wild (5)
31 Treacherous debut hit (9)
33 Give 'em enough and the Clash will hang themselves (4)
34 Super vagrant (5)
35 Drum from Japan (3)
39 Barrington's on his way (4,1,4)
40 She sings The Belle Of St Mark (6,1)
DOWN
1 It can't get worse, says Hayward (6,3,4,5,8)
2 Barbara and Elaine are both in agreement (1,4,3,2,4)
3 Group always in conversation (4,4)
4 Gang the Pretenders got back on (5)
7 The city rollers (3)
7 Early Foreigner classic (4,2,3)
8 Elvis didn't want to go there (7)
10 Spandau long player (6)
11 The Smiths' question (3,4,2,3)
13 1982 Classix Nouveaux hit that might not have been real (2,2,1,5)
15 Group found in the Sea Of Love (13)
17 Blondie's was of glass (5)
18 He can be found playing bass on 19 across (4,7)
20 I'm not sure what James Ingram's trying to say (3,2,1,5)
25 Numani's partner (5)
26 There's no chance of falling asleep when listening to XTC (4,2)
27 Maybe Billy Mackenzie had something to do with this (7)
28 Mix you'll find in the charts (5)
32 It's just another one for him (6)
36 Mr Davies lives in an Icehouse (3)
37 See 38 down
38 Hendrix classic (3,3)
LAST WEEK'S SOLUTION
ACROSS: 1 Barbara Dickson, 5 Bites, 8 You Spin Me Round, 11 Right By, 12 Deep Purple, 14 Dancing In The Dark, 16 Mark King, 18 Pride, 21 Bobby, 23 Culture Club, 27 Blue, 28 The Thin Wall, 30 Shout, 31 Bruce, 32 Sarah, 33 Rattlesnakes, 36 Love Hurts, 37 Commodores.
DOWN: 1 Bryan Adams, 2 Round And Round, 3 Apple, 4 Squeeze, 5 Bangkok, 6 Tom Tom Club, 7 Huey Lewis, 9 Rip It Up, 10 Draconia, 13 Pain, 14 Generation X, 17 Kate Bush, 19 Nancy Justice, 20 Too, 22 Your Side, 24 Creatures, 25 Power, 26 Invisible, 27 Blue Bayou, 29 Groove, 34 Lamb, 35 Shy
PERSONAL
MALE 28, requires girl to write and meet, for friendship, interests are, music film travel sport. Box No 4410.
LONDON (R) Penpals list £1 + SAE 4B Hartham Rd, London N7 9JG.
CHRISS 27, seeks young, caring female penfriends 20+ photo appreciated, 62, Royal Sussex Crescent, Eastbourne.
FOR EXCHNG new dates with compatible partner, same sex). Contact Int'l Dating (RMR) 30 Baker Street London W1 Tel 01-486 7788/9
LYNN 19, would like to write to young man, same sex. Box No 4411.
SUPER PENPAL list £1: J. Heal 14 Shorncliffe Road, Folkestone, Kent.
PENPAL MAG for lonely people. Approval copy from: Matchmaker, (A.44) Chorley Lanes
DJS Wanted
FEMALE AND male disc jockeys for top contracts in Scandinavia. Send Promotion, Music and DJ experience. Experience to Norbusiness 281 B Vigerslevsvalby Copenhagen Denmark.
Records for Sale
ABBA/AGNETHA albums Abba books, magazine SAE 5 Barnend, Henfield, Sussex.
60'S SINGLES From 60's present ideal for disco/collector 70's soul section some rarities £500 Slough 42964.
EXCELLENT OPPORTUNITY to purchase a DJ's record collection, ideal for Gay club 1200 12" many deletions, imports, 100's 7" oldies soul tamla stax etc. All £100-£1000 Box No 4414.
RECORDS BOOKS POSTAGE 30p stamps cash for list of unplayed 1976/8 45's and list of records books etc. For Auction 5 Creditor Walk, Hornchurch, Essex RM12 6GD.
BOWIE IMPORTED singles, all picture sleeves, see details for 80 Leigh Road, East Finchley, London E6.
A BARGAIN! A lusty assortment of 100 used LP's and 12" singles for £12.00 or 500 used 7" singles for £30 (numbers approx. see our section). Music & Video Exchange, 28 Pembridge Rd, London W11 (01-727 3538).
RECORD FINDER SERVICE send your "WANTED" list to: 50 Cliffe Ave, Weybridge, Surrey KT13 7AJ.
FREE CATALOGUE albums, all types SAE Jane Lawson, 3 Kent Wing, Redbourn, Fakenham, Norfolk.
RING 0273 680000 Singles from 29p - LP's from 99p 40's-80's. Free catalogue SAE. Backtrack, Baker Street, London W1.
BEAT RECORDS Specialists in 12", soul, High Energy, Northern soul, 7" rarities. U.K. and U.S. imports. 7 Cumberland Street, Dun Laoghaire, Dublin 2. Wed-Sun 9-6.
AMAZING SUPER-RARITIES!!!—Fantastic NEW Lists-Out Now!!! Rare-Pics, Picture Discs, Promos, UK Promos/US Radio Station Promos/Autographed LPs/Concert Programmes/Japanese Picture Discs/Limited Editions. 10,000 Ultra Collectors Items. U-2, Jam, The Who, Genesis, Deep Purple, Police, Hawkwind, Floyd, Gary Numan, Bowie, AC/DC/Iron Maiden, Rush, Siouxie, The Cure, Curved Air, Pinkfloyd, Damned, Sex Pistols, T-Rex, Sweet, Slade. All new Wave/Heavy Metal/70s Groups!! Must, Much More!!!—Collected by Don M. Mail order only. Large SAE—(Dept—RS). RS Records, 21 Silver Street, Wiveliscombe, Somerset.
FREE CATALOGUE! deletions! Oldest! £5.00 post paid. 40 items. SAE 24 Southwark, Middletown, Sussex.
25,000 GREAT SOUNDS including punk, metal, disco oldies, pop etc, plus 100 tapes 12", 8" & 12" singles, plus many collectors items. Pop in shop or send large s.a.e for massive fortnightly lists. Greg's Records & Tapes, 126 Merton Park Road, Mitcham. ALSO RECORDS PURCHASED.
ORIGINAL UK/45's — see 3 Woodland Street, Hertford, Herts.
FREE CATALOGUES, over 1,500 to choose from. all new. Send 9½ x 6½ in s/a to Chris Foss Records (R). 344 Paddington Street, London W1.
RECORD FINDING SERVICE Having trouble finding that record? Try us. Send enquiries with SAE to 59 rockall, Southend-On-Sea, Essex, Personal, efficient service.
CONTINUED ON PAGE 35 & 36
CABINET FITTINGS
Fretcloths, Coverings, Handles, Castors, Flight Case Locks & Parts, Jacks, XLRs, Bulgins, Reverb Trays, P & N mic Stands, SS Glassibre Horns, CEASTION POWER Speakers.
Send 30p cheque/PO for illustrated catalogue
Adam Hall Supplies, Unit H,
Carlton Court, Granger Road,
Southend-on-Sea.
LEAMINGTON SIGHT & SOUND
Suppliers of Professional Sound, Lighting and Special Effects Equipment
MON to FRI 10am-5.30pm. SAT. 4.30pm
Telephone: (0926) 833617
Supply of all equipment to the new "Bears" Disco in Warwick. All the best to Tony Hall (The Bear) for the future.
Remember all leading brands stocked and on demo. A complete Mobile Disco for £950. Yes, £950! All equipment supplied with guaranteed equipment. No hidden extras like Stands, Leads & VAT £950 the lot. Catch up with the rest. Check out our Special Offers. To callers and all our other facilities. For your HIRE, SUPPLY, REPAIR, SPARES and INSTALLATION.
Unit 17, Rigby Cl, Heathcote Ind Est, Leamington Spa
PERSONALISED A'CAPELLAS
A MUST FOR EVERY MOBILE & RADIO DJ
(i) Your custom name indent for only £35 + VAT.
(ii) 2 custom name indents + 1 shout only £80 + VAT.
* INCLUDES *
Free loading onto cart, cassette or reel to reel.
SEND YOUR ORDER & REMITTANCE TO
eastasian productions
Studio House, 21-23 Winton Road
Frinton-on-Sea, Essex. CO15 6AA
Telephone Frinton (025 56) 6525
OR; PHONE QUOTING YOUR ACCESS/BARCLAYCARD NUMBER
GRANGE DISCO CENTRE LTD
483, OXFORD ROAD, READING.
(0734) 509969
JUST ARRIVED
150 WATT BUDGET PRICE SPEAKERS
EXCELLENT VALUE AT ONLY £250 per pair
+ LIGHTING EFFECTS FROM FAL & PULSAR
NEW BARGAIN PRICES
Please check our prices before purchasing
Mail Order available. No charge for P&P
CONTINUED ON PAGE 36
LUTON SOUND & LIGHTING
SOUND, LIGHTING & SPECIAL EFFECTS
FOR ENTERTAINMENT LEISURE AND PROMOTION
U.K. & WORLDWIDE SUPPLIES —
EXPRESS MAIL ORDER & EXPORT SERVICE
ALL PRICES INCLUDING VAT — DELIVERY EXTRA
COME AND SEE IT ALL IN OUR SUPER SHOWROOMS
Citronic Severn ........................................... 296.00
Citronic Hawaii ............................................. 449.00
Jammin' ...................................................... 399.00
Thames Hi Mains ........................................... 889.00
1000 Watt Slave ........................................... 399.00
100 + 100 Stereo Slave .................................. 139.00
Citronic SM440 inc PSU ................................. 169.00
Citronic SM440 inc PSU ................................. 249.00
ADC SS115 10 Band Graphic ......................... 119.00
Stanton 500 Mic inc Lead ............................... 19.90
Electro-Voice PL68 Mic inc Lead ..................... 66.95
Squire B30 Pinspot ....................................... 15.90
Stanton 500 Stylus ........................................ 11.95
Stanton 500 Triple Pack ................................. 29.95
Ortofon OM Pro Cartridge ............................. 19.95
Pulsar Zero 2250 .......................................... 34.95
Pulsar Zero 2500 .......................................... 39.95
Pulsar Zero 4000 .......................................... 59.00
Soundcraft 4000 .......................................... 44.95
Computer Chaser 4000 ................................. 69.00
Rapport 7m inc lead ..................................... 49.90
Futuright .................................................... 29.95
Hendy Budget Pinspot ................................. 13.99
Hendy Scanner ............................................. 39.95
Handy Spinner ............................................. 49.95
Le Maire Mini Mist ....................................... 295.00
Le Maire Mini Mist Canister ......................... 3.95
Mic Boom Stand .......................................... 18.99
Guitar Stand ............................................... 26.99
Speaker Stand .............................................. 26.99
Technics SL 1200 MK II ................................. 259.00
Technics SL 1200 Bridge ............................... 259.00
Citronic Trent II ........................................... 1199.00
Citronic PPX 450 ......................................... POA
Citronic PPX 460 ......................................... POA
Citronic PPX 1000 ....................................... 459.00
Cloud Disco Master 600 ............................... 339.00
MXR Dual Octave Equaliser ........................... 289.00
MXR Phase Shifter ....................................... 199.00
Electro-Voice PL68 Mic inc lead ..................... 129.00
Ortofon OM Pro Cartridge ............................. 29.95
Ortofon Concorde Pro Cartridge .................... 29.95
Pulsar Modulator IV c/w Dim Pack ................. 459.00
Pulsar Modulator IV c/w Dim Pack .................. 299.00
Pulsar Pinspot ............................................. 19.99
4 Head Helicopter ....................................... 119.90
4 Head Helicopter ....................................... 149.00
Le Maire Optimus ....................................... 379.00
MAIN DEALERS FOR MOST LEADING MANUFACTURERS
SPECIALISTS IN CLUB & MOBILE SUPPLIES
OR GET IT THROUGH OUR EXPRESS DELIVERY SERVICE
MAKE SURE YOU'VE GOT A COPY OF THE CATALOGUE FOR THE DISCO INDUSTRY — THE LUTON SOUND & LIGHTING GUIDE — 64 Pages including equipment for Installation and Mobiles
SEND THE COUPON FOR YOUR COPY NOW
LUTON SOUND & LIGHTING LTD.
75 & 82-88 STANMORE AVENUE STREET
LUTON LU1 5AA ENGLAND
Telephone 0582 391021/411733
Telex 82556 CHACOM G LITECO
OPEN MON-FRI 10AM-6PM, SAT 10AM-4.30PM
Please send me a copy of the Luton Sound & Lighting Product Guide
Name .......................................................
Address .......................................................
...............................................................
...............................................................
Type of Business ...........................................
Please include UK Postcode or area code if overseas
RM9/3
Please send me your latest Mobile Disco Catalogue + Professional Range Catalogue. Free if you send your Disco Card. Otherwise enclose £1
Name .......................................................
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Send to Squire's, 176 Junction Rd, London N19 5QQ
Jingles
PAMS CARAT 100 a collection of non-custom jingles from the 1960s and 1970s. A must for DJ's and collectors alike. Send PO's or cheques for £7.99 payable to BL Studio, 4 Hansol Road, Bexleyheath, Kent DA5 8UG, or phone card or 01-304 9088 with your credit card number.
Mobile Discos
MIKE DEE discotheques 01-460 4653.
NIGHTFUE, 061 793 4106.
LEISURE DISCO, 061 793 938.
DAVE JANSEN — 01-690 7636.
JUSS'JAZZ Mobile Disco — 800 5811
Disco Equipment
CITRONIC HAWAII II HM pro 200w c/ With 100 W.P.C. amplifier lighting archway Tutor II projectors excellent sound £760 Slough 429564.
COMET 100w 100w 100w lights for sale 200w ring Uckfield 4418.
I HAVE a sony twin tube projector with five foot screen complete with leads also 100w off-air radios £1000.00ono telephone Michael at Steels Hants 872834.
VIDEOL ROADSHOW complete video disco for sale. Big screen 200w x PA big light show smoke pyrotechnics 0653 829278 gigs nationwide contacts after 6pm.
ELECTRO-VOICE UNBEATABLE quality, unbeatable prices. 01-317 0494.
DJ IMPERIAL Wizard's complete roadshow for hire. Items too numerous to mention. Will consider offers. £1,000.00ono telephone 540-3503 after 2pm no time wasters please.
SECONHAND DISCO equipment bought and sold 01-368 9852.
Record Fairs
POOLE 16-8-88. Saturday, Arts Centre, Pre-entry £1, 12.30pm. 1.30-4.30, 40p.
1960's BEATLES Record Fair Sunday April 21st, Clarendon, Hammersmith entrance 11am-5pm.
WAKEFIELD RECORD Fair Saturday 16th March (10-5pm) Unity Hall Westgate Wakefield (25 stalls 100yds rail station).
CROYDON RECORD collectors fair on Sunday 10th March at the Aerodrome Hotel Purley Way Croydon 11.00-12.00 £1 12noon-5:00 50p. Next Fair Sunday 24th March Dog & Fox, Wimbledon Hill SW11.
BASILDON RECORD Fair — Saturday 16th March, Basilsye P.H. Market Place — 10am-4.30pm.
LEICESTER. Y.M.C.A. Saturday 9th March. Simply one of the best of its kind. Check it out.
LONDON'S BEST RECORD FAIR? Why not come and find out for yourself & join up to 100 dealers from all over the place. 100% Electric Ballroom, Camden High Street, Saturday, March 16th Tube — Camden High Street. Adm only a quid 10am-4pm. All kinds of music from 1960s to 1980s. A lot of work put into getting all these dealers under one roof. Don't miss on this extravaganza!! Info V.I.P. Fairs 0533 704200.
LONDON'S SECONHAND NEW WAVE FAIR Hammersmith Clarendon. Sunday 17-3-88. Entry £1 11-4 pm. 500,000 records 100 stalls. New wave, psychobilly, factory, Stranglers, Frankie, Cure, Costello (0734) 588070.
LINCOLN SATURDAY March 9th Drill Hall Broadgate 11am-5pm 40p (10am — £1)
COLCHESTER RECORD Fair — Saturday 19th March, Larks Club St. Johns Street Colchester 10.30 - 4.30pm.
UPMINTER RECORD Fair — Sunday 3rd March — Old Windmill Hall, St. Mary's Lane, 11am-4.30pm.
OXFORD 9.3.88 Saturday. Fifty stalls at the Town Hall. Pre-entry £1, 11am, 12-4pm 40p.
Birthday Greetings
COLEEN NOLAN — Happy Birthday 12th March. Lots of love to my favourite Nolan from your number one fan David, Leatherhead (135).
COLEEN NOLAN — Have a great Birthday on 12th March. Thinking of you David, Leatherhead (135).
COLEEN NOLAN — You are always "Dreamed to Kiss" Happy Birthday Coleen with lots of love David Leatherhead (135).
COLEEN NOLAN — Happy birthday to the world's greatest singer. With lots of love David, Leatherhead (135).
COLEEN NOLAN — Happy Birthday 12th March. Hoping that this day will be the first of a fantastic year for you. With Lots of love David, Leatherhead (135).
COLEEN NOLAN — Happy Birthday for 12th March. Hope to see you again soon. With lots of love David, Leatherhead (135).
Special Notice
LASER 258 Caroline set or six quality colour prints (5 x 7) send cheque PO's £9.40 inc P&P to Des Sheffield, 21 Hardeville Drive, Witham, Essex.
OFFSHORE ECHO's magazine now out featuring Chas and Dave, Kenny Blackburn interviews free radio nags and more. Write DEM Box No 4409.
GAR NUMBERS — We are not hunters, we are very patient. "We'll wait just for you". Happy Birthday with love Donna 8086 and Tracey 6174.
Situations Vacant
LYRIC WRITERS required by recording company. Details. SAE Robert Noakes, 30 Snead Hall Road, Bloxwich, Walsall, Midlands.
EUROPEAN pop — magazine looking for young bright writers. Experienced photo-reporter. Send items and personal portrait to: Moderne Media, A M Marstall 6 3000 Hannover 1, West Germany.
BUSINESS — ACHIEVE FINANCIAL independence, operating your own finance broking company from home or office, full/part time. No capital/experience. Free advisory Service. 01-344 2266. Now £5.00 from: R. W. Projects 16, Glenkingslass Rd, Greenock, Scotland PA16 9NW
Records Wanted
DEREGHE MODE records imports live tapes demos interviews artists etc. state total cost Steven Cooper 7 Kentmere Road, Berwick Hills Middleborough Cleveland 0724 833 701.
WANTED IMPORTED 12" big country — Heart and Soul in Mint VGC your price paid Also Big Country Fan Club Magazine issues one and five contact first. A. Smith Uplands Arley Lane, Fillongley, Warcs.
RARE RECORDS WANTED!!! Test Pressings/Picture Discs/Demo/Promo Material/Coloured Vinyl/Autographed LPs & Programmes. All your usual wants. Foreign Singles. Special Request for — Kiss/Queen/Bowie/Policeman/AD/DC/Iron Maiden/Gary Numan/Japan/Happy Metal/New Wave/Patti Smith Band/Police/Lists/State Price — (Dept W). RS Records, 21 Silve St., Wiveliscombe, Somerset.
ABSOLUTELY ALL your records, cassette tapes, LPs, compact discs, musical instruments bought or exchanged. NONE REFUSED!! Bring ANY quantity in ANY condition to: Record, Tape and Video Emporium, 100 Littlehampton Hill Gate, London W11 (shop open 7 days, 10-8; tel: 01-243 8573). Or SEND any quantity by post with SAE for cash — we will call once sent and decide fair price. (large quantities collected — phone 01-727 3538 10am-8pm).
Wanted
URGENT! WANTED! Alan Horne/Alan Tucker/Christie Test February 18th on TVG Video. Urgent price paid. Phone Cathy 01-761 2871.
ALTERED IMAGES — videos (beta) live + promo. Send anything — State price. Dave 49 Ibsley Grove Havant Hants PO9 3DP.
WANTED POP magazine N.M.E. Record Mirror's 80's, s.a.e chris cugging also wanted singles, albums, M.O.G. 200m 89 Barrett Road, Lakenham Norwich, Norfolk NR1 2LL.
SLASH VIDEOS 'Seven Year Itch' and pre 83 'Return J. Newing' 69 Kingston Avenue, Carlinge Margate Kent.
For Sale
THOUSANDS OF NAMES and addresses in the music business are contained in the 1985 edition of the Music Week Directory including record companies, music publishers, recording studios, record producers and concert promoters. Price £10.00 plus £1.00 delivery. (Dept RM), Music Week Directory, 40 Beresford Street, London SE18 6BB.
T.T. TICKET B'Ham 11.4.85. Offers? Phil 021-360-1455.
JAPAN SCENE magazine No.3 and No.4 available now photos, reviews, interviews, news, etc. Send s.a.e. to Japan Scene 20 Long Row Ditherington, Shrewsbury Shropshire. S.Y.4DF Enclose SAE.
CUTTINGS HUNDREDS available, see (stating interests) 66 Victoria Road, Thornhill-Lees Dewsbury.
STARSKY & HUTCH items, many American rarities. SAE for lists Box No 4413.
Equipment Wanted
DJ REQUIRES used equipment. 01-209 1109.
WE BUY all branded, used disco equipment. 01-354 2254.
Musical Services
LYRICS SET to music and recorded for promotion. 25 weeks. 100% ballroom and MOR 60's and 70's music. DJ's must have bright personalities. Plus nice equipment reasonable rates paid. Interested? then write to: Professional Discos, 100 Littlehampton Hill, Clifton 74 Lavender Hill, Erdleid, Middx.
HOW TO MAKE A HIT RECORD — THE VIDEO. An instructional guide on studio procedures and techniques for anyone interested in music. Contains — the basic song, studio contact, studio equipment, multitrack recording, overdubbing, mixing, the finished stereo master, what's on the tape, for your copy (VHS) Beta enclosed cheque/po for £22.95 plus £1.00 P&P mailable. HOW TO MAKE PRODUCTIONS. Please allow 28 days delivery. Kingdom Promotions/HTP, 83 High St. Ellie, Fife, Scotland, REF. RM.
LYRICS WANTED by Music Publishing House, 11 St Albans Avenue, London W4.
ABSOLUTELY FREE "Twenty Songwriting Tips" by Alan Culling. Includes copyright, royalties, publishing contracts, recording agreements etc. Absolutely free without obligation from International Songwriters Association (RM) Limerick, Ireland.
Send completed form with cheque/postal order to Small Ads Dept Record Mirror 40 Long Acre London WC2 E9JT
Please write in BLOCK CAPITALS
Personal. For Sale records wanted — Situations Vacant. Record Fairs. Send Names and other private/trade. Announcements 20p word. Advert in Bold. First two 30p word. Box Numbers add £1 extra. Advert closes 10 days prior to issue date.
Heading required (as personal, for sale etc)
Number of words/insertions
Commencing date
NAME........................................ADDRESS...........................................
I enclose cheque/postal order for
Name & address when included in advert must be paid for
## UK ALBUMS
| THIS WEEK | LAST WEEK | WEEKS IN CHART |
|-----------|-----------|----------------|
| 1 | 2 | NO JACKET REQUIRED, Phil Collins, Virgin V2345 □ |
| 2 | — | SONGS FROM THE BIG CHAIR, Tears For Fears, Mercury MERH58 |
| 2 | 39 | BORN IN THE USA, Bruce Springsteen, CBS |
| 4 | 5 | HITS OUT OF HELL, Meat Loaf, Epic □ |
| 4 | 17 | ALL ALISON MOYET, CBS △ |
| 5 | 3 | MEAT LOAF UNDER, Meat Loaf, Rough Trade □ |
| 6 | 2 | RECKLESS, Bryan Adams, A&M AMA5013 |
| 7 | 9 | ELIMINATOR, ZZ Top, Warner Bros ▲ |
| 8 | 36 | PRIDE RAIN, Prince And The Revolution, Warner Bros □ |
| 9 | 34 | DIAMOND, Michael Jackson, Epic ▲ |
| 10 | 8 | NIGHT TIME, Killing Joys, EG EGLP61 |
| 11 | — | PRIVATE DANCER, Tina Turner, Capitol ▲ |
| 12 | 37 | MODERN LOVE, Various, K-Tel NE1286 □ |
| 13 | 2 | THE KING OF CONGA, Tito Puente, Epic □ |
| 14 | 21 | MAKE IT BIG, Wham!, Epic ▲ |
| 15 | 6 | BUILDING THE PERFECT BEAST, Don Henley, Geffen GEF25939 |
| 16 | — | CHESS, Various, RCA □ |
| 17 | 10 | VARIOUS, DREAMSONGBOOK, Barbara Dickson, CBS |
| 18 | 5 | STEPS IN TIME, King, CBS □ |
| 19 | 5 | NIGHTSHIFT, Commodores, Motown |
| 20 | 11 | AGENT OF INOCULEUS, Weinger, Atlantic □ |
| 21 | 12 | THE 12" ALBUM, Howard Jones, WEA □ |
| 22 | 16 | LIKE A VIRGIN, Madonna, Sire □ |
| 23 | 37 | BEVERLY HILLS COP, Original Soundtrack, MCA |
| 24 | 32 | THE COLLECTION, Ultravox, Chrysalis ▲ |
| 25 | 18 | THE TRUTH, Firm, Atlantic □ |
| 26 | 15 | STREET SOUNDS ELECTRO 6, Various, Streetsounds ELCST6 |
| 27 | — | VERY BEST OF CHRIS DE BURGH, Chris De Burgh, Telstar □ |
| 28 | 11 | THE HIT ALBUM / THE HITS TAPE, Various, CBS/WEA ▲ |
| 29 | 15 | 2001, Queen, Bad Boy Records □ |
| 30 | 2 | HATFUL OF HOLLOW, Smiths, Rough Trade □ |
| 31 | 16 | THE BAD AND LOWDOWN WORLD OF THE . . . , Kane Gang, Kitchenware |
| 32 | 26 | COME TO THE PLEASUREDOME, Frankie Goes To Hollywood, ZTT ▲ |
| 33 | 23 | FULL MOON, Chicago, Epic ▲ |
| 34 | 7 | CANCER, Michael Jackson, Motown ▲ |
| 35 | 72 | ROW THAT'S WHAT I CALL MUSIC 4, Various, EMI/Virgin ▲ |
| 36 | 14 | SECRET SECRETS, Joan Armatrading, A&M |
| 37 | 5 | FACE VALUE, Phil Collins, Virgin ▲ |
| 38 | 8 | MY PARLEY, Paul Young, CBS □ |
| 39 | 85 | UNIVERSE, BLOOD RED SKY, U2, Island ▲ |
| 40 | 67 | I CAN'T STOP THE LOVE, Maze Featuring Frankie Beverly, Capitol Maze ▲ |
| 41 | — | FANTASTIC, Wham! Innervision ▲ |
| 42 | 16 | ARTIST, Duran Duran, EMI ▲ |
| 43 | 36 | WHO'S AFRAID OF THE ART OF NOISE, Art Of Noise, ZTT |
| 44 | 8 | PERHAPS, Associates, WEA |
| 45 | 4 | 1999, Prince, Warner Bros □ |
| 46 | 21 | CENTREFIELD, John Fogerty, K-Tel ▲ |
| 47 | 9 | BREAKOUT 2 – ELECTRIC BOOGALOO, Original Soundtrack, Polydor |
| 48 | 43 | THE VIDEO, Wham!, CBS/Fox |
| 49 | 4 | SQUAD, The Police, Capitol ▲ |
| 50 | 22 | THE UNFORGETTABLE FIRE, U2, Island ▲ |
| 51 | 8 | CENTREFIELD, John Fogerty, Warner Bros |
| 52 | 83 | VARIOUS POSITIONS, Leonard Cohen, CBS |
| 53 | 5 | HUNGRY DRIVING, Kool & The Gang, MCA ▲ |
| 54 | 6 | LIFE'S A HOT MESS, Kool & The Gang, MCA ▲ |
| 55 | 6 | STAGES, Elaine Paige, K-Tel ▲ |
| 56 | 4 | WHOSE SIDE ARE YOU ON, Matt Bianco, WEA □ |
| 57 | 1 | VULTURE CULTURE, Alan Parsons Project, Arista |
| 58 | 15 | THE BODY SHOP, Kenzo, RCA ▲ |
| 59 | 7 | SUDDENLY, Billy Ocean, Jive |
| 60 | 74 | BAT OUT OF HELL, Meat Loaf, Epic/Cleveland ▲ |
| 61 | 2 | THE WORKS, Queen, EMI ▲ |
| 62 | 5 | HELLO, I MUST BE GOING, Phil Collins, Virgin ▲ |
| 63 | 5 | VU, Velvet Underground, Polydor |
| 64 | 4 | LEGENDS, Bob Marley And The Wailers, Island ▲ |
| 65 | 49 | STAY MAJOR, SENSE, Talking Heads, EMI |
| 66 | 45 | BREAKOUT, Pussycat, Island |
| 67 | 36 | PARADE, Spandau Ballet, Chrysalis ▲ |
| 68 | 12 | ALCHEMY, Dire Straits, Vertigo ▲ |
| 69 | 4 | BURNING ROCK, Billy Joel and John Oates, RCA |
| 70 | 53 | THE WORKS, Queen, EMI ▲ |
| 71 | 20 | STEELTOWN, Big Country, Mercury □ |
| 72 | 62 | QUEEN GREATEST HITS, Queen, EMI ▲ |
| 73 | 8 | A SONG FOR ANDERSON, Van Morrison, Mercury |
| 74 | 55 | THRILLER, Michael Jackson, Epic ▲ |
| 75 | — | YESTERDAY ONCE MORE, Carpenters, EMI SING1 |
| 76 | 84 | TWELVE GOLD BARS VOL 1 AND 2, Status Quo, Vertigo □ |
### MUSIC VIDEO
| 1 | THE VIDEO, Wham!, CBS/Fox |
| 2 | FEED THE WORLD COMPILATION, Video Aid, Virgin |
| 3 | THE SONG REMAINS THE SAME, Led Zeppelin, WHV |
| 4 | THE VIDEO, The Police, Vertigo |
| 5 | PRIVATE DANCER, Tina Turner, PMI |
| 6 | HITS OUT OF HELL, Meat Loaf, CBS/Fox |
| 7 | "UNDER A BLOCK RED SKY", LIVE AT REDROCK, U2, Virgin/PVG |
| 8 | THEY DON'T KNOW IT'S XMAS, Boney M., Polygram |
| 9 | THE COLLECTION, Ultravox, Palace/PV |
| 10 | VIDEO REWIND, the Rolling Stones, Vestron/PVG |
| 11 | A NIGHT WITH MORRISON, the Doors, WHV |
| 12 | ON VIDEO, Marc Bolan, Video/MV |
| 13 | LLOYD COLE AND THE COMMOTIONS, Polygram |
| 14 | SYNCHRONICITY CONCERT, The Police, A&M/PVG |
| 15 | "KILL DEVO", Devo, Virgin/PVG |
| 16 | ALCHEMY, Dire Straits, PolyGram |
| 17 | SING BLUE SILVER, Duran Duran, PMI |
| 18 | THE WORKS, Queen, Peppermint/Guild |
| 19 | WE WILL ROCK YOU, Queen, Peppermint/Guild |
| 20 | END OF THE ROAD '84, Status Quo, Videoform |
Compiled at considerable expense by Video Week
## UK SINGLES
| THIS WEEK | LAST WEEK | CHART |
|-----------|-----------|-------|
| 1 | 2 | YOU SPIN ME ROUND (LIKE A RECORD), Dead Or Alive, EMI |
| 2 | 1 | I KNOW HIM SO WELL, Elaine Paige and Barbara Dickson, RCA O |
| 3 | 6 | NIGHTSHIFT, Commodores, Motown |
| 4 | 2 | KISS ME, I'M THE TIN MAN, 10 Records |
| 5 | 2 | MAMMAL GIRL, Madonna, Sire |
| 6 | 4 | SOLID, Ashford and Simpson, Capitol |
| 7 | 9 | LET'S GO CRAZY, Prince And The Revolution, Warner Bros |
| 8 | 3 | LOVIN' AND HAVIN', Billy Ocean, CBS |
| 9 | 5 | DANCING IN THE DARK, Bruce Springsteen, CBS |
| 10 | 7 | THINGS CAN ONLY GET BETTER, Howard Jones, WEA |
| 11 | 34 | THE LAST KISS, David Cassidy, Arista |
| 12 | 13 | THE BOYFRIEND OF SUMMER, Don Henley, Geffen |
| 13 | 3 | SHARP, Eddie And The Soul Brothers Club |
| 14 | 8 | A NEW ENGLAND, Kirsty MacColl, Stiff |
| 15 | 30 | BREAKING UP MY HEART, Shakin' Stevens, Epic |
| 16 | 20 | LEBELZ, Top, Warner Bros |
| 17 | 11 | RUN TO ME, Bryan Adams, A&M |
| 18 | 39 | DO WHAT YOU DO, Jermaine Jackson, Arista |
| 19 | 6 | LOVE LIKE BLOOD, Killing Joke, EG |
| 20 | 1 | EARLY LOVE, Phil Collins, CBS/Virgin A4915 |
| 21 | 10 | CLOSE (TO THE EDIT), Art Of Noise, ZTT |
| 22 | 12 | THINKING OF YOU, Colour Field, Chrysalis |
| 23 | 14 | YOU'RE THE INSPIRATION, Chicago, Full Moon |
| 24 | 17 | CHANGING MY LIFE, Shirley Amos and Ray Taylor |
| 25 | 33 | METHOD OF MODERN LOVE, Cheryl Hall and John Oates, RCA |
| 26 | — | EVERY TIME YOU GO AWAY, Paul Young, CBS A6300 |
| 27 | 18 | LOVERBOY, Billy Ocean, Jive |
| 28 | 15 | LITTLE RED CORVETTE/NEVER, Prince, Warner Bros O |
| 29 | 25 | THIS IS NOT AMERICA, David Bowie, EMI America |
| 30 | 21 | I WANT TO KNOW WHAT LOVE IS, Foreigner, Atlantic O |
| 31 | 44 | WE CLOSE OUR EYES, Go West, Chrysalis |
| 32 | 35 | JUST ANOTHER NIGHT, Mick Jagger, CBS |
| 33 | 36 | WHO COMES TO BOOGIE, Little Benny And The Masters, Bluebird/10 |
| 34 | 25 | SUMMER, Phil Collins, Virgin |
| 35 | 20 | ATMOSPHERE, Russ Abbott, Spirit |
| 36 | 27 | THIS HOUSE, Big Sound Authority, MCA |
| 37 | 42 | HANGIN' ON A STRING, Loose Ends, Virgin |
| 38 | 38 | FALLING IN LOVE, George Michael, Mercury |
| 39 | 56 | THE BELLE OF ST MARK, Sheila E, Warner Bros |
| 40 | 47 | MR TELEPHONE MAN, New Edition, MCA |
| 41 | 67 | NO FOOL FOR LOVE, Hazell Dean, Proto |
| 42 | 32 | MIND, Kool And The Gang, De-lite |
| 43 | 43 | ABSOLUTE REALITY, Alain JEAN |
| 44 | — | STARVATION/TAM TAM POUR L'ETHIOPIE, Starvation, Zarjaz/JAZZ3 |
| 45 | 66 | THIS HEART OF MINE, Glenn Frey, MCA |
| 46 | 28 | HOW SOON IS NOW?, Smiths, Rough Trade |
| 47 | 52 | NEVER UNDERSTAND, Jesus And Mary Chain, Blanco y Negro |
| 48 | 58 | MORE CLOSER, Phyllis Nelson, Carrere |
| 49 | 29 | SHOUT, Tears For Fears, Mercury O |
| 50 | 31 | GHOSTBUSTERS, Ray Parker Jr, Arista O |
| 51 | 50 | WORLD DESTRUCTION, Time Zone, Celluloid |
| 52 | 48 | 20/20, Culture Beat, Warner Bros |
| 53 | 17 | LIKE A VIRGIN, Madonna, Sire O |
| 54 | 37 | WE BELONG, Pat Benatar, Chrysalis |
| 55 | 45 | TAINTED LOVE, Soft Cell, Some Bizzare O |
## THE NEXT 25
| 76 | 61 | I'M SO HAPPY, Julia And Co, London/Next |
| 77 | 68 | FUTURE PICTURE, Eric Clapton, Warner Bros W9069 |
| 78 | 69 | OPERATOR, Midnight Oil |
| 79 | — | I'M NOT FOOLED/THE PILLOW, U840, Dep International DEP-16 |
| 80 | 68 | CALIFORNIA GIRLS, David Lee Roth, Warner Bros |
| 81 | 77 | BUSTIN' LOOSE, Chuck Brown And Soul Searchers, Source |
| 82 | 87 | BELFAST, Barrbrack, Homespun |
| 83 | 53 | DO YOU REALLY (WANT MY LOVE), Junior, London |
| 84 | 74 | NO WAY FOR, Robert Ralston, The Magnet |
| 85 | 56 | TEMPTATION, Joan Armatrading, A&M |
| 86 | 81 | JACOB'S LADDER, Monochrome Set, Blanco y Negro |
| 87 | 100 | CAN'T FIGHT THIS FEELING, Reo Speedwagon, Epic |
| 88 | 79 | BACK IN THE U.S.A., Mott featuring Frankie Beverly, Capitol |
| 89 | — | HOWLING AT THE MOON/CHASING THE NIGHT, Ramones, Beggars Banquet BEG128 |
| 90 | — | IN MY HOUSE, Mary Jane Girls, Gordy TMG1377 |
| 91 | 76 | SUMMER OF '69, Copper Mercury |
| 92 | 92 | I JUST CALLED TO SAY I LOVE YOU, Stevie Wonder, Motown |
| 93 | 98 | IN THE SAND, I Level, Virgin |
| 94 | 85 | ON THE AIR TONIGHT, Willy Finlayson, PRT |
| 95 | 80 | MOVIN' AND GROOVIN', Redds And The Boys, DETT/4th & Broadway |
| 96 | — | MAGIC FLY, Space, Record Shack RMX1 |
| 97 | — | TEARSABLE TEARS, Vision, Polydor TP2320 |
| 98 | — | BAD, The Human League, Atlantic A9583 |
| 99 | 91 | THE OLD MAN DOWN THE ROAD, John Fogerty, Warner Bros |
| 100 | — | INNER CITY BLUES, Working Week, Virgin VS745 |
*Platinum (one million sales) □Gold (500,000 sales) ○Silver (250,000 sales)*
Those fun loving flower children the Smiths deservedly tripped to the top of the album charts a fortnight ago with their stunning new selection of ditties, 'Meat Is Murder'.
From being a cult band a little over a year ago, the Smiths have evolved rapidly to become one of the brightest lights on the chart landscape. Though Morrissey's posturing still alienates more than it endears, there's no doubting their musical ability, particularly Johnny Marr's composing skills; his complex intertwining of melodies and arrangements providing a perfect foil for Morrissey's more vulnerable vocals.
The Smiths' first album, titled simply 'The Smiths' peaked at number two last March, and 'Hatful Of Hollow' — a tasty grab-bag of singles and BBC sessions — reached number seven in November. 'Meat Is Murder' was the first number one for the Smiths' label Rough Trade, at their 81st attempt, and the first number one distributed to the record trade entirely by the indie cartel.
It's ironic that on Radio One's 'Round Table', Morrissey recently said "I don't understand Phil Collins, and I don't suppose I ever will"; record buyers obviously do, and last week they placed Collins' new long player 'No Jacket Required' at number one ahead of both the Smiths and the current incumbent Bruce Springsteen.
Collins has an unmatched record of success in the album charts in the Eighties. Solo, and as a member of Genesis, he has featured on seven albums this decade garnering five number ones and two number twos.
Genesis have released four albums, 'Duke', 'Abacab' and 'Genesis' all reached number one, the double '3 Sides Live' stopped at number two.
Solo, Collins debuted in 1981 with the instant number one 'Face Value', and followed up a year later with the number two album 'Hello, I Must Be Going!'.
Elvis Presley and the Sweet recently joined the small band of hitmakers to find chart success with medleys/megamixes cobbled together from their earlier hits.
This peculiarly Eighties phenomenon has now yielded a total of eight hits, though only the Beatles' 'Movie Medley' has reached the top 10.
Apart from the hits, which you'll find detailed elsewhere on this page, many have created artificial medleys for a wide variety of acts including the Crusaders, Herbie Hancock, the Thompson Twins, Boney M, Shakin' Stevens, Jeffrey Osborne, the Rubettes, Roberta Flack, Shalamar and Change. Both Shalamar's 'A Mix To Remember' and 'The Change Medley' contributed more to the acts' latest success than their nominal A-sides.
Motown has even gone so far as to release an album called 'The Incredible Medleys' (STMS 5106) comprising technically superior medleys of hits by the Four Tops, Martha And The Vandellas, Gladys Knight And The Pips, The Jackson Five, Diana Ross And The Supremes, and a spirited 'Temptations And Four Tops Medley' which was recorded at the NBC TV special celebrating 25 years of Motown.
Top minds like Alan Coubert and Sunny X will undoubtedly be compiling more disco megamixes before long, but the immediate prospect is for an avalanche of Seventies medleys. Anagram is already planning a second Sweet medley, with the band's cooperation, and is negotiating for medleys by two of the Sweet's glamrock contemporaries, though my name would be mud if I dared to suggest which glittering stars are involved. There's also a distinct possibility of a T Rex medley single from the Marc Bolan fan club...
When Lionel Richie recently became the first act to spend a year in the US top 40 with singles from the same album it was widely reported. Now, Cyndi Lauper has repeated the feat — and nobody's mentioned it.
Wacky Cyndi maintained top 40 status for a total of 56 weeks with 'Girls Just Want To Have Fun', 'Time After Time', 'She Bop', 'All Through The Night' and 'Money Changes Everything', all from her first solo album 'She's So Unusual'. The first four singles all reached the top five, but 'Money Changes Everything' peaked in the Seventies and bowed out of the 40 a couple of weeks ago to bring Cyndi's run to an end...
AND THAT'S AFACT
'Save A Prayer' is, belatedly, Duran Duran's eighth consecutive American top 40 hit, a sequence that started in 1981 with 'Planet Earth Wolf' ... 'Sussudio' is the second nonsensical song title coined by Phil Collins, the song's single 'Abacab' being similarly bereft of meaning ... Professional weirdo Andy Warhol has designed album sleeves for the Rolling Stones, Diana Ross and Billy Squier. Rumour has it he'll design the next Cars sleeve too ... The only song to make the top 40 in two different versions in the Eighties is 'Sweet Love'. Recently a teen-ager hit his Robert Plant's version on the Honeydrippers, it was also a number 33 record for Del Shannon in 1982. The original version, by Paul Phillips, was an American number two in 1959. Meanwhile, the Honeydrippers' latest US chart climber is 'Rockin' At Midnight', first recorded in 1949 by Ray Brown ... James Brown's next US soul hit will be his 100th — more than any other artist. The only way to break it is George Jones, whose current hit 'She's My Rock' is his 122nd. But the hottest country act of the last few years is indisputably Alabama, who're heading for their sixteenth number one in a row with 'There's No Way' ... According to Billboard magazine, Kim Carnes recently scored a unique double, appearing simultaneously on the US singles chart with 'Imitation Of Life' as a duet with Barbra Streisand ('Make No Mistake, He's Mine'), and as part of a trio with Kenny Rogers and James Ingram ('What About Me?') ... And how many noticed that Kirsty MacColl, Pat Benatar and Alison Moyet all featured in the top 30 at the same time recently, whilst all were heavily pregnant ... On BBC2's 'The Money Programme', Ray Parker Junior revealed that he had been offered the role of the lead actor for the movie of the same name after 60 others had been rejected ... Her latest is Hazell Dean's fourth top 50 hit — and all have had part of their title in brackets. First to hit was 'Searchin' (I Gotta Find A Man)', followed by 'Whatever I Do (Wherever I Go)', 'Back In My Arms (Once Again)' and now 'No Fool (For Love)'. . .
TAKE TWO boisterous boys, full of life, young but wise, black and white, not yet crushed by the wheels of injustice. Give them a highly enlightened upbringing, a cross-cultural fusion of musical influences, and you have Dark City — Cass (left), and Amos — and their pop rock toasty jazz funk debut 45 'False Alarm'.
Broader than Broadway... But their story started in London's Shepherd's Bush, where Amos's mum, Erin Pizsey, had her refuge for battered wives. Amos got a guitar and wrote his first song at eight, and by the age of 12 had a wild and free basement to himself. When his posse moved in, including Cass...
The kids got heavily into rasta stuff through a street cred phase, Amos running a local Sound System. At age 15, Ma upped and dragged a screaming and kicking Amos to Santa Fe, New Mexico; the streetbrat ended up in the desert learning to sing opera.
Then Ma called for Cass... Amos takes up his story: "So we said yeah, let's take America by storm with our brand of new music. We had vocals and bass. We auditioned for a drummer and we had vocals and bass... Santa Fe has the population of Hammersmith, and 98 per cent are Chicano, and when it comes to playing black funk...
"We went into funk and reggae but becoming heavily influenced by rock 'cos of the whole American thing.
"Being in America really opened us up to a lot of MOR stuff — now I'll listen to everything and get into all kinds of music. So they lost the tunnel vision. London clique life brings, came home to rest, and now follow this general trend of threads coming together: AOR, guitar rock, Smiley Culture. It all melted together cosmopolitan style."
How many people have mentioned those dreaded words Culture Club? Amos: "Not that many. People have mentioned George, but also Eartha Kitt. 'False Alarm' has a sleazy backstreet vocal, I suppose. When I sing ballads I croon 'cos my influences are people like Luther Vandross, so people are going to put me alongside George, and it doesn't help that I had an early adventure with Culture Club." Other influences? Cass: "Prince is our main man, he's hard. A very strong influence, someone we look to, for the way he does things." Amos: "He's closer to us, he's pretty raw."
Dark City say yes to success, no to street cred, yes to learning to live with yourself, no to arrogance, and yes to Ninesse, music cred, Charles Mingus, Carlos Castaneda and having a good time. Cass: "We are having a very good time doing this — that's very important. We are having FUN!"
— BETTY PAGE
|
Gregory L. Lippetz (State Bar No. 154228)
firstname.lastname@example.org
Laurie M. Charrington (State Bar No. 229679)
email@example.com
JONES DAY
Silicon Valley Office
1755 Embarcadero Road
Palo Alto, CA 94303
Telephone: 650-739-3939
Facsimile: 650-739-3900
Attorneys for Plaintiffs
INTERSIL CORPORATION AND ELANTEC SEMICONDUCTOR, INC.
UNITED STATES DISTRICT COURT
NORTHERN DISTRICT OF CALIFORNIA
INTERSIL CORPORATION, AND ELANTEC SEMICONDUCTOR, INC.,
Plaintiffs,
v.
TECHNOLOGY LICENSING CORPORATION, PIXEL INSTRUMENTS CORPORATION, AND J. CARL COOPER,
Defendants.
Case No. C09-02386-JCS
COMPLAINT FOR DECLARATORY JUDGMENT OF PATENT NON-INFRINGEMENT, INVALIDITY AND UNENFORCEABILITY
Plaintiffs Intersil Corporation ("Intersil") and Elantec Semiconductor, Inc. ("Elantec"), hereby allege as follows:
NATURE OF THE ACTION
1. This is an action for a declaratory judgment of non-infringement, invalidity, and unenforceability of United States Patent Nos. U.S. RE40,411E (the "411 patent"); RE40,412E (the "412 Patent"); 5,486,869 (the "869 Patent") and 5,754,250 (the "250 Patent") (collectively, the "Cooper Patents").
PARTIES
2. Plaintiff Intersil is a corporation organized and existing under the laws of Delaware, and has its headquarters and principal place of business in this District at 1001 Murphy Ranch Road, Suite 1, Milpitas, California. Intersil is engaged in the business of, among other things, designing, manufacturing, and selling, integrated circuits.
3. Plaintiff Elantec is a corporation organized and existing under the laws of the state of California. Elantec is wholly owned by Intersil. Elantec is engaged in the business of, among other things, designing, manufacturing and selling integrated circuits.
4. Defendant Technology Licensing Corporation ("TLC") is a Nevada corporation with its principal place of business in Carson City, Nevada. TLC is in the business of enforcing the Cooper Patents and suing companies for infringement of those patents. TLC does not practice any of the Cooper Patents.
5. On information and belief, Defendant Pixel Instruments Corporation ("Pixel") is a California corporation with its principal place of business in Los Gatos, California.
6. On information and belief, Defendant Cooper is an individual residing in Monte Sereno, California.
JURISDICTION AND VENUE
7. This action arises under the Declaratory Judgment Act, 28 U.S.C. §§ 2201 et. seq., and under the patent laws of the United States, Title 35 of the United States Code. The Court has jurisdiction over this action pursuant to 35 U.S.C. §§ 271, et. seq., and 28 U.S.C. §§ 1331, 1338, and 2201-2202.
8. Venue is proper in this District under 28 U.S.C. §§ 1391 and 1400(b) because a substantial part of the events giving rise to the claims at issue occurred in this District.
9. This Court has personal jurisdiction over each of the Defendants by virtue of the business activities it conducts within the State of California and within this District, resulting in sufficient minimum contacts with this forum.
INTRADISTRICT ASSIGNMENT
10. This case is an Intellectual Property Action under Civil Local Rule 3-2(c) and, pursuant to Civil Local Rule 3-5(b), shall be assigned on a district-wide basis.
THE COOPER PATENTS
11. United States Patent Application No. 08/165,688, was filed on December 13, 1993, and issued as U.S. Patent No. 5,486,869. A true and correct copy of the ‘869 Patent, which is entitled “Synchronizing Signal Separating Apparatus And Method,” is attached as Exhibit 1 to this Complaint.
12. United States Patent Application No. 08/566,484, which is a continuation-in-part application of Application No. 08/165,688, was filed on December 4, 1995, and issued as U.S. Patent No. 5,754,250 on May 19, 1998. A true and correct copy of the ‘250 Patent, which is also entitled “Synchronizing Signal Separating Apparatus And Method,” is attached as Exhibit 2 to this Complaint.
13. The ‘411 Patent is a reissue of U.S. Patent No. 5,754,250, reissued on July 1, 2008. A true and correct copy of the ‘411 Patent is attached as Exhibit 3 to this Complaint.
14. The ‘412 Patent is a reissue of U.S. Patent No. 5,486,869, reissued on July 1, 2008. A true and correct copy of the ‘412 Patent is attached as Exhibit 4 to this Complaint.
15. Plaintiffs are informed and believe that each of the Defendants has an ownership interest in the Cooper Patents.
DEFENDANTS’ ENFORCEMENT OF THE COOPER PATENTS
16. One line of integrated circuit products designed by Elantec and sold through Intersil are video sync separators. Defendants have accused certain of these video sync separators of infringing the Cooper Patents (“the Accused Video Sync Separators”).
17. Over the last several years, Defendants have sent numerous letters to Plaintiffs’ customers charging that their use of certain of the Accused Video Sync Separators infringe the ‘250 and ‘869 Patents. On information and belief, Defendants’ allegations of infringement are based entirely on the operation/functionality of the Accused Video Sync Separators. By its letters, Defendants have engaged in a concerted campaign of enforcement, threatening to assert or
asserting the Cooper patents against Plaintiffs’ customers based on their purchase of the Accused Video Sync Separators and incorporation of the chips into the customer’s own products.
18. On February 9, 2009, Defendant TLC filed suit against Harris in the United States District Court, Northern District of Illinois, Case No. 1:09-cv-00820 (the “Harris Action”), alleging that various of Harris’ products infringe the ‘411 and ‘412 Patents. Harris is a customer of Plaintiffs, and purchases the Accused Video Sync Separators for incorporation into its own products. On information and belief, Defendants’ allegations of infringement in the Harris Action against those Harris products using the Accused Video Sync Separators are based entirely on the operation/functionality of the Accused Video Sync Separators.
19. As a distributor of the Accused Video Sync Separators, Intersil has certain contractual indemnity obligations to its customers relating to claims of infringement. Pursuant to those obligations, certain customers, including Harris, have tendered to Intersil claims made by Defendants against such customers for infringement based on such customers’ use of the Accused Video Sync Separators.
COUNT I
(By Plaintiffs Intersil and Elantec Against All Defendants)
(Declaratory Judgment of Non-infringement of the Cooper Patents)
20. Plaintiffs reallege and incorporate by reference paragraphs one through nineteen as if fully set forth herein.
21. Defendants have stated that the Accused Video Sync Separators, and products incorporating such chips, infringe the Cooper Patents.
22. Plaintiffs have not infringed and do not infringe, either directly or indirectly, any valid and enforceable claim of the Cooper Patents.
23. As a result of the acts described in the foregoing paragraphs, there exists a substantial controversy of sufficient immediacy and reality to warrant the issuance of a declaratory judgment.
24. An actual and justiciable controversy exists between Plaintiffs on the one hand, and Defendants on the other, as to whether the Cooper Patents are infringed by Plaintiffs and/or
their customers. A judicial declaration is necessary and appropriate so that Plaintiffs may ascertain their rights regarding the Cooper patents.
COUNT II
(By Plaintiff Intersil against All Defendants)
(Declaratory Judgment of Invalidity of the Cooper Patents)
25. Intersil realleges and incorporates by reference paragraphs one through twenty-four above as if fully set forth herein.
26. Intersil contends that, to the extent any claims of the Cooper Patents could be construed to apply to the Accused Video Sync Separators, the Cooper Patents are invalid for failure to meet the conditions of patentability and/or otherwise comply with one or more Sections of 35 U.S.C. § 101 et seq.
27. An actual and justiciable controversy exists between Intersil on one hand, and Defendants on the other, as to whether the Cooper Patents are valid. A judicial declaration is necessary and appropriate so that Intersil may ascertain its rights regarding the Cooper patents.
COUNT III
(By Plaintiffs Intersil and Elantec against All Defendants)
(Declaratory Judgment of Unenforceability of the Cooper Patents)
28. Plaintiffs re-allege and incorporate by reference Paragraphs one through twenty-seven above as if fully set forth herein.
29. During prosecution of the applications for the ‘869 and ‘250 Patents, J. Carl Cooper, and/or the prosecuting attorneys, and/or others associated with the filing and prosecution of the applications for the ‘869 and ‘250 Patents had knowledge of and/or were in possession of the National Semiconductor LM1881 video sync separator and/or datasheets regarding the LM1881 video sync separator.
30. The National Semiconductor LM1881 video sync separator and/or datasheets regarding the LM1881 video sync separator are prior art that was material to the patentability of one or more claims of the ‘869 and ‘250 Patents.
31. The National Semiconductor LM1881 video sync separator and/or datasheets regarding the LM1881 video sync separator were not disclosed to the United States Patent and Trademark Office during the filing and prosecution of the applications for the ‘869 and ‘250
Patents. The named inventor, and/or the prosecuting attorneys, and/or others associated with the filing and prosecution of the applications for the ‘869 and ‘250 Patents, withheld information regarding the National Semiconductor LM1881 video sync separator and datasheets regarding the LM1881 video sync separator with an intent to deceive the United States Patent and Trademark Office.
32. During prosecution of the applications for the ‘869 and ‘250 Patents, J. Carl Cooper, and/or the prosecuting attorneys, and/or others associated with the filing and prosecution of the applications for the ‘869 and ‘250 Patents had knowledge of the VSG200 sync separator.
33. The VSG200 sync separator was prior art that was material to the patentability of one or more claims of the ‘869 and ‘250 Patents.
34. The VSG200 sync separator was not disclosed to the United States Patent and Trademark Office during the filing and prosecution of the applications for the ‘869 and ‘250 Patents. The named inventor, and/or the prosecuting attorneys, and/or others associated with the filing and prosecution of the applications for the ‘869 and ‘250 Patents withheld information regarding the VSG200 sync separator with an intent to deceive the United States Patent and Trademark Office.
35. An actual and justiciable controversy exists between Plaintiffs on the one hand, and Defendants on the other, as to whether the Cooper Patents are unenforceable. A judicial declaration is necessary and appropriate so that Plaintiffs may ascertain its rights regarding the Cooper Patents.
**PRAYER FOR RELIEF**
WHEREFORE, Plaintiffs respectfully requests that judgment be entered in their favor and prays that the court grant the following relief:
A. A declaration that the Accused Video Sync Separators have not infringed and do not infringe, either directly or indirectly, any valid and enforceable claim of the Cooper Patents;
B. A declaration that the claims of the Cooper Patents are invalid;
C. A declaration that the Cooper Patents are unenforceable;
D. An order enjoining Defendants, their officers, directors, agents, counsel, servants,
and employees, and all persons in active concert or participation with any of them, from charging infringement of or instituting any action for infringement of the Cooper Patents against Intersil, Elantec and/or any of their customers;
E. An order declaring that Plaintiffs are the prevailing party and that this is an exceptional case under 35 U.S.C. § 285 and award Plaintiffs their reasonable attorneys fees, expenses, and costs in this action; and
F. Such other and further relief as this Court may deem just and proper.
Dated: May 28, 2009
JONES DAY
By: ____________________________
Gregory L. Lippetz
Attorneys for Plaintiffs
INTERSIL CORP. AND ELANTEC SEMICONDUCTOR, INC.
|
Development of diagnostic tools and analysis of the density functional theory zoo
Pierpaolo Morgante
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Part of the Chemistry Commons
Development of diagnostic tools and analysis of the density functional theory zoo.
by
Pierpaolo Morgante
A dissertation submitted to the Department of Biomedical and Chemical Engineering and Science of Florida Institute of Technology in partial fulfillment of the requirements for the degree of
Doctor of Philosophy in Chemistry
Melbourne, Florida May 2021
We the undersigned committee hereby approve the attached dissertation,
Development of diagnostic tools and analysis of the density functional theory zoo.
by
Pierpaolo Morgante
______________________________
Roberto Peverati, Ph.D.
Assistant Professor
Biomedical and Chemical Engineering and Sciences
Major Advisor
______________________________
Alan B. Brown, Ph.D.
Associate Professor
Biomedical and Chemical Engineering and Sciences
Committee Member
______________________________
Nasri Nesnas, Ph.D.
Professor
Biomedical and Chemical Engineering and Sciences
Committee Member
______________________________
Jean Carlos Perez, Ph.D.
Associate Professor
Aerospace, Physics and Space Sciences
Committee Member
______________________________
D. Andrew Knight, Ph.D.
Professor and Department Head
Biomedical and Chemical Engineering and Sciences
The main goal of this dissertation is to give an overview of existing and new databases for density functional theory (DFT). It describes the procedures used to introduce A Collection of Chemistry DataBases (ACCDB) and its reduction to a smaller and statistically significant database called A Small(er) Collection of DataBase (ASCDB). Also, it presents an application of such databases in chemical education through the introduction of a notebook of twelve exercises that can easily complement a computational chemistry class. In addition, a new database of molecules having carbon-carbon long bonds (CLB18) is presented. Last, the databases are used in the selection of a suitable exchange-correlation functional approximation utilized to shed light on the mechanism of uncaging of 4-methoxy-5,7-dinitroindolinyl glutamate (MDNI-Glu) and its closely-related analogues.
Experiments 4–6: The choice of a basis set. ........................................... 41
Experiment 7: The origin of the popularity of B3LYP/6-31G*. .................. 45
Experiments 8 and 9: The choice of an integration grid. .......................... 46
Experiment 10: Do my calculations need stability analysis? ...................... 48
Experiment 11: How to compare calculated and experimental results. ........ 48
Experiment 12: Self-assessment through a transition-metal catalyzed reaction. 50
2.5 Conclusions. ..................................................................................... 50
Chapter 3: The development of A Small(er) Collection of DataBases (ASCDB) through statistical analysis. ................................................................. 52
3.1 Introduction. ....................................................................................... 52
3.2 Reducing the number of datapoints in ACCDB through data science. . 55
Step 1: Grouping the datapoints together in an unbiased manner. ....... 60
Step 2: Reducing the number of datapoints. .......................................... 60
Step 3: The *a posteriori* analysis of the new database and the definition of ASCDB. ................................................................. 61
3.3 Testing ASCDB: Results and discussion. ............................................ 65
Validation. .......................................................................................... 67
3.4 Conclusions and future applications. .................................................. 72
Chapter 4: The definition of CLB18, a new database for geometries, and insights for *xc* functional developers. ......................................................... 74
4.1 Introduction. ....................................................................................... 74
4.2 Structure of the CLB18 database. ...................................................... 75
4.3 Computational details. ....................................................................... 76
4.4 Basis set and grid studies. .................................................................. 78
4.5 Results. .............................................................................................. 81
4.6 Discussion. ......................................................................................... 83
4.6.1 Comparison with other databases .............................................. 83
4.6.2 Effect of the Hartree-Fock exchange, multi-reference character, and comparison with metal-organic interactions ........................................ 83
4.6.3 Comparison with transient long bonds ....................................... 86
4.7 Conclusions ....................................................................................... 93
Chapter 5: Uncaging glutamate using DFT: A case study. ........................................... 94
5.1 Introduction ................................................................................................................. 94
5.2 Computational details ................................................................................................. 97
5.3 Results and discussion. .............................................................................................. 98
5.3.1 The cyclic intermediate. ..................................................................................... 99
5.3.2 The migration pathway. .................................................................................... 104
5.3.3 The mechanism of uncaging of MDNI-Ac. ....................................................... 106
5.4 The origin of the quantum yield in the MDNI-family of cages. .......................... 107
5.5 Conclusions ............................................................................................................... 109
Appendix A ..................................................................................................................... 111
A1: Regression coefficients used for MGCDB84, GMTKN55, and Minnesota 2015B. ................................................................. 111
A2: Correlation plots for three basis sets (6-31G*, def2-SVPD, def2-TZVPPD) and three functionals (PBE-D3(BJ), B3LYP-D3(BJ), ωB97M-V). ................................................................. 112
Appendix B. ..................................................................................................................... 114
General note. .................................................................................................................. 114
B.1: General performance of B3LYP and B3LYP-D3(BJ) with small basis sets. ........ 114
B.2: Convergence issues for B3LYP and B3LYP-D3(BJ). ........................................... 116
B.3: Convergence of the integration grids. .................................................................. 117
B.4: Data used to generate Figure 4.2. ........................................................................ 118
Appendix C. ..................................................................................................................... 122
General note. .................................................................................................................. 122
C.1: Multi-reference character of the transition structures in the MDNI-Ac reaction mechanisms. ......................................................... 122
C2: Other reaction mechanisms involving MDNI-Ac. ................................................ 124
The Cyclization Pathway—D (CP—D). ..................................................................... 124
The Cyclization Pathway CP–B and Singlet Reactivity. .......................................... 125
C3: Reaction mechanisms involving MNI-Ac. ............................................................. 126
Main mechanism. ....................................................................................................... 126
C4: Multi-reference Character of the Relevant Transition Structures of the Main Mechanism for MNI-Ac. .......................................................... 127
C5: Other reaction mechanisms involving MNI-Ac. ........................................... 128
The Cyclization Pathway–D (CP–D). ................................................................. 128
Pathway CP–B and Singlet Reactivity. ................................................................. 129
References. ............................................................................................................. 131
2.9 Dissociation curve of the argon dimer calculated with the MN15-L/def2-QZVPPD level of theory with four different integration grids: SG1; 75,302; 99,590; 175,974. The oscillations in the curves calculated with the coarser grids are unphysical artifacts due to an insufficient grid choice. The energy is reported in kcal mol$^{-1}$, while the distance is in Å.
2.10 Dissociation reaction of the diphosphinohexatrienepalladium(0) complex. The carbon atoms are black, the phosphorus atoms are orange, the hydrogen atoms are white, the palladium atom is light blue. The dotted lines in the skeletal formulas show dative bonds. Structure taken from the PdBE2 subset of the Minnesota 2015B database.
3.1 Summary of the clusters in the ASCDB database with the “a posteriori” assignment of chemical properties. The average relative absolute energies $|\Delta E|$, and the average MUE for the 50 functionals, are also reported in the last two columns (in kcal mol$^{-1}$).
3.2 Visual summary of the properties of the small databases MG8, dietGMTKN55-30, dietGMTKN55-100, dietGMTKN55-150, and ASCDB.
3.3 Correlation plots between the MUE calculated on ASCDB and the total MUE calculated using all the datapoints from the three major databases (top-left panel, black diamonds), the MUE calculated with GMTKN55 (top-right panel, red squares), MGCDB84 (bottom-left panel, green triangles), and Minnesota 2015B (bottom-right panel, green triangles). Units on the axes are kcal mol$^{-1}$.
3.4 Correlation plots between the estimated MUEs (eMUEs) calculated using the regression formulas and the datapoints in ASCDB and the MUEs calculated with the parent databases GMTKN55 (red squares, left panel), MGCDB84 (blue circles, middle panel) and Minnesota 2015B (green triangles, right panel). Units on the axes are kcal mol$^{-1}$.
3.5 Correlation plot between the MUEs of the GMTKN55 and the eMUEs of the ASCDB database for 21 double-hybrid functionals. The units on the axes are kcal mol$^{-1}$.
3.6 Correlation plot between the calculated MUEs of selected subsets of the ASCDB database and the corresponding ones in the GMTKN55 database with the B2-PLYP-D3(BJ) (green squares) and DSD-PBEP86-D3(BJ) (blue diamonds) double-hybrid functionals. Units on the axes are kcal mol$^{-1}$.
3.7 Correlation plots between the RMSEs of ASCDB and those of the parent databases: GMTKN55 (left panel, red squares); MGCDB84 (middle panel, blue circles); Minnesota 2015B (right panel, green triangles). Some points—reported as red crosses—have been excluded from the calculations of the correlation coefficients. Units on the axes are kcal mol$^{-1}$. ........................................... 70
3.8 Mean signed errors (MSEs) for 10 methods that present a systematic error for non-covalent interactions (NC, top panel) and non-local effects (bottom panel) using ASCDB and selected datasets of the parent databases. Units on the axes are kcal mol$^{-1}$. ................................................................. 72
4.1 Performance of the 26 approximations on the CLB18 database. All results (in Å) are listed in order of increasing overall MUE. Columns 3—5 list the MUE for each group of molecules. The worst result for each method/approximation is reported in bold. .................................................................................. 82
4.2 Effect of the HF exchange on the performance of the PBE0 functional for the CLB18 database (black, solid). Performance for the three constituent group are also reported (orange, dashed: group 1; blue, dotted: group 2, green, dot-dashed: group 3). The data used to generate this figure can be found in Table B.6 in Appendix B. .................................................................................................. 84
4.3 Values of the $A_\lambda$ MR diagnostics for all structures in the CLB18 database. Problematic values according to ref.$^{382}$ are reported in bold. .................................................. 87
4.4 Comparison of the performance of the functionals on the CLB18 (MUE) and BH76 (ARMSG) databases. A green background indicates satisfactory performance, while a red background indicates poor performance. ............................................ 89
4.5 Effect of the HF exchange on the performance of the PBE0 functional for the BH76 database (green curve). The non-hydrogen transfer (NHTBH38) and hydrogen transfer (HTBH38) subsets are also reported (orange and blue curves, respectively). The data used to generate this figure can be found in Table 4.7 below. .................................................. 90
4.6 Values of the $A_\lambda$ MR diagnostics for all transition structures in the BH76 database. Problematic values according to ref.$^{382}$ are reported in bold, while the highest value is reported in red. The transition structures nomenclature (first column) is based on the names used in the original publication$^{177}$ and reported in our ACCDB database.$^{79}$ ........................................................................................................ 92
5.1 The two proposed mechanisms for the uncaging of MDNI-Glu (R=CH$_2$CH$_2$CH(NH$_2$)COOH): Migration pathway (MP), the computational mechanism reported by Pálfi et al. in ref.$^{430}$ Cyclization pathway (CP), the mechanism presumed by Ellis-Davies et al.$^{126}$ based on the data gathered by Morrison et al.$^{431}$ ........................................ 95
5.2 Schematics of the main reaction pathways for MDNI-Ac: Migration Pathway (MP)—mechanism of Pálfi et al., verified by our improved computational results; Cyclization Pathway—C (CP-C) mechanism via cyclic intermediate (see text for a detailed explanation of the pathways). Note that both mechanisms converge towards intermediate 13. The hydrolysis pathway on the singlet surface is also shown. The structures of the relevant transition states are reported in a box, while significant intermediates and products are reported without a box. .................. 96
5.3 The cyclic intermediate for MDNI-Ac. This structure is similar to structure 5 in Figure 5.1, but instead of glutamate, it is bound to acetate. ......................... 99
5.4 Key electronic effects for the cyclization and migration pathways (CP and MP). The oxygen radical in (a) is not positioned at the ideal Bürgi–Dunitz angle of 107° relative to the plane of the molecule, and it is far away from the unoccupied orbital of the carbonyl group. Both pathways necessitate deplanarization of the amide bond via a rotation (b) in either sense, leading to two enantiomers (only one shown; see Table 5.2 for more details) in the case of the CP, or directly to the following intermediate in the case of MP. .................................................. 102
5.5 Schematics of the Norrish type reactions (a). The MP pathway combines both Norrish type I and type II characteristics (b). Breaking the Z–R bond (Z=N, R=COCH$_3$) results in a Norrish type I reaction, while the adjacent Z–O bond is in a 1,6-relationship (Norrish type II) and facilitates the cleavage of Z–R. Panel (c) shows the key migration transition state. If R=CH$_2$CH$_3$, the reaction loses the Norrish type I characteristic, and hence, not surprisingly, higher in energy by about 15.5 kcal mol$^{-1}$. ................................................................. 105
5.6 The reaction mechanism for the uncaging of MDNI-Ac. The singlet surface is indicated in blue, while the triplet surface is indicated in green. The CP–C mechanism is outlined with the dashed line, while the MP mechanism follows the dotted line. They both converge towards structure 22, and then they proceed towards the products in the same way. The reported numerical values are Gibbs free energies calculated with respect to MDNI-Ac in the singlet state (structure 16), and they have been obtained at the ωB97X-D/def2-TZVP level of theory in water (C-PCM). All values are in kcal mol$^{-1}$. Structures 16, 18, 19, 20, 22, 24, and 26 have been renumbered for better clarity, and they correspond to structures 9, 11, 12, 10, 13, 14, and 8, respectively, in Figure 5.2.
5.7 Comparison of the experimental (green, solid) and calculated (red, dashed) UV–Vis spectra of (a) MNI (top panel) and (b) MDNI (bottom panel). The calculated spectra are obtained by applying a Lorentzian broadening to the ωB97X-D/def2-TZVP electronic transitions, also reported as red vertical lines. The canonical π* lowest unoccupied molecular orbitals involved in the bright electronic transitions are also reported. The experimental spectra have been obtained in water (pH = 7.39 with NaHCO$_3$), at a concentration of 0.074 mM and using radiation at 350 nm as explained in Ref. 428
A.1 Regression coefficients for the estimated MUEs of the MGCD84 (top panel), GMTKN55 (middle panel), and Minnesota 2015B (bottom panel) databases. Only the 54 datapoints reported in this figure have a non-zero coefficient.
A.2 Correlation plot for the 6-31G* basis set with the PBE-D3(BJ) (in blue), B3LYP-D3(BJ) (in red), and ωB97M-V (in green) functionals. The units on the axes are kcal mol$^{-1}$.
A.3 Correlation plot for the def2-SVPD basis set with the PBE-D3(BJ) (in blue), B3LYP-D3(BJ) (in red), and ωB97M-V (in green) functionals. The units on the axes are kcal mol$^{-1}$.
A.4 Correlation plot for the def2-TZVPPD basis set with the PBE-D3(BJ) (in blue), B3LYP-D3(BJ) (in red), and ωB97M-V (in green) functionals. The outlier for the ωB97M-V functional is reported as a green x, and not considered in the correlation. The units on the axes are kcal mol$^{-1}$.
C.1 The CP–D reaction mechanism for MDNI-Ac. The triplet surface is reported in green, while the singlet surface is in blue. The reported numerical values are Gibbs free energies calculated with respect to MDNI-Ac in the singlet state (structure S1), and they have been obtained at the $\omega$B97X-D/def2-TZVP level of theory in water (C-PCM). All values are in kcal mol$^{-1}$. ................................. 125
C.2 The reaction mechanism for the uncaging of MNI-Ac. The singlet surface is indicated in blue, while the triplet surface is indicated in green. The CP–C mechanism is outlined with the dashed line, while the MP mechanism follows the dotted line. They both converge towards structure S19, and then they proceed towards the products in the same way. The reported numerical values are Gibbs free energies calculated with respect to MNI-Ac in the singlet state (structure S13), and they have been obtained at the $\omega$B97X-D/def2-TZVP level of theory in water (C-PCM). All values are in kcal mol$^{-1}$. ........................................... 126
C.3 The reaction mechanism for the uncaging of MNI-Ac. The singlet surface is indicated in blue, while the triplet surface is indicated in green. The CP–C mechanism is outlined with the dashed line, while the MP mechanism follows the dotted line. They both converge towards structure S19, and then they proceed towards the products in the same way. The reported numerical values are Gibbs free energies calculated with respect to MNI-Ac in the singlet state (structure S13), and they have been obtained at the $\omega$B97X-D/def2-TZVP level of theory in water (C-PCM). All values are kcal mol$^{-1}$. ........................................... 127
C.4 The CP–D reaction mechanism for MNI-Ac. The triplet surface is reported in green, while the singlet surface is in blue. The reported numerical values are Gibbs free energies calculated with respect to MNI-Ac in the singlet state (structure S17) and they have been obtained at the $\omega$B97X-D/def2-TZVP level of theory in water (C-PCM). All values are in kcal mol$^{-1}$. ........................................... 129
C.5 The CP–B mechanism (upper section) and the hydrolysis mechanism (lower section) for MNI-Ac. The triplet surface is reported in green, while the singlet surface is in blue. The reported numerical values are Gibbs free energies calculated with respect to MNI-Ac in the singlet state (structure S13) and they have been obtained at the $\omega$B97X-D/def2-TZVP level of theory in water (C-PCM). All values are in kcal mol$^{-1}$. 130
2.1 Summary of the databases included in ACCDB, listed by decreasing size. RE stands for “reaction energies.” ................................................................. 24
2.2 Unique subsets in the ACCDB collection of chemical databases, listed in alphabetical order. ........................................................................................................... 25
3.1 The 50 $xc$ functionals used to generate a population of data large enough to be analyzed through statistical tools. The following acronyms have been used: LSDA=Local Spin-Density Approximation; GGA=generalized gradient approximation; NGA=non-separable gradient approximation; mGGA=meta-generalized gradient approximation; mNGA=meta-non-separable gradient approximation; GH=Global hybrid; RSH=range-separated hybrid. ............................................. 56
3.2 Basis sets used in the calculation of the datapoints for the three core databases of the ACCDB collection. MN refers to the Minnesota 2015B database. ............ 59
3.3 Estimated RMSEs from ASCDB compared to the calculated RMSEs (in parenthesis) for the main subsets of MGCDB84 for three $xc$ functionals and three basis sets not used in the data analysis. All data are in kcal mol$^{-1}$. ........................................ 68
3.4 Comparison of the MUEs between significant chemical properties of ASCDB and GMTKN55 using doubly hybrid functionals. All data are in kcal mol$^{-1}$. .... 69
4.1 Molecules included in the CLB18 database. The reference bond lengths are listed in Å, and they refer to the bond highlighted in red in the corresponding formula. 75
4.2 Computational methods used in this study. We listed the functional type (Generalized Gradient Approximation (GGA), meta-GGA, global and range-separated hybrid GGA or meta-GGA) together with the quantum chemistry program used, the percentage of HF exchange (%HF X) included in the approximation, and the respective references. ....................................................................................... 77
4.3 Calculated bond length (in Å) for the CLB18_15 molecule with the B3LYP and B3LYP-D3(BJ) functionals and 17 different basis sets. The reference experimental value is 1.830 Å. ....................................................................................... 80
4.4 Calculated bond length (in Å) for the CLB18_15 molecule with different integration grids using the B3LYP and B3LYP-D3(BJ) functionals and the 6-31G* basis set. The computation time (in s) of a single SCF cycle on a single thread is also reported as an estimation of the computational cost of each grid. ........................................... 81
4.5 Calculated bond lengths and mean unsigned errors (MUEs) for the HF method using the restricted and unrestricted formalism. Bond lengths and MUEs are in Å, while the energy differences (fourth column) are in kcal mol$^{-1}$. ........................................... 85
4.6 The level of theory (functional/method and basis set) used to obtain the reference geometries in the BHs subsets of the MGCDB84 and GMTKN55 databases. We reported in bold the non-DFT approaches. ................................................................. 88
4.7 Average root-mean square gradient (ARMSG, Hartrees) for the BH76 database (and its subsets NHTBH38 and HTBH38) calculated with the PBE functional and its hybrids. .................................................................................................................. 90
5.1 Gibbs free energies of the relevant transition structures (TSs) obtained at the ωB97X-D/def2-TZVP level of theory. The number in parenthesis represents the activation (free) energies calculated with respect to the compound in the triplet state (column 2). All values are in kcal mol$^{-1}$. ................................................................. 98
5.2 The four cyclic structures taken into account based on the configuration of the stereocenter (R or S) and the conformation (boat or chair) of the cyclohexene ring for MDNI-Ac. The carbon atoms are in black, hydrogens in white, nitrogens in blue and oxygens in red. Skeletal formulas (hydrogen atoms are omitted) are included for clarity. ........................................................................................................ 101
5.3 Calculated bond lengths (in Å) and bond angles (°) in the cyclization and migration transition structures (TSs) compared with the (R-Chair) cyclic intermediate. ................................................................. 103
5.4 Details of the excitation types for the singlet and triplet states in the experimental range (250–500 nm) together with the excitation energy (in eV) for MNI-Glu and MDNI-Glu. .................................................................................................................. 109
B.1 Performance of B3LYP and B3LYP-D3(BJ) with some common basis sets of double-ζ quality against the CLB18 database. The results are reported in order of increasing basis set size. All values are in Å. MUE = Mean unsigned error. ........ 114
B.2 Performance of B3LYP and B3LYP-D3(BJ) with some common basis sets of double-ζ quality against the CLB18 database. The results are reported in order of increasing basis set size. All values are in Å. MSE = Mean signed error. ........ 115
First and foremost, I would like to thank Roberto Peverati for being a great advisor and mentor. I consider myself lucky that I got the opportunity to join his research group four years ago. I am grateful for his support and encouragement, as well as the freedom he gave me to work on so many different projects. I have learned so much from him that it is hard to describe in a few lines. I am honored he chose me as his first Ph.D. student, and I believe I am fortunate to know that I will always have his guidance, advice and friendship. I would like to thank Dr. Nasri Nesnas for his kindness, support, and for always challenging me, in class as well as outside. The uncaging of MDNI-Glu was one of the most challenging reactions I ever worked on, but undoubtedly one of the most rewarding. I want to thank Dr. Alan Brown for his Advanced Organic Chemistry class, and for his helpful comments on the progress of my Ph.D. work. I want to thank Dr. Jean Perez for accepting to be in my Ph.D. committee, and for his helpful remarks on my proposals. I want to thank Dr. Norito Takenaka for being a long-time collaborator, and for always being kind and supportive.
I also want to thank everyone in the Chemistry program at Florida Tech. My first special shout-out goes to Dr. Jessica Smeltz, an amazing supervisor. I wish to thank Dr. Rudi for collaborating with us, Christie Gates for her amazingly delicious brownies, as well as for her helpfulness in dealing with the bureaucracy, and Wilma Cohen, the kindest person I know.
I would not be where I am today without the love and support of my parents, Anna Rita and Domenico. Even if they (still) do not understand what I do, they tried to listen to me, and even pretended to find it interesting. I want to thank my sister, Cinzia, for being the most amazing sister one could possibly imagine. I love you with all my heart.
I want to thank Kelly for being the most amazing partner one might possibly want. You are my sunshine. I want to thank Debbie, Dennis, Emily, Jinx, Nancy and Maria for treating me like a second (or third) son (or brother). I want to thank Pebbles, Lola, Daisy and Shadow—our lovable fur babies—for all the cuddles and kisses (even if they are stinky).
I am grateful to (in rigorous random order) Matteo, Elena, Alberto, Luigi, Simone, Fiorenzo,
Silvia, Pietro Marchetti, Livia Conte, Andrea, Giovanni (Doc), Chiara and Michela for their love, support and patience over the past decade (if not more).
Since I am a classical music fanatic, I want to acknowledge (the living) Martha Argerich, Daniel Barenboim, Maria João Pires, Gustavo Dudamel, Yuja Wang, and (the dead) Bach, Mozart, Beethoven, Rachmaninoff, Schubert, Schumann and Liszt.
Chapter 1: Foundations of Quantum Mechanics.
1.1 Introduction.
At the beginning of the twentieth century, many fields of science already achieved some of their greatest accomplishments. In chemistry, the concept of the molecule was introduced and later accepted, and Mendeleev proposed the periodic table in its modern form. In physics, the whole field of thermodynamics was born, and many contributed to the growth and maturation of optics and electromagnetic theory. However, despite such improvements and enthusiasm, classical physics still failed to describe a few observed properties of condensed matter, most notably the blackbody radiation and the photoelectric effect. Both problems were solved—by Max Planck and Albert Einstein respectively—by making the assumption that the energy, rather than being a continuous quantity, was instead quantized, i.e. had discreet values. This revolutionary idea contributed to give birth to a new field of physics, called quantum mechanics, which describes the behavior of microscopic objects such as atoms and molecules.
The discipline of quantum mechanics is built upon the time-dependent Schrödinger equation, published in 1926. It is a partial differential equation that can be formulated as an eigenvalue problem in the form:
\[
\hat{H}|\Psi_i(\mathbf{r}, \mathbf{R})\rangle = E_i|\Psi_i(\mathbf{r}, \mathbf{R})\rangle
\]
(1.1)
In equation (1.1), $\hat{H}$ symbolizes the Hamiltonian operator, $E_i$ is the energy of a particle or collection of particles (a state) described by the wave function (WF) $|\Psi_i(\mathbf{r}, \mathbf{R})\rangle$. $|\Psi_i(\mathbf{r}, \mathbf{R})\rangle$ represents an eigenfunction of $\hat{H}$, and $E_i$ is the associated eigenvalue. The variational principle implies that the state with $i = 0$ will always have the lowest energy. As a consequence, the relationship among the different eigenvalues can be described as $E_0 \leq E_1 \leq E_2 \leq \cdots \leq E_i \leq \cdots \leq E_\infty$. Finding $E_0$ corresponds to solving equation (1.2)
\[
\hat{H}|\Psi_0\rangle = E_0|\Psi_0\rangle
\]
(1.2)
where \(|\Psi_0\rangle\) is the ground state WF and \(E_0\) represents the ground state energy.
We can apply the Hamiltonian operator to find the total energy of atoms and molecules. More specifically, we can use the Hamiltonian to calculate the five different contributions to the total energy, expressed in equation 1.3 using atomic units:
\[
\hat{H} = -\frac{1}{2} \left[ \sum_i^N \nabla_i^2 \right] - \left[ \sum_a^M \frac{1}{2m_a} \nabla_a^2 \right] - \left[ \sum_i^N \sum_a^M \frac{Z_a}{|\vec{r}_i - \vec{R}_a|} \right] + \\
\left[ \sum_i^N \sum_{j>i}^N \frac{1}{|\vec{r}_i - \vec{r}_j|} \right] + \left[ \sum_a^M \sum_{b>a}^M \frac{Z_a Z_b}{|\vec{R}_a - \vec{R}_b|} \right]
\]
(1.3)
The \(N\) electrons in the system are indicated with the indeces \(i\) and \(j\), while the \(M\) nuclei are described using \(a\) and \(b\). \(\nabla^2\) is the Laplacian operator, and it corresponds to the second derivative with respect to the spatial coordinates, namely \([\frac{\partial^2}{\partial x^2} + \frac{\partial^2}{\partial y^2} + \frac{\partial^2}{\partial z^2}]\), for either the electrons (\(i\)) or the nuclei (\(a\)). \(\vec{r}_i\) is the vector that indicates the position of the electrons, and \(\vec{R}_a\) is the vector that describes the position of the nuclei. \(Z_a\) indicates the atomic number.
By using the operators only, we can write the previous equation in a more compact way as:
\[
\hat{H} = \hat{T}_e + \hat{T}_n + \hat{V}_{en} + \hat{V}_{ee} + \hat{V}_{nn} \xrightarrow{\text{Used to calculate}} E
\]
(1.4)
The subscripts \(e\) and \(n\) refer to electrons and nuclei, respectively. The first and second terms of equation 1.4 correspond to the kinetic energy of the electrons (equation 1.4.1) and the nuclei (equation 1.4.2). The third, fourth and fifth term are instead associated with the potential energy of the system. More specifically, the third term in equation 1.3 (equation 1.4.3) arises from the attractive interactions between electrons and nuclei, while the fourth (equation 1.4.4) and fifth (equation 1.4.5) are the repulsive nuclear-nuclear and electron-electron interaction terms. By applying these operators, we can get access to the associated kinetic (\(T_e, T_n\)) and potential (\(V_{ee}, V_{ne}, V_{nn}\)) energies (the operators are indicated through the \(\hat{}\)) sign, while the respective energies are not).
\[
\hat{T}_e = -\frac{1}{2} \left[ \sum_i^N \nabla_i^2 \right] \xrightarrow{\text{Used to calculate}} T_e
\]
(1.4.1)
\[
\hat{T}_n = - \left[ \sum_a^M \frac{1}{2m_a} \nabla_a^2 \right] \quad \text{Used to calculate } T_n
\]
(1.4.2)
\[
\hat{V}_{en} = - \left[ \sum_i^N \sum_a^M \frac{Z_\alpha}{|\vec{r}_i - \vec{R}_a|} \right] \quad \text{Used to calculate } V_{en}
\]
(1.4.3)
\[
\hat{V}_{ee} = \left[ \sum_i^N \sum_{j>i}^N \frac{1}{|\vec{r}_i - \vec{r}_j|} \right] \quad \text{Used to calculate } V_{ee}
\]
(1.4.4)
\[
\hat{V}_{nn} = \left[ \sum_a^M \sum_{b>a}^M \frac{Z_\alpha Z_b}{|\vec{R}_a - \vec{R}_b|} \right] \quad \text{Used to calculate } V_{nn}
\]
(1.4.5)
Overall, the Schrödinger equation written in equation 1.1 depends on $3N$ electronic and $3M$ nuclear coordinates. Solving this problem, which depends on $3(N + M)$ variables overall, is such an extraordinary task that it would prove almost impossible for everything besides a handful of simple systems. To reduce the complexity of the problem, the Born-Oppenheimer approximation is usually invoked. Its physical foundation is built on the fact that nuclei are $\sim 1,800$ times heavier than the electrons. If nuclei move, the electrons accommodate this change almost instantaneously. As a result, we can consider the nuclear arrangement as fixed, and the electrons move in the field of the (fixed) nuclei. Then, we can treat separately the terms that depend only on the nuclear coordinates from the terms that depend on electronic coordinates. Equation 1.4 is then simplified by disregarding the nuclear terms $\hat{T}_n$ and $\hat{V}_{nn}$, and the resulting *electronic* Schrödinger equation has the form:
\[
\hat{H}_e |\Psi_e\rangle = [\hat{T}_e + \hat{V}_{en} + \hat{V}_{ee}] |\Psi_e\rangle = E_e |\Psi_e\rangle
\]
(1.5)
Each of the terms in equation 1.5 is now dependent only on the electronic coordinates, while it depends parametrically on the coordinates of the nuclei. The electronic energy is then calculated for a given nuclear configuration. The nuclear terms from equations 1.4.2 and 1.4.5 are known, and once the corresponding energies are calculated, they can be added to the electronic energy from equation 1.5 to yield the total energy of the system as written in equation 1.4. It is also worth noting at this point that the wave function $|\Psi_i\rangle$ used in equation 1.1 depends on the coordinates of both electrons and nuclei, while the electronic WF used in equation 1.5 only depends on the
coordinates of the electrons.
The purely electronic terms in equation 1.5, *i.e.* the kinetic and potential energy of the electrons ($T_e$ and $V_{ee}$), can be directly calculated, while the nuclei-electrons potential ($V_{en}$) can be treated as a parameter. Despite these simplifications, the electronic Schrödinger equation is still a formidable task to solve analytically, primarily because of the electron-electron interaction term $V_{ee}$. Systems with just one electron can be treated *exactly* (*i.e.* equation 1.5 can be solved), while systems having two (or more) electrons, such as the helium atom, cannot. Approximations to the electronic wave function $|\Psi_e\rangle$ need to be made, and they usually involve one-electron wave functions called *spin orbitals*, $|\phi(\mathbf{x}_i)\rangle$, that depend on the spin and position of the electrons, here collectively indicated as $\mathbf{x}_i$. The simplest approximation to $|\Psi_e\rangle$ is called the *Hartree product* (HP),\textsuperscript{1,2} which simply corresponds to the product of $N$ spin orbitals.
$$|\Psi_e\rangle = \prod_i^N |\phi(\mathbf{x}_i)\rangle = |\Psi_{\text{HP}}\rangle \quad (1.6)$$
Simplicity is the major advantage of the HP. However, this comes at the cost of completely neglecting electron-electron interactions. Electrons are treated as completely independent from each other. In reality, they are charged particles whose motion is correlated, and the position of one electron influences the position of another. In other words, two electrons cannot coexist in the same space, as stated by the Pauli exclusion principle. The HP violates this physical law.
The electron WF should be anti-symmetric with respect to the exchange of two particles. This property originates from the fermionic nature of the electrons. The HP does not change sign when two particles are interchanged, and it lacks this second requirement as well. In 1930, Fock\textsuperscript{3} and Slater\textsuperscript{4} were the first to identify this limitation, and they corrected the problem by proposing a different mathematical form for the (approximated) WF, namely a *Slater determinant* of spin orbitals ($|\Psi_{\text{SD}}\rangle$, equation 1.7 below):
$$|\Psi_{\text{SD}}\rangle = \frac{1}{\sqrt{N!}} \begin{vmatrix}
|\phi_1(\mathbf{x}_1)\rangle & |\phi_2(\mathbf{x}_1)\rangle & \cdots & |\phi_N(\mathbf{x}_1)\rangle \\
|\phi_1(\mathbf{x}_2)\rangle & |\phi_2(\mathbf{x}_2)\rangle & \cdots & |\phi_N(\mathbf{x}_2)\rangle \\
\vdots & \vdots & \ddots & \vdots \\
|\phi_1(\mathbf{x}_N)\rangle & |\phi_2(\mathbf{x}_N)\rangle & \cdots & |\phi_N(\mathbf{x}_N)\rangle
\end{vmatrix} \quad (1.7)$$
Slater determinant wave functions are anti-symmetric, and at the same time they treat the electrons
as indistinguishable, as each of them appears in every spin orbital. Using Slater determinants to solve the Schrödinger equation corresponds to using the Hartree–Fock approximation, which will be the subject of the following section.
1.2 The Hartree–Fock approximation.
Within the theoretical framework of the Hartree–Fock (HF) approximation (or method), we can recast the eigenvalue problem of equation 1.1 as
\[
E_e^{\text{HF}}[\Phi_{\text{HF}}] = \min_{\Psi_{\text{SD}}} \left\{ \langle \Psi_{\text{SD}} | \hat{H}_e | \Psi_{\text{SD}} \rangle \right\}
\]
(1.8)
Equation 1.8 states that the HF energy (\(E_e^{\text{HF}}\)) results from the minimization of the right hand side of equation 1.8, the expectation value \( \langle \Psi_{\text{SD}} | \hat{H} | \Psi_{\text{SD}} \rangle \). The determinant that gives the lowest energy is the HF wave function, \(|\Phi_{\text{HF}}\rangle\). \(|\Psi_{\text{HF}}\rangle\) has the important property of being the best monodeterminantal approximation to the exact WF of the system, and usually it is the branching point either towards more accurate (coupled cluster, CC) or less accurate (semi-empirical) approaches.
The electronic Hamiltonian of 1.5 is composed of two terms (\(\hat{T}_e\) and \(\hat{V}_{en}\)) that depend on the coordinates of only one electron, and one term (\(\hat{V}_{ee}\)) that depends on the coordinates of two electrons. It is convenient to re-write their expression as the sum of a one-electron operator \(\hat{h}\) (equation 1.9) and a two-electron operator \(\hat{g}\) (equation 1.10)
\[
\hat{h} = \sum_i^N \hat{h}(\mathbf{r}_i) \longrightarrow \hat{h}(\mathbf{r}_i) = \hat{t}(\mathbf{r}_i) + \hat{v}(\mathbf{r}_i) = -\frac{1}{2} \nabla_i^2 - \sum_a^M \frac{Z_a}{|\mathbf{r}_i - R_a|}
\]
(1.9)
\[
\hat{g} = \sum_i^N \sum_{j>i}^N \hat{g}(\mathbf{r}_i; \mathbf{r}_j) \longrightarrow \hat{g}(\mathbf{r}_i; \mathbf{r}_j) = \frac{1}{|\mathbf{r}_i - \mathbf{r}_j|} = \frac{1}{r_{ij}}
\]
(1.10)
Using equations 1.9 and 1.10, the electronic Hamiltonian becomes:
\[
\hat{H}_e = \hat{h} + \hat{g} = \sum_i^N \left\{ \hat{h}(\mathbf{r}_i) + \sum_{j>i}^N \hat{g}(\mathbf{r}_i, \mathbf{r}_j) \right\}
\]
(1.11)
From now on, we will use the bra-ket notation introduced by Paul Dirac in the 1930s to describe the integrals resulting from the evaluation of \(\hat{h}\) and \(\hat{g}\) with the spin orbitals \(|\phi_i(\mathbf{x}_i)\rangle\), as in equations 1.12.1–1.12.5.
\[ |\phi_i\rangle \equiv \phi(x_i) \]
(1.12.1)
\[ \langle \phi_i | \equiv \phi^*(x_i) \]
(1.12.2)
\[ \langle \phi_i | \phi_i \rangle \equiv \int \phi^*(x_i) \phi(x_i) \, dx_i \]
(1.12.3)
\[ \langle \phi_i | \hat{O} | \phi_i \rangle \equiv \int \phi^*(x_i) \hat{O} \phi(x_i) \, dx_i \]
(1.12.4)
\[ \langle \phi_i \phi_j | \phi_i \phi_j \rangle \equiv \int \int \phi^*(x_i) \phi^*(x_j) \frac{1}{r_{ij}} \phi(x_i) \phi(x_j) \, dx_i \, dr_j \]
(1.12.5)
We used the general notation for a quantum mechanical operator, \( \hat{O} \), in equation 1.12.4.
The total electronic energy is then evaluated as
\[ E_e[\{\phi_i\}] = \sum_i^N \langle \phi_i | \hat{h} | \phi_i \rangle + \frac{1}{2} \sum_i^N \sum_j^N \langle \phi_i \phi_j | \phi_i \phi_j \rangle - \langle \phi_i \phi_j | \phi_j \phi_i \rangle = \]
\[ = \sum_i^N \langle \phi_i | \hat{h} | \phi_i \rangle + \frac{1}{2} \sum_i^N \sum_j^N J_{ij} - K_{ij} \]
(1.13)
In equation 1.13, the \( J_{ij} \) and \( K_{ij} \) correspond to the Coulomb and exchange integrals, respectively. One describes the electrostatic interaction between two electrons, while the other describes the purely quantum mechanical “exchange” interaction, which has no classical analogue.
In order to calculate the HF energy of equation 1.8, we need to minimize the energy with respect to the spin orbitals. We perform this task as a constrained optimization, since the spin orbitals have to be orthogonal and normalized, \( \langle \phi_i | \phi_j \rangle = \delta_{ij} \). The problem can be reformulated invoking the use of Lagrange multipliers, and the energy expression becomes:
\[ \mathcal{L}[\{\phi_i\}] = E_e[\{\phi_i\}] - \sum_i^N \sum_j^N \epsilon_{ij} [\langle \phi_i | \phi_j \rangle - \delta_{ij}] \]
(1.14)
It is possible to choose a set of spin orbitals, usually by means of unitary transformations, so that only the diagonal Lagrangian multipliers, $\epsilon_{ii}$, are non-zero. The first variation of the Lagrangian $\delta L$ is set to zero, which allows the calculation of the variation of the energy $\delta E_e$, which can be written in terms of an additional one-electron operator called the Fock operator, $F_i$:
$$\hat{F}_i = \hat{h}_i + \sum_j^N \hat{J}_{ij} - \hat{K}_{ij}$$ \hspace{1cm} (1.15)
Through the Fock operator, all the one- and two-electron contributions to the total energy can be evaluated. From the inspection of the right hand side of equation 1.15, we see that each electron interacts with the average field generated by the $N - 1$ other electrons in the system through the Coulomb and exchange operators. The electron-electron interactions are treated in a mean-field manner.
So far, we considered the spin orbitals as the building blocks to evaluate the HF energy. They are composed of a spatial and a spin part, and they can be indicated as
$$|\phi_i(x_i)\rangle = |\psi_i(r_i)\rangle|\alpha_i(s_i)\rangle$$ \hspace{1cm} (1.16)
$$|\phi_i(x_i)\rangle = |\psi_i(r_i)\rangle|\beta_i(s_i)\rangle$$ \hspace{1cm} (1.17)
where the dependence of the spin orbitals on the spatial ($r$) and spin (either up, $\alpha(s)$, or down, $\beta(s)$) components is made explicit. For simplicity, we will only consider a closed-shell case where there are $\frac{N}{2}$ electrons with $\alpha$ (up) spin, and $\frac{N}{2}$ electrons with $\beta$ (down) spin. The theoretical framework is called restricted HF (RHF), and it used for closed-shell systems as a default in most computational chemistry programs. There are $\frac{N}{2}$ occupied orbitals in a closed-shell system, and they are all doubly occupied. The RHF framework allows us to focus on the spatial part only, since the spin component is integrated out. The spatial part of the spin orbitals in equations 1.16 and 1.17 is usually expanded through a set that includes $K$ (atomic) basis functions $|\chi_\mu\rangle$.
$$|\psi_i(r_i)\rangle = \sum_{\mu=1}^{K} c_{\mu i} |\chi_\mu(r_i)\rangle$$ \hspace{1cm} (1.18)
The basis functions are used as building blocks in all the integrals needed to evaluate the
Coulomb and exchange integrals, as well as the kinetic energy. The eigenvalue problem can then be recast in matrix form:
\[
F_i \sum_{\mu}^{K} c_{\mu i} | \chi_{\mu} \rangle = \varepsilon_i \sum_{\mu}^{N} c_{\mu i} | \chi_{\mu} \rangle
\]
(1.19)
or equivalently, in the Roothaan–Hall formalism,\textsuperscript{5,6}
\[
\mathbf{F C} = \mathbf{S C} \varepsilon
\]
(1.20)
where $\mathbf{F}$ represents the Fock matrix, $\mathbf{S}$ is the matrix of the overlap between the basis functions, $\mathbf{C}$ is the matrix containing all the coefficients of the basis functions, and $\varepsilon$ is the matrix containing all the energy (eigen)values.
The Fock matrix allows the determination of both the coefficients used in the basis set expansion and the energy of the system. However, the coefficients are also needed for the initial evaluation of the Fock matrix. The minimization of the energy is then carried out using a procedure called self-consistent field (SCF). The SCF consists of different steps, that collectively form a cycle. The first step corresponds to the calculation of all the (one- and two-electron) integrals in the Fock matrix using an initial guess (the initial matrix of the coefficients $\mathbf{C}$, also called density matrix) provided at the beginning. After the Fock matrix is formed, it is subsequently diagonalized to obtain a new set of coefficients, and a new density matrix ($\mathbf{C}'$) is then determined. The cycle is repeated until the initial and final density matrices are similar to each other within a certain threshold. If this condition is met, the cycle is concluded and \textit{convergence is reached}. The coefficients obtained in this way, together with the basis functions used to approximate the WF define the \textit{canonical orbitals}, \textit{i.e.} the orbitals that give the ground state energy $E_0^{\text{HF}}$.
### 1.3 Density functional theory.
The central quantity in the theoretical framework built upon the Schrödinger equation is the wave function, a mathematical object that encloses all the information about a system. By interrogating the WF using quantum mechanical operators, one can obtain all the relevant quantities of interest, from the total energy of a system to its dipole moment. The HF method and its improvements are collectively called wave function theory, or WFT.
Using the WF of a system to obtain its total energy is a tedious and expensive task. Already a few years after the publication of the Schrödinger equation, many physicists started looking at other, more accessible quantities that they could use to calculate the total energy of a system and other properties. Scientists such as Enrico Fermi, Llewellyn Thomas, and Paul Dirac focused their efforts on using the electron density of a system, $\rho(\mathbf{r})$, as basic variable. The electron density, an observable, depends only on 3 spatial variables, becoming a cheaper alternative to the more sophisticated wave function. In terms of the WF, the electron density can be defined as
$$\rho(\mathbf{r}_1) = N \int \int \cdots \int \Psi_{e,0}^*(\mathbf{r}_1, s, \mathbf{x}_2, \cdots, \mathbf{x}_N) \Psi_{e,0}(\mathbf{r}_1, s, \mathbf{x}_2, \cdots, \mathbf{x}_N) ds_1 d\mathbf{x}_2 \cdots d\mathbf{x}_N \quad (1.21)$$
It is defined as “the number of electrons per unit volume in a given state,” or as “the probability of finding an electron within the volume element $d\mathbf{r}_1$.” Integration of the electron density over all space gives the number of electrons in a system, $\int \rho(\mathbf{r}) d\mathbf{r} = N$.
Historically, the first definition of a functional of the density, i.e. a mathematical framework that delivers a number when it is fed a function, was proposed in 1927, when both Thomas and Fermi tried to calculate the electronic energy of a system given its density. Formally, the problem can be stated as
$$E_e[\rho(\mathbf{r})] = T[\rho(\mathbf{r})] + V_{en}[\rho(\mathbf{r})] + V_{ee}[\rho(\mathbf{r})] \quad (1.22)$$
In equation 1.22, all quantities have been expressed as functionals of the electron density. The electrostatic interaction terms $V_{en}$ and $V_{ee}$ can be calculated exactly using equations 1.23 and 1.24
$$V_{en}[\rho(\mathbf{r})] = - \int \frac{Z_a \rho(\mathbf{r})}{|\mathbf{r} - R_a|} d\mathbf{r} \quad (1.23)$$
$$V_{ee}[\rho(\mathbf{r})] = + \frac{1}{2} \int \frac{\rho(\mathbf{r}_i) \rho(\mathbf{r}_j)}{|\mathbf{r}_i - \mathbf{r}_j|} d\mathbf{r}_i d\mathbf{r}_j \quad (1.24)$$
To calculate the kinetic energy as a functional of the electron density, an approximation is needed. According to the Thomas–Fermi (TF) model, the kinetic energy can be calculated as
\[ T_e^{\text{TF}}[\rho(\mathbf{r})] = \frac{3}{10} (3\pi^2)^{\frac{2}{3}} \int \rho(\mathbf{r})^{\frac{5}{3}} d\mathbf{r} \tag{1.25} \]
A few years later, Dirac\textsuperscript{11} proposed to include an exchange term to the TF ansatz. The total energy of the system becomes
\[ E_e^{\text{TFD}}[\rho(\mathbf{r})] = T_e^{\text{TF}}[\rho(\mathbf{r})] + V_{en}[\rho(\mathbf{r})] + V_{ee}[\rho(\mathbf{r})] - E_x^{\text{D}}[\rho(\mathbf{r})] \tag{1.26} \]
The last term in equation 1.26, indicated here as \( E_x^{\text{D}}[\rho(\mathbf{r})] \), has the form
\[ E_x^{\text{D}}[\rho(\mathbf{r})] = \frac{3}{4} \left( \frac{3}{\pi} \right)^{\frac{1}{3}} \int \rho(\mathbf{r})^{\frac{4}{3}} d\mathbf{r} \tag{1.27} \]
Unfortunately, both the TF and the TFD models suffer from a major drawback: they do not bind molecules, as they predict them to be less stable than their constituent atoms.\textsuperscript{12} The origin of this behavior was later identified in the poor description of the kinetic energy, an issue that still affects the approaches known as orbital-free DFT.
It took more than thirty years to re-establish the utility of the electron density as a variable of interest. In 1964, Pierre Hohenberg and Walter Kohn established the theoretical foundation of density functional theory (DFT) through two theorems.\textsuperscript{13} The first Hohenberg–Kohn theorem (HK-I) is formulated to establish the importance of the density as the basic variable, and to validate its use. The first theorem states that the external potential “\( v(\mathbf{r}) \) is (to within a constant) a unique functional of \( \rho(\mathbf{r}) \).”\textsuperscript{13} This means that the density can be used as basic variable, since it uniquely defines an external potential, and to an external potential corresponds one (and one only) electron density. In order to understand the first theorem, we will refer back to the electronic Hamiltonian of equation 1.5. In that case, the external potential is represented by the interaction between the electrons and the nuclei (equation 1.4.3). If we define \( v(\mathbf{r}_i) = \sum_a^M \frac{Z_a}{|\mathbf{r}_i - \mathbf{R}_a|} \), we can re-write equation 1.5 as
\[ \hat{H}_e = -\frac{1}{2} \sum_i^N \nabla_i^2 - \sum_i^N v(\mathbf{r}_i) + \sum_i^N \sum_{j>i}^N \frac{1}{|\mathbf{r}_i - \mathbf{r}_j|} \tag{1.28} \]
The electronic Hamiltonian is then completely defined given the total number of electrons \( N \), as well as the charge (\( Z_a \)) and position (\( \mathbf{R}_a \)) of the nuclei. Once we define the Hamiltonian, we can
Using the functional of the electron density described in the right hand side of equation 1.32, it is possible to calculate the total energy. For any trial density $\tilde{\rho}(\mathbf{r})$, the expression becomes
$$E_{e,0}[\tilde{\rho}(\mathbf{r})] = F_{HK}[\tilde{\rho}(\mathbf{r})] + \int \hat{v}(\mathbf{r})\tilde{\rho}(\mathbf{r})d\mathbf{r}$$ \hspace{1cm} (1.33)
Accordingly, HK showed that if the trial density is not the ground state density, then
$$E_{e,0}[\tilde{\rho}(\mathbf{r})] \geq E_{e,0}[\rho(\mathbf{r})]$$ \hspace{1cm} (1.34)
The equality holds only if the trial density is the ground state density. The second theorem establishes that it is necessary to search through the valid trial densities $\tilde{\rho}(\mathbf{r})$ in order to find the one that returns the lowest value of the total energy. The $v$-representability of the density is the property that allows the selection of a valid $\tilde{\rho}(\mathbf{r})$. It is however a very strict requirement. If the densities could satisfy the $N$-representability condition, instead, the selection of a trial density would be more convenient. A density is defined as $N$-representable if it is positive, continuous and if it satisfies the condition $\int \rho(\mathbf{r})d\mathbf{r} = N$. In 1979, Mel Levy extended the HK theorems to $N$-representable densities, thus eliminating the requirement for $v$-representability.\textsuperscript{14,15}
The formalism used by Levy is a two-step constrained search that makes use of the one-to-one correspondence between the ground state electron density and the ground state wave function as established by HK-I. Rather than searching through the whole Hilbert space as in the original formulation of HK-I, the HK functionals $F_{HK}$ is minimized under the constraint that only the ground state wave functions that yield the reference density $\rho(\mathbf{r})$ are taken into account. In mathematical form, this can be written as
$$F_{HK}[\rho(\mathbf{r})] = \min_{\Psi \rightarrow \rho(\mathbf{r})} \left\{ \langle \Psi | \hat{T} + \hat{V}_{ee} | \Psi \rangle \right\}. $$ \hspace{1cm} (1.35)
When calculating the ground state energy, $E_0$, the search is done among all the (valid) input densities, a condition that could be written as $E_0 = \min_{\rho(\mathbf{r})} E[(\rho(\mathbf{r})]$. The two-step procedure can then be summarized as
$$E_0 = \min_{\rho(\mathbf{r})} \left\{ \min_{\Psi \rightarrow \rho(\mathbf{r})} \left[ F_{HK}[\rho(\mathbf{r})] + \int \hat{v}(\mathbf{r})\rho(\mathbf{r})d\mathbf{r} \right] \right\}$$ \hspace{1cm} (1.36)
The equation enclosed in square brackets corresponds to the definition of the functionals written in equation 1.35. When looking for a minimum of the energy functional, we first search through all wave functions that yield the correct density $\rho(\mathbf{r})$, and then we lift this constraint by looking at all the densities until we find the ground state density $\rho_0(\mathbf{r})$.
The Hohenberg–Kohn theorems and their extension provided by Levy are certainly the cornerstones of the theoretical framework upon which modern DFT is built. However, HK did not give any prescriptions on how to actually use the density as central quantity. In 1965, only a year after the publication of the HK theorems, Kohn and Lu S. Sham introduced the practical framework that would later be known as Kohn–Sham DFT (KS-DFT).\textsuperscript{16}
In order to overcome the major limitation of the TFD theory, Kohn and Sham realized that a better approximation to the kinetic energy was needed. Instead of using an integral form like equations 1.25 or 1.26, they calculate the kinetic energy using a Slater determinant of spin orbitals, as in
\begin{equation}
\hat{T}_s = -\frac{1}{2} \sum_i^N \langle \phi_i(\mathbf{x}_i) | \nabla_i^2 | \phi_i(\mathbf{x}_i) \rangle .
\end{equation}
(1.37)
The previous equation holds true for a system of $N$ non-interacting electrons. A more general description of the kinetic energy, for example for an interacting system, would involve an infinite amount of terms ($N = \infty$), which is impossible to calculate in practice. Given that electron-electron interactions are ignored when the kinetic energy is calculated through equation 1.37, KS called this term $T_s$ to distinguish it from the exact (electronic) kinetic energy $T_e$. The shift from an interacting to a non-interacting reference system is the key in the success of KS-DFT. The wave function approximated as a single Slater determinant describes exactly a (fictitious) system of $N$ non-interacting electrons, whose density can be defined as $\rho(\mathbf{r}) = \sum_i^N |\phi(\mathbf{x}_i)|^2$, which corresponds to the major part of the kinetic energy of the electrons in a real system. In fact, the error made by approximating the kinetic energy using spin orbitals is smaller than the one resulting from the TFD approach.
The HK functional $F_{HK}$ in equation 1.32 can be factorialized as
\begin{equation}
F_{HK} = T_e[\rho(\mathbf{r})] + V_{ee}[\rho(\mathbf{r})] = T_s[\rho(\mathbf{r})] + J[\rho(\mathbf{r})] + E_{xc}[\rho(\mathbf{r})]
\end{equation}
(1.38)
The last term in equation 1.38 is called the *exchange-correlation* (*xc*) energy, $E_{xc}[\rho(\mathbf{r})]$. It represents the central quantity in KS-DFT, and it includes contributions from the kinetic energy as well as from the nonclassical interactions between the electrons of the system. Its mathematical form we can write
$$E_{xc}[\rho(\mathbf{r})] = T_e[\rho(\mathbf{r})] - T_s[\rho(\mathbf{r})] + V_{ee}[\rho(\mathbf{r})] - J[\rho(\mathbf{r})] \tag{1.39}$$
By using the *xc* energy, the KS ansatz yields the same results as conventional DFT when applied to a system of $N$ non-interacting particles moving in the field represented by $v_{eff}(\mathbf{r}) = v(\mathbf{r}) + \frac{\delta J[\rho(\mathbf{r})]}{\delta \rho(\mathbf{r})} + \frac{\delta E_{xc}[\rho(\mathbf{r})]}{\delta \rho(\mathbf{r})}$. In other words, KS-DFT is an *exact* theory because the *xc* energy term includes contributions to the exact kinetic energy and to the exchange and correlation effects that are found in the *real* system.
The framework introduced by KS can be used to solve many problems in electronic structure theory, provided that the functional needed to calculate the *xc* energy is known. Unfortunately, no information is given to design the *exact* functional, which is essentially unknowable,\textsuperscript{17,18} and only some of its mathematical properties have been discovered.\textsuperscript{19} It is with the introduction of many *approximations* to the exact functional that KS-DFT has become the workhorse of electronic structure theory. After the development of the first density functional approximations (from now on, simply functionals or density functionals) in the 1980s,\textsuperscript{20–23} the popularity of DFT rose after the release of Gaussian92/DFT,\textsuperscript{24} the first version of the popular quantum chemistry package that included some of the early functionals such as BP86\textsuperscript{21,22} and the popular B3LYP.\textsuperscript{22,23,25,26} [For a historical perspective, see ref.]\textsuperscript{27} We will look at how the different approximations to the exact functionals have been introduced in the next section.
### 1.4 The approximations to the *xc* functional: nature and classification.
In the previous section, we reviewed the KS-DFT framework, one of the many practical implementations of DFT. From now on and for the rest of this work, we will use the acronym DFT to refer to KS-DFT, thus conforming to the widespread habit in chemistry, physics and material science. Since its inception, density functional development could be divided into two major philosophies, one called non-empirical, and the other called semi-empirical. John P. Perdew and Axel D. Becke started perfecting each approach since the early 1980s, and almost all the approximations in use today can be ascribed to either philosophy. Many other scientists—like
Stefan Grimme, Nicholas C. Handy, Martin Head-Gordon, Jan M. L. Martin, Robert G. Parr, Donald G. Truhlar, Weitao Yang and their collaborators, just to name a few—contributed to the growth of the field. To date, more than 300 approximations to the exact $xc$ functional have been proposed, and this number is destined to increase even more in the next decades.\textsuperscript{28}
The $xc$ energy is usually given as the sum of two independent contributions, the exchange energy $E_x$ calculated through an exchange functional, and the correlation energy $E_c$ calculated using a correlation functional. They can be functionals of the total electron density $\rho(\mathbf{r})$, or of the two spin densities $\rho_\alpha(\mathbf{r})$ and $\rho_\beta(\mathbf{r})$ separately. We should keep in mind that $\rho(\mathbf{r}) = \rho_\alpha(\mathbf{r}) + \rho_\beta(\mathbf{r})$. From now on, we shall use the notation $\rho_\sigma = \rho_\sigma(\mathbf{r})$ (with $\sigma = \alpha$ or $\beta$), thus dropping the dependence of the density to the coordinates $\mathbf{r}$ for better readability. Regardless of the approach used to introduce a new approximation, all exchange functionals can be expressed in a form like equation 1.40, while all correlation functionals can be defined according to equation 1.41.
\begin{equation}
E_x \left[ \rho_\alpha, \rho_\beta, |\nabla \rho_\alpha|^2, |\nabla \rho_\beta|^2, \tau_\alpha, \tau_\beta \right] = \sum_{\sigma}^{\alpha,\beta} \int \epsilon_{x,\sigma}(\rho_\sigma, |\nabla \rho_\sigma|^2, \tau_\sigma) d\mathbf{r}
\end{equation}
\begin{equation}
E_c \left[ \rho_\alpha, \rho_\beta, |\nabla \rho_\alpha|^2, |\nabla \rho_\beta|^2, \nabla \rho_\alpha \cdot \nabla \rho_\beta, \tau_\alpha, \tau_\beta \right] =
\end{equation}
\begin{equation}
\sum_{\sigma}^{\alpha,\beta} \int \epsilon_{c,\sigma}(\rho_\alpha, \rho_\beta, |\nabla \rho_\alpha|^2, |\nabla \rho_\beta|^2, \nabla \rho_\alpha \cdot \nabla \rho_\beta, \tau_\alpha, \tau_\beta) d\mathbf{r}
\end{equation}
$\epsilon_{x,\sigma}$ and $\epsilon_{c,\sigma}$ correspond to the exchange and correlation energy densities, respectively. Equations 1.40 and 1.41 list some of the quantities, or “ingredients,” used in an $xc$ functional definition. A density functional might depend on the density, $\rho_\sigma$, on the first derivative of the density (its gradient), $\nabla \rho_\sigma$, or on the kinetic energy density $\tau_\sigma = \sum_{i=1}^{N} |\nabla \phi_{i,\sigma}|^2$ (as an alternative, it could depend on the Laplacian of the density, $\nabla^2 \rho_\sigma$). Perdew and Schmidt introduced a convenient tool, the Jacob’s ladder of density functional approximations (DFAs),\textsuperscript{29} to classify the different approximations. They identified five classes, corresponding to five rungs on the ladder, depending on the ingredients used in an approximation. The rationale behind the ladder’s hierarchy relies on a simple axiom: the more ingredients used in a functional definition, the more accurate the functional. However, the higher accuracy comes at the cost of a higher computational cost as well.
Using only the density as building block yields functionals belonging to the local spin-density
approximation (LSDA) class (rung one). The TFD functional\textsuperscript{4,9,10} or the Slater functional (S) are LSDA functionals for exchange, while the Vosko-Wilk-Nusair (VWN),\textsuperscript{20} or the Perdew-Wang 1992 (PW92)\textsuperscript{30,31} are LSDA correlation functionals. While LSDA improves dramatically over the HF method, it still results in major overbinding. In order to improve upon LSDA, the gradient $\nabla \rho_\sigma$ of the density has to be included to account for inhomogeneities in the density. This results in a new class, called generalized gradient approximation (GGA), belonging to the second rung. The most popular GGA functionals for exchange include B88 (B),\textsuperscript{22} PBE,\textsuperscript{32} HCTH,\textsuperscript{33} while the most popular GGA correlation functionals include LYP,\textsuperscript{23} P86,\textsuperscript{21} PBE,\textsuperscript{32} and PW91.\textsuperscript{30} Usually, exchange and correlation functionals are indicated through acronyms including the name of the developer and the year of introduction. For example, the B88 functional for exchange is called after Becke and it was published in 1988. In a similar way, an $xc$ functional is named by combining an exchange and a correlation functional. The BLYP functional uses B88 exchange (also called B) and LYP correlation. An exception to this rule comes with the PBE functional. While it is referred to as PBE, in some quantum chemistry packages it is called PBEPBE.
With the addition of the kinetic energy density, or the Laplacian of the density, we reach the meta-GGA class (rung three). Functionals developed by Perdew and coworkers such as PKZB,\textsuperscript{34} TPSS,\textsuperscript{35} SCAN,\textsuperscript{36} and r$^2$SCAN\textsuperscript{37} belong to this rung, together with the Minnesota functionals M06-L,\textsuperscript{38} M11-L,\textsuperscript{39} MN12-L,\textsuperscript{40} MN15-L,\textsuperscript{41} and revM06-L\textsuperscript{42} of Truhlar, Zhao, Peverati, Yu and coworkers. Other popular functionals are the VSXC functional\textsuperscript{43} of Van Voorhis and Scuseria, the $\tau$-HCTH of Handy and coworkers,\textsuperscript{44} and the recent B97M-V\textsuperscript{45,46} and B97M-rV\textsuperscript{47,48} of Mardirossian and Head-Gordon. The functionals belongings to rungs 1–3 are also known as local functionals, since they depend on local quantities (the density at a given point, or in its closest proximity through either the gradient or the Laplacian/$\tau$).
In his continuous quest to improve the performance of the different approximations, Becke introduced in 1993 the first “hybrid” functional, \textit{i.e.} an approximation in which a certain percentage of local (DFT) exchange is replaced by HF exchange (called “exact exchange” in the original publications).\textsuperscript{25,49} Using the adiabatic connection formula,\textsuperscript{50–53} Becke reasoned that a certain percentage of HF exchange is needed to improve the performance of local functionals, since the exact functional is by definition nonlocal. This led him to the introduction of a three-parameter scheme using the B88 GGA functional and the PW91 correlation functional as in equation 1.42 below:
\[ E_{xc}^{\text{B3PW91}} = a_0 E_x^{\text{HF}} + (1 - a_0) E_x^{\text{LSDA}} + a_x E_x^{\text{B88}} + E_c^{\text{LSDA}} + a_c E_c^{\text{PW91}} \]
(1.42)
The empirical parameters \(a_0\), \(a_x\), and \(a_c\) have been determined through a fit to a series of atomization energies, ionization potentials and proton affinities, which yield a value of 0.20, 0.72, and 0.81 respectively.\(^{25}\)
A few years later, Becke developed a simpler, one-parameter functional scheme,\(^{54}\) here exemplified using the B88 exchange functional and the LYP correlation functional, which takes the form
\[ E_{xc}^{\text{B1LYP}} = a_0 E_x^{\text{HF}} + (1 - a_0) E_x^{\text{B88}} + E_c^{\text{LYP}} \]
(1.43)
The popular B3LYP approximation is a hybrid functional obtained by replacing the PW91 correlation functional with the LYP functional following equation 1.42, while the PBE0 functional\(^{55,56}\) is instead obtained using the PBE functional for exchange and correlation according to equation 1.43 (with \(a_x = 0.25\)).
If a meta-GGA functional is used for the local part in either equation 1.42 or 1.43, a hybrid meta-GGA is obtained. Popular hybrid meta-GGAs are TPSSh,\(^{57}\) M06,\(^{58}\) M06-2X,\(^{58}\) M06-HF,\(^{59}\) and MN15.\(^{60}\) Another “trick of the trade” used for the definition of hybrid (GGA or meta-GGA) functionals consists in changing the percentage of HF exchange. All the hybrid functionals named so far are called “global” hybrid, since the HF percentage used in the functional definition is kept constant. For range-separated hybrids (RSH), instead, the Coulomb operator \(\hat{J}\) can be split into a short-range (SR) and a long-range (LR) part, usually through an equation as
\[ \frac{1}{r_{12}} = \frac{\text{erf}c(\omega r_{12})}{r_{12}^{\text{SR}}} + \frac{\text{erf}f(\omega r_{12})}{r_{12}^{\text{LR}}} \]
(1.44)
where \(r_{12}\) is the interelectronic distance, and \(\text{erf}(c)\) is used to indicate an error function (or the complementary error function, hence the c) that “switches” between the short and long range based on the parameter \(\omega\). RSH-GGA functionals include CAM-B3LYP,\(^{61}\) \(\omega\)B97X-D,\(^{62}\) and \(\omega\)B97X-V.\(^{46,63}\) RSH-meta-GGA functionals include M11,\(^{64}\) revM11,\(^{65}\) and \(\omega\)B97M-V.\(^{46,66}\)
The fifth and last rung of Jacob’s ladder of DFAs hosts double-hybrid\(^{67}\) (or doubly hybrid)\(^{68}\) \(xc\) functionals. In addition to replacing a certain percentage of local exchange with HF exchange,
double-hybrid (DH) functionals have a certain amount of correlation calculated in a manner similar to second-order Møller-Plesset (MP) perturbation theory (PT). In mathematical form, a DH functional expression becomes
\[
E_{xc}^{\text{DH}} = a_x E_x^{\text{HF}} + (1 - a_x) E_x^{\text{DFT}} + a_c E_c^{\text{PT2}} + (1 - a_c) E_c^{\text{DFT}}
\]
(1.45)
B2PLYP was the first double-hybrid functional to be proposed,\textsuperscript{67} and it comes from using the B88 exchange and the LYP correlation functionals in equation 1.45. Other popular approximations are DSD-PBEP86-D3(BJ),\textsuperscript{69} PWPB95,\textsuperscript{70} and ωB97M(2).\textsuperscript{71}
### 1.5 Practical consideration and final remarks.
When performing DFT calculations, there are a few methodological and technical issues to take into account.\textsuperscript{28} First, before setting up a computational experiment, a functional and an atomic orbital basis set should be chosen. Choosing a functional is not an easy task, and part of this work is devoted to the use of databases as tools to help in this selection. At the same time, we will also explore how databases have been used to develop new functional approximations. As we delve deeper into these issues, we should keep in mind that the best results are usually obtained with the most modern functionals.
The choice of a basis set is somehow slightly easier to approach than the choice of a functional. For basis sets, in fact, the simple axiom “the larger, the better” solves every issue.\textsuperscript{28} However, the size of the system under investigation and the computational capabilities available influence this choice, usually towards smaller basis sets. While WFT approaches are guaranteed to converge to the so-called basis set limit, the same is not true for DFT. The basis set requirements can be dependent on the functionals, on the tasks that need to be performed, or on both. We should always be aware that a fortuitous error cancellation can always happen,\textsuperscript{28,72} which might lead to finding the wrong answer for the wrong reason. The most subtle technical issues behind a DFT calculation is the choice of an integration grid. The evaluation of the \(xc\) energy is performed as an integral over all space of the \(xc\) energy density (equations 1.40 and 1.41). In practice, this is achieved by calculating these quantities on a three-dimensional, real space grid, which results from the partition of the integral into weighted atomic contributions.\textsuperscript{73} Not all functionals have the same grid requirements, and it has been shown that meta-GGAs are more sensitive to the choice of integration grids than GGAs.\textsuperscript{74–78} Larger grids usually yield better results, but as for
basis sets, they require longer computational times. We will cover all these issues at the end of Chapter 2.
1.6 Outline.
The major goal of this dissertation is to elucidate the modern usage of computational chemistry databases as useful tools for the parameterization and evaluation of existing and new exchange-correlation functional approximations. In the second chapter, a large collection of computational databases named ACCDB is presented, and its components are thoroughly discussed. At the end of the chapter, computational databases will be used in the context of chemical education for the first time. The third chapter deals with the statistical reduction of the ACCDB collection introduced in the second chapter of this dissertation. The procedures used in this process will be described in great detail, and all the data gathered in support of the soundness of the statistical approach are presented and discussed as well. In the fourth chapter, a new database of experimental geometries called CLB18 is introduced. Its usage to assess the performance of low-cost and semi-empirical methods, as well as a few functionals with a small basis set is reported. In the last chapter, the mechanism of uncaging of 4-methoxy-5,7-dinitroindolinyl glutamate (MDNI-Glu) is unraveled using state-of-the-art density functional theory calculations. The origin of the better quantum yield of the dinitro species is justified using time-dependent DFT calculations.
Chapter 2.
The importance of databases of reliable and accurate data in chemistry has substantially increased in the past two decades. Their main usage is to parametrize electronic structure theory methods, and to assess their capabilities and accuracy for a broad set of chemical problems. The collection we present here—ACCDB—includes data from 16 different research groups, for a total of 44,931 unique reference data points, all at a level of theory significantly higher than density functional theory, and covering most of the periodic table. It is composed of five databases taken from literature (GMTKN, MGCDB84, Minnesota2015, DP284, and W4-17), two newly developed reaction energy databases (W4-17-RE and MN-RE), and a new collection of databases containing transition metals. A set of expandable software tools for its manipulation is also presented here for the first time. As a case study, we selected a few reactions from the main databases of the ACCDB collection (GMTKN55, MGCDB84, and Minnesota 2015B) to introduce a series of computational experiments in the form of a laboratory notebook. We tackle many different issues, starting with
the selection of an appropriate approximation to the exact exchange-correlation functional for a specific (chemical) application. We also cover the choices of a basis set and of an integration grid, that are often overlooked by occasional practitioners and by more experienced users as well. This notebook contains 12 experiences that can be run on a simple computer in just a few hours. We propose this review as a primary source for those who are willing to include DFT in their everyday research or teaching activities in a way that reflects the research advances of the field in the last couple of decades. The first part of this work has been published in the *Journal of Computational Chemistry*, while the laboratory notebook is published as a tutorial review in the *International Journal of Quantum Chemistry*.
**Chapter 3.**
The amount of data and number of databases for the assessment and parameterization of density functional theory methods has grown substantially in the past two decades. In this work, we introduce a novel cluster analysis technique for density functional theory calculations of the electronic structure of atoms and molecules with the goal of creating new statistically significant databases with broad chemical scope, and a manageable number of data-points. By analyzing without a priori chemical assumptions a population of almost 350k data-points, we create a new database called ASCDB containing only 200 data-points. This new database holds the same chemical information as the larger population of data from which it is obtained, but with a computational cost that is reduced by several orders of magnitude. The labelling of the significant chemical properties is performed a posteriori on the resulting 16 subsets, classifying them into four areas of chemical importance: non-covalent interactions, thermochemistry, non-local effects, and unbiased calculations. The analysis of the results and their transferability shows that ASCDB is capable of providing the same information as that of the larger collection of data—such as GMTKN55, MGCDB84, and Minnesota 2015B—for several density functional theory methods and basis sets. In light of these results, we suggest the use of this new small database as a first inexpensive tool for the evaluation and parameterization of electronic structure theory methods. This work has been published in *Physical Chemistry Chemical Physics*.
**Chapter 4.**
A new database called Carbon Long Bond 18 (CLB18), composed of 18 structures with one long C–C bond, is introduced. We use this new database to evaluate the performance of several low-cost methods commonly used for geometry optimization of medium and large molecules. We
found that the long bonds in CLB18 are electronically different from those found in barrier heights databases. We also report the unexpected correlation between the results of CLB18 and those of the energetics of spin states in transition-metal complexes. Given this unique property, CLB18 can be a useful tool for assessing existing electronic structure calculation methods and developing new ones. The subject of this chapter has been recently published in *Chemical Physics Letters*.\(^{81}\)
**Chapter 5.**
The last chapter of this dissertation is a case study. The 7-nitroindolinyl family of caging chromophores has received much attention in the past two decades. However, its uncaging mechanism is still not clearly understood. In this study, we performed state-of-the-art density functional theory calculations to unravel the photo-uncaging mechanism in its entirety, and we compared the probabilities of all plausible pathways. We found competition between a classical cyclization and an acyl migration pathway, and here we explain the electronic and steric reasons behind such competition. The migration mechanism possesses the characteristics of a combined Norrish type I and a 1,6-nitro-acyl variation of a Norrish type II mechanism, which is reported here for the first time. We also found negligible energetic differences in the uncaging mechanisms of the 4-methoxy-5,7-dinitroindolinyl (MDNI) cages and their mononitro analogues (MNI). We traced the experimentally observed improved quantum yields of MDNI to a higher population of the reactants in the triplet surface. This fact is supported by a more favorable intersystem crossing due to the availability of a higher number of triplet excited states with the correct symmetry in MDNI than in MNI. Our findings may pave the way for improved cage designs that possess higher quantum yields and a more efficient agonist release. This work has been recently published in *Scientific Reports*.\(^{82}\)
Chapter 2: The development of A Collection of Chemistry DataBases (ACCDB).
2.1 Introduction.
In Chapter 1, we briefly examined a few of the most popular $xc$ functional approximations from the perspective of the ingredients used in the functional form. We also mentioned the two philosophies of functional development, either nonempirical or semi-empirical. Independently of how a functional is developed, or what ingredients are included in its definition, it is necessary to test it against well-known reference data to understand its strengths and limitations. This is possible thanks to the many databases of chemical data that have been published in the last fifty years. Databases are usually made of reference geometries and the corresponding reference (reaction) energies or molecular properties (dipole moments, polarizabilities, NMR chemical shifts...). Our focus for this and the next chapters will be on energetic databases, while we will introduce a new database for molecular geometries in Chapter 4.
Since the mid-1970s, Dewar and coworkers used databases of chemical data to parametrize semi-empirical methods.\textsuperscript{83–86} They used experimental geometries and heat of formations of small organic molecules as the training set (a small subset of a database used to determine the parameters of the method) for the MINDO/3,\textsuperscript{83} MDNO\textsuperscript{84,85} and AM1\textsuperscript{86} semi-empirical methods. For a historical perspective, see ref.\textsuperscript{87} Another early contribution to the development of databases of chemical data came from Pople and coworkers. While working on the introduction of the composite methods that would later be called G1\textsuperscript{88,89} and G2 theories,\textsuperscript{90} they introduced a collection of heat of formations, ionization potentials and atomization energies.\textsuperscript{88,91} This collection became known as the G2 test set of chemical data.\textsuperscript{90} It contains 125 reference energies from accurate experimental data for small molecules ranging from ethane to water, including neutral species and radicals. Additional energies were added a few years later, bringing the total to 298 for the G2/97 dataset.\textsuperscript{92,93} The original G2 dataset has been introduced to assess the performance of the G1 and G2 theories, but it became a popular tool for the parametrization and the evaluation of $xc$ functionals as well. In fact, Becke proposed the B3PW91 approximation after determining
its parameters through a least-square fit against the energies contained in it.\textsuperscript{25} In the following years, many functionals were parametrized against this subset. Notable examples are Becke’s B97 functional (the first to be systematically parametrized against a database),\textsuperscript{94} the HCTH,\textsuperscript{33} $\tau$-HCTH\textsuperscript{44} and B97-1\textsuperscript{33} functionals of Handy and coworkers, and the VSXC functional of Van Voorhis and Scuseria.\textsuperscript{43}
As the computational cost of high-level \textit{ab initio} methodologies decreased due to more efficient implementations and the progress in computer hardware, databases of \textit{calculated} energies became the focus for both the development and the assessment of density functional approximations. Using databases of theoretical data has many advantages over experimental ones, and the most important is a better comparability of the results. Once calculations are performed, there is no need to correct for experimental conditions through zero-point energies, thermal corrections to enthalpies or heats of formation, spin-orbit coupling contributions, or anharmonicity corrections for frequencies. When only electronic energies are involved, there is a practical advantage as well. The computational effort required to calculate electronic energies is smaller than that required to compute any of the previous properties. In addition, experimental errors are removed, and while theoretical values still have an associated error, mainly due to the level of theory (\textit{i.e.} method and basis set) employed, it is usually easier to assess and control it. Truhlar and coworkers were the first to use databases of high-level theoretical energies, which were not limited to thermochemical data only. For example, the database used to develop the popular M06-2X functional\textsuperscript{58} included thermochemical data (atomization energies, ionization potentials), as well as non-covalent interactions, barrier heights and charge transfer subsets. The following three generations of Minnesota functionals have all been parametrized against an expanded and updated version of this original database.
Given the increased popularity and importance of theoretical databases of accurate, high-level computational data, we decided to group them in the largest collection presented so far. We named this collection ACCDB, “A Collection of Chemistry DataBases.” While other large databases of computational data have been presented in recent years,\textsuperscript{95–100} they all involve data calculated at either the DFT level or lower. All the data in ACCDB instead comes from higher levels of theory, such as coupled cluster with single, double and perturbative triple excitations (CCSD(T)), or complete active space self-consistent field/complete active space second order perturbation theory (CASSCF/CASPT2). We will analyze the subsets of ACCDB in more details in the following section.
2.2 Description of the subsets included in ACCDB.
Since the development of the G2 test set, many additional databases have been developed and published as independent datasets in the literature. Before the publication of ACCDB, the three largest collections of chemical data were the Minnesota 2015B database of Truhlar and coworkers,\textsuperscript{60} a collection of General Main-group Thermochemistry, Kinetics and Non-covalent interactions database (GMTKN55) by Grimme, Goerigk and coworkers,\textsuperscript{101} and the Main Group Chemistry DataBase (MGCDB84) by Mardirossian and Head-Gordon.\textsuperscript{102} In turn, these databases collected more than 120 datasets from the literature. ACCDB incorporates them all, together with many new subsets including mostly transition metals (TMs). Throughout the rest of this discussion, we will use the term dataset to refer to an independent group of structures (and the associated reference energies). When the dataset is collected within a larger database, we will use the term subset (of the larger database). In total, ACCDB includes 191 datasets from the literature, 44,931 reference energies, and 10,049 reference structures. A visual description of ACCDB is shown in Table 2.1 below.
Table 2.1: Summary of the databases included in ACCDB, listed by decreasing size. RE stands for “reaction energies.”
| Database | Brief description | # of structures | # of datapoints | References |
|---------------------------|-----------------------------------------------------------------------------------|-----------------|-----------------|------------|
| MGCDB84 | Main-group chemistry DataBase with 84 subsets | 5,931 | 4,985 | 102 |
| GMTKN | General, main-group, thermochemistry, kinetics and non-covalent interactions database with 55 subsets and Mindless benchmarks. | 2,639 | 1,664 | 101,103 |
| Minnesota 2015 | Thermochemistry, kinetics, non-covalent interactions (Three versions: Minnesota 2015, Minnesota 2015A, Minnesota 2015B) | 719 | 471 | 41,60,104 |
| Metals&EE | Collection of subsets containing metals and excitation energies | 364 | 210 | 79 |
| W4-17 | Total atomization energies | 215 | 1,042 | 105 |
| Database | Brief description | # of structures | # of datapoints | References |
|--------------|--------------------------------------------------------|-----------------|-----------------|------------|
| DP284 | Dipole moments and polarizabilities | 181 | 284 | 106,107 |
| W4-17-RE | Automatically generated reaction energies from the W4-17 database | – | (27,140) | 79 |
| MN-RE | Automatically generated reaction energies from the Minnesota 2015B database | – | (9,135) | 79 |
| ACCDB | A Collection of Chemistry DataBases | 10,049 | 8,656 (44,931)\(^a\) | 79 |
\(^a\)The number in parenthesis includes the “non-traditional” RE subsets.
**Table 2.2** includes the 163 unique datasets from the literature, and the corresponding references, collected in ACCDB (excluding some redundancies, *vide infra*). They include up to 63 different elements from the \(s\), \(p\), \(d\), and \(f\) blocks of the periodic table, as shown in **Figure 2.1**.
**Table 2.2:** Unique subsets in the ACCDB collection of chemical databases, listed in alphabetical order.
| Dataset | Reference | Dataset | Reference | Dataset | Reference |
|---------------|-----------|-----------|-----------|-----------|-----------|
| 2pISOE4 | 108 | Dip152 | 107 | NGDWI21 | 18,109 |
| 3B-69-TRIM | 110 | DIPCS10 | 101 | p-VR17 | 111 |
| 3B-69-DIM | | DS14 | 113 | PA26 | 101,114,115 |
| 3d-SSIP30 | 112 | EA13 | 18,118–121| PA8 | 18,115 |
| 3dAEE8 | 112,116,117| EIE22 | 123 | PARel | 101 |
| 3dMRBE6 | 122 | FeCl | 124 | PCONF21 | 125,126 |
| 3dSRBE2 | 122 | FH51 | 127,128 | PdBE2 | 129 |
| 3dSRBE4 | 122 | FmH2O10 | 131,132 | pEE5 | 111 |
| 4d-SSIP24 | 130 | G21EA | 90,133 | Pentane14 | 134 |
| 4dAEE5 | 130 | G21IP | 90,133 | PlatonicHD6 | 135 |
| 4pISOE4 | 108 | G2RC | 92,101 | PlatonicID6 | 135 |
| A21x12 | 136 | H2O16Rel5 | 138 | PlatonicIG6 | 135 |
| A24 | 137 | | | | |
| Dataset | Reference | Dataset | Reference | Dataset | Reference |
|--------------|-----------|---------------|-----------|---------------|-----------|
| ABDE13 | 139 | H2O20Bind10 | 132 | PlatonicTAE6 | 135 |
| ACONF | 133,140 | H2O20Bind4 | 133,141–143 | PNICO23 | 101,144 |
| ADIM6 | 101,145 | H2O20Rel10 | 132 | Pol132 | 106 |
| AE18 | 18,58,146 | H2O20Rel4 | 133,141–143 | Por21 | 79,147,148|
| AHB21 | 149 | H2O6Bind8 | 131,132 | πTC13 | 18,38,115,119 |
| AIP28 | 150 | HAL59 | 151,152 | PX13 | 153,154 |
| AL2x6 | 101 | HAT707 | 155 | RC21 | 101 |
| ALK8 | 101 | HB15 | 156 | RG10 | 109 |
| AlkAtom19 | 157 | HB49 | 158–160 | RG18 | 101 |
| ALKBDE10 | 161 | HC7/11 | 18,162 | RSE43 | 101,163 |
| AlkBInd12 | 164 | HEAVY28 | 101,145 | S22 | 165,166 |
| AlkIsod14 | 157 | HEAVYSB11 | 101 | S66 | 167,168 |
| AlkIsomer11 | 157 | HNBrBDE18 | 169 | S66x8 | 167 |
| AMINO20x4 | 170 | HSG | 166,171 | S6x6 | 167 |
| Bauza30 | 172,173 | HW30 | 174 | SCONF | 133,175 |
| BDE99 | 155 | HW6Cl | 131,132 | Shields38 | 176 |
| BH76 | 101,177,178 | HW6F | 131,132 | SIE4x4 | 101 |
| BH76RC | 101,133,177,178 | ICONF | 101 | SMAE3 | 41,60,179 |
| BHDIV10 | 101 | IDISP | 133,180–183 | SN13 | 155 |
| BHPERI | 101,133,184–187 | IL16 | 149 | SR-MGN-BE107 | 18,64,121 |
| BHROT27 | 101 | INV24 | 188 | SRM2 | 18 |
| BSR36 | 101,189,190 | Ionic43 | 149 | SRMBE10 | 18 |
| BUT14DIOL | 191 | IP23 | 18,40,118–121 | SRMGD5 | 18,192,193 |
| BzDC215 | 194 | ISO34 | 181 | Styrene45 | 195 |
| C20C24 | 196 | ISOL24 | 183,197 | SW49Bind345 | 198 |
| C60ISO | 199 | ISOL6/11 | 18,200 | SW49Bind6 | 198 |
| CARBHB12 | 101 | ISOMERIZATION20 | 155 | SW49Rel345 | 198 |
| CDIE20 | 201 | LTMBH26 | 202 | SW49Rel6 | 198 |
| CE20 | 153,154 | MB08-165 | 103 | TA13 | 203 |
| CHB6 | 149 | MB16-43 | 101,103 | TAUT15 | 101 |
| Dataset | Reference | Dataset | Reference | Dataset | Reference |
|------------------|-----------|------------------|-----------|------------------|-----------|
| CO2Nitrogen16 | 204 | MCONF | 205 | TMBH23 | 206–208 |
| CR20 | 209 | Melatonin52 | 205 | UPU23 | 210 |
| CRBH20 | 193 | MN-RE | 79 | W4-11 | 155 |
| CT20 | 211 | MOR41 | 212 | W4-17 | 105 |
| CT7 | 18 | MR-MGM-BE4 | 161 | W4-17-RE | 79 |
| CuH, VO, CuCl, NiCl | 124 | MR-MGN-BE17 | 18 | WATER27 | 133,142,213 |
| CYCONF | 133,214 | MR-TMD-BE3 | 18,116 | WCPT27 | 215 |
| DARC | 101,133,216 | MRBE3 | 18 | X40 | 152 |
| DBH24 | 217,218 | NBC10 | 166,219–221 | XB18 | 151 |
| DC13 | 58,67,101,133,195,196,222–228 | NBPRC | 133,183,229 | XB51 | 151 |
| DC9/12 | 18,230 | NC15 | 231 | YBDE10 | 101,232 |
| DIE60 | 201 | NCCE23 | 18,166,233–235 | YMPJ519 | 170 |
Figure 2.1: The 63 elements of the periodic table included in ACCDB (in green).
We will now focus our attention on the description of each database included in ACCDB, with particular emphasis on their size, the number and kind of chemical properties included, and on their use as benchmarking tools for density functional approximations.
MGCDB84.
The Main Group Chemistry DataBase has been introduced by Mardirossian and Head-Gordon for the development of the (ω)B97M-V family of functionals.\textsuperscript{45,63,66} It includes 84 subsets, 4,985 reference reaction energies, and a total of 5,931 single-point calculations are needed for this database. In its entirety, it has been used for the evaluation of the Minnesota functionals,\textsuperscript{76,77} as well as for 200 additional $xc$ functional approximations.\textsuperscript{102} It is the largest database in ACCDB, and while its main focus are non-covalent interactions (2,647 datapoints out of 4,985, or 53.1%), it includes 1,205 datapoints (24.2%) belonging to thermochemistry chemical properties, 910 datapoints (18.3%) for isomerization energies, and 206 datapoints (4.1%) for barrier heights. It also includes the electronic energies of the first 18 atoms, H through Ar. Given its large size, a smaller version has been introduced in 2018 using statistical analysis.\textsuperscript{236} Chan called this new database MG8, and it includes 64 datapoints and a total of 166 geometries. Through a regression formula, it is possible to recover the performance obtained against the MGCDB84 database. As its larger parent, MG8 focuses mostly on non-covalent interactions (44 datapoints, 68.8%), followed by thermochemistry (nine datapoints, 14.0%), barrier heights (seven datapoints, 10.9%), and isomerization energies (four datapoints, 6.3%).
GMTKN.
This database is composed of the general, main group, thermochemistry, kinetics, non-covalent interaction database (GMTKN55)\textsuperscript{101} and the “mindless benchmark” MB08-165 database.\textsuperscript{103} GMTKN55 is the second largest database in ACCDB, with 55 subsets, 1,499 relative energies, and 2,459 geometries. It is the third generation of the database developed by the Goerigk and Grimme groups after the introduction of GMTKN24\textsuperscript{133} and GMTKN30.\textsuperscript{183} It includes many different chemical properties grouped in five different areas: non-covalent interactions, with a total of 595 datapoints—corresponding to 39.7% of the database—almost equally divided between intra- and intermolecular (304 and 291 datapoints respectively); basic properties including atomization energies, electron and proton affinities, dissociation energies of various compounds for a total of 467 datapoints (31.2%); thermochemistry, including reaction energies and isomerization reactions, with a total of 243 data (16.2%); kinetics, with barrier heights subsets having 194 datapoints (12.9% of the database). In addition to GMTKN55, ACCDB includes the MB08-165 database, which contains 165 datapoints of randomly generated artificial molecules having eight atoms. The procedure that Korth and Grimme named “mindless benchmark” aims at introducing new
molecules that do not span the same chemical space as the other subsets in other databases. Since the molecules are not “real” molecules, it is not possible to know in advance the outcome of the calculations, an additional advantage that results in the (partial) removal of the developer’s bias. MB08-165 was included as subset in both GMTKN24 and GMTKN30, and it was later replaced in GMTKN55 by the MB16-43 subset, which includes 43 artificial molecules with 16 atoms each. Because of its significance as “chemically unbiased” dataset, we decided to keep MB08-165 in the ACCDB collection. To date, more than 250 functionals have been tested against the GMTKN55 database. In the original publication, 217 functionals were tested,\textsuperscript{101} and a few years later 39 more have been added by the Goerigk group.\textsuperscript{237–239} Jan Martin introduced also three double-hybrids parametrized against this database,\textsuperscript{240} and tested the $\omega$B97M(2) double-hybrid,\textsuperscript{71} bringing the total to 260 functional approximations. Similarly to what has been done for the MGCDB84 database, GMTKN55 has been reduced in size as well.\textsuperscript{241} Gould introduced three different versions of a “dietGMTKN55” database including 30, 100 and 150 datapoints and 84, 270 and 382 different geometries. In this case, no regression formulas are needed, and the full results of GMTKN55 can be replicated with any of these smaller versions.
**Minnesota.**
Among the databases collected in ACCDB, the Minnesota database is probably the oldest. Its first version appeared with the development of the M06 family of functionals,\textsuperscript{58} and it was later updated when used to develop the M11 and (M)N12 family of Minnesota functionals.\textsuperscript{39,40,192,242,243} The Minnesota 2015 database collected in ACCDB includes the Minnesota 2015,\textsuperscript{104} Minnesota 2015A,\textsuperscript{41} and Minnesota 2015B versions.\textsuperscript{60} These three different versions have been used to develop the GAM, MN15-L and MN15 functionals respectively. The most recent version is the Database2019,\textsuperscript{65} published a few years after the development of our collection, and used for the development of the revM11 functional. The largest database among those published in 2015 is the Minnesota 2015B. It has 34 subsets, 471 reference energies, and 719 geometries. The datapoints are divided into different areas: bond energies of main-group elements and transition metal compounds (183 datapoints, corresponding to 38.9\% of the reference data); non-covalent interactions (87 datapoints, 18.5\% of the data); barrier heights (76 datapoints, 16.1\% of the data); thermochemistry, including isomerization energies, excitation energies, hydrocarbon thermochemistry (73 datapoints, 15.5\% of the data); and basic properties, including ionization potentials and atomic energies (53 datapoints, 11.0\% of the data). While it is still used as training set for new approximations, the Minnesota 2019 database has been expanded to include some
subsets of GMTKN55, as well as new databases such as WCCR10\textsuperscript{244,245} and the metal-catalyzed reactions of Chen and coworkers.\textsuperscript{206–208}
**Metals and EE.**
The Metals and Excitation Energies (EE) database is one of the new additions that came with the definition of ACCDB. The main goal of this database is to introduce transition metal excitation energies from the first-, second- and third-row of the periodic table, as well as from the lightest lanthanides (Th–Cm). All the data have been taken from the literature. We advise particular care when dealing with these subsets, mostly when selecting an appropriate basis set, and towards the stability of the self-consistent field solution.\textsuperscript{246,247} In most cases, a pseudopotential should be used, or a relativistic approach has to be considered when all-electron basis sets are employed. This database has eight subsets.
**3d-SSIP30**
This subset includes spin-state (SS) energetics and ionization potentials of first-row transition metals (hence the prefix “3d”) from scandium to zinc. Spin-state energetics correspond to the energy differences between the lowest-energy multiplicity-changing transition for each species, and it includes data for both the neutral atom and the (singly charged) cations. It consists of 30 datapoints, and 40 single-point calculations are needed for this subset. Ten datapoints are for the spin-state transitions for the neutral atoms, ten datapoints for the spin-state transitions for the cations, and additional ten represent the ionization potentials. The reference energies are experimental energies with spin-orbit coupling removed.\textsuperscript{112}
**4d-SSIP24**
This subset includes spin-state energetics and ionization potentials of second-row transition metals, hence the prefix “4d.” It is similar in composition to the 3d-SSIP30 subset presented above. It includes 24 datapoints, equally divided into spin-state transitions for neutral atoms, their (singly-charged) cations, and the corresponding ionization potentials. 32 single-point calculations are needed for this subset. The atoms included are Y, Zr, Nb, Mo, Tc, Ru, Rh, and Pd.\textsuperscript{130}
**AIP28**
This third subset of ionization potentials includes mono- and dioxides of the seven actinides Th, Pa, U, Np, Pu, Am, and Cm. It includes 28 datapoints, and 42 geometries. All data are calculated at the CASPT2/ANO-RCC (an all-electron relativistic triple-\(\zeta\) basis set) level of theory.
An active space of 16 orbitals (14 orbitals for the metals, 2 for oxygen) for 14 electrons has been used for the monoxides, while an active space of 14 orbitals for 14 electrons has been used for the dioxides. The reference geometries have been obtained at the CASPT2 level of theory as well.\textsuperscript{150}
**TMBH23**
The transition metal barrier heights database has been introduced by Chen and coworkers starting in 2013.\textsuperscript{206–208} It involves many metal-catalyzed reactions, more specifically the reactants and transition structures involved in the first step of these reactions. The metals included in this subset are Zr, Mo, W, and Re. Five of the reactions in this subset are catalyzed by Zr, five by Mo, seven by W, and six by Re, for a total of 23 datapoints and 49 structures. The geometries included in this subset correspond to the structures of the reactants and the transition states (TSs). They have been obtained with the PBE0 functional\textsuperscript{55,56} and either the cc-pVTZ (Zr, Re) or the cc-pVDZ (Mo, W) basis sets,\textsuperscript{248} with the inclusion of a pseudopotential for the heaviest metal atoms.\textsuperscript{249}
**LTMBH26**
The late transition metal barrier heights subset is another database introduced by Chen and coworkers in 2012,\textsuperscript{202} and it is the heavier analogue of the TMBH23 subset. The original authors included 26 reactions catalyzed by Au, Pt, and Ir, three metals that are fundamental in organic synthesis. It is mostly focused on gold, which is involved in 22 out of the 26 reactions of this subset, while Pt and Ir are involved in two reactions each. It includes 40 structures, corresponding to reactants and TSs. The geometries have been obtained at the PBE0/cc-pVTZ level of theory, with the addition of a pseudopotential for the metal atoms.
**MOR41**
The metal organic reaction database was introduced by Grimme and coworkers in 2018.\textsuperscript{212} It is a dataset of reaction energies involving thirteen different TMs: Ti, Cr, Mn, Fe, Co, Ni, Mo, Ru, Rh, Pd, W, Ir, Pt, and Al. The original authors selected these systems very carefully among 120 candidates to focus their attention on catalytically relevant metals only. In addition, they intentionally avoided multi-reference (MR) systems. Only single-reference structures are included in this database, and all the TM-containing complexes are neutral, closed-shell systems. There are 41 datapoints and 95 geometries for this subset. The geometries have been obtained at the TPSS-D3(BJ)/def2-TZVP level of theory.\textsuperscript{250,251} Ten reactions are catalyzed by 3d elements (one by Ti—with Al—, Mn, Fe, and Co, three by Cr and Ni), 21 by 4d elements (three by Mo, two
by Pd, ten by Ru, and six by Rh), and ten by 5d elements (one by W, six by Ir, and three by Pt). The size of the molecules included ranges between 2 and ~120 atoms, making it a costly exercise. For this reason, Chan introduced a reduced version of this database as well, calling it MOR13.\textsuperscript{236} MOR13 includes 13 datapoints, and 37 single-point calculations are required to compute the reaction energies. Analogously to MG8, this sub-database comes with regression formulas that help in recovering the performance of the original set of data.
**p-VR17**
This database includes valence and Rydberg excitations of p-block elements and their singly-charged cations. A valence excitation usually involves the transition of an electron to a $ns$ orbital to a $np$ orbital, while in a Rydberg excitation an electron is excited from a $np$ orbital to a $(n+1)s$ orbital. The atoms included are B, F, Ne, Al, Cl, Ar, Ga, Br, and Kr, and the cations are B$^+$, C$^+$, Ne$^+$, Al$^+$, Si$^+$, Ga$^+$, Ge$^+$, and Kr$^+$. This subset includes 17 reference energies (9 from valence and 8 from Rydberg excitations), and 34 atomic structures.\textsuperscript{111}
**Por21**
This database was introduced for the first time with the creation of ACCDB. It includes spin states and binding energies of metal porphyrins. An example is reported in \textbf{Figure 2.2}. Given their ubiquitous presence in nature, as well as their central role in enzymatic catalysis, like the famous heme group, we decided to collect data from different sources from the literature and group them in a new database. This subset includes 21 datapoints divided in two groups. The eleven reference energies for spin states are grouped in the PorSS11 subset. The ten additional datapoints correspond to binding energies are collected in the PorBE10 subset. The reference spin states involve one Mn porphyrin, a Co porphyrin, the unsubstituted Fe porphyrin, and an iron porphyrin bound to four different substituents, namely NH$_3$, OH$^-$ and SH$^-$. \textbf{Figure 2.2} shows an iron porphyrin bound to the hydride anion (panel a). The binding energies involve either a free iron porphyrin or an iron porphyrin bound to an imidazole ring, a common model system for the heme group, and either NO, CO or O$_2$. An example is reported in \textbf{Figure 2.2}, panel b. The reference spin splittings have been calculated at the CASPT2 level, with different active spaces depending on the system taken into account.\textsuperscript{147,148} A total of 32 single-point calculations are needed for this subset, with the corresponding geometries optimized at either the PBE0/def2-TZVP (iron porphyrin in PorSS11 and PorBE10, the diatomic gases in PorBE10) or BP86/def2-TZVP (all other structures) levels of theory.
Figure 2.2: An example of the structures included in the Por21 database. Panel a: A structure from the PorSS11 subset representing a porphyrin ring bound to an OH$^-$ group; Panel b: A structure from the PorBE10 subset showing a porphyrin ring bound to imidazole (below the plane of the ring) and carbon monoxide (above the plane of the ring).
**W4-17**
The W4-17 database is the latest update to the W4-family of databases.\textsuperscript{105} Previously published versions of this database are W4-08\textsuperscript{218} and W4-11,\textsuperscript{155} the latter included in both MGCDB84 and GMTKN55. W4-17 includes 203 total atomization energies of 215 reference structures, mostly small molecules. Each reference energy is obtained with the Weizmann-4 composite method, one of the most accurate available to date, with a guaranteed confidence interval within 1 kJ mol$^{-1}$. For a review of the W4 procedure, we refer the interested reader to ref.\textsuperscript{252} In addition to the new calculated data, we also included in this database additional datapoints from W4-11. We added 99 reference energies from the bond dissociation energy subset (BDE99), 707 reference energies for hydrogen atom transfer reactions (HAT707), 20 reference energies for isomerization reactions (ISOMERIZATION20), and 13 reference energies for nucleophilic substitutions (SN13). The inclusion of these additional subsets expands the database to a total of 1,042 reference data without the need for additional reference geometries, since both W4-11 and W4-17 are based on the same structures.
**DP284**
This database is a collection of 152 reference dipole moments\textsuperscript{107} and 132 polarizabilities of small molecules.\textsuperscript{106} It includes 181 structures, either obtained from accurate experimental measurements, or at the MP2/cc-pVTZ level of theory. The reference values have instead been obtained at the CCSD(T)/CBS level of theory.
W4-17-RE and MN15-RE
These two databases have been introduced within ACCDB for the first time. They have been automatically generated from the W4-17 and Minnesota 2015B databases using the autoRE script found in ref.\textsuperscript{253} This program selects reactions having the form
\begin{equation}
A + B \rightarrow C + D
\end{equation}
using a list of molecules and their total atomization energies. We started from the W4-17 and the atomization energies in the Minnesota 2015B databases. The program automatically filtered out redundancies, duplicates, or trivial isomerizations, and its application resulted in these two new databases, which have 27,140 (W4-17-RE) and 9,175 (MN15-RE) datapoints respectively, for a total of 36,275 reference data. They have never been used for the evaluation of $xc$ functionals, and we expect these unbiased, “non-traditional” databases to be useful for future applications.
Additional considerations.
The three major databases MGCDB84, GMTKN55, and Minnesota 2015, although smaller than ACCDB, can be regarded as a collection of other databases from the literature. In some cases, the original sources are the same, which resulted in the overlap among some of the subsets in the three databases. For instance, GMTKN55 and MGCDB84 include the database W4-11, which is the previous version of W4-17. While the geometries are the same, the latter has the most up-to-date reference energies. In addition, MGCDB84 heavily relies on data taken from GMTKN30, a preceding version of GMTKN55. Both databases include the S22 and S66 subsets of non-covalent interactions, as well as subsets such as G21IP, G21EA, ACONF, and CYCONF. Another situation of substantial overlap comes with the BH76 subset of barrier heights. It is present in Minnesota 2015B, in MGCDB84 (as the two separate sets HTBH38 and NHTBH38), and in GMTKN55, which has the most recent—and most accurate—reference data. For consistency purpose, we kept the values in MGCDB84 and Minnesota to the values introduced by the original authors. As last example, we want to discuss the DC13 subset in GMTKN55. It includes 13 problematic reactions for DFT approximations, and it is an extension of the DC9 subset of Truhlar and coworkers (included in Minnesota 2015B). The new version shares only one reaction with DC9, and it includes one reaction from the Styrene45 subset, and one reaction from the C20C24 subsets of the MGCDB84 database. The other datapoints come instead from the literature. A visual
representation of the overlaps is reported in Figure 2.3 below.
We advise particular care when handling the entire collection, most importantly if the reduction of the number of single-point calculations is a priority, for example due to limited computational resources. Our choice of keeping the databases as they are in the literature was dictated by practical considerations. In our opinion, it is better to use the original databases despite the overlap among the subsets. This allows the comparability with the results already published in benchmark studies, which would not be possible if the reference data are mixed, for example by using different (more up-to-date) reference energies not included in the original publications.
2.3 Instructions about how to use ACCDB: Automation software.
ACCDB is organized in many different folders according to their content. For example, the “Geometries” folder contains the 10,049 molecular geometries—in xyz format, all appropriately named and labeled with charge and multiplicity—needed for the whole collection. Each structure necessitates a single-point calculation, which then results in 44,931 calculated energies that can be compared with the respective references provided as .csv (comma-separated value) files. To help in the management of this large number of structures, ACCDB comes with a set of automation tools in the form of a snakemake workflow.\textsuperscript{254} Starting from the creation of the input files, snakemake takes care of each step from the submission of the jobs (either to a workstation, or a computer cluster) to collecting the final results. The user needs to provide a template input file for the quantum chemistry program of choice (we included examples written for the Gaussian\textsuperscript{255} and Q-Chem\textsuperscript{256} programs in the “Automation” folder). In addition, they have to choose the molecules to include in the workflow through one of the lists provided in the “Databases” folder. We provide lists covering the whole collection, entire databases, groups of subsets, or single subsets. For example, the list called “MGCDB84_ALL” includes the 5,931 structures needed for the MGCDB84 database, while the one named “MGCDB84_ID” lists the 248 structures needed for the subsets labeled “Isomerization Difficult” (ID) in the original publication.\textsuperscript{102} Last, the list called “MGCDB84_S22” includes the 66 structures needed for this dataset only. The lists can be combined regardless of their origin and their number. It is possible to run calculations using different subsets from different databases, and there is virtually no limit to the number of lists—and therefore subsets—that can be combined. For example, one can choose to run calculations using the BH76 subset of the Minnesota database together with the DC13 subset of GMTKN55. The customization of the procedure is made possible using a configuration file
| GMTKN55<sup>a,b</sup> | MGCDDB84<sup>a</sup> | W4-17 | Minnesota | Metals&EE |
|----------------------|---------------------|-------|-----------|----------|
| W4-11 (TAE140) | TAE140 | TAE203<sup>c</sup> | | |
| | BDE99, HAT707, | BDE99, HAT707, | | |
| | ISOMERIZATION20, SN13| ISOMERIZATION20, SN13| | |
| G21EA | G21EA | | | |
| G21IP | G21IP | | | |
| PA26 | | | PA8 | |
| NBPRC | NBPRC | | | |
| BH76RC | BH76RC | | | |
| DC13<sup>d</sup> | C20C24, Styrene45<sup>d</sup> | | DC9<sup>d</sup> | |
| (DC9 in GMTKN30) | | | | |
| MB16-43<sup>e</sup> | | | | |
| (similar to MB08-165)| | | | |
| BSR36 | BSR36 | | | |
| CDIE20 | DIE60 | | | |
| ISOL24 | | IsoL6/11 | | |
| (ISOL22 in GMTKN30) | | | | |
| BH76 | HTBH38, NHTBH38 | | BH76 | |
| BHPERI26 | BHPERI | | | |
| PX13 | CE20, PX13 | | | |
| WCPT18 | WCPT6, WCPT27 | | | |
| S22 | S22, HSG, NBC10 | | NCCE23 | |
| S66 | S66, S66x8 | | S6x6 | |
| WATER27 | WATER27, H2O20Bind4, H2O20Rei4 | | | |
| HAL59 | X40, XB51, XB18 | | | |
| AHB21, CHB6, IL16 | Ionic43 | | | |
| ACONF | ACONF | | | |
| Amino20x4 | YMPJ519 | | | |
| CYCONF (GMTKN30) | CYCONF (from GMTKN30) | | | |
| MCONF | Melatonin52 | | | |
| But14diol | Butanediol165 | | | |
| RG10 | | NGDW121 | | |
| IP13 | | IP23 | | |
| EA13 | | EA13 | | |
| AE18 | | AE17 | | |
| DBH24 | W4-08<sup>f</sup> | 3dEE8 | 3d-SSIP30 | |
| | | 4dAE5 | 4d-SSIP24 | |
| | | pEE5 | p-VR17 | |
[a] MGCDDB84 and GMTKN55 share a considerable number of subsets since some of the subsets in MGCDDB84 are taken from GMTKN30. [b] For GMTKN55, all reference energies have been recalculated, and they differ (in general) from the ones reported in MGCDDB84, or in Minnesota. [c] TAE203 replaces TAE140. The latter is taken from W4-11, together with BDE99, HAT707, ISOMERIZATION20, and SN13. [d] One of the reactions included in DC13 comes from C20C24, one from Styrene45, one from DC9, the others from literature. [e] MB16-43 substitutes MB08-165. [f] The reference energies for DBH24 are taken from W4-08, but they are not included in our version of W4-17.
Figure 2.3: A detail of the superposition among the different databases in ACCDB. A yellow background indicates partial overlap (geometries or reference energies), while an orange background indicates complete overlap (geometries and reference energies).
written in `yaml` syntax. The user can specify the source directory for the geometries, the name of the directory in which the input files are created, the name of the directory in which the output files can be found at the end of the process, and the methods and basis sets to be included. `Snakemake` will create as many input files as the combinations of methods and basis sets indicated, each one with a unique name. For example, if we want to run calculations with $n$ functionals and $m$ basis sets for $z$ structures, the workflow will generate $n \cdot m \cdot z$ input files grouped by methods and basis sets. Once these files are created, they will be submitted, and the results collected at the end of the process, which is visually summarized in Figure 2.4.
Figure 2.4: Schematic representation of the ACCDB workflow.
### 2.4 Using databases in chemical education: A case study.
The computational chemistry databases collected in ACCDB are important diagnostic tools in DFT development and in its applications. Since they span a wide chemical space, they test the response of the many approximations to the $xc$ energy in different situations. DFT benchmark studies are usually accompanied by guidelines on which functional to use for a given application, and they represent the best practices in the field at a given point in time. Theoreticians can understand the strengths and weaknesses of each functional, and they can gather
important information to introduce more robust and improved approximations. Practitioners can use databases instead of citation counts or hearsay when selecting a functional for a given chemical application.\textsuperscript{257} One can understand in advance if an approximation is trustworthy for the description of a chemical problem through comparison of the specific reaction with those in the databases. In addition, it is also possible to get an idea about the expected accuracy of the functionals across a broad spectrum of chemical reactivity. We recently introduced a tutorial review to help users with a few issues that arise when running DFT calculations.\textsuperscript{28} Our review represents the first application of computational databases in chemical education, and it is accompanied by a commented laboratory notebook with 12 different experiences. Each experience contains one or more chemical reactions coming from a different subsets of the ACCDB collection, and it includes many of the challenges that could arise in every day research. The notebook is divided into three parts. The first covers the foundations of ground-state DFT (as in Chapter 1 of this dissertation); the second deals with shortcomings in the choice of the method, \textit{i.e.} functionals and basis sets; the third part instead deals with technical details such as the choice of integration grids, stability analyses, and comparisons with experimental results. As last exercise, we included a self-assessment test. We will review each experiment in the following paragraphs.
\textbf{Experiments 1–3: The choice of a functional.}
Given the sheer amount of options available, the main issue in (KS-)DFT is choosing a $xc$ functional. Among other things, the reliability of the results is heavily dependent on the functional chosen to describe the chemical system. If one uses a functional that is inadequate for the chemical problem at hand, the results will not be reliable. This is not a failure of DFT as a whole, but rather of the specific approximations. In this context, we can think of $xc$ functionals as a tool needed to perform a specific task. “If the tool is not adequate, we should choose a different tool or a different task.”\textsuperscript{28} If our goal is to put a nail on the wall, we should pick a hammer, regardless of how many screwdrivers we have at our disposal. Another take on this stance has been proposed by Kathryn Hopmann. She advised “Do not use DFT for mechanisms it cannot handle.”\textsuperscript{258} As the first three experiments show, it is important to be aware of the strengths and limitations of a given approximation. They have been designed with the goal of showing that even seemingly simple reactions can be daunting for DFT, and chemical intuition \textit{alone} should not be used as a guideline.
In the first experiment, we use four familiar reactions that include conformational and rotational
barriers, as well as a *cis-trans* isomerism (Figure 2.5, panel A). The first reaction is the rotational barrier of ethane, the second is the rotational barrier of ethanol, the third is the conformational change of two isomers of butane-1,4-diol, and the last is the *cis-trans* isomerization of pent-2-ene. These reactions come from the BHROT27\textsuperscript{101}, BUT14DIOL\textsuperscript{191} and FH51\textsuperscript{127,128} subsets of the GMTKN55 database.\textsuperscript{101} They are “easy” in the sense that most of the approximations we tried give good results, with errors below 1 kcal/mol (see Figure 2.6 below). The reason behind this good performance relies in the similarity between the reactants and the products. The electron densities of both molecules are very similar to each other, and they do not change too much throughout the reaction. Even if the density obtained for the reactants is completely wrong, it is highly likely that the density of the products will be equally wrong. As a result, the errors will cancel each other. As long as the functional is consistent, independently of whether it gives an accurate description or not, the results will still be acceptable, and the approximation will provide the right answer for the wrong reason. This compensation is called “error cancellation.” While it might seem beneficial at first, we should keep in mind that it is unpredictable, and it is not possible to know if it will work to our advantage \textit{a priori}. Databases can help us in this sense, as they can be used as diagnostic tools to identify cases that are affected by error cancellation and those that are not.
In the second experiment, we collected Diels–Alder and isomerization reactions that can be found in introductory organic chemistry textbooks (Figure 2.5, panel B). The first reaction
in this second set is the prototypical Diels–Alder reaction between ethene and butadiene; the second reaction is the isomerization of $n$-hexane to neohexane; the third and fourth reactions correspond to the formation of the ethane and methanol dimers respectively. These structures come from the DAR\textsubscript{C}\textsuperscript{101,133,216} and ADIM\textsubscript{6}\textsuperscript{101,145} subsets of the GMTKN55 database,\textsuperscript{101} and from the AlkIsomer11\textsuperscript{157} and S66\textsuperscript{167,168} subsets of MGCDB84.\textsuperscript{102} In all these cases, the electron densities of the products are drastically different from the electron densities of the reactants. Error cancellations does not work as well as in the previous cases, as shown in \textbf{Figure 2.6} below. The results we collected using some of the most popular functionals show that some approximations can be quite inaccurate, and more care is needed to handle these reactions. The two dimerization reactions have been included as examples of molecules bound together by dispersion interactions. Most functionals (especially older ones) are incapable of describing species bound through Van der Waals interactions, and in 2012 the accurate description of such molecules was still listed as a major challenge for DFT.\textsuperscript{259} Many approaches have been designed to specifically deal with this problem, and it is widely recognized that the application of a “dispersion correction” is usually beneficial. Two of the most popular corrections are the “-D” approach of Grimme and coworkers\textsuperscript{145,260–262} and the VV10 functional of Vydrov and Van Voorhis.\textsuperscript{46} The -D approach comes in four different flavors, and it has the advantage of adding no cost to the calculation. The VV10 functional is more reliable, but it is more expensive than the -D approach. It is now generally accepted (and strongly recommended) to use dispersion corrections with most functionals to greatly increase their accuracy when dealing with dispersion-bound systems. The Minnesota functionals represent an important exception: since they have been designed to properly account for the bonding in the Van der Waals region, they do not require the use of a dispersion correction in the majority of cases, although the corrections have been proposed.\textsuperscript{263}
In the third experiment, we deal with reactions that are very hard to describe for DFT. The first reaction corresponds to the first step of the ozonolysis process involving ozone and ethene; the second reaction is the dissociation of the CaO molecule; the third reaction is the atomization energy of sulfuric acid; the fourth reaction involves the conversion of ozone to molecular oxygen (\textbf{Figure 2.5}, panel C). The first reaction comes from the DC13 subset\textsuperscript{[222,,223,224,67,225,226,227,58,228,133,195,196,101]} of the GMTKN55 database,\textsuperscript{101} while the second and third reactions come from the MR-MGM-BE4\textsuperscript{161} and DC9\textsuperscript{18,230} subsets of the Minnesota 2015B database respectively.\textsuperscript{41} The last reaction has been obtained combining two datapoints of the W4-17 database.\textsuperscript{105} Each of the species involved (but the ozonolysis
intermediate) are usually encountered very early in chemical education. They are the best example to show that we cannot judge the reliability of a calculation based on chemical intuition alone. Many approximations fail badly in the description of these molecules despite their simplicity, and we recommend consulting with experts for advice and guidance when dealing with these situations.
| Functional/method | 1a | 1b | 1c | 1d | 2a | 2b | 2c | 2d | 3a | 3b | 3c | 3d |
|-------------------|------|------|------|------|------|------|------|------|------|------|------|------|
| B3LYP/def2-QZVP | 0.12 | 0.02 | 0.29 | 0.24 | 11.41| 1.57 | 3.77 | 0.91 | 3.50 | 7.20 | 23.63| 19.44|
| B3LYP-D3(BJ)/def2-QZVP | 0.10 | 0.02 | 0.23 | 0.07 | 5.92 | 0.63 | 1.13 | 0.04 | 1.22 | 8.41 | 18.09| 0.71 |
| MN15/def2-QZVP | 0.10 | 0.07 | 0.15 | 0.07 | 1.03 | 0.44 | 0.58 | 0.19 | 10.63| 0.14 | 1.88 | 8.40 |
| wB97M-V/def2-QZVP | 0.16 | 0.05 | 0.03 | 0.05 | 1.34 | 0.12 | 0.21 | 0.08 | 7.88 | 1.54 | 5.30 | 14.50|
| PBE/def2-QZVP | 0.24 | 0.05 | 0.16 | 0.06 | 2.95 | 1.16 | 2.78 | 0.43 | 7.62 | 27.11| 19.71| 31.37|
| PBE-D3(BJ)/def2-QZVP | 0.23 | 0.05 | 0.48 | 0.14 | 0.37 | 0.17 | 1.19 | 0.17 | 6.26 | 27.64| 22.85| 0.42 |
| PBE0/def2-QZVP | 0.08 | 0.01 | 0.01 | 0.09 | 3.52 | 1.22 | 2.68 | 0.49 | 4.22 | 5.65 | 14.29| 27.43|
| SCAN/def2-QZVP | 0.13 | 0.09 | 0.42 | 0.12 | 2.73 | 0.58 | 0.88 | 0.18 | 5.09 | 19.59| 1.12 | 2.14 |
| B97M-V/def2-QZVP | 0.12 | 0.11 | 0.17 | 0.12 | 1.62 | 0.15 | 0.21 | 0.03 | 0.06 | 10.06| 2.29 | 9.79 |
| M06-2X/def2-QZVP | 0.07 | 0.02 | 0.04 | 0.04 | 0.42 | 0.21 | 0.11 | 0.11 | 11.73| 6.03 | 10.96| 10.74|
| M11/def2-QZVP | 0.27 | 0.12 | 0.12 | 0.16 | 0.25 | 0.26 | 0.23 | 0.22 | 5.32 | 11.55| 8.56 | 11.92|
| BP86/def2-QZVP | 0.26 | 0.05 | 0.13 | 0.11 | 7.16 | 2.52 | 3.28 | 1.30 | 9.41 | 27.34| 19.19| 22.92|
| BLYP/def2-QZVP | 0.27 | 0.01 | 0.34 | 0.27 | 19.37| 2.47 | 4.28 | 1.19 | 15.65| 21.51| 12.99| 5.25 |
| B3PW91/def2-QZVP | 0.15 | 0.04 | 0.26 | 0.23 | 2.62 | 2.48 | 3.64 | 1.21 | 0.00 | 11.15| 15.57| 20.70|
Figure 2.6: Results obtained for the reactions in experiments 1–3 for a selection of functionals with the def2-QZVP basis set. All results are in kcal mol$^{-1}$. A green background means satisfactory results, while a red background implies an inadequate description.
Experiments 4–6: The choice of a basis set.
The right choice of functional for a given application cannot guarantee a successful outcome on its own. A reliable calculation depends also on the selection of a suitable basis set. This issue has a more general nature, and it applies to both WFT (HF and post-HF approaches) and DFT, albeit having slightly different caveats depending on which theory is used. In this context, we will deal with the issues that are distinctive of DFT. In the theoretical chemistry literature the basis sets are grouped into “families.” The most popular basis sets for electronic structure calculations have been developed by Pople and coworkers,\textsuperscript{264–272} by Dunning and coworkers,\textsuperscript{248,273–276} by Weigend and Ahlrichs,\textsuperscript{251} and by Jensen.\textsuperscript{277–284} The choice of a basis set is as intimidating as the choice of a functional, especially when considering the many different acronyms used to identify the members of the different families. To complicate things even further, there is no universal nomenclature for basis sets, and each family requires its own Rosetta stone. For example, the Dunning family of basis sets has the shorthand notation cc-pV$n$Z, where “cc” stands for “correlation consistent,” p means polarized, V indicates “split-valence,” and $n$ refers to the size of the basis set (either D for double-$\zeta$, T for triple-$\zeta$, Q for quadruple-$\zeta$, 5 for quintuple-$\zeta$, and 6 for sextuple-$\zeta$). The Ahlrichs basis sets instead adopt the notation def2-$n$ZVP, where $n$ can be either T or Q (for triple- and quadruple-$\zeta$), while the double-$\zeta$ basis set is indicated as def2-SVP. The prefix “def2” is included because these basis sets have been redefined, while the letters P and V
have the same meaning as in the Dunning’s basis sets. As a general rule, a polarized basis set includes an additional function having higher angular momentum than the valence orbitals. For instance, a double-$\zeta$ polarized basis set for carbon would include a $d$ function, while for iron it would include an $f$ function. Split-valence implies that the basis set includes one set of functions for the core orbitals, while the number of functions for the valence orbitals depends on the size of the basis set, i.e. on the “zeta.” A double-$\zeta$ has two sets of valence orbitals, a triple-$\zeta$ has three, and so on. The Pople basis sets adopt a completely different nomenclature. These basis sets include 3-21G, 6-31G, and 6-311G. The first number indicates the number of functions used for the core electrons, while the two (or three) numbers after the hyphen indicate the number of functions used for the valence orbitals. G instead stands for Gaussian function(s). 3-21G and 6-31G are both double-$\zeta$ basis sets, while 6-311G is a triple-$\zeta$ basis set. In Pople’s notation, polarized functions are added using a * at the end of the name. The popular 6-31G* basis set is double-$\zeta$, split-valence and polarized for all elements but hydrogen. 6-31G** would instead have polarization functions on this element as well. Last, the Jensen family of basis sets is designed as pc-$n$, with pc meaning “polarization consistent,” and $n$ equals 0, 1, 2, 3 and 4 to indicate a non-polarized double-, a polarized double-, triple-, quadruple- and quintuple-$\zeta$ basis sets. Basis sets can also be enriched with “diffuse” functions, most importantly when describing the electron density in regions that are far away from the nuclei. An augmented basis set is denoted by the prefix “aug-” in the Dunning and Jensen’s families (aug-cc-pVDZ, or aug-pc-1), by the suffix “D” for the Ahlrichs basis sets (def2-SVPD), and by a “+” sign in the Pople’s family (6-31+G*, or 6-31++G* if augmented functions are included on hydrogen).
While the simple principle “the larger, the better” is a good guideline to keep in mind when choosing an adequate basis set, one usually has to make a compromise between the accuracy of the results and the computational cost. Especially with limited computational resources, large molecules become too expensive to be described with a large basis set, and routine calculations are usually performed with small basis sets (double-$\zeta$ quality or smaller). In such situations, the basis set introduces an error that can be traced back to two distinct components. One is called basis set incompleteness error (BSIE),\textsuperscript{285,286} and the other is called basis set superposition error (BSSE).\textsuperscript{287,288} The BSIE is defined as the difference between the results obtained with a very large basis set and those obtained with a small basis set. This source of error is largest when double-$\zeta$ (or smaller) basis sets are employed, but it can be easily minimized if larger basis sets are used. We deal with BSIE specifically in experiment 4 through the calculation of the interaction energies.
of the HCl dimer. The BSSE is an artificial effect that arises when comparing the energy of a molecule with the energy of the constituent fragments at infinite separation. Typical chemical applications that might suffer from BSSE are the calculations of binding energies, or dimerization reactions. The basis set of the complete system (A-B) and those of the separated components (A and B) are different, and the former is larger than the latter. The united system can benefit from an artificial increase of the basis set size due to the simultaneous presence of the two fragments. The basis set from A can be used to describe B, and vice-versa. These extra contributions are absent in the separated fragment, resulting in the fictitious overestimation of the binding energy of the system. Similarly to the BSIE, the BSSE can also be minimized when using a large basis set. Many different (approximate) approaches have been proposed to solve this issue, and the one we employ in experiment 5 is the counterpoise (CP) correction of Boys and Berardi.\textsuperscript{289} While other schemes have been designed as an alternative to it,\textsuperscript{290–292} the CP correction is still widely used, and its inclusion is generally recommended. For these calculations, we used the dimerization reaction of HCl from the NCCE23 subset\textsuperscript{18,166,233–235} of the Minnesota 2015B database\textsuperscript{60} as an example (equation 2.1 below).
\[(\text{HCl})_2 \rightarrow 2 \text{HCl}\]
(2.1)
The last experiment in this group is designed as a review experience on the usage of modern basis sets. The reactions used as an example include the proton affinity of ammonia from the PA8\textsuperscript{18,115} subset of the Minnesota 2015B database,\textsuperscript{60} the ionization energy of acetylene and the electron affinity of the methyl radical coming from the G21IP and G21EA subsets\textsuperscript{93} of the GMTKN55 database.\textsuperscript{101} Charged species require a different treatment than neutral species when it comes to the basis set. More specifically, anions usually require augmented basis sets for a more reliable description, while cations do not. Using augmented basis sets with cations usually worsens the results, while they are needed to achieve better accuracy for anions, as shown in Figure 2.7. For neutral species, the results usually improve when augmented basis sets are employed. The reason behind the better performance is in an increased amount of basis functions available, rather than in the better description of the system. For such cases, it would be better to use a larger basis set (for example, a triple-\(\zeta\)) rather than an augmented one (augmented double-\(\zeta\), for instance) of the same size.
| IP of HC≡CH | def2-SVP | def2-SVPD | def2-TZVP | def2-TZVPD | def2-QZVP | def2-QZVPD |
|-------------|----------|-----------|-----------|------------|-----------|------------|
| BP86 | 4.64 | 3.57 | 3.5 | 3.38 | 3.08 | 3.07 |
| PBE | 6.4 | 5.23 | 5.25 | 5.08 | 4.83 | 4.8 |
| SCAN | 8.89 | 8.54 | 8.91 | 8.82 | 8.68 | 8.67 |
| B97M-V | 7.46 | 6.82 | 8.27 | 8.05 | 7.85 | 7.84 |
| PBE0 | 7.06 | 6.46 | 6.96 | 6.84 | 6.66 | 6.65 |
| M06-2X | 5.51 | 5.03 | 4.73 | 4.67 | 4.25 | 4.25 |
| B3LYP | 6.37 | 5.1 | 5.11 | 4.97 | 4.68 | 4.67 |
| ωB97M-V | 7.47 | 6.56 | 6.69 | 6.59 | 6.42 | 6.41 |
| HF | 36.84 | 37.03 | 38.67 | 38.62 | 38.62 | 38.63 |
| SPW92 | 1.23 | 2.6 | 2.92 | 3.03 | 3.42 | 3.43 |
| Average | 9.19 | 8.69 | 9.10 | 9.01 | 8.85 | 8.84 |
| EA of CH₃⁻ | def2-SVP | def2-SVPD | def2-TZVP | def2-TZVPD | def2-QZVP | def2-QZVPD |
|------------|----------|-----------|-----------|------------|-----------|------------|
| BP86 | 18.77 | 3.06 | 4.72 | 0.29 | 0.4 | 0.52 |
| PBE | 21.65 | 6.16 | 8.01 | 3.25 | 3.44 | 2.38 |
| SCAN | 25.95 | 12.37 | 13.67 | 9.96 | 9.88 | 9.14 |
| B97M-V | 19.55 | 6.49 | 9.01 | 4.12 | 4.48 | 3.46 |
| PBE0 | 23.33 | 9.93 | 11.23 | 7.39 | 7.45 | 6.63 |
| M06-2X | 20.7 | 7.57 | 8.12 | 5.02 | 4.63 | 3.77 |
| B3LYP | 21.38 | 6.35 | 8.09 | 3.81 | 3.97 | 3.02 |
| ωB97M-V | 21.14 | 6.35 | 7.96 | 4.11 | 4.27 | 3.47 |
| HF | 51.06 | 40.58 | 41.51 | 38.61 | 38.63 | 38.02 |
| SPW92 | 16.11 | 0.84 | 0.82 | 3.87 | 3.7 | 4.75 |
| Average | 23.96 | 9.97 | 11.31 | 8.04 | 8.09 | 7.52 |
| PA of NH₃ | def2-SVP | def2-SVPD | def2-TZVP | def2-TZVPD | def2-QZVP | def2-QZVPD |
|-----------|----------|-----------|-----------|------------|-----------|------------|
| BP86 | 5.49 | 1.2 | 0.32 | 0.94 | 0.43 | 0.69 |
| PBE | 5.41 | 1.43 | 0.17 | 1.33 | 0.75 | 1.09 |
| SCAN | 6.07 | 0.36 | 1.54 | 0.5 | 1.07 | 0.87 |
| B97M-V | 4.24 | 1.1 | 0.58 | 0.77 | 0.18 | 0.04 |
| PBE0 | 6.44 | 0.7 | 1.95 | 0.78 | 1.3 | 1.07 |
| M06-2X | 3.77 | 1.88 | 1.22 | 2.17 | 1.91 | 2.11 |
| B3LYP | 5.44 | 1.22 | 0.32 | 0.94 | 0.35 | 0.61 |
| ωB97M-V | 3.16 | 3.34 | 1.68 | 2.89 | 2.24 | 2.51 |
| HF | 7.64 | 3.42 | 4.19 | 3.5 | 4.05 | 3.94 |
| SPW92 | 2.37 | 5.43 | 4.02 | 5.44 | 4.95 | 5.26 |
| Average | 5.00 | 2.01 | 1.60 | 1.93 | 1.72 | 1.82 |
Figure 2.7: Unsigned errors obtained with a selection of functionals for the reactions in experiment 6 (ionization potential—IP—of acetylene, electron affinity—EA—of the methyl radical, and the proton affinity—PA—of ammonia) with the Ahlrichs family of basis sets. Augmented basis sets yield the worst results for the positively charged species, while they are necessary for anions. A green background means satisfactory results, while a red background implies an inadequate description. All values are in kcal mol$^{-1}$.
Experiment 7: The origin of the popularity of B3LYP/6-31G*.
The B3LYP functional\textsuperscript{22,23,25,26} is undoubtedly the most used \(xc\) functional of all times.\textsuperscript{293,294} When coupled with the 6-31G* basis set of Pople and coworkers, it has become the \textit{de facto} standard of the organic chemistry community. B3LYP was introduced in 1994\textsuperscript{26} in a study involving the \textit{in silico} modeling of circular dichroism spectra. Since then, it has been applied to almost all areas of chemistry, and over the years it became almost a synonym for DFT, especially in the non-specialized literature and in chemical education.\textsuperscript{295–297} As the method grew in popularity, many shortcomings have been reported at the same time.\textsuperscript{72,298–300} We specifically designed experiment 7 to show the limitations of the B3LYP/6-31G* level of theory. As others have shown,\textsuperscript{72} this method and basis set heavily benefit from the cancellation of errors between the functional and the basis set. If we change the basis set, this “magic” does not work anymore. Figure 2.8 lists some selected results obtained for the same reactions in experiments 1–3. In some cases, most notably the Diels–Alder reaction, the error cancellation is very beneficial. In others, as the atomization reaction of sulfuric acid, it is catastrophic. This behavior is totally unpredictable, but fortunately it is easy to diagnose through comparison between the results obtained with a larger basis set. If the error made with 6-31G* is smaller than the one obtained with, for example, def2-QZVP, then the datapoint we are analyzing is affected by error cancellation. Instead of looking for systems where B3LYP/6-31G* is trustworthy, we encourage practitioners (as other did before us)\textsuperscript{72} to move away from this level of theory in favor of other functionals that are more reliable for problematic cases. A few years ago, Cohen, Mori-Sánchez and Yang said that uniformly performing better than B3LYP was still a challenge for \(xc\) functionals.\textsuperscript{259} Recent reviews in the field proved that this is no longer true,\textsuperscript{18,60,101,102} and it is possible to perform more accurate calculations at a similar cost, for example when using the most modern hybrid functionals (\(\omega\)B97M-V, or MN15) and basis sets as def2-SV(P) or pc-0. We believe that the real challenge is to get rid of B3LYP/6-31G* altogether, but as Kruse, Goerigk and Grimme said,\textsuperscript{72} we do not expect this to happen anytime soon.
Experiments 1–7 make up the first part of the laboratory notebook we developed using databases that specifically deal with methodological components, \textit{i.e.} the choice of a given functional and basis set. The main messages of this section are:
- Always follow the recommendations coming from the experts in the field.
- Avoid choosing a functional/basis set because of their popularity or because of hearsay.
| Functional | Basis Set | 1a | 1c | 2a | 2c | 3a | 3c |
|---------------|-------------|-----|-----|-----|-----|-----|-----|
| B3LYP | def2-QZVP | 0.12| 0.29| 11.41| 3.77| 3.50| 23.63|
| B3LYP | 6-31G* | 0.05<sup>b</sup> | 1.52 | 2.45<sup>b</sup> | 3.64 | 3.63 | 73.88 |
| B3LYP | def2-SVP | 0.12| 1.89| 2.73| 4.00| 1.49<sup>a</sup> | 70.11 |
| B3LYP-D3(BJ) | def2-QZVP | 0.10| 0.23| 5.92| 1.13| 1.22| 18.09 |
| B3LYP-D3(BJ) | 6-31G* | 0.06<sup>a</sup> | 2.04 | 3.04<sup>b</sup> | 1.01<sup>a</sup> | 5.91 | 68.34 |
| B3LYP-D3(BJ) | def2-SVP | 0.14| 2.41| 2.76| 1.37| 3.77| 64.57 |
| ωB97M-V | def2-QZVP | 0.16| 0.03| 1.34| 0.21| 7.88| 5.30 |
| ωB97M-V | 6-31G* | 0.06<sup>a</sup> | 1.78 | 9.91 | 0.35 | 14.64| 51.60 |
[a] mildly affected by error cancellation; [b] heavily affected by error cancellation.
Figure 2.8: General performance of the B3LYP, B3LYP-D3(BJ) and ωB97M-V functionals with the 6-31G*, def2-SVP, and def2-QZVP basis sets for some of the reactions used in experiments 1–3 (Figure 2.5). All values are in kcal mol<sup>-1</sup>.
- The best way to select a functional is through comparison with databases of computational data.
- Basis set errors can be grouped into BSIE and BSSE. Both can be alleviated by using a suitably large basis set, or by using appropriate techniques (CP for BSSE, for example).
- Do not use B3LYP\6-31G* blindly. It cannot be universally applied or trusted.
Experiments 8 and 9: The choice of an integration grid.
With experiment 8, we delve deeper into technical issues underlying virtually any KS-DFT calculation. As explained in Chapter 1, the central quantity in KS-DFT is the exchange-correlation energy, which is usually obtained as the integral of the exchange-correlation energy density over all space (see equations 1.40 and 1.41). This integration is performed on a real-space grid, and most software uses a grid construction algorithm first introduced by Becke in 1988. The integrals are calculated as weighted sums over a finite amount of grid points generally defined on the surface of a sphere. The atomic grids are separated into a radial and angular parts. A grid is usually labeled as “n,m,” where n denotes the number of radial points, and m denotes the number of angular points. For example, the 99,590 grid has 99 radial and 590 angular points, and it corresponds to the ultrafine grid in the Gaussian program. The quality of the integration grid can be selected according to the same principle as the basis set (“the larger, the better”), and once again one has to balance between accuracy and computational cost. The issue of choosing the most appropriate integration grid is often overlooked, and many computational practitioners are unaware of it. Many recent studies have shown that the wrong integration grid not only affects the electronic energies, but also thermodynamic quantities such as the vibrational frequencies and Gibbs free energies. In addition, grid issues might be functional-specific, and it
has been shown that meta-GGAs are more sensitive to the integration grids than GGAs.\textsuperscript{74–77} The Minnesota functionals, for example, require at least a 99,590 grid, while some GGAs like PBE, PBE0 or B3LYP can be considered converged already at the 75,302 level. \textbf{Figure 2.9} shows the performance of the MN15-L functional with the def2-QZVPPD basis set for the dissociation curve of the argon dimer, taken from the RG10\textsuperscript{100} subset of the MGCDB84 database.\textsuperscript{102} It is evident that this erratic behavior is unphysical, and that the many minima and maxima on the energy curve are artifacts. With a large enough grid, the good performance of this functional is recovered, and the artifacts—either in the electronic energies or in the thermodynamic quantities—usually vanish.
Unless specified otherwise, integration grids are set automatically by the computational software, and unfortunately there is no guarantee that the different quantum chemistry programs have the same default for integration grids. For this reason, it is always better to set the integration grid explicitly, as well as to report the grid setting used in a molecular calculation for better reproducibility. As experiments 8 and 9 show, the 99,590 grid is the minimum requirement to guarantee the convergence of the results with respect to the grid size, and it represents a good compromise between accuracy, reliability and cost.
\begin{figure}[h]
\centering
\includegraphics[width=\textwidth]{dissociation_curve.png}
\caption{Dissociation curve of the argon dimer calculated with the MN15-L/def2-QZVPPD level of theory with four different integration grids: SG1; 75,302; 99,590; 175,974. The oscillations in the curves calculated with the coarser grids are unphysical artifacts due to an insufficient grid choice. The energy is reported in kcal mol$^{-1}$, while the distance is in Å.}
\end{figure}
Experiment 10: Do my calculations need stability analysis?
The iterative self-consistent field procedure used in all quantum chemical calculations is used to optimize the coefficients of the atomic basis sets. At the end of an SCF cycle, one obtains the coefficients that correspond to a stationary point of either the HF or KS energy functional. Chemists usually refer to this solution as the converged solution of the SCF procedure. In order to ensure that we really get the lowest energy, we need to characterize the stationary point, and correct it if necessary. To do so, we need to calculate the second derivatives of the wavefunction using a procedure called stability analysis. To lower the energy if an unstable solution is found, there are two different constraints we can lift: we could either change the theoretical framework we are adopting (restricted or unrestricted), or we could use complex orbitals (rather than real ones).\textsuperscript{247,301} We could perform stability analysis more than once if necessary, and the wave function we obtain at the end is said to be stable, and it delivers a lower energy than an unstable solution. For molecules including main-group elements only, the converged solution usually corresponds to the stable, lowest-energy solution as well, but there is no guarantee that this is universally true.\textsuperscript{246} For poorly chosen initial guesses, or for some problematic cases, the SCF could end on an excited state, rather than on the ground state. Stability analysis becomes crucial when studying systems having low-lying excited states, such as transition metal systems. The majority of quantum chemistry programs do not include stability analysis as default procedure at the end of a SCF calculations (it is unfortunately computationally demanding), and a small minority lacks such algorithms altogether. Sometimes, even experienced users can overlook the issue of the stability of the final solution, and extreme caution should be exerted for cases where it is not clear which functionals give the right results.\textsuperscript{302}
Experience 10 uses two low-gap systems, Fe and Ru$^+$, to remind the users of the importance of the stability analysis. These two transition metals come from the 3dAEE8\textsuperscript{112,116,117} and the 4dAEE5\textsuperscript{130} subsets of the Minnesota 2015B database.\textsuperscript{60} Stability analysis is also required when dealing with the six systems in the following experiment 11.
Experiment 11: How to compare calculated and experimental results.
Dissociating a bond is conceptually a very simple task. Taking the H$_2$ molecule as an example, the dissociation of the H–H bond yields two hydrogen atoms. However, even this straightforward process involving the smallest molecule is very hard to describe computationally. The products of this reaction are two radicals, species that are notoriously difficult to describe for single-reference
approaches such as HF and KS-DFT. Since most chemical reactions might include partially- or fully-dissociated bonds, bond dissociation and total atomization energies (BDEs and TAEs respectively) are ubiquitous in computational databases. A bond dissociation energy is the amount of energy needed to dissociate one bond. The total atomization energy of a molecule is the energy required to completely dissociate a molecule into its constituent atoms. For the H\textsubscript{2} molecule described above, these two quantities are identical, but for molecules including three or more atoms, they are substantially different.
BDEs and TAEs are conceptually different from the enthalpies of formation of a molecule, and we should be very careful when comparing calculated and experimental data. This is not due to a limitation of the computational or experimental settings. Instead, these differences should be attributed to the fact that the computational and practical experiments are measuring \textit{different} properties. There are a few corrections that need to be accounted for (zero-point energies, spin-orbit couplings,\ldots) when comparing the experimental and theoretical results.\textsuperscript{122,124,252} A direct comparison is always discouraged, even when performing thermochemistry calculations for calculated enthalpies or Gibbs free energies,\textsuperscript{303} or for the dissociation constants of acids and bases (pK\textsubscript{a} or pK\textsubscript{b}).\textsuperscript{304} In this experiment, the dissociation energies of the C–O bond in \textit{tert}-butyl alcohol and the Fe–Cl bond in FeCl are considered together with the total atomization energies of H\textsubscript{2}, CH\textsubscript{4}, NH\textsubscript{3}, H\textsubscript{2}O and O\textsubscript{3}. The first two molecules come from the SR-MGN-BE107\textsuperscript{18,64,121} and SR-TM-BE17\textsuperscript{18,39,122,124,129} subsets of the Minnesota 2015B database,\textsuperscript{60} while the atomization reactions come from the W4-11 subset\textsuperscript{155} of the GMTKN55 database.\textsuperscript{101}
Experiment 11 concludes the last section of the laboratory notebook. The take-home messages from experiments 8–11 are:
• Integration grids are a subtle but important technical detail. One should use at least a 99,590 grid when dealing with meta-GGAs, especially if they are Minnesota functionals, to avoid any issues.
• Stability analysis is required when dealing with potential low-gap cases, especially transition metals and their complexes.
• Avoid comparing calculated and experimental values. Computational results should always be corrected if compared with experimental data.
Experiment 12: Self-assessment through a transition-metal catalyzed reaction.
As last experiment, we included a palladium-catalyzed reaction (Figure 2.10) from the PdBE2 subset of the Minnesota 2015B database.\textsuperscript{129} The key we provided for the exercise stresses a few points, that can be summarized as follows:
- A choice of a functional should be supported by suggestions coming from the experts in the field. Before setting up a computational experiment, it is good practice to look for benchmark studies dealing with the chemical properties of interest.
- When choosing the basis set, refer to benchmark studies in the literature. We use the Ahlrichs basis sets because they are defined for most of the periodic table, but other choices (Dunning’s or Jensen’s basis sets) are also possible, and equally valid.
- The size of the integration grid should always be reported.
- Since this particular reaction deals with transition metals, stability analysis should be included.
2.5 Conclusions.
In the first chapter of this dissertation, we introduced the ACCDB collection of databases, the software needed to interact with it, as well as a case study involving the application of such databases in chemical education. ACCDB includes 44,931 datapoints of high-level computational data that cover most of the periodic table. It includes the first 36 elements, many transition metals belonging to the 5\textsuperscript{th} and 6\textsuperscript{th} rows of the periodic table, and even the actinides from Th to Cm. ACCDB includes more than 150 different subsets taken from the literature, plus two newly
developed reaction energy databases, called W4-17-RE and MN-RE, and a collection of databases containing TMs, also introduced here for the first time. All the data in ACCDB can be easily handled through a \textit{snakemake} script that creates the input files, submits calculations, collects the results and formats them in different ways. We used some of the subsets from ACCDB to present a set of 12 experiments, which can ideally complement a computational chemistry class, in which many key issues in modern DFT research are analyzed and discussed. Our main goal is to raise awareness about the struggles underlying successful calculations, while at the same time fighting the most common misconceptions around DFT and its applications.
Chapter 3: The development of A Small(er) Collection of DataBases (ASCDB) through statistical analysis.
3.1 Introduction.
Since the early 1980s, several large databases of chemical data have been published and extensively used for the evaluation and parametrization of electronic structure theory methods ranging from semi-empirical\textsuperscript{83–86} to composite methods,\textsuperscript{88–90,305} and later extended to DFT as well.\textsuperscript{60,65,101,102} In the field of approximated $xc$ functionals, three databases rose to prominence given their size, the range of chemical space spanned, and their popularity as benchmarking tools. The General Main-group Thermochemistry, Kinetics and Non-covalent interactions (GMTKN55) database of Goerigk, Grimme and coworkers,\textsuperscript{101} the Main-Group Chemistry DataBase (MGCDB84) of Mardirossian and Head-Gordon,\textsuperscript{102} and the Minnesota 2015B database of Truhlar and coworkers\textsuperscript{60} are the most valuable contribution in this sense. Their importance for the evaluation\textsuperscript{60,101,102,237–240} and parametrization\textsuperscript{11,60,102,104,240} of $xc$ functionals cannot be overstated. Their expansion is the center of the development and assessment of methods with broad applicability in chemistry and physics, as well as the driving force towards improved and more reliable approximations. We recently collected these three major databases, together with many smaller ones, as the core of a larger collection that we named ACCDB (A Collection of Chemistry DataBases). The ACCDB collection contains more than 10,000 geometries and a total of 8,656 unique reference datapoints (6,953 coming from the three major databases listed above), covering a broad chemical space, as well as an open-source repository that is easy to maintain, share, access, and expand. However, the inclusion of as many properties and data as possible resulted in three major drawbacks:
1. there is extensive, and often unclear, overlap between several data;
2. the collection is heavily unbalanced when the number of subsets is taken into account;
3. the computational cost is very high.
The first drawback of the ACCDB collection is directly inherited from its constituents databases.
In all the databases, the classification of the chemical properties has been done *a priori* following chemical intuition, previous examples in the literature, or simply according to the generally accepted definitions. Sometimes, however, it is not easy to separate the chemical space into distinct regions, and some of the labels can be inherently fuzzy.\textsuperscript{306–308} For example, it is relatively straightforward to separate groups of data that represent non-covalent interactions from bond dissociation energy datapoints. While the former is dominated by dispersion interactions (an intermolecular force), the latter deals instead with covalent bonds. The two belong to two separate categories. This classification becomes harder when more complex properties are taken into account. Barrier heights (BH) subsets and datasets including structures dominated by strong correlation effects are an example. The main focus of BH datasets is on transition structures, stationary points on the potential energy surface of a reaction where bonds are partially dissociated. These structures usually need a multi-reference description due to the nature of the interaction, and they are highly likely characterized by a certain degree of strong correlation. In this situation, the labeling of the chemical properties is not as clear as in the first example, and similar other cases are undoubtedly present. Older subsets might also suffer from another inherent bias, especially those derived from back-corrected experimental data. The majority of them is limited to very stable compounds that are easy to handle and analyze, and whose reference data have been collected with high accuracy. This limits the chemical space spanned by those databases, leading to an excess of information on easily accessible compound and a lack of information for more diverse chemical species.\textsuperscript{103} Korth and Grimme introduced the expression “developer’s bias” when they identified this issue, indicating “availability, chemical intuition, and the professional interest in good results” as the possible causes.\textsuperscript{103} They also introduced a procedure, called “mindless benchmarking,” whose goal is to expand the chemical space of the databases through the creation of diversity-oriented datasets.
The second drawback of ACCDB and its core databases MGCDB84, GMTKN55, and Minnesota 2015B lies in the fact that the chemical properties are not balanced in terms of number of datapoints per property. The datasets have a significant size bias. In fact, 48% of the datapoints in ACCDB deal with non-covalent interactions, while only 8.3% are for bond energies. Less than 1% of the datapoints include transition metals (TMs). This is a worrisome under-representation, especially when considering the fact that countless applications of electronic structure theory to TM-containing complexes involve DFT.\textsuperscript{309} This situation is exacerbated even further by the inherent difficulties of obtaining reliable reference data for the different properties.
Although it is extremely expensive, it is relatively straightforward to obtain reliable computational data for molecules including main-group elements only. Most of the data in ACCDB dealing with non-covalent interactions have been obtained with the golden standard of computational chemistry, the coupled cluster with single, double and perturbative triple excitations (CCSD(T)) approach at the complete basis set limit. Despite being one of the most reliable and expensive computational approaches, it is not suitable for transition-metal-containing systems, which usually necessitate multi-reference approaches like multi-configuration self-consistent field (MC-SCF) and CASPT2. These approaches are even more expensive than CCSD(T), and they are not exempt from additional drawbacks (how to properly choose the size of the active space being the most prominent issue). Databases coming from experimental data have their own issues as well, most importantly when the experimental measurement comes with a large uncertainty. All of these aspects make obtaining reference data very challenging, if not impossible.
The last drawback in ACCDB is purely computational in nature. With 10,049 different geometries, each requiring a single-point calculation to obtain the corresponding energy, ACCDB is very expensive. Based on our own experience, the calculations of all the datapoints through a modern quantum chemistry program on a recently acquired machine with a hybrid functional and a quadruple-$\zeta$ basis set requires at least $10^7$ single-core hours.\textsuperscript{79} It would surely be beneficial to reduce the number of calculations needed for the datapoints, especially if this can be done without losing any information provided by the whole collection. Several strategies for the reduction of the number of datapoints of a large chemical database have been used in the past, starting with the pioneering work of Truhlar and coworkers. For example, they reduced the number of datapoints in the BH76 database to just six, the BH6 subset,\textsuperscript{310} or the 17 datapoints of the p-VR17 database to just eight, the R4 and V4 subsets.\textsuperscript{299} The most recent example involves the reduction of the large MGCDB84 and GMTKN55 to smaller datasets, called MG8 and dietGMTKN55.\textsuperscript{236,241} Through the application of regression techniques, Chan reduced the largest database in ACCDB to only 64 datapoints (from 4,985) and 166 geometries (starting from 5,931), thus drastically reducing the computational effort. Gould used a different strategy, and presented three diet versions of GMTKN55. Each includes 30, 100 or 150 datapoints respectively, and a total of 84, 270 or 382 different geometries. Both approaches are statistically solid, and they certainly solve the issue of computational manageability. However, they still inherit the limitations of their respective parents, as they do not contain transition metal systems and they are primarily based on non-covalent interactions. In addition, they do not include atomic energies, either because
they are not included in the parent database (GMTKN55), or because they have been excluded *a priori* from the definition of the new database (MG8).
To overcome all the limitations listed above, we identified four requirements that a modern collection of data used for the development and the evaluation of electronic structure methods should satisfy:
1) it should include various chemical properties, and span the largest accessible chemical space;
2) it should have the data uniquely and impartially divided into subsets;
3) it should have a balanced number of data for each subset. No particular property should be either overemphasized or downplayed;
4) it should be computationally tractable without losing statistical significance.
MGCDB84, GMTKN55, and Minnesota 2015 certainly succeed in addressing point 1. It is also possible to find *ad hoc* solutions to balance the results of the large databases to satisfy the requirement in point 3, for example through weighting schemes. Previous work in the literature helped in addressing point 4, at least for two of the core databases.\textsuperscript{236,241} However, there is currently no database that is specifically designed to meet all four requirements at the same time. We hereby present a new database, obtained using statistical analysis tools borrowed from data science, that will fill this gap. We are confident that our approach will give insight on the validity of fuzzy chemical concepts, and it will help in the definition of new problems that must be addressed in the future.
### 3.2 Reducing the number of datapoints in ACCDB through data science.
The datapoints present in ACCDB readily satisfy the chemical requirement (requirement 1) of being a large and diverse collection of chemical properties. In order to address requirements 2 and 3 in an unbiased way, we need a tool that is capable of grouping and sorting the data without any external inputs. Statistical analyses represent the best solution, but they require a large amount of data to yield meaningful results. We decided to use all the data in the three core databases of ACCDB, and we calculated the mean unsigned errors (MUEs) of all the datapoints using 50 $xc$ functional approximations with basis sets that are close to the complete basis set limit. This generated a population of 347,650 datapoints, which is large enough to enable the usage of data analysis algorithms.
As explained in chapter 2 (see Section 2.2), some subsets in the three core databases overlap with each other for a total of about 900 datapoints. We carefully analyzed these overlaps, and we saw that 90 datapoints coming from Minnesota 2015B are found in either MGCDB84 or GMTKN55. The overlap is more substantial for these two databases, since MGCDB84 incorporates datapoints from GMTKN30,\textsuperscript{183} a previous version of Grimme and Goerigk’s database. A total of 810 datapoints are found in both databases. However, some subsets have updated reference values in GMTKN55. The average difference between the old and the new values—taken over 337 datapoints—is about 0.4 kcal mol\(^{-1}\). In our opinion, this is enough to consider the datapoints different from each other, at least from the statistical analysis point of view, thus bringing down the overlap to only 563 datapoints, or 8% of the total data. After we collected and analyzed the results (\textit{vide infra}), we found that the statistical tool does not favor the old nor the new references.
We selected the 50 functionals in order to cover a heterogeneous mixture of approximations. We considered modern functionals like SCAN,\textsuperscript{19} MN15,\textsuperscript{60} and \(\omega\)B97M-V\textsuperscript{46,66} as well as historically significant approximations like B3LYP\textsuperscript{22,23,25,26} and BP86.\textsuperscript{21,22} We included different flavors of dispersion corrections, like -D3(0),\textsuperscript{145} -D3(BJ),\textsuperscript{261} VV10,\textsuperscript{46} and rVV10,\textsuperscript{48} as well as dispersion uncorrected functionals. We considered functionals that have been parametrized through least-square fittings, like the Minnesota functionals or the B97-family, as well as functionals obtained through constraints satisfaction (PBE, TPSS, SCAN). In addition, we included functionals from the first four rungs of the Jacob’s Ladder of density functional approximations\textsuperscript{29} that combine different ingredients, especially for the hybrid functionals. For this rung, we considered global hybrid generalized gradient approximations (GGAs) or meta-GGAs, as well as range-separate hybrids with different “flavors” or range-separation (HSE-HJS,\textsuperscript{32,311,312} M11,\textsuperscript{242} \(\omega\)B97M-V).\textsuperscript{46,66}
The main goal of this selection was to reduce the developer’s bias by sampling the largest possible functional space. A list of all the functionals considered is given in Table 3.1.
Table 3.1: The 50 \(xc\) functionals used to generate a population of data large enough to be analyzed through statistical tools. The following acronyms have been used: LSDA=Local Spin-Density Approximation; GGA=generalized gradient approximation; NGA=non-separable gradient approximation; mGGA=meta-generalized gradient approximation; mNGA=meta-non-separable gradient approximation; GH=Global hybrid; RSH=range-separated hybrid.
| Name | Form | Dispersion corrected? | Refs | Name | Form | Dispersion corrected? | Refs |
|----------|--------|-----------------------|------|----------|------------|-----------------------|------|
| SVWN5 | LSDA | No | 20,313 | B3PW91 | GH - GGA | No | 22,25,30,31 |
| Name | Form | Dispersion corrected? | Refs |
|---------------|----------|-----------------------|--------|
| BP86 | GGA | No | 21,22 |
| PW91 | GGA | No | 30 |
| B97-D | GGA | D3(0) | 260,261|
| PBE | GGA | No | 32 |
| PBE-D3(BJ) | GGA | D3(BJ) | 32,261 |
| revPBE | GGA | No | 314 |
| revPBE-D3(BJ) | GGA | D3(BJ) | 261,314|
| RPBED | GGA | No | 316 |
| HCTH/407 | GGA | No | 318 |
| BLYP | GGA | No | 22,23 |
| OLYP | GGA | No | 23,319 |
| N12 | NGA | No | 192 |
| SCAN-D3(BJ) | mGGA | D3(BJ) | 19,261 |
| B97M-V | mGGA | VV10 | 45,46 |
| B97M-rV | mGGA | rVV10 | 47,48 |
| τ-HCTH | mGGA | No | 44 |
| Name | Form | Dispersion corrected? | Refs |
|---------------|----------|-----------------------|--------|
| B3LYP | GH - GGA | No | 22,23,25,26 |
| B3LYP-D3(BJ) | GH - GGA | D3(BJ) | 22,23,25,26,261 |
| PBE0 | GH - GGA | No | 55,56 |
| PBE0-D3(BJ) | GH - GGA | D3(BJ) | 55,56,261|
| SOGGA11-X | GH - GGA | No | 64 |
| B97-1 | GH - GGA | No | 33 |
| B97-2 | GH - GGA | No | 315 |
| BMK | GH - mGGA| No | 317 |
| PW6B95 | GH - mGGA| No | 120 |
| PW6B95-D3(BJ) | GH - mGGA| D3(BJ) | 120,261 |
| M05 | GH - mGGA| No | 119 |
| M05-2X | GH - mGGA| No | 121 |
| M06 | GH - mGGA| No | 58 |
| M06-2X | GH - mGGA| No | 58 |
| M08-HX | GH - mGGA| No | 320 |
| τ-HCTHh | GH - mGGA| No | 44 |
| Name | Form | Dispersion corrected? | Refs |
|---------------|----------|-----------------------|-------|
| TPSS | mGGA | No | 35 |
| revTPSS | mGGA | No | 250 |
| M06-L | mGGA | No | 38 |
| M06-L-D3(0) | mGGA | D3(0) | 38,145|
| M11-L | mGGA | No | 39 |
| MN12-L | mNGA | No | 40 |
| MN15-L | mNGA | No | 41 |
| HF | | No | 1–3 |
The only notable exclusion is rung five functionals, i.e. double-hybrids. We were incapable of collecting all the necessary data due to the inherent limitations of the PT2-like correlation for some of the datapoints, either due to unclear basis set convergence,\textsuperscript{321,322} erroneous dissociation limits,\textsuperscript{323} or the divergent behavior of the virtual orbital-dependent terms.\textsuperscript{324,325} The transition-metal-containing subsets of the Minnesota 2015B database are the most notable example in this sense, especially those labeled as “multi-reference.” If we used incomplete results, the cluster analysis would have automatically excluded the double-hybrid functionals, and we would have introduced a bias against them. Instead, we decided to use them for validation and proof of reliability of our database. The data for the MGCDDB84 database have been taken from the supporting information of ref.\textsuperscript{102} while some data relative to GMTKN55 have been taken from Prof. Grimme’s website associated with the work in ref.\textsuperscript{101} We have calculated the remainder of the data—including those for the Minnesota 2015B database—with Q-Chem 5.1.\textsuperscript{256} We used the same basis sets used in the parent articles, which are mostly of quadruple-\(\zeta\) quality. In this way, the results are close to the complete basis set (CBS) limit, but for several molecules in the Minnesota database, a triple-\(\zeta\) basis set was used, as detailed in Table 3.2. We performed
stability analysis for all the converged solutions, allowing re-optimization to the most stable (symmetry-broken) one when necessary. In all cases (but HF), we used a Lebedev grid with 99 radial and 590 angular points for the integration of the $xc$ functional. The coarser SG-1 grid\textsuperscript{326} was used for the integration of the non-local (r)VV10 contribution for functionals that include it. The entirety of the statistical analysis has been performed with the JMP Pro 14 program.\textsuperscript{327}
Table 3.2: Basis sets used in the calculation of the datapoints for the three core databases of the ACCDB collection. MN refers to the Minnesota 2015B database.
| Data-point | Basis set |
|-----------------------------|------------------------------------------------|
| All MGCD84 | def2-QZVPPD\textsuperscript{251} |
| All GMTKN55 | def2-QZVP\textsuperscript{251} |
| NCCE30_16 | MG3S\textsuperscript{118,330,331} |
| PA8_2 | MG3S\textsuperscript{118,330,331} |
| SR-MGN-BE107_76 | MG3S\textsuperscript{118,330,331} |
| NCCE30_8 | MG3S\textsuperscript{118,330,331} |
| 4pIsoE4_3 | cc-pVQZ\textsuperscript{333} |
| MR-TM-BE13_13 | aug-cc-pwCVTZ (metal)\textsuperscript{334,335}|
| | aug-cc-pVTZ (non-metal)\textsuperscript{273,336}|
| IP23_20 | cc-pVTZ-DK\textsuperscript{249} |
| SR-TM-BE17_1 | def2-TZVP(metal)\textsuperscript{251} |
| | ma-TZVP (non-metal)\textsuperscript{251,337} |
| SR-TM-BE17_9 | def2-TZVP\textsuperscript{251} |
| 4dAEE5_4 | cc-pVTZ-DK\textsuperscript{249} |
| SR-MGN-BE107_43 | MG3S\textsuperscript{118,330,331} |
| ABDE13_6 | ma-TZVP\textsuperscript{251,337} |
| MR-MGN-BE17_7 | MG3S\textsuperscript{118,330,331} |
| HTBH38_3 | MG3S\textsuperscript{118,330,331} |
| MR-MGM-BE4_1 | cc-pCVQZ (metal)\textsuperscript{276,328} |
| | aug-cc-pCVQZ (non-metal)\textsuperscript{329} |
| NHTBH38_10 | MG3S\textsuperscript{118,330,331} |
| NHTBH38_12 | MG3S\textsuperscript{118,330,331} |
| pTC13_13 | MG3S\textsuperscript{118,330,331} |
| HC7_6 | 6-311+G(2df,2p)\textsuperscript{332} |
| MR-MGM-BE4_3 | cc-pCVQZ (metal)\textsuperscript{276,328} |
| | aug-cc-pCVQZ (non-metal) |
| IP23_14 | cc-pVTZ-DK\textsuperscript{334,335} |
| AE17_6 | cc-pCVQZ\textsuperscript{329} |
| MR-TM-BE13_12 | aug-cc-pwCVTZ (metal)\textsuperscript{334,335}|
| | aug-cc-pVTZ (non-metal)\textsuperscript{273,336}|
| MR-MGN-BE17_15 | MG3S\textsuperscript{118,330,331} |
| MR-TMD-BE3_1 | def2-TZVP\textsuperscript{251} |
| MR-TMD-BE3_2 | def2-TZVP\textsuperscript{251} |
| MR-TMD-BE3_3 | def2-TZVP\textsuperscript{251} |
| SR-TM-BE17_7 | def2-TZVP\textsuperscript{251} |
| SR-MGN-BE107_12 | MG3S\textsuperscript{118,330,331} |
| DC9_6 | MG3S\textsuperscript{118,330,331} |
| Data-point | Basis set |
|------------|-----------|
| SR-MGN-BE107_11 | MG3S$^{118, 330, 331}$ |
**Step 1: Grouping the datapoints together in an unbiased manner.**
Cluster analysis is the first technique that we applied to the data we collected. Its main goal is to divide the data into different groups—or clusters—without previous knowledge of the nature of the datapoints. We imposed no constraints or limitations on the number of clusters that the analysis could output, nor that could be grouped together. The starting point is the extreme situation where all the data are their own cluster, i.e. 347,650 different groups. The analysis proceeds by locating the two clusters with the shortest Euclidean distance in the hyper-space of all functionals, replacing them with a new cluster of two datapoints located in the mean distance between the two original points. The procedure is repeated until the number of clusters is sufficiently small. The whole process is accompanied by a cost function that can be used as indication of the clusters’ similarity. When two clusters that are far away from each other are merged together, the cost function grows indiscriminately, indicating that the procedure is merging clusters that are too different, and independent of each other. In the opposite case, i.e. when two similar clusters are merged together, there is only a small penalty associated with this action. From our analysis, the stopping point emerged quite clearly at 16 clusters. Any further reduction was too costly in terms of the associated cost function.
**Step 2: Reducing the number of datapoints.**
After obtaining the 16 clusters from cluster analysis, the goal became to reduce the number of datapoints in a statistically significant manner. Even after the first step, the number of datapoints in the collection did not change. The next step we considered was the application of regression techniques to perform the reduction. Following the examples set by Chan$^{236}$ and Gould$^{241}$ we explored the use of the least absolute shrinkage and selection operator (LASSO)$^{338}$ as well as stepwise regression algorithms$^{339}$ for the overall reduction of the data within each cluster. In the end, we used the method that allowed us to maintain a regression coefficient, $R^2$, $\geq 0.99$ for each cluster. The relationship between the clusters with the reduced number of datapoints and the corresponding original one is linear. Through a simple regression formula, it is possible to recover the performance of the full cluster of datapoints.
We obtained a total of 200 datapoints in the reduced clusters. 102 of the datapoints come from the MGCD84 database, 67 come from GMTKN55, and 31 come from Minnesota 2015B. This division reflects the original size of the three major databases, and they can all be used to calculate the estimated MUE (eMUE) of each individual parent. This step is possible using a set of linear regression formulas containing 57 coefficients. The 200 datapoints in the reduced database represent less than 3% of the total original data points. The regression formulas retain a striking statistical significance, with values of $R^2 \geq 0.92$. The 16 resulting clusters are summarized in Figure 3.1. The regression formulas are reported in Appendix A (Figure A.1) at the end of this dissertation.
| Cluster | Total data-points | Reduced data-points | Name of subset | Average $|\Delta E|$ | Average MUE |
|---------|-------------------|---------------------|---------------------------------------------------------------------------------|----------------------|-------------|
| 1 | 2,582 | 15 | Non-covalent: A (NCA15) | 17.63 | 0.43 |
| 2 | 2,295 | 21 | Non-covalent: B (NCB21) | 94.78 | 2.10 |
| 3 | 610 | 15 | Non-covalent: cluster (NCC21) | 42.45 | 4.37 |
| 4 | 18 | 3 | Non-covalent: water (NCW3) | 205.33 | 26.16 |
| 5 | 374 | 19 | Thermochemistry: atomization and reaction energies (TARE19) | 116.83 | 3.97 |
| 6 | 531 | 20 | Thermochemistry: barrier heights A (TBHA20) | 165.88 | 5.37 |
| 7 | 139 | 16 | Thermochemistry: barrier heights B (TBHB16) | 133.84 | 9.95 |
| 8 | 22 | 5 | Thermochemistry: hydrocarbons reactions (THR5) | 2204.76 | 21.43 |
| 9 | 242 | 15 | Non-local effects: mixed A (NLMA15) | 1701.33 | 7.24 |
| 10 | 34 | 7 | Non-local effects: mixed B (NLMB7) | 348.46 | 10.49 |
| 11 | 15 | 11 | Non-local effects: multi-reference (NLMR11) | 147.24 | 24.57 |
| 12 | 33 | 9 | Non-local effects: self-interaction-error (NLSIE9) | 123.97 | 18.92 |
| 13 | 22 | 8 | Unbiased calculations: mindless benchmarks A (UMBAA8) | 445.17 | 30.92 |
| 14 | 8 | 5 | Unbiased calculations: mindless benchmarks B (UMBBS5) | 352.91 | 36.38 |
| 15 | 10 | 7 | Unbiased calculations: mindless benchmarks C (UMBCC7) | 726.35 | 53.06 |
| 16 | 18 | 18 | Unbiased calculations: atomic energies per electron (UAE18) | 8201.08 | 1.77 |
| **Total** | **6,953** | **200** | | **15028.01** | |
Figure 3.1: Summary of the clusters in the ASCDB database with the “a posteriori” assignment of chemical properties. The average relative absolute energies $|\Delta E|$, and the average MUE for the 50 functionals, are also reported in the last two columns (in kcal mol$^{-1}$).
**Step 3: The a posteriori analysis of the new database and the definition of ASCDB.**
Through the application of cluster analysis and regression techniques in points 1 and 2, we introduced a statistically significant database that is substantially smaller than the parent collection, and we reported the regression formulas needed to successfully use it to recover the performance of the larger collection. We stress here one more time that the division into the different clusters, as well as their reduction have been performed without any biases or chemical assumptions. We now turn to the analysis of the data in each cluster more closely. Our goal is to check whether there is any chemical significance that justifies the division performed by the statistical tools or not.
To our surprise, the classification of each cluster according to chemically meaningful labels was straightforward. We will use cluster 1 as an example. The first cluster is composed of 2,582
datapoints, and it is the largest identified by the cluster analysis procedure. The data come from the following subsets of the parent databases: A21x12,\textsuperscript{136} BzDC215,\textsuperscript{194} RG10\textsuperscript{109} and S66x8\textsuperscript{167} from MGCDB84, Amino20x4,\textsuperscript{170} BUT14DIOL,\textsuperscript{191} HAL59,\textsuperscript{151,152} MCONF,\textsuperscript{205} and SCONF\textsuperscript{133,175} from GMTKN55, and NCCE30, NGDWI21, and a small number from SR-MGN-BE107\textsuperscript{18,64,121} from Minnesota 2015B. The majority of datapoints in this cluster can be easily attributed to the class of non-covalent interactions, with only a limited amount of exceptions. The presence of these (apparent) outliers can be explained considering that they behave as non-covalent interactions from the functionals’ point-of-view. In other words, functionals that have a good overall performance for non-covalent interactions provide a good description of such cases as well. This is not surprising, as it is common during the optimization of a new $xc$ functional to find that a new set of parameters might improve the performance not only for property A, but also for molecules dominated by property B. At the same time, this could also lead to the systematic worsening of some other property C, even when no datapoints for property B or C are included in the optimization function. There might be a correlation on a density standpoint between properties that seem chemically disconnected.
A similar \textit{a posteriori} analysis can be repeated for the other 15 clusters, yielding the following results:
- Three more clusters (2, 3, and 4) contain mostly non-covalent interaction datapoints;
- Four clusters (5–8) contain mostly thermochemical data;
- Four other clusters (9–12) contain systems that are dominated by strong-correlation effects;
- Three clusters (13–15) contain data from the “mindless benchmark” subset of the GMTKN55 database;
- The last cluster (16) contains the atomic energies of the first 18 elements (H–Ar), which we decided to report on a per-electron basis to avoid biases towards the heavier elements.
We used labels such as A, B, and C to indicate one or more clusters that describe the same property, especially when it is difficult to differentiate between clusters that belong to the same group. In clearer cases, we decided to adopt a more descriptive label. In the non-covalent group, for example, we have the “non-covalent A” and “non-covalent B” subsets that are mixed cases, with both dimers and trimers. In the “non-covalent clusters” subset, instead, we only have clusters (in the chemical sense of the terms, as in Mardirossian and Head-Gordon’s work)\textsuperscript{66} of
molecules. In the “non-covalent water” subset, we only have clusters of water molecules, hence the use of this label. The subsets labeled “thermochemistry” include data for “atomization and reaction energies” in cluster 5, “barrier heights A” and “barrier heights B” (using the labels A and B in a similar way to clusters 1 and 2), and “alkane reactions” (cluster 8). The third group, labeled “non-local effects,” mainly deals with systems that suffer from errors in the description of non-local effects either in the exchange, i.e. cases with significant self-interaction error (SIE, cluster 11), or in the correlation, i.e. multi-reference (MR) species as in cluster 12. Clusters 9 and 10 are harder to classify, and it is not clear (at least for us) which effect is dominating. To distinguish them, we decided to use the label “mixed A” and “mixed B.” We assigned the label MR to cluster 11 as it includes species that are not properly described by a single-determinant treatment as the one used for HF or KS-DFT. Species having a small HOMO–LUMO gap belong to this category. In general, \( xc \) functionals that contain a high percentage of non-local exact (“HF-like”) exchange fail badly for such systems. The label “SIE” used for cluster 12 denotes systems that require a non-local exchange functional for their accurate description. For these systems the HF method gives reasonable results, and so do \( xc \) functionals with a high percentage of exact exchange. Instead, local functionals perform particularly badly. The last group of clusters (13–16) has been called “unbiased calculations.” It includes data that has been developed using a procedure (“mindless benchmarking”) that does not suffer from chemical biases (clusters 13–15), and cluster 16 contains atomic energies. The data in clusters 13–15 come from the MB16-43 subset of the GMTKN55 database,\(^{101} \) which in turn is based on the procedure developed by Korth and Grimme in 2009.\(^{103} \) For this reason, we decided to keep the original name for these clusters. Because of their random nature, it is difficult to classify these molecules according to a chemical rationale, and it is interesting to see that the cluster analysis algorithm divided these molecules in three different groups (despite belonging to the same subset in the original publication). This suggests that these molecules span a chemical space that is substantially unexplored, and only highly robust approximations can perform acceptably for these systems. These datapoints contain decomposition reactions of molecules including the first 16 elements into hydrides, or simple diatomic molecules based on the atomic composition of the system taken into account. For example, one of the reactions included in cluster 13 has the stoichiometry (equation 3.1 below)
\[
\text{H}_2\text{O}_2 + \text{H}_2\text{O} = 2\text{H}_2\text{O}
\]
Given the nature of these reactions, it is highly unlikely that error cancellation plays a role, as the reactants and products are extremely different. This is reflected in the largest average MUE among the clusters, also found for cluster 16. In fact, atomic energies are by definition exempt from error cancellation, which further motivated us in choosing the “unbiased calculations” label for this group of clusters.
At the end of this analysis, we realized that the 200 datapoints, grouped into four groups of four clusters, for a total of 16, are a reasonably small collection of diverse chemical properties. In other words, the smaller collection we created can be treated as a standalone database, which we named “ASCDB: A Smaller Chemistry DataBase” (when compared with the larger ACCDB). ASCDB meets all the requirements we listed for a useful chemistry database. In fact, it spans a broad chemical space, and its datapoints are almost equally divided among the four different properties (non-covalent, thermochemistry, non-local effects, unbiased calculations) that we introduced earlier. The non-covalent subsets have a total of 60 datapoints, and so do the thermochemistry subsets. The non-local effects property includes 42 datapoints, and the unbiased calculations group has 32 datapoints. In addition, the cluster analysis algorithm selected a heterogeneous spectrum of molecular sizes, ranging from atoms to small and large systems with up to 80 atoms. The majority of systems ranges between 1 and 30 atoms, extensively covering the range of molecular sizes studied in routine calculations, but molecules including more than 30 atoms represent 6% of the database. As a comparison, the number of molecules with more than 30 atoms represents 11% of GMTKN55, about 7% for MGCD84, less than 1% for the Minnesota 2015B database, about 9% of the MG8 database, 6% of the dietGMTKN55-30 database, and 8% of the dietGMTKN55-100 and dietGMTKN55-150 respectively. ASCDB offers even more advantages when compared to the other four small databases. In fact, it is the only one that includes transition metals and atomic energies, while at the same time offering a balanced description of all the chemical properties included. The vast majority of MG8 or the dietGMTKN55 databases are in fact focused on non-covalent interactions, which represent 42% of MG8 and dietGMTKN55-150. ASCDB requires 350 single-point calculations to obtain all the reference energies for all datapoints, and it is the largest among the “diet” databases. However, based on our own benchmarks obtained
at the HF/3-21G level, it is the second least expensive database, making it one of the most computationally affordable (only the smallest database, dietGMTKN55-30, outperforms ASCDB in terms of computational time). These comparisons are visually summarized in Figure 3.2.
| Database | Reliable? | Transition metals? | Atomic Energies? | Size (# of structures) | Computational time (s) | Balanced? |
|-------------------|-----------|--------------------|------------------|------------------------|------------------------|-----------|
| MG8 | ✓ | X | X | 140 | 723 | X |
| dietGMTKN55-30 | ✓ | X | X | 82 | 150 | X |
| dietGMTKN55-100 | ✓ | X | X | 238 | 837 | X |
| dietGMTKN55-150 | ✓ | X | X | 336 | 2772 | X |
| ASCDB | ✓ | ✓ | ✓ | 350 | 537 | ✓ |
Figure 3.2: Visual summary of the properties of the small databases MG8, dietGMTKN55-30, dietGMTKN55-100, dietGMTKN55-150, and ASCDB.
Before recommending ASCDB for chemical applications, we show some of its most important results, and we validate it using methodologies that have not been used in its definition, such as different $xc$ functionals, basis sets, and statistical indicators.
### 3.3 Testing ASCDB: Results and discussion.
The first step in ASCDB’s testing phase is the assessment of how much information is lost with respect to the three parent databases. Our interest is in evaluating the performance of ASCDB using both the estimated MUEs (eMUEs), i.e. the mean unsigned errors calculated using the regression formulas and the 200 datapoints in ASCDB, and the MUEs. We will compare both indicators with the MUEs calculated when considering the original 6,953 datapoints. The visual results are reported in Figure 3.3 and 3.4, which show a striking correlation in all cases. Figure 3.3 shows the data calculated using ASCDB only, while Figure 3.4 shows the results calculated through the regression formulas. These latter plots show the strength of the data-science techniques we used to develop ASCDB because the points align very well on the diagonal. The correlation coefficients we got are excellent in all cases, with an $R^2$ value always greater than 0.900.
The best advantage of ASCDB, even when compared with its direct competitors MG8 and dietGMTKN55, is that it provides information on all the parent databases simultaneously and at a fraction of the cost. For example, while only the Minnesota 2015B contains transition metals, ASCDB can provide information on MGCDB84 and GMTKN55 as well. If one needs more information on a specific property, then it is always possible to run additional calculations using the subsets in the parent databases, or the parent databases in their entirety.
Figure 3.3: Correlation plots between the MUE calculated on ASCDB and the total MUE calculated using all the datapoints from the three major databases (top-left panel, black diamonds), the MUE calculated with GMTKN55 (top-right panel, red squares), MGCDB84 (bottom-left panel, green triangles), and Minnesota 2015B (bottom-right panel, green triangles). Units on the axes are kcal mol$^{-1}$.
Figure 3.4: Correlation plots between the estimated MUEs (eMUEs) calculated using the regression formulas and the datapoints in ASCDB and the MUEs calculated with the parent databases GMTKN55 (red squares, left panel), MGCDB84 (blue circles, middle panel) and Minnesota 2015B (green triangles, right panel). Units on the axes are kcal mol$^{-1}$.
Validation.
The results shown in the previous figures demonstrate the utility of ASCDB as a proxy to get access to the results of the largest databases in ACCDB. However, we are interested in its performance as standalone database for the evaluation of electronic structure theory methods. In other words, we want to evaluate ASCDB independently of the parent databases or the ACCDB collection.
Our first validation is with respect to the choice of basis set. We obtained the data for cluster analysis employing the largest basis sets we could afford (mostly quadruple-\(\zeta\) basis sets, with triple-\(\zeta\) basis sets used occasionally as seen in Table 3.2). We should keep in mind that some unwanted sources of error might still be present even when performing calculations as close as possible to the basis set limit. Possible sources of errors might include the triple-\(\zeta\) basis sets we used for some of the largest or most problematic cases, the residual incompleteness error found even at the quadruple-\(\zeta\) level, or the intrinsic inability of some functionals to reach the basis set limit. To validate the stability of our database with respect to the basis set size, we chose three basis sets not included in the definition of the original data as test cases. Since ASCDB was defined using the MUEs, we decided to include a different statistical indicator, namely the root-mean-square error (RMSE) to remove any bias. We calculated it for ASCDB employing three basis sets, namely 6-31G*, def2-SVPD, and def-TZVPPD, and three functionals, PBE-D3(BJ), B3LYP-D3(BJ), and \(\omega\)B97M-V. We considered the four chemical categories of the parent databases—non-covalent interactions (NC, most data coming from MGCDB84), isomerization energies (ISO, most data coming from GMTKN55), thermochemistry (TC, data equally divided), and barrier heights (BH, mostly coming from Minnesota 2015B)—for which RMSEs are already reported in the literature,\(^{66}\) and we compared the calculated and estimated RMSEs (eRMSEs). The calculated and estimated data are reported in Table 3.3, and a visual representation can be found in Figures A.2—A.4 in Appendix A. The trends in MUEs of the parent databases are very well reflected in the eRMSEs obtained through the ASCDB results, despite a limited amount of outliers (only one is clear in Table 3.3 for the \(\omega\)B97M-V/def2-TZVPPD results). The results of Table 3.3 are enough to establish that the stability of ASCDB with respect to changes in the basis set size is similar to that of the parent databases.
Table 3.3: Estimated RMSEs from ASCDB compared to the calculated RMSEs (in parenthesis) for the main subsets of MGCD84 for three $xc$ functionals and three basis sets not used in the data analysis. All data are in kcal mol$^{-1}$.
| Functional | Basis set | NC | ISO | TC | BH |
|----------------|-------------|-------|-------|-------|-------|
| PBE-D3(BJ) | 6-31G* | 7.38 | 1.43 | 14.33 | 8.26 |
| | | (8.35)| (2.77)| (14.92)| (15.88)|
| | def2-SVPD | 1.63 | 1.16 | 13.72 | 10.35 |
| | | (2.90)| (1.97)| (15.23)| (11.28)|
| | def2-TZVPPD | 1.23 | 1.57 | 11.61 | 10.14 |
| | | (1.27)| (1.48)| (11.42)| (10.09)|
| B3LYP-D3(BJ) | 6-31G* | 6.77 | 1.58 | 6.37 | 5.02 |
| | | (7.47)| (11.96)| (9.45)| (10.96)|
| | def2-SVPD | 2.41 | 1.30 | 6.72 | 7.78 |
| | | (2.46)| (1.83)| (8.49)| (6.29)|
| | def2-TZVPPD | 0.77 | 1.46 | 4.32 | 6.75 |
| | | (0.75)| (1.84)| (4.32)| (5.71)|
| $\omega$B97M-V | 6-31G* | 10.55 | 2.74 | 12.83 | 2.80 |
| | | (6.82)| (2.07)| (9.63)| (8.84)|
| | def2-SVPD | 2.15 | 1.09 | 9.31 | 4.09 |
| | | (2.14)| (0.87)| (9.65)| (3.21)|
| | def2-TZVPPD | 0.31 | 0.57 | 2.79 | 3.56 |
| | | (0.30)| (0.61)| (3.04)| (1.78)|
A second validation of the robustness of the ASCDB database comes from the expansion of the functional space when double-hybrids functionals are considered. As explained above, we originally excluded rung-five functionals because it was not possible to obtain reliable PT2-like correlation energies for several low-gap, MR datapoints coming from the Minnesota 2015B database. We can however use double-hybrid functionals as a validation tool between the ASCDB and the GMTKN55 databases, since the latter does not include TMs and it has been used for the extensive assessment of this kind of functionals.\cite{101,183,238,240} As first step, we selected the double-hybrid data from ref.\cite{101} which include a total of 21 functionals with different flavors of dispersion corrections, and we calculated the correlation plots between the original MUEs and the estimated MUEs from ASCDB. The results are visually reported in Figure 3.5. We obtained a very good correlation ($R^2=0.94$) and a small systematic overestimation of about 0.2 kcal mol$^{-1}$.
We also decided to test two of the most popular double-hybrid functionals—B2-PLYP-D3(BJ)\textsuperscript{67,183} and DSD-PBEP86-D3(BJ)\textsuperscript{69}—even further. We performed calculations with these two functionals to include all the datapoints in ASCDB without using the correlation formulas, and the results are collected in Table 3.4. The corresponding correlation plot is instead reported in Figure 3.6.
Table 3.4: Comparison of the MUEs between significant chemical properties of ASCDB and GMTKN55 using doubly hybrid functionals. All data are in kcal mol\textsuperscript{-1}.
| Chemical property | B2-PLYP-D3(BJ) | DSD-PBEP86-D3(BJ) |
|------------------------------------------|---------------|-------------------|
| Non-covalent (ASCDB) | 1.52 | 1.28 |
| Total non-covalent (GMTKN55) | 0.34 | 0.36 |
| Thermochemistry (ASCDB) | 3.44 | 3.01 |
| Basic properties (GMTKN55) | 2.17 | 2.00 |
| Non-locality-error-dominated (ASCDB) | 5.22 | 5.77 |
| W4-11MR (GMTKN55) | 3.04 | 6.55 |
| Unbiased calculations (ASCDB) | 9.79 | 5.05 |
| Mindless benchmark (GMTKN55) | 16.62 | 6.46 |
| Total MUE (ASCDB) | 4.60 | 3.51 |
| Total MUE (GMTKN55) | 1.79 | 1.29 |
The analysis of Table 3.4 and Figure 3.6 shows a direct correlation between the four areas of ASCDB (non-covalent interactions, thermochemistry, non-local effects, and unbiased calculations) and a corresponding property in GMTKN55 (total non-covalent interactions, basic properties, multi-reference cases in the W4-11 dataset, and mindless benchmarks). These results validate our database as a robust tool, since none of these methods are used in the definition of the database itself.
As additional validation, we considered other statistical indicators besides the mean-unsigned error. Figure 3.7 shows the correlations between the root-mean-square errors calculated using ASCDB and the parent databases. These plots unravel reasonably good correlations, with the exception of a few outliers that can be easily identified and excluded from the trends.
The last statistical indicator we considered is the mean signed error, or MSE. The MSE is used to evaluate the systematic over- or underestimation trends when a specific property is considered.
For example, a method that systematically underestimates the reference data will have MUEs and MSEs very close in magnitude, but with opposite signs. On the other hand, methods that systematically overestimate the reference data will have comparable MUEs and MSEs both in magnitude and sign. Cases with no systematic over- or underestimation will instead have MSEs close to zero, independently of the value of the MUEs. The striking majority of cases where there is either systematic over- or underestimation of the reference properties involves three chemical categories: non-covalent interactions (NC), non-local effects (NL), and unbiased calculations (UC). We report a comparison between the results obtained for two relevant groups of properties of the ASCDB database and those obtained from the parent databases (Figure 3.8). We used 10 functionals that are well-known for having systematic errors for the cases considered.\textsuperscript{60,101,102} The plot involving UC does not provide any additional information when compared with the mindless benchmark subset of GMTKN55, since the majority of the data comes from MB16-43, and the MSEs are overlapping. All functionals that underestimate a property using the parent database will also present a negative sign of the corresponding subsets of ASCDB, with magnitudes of the errors that are also very comparable. At the same time, all functionals that systematically overestimate the reference data have a positive MSE for the corresponding ASCDB subset, with magnitudes that are also equally well correlated.
Overall, our validation process shows a very good correspondence between the results obtained with ASCDB and those obtained from the parent databases. We established the validity of ASCDB as a standalone tool for the evaluation of electronic structure theory methods independently of the basis sets, methods, and statistical indicators used.
3.4 Conclusions and future applications.
In the previous paragraphs, we introduced the first database that is:
- representative of a large set of chemical properties;
- unbiased, since the subdivision of the subsets is performed *a posteriori* and after the application of unbiased methodologies;
- well-balanced, since the datasets or groups of datasets have comparable size;
- computationally affordable (*i.e.* it has a small number of data).
This database is called ASCDB (A Smaller Chemistry DataBase) and it represents the first database for the parametrization and evaluation of electronic structure theory methods that has been introduced using statistical analysis tools, namely cluster analysis and regression techniques. ASCDB is representative of the three largest databases (MGCDB84, GMTKN55, Minnesota 2015B) in the ACCDB collection. It contains 200 datapoints, for a total of 350 geometries, that are impartially divided into 16 subsets and grouped into four different chemical categories: non-covalent interactions, thermochemistry, non-local effects, and unbiased calculations. It can be also used to estimate two statistical indicators (MUEs and RMSEs) for the parent databases with a remarkable accuracy and with minimal loss of information.
When compared with the parent databases, or with their reduced versions (MG8 or di-etGMTKN55), our database offers the advantage of being computationally affordable, and it offers two kinds of (complementary) information: a) a statistical estimate of the performance of the method against much larger databases (through regression formulas); b) a new unbiased, independent and well-balanced evaluation via its subsets. The reliability of ASCDB has been established after a thorough analysis involving basis sets, methods and statistical indicators not used in its definition. We recommend using ASCDB as a first tool for the evaluation and parametrization of electronic structure theory methods. Larger collections such as GMTKN55, MGCDB84, and Minnesota 2015B can subsequently be used when further validation or more granular data are needed.
Chapter 4: The definition of CLB18, a new database for geometries, and insights for \(xc\) functional developers.
4.1 Introduction.
Density functional theory is the workhorse of electronic structure calculations. Due to its good accuracy-to-cost ratio, it is currently employed for countless routine applications in chemistry, physics and material sciences. The computational protocols usually involve procedures such as single-point calculations, geometry optimizations, and frequency calculations. They are all used for the rationalization, justification and prediction of observed properties, most notably in organic and inorganic chemistry. Organic molecules with long covalent carbon-carbon bonds have been fascinating chemists for more than forty years. The crystal structure of 1,1,1,2,2,2-hexakis(3,5-di-tert-butylphenyl)ethane (CLB18_1 in Table 4.1 below) has been reported in 1986 for the first time.\(^{340}\) It shows a C—C bond length of 1.67 Å, which is 0.13 Å longer than the one reported for ethane (1.54 Å). Many other structures with unusually long C—C bonds have been successfully synthesized and characterized through X-ray diffraction in the subsequent years.\(^{341-344}\) For these compounds, the bond distance ranges between 1.60 Å and 1.99 Å. These molecules have also attracted the attention of the computational community as well, with DFT at the center of many applications. For example, TPSS-D3(BJ) has been applied to understand the stability of 1,1,1,2,2,2-hexakis(3,5-di-tert-butylphenyl)ethane and the instability of its analogue hexaphenylethane.\(^{345}\) In other studies, hybrid exchange-correlation (\(xc\)) functional approximations have successfully reproduced the experimental bond lengths of the C—C bond in these molecules.\(^{341,342,344,346}\)
In this Chapter, we introduce a new database of molecular geometries called Carbon Long Bond 18 (CLB18), composed of 18 structures with one long C—C bond. We will use it to test and evaluate the performance of several low-cost methods commonly used for geometry optimization of medium and large molecules. We establish a connection with the 34 transition states structures from the BH76 database of barrier heights,\(^{18,177,178,347}\) and we show that CLB18 can be used to obtain information not included in any other BH database. CLB18 thus represents a valuable
addition to the $xc$ functional development toolbox.
4.2 Structure of the CLB18 database.
The CLB18 database is composed of 18 minimum-energy structures. The reference bond distance for the long C–C bond in each molecule is taken from the experimental crystal structure. In fifteen cases, they are taken from the Cambridge Structural Database (CSD), while for the remaining three they come from ref..343 The shortest C–C bond length is 1.627 Å (in CLB18_3), while the longest is 1.987 Å (in CLB18_18). The average C–C bond length is 1.766 Å. The experimental accuracy of the first three structures (CLB18_1, CLB18_2, CLB18_3) is around $10^{-3}$ Å, while for all others is around $10^{-4}$ Å. All the structures and the reference C–C bond lengths are reported in Table 4.1. The molecules in CLB18 can also be divided into three different groups of six molecules according to their chemical composition. The first group includes hydrocarbons, the second group includes halogen-substituted hydrocarbons, and the third group contains differently substituted carboranes.
Table 4.1: Molecules included in the CLB18 database. The reference bond lengths are listed in Å, and they refer to the bond highlighted in red in the corresponding formula.
| Datapoint | Structure’s formula & CSD Identifier | C–C bond length, Å | Datapoint | Structure’s formula & CSD Identifier | C–C bond length, Å |
|-----------|--------------------------------------|-------------------|-----------|--------------------------------------|-------------------|
| CLB18_1 |  | 1.67(3) | CLB18_10 |  | 1.733(6) |
| CLB18_2 |  | 1.70(4) | CLB18_11 |  | 1.743(3) |
| CLB18_3 |  | 1.71(3) | CLB18_12 |  | 1.8029 |
| CLB18_4 |  | 1.723(4) | CLB18_13 |  | 1.627(3) |
| Datapoint | Structure’s formula & CSD Identifier$^a$ | C–C bond length, Å |
|-----------|------------------------------------------|-------------------|
| CLB18_5 |  | 1.770(8) |
| CLB18_6 |  | 1.806(5) |
| CLB18_7 |  | 1.696(1) |
| CLB18_8 |  | 1.711(9) |
| CLB18_9 |  | 1.723(9) |
| CLB18_14 |  | 1.719(9) |
| CLB18_15 |  | 1.829(7) |
| CLB18_16 |  | 1.893(4) |
| CLB18_17 |  | 1.931(5) |
| CLB18_18 |  | 1.987(0) |
$^a$For the datapoints CLB18_4, CLB18_5, and CLB18_6, the CSD identifier is not available (see text and ref.).$^{343}$
### 4.3 Computational details.
To validate CLB18 as useful database for density functional theory and semi-empirical methods, we used 26 approximations to perform geometry optimizations for all the structures listed in Table 4.1. We tested seven generalized gradient approximation (GGA) functionals, namely BLYP,$^{22,23}$ BLYP-D3(BJ),$^{22,23,261}$ BP86,$^{21,22}$ BP86-D3(BJ),$^{21,22,261}$ PBE,$^{32}$ PBE-D3(BJ),$^{32,261}$ B97-D3(BJ),$^{260,261}$ one meta-GGA functional (TPSS-D3(BJ),$^{35,261}$), four hybrid GGA functionals, B3LYP,$^{22,23,25,26}$ B3LYP-D3(BJ),$^{22,23,25,26,261}$ PBE0,$^{32,55,56}$ PBE0-D3(BJ),$^{32,55,56,261}$, three
hybrid meta-GGA functionals, (M06-2X,\textsuperscript{58} M06-2X-D3(0),\textsuperscript{58,263} TPSH-D3(BJ)\textsuperscript{57,261}), two range-separated hybrid GGA functionals (CAM-B3LYP\textsuperscript{61} and CAM-B3LYP-D3(BJ)\textsuperscript{61,261}), and the Hartree-Fock (HF) method.\textsuperscript{1–3} In addition, we tested four semi-empirical methods (AM1,\textsuperscript{86} PM3,\textsuperscript{348,349} PM6,\textsuperscript{350} PM7\textsuperscript{351}). For these approximations, we used the Gaussian program.\textsuperscript{255} We tested also two low-cost methods (HF-3c\textsuperscript{286} and PBEh-3c\textsuperscript{352}) using the Q-Chem program,\textsuperscript{256} and two tight-binding approximations (GFN-xTB1\textsuperscript{353} and GFN-xTB2\textsuperscript{354}) with the xTB program.\textsuperscript{355}
A visual summary is reported in Table 4.2 below. For all functionals, we used the 6-31G* basis set\textsuperscript{266,267,269,270,272,356,357} and the fine integration grid (ultrafine for meta-GGA functionals). We adopted this basis set and integration grid after a careful basis set study that included the most common double-\(\zeta\) basis sets. We considered the following: STO-3G\textsuperscript{264,265,358,359}, SBKJC\textsuperscript{360–362}, 3-21G,\textsuperscript{269,360,363} 6-31G*,\textsuperscript{266,267,269,270,272,356,357} def2-SV(P),\textsuperscript{251} def2-SVP,\textsuperscript{251} def2-SVPD,\textsuperscript{251} cc-pVDZ,\textsuperscript{248,273,333} aug-cc-pVDZ,\textsuperscript{248,273,333,336} pc-0,\textsuperscript{277,278,280,281,283} pc-1,\textsuperscript{277,278,280,281,283} aug-pc-0,\textsuperscript{277–281,283} and aug-pc-1\textsuperscript{277–281,283}. The def2-TZVP,\textsuperscript{251} def2-TZVPD,\textsuperscript{251} def2-QZVP,\textsuperscript{251} and def2-QZVPD\textsuperscript{251} were only considered for the CLB18_15 structure. For the grid study, we considered the B3LYP and B3LYP-D3(BJ) functionals with the 6-31G* basis set and a selection of integration grids (Coarse, SG1,\textsuperscript{326} Fine, Ultrafine, and Superfine).
Table 4.2: Computational methods used in this study. We listed the functional type (Generalized Gradient Approximation (GGA), meta-GGA, global and range-separated hybrid GGA or meta-GGA) together with the quantum chemistry program used, the percentage of HF exchange (%HF X) included in the approximation, and the respective references.
| Functional/Method | Type | Program | %HF X | Ref. |
|-------------------|-------------------------------------------|---------|-------|------|
| HF-3c | Low cost, corrected HF | Q-Chem | 100 | 286 |
| PBEh-3c | Low cost, hybrid GGA | Q-Chem | 42 | 352 |
| AM1 | Semi-empirical | Gaussian| N/A | 86 |
| PM3 | Semi-empirical | Gaussian| N/A | 348,349 |
| PM6 | Semi-empirical | Gaussian| N/A | 350 |
| PM7 | Semi-empirical | Gaussian| N/A | 351 |
| GFN-xTB1 | Tight-binding | xTB | N/A | 353 |
| GFN-xTB2 | Tight-binding | xTB | N/A | 354 |
| HF | Hartree-Fock | Gaussian| 100 | 1–3 |
| BLYP | GGA | Gaussian| 0 | 22,23 |
| BLYP-D3(BJ) | Dispersion-corrected GGA | Gaussian| 0 | 22,23,261 |
| Functional/Method | Type | Program | %HF X | Ref. |
|---------------------------|-------------------------------------------|----------|-------|------|
| BP86 | GGA | Gaussian | 0 | 21,22|
| BP86-D3(BJ) | Dispersion-corrected GGA | Gaussian | 0 | 21,22,261 |
| PBE | GGA | Gaussian | 0 | 32 |
| PBE-D3(BJ) | Dispersion-corrected GGA | Gaussian | 0 | 32,261 |
| B97-D3(BJ) | Dispersion-corrected GGA | Gaussian | 0 | 260,261 |
| TPSS-D3(BJ) | Dispersion-corrected meta-GGA | Gaussian | 0 | 35,261 |
| B3LYP | Global hybrid GGA | Gaussian | 20 | 22,23,25,26 |
| B3LYP-D3(BJ) | Dispersion-corrected global hybrid GGA | Gaussian | 20 | 22,23,25,26,261 |
| PBE0 | Global hybrid GGA | Gaussian | 25 | 32,55,56 |
| PBE0-D3(BJ) | Dispersion-corrected global hybrid GGA | Gaussian | 25 | 32,55,56,261 |
| M06-2X | Global hybrid meta-GGA | Gaussian | 54 | 58 |
| M06-2X-D3(0) | Dispersion corrected global hybrid meta-GGA | Gaussian | 54 | 58,263 |
| TPSSh-D3(BJ) | Dispersion corrected global hybrid meta-GGA | Gaussian | 10 | 57,261 |
| CAM-B3LYP | Range-Separated hybrid GGA | Gaussian | 19–100 | 61 |
| CAM-B3LYP-D3(BJ) | Dispersion corrected range-separated hybrid GGA | Gaussian | 19–100 | 61,261 |
### 4.4 Basis set and grid studies.
The good performance of the Hartree-Fock (HF) method and small basis sets has been known since the 1970s.\textsuperscript{364} There is enough evidence in the literature of the fact that this behavior translates to DFT as well,\textsuperscript{8,205,365–373} and many approaches that use small basis sets have been proposed in the past few years.\textsuperscript{286,352,374} For these reasons, molecular structures are usually considered converged at the double-\(\zeta\) level. In routine calculations, in fact, it is customary to run geometry optimizations using a small basis set. After the optimized geometries are obtained, they are used for more refined single-point energy calculations, usually employing a larger basis set and a better method. This approach is often used in energetic benchmark databases as, for example,
the BHDIV10 subset in GMTKN55, or some structures in the L7 subset of Hobza et al.
To validate the assumption that the long bonds in CLB18 do not require basis sets larger than double-\(\zeta\) quality, we focused our attention on the smallest molecule in the database (CLB18_15). We wanted to understand the basis set convergence and the dependence of the results on the size of the integration grid used to calculate the exchange-correlation energy. We chose the B3LYP and B3LYP-D3(BJ) functionals and a selection of basis sets of different sizes, with a specific focus on low-cost methods. We included two minimal basis sets (STO-3G and SBKJC), as well as 11 double-\(\zeta\) basis sets (3-21G, 6-31G*, def2-SV(P), def2-SVP, def2-SVPD, cc-pVDZ, aug-cc-pVDZ, pc-0, pc-1, aug-pc-0, and aug-pc-1), and the def2-TZVP, def2-TZVPD, def2-QZVP, and def2-QZVPD larger basis sets. The last three basis sets are prohibitive for the largest molecules, but we included them to shed light on the convergence of the results with respect to the number of basis functions. The pseudopotential associated with each basis was used for the molecules containing iodine. For the basis sets that do not have an associated pseudopotential, we used the def2 pseudopotential from the corresponding Ahlrichs basis set. Results are reported in Table 4.3. The B3LYP functional shows good convergence of the bond length as a function of the basis set size to a value of 1.880 Å, which is fairly close to the reference value of 1.830 Å. The B3LYP-D3(BJ) results also show a similar trend, with errors for the bond length obtained with the double-\(\zeta\) bases within 3% of the experimental reference.
Interestingly, the semi-empirical -D3(BJ) dispersion correction causes a more erratic behavior for the B3LYP-D3(BJ) functional. Its convergence with respect to the number of basis functions is slightly worse than that of the uncorrected B3LYP functional, despite a lower absolute error for the bond length. The -D3(BJ) term is correctly reducing the error (because of better treatment of dispersion effects), but it complicates the convergence at the same time. Since the parameterization was done with a quadruple-\(\zeta\) basis set, it might be overcorrecting when the basis set is too small, thus resulting in a less predictable convergence pattern.
Similar trends are observed for the other molecules in the database and can be seen on the mean signed and mean unsigned errors (MSE and MUE) for the entire CLB18 database, as reported in Appendix B (Tables B.1 and B.2). These results confirm an overall weak dependence of the bond lengths on the basis set size, and support the usage of double-\(\zeta\) bases to keep the computational cost under control. We decided to use the 6-31G* basis set for the remainder of this work given its historical importance and prevalence in the literature on geometry optimizations. Table 4.3 shows that other double-\(\zeta\) basis sets of similar size or larger are mostly within 0.02 Å of the
6-31G* ones (with a few exceptions, such as pc-0 and aug-pc-0, see also Tables B.1 and B.2 in Appendix B).
Table 4.3: Calculated bond length (in Å) for the CLB18_15 molecule with the B3LYP and B3LYP-D3(BJ) functionals and 17 different basis sets. The reference experimental value is 1.830 Å.
| Basis set | # of basis functions | B3LYP | B3LYP-D3(BJ) |
|-----------------|----------------------|---------|--------------|
| STO-3G | 84 | 2.023 | 1.995 |
| SBKJC | 140 | 2.113 | 2.076 |
| 3-21G | 154 | 2.080 | 2.033 |
| pc-0 | 154 | 2.012 | 1.991 |
| aug-pc-0 | 224 | 1.976 | 1.955 |
| def2-SV(P) | 224 | 1.901 | 1.873 |
| 6-31G* | 238 | 1.883 | 1.859 |
| cc-pVDZ | 266 | 1.864 | 1.838 |
| pc-1 | 266 | 1.880 | 1.850 |
| def2-SVP | 266 | 1.882 | 1.861 |
| def2-SVPD | 392 | 1.880 | 1.854 |
| aug-cc-pVDZ | 448 | 1.863 | 1.838 |
| aug-pc-1 | 448 | 1.865 | 1.841 |
| def2-TZVP | 518 | 1.885 | 1.858 |
| def2-TZVPD | 644 | 1.880 | 1.854 |
| def2-QZVP | 1218 | 1.880 | 1.853 |
| def2-QZVPD | 1344 | 1.880 | 1.852 |
Understanding the convergence of the results with respect to the exchange-correlation integration grids is often a neglected detail, but it might be relevant for medium and large molecules. For such cases, the calculation of the $xc$ energy might become as expensive as, if not more than, the construction of the Fock matrix, the bottleneck of a calculation involving small(er) molecules. Given the size of the molecules in CLB18, we decided to investigate this issue by performing full geometry optimizations with five popular integration grids with the B3LYP and B3LYP-D3(BJ) methods and the 6-31G* basis set. We report our results in Table 4.4 and Tables B.4 and B.5 in Appendix B. The grids that we explored are, in order of increasing size:
“coarse,” with 35 radial and 110 angular points, “SG1 grid,” analogous to a grid with 50 radial and 194 angular points, “fine,” with 75 radial and 302 angular points, “ultrafine,” with 99 radial and 590 angular, and finally, “superfine,” with 175 radial and 974 angular points for hydrogen and 250 radial and 974 angular points for all other elements.
The convergence of the results with respect to the integration grid is generally a functional-dependent issue, and while meta-GGA functionals usually require finer grids,\textsuperscript{74–77} our results on GGA functionals are essentially converged at the fine level, with a substantial saving in computational cost (Tables 4.4, B.4 and B.5). We chose the fine grid for all GGA functionals in the remainder of the work, while we opted for the ultrafine grid for all meta-GGA functionals.\textsuperscript{74–76}
Table 4.4: Calculated bond length (in Å) for the CLB18_15 molecule with different integration grids using the B3LYP and B3LYP-D3(BJ) functionals and the 6-31G* basis set. The computation time (in s) of a single SCF cycle on a single thread is also reported as an estimation of the computational cost of each grid.
| Integration Grid | Computation time, s | B3LYP | B3LYP-D3(BJ) |
|------------------|---------------------|---------|--------------|
| Coarse | 324 | 1.8299 | 1.8131 |
| SG1 | 360 | 1.8999 | 1.8744 |
| Fine | 432 | 1.8824 | 1.8595 |
| Ultrafine | 840 | 1.8831 | 1.8592 |
| Superfine | 1272 | 1.8819 | 1.8592 |
4.5 Results.
The mean unsigned errors (MUEs) calculated with respect to the 18 experimental reference bond lengths for each of the 26 approximations listed in Table 4.2 are reported in Figure 4.1.
Fourteen methods have a very good overall performance and MUE < 0.1 Å. Among these approximations, the best performers (TPSSh-D3(BJ) and B3LYP-D3(BJ)) have an MUE of 0.017 and 0.018 Å, respectively, followed by nine methods with MUE smaller than 0.04 Å. The accuracy of these methods for the medium-sized molecules in CLB18 is similar to their accuracy for smaller molecules, as reported in the literature.\textsuperscript{18,352} The Minnesota functionals (M06-2X and M06-2X-D3(0)), as well as the PBEh-3c approximation, the BP86 functional, and the HF method all have acceptable performance for CLB18, with MUEs between 0.040 Å and 0.060 Å. Grimme and coworkers introduced a database of 12 molecules having long bonds (LB12) to
| Level of theory | Overall MUE, Å | Group 1 MUE, Å | Group 2 MUE, Å | Group 3 MUE, Å |
|-----------------|----------------|----------------|----------------|----------------|
| TPSSh-D3(BJ) | 0.017 | 0.014 | 0.014 | 0.022 |
| B3LYP-D3(BJ) | 0.018 | 0.017 | 0.010 | 0.026 |
| PBE0 | 0.024 | 0.014 | 0.026 | 0.032 |
| TPSS-D3(BJ) | 0.026 | 0.020 | 0.021 | 0.038 |
| PBE-D3(BJ) | 0.029 | 0.027 | 0.015 | 0.046 |
| B3LYP | 0.030 | 0.033 | 0.012 | 0.045 |
| CAM-B3LYP | 0.030 | 0.015 | 0.029 | 0.046 |
| PBE0-D3(BJ) | 0.034 | 0.021 | 0.029 | 0.052 |
| CAM-B3LYP-D3(BJ)| 0.037 | 0.022 | 0.031 | 0.058 |
| BP86-D3(BJ) | 0.038 | 0.039 | 0.023 | 0.052 |
| PBE | 0.040 | 0.034 | 0.015 | 0.069 |
| M06-2X | 0.044 | 0.016 | 0.030 | 0.088 |
| M06-2X-D3(0) | 0.045 | 0.016 | 0.029 | 0.088 |
| PBEh-3c | 0.049 | 0.034 | 0.041 | 0.074 |
| BP86 | 0.054 | 0.045 | 0.025 | 0.091 |
| HF | 0.061 | 0.027 | 0.031 | 0.125 |
| GFN-xTB2 | 0.104 | 0.087 | 0.078 | 0.148 |
| GFN-xTB1 | 0.112 | 0.076 | 0.078 | 0.182 |
| B97-D3(BJ) | 0.119 | 0.286 | 0.033 | 0.038 |
| HF-3c | 0.128 | 0.072 | 0.065 | 0.246 |
| PM3 | 0.132 | 0.049 | 0.035 | 0.310 |
| PM6 | 0.145 | 0.078 | 0.070 | 0.286 |
| BLYP-D3(BJ) | 0.150 | 0.307 | 0.050 | 0.092 |
| PM7 | 0.165 | 0.062 | 0.040 | 0.395 |
| AM1 | 0.172 | 0.079 | 0.052 | 0.386 |
| BLYP | 0.194 | 0.379 | 0.059 | 0.145 |
Figure 4.1: Performance of the 26 approximations on the CLB18 database. All results (in Å) are listed in order of increasing overall MUE. Columns 3—5 list the MUE for each group of molecules. The worst result for each method/approximation is reported in bold.
test the PBEh-3c method.\textsuperscript{352} The LB12 database, however, includes bonds that are generally dominated by non-covalent interactions, while CLB18 is composed of mostly covalent bonds. Nevertheless, the accuracy of PBEh-3c for LB12 (0.039 Å) is similar to what we found here for CLB18 (0.049 Å). The remaining ten methods all have an MUE higher than 0.1 Å. The low-cost HF-3c method performs similarly to the tight-binding approximations, which in turn outperform the semi-empirical Hamiltonian methods. GFN-xTB1 and GFN-xTB2 have been tested against the LB12 dataset as well. Their reported accuracies for LB12 (0.14 and 0.12 Å, respectively)\textsuperscript{354} are once again similar to the results that we obtained for CLB18 (0.11 and 0.10 Å, respectively). Surprisingly, we also found three DFT functionals that perform worse than the semi-empirical methods, namely B97-D3(BJ), BLYP-D3(BJ), and BLYP. They all make high errors in the description of two structures (CLB18_5 and CLB18_6) that belong to group 1, and for which all other methods have no issues. We also notice that all the dispersion-corrected functionals besides M06-2X-D3(0) perform better than their uncorrected counterparts, in line with previous findings and recommendations.\textsuperscript{101,102,263} When looking at the performance for the three constituent groups, it is interesting to note that the carborane molecules (group 3) are the most problematic structures for the vast majority of the methods.
4.6 Discussion.
4.6.1 Comparison with other databases
Figure 4.1 shows that most of the best performers for the CLB18 database have a percentage of HF exchange of 25% or lower. Such behavior is not uncommon, and it is found, for example, when describing the energetics of metal-organic interactions, such as in the study of spin states of transition-metal complexes.\textsuperscript{376–379} For example, Reiher and coworkers proposed the B3LYP* functional by decreasing the percentage of HF exchange from 20 (as in the original B3LYP approximation) to 15% to improve its performance for spin states.\textsuperscript{376} Kepp, Cirera, and coworkers identified TPSSh—a functional containing 10% of HF exchange—as the best performer for applications involving spin-crossover complexes.\textsuperscript{377–379} Although CLB18 exclusively contains structural data, the fact that TPSSh and B3LYP are the top two performers might suggest a connection between the effects that are necessary to describe the structure of C–C long bonds in CLB18 and the energetics of metal-organic interactions.
Similarly, a percentage of HF exchange above 40% seems detrimental to the performance on CLB18. This finding is also unexpected since functionals with a higher percentage of HF exchange (such as M06-2X and PBEh-3c) are usually suggested for calculations on systems with partially dissociated bonds, such as transition states, or barrier heights. In the following sections, we explore the potential connection between the information contained in CLB18 and those in other databases that are commonly used for the benchmark and parametrization of computational methods.
4.6.2 Effect of the Hartree-Fock exchange, multi-reference character, and comparison with metal-organic interactions
To further understand the effect of the percentage of HF exchange on the results of CLB18, we started from the PBE0 functional form:
\begin{equation}
E_{xc}^{\text{PBE0}} = \lambda E_{HF} + (1 - \lambda) E_x^{\text{PBE}} + E_c^{\text{PBE}},
\end{equation}
and we increased the percentage of HF exchange ($\lambda$) from 0 to 100% in increments of 10%. We used the resulting functionals to perform full geometry optimization on the entire CLB18 database. The results are visually summarized in Figure 4.2. The curve for the overall MUE as a function
of the percentage of HF exchange shows an optimal value of around 15%. The minima for each constituent group is within a range between 10 and 20%: the minimum for group 1 is at 20%, for group 2 it is at 10%, while for group 3 it is at 15%. With percentages of HF exchange higher than 30%, the performance deteriorates significantly. Above the 35% threshold, the overall mean unsigned error of the resulting hybrid functional becomes higher than local PBE (0.040 Å), while above the 50% threshold, it becomes worse than HF (0.061 Å). This fact explains, for example, the unexpectedly bad performance of M06-2X and PBEh-3c, which have 54 and 42% HF exchange, respectively.
**Figure 4.2:** Effect of the HF exchange on the performance of the PBE0 functional for the CLB18 database (black, solid). Performance for the three constituent group are also reported (orange, dashed: group 1; blue, dotted: group 2, green, dot-dashed: group 3). The data used to generate this figure can be found in **Table B.6** in Appendix B.
A long bond length might be electronically similar to a partially dissociated bond, and it might imply some degree of strong correlation that requires a multi-reference (MR) treatment. After performing stability analysis on the structures of the CLB18 database, we found that 15 out of the 18 molecules present a stable broken-symmetry (spin-contaminated) solution at the HF level. The only stable structures are CLB18_2, CLB18_12, and CLB18_15. The restricted-unrestricted instability we found might be due to some MR character. In general, for molecules exhibiting MR character, the use of either local or hybrid functionals with a small percentage of exact exchange is usually recommended.\textsuperscript{309,380,381} The results reported in **Figure 4.2** appear consistent with
this picture. To establish whether the molecules in the CLB18 database have a certain degree of MR character or not, we used the $A_\lambda$ diagnostics of Martin and coworkers\textsuperscript{382} and the $N_{FOD}$ diagnostics of Grimme and Hansen.\textsuperscript{383} The formula used to calculate the $A_\lambda$ parameter is
$$A_{\lambda*100} = \frac{1 - \frac{\text{TAE}[\lambda]}{\text{TAE}[0]}}{\lambda},$$
(4.2)
while the $N_{FOD}$ parameter is obtained according to ref.\textsuperscript{383} using the corresponding keyword (FOD) in the Orca program.\textsuperscript{384}
The calculated values of $A_\lambda$ for most molecules are essentially constant with respect to changes of $\lambda$, and values are within the unproblematic cases threshold set by Fogueri et al.\textsuperscript{382} at $A_\lambda < 0.10$ (\textbf{Figure 4.3}). The values of $N_{FOD}$ are also providing similar results, showing that most molecules should be unproblematic (\textbf{Table 4.5}). There are a few outliers to these trends—such as CLB18_16 according to $A_\lambda$ and CLB18_4, CLB18_5, and CLB18_6 according to $N_{FOD}$—which might suggest some moderate MR character for those structures. We also investigated the performance of the geometry optimization on the spin-contaminated unrestricted Hartree-Fock (UHF) surface (\textbf{Table 4.5}). The largest change when the unrestricted framework is used involves mainly the energies of the structures, which are more stable by 11.4 kcal mol$^{-1}$ on average. When it comes to the reference bond lengths, the UHF geometries are essentially identical to those obtained on the unstable restricted HF (RHF) solution, differing by only 1 mÅ. These results indicate that an MR treatment is not required to obtain consistent geometries for the molecules in CLB18. The optimal performance of hybrid functionals with a low percentage of HF exchange is likely not due to intrinsic strong correlation in the molecules, but it must be attributed to more subtle exchange–correlation effects that are harder to quantify.
\begin{table}[h]
\centering
\caption{Calculated bond lengths and mean unsigned errors (MUEs) for the HF method using the restricted and unrestricted formalism. Bond lengths and MUEs are in Å, while the energy differences (fourth column) are in kcal mol$^{-1}$.}
\begin{tabular}{l c c c c c}
\hline
\textbf{Structure} & \textbf{RHF}\textsuperscript{a} & \textbf{UHF}\textsuperscript{a} & \textbf{Energy difference}\textsuperscript{b} & \textbf{S}\textsuperscript{2} & \textbf{N\_FOD} \\
\hline
CLB18_1, FEBMII & 1.708 & 1.708 & 0.0 & 0.0000 & 0.208 \\
CLB18_2, UBEQAV & 1.714 & 1.714 & 0.0 & 0.0000 & 0.011 \\
CLB18_3, RIRTUH & 1.673 & 1.691 & -15.9 & 3.2371 & 0.154 \\
CLB18_4, dihydropyracilene_10a & 1.7170 & 1.7136 & -27.3 & 4.0319 & 0.390 \\
CLB18_5, dihydropyracilene_10b & 1.7511 & 1.7463 & -26.9 & 4.0086 & 0.373 \\
\end{tabular}
\end{table}
| Structure | RHF$^a$ | UHF$^a$ | Energy difference$^b$ | $S^2$ | N_FOD |
|---------------------------|---------|---------|-----------------------|--------|-------|
| CLB18_6, dihydropyracilene_10c | 1.7556 | 1.7580 | -32.7 | 4.7537 | 0.528 |
| CLB18_7, HOLKEY01 | 1.6839 | 1.6934 | -14.6 | 2.3285 | 0.155 |
| CLB18_8, QOMYIA | 1.6868 | 1.6799 | -18.8 | 3.3294 | 0.218 |
| CLB18_9, QOMYUM | 1.6616 | 1.6765 | -11.4 | 2.4228 | 0.351 |
| CLB18_10, QOMYOG | 1.6854 | 1.6785 | -18.7 | 3.3300 | 0.265 |
| CLB18_11, HOLKOI01 | 1.7161 | 1.7217 | -14.7 | 2.3469 | 0.169 |
| CLB18_12, VUFXUP | 1.8221 | 1.8221 | -0.1 | 0.1608 | 0.251 |
| CLB18_13, YISSOL | 1.5857 | 1.5856 | -4.1 | 0.9812 | 0.067 |
| CLB18_14, YISSAX | 1.6706 | 1.6715 | -8.4 | 1.6405 | 0.156 |
| CLB18_15, YISRUQ | 1.7247 | 1.7247 | 0.0 | 0.0000 | 0.021 |
| CLB18_16, YISSEB | 1.7510 | 1.7511 | -4.0 | 0.9775 | 0.052 |
| CLB18_17, YISSIF | 1.7060 | 1.7062 | -3.9 | 0.9848 | 0.075 |
| CLB18_18, JOWROF | 1.8021 | 1.7983 | -4.1 | 1.0190 | 0.078 |
$^a$ Values in Å; $^b$ Values in kcal mol$^{-1}$, calculated as UHF–RHF.
### 4.6.3 Comparison with transient long bonds
It is also worth exploring if CLB18 provides information similar to other databases with stretched bonds, most importantly the barrier heights databases of transition structures. As term of comparison, we selected the BH76 database of Truhlar and coworkers. Its geometries have been obtained at the QCISD/MG3 level of theory, and they are the only suitable option for benchmarking DFT (Table 4.6), despite a noticeable difference in size with the molecules in CLB18. We reduced the numbers of methods tested to 24, excluding the GFN-xTB1 and GFN-xTB2 methods, because their application to very small molecules such as those in the BH76 database is not recommended. In addition, a reliable transition-state search algorithm is not implemented in the programs that we have available for these calculations.
| Structure | $A_{10}$ | $A_{20}$ | $A_{25}$ | $A_{30}$ | $A_{40}$ | $A_{50}$ | $A_{60}$ | $A_{70}$ | $A_{80}$ | $A_{90}$ | $A_{100}$ |
|-----------|----------|----------|----------|----------|----------|----------|----------|----------|----------|----------|-----------|
| CLB18_1 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 0.05 | 0.05 | 0.05 | 0.05 |
| CLB18_2 | 0.04 | 0.04 | 0.04 | 0.04 | 0.04 | 0.04 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 |
| CLB18_3 | 0.09 | 0.09 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.07 | 0.07 | 0.07 |
| CLB18_4 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.07 | 0.07 | 0.07 | 0.07 |
| CLB18_5 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.07 | 0.07 | 0.07 | 0.07 | 0.07 |
| CLB18_6 | 0.09 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.07 | 0.07 | 0.07 |
| CLB18_7 | 0.07 | 0.07 | 0.07 | 0.07 | 0.07 | 0.06 | 0.06 | 0.06 | 0.06 | 0.02 | 0.06 |
| CLB18_8 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 |
| CLB18_9 | 0.09 | 0.09 | 0.09 | 0.09 | 0.09 | 0.09 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 |
| CLB18_10 | 0.09 | 0.09 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.07 | 0.07 |
| CLB18_11 | 0.07 | 0.07 | 0.07 | 0.07 | 0.07 | 0.07 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 |
| CLB18_12 | 0.09 | 0.09 | 0.09 | 0.09 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.07 |
| CLB18_13 | 0.06 | 0.06 | 0.06 | 0.06 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 |
| CLB18_14 | 0.07 | 0.07 | 0.07 | 0.07 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 |
| CLB18_15 | 0.05 | 0.04 | 0.05 | 0.04 | 0.04 | 0.04 | 0.04 | 0.04 | 0.04 | 0.04 | 0.03 |
| CLB18_16 | 0.50 | 0.28 | 0.23 | 0.20 | 0.16 | 0.14 | 0.12 | 0.11 | 0.10 | 0.09 | 0.09 |
| CLB18_17 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.00 | 0.04 | 0.04 |
| CLB18_18 | 0.06 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.04 |
| AVERAGE | 0.09 | 0.08 | 0.08 | 0.07 | 0.07 | 0.07 | 0.07 | 0.06 | 0.06 | 0.06 | 0.06 |
Figure 4.3: Values of the $A_\lambda$ MR diagnostics for all structures in the CLB18 database. Problematic values according to ref.\textsuperscript{382} are reported in bold.
Table 4.6: The level of theory (functional/method and basis set) used to obtain the reference geometries in the BHs subsets of the MGCDB84 and GMTKN55 databases. We reported in bold the non-DFT approaches.
| Dataset | Origin | Optimization at | References |
|-------------|----------|-----------------|------------|
| BHPERI26\(^a\) | MGCDB84 | B3LYP/A’TZVP\(^b\) | 133 |
| CRBH20 | MGCDB84 | B3LYP/A’TZVP\(^b\) | 193 |
| DBH24\(^c\) | MGCDB84 | QCISD/MG3 | 389 |
| HTBH38\(^d,e\) | MGCDB84 | QCISD/MG3 | 178 |
| NHTBH38\(^d,e\) | MGCDB84 | QCISD/MG3 | 177 |
| PX13\(^f\) | MGCDB84 | B3LYP/A’TZVP\(^b\) | 153 |
| WCPT27\(^g\) | MGCDB84 | B3LYP/A’TZVP\(^b\) | 215 |
| BH76\(^d\) | GMTKN55 | QCISD/MG3 | 177,178 |
| BHPERI\(^a\) | GMTKN55 | B3LYP/A’TZVP\(^b\) | 133,187 |
| BHDIV10 | GMTKN55 | PBEh-3c | 101 |
| INV24 | GMTKN55 | B3LYP-D3(BJ)/def2-TZVPP | 188 |
| BHROT27 | GMTKN55 | TPSS-D3(BJ)/def2-TZVP | 101 |
| PX13\(^f\) | GMTKN55 | B3LYP/A’TZVP\(^b\) | 153 |
| WCPT18\(^g\) | GMTKN55 | B3LYP/A’TZVP\(^b\) | 215 |
\(^a,f,g\)These two datasets are the same. The values in GMTKN55 have been updated according to ref.\(^{187}\)
\(^b\)The A’TZVP basis set corresponds to the cc-pVTZ basis set on H, aug-cc-pVTZ basis set for first-row atoms (Li–Ne), and the aug-cc-pV(n+d)Z basis set for second-row atoms (Na–Ar).
\(^c\)DBH24 is a dataset curated from HTBH38 and NHTBH38.
\(^d\)These datasets are the same. In GMTKN55, the reference values have been recalculated.
\(^e\)These subsets are taken from the Minnesota 2015B database, and they are used in this work.
The differences in geometries in the BH76 can not be evaluated using just a single bond length. As an indicator for the geometric performance, we calculated the average of the root-mean-square gradients of the energy (ARMSG) at the reference molecular structure. Ideally, the gradient should be zero at each reference transition state. Instead, the larger the ARMSG gradient is, the further the method predicts the transition state from that reference structure, and consequently
the error will be larger too. Results are reported in Figure 4.4, alongside the MUE for CLB18 copied from Figure 4.1.
| Level of theory | MUE, CLB18 (Å) | ARMSG, BH76 (E_r) |
|-----------------|----------------|--------------------|
| TPSSh-D3(BJ) | 0.017 | 0.0067 |
| B3LYP-D3(BJ) | 0.018 | 0.0059 |
| PBE0 | 0.024 | 0.0047 |
| TPSS-D3(BJ) | 0.026 | 0.0085 |
| PBE-D3(BJ) | 0.029 | 0.0097 |
| B3LYP | 0.030 | 0.0059 |
| CAM-B3LYP | 0.030 | 0.0044 |
| PBE0-D3(BJ) | 0.034 | 0.0047 |
| CAM-B3LYP-D3(BJ)| 0.037 | 0.0046 |
| BP86-D3(BJ) | 0.038 | 0.0102 |
| PBE | 0.040 | 0.0095 |
| M06-2X | 0.044 | 0.0040 |
| M06-2X-D3(0) | 0.045 | 0.0040 |
| PBEh-3c | 0.049 | 0.0038 |
| BP86 | 0.054 | 0.0101 |
| HF | 0.061 | 0.0102 |
| GFN-xTB2 | 0.104 | N/A |
| GFN-xTB1 | 0.112 | N/A |
| B97-D3(BJ) | 0.119 | 0.0091 |
| HF-3c | 0.128 | 0.0175 |
| PM3 | 0.132 | 0.0145 |
| PM6 | 0.145 | 0.0139 |
| BLYP-D3(BJ) | 0.150 | 0.0105 |
| PM7 | 0.165 | 0.0142 |
| AM1 | 0.172 | 0.0166 |
| BLYP | 0.194 | 0.0104 |
Figure 4.4: Comparison of the performance of the functionals on the CLB18 (MUE) and BH76 (ARMSG) databases. A green background indicates satisfactory performance, while a red background indicates poor performance.
A few general conclusions can be drawn from the data in Figure 4.4. Functionals that generally perform well for CLB18—such as TPSSh-D3(BJ), B3LYP-D3(BJ), and PBE0—also have a good performance on the geometries of the BH76 database. At the same time, functionals that perform poorly for CLB18—BLYP-D3(BJ), B97-D3(BJ), and BLYP—are also bad performers against BH76. However, we can also notice that the top performers for CLB18 (TPSSh-D3(BJ), B3LYP-D3(BJ)) are only average performers for BH76. The same is true for the top performers for BH76 (PBEh-3c, M06-2X, and M06-2X-D3(0)), which are only moderately good for CLB18. Looking at these functionals in terms of the percentage of HF exchange, it appears that a higher $\lambda$ is detrimental to the performance for CLB18, while it seems to provide the best performance for BH76.
These results suggest that the bonds in the CLB18 database are electronically different from the stretched bonds in the transition structures of the BH76 database. It is important to stress
that while the former are minima structures in the corresponding potential energy surface, the latter are saddle point structures. This radically different nature might explain the differences that are observed in the exchange–correlation effects. To further investigate the issue, we also calculated the optimal percentage of HF exchange for all the molecules in BH76 and the $A_\lambda$ MR diagnostics. Results are reported in Figure 4.5 and Table 4.7.
Table 4.7: Average root-mean square gradient (ARMSG, Hartrees) for the BH76 database (and its subsets NHTBH38 and HTBH38) calculated with the PBE functional and its hybrids.
| Functional | ARMSG BH76 | ARMSG NHTBH38 | ARMSG HTBH38 | $\lambda$ % |
|-----------------------------|------------|---------------|--------------|-------------|
| PBE | 0.0095 | 0.0128 | 0.0069 | 0 |
| PBE+10% HF exchange | 0.0074 | 0.0098 | 0.0055 | 10 |
| PBE+20% HF exchange | 0.0054 | 0.0070 | 0.0042 | 20 |
| PBE0 | 0.0047 | 0.0062 | 0.0036 | 25 |
| PBE+30% HF exchange | 0.0039 | 0.0050 | 0.0030 | 30 |
| PBE+40% HF exchange | 0.0032 | 0.0046 | 0.0020 | 40 |
| PBE+50% HF exchange | 0.0043 | 0.0061 | 0.0030 | 50 |
| PBE+60% HF exchange | 0.0058 | 0.0083 | 0.0039 | 60 |
| Functional | ARMSG BH76 | ARMSG NHTBH38 | ARMSG HTBH38 | $\lambda$ % |
|----------------------------|------------|---------------|--------------|-------------|
| PBE+70% HF exchange | 0.0075 | 0.0105 | 0.0051 | 70 |
| PBE+80% HF exchange | 0.0092 | 0.128 | 0.0063 | 80 |
| PBE+90% HF exchange | 0.0109 | 0.149 | 0.0077 | 90 |
| PBE+100% HF exchange | 0.0125 | 0.170 | 0.0089 | 100 |
The best results for the BH76 database are obtained when a minimum of 40% HF exchange is included, a value that is drastically different from the optimal value for CLB18. Both of its subsets—HTBH38, for hydrogen transfers, and NHTBH38, for non-hydrogen transfers—also share the same minimum at 40%. The best performances are achieved by functionals with percentages of HF exchange between 30 and 50%, such as with M06-2X and PBEh-3c. This is also consistent with the fact that exact exchange is needed to improve the performance on barrier heights.\textsuperscript{58,119,177,390–392} Noticeably, we can now add the important information that high percentages of HF exchange are required for describing both energies and geometries of transition states. Such a high optimal percentage of HF exchange is even more surprising considering that the $A_\lambda$ diagnostic indicates that the structures in BH76 are more MR than those in CLB18 (Figure 4.6 below). Given the higher MR character in BH76, one might naively expect a functional with a lower percentage of HF exchange to be more suitable, but this is not observed from the data. These results prove that the simple suggestion of using functionals with a low percentage of HF exchange for potentially MR cases and using functionals with a high percentage of HF exchange for non-MR cases—while statistically motivated—is not valid in general.
| Transition structure | $A_{10}$ | $A_{20}$ | $A_{30}$ | $A_{40}$ | $A_{50}$ | $A_{60}$ | $A_{70}$ | $A_{80}$ | $A_{90}$ | $A_{100}$ |
|----------------------|---------|---------|---------|---------|---------|---------|---------|---------|---------|---------|
| BH76_13_c2h5ts | 0.07 | 0.07 | 0.07 | 0.07 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 |
| BH76_17_c3h7ts | 0.07 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 0.05 | 0.05 | 0.05 | 0.05 |
| BH76_21_ch3fctts | 0.22 | 0.22 | 0.21 | 0.21 | 0.21 | 0.20 | 0.20 | 0.19 | 0.19 | 0.18 |
| BH76_28_clch3clts | 0.09 | 0.08 | 0.08 | 0.08 | 0.08 | 0.07 | 0.07 | 0.07 | 0.07 | 0.06 |
| BH76_37_fch3clts | 0.13 | 0.13 | 0.13 | 0.12 | 0.12 | 0.11 | 0.11 | 0.11 | 0.10 | 0.10 |
| BH76_39_fch3fcts | 0.16 | 0.15 | 0.15 | 0.14 | 0.14 | 0.14 | 0.13 | 0.13 | 0.12 | 0.12 |
| BH76_45_hchlts | 0.28 | 0.27 | 0.27 | 0.26 | 0.26 | 0.25 | 0.25 | 0.25 | 0.24 | 0.24 |
| BH76_49_hcmtts | 0.25 | 0.25 | 0.25 | 0.24 | 0.24 | 0.23 | 0.23 | 0.23 | 0.23 | 0.23 |
| BH76_51_hcots | 0.25 | 0.25 | 0.25 | 0.24 | 0.24 | 0.23 | 0.23 | 0.22 | 0.22 | 0.22 |
| BH76_52_hfzts | **1.52**| **1.49**| **1.47**| **1.45**| **1.41**| **1.36**| **1.31**| **1.26**| **1.21**| **1.15**| **1.10**|
| BH76_53_hfch3ats | 0.16 | 0.16 | 0.16 | 0.16 | 0.15 | 0.15 | 0.15 | 0.15 | 0.15 | 0.14 |
| BH76_54_hfhts | 0.46 | 0.45 | 0.45 | 0.45 | 0.44 | 0.44 | 0.43 | 0.43 | 0.43 | 0.42 |
| BH76_59_hnztts | **0.39**| **0.39**| **0.38**| **0.38**| **0.37**| **0.37**| **0.36**| **0.36**| **0.35**| **0.35**|
| BH76_63_hoch3fhts | 0.16 | 0.16 | 0.16 | 0.15 | 0.15 | 0.14 | 0.14 | 0.13 | 0.13 | 0.13 |
| BH76_68_n2ohzts | **0.57**| **0.56**| **0.56**| **0.55**| **0.54**| **0.54**| **0.53**| **0.52**| **0.51**| **0.50**|
| BH76_78_RKT01 | 0.17 | 0.17 | 0.17 | 0.16 | 0.16 | 0.16 | 0.15 | 0.15 | 0.14 | 0.14 |
| BH76_79_RKT02 | 0.21 | 0.21 | 0.20 | 0.20 | 0.19 | 0.19 | 0.18 | 0.18 | 0.18 | 0.17 |
| BH76_80_RKT03 | 0.06 | 0.06 | 0.06 | 0.06 | 0.06 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 |
| BH76_81_RKT04 | 0.11 | 0.11 | 0.11 | 0.11 | 0.10 | 0.10 | 0.10 | 0.10 | 0.10 | 0.09 |
| BH76_82_RKT06 | 0.12 | 0.12 | 0.12 | 0.12 | 0.11 | 0.11 | 0.11 | 0.11 | 0.11 | 0.11 |
| BH76_83_RKT07 | 0.22 | 0.21 | 0.21 | 0.20 | 0.20 | 0.20 | 0.19 | 0.19 | 0.19 | 0.18 |
| BH76_84_RKT08 | 0.08 | 0.08 | 0.07 | 0.07 | 0.07 | 0.07 | 0.07 | 0.07 | 0.07 | 0.06 |
| BH76_85_RKT09 | 0.09 | 0.09 | 0.09 | 0.09 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.07 |
| BH76_86_RKT10 | 0.29 | 0.27 | 0.27 | 0.26 | 0.25 | 0.24 | 0.21 | 0.20 | 0.19 | 0.19 |
| BH76_87_RKT11 | 0.11 | 0.11 | 0.11 | 0.10 | 0.10 | 0.10 | 0.10 | 0.09 | 0.09 | 0.09 |
| BH76_88_RKT12 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.07 | 0.07 | 0.07 | 0.06 | 0.06 |
| BH76_89_RKT14 | **0.31**| **0.31**| **0.30**| **0.30**| **0.29**| **0.28**| **0.28**| **0.27**| **0.27**| **0.26**|
| BH76_90_RKT16 | 0.12 | 0.12 | 0.12 | 0.12 | 0.11 | 0.11 | 0.11 | 0.11 | 0.10 | 0.10 |
| BH76_91_RKT17 | **0.63**| **0.58**| **0.56**| **0.55**| **0.52**| **0.49**| **0.47**| **0.45**| **0.44**| **0.41**|
| BH76_92_RKT18 | 0.11 | 0.10 | 0.10 | 0.10 | 0.10 | 0.10 | 0.09 | 0.09 | 0.09 | 0.09 |
| BH76_93_RKT19 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.07 | 0.07 | 0.07 |
| BH76_94_RKT20 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.07 | 0.07 | 0.07 | 0.07 |
| BH76_95_RKT21 | 0.10 | 0.10 | 0.10 | 0.10 | 0.09 | 0.09 | 0.09 | 0.09 | 0.09 | 0.09 |
| BH76_96_RKT22 | 0.09 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 | 0.08 |
| **AVERAGE** | **0.23**| **0.23**| **0.22**| **0.22**| **0.21**| **0.21**| **0.20**| **0.19**| **0.19**| **0.18**|
Figure 4.6: Values of the $A_\lambda$ MR diagnostics for all transition structures in the BH76 database. Problematic values according to ref.\textsuperscript{382} are reported in bold, while the highest value is reported in red. The transition structures nomenclature (first column) is based on the names used in the original publication\textsuperscript{177} and reported in our ACCDB database.\textsuperscript{79}
4.7 Conclusions
In this chapter, we have introduced the CLB18 database of 18 large molecules having long C–C bonds, and we used it to benchmark the performance of 26 computational methods commonly used for geometry optimizations. Our results show that DFT exchange–correlation functionals with a low percentage of HF exchange are the best performers for CLB18. Comparison with results from other databases—including the BH76 database of barrier heights and its subsets—suggests that the long bonds in CLB18 are electronically different from the elongated bonds in transition structures. We found out that functionals that are successful for the long bonds in CLB18 are also those suggested to describe the energetics of spin states in transition-metal complexes. We also report other indicators that point towards a correlation between these two seemingly different properties. As such, CLB18 represents a valuable tool for the parametrization of new electronic structure methods with broader chemical applicability.
Chapter 5: Uncaging glutamate using DFT: A case study.
5.1 Introduction.
Spatio-temporal control, i.e. the ability to deliver or activate a compound to a specific site at a specific timing, is of uttermost importance.\textsuperscript{393–396} Techniques that exhibit spatio-temporal advantages have been widely applied in many different fields of science, from medicinal chemistry to photo-targeted therapeutics\textsuperscript{393,395}—cancer treatments\textsuperscript{397–400} and photoinitiated catalysis\textsuperscript{400} being the most relevant—as well as in the neurosciences, with the target goal of activating selected neurons to study specific pathways.\textsuperscript{393,401–404} Thanks to the most recent achievements in the field of optogenetics, it is possible to genetically engineer light-sensitive channel rhodopsin receptors in selected neurons, thus rendering them light-responsive.\textsuperscript{395,403,405–411} Additional methods to fire specific neurons make use of a broad spectrum of photocleavable protecting groups attached to critically active moieties of agonists.\textsuperscript{412–415} These photocleavable protecting groups acquired the terms photocages, or simply cages. Their attachment to an agonist necessarily incapacitates it, much like caging a ferocious animal.
Caged molecules are hemically inert, and they need an external agent—a physical, chemical, or mechanical force\textsuperscript{400,416–419}—to induce the release of the biologically active compound they are protecting. This process is called “uncaging,” independently of how this release mechanism is triggered. Light is certainly the most advantageous activator since it gives the possibility to exert control on both the time and the location of the release.\textsuperscript{393–396} Regardless of the class they belong to, the different cages share the common feature of being cromophores for radiations of specific wavelength. This leads to the excitation from the singlet ground state (S\textsubscript{0}) to an excited singlet state (S\textsubscript{1,2,3…}). Subsequently, the molecules relax to the lowest-lying singlet state (S\textsubscript{1}) \textit{via} internal conversion,\textsuperscript{120} and some systems can undergo intersystem crossing (ISC) to the triplet state manifold, with the consequent relaxation to the lowest triplet state (T\textsubscript{1}).\textsuperscript{413,421} For caging compounds, this process usually results in the release of the active compound, accomplished through the cleavage of the most labile bond (shown in red for 4-methoxy-5,7-dinitroindoliny glutamate (MDNI-Glu) 1 in Figure 5.1). The elementary steps in these reaction mechanisms are sometimes not clearly understood, as they depend on the class of protecting groups employed.
In addition, since they occur in excited electronic states, they may not resemble the familiar mechanistic pathways commonly accepted for ground states.\textsuperscript{413}
The 7-nitroindolinyl family of cages gained popularity at the turn of the century due to their optimal quantum yields and efficient release of the agonists.\textsuperscript{422–424} Such systems have been extensively studied and improved through the installation of a second nitro group at the 5 position. This slight modification of the substitution pattern of the cage resulted in a dramatical increase in both quantum yields and efficiency of the release.\textsuperscript{425,426} According to experimental data, the quantum yield of MDNI-Glu is at least fivefold the quantum yield of its corresponding mono-nitro analog,\textsuperscript{427–429} even though the second nitro group at C-5 is not directly participating in the reaction mechanism. The uncaging for the 7-indolinyl system was studied by at least three groups using kinetic,\textsuperscript{425} fast pulse IR,\textsuperscript{[cohen2005]} and computation.\textsuperscript{430} The mechanism of uncaging for the 7-indolinyl system was studied by at least three groups using kinetic, fast pulse IR, and computation. However, the most recent computational study does not explain the difference in quantum yield, which is an important parameter, especially for consideration in future improved designs. We herein report a thorough computational analysis focusing on the electronic effects behind the better quantum yields of the dinitroindolinyl systems. Our study clarifies the mechanism with respect to the traditionally accepted formation of a cyclic intermediate.
\begin{figure}[h]
\centering
\includegraphics[width=\textwidth]{figure5_1.png}
\caption{The two proposed mechanisms for the uncaging of MDNI-Glu (R=CH$_2$CH$_2$CH(NH$_2$)COOH): Migration pathway (MP), the computational mechanism reported by Pálfi et al. in ref.\textsuperscript{430} Cyclization pathway (CP), the mechanism presumed by Ellis-Davies et al\textsuperscript{426} based on the data gathered by Morrison et al.\textsuperscript{431}}
\end{figure}
The first mechanism for the light-initiated uncaging of MDNI-Glu (\textbf{1} in \textbf{Figure 5.1}) has been proposed by Ellis-Davies and coworkers in 2005,\textsuperscript{426} and is based on kinetic data collected by
Morrison et al.\textsuperscript{431} for another member of the 7-nitroindoline family. We will label this mechanism “cyclization pathway,” and we will refer to it as CP from here on for simplicity. According to this proposed mechanism, after irradiation, the reaction proceeds on the triplet surface via a cyclic intermediate (5). Subsequently, the system is deprotonated (3), and finally, it delivers the free glutamate (7) to the reaction medium. According to the CP mechanism, the increased reactivity of MDNI is due to the influence of the nitro group in position 5 on the overall electronic structure of the indoline scaffold.\textsuperscript{426} The subsequent computational study by Pálfi et al. (the migration pathway, MP),\textsuperscript{430} however, did not report any cyclic intermediate, and it collected no evidence about a possible cyclization pathway. Also, Pálfi et al. attributed the increased reactivity of MDNI to a smaller difference in the enthalpy of activation ($\Delta \Delta H$) in one of the steps of the uncaging reaction on the triplet surface. The most notable difference between the proposed CP mechanism and the MP mechanism is the formation of the cyclic intermediate 5, and to which extent the substituents of the indoline scaffold affect each pathway.
Figure 5.2: Schematics of the main reaction pathways for MDNI-Ac: Migration Pathway (MP)—mechanism of Pálfi et al., verified by our improved computational results; Cyclization Pathway—C (CP-C) mechanism via cyclic intermediate (see text for a detailed explanation of the pathways). Note that both mechanisms converge towards intermediate 13. The hydrolysis pathway on the singlet surface is also shown. The structures of the relevant transition states are reported in a box, while significant intermediates and products are reported without a box.
5.2 Computational details.
In this work, we aim to shed light on the two mechanisms, and finally establish whether the cyclic intermediate proposed by Ellis-Davies and coworkers forms or not. We performed state-of-the-art density functional theory (DFT) calculations using the range-separated hybrid generalized gradient approximation (GGA) functional $\omega$B97X-D\textsuperscript{62} and the def2-TZVP basis set\textsuperscript{251} on top of geometries optimized at the B3LYP-D3(BJ)/def2-SVPD level of theory.\textsuperscript{22,23,25,26} We performed calculations using six other exchange-correlation functional approximations. We used the two range-separated hybrid GGA functionals CAM-B3LYP\textsuperscript{61} and LC-$\omega$HPBE,\textsuperscript{432} the global hybrid meta-GGA MN15,\textsuperscript{60} the range-separated meta-GGAs M11\textsuperscript{242} and $\omega$B97M-V,\textsuperscript{66} and the double-hybrid GGA functional DSD-PBEP86-D3(BJ).\textsuperscript{69,261} These functionals, especially the Minnesota- and $\omega$B97-families, are known to work well for both ground-state\textsuperscript{60,101,102} and excited-state properties.\textsuperscript{299,433–435} Our preference for $\omega$B97X-D was dictated by the excellent agreement between the experimental UV-VIS spectrum with the one calculated at this level of theory (see Figure 5.7 below). We used the B3LYP-D3(BJ)/def2-SVPD level of theory for geometry optimization because it yields reliable structures according to previous studies.\textsuperscript{136,436,437} To address the potential multi-reference (MR) character of some of the molecules, most notably the relevant transition structures in the CP and MP mechanisms, we also used the $B_1$\textsuperscript{438} and $A_\lambda$\textsuperscript{382} diagnostics. This analysis showed a lack of strong MR for all structures considered (see Section C1 in Appendix C).
We calculated and also report the Gibbs free energy profile of the reaction for the first time, including enthalpic and entropic corrections. All calculations have been performed in water using the Conductor-like Polarizable Continuum Model (C-PCM) framework.\textsuperscript{439,440}
For computational efficiency, we focused our attention on two maller molecules, MDNI-acetate (MDNI-Ac) and 4-methoxy-5-nitroindolynil acetate (MNI-Ac), that closely resembles the larger analogs. We found competition between the cyclization and migration pathways, but they do not significantly differ from each other. The extension of the study to the relevant structures of the larger MDNI-Glu and MNI-Glu systems shows very little difference in the reactivity of the glutamate with respect to the acetate.
5.3 Results and discussion.
We found that the cyclic intermediate proposed by Ellis-Davies forms, and the CP represents the lowest-energy pathway for the uncaging of MDNI-Ac. The difference with the migration mechanism is almost negligible, being around 2.6 kcal mol$^{-1}$ for MDNI-Ac at the $\omega$B97X-D/def2-TZVP level of theory. Additional calculations on the MDNI-Glu and $\alpha$-MDNI-Glu systems suggest that the different caged moieties do not affect the mechanism on the triplet surface, contradicting what has been previously reported.\textsuperscript{430} The cyclization and migration steps are within 2.3 kcal/mol from each other independently of how the glutamate is bound to the cage scaffold. This amount is very close to the one we obtained for MDNI-Ac, so we concluded that the uncaging mechanism is not affected by the length of the side chain, nor by its nature.
The second nitro group in position 5 does not affect the pathway significantly. The differences we found are within $\sim 4.0$ kcal/mol of each other, and the highest activation energy for the migration pathway is 10.8 kcal/mol, while it is 8.2 kcal/mol for the cyclization pathway (Table 5.1). These barriers are easily overcome at room temperature, and they do not represent an obstacle to the reactivity of the molecule.
Table 5.1: Gibbs free energies of the relevant transition structures (TSs) obtained at the $\omega$B97X-D/def2-TZVP level of theory. The number in parenthesis represents the activation (free) energies calculated with respect to the compound in the triplet state (column 2). All values are in kcal mol$^{-1}$.
| Compound | Relative energy, Triplet\textsuperscript{a} | Relative energy, Cyclization TS\textsuperscript{a} | Relative energy, Migration TS\textsuperscript{a} | Migration/Cyclization difference |
|--------------|---------------------------------------------|--------------------------------------------------|-------------------------------------------------|----------------------------------|
| MDNI-Glu | 54.1 | 58.3 (4.2)\textsuperscript{b} | 60.6 (6.5)\textsuperscript{b} | 2.3 |
| MNI-Glu | 53.2 | 61.4 (8.2)\textsuperscript{b} | 64.0 (10.8)\textsuperscript{b} | 2.6 |
| $\alpha$-MDNI-Glu | 52.5 | 57.8 (5.3)\textsuperscript{b} | 58.9 (6.4)\textsuperscript{b} | 1.1 |
| $\alpha$-MNI-Glu | 51.9 | 57.6 (5.7)\textsuperscript{b} | 61.9 (10.0)\textsuperscript{b} | 4.3 |
| MDNI-Ac | 54.8 | 58.1 (3.3)\textsuperscript{b} | 60.6 (5.8)\textsuperscript{b} | 2.5 |
| MNI-Ac | 57.8 | 60.0 (2.4)\textsuperscript{b} | 63.3 (5.7)\textsuperscript{b} | 3.3 |
\textsuperscript{a}The relative energies have been calculated with respect to the compound on the singlet surface.
\textsuperscript{b}The values in parenthesis represent activation energies calculated with respect to the compounds.
Since we excluded any significant effect from the substituents to the uncaging mechanism, other factors may be contributing to the observed differences in quantum yields. For instance, the increased reaction quantum yield of the MDNI-based cages relative to their mononitro analogues could be attributed to a better photosensitivity due to the second nitro group. It has been shown that rates of ISC are increased upon substitution with heavy atoms (such as Br, I) or with functional groups that have low-lying n, π* states (e.g. carbonyls, nitro groups).\textsuperscript{441} As the reaction takes place in the triplet excited state, an improvement in the intersystem crossing of the excited singlet state of the molecule (carrying the additional nitro group) populates the triplet state more efficiently, and thus could be responsible for the overall improvement of the reaction, hence its quantum yield. We investigated this using time-dependent density functional theory (TD-DFT) calculations to analyze the electronic structure of the excited states manifold. This approach can be used to screen potential candidates for future applications, and it can help identify and select the compounds with the best light-absorbing and light-responsive properties.
5.3.1 The cyclic intermediate.
The reaction pathway from MDNI-Ac (9 in Figure 5.2) to 4-methoxy-5-nitro-7-nitrosoindole (8) and acetic acid is thermodynamically favored, with a calculated ΔG of −35.8 kcal mol\(^{-1}\) (Figure 5.2).
According to the cyclization pathway, the cyclic intermediate 10 is the key structure in the reaction mechanism of MDNI- and MNI-Ac. A new stereocenter is formed at the carbonyl carbon, and its configuration (R or S) depends on the geometry of attack. At the same time, the orientation of the attack can also lead to two different conformations of the ring, which can be
either chair- or boat-like (it is not properly a chair or a boat because the aromatic ring is planar). In total we have a combination of four structures, which come from either configuration of the stereocenter with either conformation of the ring. Namely, we have R-Chair, R-Boat, S-Chair and S-Boat (\textbf{Table 5.2} below). One might expect that the two structures labeled R-Boat and S-Chair are more stable than the others because of the position of the methyl group. In fact, in these two cases it lies outside the rest of the ring, in an \textit{exo} position, while in the two conformers R-Chair and S-Boat the methyl group is in an \textit{endo} position. Surprisingly, all four intermediates have almost the same energy, as indicated in \textbf{Table 5.2} by the relative differences in Gibbs free energy ($\Delta \Delta G$). The position of the methyl substituent therefore is not as important as we first expected. Since the energy difference ($-2.1$ kcal mol$^{-1}$) we found is below the accuracy of the method we used, we can conclude that the structures are practically equivalent. We focused only on the R-Chair structure (structure C in \textbf{Table 5.2}). The consideration we made for the cyclic intermediate of MDNI-Ac can be applied to the MNI-Ac case as well.
Table 5.2: The four cyclic structures taken into account based on the configuration of the stereocenter (R or S) and the conformation (boat or chair) of the cyclohexene ring for MDNI-Ac. The carbon atoms are in black, hydrogens in white, nitrogens in blue and oxygens in red. Skeletal formulas (hydrogen atoms are omitted) are included for clarity.
Close inspection of structure 10 reveals that there are at least four bonds that can be broken, leading to the collapse of the cyclic intermediate. We labeled those bonds A, B, C, and D in Figure 5.3. We will examine all four routes, which we will label according to the bond that breaks first. Breaking the bond between carbon and oxygen (bond A in Figure 5.3) reverts the cyclic intermediate to the reactant. It does not lead to uncaging. Breaking bond B resulted in an unstable intermediate that reverted to the cyclic structure itself during geometry optimization. Coincidentally, it also leads to an intermediate that has not been previously characterized experimentally. For these reasons, we did not explore this pathway any further. The CP mechanism, as proposed initially by Ellis-Davies, involves breaking bond C first, followed by a concerted process initiated by the deprotonation that leads to the reaction products, as
seen in Figure 5.1. Our calculations show that bond C can indeed be broken, but there is no deprotonation of the resulting intermediate. Instead, once the nitronic anhydride (13 in Figure 5.2) is formed, the leaving group departs, and then deprotonation occurs before the spent cage decays back to the singlet state. Last, we found that the process starting from breaking bond D, followed by the collapse of the cyclic intermediate, is also possible but energetically disfavored compared to the other pathways described above. Breaking bond D, which corresponds to a hydrogen atom abstraction, requires 22.3 kcal mol$^{-1}$ for MDNI-Ac.
The MP mechanism involves a migration process that occurs as an alternative to the formation of the cyclic intermediate. According to our results, the migration mechanism is in direct competition with the CP–C cyclization pathway. Both these pathways are consistent with the experimental observations since they both yield the same intermediate—the nitronic anhydride 13—identified by Morrison et al. in their kinetic study\textsuperscript{431} and by Cohen et al. in their fast IR spectroscopy study.\textsuperscript{442}
![Figure 5.4: Key electronic effects for the cyclization and migration pathways (CP and MP). The oxygen radical in (a) is not positioned at the ideal Bürgi–Dunitz angle of 107° relative to the plane of the molecule, and it is far away from the unoccupied orbital of the carbonyl group. Both pathways necessitate deplanarization of the amide bond via a rotation (b) in either sense, leading to two enantiomers (only one shown; see Table 5.2 for more details) in the case of the CP, or directly to the following intermediate in the case of MP.]
The migration pathway necessitates only one step (structure 11 in Figure 5.2) to yield the nitronic anhydride 13, while the cyclization pathway requires two consecutive steps—the formation and the subsequent opening of the cyclic intermediate 10. The overall energy required to go through these steps is similar, albeit slightly smaller for the cyclization, as seen in Figure 5.2. To rationalize this discovery, we looked at a few structural features of the corresponding transition structures (TSs, structures 11 and 12), as reported in Table 5.3. Specifically, we considered the bond lengths of bond A and bond C (see Figure 5.3), in conjunction with the bond angle they describe. On the one hand, the bond angle is very similar for the two TSs, differing by only 3.7 degrees. This fact shows that the molecule necessitates deplanarization
before undergoing any additional change, which exploits the favorable interaction between the available unoccupied orbital of the carbonyl and the singly occupied orbital of the oxygen radical regardless of the pathway (Figure 5.4). If the carbonyl group is substituted with an ethyl group, the cyclization pathway is no longer accessible. The migration becomes the only possible pathway, although the electronic effects that make it more accessible are lost (vide infra).
Table 5.3: Calculated bond lengths (in Å) and bond angles (°) in the cyclization and migration transition structures (TSs) compared with the (R-Chair) cyclic intermediate.
| Structure | N-C bond length (Å) | C-O bond length (Å) | N-C-O angle (degrees) |
|--------------------|---------------------|---------------------|-----------------------|
| Cyclization TS (12)| 1.537 | 1.787 | 95.4 |
| Cyclic intermediate (10) | 1.559 | 1.641 | 98.4 |
| Migration TS (11) | 1.709 | 1.910 | 91.7 |
On the other hand, the two bond lengths differ slightly in the two transition structures. They are shorter for the cyclization TS, which justifies why the cyclization process is slightly more favored than the migration process. The acetyl group needs to move farther away from the cage when the migration process occurs, while in the cyclization pathway, the oxygen radical can readily attack the carbon atom without undergoing substantial modifications. The energy requirements for the two reactions are consistent with this concept. The migration process has a slightly larger barrier (5.8 kcal/mol) because the transition structure requires a more significant rearrangement than the cyclization process, which has shorter bonds and a less strict geometric requirement. The cyclization requires 4.7 kcal/mol overall (3.3 kcal mol$^{-1}$ for the first step and an additional 1.4 kcal mol$^{-1}$ for the ring-opening).
5.3.2 The migration pathway.
The migration of the acyl unit is facilitated by the inherent formation of the stabilized acyl radical, typically formed within classical Norrish\textsuperscript{443} type I alpha cleavages of excited carbonyl of ketones (Figure 5.5, panel a).\textsuperscript{444–447} The 1,6-acyl migration effectively embodies characteristics of both Norrish type I and type II reactions in which the acyl undergoes an $\alpha$-cleavage as it transfers to the O radical of the nitro unit similar to the $\gamma$-H abstraction by triplet carbonyl in Norrish type II positioned at a 1,6-relationship (Figure 5.5, panel b).\textsuperscript{443,447} In order to better understand this migration pathway, we computed the energy for an ethyl group migration (in place of the acetyl) and noted that it was higher in energy (21.3 kcal mol$^{-1}$, Figure 5.5, panel c), despite being positioned in a 1,6-relationship to the nitro oxygen radical. This reflects the stabilizing effect of an acyl radical in the transition state of the 1,6-migration. The energy difference between the migration and the cyclization pathways is calculated at 1.1 kcal/mol, and since the expected accuracy of our calculations is $\pm$ 1.0 kcal mol$^{-1}$,\textsuperscript{60,80,101,102,435} we can conclude that the two mechanisms are competitive. As such, given the small activation barriers, both mechanisms can co-occur. We found the same to be true for MNI-Ac as well, with the cyclization requiring 4.8 kcal mol$^{-1}$ overall and the migration needing 5.7 kcal mol$^{-1}$.
The inclusion of the entropic contribution to calculate the Gibbs free energy profile of the reaction proves that the substitution pattern on 7-nitroindoline is not as important as initially thought. MDNI and MNI require activation energies that are within $\sim$2.0 kcal/mol from each other, independently of the side chain of the compound they cage. These similar barriers are evidence for the little influence of the substituents on the crucial steps of the reaction. The energy difference of $\Delta \Delta H = 2.0$ kcal mol$^{-1}$ (calculated with B3LYP/6-31G*, which has an estimated error bar of $\sim \pm$ 2.0 kcal mol$^{-1}$) in favor of the MDNI-Glu pathway over the MNI-Glu one reported by Pálfi et al. becomes $\sim$4.0 kcal mol$^{-1}$ (see Table 5.1) when the entropic corrections are appropriately included. In the case of acetate as a caged compound, this difference is almost zero ($\Delta \Delta G = 0.10$ kcal mol$^{-1}$ in favor of MNI-Ac for the migration process and $\sim$0.10 kcal mol$^{-1}$ in favor of MDNI-Ac for the cyclization). All of these values are too small to result in a significant difference in reactivity. In our opinion, the values of the reaction barriers found for both MDNI- and MNI-based cages are also consistent with their current usage as efficient caging agents. Once activated, both structures release the caged compound efficiently, as none of the activation energies are prohibitive. In fact, despite noticeable minimal numerical differences between pathways, the energetic requirements are readily met at room temperature in all cases.
Figure 5.5: Schematics of the Norrish type reactions (a). The MP pathway combines both Norrish type I and type II characteristics (b). Breaking the Z–R bond (Z=N, R=COCH$_3$) results in a Norrish type I reaction, while the adjacent Z–O bond is in a 1,6-relationship (Norrish type II) and facilitates the cleavage of Z–R. Panel (c) shows the key migration transition state. If R=CH$_2$CH$_3$, the reaction loses the Norrish type I characteristic, and hence, not surprisingly, higher in energy by about 15.5 kcal mol$^{-1}$.
Besides, the results of Pálfi et al. show that the largest barrier in the reaction between MNI-Glu and MDNI-Glu is different from the one for α-MNI-Glu and α-MDNI-Glu. The only difference between these two classes of compounds is in the linking point of the cage, as also noted in our previous work in Ref. \(^429\). In the first case, it is bound to the ω-substituent of the glutamate chain, while in the second case, the cage is bound to the carbon in the α-position, hence the α-M(D)NI notation. Pálfi et al. referred to the two α-structures as MNI-Ulg and MDNI-Ulg, respectively. \(^430\) However, according to their reported data (see Table 1 in Ref.), \(^430\) the enthalpy difference for the highest barrier in the α-case is \(ΔΔH = 2.3 \text{ kcal mol}^{-1}\), a value within the accuracy of the method from the Glu case. For this reason, it is safe to assume that the effect of the substituents on the α-mechanism will be similarly inconsequential, while the origin of the different quantum yields has to be attributed elsewhere. Another significant difference between the results of Pálfi et al. and ours is that we did not find any reaction occurring on the singlet ground state. Pálfi et al. claim that hydrolysis and other side reactions are problematic, as they can readily occur on the singlet ground state (despite a reported ΔH for the hydrolysis reaction barriers of at least 44.6 kcal mol\(^{-1}\)). \(^430\) Our new computational results, in conjunction with the experimental findings of Cohen et al., \(^442\) contradict the hypothesis of other side reactions occurring on the singlet surface. It is noteworthy that if hydrolysis occurred, then the cage would lead to the uncontrolled release of the active compound, invalidating its application as a reliable cage, and additionally would have been experimentally observed by Cohen et al. and in our laboratory. Furthermore, the hydrolysis product (15) and the uncaging product (8) are different in that the former retains the nitro group while the photo uncaging leads to the nitroso. However, both processes result in the release of acetic acid (from 9 and 13, respectively).
### 5.3.3 The mechanism of uncaging of MDNI-Ac.
In summary, our improved computational protocol on the uncaging mechanism shows that the lowest energy path proceeds as follows. After irradiation, the first step is either the migration of the acetyl group from the nitrogen atom of indoline to one of the oxygen atoms of the neighboring nitro group (dotted line in Figure 5.6) or the formation of the cyclic intermediate and its subsequent opening (dashed line in Figure 5.6). The migration transition structure (structure 18) is only 5.8 kcal mol\(^{-1}\) higher than the reactant, and it is accessible at room temperature. The cyclization step involves two subsequent reactions having an activation barrier of 3.3 and 1.4 kcal mol\(^{-1}\), respectively (structures 19 and 21 via intermediate 20 in Figure 5.6). In all cases, the energy barriers are non-prohibitive, making both routes plausible at room temperature. Both
pathways converge towards the formation of the nitronic anhydride (structure 22). The subsequent step (23) has a barrier of 8.1 kcal mol$^{-1}$. After the leaving group (acetate in this case) is released (23), a charged nitroso intermediate then forms (24), which readily deprotonates forming 25, which then dis-excites, going back to the singlet surface. This is also consistent with the fact that molecules including an aromatic ring are more acidic in the excited state than in the ground state.\textsuperscript{448–451} Subsequently, the non-aromatic five-membered ring in 25 spontaneously tautomerizes to the fully aromatic indole system of the spent cage, 4-methoxy-5-nitro-7-nitrosoindole (26).
The overall reaction mechanism for MDNI-Ac is outlined in \textbf{Figure 5.6}.
\begin{figure}[h]
\centering
\includegraphics[width=\textwidth]{reaction_mechanism.png}
\caption{The reaction mechanism for the uncaging of MDNI-Ac. The singlet surface is indicated in blue, while the triplet surface is indicated in green. The CP–C mechanism is outlined with the dashed line, while the MP mechanism follows the dotted line. They both converge towards structure 22, and then they proceed towards the products in the same way. The reported numerical values are Gibbs free energies calculated with respect to MDNI-Ac in the singlet state (structure 16), and they have been obtained at the $\omega$B97X-D/def2-TZVP level of theory in water (C-PCM). All values are in kcal mol$^{-1}$. Structures 16, 18, 19, 20, 22, 24, and 26 have been renumbered for better clarity, and they correspond to structures 9, 11, 12, 10, 13, 14, and 8, respectively, in \textbf{Figure 5.2}.}
\end{figure}
\subsection*{5.4 The origin of the quantum yield in the MDNI-family of cages.}
Having excluded the uncaging mechanism on the triplet surface as a potential explanation of the quantum yield, the most likely steps that can affect the behavior of this family of molecules are the response towards the incident light and how readily the triplet state is populated. The true reactant is the molecule in the triplet state. Motivated in a slight redshift observed experimentally in the UV–Vis absorption spectrum going from MNI-Glu to MDNI-Glu, we contend that controlling the amount of reactant in the triplet state is the key to achieving higher quantum yields. Such control can be achieved by introducing different substituents on the cage moiety.
To computationally characterize the excited states of the molecules, we performed timedependent density functional theory calculations with the Tamm–Dancoff approximation\textsuperscript{452} using several exchange–correlation functionals, and we compared them with the experimental UV–Vis spectra of MNI-Ac and MDNI-Ac. The range-separated $\omega$B97X-D showed the best agreement with the experimental spectra, as shown in \textbf{Figure 5.7}, and therefore it was chosen as the reference exchange–correlation functional for the entire study.
\textbf{Figure 5.7:} Comparison of the experimental (green, solid) and calculated (red, dashed) UV–Vis spectra of (\textbf{a}) MNI (top panel) and (\textbf{b}) MDNI (bottom panel). The calculated spectra are obtained by applying a Lorentzian broadening to the $\omega$B97X-D/def2-TZVP electronic transitions, also reported as red vertical lines. The canonical $\pi^*$ lowest unoccupied molecular orbitals involved in the bright electronic transitions are also reported. The experimental spectra have been obtained in water (pH = 7.39 with NaHCO$_3$), at a concentration of 0.074 mM and using radiation at 350 nm as explained in Ref.\textsuperscript{428}
A singlet–triplet transition is strictly forbidden in non-relativistic quantum mechanics, but it can happen, in practice, because of intersystem crossing (ISC). Higher reaction quantum yields generally correspond to higher rates of ISC, as established, for example, for the boron-dipyromethene (BODIPY) family of cages.\textsuperscript{415,453} Both MDNI and MNI have three singlet–singlet transitions in the relevant region of the UV–Vis spectrum (250–500 nm, \textbf{Figure 5.7}), but it is sufficient to focus on the first singlet excited state ($S_1$), as the internal conversion between any higher singlet state and the $S_1$ state is usually faster than any other process.\textsuperscript{454} Our TD-DFT results reported in \textbf{Table 5.4} show that MDNI-Glu has five triplet states below the first singlet, while MNI-Glu has only three. According to El-Sayed’s rule,\textsuperscript{455} the rate of ISC is larger for
transitions that involve a change of molecular orbital type. The orbital analysis of the relevant triplet states of MNI- and MDNI-Glu (Table 5.4) reveals that the latter has two electronic transitions presenting this characteristic ($S_1 \rightarrow T_4$ and $S_1 \rightarrow T_5$, which go from a $^1(\pi \rightarrow \pi^*)$ state to a $^3(\sigma \rightarrow \pi^*)$ state), while the former has only one (the ($S_1 \rightarrow T_3$), which is a $^3(\sigma \rightarrow \pi^*)$ transition). These results qualitatively explain the faster ISC for MDNI compared to MNI since it has access to twice as many triplet states. More details on the analysis of the transitions between the S1 state and the triplet states are also reported in Table S5 in the Supplementary Information. The introduction of better chromophores represents the key to achieving better quantum yields since modifications of the electronic structure of the cages can be achieved by changing the substituents on their scaffolds.
Table 5.4: Details of the excitation types for the singlet and triplet states in the experimental range (250–500 nm) together with the excitation energy (in eV) for MNI-Glu and MDNI-Glu.
| Molecule | State | Energy (eV) | Orbital character |
|------------|-------|-------------|-------------------|
| MNI-Glu | $S_1$ | 3.62 | $^1(\pi \rightarrow \pi^*)$ |
| | $S_2$ | 3.93 | $^1(\sigma \rightarrow \pi^*)$ |
| | $S_3$ | 4.35 | $^1(\pi \rightarrow \pi^*)$ |
| | $T_1$ | 3.01 | $^3(\pi \rightarrow \pi^*)$ |
| | $T_2$ | 3.18 | $^3(\pi \rightarrow \pi^*)$ |
| | $T_3$ | 3.54 | $^3(\sigma \rightarrow \pi^*)$ |
| | $T_4$ | 3.66 | $^3(\sigma \rightarrow \pi^*)$ |
| | $T_5$ | 4.02 | $^3(\pi \rightarrow \pi^*)$ |
| | $T_6$ | 4.20 | $^3(\sigma \rightarrow \pi^*)$ |
| MDNI-Glu | $S_1$ | 3.64 | $^1(\pi \rightarrow \pi^*)$ |
| | $S_2$ | 3.91 | $^1(\sigma \rightarrow \pi^*)$ |
| | $S_3$ | 4.00 | $^1(\sigma \rightarrow \pi^*)$ |
| | $T_1$ | 2.96 | $^3(\pi \rightarrow \pi^*)$ |
| | $T_2$ | 3.30 | $^3(\pi \rightarrow \pi^*)$ |
| | $T_3$ | 3.42 | $^3(\pi \rightarrow \pi^*)$ |
| | $T_4$ | 3.47 | $^3(\sigma \rightarrow \pi^*)$ |
| | $T_5$ | 3.48 | $^3(\sigma \rightarrow \pi^*)$ |
| | $T_6$ | 3.69 | $^3(\sigma \rightarrow \pi^*)$ |
| | $T_7$ | 3.75 | $^3(\sigma \rightarrow \pi^*)$ |
5.5 Conclusions.
In conclusion, we present a detailed description of the reaction mechanism of the photouncaging of 4-methoxy-5,7-dinitroindolinyl acetate (MDNI-Ac) and 4-methoxy-7-nitroindolinyl acetate (MNI-Ac). We show that the mechanism proceeds in the triplet state through two competitive pathways via the formation of a cyclic intermediate or through a combined Norrish type I and type II 1,6-acyl migration to the adjacent nitro group. We analyze the geometric and electronic
features of the competitive routes. Both pathways yield a nitronic anhydride, which subsequently releases the leaving group of the nitronic ester, while the spent cage is recovered in the singlet ground state. The reason behind the superior photoproperties of MDNI as a better caging group does not lie in lower activation energies but rather in a better sensitivity towards light due to an increased intersystem crossing rate. This is possible because the second nitro group on the MDNI cages lowers the energy of the triplet states with respect to the first singlet state, thus increasing their availability to undergo ISC. Our findings provide additional insights into the reactivity of the MDNI family of photocages and guidance for the future synthesis of improved cage designs.
Appendix A
A1: Regression coefficients used for MGCD84, GMTKN55, and Minnesota 2015B.
| MGCD84 regression | Intercept | 0.4159594 |
|-------------------|-----------|-----------|
| Data-point | Coefficients | Data-point | Coefficients |
| MGCD84_AE18_7 | 0.0022507 | MGCD84_HAT707nonMR_466 | 0.0144524 |
| MGCD84_AikAtom19_17 | 0.0081029 | MGCD84_PlatonicHDG_5 | 0.0040497 |
| MGCD84_Bauza30_9 | 0.0385295 | MGCD84_PlatonicCI6G_4 | 0.0024323 |
| MGCD84_BH76RC_10 | 0.0340625 | MGCD84_S66x8_35 | 0.1704662 |
| MGCD84_BSRA36_22 | 0.0246638 | MGCD84_S66x8_108 | 0.2310747 |
| MGCD84_FmH2O10_7 | 0.0105061 | MGCD84_TA13_6 | 0.0235536 |
| MGCD84_G21IP_28 | 0.0033971 | MGCD84_TAEL40nomMR_4 | 0.0057926 |
| MGCD84_HAT707MR_484 | 0.0064691 | MGCD84_TAEL40nomMR_78 | 0.0122523 |
| MGCD84_HAT707nonMR_194 | 0.0042939 | MGCD84_TAEL40nomMR_120 | 0.0188418 |
| MGCD84_HAT707nonMR_219 | 0.0116288 | MGCD84_TAEL40MR_132 | 0.0082053 |
| MGCD84_HAT707nonMR_302 | 0.0041668 | MGCD84_WATER27_23 | 0.0050195 |
| GMTKN55 regression | Intercept | 0.7364613 |
|-------------------|-----------|-----------|
| Data-point | Coefficients | Data-point | Coefficients |
| GMTKN55_BH76_45 | 0.0148906 | GMTKN55_PCONF21_11 | 0.0573541 |
| GMTKN55_DC13_2 | 0.0122999 | GMTKN55_W4-11_7 | 0.0755413 |
| GMTKN55_DC13_4 | 0.0227417 | GMTKN55_W4-11_11 | 0.0169214 |
| GMTKN55_ISOL24_4 | 0.0069133 | GMTKN55_W4-11_34 | 0.0299297 |
| GMTKN55_MB16-43_2 | 0.0148394 | GMTKN55_W4-11_114 | 0.0217445 |
| GMTKN55_MB16-43_5 | 0.0030353 | GMTKN55_W4-11_115 | 0.0002151 |
| GMTKN55_MB16-43_13| 0.0043958 | GMTKN55_W4-11_139 | 0.0101862 |
| GMTKN55_MB16-43_28| 0.0026937 | GMTKN55_WATER27_14 | 0.0048557 |
| GMTKN55_MB16-43_38| 0.0054141 | GMTKN55_WATER27_19 | 0.0186607 |
| GMTKN55_MB16-43_40| 0.0019886 | GMTKN55_WCPT18_2 | 0.1183882 |
| Minnesota 2015B regression | intercept | 2.3968004 |
|-----------------------------|-----------|-----------|
| Data-point | Coefficients | Data-point | Coefficients |
| MN_SR-MGN-BEI07_11 | 0.0055525 | MN_MR-TM-BEI3_12 | 0.0028237 |
| MN_SR-MGN-BEI07_43 | 0.1637088 | MN_MR-TMD-BE3_1 | 0.0044447 |
| MN_SR-MGN-BEI07_76 | 0.0985072 | MN_MR-TMD-BE3_3 | 0.0038915 |
| MN_ABE13_6 | 0.1119293 | MN_HC7_6 | 0.0089449 |
| MN_MR-MGN-BEI17_7 | 0.2089518 | MN_AE17_6 | 0.0762471 |
| MN_SR-TM-BEI7_9 | 0.0111902 | MN_DC9_6 | 0.0052714 |
Figure A.1: Regression coefficients for the estimated MUEs of the MGCD84 (top panel), GMTKN55 (middle panel), and Minnesota 2015B (bottom panel) databases. Only the 54 datapoints reported in this figure have a non-zero coefficient.
A2: Correlation plots for three basis sets (6-31G*, def2-SVPD, def2-TZVPPD) and three functionals (PBE-D3(BJ), B3LYP-D3(BJ), ωB97M-V).
Figure A.2: Correlation plot for the 6-31G* basis set with the PBE-D3(BJ) (in blue), B3LYP-D3(BJ) (in red), and ωB97M-V (in green) functionals. The units on the axes are kcal mol$^{-1}$.
Figure A.3: Correlation plot for the def2-SVPD basis set with the PBE-D3(BJ) (in blue), B3LYP-D3(BJ) (in red), and ωB97M-V (in green) functionals. The units on the axes are kcal mol$^{-1}$.
Figure A.4: Correlation plot for the def2-TZVPPD basis set with the PBE-D3(BJ) (in blue), B3LYP-D3(BJ) (in red), and $\omega$B97M-V (in green) functionals. The outlier for the $\omega$B97M-V functional is reported as a green x, and not considered in the correlation. The units on the axes are kcal mol$^{-1}$.
Appendix B.
General note.
All optimized geometries (for all methods) can be found in our group’s GitHub page at https://github.com/peverati/CLB18.
B.1: General performance of B3LYP and B3LYP-D3(BJ) with small basis sets.
The results reported in Tables B.1 and B.2 detail the basis set study we performed for the CLB18 database. We calculated the mean unsigned errors (MUEs, Table B.1) and the mean signed errors (MSEs, Table B.2). From the analysis of both tables, we see that there is no substantial difference among the results. Excluding basis sets with less than 10,000 basis functions in total (the minimal basis sets STO-3G, SBKJC, 3-21G, and pc-0), the B3LYP and B3LYP-D3(BJ) functionals can achieve a very good performance, with MUE < 0.04 Å, in all cases.
Table B.1: Performance of B3LYP and B3LYP-D3(BJ) with some common basis sets of double-ζ quality against the CLB18 database. The results are reported in order of increasing basis set size. All values are in Å. MUE = Mean unsigned error.
| Basis set | Total number of basis functions | MUE (Å), B3LYP | MUE (Å), B3LYP-D3(BJ) |
|-----------------|---------------------------------|----------------|-----------------------|
| aug-cc-pVDZ\(^a\) | 19,846 | 0.022 | 0.013 |
| aug-pc-1\(^a\) | 19,846 | 0.024 | 0.016 |
| def2-SVPD | 17,413 | 0.037 | 0.017 |
| def2-SVP | 11,857 | 0.032 | 0.028 |
| cc-pVDZ\(^b\) | 11,847 | 0.032 | 0.020 |
| pc-1\(^b\) | 11,847 | 0.034 | 0.019 |
| 6-31G*\(^a\) | 10,740 | 0.028 | 0.017 |
| def2-SV(P) | 10,144 | 0.035 | 0.022 |
| aug-pc-0\(^b\) | 10,085 | 0.039 | 0.036 |
| 3-21G | 7,021 | 0.066 | 0.057 |
The MSEs reveal the most striking difference in the behavior of the two functionals. In fact, the B3LYP approximation always (slightly) overestimates the bond lengths, as shown by its positive MSE with all basis sets. The addition of the -D3(BJ) empirical correction corrects this trend, most notably for the smallest basis sets considered, and it represents an improvement for most cases. In certain situations, the -D3(BJ) scheme overcorrects, thus resulting in a negative MSE, as in the case of the def2-SVP and the cc-pVDZ basis sets. We observed similar trends also for the CLB18_15 structure, as shown in Table 4.3.
Table B.2: Performance of B3LYP and B3LYP-D3(BJ) with some common basis sets of double-$\zeta$ quality against the CLB18 database. The results are reported in order of increasing basis set size. All values are in Å. MSE = Mean signed error.
| Basis set | Total number of basis functions | MUE (Å), B3LYP | MUE (Å), B3LYP-D3(BJ) |
|--------------------|---------------------------------|----------------|-----------------------|
| pc-0$^c$ | 6,984 | 0.051 | 0.055 |
| SBKJC | 6,222 | 0.096 | 0.073 |
| STO-3G | 3,898 | 0.055 | 0.068 |
$^a$The def2-SVPD basis set and the associated pseudopotential were used for I.
$^b$The def2-SVP basis set and the associated pseudopotential were used for I.
$^c$The def2-SV(P) basis set and the associated pseudopotential were used for I.
| Basis set | Total number of basis functions | MUE (Å), B3LYP | MUE (Å), B3LYP-D3(BJ) |
|-----------|---------------------------------|-----------------|------------------------|
| pc-0 | 6,984 | 0.043 | 0.018 |
| SBKJC | 6,222 | 0.099 | 0.077 |
| STO-3G | 3,898 | 0.028 | 0.022 |
\(^a\)The def2-SVPD basis set and the associated pseudopotential were used for I.
\(^b\)The def2-SVP basis set and the associated pseudopotential were used for I.
\(^c\)The def2-SV(P) basis set and the associated pseudopotential were used for I.
**B.2: Convergence issues for B3LYP and B3LYP-D3(BJ).**
We experienced some convergence issues when the augmented basis sets were used, most often with the B3LYP-D3(BJ) approximation. Despite multiple attempts with both the Gaussian and Q-Chem programs using different algorithms, some structures are not completely converged within the usual energetic threshold \((10^{-8} E_h)\), but results do not appear biased because of them. The least problematic basis set for both approximations was aug-cc-pVDZ, while the most problematic was aug-pc-1 for B3LYP-D3(BJ), and def2-SVPD for B3LYP (Table B.3).
Table B.3: Convergence issues with B3LYP and B3LYP-D3(BJ) and the augmented basis sets with 13 structures (out of the 18) in the CLB18 database.
| Structure | def2-SVPD | aug-cc-pVDZ | aug-pc-1 |
|-------------|--------------------|-------------------|---------------------|
| CLB18_1 | B3LYP, B3LYP-D3(BJ)| B3LYP, B3LYP-D3(BJ)| B3LYP, B3LYP-D3(BJ) |
| CLB18_2 | | | B3LYP-D3(BJ) |
| CLB18_4 | B3LYP, B3LYP-D3(BJ)| | B3LYP-D3(BJ) |
| CLB18_5 | B3LYP, B3LYP-D3(BJ)| B3LYP, B3LYP-D3(BJ)| B3LYP-D3(BJ) |
| CLB18_6 | B3LYP, B3LYP-D3(BJ)| B3LYP, B3LYP-D3(BJ)| B3LYP-D3(BJ) |
| CLB18_7 | B3LYP | B3LYP-D3(BJ) | B3LYP-D3(BJ) |
| CLB18_8 | B3LYP, B3LYP-D3(BJ)| | |
| CLB18_9 | B3LYP-D3(BJ) | | B3LYP-D3(BJ) |
| CLB18_10 | B3LYP, B3LYP-D3(BJ)| | |
| CLB18_11 | B3LYP | | B3LYP-D3(BJ) |
| CLB18_13 | | | B3LYP-D3(BJ) |
B.3: Convergence of the integration grids.
We report the calculated bond lengths (Tables B.4 and B.5) using the B3LYP and B3LYP-D3(BJ) functionals with the 6-31G* basis set and a selection of the most common integration grids (Coarse, SG1,\textsuperscript{326} Fine, Ultrafine, and Superfine).
Table B.4: Bond lengths (in Å) calculated with the B3LYP/6-31G* level of theory with five popular integration grids. The mean signed error (MSE), and the average computational time (in s) are reported as well for each grid (last two rows).
| Structure | Coarse | SG1 | Fine | Ultrafine | Superfine |
|-----------|----------|---------|---------|-----------|-----------|
| CLB18_1 | 1.699 | 1.709 | 1.710 | 1.710 | 1.711 |
| CLB18_2 | 1.719 | 1.733 | 1.731 | 1.731 | 1.731 |
| CLB18_3 | 1.712 | 1.724 | 1.724 | 1.724 | 1.724 |
| CLB18_4 | 1.7743 | 1.7639 | 1.7673 | 1.7658 | 1.7658 |
| CLB18_5 | 1.8087 | 1.8266 | 1.8230 | 1.8247 | 1.8246 |
| CLB18_6 | 1.8518 | 1.8358 | 1.8322 | 1.8337 | 1.8335 |
| CLB18_7 | 1.7061 | 1.6931 | 1.6986 | 1.6997 | 1.7000 |
| CLB18_8 | 1.7462 | 1.7315 | 1.7313 | 1.7312 | 1.7313 |
| CLB18_9 | XXX\textsuperscript{a} | 1.7370 | 1.7295 | 1.7321 | 1.7300 |
| CLB18_10 | 1.7412 | 1.7304 | 1.7299 | 1.7306 | 1.7306 |
| CLB18_11 | 1.7483 | 1.7516 | 1.7497 | 1.7495 | 1.7498 |
| CLB18_12 | 1.8316 | 1.8285 | 1.8277 | 1.8278 | 1.8278 |
| CLB18_13 | 1.6348 | 1.6215 | 1.6145 | 1.6140 | 1.6141 |
| CLB18_14 | 1.7256 | 1.7337 | 1.7320 | 1.7299 | 1.7304 |
| CLB18_16 | 1.8842 | 1.9778 | 1.9769 | 1.9732 | 1.9730 |
| CLB18_17 | 1.9235 | 2.0139 | 2.0096 | 1.9745 | 1.9743 |
| CLB18_18 | 2.0041 | 2.0025 | 2.0203 | 2.0138 | 2.0143 |
| MSE | 0.016 | 0.029 | 0.028 | 0.025 | 0.025 |
| Average time for one SCF cycle, s\textsuperscript{b} | 252 | 288 | 240 | 540 | 564 |
Table B.5: Bond lengths (in Å) calculated with the B3LYP-D3(BJ)/6-31G* level of theory with five popular integration grids. The mean signed error (MSE), and the average computational time (in s) are reported as well for each grid (last two rows).
| Structure | Coarse | SG1 | Fine | Ultrafine | Superfine |
|-------------|---------|---------|---------|-----------|-----------|
| CLB18_1 | 1.647 | 1.659 | 1.660 | 1.660 | 1.660 |
| CLB18_2 | 1.689 | 1.703 | 1.702 | 1.702 | 1.702 |
| CLB18_3 | 1.712 | 1.721 | 1.722 | 1.723 | 1.723 |
| CLB18_4 | 1.7307 | 1.7590 | 1.7536 | 1.7511 | 1.7514 |
| CLB18_5 | 1.7763 | 1.8235 | 1.8091 | 1.8080 | 1.8073 |
| CLB18_6 | 1.7811 | 1.8340 | 1.8168 | 1.8160 | 1.8156 |
| CLB18_7 | 1.6824 | 1.6953 | 1.6915 | 1.6895 | 1.6895 |
| CLB18_8 | 1.7354 | 1.7307 | 1.7296 | 1.7289 | 1.7289 |
| CLB18_9 | XXX\(^a\) | 1.7244 | 1.7206 | 1.7243 | 1.7238 |
| CLB18_10 | 1.7292 | 1.7263 | 1.7258 | 1.7262 | 1.7263 |
| CLB18_11 | 1.7309 | 1.7338 | 1.7290 | 1.7292 | 1.7292 |
| CLB18_12 | 1.8274 | 1.8246 | 1.8236 | 1.8238 | 1.8238 |
| CLB18_13 | 1.6321 | 1.6218 | 1.6128 | 1.6120 | 1.6119 |
| CLB18_14 | 1.7107 | 1.7143 | 1.7122 | 1.7104 | 1.7111 |
| CLB18_16 | 1.8650 | 1.9310 | 1.9328 | 1.9302 | 1.9305 |
| CLB18_17 | 1.8748 | 1.9544 | 1.9478 | 1.9487 | 1.9484 |
| CLB18_18 | 1.9277 | 1.9430 | 1.9406 | 1.9434 | 1.9424 |
| MSE | -0.009 | 0.012 | 0.008 | 0.007 | 0.007 |
| Average time for one SCF cycle, s\(^b\) | 444 | 312 | 252 | 492 | 600 |
\(^a\)This structure could not be converged.
\(^b\)This value is calculated per single thread.
**B.4: Data used to generate Figure 4.2.**
Table B.6: Calculated bond lengths and mean unsigned errors (MUEs) for the PBE functional and its hybrids, obtained by increasing the percentage of HF exchange from 0% to 100% according to equation 4.1. The PBE0 functional (25%) and the hybrid with 15% HF exchange are also included.
| Structure | PBE | PBE+10%HF | PBE+15%HF | PBE+20%HF | PBE0 | PBE+30%HF |
|-------------|---------|-----------|-----------|-----------|----------|-----------|
| CLE18_1 | 1.699 | 1.687 | 1.682 | 1.678 | 1.674 | 1.670 |
| CLE18_2 | 1.724 | 1.712 | 1.706 | 1.701 | 1.696 | 1.692 |
| CLB18_3 | 1.732 | 1.714 | 1.706 | 1.700 | 1.693 | 1.687 |
| CLB18_4 | 1.7683 | 1.7458 | 1.7367 | 1.7284 | 1.7208 | 1.7140 |
| CLE18_5 | 1.8362 | 1.7977 | 1.7844 | 1.7729 | 1.7626 | 1.7534 |
| CLE18_6 | 1.8435 | 1.8039 | 1.7891 | 1.7766 | 1.7657 | 1.7562 |
| CLB18_7 | 1.6951 | 1.6836 | 1.6783 | 1.6734 | 1.6687 | 1.6642 |
| CLB18_8 | 1.7390 | 1.7199 | 1.7120 | 1.7047 | 1.6977 | 1.6918 |
| CLE18_9 | 1.7506 | 1.7175 | 1.7051 | 1.6964 | 1.6851 | 1.6770 |
| CLE18_10 | 1.7391 | 1.7197 | 1.7116 | 1.7043 | 1.6975 | 1.6913 |
| CLE18_11 | 1.7449 | 1.7296 | 1.7227 | 1.7164 | 1.7105 | 1.7048 |
| CLE18_12 | 1.8256 | 1.8214 | 1.8194 | 1.8174 | 1.8155 | 1.8136 |
| CLE18_13 | 1.6294 | 1.6198 | 1.6154 | 1.6113 | 1.6074 | 1.6038 |
| CLB18_14 | 1.7485 | 1.7238 | 1.7137 | 1.7047 | 1.6966 | 1.6892 |
| CLB18_15 | 1.9039 | 1.8495 | 1.8265 | 1.8062 | 1.8084 | 1.7732 |
| CLB18_16 | 2.0181 | 1.9559 | 1.9261 | 1.8971 | 1.8708 | 1.8470 |
| CLB18_17 | 2.0630 | 1.9908 | 1.9625 | 1.9350 | 1.9084 | 1.8835 |
| CLB18_18 | 2.0381 | 1.9818 | 1.9540 | 1.9272 | 1.9019 | 1.8782 |
| Total MUE, Å| 0.040 | 0.017 | 0.015 | 0.017 | 0.024 | 0.034 |
| Group 1 MUE, Å| 0.035 | 0.013 | 0.010 | 0.010 | 0.013 | 0.020 |
| Group 2 MUE, Å| 0.016 | 0.011 | 0.014 | 0.020 | 0.025 | 0.030 |
| Group 3 MUE, Å| 0.069 | 0.026 | 0.020 | 0.020 | 0.033 | 0.052 |
| Structure | PBE+40%HF | PBE+50%HF | PBE+60%HF | PBE+70%HF | PBE+80%HF |
|------------|-----------|-----------|-----------|-----------|-----------|
| CLB18_1 | 1.662 | 1.655 | 1.648 | 1.642 | 1.636 |
| CLB18_2 | 1.683 | 1.674 | 1.666 | 1.659 | 1.652 |
| CLB18_3 | 1.675 | 1.665 | 1.656 | 1.647 | 1.640 |
| CLB18_4 | 1.7018 | 1.6913 | 1.6820 | 1.6736 | 1.6660 |
| CLB18_5 | 1.7376 | 1.7244 | 1.7131 | 1.7031 | 1.6942 |
| CLB18_6 | 1.7400 | 1.7270 | 1.7155 | 1.7054 | 1.6966 |
| CLB18_7 | 1.6556 | 1.6481 | 1.6375 | 1.6307 | 1.6241 |
| CLB18_8 | 1.6806 | 1.6708 | 1.6619 | 1.6540 | 1.6467 |
| CLB18_9 | 1.6625 | 1.6500 | 1.6392 | 1.6297 | 1.6209 |
| CLB18_10 | 1.6802 | 1.6704 | 1.6615 | 1.6535 | 1.6463 |
| CLB18_11 | 1.6944 | 1.6851 | 1.6764 | 1.6684 | 1.6609 |
| CLB18_12 | 1.8099 | 1.8062 | 1.8027 | 1.7993 | 1.7960 |
| CLB18_13 | 1.5969 | 1.5906 | 1.5849 | 1.5795 | 1.5745 |
| CLB18_14 | 1.6762 | 1.6650 | 1.6552 | 1.6464 | 1.6385 |
| CLB18_15 | 1.7215 | 1.7026 | 1.6874 | 1.6748 | 1.6638 |
| CLB18_16 | 1.8067 | 1.7752 | 1.7500 | 1.7291 | 1.7115 |
| CLB18_17 | 1.8388 | 1.8027 | 1.7739 | 1.7504 | 1.7313 |
| CLB18_18 | 1.8375 | 1.8051 | 1.7792 | 1.7580 | 1.7404 |
| Total MUE, Å | 0.052 | 0.066 | 0.077 | 0.088 | 0.097 |
| Group 1 MUE, Å | 0.032 | 0.042 | 0.052 | 0.060 | 0.068 |
| Group 2 MUE, Å | 0.039 | 0.046 | 0.054 | 0.061 | 0.068 |
| Group 3 MUE, Å | 0.085 | 0.108 | 0.126 | 0.142 | 0.155 |
| Structure | PBE+90%HF | PBE+100%HF |
|------------|-----------|------------|
| CLB18_1 | 1.630 | 1.625 |
| CLB18_2 | 1.645 | 1.639 |
| CLB18_3 | 1.633 | 1.626 |
| CLB18_4 | 1.6591 | 1.6527 |
| CLB18_5 | 1.6863 | 1.6790 |
| CLB18_6 | 1.6885 | 1.6812 |
| CLB18_7 | 1.6177 | 1.6117 |
| CLB18_8 | 1.6400 | 1.6338 |
| CLB18_9 | 1.6131 | 1.6058 |
| CLB18_10 | 1.6396 | 1.6333 |
| CLB18_11 | 1.6539 | 1.6474 |
| CLB18_12 | 1.7927 | 1.7896 |
| CLB18_13 | 1.5699 | 1.5656 |
| CLB18_14 | 1.6313 | 1.6246 |
| CLB18_15 | 1.6545 | 1.6461 |
| CLB18_16 | 1.6965 | 1.6836 |
| CLB18_17 | 1.7151 | 1.7011 |
| CLB18_18 | 1.7254 | 1.7123 |
| Total MUE, Å | 0.105 | 0.112 |
| Group 1 MUE, Å | 0.075 | 0.081 |
| Group 2 MUE, Å | 0.074 | 0.080 |
| Group 3 MUE, Å | 0.166 | 0.176 |
Appendix C.
General note.
All optimized geometries can be found in our group’s GitHub page at https://github.com/peverati/MDNI-MNI_Uncaging_Mechanism. The Geometries folder is organized in four sub-folders named Acetate, Glutamate, MDNI-Et, and TD-DFT. In the Acetate folder, we collected all the structures belonging to the pathways described in the main text and here for MDNI-Ac and MNI-Ac. We named them according to the structure’s number in the main text or the Supplementary Information of the original article.\textsuperscript{82} For example, MDNI-Ac in the singlet state (structure 16 from Figure 6 of the main text, and also S1 in Supplementary Figures S3) has been named Structure16_S1.xyz. All the other structures have been named following this scheme. A key is also provided in the Excel file where all the calculated electronic and Gibbs free energy values have been reported. The geometries used for the TD-DFT calculations are instead collected in the folder called TD-DFT. They are named according to the compound’s name. The geometries used for MDNI-Et are collected in the MDNI-Et folder. As for the TD-DFT structures, they are named according to the compound’s name. The geometries for caged glutamate, instead, are collected in sub-folders depending on the how the compound is bonded to the cage.
The calculated electronic and Gibbs free energies are reported in the Excel files, together with the numerical results for the multi-reference diagnostics, and the quantum yield calculations.
C.1: Multi-reference character of the transition structures in the MDNI-Ac reaction mechanisms.
To make sure that density functional theory (DFT) is indeed capable of providing an accurate answer for the cyclization and migration pathways, we assessed the multi-reference (MR) character of the relevant transition structures for MDNI-Ac. We used the $B_1$\textsuperscript{438} and $A_\lambda$\textsuperscript{382} diagnostics. In order to calculate the $B_1$ parameter, we need to compute the bond dissociation energy (BDE) with the BLYP\textsuperscript{22,23} and B1LYP\textsuperscript{22,23,32,456} functionals according to the formula
$$B_1 = \frac{\text{BDE(BLYP)} - \text{BDE(B1LYP)}}{n}$$
while the $A_\lambda$ indicator has been calculated with the same functionals according to the formula
$$A_\lambda = \frac{1 - \frac{\text{TAE}(\lambda)}{\text{TAE}(0)}}{\lambda} \rightarrow 4 * (1 - \frac{\text{TAE(B1LYP)}}{\text{TAE(BLYP)}})$$ \hspace{1cm} (B.2)
In this second case, we need to calculate the total atomization energies (TAEs) of the structures under investigation. In the right hand side of equation B.2 we made explicit the usage of the BLYP and B1LYP functionals and the fact that the percentage of Hartree-Fock (HF) exchange $\lambda * 100$ is equal to 25% (or, equivalently, $\lambda=0.25$). Following the indications of the original papers,\cite{382,438} we used the MG3 basis set\cite{93,330,332,387,388,457} for the $B_1$ diagnostic, and the cc-pVTZ basis set\cite{248} for the $A_\lambda$ parameter.
For the $B_1$ diagnostics, we computed the bond dissociation energy of the reaction from structure 17 (Figure 5.6) to structure 22, from structure 17 to structure 20, from structure 20 to structure 22, and from structure 22 to structure 24 (refer to Figure 5.6 for the structures' numbers). The numerical values are reported in Table C.1 below. All of them are below the threshold of 10.0 kcal mol$^{-1}$ indicated by Schultz, Zhao and Truhlar.\cite{438} Therefore, all steps are unproblematic cases.
This result has been cross validated with the $A_\lambda$ diagnostic. In this case, we directly applied it to the transition structures (TSs) 18, 19, 21 and 23, with the numerical values reported in the table below. All cases fall within the range of unproblematic values, and we concluded that DFT is reliable for the description of this reaction mechanism.
Table C.1: $B_1$ and $A_\lambda$ diagnostics on relevant transition structures and steps of the migration and cyclization pathways for MDNI-Ac. The structure numbers refer to Figure 5.6.
| Structure$^a$ | $A_\lambda$ diagnostics |
|---------------|-------------------------|
| Migration TS (18) | 0.154 |
| Cyclization TS (19) | 0.151 |
| Ring-opening TS (21) | 0.150 |
| Leaving group departure TS (23) | 0.153 |
| Structure$^a$ | $B_1$ diagnostics$^b$ |
|---------------|------------------------|
| Formation of 22 from 17 through 18 | 8.26 |
| Formation of 20 (R-Chair) from 17 | 1.91 |
| Structure$^a$ | $A_\lambda$ diagnostics |
|---------------|--------------------------|
| Formation of 22 from 20 | 6.35 |
| Formation of 24 from 22 | 9.43 |
$^a$ Refer to Figure 5.6 to identify the structures from their numbers. $^b$ Units are kcal mol$^{-1}$
**C2: Other reaction mechanisms involving MDNI-Ac.**
**The Cyclization Pathway—D (CP—D).**
In 2005, Ellis-Davies proposed a mechanism for the uncaging of 4-methoxy-5,7-dinitroindolinyl glutamate (MDNI-Glu) following a previously reported pathway for the decay of the methyl ester of 4-carboxymethoxy-7-nitroindolinyl acetate.\textsuperscript{431} We named this mechanism Cyclization Pathway (CP, Figure 5.1). CP involves the formation of the cyclic intermediate, and then its subsequent collapse via a concerted process that involves deprotonation and the departure of the leaving group. We found that this concerted mechanism is actually split into two separate steps, as reported in Figure 5.6, and proceeds through the formation of the cyclic intermediate, its breakdown, and the subsequent deprotonation. As noted in the main text, we labeled the different pathways according to the bond that breaks first in that pathway. We reported the cyclization pathway–C (CP–C) in Figure 5.6, together with the migration pathway (MP). The other mechanism that is consistent with Ellis-Davies and Morrison et al. idea is reported in Figure C.1, and we label it CP–D. The reason behind this name is because it starts with the breakage of bond D (see structure 9, Figure 5.2). In this case, MDNI-Ac cyclizes (structures S2–S4 in Figure C.1), and then it undergoes a hydrogen atom abstraction (S5). This step yields the formation of a “protonated” intermediate (S6), which then collapses in an almost concerted way to yield acetate and the cage. It makes sense to represent this step as concerted for two reasons. First, the second transition structure (S7) is lower in energy than the first (S8); second, there is no intermediate between them, and the two transition structures are subsequent to each other. The cage then undergoes decay to the singlet surface (S9) in an analogous manner to CP–C and MP, and then it tautomerizes (S10). Given the high barrier (22.3 kcal mol$^{-1}$), this pathway is less likely to happen than those reported in Figure 5.6.
The Cyclization Pathway CP–B and Singlet Reactivity.
We grouped here the last two side-mechanisms that we explored. The first is another “branch” of the pathway, starting from the cyclic intermediate on the triplet surface. Since it involves breakage of bond B of structure 9 in Figure 5.2, we call this mechanism CP–B. The formation of S4 is the same that we described before (Figure 5.6, structures 17, 19 and 20, and Figure C.1), but in this case, it evolves by breaking the N–O bond (structure S11 in Figure C.2). The activation energy needed for this process is 22.2 kcal mol$^{-1}$, which is as high as the hydrogen abstraction described in Figure C.1. However, we could not locate any other structure for the intermediate immediately following this transition state, despite multiple attempts with different search algorithms. During geometry optimization, the structure kept reverting to the cyclic structure S4. Due to the prohibitive reaction barrier, this pathway is not likely to occur, and due to the fact that it would not lead to an experimentally characterized intermediate, we did not explore it any further.
We located two interesting transition structures on the singlet surface as well. We wanted to make sure that the cyclization process would not occur on the singlet surface. We explored the potential energy surface, and we located the cyclization transition structure (analogous to structure 19 in Figure 5.6 of the main text) at 35.1 kcal mol$^{-1}$ above the reactant. This is enough to ensure that there is no reaction on the singlet surface. The second transition structure we wanted to locate is related to the hydrolysis process, as according to Pálfi et al. it
represents a problem for MDNI- or MNI-Glu.\textsuperscript{430} The activation energy of this process is 43.7 kcal mol\textsuperscript{-1} (the hydrolysis transition structure is structure S12 in Figure C.2). We do not believe hydrolysis represents an important side-process, as it has not been observed experimentally.\textsuperscript{442} Our calculations confirm that it requires too much energy to occur in any appreciable way.
**C3: Reaction mechanisms involving MNI-Ac.**
**Main mechanism.**
The reaction mechanism reported in Figure C.3 below can be directly compared to the reaction mechanism in Figure 5.6. For MNI-Ac, we see that the reaction happens in the triplet surface, as for MDNI-Ac. We observe a competition between the migration and cyclization pathways as we did for MDNI-Ac. The first step is either the acyl transfer between the nitrogen atom of the indoline scaffold (S15 in Figure C.3) and one of oxygen atoms of the nitro group, or the cyclization process. The migration requires 5.7 kcal mol\textsuperscript{-1} (it requires 5.8 kcal mol\textsuperscript{-1} for MDNI-Ac, structure 18 in Figure 5.6), while the cyclization transition structure (S16) requires only 2.4 kcal/mol\textsuperscript{-1}, thus representing the lowest energy pathways. The cyclic structure S17
then open up yielding the nitronic anhydride S21 after overcoming a barrier of 2.5 kcal mol$^{-1}$. Overall, the energy required for the cyclization is 4.9 kcal mol$^{-1}$, only 0.8 kcal mol$^{-1}$ less than the migration pathway. For MNI-Ac as well, the migration and cyclization processes are equivalent, and they yield the same intermediate (S17). Once the intermediate S17 is formed, the leaving group departs after a barrier of 7.9 kcal mol$^{-1}$ is overcome (S22). This step requires slightly less energy than MDNI-Ac (which needs 8.1 kcal mol$^{-1}$, structure 23 in Figure 5.6). The charged intermediate S21 then deprotonates and goes back to the singlet surface (S22) and it yields the spent cage S23 after tautomerization. The Gibbs free energy of the reaction amounts to $-30.6$ kcal mol$^{-1}$. Overall, MNI-Ac and MDNI-Ac react in a very similar way, and the differences in the mechanisms are negligible.
Figure C.3: The reaction mechanism for the uncaging of MNI-Ac. The singlet surface is indicated in blue, while the triplet surface is indicated in green. The CP–C mechanism is outlined with the dashed line, while the MP mechanism follows the dotted line. They both converge towards structure S19, and then they proceed towards the products in the same way. The reported numerical values are Gibbs free energies calculated with respect to MNI-Ac in the singlet state (structure S13), and they have been obtained at the $\omega$B97X-D/def2-TZVP level of theory in water (C-PCM). All values are kcal mol$^{-1}$.
**C4: Multi-reference Character of the Relevant Transition Structures of the Main Mechanism for MNI-Ac.**
As we did for MDNI-Ac, we tested the reliability of the single-determinant treatment for the main intermediates and transition structures in the main mechanism of MNI-Ac as well. We analyzed the same steps we tested for MDNI-Ac (the cyclization, migration and leaving group departure steps). For the B$_1$ diagnostic$^{438}$ of Schultz, Zhao and Truhlar we computed the bond dissociation energy of the reaction from structure S14 to structure S19, from structure S14 to S17, from structure S17 to structure S19, and from structure S19 to S21. The values we
obtained are reported in Table C.2 below. All cases are below the threshold of 10.0 kcal mol$^{-1}$ indicated by Schultz, Zhao and Truhlar to characterize unproblematic cases.\textsuperscript{438} These results have been cross validated with the A$_\lambda$ diagnostic\textsuperscript{382} of Martin and coworkers. In this case, we directly applied it to the transition structures S15, S16, S18, and S20. The values we obtained have been reported in the table below. All cases fall within the range of unproblematic values, and we concluded that DFT is reliable for the description of this reaction mechanism.
Table C.2: B$_1$ and A$_\lambda$ diagnostics on relevant transition structures and steps of the migration and cyclization pathways for MNI-Ac. The structure numbers refer to Figure C.3.
| Structure$^a$ | A$_\lambda$ diagnostics |
|---------------|-------------------------|
| Migration TS (S15) | 0.118 |
| Cyclization TS (S16) | 0.115 |
| Ring-opening TS (S18) | 0.115 |
| Leaving group departure TS (S20) | 0.121 |
| Structure$^a$ | B$_1$ diagnostics$^b$ |
|---------------|------------------------|
| Formation of S19 from S14 through S15 | 8.36 |
| Formation of S17 (R-Chair) from S14 | 3.02 |
| Formation of S19 from S17 | 5.34 |
| Formation of S21 from S19 | 8.85 |
$^a$ Refer to Figure C.3 to identify the structures from their numbers. $^b$ Units are kcal mol$^{-1}$
C5: Other reaction mechanisms involving MNI-Ac.
The Cyclization Pathway–D (CP–D).
According to the CP–D mechanism (Figure C.4, which we will compare to Figure C.1 for MDNI-Ac) once S17 is formed it can undergo hydrogen atom abstraction through S24, which requires 25.1 kcal mol$^{-1}$. This step has the highest energy requirement for MNI-Ac, and therefore it is the less likely to happen. The following cyclic intermediate S25 opens up through breakage of the N—O bond (S26), followed by the departure of the leaving group (S27). Dis-excitation to the singlet surface (S22) and tautomerization (S23) are the same as in the mechanism discussed previously. The highest activation energy for this pathway is slightly higher in energy than the corresponding step for MDNI-Ac by $\sim$3.0 kcal mol$^{-1}$. Otherwise, as we saw for all the other
pathways, the differences between MDNI-Ac and MNI-Ac are minimal.
**Pathway CP–B and Singlet Reactivity.**
As last pathway (CP–B), we analyze the outcome of breaking the N–O bond of the cyclic intermediate S17. The cyclic intermediate S17 is formed in the same way as for the previous pathways (Figures C.3 and C.4). In this case, the intermediate opens up after overcoming an activation barrier of 22.6 kcal mol$^{-1}$ (structure S28, Figure C.5). This value is almost identical to the 22.2 kcal mol$^{-1}$ reported for MDNI-Ac, confirming one more time that the substitution pattern on the cage does not influence the reactivity of the molecule on the triplet surface. After locating the transition structure S28, we could not locate the corresponding intermediate. As for MDNI-Ac, the structure would revert to the cyclic intermediate S17 during geometry optimization. In addition, this high-energy pathway does not lead to an experimentally observed intermediate, so we did not explore its reactivity any further. The hydrolysis side-reaction has been taken into account as well. This process requires 50.3 kcal mol$^{-1}$ (structure S29), compared to the 43.7 kcal mol$^{-1}$ required by MDNI-Ac (S12). This process is the least favored, and it does not represent a problem for MNI-Ac. Last, we located the cyclization transition structure on the singlet surface for MNI-Ac as well. The cyclization process for MNI-Ac requires 39.0 kcal mol$^{-1}$, which is 3.9 kcal mol$^{-1}$ higher than MDNI-Ac.
Figure C.5: The CP–B mechanism (upper section) and the hydrolysis mechanism (lower section) for MNI-Ac. The triplet surface is reported in green, while the singlet surface is in blue. The reported numerical values are Gibbs free energies calculated with respect to MNI-Ac in the singlet state (structure S13) and they have been obtained at the $\omega$B97X-D/def2-TZVP level of theory in water (C-PCM). All values are in kcal mol$^{-1}$.
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Avoidance in Nonpayment Situations and Fundamental Breach Under the 1980 U.N. Convention on Contracts for the International Sale of Goods
Olof Clausson
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Recommended Citation
Clausson, Olof (1984) "Avoidance in Nonpayment Situations and Fundamental Breach Under the 1980 U.N. Convention on Contracts for the International Sale of Goods," NYLS Journal of International and Comparative Law: Vol. 6 : No. 1 , Article 4. Available at: https://digitalcommons.nyls.edu/journal_of_international_and_comparative_law/vol6/iss1/4
AVOIDANCE IN NONPAYMENT SITUATIONS AND FUNDAMENTAL BREACH UNDER THE 1980 U.N. CONVENTION ON CONTRACTS FOR THE INTERNATIONAL SALE OF GOODS
OLOF CLAUSSON*
INTRODUCTION
The United Nations Convention on Contracts for the International Sale of Goods¹ (the Convention) provides, as a general rule, that a party may avoid or terminate a contract only in response to a "fundamental breach" by the other party to the contract.² The Convention defines fundamental breach in article 25.³ This article identifies guidelines for determining when a buyer's nonpayment amounts to a fundamental breach entitling the seller to avoid the contract. The basic premise upon which this analysis rests is that only substantive law can give meaning to a theoretical concept such as fundamental breach.
Initially, to provide a general understanding of the rule, a historical overview of its development at the United Nations Commission on International Trade Law (UNCITRAL)⁴ will be provided. Article 25⁵ will then be compared with its counterpart in the Uniform Law on the International Sale of Goods (ULIS).⁶
* LL.B., Stockholm University, 1981; LL.M., University of Pennsylvania, 1984. The author is currently associated with the firm of Carl Swartling, Advokatbyra in Stockholm, Sweden.
1. Final Act of The United Nations Conference on Contracts for the International Sale of Goods (the Convention), U.N. CONF. ON CONT. FOR THE INT'L SALE OF GOODS—OFFICIAL RECS., U.N. Doc. A/CONF. 97/19 (1981), [hereinafter cited as OFFICIAL RECORDS]. The Convention is reprinted in 19 I.L.M. 668 (1980).
2. Id. arts. 49 (1)(a), 64 (1)(a).
3. Id. art. 25.
4. See infra notes 8-24 and accompanying text. For a more comprehensive understanding of the history and goals of the Convention, see OFFICIAL RECORDS, supra note 1, at 230. The Official Records comprise the documents reviewed at and generated by the Conference. Included among these materials are prior drafts of the Convention, the Secretary General's appraisal of the penultimate draft, the comments of governments and internal organizations on various drafts, amendments proposed at the Conference and committee reports. See also Farnsworth, The Vienna Convention: History and Scope, 18 INT'L LAW. 17, 17-19 (1984).
5. Convention, supra note 1, art. 25.
6. Convention Relating to a Uniform Law on the International Sale of Goods, 834 U.N.T.S. 107 (1972), reprinted in 3 I.L.M. 854 (1964). [hereinafter cited as ULIS].
Since no case law interpreting article 25 of the Convention exists, other less authoritative sources must be examined. Hence, statutes and decisions prescribing commercial law in England, the United States and Sweden, which maintain the basic rule that not every breach of contract is enough to give an aggrieved party the right to avoid a contract, will be appraised. From this study it may be possible to derive guidelines pertaining to the definition of fundamental breach in article 25 of the Convention and its application.
Since the Convention gives legal effect to commercial usages, the manner in which international traders have developed the concept of fundamental breach will then be examined. For example, standard contracts used in international trade usually indicate a time period for a reasonable delay in payment, and this period differs with the type of transaction and the goods involved. Thus, these contracts may also prove helpful in determining how the definition of fundamental breach in article 25 will develop and be applied in practice.
I. THE CONVENTION
A. Avoidance: Fundamental Breach
In a sales contract governed by the Convention, the buyer is obligated to pay the price and to take delivery. Since the parties may deviate from the Convention's provisions, the contract itself may im-
7. Avoidance in installment contracts is not discussed herein. But see Convention, supra note 1, art. 73, which reads:
(1) In the case of a contract for delivery of goods by instalments, if the failure of one party to perform any of his obligations in respect of any instalment constitutes a fundamental breach of contract with respect to that instalment, the other party may declare the contract avoided with respect to that instalment.
(2) If one party's failure to perform any of his obligations in respect of any instalment gives the other party good grounds to conclude that a fundamental breach of contract will occur with respect to future instalments, he may declare the contract avoided for the future, provided that he does so within a reasonable time.
(3) A buyer who declares the contract avoided in respect of any delivery may, at the same time, declare it avoided in respect of deliveries already made or of future deliveries if, by reason of their interdependence, those deliveries could not be used for the purpose contemplated by the parties at the time of the conclusion of the contract.
Id.
It is to be noted that this article bears a strong resemblance, both in form and substance, to § 2-612 of the Uniform Commercial Code.
8. Convention, supra note 1, art. 53.
9. Id. art. 6.
pose additional obligations upon the buyer. In the case of a buyer's breach of any of his obligations, the Convention provides the seller with an assortment of remedies.\textsuperscript{10}
Under article 64 the seller may, under certain circumstances, declare the contract "avoided,"\textsuperscript{11} i.e., the seller may cancel or terminate the contract, or may be discharged from further obligations under the contract.\textsuperscript{12} Not every breach, however, entitles the seller to avoid the contract.\textsuperscript{13} As a general rule, the breach must amount to a "fundamental breach" of contract.\textsuperscript{14}
Article 25 provides a definition of fundamental breach:
A breach of contract committed by one of the parties is fundamental if it results in such detriment to the other party as substantially to deprive him of what he is entitled to expect under the contract, unless the party in breach did not foresee and a reasonable person of the same kind in the same circumstances would not have foreseen such a result.\textsuperscript{15}
Over the years, the international sales law's definition of funda-
\begin{itemize}
\item \textsuperscript{10} \textit{Id.} arts. 61-65.
\item \textsuperscript{11} \textit{Id.} art. 64. Article 64 provides in part:
(1) The seller may declare the contract avoided: (a) if the failure by the buyer to perform any of his obligations under the contract or this Convention amounts to a fundamental breach of contract; or (b) if the buyer does not, within the additional period of time fixed by the seller in accordance with paragraph (1) of article 63, perform his obligation to pay the price or take delivery of the goods, or if he declares that he will not do so within the period so fixed. \textit{Id.}
\item \textsuperscript{12} Other terms describing the same effect are rescission, repudiation and the setting aside of the contract. See, e.g., J. CALAMARI & J. PERILLO, \textit{CONTRACTS} §§ 21-22 (2d ed. 1978).
\item \textsuperscript{13} Convention, \textit{supra} note 1, art. 64(2). Article 64(2) states in part: "in cases where the buyer has paid the price, the seller loses the right to declare the contract avoided. . . ." \textit{Id.}
\item \textsuperscript{14} \textit{Id.} art. 64 (1)(a). The Convention incorporated what seems to be an exception to this general rule adapted from German law. \textit{Id.} art. 64(1)(b). In the event of a delay in the performance of an obligation, the aggrieved, waiting obligee may notify the delinquent obligor that he must perform within a specified reasonable time period. If the delaying obligor does not comply with the \textit{Nachfrist} ("or else") notice, the obligee is entitled to avoid the contract, notwithstanding the delaying obligor's failure to fulfill the requisites for fundamental breach in article 25. \textit{Id.} See Honnold, \textit{The New Uniform Law for International Sales and the UCC: A Comparison}, 18 \textit{INT'L LAW} 27-28 (1984).
Even though the \textit{Nachfrist} provision in article 64 (1)(b) appears to state an exception to the general rule, one would hope that international merchants would regularly exploit this provision. If so, litigation as to whether a breach is fundamental will arise with less frequency.
\item \textsuperscript{15} \textit{Id.} art. 25.
mental breach has been modified a number of times. The 1964 Hague Convention on Uniform Law on the International Sale of Goods, section 10, defined a breach as "wherever a party in breach knew, or ought to have known, at the time of the conclusion of the contract, that a reasonable person in the same situation as the other party would not have entered into the contract if he had foreseen the breach and its effects."16 This definition was highly criticized;17 especially its "subjective" requisites, which were viewed as causing problems in its application.18
The effect of the Article is that it depends on the actual or required knowledge on the part of the party in breach at the time of the conclusion of the contract, whether a breach is fundamental or not, but this knowledge refers in part to a hypothetical situation of fact prevailing partly at the time of the breach, i.e., the breach itself and its effect.19
Another commentator stated: "Analysis of this provision in UNCTRAL made it clear that 'fundamental breach' must be redefined in terms of the materiality of the breach."20
Article 25 of the Convention sets up three criteria to assist in defining "fundamental breach." First, the breach must result in a detriment to the innocent party; second, it must substantially deprive the innocent party of what he is entitled to expect under the contract; and third, the result must have been foreseeable.21 These are objective criteria. The objective approach is evident in the second requisite's reference to what the innocent party is "entitled" to expect and in the third requisite's incorporation of the "reasonable person" test.22
As previously mentioned, the definition of fundamental breach has undergone substantial change over the years during the development of an international sales law. A number of scholars have scrutinized
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16. ULIS, supra note 6, art. 10.
17. See supra note 4.
18. Conversely, Huber labels article 25 a failure, "sie ist nichtssagend und überdies missverständlich." Huber, Der Uncitral-Entwurf eines Überinkommens über internationale Warenkaufverträge, 43 Rabels Zeitschrift für ausländisches und internationales Privatrecht 462 (1969).
19. R.H. Graveson, The Uniform Laws of International Sales Act (1967): A Commentary 55 (1968).
20. J. Honnold, Uniform Law for International Sales Under the United Nations Convention 212 (1982).
21. Convention, supra note 1, art. 25. In order to avoid the contract, however, notice must be given to the other party. Id. art. 26. This is a change from the ULIS and a rejection of ipso facto avoidance. See supra note 6.
22. Id.
each definition in an attempt to determine which one most accurately reflects the concept's application in practice.\textsuperscript{23} The prevailing view remains that judges have not yet articulated a principled standard for determining what constitutes a fundamental breach:
Only practice has been able to define [the] meaning with reasonable certainty over the decades. In applying [the] concept the judge forms an opinion, taking all circumstances into consideration whether the breach of contract is so significant as to justify avoidance or not. If it is, he then comes to the conclusion that the statutory definition has been fulfilled; otherwise, that it has not. In other words, he does not form his conclusion through confronting the facts and the statutory text. He resorts to the statutory text merely to support his already existing instinctive conclusion.\textsuperscript{24}
If we accept this view, it may be advisable to study the interpretation of similar provisions in national legal systems, bearing in mind the international character of the sales convention and other limitations on the authoritativeness of these sources.
\textit{B. Payment of the Price}
Before commencing this analysis, a few words regarding the buyer's obligation to pay the price are appropriate. Article 58\textsuperscript{25} establishes when the buyer must pay for the goods. If a date is not agreed upon in the contract, the buyer generally must pay when the seller ten-
\begin{itemize}
\item \textsuperscript{23} One scholar views the doctrine of fundamental breach of contract in terms of a method of escape from the most carefully drafted exemption clauses. He claims the cases seem to establish that no matter how extensive an exemption clause might be, it could not exclude liability in respect of the breach of a fundamental term or of a fundamental breach. See A. Guest, Anson's Law of Contract 149 (26th ed. 1984).
\item \textsuperscript{24} Eorsi, A Propos the 1980 Vienna Convention on Contracts for the International Sale of Goods, 31 Am. J. Comp. L 333, 336 (1983).
\item \textsuperscript{25} Convention, supra note 1. Article 58 reads:
(1) If the buyer is not bound to pay the price at any other specific time, he must pay it when the seller places either the goods or documents controlling their disposition at the buyer's disposal in accordance with the contract and this Convention. The seller may make such payment a condition for handing over the goods or documents. (2) If the contract involves carriage of the goods, the seller may dispatch the goods on terms whereby the goods, or documents controlling their disposition, will not be handed over to the buyer except against payment of the price. (3) The buyer is not bound to pay the price until he has had an opportunity to examine the goods, unless the procedures for delivery or payment agreed upon by the parties are inconsistent with his having such an opportunity.
ders either the goods or the documents controlling the goods.\textsuperscript{26} The buyer's obligation to pay the price includes compliance with the terms concerning the manner of payment,\textsuperscript{27} e.g., the issuance of a letter of credit,\textsuperscript{28} or the establishment of a banker's acceptance.\textsuperscript{29}
\section*{II. ENGLISH LAW}
Under English law the seller's right to avoid\textsuperscript{30} the contract due to the buyer's failure to pay the price arises in two types of cases. The first, which is not dealt with here, involves the buyer's repudiation of the contract.\textsuperscript{31} The second involves a breach amounting to a "fundamental breach."\textsuperscript{32} The second group may be sub-categorized as follows:
\begin{enumerate}
\item \textit{Id.} art. 58 (1).
\item \textit{Id.} art. 58.
\item For an overview of the purposes and mechanics of establishing a letter of credit, see P. Oppenheim, \textit{International Banking} 73-104 (3d ed. 1979). For general guidelines concerning international letters of credit, see Uniform Customs and Practices for Documentary Credits, I.C.C. No. 400.
\item See generally P. Oppenheim, \textit{supra} note 28, at 105-113.
\item See \textit{The Common Law Library}, Benjamin's Sales of Goods §§ 990-94 (2d ed. 1981); see also \textit{infra} notes 33, 45 & 67.
\item \textit{Cf.} Convention, \textit{supra} note 1, art. 71. Article 71 provides:
\begin{enumerate}
\item A party may suspend the performance of his obligations if, after the conclusion of the contract, it becomes apparent that the other party will not perform a substantial part of his obligations as a result of: (a) a serious deficiency in his ability to perform or in his creditworthiness; or (b) his conduct in preparing to perform or in performing the contract. (2) If the seller has already dispatched the goods before the grounds described in the preceding paragraph become evident, he may prevent the handing over of the goods to the buyer even though the buyer holds a document which entitles him to obtain them. The present paragraph relates only to the rights in the goods as between the buyer and the seller. (3) A party suspending performance, whether before or after dispatch of the goods, must immediately give notice of the suspension of the other party and must continue with performance if the other party provides adequate assurance of his performance.
\end{enumerate}
\item \textit{Id.}
\item The doctrine of fundamental breach in English law is confused:
The terminology of "fundamental breach" and "breach of a fundamental term", which was originally adopted to deal with problems created by wide exclusion clauses, has also been brought into service to determine whether a breach of contract is sufficiently serious to justify the innocent party in treating himself as discharged from further obligations under the contract.
\textit{Halsbury's Laws of England} § 545 (Lord Hailsham ed. 1974). The House of Lords has recently addressed the doctrine in connection with wide exemption clauses in Photo Prod. v. Securicor Transp., [1980] 2 All E.R. 556. "Insofar as the doctrine survives the Securicor case, it is therefore one of interpretation, that exemption clauses will not be interpreted so as to cover fundamental breach of contract." Benjamin's Sales of Goods \textit{supra} note 30, § 992.
\end{enumerate}
first, where the contract stipulates a right to terminate; second, where the statute defines the right to terminate the contract; third, where termination is a common law right; and fourth, where a right to terminate arises as a result of the buyer's failure to comply with contract provisions as to the method of payment.
A. Private, Statutory and Common Law Rights to Terminate
A contract may stipulate a right to terminate in the event of any breach. In *Mardorf Peach & Co., Ltd. v. Attica Sea Carriers Corporation of Liberia*,\(^{33}\) where such a clause was at issue, the court upheld the obligee's right to rescind the contract upon the obligor's failure to make timely installment payments.\(^{34}\) Further, the English Sale of Goods Act provides: "[u]nless a different intention appears from the contract, stipulations as to time of payment are not deemed to be of the essence of a contract of sale."\(^{35}\) The seller's right to resell the goods is therefore limited to cases "where the buyer does not within a reasonable time pay or tender the price."\(^{36}\)
Even if such is the case, the doctrine might still be applicable to determine whether the breach justifies termination or not. In *Suisse Atlantique Societe d'Armement Maritime, S.A. v. N.V. Rotterdamsche Kolen Centrale*, [1966] 2 All E.R. 61, Lord Wilberforce wrote:
For, though it may be true generally, if the contract contains a wide exemptions clause, that a breach sufficiently serious to take the case outside the clause will also give the other party the right to refuse further performance, it is not the case, necessarily, that a breach of the latter character has the former consequence. An act which apart from the exemptions clause, might be a breach sufficiently serious to justify refusal or further performance, may be reduced in effect, or made not a breach at all, by the terms of the clause.
*Id.* at 91-92. Lord Reid stated to the contrary: "General use of the term 'fundamental breach' is of most recent origin, and I can find nothing to indicate that it means either more or less than the well known type of breach which entitles the innocent party to treat it as repudiatory and to rescind the contract." *Id.* at 70.
\(^{33}\) [1977] 1 All E.R. 545.
\(^{34}\) *Id.* at 548.
\(^{35}\) *Sale of Goods Act § 10(1) (1893).* Section 10(1) provides:
Unless a different intention appears from the contract, stipulations as to time of payment are not deemed to be of the essence of a contract of sale. Whether any other stipulation as to time is of the essence of the contract or not depends on the terms of the contract.
*Id.*
\(^{36}\) *Id.* § 48(3). This section states:
Where the goods are of a perishable nature, or where the unpaid seller gives notice to the buyer of his intention to re-sell, and the buyer does not within a reasonable time pay or tender the price, the unpaid seller may re-sell the goods and recover from the original buyer damages for any loss occasioned by his breach of contract.
These principles were applied in *Ryan v. Ridley and Co.*, where the plaintiff sold a cargo of codfish to the defendant and payment was to be made "by cash in exchange for" the bill of lading and insurance policy. The cargo arrived by ship on October 24, 1901, and "soon" thereafter the plaintiff tendered the shipping documents and requested payment. The plaintiff, not having been paid by November 1, sold the cargo a day or two later and sued to recover the loss caused by the resale. In holding for the plaintiff, the court reasoned:
The words in the clause must be given their natural meaning, and 'cash against documents' meant that when the documents were tendered the cash must be ready. But a practical meaning must be given to the words . . . . The clause meant that the payment against the documents must be made within a reasonable time—*e.g.*, if the buyer said, 'You shall have a cheque tomorrow morning,' that would be sufficient, or even a greater delay would not be unreasonable if it would not affect the rights of the parties. If ever there was a case in which the nature of the cargo demanded promptitude, that of the cargo in the present case did.
*Ryan* illustrates that under the English Sale of Goods Act a failure by the buyer to pay the price on the date stipulated is not per se a breach entitling the seller to terminate the contract. The common law provided, "however, [that] a repeated failure to pay the price when the seller demands payment on several occasions, would . . . it is submitted, amount to a breach sufficiently serious to entitle the seller to terminate the contract."
---
37. [1902] 19 T.L.R. 45; 8 Com. Cas. 105.
38. *Id.* at 45-46.
39. Stipulations of time involving subjects other than payment are treated as being of the essence of the contract. *See* Charles Rickards Ltd. v. Oppenheim, [1950] I K.B. 616; Hartley v. Hymans [1920] 3 K.B. 475, 494-95; Crawford v. Toogood (1879) 13 Ch. D. 153 (As a condition of performance, time is of the essence in a contract for the sale of goods. On the lapse of the stipulated time, if the buyer continues to press for delivery, he waives his right to cancel the contract. The buyer then has a right to give notice fixing a reasonable time for delivery, again making time of the essence. If this new stipulation is not fulfilled the buyer may cancel the contract). *Id.*
40. *Benjamin's Sales of Goods*, *supra* note 30, § 1445.
B. Breach of the Terms of a Documentary Credit Clause
In international commerce it is common practice to have a documentary credit clause in a sales contract.\textsuperscript{41} These clauses obligate the buyer to open a documentary credit in favor of the seller.\textsuperscript{42} Usually, no time is specified for the furnishing of the documentary credit;\textsuperscript{43} the only time specified in the contract is the period of shipment.\textsuperscript{44} In these instances, it is necessary to determine when the buyer was supposed to open the documentary credit; only then can consideration be given as to whether there was a breach entitling the seller to terminate the contract.
This issue was raised in \textit{Pavia & Co., S.P.A. v. Thurmann-Nielsen},\textsuperscript{45} where, pursuant to a c.i.f. contract,\textsuperscript{46} the plaintiff sold groundnuts for shipment from Brazil to Genoa, with delivery to be made in February, March or April of 1949. Payment was to be made by letter of credit in favor of the sellers and utilizable by them against delivery of shipping documents.\textsuperscript{47} The buyers did not make the credit available until April 22.\textsuperscript{48} The court held that in the absence of an express stipulation in the contract, the credit should have been opened not when the sellers were ready to ship the goods, but at the beginning of the shipment period. Lord Denning stated the rationale for this holding as follows:
The reason is because the seller is entitled, before he ships the goods, to be assured that, on shipment, he will get paid. The seller is not bound to tell the buyer the precise date when he is going to ship, and whenever he does ship the goods he must be able to draw on the credit. He may ship on the very first day of the shipment period. If the buyer is to fulfill his obligations, he must, therefore, make the credit available to the seller at the very first date when the goods may be lawfully shipped in compliance with the contract.\textsuperscript{49}
The rule in \textit{Pavia & Co.} was slightly reformulated, to the advan-
\begin{itemize}
\item[41.] See generally id. §§ 2131-2294.
\item[42.] See id. § 2133.
\item[43.] See id. § 2167.
\item[44.] Id.
\item[45.] [1952] 1 All E.R. 492.
\item[46.] In a c.i.f. sales contract, the contract price includes in one lump sum the cost of the goods as well as the insurance and freight costs to the given destination. \textit{Black's Law Dictionary} 220 (5th ed. 1979).
\item[47.] \textit{Pavia & Co.}, 1 All E.R. at 493.
\item[48.] \textit{Id.} at 492.
\item[49.] \textit{Id.} at 495.
tage of the sellers, in *Sinason-Teicher Inter-American Grain Corporation v. Oilcakes and Oilsseeds Trading Co., Ltd.*\(^{50}\) In *Sinason-Teicher*, the c.i.f. contract called for shipment of barley during October or November 1952. Payment was to be net cash against documents, and the buyers were to issue a guarantee to the sellers through their bank that the documents would be taken up on first presentation. No date was provided with respect to when this guarantee was to be furnished. The court, in this case, adopted a rule which obligated the buyers to furnish the guarantee within a reasonable time prior to the first date for shipment. Before the first date for shipment, the seller cancelled the contract on September 10, 1952, and therefore the sellers rather than the buyers were liable.\(^{51}\)
In *Ian Stach, Ltd. v. Baker Bosley, Ltd.*, the court rejected the rule applied in *Sinason-Teicher* and found the rule laid down in *Pavia & Co.* controlling.\(^{53}\) In *Ian Stach*, the plaintiff sold steel to the defendant under a f.o.b. contract. Delivery was to be made in August or September 1956, and payment was to be made by letter of credit.\(^{54}\) The buyer had the right to select the date of shipment and also the responsibility of making the arrangements for shipment.\(^{56}\) When the buyer had not opened the letter of credit by August 14, the seller treated the contract as repudiated and resold the goods.\(^{56}\) The buyer argued that since he had the right to determine the date of shipment, the credit had to be opened at a reasonable time before that date.\(^{57}\) The court, however, rejected this notion and held that the prima facie rule requires opening of the credit, at the latest, by the earliest shipping date.\(^{58}\)
Hence, although no general rule can be cited as controlling the time in which the buyer must comply with specifications of the method of payment (i.e., the issuance of a letter of credit or the establishment of a bank guarantee), compliance is required, at the latest, by the first date of the shipping period. In some cases, however, this would not be sufficient and compliance would have to be accomplished within a reasonable time before the first date for shipment.\(^{59}\)
---
\(^{50}\) [1954] 3 All E.R. 468.
\(^{51}\) *Id.* at 472.
\(^{52}\) [1958] 2 Q.B. 130.
\(^{53}\) [1958] 1 All E.R. 547.
\(^{54}\) *Id.*
\(^{55}\) *Id.* at 542.
\(^{56}\) *Id.*
\(^{57}\) *Id.*
\(^{58}\) *Id.*
\(^{59}\) The argument could be made, however, that the Queen's Bench decision in *Ian Stach* does not have any impact upon the decisions in the Court of Appeals. *But see*
C. A Seller's Right to Rescind Under a Documentary Credit Clause
With respect to c.i.f. contracts, Benjamin states: "The seller can rescind if the buyer fails to comply with a requirement of the contract to provide a bank guarantee for payment, or a confirmed credit,"\(^{60}\) and, with respect to f.o.b. contracts: "[T]he general rule is that any failure of the buyer, unless waived, as to the time and place . . . justifies rescission . . . ."\(^{61}\) A literal reading of Benjamin's restatement of the rule would imply that stricter compliance with the terms of a f.o.b. contract is required. Thus, it may be inferred that the range of instances which would trigger a seller's right to immediately rescind is broader under f.o.b. contracts than it is under c.i.f. contracts since "any failure" entitles the seller to rescind the contract. The case law, however, does not appear to recognize this distinction, and it is doubtful whether Benjamin intended such a literal interpretation.\(^{62}\)
In *Trans Trust S.P.R.L. v. Danubian Trading Co., Ltd.*, \(^{63}\) the seller sued for damages caused by the buyer's failure to establish a letter of credit. Without reference to whether the contract was c.i.f. or f.o.b., Lord Denning observed:
[T]he seller stipulates that the credit should be provided at a specified time well in advance of the time for delivery of the goods. What is the legal position of such a stipulation? Sometimes it is a condition precedent to the formation of a contract, that is, it is a condition which must be fulfilled before any contract is concluded at all. In those cases the stipulation 'subject to the opening of a letter of credit' is rather like a stipulation 'subject to the contract.' If no credit is provided, there is no
---
\(^{60}\) *Benjamin's Sales of Goods*, *supra* note 30, § 2170, which states that the cases do not lay down a conclusive rule either in the case of f.o.b. contracts (*see infra* note 61) or in the case of c.i.f. contracts:
A documentary credit furnished at a reasonable time before the commencement of the shipping period enables him to prepare the goods for punctual shipment. He can also use the credit in order to furnish, right at the beginning of the shipping period, a back-to-back credit in favour of an ultimate supplier of the goods.
*Id.*
\(^{61}\) *Id.* § 1751.
\(^{62}\) *Id.* § 1863. The meaning of f.o.b. is free on board at some location, such as f.o.b. shipping point or f.o.b. destination. The invoice price includes delivery at the seller's expense to that designated location. *Black's Law Dictionary* 578 (5th ed. 1979).
\(^{63}\) *See Benjamin's Sales of Goods*, *supra* note 30, § 1863. In fact, Benjamin notes that "the rate is not utterly inflexible . . . ." *Id.*
\(^{64}\) [1952] 1 All E.R. 970.
contract between the parties. In other cases a contract is concluded and the stipulation for a credit is a condition which is an essential term of the contract. In those cases the provision of the credit is a condition precedent, not to the formation of a contract, but to the obligation of the seller to deliver the goods. If the buyer fails to provide the credit, the seller can treat himself as discharged from any further performance of the contract. . . .
The facts in *A.E. Lindsay & Co., Ltd. v. Cook* involved a buyer's failure to satisfy what was characterized as an implied condition precedent to the contract. A seller in Australia contracted to sell frozen chickens to a buyer in England. Delivery was to be made by a ship due to sail on September 28, 1951. Payment was to be made by credit, to be opened "immediately" in favor of the seller at the Rural Bank of Sydney. Although the buyer instructed his bank to open the credit with the Rural Bank on September 24, the credit was not opened until October 6. By that time the ship had already set sail without the buyer's shipment. The buyer sued for damages due to non-delivery. Although the court voiced its sympathy toward the plaintiffs, who did their best to open the credit but failed to do so because of an "unfortunate conjunction of circumstances," it held that it was an implied condition precedent to the contract that the buyer would open the credit before the seller shipped the goods. The court found that the condition precedent was not satisfied, as a result of which the seller repudiated the contract and sold the chickens to a third party. The court reasoned that "[i]t is quite clear . . . that the defendant was entitled to do what he did."
The rule applied in *A.E. Lindsay & Co.* has implicitly controlled later decisions, including those where the buyer's delay in opening the letter of credit was characterized merely as a failure to satisfy a condi-
---
64. *Id.* at 976. Lord Denning further noted that he regarded "the provision of a credit as different from the payment of money and not subject to the special rules . . . relating thereto." *Id.* at 977.
65. [1953] 1 Lloyd's List L.R. 328.
66. *Id.* at 331.
67. *Id.*
68. *Id.*
69. *Id.* The Rural Bank was not known to buyer's bank, which instead opened the credit with the New South Wales Bank in Sydney. The New South Wales Bank was then, according to its contract with buyer's bank, required to open the credit at Rural Bank. The credit, however, was not opened at the New South Wales Bank until September 28, and the Rural Bank was not notified until October 6. *Id.* at 332.
70. *Id.* at 335.
71. *Id.*
tion precedent to delivery of the goods. In *Etablissements Chainbaux S.A.R.L. v. Harbormaster, Ltd.*,72 the buyer sued the seller for failing to deliver the goods. The court determined that: “[t]he furnishing of a letter of credit was a condition precedent to the delivery of the goods, and therefore *prima facie* if they want to establish the breach of delivering the goods they must allege that they have fulfilled the necessary condition precedent, that they have furnished the letter of credit.”73
The contract indicated that the buyer was to open the credit within a few weeks.74 The court estimated a reasonable time therefrom and held that since the buyer had not opened the letter of credit within that time the sellers were justified in canceling the contract.75
In *Enrico Furst & Co. v. W.E. Fischer, Ltd.*,76 one of the issues raised was whether the buyer’s breach of his obligation to open the letter of credit within the time agreed upon was waived by the seller. With regard to this issue, the court stated:
If by his conduct the seller leads the buyer to suppose, and to act upon the supposition, that he, the seller, will not insist upon the opening of the credit within the specified time, he waives his right to treat the failure to open the credit within the specified time as a breach of condition entitling him to repudiate the contract but he does not thereby waive his right to have the credit of the particular kind opened within a reasonable time, and to treat failure to open it as a breach of condition if it is not opened in a reasonable time.77
**D. English Law in Comparison with the Convention**
As noted above, four types of cases relevant to this inquiry arise under English law. Each group offers guidance in refining the Convention’s definition of fundamental breach in article 2578 and the right to the remedy provided in article 64(1)(a)79 of the Convention.
Freedom of contract is the prevailing principle under both English law and the Convention.80 Thus, merchants can, in most circumstances, explicitly provide for their rights and remedies in the event of a breach
---
72. [1955] 1 Lloyd’s List L.R. 303.
73. *Id.* at 310.
74. *Id.* at 311.
75. *Id.*
76. [1960] 2 Lloyd’s List L.R. 340.
77. *Id.* at 345.
78. Convention, *supra* note 1, art. 25.
79. *Id.* art. 25.
80. Convention, *supra* note 1, art. 6.
of the contract. Additionally, the English Sale of Goods Act and English common law remedies reflect the same basic ideas that underlie the definition of fundamental breach in article 25.\textsuperscript{81} Under both laws, three factors appear determinative of whether the breach is fundamental or not: the perishability of the goods; whether the buyer repeatedly fails to pay on seller's demand; and whether the buyer is given a reasonable time.\textsuperscript{82} Thus, in these cases, determining if and when a seller may avoid the contract is particularly dependent on the configuration of facts presented by the individual case.\textsuperscript{83}
The cases under English law, where a right to terminate arises as a result of the buyer's failure to comply with contract provisions as to method of payment, however, do not fit into this system of case by case judgments.\textsuperscript{84} A "per se" rule of fundamental breach is applied to the cases that fall within this group.\textsuperscript{85} Any breach as to the opening of a letter of credit (or the establishment of a bank guarantee) is in itself so important that it gives the seller the right to cancel the contract, or in the words of the Convention, any such breach is "fundamental."\textsuperscript{86} The Convention's language, however, does not provide the seller with an automatic right to rescind in these circumstances.\textsuperscript{87} In fact, article 54 of the Convention obligates the buyer to pay the price and complete the "steps" and "formalities" required to enable payment to be made, and thus clarifies that there should not be a specific, independent approach to these steps and formalities.\textsuperscript{88} Of course, there might also be cases under the Convention where \textit{any} delay in establishing a letter of credit will be considered a fundamental breach, but then the judgment will be based upon the terms of the contract and the factual situation of the case, and not on a per se rule.
\begin{itemize}
\item[81.] \textit{Id.} art. 25.
\item[82.] \textit{See supra} notes 36-77 and accompanying text. \textit{See also} Benjamin's Sales of Goods, \textit{supra} note 30, §§ 1231-1256 (discussing fundamental breach as defined by English common law and also reviewing the statutory remedy under the Sale of Goods Act); J. Honnold, Uniform Law For International Sales § 185 (1982); Parker Sch. of Foreign & Comp. Law of Colum. U. Int'l Sales: The U.N. Convention on Contracts For The International Sale of Goods § 9.03(d) (1984) [hereinafter cited as Parker School].
\item[83.] \textit{See generally supra} note 82.
\item[84.] \textit{See} Benjamin's Sales of Goods, \textit{supra} note 30, § 1751 (1981).
\item[85.] \textit{Id.}
\item[86.] Convention, \textit{supra} note 1, art. 25.
\item[87.] \textit{Id.} art. 54.
\item[88.] J.Honnold, \textit{supra} note 86, § 323: "The failure to take one of the required steps 'to enable payment to be made' itself constitutes a breach of contract." \textit{Id. See also} Parker School, \textit{supra} note 82, § 7.02.
III. The Law Under the Uniform Commercial Code
A. In General
The Uniform Sales Act, in section 65, the precursor to the Uniform Commercial Code (U.C.C.), stated: "where the goods have not been delivered to the buyer, and the buyer has . . . committed a material breach [of the contract], the seller may totally rescind the contract or the sale."89
The question of "whether a breach [was] material or substantial [under § 65 depended] on the terms of the contract and on the facts and circumstances of the case."90 The terms of the contract stated, e.g., whether or not time was of the essence. If payment was to be made in advance or concurrent with delivery, it was inferred that time was of the essence in the contract. If, however, the sale was on credit the opposite inference was taken.
The U.C.C. provides that payment is due in accordance with the contract, or otherwise at the time of delivery.91 If payment is due on or before delivery, and the buyer fails to make the payment when due, the remedies afforded the seller are stated in Section 2-703.92 Section
---
89. Uniform Sales Act § 65, 1A U.L.A. 233 (1950).
90. 77 C.J.S. Sales § 233 (1952).
91. U.C.C. § 2-301 (1983) states: "The obligation of the seller is to transfer and deliver and that of the buyer is to accept and pay in accordance with the contract." Id. § 2-310 provides:
Unless otherwise agreed
(a) payment is due at the time and place at which the buyer is to receive the goods even though the place of shipment is the place of delivery; and
(b) if the seller is authorized to send the goods he may ship them under reservation, and may tender the documents of title, but the buyer may inspect the goods after their arrival before payment is due unless such inspection is inconsistent with the terms of the contract (Section 2-513); and
(c) if delivery is authorized and made by way of documents of title otherwise than by subsection (b) then payment is due at the time and place at which the buyer is to receive the documents regardless of where the goods are to be received; and
(d) where the seller is required or authorized to ship the goods on credit the credit period runs from the time of shipment but post-dating the invoice or delaying the starting of the credit period.
Id.
92. Id. § 2-703 provides:
Where the buyer wrongfully rejects or revokes acceptance of goods or fails to make a payment due on or before delivery or repudiates with respect to a part or the whole, then with respect to any goods directly affected and, if the breach is of the whole contract (Section 2-612), then also with respect to the whole un2-703 states that failure to make payment includes the dishonor of a check, the nonacceptance of a draft and the failure to furnish an agreed upon letter of credit.\textsuperscript{93} The section further states that: "[w]here the buyer . . . fails to make a payment due on or before delivery," the seller may, among other remedies, re-sell the goods or cancel the contract.\textsuperscript{94} Hence, it seems as if the seller would have an absolute right to re-sell or cancel, independent of whether the breach is material. Nothing in the comment to the U.C.C., however, indicates that such a radical change in the law was intended. One commentator has observed, "the mere fact that section 2-703 states that the seller may cancel, in absolute terms, would be immaterial and really ineffective to provide any substantive right."\textsuperscript{95}
The question of materiality was dealt with in \textit{Nora Springs Cooperative v. Braudau},\textsuperscript{96} where the alleged breach was the buyer's nonacceptance of delivery. One of the buyer's defenses was that the breach was too insubstantial to give the seller the right to cancel the contract.\textsuperscript{97} The court stated that under U.C.C. 2-703(f):
where the buyer breaches the contract the aggrieved seller may "cancel." "Cancellation" occurs when either party puts an end to the contract for breach by the other. Nothing is mentioned about \textit{materiality} of the breach as argued by [the buyer]. Thus, when read together with [1-106] which mandates that remedies are to be "liberally administered to the end that the aggrieved party may be put in as good a position as if the other party has fully performed," we might very well hold that \textit{materiality} need not be shown to warrant cancellation . . . .
However, even if, arguendo, materiality is a requirement for cancellation under the theory that the general principles of law merchant
\begin{itemize}
\item delivered balance, the aggrieved seller may
\begin{itemize}
\item withhold delivery of such goods;
\item stop delivery by any bailee as hereinafter provided (Section 2-705);
\item proceed under the next section respecting goods still unidentified to the contract;
\item resell and recover damages as hereafter provided (Section 2-706);
\item recover damages for nonacceptance (Section 2-708) or in a proper case the price (Section 2-709);
\item cancel
\end{itemize}
\end{itemize}
\textsuperscript{93} \textit{Id.} comment 3.
\textsuperscript{94} \textit{See supra} note 92 and accompanying text.
\textsuperscript{95} [3A Sales & Bulk Transfers] U.C.C. Serv. (Bender) § 13.02, at 1 (1984).
\textsuperscript{96} 247 N.W.2d 744 (Iowa 1976).
\textsuperscript{97} \textit{Id.} at 747.
\textsuperscript{98} \textit{Id.} at 749.
supplement the specific provisions of the [U.C.C.] section [1-103], [the buyer's] contention is still unconvincing.
The decisions in *Mott Equity Elevator v. Svihovec* and *Ziebarth v. Kalenze* are in accord with this theory. Both cases involve the buyer's non-acceptance of delivery of the goods. In *Mott*, the court held that "the buyer breached the agreement in not accepting delivery within a reasonable time, giving rise to [the seller's] right to cancel under [2-703]." In *Ziebarth*, the court reaffirmed this reasoning and held that the buyer "breached the agreement by not picking up [the goods] within a reasonable time."
The reasoning in these cases, which define breach as the buyer's nonacceptance of delivery within a reasonable time, clearly indicates that the court incorporated a material breach requirement into section 2-703(f).
Since, as a practical matter, resale and cancellation have the same effect on a contract, it is also helpful to review cases involving resale upon a buyer's failure to pay the price. In *Mott*, the court said: "[t]he only condition precedent to the seller's right to resell is a breach by the buyer within section 2-703." The same rule is expressed in other decisions. The result in *Mott* and its successors cannot, however, have been intended by the legislators. The comment to section 2-703 specifically states that the article rejects any doctrine of election of remedy, and that the remedies in the section are cumulative. Consequently, it would be quite possible for a seller, in the case of a buyer's failure to pay, to first cancel the contract and then resell the goods and recover damages. If that were the case, then a material breach would be required in order to cancel the contract, whereas any breach would justify a resale of the goods under the same contract. Clearly, this result could not have been intended. If the requirement for cancellation is a
---
99. 236 N.W.2d 900 (N.D. 1975).
100. 238 N.W.2d 261 (N.D. 1976).
101. 236 N.W.2d at 909 [emphasis added].
102. 238 N.W.2d at 269 [emphasis added].
103. 236 N.W.2d at 908.
104. See, e.g., Desbien v. Penokee Farm Union, 220 Kan. 358, 552 P.2d 917 (1976); Clock v. Missouri-Kansas-Texas Railroad, 407 F. Supp. 448 (E.D. Mo. 1976).
105. U.C.C. § 2-703 (1983) (comment 1):
This section . . . gathers together in one convenient place all of the various remedies open to a seller for any breach by the buyer. This Article rejects any doctrine of election of remedy as a fundamental policy and thus the remedies are essentially cumulative in nature and include all of the available remedies for breach. Whether the pursuit of one remedy bars another depends entirely on the facts of the individual case.
material breach, then in order to justify a resale the breach would also have to be material.
B. The U.C.C. in Comparison with the Convention
It is not clear whether the Convention's concept of fundamental breach is interchangeable with the U.C.C.'s definition of material breach. As mentioned previously, the Convention's definition of fundamental breach is interpreted of late in a more objective manner. The U.C.C. does not make certain whether a material breach is always required and, if so, whether the materiality of the breach is meant to be a subjective standard. Some courts have interpreted a materiality requirement into U.C.C. section 2-703; those decisions are the only available evidence of the standards used in applying the requirement. Unfortunately, these decisions do not clarify whether an objective or subjective standard governs under the Code. If subjective criteria determine whether a material breach has occurred, it may be impossible to use cases arising under the Code as guidance for the interpretation of the Convention's objectively determined concept of fundamental breach.
IV. Swedish Law
A. In General
The Swedish Sale of Goods Act of 1905\textsuperscript{106} was prepared in consultation with Denmark and Norway; these countries enacted sales acts nearly identical to Sweden's shortly after promulgation of the Swedish Act.\textsuperscript{107} Hence, the following analysis can be analogously applied to all three countries.
Section 28 of the Swedish Act states in part:
Where the price is not paid in due time or the buyer fails to take a measure on which the payment of the price depends, the seller may, at his option, avoid the contract of purchase or demand its performance; if the delay is insignificant, the purchase may not be repudiated, unless it is a commercial sale.\textsuperscript{108}
The applicability of the section is limited to cases where the goods are
\begin{itemize}
\item \textsuperscript{106} Swedish Sale of Goods Act of 1905, 1961-62 Int'l Inst. for Unification of Private Law, Unification of Law Y.B. 203 (UNIDROIT).
\item \textsuperscript{107} Id. at 203 n.1.
\item \textsuperscript{108} Id. § 28.
not yet in the buyer's possession.\textsuperscript{109}
The scope of the provision encompasses not only cash payments, but "measure[s] on which the payment of the price depends."\textsuperscript{110} These measures might include, for example, the acceptance of a draft or the issuance of a letter of credit.\textsuperscript{111} The seller's right under section 28 is also limited by the requirement that the delay must be significant, unless the sale is a commercial sale (i.e., between two merchants).\textsuperscript{112} In a commercial sale or trade purchase, any delay entitles the seller to avoid the contract.\textsuperscript{113} According to the commentary, in commercial trade even an insignificant delay of payment is of great importance.\textsuperscript{114} "Thus, [a] seller may . . . lawfully discharge himself from further performance under the contract even if payment is tendered the day after it is due."\textsuperscript{115}
Today, this absolute right of the seller is considered too harsh. In a draft for a new Swedish Sale of Goods Act (the Draft),\textsuperscript{116} "the seller [may] declare the contract avoided if the breach is 'fundamental' but not, as under the present Act, [for] any delay in a commercial sale."\textsuperscript{117} As an exception to this provision, the Draft presents another "new" rule. When the buyer is obliged to pay against documents representing the goods, the seller may declare the contract avoided even though the breach is not fundamental.\textsuperscript{118} "This rule follows commercial practice."\textsuperscript{119}
Even though the Draft has not yet led to legislation, there is no reason to neglect these "new influences" in Swedish law. Rather, one might assume that these new rules will be kept intact, especially since they reflect modern commercial practice.\textsuperscript{120} If this assumption is correct, Swedish sale of goods law will provide that the buyer's delay in
\begin{itemize}
\item \textsuperscript{109} \textit{Id.} Paragraph 2 of § 28 reads: "After the goods have come into the buyer's possession, the seller is not entitled to repudiate the purchase . . . unless he has reserved his right to do so or the buyer has rejected the goods." \textit{Id.}
\item \textsuperscript{110} \textit{Id.}
\item \textsuperscript{111} See generally notes 28-29 and accompanying text.
\item \textsuperscript{112} Swedish Sale of Goods Act, art. 28. As defined by the Swedish Act, a "trade purchase" is a purchase "concluded between merchants in the course of their trade." \textit{Id.} art. 3.
\item \textsuperscript{113} \textit{Id.} art. 28.
\item \textsuperscript{114} Almen, \textit{Om Köp och byte av lös egendom}, 1960 P.A. Norstedts och Söners Förlag 402.
\item \textsuperscript{115} \textit{Id.} at 402.
\item \textsuperscript{116} Köplag. Slutbetänkande av Köplagsutredningen. 1976:66, § 6.3.3.
\item \textsuperscript{117} Hellner, \textit{The Draft to a New Swedish Sale of Goods Act}, in 1978 Scandinavian Studies in Law 69 (1978).
\item \textsuperscript{118} \textit{Id.}
\item \textsuperscript{119} \textit{Id.}
\item \textsuperscript{120} \textit{Id.}
payment must be significant or fundamental in order to entitle the seller to avoid the contract. In the application of such a rule, case law and other authorities concerning noncommercial sales contracts under section 28 may be helpful, because traditionally only a material breach by a noncommercial buyer would entitle a seller to avoid the contract.\textsuperscript{121}
Section 28 provides that the delay is to be considered from the seller's perspective.\textsuperscript{122} The commentary stresses that the judgment should be made as to the effect of the breach "in concreto."\textsuperscript{123} According to the commentary, primary considerations are the length of the delay and whether the seller made any demand of payment or other indicia of the necessity of prompt payment.\textsuperscript{124}
Two Norwegian cases have turned on whether the buyer's breach was fundamental. In \textit{N.R.T. 1921 s. 513},\textsuperscript{125} the court emphasized the type of goods (shares of stock) and the seller's continued demand for payment as important factors giving the seller the right to avoid the contract. In \textit{N.R.T. 1925 s. 566},\textsuperscript{126} where the court also held the delay significant, the length of the delay, the buyer's knowledge of the importance of timeliness and the seller's interest in closing the contract were determinative factors in the decision.
\textit{B. Swedish Law in Comparison with the Convention}
Currently, Swedish law provides that any delay in a commercial sale entitles the seller to avoid the contract.\textsuperscript{127} This, of course, differs greatly from the rule of the Convention.\textsuperscript{128} As pointed out above, however, this is a Swedish regulation born at the beginning of the century, and hence the difference is more the result of archaic legislation than an intended approach toward this type of buyer's breach.\textsuperscript{129}
Apart from the exception for payment against documents, the rules in the Draft largely follow the Convention as to materiality under section 28. The commentary suggests that the issue should be determined according to subjective criteria. The case law indicates as determinative the length of the delay, the seller's demand or other indica-
\begin{itemize}
\item \textsuperscript{121} See supra note 108 and accompanying text.
\item \textsuperscript{122} Bengtsson, \textit{Hävningsrätt och uppsägningsrätt vid Kontraktsbrott}, 1967 P.A. Norstedts och Söners Förlag 270.
\item \textsuperscript{123} Almen, \textit{supra} note 114, at 402.
\item \textsuperscript{124} \textit{Id.} at 139.
\item \textsuperscript{125} See Bengtsson, \textit{supra} note 122, at 270.
\item \textsuperscript{126} \textit{Id.} Hellner, \textit{supra} note 117, at 69.
\item \textsuperscript{127} See supra notes 108 & 114-15 and accompanying text.
\item \textsuperscript{128} See Convention, \textit{supra} note 1, arts. 25 & 64.
\item \textsuperscript{129} See supra notes 116-20 and accompanying text.
tions that time is of the essence, and the type of goods involved. These factors may also be of great importance in decisions under article 25 of the Convention. However, due to the subjective approach under section 28, the potential influence Swedish law could have on the development of a working definition of fundamental breach under the Convention may be vastly diluted.
V. STANDARD CONTRACTS
A review and appraisal of domestic legal decisions and laws is helpful in interpreting article 25 of the Convention; it gives the concept of fundamental breach a more practical meaning. Guidelines deduced from this appraisal indicate the specific factors which national courts have emphasized as important to their decisions.
Another way of approaching the problem is to study provisions in contracts normally used in international sales. The Convention gives legal effect to commercial usages and practices. Under article 9(2) customary usages are read into the contract.\textsuperscript{130} The usage must be one "of which the parties knew or ought to have known and which in international trade is widely known to, and regularly observed by, parties to contracts of the type involved in the particular trade concerned."\textsuperscript{131}
If the standard contracts\textsuperscript{132} used in international trade fulfill the requirements of article 9(2)\textsuperscript{133} then these contracts may be helpful in defining "fundamental breach" as used in the Convention. Of course, a provision in a standard contract does not in itself establish "a usage," but it does provide a strong indication of custom and usage. Moreover, even if the provision (or the contract) is such that it does not meet the required standards for "a usage," it is still possible that a court would take it into consideration when determining whether the buyer's delay was reasonable. In addition, comparison of standard contracts indicates how merchants treat delays by a buyer and how such treatment differs with the type of transaction and goods which are sold.
Following are a few examples of provisions in standard contracts concerning this issue. The ECE General Conditions for the Supply of Plant and Machinery for Export (No. 188) states at article 8.7: "if at the end of the period fixed in paragraph F of the Appendix, the Purchasers still have failed to pay the sum due, the Vendor shall be enti-
\begin{itemize}
\item[130.] Convention, \textit{supra} note 1, art. 9(2).
\item[131.] \textit{Id}.
\item[132.] These agreements are normally drafted by an international organization (such as the United Nations), an organization of sellers in the same field or by a seller with substantial sales.
\item[133.] Convention, \textit{supra} note 1, art. 9(2).
tled by notice in writing to the Purchaser, and without requiring the consent of any court, to terminate the contract . . . ."134 Paragraph F of the appendix allows the parties to state the "period of delay in payment authorizing termination of vendor [in] . . . months."135
The ECE General Conditions for the Supply and Erection of Plant and Machinery for Import and Export (No. 188A)136 are similar to the provision in No. 188.137 The period of delay, however, is set at three months.138
Both of these contracts allow the buyer a long period of delay before the seller may cancel.139 This is, of course, due to the type of goods and kind of transaction concerned. Undoubtedly, this could be used as a guideline for a court in its interpretation of fundamental breach in article 25.
Conversely, when a transaction involves perishable goods, the period of permissible delay is shorter. Article 56 of the ECE General Conditions for Fresh Fruit and Vegetables Including Citrus Fruits, for instance, states:
failure to comply with the payment clauses contained in the contract shall give the seller the right to serve notice on the purchaser calling upon him to make payment within 24 hours, and notifying him that at the end of such period he reserves the right to suspend subsequent deliveries or to terminate the contract . . . .140
Moreover, article 44(i) of the ECE General Conditions for Dry and Dried Fruits advises:
If the documents are not taken up by the buyer within the prescribed time limit [24 hours following the presentation of the invoice or the documents], the seller shall be entitled, after giving notice within the two working days following the expiry of the said time limit, to terminate the contract . . . .141
---
134. U.N./ECE General Conditions for the Supply of Plant and Machinery for Export, 1953 Econ. Commission Euro. No. 188, U.N. Doc. E/ECE/ME88.
135. Id.
136. U.N./ECE General Conditions for the Supply and Erection of Plant and Machinery for Import and Export, No. 188A, U.N. Doc. E/ECE/ME/188A (1957).
137. Id. appendix, para. F. See also ECE No. 188, supra note 134, para. F.
138. See supra note 136, appendix, para. F.
139. See supra note 137 and accompanying text.
140. U.N./ECE General Conditions for Fresh Fruit and Vegetables Including Citrus Fruit, U.N. Doc. ECE (1979).
141. U.N./ECE General Conditions of Sale for Dry (Shelled and Unshelled) and Dried Fruit, U.N. Doc. ECE/AGRI/41 at 23 (1979). The General Conditions of Sale reFurthermore, the ECE General Conditions for Potatoes declares in article 47(i):
any delay in payment, non-payment or failure to open a bank credit by the date specified in the contract shall entitle the seller to call upon the buyer to perform his obligation within three working days after receipt of the communication from the seller, warning him that, on the expiry of the time limit, the seller may suspend deliveries or terminate the contract.\(^{142}\)
It should be noted that in none of these three provisions is any delay, per se, a breach entitling the seller to avoid the contract. Even when the goods are of such a perishable nature as fresh fruit, the buyer is permitted twenty-four hours to tender payment.\(^{143}\)
The CMEA General Conditions provide in sections 67(2) & (3): “The seller shall be obliged to give the buyer additional time to open a letter of credit... If the buyer fails to open the letter of credit even within the additional time, the seller shall have the right to cancel the contract.”\(^{144}\)
This provision is close to a requirement of fundamental breach. The requirement that the seller must give the buyer additional time is another way of saying that not just *any* delay is enough to give the seller the right to cancel the contract. The difference is that, under the CMEA provision, additional time must always be granted, whereas under article 25, at least theoretically, any delay might be a fundamental breach.\(^{145}\)
Article 24 of General Contract (No. 1) of the Grain and Feed Trade Association provides: “[i]n default of fulfillment of contract by either party, the other, at his discretion, shall, after giving notice by letter, telegram, or telex, have the right to sell or purchase as the case
---
ferred to herein for various agricultural products are prepared by “the Group of Experts on International Trade Practices Relating to Agricultural Products, a subsidiary organ of the Working Party on Standardization of Perishable Produce, which is itself a subsidiary body of the Committee on Agricultural Problems of the United Nations Economic Commission for Europe.” *Id.* at 1.
142. U.N./ECE General Conditions of Sale for Potatoes, U.N. Doc. ECE/AGRI/42 at 28 (1980).
143. See U.N./ECE General Conditions for Fresh Fruit, *supra* note 140.
144. U.N./ECE General Conditions of Delivery of Goods Between Organizations of the Member Countries of the Counsel for Mutual Economic Assistance, U.N. Doc. ECE (1968).
145. Convention, *supra* note 1. Article 25 does not require additional time to be given to the buyer, but only that the three criteria be met. See *supra* note 21 and accompanying text.
may be, against the defaulter . . . ."146 This right to resell the goods affords the seller the same practical result as if he had a right to cancel the contract. Resale, however, must be preceded by notification to the buyer by letter, telegram or telex.147 This provision grants the seller a surprisingly extensive right, as the mere transmittal of a telex is a simple procedure. Thus, this provision provides the seller with nearly an absolute right to resell.
The Official Contract Form for Hide and Skin Sellers and Tanners provides in article 12:
if the buyer fails to make payment in accordance with the terms laid down in this contract and such failure is fairly attributable to government restrictions imposed after the signing of this contract, the seller may, at any time before tender of payment in full by the buyer, and after giving seven days' notice to the buyer, cancel this contract . . . .148
Under this contract, the seller's right to cancel because of nonpayment is limited to cases where the failure to pay is due to governmental restrictions imposed after the signing of the contract.
The governmental restrictions provision raises another issue. Suppose that such a restriction of the seller's right would be a usage covered by article 9(2).149 The restriction is then applicable to the contract and prohibits the seller from canceling when the delay of payment occurs for any reason other than governmental restrictions. If, however, the delay is not due to governmental restrictions, whether the requirements of article 25 would require a fundamental breach is unclear. In this case the situation is governed by both usage—not entitling the seller to cancel—and article 25—entitling avoidance. The usage prevails here, however, since it is impliedly made applicable to the contract (article 9(2)) and under article 6 the parties are permitted to derogate from the provisions of the Convention.150
146. The Grain and Feed Trade Association, General Contract No. 1 (Aug. 1, 1976) (available at Baltic Exchange Chambers, 28 St. Mary Ave., London, EC3A 8EF).
147. Id.
148. The International Counsel of Hide and Skin Sellers Associations and the International Council of Tanners, Official Contract Form No. 5 (1973).
149. Convention, supra note 1, art. 9(2).
150. J. HONNOLD, supra note 20, at 149. Articles 9(2) and 6 provide that a party may derogate from the provisions of the Convention. See supra notes 9, 79, 130, 133, 149 and accompanying text for a discussion of these provisions.
CONCLUSION
The basic rule of the Convention, that a seller may avoid the contract if the buyer's breach amounts to a fundamental breach, has its counterpart in the legal systems of England, the United States and Sweden. Although the basic rule is the same, the differences in commercial practice and behavior are influential for each nation's interpretation of fundamental breach under article 25. The major factor the courts of England, the United States and Sweden have considered in determining whether a breach is fundamental (material or significant) is the length of the delay. But the length of the delay is not meaningful until the reasonableness of the delay is established. Reasonableness, in turn, depends upon other factual circumstances.
Both English and Scandinavian courts have emphasized the importance of the type of goods concerned. In a transaction involving goods, such as shares of stock or codfish, the buyer will be required to meet his obligations expeditiously. Moreover, English law provides that repeated failure by the buyer to pay on demand might give the seller an immediate right of cancellation. Even when this rule is not applied, the seller, by repeating his demand for payment or by otherwise showing the buyer that time is of the essence, can shorten the payment period to the extent a court will deem reasonable.
Finally, standard commercial contracts also clearly indicate how time periods differ depending on the goods sold under the agreement. In the case of perishable goods, the seller may avoid the contract after a twenty-four hour delay; the time period for a seller of plants and machinery could be months or years.
While the determination of whether a breach is fundamental will be decided on a case by case basis, depending on the factual circumstances, domestic and private law consistently recognize as important certain factors. Given the ambiguity in article 25 and the international community's interest in promoting commercial stability, courts should consider these general norms in interpreting and applying article 25 of the Convention.
|
CUSTOMS MEMORANDUM CIRCULAR
NO. 150-2019
SUBJECT: Intellectual Property Recordation of JUUL LABS INC. Trademarks
Attached are copies of Certificates of Registration No. 4-2018-011466, 4-2018-022134 & 4-2018-022133 certified as true copies by the Intellectual Property Office on 26 April 2019, issued by the Bureau of Trademarks. The subject trademarks as submitted to this Office by Lee Bumgarner of BETITA CABILAO CASUELA SARMIENTO, are in the records of the above-mentioned agencies, as follows:
a. Mark
Registration No.: 4-2018-00022133
Date of Registration: 17 March 2019
Date of Expiration: 17 March 2029
Goods/Services: (CLASS 09) Downloadable software for mobile devices for use with an oral vaporizer for smoking purposes, namely, software for remotely adjusting and saving vaporizer temperature settings and updating vaporizer firmware; accessories
for electric vaporizers for the vaporization of herbal and plant matter for household purposes, namely, charging cases, electrical adapters, and power cords; batteries and electric accumulators for electric and electronic cigarettes; battery chargers for use with electronic and electric cigarettes; USB chargers for electric and electronic cigarettes; car chargers for electric and electronic cigarettes; electronic devices and appliances for charging and carrying electronic and electric cigarettes; computer software for use in posting, transmitting, retrieving, receiving, reviewing, organizing, searching and managing text, audio, visual and multimedia data and content via computers, mobile phones, wired and wireless communication devices, and optical and electronic communications networks; computer software for calculating, mapping, transmitting and reporting information relating to the location, movement, proximity, departure and arrival of individuals and objects via computers, mobile phones, wired and wireless communication devices, and optical and electronic communications networks; as far as included in Class 9 and not in other classes
(CLASS 34) Nicotine-based liquid, namely, liquid nicotine used to refill electronic cigarettes; cartridges sold filled with liquid nicotine for electronic cigarettes; electronic cigarette refill liquids, namely, chemical flavorings in liquid form used to refill electronic cigarettes; cartridges sold filled with chemical flavorings in liquid form for electronic cigarettes; electronic cigarettes; electronic smoking vaporizers, namely, electronic cigarettes; tobacco substitutes in liquid solution form other than for medical purposes for electronic cigarettes;
as far as included in Class 34 and not in other classes
b. Mark: JUUL
Registration No.: 4-2018-011466
Date of Registration: 22 November 2018
Date of Expiration: 22 November 2028
Goods/Services:
(CLASS 03) Downloadable software for mobile devices for use with an oral vaporizer for smoking purposes, namely, software for remotely adjusting and saving vaporizer temperature settings and updating vaporizer firmware; batteries and electric accumulators for electric and electronic cigarettes; battery chargers for use with electronic and electric cigarettes; usb chargers for electric and electronic cigarettes; car chargers for electric and electronic cigarettes; electronic devices and appliances for charging and carrying electronic and electric cigarettes; as far as included in Class 9 and not in other classes
(CLASS 11) Electric vaporizers for the vaporization of herbal and plant matter for household purposes and related accessories therefore, namely, covers, charging cases, adapters, and power cords; as far as included in Class 11 and not in other classes
(CLASS 34) Nicotine-based liquid, namely, liquid nicotine used to refill electronic cigarettes; cartridges sold filled with liquid nicotine for electronic cigarettes; electronic cigarette refill liquids, namely, chemical flavorings in liquid form used to refill
electronic cigarettes; chemical flavorings in liquid form, and tobacco substitutes in liquid solution form other than for medical purposes; cartridges sold filled with chemical flavorings in liquid form for electronic cigarettes; electronic cigarettes; electronic smoking vaporizers, namely, electronic cigarettes; tobacco substitutes in liquid solution form other than for medical purposes for electronic cigarettes; refill cartridges sold empty for electronic cigarettes; electric vaporizers, namely, smokeless vaporizer pipes for the ingestion and inhalation of tobacco and other herbal matter; electric vaporizers for the vaporization of tobacco; processed tobacco pods; pipe tobacco, namely, tobacco for use in electric vaporizers; tobacco, whether manufactured or unmanufactured; smoking tobacco, pipe tobacco, hand rolling tobacco, snus tobacco; tobacco sold in pods; smoker's articles for electric and electronic cigarettes namely, cases for electronic cigarettes and electronic cigarette accessories and boxes for electronic cigarettes and electronic cigarette accessories; components for electric and electronic cigarettes, namely, atomisers for tobacco substitutes, cartomisers for tobacco substitutes, clearomisers for tobacco substitutes; electric coils sold as a component of electric and electronic cigarettes and electronic smoking devices; as far as included in Class 34 and not in other classes
(CLASS 35) Retail store services featuring electric vaporizers and related consumer electronic devices, downloadable software for mobile devices for use with an oral vaporizer for smoking purposes, nicotine-based liquid, namely, liquid nicotine used to refill electronic cigarettes, cartridges
sold filled with liquid nicotine for electronic cigarettes, electronic cigarette refill liquids, namely, chemical flavorings in liquid form used to refill electronic cigarettes, chemical flavorings in liquid form, and tobacco substitutes in liquid solution form other than for medical purposes, cartridges sold filled with chemical flavorings in liquid form for electronic cigarettes, electronic cigarettes, electronic smoking vaporizers, namely, electronic cigarettes, tobacco substitutes in liquid solution form other than for medical purposes for electronic cigarettes, refill cartridges sold empty for electronic cigarettes, electric vaporizers, namely, smokeless vaporizer pipes for the ingestion and inhalation of tobacco and other herbal matter, electric vaporizers for the vaporization of tobacco, processed tobacco pods, pipe tobacco, namely, tobacco for use in electric vaporizers, tobacco, whether manufactured or unmanufactured, smoking tobacco, pipe tobacco, hand rolling tobacco, snus tobacco, tobacco sold in pods, smoker's articles for electric and electronic cigarettes namely, cases for electronic cigarettes and electronic cigarette accessories and boxes for electronic cigarettes and electronic cigarette accessories, components for electric and electronic cigarettes, namely, atomisers for tobacco substitutes, cartomisers for tobacco substitutes, clearomisers for tobacco substitutes, electric coils sold as a component of electric and electronic cigarettes and electronic smoking devices and demonstration of products relating thereto; providing consumer information in the field of electric vaporizers and related consumer electronic devices, downloadable software for mobile devices for use with an oral vaporizer for smoking.
purposes, nicotine-based liquid, namely, liquid nicotine used to refill electronic cigarettes, cartridges sold filled with liquid nicotine for electronic cigarettes, electronic cigarette refill liquids, namely, chemical flavorings in liquid form used to refill electronic cigarettes, chemical flavorings in liquid form, and tobacco substitutes in liquid solution form other than for medical purposes, cartridges sold filled with chemical flavorings in liquid form for electronic cigarettes, electronic cigarettes, electronic smoking vaporizers, namely, electronic cigarettes, tobacco substitutes in liquid solution form other than for medical purposes for electronic cigarettes, refill cartridges sold empty for electronic cigarettes, electric vaporizers, namely, smokeless vaporizer pipes for the ingestion and inhalation of tobacco and other herbal matter, electric vaporizers for the vaporization of tobacco, processed tobacco pods, pipe tobacco, namely, tobacco for use in electric vaporizers, tobacco, whether manufactured or unmanufactured, smoking tobacco, pipe tobacco, hand rolling tobacco, snus tobacco, tobacco sold in pods, smoker's articles for electric and electronic cigarettes namely, cases for electronic cigarettes and electronic cigarette accessories and boxes for electronic cigarettes and electronic cigarette accessories, components for electric and electronic cigarettes, namely, atomisers for tobacco substitutes, cartomisers for tobacco substitutes, clearomisers for tobacco substitutes, electric coils sold as a component of electric and electronic cigarettes and electronic smoking devices and demonstration of products relating thereto; as far as included in Class 35 and not in other classes
(CLASS 37) Maintenance, installation and repair of electric vaporizers, and related consumer electronic devices; as far as included in Class 37 and not in other classes
(CLASS 42) Troubleshooting in the nature of diagnosing problems with consumer electronics, namely, electric vaporizers, vaporizer software and peripherals, and related consumer electronic devices; consulting in the field of troubleshooting in the nature of diagnosing problems with consumer electronics, namely, electric vaporizers, vaporizer software and peripherals, and related consumer electronic devices; maintenance, installation and repair of software; as far as included in Class 42 and not in other classes
c. Mark: JUUL
Registration No.: 4-2018-022134
Date of Registration: 17 March 2019
Date of Expiration: 17 March 2029
Goods/Services: (CLASS 09) Downloadable software for mobile devices for use with an oral vaporizer for smoking purposes, namely, software for remotely adjusting and saving vaporizer temperature settings and updating vaporizer firmware; accessories for electric vaporizers for the vaporization of herbal and plant matter for household purposes, namely, charging cases, electrical adapters, and power cords; batteries and electric accumulators for electric and electronic cigarettes; battery chargers for use with electronic and
electric cigarettes; USB chargers for electric and electronic cigarettes; car chargers for electric and electronic cigarettes; electronic devices and appliances for charging and carrying electronic and electric cigarettes; computer software for use in posting, transmitting, retrieving, receiving, reviewing, organizing, searching and managing text, audio, visual and multimedia data and content via computers, mobile phones, wired and wireless communication devices, and optical and electronic communications networks; computer software for calculating, mapping, transmitting and reporting information relating to the location, movement, proximity, departure and arrival of individuals and objects via computers, mobile phones, wired and wireless communication devices, and optical and electronic communications networks; as far as included in Class 9 and not in other classes
(CLASS 34) Nicotine-based liquid, namely, liquid nicotine used to refill electronic cigarettes; cartridges sold filled with liquid nicotine for electronic cigarettes; electronic cigarette refill liquids, namely, chemical flavorings in liquid form used to refill electronic cigarettes; cartridges sold filled with chemical flavorings in liquid form for electronic cigarettes; electronic cigarettes; electronic smoking vaporizers, namely, electronic cigarettes; tobacco substitutes in liquid solution form other than for medical purposes for electronic cigarettes; as far as included in Class 34 and not in other classes
Having complied with the requirements of CAO-9-2008, the above-mentioned trademarks had been recorded in the IPR (Intellectual Property Rights)
Registry, Bureau of Customs, thus shall be accorded the rights and benefits emanating from such registration as therein provided.
The recordation of said trademarks shall be valid for two (2) years from signing hereof, and renewable every two years thereafter.
Date signed: 24/6/19
RANIEL T. RAMIRO
Deputy Commissioner, IG
THIS IS TO CERTIFY THAT THE ATTACHED DOCUMENT IS A TRUE COPY of the original
Certificate of Registration No. : 4-2018-011466
Trademark : JUUL
Registrant : JUUL LABS, INC.
Date of Registration : November 22, 2018
Taguig City, Philippines, this 26th day of April 2019
By Authority of the Director of the Bureau of Trademarks
MARY ANNE MUDLONG
Administrative Officer V
Bureau of Trademarks
THIS IS TO CERTIFY THAT THE ATTACHED DOCUMENT IS A TRUE COPY of the original
Certificate of Registration No. : 4-2018-022134
Trademark : JUUL
Registrant : JUUL LABS, INC.
Date of Registration : March 17, 2019
Taguig City, Philippines, this 26th day of April 2019
By Authority of the
Director of the Bureau of Trademarks
MARY ANNE MUDLONG
Administrative Officer V
Bureau of Trademarks
THIS IS TO CERTIFY THAT THE ATTACHED DOCUMENT IS A TRUE COPY of the original
Certificate of Registration No. : 4-2018-022133
Trademark :
Registrant : JUUL LABS, INC.
Date of Registration : March 17, 2019
Taguig City, Philippines, this 26th day of April 2019
By Authority of the
Director of the Bureau of Trademarks
MARY ANNE MUDLONG
Administrative Officer V
Bureau of Trademarks
748-2019 COR (2)
REPUBLIC OF THE PHILIPPINES
DEPARTMENT OF FINANCE
BUREAU OF CUSTOMS
Port of ____________________________
RECEIVED from _______________________
(Name of Payor)
BP
VAT No. ____________________________
(Address) ____________________________
Nature of Collection: Amount
1. Import Duty P _____________________
2. BIR Taxes _________________________
2.1 VAT ____________________________
2.2 Specific/Excise Tax ______________
3. Proceeds-Auction Sale ______________
4. Penalties/Surcharges Advance Duty ____
5. Miscellaneous (Specify) IPE __________
CBS __________
DISPER __________
6. Trust Liabilities REG __________
Postal Research Fee __________
TOTAL P ___________________________
Payment in cash payment of P200.00
DISTRIBUTION: Original and three copies
Original White - Duplicate (Green) - Triplicate (Yellow) - Quadruplicate (Blue)
JESUSA P. DELA CRUZ
Collecting Officer
(sign above printed name)
Date ____________________________
|
**CULTURAL DATA BANK**
**BARTLETT**
**KAOS PIRATE TV LIBRARY**
**POLITICAL:**
- ABC CBS NBC
- Kenya, Laos, Cambodia May 1970 2 hrs
- Demetrius & The Gladiators 2 hrs
- Samson & Delilah 2 hrs
- % Paths of Glory 1 hr
- % Nami The Killer Whale 1 hr
**ARTS/CULTURE:**
- How Life Begins 1 hr
**MISCELLANEOUS:**
- Current Events
- Riots, R. Reagan, B. Panthers . . .
- 3 Films in Production:
- The Serpent About the Sword
- Heavy Metal About the Gun
- Outerspace About the Rocket
- All Film from Broadcast TV and about American Culture. 3 Months of Taping—Filming America's Insanity. Because My Insanity & the Construction of these Films has been my Unraveling—Reinventing—Back to Earth.
**SCOTT AND FREUDE BARTLETT, 57 Harriet Alley, San Francisco**
**JACKIE CASSEN**
**PERFORMANCE:**
- Boston Symphony Video Experiment at WGBH. 2" Quadraplex
- Children's show. Color
- Messian "Quartet for End of Time" 3rd, 4th, & 8th movements. Oscilloscope patterns, sculptures, people. 1" Ampex
- Crumb--12 Shades 12 nudes choreographed. Sony ½"
- Faces 11 portraits. Sony ½"
- Wesson Oil Satire on video underground. Sony ½"
- Crumb Three tape interface on 4 monitors. Sony ½"
**PROCESS:**
- Nam June Paik Paik broadcasting at WGBH plus interviews. Sony ½"
- People's Video Community service program on 14th St. Interviews with Ken Burns and Robert Glass. Sony ½"
- Don Snyder Multi media "Rebirth of Planet Earth". Interviews with Jean Huston and a classmate of Hitler who happened to be Jewish. Sony ½"
**DOCUMENTATION:**
- Gibson Campaign New York episodes, video van, interviews. Sony ½"
- Street Music Six different groups of musicians of streets of NY. 1" Ampex or Sony ¾"
- Times Square New Years Eve 1970. Sony ¾"
**STEVE CHRISTIANSEN**
**OFF THE AIR:**
- Jerry Rubin On the Phil Donahue Show, April 1, 1970
- Bill Kunstler In debate with Henry L. Pitts, President of Illinois Bar Assoc. on the David Susskind Show
- Eldridge Cleaver CBS 80 Minutes
- Nixon Fairly complete set of speeches and press conferences
**DOCUMENTS:**
- Ann Arbor Blues Festival 1970
- Gary Snyder at Antioch
- Panther Doug Miranda at Antioch
- Dancing and Singing Choreography by Michael Fajans.
- Omega Point Omega Choreography by Brooke Higdon.
- Where is Columbia? Semi-documentary on Antioch's field center in Columbia, Md.
**CREATIONS:**
- Massage Montage—Mideke, Hilton, Cohen
- AutoPurge
- Consumption—Pollution
- La Dome of Fire A day of dome building
- Shopping Center Interviews
- Systems Bao (Forthcoming)
- Midwest Cultural Mosiac (Forthcoming)
**STEVE CHRISTIANSEN, Dept. of Instructional Systems, Antioch College, Yellow Springs, Ohio 45387 (513) 767-7331**
All tapes on either ½" Shiladen, or 2" Ampex format. Most of the Originals were made on Shiladen.
**TOM DeWITT**
Intraview 30 min Camera: Three broadcast Sept. 29, 1969. At WCBS TV 525 W. 57th., NYC. Contact office of Merrill Brookley for viewing.
Instant 20 min Excerpts from the Spring of '68, TV Doc. San Francisco State College. Contact Herb Zettl for viewing.
**ELECTRIC EYE**
Dick Gregory 2 hrs. Speaking at San Jose State College, November 1968.
Philo T. Farnsworth III 3 hrs. Interview with the son of the inventor of television, Philo T. Farnsworth II.
System of Teaching several Work Dr. Cattegato at San Jose State
Reading by Color hours
Coding Words 1 hr. Includes some of alpha's Zen and Yogi implications. Electric Zen?
Alpha Wave Research 30+ hrs. Hardly a Working Title! Political scene in the Pacific Northwest.
is Stanford
Showdown in Portland Meets the Sky River
Festival of Life and Lighter than Air Fair
**ELECTRIC EYE, 584 Park Court, Santa Clara, California 95050 (408) 244-3014; in San Francisco: (415) 826-7892**
Tim Barger, Skip Sweney, Jon Mandis, Jim Murphy, Michael Newman
**FOBILE MUCK TRUCK**
**NEW YORK:**
Woodstock on Broadway Jun '70
Airplane at the Fillmore Jun '70
Trinity Church—Business/rock symposium Jun '70
**VERMONT:**
Goddard Alternate Media Free Out Jun '70
Hackett's Garage (or the old fashioned toilet) Jun '70
**SAN FRANCISCO:**
North Beach (roadway tossles) Aug '70
North Beach, Grant Ave. Aug '70
Talk back, Black parolees involved in art program Aug '70
Fresh Unions, S.F. Aug '70
San Simeon, Calif. Aug '70
grape pickers interviews, gallery opening. Aug '70
**FOBILE MUCK TRUCK, 406 Gates, San Francisco, Calif. Dudley Dickinson, Dean Evenson, Janina Carroll.**
**PHIL GIETZEN**
Palace Theater (remote) 4 hr. originals 1" Craig
- With Dennis Combs Morrison
- Oh Happy Day, Lamb, Mad
- River, Red Lab Moog Synthesizer,
- James Smith, Demensia, James Rheinhart, MC.
Video LP 1 hr. Studio Mix 1" from each of above.
Post Palace Video 2 hr. original 1" IVC
- Media Mind Massage 40 min. mix effects over recordings.
- Upper Grant Street Fair
- Soiled Brothers Documentary
- Prisoners Drawnup Documentary
- Pickets are Witches
Additional listings on request.
**PHIL GIETZEN, 47 Clarion St., San Francisco, California**
**GLOBAL VILLAGE**
Abbie Hoffman—Katzman Interview on Trial Nov '69
Press Conference March '70
Rally-Panthers March '70
Jerry Rubin's Brew Before Dinner
Global Village with Rosemary Leary
CBS Demonstration on Censorship of Abbie Hoffman
Peace March—18 Minute Documentary March '70
Street Scenes—Bowery—Harlem
NYU Rally
Aferi Shakur—Black Panthers
Allan Ginsberg
Phil Ochs
Jimi Hendrix
Johnny Winters
L.A. Love Riots
Peter Fonda—UCLA
Rosemary Leary at WBAI
Womans Lib March
Washington Peace March—Cambodia
Student Worker Rally
Paul Krassner
Barbara Solomon—Studio Interview
Rip Torre, Richard III and Others
Women Lib Interview
Gay Lib Interview—Jim Fournat
Jim Higgins—Urban Media
David Silver—Media Radical Style
Woodstock
Woody
Janis Joplin
Richie Havens
Jefferson Airplane
Grateful Dead
Sly & Family Stone
Panther Rally August '70
Lee Kaminski—on Electric Eye—San Francisco Video Group
Paul Krassner August '70
Ned Reger—NYU Radical Student Leader August '70
East Village Street Interviews August '70
Kinetic Compositions:
Faces
Black and White Circle Composition
Experimental Neo Compositions
Ruby Tuesday
Raga Light Projections
Self-satisfaction Composition
Kinetic Laser Compositions with Lloyd Cross
Barbara Experimental Compositions
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Rudi Steen, John Reilly, Joe Davidson, Laura Asakoa, Dean Velaz, Carimir Niedzwiedz, Harry Heinisch, Jim Sheldon.
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a 3-hour Sony 1" tape (3 reels)
**ATASCADERO STATE HOSPITAL FOR THE CRIMINALLY INSANE ALIVE ON THE OTHER SIDE** Visit by Cleveland Wrecking Company Band, Free Family, Noel Jewkes, Food, Guards, Outmates
**HOMESKIN, Peter Berg, P.O. 31251, San Francisco 94131**
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More than 100 hours of experimental videotape in color and black and white. Two-inch, one-inch, and half-inch including:
The Bridge By Cinematographer Phillip Greene
Linearity Electronic composer Richard Feliciano
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Descartes By poet Joanne Kyger
Feedback and Tape Featuring dancer John Graham
Delay
Heimwehla 90 minute color production for NET; both & West Pole with videotape mix by Robert N. Zagone
Dawn Experimental piece by Bruce Howard
Color
Growing By sculptor Benedic Tatti / Color
Recent tapes include: a series of explorations in color and black and white of light forms, largely the work of William Rosener; and of feedback graphics by painter William Gwin. The Center also holds copies of experimental videotapes produced by a number of the fourteen interns from public television stations who have studied at the center over the past year.
**NATIONAL CENTER FOR EXPERIMENTS IN TELEVISION, 425 Brennin St., San Francisco, California 94107 (415) 362-4580.**
**PVT**
**MEDIATION DOCUMENTS:**
15th Street Squatters
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**IN PRODUCTION:**
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**PEOPLE'S VIDEO THEATER, 544 Ave. of Americas, NYC**
Ken Merzb and Elliot Glass.
All tapes: Sony ¾" Old Generation
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**PART I:**
St. Marks Tapes (street Rapping)
Summer '68
Antioch tapes (midwestern American subculture; interviews and experimental video entertainments)
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March '70
Loud Attack Chicago
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May '70
Interview with R. Buckminster Fuller, NYC
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Port Kent State—Washington DC Peace Demonstration
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City Hall labor and student anti-administration demonstration
plus
New tapes from TV during the week of the Kent State killings and Cambodias protest demonstrations
President Nixon's State of the Union Messages
The Party the President threw for the Astronauts
Keep: composition for four synchronized screens
Loop Switch: an abstract tape composed of feedback patterns
Computer: document on the home computer
Weekend at White Tank: two-part tape of meeting of video artists
Rose Art Museum show—Vision and Television documentary, Jan. '70
Moon: off-air collage
Ira Schneider, Frank Gillette, Michael Shambeg, Paul Ryan.
All tapes Sony ¾" old generation.
**PART II**
Mountaintide Festival (Frank Gillette)
Year of the Mushroom—Upstate N.Y. Commune at home and on stage with musical acts (Frank Gillette and Paul Ryan)
Roberts School street demonstration
Nam June Paik at WGBH (Frank Gillette)
Interviews with Levittown, N.Y., housewives and newlyweds about their television viewing tastes
Lower East Side, NYC
Warren Brody—interviews and verite video of Ecological design commune in New Hampshire
N.Y. State CATV operators convention
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Clinton Program Tapes of and by Junior High School Students in Clinton Program
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Nicholas Johnson at Raindance loft
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Ira Schneider, Michael Shambeg, Paul Ryan
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|
Loudspeakers in Vented Boxes: Part II*
A. N. THIELE
Australian Broadcasting Commission, Sydney, N.S.W. 2001, Australia
Editor's Note: Part I of *Loudspeakers in Vented Boxes* was published in the May, 1971 issue of the Journal.
VIII. LOUDSPEAKER EFFICIENCY
In Eq. (12) an expression was derived for the efficiency of a loudspeaker in a box, which consists of three parts. We have considered, in the meantime, the third part which varies with frequency. We now consider the first two parts. Thus the basic efficiency
\[
\eta_{ob} = \left( \frac{\rho_0}{4\pi c} \right) \left( \frac{B^2 P S_d^2}{R_e M^2 ms} \right). \tag{66}
\]
If this experience is compared with Beranek's Eq. (7.19) it will be seen to give one quarter of his value, after the differences in notation are allowed for.
1) Multiplication by 100 to give percentage.
2) The definition of "nominal input power" in Eq. (10) of this paper as the power delivered by the amplifier into the nominal speaker impedance \( R_e \). Beranek's treatment is based on the idea of maximum power transfer when the load impedance is equal to the generator impedance, as in his Eq. (7.14). If this condition, \( R_g = R_e \), is substituted in his Eq. (7.19), one of the conditions for agreement with Eq. (66) is satisfied. However, in dealing with the output power from an amplifier, the writer prefers to consider the power delivered into the load without regard to the output impedance \( R_g \), for the relationship of \( R_g \) to the optimum load impedance depends in the first place on the nature of the output device, transistor, pentode, or triode. Furthermore, \( R_g \) can be manipulated by feedback techniques (see Section XII) to almost any desired value without affecting the condition for optimum output power. Hence the treatment in this paper.
3) The lumping in this paper of all mechanical mass into \( M_{ms} \).
The additional multiplication factor of one quarter arises from the following.
4) Beranek's figure being for the radiation from both sides of the diaphragm, giving twice the output from one side.
5) The assumption in this paper that the radiation resistance in a box is that of a piston at the end of a long tube [3, p. 216]. This radiation resistance is one half of that of a piston in an infinite baffle.
Thus the results are consistent. We will continue here to use \( \eta_{ob} \), unless stated otherwise. But it is important to define efficiency in terms of actual use and to remember that the value of \( \eta_{ob} \), being the basic efficiency in a box, is one half the efficiency on an infinite baffle and one quarter of the efficiency, if radiation from both the front and back of a speaker in an infinite baffle is considered.
To simplify the understanding of Eq. (66), we make a further substitution. It can be shown that
\[
l^2 / R_e = V_{cu} / 2\sigma \tag{67}
\]
where \( \sigma \) is the resistivity of the conductor and \( V_{cu} \) is the volume of the conductor assumed to be completely within the air gap. In so far as the conductor overlaps the air gap a correction factor would be applied. Then
Eq. (66) becomes
\[ \eta_{ab} = \left( \rho_o / 8\pi c \sigma \right) \left( B^2 S_d^2 V_{cu} / M_{ms}^2 \right) \]
(68)
that is, once the voice coil conductor material, and therefore \( \sigma \), is chosen, the loudspeaker efficiency depends on the four parameters in the second bracket. Without digressing too far into the problem of loudspeaker design, it is noted that this shows the two basic questions in loudspeaker design for good efficiency at low frequencies.
1) How to make the product \( B^2 V_{ru} \) a maximum for a given magnet, since the larger \( V_{ru} \) is made, the wider and/or deeper is the air gap, and hence the lower is \( B \).
2) How to make \( S_d^2 / M_{ms}^2 \) a maximum, since the larger the area the greater the mass for a given cone thickness. If thickness is reduced, break-up problems increase due to nonlinearity of the piston drive. In conventional designs the mass of the voice coil is small (less than 20%) compared with the mass of the cone, so there is little interaction between \( V_{cu} \) and \( M_{ms} \).
The writer prefers to express efficiency as an electro-acoustic conversion loss
\[ dB_{ea} = 10 \log_{10} \eta. \]
(69)
For example, 1% efficiency is equivalent to 20-dB electro-acoustic conversion loss. This facilitates comparisons between different designs and estimations of the acoustic level (in phons) which a speaker will provide with a given amplifier and listening room (see Appendix).
**IX. RELATIONSHIP OF EFFICIENCY \( \eta \), Q, AND BOX VOLUME**
First we take Eq. (57) and break \( Q_t \) into two component parts, one due to the acoustic resistances and the other due to electrical damping, so that
\[ 1/Q_t = 1/Q_a + (1/Q_r) [R_e/(R_g + R_e)]. \]
(70)
Then from Eqs. (8) and (57), the acoustic \( Q \) of the loudspeaker
\[ Q_a = \omega_s M_{as}/R_{as} \]
(71)
and the electrical \( Q \) of the loudspeaker
\[ Q_r = \omega_s M_{as} R_e S_d^2 / B^2 l^2 \]
(72)
i.e.,
\[ Q_r = 2\sigma \omega_s M_{ms}/B^2 V_{cu}. \]
(73)
Again if we consider the approximate relationship established in Table I that
\[ C_{aa} f_3^2 / C_{ab} f_3^2 \approx \sqrt{2} \]
(74)
thus, converting the acoustic compliance of the box into the equivalent volume of air, the box volume
\[ V_b \cong (\rho_o c^2/\omega_s^2 \sqrt{2}) (S_d^2 / M_{ms}) \]
(75)
remembering that this approximate relationship holds only in the absence of amplifier assistance.
Now considering together Eqs. (68), (73), and (75), the following points emerge.
1) The same considerations that ensure high efficiency also ensure a low \( Q_r \), except that \( Q_r \) is independent of the projected piston area \( S_d \) and depends only on the first power of the cone mass \( M_{ms} \) instead of the second power.
2) The box volume depends, apart from the choice of cutoff frequency \( f_3 \), only on \( S_d^2 \) and \( M_{ms} \). Reduction of box volume by reduction of \( S_d \) involves an increased cone excursion, which is inversely proportional to \( S_d \) and \( \omega_b^2 \), for a given acoustic power. If the box volume is reduced by increasing \( M_{ms} \), \( \eta \) is decreased even more (see Eq. (68)), necessitating increased amplifier power. It would seem that the well-known R–J enclosure works this way. The opening in front of the cone is restricted, and this increases the air mass loading \( M_{m1} \) of Fig. 1 in the same manner as a vent. Thus \( M_{ms} \) is increased and the box volume \( V_b \), i.e., \( C_{ab} \), for a given low-frequency cutoff is reduced, but at the price of reduced efficiency throughout the piston range.
3) The best way of increasing \( \eta \) and lowering \( Q_c \) is to increase the flux density \( B \). But if one starts with a reasonably high value of \( B \) in the first place, the cost of obtaining an extra decibel of efficiency increases rapidly. So again to obtain a given amount of acoustic power at a given price, a compromise must be struck between the sizes of magnet, box, and amplifier. However, this discussion does show the reason for the large magnet, long throw, heavy cone designs used overseas in small “bookshelf boxes.”
Note that \( Q_a \) in Eq. (71) depends only on acoustic reactance and resistance, that is, \( Q_a \) is independent of \( B \).
Substituting Eqs. (58) and (73) in (68), we obtain the interesting relationship
\[ \eta_{ab} = \omega_s^3 V_{as}/4\pi c^3 Q_r \]
(76)
where \( V_{as} \) is the volume of air equivalent to the acoustic compliance of the loudspeaker, or
\[ \eta_{ab} = 8.0 \times 10^{-12} f_s^3 V_{as}/Q_r \]
(77)
where \( V_{as} \) is in cubic inches. *Thus the basic efficiency of the speaker can be calculated from the three parameters which are used for the design of the box.* A physical explanation of the variation of \( \eta \) and \( Q_r \) is given at the end of Section XII.
**X. EXCURSION OF LOUDSPEAKER CONE**
In the derivation of Eq. (12) it was found that
\[ U_c/(U_c - U_p) = 1 - 1/\omega^2 M_{a1} C_{ab} \]
\[ = 1 - (\omega_b/\omega)^2. \]
(78)
Thus the acoustic output power radiated by the cone alone is
\[ W_{aoc} = W_{cu} \eta_{ab} [1 - (\omega_b/\omega)^2]^2 |E(j\omega)|^2. \]
(79)
Now starting from the relationship
\[ W_{aoc} = (R_{ma} \dot{x}^2) 10^{-7} \]
(80)
which is [4, Eq. 6.13], where \( R_{ma} \) is the mechanical radiation resistance and \( \dot{x} \) is the rms velocity of the piston in cm/s, it is possible to derive an expression for peak cone movement,
\[ x_{pk} = 1.31 \times 10^5 \sqrt{W_{aoc}/f^2 S_d} \]
(81)
or
\[ x_{pk} = 5.17 \times 10^6 \sqrt{W_{aoc}/\omega^2 S_d} \]
(82)
where \( x_{pk} \) is in inches (note that this \( x \) which stands for excursion is unrelated to the shape parameter \( x \) of Eq.
Fig. 10. Normalized cone excursion versus normalized frequency for various orders of Butterworth response with loudspeaker in vented box (solid curves) and in infinite Baffle (dashed curves). Curves are numbered for order of response. Normalized excursion is \( \left| \left( f_s / f \right)^2 - \left( f_b / f \right)^4 \right| \cdot |E(j\omega)| \), part of Eq. (84).
(21) et seq.), \( S_d \) is in square inches, and \( W_{\text{rms}} \) is in watts. Again allowance is made for the fact that the loudspeaker is mounted in a box so that the radiation resistance is half the value for an infinite baffle. Thus Eqs. (81) and (82) will give values for displacements which are \( \sqrt{2} \) times those given in [4, Fig. 6.9]. Thus
\[
x_{pk} = 5.17 \times 10^6 (\eta_{nb} W_{ei})^{1/2} \left[ 1 - \left( \omega_h / \omega \right)^2 \right] |E(j\omega)| / \omega^2 S_d.
\]
If we write this expression as
\[
x_{pk} = \left[ 1.31 \times 10^5 (\eta_{nb} W_{ei})^{1/2} / f_h^2 S_d \right] \left[ \left( \omega_h / \omega \right)^2 - \left( \omega_h / \omega \right)^4 \right] |E(j\omega)|,
\]
it is apparent that there are two parts, one fixed for a given speaker and box (note frequency \( f_h \) in this expression) and one that varies with frequency. This latter expression is plotted in Fig. 10 for various Butterworth responses, in which box, speaker, and cutoff frequencies are identical. The solid curve 4 gives the excursion of the classical fourth-order Butterworth alignment no. 5 of Table I. Solid curve 5 refers to the fifth-order Butterworth alignment no. 10, which includes a simple auxiliary filter. Solid curve 6 refers to the sixth-order Butterworth alignment which is identical for nos. 15, 20, and 26, since both frequency response and box resonant frequency are the same in each. For comparison, the dotted curves give the excursions for the same speaker in an infinite baffle (totally enclosed box) with the same power. Dotted curve 2 applies to a speaker with a second-order Butterworth response (\( Q_t = 0.707 \)). Dotted curve 3 applies to a third-order Butterworth response (\( Q_t = 1 \), with a simple auxiliary filter). Dotted curve 4 applies to a fourth-order Butterworth response (\( Q_t = 1.307 \), with a second-order auxiliary filter). The frequency response is the same as solid curve 4, but it is obtained by different means. The curves show the following.
1) The excursion below resonance is reduced greatly in both vented box and infinite baffle when an auxiliary highpass filter is used. The first-order auxiliary filter gives a good improvement especially in view of its simplicity. The second-order auxiliary filter not only allows a greater reduction of cone excursion, it also allows the use of three separate box alignments for the same response and allows box volume to be traded for amplifier power in the case of the vented box. The Butterworth curves with second-order auxiliary filters are symmetrical about the center frequency. There seems little need therefore to use more elaborate filtering.
2) Even more important, the excursion of the cone is reduced greatly when the loudspeaker is placed in a vented box. The curve predicts zero excursion at the box frequency. This arises from the assumption that the \( Q \) of the box circuit is infinite. While this cannot be achieved completely in practice, the excursion at the box frequency will be low so long as the ratio of \( Q \) of the box to \( Q \) of the speaker is high, as demonstrated in Section II.
Of course, if resistance is deliberately introduced into the box circuit, as by making the vent from a number of small holes or by stretching fabric across the vent, the \( Q \) will be greatly reduced and some of the advantage of the vented box will be lost, as shown in the next section. Fig. 10 refers only to Butterworth responses. In Fig. 11, a plot is made of the function \( \left| \left( \omega_h / \omega \right)^2 - \left( \omega_h / \omega \right)^4 \right| \) against frequency. If, for example, in a Chebyshev response the frequency response is known, the excursion at different frequencies can be found by reading off the function at a given frequency on Fig. 11 and multiplying it with the frequency response. The rapid rise of the function between normalized frequencies of 1 and 0.71 shows why responses should be preferred in which \( f_h \) is not too much greater than \( f_3 \). Thus with respect to cone excursion, an alignment in the group 20–25 would be preferred to its counterpart in the group 15–19 which has a lower value of \( f_3 / f_h \).
It would seem that in published ratings of loudspeakers, the maximum excursion \( x_{\text{max}} \) would be more useful than the conventional rating of maximum input power. The latter might save the loudspeaker from a melted voice coil, but when mechanical damage or undistorted acoustic output are of interest, \( x_{\text{max}} \), along with the kind of baffle and the alignment, determine the performance.
XI. BOXES WITH RESISTIVE LOADING OF VENT
Good results have been reported with resistively loaded vents [1]. These were therefore investigated using both series and parallel loading of the vent as shown in Fig. 12. In both cases, the resistance was assumed to be constant with respect to frequency and the response function was found to be of third order.
This, by the way, explains a discrepancy between the statements in [3, p. 244] and in [2, p. 11] that the drop in response below cutoff is 18 dB per octave, even though [2, Eq. 15)], which is equivalent to Eq. (20) of this
Fig. 12. Equivalent acoustic circuit of loudspeaker and box showing added acoustic damping in series or parallel with vent.
paper, obviously has an asymptotic slope of 24 dB per octave. In the practical case, where resistance loading of the vent however small will be encountered, the asymptotic slope will eventually be 18 dB per octave; but so long as the original simplifying assumptions hold, the response in the region that concerns us will be effectively 24 dB per octave.
The expressions are, for the case of series resistance loading,
\[ E(p) = \frac{1}{\{1 + (1/p)(1/Q_b T_b + Q_s T_b / T_s^2) + (1/p^2)} \\
(1/T_s^2 + 1/T_b^2 + C_{as}/C_{ab} T_s^2) + Q_b / p^3 T_s^2 T_b \} \quad (85) \]
when
\[ 1/Q_t = T_s / Q_b T_b + Q_b T_b / T_s \quad (86) \]
and \( Q_b \) is defined as the ratio of acoustic mass resistance to series acoustic resistance of the vent at the box resonant frequency.
For the case of parallel resistance loading,
\[ E(p) = \frac{1}{\{1 + (1/p)(Q_b T_b / T_s^2 + 1/Q_b T_b + C_{as} T_b / C_{ab} T_s^2 Q_b) + (1/p^2)} \\
(1/T_s^2 + 1/T_b^2 + C_{as}/C_{ab} T_s^2) + Q_b / p^3 T_s^2 T_b \} \quad (87) \]
when
\[ 1/Q_t = Q_b T_b / T_s + T_s / Q_b T_b + C_{as} T_b / C_{ab} T_s Q_b \quad (88) \]
and \( Q_b \) in this case is the ratio of parallel acoustic resistance across the vent (series resistance being assumed negligible) to acoustic mass reactance. Note the inversion of the expression for parallel \( Q_b \) compared with that for series \( Q_b \). Since these equations are of third order and there is one extra variable \( Q_b \), there are two extra degrees of freedom in the design. However, one is removed if an all-pole function is desired, hence Eqs. (86) and (88). Before an alignment is commenced, one other parameter must be fixed arbitrarily. The ratio \( C_{as}/C_{ab} \) seems the easiest to handle for this purpose. Thus in a third-order Butterworth alignment, if \( C_{as}/C_{ab} \) is made 1.414, for comparison with the fourth-order Butterworth alignment no. 5 of Table I, the results are as given in Table II.
Table II. Parameters for third-order Butterworth alignment with resistive vented loading.
| Method of Loading | \( f_3 / f_s \) | \( f_3 / f_b \) | \( C_{as}/C_{ab} \) | \( Q_t \) | \( Q_b \) |
|-------------------|-----------------|-----------------|------------------|----------|----------|
| Series Resistance | 1.317 | 1.285 | 1.414 | 0.379 | 2.22 |
| Parallel Resistance | 1.420 | 1.120 | 1.414 | 0.352 | 2.25 |
| No Resistance (Alignment No. 5, for comparison) | 1.000 | 1.000 | 1.414 | 0.383 | ∞ |
It will be seen that although the box had the same volume, the cutoff frequencies for the resistively loaded alignments are 1.32 and 1.42 times higher than no. 5 of Table I. Compared with previous alignments (no. 1–9 of Table I) those of Table II are most inefficient in utilization of box volume, there is no compensating freedom to use a larger value of \( Q_t \), in fact it needs to be a little smaller, finally and more important, the excursion of the speaker near cutoff frequency is greatly increased. For these reasons, the use of acoustic damping seems to be unjustified. It is realized that the cases treated here use resistances which are constant with frequency. Some acoustic resistances, as described for example in [3, Eqs. (5.54) and (5.56)], vary with frequency and might have a somewhat different effect. However, the use of added damping with the attendant dissipation of input power seems to be wrong in principle, unless a suitable alternative cannot be found. It is believed that the method outlined already provides the suitable alternative.
**Effect of Losses in Box and Vent**
Having established that intentional loading of the vent is undesirable, it is of interest to know the effect on the ideal response, obtained by assuming zero loss, of small unavoidable losses in the box and vent. We will only consider performance at the box resonant frequency, since at this frequency 1) the box circuit contributes most, in the ideal case all, of the acoustic output, and 2) the losses in the box circuit are greatest.
In the ideal case, the transfer impedance connecting the input force \( E_g B l / S_n (R_p + R_r) \) with the vent volume velocity \( U_p \) in Fig. 2, at the box resonant frequency \( \omega_b \), is \( j \omega_b M_{av} \). If now we express all the losses in the vent and the box as \( Q_b \), the “\( Q \) of the box and vent circuit,” the transfer impedance, and thus the frequency response at \( \omega_b \) is reduced by a factor which we will call the maximum box loss \( (A_b)_{max} \). Then, to a close approximation,
\[ (A_b)_{max} = \frac{1}{[1 + (1/Q_t Q_b) (C_{ab}/C_{as}) (\omega_b/\omega_s)]}. \quad (89) \]
If we apply the approximations of parts 1) and 2) of Section VI for the “unassisted” alignments no. 1–9 of Table I, Eq. (89) is simplified to
\[ (A_b)_{max} = \frac{1}{[1 + (1.85/Q_b) (f_b^2/f_s^2)]} \quad (90) \]
that is, for a given value of \( Q_b \), the box loss increases with higher values of \( f_b/f_s \) and thus, larger box sizes.
To illustrate the effect of box loss, Eq. (89) is applied to various alignments. Taking first the classical alignment, no. 5 of Table I, the maximum box loss is 0.5 dB when \( Q_b \) is 30 and 1.5 dB when \( Q_b \) is 10. Taking other, extreme, alignments when \( Q_b \) is 30, the losses for alignments no. 1, 9, 19, and 25 are 0.3 dB, 0.7 dB, 0.5 dB, and 0.7 dB, respectively. Thus it can be seen that a \( Q_b \) of 30 will have little effect on any alignment. With a \( Q_b \) of 10, the losses are 0.9 dB, 1.9 dB, 1.5 dB, and 2.2 dB, respectively, i.e., when the box \( Q \) is reduced three times, the maximum box loss is increased approximately three times in each case. A method of measuring \( Q_b \) is given at the end of Section XIV and illustrated in the Appendix.
Table III. Change of output impedance $R_g$ with type of feedback.
| | Negative | Positive |
|------------------|----------------|---------------|
| Voltage Feedback | $R_g$ Decreases| $R_g$ Increases|
| Current Feedback | $R_g$ Increases| $R_g$ Decreases|
XII. AMPLIFIER CIRCUITS
Negative Output Impedance
It is essential to the method that the overall $Q_t$ of the loudspeaker plus amplifier be properly controlled within $\pm 10\%$ for $\pm 1$ dB accuracy of response. As explained in Section V, if $Q_t$ is twice the optimum value, a 6-dB peak results. Similarly if $Q_t$ is too small, there will be a dip in the response. Thus it is important that the speaker $Q_e$ be known, either from information supplied by the manufacturer or by measurement, and that the amplifier output impedance be then adjusted to give the required overall value of $Q_t$. It is assumed in the following that the available speaker $Q_e$ is larger than the required $Q_t$. This is the more usual case, especially with lower priced loudspeakers. But if it is smaller, a suitable adjustment can easily be made, for example, by changing the positive current feedback to negative current feedback.
The subject of amplifier output impedance control properly requires another paper, which it is hoped will be presented later. For the present only some general results will be given.
If feedback is applied to an amplifier, not only does its gain change, but its effective output impedance $R_g$ changes also; not its optimum load impedance which remains unchanged by feedback but the impedance which is seen when looking back into the amplifier output terminals. The effect of applying different kinds of feedback is shown in Table III.
The terms voltage feedback and current feedback refer of course to feedback of a voltage which is proportional to output voltage and output current, respectively. In the latter case, this is usually achieved by placing a small resistor in series with the load, and taking the voltage drop across it for feedback. It will be seen that not only does negative voltage feedback reduce the output impedance $R_g$, positive current feedback reduces $R_g$ also, and to the greater extent that $R_g$ can be made zero or negative.
Negative output impedance is characteristic of oscillators; one therefore tends to be wary of it as tending to instability. But this can only happen when the positive output impedance presented by the load is less than the negative impedance presented by the amplifier. Now the impedance of a loudspeaker in a box, typified by Fig. 5, can never be less than its dc resistance $R_e$ of Fig. 4. The only exception is at very high frequencies, where the shunt capacitance of the connecting leads takes effect. But unless the leads are very long and the nominal impedance of the speaker is high, this will not usually take effect within the bandwidth of the amplifier. And in any case, we will want to eliminate the negative impedance characteristic at the higher audio frequencies for reasons that will be discussed later. Thus a negative impedance amplifier can be made completely stable apart from gross misadjustment, such as connecting a loudspeaker of much lower impedance than the design figure or short-circuiting the output leads.
The method of applying mixed feedback is shown in Fig. 13. It will be seen that if the sense of the voltage developed across the potential divider $R_3$ and $R_4$ is negative, then the voltage developed across the current feedback resistor $R_2$, usually made less than 1/10 the nominal impedance of the speaker to minimize power loss, will be positive. The circuit shows why this method is sometimes described as bridge feedback. Usually the circuit is arranged to be unbalanced at all frequencies so that the net feedback is always negative, but it need not necessarily be so. For example, if no net negative feedback is desired, so that there is no overall gain reduction with nominal load, the bridge will be balanced at nominal load.
Physically, the circuit can be thought of as having a certain amount of feedback with nominal load, in which the negative voltage feedback is partially neutralized by the voltage from the positive current feedback resistor. If the impedance $Z_1$ is open-circuited, the current feedback from $R_2$ disappears leaving a greater amount of negative feedback. Thus the output voltage may be less on open circuit than on nominal load. This is the effect we describe as negative output impedance. Its extent, or whether it is seen at all, will depend on the original gain and output impedance of the amplifier and the value of the feedback resistor $R_2$. Thus if we have, as in Fig. 4, a loudspeaker resistance $R_e$, and make the effective output impedance of the amplifier $R_g$ equal to, say, $-0.6R_e$, the total effective impedance of $R_g + R_e$ becomes $+0.4R_e$. And if the $Q_e$ of the loudspeaker is 1.0, this will make the overall $Q_t$ a value of 0.4 by applying a maximum of 1.0/0.4 times, i.e., 8.0 dB, extra gain reduction by negative feedback when the impedance of the speaker becomes high, as at $f_h$ and $f_l$ of Fig. 5. (Need it be emphasized that this form of damping does not dissipate amplifier output power, except in the small current feedback resistor. It reduces power by feedback at the source.)
This fact necessitates a degree of additional care in the design of negative impedance amplifier. For when the load is open-circuited, the negative feedback rises to the maximum; in this case a gain reduction of 8 dB above the nominal value, and the stability margin will be reduced. The size of the negative impedance will in practice be limited either by this consideration or by the need for a feedback resistor so large that it dissipates an appreciable part of the output power.
An alternative method of control damping uses a feedback winding closely coupled to the voice coil. In this way, feedback can be taken effectively from the junction of $R_e$ and $L_e$ in Fig. 4. Simple negative feedback
then reduces an effective output impedance which is the sum of $R_g + R_e$. Thus $Q_t$ is reduced in the same way as before. Since the impedance of the feedback circuit is usually high compared with the voice coil impedance, the feedback winding can be made of very fine wire. In fact, if it is wound bifilar with the main winding with wire 16 B&S gauges smaller, it will fit into the air spaces between the larger wires. It thus takes up no more space in the air gap and adds less than 3% to the mass of the copper in the voice coil. Unfortunately, such a winding is difficult to achieve in production and is thus rarely, if ever, used.
If negative impedance is applied, it reduces the output voltage whenever the load impedance is high, i.e., not only in the region of $f_l$ and $f_h$ in Fig. 5, but also at frequencies above $f_n$ where the impedance rise is due to the inductance $L_e$ of Fig. 4. At high frequencies, this contributes nothing to the acoustic damping of the speaker, but simply reduces the high-frequency response, in the case quoted above, a maximum of 8 dB. This is usually undesirable, so the negative impedance should be eliminated at the higher audio frequencies. One method among several possible is shown in Fig. 14a. Here an inductance $L_2$ is added to the feedback resistor $R_2$ with a time constant $L_2/R_2$ matching that of the speaker, usually in the range of 30–60 $\mu$s. This can be easily done by winding a solenoid of copper wire which combines resistance $R_2$ and inductance $L_2$. However, since this achieves its result by feeding back an increasing positive voltage to neutralize an increasing negative voltage, quite small unbalance between the two can cause instability at high frequencies.
On the other hand, consider the circuit of Fig. 14b where the lower resistor of the negative feedback potential divider $R_4$ becomes two resistors $R_5$ and $R_6$ in series. Suppose that a suitable set of resistors $R_2$, $R_3$, and $R_4$ has been found to give the correct gain and output impedance for low frequencies with the dotted connection open-circuited. It is then possible to find a tapping point on $R_4$ (i.e., the junction of $R_5$ and $R_6$) such that the same gain is obtained on nominal load whether the dotted connection is open circuit or short circuit. This is done by connecting the nominal load and making $R_5$ and $R_6$ a potentiometer whose wiper is grounded through a switch. The wiper is adjusted until the gain is the same with the switch open or closed. In the open-circuit condition, the output impedance will be the value originally chosen, but on short circuit, most of the positive current feedback will be eliminated. If then a capacitor is substituted for the switch as shown in Fig. 14b, the output impedance will change from a negative value at low frequencies to a small value, either positive or negative depending on the particular circuit. The frequency of changeover, which should be, say, two octaves above $f_h$, depends on the capacitance $C$ and the resistances $R_5$ and $R_6$. At the same time, the gain of the amplifier on nominal load stays constant over the whole audio range.
**Auxiliary Filters**
The auxiliary filtering needed for sixth-order alignments is best provided by circuits using $RC$ networks in a feedback loop ahead of the main amplifier. In general it is unwise to use the main amplifier feedback loop to provide both negative impedance and high-pass filtering. It is hoped to deal with this in a later paper, but for the moment the reader's attention is directed to the extensive literature, of which [9] and [10] are examples, concerning low-frequency filters without inductors, which use resistors, capacitors, and tubes in comparatively inexpensive combinations.
**Maximum Power at Maximum Impedance**
The electrical impedance seen at the terminals of a loudspeaker varies greatly with frequency, but output stages deliver maximum power into a comparatively narrow range of impedances. To consider the maximum acoustic power that can be delivered by an amplifier through a loudspeaker, we return to the equivalent electrical circuit of Fig. 4, together with the impedance curve of Fig. 5. For this purpose, we ignore for the moment the inductance $L_e$ with its electrical shunt loss $R_{sh}$ and assume that the curve of Fig. 5 reaches a final value of $R_e$ above $f_n$.
The acoustic output depends on the voltage across $R_{rs}$, which includes the electrical equivalent of the radiation resistance $R_{ar1}$. Since $R_{ar1}$ varies with frequency squared, the voltage across $R_{rs}$ needs to vary inversely with frequency to maintain constant acoustic power. At the higher frequencies the motional impedance is much lower than $R_e$ and is controlled by the reactance of $C_{mres}$, which is equal to $B^2/2/M_{ms}$. Thus the condition for flat response is achieved, often described as mass control.\footnote{This should not be confused with the technique of mass control practiced by politicians and advertising people. In that context, the reactance is usually assumed to result from the equivalent of a compliance, and hence to decrease with signal frequency.}
If $B$ is varied while $R_e$ remains constant, the motional impedance at any given high frequency within the piston range will increase with $B^2$. The electrical equivalent of radiation resistance, though small, will increase and with it the ratio, again small, of acoustic power radiated to electrical power input. Thus efficiency varies with $B^2$. At the same time the increase of motional impedance while the resistance $R_e$ remains constant causes $Q_e$, the electrical $Q$, to decrease inversely with increasing $B^2$.
But as the frequency decreases, the motional impedance rises, reaching at $f_h$ and again at $f_l$ a maximum value of $R_{rs}$ which is usually several times the resistance $R_e$. Thus at these peaks the motional impedance, which at high frequencies was negligible compared with $R_e$, is now the major part of the total impedance. Suppose for simplicity that it comprises all of the speaker impedance. This time when $B$ is varied and the motional impedance...
varies as $B^2$, then for a given acoustic power output the voltage across $R_{es}$, which is virtually the input voltage, will need to increase with increasing $B$. Summarizing, for a fixed acoustic power output, an increase of $B$ will decrease the input voltage required at high frequencies, and increase the input voltage required at the impedance peaks. Also $Q_e$ will decrease.
With a load impedance much larger than nominal, the criterion of performance of the amplifier becomes, not output power, but the undistorted output voltage on open circuit. This will always be larger than the undistorted output voltage at nominal load; how much larger will depend on the design of the amplifier.
Now if the $Q_t$ required for a flat frequency response is identical with the $Q_r$ of the loudspeaker, then if we ignore $Q_a$, the generator impedance $R_g$ must be zero. Thus for a constant acoustic power output the same voltage will be required at the loudspeaker terminals at all frequencies, and all impedances, so that at the frequency $f_h$ somewhat more maximum acoustic power is available than at higher frequencies.
If the $Q_t$ required is less than $Q_r$, $R_g$ will need to be negative, and for constant acoustic power and amplifier output voltage, at the junction of $R_g$ and $R_v$ in Fig. 4, will fall at $f_h$. But if the $Q_t$ required is greater than $Q_r$, $R_g$ will need to be positive, and the amplifier output voltage for constant acoustic power will rise at $f_h$. If the ratio of increase of voltage required is greater than the ratio of amplifier undistorted output voltages on open circuit to on-load, it is possible for less maximum acoustic power to be available in the region of $f_h$ than at other frequencies in the useful band. But since low values of $Q_r$ are normally associated with high efficiency, this is only likely to occur with high-efficiency, usually high-quality speakers. It should not cause trouble until $Q_r$ is less than half $Q_t$, and even then the maximum acoustic power in most program material is less at frequencies below 100 Hz than around 400 Hz.
Thus there is a paradox that a highly efficient speaker may deliver less power around $f_h$ than at higher frequencies, while a less efficient speaker delivers more. This will depend on the ratio of $Q_r$ to $Q_t$ and of amplifier undistorted output voltage off-load to on-load.
Related to this topic is the flattening of the impedance characteristic which is usually considered to be a good feature of vented boxes. Reference to Fig. 5, and comparison with Fig. 16, shows that, with the simplifying assumption that the resistive losses in the box and vent are negligible, the height of the impedance peak $R_v + R_{es}$ peaks at $f_h$ and $f_l$ and raise the minimum impedance at $f_b$. But this is incidental, and the relative heights are of little importance. Thus the idea of tuning the box so that the impedance peaks at $f_h$ and $f_l$ are equal, misses the real point. In the impedance curve of a loudspeaker in a box, the most useful information is not the values of the impedances, so long as box and vent damping is not too severe, but the values of the frequencies $f_h$, $f_b$, and $f_l$. Knowledge of these three frequencies alone enables a box alignment to be checked by Eqs. (105) and (106).
It should be clear that flatness of the impedance characteristic is no indication of flatness of acoustic response. Take as an analogy a coupled pair of tuned circuits. When the output voltage, or more exactly the transfer impedance, is maximally flat, the input impedance has two peaks. If one parameter is known, say the ratio of primary to secondary $Q$, the transfer impedance can be deduced from the input impedance, just as we do for loudspeakers in Eqs. (105) and (106). But a flat input impedance characteristic does not indicate a flat transfer impedance. In a loudspeaker, the impedance characteristic has greater peaks, whose height depends purely on the acoustic damping, though this contributes little to the overall system damping, and thus the overall frequency response.
XIII. EFFECTIVE REVERBERATION TIME
An objection sometimes made to the use of vented boxes is that the slope of attenuation beyond cutoff, 24 dB per octave, is much steeper than the 12 dB per octave of a speaker on an infinite baffle, and therefore the transient response is worse. In a low-pass filter, the ringing associated with steep attenuation slope is virtually removed by the use of Thompson or critically damped responses. But in high-pass filters such as are considered here, there is always some overshoot with filters of order two or more. To estimate its effect on a listener we use the concept of "effective reverberation time."
Imagine that we have a source of sound in a room which has built up a steady field. The source is then stopped. The sound in the room does not stop immediately, but dies away gradually. The time taken for the sound to decay is called the reverberation time, defined as the time taken for the sound pressure in the room to fall 60 dB from its original value. In small rooms the reverberation time will probably lie between 400 ms for a highly damped room to 1 s or more for a live one.
When the sound passes through two reverberant rooms in cascade, the law of the resulting overall reverberation time is not well established, but calculations on cascaded high-pass filters suggest that rms addition gives at least a guide. In any case it would appear that an added reverberation time of 200–300 ms should not appreciably color the reproduction.
When a transient is applied to a filter and it rings, the effect is perceived by the ear, or brain, as an extension of the transient event in time. Hence the expression "hang-over." To express the effect of the ringing then, an idea is borrowed from architectural acoustics, and the effective reverberation time of a filter is defined as the time taken, after a step function is applied, for the amplitude of the envelope of ringing to fall 60 dB below the amplitude of the original step function.
For the higher order filter functions, with two or more second-order factors, only the most lightly damped factor need be considered. For, by the time the ringing due to the most lightly damped factors is 60 dB down, the ringing due to the more heavily damped factors is negligible. This eases computation greatly.
Actually, at low frequencies the reverberation time defined above will be rather longer than the time the sound is perceived by the listener. To see why, we consult the much abused Fletcher–Munson curves [4, Fig. 12.11].
Suppose, for example, that the original sound is at 100-phon level. This is probably the maximum a system could reproduce, or a listener tolerate. Now at 50 Hz the threshold of hearing is 51 dB above reference level, that is, 49 dB below our arbitrary listening level. At 25 Hz the threshold of hearing is 67 dB above reference
Table IV. Reverberation times for various alignments.
| Type of response | $B_2$ | $C_2$ | $C_2$ | $B_4$ | $C_4$ | $B_6$ | $C_6$ | $C_6$ | $C_8$ |
|------------------|-------|-------|-------|-------|-------|-------|-------|-------|-------|
| $Q_t$ (for second order alignments) | 0.707 | 1.000 | 1.414 | — | — | — | — | — | — |
| $k$ (for sixth order alignments) | — | — | — | — | — | 1.000 | 0.600 | 0.414 | 0.268 |
| Alignment numbers | — | — | — | 5 | 8 | 15, 20, 26 | 17, 22 | 19, 24 | 25, 27 |
| Time (in periods of cutoff frequency) | 1.63 | 2.24 | 3.17 | 2.87 | 7.09 | 4.77 | 6.79 | 9.67 | 14.86 |
| Time for 50 c/s cutoff (mS) | 33 | 45 | 63 | 57 | 142 | 95 | 136 | 193 | 297 |
level, that is, only 33 dB below our arbitrary listening level. At 25 Hz, therefore, the effective reverberation time for the listener cannot be greater than the time in which the sound level falls 33 dB, i.e., about half the reverberation time as defined conventionally. Thus at low frequencies in general, the conventional definition based on a 60-dB fall in level yields a reverberation time rather longer than a listener will hear. (This is probably the reason for the observed increase in optimum reverberation time at low frequencies, see [4, Fig. 11.11].)
In a filter which cuts off sharply, the major ringing frequency will be close to the cutoff frequency. Also for a given shape of response curve the reverberation time can be expressed as a certain number of cycles of the cutoff frequency (see Table IV), i.e., the reverberation time increases with decreasing cutoff frequency. On the other hand, below, say, 50 Hz, its effect on the listener will decrease at approximately the same rate. Thus for all filters of a given response curve shape, the figure for 50 Hz should give a rough idea of the maximum reverberation time, as perceived by the listener.
Calculated reverberation times are given in Table IV. The first three alignments are of second order, corresponding to a loudspeaker on an infinite baffle. For these, the values of $Q_t$ are shown. Note that the reverberation time, though low, doubles as $Q_t$ increases from 0.707 to 1.414, that is, when the frequency response goes from maximally flat to a 4-dB peak. The times for 50-Hz cutoff are all below 200 ms, except for the last ($k = 0.268$), which is the very steepest.
It thus appears that a properly adjusted vented box, even with amplifier assistance (auxiliary filtering), need cause no perceptible coloration due to ringing. But it is important to emphasize that the adjustment must be correct. Table IV shows that the addition of a 4-dB peak to the response of a speaker on an infinite baffle can double the reverberation time. Being low in the first place it remains tolerable. But in the case of a vented box, particularly with an auxiliary filter, a doubled reverberation time would be more serious. Again, this emphasizes the importance of adequate damping (for correct value of $Q_t$) by the amplifier.
XIV. MEASUREMENT OF LOUDSPEAKER PARAMETERS
In earlier sections it was shown how the required response can be obtained from a loudspeaker and box if several parameters are known. The question remains, how are these parameters found?
Properly, this information should be available from the loudspeaker manufacturer. This is particularly important for equipment produced in quantity, where it is important to know not only the mean values but also the tolerances. However, in the absence of published figures, or to check them, the following procedure will provide the information.
Procedures for measuring $Q$ are given in [2, p. 13], but the method used seems too laborious and inaccurate. The method outlined hereafter can be understood by considering Figs. 15 and 16. Figure 15 is derived from Fig. 4; only this time we omit the vented box and we ignore $L_c$ and $R_{ch}$ which take effect at much higher frequencies. Now
$$Q_a = \omega_s C_{mes} R_{es}$$ \hspace{1cm} (91)
$$Q_e = \omega_s C_{mes} R_e.$$ \hspace{1cm} (92)
These quantities, defined earlier in Eqs. (71) and (72) in terms of the acoustic equivalent circuit, are defined here in terms of the electrical equivalent circuit. We define $r_0$ as the ratio of the impedance at resonance, $R_{es} + R_e$, to the dc resistance of the voice coil $R_e$. Now we take another arbitrary impedance which is presented at two other frequencies $f_1$ and $f_2$ on the flanks of the curve, and we call its ratio to the dc resistance $r_1$. Then
$$f_1 f_2 = f_s^2.$$ \hspace{1cm} (93)
Physically, this means that the curve is symmetrical on a logarithmic frequency scale. In experimental work it provides a handy check. Now we can find
$$Q_a = \left[ f_s / (f_2 - f_1) \right] \left[ r_0^2 - r_1^2 \right] / (r_1^2 - 1) \right]^{1/2}$$ \hspace{1cm} (94)
and
$$Q_e = Q_a / (r_0 - 1).$$ \hspace{1cm} (95)
If additionally we choose $r_1$ such that
$$r_1 = \sqrt{r_0}$$ \hspace{1cm} (96)
then Eq. (94) is simplified to
$$Q_a = \sqrt{r_0} f_s / (f_2 - f_1).$$ \hspace{1cm} (97)
The interesting feature of these expressions is that they involve no approximations, and thus hold for all values of $Q$. Furthermore around the value $\sqrt{r_0}$ the curve has its greatest slope. Thus the frequencies $f_1$ and $f_2$ can be
found most accurately. This is especially important since the calculation involves a comparatively small difference between large numbers $f_2 - f_1$.
Usually $Q_a$ takes account of the acoustic resistances in the loudspeaker. But if the voice coil has a short-circuited turn by accident or design, e.g., an aluminum former, this will appear in $Q_a$, even though its physical nature is similar to $Q_e$. (But eddy current losses in the pole piece or front plate appear in $R_{sh}$.)
Fig. 17 shows the test circuit. $V$ is a voltmeter of impedance much higher than the loudspeaker. Throughout the readings, the generator is adjusted so that the reading of $V$ is constant. The value is not of great importance, but a standard test figure is one volt. The accuracy of this voltmeter is not important so long as it is independent of frequency. $A$ is an ac ammeter which reads the current into the speaker with the fixed voltage across its terminals. Again, since we are interested only in the shape of the impedance curve, the absolute accuracy of this instrument is not important so long as the meter reading is linear. However, to set the relative current due to $R_p$, first we measure $R_g$ with dc on a Wheatstone bridge, and then a calibrating resistor $R_c$ of similar value. Connecting $R_c$ to the test terminals and applying the standard test voltage at say, $f_s$, a current value $I_c$ is found on the ammeter $A$. Then the current $I_e$ which corresponds to $R_e$ is found by
$$I_e = I_c R_c / R_e.$$
(98)
Now the loudspeaker is suspended in air as far from reflecting surfaces as is practical and connected to the test terminals instead of $R_c$. The generator is adjusted to the speaker resonant frequency $f_s$, indicated by minimum current $I_o$. Thus $r_o$ is found:
$$r_o = I_e / I_o.$$
(99)
Now the current $\sqrt{(I_e I_o)}$ is found corresponding to the ratio $\sqrt{r_o}$ and the frequencies either side of resonance, where this current value is read. These are $f_1$ and $f_2$ and they should be read to as close an accuracy as the test gear will allow. Eq. (93) provides a check on the method, and Eqs. (97) and (95) give $Q_a$ and $Q_e$.
The next problem is to find the value of $V_{as}$, the volume of air equivalent to the loudspeaker compliance. For this, the loudspeaker is placed in a totally enclosed unlined box whose internal volume $V_b$ is known, remembering that allowance must be made for bracing and the volume displaced by the speaker. It is important that this box be free of air leaks. If these occur we will read part of the curve of Fig. 5, around $f_h$. Thus care should be taken, not only in the construction of the box and in the mounting of the speaker, but also in the way the speaker leads are taken through the walls of the box. Solid terminals are preferred.
Another precaution may be necessary. In Figs. 15 and 16, from which we derived Eqs. (93), (94), (95), and (97), we assumed that the effect of the inductance $L_e$ is negligible. In fact, $L_e$ interacts with the parallel combination of $L_{ces}$ and $C_{mes}$ to produce a series resonance at $f_n$ in Fig. 5, where the nominal impedance is measured. If this frequency, usually 400–600 Hz, is well above the speaker resonance $f_s$, so that there is little disturbance of the curve at $f_2$ of Fig. 16, the accuracy of the measurements will be unaffected. But if $f_n$ is above 150 Hz, which can occur with small speakers and becomes even more likely when the speaker is placed in the box for the last test, the likelihood of inaccurate results increases.
This could be avoided by connecting in the circuit of Fig. 18 a bifilar inductance whose value $L_e$ in each half is equal to the inductance of the voice coil. It is preferable, and not difficult, to wind this with an air core. In measuring $L_e$ of the loudspeaker, it is important to measure it at a frequency well away from $f_n$, say, 10 kHz. Also it is important to measure it as an inductance in parallel with a resistance ($D$ or tan $\delta$ scale, \textit{not} the $Q$ scale of a bridge), for the $Q$ of the inductance at 10 kHz is usually of the order of one which can lead to serious error if the measurement is made as an inductance in series with a resistance. With a high-impedance voltmeter $V$, error due to series resistance of the inductor should be negligible.
If the new resonant frequency in the closed box $f_{sc}$ is found, the ratio of volume is usually given as
$$V_{as} / V_b = (f_{sc} / f_{sa})^2 - 1$$
(100)
where $f_{sa}$ is the resonant frequency of the speaker in air which we previously called $f_s$. However, this expression ignores the change in the acoustic mass $M_{as}$ of 1.05 to 1.25 times which results from placing the speaker in the box. A more accurate method is to repeat the previous procedures for finding $Q_e$. Then if we call $Q_{ea}$ and $Q_{eo}$ the values of $Q_e$ measured in air and in the closed box, respectively, then
$$V_{as} / V_b = [(f_{sc} Q_{ec}) / (f_{sa} Q_{ea})] - 1.$$
(101)
Also the ratio of the acoustic masses in air and in the closed box
\[ M_{asb}/M_{asf} = f_{sa}Q_{ea}/f_{sa}Q_{ec} \]
(102)
should lie between 0.8 and 0.95.
With \( V_b \) known, \( V_{ns} \) can be calculated. The size of \( V_b \) is not critical, but should not be too large, otherwise the ratio \( f_{sr}/f_{sa} \) becomes close to unity, and the accuracy of the \( V_{ns}/V_b \) calculation falls. This can be seen from Eq. (100). Finally the values of \( f_{sa} \) and \( Q_{ea} \) are adjusted to take account of the change in \( M_{as} \) when the speaker is placed in the box. Thus
\[ f_{sb} = f_{sa}(M_{asb}/M_{asf})^{1/2} \]
(103)
\[ Q_{rb} = Q_{sa}/(M_{asb}/M_{asf})^{1/2}. \]
(104)
Thus the efficiency \( \eta_{ob} \) can be calculated from Eq. (77). This gives the result, rather surprising at first sight, that the electroacoustic conversion efficiency of a loudspeaker in the piston range can be calculated from electrical measurements alone.
The following alternative method is useful, particularly when the loudspeaker has to be placed in a box whose size is already determined or as a final check on a previously calculated box, or again if it becomes too difficult to seal the loudspeaker in the test box.\(^5\)
First the vent, if adjustable, is made to resonate with the box somewhere near the speaker resonant frequency, but this is not very important. Then the three frequencies \( f_l, f_b, \) and \( f_h \) of Fig. 5 are found as accurately as possible. Special care is needed in reading \( f_b \) as the curve has a flat bottom.
From these readings we find \( f_{sb} \), the resonant frequency of the speaker when mounted in the box,
\[ f_{sb} = f_h f_l/f_b \]
(105)
and the compliance ratio \( C_{as}/C_{ab} \), i.e.,
\[ V_{ns}/V_b = (f_h^2 - f_b^2)(f_b^2 - f_l^2)/f_h^2 f_l^2. \]
(106)
With the speaker resonant frequency in air \( f_{sa} \) already known and \( f_{sb} \) known from Eq. (105), we find the mass ratio \( M_{asb}/M_{asf} \) from Eq. (103), and then \( Q_{eb} \) from Eq. (104). \( Q_a \) is adjusted to \( Q_{ab} \) in a similar manner. By reference to Table I and Fig. 7, a suitable alignment can be found, thus setting the final values of \( f_h \) and \( Q_r \). Note that \( Q_t \) is due to the parallel combination of 1) \( Q_{ab} \) and 2) \( Q_{rb} \) modified by the amplifier.
To estimate the value of \( Q_b \), the “\( Q \) of the box and vent circuit,” we measure \( I_b \), the current through the speaker at \( f_h \) with the input voltage held constant as before. Then
\[ Q_b = (\omega_b/\omega_s)(C_{ab}/C_{as})[(1/Q_r) + (1/Q_a)][(I_b - I_o)/(I_r - I_b)]. \]
(107)
Note that because the difference between \( I_r \) and \( I_b \) will be small, the readings must be taken carefully.
Comparing Eq. (107) with Eq. (89), it can be seen that
\[ (A_b)_{max} = 1/[1 + [Q_0Q_r/Q_t(Q_a + Q_e)][(I_r - I_b)/(I_b - I_o)]]. \]
(108)
This greatly simplifies the estimation of \( (A_b)_{max} \).
A worked example of this method is given in the Appendix.\(^6\)
**XV. EXPERIMENTAL WORK**
When the work was started from which this paper derived, it was necessary first to find the parameters for a number of loudspeakers. To date about fifty have been measured. In the case of one speaker, the effect of a number of modifications was observed; in the rest, usually one and occasionally two or three samples have been checked. The results obtained give confidence in the method. For example, from the readings and knowing other parameters, it is possible to calculate the flux density, and the values obtained give good correlation with readings on a flux meter. Changes of parameters during production can also be detected.
Some generalizations from the results have been mentioned earlier. For example, it was found that \( Q_a \) varies between about 3 and 10, which is high compared with the \( Q_t \) values of 0.2 to 0.6 required in Table I. Thus it was apparent that acoustic resistance usually has little effect on the damping of a speaker in a well-designed system. Values of \( Q_r \) varied from 0.2 to 0.5 in the case of high-quality speakers, through 0.5 to 1.0 in the better commercial grades of speakers, to 2 and even 3 in the case of some low-priced speakers.
Similarly efficiencies, for radiation from one side of an infinite baffle, ranged from \(-24\) dB (0.4%) for low-priced speakers through \(-20\) dB (1%) for medium-grade to \(-14\) dB (4%) for high-quality speakers.
However, one must resist the tempting generalization that it is possible to rate the overall quality of a speaker by its \( Q_r \) or even its efficiency. For example, if efficiency is made higher and \( Q_r \) lower by reducing the cone mass \( M_{ms} \), trouble with “break up” may result at middle frequencies. In fact while the best 8-in speaker tested had a \( Q_r \) of 0.33, there was one sample with good clean response at high frequencies with a high \( Q_r \) of 1.7 and another with \( Q_r \) below 1 which was less acceptable. It must be remembered that these readings, and the paper in general, are concerned only with low-frequency performance.
As a result of the design theory, a number of boxes have been made. In the absence of reliable measurements of sound pressure, all that can be said is that they gave a good improvement in clean low-frequency response, and that the cutoff frequencies are near the predicted values. Some particularly gratifying results have been obtained
\(^5\) Experience gained since the writing of this paper shows that accurate results are more easily obtained with this second method. Using a vented box is especially preferred if the speaker being measured has a low resonant frequency and if the testing box is fairly small. In such cases, small leaks in the “totally enclosed” box or around the loudspeaker pad ring can produce a virtual vent which produces the familiar twin peaks of loudspeaker impedance. But if the lower peak is below the limit of measurement, say, below 10 or 15 Hz, it could easily happen that the remaining upper peak would be taken as the single peak of a closed-box system with dire results.
\(^6\) Experimental work using the above method indicates that in practical boxes \( Q_r \) is often of the order of 10. This difference from the calculated values of 30 or more may be due to frictional losses in the timber. It is shown in Section XI that when \( Q_b \) is 10, the frequency response error is still only 1 to 2 dB. However, if there are sufficient air leaks, or if the cavity damping is excessive, as when the box is completely stuffed with underfelt, \( Q_b \) can fall below 5.
with 5-in speakers in modest boxes with response down to 80 Hz.
XVI. CONCLUSION
The work described herein was begun as an advanced development project in an attempt to obtain good low-frequency response from loudspeakers in small boxes. Unfortunately, no "revolutionary concept" was uncovered that offers something for nothing. On the other hand, it has provided a reasonably precise method of design that was previously lacking.
In general, a system with good flat response down to a predictable cutoff frequency can be designed, if the necessary parameters $Q_s$ (and $Q_n$), $V_{ns}$, and $f_s$ are known for the loudspeaker. The box volume is closely proportional to the inverse square of cutoff frequency, which can be varied over a wide range. The output impedance $R_g$ of the amplifier has a large effect in controlling the response, especially at $f_h$, the higher frequency of maximum impedance. Whether $R_g$ needs to be positive, zero, or negative depends on the type of alignment and the $Q$ parameters of the speaker. On the evidence available, acoustic resistance damping of the vent has no advantage, and is wasteful of box volume or bandwidth.
The advantages accruing from a predictable design include the possibility of optimum design of "rumble" filters. At frequencies below cutoff where negligible acoustic output is produced, these relieve the amplifier and loudspeaker of high signal amplitudes and thus minimize an annoying source of intermodulation distortion. Carried a step further, the use of auxiliary electrical filters makes it possible to trade box volume for low-frequency power capability of the amplifier.
Another way of reducing box volume is to increase the mass of the loudspeaker cone. But since this also reduces efficiency, it may be considered as a further example of trading amplifier size for box size, only this time the amplifier must deliver increased power over the whole audio spectrum. Again, the box volume may be reduced if a smaller diameter loudspeaker is used. The danger here is that the speaker excursion increases, but it is a good solution if the speaker is capable of a long linear excursion, or if the power output and/or low-frequency response is restricted.
The size of the magnet, or more precisely the flux density $B$, has a great influence on performance. Both efficiency, hence acoustic output, and $Q_s$ vary with $B^2$; so it is clear that the saving of pennies on a smaller magnet can be poor economy.
The parameters needed for vented-box design can be measured with normal electrical measuring equipment together with a test box of known net internal volume. Nevertheless it is suggested to loudspeaker manufacturers that it is in their interest, as well as the user's, to publish typical values of $Q_s$, $Q_n$, $V_{ns}$, and $x_{max}$, as well as $f_s$. These parameters are more useful to the system designer than, for example, flux density or total flux. Their publication would help ensure that the manufacturer's product is used to the best possible advantage.
The totally enclosed box has been mentioned only in passing, since it is well covered in [2]. But it should be noted that if a totally enclosed box is chosen with the same volume as that of alignment no. 5, the cutoff frequency is 1.55 times higher. With smaller boxes, the advantage decreases, though with practical sizes it is still appreciable. With larger totally enclosed boxes, the cutoff frequency can never fall below $f_s$, while the Chebyshev vented box alignments can extend the response considerably below $f_s$.
The greatest advantage of a vented box over an infinite baffle is the reduction of loudspeaker excursion, permitting higher power output or lower distortion. To this advantage, the present paper adds, it is hoped, a greater flexibility in design. The only apparent disadvantage of a vented box is in the transient response, but in fact the ringing is only perceptible with a misadjusted alignment. With proper adjustment, the effective reverberation time, though longer than that of a properly adjusted infinite baffle, is not long enough to appreciably color the sound in the listening room.
Finally, it is emphasized again that the acoustic response is due to the combination of speaker plus box plus amplifier as an integrated whole.
APPENDIX: WORKED EXAMPLE
This refers to a purely imaginary speaker, the readings being chosen to simplify the calculations. However, the readings would be typical of a medium-quality 8-in speaker.
Measurement of Speaker Parameters $Q_o$, $Q_s$, $V_{ns}$, and $f_s$
With a Wheatstone bridge we find
dc resistance of speaker $R_s = 4.00$ ohms
dc resistance of calibrating resistor $R_c = 5.00$ ohms.
Now we place $R_s$ in the test circuit of Fig. 17 and find that when $V$ reads 1 volt.
$$I_v = 180 \text{ mA}.$$
Now
$$I_v R_c = 0.180 \times 5.00 = 0.900.$$
Since this is 10% below the observed reading of 1 volt, one or both of the meters is inaccurate, but this is unimportant so long as their readings are constant with frequency and the reading of ammeter $A$ is linear.
Then from Eq. (98),
$$I_v = I_v R_c / R_s = (0.180 \times 5.00) / 4.00 = 225 \text{ mA}.$$
We now suspend the loudspeaker in air as far from reflecting surfaces as possible and read the minimum current $I_0$ which is 25 mA at 55.0 Hz ($f_{sa}$, the speaker resonant frequency in air).
Then from Eq. (99),
$$r_0 = I_v / I_0 = 225 / 25 = 9$$
$$\sqrt{r_0} = \sqrt{9} = 3$$
$$\sqrt{(I_0 I_v)} = \sqrt{(225 \times 25)} = 75 \text{ mA}.$$
With the voltmeter $V$ reading a constant 1 volt, the ammeter $A$ reads 75 mA at 44.0 and 68.75 Hz.
First we use this reading to check $f_{sa} = \sqrt{(44.0 \times 68.75)}$ from Eq. (93) = 55.0 Hz as before. Then from Eq. (97),
$$Q_s = f_u \sqrt{r_0} / (f_2 - f_1) = (55 \times 3) / (68.75 - 44) = 6.67$$
and from Eq. (95),
\[ Q_e = Q_a / (r_o - 1) = 6.67 / (9 - 1) = 0.833. \]
The speaker is now placed in a vented box whose net volume is 1000 in\(^3\) and we read the frequencies defined in Fig. 5,
\[ f_h = 100 \text{ Hz}; \quad f_b = 60 \text{ Hz}; \quad f_l = 30 \text{ Hz}. \]
Then from Eq. (105),
\[ f_{sb} = f_h f_l / f_b = (100 \times 30) / 60 = 50 \text{ Hz} \]
and from Eq. (106),
\[ V_{as} / V_b = (f_h^2 - f_b^2)(f_b^2 - f_l^2) / f_h^2 f_l^2. \]
Computation is easier if we rewrite Eq. (106) as
\[ V_{as} / V_b = (f_h + f_b)(f_h - f_b)(f_b + f_l)(f_b - f_l) / f_h^2 f_l^2 \]
i.e.,
\[ V_{as} / V_b = (100 + 60)(100 - 60)(60 + 30)(60 - 30) / 100^2 \times 30^2 \]
\[ = (160 \times 40 \times 90 \times 30) / (100 \times 30 \times 100 \times 30) \]
\[ = 1.92 \]
i.e.,
\[ V_{as} = 1.92 \times 1000 = 1920 \text{ in}^3. \]
In the vented box, the speaker resonant frequency has dropped \( f_{sb} / f_{sa} = 50 / 55 = 0.909 \) times. Thus from Eq. (103),
\[ M_{asa} / M_{asb} = (0.909)^2 = 0.826 \]
and from Eq. (104),
\[ Q_{ab} = 6.67 / 0.909 = 7.33 \]
while
\[ Q_{cb} = 0.833 / 0.909 = 0.917. \]
At \( f_b \) the current \( I_b \) was read as 220 mA. Then from Eq. (107), the \( Q \) of the box plus vent
\[ Q_b = (f_b / f_s)(C_{ab} / C_{as})[(Q_a + Q_e) / Q_a Q_e] \]
\[ = [60 \times (7.333 + 0.917) \times (220 - 25)] / \]
\[ [50 \times 1.92 \times 7.33 \times 0.917 \times (225 - 220)] \]
\[ = (60 \times 8.25 \times 195) / (50 \times 1.92 \times 7.33 \times 0.917 \times 5) \]
\[ = 29.9. \]
From Eq. (108) the maximum box loss in the quasi-Butterworth alignment described below, where \( Q_t = 0.347 \), is
\[ (A_b)_{max} = 1 / \{1 + [Q_a Q_e / Q_t(Q_a + Q_e)] \}
\[ [(I_e - I_o) / (I_b - I_o)]\} \]
\[ = 1 / \{1 + (7.33 \times 0.917 \times 5) / (0.347 \times 8.25 \times 195)\} \]
\[ = 1 / 1.060 \]
which is equivalent to 0.5 dB.
**Efficiency \( \eta \) from Eq. (77)**
\[ \eta_{ob} = 8.0 \times 10^{-12} f_s^3 V_{as} / Q_e \]
\[ = (8.0 \times 50^3 \times 1920) / (10^{12} \times 0.917) \]
\[ = 2.09 \times 10^{-3} \]
which is equivalent to \(-26.6 \text{ dB}\) in a box, or \(-23.6 \text{ dB}\) on an infinite baffle (i.e., a true infinite baffle, not a totally enclosed medium-sized box which gives the same efficiency as a vented box), or \(-20.6 \text{ dB}\) on a true infinite baffle, taking into account radiation from both front and back.
Thus if the speaker is mounted in a box and fed by a 5-watt amplifier, the acoustic power output will be
\[ W_{ao} = \eta_{ob} W_{ei} = 5 \times 2.09 \times 10^{-3} = 0.0104 \text{ Watts}. \]
If we assume a listening room of \( 16 \times 12\frac{1}{2} \times 10 = 2000 \text{ ft}^3 \), then from [4, p. 418, Fig. 11.12] an acoustic power of 0.003 watt provides \(+80 \text{ dB}\) intensity level. Our output is \(10.4/3\) times, i.e., 5.4 dB greater than this; therefore the system is capable of a peak \(+85 \text{ dB}\) intensity level.
**Peak Excursion \( x_{pk} \)**
We assume an alignment where the box is tuned to the same frequency as the loudspeaker, i.e., 50 Hz. This is typical of Butterworth alignments. Then the fixed part of the expression for \( x_{pk} \) in Eq. (84) is
\[ (1.31 \times 10^5 \times \sqrt{W_{ao}}) / f_b^2 S_d. \]
Now if the effective piston diameter is 7 in, i.e.,
\[ S_d = \pi \times 3.5^2 = 38.5 \text{ in}^2 \]
then the expression becomes
\[ 1.31 \times 10^5 \times \sqrt{0.104/(50^2 \times 38.5)} = 0.139 \text{ in}. \]
Now the maximum value of the frequency-sensitive expression for a vented box in the useful band (above \( f_b \)) in Fig. 10 is approximately one quarter. Thus
\[ x_{pk} = 0.139 / 4 = \pm 0.035 \text{ in} \]
compared with \( \pm 0.098 \text{ in} \) in a totally enclosed box (infinite baffle).
**Box Design**
First suppose we wish to obtain the best results with the original 1000-in\(^3\) box. Allowing 10% for the bracing and volume displaced by the speaker, the optimum inside dimensions would be \( \frac{3}{4} \times 1100 \times (0.8, 1.0, 1.25) \) in, i.e., 8.28 \times 10.33 \times 12.9 \text{ in}, say 8\(\frac{1}{4} \times 10\frac{1}{4} \times 13 \text{ in}\). This would need to be checked in case the original assumption of 10% was incorrect. Assuming that the dimensions are
| Type of alignment | QB\(_2\) | B\(_4\) | B\(_5\) | B\(_6(i)\) |
|-------------------|---------|---------|---------|-----------|
| C\(_a\) / C\(_ab\) | 1.92 | 1.414 | 1.000 | 2.732 |
| V\(_b\) (cubic inches) | 1000 | 1358 | 1920 | 704 |
| Box Height (in.) | 13 | 14 | 16 | 11\(\frac{1}{2}\) |
| Width (in.) | 10\(\frac{1}{2}\) | 11\(\frac{1}{2}\) | 13 | 9 |
| Depth "d" (in.) | 8\(\frac{1}{4}\) | 9 | 10 | 7\(\frac{1}{2}\) |
| Cutoff frequency \( f_3 \) (c/s) | 58.5 | 50 | 50 | 50 |
| Box frequency \( f_b \) (c/s) | 54.7 | 50 | 50 | 50 |
| L\(_v\) / S\(_v\) (in.\(^{-1}\)) | 1.56 | 1.37 | 0.97 | 2.65 |
| S\(_s\) (in.\(^2\)) | 7.69 | 10.07 | 16.25 | 4.50 |
| Vent height "1" (in.) | \(\frac{3}{4}\) | \(\frac{7}{8}\) | 1\(\frac{1}{4}\) | \(\frac{1}{2}\) |
| Q\(_t\) | .347 | .383 | .447 | .299 |
| (Q\(_s\))\(_{total}\) | .364 | .404 | .476 | .312 |
| R\(_s\) / R\(_s\) | \(-.600\) | \(-.560\) | \(-.481\) | \(-.860\) |
Table V. Computation of three Butterworth alignments for imaginary speaker.
then in a box similar to Fig. 9, the width of will be $10\frac{1}{4}$ in. The length of the tunnel will be $3\frac{3}{4}$ in, together with two thicknesses of timber (say 1 in each) plus a $\frac{1}{2}$-in square stiffener on the top rear edge of the shelf, giving a total tunnel length of $9\frac{3}{4}$ in.
The simplest alignment for $C_{as}/C_{ab} = 1.92$ is a third-order quasi-Butterworth between alignments no. 4 and 5. From Fig. 7 (b),
$$f_3/f_s = 1.17,$$ thus $$f_3 = 50 \times 1.17 = 58.5 \text{ Hz}$$
$$f_3/f_b = 1.07,$$ thus $$f_b = 58.5/1.07 = 54.7 \text{ Hz}.$$
Thus
$$\omega_b^2 = 1.18 \times 10^5$$
and for the tunnel, from Eq. (61),
$$(L_v/S_v)_{required} = 1.84 \times 10^8/\omega_b^2 V_h$$
$$= 1.84 \times 10^8/1.18 \times 10^5 \times 10^3$$
$$= 1.56 \text{ in}^{-1}.$$
Now if the tunnel height $l = \frac{3}{4}$ in, then area
$$S_v = 10\frac{1}{4} \times \frac{3}{4} = 7.69 \text{ in}^2$$
and
$$(L_v/S_v)_{end} = 0.958/\sqrt{S_v}$$
$$= 0.958/\sqrt{7.69}$$
$$= 0.34 \text{ in}^{-1}$$
$$(L_v/S_v)_{tunnel} = 9.75/7.69$$
$$= 1.27 \text{ in}^{-1}.$$
Thus
$$(L_v/S_v)_{available} = 1.61 \text{ in}^{-1}$$
which is about as close as can be obtained with the tolerances on the small dimension ($\frac{3}{4}$ in) of $l$.
**Amplifier Output Impedance $R_g$**
Now by interpolation,
$$Q_t = 0.347$$
and since $Q_{ab} = 7.33$, $Q_{eb} = 0.917$, and from Eq. (70),
$$1/Q_t = 1/Q_a + 1/Q_e(1 + R_g/R_e).$$
Thus
$$1/0.347 = 1/7.33 + 1/0.917(1 + R_g/R_e).$$
Hence
$$R_g/R_e = -0.60.$$
**Notes**
1) $(L_v/S_v)_{end}$ is small compared with $(L_v/S_v)_{tunnel}$, and since the vent area is already small compared with the piston area, a simple hole in the front panel would be quite impractical as a vent. Its area would need to be about 1 in$^2$.
2) The dimension $l$ ($\frac{3}{4}$ in) is fairly critical.
3) $Q_a$ has little effect on $Q_t$. The negative impedance required is fairly high but quite practical.
For comparison three Butterworth alignments have also been computed for this imaginary speaker so that the effect of amplifier filtering can be assessed (Table V). All three have cutoff frequencies of 50 Hz. But while $B_4$ has no filtering, $B_5$ has a simple CR filter which is $-3$ dB at 50 Hz ($CR = 3180 \mu s$), and $B_6$ has a peak 6 dB high at 53.5 Hz before it falls off at the rate of 12 dB per octave ($y = -1.732, f_{aux} = 50$ Hz).
**ACKNOWLEDGMENT**
The writer must acknowledge his heavy indebtedness to Novak's original paper [2], as well as to a number of his colleagues, including W. Buckland, D. A. Drake, J. G. Elder, M. C. Plumley, and N. K. Snow, who in many discussions have helped to hammer out the ideas presented. Special thanks are due to J. A. Lane who carried out most of the early experimental work and to K. W. Titmuss who continued it.
**REFERENCES**
1. E. J. Jordan, "Loudspeaker Enclosure Design," *Wireless World*, vol. 62, pp. 8-14 (Jan. 1956); pp. 75-79 (Feb. 1956).
2. J. F. Novak, "Performance of Enclosures for Low-Resonance High-Compliance Loudspeakers," *IRE Trans. Audio*, vol. AU-7, pp. 5-13 (Jan.-Feb. 1959).
3. L. L. Beranek, *Acoustics* (McGraw-Hill, London, 1959).
4. H. F. Olson, *Elements of Acoustical Engineering*, 2nd ed. (Van Nostrand, Princeton, N.J., 1947), pp. 154-156.
5. O. W. Eshbach, Ed., *Handbook of Engineering Fundamentals* (John Wiley, New York, 1936), p. 8-60.
6. A. N. Thiele, "Television IF Amplifiers with Linear Phase Response," *Proc. IRE (Aust.)*, vol. 19, p. 655 (Nov. 1958).
7. J. L. Stewart, *Circuit Theory and Design* (John Wiley, New York, 1956), pp. 159-163.
8. F. Langford-Smith, *Radiotron Designer's Handbook*, 4th ed. (A.W.V. Co., Sydney, 1952), p. 847.
9. R. P. Sallen and B. L. Key, "A Practical Method of Designing RC Active Filters," *IRE Trans. Circuit Theory*, vol. CT-2, No. 1, pp. 74-85 (Mar. 1955).
10. A. N. Thiele, "The Design of Filters Using only RC Sections and Gain Stages," *Electron. Eng.*, pp. 31-36 (Jan. 1956); pp. 80-82 (Feb. 1956).
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Causal Inference
Nancy Cartwright
1. Problems for Causation
Causation has long been a vexed notion across the sciences, so much so that there have been repeated cries that it must be banished forever from both the natural and the social sciences. Yet, whatever is the case with the natural sciences, causation remains at the heart of the social sciences. For example, the London School of Economics and Political Science (LSE), which is one of the foremost social science institutions in the world, has as its motto, *Rerum cognoscere causas*: ‘To know the causes of things’.
Why is causality so central? Because, it is widely believed, it is knowledge of the causes of things that allows us to understand the world, to predict the future, to build better social systems, to change the world we live in. Why, then, try to banish causation? Because the notion faces serious problems and there is no widespread agreement how, or even that, the problems can be solved. The problems can be grouped under three headings: meaning, method, and metaphysics.
1.1 Meaning
The eighteenth-century Scottish philosopher David Hume argued that our notion of causation is not what we think it is (see e.g. Garrett 2009). He claimed that all of our concepts (he called them ‘ideas’) come from experience. They are copies of the impressions that make up our experiences. Where, then, in our experience of happenings in the world is the impression of causation? Nowhere, he argued. Look as hard as you will. You only really see shapes, and colours, and motions, never a ‘making something happen’. So where does the concept come from?
What we call causes are regularly followed by what we label as their effects, said Hume. Human beings, he believed, are deeply prone to forming habits. So, having observed a regular association between two kinds of events, we come to expect the second when we see the first. Looking inwards at ourselves, we notice this feeling of expectation; we get an impression of it. Our concept of causation, Hume claimed, is a copy of that impression of expectation. All that is happening in the external world that contributes to our coming to have this concept is a regular association of events. The concept itself derives from an impression of our own internal state.
Although no one nowadays subscribes to Hume’s theory of concept formation, his conclusion still has a powerful grip, and it is not uncommon to find contemporary thinkers who hold what is called a *regularity theory* of causality, that all there is to causality is association, though many now would allow that the association may be merely probabilistic, not one that holds with full regularity.
Independent of the influence of thinking like Hume’s, there is good reason to care about just what the concept of causation is if we are to employ it in social science. For it is one of the central norms of science, whether it be natural science or social science, that its claims be clear and unambiguous and that the concepts employed in them be well defined and well understood. When we talk in science, we are supposed to be clear exactly what we are saying. So philosophers and social scientists alike have invested a great deal of effort trying to get straight the meaning of ‘cause’. We shall review some of the central recent attempts in section 2.
1.2 Method
Just as good science demands clarity about the meaning of the concepts it employs, it also demands that there be clear, explicit methods for determining just when a concept applies to something in the world and when it does not. Without such methods, claims involving the concept will be untestable. There will be no way to judge whether they are true of the world or not. It will also be difficult to use the concepts to make predictions or to provide instructions for how to bring about change in the world. So much effort is also put into devising sound methods for establishing when relations hold and when they do not. We will look at some of these in section 2.
As Chapter 14 of this book stresses, the two endeavours of characterizing or defining concepts, on the one hand, and of devising methods for determining when they obtain, on the other, must go hand-in-hand. The methods must be geared to showing whether the very concept that has been defined holds, so that we can be sure that our methods are teaching us about what we think we are learning about. You will not be surprised then that some of
the characterizations of causation you will see are almost read directly off from some of our favoured methods for establishing causal relations, and vice versa.
1.3 Metaphysics
The problems under this heading concern the nature of the causal relations themselves. Social science studies causal relations at two levels. The first is the singular: what are the causes and effects of specific single events? Using an example from Chapter 14, what caused the civil war in Angola in 1975? The second is the general: what kind of features are generally connected as cause and effect? For instance, ‘Ethnic diversity is among the causes of ethnic civil war’, or ‘Skill loss during periods of unemployment causes persistence of high levels of unemployment.’ Claims in social sciences about general causal relations are often called causal principles.
Probably the most central metaphysical problems facing singular causal relations arise from questions about how they behave in time. Are they instantaneous or extended in time? Is the cause temporally contiguous with the effect or is there a gap between them? Bertrand Russell (1912–13) argued that there cannot be a gap. Otherwise what would make the effect pop into existence when the cause was no longer there to produce it? This implies, he argued, that neither the cause nor the effect can be extended in time because the earlier parts of the cause, if they were to matter, would have to have an influence across a time gap; and so too with later parts of the effect. So causes must be instantaneous and contiguous with their effects. But that is impossible because time is continuous. Between any two instants there are infinitely many more, no matter how close together those two instants are. So, said Russell, the notion of one thing causing another does not make sense. We can sensibly talk of what values quantities have at each instant through continuous time, as when we use a line to represent the changing locations or changing velocity of the centre of mass of an object; but not of causality. That’s part of why the concept should be banned from both natural and social science.
Social scientists have not worried much about Russell’s problems. But they do worry about similar ones. There is a general consensus that in the single case there should be some continuous causal process connecting cause and effect. But the causes represented in social science studies are often aggregates, like GDP; they are often characteristics of institutions, like the managerial structure of Enron; and they are often norms or practices, like tax evasion. For causes like these, it can be hard to identify a causal process connecting cause and effect. What, for instance, might connect the high ethnic diversity in Angola in the period before 1975 with the ethnic civil war there in 1975 that it is supposed to have helped cause? Or suppose we want to argue that the GDP in a country in one quarter caused the rise in consumer spending there next quarter. What temporally continuous causal process connects these aggregate quantities? How even should we think about GDP itself with respect to time? Suppose all the production processes in a country are stopped for a day to mourn a dead leader. Does GDP go to zero then? Although these are live questions from social science, there has not been much headway on them recently and we shall not discuss them further here.
The dominant questions at the general level have to do with, first, the relation between the singular and the general. Must every singular causing fall under a general principle? We may say that the event reported in paragraph three of the lead story in today’s *New York Times* caused the event reported in the first paragraph on page five. Surely we do not think there is a general principle that ‘Events reported in paragraph three of the lead story of the *New York Times* cause events reported in the first paragraph of page five.’ But must there be some feature of the event reported in the lead story—say, that it involves a high level of ethnic diversity—and some feature of the event reported in page five—say that it is an ethnic civil war—which are connected by a general principle? This is an important question for how we approach the understanding of singular events in the social sciences, which is the meat of what is done in anthropology and in policy evaluation (did the policy really produce the improved outcomes we observe?). Philosophers over the last two and a half decades have tended to answer ‘yes’ to this question. Although there was a lively debate about the question involving both philosophers and social scientists in the 1960s (see e.g. Roberts 1995) concerning historical explanation, social scientists have not had much to say about it lately. The exception is the recent literature on evidence-based policy, to which we will turn.
The second dominant question at the general level is one that confronts all principles in science whether they are causal principles or some other kind of law and whether they are in natural or in social science. What makes a general truth a law? Don’t some facts just happen to be true generally? They hold by accident and we would not want to include them as principles in our scientific theories. For instance it is true that Venetian sea levels rise and so do bread prices. But this general association does not seem to be a law. Though this can be an important question, it is widely—and well—addressed in texts on philosophy of science in general, so we shall not pursue it here.
In what follows we shall discuss first general causation and then singular and, because of the close connection between the two, we shall discuss meaning and method together.
2. Meaning and Method
What does it mean to say, at the general level, ‘Ethnic diversity causes (or is among the causes of) ethnic civil war?’ Or ‘Rising inflation causes decreases in unemployment?’ Or, as Chapter 1 discusses, that wives slimmer than their husbands make for happy marriages? Most recent attempts to answer this question are not reductive. They do not attempt to define causation in totally non-causal terms. They suppose, to the contrary, that causation is a basic fact of nature that cannot be properly characterized in totally non-causal terms any more than central physics concepts, such as electron, can be characterized without using other concepts from physics theory, like proton and electromagnetic field. The view that causation cannot be characterized in entirely non-causal language and correlatively that the validity of our methods to test causal claims will always rest on some specific causal assumptions gets summed up in the slogan, ‘No causes in, no causes out.’ Nevertheless, even if a reductive characterization is not possible, if the concept is to play a role in social science, it is still essential that it have clear unambiguous meaning and that our methods for testing for it be appropriate to the concept as thus understood. Four main ideas are used in recent attempts to characterize causation, which we address in turn: probabilistic association; manipulation or intervention; mechanism; powers.
How do these four different approaches to causation relate? Are they all different theories about the very same thing, perhaps a special kind of relation that holds between events or a special kind of general truth or a special kind of law of nature? If so which, if any, is correct; and might they all, or at least more than one, be correct, perhaps by focusing on different aspects of this one thing? If they are not about the same thing, are each of them—or at least some of them—correct about something? Perhaps they focus on somewhat different kinds of relations in the social world, though are similar enough to go under the same loose title ‘causal’? These are important questions requiring both philosophical and empirical input. We will not discuss these here but leave them as topics in a more advanced course since they cannot be tackled till after we have a good idea what the different approaches are, which we do aim to provide here.
We shall also not discuss the relation between causation and explanation, on which there is a large philosophical literature (see e.g. Reiss 2013: chs 5–7).
2.1 Probabilistic Association
When a cause is present there should be more of the effect than if it were absent. That is the root idea of the probabilistic theory of causation. If C-type events occurring at some arbitrary time t cause E type of events at a time t later, then we should expect $\text{Prob}(E_v \mid C_v) > \text{Prob}(E_v \mid \neg C_v)$. But that’s similar to saying $E_v$ and $C_v$ are correlated—they tend to occur together; and it is a well-known mantra that correlation is not causation. High candy consumption in one month is correlated with low divorce rate the next. But eating candy does not prevent divorce. The correlation is due to the fact that candy eating is correlated with being young and young people have generally not been married long enough to get divorced. So, something more needs to be said.
Suppose you were able to control for all the other factors causing or preventing $E_v$.\(^1\) Imagine you could look in a population where all of these took some fixed value. So everyone is the same age, the same religion, is undergoing the same stress at work, has the same number of children, etc. In this population it seems reasonable to expect that if candy consumption does cause low divorce rate, it will increase the probability; and the probability will increase only if it does so since in this population there’s no other way to account for an increase in probability. Somewhat more formally, let $K_i$ represent a population where all the causes of $E_v$ other than $C_i$ take some fixed value, where $i$ ranges over all the sets of values these causes can take. Then $C_i$ causes $E_v$ in $K_i$ if and only if $\text{Prob}(E_v \mid C_i + K_i) > \text{Prob}(E_v \mid \neg C_i + K_i)$.
This is the gist of the probabilistic theory of causation, though more details need to be ironed out. For instance, note that this characterization is relative to the population satisfying $K_i$. What then about a larger population that contains $K_i$? That is one of the points of dispute. Some philosophers and social scientists insist on keeping the relativization to the $K_i$. Others allow that $C_i$ causes $E_v$ in any population that contains $K_i$, though this can lead to it being true both that $C_i$ causes $E_v$ in a population and also prevents $E_v$ there in cases where in one subpopulation $C_i$ increases the probability of $E_v$ and in another it decreases it. Under any circumstances this kind of characterization will always be relativized to a population since different populations will have different probabilities over the same variables and also they may well have other general causal relations holding, hence different factors for determining the $K_i$s. Chapter 3 worries about taking studies that show that a policy worked—i.e. caused the targeted outcomes—in one situation as evidence that it will have the same effects elsewhere. It is just this kind of relativity of causal claims to other causal factors (those that pick out the $K_i$s) that creates this worry: whether $C_i$ causes $E_v$ or prevents it or does nothing to $E_v$ at all in a given situation can depend very much on what other causal factors are there in the situation along with $C_i$.
\(^1\) Except those on the causal pathway between $C_i$ and $E_v$ if it exists.
Note also that seen as a characterization of general level causality—as a way of providing clear unambiguous sense for the concept—the probabilistic theory of causation is not reductive since we need to refer to general causal relations in specifying what $K_i$ is and thus we refer to other general causal relations in explaining what is required for any given general causal relation to hold. It is, however, very constraining. Each of the factors in $K_i$ must satisfy a similar formula with respect to $C_i$ and the remaining factors in $K_i$ since each of these is itself meant to be a cause of $E_v$. Although this may not narrow the choice of causes for $E_v$ to a single choice, it will rule out a huge number of alternatives. And adding some information about a few factors, that they are indeed a cause of $E_v$ or that they are not, can sometimes fix the set entirely given the probabilities.
This way of characterizing causality has the advantage that it connects immediately with standard statistical methods used in the social sciences to test for causal relations. These methods are used in what are called ‘observational studies’, which means that the data come from populations in their natural settings and not from specially selected populations enrolled in experiments. In the populations under study, social scientists measure correlations or regressions between factors to begin to test whether there is a causal connection between them in that population. These are weaker notions than conditional probabilities, like $\text{Prob}(E_v \mid C_i)$ and $\text{Prob}(E_v \mid \neg C_i)$, but are closely related. In a better test, they measure partial correlations or partial regressions, holding fixed other variables that they hope represent the other causes. This is akin to the partial conditional probabilities $\text{Prob}(E_v \mid C_i + K_j)$ and $\text{Prob}(E_v \mid \neg C_i + K_j)$. Similar kinds of ideas are used in econometrics, in estimating the coefficients that appear in economic equations that will hopefully represent causal relations.
The two assumptions of the probabilistic theory—that an effect is always probabilistically dependent on its cause and that this kind of dependency disappears when other causes are held fixed—are also at the core of what are called ‘Bayes nets methods’ for causal inference, for which computer programmes can generate all sets of causal relations among a given set of variables that are possible, given information about probabilistic dependencies among the variables, supposing the fundamental assumptions linking causes and probabilities are satisfied.
These two assumptions are also at the core of the reasoning behind randomized controlled trials (RCTs), which are highly touted as the gold standard for causal testing in medicine and, as we see in Chapter 3, in evidence-based policy, and are being pushed throughout the social sciences now, especially in development economics. The neat thing about RCTs is that they help with one of the central problems that the statistical methods discussed so far face: that we generally don’t know what the other causal factors are and so don’t know what to hold fixed.
In an RCT, the individuals in a population enrolled in the experiment—which could be individuals, schools, countries, etc.—are randomly assigned either to the treatment group, which will be subject to the cause ($C_i$), or to the control group, which will not have the cause ($\neg C_i$), but may perhaps receive a placebo. There should be as much masking as possible: the individuals in the experiment should not know which group they are in, nor should anyone involved in further monitoring or treatment, or in reading out the results to see if $E_v$ obtains or not, or in carrying out the statistical analysis. This is to guard against conscious or unconscious bias that may influence the results that are finally recorded. The aim is to ensure that both groups have the same distribution for all the other causal factors influencing the outcome, so that every arrangement of them—every $K_i$—has the same frequency in both groups. This aim can very rarely be achieved and moreover, we won’t know when it has been: statistics can tell us how often in the long run random assignment will produce any particular imbalance but we do not have even that kind of assessment when it comes to how well the masking has succeeded. So, as always in scientific work, we must not place too much confidence in the results of a single study, even one that has been very well conducted.
In order to see the logic of the causal inference, let us suppose, though, that the other causal factors have the same frequency in the treatment and control groups. The $\text{Prob}(E_v)$ in the treatment group will then be an average across the probability of $E_v$ given $C_i$ in each of the subpopulations in it—i.e. each of the $K_i$. So it will be an average over $\text{Prob}(E_v \mid C_i + K_j)$. Similarly, the $\text{Prob}(E_v)$ in the control is the average of the probability of $E_v$ given $\neg C_i$, in each of the subpopulations represented there—each of the $K_i$. So it is an average over $\text{Prob}(E_v \mid \neg C_i + K_j)$. By our hypothesis that the other causal factors have the same distribution in the two groups, the frequency of each $K_i$ subpopulation is the same in both. So if the probability of $E_v$ is greater in the treatment group than in the control group, that implies that for one of the $K_i$ subpopulations, $\text{Prob}(E_v \mid C_i + K_j) > \text{Prob}(E_v \mid \neg C_i + K_j)$. It follows that there is at least one subpopulation—one $K_i$—of the experimental population in which ‘$C_i$ causes $E_v$ in $K_i$’ is true, or at least is true under the probabilistic theory’s way of characterizing causality.
Can we conclude from the experiment ‘$C_i$ causes $E_v$ in the experimental population’? That depends on the decision referred to earlier about what to say about ‘$C_i$ causes $E_v$’ in a population given that $C_i$ causes $E_v$ in one of its subpopulations. What matters is that, however this decision is made, those using the claim should understand it and not read more out of it than the study supports. In particular, the higher probability in the treatment over the control group shows only that $C_i$ causes $E_v$ in some subpopulation of the population enrolled in the experiment. It may hold in specific other populations
or even across most. But $C_t$ may have exactly the opposite effect on $E_v$ in some subpopulations than it does in others. Finding out whether that is true requires a great deal more social science work.
2.2 Intervention and Manipulation
The manipulation view of causation revolves around the idea that causes give us effective strategies for producing effects we want, or preventing those we do not; by manipulating the cause we can manipulate the effect in a predictable way. So manipulation theories characterize general-level causation roughly this way, where again there is a dispute about the exact details of the formulation: for any two times $t$ and $t'$, ‘$C_t$ causes $E_{v'}$ in situations of type $S'$ just in case manipulating $C$ at $t$—making it bigger or smaller, or bringing it in or taking it away—is regularly followed by appropriate changes in $E$ at $t'$. For this to be true, we must be careful how these manipulations are carried out. ‘Reducing class sizes causes improved reading scores’ is true in many populations. But not if you lower teacher quality at the same time as reducing class size. Alternatively the manipulation of one factor may be followed by improvement in another without any causal connection. For instance new programmes for teaching reading may be followed by better reading scores not because the programmes cause better scores but because the teachers adopting them are regularly the better teachers or they become more enthusiastic when trying out new programmes.
This is where the concept of intervention comes in. ‘Intervention’ has been given a variety of formal definitions, but the basic idea is that an intervention is a manipulation that is done in the ‘right way’ to make the causal relation, or lack of it, apparent. The right way will be one in which neither other causes nor other preventative of the effect change, an idea we are familiar with from the probabilistic theory. Nor can any causal relations involving the effect change during the intervention. That’s because it is not a fair test of a causal relation if suddenly there are a lot of new causal relations produced between the effect and other factors that were not causes before. This includes changes in the very relation under test. One familiar way in which the requirement that causal relations involving the effect not change during intervention is violated is when the manipulation of the cause is so ham-fisted that it busts up the causal relation we are trying to test. One familiar example is when we wind up the toy soldier to see if that will make it march, but we wind too tightly and break the mechanism that makes it work.
Nobel-prize-winning Chicago School economist Robert Lucas claims this is frequently true with attempts to manipulate economic variables to bring about desired change. For instance, inflation, when it occurs naturally, can in the short run cause reductions in unemployment. That’s because, so the story goes, entrepreneurs mistakenly see the universal rise in prices as a real rise in prices in their sector and so hire more workers to produce more goods to meet increased demand. If, though, the government manipulates inflation to improve unemployment, entrepreneurs will recognize the rise in prices for what it is—just inflation—and will not open new jobs. The government’s very attempt to use the causal relation between inflation and unemployment will break it. Or at least so says Lucas’s model.
As with probabilistic theory, the characterization of general causation using the concept of manipulation is not reductive since the definition of an intervention, however the details are worked out, will have to refer to other general causal relations.
Also, as with the probabilistic theory, the characterization of causality in terms of manipulation is closely linked with familiar methods for testing, in this case with real non-statistical experiments where other causes are held fixed and only the cause under test is varied. Here too we are often stymied by our lack of knowledge of what these other causes are. This is the problem that plagues standard before-and-after studies. In before-and-after studies the cause is administered and we look to see if the effect changes. But, are we sure that nothing else changed at the same time as the cause? One of the tricks of social science is to find situations where we can have reasonable confidence that nothing else that could influence the effect has changed even if we do not have a catalogue of what all the other influences might be to check on them. This is the strategy that is used in what are called ‘instrumental variables’ models in economics. For instance Joshua Angrist in a classic paper uses the Vietnam era draft lottery to measure the effect of veteran status on earnings (Angrist 1990). Since whether an individual was drafted was determined on the basis of a randomly assigned number, we can be fairly confident that factors that affect earnings, like education, were equally distributed between the group of individuals who were drafted and the group of individuals who were not, as in an RCT. And so we can be fairly confident that the comparison between the average earnings in one group and the average earnings in the other tells us something about the effect of veteran status on earnings.
The manipulation theory of general-level causation is closely akin to the counterfactual theory of singular causation, which is based on the idea that one specific event, $c$, is a cause of a later event, $e$, just in case $e$ would not have occurred unless $c$ had. You can see immediately that this kind of claim will be hard to test since $c$ and $e$ are specific events that either occur or do not; so we can never observe what would have happened had things been different. There are also huge problems of formulation. There is a vast literature available in philosophy on counterfactuals and causes in which you can read more about these (see e.g. Paul 2009 and references therein).
Similar counterfactuals have come to the fore in social science lately because of the rise of the evaluation industry for social policy. As Chapter 3 explains, there is a great demand to know whether the policies we have implemented have ‘worked’: Has the policy genuinely produced the change intended? To answer that, it helps to know what would have happened otherwise. RCTs are often employed to this end. But of course they cannot tell us of a specific case whether the policy brought about the effect there, since they only look at groups. If an RCT shows more positive results in the treatment group, the group in which the policy was implemented, than in the group where it was not, we can conclude that in some of the cases in the treatment group the policy produced the effect. But we do not know which; we have no way to sort cases where the policy brought about the effect from those where something else was responsible. So for reliable evaluation of the single case, some different way of reasoning is required. This often involves process tracing which we shall discuss in the next section.
Not only do we not know from the RCT for which individuals the policy worked. We do not know what sets of characteristics matter. We know that there is at least one arrangement of characteristics that individuals might have—one $K_i$—for which the policy promotes the desired outcome. We call these ‘support factors’. But we do not know which it is. This matters when it comes to putting the claims established in RCTs to use in predicting what will happen if the policy were to be implemented elsewhere. If the population elsewhere has no individuals with just the right arrangement of support factors (no individuals described by the successful $K_i$s), or not enough to make it pay, the policy will not produce the desired outcomes in the new setting. Given good reason to think that the new setting does have enough individuals with the right arrangements, we have good reason to suppose the policy will work for some individuals there. But without good reason to suppose this, the RCT results will not be much use in predicting results in the new setting.
Consider for instance Oportunidades, a Mexican programme for poverty reduction. Because Oportunidades was itself designed as an RCT, there is very good evidence that it did contribute to reducing poverty where it was implemented. Does the success of Oportunidades provide evidence supporting predictions regarding what would happen were a similar programme implemented in some other population? Only if enough individuals in this target population have the right arrangement of support factors. And one cannot assume without evidence that this will be so. This point is illustrated by the case of Opportunity NYC, a poverty reduction programme modelled—and named—after Oportunidades which was implemented in New York City in 2007 but discontinued in 2010 because it failed to produce the expected effects. The success of Oportunidades in Mexico did not, by itself, provide sufficient evidence to support the prediction that Opportunity NYC would produce similar results, and the mistaken assumption that it did led to a poor policy decision.
### 2.3 Mechanisms
There are three senses of the term ‘mechanism’ in play currently in work on causation in philosophy and in the social and in the biomedical sciences. These include notions of *causal process*, *invariance*, and *underlying structure*.
**CAUSAL PROCESSES**
For singular causation it is widely held that a cause must be connected with its effect by some spatio-temporally continuous process between them. So tracing the causal process between them can be a good way to tell whether two events are related as cause and effect, a good way IF we know how to tell a causal process when we see one. So what distinguishes a continuous sequence of events that constitutes a causal process from one that does not? One popular philosophical account answers that in causal processes energy is transferred at each step in between. But this is not of much help in social science (see e.g. Dowe 2009).
A standard procedure in social science and in policy evaluation is to break the sequence into small steps where it is easier to determine for each step whether it is a genuine cause of the next. For instance, the Global Environment Facility evaluated the effectiveness of one of its programmes, the end-goal of which was to ‘establish a long-term conservation finance mechanism to support biodiversity conservation’ in the Bwindi national park (Uganda), by determining whether this programme had an effect on four intermediate outcomes (e.g. the establishment of a Bwindi Trust) which in turn were supposed to contribute to achieving the end-goal of the program (GEF 2007: 6).
Process tracing necessarily focuses on singular causation. But it can be a tool for general causation as well. If in situations of kind $S$, an event of kind $C$ at $t$ is regularly followed by an event of kind $E$ at $t'$ and it regularly happens that the same kind of causal process connects the individual events, then we can conclude at the general level that ‘In $S$-type situations, $C_t$ causes $E_{t'}$.’ This kind of reasoning has played a significant role in the biomedical sciences. For instance, tracing the process by which the chemicals in cigarette smoke lead to lung cancer provided a central piece of the evidence that showed that smoking causes lung cancer.
**INVARIANCE**
Suppose we observe that in a particular kind of situation $S$ one kind of feature $F_2$ regularly changes after another, $F_1$, changes. That is mere correlation and we know that correlation does not equal causation. But suppose that $F_1$
changes by intervention. If F1 causes F2 we expect that F2 will change following the intervention in the way that it always has; but not, if F1 does not. Changing levels of candy consumption may be regularly followed by changing probabilities of divorce. But this relationship is not invariant under intervention. If we intervene to change candy consumption—‘intervene’, so the only change is in the amount of candy consumed—the divorce rate will not change in tandem. This observation has given rise to the invariance account of causality now in fashion in philosophy of science. The basic idea is that, supposing C\textsubscript{i} and E\textsubscript{t} are regularly associated in S, then ‘C\textsubscript{i} causes E\textsubscript{t} in S’ just in case the association between C\textsubscript{i} and E\textsubscript{t} is invariant under interventions on C\textsubscript{i}.
This account is clearly closely connected with the manipulation account, and it shares one major drawback with that account that we have not yet discussed. The manipulation account is grounded in the idea that causes provide strategies for changing the world. Suppose we do characterize causation as invariance under intervention. Then changing C\textsubscript{i} will be a good way to change E\textsubscript{t}—but that follows only if C\textsubscript{i} is changed by intervention. But interventions are hard to come by. We are in a far more powerful position to predict what will happen and to engineer what we want if we know about a regular association that is invariant under the method of implementation that we will in fact employ. This gives rise to another, different characterization of general-level causation, one that we can find in contemporary economics (see e.g. Hoover 2001: ch. 2). Roughly, if C\textsubscript{i} and E\textsubscript{t} are regularly associated in S, then C\textsubscript{i} causes E\textsubscript{t} in S relative to φ just in case the association between C\textsubscript{i} and E\textsubscript{t} is invariant when C\textsubscript{i} is changed by φ, where φ can then represent the method by which we will in fact change C\textsubscript{i}. Knowing that C\textsubscript{i} causes E\textsubscript{t} under this characterization of causation immediately provides an effective strategy for changing E at t’.
Notice that these different versions of the invariance account can give different verdicts about whether C\textsubscript{i} causes E\textsubscript{t} in any given situation. This underlines the importance of being clear exactly what we mean when we make causal claims in social science.
**UNDERLYING STRUCTURES**
Different causal relations hold in different social, cultural, and economic settings. What is a required politeness in one culture can cause insult in another. Why? What causal relations hold in a social setting depends on the underlying system, the underlying social, economic, and cultural structure. It is now standard in philosophy of biology to call the structures that underlie causal relations there ‘mechanisms’, and sometimes this usage is adopted in the social sciences as well. Though there is no widely used mechanistic characterization of causation, there is widespread sense that, if we are to follow the injunction of the LSE motto, to know the causes of things, we should come to understand the underlying mechanism that make the particular causal principles that hold for a given population possible.
This is clearly of central importance in social planning and policy. Consider recent problems in child welfare in the UK, where attention tended to focus on causal processes. When a tragic child death occurs, ‘The standard response is to hold an inquiry…trying to get a picture of the causal sequence of events that ended in the child’s death…We are tracing a chain of events back in time to understand how it happened.’ So says Eileen Munro (2005: 377), author of Chapter 3 and of the 2012 UK government report on child protection. Munro recommends instead a ‘systems’ approach. That means focusing on the underlying mechanism or social structure that makes these kinds of processes possible, or even probable. As a US National Academy of Science report on building safer health systems explains, ‘The focus must shift from blaming individuals for past errors to a focus on preventing future errors by designing safety into the system’ (Kohn et al. 2000: 5).
So it is clear that it is important to study social mechanisms in social science. What is not clear is what kinds of concepts should be used to describe these mechanisms and their operation, nor what tool kit of methods will help us to understand them. This is one of the central tasks right now on which philosophy and social science intersect.
### 2.4 Powers
John Stuart Mill argued that women have the same powers of leadership and imagination as men, and we have these powers even should they be seldom displayed. Give women the same education, upbringing, situation, and opportunities as men and they will display these qualities equally with men. And he used this claim to argue for dramatic changes in the social policies and practices in play at the time regarding the role of women in society.
As with all concepts, if the concept *power* is to play an important role like this in social science or in social policy deliberation, we should be clear what is meant by it. This is a big topic in metaphysics nowadays and, to a lesser extent, in philosophy of science (see e.g. Mumford 2009). It is usual to make at least four assumptions about powers. First, a system—an object, a person, an institution—can have a power without displaying it. Second, the power will be displayed if the conditions are right. Third, what actually happens when a power is displayed depends on the setting. So what happens when a woman displays her imagination will depend on whether she is at a laboratory work bench or addressing Parliament or tending her children. Fourth, when a power operates in a situation, what happens there will depend in some intelligible way on the display of the power (as when a heavy metal object does not fall to the ground because the power of a magnet is displayed).
The most typical characterizations of powers ‘back’ define them from their displays. Roughly, A has the power to $\alpha$ just in case when properly enabled and failing something stopping it doing so (which we sometimes express as ‘nothing interferes with the display’), A does $\alpha$. This characterization is dramatically non-reductive because of the concepts of *enablement* and *interference*. Contrast this with an alternative: ‘A has the power to $\alpha$ just in case there are some circumstances $C\alpha$ such that it is a law of nature that in $C\alpha$, A does $\alpha'$, where $C\alpha$ is to be filled in by some possibly very long list of features that can be characterized without any power-related notions, like ‘is schooled at Eton’, ‘learns Latin’, ‘does not play with dolls’, etc. Many powers advocates do not adopt this alternative because, they argue, there is never a list that does the job. In each actual situation there is a fact of the matter about whether the circumstances that obtain there constitute an interference and whether they are enabling, but there is nothing these facts share in common that we could fill in for $C\alpha$ other than that they are enabling or they interfere. This is taken by many to be particularly problematic since philosophers have not succeeded in having much enlightening to say about interferences and enablers at the general level.
Why then take powers seriously? Philosophers offer a panoply of metaphysical arguments. But for social science the answer seems to lie in the usefulness of the concept, as with Mill, who argued for the centrality of the related notion of *tendency* in political economy. Another word used for much the same idea is ‘capacity’. In order to link it with our contemporary discourse, I shall use the word ‘power’ throughout for all these related notions. Mill’s model for powers comes from Newtonian mechanics. The sun has the power to attract the planets. Nothing ever interferes with it so the sun constantly displays this power (called ‘gravity’). What happens when it displays it? The planets move around it in elliptical orbits. By contrast, when the earth displays its power to attract a cannonball shot at an enemy ship, the cannonball moves along a parabola. What actually happens when the power of gravitational attraction is displayed (i.e. when the sun or earth attract other objects) depends on the setting. To make a link with our immediately previous discussion, we may think of the earth–cannonball pair as a mechanism, in the sense of an underlying structure, giving rise to the causal principle, ‘The earth displaying its power of gravitational attraction causes cannonballs to move along parabolas’; and the planetary system as a mechanism giving rise to the causal principle ‘The sun displaying its power of gravitational attraction causes the planets to move along ellipses around it’.
How do we know what happens when a power is displayed in a given setting? That’s easy, at least in principle, in Newtonian mechanics. All that’s relevant to how a power affects motions is what other powers are displayed in the situation. The display of the power is represented by what we call a force, represented by a vector. When several forces are displayed at once, we add these vectors, call the result ‘the total force, $F_{\text{tot}}$’, and calculate the acceleration of any object in the situation by the familiar formula $F_{\text{tot}} = ma$, where $m$ is the mass of the object. That is what we do, for instance, when we want to know how a pin will behave when a magnet attracts it upward and the earth attracts it downward, or how an electron in an atom behaves when it is attracted by both the mass of the nucleus and the charge on the protons there.
That’s all physics. Life is not so easy in social science, which may have some knowledge of powers but little knowledge of methods for calculating just what happens in different situations when the powers are displayed. Consider an economics example already mentioned, the one involving skill loss during unemployment. Earlier I suggested that economics entertains a causal principle that skill loss perpetuates high levels of unemployment. That is probably a mistaken way to look at it. It is seldom the case that skill loss is the only thing going on that affects unemployment. There are, for instance, sometimes vigorous successful government efforts to combat unemployment. In these cases skill loss may be followed by improving levels of employment, contrary to what is suggested by this causal principle. A better way to put it might be ‘Skill loss has the power to cause continuing high unemployment’. This will not lead us to expect that skill loss actually produces unemployment whenever it occurs, but only that, to understand the levels of employment that actually occur, we shall have to take the downward push of skill loss into account.
So, we return to the question, ‘How do we take it into account?’ How do we predict what happens when a socio-economic power is displayed? There is no systematic answer. This is one of the problems social scientists constantly confront in trying to put their isolated parcels of hard-won knowledge—like the knowledge of specific powers such as the power of skill loss to perpetuate unemployment—to use.
### 3. Putting Causal Knowledge to Use
Chapter 14 in this book teaches us that the meaning we assign a scientific concept must be closely paired with our methods for finding out whether it obtains. We should have good arguments to show that the methods we employ provide a good way to find out about just what we claim we are finding out about. That message has been echoed throughout this chapter with respect to the concept of causation when it is used in the social sciences. It is not just meaning and method that must match, however. So too must use. The use to which we put our social scientific claims—the inferences we draw from them and the practices we adopt on the basis of them—should be
supported by what those claims actually say; and what they say that supports our inference should in turn be supported by our methods for deciding that what they say is true. It is no good making a very broad claim whose first half is well supported by the evidence but then draw our inferences from the second half, which is unsupported.
We have looked at a number of accounts of what causal claims say. The claims say different things because they employ different concepts of causation. These concepts may all go under the same title, ‘general causal relation’, but they require different conditions to be met in order to obtain. So different methods must be used to find out about them and different uses made of them. So, of what use is knowledge of the causes of things? Knowing causes helps us to understand how things work. But it is also supposed to be of practical value. Knowing the causes of things should help us to change the world.
Here we must be cautious though. There is a tendency to too much haste, to try to read off directly from our causal claims just what we must do to bring about the change we desire. That will work with some kinds of causal knowledge but not most. Consider the manipulation concept of causation found in economics that I mentioned in discussing invariance: $C_t$ causes $E_t$ relative to $\phi$ just in case manipulating $C_t$ by $\phi$ is followed by $E'_t$, where $\phi$ is one of the methods available to us to change $C$ at $t$. Suppose we know that. Then we are in a powerful position to change $E$ at $t'$.
But suppose we have established instead a different causal claim, say a manipulationist claim of the kind favoured in philosophy—$C_t$ causes $E_t$ just in case if $C_t$ changes by intervention $E_t$ changes—or in invariance-type claim—$C_t$ causes $E_t$ just in case the association between them is invariant when $C_t$ changes by intervention. That too can be of immediate use—if we have an intervention to hand, which we usually do not. We have seen the same kind of problem with knowledge of powers. Knowing about what powers a system has will not by itself tell us what to expect to happen in particular circumstances nor how to build circumstances to get what we want. Nor does knowing the causal relations, on the probabilistic theory, that hold in one population, perhaps the population enrolled in an RCT, give us immediate guidance about how to bring about change in a different population.
This does not, however, in any way make causal knowledge in social science useless. It just puts it more on a par with similar knowledge in the natural sciences, where strategies are built by splinting together a very great many different pieces of knowledge and often undertaking designated new researches and developing new theory to fill in gaps. The great nineteenth-century physicist Lord Kelvin laid the first Atlantic cable in 1866. His knowledge of causes in physics was essential to his success but it definitely did not tell him just what to do. Similarly, the large team of physicists, engineers, mathematicians, and technicians who developed the US Second World War radar took many months to do so, despite the vast repository of causal knowledge the team members brought to the project. The conclusion I propose is that the causal knowledge we work so hard to gain in the social sciences can be of immense use. But there is no reason to think it should be easier to put our social science knowledge to use than it is to use our knowledge in the natural sciences.
References
Angrist, J. (1990). ‘Lifetime Earnings and the Vietnam Era Draft Lottery: Evidence from Social Security Administrative Records’, *American Economic Review*, 80(3): 313–36.
Dowe, P. (2009). ‘Causal Process Theories’ in H. Beebee et al. (eds), *Oxford Handbook of Causation*. Oxford: Oxford University Press, 213–33.
Garrett, D. (2009). ‘Hume’, in H. Beebee et al. (eds), *Oxford Handbook of Causation*. Oxford: Oxford University Press, 73–91.
GEF (2007). *Case Study: Bwindi Impenetrable National Park and Mgahinga Gorilla National Park Conservation Project*. Impact Evaluation Information Document, 7. Washington, DC: Global Environment Facility, Evaluation Office.
Hoover, K. (2001). *Causality in Macroeconomics*. Cambridge: Cambridge University Press.
Kohn, L., Corrigan, J., and Donaldson, M., eds (2000). *To Err is Human: Building a Safer Health System*. Washington, DC: Committee on Quality of Health Care in America, Institute of Medicine.
Mumford, S. (2009). ‘Causal Powers and Capacities’, in H. Beebee et al. (eds), *Oxford Handbook of Causation*. Oxford: Oxford University Press, 265–78.
Murro, E. (2005). ‘Improving Practice: Child Protection as a Systems Problem’, *Child and Youth Services Review*, 27: 375–91.
Paul, L. A. (2009). ‘Counterfactual Theories’, in H. Beebee et al. (eds), *Oxford Handbook of Causation*. Oxford: Oxford University Press, 158–84.
Reiss, J. (2013). *Philosophy of Economics: A Contemporary Introduction*. London: Routledge.
Roberts, C. (1995). *The Logic of Historical Explanation*. University Park, PA: Pennsylvania State University Press.
Russell, B. (1912–13). ‘On the Notion of Cause’, *Proceedings of the Aristotelian Society*, 13: 1–26.
Further Readings
Beebee, H., Hitchcock, C., and Menzies, P., eds. (2009). *Oxford Handbook of Causation*. Oxford: Oxford University Press.
Campaner, R., and Galavotti, M. C. (2007). ‘Plurality in Causality’, in P. Machamer and G. Wolters (eds), *Thinking about Causes: From Greek Philosophy to Modern Physics*. Pittsburgh: University of Pittsburgh Press. 178–99.
Cartwright, N. (2007). *Hunting Causes and Using Them*. Cambridge: Cambridge University Press.
McKim, V., and Turner, S., eds (1997). *Causality in Crisis: Statistical Methods and the Search for Causal Knowledge in the Social Sciences*. South Bend, IN: University of Notre Dame Press.
Russo, F. (2009). *Causality and Causal Modelling in the Social Sciences*. New York: Springer.
**Index**
aggression 104–5, 107, 114–16, 119
agmatology 259
Anany, Roger 71–2
archaeology 68–77, 79–82
behavioural traits 115, 118
environmental factors 102–3, 106–7, 118
heritable factors 107–8
individual differences 104, 116–17, 119
pluralism in the study of 113, 115–17
population differences 115–17, 119
population-level factors 109–10, 116
sex differences in heritability 115
behaviour genetics:
molecular 105, 108, 112–13
quantitative 104–5, 107–8, 112
Boghossian, Paul 71–3
boundary objects 238–9
Brannan, Michael 93
Burt, Cyril 172–3
Campbell, Donald 294–5
Cartwright, Nancy 61–2
case studies 288–307
combinath laboratory experiments 291–2, 297, 301, 304
critiques of 292–3
categories:
characterization of 267–70, 272–3, 276, 278–81, 284–5
procedures for measurement 276–8, 280–1, 284–5
representation of 271–6, 279–80, 284–5
causal principles 62, 310–11, 322–3
causal processes 63, 305, 319
causal spaces 111–13, 115
causation 102–6, 115, 273, 308–10, 312–13, 319–20, 323–5
manipulation view 316–18
probabilistic theory of 312–14, 324
singular 317, 319
choice models 185–7, 189, 191, 193, 195, 197–9, 201, 203, 205–6
EU models 199, 201
game models 191–201, 203–5
individual decision 186–91
civil war 265–73, 277–8, 284
ethnicity 270, 282–4, 311–12
civil war studies 265, 275–6
Clark, Geoff 71, 73, 81
class sizes 55–7, 61–2
climate change 2, 31–46, 133–4, 253
adaptation 39–41, 43
adaptive capacity 39–41
cost of future generations 42, 45, 130–3
integrated assessment models 38, 41
mitigation 41–3
scenarios 37–41
vulnerability to 39–41
climate change impacts 38–9, 43
collectivism 7–9, 20, 95
community-based participatory research (CBPR) 68, 79–80
concepts 1, 97, 238, 268–72, 274, 277–8, 283–4, 308–9
Ballung 268–72, 273–8, 284
conventional networks 96–7, 100
constraint satisfaction network 97
consensus conferences 251–3, 261
conventions 215–18, 221–6, 229
crime 103–4, 108–10, 119
Crombie, Alistair 288–9
Dasgupta, Partha 24
Daston, Lorraine 127, 155
Davies, Huw 52, 64
decision theory 185, 201, 203
degrees of freedom 295
Deleuze, Gilles 139–40
DeVault, Marjorie 192–3
developmental systems theory (DST) 106, 112–14
disability-adjusted life year (DALY) 276, 280–1
discipline 234–5, 237–40, 244
discount rate 42, 130–1, 133
discovery 299–300, 302, 306
doctrine 234–5, 242, 244
Douglas, Heather 70
Douglas, Jack 137–8
Dupré, John 179
Durkheim, Émile 136–8
|
NEUROENDOCRINE REGULATION OF GASTRIC ENDOCRINE CELL FUNCTION
Thesis submitted in accordance with the requirements of The University of Liverpool for the degree of Doctor of Philosophy by Andrew Damian Higham B.Sc., M.B. Ch.B., M.R.C.P.
March 1998
LIVERPOOL UNIVERSITY LIBRARY
LATIVX
The last decade has seen major advances in the understanding of diseases of the proximal gastrointestinal tract. Not least of these has been the recognition of the significance of *Helicobacter pylori* (*H. pylori*) gastritis in the pathogenesis of peptic ulceration, chronic atrophic gastritis and gastric carcinoma. In addition the development of proton pump inhibitors (PPI) has been heralded as a major therapeutic advance. However both *H. pylori* gastritis and PPI therapy elevate plasma gastrin concentrations and this has led to increasing interest in the physiological role of the antral hormone gastrin.
In this thesis, the role of intrinsic antral neurons in the regulation of gastrin release in response to a variety of stimuli has been examined. In addition, the effects of antral denervation on gastric emptying were clearly apparent and prompted investigation of the relationship between antral neurons and the cholecystokinin-independent mechanisms that have been well described as regulators of gastric emptying in the intact rat. It was found that antral neurons are essential for normal gastric emptying but are not required for the CCK-mediated inhibition of gastric emptying.
In addition, following antral denervation there was elevated fasting and meal stimulated plasma gastrin, the post-prandial rise in gastrin release occurring by a non-gastrin releasing peptide mediated mechanism. Moreover, non-nutrient
distension of the stomach was an adequate stimulus for gastrin release following antral denervation, suggesting that in the intact rat, antral neurons normally inhibit gastrin release to non-nutrient distension.
The effects of antral denervation on the endocrine cells of the stomach were also studied as were the effects of hypergastrinaemia on the Enterochromaffin-like (ECL) cells of the gastric corpus. Of particular relevance was the demonstration by Ashahara et al., (1996) of the *reg* gene in the ECL cells of the rat. Expression of this gene has been associated with pancreatic endocrine cell growth and differentiation. Data is presented that indicates that expression of this gene is regulated by gastrin and that it may play a role in promoting the differentiation of endocrine cells of the proximal gastrointestinal tract. Mutations of *reg* were associated with the development of gastric carcinoid (ECL cell) tumours in patients with hypergastrinaemia.
Overall it is clear that the antral innervation plays a central role in the control of gastrin release and that loss of the normal innervation of the G-cell leads to fasting and meal-stimulated hypergastrinaemia. The effects of persistently elevated gastrin on other cell populations of the stomach are likely to be of increasing importance with respect to understanding the mechanisms of tumour development.
The work presented in this thesis has appeared in part in the following publications:
**Papers**
Higham A.D., Vaillant C., Yegen B., Thompson D.G., Dockray G.J.: Relationship between CCK and antral innervation in the control of gastric emptying in the rat. *Gut* 41:24-32 1997
Higham A.D., Noble P., Thompson D.G., Dockray G.J.: Increased sensitivity of gastrin cells to gastric distension following antral denervation in the rat. *J Physiol* 503(1):169-175 1997
Higham A.D., Dimaline R., Varro A., Attwood S, Armstrong G, Dockray G.J., Thompson D.G.: Atrophic gastritis and large carcinoid nodules: Octreotide suppression test predicts beneficial outcome from antrectomy. *Gastroenterology (In press)*.
**Abstracts**
Higham, A.D. Thompson, D.G., Dockray, G.J.: Antral denervation by benzalkonium chloride leads to gastric retention of solids in the rat. *Gut* 36(1):A52 1995.
Higham, A.D. Thompson, D.G., Dockray, G.J.: Peptidergic afferent and intrinsic innervation of the rat stomach following antral denervation. *Neurogastroenterol Mot* 7:9 1995.
Higham, A.D. Valliant, C. Thompson, D.G., Dockray, G.J.: Complete loss of peptidergic afferent fibre innervation of the rat stomach following antral denervation. *Reg Peptides* 57:199 1995.
Higham, A.D., Thompson, D.G., Dockray, G.J.: Post-prandial gastrin release after antral denervation: no role for gastrin-releasing peptide. *J Physiol* 487:34P 1996.
Higham A.D., Noble P.J., Thompson D.G., Dockray G.J. Increased gastrin release following distension after antral denervation. *J Physiol* 499: 104P 1997.
Higham A., Thompson D.G., Dimaline R. & Dockray G.J.: Increased *Reg* gene expression in the rat gastric corpus following omeprazole treatment. *Reg Peptides*. 71:55 1997.
**Chapters and Invited Reviews:**
Higham A.D.: Clinical endocrinology of gastric function. *Surgery* (in press) 1997.
Statement of Originality
All of the data in this thesis that is presented as results has arisen wholly from my own work apart from the following contributions from colleagues within the Department of Physiology, University of Liverpool. The tissue culture of AR42J and HIT cells was performed by Dr Lisa Bishop as was the permanent transfection of the HIT cells. The plasma CCK assay was performed by Mrs C McLean. On occasion, the preparative surgery of the gastric fistula rats was performed by myself in conjunction with P J Noble.
| Abbreviation | Description |
|--------------|-------------|
| BAC | Benzalkonium chloride |
| CAG | Chronic atrophic gastritis |
| CCK | Cholecystokinin |
| CGRP | Cacitonin gene-related peptide |
| CRF | Chronic renal failure |
| EC | Enterochromaffin |
| ECL | Enterochromaffin-like |
| EGF | Epidermal growth factor |
| GRP | Gastrin-releasing peptide |
| HGF | Hepatocyte growth factor |
| LOH | Loss of heterozygosity |
| MEN1 | Type I multiple endocrine neoplasia syndrome |
| NO | Nitric oxide |
| PA | Pernicious anemia |
| PAP | Pancreatitis associated protein |
| PPI | Proton pump inhibitor |
| PSP | Pancreatic stone protein |
| PTP | Pancreatic thread protein |
| SP | Substance P |
| TGF-α | Transforming growth factor alpha |
| VIP | Vasoactive intestinal polypeptide |
| ZE | Zollinger Ellison syndrome |
# Contents
## Chapter 1
### Introduction
- Historical review of gastrointestinal peptide physiology: 2
- Innervation of the stomach: 5
- Neuroendocrine regulation of gastric emptying: 15
- Neuroendocrine regulation of gastric acid secretion: 16
- Clinical significance of hypergastrinaemia: 22
- Gastrin, endocrine cell growth and differentiation in the stomach and pancreas: 26
- Aims and objectives: 34
## Chapter 2
### Materials and methods
- Materials: 37
- Methods: 39
## Chapter 3
### Chemical denervation of antral neurons
- Introduction: 57
- Methods: 59
- Results: 60
- Discussion: 69
- Summary: 72
Chapter 4
Antral denervation and gastric emptying
Introduction ................................................................. 75
Methods .................................................................. 77
Results .................................................................... 78
Discussion ................................................................. 85
Summary .................................................................. 88
Chapter 5
Antral denervation and G-cell function
Introduction ................................................................. 91
Methods .................................................................. 93
Results .................................................................... 94
Discussion ................................................................. 105
Summary .................................................................. 110
Chapter 6
Control of reg expression in rat corpus mucosa and AR42J cells
Introduction ................................................................. 112
Methods .................................................................. 113
Results .................................................................... 114
Discussion ................................................................. 124
Chapter 7
Reg gene expression in human gastric mucosa
Introduction 131
Methods 133
Results 136
Discussion 145
Summary 152
Chapter 8
Conclusion and Implications
Conclusions 155
Implications and future studies 156
Chapter 9
Bibliography 162
Acknowledgements 188
Chapter 1
Introduction
At the beginning of this century two schools of thought were prominent with regard to the mechanisms that controlled visceral function. Langley provided evidence that responses to feeding were mediated by nervous reflexes (Langley, 1898) and his conclusions were supported by the work of Sherrington (1899) and Pavlov (1910). Set against this were the experiments of Bayliss and Starling (1902) and Edkins (1905) who proposed that blood borne agents (named secretin and gastrin respectively) were responsible for stimulating digestive secretions from the pancreas and stomach. Although Bayliss and Starling (1899) previously had reported the reciprocal effects of the vagal and splanchnic innervation on gastric motility, it took almost forty years before it was considered that the nervous and endocrine systems might interact closely to regulate visceral functions such as gastric acid secretion (Uvnas 1942).
Whilst the work of Bayliss and Starling was readily accepted, Edkins’ proposal that an antral hormone stimulated gastric acid secretion remained unconfirmed until the work of Komarov in the late 1930’s and of Grossman et al., a decade later (1948). Meanwhile several groups attempted to purify to homogeneity secretin, the hormone proposed by Bayliss and Starling to stimulate pancreatic secretion in response to a meal. This was achieved in 1961 by Jorpes and Mutt who isolated secretin and at the same time Gregory and Tracy isolated and characterised the peptide hormone gastrin (Gregory and Tracy, 1964). Within a few years it was shown conclusively that gastrin was produced by and localised within a defined
population of endocrine cells in the gastric antral mucosa (McGuigan, 1968). Not surprisingly at the time, gastrin was considered to be strictly a gastrointestinal hormone. Furthermore, the similarities between gastrin and secretin seemed to confirm the prevailing view that peptides acted as classical hormones.
Almost coincidentally however, Burnstock et al., (1963) described neurotransmission in the gut that was mediated by a nonadrenergic and noncholinergic agent. Morphological studies of the myenteric plexus suggested the existence of peptide-containing nerve fibres (Baumgarten et al., 1970; Cook and Burnstock 1976). Subsequently, considerable evidence has been provided that has generated widespread support for the idea that peptides act as neurotransmitters in neurons of the enteric, sympathetic, parasympathetic and central nervous systems.
Although von Euler and Gaddum as early as 1931, had suggested that agents with identical biological activity were present in extracts of both gut and brain, the work of Vanderhaeghen et al., (1975) raised again the idea that a peptide previously considered to be a classic gut hormone (gastrin) might be present within neurons of the central nervous system. The technical and analytical advances in the last two decades have now established fundamental new concepts in the distribution of regulatory peptides. It is now clear that any one peptide may have a specific function at a specific site, determined by the cell type responsible for its synthesis, mode of release and subsequent tissue distribution, but the same peptide may occur at another independent site within the body and have a separate function and mode of action determined again by the local environment. Hence peptides such as
cholecystokinin (CCK) and gastrin, previously considered as hormones may act elsewhere as neurotransmitters or paracrine mediators, the converse also being true.
With the general acceptance of similarity between the spectrum of peptides that function as hormones and as neurotransmitters, it is perhaps less surprising that there is such close interaction between neurons and endocrine cells in the regulation of visceral functions such as gastric acid secretion. The main objective of the studies described in this thesis was to determine the role of antral neurons in the control of antral G-cell responses to physiological stimuli. The experimental model of antral denervation that was developed to investigate the role of intrinsic neurons in the regulation of the G-cell produced chronic hypergastrinaemia and delayed gastric emptying. This prompted further studies on the role of antral neurons in the regulation of gastric emptying and the effects of sustained elevations in plasma gastrin on endocrine cell gene expression with particular relevance to enterochromaffin-like (ECL) cell growth and carcinoid tumour development.
The following account will describe the normal gastric innervation and its role in regulating G cell function and gastric emptying, review the physiology of gastrin and the pathophysiology of hypergastrinaemia in particular the development of ECL cell tumours, review the evidence for the role of gastrin in endocrine cell growth and differentiation and finally introduce putative regulators of cell growth and differentiation in gastroentero-pancreatic endocrine cells.
Langley (1921) first recognised the importance of the intrinsic nervous system of the gut, considering it a separate division of the autonomic nervous system in addition to the sympathetic and parasympathetic divisions. Considerable evidence in support of this has been presented (Cooke, 1987; Furness and Costa, 1987; Wood, 1987) and it is clear that processes such as absorption, secretion and coordinated motor activity may be regulated wholly by the enteric nervous system. The extrinsic innervation of the gut interacts with enteric neurons exerting an overriding influence when necessary so that gut function may be integrated with the prevailing needs of the body as a whole. Extrinsic neuronal inputs are supplied by vagal and spinal nerve trunks each providing both afferent and efferent nerve fibres.
i) Vagal innervation of the stomach
The majority of nerve fibres in the vagi are afferent fibres having their nerve cell bodies in the nodose ganglia. These afferent fibres have their peripheral endings within the muscle and mucosal layers of the gut wall and act as mechano-, thermo- and chemo-receptors conveying signals to the central nervous system (Andrews, 1986; Blackshaw and Grundy, 1990; El Ouazziani and Mei, 1979; 1891). Almost all vagal afferent fibres are unmyelinated (Andrews, 1986) but a small population of nerve fibres within the vagus are small diameter myelinated fibres believed to be of sympathetic origin (Lundberg et al., 1976).
Using retrograde tracing and immunohistochemistry it has been shown that vagal afferent fibres projecting from the anterior wall of the stomach have their cell bodies in the left nodose ganglion and those that project from the posterior gastric wall have their cell bodies in the right nodose ganglion (Dockray et al., 1988; Green 1988). A number of peptides have been identified in vagal afferent fibres including substance P (SP) (Katz and Karten, 1980; Lundberg et al., 1978; 1983; 1985) CCK (Mantyh and Hurt, 1984), vasoactive intestinal polypeptide (VIP) (Lundberg, 1978; Said and Rosenberg, 1976) and gastrin (Dockray et al., 1981). Despite this variety, the majority of peptide-containing vagal nerve fibres project to the thoracic viscera (Lundberg, 1983; Mulderry 1985; Cadieux 1986) such that less than 10% of gastric vagal afferent fibres are peptidergic (Green, 1988).
The vagus also carries efferent fibres to the stomach but these account for only 10% of the total number of vagal fibres. The majority of vagal efferent fibres are excitatory cholinergic fibres that are involved with the normal regulation of gastric tone as well as secretion of gastrin and gastric acid. In addition however, there are also inhibitory pathways such as those causing release of nitric oxide (NO) (Desai et al., 1991) and VIP (Grider et al., 1985).
ii) Spinal afferent innervation of the stomach
Similar to the vagal innervation of the stomach, spinal afferent neurons for the most part are unmyelinated (Kuo et al., 1982). Gastric spinal afferents have their nerve cell bodies in the thoracolumbar dorsal root ganglia (Green and Dockray, 1988). Conversely however, there is no lateralisation of innervation such that
retrograde tracing from one surface of the stomach labels equal numbers of nerve cell bodies in the right and left dorsal root ganglia. The spinal efferent innervation of the stomach is approximately 95% adrenergic and projects from cell bodies contained within the coeliac ganglion (Macrae et al., 1986).
Several different peptides have been identified within spinal afferent neurons but the most important are calcitonin gene-related peptide (CGRP) (Gibson et al., 1984; Lee et al., 1987; Rosenfeld et al., 1983) and substance P (Hokfelt et al., 1976; Price 1985; Seybould and Elde, 1980). CGRP is of primary interest because of its specific distribution within the stomach and is relevant to the work contained within this thesis. A brief review of the physiology of CGRP is given below before describing the intrinsic innervation of the stomach.
**Discovery and distribution of CGRP:**
The existence of CGRP was predicted on the basis of the discovery of a novel cDNA sequence corresponding to an mRNA transcript that was shown to arise from alternative splicing of the gene encoding calcitonin (Amara et al., 1982; Rosenfeld et al., 1983). Subsequently a related peptide β-CGRP was isolated (Amara et al., 1985; Steenbergh et al., 1985) whose primary gene does not encode for calcitonin. The distribution of CGRP within the stomach has important implications. CGRP-immunoreactive fibres may be found in all layers of the stomach (Clague et al., 1985; Green and Dockray 1988; Sternini et al., 1987) but there are no intrinsic neurons or endocrine cells that contain CGRP in the rat stomach. In fact in rodent stomach almost all CGRP is contained within spinal
afferent extrinsic nerve fibres (Green and Dockray, 1988; Sternini et al., 1987; Varro et al., 1988). This makes CGRP a useful marker for an intact spinal afferent gastric innervation.
**Biological actions of CGRP in the stomach:**
It is well established that CGRP acts as a peripheral neurotransmitter from spinal afferent nerve terminals (Holzer, 1992). Much of the available experimental evidence points to a role for CGRP in gastric mucosal protection. Hence, installation of noxious chemicals into the gastric lumen results in increased mucosal damage if the spinal afferent innervation has been lesioned (Holzer and Sametz, 1986) or the action of CGRP is blocked by immunoneutralisation (Forster and Dockray, 1991). Conversely, stimulation of CGRP release protects against mucosal damage (Holzer and Lippe, 1988; Holzer et al., 1989; 1990) induced by installation of noxious chemicals.
The gastroprotective effect of CGRP acts partly by increasing gastric mucosal blood flow possibly by the release of NO (Lambrecht et al., 1993). In addition however, CGRP has been shown to be a potent inhibitor of gastric acid secretion (Tache et al., 1984; Pappas et al., 1986; Helton et al., 1989), a factor that also contributes to the gastroprotective effects of this peptide. The inhibition of acid secretion is mediated in part by release of somatostatin from D cells of the gastric corpus (Dunning and Taborsky, 1987; Inui et al., 1991). In addition it has been shown that CGRP regulates somatostatin synthesis by corpus D cells at the level of gene transcription (Sandvik et al., 1993).
iii) Intrinsic innervation of the stomach
The intrinsic nerve supply of the gastrointestinal tract, collectively termed the enteric nervous system, comprises two main plexi of nerves described first by Meissner (1857) and Auerbach (1864). Meissner described a submucous plexus and Auerbach a plexus of nerves lying between the circular and longitudinal muscle layers generally referred to as the myenteric plexus. The structure and distribution of the enteric nervous system were described in detail by Gaskell (1886) and the functional significance of the enteric nervous system as a separate branch of the autonomic nervous system was proposed later by Langley (1921).
Studies of the anatomy of intrinsic gut neurons were facilitated greatly by the techniques of myotomy and myomectomy coupled with immunohistochemistry first described by Furness and Costa (1979). The variety and colocalisation of neuropeptides present in enteric neurons, their projections and putative actions have received much interest over the past two decades (for review see Dockray 1994; Furness and Costa 1987). Generally speaking, the tachykinin/substance P family of peptides are present in intrinsic excitatory neurons (Katayama and North, 1978; Bartho et al., 1982; Bartho and Holzer, 1985; Holzer, 1989) and the VIP/peptide histidine isoleucine family is present in intrinsic inhibitory neurons (Brookes et al., 1991; Ekblad et al., 1987; Shultzberg et al., 1989; Grider, 1989). Gastrin-releasing peptide (GRP) is present in secretor motor neurons and this is of particular relevance as GRP-containing neurons stimulate the release of gastrin from antral G
cells (Bunnett et al., 1985; Kovacs et al., 1995; Varner et al., 1991). In the lesioning experiments described later in this thesis, substance P, VIP, GRP and CGRP were assayed as markers of different classes of intrinsic or extrinsic neurons to enable confirmation of denervation. There follows a brief review of substance P, VIP and GRP in relation to the enteric nervous system.
**Substance P**
Substance P (SP) was the first gut neuropeptide to be discovered. Although extracts of horse intestine had been shown to stimulate atropine resistant contraction of rabbit ileum as early as 1931 (von Euler and Gaddum, 1931), SP was isolated and sequenced first from the bovine hypothalamus (Chang and Leeman, 1970; Chang and Leeman, 1971). The identical peptide was shown later to be present in small intestine (Carraway and Leeman, 1979). Evidence for its neuronal localisation was suggested by Ehrenpreis and Pernow (1953) who reported reduced levels of SP bioactivity in the aganglionic segment of colon affected by Hirschsprung’s disease. Lembeck (1953) suggested that SP was located within the primary afferent neurons on the basis of findings of higher concentrations of peptide in dorsal compared with ventral spinal roots. Although there is compelling evidence that a subset of primary afferent neurons contain both SP and CGRP (Gibbins et al., 1985; Green and Dockray, 1988), lesioning experiments using capsaicin, have shown that in the rat stomach and small intestine the majority (>90%) of SP is present in intrinsic neurons (Holzer et al., 1980).
The presence of SP within primary afferent neurons has prompted studies that suggest that SP released from these neurons may act as a central neurotransmitter of pain and may play a role as a peripheral transmitter during inflammatory reactions in the gut (Dockray, 1994; Holzer, 1988; Sharkey, 1992). The predominant localisation of SP within intrinsic gut neurons has directed several groups towards identifying its possible role within the gut. There is evidence that SP directly stimulates intestinal smooth muscle contraction (Taylor and Bywater, 1986) and that SP release is involved in the contraction that occurs on the oral side of a distending bolus as part of the peristaltic reflex (Bartho et al., 1982; Holzer, 1989; Yokayama and North, 1983). In the rat colon, SP release appears to mediate only the noncholinergic contraction that occurs with high pressure distension as this could be inhibited by immunoneutralisation (Grider, 1989b).
In addition to the direct effects of SP on smooth muscle, there is also evidence that release of SP may stimulate other myenteric plexus neurons (Yan and Youther, 1982; Fosberg et al., 1984). Regardless of the precise physiological role of SP in the control of motility, intrinsic SP neurons generally can be considered to be excitatory and SP is the most useful marker of this class of neuron.
**Vasoactive intestinal polypeptide**
Vasoactive intestinal polypeptide (VIP) was discovered by Said and Mutt by screening of extracts of gut and lung for vasodilator activity (Said and Mutt, 1970; 1972). Vasodilatation induced by VIP is only one manifestation of its ability to relax smooth muscle. In the gut VIP is localised to a population of myenteric neurons that
project distally and innervate the circular muscle (Brookes et al., 1991; Ekblad et al., 1987), and to a population of submucosal neurons that project to the mucosa (Schultzberg et al., 1980; Costa and Furness, 1983). There is evidence from immunoneutralisation studies that VIP may mediate the descending relaxation of the peristaltic reflex (Grider and Makhlouf, 1986; Grider et al., 1985; Grider, 1989a). In addition, VIP may be one of the final mediators of the vagovagal, non-adrenergic, non-cholinergic relaxation of the stomach (Faherenkug et al., 1978; Ito et al., 1988), and play a role in the control of gastric emptying (Forster et al., 1991; D’Amato et al., 1992).
Submucosal VIP-containing neurons that project to the mucosa stimulate secretion from enterocytes (Cooke, 1992). In terms of gastric endocrine cell function, VIP has been shown to inhibit gastric acid secretion by stimulating the release of somatostatin from mucosal D cells (Saffouri et al., 1984). From the above description therefore, in contrast to SP, VIP can be considered as a marker of inhibitory myenteric plexus neurons.
**Gastrin-releasing peptide**
In 1971, Espramer and Anastasi isolated bombesin from amphibian skin (Anastasi et al., 1971). A major effect of this peptide was stimulation of acid secretion that was shown in the dog to be mediated by gastrin (Bertaccini et al., 1974). The mammalian counterpart of bombesin was identified using C-terminally directed antibodies to the amphibian peptide (Walsh et al., 1979). Coincidentally, McDonald and co-workers discovered that extracts of the non-antral porcine
stomach were a potent stimulus to gastrin release (McDonald et al., 1978; McDonald et al., 1979). They proposed that the peptide responsible was gastrin-releasing peptide (GRP) and subsequently the C-terminal sequence of GRP and bombesin have been shown to be almost identical. However, GRP is not strictly the mammalian equivalent of amphibian bombesin as amphibia have separate genes encoding GRP and bombesin (Nagalla et al., 1992).
In the mammalian gut, GRP-immunoreactivity is found exclusively within neurons (Dockray et al., 1979). The majority of GRP immunoreactive fibres are present in the mucosa with some fibres present in the circular muscle and myenteric plexus. (Dockray et al., 1979). Most GRP-containing neurons have their cell bodies in the myenteric plexus and project their axons distally (Costa et al., 1984; Ekblad et al., 1984). There is good evidence that the major physiological role of this neuropeptide in the stomach is to stimulate gastric acid secretion via release of gastrin from antral G cells (Bunnett et al., 1985; Hildebrand et al., 1991; Hirschowitz and Molina, 1983; Kovacs et al., 1995).
A schematic representation of the major classes of neuron present within the gastric antrum is shown in Figure 1.1.
Figure 1.1: Schematic representation of the major classes of intrinsic and extrinsic neurons that innervate the gastric antrum in the rat and are susceptible to lesioning by serosal application of the neurotoxin BAC. The shaded area depicts the likely depth of penetration of BAC. LM - longitudinal muscle; CM - circular muscle; SM - submucosa. Excitatory (SP) and inhibitory (VIP) myenteric neurons are shown and receive inputs from spinal afferent (CGRP) and vagal nerve fibres. GRP is present predominantly in secretomotor nerves that supply the mucosa (see text for full details).
In recent years a substantial body of evidence has emerged detailing the interactions between the intestinal hormone cholecystokinin (CCK) and vago-vagal reflexes that together mediate the delayed gastric emptying in response to fat- and protein-rich meals (Dockray, 1988; Mayer, 1994). Exogenous CCK inhibits gastric emptying in both rat (Conorver et al., 1988; Green et al., 1988) and man (Chey et al., 1969; Liddle et al., 1986). Protein-rich meals are known to stimulate secretion of endogenous CCK (Liddle et al., 1984; 1986) and delay gastric emptying (Green et al., 1988; Moran and McHugh, 1981), an effect that is reversed by specific CCK-A receptor antagonists (Green et al., 1988; Liddle et al., 1989). In addition there is good evidence that vagal afferent fibres express CCK-A receptors (Corp et al., 1993; Moriarty et al., 1997) and that the CCK-mediated delay in gastric emptying is initiated by stimulation of vagal afferent fibres (Raybould and Tache, 1988; Forster et al., 1990; Forster et al., 1991; Holzer et al., 1994). Furthermore, the efferent limb of the reflex that results in relaxation of the gastric body also requires an intact vagus nerve (Raybould and Tache, 1988; Raybould et al., 1987). Vagal efferent relaxation of the gastric corpus is mediated by non-adrenergic and non-cholinergic neurotransmission that probably involves both NO (Desai et al., 1991; Forster et al., 1991; D’Amato et al., 1992) and VIP (Forster et al., 1991; D’Amato et al., 1992).
In addition to the CCK-vagal pathway that regulates gastric relaxation in response to feeding, gastric emptying may also be affected by spinal afferent innervation, since lesioning of spinal afferent neurons by capsaicin pre-treatment
reduces the emptying rate of liquid test meals (Forster et al, 1990; Forster and Dockray, 1992).
Overall the rate of gastric emptying depends on the pressure gradient between the duodenum and stomach and the resistance to flow across the pylorus and antrum (Mayer, 1994). The content of the ingested meal determines the rate of emptying and CCK-mediated vagovagal reflexes play an important role in delaying gastric emptying by relaxation of the gastric corpus. The importance of antropyloric motility in regulating gastric emptying has received less attention although the pulsatile manner of delivery of gastric content to the duodenum can be attributed to the phasic motor pattern of the antrum (Houghton et al., 1988; Malbert et al., 1991; Prather et al., 1993). There is some evidence that CCK may act on the distal stomach both by a direct action on smooth muscle and indirectly via release of neurotransmitters (Scheurer et al., 1983; Yamagishi and Debas, 1978). However the relationships between CCK and antral neurons in the regulation of gastric emptying has not been examined previously in any detail and represents one of the objectives of the studies presented in this thesis.
**Neuroendocrine Regulation of Gastric Acid Secretion.**
The G cells, D cells and ECL cells of the stomach together with the extrinsic and intrinsic innervation of the stomach, provide an intricate regulatory mechanism for the control of gastric pH (Fig 1.2).
Figure 1.2: Following meal ingestion, intraluminal pH rises and somatostatin (SOM) release from the D cell is inhibited. The G cell secretes gastrin that leads to histamine release from the ECL cell and H⁺ release from the parietal cell. Vagal neurons also stimulate the parietal cell (1.2a). As luminal pH then falls, antral D cells release SOM that inhibits further gastrin release. Spinal afferent neurons regulate SOM release from corpus D cells. SOM inhibits the ECL cell and parietal cell (1.2b). See text for full description.
G cells
These cells are present in the antrum of the stomach and are also found in lesser numbers in the proximal duodenum (McGuigan, 1968; Solcia et al., 1975). Their primary function is to synthesise, store and release the amidated 17 amino acid peptide gastrin. G cells process the prohormone precursor peptide progastrin by sequential steps involving sulfation, phosphorylation, cleavage, C-terminal amidation or glycine extension and further cleavage (for review see Dockray et al., 1996). The acid secretory activity of gastrin is dependent on the presence of the amidated carboxyl terminus (Soll et al., 1984; Takeuchi et al., 1980). Alternative forms of gastrin, extended at their C-terminus by glycine also have been reported to possess biological activity other than stimulation of acid secretion, namely stimulation of expression of the gene encoding the H⁺-K⁺-ATPase present in parietal cells (Kaise et al., 1995). Furthermore, recent studies suggest that amidated gastrin, glycine extended gastrin and progastrin itself are all putative growth factors (Seva et al., 1994; Singh et al., 1996; Wang et al., 1996).
G cells have a luminal brush border and secrete gastrin in response to the presence of protein and amino-acids within the gastric lumen (Dockray and Gregory, 1989; Walsh, 1994). In keeping with these observations are the reduction in gastrin mRNA reported with fasting and increases in gastrin mRNA abundance seen with feeding (Wu et al., 1991). In addition G cells respond to changes in luminal pH although this may be indirect, being mediated by changes in somatostatin release from adjacent antral D cells (Karnik et al., 1989). G cells
express several different receptors on their basolateral membrane including receptors for gastrin releasing peptide (Vigna et al., 1990; Weigart et al., 1992).
**ECL cells**
These cells are present only in the gastric corpus and their main function is to synthesise histamine by the decarboxylation of the amino acid histidine (Rubin and Schwartz, 1979). This reaction is catalysed within the cell cytosol by the enzyme histidine decarboxylase (Kubota et al., 1984). Histamine is then transported into secretory vesicles probably by the vesicular monoamine transporter VMAT$_2$ (Merickel and Edwards., 1995). Unequivocal evidence for the stimulation of acid secretion by histamine was provided by the discovery of H$_2$ receptor antagonists and their ability to block acid secretion (Black et al., 1972). Gastrin stimulates the ECL cell to release histamine (Chuang et al., 1992) and if the gastrin stimulus is sustained, ECL cells increase their production and storage capacity of histamine, in part by upregulating HDC and VMAT$_2$ gene expression (Dimaline and Struthers, 1996; Swarovsky et al., 1994). The secretion of histamine by the ECL cell can be inhibited by release of somatostatin from corpus D cells (Chuang et al., 1993). Finally ECL cells increase in number in response to continued gastrin stimulation (Hava, 1986; Poynter and Selway, 1991; Wangberg et al., 1995).
**D cells**
D cells are present in both the antrum and corpus of the stomach. In both sites, their function is to synthesise, store and release somatostatin (Dockray et al., 1996). Antral D cells have a luminal brush border and respond to changes in pH in
the stomach (Gustavsson and Lundquist, 1978; Holst et al., 1987; Schubert et al., 1988). In contrast, corpus D cells do not have a luminal brush border and somatostatin mRNA abundance in corpus D cells is much less affected by changes in luminal pH that occur with feeding or treatment with omeprazole (Sandvik et al., 1993). It appears that corpus D cell somatostatin gene transcription is regulated primarily by neurohumoral influences of which the most significant may be those of the CGRP-containing spinal afferent neurons (Sandvik et al., 1993).
**Regulation of acid secretion**
The parietal cells are responsible for the release of acid ($\text{H}^+$) into the gastric lumen. They can be stimulated to release $\text{H}^+$ by cholinergic neurons and by histamine (Black et al., 1972; Black and Shankley, 1987; Kabekar et al., 1969; Main and Pearce, 1982; Soll, 1978). Gastrin receptors have been demonstrated on parietal cells (Soll et al., 1984) suggesting that gastrin also may act directly on these cells to induce acid secretion. As intragastric pH rises (such as with ingestion of a meal) antral G cells release gastrin in response to a variety of subsequent stimuli. These include rising luminal pH (Dockray et al., 1991), increased cholinergic stimulation (Kabekar et al., 1969; Main and Pearce, 1982), release of GRP from non-cholinergic neurons (Saffouri et al., 1984; Schubert et al., 1992), the presence of intraluminal protein and amino-acids (Chiba et al., 1980; Saffouri et al., 1980; Walsh, 1994), and a reduction in somatostatin release from adjacent antral D cells that are themselves inhibited by the rising luminal pH (Gustavsson and Lundquist, 1978; Holst et al., 1987; Schubert et al., 1988).
Gastrin passes via the circulation to the ECL cell and stimulates these cells to release histamine (Chuang et al., 1992). ECL cells may also respond to the increased cholinergic drive that accompanies meal ingestion (Maine and Pearce, 1982; Soll, 1982). Histamine released from ECL cells acts via parietal cell H\textsubscript{2} receptors causing H\textsuperscript{+} release (Black et al., 1972, Black and Shankley, 1987). This co-ordinated response provides the acid pH required for gastric digestion by pH dependent enzymes and as digestion and gastric emptying proceeds, returns gastric pH to its normal basal level (Fig 1.2a).
As the gastric pH falls, antral D cells respond by releasing somatostatin that in turn inhibits gastrin release from G cells (Gustavsson and Lundquist, 1978; Holst et al., 1987; Schubert et al., 1988). As a result of reduced intraluminal pH, gastrin levels fall, permitting somatostatin release from corpus D cells which in turn prevents further secretion of histamine from the ECL cell and H\textsuperscript{+} from the parietal cell (Fig. 2b). The spinal afferent innervation of the corpus may also stimulate corpus D cell somatostatin release as it has been shown to functionally regulate corpus rather than antral D cell somatostatin gene expression (Sandvik et al., 1993).
It may be readily appreciated from the above description that disorders of gastric acid secretion are likely to be associated with elevated plasma gastrin levels. Hence, primary disorders of the G cell or ectopic gastrin secretion that result in hypergastrinaemia will result in increased acid output. Conversely, decreased acid output from whatever cause will result in elevated plasma gastrin as antral G cells
respond to the high intragastric pH. There follows a discussion of conditions associated with hypergastrinaemia and the related pathophysiology of the ECL cell.
**Clinical significance of chronic hypergastrinaemia**
From a clinical viewpoint the major conditions of the stomach that are associated with altered endocrine cell function are encompassed by the conditions associated with hypergastrinaemia (Figure 1.3). The commonest of these are the conditions associated with *H pylori* infection with or without concurrent treatment with a proton pump inhibitor (PPI). However, Zollinger Ellison syndrome (ZE) and pernicious anaemia are the conditions associated with the highest and most sustained elevations in plasma gastrin. In these conditions ECL cell (carcinoid) tumours have been described (see below) and it appears that it is the trophic action of gastrin on the ECL cell that is of pathophysiological importance. The elevations of plasma gastrin in *H pylori* or with PPI therapy are of lesser magnitude and rarely sustained and ECL cell tumours have not been reported. However, *H pylori* infected patients treated with a PPI may develop significant elevations in plasma gastrin and ECL cell hyperplasia has been documented in up to a third of such patients taking a PPI for five years (Eiselle et al., 1997). Although hyperplasia precedes dysplasia and neoplasia, the time course of this progression is not known and it may take several years before carcinoid tumours develop. Hence carcinoid tumours developing in patients rendered hypergastrinaemic by *H pylori* and PPI therapy remains a remote possibility.
| Associated Condition | Representative Range of Plasma Gastrin* (pM) | Causal mechanism for hypergastrinaemia |
|----------------------|---------------------------------------------|----------------------------------------|
| Pernicious Anaemia (PA) | 200 - >1000 | Atrophic gastritis leads to achlorhydria and secondary rise in plasma gastrin |
| Zollinger Ellison (ZE) Syndrome | Normal - >1000 | Functioning gastrin secreting tumour usually of the duodenum or pancreas causes increased acid output. |
| Chronic Renal Failure. (CRF) | Normal - 600 | Kidneys are the major site of breakdown of circulating gastrins. |
| Proton Pump Inhibitor Treatment (PPI) # | Normal - 350 | Reduces acid output by direct action on the parietal cells’ H⁺/K⁺ ATPase. |
| H. pylori infection | Normal - 200 | a) antral gastritis reduces antral somatostatin with loss of inhibition of gastrin release by gastric acid.
b) Corpus gastritis leads to atrophy and hypochlorhydria |
Figure 1.3: Conditions associated with hypergastrinaemia and the primary causal mechanism for the elevated plasma gastrin.
* Amidated gastrin; normal fasting <30pM; normal post-prandial <90pM.
# depends on duration of treatment and presence of *H pylori*. (6-8 week treatment with PPI raises plasma gastrin up to twofold).
ECL cell (Carcinoid) tumours and Hypergastrinaemia.
Chronic atrophic gastritis (CAG) with or without pernicious anemia (PA) is the commonest cause of ECL cell hyperplasia and in 5-10% of patients there is a progression to gastric carcinoid tumors (Borch et al., 1985; Bordi et al., 1991; 1995). The achlorhydria of CAG leads to hypergastrinemia which is generally accepted to be a stimulus to ECL cell growth (Bordi et al., 1995; Creutzfeld, 1988; Hakanson and Sundler, 1990; Solcia et al., 1986). The natural history of these tumors in man has been extensively reviewed (Modlin et al., 1995; Rindi, 1995; Solcia et al., 1991; Thomas et al., 1994), prompted at least in part by the demonstration that in rodents hypergastrinemia secondary to long-term treatment with H$_2$-receptor antagonists or proton pump inhibitors, may lead to gastric carcinoid tumors (Hava, 1986; Poynter and Selway, 1991; Wangberg et al., 1995).
Enterochromaffin-like cell tumors associated with CAG are usually small (<1cm), may be single or multiple, are most often present incidentally in patients over the age of 60, and rarely progress to metastatic disease (Bordi et al., 1995; Solcia et al., 1991; Modlin et al., 1995). This has led to conservative management for the majority of patients with or without endoscopic screening. Some authors have advocated twice daily subcutaneous injections of the long acting somatostatin analogue octreotide (Ferraro et al., 1996; Bordi et al., 1993). This therapy has been shown to reverse ECL cell hyperplasia in patients with CAG without ECL cell tumors, and to inhibit gastrin release from the antral gastrin (G-) cell (Ferraro et al., 1996; Bordi et al., 1993). Surgical antrectomy to remove G-cells is also effective in
reducing plasma gastrin levels and is reported to result in complete resolution of small ECL cell tumors (Hirschowitz et al., 1992; Modlin et al., 1995; Scully et al., 1997). In contrast, the management of large ECL cell tumours (>2cm) associated with CAG is less well defined. Antrectomy has been advocated since it may induce tumor regression, or at least halt metastatic spread (Eckhauser et al., 1988; Scully et al., 1997) However, reports of a metastasis rate of >20% for primary tumours that are >2cm in diameter, together with the demonstration that in a few cases the tumors fail to regress following antrectomy, has persuaded others to recommend total gastrectomy (Borch et al., 1985; Thomas et al., 1995).
Although all PA patients are hypergastrinaemic and the majority show ECL cell hyperplasia some other factor(s) must also be involved in the progression of hyperplasia to neoplasia as the incidence of carcinoid tumours in PA patients is 5-10% (Borch et al., 1985; Bordi et al., 1991; 1995). Azzoni has suggested that the expression of BCL-2 by hyperplastic ECL cells may be of importance as it may prolong exposure to other oncogenic factors (Azzoni et al., 1996). However relatively little is known of the underlying genetic risk factors for ECL cell tumour development in CAG.
In the sporadic form of ZE the incidence of carcinoid tumours is similar to that in PA. In contrast patients with ZE as part of the type I multiple endocrine neoplasia syndrome (MEN1) have a much higher incidence of carcinoid tumour development (up to 35%) presumably due to an inherited susceptibility to endocrine cell tumour development (Lehy et al., 1992). The recent positional cloning of the
MEN1 gene (Chandrasekharappa et al., 1997) has facilitated the study of abnormalities of this gene in the development of carcinoid tumours. Loss of heterozygosity (LOH) at the MEN1 locus could be demonstrated in the majority of carcinoid tumours arising in patients with ZE as part of the MEN1 syndrome (Debelenko et al., 1997). However very few gastric carcinoids arising from CAG had associated LOH at the MEN1 gene locus suggesting other genes may be important in the pathogenesis of these tumours. Data is presented in this thesis that is in favour of a potential candidate gene, reg, being of importance to the development of carcinoid tumours. Before reviewing the available information on the reg family of genes, the role of gastrin in the differentiation and growth of endocrine cells of the stomach and pancreas is discussed.
Gastrin, endocrine cell growth and differentiation in the stomach and pancreas.
In addition to the well established role of amidated gastrin in regulating gastric acid output, there is an increasing body of evidence to suggest that gastrin also regulates the differentiation of some gut endocrine cells and the growth and overall numbers of certain endocrine and epithelial cell populations within the stomach.
The first direct evidence of the trophic effect of amidated gastrin came from the observation that pentagastrin administration to rats increased the functioning
parietal cell mass thereby contributing to the long term increase in acid output. (Crean et al., 1969). Prior to this, gland atrophy following antrectomy had been reported (Lees and Grandjean, 1958), indirectly suggesting a trophic role for gastrin in maintaining the acid secreting mucosa. Gastrin also stimulates the growth of ECL cells (Bordi et al., 1995; Tielemans et al., 1990), a subject that has received much interest due to the theoretical risk of ECL cell tumour development in patients receiving long term pharmacological acid suppression.
More recent studies using transgenic mice clearly demonstrate the importance of amidated gastrin acting via the CCK-B receptor in the maintenance of normal cell populations within the gastric mucosa. Hence, overexpression of amidated gastrin leads to increased proliferation of precursor cells in the proliferative zone of the gastric mucosa and increased parietal and ECL cell numbers (Wang et al., 1993). Conversely, targeted disruption of the CCK-B receptor produced marked gastric mucosal atrophy with reductions in the numbers of parietal and ECL cells (Langhans et al., 1997; Nagata et al., 1996). Similarly, targeted disruption of the gastrin gene resulted in a marked reduction in ECL cell and parietal cell number but did not affect the proliferative index in the stomach (Koh et al., 1997). The latter study is of particular interest as it highlights the importance of gastrin in regulating the differentiation of gastric parietal and ECL cells independent of the degree of stem cell proliferation.
There is also good evidence that gastrin is responsible for the development in the embryo of endocrine cells of the pancreas (Wang et al., 1993). In the case of
pancreatic endocrine cell development candidate genes have been proposed to be involved in the growth and differentiation of islet β-cells. (Terazono et al., 1988; Otonkoski et al., 1994; Watanabe et al., 1994). The *reg* gene was first isolated by Terazono and colleagues in 1988 by differential screening of a rat regenerating pancreatic islet cDNA library. This gene has been renamed *reg Iα*, following the discovery of several other homologous genes that appear to comprise a multigene family.
**The *reg* gene family**
The rat *reg Iα* cDNA contained a single open reading encoding for a 165 aminoacid protein, of which the first 21 aminoacids appeared to constitute a signal peptide. The human homologue of rat *reg Iα* was identified by screening of a human pancreatic cDNA library with a 60 base pair complement of the rat cDNA (Terazono et al., 1988). Human *reg Iα* encodes a 166 aminoacid protein with a 22 aminoacid signal peptide. Human *reg Iα* normally is expressed in the pancreas, gastric mucosa and kidney; ectopic expression of *reg* has been described in tumours of the colon and rectum (Watanabe et al., 1990). The homology between rat and human *reg* cDNA was 75% (68% in AA sequence) including similar homology in the 5’ untranslated region. Perhaps of significance was the observation that the position of all seven cysteine residues within the predicted protein were conserved. Subsequent cloning of the respective genes indicated another common feature, each consisting of 6 exons and 5 introns. These two features are common to all members of the *reg* gene family.
Following the cloning and sequencing of *reg* it became clear that the previously described pancreatic stone protein (PSP) was a truncated product of the same gene (Watanabe *et al.*, 1990). In fact PSP consists of 144 amino acids (23-166 of the parent *reg*) and has been renamed PSP$_I$. A shorter variant also has been identified and is 11 amino acids shorter (34-166 of the parent *reg*). This protein is identical to the pancreatic thread protein (PTP) also referred to as PSP$_{II}$ (Watanabe *et al.*, 1990).
Other members of the human *reg* family that have been identified so far, include *reg Iβ* (Moriizumi *et al.*, 1994), *reg* related sequence (Watanabe *et al.*, 1990) and pancreatitis associated protein (Iovanna *et al.*, 1991; Morizumi *et al.*, 1993). Confusingly, pancreatitis associated protein (PAP) has an identical aminoacid sequence to peptide 23 (Katsumata *et al.*, 1995) and HIP, a protein that was first identified by its expression ectopically in primary hepatocellular carcinoma (Lasserre *et al.*, 1992). These members of the gene family are tandemly ordered in a 95kbp DNA region of human chromosome 2p12 as determined by fluorescence in situ hybridisation (Miyashita *et al.*, 1995).
The murine *reg* gene family has also been studied but reveals unexpected differences. The murine homologue of *reg Iα* has been identified and localised to chromosome 12 (Unno *et al.*, 1993). A second member of the family *reg II* has also been cloned. Although the gene consists of 5 introns and 6 exons with conservation of all cysteine residues, the whole sequence is <60% homologous to *reg I*, has no human homologue and maps to mouse chromosome 3 (Unno *et al.*, 1993). Further
members of the mouse *reg* gene family have recently been described. Hence murine *reg IIIα*, *reg IIIβ* and *reg IIIγ* bear reasonable homology to each other but represent a separate gene group and are localised on Chromosome 6 (Narushima et al., 1997). Unlike other members of the *reg* family, the *reg III* genes are preferentially expressed in the intestine and are expressed only at low levels in regenerating pancreatic islets.
The available evidence suggests that members of the *reg* gene family are expressed within endocrine cells of the stomach and pancreas. In particular, *reg Iα* (*reg*) expression appears to be markedly upregulated during regeneration following surgical resection (Terazono et al., 1988; or mucosal ulceraton induced by waster immersion stress (Ashahara et al., 1997) or treatment with non-steroidal anti-inflammatory drugs (Kawanami et al., 1997). However, the potential relationship between gastrin and expression of the *reg* gene has not been explored.
**Reg gene expression and endocrine cell growth and differentiation**
The *reg* gene was first proposed as a regulator of pancreatic islet cell growth following its isolation from regenerating islets (Terazono et al., 1988). In mature adult pancreas, *reg* expression is limited to acinar cells. The Reg protein is secreted into pancreatic juice where it has been suggested to act as an inhibitor of pancreatic stone formation (Patard et al., 1996; Bertrand et al., 1996). During islet cell regeneration the Reg protein also appears to be produced by endocrine cells as it has been shown to colocalise almost exclusively with insulin in β-cell secretory granules (Terazono et al., 1990).
Other models of islet cell growth such as hyperplasia following surgical wrapping of the pancreas (Zenilman et al., 1996) and islet cell regeneration following growth suppression by insulinoma (Miyaura et al., 1991), have shown a consistent relationship between expression of reg and increasing islet cell number. None of these studies attempted to determine whether reg may act as a growth factor or as a promoter of differentiation.
Otonkoski and colleagues (1994b) proposed that reg expression was a marker of proliferation rather than differentiation based on observations using nicotinamide to stimulate differentiation and hepatocyte growth factor (HGF) to stimulate proliferation in 7 day cultures of human foetal pancreatic cells. Reg expression was significantly increased by HGF. However, the effects of HGF are not purely trophic. In fact, the in vitro mitogenic effect of HGF is limited to the first three to four days of culture (Beattie et al., 1996). Thereafter, HGF has been shown to increase the formation of islet cell clusters in 6 day cultures of human foetal pancreatic cells and increase their insulin content (Otonkoski 1994a). Thus HGF appears to stimulate both growth and differentiation in this model. HGF also stimulates the differentiation of AR42J cells into insulin secreting cells (Mashima et al., 1996). The pancreatic cell line AR42J has recently been shown to express reg (Zenilman et al., 1996) and can be manipulated by appropriate culture conditions to differentiate towards either acinar or endocrine cell phenotype (Christophe, 1994; Mashima et al., 1996). Interestingly, differentiation towards an acinar cell phenotype is associated with reduced reg expression (Zenilman et al., 1997).
In 1994, Watanabe et al., described the amelioration of diabetes (induced surgically in rats by 90% pancreatectomy) by daily administration of recombinant rat Reg protein. Administration of Reg protein led to a significant increase in islet cell size, insulin positive β-cell number and amelioration of diabetes. They also demonstrated a threefold increase in the incorporation of $^{3}$H-thymidine into cultured rat islet cells exposed to recombinant rat Reg protein. Autoradiography demonstrated a limited increase in the proliferative index of insulin positive cells from 0.1% in controls to 0.2% in Reg treated cultures. Based on these experiments, Watanabe et al., claimed that the trophic effects of Reg protein explain its ability to stimulate β-cell regeneration and ameliorate diabetes.
In contrast, Smith et al., (1994) argue against reg playing a role in islet cell growth from experimental evidence employing a model of selective β-cell growth induced by glucose infusion for 96hrs. Such a procedure produces a 50% increase in the rat islet β-cell mass with a fivefold increase in the proliferative index of islet β-cells (Bonner-Weir et al., 1989). Using this model Smith et al., (1994) could find no evidence of increased reg expression during or after the glucose infusion. Isolated rat islets in culture however, do respond to glucose exposure, both by increased reg expression and increased $^{3}$H-thymidine incorporation (Francis et al., 1992).
**Reg expression in endocrine cells of the stomach**
In 1996, Ashahara and colleagues reported the presence of reg in the endocrine cells of the rat stomach. In fact, by using the techniques of in situ
hybridisation and immuno-histochemistry they clearly demonstrated that the large majority of *reg*-positive cells were the ECL cells. Furthermore, *reg* mRNA abundance was upregulated during the period of mucosal repair following stress ulceration induced by water immersion. Plasma gastrin concentrations and acid output were not measured. Nevertheless the presence of *reg* in ECL cells and its upregulation during ECL cell regeneration suggest that *reg* may play a role in the growth and/or differentiation of the gastric ECL cell.
In summary, *reg Iα* is a major member of the *reg* family and is expressed during pancreatic islet cell regeneration and in the rat gastric ECL cell. The protein product of the gene stimulates the formation of mature endocrine cells in the regenerating pancreas. Expression of *reg* is associated with growth and differentiation of endocrine cells but its exact function as trophic factor or promoter of differentiation remains to be determined. The ectopic expression of *reg* and related family members in tumours of the gastrointestinal tract and liver justifies the further studies required to elucidate the exact role of the *reg* gene and its protein product. The possibility that the *reg* gene is involved in the development of gastric carcinoid tumours in patients with hypergastrinaemia is examined in this thesis.
Aims and Objectives
1. To develop a model of localised denervation of the gastric antrum in the rat in order to study the role of intrinsic antral neurons in the regulation of gastric function.
2. To examine the effects of lesioning of antral denervation on gastric emptying in the rat to test the hypothesis that intrinsic neurons of the gastric antrum regulate gastric emptying and mediate the actions of extrinsic reflexes already known to influence gastric emptying.
3. To examine the effects of antral denervation on the control of gastrin release in the rat in order to test the hypothesis that antral neurons regulate the responses of the G-cell to feeding and gastric distension.
4. To investigate *reg* gene expression in the pancreatic cell line AR42J in order to test the hypothesis that gastrin and EGF may regulate *reg* expression.
5. To determine the relationship between *reg* expression and hypergastrinaemia in both rat and man to test the hypothesis that gastrin stimulation of ECL cell function and growth are associated with upregulation of *reg* expression.
6. To investigate the possibility that mutations of the *reg* gene (a putative regulator of endocrine cell differentiation) are associated with carcinoid tumour development as a consequence of chronic hypergastrineamia.
Chapter Two
Materials and Methods
Materials
All laboratory chemicals that can be considered as in routine use were obtained from either BDH chemicals, Poole U.K., or from Sigma Chemicals limited, Poole, U.K.. The materials used for routine molecular biology experiments that were not available from the above companies were obtained from either Gibco GBRL, Renfrewshire, Scotland, or from Severn Biotech, Worcester, U.K.. All radioactive materials were obtained from Amersham Life Sciences, Buckinghamshire, U.K.. Other specific items are listed below.
Drugs/Chemicals:
- Benzalkonium chloride: Sigma Chemicals Ltd.
- Bovumin: First link, West Midlands, U.K.
- BW2258U89: Dr J Leban, Burroughs Wellcome, U.S.A.
- Capsaicin: Sigma Chemicals Ltd.
- Diazepam (Valium): Roche products Ltd., Derbyshire U.K.
- Enrofloxacin: Bayer Health Business Group, Suffolk, U.K.
- Fentanyl/Fluanisone (Hypnorm): Janssen Animal Health, Buckinghamshire, U.K.
- L-740093: Merk Sharp and Dohme, Harlow, U.K.
Peptides:
Gastrin Peninsula Laboratories, St Helens, UK.
porcine GRP\textsubscript{14-28} Bachem (uk) Ltd., Saffron Walden, U.K.
rat Tyr\textsuperscript{0}\alpha-CGRP\textsubscript{28-37} Bachem (uk) Ltd., Saffron Walden, U.K.
Substance P Peninsula Laboratories, St Helens, UK.
porcine VIP Peninsula Laboratories, St Helens, UK
Restriction endonucleases:
Apa I Promega, Southampton, U.K.
Not I Gibco GBRL, Renfrewshire, Scotland
Nru Gibco GBRL, Renfrewshire, Scotland
Spe I Gibco GBRL, Renfrewshire, Scotland
Xba Promega, Southampton, U.K.
Cloning Vectors:
pGEMT-Easy Vector Promega, Southampton, U.K.
pcDNA3.1 ZEO Invitrogen, De Schelp 12, Netherlands
Methods
Animals
Adult Wistar rats (approximately 250g) were kept on a 12hr light/dark cycle and fed standard laboratory rat chow. Rats were fasted overnight before operations and refeeding experiments but allowed free access to water. Rats were anaesthetized by i.m. injection of a mixture of fentanyl citrate (0.32 mg.kg$^{-1}$), fluanisone (4 mg.kg$^{-1}$) and diazepam (2mg.kg$^{-1}$). Post-operatively, rats were given buprenorphine (0.1mg.kg$^{-1}$) for analgesia, antibiotic prophylaxis (enrofloxacin 5mg.kg$^{-1}$) and 10ml of saline by subcutaneous injection.
Benzalkonium chloride treatment
The stomach was exteriorised through a midline upper abdominal incision. The serosal surface of the region to be denervated was covered with a layer of gauze cut to size. Benzalkonium chloride (0.02%, 0.1%, or 0.5% w/v) dissolved in 0.9% NaCl was painted onto the gauze, which was kept moist with the BAC solution for 3 minutes, and then removed. The serosal surface of the stomach was then rinsed thoroughly with normal saline and returned to the peritoneal cavity and the incision closed in layers. The two faces of the stomach were treated separately; in some experiments only the anterior face of the antrum was treated. A similar method was used for denervation of the corpus employing a strip of gauze (approximately 5mm wide) placed over the corpus alongside the border of the glandular and non-glandular regions. In order to selectively lesion the afferent innervation of the stomach, the same experimental approach was employed using
the sensory neurotoxin capsaicin (1% w/v in olive oil) in place of BAC. Control animals underwent a sham operation with serosal application of normal saline (or olive oil). Animals were allowed to recover for at least 11 days before experiments.
**Refeeding experiments**
Control and BAC-treated rats were fasted for 24hrs on wire-bottomed cages, and were then either maintained as fasted controls or allowed to refeed for 30 minutes. In some experiments rats were injected subcutaneously 30 minutes before refeeding with either a GRP antagonist BW2258U89 at a dose of 2mg.kg$^{-1}$ (Moody et al., 1995) or with vehicle (0.14M saline). Rats were killed by cervical dislocation, decapitated and trunk blood was taken for assay of plasma gastrin. Samples of antral and corpus mucosa were taken for mRNA extraction (see below) and the stomach content was removed. Gastric retention was determined by the dry weight of solid material in the stomach.
**Gastric fistula rats**
Rats were treated by application of either BAC or saline to the antral serosa as described above and a small stainless steel Gregory cannula was installed in the corpus as previously described (Dimaline et al., 1986). The cannula was exteriorised through a midline stab incision and the incision closed in layers. Rats began training in Bollman cages on the tenth post operative day.
Gastric emptying studies
Gastric fistula rats were allowed at least 11 days to recover from surgery before experiments. Prior to experiments the stomach was washed with warm 0.14M NaCl to remove solids. On the experimental day, the emptying of test solutions (saline, 50mM HCl, 4.5% peptone, containing phenol red, 60mg.l\(^{-1}\)) instilled into the stomach via the gastric fistula, was determined as previously described (Forster et al., 1990).
Gastric distension studies
Prior to experiments the stomach was washed extensively via the gastric cannula with warm 0.14M NaCl to remove solids. Rats were left for at least 90 minutes before experiments began. Plasma gastrin responses were determined after gastric distension with the following test solutions: 2% (w/v) methyl cellulose (2%MC); 2%MC at pH6 (50mM ammonium acetate); 2% MC with 50mM HCl; 4.5% w/v peptone. In each case the stomach was distended by connecting the cannula to a reservoir of test solution and elevating the reservoir to 5cm H\(_2\)O pressure for 30mins. Immediately following distension, rats were killed by cervical dislocation and decapitation and 1.5ml of trunk blood was then taken for gastrin radioimmunoassay. Samples of antrum from control and BAC-treated rats were taken to confirm denervation.
Measurement of acid output
Rats fitted with a gastric cannula were trained in Bollman cages to permit the required handling of the cannula before acid output could be measured. Prior to experiments, rats were fasted overnight. On the day of experiment the stomach was
washed extensively with warm 0.14M NaCl to remove solids. Rats were left for at least 90 minutes before experiments began. Gastric acid output was determined by collecting gastric secretion over four sequential 15 min periods and titrating to pH 7.0 with 20mM NaOH using a pH autotitrator as previously described (Dimaline, et al., 1986).
**Omeprazole treatment**
Rats were given omeprazole (400μmol.kg\(^{-1}\)) by gavage under light Halothane anaesthesia. Rats were fed *ad libitum* and received omeprazole daily for five days. On the fifth day rats were killed by cervical dislocation and decapitation and 1.5ml of trunk blood was taken for gastrin radioimmunoassay. Samples of gastric corpus were taken for total RNA extraction (see below).
**Radioimmunoassay**
The neuropeptide content of tissues was determined after extraction with boiling water (0.1 g.ml\(^{-1}\)), homogenizing and centrifuging and re-extraction of the pellet with 0.5M acetic acid, as previously described (Hutchison et al., 1981). Water or acid extracts were assayed at the appropriate dilution for the following peptides: Gastrin releasing peptide (GRP) was assayed using antibody 1078, with porcine GRP 14-28 as standard and iodinated Tyr\(^1\) COOH-terminal decapeptide of bombesin as label, as previously described (Dockray et al., 1979). Substance P was assayed with antibody L83, using undecapeptide substance P as standard and \(^{125}\)I-Bolton Hunter labelled substance P as label. Vasoactive intestinal polypeptide (VIP) was assayed using antibody L25, with porcine VIP as standard and \(^{125}\)I labelled VIP as label as previously described (Hutchison
Calcitonin gene related peptide (CGRP) was assayed with antibody L273, using rat Tyr\textsuperscript{0}a-CGRP 28-37 as standard and the same peptide labelled with \textsuperscript{125}I as label as previously described (Varro et al., 1988). The concentration of amidated gastrins in plasma gastrin was assayed using antibody L-2, using synthetic human non-sulphated G-17 as standard and for labelling with \textsuperscript{125}I as previously described (Dockray et al., 1991). The standard conditions for each assay are shown in Figure 2.1
**Extraction of RNA**
**Extraction of Tissue RNA:**
Tissue mRNA was extracted and quantified as described previously (Dimaline et al., 1991). Samples of antral or corpus mucosa were homogenised in 3mls of 4M guanadinium isothiocyanate buffer containing 25mM sodium acetate pH 6.0 and 0.84% (vol/vol) β-mercaptoethanol and frozen at -80°C until required. After thawing at room temperature, samples were pipetted onto a 5.7M caesium chloride cushion (1.3ml) in an ultracentrifuge tube. Samples were centrifuged overnight at 41,100 rpm to pellet the RNA. The mRNA pellet was dissolved in 300µl of Tris-EDTA (TE) buffer with 0.2% SDS and precipitated with 0.3M sodium acetate under 100% ethanol overnight.
**Extraction of Cell RNA**
RNA extraction from cultured cells was performed using the Quickprep™ total RNA extraction kit (Pharmacia Biotech, Herts, U.K.) according to the manufacturers instructions. The media (2ml) was removed from cultured cells and replaced by 900µl of a mixture of lithium chloride buffer, β-mercaptoethanol and extractoin buffer. Cells were
| Peptide Assay | Antibody/Dilution | BUFFER | INCUBATION | SEPARATION |
|---------------|-------------------|--------|------------|------------|
| | | | | Ch:Dx:MPr* |
| GASTRIN | L2 - 1:100,000 | Na⁺Bicarbonate pH 8.4, 0.5% Bovumin, 10mM Na⁺ Azide | 72hrs : 4 °C | 10 : 1 : 0.5 |
| CGRP | L271 - 1:80,00 | 50mM PO₄, pH 7.4, 0.5% Bovumin, 0.25% EDTA, 0.14M NaCl | 72hrs : 4 °C | 10 : 1 : 1 |
| GRP | 1078 - 1:80,000 | Na⁺Bicarbonate pH 8.4, 0.5% Bovumin, 10mM Na⁺ Azide | 48hrs : 4 °C | 10 : 1 : 0.5 |
| SUBSTANCE P | L-83 - 1:400,000 | 50mM PO₄, pH 7.4, 0.5% Bovumin, 0.14M NaCl, 10mM Na⁺ Azide | 48hrs : 4 °C | 10 : 1 : 0.5 |
| VIP | L-25 - 1:40,000 | 20mM PO₄, pH 6.0, 0.4% BSA, 0.1% EDTA, 10mM Na⁺ Azide, 0.025% w/v polybrene | 48hrs : 4 °C | 10 : 1 : 0.5 |
Fig 2.1 Conditions for each radioimmunoassay are provided in the table. *For separation of the assay a mixture of charcoal dextran and milk protein (Marvel) in the above ratio (g/100ml) was added to each tube.
resuspended in the solution by scraping and then pipetted into a universal container. The cells were then homogenised and the lysate transferred to a 1.5ml eppendorf containing 600µl of caesium trifluoroacetate solution and left to stand on ice for ten minutes. RNA was pelleted by centrifugation at 15000rpm for 15 minutes in a microcentrifuge. The RNA pellet was washed thoroughly, allowed to dry and resuspended in 10-20µl of diethyl pyrocarbonate-treated (DEPC) water before quantification. Any unused RNA was precipitated with 0.3M sodium acetate under 100% ethanol and stored at -80°C.
**RNA electrophoresis**
Stored precipitated RNA was pelleted by centrifugation at 13000rpm for 15 minutes, washed twice in 70% ethanol/DEPC H₂O and once in 100% ultrapure ethanol. Pellets were air dried at room temperature and dissolved in 10-50µl of DEPC H₂O. RNA samples were quantified by spectrophotometer and 10µg aliquots added to denaturing buffer (formamide: 37% formaldehyde: 10 x MOPS (3-(4-Morpholino) propane sulfonic acid) in ratio 4:1.75:1 and heated to 55 °C for 15 minutes. Samples were then placed on ice and 2µl of RNA loading buffer added to each sample. The denatured RNA was then electrophoresed in a 1x MOPS/2% formaldehyde 1% agarose gel.
**Northern Blot and Hybridisation**
After electrophoresis, RNA was electroblotted to nylon membranes (Nytran 0.5) overnight in 0.5% TAE buffer (containing 0.2M Tris base, 1M acetate and 0.1MEDTA) at 4 °C. RNA was crosslinked to the nylon membrane by ultraviolet light (UV Cross Linker, Hoeffer scientific instruments, San Francisco, USA). Membranes were placed in
hybridisation buffer (50% formamide containing 5 x Denhardts solution, 5 x sodium chloride sodium phosphate EDTA buffer (SSPE), 0.5% SDS and sonicated salmon sperm DNA (200μg.ml\(^{-1}\)) and warmed to 65 °C for 4 hours prior to the addition of \(^{32}\text{P}\)-labelled cRNA probe (2x10\(^6\) cpml\(^{-1}\)). Membranes were hybridised overnight and then washed twice with 2xSSPE containing 0.1% SDS at room temperature for 20min, and once with 0.1xSSPE/0.1% SDS at 65°C for 20min. Signals were removed from membranes using boiling 0.1% SDS and membranes rehybridized with alternative specific probes as required. Signals were quantified using a Phosphor Imager.
**Reverse transcription/PCR**
*Reverse transcription* of RNA was performed by first annealing 1μl oligo dTs (500μg.ml\(^{-1}\)) to ≈50μg of total RNA by heating to 55°C for 5 minutes and cooling slowly. cDNA was generated by incubating the following reaction at 42°C for 1 hour.: oligo dT and RNA (10μl), 2μl DTT(100mM), 1μl dNTPs (25 mM), 1μl RNAsin, 4μl 5x AMV RT Buffer and 2μl AMV reverse transcriptase. The cDNA generated was purified by phenol/chloroform extraction, precipitated with 0.5M ammonium acetate and washed with 100% ethanol. The cDNA was then resuspended in 100μl of TE. 5μl of this solution was used as template for PCR.
Human *reg* cDNA was generated by reverse transcription of total RNA extracted from gastric mucosal biopsies taken from either normal corpus or from carcinoid nodules. Two different size products were generated by separate pairs of primers chosen to include either a 297bp central portion of the coding region.
containing an *EcoR* I restrictiton site, or the full length coding sequence. The smaller fragment was used as template for riboprobe synthesis (see below) and the larger fragment (557bp) used for cloning and sequencing (see below). Primers were chosen based on the full coding sequence of the published human *reg* mRNA (Terazono *et al.*, 1988) as follows: The 297 bp fragment: sense - TCTCCTGCCTGATGTTTCTGTCTC antisense - GGCGGTTCTTTTTGGGGTCAT, The 557bp fragment: sense GATTGTTGATTTGCCCTCTTA, antisense - TCCAGCTGCCTCTAGTTTTTGAA.
Rat *reg* cDNA was generated by reverse transcription of total RNA extracted from rat pancreas. PCR primers were chosen to include the full coding sequence of the published rat *reg* mRNA (Terazono *et al.*, 1988) as follows: sense - AGCCTGCAGAGATTGTTGACTTTG; antisense - AGGGGGTTGACTTTGCTTTTGATA and gave a predicted product size of 651bp (Figure 2.2). Restriction digestion of the 651bp PCR product with *EcoR*1 cleaved the product into 253 & 398bp fragments.
*Polymerase chain reaction* was performed under standard conditions in a final volume of 100µl containing 10x *taq* polymerase buffer (10µl), MgCl (1.5mM), dATP (0.2mM), dCTP (0.2mM), dGTP (0.2mM), dTTP (0.2mM), template cDNA (5µl), sense and antisense primers (5ng. µl$^{-1}$) and *taq* polymerase (0.5µl). 60µl of mineral oil was layered over the reaction mixture which was then placed into a thermal cycler (Hybaid, Middlesex UK). After a 5 minute denaturation at 95°C, thirty cycles consisting of annealing (1 minute at 58 °C), extension (2 minutes at 72°C) and denaturation (1 minute at 95°C) were performed. A final extension step (72 °C) was performed for ten minutes before determining the outcome by agarose gel electrophoresis.
Figure 2.2 PCR products of human and rat *reg* cDNA using human carcinoid tumour RNA and rat pancreatic RNA as template for reverse transcription. The predicted sizes of the human PCR products were 557bp (lane 1) and 297bp (lane 2) depending on the primers used. The predicted size of the rat PCR product was 651bp (lane 3). See text for full details.
DNA Ligation
The PCR fragments of human and rat *reg* cDNA were ligated into the pGEM-T Easy vector by incubating approximately equimolar concentrations of PCR product and vector in the presence of DNA ligase at 4°C overnight according to the manufacturer's instructions.
Cloning and Sequencing
*Bacterial Transformation:* Chemically competent JM109 bacterial cells were stored at -80°C. Prior to transformation, cells were thawed on ice for 20 minutes before addition of 2 µl of 8.7M β-mercaptoethanol and 1 µl of DNA ligation mixture. Incubation on ice was continued for a further 30 minutes. The cells were then heated to 42°C for 45 seconds and immediately placed on ice for a further 2 minutes. Thereafter, 450 µl of SOC medium (containing 2% Tryptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM Kcl, 10 mM MgCl₂·6H₂O and 20 mM glucose) was added and the reaction incubated at 37°C for one hour with constant gentle agitation.
After this incubation a 100 µl aliquot was spread onto petri dishes containing 20 mls of LB/agar (1% Tryptone, 0.5% Yeast extract, 1.0% NaCl, 1.5% agar) that had been treated previously with 100 µl isopropyl-β-D-thiogalactosidase (IPTG 0.1 M), 30 µl 5-bromo-4-chloro-3-indolyl-β-D-galactosidase (XGAL 0.2 M) and 200 µl of ampicillin (10 mg.ml⁻¹). The remaining cell suspension was pelleted by centrifugation (5000 rpm for 3 minutes) and the cells resuspended in 200 µl of SOC medium. A further 100 µl aliquot
of cells, now at a higher cell density, was plated onto a LB/agar plate. Plates were then incubated overnight at 37°C and selection of successfully transformed colonies was by colour selection and antibiotic resistance. Successfully transformed colonies (white) were sub-cloned onto a second LB/agar plate and into 3ml of LB medium containing ampicillin (0.05mg.ml\(^{-1}\)). Following a further overnight incubation at 37 °C, the amplified plasmid DNA was extracted and purified for restriction digestion (to confirm presence of the expected cDNA sequence insert) and sequencing.
**Plasmid DNA extraction** Small, medium and large scale plasmid DNA extraction was performed using commercially available kits according to manufacturers instructions (Wizard Minipreps, Midipreps and Maxipreps). The protocol is similar for each with adjustments for scale depending on the yield of plasmid DNA required. In general, small scale preparations were sufficient for sequencing purposes. Medium scale preparations were used to generate templates for cRNA probe synthesis and large scale preparations were used for generating linearised plasmid DNA for permanent cell transfection (see below).
Following overnight culture at 37 °C, bacterial cells were pelleted by centrifugation, the supernatant discarded and the cells resuspended in resuspension buffer. The cells were then lysed by addition of lysis solution (NaOH/SDS) and protein and bacterial DNA were precipitated by addition of a neutralising solution (potassium acetate). The precipitate was removed by centrifugation and the plasmid DNA extracted from the supernatant by addition of a high affinity resin. This resin with bound DNA was
immobilized to a column by vacuum suction. The column was then washed twice (80% ethanol) and the DNA eluted by addition of TE buffer (68 °C) followed by centrifugation with collection of the eluate. The DNA content and purity was measured by spectrophotometry.
**Riboprobe synthesis**
Templates for generating cRNA probes for gastrin, HDC, somatostatin and GAPDH were provided by Dr R. Dimaline and contained the appropriate template as described previously (Dimaline *et al.*, 1991; 1993a; 1994). The template for generating the cRNA probes for rat and human *reg* were prepared by restriction fragment digestion of midiprep purified pGEM t-Easy vector containing the full length rat *reg* sequence or the 253bp human *reg* sequence as described above. After sequencing the vector to determine orientation of the insert, 10µg of each sample was digested with restriction enzyme *ApaI* in appropriate enzyme buffer in a volume of 20µl for 3 hours at 37°C. Complete linearisation of plasmid DNA was confirmed by gel electrophoresis. 0.5µl of template was used for riboprobe synthesis. The reaction conditions were as follows: 2µl 5x SP6 buffer, 1µl DTT(10mM), 2.5µl DNTPs (100µM each ATP, UTP, GTP), 1µl CTP (10µM), 0.5µl RNase, 0.5µl template DNA, 0.5 µl RNA polymerase (1-5 units) and 2.5µl $^{32}$P-CTP ($\approx$ 5µM). The reaction mixture was incubated at 37°C for 1 hour and then RNase free DNase added for a further 15 minutes. The synthesised probe was purified by size exclusion and 1µl of purified probe placed in 5ml of scintillation fluid and
incorporated radioactivity counted. Labelled probe was used at a concentration of $2 \times 10^6$ cpm per ml of hybridisation buffer.
**Preparation of DNA for permanent transfection**
Two sequences of the human *reg* gene were chosen for permanent transfection into the hamster insulinoma tumour cell (HIT) line. These were the full length coding sequence of the normal (wild type) *reg* and the sequence in which the first methionine residue (initiator methionine) had been mutated to a valine (ATG→GTG). Representative clones containing each sequence were prepared by small scale DNA extraction (Miniprep). The purified plasmid DNA (>600ng in 15μl) was then restriction digested with the combined endonucleases *Spe*I and *Not*I at 37 °C for 3 hours. The restriction fragment containing the *reg* sequence was then purified by gel electrophoresis (1.5% agarose). The DNA band of correct size (∼600bp) was cut from the gel and DNA removed from the gel onto a column by centrifugation (13,000rpm for 60 seconds), and eluted from the column by the addition of 20μl sterile water followed by centrifugation at 13,000rpm for 20 seconds. A small aliquot of the DNA was again gel electrophoresed to assess purity and concentration (20ng.μl$^{-1}$).
Each restriction fragments was then ligated into the mammalian expression vector pcDNA3.1 Zeo. The vector circular DNA was linearised by concomitant digestion with the restriction endonucleases *Xba*I and *Not*I. (The *Xba*I restriction leaves an identical sequence overhang to that of *Spe*I used to create the insert DNA). Complete digestion by both enzymes was presumed by serial examination of control vector digests.
by each restricton endonuclease separately. After 4 hours, the linearised vector was gel purified and eluted. The concentration of vector and its purity was estimated by gel electrophoresis.
Ligation of the wild type and mutant *reg* inserts was achieved by addition of equimolar concentrations of insert and vector DNA in the presence of DNA ligase and appropriate buffer. The ligation reaction was incubated overnight at 14°C. In addition, a control ligation reaction was also performed to ensure that complete digestion by both endonucleases occurred whilst preparing the vector DNA. This consisted of linearised vector without any insert. As the *Xba* and *Not I* restriction site overhangs are incompatible, re-ligation should not occur without the presence of insert DNA. Hence no colonies should form with cloning of the control ligation. Conversely, any colonies present from the *reg* insert ligations should all be positive for insert DNA. This selection process was required as the pcDNA3.Zeo vector does not incorporate the lac operon gene that allows for colour selection.
Following cloning into JM109 cells, colonies from each of the wild type and mutant *reg* transformations were selected for small scale DNA preparation. The presence of a *reg* insert was shown by restriction digestion with *EcoRI* and then confirmed by sequencing. Positive clones for each sequence were then processed for large scale DNA preparation to yield $\approx 300 \mu l$ of concentrated DNA (3-5 $\mu$cg.$\mu l^{-1}$). For each permanent transfection, 10 $\mu g$ of linearised DNA (in <20 $\mu l$) was required. Therefore, the appropriate volume of each vector containing wild type and mutant *reg* inserts were linearised by
digeston using the restriction endonuclease *Nru*. Linearised DNA was incorporated into cultured HIT cells by electroporation (see below).
**Tissue culture and permanent transfection**
The hamster insulinoma cell line HIT T15 was grown in RPMI 1640 medium supplemented with 11mM glucose, 10% v/v foetal calf serum, 2.5ug.ml\(^{-1}\) fungizone, 100U.ml\(^{-1}\) penicillin and 100µg.ml\(^{-1}\) streptomycin (all from GIBCO BRL, Life Technologies, Paisley, Scotland). Stably transfected cell lines expressing wild type or mutant human *reg* were produced by electroporation of \(5 \times 10^6\) cells in 0.8ml at 200V and 1070µF in incomplete medium containing 10µg of linearised pcDNA *reg* construct. Cells were grown in complete RPMI in 10cm dishes for 48 hours prior to selection with zeocin (750µg/ml\(^{-1}\)) (Invitrogen). Six colonies expressing each construct were cloned and screened for *reg* expression by northern blot.
The rat pancreatic cell line AR42J was grown in RPMI 1640 medium supplemented with 10% v/v foetal calf serum. For experiments, cells were plated out at \(1.5 \times 10^6\) cells in 5ml culture medium / 60ml dish and grown at 37°C/ 5% CO2. Following a 24 hour growth period, the cells were washed and their media replaced with incomplete RPMI with the following stimulii or appropriate vehicle (control). Human gastrin-17 (HG17), rat gly-extended gastrin-9 (G-Gly) in 50mM ammonium bicarbonate, wortmannin, mouse salivary gland extract (EGF) in RPMI, L740093 in 1x10\(^{-6}\)% DMSO. In all cases except for wortmannin, the cells were incubated for 24
hours at 37°C / 5%CO₂ prior to RNA extraction. In the case of wortmannin, the cells were incubated for 4 hours prior to RNA extraction.
**Statistical analysis**
The statistical test used for each experiment is indicated in the appropriate figure legend. In general, for the experiments involving denervation, the data was analysed using Mann Whitney U test and for other analyses Student’s t test was used.
Chapter Three
Chemical denervation of antral neurons
Introduction
The gastric emptying of liquids is dependent on the pressure gradient between the lumen of the stomach and proximal duodenum, and the resistance to flow between the two (Mayer et al., 1994). It has long been known that the rate of emptying depends on the nature of the luminal contents; thus, acid, hyperosmolal solutions, high viscosity, protein- and fat-rich meals all delay gastric emptying (Mayer et al., 1994). The emptying of liquids is pulsatile in nature and this can be attributed to phasic contraction of the pyloric antral region of the stomach (Houghton et al., 1988; Malbert et al., 1991; Prather et al., 1993). Alterations in the flow of gastric content across the antrum can be effected by changing either resistance to flow generated by antral contractions or the overall tone of the gastric body and the duodenum. There is a significant body of evidence to indicate the importance of vagal afferent innervation of the proximal gut (Raybould et al., 1988; Forster et al., 1990; Holzer et al., 1994) and vagal efferent innervation of the gastric corpus (Forster et al., 1991; D’Amato et al., 1992) in regulating delayed gastric emptying by inducing gastric corpus relaxation in response to hyperosmolal, protein-rich or fat-rich solutions in the duodenum. However, much less is known of the role of intrinsic antral neurons in the control of pyloric flow.
Antral neurons however are known to innervate both the muscle layers of the antrum and the mucosal epithelial cells including endocrine cells (Gabella, 1987; Furness and Costa, 1987; Ekblad et al., 1987). The clearest example of this and the most relevant in terms of digestive physiology of the stomach is the innervation of Gcells of the gastric antrum by GRP-immunoreactive neurons that have their cell bodies in the myenteric plexus (Dockray et al., 1979; Miller et al., 1989). Systemically administered GRP has been shown to stimulate gastric acid output by the release of gastrin from G-cells in both animals and man (Varner et al., 1981; Bunnett et al., 1985). Further evidence that GRP exerts physiologically regulates the G-cell comes from the demonstration that G-cells express GRP receptors (Vigna et al., 1990) and GRP is capable of stimulating the release of gastrin from G cells in primary culture (Giraud et al., 1987).
The experiments described in this chapter were undertaken to define the anatomical lesion induced by chemical denervation of the rat antrum. This would then allow potential study of the role of the antral innervation in gastric emptying and the regulation of mucosal endocrine cell function. To this end I used the detergent benzalkonium chloride (BAC) which has already been used to lesion small intestinal myenteric neurons (Fox et al., 1983; Dahl et al., 1987). In keeping with the observations of others (Fox et al., 1983; Dahl et al., 1987) I was able to demonstrate that BAC application to the rat stomach produces a highly specific, reproducible and permanent loss of the all neurons in the treated region. Following antral denervation, the intrinsic innervation of the corpus is spared, but there was loss of the spinal afferent innervation of the gastric corpus. Conversely, when a band of proximal gastric corpus was denervated, intrinsic and extrinsic antral innervation was preserved.
Methods
All operations were performed on Wistar rats. For antral denervation rats were starved overnight, but for corpus denervation rats were fed *ad libitum*. Following exposure of the stomach, BAC (0.02%, 0.1%, or 0.5% w/v) dissolved in 0.9% NaCl was painted onto gauze, which was kept in contact with the region to be treated and kept moist with the BAC solution for 3 minutes, and then removed. The two faces of the stomach were treated separately; in some experiments only the anterior face of the antrum was treated. In order to selectively lesion the afferent innervation of the stomach, the same experimental approach was employed using the sensory neurotoxin capsaicin (1% w/v in olive oil) in place of BAC. Control animals underwent a sham operation with serosal application of vehicle (normal saline or olive oil). Animals were allowed to recover for at least 11 days before experiments. Denervation was assessed by radioimmunoassay of neuropeptides in extracts of BAC-treated and adjacent tissues.
Results
Dose-Response Relationship.
Initial experiments were directed at determining the optimal dose of BAC for antral denervation. The tissue concentrations of GRP, CGRP, substance P and VIP measured by radioimmunoassay of antral extracts were reduced in a dose dependent manner 11 days after prior administration of BAC (Fig 3.1). At a concentration of 0.02% BAC had no effect; 0.1% BAC produced a 25% loss of CGRP, but following 0.5% w/v BAC all neuropeptides were depleted. The concentrations of substance P, VIP and GRP in extracts of the gastric corpus were not affected by prior application of BAC to the antrum (Fig 3.2). However, the concentration of CGRP in the corpus was significantly reduced by application of 0.5% BAC to the antrum. (Fig 3.2)
Time Dependency of Responses
In order to determine the duration of responses to 0.5% BAC, neuropeptide concentrations were examined in extracts of tissues sampled 11 to 25 days after treatment. All neuropeptides assayed were depleted in antral extracts 11 days after BAC, and remained substantially reduced at all times up to 25 days (Fig 3.3). Similarly there was loss of CGRP in both antrum and corpus in rats treated with BAC on the antrum at all times examined (Fig 3.4).
Fig 3.1 Neuropeptide concentrations (CGRP, GRP, substance P and VIP) in extracts of antrum following serosal application to the antrum of BAC or vehicle (control). Only at 0.5% w/v BAC was there a significant loss of neuropeptides derived from both intrinsic and extrinsic afferent neurons of the antrum. (n = 6 all groups; * p<0.05, compared with control, Mann Whitney U test; in this and subsequent figures, values are means ± SEM).
Fig 3.2 Neuropeptide concentrations (CGRP, GRP, substance P and VIP) in extracts of the antrum 11 (n=5), 18 (n=5) and 25 (n=3) days after application of 0.5% w/v BAC or vehicle (control, n=6) to the serosa of the antrum. The loss of all neuropeptides was complete at day 11 and unchanged thereafter. (* p<0.05, compared with control, Mann Whitney U test)
Fig 3.3 Neuropeptide concentrations (CGRP, GRP, substance P and VIP) in extracts of corpus following serosal application to the antrum of BAC or vehicle (control). Even at 0.5% w/v BAC there was no significant loss of neuropeptides derived from intrinsic neurons of the Corpus. However at 0.5% w/v BAC there is a significant loss of corpus afferent innervation as shown by the significant reduction in corpus CGRP levels. (n = 6 all groups; * p<0.05, compared with control, Mann Whitney U test)
Fig 3.4 Neuropeptide concentrations (CGRP, GRP, substance P and VIP) in extracts of corpus 11 (n=5), 18 (n=5) and 25 (n=3) days after application of 0.5% w/v BAC or vehicle (control, n=6) to the serosa of the antrum. As with the results for the antrum, the selective loss of corpus CGRP after antral BAC was complete at day 11 and unchanged thereafter (* p<0.05, compared with control, Mann Whitney U test)
Specificity of Lesion
To examine the specificity of the effects described above, we studied the effect of BAC applied to a circumferential band (approximately 5mm) of the gastric corpus immediately distal to the non-glandular part of the stomach. In the BAC-treated region there was substantial depletion of all the neuropeptides studied (Fig 3.5). In the untreated corpus, and in the antrum, the tissue concentrations of VIP, substance P, CGRP and GRP were similar to control (Fig 3.5). It is worth emphasising that following BAC treatment of the corpus, antral CGRP was unaffected. Given that CGRP is present only in spinal afferent neurons, this result seemed to indicate a specific lesion in the corpus projections of these neurons induced by antral BAC treatment. To confirm this, the effects of the sensory neurotoxin capsaicin applied in a similar fashion to BAC, were examined as this neurotoxin is known to lesion only small diameter afferents leaving intrinsic innervation unaffected. Serosal application of 1% w/v capsaicin to the antrum, produced complete loss of CGRP in extracts of antrum and corpus (Fig 3.6). In contrast, neuropeptides found in intrinsic neurons (VIP and GRP) were not changed by treatment of the antrum with capsaicin (Fig 3.6).
Finally, in some experiments BAC was applied to a single side of the antrum. In this case, there was depletion of neuropeptides on the treated side as already noted, but the concentrations in the untreated side of the antrum were similar to controls (Fig 3.7).
Fig 3.5 Neuropeptide concentrations (CGRP, GRP, substance P and VIP) in extracts of antrum, control corpus and a band of proximal corpus treated with either 0.5% BAC (hatched bars) or vehicle (open bars). There was complete loss of the neuropeptides in the band of corpus exposed to BAC but not in the remainder of the corpus, or the antrum. Note in particular the preservation of CGRP in the antrum. (* p<0.05, compared with vehicle, Mann Whitney U test; n = 6 all groups).
Fig 3.6 Neuropeptide concentrations (CGRP, GRP and VIP) in extracts of antrum and corpus following topical 0.1% w/v capsaicin (hatched bars), or vehicle (open bars), to the serosal surface of the antrum. The spinal afferent innervation of both the antrum and corpus was lost as shown by CGRP concentrations, while neuropeptides of the intrinsic innervation (GRP, VIP) were unaffected in both regions. (n = 4, both groups; * p<0.05, compared with vehicle, Mann Whitney U test).
Fig 3.7 Neuropeptide concentrations (CGRP and GRP) in extracts of either the anterior (a) or posterior (p) surfaces of the antrum (A) and ipsilateral corpus (C) following topical 0.5% w/v BAC (hatched bars) or vehicle (open bars) to the anterior serosal surface of the antrum. Neuropeptides of the intrinsic innervation (GRP, VIP) were reduced only in the BAC treated region. The spinal afferent innervation of both the antrum and corpus was lost as shown by CGRP concentrations predominantly on the side of the stomach that received antral BAC (n = 4, both groups; * p<0.05, compared with vehicle, Mann Whitney U test).
The experimental data obtained in these experiments provided substantial evidence that application of 0.5% w/v BAC to the serosal surface of the antrum for 5 minutes produced a chronic, profound and sustained loss of both the intrinsic and extrinsic afferent innervation. Although this is in keeping with the studies that have reported the effects of BAC on the rat small intestine (Fox et al., 1983; Dahl et al., 1987; Holle et al., 1990; Jodal et al., 1993), rat colon (Sato et al., 1978, Sakata et al., 1979) and opossum oesophagus (Gaumnitz et al., 1993) the duration of exposure and concentration of BAC used was different. In the previous reports in the rat, the preferred method has been that described by Fox et al. (1983) requiring 0.062% w/v BAC and an exposure time of 30 minutes for each surface of the intestine. Others have used higher concentrations of BAC (0.2%) with the same exposure time (Zuculoto et al., 1991). Fox et al. (1983) demonstrated clearly that the degree of denervation produced by BAC was concentration dependent. Extending these observations, See et al. (1988) confirmed that BAC induces myenteric denervation by disrupting the outer layers of the intestine following application to the serosa. The depth of damage to the intestine was dependent on the concentration used and the length of exposure time.
In order to reduce the time under anaesthetic, it seemed reasonable to use a higher concentration of BAC and to determine the appropriate dose by a dose response study. The effective dose to induce comprehensive denervation with a 5
minute exposure time was found to be 0.5% w/v BAC as this dose significantly reduced neuropeptide levels to less than 10% of control values.
The neuropeptides assayed were GRP, CGRP, VIP and substance P. These neuropeptides are representative markers of separate populations of intrinsic and spinal afferent neurons although there is a degree of overlap. Hence GRP is a good and specific marker in the rat antrum for intrinsic myenteric neurons that are secretomotor and stimulate release of gastrin from G cells (Dockray et al., 1979). A proportion of these neurons also contain VIP which, in addition is also found separately in inhibitory motor neurons of the rat myenteric plexus (Ekblad et al., 1987). CGRP is present almost exclusively in spinal afferent neurons (Varro et al., 1988; Sternini et al., 1986; Sternini et al., 1987) in the rat although a proportion of these neurons also contain substance P (Green and Dockray, 1988). However the majority of substance P is found in intrinsic neurons of the myenteric and submucosal plexi (Holzer et al., 1980).
The distribution of neuropeptides within the wall of the stomach explains to some extent the pattern of loss of as determined by radioimmunoassay. Thus, GRP present predominantly in myenteric neurons was reduced to undetectable levels. Similarly CGRP present in spinal afferent neurons that innervate the serosa and muscle layers of the gut also was reduced to undetectable levels. However VIP and substance P are known to be present also in submucosal neurons and the latter also
may be found in inflammatory cells. Although there was a significant reduction in VIP and substance P, these peptides were still detectable in tissue extracts.
The earliest time point that denervation was assessed was 11 days and the latest time point was 27 days. Antral denervation was complete at 11 days and there was no change at the later times studied. This observation is in agreement with previously published experiments on the rat small intestine that have shown complete denervation as early as 24 hours after exposure to BAC (See et al., 1988) and persistence of denervation for up to 160 days (Zuculoto et al., 1991; See et al., 1988). In all the published studies on denervation induced by BAC it has been assumed that the effect was localised to the region of exposure. The data presented here indicate clearly that the spinal afferent innervation of the whole stomach is lesioned by antral BAC treatment. This was shown by loss of CGRP in antral and corpus tissue extracts following antral BAC.
Three lines of evidence suggest that this observation was not due to inadvertent exposure of the corpus to BAC. Firstly, other neuropeptide levels in corpus tissue extracts remained unaffected by antral BAC treatment. Secondly, in the reverse experiment lesioning a circumferential band of proximal corpus did not produce a loss of antral CGRP. Thirdly, exposure to BAC of only the anterior surface of the antrum caused a significant reduction of intrinsic neuropeptides only in tissue extracts of the anterior and not posterior antrum.
The validity of the observation that lesioning antral spinal afferents removes corpus spinal afferent innervation was confirmed by using the specific small diameter afferent fibre neurotoxin capsaicin. Again exposure of antral spinal afferents to capsaicin reduced CGRP levels in antrum and corpus leaving other neurotransmitters levels unaffected. Taken together these observations suggest that CGRP-containing nerve fibres that supply the gastric corpus also supply the antrum, either by passage over the antrum or by projection of branches to both regions.
Finally, confirmation of the effect of BAC on the gastric innervation described above were supported by immunohistochemical studies performed by Dr. C Vaillant. Samples of antrum and corpus were taken from rats that had either received antral BAC or BAC to a circumferential band of proximal corpus. The results of Dr. Vaillant’s studies essentially confirm the data obtained by radioimmunoassay. Hence there was a complete loss of immunoreactive nerve cell bodies and fibres only in the treated region following exposure to 0.5% BAC. However, specific staining for CGRP demonstrated that following antral application of BAC there was loss of CGRP-immunoreactive fibres from both the antrum and corpus whereas denervation of a band of corpus with BAC resulted in complete loss of immunoreactive fibres in the treated region, but not in the untreated antrum (Higham et al., 1997).
Summary
Antral BAC treatment effectively lesions all myenteric neurons and spinal afferent fibres of the antrum, as well as the spinal afferent innervation of the
remainder of the stomach. The intrinsic innervation of the proximal stomach is unaffected. Consequently there is a loss of innervation of the antral musculature and antral mucosal endocrine cells. The primary aim of developing such a model of complete antral denervation was to study the effects that this would have on gastric motility and antral endocrine cell function. The results of these studies are presented in the next two chapters.
Chapter Four
Antral denervation and Gastric Emptying
For many years, antro-pyloric motility has been recognised to be an important factor controlling the rate of gastric emptying (Canon 1911). More recently, the pulsatile nature of antral contractions and the resting pressure of the pyloric sphincter have been shown to be significant factors governing the delivery of gastric content to the duodenum (Houghton et al., 1988; Malbert and Ruckebusch, 1991; Prather et al., 1993; Dent et al., 1994; Mayer et al., 1994). The likely role of the intrinsic antral innervation in determining the pattern of antral motor contractions has received little attention. This has been primarily because of the lack of available methodology to selectively lesion only antral neurons.
In contrast the extrinsic innervation of the stomach has been more amenable to study but have been shown to have little long term effects on the pattern of antro-pyloric motility (Quigley and Louckes, 1951; Stoddard et al., 1973). Indeed the majority of studies on the extrinsic reflex control of gastric emptying have concentrated on duodeno-gastric reflexes that regulate gastric emptying by effecting changes in gastric corpus tone either through vagally mediated (Raybould and Tache, 1988; Raybould et al., 1987; Holzer et al., 1994) or spinal afferent pathways (Forster et al., 1990; Forster et al., 1991).
However, BAC has been shown to lesion myenteric neurons in a defined segment of rat jejunum and disrupt motor patterns that are dependent only upon an intact intrinsic innervation (Holle and Forth, 1990). In this chapter the data presented are from experiments that have used BAC to lesion the antral innervation to determine the role of antral innervation on the control of gastric emptying. Initially the retention of solid food was assessed and compared to the effects of the lesion produced by antral application of capsaicin. Finally the gastric emptying of a variety of liquid test meals was determined in control and BAC treated rats. The results demonstrate the requirement for an intact antral innervation for normal gastric emptying.
Methods
Antrally denervated and capsaicin treated rats were prepared and after each experiment denervation confirmed by radioimmunoassay of appropriate neuropeptide markers (see methods and chapter 3). Gastric emptying was determined either by studies of the retention of solid material in the stomach of fasted rats or by the determination of the emptying of liquids instilled into the stomach of rats with a gastric cannula. Gastric cannulas were placed at the time of antral denervation and all rats were allowed at least 11 days to recover after surgery.
For the determination of gastric emptying of solids, rats were fasted for 48 hrs on wire bottomed cages, killed by cervical dislocation and the stomach content weighed wet and again after prolonged drying at $50^0C$. For the determination of gastric emptying of liquids in gastric fistula rats, the stomach was washed extensively with warm 0.14M NaCl to remove solids prior to experiments and then the emptying of test solutions (saline, HCl, peptone: containing phenol red, 60mg.l$^{-1}$) instilled into the stomach via the gastric fistula was determined.
Results
In fasted rats previously treated with 0.5% BAC on the antrum, there was a striking retention of solids in the stomach compared to controls (Fig 4.1). The wet and dry weight of gastric content was variable in BAC treated rats but all 0.5% BAC-treated rats exhibited overtly distended stomachs (by solid, gas or liquid) after a 48 hr fast. The retention of solid was not apparent after treatment with lower concentrations of BAC (Fig 4.1). However with 0.5% BAC the retention of solids was seen at all time points examined (Fig 4.2). In 48 hr fasted BAC treated rats that were allowed to refeed for 30 minutes, the dry weight of gastric content increased further (Fig 4.3). The dry weight of gastric content was similar to refed control rats.
Given that BAC causes loss of intrinsic and extrinsic afferent innervation whereas capsaicin lesions only extrinsic afferents (see chapter 3), the latter was used to determine whether the effects of BAC might simply reflect the loss of afferent innervation. Rats that had been pre-treated with capsaicin on the antrum did not exhibit gastric retention after fasting (Fig 4.4). To confirm the importance of the whole antrum in regulating gastric emptying the effects of denervation of a circumferential band of corpus and of one antral surface was also examined. Interestingly, these lesions had little effect on gastric retention of solid (Fig 4.4)
The emptying of several different types of test meal was significantly delayed in rats previously treated with 0.5% BAC on the antrum (Fig 4.5). It is worth noting
that in control rats, the emptying of HCl and peptone was inhibited compared with saline. However, in BAC-treated rats there was no significant difference in the emptying of saline, HCl and peptone, even though all of these emptied less rapidly than the corresponding rates in control animals.
It is known that in intact rats, prior administration of peptone just before an emptying trial with peptone (ie peptone plus preload) produces an exaggerated inhibition of emptying due to enhanced CCK release (Forster and Dockray, 1992). The same effect was observed in the present control rats; in addition, however, peptone plus preload produced a significant inhibition of emptying compared with saline in BAC-treated rats (Fig 4.5). This suggests that CCK might be mediating this effect in BAC-treated rats implying an intact vagally mediated duodeno-gastric reflex. To ensure that the gastric retention in BAC-treated rats was not primarily due to enhanced CCK release, plasma CCK was measured in fasted BAC and control rats. The concentration in control rats ($1.0 \pm 0.1$ pM) was not significantly different from that in BAC-treated rats ($1.2 \pm 0.1$ pM).
Fig 4.1 Gastric retention after a 48 hr fast in control rats (open bars, n=6) and in rats treated with increasing concentrations of BAC to the antrum (hatched bars, n=6). Only at the highest dose of 0.5% w/v BAC was there significant retention of solid in the stomach (*p < 0.05, Mann Whitney U test).
Fig 4.2. Gastric retention at various time points following antral BAC (hatched bars, n=6) or sham operation (open bars, n=6) and after a 48 hr fast. At all time points there was a significant retention of solid within the stomach only in rats treated with BAC to the antrum (*p < 0.05, Mann Whitney U test).
Fig 4.3 Gastric retention in control rats (open bars, n=6) and in rats treated with 0.5% w/v BAC to the antrum (hatched bars, n=6) after a 48 hr fast and following a 30 minute refeed. There was a significant retention of solid in the stomach of fasted BAC treated rats compared to controls but after refeeding the dry weight of gastric content was similar in both groups (*p < 0.05, Mann Whitney U test).
Fig 4.4 Gastric retention after a 48 hr fast in control rats and rats treated with BAC either to the whole of the antrum, or to the anterior surface of the antrum only, or to a band of corpus adjacent to the non-glandular stomach, or with capsaicin to the antrum.
Data shown are for wet weight (hatched bars) and dry weight (open bars). Only BAC treatment of the whole antrum leads to gastric retention (*p < 0.05, Mann Whitney U test).
Fig 4.5 Gastric emptying rates for different liquids in gastric fistula rats pre-treated with either 0.5% w/v BAC (n=8, hatched bars) or vehicle (n=6, open bars). For all liquids shown emptying rates are slower in BAC treated rats. In control rats, peptone and HCl empty more slowly than saline, and peptone after a peptone preload empties more slowly than peptone alone. In BAC treated rats, saline, HCl and peptone empty at the same rate, but peptone after a peptone preload induces further delay in gastric emptying.
The present data provides evidence that the integrity of the intrinsic innervation of the rat antrum is obligatory for the normal emptying of solids and liquids from the rat stomach. Specifically, it was found that the loss of intrinsic antral neurons caused by the detergent BAC resulted in retention of solids, and delayed emptying of both nutritive (peptone) and non-nutritive (saline, HCl) solutions. The retention of solids and liquids could not be attributed to changes in plasma CCK, which is the hormone best known for its ability to inhibit gastric emptying (Dockray 1988) since plasma CCK was normal. In a further series of experiments performed on antrally denervated gastric fistula rats, Dr B Yegen studied the effects of the specific CCK-antagonist (Green et al., 1988) on the emptying of liquid test meals. In these experiments there was no significant difference in the emptying rates of saline, HCl and peptone after L-364,718 in BAC-treated rats compared to BAC rats that did not receive the antagonist. Interestingly, however, the delayed emptying of peptone after a preload was reversed and was comparable to the emptying of saline. It would appear therefore that the action of CCK in controlling gastric emptying can be exerted independently of the antral innervation.
The application of BAC to the antrum lesioned both the intrinsic and extrinsic afferent innervation; however loss of the latter cannot explain the retention of solids, since there was no retention in rats treated with capsaicin which lesions only extrinsic afferent fibres. Taken as a whole, therefore, the present data lead to the conclusion that
the antral innervation is essential for the gastric emptying of solid and of both nutritive and non-nutritive solutions, but not for the delaying actions of CCK on gastric emptying.
The neuroendocrine mechanisms that determine gastric emptying have attracted increasing attention in recent years. In the case of CCK such studies have suggested a primary site of action at receptors on vagal afferent nerve fibres, the activation of which evokes a vago-vagal reflex leading to relaxation of the gastric corpus (Raybould and Tache, 1988; Tache et al., 1988; Forster et al., 1990). The possible involvement of the distal stomach in mediating the effect of CCK on gastric emptying has been suggested by previous studies (Yamagishi and Debas, 1978) but little has been done to determine whether - in this case - CCK acts directly on smooth muscle or indirectly via the innervation. The findings here suggest that any effects of CCK on the innervation of the antro-pyloric region are not of central importance in its role as an inhibitor of gastric emptying.
The effect of antral denervation on emptying can be considered specific since denervation of a fully circumferential band of proximal corpus (which would also lesion vagal efferent fibres) did not lead to gastric retention. In part the retention of solids after antral denervation may be attributable to a decreased capacity of the antrum for trituration and therefore the reduction of solids to a form suitable for emptying. In addition, however, there is also plainly a marked delay in emptying of liquids. This effect is distinguishable from the reduced emptying of liquids seen after
lesioning of afferent neurons by capsaicin (Forster et al., 1990; Forster et al., 1991) since it is only in antrally denervated rats that there is a substantial delay in the emptying of isosmotic saline.
The pumping action of the antrum is well known, and contributes to the episodic delivery of gastric contents to the duodenum (Malbert and Ruckebusch, 1991; Dent et al., 1994). We suggest that the co-ordination of this activity requires intrinsic antral neurons and possibly intact efferent vagal neurons and that in the absence of these neurons the antrum generates an unremitting high-resistance to the passage of both liquids and solids. In view of the non-specific effect of BAC in lesioning all neuron populations within the area of exposure (excluding the submucosal neurons) it is not possible to speculate which if any neuron population is essential to maintain normal gastric emptying. It is interesting to note however that targeted disruption of the neuronal nitric oxide synthase gene leads to a phenotype essentially indistinguishable from the BAC treated antrally denervated rat (Huang et al., 1993). NO has been shown to mediate the nonadrenergic, noncholinergic smooth muscle relaxation throughout the gastrointestinal tract (Boeckxstaens et al., 1991) including relaxation of the gastric corpus (Desai et al., 1991). In the NOS knockout mouse, the presumed loss of smooth muscle relaxation in the region of the antrum and pylorus had similar effects to those documented here.
A further possibility that may explain the high pressure resistance in the BAC treated antrum is that the BAC treatment has a direct effect on the muscle itself. It has
been apparent from early studies with BAC that following denervation of the small intestine there is significant thickening of both longitudinal and circular muscle layers of the intestine (Fox et al., 1983; See et al., 1988). In addition the muscle became spontaneously contractile with altered mechanical properties including the ability to generate three times the active stress of control smooth muscle in response to muscarinic agonists (Osinski and Bass, 1994). This was later shown to be a consequence of an inability to resequester Ca$^{2+}$ in the sarcoplasmic reticulum leading to increased intracellular calcium (Osinski and Bass, 1995). Although it has been impossible to distinguish between the direct effect of BAC and the secondary effects of loss of normal innervation on smooth muscle function, Hadzijahic et al. (1993) have compared different methods of inducing myenteric denervation (Surgical vs BAC) and inflammation (intraluminal stasis and 5% acetic acid) to determine the underlying stimulus to the increased muscle thickness. Their results together with the observation of muscle thickening in the NOS knockout mice indicate that the changes in muscle thickness at least are a consequence of denervation and not a direct effect of BAC on smooth muscle.
**Summary**
Antral BAC produces selective lesions of defined populations of neurons in the stomach and leads to dramatic changes in gastric emptying. After antral BAC treatment there was retention of solids in rats fasted for 48 hr. Moreover, in antrally denervated rats with a gastric fistula, the emptying of saline, acid and peptone was substantially delayed. The cholecystokinin-dependent inhibition of gastric emptying
of peptone was preserved after antral BAC. Hence it appears that the innervation of the antrum is essential for normal emptying of both liquids and solids, but the inhibition of gastric emptying produced by cholecystokinin is not dependent on antral neurons.
Chapter Five
Antral denervation and G-cell Function
Introduction
The release of the antral hormone gastrin is controlled by the luminal contents of the stomach and by neurohumoral agents acting at the basolateral membrane of the G-cell. The main luminal stimulus is the presence of protein and protein-digestion products while the main luminal inhibitory factor is gastric acid (Dockray & Gregory, 1989; Walsh, 1994). The available evidence suggests that gastric acid releases somatostatin from antral D-cells which in turn inhibits gastrin release by a paracrine mechanism (Saffouri et al., 1980; Chiba et al., 1980; Walsh, 1994). The mechanisms by which luminal nutrients stimulate the G-cell are less certain. The luminal surface of the G-cell consists of tufts of microvilli that project into the lumen and this has led to the idea that luminal nutrients might act directly on the cell (Lichtenberger et al., 1982; Delvalle & Yamada, 1990; Lichtenberger, 1982). However, it is also clear that luminal stimulation of the G-cell can be indirectly mediated by nervous pathways (Saffouri et al., 1984; Schubert et al., 1992).
It is well recognised that vagal efferent neurons stimulate gastrin secretion in the cephalic phase of digestion and in addition, there are local intrinsic nervous reflexes controlling the G-cell. There is evidence for cholinergic-muscarinic stimulation of the G-cell, particularly in the rat (Schubert & Makhlouf, 1993; Schubert et al., 1992; Saffouri et al., 1984), but in dog and man atropine typically has no effect on nerve-mediated gastrin release, or stimulates it - indicating the importance of non-cholinergic inputs to the G-cell (Dockray & Tracy, 1980; Feldman et al., 1979;
Schiller *et al.*, 1980; Farooq & Walsh, 1975). The neuropeptide gastrin releasing peptide (GRP) which is well represented in antral mucosal nerve fibres (Dockray *et al.*, 1979), is thought to be the main non-cholinergic neurotransmitter stimulating the G-cell. In keeping with this, both antibodies and more recently selective antagonists to GRP have been shown to inhibit gastrin secretion in response to either luminal peptone, or electrical field stimulation of the antrum or vagal nerve stimulation (Schubert, *et al.*, 1985; Holst, *et al.*, 1990; Schubert, *et al.*, 1992). The experiments described in this chapter were performed in an attempt to define the relative importance of neural mechanisms compared to the direct stimulation by luminal factors on gastrin release. To this end I have studied the luminal factors that influence gastrin release in control and antrally denervated rats. The results indicate increased sensitivity to non-nutrient distension after antral denervation and suggest the existence of an antral inhibitory innervation in the rat that normally suppresses G-cell responses to non-nutrient distension.
Methods
Antrally denervated rats were prepared and after each experiment denervation confirmed by radioimmunoassay of appropriate neuropeptide markers in tissue extracts of antral muscle (see Methods). Gastric cannulas were placed at the time of antral denervation and all rats were allowed at least 11 days to recover after surgery. For the refeeding experiments, rats were fasted for 48 hrs on wire-bottomed cages and were then allowed to refeed for 30 minutes. The GRP antagonist (BW2258U89) {Moody et al Life Sci 56: 521-529} (2mg.kg$^{-1}$ in 0.5ml of 0.14M saline) or saline, were injected subcutaneously 30mins before refeeding and then trunk blood was taken for assay of plasma gastrin. Samples of antral and corpus mucosa were taken for RNA extraction for analysis of gastrin, somatostatin, HDC and GAPDH mRNA abundance by Northern blot.
Gastric fistula rats:
As before, (chapter 4) the stomach was washed with warm 0.14M NaCl to remove solids at least one hour prior to experiments. Gastric acid output was determined by collecting gastric secretion over four sequential 15 min periods and titrating to pH 7.0 with 20mM NaOH using a pH autotitrator. Plasma gastrin responses were determined after gastric distension with the following test solutions: 2% (w/v) methyl cellulose (2%MC); 2%MC at pH6 (50mM ammonium acetate); 2% MC with 50mM HCl; 4.5% w/v peptone. In each case the stomach was distended by connecting the cannula to a reservoir of test solution and elevating the reservoir to 5cm H$_2$O pressure for 30mins.
Results
Basal plasma gastrin
Basal plasma gastrin concentrations were similar in control, fasted rats (7.6 ± 1 pM; Fig5.1) and control rats with a gastric fistula (11.8 ± 2 pM; Fig5.4). In BAC treated rats fasted for 48hrs plasma gastrin was significantly elevated (28.4 ± 7pM, p<0.05 vs control; Fig 5.1). In these animals there was also marked gastric retention compared to controls (see Chapter 4). It seemed likely that gastric retention in BAC-treated rats accounted for the increased plasma gastrin, since in fasted BAC-treated rats with a gastric fistula, plasma gastrin was similar to control (13.5 ± 3.2pM; Fig 5.4) when the gastric contents were removed by lavage via the cannula.
Refeeding experiments
When control rats were fed for 30min after fasting for 48hr, plasma gastrin increased approximately 3-fold. There was a comparable increase in plasma gastrin in BAC-treated rats but the absolute increase after refeeding was higher than that in control rats, due to the elevation of basal plasma gastrin concentration (Fig 5.1). In keeping with the idea that GRP mediates food-stimulated release of gastrin in the rat, I found that the GRP antagonist BW2258U89 (0.5 mg.kg⁻¹) inhibited the rise in plasma gastrin in control rats seen after refeeding for 30min following a 48 hr fast. In contrast, in BAC-treated rats, the post-prandial rise in gastrin was not dependent on GRP since there was no significant difference compared with BAC-treated rats that received vehicle in place of the GRP antagonist (Fig 5.2).
Figure 5.1 Plasma gastrin in fasted and refed rats 11 days after either sham operation (open bars) or antral denervation by 0.5% BAC (hatched bars). In fasted BAC-treated rats there was elevated fasting plasma gastrin. After refeeding, plasma gastrin increased approximately three-fold in both control and BAC-treated rats. (n=>6 for each group, * p<0.05 vs control response, Mann Whitney U test).
Figure 5.2 Plasma gastrin concentrations in control and antrally-denervated rats after administration of the specific GRP antagonist BW225U86 (hatched bars) or saline (open bars) 30mins prior to refeeding. In control rats, but not antrally denervated rats, the GRP antagonist significantly attenuated the post-prandial rise in plasma gastrin. (n=6 for all groups, * p<0.05 Mann Whitney U test).
Basal acid output:
Under normal circumstances gastrin release is inhibited by luminal acid so that one potential explanation for the elevated plasma gastrin in BAC-treated rats was that BAC treatment of the antrum influenced acid secretion. In fact the range of basal acid output in BAC-treated rats overlapped with that in controls but the mean was approximately 3-fold lower (Fig 5.3). Complete antral denervation was required to reduce mean basal acid output as rats that received BAC only to the anterior surface of the stomach had a mean basal acid output comparable with controls.
Distension experiments
In control rats distension with the non-nutritive solution 2% MC had no effect on plasma gastrin. In contrast, distension of BAC-treated rats with 2% MC produced a significant rise in plasma gastrin (Fig 5.4). Although this appeared to substantiate the view that distension stimulates gastrin release in antrally denervated rats, there was also the possibility that alterations in pH may play a part in the response given the reduced acid output in BAC-treated rats. Therefore distension experiments were performed with buffered 2% MC (acidified and pH6) and with peptone as a positive control. In control rats distension with 4.5% w/v peptone increased plasma gastrin 3-fold but distension with the non-nutritive solutions 2 % methylcellulose (2%MC), or 2%MC pH 6 or 2%MC with 50 mM HCl did not increase plasma gastrin (Fig 5.5).
In BAC-treated rats, the effects of distension with peptone and 2%MC with 50mM HCl in methylcellulose were similar to control. However distension by either 2%MC or 2%MC pH 6.0 did stimulate gastrin release, the increase in plasma gastrin being comparable to that produced by peptone (Fig 5.5).
Figure 5.3 Mean basal acid output (mmol.15min$^{-1}$) in control and antrally denervated rats. Two groups of denervated rats were studied: either total antral denervation, or denervation of the anterior surface only (hemi-BAC). Each data point represents the mean acid output of four sequential 15min collections from one rat. Only in the rats with complete antral denervation was there a significant reduction in basal acid output. (* p<0.05 ANOVA).
Figure 5.4 Plasma gastrin responses in control and BAC-treated rats after gastric lavage to remove residual gastric content. Rats were either left undistended (open bars) or were distended with the non-nutritive solution 2%MC for 30 minutes at a pressure of 5cm of H$_2$O (hatched bars). In BAC-treated rats but not controls, plasma gastrin was increased by distension with 2%MC. (n=6 or more for each group, * p<0.05 vs control response).
Figure 5.5 Plasma gastrin responses in control (open bars) and BAC-treated rats (hatched bars). Rats were either left undistended or distended for 30 mins with either 2%MC (buffered with 50mM HCL or to pH6) or 4.5% w/v peptone. Plasma gastrin after distension with 4.5% peptone was similar in control and antrally-denervated gastric fistula rats. In BAC-treated rats, but not controls, plasma gastrin was also increased by distension with 2%MC buffered to pH6.0. Neither group showed an increase in plasma gastrin after distension with 2%MC with 50mmol HCl. (n=6 or more for each group, * p<0.05 vs control response, Mann Whitney U test).
Gastrin, somatostatin HDC and GAPDH mRNA abundance
It is known that gastric endocrine cell responses frequently involve altered changes in the synthesis of active products, as seen by changes in mRNA abundance (Dockray et al. 1996). To determine whether antral denervation might alter the synthesis of regulatory molecules in gastric endocrine cells Northern blots of mRNA’s encoding gastrin, somatostatin, HDC and GAPDH were analysed (Figures 5.6 & 5.7). There was no difference in the expression of GAPDH between control and BAC treated rats. In the antrum there was a significant reduction in antral mucosal somatostatin mRNA abundance in BAC-treated rats compared with controls and a similar decrease in gastrin mRNA abundance. In the corpus however, somatostatin mRNA abundance was unchanged in BAC-treated rats compared with controls, while HDC mRNA was variably increased. The latter is compatible with the sustained increase in plasma gastrin in these animals.
Figure 5.6 Representative Northern blots of total antral mRNA hybridised with specific cRNA probes for gastrin (upper panel) and somatostatin (lower panel). Lanes 1-6 are control and lanes 7-12 BAC-treated rats. BAC treatment significantly lowered gastrin and somatostatin mRNA abundance. These changes were considered specific as BAC treatment had no effect on GAPDH mRNA abundance (results not shown).
Fig 5.7 Relative mRNA abundance in the antrum of GAPDH, gastrin and somatostatin, and in the corpus of GAPDH, somatostatin and HDC in control (open bars) and BAC-treated rats (hatched bars). The relative abundance of each mRNA species has been normalised by taking as 100% the abundance in controls. Note the abundance of the non-regulated marker GAPDH was similar in control and BAC treated rats. In the antrum BAC decreased gastrin and somatostatin mRNA (n=6 in each group; * p<0.05, Students t test).
Discussion
In previous studies that have examined the neuronal regulation of G-cell function the extrinsic innervation has been manipulated by stimulation, lesioning or pharmacological blockade (Dockray & Gregory, 1989; Walsh, 1994); the role of intrinsic neurons has been largely determined by pharmacological approaches using neurotransmitter antagonists or antibodies (Schubert et al., 1985; Schubert et al., 1992). In the present experiments antral denervation by BAC was used to lesion both the intrinsic and extrinsic innervation of the rat antrum. The subsequent experimental data support the conclusions that the post-prandial gastrin response in control rats is largely mediated by the neuropeptide transmitter GRP; in antrally denervated rats, a post-prandial gastrin response is maintained but this is no longer inhibited by a GRP antagonist. Instead, it appears that non-nutritive distension becomes a strong stimulus for the denervated G-cell and this explains for the most part the fasting and meal stimulated hypergastrinaemia seen in the antrally denervated rat. The results are unexpected given that it is generally thought that G-cell responses to distension are mediated by nervous reflexes (Dockray & Gregory, 1989; Walsh, 1994; Schubert & Makhlouf, 1993).
Since gastrin release is inhibited by luminal acid, one potential explanation for the elevated plasma gastrin in antrally denervated rats might be reduced acid output. In fact there was a decrease in basal acid secretion after denervation. However, the capacity of the denervated G-cell to respond to distension was increased even when intragastric pH was buffered at pH 6.0 suggesting that G-cell responses secondary to
changes in acid output are unlikely to be important in the response to denervation. Furthermore, in undistended BAC-treated rats with a gastric fistula plasma gastrin was comparable to control rats suggesting that basal acid output was sufficient to maintain inhibition of gastrin release. In man, gastric distension is a good stimulus to acid secretion (Soares et al., 1977), but in the conscious rat, it has been reported that non-nutritive distension of the stomach inhibits acid secretion by activating a sympathetic reflex (Dimaline et al., 1986). Hence the chronic gastric retention after antral denervation may lead to inhibition of acid output by this mechanism.
In support of this argument, it is clear that after lesioning the antral innervation there was a pronounced gastric retention of solids, even after 48hr fasting. After complete emptying of the stomach in antrally denervated rats fitted with a gastric fistula, basal plasma gastrin was similar to control levels, suggesting that the hypergastrinaemia in denervated rats without a gastric cannula was attributable to the gastric retention.
Studies in the isolated perfused rat stomach have provided evidence that GRP mediates the release of gastrin in response to luminal peptone (Schubert et al., 1992). The observation that the post-prandial increase in plasma gastrin concentration was blocked by a GRP antagonist suggests that a similar mechanism operates in conscious rats *in vivo*. However, the post-prandial increase in gastrin in antrally denervated rats was not blocked by a GRP antagonist suggesting that an additional mechanism comes into play after denervation. Interestingly, the gastrin response to gastric distension
with peptone in antrally denervated rats was no greater than that to non-nutritive distension. Two inferences may be drawn from this: First, that the stimulatory effect of nutrient is normally mediated by antral neurons, and second that antral neurons normally inhibit G-cell responses to non-nutrient distension. Studies in the isolated perfused rat stomach have shown that antral neurons mediate stimulation of gastrin release after distension at low pressures, and inhibition at high pressures (Schubert & Makhlouf, 1993). The latter may explain why methylcellulose at pH6 did not stimulate gastrin release in the intact stomach. The novel finding in the present experimental series is that there exists a mechanism for distension-evoked release of gastrin which is independent of the antral innervation.
It is generally thought that gastrointestinal responses to mechanical distension are mediated by nervous reflexes. Two possible mechanisms might account for the unexpected observation that the denervated G-cell responds to non-nutritive distension. First, the G-cell or a nearby cell in the antral mucosa might be mechanosensitive (which is suppressed by the antral innervation), or develops such sensitivity after denervation. In this context it has been recognised for many years that enterochromaffin cells (EC) cells are mechano-sensitive (Bulbring and Lin, 1958; Kirchgessner et al., 1992); denervated G-cells might therefore take on EC-cell like properties, or alternatively antral EC cells might regulate G-cell function for example through a paracrine mechanism. Second, non-nutritive distension of the stomach could release an endocrine factor from the gastric corpus, which increases gastrin release.
from the denervated, but not innervated, antrum. In the dog, Debas *et al.* (1975) showed that distension of the corpus increased gastrin release from an extrinsically denervated antral pouch suggesting the existence of an oxynto-pyloric endocrine reflex. The existence of a similar reflex in the rat might explain the present findings. If so, however, it would appear that such a reflex is not normally activated in the innervated antrum.
Hypergastrinaemia stimulates the enterochromaffin-like (ECL) cells of the gastric corpus which are an important source of histamine. The increased HDC mRNA abundance observed in BAC-treated rats is compatible with the hypergastrinaemia of antral denervation. Increased histamine synthesis and secretion might be expected to stimulate increased acid output, and the reduced acid secretion observed would therefore appear not to be secondary to inhibition at the level of the ECL cell. In the antrum we found marginally reduced gastrin and somatostatin mRNA abundance. Gastrin synthesis is regulated both at the level of gene transcription and at the level of mRNA translation (Bate *et al.*, 1996). The present data raise the possibility that after denervation there is independent control of release of synthesis at a post-transcriptional level. Furthermore, given that somatostatin and gastrin mRNA abundance appeared to be reduced following denervation without an overall depression in gene transcription (indicated by similar abundance of GAPDH in control and BAC treated rats) it seems possible that the innervation of the antral mucosa normally may regulate transcription of specific genes in target endocrine cells. Taken together with the possibility that following denervation G-cells may themselves
take on the EC-cell property of mechanosensitivity, it would appear that antral neurons play some role in the maintenance of the normal differentiated state of the G-cell.
Unfortunately the factors that regulate the differentiation of G-cells have proved difficult to study not least because of the problems of generating G-cell lines. However the mechanisms of differentiation in endocrine cells per se have been studied in pancreatic islet cells. Interestingly, gastrin is expressed in the foetal pancreas at the time of endocrine cell development (Brand and Fuller, 1988) and there is good evidence to suggest that gastrin acts in conjunction with TGF-α to promote normal pancreatic endocrine cell differentiation (Wang et al., 1993). In adult rats, following partial pancreatectomy, pancreatic islet cell regeneration occurs and is associated with expression of the Reg gene (reg) (Terazono et al., 1988) implying that reg expression may be involved in differentiation. The latter is of interest because the reg gene has been shown recently to be present in the gastric ECL cell (Ashahara et al., 1996), a cell whose gene expression is well known to be regulated by gastrin. In addition reg expression is increased during the regeneration phase following acute gastric mucosal injury (Ashahra et al., 1996).
It is plausible to suppose therefore that the remodelling of gastric mucosal function after denervation involves gastrin itself, acting in conjunction with other factors to regulate differentiation of endocrine cells, including the ECL cell. The
studies detailed in the next chapter begin to investigate the possibility that gastrin regulates *reg* expression.
**Summary**
In the intact rat, luminal nutrient releases gastrin via activation of neurons secreting GRP whilst the antral innervation normally inhibits gastrin release in response to non-nutrient distension. After antral denervation, post-prandial gastrin release is maintained but by a mechanism independent of neurons secreting GRP. Moreover, gastric distension with a non-nutrient solution becomes an adequate stimulus for gastrin release. Despite reductions in gastrin and somatostatin mRNA abundance following BAC treatment, hypergastrinaemia occurred and produced changes in ECL-cell gene expression although basal acid output was reduced. The altered G-cell function that occurred after denervation and the possibility that this effected changes in gastric endocrine cell differentiation remains to be determined.
Chapter Six
Control of reg expression in rat corpus mucosa and AR42J cells
The work of several laboratories indicates that the *reg* gene is expressed during pancreatic or gastric mucosal regeneration (Terazono et al., 1988; Miyaura et al., 1991; Ashahara et al., 1996). Since *reg* is expressed in pancreatic β cells and gastric ECL cells it is reasonable to suppose that it may be implicated in endocrine cell growth or differentiation. Gastrin is known to stimulate growth of ECL cells both in the human (Borch et al., 1985; Bordi et al., 1986; Roucayrol and Cattan, 1989; Solcia et al., 1991) and the rat (Havu 1986; Hakanson and Sundler, 1990) and is essential for normal gastric (Koh et al., 1997) and pancreatic (Wang et al., 1993) endocrine cell development. In the latter case, gastrin must act in conjunction with growth factors for normal differentiation to occur. However, the possible interactions between gastrin, growth factors and *reg* expression have not been directly studied.
AR42J cells are an amphicrine cell line with the capability to differentiate into either acinar or endocrine cells depending on the tissue culture conditions (Christophe, 1994). These cells express both the gastrin/CCK-B receptor and the *reg* gene (Lambert et al., 1991; Zenilman et al., 1996), suggesting that they might be a useful system in which to investigate the relationships outlined above. The experiments performed in this chapter were undertaken first to determine whether gastrin might regulate *reg* gene expression in rat gastric corpus and second to study in the AR42J cell line some of the features of gastrin-induced *reg* expression. The results indicate that *reg* expression is regulated by gastrin acting via the CCK-B receptor. In addition,
although EGF potentiated gastrin-stimulated *reg* expression the reported data suggest that *reg* expression is associated with endocrine cell differentiation and not growth.
**Methods**
**Animal Studies**
Rats were either fasted for 48 hrs on wire-bottomed cages or fed ad libitum and given omeprazole (400μmol.kg$^{-1}$) by gavage for 5 days. Rats fed *ad libitum* were used as controls. At the end of treatment, trunk blood was taken for assay of plasma gastrin and samples of corpus mucosa were taken for RNA extraction and analysis of HDC and *reg* mRNA abundance by Northern blot.
**Tissue Culture**
Culture of AR42J cells and treatment with amidated and gly-extended gastrin, L740093 (1nM), EGF (10pM-1μM) and wortmannin (5 x 10$^{-8}$M) was performed by Dr. LA Bishop. Cells were cultured at a density of 1x10$^6$ for 24 hrs in depleted media before appropriate treatment. All treatments were given for 24 hrs except for wortmannin (4 hrs). At the end of the culture period total cell mRNA was extracted by the lithium chloride precipitation method and *reg* mRNA abundance analysed by Northern blot (see Chapter 2).
Results
Reg expression in rat gastric corpus
In keeping with previously published observations (Dimaline et al., 1993; Swarovsky et al., 1994), treatment of Wistar rats with omeprazole (400μmol.kg\(^{-1}\)) for five days lead to a five to ten-fold increase in plasma gastrin and an associated three-fold increase in HDC mRNA in gastric corpus mucosa, compared to rats fed ad libitum. Conversely, fasting significantly reduced plasma gastrin and HDC mRNA abundance (Fig 6.1). In addition however, omeprazole-induced hypergastrinaemia also resulted in altered reg gene expression in the gastric mucosa, the changes in magnitude of reg mRNA abundance being almost identical to those of HDC (Fig 6.1).
Reg expression in AR42J cells
To examine in more detail how gastrin might regulate reg gene expression, AR42J cells were cultured in the presence of increasing concentrations of both amidated (G17-NH\(_2\)) and glycine-extended gastrin (G-gly). Amidated and glycine-extended gastrin both produced dose-dependent increases in reg expression with G-gly being approximately 1000-fold less potent than its amidated counterpart (Fig 6.2 & 6.3). To determine the relevant gastrin receptor mediating the reg response, AR42J cells were treated with either G17-NH\(_2\) (10\(^{-8}\) M) or G-gly (10\(^{-6}\) M) in the presence of the specific CCK-B receptor antagonist L-740093. The increase in reg mRNA abundance stimulated by G17-NH\(_2\) was completely inhibited by the CCK-B receptor antagonist (Fig 6.4). The response to G-gly, although significantly attenuated by L-740093, remained increased by approximately two-fold (Fig 6.5).
Figure 6.1 **upper panel**: Effect of fasting and omeprazole treatment on plasma gastrin (open bars), *reg* mRNA (hatched bars) and HDC mRNA abundance (stippled bars). Note *reg* mRNA abundance was decreased by fasting and increased by omeprazole treatment, the magnitude of the change paralleling that of HDC mRNA. (*n=6 for each group, * p<0.05 vs control response, Mann Whitney U test). **lower panel**: Representative Northern blot of *reg* mRNA abundance in 48 hr fasted (lanes 1-4), fed ad lib. (lanes 5-8) and omeprazole treated rats (lanes 9-12).
Figure 6.2 Representative Northern blots for *reg* mRNA abundance in total RNA extracted from duplicate cultures of AR42J cells treated with increasing molar concentrations of either G17-NH$_2$ (upper panel) or G-gly (lower panel). Note the dose range of the response is approximately 3 log units higher for G-gly.
Figure 6.3 Dose response relationship between G17-NH$_2$ (closed circles) and G-gly (open circles) and $reg$ mRNA abundance in AR42J cells. The $A_{50}$ for G17-NH$_2$ was approximately $10^{-8}$M and that for G-gly approximately $5 \times 10^{-5}$M.
Figure 6.4 Effect of $10^{-9}$M L-740093 (hatched bars) on the *reg* mRNA response to $10^{-8}$M G17-NH$_2$ (upper panel) and a representative Northern blot (lower panel) of *reg* mRNA abundance in total RNA extracted from duplicate cultures of AR42J cells treated with vehicle (ammonium bicarbonate; lanes 1&2), L-740093 (lanes 3&4), G17-NH$_2$ (lanes 5&6) and G17-NH$_2$ and L-740093 (lanes 7&8). The twenty five-fold increase in mRNA abundance produced by G17-NH$_2$ was completely reversed by L-740093.
Figure 6.5 Effect of $10^{-9}$M L-740093 (hatched bars) on the *reg* mRNA response to $10^{-6}$M G-gly (upper panel) and a representative Northern blot (lower panel) of *reg* mRNA abundance in total RNA extracted from cultures of AR42J cells treated with ambic (lanes 1&2) G-gly (lanes 3&4) and G-gly and L-740093 (lanes 5-7). Although the response to G-gly was significantly attenuated by L-740093, *reg* mRNA abundance remained increased by approximately two-fold.
EGF and *Reg* expression in AR42J cells
The effects of EGF on *reg* expression is shown in Fig 6.6. At the highest dose of EGF ($10^{-6}$M) there is no significant upregulation of *reg* expression even though this concentration of EGF has been shown to induce proliferation of AR42J cells. However, EGF, at a concentration of $10^{-9}$M potentiated the effects of amidated and glycine-extended gastrin on *reg* expression (Fig 6.7). Hence the ten to twenty-fold increase in *reg* expression induced by $10^{-8}$M G17-NH$_2$ was further enhanced ten-fold by EGF ($10^{-9}$M).
Wortmannin and *Reg* expression in AR42J cells
Inhibition of the PI3 kinase in cultured human foetal pancreatic cells has been shown to induce differentiation towards an endocrine cell phenotype (Ptasznik et al., 1997). Treatment of AR42J cells with the PI3 kinase inhibitor wortmannin ($5 \times 10^{-8}$ M) for four hours, increased *reg* expression in both unstimulated and G17-NH$_2$-stimulated AR42J cells (Fig 6.8).
Figure 6.6 **upper panel**: Relationship between increasing doses of EGF and *reg* mRNA abundance in AR42J cells. Results are expressed relative to mRNA expression in control cultures arbitrarily defined as 5% of a predicted maximum response. Even at $10^{-6}$M EGF there is no increase in *reg* mRNA abundance.
**lower panel**: Representative Northern blot of *reg* mRNA in duplicate cultures of AR42J cells exposed to increasing concentrations of EGF.
Fig 6.7 **upper panel**: relationship between G17-NH$_2$ (10$^{-8}$M) or G-gly (10$^{-7}$M), EGF (10$^{-9}$M) and *reg* mRNA abundance in AR42J cells. Results have been normalised to the response to 10$^{-8}$M G17-NH$_2$ taken as 100%. The response of AR42J cells to either G-gly or EGF alone was approximately 10% of the G17-NH$_2$ response. However, EGF increased the response of AR42J cells to G17-NH$_2$ by fifteen-fold and to G-gly by seven-fold.
**lower panel**: Representative Northern blots for *reg* mRNA in duplicate cultures of AR42J cells treated with G17-NH$_2$ (lanes 1&2), G-gly (lanes 3&4), EGF (lanes 5&6), G17-NH$_2$ and EGF (lanes 7&8) and G-gly and EGF (lanes 9&10).
Fig 6.8 **upper panel**: relationship between G17-NH$_2$ (10$^{-8}$M), wortmannin (5 x 10$^{-8}$M) and *reg* mRNA abundance in AR42J cells. The gastrin-stimulated response in *reg* mRNA is further increased two-fold by wortmannin. Note that wortmannin also stimulates *reg* mRNA expression in control cultures. **lower panel**: Representative Northern blots for *reg* mRNA in duplicate cultures of AR42J cells treated with Ambic (lanes 1&2), wortmannin (lanes 3&4), G17-NH$_2$ (lanes 5&6) and G17-NH$_2$ and wortmannin (lanes 7&8).
The data presented in this chapter provide evidence that *reg* expression in rat corpus mucosa responds to changes in plasma gastrin and in AR42J cells is regulated by amidated gastrin acting through the gastrin/CCK-B receptor. In addition, whilst EGF alone had no effect on *reg* expression it markedly potentiated gastrin stimulated *reg* mRNA abundance. Furthermore, in culture conditions that have been shown to favour differentiation of AR42J cells into insulin-secreting endocrine cells, *reg* gene expression was increased.
Regulation of ECL cell gene expression by plasma gastrin has been demonstrated for a variety of genes including HDC (Dimaline *et al.*, 1991; Swarovsky *et al.*, 1994;). The observed threefold increase in HDC mRNA abundance following 5 day omeprazole treatment was in keeping with previously reported data. The presence of *reg* in rat ECL cells has been reported only recently (Ashahara *et al.*, 1996) and the results presented here demonstrate for the first time that *reg* gene expression in vivo can be regulated by plasma gastrin.
The present results contrast with those of Zenilman *et al.*, (1997) who showed that gastrin, CCK and glucagon (all 10nM) had no effect on reg mRNA abundance in AR42J cells. However, the tissue culture protocol in the experiments reported by Zenilman did not incorporate 24 hrs of culture in serum depleted media prior to the period of treatment. AR42J cells have been reported to synthesise and secrete gastrin
that may then act in an autocrine manner (Blackmore and Hirst, 1992) and this may explain the discrepancy between the above findings and the lack of effect of gastrin seen by Zenilman and colleagues. An additional explanation of the results of Zenilman et al., is the influence of the cell density of AR42J cells on *reg* gene expression. In preliminary experiments it was found that *reg* gene expression was upregulated (approximately tenfold) as cell density reached confluence. Zenilman et al. conducted their experiments on confluent cells unlike the experiments reported here.
The gastrin stimulated *reg* expression in AR42J cells appears to act via the gastrin/CCK-B receptor known to be present on these cells (Lambert et al., 1991). The reported dissociation constant for gastrin at this receptor in AR42J cells is 4nM (Zhou et al., 1992). The $A_{50}$ of the *reg* response to G17-NH$_2$ was $\approx 10$ nM and the complete inhibition of the response by the specific gastrin/CCK-B receptor antagonist L-740093 indicates that this response is mediated via the gastrin/CCK-B receptor. The *reg* response to G-gly also was inhibited by L-740093 but only by $\approx 80\%$. The dose of G-gly required to increase *reg* expression was much higher than that for G17-NH$_2$ and given that L-740093 is a competitive antagonist at this receptor it seems likely that the effects of G-gly on *reg* expression also are mediated via the gastrin/CCK-B receptor. The parallel rightward displacement of the G-gly dose-response curve compared to that of G17-NH$_2$ also suggested that G-gly was acting at the same receptor as G17-NH$_2$ but with lower affinity.
Perhaps by coincidence, the magnitude of change in *reg* mRNA abundance in the rat gastric mucosa was identical almost to that of HDC. Given that gastrin regulates HDC gene expression via the gastrin/CCK-B receptor (Hollande et al., 1994), this might suggest a common intracellular pathway regulating HDC and *reg* gene expression. The rat and human HDC promoter has been shown to be regulated by a protein kinase C dependent pathway (Hocker et al., 1996; Zhang et al., 1996). For the human HDC gene a palindromic sequence (5'-CCCTTTAAATAAAGGG-3') that confers transcriptional responsiveness to gastrin has been identified and defined as a gastrin response element. A similar sequence is present within the rat HDC promoter region (5'-CCCTTAAATAAGAGGG-3'). In each case the putative response element lies within 25 base pairs of the transcriptional start site. The 5'-regulatory region of the rat *reg* gene has also been described in AR42J cells (Miyashita et al., 1994). The regulatory sequence of most significance indicates a region -256 to -237 upstream of the transcriptional start site that has significant sequence homology with the consensus sequence of the pancreatic exocrine enhancer (Boulet et al., 1986). Although this would suggest a separate regulatory mechanism for *reg* gene transcription in AR42J cells, the response to gastrin was not studied. However, there does not appear to be a sequence similar to the putative gastrin response element of the rat HDC gene within the reported promoter region of the rat *reg* gene. Nevertheless the data clearly indicate that gastrin regulates *reg* expression in AR42J cells via a gastrin/CCK-B receptor mediated pathway.
Other effects of gastrin on AR42J cells that have been shown to depend on a gastrin/CCK-B receptor mediated pathway include stimulation of amylase secretion (Lambert et al., 1991) and stimulation of growth (Pradel et al., 1993; Seva et al., 1994; Zenilman et al., 1997). This raises the possibility that *reg* gene expression may be part of the growth response of AR42J cells to gastrin. This seems unlikely for two reasons: Firstly, the growth promoting effects of G17-NH$_2$ on AR42J cells is relatively weak compared to that of EGF (Watson et al., 1993). Despite this, the data above demonstrate that $10^{-6}$M EGF had no effect on *reg* gene expression although this dose would be expected to produce a large trophic response. This indicates that *reg* expression, although present in AR42J cells, is not required for cell growth. Secondly, treatment of AR42J cells with the PI3 kinase inhibitor wortmannin which has been shown to inhibit cell growth and promote endocrine cell differentiation (Ptasznick et al., 1997) was found to increase *reg* gene expression. The effect of wortmannin on AR42J cell *reg* expression was not confined to unstimulated cells as it also potentiated the effects of gastrin on *reg* expression.
Several other lines of evidence indicate that *reg* gene expression is associated with differentiation towards an endocrine cell phenotype. Treatment of AR42J cells with dexamethasone has been shown to promote differentiation of an acinar cell phenotype and concurrently reduced *reg* expression (Zenilman et al., 1997). Conversely, treatment with activin A, betacellulin or HGF stimulates differentiation of AR42J cells towards an endocrine cell phenotype (Mashima et al., 1996a; Ptasznik et al., 1997; Mashima et al., 1996b) and HGF has been shown to increase *reg* expression.
in human foetal pancreatic cells (Otonkoski et al., 1994). Taken together, the experimental evidence suggests that *reg* expression in AR42J cells is associated with differentiation towards an endocrine cell phenotype. If so, then this process would be expected to be favoured by treatment with gastrin and potentiated by either EGF or wortmannin. The data are of interest as AR42J cells may provide a useful model to elucidate the intracellular pathways that mediate these effects.
The interaction between growth factors, gastrin and the differentiation of endocrine cells has been described in the mouse pancreas (Wang et al., 1993). Here at least, both are required in conjunction for normal development of the endocrine pancreas. In the stomach, gastrin does not seem to be essential for normal ECL cell development as these cells can still be found in the gastric mucosa of gastrin knockout mice (Koh et al., 1997). However, in this model ECL cell numbers were significantly reduced implying that gastrin regulates the number of mature ECL cells in the stomach perhaps by influencing the growth and differentiation of precursor cells. There are no data as yet on *reg* expression in such experimental situations but the provisional data presented thus far indicate that *reg* expression may be a marker for ECL cell function and more specifically may play a role in the maintenance of a normal ECL population within the gastric mucosa. The clinical implications of this are the subject of the next chapter.
Summary
Reg expression in rat gastric corpus was increased by 5 day omeprazole treatment. In AR42J cells reg expression was shown to be regulated by amidated gastrin acting through the CCK-B receptor. EGF alone had no effect on reg expression but markedly potentiated the gastrin stimulated response. In culture conditions that favour differentiation of AR42J cells into insulin-secreting endocrine cells, reg gene expression was increased. The observations of others clearly indicate that growth factors acting together with gastrin may regulate endocrine cell growth and differentiation in the gastrointestinal tract. AR42J cells may provide a useful model to elucidate the intracellular pathways that mediate these effects.
Chapter Seven
Reg gene expression in human gastric mucosa.
In rodents hypergastrinemia secondary to long-term treatment with H$_2$-receptor antagonists or proton pump inhibitors (PPI) may lead to gastric carcinoid tumours (Hava, 1986; Poynter and Selway, 1991; Wangberg et al., 1995). This has prompted extensive reviews of the natural history of these tumours in man (Solcia et al., 1991; Thomas et al., 1994; Rindi, 1995; Modlin et al., 1995). It is now clear that hypergastrinaemia from whatever cause may result in hyperplasia of the gastric ECL cell. Significant ECL-cell hyperplasia has been demonstrated in one third of patients rendered hypergastrinaemic by *H pylori* infection and coincident treatment with a PPI (Eiselle et al., 1997). The majority of patients with CAG display some degree of ECL cell hyperplasia, the achlorhydria of CAG leading to hypergastrinemia that is generally accepted to be a stimulus to ECL cell growth (Creutzfeld, 1988; Hakanson and Sundler, 1990; Bordi et al., 1995). In fact, chronic atrophic gastritis (CAG) with or without pernicious anaemia is the commonest cause of enterochromaffin-like (ECL-) cell hyperplasia and in 5-10% of patients there is a progression to gastric carcinoid tumours (Borch et al., 1985; Bordi et al., 1991; Bordi et al., 1995).
The recent demonstration of *reg* gene expression in rat ECL cells together with the evidence suggesting that the reg protein may be important for pancreatic endocrine cell growth and differentiation imply a possible role for *reg* in the control of
ECL cell growth and differentiation. Gastrin has been shown to be important for entero-pancreatic endocrine cell development (Wang et al., 1993; Koh et al., 1997), acting in concert with growth factors such as TGF-α. In keeping with these observations, the results presented in the previous chapter showed gastrin stimulation of *reg* expression, potentiation of this response by EGF and upregulation of *reg* mRNA in AR42J cells exposed to culture conditions that favour differentiation towards an endocrine cell phenotype. The experiments performed in this chapter attempt to determine whether *reg* expression in the human gastric corpus is affected by plasma gastrin concentration and whether *reg* may have a pathophysiological role in ECL cell tumour development in patients with chronic hypergastrinaemia.
Human studies
The details of the patients that provided samples for the experimental studies described in this chapter are shown in Table 7.1 Plasma gastrin was assayed by RIA using antibody L2. Endoscopic biopsies were taken from the gastric corpus and from any corpus nodules if present from hypergastrinaemic patients that required a gastroscopy. Duplicate biopsies were sent to Dr G Armstrong (Consultant Histopathologist, Hope Hospital) for Histology. Total mRNA was extracted from biopsies and reg mRNA analysed by Northern blot and RT/PCR, cloning and sequencing (see Chapter 2). For the Northern blots, reg mRNA is expressed relative to the non-regulated mRNA for GAPDH.
Octreotide Suppression Test
Two of the patients with carcinoid tumours (JH & KD) consented to an octreotide suppression test. The intended study was explained and informed consent obtained from both patients. Endoscopic biopsies were taken from ECL cell nodules and apparently normal corpus prior to and at the end of a 72hr infusion of octreotide (25mg.hr\(^{-1}\)). Plasma samples were taken before, during and after the octreotide infusion for assay of gastrin.
| PATIENT | AGE | SEX | DIAGNOSIS | PLASMA GASTRIN (pM) | ECL Cell Polyps | Polyp size |
|---------|-----|-----|------------------------------------------------|---------------------|-----------------|------------|
| J.H. | 44 | F | P.A. Nodules | 850 | >50 | >1cm |
| K.D. | 42 | M | P.A. Nodules | 600 | 2 | 1cm + <5mm |
| K.W. | 72 | F | P.A. Nodules | 1200 | >20 | <1cm |
| P.S. | 47 | M | Chronic Renal Failure Diabetes Mellitus | 650 | <10 | <5mm |
| M.B. | 56 | F | Z.E./MEN1 | 280 | <10 | <5mm |
| P.G. | 65 | F | P.A. | 450 | <10 | <5mm |
| P.M. | 61 | F | P.A. | 400 | <10 | <5mm |
| W.S. | 72 | M | P.A. | 480 | <10 | <5mm |
| J.C. | 47 | M | Z.E. | 250 | Nil | |
| T.O. | 72 | M | P.A. | 570 | Nil | |
| D.K. | 46 | M | P.A. | 440 | Nil | |
| H.W. | 75 | F | P.A. | 180 | Nil | |
| B.C. | 62 | M | P.A. | 250 | Nil | |
| S.B. | 47 | F | P.A. | 1300 | Nil | |
| E.H. | 84 | F | P.A. | 850 | Nil | |
| P.T. | 56 | F | Cystic fundic gland polyps | 7 | Nil | <5mm |
| M.L. | 70 | F | Cystic fundic gland polyps | 28 | Nil | upto 1cm |
| M.L. | 66 | F | Cystic fundic gland polyps | 20 | Nil | <5mm |
| H.B. | 86 | F | Cystic fundic gland polyps | 14 | Nil | <5mm |
| J.T. | 43 | F | Cystic fundic gland polyps | 20 | Nil | <5mm |
| A.M. | 63 | F | Dyspepsia, Normal OGD | 15 | Nil | |
| E.H. | 72 | F | Iron Deficiency, Normal OGD | 26 | Nil | |
| E.Q. | 50 | F | Dyspepsia, Normal OGD | 13 | Nil | |
| T.B. | 43 | M | Dyspepsia, Normal OGD | 25 | Nil | |
| A.L. | 63 | M | Dyspepsia, Normal OGD | 12 | Nil | |
Table 7.1 Details for all patients that provided tissue biopsies for either Northern blotting of *reg* mRNA abundance or RT/PCR to generate cDNA transcripts of *reg* mRNA for sequencing purposes. Hypergastrinaemic patients are shown above the control subjects that were selected on the basis of a normal oesophago-gastric-duodenoscopy (OGD) and normal plasma gastrin.
Permanent cell transfection
Two constructs of human *reg* were selected for permanent transfection. These were the normal full length construct and the *reg* sequence that included a missense mutation at the initiator methionine residue (ATG→GTG). Stably transfected hamster insulinoma tumour (HIT) cell lines expressing wild type or mutant human *reg* were produced by electroporation of $5 \times 10^6$ cells in 0.8ml at 200V and 1070μF in incomplete medium containing 10μg of linearised pcDNA *reg* construct. Cells were grown in complete RPMI in 10cm dishes for 48 hours prior to selection with zeocin ($750\mu g/ml^{-1}$) (Invitrogen). Six colonies expressing each construct were cloned and screened for *reg* expression by northern blot.
Results
Reg expression in human gastric corpus.
Reg mRNA abundance was determined in hypergastrinaemic patients and compared to that of control subjects (figure 7.1). In patients with sustained hypergastrinaemia (plasma gastrin <400pM) reg mRNA abundance was increased approximately threefold. In addition, suppression of plasma gastrin by a 72hr infusion of the somatostatin analogue octreotide, was associated with a significant downregulation of HDC (n=2) and reg mRNA (n=1) in tumour biopsies. The effect of octreotide on plasma gastrin and HDC mRNA abundance are shown for patient JH in Figure 7.2. The effect of octreotide on reg expression was determined in patient KD and is shown in Figure 7.3.
Reg expression in endocrine and non-endocrine cell polyps
Total RNA extracted from biopsies that were confirmed histologically to be non-endocrine in type (fundic cystic gland hyperplasia) showed a reduction in reg mRNA abundance compared to controls. Conversely, reg mRNA abundance was greatly increased in some ECL cell nodules compared to the patient’s own corpus but not in all cases. In fact the abundance of reg mRNA in ECL cell tumour biopsies was very variable (Figure 7.4) and did not relate directly to plasma gastrin concentrations.
Fig 7.1 *Upper panel* Reg mRNA abundance in patients with hypergastrinaemia (plasma gastrin >400pM) compared to control subjects (plasma gastrin <100pM). There is a threefold increase in reg mRNA in patients with hypergastrinaemia (n = 10 for each group; * p<0.05, compared with control, Mann Whitney U test). *Lower panel* Representative Northern blot of reg mRNA in controls (lane 1-4) and hypergastrinaemic patients (lanes 5-8) demonstrating the increased reg mRNA in hypergastrinaemic corpus.
Fig 7.2 *Upper panel* Effect of octreotide infusion on plasma concentrations of amidated gastrin in patient JH. Plasma gastrin concentrations decreased during the infusion but remained above the upper limit of normal (indicated by dashed horizontal lines). The dose of octreotide is indicated by the black bar graph. *Lower panel* Northern blot for HDC mRNA in total RNA extracted from biopsies of carcinoid nodule (lane 1) and corpus (lane 2) pre-octreotide and from carcinoid nodule (lane 3) and corpus (lane 4) post-octreotide. HDC mRNA was reduced in both nodule and corpus following the octreotide infusion.
Fig 7.3 Upper panel Effect of octreotide infusion on plasma concentrations of amidated gastrin in patient KD. Plasma gastrin concentrations decreased during the infusion falling to within the normal range (indicated by dashed horizontal lines). The dose of octreotide is indicated by the black bar graph. Lower panel a) Northern blot for reg mRNA in total RNA extracted from biopsies of carcinoid nodule (lane 1) and corpus (lane 2) pre-octreotide and from carcinoid nodule (lane 3) and corpus (lane 4) post-octreotide. Reg mRNA was reduced in carcinoid nodule following the octreotide infusion. b) Northern blot of the same membrane for GAPDH mRNA.
Fig 7.4 Upper panel Reg mRNA abundance in fundic cystic gland polyps (FCGP n=5) compared to controls (n=6). Reg mRNA was reduced by $\approx$50% in the non-endocrine polyps (* p<0.05 Mann Whitney U test). To the right of the axis break is shown the reg mRNA abundance in ECL cell tumours in individual patients (initials on X axis) relative to their own corpus reg mRNA abundance normalised to 100 arbitrary units. Note the wide variation in tumour reg mRNA abundance Lower panel Representative Northern blot for reg mRNA in total RNA extracted from biopsies of normal corpus (lanes 1,3 and 5) non-endocrine cell polyps (lanes 2,4 and 6) hypergastrinaemic corpus (lane 7) and ECL cell nodule (lanes 8).
Reg Sequence and ECL cell tumours
The cDNA sequence of clones derived from RT/PCR of reg mRNA was determined in six hypergastrinaemic patients without ECL cell tumours and five patients with tumours. At least two clones were sequenced and data on the full sequence length obtained. All patients without ECL cell tumours had a normal reg sequence. Three of the five patients with nodules had at least one mutation of their reg sequence in either corpus or nodule. The sequence information from patients with ECL cell nodules is presented in Table 7.2. JH had multiple presumably acquired mutations identified from the biopsies of her nodules. PS had a single mutation identified in both corpus and nodule suggesting a germ line mutation. Interestingly, this mutation also occurred in JH and immediately appeared to suggest a pathophysiological mechanism. The mutation was a point mutation in the codon for the initiator methionine (ATG → GTG) and this would result in either the failure of initiation of protein translation or initiation of protein translation at a suitable upstream methionine residue. If protein translation commenced at the next upstream methionine, this would result in loss of the first eight amino acids of the signal peptide with a failure of the protein translated to enter the secretory pathway (Fig 7.5).
Table 7.2 Sequence data from cDNA transcripts of *reg* mRNA extracted from hypergastrinaemic patients with ECL cell tumours. Three of the five patients with nodules had at least one mutation in their *reg* gene identified from either nodule or corpus. All patients without nodules had normal *reg* sequence. PS had a single mutation identified from both nodule and corpus suggesting a germ line mutation. Patient JH had multiple, presumably acquired mutations identified from her tumour RNA. Note the same mutation in the initiator methionine residue occurred in both JH and PS.
Fig 7.5 Upper panel Hydrophobicity plot of normal Reg protein showing the hydrophobic tail of the signal peptide (bold line) and below this the first ten amino acids of the protein sequence. Lower panel Initiator methionine mutant reg showing the point mutation resulting in the initiator methionine residue being substituted by valine. Above this is shown the predicted hydrophobicity plot assuming protein translation occurs at the next upstream methionine. Note loss of the membrane spanning hydrophobic signal peptide.
Fig 7.6 Northern blot of *reg* mRNA abundance in 5 clones of HIT cells permanently transfected with mutant *reg* (lanes 1-5) and 6 clones of HIT cells permanently transfected with wild type *reg* (lanes 6-11). Although there is variability, all clonal cell lines express *reg* mRNA.
The data presented in this chapter demonstrate for the first time that *reg* expression in the human gastric corpus is increased in patients with hypergastrinaemia and may be suppressed in carcinoid tumour nodules by a 72hr infusion of octreotide. Perhaps of more significance was the demonstration of an association between mutations of *reg* and the development of carcinoid tumours in patients with hypergastrinaemia.
It is now generally accepted that gastrin has a trophic action on the ECL cell and high circulating levels of gastrin promotes ECL cell hyperplasia. Hypergastrinaemia induced by H$_2$-receptor antagonists leads to the progressive development of ECL cell hyperplasia and gastric carcinoid tumours in rodents (Hava 1986; Poynter and Selway, 1991; Wangberg et al., 1995). Anti-secretory therapy in man may lead to significant ECL cell hyperplasia (Eiselle et al., 1997) but no cases of carcinoid tumour have been reported. This may be because the hypergastrinaemia induced by such therapy is rarely high enough or sustained. Conversely, conditions associated with persistent and large elevations in plasma gastrin are associated with carcinoid tumour development.
Several genes expressed by ECL cells, including those for HDC, VMAT2 and chromogranin A, are upregulated by an elevated plasma gastrin (Dimaline et al., 1993a; Dimaline and Struthers, 1996; Dimaline et al., 1993b). The ability of gastrin to
stimulate new protein synthesis and growth of ECL cells can be blocked by specific gastrin/CCK-B receptor antagonists or by somatostatin and its analogues (Dimaline and Sandvik, 1991; Bordi et al., 1993; Ferraro et al., 1996). The data presented here confirm that HDC gene expression within the human gastric corpus mucosa was increased in patients with hypergastrinaemia and may be downregulated by a 72hr infusion of octreotide. Reg mRNA was downregulated in carcinoid nodule but not in normal corpus by octreotide (see below). In both cases, plasma gastrin fell during octreotide treatment although in the case of JH, plasma gastrin remained elevated above the upper limit of normal. Despite this there was profound depression in ECL cell function in the face of elevated plasma gastrin. This observation suggests that in addition to reducing the stimulus to ECL cell function mediated by gastrin, octreotide may itself exert direct inhibitory effects on ECL cell function. The ability of octreotide to influence gene expression within carcinoid tumour tissue suggests that this tissue is still under normal regulatory control.
In patient KD, reg expression changed in parallel with HDC. There was a marked increase in reg mRNA abundance in the patient’s carcinoid nodule compared to his corpus mucosa and in his nodule at least, this was reduced by the octreotide infusion. Perhaps surprisingly, octreotide appeared to increase reg mRNA in gastric corpus. One possible explanation for this may be the variation in tissue sampling (see below). The elevated reg mRNA in gastric carcinoid tissue seen in KD was clearly not the situation for all patients with nodules as the abundance of reg mRNA in nodule
tissue was highly variable between patients. There may be several reasons for this. In rodents it has been suggested that as carcinoid tumours develop and progress, they develop autonomy and become less dependent on gastrin as a trophic stimulus. The variability in *reg* expression in patients perhaps may indicate patients at a different stage of their disease. Alternatively, the degree of inflammation in the surrounding corpus was not taken into account. The ability of growth factors to potentiate the gastrin stimulated *reg* mRNA response also may explain the observed variability in nodule *reg* mRNA abundance. Hence, perhaps as CAG progresses and the parietal cell mass falls, the degree of inflammation also subsides. Tissue sampling introduces another unknown variable although this maybe more relevant to corpus mRNA as macroscopic nodules represent reproducible sites to biopsy. Although biopsies were taken from corpus mucosa that appeared macroscopically normal through an endoscope, the presence of microscopic carcinoids within biopsies that were processed for corpus mRNA cannot be excluded. This probably explains not only the variability in *reg* expression between patients but also the somewhat surprising increase in corpus *reg* mRNA in KD after his octreotide infusion. Furthermore, part or all of the increase in corpus *reg* mRNA in patients with hypergastrinaemia may be explained by an increase in ECL cell number as a consequence of hyperplasia and also due to concurrent atrophy of other mucosal cell types in patients with CAG.
Carcinoid tumours occur most commonly in patients with CAG, but are also associated with ZE and CRF. The incidence of carcinoid nodules in CAG is
approximately 5% and is similar to the incidence of carcinoid tumour occurring in the sporadic form of ZE syndrome. The incidence of carcinoid nodules in ZE associated with MEN1 syndrome rises to $\approx 30\%$ (Lehy et al., 1992). This suggests a genetic predisposition to carcinoid tumour development in this patient subgroup and is in keeping with the recently published observations of (Debelenko et al., 1997). They demonstrated that 75% of ZE patients with MEN1 syndrome have loss of heterozygosity at the MEN1 gene locus with deletion of the wild type allele. The MEN1 gene has recently been sequenced (Chandrasekharappa et al., 1997) and is the tumour suppressor gene that carries the inherited risk for developing endocrine tumours that are the feature of MEN1 syndrome. Of interest also however, was the observation of a low incidence of loss of heterozygosity at the MEN1 gene locus (1 of 6 patients) in patients with CAG and carcinoid nodules (Debelenko et al., 1997). This implies that another gene may be responsible for the risk of tumour development in these patients.
There is a growing body of evidence to suggest that the *reg* gene may play a role in the growth and differentiation of entero-pancreatic endocrine cells. It seemed reasonable to examine therefore whether mutations of *reg* may be associated with the development of carcinoid tumours in patients with hypergastrinaemia. The sequence data presented show that in 3 of 5 patients with hypergastrinaemia and carcinoid tumours there was at least one identifiable mutation in *reg* whereas in all hypergastrinaemic patients without carcinoid nodules the wild type *reg* coding
sequence was found. This identifies an association between mutations of *reg* and a risk of developing carcinoid tumours in the presence of hypergastrinaemia. However, a significant number of normal *reg* sequences were also obtained from both the corpus and the nodules of patients with tumours. Again this may reflect sampling error as each biopsy of tumour tissue will also have normal mucosal cells within it. To overcome this, microscopic dissection of fresh biopsy material to obtain clonal tumour cells will be required. It is also likely that the *reg* gene is not the only genetic mutation required or necessary to develop carcinoid tumours just as mutations of menin do not appear to be solely responsible for the development of carcinoid tumours in ZE associated with MEN1 (Debelenko *et al.*, 1997).
It seems likely that both acquired and germ line mutations of *reg* may occur in association with carcinoid tumours. Patient JH had multiple mutations of *reg* in several clones sequenced indicating multiple acquired mutations. Patient PS had only one mutation of his *reg* but it occurred in three of four sequences in both his corpus and nodule. This is suggestive of a germ line mutation. Interestingly the mutation identified in PS also occurred in patient JH and occurred at the methionine residue that initiates protein synthesis. The point mutation of A → G resulted in a loss of the initiator methionine, it being replaced by a valine residue. The possible results of this mutation include; i) a complete failure of protein translation, ii) protein translation occurs at the next upstream methionine that is “in frame”, iii) protein translation occurs at the most appropriate methionine residue that may be “out of frame”. In the case of the first and last instance either no protein or a
different protein will be produced with an expected loss of the normal function of *reg*. In the case of the second instance the protein produced will be missing eight amino acids of the N-terminal hydrophobic signal peptide. This should result in failure of the protein product to enter the secretory pathway as the signal peptide will be too short to span the hydrophobic membrane domain. The subsequent protein product may then either be degraded within the cytosol or act intracellularly. This could result theoretically in either a loss or gain in function.
According to the scanning model of mRNA translation proposed by Kozac (1978 and 1989), the sequence immediately adjacent to the initiator methionine codon determine the likelihood of protein translation occurring. The most important requirement is for a purine at position -3. In the absence of a purine at position -3, a G at +4 is essential for efficient translation (Kozak, 1989 & 1991; Iida and Masuda, 1996). Examination of the transfected mutant *reg* sequence fails to identify any alternative ATG codon with a surrounding sequence that meets these criteria. This would suggest that translation is unlikely to occur at an alternative start site and if it does occur at the next upstream methionine residue then translation will be inefficient. To confirm that the initiator methionine mutant *reg* resulted in failure of the translated protein product to enter the secretory pathway, constructs of human *reg* were inserted into a mammalian expression vector (pcDNA3-ZEO$^+$). The constructs were then permanently transfected into the hamster insulinoma tumour cell line (HIT) by Dr L Bishop. Cells were cultured
Fig 7.7 Photomicographs of HIT cells permanently transfected with either wild type (a) or mutant (b) *reg* constructs and stained sequentially with monoclonal anti-human *reg* antibody (1:2000; gift from Dr J Wand, Boston USA) and FITC conjugated horse anti mouse antibody (1:50). Note, only in the cells expressing wild type *reg* is there clearly discernible staining within the golgi apparatus and secretory granules. (x 400 approx., Courtesy of Colin Blackmore)
under zeocin selection and surviving cell lines were confirmed as expressing the *reg* gene by Northern blot of total mRNA extracted from $10^6$ cells (Fig 7.6). Clonal cell lines were then stained immuno-histochemically with a monoclonal anti-human reg antibody followed by FITC rabbit-anti-human secondary antibody. Intracellular distribution of reg protein was determined by immunofluorescence microscopy. (This work was performed by C. Blackmore, Dept of Physiology, Liverpool). The results of this work demonstrate that in the cell lines expressing wild type *reg* the protein enters the secretory pathway with clear staining of the Golgi apparatus and secretory granules (see photomicrograph 7.7a). In contrast, in cells expressing mutant *reg* there is no discernible pattern of staining (see photomicrograph 7.7b). Although this suggests that the protein either is not produced or is rapidly degraded, it remains possible that the shortened signal peptide remains attached to the N-terminal of the protein product and prevents the anti-human reg antibody from recognising the normal N-terminus of the reg protein. The specificity of the human reg antibody is being determined.
**Summary**
The *reg* gene is known to be present in the human stomach and there is an increasing body of evidence to suggest that it may play a role in the growth and differentiation of endocrine cells in the gut including the ECL cell. Gastrin is also known to influence enteropancreatic endocrine cell differentiation and has a trophic effect on the ECL cell. The data presented demonstrate that plasma gastrin influences *reg* gene expression in human gastric corpus mucosa. In addition, the development of
gastric carcinoid tumours in patients with hypergastrinaemia is associated with mutations of the *reg* gene. This raises the possibility of identifying patients at risk of carcinoid tumour development in the presence of sustained hypergastrinaemia.
Chapter Eight
Conclusions and Implications
The main objectives of the work contained within this thesis were to examine the role of antral neurons in the control of gastric emptying and the mechanisms that regulate gastrin release from G cells of the antrum, using a model of antral denervation in the rat. In addition, the significance of *reg* gene expression and carcinoid tumour development was examined by studying the relationships between gastrin and *reg* expression in the ECL-cells of the rat, in human gastric corpus and in a rat pancreatic cell line (AR42J).
**Conclusions**
1. Serosal application of BAC to the gastric antrum effectively lesions all myenteric and spinal afferent fibres of the antrum and the spinal afferent innervation of the gastric corpus. The intrinsic innervation of the proximal stomach is unaffected. Such chemically induced denervation provides a model for experimental studies of the intrinsic innervation of antral muscle and the antral mucosal endocrine cells of which the major cell of interest is the G cell.
2. In the antrally denervated rat there is retention of solid within the stomach that is apparent after a 48hr fast. In antrally denervated rats fitted with a gastric fistula, the emptying of liquid test meals such as saline, acid and peptone was delayed. However, the CCK-dependent inhibition of gastric emptying of peptone was preserved after antral denervation by BAC. It appears that innervation of the antral musculature is
essential for normal gastric emptying of liquids and solids but not CCK-mediated inhibition of gastric emptying.
3. In the intact rat, antral neurons normally inhibit gastrin release in response to non-nutrient distension of the gastric corpus and luminal nutrient produces gastrin release via activation of neurons that release GRP. Following antral denervation, post-prandial gastrin release was maintained but was no longer dependent on either antral neurons or release of GRP. Moreover, non-nutrient distension of the stomach becomes an adequate stimulus for gastrin release.
4. In AR42J cells *reg* mRNA expression was stimulated, in a dose-dependent manner, by amidated gastrin acting through the CCK-B receptor. *Reg* expression in rat and human gastric corpus was also dependent on plasma gastrin concentrations. Mutations of *reg* (a putative regulator of differentiation of gut endocrine cells) were associated with gastric carcinoid tumour development in patients with hypergastrinaemia. A major limitation of the studies thus far are that mRNA only has been measured and quantification of the protein product has not been performed.
**Implications and future studies**
Myenteric denervation by BAC was described first in the rat colon (Sato *et al.*, 1988), where its serosal application produced a phenotype similar to that of Hirschsprung’s disease. The effects of small intestinal denervation by BAC have also
been described both by serosal application (myenteric plexus ablation) and luminal application (submucosal plexus ablation). The particular advantages that have been described for BAC are that it can be applied to a strictly defined area of the gut and its application lesions only the myenteric intrinsic neurons and the extrinsic innervation of the region when applied to the serosal surface. Clearly, the effects of BAC on extrinsic afferent fibres may be more widespread depending on the anatomy of innervation. Nevertheless the lesion produced relies upon the ability of intestinal smooth muscle to recover whilst neuronal loss persists. There is a significant inflammatory element to the lesion during the first 7 days (See et al., 1988). Thereafter, histologically inflammation subsides but muscle hypertrophy begins, although active stress generation by hypertrophied muscle does not exceed normal values until after the second week postoperatively (Herman and Bass, 1990).
Most of the studies performed in this thesis used animals towards the end of their second post-operative week. However, the possible confounding factors of continued inflammation and antro-pyloric muscle hypertrophy could conceivably contribute to some of the observations described. In addition, because of a lack of a neuropeptide marker for extrinsic vagal fibres, no attempt was made to determine the extent of vagal damage induced by BAC. Although antral BAC treatment almost certainly disrupts the antral vagal innervation, the preservation of CCK-mediated delay in gastric emptying suggests that the duodenal vagal afferent and gastric vagal efferent fibres were intact.
Inflammation of the gastric mucosa is known to result in increased levels of growth factors such as TGF-α (Polk et al., 1992). Growth factors such as TGF-α and EGF have been shown to both stimulate gastrin release and inhibit acid secretion (Konturek et al., 1984; Rhodes et al., 1986). Furthermore, chronic gastric retention and delayed gastric emptying are well recognised complications of hypertrophic pyloric stenosis. In fact, chronic gastric retention is also associated with fasting and meal-stimulated hypergastrinaemia in the myenteric neuropathy that occurs as a result of *Trypanosomal* infection (Oliveira et al., 1980; Troncon et al., 1984).
Perhaps of more immediate relevance may be the mucosal inflammation produced by *H.pylori*. There have been reports that antral gastritis due to *H.pylori* may also induce loss of mucosal nerve fibres and hence lead to denervation (Stead et al., 1996). Interestingly, and in keeping with the observations here, *H. pylori* infection produces basal and meal stimulated increases in plasma gastrin (Levi et al., 1989; McKoll et al., 1989; Tarnasky et al., 1993) and reduces antral somatostatin mRNA levels (Kaneko et al., 1990; Moss et al., 1992) although gastrin mRNA apparently is unaffected. The exaggerated response to GRP in *H. pylori* infection (Graham et al., 1991; McKoll and El-Omar, 1995) is in keeping with increased sensitivity seen following chronic denervation. The potential role of inflammation and growth factors in the BAC-treated rat was not examined.
Of more concern however, may be the potential relationship between trophic factors, elevated plasma gastrin and the growth of gastric mucosal cells given the association between *H. pylori* and the development of gastric tumours. Certainly, the
marked potentiation by EGF of the gastrin-stimulated expression of *reg* in AR42J cells is preliminary evidence for a similar potentiation occurring in gastric mucosal endocrine cells or their precursors. The wide variability of *reg* expression in hypergastrinaemic patients may indicate different degrees of mucosal inflammation, independent of the level of their plasma gastrin and was a variable that was not controlled for in these studies.
Finally, the role of gastrin and growth factors in the control of differentiation of gastric epithelial (endocrine and non-endocrine) cells is likely to become of increasing importance given the relationship between these factors, PPI therapy, *H. pylori* gastritis and gastric malignancy. The possible role of *reg Iα* as a tumour suppressor gene for endocrine cells of the stomach remains to be clarified but further studies of the *reg* gene family are likely to provide new insights into the cellular and molecular mechanisms that regulate the fundamental processes that lead to the differentiation and maintenance of cell phenotype. The role of *reg Iα* in the stomach and in AR42J cells provides a model system for testing the following hypotheses:
i. expression of the *reg* gene is regulated by gastrin,
ii. expression of the *reg* gene influences the progressive differentiation of precursor endocrine cells towards in the gastric mucosa,
iii. mutations of *reg* result in failure of differentiation and may cause endocrine cell tumour development in the gastric mucosa.
The following provides an experimental outline of the future studies that will test these hypotheses:
i) Does gastrin directly control *reg* expression?
a) Using the same techniques of tissue culture, mRNA extraction and Northern blot it would be possible to study gastrin receptor mediated events in AR42J cells using recognised gastrin receptor agonists, antagonists and inhibitors. The effects of somatostatin on *reg* expression could also be examined.
b) Gastrin response elements in the promoter region of human *reg* could be identified by transfection of AR42-J cells with different constructs of the promoter region coupled to a reporter luciferase gene. Exact localisation of the relevant sequences would then require scanning mutagenesis and DNA footprinting.
ii) Is an intact *reg* essential for normal differentiation of an endocrine cell?
a) The ability of *reg* to induce differentiation will be assessed. The ability of recombinant reg protein to induce differentiation in responsive precursor cell lines will be studied and in addition, transfection of undifferentiated but responsive cells with wild type *reg* will also be performed. Expression of markers of differentiation will be assessed following stimulation with gastrin.
b) Immunohistochemical markers of differentiation (CGA, HDC) and proliferation (PCNA, MIB) will be correlated with *reg* expression in precursor stem cells and mature ECL cells in patients with and without hypergastrinaemia.
iii Does a *reg* mutation produce abnormal physiology?
a) The normal biosynthesis of the Reg protein will be assessed *in vitro* in gastric biopsies by pulse chase experiments incorporating $^{35}$S methionine. Comparisons will be made between patients with and without mutations of *reg*.
b). The processing of wild type and mutant *reg* by Hamster Insulinoma Tumour (HIT) cells will be assessed by morphological and biosynthetic analysis. We have already permanently transfected wild type and mutant *reg* into HIT cells that are capable of processing and secreting transfected proteins.
c). Genomic DNA from hypergastrinaemic subjects with and without ECL cell dysfunction will be screened for mutant *reg*.
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WECKBECKER G, RAULF F, TOLSCAVI L & BURNS C. (1996) Potentiation of the anti-proliferative effects of anti-cancer drugs by octreotide in vitro and in vivo. Digestion 57:22-28.
WEIGART N, MADAUS S, ALEXIOU C, SCHEPP W, COY DH, CLASSEN M & SCHUSDZIARRA V. (1992) Effect of bombesin antagonist D-Phe$^6$-BN(6-13)OMe on vagally induced gastrin release from perfused rat stomach. Life Sci 52: 725-732.
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YAMAGISHI T & DEBAS HT. (1978) Cholecystokinin inhibits gastric emptying by acting on both proximal stomach and pylorus. Am J Physiol 234: E375-E378.
YAN WM & YOUTHER ML. (1982) Direct evidence for the release of acetylcholine from the myenteric plexus of guinea pig small intestine by substance P. Eur J Pharmacol 81: 665-668.
YOKOYAMA S & NORTH RA. (1983) Electrical activity of longitudinal and circular muscle during peristalsis. Am J Physiol 244: G83-G88.
YUAN S, ROSENBERG L, PARASKEVAS S, AGAPITOS D & DRUGGED W P. (1996) Transdifferentiation of human islets to pancreatic ductal cells in collagen matrix culture. Differentiation 61: 67-75
ZENILMAN ME, MAGNUSON TH, PERFETTI R, CHEN J & SCHULDINER AR. (1997) Pancreatic Reg gene expression is inhibited during cellular differentiation. Annals Surgery 225: 327-332.
ZENILMAN ME, MAGNUSON TH, SWINSON K, EGAN J, PERFETTI R & SHULDINER AR. (1996) Pancreatic thread protein is mitogenic to pancreatic-derived cells in culture. Gastroenterology 110: 1208-1214.
ZENILMAN ME, PERFETTI R, SWINSON K, MAGNUSON T & SHULDINER MD. (1996) Pancreatic regeneration (reg) gene expression in a rat model of islet hyperplasia. Surgery 119: 576-584.
ZHANG Z, HOCKER M, KOH T & WANG TC. (1996) The human histidine decarboxylase promoter is regulated by gastrin and phorbol 12-myristate 13-acetate through a downstream cis-acting element. J Biol Chem 271: 14188-14197.
ZHOU W, POVOSKI SP, LONGNECKER DS & BELL RH. (1992) Novel expression of gastrin (cholecystokinin-B) receptors in azaserine-induced rat pancreatic carcinoma: receptor determination and characterisation. Cancer Res 52: 6905-6911.
ZUCOLOTO S, SILVA JC, OLIVIERA SM & MUCCILLO G. (1991) The chronological relationship between the thickening of smooth muscle, epithelial cell proliferation and myenteric neural denervation in the rat jejunum. Cell Prolif 24: 15-20.
Acknowledgements
I would like to acknowledge Professor Graham Dockray for his supervision of my project and for his advice, support and encouragement over the last three years. I also wish to thank Professor David Thompson and Drs. Rod Dimaline and Andrea Varro for their help and collaboration. I am grateful for the technical assistance of Mrs Cath MacLean, Mark Houghton and particularly David Trafford whose patience and willingness to help seemed infinite. I also would like to recognise the MRC for their financial support of this research.
On a personal note, I wish to offer special thanks to my colleagues in the Department of Physiology (University of Liverpool) for their helpful advice and friendship and in particular to Lisa Bishop, PJ Noble, Atsushi Murai, Peter Djali and Colin Blackmore each of whom in their own way have made the last three years unforgettable.
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AN ORDINANCE APPROVING, ADOPTING AND ENACTING AMERICAN LEGAL PUBLISHING'S OHIO BASIC CODE, 2017 EDITION, AS THE CODE OF ORDINANCES FOR THE MUNICIPALITY OF Amanda Village, OHIO, AND DECLARING AN EMERGENCY.
WHEREAS, the present general and permanent ordinances of the municipality are inadequately arranged and classified and are insufficient in form and substance for the complete preservation of the public peace, health, safety and general welfare of the municipality and for the proper conduct of its affairs.
WHEREAS, American Legal Publishing Corporation publishes a Code of Ordinances suitable for adoption by municipalities in Ohio.
WHEREAS, it is necessary to provide for the usual daily operation of the municipality and for the immediate preservation of the public peace, health, safety and general welfare of the municipality that this ordinance take effect at an early date.
NOW, THEREFORE, BE IT ORDAINED BY THE LEGISLATIVE AUTHORITY OF THE MUNICIPALITY OF Amanda Village, OHIO:
Section 1. American Legal Publishing's Ohio Basic Code, 2017 Edition, as reviewed and approved by the Legislative Authority, is hereby adopted and enacted. Any prior version of the Ohio Basic Code which may have been previously adopted by the municipality is hereby repealed as obsolete and is hereby replaced in its entirety by this Ohio Basic Code, 2017 Edition.
Section 2. One copy of American Legal Publishing's Ohio Basic Code, 2017 Edition, certified as correct by the Mayor and Clerk of the Legislative Authority, as required by Ohio Revised Code § 731.23, shall be kept in its initial form on file in the office of the Clerk of the municipality and retained as a permanent ordinance record of the municipality. The Clerk of the municipality is authorized and directed to publish a summary of all new matters contained in the Code of Ordinances as required by Ohio Revised Code § 731.23. Such summary is attached hereto and marked as "Exhibit A".
Section 3. All ordinances and resolutions or parts thereof which are in conflict or inconsistent with any provision of the Ohio Basic Code, 2017 Edition, as adopted in Section 1 hereof, are hereby repealed as of the effective date of this ordinance, except as follows:
(A) The enactment of the Ohio Basic Code, 2017 Edition, shall not be construed to affect a right or liability accrued or incurred under any legislative provision prior to the effective date of such enactment, or an action or proceeding for the enforcement of such right or liability. Such enactment shall not be construed to relieve any person from punishment for an act committed in violation of any such legislative provision, nor to affect an indictment or prosecution therefor. For such purposes, any such legislative provision shall continue in full force notwithstanding its repeal for the purpose of revision and codification.
(B) The repeal provided above shall not affect:
(1) The grant or creation of a franchise, license, right, easement or privilege;
(2) The purchase, sale, lease or transfer of property;
(3) The appropriation or expenditure of money or promise or guarantee of payment;
(4) The assumption of any contract or obligation;
(5) The issuance and delivery of any bonds, obligations or other instruments of indebtedness;
(6) The levy or imposition of taxes, assessments or charges;
(7) The establishment, naming, vacating or grade level of any street or public way;
(8) The dedication of property or plat approval;
(9) The annexation or detachment of territory;
(10) Any legislation enacted subsequent to the adoption of this ordinance.
(11) Any legislation specifically superseding the provision of the Ohio Basic Code.
Section 4. Whenever reference is made in any documents, publications, or signs of the municipality, including but not limited to traffic tickets and traffic-control signs, to a section as it existed in a former edition of the Ohio Basic Code, the reference shall extend and apply to the section referred to as subsequently amended, revised, recodified, or renumbered.
Section 5. This ordinance is declared to be an emergency measure necessary for the immediate preservation of the peace, health, safety and general welfare of the people of this municipality, and shall take effect at the earliest date provided by law.
Date Passed: 2-6-17
Attest:
Mayor
Clerk of the Legislative Authority
Notice is hereby given that on the 6th day of February, 2017, there was enacted by the Legislative Authority of the Municipality of Amanda Village, Ohio, an ordinance entitled “An Ordinance Approving, Adopting and Enacting American Legal Publishing’s Ohio Basic Code, 2017 Edition, as the Code of Ordinances for the Municipality of Amanda Village, Ohio.”
A summary of the subjects, including all new matters contained in the Code of Ordinances, as adopted, are as follows. The majority of Basic Code provisions are based directly on state law.
**TITLE I: GENERAL PROVISIONS**
**Chapter 10: General Provisions**
| Section | Title |
|---------|--------------------------------------------|
| 10.01 | Short titles |
| 10.02 | Definitions |
| 10.03 | Rules of construction |
| 10.04 | Revivor; effect of amendment or repeal |
| 10.05 | Construction of section references |
| 10.06 | Conflicting provisions |
| 10.07 | Severability |
| 10.08 | Reference to offices |
| 10.09 | Errors and omissions |
| 10.10 | Ordinances repealed |
| 10.11 | Ordinances unaffected |
| 10.12 | Ordinances saved |
| 10.13 | Application to future ordinances |
| 10.14 | Interpretation |
| 10.15 | Amendments to code; amendatory language |
| 10.16 | Statutory references |
| 10.17 | Preservation of penalties, offenses, rights and liabilities |
| 10.18 | Determination of legislative intent |
| 10.99 | General penalty |
**TITLE III: ADMINISTRATION**
**Chapter 30: General Provisions**
| Section | Title |
|---------|--------------------------------------------|
| 30.01 | Application of Title III |
| 30.02 | Qualifications; oaths |
| 30.03 | Bonds of officers and employees; amount |
| 30.04 | Additional bond; where bonds recorded and kept |
| 30.05 | Approval of bonds |
| 30.06 | Sufficiency of form of bond |
| 30.07 | Filling vacancies in offices |
| 30.08 | Public records available |
| 30.09 | Records Commission |
| 30.10 | Meetings of public bodies to be open; exceptions; notice |
| 30.11 | Municipal officers may attend conference or convention; expenses |
| 30.12 | Residency requirements prohibited; exceptions |
**Chapter 31: Executive Authority**
**General Provisions**
| Section | Title |
|---------|--------------------------------------------|
| 31.001 | Executive power; where vested |
**Mayor**
31.015 Term of Mayor; power and duties
31.016 General duties of the Mayor
31.017 Communications to the Legislative Authority
31.018 Protest against excess of expenditures
31.019 Supervision of conduct of officers
31.020 Annual report to the Legislative Authority
31.021 Mayor to file charges against delinquent officers
31.022 Vacancies in office of Mayor
31.023 Disposition of fines and other moneys
**Clerk**
31.040 Election, term, qualifications of the Clerk
31.041 Powers and duties of Clerk
31.042 Books and accounts; merger of offices
31.043 Seal of Clerk
31.044 Combined offices of Clerk and Treasurer; Fiscal Officer
**Treasurer**
31.060 Election, term, qualifications of the Treasurer
31.061 Accounts of Treasurer
31.062 Powers and duties
31.063 Quarterly account; annual report
31.064 Receipt and disbursement of funds
31.065 Duty of delivering money and property
**Street Commissioner**
31.080 Qualifications
31.081 General duties
31.082 Assistants
**Other Officials**
31.100 Legal counsel
31.101 Administrator
31.102 Board of Trustees of Public Affairs
31.103 Fire Engineer, Engineer and Superintendent of Markets
---
**Chapter 32: Legislative Authority**
**General Provisions**
32.001 Members of the Legislative Authority; election; terms of office
32.002 President Pro Tempore; employees
32.003 Vacancy when President Pro Tempore becomes Mayor
32.004 Qualifications of members of the Legislative Authority
32.005 Compensation and bonds of municipal officers and employees
32.006 Vacancy
32.007 Judge of election and qualification of members; quorum and special meetings
32.008 Rules; journal; expulsion of members
32.009 Meetings
32.010 General powers
32.011 Failure to take oath or give bond
32.012 Notice when new bond required
32.013 Care, supervision and management of public institutions
Contracts, Bids and Proceedings
32.025 Contracts by the Legislative Authority or Administrator
32.026 Bids and proceedings
32.027 Alterations or modifications of contract
32.028 Contract restrictions
32.029 Award to lowest responsive and responsible bidder
Ordinances and Resolutions
32.040 Ordinances and resolutions as evidence
32.041 Passage procedure
32.042 Style of ordinances
32.043 Subject and amendment of ordinances and resolutions
32.044 Authentication and recording of ordinances and resolutions
32.045 Publication of ordinances and resolutions; proof of publication and circulation
32.046 Notice for proposed amendments to the municipal Charter
32.047 Times of publication required
32.048 Publication and certification of ordinances in book form
32.049 Adoption of technical ordinances and codes
32.050 Certificate of Clerk as to publication
32.051 Publication when no newspaper published in municipality
32.052 Effect of not making publication
32.053 Ordinances providing for appropriations or street improvements; emergency ordinances
Initiative and Referendum
32.070 Initiative petitions
32.071 Referendum petitions
32.072 More than one ordinance required; application of subchapter
32.073 Presentation of petitions
32.074 Copy of proposed ordinance or measure to be filed with Clerk
32.075 Words to be printed in red
32.076 Designation of committee filing petition; public inspection of petitions; ordinances passed or repealed prior to election
32.077 Itemized statement by petition circulator
32.078 Prohibited practices relative to petitions
32.079 Accepting premiums for signing
32.080 Threats in securing signatures
32.081 Application of subchapter if Charter adopted
Chapter 33: Judicial Authority
General Provisions
33.01 Jurisdiction in ordinance cases and traffic violations
33.02 Powers of Mayor and Mayor's Court Magistrate in criminal matters
33.03 Duties of Mayor and Mayor's Court Magistrate; fees; office; seal
33.04 Mayor's Court Magistrate
33.05 Powers to suspend driver's license in OVI cases
Contempt of Court
33.20 Summary punishment for contempt
33.21 Acts in contempt of court
33.22 Hearing
33.23 Contempt action for failure to pay support, failure to comply or interference with a visitation order; summons
33.24 Right of accused to bail
33.25 Hearing on contempt; penalties; support orders; failure to withhold or deduct money pursuant to support order
33.26 Imprisonment until order obeyed
33.27 Proceedings when party released on bail fails to appear
33.28 Release of prisoner committed for contempt
33.29 Judgment final
33.30 Alternative remedy
Chapter 34: Police Department
Section
34.01 Marshal and Police Chief synonymous
34.02 Appointment of Marshal
34.03 Deputy marshals and police officers
34.04 Auxiliary police units
34.05 Offenses affecting employment of law enforcement officers; probationary period; final appointment
34.06 Removal proceedings; suspension; appeals
34.07 General powers
34.08 Powers and duties of Marshal
34.09 Disposition of fines and penalties
34.10 Property recovered by police
34.11 Disposition to claimant
34.12 Sale of unclaimed property; disposition of proceeds
34.13 Expenses of storage and sale; notice
34.14 Contracts for police protection; nonresident service without contract
34.15 Peace officer administering oaths; acknowledging complaints, summonses, affidavits and returns of court orders
Chapter 35: Fire Department
Section
General Provisions
35.01 Municipal fire regulations; fire department
35.02 Fire Chief; Fire Prevention Officer; employment of firefighters; criminal records check for firefighters
35.03 Schooling of officers and firefighters of fire department
35.04 Legislative Authority may purchase engines and equipment
35.05 Buildings for department
35.06 Records
35.07 Maximum consecutive hours for firefighters on duty
35.08 Investigation of cause of fire
35.09 Right to examine buildings, premises, and vehicles
35.10 Burning buildings for firefighting instruction or research
35.11 Impersonating fire safety inspector
35.12 Standards for equipment
35.13 Persons entitled to be known as firefighters
35.14 Firefighting and emergency services agreements
35.15 Regulation of construction in fire limits
Volunteer Firefighters’ Dependents Fund Board
35.30 Definitions
35.31 Establishment
35.32 Membership; vacancies
35.33 Election and term of members
35.34 Organization; rules and regulations; roster
35.35 Compensation and expenses of Board; legal advisor
Chapter 36: Civil Actions Against the Municipality
Section
36.01 Definitions
36.02 Nonliability of municipality; exceptions
36.03 Defenses and immunities
36.04 Limitation of actions
36.05 Damages
36.06 Satisfaction of judgments
36.07 Provision of employees’ defense; consent judgments
36.08 Liability insurance
36.09 Certain actions unaffected
36.10 Certain charges against municipal officers filed with Probate Judge; proceedings
TITLE VII: TRAFFIC CODE
Chapter 70: General Provisions
General Provisions
70.01 Definitions
70.02 Compliance with order of police officer
70.03 Emergency vehicles to proceed cautiously past red or stop signal
70.04 Exceptions generally; emergency, public safety and coroner vehicles exempt
70.05 Persons riding or driving animals upon roadways
70.06 Prohibitions against pedestrians and slow-moving vehicles on freeways
70.07 Use of private property for vehicular travel
70.08 Names of persons damaging real property by operation of vehicle to be provided to owner
70.09 Limited access highways; barriers along; vehicles to enter and leave at designated intersections
70.10 Through highways
70.11 Officer may remove ignition key
70.12 Removal of vehicles after accidents
Traffic-Control Devices
70.30 Obeying traffic-control devices
70.31 Signal lights
70.32 Signals over reversible lanes
70.33 Ambiguous or non-working traffic signals
70.34 Pedestrian-control signals
70.35 Unauthorized signs and signals prohibited
70.36 Alteration, defacement, or removal prohibited
70.37 Unauthorized possession or sale of devices
70.38 Signal preemption devices; prohibitions
70.99 Penalty
Chapter 71: Licensing Provisions
Motor Vehicle Licensing
71.01 Display of license plates or validation stickers; registration
71.02 Improper use of noncommercial motor vehicle
71.03 Operating motor vehicle ordered immobilized; forfeiture
71.04 Operation or sale without certificate of title
71.05 Display of certificate of registration
71.06 Use of unauthorized plates
71.07 Operating without dealer or manufacturer license plates
Driver’s Licenses
71.20 Prohibited acts
71.21 Permitting minor to operate vehicle prohibited; temporary instruction permit; probationary license
71.22 License required as driver or commercial driver on public or private property; nonresident exemption
71.23 Employment of a minor to operate a taxicab prohibited
71.24 Restriction against owner lending vehicle for use of another
71.25 Suspension of driver’s licenses; license suspended by court of record
71.26 Display of license
71.27 Prohibition against false statements
71.28 Driving under suspension or in violation of license restriction
71.29 Operating motor vehicle or motorcycle without valid license
71.30 Driving under OVI suspension
71.31 Driving under financial responsibility law suspension or cancellation; driving under a nonpayment of judgment suspension
71.32 Failure to reinstate license
**Commercial Driver's Licenses**
71.45 Definitions
71.46 Use of actual gross weight in lieu of rating
71.47 Prohibited acts
71.48 Prerequisites to operation of commercial motor vehicle
71.49 Physical qualification to operate commercial motor vehicles
71.50 Criminal offenses
71.51 Application of federal regulations
71.52 Employment of drivers of commercial vehicles
71.99 Penalty
**Chapter 72: Traffic Rules**
**General Provisions**
72.001 Lanes of travel upon roadways
72.002 Driving through safety zone
72.003 Vehicles traveling in opposite directions
72.004 Rules governing overtaking and passing of vehicles
72.005 Permission to overtake and pass on the right
72.006 Driving to left of center line
72.007 Prohibition against driving upon left side of roadway
72.008 Hazardous zones
72.009 One-way highways and rotary traffic islands
72.010 Rules for driving in marked lanes
72.011 Space between moving vehicles
72.012 Divided roadways
72.013 Rules for turns at intersections
72.014 U-turns and turning in roadway prohibited
72.015 Starting and backing vehicles
72.016 Turn and stop signals
72.017 Hand and arm signals
**Right-of-Way**
72.030 Right-of-way at intersections
72.031 Right-of-way when turning left
72.032 Right-of-way at through highways; stop signs; yield signs
72.033 Stop at sidewalk area; stop signs on private roads and driveways
72.034 Right-of-way on public highway
72.035 Pedestrian on sidewalk has right-of-way
72.036 Right-of-way of public safety vehicles
72.037 Funeral procession has right-of-way
72.038 Pedestrians yield right-of-way to public safety vehicle
72.039 Pedestrian on crosswalk has right-of-way
72.040 Right-of-way yielded to blind person
72.041 Right-of-way yielded by pedestrian
**Pedestrians**
72.055 Pedestrian movement in crosswalks
72.056 Pedestrian walking along highway
72.057 Prohibition against soliciting rides; riding on outside of vehicle
72.058 Pedestrian on bridge or railroad crossing
72.059 Persons operating motorized wheelchairs
72.060 Intoxicated or drugged pedestrian hazard on highway
72.061 Operation of electric personal assistive mobility devices
**Grade Crossings**
72.075 Stop signs at grade crossings
72.076 Driving vehicle across railroad grade crossing
72.077 Vehicles required to stop at grade crossings
72.078 Slow-moving vehicles or equipment crossing railroad tracks
**School Buses**
72.090 Regulations concerning school buses
72.091 Violation of regulations; report; investigation; citation; warning
72.092 Restrictions on the operation of school buses
72.093 School bus inspection
72.094 School bus not used for school purposes
72.095 Licensing by Department of Public Safety
72.096 Registration and identification of school buses
72.097 School bus marking
72.098 Flashing light signal lamps
72.099 Occupant restraining device for operator
**Prohibitions**
72.115 Obstruction and interference affecting view and control of driver
72.116 Occupying travel trailer while in motion
72.117 Driving upon closed highway prohibited
72.118 Driving upon sidewalk area or paths exclusively for bicycles
72.119 Obstructing passage of other vehicles
72.120 Following an emergency or public vehicle prohibited; approaching stationary public safety vehicle with caution
72.121 Driving over unprotected fire hose
72.122 Placing injurious material on highway or depositing litter from motor vehicle
72.123 Transporting child not in child-restraint system prohibited
72.124 Occupant restraining devices
72.125 Use of engine brakes prohibited
72.126 Operating motor vehicle while wearing earphones or earplugs
72.127 Chauffeured limousines and livery services
72.128 Operating traction engine upon improved highway
72.129 Cracking exhaust noises; peeling out
72.130 Shortcutting across private property
72.131 Texting while driving prohibited
72.132 Use of electronic wireless communication devices by minors or probationary drivers while driving prohibited
**Chapter 73: Motor Vehicle Crimes**
**General Provisions**
73.01 Driving under the influence of alcohol or drugs
73.02 Implied consent
73.03 Physical control of vehicle while under the influence
73.04 Driving commercial vehicle with impaired alertness or ability; use of drugs
73.05 Reckless operation of vehicles
73.06 Reckless operation off streets and highways; competitive operation
73.07 Operator to be in reasonable control
73.08 Immobilizing or disabling device violation
73.09 Street racing defined; prohibited on public highways
73.10 Speed limits
73.11 Slow speed or stopping
73.12 Emergency vehicles excepted from speed limitation
73.13 Speed regulations on bridges
73.14 Presenting false name or information to officer
73.15 Prohibition against resisting officer
73.16 Operation restricted for mini-trucks and low-speed, under-speed, or utility vehicles
**Stopping After Accident**
73.30 Failure to stop after accident
73.31 Stopping after accident on other than public roads or highways
73.32 Accident involving damage to realty
73.33 Failure to report accident
Chapter 74: Equipment and Loads
**Equipment**
74.01 Unsafe vehicles, prohibition against operation
74.02 Bumpers on motor vehicles
74.03 Lighted lights required
74.04 Headlights
74.05 Tail lights and illumination of rear license plate
74.06 Red reflectors required
74.07 Safety lighting of commercial vehicles
74.08 Stoplight regulations
74.09 Obscured lights on vehicles
74.10 Red light or flag required
74.11 Lights on parked vehicles
74.12 Lights and emblem on slow-moving vehicles; lights and reflectors on multi-wheel agricultural tractors or farm machinery
74.13 Spotlight and auxiliary driving lights
74.14 Cowl, fender, and back-up lights
74.15 Two lights displayed
74.16 Headlights required
74.17 Lights of less intensity
74.18 Number of lights permitted; red and flashing lights
74.19 Standards for lights on snow removal equipment and oversize vehicles
74.20 Flashing lights permitted for certain types of vehicles
74.21 Lights and sign on transportation for preschool children
74.22 Focus and aim of headlights
74.23 Brake equipment; specifications
74.24 Brake fluid
74.25 Minimum standards for brakes and components
74.26 Horns, sirens, and warning devices
74.27 Mufflers; excessive smoke or gas
74.28 Rearview mirrors
74.29 Windshields and wipers
74.30 Solid tire requirements
74.31 Requirements for safety glass in motor vehicles; use of tinted glass or reflectorized material
74.32 Directional signals
74.33 Installation and sale of seat safety belts required; definition
74.34 Requirements for extra signal equipment
74.35 Display of warning devices on disabled vehicles
74.36 Requirements for vehicles transporting explosives
74.37 Studded tires; seasonal use permitted
74.38 Safety inspection decals for buses
74.39 Air bags
**Loads**
74.50 Permit required to exceed load limits
74.51 Limitation of load extension on left side of vehicle
74.52 All loads shall be properly secured
74.53 Towing requirements; exception to size and weight restrictions
74.54 Weighing of vehicle; removal of excess load
74.55 Operation of vehicle exceeding weight limits prohibited
74.56 Load limits
74.57 Maximum width, height, and length
74.58 Statement of gross vehicle weight
74.59 Wheel protectors required on heavy commercial vehicles
74.60 Liability for damages; prosecution; application of monies
74.61 Weight exceptions for certain vehicles
Chapter 75: Bicycles, Motorcycles and Off-Road Vehicles
General Provisions
75.01 Bicycles; application of Title VII
75.02 Operation of motorized bicycle
75.03 Rules for bicycles, motorcycles, and snowmobiles
75.04 Prohibition against attaching bicycles and sleds to vehicles
75.05 Riding bicycles; motorcycles abreast
75.06 Equipment of bicycles
Snowmobiles, Off-Highway Motorcycles, and All-Purpose Vehicles
75.25 Definitions
75.26 Equipment
75.27 Code application; prohibited operation
75.28 Permitted operation
75.29 Licensing requirements of operator
75.30 Maintenance of vehicles for hire
75.31 Accident reports
75.32 Impounding of vehicle
75.33 Local control within police power
75.34 Registration of vehicles
75.35 Certificate of title; prohibitions
Chapter 76: Parking Regulations
Section
76.01 Prohibition against parking on highways
76.02 Condition when motor vehicle left unattended
76.03 Police may remove illegally parked vehicle
76.04 Parking prohibitions
76.05 Parking near curb; privileges for persons with disabilities
76.06 Parking on private property in violation of posted prohibition
76.07 Selling, washing or repairing vehicle upon roadway
76.08 Truck loading zones
76.09 Bus stops and taxicab stands
76.10 Parking in alleys and narrow streets; exceptions
76.11 Registered owner prima facie liable for unlawful parking
76.12 Waiver
TITLE IX: GENERAL REGULATIONS
Chapter 90: Animals
Animals Running at Large
90.01 Dogs or other animals running at large; nuisance, dangerous or vicious dogs; hearings
90.02 Confining animal found at large; publication of notice; lien
90.03 Unavoidable escapes
90.04 Fees
90.05 Rabies quarantine orders of Mayor
90.06 Interfering with enforcement of quarantine orders
90.07 Dogs may be killed for certain acts
Offenses Relating to Animals
90.20 Abandoning animals
90.21 Injuring animals
90.22 Poisoning animals
90.23 Cruelty to animals; cruelty to companion animals
90.24 Animal fights
90.25 Trapshooting
90.26 Loud dog
90.27 Dog tags
90.28 Nuisance, dangerous and vicious dog defined; transfer of ownership certificate; form stating dog's prior behavior
90.29 Failure to register dog or dog kennel
90.30 Hindering the capture of unregistered dog
90.31 Unlawful tag
90.32 Rights of blind, deaf or hearing impaired, or mobility impaired person, or trainer with assistance dog
90.33 Retail sale and transportation of dogs
90.34 Coloring rabbits or baby poultry; regulation of sale and display
90.35 Jacklighting prohibited
90.36 Restrictions on dog ownership for certain convicted felons
90.99 Penalty
Chapter 91: Fireworks, Explosives, Fire Prevention
Fireworks and Explosives
91.01 Definitions
91.02 Possession, sale, and use of fireworks
91.03 Permit to use fireworks
91.04 Manufacturing or wholesale sale without a license; prohibitions
91.05 Purchasers to comply with law; unauthorized purchases
91.06 Exhibition without a license; prohibitions
91.07 Unauthorized transportation or shipping
91.08 Application of subchapter
91.09 Arrest of offender; seizure and forfeiture of fireworks; distribution of fines
91.10 Safety requirements for fireworks showroom structures
91.11 Storage of explosives
91.12 Blasting permit
Fire Prevention
91.30 Removal of flammable materials or obstructions
91.31 Protective appliances
91.32 Compliance with order
91.33 Waste receptacles
91.34 Hotel to have fire warning device producing visible signal
91.35 Fire suppression systems
91.36 Violations of State Fire Code prohibited
91.37 Posting arson notices in hotels, motels and other places
91.38 Negligent burning
91.39 Spreading alarm of unfriendly fire
91.40 Unvented heaters
Open Burning
91.55 Definitions
91.56 Relations to other prohibitions
91.57 Open burning in restricted areas
91.58 Permission and notice to open burn
91.99 Penalty
Chapter 92: Intoxicating Liquors
Section
92.01 Definitions
92.02 Exemptions from chapter
92.03 Restrictions applicable to sale of beer and intoxicating liquor for consumption on the premises
92.04 Restrictions on sale of beer and liquor
92.05 Permit required; activities prohibited without permit
92.06 Illegal transportation prohibited
92.07 Open container prohibited; exception
92.08 Underage person shall not purchase intoxicating liquor or beer
92.09 Prohibitions; minors under 18 years; low-alcohol beverages
92.10 Alcohol vaporizing devices prohibited
92.11 Misrepresentation to obtain alcoholic beverage for a minor prohibited
92.12 Misrepresentation by a minor under 21 years
92.13 Sale to underage persons prohibited
92.14 Posting of card
92.15 Good faith acceptances of spurious identification
92.16 Consumption in motor vehicle prohibited
92.17 Hours of sale or consumption
92.18 Obstructing search of premises prohibited
92.19 Illegal possession of intoxicating liquor prohibited
92.20 Sale or possession of diluted liquor and refilled containers prohibited
92.21 Keeping place where beer or intoxicating liquors are sold in violation of law
92.22 Intoxicating liquors shall not be sold in brothels
92.23 Use of intoxicating liquor in a public dance hall prohibited; exceptions
92.24 Poisonously adulterated liquors
92.25 Tavern keeper permitting rioting or drunkenness
92.26 Notice of action to prohibit liquor business
92.27 Procedure when injunction violated
92.28 Liquor transaction scans
92.29 Affirmative defenses
92.99 Penalty
Chapter 93: Nuisances
Section
General Provisions
93.01 Application of the chapter
93.02 Definitions
93.03 Nuisances generally; injunctions; violation; contempt
93.04 Maintaining certain nuisances
93.05 Collection of cost of abating dangerous property condition; injunction; rehabilitation
93.06 Trimming of trees and shrubbery to prevent obstruction
Septic Tanks, Cesspools, and Refuse
93.20 Location of privy vaults, cesspools, and septic tanks
93.21 Unsanitary vaults
93.22 Removal of contents of vault
93.23 Deposit of dead animals, offal upon land or water
93.24 Defiling spring or well prohibited
93.25 Dumping of refuse in municipality prohibited
93.26 Abandoned refrigerators
93.27 Discarding litter prohibited
93.28 Power of municipality to fill or drain land
Weeds and Litter on Private Property
93.40 Keeping down weeds
93.41 Notice to owner to cut noxious weeds, remove litter; service
93.42 Fees for service and return
93.43 Procedure when owner fails to comply with notice
93.44 Written return to County Auditor; amount as a lien upon property
Unclean Habitations
93.60 Permitting unclean habitations
93.61 When habitations are deemed unsanitary
93.62 Order for abatement or vacation of premises
93.63 Enforcement of vacation order by Fire Chief or Police Chief
93.64 Enforcement through court proceedings
93.99 Penalty
Chapter 94: Streets and Sidewalks
General Provisions
94.01 Conditions precedent to improving streets
94.02 Opening permit required
94.03 Application and cash deposit
94.04 Restoration of pavement
94.05 Barriers around excavations
94.06 Warning lights
94.07 Sidewalk construction by the municipality
94.08 Unloading on street or sidewalk
94.09 Street or sidewalk obstruction
94.10 Materials on street or sidewalk
94.11 Duty to keep sidewalks in repair and clean of ice and snow
94.12 Ramped curbing for persons with disabilities
94.13 Flagpole along right-of-way
94.14 Altering or injuring marker or monument
Construction and Repair
94.25 Construction and repair may be required
94.26 Resolution of necessity
94.27 Notice to construct or repair
94.28 Assessments of costs against owner
94.29 Proceedings may include different owners
94.30 Making and levying assessments
Changes in Streets
94.40 Change of name, vacating or narrowing streets by petition
94.41 Change of name, vacating or narrowing streets without petition
94.42 Notice; exception
94.43 Publication of notice
94.44 Effect of order of vacation
94.45 Effect on public utility easements
94.99 Penalty
Chapter 95: Unclaimed and Abandoned Vehicles
Section
95.01 Impounding motor vehicle on private property; requirements
95.02 Impounding abandoned motor vehicle on public property; notice; disposition
95.03 Disposition of vehicle ordered into storage
95.04 Disposition of abandoned junk motor vehicles
95.05 Abandonment of junk motor vehicle prohibited
95.06 Junk motor vehicle; order to cover or remove; notice; exceptions
Chapter 96: Watercraft
Section
96.01 Definitions; applicability
96.02 Failure to comply with law enforcement order; fleeing
96.03 Duty upon approach of law enforcement vessel
96.04 Flashing lights prohibited; exceptions
96.05 Siren prohibited; exceptions
96.06 Regulations for operation of powercraft of more than ten horsepower
96.07 Restrictions on child operators; duty of supervisory adult
96.08 Reckless operation; maintaining sufficient control; wakes restricted
96.09 Unsafe conditions
96.10 Marking of bathing and vessel areas
96.11 Mooring prohibited in certain areas
96.12 Operating under influence of alcohol or drugs prohibited
96.13 Implied consent
96.14 Incapacitated operators prohibited
96.15 Water skiing confined to ski zones
96.16 Observer required when towing skier
96.17 Water skiing after dark prohibited
96.18 Personal flotation device required for towed person
96.19 Ski jumps prohibited
96.20 Permit for special water events
96.21 Sale of single celled inflatable vessels prohibited
96.22 Sitting, standing, walking on moving vessels restricted
96.23 Engine warm-up required
96.24 Personal flotation devices for children under ten
96.25 Operation without personal flotation devices prohibited
96.26 Distress signal or flag required
96.27 Anchor requirements
96.28 Specification for fire extinguishers
96.29 Backfire flame control device required
96.30 Ventilation requirement on powercraft
96.31 Abandonment of junk vessels or outboard motors
96.32 Exhaust muffler required; noise levels; exceptions
96.33 Safety equipment on rental vessels
96.34 Capacity plate
96.35 Littering prohibited
96.36 Dwellings; sanitary systems
96.37 Prima facie evidence of negligence
96.38 Requirements for operating personal watercraft
96.39 Numbering
96.40 Registration
96.41 Tags indicating expiration date; attachment of identification number
96.42 Altering of serial numbers; false information prohibited
96.43 Accident reports
96.44 Enforcement
96.45 Firearms offenses; signaling devices
96.46 Tampering with navigation aid or vessel prohibited
96.47 Certificate of title; exceptions
96.48 Manufacturer's or importer's certificate
96.49 Prohibitions relating to certificates of title
96.50 Permanently displayed hull identification number
96.99 Penalty
TITLE XI: BUSINESS REGULATIONS
Chapter 110: General Provisions
Section
110.01 Licenses required to engage in certain businesses; exceptions
110.02 Application for license
110.03 Issuance of license
110.04 Date and duration of license
110.05 License not transferable
110.06 License certificate to be displayed
110.07 Revocation or suspension
110.08 Appeal and review
110.99 General penalty for Title XI
Chapter 111: Taxicabs
Section
111.01 Definitions
111.02 Certificate of public convenience and necessity required
111.03 Application for certificate
111.04 Issuance of certificate
111.05 Liability insurance required
111.06 License fees
111.07 Transfer of certificates and licenses
111.08 Suspension and revocation of certificates
111.09 Taxicab driver's license
111.10 Application for driver's license
111.11 Examination of applicant; motor vehicle operator's permit required
111.12 Police investigation of applicant; traffic and police record
111.13 Consideration of application
111.14 Issuance of license; duration; annual fee
111.15 Display of license
111.16 Suspension and revocation of license
111.17 Failure to comply with federal, state and municipal laws
111.18 Vehicle equipment and maintenance
111.19 Designation of taxicabs
111.20 Taximeter and display of rates required
111.21 Number of passengers allowed
111.22 Articles left in vehicles
111.23 Vehicles from other municipalities
111.24 Receipts
111.25 Refusal of passenger to pay legal fare
111.26 Solicitation, acceptance and discharge of passengers
111.27 Open stands; use
111.28 Taxicab service
111.29 Manifests
111.30 Records and reports of holders
111.31 Police department; duty to enforce chapter
111.32 Disposition of vehicle license fees
Chapter 112: Peddlers, Itinerant Merchants, and Solicitors
Section
112.01 Definitions
112.02 License requirement
112.03 Application procedure
112.04 Standards for issuance
112.05 Revocation procedure
112.06 Standards for revocation
112.07 Appeal procedure
112.08 Exhibition of identification
112.09 Municipal policy on soliciting
112.10 Notice regulating soliciting
112.11 Duty of solicitors
112.12 Uninvited soliciting prohibited
112.13 Time limit on soliciting
Chapter 113: Commercial Amusements
Section
113.01 Bowling; billiards and pool
113.02 Circuses, carnivals, shows and other such entertainment
113.03 Deposit required
113.04 License fee for public entertainment or exhibition
113.05 License fee may be waived for civic interest
Chapter 114: Tattooing and Body Piercing Services
Section
114.01 Definitions
114.02 Prohibitions
114.03 Application for license; fees; issuance
114.04 Inspection of facilities
114.05 Suspension or revocation of license
114.06 Consent for performing procedures on persons under 18
114.07 Prohibitions relating to persons under 18
114.08 Defenses to violations
114.09 Training standards; records; safety and sanitation; equipment
114.10 Application of local regulation on chapter
114.99 Penalty
TITLE XIII: GENERAL OFFENSES
Chapter 130: General Provisions
Section
130.01 Application of Title XIII
130.02 Definitions
130.03 Classification of offenses
130.04 Common law offenses abrogated
130.05 Rules of construction
130.06 Limitation of criminal prosecutions
130.07 Requirements for criminal liability; voluntary intoxication
130.08 Culpable mental states
130.09 Organizational criminal liability
130.10 Personal accountability for organizational conduct
130.11 Attempt
130.12 Complicity
130.13 Presumption of innocence; proof of offense; affirmative defense
130.14 Battered woman syndrome
130.15 Delinquency adjudications deemed convictions
130.16 Criminal law jurisdiction
130.17 Disposition of unclaimed or forfeited property held by Police Department
130.18 Imposing sentence for misdemeanor
130.19 Multiple sentences
130.20 Apprehension, detention, or arrest of persons on bond
130.21 Self defense: limitations on duty to retreat prior to using force
130.99 Penalty for Title XIII
Chapter 131: Offenses Against Property
Section
131.01 Definitions
131.02 Arson; determining property value or amount of physical harm
131.03 Criminal damaging or endangering; vehicular vandalism
131.04 Criminal mischief
131.05 Damaging or endangering aircraft or airport operations
131.06 Criminal trespass; aggravated trespass
131.07 Tampering with coin machines
131.08 Theft
131.09 Unauthorized use of a vehicle
131.10 Unauthorized use of property, including telecommunication property and computers; possession of municipal property
131.11 Passing bad checks
131.12 Misuse of credit cards
131.13 Making or using slugs
131.14 Prima facie evidence of purpose to defraud
131.15 Tampering with records
131.16 Securing writings by deception
131.17 Defrauding creditors
131.18 Receiving stolen property
131.19 Value of stolen property
131.20 Degree of offense when certain property involved
131.21 Injuring vines, bushes, trees, or crops
131.22 Detention and arrest of shoplifters and those committing motion picture piracy; protection of institutional property
131.23 Insurance fraud; workers' compensation fraud; Medicaid fraud
131.24 Injury to property by hunters
131.25 Evidence of intent to commit theft of rented property; evidence of lack of capacity to consent
131.26 Forgery of identification cards
131.27 Criminal simulation
131.28 Personating an officer
131.29 Trademark counterfeiting
131.30 Diminishing or interfering with forfeitable property
131.31 Recording credit card, telephone or Social Security numbers
131.32 Prosecutions for theft of utilities
131.33 Motion picture piracy
Chapter 132: Offenses Against Public Peace
Section
132.01 Riot
132.02 Failure to disperse
132.03 Justifiable use of force to suppress riot
132.04 Disorderly conduct
132.05 Disturbing a lawful meeting
132.06 Misconduct at an emergency
132.07 Telecommunications harassment
132.08 Inducing panic
132.09 Making false alarms
132.10 Inciting to violence
132.11 Unlawful display of law enforcement emblem
132.12 Impersonating a peace officer
132.13 Safety of crowds attending live entertainment performances
132.14 Misconduct involving a public transportation system
Chapter 133: Sex Offenses
Section
133.01 Definitions
133.02 Unlawful sexual conduct with a minor
133.03 Sexual imposition
133.04 Public indecency
133.05 Voyeurism
133.06 Polygraph examinations for victims: restrictions on use
133.07 Procuring
133.08 Soliciting; loitering to engage in
133.09 Prostitution
133.10 Disseminating matter harmful to juveniles
133.11 Displaying matter harmful to juveniles
133.12 Deception to obtain matter harmful to juveniles
133.13 Rules of evidence
133.14 Declaratory judgment
133.15 Injunction; abatement of nuisance
133.16 Unlawful operation of viewing booths depicting sexual conduct
133.17 Juveniles on the premises of adult entertainment establishments prohibited
133.18 Sexually oriented businesses; illegal operation and activity
133.19 Unlawful advertising of massage
133.99 Sentencing for sexually oriented offenses; sexual predators; registration
Chapter 134: Gambling Offenses
Section
134.01 Definitions
134.02 Prohibitions against gambling; exception
134.03 Operating a gambling house
134.04 Public gaming
134.05 Cheating
134.06 Regulations concerning operation of licensed bingo game
134.07 Records to be kept
134.08 Requirements for bingo game operators
134.09 Bingo games for amusement only
134.10 Prohibitions where instant bingo game is conducted
134.11 Raffle drawings
134.12 Instant bingo other than at bingo sessions
134.13 Restrictions on owner or lessor of location at instant bingo
134.14 Skill-based amusement machines; prohibited conduct
Chapter 135: Offenses Against Persons
Section
135.01 Definitions
135.02 Negligent homicide
135.03 Vehicular homicide; vehicular manslaughter
135.04 Assault; negligent assault
135.05 Injury to persons by hunters
135.06 Menacing; aggravated menacing; menacing by stalking
135.07 Unlawful restraint
135.08 Criminal child enticement
135.09 Coercion
135.10 Bigamy
135.11 Unlawful abortion; failure to perform viability testing
135.12 Abortion trafficking
135.13 Nonsupport of dependents
135.14 Endangering children
135.15 Interference with custody; interference with support orders
135.16 Domestic violence
135.17 Hazing prohibited
135.18 Contributing to unruliness or delinquency of a child
135.19 Failure to provide for functionally impaired person
135.20 Patient abuse or neglect; patient endangerment; exceptions; false statements; retaliation
135.21 Interference with right of person to engage in housing transactions because of race, religion, or the like
135.22 Ethnic intimidation
135.23 Violating a protection order, consent agreement, anti-stalking protection order or order issued by a court of another state
135.24 Adulteration of food
135.25 Illegal distribution of cigarettes, other tobacco products, or alternative nicotine products; transaction scans
135.26 Nonsmoking areas in places of public assembly
135.27 Spreading contagion
135.28 Abuse of a corpse
135.29 Unlawful collection of bodily substances
Chapter 136: Offenses Against Justice and Administration
Section
136.01 Definitions
136.02 Falsification
136.03 Compounding a crime
136.04 Failure to report a crime
136.05 Failure to aid a law enforcement officer
136.06 Obstructing official business
136.07 Obstructing justice
136.08 Resisting arrest
136.09 Having an unlawful interest in a public contract
136.10 Soliciting or receiving improper compensation
136.11 Dereliction of duty
136.12 Interfering with civil rights
136.13 Illegal conveyance of prohibited items onto grounds of a detention facility or other specified governmental facilities
136.14 False report of child abuse or neglect
136.15 Assaulting police dog or horse, or assistance dog
136.16 Disclosure of confidential peace officer information
136.17 Intimidation of crime victim or witness
136.18 Using sham legal process
136.19 Making false allegation of peace officer misconduct
136.20 Misuse of 9-1-1 system
136.21 Failure to disclose personal information
Chapter 137: Weapons Control
Section
| Section | Description |
|---------|-------------|
| 137.01 | Definitions |
| 137.02 | Carrying concealed weapons |
| 137.03 | Using weapons while intoxicated |
| 137.04 | Improperly handling firearms in a motor vehicle |
| 137.05 | Possessing criminal tools |
| 137.06 | Failure to secure dangerous ordnance |
| 137.07 | Unlawful transactions in weapons |
| 137.08 | Underage purchase of firearm or handgun |
| 137.09 | Pointing and discharging firearms and other weapons |
| 137.10 | License or permit to possess dangerous ordnance |
| 137.11 | Possession of an object indistinguishable from a firearm in a school safety zone |
| 137.12 | Possession of deadly weapon while under detention |
| 137.13 | Concealed handgun licenses: possession of a revoked or suspended license; additional restrictions; posting of signs prohibiting possession |
| 137.14 | Defaced firearms |
Chapter 138: Drug Offenses
Section
| Section | Description |
|---------|-------------|
| 138.01 | Definitions |
| 138.02 | Trafficking in controlled substances; gift of marihuana |
| 138.03 | Drug possession offenses |
| 138.04 | Possessing drug abuse instruments |
| 138.05 | Permitting drug abuse |
| 138.06 | Illegal cultivation of marihuana |
| 138.07 | Abusing harmful intoxicants |
| 138.08 | Illegal dispensing of drug samples |
| 138.09 | Federal prosecution bar to municipal prosecution |
| 138.10 | Nitrous oxide: improper dispensing or distribution; possession in a motor vehicle |
| 138.11 | Laboratory report required |
| 138.12 | Counterfeit controlled substances |
| 138.13 | Use, possession, or sale of drug paraphernalia |
| 138.14 | Controlled substance or prescription labels |
| 138.15 | Possession, sale and disposal of hypodermics |
| 138.16 | Controlled substance schedules |
| 138.17 | Unlawful furnishing of prescription to enable persons to be issued handicapped parking placards or license plates |
| 138.18 | Pseudoephedrine sales |
TITLE XV: LAND USAGE
Chapter 150: General Provisions
Section
Parks and Recreation
| Section | Description |
|---------|-------------|
| 150.01 | Recreation Board |
| 150.02 | Board of Park Trustees |
Planning and Zoning
| Section | Description |
|---------|-------------|
| 150.15 | Planning Commission |
| 150.16 | Board of Zoning Appeals |
PARALLEL REFERENCES
Ohio Legislative History References – Master Table
INDEX
This summary of contents has been verified and authorized for publication by the Legislative Authority of the Municipality of Amanda Village, Ohio.
Signed:
Mayor
Clerk of the Legislative Authority
CERTIFICATION OF CODIFIED ORDINANCES
We, Mark A Moore, Mayor, and Carrie A Ayers, Clerk of the Legislative Authority, of the Municipality of Amanda Village, Ohio, pursuant to Ohio Revised Code §§ 731.23 and 731.42, hereby certify that the general and permanent ordinances of the Municipality, as revised, rearranged, compiled, renumbered as to sections, codified and printed herewith in component codes and titles are correct as and constitute the Code of Ordinances for the Municipality of Amanda Village, Ohio.
Mark A Moore
Mayor
Clerk of the Legislative Authority
Ordinance to set rates for outside of village limits
Amend Ordinance #1-2014
Whereas the legislative authority deems it necessary to amend water rates.
Whereas the village would like to set rates for outside the village limits.
Whereas the legislative authority would like to set a 20% monthly surcharge for water on any tap outside the village limits.
Whereas the legislative authority would like to set a 20% monthly surcharge for sewer on any tap outside the village limits.
Now therefore let it be ordained by VILLAGE OF AMANDA, OHIO:
That the village Legislative Authority does hereby amend the water rates listed above. To add the surcharge fees for water and sewer for any tap outside of the village limits. This surcharge will be a 20% surcharge on water and sewer. That this ordinance shall take effect upon the earliest date allowed by law.
Mayor Mark A. Moore
Vote: 4 yes 0 no
Attest: Carrie Ayers Fiscal Officer
The three reading requirement was waived/not waived:
Yeas: 4 nays: 0
The undersigned, Fiscal Officer of the Legislative Authority, does hereby certify that the foregoing legislation was posted in not less than five (5) public places, as determined by the legislative authority, for a period of not less than fifteen (15) days prior to the effective date thereof.
Fiscal Officer
1st 4-3-17
2nd Read 5-1-17
3rd Wnd 5-1-17
ORDINANCE TO AMEND PROPERTY MAINTENANCE CODE
WHEREAS THE VILLAGE COUNCIL DESIRES TO AMEND ORDINANCE 02-2013 THE INTERNATIONAL PROPERTY MAINTENANCE CODE
WHEREAS THE LEGISLATIVE AUTHORITY DEEMS IT NECESSARY TO ADD ADDITIONAL PROVISIONS TO THE EXISTING CODE
WHEREAS THE ATTACHED EXHIBITS B, C, AND D ARE TO BE AMENDED TO THE EXISTING CODE 02-2013
NOW THEREFORE, LET IT BE ORDAINED BY THE VILLAGE OF AMANDA, OHIO:
THAT THE VILLAGE COUNCIL AUTHORIZES AMENDMENTS TO THE INTERNATIONAL PROPERTY MAINTENANCE CODE 02-2013, WITH THE ADDITIONS OF EXHIBIT B, C, AND D
THE ORDINANCE SHALL BE IN EFFECT AND BE IN FULL FORCE FROM AND AFTER THE EARLIEST PERIOD ALLOWED BY LAW.
Mark A. Moore
MAYOR
VOTE: YES 5 NO 0
Carrie Ayers
FISCAL OFFICER
THE THREE READING REQUIREMENT WAS WAIVED / NOT WAIVED:
YEAS: 5 NAYS: 0
FISCAL OFFICER
THE UNDERSIGNED, FISCAL OFFICER OF THE LEGISLATIVE AUTHORITY, DOES HEREBY CERTIFY THAT THE FOREGOING LEGISLATION WAS POSTED IN NOT LESS THAN FIVE (5) PUBLIC PLACES, AS DETERMINED BY THE LEGISLATIVE AUTHORITY FOR THE PERIOD OF NOT LESS THAN FIFTEEN (15) DAYS PRIOR TO THE EFFECTIVE DATE THEREOF.
Carrie Ayers
FISCAL OFFICER
6-5-17 1st Reading
SECTION 302
EXTERIOR
PROPERTY AREAS
302.13 STORAGE OF JUNK VEHICLES AND OTHER MOTOR VEHICLES:
DEFINITIONS
(A) "Junk motor vehicles" means any motor vehicle which meets any three of the following requirements:
1. Is five years old or older;
2. Is extensively damaged, such as damage including but not limited to any of the following: missing wheels, tires, motor, or transmission;
3. Is not able to be legally operated upon a public street, road, or highway for any reason, whether mechanical, operational, or otherwise;
4. Does not have validly issued license plates and a valid registration which allows it to be operated legally upon public streets, roads, or highways;
5. Has a fair market value of fifteen hundred dollars ($1,500.00) or less;
6. Is not running and not operable ("operable" meaning able to be started and driven under its own power).
(B) A junk motor vehicle left in the open for 72 hours is declared a nuisance and shall be abated in accordance with provisions of this chapter.
(C) "Motor vehicle" means every vehicle propelled or drawn by power other than muscular power, except motorized bicycles, road rollers, traction engines, power shovels, power cranes, and other equipment used in construction work and not designed for or employed in general highway transportation, hole-digging machinery, well-drilling machinery, ditch-digging machinery, farm machinery, and trailers designed and used exclusively to transport a boat between a place of storage and a marina, or in and around a marina, when drawn to towed on a street or highway for a distance of no more than ten miles.
and at a speed of twenty-five miles per hour or less
(See ORC. Section 4511.OI(B).
(D) "In the open" shall be defined as not garaged.
PROHIBITONS.
(A) No person shall willfully permit a junk motor vehicle to remain in the open on private property which the person owns, occupies, or controls after receipt of an order to remove the junk motor vehicle. The order shall state that a hearing appealing the order to remove the junk motor vehicle maybe had, shall describe the vehicle to be removed, and shall be served by a Code Enforcement Officer or Police Officer.
(B) If any recipient of an order to remove a junk motor vehicle shall fails to appeal the same to the Mayor in writing within ten (10) days after the receipt of the order, it shall be conclusively presumed to establish the junk motor vehicle as a nuisance and the junk motor vehicle shall be removed immediately upon order by a code official designated by the Mayor, the Fire Department, Police Department, or their designee. The fact that a junk motor vehicle is left on private property without the filing of an appeal by the recipient of an order to remove a junk motor vehicle is prima facie evidence of willful failure to comply with the order.
(C) No person shall leave a junk motor vehicle for any period of time on private property to which such person does not have the right of possession without the authorization of the person having the right of possession of such property.
COLLECTOR'S VEHICLE
A collector's vehicle as defined by O.R.C. Section 4501.01 is a junk motor vehicle for purposes of this section, regardless if it is licensed or unlicensed, if the collector's vehicle meets the definition of a junk motor vehicle as set forth herein.
STORAGE AND DISPOSAL OF JUNK MOTOR VEHICLES.
(A) After a motor vehicle has been determined to be a junk motor vehicle, as provided herein, the Health Department, Fire Department: Code Enforcement Officer, Police Officer, or their designee shall immediately cause the junk motor vehicle to be removed from the property where the junk motor vehicle is located. The place of storage for any junk motor vehicle which has been removed by this section shall be designated by the Mayor.
(B) Any junk motor vehicle which has been stored by the Police Department pursuant to this chapter, shall be disposed of in accordance with the procedures set forth in O.R.C. Section 4513.63.
**APPEALS**
(A) The Board of Zoning Appeals (BZA) Shall hear any appeals within thirty (30) days of the filing of an appeal by the recipient of an order to remove a junk motor vehicle left in the open. The BZA shall rule whether or not the motor vehicle is a junk motor vehicle left in the open. If the vehicle is declared by the BZA to be a junk motor vehicle left in the open, the BZ shall order the removal of the particular vehicle by the Police Department.
(B) Any appeal from a decision of the BZA shall be made pursuant to O.R.C. Section 2506.
**EXCEPTIONS**
This chapter shall not apply to vehicles stored inside a garage, in a licensed towing service, in a licensed motor vehicle salvage facility, in a licensed scrap processing yard, in a licensed auto repair garage yard, in a licensed paint spray shop yard, in a licensed gasoline station yard, or in a licensed vehicle dealership yard, or any other lawful storage area not within the public view as prescribed by O.R.C. Section 4737.09.
**PENALTY**
Whoever fails to remove a junk motor vehicle left in the open, after receipt of a proper order to remove the same, shall be subject to the penalties provided here in. Each junk motor vehicle left in the open in violation here of shall constitute a separate offense: Every twenty (20) days that this section is violated shall constitute a separate offense.
SECTION 302
EXTERIOR PROPERTY AREAS
302.10 MISCELLANEOUS PROVISIONS:
(A) All exterior parts of every dwelling or structure, including exterior walls, parapet walls, decorative additions, chimneys and all other exterior structures, either above or below the roof line, shall be maintained in a safe condition, weather-tight and so as to resist decay or deterioration.
(B) Any dwelling, structure or accessory building whose exterior surface is bare, deteriorated, ramshackle, tumble-down, decaying, disintegrating or in poor condition shall be repaired or razed.
(C) All buckled, rotted or decayed walls, doors, windows, porches, floors, steps, railing trim and their missing members shall be replaced and put in good condition. All replacements shall match and conform to current design or be replaced completely.
(D) All exterior wood or unfinished surface shall be sealed and painted or the surface covered with other protective coating or treated to prevent rot and decay and to conform and match the existing paint or surface covering and original design or replacement thereof. All exterior walls and surfaces shall be properly protected against the weather where such are defective or lack weather protection, including lack of paint or surface covering or have weathered due to lack of proper protective coating.
(E) Any dwelling structure or secondary or appurtenant structure whose exterior surface is deteriorated, decaying, disintegrating, or whose exterior surface has weathered with dirt and grime or has been impaired through the peeling or flaking of paint or other protective coating, and which exterior surfaces consist of an area of twenty-five percent (25%) or more of the external surface of the structure, shall be repaired, repainted or recovered with an approved protective coating or surface. All exterior surfaces shall be replaced or repaired in good condition preparatory to painting or coating. All bare exterior surfaces, which are flaking or crumbling shall be replaced or sealed in a good and workmanlike manner. All new or repaired bare surfaces shall be painted or coated. All exterior surfaces weathered
with dirt or grime, which are peeling or flaking, shall be painted or covered with approved protective coating or surface.
(F) All repairs shall be completed in a timely manner consistent with accepted construction practices.
(G) All dwelling structures and the premises thereof shall be maintained free from sources of breeding, harborage and infestation by insects, vermin or rodents.
(H) No owner, operator or resident agent of any premises shall maintain or permit to be maintained at or on the exterior areas of any such premises any condition which deteriorates or debases the appearance of the neighborhood, reduces property values in the neighborhood, adversely alters the appearance and general character of the neighborhood, creates a fire, safety or health hazard or which is a public nuisance. Such conditions include but are not limited to the following:
1. Broken or dilapidated fences, walls or other structures;
2. Out-of-use or non-usable appliances and machinery;
3. Rugs, rags or other materials hung on lines or in other places on the premises, which materials are not being used for general household or housekeeping purposes;
4. Broken, dilapidated or unusable furniture, mattresses or other household furniture, broken glass, plastic materials, paints, miscellaneous coverings and/or any other materials, including those described in this section, placed at or on the premises in such a manner as to be patently unsightly, grotesque or offensive to the senses;
5. No unregistered motor vehicles and/or junk vehicles shall be parked on any property for more than thirty (30) days;
6. Lawns and landscaping shall be so maintained so as not to constitute a blighting or deteriorating effect on the neighborhood. Grass brush, briers, burrs shall be maintained at a height not to exceed twelve (12) inches. This provision shall not apply to vegetable gardens, bushes, vines, shrubs or trees that produce food for human consumption;
7. No building materials or materials, earth, sand or dirt intended for use in landscaping, gardening or construction shall be left standing open or covered upon any premises for a period of time exceeding two (2) months without specific written authorization from the Village Zoning Department.
(I) New construction initiated on the exterior of any structure or surrounding yard, after the effective date of the adoption of this provision, shall be completed within one (1) year. In the event of unforeseeable delays in construction, the Code Enforcement Officer is authorized to extend the one (1) year time period for an additional time period of six (6) months or less.
Section 302 Exterior Property Areas
302.11 STORAGE OF CERTAIN MATERIALS DECLARED A NUISANCE:
(A) Definitions
1. Automobile parts" means and includes any portion or parts of any motor driven vehicle as detached from the vehicle as a whole.
2. Motor vehicle in an inoperative condition" means and includes any style or type of motor-driven vehicle used or useful for the conveyance of persons or property which is unable to move under its own power due to defective or missing parts, and which have remained in such condition for a period of not less than 30 consecutive days.
3. Motor vehicle unfit for further use" means and includes any style type of motor-driven vehicle used for the conveyance of persons or property, which is in a dangerous condition, has defective or missing parts, or is in such a condition generally as to be unfit for further (1se as a conveyance.
4. Refuse" embraces only such matter as was either in fact noxious or has been refused and abandoned by its owner as worthless.
5. Rubbish" means and includes wire, chips, shavings, bottles, broken glass, crockery, tin, cast or wooden ware, boxes, rags, dead weeds, paper circulars, handbills, boots, shoes, ashes or any waste material other than garbage or offal.
6. Scrap metal" means and includes pieces of or parts of steel, iron, tm, zinc, copper, aluminum, or any alloy thereof, whether covered with porcelain
or any other material, whether intact or in parts, which has served its usefulness in its original form and can no longer be used or useful for its originally intended purpose.
7. "Used building materials" means and includes any materials, such as wood, stone, brick, cement block, asphalt, blacktop, concrete, construction debris, or any composition thereof, used or useful in the erection of any building or structure, which have been used previously for such erection or construction, by the same persons or by any other person.
(B) Storage of Certain Materials Declared a Nuisance.
It shall be unlawful and is hereby declared a nuisance for any reason to store, place or allow to remain, automobile parts, motor vehicles in an inoperative condition, motor vehicles unfit for further use, refuse, rubbish, scrap metal, or used building materials, as defined herein, on any lot, lots, parts of lots, or parcel of land, within the corporate limits if the Village.
(C) Order for Removal.
(1) In the event of a violation of division (b) of this section, the Mayor or Property Maintenance Code Officer shall give notice to the owner, occupant or person having charge of the premises upon which the violation occurs to cease such violation. Such notice shall be in writing and shall be served upon the owner, occupant or person having charge of the premises either personally or at the usual place of residence of such owner, occupant or person having charge of such premises or by registered or certified mail addressed to such person's last known place of residence.
(2) If the person served with the notice as provided in division (1) fails to cause such violation to cease within ten days of the date upon which the notice w
issued, he or she shall be subject to the penalties provided herein, and a separate offense shall be deemed committed upon each day during or on which the violation occurs or continues beyond such ten-day period; provided that no additional notice of violation is required to be given.
(D) Exception
Notwithstanding the provisions of division (b) of this section, it shall not be unlawful for any person to purchase used building materials and place or store them on any lot, lots parts of lots, or parcel of land, when such materials are stored within the confines of a closed garage, or building or are to a used by the purchaser or owner in later construction on the same lot or any lot owned or controlled by such person; provided that such materials shall not remain on the lot, lots, parts of lots, or parcel of land, for a period of more than 30 days, unless the construction or erection planned for the use of the materials has commenced; and provided further that such materials are used or consumed in the construction or removed from the premises within a period of four months from the time the materials are first placed on the lot, lots, parts of lots or parcel off and. It shall be unlawful for any person or persons to move any materials so stored or placed to another location within the village for the purpose of avoiding the intent of this section, except that any such materials may be moved to another lot, lots, parts of lots or parcel of land, when the same have been sold to a bona fide purchaser for value for such purchaser's own use.
SECTION 302
EXTERIOR
PROPERTY AREAS
302.13 STORAGE OF JUNK VEHICLES AND OTHER MOTOR VEHICLES:
DEFINITIONS:
(A) "Junk motor vehicles" means any motor vehicle which meets any three of the following requirements:
1. Is five years old or older;
2. Is extensively damaged, such as damage including but not limited to any of the following: missing wheels, tires, motor, or transmission;
3. Is not able to be legally operated upon a public street, road, or highway for any reason, whether mechanical, operational, or otherwise;
4. Does not have validly issued license plates and a valid registration which allows it to be operated legally upon public streets, roads, or highways;
5. Has a fair market value of fifteen hundred dollars ($1,500.00) or less;
6. Is not running and not operable ("operable" meaning able to be started and driven under its own power);
(B) A junk motor vehicle left in the open for 72 hours is declared a nuisance and shall be abated in accordance with provisions of this chapter.
(C) "Motor vehicle" means every vehicle propelled or drawn by power other than muscular power, except motorized bicycles, road rollers, traction engines, power shovels, power cranes, and other equipment used in construction work and not designed for or employed in general highway transportation, hole-digging machinery, well-drilling machinery, ditch-digging machinery, farm machinery, and trailers designed and used exclusively to transport a boat between a place of storage and a marina, or in and around a marina, when drawn to towed on a street or highway for a distance of no more than ten miles.
and at a speed of twenty-five miles per hour or less
(See ORC. Section 451I.Ol(B).
(D) "In the open" shall be defined as not garaged.
PROHIBITIONS.
(A) No person shall willfully permit a junk motor vehicle to remain in the open on private property which the person owns, occupies, or controls after receipt of an order to remove the junk motor vehicle. The order shall state that a hearing appealing the order to remove the junk motor vehicle maybe had, shall describe the vehicle to be removed, and shall be served by a Code Enforcement Officer or Police Officer.
(B) If any recipient of an order to remove a junk motor vehicle shall fails to appeal the same to the Mayor in writing within ten (10) days after the receipt of the order, it shall be conclusively presumed to establish the junk motor vehicle as a nuisance and the junk motor vehicle shall be removed immediately upon order by a code official designated by the Mayor, the Fire Department, Police Department, or their designee. The fact that a junk motor vehicle is left on private property without the filing of an appeal by the recipient of an order to remove a junk motor vehicle is prima facie evidence of willful failure to comply with the order.
(C) No person shall leave a junk motor vehicle for any period of time on private property to which such person does not have the right of possession without the authorization of the person having the right of possession of such property.
COLLECTOR'S VEHICLE
A collector's vehicle as defined by O.R.C. Section 4501.01 is a junk motor vehicle for purposes of this section, regardless if it is licensed or unlicensed, if the collector's vehicle meets the definition of a junk motor vehicle as set forth herein.
STORAGE AND DISPOSAL OF JUNK MOTOR VEHICLES.
(A) After a motor vehicle has been determined to be a junk motor vehicle, as provided herein, the Health Department, Fire Department: Code Enforcement Officer, Police Officer, or their designee shall immediately cause the junk motor vehicle to be removed from the property where the junk motor vehicle is located. The place of storage for any junk motor vehicle which has been removed by this section shall be designated by the Mayor.
(B) Any junk motor vehicle which has been stored by the Police Department pursuant to this chapter, shall be disposed of in accordance with the procedures set forth in O.R.C. Section 4513.63.
APPEALS
(A) The Board of Zoning Appeals (BZA) Shall hear any appeals within thirty (30) days of filing an appeal by the recipient of an order to remove a junk motor vehicle left in the open. The BZA shall rule whether or not the motor vehicle is a junk motor vehicle left in the open.
If the vehicle is declared by the BZA to be a junk motor vehicle left in the open, the BZ shall order the removal of the particular vehicle by the Police Department.
(B) Any appeal from a decision of the BZA shall be made pursuant to O.R.C. Section 2506.
EXCEPTIONS
This chapter shall not apply to vehicles stored inside a garage, in a licensed towing service, in a licensed motor vehicle salvage facility, in a licensed scrap processing yard, in a licensed auto repair garage yard, in a licensed paint spray shop yard, in a licensed gasoline station yard, or in a licensed vehicle dealership yard, or any other lawful storage area not within the public view as prescribed by O.R.C. Section 4737.09.
PENALTY
Whoever fails to remove a junk motor vehicle left in the open, after receipt of a proper order to remove the same, shall be subject to the penalties provided here in. Each junk motor vehicle left in the open in violation here of shall constitute a separate offense. Every twenty (20) days that this section is violated shall constitute a separate offense.
ORDINANCE TO ADOPTING AN UPDATED VERSION OF THE (2018) INTERNATIONAL PROPERTY MAINTENANCE CODE, AS AMENDED, TOGETHER WITH ALL UPDATED REVISIONS OF SAID CODE.
WHEREAS THE VILLAGE COUNCIL DESIRES TO AMEND ORDINANCE 02-2013 THE INTERNATIONAL PROPERTY MAINTENANCE CODE TO ADD ALL REVISIONS AND UPDATED VERSIONS;
WHEREAS THE VILLAGE COUNCIL DESIRES TO ADOPT THE UPDATED 2018 INTERNATIONAL PROPERTY MAINTENANCE CODE;
WHEREAS THE LEGISLATIVE AUTHORITY DEEMS IT NECESSARY TO ADD ADDITIONAL PROVISIONS TO THE EXISTING CODE IN ORDER TO MAINTAIN THE SAFETY AND WELLBEING OF THE CITIZENS;
NOW THEREFORE, LET IT BE ORDAINED BY THE VILLAGE OF AMANDA, OHIO:
THAT THE VILLAGE COUNCIL AUTHORIZES AMENDMENTS AND MOVES TO ADOPT THE 2018 INTERNATIONAL PROPERTY MAINTENANCE CODE,
THE ORDINANCE SHALL BE IN EFFECT AND BE IN FULL FORCE FROM AND AFTER THE EARLIEST PERIOD ALLOWED BY LAW.
MAYOR
VOTE: YES 5 NO 0
CARRIE AYERS
FISCAL OFFICER
THE THREE READING REQUIREMENT WAS WAIVED / NOT WAIVED:
YEAS: 5 NAYS: 0
FISCAL OFFICER
THE UNDERSIGNED, FISCAL OFFICER OF THE LEGISLATIVE AUTHORITY, DOES HEREBY CERTIFY THAT THE FOREGOING LEGISLATION WAS POSTED IN NOT LESS THAN FIVE (5) PUBLIC PLACES, AS DETERMINED BY THE LEGISLATIVE AUTHORITY FOR THE PERIOD OF NOT LESS THAN FIFTEEN (15) DAYS PRIOR TO THE EFFECTIVE DATE THEREOF.
FISCAL OFFICER
To: Village Officials
Re: Subscription to the Ohio Basic Code, 2018 Edition
Thank you for your subscription to the *Ohio Basic Code, 2018 Edition*. This model Code of Ordinances has been adopted by the Village as their own Code, and the 2018 Edition replaces any previous editions of the Village Code. Please note, however, that if you have received a looseleaf “Title 17: Municipal Regulations,” this **Title 17 is not affected by this 2018 Edition and should be retained**. Please mail any Village ordinances to update your Title 17 to our office at your earliest convenience.
Enclosed you will also find a sample adopting ordinance, a summary of new matter (for publication notification), and a certification of codified ordinances, all provided for your convenience in adopting the *Ohio Basic Code, 2018 Edition*.
We are pleased to provide the *Ohio Basic Code, 2018 Edition*, and we look forward to continuing to serve Ohio villages.
Very truly yours,
Todd Paul Myers, J.D.
Vice President, Major Client Services
email@example.com
CERTIFICATION OF CODIFIED ORDINANCES
We, ________________, Mayor, and ________________, Clerk of the Legislative Authority, of the Municipality of ________________, Ohio, pursuant to Ohio Revised Code §§ 731.23 and 731.42, hereby certify that the general and permanent ordinances of the Municipality, as revised, rearranged, compiled, renumbered as to sections, codified and printed herewith in component codes and titles are correct as and constitute the Code of Ordinances for the Municipality of ________________, Ohio.
__________________________
Mayor
__________________________
Clerk of the Legislative Authority
AN ORDINANCE APPROVING, ADOPTING AND ENACTING AMERICAN LEGAL PUBLISHING’S OHIO BASIC CODE, 2018 EDITION, AS THE CODE OF ORDINANCES FOR THE MUNICIPALITY OF ________, OHIO, AND DECLARING AN EMERGENCY.
WHEREAS, the present general and permanent ordinances of the municipality are inadequately arranged and classified and are insufficient in form and substance for the complete preservation of the public peace, health, safety and general welfare of the municipality and for the proper conduct of its affairs.
WHEREAS, American Legal Publishing Corporation publishes a Code of Ordinances suitable for adoption by municipalities in Ohio.
WHEREAS, it is necessary to provide for the usual daily operation of the municipality and for the immediate preservation of the public peace, health, safety and general welfare of the municipality that this ordinance take effect at an early date.
NOW, THEREFORE, BE IT ORDAINED BY THE LEGISLATIVE AUTHORITY OF THE MUNICIPALITY OF ________, OHIO:
Section 1. American Legal Publishing’s Ohio Basic Code, 2018 Edition, as reviewed and approved by the Legislative Authority, is hereby adopted and enacted. Any prior version of the Ohio Basic Code which may have been previously adopted by the municipality is hereby repealed as obsolete and is hereby replaced in its entirety by this Ohio Basic Code, 2018 Edition.
Section 2. One copy of American Legal Publishing’s Ohio Basic Code, 2018 Edition, certified as correct by the Mayor and Clerk of the Legislative Authority, as required by Ohio Revised Code § 731.23, shall be kept in its initial form on file in the office of the Clerk of the municipality and retained as a permanent ordinance record of the municipality. The Clerk of the municipality is authorized and directed to publish a summary of all new matters contained in the Code of Ordinances as required by Ohio Revised Code § 731.23. Such summary is attached hereto and marked as “Exhibit A”.
Section 3. All ordinances and resolutions or parts thereof which are in conflict or inconsistent with any provision of the Ohio Basic Code, 2018 Edition, as adopted in Section 1 hereof, are hereby repealed as of the effective date of this ordinance, except as follows:
(A) The enactment of the Ohio Basic Code, 2018 Edition, shall not be construed to affect a right or liability accrued or incurred under any legislative provision prior to the effective date of such enactment, or an action or proceeding for the enforcement of such right or liability. Such enactment shall not be construed to relieve any person from punishment for an act committed in violation of any such legislative provision, nor to affect an indictment or prosecution therefor. For such purposes, any such legislative provision shall continue in full force notwithstanding its repeal for the purpose of revision and codification.
(B) The repeal provided above shall not affect:
(1) The grant or creation of a franchise, license, right, easement or privilege;
(2) The purchase, sale, lease or transfer of property;
(3) The appropriation or expenditure of money or promise or guarantee of payment;
(4) The assumption of any contract or obligation;
(5) The issuance and delivery of any bonds, obligations or other instruments of indebtedness;
(6) The levy or imposition of taxes, assessments or charges;
(7) The establishment, naming, vacating or grade level of any street or public way;
(8) The dedication of property or plat approval;
(9) The annexation or detachment of territory;
(10) Any legislation enacted subsequent to the adoption of this ordinance.
(11) Any legislation specifically superseding the provision of the Ohio Basic Code.
Section 4. Whenever reference is made in any documents, publications, or signs of the municipality, including but not limited to traffic tickets and traffic-control signs, to a section as it existed in a former edition of the Ohio Basic Code, the reference shall extend and apply to the section referred to as subsequently amended, revised, recodified, or renumbered.
Section 5. This ordinance is declared to be an emergency measure necessary for the immediate preservation of the peace, health, safety and general welfare of the people of this municipality, and shall take effect at the earliest date provided by law.
Date Passed: 2-5-17
Attest:
Mayor
Clerk of the Legislative Authority
Notice is hereby given that on the 5th day of Feb., 2018, there was enacted by the Legislative Authority of the Municipality of Village of Avon, Ohio, an ordinance entitled “An Ordinance Approving, Adopting and Enacting American Legal Publishing’s Ohio Basic Code, 2018 Edition, as the Code of Ordinances for the Municipality of Village of Avon, Ohio.”
A summary of the subjects, including all new matters contained in the Code of Ordinances, as adopted, are as follows. The majority of Basic Code provisions are based directly on state law.
TITLE I: GENERAL PROVISIONS
Chapter 10: General Provisions
Section
10.01 Short titles
10.02 Definitions
10.03 Rules of construction
10.04 Revivor; effect of amendment or repeal
10.05 Construction of section references
10.06 Conflicting provisions
10.07 Severability
10.08 Reference to offices
10.09 Errors and omissions
10.10 Ordinances repealed
10.11 Ordinances unaffected
10.12 Ordinances saved
10.13 Application to future ordinances
10.14 Interpretation
10.15 Amendments to code; amendatory language
10.16 Statutory references
10.17 Preservation of penalties, offenses, rights and liabilities
10.18 Determination of legislative intent
10.99 General penalty
TITLE III: ADMINISTRATION
Chapter 30: General Provisions
Section
30.01 Application of Title III
30.02 Qualifications; oaths
30.03 Bonds of officers and employees; amount
30.04 Additional bond; where bonds recorded and kept
30.05 Approval of bonds
30.06 Sufficiency of form of bond
30.07 Filling vacancies in offices
30.08 Public records available
30.09 Records Commission
30.10 Meetings of public bodies to be open; exceptions; notice
30.11 Municipal officers may attend conference or convention; expenses
30.12 Residency requirements prohibited; exceptions
Chapter 31: Executive Authority
Section
General Provisions
31.001 Executive power; where vested
**Mayor**
31.015 Term of Mayor; power and duties
31.016 General duties of the Mayor
31.017 Communications to the Legislative Authority
31.018 Protest against excess of expenditures
31.019 Supervision of conduct of officers
31.020 Annual report to the Legislative Authority
31.021 Mayor to file charges against delinquent officers
31.022 Vacancies in office of Mayor
31.023 Disposition of fines and other moneys
**Clerk**
31.040 Election, term, qualifications of the Clerk
31.041 Powers and duties of Clerk
31.042 Books and accounts; merger of offices
31.043 Seal of Clerk
31.044 Combined offices of Clerk and Treasurer; Fiscal Officer
**Treasurer**
31.060 Election, term, qualifications of the Treasurer
31.061 Accounts of Treasurer
31.062 Powers and duties
31.063 Quarterly account; annual report
31.064 Receipt and disbursement of funds
31.065 Duty of delivering money and property
**Street Commissioner**
31.080 Qualifications
31.081 General duties
31.082 Assistants
**Other Officials**
31.100 Legal counsel
31.101 Administrator
31.102 Board of Trustees of Public Affairs
31.103 Fire Engineer, Engineer and Superintendent of Markets
---
**Chapter 32: Legislative Authority**
**General Provisions**
32.001 Members of the Legislative Authority; election; terms of office
32.002 President Pro Tempore; employees
32.003 Vacancy when President Pro Tempore becomes Mayor
32.004 Qualifications of members of the Legislative Authority
32.005 Compensation and bonds of municipal officers and employees
32.006 Vacancy
32.007 Judge of election and qualification of members; quorum and special meetings
32.008 Rules; journal; expulsion of members
32.009 Meetings
32.010 General powers
32.011 Failure to take oath or give bond
32.012 Notice when new bond required
32.013 Care, supervision and management of public institutions
### Contracts, Bids and Proceedings
32.025 Contracts by the Legislative Authority or Administrator
32.026 Bids and proceedings
32.027 Alterations or modifications of contract
32.028 Contract restrictions
32.029 Award to lowest responsive and responsible bidder
### Ordinances and Resolutions
32.040 Ordinances and resolutions as evidence
32.041 Passage procedure
32.042 Style of ordinances
32.043 Subject and amendment of ordinances and resolutions
32.044 Authentication and recording of ordinances and resolutions
32.045 Publication of ordinances and resolutions; proof of publication and circulation
32.046 Notice for proposed amendments to the municipal Charter
32.047 Times of publication required
32.048 Publication and certification of ordinances in book form
32.049 Adoption of technical ordinances and codes
32.050 Certificate of Clerk as to publication
32.051 Publication when no newspaper published in municipality
32.052 Effect of not making publication
32.053 Ordinances providing for appropriations or street improvements; emergency ordinances
### Initiative and Referendum
32.070 Initiative petitions
32.071 Referendum petitions
32.072 More than one ordinance required; application of subchapter
32.073 Presentation of petitions
32.074 Copy of proposed ordinance or measure to be filed with Clerk
32.075 Words to be printed in red
32.076 Designation of committee filing petition; public inspection of petitions; ordinances passed or repealed prior to election
32.077 Itemized statement by petition circulator
32.078 Prohibited practices relative to petitions
32.079 Accepting premiums for signing
32.080 Threats in securing signatures
32.081 Application of subchapter if Charter adopted
### Chapter 33: Judicial Authority
#### General Provisions
33.01 Jurisdiction in ordinance cases and traffic violations
33.02 Powers of Mayor and Mayor’s Court Magistrate in criminal matters
33.03 Duties of Mayor and Mayor’s Court Magistrate; fees; office; seal
33.04 Mayor’s Court Magistrate
33.05 Powers to suspend driver’s license in OVI cases
#### Contempt of Court
33.20 Summary punishment for contempt
33.21 Acts in contempt of court
33.22 Hearing
33.23 Contempt action for failure to pay support, failure to comply or interference with a visitation order; summons
33.24 Right of accused to bail
33.25 Hearing on contempt; penalties; support orders; failure to withhold or deduct money pursuant to support order
33.26 Imprisonment until order obeyed
33.27 Proceedings when party released on bail fails to appear
33.28 Release of prisoner committed for contempt
33.29 Judgment final
33.30 Alternative remedy
Chapter 34: Police Department
Section
34.01 Marshal and Police Chief synonymous
34.02 Appointment of Marshal
34.03 Deputy marshals and police officers
34.04 Auxiliary police units
34.05 Offenses affecting employment of law enforcement officers; probationary period; final appointment
34.06 Removal proceedings; suspension; appeals
34.07 General powers
34.08 Powers and duties of Marshal
34.09 Disposition of fines and penalties
34.10 Property recovered by police
34.11 Disposition to claimant
34.12 Sale of unclaimed property; disposition of proceeds
34.13 Expenses of storage and sale; notice
34.14 Contracts for police protection; nonresident service without contract
34.15 Peace officer administering oaths; acknowledging complaints, summonses, affidavits and returns of court orders
Chapter 35: Fire Department
General Provisions
Section
35.01 Municipal fire regulations; fire department
35.02 Fire Chief; Fire Prevention Officer; employment of firefighters; criminal records check for firefighters
35.03 Schooling of officers and firefighters of fire department
35.04 Legislative Authority may purchase engines and equipment
35.05 Buildings for department
35.06 Records
35.07 Maximum consecutive hours for firefighters on duty
35.08 Investigation of cause of fire
35.09 Right to examine buildings, premises, and vehicles
35.10 Burning buildings for firefighting instruction or research
35.11 Impersonating fire safety inspector
35.12 Standards for equipment
35.13 Persons entitled to be known as firefighters
35.14 Firefighting and emergency services agreements
35.15 Regulation of construction in fire limits
Volunteer Firefighters’ Dependents Fund Board
Section
35.30 Definitions
35.31 Establishment
35.32 Membership; vacancies
35.33 Election and term of members
35.34 Organization; rules and regulations; roster
35.35 Compensation and expenses of Board; legal advisor
Chapter 36: Civil Actions Against the Municipality
Section
36.01 Definitions
36.02 Nonliability of municipality; exceptions
36.03 Defenses and immunities
36.04 Limitation of actions
36.05 Damages
36.06 Satisfaction of judgments
36.07 Provision of employees’ defense; consent judgments
36.08 Liability insurance
36.09 Certain actions unaffected
36.10 Certain charges against municipal officers filed with Probate Judge; proceedings
TITLE VII: TRAFFIC CODE
Chapter 70: General Provisions
General Provisions
| Section | Description |
|---------|-------------|
| 70.01 | Definitions |
| 70.02 | Compliance with order of police officer |
| 70.03 | Emergency vehicles to proceed cautiously past red or stop signal |
| 70.04 | Exceptions generally; emergency, public safety and coroner vehicles exempt |
| 70.05 | Persons riding or driving animals upon roadways |
| 70.06 | Prohibitions against pedestrians and slow-moving vehicles on freeways |
| 70.07 | Use of private property for vehicular travel |
| 70.08 | Names of persons damaging real property by operation of vehicle to be provided to owner |
| 70.09 | Limited access highways; barriers along; vehicles to enter and leave at designated intersections |
| 70.10 | Through highways |
| 70.11 | Officer may remove ignition key |
| 70.12 | Removal of vehicles after accidents |
Traffic-Control Devices
| Section | Description |
|---------|-------------|
| 70.30 | Obeying traffic-control devices |
| 70.31 | Signal lights |
| 70.32 | Signals over reversible lanes |
| 70.33 | Ambiguous or non-working traffic signals |
| 70.34 | Pedestrian-control signals |
| 70.35 | Unauthorized signs and signals prohibited |
| 70.36 | Alteration, defacement, or removal prohibited |
| 70.37 | Unauthorized possession or sale of devices |
| 70.38 | Signal preemption devices; prohibitions |
| 70.99 | Penalty |
Chapter 71: Licensing Provisions
Motor Vehicle Licensing
| Section | Description |
|---------|-------------|
| 71.01 | Display of license plates or validation stickers; registration |
| 71.02 | Improper use of noncommercial motor vehicle |
| 71.03 | Operating motor vehicle ordered immobilized; forfeiture |
| 71.04 | Operation or sale without certificate of title |
| 71.05 | Display of certificate of registration |
| 71.06 | Use of unauthorized plates |
| 71.07 | Operating without dealer or manufacturer license plates |
Driver’s Licenses
| Section | Description |
|---------|-------------|
| 71.20 | Prohibited acts |
| 71.21 | Permitting minor to operate vehicle prohibited; temporary instruction permit; probationary license |
| 71.22 | License required as driver or commercial driver on public or private property; nonresident exemption |
| 71.23 | Employment of a minor to operate a taxicab prohibited |
| 71.24 | Restriction against owner lending vehicle for use of another |
| 71.25 | Suspension of driver’s licenses; license suspended by court of record |
| 71.26 | Display of license |
| 71.27 | Prohibition against false statements |
| 71.28 | Driving under suspension or in violation of license restriction |
71.29 Operating motor vehicle or motorcycle without valid license
71.30 Driving under OVI suspension
71.31 Driving under financial responsibility law suspension or cancellation; driving under a nonpayment of judgment suspension
71.32 Failure to reinstate license
**Commercial Driver's Licenses**
71.45 Definitions
71.46 Use of actual gross weight in lieu of rating
71.47 Prohibited acts
71.48 Prerequisites to operation of commercial motor vehicle
71.49 Physical qualification to operate commercial motor vehicles
71.50 Criminal offenses
71.51 Application of federal regulations
71.52 Employment of drivers of commercial vehicles
71.99 Penalty
**Chapter 72: Traffic Rules**
**General Provisions**
72.001 Lanes of travel upon roadways
72.002 Driving through safety zone
72.003 Vehicles traveling in opposite directions
72.004 Rules governing overtaking and passing of vehicles
72.005 Permission to overtake and pass on the right
72.006 Driving to left of center line
72.007 Prohibition against driving upon left side of roadway
72.008 Hazardous zones
72.009 One-way highways and rotary traffic islands
72.010 Rules for driving in marked lanes
72.011 Space between moving vehicles
72.012 Divided roadways
72.013 Rules for turns at intersections
72.014 U-turns and turning in roadway prohibited
72.015 Starting and backing vehicles
72.016 Turn and stop signals
72.017 Hand and arm signals
**Right-of-Way**
72.030 Right-of-way at intersections
72.031 Right-of-way when turning left
72.032 Right-of-way at through highways; stop signs; yield signs
72.033 Stop at sidewalk area; stop signs on private roads and driveways
72.034 Right-of-way on public highway
72.035 Pedestrian on sidewalk has right-of-way
72.036 Right-of-way of public safety vehicles
72.037 Funeral procession has right-of-way
72.038 Pedestrians yield right-of-way to public safety vehicle
72.039 Pedestrian on crosswalk has right-of-way
72.040 Right-of-way yielded to blind person
72.041 Right-of-way yielded by pedestrian
**Pedestrians**
72.055 Pedestrian movement in crosswalks
72.056 Pedestrian walking along highway
72.057 Prohibition against soliciting rides; riding on outside of vehicle
72.058 Pedestrian on bridge or railroad crossing
72.059 Persons operating motorized wheelchairs
72.060 Intoxicated or drugged pedestrian hazard on highway
72.061 Operation of electric personal assistive mobility devices
72.062 Operation of personal delivery device on sidewalks and crosswalks
**Grade Crossings**
72.075 Stop signs at grade crossings
72.076 Driving vehicle across railroad grade crossing
72.077 Vehicles required to stop at grade crossings
72.078 Slow-moving vehicles or equipment crossing railroad tracks
**School Buses**
72.090 Regulations concerning school buses
72.091 Violation of regulations; report; investigation; citation; warning
72.092 Restrictions on the operation of school buses
72.093 School bus inspection
72.094 School bus not used for school purposes
72.095 Licensing by Department of Public Safety
72.096 Registration and identification of school buses
72.097 School bus marking
72.098 Flashing light signal lamps
72.099 Occupant restraining device for operator
**Prohibitions**
72.115 Obstruction and interference affecting view and control of driver
72.116 Occupying travel trailer while in motion
72.117 Driving upon closed highway prohibited
72.118 Driving upon sidewalk area or paths exclusively for bicycles
72.119 Obstructing passage of other vehicles
72.120 Following an emergency or public vehicle prohibited; approaching stationary public safety vehicle with caution
72.121 Driving over unprotected fire hose
72.122 Placing injurious material on highway or depositing litter from motor vehicle
72.123 Transporting child not in child-restraint system prohibited
72.124 Occupant restraining devices
72.125 Use of engine brakes prohibited
72.126 Operating motor vehicle while wearing earphones or earplugs
72.127 Chauffeured limousines and livery services
72.128 Operating traction engine upon improved highway
72.129 Cracking exhaust noises; peeling out
72.130 Shortcutting across private property
72.131 Texting while driving prohibited
72.132 Use of electronic wireless communication devices by minors or probationary drivers while driving prohibited
**Chapter 73: Motor Vehicle Crimes**
**General Provisions**
73.01 Driving under the influence of alcohol or drugs
73.02 Implied consent
73.03 Physical control of vehicle while under the influence
73.04 Driving commercial vehicle with impaired alertness or ability; use of drugs
73.05 Reckless operation of vehicles
73.06 Reckless operation off streets and highways; competitive operation
73.07 Operator to be in reasonable control
73.08 Immobilizing or disabling device violation
73.09 Street racing defined; prohibited on public highways
73.10 Speed limits
73.11 Slow speed or stopping
73.12 Emergency vehicles excepted from speed limitation
73.13 Speed regulations on bridges
73.14 Presenting false name or information to officer
73.15 Prohibition against resisting officer
73.16 Operation restricted for mini-trucks and low-speed, under-speed, or utility vehicles
**Stopping After Accident**
73.30 Failure to stop after accident
73.31 Stopping after accident on other than public roads or highways
73.32 Accident involving damage to realty
73.33 Failure to report accident
**Chapter 74: Equipment and Loads**
**Equipment**
74.01 Unsafe vehicles, prohibition against operation
74.02 Bumpers on motor vehicles
74.03 Lighted lights required
74.04 Headlights
74.05 Tail lights and illumination of rear license plate
74.06 Red reflectors required
74.07 Safety lighting of commercial vehicles
74.08 Stoplight regulations
74.09 Obscured lights on vehicles
74.10 Red light or flag required
74.11 Lights on parked vehicles
74.12 Lights and emblem on slow-moving vehicles; lights and reflectors on multi-wheel agricultural tractors or farm machinery
74.13 Spotlight and auxiliary driving lights
74.14 Cowl, fender, and back-up lights
74.15 Two lights displayed
74.16 Headlights required
74.17 Lights of less intensity
74.18 Number of lights permitted; red and flashing lights
74.19 Standards for lights on snow removal equipment and oversize vehicles
74.20 Flashing lights permitted for certain types of vehicles
74.21 Lights and sign on transportation for preschool children
74.22 Focus and aim of headlights
74.23 Brake equipment; specifications
74.24 Brake fluid
74.25 Minimum standards for brakes and components
74.26 Horns, sirens, and warning devices
74.27 Mufflers; excessive smoke or gas
74.28 Rearview mirrors
74.29 Windshields and wipers
74.30 Solid tire requirements
74.31 Requirements for safety glass in motor vehicles; use of tinted glass or reflectorized material
74.32 Directional signals
74.33 Installation and sale of seat safety belts required; definition
74.34 Requirements for extra signal equipment
74.35 Display of warning devices on disabled vehicles
74.36 Requirements for vehicles transporting explosives
74.37 Studded tires; seasonal use permitted
74.38 Safety inspection decals for buses
74.39 Air bags
**Loads**
74.50 Permit required to exceed load limits
74.51 Limitation of load extension on left side of vehicle
74.52 All loads shall be properly secured
74.53 Towing requirements; exception to size and weight restrictions
74.54 Weighing of vehicle; removal of excess load
74.55 Operation of vehicle exceeding weight limits prohibited
74.56 Load limits
74.57 Maximum width, height, and length
74.58 Statement of gross vehicle weight
74.59 Wheel protectors required on heavy commercial vehicles
74.60 Liability for damages; prosecution; application of monies
74.61 Weight exceptions for certain vehicles
Chapter 75: Bicycles, Motorcycles and Off-Road Vehicles
Section
General Provisions
75.01 Bicycles; application of Title VII
75.02 Operation of motorized bicycle
75.03 Rules for bicycles, motorcycles, and snowmobiles
75.04 Prohibition against attaching bicycles and sleds to vehicles
75.05 Riding bicycles; motorcycles abreast
75.06 Equipment of bicycles
Snowmobiles, Off-Highway Motorcycles, and All-Purpose Vehicles
75.25 Definitions
75.26 Equipment
75.27 Code application; prohibited operation
75.28 Permitted operation
75.29 Licensing requirements of operator
75.30 Maintenance of vehicles for hire
75.31 Accident reports
75.32 Impounding of vehicle
75.33 Local control within police power
75.34 Registration of vehicles
75.35 Certificate of title; prohibitions
Chapter 76: Parking Regulations
Section
76.01 Prohibition against parking on highways
76.02 Condition when motor vehicle left unattended
76.03 Police may remove illegally parked vehicle
76.04 Parking prohibitions
76.05 Parking near curb; privileges for persons with disabilities
76.06 Parking on private property in violation of posted prohibition
76.07 Selling, washing or repairing vehicle upon roadway
76.08 Truck loading zones
76.09 Bus stops and taxicab stands
76.10 Parking in alleys and narrow streets; exceptions
76.11 Registered owner prima facie liable for unlawful parking
76.12 Waiver
TITLE IX: GENERAL REGULATIONS
Chapter 90: Animals
Section
Animals Running at Large
90.01 Dogs or other animals running at large; nuisance, dangerous or vicious dogs; hearings
90.02 Confining animal found at large; publication of notice; lien
90.03 Unavoidable escapes
90.04 Fees
90.05 Rabies quarantine orders of Mayor
90.06 Interfering with enforcement of quarantine orders
90.07 Dogs may be killed for certain acts
Offenses Relating to Animals
90.20 Abandoning animals
90.21 Injuring animals
90.22 Poisoning animals
90.23 Cruelty to animals; cruelty to companion animals
90.24 Animal fights
90.25 Trapshooting
90.26 Loud dog
90.27 Dog tags
90.28 Nuisance, dangerous and vicious dog defined; transfer of ownership certificate; form stating dog's prior behavior
90.29 Failure to register dog or dog kennel
90.30 Hindering the capture of unregistered dog
90.31 Unlawful tag
90.32 Rights of blind, deaf or hearing impaired, or mobility impaired person, or trainer with assistance dog
90.33 Retail sale and transportation of dogs
90.34 Coloring rabbits or baby poultry; regulation of sale and display
90.35 Jacklighting prohibited
90.36 Restrictions on dog ownership for certain convicted felons
90.37 Sexual conduct with an animal
90.99 Penalty
Chapter 91: Fireworks, Explosives, Fire Prevention
Fireworks and Explosives
91.01 Definitions
91.02 Possession, sale, and use of fireworks
91.03 Permit to use fireworks
91.04 Manufacturing or wholesale sale without a license; prohibitions
91.05 Purchasers to comply with law; unauthorized purchases
91.06 Exhibition without a license; prohibitions
91.07 Unauthorized transportation or shipping
91.08 Application of subchapter
91.09 Arrest of offender; seizure and forfeiture of fireworks; distribution of fines
91.10 Safety requirements for fireworks showroom structures
91.11 Storage of explosives
91.12 Blasting permit
Fire Prevention
91.30 Removal of flammable materials or obstructions
91.31 Protective appliances
91.32 Compliance with order
91.33 Waste receptacles
91.34 Hotel to have fire warning device producing visible signal
91.35 Fire suppression systems
91.36 Violations of State Fire Code prohibited
91.37 Posting arson notices in hotels, motels and other places
91.38 Negligent burning
91.39 Spreading alarm of unfriendly fire
91.40 Unvented heaters
Open Burning
91.55 Definitions
91.56 Relations to other prohibitions
91.57 Open burning in restricted areas
91.58 Permission and notice to open burn
91.99 Penalty
Chapter 92: Intoxicating Liquors
Section
| Section | Description |
|---------|-------------|
| 92.01 | Definitions |
| 92.02 | Exemptions from chapter |
| 92.03 | Restrictions applicable to sale of beer and intoxicating liquor for consumption on the premises |
| 92.04 | Restrictions on sale of beer and liquor |
| 92.05 | Permit required; activities prohibited without permit |
| 92.06 | Illegal transportation prohibited |
| 92.07 | Open container prohibited; exception |
| 92.08 | Underage person shall not purchase intoxicating liquor or beer |
| 92.09 | Prohibitions; minors under 18 years; low-alcohol beverages |
| 92.10 | Alcohol vaporizing devices prohibited |
| 92.11 | Misrepresentation to obtain alcoholic beverage for a minor prohibited |
| 92.12 | Misrepresentation by a minor under 21 years |
| 92.13 | Sale to underage persons prohibited |
| 92.14 | Posting of card |
| 92.15 | Good faith acceptances of spurious identification |
| 92.16 | Consumption in motor vehicle prohibited |
| 92.17 | Hours of sale or consumption |
| 92.18 | Obstructing search of premises prohibited |
| 92.19 | Illegal possession of intoxicating liquor prohibited |
| 92.20 | Sale or possession of diluted liquor and refilled containers prohibited |
| 92.21 | Keeping place where beer or intoxicating liquors are sold in violation of law |
| 92.22 | Intoxicating liquors shall not be sold in brothels |
| 92.23 | Use of intoxicating liquor in a public dance hall prohibited; exceptions |
| 92.24 | Poisonously adulterated liquors |
| 92.25 | Tavern keeper permitting rioting or drunkenness |
| 92.26 | Notice of action to prohibit liquor business |
| 92.27 | Procedure when injunction violated |
| 92.28 | Liquor transaction scans |
| 92.29 | Affirmative defenses |
| 92.99 | Penalty |
Chapter 93: Nuisances
Section
General Provisions
| Section | Description |
|---------|-------------|
| 93.01 | Application of the chapter |
| 93.02 | Definitions |
| 93.03 | Nuisances generally; injunctions; violation; contempt |
| 93.04 | Maintaining certain nuisances |
| 93.05 | Collection of cost of abating dangerous property condition; injunction; rehabilitation |
| 93.06 | Trimming of trees and shrubbery to prevent obstruction |
Septic Tanks, Cesspools, and Refuse
| Section | Description |
|---------|-------------|
| 93.20 | Location of privy vaults, cesspools, and septic tanks |
| 93.21 | Unsanitary vaults |
| 93.22 | Removal of contents of vault |
| 93.23 | Deposit of dead animals, offal upon land or water |
| 93.24 | Defiling spring or well prohibited |
| 93.25 | Dumping of refuse in municipality prohibited |
| 93.26 | Abandoned refrigerators |
| 93.27 | Discarding litter prohibited |
| 93.28 | Power of municipality to fill or drain land |
Weeds and Litter on Private Property
| Section | Description |
|---------|-------------|
| 93.40 | Keeping down weeds |
| 93.41 | Notice to owner to cut noxious weeds, remove litter; service |
| 93.42 | Fees for service and return |
| 93.43 | Procedure when owner fails to comply with notice |
| 93.44 | Written return to County Auditor; amount as a lien upon property |
Unclean Habitations
93.60 Permitting unclean habitations
93.61 When habitations are deemed unsanitary
93.62 Order for abatement or vacation of premises
93.63 Enforcement of vacation order by Fire Chief or Police Chief
93.64 Enforcement through court proceedings
93.99 Penalty
Chapter 94: Streets and Sidewalks
General Provisions
94.01 Conditions precedent to improving streets
94.02 Opening permit required
94.03 Application and cash deposit
94.04 Restoration of pavement
94.05 Barriers around excavations
94.06 Warning lights
94.07 Sidewalk construction by the municipality
94.08 Unloading on street or sidewalk
94.09 Street or sidewalk obstruction
94.10 Materials on street or sidewalk
94.11 Duty to keep sidewalks in repair and clean of ice and snow
94.12 Ramped curbing for persons with disabilities
94.13 Flagpole along right-of-way
94.14 Altering or injuring marker or monument
Construction and Repair
94.25 Construction and repair may be required
94.26 Resolution of necessity
94.27 Notice to construct or repair
94.28 Assessments of costs against owner
94.29 Proceedings may include different owners
94.30 Making and levying assessments
Changes in Streets
94.40 Change of name, vacating or narrowing streets by petition
94.41 Change of name, vacating or narrowing streets without petition
94.42 Notice; exception
94.43 Publication of notice
94.44 Effect of order of vacation
94.45 Effect on public utility easements
94.99 Penalty
Chapter 95: Unclaimed and Abandoned Vehicles
Section
95.01 Impounding motor vehicle on private property; requirements
95.02 Impounding abandoned motor vehicle on public property; notice; disposition
95.03 Disposition of vehicle ordered into storage
95.04 Disposition of abandoned junk motor vehicles
95.05 Abandonment of junk motor vehicle prohibited
95.06 Junk motor vehicle; order to cover or remove; notice; exceptions
Chapter 96: Watercraft
Section
96.01 Definitions; applicability
96.02 Failure to comply with law enforcement order; fleeing
96.03 Duty upon approach of law enforcement vessel
96.04 Flashing lights prohibited; exceptions
| Section | Description |
|---------|-------------|
| 96.05 | Siren prohibited; exceptions |
| 96.06 | Regulations for operation of powercraft of more than ten horsepower |
| 96.07 | Restrictions on child operators; duty of supervisory adult |
| 96.08 | Reckless operation; maintaining sufficient control; wakes restricted |
| 96.09 | Unsafe conditions |
| 96.10 | Marking of bathing and vessel areas |
| 96.11 | Mooring prohibited in certain areas |
| 96.12 | Operating under influence of alcohol or drugs prohibited |
| 96.13 | Implied consent |
| 96.14 | Incapacitated operators prohibited |
| 96.15 | Water skiing confined to ski zones |
| 96.16 | Observer required when towing skier |
| 96.17 | Water skiing after dark prohibited |
| 96.18 | Personal flotation device required for towed person |
| 96.19 | Ski jumps prohibited |
| 96.20 | Permit for special water events |
| 96.21 | Sale of single celled inflatable vessels prohibited |
| 96.22 | Sitting, standing, walking on moving vessels restricted |
| 96.23 | Engine warm-up required |
| 96.24 | Personal flotation devices for children under ten |
| 96.25 | Operation without personal flotation devices prohibited |
| 96.26 | Distress signal or flag required |
| 96.27 | Anchor requirements |
| 96.28 | Specification for fire extinguishers |
| 96.29 | Backfire flame control device required |
| 96.30 | Ventilation requirement on powercraft |
| 96.31 | Abandonment of junk vessels or outboard motors |
| 96.32 | Exhaust muffler required; noise levels; exceptions |
| 96.33 | Safety equipment on rental vessels |
| 96.34 | Capacity plate |
| 96.35 | Littering prohibited |
| 96.36 | Dwellings; sanitary systems |
| 96.37 | Prima facie evidence of negligence |
| 96.38 | Requirements for operating personal watercraft |
| 96.39 | Numbering |
| 96.40 | Registration |
| 96.41 | Tags indicating expiration date; attachment of identification number |
| 96.42 | Altering of serial numbers; false information prohibited |
| 96.43 | Accident reports |
| 96.44 | Enforcement |
| 96.45 | Firearms offenses; signaling devices |
| 96.46 | Tampering with navigation aid or vessel prohibited |
| 96.47 | Certificate of title; exceptions |
| 96.48 | Manufacturer’s or importer’s certificate |
| 96.49 | Prohibitions relating to certificates of title |
| 96.50 | Permanently displayed hull identification number |
| 96.99 | Penalty |
---
**TITLE XI: BUSINESS REGULATIONS**
**Chapter 110: General Provisions**
| Section | Description |
|---------|-------------|
| 110.01 | Licenses required to engage in certain businesses; exceptions |
| 110.02 | Application for license |
| 110.03 | Issuance of license |
| 110.04 | Date and duration of license |
| 110.05 | License not transferable |
| 110.06 | License certificate to be displayed |
| 110.07 | Revocation or suspension |
| 110.08 | Appeal and review |
| 110.99 | General penalty for Title XI |
Chapter 111: Taxicabs
Section
111.01 Definitions
111.02 Certificate of public convenience and necessity required
111.03 Application for certificate
111.04 Issuance of certificate
111.05 Liability insurance required
111.06 License fees
111.07 Transfer of certificates and licenses
111.08 Suspension and revocation of certificates
111.09 Taxicab driver's license
111.10 Application for driver's license
111.11 Examination of applicant; motor vehicle operator's permit required
111.12 Police investigation of applicant; traffic and police record
111.13 Consideration of application
111.14 Issuance of license; duration; annual fee
111.15 Display of license
111.16 Suspension and revocation of license
111.17 Failure to comply with federal, state and municipal laws
111.18 Vehicle equipment and maintenance
111.19 Designation of taxicabs
111.20 Taximeter and display of rates required
111.21 Number of passengers allowed
111.22 Articles left in vehicles
111.23 Vehicles from other municipalities
111.24 Receipts
111.25 Refusal of passenger to pay legal fare
111.26 Solicitation, acceptance and discharge of passengers
111.27 Open stands; use
111.28 Taxicab service
111.29 Manifests
111.30 Records and reports of holders
111.31 Police department; duty to enforce chapter
111.32 Disposition of vehicle license fees
Chapter 112: Peddlers, Itinerant Merchants, and Solicitors
Section
112.01 Definitions
112.02 License requirement
112.03 Application procedure
112.04 Standards for issuance
112.05 Revocation procedure
112.06 Standards for revocation
112.07 Appeal procedure
112.08 Exhibition of identification
112.09 Municipal policy on soliciting
112.10 Notice regulating soliciting
112.11 Duty of solicitors
112.12 Uninvited soliciting prohibited
112.13 Time limit on soliciting
Chapter 113: Commercial Amusements
Section
113.01 Bowling; billiards and pool
113.02 Circuses, carnivals, shows and other such entertainment
113.03 Deposit required
113.04 License fee for public entertainment or exhibition
113.05 License fee may be waived for civic interest
Chapter 114: Tattooing and Body Piercing Services
Section
114.01 Definitions
114.02 Prohibitions
114.03 Application for license; fees; issuance
114.04 Inspection of facilities
114.05 Suspension or revocation of license
114.06 Consent for performing procedures on persons under 18
114.07 Prohibitions relating to persons under 18
114.08 Defenses to violations
114.09 Training standards; records; safety and sanitation; equipment
114.10 Application of local regulation on chapter
114.99 Penalty
TITLE XIII: GENERAL OFFENSES
Chapter 130: General Provisions
Section
130.01 Application of Title XIII
130.02 Definitions
130.03 Classification of offenses
130.04 Common law offenses abrogated
130.05 Rules of construction
130.06 Limitation of criminal prosecutions
130.07 Requirements for criminal liability; voluntary intoxication
130.08 Culpable mental states
130.09 Organizational criminal liability
130.10 Personal accountability for organizational conduct
130.11 Attempt
130.12 Complicity
130.13 Presumption of innocence; proof of offense; affirmative defense
130.14 Battered woman syndrome
130.15 Delinquency adjudications deemed convictions
130.16 Criminal law jurisdiction
130.17 Disposition of unclaimed or forfeited property held by Police Department
130.18 Imposing sentence for misdemeanor
130.19 Multiple sentences
130.20 Apprehension, detention, or arrest of persons on bond
130.21 Self defense: limitations on duty to retreat prior to using force
130.99 Penalty for Title XIII
Chapter 131: Offenses Against Property
Section
131.01 Definitions
131.02 Arson; determining property value or amount of physical harm
131.03 Criminal damaging or endangering; vehicular vandalism
131.04 Criminal mischief
131.05 Damaging or endangering aircraft or airport operations
131.06 Criminal trespass; aggravated trespass
131.07 Tampering with coin machines
131.08 Theft
131.09 Unauthorized use of a vehicle
131.10 Unauthorized use of property, including telecommunication property and computers; possession of municipal property
131.11 Passing bad checks
131.12 Misuse of credit cards
131.13 Making or using slugs
131.14 Prima facie evidence of purpose to defraud
131.15 Tampering with records
131.16 Securing writings by deception
131.17 Defrauding creditors
131.18 Receiving stolen property
131.19 Value of stolen property
131.20 Degree of offense when certain property involved
131.21 Injuring vines, bushes, trees, or crops
131.22 Detention and arrest of shoplifters and those committing motion picture piracy; protection of institutional property
131.23 Insurance fraud; workers' compensation fraud; Medicaid fraud
131.24 Injury to property by hunters
131.25 Evidence of intent to commit theft of rented property; evidence of lack of capacity to consent
131.26 Forgery of identification cards
131.27 Criminal simulation
131.28 Personating an officer
131.29 Trademark counterfeiting
131.30 Diminishing or interfering with forfeitable property
131.31 Recording credit card, telephone or Social Security numbers
131.32 Prosecutions for theft of utilities
131.33 Motion picture piracy
Chapter 132: Offenses Against Public Peace
Section
132.01 Riot
132.02 Failure to disperse
132.03 Justifiable use of force to suppress riot
132.04 Disorderly conduct
132.05 Disturbing a lawful meeting
132.06 Misconduct at an emergency
132.07 Telecommunications harassment
132.08 Inducing panic
132.09 Making false alarms
132.10 Inciting to violence
132.11 Unlawful display of law enforcement emblem
132.12 Impersonating a peace officer
132.13 Safety of crowds attending live entertainment performances
132.14 Misconduct involving a public transportation system
Chapter 133: Sex Offenses
Section
133.01 Definitions
133.02 Unlawful sexual conduct with a minor
133.03 Sexual imposition
133.04 Public indecency
133.05 Voyeurism
133.06 Polygraph examinations for victims: restrictions on use
133.07 Procuring
133.08 Soliciting; loitering to engage in
133.09 Prostitution
133.10 Disseminating matter harmful to juveniles
133.11 Displaying matter harmful to juveniles
133.12 Deception to obtain matter harmful to juveniles
133.13 Rules of evidence
133.14 Declaratory judgment
133.15 Injunction; abatement of nuisance
133.16 Unlawful operation of viewing booths depicting sexual conduct
133.17 Juveniles on the premises of adult entertainment establishments prohibited
133.18 Sexually oriented businesses; illegal operation and activity
133.19 Unlawful advertising of massage
133.99 Sentencing for sexually oriented offenses; sexual predators; registration
Chapter 134: Gambling Offenses
Section
134.01 Definitions
134.02 Prohibitions against gambling; exception
134.03 Operating a gambling house
134.04 Public gaming
134.05 Cheating
134.06 Regulations concerning operation of licensed bingo game
134.07 Records to be kept
134.08 Requirements for bingo game operators
134.09 Bingo games for amusement only
134.10 Prohibitions where instant bingo game is conducted
134.11 Raffle drawings
134.12 Instant bingo other than at bingo sessions
134.13 Restrictions on owner or lessor of location at instant bingo
134.14 Skill-based amusement machines; prohibited conduct
Chapter 135: Offenses Against Persons
Section
135.01 Definitions
135.02 Negligent homicide
135.03 Vehicular homicide; vehicular manslaughter
135.04 Assault; negligent assault
135.05 Injury to persons by hunters
135.06 Menacing; aggravated menacing; menacing by stalking
135.07 Unlawful restraint
135.08 Criminal child enticement
135.09 Coercion
135.10 Bigamy
135.11 Unlawful abortion; failure to perform viability testing
135.12 Abortion trafficking
135.13 Nonsupport of dependents
135.14 Endangering children
135.15 Interference with custody; interference with support orders
135.16 Domestic violence
135.17 Hazing prohibited
135.18 Contributing to unruliness or delinquency of a child
135.19 Failure to provide for functionally impaired person
135.20 Patient abuse or neglect; patient endangerment; exceptions; false statements; retaliation
135.21 Interference with right of person to engage in housing transactions because of race, religion, or the like
135.22 Ethnic intimidation
135.23 Violating a protection order, consent agreement, anti-stalking protection order or order issued by a court of another state
135.24 Adulteration of food
135.25 Illegal distribution of cigarettes, other tobacco products, or alternative nicotine products; transaction scans
135.26 Nonsmoking areas in places of public assembly
135.27 Spreading contagion
135.28 Abuse of a corpse
135.29 Unlawful collection of bodily substances
Chapter 136: Offenses Against Justice and Administration
Section
136.01 Definitions
136.02 Falsification
136.03 Compounding a crime
136.04 Failure to report a crime
136.05 Failure to aid a law enforcement officer
136.06 Obstructing official business
136.07 Obstructing justice
136.08 Resisting arrest
136.09 Having an unlawful interest in a public contract
136.10 Soliciting or receiving improper compensation
136.11 Dereliction of duty
136.12 Interfering with civil rights
136.13 Illegal conveyance of prohibited items onto grounds of a detention facility or other specified governmental facilities
136.14 False report of child abuse or neglect
136.15 Assaulting police dog or horse, or assistance dog
136.16 Disclosure of confidential peace officer information
136.17 Intimidation of crime victim or witness
136.18 Using sham legal process
136.19 Making false allegation of peace officer misconduct
136.20 Misuse of 9-1-1 system
136.21 Failure to disclose personal information
Chapter 137: Weapons Control
Section
| Section | Description |
|---------|-------------|
| 137.01 | Definitions |
| 137.02 | Carrying concealed weapons |
| 137.03 | Using weapons while intoxicated |
| 137.04 | Improperly handling firearms in a motor vehicle |
| 137.05 | Possessing criminal tools |
| 137.06 | Failure to secure dangerous ordnance |
| 137.07 | Unlawful transactions in weapons |
| 137.08 | Underage purchase of firearm or handgun |
| 137.09 | Pointing and discharging firearms and other weapons |
| 137.10 | License or permit to possess dangerous ordnance |
| 137.11 | Possession of an object indistinguishable from a firearm in a school safety zone |
| 137.12 | Possession of deadly weapon while under detention |
| 137.13 | Concealed handgun licenses: possession of a revoked or suspended license; additional restrictions; posting of signs prohibiting possession |
| 137.14 | Defaced firearms |
Chapter 138: Drug Offenses
Section
| Section | Description |
|---------|-------------|
| 138.01 | Definitions |
| 138.02 | Trafficking in controlled substances; gift of marihuana |
| 138.03 | Drug possession offenses |
| 138.04 | Possessing drug abuse instruments |
| 138.05 | Permitting drug abuse |
| 138.06 | Illegal cultivation of marihuana |
| 138.07 | Abusing harmful intoxicants |
| 138.08 | Illegal dispensing of drug samples |
| 138.09 | Federal prosecution bar to municipal prosecution |
| 138.10 | Nitrous oxide: improper dispensing or distribution; possession in a motor vehicle |
| 138.11 | Laboratory report required |
| 138.12 | Counterfeit controlled substances |
| 138.13 | Use, possession, or sale of drug paraphernalia |
| 138.14 | Controlled substance or prescription labels |
| 138.15 | Possession, sale and disposal of hypodermics |
| 138.16 | Controlled substance schedules |
| 138.17 | Unlawful furnishing of prescription to enable persons to be issued handicapped parking placards or license plates |
| 138.18 | Pseudoephedrine sales |
TITLE XV: LAND USAGE
Chapter 150: General Provisions
Section
Parks and Recreation
| Section | Description |
|---------|-------------|
| 150.01 | Recreation Board |
| 150.02 | Board of Park Trustees |
Planning and Zoning
| Section | Description |
|---------|-------------|
| 150.15 | Planning Commission |
| 150.16 | Board of Zoning Appeals |
PARALLEL REFERENCES
Ohio Legislative History References – Master Table
INDEX
This summary of contents has been verified and authorized for publication by the Legislative Authority of the Municipality of __________, Ohio.
Signed: _________________________ _________________________
Mayor Clerk of the Legislative Authority
Ordinance #4- 2018
Ordinance to Increase Trash Rates
Whereas the legislative authority deems it necessary to increase trash rates.
Whereas the new contract price has increased deeming it necessary to increase rates.
Whereas the legislative authority would like increase residential rates from 13.47 to 15.37 and senior rates from 12.47 to 14.37
Whereas the legislative authority would like increase dumpster rates 2 yd to $42.00 and 4 yd to $65.00
Whereas all other trash rates will remain the same.
Now therefore let it be ordained by VILLAGE OF AMANDA, OHIO:
That the village Legislative Authority does hereby pass the trash rates listed above. That this ordinance shall take effect upon the earliest date allowed by law.
Mayor Mark A. Moore
Vote 5 yes 0 no
Attest: Carrie Ayers Fiscal Officer
The three reading requirement was waived/not waived:
Yeas 5 nays 0
The undersigned, Fiscal Officer of the Legislative Authority, does hereby certify that the foregoing legislation was posted in not less than five (5) public places, as determined by the legislative authority, for a period of not less than fifteen (15) days prior to the effective date thereof.
Fiscal Officer
ORDINANCE TO RESTRICT PARKING ON PARTS OF LUTZ ST.
Whereas the village legislative authority wishes to restrict parking on the north side of Lutz St from Halderman St to the side doors of Midwest Fabricating to state no parking zone from 7am to 2pm.
Whereas the legislative authority deems it in the best interest of the residents.
NOW THEREFORE BE IT ORDAINED BY THE VILLAGE OF AMANDA, OHIO:
That the area along side of Midwest Fabricating on the north side of Lutz St. be restricted parking-- no parking zone during 7am-2pm from Halderman St to side doors of Midwest Fabricating.
That this ordinance shall take effect upon the earliest date allowed by law.
Mayor Mark A. Moore
Vote 5 yes 0 no
Attest:
Carrie Ayers Fiscal Officer
The three reading requirement was waived/not waived:
Yea 5 nays 0
The undersigned, Fiscal Officer of the Legislative Authority, does hereby certify that the foregoing legislation was posted in not less than five (5) public places, as determined by the legislative authority, for a period of not less than fifteen (15) days prior to the effective date thereof.
Fiscal Officer
ORDINANCE TO RAISE EMPLOYEES SALARY
WHEREAS the village Legislative Authority give employees a raise.
WHEREAS a 3% increase in wages for salary and hourly employees.
NOW THEREFORE, BE IT ORDAINED BY THE VILLAGE OF AMANDA, OHIO:
That the village council feel a 3% increase in salary is warranted and shall be implemented on the current pay cycle. For hourly and salary employees.
That this ordinance shall take effect and be in force from and after the earliest period allowed by law.
Mayor Mark A. Moore
Vote: Yes 5 No 0
Attest:
Carrie Ayers
Fiscal Officer
The undersigned, Fiscal Officer of the Legislative Authority, does hereby certify that the foregoing legislation was posted in not less than (5) five public places as determined by the Legislative Authority, for a period of not less than (15) fifteen days prior to the effective date thereof.
Fiscal Officer
Ordinance to regulate all complaints
Whereas the legislative authority deems it necessary to regulate incoming complaints.
Whereas the legislative authority following legal counsel and Ohio Municipal League advise, would like to require any complaints coming into the village office whether for zoning, property maintenance, or any other village business identify themselves by name and address.
Whereas identifying the complainant can serve to establish that complaints are made in good faith.
Now therefore let it be ordained by VILLAGE OF AMANDA, OHIO:
That the village Legislative Authority does hereby regulate incoming complaints. That all incoming complaints are required to identify themselves by name and address. That this ordinance shall take effect upon the earliest date allowed by law.
Mayor Mark A. Moore
Vote 5 yes 0 no
Attest:
Carrie Ayers Fiscal Officer
The three reading requirement was waived/not waived:
Yeas 5 nays 0
The undersigned, Fiscal Officer of the Legislative Authority, does hereby certify that the foregoing legislation was posted in not less than five (5) public places, as determined by the legislative authority, for a period of not less than fifteen (15) days prior to the effective date thereof.
Fiscal Officer
Ordinance to regulate all record requests
Whereas the legislative authority deems it necessary to regulate incoming requests as well as public record request.
Whereas the legislative authority following legal counsel and Ohio Municipal League advise, would like to require any requests coming into the village office whether for zoning, property maintenance, or any other village business identify themselves by name and address.
Whereas identifying the requests can serve to establish that request are made in good faith.
Now therefore let it be ordained by VILLAGE OF AMANDA, OHIO:
That the village Legislative Authority does hereby regulate all incoming requests. That all incoming requests are required to identify themselves by name and address. That this ordinance shall take effect upon the earliest date allowed by law.
Mayor Mark A. Moore
Vote 4 yes 0 no
Attest:
Carrie Ayers Fiscal Officer
The three reading requirement was waived/not waived:
Yea 4 nays 0
The undersigned, Fiscal Officer of the Legislative Authority, does hereby certify that the foregoing legislation was posted in not less than five (5) public places, as determined by the legislative authority, for a period of not less than fifteen (15) days prior to the effective date thereof.
Fiscal Officer
Ordinance to Set Garbage Guidelines
Whereas the Village Legislative Authority would like to set guidelines to regulate the garbage collection.
Whereas the guidelines will regulate the garbage pick-up dates and times, as well as set limits how much refuse is allowed.
Whereas the guidelines further address required containers, in addition to what items are suitable/not suitable for collection.
Now Therefore let it be ordained by VILLAGE OF AMANDA, OHIO:
That the village Legislative Authority does hereby set Garbage Guidelines set forth in the attached appendix A, to regulate garbage collection.
That this ordinance shall take effect upon the earliest date allowed by law.
Mayor Mark A. Moore
Vote 5 yes 0 no
Attest:
Carrie Ayers Fiscal Officer
The three reading requirement was waived/not waived:
Yea 5 nays 0
The undersigned, Fiscal Officer of the Legislative Authority, does hereby certify that the foregoing legislation was posted in not less than five (5) public places, as determined by the legislative authority, for a period of not less than fifteen (15) days prior to the effective date thereof.
Fiscal Officer
1st Reading 11/05/18
2nd Reading 12-3-18
Garbage Guidelines
- Garbage is collected 1 day per week on your scheduled pick up day (Tuesday). Selective dumpsters are set up on a 2 day a week pick up the alternate day is (Friday). Pick up times runs between 6 A.M. and 5 P.M.
- Garbage must be set out no earlier than the night before pick-up. Containers are to be removed the same day.
- All garbage must be placed within 5 feet of curb or alley. (Do not block road or sidewalk).
- Major holiday of Thanksgiving and Christmas collection will run one day behind.
- City residents are limited to 8-45 gallon cans or bags or 2-96-gallon rollout containers a week. Weighing no more than 50 pounds per container. Excluding yard waste. Cardboard boxes and burning barrels are not acceptable containers. Ashe containers shall be made of metal; ash and rubbish containers shall be of a kind suitable for collection purposes and shall be of such weight that can be handled by one person.
- Home cleanouts are in excess of regular trash service and will be subject to additional charges.
- Brush and tree limbs, carpets, etc. must be bundled and tied in lengths no longer than 3 to 4 feet.
- One large item a month at no cost. Any additional large items will be charged. 24-hour advance notice to notify drivers of large item pick-up.
- Special and Hazardous waste including but not limited to construction material, rocks, dirt, metal pipe, concrete, liquids, paint, batteries, tires, hazardous chemicals, anything with Freon (refrigerators and air conditioners), or any Epa prohibited items will NOT be collected.
- If animals or weather scatter trash from your trash cans it is NOT the trash haulers responsibility to clean up, Residents are responsible for cleaning their area. To avoid this make sure all refuse is in a lidded container.
January 2019
Re: The Ohio Basic Code, 2019 Edition
Enclosed please find your subscription order for the *Ohio Basic Code, 2019 Edition*. Please note that the *Ohio Basic Code, 2019 Edition*, has been designed to replace, in its entirety, any previous editions of the Ohio Basic Code.
We are pleased to provide the *Ohio Basic Code, 2019 Edition*. Should you have any questions, please do not hesitate to contact our office.
Very truly yours,
Todd Paul Myers, J.D.
Vice President, Major Client Services
firstname.lastname@example.org
AN ORDINANCE APPROVING, ADOPTING AND ENACTING AMERICAN LEGAL PUBLISHING’S OHIO BASIC CODE, 2019 EDITION, AS THE CODE OF ORDINANCES FOR THE MUNICIPALITY OF Village of Amanda, OHIO, AND DECLARING AN EMERGENCY.
WHEREAS, the present general and permanent ordinances of the municipality are inadequately arranged and classified and are insufficient in form and substance for the complete preservation of the public peace, health, safety and general welfare of the municipality and for the proper conduct of its affairs.
WHEREAS, American Legal Publishing Corporation publishes a Code of Ordinances suitable for adoption by municipalities in Ohio.
WHEREAS, it is necessary to provide for the usual daily operation of the municipality and for the immediate preservation of the public peace, health, safety and general welfare of the municipality that this ordinance take effect at an early date.
NOW, THEREFORE, BE IT ORDAINED BY THE LEGISLATIVE AUTHORITY OF THE MUNICIPALITY OF Village of Amanda, OHIO:
Section 1. American Legal Publishing’s Ohio Basic Code, 2019 Edition, as reviewed and approved by the Legislative Authority, is hereby adopted and enacted. Any prior version of the Ohio Basic Code which may have been previously adopted by the municipality is hereby repealed as obsolete and is hereby replaced in its entirety by this Ohio Basic Code, 2019 Edition.
Section 2. One copy of American Legal Publishing’s Ohio Basic Code, 2019 Edition, certified as correct by the Mayor and Clerk of the Legislative Authority, as required by Ohio Revised Code § 731.23, shall be kept in its initial form on file in the office of the Clerk of the municipality and retained as a permanent ordinance record of the municipality. The Clerk of the municipality is authorized and directed to publish a summary of all new matters contained in the Code of Ordinances as required by Ohio Revised Code § 731.23. Such summary is attached hereto and marked as “Exhibit A”.
Section 3. All ordinances and resolutions or parts thereof which are in conflict or inconsistent with any provision of the Ohio Basic Code, 2019 Edition, as adopted in Section 1 hereof, are hereby repealed as of the effective date of this ordinance, except as follows:
(A) The enactment of the Ohio Basic Code, 2019 Edition, shall not be construed to affect a right or liability accrued or incurred under any legislative provision prior to the effective date of such enactment, or an action or proceeding for the enforcement of such right or liability. Such enactment shall not be construed to relieve any person from punishment for an act committed in violation of any such legislative provision, nor to affect an indictment or prosecution therefor. For such purposes, any such legislative provision shall continue in full force notwithstanding its repeal for the purpose of revision and codification.
(B) The repeal provided above shall not affect:
(1) The grant or creation of a franchise, license, right, easement or privilege;
(2) The purchase, sale, lease or transfer of property;
(3) The appropriation or expenditure of money or promise or guarantee of payment;
(4) The assumption of any contract or obligation;
(5) The issuance and delivery of any bonds, obligations or other instruments of indebtedness;
(6) The levy or imposition of taxes, assessments or charges;
(7) The establishment, naming, vacating or grade level of any street or public way;
(8) The dedication of property or plat approval;
(9) The annexation or detachment of territory;
(10) Any legislation enacted subsequent to the adoption of this ordinance.
(11) Any legislation specifically superseding the provision of the Ohio Basic Code.
Section 4. Whenever reference is made in any documents, publications, or signs of the municipality, including but not limited to traffic tickets and traffic-control signs, to a section as it existed in a former edition of the Ohio Basic Code, the reference shall extend and apply to the section referred to as subsequently amended, revised, recodified, or renumbered.
Section 5. This ordinance is declared to be an emergency measure necessary for the immediate preservation of the peace, health, safety and general welfare of the people of this municipality, and shall take effect at the earliest date provided by law.
Date Passed: 2-1-19
Attest:
Mayor
Clerk of the Legislative Authority
Notice is hereby given that on the 4th day of Feb., 2019, there was enacted by the Legislative Authority of the Municipality of Village of Avon, Ohio, an ordinance entitled “An Ordinance Approving, Adopting and Enacting American Legal Publishing’s Ohio Basic Code, 2019 Edition, as the Code of Ordinances for the Municipality of Village of Avon, Ohio.”
A summary of the subjects, including all new matters contained in the Code of Ordinances, as adopted, are as follows. The majority of Basic Code provisions are based directly on state law.
---
**TITLE I: GENERAL PROVISIONS**
Chapter 10: General Provisions
| Section | Title |
|---------|--------------------------------------------|
| 10.01 | Short titles |
| 10.02 | Definitions |
| 10.03 | Rules of construction |
| 10.04 | Revivor; effect of amendment or repeal |
| 10.05 | Construction of section references |
| 10.06 | Conflicting provisions |
| 10.07 | Severability |
| 10.08 | Reference to offices |
| 10.09 | Errors and omissions |
| 10.10 | Ordinances repealed |
| 10.11 | Ordinances unaffected |
| 10.12 | Ordinances saved |
| 10.13 | Application to future ordinances |
| 10.14 | Interpretation |
| 10.15 | Amendments to code; amendatory language |
| 10.16 | Statutory references |
| 10.17 | Preservation of penalties, offenses, rights and liabilities |
| 10.18 | Determination of legislative intent |
| 10.99 | General penalty |
---
**TITLE III: ADMINISTRATION**
Chapter 30: General Provisions
| Section | Title |
|---------|--------------------------------------------|
| 30.01 | Application of Title III |
| 30.02 | Qualifications; oaths |
| 30.03 | Bonds of officers and employees; amount |
| 30.04 | Additional bond; where bonds recorded and kept |
| 30.05 | Approval of bonds |
| 30.06 | Sufficiency of form of bond |
| 30.07 | Filling vacancies in offices |
| 30.08 | Public records available |
| 30.09 | Records Commission |
| 30.10 | Meetings of public bodies to be open; exceptions; notice |
| 30.11 | Municipal officers may attend conference or convention; expenses |
| 30.12 | Residency requirements prohibited; exceptions |
Chapter 31: Executive Authority
Section
*General Provisions*
31.001 Executive power; where vested
**Mayor**
31.015 Term of Mayor; power and duties
31.016 General duties of the Mayor
31.017 Communications to the Legislative Authority
31.018 Protest against excess of expenditures
31.019 Supervision of conduct of officers
31.020 Annual report to the Legislative Authority
31.021 Mayor to file charges against delinquent officers
31.022 Vacancies in office of Mayor
31.023 Disposition of fines and other moneys
**Clerk**
31.040 Election, term, qualifications of the Clerk
31.041 Powers and duties of Clerk
31.042 Books and accounts; merger of offices
31.043 Seal of Clerk
31.044 Combined offices of Clerk and Treasurer; Fiscal Officer
**Treasurer**
31.060 Election, term, qualifications of the Treasurer
31.061 Accounts of Treasurer
31.062 Powers and duties
31.063 Quarterly account; annual report
31.064 Receipt and disbursement of funds
31.065 Duty of delivering money and property
**Street Commissioner**
31.080 Qualifications
31.081 General duties
31.082 Assistants
**Other Officials**
31.100 Legal counsel
31.101 Administrator
31.102 Board of Trustees of Public Affairs
31.103 Fire Engineer, Engineer and Superintendent of Markets
**Chapter 32: Legislative Authority**
**General Provisions**
32.001 Members of the Legislative Authority; election; terms of office
32.002 President Pro Tempore; employees
32.003 Vacancy when President Pro Tempore becomes Mayor
32.004 Qualifications of members of the Legislative Authority
32.005 Compensation and bonds of municipal officers and employees
32.006 Vacancy
32.007 Judge of election and qualification of members; quorum and special meetings
32.008 Rules; journal; expulsion of members
32.009 Meetings
32.010 General powers
32.011 Failure to take oath or give bond
32.012 Notice when new bond required
32.013 Care, supervision and management of public institutions
### Contracts, Bids and Proceedings
| Section | Description |
|---------|-------------|
| 32.025 | Contracts by the Legislative Authority or Administrator |
| 32.026 | Bids and proceedings |
| 32.027 | Alterations or modifications of contract |
| 32.028 | Contract restrictions |
| 32.029 | Award to lowest responsive and responsible bidder |
### Ordinances and Resolutions
| Section | Description |
|---------|-------------|
| 32.040 | Ordinances and resolutions as evidence |
| 32.041 | Passage procedure |
| 32.042 | Style of ordinances |
| 32.043 | Subject and amendment of ordinances and resolutions |
| 32.044 | Authentication and recording of ordinances and resolutions |
| 32.045 | Publication of ordinances and resolutions; proof of publication and circulation |
| 32.046 | Notice for proposed amendments to the municipal Charter |
| 32.047 | Times of publication required |
| 32.048 | Publication and certification of ordinances in book form |
| 32.049 | Adoption of technical ordinances and codes |
| 32.050 | Certificate of Clerk as to publication |
| 32.051 | Publication when no newspaper published in municipality |
| 32.052 | Effect of not making publication |
| 32.053 | Ordinances providing for appropriations or street improvements; emergency ordinances |
### Initiative and Referendum
| Section | Description |
|---------|-------------|
| 32.070 | Initiative petitions |
| 32.071 | Referendum petitions |
| 32.072 | More than one ordinance required; application of subchapter |
| 32.073 | Presentation of petitions |
| 32.074 | Copy of proposed ordinance or measure to be filed with Clerk |
| 32.075 | Words to be printed in red |
| 32.076 | Designation of committee filing petition; public inspection of petitions; ordinances passed or repealed prior to election |
| 32.077 | Itemized statement by petition circulator |
| 32.078 | Prohibited practices relative to petitions |
| 32.079 | Accepting premiums for signing |
| 32.080 | Threats in securing signatures |
| 32.081 | Application of subchapter if Charter adopted |
### Chapter 33: Judicial Authority
#### General Provisions
| Section | Description |
|---------|-------------|
| 33.01 | Jurisdiction in ordinance cases and traffic violations |
| 33.02 | Powers of Mayor and Mayor's Court Magistrate in criminal matters |
| 33.03 | Duties of Mayor and Mayor's Court Magistrate; fees; office; seal |
| 33.04 | Mayor's Court Magistrate |
| 33.05 | Powers to suspend driver's license in OVI cases |
#### Contempt of Court
| Section | Description |
|---------|-------------|
| 33.20 | Summary punishment for contempt |
| 33.21 | Acts in contempt of court |
| 33.22 | Hearing |
| 33.23 | Contempt action for failure to pay support, failure to comply or interference with a visitation order; summons |
| 33.24 | Right of accused to bail |
| 33.25 | Hearing on contempt; penalties; support orders; failure to withhold or deduct money pursuant to support order |
| 33.26 | Imprisonment until order obeyed |
| 33.27 | Proceedings when party released on bail fails to appear |
33.28 Release of prisoner committed for contempt
33.29 Judgment final
33.30 Alternative remedy
Chapter 34: Police Department
Section
34.01 Marshal and Police Chief synonymous
34.02 Appointment of Marshal
34.03 Deputy marshals and police officers
34.04 Auxiliary police units
34.05 Offenses affecting employment of law enforcement officers; probationary period; final appointment
34.06 Removal proceedings; suspension; appeals
34.07 General powers
34.08 Powers and duties of Marshal
34.09 Disposition of fines and penalties
34.10 Property recovered by police
34.11 Disposition to claimant
34.12 Sale of unclaimed property; disposition of proceeds
34.13 Expenses of storage and sale; notice
34.14 Contracts for police protection; nonresident service without contract
34.15 Peace officer administering oaths; acknowledging complaints, summonses, affidavits and returns of court orders
Chapter 35: Fire Department
Section
General Provisions
35.01 Municipal fire regulations; fire department
35.02 Fire Chief; Fire Prevention Officer; employment of firefighters; criminal records check for firefighters
35.03 Schooling of officers and firefighters of fire department
35.04 Legislative Authority may purchase engines and equipment
35.05 Buildings for department
35.06 Records
35.07 Maximum consecutive hours for firefighters on duty
35.08 Investigation of cause of fire
35.09 Right to examine buildings, premises, and vehicles
35.10 Burning buildings for firefighting instruction or research
35.11 Impersonating fire safety inspector
35.12 Standards for equipment
35.13 Persons entitled to be known as firefighters
35.14 Firefighting and emergency services agreements
35.15 Regulation of construction in fire limits
Volunteer Firefighters’ Dependents Fund Board
35.30 Definitions
35.31 Establishment
35.32 Membership; vacancies
35.33 Election and term of members
35.34 Organization; rules and regulations; roster
35.35 Compensation and expenses of Board; legal advisor
Chapter 36: Civil Actions Against the Municipality
Section
36.01 Definitions
36.02 Nonliability of municipality; exceptions
36.03 Defenses and immunities
36.04 Limitation of actions
36.05 Damages
36.06 Satisfaction of judgments
36.07 Defending and indemnifying employees
36.08 Liability insurance
36.09 Certain actions unaffected
36.10 Certain charges against municipal officers filed with Probate Judge; proceedings
TITLE VII: TRAFFIC CODE
Chapter 70: General Provisions
General Provisions
| Section | Description |
|---------|-------------|
| 70.01 | Definitions |
| 70.02 | Compliance with order of police officer |
| 70.03 | Emergency vehicles to proceed cautiously past red or stop signal |
| 70.04 | Exceptions generally; emergency, public safety and coroner vehicles exempt |
| 70.05 | Persons riding or driving animals upon roadways |
| 70.06 | Prohibitions against pedestrians and slow-moving vehicles on freeways |
| 70.07 | Use of private property for vehicular travel |
| 70.08 | Names of persons damaging real property by operation of vehicle to be provided to owner |
| 70.09 | Limited access highways; barriers along; vehicles to enter and leave at designated intersections |
| 70.10 | Through highways |
| 70.11 | Officer may remove ignition key |
| 70.12 | Removal of vehicles after accidents |
Traffic-Control Devices
| Section | Description |
|---------|-------------|
| 70.30 | Obeying traffic-control devices |
| 70.31 | Signal lights |
| 70.32 | Signals over reversible lanes |
| 70.33 | Ambiguous or non-working traffic signals |
| 70.34 | Pedestrian-control signals |
| 70.35 | Unauthorized signs and signals prohibited |
| 70.36 | Alteration, defacement, or removal prohibited |
| 70.37 | Unauthorized possession or sale of devices |
| 70.38 | Signal preemption devices; prohibitions |
| 70.99 | Penalty |
Chapter 71: Licensing Provisions
Motor Vehicle Licensing
| Section | Description |
|---------|-------------|
| 71.01 | Display of license plates or validation stickers; registration |
| 71.02 | Improper use of noncommercial motor vehicle |
| 71.03 | Operating motor vehicle ordered immobilized; forfeiture |
| 71.04 | Operation or sale without certificate of title |
| 71.05 | Display of certificate of registration |
| 71.06 | Use of unauthorized plates |
| 71.07 | Operating without dealer or manufacturer license plates |
Driver's Licenses
| Section | Description |
|---------|-------------|
| 71.20 | Prohibited acts |
| 71.21 | Permitting minor to operate vehicle prohibited; temporary instruction permit; probationary license |
| 71.22 | License required as driver or commercial driver on public or private property; nonresident exemption |
| 71.23 | Employment of a minor to operate a taxicab prohibited |
| 71.24 | Restriction against owner lending vehicle for use of another |
| 71.25 | Suspension of driver's licenses; license suspended by court of record |
| 71.26 | Display of license |
| 71.27 | Prohibition against false statements |
| 71.28 | Driving under suspension or in violation of license restriction |
71.29 Operating motor vehicle or motorcycle without valid license
71.30 Driving under OVI suspension
71.31 Driving under financial responsibility law suspension or cancellation; driving under a nonpayment of judgment suspension
71.32 Failure to reinstate license
**Commercial Driver's Licenses**
71.45 Definitions
71.46 Use of actual gross weight in lieu of rating
71.47 Prohibited acts
71.48 Prerequisites to operation of commercial motor vehicle
71.49 Physical qualification to operate commercial motor vehicles
71.50 Criminal offenses
71.51 Application of federal regulations
71.52 Employment of drivers of commercial vehicles
71.99 Penalty
---
**Chapter 72: Traffic Rules**
**General Provisions**
72.001 Lanes of travel upon roadways
72.002 Driving through safety zone
72.003 Vehicles traveling in opposite directions
72.004 Rules governing overtaking and passing of vehicles
72.005 Permission to overtake and pass on the right
72.006 Driving to left of center line
72.007 Prohibition against driving upon left side of roadway
72.008 Hazardous zones
72.009 One-way highways and rotary traffic islands
72.010 Rules for driving in marked lanes
72.011 Space between moving vehicles
72.012 Divided roadways
72.013 Rules for turns at intersections
72.014 U-turns and turning in roadway prohibited
72.015 Starting and backing vehicles
72.016 Turn and stop signals
72.017 Hand and arm signals
**Right-of-Way**
72.030 Right-of-way at intersections
72.031 Right-of-way when turning left
72.032 Right-of-way at through highways; stop signs; yield signs
72.033 Stop at sidewalk area; stop signs on private roads and driveways
72.034 Right-of-way on public highway
72.035 Pedestrian on sidewalk has right-of-way
72.036 Right-of-way of public safety vehicles
72.037 Funeral procession has right-of-way
72.038 Pedestrians yield right-of-way to public safety vehicle
72.039 Pedestrian on crosswalk has right-of-way
72.040 Right-of-way yielded to blind person
72.041 Right-of-way yielded by pedestrian
**Pedestrians**
72.055 Pedestrian movement in crosswalks
72.056 Pedestrian walking along highway
72.057 Prohibition against soliciting rides; riding on outside of vehicle
72.058 Pedestrian on bridge or railroad crossing
72.059 Persons operating motorized wheelchairs
72.060 Intoxicated or drugged pedestrian hazard on highway
72.061 Operation of electric personal assistive mobility devices
72.062 Operation of personal delivery device on sidewalks and crosswalks
**Grade Crossings**
72.075 Stop signs at grade crossings
72.076 Driving vehicle across railroad grade crossing
72.077 Vehicles required to stop at grade crossings
72.078 Slow-moving vehicles or equipment crossing railroad tracks
**School Buses**
72.090 Regulations concerning school buses
72.091 Violation of regulations; report; investigation; citation; warning
72.092 Restrictions on the operation of school buses
72.093 School bus inspection
72.094 School bus not used for school purposes
72.095 Licensing by Department of Public Safety
72.096 Registration and identification of school buses
72.097 School bus marking
72.098 Flashing light signal lamps
72.099 Occupant restraining device for operator
**Prohibitions**
72.115 Obstruction and interference affecting view and control of driver
72.116 Occupying travel trailer while in motion
72.117 Driving upon closed highway prohibited
72.118 Driving upon sidewalk area or paths exclusively for bicycles
72.119 Obstructing passage of other vehicles
72.120 Following an emergency or public vehicle prohibited; approaching stationary public safety vehicle and certain other vehicles with caution
72.121 Driving over unprotected fire hose
72.122 Placing injurious material on highway or depositing litter from motor vehicle
72.123 Transporting child not in child-restraint system prohibited
72.124 Occupant restraining devices
72.125 Use of engine brakes prohibited
72.126 Operating motor vehicle while wearing earphones or earplugs
72.127 Chauffeured limousines and livery services
72.128 Operating traction engine upon improved highway
72.129 Cracking exhaust noises; peeling out
72.130 Shortcutting across private property
72.131 Texting while driving prohibited
72.132 Use of electronic wireless communication devices by minors or probationary drivers while driving prohibited
**Chapter 73: Motor Vehicle Crimes**
**General Provisions**
73.01 Driving under the influence of alcohol or drugs
73.02 Implied consent
73.03 Physical control of vehicle while under the influence
73.04 Driving commercial vehicle with impaired alertness or ability; use of drugs
73.05 Reckless operation of vehicles
73.06 Reckless operation off streets and highways; competitive operation
73.07 Operator to be in reasonable control
73.08 Immobilizing or disabling device violation
73.09 Street racing defined; prohibited on public highways
73.10 Speed limits
73.11 Slow speed or stopping
73.12 Emergency vehicles excepted from speed limitation
73.13 Speed regulations on bridges
73.14 Presenting false name or information to officer
73.15 Prohibition against resisting officer
73.16 Operation restricted for mini-trucks and low-speed, under-speed, or utility vehicles
**Stopping After Accident**
73.30 Failure to stop after accident
73.31 Stopping after accident on other than public roads or highways
73.32 Accident involving damage to realty
73.33 Failure to report accident
**Chapter 74: Equipment and Loads**
**Equipment**
74.01 Unsafe vehicles, prohibition against operation
74.02 Bumpers on motor vehicles
74.03 Lighted lights required
74.04 Headlights
74.05 Tail lights and illumination of rear license plate
74.06 Red reflectors required
74.07 Safety lighting of commercial vehicles
74.08 Stoplight regulations
74.09 Obscured lights on vehicles
74.10 Red light or flag required
74.11 Lights on parked vehicles
74.12 Lights and emblem on slow-moving vehicles; lights and reflectors on multi-wheel agricultural tractors or farm machinery
74.13 Spotlight and auxiliary driving lights
74.14 Cowl, fender, and back-up lights
74.15 Two lights displayed
74.16 Headlights required
74.17 Lights of less intensity
74.18 Number of lights permitted; red and flashing lights
74.19 Standards for lights on snow removal equipment and oversize vehicles
74.20 Flashing lights permitted for certain types of vehicles
74.21 Lights and sign on transportation for preschool children
74.22 Focus and aim of headlights
74.23 Brake equipment; specifications
74.24 Brake fluid
74.25 Minimum standards for brakes and components
74.26 Horns, sirens, and warning devices
74.27 Mufflers; excessive smoke or gas
74.28 Rearview mirrors
74.29 Windshields and wipers
74.30 Solid tire requirements
74.31 Requirements for safety glass in motor vehicles; use of tinted glass or reflectorized material
74.32 Directional signals
74.33 Installation and sale of seat safety belts required; definition
74.34 Requirements for extra signal equipment
74.35 Display of warning devices on disabled vehicles
74.36 Requirements for vehicles transporting explosives
74.37 Studded tires; seasonal use permitted
74.38 Safety inspection decals for buses
74.39 Air bags
**Loads**
74.50 Permit required to exceed load limits
74.51 Limitation of load extension on left side of vehicle
74.52 All loads shall be properly secured
74.53 Towing requirements; exception to size and weight restrictions
74.54 Weighing of vehicle; removal of excess load
74.55 Operation of vehicle exceeding weight limits prohibited
74.56 Load limits
74.57 Maximum width, height, and length
74.58 Statement of gross vehicle weight
74.59 Wheel protectors required on heavy commercial vehicles
74.60 Liability for damages; prosecution; application of monies
74.61 Weight exceptions for certain vehicles
Chapter 75: Bicycles, Motorcycles and Off-Road Vehicles
Section
General Provisions
75.01 Bicycles; application of Title VII
75.02 Operation of motorized bicycle
75.03 Rules for bicycles, motorcycles, and snowmobiles
75.04 Prohibition against attaching bicycles and sleds to vehicles
75.05 Riding bicycles; motorcycles abreast
75.06 Equipment of bicycles
Snowmobiles, Off-Highway Motorcycles, and All-Purpose Vehicles
75.25 Definitions
75.26 Equipment
75.27 Code application; prohibited operation
75.28 Permitted operation
75.29 Licensing requirements of operator
75.30 Maintenance of vehicles for hire
75.31 Accident reports
75.32 Impounding of vehicle
75.33 Local control within police power
75.34 Registration of vehicles
75.35 Certificate of title; prohibitions
Chapter 76: Parking Regulations
Section
76.01 Prohibition against parking on highways
76.02 Condition when motor vehicle left unattended
76.03 Police may remove illegally parked vehicle
76.04 Parking prohibitions
76.05 Parking near curb; privileges for persons with disabilities
76.06 Parking on private property in violation of posted prohibition
76.07 Selling, washing or repairing vehicle upon roadway
76.08 Truck loading zones
76.09 Bus stops and taxicab stands
76.10 Parking in alleys and narrow streets; exceptions
76.11 Registered owner prima facie liable for unlawful parking
76.12 Waiver
TITLE IX: GENERAL REGULATIONS
Chapter 90: Animals
Section
Animals Running at Large
90.01 Dogs or other animals running at large; nuisance, dangerous or vicious dogs; hearings
90.02 Confining animal found at large; publication of notice; lien
90.03 Unavoidable escapes
90.04 Fees
90.05 Rabies quarantine orders of Mayor
90.06 Interfering with enforcement of quarantine orders
90.07 Dogs may be killed for certain acts
Offenses Relating to Animals
90.20 Abandoning animals
90.21 Injuring animals
90.22 Poisoning animals
90.23 Cruelty to animals; cruelty to companion animals
90.24 Animal fights
90.25 Trapshooting
90.26 Loud dog
90.27 Dog tags
90.28 Nuisance, dangerous and vicious dog defined; transfer of ownership certificate; form stating dog's prior behavior
90.29 Failure to register dog or dog kennel
90.30 Hindering the capture of unregistered dog
90.31 Unlawful tag
90.32 Rights of blind, deaf or hearing impaired, or mobility impaired person, or trainer with assistance dog
90.33 Retail sale and transportation of dogs
90.34 Coloring rabbits or baby poultry; regulation of sale and display
90.35 Jacklighting prohibited
90.36 Restrictions on dog ownership for certain convicted felons
90.37 Sexual conduct with an animal
90.99 Penalty
Chapter 91: Fireworks, Explosives, Fire Prevention
Section
Fireworks and Explosives
91.01 Definitions
91.02 Possession, sale, and use of fireworks
91.03 Permit to use fireworks
91.04 Manufacturing or wholesale sale without a license; prohibitions
91.05 Purchasers to comply with law; unauthorized purchases
91.06 Exhibition without a license; prohibitions
91.07 Unauthorized transportation or shipping
91.08 Application of subchapter
91.09 Arrest of offender; seizure and forfeiture of fireworks; distribution of fines
91.10 Safety requirements for fireworks showroom structures
91.11 Storage of explosives
91.12 Blasting permit
Fire Prevention
91.30 Removal of flammable materials or obstructions
91.31 Protective appliances
91.32 Compliance with order
91.33 Waste receptacles
91.34 Hotel to have fire warning device producing visible signal
91.35 Fire suppression systems
91.36 Violations of State Fire Code prohibited
91.37 Posting arson notices in hotels, motels and other places
91.38 Negligent burning
91.39 Spreading alarm of unfriendly fire
91.40 Unvented heaters
Open Burning
91.55 Definitions
91.56 Relations to other prohibitions
91.57 Open burning in restricted areas
91.58 Permission and notice to open burn
91.99 Penalty
Chapter 92: Intoxicating Liquors
Section
| Section | Description |
|---------|-------------|
| 92.01 | Definitions |
| 92.02 | Exemptions from chapter |
| 92.03 | Restrictions applicable to sale of beer and intoxicating liquor for consumption on the premises |
| 92.04 | Restrictions on sale of beer and liquor |
| 92.05 | Permit required; activities prohibited without permit |
| 92.06 | Illegal transportation prohibited |
| 92.07 | Open container prohibited; exception |
| 92.08 | Underage person shall not purchase intoxicating liquor or beer |
| 92.09 | Prohibitions; minors under 18 years; low-alcohol beverages |
| 92.10 | Alcohol vaporizing devices prohibited |
| 92.11 | Misrepresentation to obtain alcoholic beverage for a minor prohibited |
| 92.12 | Misrepresentation by a minor under 21 years |
| 92.13 | Sale to underage persons prohibited |
| 92.14 | Posting of card |
| 92.15 | Good faith acceptances of spurious identification |
| 92.16 | Consumption in motor vehicle prohibited |
| 92.17 | Hours of sale or consumption |
| 92.18 | Obstructing search of premises prohibited |
| 92.19 | Illegal possession of intoxicating liquor prohibited |
| 92.20 | Sale or possession of diluted liquor and refilled containers prohibited |
| 92.21 | Keeping place where beer or intoxicating liquors are sold in violation of law |
| 92.22 | Intoxicating liquors shall not be sold in brothels |
| 92.23 | Use of intoxicating liquor in a public dance hall prohibited; exceptions |
| 92.24 | Poisonously adulterated liquors |
| 92.25 | Tavern keeper permitting rioting or drunkenness |
| 92.26 | Notice of action to prohibit liquor business |
| 92.27 | Procedure when injunction violated |
| 92.28 | Liquor transaction scans |
| 92.29 | Affirmative defenses |
| 92.99 | Penalty |
Chapter 93: Nuisances
Section
General Provisions
| Section | Description |
|---------|-------------|
| 93.01 | Application of the chapter |
| 93.02 | Definitions |
| 93.03 | Nuisances generally; injunctions; violation; contempt |
| 93.04 | Maintaining certain nuisances |
| 93.05 | Collection of cost of abating dangerous property condition; injunction; rehabilitation |
| 93.06 | Trimming of trees and shrubbery to prevent obstruction |
Septic Tanks, Cesspools, and Refuse
| Section | Description |
|---------|-------------|
| 93.20 | Location of privy vaults, cesspools, and septic tanks |
| 93.21 | Unsanitary vaults |
| 93.22 | Removal of contents of vault |
| 93.23 | Deposit of dead animals, offal upon land or water |
| 93.24 | Defiling spring or well prohibited |
| 93.25 | Dumping of refuse in municipality prohibited |
| 93.26 | Abandoned refrigerators |
| 93.27 | Discarding litter prohibited |
| 93.28 | Power of municipality to fill or drain land |
Weeds and Litter on Private Property
| Section | Description |
|---------|-------------|
| 93.40 | Keeping down weeds |
| 93.41 | Notice to owner to cut noxious weeds, remove litter; service |
| 93.42 | Fees for service and return |
| 93.43 | Procedure when owner fails to comply with notice |
| 93.44 | Written return to County Auditor; amount as a lien upon property |
Unclean Habitations
93.60 Permitting unclean habitations
93.61 When habitations are deemed unsanitary
93.62 Order for abatement or vacation of premises
93.63 Enforcement of vacation order by Fire Chief or Police Chief
93.64 Enforcement through court proceedings
93.99 Penalty
Chapter 94: Streets and Sidewalks
General Provisions
94.01 Conditions precedent to improving streets
94.02 Opening permit required
94.03 Application and cash deposit
94.04 Restoration of pavement
94.05 Barriers around excavations
94.06 Warning lights
94.07 Sidewalk construction by the municipality
94.08 Unloading on street or sidewalk
94.09 Street or sidewalk obstruction
94.10 Materials on street or sidewalk
94.11 Duty to keep sidewalks in repair and clean of ice and snow
94.12 Ramped curbing for persons with disabilities
94.13 Flagpole along right-of-way
94.14 Altering or injuring marker or monument
Construction and Repair
94.25 Construction and repair may be required
94.26 Resolution of necessity
94.27 Notice to construct or repair
94.28 Assessments of costs against owner
94.29 Proceedings may include different owners
94.30 Making and levying assessments
Changes in Streets
94.40 Change of name, vacating or narrowing streets by petition
94.41 Change of name, vacating or narrowing streets without petition
94.42 Notice; exception
94.43 Publication of notice
94.44 Effect of order of vacation
94.45 Effect on public utility easements
94.99 Penalty
Chapter 95: Unclaimed and Abandoned Vehicles
Section
95.01 Impounding motor vehicle on private property; requirements
95.02 Impounding abandoned motor vehicle on public property; notice; disposition
95.03 Disposition of vehicle ordered into storage
95.04 Disposition of abandoned junk motor vehicles
95.05 Abandonment of junk motor vehicle prohibited
95.06 Junk motor vehicle; order to cover or remove; notice; exceptions
Chapter 96: Watercraft
Section
96.01 Definitions; applicability
96.02 Failure to comply with law enforcement order; fleeing
96.03 Duty upon approach of law enforcement vessel
96.04 Flashing lights prohibited; exceptions
96.05 Siren prohibited; exceptions
96.06 Regulations for operation of powercraft of more than ten horsepower
96.07 Restrictions on child operators; duty of supervisory adult
96.08 Reckless operation; maintaining sufficient control; wakes restricted
96.09 Unsafe conditions
96.10 Marking of bathing and vessel areas
96.11 Mooring prohibited in certain areas
96.12 Operating under influence of alcohol or drugs prohibited
96.13 Implied consent
96.14 Incapacitated operators prohibited
96.15 Water skiing confined to ski zones
96.16 Observer required when towing skier
96.17 Water skiing after dark prohibited
96.18 Personal flotation device required for towed person
96.19 Ski jumps prohibited
96.20 Permit for special water events
96.21 Sale of single celled inflatable vessels prohibited
96.22 Sitting, standing, walking on moving vessels restricted
96.23 Engine warm-up required
96.24 Personal flotation devices for children under ten
96.25 Operation without personal flotation devices prohibited
96.26 Distress signal or flag required
96.27 Anchor requirements
96.28 Specification for fire extinguishers
96.29 Backfire flame control device required
96.30 Ventilation requirement on powercraft
96.31 Abandonment of junk vessels or outboard motors
96.32 Exhaust muffler required; noise levels; exceptions
96.33 Safety equipment on rental vessels
96.34 Capacity plate
96.35 Littering prohibited
96.36 Dwellings; sanitary systems
96.37 Prima facie evidence of negligence
96.38 Requirements for operating personal watercraft
96.39 Numbering
96.40 Registration
96.41 Tags indicating expiration date; attachment of identification number
96.42 Altering of serial numbers; false information prohibited
96.43 Accident reports
96.44 Enforcement
96.45 Firearms offenses; signaling devices
96.46 Tampering with navigation aid or vessel prohibited
96.47 Certificate of title; exceptions
96.48 Manufacturer’s or importer’s certificate
96.49 Prohibitions relating to certificates of title
96.50 Permanently displayed hull identification number
96.99 Penalty
TITLE XI: BUSINESS REGULATIONS
Chapter 110: General Provisions
Section
110.01 Licenses required to engage in certain businesses; exceptions
110.02 Application for license
110.03 Issuance of license
110.04 Date and duration of license
110.05 License not transferable
110.06 License certificate to be displayed
110.07 Revocation or suspension
110.08 Appeal and review
110.99 General penalty for Title XI
Chapter 111: Taxicabs
Section
111.01 Definitions
111.02 Certificate of public convenience and necessity required
111.03 Application for certificate
111.04 Issuance of certificate
111.05 Liability insurance required
111.06 License fees
111.07 Transfer of certificates and licenses
111.08 Suspension and revocation of certificates
111.09 Taxicab driver's license
111.10 Application for driver's license
111.11 Examination of applicant; motor vehicle operator's permit required
111.12 Police investigation of applicant; traffic and police record
111.13 Consideration of application
111.14 Issuance of license; duration; annual fee
111.15 Display of license
111.16 Suspension and revocation of license
111.17 Failure to comply with federal, state and municipal laws
111.18 Vehicle equipment and maintenance
111.19 Designation of taxicabs
111.20 Taximeter and display of rates required
111.21 Number of passengers allowed
111.22 Articles left in vehicles
111.23 Vehicles from other municipalities
111.24 Receipts
111.25 Refusal of passenger to pay legal fare
111.26 Solicitation, acceptance and discharge of passengers
111.27 Open stands; use
111.28 Taxicab service
111.29 Manifests
111.30 Records and reports of holders
111.31 Police department; duty to enforce chapter
111.32 Disposition of vehicle license fees
Chapter 112: Peddlers, Itinerant Merchants, and Solicitors
Section
112.01 Definitions
112.02 License requirement
112.03 Application procedure
112.04 Standards for issuance
112.05 Revocation procedure
112.06 Standards for revocation
112.07 Appeal procedure
112.08 Exhibition of identification
112.09 Municipal policy on soliciting
112.10 Notice regulating soliciting
112.11 Duty of solicitors
112.12 Uninvited soliciting prohibited
112.13 Time limit on soliciting
Chapter 113: Commercial Amusements
Section
113.01 Bowling; billiards and pool
113.02 Circuses, carnivals, shows and other such entertainment
113.03 Deposit required
113.04 License fee for public entertainment or exhibition
113.05 License fee may be waived for civic interest
Chapter 114: Tattooing and Body Piercing Services
Section
114.01 Definitions
114.02 Prohibitions
114.03 Application for license; fees; issuance
114.04 Inspection of facilities
114.05 Suspension or revocation of license
114.06 Consent for performing procedures on persons under 18
114.07 Prohibitions relating to persons under 18
114.08 Defenses to violations
114.09 Training standards; records; safety and sanitation; equipment
114.10 Application of local regulation on chapter
114.99 Penalty
TITLE XIII: GENERAL OFFENSES
Chapter 130: General Provisions
Section
130.01 Application of Title XIII
130.02 Definitions
130.03 Classification of offenses
130.04 Common law offenses abrogated
130.05 Rules of construction
130.06 Limitation of criminal prosecutions
130.07 Requirements for criminal liability; voluntary intoxication
130.08 Culpable mental states
130.09 Organizational criminal liability
130.10 Personal accountability for organizational conduct
130.11 Attempt
130.12 Complicity
130.13 Presumption of innocence; proof of offense; affirmative defense
130.14 Battered woman syndrome
130.15 Delinquency adjudications deemed convictions
130.16 Criminal law jurisdiction
130.17 Disposition of unclaimed or forfeited property held by Police Department
130.18 Imposing sentence for misdemeanor
130.19 Multiple sentences
130.20 Apprehension, detention, or arrest of persons on bond
130.21 Self defense: limitations on duty to retreat prior to using force
130.99 Penalty for Title XIII
Chapter 131: Offenses Against Property
Section
131.01 Definitions
131.02 Arson; determining property value or amount of physical harm
131.03 Criminal damaging or endangering; vehicular vandalism
131.04 Criminal mischief
131.05 Damaging or endangering aircraft or airport operations
131.06 Criminal trespass; aggravated trespass
131.07 Tampering with coin machines
131.08 Theft
131.09 Unauthorized use of a vehicle
131.10 Unauthorized use of property, including telecommunication property and computers; possession of municipal property
131.11 Passing bad checks
131.12 Misuse of credit cards
131.13 Making or using slugs
131.14 Prima facie evidence of purpose to defraud
131.15 Tampering with records
131.16 Securing writings by deception
131.17 Defrauding creditors
131.18 Receiving stolen property
131.19 Value of stolen property
131.20 Degree of offense when certain property involved
131.21 Injuring vines, bushes, trees, or crops
131.22 Detention and arrest of shoplifters and those committing motion picture piracy; protection of institutional property
131.23 Insurance fraud; workers' compensation fraud; Medicaid fraud
131.24 Injury to property by hunters
131.25 Evidence of intent to commit theft of rented property; evidence of lack of capacity to consent
131.26 Forgery of identification cards
131.27 Criminal simulation
131.28 Personating an officer
131.29 Trademark counterfeiting
131.30 Diminishing or interfering with forfeitable property
131.31 Recording credit card, telephone or Social Security numbers
131.32 Prosecutions for theft of utilities
131.33 Motion picture piracy
Chapter 132: Offenses Against Public Peace
Section
132.01 Riot
132.02 Failure to disperse
132.03 Justifiable use of force to suppress riot
132.04 Disorderly conduct
132.05 Disturbing a lawful meeting
132.06 Misconduct at an emergency
132.07 Telecommunications harassment
132.08 Inducing panic
132.09 Making false alarms
132.10 Inciting to violence
132.11 Unlawful display of law enforcement emblem
132.12 Impersonating a peace officer
132.13 Safety of crowds attending live entertainment performances
132.14 Misconduct involving a public transportation system
Chapter 133: Sex Offenses
Section
133.01 Definitions
133.02 Unlawful sexual conduct with a minor
133.03 Sexual imposition
133.04 Public indecency
133.05 Voyeurism
133.06 Polygraph examinations for victims: restrictions on use
133.07 Procuring
133.08 Soliciting; loitering to engage in
133.09 Prostitution
133.10 Disseminating matter harmful to juveniles
133.11 Displaying matter harmful to juveniles
133.12 Deception to obtain matter harmful to juveniles
133.13 Rules of evidence
133.14 Declaratory judgment
133.15 Injunction; abatement of nuisance
133.16 Unlawful operation of viewing booths depicting sexual conduct
133.17 Juveniles on the premises of adult entertainment establishments prohibited
133.18 Sexually oriented businesses; illegal operation and activity
133.19 Unlawful advertising of massage
133.99 Sentencing for sexually oriented offenses; sexual predators; registration
Chapter 134: Gambling Offenses
Section
134.01 Definitions
134.02 Prohibitions against gambling; exception
134.03 Operating a gambling house
134.04 Public gaming
134.05 Cheating
134.06 Regulations concerning operation of licensed bingo game
134.07 Records to be kept
134.08 Requirements for bingo game operators
134.09 Bingo games for amusement only
134.10 Prohibitions where instant bingo game is conducted
134.11 Raffle drawings
134.12 Instant bingo other than at bingo sessions
134.13 Restrictions on owner or lessor of location at instant bingo
134.14 Skill-based amusement machines; prohibited conduct
Chapter 135: Offenses Against Persons
Section
135.01 Definitions
135.02 Negligent homicide
135.03 Vehicular homicide; vehicular manslaughter
135.04 Assault; negligent assault
135.05 Injury to persons by hunters
135.06 Menacing; aggravated menacing; menacing by stalking
135.07 Unlawful restraint
135.08 Criminal child enticement
135.09 Coercion
135.10 Bigamy
135.11 Unlawful abortion; failure to perform viability testing
135.12 Abortion trafficking
135.13 Nonsupport of dependents
135.14 Endangering children
135.15 Interference with custody; interference with support orders
135.16 Domestic violence
135.17 Hazing prohibited
135.18 Contributing to unruliness or delinquency of a child
135.19 Failure to provide for functionally impaired person
135.20 Patient abuse or neglect; patient endangerment; exceptions; false statements; retaliation
135.21 Interference with right of person to engage in housing transactions because of race, religion, or the like
135.22 Ethnic intimidation
135.23 Violating a protection order, consent agreement, anti-stalking protection order or order issued by a court of another state
135.24 Adulteration of food
135.25 Illegal distribution of cigarettes, other tobacco products, or alternative nicotine products; transaction scans
135.26 Nonsmoking areas in places of public assembly
135.27 Spreading contagion
135.28 Abuse of a corpse
135.29 Unlawful collection of bodily substances
Chapter 136: Offenses Against Justice and Administration
Section
136.01 Definitions
136.02 Falsification
136.03 Compounding a crime
136.04 Failure to report a crime
136.05 Failure to aid a law enforcement officer
136.06 Obstructing official business
136.07 Obstructing justice
136.08 Resisting arrest
136.09 Having an unlawful interest in a public contract
136.10 Soliciting or receiving improper compensation
136.11 Dereliction of duty
136.12 Interfering with civil rights
136.13 Illegal conveyance of prohibited items onto grounds of a detention facility or other specified governmental facilities
136.14 False report of child abuse or neglect
136.15 Assaulting police dog or horse, or assistance dog
136.16 Disclosure of confidential peace officer information
136.17 Intimidation of crime victim or witness
136.18 Using sham legal process
136.19 Making false allegation of peace officer misconduct
136.20 Misuse of 9-1-1 system
136.21 Failure to disclose personal information
Chapter 137: Weapons Control
Section
137.01 Definitions
137.02 Carrying concealed weapons
137.03 Using weapons while intoxicated
137.04 Improperly handling firearms in a motor vehicle
137.05 Possessing criminal tools
137.06 Failure to secure dangerous ordnance
137.07 Unlawful transactions in weapons
137.08 Underage purchase of firearm or handgun
137.09 Pointing and discharging firearms and other weapons
137.10 License or permit to possess dangerous ordnance
137.11 Possession of an object indistinguishable from a firearm in a school safety zone
137.12 Possession of deadly weapon while under detention
137.13 Concealed handgun licenses: possession of a revoked or suspended license; additional restrictions; posting of signs prohibiting possession
137.14 Defaced firearms
Chapter 138: Drug Offenses
Section
138.01 Definitions
138.02 Trafficking in controlled substances; gift of marihuana
138.03 Drug possession offenses
138.04 Possessing drug abuse instruments
138.05 Permitting drug abuse
138.06 Illegal cultivation of marihuana
138.07 Abusing harmful intoxicants
138.08 Illegal dispensing of drug samples
138.09 Federal prosecution bar to municipal prosecution
138.10 Nitrous oxide: improper dispensing or distribution; possession in a motor vehicle
138.11 Laboratory report required
138.12 Counterfeit controlled substances
138.13 Use, possession, or sale of drug paraphernalia
138.14 Controlled substance or prescription labels
138.15 Possession, sale and disposal of hypodermics
138.16 Controlled substance schedules
138.17 Unlawful furnishing of prescription to enable persons to be issued handicapped parking placards or license plates
138.18 Pseudoephedrine sales
TITLE XV: LAND USAGE
Chapter 150: General Provisions
Section
Parks and Recreation
150.01 Recreation Board
150.02 Board of Park Trustees
Planning and Zoning
150.15 Planning Commission
150.16 Board of Zoning Appeals
PARALLEL REFERENCES
Ohio Legislative History References – Master Table
INDEX
This summary of contents has been verified and authorized for publication by the Legislative Authority of the Municipality of Village of Avon, Ohio.
Signed: [Signature] Mayor
Clerk of the Legislative Authority
CERTIFICATION OF CODIFIED ORDINANCES
We, Mark A. Mear, Mayor, and Carrie Ayers, Clerk of the Legislative Authority, of the Municipality of Village of Anarole, Ohio, pursuant to Ohio Revised Code §§ 731.23 and 731.42, hereby certify that the general and permanent ordinances of the Municipality, as revised, rearranged, compiled, renumbered as to sections, codified and printed herewith in component codes and titles are correct as and constitute the Code of Ordinances for the Municipality of Village of Anarole, Ohio.
[Signature]
Mayor
[Signature]
Clerk of the Legislative Authority
|
Project Overview
Whether traveling to school, running errands, or enjoying a stroll through the park, many people in Pekin choose to walk and bike, and many more would choose to do so if conditions were better.
A safe and connected system of sidewalks, trails, and bikeways not only supports people to traveling by bike and foot, but also contributes to the safety of all road users and to the quality of life for Pekin residents.
The Pekin Bicycle and Pedestrian Master Plan will serve as the City’s vision and blueprint for creating a safer, more comfortable transportation environment for people of all ages, abilities, and backgrounds, especially for people who walk, bike, and use mobility assistance devices like wheelchairs or walkers.
This long-range planning document will fuse inspiration and ideas from Pekin residents and community stakeholders with technical analysis of current conditions and best practices in bicycle and pedestrian planning and facility design.
The final plan will include recommendations for new trails, bikeways, sidewalks, and intersection improvements, as well as supporting policies, programs, and implementation strategies. These recommendations will guide future investments in infrastructure projects and help the City secure funding from state, federal, and other sources.
Planning Inputs
- Field Analysis of Current Conditions
- Public Workshops and Survey Forms
- Existing & Previously Planned Facilities
- Network Quality (LTS) Analysis
- Existing Bike/Ped Activity & Demand Analysis
- Direction from Municipalities and MPO
- Steering Committee & Stakeholder Input
Project Timeline
**PHASE 1: Existing Conditions, Needs Assessment, Visioning**
- FEB
- MAR
- APR
- MAY
- JUN
The first phase of the plan focuses on creating a shared understanding of current conditions for walking and biking and learning from Pekin residents about community needs, issues, and aspirations relating to active transportation. The project team will establish a vision for the Bicycle and Pedestrian Master Plan and supporting goals to help bring the vision to life. These early stages in the planning process will form the foundation for identifying and prioritizing recommendations for new trails, sidewalks, bikeways, and supporting programs.
**PHASE 2: Plan Recommendations & Implementation Strategies**
- JUL
- AUG
- SEP
The second phase of the project focuses on developing recommendations for projects that improve walking and bicycling safety, connectivity, and access, like new sidewalks, bike lanes, and trails. The project team will also identify policy, programming, and project development strategies to support the plan’s vision and integrate walking and biking into transportation decision-making and projects.
**PHASE 3: Draft Plan Development & Review**
- OCT
- NOV
- DEC
The third and final phase of the project focuses on refining the plan recommendations, prioritization, and implementation strategies based on feedback from the open house public meeting and creating a draft plan document for review. After final revisions, the plan will be considered for official adoption.
Pekin Bicycle & Pedestrian Master Plan
September 28, 2023
Open House #2
THE VISION: The City of Pekin strives to make walking and bicycling safe, accessible, convenient, and equitable transportation options that connect people to places, foster recreational and economic development opportunities, support healthy and active living, and elevate quality of life.
GOAL #1 - SAFETY: Create a safe travel environment for everyone, especially vulnerable road users like people walking, rolling, and bicycling.
Objective 1.1. Improve pedestrian and bicycle safety through the installation of sidewalks, trails, dedicated bikeways, and intersection enhancements.
Objective 1.2. Create a safety action plan to evaluate crash history, identify trends, and develop a high-injury network for future safety investments.
Objective 1.3. Reduce fatal and serious injury crashes through proven safety countermeasures.
Objective 1.4. Support safe and responsible travel through education programs and campaigns.
GOAL #2 - NETWORK: Develop a complete, convenient, and interconnected active transportation network.
Objective 2.1. Expand the Pekin Bike Trail to increase community access to one of the community’s most valued recreation and active transportation amenities.
Objective 2.2. Target sidewalk infill, sidewalk system expansion, and crossing improvements on pedestrian priority corridors.
Objective 2.3. Develop a low-stress on-street bikeway system to support access to everyday destinations throughout Pekin.
Objective 2.4. Coordinate with community partners and local, regional, and state agencies to increase regional connectivity and access to destinations in neighboring communities.
Objective 2.5. Develop a community-wide active transportation wayfinding system to guide people to schools, parks, Downtown Pekin, the Pekin Bike Trail, and other major destinations.
Objective 2.6. Prioritize and pursue projects in a manner that balances community impact, project feasibility, and available resources and staff capacity.
GOAL #3 - DESIGN: Design active transportation facilities that are accessible and comfortable for people of all ages and abilities.
Objective 3.1. Apply current standards and best practices for low-stress bicycle and pedestrian facility design.
Objective 3.2. Prioritize projects that address known ADA deficiencies, align with the recommendations in the ADA Transition Plan, and promote safe and accessible pathways for people with disabilities, children, seniors, and people with limited mobility.
Objective 3.2. Evaluate Pekin Bike Trail crossings to improve user safety and to increase motorist awareness.
GOAL #4 - POLICY: Develop policies that integrate active transportation into city decision-making processes.
Objective 4.1. Develop internal procedures to consider and integrate active transportation facilities in capital improvement planning, programming, and project development.
Objective 4.2. Support active transportation through revisions to the zoning ordinance, subdivision regulations, and street standards.
Objective 4.3. Establish baseline community outreach and engagement protocols for transportation projects.
GOAL #5 - PROGRAMS: Foster an environment that supports and embraces walking and biking through community partnerships and education, encouragement, and awareness programs.
Objective 5.1. Foster collaboration and partnerships with community organizations and agencies throughout the region to identify and implement education, encouragement, and awareness programs and campaigns to increase walking and bicycling activity and safety.
Objective 5.2. Develop Safe Routes to School program in partnership with Pekin Public Schools to increase walking and bicycling to school.
Objective 5.3. Create an awareness campaign to raise the profile of active transportation, promote safe travel behaviors, and highlight the benefits of walking and bicycling.
Online Survey Results
Walking-Related Questions
How easy is it to walk in your neighborhood and to nearby destinations?
- Very Easy: 11%
- Moderately Easy: 31%
- Neutral: 15%
- Moderately Difficult: 24%
- Very Difficult: 10%
- Other (please specify): 6%
How far do you typically travel when walking?
- Less than 0.5 miles: 10%
- 0.5 to 1 mile: 17%
- 1 to 2 miles: 36%
- 2 to 5 miles: 31%
- 5 to 10 miles: 3%
- Over 10 miles: 0%
- N/A: 3%
For which of the following reasons do you choose to walk for work or recreation trips? (Check all that apply)
- Safety/security: 23%
- Convenience: 24%
- Health benefits/fitness: 95%
- Environmental benefits: 39%
- For fun/enjoyment: 72%
- Don't have access to a vehicle: 1%
- Don't have access to public transit: 0%
- Other (please specify): 4%
Which of the following barriers prevent you from walking more? (Please select all that apply)
- I do not feel safe and worry about: 55%
- I am physically unable to walk: 1%
- I have no place to park: 68%
- Lack of sidewalks: 18%
- Obstruction to vision: 15%
- Lack of lighting: 43%
- I don't like walking: 27%
- I don't like walking because of children: 8%
- I don't like walking because of crime: 4%
- I don't like walking because of weather: 16%
- Noise: 6%
- I walk too much as it is: 4%
- Other (please specify): 7%
Please evaluate the following sentence based on how it represents you: "I would like to walk more than I do now for my daily commute, errands, and other activities."
- Agree: 42%
- Somewhat Agree: 24%
- Neutral: 21%
- Somewhat Disagree: 7%
- Disagree: 6%
Please evaluate the following sentence based on how it represents you: "I would like to walk more than I do now for fun, exercise, and other recreational activities."
- Agree: 78%
- Somewhat Agree: 16%
- Neutral: 5%
- Somewhat Disagree: 1%
- Disagree: 1%
What destinations would you like to be able to walk to? (Choose all that apply)
- Work: 12%
- Bus Stop: 4%
- School: 11%
- Trails: 75%
- Parks, playgrounds, and recreation centers: 75%
- Grocery stores, local shops, and restaurants: 59%
- Government services (city hall, police station, social services, library, office, etc.): 23%
- Place of worship (church, synagogue, mosque, etc.): 21%
- Other (please specify): 5%
Pekin Bicycle & Pedestrian Master Plan
September 28, 2023
Open House #2
Online Survey Results
Bicycling-Related Questions
How easy is it for you to bike in your neighborhood and to nearby destinations?
- Very easy: 8%
- Moderately Easy: 21%
- Neutral: 24%
- Moderately Difficult: 31%
- Very difficult: 16%
How far do you typically travel when biking?
- Less than 0.5 miles: 5%
- 0.5 to 1 mile: 2%
- 1 to 2 miles: 11%
- 2 to 5 miles: 25%
- 5 to 10 miles: 23%
- Over 10 miles: 11%
- N/A: 22%
For which of the following reasons do you choose to bike for work, school, or recreation trips? (Check all that apply)
- Safety concerns: 28%
- Convenience: 20%
- Health benefits/exercise: 92%
- Environmental benefits: 49%
- For fun/entertainment: 72%
- Don’t have access to a vehicle: 1%
- Don’t have access to public transit: 1%
- Other (please specify): 2%
What type of cyclist best describes you? (One selection allowed)
- Strong and Fearless (Comfortable biking on major streets without bike lanes): 16%
- Enthusiastic and Confident (Comfortable biking on major streets only with bike lanes): 16%
- Interested but Concerned (Only comfortable biking if separated from cars and/or major streets): 54%
- No Way, No How (Not interested in biking at all): 15%
Please evaluate the following sentence based on how it represents you: “I would like to bike more than I do now for my daily commute, errands, and other activities.”
- Agree: 38%
- Somewhat Agree: 24%
- Neutral: 21%
- Somewhat Disagree: 6%
- Disagree: 11%
Please evaluate the following sentence based on how it represents you: “I would like to bike more than I do now for fun, exercise, and other recreational activities.”
- Agree: 63%
- Somewhat Agree: 21%
- Neutral: 8%
- Somewhat Disagree: 5%
- Disagree: 4%
Which of the following bikeway types would you like to see in Pekin? (Check all that apply)
- Shared Lane: 6%
- Painted Shoulder/Shoulder Bike Lane: 50%
- Marked and Signed Bike Route: 49%
- Bike Lane: 59%
- Buffered Bike Lane: 47%
- Separated Bike Lane (Cycle Track): 65%
- Shared Use Path / Speed Hump: 84%
Which of the following barriers prevent you from biking more? (Please select all that apply)
- I don't have time/space to ride: 11%
- Distractions from traffic: 10%
- I live too far from the places I need to go: 4%
- Lack of dedicated bike lanes: 52%
- Lack of safe places to park: 69%
- There isn't an entire place to ride: 58%
- Not enough parking: 32%
- Too much traffic: 22%
- I don't feel safe biking around pedestrians: 22%
- Disability or other health issues: 19%
- Weather prevents me: 17%
- None. I like as much as I want to bike (like I currently do): 4%
- Other (please specify): 15%
What destinations would you like to be able to bike to? (Choose all that apply)
- Work: 27%
- Bus Stop: 3%
- School: 12%
- Trails: 94%
- Parks, playgrounds, and recreation centers: 77%
- Grocery stores, local shops, and restaurants: 55%
- Government services (city hall, library, social services, post office, etc.): 28%
- Place of worship (church, temple, mosque, etc.): 17%
- Other (please specify): 6%
Bicycle Improvements Toolkit
There is no one-size-fits-all approach to bicycle improvements. Each bicycle facility or intersection improvement must be compatible with roadway characteristics, traffic patterns, and adjacent land uses. The following bicycle facilities and intersection improvements highlight the diversity of options available to better support bicycling in Pekin.
**BICYCLE FACILITY TYPES**
- **Shared Travel Lanes**
- **Paved Shoulders**
- **Separated Bicycle Lanes**
- **Bicycle Wayfinding & Direction Signs**
- **Bicycle Boulevards**
- **Bicycle Lanes / Buffered Bicycle Lanes**
- **Shared Use Paths & Trails**
- **Bicycle Parking**
**INTERSECTION IMPROVEMENTS**
- **Bike Boxes**
- **Bike Lane Markings/Conflict Markings/Green Paint**
- **Bicycle Signals**
- **Protected Intersections**
Pekin Bicycle & Pedestrian Master Plan
September 28, 2023
Open House #2
Proposed Bicycle Network
Pekin Bicycle & Pedestrian Master Plan
Community Features
- City Hall
- Hospital
- Library
- Post Office
- School
- Place of Worship
- Full Service Grocery Store
- Park
- CityLink Bus Stop
Bicycle Recommendations
- Existing Trail
- Shared Use Path
- Side Path
- Separated Bike Lane
- Bike Lane
- Bike Blvd
- Shared Travel Lane
- Crossing Improvements
Pedestrian Improvements Toolkit
Creating a safe, comfortable, accessible, and attractive environment for walking does not happen overnight. It takes years to develop a high-quality pedestrian system. With the right plans and policies in place, the City of Pekin can direct public infrastructure investments and private development to support people walking, regardless of their age, ability, or use of mobility assistance devices.
**NETWORK ENHANCEMENTS**
- **Sidewalks**
- New Construction
- Sidewalk Infill & Repairs
- **Street Furniture**
- Benches
- Planters
- **Pedestrian-Scale Lighting**
- Streetlights
- Pathway Lights
- **Shade Trees**
- Trees along sidewalks
- Trees in parks
**INTERSECTION IMPROVEMENTS**
- **Curb Extensions**
- Curbs extended into intersections
- **ADA-Accessible Curb Ramps**
- Ramps for wheelchair access
- **Mid-Block Crosswalks**
- Crosswalks at intersections
- **Pedestrian Signals & Countdown Timers**
- Traffic signals with pedestrian signals
- **Marked Crosswalks**
- Painted crosswalks
- **Raised Crosswalks**
- Elevated crosswalks
- **Leading Pedestrian Interval**
- Traffic lights timed for pedestrians
- **Median Refuge Islands**
- Islands in the middle of intersections
---
Pekin Bicycle & Pedestrian Master Plan
September 28, 2023
Open House #2
The ADA Transition Plan will be the primary driver of investments in sidewalks and intersection improvements.
With that in mind, the pedestrian network in the Pekin Bicycle and Pedestrian Master Plan focuses on pedestrian priority corridors.
Pedestrian priority corridors consist primarily of arterial and collector roadways that connect neighborhoods to retail, shopping, schools, parks, and other vital destinations.
This approach acknowledges that not all roads are created equal. Pedestrian priority corridors generally carry higher volumes of motor vehicle traffic at higher speeds, and safe and accessible pedestrian facilities are necessary to support pedestrian activity.
Also included in the pedestrian network are shared use paths, sidepaths, and the existing Pekin Bike Path.
Community Features
- City Hall
- Hospital
- Library
- Post Office
- School
- Full Service Grocery Store
- Place of Worship
- Park
- CityLink Bus Stop
Pedestrian Recommendations
- Pedestrian Priority Corridor
- Existing Trail
- Shared Use Path
- Side Path
- Crossing Improvements
Project Prioritization
Active transportation infrastructure projects must compete with other capital improvements and municipal services, as well as with one another, for limited internal and external resources. In order to maximize investment and provide the greatest benefit, the City of Pekin should take a prioritized approach to invest in active transportation infrastructure and plan implementation.
Impact Factors:
- Access to schools, parks, commercial districts/hubs, transit
- Low stress/all ages and abilities
- Connections to existing facilities
- Safety (addressing high-crash corridors & intersections)
- Public support
Effort Factors:
- Project Cost
- Project Complexity
- Ownership
- Coordination with Programmed Improvements
Implementation Categories:
| Priority | Feasibility | Description |
|----------|-------------|-------------|
| High | High | Long Term Improvement |
| | | Projects for further study and evaluation. Seek grant funding to advance projects. |
| Low | Low | Short Term Improvement |
| | | High priority and easy to implement projects for short term development. |
| Low | High | Opportunity Improvement |
| | | Lower priority projects that may become an opportunity with the necessary funding or partnerships. |
What are your Priorities?
Supporting Programs
EARN-A-BIKE PROGRAM
Earn-a-Bike programs give students the opportunity to learn basic bike maintenance and bicycling skills, as well as route selection and mapping. Students who participate in these programs typically learn to build a bicycle from the ground up, and upon completion of the program receive a refurbished bike (usually the one they build), along with a helmet, bike lock, and bike lights.
TRAIL USER ETIQUETTE CAMPAIGN
Statewide, local parks and trails are available for use by bicyclists, walkers, runners, skateboarders, rollerbladers, parents with strollers, dogs, children, and, in some cases, equestrians, as well as other modes. A campaign for trail user etiquette works to ensure that all users safely and responsibly share the trail. The campaign may include media advertisements, trail signage, brochures, an “ambassador” program, etc.
HAPPY TRAILS TO HEALTHY FOODS
Many communities have developed programs that promote physical activity and healthy eating goals in improving overall public health and wellness. This important link can be highlighted in a fun and interactive manner through promoting healthy food outlets along the bicycle, pedestrian, trail, and transit network and partnering with health food providers to identify safe routes for active transportation to their locations.
CAR-FREE STREET EVENTS
Car-free street events are temporary “openings” that create a temporary park that is open to the public for walking, bicycling, dancing, etc. The purpose of the event is to encourage biking and other forms of physical activity to the general public by providing a fun, welcoming environment for activity. Car-free street events have been very successful internationally and are rapidly becoming popular in the U.S.
FAMILY BIKING PROGRAMS
Family Biking programs help parents figure out how to safely transport children by bicycle and help children learn bicycling skills. Activities may include bicycle safety checks, a group ride or parade, “freedom from training wheels” clinics, and opportunities to try out different ways to transport children (e.g., trailers, cargo bicycles, kid seats, etc.).
SAFE ROUTES TO SCHOOL PROGRAM
The City of Pekin can benefit from a comprehensive Safe Routes to School program that combines local facility, safety, and route planning with the development of school and travel events, like walking school buses or bike trains. Both of these strategies require a commitment and support from school leadership and staff, the City of Pekin, and families with school-aged children.
SILVER SNEAKERS SENIORS PROGRAM
Interested agencies, nonprofits, health departments and senior centers can partner to develop an active lifestyles program for senior citizens utilizing the bicycle, pedestrian, and greenways network. Activities could include adult tricycle or bicycle rides, nature walks, walks to lunch, and safety education.
OUTDOOR PUBLIC ART
Outdoor art along a bicycle, pedestrian, and trail network can bring attention to the network and attract newcomers to bicycling and walking, while also enhancing outdoor space. By adding art to trail facilities, the community is creating a unique interactive amenity for both residents and visitors. Such programs can also attract new partners, promoters, and sponsors of the active transportation network.
INTERPRETIVE SIGNAGE
Interpretive signage along a trail and greenway network serves as an education tool. Information related to the history of an area, its cultural significance, or natural features is provided on a graphically appealing sign. Topics could range from native species of plants to river currents to famous historical figures.
BICYCLE FRIENDLY BUSINESS PROGRAM
Businesses in Pekin have an important role to play in fostering a bicycle friendly community. From supporting employees with secure bicycle storage facilities, showers, and lockers to incentivizing customers that arrive on bike with discounts or other promotions, there are many ways businesses can promote bicycling. The League of American Bicyclists Bicycle Friendly Business program will be a valuable resource, and businesses should strive to achieve recognition as a Bicycle Friendly Business.
BIKE MONTH EVENTS
Cities and towns across the country participate in National Bike Month annually, during May. The League of American Bicyclists (LAB) hosts a website for event organizers. The website contains information on nationwide and local events, an organizing handbook, and promotional materials. Events can vary widely, including events to promote cycling like morning-commute energizer stations and an end-of-day rally or celebration, a group ride with the mayor, discounts at local businesses for bicycle commuters, short, themed community bicycle rides, mountain bike skills clinics, and commuter courses for adults.
PERSONAL TRAVEL ENCOURAGEMENT
PTE programs are designed to educate the public on how to approximately 10% and increase bicycling, walking and transit use within a target area. The program:
- Delivers customized travel information packets;
- Hosts fun events such as guided rides, walks, and classes; and
- Sends trained outreach staff to farmers’ markets and other community events
BICYCLE COUNT PROGRAM
By utilizing both a manual and automated counting practices, Pekin can better measure and understand how people travel in the community, which can provide insight into heavily utilized, and where to invest in future transportation improvements. The Pedestrian and Bicycle Information Center (PBIC) has a wealth of information and resources to help the City develop a comprehensive counting program.
TARGETED TRAFFIC ENFORCEMENT
By focusing traffic enforcement efforts on areas of high travel around schools, senior centers, business districts, and high-crash locations, the Pekin Police Department can support bicycle and pedestrian safety, especially for the most vulnerable road users in our community.
THEMED WALKING/BIKING MAPS & GUIDES
Walking and biking maps and guides can be used to explore the city’s rich history, unique character, and beautiful parks. The guides would be targeted to a variety of ages and abilities, offering varying routes for families, touring cyclists, and others. Themes can include history, architecture, parks, and culture and suited for both Pekin residents and visitors.
TRAFFIC TICKET DIVERSION CLASS
A diversion class is offered to those offenders of certain bicycle-related traffic violations, such as running a stop sign on a bike. It can be aimed just at bicyclists or at bicyclists and motorists, or pedestrians. In lieu of a citation and/or fine, individuals can take a one-time, free or inexpensive class.
CREATE-A-COMMUTER PROGRAM
Create-A-Commuter programs equip people with the tools to overcome common barriers to bicycling to work, school, and other everyday destinations. A successful program often includes bicycle skills and maintenance courses, mentorship pairing with experienced cyclists, and Guaranteed Ride Home (GRH) element to provide participants with taxi, rideshare, or transit service if an unexpected need arises.
BICYCLE + PEDESTRIAN RESOURCE WEBSITE
The City could create a website that serves as a one-stop resource for bicyclists, pedestrians, and trail users of all types. Information could include tips for commuters, route planning services, community events, message boards, and more.
BICYCLE PARKING PROGRAM
People traveling by bicycle rely on safe and secure bicycle parking facilities to support their trips. To incentivize bicycle parking installation, the City of Pekin should consider the development of a bicycle parking program that provides reduced-cost bicycle racks for local businesses and community destinations.
KEY QUESTION: ARE THERE OTHER PROGRAM IDEAS YOU WOULD LIKE TO SEE IN PEKIN?
(write your thoughts or place a dot beside an existing idea to show your support)
LAUNCH PARTIES FOR NEW BIKEWAYS
The area’s cities and counties should partner with local advocacy groups to generate media attention and engage local citizens in each incremental expansion of the bikeway network. Popular launch parties in other jurisdictions have included bicycle-handling workshops, free bike mechanic services, live music, bicycle giveaways, and other activities.
We Need Your Input!
What will make this plan a success?
What’s your top priority or project?
What else do we need to know?
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Cardiac Myocyte Diversity and a Fibroblast Network in the Junctional Region of the Zebrafish Heart Revealed by Transmission and Serial Block-Face Scanning Electron Microscopy
Pascal J. Lafontant
*DePauw University*, email@example.com
Follow this and additional works at: [https://scholarship.depauw.edu/bio_facpubs](https://scholarship.depauw.edu/bio_facpubs)
Part of the Biology Commons
**Recommended Citation**
Lafontant PJ, Behzad AR, Brown E, Landry P, Hu N, et al. (2013) Cardiac Myocyte Diversity and a Fibroblast Network in the Junctional Region of the Zebrafish Heart Revealed by Transmission and Serial Block-Face Scanning Electron Microscopy. *PLoS ONE* 8(8): e72388. doi:10.1371/journal.pone.0072388
This Article is brought to you for free and open access by the Biology at Scholarly and Creative Work from DePauw University. It has been accepted for inclusion in Biology Faculty publications by an authorized administrator of Scholarly and Creative Work from DePauw University.
Cardiac Myocyte Diversity and a Fibroblast Network in the Junctional Region of the Zebrafish Heart Revealed by Transmission and Serial Block-Face Scanning Electron Microscopy
Pascal J. Lafontant¹*, Ali R. Behzad², Evelyn Brown³, Paul Landry³, Norman Hu⁴, Alan R. Burns³,⁵
¹ Department of Biology, DePauw University, Greencastle, Indiana, United States of America, ² Imaging and Characterization Core Lab, King Abdullah University of Science and Technology, Thuwal, Kingdom of Saudi Arabia, ³ College of Optometry, University of Houston, Houston, Texas, United States of America, ⁴ Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, Utah, United States of America, ⁵ Department of Pediatrics, Baylor College of Medicine, Houston, Texas, United States of America
Abstract
The zebrafish has emerged as an important model of heart development and regeneration. While the structural characteristics of the developing and adult zebrafish ventricle have been previously studied, little attention has been paid to the nature of the interface between the compact and spongy myocardium. Here we describe how these two distinct layers are structurally and functionally integrated. We demonstrate by transmission electron microscopy that this interface is complex and composed primarily of a junctional region occupied by collagen, as well as a population of fibroblasts that form a highly complex network. We also describe a continuum of uniquely flattened transitional cardiac myocytes that form a circumferential plate upon which the radially-oriented luminal trabeculae are anchored. In addition, we have uncovered within the transitional ring a subpopulation of markedly electron dense cardiac myocytes. At discrete intervals the transitional cardiac myocytes form contact bridges across the junctional space that are stabilized through localized desmosomes and fascia adherent junctions with adjacent compact cardiac myocytes. Finally using serial block-face scanning electron microscopy, segmentation and volume reconstruction, we confirm the three-dimensional nature of the junctional region as well as the presence of the sheet-like fibroblast network. These ultrastructural studies demonstrate the previously unrecognized complexity with which the compact and spongy layers are structurally integrated, and provide a new basis for understanding development and regeneration in the zebrafish heart.
Citation: Lafontant PJ, Behzad AR, Brown E, Landry P, Hu N, et al. (2013) Cardiac Myocyte Diversity and a Fibroblast Network in the Junctional Region of the Zebrafish Heart Revealed by Transmission and Serial Block-Face Scanning Electron Microscopy. PLoS ONE 8(8): e72388. doi:10.1371/journal.pone.0072388
Editor: Leonard Eisenberg, New York Medical College, United States of America
Received March 1, 2013; Accepted July 9, 2013; Published August 23, 2013
Copyright: © 2013 Lafontant et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: PJL is supported by the Fisher and Faculty Development Funds at DePauw University. A. Burns is supported National Institutes of Health/National Eye Institute grants EY017120 and P30EY007551. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Introduction
The zebrafish heart consists of a spongy ventricular myocardium made of trabecular bundles projecting radially into the ventricular lumen, and of an outer compact heart layer that encases the spongy trabeculae. The proportion of spongy to compact heart varies significantly between fish and appears to strongly correlate to each species particular ecological physiology [1,2,3,4]. The hearts of fish with relatively low activity consist primarily or exclusively of a spongy myocardium with a rudimentary compact myocardium fed by deoxygenated luminal blood flow (type-1 heart). More active fish display a thicker compact myocardium invested with vessels carrying oxygenated blood (type-2 heart). The two myocardial segments have been historically and empirically considered distinct anatomical structures that form a functional unit supporting the physiological demands of fish. Because of its hypothesized importance in fish ventricular function, the characteristics of the spongy-compact interface (SCI) between the two ventricular layers have been an area of considerable interest and controversy.
The zebrafish has emerged as an important model of heart development and regeneration. The adult zebrafish heart is a type-2 heart with a compact layer perfused by capillaries. An extensive number of studies have provided insights into the molecular mechanisms underlying the development of the zebrafish ventricle [5,6,7] and its spongy trabeculae [8,9]. Other studies have explored the molecular mechanisms that orchestrate the zebrafish’s remarkable ability to regenerate [10,11]. More recently, the emergence of the mature zebrafish ventricle has been demonstrated by genetic approach to be driven by clonally-dependent cardiac myocytes, illustrating diversity within the ventricular myocyte population [12]. A number of studies have also provided insight into the ultrastructural characteristics of the developing and adult zebrafish heart [13,14,15]. However to date the nature of SCI in the zebrafish heart has received little attention.
Studies in a variety of fish using different approaches have resulted in markedly diverse understandings and models of this
interface region. Early studies in the albacore tuna suggested that the two myocardial layers were attached by a connective tissue layer [16,17]. Transmission electron microscopy studies in four different fish species, not including the tuna, provided the first detailed evidence not only for connective tissue at the interface of the two myocardial layers [18,19], but of the presence of fibroblasts and a distinct set of flattened transitional cardiac myocytes occupying this complex junctional region (JR). On the other hand, in a more recent study of the sockeye salmon and rainbow trout, similar structures in the interface region were not observed [20]. Whether these studies underscore the structural diversity of the JR in fish, or reflect differences in study methodologies is not clear. The different findings in the salmon and trout heart may be indicative of the intra- and inter-species diversity in the architectural arrangements of the two layers.
The purpose of our study was to ascertain the cellular and ultrastructural nature of the interface region between compact and spongy heart of adult zebrafish using light and transmission electron microcopy (TEM). Here, we provide evidence of a complex and previously unrecognized JR containing a network of fibroblasts, a subpopulation of phenotypically transitional cardiac myocytes at the base of the spongy myocardium, and describe the spatial distribution of adherens junctions linking the spongy and compact layers. The three-dimensional structure of the JR was confirmed by serial block-face scanning electron microscopy (SBF-SEM) and computerized segmentation. To our knowledge, this is the first detailed description of the JR of the zebrafish ventricle, as well as the first ultrastructural 3D reconstruction of this region using SBF-SEM.
**Materials and Methods**
**Animals**
Zebrafish were obtained from Aquatic Research Organisms (Hampton, NH), and maintained in 10-gallon tanks, with 15 to 20 fish per tank, at 28 degrees Celsius on a 14/10 hour day/night cycle. Experimental procedures were approved by the Committee for the Care and Use of Laboratory Animals at DePauw University.
**Transmission Electron Microscopy**
For studies of the myocardium in plastic sections and by transmission electron microscopy tissue samples were processed as described previously [21]. Zebrafish were euthanized using 0.2% MS Tricaine. Once all body and opercula movements had ceased, the heart was removed by grasping and pulling the aorta proximal to the bulbus arteriosus. The hearts were fixed in 100 mM cacodylate buffer containing 2.5% glutaraldehyde overnight at 4 degrees C. The hearts were washed the next day in cacodylate buffer and stored at 4 degrees C for later processing. Hearts were post-fixed in 1% tannic acid and transferred to 1% osmium tetroxide, dehydrated in an acetone series, and then embedded in Embed-812 resin (Electron Microscopy Sciences, Hatsfield, PA). Two-micron thick sagittal sections were cut and toluidine-blue stained for light microscopy analysis. Stained sections were visualized and imaged using a Nikon Optiphot (Nikon Instruments Inc., Melville, NY). For electron microscopy analysis, ultra-thin sections 100 nm thick were cut, set on single slot or 200-mesh copper or nickel grids, and imaged on a Tecnai G2 Spirit BioTWIN electron microscope (FEI Company, Hillsboro, OR). Additional fixed and embedded hearts (n = 9) were obtained from the University of Utah and processed as previously described [14]. Briefly, these hearts were perfusion-fixed with 2.5% glutaraldehyde-2% paraformaldehyde in 0.1 M cacodylate buffer, and 4×10⁻⁴ g/kg dose of diltiazem for 4 hours. The hearts were then post-fixed in 2% osmium tetroxide, dehydrated, and embedded in Spurr’s epoxy resin. Ultra-thin sections 100 nm thick were cut and mounted on single slot or 200-mesh copper or nickel grids and imaged on a Tecnai G2 Spirit BioTWIN electron microscope (FEI Company, Hillsboro, OR).
**Immunofluorescence Imaging**
Following isolation, zebrafish hearts were cryoprotected in 30% sucrose in PBS overnight. The following day, the hearts were embedded in freezing medium (Tissue-tek OCT, Torrance, CA), kept at −80 degrees C and later sectioned using a Leica CM 1900 cryostat (Leica Microsystems, Bannockburn, IL). Ten-micron sagittal sections were fixed for 3 minutes in 4% paraformaldehyde, permeabilized with 0.5% triton X in PBS, blocked with 3% BSA for 2 hours, and immunoreacted overnight with anti-MYH1 antibody or MEF antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA), followed by Texas-Red conjugated anti-mouse or anti-rabbit secondary antibody. Next, the sections were incubated with FITC-labeled wheat germ agglutinin (WGA, Triticum vulgaris, Sigma-Aldrich, Saint Louis, MO) for 2 hours. Other sections were stained with TRITC-labeled WGA. Following Hoechst staining, sections were imaged on a Zeiss Axio Imager.M (Carl Zeiss Microscopy, Thornwood, NY) or a Leica TCS SP5 confocal microscope (Leica Microsystems, Bannockburn, IL).
**Serial Block-face Scanning Electron Microscopy Imaging**
Tissue fixation and embedding were performed using the previously published protocols with some modifications [22]. Briefly, excised zebrafish hearts were fixed in 2.5% glutaraldehyde in 100 mM sodium cacodylate buffer at 4 degrees C overnight. The hearts were rinsed the next day in 100 mM sodium cacodylate. The hearts were post-fixed and heavy metal-contrasted with potassium ferrocyanide, osmium tetroxide, thiocarbohydrazide, uranyl acetate and lead aspartate. Next, the hearts were dehydrated in ethanol followed by acetone and embedded in Durcupan resin. Approximately 2×2 mm, 100 nm thick sections were obtained, mounted on slot grids and surveyed by TEM. Blocks with optimal tissue orientation and easily identifiable landmarks in the region of interest were selected for further trimming and imaging. Serial block-face sectioning (at 50 or 100 nm imaging intervals) and imaging were performed using a Gatan 3View system (Gatan, Pleasanton, CA) mounted in an FEI Quanta FEG 200 SEM (FEI Company, Hillsboro, OR). The SEM was operated at an accelerating voltage of 2.0 kV and 30 pascals pressure in low vacuum mode. A solid state backscatter detector was used to acquire serial section images (stacks of 800, 632, and 500 sections) from three regions of interest. Three-dimensional segmentation and reconstruction of fibroblasts and cardiac myocytes was performed using Amira 5.2 software.
**Results**
I. **Electron Translucent and Electron Dense Cardiac Myocytes Are Organized into a Transitional Cell Ring**
To determine the nature of the interface region between the compact and spongy heart of the adult zebrafish, we first studied the ventricle under light microscopy. Using two-micron thick toluidine blue-stained plastic embedded sections, we observed the expected organization: a relatively thin layer of the compact heart (approximately 10–20 um thick) framing the trabeculae which projected radially into the ventricular lumen (Fig. 1. A). However closer inspection revealed an unexpected number of darkly-stained elongated structures, at irregular intervals at the interface between
Figure 1. Low magnification of the adult zebrafish heart by light and TEM. (A), light microscopy view of the apical region of the zebrafish ventricle showing a thin layer compact heart (CH) with trabeculae (Tr) projecting within the lumen (Lu). (B), higher magnification of the apex showing darkly stained linear structures (arrows) close to the spongy-compact interface of the ventricular myocardium. (C), low magnification TEM of compact ventricular myocardium constituted of four to five overlapping cardiac myocytes (CM) layers from epicardium (Epi) to lumen (Lu), with electron dense cells (arrow) within a complex junctional region (JR, brackets) at the interface of compact and spongy myocardium. (D), higher magnification show mitochondria (Mt) filled section of electron dense transitional cardiac myocyte (EDTCM). EDTCM are apposed to trabecular cardiac myocytes and endocardial cells (Endo) on the luminal side. EDTCMs are separated from the compact cardiac myocytes (CCM) by a junctional space, an interstitial space within the JR. (E), low magnification TEM of the JR (brackets) showing flattened electron translucent transitional cardiac myocytes (ETTCM) in contact with trabeculae and in proximity to endocardium (Endo) on the luminal side. (F), higher magnification of a flattened cells show presence of actin and myosin filaments, apposition to trabeculae and endocardial cells as well as separation from the compact myocardium by the junctional space.
doi:10.1371/journal.pone.0072388.g001
Figure 2. Electron dense and electron translucent transitional cardiac myocytes interactions. (A), example of end-to-end interaction between EDTCM and electron translucent cardiac myocytes (ETTCM) forming a continuum at the spongy-compact interface. (B), higher magnification of contact area with complex adhesion junctions including desmosomes between the two cells. (C), example of interaction between mitochondria filled and actin-myosin filament (AMF) containing EDTCM cells and ETTCM via adhesion junctions (arrows). (D), higher magnification of EDTCM and ETTCM showing gap junctions (arrows), and (E), desmosomes. (F), AMF in a transitional cell. (G), higher magnification (inset in F, rotated) showing the hexagonal array of myosin in cardiac myocytes. (F) sarcomere and z-band in the same cell. AMF, actin-myosin filament; CCM, compact cardiac myocytes; CW, cardiac myocytes; EDTCM, electron dense transitional cardiac myocytes; ETTCM, electron translucent transitional cardiac myocytes; Endo, endocardium; Epi, IF, intermediate filaments; Lu, lumen; Mt, mitochondrion; Tr, trabeculae.
doi:10.1371/journal.pone.0072388.g002
the compact and spongy segments of the ventricle (Fig. 1, B). To elucidate the nature of these darkly-stained structures, we studied their characteristics and precise location within the zebrafish junctional region (JR) using transmission electron microscopy (TEM). Low magnification TEM reveals a compact myocardium of 4–5 layers of cardiac myocytes (CM) (Fig. 1, C). The compact and spongy interface is revealed as a complex JR containing a set of flattened CM as compared to the compact or trabecular CM. Ultrastructurally, the darkly stained structures appear as electron dense CM running along the adjacent base of the trabeculae (Fig. 1, D). We have called these cells: “dark cells” or electron dense transitional cardiac myocytes (EDTCM). Except for their high electron density, these cells display similar flattened and rectangular phenotypes as the transitional CM described previously [18]. In adjacent regions, the ventricle displays a similar arrangement but with electron translucent transitional cardiac myocytes (ETTCM) (Fig. 1, E). Actin-myosin filament bundles were easily identified within the ETTCM confirming their CM phenotype (Fig. 1 F), whereas the cytoskeletal contents of the EDTCM were obscured by the electron dense material contained in these cells.
II. Transitional Cell Ring and Trabeculae Contacts
The EDTCM were in direct contact with the ETTCM (Fig. 2, A). Within the plane of observation, shorter segments of EDTCM alternated with longer segments of ETTCMs, and together these two cell populations appear to form a continuous ring of transitional CM within the JR. The transitional CM could be seen linked at their tapered ends through complex adherent junctions resembling fascia adherentes (Fig. 2, B). Additionally, the transitional CM were in direct contact or in close apposition to the base of CM sheets of projecting spongy trabeculae (Fig. 2, A, C), and in the intertrabecular lacunar spaces they were bounded in their luminal aspect by the endocardium (Fig. 2, C). However in contrast to their connections to trabeculae, no contacts were observed between transitional CM of the transitional ring and the adjacent endocardial cells. In some instances, EDTCM and ETTCM minimally overlapped at their tapered ends (Fig. 2, C), with connections occurring through discrete gap junctions (Fig. 2, D) and desmosomes (Fig. 2, E). Detailed observations of the cytoplasm of these cells show the presence of actin-myosin filaments oriented in different planes (Fig. 2, F). The presence of caveolae and the hexagonal array of equidistant thick myosin filaments characteristic of cardiac muscle cells can be appreciated (Fig. 2, G). Z-bands bordering sarcomeres oriented perpendicularly or obliquely in the plane of observation (Fig. 2, H) can also be seen in these cells, further confirming their cardiac myocyte phenotype. Finally the cardiac nature of these narrow width transitional cells was also confirmed using immunostaining with anti-myosin heavy chain-1 (MYH1) and anti-myocyte enhancer factor-2 (MEF) antibodies to specifically identify cardiac myocytes (Fig. 3).
We further investigated the nature of the arrangement between the transitional CM ring and the CM forming the trabeculae. Transitional CM can be found linked to trabecular myocytes through a variety of adherens junctions. These junctions included fascia adherentes (Fig. 4, A, B) that link actin-myosin filaments of adjacent cells. Various amounts of amorphous material were observed along transitional CM and trabecular CM areas of apposition (Fig. 4 C, D). However discrete electron dense areas...
with a sub-domain containing desmosomes were also observed (Fig. 4 E, F).
III. Fibroblast Network and Collagen in the Junctional Region
The transitional ring of myocytes on its abluminal side is bounded by a narrow (0.5–1 um) connective tissue space. This space separates the EDTCM and ETTCM from the adjacent CM of the compact layer, creating a junctional space within the JR (Fig. 5. A). The junctional space appears to create an extensive structural discontinuity between the spongy and the compact myocardium. It contains numerous cells arranged longitudinally and running parallel to the transitional CM ring. The absence of basement membranes, their elongated shape, and their location within this interstitial space suggest they are fibroblasts (Fig. 5. B, C). These fibroblasts contained ovoid nuclei with scant perinuclear cytoplasm that appeared mostly devoid of organelles, except for occasional vesicles and polyribosomes. In the perinuclear region of the fibroblasts, the junctional space widens to 2–3 microns. The cytoplasm of these fibroblasts is stretched bi-directionally into long thin filopodia-like processes. These fibroblasts mostly form a single layer in the junctional space with little to minimal overlap of their cytoplasm. The fibroblasts and their cytoplasmic processes fit tightly within the junctional space, however a measurable space was always observed between the fibroblast and the adjacent transitional and the compact ventricular CM.
The fibroblasts were also found in close proximity or in direct contact with parallel or perpendicularly-oriented collagen fibers (Fig. 5. B, C). At irregular intervals, terminal profiles displaying long cytoplasmic extensions were in close proximity with the membranes of long cytoplasmic processes originating from another fibroblast (Fig. 5. D). In some instances, cytoplasmic terminal ends of fibroblasts overlapped, or could be seen in contact with the same bundle of collagen. Occasionally fibroblasts terminal ends were found in close contact; however we could not unequivocally confirm specialized adhesion junctions between interacting terminal ends. Occasionally close approximation between fibroblast filopodia and CM could be seen; however no direct contact between fibroblasts and CM and no specialized myocytes-fibroblasts junctions were observed. Close observation of many of the transitional CM however revealed the presence of caveolae within the membranes orientated toward the fibroblasts within the junctional space (Fig. 5. C). Across the junctional space, the compact CM membranes were observed to have numerous caveolae facing toward the fibroblasts (Fig. 5. D, E). More examples of caveolae could be seen on the membranes of CM facing the junctional space at a distance from the fibroblast processes (Figure S1). While most fibroblast nuclei and cytoplasmic processes resided within the JR, some cytoplasmic projections could be seen invested in the compact myocardium adjacent to small vessels, suggesting that fibroblasts may not be locally restricted to the junctional space.
Figure 5. Fibroblasts network in the junctional region of the zebrafish ventricle. (A), TEM of a fibroblast (Fb) cytoplasm and its thin extended process/filopodium (FP) 100 to 200 nm thick spanning the junctional space (JS) in between the transitional CM ring and the adjacent compact cardiac myocyte (CCM). Note vesicle (*) in an enlarged region (~300 nm) of the fibroblasts filopodium (FP). (B), fibroblast profile with nucleus (Nu) and its cytoplasm extending into a long thin process, and collagen fiber (CF) bundles running perpendicular to the section’s plane. (C), another fibroblast and its nucleus (Nu) and with cytoplasm extending into a long process, and collagen fiber (CF) bundles running parallel to the section’s plane. (D), fibroblast filopodial termini in close approximation. Electron dense region in the close approximation area suggestive of specialized adhesion junction (arrow). Note the presence of caveolae (*) on the membrane of the CCM facing the fibroblast in the junctional space.
doi:10.1371/journal.pone.0072388.g005
Figure 6. Transitional CM ring and compact CM contacts. (A), ETTCM and Compact CM direct contacts mediated by adhesion junctions (arrows). Note the JS with fibroblast (Fb) on the right and narrow JS of the left of the contact region. (B), higher magnification of inset in (A) showing adhesion junctions associated with fascia adherentes (thick arrow), and also with desmosomes (thin arrow). Note the abundance of caveolae (*) in the transitional CM membrane in the contact region. (C), another narrow region of contact (arrow) between a transitional and a compact cardiac myocyte.
forming a bridge between the two cells and adjacent Z-band in the CCM. A fibroblast process is seen on each side of the junction. Note a vesicle (V) in the fibroblast process. (D), higher magnification of the contact area (arrow) showing a desmosome associated with actin, myosin, and intermediate filaments on the ETTCM and abundant ribosomes (rb) in the CCM. (E), a tri-cellular junction between two transitional cardiac CM and one compact CM. (F), higher magnification of (E, inset) showing desmosomes between the ETTCM and each ETTCM with the compact CM (arrows). (G), discrete adhesion junction between a transitional and compact CM (arrows). (H), higher magnification of inset 1 of (F). (I), higher magnification of inset 2 of F, showing compact CM contact within an invagination of the transitional CM.
doi:10.1371/journal.pone.0072388.g006
IV. Transitional Cardiac Myocytes Contacts across the Junctional Space
From our observations, we estimated that the junctional space containing fibroblasts and collagen occupies 80 to 90% of the junctional region between compact and spongy heart, so that the majority of the CM membranes in the transitional ring are not directly linked to the compact heart. The remaining portion of the spongy-compact interface consisted of cell membrane appositions between transitional CM and the most adjacent CM of the compact heart (Fig. 6. A). These regions of apposition contained specialized adherens junctions including closely spaced desmosomes and fascia adherents (Fig. 6. B). These areas of contact between the transitional ring and compact myocardium circumscribed the junctional space and interrupted the course of the fibroblasts and their filopodia in the plane of the sections (Fig. 6. C, D). Occasionally a region of tri-cellular contact was observed, where a compact myocyte was joined at the end-to-end contact of two transitional CM through desmosomes (Fig. 6. E, F). However most transitional ring myocytes and compact CM contacts occurred through apposition of their lateral borders via desmosomes (Fig. 6. G, H), membrane protrusions, and interdigitations (Fig. 6. I).
V. Serial Block-face SEM, Segmentation, and Volume Reconstruction of the Junctional Region
To better assess the three-dimensional architecture of the junctional region and confirm the three-dimensional nature of the fibroblast network, we performed serial block-face scanning electron microscopy (SBF-SEM) of the adult zebrafish heart. A preparatory survey of the fibroblast network in the JR of the zebrafish ventricle was performed by TEM in 100 nm sections from heavy metal-contrasted Durcupan-embedded hearts. The junctional region and the fibroblasts were easily identifiable. Image stacks generated with the FEI Quanta 200 FEG SEM equipped with the Gatan 3View (Fig. 7. A) were used to generate three-dimensional reconstructions of the JR architecture (Fig. 7. B, Movie S1), and confirm the 3D projection of fibroblasts in the junctional volume space. Observation of a subset of serial sections demonstrated the predicted discontinuous nature of cellular contacts between the compact heart and transitional myocytes that anchor the luminal trabeculae, and confirmed fibroblast-fibroblast contacts in the junctional space (Fig. 7. C-I, Movie S2). Segmentation of fibroblasts from serial sections followed by three-dimensional reconstruction demonstrated the sheet-like nature of fibroblasts arranged in a network occupying the junctional space of the zebrafish heart (Fig. 7. J, K). Additional segmentation of transitional and compact cardiac myocytes demonstrated contact surfaces between compact and spongy heart through the fibroblast network layer (Fig. 7. L).
Discussion
This paper documents a previously unrecognized connective tissue space located at the interface of the spongy and compact myocardial of the adult zebrafish heart. It extends our understanding of the cellular and ultrastructural nature of the ventricular JR of an important research model. The existence of a connective tissue layer in the junctional region of type-2 fish hearts has remained controversial. A continuous fibrous membrane interposed between the compact and spongy layers has been reported in the hearts of tuna [16,17], atlantic salmon and rainbow trout [23]. These observations led to the hypothesis of the connective tissue as an important adhesive substrate between the two myocardial layers. In these early studies, detailed composition of the connective tissue layer was not described. Midttun using TEM demonstrated in four type-2 fish hearts species the presence of fibroblasts and collagen fibers within a 6 to 7 um wide space existing in the junctional region [18]. By contrast in a study by Pieperhoff et al., using light and scanning electron microscopy, a connective tissue layer at the interface of the two myocardial layers in the sockeye salmon and trout was not observed [20], beyond small focal regions of extracellular matrix enrichment. Unlike the previous studies, these authors challenged the hypothesis of a connective tissue layer separating the two myocardial layers. In both Midtun’s and our studies, the junctional space and its content were not readily detected by light microscopy; however, they could be observed by transmission electron microscopy, suggesting that ultrastructural thin sections imaging may be necessary to describe the structure of the JR of fish ventricles.
Our ultrastructural observations reveal a junctional space measuring for the most part less than one micrometer wide, populated by a single layer of fibroblasts organized into a network, and framed by CM belonging the compact myocardium distally and the spongy myocardium proximally. Fibroblasts are an important component of the mammalian heart and play an essential role in heart structure and physiology. They are a source of extracellular matrix molecules and growth factors, and they serve as sensors for mechanical signals [24,25,26,27,28]. While it has been suggested in early studies that collagen accumulation modulates regeneration in the zebrafish ventricle [10], fibroblasts in the fish heart have received little attention. More recently studies of cardiac injury in the zebrafish [29,30,31] and giant danio [21] demonstrated accumulation and resorption of collagen during fish ventricular regeneration, suggesting an important role for fibroblasts. Indeed the presence of activated fibroblasts was observed during ventricular remodeling in the zebrafish [31] and giant danio heart [21]. The presence of fibroblasts has previously been noted in the sub-epicardial space of fish species, including the zebrafish [14]. In the present study, the fibroblasts’ arrangement suggests a highly organized three-dimensional network exists at the interface of the compact and spongy ventricle. The function of this fibroblast network is presently unclear.
Fibroblast networks have been documented in a variety of organs and species, with direct fibroblast-fibroblast contacts being observed in mammalian corneas [32,33]. Fibroblast interactions suggestive of a network have also been reported in the mammalian heart [34]. Fibroblasts have also been implicated in physiological mammalian cardiac muscle growth [27] as well as in injury responses [35,36,37,38]. Our study by TEM and the reconstruction following segmentation of the SBF-SEM micrographs demonstrate that fibroblasts form a three-dimensional network in the JR of the adult zebrafish heart. In addition to fibroblastfibroblast interactions, direct CM and fibroblast contacts have also been reported in normal and diseased hearts and in culture [39,40,41]. In this study however, we did not uncover direct CM-fibroblasts contacts in the uninjured zebrafish heart. When fibroblast processes were seen in close proximity and in apparent apposition to myocytes, the intervening myocyte basement membrane was always present. Yet we cannot rule out that these contacts exist and may occur at a frequency too low to be easily...
detected. Nevertheless, we found a remarkable number of caveolae in the sarcolemma of CM bordering the junctional space and facing adjacent fibroblasts. This suggests possible mechanisms by which paracrine signaling may take place between adjacent CM and fibroblasts occupying the junctional space.
The present study also establishes that the junctional space occupies the majority of the overall SCI, and creates an extensive volume of non-contact between the two myocardial layers. Given the sparsity of the collagen fibers observed in the junctional space, we question whether in the zebrafish, the connective tissue would provide sufficient support to maintain the structural integrity of the two layers. We speculate that the connective tissue compartment may not be the primary mechanism by which adhesion between the two myocardia is maintained. Indeed, the junctional space is interrupted by contact bridges between the two layers at discrete intervals through membrane apposition of adjacent CM, and that desmosomes and fascia adherentes are frequently found within the contact regions between transitional zebrafish CM and compact CM. These findings are consistent with the demonstration of highly enriched adhesion junction molecules between the two myocardial layers observed in salmon and trout [20].
An important finding is the transitional ring of cardiac myocytes similar to that previously described by Midrunn, that underscores the phenotypic diversity of cardiac myocytes present in the zebrafish heart, suggesting specific structure-function relationship. The notion of cardiac myocyte diversity is apparent in recent work demonstrating the clonal contribution of cardiac myocytes to the emergence of the zebrafish heart [12]. In that study two populations of peripheral cardiac myocytes were described by genetic labeling and spatio-temporal characteristics: a primordial and a cortical set. The localization of the primordial layer suggests it occupies a space similar to the transitional ring population described in the present study. Consequently, it would be interesting to determine whether these two populations spatially interact or are one and the same. Another intriguing finding is the discovery of a subset of “dark” electron dense myocytes within the transitional CM ring that supports the trabeculae. To our knowledge, these electron dense cardiac myocytes have not been reported in fish or mammalian hearts. A set of dark cells was observed in the ventricle of icefish; however, they were believed to be non-contractile in nature [42]. Observations of the zebrafish electron dense cells at high magnification show clear evidence of myofibrils. In addition these cells form a continuum with adjacent electron translucent CM as well as other electron dense CM in the transitional ring and myocytes of adjacent trabeculae via adhesive junctions. These observations raise the intriguing possibility of further phenotypic specialization within the transitional CM population bordering the JS. We speculate this particular phenotypic change may be regulated by regional stress distribution within the myocardium or may reflect their metabolic state. The understanding of the functional and the molecular differences between the dark and light cells within the transitional ring warrants further study.
Conclusions
In conclusion, this study sheds light on the phenotypic diversity of CM at the JR of the adult zebrafish heart. It documents the existence of a fibroblast network that contributes to the architectural complexity of the region (Fig. 8). We suggest the transitional CM form the main anchoring substrate for the trabecular and compact heart. It is possible that in type-2 fish hearts, both the connective tissue layer and the adherens junctions account for the integrity of the spongy-compact interface. It is also possible that in fish, the ratio of connective tissue and direct CM adhesive contact, as well as the distribution of adherens junctions might be dependent on the species, their age and ecological
physiology. In the end it is intriguing to consider how the articulation of the compact and spongy interface progresses during development and how the re-articulation occurs during regeneration of the zebrafish heart.
Supporting Information
Figure S1 Transitional myocyte abluminal caveolae. (A), Junctional space (JS) with a fibroblast process (FP). (B), Higher magnification of inset in (A) showing caveolae (*) on transitional CM facing the JS. (TIF)
Movie S1 Architecture of the junctional region of zebrafish heart. The sheet-like nature of fibroblasts can be appreciated. (MP4)
Movie S2 Segmented fibroblasts and transitional CM. (MP4)
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Acknowledgments
We thank Margaret Gondo for TEM help, Lynn Bedard and Tamara Beauboeuf for editorial suggestions, Tanmoy Das and Henning Schneider for valuable discussions, the Prindle Institute for Ethics at DePauw University for dedicated space for scholarly work, and also John Eskew, Wendy Tomamichel, and Clay Higginbotham for technical help and with animal care. We thank Jayne Williams for logistical help. We thank Barry Stein of The Electron Microscopy Center and Jim Powers of the Light Microscopy Imaging Center at Indiana University-Bloomington for their help.
Author Contributions
Conceived and designed the experiments: PJL A. Behzad EB PL NH A. Burns. Performed the experiments: PJL A. Behzad EB PL NH A. Burns. Analyzed the data: PJL PL A. Burns. Contributed reagents/materials/analysis tools: PJL A. Behzad EB PL NH A. Burns. Wrote the paper: PJL. Designed and performed TEM experiments: PJL A. Burns EB NH. Designed and performed SBF-SEM experiments: PJL A. Behzad A. Burns EB. Performed segmentation and 3D reconstruction: PJL PL.
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Cellular characterization of the outgrowth and connectivity of the brain serotonin system
Jeroen Dudok
The research described in this thesis was conducted at the Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, The Netherlands, and was supported by a grant from the Dutch Organization of Scientific Research (NWO-MW-PIO900-01-001).
Publication of this thesis was financially supported by:
CNCR
Department of Functional Genomics
Vrije Universiteit Amsterdam
Cover: Serotonergic neurons in the dorsal raphe nucleus
Print: Wöhrmann Print Service
ISBN: 978-90-8570-585-7
© Jeroen Dudok 2010. All rights reserved. No part of this thesis may be reproduced, stored in a retrieval system, or transmitted in any form or by any means without prior permission of the author.
Cellular characterization of the outgrowth and connectivity of the brain serotonin system
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad Doctor aan
de Vrije Universiteit Amsterdam,
op gezag van de rector magnificus
prof.dr. L.M. Bouter,
in het openbaar te verdedigen
ten overstaan van de promotiecommissie
van de faculteit der Aard- en Levenswetenschappen
op woensdag 29 september 2010 om 15.45 uur
in de aula van de universiteit,
De Boelelaan 1105
door
Jacobus Johannis Dudok
geboren te Zierikzee
promotor: prof.dr. M. Verhage
copromotor: dr. A.J.A. Groffen
Aan mijn ouders
# Contents
| Chapter | Title | Page |
|---------|----------------------------------------------------------------------|------|
| 1 | General introduction | 11 |
| 2 | Chronic activation of the 5-HT$_2$ receptor reduces 5-HT neurite density as studied in organotypic slice cultures | 37 |
| 3 | Dynamic vesicular trafficking of the serotonin reuptake transporter in hippocampal neurons | 57 |
| 4 | Presynaptic localization of tryptophan hydroxylase 2-EGFP in mature hippocampal neurons | 73 |
| 5 | The effect of a polymorphism in Piccolo on localization and trafficking dynamics of the serotonin reuptake transporter | 85 |
| 6 | Deletion of Munc18-1 in 5-HT neurons results in rapid degeneration of the 5-HT system and early postnatal lethality | 105 |
| 7 | Towards a genetic approach to study serotonergic outgrowth and connectivity in vivo | 127 |
| 8 | General discussion | 140 |
| | Nederlandse samenvatting | 154 |
| | List of abbreviations | 159 |
| | Dankwoord | 161 |
| | Curriculum Vitae | 164 |
Chapter 1
General introduction
General introduction
The mammalian brain is by far the most complex system in the body. The human brain consists of approximately 100 billion nerve cells (neurons), which form a highly complex wiring scheme. How this complex wiring is achieved is still poorly understood. The brain contains several clusters of cells which contain a certain neurotransmitter, such as dopaminergic cells in the substantia nigra or acetylcholinergic cells in ventral and dorsal striatum.
Another example of such clusters are the raphe nuclei in the midbrain that secrete serotonin (5-hydroxytryptamine, 5-HT). 5-HT has diverse modes of action in the central nervous system and is involved in brain development as will be described in this introduction. There are a few remarkable characteristics of the 5-HT system. First of all, the 5-HT system consists of only a few neurons (~1000 in rodents, ~0.5 million in humans) compared to the total number of neurons. Nevertheless, these neurons send projections to virtually every other brain area. It is estimated that each 5-HT neuron makes more than $1 \times 10^6$ connections. Secondly, 5-HT is predominantly released extrasynaptically (so called volume or paracrine transmission), which implies that 5-HT acts on a slower and longer-lasting time scale compared to classical neurotransmitters like glutamate. Thirdly, emerging evidence shows that 5-HT has a role during brain development. Finally, the 5-HT system is involved in certain behavioural and psychopathological processes including anxiety, depression, aggression and obsessive-compulsive disorder (OCD).
In this chapter the main aspects of the 5-HT system will be introduced and the diverse modes of action which 5-HT has are described. First, there will be a description about the anatomical organization and development of the 5-HT system. Subsequently, the role of 5-HT in brain development will be introduced and the relation between the 5-HT system and behavioural and psychopathological processes will be discussed. Finally, the aim and scope of this thesis will be outlined.
Anatomical organization of 5-HT cell clusters
After the initial discovery of 5-HT (see box 1), it took about 20 years before the cellular distribution of 5-HT was described by Dahlstrom and Fuxe using the Falck-Hillarp technique. They showed that 5-HT cell bodies are clustered in nine cell groups close to the midline in the rhombencephalon, called the raphe nuclei. The nine nuclei are divided in two groups, the rostral and caudal group. The caudal group consists of clusters B1 to B5 and send projections predominantly in the midbrain itself and towards the spinal cord. On the other hand, the rostral
cell group consists of clusters B6 to B9 and sends projections to the forebrain. The largest clusters of cells are the dorsal raphe nucleus (DRN) and the median raphe nucleus (MRN) (B7 and B8 respectively). These raphe nuclei also contain several types of non-5-HT neurons, such as GABA-ergic and noradrenergic neurons. The 5-HT neurons densely innervate virtually every brain area, with the most dense 5-HT innervations in the cortex and limbic structures including the hippocampus and basal ganglia.
**Box I**
**The discovery of 5-HT**
In the 1930’s Dr. Vittorio Erspamer tried to extract chemical compounds from enterochromaffin cells capable of causing smooth muscle contraction in the gut. In an extract of rabbit gastric mucosa, he identified a substance that could cause smooth muscle contraction. He called this substance enteramine, since it was an amino acid derivative (amine) found in enterochromaffin cells.
In the 1940’s another group identified a substance which had vasoconstrictive activity in serum. This serum influenced the tone, hence the name serotonin. When analyzing both substances, it was found that enteramine and serotonin were the same substance.
The first indication for a role of 5-HT in the brain came from Woolley, who showed that 5-HT and the hallucinogenic compound lysergic acid diethylamide (LSD) had comparable actions in the cortex of the cat. This eventually led to the discovery of 5-HT in the vertebrate brain like man and dog.
Dahlstrom and Fuxe were the first to describe the distribution of 5-HT cell bodies in the brain by histochemical detection of the 5-HT derivative indoleamine. The description of 5-HT projections from the 5-HT cell bodies towards the telencephalon and the diencephalon followed one year later. It was found that in the brain stem a relatively small population of 5-HT neurons was present, with the largest clusters in the dorsal and median raphe nuclei.
**The development of the 5-HT system**
In the mouse brain, 5-HT neurons are generated around embryonic day (E) 10, and these cells are among the first neurons expressing a specific neurotransmitter. In the neural tube, 5-HT precursor cells are formed by the combined action of sonic Hedgehog, Fgf4 and Fgf8. The proper position of these 5-HT precursor cells is defined by the midbrain-hindbrain organizing centre (MHO), Shh signaling and the transcription factor Nkx2.2 (see figure 1).
In a knockout (KO) mouse for Nkx2.2, no 5-HT neurons are present in the midbrain raphe nuclei, except in the DRN, showing that Nkx2.2 is essential for 5-HT neuron differentiation (Briscoe et al., 1999). Another transcription factor that is involved in the initial development of 5-HT neurons is GATA3 (van Doorninck et al., 1999). After 5-HT precursor cells are formed, other
factors are required to establish mature 5-HT neurons. Among these factors are two transcription factors, LIM homeobox transcription factor 1 (Lmx1) and PC12 Ets factor (Pet1).
Lmx1b expression starts at E10.75 in the ventral part of the hindbrain including the floor plate and from E13.5 Lmx1b is present in the raphe nuclei (Ding et al., 2003). Lmx1b KO mice die within 24 hours after birth (Chen et al., 1998). Lmx1b-deficient mice lack all 5-HT neurons, even when Lmx1b was only deleted in 5-HT neurons, (Ding et al., 2003; Zhao et al., 2006). These studies show that Lmx1b is not required for the initial generation of 5-HT neurons, but is necessary for the differentiation and survival of 5-HT neurons during postnatal development.
Pet1 was found in a screen in PC12 cells to identify novel ETS domain transcription factors (Fyodorov et al., 1998). Pet1 expression is restricted to the raphe nuclei and precedes the expression of 5-HT by approximately half a day (Hendricks et al., 1999). Furthermore, several genes specific for 5-HT neurons, such as the rate limiting enzyme tryptophan hydroxylase (Tph) and the 5-HT reuptake transporter (SERT) have a conserved Pet1 binding site in their promoter (Hendricks et al., 1999). This suggests that Pet1 is involved in the final
differentiation of 5-HT neurons. In a Pet1 KO mouse, there is an approximately 70% reduction in number of 5-HT neurons. The fact that there are still 30% of 5-HT neurons remaining suggests that these are either independent of Pet1 or there is another unknown transcription factor.
After this genetic cascade, at approximately E11.5, 5-HT neurons start expressing the enzymes necessary for 5-HT synthesis (see box II) and proteins necessary for reuptake (SERT) and degradation of 5-HT (Monoamine-A (MAO-A)). Shortly after initiation of expression of these genes, 5-HT neurons start to
**Box II**
**Synthesis of 5-HT**
5-HT is a neurotransmitter which belongs to the group of the biogenic amines, to which also the catecholamines (dopamine, adrenaline and noradrenaline) belong. All biogenic amines are derived from amino acids: the catecholamines from tyrosine, and 5-HT from tryptophan. 5-HT and the precursor tryptophan belong to a group of chemical compounds called the indoles, with a benzene ring joined to a five-member ring containing nitrogen. Tryptophan is an essential amino acid, which means that this amino acid cannot be made in the body from other amino acids and must therefore be ingested through the diet.
For synthesis of 5-HT in the brain, tryptophan is actively transported through the blood brain barrier via the large neutral amino acid transporter (LAT1). Once in the brain, tryptophan is metabolized to 5-HT using two different enzymes. First, tryptophan is hydroxylated in the 5-position to generate 5-hydroxy-L-tryptophan. This reaction is catalyzed by Tph, which is the rate limiting enzyme in 5-HT synthesis. Subsequently 5-hydroxy-L-tryptophan is converted to 5-hydroxytryptamine (5-HT) by the enzyme 5-hydroxytryptophan decarboxylase. 5-HT is stored in synaptic vesicles and large dense cored vesicles and transported into these vesicles by the enzyme Vesicular Monoamine Transporter 2 (VMAT2). After being released, 5-HT is transported back into the terminal using SERT, and either recycled or converted to 5-hydroxyindolacetic acid (5-HIAA) by MAO-A.
The rate limiting enzyme Tph has a (6R)-L-erythro-5,6,7,8-Tetrahydrobiopterin (Tetrahydrobiopterin, BH4) binding site. BH4 is an essential cofactor for the proper functioning of Tph. Without BH4, Tph cannot hydroxylate its substrate and thus 5-HT cannot be synthesized. BH4 is also required for dopamine synthesis by acting as a cofactor for tyrosine hydroxylase. Mice deficient for the BH4 gene die within 48 hours after birth and have extremely low levels of 5-HT and dopamine.
It has long been thought that only one Tph gene was present in the mammalian brain, although in zebrafish, two Tph genes were identified. However, in a mouse deficient for the Tph gene, no difference in brain 5-HT levels was found compared to control mice. This led to the discovery of the second Tph gene, called neuronal Tph or Tph2 (Walther et al., 2003). By using immunohistochemical analysis and in situ hybridization, it has been shown that Tph1 is expressed in the periferic system in the enterochromaffin cells and in the pineal gland in the brain, whereas Tph2 is exclusively expressed in the raphe nuclei in brain and in the myenteric plexus in the gut (Patel et al., 2004; Malek et al., 2005; Gutknecht et al., 2009).
release 5-HT and start to send axon branches towards the spinal cord and via the medial forebrain bundle towards the cortex. Only after birth the 5-HT network is fully matured.
In the human brain, 5-HT neurons are for the first time detected when the embryo is five weeks old (Sundstrom et al., 1993). At ten weeks of age, 5-HT neurons are detected in all nuclei of the reticular formation and after 15 weeks, the 5-HT neurons are clustered in the raphe nuclei (Shen et al., 1989). After birth, 5-HT levels increase during the first two years and then decline to adult levels at the age of five years (Hedner et al., 1986).
**5-HT release is both synaptic and extrasynaptic**
Several classical neurotransmitters such as GABA, acetylcholine and glutamate are released from small synaptic vesicles (SSVs) at synaptic sites with a postsynapse in close proximity. However, neurotransmitters such as dopamine and 5-HT are released not only synaptically, but there is also so-called extrasynaptic release (also called paracrine transmission or volume transmission; here there will be referred to this type of release as volume transmission). A major difference between synaptic release and volume transmission is that there is no postsynapse present, so upon release the neurotransmitters diffuse away and can mediate effects distant from the release site. In this manner volume transmission can produce paracrine effects. In contrast to synaptic release, where SSVs are only released from axonal terminals, volume transmission of 5-HT occurs also in the soma and in dendrites, the so-called somatodendritic release. In 5-HT neurons, vesicles storing 5-HT are not only present in axonal terminals, but also in somata, dendrites and axonal varicosities (Hery and Ternaux, 1981). It was shown in Retzius neurons of the leech that 5-HT is released from both SSVs and large dense core vesicles (LDCVs), where there is an average discharge of 4700 and 80,000 molecules of 5-HT for SSVs and LDCVs respectively (Bruns and Jahn, 1995). In another study of the same authors, several differences between SSV and LDCV release of 5-HT were shown. First of all there was a difference in distribution: while SSVs were exclusively found in axons, LDCVs were also found at the soma. Furthermore LDCV release was slower compared to SSV release (Bruns et al., 2000).
There are several lines of evidence for volume transmission of 5-HT, some of which are indirect. First of all it has been shown that 5-HT varicosities contain 5-HT vesicles, but lack a postsynaptic specialization in close proximity (Moukhles et al., 1997). 5-HT receptors and the SERT are localized at sites away from release sites, indicating that 5-HT diffuses over larger distances to activate these receptors (Ridet et al., 1993). The presence of SERT distant from release sites
suggests that these are required to transport 5-HT back into the cells to terminate the action of 5-HT in the extracellular space. Secondly, it has been shown by carbon fiber amperometry that upon stimulation an increase of 5-HT levels in the extracellular medium occurs (Bunin and Wightman, 1998). This suggests that upon stimulation, not all 5-HT is buffered by synaptic receptors and transporters, but diffuses away into the extracellular medium (Bunin and Wightman, 1998).
In contrast to extrasynaptic release, synaptic release occurs predominantly from SSVs. Apart from vesicular release of 5-HT, there is also evidence for non-vesicular release of 5-HT using a carrier-mediated mechanism (Levi and Raiteri, 1993).
The ratio between volume transmission and synaptic transmission of 5-HT depends on the location in the brain. In the DRN itself, 5-HT release is predominantly paracrine, and here somatodendritic 5-HT release activates 5-HT\textsubscript{A} autoreceptors which are present at the somata and dendrites. These activated receptors activate a G protein-activated inwardly rectifying K\textsuperscript{+} (GIRK) current. In target areas of the 5-HT system, the ratio between volume transmission and synaptic transmission is still under debate. However, there are electron microscopy studies that quantified the percentage of 5-HT varicosities which formed a complex with a postsynaptic specialization. This revealed that ~25%, 48% and 38% of 5-HT varicosities formed a synaptic complex in rat hippocampus, suprachiasmatic nuclei and supraoptic nuclei of the hypothalamus respectively (Oleskevich and Descarries, 1990; Boulaiach et al., 1994). It is noteworthy that 5-HT is not released exclusively from 5-HT neurons. During development, thalamocortical fibers transiently express SERT, capture 5-HT and use it as a borrowed transmitter (Lebrand et al., 1996). In the striatum, dopamine transporters can take up 5-HT and subsequently co-release dopamine and 5-HT from dopaminergic terminals (Zhou et al., 2005a).
**5-HT has several effects on synaptic transmission**
5-HT not only functions to activate postsynaptic receptors, but also has diverse functions in the presynapse. Several studies were performed on invertebrates, such as crayfish, lamprey and aplysia. It was observed at the crayfish neuromuscular junction that a brief exposure to 5-HT resulted in increased release of glutamate (Dixon and Atwood, 1989a). Injection of a protein kinase A inhibitor or an adenylate cyclase inhibitor abolished this effect, suggesting that the facilitatory effect of 5-HT required the action of both the phosphatidylinositol and adenylate cyclase second messenger pathway (Dixon and Atwood, 1989b). In a study by Wang and colleagues, they made use of the styryl dye FM1-43, a fluorescent dye which is taken up by endocytosis following vesicle release, thus
specifically labeling recycled vesicles (Betz and Bewick, 1992). Using this approach, application of 5-HT at the crayfish neuromuscular junction did not affect the rate of recycling. Instead, 5-HT increased the number of vesicles which are available for release, the ready-releasable pool (RRP) of vesicles (Wang and Zucker, 1998).
In contrast to this stimulatory effect in the crayfish neuromuscular junction, 5-HT has an inhibitory action on synaptic transmission in the lamprey giant synapse (Buchanan and Grillner, 1991). This effect of 5-HT is likely mediated via G protein βγ subunits, since injection of these subunits mimic the effect of 5-HT (Blackmer et al., 2001). Furthermore the presynaptic protein SNAP25 is involved in this effect, since injection of Botulimum toxin A, which cleaves SNAP25, abolished the effect of 5-HT (Gerachshenko et al., 2005). Again using FM-dyes it was demonstrated that the inhibitory effect of 5-HT on the lamprey giant synapse is mediated via a change in vesicle fusion properties since 5-HT seemed to prevent full fusion of vesicles (Photowala et al., 2006).
In aplysia the effect of 5-HT on the gill-withdrawal reflex is well studied. In the 70’s it was shown for the first time that local application of 5-HT sensitized the gill-withdrawal reflex. This effect was also shown after application of cyclic adenosine monophosphate (cAMP), suggesting that this effect of 5-HT is mediated via an increase in cAMP (Brunelli et al., 1976). Both 5-HT and cAMP can close single potassium channels in aplysia sensory neurons, resulting in an increased transmitter release (Siegelbaum et al., 1982). This K⁺ channel, called the S (serotonin)-type K⁺ channel is modulated by 5-HT, cAMP and protein kinase A (PKA) (Shuster et al., 1985). Using calcium imaging on isolated synapses in culture, it was shown that 5-HT application results in a calcium influx in the terminal (Eliot et al., 1993). A brief single pulse of 5-HT resulted in short-term facilitation lasting minutes, whereas five subsequent pulses of 5-HT over 1.5 hours resulted in long-term facilitation lasting more than one day (Montarolo et al., 1986). 5-HT does not only affect release characteristics at single synapses, but upon application also activates silent presynaptic terminals. This results in the generation of new functional release sites within hours after application (Kim et al., 2003). This 5-HT-related growth of new synapses requires Cdc42, N-WASP and PAK (Udo et al., 2005).
In summary, 5-HT affects synaptic transmission in several ways. In crayfish neuromuscular junctions, 5-HT increases synaptic transmission by increasing the size of the RRP of vesicles. In the lamprey giant synapse 5-HT inhibits transmission by affecting single vesicle release kinetics. Finally, in aplysia sensory neurons, application of 5-HT can induce both short term facilitation and long term facilitation via an increase in transmitter release and the formation of new functional release sites.
5-HT has a role during development in maturation of the brain.
An increasing amount of evidence points towards a role of 5-HT in brain development and maturation. Additionally, several studies show that 5-HT has an important role during early embryogenesis. 5-HT is present at early stages in sea urchins, chick embryos and rodents (Emanuelsson et al., 1988; Buznikov, 1991). Application of a 5-HT antagonist to sea urchin embryos significantly delayed cell division, implicating a role in the regulation of cell division (Renaud et al., 1983). In sea urchin blastulae, 5-HT, but also dopamine and noradrenaline stimulate ciliary activity. Application of these monoamines was associated with changes in intracellular calcium and adenyl cyclase activity (Soliman, 1983). In the chick embryo, 5-HT is synthesized by the notochord (Wallace, 1982). Monoamine inhibitors cause malformation in the chick embryo such as neural tube defects. Exposure of mouse embryos to 5-HT resulted in craniofacial malformations and increased cell death in craniofacial and cardiac mesenchyme (Yavarone et al., 1993); (Shuey et al., 1993). Several studies in rodents used injections of either p-chlorophenylalanine (PCPA) to inhibit Tph or the 5-HT-specific neurotoxin 5,7-dihydroxytryptamine. Injection of PCPA in mouse embryos from E12-E17 resulted in subtle alterations in pyramidal and non-pyramidal neuronal populations (Vitalis et al., 2007). 5,7-dihydroxytryptamine was injected at the day of birth in mice embryos in the medial forebrain bundle. These injected mice had a decreased 5-HT innervation towards the cortex and hippocampus and male mice showed a decreased novelty-induced exploration, suggesting an anxiety-like phenotype (Hohmann et al., 2007).
KO mice have shown to be a valuable tool in studying the role of the 5-HT system in development. KO mice for tryptophan hydroxylase 1 (Tph1), the rate limiting enzyme involved in periferic 5-HT synthesis, lack periferic 5-HT (Walther et al., 2003). Although these KO mice do not have an overt phenotype, approximately 80% of pups born from homozygous mothers are smaller and show developmental abnormalities compared to embryos born from heterozygous or wildtype mothers (Cote et al., 2007).
This phenotype was only observed in Tph1 KO embryos born from Tph1 KO mothers, showing that maternal 5-HT is crucial for normal embryonic development. Tryptophan hydroxylase 2 (Tph2) is the Tph isoform that is exclusively expressed in the raphe nuclei (Gutknecht et al., 2009). Deletion of Tph2 does not result in an overt phenotype, nor does deletion of both Tph1 and Tph2, although in another study a reduced viability in these mice was observed (Savelieva et al., 2008; Alenina et al., 2009). Thus it appears that maternal 5-HT is indispensable for proper embryonic (brain) development, but 5-HT synthesis in the embryo is not.
5-HT as a neurotrophic factor
In addition to the role of 5-HT in (early) neurodevelopment, 5-HT also functions as a neurotrophic factor. Application of 5-HT to buccal ganglion neuron 19 in helisoma caused a cessation in filopodial motility and an inhibition of neurite outgrowth (Haydon et al., 1984). Application of 5-HT results in a rise in intracellular calcium levels in the growth cone which probably links directly to the inhibition in outgrowth (McCobb et al., 1988). Application of 5-HT to 5-HT neurons also resulted in decreased outgrowth (Whitaker-Azmitia and Azmitia, 1986). In contrast to this inhibitory effect of 5-HT on growth of 5-HT neurons, 5-HT acts as a stimulatory signal on thalamic neurons which display more processes per cell and increased length of processes upon 5-HT administration (Lieske et al., 1999).
Mouse models with an impaired 5-HT system
Since the introduction of the knockout technique in mice, several genes of the 5-HT system have been assessed for their effect on development and behaviour. The most important genes with striking effects on brain development or behaviour will be mentioned here. Mice lacking the MAO-A gene involved in the degradation of 5-HT to 5-hydroxyindolacetic acid (5-HIAA) display increased levels of 5-HT (Cases et al., 1996). These mice have a lack of barrels in the somatosensory cortex that is restored by application of PCPA, suggesting that this effect is due to excess levels of 5-HT (Cases et al., 1996). Additionally, mice lacking MAO-A display increased aggressive behaviour (Cases et al., 1995).
On the contrary, deleting the Vesicular Monoamine Transporter 2 (VMAT2) which is necessary for 5-HT packaging in vesicles, results in mice with brain 5-HT levels of ~1% compared to control (Wang et al., 1997; Fon et al., 1997). These KO mice are significantly smaller compared to wildtype littermates and the majority dies within a few days after birth. 5-HT fibers are almost completely absent in the cortex at that age. VMAT2 heterozygotes display prolonged immobility times in the forced swim test, suggesting depressive-like behaviour (Fukui et al., 2007). This behaviour was normalized by the antidepressant imipramine.
Deletion of the SERT which is essential for 5-HT reuptake results in mice with increased extracellular 5-HT levels (Mathews et al., 2004). These mice have a reduced number of 5-HT neurons in the DRN, a reduced firing rate of these neurons and these mice display behavioural alterations (Lira et al., 2003). Notably, these behavioural effects were mimicked by applying the SERT blocker fluoxetine to young control mice between postnatal day 4 and 21 (Ansorge et al., 2004).
In addition to these genes involved in 5-HT synthesis, packaging, breakdown or reuptake, several 5-HT receptor genes have also been ablated in mice (for an overview of 5-HT receptors see Box III). The 5-HT\textsubscript{1A} receptor acts as a heteroreceptor on non-5-HT neurons, but also as a 5-HT autoreceptor in the DRN. Mice with a deletion of the 5-HT\textsubscript{1A} receptor spent less time in the center of the open field in the open field test and avoid the open arms in the elevated plus maze, suggesting that these mice display an increased anxiety-related behaviour (Parks et al., 1998; Ramboz et al., 1998). A very elegant study demonstrated that this effect on behaviour in the 5-HT\textsubscript{1A} receptor KO occurs during development. In this study a second mouse line was made in which the expression of the 5-HT\textsubscript{1A} receptor could be restored specifically in the forebrain. Rescue of 5-HT\textsubscript{1A} receptor expression in the forebrain was sufficient to restore normal anxiety-related behaviour (Gross et al., 2002). In contrast, deletion of the 5-HT\textsubscript{1A} receptor during development and rescue of the receptor after postnatal day 21 did not restore normal anxiety-related behaviour (Gross et al., 2002). This clearly shows that the 5-HT\textsubscript{1A} receptor is required during development to establish normal anxiety-related behaviour.
**Box III**
**5-HT receptors**
At least 14 different 5-HT receptor subtypes are known which are divided into 6 different groups. The first and largest group of 5-HT receptors is the 5-HT\textsubscript{1} group consisting of the 1A, 1B, 1D, 1E, 1F and 1P receptors. All receptors in this group are coupled to G proteins, and activation results in a decrease in cAMP levels. The most important receptor of this group is the 5-HT\textsubscript{1A} receptor, which is expressed in the raphe nuclei on 5-HT cell bodies and functions as a 5-HT autoreceptor. Binding of 5-HT to the 5-HT\textsubscript{1A} receptor results in activation of a GIRK channel which results in hyperpolarization of the 5-HT neuron; in this way, the 5-HT\textsubscript{1A} receptor is involved in fine-tuning 5-HT release.
To the 5-HT\textsubscript{2} receptor group belong the 2A, 2B and 2C receptors. These receptors are also coupled to G proteins, and activation of these receptors results in an increase of inositol 3-phosphate (IP3). The 5-HT\textsubscript{3} receptor is the only 5-HT receptor which is a (Na\textsuperscript{+}/K\textsuperscript{+}) ion channel. The 5-HT\textsubscript{4} and 5-HT\textsubscript{6,7} receptors are coupled to Gs proteins and activation results in an increase of cAMP. Finally, activation of the 5-HT\textsubscript{5A,B} receptor results in cAMP decrease. For the majority of these 5-HT receptors there are agonists and antagonists known. For example, the 5-HT\textsubscript{1A} receptor is agonized by 8-OH-DPAT whereas it is antagonized by WAY-100635.
Mice lacking the 5-HT\textsubscript{1B} receptor display an increased aggressive-related behaviour: they have a shorter latency to attack in the resident intruder paradigm (Saudou et al., 1994). The 5-HT innervation is increased in the hippocampus and the amygdala of these mice, possibly contributing to the aggressive phenotype of these mice (Ase et al., 2001). Another receptor known to
be localized in the DRN is the 5-HT\textsubscript{2C} receptor (Clemett et al., 2000). This receptor is localized on GABA-ergic neurons and activation of this receptor results in a decreased firing of 5-HT neurons (Boothman et al., 2006). Mice that lack the 5-HT\textsubscript{2C} receptor are overweight and suffer from seizures (Tecott et al., 1995). Moreover, studies have been performed to show that these mice display compulsive-like behaviour (Chou-Green et al., 2003).
An additional study showed that 5-HT\textsubscript{2C} KO mice display decreased inhibition in the conflict anxiety paradigm (Weisstaub et al., 2006). This decreased inhibition was rescued by selective restoration of the 5-HT\textsubscript{2C} receptor in the cortex, suggesting that 5-HT\textsubscript{2C} receptors in the DRN are not involved in this effect (Weisstaub et al., 2006).
Thus, 5-HT receptor KO mice do not exhibit overt developmental defects, but anxiety- or aggressive-related behaviour of these animals is affected as assessed in various behavioural paradigms.
**The 5-HT system in relation to psychopathological processes**
**Anxiety & depression**
The notion that the 5-HT system could be involved in psychopathological conditions such as major depression and anxiety-related disorders came only after the discovery that drugs used to treat these conditions exerted their function (partially) via the 5-HT system. In the late 50’s, compounds such as iproniazide and G22355 (later named imipramine) were prescribed to patients suffering from tuberculosis. However, serendipitously it was found that these compounds were also effective in treating depression (Bloch et al., 1954; Kuhn, 1958). Only several years after it was found that these drugs had an antidepressant response, the mode of action of these drugs was elucidated; iproniazide inhibited the degradation of mono-aminergic compounds (5-HT, noradrenaline and dopamine), whereas imipramine blocked the reuptake of 5-HT and noradrenaline into the terminal. These findings led to the postulation of the monoamine hypothesis of depression in the late 60’s, which stated that a decreased concentration of 5-HT and noradrenaline in the extracellular space might be responsible for depression. It was poorly understood, however, why there was such a discrepancy between the immediate modes of action of these compounds in contrast to the delayed therapeutic effects. Also, it was not clear why only $\sim$2/3 of all patients responded to these drugs.
These considerations abandoned the monoamine hypothesis and the so-called network hypothesis of depression was postulated. This hypothesis states that depression results from an alteration in certain neuronal networks, and antidepressants restore these networks, which explains the time lag between
starting antidepressant administration and the onset of therapeutic actions. Evidence for this hypothesis, however, is still limited and mostly indirect. Human studies using functional magnetic resonance imaging (fMRI) or positron emission tomography (PET) imaging on patients suffering from major depression have shown volume reductions in cortex and hippocampus. Although farfetched, one could argue that this reflects a decreased network density.
The last few years a new hypothesis was proposed, largely based on the work by Santarelli and colleagues: the neurogenesis hypothesis. It had already been shown that treatment with antidepressants increased adult neurogenesis in the dentate gyrus (Malberg et al., 2000; Perera et al., 2007). Santarelli and colleagues showed that blocking neurogenesis in the dentate gyrus by X-irradiation blocked the behavioural effects of the antidepressant fluoxetine (Prozac) (Santarelli et al., 2003). In other words neurogenesis is required for the behavioural effects of antidepressants. However, several studies have shown that neurogenesis is not involved in the etiology of depression.
Thus, although it is clear that there is a link between the 5-HT system and anxiety and depression, it is still far from clear what this exact link is.
**Aggression**
Aggressive behaviour is defined as violent and impulsive behaviour often resulting in breaking the law. 5-HT is one of the neurotransmitters which is implicated in the etiology of aggression. A mutation in the MAO-A gene caused aggression-related abnormal behaviour (impulsive aggression, arson, attempted rape, and exhibitionism) in five males of a large kindred (Brunner et al., 1993). Several studies have shown a reduced concentration of the 5-HT metabolite 5-HIAA in cerebrospinal fluid (CSF) of individuals suffering from aggressive behaviour (Asberg et al., 1976; Coccaro et al., 1997). In rhesus monkeys, there was a negative relation found between high aggression and CSF 5-HIAA levels (Higley et al., 1992). A number of studies implicate the 5-HT$_2$ receptor subtypes in aggressive behaviour. A study by Winstanley and colleagues showed that 5-HT depletion increased the number of premature responses in the 5-choice serial reaction time task in rats (Winstanley et al., 2004). Also, in this study it was found that application of a 5-HT$_{2A}$ antagonist decreased impulsive behaviour, whereas application of a 5-HT$_{2C}$ antagonist had the opposite effect (Winstanley et al., 2004). A clinical study showed that antipsychotic drugs which had anti-aggressive properties were 5-HT$_{2A}$ antagonists (Krakowski et al., 2006). PET imaging studies show an increased 5-HT$_{2A}$ receptor binding in aggressive patients or in borderline personality disorder patients (Siever et al., 2002; Soloff et al., 2007). These abovementioned studies indicate increased 5-HT$_{2A}$ receptor sensitivity in aggressive behaviour patients.
Autism
Autism is a behavioural disorder with unknown etiology, although impaired brain development may underlie this disorder. The link between autism and 5-HT was made after the discovery that autistic patients had elevated platelet 5-HT levels (Boullin et al., 1970; Ritvo et al., 1970). Additionally, depletion of the 5-HT precursor tryptophan resulted in a deterioration in autistic patients (McDougle et al., 1993). Chugani and colleagues used alpha-[11C]methyl-L-tryptophan as a tracer for PET to measure 5-HT synthesis. They showed that in autistic boys there is a decreased 5-HT synthesis in frontal cortex, thalamus, and dentate nucleus of the cerebellum, although there is an increased 5-HT synthesis in the contralateral dentate nucleus (Chugani et al., 1997). Possibly increased levels of 5-HT during early brain development result in the loss of 5-HT terminals which could explain why several drugs which show great efficacy in treating autism enhance 5-HT neurotransmission (Brodkin et al., 1997; Fatemi et al., 1998). More evidence for this hypothesis is the fact that prenatal exposure to cocaine, which is a releaser of 5-HT, is a risk factor for developing autism (Davis et al., 1992). Furthermore, several studies using the 5-HT agonist 5-methoxytryptamine during development in rodents show that this results in autistic-like behaviour (Winslow and Insel, 1990; Kahne et al., 2002).
Polymorphisms in genes involved in the 5-HT system
SERT
In several genes involved in the 5-HT system, polymorphisms and short nucleotide polymorphisms (SNPs) have been found. An example of a gene in which polymorphisms have been found which correlates with psychopathological processes is SERT. Soon after the cloning of the human SERT, several polymorphisms have been found (Furlong et al., 1998; Battersby et al., 1999). These include two SNPs in the promoter region, a variable number of tandem repeats polymorphism in intron 2, and rare Ile-425-Val and Pro-339-Leu SNPs which affects function and surface expression (Kilic et al., 2003; Prasad et al., 2005). One polymorphism has been found in the promoter region. This polymorphism, a 44 bp insertion has been called the 5-HTT gene-linked polymorphic region, 5-HTTLPR. The two polymorphisms, called the short (s) and long (l) variant have been shown to modulate SERT transcriptional activity (Lesch et al., 1996). In a luciferase assay in human placental choriocarcinoma cells it was shown that the l variant has approximately a threefold higher transcriptional activity compared to the s variant (Heils et al., 1996). Additionally, cells expressing the l/l variant of the SERT displayed a twofold
higher uptake rate of 5-HT compared to cells expressing the s/s variant. In human postmortem brain material, reduced mRNA concentrations of SERT were found in the raphe nuclei from subjects having the s/s variant of the SERT promoter polymorphism. This shows that both in vitro and in vivo, the s/s variant is associated with a decreased SERT expression and reduced 5-HT reuptake (Little et al., 1998).
In 1996, the first study was published which demonstrated a link between the SERT promoter polymorphism and anxiety, aggression and depression traits (Lesch et al., 1996). Since then, several studies have demonstrated a link between the s/s promoter variant and aggression, anxiety and depression. Moreover, several studies show that individuals carrying the s/s variant have a poorer response to antidepressant treatment than individuals carrying the l/l or l/s variant (Smeraldi et al., 1998; Zanardi et al., 2000).
**MAO-A**
MAO is involved in the degradation of 5-HT to 5-HIAA, and there are 2 isoforms encoded by different genes, MAO-A and MAO-B. MAO-A has a higher affinity for 5-HT than MAO-B. These genes are both localized on the X chromosome (Lan et al., 1989). In humans a point mutation in the MAO-A gene has been found in the 8th exon, resulting in a truncated MAO-A protein. The family in which the point mutation was identified suffered from abnormal behaviour including disturbed regulation of impulsive aggressiveness (Brunner et al., 1993). In the promoter region of MAO-A a 30 bp repeat polymorphism was found. The low-activity 3-repeat allele was associated with antisocial behaviour in a German population (Samochowiec et al., 1999). In the rhesus macaque, a repeat polymorphism in the promoter affected transcription of the MAO-A gene and was associated with aggressive behaviour (Newman et al., 2005).
**Tph2**
Tph1 and Tph2 are the rate limiting enzymes for 5-HT synthesis. Tph1 is expressed in the enterochromaffin cells in the gut and in the pineal gland, whereas Tph2 is the neuronal form and exclusively expressed in the raphe nuclei (Patel et al., 2004; Zill et al., 2004a). In Tph2 a C1473G mutation, resulting in a Pro-447-Arg substitution results in a reduction in 5-HT synthesis. BALB/cJ mice, which are homozygous for the 1473G mutation, have a 50 to 70% reduction in 5-HT synthesis in the frontal cortex resulting in ~40% reduction in 5-HT content in the frontal cortex (Zhang et al., 2004). In the human Tph2 gene, a G1463A SNP resulting in a Arg441His replacement was identified which, when transfected in PC12 cells, resulted in a ~80% loss of function in 5-HT synthesis.
(Zhang et al., 2005). In a cohort of 87 patients suffering from major depression, 9 were found to carry this SNP, while among 219 controls, only 3 subjects had this SNP (Zhang et al., 2005). Knockin mice carrying this human mutation displayed behavioural abnormalities (Beaulieu et al., 2008). In contrast to SNPs found in exons, also SNPs in introns of the Tph2 gene have been found. In intron 5, a SNP has been found which was associated with major depression and suicide (Zill et al., 2004b; Harvey et al., 2004).
As in the SERT promoter, also in the Tph2 promoter polymorphisms have been found. One of these polymorphisms, rs4570625, has been associated with increased amygdala reactivity to emotional stimuli (Canli et al., 2005). Two polymorphisms, rs4570625 and rs11178997, were linked to anxiety disorder and major depression (Zhou et al., 2005b). As shown by a luciferase assay in 5-HT neurons, the polymorphism rs11178997 significantly reduced Tph2 transcriptional activity by 22% (Scheuch et al., 2007).
**Conclusions**
5-HT is a transmitter that has several modes of action in the central nervous system. First of all, 5-HT has a role during brain development and maturation. Secondly, due to the volume transmission, 5-HT can influence several aspects of behaviour, depending on the receptor(s) to which 5-HT binds. 5-HT can also regulate synaptic transmission. KO mice for the 5-HT\textsubscript{1A}, 5-HT\textsubscript{1B} or SERT gene display altered anxiety-related, depression-related and aggression-related behaviour. Finally, polymorphisms in several genes functioning in the 5-HT system are implicated in depression, anxiety, aggression and autism.
**Aim and outline of this thesis**
In this thesis the outgrowth and connectivity of the brain 5-HT system is studied. To study these aspects we used a number of different approaches. *First* of all, we used organotypic slice cultures to study the outgrowth of the 5-HT system in vitro. *Secondly*, we used neuronal cultures to study trafficking of two components of the 5-HT system, SERT and Tph2. *Thirdly*, we investigated whether a SNP in a presynaptic gene which associates with major depressive disorder, affects trafficking of the SERT. *Fourthly*, we used a genetic approach in mice to study the effect of a silenced 5-HT system on mouse development. *Finally*, we describe our approach to generate a mouse model in which the 5-HT system is labeled with fluorescent reporter genes to study the outgrowth and connectivity in vivo.
In chapter 2, we used organotypic slice cocultures of the dorsal raphe nucleus and the hippocampus as a target area. We have used this model system to study the outgrowth of the 5-HT system in vitro. Moreover, we investigated whether this system could be used to assess the effect of pharmacological manipulations on the outgrowth of the 5-HT system.
In chapter 3, we tagged SERT with the red fluorescent protein mCherry, and expressed this protein in hippocampal neurons to investigate the trafficking dynamics of SERT.
In chapter 4, we tagged Tph2 with EGFP and took the same approach as in chapter 3 to investigate the subcellular distribution and trafficking of Tph2 in neurons.
In chapter 5, we studied whether knockdown of the presynaptic gene Piccolo affects SERT trafficking and incorporation into the membrane. A SNP in the C2A domain of this protein was found which associated with major depressive disorder. We expressed these two variants and investigated whether there is a difference in SERT localization. Also, we investigated whether knockdown of Piccolo affects SERT trafficking.
In chapter 6, we used a genetic approach to silence the 5-HT system. We crossed floxed Munc18-1 mice with mice expressing Cre recombinase in SERT expressing neurons, which include 5-HT neurons. We used these mice to study the effect of Munc18-1 removal in 5-HT neurons on the development of the 5-HT system and viability. We used heterozygous Munc18-1\textsuperscript{+/lox} mice to study whether deletion of one allele of Munc18-1 in the 5-HT system affects mouse behaviour.
In chapter 7, we describe an approach to specifically label 5-HT neurons and projections with fluorescent markers which could eventually be used to study 5-HT outgrowth and connectivity in vivo.
Finally, in chapter 8, the main findings of this thesis will be summarized, discussed and future directions are suggested.
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Chapter 2
Chronic activation of the 5-HT$_2$ receptor reduces 5-HT neurite density as studied in organotypic slice cultures
J.J. Dudok$^1$, A.J.A. Groffen$^1$, M.P. Witter$^{a,*}$, P. Voorn$^2$ and M. Verhage$^1$
$^1$Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, the Netherlands
$^a$Department of Anatomy and Neurosciences, Vrije Universiteit Medical Center, Amsterdam, the Netherlands
$^b$Present address: Kavli Institute for Systems Neuroscience and Centre for the Biology of Memory, Norwegian University of Science and Technology NTNU, Trondheim, Norway
Published in Brain Research (2009) Dec 11;1302:1-9
Abstract
The serotonin system densely innervates the brain and is implicated in psychopathological processes. Here we studied the effect of serotonin and serotonin pharmacological compounds on the outgrowth of serotonergic projections using organotypic slice co-cultures of hippocampus and dorsal raphe nuclei. Immunohistochemical analysis showed that several serotonergic neurites had grown into the target slice within 7 days in culture, after which the neurite density stabilized. These projections expressed the serotonin-synthesizing enzyme tryptophan hydroxylase and the serotonin transporter and contained several serotonin positive varicosities that also accumulated presynaptic markers. Chronic application of a 5-HT$_2$ agonist reduced the serotonergic neurite density, without effects on survival of serotonergic neurons. In contrast, application of a 5-HT$_{1A}$ agonist or the serotonin transporter inhibitor fluoxetine did not affect serotonergic neurite density. We conclude that serotonergic connectivity was reproduced in vitro and that the serotonin neurite density is inhibited by chronic activation of the 5-HT$_2$ receptor.
Introduction
The serotonin (5-hydroxytryptamine (5-HT)) system has its cell bodies clustered in the midbrain raphe nuclei and sends abundant projections to virtually every brain area (Hornung, 2003; Rubenstein, 1998). The 5-HT system influences a wide variety of physiological processes, such as sleep-wake rhythm, feeding, sexual behaviour and nociception (Jacobs and Azmitia, 1992). Moreover, the 5-HT system is implicated in several psychopathological processes, such as anxiety, aggression, obsessive compulsive disorder and depression (Castren, 2005).
During brain development, the 5-HT neurons are one of the first groups of cells expressing a specific neurotransmitter (Lauder, 1990; Gaspar et al., 2003). Accumulating evidence shows that alterations in brain 5-HT levels during development could result in alterations in brain development and behavioural alterations. 5-HT reuptake transporter (SERT) knockout (KO) mice have an increase in extra neuronal 5-HT but a reduction in overall brain 5-HT levels (Bengel et al., 1998; Mathews et al., 2004). They exhibit behavioural alterations, reduced number of 5-HT neurons in the dorsal raphe nuclei (DRN) and reduced firing of 5-HT neurons in the DRN (Lira et al., 2003). These behavioural alterations observed in SERT KO mice can be mimicked by application of the
SERT inhibitor fluoxetine during development between postnatal day 4 and 21 (Ansorge et al., 2004).
In contrast to reduction in brain 5-HT content after disruption of the SERT gene, there is a significant increase in brain 5-HT levels after inactivation of the Monoamine Oxidase-A (MAO-A) gene (Cases et al., 1996). The chronic elevation of 5-HT levels during brain development results in lack of barrels in the barrel cortex (Cases et al., 1996). These data suggest that either reductions in brain 5-HT levels or excess 5-HT levels during a critical period in the development of the brain can lead to abnormalities in brain development and behavioural alterations. In vitro experiments have shown that 5-HT can inhibit the outgrowth of neurites or even cause growth cone collapse on 5-HT neurons (Haydon et al., 1984; Koert et al., 2001). Whether alterations in brain 5-HT levels during development of the brain result in alterations in 5-HT outgrowth, branching or connectivity, or whether the behavioural alterations observed are a result of alterations in the 5-HT outgrowth and connectivity is currently unknown. Organotypic slice cultures of the DRN and a target slice could be a valuable reduced model system to study 5-HT outgrowth in vitro.
Therefore the aim of this study was to investigate whether organotypic slice co-cultures of DRN and hippocampus, a method that has previously been characterized, can be used to study 5-HT outgrowth and the effect of pharmacological manipulations in vitro (Papp et al., 1995; Guthrie et al., 2005). We show that within seven days of culturing several 5-HT neurites have grown into the hippocampal slice. Using these slice co-cultures, we studied whether chronic application of 5-HT pharmacological compounds affects the outgrowth of 5-HT neurites from the DRN. To this end we used a 5-HT\textsubscript{1A} receptor agonist, a 5-HT\textsubscript{2} receptor agonist and the SERT inhibitor fluoxetine since it has been shown before, also on non-5-HT cells, that (chronic) activation of a 5-HT\textsubscript{1A} or 5-HT\textsubscript{2} receptor or blockade of the SERT can affect (5-HT) outgrowth or synaptogenesis (Wilson et al., 1998; Kondoh et al., 2004; Fricker et al., 2005; Zhou et al., 2006). We show here that chronic application of a 5-HT\textsubscript{2} agonist results in a reduction in 5-HT neurite density. We conclude that these organotypic slice co-cultures can be used to study 5-HT outgrowth and the effect of 5-HT pharmacological compounds on this outgrowth in vitro.
**Results**
*Development and outgrowth of 5-HT neurons in organotypic slice co-cultures*
To study the outgrowth of 5-HT neurites ex vivo, we used organotypic slice co-cultures of the DRN and a hippocampal slice. We positioned the slices close to each other to allow 5-HT neurites to grow into the hippocampal slice. Within
seven days of culturing the slices flattened out and several non-neuronal cells migrated away from the slices (Fig. 1).
**Figure 1. Overview of the culturing method**
The DRN and hippocampal slice were positioned close to each other to allow 5-HT neurites to grow into the hippocampal slice. After culturing for 7 days, the slices flatten and several cells (e.g. macrophages) migrate away from the slices. Scale bar: 500 µm.
We performed immunohistochemistry for 5-HT to investigate whether 5-HT neurons survived in the slices. This showed that in several DRN slices 5-HT neurons were present, which resembled the topology of 5-HT neurons in the DRN in vivo (Fig. 2A). However, there were also DRN slices which contained only few 5-HT neurons, possibly resulting from a variation in the dissection of the DRN slice. A Z-stack of 5-HT cell somata showed that these cells have several primary neurites which contain numerous varicosities (Fig. 2B). In the growth cones of 5-HT neurites, 5-HT was also detected (Fig. 2C). Next, we focussed on the expression of tryptophan hydroxylase (Tph) and SERT, two markers for the 5-HT system. In 5-HT immunopositive cell bodies and in the 5-HT neurites and varicosities Tph was expressed (Fig. 2D,E). We found that all the 5-HT neurites were positive for SERT labelling, with the highest level of SERT staining on the soma and in the 5-HT varicosities (Fig. 2F,G). Moreover, SERT was also expressed in a punctate pattern in the 5-HT growth cones. (Fig. 2H). In vivo, 5-HT is released from varicosities in 5-HT axons and dendrites and 5-HT release is predominantly paracrine (Bunin and Wightman, 1999). In the slices, the 5-HT neurites contain several round and fusiform shaped varicosities (Fig. 3A,B).
Figure 2. 5-HT neurons survive in the slice and grow out several 5-HT neurites
(A) A DRN slice cultured for 7 days which is stained for 5-HT shows that several 5-HT cells are present in the slice. Note the presence of the lateral wings of the DRN (arrows) and the group of 5-HT neurons in the ventral part of the DRN (arrowhead). (B) A Z stack of two cells shows that the cells project several neurites that are 5-HT positive. (C) The growth cones also contain 5-HT, both in the core (arrowhead) and in the filopodia (arrows). (D,E) Staining for the rate limiting enzyme Tph shows that Tph is present both in the cell bodies and in the varicosities in the neurites. (F,G) The other marker for the 5-HT system, SERT, is present in 5-HT cell bodies and 5-HT neurites. (H) Moreover, there is also a punctate SERT staining in 5-HT growth cones, both in the core (arrowhead) and in the filopodia (arrows). Scale bars: 200 µm in A, 50 µm in B and D, 20 µm in F and G, 5 µm in C and E, 2 µm in H.
Immunohistochemistry revealed that in these varicosities the calcium sensor Synaptotagmin and the active zone marker Bassoon are present, suggesting that the varicosities are presynaptic sites where calcium dependent 5-HT release occurs (Fig. 3C,D) (Perin et al., 1991; tom Dieck et al., 1998). This showed that in organotypic slice co-cultures of DRN and hippocampus 5-HT neurons survive and grow out neurites that contain 5-HT varicosities with immunoreactivity for all tested components of the 5-HT release machinery.
Dense ingrowth of 5-HT neurites into hippocampal slices
To study the outgrowth, we fixed slice cultures at different time points and quantified the 5-HT outgrowth. To this end the images were converted to binary images and the 5-HT neurite density in the slices was quantified (Fig. 4A-C).
Figure 3. 5-HT neurites contain several varicosities which are positive for presynaptic markers
(A) A Z-stack in the hippocampal slice shows that several thin, highly branching 5-HT neurites have grown into the slice. (B) A blow-up of the red box in A shows that these neurites contain several varicosities. (C,D) These varicosities are positive for the protein involved in calcium dependent secretion, Synaptotagmin (Syt), and for the active zone marker Bassoon. Scale bars: 50 µm in A, 10 µm in B and D and 2 µm in C.
We quantified the 5-HT neurite density as the area in the slice occupied by 5-HT neurites compared to the total area of the slice. Immunohistochemistry for 5-HT on hippocampal slices cultured without the DRN for seven days revealed that there were no 5-HT neurites present (Fig. 4E). Thus, 5-HT neurites observed in the hippocampal slice in co-cultures are the result of re-growth of 5-HT neurites from the DRN slice. In co-cultures fixed at four days in vitro (DIV) we observed that the DRN slice already contained several 5-HT neurites, and the first 5-HT neurites started to grow into the hippocampal slice (Fig. 4F,I). At DIV7, also the
hippocampal slice contained several 5-HT neurites (Fig. 4G,J). Quantification of the 5-HT neurite density in the slices at DIV14 and DIV21 revealed that this was not significantly different from DIV7 (Fig. 4D,H; DIV7 DRN 8.42 ± 0.8%, HIP 6.31 ± 0.84% n=16; DIV14 DRN 9.49 ± 1.42%, HIP 5.12 ± 0.9% n=12; DIV21 DRN 9.26 ± 1.16%, HIP 6.61 ± 1.43% n=4). We investigated whether there is regional variation in the 5-HT neurite distribution in the hippocampus. At DIV4 the first 5-HT neurites which start growing into the hippocampal slice do not appear to display a preference for a certain region. (Fig. 4K). At DIV7, 5-HT neurites have grown in a uniform distribution into the hippocampus, and there are no regions which contain a higher density of 5-HT neurites than other regions (Fig. 4L). Thus, the initial outgrowth of the 5-HT neurites occurs in the first 7 days, after which the network stabilizes and the homogenous distribution suggests that 5-HT neurites do not have any preferential target region within the hippocampal slice in vitro.
**The effect of 5-HT pharmacological compounds on the outgrowth of 5-HT projections**
Finally, we studied whether this culture model system can be used to study the effect of chronic application of 5-HT pharmacological compounds on the outgrowth of 5-HT neurites. There are several 5-HT receptors which are expressed in both the DRN and the hippocampus.
**Figure 4 next page. Dense ingrowth of 5-HT neurites into hippocampal slices within seven days of culturing**
An original image of a hippocampal slice co-cultured with a DRN slice shows that there is abundant ingrowth of 5-HT neurites, but there is also a lot of red background staining. (B) When this image is converted to a binary image using the steps described in experimental procedures, the 5-HT neurites are positively labelled (black), whereas the background is removed (white). (C) A blow up of part of the binary image shows that all 5-HT neurites are detected, and the red background is removed. (D) Quantification of the 5-HT neurite density (Y-axis) in the DRN and hippocampal slices at DIV7, DIV14 and DIV21. (E) A hippocampal slice cultured without the DRN contains only background staining, and no 5-HT neurites. (F,I) Maximal 5-HT ingrowth of the slices is reached in the first seven days of culturing. At DIV4, the DRN slice already contains several 5-HT neurites, and the first 5-HT neurites start to grow into the hippocampal slice. (G,J) At DIV7, both the DRN and hippocampal slice contain abundant 5-HT neurites. (H) At DIV21 the 5-HT neurite density in the slices has not changed. (K,L) No regional variation in 5-HT neurite density in the hippocampal slice at DIV4 (K) and DIV7 (L). Figures I and J are a blow up of the black box in F and G respectively. Figures K and L are blow ups of the hippocampal slices in figure F and G respectively. Scale bars: 500 µm in A, B, E, F, G, H, I, J, K and L, 20 µm in C.
These include the 5-HT\textsubscript{1A} receptor, the 5-HT\textsubscript{2} receptor and the 5-HT\textsubscript{7} receptor (Gustafson et al., 1996; Clemett et al., 2000; Xu and Pandey, 2000; Bickmeyer et al., 2002; Garcia-Alcocer et al., 2006). Since it has been shown before, also on non-5-HT cells, that (chronic) activation of a 5-HT\textsubscript{1A} or 5-HT\textsubscript{2} receptor or blockade of the SERT can affect (5-HT-ergic) outgrowth or synaptogenesis, we decided to use a 5-HT\textsubscript{1A} receptor agonist 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT), a 5-HT\textsubscript{2} receptor agonist (+)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI hydrochloride) and the SERT inhibitor fluoxetine (Wilson et al., 1998; Kondoh et al., 2004; Fricker et al.,
We chronically applied the pharmacological compounds in 10 µM concentrations during the first seven days of outgrowth. First of all, we studied the effect of chronic blockade of the SERT by fluoxetine. Fluoxetine did not affect 5-HT neurite density (Fluoxetine: DRN: 8.44 ± 0.72%, HIP: 6.12 ± 1.12% n=10) compared to control condition (Fig. 5A,B,E,F). Subsequently, we tested the effect of chronically activating the 5-HT\textsubscript{1A} and 5-HT\textsubscript{2} receptor, two receptors which are present on 5-HT neurons. Moreover, it has been shown previously that activation of these receptors can affect outgrowth of Purkinje cells (Kondoh et al., 2004). First we investigated the effect of the 5-HT\textsubscript{1A} receptor agonist 8-OH-DPAT. Activation of the autoreceptor by 5-HT or an agonist results in a reduction in 5-HT release (Quick, 2003). However, 8-OH-DPAT did not affect 5-HT neurite density (DRN 10.67 ± 0.79%, HIP 7.21 ± 0.9% n=9) (Fig. 5 C,E,F).
**Figure 5. Chronic application of a 5-HT\textsubscript{2} agonist reduces 5-HT neurite density in the slices**
(A) In control condition, after seven days of culturing several 5-HT neurites have grown into the slices. (B) Chronic blockade of the SERT with the SERT inhibitor fluoxetine did not affect 5-HT neurite density in the slices. (C) Chronic activation of the 5-HT\textsubscript{1A} receptor with 8-OH-DPAT also did not affect 5-HT neurite density in the slices. (D) However, chronic activation of the 5-HT\textsubscript{2} receptor with DOI significantly reduced 5-HT neurite density both in the DRN and hippocampal slice. (E,F) Quantification of 5-HT neurite density in the DRN slice and hippocampal slice, respectively. Scale bar: 500 µm. * p< 0.05, ** p<0.01, *** p<0.001.
Next, we investigated the effect of chronic 5-HT\textsubscript{2} receptor activation. Activation of the 5-HT\textsubscript{2} receptor results in an increased protein kinase C activity and release of calcium from internal stores. We chronically activated the 5-HT\textsubscript{2} receptor by applying the 5-HT\textsubscript{2} receptor agonist DOI hydrochloride. Chronic application of the 5-HT\textsubscript{2} receptor agonist DOI hydrochloride resulted in a significantly decreased 5-HT neurite density (Fig. 5 D,E,F) (DRN 2.94 ± 1.01%, HIP 2.29 ± 1.05% n=5). To exclude the possibility that pharmacological treatment affected the survival of 5-HT neurons, we counted the number of 5-HT cells per group. This showed that treatment with fluoxetine, 8-OH-DPAT or DOI hydrochloride did not affect the number of 5-HT neurons in the slices (data not shown). Hence, changes in viability of 5-HT neurons cannot explain the effect of DOI hydrochloride. We conclude that these organotypic slice co-cultures can be used to study the 5-HT outgrowth and ingrowth into a target area in vitro and that chronic activation of the 5-HT\textsubscript{2} receptor results in a decreased 5-HT neurite density.
**Discussion**
*An organotypic co-culture model system to study 5-HT outgrowth ex vivo*
We used organotypic slice co-cultures to study the outgrowth of the 5-HT system ex vivo. Neurite projections of 5-HT neurons grew into the slices and at 7 days a dense network had formed with many serotonergic boutons that had accumulated presynaptic markers. The regional differences in 5-HT innervation observed in the hippocampus in vivo were not reproduced ex vivo and it has been observed that fewer synapses form in slices than in vivo (see discussion in Papp et al., 1995). However, also in vivo the majority of the 5-HT release sites (70-80%, (Oleskevich and Descarries, 1990), are not classical synapses, i.e. with a juxtaposed postsynaptic specialization. Hence, the in vivo connectivity of the 5-HT system appears to depend largely on non-synaptic release. Therefore, despite some discrepancies compared to the in vivo situation, the slice co-culture system is an excellent, reduced model system to unravel the basic principles that orchestrate 5-HT neurite outgrowth and connectivity. Such a reduced system is a valuable supplementation of in vivo studies, which are often complicated by the exceptionally complex and dynamic connectivity of the 5-HT system, with for instance large fluctuations in innervation over the course of a few days (transient hyper-innervation, see (Fujimiya et al., 1986; D’Amato et al., 1987).
*Chronic activation of the 5-HT\textsubscript{2} receptor decreased 5-HT neurite density*
We found that chronic application of a 5-HT\textsubscript{2} agonist decreased the neurite density of 5-HT neurites. Since DOI hydrochloride treatment did not affect 5-HT neuron survival, 5-HT mediated cytotoxicity cannot explain the decrease in 5-HT
neurite density. Hence, the decreased neurite density appears to be the result of a specific effect of the activation of the 5-HT$_2$ receptor on the neurites (decreased outgrowth/increased pruning). In contrast, chronic activation of the 5-HT$_{1A}$ receptor did not mimic the effects of 5-HT, although the 5-HT$_{1A}$ receptor has often been implicated in shaping 5-HT connectivity (Gross et al., 2000; Gross et al., 2002). Activation of somatodendritic 5-HT$_{1A}$ receptors results in hyperpolarisation and a decrease in 5-HT release (Quick, 2003). However, chronic activation results in rapid desensitization (Assie et al., 2006) which may explain why we do not observe an effect of chronic 5-HT$_{1A}$ receptor activation. On the other hand, 5-HT$_2$ receptor activation increases intracellular calcium and activates protein kinase C (Tamir et al., 1992). This suggests that (chronic) activation of the 5-HT$_2$ receptor affects outgrowth, since elevation of calcium levels in growth cones reduces outgrowth (Gomez and Spitzer, 1999). Since DOI hydrochloride has a comparable affinity for the 5-HT$_{2A}$ and 5-HT$_{2C}$ receptor subtype, based on these data we cannot conclude which of the two subtypes is involved in the effect of DOI hydrochloride.
However, although the 5-HT$_{2A}$ receptor is widely expressed throughout the brain, the receptor seems not to be expressed in the DRN (Xu and Pandey, 2000). In contrast, the 5-HT$_{2C}$ receptor is present in the DRN (Clemett et al., 2000; Serrats et al., 2005). Moreover, it has been shown previously, that activation of the 5-HT$_{2C}$ receptor in the DRN results in a reduction in 5-HT neuron firing (Boothman et al., 2006). Therefore, based on the anatomical distribution we speculate that the effect of DOI hydrochloride is predominantly mediated via the 5-HT$_{2C}$ receptor. Fluoxetine did not affect 5-HT neurite density. It could be expected that fluoxetine application mimics a 5-HT$_{1A}$ receptor agonist, i.e. SERT blockade results in an increase in extracellular 5-HT which could activate 5-HT$_{1A}$ autoreceptors. However, possibly upon application of fluoxetine extracellular 5-HT is rapidly diluted in the culture medium in vitro, and therefore extracellular 5-HT will not reach high enough concentration levels to mimic the effect of a 5-HT$_{1A}$ receptor agonist. Also chronic fluoxetine treatment in vivo failed to produce effects similar to chronic 5-HT application (Zhou et al., 2006). In fact, chronic fluoxetine enhanced neurite density in this case (Zhou et al., 2006).
**Conclusions**
We have shown here that in DRN – hippocampus organotypic slice co-cultures 5-HT projections grow into the target slice. After four days of culturing in the DRN slice already several 5-HT projections were present, and the first 5-HT projections started to grow into the target slice. Within seven days of culturing also the target slice contained several 5-HT neurites. These 5-HT neurites,
contained several varicosities, which are presumably sites of 5-HT release, since presynaptic markers were present in these varicosities. Finally, we have shown that this culture model system can be used to study the effect of pharmacological manipulations on the outgrowth of 5-HT neurites, and that application of a 5-HT$_2$ agonist results in a reduction in 5-HT neurite density in the slices.
**EXPERIMENTAL PROCEDURES**
*Laboratory animals*
Wildtype C57BL/6 mouse fetuses were obtained from caesarean section at embryonic day 18 or postnatal day 1. Animals were housed and bred according to institutional, Dutch and U.S. governmental guidelines.
*Pharmacological compounds and antibodies*
Mouse monoclonal anti-Tph antibody, which detects both Tph1 and Tph2, was obtained from Sigma-Aldrich and used in a 1:1000 dilution. Rabbit anti 5-HT polyclonal and mouse anti-SERT monoclonal were obtained from Immunostar/Diasorin and used in a 1:1000 dilution. Bassoon and Synaptotagmin monoclonal antibodies were used in a 1:500 and 1:1000 dilutions respectively and obtained from Stressgen. As secondary antibodies Alexa593-conjugated goat anti rabbit and Alexa488-conjugated goat anti mouse were used in a 1:1000 dilution (Molecular probes). 8-OH-DPAT and DOI hydrochloride were obtained from Sigma-Aldrich. Fluoxetine was obtained from Tocris.
*Organotypic slice co-cultures*
Organotypic slice cultures were made as follows. After decapitation of the fetuses, the heads were immediately transferred to ice-cold dissection Gey’s balanced salt solution (dGBSS, Invitrogen, supplemented with 0.65 g/l glucose and 200 µM kynurenic acid). For isolation of a DRN slice, the brains were dissected in dGBSS and the midbrain cut into 400 µm thick slices using a McIlwain tissue chopper (Mickle Engineering, Gomshall, UK). Individual slices were separated in dGBSS and the hindbrain slice containing the rostral DRN was identified by visual inspection. The DRN was dissected out using a dissection knife. The entire hippocampus was dissected out of the brain and individual slices (400 µm) were sagitally cut perpendicular to the hippocampus’ longitudinal axis. The hippocampal and DRN slices were allowed to recuperate in dGBSS at 4°C for 60 minutes. A hippocampal and DRN slice were cultured in close proximity on a poly-D-lysine coated 12x24 glass coverslip (O. Kindler GmbH & Co. Mikroskopische Gläser, Freiburg, Germany) in a plasma clot (chicken plasma, Cocalico Biologicals Inc. Reamstown, U.S.) which was coagulated with thrombin (Merck, Darmstadt, Germany). Culturing medium
consisted of 50% BME Hanks, 25% Hanks balanced salt solution and 25% horse serum supplemented with 1.3 g/L glucose and 200 µM glutamine. For culturing 700 µL of culturing medium was added to the slices in a Nunc flat-bottomed tube. The slices were incubated in a roller drum at 36°C. After the first week, medium was changed twice a week.
**Pharmacological treatments**
All pharmacological compounds were dissolved in ddH$_2$O, filter sterilized and aliquots stored at -80°C. After DIV1 10 µM of the pharmacological compound was added to the slices. Every day a new aliquot of pharmacological compound was added to the slices. In control slices only vehicle was added (sterile ddH$_2$O). At DIV7 the slices were fixed, processed and analyzed as described below.
**Immunohistochemistry**
Slices were fixed for 20 minutes in 4% paraformaldehyde dissolved in phosphate buffered saline. After fixation the slices were washed three times five minutes in phosphate buffered saline (PBS) and nonspecific binding was blocked by incubating the slices in PBS containing 0.1% Triton X-100 and 2% normal goat serum for two hours. The slices were incubated overnight with the primary antibodies diluted in PBS containing 0.1% Triton X-100 supplemented with 2% normal goat serum at 4°C. After incubation in the primary antibodies, the slices were washed three times two hours in PBS on a shaking platform. The secondary antibodies were diluted in PBS supplemented with 2% normal goat serum and incubated for one hour. After incubation in the secondary antibodies, the slices were washed again three times two hours in PBS and mounted in Dabco-Mowiol (Sigma) on glass coverslips. All reactions were carried out at room temperature unless otherwise stated. No labeling was observed when omitting the primary antibodies.
**Confocal analysis**
Confocal analysis of the slices was performed on a LSM 510 microscope (Carl Zeiss b.v. Weesp, the Netherlands) and a 63x Plan-Neofluar lens (Numerical aperture 1.4, Carl Zeiss). To excite the Alexa 488 antibody a HeNe1 laser was used and for excitation of the Alexa 593 antibody a HeNe2 laser was used. For analysis of the 5-HT neuron morphology and the 5-HT neurites, Z-stacks of 1 µm were made. Images were analyzed and further processed in Zeiss CLSM software.
**Histological quantification of 5-HT neurite density**
Quantification of 5-HT immunopositive neurites and cells was performed using an MCID Elite imaging system (Imaging Research Inc., Ont., Canada). Images of the immunostained co-cultures were digitized using an objective magnification
of 20x on a Leica DM/RBE photo-microscope with a Sony (DXC-950P, 640 x 512 pixels) camera using epifluorescence microscopy. The 5-HT-immunopositive neurites were segregated from background using several point operators and spatial filters combined in an algorithm designed to detect local changes in relative optical density. Briefly, images underwent histogram equalization and smoothing (low-pass filter, kernel size 7x7). The unfiltered image was subtracted from the smoothed image, followed by a series of steps to optimize the processed image and make it a suitable measuring template for detecting objects the size and shape of 5-HT neurites or cell bodies. This algorithm was preferred over relative optical density thresholding since it does not involve an observer-dependent operation. Finally, a line was manually drawn around the DRN or hippocampal slice and the number of positive pixels (i.e. pixels representing 5-HT neurites) compared to the total number of pixels was calculated. The number of 5-HT neurons was counted manually in the slices.
Data analysis
In order to analyze differences in neurite density between different pharmacological treatments, ANOVA was used with the Bonferonni test for post hoc analysis. Data shown are mean values ± standard error of the mean (SEM). Significance levels were set at <0.05.
Acknowledgements
This work was supported by the Dutch Organization for Scientific Research (Zon-MW Pionier MW-PIO900-01-001).
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Chapter 3
Dynamic vesicular trafficking of the serotonin reuptake transporter in hippocampal neurons
J.J. Dudok\textsuperscript{1}, A.J.A. Groffen\textsuperscript{1} and M. Verhage\textsuperscript{1}
\textsuperscript{1}Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, the Netherlands
\textit{In preparation}
Abstract
The serotonin (5-HT) reuptake transporter (SERT) has an important role in 5-HT transmission by terminating the action of 5-HT in the synaptic cleft. Here we address the question how SERT is transported in neuronal cells. We studied the trafficking of SERT in hippocampal neurons by tagging SERT with a fluorescent protein. We show that SERT displays a punctate distribution in axons and a more uniform distribution in dendrites. The majority of SERT puncta do not co-localize with the synaptic marker Synapsin, showing that SERT is predominantly localized extra-synaptically. SERT is transported in secretory vesicles as SERT co-localizes with the secretory vesicle marker tissue plasminogen activator (tPA). Live cell imaging revealed a highly dynamic trafficking of SERT along the neurites. Approximately 35% of the vesicles moved, with an average velocity of $\sim$1.2 µm/s. SERT vesicles do not display a preference for direction. We conclude that SERT, tagged with a fluorescent protein, displays a highly dynamic character in hippocampal neurons. These data are important to achieve a better understanding of the dynamics of treatment with 5-HT reuptake inhibitors.
Introduction
The projections of the 5-HT system abundantly innervate several brain areas. 5-HT is released both synaptically and extra-synaptically. After being released, 5-HT is cleared from the extracellular space by the SERT. Since this protein regulates the termination of the action of 5-HT, it is very important in regulating efficacy of 5-HT transmission. Moreover, SERT is the primary target for the selective serotonin reuptake inhibitors (SSRIs) class of antidepressants, several tricyclic antidepressants, and may mediate the effects of amphetamines such as 3,4-methylenedioxy-N-methylamphetamine (MDMA).
SERT is a sodium and chloride neurotransmitter symporter and belongs to the solute carrier family 6, to which also transporters for dopamine, noradrenaline/adrenaline, glycine and GABA belong (Chen et al., 2004). In the adult brain, SERT is expressed in 5-HT neurons, but during development also transiently in the cortex, hippocampus, thalamocortex and the lateral geniculate nucleus (Lebrand et al., 1996; Zhuang et al., 2005). In 5-HT neurons, SERT is predominantly localized in axons but also in the cytoplasm of the soma and dendrites (Zhou et al., 1998; Tao-Cheng and Zhou, 1999). In axons SERT is not only associated with varicosities, sites where 5-HT is released, but also with the plasma membrane of the axons (Zhou et al., 1998).
Several lines of evidence indicate that 5-HT has a role during brain development. In mice, deletion of SERT results in a 60-80% reduction in total
brain tissue 5-HT level, although there is a 7-8 fold increase in extracellular 5-HT levels in the striatum and substantia nigra (Fabre et al., 2000; Mathews et al., 2004). Analysis of emotional behaviour of these mice revealed that deletion of SERT results in abnormalities in anxiety-related behaviour (Holmes et al., 2003a; Holmes et al., 2003b). Additionally, blockade of SERT during development with the SSRIs fluoxetine, clomipramine or citalopram results in mice which display altered emotional behaviour in adulthood (Ansorge et al., 2004; Ansorge et al., 2008). These studies show that SERT is important in establishing normal emotional behaviour during development, presumably by regulating the extracellular concentration of 5-HT in the brain.
The level of SERT present at the cell membrane at synaptic boutons determines the rate of 5-HT reuptake and hence the efficacy of 5-HT transmission. Therefore, there is extensive regulation of the level of cell-surface expressed SERT. The trafficking and regulation of SERT cell-surface expression has been studied in many different cell types, including HEK293 cells, iC11 cells, basophilic leukemia 2H3 cells and 5-HT neurons (Zhu et al., 2004; Lau et al., 2008a; Lau et al., 2008b). A study using biotinylated SERT in 5-HT neurons showed that citalopram reduced the amount of cell surface-expressed SERT (Lau et al., 2008b) However, live cell imaging to investigate the trafficking dynamics of SERT has not been studied in neurons before.
Here we investigated the trafficking of SERT in hippocampal neurons. To this end we expressed SERT tagged with the fluorescent protein mCherry in hippocampal neurons. SERT displays a polarized distribution, as in axons SERT is distributed in a punctate manner, whereas in dendrites SERT is present in a uniform distribution. SERT is transported in secretory vesicles through the neurites, as it co-localized with the secretory vesicle marker tPA. Live cell imaging revealed that SERT displays a highly dynamic vesicular trafficking. Analysis of direction of movement showed that SERT does not display a preference for a direction of movement. In future experiments, live cell imaging of mCherry-SERT can be used to address whether 5-HT reuptake inhibitors affect the highly dynamic SERT trafficking.
**Results**
*Localization of mCherry-SERT in hippocampal neurons differs between axons and dendrites*
To study the trafficking we tagged the N-terminus of SERT with the red fluorescent protein mCherry, which is an improved variant of monomeric red fluorescent protein in terms of maturation and photostability (Shaner et al., 2004). Tagging the N-terminus of SERT with a fluorescent protein does not interfere with the function of SERT (Schmid et al., 2001; Just et al., 2004;
We used hippocampal neurons that do not express SERT, although it is expressed transiently in a subpopulation of hippocampal neurons during development (Narboux-Neme et al., 2008).
The expression of mCherry-SERT in dissociated hippocampal neurons was analyzed between 9-12 days in vitro (DIV) using live cell imaging and immunocytochemistry on fixed neurons. This revealed that the whole neuron was extensively labelled with mCherry-SERT (Fig. 1A). Immunocytochemistry for the dendritic marker Microtubule Associated Protein 2 (MAP2) was used to discern between the dendrite and axon (Fig. 1B,C). SERT localization differed between axons and dendrites. In dendrites SERT is predominantly uniformly distributed where only occasionally a puncta of SERT is observed. However in axons, SERT displays a more punctate distribution suggesting that SERT is transported in vesicles through the axon (Fig. 1D,E,F). Alternatively, the punctate pattern may reflect local accumulation of SERT in varicosities. To further explore whether these puncta are varicosities or vesicles, we used immunocytochemistry and live cell imaging.
**Figure 1. Distribution of mCherry-SERT differs between axons and dendrites**
(A-C) Expression of mCherry-SERT in a hippocampal neuron (DIV11). (A) mCherry-SERT is present in all the neurites and the cell body. (B) MAP2 labelling to discern between axons and dendrites. (C) Merged image. (D-F) Blow up of the white box in C. (D) mCherry-SERT distribution differs between axons and dendrites, as in the axon mCherry-SERT displays a punctate distribution, whereas in dendrites mCherry-SERT displays a more uniform distribution where only occasionally a mCherry-SERT puncta is observed. (E) MAP2 labelling. (F) Merged image. Scale bars in C and F are 10 µm.
SERT is transported in secretory vesicles
Since SERT displayed a punctate distribution in neurites, we investigated whether these puncta are secretory vesicles. To this end mCherry-SERT was co-expressed with tPA-GFP, which is transported in secretory vesicles in hippocampal neurons (Lochner et al., 1998; Silverman et al., 2005). In neurons expressing both mCherry-SERT and tPA-GFP, the majority of SERT puncta co-localized with tPA-GFP (Fig. 2A-F). Therefore, these data strongly suggest that SERT is transported in secretory vesicles along the axons in hippocampal neurons.
Figure 2. mCherry-SERT is transported in secretory vesicles
(A-C) Co-expression of mCherry-SERT and tPA-GFP in a hippocampal neuron (DIV11). (A) mCherry-SERT displays a punctate distribution. (B) tPA-GFP is transported in secretory vesicles. (C) The majority of mCherry-SERT puncta co-localize with tPA-GFP, suggesting that mCherry-SERT is transported in secretory vesicles. (D-F) Blow-up of the white box in C. (D) mCherry-SERT expression. (E) tPA-GFP expression. (F) Merged image. Arrows show the co-localization between mCherry-SERT and tPA-GFP. Scale bars in C and F are 10 μm.
To investigate whether vesicles of SERT co-localized with presynaptic terminals, we performed immunocytochemistry for the synaptic marker Synapsin on neurons transfected with mCherry-SERT. Although several mCherry-SERT puncta are in close vicinity of Synapsin puncta (Fig. 3 A-F), only few mCherry-SERT puncta co-localized with Synapsin. Thus, mCherry-SERT is transported in secretory vesicles and several of those vesicles localize to the vicinity of presynaptic terminals.
Figure 3. Several mCherry-SERT vesicles are localized in the vicinity of presynaptic terminals
(A-C) Expression of mCherry-SERT in a hippocampal neuron (DIV11). (A) mCherry-SERT expression, (B) Synapsin labelling, (C) Merged image. Several mCherry-SERT vesicles are localized in the vicinity of presynaptic terminals, and some co-localized with the presynaptic marker Synapsin. (D-F) Blow up of the white box in C. (D) mCherry-SERT expression, (E) Synapsin labelling, (F) Merged image. Arrows in F show mCherry-SERT vesicles which are localized in the vicinity of presynaptic terminals. Arrowhead shows a mCherry-SERT vesicle which co-localizes with a Synapsin puncta. Scale bars in C and F are 10 µm.
Figure 4 next page. Trafficking dynamics of mCherry-SERT secretory vesicles
Live cell imaging of mCherry-SERT trafficking dynamics in hippocampal neurons (DIV 9-11). (A) Two frames 5 seconds apart (green is 1 s, red is 5 s) from a 1 minute movie reveal the different modes of trafficking of mCherry-SERT. There are mCherry-SERT vesicles which move in the anterograde direction (arrow), in the retrograde direction (arrowhead) or are stationary (yellow puncta, asterisks). (B) Corresponding kymograph of part of the neurite displays the different trafficking modes of mCherry-SERT. Vesicle 1 is a stationary vesicle, vesicle 2 moves in the anterograde direction, whereas vesicle 4 moves in the retrograde direction. Vesicle 3 is a vesicle which pauses for a while and then continues moving. Vesicle numbers are shown in the individual images taken 10 seconds apart and vesicle trajectories are highlighted in the kymograph. (C) No difference between average anterograde and retrograde velocity as can be observed in the histogram of the average velocity. (D) No difference between average velocity of mCherry-SERT vesicles which move in the anterograde or retrograde direction or which display bidirectional movements. (E) Percentage moving vesicles does not differ between vesicles which move anterogradely, retrogradely or bidirectionally. (F) Also, percentage pausing time for moving vesicles is not different between vesicles which move anterogradely, retrogradely or bidirectionally. Scale bars in A and B are 10 µm.
SERT trafficking is highly dynamic
We analyzed the trafficking dynamics of SERT using live-cell imaging of hippocampal neurons expressing mCherry-SERT. We observed rapid movements of mCherry-SERT vesicles in both the anterograde and retrograde direction. Additionally, also stationary mCherry-SERT puncta were observed (Fig. 4A). To illustrate different types of movement we show part of a neurite with 10 second time intervals. In this neurite a stationary vesicle can be observed (vesicle 1), but also a vesicle which moves in an anterograde direction (vesicle 2) and a vesicle which moves in a retrograde direction (vesicle 4). Additionally, vesicles are...
observed which are temporarily stationary but then continue moving (vesicle 3 in Fig. 4B). In the corresponding kymograph of this part of the neurite the trajectories of the numbered vesicles are shown (Fig. 4B). We quantified the direction of vesicle movement, percentage of moving vesicles, average velocity of moving vesicles and percentage pausing time of moving vesicles using the automated vesicle detection program Fluotrack (Broeke et al., 2008). First we measured the average velocity of anterogradely and retrogradely moving vesicles. The frequency histogram plotting the velocities of mCherry-SERT vesicles moving in the anterograde or retrograde direction showed that the velocity distributions for anterogradely and retrogradely moving vesicles did not differ (Fig. 4C). The average velocity of all moving vesicles was $1.22 \pm 0.03$ µm/s. The average velocity of anterograde vesicles was $1.31 \pm 0.05$ µm/s compared to $1.34 \pm 0.06$ µm/s in retrograde direction. Vesicles moving bidirectional (vesicles that changed direction during imaging) moved with an average velocity of $1.12 \pm 0.055$ µm/s (Fig. 4D). Next, we determined the percentage of puncta which moved in the anterograde or retrograde direction, moved bidirectional or were stationary. This revealed that on average $34.52 \pm 5.31\%$ of mCherry-SERT puncta were moving during one minute of imaging. Further analysis of fraction of moving mCherry-SERT vesicles which moved in the anterograde or retrograde direction or moved bidirectional, revealed that there was no difference (Fig. 4E, see also table 1). Finally, we calculated the pausing time for the moving vesicles, which is the fraction of time moving vesicles pause during the 1 minute movie. There was no difference in percentage pausing time between mCherry-SERT vesicles which moved anterogradely, retrogradely or bidirectionally (Fig. 4F, see also table 1).
We conclude that mCherry-SERT displays highly dynamic vesicular trafficking in hippocampal neurons and that these mCherry-SERT vesicles do not have a preference for a direction of movement.
**Table 1: Overview of mCherry-SERT vesicle dynamics**
| | Velocity (µm/s) | Percentage of total | Percentage pausing time |
|------------------|-----------------|---------------------|-------------------------|
| Anterograde | $1.31 \pm 0.05$ | 13.83 | 42.13 |
| Retrograde | $1.34 \pm 0.06$ | 12.06 | 39.21 |
| Bidirectional | $1.12 \pm 0.06$ | 8.63 | 41.04 |
| Stationary | - | 65.48 | - |
Velocity data are shown as mean ± standard error of the mean (SEM). Data of 927 mCherry-SERT puncta in 6 neurons.
Discussion
Here we studied the trafficking of SERT in hippocampal neurons. We used human SERT cDNA tagged at its N-terminus with the red fluorescent protein mCherry. Overexpression of mCherry-SERT in hippocampal neurons resulted in a distinct pattern of expression between axons and dendrites; in dendrites SERT displayed a uniform distribution, whereas in axons SERT displayed a punctate distribution. Using electron microscopy, it has been shown that in 5-HT neurons endogenous SERT predominantly localized to varicosities and the membrane in axons, and in soma and dendrites SERT localized predominantly in the cytoplasm (Zhou et al., 1998; Tao-Cheng and Zhou, 1999). Thus, although we are using hippocampal neurons, mCherry-SERT distribution seems to mimic the distribution of endogenous SERT in 5-HT neurons.
Ultrastructural studies revealed that SERT labeling was associated with vesicular organelles, which possibly are large dense cored vesicles (LDCVs) (Pickel and Chan, 1999). Here, we co-transfected mCherry-SERT with tPA-GFP for which it has been shown that these are transported in LDCVs (also called secretory vesicles) (Silverman et al., 2005). The majority of mCherry-SERT puncta co-localized with tPA-GFP, strongly suggesting that SERT is transported in secretory vesicles. Several mCherry-SERT vesicles are localized in close proximity (~1 µm), and some co-localized, with presynaptic terminals. This suggests that the majority of SERT is not localized in the presynaptic terminal itself, but rather is localized close to a synapse. Indeed, previous research found that presynaptic membranes lacked SERT, but SERT was present on extrasynaptic domains, either in close proximity or at some distance away from synapses (Zhou et al., 1998).
Live cell imaging revealed that mCherry-SERT vesicles display highly dynamic transport, i.e. there are vesicles which move anterogradely or retrogradely without interruptions, stationary vesicles or vesicles which move bidirectional. Analysis of average velocity revealed that both anterograde and retrograde vesicles moved with an average velocity of ~1.3 µm/s. The average velocity is in analogy with other transport or transport associated proteins which are transported in secretory vesicles, like the p75 neurotrophin receptor and Gephyrin (Maas et al., 2006; Formaggio et al., 2008). Surprisingly, we found that mCherry-SERT vesicles did not display a preference for direction of movement. For other releasable fluorescent cargos that are transported in secretory vesicles, such as Sema3A, BDNF and CCL21 it has been shown that transport is predominantly anterogradely (Adachi et al., 2005; de Wit et al., 2006; de Jong et al., 2008). In contrast, the p75 neurotrophin receptor, which is transported in vesicular structures, does not display a preference for direction of movement (Formaggio et al., 2008). Moreover, the scaffold protein Gephyrin,
which is associated and co-transported with the glycine receptor also does not display a preference for anterograde or retrograde movement (Maas et al., 2006). Therefore, it appears that proteins which can be secreted and are transported in secretory vesicles predominantly move in the anterograde direction, whereas receptor/transporter proteins move both in the anterograde and retrograde direction.
An explanation for this could be that cargos which can be secreted are transported towards synaptic release sites distant from the cell soma. After a vesicle has been released, the secreted compounds either diffuse in the extracellular space (as is the case for Sema3A) or are taken up again and degraded in the presynaptic terminal. Hence, releasable cargos are hardly transported back towards the cell soma. On the other hand, the level of membrane expression for receptor and transporter protein is tightly regulated, and these proteins can be rapidly internalized (Loder and Melikian, 2003; Melikian, 2004; Zhu et al., 2004). Upon internalization, transporters are packaged in vesicles again and are recycled. Finally, receptors and transporters can maintain functioning without a need for intracellular transport, whereas secreted components are dependent on a new supply of vesicles. Therefore, vesicles containing transporters or receptors can be transported both in anterograde and retrograde directions.
In conclusion, expression of mCherry-SERT in hippocampal neurons differs between axons and dendrites. In axons mCherry-SERT is distributed in puncta, which co-localize with tPA-GFP and therefore presumably are secretory vesicles. Several of these mCherry-SERT vesicles are localized in close proximity of presynaptic terminals, but only few mCherry-SERT vesicles co-localized with presynaptic terminals. Live cell imaging revealed that mCherry-SERT displays highly dynamic trafficking behaviour with no preference for direction of movement.
**EXPERIMENTAL PROCEDURES**
*Plasmids*
Construction of the human SERT cDNA with ECFP at the 5’ end (ECFP-SERT in the pECFP-C1 backbone (Clontech)) has been described before ((Schmid et al., 2001); a kind gift of Dr. H. Sitte). To construct mCherry-SERT, ECFP was excised with AseI and BsrGI and replaced for mCherry.
*Neuronal culturing and transfection*
Hippocampi from embryonic day 18 C57/Bl6 mouse embryos were dissected free of meninges and collected in Hanks Balanced Salt Solution (HBSS; Invitrogen) supplemented with 7 mM HEPES (Invitrogen). Hippocampi were digested with
0.25% trypsin (Invitrogen) in HBSS supplemented with 7 mM HEPES for 15 minutes at 37°C. After digestion tissue was washed three times with HBSS supplemented with 7 mM Hepes and subsequently triturated with a fire-polished glass pipette. Dissociated neurons were plated at a density of 25,000 cell/well on rat glial cells on 18 mm glass coverslips (Menzel Glaser, Braunschweig, Germany) in 12 well plates. Cells were cultured in neurobasal medium supplemented with B27 supplement, 18 mM HEPES, 0.5 mM Glutamax and penicillin/streptomycin (Invitrogen). Once a week half of the medium was replaced. The hippocampal cultures were transfected using Lipofectamine 2000 (Invitrogen). Before transfection, half of the neurobasal medium was collected from each well and replaced with new neurobasal medium. For one well, 1 µl of Lipofectamine 2000 was added to 25 µl of serum free glutamax medium (Invitrogen) and incubated for 5 minutes. Subsequently, from each construct 1 µg of DNA was added to 25 µl of serum free glutamax and immediately mixed with the 25 µl of serum free glutamax containing Lipofectamine 2000. This mixture was incubated for 30 minutes at room temperature before adding to the cells. After 4-6 hours of incubation on the cells at 37°C, half of the neurobasal medium was replaced again with the earlier collected neurobasal medium. Cells were routinely transfected at DIV2-7 and imaged at DIV9-12.
**Live cell imaging**
Cells were imaged in a chamber perfused with Tyrode’s solution (119 mM NaCl, 2.5 mM KCl, 2 mM MgCl$_2$, 2 mM CaCl$_2$, 30 mM glucose, 25 mM HEPES, pH 7.4) on an Axiovert II Microscope (Zeiss, Oberkochen, Germany) equipped with a Coolsnap HQ camera (Photometrics, Tucson, Arizona, USA) and a Polychrome IV illumination unit (TILL photonics, Grafelfing, Germany). Images were acquired in MetaMorph 6.2 software (Universal Imaging, Downingtown, Philadelphia, USA) using a 100x oil objective.
**Immunocytochemistry and confocal microscopy**
For immunocytochemistry, neurons were fixed by incubation in 4% paraformaldehyde in phosphate buffered saline (PBS) for 20 minutes. Subsequently, to block aspecific binding and for permeabilization, the neurons were incubated in PBS containing 0.2% Triton X-100 and 4% fetal calf serum for 20 minutes. Neurons were incubated in primary antibody diluted in PBS containing 0.2% Triton X-100 for one hour. After washing three times in PBS, neurons were incubated in secondary antibodies in PBS for one hour. Neurons were washed again three times in PBS and mounted in Dabco-Mowiol. All reactions were carried out at room temperature. As primary antibodies, monoclonal MAP2 (Chemicon) and polyclonal Synapsin (E028) were used. As secondary antibodies Alexa fluor-conjugated antibodies were used (Invitrogen).
For confocal analysis, neurons were examined on a confocal LSM510 microscope (Zeiss, B.V. The Netherlands)
**Trafficking analysis**
In order to analyze the trafficking dynamics of SERT, one minute movie image stacks with 1 s interval between images, were analyzed in the custom written software program Fluotrack (Broeke et al., 2008). To this end, metamorph files (.stk) were converted to .avi files using ImageJ, and analyzed using Fluotrack. Data analysis was performed using the SPSS statistical package. Data shown are mean values ± SEM.
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Chapter 4
Presynaptic localization of tryptophan hydroxylase 2 in mature hippocampal neurons
J.J. Dudok\textsuperscript{1}, A.J.A. Groffen\textsuperscript{1} and M. Verhage\textsuperscript{1}
\textsuperscript{1}Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, the Netherlands
\textit{In preparation}
Abstract
Tryptophan hydroxylase (Tph) is the rate limiting enzyme in serotonin (5-HT) synthesis. Two genes named Tph1 and Tph2 synthesize 5-HT in peripheral tissues and the central nervous system, respectively. When expressed in neurons, Tph1 displays a cytoplasmic distribution in the cell body and is associated with microtubules in the axon and dendrites. Axonal Tph1 is associated with 5-HT varicosities. Here, we studied the subcellular distribution of Tph2. To this end we expressed Tph2-EGFP in hippocampal neurons. In young neurons, before synaptogenesis has occurred, Tph2-EGFP displayed a cytoplasmic distribution throughout the neurites. Live cell imaging revealed that Tph2-EGFP is not transported in vesicles. In mature neurons, Tph2-EGFP accumulated in presynaptic terminals of axons, whereas in dendrites it was distributed throughout the cytoplasm, also in spines. In live cell imaging, Tph2-EGFP hardly displayed any movement. These results suggest that Tph2 is freely diffusible in the dendrites and soma, but accumulates at the synaptic terminal by a mechanism that deserves further study.
Introduction
The neurons of the 5-HT system are among the first neurons in the brain synthesizing a specific neurotransmitter. Two enzymes are necessary to synthesize 5-HT from its precursor L-tryptophan. Of these, the rate limiting enzyme is Tph which converts L-tryptophan to 5-hydroxy-L-tryptophan. For decades only one Tph isoform was known in the mammalian brain. However, deletion of this gene did not result in a significant decrease in brain 5-HT levels, which led to the discovery of a second Tph gene called Tph2 (Walther et al., 2003). Tph2 is exclusively expressed in the raphe nuclei, whereas Tph1 is expressed in the pineal gland and in the peripheral 5-HT system (Patel et al., 2004; Gutknecht et al., 2009).
Since 5-HT projections are distributed throughout the brain, it is unlikely that Tph2 is only present in the cell body to synthesize 5-HT. Indeed, previous research showed that Tph was present in the cytoplasm of the cell body, and in axons and dendrites where it was associated with microtubules (Joh et al., 1975). Additionally, Tph was associated with 5-HT varicosities, suggesting local synthesis of 5-HT (Pickel et al., 1976; Pickel et al., 1977).
Although the subcellular distribution of Tph1 is known, it is currently unknown how Tph is transported to synaptic terminals and whether Tph2 also
localizes to presynaptic terminals. Therefore we tagged Tph2 with EGFP, and expressed this chimaeric protein in hippocampal neurons. We show here that in young neurons (before synaptogenesis), Tph2-EGFP is homogenously distributed throughout the neurites. However, in mature neurons, Tph2-EGFP accumulated at presynaptic terminals in the axon. In the dendrites of mature neurons, Tph2-EGFP displayed a homogenous distribution, and also localized to spines. Live cell imaging suggested that Tph2 is a soluble protein and is not transported in vesicles to presynaptic terminals. We conclude that Tph2-EGFP accumulates at presynaptic terminals in mature hippocampal neurons.
**Results**
*Cytoplasmic distribution of Tph2-EGFP in young neurons*
To study the distribution of Tph2 in neurons, we inserted EGFP at the N-terminus of Tph2 and expressed Tph2-EGFP in hippocampal neurons. First, we started with imaging Tph2-EGFP in neurons at 9 days in vitro (DIV), which are still immature neurons in terms of synapse development (Basarsky et al., 1994) Transfected neurons showed a uniform distribution of Tph2-EGFP through the whole neuron, indicating that Tph2-EGFP is cytoplasmic (Fig. 1A). We did not observe Tph2-EGFP puncta, suggesting that Tph2-EGFP is not transported in
Tph2-EGFP was expressed in young hippocampal neurons before synaptogenesis and imaged at DIV9. (A) Tph2-EGFP displays a cytoplasmic distribution throughout all the neurites. (B) Live cell imaging revealed that Tph2-EGFP is not transported in vesicles, and only little movement is detected. Images are taken 10 seconds apart, first image is green and image 10 seconds later is in red. (C) Zooming in on the axon (arrow in A), as identified by its smaller diameter compared to dendrites shows that already puncta are observed which might be immature synapses or varicosities (arrows). (D) In the dendrites, Tph2-EGFP distribution is cytoplasmic, and Tph2-EGFP also localized to dendritic filopodia. Scale bars: 10 µm in B, 5 µm in C and D.
A
Tph2-EGFP
B
Synaptophysin-mCherry
C
Merge
D
*
***
E
F
G
H
I
J
*
*
K
L
M
1s 30s
N
1s 30s
Scale bars: A, B, C = 5 μm; D, E, F = 10 μm; G, H, I = 20 μm; J, K, L = 20 μm; M, N = 10 μm.
Neurons expressing Tph2-EGFP and the presynaptic marker Synaptophysin-mCherry were imaged at DIV23. (A) Tph2-EGFP was expressed in the cytoplasm of dendrites. (B) Expression of Synaptophysin-mCherry in the same neuron. (C) Merged image. (D) Blow up of the white box in C shows that some Tph2-EGFP appears to accumulate in the tip of dendritic spines (asterisks). (E) Synaptophysin-mCherry expression. (F) Merged image. (G) Axon of a neuron expressing Tph2-EGFP. Tph2-EGFP displays a punctate expression, indicative of presynaptic localization. (H) Indeed, Tph2-EGFP co-localizes with Synaptophysin-mCherry. (I) Merged image. (J) A EGFP expressing neuron as a control shows that EGFP is homogenously distributed in the dendrites (asterisks) and in the axon as identified by the absence of MAP2 staining (arrows). (K) MAP2 staining. (L) Merged image. (M) Live cell imaging revealed that Tph2-EGFP is static. Images are taken 30 seconds apart, with the green image after 1 second, and the red image after 30 seconds. (N) Blow up of the white box in J shows that some dendrites which contain Tph2-EGFP displayed some movement (arrows). Scale bars: 10 µm in C, 5 µm in F, 10 µm in I, 10 µm in L, 10 µm in M and 5 µm in N.
vesicles, but instead is a soluble protein. Live cell imaging revealed that Tph2-EGFP is rather static, and no Tph2-EGFP puncta or other movements were observed (Fig. 1B). The axon, as identified by its smaller diameter and larger length as compared to dendrites, also had a uniform distribution of Tph2-EGFP, although some Tph2-EGFP appeared to accumulate in puncta along the axon (arrows in Fig. 1C). As in axons, in dendrites Tph2-EGFP displayed a uniform distribution and also filopodia were labelled (Fig. 1D).
**Tph2-EGFP localizes to presynaptic terminals in mature neurons**
Next, we investigated the distribution of Tph2-EGFP in mature neurons after synaptogenesis had occurred. To this end we transfected hippocampal neurons with Tph2-EGFP at DIV18 and imaged Tph2-EGFP at DIV23. Tph2-EGFP was uniformly distributed in the cell body, dendrites and dendritic spines (Fig. 2A-C). We expressed EGFP in neurons as a control to compare the distribution of EGFP with Tph2-EGFP (Fig. 2J-L). In contrast to EGFP, which labels the whole dendritic spine, it appeared that in neurons transfected with Tph2-EGFP in some spines Tph2-EGFP accumulated in the tip of the spine, suggesting that Tph2-EGFP was enriched in postsynaptic structures (Fig. 2D-F). We co-transfected Tph2-EGFP with Synaptophysin-mCherry, which is a marker for synaptic vesicles and presynaptic terminals (Jahn et al., 1985; Granseth et al., 2006). In the axon, identified by the punctate labelling of Synaptophysin-mCherry, Tph2-EGFP displayed a punctate distribution (Fig. 2G). The Tph2-EGFP puncta co-localized with Synaptophysin-mCherry puncta, strongly suggesting that Tph2-EGFP accumulated at presynaptic terminals (Fig. 2H,I). In neurons transfected
with EGFP as a control, EGFP displayed a uniform distribution throughout the axon (Fig. 2J-L). Next, we imaged the neurons expressing Tph2-EGFP during 1 minute. Hardly any Tph2-EGFP movement could be observed (Fig. 2M). The only movement which could be observed was some motility in postsynaptic spines (Fig. 2N).
To further test if Tph2-EGFP is localized to presynaptic terminals in mature neurons, we fixed neurons expressing Tph2-EGFP at DIV23 and performed immunocytochemistry for the synaptic marker Synapsin and the dendritic marker MAP2 to discriminate dendrites and axons (Fig. 3A-C). In the axon, as identified by the absence of MAP2 labelling, Tph2-EGFP displayed a punctate distribution (Fig. 3D-F). Immunocytochemistry for Synapsin showed that in dendrites of neurons expressing Tph2-EGFP, spines were labelled, and some spine heads were localized in close proximity with Synapsin puncta, suggesting that this is a synapse (Fig. 3G-I). Finally, we focussed on the axon of a neuron expressing Tph2-EGFP which we stained for Synapsin. The boutons with Tph2-EGFP co-localized with Synapsin, strongly suggesting that Tph2-EGFP localized to presynaptic terminals (Fig. 3J-L).
**Discussion**
We have shown here that Tph2, the rate limiting enzyme in 5-HT synthesis localized to presynaptic terminals in mature hippocampal neurons. Moreover, in dendrites and in the cell body Tph2-EGFP had a cytoplasmic distribution and appeared to accumulate in the tip of some dendritic spines. Thus, it appears that Tph2-EGFP displayed a polarized distribution in mature hippocampal neurons. Live cell imaging revealed that hardly any Tph2-EGFP movement could be observed, and no Tph2-EGFP vesicles were observed, indicating that Tph2 is a soluble protein. Accordingly, Tyrosine hydroxylase, an enzyme related to Tph2, also was shown to be a soluble protein (Mockus et al., 1997). Electron microscope studies on Tph and TH localization revealed that these proteins were enriched on presynaptic terminals, and were associated with microtubules in somatodendritic compartments (Pickel et al., 1976). Therefore, Tph2-EGFP seems to mimic the endogenous distribution of Tph in 5-HT neurons.
Tph2-EGFP displayed a polarized distribution in mature hippocampal neurons, i.e. in dendrites the protein is distributed throughout the cytoplasm, whereas in the axon the protein accumulated in presynaptic terminals. This suggests that in presynaptic terminals and in 5-HT varicosities, there is local synthesis of 5-HT. In dendrites, Tph2-EGFP localized to spines, which prompts the question whether 5-HT could be synthesized in dendrites.
Figure 3. Immunocytochemistry on a mature neuron expressing Tph2-EGFP
Neurons (DIV23) expressing Tph2-EGFP were fixed and immunocytochemistry was performed for MAP2 and Synapsin. (A) Tph2-EGFP expression. (B) MAP2 immunostaining to distinguish between dendrites and the axon. (C) Merged image. (D) Blow up of the white box in C shows a dendrite (asterisk) which has Tph2-EGFP through the whole cytoplasm, and the axon (arrows) in which Tph2-EGFP displays a punctate distribution. (E) MAP2 staining. (F) Merged image. (G) Tph2-EGFP expressing neurons were stained for the presynaptic marker Synapsin. (H) Synapsin staining. (I) Merged image, which shows that in the close proximity of some dendritic spines there is a Synapsin puncta, suggesting that these are true synapses (asterisks). (J) Distribution of Tph2-EGFP in an axon. (K) Synapsin staining. (L) Tph2-EGFP puncta were positive for Synapsin (arrows), showing that these are presynaptic terminals. Scale bars: 20 µm in C, 10 µm in F, 5 µm in I and L.
Tph is localized in dendrites, where it associated with microtubules (Joh et al., 1975). Correspondingly, 5-HT vesicles are detected in dendrites, either associated with a synaptic specialization or with the membrane (Chazal and Ralston, 1987). After stimulation of Retzius neurons in the leech, which contain large dense core vesicles (LDCVs) filled with 5-HT, several vesicles are localized close to the cell membrane as studied by electron microscopy (Trueta et al.,
Therefore, this strongly suggests that 5-HT release can occur from the somatodendritic compartment. Our results indicate that 5-HT can also be synthesized in the somatodendritic compartment.
Tph2-EGFP was not associated with vesicles and the protein interactions for its presynaptic accumulation remain unclear. Tph is associated with microtubules, suggesting it could interact with molecular motors of the dynein and kinesin family which move along microtubules (Hirokawa and Takemura, 2005). For Tph2, several polymorphisms are known, some of which associate with neuropsychiatric conditions (Zhang et al., 2006). Both in humans and in mice a polymorphism in the Tph2 gene is found which results in a marked reduction in Tph2 activity (Zhang et al., 2004; Zhang et al., 2005). Finally, it would be interesting to investigate whether these polymorphisms not only affect Tph2 activity, but could also affect Tph2 transport and localization.
**EXPERIMENTAL PROCEDURES**
*Plasmids*
Tph2 cDNA in pCR4-TOPO vector was obtained from Geneservice. To clone Tph2 in an expression vector, EGFP was digested using PstI and cloned in the PstI site of pBiotag-EGFP-C1. Synaptophysin-mCherry was a kind gift from Dr. A. Jeromin (Allen Brain Institute).
*Neuronal culturing and transfection*
Hippocampi from embryonic day 18 C57/Bl6 mouse embryos were dissected free of meninges and collected in Hanks’ Balanced Salt Solution (HBSS; Invitrogen) supplemented with 7 mM HEPES (Invitrogen). Hippocampi were digested with 0.25% trypsin (Invitrogen) in HBSS supplemented with 7 mM HEPES for 15 minutes at 37°C. After digestion tissue was washed three times with HBSS supplemented with 7 mM Hepes and subsequently triturated with a fire-polished glass pipette. Dissociated neurons were plated at a density of 25,000 cell/well on rat glial cells on 18 mm glass coverslips (Menzel Glaser, Braunschweig, Germany) in 12 well plates. Cells were cultured in neurobasal medium supplemented with B27 supplement, 18 mM HEPES, 0.5 mM Glutamax and penicillin/streptomycin (Invitrogen). Once a week half of the medium was replaced. The hippocampal cultures were transfected using Lipofectamine 2000 (Invitrogen). Before transfection, half of the neurobasal medium was collected from each well and replaced with new neurobasal medium. For one well, 1 µl of Lipofectamine was added to 25 µl of serum free glutamax medium and incubated for five minutes. Subsequently, from each construct 1 µg of DNA was added to 25 µl of serum free glutamax and immediately mixed with the 25 µl of serum free
glutamax containing Lipofectamine. This mixture was incubated for 30 minutes before adding to the cells. After 4-6 hours of incubation on the cells, half of the neurobasal medium was replaced again with the earlier collected neurobasal medium. Cells were routinely transfected at DIV2-7 and DIV18 and imaged at DIV10-14 and DIV23.
**Live cell imaging**
Cells were imaged in a chamber perfused with Tyrode’s solution (119 mM NaCl, 2.5 mM KCl, 2 mM MgCl$_2$, 2 mM CaCl$_2$, 30 mM glucose, 25 mM HEPES, pH 7.4) on an Axiovert II Microscope (Zeiss, Oberkochen, Germany) equipped with a Coolsnap HQ camera (Photometrics, Tucson, Arizona, USA) and a Polychrome IV illumination unit (TILL photonics, Grafelfing, Germany). Images were acquired in MetaMorph 6.2 software (Universal Imaging, Downingtown, Philadelphia, USA) using a 100x oil objective.
**Immunocytochemistry**
For immunocytochemistry, primary antibodies against MAP2 (monoclonal, Chemicon) and synapsin (polyclonal Eo28) were used in a 1:1000 dilution. As secondary antibodies, goat-anti-mouse Alexa488 and goat-anti-rabbit Alexa 543 were used (Invitrogen). To perform immunocytochemistry, cells were incubated in 4% paraformaldehyde dissolved in phosphate buffered saline (PBS, pH 7.4), for 20 minutes. Subsequently, cells were washed two times with PBS and incubated in PBS containing 0.2% Triton X-100 and 4% fetal calf serum for 20 minutes. Cells were incubated in primary antibody in PBS containing 0.2% Triton X-100 for one hour, washed three times with PBS and incubated in the secondary antibody in PBS for one hour. After washing the cells three times with PBS, coverslips were mounted using Dabco-Mowiol. All reactions were performed at room temperature. Cells were analyzed on a confocal LSM510 microscope (Zeiss, B.V. The Netherlands) using a 63x oil immersion lens and appropriate lasers and filters to visualize EGFP fluorescence and secondary antibodies.
**References**
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Chapter 5
The effect of a polymorphism in Piccolo on localization and trafficking dynamics of the serotonin reuptake transporter
J.J. Dudok\textsuperscript{1}, A.J.A. Groffen\textsuperscript{1} and M. Verhage\textsuperscript{1}
\textsuperscript{1}Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, the Netherlands
Abstract
The active zone protein Piccolo is involved in formation of synapses and stabilization of existing synapses. Recently, a short nucleotide polymorphism (SNP) in the C2A domain of Piccolo was found in association with depression. In this study we addressed whether knockdown of Piccolo affected the localization and trafficking of the serotonin reuptake transporter (SERT) in hippocampal neurons. We co-expressed a short nucleotide RNA which resulted in an efficient knockdown of Piccolo with mCherry-SERT to evaluate the effect of knockdown of Piccolo on SERT localization and trafficking in hippocampal neurons. We found that knockdown of Piccolo did not alter SERT trafficking dynamics. Subsequently, we expressed the two variants of the Piccolo C2A domain together with SERT in heterologous cells. SERT displayed a uniform distribution at the membrane upon co-expression of the wildtype C2A variant. However, upon co-expression of the mutant C2A variant, SERT displayed a more patchy distribution at the membrane, indicating that membrane levels of SERT might be decreased. These data suggest that knockdown of Piccolo does not alter SERT trafficking dynamics, but the mutant variant of the Piccolo C2A domain might alter SERT membrane localization.
Introduction
Major depression is a neuropsychiatric disorder affecting millions of people world wide each year. The serotonin (5-HT) system is implicated in depression. An important protein in the 5-HT system is the SERT which regulates the transport of 5-HT back into the synapse after its release, thus regulating the extracellular 5-HT concentration. Therefore, SERT is an important factor in determining efficacy of 5-HT neurotransmission. Several polymorphisms have been described in SERT which associate with psychopathological processes such as depression, anxiety, aggression and autism (Murphy et al., 2004; Serretti et al., 2006).
Several decades ago it was hypothesized that psychopathological processes such as depression and anxiety were the result of an alteration in extracellular 5-HT content. This hypothesis, the so-called monoamine hypothesis, was based on the fact that blockade of the SERT by selective 5-HT reuptake inhibitors (SSRIs) resulted in an alleviation in depressive symptoms, possibly due to the increase in extracellular 5-HT levels. However, between starting SSRI treatment and alleviation of depressive symptoms was a time period of 3-4 weeks, whereas 5-
HT levels increased immediately after SSRI intake. Therefore the monoamine hypothesis was abandoned and shifted towards the so-called network hypothesis, which stated that psychopathological processes such as anxiety and depression could be the result of an altered 5-HT network connectivity (Castren, 2005). The evidence for this hypothesis was based on human imaging studies which showed that humans with depression had a reduced grey matter volume in cortex and hippocampus. However, evidence for this hypothesis on a cellular level is still lacking. Recently, a SNP in the presynaptic gene Piccolo was found which might be associated with major depressive disorder (Sullivan et al., 2008). Therefore, this revitalizes the network hypothesis for depression and opens up new opportunities to study the relation between the presynaptic terminal, proper formation of networks, and the relation between 5-HT network (formation) and psychopathological processes such as depression.
Piccolo is a 420 kDa protein which is enriched in presynaptic terminals where it is a structural component of the presynaptic cytoskeletal cytomatrix (PCM) (Cases-Langhoff et al., 1996). Piccolo is a multidomain zinc finger protein which is structurally related to Bassoon, another component of the PCM (tom Dieck et al., 1998; Fenster et al., 2000). Piccolo contains several coiled coil domains, a PDZ domain and two C2 domains (Fenster and Garner, 2002). The C2A domain functions as a low-affinity calcium sensor in presynaptic terminals (Gerber et al., 2001). Piccolo and Bassoon, along with other presynaptic constituents, are transported in 80 nm large dense core vesicles (LDCVs) to nascent synapses, and 2 or 3 of these vesicles are sufficient to form an active synapse (Zhai et al., 2001; Shapira et al., 2003; Tao-Cheng, 2007). The recent described nonsynonymous SNP rs2522833 in Piccolo encodes a common variation in the C2A domain, where a serine is substituted for an alanine (Sullivan et al., 2008).
Piccolo is involved in the regulation of the internalization of the dopamine transporter (DAT) in heterologous cells. Specifically, expression of the C2A domain of Piccolo reduces methamphetamine-induced DAT internalization. (Cen et al., 2008). DAT and SERT are structurally related proteins, which belong to the solute carrier superfamily. It could be likely that membrane incorporation and internalization of these proteins is regulated in the same manner. Additionally, Piccolo influences synaptic function by negatively regulating synaptic vesicle exocytosis (Leal-Ortiz et al., 2008). These data led us to hypothesize that possibly, Piccolo might contribute to the overall risk of depression by affecting the membrane incorporation of SERT.
To test this hypothesis we expressed mCherry-SERT in hippocampal neurons and investigated whether knockdown of Piccolo or the variation rs2522833 affected the localization and trafficking of SERT. We show here that
knockdown of Piccolo in neurons did not affect SERT trafficking. However, expression of the Piccolo C2A domain variation rs2522833 in heterologous cells might affect membrane localization of SERT.
Results
*Piccolo localized to neurotransmitter release sites in hippocampal and 5-HT neurons*
First, we analyzed the cellular localization of Piccolo in hippocampal neurons and 5-HT neurons. Hippocampal neurons (14 days in vitro (DIV)) were fixed and immunocytochemistry was performed for Piccolo and for the presynaptic terminal marker Synaptobrevin-2 (VAMP2). Piccolo immunoreactivity was distributed in a punctate pattern, indicative for labeling of presynaptic terminals (Fig. 1A). Indeed, the majority of Piccolo puncta co-localized with VAMP2 puncta, showing that these represented presynaptic terminals (Fig. 1B,C). Zooming in on part of a neurite showed that >90% of Piccolo puncta co-localized with VAMP2 (Fig. 1D-F). Thus, Piccolo was present in presynaptic terminals.
Next, we investigated the localization of Piccolo in 5-HT neurons. We used a coronal brain slice from an adult mouse through the dorsal raphe nucleus (DRN), the region in the brain where the majority of 5-HT neurons is localized. We performed immunocytochemistry for tryptophan hydroxylase (Tph), the rate limiting enzyme for 5-HT synthesis and a marker for the 5-HT system. In a DRN slice, several 5-HT neurons were present (Fig. 1G). Immunocytochemistry for Piccolo resulted in a punctate labeling in the DRN slice (Fig. 1H). Piccolo staining was barely detectable in the 5-HT cell bodies (Fig. 1I). However, Piccolo puncta co-localized with Tph puncta. Tph is present in varicosities and presynaptic terminals (chapter 4 of this thesis) (Pickel et al., 1976).
**Figure 1 next page. Piccolo localizes to presynaptic terminals in neurons**
(A-C) Hippocampal neurons were fixed at DIV14, and immunocytochemistry was performed for Piccolo (A) and the presynaptic marker VAMP2 (B). (C) Merged image shows that several Piccolo puncta co-localized with VAMP2 puncta. (D-F) Blow up of the white box in C shows that almost every Piccolo puncta co-localizes with a VAMP2 puncta. (D) Piccolo staining. (E) VAMP2 staining. (F) Merged image. (G) A coronal brain slice through the DRN from an adult mouse was stained against the 5-HT marker Tph. This shows that 5-HT cell bodies and fibers are present. (H) Piccolo staining on this slice shows that Piccolo expression is low in 5-HT cell bodies. (I) Merged image. (J) Blow up of the white box in I shows a 5-HT cell body and several Tph puncta, which might be 5-HT varicosities. (K) Piccolo staining shows that in several of the Tph puncta Piccolo is present. (L) Merged image. Scale bars: 20 µm in C and I, 10 µm in F and L.
These Tph puncta might therefore represent 5-HT varicosities, axonal sites where 5-HT release occurs, often without a post-synaptic specialization. Therefore, it seems that also in 5-HT neurons Piccolo is localized in release sites (Fig 1J-L).
**Efficient knockdown of Piccolo in hippocampal neurons**
Next, we investigated whether knockdown of Piccolo affected trafficking of mCherry-SERT. In hippocampal neurons, mCherry-SERT is transported in secretory vesicles which move through the axon with no preference for direction of movement (Chapter 3, this thesis). To achieve efficient knockdown of Piccolo,
we designed six short hairpin RNA (shRNA) nucleotides, targeted towards the N-terminus of Piccolo. These short hairpins were inserted in a vector containing also an internal ribosomal entry site (IRES) and EGFP, as a control for transfected neurons. We tested the shRNAs for their Piccolo knockdown efficiency. To this end we transfected the shRNA constructs in hippocampal neurons, and four days later fixed and processed the neurons for immunocytochemistry. Several constructs tested did not show any knockdown of Piccolo. We decided to use shRNA#5 as a negative control. Analysis of Piccolo expression in MAP2 negative axons of neurons transfected with shRNA#5 was not reduced compared to untransfected cells (Fig. 2A-D). The axon had a punctate labeling of Piccolo, which was indistinguishable from untransfected neurons. On the other hand, transfection of shRNA#6 caused a reduction in levels of Piccolo in axons (Fig. 2E-H, 2I-L).
Quantification of the number of Piccolo puncta per μm neurite revealed that in control condition there was on average one Piccolo puncta per two μm neurite, whereas in shRNA#6 mediated knockdown there was on average one Piccolo puncta per ten μm neurite (control $0.49 \pm 0.11$ Piccolo puncta per μm axon; knockdown $0.11 \pm 0.02$ Piccolo puncta per μm axon, $p=0.002$) (Fig. 2M). Thus, shRNA#6 resulted in an efficient, approximately 80%, reduction in number of Piccolo puncta. Therefore, we used this construct to test the effect of Piccolo knockdown on mCherry-SERT localization and trafficking.
**Knockdown of Piccolo does not affect SERT trafficking**
We used shRNA#6 to investigate the effect of Piccolo knockdown on mCherry-SERT localization and trafficking. SERT tagged with mCherry is transported in secretory vesicles, which move in anterograde and retrograde direction, with no preference for direction of movement (chapter 3 of this thesis). We transfected mCherry-SERT and shRNA#6 in hippocampal neurons, and used EGFP as a control. One minute movies of mCherry-SERT trafficking were made, and the movies were analyzed using the custom written Fluotrack software program (Broeke et al., 2008). The velocity of moving puncta was quantified, as well as the direction of movement. For direction we discerned anterograde direction, retrograde direction and bidirectional, vesicles which changed direction during imaging. Both in neurons expressing shRNA#6 and EGFP, we observed several puncta of mCherry-SERT, which displayed rapid movements in both the anterograde and retrograde direction. Additionally, also stationary vesicles were observed (Fig. 3A-D). We quantified the average velocity of mCherry-SERT moving puncta in control neurons expressing EGFP, and neurons expressing shRNA#6. A velocity histogram shows the distribution of the average velocity and direction of movement between control and knockdown (Fig. 3E).
Figure 2. Efficient shRNA-mediated knockdown of Piccolo
Several shRNA constructs against Piccolo were tested for their efficacy to reduce Piccolo expression in cultured hippocampal neurons. ShRNA#5 did not result in Piccolo knockdown. (A) EGFP expression, showing that the shRNA construct is expressed. (B) Piccolo staining. (C) Neurite is MAP2 negative, showing that this is an axon. (D) Merged image. (E) EGFP expression in a neuron transfected with shRNA#6-EGFP. (F) Piccolo staining. (G) MAP2 staining. (H) Merged image, showing that shRNA#6 results in efficient knockdown of Piccolo in the axon. (I-L) Blow up of the white box in H, with EGFP, Piccolo staining, MAP2 staining and merged image respectively. (M) Quantification of the level of Piccolo knockdown shows that shRNA#6 resulted in a ~80% reduction in Piccolo puncta (control $0.49 \pm 0.11$ Piccolo puncta per $\mu m$ axon ($n=7$); knockdown $0.11 \pm 0.02$ Piccolo puncta per $\mu m$ axon ($n=8$)). Scale bars: 5 $\mu m$ in D and L, 10 $\mu m$ in H.
Trafficking of mCherry-SERT did not differ between control and knockdown (average velocity control 1.30 ± 0.042 µm/s (n=8 neurons); knockdown 1.36 ± 0.039 µm/s, (n=18 neurons) (Fig. 3F). Also, percentage of moving vesicles was not different between control and knockdown (control 46.69 ± 5.03%; knockdown 39.88 ± 3.39%) (Fig. 3G). Additionally, percentage of vesicles moving anterogradely, retrogradely or bidirectionally did not differ between control and knockdown (data not shown).
Figure 3 previous page. Piccolo knockdown does not affect SERT trafficking dynamics
(A) Neurons transfected with EGFP and mCherry-SERT. Images are taken 5 seconds apart. Several mCherry-SERT puncta are observed which are either stationary (yellow), or are moving anterogradely or retrogradely. (B) A neuron transfected with shRNA#6EGFP and mCherry-SERT. Images taken 5 seconds apart show that after Piccolo knockdown also several mCherry-SERT puncta are observed, some of which are stationary, and other which display anterograde or retrograde movements. (C) and (D) are blow ups of the white boxes in A and B, respectively. (E) Velocity histogram of mCherry-SERT puncta in control neurons and Piccolo knockdown neurons. Negative values are retrograde movements, positive values are anterograde movements. (F) Average velocity of mCherry-SERT puncta in control neurons and Piccolo knockdown neurons. (G) Percentage moving mCherry-SERT puncta in control neurons and Piccolo knockdown neurons. Scale bars: 10 µm in A and B, 5 µm in C and D.
Expression of C2A variant rs2522833 alters SERT distribution at the membrane in heterologous cells
Finally, we were interested whether the SNP in the C2A domain of Piccolo which was found to associate with major depression, affected the membrane localization of SERT. Therefore, we constructed the C2A\textsubscript{wt} domain of Piccolo in an IRES2-EGFP vector and a C2A\textsubscript{mut} domain with the rs2522833 SNP which results in a serine to alanine substitution at amino acid 4684. We transfected these constructs, together with mCherry-SERT, in heterologous Neuro2A cells, which do not express endogenous Piccolo. In Neuro2A cells transfected with mCherry-SERT together with Piccolo C2A\textsubscript{wt}, SERT was localized predominantly at the cell membrane. The majority of cells displayed this SERT distribution, although also in some cells mCherry-SERT was homogenously distributed. This may be due to the level of expression, since in several cells which showed this mCherry-SERT distribution, also EGFP fluorescence intensity was increased compared to other cells (Fig. 4A-C). Zooming in on a few cells expressing EGFP and mCherry-SERT showed that SERT is localized at the membrane, as mCherry fluorescence appeared like a ring at the membrane. Moreover, mCherry-SERT also seemed to associate with intracellular organelles, as some mCherry-SERT appeared as puncta in the cytoplasm. (Fig. 4D-F). Expressing mCherry-SERT together with Piccolo C2A\textsubscript{mut} resulted in cells in which mCherry-SERT was localized at the membrane, but also some cells in which mCherry-SERT displayed a more uniform distribution in the cytoplasm which might again be due to the level of expression (Fig. 4G-I). A typical example of three cells expressing both constructs showed that in one cell mCherry-SERT was localized at the membrane, and in the other cells mCherry-SERT displayed a more uniform distribution in the cytoplasm (Fig. 4J-L). Strikingly, membrane bound mCherry-SERT in Piccolo C2A\textsubscript{mut} expressing cells displayed a more patchy
distribution than in Piccolo C2Awt expressing cells (Fig. 4M-O). A line scan profile of a typical cell co-expressing mCherry-SERT with Piccolo C2Awt showed that the highest level of fluorescence was at the membrane (Fig. 4P). In contrast, a line scan profile of a cell co-expressing mCherry-SERT with Piccolo C2Amut showed that fluorescence levels in the cytoplasm were comparable with fluorescence levels at the membrane (Fig. 4Q).
Thus, it appeared that co-expression of the Piccolo C2Amut with the rs2522833 SNP resulted in an altered distribution of mCherry-SERT at the membrane. These data suggest that Piccolo is not involved in the trafficking of SERT, but might be involved in regulating SERT membrane levels.
**Discussion**
Piccolo is a large protein, which is part of the active zone in the presynaptic terminal. There, Piccolo probably has a scaffolding role, but Piccolo also contains a PDZ domain and two C2 domains. Piccolo can be alternatively spliced, resulting in absence or presence of the C2B domain (Wang et al., 1999). Additionally, in the C2A domain a nine-residue sequence has been found which is subject of alternative splicing, resulting in variants of the C2A domain with different calcium affinities (Garcia et al., 2004). In this study we focused exclusively on the long C2A variant. The gene region in which Piccolo is localized has been linked to autism (Nabi et al., 2003). The SNP rs2522833 is localized in the C2A domain (Sullivan et al., 2008). The C2A domain has a low calcium affinity, suggesting an involvement in presynaptic plasticity processes (Gerber et al., 2001). It is currently unknown whether the SNP affects PIP$_2$ or Ca$^{2+}$ binding to the C2A domain.
*Figure 4 next page. Expression of Piccolo C2Awt and C2Amut with mCherry-SERT in heterologous cells*
(A-C) Expression of C2Awt–IRES-EGFP and mCherry-SERT in Neuro2A cells. (D-F) Blow up of white box in C shows that in the majority of cells mCherry-SERT is localized at the membrane. In the cytoplasm, mCherry-SERT displays a punctate distribution, suggesting association with intracellular organelles. (G-I) Expression of C2Amut–IRES-EGFP and mCherry-SERT in heterologous cells. (J-L) Blow up of white box #1 in I shows that in several Neuro2A cells expressing C2Amut–IRES-EGFP, mCherry-SERT seems to have a more uniform cytoplasmic distribution. (M-O) Blow up of white box #2 in I shows that in cells in which mCherry-SERT is localized at the membrane, mCherry-SERT is not uniformly distributed at the membrane, but instead is distributed in patches along the membrane. (P,Q) Line scan profiles of a typical cell expressing mCherry-SERT with C2Awt–IRES-EGFP or C2Amut–IRES-EGFP (lines in figures E and K respectively). Arrows indicate cell membrane. Scale bars: 20 µm in C and I, 10 µm in F and L and 5 µm in O.
**EGFP**
- **A**: EGFP expression in cells.
- **B**: mCherry-SERT expression in cells.
- **C**: Merge of EGFP and mCherry-SERT expressions.
**C2Awt-IRESGFP**
- **D**: EGFP expression in cells.
- **E**: mCherry-SERT expression in cells.
- **F**: Merge of EGFP and mCherry-SERT expressions.
**C2Amut-IRESGFP**
- **G**: EGFP expression in cells.
- **H**: mCherry-SERT expression in cells.
- **I**: Merge of EGFP and mCherry-SERT expressions.
**P**: Fluorescence intensity profile across a cell with a nucleus.
**Q**: Fluorescence intensity profile across a cell without a nucleus.
The regulation of SERT membrane localization and internalization is important in determining efficacy of 5-HT transmission. Increases in membrane localization of SERT increase 5-HT reuptake and thereby reduce extracellular levels of 5-HT and binding of 5-HT to postsynaptic 5-HT receptors. In contrast, internalization of SERT results in increased extracellular levels of 5-HT and increased postsynaptic activation of 5-HT receptors.
Protein kinase C (PKC) reduces the expression level of SERT, presumably by altering the membrane localization of SERT (Qian et al., 1997). Indeed, application of phorbol esters results in increased internalization of SERT. In contrast, substrates of SERT, such as 5-HT, reduce PKC dependent SERT internalization, whereas SERT antagonists, such as antidepressants, block this 5-HT mediated reduction in SERT internalization (Ramamoorthy and Blakely, 1999). On the other hand, protein kinase G (PKG) activity increases the membrane localization of SERT (Zhu et al., 2004). Therefore, there is an extensive level of regulation of membrane expression of SERT. There is an indirect link between Piccolo and SERT; Syntaxin1A is known to interact with SERT, and to regulate SERT activity (Quick, 2003). The C2A domain of Synaptotagmin, which shares similarity with the C2A domain of Piccolo, interacts with Syntaxin1A (Shao et al., 1997). Therefore this makes it likely that also the C2A domain of Piccolo is able to interact with Syntaxin1A, and thus possibly with SERT.
We used heterologous Neuro2A cells, which do not express Piccolo endogenously, to study the effect of expression of both C2A variants on SERT localization. We showed that distribution of SERT at the membrane might be altered after co-expression with the mutant C2A variant. Previously it has been shown that DAT trafficking and internalization is not altered in heterologous cells upon Piccolo knockdown. However, upon treatment of cells with 3,4-methylenedioxy-N-methylamphetamine (MDMA), there is a significant increase in DAT internalization. Thus, Piccolo seems to regulate the MDMA dependent internalization of DAT. For SERT, it has been shown that application of antidepressants and the PKC activator β-phorbol 12-myristate 13-acetate (β-PMA) results in an increased internalization of SERT (Lau et al., 2008a; Lau et al., 2008b). In view of this, it would be interesting to challenge SERT with an antidepressant after expression of both C2A variants, to investigate whether this results in an altered distribution of SERT between the two C2A variants.
Several studies show that the expression level, membrane level, or transport activity of SERT might be affected in psychopathological processes. Levels of SERT expression in DRN are reduced in brains of depressed suicide victims (Arango et al., 2001). In contrast, long-term administration of antidepressants seemed to also reduce SERT surface expression (Hirano et al., 2005). Recent
studies showed that a SNP in SERT which associated with obsessive compulsive disorder resulted in increased transport activity of SERT (Kilic et al., 2003). Thus, further studies are required to investigate the relation between the SNP in Piccolo, SERT expression and activity, and major depression disorder.
EXPERIMENTAL PROCEDURES
Plasmids
Human SERT (hSERT) cDNA tagged with ECFP at the 5' end in the Clontech ECFP-C1 vector was a kind gift from Dr. H. Sitte. To clone a red fluorescent version of hSERT we replaced ECFP for the red fluorescent protein mCherry. To this end mCherry was digested out of the vector using AseI and BsrGI and cloned sticky in the ECFP-hSERT vector from which ECFP had been excised. For construction of pSuper-CMV-EGFP-shRNA-Piccolo, pSuper was digested using EcoRI and PstI and CMV-EGFP was inserted after isolation of this fragment from pEGFP-C3 (Clontech) using BsrGI and NsiI. The shRNA against Piccolo was constructed using the following primers: forward 5'-GATCCCCACCCCAAAATATAAAGCTTTCAAGAGAAGCCTTATATTITGTGGGTTTTTTTGGAAGA-3' and reverse 5'-AGCTTTTCTCAAAGCTTTATATTGTGGGGTGGG-3'. These primers were annealed by heating to 100°C, cooling down to room temperature and inserted in pSuper-CMV-EGFP by digesting this vector with BglII and HindIII. For construction of C2Awt, this fragment was isolated from a Yeast-2-Hybrid mouse cDNA bank using the following primers: forward 5'-GGCGTGGATCCCAAGCAGCTG-3' and reverse 5'-GTTTTGACTGGAGTGGAGACTG-3' and subsequently a PCR was performed using the following primers: forward 5'-AAGGATCCGCCTCTCACCCAATTACAGGA-3' and reverse 5'-TTTCGGGCCGATGCTTTCACTGCTGTITTTTCAG-3' to add BamHI and NotI sites. For C2Amut (S468A), the C2A domain was isolated using two PCR reactions using the following primers: forward 5'-AAGGATCCGCCTCTCACCCAATTACAGGA-3' and reverse 5'-CGAGGAGTGTTGTCCAGATGAGCAGTACTAGATAAAATCAATCAATAC-3' and forward 5'-GTATGATGTGATTATCTAGTACTGCTCATCTGGACAACACTCCTCG-3' and reverse 5'-TTTCGGGCCGATGCTTTCACTGCTGTITTTTCAG-3'. These two PCR products were annealed and a PCR was performed on this product using primers: forward 5'-AAGGATCCGCCTCTCACCCAATTACAGGA-3' and reverse 5'-TTTCGGGCCGATGCTTTCACTGCTGTITTTTCAG-3' to add BamHI and NotI sites and C2Awt and C2Amut fragments were ligated in pGEX. To construct pCMV-C2A-IRES2EGFP, both fragments were isolated from pGEX by
PCR using the following primers: forward 5'-AAAGGATCCACATGGCCTCTCACCCCAATTACAGG-3' and reverse 5'-TTTGAATTCTCAGCTTTTCAGTCTGTCTTTTCAG-3' to add BamHI and EcoRI sites and a Kozak and stop sequence. Fragments were inserted in pIRES2EGFP by ligating this vector with BglII and EcoRI.
**Cell culture and transfections**
Hippocampi from embryonic day 18 C57/Bl6 mouse embryos were dissected free of meninges and collected in Hanks’ Balanced Salt Solution (HBSS; Invitrogen) supplemented with 7 mM HEPES (Invitrogen). Hippocampi were digested with 0.25% trypsin (Invitrogen) in HBSS supplemented with 7 mM HEPES for 15 minutes at 37°C. After digestion tissue was washed three times with HBSS supplemented with 7 mM Hepes and subsequently triturated with a fire-polished glass pipette. Dissociated neurons were plated at a density of 25,000 cell/well on rat glial cells on 18 mm glass coverslips (Menzel Glaser, Braunschweig, Germany) in 12 well plates. Cells were cultured in neurobasal medium (Invitrogen) supplemented with B27 supplement (Invitrogen), 18 mM HEPES, 0.5 mM Glutamax (Invitrogen) and penicillin/streptomycin (Invitrogen). Once a week half of the medium was replaced. The hippocampal cultures were transfected using Lipofectamine 2000 (Invitrogen). Before transfection, half of the neurobasal medium was collected from each well and replaced with new neurobasal medium. For one well, 1 µl of Lipofectamine was added to 25 µl of serum free glutamax (Invitrogen) medium and incubated for 5 minutes. Subsequently, from each construct 1 µg of DNA was added to 25 µl of serum free glutamax and immediately mixed with the 25 µl of serum free glutamax containing Lipofectamine. This mixture was incubated for 30 minutes before adding to the cells. After 4–6 hours of incubation on the cells, half of the neurobasal medium was replaced again with the earlier collected neurobasal medium. Cells were routinely transfected at DIV2–7 and imaged at DIV10–14.
**Immunocytochemistry**
For immunocytochemistry, neuronal cells were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) (pH 7.4) for 20 minutes at room temperature. Cells were washed in PBS and blocked in PBS containing 4% fetal calf serum and 0.2% Triton X-100 for 20 minutes at room temperature. Cells were incubated overnight with primary antibodies at 4°C in PBS containing 0.2% Triton X-100. As primary antibodies monoclonal MAP2 (Chemicon), and polyclonal Piccolo (Synaptic Systems) and Synapsin (Eo28) were used. After incubation in primary antibodies, cells were washed three times using PBS. Subsequently, cells were incubated in Alexa Fluor-conjugated secondary antibodies (Invitrogen) in PBS
for one hour at room temperature. All antibodies were used in a 1:1000 dilution. Finally, cells were washed three times in PBS and mounted using Dabco-Mowiol.
For immunohistochemistry on mouse brain slices, mice were anaesthetized and transcardially perfused with 4% paraformaldehyde (PFA) in PBS (pH 7.4). Brains were post fixed in PFA for one day, and subsequently cryoprotected in increasing concentrations of sucrose. Thirty μm coronal adult brain slices were made using a Cryostat (Leica). Midbrain slices containing the dorsal and medial raphe nucleus were blocked for two hours in PBS containing 10% normal goat serum and 0.5% Triton X-100. Subsequently, the slices were incubated overnight in primary antibodies in PBS containing 0.5% Triton X-100 at 4°C. Brain slices were washed three times two hours in PBS and incubated in secondary antibodies in PBS overnight at 4°C. After washing three times two hours, slices were mounted using Dabco-Mowiol. As primary antibodies monoclonal Tph (Sigma, recognizing both Tph2 and Tph2) and polyclonal Piccolo (Synaptic systems) were used in a 1:1000 dilution. Alexa Fluor-conjugated secondary antibodies (Invitrogen) were used in a 1:1000 dilution. Cells and slices were examined on a Zeiss LSM 510 confocal laser scanning microscope (Zeiss, BV The Netherlands).
**Live cell imaging**
Cells were imaged in a chamber perfused with Tyrode’s solution (119 mM NaCl, 2.5 mM KCl, 2 mM MgCl$_2$, 2 mM CaCl$_2$, 30 mM glucose, 25 mM HEPES, pH 7.4) on an Axiovert II Microscope (Zeiss, Oberkochen, Germany) equipped with a Coolsnap HQ camera (Photometrics, Tucson, Arizona, USA) and a Polychrome IV illumination unit (TILL photonics, Graefling, Germany). Images were acquired in MetaMorph 6.2 software (Universal Imaging, Downingtown, Philadelphia, USA) using a 100x oil objective.
**Image analysis**
To analyze the trafficking of mCherry-hSERT, image stacks were converted to .avi movie files using ImageJ software. Movies were analyzed using the Fluotrack software program (Broeke et al., 2008) to quantify the average number of moving puncta, the average velocity of moving puncta, the average pausing time of moving puncta and the direction of moving puncta.
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Chapter 6
Deletion of Munc18-1 in 5-HT neurons results in rapid degeneration of the 5-HT system and early postnatal lethality
J.J. Dudok\textsuperscript{1}, A.J.A. Groffen\textsuperscript{1}, R.F.T. Toonen\textsuperscript{1}, Neuro-BSIK Mouse Phenomics consortium and M. Verhage\textsuperscript{1}
\textsuperscript{1}Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, the Netherlands
Submitted
Abstract
The serotonin (5-HT) system densely innervates many brain areas and is important for proper brain development. To specifically ablate the 5-HT system we generated mutant mice carrying a floxed Munc18-1 gene and Cre recombinase driven by the 5-HT-specific serotonin reuptake transporter (SERT) promoter. Mutant mice were smaller than their control littermates and the majority died within a few days after birth. Immunohistochemical analysis of brains of these mice showed that initially 5-HT neurons are formed and the cortex is innervated with 5-HT projections. From embryonic day (E) 16 onwards, however, 5-HT neurons started to degenerate and at postnatal day (P) 2 hardly any 5-HT projections were present in the cortex. The 5-HT system of mice heterozygous for the floxed Munc18-1 allele was indistinguishable from control mice. Behavioural analysis of these heterozygous mice showed that there is no difference in basal behaviour, conditioning and anxiety-related behaviour compared to control mice. These data show that deletion of Munc18-1 in 5-HT neurons results in rapid degeneration of the 5-HT system and suggests that the 5-HT system is important for postnatal survival.
Introduction
The 5-HT system consists of clusters of cell bodies in the midbrain raphe nuclei, with the largest clusters in the median raphe nucleus (MRN) and the dorsal raphe nucleus (DRN). Several brain areas receive dense 5-HT innervation and 5-HT is released both synaptically and as volume transmission (Ridet et al., 1993; Bunin and Wightman, 1998). Due to this and to the several 5-HT receptor subtypes which are present in the brain, 5-HT has many roles and influences many processes in the brain (Jacobs and Azmitia, 1992).
Neurogenesis of 5-HT neurons in the mouse brain occurs in the ventral rhombencephalon around E10 (Wallace and Lauder, 1983). One day later, 5-HT neurons begin to synthesize and secrete 5-HT and start growing out axons. Around birth, target areas such as the forebrain and the hippocampus are densely innervated with 5-HT projections. Only after birth, the maturation of the 5-HT network is completed.
Several studies have addressed the role of 5-HT on the development of the 5-HT system and brain development. In a conditional Lmx1b knockout (KO) mouse, almost all 5-HT neurons fail to survive, resulting in a significant decrease in brain tissue 5-HT levels (Zhao et al., 2006). However, these mice do not show
an overt phenotype and survive to adulthood (Zhao et al., 2006). In contrast, it was shown that maternal 5-HT is required for embryonic development (Cote et al., 2007). Furthermore, in tryptophan hydroxylase 2 (Tph2) KO mice 5-HT neurons are completely devoid of 5-HT, but the morphology and neurite distribution of the 5-HT system is not affected, and these mice do not show any behavioural phenotype (Gutknecht et al., 2008). Neonatal depletion of 5-HT by the neurotoxin 5,7-Dihydroxytryptamine results in rather subtle changes in behavioural response and brain development (Hohmann et al., 2007; Bennett-Clarke et al., 1995).
In this study we silenced the 5-HT system by conditional deletion of Munc18-1 in 5-HT neurons. Neurons that lack the presynaptic protein Munc18-1 have a complete absence of neurotransmitter secretion and these mice are born paralyzed and die immediately after birth (Verhage et al., 2000). In these mice, initially synapses are formed and the assembly of the brain is normal. However, in later stages of brain development there is massive neuronal cell death and brain degeneration (Verhage et al., 2000).
In SERT-Cre\textsuperscript{wt} Munc18-1\textsuperscript{lox/lox} mice 5-HT neurons were initially generated and 5-HT projections innervated the midbrain and cortex, later followed by degeneration and loss of 5-HT projections in the cortex. The majority of these mice died within a few days after birth. These data suggest that the 5-HT system contributes importantly to postnatal brain development.
**Results**
*Deletion of Munc18-1 in SERT expressing neurons results in postnatal lethality*
We have shown previously that Munc18-1 mutant mice lack regulated secretion and have a lethal phenotype (Verhage et al., 2000). Therefore, we have generated Munc18-1\textsuperscript{lox/lox} mutant mice in order to conditionally delete Munc18-1. Crossing these mice with a L7-Cre line, with Cre expressed in Purkinje neurons in the cerebellum, resulted in mice which developed severe ataxia, suggesting a cerebellar phenotype (Heeroma et al., 2004). Here we crossed Munc18-1\textsuperscript{lox/lox} mice with SERT-Cre mice, which express Cre in SERT expressing neurons. These are the 5-HT neurons in the raphe nuclei, but also some hippocampal neurons and thalamocortical neurons which express SERT transiently during development (Zhuang et al., 2005; Narboux-Neme et al., 2008). Crossing SERT-Cre mice with Munc18-1\textsuperscript{lox/lox} mice results in mice in which Munc18-1 is specifically removed in SERT expressing neurons (Fig. 1A). We crossed SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/wt} mice with SERT-Cre\textsuperscript{wt/wt} Munc18-1\textsuperscript{lox/wt} mice which should result in 12.5% of offspring which have SERT-Cre\textsuperscript{cre/wt} and Munc18-1\textsuperscript{lox/lox}
genotypes. Genotyping mice sacrificed at E16 or E18 revealed that there were more SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} and SERT-Cre\textsuperscript{wt/wt} Munc18-1\textsuperscript{lox/lox} mice and less SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/wt} mice than expected (p = 0.007) (Fig. 1B). Genotyping 101 mice three weeks after birth revealed that genotype frequencies were not distributed at Mendelian ratio (p = 0.027). Only three SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} mice were found, whereas based on Mendelian ratio the expected number of SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} mice should be 13, giving a 77% mortality rate in SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} mice (Fig. 1C). Also, SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} mice were smaller compared to littermates (data not shown). These three remaining SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} mice all died in the fourth postnatal week. This shows that Munc18-1 deletion in SERT expressing neurons results in postnatal lethality.
Figure 1. Postnatal lethality in SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} mice
(A) Overview of breeding approach. Crossing SERT-Cre with Munc18-1\textsuperscript{lox/lox} mice results in deletion of Munc18-1 only and specifically in SERT expressing neurons, whereas all other neurons still express Munc18-1.
(B) Genotypes of mice sacrificed at E16 or E18 differed from the expected Mendelian ratio. (C) Genotyping mice after weaning at 3 weeks of age revealed that only few SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} mice survived up to three weeks.
5-HT neurons are initially generated but then quickly degenerate
To assess the effect of Munc18-1 deletion in 5-HT neurons on the development of the 5-HT system, we made coronal sections of paraformaldehyde (PFA) fixed brains at different developmental stadia and performed immunohistochemistry for 5-HT. As developmental time points we chose E16, E18 and P2. Expression of SERT, and thus of Cre recombinase, starts at E11 and the earliest recombination observed in the DRN in SERT-Cre mice was at E12.5 (Narboux-Neme et al., 2008). Since it will take some days before all remaining Munc18-1 mRNA and protein is degraded, we chose E16 as the first time point. We first focused on the 5-HT cell bodies in the DRN. Immunohistochemistry for 5-HT on brain slices from E16 SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} and control mice showed that in the DRN 5-HT neurons are present and these showed the characteristic DRN topology (Fig. 2A,B). The number of 5-HT neurons did not differ from control (control 312 ± 17.67, SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} 305 ± 25.43; Fig. 2G,H,O). However, morphological analysis of the neurons showed that these were already degenerating, as assessed by the round morphology and reduced number of primary neurites. At E18, there was a ~80% decrease in the number of 5-HT neurons in SERT-Cre\textsuperscript{cre/wt} M18\textsuperscript{lox/lox} mice compared to control (control 358 ± 10.82, SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} 59 ± 4.84, Fig. 2C,D,O). At P2 there were only few 5-HT neurons left in the DRN (control 364 ± 19.68, SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} 25 ± 2.89, Fig. 2E,F,O). At E18 and P2 the remaining 5-HT neurons in SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} brain sections displayed an altered morphology compared to control 5-HT neurons (Fig. 2I,J,K,L). 5-HT neurons in control brains had a characteristic fusiform or ovoid shape and grew out several primary neurites (Fig. 2M). In contrast, the mutant 5-HT neurons had a rounded morphology and the majority did not contain primary neurites, indicative of degenerating neurons (Fig. 2N). This showed that between E16 and P2 there is a massive degeneration of 5-HT neurons in the DRN.
5-HT projections innervate the DRN and cortex but are degenerated at P2
Next, we focused on the innervation of the midbrain and cortex with 5-HT projections. Midbrain and cortex are the first regions which are densely innervated with 5-HT projections during development. We quantified 5-HT neurite density first in DRN midbrain sections at E16, E18 and P2. At E16 the sections from both control and SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} mice contained several 5-HT neurites and there was no significant difference in 5-HT innervation density (1.31 ± 0.51% and 0.63 ± 0.24% respectively, Fig. 3A,B). At E18, in control sections the 5-HT innervation density was slightly increased compared to E16 (Fig. 3C). However, in SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} sections the 5-HT innervation density was decreased compared to control (control 2.32 ± 0.86%, SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} 0.31 ± 0.27%, Fig. 3D).
Figure 2. Rapid degeneration of 5-HT neurons in SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} mice
We compared the number of 5-HT cell bodies in the DRN in control and SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} mice at E16, E18 and P2. (A-F) At E16, in both control mice (A) and SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} (lox/lox) mice (B) the 5-HT cell bodies are distributed in the DRN topology. At E18 and P2, however, in midbrain sections from SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} brains (D, F respectively) only few 5-HT neurons are present compared to control sections (C, E respectively). (G-L) Zooming in on 5-HT cell bodies shows that in control sections at E16 (G), E18 (I) and P2 (K) the cell bodies grow out several neurites and have a fusiform or ovoid morphology. However, remaining 5-HT cell bodies in SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} sections at E16 (H), E18 (J) and P2 (L) have a round morphology with hardly any neurites. (M,N) Blow up of some 5-HT cell bodies in control and SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} mice sections shows the differences in 5-HT cell body morphology. (O) Analysis of the number of 5-HT cell bodies in the sections showed that there is no difference at E16, but at E18 and P2 there is a decrease of 70% to 80% in SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} sections. Scale bars: 200 µm in F, 50 µm in L and 20 µm in N. Data shown are mean ± standard error of the mean (SEM). * p < 0.05.
At P2 only few 5-HT neurites are left in SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} sections (control 1.6 ± 0.41%, SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} 0.33 ± 0.14%, Fig. 3E,F). Next, we focused on the 5-HT innervation density in the cortex. This revealed that already at E16 there is a reduction in 5-HT innervation density in SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} sections (control 3.27 ± 1.14%, SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} 0.65 ± 0.19%, Fig. 3G,H). At E18 and P2 the reduction in 5-HT innervation density is augmented, with only very few 5-HT neurites left at P2 in the cortex (E18 control 1.73 ± 0.45%, SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} 0.36 ± 0.18%, P2 control
1.78 ± 0.52%, SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsubscript{lox/lox} 0.19 ± 0.04%, Fig. 3I,J,K,L). Quantification showed that in the midbrain sections the 5-HT innervation density was reduced to ~20% and ~10% at E18 and P2 respectively (Fig. 3M). In the cortex, the 5-HT innervation density was reduced to ~20%, ~10% and ~5% at E16, E18 and P2 respectively (Fig. 3N).
In summary, deletion of Munc18-1 in 5-HT neurons resulted in rapid degeneration of 5-HT cell bodies and the absence of 5-HT projections in the postnatal brain.
**Figure 3. Degeneration of 5-HT neurites in DRN and cortex**
(A-F) At E16 in the midbrain containing the DRN, there was no difference in 5-HT neurite density between control (A) and SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsubscript{lox/lox} (B) mice. In control sections at E18 (C) and P2 (E) several 5-HT neurites were present. However, in SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsubscript{lox/lox} sections at E18 (D) and P2 (F) only very few 5-HT neurites are left. (G-L) In the cortex already at E16 there is a reduction in 5-HT neurite density in sections from SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsubscript{lox/lox} (H) mice compared to control (G). At E18 (J) and P2 (L), in cortical sections from SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsubscript{lox/lox} mice only very few remaining 5-HT neurites are present, in contrast to E18 (I) and P2 (K) control cortical sections. (M) Quantification of the 5-HT neurite density in the DRN revealed that at E16 there is no difference, but at E18 and P2 5-HT neurite density is reduced to ~20% and ~10% respectively. (N) In cortical sections, at E16 the 5-HT neurite density is reduced to ~20% and at E18 and P2 the 5-HT neurite density is reduced to ~10% and ~5% respectively. Scale bar 50 µm in L. Data shown are mean ± SEM * p < 0.05.
Basal behaviour is not affected in SERT-Cre\textit{er/cut} Munc18-1\textit{fl/ut} mice
Finally, we were interested whether deletion of one allele of Munc18-1 in SERT expressing cells affected mouse behaviour. Previously, we have shown that Munc18-1 expression levels regulate the readily releasable pool (RRP) size. In Munc18-1 heterozygous mice there is a faster synaptic rundown during episodes of high activity, a decreased RRP size and a decrease in docked vesicles at the synapse (Toonen et al., 2006). Therefore it is likely that deletion of one functional Munc18-1 allele in SERT expressing neurons affects the efficacy of 5-HT neurotransmission.
Figure 4. Deletion of one functional allele of Munc18-1 in SERT expressing neurons does not affect number of 5-HT cell bodies and 5-HT innervation density
(A-D) Midbrain sections containing the DRN from control (A) and hz (B) mice shows that 5-HT neurons are distributed in the characteristic DRN topology. Zooming in on 5-HT cell bodies show that both in control (C) and hz sections (D) these neurons have a fusiform or ovoid morphology and grow out several neurites. (E-H) 5-HT neurite density is not different between control and hz mice in the midbrain containing the DRN (E and F respectively) or in cortical sections from control and hz mice (G and H respectively). (I) Number of 5-HT cell bodies per section does not differ between control and hz. (J) 5-HT neurite density in midbrain containing DRN and cortical sections is not different between control and hz. Scale bars: 200 µm in B, 50 µm in D and H.
First, we investigated whether deletion of one allele of Munc18-1 in SERT expressing neurons affected the number of 5-HT cell bodies in the DRN, or the 5-HT fiber density in the DRN and cortex. Analysis of midbrain sections from control and SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/wt} (hz) mice showed that 5-HT neurons are distributed in the characteristic DRN topology, and control and hz are indistinguishable (Fig. 4A,B). 5-HT cell bodies in both control and hz had a fusiform or ovoid shape (Fig. 4C,D). Quantification of the number of 5-HT cell bodies in the DRN revealed no difference between control and hz (control 364 ± 14.78, hz 361 ± 15.01, Fig. 4I). Likewise, the 5-HT neurite density in the midbrain and cortex were similar (midbrain control 4.38 ± 0.93%, hz 4.67 ± 0.55%; cortex control 2.18 ± 0.35%, hz 2.87 ± 0.99%, Fig. 4E-H,I,J). Thus, deletion of one allele of Munc18-1 does not affect 5-HT neuronal survival, 5-HT neurite outgrowth or 5-HT projection density.
We tested several behavioural characteristics of hz mice by monitoring them for six days in home cages equipped with a video camera on top to visualize and analyze mouse behaviour. We analyzed the behaviour of eleven hz mice and compared several behavioural parameters with eleven control mice. First, we analyzed whether there was a difference in total distance moved in the open space and time spent under the shelter. Control and hz mice moved a similar total distance in the open space (control dark phase 2231.78 ± 139.39 cm/hour, light phase 483.91 ± 32.30 cm/hour; hz dark phase 2150.61 ± 112.70 cm/hour, light phase 433.69 ± 26.15 cm/hour; Fig. 5A,B), spent a similar proportion of time in a sheltered compartment (control dark phase 58.99 ± 2.03%, light phase 86.37 ± 2.36%; hz dark phase 60.04 ± 1.11%, light phase 90.38 ± 0.45%; Fig. 5C) and spent a similar time drinking (control dark phase 33.92 ± 1.99 s/hour, light phase 7.50 ± 0.52 s/hour; hz dark phase 41.06 ± 4.72 s/hour, light phase 8.06 ± 0.79 s/hour; Fig. 5D). Also, time spent in the feeder was indistinguishable between control and hz (control dark phase 476.77 ± 15.17 s/hour, light phase 160.55 ± 6.58 s/hour; hz dark phase 497.73 ± 18.41 s/ hour, light phase 165.33 ± 6.99 s/hour; Fig. 5E). Thus, basal behavioural parameters were indistinguishable between control and hz.
\textit{Conditioning and anxiety related behaviour is not affected in SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/wt} mice}
To investigate whether hz mice display altered conditioning behaviour, a free operant conditioning task was performed, where a jump on the shelter resulted in the drop of a sucrose pellet. The number of directed short movement bouts (SMBs) increased during day four and five compared to day 1-3, but this conditioning behaviour did not differ between control and hz.
Figure 5. Hz mice do not display altered basal behaviour
(A) Six day plot of average distance moved (cm / 12 hours) during dark and light phase. Note the increase in distance moved during the dark phase at day 4 due to the free operant conditioning test. (B) No difference in average distance moved (cm/hour) between control and Hz mice during the dark and light phase. (C) Proportion of time spent under the shelter does not differ between control and Hz mice. (D) There is no difference in time spent drinking (s/hour) between control and Hz mice. (E) Also, there is no difference in time spent in the feeder (s/hour). Data shown are mean ± SEM.
(day 1-3 control $17.07 \pm 2.12$, hz $16.33 \pm 2.17$; day 4-5 control $45.3 \pm 5.27$, hz $46.32 \pm 5.78$; Fig. 6A). Number of pellets dropped during pre-training phase (session 1-10, day four) did not differ between control and hz mice (control $4.88 \pm 0.43$, hz $5.99 \pm 0.45$; Fig. 6B). Also during the sessions at day five (session 11-12), there was no difference in the number of pellets dropped (control $20.36 \pm 2.81$, hz $19.95 \pm 2.34$; Fig. 6B).
To assess whether hz mice displayed altered anxiety related behaviour, a one trial anxiety task was performed at day six. To this end a lightspot was created at the feeder place, and the duration of time the animal spent in the lightspot and the feeder was recorded and compared with the time the animal spent at the feeder without a lightspot on. At day five (baseline) and day six (test) there was no difference between control and hz, but both groups spent less time in the lightspot at day six (day five control $1798.72 \pm 208.1$ s, hz $2108.01 \pm 284.63$ s; day six control $668.76 \pm 61.28$ s, hz $682.19 \pm 83.98$ s; Fig. 6C). Next, we investigated whether the frequency of entering the feeder zone at day five (baseline) and day six (test) was different. Frequency of entering the feeder zone did not differ between control and hz mice, but both groups displayed a decreased frequency during the lightspot test during day six (day five control $16.64 \pm 2.51$, hz $23.09 \pm 3.51$; day six control $7.15 \pm 1.40$, hz $8.12 \pm 1.56$; Fig. 6D). Also, time spent in the feeder zone during day five and day six did not differ between control and hz (day five control $528.15 \pm 50.21$ s, hz $621.91 \pm 65.99$ s; day six control $191.01 \pm 30.36$ s, hz $198.46 \pm 33.53$ s; Fig. 6E). Finally, analysis of latency to enter the lightspot zone after lightspot off revealed that this did not differ between control and hz (control $1567.71 \pm 377.86$ s; hz $1293.87 \pm 329.17$ s; Fig. 6F). Thus, mice in which one allele of Munc18-1 is deleted in SERT expressing neurons do not display altered basal, conditioning or anxiety-related behaviour.
**Discussion**
In this study, we showed that SERT-Cre$^{cre/cre}$wt Munc18-1$^{lox/lox}$ mice display a postnatal lethality phenotype, accompanied by a rapid degeneration of the 5-HT system. Inactivation of one allele of Munc18-1 in SERT expressing neurons does not affect 5-HT cell number or 5-HT innervation density, and does not affect anxiety related behaviour or conditioning related behaviour.
Crossing Munc18-1$^{lox/lox}$ mice with SERT-Cre mice results in deletion of Munc18-1 from SERT expressing cells. It was shown previously that crossing SERT-Cre mice with loxSTOPlox-EYFP mice results in >99% of 5-HT neurons in the DRN which are EYFP positively labeled (Zhuang et al., 2005).
Figure 6. Hz behaviour on free operant conditioning test and one trial anxiety test does not differ from control
(A) The number of directional SMB’s is increased at day 4 and 5 compared to day 1-3, but does not differ between control and hz. (B) Number of sucrose pellets acquired is increased at session 11 and 12 (day 5), compared to session 1-10 (day 4, pre-training day), but does not differ between control and hz. (C) Time spent in the lightspot (s) is decreased at day 6 (lightspot on) compared to day 5 (baseline, without lightspot on), but does not differ between control and hz. (D) Frequency of entering the feeder zone is decreased at day 6 compared to day 5, but there is no difference between control and hz mice. (E) During lightspot on on day 6 mice spent less time in the feeder (s) which does not differ between control and hz. (F) No difference in latency (s) to enter the lightspot zone after lightspot off. Data shown are mean ± SEM.
However, SERT is also transiently expressed in some other brain regions during development, such as the thalamocortical neurons (Lebrand et al., 1996). In SERT-Cre ROSA-loxstoplox-LacZ double positive mice, strong LacZ expression was not only observed in the DRN, but also in the thalamus, cingulate cortex and
in the CA3 region of the hippocampus (Zhuang et al., 2005; Narboux-Neme et al., 2008). Therefore, in SERT-Cre Munc18-1\textsuperscript{lox/lox} mice also in some non-5-HT neurons Munc18-1 will be deleted. Genotyping sacrificed mice at E16 or E18 revealed that there were more SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} mice than expected, showing that these mice do not die during embryonic development. However, genotyping mice three weeks after birth showed that the majority of SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} mice had died, and the remaining SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} mice died within the fourth postnatal week. SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} mice were smaller compared to control mice which is in analogy with the VMAT2 KO mice which are hypoactive, smaller compared to control littermates, do not feed and die within a few days after birth (Fon et al., 1997; Wang et al., 1997).
Previously we showed that in Munc18-1 KO mice, the neuromuscular junction initially develops, but at later stages most of the motor neuronal cell bodies in the spinal cord degenerate (Heeroma et al., 2003). In analogy with this, in the absence of regulated secretion the hypothalamo-neurohypophysial system is initially normally formed, but in later stages of development degenerates (Korteweg et al., 2004). This is consistent with our observation that Munc18-1 is dispensable for initial generation and outgrowth of 5-HT neurons, but required for their survival.
Since also in some other neurons Munc18-1 is deleted, it is unclear whether the observed phenotypes can be attributed to the degeneration of the 5-HT system. In Tphz2 KO mice, 5-HT neurons are completely devoid of 5-HT, yet there are no alterations in 5-HT neuron morphology and 5-HT neurite distribution (Gutknecht et al., 2008). Although these mice can survive into adulthood, approximately 50% does not survive the first 4 weeks and surviving mice have growth retardation (Alenina et al., 2009). Additionally, in a conditional Lmx1b KO mouse, 5-HT neurons are initially formed but almost all central 5-HT neurons fail to survive and brain 5-HT levels are reduced to ~10% (Zhao et al., 2006). Remarkably, these mice display normal locomotor behaviour and show no abnormalities in gross brain morphology. Recently, it was shown that these mice have severe apnea and have a ~20% mortality rate during the neonatal period, although the majority of these mice survive beyond P28 (Hodges et al., 2008; Hodges et al., 2009).
Apparently, even the absence of central 5-HT synthesis or an almost complete removal of 5-HT neurons does not result in a severe (postnatal) lethality phenotype. Thus, although several of these mice display high (postnatal) mortality, the phenotypes observed are not as severe as in the SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} mice.
On the other hand, mice which lack GAP43, display disrupted barrel cortex formation and thalamocortical connections fail to form. These mice display a
postnatal lethality phenotype, with 50% of homozygotes which die between P0 and P2, and >95% of homozygotes which die before P21 (Maier et al., 1999). Although these mutant mice display a reduced 5-HT innervation in the cortex and hippocampus, they have no significant difference in the number of 5-HT neurons, and other areas such as the piriform cortex and amygdale receive normal 5-HT innervation (Donovan et al., 2002). Thus, based on these data it is unclear whether the postnatal lethality phenotype observed in this study is attributable to a degeneration of thalamocortical or 5-HT neurons. It would be very interesting to delete the Munc18-1 gene specifically in 5-HT neurons using the Pet-Cre transgenic line, in which expression of Cre is restricted to 5-HT neurons (Scott et al., 2005). This approach could be used to investigate whether the phenotype we observed could indeed be attributed to degeneration of the 5-HT system rather than degeneration of thalamocortical neurons.
We have shown that mice in which one allele of Munc18-1 is deleted in SERT expressing neurons did not display altered basal, conditioning or anxiety-related behaviour. In these mice number of 5-HT neurons and 5-HT neurite density was indistinguishable from control mice. However, we have shown previously in neurons cultured from Munc18-1 heterozygous mice that there is a subtle secretion phenotype (Toonen et al., 2006). Thus, one could argue that possibly deletion of one functional allele of Munc18-1 affects 5-HT release and thus efficacy of 5-HT neurotransmission.
There could be a few explanations why reduced 5-HT neurotransmission in the heterozygous mice used in this study does not affect behaviour. First of all, the reduction in 5-HT neurotransmission might just be too subtle to affect mouse behaviour. In Munc18-1\(^{-/-}\) neuronal cultures, there is a ~25% reduction in synaptic vesicle release (Toonen et al., 2006). In contrast, expression of tetanus toxin light chain in 5-HT neurons in vivo results in a robust (>90%) inhibition in 5-HT neurotransmission and these mice display decreased anxiety-related behaviour and enhanced associative learning (Kim et al., 2009). This suggests that a substantial reduction in 5-HT release is required to affect behaviour and that deletion of only one allele of Munc18-1 is not sufficient to achieve this reduction in 5-HT release.
Secondly, in Munc18-1 null mice synaptic vesicle release is completely abolished. However, at least in chromaffin cells lacking Munc18-1, there is still some large dense core vesicle (LDCV) release, although this is reduced to ~10% (Voets et al., 2001). In Munc18-1 heterozygous mice LDCV release is presumably largely unaffected. In the 5-HT system, the bulk amount of 5-HT is released from LDCVs, which contain up to ~17 times as much 5-HT molecules as a single synaptic vesicle (Bruns and Jahn, 1995). Thus, if 5-HT released from synaptic vesicles is more severely affected than 5-HT released from LDCVs, there is only a
minor reduction in release of 5-HT and thus in efficacy of 5-HT neurotransmission. Finally, (minor) reductions in extracellular 5-HT levels might be compensated for, such as by reduced SERT levels at the synaptic membrane and/or increased expression of postsynaptic 5-HT receptors, to guarantee proper levels of 5-HT neurotransmission.
In conclusion, deletion of one allele of Munc18-1 in 5-HT neurons does not affect mouse behaviour presumably due to the fact that there is a too subtle reduction in 5-HT neurotransmission in these mice. However, deletion of both alleles of Munc18-1 in SERT expressing neurons results in a rapid degeneration of the 5-HT system and postnatal lethality.
**EXPERIMENTAL PROCEDURES**
*Laboratory animals*
Generation and characterization of SERT-Cre mice has been described (Zhuang et al., 2005). Briefly, immediately upstream of the SERT translational start codon a cassette containing Cre and a FRT-flanked neomycin cassette was inserted. To generate conditional Munc18-1 mice (Heeroma et al., 2004), floxed Munc18-1 mice were generated by insertion of LoxP sites flanking the second exon using homologous recombination. To obtain SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} mice, SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/wt} mice were crossed with SERT-Cre\textsuperscript{wt/wt} Munc18-1\textsuperscript{lox/wt} mice. Pregnant females were sacrificed by cervical dislocation. Mouse embryos were obtained by caesarean section of pregnant females from timed matings. To investigate the effect of deletion of Munc18-1 in the 5-HT system on the development of the 5-HT system, SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{lox/lox} mice were used. SERT-Cre\textsuperscript{wt/wt} Munc18-1\textsuperscript{lox/lox}, SERT-Cre\textsuperscript{wt/wt} Munc18-1\textsuperscript{lox/wt}, or SERT-Cre\textsuperscript{cre/wt} Munc18-1\textsuperscript{wt/wt} littermates were not different from SERT-Cre\textsuperscript{wt/wt} Munc18-1\textsuperscript{wt/wt} mice and were used as controls. SERT-Cre mice were genotyped as has been previously described (Zhuang et al., 2005). For genotyping of Munc18\textsuperscript{lox/lox} mice the following primers were used: 5’-ttgtggtcgaatggccagtag-3’, 5’-cctgtatgggtactgttcgttcaactaaataa-3’ and 5’-tctgaaccttgaggccagtcgagacacag-3’. Animals were housed and bred according to institutional and Dutch guidelines.
*Immunohistochemistry*
For immunohistochemical analysis of 5-HT, embryonic mouse brains were dissected and immediately fixed by immersion in 4% PFA in phosphate buffered saline (PBS, pH 7.4). For analysis of 5-HT neurons and projections in adult brains, mice were anaesthetized and transcardially perfused with 4% PFA in PBS. For post-fixation, brains were incubated in 4% PFA in PBS overnight. For cryoprotection, brains were incubated in increasing concentrations of sucrose.
Subsequently, coronal slices of 40 µm were made. For immunohistochemistry, brain slices were first incubated in blocking buffer containing PBS supplemented with 0.5% Triton X-100 and 10% normal goat serum for two hours. Subsequently, slices were incubated overnight with primary antibodies in PBS containing 0.5% Triton X-100 at 4°C in under gentle agitation. The next day, the slices were washed three times two hours in PBS and incubated with secondary antibodies in PBS for one hour under gentle agitation. Finally, slices were washed again three times two hours in PBS and mounted in Dabco-Mowiol for analysis. All procedures were performed at room temperature unless otherwise stated. Polyclonal anti-5-HT (1:1000) (Immunostar/Diasorin) was used as the primary antibody, and as a secondary antibody GAR-546 (1:1000) was used (Invitrogen). To analyze number of 5-HT cell bodies in DRN sections, at least three sections per experimental group were imaged using a CLSM 510 microscope. Number of 5-HT cell bodies was manually counted in the slices. For analysis of 5-HT fiber density in DRN and cortex a Z-stack of at least three positions per group was made. To analyze the 5-HT fiber density, the Z-stacks were projected in a single image and binarized. The 5-HT fiber density was quantified as the area in the image occupied by 5-HT neurites compared to the total area.
Mouse behaviour
Behavioural analysis of mice was tested in the Phenotyper® (Noldus Information Technology, Wageningen, The Netherlands). For the home cage test, one animal per cage was tested for six days. Eleven SERT-Cre\textsuperscript{wt/wt} Munc18-1\textsuperscript{lox/wt} mice were used in the experimental group and eleven SERT-Cre\textsuperscript{wt/wt} Munc18-1\textsuperscript{lox/wt} mice were used as a control group. Each cage contains a top unit equipped with built-in hardware for videotracking, containing a digital infrared sensitive video camera and infrared lights. Using this method the animal behaviour can be continuously assessed, both in the light and in the dark phase. Ethovision 3.0 software was used as videotracking software. The phenotyper cages (30x30x35 cm) were made of transparent Perspex walls with an aluminium floor, and a feeding station and a water bottle attached to the outside. To the waterspout a lickometer was attached to determine the total duration the animal is touching the waterspout. A shelter unit (diameter: 9 cm, height: 10 cm, non-transparent material) was placed in the cage, which was defined as a hidden zone in Ethovision, and the software program could distinguish between in the shelter and on the shelter. Videotracking was performed at a rate of 12.5 samples per second and the following parameters were determined: average velocity of movement, total distance moved in the open space, proportion of time spent in the feeder, proportion of time spent in the shelter, and time spent drinking.
These parameters were calculated in two hour bins for both the dark and light phase. For the analysis of movement and velocity, only movement was scored when the mouse moved at a velocity of 3.5 cm/s and higher, averaged over twelve samples. Furthermore, total proportion of time spent in a zone of the home cage (on shelter, in shelter, feeder, pellet or waterspout), frequency of zone visits and frequency on the shelter during the dark period was quantified.
On day four and five a free operant conditioning task was performed as follows. The number of SMBs was quantified, which is a directed movement of the animal from one point in the cage to another. A distinction is made between raw and directional SMBs; When the animal moves with a higher velocity than the threshold value, which is determined by dividing the total distance moved by the total duration of movement, the SMB is considered to be a directional SMB. When the animal is moving at a lower velocity, it is considered as a raw SMB. Thus, in a directional SMB the animal is either moving with a greater velocity, or is taking a shorter route. At day four and five, when the mice gets on its shelter, this will result in the drop of a sucrose pellet. Thus, the mouse ‘learns’ that it has to jump onto its shelter in order to receive a new sucrose pellet.
One day six a one trial anxiety test was performed as follows. Fifteen minutes after the change from light to dark phase, a lightspot is on the feeder, thus creating a lightspot zone. The lightspot lasts for three hours, and the total amount of time the animal is in the lightspot zone in this period is recorded and compared with a baseline reference. The baseline reference is acquired by recording the amount of time the animal is in the feeder zone at day five at the corresponding hours, without a lightspot on.
**Statistical analysis**
Data were analyzed using SPSS 17.0. For analysis of number of 5-HT cell bodies in DRN and 5-HT neurite density in DRN and cortex, data was analyzed using the independent samples t-test. To analyze whether observed genotype frequencies differed significantly from a Mendelian ratio, a one-way chi-square test was performed. Mouse behaviour parameters were analyzed using the non-parametric Wilcoxon test after checking for normal distribution. Data shown are mean ± SEM. Significance level was set at p <0.05.
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Chapter 7
Towards a genetic approach to study serotonergic outgrowth and connectivity in vivo
J.J. Dudok\textsuperscript{1}, A.J.A. Groffen\textsuperscript{1}, J. Wortel\textsuperscript{1} and M. Verhage\textsuperscript{1}
\textsuperscript{1}Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, the Netherlands
Abstract
Altered connectivity of the serotonin (5-HT) system might underlie psychopathological processes such as anxiety and depression. Therefore, it would be very interesting to be able to visualize all 5-HT fibers, axons or presynaptic terminals/varicosities in living mouse brain. To specifically label 5-HT neurons and fibers, our aim is to express Cre recombinase in the 5-HT system. We describe here the generation of a tryptophan hydroxylase 2 (Tph2)-iCre targeting construct, the targeting of mouse embryonic stem (ES) cells and the blastocyst injections of positively targeted clones. Moreover, to study 5-HT connectivity in vivo, we generated several transgenic lines using the loxSTOPlox (LSL) methodology.
Eventually, we hope to use these techniques to create mice in which specifically in the 5-HT system reporter genes are expressed, so that these can be used to study the 5-HT outgrowth and connectivity in vivo.
Introduction
The 5-HT system has its cell bodies in the midbrain raphe nuclei. Although only few 5-HT neurons are present, virtually every brain area receives dense 5-HT innervation which triggers signaling pathways through several different 5-HT receptors. For these reasons the 5-HT system influences a wide variety of processes. Moreover, the 5-HT system is implicated in major depressive disorder and anxiety. One of the current hypotheses for psychopathological processes such as anxiety and depression is that 5-HT connectivity might be affected. Therefore, it would be very interesting to study the connectivity of the 5-HT system in vivo.
To study 5-HT outgrowth and connectivity in vivo, our aim is to generate a mouse line with expression of improved Cre recombinase (iCre) specifically in 5-HT neurons. As locus for the iCre knockin, we selected the Tph2 gene, which is selectively expressed in the 5-HT neurons in the raphe nuclei (Patel et al., 2004; Zhang et al., 2005; Clark et al., 2006). To be able to specifically express transgenes in the 5-HT system, we relied on the Cre-loxP technique (Tsien et al., 1996; Nagy, 2000). We selected to use iCre instead of Cre, since iCre gives improved Cre expression due to the application of mammalian codon usage (Shimshek et al., 2002).
In addition we generated several transgenic lines that carry a ubiquitous promoter, a LSL sequence and synaptically or axonally targeted fluorescent fusion proteins. Our ultimate goal is to combine the Tph2-iCre knockin allele with these transgenes, which would constitute an ideal model to study 5-HT
connectivity and dynamics in the living mouse brain. Here we describe the generation of the Tph2-iCre targeting construct, and the generation of Tph2-iCre chimaeric mice. Additionally, we describe the generation of several LSL transgenic lines.
Results
Construction and design of the Tph2-iCre targeting construct
In order to specifically express iCre in 5-HT neurons, we selected the locus for Tph2 which is localized at chromosome 10 D2. Tph2 is the rate limiting enzyme in 5-HT synthesis and is specifically expressed in 5-HT neurons in the brain. To generate mice with expression of iCre specifically in 5-HT neurons, our approach was to generate a targeting construct in which iCre cDNA is inserted at the endogenous Tph2 start codon. We designed the targeting construct in a way that the 1.2 kb short arm at the 5’ side of the construct stops just upstream of the endogenous Tph2 start codon. The 5.5 kb long arm was designed to start 53 bp downstream of the short arm, so that only 53 bp of endogenous DNA, including the endogenous Tph2 start codon, was deleted in case of homologous recombination (Fig. 1A). In between the short and long arm the iCreVenus cDNA was inserted and a neomycin phosphotransferase cassette (neo) for positive selection, flanked by FRT sites. These can be used to later on remove the neo cassette using the flippase/FRT system (O’Gorman et al., 1991).
In case of homologous recombination of the targeting construct in ES cells and subsequent generation of Tph2-iCre mice our approach was to use heterozygous Tph2-iCre mice. These contain one functional allele with Tph2 expression, whereas in the other allele, part of the first exon of Tph2 including the start codon is replaced for iCreVenus. In this case these mice will still have a functional Tph2 allele and expression of iCreVenus from the Tph2 promoter (Fig. 1B).
Generation of Tph2-iCre chimaeric mice
After designing and constructing the Tph2-iCre targeting construct, the construct was sequence verified. This showed that the construct had been correctly made and that the iCreVenus cDNA contained no mutations. The linearized targeting construct was used for ES cell transfection. The antibiotic G418 was used for positive selection of the neomycin resistance marker (Gossler et al., 1986). Eventually five clones were found in which homologous recombination had occurred. Blastocyst injection of clone 1C11 resulted in several chimaeric mice. Although we performed several rounds of chimaeric breeding, we have unfortunately not achieved germline transmission of the Tph2-iCre allele until now.
**Figure 1. Tph2-Cre knockin approach**
(A) The targeting construct was created to replace the start codon of the first exon of Tph2 with a iCre cassette and a neomycin resistance cassette flanked with FRT sites. The targeting construct consisted of a short arm of 1.2 kb, a long arm of 5.5 kb and a iCreVenus and neo cassette. In case of homologous recombination only 53 bp of endogenous DNA is deleted and replaced for the iCreVenus cassette and the neomycin resistance cassette. To identify ES cell clones in which homologous recombination occurred, a PCR screening approach was used. A forward primer (arrow) outside the short arm of the targeting construct and a reverse primer (arrow) in the iCre cassette were used to amplify a 1.5 kb fragment of DNA in case of homologous recombination (dotted line). (B) Approach for Southern blot screening of ES cell clones. An inside and an outside probe were used (grey boxes). For the inside probe, genomic DNA is digested with BsrGI. This will result in a 5.3 kb fragment in WT DNA, but in a 3.3 kb fragment in clones in which homologous recombination occurred due to the presence of an additional BsrGI site in the targeting construct. For the outside probe, genomic DNA is digested with SwaI, which will result in a 14.2 kb fragment in WT DNA, but in a 9.8 kb fragment in clones in which homologous recombination occurred, due to the presence of an additional SwaI site in the targeting construct.
**Generation of LSL transgene lines**
For construction of LSL transgene lines we used several marker genes tagged with either EGFP or mCherry, which is an improved variant of mRFP that matures more completely and is more photostable (Shaner et al., 2004). As marker genes we used SynaptophysinpHluorin (SypHy), TMSyxEGFP and TMSyxmCherry, which all specifically label axons and presynaptic terminals, and VenusKras4b which is a general membrane marker.
**Figure 2. Validation of transgene constructs**
To investigate whether the LSL approach worked, we expressed LSL transgene construct with or without iCre cDNA in HEK293 cells. (A,B) No Expression of SypHy or TMSyxEGFP in HEK293 cells after transfection of LSL-SypHy or LSL-TMSyxEGFP without iCre. (C,D) After transfection of LSL-SypHy or LSL-TMSyxEGFP with iCre, SypHy and TMSyxEGFP were expressed in HEK293 cells respectively. (E,F) In neurons, VenusKras4b or TMSyxEGFP are not expressed when transfected without iCre. (G) Upon transfection of LSL-VenusKras4b with iCre, VenusKras4b is expressed in neurons, and the membrane is labeled, also in growth cones (inset). (H) Upon transfection of LSL-TMSyxEGFP with iCre, TMSyxEGFP is expressed in axons, as shown by the absence of MAP2 staining. Immunocytochemistry for the synaptic marker Synapsin shows that there is colocalization between TMSyxEGFP and Synapsin, showing that TMSyxEGFP is targeted to synapses. Scale bars: A-G 10 µm, inset in G 2 µm, H 5 µm.
SypHy is the improved variant of Synaptophysin and detects secretion events due to the increase in pH in synaptic vesicles after exposure to the extracellular medium (Miesenbock et al., 1998; Granseth et al., 2006). TMSyxEGFP contains part of the transmembrane domain of SyntaxinA coupled to EGFP; TMSyxmCherry is the same but with a red fluorophore. VenusKras4b is the EYFP derived Venus fused with the 20 amino acid C-terminal plasma membrane targeting domain of Kras4b (Welman et al., 2000). As ubiquitous promoter either the chicken β-actin promoter or the Thy1 promoter was used (Aigner et al., 1995; Ludin et al., 1996). In between the promoter and the transgene, we inserted a floxed stop sequence in order to acquire iCre mediated transgene expression (Lakso et al., 1992). Some of the constructs were expressed in heterologous cells or neurons to test whether the floxed stop sequence prevented expression and whether co-transfection of iCre resulted in expression of the transgene. Transfection of β-actin-LSL-SypHy or Thy1-LSL-TMSyxEGFP alone in heterologous cells did not result in expression of the transgene (Fig. 2A,B). In contrast, after co-transfection with iCre, there was strong expression of
both transgenes (Fig. 2C,D). Also in neurons, expression of β-actin-LSL-VenusKras4b or Thy1-LSL-TMSyxEGFP alone did not result in detectable transgene expression (Fig. 2E,F). However, after co-expression with iCre, VenusKras4b expression was detected (Fig. 2G). After co-expression of iCre and Thy1-LSL-TMSyxEGFP neurons were found which expressed TMSyxEGFP and this localized exclusively to axons and synapses as shown by the absence of staining for the dendritic marker MAP2 and co-localization with the synaptic marker Synapsin (Fig. 2H).
These transgenic constructs were linearized and injected in oocytes to generate seven founder lines of β-actin-LSL-SypHy, five founder lines of β-actin-LSL-VenusKras4b, five founder lines of Thy1-LSL-TMSyxEGFP and eleven founder lines of Thy1-LSL-TMSyxmCherry.
**Screening of expression of transgenes in founder lines**
In order to screen the expression of the transgenes per founder line, and to find out whether there are lines with expression of the transgene in the raphe nuclei, we first removed the LSL cassette by crossing the founder lines with the CMV-Cre mouse line, a line in which Cre is ubiquitously expressed (Schwenk et al., 1995).
This breeding strategy resulted in mice in which the LSL cassette was removed in all cells (Fig. 3A). This in contrast to crossing with Tph2-iCre mice, which is expected to result in removal of the LSL cassette only and specifically in 5-HT neurons. Thus, in 5-HT neurons the transgene will be expressed, whereas in all other cells the LSL is still present and the transgene is not expressed (Fig. 3B).
After screening all transgenic founder lines, we found several lines in which the transgene was very low and mosaically expressed. For β-actin-LSL-SypHy we found one founder line with low expression in the Purkinje cell layer of the cerebellum, and two founder lines with expression in the hippocampal CA1-CA3 region and dentate gyrus (data not shown). For β-actin-LSL-VenusKras4b we only found very low and mosaic expression of the transgene (data not shown). For Thy1-LSL-TMSyxEGFP and TMSyxmCherry, we found one line with TMSyxEGFP ubiquitously expressed in neocortex, and one line with TMSyxmCherry mosaically expressed in cortex and hippocampus (data not shown). Unfortunately, not one founder line did show expression of the transgene in midbrain or raphe nuclei areas.
**Discussion**
Here we have described the generation of Tph2-iCre chimaeric mice and several LSL transgenic mouse lines. We would like to eventually create mice with expression of a marker gene specifically in 5-HT neurons. This would allow us to
Figure 3. Approach for screening expression pattern of transgenic mice
(A) To screen the expression pattern of LSL transgenic mice, these mice were crossed with a CMV-Cre mouse, which ubiquitously expressed Cre recombinase. This results in mice in which the LSL is removed in all cells, thus allowing an analysis of transgene expression pattern. (B) Once LSL mice are identified with expression in the 5-HT system, they can be used to cross with Tph2-Cre mice to drive transgene expression specifically in the 5-HT system.
We choose to target iCreVenus to the first exon of Tph2, so that the endogenous start codon and small part of the first exon of Tph2 is replaced for iCreVenus. In this way we anticipated that Tph2 expression relies on endogenous Tph2 promoter activity and is specifically expressed in 5-HT neurons in which Tph2 is expressed. In contrast to this approach we could also have chosen to target iCreVenus to the 3’ side of the Tph2 open reading frame using an internal ribosomal entry site (IRES). We did, however, not use this approach because the expression of a reporter gene behind an IRES is often rather low. Although using our approach we sacrifice one functional Tph2 allele, we expect that this will not significantly affect brain development and behaviour in Tph2 heterozygotes. Deletion of both Tyrosine hydroxylase alleles results in mid-gestational lethality.
However, TH heterozygotes showed no obvious anatomical or behavioural defects (Zhou et al., 1995). Therefore, we reason that our approach will not significantly affect brain development or behaviour.
Using a transgenic approach with a LSL cassette to allow transgene expression only in cells in which iCre is also present, we aimed to express these marker genes specifically in 5-HT neurons. However, none of the founder lines we screened showed expression of the transgene construct in the midbrain or raphe nuclei. It has been shown for both the chicken β-actin and mouse Thy1 promoter that these are able to induce expression of transgenes in the cortex and hippocampus (Feng et al., 2000; Roelandse et al., 2003). However, it appears more difficult to also induce expression in midbrain and raphe nuclei for these promoters, since none of our transgenic lines expressed the transgene in these regions. Recently, it was shown that a 1.8 kb region immediately upstream the Pet-1 gene is sufficient to acquire 5-HT neuron specific expression of a transgene (Scott et al., 2005a; Scott et al., 2005b). Therefore, in new experiments we could try to rely on the Pet-1 promoter to express transgenes in the 5-HT neurons. Possibly, future experiments with novel blastocyst injections of Tph2-iCre targeted ES clones and transgene injections using the Pet-1 promoter will result in a Tph2-iCre mouse line and transgenic mouse lines with expression of reporter genes in 5-HT neurons. These mouse lines can then be used to create mouse lines with expression of reporter genes specifically in 5-HT neurons to possibly resolve the longstanding question whether psychopathological processes are a result of altered 5-HT network connectivity.
**EXPERIMENTAL PROCEDURES**
*Generation of Tph2-iCre targeting construct*
The Tph2-iCre targeting construct was designed in such a way that in the wildtype Tph2 locus, 53 bp in exon 1 are deleted, including the start codon, and replaced for a iCreVenus cassette. The Tph2-iCre targeting construct was generated as follows. The 1.2 kb short arm upstream the endogenous start codon of Tph2 was generated with PCR primers forward 5’CATGCATAGAAAGTGTCCAG3’ reverse 5’TTCTITCTCAGCAGCAGGGG3’. The 5.5 kb long arm starting 34 bp behind the Tph2 start codon was generated with PCR primers forward 5’GGGCCAGGAGAGGGTTGTCTCTTGGAITICTG3’ and reverse 5’CCGTACATGAGGACTCGGTGAGAGCATCTG3’. Both arms were subcloned in the pGEM-T easy vector (Promega). In the piCreVenus vector the NotI site behind Venus was removed and a linker containing SwaI, BsiWI, MluI and NotI sites was placed in the AflII site behind the polyA tail. The short arm was cloned sticky using EcoRI in front of iCreVenus in the modified iCreVenus vector. A neomycin resistance cassette flanked by FRT sites was removed from
the ploxPFRTNEOFRTloxP vector (a kind gift from Jeroen Pasterkamp, Utrecht) using partial digestion with BamHI and cloned blunt in the unique SpeI site behind the long arm in the pGEM-T easy vector. Finally, the FRTNEOFRT long arm Tph2 was cloned sticky behind the short arm iCreVenus using NotI. The construct was verified by sequence analysis. The construct was linearized with KspI for targeting.
**Targeting ES cells**
In order to target ES cells with the Tph2-iCre targeting construct, approximately $1 \times 10^6$ ES cells (line E14.1) were electroporated with 100 µg of linearized Tph2-Cre targeting construct and plated on murine embryonic feeders. The subsequent day, neomycin selection was started by adding 400 µg/ml G418. After 10 days, non targeted clones had died and 700 surviving clones were picked and individually grown. DNA of all these clones was screened to distinguish between clones in which the targeting construct was randomly inserted and clones in which homologous recombination had occurred.
To select for clones in which homologous recombination occurred, the clones were screened with a nested PCR approach using the following primers: 1st PCR forward 5’ CACTTTTCTTCACACCCITCTC3’ and reverse 5’ CCTGACTTTCATCAGAGGTGGCATC3’ nested PCR forward 5’ TTATTTGTGTGAGACACGTGA3’ and reverse 5’ TTTTGGTGCAACAGTCAGCAG3’ which results in a 1.6 kb PCR fragment in positively targeted clones. From the 700 targeted clones, eventually 5 clones were selected in which homologous recombination occurred designated 2F11, 1A1, 1C11, 4E1 and 2E11. Homologous recombination was verified using a 5’ primer outside the forbidden construct and sequence analysis of this region. ES cell clones 1C11 and 4E1 were injected in blastocysts to generate chimaeric mice.
**Generation of transgenic mice**
In order to generate transgenic mice for conditional expression of reporter genes, the LSL cassette derived from the Pbs302 vector was used (Sauer, 1993). As promoter, the chicken β-actin and regulatory elements of the mouse Thy1 gene were used. As reporter genes, SypHy (a kind gift from Andreas Jeromin), VenusKras4b, TMSyx (transmembrane Syntaxin1A) EGFP and TMSyxmCherry were used. The constructs were cloned as follows. For β-actin-LSL-SypHy, the LSL cassette was excised from the Pbs302 vector using EcoRI and SpeI and cloned blunt in the unique XbaI site in the pβ-actin-SypHy vector. For β-actin-LSL-VenusKras4b, the LSL cassette was excised from the Pbs302 vector using EcoRI and SpeI and cloned blunt in the BglII site in the pVenusKras4b vector. Subsequently, LSL-VenusKras4b was excised using NheI and XbaI and cloned blunt in BlnI and XbaI in pβ-actin-EGFP to replace EGFP. Both β-actin-LSLSypHy and β-actin–LSL-VenusKras4b were linearized with AatII for transgene injection. For Thy1-LSL-TMSyxEGFP, TMSyxEGFP was excised with NheI and SspI and cloned blunt in the unique NotI site in pThy1-LSL (a kind gift from Christiaan Levelt, Amsterdam). To clone TMSyxmCherry, TMSyx was excised from pTMSyxEGFP using NheI and EcoRI and cloned blunt in pmCherry-N3 in the EcoXI site. Then TMSyxmCherry was excised using NheI and SspI and cloned blunt in the NotI site the in pThy1-LSL vector. Both Thy1-LSL-TMSyxEGFP and Thy1-LSL-TMSyxmCherry were linearized with AatII for transgene injection. To create transgenic mice, linearized DNA constructs were injected in the pronucleus of fertilized C57BL/6 oocytes.
**Transfection of transgene plasmids in heterologous cells and neurons**
Transgene plasmids were transfected in HEK293 cells or neurons using Lipofectamine 2000 reagent according to the manufacturer’s protocol. HEK293 cells and neurons were cultured as has been published before (Groffen et al., 2004; de Wit et al., 2006). In HEK293 cells, one day post-transfection expression of transgene constructs was visualized using an inverted microscope fitted with fluorescence filters. In neurons and HEK293 cells, expression of transgene constructs was analyzed using a LSM510 confocal laser scanning microscope (Zeiss, B.V. The Netherlands). Immunocytochemical analysis of neurons was performed as has been described before using monoclonal MAP2 (Chemicon) and polyclonal Synapsin I (E028) as primary antibodies and goat-anti-mouse Alexa 543/644 and goat-anti-rabbit Alexa 543 (Molecular Probes) (de Wit et al., 2006).
**Screening of expression pattern**
In order to investigate the expression pattern of the transgene construct per transgenic line, the founder lines were crossed with the 129-Cre mouse line, which has ubiquitous expression of Cre (Schwenk et al., 1995). Pups positive for Cre and the transgene were used for in vivo paraformaldehyde (PFA) perfusion between two and four months age. For in vivo PFA fixation, mice were anaesthetized and transcardially perfused with 4% PFA dissolved in phosphate buffered saline (PBS) (pH 7.4). After fixation, brains were removed and postfixed in 4% PFA for 24 hours at 4°C. Subsequently, brains were cryoprotected in increasing concentrations of sucrose dissolved in PBS at 4°C. For screening of expression pattern of the transgenes, the fixed and cryoprotected brains were sagitally cut in 30 µm slices using a microtome. In case the EGFP fluorescence was too low, Immunohistochemistry was performed using the free floating method. To this end, slices were first blocked using blocking solution which contained 0.1% Triton X-100 and 10% normal goat serum for two hours under gentle agitation. Slices were incubated overnight at 4°C using primary antibody,
washed three times two hours the next day with PBS 0.1% Triton X-100 and incubated with the secondary antibody for two hours. After washing for three times two hours in PBS, slices were positioned on a glass slide, allowed to dry and overlaid with a glass coverslip using Dabco Mowiol. All reactions were carried out at room temperature unless otherwise stated.
**Analysis of transgene expression**
To analyze the expression pattern of the transgene constructs, brain slices were analyzed using an inverted microscope equipped with fluorescence filters and a Sony camera. A LSM510 confocal laser scanning microscope was used to investigate the expression of the transgene constructs in individual neurons.
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Chapter 8
General discussion
General discussion
The aim of the studies described in this thesis was to investigate different cellular aspects of the outgrowth and connectivity of the brain 5-HT system. The 5-HT system has the remarkable feature that although there are only few 5-HT neurons, localized in the midbrain raphe nuclei, virtually every brain area receives dense 5-HT innervations. These innervations contain several 5-HT varicosities, where 5-HT is released both synaptically and as volume transmission. Due to several 5-HT receptors which are present in the brain, 5-HT has many functions and influences a wide variety of processes. Moreover, the 5-HT system is implicated in several psychopathological processes such as anxiety and depression. Despite the high social and economical importance of these conditions, there is a lot of uncertainty about the involved mechanisms. Therefore, a further cellular characterization of the outgrowth and connectivity of the 5-HT system is of high interest. We used several experimental approaches to study different cellular aspects of the outgrowth and connectivity of the 5-HT system.
In chapter 2 we used organotypic slice cultures to study 5-HT outgrowth and connectivity and the effect of pharmacological manipulations in vitro. This revealed that the 5-HT neurite density was not affected by blocking the SERT or application of a 5-HT$_{1A}$ agonist, but the density was reduced by application of a 5-HT$_2$ agonist. This was presumably due to inhibited outgrowth of the 5-HT system.
In chapter 3, we investigated the trafficking of SERT. We expressed SERT tagged with the fluorescent protein mCherry in hippocampal neurons. We showed that mCherry-SERT is transported in vesicles which displayed a dynamic trafficking.
In chapter 4 we used the same approach as in chapter 3, but now we studied the localization and trafficking of Tph2, the rate limiting enzyme in 5-HT synthesis. We tagged Tph2 with EGFP and studied the localization of Tph2-EGFP in hippocampal neurons. In axons Tph2-EGFP displayed a punctate distribution and co-localized with Synaptophysin-mCherry, a marker for synaptic terminals, suggesting that Tph2-EGFP localized to synaptic terminals.
In chapter 5 we studied the effect of a SNP in the presynaptic gene Piccolo on the trafficking and localization of SERT. Knockdown of Piccolo in neurons did not affect mCherry-SERT trafficking. The SNP in Piccolo possibly affects the localization of SERT, although future research is needed to further resolve this issue.
In chapter 6 we crossed SERT-Cre mice with floxed Munc18-1 mice to specifically abolish Munc18-1 in SERT expressing neurons. This resulted in early postnatal lethality and a rapid degeneration of the 5-HT system. We used heterozygous mice to study the effect of removal of one Munc18-1 allele in SERT expressing neurons on behaviour using a phenotyping experiment. Basal behaviour, conditioning and anxiety related behaviour were not affected.
In chapter 7 we describe our approach to create mice which have Cre expression specifically in 5-HT neurons. Moreover, we show that we have generated transgenic lines with a floxed stop cassette which can be used for spatial regulation of transgene expression upon Cre mediated recombination. In the future, these mice can be used to specifically label 5-HT neurons, or to specifically remove a gene in 5-HT neurons.
Roles of 5-HT in outgrowth, connectivity and behaviour
One of the current hypotheses for psychopathological processes such as depression is that altered 5-HT network connectivity might be involved in these processes. Furthermore, it is hypothesized that antidepressants such as fluoxetine might restore aberrant 5-HT network connectivity (Castren, 2005). We used organotypic slices to study these processes in an in vitro model system. Our data showed that fluoxetine did not affect 5-HT innervation density.
In adult rats, chronic fluoxetine application did result in an increased density of 5-HT projections in the forebrain (Zhou et al., 2006). Interestingly, an in vivo study using chronic fluoxetine treatment either during development or during adulthood, showed that when applied during adulthood, fluoxetine did not affect behaviour. In contrast, fluoxetine treatment during development resulted in increased anxiety-related behaviour during adulthood (Ansorge et al., 2004; Ansorge et al., 2008). Our data suggests, although it is in vitro, that this effect of fluoxetine on behaviour does not result from an altered innervation of 5-HT projections. However, in our model system we were unable to investigate whether the number of 5-HT varicosities was affected by fluoxetine application. Thus, although 5-HT projection density is unaffected, connectivity of the 5-HT system might be affected due to an altered number of 5-HT varicosities or an altered rate of 5-HT release.
At least in the snail Helisoma, 5-HT acts as an autotrophic factor on its own outgrowth; 5-HT applied at µM concentrations to the growth cones of 5-HT neurons resulted in an abrupt cessation of growth cone motility and outgrowth (Haydon et al., 1984; Haydon et al., 1987).
The 5-HT\textsubscript{IA} and 5-HT\textsubscript{3C} receptor might mediate this effect of 5-HT, as these receptors are expressed in the DRN (Wright et al., 1995; Clemett et al., 2000). Our data showed that 5-HT\textsubscript{IA} receptor activation slightly increased 5-HT neurite
density whereas chronic activation of the 5-HT\textsubscript{2} receptor decreased 5-HT neurite density. It is tempting to speculate that these receptors act in concert during outgrowth to regulate a proper development of 5-HT projections towards target areas mediated by 5-HT. Interestingly, 5-HT\textsubscript{1A} and 5-HT\textsubscript{2} receptors have different affinities for 5-HT; 5-HT\textsubscript{1A} receptors are high affinity 5-HT receptors which have an EC\textsubscript{50} value in the nM range, whereas 5-HT\textsubscript{2} receptors are low affinity 5-HT receptors, which have an EC\textsubscript{50} value in the \(\mu\)M range (Fargin et al., 1989; Marek and Aghajanian, 1994; Azmitia, 2001). The 5-HT\textsubscript{3C} receptor is present on GABA-ergic cells in the DRN, and it is unknown whether this receptor is present on 5-HT neurons (Serrats et al., 2005). This suggests that the inhibitory effect of 5-HT\textsubscript{3C} receptor activation is not a direct effect on 5-HT neurons, but might be an indirect effect via GABA-ergic neurons. Indeed, in vivo data supports that 5-HT\textsubscript{3C} receptor activation inhibits neuronal firing of 5-HT neurons via GABA-ergic neurons (Boothman et al., 2006). Thus, in the DRN \(\mu\)M levels of 5-HT might inhibit outgrowth, whereas nM levels might stimulate outgrowth of 5-HT innervations.
Mice in which the 5-HT\textsubscript{1A} receptor is deleted have increased anxiety-related behaviour, as they spend less time in the center in the open field test and avoid the open arms in the elevated plus maze (Parks et al., 1998; Ramboz et al., 1998). Overexpression of the 5-HT\textsubscript{1A} receptor, on the other hand, results in reduced anxiety-related behaviour (Deng et al., 2007). A very elegant study was performed by Gross and colleagues, who showed that restoring 5-HT\textsubscript{1A} receptor expression in the forebrain during development, but not during adulthood rescued the behavioural phenotype of 5-HT\textsubscript{1A} KO mice. (Gross et al., 2002). Clearly, the 5-HT\textsubscript{1A} receptor is important during development to establish normal anxiety-like behaviour. Unfortunately, no studies were performed to resolve whether 5-HT connectivity is affected in these mice.
Deletion of the 5-HT\textsubscript{3C} receptor, on the other hand, results in compulsive-like behaviour, and these mice are obese and suffer from seizures (Tecott et al., 1995; Chou-Green et al., 2003). Unfortunately, also for these mice it is not known whether 5-HT connectivity is affected.
**Trafficking of SERT and Tph2 in neurons**
As the 5-HT system has long projections to several brain areas, proteins involved in 5-HT synthesis and reuptake are transported over large distances towards axonal growth cones and varicosities. In chapter 3 and 4 we focused on SERT and Tph2 transport in hippocampal neurons and observed that SERT and Tph2 displayed a distinct distribution. SERT was predominantly localized extrasynaptically and some SERT puncta localized in the vicinity of presynaptic terminals. In contrast, Tph2 accumulated in puncta which colocalized with
synaptic markers. Obviously, Tph2 is required at presynaptic terminals and varicosities for local synthesis of 5-HT. Indeed in 5-HT neurons Tph is enriched in presynaptic terminals (Pickel et al., 1976; Pickel et al., 1977). On the other hand, SERT is localized extra-synaptically to transport extracellular 5-HT which has diffused away from its release sites back into the neuron. An electron microscopy study showed that the majority of SERT in 5-HT neurons was located outside synapses (Zhou et al., 1998). Thus, the distribution of Tph2 and SERT which we found in hippocampal neurons seems to mimic the distribution of endogenous SERT and Tph2 in 5-HT neurons.
We also showed that SERT and Tph2 are transported in distinct ways through the neuron. In contrast to Tph2, SERT displayed a vesicular trafficking. Apparently, SERT contains a signal peptides which mediates the transport in vesicles. Alternatively, SERT contains transmembrane domains, which requires that SERT is transported in vesicles as these lipophilic domains are insoluble.
As SERT is transported in secretory vesicles, there should be one or more motor proteins responsible for this transport. There are several families of motor proteins which are involved in the transport of several different proteins through neurites (for a review see (Schlager and Hoogenraad, 2009). The average velocity of SERT transport vesicles and the fact that these do not display a preference for direction of movement is in accordance with the average velocity of transport vesicles containing glycine (Maas et al., 2006). Also in this study, it was shown that dynein motors are responsible for the retrograde transport of glycine (Maas et al., 2006). Dynein motors are involved in transporting other cargo molecules through neurons, such as the Trkb receptor and Bassoon (Yano et al., 2001; Fejtova et al., 2009). The trafficking dynamics of Trkb and Bassoon are comparable with the trafficking dynamics of SERT, strongly suggesting that the dynein motor protein is responsible for SERT transport (Ha et al., 2008; Fejtova et al., 2009).
**A SNP in Piccolo associates with depression**
Recently, a SNP in Piccolo was found which was associated with depression (Sullivan et al., 2008; Bochdanovits et al., 2009). Piccolo is a structural component of the presynaptic cytoskeletal cytomatrix (PCM) and structurally related to Bassoon, another component of the PCM (Cases-Langhoff et al., 1996; tom Dieck et al., 1998; Fenster et al., 2000). Piccolo is a multidomain zinc finger protein which contains several coiled coil domains and a PDZ domain (Fenster and Garner, 2002). Piccolo also contains two C2 domains, C2A and C2B, of which C2A functions as a low-affinity calcium sensor in presynaptic terminals (Gerber et al., 2001). The SNP in Piccolo was found to be closely localized to the C2A domain (Sullivan et al., 2008).
In the presynaptic terminal, Piccolo is involved in different processes. First of all, Piccolo, together with Bassoon, is involved in presynaptic terminal formation. Piccolo and Bassoon are transported in Piccolo-Bassoon transport vesicles together with other constituents of presynaptic terminals, and two or three of these vesicles are sufficient to form an active synapse (Zhai et al., 2001; Shapira et al., 2003; Tao-Cheng, 2007). In chapter 5 we have shown that 5-HT neurons contain Piccolo. Thus, in 5-HT varicosities Piccolo might be involved in formation of varicosities, regulating 5-HT secretion and regulation of SERT internalization. We have shown that knockdown of Piccolo did not affect SERT trafficking dynamics, suggesting that Piccolo is not involved in SERT transport. Secondly, since Piccolo is present in the presynaptic terminal, Piccolo might be involved in the modulation of synaptic vesicle exocytosis. Indeed, Piccolo influences synaptic function by negatively regulating synaptic vesicle exocytosis, possibly via a calmodulin kinase II-dependent mechanism, mediated through the modulation of Synapsin1A dynamics (Leal-Ortiz et al., 2008). Thus, Piccolo might be involved in modulating 5-HT release from synaptic vesicles. However, the majority of 5-HT is secreted as bulk release from large dense core vesicles (LDCVs) (Bruns and Jahn, 1995). Therefore, although it might be that a SNP in Piccolo could modulate 5-HT release from synaptic vesicles, this would probably only have minor effects on extracellular 5-HT levels.
Thirdly, it was shown in heterologous cells that Piccolo is involved in the regulation of DAT internalization. Specifically, the C2A domain was involved in this process (Cen et al., 2008). As DAT and SERT belong to the same gene family and are structurally related it is conceivable that Piccolo could be involved in SERT internalization. In chapter 5, we have investigated in heterologous cells whether the SNP in the C2A domain of Piccolo alters the localization of mCherry-SERT. This showed that expression of the C2A domain of Piccolo containing the SNP seemed to alter the membrane distribution of SERT compared to the control C2A variant. It would be very interesting to further investigate whether the SNP in the C2A domain affects varicosity density of 5-HT projections or regulation of SERT membrane localization. If so, this would be the first cellular evidence that altered 5-HT network connectivity might be involved in the etiology of depression.
**Deletion of Munc18-1 in SERT expressing neurons results in early postnatal lethality**
Our data in chapter 6 showed that deletion of Munc18-1 in SERT expressing neurons resulted in early postnatal lethality. Although neurons are initially formed and there is normal brain development, deletion of Munc18-1 eventually results in degeneration (Verhage et al., 2000; Heeroma et al., 2003; Korteweg et
Our data showed that also 5-HT neurons in which Munc18-1 is deleted initially develop and grow out projections which innervated the cortex. However, after the initial generation, the 5-HT neurons undergo rapid degeneration, and at P2 only very few 5-HT neurons were left. This is the first mouse model which has a complete removal of the 5-HT system. This in contrast to the Tph2 KO mouse, which has no 5-HT synthesis in the central nervous system, but still 5-HT neurons and projections are unaffected.
Deletion of Tph2 resulted in ~50% lethality in the first four weeks (Alenina et al., 2009). Surprisingly, however, another research group did not find any increased lethality in Tph2−/− mice ((Savelieva et al., 2008) and personal communication). Deletion of Pet1 and Lmx1b results in mice in which ~70% and >99% of 5-HT neurons fail to survive respectively. Concerning the marked reduction in 5-HT neurons and projections in these mice, it is surprising that these mice only display subtle phenotypes (Ding et al., 2003; Hendricks et al., 2003; Zhao et al., 2006)
Unfortunately, as also thalamocortical neurons and some hippocampal neurons briefly express SERT, and thus Cre recombinase, during development, it is difficult to assess whether the observed phenotype is attributable to a degenerating 5-HT system. Therefore, crossing floxed Munc18-1 mice with Pet-Cre or Tph2-Cre mice which express Cre exclusively in 5-HT neurons should finally resolve whether complete removal of the 5-HT system affects brain development and survival.
**Altered connectivity of the 5-HT system in human psychopathological processes?**
Descriptive studies on the effect of psychopathological processes on human 5-HT connectivity rely on postmortem brain tissue. The last decade, however, more and more human imaging studies are performed using functional magnetic resonance imaging (fMRI) and positron emission tomographic (PET) imaging to resolve whether brain circuitry is affected in psychopathological processes associated with the 5-HT system.
Research on postmortem brain material from depressive people who committed suicide predominantly focused on mRNA expression levels or binding of radiolabeled 5-HT to 5-HT receptors. Several studies showed that there is an elevated 5-HT2A and 5-HT2C receptor abundance in the frontal cortex of depressed suicide victims (Arango et al., 1990; Xu and Pandey, 2000; Prasad et al., 2005). Other studies reported either reduced or increased binding or availability of 5-HT1A receptors, depending on the brain region studied (Drevets et al., 2007). From these data, however, it cannot be deduced whether the altered
levels of mRNA expression or altered levels of receptor binding are the result of an alteration in expression levels or of an altered 5-HT projection density.
PET imaging is used to measure cerebral blood flow and rate of glucose metabolism as a measure for brain activity. Functional MRI is used to measure blood oxygen level dependent (BOLD) fluctuations which are a measure for activity and connectivity of brain regions. PET and fMRI studies have reported a decreased activation of cortical areas such as the dorsolateral prefrontal cortex and the anterior cingulate cortex in depressed subjects (Drevets et al., 1997; Mayberg et al., 1999). On the other hand, other studies reported an increased activation of limbic areas such as the amygdala in depression (Sheline et al., 2001; Siegle et al., 2002). Several studies have explored whether antidepressants restore aberrant activation patterns observed in depression. Antidepressant treatment increases glucose metabolism in prefrontal cortex and reduces activity in limbic areas (Sheline et al., 2001; Harvey et al., 2004b).
Although it is difficult to conclude from these imaging studies if altered brain network connectivity is a cause or consequence of the depression, one could at least speculate that connectivity changes occur at some stage. Clearly, more research is needed to further elucidate whether network connectivity alterations underlie psychopathological processes associated with the 5-HT system.
**Future perspectives**
There are a number of approaches which could be taken to further elucidate the connectivity of the 5-HT system, the relation between the 5-HT system and behaviour, and the involvement of the 5-HT system in psychopathology. First of all, it would be of great interest to label all 5-HT neurons and neurites with fluorescent markers. This could be achieved by creating knockin mice using the Tph2 promoter to drive expression of Cre and to cross these mice with floxed stop fluorescent reporter mice. Additionally, it has already been shown that Pet-Cre mice express Cre specifically in 5-HT neurons, and crossing with reporter mice showed that >99% of 5-HT neurons were labeled (Scott et al., 2005). These mice could then be used to reconstruct the connectivity of the 5-HT system. Secondly, there are several subpopulations of 5-HT neurons that can be distinguished based upon their precursor genes (Jensen et al., 2008). If Cre mouse lines could be made for these specific subpopulations, these could be crossed with reporter mice or with the so-called brainbow mice (Livet et al., 2007). It would be very interesting to find out whether different subpopulations of 5-HT neurons innervate different brain regions, and are involved in different aspects of behaviour.
Additionally, these mouse lines could be used to specifically inhibit neurotransmission from these populations of 5-HT neurons, or to inhibit neurotransmission from all 5-HT neurons using Tph2-Cre mice. Expressing tetanus toxin in neurons blocks synaptic activity since tetanus toxin cleaves VAMP2 which is a protein necessary for vesicle fusion. It has been shown that this approach works in vivo, e.g. in the retina, but also for 5-HT neurons (Kerschensteiner et al., 2009; Kim et al., 2009). The work of Kim et al showed that silencing a subset of 5-HT neurons resulted in mice with behavioural alterations (Kim et al., 2009). These mice could be used to further explore the role of the 5-HT system and subsets of the 5-HT system on behaviour and to what extend these contribute to pathophysiological processes such as anxiety and depression. The other way around, these mice could be used to study whether silencing of 5-HT neurons affects 5-HT connectivity, i.e. 5-HT projection and varicosity density.
A third interesting future direction is to use mice which express Cre specifically in 5-HT neurons to inactivate 5-HT autoreceptors only in 5-HT neurons. A lot of research has been conducted in elucidating the role of 5-HT autoreceptors in determining 5-HT transmission and behaviour. The 5-HT\textsubscript{IA} and 5-HT\textsubscript{IB} knockout mice display altered anxiety-related and aggression-related behaviours. However, in these mice it is difficult to assess whether these phenotypes observed are due to deletion of 5-HT autoreceptors, or deletion of these receptors from non-5-HT neurons. A far more elegant approach, however, would be to generate floxed 5-HT\textsubscript{IA} and 5-HT\textsubscript{IB} mice and cross these with Pet-Cre or Tph2-Cre mice to selectively inactivate these receptors only in 5-HT neurons. This will allow to distinguish between the role of 5-HT autoreceptors and heteroreceptors, and to investigate to what extend the deletion of 5-HT autoreceptors affects mouse behaviour.
Recently, also a tamoxifen inducible Tph2-Cre mouse line has been generated, which allows to temporally regulate Cre expression in 5-HT neurons (Weber et al., 2009). This mouse line allows to investigate whether deleting a gene in the 5-HT system either during development or during adulthood affects 5-HT outgrowth, connectivity or mouse behaviour. Finally, it would be interesting to further investigate whether connectivity is affected in anxiety and depression in human subjects. Novel brain imaging techniques, such as pharmaco MRI are being developed to further explore this relationship (Windischberger et al.)
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Nederlandse samenvatting
Cellulaire karakterisatie van de uitgroei en connectiviteit van het serotonine systeem in het brein
Nederlandse samenvatting
De menselijke hersenen bestaan uit ongeveer 100 miljard zenuwcellen (neuronen). Neuronen zijn de basis elementen die bijvoorbeeld gedrag en geheugen mogelijk maken. Neuronen hebben een aantal uitlopers waarmee ze met elkaar kunnen communiceren. Een neuron heeft een axon, waarmee het signalen naar een ander neuron kan versturen, en meerdere dendrieten, waarmee een neuron signalen van andere neuronen kan ontvangen. Neuronen communiceren met elkaar door het uitscheiden van signaalstoffen, neurotransmitters genaamd. Deze neurotransmitters kunnen binden aan bepaalde receptoren in een ontvangende cel, waardoor een signaal doorgegeven kan worden. De punten waar twee cellen met elkaar communiceren worden synapsen genoemd. Hier wordt door de zendende cel een presynaptische terminal gevormd op de ontvangende cel. Vanuit deze presynaptische terminal worden de neurotransmitters uitgescheiden, en deze worden opgevangen door receptoren in de postsynaptische terminal in de ontvangende cel.
Er zijn veel verschillende soorten neuronen, deze worden onder andere in groepen ingedeeld door de neurotransmitter die ze uitscheiden. Een van de groepen neuronen zijn de zogenaamde serotonerge neuronen, neuronen die de neurotransmitter serotonine uitscheiden. Deze neuronen zijn in een aantal clusters gelokaliseerd in de hersenstam. In tegenstelling tot vele andere groepen neuronen, hebben de serotonerge neuronen een aantal opvallende kenmerken. Ten eerste is het een erg kleine groep neuronen, in de menselijke hersenen gaat het ongeveer om een half miljoen cellen. Ten tweede zijn ondanks dit kleine aantal in bijna alle andere hersengedeelten uitlopers van deze serotonerge neuronen aanwezig. Ten derde wordt deze neurotransmitter in deze hersengebieden in grote hoeveelheden uitgescheiden, waardoor grote groepen andere neuronen door deze serotonerge neuronen beïnvloed kunnen worden. Ten vierde zijn deze serotonerge neuronen, het zogenaamde serotonerge systeem, betrokken bij het beïnvloeden van verschillende soorten gedrag. Als laatste is het serotonerge systeem betrokken bij aandoeningen zoals depressiviteit, angststoornissen, autisme en obsessief compulsief gedrag. Een welbekend antidepressivum zoals Prozac grijpt bijvoorbeeld aan op de serotonerge neuronen, door de heropname van uitgescheiden serotonine terug het neuron in te blokkeren.
Het idee achter aandoeningen zoals depressie is dat er een verandering ontstaat in de uitlopers van deze serotonerge neuronen naar andere hersengebieden en dat antidepressiva de uitlopers weer in de oude, gezonde staat terugbrengen. In dit proefschrift hebben we op een aantal verschillende
manieren geprobeerd te kijken naar de dynamiek van de uitgroei van deze serotonerge neuronen.
In hoofdstuk 2 hebben we uit muizenhersenen, die we als modelsysteem hebben gebruikt, de serotonerge neuronen gesneden in een hersenplakje, een zogenaamde organotypische slice. Dit plakje hebben we naast een plakje van een ander hersengebied, de hippocampus, gelegd om te kijken of in dit kweeksysteem de serotonerge neuronen uitlopers vormden het hippocampale plakje in. Inderdaad bleken de uitlopers van de serotonerge neuronen binnen vier dagen het andere plakje in te groeien. Vervolgens hebben we onderzocht of door toediening van stoffen zoals het hierboven genoemde Prozac de uitgroei van de serotonerge neuronen geremd of juist gestimuleerd werd. Voor de stof Prozac bleek dat het toedienen geen effect had op de uitgroei van de serotonerge neuronen. Echter, toediening van een andere stof die aan een bepaalde serotonerge receptor bindt, bleek een effect te hebben op de serotonerge uitlopers. Na zeven dagen waren er namelijk minder serotonerge uitlopers in het hippocampale plakje dan zonder de toediening van deze stof. Hieruit blijkt dat chronische activatie van deze serotonerge receptor leidt tot een remming in de uitgroei van de serotonerge neuronen.
In hoofdstuk 3 hebben we een belangrijk eiwit in serotonerge neuronen, het eiwit dat zorgt voor heropname van serotoninine in het neuron na afgifte (de serotoninine heropname transporter, SERT) voorzien van een rood fluorescerend eiwit (mCherry). Dit zogenaamde fusie-eiwit, mCherry-SERT genaamd, hebben we tot expressie gebracht in neuronen om te onderzoeken hoe de verdeling van dit eiwit er in neuronen uitziet. Het bleek dat dit eiwit in het neuron aanwezig was in vesicles. Dit zijn pakketjes waarmee eiwitten in een neuron getransporteerd worden. Vervolgens hebben we door middel van microscopie ‘live’ naar de beweging van pakketjes mCherry-SERT gekeken. Uit de analyse van de snelheid en de richting van de beweging bleek dat de pakketjes zich voortbewogen met een gemiddelde snelheid van 1.2 µm/s (=4.32 mm/uur) en dat de pakketjes geen voorkeur hadden voor de richting waarin ze zich voortbewogen in de uitlopers.
In hoofdstuk 4 hebben we een soortgelijke aanpak gekozen als in hoofdstuk 3. Echter, nu hebben we gekeken naar het eiwit dat belangrijk is voor de aanmaak van serotoninine, tryptophan hydroxylase 2 (Tph2). Dit eiwit hebben we voorzien van een groen fluorescerend eiwit, (Enhanced Green Fluorescent Protein, EGFP). Dit fusie-eiwit hebben we tot expressie gebracht in neuronen om de verdeling van dit eiwit, ook in vergelijking met mCherry-SERT, te onderzoeken. Hieruit bleek dat, in tegenstelling tot mCherry-SERT, Tph2-EGFP zich door de hele cel bevond, en niet in vesicles zat. In jonge neuronen had Tph2-EGFP geen voorkeur voor een bepaald deel van de cel. In oudere neuronen echter, bleek dat Tph2-EGFP voornamelijk aanwezig was in presynaptische
terminals, maar tegelijkertijd ook in de dendrieten. Ook in deze cellen hebben we door middel van microscopie live naar de beweging van Tph2-EGFP gekeken. Hieruit bleek dat Tph2-EGFP, in tegenstelling tot mCherry-SERT, zich niet in vesicles bevond en daardoor ook geen beweging liet zien.
In hoofdstuk 5 hebben we onderzocht of een variatie in een gen dat codeert voor het eiwit Piccolo, effect heeft op de verdeling en dynamiek van het eiwit SERT. Deze variatie in het gen wordt geassocieerd met depressie, waardoor het erg interessant is om te onderzoeken of de variatie in dit gen verantwoordelijk is voor een verandering in de dynamiek van het serotonine systeem.
Hiertoe hebben we eerst onderzocht of het eiwit Piccolo voorkwam in de serotonerge neuronen en uitlopers, en dit bleek inderdaad het geval. Vervolgens hebben we een zogenaamde knockdown construct gemaakt waarmee de hoeveelheid Piccolo in een cel grotendeels gereduceerd kan worden. Na het tot expressie brengen van dit construct in neuronen bleek inderdaad dat de hoeveelheid Piccolo in een cel met meer dan 80% gereduceerd was. Vervolgens hebben we onderzocht of een vermindering in de hoeveelheid Piccolo van invloed is op de beweging en dynamiek van het eiwit SERT. Om dit te onderzoeken hebben we mCherry-SERT tot expressie gebracht in neuronen waarin de hoeveelheid Piccolo gereduceerd was en hebben we door middel van live microscopie naar de dynamiek van het eiwit mCherry-SERT gekeken. Dit hebben we vergeleken met neuronen waarin de hoeveelheid Piccolo niet gereduceerd was. We concludeerden uit deze vergelijking dat een vermindering in de hoeveelheid Piccolo geen effect heeft op de beweging en snelheid van bewegen van mCherry-SERT. Als laatste hebben we gekeken, in een ander cel type, of de mutante variant van het eiwit Piccolo, in vergelijking met de normale variant de verdeling van het eiwit SERT beïnvloedt. Om dit te onderzoeken hebben we de mutante variant van Piccolo en mCherry-SERT in cellen tot expressie gebracht en gekeken of de verdeling van mCherry-SERT veranderd was. In cellen waarin mCherry-SERT tot expressie gebracht was met de normale variant van Piccolo was mCherry-SERT, zoals verwacht, gelocaliseerd aan het membraan. Na expressie van mCherry-SERT met de mutante variant van Piccolo bleek echter dat de hoeveelheid mCherry-SERT aan het membraan verminderd was. Hieruit concludeerden wij dat mogelijk in serotonerge neuronen de mutante variant van het eiwit Piccolo ervoor zorgt dat er een verandering is in de hoeveelheid SERT aan het membraan. Hierdoor zou de signaaldoorgifte van serotonerge neuronen naar andere neuronen verstoord kunnen zijn.
In hoofdstuk 6 hebben we onderzocht wat het effect is van het uitschakelen van het serotonine systeem in de muis. Om dit te onderzoeken hebben we door muizen te kruisen een muis verkregen waarbij een gen in het serotonine systeem uitgeschakeld is. Uit onderzoek naar de embryonale ontwikkeling van het serotonerge systeem in deze muizen bleek dat het serotonerge systeem in eerste
instantie tot ontwikkeling kwam. De serotonerge neuronen waren aanwezig, en serotonerge vezels groeiden uit naar andere hersengebieden. We hebben het serotonerge systeem ook bestudeerd iets later in de hersenontwikkeling van deze muizen. Dit toonde aan dat de serotonerge neuronen een veranderde morfologie vertoonden, waaruit afgeleid kan worden dat deze neuronen degenereren. Uiteindelijk zijn bijna al de serotonerge neuronen verdwenen. Ook de projecties van deze neuronen verdwenen later tijdens de hersenontwikkeling. Hieruit concludeerden wij dat het uitschakelen van dit gen uiteindelijk leidt tot een verdwijning van de serotonerge neuronen. Vervolgens hebben we gekeken wat het effect hiervan is op de ontwikkeling en het overleven van deze muizen. Dit toonde aan dat de meeste muizen vlak na de geboorte doodgingen. Enkele muizen die wel overleefden waren kleiner dan controle muizen die dit gen nog wel hadden, en gingen uiteindelijk ook binnen drie weken dood. We waren ook geïnteresseerd of het gedrag van de muizen veranderd was. Echter, aangezien de meeste muizen al snel na de geboorte dood gingen, was dit niet mogelijk. Daarom hebben we het gedrag geanalyseerd van muizen die nog een kopie van dit gen hadden, en dit vergeleken met controle muizen. Om het gedrag van deze muizen te analyseren hebben we deze muizen zeven dagen in een speciale kooi gehouden. Deze kooien bevatten een videocamera zodat later een groot aantal parameters geanalyseerd kan worden, zoals de hoeveelheid beweging, hoe vaak deze muizen drinken of hoeveel deze muizen eten. Verder konden we ook nog het angst gerelateerde gedrag en het leergedrag van deze muizen analyseren. Uit analyses van muizen met een kopie van het gen bleek dat deze geen veranderd gedrag vertoonden ten opzichte van controle muizen.
Het doel in hoofdstuk 7 was om een muis te maken waarbij alle serotonerge neuronen en uitlopers naar andere hersengebieden gelabeld waren. Om dit doel te bereiken hebben we een gen gebruikt dat specifiek tot expressie komt in serotonerge neuronen, Tph2. Een stuk DNA voor een gen, een promoter genoemd, is verantwoordelijk voor waar en wanneer het gen afgeschreven wordt. Achter de promoter van Tph2 hebben een eiwit genaamd Cre recombinase gezet, zodat de expressie van Cre recombinase wordt gereguleerd door de Tph2 promoter en zodoende alleen tot expressie komt in serotonerge neuronen. In dit hoofdstuk hebben we beschreven hoe we embryonale stamcellen gemanipuleerd hebben en hoe we deze hebben geïnjecteerd in muizen blastocysten. Helaas is het nog niet gelukt om deze muis te genereren. Om specifiek serotonerge neuronen te labelen hebben we nog een aantal andere muizen gemaakt, waarbij het eiwit alleen aangemaakt wordt als in de cell Cre recombinase tot expressie komt. Door deze muizen met elkaar te kruisen zou Cre recombinase alleen in serotonerge neuronen ‘aan’ staan, en zou alleen in deze neuronen een zogenaamd reporter eiwit aangezet worden. Op deze manier hopen we uiteindelijk muizen te hebben waarbij specifiek in serotonerge neuronen reporter genen tot expressie komen,
waardoor we deze uiteindelijk kunnen gebruiken om in levende muizen naar de dynamische uitgroei en connectiviteit van het serotonerge systeem te kijken. Verder kunnen we dan onderzoeken of manipulatie aan dit systeem leidt tot een verandering in uitgroei van serotonerge neuronen en tot muizen die bepaalde aspecten van humane depressiviteit nabootsen. Aan de andere kant kunnen we symptomen van humane depressiviteit in muizen induceren, en onderzoeken of dit leidt tot veranderingen in het serotonerge systeem. Uiteindelijk zouden we op deze manier ook kunnen onderzoeken of medicatie tegen depressiviteit leidt tot een verandering in de connectiviteit van het serotonerge systeem en deze weer terugbrengt in de oude staat.
| Abbreviation | Description |
|--------------|-------------|
| 5-HIAA | 5-hydroxyindolacetic acid |
| 5-HT | serotonin |
| 5-HT | 5-hydroxytryptamine |
| 8-OH-DPAT | 8-hydroxy-2-(di-n-propylamino)tetraline |
| BOLD | blood oxygen level dependent |
| cAMP | cyclic adenosine monophosphate |
| CSF | cerebrospinal fluid |
| DAT | dopamine transporter |
| dGBSS | dissection Gey’s balanced salt solution |
| DIV | days in vitro |
| DOI hydrochloride | (+)-2,5-dimethoxy-4-iodoamphetamine hydrochloride |
| DRN | dorsal raphe nucleus |
| E | embryonic day |
| EGFP | enhanced green fluorescent protein |
| ES | embryonic stem |
| fMRI | functional magnetic resonance imaging |
| GIRK | G protein-activated inwardly rectifying K⁺ |
| HBSS | Hanks Balanced Salt Solution |
| iCre | improved Cre recombinase |
| IP₃ | inositol 3-phosphate |
| IRES | internal ribosomal entry site |
| KO | knockout |
| l | long |
| LAT₁ | large neutral amino acid transporter |
| LDCV | large dense core vesicle |
| Lmx1 | LIM homeobox transcription factor 1 |
| LSD | lysergic acid diethylamide |
| LSL | loxSTOPlox |
| MAO-A | monoamine Oxidase-A |
| MAP2 | microtubule associated protein 2 |
| MDMA | 3,4-methylenedioxy-N-methylamphetamine |
| MHO | midbrain-hindbrain organizing centre |
| MRN | median raphe nucleus |
| neo | neomycin phosphotransferase cassette |
| OCD | obsessive-compulsive disorder |
| p | postnatal day |
| PBS | phosphate buffered saline |
| PCM | presynaptic cytoskeletal cytomatrix |
| Abbreviation | Description |
|--------------|-------------|
| PCPA | p-chlorophenylalanine |
| PET | positron emission tomography |
| Pet1 | PC12 Ets factor |
| PFA | paraformaldehyde |
| PKA | protein kinase A |
| PKC | Protein kinase C |
| PKG | protein kinase G |
| β-PMA | β-phorbol 12-myristate 13-acetate |
| RRP | readily releasable pool |
| s | short |
| SEM | standard error of mean |
| SERT | 5-HT reuptake transporter |
| SMB | short movement bouts |
| SNP | short nucleotide polymorphism |
| SSRI | selective serotonin reuptake inhibitor |
| SSV | small synaptic vesicle |
| Tetrahydrobiopterin BH4 (6R)-L-erythro-5,6,7,8-Tetrahydrobiopterin | |
| TH | tyrosine hydroxylase |
| tPA-GFP | tissue plasminogen activator-GFP |
| Tph | tryptophan hydroxylase |
| Tph1 | tryptophan hydroxylase 1 |
| Tph2 | tryptophan hydroxylase 2 |
| VAMP2 | synaptobrevin-2 |
| VMAT2 | Vesicular Monoamine Transporter 2 |
Dankwoord
Nu ik aan het einde van mijn promotietraject ben gekomen en het boekje af is, besef ik dat ik een ontzettend leuke en leerzame tijd heb gehad, en daar wil ik graag een aantal mensen voor bedanken.
In de eerste plaats mijn promotor Matthijs. Beste Matthijs, ik wil je bedanken voor het feit dat ik dit project op je lab heb mogen uitvoeren, de begeleiding en de dingen die ik van je geleerd heb. Verder ben ik je dankbaar dat ik ook de ruimte kreeg om mijn eigen invulling aan dit project te geven.
In de tweede plaats mijn copromotor Sander: Beste Sander, door een stage die ik bij jou gelopen heb ben ik op dit lab terecht gekomen, ik ben je daar nog steeds dankbaar voor. Tijdens mijn promotieproject dank ik je voor de begeleiding en het nakijken van mijn manuscripten.
De leescommissie dank ik voor de aandacht die ze aan mijn proefschrift hebben willen besteden.
Verder heb ik veel gehad aan de suggesties en input van Ruud en Joris tijdens onze meetings, mijn dank daarvoor. Oliver, ik zou je graag willen bedanken voor de suggesties met betrekking tot gedragsproeven in muizen en de hulp met de perfusies. Ik dank Pieter Voorn en Menno Witter voor het meedenken met het slice hoofdstuk en Pieter en Allert tevens voor alle hulp op de MCID microscoop.
Mijn dank gaat uit naar mijn beide paranimfen. Beste Joost, bedankt voor al die keren dat je me op het lab een hoetje(s) liet schrikken 😂 Ik heb altijd met heel veel plezier met jou samengewerkt op het lab, maar we hebben ook heel veel lol gehad buiten het werk om.
Beste Arthur, ondanks dat je pas in mijn laatste jaar mijn kamergenoot werd, bleek al snel dat we het qua humor goed met elkaar konden vinden. Behalve onze liefde voor flauwekul bleek ook onze liefde voor (speciale) bier(en) overeen te komen (zou daar een relatie tussen zitten?). Ik wens je alle succes met jouw verdere afronding van je promotietraject en met de voortzetting van het door ons opgezette ‘bureau voor flauwekul’.
Beste Ineke, ik wil je danken voor alle gezelligheid, maar ook voor alle goede gesprekken over de wetenschap en aanverwante zaken. Ik wens je heel veel succes met de verdere afronding van je opleiding tot klinisch chemicus.
Buiten het feit dat ik veel geleerd heb, heb ik ook de sfeer op de afdeling en het lab altijd als zeer positief ervaren. Op het DNA lab, het kloppend hart van de afdeling, heb ik altijd met heel veel plezier gewerkt. Niet in de laatste plaats door
alle gezelligheid die daar altijd te vinden was. Voor al deze gezelligheid, maar zeker ook voor alle hulp ben ik de analisten grote dank verschuldigd.
Beste Robbie, Mr. Salmon, ik ben je dankbaar voor de stage die ik bij jou gelopen heb waarin je me hebt ingewijd in de geheimen van het kloneren. Hoewel ik nog niet eens jouw pipetten mag vasthouden denk ik dat ik inmiddels aardig kan kloneren.
Beste Ingrid, bedankt voor je humor op het lab en voor de hulp met de kloneringen. N.B. Ik heb in de wandelgangen vernomen dat zelfs *jij* nu getrouwd bent :-)
Ik ben ook veel dank verschuldigd aan Desiree en later Boukje voor alle hulp met de neuronen kweek. Bastijn, jou dank ik voor het uitwerken van de Phenotyper data.
Beste Joke, bedankt voor alle hulp met de muizen, maar zeker ook voor je altijd vriendelijke houding.
Dit proefschrift was ook zeker niet mogelijk geweest zonder alle hulp vanuit de muizenstallen. Dus Chris, Bert en Lieselotte, ook jullie hartelijk dank voor alle hulp met de muizen.
Mijn mede aio’s Jurjen, Juliane, Sabine, Marieke, Asiya, Rhea, Jiun, Tony en Gregoire dank ik voor alle discussies en ik wens jullie allen veel succes met de verdere voortzetting van jullie project en de afronding van jullie proefschrift.
Tina en Els jullie dank ik voor alle administratieve ondersteuning. Els, als ik een vraag had wist je die altijd binnen no time te beantwoorden.
Alle andere nog niet genoemde collega’s van het lab dank ik voor alle discussies tijdens labmeetings, gezelligheid tijdens de dagelijkse lablunch en het jaarlijkse labuitje.
Roel, ik dank je voor alle gezelligheid tijdens de treinreizen vanuit Hoofddorp, en ik vind het erg leuk dat we elkaar nog weleens tegenkomen.
Mijn nieuwe lab Neuromedische Genetica op het NIN dank ik voor de gastvrijheid waarmee ik in hun lab ben opgenomen en de plezierige werksfeer.
Gijs, Ray en Robert, we hebben altijd ontzettend veel lol gehad tijdens onze studieperiode. Ik ben blij dat we nog weleens afspreken. Wanneer gaan we weer poolen (of was het nou snooker...?).
Als laatste zou ik graag een aantal mensen willen bedanken uit mijn persoonlijke omgeving.
De mannen uit Renesse: Alex, Bart, Kenneth, Michael; bedankt voor alle nachtelijk stapuurtjes en (wintersport) vakanties.
Beste familie Visser, ik dank jullie voor alle gezelligheid en alle borreltjes die we al met elkaar gedronken hebben. Wat is het bij jullie toch altijd lekker rustig... :-)
Ik dank mijn schoonouders, Jan en Christien, mijn schoonzussen en zwagers voor de interesse in mijn proefschrift en alle feesten, familiedagen en andere gezellige activiteiten. Is die Eiffeltoren nou al gevonden?
Mijn broer(tje) en zus(je), ik dank jullie voor de interesse in mijn proefschrift, de gezelligheid tijdens het meerijden naar Renesse en ik wens jullie alle succes met de afronding van jullie studie.
Mijn ouders, ‘promoveren zit dat erin dan?’, Frans en Lia, ik ben heel trots op jullie en ik ben blij jullie zoon te zijn. Ik realiseer me maar al te vaak wat een rijkdom het was om zo te mogen opgroeien. Ik dank jullie voor alle steun en vertrouwen, maar ook voor alle gezelligheid. Als we elkaar zien is het altijd erg gezellig en vaak ineens midden in de nacht als we zitten te kletsen onder het genot van een wijntje.
Als laatste dank ik natuurlijk mijn lieve vrouw Judith. Sinds die kapotte printer zijn we alweer bijna 7 jaar bij elkaar, wonen we al ruim 3 jaar in Hoofddorp en zijn we al ruim 1 jaar getrouwd. Time flies when having fun, wil ik daar alleen maar even mee zeggen. Ik wens jou ook alle succes met de verdere afronding van jouw promotieproject en boekje, en heb zin om samen de toekomst tegemoet te gaan!
Jeroen
Hoofddorp, Juli 2010
Curriculum vitae
Jacobus Johannis Dudok werd geboren op 13 juni 1981 te Zierikzee. In 1993 begon hij aan zijn VWO opleiding aan het Buys Ballot College te Goes, waar hij in 1999 zijn Gymnasium Beta diploma haalde. In augustus 1999 begon hij aan de Bachelor Biomedische Wetenschappen aan de Vrije Universiteit te Amsterdam. In deze opleiding liep hij zijn Bachelor stage van februari 2002-juni 2002 aan de vakgroep pathologie in het VU medisch centrum. Hier bestudeerde hij, begeleidt door Dr. R. Veerhuis en Ing. E. van Elk, de verschillende aggregatie vormen van Aβ1-42, het eiwit betrokken bij de ziekte van Alzheimer. In september 2002 begon hij aan de Master of Neurosciences aan de Vrije Universiteit te Amsterdam. Tijdens deze master deed hij twee stages bij de afdeling Functionele Genoomanalyse. Tijdens de eerste masterstage deed hij onderzoek naar de calcium afhankelijke translocatie van het presynaptische eiwit Doc2B, begeleidt door Dr. S. Groffen. Tijdens zijn tweede masterstage, begeleidt door Ing. R. Zalm, cloneerde hij een targeting construct en gebruikte deze om muis embryonale stamcellen mee te targetten. Zijn masterscriptie schreef hij onder leiding van Dr. J. Pronk en ging over de in silico clonering en karakterisatie van het eiwit Tph2. In augustus 2004 studeerde hij af aan de Vrije Universiteit te Amsterdam. Van oktober 2004 tot april 2009 deed hij zijn promotieonderzoek naar de uitgroei en connectiviteit van het serotonerge systeem. Dit onderzoek deed hij onder begeleiding van professor Verhage aan de vakgroep Functionele Genoomanalyse aan de Vrije Universiteit te Amsterdam. De resultaten van dit promotieonderzoek zijn in dit proefschrift beschreven. Vanaf april 2009 werkt hij als postdoctoraal onderzoeker op het Nederlands Instituut voor Neurowetenschappen (NIN) bij de vakgroep Neuromedische Genetica onder leiding van Dr. J. Wijnholds. Hier doet hij onderzoek naar de functionele rol van het eiwit MPP3 in het Crumbs complex en in neuronale cellen.
Jacobus Johannis Dudok was born on the 13th of June 1981 in Zierikzee, the Netherlands. In 1993 he started his secondary education at the Buys Ballot College in Goes, where he graduated in 1999. In August 1999 he started the Bachelor Medical Biology at the Vrije Universiteit Amsterdam. During this Bachelor he carried out a research project from February 2002 to June 2002 at the research department of pathology at the VU medical center. Here he studied the different aggregation states of Aβ1-42, the peptide involved in Alzheimer’s disease, supervised by Dr. R. Veerhuis and Ing. E. van Elk. In September 2002 he started the Master of Neuroscience at the Vrije Universiteit Amsterdam. During this master he carried out two research projects at the department of Functional Genomics. During his first research project he studied the calcium dependent translocation of the presynaptic protein Doc2B, supervised by Dr. S. Groffen. During his second research project he constructed a targeting construct and used this to target mouse embryonic stem cells, supervised by Ing. R. Zalm. He wrote his Master Thesis under supervision of Dr. J. Pronk about the in silico cloning and characterization of the protein Tph2. In August 2004 he graduated at the Vrije Universiteit Amsterdam. From October 2004 until April 2009 he did his PhD project on the outgrowth and connectivity of the serotonin system. This PhD project was supervised by professor Verhage and carried out at the department of Functional Genomics at the Vrije Universiteit Amsterdam. The results of that study are described in this thesis. He is currently working as a postdoctoral fellow at the Netherlands Institute for Neuroscience (NIN) in the department of Neuromedical Genetics headed by Dr. J. Wijnholds. Here he studies the functional role of the protein MPP3 in the Crumbs complex and in neuronal cells.
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